Pharmacology of Drug Stereoisomers (Progress in Drug Research, 76) 9811923191, 9789811923197

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Progress in Drug Research 76

Alexander A. Spasov Igor N. Iezhitsa Pavel M. Vassiliev Alexander A. Ozerov Renu Agarwal

Pharmacology of Drug Stereoisomers

Progress in Drug Research Volume 76

Series Editor K.D. Rainsford, Sheffield Hallam University, Sheffield, UK

Progress in Drug Research is a prestigious book series which provides extensive expert-written reviews on highly topical areas in current pharmaceutical and pharmacological research. Founded in 1959 by Ernst Jucker, the series moved from its initial focus on medicinal chemistry to a much wider scope. Today it encompasses all fields concerned with the development of new therapeutic drugs and the elucidation of their mechanisms of action, reflecting the increasingly complex nature of modern drug research. Invited authors present their biological, chemical, biochemical, physiological, immunological, pharmaceutical, toxicological, pharmacological and clinical expertise in carefully written reviews and provide the newcomer and the specialist alike with an up-to-date list of prime references. Starting with volume 61, Progress in Drug Research is continued as a series of monographs and contributed volumes.

Alexander A. Spasov · Igor N. Iezhitsa · Pavel M. Vassiliev · Alexander A. Ozerov · Renu Agarwal

Pharmacology of Drug Stereoisomers

Alexander A. Spasov Department of Pharmacology and Bioinformatics Volgograd State Medical University Volgograd, Russia Pavel M. Vassiliev Department of Pharmacology and Bioinformatics Volgograd State Medical University Volgograd, Russia

Igor N. Iezhitsa Department of Pharmacology and Therapeutics School of Medicine, International Medical University Kuala Lumpur, Malaysia Alexander A. Ozerov Pharmaceutical and Toxicological Chemistry Volgograd State Medical University Volgograd, Russia

Renu Agarwal Department of Pharmacology and Therapeutics School of Medicine, International Medical University Kuala Lumpur, Malaysia

ISSN 0071-786X ISSN 2297-4555 (electronic) Progress in Drug Research ISBN 978-981-19-2319-7 ISBN 978-981-19-2320-3 (eBook) https://doi.org/10.1007/978-981-19-2320-3 © Springer Nature Singapore Pte Ltd. 2022 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

The optical activity of substances in solution was first discovered by French physicist Jean-Baptiste Biot in 1815, and optically active compounds from inactive racemic mixtures were first obtained by Louis Pasteur in 1848. These fundamental discoveries promoted the development of research that focused on investigating the role of steric factors in biology. So far, it has been established that optical, geometric and spatial stereovariety underlie the existence of all living things and differentiate them from non-living things. Many biologically active molecules such as amino acids, sugars and nucleic acids exist in the form of one predominant stereoisomer. Naturally occurring proteins consist of L-amino acids and are known as left-handed proteins. The biologically active centres of enzymes and receptors are stereoselective in their spatial arrangement to correspond with receptor agonists and substrates of natural biochemical reactions. Similarly, there are molecules with D-configuration in the DNA and RNA. It should be mentioned, however, that biologically active molecules and substrates of biochemical reactions in the humans and animals are homochiral (either L- or D-optic form, etc.). The origin of homochirality in biology is an extensively debated issue. Most authors are of the opinion that the choice of chirality in the process of emergence of living matter was purely accidental. At the same time, research into fundamental causes of the choice of chirality on Earth is under way. Fundamental discoveries in the field of stereochemistry by scientists such as Louis Pasteur, Edmond Henri Fischer, Vladimir Prelog, William S. Knowles, Ry¯oji Noyori, Christopher Kelk Ingold and Karl Barry Sharpless gave insight into the nature of stereoselectivity in chemical and biochemical reactions and made it possible to develop methods for isolating pure monochiral substances. When the synthesis of biologically active molecules is designed, pharmacologists try to achieve structures resembling that of active protein molecules (receptors, enzymes etc.) and endogenous ligands. Pharmacological and toxicological properties of drug stereoisomers became an issue when the infamous thalidomide disaster was investigated. This drug was produced as a racemate, where one optical isomer provided its pharmacological action, and the other one was found to have a teratogenic effect. This triggered an initiation of regulatory affairs for preclinical and clinical research v

vi

Preface

of both racemates and the isomers composing the racemates. Several thousand chiral synthetic pharmaceutical substances and over 2,000 chiral drugs have now been synthesized and described. This approach to optimization of drug molecules is known as “chiral switches” (Tucker 2000). A significant contribution to the study of pharmacological properties of optical isomers and to stereochemistry was made by Russian scientists under the guidance of academician Sergei Nikolaevich Golikov. The monograph “Stereospecificity of drugs action” (Golikov et al. 1973) summarises the experience of developing substances affecting the cholinergic and adrenergic systems, as well as of developing local anaesthetic drugs. In recent years, there has been considerable progress in studying biological properties of virtually all components of many racemates. The study of biological properties (pharmacological and toxicological) of all components of many racemic drugs has led to the development of new drugs based on pure optical isomers. The authors of the present work studied the pharmacological properties of optical isomers of carnitine, potassium and magnesium salts of aspartic acid when developing Elcar and Asparcam-L drugs, and this experience provided the basis for the subject matter presented in this book. Chapter 1 of the book sets general principles of molecular chirality and IUPAC classification. Chapter 2 describes the methods of separating isomers, asymmetric synthesis and analysis. Chapter 3 outlines general issues of chirality in pharmacology. Chapter 4 addresses the significance of chirality in pharmacological and toxicological properties of drugs. Chapter 5 describes pre-experimental computerised methods of in silico search for chiral drug compounds. The authors did not assign themselves the task of describing all racemic mixtures or pure isomers of drugs. In this book, the authors draw the attention of specialists developing or using drugs to the problems due to differences in the pharmacology of isomers and present an analysis of research in this field. Volgograd, Russia Kuala Lumpur, Malaysia Volgograd, Russia Volgograd, Russia Kuala Lumpur, Malaysia

Alexander A. Spasov Igor N. Iezhitsa Pavel M. Vassiliev Alexander A. Ozerov Renu Agarwal

References Golikov SN, Kuznetsov SG, Zatsepin EP (1973) Stereospecificity of drug action. Meditsina, Leningrad, 184 p Tucker GT (2000) Chiral switches. Lancet 355(9209):1085-1087. https://doi.org/10.1016/S01406736(00)02047-X

Contents

1 General Principles of Molecular Chirality . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Types of Isomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Stereoisomers with Different Chirality Types . . . . . . . . . . . . . . . . . . 1.3 Conformational Isomerism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 3 15 19 21

2 Separation, Asymmetric Synthesis and Analysis of Stereoisomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Separation of Stereoisomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Quantitative Methods of Analysing Chiral Drugs . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

23 23 29 31

3 General Issues of Chirality in Pharmacology . . . . . . . . . . . . . . . . . . . . . . 3.1 Chirality and Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Chirality and Pharmacodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Chirality and Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Classification of Drugs from Stereochemical Point of View . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

35 36 56 61 63 67

4 The Significance of Chirality in Pharmacological and Toxicological Properties of Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Drugs Regulating the Function of Peripheral Nervous System . . . . 4.2 Drugs Affecting the Central Nervous System Function . . . . . . . . . . 4.3 Drugs Regulating the Functions of Major Organs and Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Drugs Affecting the Immunity Processes . . . . . . . . . . . . . . . . . . . . . . 4.5 Drugs Used in Endocrine Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 Antibacterial Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7 Antitumor Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8 Pros and Cons of Administering Drugs in the Form of Racemic Mixtures or Single Stereoisomers in Clinical Practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

75 75 100 126 144 149 153 163

164

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Contents

4.9 Rules of Developing and Administering Chiral Drugs . . . . . . . . . . . 166 4.10 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 5 In Silico Search for Chiral Drug Compounds . . . . . . . . . . . . . . . . . . . . . 5.1 Ways of Describing Chirality of Compounds for in Silico Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2 2D in Silico Analysis of Chiral Compounds . . . . . . . . . . . . . . . . . . . 5.3 3D in Silico Analysis of Chiral Compounds . . . . . . . . . . . . . . . . . . . 5.4 Molecular Docking of Chiral Compounds . . . . . . . . . . . . . . . . . . . . . 5.5 Prediction of Pharmacological Activity of Chiral Compounds Using IT Microcosm . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

193 193 194 196 198 200 204 205

Abbreviation

(–):(+) (+):(–) 2D3D5-HT ab initio AMP AMPA-receptor AP ATP AUC CAPE CCK Cl Cmax CNS CoA CoMFA CoMSIA COX CYP DAO DHP DNA DOPA EC50 ECG

Enantiomer drug index Enantiomer drug index Two-dimensional Three-dimensional 5-hydroxytriptamine, serotonin “From the beginning”, a general name for computational chemistry methods based on precise high-level quantum chemistry Adenosine monophosphate α-amino methylisoxazol propionic acid sensitive receptor Arterial pressure Adenosine triphosphate Area under the curve: area under the plasma concentration versus time curve Circadian Anti-ischemia Program in Europe Cholecystokinin receptor Total clearance of a drug Maximum drug concentration in blood plasma Central nervous system A coenzyme Comparative Molecular Field Analysis, one of the in silico methods Comparative Molecular Similarity Index Analysis, one of the in silico methods Cyclooxigenase Enzymes of cytochrome P450 system Oxidase of D-amino acids Dehydropeptidase Deoxyribonucleic acid Dihydroxyphenylalanine Half maximal effective concentration Electrocardiogram ix

x

ED50 EEG ex vivo

F0 Fa FDA Fn GABA HIV hOCT hPepT HPLC HQSAR HR IC50 IHD in silico in vitro in vivo IUPAC Ki Km LD50 MAO mcM MDDR mM nM NMDA-receptor NSAID OAT OATP OCT PDB PDE per os pH pKi

Abbreviation

Median Effective Dose (produces desired effect in 50% of population) Electroencephalogram Experimentation or measurements done in or on tissue in an artificial environment outside the organism with the minimum alteration of natural conditions General accuracy of a prediction Accuracy of a prediction of active compounds US Food and Drug Administration Accuracy of a prediction of inactive compounds γ-aminobutyric acid Human immunodeficiency virus Human Organic Cation Transporter Human oligopeptide transporter High-performance liquid chromatography Hologram QSAR, one of the in silico methods Heart rate Concentration with 50% inhibition Ischemic heart disease “In silicon”, a general name for computerized methods of drug research A general name for drug research performed not in a living organism but in a controlled environment A general name for drug research methods using a whole, living organism International Union of Pure and Applied Chemistry Constant bonding the ligand with biotarget Michaelis constant Lethal dose-50 Monoamino oxidase Micromole MDL Drug Data Report, database on structure and biological activity of chemical compounds Millimole Nanomole Receptor sensitive to N-methyl-D-aspartate Nonsteroid anti-inflammatory drug Organic Anion Transporter Organic Anion Transport Protein Organic Cation Transporter Protein Data Bank Phosphodiesterase By the mouth A measure of acidity Binding affinity of ligand for biotarget

Abbreviation

Q2 QL QSAR R:S R2 RNA RXR S:R SAP t1/2 Vd Vmax Vss

xi

Squared coefficient of multiple correlation in cross validation QSAR Language Quantitative Structure-Activity Relationships Enantiomer drug index Squared coefficient of multiple correlation Ribonucleic acid Retinoid X-receptor Enantiomer drug index Systemic arterial pressure Elimination half-life Apparent volume of drug distribution Maximum rate of product formation in enzymatic reaction Steady state volume of distribution

Chapter 1

General Principles of Molecular Chirality

Several aspects of stereochemistry are discussed in detail in the following chapters of this book and therefore, for better understanding we find it necessary to describe here the definitions of stereochemical terms used in scientific literature according to IUPAC nomenclature (Moss 1996; IUPAC 2014). Stereochemistry, a subdiscipline of chemistry, involves the study of the relative spatial arrangement of atoms within molecules and its effect on physical and chemical properties of substances. Chirality is a property of molecules having a non-superimposable mirror image. The opposite property is called achirality. The main types of molecular chirality are central, axial, planar, helical and topological depending on the chirality element. Chirality element is the smallest structural fragment of a molecule that possesses chirality. It can be a centre, axis, plane, helical or a topological bond. Chiral molecule has one or several chirality elements. Such a molecule does not have Sn rotoflexion axes of symmetry. Symmetry element is a geometrical entity making an object (a molecule) achiral. These can be a symmetry centre, axis of symmetry of nth order, plane of symmetry, rotoflexion axis of symmetry of nth order. Isomerism refers to the existence of molecules that have the same numbers of the same kind of atoms (and hence the same formula) but differ in chemical and physical properties (isomers). Molecular topological form is a geometric figure (in the topological sense of the word) characterizing the spatial arrangement of the nuclei of the given object in combination with specific points. Stereochemical configuration characterizes the arrangement of atoms or groups of atoms of a molecular entity in space (when there is chirality, a molecular topological form is preserved). © Springer Nature Singapore Pte Ltd. 2022 A. A. Spasov et al., Pharmacology of Drug Stereoisomers, Progress in Drug Research 76, https://doi.org/10.1007/978-981-19-2320-3_1

1

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1 General Principles of Molecular Chirality

Stereoisomerism (stereochemical, spatial isomerism) is the isomerism due to differences in the spatial arrangement of atoms without any differences in connectivity or bond multiplicity between the isomers; these are called stereoisomers (stereochemical, spatial isomers). Optical activity is the ability of the material to rotate the plane of a beam of transmitted plane-polarized light. It is the characteristic of only chiral compounds. The dextrorotatory isomers causing rotation in a clockwise direction (+)- need to be distinguished from those causing rotation in the opposite direction and hence are laevorotatory and designated by the prefix (−)-. Enantiomers are a pair of molecular entities which are mirror images of each other. In this case asymmetric atom is the center of chirality (an asymmetric carbon atom, as a rule). Racemate is an equimolar mixture of a pair of enantiomers. Diastereomers are stereoisomers not related as mirror images. Geometric isomers are stereoisomers obtained as a result of cis–trans isomerism. Topological stereoisomers are stereoisomers obtained as a result of a topological link. Conformation refers to any relative arrangement of molecular nuclei in space achievable without disrupting the integrity of the molecule. Each particular conformation is matched by particular energy. Conformer is any set of stereoisomers characterized by a conformation that corresponds to a distinct potential energy minimum. The existence of conformers is referred to as conformational isomerism. Chiral separation is a process for the separation of racemic compounds into their enantiomers. Chiral selector is an enantiomer used to obtain diastereomers from a racemate separated into optical isomers of a compound. Chiral phase is a phase in chromatography containing a chiral compound. Epimers are diastereoisomers that have the opposite configuration at only one chiral centre. A correct understanding of the essentials of biochemical interactions is facilitated by stereochemical theory developed by the great chemists in the nineteenth century. Major contribution in the field of stereochemistry were made by Louis Pasteur in France, Friedrich August Kekulé in Prussia, Jacobus Henricus van’t Hoff in the Netherlands, and Josef Le Bel in France (Golikov et al. 1973).

1.1 Types of Isomers

3

1.1 Types of Isomers Structural isomers In the early nineteenth century the chemists were baffled by the fact that compounds with the same empirical formula can differ in their properties. For instance, dimethyl ether and ethyl alcohol have the same empirical formula of C2 H6 O and molecular weight of 46. However, their physical properties are discernibly different. Dimethyl ether is a gas with a boiling temperature of −24 °C, while ethyl alcohol is a liquid with a boiling temperature of 79 °C. In his classical work published in 1858 German chemist August Kekulé proposed explanation for the differences in the properties of isomers. Taking into account the observation made by the English chemist Edward Franklin that every atom can only attach to a certain amount of other atoms, Kekulé stated that the valency of carbon should be four. He also proposed that bonds can form between carbon atoms leading to chain formation. That is how the concept of chemical bond came about. Kekulé began to represent the bonds between atoms with lines and stated that isomers emerge as a result of atom bonding in different ways. At present such isomers are referred to as structural isomers. For example, formulas of structural isomers, ethyl alcohol and dimethyl ether, can be represented in the following way (Fig. 1.1). To explain the structure of certain molecules, Kekulé introduced the notion of double and triple bonds. Accordingly, carbon dioxide should have two double bonds and the nitrogen atom in hydrogen cyanide should be attached to the carbon atom with a triple bond (Fig. 1.2). Kekulé’s structural formulas provided two-dimensional representation of a molecule. In 1874, two chemists, Jacobus Henricus van ’t Hoff and Joseph Achille Le Bel working independently, suggested considering a carbon atom as a threedimensional object. According to this notion, carbon is represented as a tetrahedron with the bonds directed at its vertices. Thus single, double and triple carbon-to-carbon bonds can be represented as tetrahedrons connected with their vertices, edges and faces with a carbon atom at the centre of each (Fig. 1.3). Another problem perplexing the nineteenth century chemists was the presence of two strikingly different classes of organic compounds, fatty (aliphatic) and aromatic Fig. 1.1 Structural isomers H

H

H

C

C

H

H

O

H

H

C

O

H

Ethyl alcohol

Fig. 1.2 Compounds with multiple bonds

H

H

O

C

H

H

Dimethyl ether

C

O

Hydrogen dioxide

H

C

N

Hydrogen cyanide

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1 General Principles of Molecular Chirality

Fig. 1.3 Tetrahedral representation of carbon-to-carbon bonds C C

Fig. 1.4 Structural formula of benzene

C C

C C

H C

H C

H

C

C C H

C

H

C

C H

H

C H

H

H C C

C

H

H

Fig. 1.5 Simplified structural formula of benzene

ones. Compounds of the first group are comparatively stable and include soaps, alcohols and lubricants. Aromatic compounds are often easily volatile and have an odor. They always contain at least six carbon atoms and as compared to aliphatic compounds have a higher content of carbon atoms compared to other atoms. In 1864, Kekulé proposed an explanation for the structure of aromatic compounds. He stated that all aromatic compounds consist of 6-carbon atom benzene ring. Since the empirical formula of benzene C6 H6 was known then, Kekulé assumed that its structural formula could be represented in either of the two ways (Fig. 1.4). For the sake of simplicity, it is often shown in a shorter form (Fig. 1.5). According to Kekulé, since all carbon atoms in the benzene ring are equal, substitution of any hydrogen atom by any group (e.g. OH, NH2 , CH3 , COOH) yields the same monosubstituted product. However, the disubstituted products can be of three types depending on the relative position of the substituent groups. The substituent groups can be next to each other, at the nearest carbon atoms (ortho position), at alternate carbon atoms (meta position) or at opposite carbon atoms (para position). Different types of disubstituted benzene derivatives are given below. It is of particular interest to note the difference in physical properties (melting points) of the three disubstituted products, all of which have the same empirical formula of C7 H6 O3 (Fig. 1.6). This type of structural isomerism is called the position isomerism. It is worthwhile to say that position isomers are found in all classes of organic compounds. Valence isomerism is an interesting type of structural isomerism where the isomers can only interconvert through redistribution of bonds. For example, benzene and prismane are valence isomers sharing the same empirical formula C6 H6 (Katz, 1973) (Fig. 1.7).

1.1 Types of Isomers

5

COOH

COOH

COOH

OH

OH OH

o-Oxybenzoic acid (salicylic acid) Melting point 158-161 оС

m-Oxybenzoic acid

p-Oxybenzoic acid

Melting point 201-203 оС

Melting point 215-217 оС

Fig. 1.6 Position isomers

Fig. 1.7 Valence isomers

Benzene

Prismane

Geometric isomers Geometric, or cis–trans, isomerism is a type of stereoisomerism typical of compounds with a double (ethylene) bond or a cyclic structure, polymethylene in particular. As free rotation about carbon-to-carbon bonds is impossible, the structure of molecules of these compounds is quite rigid. A different position of any two atoms or groups in relation to these rigid structures yields a geometric isomer. In a cis isomer two substituents are placed on the same side of the plane of double bond or ring whereas in a trans isomer they are placed on opposite sides (Fig. 1.8). Geometric isomers have the same sequence of bonds in the molecule. They only differ in the spatial arrangement of atoms or the groups of atoms. However, in contrast to optical isomers they have different distances between the rows of mutually unattached atoms and groups of atoms, and these distances are quite rigidly fixed. In this respect cis and trans isomers significantly differ in the nature of the mutual effect of atoms on one another, hence different physical and chemical properties. Geometric isomers also show considerably greater difference in the shape of their molecules than optical isomers do (Goodman and Morehouse 1973). The difference in the chemical properties of geometric isomers is illustrated by the difference in the constants of consecutive dissociation of ethylenedicarbonic acid. For maleic acid (cis isomer) K1 = 1,3 · 10–2 and K2 = 3,2 · 10–7 while for fumaric acid (trans isomer) K1 = 1,0 · 10–3 and K2 = 3,2 · 10–5 . Besides, out of these two, only maleic acid can convert to an anhydride (Fig. 1.9).

6

1 General Principles of Molecular Chirality

OH

C2H5

C2H5

C2 H5

C2H5

HO

HO

OH cis-Diethylstilbestrol

O

H

trans-Diethylstilbestrol (synthetic estrogen)

H

CH3

CH3

O

H

H

P(O)(OH)2

cis-(1,2-Epoxypropyl)phosphonic acid (phosphomycin antibiotic)

P(O)(OH)2

trans-(1,2-Epoxypropyl)phosphonic acid (low-activity compound)

Fig. 1.8 Cis–trans isomers HOOC

COOH 150o C

H

O

O

HOOC

H

Maleic acid

H

O H

Maleic anhydride

COOH

Fumaric acid

Fig. 1.9 Difference in the chemical properties of cis and trans isomers

In complex cases, for instance, when there are more than two heterogeneous substituents around a double bond, cis–trans nomenclature is not applicable. Instead, E-Z nomenclature is used as described below. 1.

With the help of Cahn-Ingold-Prelog system (see below) the relative priority of substituents is established for each pair in the ethylene unit of the molecule or at carbon atoms of the cyclic structure. The highest priority substituent in each pair is assigned—number 1, the lowest priority substituent—number 2.

1.1 Types of Isomers

7

(2) H

Fig. 1.10 Use of E-Z-nomenclature in the names of geometric isomers

Cl (1)

C(O)H (2) (1)

O2N

(Е)-para-Nitro-α-chlorocinnamic aldehyde

2.

If the highest priority groups in each pair of substituents are found on the same side from the plane of double bond or cyclic structure, the configuration is represented with the symbol Z (from German word “zusammen” i.e. together). If the highest priority substituents are found on opposite sides, the configuration is represented with the symbol E (from German word “entgegen” i.e. opposite).

For instance, in para-nitro-α-chlorocinnamic aldehyde (Ciminalum, antiseptic and wound-healing medication), in the right pair of substituents the chlorine atom has a higher priority than the aldehyde group and in the left pair of substituents paranitrophenyl radical has a higher priority order than hydrogen. That is why the configuration of this acid is designated as E despite the fact that the largest substituents (phenyl and the aldehyde group) are in cis relation to each other (Fig. 1.10). This system is also applicable to alkenes and cyclic compounds, for which cis– trans nomenclature is commonly used. Thus, due to its universal applicability, E-Z system is gradually ousting the cis–trans nomenclature. The difference in physical and chemical properties and geometric parameters of molecules determines the difference in the physiological activity of geometric isomers. Geometric isomerism is of considerable importance in the study of drug’s interactions with receptors and active centers of enzymes. Optical isomers Another type of isomerism, optical isomerism, is due to the ability of some substances to rotate the plane of polarized light passing through it. Optical activity was first discovered by a French physicist Jean-Baptiste Biot in 1815. He discovered that solutions of some naturally occurring compounds are optically active. After this discovery chemists routinely studied optical activity of new compounds. It turned out that substances synthesized in laboratory conditions are optically inactive, but many naturally occurring compounds possess optical activity. Some substances rotated the plane of polarized light to the right, and these were called dextrorotatory (d-forms); others rotated the plane of polarization to the left, so they were called laevorotatory (l-forms). Louis Pasteur working with tartaric and paratartaric acid explained the phenomenon of optical activity. His research played a key role in the understanding

8

1 General Principles of Molecular Chirality

of chemical structure and properties of substances. What impresses one even more is the fact that Pasteur’s work anticipated the ideas of Kekulé, van’t Hoff, and Le Bel about chemical bonds. Pasteur knew that tartaric and paratartaric acids share the same empirical formula of C4 H6 O6 . It was known that tartaric acid is optically active while paratartaric acid is optically inactive and much less soluble than tartaric acid. In 1848 Pasteur studied the size of crystals of nineteen different salts of tartaric acid. He noticed what was overlooked by other researchers before him—the crystals of all 19 salts turned out to be hemihedral, i.e. they had only half of the symmetry. Asymmetrical crystals had two hemihedral forms, right and left. Different hemihedral crystals for asymmetrical molecules were mirror images of each other like the right and left hands of a human are mirror images of each other. Pasteur also noticed that all nineteen salts of tartaric acid were hemihedral in one direction. This is of no wonder as solutions of these salts rotate the plane of polarized light in one direction (to the right). Since paratartaric acid was optically inactive, Pasteur assumed that crystals of the salts of paratartaric acid, sodium ammonium racemate, would be symmetrical. To his utter surprise, after performing crystallization at a temperature below 28 °C Pasteur found that some of the crystals were hemihedral to the right and some to the left. Pasteur managed to divide the two types of crystals with pincers. Converting the right sided crystals of sodium ammonium racemate into an acid, he discovered that he had obtained tartaric acid. On the basis of this fact Pasteur came to the conclusion that paratartaric acid is a mixture of dextrorotatory and laevorotatory tartaric acids. Optical isomers like d- and l-forms of tartaric acid are called enantiomorphs, enantiomers or optical antipodes. Their physical and chemical properties are identical, except for the direction of rotation of the plane of polarized light. At the time when Pasteur performed his research, the notion of structural formulas was not known. Nevertheless, Pasteur arrived to the correct conclusion that there should be stereochemical difference between the molecules of d- and l-tartaric acids. Later it was demonstrated that tartaric acid can be represented with the structural formula shown in Fig. 1.11. Mesotartaric acid is optically inactive due to the fact that there is a plane of symmetry between the inferior and superior halves of the molecule. Paratartaric acid is a racemic mixture of d- and l-forms of tartaric acid. COOH H

COOH

COOH

OH

HO

H

H

HO

H

HO

OH

COOH HO

H

= H

OH COOH

COOH

Mesotartaric acid (optically inactive)

Fig. 1.11 Stereoisomers of tartaric acid

H

H

OH

COOH

COOH

d-Tartaric acid (dextrorotatory)

l-Paratartaric acid (laevorotatory)

1.1 Types of Isomers

9

CH3

CH3

CH3

H3 CHN

H

H

NHCH3

OH

H

H

HO

C6H5

erythro-(+)Ephedrine

H3 CHN

C6H5

erythro-(–)Ephedrine

CH3 H

H

HO C6H5

threo-(–)Ephedrine

NHCH3

H

H

OH C6H5

threo-(+)Ephedrine

Fig. 1.12 Ephedrine enantiomers

At present symbols (+)- and (–)- rather than d- and l- are employed to show dextroand laevorotatory forms of optical isomers, correspondingly. When the number of asymmetrical atoms in the molecule of a substance increases, the possible number of stereoisomers increases exponentially. If a compound with a single chiral centre can exist in the form of two stereoisomers, a compound with two asymmetrical atoms can exist in the form of four and the one with three in eight forms. Generally, the number of stereoisomers for a compound with n number of asymmetrical atoms equals 2n . For exmple, ephedrine with 2 asymetric C-atoms has 4 isomers (Fig. 1.12). Erythro and threo isomers in pairs are also optical antipodes (enantiomers). In each pair of enantiomers, (erythro-(+)- and erythro-(−)-), the configurations of both asymmetrical carbon atoms are mutually converse. At the same time, if we correlate any erythro isomer with any threo isomer, it is evident that their configurations at one centre of asymmetry coincide, while configurations at the other centre are converse. Such pairs of stereoisomers are not mirror images of each other and they are called as diastereoisomers or diastereomers. Unlike enantiomers, diastereomers have different physical properties, as a rule. Due to the difference in interaction with mutually unattached atoms, diastereomers also differ from one another in their chemical properties. Fischer projections (D-L-nomenclature) In 1891, Emil Fischer proposed a system based on the configuration of saccharic acid for representation of carbohydrates with asymmetrical carbon atoms (Fig. 1.13). When depicting a projection formula of saccharic acid, Fischer arbitrarily placed the hydroxyl group at the fifth carbon atom, on the right side. Since dextrorotatory glucose can be converted into dextrorotatory saccharic acid, Fischer proposed that dextrorotatory glucose should have the same spatial configuration as dextrorotatory saccharic acid. He called this glucose, D-glucose. Other groups at other asymmetrical carbon atoms of glucose were arranged in a certain way in relation to C-5. Mirror image of D-glucose was designated as L-glucose. Not all sugars classified as D-family can rotate the plane of polarized light to right. Some of them rotate the plane of polarized light to left. That is why for a complete

10

1 General Principles of Molecular Chirality

H

OH

HO

H

H H

5

C(O)H

C(O)H

COOH H HO

OH H

HO H

H OH

OH

H

OH

HO

H

OH

H

OH

HO

H CH2 OH

CH2 OH

COOH

D-Glucose

Saccharic acid

L-Glucose

Fig. 1.13 Use of saccharic acid in D-L-nomenclature of optical isomers

characterization of a sugar the (+) and (–) symbols are used in addition to D and L symbols. For instance, D-(–)-ribose is laevorotatory, but belongs to the D-family due to its spatial configuration at the penultimate carbon atom C-4. Glycerine aldehyde is the chosen standard reference for establishing relative configurations of various compounds because it has only one asymmetric carbon atom compared to glucose which has four (Fig. 1.14). Compounds for which D-(+)-glycerin aldehyde is a parent substance are classified as belonging to the D-family. Those which have L-(–)-glycerin aldehyde as a parent substance, belong to the L-family. The application of Fischer projections (DL-nomenclature) can be extended to the non-carbohydrate compounds systematically as described below. For instance, alanine amino acid can be classified as belonging to the D-family or L-family depending on spatial configuration of a particular alanine under consideration. C(O)H H

OH

H

OH

H

4

OH

C(O)H H

OH

C(O)H HO

CH2OH

H CH2 OH

CH2OH D-(–)-Ribose

D-(+)-Glycerin aldehyde

L-(–)-Glycerin aldehyde

Fig. 1.14 Use of glycerin aldehyde in D-L-nomenclature of optical isomers

1.1 Types of Isomers

11

It should be highlighted that the D-L-nomenclature and (+, –)- system of notation do not have a one-to-one correlation. Rules of working with Fischer projections For an unambiguous structural correlation, one should observe the following rules: 1.

2. 3. 4.

5. 6.

Bonds depicted with vertical lines are placed under the plane of molecule representation (paper, screen), and the bonds depicted with horizontal lines—above this plane. At the same time, the asymmetrical carbon atom found at cross sections of horizontal and vertical bonds may not be depicted. The molecule is placed in such a way that hydrogen and heteroatom at the asymmetrical centre should be in horizontal plane. “Vertical groups” are placed in such a way that the most oxidized group is uppermost. An exchange of any two substituents at the asymmetric centre, as well as rotation of the molecule in the plane of its depiction by 90° or 270° changes the configuration of this asymmetrical carbon atom converting the molecule to its enantiomer. Exchange of any three substituents at the asymmetric centre or rotation of the molecule by 180° does not lead to a change in the configuration. As the projection represents the orientation of the molecule in three-dimensional space, we cannot draw Fischer model from the plane of its representation.

D-L-nomenclature is the oldest system of notation of optical antipodes and it is still in use. It is based on comparison of the projection formula of the stereoisomer under consideration with the projection formula of a certain standard substance chosen as “key”. Thus for alpha-hydroxy acids (X = OH) and alpha-amino acids (X = NH2 ) the key is the superior part of their Fischer projection formula (Fig. 1.15). That is why the configuration of all alpha-hydroxy acids with a hydroxyl group on the left in the standard Fisher projection formula is designated with the L sign; if the hydroxyl is on the right in the projection formula, it is designated as D. Glycerin aldehyde is the key for representing the configuration of sugars. Therefore, for sugar molecules the designation D or L refers to the configuration of the lower asymmetry centre. The D-L-system can also be applied to compounds of other classes possessing more than one chiral centre. In this case, however, the position of substituents at other chiral centers needs to be specified in accordance with resemblance to sugars threose (threo) or erythrose (erythro) (Fig. 1.16). For example, chloramphenicol Fig. 1.15 Key of D-L-designations of optical isomers of α-hydroxy- and α-amino acids

COOH H

R X

D-Isomers

COOH X

H R

L-Isomers

12

1 General Principles of Molecular Chirality NO2

NO2

HO

H NHC(O)CHCl 2

H

CH2OH D-(– )-threo-

H

OH

Cl2 CHC(O)NH

H

CH2OH L-(+)-threo-

NO2

NO2

H

OH

H

NHC(O)CHCl2

HO

H

Cl2CHC(O)NH

H

CH2OH

CH2OH

D-(+)-erythro-

L-(–)-erythro-

Fig. 1.16 Chloramphenicol enantiomers

has 4 stereoisomers designated as D-threo-, L-threo-, D-erythro- and L-erythro- due to their structural similarity (similar position of substituents at chiral centers) to sugars threose and erythrose (Fig. 1.17). Among all four, only D-(−)-threo-isomer is biologically active. D-L-system has considerable disadvantages: first, D or L designation indicates the configuration of only one asymmetrical atom; second, the system yields different representations for some compounds depending on whether it was glycerin aldehyde or oxyacid structure that was chosen as the key. For example, see Fig. 1.18. At present these shortcomings of the system of keys limit the extent of its application to three classes of optically active substances: sugars, amino acids and hydroxy acids. Therefore, general recognition was accorded to the R-S-system of nomenclature proposed by Cahn-Ingold-Prelog. Cahn-Ingold-Prelog notation system (R-S-nomenclature) D-L-Nomenclature can only be applied after first establishing the orientation of projection formula. Since many compounds contain more than one asymmetrical

1.1 Types of Isomers

13

C(O)H HO

C(O)H

H

H

H

OH

OH

HO

H

CH2 OH

CH2 OH

D-Threose

L-Threose

C(O)H

C(O)H H

OH

HO

H

H

OH

HO

H CH2 OH

CH2OH D-Erythrose

L-Erythrose

Fig. 1.17 Enantiomers of threose and erythrose D – with hydroxy acid key COOH H HO

OH H CH2OH

L – with glyceraldehyde key

Fig. 1.18 Ambiguity of D-L-nomenclature of optical isomers

carbon, in 1966 R.S. Cahn, C.K. Ingold and V. Prelog developed R-S notation system for spatial configuration of compounds where R stands for right (rectus in Latin) and L—for left (sinister) enantiomer. R-S system is based on assigning priority to substituents around the asymmetrical carbon atom (or another chiral centre) following the “sequence rules”. According to these rules, four substituents receive numbers from one to four, and substituent number one has the highest priority while substituent number four (most commonly it is a hydrogen atom) gets the lowest priority. In practice the following algorithm has to be observed when assigning priorities to substituents and determining the spatial configuration. 1.

In each substituent, note the atomic number of the atom attached directly to the asymmetrical carbon atom under consideration (“first atomic layer” of

14

2.

3.

4.

5.

1 General Principles of Molecular Chirality

substituents). The highest priority is assigned to the substituent with an atom with highest atomic number attached with asymmetric carbon atom. If the substituents consist of atoms with the same atomic number closest to the asymmetrical carbon atom (e.g., other carbon atoms), take into consideration the sum of atomic numbers of the attached atoms (“second atomic layer” of substituents). Atoms attached with double and triple bonds are counted twice and thrice, respectively. If necessary, the comparison procedure can be continued for the “third atomic layer” and so on to establish all the priorities. The order of priority for the most common substituents at the asymmetrical carbon atom is as follows: I, Br, Cl, SR, SH, F, OR, OH, NO2 , NH2 , COOR, COOH, C(O)NH2 , CN, C(O)R, CR2 OH, C6 H5 , C(O)H, CHROH, CH2 OH, CH2 R, CH3 , D, H (where R is the alkyl substituent). The asymmetric carbon atom is placed in a way that the lowest priority substituent (most commonly H) is on the side opposite to the viewer; that is, behind the chirality centre. Compare the remaining substituents with the highest priority found in front of the asymmetrical carbon atom. If going clockwise around the axis connecting the asymmetrical carbon atom with the fourth substituent, the sequence 1 → 2 → 3 is preserved, the configuration of the chiral centre is R. If 3 → 2 → 1 sequence is preserved, the system is of S configuration. If the molecule has several chiral centers, its absolute configuration for each one is determined independently.

For example, when depicting a D-(–)-amphetamine molecule (Fig. 1.19) using wedge-shaped bonds, the asymmetrical carbon atom, CH3 methyl group and hydrogen are in the plane of the drawing; benzyl substituent CH2 C6 H5 is found in front of it, and NH2 amino group is behind the plane. All the substituents at the asymmetrical carbon atom are at vertices of a nearly regular tetrahedron. When assigning the order of priorities, analysis of the “first atomic layer” of substituents reveals that the one with the highest priority is amino group (attached to the chiral centre by a nitrogen atom, atomic number 7) and the one with the lowest priority Viewer (3) CH3

(3) CH3 (4) H

(1) NH2 CH2C6H5 (2)

H C6 H5 CH2

NH2

(2)

(1)

D-(–)-Amphetamine, R-Amphetamine Fig. 1.19 Use of R-S-nomenclature for optical isomers

1.2 Stereoisomers with Different Chirality Types

15

is hydrogen (atomic number 1). The methyl and the benzyl groups are attached to the chiral centre in the same way i.e. through carbon atoms (atomic number 6), and therefore, require an analysis of the “second atomic layer” for them. The methyl group has three hydrogen atoms in the “second atomic layer” (the sum of atomic numbers is 3); the benzyl substitute has 2 hydrogen atoms and 1 carbon atom (the sum of atomic numbers is 8). Thus, the benzyl substitute has a higher priority. If we view a D-(–)-Amphetamine molecule along the axis connecting the chiral centre with the substitute of the lowest priority, the hydrogen atom, the sequence of substitutes clockwise is 1 → 2 → 3, which means this molecule is of R-configuration (Fig. 1.19) (Soldatenkov et al. 2011).

1.2 Stereoisomers with Different Chirality Types Central chirality Conventionally, chiral compounds containing asymmetrical carbon atoms are referred to as “optical isomers”. However, other atoms, nitrogen or silicon for instance, can act as chiral centers. Besides, the chiral centre may not be related to any atom at all. Adamantanes in particular, whose tertiary carbon atoms display four different substituents are optically active; their chirality centre is inside the adamantane nucleus (Fig. 1.20) (Applequist et al. 1969). Axial chirality This type of chirality can be seen when an axis acts as a chirality centre. Such stereoisomers include allenes that are asymmetrically substituted at terminal carbon atoms (Fig. 1.21). Synthesis of such compounds has been described in one of the studies (Periasamy et al. 2014). CH3

Fig. 1.20 Adamantane derivatives with central chirality

CH3

H

H COOH

HOOC Br

Br

Fig. 1.21 Substituted allenes with axial chirality

H

H3C C H

C

CH3

H C

C CH3

H3C

C

C H

16

1 General Principles of Molecular Chirality CHO

Br

Br

Br

OHC

OHC

CHO

Br

Fig. 1.22 Substituted biphenyls with axial chirality

Due to steric difficulties in asymmetrical bis-2,6-substituted biphenyls, free rotation around the central link is restricted by an energy barrier, that is why the two aromatic rings lie in different planes. In this case also axial chirality is displayed (Fig. 1.22) (Wencel-Delord et al. 2015). Planar chirality In some molecules a plane can act as an element of chirality. Ferrocene derivatives are the best-known compounds of this type (Fig. 1.23). The synthesis of compounds with planar chirality such as ferrocene derivatives and metacyclophanes has been described (Pi et al. 2014; Ishida et al. 2014). Helical chirality A helix acting as an element of chirality can be twisted to the right or to the left. Hexahelicene in particular forms helical enantiomers, due to steric difficulties (Fig. 1.24) (Steed and Atwood 2009; Rickhaus et al. 2016). Helical chirality is of great importance in determining the functions of the biomolecules. In most proteins α-helix is twisted to the left. However, there are other polypeptides with a structure twisted to the right, for instance those in cobra venom.

X

X Y Fe

Fig. 1.23 Ferrocene derivatives with planar chirality

Y Fe

1.2 Stereoisomers with Different Chirality Types

H

H

17

H

H

Fig. 1.24 Helical enantiomers of hexahelicene

Moebius band

Trefoil knot

Fig. 1.25 Topostereoisomers of the first type

Topological chirality Topological chirality can be observed due to topological links in one molecule or between several molecules. At present, the molecules with several types of topological chirality have attracted increasing attention (Dobrowolski 2003). Moebius bands and knots are the examples of topostereoisomers of the former type (Fig. 1.25). The Moebius band has only one side. A molecular Moebius band is formed by connecting the ends of a flat molecule while one edge is twisted at 180°. To transform the Moebius band into a flat molecule, two chemical bonds must be severed. Right and left Moebius bands are distinguished (King 1983). A knot is a linear molecule having ends that are joined in such a way that it forms a non-self-intersecting closed curve. A knot cannot be transformed into a linear molecule without opening a chemical bond. Knots, like Moebius bands, form right and left enantiomers (King 1983; Steed and Atwood 2009). Synthesis of the enantiomers of a molecular 819 knot has been described (Fig. 1.26) (Danon et al. 2017). Nucleic acids were specifically shown to form knots. Topostereoisomers of the latter type include catenanes and rotaxanes (Fig. 1.27) (Steed and Atwood 2009). A catenane is a mechanically-interlocked molecular structure consisting of two or more interlocked macrocycles. The interlocked rings cannot be separated without breaking the covalent bonds of the macrocycles. Catenanes can function as molecular switches. For example, a bifunctional chiral (Bartuzi et al. 2017) catenane with 1,1’-binaphthyl-phosphate moiety was synthesized as artificial receptors for dicationic guest molecules (Mitra et al. 2016). Rotaxanes are

18

1 General Principles of Molecular Chirality

Fig. 1.26 Molecular 819 knot

Catenane

Rotaxane

Fig. 1.27 Topostereoisomers of the second type

compounds whose molecules consist of a macrocycle and a long open chain threaded through this cycle. For stabilization, the linear part of rotaxane contains voluminous terminal groups. Like catenanes, rotaxanes are of interest as components of molecular machines. In particular, they can also be molecular switches. Thus, new rotaxanes have been synthesized for various purposes (Powers and Smithrud 2016; Chen et al. 2021).

1.3 Conformational Isomerism

19

1.3 Conformational Isomerism Since rotation around ordinary (single) bonds is possible, molecules of saturated acyclical compounds can exist in a variety of forms, easily convertible from one to another. In contrast to ethylene derivatives, these compounds cannot be isolated in the form of stable cis or trans isomers, and so for a long time it was thought that rotation around carbon-to-carbon and other single bonds is perfectly free. It turned out, however, that rotation around single bonds is restricted to a greater or lesser degree. Even in the case of ethane, the simplest compound of this type, there is an energy barrier of approximately 12.5 kJ/mole preventing free rotation. This barrier is due to the fact that when methyl groups rotate around one another, their hydrogen atoms are alternately placed either in checkerboard fashion (“staggered”) or eclipsing each other (Fig. 1.28). The shape of a molecule in the “eclipsed” state is unstable as the molecule has high potential energy due to repulsion among the most proximate hydrogen atoms. On the contrary, the shape of a molecule in “staggered” form is more stable due to minimum potential energy. Particular shapes that the molecule can assume in space upon rotation of its parts around single bonds are called conformations. Conformations meeting a relative minimum of potential energy (stable or preferred conformations) are called conformational isomers or conformers. In the case of open chain structures such as ethane and other acyclic compounds, conformational isomers are not stable enough to be isolated as such due to relatively low energy barrier. However, in case of cyclic structures such as cyclohexane derivatives and condensed polycyclic systems like steroids, conformational isomers show significantly higher stability. Usually the existence of conformational isomers can only be displayed through some physical methods such as thermodynamic and spectral methods. Modern research has shown that at normal temperature cyclohexane almost exclusively exists in the form of a chair conformation with two types of hydrogen atoms. Six placed in the ‘middle’ plane of the ring at its outside, the equatorial hydrogens (e), and the other six (alternately in threes at the top and at the bottom of the ring) are beyond the plane, the axial hydrogens (a) (Fig. 1.29). Equatorial position of the large substituent is more advantageous energy-wise than axial position. Apparently, this is the reason why D-glucose (in β-D-Glucopyranose form with all substituents in equatorial position) is the most common monosaccharide and is also the main structural unit of most natural polysaccharides (Fig. 1.30). Fig. 1.28 Conformations of ethane

H

H

H

H

«Staggered»

H

H

H

H

H H

«Eclipsed»

H

H

20

1 General Principles of Molecular Chirality

Fig. 1.29 Chair conformation of cyclohexane

H

H H

(a)

H (e) H

H H

H H

H H

Fig. 1.30 β-D-Glucopyrano form of D-Glucose

H H

H H

HOCH2 HO HO H

O

OH OH H

It should be noted that conformation of a molecule not only determines its shape but in many cases it determines its reaction abilities as well. For some physiologically active substances, strict complementarity of structure is of exceptional importance to allow interacton with enzymes and receptors. Thus, biological activity of many substances essentially depends on the ability of their molecules to exist in the form of certain conformations most favorable for interaction with corresponding biotargets. In crystalline substances the conformation of molecules can be determined experimentally with the help of X-ray diffraction technique. It should be born in mind, however, that conformation of molecules in one and the same compound can differ in solid state and in solution. The conformation can also change significantly upon interaction with biotarget. Therefore, in addition to Xray diffraction technique, quantum-chemical calculations are also used to determine the most probable conformations of a molecule (Minkin et al. 1997, 2002; Lewars 2016; Blundell et al. 2016). The findings of conformational analysis give an insight into the mechanism of primary pharmacological reaction of many substances and provides additional information on the structure of corresponding biotarget binding sites (Granik 2001; Tikhonova et al. 2002; Ivanov et al. 2005; Baskin et al. 2009; Pietra et al. 2015; Bartuzi et al. 2017; Oparin et al. 2020).

References

21

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Periasamy M, Reddy PO, Sanjeevakumar N (2014) Convenient methods for the synthesis of highly functionalized and naturally occurring chiral allenes. Tetrahedron Asymmetry 25(24):1634–1646. https://doi.org/10.1016/j.tetasy.2014.11.002 Pi C, Cui X, Liu X, Guo M, Zhang H, Wu Y (2014) Synthesis of ferrocene derivatives with planar chirality via palladium-catalyzed enantioselective C-H bond activation. Org Lett 16(19):5164– 5167. https://doi.org/10.1021/ol502509f Pietra D, Borghini A, Bianucci AM (2015) Binding sites on G-protein coupled receptors: implications in design of bivalent ligands and allosteric modulation, Edizioni Accademiche Italiane, 96 p Powers LA, Smithrud DB (2016) A versatile axle for the construction of disassemblage rotaxanes. Molecules 21(8):E1043. https://doi.org/10.3390/molecules21081043 Rickhaus M, Mayor M, Juríˇcek M (2016) Strain-induced helical chirality in polyaromatic systems. Chem Soc Rev 45(6):1542–1556. https://doi.org/10.1039/C5CS00620A Soldatenkov AT, Kolyadina NM, Shendrik IV (2001) Osnovy organicheskoi khimii lekarstvennykh sredstv (Principles of the Organic Chemistry of Drugs), Moscow: Khimiya, 112 p Steed JW, Atwood JL (2013) Supramolecular chemistry, 2nd ed. Wiley, 1056 p Tikhonova IG, Baskin II, Palyulin VA, Zefirov NS, Bachurin SO (2002) Structural basis for understanding structure-activity relationships for the glutamate binding site of the NMDA receptor. J Med Chem 45(18):3836–3843. https://doi.org/10.1021/jm011091t Wencel-Delord J, Panossian A, Leroux FR, Colobert F (2015) Recent advances and new concepts for the synthesis of axially stereoenriched biaryls. Chem Soc Rev 44(11):3418–3430. https://doi. org/10.1039/C5CS00012B

Chapter 2

Separation, Asymmetric Synthesis and Analysis of Stereoisomers

2.1 Separation of Stereoisomers A molecule of a medicinal substance displays its pharmacological properties due to interaction with biotargets in a living organism such as enzymes, receptors, various elements of cellular membranes and endogenous macromolecules. The pharmacological effects of several isomers of a substance may be different due to steric differences. For example, enantiomers of some amino acids (D-isomers) are biologically inert and are excreted from the body unchanged. Synthomycine, an antibiotic for external application, is represented by two stereoisomers. D-(–)-threo-form (naturally occurring chloramphenicol) is the active form and L-(+)-threo-form is practically inactive (Albert 1979). In other cases, especially with diastereomers, both isomers can possess biological properties but these properties are different. For instance, quinine is an anti-malarial drug while its stereoisomer quinidine is an effective anti-arrhythmic compound (Vaughan 1970, 1992). These examples demonstrate the importance and urgency of undertaking the task of obtaining single stereoisomers of medicinal substances so as to enhance their effectiveness and safety. This goal can be achieved in two drastically different ways. The first one is to synthesize a mixture of stereoisomers and then separate the components by various chemical or physicochemical methods. The second way is to perform chemical synthesis so as to obtain only one of the stereoisomers (stereoselective, or absolute asymmetric synthesis), or to get predominantly one stereoisomer (partial asymmetric synthesis). Here we especially highlight the methods of biological synthesis. Since, most naturally occurring compounds possess chirality, synthesis of medicinal substances by biotechnology in optically pure form is highly relevant. The task of separating a racemic mixture of enantiomers into optical antipodes is simpler. In this case the following methods may be used.

© Springer Nature Singapore Pte Ltd. 2022 A. A. Spasov et al., Pharmacology of Drug Stereoisomers, Progress in Drug Research 76, https://doi.org/10.1007/978-981-19-2320-3_2

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Resolution by crystallization or spontaneous crystallization: The method is based on the fact that when racemate solutions crystallize, crystals of single enantiomers can form and they can be sorted mechanically. Additionally, placing the seed crystal of only one enantiomer into the supersaturated solutions of racemates can trigger crystallization of this particular enantiomer in racemic mixture. Resolution using optically active solvents: This method is based on differences in the behavior (eg. solubility) of stereoisomers in a solvent that also possesses chirality. The most common solvents in this case are esters of tartaric acid. Resolution after conversion to diastereomers: This method converts a mixture of enantiomers with identical chemical and physical (but not optical) properties, into diastereomers, most commonly by salt formation. The obtained diastereomers differ in their solubility, chromatographic mobility and other characteristics. This difference underlies further separation. The seprartion method allows the active chemical group of the component isomer in a racemate to react with another optically active substance, known as chiral selector, resulting in the formation of ionic or covalent bonds. The mixture is subjected to further resolution to obtain pure enantiomers. For this purpose, a racemate having an active chemical group is acted upon by another optically active substance (so-called chiral selector) which forms ionic or covalent bonds with the molecules of the racemate. For resolution of racemic acids, common chiral selectors such as alkaloids (quinine, brucine), α-phenylethylamine or its homologs, and optically active alcohols (menthol) are employed to obtain diastereomeric alcohol esters. On the contrary, for resolution of racemic amines, chiral carbonic acids, especially the cheap tartaric acid as well as pyroglutamic acid and acidic derivatives of natural terpenoids, are used. The obtained diastereomers are separated by fractional crystallization or filtration (if one of them is poorly soluble) and further resolution is done by inorganic bases or acids thus yielding pure enantiomers. Diastereomeric salts can also be separated by chromatographic methods (Aboun-Enein and Ali 2003) Such as in the case of enantiomers of α-methyl-DOPA, L-asparagic and L-glutamic acids, and naproxen (Burke and Henderson 2002). Adsorptive fractionation (chromatography): For preparative separation, high performance liquid chromatography (HPLC) is employed. Conventional chromatography can be used to separate diastereomers (Toyo’oka 2002) or alternatively a chiral carrier can be used for separation (Aboun-Enein and Ali 2003). The most common method is using chromatography with chiral stationary phase. Lactose, starch, cellulose and amylase can serve as organic asymmetrical adsorbents. Polymers like polyacrylamide containing optically active residual amines or acids are also a possibility. Chirobiotic phases like brucine, vancomycin, teicoplanin are also used (Andersson and Allenmark 2002). Nowadays, preparative enantiomeric separation with liquid chromatography continues to dominate the field. In addition, major advances were achieved with introduction of simulated moving bed (SMB) chromatography, supercritical fluid chromatography (SFC) and counter-current chromatography (CCC) (Pinto et al. 2020).

2.1 Separation of Stereoisomers

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Fig. 2.1 Current approaches to obtain enantiomerically pure compounds (from Pinto et al. 2020)

Chiral stationary phases for HPLC continue to evolve since their introduction in 1938 (Teixeira et al. 2019; Zhao et al. 2021). The most popular phases are based on polysaccharides, cyclodextrines and macrocyclic antiobiotics, while crown ethers and peptides are rarely used (Grybinik and Bosakova 2021). One of the most common modern varieties of chiral HPLC is an approach where one uses silica gel with chiral selectors grafted onto it. For example, in case of proteins, oligo- and polysaccharides, and some low-molecular weight natural compounds like alkaloids and antibiotics. Whelk-O1 is one example of such chiral stationary phase (Fig. 2.1, 2.2). Mesoporous three-dimensional graphene nanosheets, functionalized with tetracyanoethylene oxide and (S)-(+)-2-pyrrolidinemethanol, were recently proposed as an effective and inexpensive alternative to silica-based chiral stationary phase (Candelaria et al. 2018). Optically active adsorbents, which act as chiral selectors, adsorb optical antipodes. Consequently, enantiomers and the chiral selector form intermediate molecular complexes with different interaction energy. The process allows elution of different stereoisomers at a different rate. To achieve the most effective resolution, it is important to choose a selector that possesses maximum complementarity to the enantiomers to be resolved. Apart form this, the composition of the mobile phase (pH, nature of the solvent and the electrolytes dissolved in it), length of the column and temperature also play an important role in the selectivity of the resolution process. That is why there is no universal chirality phase, although hundreds of chiral sorbents are available in the market. The choice of an optimal stationary chiral carrier is a difficult task on the whole, and the most effective phase in each particular case is chosen empirically.

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CH3 O

Si

(R) (S)

N

CH3

H

O

NO2

O2N Fig. 2.2 Surface structure of Whelk-O1 chiral chromatographic carrier (Didier et al., 2008)

When resolving racemates with the help of HPLC, one can use liquid chiral phases containing chiral selectors as additives. The quality of resolution in this approach, however, is much lower than in the case with a solid chiral carrier. This method is, therefore, seldom employed. A new technique, simulated moving bed chromatography, was specially developed for industrial purposes (Johannsen et al. 2002). The solid phase is placed into separate small columns that form a closed circuit. Four valves control the flow of the liquid phase through the system of columns that are moving toward the liquid phase. This arrangement permits a high purity yield of stereoisomers. This technique was particularly employed when developing tramadol (Burke and Henderson 2002). In 2012, the 4 biggest pharmaceutical blockbusters were pure enantiomers and separation of racemic mixtures is now frequently a key step in the development of a new drug. For a long time, preparative LC was the technique of choice for the separation of chiral compounds either during the drug discovery process to get up to a hundred grams of a pure enantiomer or during the clinical trial phases that need kilograms of material. However, the advent of super critical fluid chromatography (SFC) in 1990s has brought changes. Despite some initial teething troubles, SFC is becoming the primary method for preparative chiral chromatography (Speybrouck and Lipka 2016). During the last decade, use of SFC has gained greater utility as a fast, cost-effective approach for the resolution of enantiomers (de Mas et al. 2016). In SFC the bulk of

2.1 Separation of Stereoisomers

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the solvent in the mobile phase is highly pressurized carbon dioxide (CO2 ). The low viscosity and high diffusivity of the SFC mobile phase allows higher flow rates relative to HPLC, resulting in shorter run times and increased efficiencies (Desfontaine et al. 2015; Lesellier and West 2015). A major advantage of preparative SFC versus preparative HPLC is the reduced solvent consumption and higher product concentrations post-chromatography, decreasing the time and energy expense for solvent removal and enantiomer isolation (Miller and Potter 2008). Also, the health and safety issues are highly improved by replacing solvents such as hexane or heptane used in normal-phase LC (NPLC) with the much less toxic CO2 (Lindskog et al. 2014). A strategy for large-scale chiral resolution with SFC could be illustrated by the isolation of pure enantiomer from a 5 kg batch (Leek et al. 2017). Results from SFC were presented and compared with NPLC. Solubility of the compound in the supercritical mobile phase was shown to be the limiting factor. To circumvent this, extraction injection was used but was shown not to be efficient for the particular compound. Finally, a method for chiral resolution by crystallization was developed and applied to give diastereomeric salt with an enantiomeric excess of 99% at a 91% yield. Leek and Andersson have shown that while the broad applicability of chiral stationary phases allows majority of racemates to be resolved routinely, there are some racemates that do not scale up as expected due to low solubility, poor loading or other unknown reasons (Leek and Andersson 2017). Further, the introduction of immobilized chiral stationary phases has enabled the separation of enantiomers using more unconventional solvents both in SFC and HPLC which would otherwise be very time-consuming and/or impossible due to poor compound solubility. When possible, utilization of carbon dioxide as the main solvent is preferable from a sustainability, health and economic perspective. The combination of chromatography, racemization and/or crystallization, as well as the informed choice of when to apply preparative chiral chromatography in a multi-step synthesis of a drug compound, can further facilitate accelerated drug discovery and the early clinical evaluation of drug candidates. Thus, to develop efficient synthetic routes for drug candidate compounds at a gram to kilogram scale a close partnership between synthetic chemists and separation scientists is strongly advocated to develop the most efficient strategies for positioning the resolution step within an organic synthesis rather than utilizing chromatography as a rescue technology which continues to be the most common way of working in the industry. Biochemical resolution: This process is based on the ability of microorganisms (fungi, bacteria) and their enzymes to act selectively only on one of the enaniomers of a racemic mixture. The biochemical method is especially effective for obtaining optically active amino acids. Thus, the fermentation by yeast utilizes predominantly L-forms of amino acids, while their D-antipodes are retained in the solution and can be isolated. In the presence of acylase enzyme the rate of hydrolyzation of N-acetyl-L-methionine is a thousand times faster than in production of D-methionine by acetylation. The method of biochemical resolution has been used for developing drugs like potrafiban, levofloxacin, and naproxen (Burke and Henderson 2002).

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In all the cases described above the separation of racemates into their enantiomers only takes place in the presence of other asymmetrical objects such as seed crystal, chiral selectors, optically active adsorbents or solvents, and enzymes. Asymmetric synthesis: This method is the second most important technique of obtaining optically active substances after the resolution methods, and is based on physicochemical properties of isomers. The essence of asymmetric synthesis is in performing stereoselective reactions during which optical antipodes are either formed or destroyed in unequal amounts. Kinetic resolution is a variety of asymmetric synthesis. Optical antipodes enter the same chemical reactions at different rates. This can be seen either in reactions that occur in the presence of optically active catalysts, or in reactions with other optically active substances. If a racemate is subjected to such a reaction, terminating the reaction before it is complete (e.g. by using the deficiency of a second chiral reagent) will yield predominantly one of the antipodes that has a higher rate of reaction. The other one will remain as non-reacting residue. Thus kinetic resolution of a racemate can be achieved. This type of resolution is never complete. For instance, racemic amygdalinic acid is subjected to kinetic resolution in a reaction with menthol. The ratio of reaction rates for (–)- and (+)-forms of amygdalinic acid is 0.897, so in this case, only a 20% predominance of one stereoisomer in the reaction product can be achieved by kinetic resolution. In asymmetric synthesis, auxiliary optically active substances or optically active groups are used. They can be present in the form of substrate, reagent, solvent or catalyst. Employing chiral catalysts is a most promising method of asymmetric synthesis as one molecule of the catalyst, unlike a molecule of the reagent, is capable of yielding thousands and millions of molecules of the chiral product. Homogeneous catalysts of hydration have now been developed based on complex binding of rhodium and ruthenium with phosphines where phosphine ligands are chiral molecules. When these catalysts are used in industrial synthesis of L-DOPA, the obtained product is 97.5% optically pure. Catalysts of this type were successfully used in manufacturing levofloxacin and chiral precursors of some antibiotics. In a similar way, effective catalysts of asymmetric oxidation of allyl alcohols into chiral epoxides, precursors of beta-adrenoblockers, and many other medicinal substances were obtained by combining chiral ligands and titanium as an oxidation catalyst into an active complex. A group of American and Japanese researchers (William S. Knowles, Ryoji Noyori, K. Barry Sharpless) won the Nobel Prize in Chemistry in 2001 for their work on chirally catalyzed hydrogenation reactions. The paramount importance of asymmetric organocatalysis was further acknowldeged by 2021 Nobel prize in chemistry awarded to Benjamin List and David MacMillan (NobelPrize.org 2001, 2021). At present, the asymmetric synthesis is intensively developing, for example, in the direction of using stereoselective rhodium catalysts (Chen and Xu 2017), synthesis of axially, planar and helical chiral compounds (Shirakawa et al. 2016), etc. Stereoselective synthesis of pharmaceutically important fluorine-containing and sulfinyl compounds were extensively reviewed in recent works (Zhu et al. 2018; Wojaczy´nska and Wojaczy´nski 2020).

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2.2 Quantitative Methods of Analysing Chiral Drugs So far, special methods of analyzing the concentration of stereoisomers of the same drug in the body are not a routine part of clinical practice. This procedure, however, is of great importance as patients may display different sensitivity to chiral forms of medicinal substances due to enantioselective recognition by drug transporters and biological targets (Uwai 2018; Coelho et al. 2021). If a racemate is administered, the conventional analytical methods produce distorted results as they do not show the specific concentration of the active stereoform of the drug in the human body. Thus a correct clinical use of chiral drug requires development and implementation of special analytical methods in practice. Determining the concetration of chiral compounds in biological media presents more difficulties than their analysis during the manufacturing process. The factors that interfere with analysis include the multicomponent nature of the biological material, low concentration of the drug in samples (in micro- and nanograms), conjugation of the active substance with proteins, etc. So an accurate quantitative analysis of medicinal compounds should start with highly selective extraction and then high-precision methods of chiral quantitative analysis. Extraction This procedure aims to eliminate proteins and other interfering compounds from the sample to be analyzed. To extract stereoisomers from biological material, liquid– liquid extraction and solid-phase extraction are used. In liquid–liquid extraction the appropriate solvent is of great importance and is chosen depending on the polarity of the chiral medicinal substance. As a rule, acidic (with pH below 2) or alkaline (with pH of up to 10) buffer is added prior to extraction so as to facilitate the separation of the drug from proteins. For enantiomers with identical chemical and physical properties, the recovery ratio is the same but is different for diastereomers. By calibrating the extraction conditions, one can achieve a high recovery ratio and purity of the drug. Although, conventional liquid–liquid extraction takes quite a long time, its low cost is undoubtedly a great advantage. Modern methods of liquid–liquid microextraction provide a quality of extraction that is comparable with that of conventional method. Greater simplicity and speed are other advantages (Pedersen-Bjergaard and Rasmussen 2005). Solid-phase extraction is based on chromatographic separation both with traditional sorbents (modified silica gel C8 and reverse-phase C18 ), and modern polymer carriers. The latter type of carriers is preferable as they are more universal and provide a higher recovery ratio (He et al. 2004, 2005). Unique properties of ionic liquids were also applied for the development of advanced analytical techniques. Chiral ionic liquids are successfully used as affordable and environmentally friendly solvents and chiral selectors (Flieger and Flieger 2020). Wide range of their application include liquid–liquid and solid–liquid extraction, and also stationary phase surface modification for HPLC and CCC (Flieger et al. 2020).

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Methods of Analysis Two types of analytical approaches have been developed for bioanalysis of chiral medicinal compounds: physical and immunological. Physical methods of investigating enantiomers include HPLC (Aboun-Enein and Ali 2003), liquid and gas chromatography-mass spectrometry (Xie and Yuan 2017; Awad and El-Aneed 2013; Chen et al. 2005; Martins et al. 2005; Ribeiro et al. 2018), as well as capillary electrophoresis (Wei et al. 2006). From the analytical point of view, indirect HPLC based on using chiral selectors to obtain enantiomers and diastereomers and their further separation on a standard nonchiral carrier like C8 or C18 (Toyo’oka 2002), is the most sensitive and precise method. It is described in more detail in the section “Separation of stereoisomers”, see above. Quantification of propranolol (Pham-Huy et al. 1994) and perhexiline (Davies et al. 2006) was performed with a luminescent detector. If we resort to indirect HPLC, the chiral substance must contain an active functional group (amino, carboxyl group and the like). This requirement restricts the possibilities of the wider use of the method. Direct HPLC on a chiral immobile carrier is free from this restriction (it is described in more detail in the section “Separation of stereoisomers”, see above). Many racemic drugs are effectively analyzed with the help of chiral stationary phase. Propranolol, methadone (He et al. 2005) and oxazepam (He et al. 2004) are analyzed on cyclodextrin carriers; cellulose and its derivatives are used for analyzing thalidomide (Robin et al. 1995) and tetrahydropalmatine (Hong et al. 2005); amylase in determining donepezil (Radwan et al. 2006) and chrobiotic phases like vancomycin and erhythromycin for analysis of amino acids (Staroverov et al. 2006). Enantioselective chromatography has become a favourite both at the analytical and preparative scales. HPLC and SFC are dominating the scene and are often seen as complementary techniques. Nowadays, for economic and ecologic reasons, SFC may be preferred over NPLC (normal phase liquid chromatography) as it allows significant reductions in solvent consumption. However, the transfer of NPLC methods to SFC is not always straightforward. Khater et al. (2016) have compared the retention of achiral molecules and separation of enantiomers under SFC (carbon dioxide with ethanol or isopropanol) and NPLC (heptane with ethanol or isopropanol) elution modes with polysaccharide stationary phases in order to explore the differences between the retention and enantioseparation properties. Chemometric methods (namely quantitative structure-retention relationships and discriminant analysis) were employed to compare the results obtained on a large set of analytes (171 achiral probes and 97 racemates) and gain some understanding on the retention and separation mechanisms. The results indicate that, contrary to popular belief, carbon dioxide—solvent SFC mobile phases are often weaker eluents than liquid mobile phases. It appears that SFC and NPLC elution modes provide different retention mechanisms. While some enantioseparations stay unaffected, facilitating the transfer between the two elution modes, other enantioseparations may be drastically different due to different types and strength of interactions contributing to enantioselectivity. Capillary electrophoresis with chiral selectors represents another embodiment of chromatographic separation particularly fit for analytical purposes. Its advantages

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include high efficiency and resolution, improved sensitivity and low consumption of reagents and samples (Yu and Quirino 2019; Bernardo-Bermejo et al. 2020). The immune system can effectively distinguish between racemates, selectively and specifically producing corresponding antibodies to each enantiomer. Such enantioselective antibody can recognize infinitesimal differences in the composition and configuration of a chiral drug. As these substances are essentially haptens, they should conjugate with auxiliary and transport molecules forming an immunogen so that the immune system (B-cells) could produce the corresponding antibody (Cook 1993; Got and Scherrmann 1997). Conjugation of such a hapten, as an enantiomer, with a protein is a subtle and complex process of immunogen preparation. Specificity and selectivity of the antibody to the enantiomer drug mostly depends on preparation of the hapten and conjugation technique. As Sahui-Gnassi et al. (1993) and Chikhi-Chorfi et al. (2001) reported that the asymmetry of the carbon atom should be preserved to obtain a specific antibody to a chiral compound, which means providing such conditions that hapten does not bind to transport protein through the chiral centre. It has been stated in literature that some enantioselective antibodies can be successfully used in obtaining enantiomers like antibodies to propranolol (Sahui-Gnassi et al. 1993), methadone (Chikhi-Chorfi et al. 2001), amphetamine (Cook 1993), warfarin (Cook 1993), atropine, pentobarbital (Cook 1993; PhamHuy et al. 1995; Chikhi-Chorfi et al. 2001) enantiomers. Their number, however, is not high if compared with numerous antibodies to achiral drugs. Nevertheless, use of enantioselective antibodies to analyze chiral drugs is of key importance in the methods of biochemical analysis. They can be used as specific reagents in various immunological methods like radioimmunoassay, enzymatic immunoassay, histological immunoassay, immunoaffinity chromatography, immunoextraction and liquid–liquid chromatography with antibodies serving as chiral selectors. Unlike physical methods, enantioselective immunoassay does not require a preparative extractive treatment of biomaterial. Moreover, immunological methods are of higher sensitivity and require only a small sample (no more than 10 μl). In clinical practice, however, enantioselective immunoassays are utilized extremely rarely. Such techniques as radioimmunoassay or enzymatic immunoassay are still at the stage of experimental development. In contrast to the standard immunoassays (EMIT® , FPIA or TDx® ), they are not yet automated.

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Sahui-Gnassi A, Pham-Huy C, Galons H, Warnet JM, Claude JR, Duc HT (1993) Selective antibodies to propranolol enantiomers produced from a new conjugate. Chirality 5(6):448–454. https://doi.org/10.1002/chir.530050610 Shirakawa S, Liu S, Kaneko S (2016) Organocatalyzed asymmetric synthesis of axially, planar, and helical chiral compounds. Chem Asian J 11(3):330–341. https://doi.org/10.1002/asia.201500951 Speybrouck D, Lipka E (2016) Preparative supercritical fluid chromatography: a powerful tool for chiral separations. J Chromatogr A 1467:33–55. https://doi.org/10.1016/j.chroma.2016.07.050 Staroverov SM, Kuznetsov MA, Nesterenko PN, Vasiarov GG, Katrukha GS, Fedorova GB (2006) New chiral stationary phase with macrocyclic glycopeptide antibiotic eremomycin chemically bonded to silica. J Chromatogr A 1108(2):263–267. https://doi.org/10.1016/j.chroma.2006. 01.073 Teixeira J, Tiritan ME, Pinto MMM, Fernandes C (2019) Chiral stationary phases for liquid chromatography: recent developments. Molecules 24(5):865. https://doi.org/10.3390/molecules240 50865 Toyo’oka T (2002) Resolution of chiral drugs by liquid chromatography based upon diastereomer formation with chiral derivatization reagents. J Biochem Biophys Methods 54(1–3):25–56. https:// doi.org/10.1016/S0165-022X(02)00127-6 Uwai Y (2018) Enantioselective drug recognition by drug transporters. Molecules 23(12):3062. https://doi.org/10.3390/molecules23123062 Vaughan WEM (1970) Classification of anti-arrhythmic drugs. In: Sandfte E, Flensted-Jensen E, Olesen KH (eds) Symposium on cardiac arrhythmias. AB ASTRA, Södertälje, Sweden, pp 449– 472 Vaughan WEM (1992) Classifying antiarrhythmic actions: by facts or speculation. J Clin Pharmacol 32(11):964–977. https://doi.org/10.1002/j.1552-4604.1992.tb03797.x Wei S, Guo H, Lin JM (2006) Chiral separation of salbutamol and bupivacaine by capillary electrophoresis using dual neutral cyclodextrins as selectors and its application to pharmaceutical preparation and rat blood samples assay. J Chromatogr B Analyt Technol Biomed Life Sci 832(1):90–96. https://doi.org/10.1016/j.jchromb.2005.12.044 Wojaczy´nska E, Wojaczy´nski J (2020) Modern stereoselective synthesis of chiral sulfinyl compounds. Chem Rev 120(10):4578–4611. https://doi.org/10.1021/acs.chemrev.0c00002 Xie S-M, Yuan L-M (2017) Recent progress of chiral stationary phases for separation of enantiomers in gas chromatography. J Sep Sci 40(1):124–137. https://doi.org/10.1002/jssc.201600808 Yu RB, Quirino JP (2019) Chiral selectors in capillary electrophoresis: trends during 2017–2018. Molecules 24(6):1135. https://doi.org/10.3390/molecules24061135 Zhao Y, Zhu X, Jiang W, Liu H, Sun B (2021) Chiral recognition for chromatography and membranebased separations: recent developments and future prospects. Molecules 26(4):1145. https://doi. org/10.3390/molecules26041145 Zhu Y, Han J, Wang J, Shibata N, Sodeoka M, Soloshonok VA, Coelho JAS, Toste FD (2018) Modern approaches for asymmetric construction of carbon–fluorine quaternary stereogenic centers: synthetic challenges and pharmaceutical needs. Chem Rev 118(7):3887–3964. https:// doi.org/10.1021/acs.chemrev.7b00778

Chapter 3

General Issues of Chirality in Pharmacology

Chiral compounds are present in the human body in great numbers. They are represented by various biologically active substances like hormones, neuromediators etc. Furthermore, the body is miraculously capable of recognizing them and of “turning on” appropriate metabolic or receptor mediated reactions. Understandably, the body interacts differently with different components of a racemic drug. Each enantiomer is metabolized along a separate path producing a specific pharmacological effect. Thus one isomer can produce the desired therapeutic effect while another one can be ineffective or, still worse, produce an undesired or toxic effect. When describing pharmacological properties of stereoisomers of medicinal substances, special terms are used in literature. Eutomer is a bioactive enantiomer or an enantiomer with enhanced pharmacological activity. Inactive molecules are called distomers. Enantioselectivity is a process when one enantiomer is expressed exclusively or predominantly. In pharmacology this means that there is a biological structure (enzyme, antibody or receptor) which has a higher affinity to one enantiomer compared to another. Enantioselective assay is an analytical method that makes it possible to separate and quantify enantiomers. Homochirality is biological chirality when all biological compounds of the same class possess the same chirality; for instance, all amino acids are laevorotatory isomers. Modification of chiral forms is a procedure used to transform a known racemic drug into its only active enantiomer. This new enantiomeric drug obtained by a manufacturer of pharmaceuticals may receive additional protection under the patent law. Enantiomeric (eudesmic) index is the ratio of the activity of one enantiomer to another. In specific cases more complex calculations are made.

© Springer Nature Singapore Pte Ltd. 2022 A. A. Spasov et al., Pharmacology of Drug Stereoisomers, Progress in Drug Research 76, https://doi.org/10.1007/978-981-19-2320-3_3

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36

3 General Issues of Chirality in Pharmacology

3.1 Chirality and Pharmacokinetics Absorption Stereoselectivity in biological processes emerges due to stereospecific recognition of a substrate by an acceptor. In most cases a fragment of a protein molecule represented by the binding site of the receptor or an active enzyme centre serves as an acceptor. With regards to the rate of absorption, there are not many drugs that have the changes in blood concentration over time similar to carrier-mediated transport processes (zero order kinetics). Indeed, most drugs penetrate the biological cellular membrane as a result of passive transport (first order kinetics) where carrier proteins do not play a significant role in the process of drug absorption in the intestine. Over last ten years the literature on the subject has compiled elaborate data illustrating the role of specific carrier proteins in redistribution of drugs in the body. Carrier proteins are important in the regulation of absorption, distribution and elimination of drugs. In recent years, the experimental research within this field has attracted ongoing interest. Breast cancer resistance protein (BCRP), multidrug resistance proteins (MRP), P-glycoprotein (P-gp), organic anion transporters (OATs), organic anion transporting polypeptides (OATPs), organic cation transporters (OCTs), peptide transport proteins (PepTs), human proton-coupled folate transporter (PCFT) and multidrug and toxic extrusion proteins (MATEs) have been reported to exhibit either positive or negative enantioselective substrate recognition (Zhou et al. 2014). P-glycoprotein play an important role in transmembrane transport of antitumor drugs, cardiac glycosides, glucocorticoids and many other drugs. It restricts the accumulation of digoxin, vinblastine, dexamethasone in the blood brain barrier. The gene coding for P-glycoprotein is expressed in many tissues. When its activity is not high enough, the excretion of drugs and their metabolites into urine, bile and intestinal contents is disturbed. In USA, 16% of population is carrier of OATP-C gene polymorphism, and the activity of this carrier is reduced. In this case, administration of statins is most commonly associated with hepatic lesion and rhabdomyolysis (toxic lesion of muscles). β-Adrenoblocker (±)-thalinolol (Fig. 3.1) can serve as an example of stereoselective carrier-mediated drug transport in the intestine. Gramatté and Oertel (1999) showed that the absorption of (±)-thalinolol racemate in the intestine of healthy volunteers is suppressed by the R-enantiomer of verapamil. However, in a later study (Zschiesche 2002) where stereospecific quantitative methods of analysis were utilized to determine thalinolol in intestinal aspirate, any selectivity in the mechanism of P-glycoprotein mediated transport of the drug could not be demonstrated. Currently, there is no generally accepted view on the role of P-glycoprotein in the process of stereoselective transport of drugs in the intestine. However, there are a number of chiral drugs (chlorochin, primachin, itraconazole, cisapride, fexofenadine, thalinolol) that are absorbed by P-glycoprotein mediated mechanisms, and this indicates the important role of this carrier in the stereoselective mechanism of drug absorption (Wu and Benet 2005).

3.1 Chirality and Pharmacokinetics

37

OH H3C

H N

(R)

O HN

H3 C

CH3

H N H

O

OH H3C

H N

( S)

O HN

H3 C

CH3

H N H

O

1-[4-[3-(Tert-butylamino)-2-oxypropoxy]phenyl]-3-cyclohexylurea Fig. 3.1 Thalinolol enantiomers

Following absorption, highly lipophilic drug first enters the systemic circulation through the lymphatic transport system. So the difference in concentrations of enantiomers in the lymphatic system may serve as an indicator of stereoselectivity of drug transport in the intestine. Works by Shackleford et al. (2003) are illustrative in this respect. When determining the absorption of halofantrine enantiomers (Fig. 3.2) in the lymph, the first stage of the experiment did not reveal stereoselectivity in the absorption of the racemate (Shackleford et al. 2003). In due course, however, stereoselectivity began to appear with predominance of the (+)-enantiomer over its antipode. These changes are probably associated with systemic metabolism which, as reported in other studies (Gimenez et al. 1994; Abernethy et al. 2001; Brocks 1995, 2000; Brocks and Jamali 1990; Terefe and Blaschke 1994; Gimenez et al. 1992; Brocks 2002), is more typical of this drug. Remote consequences of systemic metabolism are probably manifested by the difference in the concentration of stereoisomers in the lymph. One of the recent studies indicated that multiple transporters including P-gp, OATPs, and MRP2 play important roles in fexofenadine enantiomer pharmacokinetics (Akamine 2015). Furthermore, OATP2B1 is shown to be a key determinant of the stereoselective pharmacokinetics of fexofenadine, and drug transporters may have chiral discrimination ability. Other approaches utilized to study chirality in enantiomer-transporter interaction include experiments with inhibition of specific transporters in cell models (e.g. Caco-2 cells), studies using drug resistance cell

38

3 General Issues of Chirality in Pharmacology F F

F H3C

Cl

* Cl

N

CH3

OH

3-(Dibutylamino)-1-[1,3-dichloro-6-(trifluoromethyl)phenantren-9-yl]propane-1-ol

Fig. 3.2 Halofantrine structural formula (the chiral centre is shown by an *)

lines or transgenic cell lines expressing transporters in wild type or variant, the use of transporter knockout mice, pharmacokinetic association of single nucleotide polymorphism in transporters, pharmacokinetic interactions of racemate in the presence of specific transporter inhibitor or inducer, molecule cellular membrane affinity chromatography and pharmacophore modeling. Enantiomer-enantiomer interactions exist in chiral transport. The strength and/or enantiomeric preference of stereoselectivity may be species or tissue-specific, concentration-dependent and transporter family member-dependent. Modulation of specific drug transporter by pure enantiomers might exhibit opposite stereoselectivity (Zhou et al. 2014). Distribution It is well known that many drugs bind to various blood components including corpuscular elements as well as proteins, e.g. serum albumin, α1 -acid glycoprotein and lipoproteins (Shen et al. 2013). Many examples can be cited where stereospecific binding to plasma proteins yields a difference in the concentration of stereoisomers in the blood or plasma over time. This, in turn, leads to differences in the volume of distribution and clearance of two enantiomers (Table 3.1). Binding to plasma and tissues proteins is one of the main factors affecting the distribution of drugs in the body. Gilabert et al. (2009) established 3 steps experiment to study the stereoselective binding between enantiomers and proteins: (1) equilibration of racemic mixture and proteins, (2) separation of the unbound fraction, and (3) determination of the concentration of the enantiomers from either the free fraction or drug-protein complexes. To illustrate enantioselective drug-protein binding, classical methods, such as equilibrium dialysis, ultrafiltration and ultracentrifugation, are commonly combined with chiral separation techniques (Lammers et al. 2012; Pistolozzi and Bertucci 2008; Maddi et al. 2010). Some drugs may show interspecies stereoselective differences, in binding to the plasma proteins. For example, propafenone, an antiarrhythmic drug, has a ratio of 0.64

3.1 Chirality and Pharmacokinetics

39

Table 3.1 Degree of drug stereoselectivity according to the volume of distribution (Vdss —steady state volume of distributiona or Vdβ —apparent volume of drug distribution) upon parenteral administration in the form of a racemate (Mehvar 1991) Drug

Biological species

Enantiomer index

Albuterol

Human

1.11 [R:S]

2' -Desoxy-3' -oxa-4' -thiocitidine

Human

1.43 [(+):(–)]

Disopyramide

Human

1.82 [R:S]

Hexaconazole

Rabbit

1.03 [(+):(–)]

Ketorolac

Human

2.19 [S:R]

Mepivacaine

Human

1.55 [R:S]

Methadone

Human

1.72 [R:S]

Pimobendan

Human

1.35 [(+):(–)]

Propapfenone

Rat

1.69 [(+):(–)]

Reboxetine

Human

2.36 [(+):(–)]

Polyprane

Human

1.58 [(+):(–)]

Thalinololb

Human

1.31 [S:R]

Thiopental

Human

1.27 [R:S]

Tocainide

Human

1.02 [R:S]

Verapamil

Human

2.34 [S:R]

a Volume

of distribution in steady state from clearance values (Cl) and elimination half-life (t½)

b Calculated

for free fractions of (+)- and (–)-forms in human blood, and 1.31 in rat blood (Mehvar 1991). R-/S-2-(2-hydroxypropanamido)-5-trifluoromethyl benzoic acid (R-/S-HFBA), a COX inhibitor, showed a concentration dependent protein binding. Significant species differences were observed for R- and S-HFBA among rat, dog, and human plasma proteins (Rong et al. 2020). Stereoselective binding to the plasma proteins has been described well in the literature (Table 3.2). Here is another example illustrating the interspecies differences in the stereoselectivity of a drug binding to corpuscular elements of the blood. Following incubation of propafenone in human blood in vitro, its concentration in the blood cells was found to be higher than that in the plasma. In the plasma the (+)-enantiomer achieved a higher concentration while the (–)-enantiomer was predominant in the erythrocytes. Under similar conditions, when the drug was incubated in the rat blood, the concentration of propafenone rose higher in erythrocytes than in the plasma, and the (+)-enantiomer showed a greater concentration in the erythrocytes, than its antipode. In the plasma, on the contrary, the concentration of the (–)-enantiomer surpassed that of the (+)-enantiomer (Mehvar 1991).

40 Table 3.2 Drug stereoselectivity in relation to binding to the plasma proteins

3 General Issues of Chirality in Pharmacology Drug

Biological species

Enantiomer index

Brompheniramine E1

Human

2.8 [R:S]a

Bupivacaine

Human

1.5 [R:S]b

Carvedilol

Rat

1.5 [S:R]b

(−)Catechin

Human

1.5 [(–):(+)]a

Chlorochin

Human

1.7 [R:S]b

Chlorpheniramine E1

Human

1.8 [R:S]a

Disopyramide

Human

2.0 [R:S]b

Ethodolac

Rat

3.1 [S:R]b

Ethodolac

Human

2.5 [S:R]b

Hallopamil

Human

1.4 [S:R]b

Halofantrine

Rat

1.05 [(–):(+)]b

Hydroxychlorochin

Human

1.4 [R:S]b

Hydroxyzine E1

Human

1.2a

Ketorolac

Human

1.3 [S:R]b

Orphenadrine E1

Human

13.3a

Phenindamine E1

Human

2.5a

Promethazine E1

Human

1.4a

Propanocaine E1

Human

1.5a

Propanolol

Human

1.0–1.5 [(+):(–)]b

R-zopiclone

Human

1.95 [S:R]a

S-etodolac

human

6.06 [S:R]a

Thalinolol

human

1.01 [R:S]b

Tiamilal

human

1.67–1.76 [R:S]b

Trimeprazine E1

human

1.5a

Verapamil

human

1.3–2.0 [S:R]b

Warfarin

human

1.3 [R:S]b

a Shen

et al. (2013) (1990)

b Mehvar

Binding of enantiomers to various lipoprotein fractions of the plasma can also be stereoselective. For example, humans and dogs show a higher content of (+)halofantrine in lipoprotein-rich plasma fraction than in lipoprotein-poor plasma fraction. On the contrary, the concentration of the (–)-enantiomer is higher in lipoproteinpoor plasma fraction in comparison with lipoprotein-rich plasma fraction (Mehvar 1991). Rats have also shown a similar tendency (Mehvar 1990). Pathological conditions can also affect the stereoselective binding to corpuscular elements of the blood. For example, the absorption of R- and S- hydroxychloroquine to isolated erythrocytes in vitro (Fig. 3.3) is unremarkable in normal conditions (Brocks et al. 1994). However, when the whole blood of arthritic patients is used,

3.1 Chirality and Pharmacokinetics

41

OH N N Cl

H HN

CH3

(R)

OH

CH3 N N

CH3

H

Cl HN

(S)

CH3

2-[[4-(7-Chloroquinolin-4-ylamino)pentyl](ethyl)amino]ethanol Fig. 3.3 Enantiomers of hydroxychloroquine

the concentration of its enantiomers was different in the whole blood and serum, while no differences were noted in the plasma. As to the concentration of R- and S-forms of the drug in isolated cells, significant stereoselectivity (with the R:S index higher than 2:1) was noted for erythrocytes, thrombocytes and white blood cells. The R-enantiomer showed a higher concentration of unconjugated fraction in the plasma. The higher concentration of the R-enantiomer in blood cells may be caused by a higher concentration of unconjugated fraction of the R-enantiomer in the plasma and a faster clearance of the S-enantiomer (Brocks et al. 1994). Another factor that can affect enantioselectivity in the distribution of drugs is specific and/or nonspecific binding to tissue proteins. This problem is much more complex for study than plasma protein binding. Equilibrium dialysis of homogenized animal tissues has been used as a method to study enantioselectivity in drug distribution due to differences in tissue protein binding (Brocks and Jamali 1991). Homogenized rat tissues were used in the estimation of stereoselectivity of nonspecific binding of etodolac (Fig. 3.4). The R-enantiomer was shown to achieve greater concentrations than its antipode in all the tissues under study, with predominance in renal and cardiac tissues.

42

3 General Issues of Chirality in Pharmacology HO

HO O

H3C

O

H3C CH3

H N

(R)

H N

O

CH3 (S)

O

2-(1,8-Diethyl-1,3,4,9-tetrahydropyrano[3,4-b]indol-1-il)acetic acid

Fig. 3.4 Etodolac enantiomers

In rheumatoid arthritis, the synovial fluid is one of the targets of action of nonsteroid anti-inflammatory drugs (NSAIDs). The S:R enantiomer index of the derivatives of 2-arylpropionic acid (Fig. 3.5), ibuprofen, ketotifen, flurbiprofen, and thiaprofenic acid, in synovial fluid was found to be 2.1, 1.0, 1.3, 1.0, correspondingly (Gall et al. 1991; Foster et al. 1989; Young et al. 1991). Other studies have estimated the NSAIDs concentration in equine synovial fluid. The S:R enantiomer index for ketoprofen in inflamed and non-inflamed tissues amounted to 1.0 and 1.5, correspondingly (Armstrong et al. 1999; Verde et al. 2001). Similarly, the ratio of Sand R-enantiomers of flurbiprofen and carprofen in synovial fluid after intravenous injection of racemates was found to be 1.0 (Soraci et al. 2005) and 0.62 (Armstrong 1999), correspondingly. These differences are associated with changes in hepatic metabolism of the drug in the diseased animal. There are descriptions of cases when, upon administration of a racemic drug, one enantiomer displaced another enantiomer bound to the plasma proteins leading to differences in pharmacokinetics. Notably, it was established for the antiarrhythmic drug disopyramide (Giacomini et al. 1986) that upon administration of single enantiomers the indices S:R for clearance (Cl) and distribution volume (Vd ) amounted to 1.0, while after administration as a racemate these indices amounted to 1.6 and 1.8, correspondingly. Similar findings were obtained when experimenting on rats (Berry and Jamali 1989). Enantiomer-enantiomer interaction was also described for flurbiprofen, a NSAID, which showed displacement of the R-enantiomer by its H3 C

O

* Ar H3C

OH

O

O

F

CH3 Ar =

S

Ar =

Ibuprofen

Ar =

Ketoprofen

Ar =

Flurbiprofen

Thiaprofenic acid

Fig. 3.5 Derivatives of 2-arylpropionic acid (the chiral centre is shown with an *)

3.1 Chirality and Pharmacokinetics

43

antipode (Berry and Jamali 1989). Considering the fact that approximately 50% of drugs in clinical practice are represented by racemates, it is vital to determine the stereoselectivity of their binding to plasma proteins (Li and Hage 2017). Metabolism Stereochemical factors play a significant role in the metabolism of drugs and other xenobiotics (Yang et al. 2016). According to Testa (2015), types of metabolic stereoselectivity can be categorized as (1) substrate stereoselectivity (the differential metabolism of two or more stereoisomeric substrates) and (2) product stereoselectivity (the differential formation of two or more stereoisomeric metabolites from a single substrate). Combinations of the two categories exist as (3) substrate-product stereoselectivities, meaning that the product stereoselectivity itself is substrate stereoselective. Three-dimensional nature of the substrate-recognizing component of an enzyme allows drug metabolism to follow several possible pathways, depending on the chirality of the substrate. Several parameters such as fermentation kinetics as Km , Michaelis constant (measure of the affinity of enzyme to substrate) and Vmax (the rate of bound substrate conversion to product) (Table 3.3) may show differences between enantiomers in vitro. Such differences often translate into in vivo differences in clearance and the concentration of the drug in plasma. Sometimes the degree of stereoselectivity of a drug depends on its metabolism. In microsomes of rat liver, S-etodolac metabolizes predominantly forming acylglucoronide, while the R-enantiomer metabolizes predominantly by oxidative metabolism (Brocks and Jamali 1992). Verapamil also shows stereoselective differences in the rate of conversion to its metabolic products, norverapamil and D-617 (Tracy et al. 1999). Omeprazole, which metabolizes along several pathways including sulfoxidation, demethylation and hydroxylation, is another example of metabolic stereoselectivity in humans. Cytochrome CYP2C19 catalyzes the metabolism of Table 3.3 Stereoselectivity of drugs in clearance (Cl) upon parenteral administration (Brocks 2006)

Drug

Biological species

Enantiomer index

Albuterol

Human

1.59 [R:S]

Bisoprolol

Dog

1.45 [R:S]

Bupivacaine

Human

1.28 [R:S]

Etodolac

Rat

8.98 [S:R]

Halofantrine

Rat

1.53 [(–):(+)]

Hexaconazole

Rabbit

1.40 [(+):(–)]

Ketocorolac

Human

2.60 [S:R]

Methadone

Human

1.30 [R:S]

Propafenon

Rat

1.96 [(+):(–)]

Thalinolol

Human

1.04 [R:S]

Tocainide

Human

1.79 [R:S]

Verapamil

Human

1.77 [S:R]

44

3 General Issues of Chirality in Pharmacology

S-omeprazole predominantly by 5-O-demethylation, while R-omeprazole is metabolized by the same isoenzyme by 5-hydroxylation (Yang et al. 2016; Abelö et al. 2000) (Table 3.4). Interspecies stereoselective differences are a rather common occurrence. For example, metabolic oxidation of felodipine in humans is more intensive for the Senantiomer, while in rats and dogs it is the R-enantiomer that metabolizes intensively by this pathway (Fig. 3.6). Stereospecific metabolism of a drug can only affect the therapeutic effects of one of its enantiomers. For example, urotoxicity, one of the major dose-dependent sideeffects of ifosfamide (Fig. 3.7), is caused by acrolein, the product of dechlorethylation reaction. Dechlorethylation reaction, mediated by the cytochrome CYP2B6 and CYP3A4, is a rival metabolic pathway for 4-hydroxylation. Roy et al. (1999) established that the dechlorethylation reaction is almost 4.6 times more intensive for the S-enantiomer than for the R-enantiomer (Table 3.4). At the same time, 4-hydroxylate is formed three times faster for the R-enantiomer than for the S-enantiomer. Thus the metabolic profile of R-ifosfamide is more favorable than that of its S-isomer. Stereoselective consequences of the hepatic first pass effect are more pronounced for pharmacokinetic parameters such as area under the curve (AUC) and maximum drug concentration in the blood plasma (Cmax ) after peroral administration as compared to intravenous administration (especially for drugs with a high index of hepatic excretion). Thus in humans the ratio (–):(+) for AUC upon peroral administration of propanolol is higher than for the same doses of the drug upon intravenous administration (Mehvar and Brocks 2001). Another cardiovascular drug, verapamil, also shows a greater stereoselectivity upon peroral administration as compared to intravenous administration (Mehvar and Brocks 2001). For some drugs, hepatic first pass effect may be more pronounced for one isomer compared to other. Such stereoselectivity for 1st pass effect may result in unexpected differences in the serum concentration of its isomers especially when quantitative analysis is nonstereselective. The difference in serum concentration translates into differences in pharmacological effects. For instance, verapamil with its high rate of hepatic excretion revealed a more pronounced dependence between the concentration and the effect after intravenous administration as compared to peroral administration when the methods of quantitative analysis were nonstereoselective (Echizen et al. 1985). This is a sign of the stereoselectivity of the drug as to the first pass effect and indicates a much higher activity of the S-(–)-enantiomer upon peroral administration. The S:R index for verapamil also depends on the rate at which verapamil passes through the liver. Thus S:R index for bioavailability was 35% higher when verapamil was administered at high rates into the perfused rat liver as compared to a slower rate of administration (Mehvar and Reynolds 1995). In a similar way, Marier et al. (1998) discovered that raising the dose of propanolol from 40 to 120 mg/kg in rabbits resulted in a progressive increase in the S:R index from 1.0 to 1.7 as to the plasma concentration. It is interesting to note that a 20-fold increase of intravenous dose of propanolol (from 0.5 to 10 mg/kg) caused a drop of the S:R AUC index from 1.0 to 0.82. The gastrointestinal tract also participates in propanolol metabolism, but at high doses it is probably the slow-down of the

mono-N-desalkylation mono-N-desalkylation

CYP3A4, human

CYP3A12, dogs

Microsomal enzymes of rat liver

Microsomal enzymes of rat liver

Microsomal enzymes of rat liver

Disopyramide

Disopyramide

Etodolac

Etodolac

Felodipine

Isoproterenol

Ifosfamide

Sulfation

Human phenol sulfotransferase

Albuterol

4

Sulfation

Dechloroethylation

CYP2B6, microsomal enzymes of human liver

Human phenol sulfotransferase

Dechloroethylation

0.88 [R:S]

1.7 [R:S]

2.9 [R:S]

1.01 [R:S]

Microsomal enzymes of human liver

CYP3A4, dicrosomal enzymes of human liver

1.19 [R:S]

1.05 [R:S]

–

3.41–5.56 [R:S]

0.79 [R:S]

1.00 [R:S]

0.90 [R:S]

Enantiomer index as to maximum rate of product formation (Vmax )

Microsomal enzymes of dog liver

Oxidation

Oxidation

Acyl glucuronide formation

3 Metabolism pathway, metabolite

2

Enzyme

1

Drug

Table 3.4 Comparative stereoselectivity of drugs in the metabolism of their enantiomers 5

6.25 [R:S]

0.92 [R:S]

0.56 [R:S]

2.0 [R:S]

0.50 [R:S]

0.80 [R:S]

–

1.05–1.27 [R:S]

1.28 [R:S]

1.08 [R:S]

0.10 [R:S]

Enantiomer index as to Michaelis constant (Km )

6

0.14 [R:S]

2.5 [R:S]

4.6 [R:S]

0.52 [R:S]

1.47 [R:S]

1.35 [R:S]

~2.6 [R:S]

(continued)

3.26–4.33 [R:S]

0.62 [R:S]

0.94 [R:S]

8.88 [R:S]

Enantiomer index as to metabolic clearance (Clint )a

3.1 Chirality and Pharmacokinetics 45

hydroxylation

Norverapamil D-167 metabolite Norverapamil D-167 metabolite

Microsomal enzymes of dog liver

Human phenol sulfotransferase

Human phenol sulfotransferase

CYP3A4, microsomal enzymes of human liver

CYP3A5, microsomal enzymes of human liver

CYP2C8, microsomal enzymes of human liver

Microsomal enzymes of human liver

Propranolol

Salmeterol

Terbutalin

Verapamil

Verapamil

Verapamil

Warfarin

= Vmax /Km

0.39 [S:R]

5-O-demethylation

int

1.8 [S:R]

Sulfones formation

Microsomal enzymes of human liver

Omeprazole

a Cl

3.6 [S:R]

Sulfones formation

Human phenol sulfotransferase

4

7-hydroxylation

Norverapamil D-167 metabolite

Sulfation

Sulfation

Glucuronide formation

0.21 [R:S]

0.46 [R:S] 1.0 [R:S]

1.20 [R:S] 1.41 [R:S]

0.87 [R:S] 1.6 [R:S]

0.62 [R:S]

1.10 [R:S]

3.91 [S:R]

0.90 [R:S]

12.7 Vmax1 [(–):(+)] 3.0 Vmax2 [(–):(+)]

Metaproterenol

3 Sulfoxidation

2

Microsomal enzymes of human liver

1

Lansoprazole

Table 3.4 (continued) 5

5.3 [R:S]

0.82 [R:S] 0.69 [R:S]

0.99 [R:S] 1.43 [R:S]

1.09 [R:S] 1.76 [R:S]

0.83 [R:S]

0.28 [R:S]

1.70 [S:R]

4.0 [S:R]

0.62 [S:R]

0.82 [S:R]

2.95 [R:S]

3.39 Km1 [(–):(+)] 1.0 Km2 [(–):(+)]

6

0.043 [R:S]

0.56 [R:S] 1.46 [R:S]

1.22 [R:S] 0.98 [R:S]

0.80 [R:S] 0.91 [R:S]

0.75 [R:S]

3.99 [R:S]

2.30 [S:R]

0.10 [S:R]

3.7 [S:R]

4.6 [S:R]

0.31 [R:S]

3.8 CIint1 [(–):(+)] 3.0 CIint1 [(–):(+)]

46 3 General Issues of Chirality in Pharmacology

3.1 Chirality and Pharmacokinetics H N

H 3C

47

CH3 CH3

O

O H3C

(R)

O

H

H N

H3C

O

CH3 CH3

O

O H3C

(S)

O

H

O

Cl

Cl

Cl

Cl

3-Ethyl-5-methyl-[4-(2,3-dichlorophenyl)-2,6-dimethyl-1,4-dihydropyridine]-3,5-dicarboxylate Fig. 3.6 Felodipine enantiomers

Cl

O O

Cl

O O

(R) P

(S) P

N H

N

N H

N Cl

Cl

3-(2-Chloroethyl)-2-[(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorin-2-oxide Fig. 3.7 Ifosfamide enantiomers

S-enantiomer metabolism upon ‘first pass’ that mostly accounts for the increase in the S:R AUC ratio following oral administration (Srinivas et al. 1993). Stereoselective interaction of cytochromes with substrates has been shown for other drugs. For example, CYP2C9 metabolizes S-warfarin, S-acenocoumarol and S-ibuprofen, CYP2B6 metabolizes S-mefenitoin and CYP2C19 metabolizes R-fenitoin and S-mefenitoin. In 4-hydroxylation reaction CYP3A4 metabolizes R-isofosfamide and CYP2B6 metabolizes S-isofosfamide. CYP3A4 yields R(N 2 )-dechloroethyl-isofosfamide and R-(N 3 )-dechloroethyl-isofosfamide (from Risofosfamide), while CYP2B6 yields S-(N 3 )-dechloroethyl-isofosfamide and S-(N 2 )dechloroethyl-isofosfamide (from S-isofosfamide) (Filimonova et al. 2007). Since CYP enzymes are most versatile biocatalysts, engineering CYP450s for desired regio- and stereoselective oxidations of complex organic molecules is likely to yield useful biocatalysts in the production of several therapeutic agents as well as other chemicals inculding flavors and fragrances (Li et al. 2020). The concentration of chiral drugs in the plasma can vary due to polymorphism affecting the metabolism of drugs (Table 3.5). Various isoenzymes of the cytochrome P450 take part in the biotransformation of antidepressant drugs, but the main isoenzyme for their biotransformation is CYP2D6. Polymorphism of genes coding for this

48

3 General Issues of Chirality in Pharmacology

Table 3.5 Effect of polymorphism on drug metabolism leading to stereoselective differences in the bioavailability of chiral drugs (Fjordside et al. 1999) Drug

“Metabolizer” status

Mean value of enantiomer index for area under pharmacokinetic curve (AUC)

Chlorpheniramine

“extensive” “poor” CYP2D6

2.4 [S:R] 3.5 [S:R]

Flecainide

“extensive” “poor” CYP2D6

1,03 [R:S] 1.29 [R:S]

Fluoxetine

“extensive” “poor” CYP2D6

1.45 [S:R] 5.59 [S:R]

Lansoprazole

“extensive” “poor” CYP2C19

8.54 [R:S] 5.71 [R:S]

Metoprolol

“extensive” “poor” CYP2D6

0.60 [(+):(–)] 1.11 [(+):(–)]

Mexiletine

“extensive” “poor” CYP2D6

0.96 [R:S] 0.92 [R:S]

Omeprazole

“extensive” “poor” CYP2C19

0.62 [(+):(–)] 1.5 [(+):(–)]

Pantoprazole

“extensive” “poor” CYP2D6

0.84 [(+):(–)] 3.62 [(+):(–)]

Propafenone

“extensive” “poor” CYP2D6

1.71 [S:R] 1.48 [S:R]

Propranolol

“extensive” “poor” CYP2D6

0.70 [(+):(–)] 0.93 [(+):(–)]

Trimipramine

“extensive” “poor” CYP2D6 “poor” CYP2C19

1.15a [L:D] 0.60a [L:D] 1.66a [L:D]

a Minimum

steady-state concentration (Cssmin )

cytochrome P450 isoenzymes affects its activity. Carriers of functionally defective allele variants of CYP2D6 gene are “poor metabolizers”. Administration of standard doses of antidepressants metabolized by CYP2D6 to such patients may result in high concentrations of these drugs in the body. As has been shown, “poor metabolizers” of fluoxetine, an antidepressant, display a S:R AUC ratio that is 3.8 times higher than the corresponding parameter in “extensive metabolizers” (Fjordside et al. 1999). The antithrombotics of the tetrahydrothienopyridine series, clopidogrel and prasugrel, are prodrugs that must be metabolized in two steps to become pharmacologically active. The first step is the formation of a thiolactone metabolite. The second step involves further oxidation to a thiolactone sulfoxide, which upon hydrolytic opening forms a sulfenic acid that is eventually reduced into the corresponding active cis thiol. Very little data is available on the formation of the isomer of the active cis thiol having a trans configuration of the double bond. The most striking result in that regard is that both the cis and trans thiols were formed upon metabolism of clopidogrel by human liver microsomes in the presence of glutathione

3.1 Chirality and Pharmacokinetics

49

(GSH), however only the cis thiol was detected in the sera of patients treated with this drug. Dansette et al. (2015) have shown that trans thiols are also formed upon the microsomal metabolism of prasugrel or its thiolactone metabolite in the presence of GSH and that metabolites having the trans configuration of the double bond are only formed when microsomal incubations are done in the presence of thiols, such as GSH, N-acetyl-cysteine, and mercaptoethanol. Intermediate formation of thioesters resulting from the reaction of GSH with the thiolactone sulfoxide metabolite appears to be responsible for trans thiol formation. Addition of human liver cytosol to the microsomal incubations led to a dramatic decrease in the formation of the trans thiol metabolites. These findings suggest that cytosolic esterases would accelerate the hydrolytic opening of thiolactone sulfoxide intermediates and disfavor the formation of thioesters resulting from the reaction of these intermediates with GSH that is responsible for trans isomer formation. Chiral Inversions Some enantiomers can convert from one enantiomeric configuration to another. This phenomenon is referred as “chiral inversion”. Chiral inversion can either be spontaneous and bidirectional or be enhanced by the presence of specific proteins or enzymes and so be unidirectional. Thalidomide used in 1960s as a sedative and known to be notorious for its ability to cause grave congenital defects, is also known as a drug undergoing bidirectional chiral inversion/racemization in the blood (Eriksson et al. 1998). Oxindanac, a NSAID, also displays bidirectional chiral inversion when added to plasma in vitro and when administered to dogs (Fig. 3.8), although the equilibrium is shifted more to the side of S-enantiomer (Klebe et al. 1994). The rate of these mutual conversions increases significantly in the presence of albumin. Interestingly, the derivatives of 2-arylpropionic acid, NSAID, showed unidirectional chiral inversion (Davies 1998, 2004; Brocks and Jamali 1999). This process involves initial formation of acylCoA-thioester with a substrate, followed by its inversion, catalyzed by elimerases, and hydrolytic cleavage of the formed antipode by acyl-CoA-ligase. In addition to the substrate specificity of elimerase itself, the O

O

HO

(R)

H OH

O

H

(S)

HO O

5-Benzoyl-6-oxy-2,3-dihydro-1H-inden-1-carbonic acid Fig. 3.8 Chiral inversion of oxindanac

OH

50

3 General Issues of Chirality in Pharmacology

rival effect of some metabolic pathways predetermines the possibility of chiral inversion and of its extent (Mohri et al. 2005). It was shown experimentally that ibuprofen and especially fenoprofen are subject to considerable unidirectional chiral inversion which causes transformation of inactive R- into the active S-enantiomer both in humans and animals (Berry and Jamali 1991). For some other NSAIDs, the presence of unidirectional R → S chiral inversion and its extent are species-specific. Thus, ketoprofen and tiaprofenic acid that display minimum chiral inversion in the body of a human undergo considerable inversion in rats (Jamali et al. 1990; Foster and Jamali 1988; Singh et al. 1986; Erb et al. 1999). Ketoprofen is also intensively converted from R- to S-enantiomer in dogs and horses. Flurbiprofen undergoes chiral inversion in horses but not in humans and rats (Jamali et al. 1988). In contrast to most species, in CD-1 mice the chiral inversion of ketoprofen is bidirectional rather than unidirectional (Jamali et al. 1997). The site of chiral inversion of ibuprofen (at the organ level) was a much debated issue. Some authors proposed that inversion of ibuprofen is of exclusively systemic character (that is, hepatic by nature), while others were of the opinion that the processes of chiral conversion occurs both in liver and in the gastrointestinal tract (Hall et al. 1993; Jamali et al. 1992). Since, chiral inversion has been detected in the intestinal tissues, the second hypothesis seems to play more important role in chiral inversion (Sattari and Jamali 1994, 1997). Furthermore, studies have shown that for quickly absorbed forms of the R-enantiomer, the end concentration of the S-enantiomer formed as a result of chiral inversion decreases because the time of their stay in the intestine is minimal (Brocks and Jamali 1999). The therapeutic impact of chiral inversion of NSAIDs is that the administered dose of the racemate may be higher than the dose calculated for the content of the active ingredient in the medicinal form. This may affect several pharmacokinetic parameters such as clearance and the volume of enantiomer distribution. Chiral inversion, for most drugs, does not lead to significant changes in their concentration in the body (Brocks and Jamali 1999). Excretion Stereoselectivity can be observed in excretion of different stereoisomers of a drug through both the biliary and urinary tract. Different transport proteins like Pglycoprotein, MRP (multidrug resistance protein) and carriers of organic anions and cations are also involved in the transport of drugs and can be selective in substrate binding. Although glomerular filtration in the kidneys is not stereoselective by itself, the differences in binding to the plasma proteins can affect the rate of filtration of enantiomers. When the drug is completely excreted from the body by only one route of excretion, such as bile or urine, the excretion rate and cumulative excretion can be a good indicator of stereoselective drug excretion. When the drug is excreted by several routes, one should clearly understand that the evidence of stereoselectivity in one route of excretion may not be applicable for other routes of excretion. If one needs to determine stereoselectivity in only one route of excretion, one should assess both the concentration of the drug in the blood, and the clearance for each route of excretion

3.1 Chirality and Pharmacokinetics

51

CH3

CH3

H O

H (R)

N H

CH3

O

(S)

OH

N H

CH3

O

CH3

OH

O

CH3

O

O CH3

CH3

1-[4-[(2-Isopropoxyethoxy)methyl]phenoxy]-3-(isopropylamino)propan-2-ol Fig. 3.9 Bisoprolol enantiomers

independently. Bisoprolol (Fig. 3.9) (Horikiri et al. 1997) serves as a good example. The ratio of S:R enantiomers in the urine for this drug was found to be 1.29 despite its nonstereoselective renal clearance (S:R = 0.96). Similarly, excretion of etodolac glucuronide in bile was found to be of stereoselective nature, however, this fact can be associated with the selectivity of glucuronide synthesis rather than with the process of excretion itself (Brocks and Jamali 1990). There is indirect evidence of stereoselective binding of drug enantiomers with organic cation transporters that are found in the liver, kidneys and other tissues. A number of enantiomer compounds including disopyramide, pindolol and verapamil were shown to inhibit transmembrane transport of tetraethylammonium in renal cells stereoselectively. Similar stereoselectivity was detected in the ability of disopyramide enantiomers to inhibit the transport of tetraethylammonium by human organic cation transporter, hOCT1. In both cases the R-enantiomer inhibited the transport more actively than its antipode (Lavoie et al. 1994). Implementation of new approaches using hOCT1-containing chromatography columns has demonstrated that stereoselective binding with OCT is prominent for propranolol, atenolol and pseudoephedrine with the R:S index of 3.0, 2.1 and 1.53, correspondingly (Moaddel et al. 2005a, b). Another similar study showed that binding of R-verapamil to hOCT1 is more selective with a R:S index of 58 (Moaddel et al. 2005a, b). Stereospecific Drug Interaction The enantiomers of a racemate can interact with other drugs co-administered with the racemate (Table 3.6). This drug-drug interaction can be due to interaction in pharmacokinetic processes such as plasma protein binding, metabolism and excretion.

52

3 General Issues of Chirality in Pharmacology

Table 3.6 Pharmacokinetic interaction of drug enantiomers administered with drugs or food (Fromm et al. 1996) Drug

Biological species

Interacting drug

Plasma concentration change

Possible mechanism

Carvedilol

Human

Amiodarone

S↑ R↔

Possibly selective inhibition of CYP2C9 by desethylamiodorone

Chlorpheniramine

Human

Quinidine

R and S ↑

Inhibition of CYP2D6

Etodolac

Rat

Phenobarbital

R and S ↓

Induction of hepatic enzymes

Mexiletine

Human

Ciprofloxacin

R and S ↑

Inhibition of CYP1A2

Nicardipine

Human

Grapefruit juice

(+) and (–)↑

Inhibition of CYP3A4

Nitrendipine

Human

Cimetidine

R and S ↑

Inhibition of CYP3A4

Pindolol

Human

Cimetidine

R and S ↑

Inhibition of renal tubular secretion and hepatic metabolism

Propranolol

Human

Nicardipine, or verapamil, or cimetidine, or quinidine

R and S ↑

Inhibition of the ‘first pass’ effect

Verapamil

Human

Rifampicine

R and S ↑

Induction of hepatic and intestinal cytochrome P450

Verapamil

Human

Grapefruit juice

R and S ↑

Inhibition of cytochrome CYP3A4

Warfarin

Human

Cimetidine

R↑ S ↔

Inhibition of cytochrome CYP

Warfarin

Human

Paramidine

S↑ R↔

Inhibition of cytochrome CYP

Note ↑ increase, ↓ decrease, ↔ no change

There are several examples of drug interactions where the concentration of one enantiomer differs significantly from the concentration of the other enantiomer due to interaction with another drug. For instance, the plasma concentration of R-warfarin increases in the presence of cimetidine, while the concentration of S-warfarin remains unchanged (Choonara et al. 1986). In a similar way, the decrease of renal clearance of pindolol by cimetidine was more pronounced for the R-enantiomer, although, the difference in plasma concentrations of the isomers was minimal (47% increase for the R-enantiomer, and 38% increase for the S-enantiomer) (Somogyi et al. 1992). As general observations show, drugs with a higher hepatic excretion are more capable of interacting with enantiomers than drugs with low hepatic excretion. This can be due to the fact that the interacting drug affects enantioselectively not only hepatic excretion but also intestinal bioavailability of enantiomers. For example,

3.1 Chirality and Pharmacokinetics

53

Fig. 3.10 Mexiletine enantiomers

following peroral administration, the effect of the inhibiting interaction on the rate of metabolism is more pronounced for (+)-propranolol than for its (–)-enantiomer resulting in a considerable increase of the (+):(–) ratio in the plasma and/or serum (Vercruysse et al. 1994; Hunt et al. 1990; Donn et al. 1985; Zhou et al. 1990). Difference in the extent of induction of hepatic enzymes by different enantiomers also leads to stereoselective differences in the plasma concentration of the drug as was shown for verapamil after its co-administration with rifampicin. In this case induction of presystemic metabolism in the gastrointestinal tract was combined with hepatic induction. Consequently, the relative decrease of the AUC was more pronounced for R-verapamil than for S-verapamil (Fromm et al. 1996). Drug interaction can affect excretion by different routes. Ciprofloxacin caused a considerable decrease in the total body plasma clearance and nonrenal clearance of mexiletine enantiomers (Fig. 3.10), possibly through inhibition of CYP1A2. The same studies, however, revealed a considerable increase in renal clearance for both enantiomers of mexiletine, although these routes of excretion constituted only a small part of the total body plasma clearance. Nevertheless, the changes in the plasma concentration of mexiletine as a result of coadministration with ciprofloxacin probably do not have a great clinical importance (Labbé et al. 2004). Factors Affecting Stereospecific Pharmacokinetics Age and Sex Dependent Differences It was shown in a number of studies that patients of different age groups show differences in the sensitivity to enantiomer actions (Table 3.7). Tan et al. pointed out in their study that elderly patients were more subject to the effect of the S-enantiomer of ibuprofen, which potentially makes this drug more active in this group of patients (Tan et al. 2003). Similarly, it was also shown that S-enantiomer of amlodipine has a more pronounced vasodilating action and shows a higher R:S ratio for AUC0-24 in elderly subjects than the earlier established values for middle-aged patients (Ohmori et al. 2003). Age-related differences in stereoselectivity were also found for vigabatrin, verapamil, citalopram, ketorolac, and disopyramide.

54

3 General Issues of Chirality in Pharmacology

Table 3.7 Effect of age on pharmacokinetic stereoselectivity of (Tan et al. 2003) Drug

Group under study

Changes in comparison with teenagers

Citalopram

Elderly patients

AUC S:R ratio increased insignificantly in young patients (by 15% approximately)

Disopyramide Children aged 5–12

A higher R:S ratio for Cl/F and renal clearance in young patients

Flurbiprofen

Elderly patients

No stereoselective changes in pharmacokinetics

Ibuprofen

Elderly patients

The S-enantiomer shows an increase in such values as t½ and fu (free fraction of the drug in the blood) and a decrease in unbound clearance by 28%

Ketoprofen

Adult arthritic patients

No changes established between the group of young patients and the group of arthritic patients

Ketorolac

Children and teenagers Age-related changes in volume of distribution (Vd) and elimination half-life (t½) for the S:R index; a decrease in the S:R index for Vss from 4.6 in children to 3.1 in teenagers and 2.2 for children, without changes in clearance

Pindolol

Elderly patients

No stereoselective changes in renal clearance

Vigabatrin

Infants

Infants possibly have a lower AUC R:S ratio than children or adults

Verapamil

Elderly patients

A higher (30–81%) S:R index for Cmax and AUC in elderly men and women in comparison with young patients

Unfortunately, there is not enough evidence in literature permitting a conclusion about the stereoselectivity of drugs in relation to patients of different sexes. In the framework of a population model, a statistically significant correlation between the values of pharmacokinetic parameters of methadone and patient’s sex was established. Besides, all the obtained dependencies differ considerably in the extent of the effect on pharmacokinetic parameters of R- and S- enantiomers of methadone (Foster et al. 2004). Reboxetine is a chiral antidepressant drug used in the form of (R,R)- and (S,S)enantiomers (Fig. 3.11). After intravenous administration of the drug, no significant differences between men and women were found in Cl, Vd and Vss for two enantiomers. However, after oral administration, the (R,R):(S,S) ratio for Cmax in men (1.6) was significantly different from the same value for women (2.3). No differences whatsoever in the values of AUC enantiomer ratio were detected between men and women (Fleishaker et al. 1999). In later studies also, it was shown that the (R,R):(S,S) ratio for Cmax of the drug in the plasma was considerably higher in women than in men despite the fact that the average concentration of reboxetine enantiomers was found to be the same in male and female patients (Ohman et al. 2003).

3.1 Chirality and Pharmacokinetics

55

Fig. 3.11 Reboxetine enantiomers

Pathological States The pharmacokinetic parameters of enantiomers can be affected by the patients’ lifestyle, social habits and pathological states. Smoking is associated with induction of metabolism of many drugs. In mexiletine-receiving patients the total plasma clearance for both enantiomers was 40–60% higher in the group of smokers in comparison with non-smokers (Labbé et al. 2004). Food preferences like considerable intake of citrus fruits can result in stereoselective changes especially when the drug is a substrate for CYP3A. Some pathological states can also lead to the changes in pharmacological characteristics of enantiomers (Table 3.8). Mayo et al. report that in rheumatoid arthritis Table 3.8 Effect of a pathological state on pharmacokinetics of a stereoselective drug after peroral administration of a racemate (Mayo et al. 2000) Drug

Group under study

Average value of enantiomer ratio for Cmax

Average AUC enantiomer ratio

Healthy volunteers

3.3 [S:R]

11.6 [S:R]

Arthritic patients

3.9 [S:R]

7.0 [S:R]

Adults

1.1 [S:R]

1.6 [S:R]

Children with Cystic fibrosis

1.2 [S:R]

2.0 [S:R]

Ketoprofen

Healthy teenagers

1.05 [S:R]

1.18 [S:R]

Arthritic teenagers

1.03 [S:R]

1.06 [S:R]

Ketorolac

Adults (aged 18–44)

–

3.12 [R:S]

Fenoprofen Ibuprofen

Verapamil

Teenagers (aged 12–17)

–

3.23 [R:S]

Children (aged 6–11)

–

2.61 [R:S]

Healthy adult volunteers

4.93 [R:S]

5.6 [R:S]

Patients with rheumatoid 5.54 [R:S] arthritis

[R:S]

56

3 General Issues of Chirality in Pharmacology

the sum of bound and unbound concentrations of both enantiomers of verapamil increased almost five times (McLean et al. 2006). AUC of the less active unbound R-enantiomers increased two and a half times while that of the more active unbound S-enantiomer did not increase. Although unbound concentrations of the more active S-enantiomer did not differ in patients with inflammation from that in the control group, the R-enantiomer displayed a higher activity in relation to the increase in the P-R interval in the electrocardiogram of patients in the control group. Another study reported that the R:S index for ibuprofen as to its pain-killing action was higher when tested after surgical removal of a molar tooth than before the procedure (Jamali and Kunz-Dober 1999).

3.2 Chirality and Pharmacodynamics Pharmacodynamic features of drugs are affected by their stereoisomeric properties. Thus one of the enantiomers can be considerably more active (eutomer) in comparison with the other one that is less active or altogether inactive (distomer). The relation of the activity of the more active enantiomer to the activity of the less active one is known as eudesmic ratio. It is a measure of the extent of the effect of stereoselectivity on drug’s biological activity. The greater is this ratio, the stronger is the biological activity of only one optical isomer. This is seen more clearly when the asymmetric centre is at that place in the molecule which is responsible for its interaction with the receptor (the so-called Pfeiffer’s rule) (Alekseev 1998). The main pharmacological activity of racemic drugs is commonly associated with the action of only one enantiomer. The second one is either less active or altogether inactive or displays totally different pharmacological properties. There were cases in clinical practice when racemic drugs were administered and one of the stereoisomers had a powerful toxic effect with a fatal outcome. For example, the levorotatory enantiomer of thalidomide is a potent tranquilizer while its dextrorotatory isomer, which is present in the mixture in equal measure, produces a teratogenic effect (Alekseev 1998). Noteworthy, synergetic interaction of enantiomers is also possible. In a review on clausenamide (clau), a small molecule originally isolated from the traditional Chinese herbal medicine, Clausenalansium, Chu et al. (2016) have shown that presence of different enantiomers may result in a multitarget action of the drug. The finding of four chiral centers in clau molecules predicted the presence of 16 clau enantiomers, including (−)-clau and (+)-clau. All of the predicted enantiomers have been successfully synthesized via innovative chemical approaches, and pharmacological studies have demonstrated (−)-clau as a eutomer and (+)-clau as a distomer in improving cognitive function in both normal physiological and pathological conditions. Mechanistically, the nootropic effect of (−)-clau is mediated by its multitarget actions, which include mild elevation of intracellular Ca2+ concentrations, modulation of the cholinergic system, regulation of synaptic plasticity, and activation of

3.2 Chirality and Pharmacodynamics

57

cellular and molecular signaling pathways involved in learning and memory. Furthermore, (−)-clau suppresses the pathogenesis of Alzheimer’s disease by inhibiting multiple etiological processes: (1) beta amyloid-induced intracellular Ca2+ overload and apoptosis and (2) tau hyperphosphorylation and neurodegeneration. The nature of the multitarget actions of (−)-clau make it a promising chiral drug candidate for enhancing human cognition in normal conditions and treating memory impairment in neurodegenerative diseases. Studying the activity of stereoisomers on isolated biotargets eliminates the effect of penetration through membranes and distribution in the body and permits an evaluation of the effectiveness of stereoisomeric substances with regards to their direct reaction with the receptor. Interaction of an asymmetric molecule of a medicinal substance with the active centre of a receptor takes place according to the lockand-key principle and is determined by their contact at a number of points. Some of these contact points may be points of binding while others points of repulsion. Apparently, the existence of the former determines the affinity of the substance to the receptor. The presence of the latter can also determine the affinity as repulsion of certain groups of the drug molecule and receptor can promote a specific change in the conformation of the receptor. Let us imagine that the main forces of interaction between an asymmetric molecule of a drug substance with an active site of the receptor (or enzyme) are localized at least at three points as shown in Fig. 3.12. Only two out of three groups can have same orientation in two antipodes. Variation in the orientations of the third group can account for the differences in biological activity of the isomers. Furthermore, depending on the extent of involvement of the group in the process of interaction with the receptor, the effect of isomerism can be of greater or lesser degree. If the substance interacts with the receptor at only two points, we cannot expect any difference in biological activity of its isomers. However, in this case, if the third group in one isomer spatially prevents the contact of the substance with the receptor at two other points, there should be a difference in the biological activity of two optical antipodes. For instance, in only one of the two optical isomers of adrenaline (Fig. 3.13), all three groups are oriented in a way so as to allow binding with corresponding groups

Fig. 3.12 Differences in the orientation of the third group accounting for the difference in biological activity of optical isomers exemplified by the interaction of R-(–)-adrenaline (a) and its S-(+)antipode (b) with adrenoreceptors (Wermuth 2015)

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Fig. 3.13 Model of interaction of R-(–)-adrenalin with receptors (for amino acids their numbers are given in primary order) (Wermuth 2015)

of the receptors. This is the case with R-(–)-adrenaline, which shows maximum pharmacological activity. In S-(+)-adrenaline, incorrect orientation of the hydroxyl group allows interaction of drug molecule with receptor only at two points. As a result, S-(+)-isomer is a dozen time less potent than naturally occurring R-(–)-adrenaline. Among the opioid analgesics, morphine displays pronounced stereoselectivity (Fig. 3.14). The absolute configuration of this alkaloid has been fully established despite the presence of five asymmetrical carbon atoms (C5, C6, C9, C13 and C14). This amount of asymmetrical atoms theoretically creates possibility for the existence of 32 optical isomers of morphine. However, due to the restrictions imposed by the bridging chain of ethylamine forming a ring system (C9-C13), morphine has only sixteen optical isomers. All these isomers have been isolated and studied. Moreover, both the configuration of all asymmetric centers and their signs of rotation have been determined. Analysis showed that C5, C6 and C9 are laevorotatory centers while C13 and C14 are dextrorotatory. Morphine and similar alkaloids provide an illustration of how spatial configuration affects the pharmacological activity of compounds. Morphine obtained from the natural plant consists of laevorotatory isomers which are powerful analgesics whereas the synthesized laevorotatory isomer of morphine is totally devoid of any analgesic properties whatsoever.

3.2 Chirality and Pharmacodynamics

59

Fig. 3.14 Stereoselectivity of morphine

Similarly, the scientific evidences also indicate the difference in the activity of stereoisomers of a local anesthetic, cocaine, which has eight optical isomers. Four racemic forms of cocaine: cocaine, pseudococaine, allococaine, and allopseudococaine are known (Fig. 3.15). It is apparent from Table 3.9 that different stereoisomers of cocaine produce the local anesthetic effects of different intensity, and the most effective isomer is (–)cocaine. Among all, the most toxic one is racemic allococaine and the least toxic is—(+)-cocaine. When studying optical isomers of pseudcocaine, which is used in clinical practice labeled as psicaine, it was established that its (+)-isomer is the most active one. As described in a review (Dilruba and Kalayda 2016), cis-geometry was believed to be a necessary feature for platinum complexes to exhibit antitumor activity. However, increased repair of platinum-induced DNA damage by resistant cells turned researchers’ attention to the possibility that platinum complexes were able to build structurally different DNA adducts. Such unprecedented adducts were expected to undergo processing by cellular machinery in ways different from the processing of conventional cross-links. Due to their geometry, transplatinum complexes are predestined to interact differently with DNA than their cis-counterparts. Transplatin (trans-[Pt(NH3)2Cl2]) is known to lack antitumor activity, which has been attributed to the fast deactivation by sulfur proteins. Interestingly, if transplatin is applied to the cells irradiated with UVA light, it forms interstrand cross-links and protein–DNA adducts and exerts cytotoxic effects comparable to those of cisplatin. Introduction of the bulky ligands instead of ammonia decreased the rate of chloride exchange positively affecting the kinetics of DNA adduct formation and antitumor activity. A recent paper by Zask and Ellestad (2015) has highlighted a range of medically important protein targets for which stereoselective drugs have been identified:

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Fig. 3.15 Diastereomers of cocaine (chiral centers at which racemization takes place are shown by a *) (Findlay 1959; Villar and Loew 2004)

Table 3.9 Local anesthetic activity and toxicity of cocaine stereoisomers

Drug

Optical form

Anesthetic effect, rel. un

LD50 , mg/kg

Cocaine (natural)

(–)

1.0

205

Cocaine (synthetic)

(–)

1.9

255

Cocaine

(+)

0.22

800

Pseudococaine

Racemate

0.65

340

Allococaine

Racemate

0.47

43

heat shock protein 90 (Hsp90) inhibitors as anticancer agents; transient receptor potential vanilloid type 1 antagonists as new analgesics; stereospecific inhibition of human mutT homolog MTH1 for cancer treatment; the stereoselective binding of R- and S-propranolol by the α1 -acid glycoprotein transporter; metallohelical complexes that are nonpeptide α-helical mimetics that enantioselectively target

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61

Aβ amyloid for the treatment of Alzheimer’s disease; metallohelical assemblies with promising antimicrobial activity that enantioselectively target DNA of resistant bacteria; nonpeptide α-helical metallohelices that target the DNA of cisplatinresistant cancer cells; diastereomeric selectivity of phenanthriplatin-guanine adducts; and phenazine biosynthetic enzyme active sites that can host both enantiomers of a racemic ligand simultaneously.

3.3 Chirality and Toxicology As there are often great pharmacodynamic and pharmacokinetic differences between enantiomers, it is hardly surprising that they can also display stereoselective toxicity. Various adverse effects of chiral drugs can be associated with either one enantiomer or both. Toxicological characteristics of a pair of enantiomers may be identical or totally different. Both the pharmacologically active enantiomer and the inactive one are capable of producing side effects (Ariëns 1983; Landoni and Soraci 2001). Some of the drugs enumerated below are used in medical practice in the form of pure enantiomers for the sole reason that one of their enantiomers shows toxic properties. DOPA or hydroxy-3,4-phenylalanine is the precursor of dopamine which is effective in the treatment of Parkinson’s disease. DOPA was initially administered in racemic form (DL- DOPA, but only its laevorotatory form (L-DOPA) is now utilized in medical practice due to severe toxicity of the D-isomer (agranulocytosis). Tetramizole is an anthelminthic and immunostimulating drug. It was first used in the racemic form. Due to its numerous side effects (dizziness, headache, vomiting, abdominal pain) produced by the D-isomer, only the L-isomer labeled as levamisole is now used in practice (Fig. 3.16). The trends in modern pharmacology are such that many racemic drugs are subjected to processing to remove toxic and inactive isomers (Landoni and Soraci 2001; Waldeck 2003). Nevertheless, a number of chiral drugs are still being utilized in the form of racemates. This can be for several reasons. Either their chiral separation presents difficulty or their pharmacologic effect and toxic action are incident to one and the same enantiomer, or the cost of manufacturing the active enantiomer is too high. Only the distomer displays strong toxicity in many chiral drugs such as ketamine, penicillamine, and ethambutol. For instance, only R-(–)-ketamine Fig. 3.16 Structural formula of levamisole

S N (S)

N

H

(S)-6-Phenyl-2,3,5,6-tetrahydroimidazo [2,1-b]thiazole

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(distomer) causes anxiety, hallucinations, restlessness, in contrast to S-(+)-ketamine (eutomer) (Powell et al. 1988). Enantiomers of secobarbital (Fig. 3.17) are equally effective as anticonvulsive agents, but the S-(+)-isomer is a more effective drug for anesthesia although displaying a greater toxicity than the R-(+)-isomer (Powell et al. 1988). Two isomers of cyclophosphamide show the same toxicity (Eichelbaum 1995). Theoretically speaking, the teratogenic effect of thalidomide is only incident to the inactive S-(–)-isomer (Fig. 3.18), but in practice both isomers possess genotoxicity due to mutual conversion in vivo depending on the species specificity of the drug (Eichelbaum 1995; Scott 1993). The studies performed in 1962 on mice at first established that only one enantiomer of thalidomide (S-form) displayed teratogenic effects while the other one produced a therapeutic effect. However, the attempt to isolate the R-isomer failed to solve the problem of teratogenicity as both isomers easily undergo mutual conversion in vivo (Rajkumar 2004; Waldeck 2003). Moreover, the toxicity of thalidomide can be due to its numerous chiral and achiral metabolites. The pharmacological and toxicological properties of these metabolites have not been studied yet. CH3

CH3

H

H

O

O CH3

( R)

CH2

HN

O

N H

CH3

( S)

O

CH2

HN

N H

O

O

5-Allyl-5-(2-pentyl)pyrimidine-2,4,6(1H,3H,5H)-trione Fig. 3.17 Enantiomers of secobarbital

Fig. 3.18 Toxic enantiomer of thalidomide

O

O NH

N

(S)

O

O

(S)-2-(2,6-Dioxypiperidine-3-yl)isoindolin-1,3-dione

3.4 Classification of Drugs from Stereochemical Point of View

63

3.4 Classification of Drugs from Stereochemical Point of View Depending on the number of asymmetrical carbon atoms in the structure, drugs can be divided into the following groups. 1. 2.

Achiral drugs (do not contain asymmetrical carbon atoms). Racemic drugs: – with one main bioactive enantiomer; – with enantiomers equal in bioactivity; – with enantiomers that can undergo chiral inversion.

3.

Stereoselective (chiral) drugs: – of natural origin (analogs of neurotransmitters, endogenous hormones, enzymes, etc.); – synthetic substances obtained by the method of chiral separation.

Racemic Drugs The racemic drugs that are studied for their pharmacological effects can be divided into three main groups. The first group includes majority of the racemic drugs. They have one main bioactive enantiomer (referred to as eutomer) and the other enantiomer is inactive, less active (distomer), toxic, or displays other pharmacological effects. The second group includes drugs with enantiomers having equal activity and possessing the same pharmacodynamic properties. The third group includes racemic drugs with only one eutomer, but in vivo its distomer can turn into bioactive antipode by chiral inversion (Ariëns 1983). Group 1. Racemic Drugs with One Main Bioactive Enantiomer This group includes a number of cardiovascular drugs widely used in the treatment of arterial hypertension, cardiac insufficiency, arrhythmia and other diseases. Among these are beta-adrenoblockers, calcium channel antagonists and angiotensinconverting enzyme inhibitors. The laevorotatory isomer of all beta-adrenoblockers is more effective at blocking beta-adrenoreceptors than their dextrorotatory isomer. For instance, S-(–)-propanolol is a hundred times more effective than its R-(+)-antipode. Many beta-blockers are still administered in clinical practice in the form of racemates, for example, acebutolol, atenolol, alprenolol, betaxolol, carvedilol, metoprolol, labetalol, pindolol, sotalol and others except for timolol and penbutolol that are administered in the form of a levorotatory isomer. Pure D-isomers can also be administered on their own for activities other than beta blockade. It was shown, for example, that DL- and D-propanolol inhibit the conversion of thyroxin (T4 ) to triiodothyronine (T3 ) (Stoschitzky et al. 1992). Consequently, pure D-propanolol can be utilized as a specific drug without the beta-blocking effect to reduce the concentration of triiodothyronine in hyperthyroid

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patients who cannot receive racemic propanolol due to contraindications for betablocking effect. In such cases, different pharmacological effects of different isomers may be beneficial in different clinical scenario. Many calcium channel antagonists are used in the form of racemic drugs, for instance, verapamil, nicardipine, nimodipine, nisoldipine, felodipine, manidipine and others. Diltiazem is not used in racemate form as it is a diastereomer with two pairs of enantiomers. S-(–)-Verapamil exceeds the R-(+)-isomer by ten to twenty times as to the extent of negative chronotropic effect, atrioventricular conduction and vasodilation in humans and animals (Echizen et al. 1988; Satoh et al. 1980). On the other hand, verapamil can also be administered in cancer chemotherapy to modify resistance. Unfortunately, when used for this purpose, verapamil has to be administered in very high concentrations at which its racemate displays significant cardiotoxicity. Now it has been revealed that R-(+)-verapamil is much less cardiotoxic than S-(–)-verapamil, therefore, the R-enantiomer is preferred choice for modification of drug resistance in cancer chemotherapy while the S-enantiomer and the racemate are more effective as calcium channel blockers in the treatment of cardiovascular diseases (International Symposium on Chirality 1999). All angiotensin-converting enzyme inhibitors like captopril, benazepril, enalapril, idapril are chiral compounds forming diastereomers, and most of them are administered in the form of pure stereoisomers. Valsartan (Fig. 3.19), angiotensin II receptor H3 C

H3C

CH3 (S)

N

N

O

OH

N H

N

NH

O

(S)-2-[N-[[2'-(1H-Tetrazol-5-yl)biphenyl-4-yl]methyl]pentanamido]-3-methylbutanic acid Fig. 3.19 Structural formula of valsartan

3.4 Classification of Drugs from Stereochemical Point of View

65

antagonist, is used in the form of a pure S-enantiomer which is more effective than R-enantiomer (Patocka and Dvorak 2004). Albuterol (salbutamol), salmeterol and terbutaline are selective β2 -adrenoceptor agonists. They are administered as bronchodilators in the treatment of bronchial asthma. For a long time they were used as racemates. However, in terms of their pharmacological activity, only the laevorotatory isomer is effective, while the dextrorotatory isomer is ineffective and may also be the cause of the rare disagreeable side effects of these drugs. Levalbuterol, the pure levorotatory isomer of albuterol, was approved in the USA. However, later it was shown that in clinical practice it does not have a higher safety or effectiveness in comparison with racemic albuterol at the same dose. Moreover, the cost of levalbuterol is higher than that of its racemate (Asmus and Hendeles 2000; Nowak 2003). Several drugs used in the treatment of neurological and psychiatric diseases are also chiral compounds but are used as racemates in clinical practice. Barbiturates such as hexobarbital, secobarbital, mephobarbital, pentobarbital, thiopental, thiohexital are racemic drugs. Only their levorotatory isomer produces a sedative or hypnotic effect and the other isomer is either ineffective or stimulant. For instance, S-(–)-secobarbital is more effective as an anesthetic drug than R-(+)-secobarbital as it induces general anesthesia faster (Drayer 1986). Ketamine is an intravenous anesthetic drug and its (+)-isomer is more effective and less toxic than its (–)-antipode. Unfortunately, ketamine is still administered in the form of a racemic drug (Katzung 2004; Lee and Williams 1990). Isoflurane is an inhalational anesthetic for general anesthesia and is widely used as a racemic mixture of its two optical isomers. The (+)isomer of isoflurane is more effective than its (–)-isomer in inhibition of transmembrane ion current resulting from the action of acetylcholine on brain neurons (International Symposium on Chirality 1999). S-(+)-citalopram is a hundred times more effective as a selective inhibitor of serotonin reuptake in the treatment of depression than its R-(–)-enantiomer. Methadone, an analgesic drug of central action with a high affinity for μ-opioid receptors, is administered in the treatment of opium addiction and as an analgesic in oncology. It is a synthetic chiral compound used in the form of a racemic mixture. However, it was established that R-(–)-methadone is approximately twenty-five to sixty times more effective as an analgesic than its S-(+)-antipode (Olsen et al. 1977; Pham-Huy 1997). The list of racemic drugs with one eutomer is a long one. It includes several anticonvulsant drugs; local anesthetics like propafenone, disopyramide, prilocaine, tocainide; antibacterial drugs like ofloxacin, moxalactam; anticoagulants like warfarin, acenocoumarol; hypocholesterolemic drug atorvastatin; antihistamine drugs like amphetamine, methamphetamine and proton pump inhibitors like omeprazole, pantoprazole, lansoprazole, etc. Group 2. Racemic Drugs with Equally Active Enantiomers There are not many racemic drugs that can be included into this group. Some of the examples are cyclophosphamide (antineoplastic agent), flecainide (antiarrhythmic agent) and fluoxetine (antidepressant) (Davies and Teng 2003; Lien et al. 2006).

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Group 3. Racemic Drugs with Chiral Inversion This group includes drugs having enantiomers that can transform from one to another in vivo or in vitro (for example, conversion from R- to S-form). Due to chiral inversion the pharmacological and toxicological properties of a racemate can change. Drugs of this group include, anti-inflammatory drugs like ibuprofen, ketoprofen, fenoprofen, benoxaprofen; benzodiazepines like oxazepam, lorazepam, temazepam and some M-cholinoblockers, in particular, the pure (–)-enantiomer of hyoscyamine that turns into racemic atropine in vitro (Lien et al. 2006; Marzo and Heftmann 2002). There are two types of chiral inversion of drugs: unidirectional and bidirectional. Unidirectional inversion with an enzyme acting as a catalyst was described earlier for NSAIDs such as ibuprofen, ketoprofen, fenoprofen and benoxaprofen. Only the Senantiomer is active in this group and displays the pain-killing and anti-inflammatory action. For ibuprofen, the S-enantiomer is hundred times more effective as cyclooxygenase-1 inhibitor than the R-ibuprofen. In humans, the hepatic enzymes can only cause chiral inversion of the inactive R-enantiomer into the active S-enantiomer, and not the other way round (Landoni and Soraci 2001; Marzo and Heftmann 2002). Bidirectional chiral inversion or racemization is typical of the derivatives of 3hydroxybenzodiazepine (oxazepam, lorazepam, temazepam) and those of thalidomide. The pure R- and S-enantiomers of thalidomide can undergo racemization in an aqueous solution. However, once inside the body, thalidomide undergoes racemization but not hydroxybenzodiazepines. This is due to the structural differences of the molecular fragments adjoining the chiral carbon atom. This phenomenon was first discovered while studying the difference in the concentrations of S- and R- forms of oxazepam in rabbits (Pham-Huy 1977). The authors explained that chiral inversion of oxazepam by its tautomerisation in vivo does not take place because of the steroselective difference in the affinity for plasma albumin as in the process of transport the enantiomers of oxazepam display different affinity to albumin, the transport protein. Fragments of the albumin molecule providing stereoselective binding can impede the interaction of hydroxyl ions with oxazepam enantiomers thus preventing its racemization in vivo. Clearly, the concentrations of S- and R-oxazepam in the serum can turn out to be different. In another study, He et al. (2004) demonstrated that chiral inversion of this benzodiazepine in vitro depends on temperature and is completely suppressed once the temperature of the aqueous solution is brought down to 10 °C. It should be noted that the S-(+)-oxazepam is one to two hundred times more effective as a tranquilizer and a sedative drug than the R-(–)-oxazepam (Mohler and Richards 1983). Thalidomide is a well known racemic psychotropic drug banned from clinical use in the 1960s due to its severe teratogenic effects. Nevertheless, a limited use of thalidomide is of particular interest due to its immunomodulating, antiangiogenic and anti-inflammatory properties (Davies and Teng 2003). Moreover, thalidomide is a powerful inhibitor of tumor necrosis factor-α. Thalidomide has shown good results in the treatment of conditions such as erythema nodosum leprosum, and Behçet’s syndrome. Pure enantiomers of thalidomide and its derivative N-hydroxythalidomide were obtained by asymmetrical synthesis and their individual biological and chemical properties were studied (Flaih et al. 1999; Robin et al. 1995). Due to the fact that

References

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thalidomide undergoes chiral inversion in vivo, it is difficult to determine the pharmacological effect of each enantiomer precisely. Moreover, various pharmacological effects of thalidomide are attributed to its numerous chiral and achiral metabolites and not only to the parent molecule.

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Chapter 4

The Significance of Chirality in Pharmacological and Toxicological Properties of Drugs

The effect of differences in the stereochemistry of drugs on their pharmacokinetic and pharmacodynamic properties has been recognized for long. In recent years, technological advances in the synthesis and analysis of chiral compounds has made it possible to closely observe for such differences. As a result, a number of new drugs have been registered as pure enantiomers and the drugs used in racemic forms currently have been subjected to analysis for their enantiomers. In this chapter, we discuss various groups of drugs and highlight the chirality-based differences in their pharmacological and toxicological properties.

4.1 Drugs Regulating the Function of Peripheral Nervous System DRUGS AFFECTING AFFERENT INNERVATION Local Anesthetics In the 1960s, it was established that stereoisomers of local anesthetics in ex vivo conditions usually display similar activity (Luduena 1969). However, in vivo studies on animals demonstrated that L-prilocaine, L-mepivacaine and L-bupivacaine are more potent, have longer duration of action and more pronounced vasoconstrictor activity in comparison with their D-isomers. As early as 1969 Luduena first demonstrated that the enantiomers of the derivatives of 2’,6’-pipecoloxylidide (Fig. 4.1) differ significantly in their local anesthetic action. Although the toxicity of drugs often depends on the species of the animal, on the whole levorotatory isomers were found to be less toxic than dextrorotatory ones. For example, it was established (Akerman and Ross 1970) that S-(–)-bupivacaine (levobupivacaine, Fig. 4.2) is much less toxic than its R-(+)-isomer both upon intravenous and subcutaneous administration. Later it was also demonstrated that the S-(–)-bupivacaine has longer duration of action and does not require repeated (Mather © Springer Nature Singapore Pte Ltd. 2022 A. A. Spasov et al., Pharmacology of Drug Stereoisomers, Progress in Drug Research 76, https://doi.org/10.1007/978-981-19-2320-3_4

75

76

4 The Significance of Chirality in Pharmacological and Toxicological... CH3

CH3 H N

O

H

C

C

O

C2H5 H N

N

*

C

N

C2H5

H

H

CH3

CH3

CH3

Lidocaine N-(2,6-Dimethylphenyl)2-diethylaminoacetamide

Mepivacaine N-(2,6- Dimethylphenyl)-1-methyl2-piperidinecarboxamide CH3

H N

O

CH3

C

*

C

O

C3H7 N

H N

H3C H

H

*

C H

C4 H9

CH3

CH3

Prilocaine N-(2-Methylphenyl)2-propylaminopropanamide

N

Bumecaine N-(2,4,6-Trimethylphenyl)-1-butyl2-pyrrolidinecarboxamide

Fig. 4.1 Structural formulas of some commonly used local anesthetics: the chiral centre is in the ring in mepivacaine and bumecaine. In prilocaine, it is inside the chain while in lidocaine it is achiral

CH3

CH3

O

O H N

C H

CH3

H N

(S)

N C4H9

S-(–)-Bupivacaine (S)-N-(2,6-Dimethylphenyl)-1-butyl2-piperidinecarboxamide

(S)

C H

CH3

N C3H7

S-(–)-Ropivacaine (S)-N-(2,6-Dimethylphenyl)-1-propyl2- piperidinecarboxamide

Fig. 4.2 Structural formulas of levobupivacaine and ropivacaine

2005) administration. It was also found to be more effective, and the sensory block was more pronounced than the motor block. However, the risk of acute cardiovascular complications prompted re-evaluation of the possible advantages of levobupivacaine. It is believed that cardiotoxicity of various isomers of local anesthetics is due to the stereoselective inhibition of sodium channels in the myocardial cells. Lee-Son et al. (1992) demonstrated that the R-isomers of bupivacaine and other experimental local anesthetics blocked the sodium channels more effectively than the S-isomers. It was established that this stereoselective sodium channel blockade contributes to cardiotoxicity of some enantiomers of local anesthetics.

4.1 Drugs Regulating the Function of Peripheral Nervous System

77

Ropivacaine, the S-(–)-enantiomer of propivacaine (Fig. 4.2), and levobupivacaine have been discussed extensively in a number of reviews (McClure 1996; McLeod and Burke 2001). Butrov, Schifman and Filippovich (Butrov et al. 2003) have published one of the most comprehensive reviews devoted to comparative pharmacological characteristics of ropivacaine and bupivacaine. Ropivacaine and bupivacaine are chiral anesthetics having prolonged duration of action, although their relative effectiveness is still debated. Bader et al. (1989) compared the effect of ropivacaine and bupivacaine on type A and C fibers of the nervus vagus of a rabbit. The authors reported that bupivacaine was more potent than ropivacaine in causing block of type A fibers, but the potency of both drugs in relation to type C fibers was identical thirty minutes after administration. A conclusion was made that bupivacaine displays a more pronounced effect on motor fibers (Bader et al. 1989). Another recent review also highlights the differences in the pharmacokinetic and pharmacodynamic properties of local anesthetics such as cocaine, bupivacaine, mepivacaine, prilocaine, ropivacaine, IQB-9302, articaine, etidocaine, carbisocaine due to stereochemical ˇ differences (Cižmáriková et al. 2020). Mather et al. (1994) established in an experiment on sheep that both enantiomers of mepivacaine have same clearance and volume of distribution after intravenous bolus administration, but the apparent elimination half-life was shorter for the S-isomer. For bupivacaine there was no difference in the volume of distribution of the S- and R-isomers, but the clearance of the S-isomer was lower, its ratio of extraction by the liver was lesser, and the apparent elimination half-life was longer than that of the R-isomer. The same authors also stated that the results of pharmacokinetic measurements depended on the rate of bupivacaine administration, and the differences evened out when administration of the drug lasted for longer than 15 min (Mather et al. 1994). Similar studies were also carried out on human volunteers. Ten healthy volunteers were administered with racemic bupivacaine by infusion over 10 min (Groen et al. 1998) and it was established that the clearance of both enantiomers was the same, but the volume of distribution and apparent elimination half-life of the Sisomer was lower. They proposed that the cause of such a difference between the two stereoisomers was due to difference in the extent of their plasma protein binding as the S-isomer has a greater affinity for the proteins than the R-isomer (Groen et al. 1998). Results of studies comparing local anesthetic properties of ropivacaine and racemic bupivacaine in humans have been published (Mather 2005). In one of the studies, epidural anesthesia with 0.5, 0.75 and 1.0% ropivacaine solution produced increasingly stronger motor blockade with increasing drug concentrations. Wolff et al. (Wolff et al. 1995) compared the effectiveness of epidural anesthesia with ropivacaine (in concentrations mentioned above) with the effectiveness of bupivacaine anesthesia (0.5% solution) for surgical interventions. Their findings demonstrated that the duration of sensory block with 0.75% ropivacaine solution was the same as that produced by 0.5% bupivacaine, but the sensory block with 1.0% ropivacaine solution lasted much longer. Ropivacaine 1.0% solution also caused a considerably longer motor block (in the form of muscular relaxation which in this case is considered an advantage) than other solutions (Wolff et al. 1995).

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4 The Significance of Chirality in Pharmacological and Toxicological...

The plasma concentration of ropivacaine, even after prolonged epidural infusion, is much lower as compared to that achieved after similar infusion of other local anesthetics of the amide family (Wiedemann et al. 2000). In lower concentrations, the drug also causes a considerably lesser degree of motor block as compared to bupivacaine, and therefore, it was widely used by anesthesiologists for spinal anesthesia. An extensive utilization of the drug that ensued revealed its other properties as well. The minimum effective concentration as a local anesthetic for bupivacaine amounts to 0.093% while for ropivacaine it is 0.156%. Thus, the anesthetic effectiveness of ropivacaine is 60% of the effectiveness of bupivacaine and this may also be the reason for lesser toxicity and a less pronounced motor block produced by ropivacaine (Capogna et al. 1999). For spinal anesthesia 15 mg of ropivacaine is equivalent to 10 mg of bupivacaine as to the extent of motor block and hemodynamic effect, but ropivacaine produces a less pronounced anesthesia (Malinovsky et al. 2000). In experiments on animals, levobupivacaine (S-bupivacaine, Fig. 4.2) proved to be less toxic than bupivacaine racemate (bupivacaine). The S-isomer is less arrhythmogenic, has a higher spastic threshold and the lethal dose is approximately 1.3–1.6 times lower than that of the racemate. All these considerations make it a preferable agent for use in clinical practice (Chang et al. 2000; Huang et al. 1998). Bardsley et al. (1998) in their study on human volunteers compared the effects of levobupivacaine and bupivacaine upon intravenous administration using thoracic bioimpedance for assessment of cardiac contractility. Each drug was administered at a rate of 10 mg/min. Despite the fact that mean plasma concentration of levobupivacaine was higher than that of bupivacaine (2.38 vs. 1.87 mcg/ml), the parameters such as average stroke output per unit body surface area (medium stroke index), left ventricular contractility (increment index), and cardiac ejection fraction were affected to a lesser degree. Another study on human volunteers investigated the EEG changes upon intravenous administration of 40 mg of levobupivacaine and 40 mg of bupivacaine (Rolan 2008). Both drugs resulted in a characteristic decrease in EEG activity with a general CNS depression, but the authors concluded that levobupivacaine suppressed the activity of neurons to a lesser degree. In earlier studies, a similar method was utilized to compare the effects of ropivacaine and bupivacaine on CNS and cardiovascular system following intravenous administration in 12 healthy volunteers (Knudsen et al. 1997). Objective signs of their effect on the CNS (like muscular twitching, dysarthria) were observed in 10 ropivacaine treated volunteers whereas all volunteers treated with bupivacaine showed these signs. The average period from the end of infusion to disappearance of the drug from the plasma turned out to be shorter in the ropivacaine-receiving volunteer group despite the higher permissible dose and the concomitant increase in the concentration of the free drug in the plasma. Concentrations causing CNS symptoms and cardiovascular changes (decrease in conductance and diastolic function) were lower for ropivacaine than for bupivacaine.

4.1 Drugs Regulating the Function of Peripheral Nervous System

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Based on findings obtained from animal studies, it can be concluded that cisisomers of drugs are less toxic. Moreover, human studies have also revealed that cis-isomers are much safer than their racemates (Kopacz and Allen 1999; Korman and Riley, 1997). Drugs Affecting the Afferent Nerve Terminals Menthol is a naturally occurring compound, an important secondary metabolite of the plants of Lamiaceae family. Menthol is isolated from essential mint oil or obtained synthetically. Twelve individual stereoisomers and racemates of menthol are known, however, (−)-menthol from the natural source and the one synthesised with the same structure is the most preferred isomer. The levorotatory isomer is most common in nature, and it is the main component of essential peppermint oil (up to 50%) and essential Japanese mint (up to 90%) (PDR for Herbal Medicines 2007). (–)-Menthol, the main natural isomer, has a (1R,2S,5R)-configuration. It produces a mild local anesthetizing action and stimulates cold receptors in the skin and mucous membranes (Brain et al. 2006; Eccles 1994; Galeotti et al. 2002; Sandborn 1941). In this enantiomer the isopropyl group is transoriented in relation to the methyl and alcohol groups (PDR for Herbal Medicines 2007) (Fig. 4.3). In relation to each other all the three voluminous groups in the chair conformation are at farthest removed equatorial positions which makes (–)-menthol and its (+)enantiomer the two most stable isomers of the possible eight. DRUGS AFFECTING EFFERENT INNERVATION Drugs Affecting the Adrenergic Innervation The stereoisomerism of adrenergic substances was described as early as in 1970s (Patil et al. 1970; Sengupta et al. 1987; Garg et al. 1973; Krell and Patil 1972a, b Patil et al. 1971, 1972; Krell et al. 1972, Buckner and Patil 1971; Garg et al. 1971). Golikov et al. (1973) provided details and systematized the information. In this section, we discuss the effect of spatial factors on the pharmacological effect of adrenergic substances.

CH3

CH3

( R)

(R)

H

(R)

= ( S)

(S)

H3C H H3C

(R)

HO

OH

H3C

CH3

H3C

HO H

CH3 H

CH3

(1R,2S,5R)-2-Isopropyl-5-methylcyclohexanol

Fig. 4.3 Structural formula of (–)-menthol and (–)-menthol in chair conformation

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Table 4.1 Relative pharmacologic activity of optical isomers of noradrenaline (Golikov et al. 1973)

Organ (system)

Enantiomer ratio (–):(+)

Blood pressure (dog)

27

Isolated vessels of ear (rabbit)

18

Isolated intestine (rabbit)

60

Isolated intestine (guinea pig)

27

Isolated lung (guinea pig)

60

Isolated uterus (rat)

4

Increase in vessel permeability

10

α,β-Adrenoreceptor Agonists The presence of one asymmetrical atom in noradrenaline and adrenaline is the reason for each of them to exist in the form of two enantiomers (along with the racemic form). Absolute configurations of these stereoisomers have been established. The (–)-isomers of adrenaline and noradrenaline have the D-configuration (or R-configuration, in another notation system), while the (+)-isomers have the L-(S)-configuration, correspondingly. Several studies evaluating the optical isomers of adrenaline, noradrenaline and various sympathomimetic compounds have provided evidence that the pharmacological effects of these substances are determined by their stereoselective properties (Garg et al. 1973; Krell et al. 1972). Comparative pharmacological activity of several optical isomers of adrenergic substances was summarized in a book by Golikov et al. (1973) and summarized in Tables 4.1 and 4.2. It is clear from these data that the difference in the pharmacological action of optical isomers varies within a wide range. Among the isomers of adrenaline and noradrenaline, their natural (–)-isomers have the most pharmacological activity (Fig. 4.4). β-Adrenoreceptor Antagonists The optical isomers of isadrine (Isoproterenol), β-adrenomimetic, have shown great differences in pharmacological activity (Fig. 4.5). (–)-Isadrine exceeds the activity of the (+)-isomer 1600 times (see Table 4.3). Some authors have proposed (Beccari Table 4.2 Relative pharmacologic activity of optical isomers of adrenaline (Golikov et al. 1973)

Organ (system)

Enantiomer ratio (–):(+)

Blood pressure (dog)

15

Isolated bronchi (guinea pig)

45

Isolated heart (rabbit)

20

Isolated strip of aorta (rabbit)

45

Glucosuria (dog)

18

Hyperfibrinogenemic activity

36

Toxicity (rat)

50

4.1 Drugs Regulating the Function of Peripheral Nervous System

81

OH

H

(S)

(R)

CH2 NHCH3

CH2 NHCH3 H

OH

HO

HO

OH

OH

D-(–)-Adrenalin

L-(+)-Adrenalin

1,2-Dioxy-4-[1-oxy-2-(methylamino)ethyl]benzol

Fig. 4.4 Possible basis of pharmacodynamic differences for D-(–)-adrenaline and L-(+)-adrenaline in accordance with Preiffer’s rule. It is evident that the center of optical asymmetry of adrenaline is at that point in the molecule which is responsible for its interaction with a receptor

OH

OH ( R)

HO

H N

CH3

HO

(S)

H N

CH3

H

H

CH3

CH3 HO

HO

(–)-Isadrin

(+)-Isadrin

1,2-Dioxy-4-[1-oxy-2-(isopropylamino)ethyl]benzol

Fig. 4.5 Enantiomers of isadrine

et al. 1953) that the differences in the activity of optical isomers depend on the rate of their racemization in the body. The rate of isadrine racemization is considerably slower than the rate of noradrenaline and adrenaline racemization. Levalbuterol is a nonracemic (R-isomer, Fig. 4.6) form of albuterol (salbutamol). It is a potent beta adrenergic agonist possessing a 100 times higher affinity to β2 adrenoreceptors as compared to S-albuterol. S-albuterol can practically increase

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4 The Significance of Chirality in Pharmacological and Toxicological...

Table 4.3 Relative pharmacologic activity of isadrine optical isomers (Golikov et al. 1973)

Organ (system)

Enantiomer ratio (–):(+)

Blood pressure (cat)

600

Blood pressure (dog)

300

Blood pressure (human)

50

Isolated bronchi (guinea pig)

1000

Isolated pregnant uterus (rat)

1600

Toxicity (mice, intragastrical)

1.3

Toxicity (mice, subcutaneous)

2.2

OH

Fig. 4.6 Structural formula of levalbuterol (R-albuterol)

(R)

H N

CH3

HO H

H3 C

CH3

HO (R)-4-[2-(Tert-butylamino)-1-oxyethyl]-2-(oxymethyl)phenol

bronchial reactivity (Handley et al. 2000a). Levalbuterol has a much less pronounced effect on the heart rate than racemic albuterol, although the available evidence about the side effects is rather contradictory (Handley et al. 2000b, Lam and Chen 2003; Pancu et al. 2003, Scott and Frazee 2003). At cellular level, R-albuterol produces a dose-dependent decrease in intracellular calcium level while S-albuterol, on the contrary, increases the intracellular calcium contents in isolated smooth muscle cells. This effect was not eliminated by β2 -adrenoreceptor antagonist, but was suppressed by atropine (Mitra et al. 1998). The well-known phenomenon of paradoxical bronchoconstriction and elevated sensitivity to inhaled antigens, the increased late phase of allergic reaction and the increased eosinophil count in the sputum in response to inhaled albuterol may primarily be attributed to S-albuterol. Handley et al. (2000a), Handley (1999) systematized the results of some studies evaluating the proinflammatory effects of Salbuterol and described some probable mechanisms that may underlie the differences in the side-effects of S- and R-albuterol including eosinophil activation, increased epithelial permeability, and the effect on M2 -cholinoreceptors. Some proinflammatory effects of S-albuterol and anti-inflammatory effects of R-albuterol were studied in experiments involving sheep. The studies showed an increase in histamine release from endothelium, airway epithelial permeability, neutrophil count and interleukin8 level. Furthermore, following S-albuterol treatment, activated eosinophils induced superoxide production in response to interleukin-5, while eosinophils treated with Ralbuterol, on the contrary, inhibited the release of superoxide. Although some studies make no mention whatsoever of increased peroxide production upon stimulation of eosinophils by S-albuterol but they clearly reported about the ability of R-albuterol

4.1 Drugs Regulating the Function of Peripheral Nervous System

83

and racemic albuterol to inhibit peroxide production (Leff et al. 1997). Thus, the proinflammatory effects of S-albuterol resulting in increased bronchial hyperreactivity in allergy can be diminished by the R-enantiomer action (Handley et al. 1998). It is also interesting to note that there is greater partitioning of R-albuterol than S-albuterol into both skeletal and cardiac muscle compared to plasma. These observations may have relevance in cases of sports doping, cardiac effects, and therapeutic use in muscle wasting diseases (Jacobson et al. 2014). In clinical practice levalbuterol is effective in smaller doses and has a fewer number of side effects (like tachycardia, hyperglycemia, hypokalemia). The doses can be doubled in acute severe attacks as even a slight increase of the bronchodilating effect significantly affects the therapeutic approach (for example, use of artificial ventilation). It was shown that levalbuterol has a more pronounced bronchodilating effect in comparison with racemic albuterol in the treatment of exacerbation of bronchial asthma in adults and children (Gawchik et al. 1999; Lötvall et al. 2001; Milgrom et al. 2001; Nowak 2003). In a large study that included children with acute bronchial asthma (Carl et al. 2003), and in another study involving adults who had never received glucocorticoids (Nowak 2003). It was established that levalbuterol administration resulted in a significantly higher decrease in hospitalization rate as compared to that after treatment with racemic albuterol, although there was no significant difference in the length of hospital stay (Carl et al. 2003). Another commonly used bronchodilator with prolonged duration of action is β2 -agonist formoterol (Fig. 4.7). In clinical practice formoterol is used as a racemic mixture in the form of an aerosol. Formoterol has two chiral centers and the affinity of R,R-enantiomer for β2 adrenoreceptors is thousand times stronger than that of the S,S-isomer (Handley et al. 2002). Experiments on sensitized guinea pigs (Handley et al. 2002) demonstrated that R,R-formoterol inhibited antigen-induced bronchoconstriction and histamine OH H

H N

(R)

(R)

H CH3

H3C

OH

O NH

H

O

N-[2-Oxy-5-[(R)-1-oxy-2-[(R)-1-(4-methoxyphenyl)propyl-2-amino]ethyl]phenyl]formamide Fig. 4.7 Structural formula of R,R-formoterol

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4 The Significance of Chirality in Pharmacological and Toxicological...

Fig. 4.8 Structural formula of R-terbutaline

OH H3C H3 C

CH3 HN (R)

OH

HO H (R)-1,3-Dioxy-5-[2-(Tert-butylamino)-1-oxyethyl]benzol

production while S,S-formoterol was inactive and at times more toxic. The minimum lethal intravenous dose of the R,R-isomer was found to be twice as high as that of the S,S-isomer and it was concluded that the toxic effect of the S,S-isomer may be unrelated to its antagonism at β2 -adrenoreceptors. Terbutaline, a selective β2 adrenergic agonist, is commonly used to relieve bronchial asthma and to decrease the tone of uterus; inherently it is a chiral compound (Fig. 4.8). The R-(+)-enantiomer of terbutaline is capable of relieving carbachol-induced bronchospasm while S-(–)-terbutaline is inactive (Bielory and Leonov 2008). Other studies demonstrated an almost 1000-fold increase in the activity of the R-enantiomer following noncholinergic and nonadrenergic/cholinergic stimulation of tracheal smooth muscle cells while the S-enantiomer displayed no activity whatsoever (Källström et al. 1996). In an ovalbumin (OVA)-induced mouse model of asthma Rterbutaline efficiently ameliorated asthma responses, including airway hyperresponsiveness, eosinophils influx and expression of pro-inflammatory cytokines. The response to racemic mixture was diminished while S-terbutaline produced proasthmatic effects (Beng et al. 2019). It was established that in this case, the enantiomers change each other’s pharmacokinetics; in particular, the S-enantiomer of terbutaline affects R-terbutaline absorption and R-terbutaline affects the excretion of S-terbutaline (Borgström et al. 1989). Trimethoquinol is a bronchodilator used in Japan for the treatment of bronchial asthma and emphysema (Fig. 4.9). The S-isomer has a greater affinity to all three subtypes of β2 -adrenoreceptors (Fraundorfer et al. 1994). α-Adrenoreceptor Agonists Mesaton (phenylephrine) was obtained by replacing the 3,4-dioxyphenyl substituent of adrenaline by a 3-oxyphenyl substituent (Fig. 4.10). This change in chemical structure decreased the rate of its metabolic inactivation. It was shown that among the drugs of this group it is the (–)-isomers that possesses the most pharmacological activity (Tainter 1930; Golikov et al. 1973). Adrenomimetic substances that have no hydroxyl group in the benzene ring but contain a methyl substituent in the hydrocarbon chain possess even greater

4.1 Drugs Regulating the Function of Peripheral Nervous System

85

CH3 O O CH3 CH3 O H

(S)

HO

NH

HO (S)-1-(3,4,5-Ttrimethoxybenzyl)-1,2,3,4-tetrahydroisoquinoline-6,7-diol

Fig. 4.9 Structural formula of S-trimethoquinol

OH (R)

HO

OH H N

(S)

HO CH3

CH3

H

(–)-Mesaton

H N

H

(+)-Mesaton

1-Oxy-3-[1-oxy-2-(methylamino)ethyl]benzol

Fig. 4.10 Mesaton enantiomers

stability. These substances are not destroyed by monoamine oxidase and produce a pronounced stimulating action on the central nervous system. Sympathomimetics An ephedrine molecule contains two asymmetrical carbon atoms and exists in four optically active stereoisomeric forms, which include (–)- and (+)-ephedrine, and (–)- and (+)-pseudoephedrine. Except for (–)-pseudoephedrine, which produces a hypotensive effect, all other stereoisomers of ephedrine are vasopressor agents (Patil et al. 1965a). As to the pressor activity, (–)-ephedrine (1R,2S-isomer, Fig. 4.11) is three times more active than (+)-ephedrine (1S,2R-isomer) (Patil et al. 1965b). Although, (–)ephedrine has a faster onset of action, its effect does not last for a long duration. Upon administration of other isomers, the onset of action is slow, but the effect persists for a longer time. This peculiarity of the action of ephedrine isomers can be explained based on the fact that (–)-ephedrine rapidly demethylates to yield norephedrine, which produces the pressor action. For other ephedrine isomers, the demethylation proceeds much slower.

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4 The Significance of Chirality in Pharmacological and Toxicological... OH

Fig. 4.11 Structural formula of natural (–)-ephedrine

(R)

H ( S)

H N CH3

H CH3 (1R,2S)-2-(Methylamino)-1-phenylpropan-1-ol

When studying the mechanism of pressor action of ephedrine isomers, it was established that (+)-pseudoephedrine acts indirectly, by releasing endogenous noradrenaline while the effect of (–)-ephedrine is a mixed one (Patil et al. 1965b). Table 4.4 presents evidence of pharmacological activity of optical isomers of ephedrine and some of its analogs. α-Adrenoreceptor Blockers Prazosin was the first α-antagonist used as an effective agent in the treatment of hypertension, (Fig. 4.12). It acts by dilating peripheral blood vessels because of α-adrenoreceptor blockade. Prazosin is an achiral compound, but its structure is of great interest as a prototype for developing new antihypertensive drugs. Addition of a six-membered ring to the piperazine ring resulted in the development of an optically active selective analog of prazosin, cyclazosin (Fig. 4.13). (–)-Cyclazosin produces more pronounced blockade of all subtypes of α1 adrenoreceptors than (+)-cyclazosin. However, like prazosin it lacks selectivity for various subtypes of α1 -adrenoreceptors almost completely, except for the 12 times higher affinity to native α1B -adrenoreceptors in comparison with α1A adrenoreceptors. On the other hand, (+)-cyclazosin displayed a fine selectivity Table 4.4 Relative vasopressor activity of ephedrine isomers and its analogs (Golikov et al. 1973) Isomer

Enantiomer ratio

(–)-Ephedrine (+)-Ephedrine

(–):(+)

3

(+)-Pseudoephedrine (–)-Pseudoephedrine

(+):(–)

10

(+)-Norephedrine (–)-Norephedrine

(+):(–)

2

(+)-Norpseudoephedrine (–)-Norpseudoephedrine

(+):(–)

3

(+)-Methylephedrine (–)-Methylephedrine

(+):(–)

2

(–)-Desoxyephedrine (methamphetamine) (+)-Desoxyephedrine

(–):(+)

1.3

(–)-Desoxynorephedrine (phenamine) (+)-Desoxynorephedrine

(–):(+)

13

4.1 Drugs Regulating the Function of Peripheral Nervous System

87

NH2 O

N N

N O

N

O

CH3 O

CH3

1-(2-Furanoyl)-4-(4-amino-6,7-dimethoxyquinazolin-2-yl)-piperazine Fig. 4.12 Structural formula of prazosin

NH2 O

H

H

(S) (R)

N

N

N

O

N

O CH3 O

CH3

(4aR,8aS)-1-(2-Furanoyl)-4-(4-amino-6,7-dimethoxyquinazolin-2-yl)octahydroquinoxaline Fig. 4.13 Structural formula of (+)-cyclazosin

for cloned α1B -adrenoreceptors (pKi = 9.16), and a significantly lower affinity to α1A - and α1D -adrenoreceptors (pKi = 7.48 and 7.57, correspondingly). Besides, cyclazosin also displayed a 1100-, 19,000- and 12,000-times greater selectivity in binding to α1B -adrenoreceptors in comparison with α2 -adrenoreceptors, 5-HT1A - and D2 -receptors, correspondingly (Melchiorre et al. 1998; Minarini et al. 1998). Doxazosin (Fig. 4.14) was obtained by substituting the furane cycle in prazosin with 1,4-benzodioxane. This drug has a pronounced affinity for α1 -adrenoreceptors and is used in the treatment of hypertension and benign prostatic hyperplasia. Experiments (Hatano et al. 1996) using radioligand in isolated human tissues have shown that doxazosin and its enantiomers display a higher affinity to α1 adrenoreceptors than to α2 -adrenoreceptors. However, S-doxazosin displayed much lower affinity for α2 -adrenoreceptors as compared to its R-enantiomer. Besides, Sdoxazosin had a higher α1 /α2 selectivity index in comparison with the R-isomer. The racemate had an intermediate selectivity as compared to the enantiomers composing

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4 The Significance of Chirality in Pharmacological and Toxicological...

NH2 O

N N

N N

( S)

O

O

O

CH3 O

CH3

(S)-1-(2,3-Dihydrobenzo[b][1,4]dioxin-2-oyl)-4-(4-amino-6,7-dimethoxyquinazolin-2yl)piperazine Fig. 4.14 Structural formula of S-doxazosin

it. It has also been speculated that doxazosin pharmacokinetics are substantially stereospecific. (−)-(R)-doxazosin increases (+)-(S)-doxazosin exposure probably by inhibiting the elimination of (+)-(S)-doxazosin, and the enantiomers may be competitively absorbed from the gastrointestinal tract (Li et al. 2015). Compounds containing 1,4-benzodixane substituted at the second position in their structure display a high affinity to α1 -adrenoreceptors. WB 4101, 2-(2,6dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane, an experimental sample of this family (Fig. 4.15), shows a pronounced selectivity for α1 -adrenoreceptors but preserves considerable affinity for other receptors like α2 - and 5-HT1A -receptors. Its enantiomers have a varying affinity for α1 -adrenoreceptors. The S-isomer of WB 4101 was found to be much more active than the R-enantiomer and their affinity to α1 -adrenoreceptors amounted to 0.16 and 39.8 nM, correspondingly (Barbaro et al. 2002, Melchiorre et al. 1998). Mephendioxane is one of the analogs of WB 4101 compound (Fig. 4.16). It has a para-tolyl substituent at the third position of benzodioxane ring and is more selective to α1 -adrenoreceptors (Quaglia et al. 1996). Besides, (–)-mephendioxane (trans- or S,S-configuration) displays a much higher α1 -adrenoblocking effect than its antipode. Moreover, the (–)-enantiomer showed 12,000, 2500 and 250 times more selectivity Fig. 4.15 Structural formula of S-enantiomer of compound WB 4101

CH3 O H O

O ( S)

N H H3C

O

O

(S)-N-[(2,3-Dihydrobenzo[b][1,4]dioxine-2-yl)methyl]2-(2,6-dimethoxyphenoxy)aminoethane

4.1 Drugs Regulating the Function of Peripheral Nervous System

89 CH3 O

H O

O ( S) (S)

N H H3C O

O

CH3

2-(2,6-Dimethoxyphenoxy)-N-[[(2S,3S)-3-(п-tolyl)-2,3dihydobenzo[b][1,4]dioxine-2-yl]methyl]aminoethane

Fig. 4.16 Structural formula of (–)-mephendioxane (2S, 3S-enantiomer)

for α1 -adrenoreceptors than for α2 -, 5-HT1A - and D2 -receptors, correspondingly (Melchiorre et al. 1998). β-Adrenoreceptor Blockers β-Adrenoblockers are a group of drugs that are mainly used in the treatment of cardiovascular diseases like hypertension, cardiac arrhythmia, and ischemic heart disease. The structure of these compounds contains at least one asymmetrical atom in the alkylated side chain. This determines the high degree of their enantioselectivity in β-adrenoreceptors binding. Overall, in β-adrenoblockers with a single asymmetric centre, the S-(–)-enantiomer usually displays a higher selectivity upon interaction with β-adrenoreceptors than its antipode. The value of enantiomer S:R ratio for this activity varies from 33 to 530, according to different authors (Mehvar and Brocks 2001; Sato et al. 2002). Besides, enantiomers of some β-blockers produce additional effects such as α-adrenoreceptors antagonism and the effects similar to class III anti-arrhythmic drugs. However, for economic reasons, most β-blockers utilized in clinical practice are in the form of racemates. Propranolol is the first β-adrenoblocker that was introduced in clinical practice. S-(–)-propranolol (Fig. 4.17) is forty times more potent in its effect on β1adrenoreceptors than the R-(+)-enantiomer. Propranolol enantiomers also produce a local anesthetic and anti-arrhythmic effect. There is evidence of stereoselective interaction of propranolol with plasma proteins (Mehvar and Brocks 2001; Triggle 1997). The anti-arrhythmic activity of racemic propranolol and its two enantiomers has also been found to depend on the type of arrythmia. Moreover, it has also been observed that racemic propranolol causes higher toxicity in rats than both its pure enantiomers. And this effect of racemate can at least be partially attributed to pharmaˇ cokinetic interactions (Cižmáriková et al. 2019). Due to its strong hydrophilic property, atenolol is not subject to hepatic metabolism and produces anti-adrenergic action in an unchanged form, which is primarily attributed to the S-enantiomer (Fig. 4.18).

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4 The Significance of Chirality in Pharmacological and Toxicological...

OH

O

(S)

H

NH CH3 H3C

(S)-1-(Isopropylamino)-3-(1-naphthyloxy)propan-2-ol Fig. 4.17 Structural formula of S-(–)-propranolol

OH

O

(S)

H2 N O

H

NH CH3 H3C

(S)-2-[4-[2-Oxy-3-(Isopropylamino)propoxy]phenyl]acetamide Fig. 4.18 Structural formula of S-(–)-atenolol

Upon activation of sympathetic system due to physical exercise, secretion of the S-enantiomer by terminals of adrenergic nerves predominates (Mehvar and Brocks 2001; Stoschitzky et al. 1992). It seems of no small importance that only the S-isomer of atenolol is excreted from the body in an unchanged form, while the R-enantiomer accumulates in the body. In five randomized clinical studies (Mehvar and Brocks 2001) where a total of 250 people participated, it was revealed that S-atenolol in a dose of 25 mg produced an antihypertensive action equivalent to racemic atenolol in a dose of 50 mg, and there was no worsening of the lipid profile. In patients with ischemic heart disease, S-atenolol significantly reduced the frequency of heart attacks, decreased the intensity of ECG changes of exercise-induced myocardial ischemia, and prolonged the time of treadmill test. Pindolol is a nonselective β-blocker used in the treatment of hypertension. It is a mixture of two enantiomers that differ in their pharmacodynamic and pharmacokinetic properties. Like propranolol, the S-(–)-enantiomer of pindolol (Fig. 4.19) is a more powerful β1 -adrenoblocker than its antipode (Gonçalves et al. 2002). S-(– )-pindolol accumulates in the plasma in greater concentrations than the less active

4.1 Drugs Regulating the Function of Peripheral Nervous System

91

OH

O

(S)

HN

H

NH CH3 H3C

(S)-1-(4-Indolyloxy)-3-(isopropylamino)propan-2-ol Fig. 4.19 Structural formula of S-(–)-pindolol

R-(+)-pindolol. There are also differences between pindolol enantiomers as to their renal excretion. The S-(–)-pindolol shows a higher renal excretion (14.4 L/h) than R-(+)-pindolol (12 L/h). Metoprolol is a selective antagonist at β1 -adrenoreceptors. Its ability to block β1 adrenoreceptors is attributed primarily to the levorotatory S-enantiomer (Fig. 4.20), with R-enantiomer making only a small contribution. Studies evaluating pharmacokinetics of metoprolol in rats established that the elimination half-life (t1/2 ) of enantiomers was 35 min on an average, but for the R-enantiomer it was longer than for the S-enantiomer (Mostafavi and Foster 2000; Stoschitzky et al. 2001). It was also noted that in “poor metabolisers”, the hydroxylation of both enantiomers of metoprolol results in a new asymmetrical atom in alpha-position appearing in the R-configuration and this promotes a faster formation of α-hydroxymetoprolol (Cerqueira et al. 2003). Significant difference in the bioavailability parameters (Cmax , AUC) was shown to exist between pure enantiomers and racemic metoprolol. For S-metoprolol AUC and Cmax were higher than for its antipode. It was established that

O

OH (S)

O H3C

H

NH CH3 H3C

(S)-1-(Isopropylamino)-3-[4-(2-methoxyethyl)phenoxy]propan-2-ol Fig. 4.20 Structural formula of S-(–)-metoprolol

92

4 The Significance of Chirality in Pharmacological and Toxicological...

the S-enantiomer of metoprolol primarily produces a β1 -blocking action while the R-enantiomer causes β2 -blockade (Mehvar and Brocks 2001; Nathanson 1988). The affinity of the S-isomer for β1 -receptors is approximately 500 times higher, and the ability to prevent the increase in heart rate in response to sympathetic stimulation is 250 times higher than the corresponding ability of the R-isomer (Nathanson 1988). High lipophilicity of metoprolol triggers additional concerns while using the racemic form of the drug. In case of genetic polymorphism of cytochrome CYP2D6 that affects 5% of people, the metoprolol metabolism slows down, significantly (Benny and Adithan 2001). Since the slow-down of metoprolol clearance mainly affects R-enantiomer, the risk of developing side effects associated with β2 -receptors increases considerably even upon administration of medium doses of the racemic drug in this group of patients (Lennard 1990; Lennard et al. 1983). A number of drugs (paroxetin, cimetidine, ranitidine, ciprofloxacin, verapamil) increase the plasma concentration of the R-enantiomer of metoprolol. This is also associated with a considerable risk of side effects similar to those caused by conventional metoprolol administration, especially in “poor metabolisers” (Wahlund et al. 1990; Hemeryck et al. 2000; Kim et al. 1993; Toon et al. 1988). Administration of the levorotatory isomer of metoprolol in clinical practice makes it possible to avoid the abovementioned side effects while preserving the basic therapeutic potential of the drug in the form of adequate β1 -adrenoblockade, and this explains the potential advantage of using the levorotatory metoprolol in clinical practice. Clinical trials performed to date have largely used S-metoprolol succinate with retarded release rate and this provided stability of the pharmacodynamic parameters, when the drug was taken once a day. Thus, in SMART randomized, double blind, comparative study (SMART Trial 2005) that included 260 hypertensive patients, six weeks of observation revealed that the S-metoprolol succinate in a dose of 50 mg/day was as effective in lowering the systolic and diastolic blood pressure as the analogous retarded form of racemic metoprolol succinate at a dose of 100 mg/day. In the case of S-metoprolol, the antihypertensive effect was achieved sooner. In a specially designed clinical trial including 49 hypertensive patients with concomitant chronic broncho-obstructive disease, administration of levorotatory metoprolol succinate at a daily dose of 50 mg for 60 days resulted in a pronounced antihypertensive effect. In 92% of cases, the blood pressure was within normal limits after 30 days of treatment and in 100% of cases the same was achieved within 45 days from the start of therapy. The drug did not affect the parameters of the respiratory function. It is particularly remarkable that no patient noted an exacerbation of clinical signs of chronic obstructive pulmonary disease over the total period of observation (Mandora 2006). Findings of a comparative research using retarded forms of metoprolol succinate (levorotatory form at a dose of 50 mg/day, racemic form in a dose of 100 mg/day) in patients with stable angina have also been published. It is evident that the extent of the anti-anginal effect and the effect on hemodynamic parameters (heart rate, arterial blood pressure) is comparable for both forms of the drugs (Aneja et al. 2007).

4.1 Drugs Regulating the Function of Peripheral Nervous System

93

OH (R)

HN O H

S H3C

NH

O

CH3 H3C

(R)-N-[4-[1-Oxy-2-(isopropylamino)ethyl]phenyl]methanesulfonamide Fig. 4.21 Structural formula of R-(+)-sotalol

Sotalol, which was first regarded as a β-adrenoblocker was later classified as a class III anti-arrhythmic drug. Its S-(–)-isomer is both a β-adrenoblocker and a blocker of potassium ion channels, while the R-(+)-isomer (Fig. 4.21) blocks only the potassium ion channels. Therefore, R-(+)-sotalol is better tolerated by patients than the racemate. However, a placebocontrolled clinical trial established that mortality associated with proarrhythmogenic effect of sotalol increased after the use of its R-(+)-enantiomer (MacNeil 1997). Nebivolol is a β1 -adrenoblocking drug containing four asymmetric centers. The manufactured drug is a racemic mixture of two stereoisomers (out of the existing ten): (+)-nebivolol (S,R,R,R-isomer, Fig. 4.22) and (–)-nebivolol (R,S,S,S-isomer) (Satoh et al. 2003). (+)-Nebivolol is a potent selective β1-adrenoblocker with prolonged duration of action. The (–)-isomer is responsible for typical changes in the hemodynamics by the racemic mixture. The (–)-enantiomer at doses that do not lower the blood pressure, significantly increases the antihypertensive effect of the (+)enantiomer. The (–)-nebivolol modulates or completely eliminates the negative inotropic effect of the (+)-form and promotes the release of nitrogen oxide (NO) from endothelial cells. It has been observed that both isomers act synergistically in reducing blood pressure (MacNeil 1997; Mangrella et al. 1998; Satoh et al. 2003; Ignarro 2008). OH H O

(R)

OH H N

(R)

( S)

H O ( R)

H

H

F

F

(R)-1-[(R)-6-Fluorochromane-2-yl]-2-[(R)-2-[(S)-6- fluorochromane -2-yl]2-oxyethylamino]ethanol

Fig. 4.22 Structural formula of (+)-nebivolol (S,R,R,R-stereoisomer)

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4 The Significance of Chirality in Pharmacological and Toxicological...

O

OH (S)

H N

H2 N

H (R)

H CH3 HO S,R-Labetalol

O

OH (R)

H N

H2 N

H (R)

H CH3 HO R,R-Labetalol (dilevalol 2-Oxy-5-(1-oxy-2-[4-phenyl-2-butylamino]ethyl)benzamide Fig. 4.23 Structural formulas of labetalol stereoisomers

α,β-Adrenoreceptor Blockers Labetalol (Fig. 4.23) can serve as an illustration of a pseudohybrid drug, which is manifested in combination of two pharmacological effects. Labetalol contains two centers of asymmetry and so it has four stereoisomers. It is a potent antagonist of α1 (pKi = 7,44), β1 (pKi = 8,31) and β2 (pKi = 8,10) adrenoreceptors. β1 Adrenoblocking action is produced by the S,R-isomer (pKi = 7.18) of labetolol, while the R,R-isomer, dilevalol (Fig. 4.23), is characterized as a potent non-selective βadrenoreceptor antagonist pKiβ1 = 8.26, pKiβ2 = 8.52). Despite the fact that dilevalol does not affect the arterial pressure, it is not used as a drug due to its hepatotoxic action (Christiaans and Timmerman 1996; Triggle 1997; Tucker 2000). Like labetalol, carvedilol (Fig. 4.24) affects β–adrenoreceptors and also possesses a dual pharmacological effect. It can also be regarded as a pseudo-hybrid drug. α1– Adrenoblocking action of carvedilol is associated with its S-enantiomer, while β1 -adrenoblocking action is typical of both enantiomers. R-carvedilol is better metabolized by cytochrome CYP2D6, and this accounts for the difference in the ratio of α1 /β1 -adrenoblocking effects depending on the genotype and allele expression. Carvedilol is also a blocker of calcium channels and a modulator of sodium channels (Karle et al. 2001, Swynghedauw 2001).

4.1 Drugs Regulating the Function of Peripheral Nervous System

HN

95

H3C

OH

O

O

O

(S)

H

NH

(S)-1-(4-Carbazolyloxy)-3-[2-(2-methoxyphenoxy)ethylamino]propan-2-ol Fig. 4.24 Structural formula of S-(–)-carvedilol

Drugs Affecting Cholinergic Innervation As early as in 1973, Golikov et al. presented a thorough systematization of the evidence of stereoisomeric cholinomimetic drugs depending on the type of their isomerism (optical, geometrical, or conformational) in the monograph entitled “Stereospecificity of the action of drugs” (Golikov et al. 1973). M- and N-Cholinomimetics The naturally occurring (+)-muscarine (2S,4R,5S-isomer, Fig. 4.25) is the most active muscarinic (M)-cholinomimetic agent while its antipode possesses mild Mcholinomimetic action. Epimuscarine is an even less active compound (Golikov et al. 1973). It should be mentioned that (+)-muscarine has the same S-configuration of β-carbon atom (in relation to nitrogen) as the most active (+)-isomer of acetylβ-methylcholine. A change from the S-configuration of this atom to the opposite R-configuration, without a change in the configuration of the other two asymmetrical centers of muscarine, yields (+)-allomuscarine that only possesses mild Mcholinomimetic action. This again provides evidence regarding the significant role of spatial arrangement of radicals around the specific atom (for tetrahydrofuran ring of muscarine this is position 2; see Fig. 4.25). HO (R)

H3C

5 (S)

H3 C

3

4

2 1

O

(S)

CH3 N+

ClCH3

[(2S,4R,5S)-4-Oxy-5-methyltetrahydrofuran-2-yl]methylN,N,N-trimethylammonium chloride

Fig. 4.25 Structural formula of natural (+)-2S,4R,5S-muscarine

96

4 The Significance of Chirality in Pharmacological and Toxicological...

(S)

N CH3

N (S)-3-(1-Methylpyrrolidin-2-yl)pyridine Fig. 4.26 Structural formula of L-(–)-nicotine

When we examine the evidence of stereospecific action of substances with Ncholinomimetic effect, the picture that emerges is somewhat different. Nicotine itself occurs in nature in the form of the L-(–)-isomer (Fig. 4.26) configurationally linked to L-proline, a naturally occurring amino acid. The most detailed research into the pharmacological activities of the optical isomers of nicotine was conducted by Barlow and Hamilton (Barlow and Hamilton 1965). Their findings are shown in Table 4.5 (taken from (Golikov et al. 1973). It is clear from the table that the levorotatory isomer of nicotine is the most active one. For most of the pharmacological effects, the activity of this isomer is 3–42 times higher than the other one. Only in experiments on isolated rat diaphragm (neuromuscular block), the activity of two isomers turned out to be virtually identical. A marked difference was also detected in the action of optical isomers of nicotine on the nervous system of experimental animals where (–)-nicotine again displayed a greater activity. It caused disruption of conditioned reflex activity in rats at smaller doses than its antipode (Domino 1965).

Table 4.5 Relative pharmacological activity of optical isomers of nicotine (Golikov et al. 1973; Barlow and Hamilton 1965)

Organ (system)

Enantiomer ratio (–):(+)

Blood pressure in rats (elevated)

14.0

Blood pressure in kittens (elevated)

6.1

Ileum of guinea pigs (contracted)

42.0

Superior cervical ganglion of cats (blocked)

13.7

M. tibialis of cats (blocked)

3.1

Diaphragm of rats (blocked)

0.96

Straight muscle of frog abdomen (contracted)

10

M. biventer of chicken (contracted)

4.6

4.1 Drugs Regulating the Function of Peripheral Nervous System

97

CH3 N

H O

(S)

OH

O

(2S)-(8-Methyl-8-azabicyclo[3.2.1]octane-3-yl)-3-oxy-2-phenylpropanoate Fig. 4.27 Structural formula of L-(–)-hyoscyamine

This evidence suggests that although nicotinic (N)-cholinomimetics possess a stereospecificity that depends on optical isomerism. However, judging from the ratio of effective doses of enantiomers, this stereospecificity is pronounced to a much lesser degree as compared to M-cholinomimetics. Muscarinic Cholinoreceptor Blockers As early as in 1904 Cushny (1903) established a difference in the physiological activity of optical isomers of atropine [(+)- and (–)-hyoscyamines] in a number of tests. (–)-Hyoscyamine (Fig. 4.27) was shown to be more active than its (+)isomer. However, in other cases the difference between the activity of (+)- and (– )-hyoscyamines could not be demonstrated, especially their action on the central nervous system. Later, in numerous studies, atropine and its enantiomers were evaluated for the differences in their pharmacological effects. Table 4.6 presents some evidence obtained in these studies. As can be observed from the table, a significant difference in the action of atropine enantiomers is evident only when a cholinergic mechanism is the basis of the studied effect. It should also be highlighted that the stereospecificity of the actions of atropine is displayed not only for its peripheral effects as was proposed by Cushny (1903), but also for its action on the central nervous system. In fact, the experiments that utilized the electroencephalography methods (action of the drug on spontaneous EEG and activation reaction) for analysis of the central action, could demonstrate the difference in the central activity of the optical isomers of atropine (Bradley and Elkes 1957; Domino and Hudson 1959; Herz 1962). Stereospecificity of atropine is also displayed in its interaction with enzymes. In the experiments studying hydrolysis of optical isomers of atropine by atropinesterase, it was established that the enzyme cleaved (–)-hyoscyamine eighty times faster than its (+)-isomer. Therefore, it can be proposed that the observed difference in the

98

4 The Significance of Chirality in Pharmacological and Toxicological...

Table 4.6 Relative activity of the optical isomers of atropine (Golikov et al. 1973)

Object of study

Enantiomer ratio (–):(+)

Prevention of pilocarpine salivation

40:1

Mydriatic effect

60:1

Prevention of hypotensive effect of vagus nerve irritation

25–40:1

Relief of acetylcholine contracture of isolated intestine

30:1

Block of conduction along the superior cervical ganglion

1:1

Inhibition of blood plasma cholinesterase

3:1

Block of pressor reaction to the irritation of sciatic nerve

16:1

Prevention of arecoline-induced tremor

25:1

Prevention of tremorine-induced tremor

20:1

Prevention of nicotine-induced spasm

1.5–2.5:1

Influence on spontaneous electroencephalogram

16:1

Block of EEG activation reaction 16:1 Prevention of morphine-induced tail-flick inhibition

1.5:1

Toxicity

1:1

activity of (–)- and (+)-hyoscyamines (see Table 4.6) is underestimated due to the stereoselective action of enzymes on the (–)-isomer. Another difference in the activity of atropine isomers was observed in relation to their own effect on enzymes. It was observed that (–)-hyoscyamine inhibited serum cholinesterase much more than (+)-hyoscyamine did. A similar pattern was observed in case of optical isomers of scopolamine (hyoscine). According to Cushny (1926a; b), the (–)-hyoscine had 16–18 times higher effect (Fig. 4.28) on the salivary glands as compared to (+)-hyoscine. Its cholinolytic effect on an isolated rabbit intestine was also 10–15 times stronger than the effect produced by the (+)-isomer. Buckett and Haining (1965) demonstrated that of the two enantiomers, the (–)-isomer of hyoscine displayed a greater effect on both peripheral and central cholinergic systems. As is the case with atropine, no difference was noted in the toxicity of optical isomers of scopolamine.

4.1 Drugs Regulating the Function of Peripheral Nervous System

99

CH3 N

O

H O

(S)

OH

O

(2S)-(9-Methyl-3-oxa-9-azatricyclo[3.3.1.02,4]nonan-7-yl)-3-oxy-2-phenylpropanoate

Fig. 4.28 Structural formula of (–)-hyoscine

Neuromuscular-Blocking Drugs Pancuronium, vecuronium and rocuronium are derivatives of amino steroids and like other steroid molecules, they contain several chiral centers. However, their use in clinical practice is the same as for the isomers with one asymmetrical atom (Calvey and Williams 1997). Atracurium and mivacurium (Figs. 4.29 and 4.30) each possesses four chiral centers and thus can theoretically exist in the form of one of the sixteen possible isomers (Calvey and Williams 1997). Atracurium is synthesized by a non-selective method, but the inner symmetry brings the number of stereoisomers down to ten. Each one of them can be classified according to the configuration of two carbon atoms (R- or S-), and according to the CH3

CH3

ClO

O

CH3 H3C

H3C

O N+( R)

O

O

(R)

O O

O O H3C

( R)

Cl

CH3

O CH3

-

O

O

( R) N +

CH3

H3 C

(1R,1'R,2R,2'R)-2,2'-[3,3'-[Pentane-1,5-diylbis(oxy)]bis(3-oxopropane3,1-diyl)]bis[1-(3,4-dimethoxybenzyl)-6,7-dimethoxy-2-methyl1,2,3,4-tetrahydroisoquinolinium] dichloride

Fig. 4.29 Structural formula of cisatracurium

100

4 The Significance of Chirality in Pharmacological and Toxicological... H3 C O

CH3 O

CH3

O CH3 O

O

O

CH3 -

Cl

H3C O

O H3C

(R) +

( R)

CH3

O O

O H3C

( R) (R)

Cl-

O

CH3

N

O H3C

O

N+ CH3

(1R,1'R,2R,2'R)-(E)-2,2'-[(1,8-Dioxo-4-octen-1,8-diyl)bis(oxy3,1-propandiyl)]bis[1,2,3,4-tetrahydro-6,7-dimethoxy-2-methyl1-[(3,4,5-trimethoxyphenyl)methyl]isoquinolinium] dichloride

Fig. 4.30 Structural formula of 1R,2R,1’R,2’R-E-stereoisomer of mivacurium (trans–transconfiguration)

relative configuration of carbon–nitrogen bonds (cis- or trans-). The isomers can be divided into three groups of geometrical isomers: cis-cis-, trans–trans-, and cis–trans. Significant difference is found in their pharmacokinetic and pharmacodynamic properties. The 1R-cis,1’R-cis-isomer (cis-atracurium, Fig. 4.29) is the most potent and effective one. Although, the neuromuscular block produced by it is the same as the one produced by atracurium, the latter yields a lesser release of histamine or no release of it at all (Atherton and Hunter 1999). Mivacurium (Fig. 4.30) consists of three geometrical isomers. The predominant trans-isomers (cis–trans- and trans–trans-, 94%) are equally effective. They are characterized by a high degree of clearance (4.74 L/hr·kg) and a short elimination half-life (2 min approximately) (Atherton and Hunter 1999). On the contrary, the cis-cis-isomer displays a lesser degree of clearance, a longer elimination half-life, and is overall less effective.

4.2 Drugs Affecting the Central Nervous System Function General Anesthetic Drugs Inhalation Anesthetic Drugs The currently used inhalational anesthetics are chiral drugs, except for sevoflurane. In some studies (Franks and Lieb 1991; Harris et al. 1992; Lysko et al. 1994) explaining the mechanism of action of these drugs, stereochemical factors were shown to play a key role in determining the anesthetic activity. Although earlier theories put emphasis

4.2 Drugs Affecting the Central Nervous System Function Fig. 4.31 Structural formula of S-(+)-isoflurane

101

F

F

H O

F

F

( S)

Cl

F

(S)-2-Chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane

on physico-chemical characteristics like lipid solubility, the difference in pharmacodynamics of optical isomers of inhalational anesthetics can now be attributed to the stereoselective interaction of drug molecules with the target sites, the ion channel proteins. Franks and Lieb (1991) in their study for evaluation of the effect of isoflurane isomers on ion channels of periwinkle nerve fibers, established that the S(+)-isomer (Fig. 4.31) was twice as effective in activating potassium current as the R-(–)-enantiomer. Harris et al. (1992) established in their study on mice that the sleep time was much more longer after intraperitoneal administration of S-(+)-isomer of isoflurane as compared to the R-(–)-isomer. Later these findings were confirmed in experiments on rats performed by Lysko et al. (1994). It was found that the minimum alveolar concentration (MAC) of the S-(+)-isoflurane amounted to 1.06%, while for the R-(–)isoflurane it was 1.62%, and for the racemate 1.32%. This was one of the first studies demonstrating the role of stereoselectivity in the mechanism of action of inhalational anesthetics. Although several researchers confirmed these findings (Harris et al. 1992, 1994; Mihic et al. 1997; Moody et al. 1993), others could not demonstrate the same (Graf et al. 1994; Kendig et al. 1973; Moody et al. 1994) and the conclusions remain disputable. Moreover, although Lysko et al. (1994) and Eger et al. (1997) established that the MAC of the S-(+)-isomer, R-(–)-isomer and racemate amounted to 1.44%, 1.69%, and 1.59% respectively, the differences were not statistically significant. The evidence summarized by Burke and Henderson (Burke and Henderson 2002) (Table 4.7) testifies that the contribution of enantiomer-selective effects in the action of inhalational anesthetic drugs can be relatively poor.

Table 4.7 Comparative pharmacological characteristics of isoflurane enantiomers (Burke and Henderson 2002) Object of study Periwinkle ion channels

Parameter Total current of K+ -ions (rel. un.)

Isomer S

R

2

1

Mouse, in vivo

Sleep time (min.)

9

6

Rat, in vivo

MACa (%)

1.06

1.62

Rat, in vivo

MACa (%)

1.44

1.69

a

Maximum anesthesia inducing alveolar concentration

102

4 The Significance of Chirality in Pharmacological and Toxicological...

Non-inhalational Anesthetic Drugs Among non-inhalational anesthetic drugs, thiopental, methohexital, ketamine and etomidate are chiral compounds while propofol is not chiral. In clinical practice the first three are utilized in the form of racemates, and etomidate was obtained in the form of a pure R-(+)-isomer. S-(–)-thiopental sodium (Fig. 4.32) has a shorter elimination half-life than its R-(+)-isomer (Calvey and Williams 1997) as it undergoes a faster metabolism and clearance. This difference is attributable to its metabolite, pentobarbital. Several studies were conducted to compare the effects of two stereoisomers of thiopental. In the early 1970s, experiments on mice demonstrated that the S-enantiomer was more active (Haley and Gidley 1976). This was also noted by Mark et al. (1976) in a study involving human volunteers. Later studies performed by Mather et al. on animals (Mather et al. 1999, 2000) confirmed that S-thiopental inhibited the EEG activity more effectively (Table 4.8), but its therapeutic index was, unfortunately, much lower than that of the R-enantiomer. Recovery after inhibition of EEG was much slower and mortality in the group receiving S-thiopental was higher (Mather et al. 2000). The authors brought forward a hypothesis that the higher therapeutic index of R-thiopental can be due to a greater distribution of this isomer in the central nervous system (Mather et al. 1999). In order to explain higher efficacy of S-thiopental, its possible receptor interactions were studied in vitro. For AMPA-type of receptors (Kamiya et al. 1999) and for N-cholinoreceptors (Downie et al. 2000), this enantiomer showed minimal stereoselectivity, which ruled out role of these receptors in thiopental-induced anesthesia. H3 C

Fig. 4.32 Structural formula of S-(–)-thiopental of sodium

O

CH3

H N

(S)

CH3 Na+

-

S

N H

O

5-Ethyl-4,6-dioxo-5-[(S)-2-pentyl]-1,4,5,6-tetrahydropyrimidine2-thiolate of sodium

Table 4.8 Comparative pharmacological characteristics of enantiomers of inhalational anesthetic drugs (Burke and Henderson 2002) Object of study

Parameter

Isomer S

R

Thiopental Animal

EEG depression (% of isoline)

10

40

GABAA -receptors

EC50 (μM)

26

52.5

57

3.4

Etomidate Tadpoles

EC50 (μM)

4.2 Drugs Affecting the Central Nervous System Function

103

Studies on expressed GABAA -receptors of humans (Cordato et al. 1999) showed that the median effective concentration (EC50 ) for S-thiopental, rac-thiopental and R-thiopental was 26 μM, 35.9 μM, and 52.5 μM, respectively (Table 4.8). These values were in agreement with the established differences in the CNS-depressing effects of two enantiomers and the racemate. Thiopental displays tautomerism (dynamic isomerism) and in living organism it exists in the form of an equilibrium mixture of two isomers (Fig. 4.33). In an alkaline medium the very soluble ionized thiol form as sodium salt predominates (Fig. 4.32). In plasma, at pH 7.4, thiopental is converted to a nonionised lipophilic form, which undergoes rapid isomerization yielding an equilibrium mixture of thiol (lactim) and thione (lactam) tautomers (Burke and Henderson 2002; Hemmings and Hopkins 2006). Methohexital (brietal) is a quick-acting barbiturate anesthetic drug of ultrashort duration of action (Fig. 4.34). The structure of this compound contains two asymmetric centers, one at the 5-position in the barbituric ring and the other in 1-methyl2-pentyl side chain. Methohexital produces two pairs of stereoisomers, one represented by enantiomers and the other by diastereomers (Calvey and Williams 1997). CH3

H3C

O

H3C

O

H

CH3

H N

( S)

HN

(S)

H3C

CH3 O

N H

SH

N H

S

Thiol (lactim) form

O

Thione (lactam) form

Fig. 4.33 Thiopental tautomerism

CH3 O

N (S)

NH

H2C H3C

O

(R)

O

H3 C (S)-5-Allyl-5-[(R)-3-hexine-2-yl]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione Fig. 4.34 Structural formula of S,R-stereoisomer of methohexital

104

4 The Significance of Chirality in Pharmacological and Toxicological...

Like thiopental, methohexital displays keto-enol tautomerism in vivo (Burke and Henderson 2002; Hemmings and Hopkins 2006) and in clinical practice it is used in the form of two least stimulating isomers (Burke and Henderson 2002; Hemmings and Hopkins 2006). Steroid noninhalational anesthetic drugs are not utilized in clinical practice at present, but as they are chiral compounds, they are of interest in research. Pregnanolone and allopregnanolone are enantiomers (Fig. 4.35) with complex stereochemistry as they consist of eight chiral centers (C3 , C5 , C8 , C9 , C10 , C13 , C14 and C17 ). Covey et al. (2000) studied the effect of stereospecificity on interaction of these substances with GABAA receptors using preparations of rat neuronal membrane. It was established that the 5α-reduced steroids, in contrast to 5β-reduced steroids, displayed a higher stereoselective action both as GABAA receptor modulators and as noninhalational anesthetic drugs. It was noted that the parameters of their activity in relation to GABAA receptors correlated well with the anesthetic effect of these compounds (Covey et al. 2000). Etomidate is administered in clinical practice in the form of a cis-isomer. The anesthetic effect prevails in the R-(+)-enantiomer (Fig. 4.36) which is about five times more active than the S-(–)-isomer. Tomlin et al. (1998) estimated the median CH3

O

CH3

CH3

CH3

(S)

(S) (R)

(R)

H

(S)

(S)

CH3

(S)

(S)

H

(S)

(R)

H

CH3

O

H

(S)

(S)

(S)

(R)

H

(S)

(S)

H

HO

HO

H

H

Pregnanolon 3α-Hydroxy-5β-pregnan-20-one

Allopregnanolon 3β-Hydroxy-5α-pregnan-20-one

1-(3-Hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenatren-17-yl)ethanone

Fig. 4.35 Structural formulas of pregnanolone and allopregnanolone

Fig. 4.36 Structural formula of R-(+)-etomidate

O

O

CH3

CH3

(R)

N H N

(R)-Ethyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate

4.2 Drugs Affecting the Central Nervous System Function

105

effective concentration (EC50 ) necessary to suppress the reflexes in frog tadpoles. For R-(+)-etomidate EC50 amounted to 3.4 μM, while for S-(–)-etomidate it was 57 μM (see Table 4.8). Besides, experiments on mice fibroblasts recombined with bovine GABAA receptors using the potential fixation method studied the effect of etomidate stereoisomers on GABA-induced ion current. The R-(+)-isomer turned out to be more effective in stimulating GABA-induced current than the S-(+)-isomer, although the degree of stereoselectivity was concentration-dependent. These findings agree with the hypothesis that the mechanism of the anesthetic effect of etomidate is determined by the nature of its interaction with GABAA receptors. Belelli et al. (1997) studied the nature of interaction of etomidate with GABAA receptors of mammals. To assess the modulatory and mimetic effect of R-(+)etomidate, a system with β1 -, β2 -, β3 -, α6 - and γ2 -subunits of human GABAA receptors coexpressed in oocytes of Xenopus laevis was utilized. It was established that the point mutation of the β3 -subunit (replacement of asparagine-289 with serine at the Nterminus domain) completely suppressed the etomidate-mediated positive allosteric modulation and activation of GABAA receptors. This result clearly demonstrates the stereospecificity of the anesthetic action of etomidate. Krasowski et al. (1998) and Mihic et al. (1997) studied the role of site-specific mutagenesis in the effect of noninhalational anesthetic drugs. Specific mutations of serine-270 and alanine-291 in α-subunit of GABAA receptor were discovered. Such mutations changed the sensitivity of receptors to enflurane and isoflurane. Harrison (1998, 2000) proposed a theory that in some biotargets like NMDA and GABAA receptors, there exist specific binding pockets responsible for interaction with anesthetic agents. He explained how point mutations resulting in replacement of only one amino-acid residue are able to affect the interaction of anesthetic drugs with the binding site. Ketamine, a phencyclidine derivative (Fig. 4.37), produces dissociative anesthesia rather than a general suppressing effect observed with most intravenous anesthetics. Ketamine is unique in its way as it produces a pronounced analgesia without suppressing the cardiovascular system. However, the side effects of ketamine such as delirium and cardiovascular stimulation (increased force and rate of myocardial contraction, elevated blood pressure) impose limitations on its use in general anesthesiologic practice. Fig. 4.37 Structural formula of S-(+)-ketamine

O

CH3 HN (S)

Cl (S)-2-(2-Chlorophenyl)-2-(methylamino)cyclohexanone

106

4 The Significance of Chirality in Pharmacological and Toxicological...

In vitro studies utilizing guinea pig brain homogenate have demonstrated stereospecific differences between ketamine enantiomers with respect to their affinity for phencyclidine and opioid receptors (Hustveit et al. 1995). The highest S-(+)/R(–) stereospecific ratio for the affinity to phencyclidine receptors was found to be 2.5 in favor of the S-(+)-isomer (Fig. 4.37). This does not contradict the accepted mechanism of anesthetic analgesic effects of ketamine that is associated with NMDAreceptor blockade. It has been demonstrated that the affinity of ketamine to k-opioid receptors is 20–30 times lower than to NMDA-receptors and the stereoselectivity ratio for S-(+)/R-(–) is quite high equaling 4.2. The affinity to μ-opioid receptors is even lower and the S-(+)/R-(–) ratio amounted to 2.5. Enantiomer-specific effects of ketamine were also shown to exist for sodium channels of human brain membrane cells (Frenkel and Urban 1992). Further, it was established (Kharasch and Labroo 1992) that the rate of S-(+)ketamine metabolism in microsomes of human liver was 20% higher than for the R-(–)-enantiomer, and 10% higher than for the racemate. Thus, the anesthetic effect of S-(+)-ketamine sets on at a lower dose, and with a faster clearance. Such an advantageous pharmacological profile makes it possible to regard S-(+)-ketamine as a promising drug for future research. Adams et al. (1992) compared the effects of rac-ketamine at a dose of 2 mg/kg with the effect of S-ketamine at a dose of 1 mg/kg in human volunteers. An identical stimulating effect on the cardiovascular system was revealed, but the group of volunteers receiving S-ketamine showed a more rapid recovery. Doenicke et al. (Doenicke et al. 1992) compared the effect of different doses of the racemate and S-ketamine. They also included a third group that was premedicated with midazolam. Recovery of visual perception and sensorimotor activity was better in the S-enantiomer group, but the estimation was based on the patients’ subjective accounts. Midazolam prevented adverse effects of slow recovery from anesthesia. Pfenninger et al. (1994) compared subanesthetic doses of the racemate and S-ketamine. They discovered that S-ketamine produced the same anesthetic effect and stimulated the cardiovascular system to the same extent, but the antegrade amnesia was lesser and concentration recovery was better. Clinical trials have also supported the findings of experimental studies (Zickmann et al. 2000; Bornscheuer et al. 1997). A cardiosurgical study compared the effect of the racemate and S-ketamine during a stable state of fentanyl-midazolam general anesthesia and upon aortic compression during extracorporeal circulation. Racemate (3 mg/kg) and S-enantiomer (1.5 mg/kg) were given as single administration. Monitoring included invasive measurement of blood pressure, and right ventricular volume and pressure. The increase in heart rate and systolic blood pressure during intubation was similar in both groups. As a result, no significant difference in the hemodynamic profile of rac- and S-ketamine was found. In one of the studies, during orthopedic surgeries (knee joint surgery) anesthesia was given using midazolam and rac-ketamine at a dose of 2 mg/kg or with S-ketamine at a dose of 1 mg/kg. Throughout the surgery, continuous infusion of the racemate at a rate of 1 mg/kg/h or of the S-enantiomer at a rate of 0.5 mg/kg/h was maintained in addition to vecuronium administration and N2 O/O2 inhalation anesthesia. The authors found

4.2 Drugs Affecting the Central Nervous System Function

107

O N

N Cl

N N

(S)

O N

N

CH3

O (S)-[6-(5-Chloropyridin-2-yl)-7-oxo-6,7-dihydro-5H-pyrrolo[3,4-b]pyrazin-5-yl]- 4methylpiperazine-1-carboxylate

Fig. 4.38 Structural formula of eszopiclone

no difference with respect to the effects on cardiovascular system. In clinical practice the cardiovascular effects of S-ketamine do not differ from those of its racemate. Sedative-Hypnotic Drugs Zopiclone, a hypnotic agent, and its metabolites have been studied for stereoselective receptor interactions (Fernandez et al. 1993; Blaschke et al. 1993). It was established that the affinity of eszopiclone, the S-(+)-enantiomer (Fig. 4.38), to benzodiazepine receptors was fifty times higher than that of R-(–)-zopiclone (Blaschke et al. 1993). The pharmacokinetics of zopiclone enantiomers also turned out to be stereospecific. In volunteers receiving racemic zopiclone, the plasma elimination half-life of (+)-zopiclone was longer than that of the (–)-enantiomer (Fernandez et al. 1993). Furthermore, the urine concentration of (+)-enantiomer was higher than that of (–)-enantiomer, both for zopiclone and its metabolites (N-desmethylzopiclone and N-oxide of zopiclone) (Fernandez et al. 1993; Hempel and Blaschke 1996). Antipsychotic Agents Of all the antipsychotic agents available today, only phenothiazines with a side chain such as thioridazine (Fig. 4.39) and levomepromazine (Fig. 4.40), are chiral compounds. These drugs are used in the form of racemates and there is not enough evidence of pharmacodynamic and pharmacokinetic properties of their enantiomers. Svendsen et al. (Svendsen et al. 1988) showed that the stereoselective differences exist with regards to the affinity of thioridazine enantiomers to dopamine and α1 adrenergic receptors in rat brain. It was also reported that the (+)-enantiomer (Sisomer, Fig. 4.39) was more effective than the (–)-enantiomer in suppressing the conditioned avoidance response (Kaiser and Setler 1981). As for levomepromazine, its (–)-enantiomer (R-isomer, Fig. 4.40) has a higher affinity to dopamine receptors than the (+)-enantiomer (Boireau et al. 1980). Besides, it is four times more active in suppressing the conditioned avoidance response (Kaiser and Setler 1981). (–)-Levomeprovazine is also more effective than the (+)-enantiomer in decreasing decerebrate rigidity, in reducing vasopressor response to noradrenaline, as well as in attenuating the afferent muscle fiber discharge (Svendsen et al. 1988).

108

4 The Significance of Chirality in Pharmacological and Toxicological...

(S)

N CH3

H

N

S CH3

S

(S)-10-[2-(1-Methylpiperidin-2-yl)ethyl]-2-(methylthio)-10H-phenothiazine Fig. 4.39 Structural formula of S-(+)-thioridazine

CH3 N CH3 H

(R)

CH3 N

O CH3

S (R)-10-[2-Methyl-3-dimethylaminopropyl]-2-methoxyphenothiazine Fig. 4.40 Structural formula of R-(–)-levomepromazine

Jortani and Poklis (1993) established that patients receiving racemic thioridazine showed a ratio of (–)-thioridazine and (+)-thioridazine varying from 2.2 to 5.3, with a median value of 3.3. Mesoridazine and sulforidazine are metabolites of thioridazine that are also used as antipsychotic agents. There is not enough evidence at present about pharmacokinetic difference between the enantiomers of these two drugs, although the cytochrome CYP2D6 which has shown strong stereoselective properties (Von Bahr et al. 1991) seems to take part in the metabolism of both substances (Brosen 1990).

4.2 Drugs Affecting the Central Nervous System Function

109 CH3

Fig. 4.41 Structural formula of (Z)-isomer of chlorprothixene

N CH3 Cl

S (Z)-3-(2-Chloro-9H-thioxanten-9-yliden)-1-(dimethylamino)propane

Geometric isomerism in antipsychotic agents has also been studied. Substitution of the nitrogen atom by a carbon atom in the middle of phenothiazine ring results in thioxanthene formation (chlorprothixene, for instance, Fig. 4.41). The presence of a double bond allows formation of geometrical isomers. At present, the drugs included into the group of neuroleptic agents are manufactured in the form of mixtures of Z- and E-isomers. In all cases, the Z-isomer with its higher affinity to dopamine receptors is the one that produces therapeutic effect (Smith 1984). Numerous studies of dopaminergic systems have provided extensive evidence about the differences in pharmacological activity of geometrical isomers of thioxanthene derivatives (Smith 1984). Anxiolytic Benzodiazepines Some of the important benzodiazepines either contain a hydroxyl group at position 3, or this group appears in the process of drug metabolism. Temazepam and oxazepam (Fig. 4.42) are 3-hydroxylated metabolites of diazepam and N-desmethyldiazepam, respectively and they are marketed as independent drugs. The presence of an OH-group makes the carbon atom C3 asymmetrical in benzodiazepine nucleus, so it can be proposed that metabolic hydroxylation of these compounds makes them stereoselective, and one enantiomer predominates over the other one. Consequently, oxazepam and temazepam emerging in the process of metabolism should be optically active while the synthesized oxazepam and temazepam would be racemates. It seems reasonable to assume that the pharmacokinetics of compounds formed in the process of metabolism should differ from the pharmacokinetics of synthetically obtained compounds. In practice, however, synthetic racemic oxazepam can be separated into single enantiomers only by using an anhydrous solvent. In an aqueous medium its pure enantiomers undergo rapid racemization due to tautomerism i.e., the chiral enol form is easily converted to an optically inactive keto-form, and vice versa, and the reverse conversion is nonstereospecific (Ruelius et al. 1979; Yang and Lu 1989). O-glucuronide of oxazepam is its main metabolite, and the glucuronides of its R-(–)- and S-(+)-enantiomers are formed in unequal proportions. In humans mainly the glucuronide of S-isomer predominates and its content in urine is about three

110

4 The Significance of Chirality in Pharmacological and Toxicological...

O

H3C

O OH

N

OH

HN

* N

* H

Cl

N

H

Cl

I

7-Chloro-1,3-dihydro-3-oxy-1-methyl5-phenyl-2H-1,4-benzodiazepin-2-on

II

7-Chloro-1,3-dihydro-3-oxy5-phenyl-2H-1,4-benzodiazepin-2-on

Fig. 4.42 Structural formulas of temazepam (I) and oxazepam (II) (chiral centers are shown with an *)

times higher than the content of R-glucuronide (Sisenwine et al. 1982). There are interspecies differences in the ratio of S- and R-glucuronides, which is of importance when choosing an animal to study oxazepam. The binding of oxazepam to serum albumin in humans is also stereoselective, and the (+)-enantiomer forms a more stable complex (Müller and Wollert 1975). Such preferential binding can be regarded as a common phenomenon as the S-enantiomer of 3-methyl-1-desmethyldiazepam (an analog of oxazepam where the 3-OH-group is substituted by 3-CH3 group) also binds to human serum albumin to a greater extent than the R-enantiomer (Alebi´c-Kolbah et al. 1979). Antidepressants Tricyclic Antidepressants Protriptyline and nortriptyline are tricyclic antidepressants similar in their structure (Fig. 4.43). However, protriptyline has a double bond at position C10 –C11 , while nortriptyline has it at position C5 and the metabolic profile of two drugs differs considerably. Protriptyline is not a chiral compound, but in humans it transforms to various metabolites, for instance to protriptyline-10, 11-epoxide and its Ndemethylated homolog, nortriptyline-10, 11-epoxide that are chiral compounds (Coutts and Baker 1989). Trimipramine (Fig. 4.44) has a chiral center, but the drug used in clinical practice is a racemate. Although this drug has been in use for a long time, its single enantiomers have not been studied well. It was reported that the (+)-enantiomer of trimipramine is primarily responsible for antidepressant effect (Fig. 4.44), while the (–)-enantiomer can be a pro-depressant (Settle ahd Ayd 1980; Martindale 1982).

4.2 Drugs Affecting the Central Nervous System Function

111 11

11 10

10

5

5

CH3

CH3

I

II

NH

N-Methyl-3-(5H-dibenzo[a,d]cyclohepten-5-yl)-1-aminopropan

NH

N-Methyl-3-(10,11-dihydro-5Hdibenzo[a,d]cyclohepten-5-ylidene)-1-aminopropan

Fig. 4.43 Structural formulas of protriptyline (I) and nortriptyline (II)

H3C H3C

(S)

N

N H3C

H

(S)-N,N-Dimethyl-3-(10,11-dihydro-5H-dibenzo[b,f]azepine-5-yl)-2-methyl-1aminopropan

Fig. 4.44 Structural formula of S-(+)-trimipramine

In studies using radioligands, (–)-trimipramine showed a much higher affinity to various subtypes of biogenic amine receptors in rat brain as compared to (+)trimipramine (Gross et al. 1991). Beauchamp et al. (1992) reported that (+)trimipramine was more effective in inhibiting the potassium-induced uptake of 45 Ca by synaptosomes of rat brain cortex than (–)-trimipramine. However, later studies carried out by the same authors (Lavoie et al. 1994) demonstrated that this difference was not so pronounced for synaptosomes in other parts of the brain. Eap et al. (1992) revealed approximately equal concentrations of (+)- and (–)-enantiomers of 2-hydroxytrimipramine and 2-hydroxydesmethyltrimipramine in the plasma of three patients, while the fourth patient displayed two times higher concentrations of the (–)-enantiomer of 2-hydroxytrimipramine and (+)-enantiomer of 2-hydroxydesmethyltrimipramine. Studies on rats which received racemic trimipramine revealed that the ratio of (+)- and (–)-trimipramine concentrations in the liver and plasma was under 0.5, while the same ratio in the brain amounted to 1.4 (Lee et al. 1995).

112

4 The Significance of Chirality in Pharmacological and Toxicological...

Fig. 4.45 Structural formula of S-(+)-citalopram

N O (S)

N

CH3

H3 C F

(S)-1-[3-(Dimehtylamino)propyl]-1-(4-fluorophenyl)-5-cyano1,3-dihydroisobenzofurane

Selective Serotonin Reuptake Inhibitors Citalopram (Fig. 4.45) is marketed nowadays in the form of a racemate. In vitro studies have shown that the S-(+)-enantiomer of citalopram and its metabolites (desmethylcitalopram and didesmethylcitalopram) are more active serotonin reuptake inhibitors than its R-(–)-enantiomers (Hyttel et al. 1992). The plasma concentration of the pharmacologically active S-(+)-enantiomer of citalopram commonly amounts to one-third (varying from 24 to 49%) of the total citalopram concentration (Rochat et al. 1995). Therefore, it seems that two-thirds of the administered citalopram dose is an unnecessary drug load (Ariëns 1984). Moreover, the concentrations of the active S-(+)-enantiomers of desmethyl- and didesmethylcitalopram were also found to be considerably lower than the concentrations of inactive R-(–)-enantiomers (Baumann and Rochat, 1995). Sidhu et al. (Sidhu et al. 1997) have reported that citalopram and its two main metabolites show a slight but statistically significant stereoselectivity following 21 days administration of citalopram in human volunteers. It was suggested that (Baumann and Rochat 1995) in people with lowerthan-normal expression of CYP2C19, an enzyme involved in N-demethylation of citalopram, the plasma levels of S-(+)- and R-(–)-citalopram can be equal. Reduced expression of this enzyme is not common among Caucasian but affects up to 20% of Asiatic populations. These factors can complicate the interpretation of findings obtained solely on the basis of analyzing the plasma citalopram concentration (Evans et al. 1988). The R-(–)- and S-(+)-enantiomers of fluoxetine (Fig. 4.46) are almost equally active serotonin reuptake inhibitors (Wong et al. 1995) and the drug is used in clinical practice in the form of a racemic mixture. However, this feature does not seem to be true for its metabolites. Norfluoxetine is a demethylated metabolite of fluoxetine. There is evidence that S-(+)-norfluoxetine is a more active serotonin reuptake inhibitor than its R-(–)-enantiomer (Fuller et al. 1992). Patients treated with racemic fluoxetine have shown a higher concentration of S-(+)-norfluoxetine than of R-(–)-norfluoxetine (Torok-Both et al. 1992; Eap et al.

4.2 Drugs Affecting the Central Nervous System Function

113

F O

F F

H CH3

(S)

NH

(S)-N-Methyl-3-phenyl-3-[4-(trifluoromethyl)phenoxy]-1-aminopropane

Fig. 4.46 Structural formula of S-(+)-fluoxetine

1996), and the S-(+)- and R-(–)-fluoxetine concentration ratio varied from 1 to 3.5 (Torok-Both et al. 1992; Eap et al. 1996). It was also observed that this ratio decreases in the presence of CYP2D6 inhibitors indicating that a faster elimination of R-(–)-fluoxetine in comparison with S-(+)-fluoxetine can be mediated by cytochrome CYP2D6 (Wong et al. 1995). The predominance of R-(–)-norfluoxetine level over the S-(+)-norfluoxetine level can probably promote a recurrence of depression as in vitro studies have shown that R-(–)-norfluoxetine is much less active serotonin reuptake inhibitor than the other three enantiomers (Fuller et al. 1992). This hypothesis can possibly explain the cause of reduced therapeutic effect of fluoxetine upon long-term treatment in some patients (Fava et al. 1995). Sertraline (Fig. 4.47) and paroxetine (Fig. 4.48) are used as antidepressants only in the form of stereoisomers with the highest serotonergic activity (Baumann 1992). Sertraline and paroxetine each have two asymmetrical carbon atoms and form four different diastereomers each. At present, sertraline is marketed in the form of the 1S,4S-(+)-isomer and paroxetine—in the form of the 3S,4R-(–)-isomer. Some antidepressants can display enantioselective pharmacokinetic interaction with other racemic drugs. For instance, fluvoxamine that has an E-configuration (Fig. 4.49) reduces the metabolism of the R-(–)-enantiomer of racemic warfarin by inhibiting the CYP1A2 isoenzyme.

Cl H

H Cl

(S)

( S)

CH3 NH

(1S,4S)-1-Methylamino-4-(3,4-dichlorophenyl)-1,2,3,4-tetrahydronaphthalene

Fig. 4.47 Structural formula of 1S,4S-(+)-sertraline

114

4 The Significance of Chirality in Pharmacological and Toxicological... F

O

O

O

H (R)

(S)

H N H

(3S,4R)-3-[[(Benzo[d][1,3]dioxol-5-yl)oxy]methyl]-4-(4-fluorophenyl)piperidine

Fig. 4.48 Structural formula of 3S,4R-(–)-paroxetine

CH3 O O N

NH2

F F

F

O-(2-aminoethyl)oxime (E)-5-methoxy-1-[4-(trifluoromethyl)phenyl]pentane-1-on

Fig. 4.49 Structural formula of fluvoxamine

Although, fluvoxamine does not directly affect the clearance of the more pharmacologically effective S-(+)-enantiomer of warfarin, there is still a possibility of clinically important interaction of these two drugs. Fluvoxamine causes increased levels of R-(–)-enantiomer of warfarin (by inhibiting CYP1A2), which in turn inhibits the CYP2C9/10, an isoenzyme responsible for clearance of S-(+)-enantiomer of warfarin. Therefore, by inhibiting the clearance of R-(–)-warfarin, fluvoxamine produces a cascade effect on the metabolism of S-(+)-warfarin causing the plasma warfarin level to rise by approximately 65% (Kunze et al. 1991; Benfield and Ward 1986). In vitro studies on human liver microsomes established that the R-(–)-enantiomers of fluoxetine and norfluoxetine are weaker inhibitors of CYP2D6 isoenzyme than the corresponding S-(+)-enantiomers (Stevens and Wrighton 1993). In ex vivo studies in rats, simultaneous administration of fluoxetine and desipramine, which is also a CYP2D6 inhibitor and substrate, resulted in higher brain levels of fluoxetine and norfluoxetine as compared to those achieved after administration of fluoxetine only (Goodnough and Baker 1994; Aspeslet et al. 1994). Moreover, the above-mentioned

4.2 Drugs Affecting the Central Nervous System Function

115

drug interaction showed stereoselectivity for fluoxetine as the increase in the concentration of its R-(–)-isomer was more pronounced than that of S-(+)-isomer. Similar stereoselectivity was not observed for norfluoxetine (Aspeslet et al. 1994). Furthermore, no differences were observed in the activity of fluoxetine and norfluoxetine enantiomers in relation to other CYP isoenzymes (like CYP3A3/4) that are also inhibited by fluoxetine. It is not known at present whether citalopram and desmethylcitalopram enantiomers differ in their ability to inhibit the cytochrome P450 enzymes. Apparently, the resulting effect of citalopram should be regarded as sum of the effects of four enantiomers of these two drugs (Van Harten 1993). Similarly, fluoxetine whose effect is to a great extent due to its metabolite norfluoxetine can be regarded as a combination of four stereoisomer compounds that differ in their effectiveness and pharmacokinetic parameters (Gram 1994). Noradrenaline Reuptake Inhibitors Doxepin (Fig. 4.50) is a mixture of Z- and E-isomers in a ratio of 15:85 (Pinder et al. 1977). The doxepin isomers have extensively been studied in various neurochemical and behavioral studies such as the effect on noradrenaline and serotonin reuptake by nerve endings; antagonism to the effect of reserpine, histamine and 5-hydroxytriptamine; effect on amphetamine stereotypism; sleep potentiation and anticholinergic effects. In most studies the Z-isomer displayed a higher activity as compared to E-isomer (Hobbs 1969). However, the E-isomer turned out to be more effective in inhibiting 5-HT-uptake than the Z-isomer. Doxepin demethylation yields an active metabolite N-desmethyldoxepin. Clinical studies showed that the ratio of Z- and E-doxepin concentrations in the plasma was about 15:85, the same as in the original drug, but for N-desmethyldoxepin, the Z-isomer level turned out to be higher than the level of the E-isomer (Rosseel et al. 1978; Hrdina et al. 1990; Midha et al. 1992). O

O

Z-

ECH3

H3C N

N

CH3

CH3

N,N-Dimethyl-3-(dibenzo[b,e]oxepin-11(6H)-yliden)-1-aminopropan Fig. 4.50 Structural formulas of (Z)- and (E)-stereoisomers of doxepin

116

4 The Significance of Chirality in Pharmacological and Toxicological...

NH2

NH2 (R)

(S)

(S)

( R)

H

H H

H

(1S,2R)-(+)-Isomer (1R,2S)-(–)-Isomer trans-2-Phenyl-1-aminocyclopropane Fig. 4.51 Structural formulas of tranylcypromine enantiomers

Monoamine Oxidase Inhibitors Tranylcypromine (Fig. 4.51) is a racemic mixture of (+)- and (–)-trans-2-phenylcyclopropylamine. Tranylcypromine enantiomers vary in their pharmacological properties. 1S,2R(+)-Tranylcypromine is a more effective monoamine oxidase inhibitor while 1R,2S(–)-tranylcypromine is a more effective catecholamine reuptake inhibitor in the rat brain (Horn and Snyder 1972; Fuentes et al. 1976; Hampson et al. 1986). Tranylcypromine has shown mild therapeutic benefits in some patients suffering from Parkinson’s disease. Reynolds et al. (1981) reported that (–)-tranylcypromine produced a more pronounced benefits in correcting psychiatric disturbances while (+)-tranylcypromine improved motor activity. Other studies performed on rats and volunteers have also demonstrated a considerable difference in the pharmacological profiles of (+)- and (–)-tranylcypromine (Fuentes et al. 1976; Reynolds et al. 1981; Hampson et al. 1986). (–)-Deprenyl (selegiline, Fig. 4.52) is a R-(–)-N-propargyl derivative of methamphetamine. This compound is a selective monoamine oxidase-B inhibitor; in clinical practice, it is mostly utilized as an anti-Parkinson drug rather than an antidepressant (Gerlach et al. 1996). The (+)-Deprenyl is a weaker monoamine oxidase inhibitor than the (–)-enantiomer (Magyar et al. 1967). A greater part of the administered R-(–)-deprenyl is transformed into the (–)-isomers of N-methylamphetamine, Npropargylamphetamine and amphetamine (Reynolds et al. 1978; Gerlach et al. 1996), thus, no racemic transformation occurs during metabolism (Magyar and Tóthfalusi 1984). Fig. 4.52 Structural formula R-(–)-deprenyl

CH3 (R)

CH N

H CH3

(R)-N-Methyl-N-(propin-2-yl-1)-1-phenyl-2-aminopropan

4.2 Drugs Affecting the Central Nervous System Function Fig. 4.53 Structural formula of S-(–)-viloxazine

117

H O O

(S)

CH3

O N H

(S)-2-[(2-Ethoxyphenoxy)methyl]morpholine

Other Antidepressants Viloxazine (Fig. 4.53) is a bicyclic compound. It is a selective inhibitor of noradrenaline uptake. There is some evidence that viloxazine also facilitates the release of 5-hydroxytryptamine from neuronal storage. In clinical practice this drug is used in the form of a racemate, but animal studies have demonstrated that the S(–)-isomer has higher efficacy as antidepressant as compared to its R-(+)-isomer (Case and Reeves 1975). Although viloxazine is metabolized extensively, none of its metabolites displays an antidepressant action (Case and Reeves 1975). As viloxazine metabolites have no antidepressant properties, no in-depth research to study their stereoselectivity has been performed. Bupropion (Fig. 4.54) is an antidepressant and its mechanism of action permits its classification as noradrenaline and dopamine reuptake inhibitor. It is a derivative of 2-aminopropiophenone. It is a structural analog of cathinones (CNS stimulants obtained from the leaves of khat, Catha edulis) and diethylpropione drug family (appetite-suppressants). Bupropion is used as a racemate. It has one chiral center, but in the human body it is rapidly transformed into three main products: threo-amino alcohol, erythro-amino alcohol and bupropion hydroxylated in the side chain (Laizure et al. 1985). Threo-amino and erhythro-amino alcohols are analogs of sympathomimetic amines: pseudoephedrine, 1R,2R-(+)-2-methylamino-1-phenyl1-propanol and ephedrine, 1R,2S-(–)-2-methylamino-1-phenyl-1-propanol, correspondingly. Mianserin (Fig. 4.54), tetracyclic antidepressant, is a racemic mixture of R-(–)and S-(+)-enantiomers (Peet and Behagel 1978). S-(+)-mianserin is 200–300 times more active than the R-(–)-isomer as noradrenaline reuptake inhibitor (Schoemaker O

Fig. 4.54 Structural formula of bupropion (the chiral center is shown with an *)

H

H N

* CH3 H3C

CH3 CH3

Cl 2-(Tert-butylamino)-1-(3-chlorophenyl)propane-1-on

118

4 The Significance of Chirality in Pharmacological and Toxicological... H3C N

H

(S)

N

(S)-2-Mehtyl-1,2,3,4,10,14b-hexahydrobenzo[c,f]pyrazino[1,2-a]azepine

Fig. 4.55 Structural formula of S-(+)-mianserin

et al. 1981). Studies on animals showed that the antidepressant activity of the (+)enantiomer of mianserin is much higher than the activity of its (–)-enantiomer (Cheeta et al. 1994; Hand et al. 1991; Jancsár and Leonard 1984) (Fig. 4.55). Mianserin blocks presynaptic α2 -adrenergic autoreceptors. This effect is stereoselective and is mostly typical of the (+)-isomer (Schoemaker et al. 1981; Raiter et al. 1983). However, both enantiomers have similar antagonistic properties in relation to α2 -adrenergic heteroreceptors on 5-HT nerve endings (Raiter et al. 1983). Two mianserin enantiomers also differ considerably in their ability to bind with various subtypes of 5-HT-receptors (Alexander and Wood 1987; Wood et al. 1993; Kooyman et al. 1994). Such differences in receptor interaction of two enantiomers lead to differences in their antidepressant effects. In contrast, the sedative action that is probably due to antihistaminic properties turns out to be the same for racemic mianserin and for its single enantiomers. Thus, the antidepressant action of mianserin is stereospecific while its other properties are not. There is also a significant difference in the metabolic pathways for the two mianserin enantiomers. Heinig and Blaschke (Heinig and Blaschke, 1993) discovered in their study on mice that the R-enantiomer is mostly subject to N-demethylation while the S-enantiomer mostly undergoes 8-hydroxylation. Studying mice liver homogenate, Heinig and Blaschke (Heinig and Blaschke 1993) demonstrated that the greater part of N-oxidized metabolites is formed from S-mianserin, while the R-enantiomer yields N-demethyl derivatives. Comparable results were obtained in a study on human liver microsomes (Heinig and Blaschke 1993). In their study using rats, Heinig et al. (1993) revealed that hydroxyl-metabolites of S-mianserin enter conjugation more easily than the R-enantiomer metabolites. In a study involving human volunteers, Dahl et al. (1994) discovered that the elimination of mianserin depends on the activity of CYP2D6. This process is enantioselective to a considerable extent and proceeds faster for S-enantiomer. Tybring et al. (1995) analyzed the content of mianserin and N-desmethylmianserin enantiomers in the plasma of patients undergoing treatment for depression. It was shown that the average S:R ratio was 1.9 for mianserin but in the case of N-desmethylmianserin it was the Renantiomer that predominated. Similar findings were obtained by Eap et al. (1994) in

4.2 Drugs Affecting the Central Nervous System Function

119

their study involving human volunteers. A study on human liver microsomes utilizing eight recombinant CYP isoforms performed by Koyama et al. (1996) concluded that 8-hydroxylation of both mianserin enantiomers occurs in the presence of CYP2D6 while N-demethylation of both enantiomers and N-oxidation of S-(+)-mianserin are mainly catalyzed by CYP1A2. CNS Stimulants Amphetamine (Fig. 4.56) is a stimulant of the central nervous system. Amphetamine stereoisomers, D-(+)-isomer (dextroamphetamine) and L-(–)-isomer (levoamphetamine), produce different pharmacological effects. The D-(+)-enantiomer is more active as a psychostimulant (Golikov et al. 1973). There is some evidence that stereoisomerism also affects the toxicity of amphetamine. As shown in the Table 4.9, in mice kept separated from one another (+)-amphetamine was 1.5 times more toxic than the other isomer. In mice grouped together, the toxicity to (+)-amphetamine was drastically higher than the other isomer amounting to more than 6 times. Thus, while the (–)-isomer toxicity in grouped mice increased only 1.2 times in comparison with the toxicity of the same isomer in isolated mice, this difference in the case of (+)-amphetamine amounted to almost 5 times. Methylphenidate (meridil, ritalin), derivative of piperidine, is a psychostimulant. It exists in the form of a mixture of D- and L-isomers. Dexmethylphenidate (focalin, Fig. 4.57), a purified part of ritalin, was approved by the Food and Drug Administration for treatment of attentiondeficit/hyperactivity disorder (Quinn 2008). While ritalin contains both D- and L-isomers of methylphenidate, dexmethylphenidate only contains the more active D-(+)-isomer.

CNS stimulants

H

H CH3

CH3

(S)

(R)

NH2

NH2

D-(+)-Isomer L-(–)-isomer (Dextroamphetamine) (Levoamphetamine) 1-Phenyl-2-aminopropane Fig. 4.56 Structural formulas of amphetamine enantiomers

Table 4.9 Toxicity of amphetamine optical isomers (Golikov et al. 1973)

Isomer (+) (–)

LD50 mg/kg Isolated mice

Mice grouped together

98 150

20 125

120

4 The Significance of Chirality in Pharmacological and Toxicological...

H H N

O

(R)

CH3

(R)

H O

D-(+)-Methylphenidate (R)-Methyl[2-phenyl-2-[(R)-piperidin-2-yl]]acetate

Fig. 4.57 Structural formula of dexmethylphenidate

O H HO

HO (S)

OH

NH2 HO Levodopa (S)-2-Amino-3-(3,4-dioxyphenyl)propanic acid

NH2 HO Dopamine 1,2-Dioxy-4-(2-aminoethyl)benzene

Fig. 4.58 Metabolism of L-(–)-DOPA

Anti-parkinsonian Drugs The laevorotatory (–)-isomer (L-DOPA, levodopa, S-3,4-dihydroxyphenylalanine, Fig. 4.58) of dioxyphenylalanine (DOPA) is much more active than its antipode. L-DOPA penetrates the blood brain barrier, transforms into dopamine, and makes up for the dopamine deficiency in the brain. However, L-DOPA absorbed in the intestine undergoes decarboxylation. That is why combination drugs were proposed that contained, besides L-DOPA, a peripheral decarboxylation inhibitor that does not penetrate the blood brain barrier (Encyclopedia 2008). Analgesics Narcotic Analgesics Administration of S-ketamine (Fig. 4.37) as an analgesic drug can be more effective than when utilizing it as an anesthetic drug. S-ketamine is used for epidural anesthesia in pediatrics (Marhofer et al. 2000). It provides the same degree of postoperative analgesia as bupivacaine. It can also be administered to adults and children in critical care. Arendt-Nielsen et al. (1996) showed that S-ketamine was better than the racemate in decreasing temporal and spatial summation of pain intensity and reduced the time lag in a post-burn model on volunteers.

4.2 Drugs Affecting the Central Nervous System Function

H3 C

121

H3 C

CH3

CH3 N

N

(R)

H O

( S)

H

(R)

O

(S)

H3C

H3C OH

OH

1R,2R-(+)-Isomer 1S,2S-(–)-Isomer 2-[(Dimethylamino)methyl]-1-(3-methoxyphenyl)cyclohexanol Fig. 4.59 Structural formulas of tramadol enantiomers

Tramadol is a chiral analgesic with an unusual double mode of action (Fig. 4.59). It has a weak affinity to the μ-opioid receptor (ten times weaker than that of codeine) (Raffa 1996), and moreover, it increases central neuronal synaptic transmission of 5-hydroxytryptamine and noradrenaline. These neurotransmitters are involved in antinociceptive ascending pathways in the spinal cord. Tramadol has two chiral centers at positions 1 and 2 of the cyclohexane ring, thus providing four stereoisomers (Calvey and Williams 1997). In clinical practice it is utilized in the form of a mixture of two enantiomers: 1R,2R-(+)-tramadol and 1S,2S-(–)-tramadol. The effect of these stereoisomers was studied in animal experiments. The effect of rac-, (+)- and (–)-tramadol on noradrenaline release and reuptake, and the action potential of neurons were studied in the sections of rat brain (Dressen et al. 1993). Rac-tramadol and both its enantiomers increased noradrenaline release significantly. However, only (–)-tramadol blocked noradrenaline reuptake, and the effect of its mono-O-demethylated metabolite surpassed the effect of (–)-tramadol itself by five times. These findings agree with the results obtained in other studies (Halfpenny et al. 1999; Sevcik et al. 1993). The effect of tramadol on serotonergic system turned out to be stereoselective as well. Raffa et al. (1993) in their research involving mice showed that the (+)isomer was the most powerful serotonin reuptake inhibitor of all its diastereomers. Moreover, they found that both the (+)- and (–)-enantiomers had antinociceptive effects in experiments on acetylcholine-mediated gastrointestinal contractions. Both stereoisomers act in a synergistic way, and so rac-tramadol displays an effect higher than the total effect of two enantiomers. Antinociceptive synergism was demonstrated in rats with experimentally induced inflammation such as in Randall-Selitto test and the hot plate test. Based on experimental findings it was established that the tramadol racemate was the optimum choice for use in clinical practice. Grond et al. (1995) confirmed the choice of tramadol racemate in a study using tramadol for controlled analgesia after a major gynecological surgery. Patients received personalized doses (from a

122

4 The Significance of Chirality in Pharmacological and Toxicological...

minimum to 200 mg) and were randomized into groups receiving (+)-, (–)- or ractramadol. A reduction of pain sensation to a certain threshold on a conventional pain assessment scale was used as a measure of effectiveness. Up to 53% of the patients in the (–)-enantiomer group had analgesia below the set threshold as compared to 12% in the (+)-tramadol group and 15% in the rac-tramadol group. Patients in whom the analgesic effect reached the threshold were mostly in the (+)-enantiomer group amounting to 67% compared to 48% in the rac-tramadol group, and 38% in the (–)-tramadol group. The proportion of patients satisfied with pain relief in the first 24 h after surgery was 82, 76 and 41% in the groups of (+)-, rac- and (–)-tramadol, respectively. Nausea and vomiting were the most common side effects and were more common in the (+)-tramadol group. Thus, considering the therapeutic effects and side effects, a conclusion can be made that tramadol racemate is a preferred choice for use in clinical practice than its pure enantiomers (Grond et al. 1995). Medetomidine or dexmedetomidine (Fig. 4.60) is an imidazoline derivative. It is a full selective agonist of both pre- and postsynaptic α2 -adrenoreceptors (Virtanen 1989). It is widely used for veterinary anesthesia. An inhibition of sympathetic activity in the CNS induced by this drug is accompanied by a characteristic pharmacological picture that includes hypotension, bradycardia, sedation, decreased anxiety, analgesia, and hypothermia. These effects can be reduced or reversed by administering atipamezole, a selective and specific α2 -antagonist which is used in veterinary practice to reverse the effect of medetomidine (for instance, at the end of a surgical intervention) (Virtanen 1989). In experiments studying α adrenoreceptors binding, the α2 /α1 selectivity ratio was found to be 1620 for medetomidine, 220 for clonidine, and 160 for xylazine (Virtanen 1989). α2 -Adrenoreceptor activity of medetomidine is exclusively attributed to its D-enantiomer (dexmedetomidine). In experiments on rats (MacDonald et al. 1991), dexmedetomidine caused sedation and hypothermia, stimulated a dose-dependent release and reuptake of noradrenaline, dopamine and serotonin, which is typical of α2 -agonists. It reduces the minimum alveolar concentration (MAC) considerably when administered together with halothane for anesthesia (Vickery et al. 1988) and decreases the heart rate and the cardiac output. A study involving human volunteers confirmed this effect using isoflurane. Ebert et al. (2000) estimated the cardiovascular, respiratory, and endocrine effects of dexmedetomidine. The parameters monitored included arterial blood pressure, central venous pressure, pulmonary artery pressure, cardiac output, oxygenation, and Fig. 4.60 Structural formula of D-(+)-medetomidine (dexmedetomidine)

H3 C N

HN

H (R)

CH3

CH3

(R)-4-(1-(2,3-Dimethylphenyl)ethyl)-1H-imidazole

4.2 Drugs Affecting the Central Nervous System Function

123

catecholamine concentration. The catecholamine content was significantly lower at the initial dose and no noradrenaline content increase commonly seen in cold pressor test, took place. A dose-dependent increase in sedation, a decrease in the heart rate, systolic blood pressure, pulmonary artery pressure, cardiac output and stroke volume were noted. Respiratory parameters changed only fractionally, and the acid–base equilibrium did not change at all. The effects of dexmedetomidine were also studied both during and after surgery. Aho et al. (1992) in their randomized double-blind study involving 20 patients evaluated the effect of dexmedetomidine in comparison with placebo in patients undergoing hysterectomy. Infusion was given 10 min prior to administration of anesthesia and further throughout the surgery. In the dexmedetomidine group the need for isoflurane diminished by over 90%. In another study involving 41 patients undergoing vascular surgery, the administration of dexmedetomidine or its salt was started 20 min prior to administration of anesthesia and continued for as long as 48 h after surgery. The heart rate stability was higher in the dexmedetomidine group and the noradrenaline concentration in the early postoperative period was much lower in the same group. Venn et al. (1999) evaluated dexmedetomidine as a sedative and analgesic drug in comparison with placebo in 119 patients who were on artificial ventilation after abdominal or cardiac surgery. In the dexmedetomidine-receiving group of patients a considerably lower need for midazolam or morphine was noted. Morphine and methadone are also chiral compounds. Morphine (Fig. 4.61) and codeine (morphine 3-methyl ether) have five chiral centers. They are administered in the form of native mono-(–)-enantiomers. Methadone has one chiral center; it is usually used in the form of a racemate. R-Methadone (L-form, Fig. 4.62) is an agonist of opioid receptors, and S-methadone (D-form) is an antagonist of NMDA-receptors. Naloxone is administered in the form of the (–)-enantiomer (Fig. 4.63); while (+)naloxone is essentially inactive. Dextromethorphan is a dextrorotatory (+)-isomer of levorphanol methyl ether (Fig. 4.64). It is the antipode of levomethorphan. The drug is widely used as an antitussive agent. It has a weak affinity to μ-opioid receptors (ten times weaker than codeine) (Raffa 1996). In normal concentrations, it CH3

Fig. 4.61 Structural formula of natural morphine

H (R)

H

N (R)

(S) (S) (R)

HO

O

OH

(5R,6S,9R,13S,14R)-4,5-Epoxy-N-methyl-7-morphinan-3,6-diol

124

4 The Significance of Chirality in Pharmacological and Toxicological... CH3

Fig. 4.62 Structural formula of L-methadone

CH3

O

(R)

CH3 N

H

CH3

(R)-6-(Dimethylamino)-4,4-diphenylheptan-3-one

Fig. 4.63 Structural formula of (–)-naloxone

CH2 H (R)

HO

N (S)

(S)

(R)

O

HO

O

(5R,9R,13S,14S)-17-Allyl-4,5-epoxy-3,14-dioxymorphinan-6-one

CH3

Fig. 4.64 Structural formula of dextromethorphan

N (S)

H

(S)

(S)

H3 C

O

(9S,13S,14S)-3-Methoxy-N-methylmorphinan

produces neither analgesic and sedative effect nor suppresses the respiratory center. Besides, it is also a non-competitive antagonist of NMDA-receptors. This property permits its utilization in clinical situations such as acute and chronic pain and as a neuroprotective agent after cerebral injury. Dextromethorphan was administered prior to (Grace et al. 1998; Helmy and Bali 2001) and after (Henderson et al. 1999) surgery and was found to reduce the need for analgesia. In chronic pain, it was administered either alone or in combination

4.2 Drugs Affecting the Central Nervous System Function

125

with morphine (MorphiDex) (Katz et al. 2000). A suggestion was put forward that it not only enhances the analgesic effect of opioids but also relieves or reduces physiological tolerance to them resulting from repeated use (Elliot et al. 1994). Non-narcotic Analgesics and Nonsteroid Anti-inflammatory Drugs Nonsteroid anti-inflammatory drugs (NSAIDs) vary in their chemical structure. They inhibit the synthesis of prostaglandins at one (inhibition of cyclooxygenase-1,2) or several stages of endoperoxide biosynthesis. Some drugs of this family are chiral belonging either to a large group of 2-arylpropionic acids (2-APA) or to the propiophenone group. Hetaryl-carboxylic acid derivatives are also chiral for instance, ketorolic acid (ketorolac) and etodolic acid (etodolac) as well as drugs from other chemical classes (e.g., azapropazone). It is recognized that most or even all inhibitors of prostaglandin synthesized in vitro are S-enantiomers (Evans 1992). As a result, single isomers of NSAIDs were introduced into clinical practice such as S-naproxen (Fig. 4.65), S-ibuprofen (Fig. 4.66), and S-ketoprofen (Fig. 4.67). These drugs produce a rapid and effective analgesia and have fewer side effects. Administering S-isomers of NSAIDs has several advantages in clinical practice. The dose selection is much easier, the pharmacokinetic profile is less complicated, and interactions with other drugs occur less often. Utilization of the stereoselective properties of NSAIDs helps to promote their rational use and better understanding of nociceptive and anti-inflammatory processes Ketorolac (Fig. 4.68) has one chiral center and is used in the form of racemate like most derivatives of phenylpropionic acid or NSAIDs of propiophenone group. The characteristic of their S-enantiomer is CH3

Fig. 4.65 Structural formula of S-(+)-naproxen

(S)

O H

H3 C

OH O

(S)-2-(6-Methoxy-2-naphthyl)propanoic acid

CH3

Fig. 4.66 Structural formula of S-ibuprofen

CH3

H3C

(S)

H HO (S)-2-(4-Isobutylphenyl)propanoic acid

O

126

4 The Significance of Chirality in Pharmacological and Toxicological... CH3

O

Fig. 4.67 Structural formula of S-ketoprofen

(S)

OH H O

(S)-2-(3-Benzoylphenyl)propanoic acid

Fig. 4.68 Structural formula of S-ketorolac

O H N

O

(S)

OH

(S)-5-Benzoyl-2,3-dihydro-1H-pyrrolizine-1-carboxylic acid

that they are active cyclooxygenase inhibitors, in contrast to the R-isomers. However, one enantiomer can turn into the other as a result of metabolism, which promotes complex pharmacokinetic and pharmacodynamic interactions (Evans 1992).

4.3 Drugs Regulating the Functions of Major Organs and Systems Cardiovascular Drugs Calcium Antagonists Calcium antagonists block the influx of Ca2+ ions into the vascular smooth muscle myocytes promoting a reduction in total peripheral vascular resistance and a decrease in systolic arterial pressure. There are three main groups of calcium channel blockers: dihydropyridines, phenylalkylamines and benzothiazepines. 4-Aryl-1,4dihydropyridines (e.g. nifedipine) are the most studied family of Ca2+ channel modulators. From the moment they were introduced into clinical practice in 1975, they became indispensable in the treatment of cardiovascular conditions such as hypertensive disease and ischemic heart disease. Although nifedipine is an achiral agent, other dihydropyridines have an asymmetrical carbon atom at position 4 of the heterocyclic ring and are mostly used in the form of racemic mixtures, except for barnidipine. It was demonstrated that mostly the S-enantiomers of these compounds display the greatest activity. Their eudesmic ratios vary from 2 for nimodipine to 1000 for amlodipine (Inotsume and Nakano 2002; Kappe 1998).

4.3 Drugs Regulating the Functions of Major Organs and Systems

127

Amlodipine is a mixture of two enantiomers where the S-enantiomer (Asomex, Fig. 4.69) blocks the L-type of calcium channels while the R-enantiomer promotes the release of NO (Zhang et al. 2002). R-(+)- and S-(–)-enantiomers of amlodipine do not differ significantly in their pharmacokinetic properties. However, nicardipine enantiomers have shown differences in their pharmacokinetic properties (Fig. 4.70). The serum concentration of (+)-nicardipine playing the key role in calcium channel blockade was found to be twice the concentration of (–)-nicardipine (Inotsume and Nakano 2002; Japelj et al. 1999; Luksa et al. 1997). The enantiomers of 1,4dihydropyrimidine derivative, Bay K 8644 compound, have also displayed a difference in their pharmacological properties (Fig. 4.71). The S-(–)-form of this compound is an agonist of calcium channels while the R-(+)-isomer is the antagonist (Kubinyi 2002; Triggle 1997). H N

H3 C

NH2 O

O

O

(S)

CH3

H3C O

H

O Cl

(S)-3-Ethyl-5-methyl-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)6-methyl-1,4-dihydropyridine-3,5-dicarboxylate

Fig. 4.69 Structural formula of S-amlodipine

H N

H3C

O

CH3

O

(S)

H3C

N O

H

O

CH3 O-

N

+

O

(S)-3-{2-[Benzyl(methyl)amino]ethyl}-5-methyl-2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate

Fig. 4.70 Structural formula of (+)-nicardipine

128

4 The Significance of Chirality in Pharmacological and Toxicological... F

Cl

F

F F

H3C

F H3C O

O-

H (R)

O

H3C

N H

CH3

O

O

CH3

N

H

N+

(R)

O

H3C

N

HO

O

O

H (R)

O

S

N

CH3

H3C

F F

H3C

NH2

N

N H

O

O

Bay K 8644

SQ 32547

SQ 32926

(R)-Methyl-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-1,4-dihydropyridine-3-carboxylate

(R)-3-[1-(4-Fluorobenzyl)piperidine-4-yl]-5-(isopropoxycarbonyl)-6-methyl-2-thioxo-4-[2-(trifluoromethyl)phenyl]-3,4-dihydropyrimidine-1(2H)-carboxylic acid

(R)-Isopropyl-3-carbamoyl-4-(3-chlorophenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate

Fig. 4.71 Structural formulas of active enantiomers of new calcium channel blockers

Dihydropyridine analogs, dihydropyrimidines, have aroused great interest in recent years. Studies have shown that the antihypertensive effect of SQ 32,547 and SQ 32,926 compounds (Fig. 4.71) was primarily attributable to their R-enantiomers (Kappe 2000). In a multi-center safety and efficacy of S-amlodipine (SESA) study (2002), 314 patients with arterial hypertension and peripheral edema receiving amlodipine (at a dose of 10 mg/day) were switched to S-amlodipine (at a dose of 5 mg/day). The edema subsided in 310 patients (98.7%) although the antihypertensive effect was preserved (SESA 2003). Identical results were obtained in another clinical trial where replacement of racemic amlodipine (5 mg/day) by S-amlodipine (2.5 mg/day) in 256 patients promoted elimination of edema in 252 (98.4%) patients (Deanfield et al. 1994). In a comparative randomized study of S-amlodipine and racemic amlodipine, the rates of edema in a 12-week therapy with S-amlodipine amounted to 1.6 and 7.85, correspondingly. It follows that the S-amlodipine therapy reduced the risk of edema development by 4.8 times (Bobrov and Davydova 2007). The rates of peripheral edema development with S-amlodipine therapy in two major post registration studies amounted to 0.75% (14 of 1859 people under observation) and 0.84% (14 of 1669) only. It was shown in a four-week observational study that the antihypertensive activity of S-amlodipine at a dose of 2.5 and 5 mg/day is equivalent to the effect of racemic amlodipine taken at twice larger daily doses, 5 and 10 mg, correspondingly (SESA 2003; Deanfield et al. 1994).

4.3 Drugs Regulating the Functions of Major Organs and Systems

129

Further, like racemic amlodipine, S-amlodipine (Asomex) produces a pronounced antianginal and anti-ischemic effect as evidenced by another study using a loading test and daily Holter ECG for patient monitoring and evaluation (Bobrov and Davydova 2007). In clinical trials performed by a research branch of SESA Group (India), patients receiving amlodipine showed a significantly low rate of tachycardia development (one patient of 1859; 0.05%), as compared to those receiving racemic amlodipine (0.5%–CAPE study, 1994). This observation could be explained by a longer plasma elimination half-life of S-amlodipine as compared to racemate (Laufen and Leitold 1994). The longer half-life of S-amlodipine accounts for a potential enhancement of the pharmacological effect, a smoother profile of the pharmacokinetic curve thus reducing the possibility of reflex tachycardia (Blankfield 2005). Another important fact that the clearance of S-amlodipine is prone to lesser individual variations than that of the R-isomer, provides additional argument in favor of greater safety of therapy with the pure S-enantiomer of amlodipine (Laufen and Leitold 1994). Verapamil is an L-type calcium channel antagonist, a derivative of phenylalkylamine. This drug is used for the treatment of hypertension, cardiac arrhythmia and ischemic heart disease. Verapamil enantiomers display variations in their pharmacokinetic properties and so differ from one another in their bioavailability and the pharmacological effect. The S-(–)-enantiomer (Fig. 4.72) is approximately 20 times more active than the R-(+)-isomer. S-verapamil produces both an extracardiac effect (vasodilating) and a cardiac effect (reducing the myocardial work), while the Risomer exerts the vasodilating action predominantly. Moreover, the hepatic first-pass uptake of S-verapamil is almost twice as high as that of its R-antipode (Mori et al. 2001; Sawicki and Janicki 2002; Triggle 1997). O

O

H3C H3 C

O

O

H3 C

CH3

N

(S)

H3 C N

(S)-2-Methyl-3-cyano-3-(3,4-dimethoxyphenyl)-6-[(2-(3,4dimethoxyphenyl)ethyl)(methyl)amino]hexane Fig. 4.72 Structural formula of S-verapamil

CH3

CH 3

130

4 The Significance of Chirality in Pharmacological and Toxicological...

Gallopamil is a methoxy derivative of verapamil. Like verapamil, the Senantiomer of gallopamil (Fig. 4.73) shows the cardiovascular effects, but unlike verapamil, it is not stereoselective with regards to the hepatic first pass uptake (Gross et al. 2000). Diltiazem is a derivative of benzothiazepine consisting of two asymmetric centers. The drug is an antagonist of voltage-dependent calcium channels and is used in the treatment of arrhythmias. (+)-Diltiazem in the 2S,3S-configuration (Fig. 4.74) is a blocker of voltagedependent calcium channels producing a protective effect on the myocardium in conditions of ischemia and reperfusion. Its optical isomer (–)-diltiazem provides a 20–100 times lesser blocking effect on the slow calcium channels, but like the (+)-isomer, it exerts a cardioprotective action (Sato et al. 2002).

CH3 CH3 O

O

H3C H3 C

N

CH3

H3C

O O

H3C

CH3

( S)

N

O

(S)-2-Methyl-3-cyano-3-(3,4,5-trimethoxyphenyl)-6-[(2-(3,4dimethoxyphenyl)ethyl)(methyl)amino]hexane

Fig. 4.73 Structural formula of S-gallopamil

O CH3 H S (S) (S)

O

O

H 3C

CH3

H

N

O

N CH3

(2S,3S)-5-[2-(Dimethylamino)ethyl]-2-(4-methoxyphenyl)-4-oxo-2,3,4,5tetrahydrobenzo[b][1,4]thiazepine-3-ylacetate Fig. 4.74 Structural formula of 2S,3S-(+)-stereoisomer of diltiazem

4.3 Drugs Regulating the Functions of Major Organs and Systems

131

A Potassium-Channel Opening Vasodilator ATP-sensitive activators of potassium channels are a promising class of drugs for the treatment of cardiovascular conditions such as ischemic heart disease, hypertension, and cardiac arrhythmia. Chromakalim was one of the first drugs of this family. Its hypotensive effect is attributed to the 3S,4R-(–)-enantiomer, levchromakalim exclusively (Fig. 4.75). However, besides the pronounced vasodilating effect, chromakalim and its (–)isomer produced marked side effects such as reflex tachycardia, edema, headaches and hyperemia (Blackburn et al. 1995; Lee et al. 2000). To reduce the side effects and to improve the clinical usage of this family of drugs, various modifications of chromakalim were developed. Introduction of a hydroxymethyl group at the 5th position of pyrrolidine-2-one ring made it possible to develop a compound (–)-MJ-451 (Fig. 4.76) that has antihypertensive effects but provokes no reflex tachycardia. This substance also suppresses

O OH

N

H

H (R) (S)

CH3

N CH3

O

(3S,4R)-2,2-Dimethyl-3-oxy-4-(2-oxopyrrolidin-1-yl)-6-cyanochroman

Fig. 4.75 Structural formula of levchromakalim

O H

(S)

OH

N HO

H

H (R) (S)

CH3

N O

CH3

(3S,4R)-2,2-Dimethyl-3-oxy-4-[(S)-2-(oxymethyl)-5-oxypyrrolidin-1-yl]6-cyanochroman

Fig. 4.76 Structural formula of (–)-MJ-451 compound

132

4 The Significance of Chirality in Pharmacological and Toxicological... H

H2C

H

H2C (R)

(R)

N

N (S)

HO

(R)

HO H

(R)

H

(S)

H

H O

O H3 C

H3 C

N

N

Quinine Quinidine 8S,9R-(–)-Enantiomer 8R,9S-(+)-Enantiomer (6-Methoxyquinolin-4-yl)(5-vinylquinuclidin-2-yl)methanol

Fig. 4.77 Structural formulas of quinine and quinidine

ventricular arrhythmia caused by myocardial ischemia and reduces the size of infarct zone after reperfusion. Thus, the compound (–)-MJ-451 produces a cardioprotective effect without affecting the hemodynamic indices (Lee et al. 2000). Antiarrhythmic Drugs–Sodium Channel Blockers Quinine (Fig. 4.77) can serve as an example of a chiral compound that was one of the first in history to be administered in clinical practice. The (+)-enantiomer of quinine, quinidine (Fig. 4.77) was first used in medicine in 1918. Now it is classified as a class IA antiarrhythmic agent according to the Vaughan-Williams classification (Vaughan 1970; 1992). Quinine exerts complex and multiple pharmacological effects in humans and animals. It shows an antiarrhythmic action, slows down conduction, and reduces excitability and heart muscle automatism at the same time producing a weak atropinelike effect. However, as compared to its isomer quinidine, it is less efficacious as an antiarrhythmic agent but has more side effects. Therefore, at present quinine is completely replaced by quinidine as an antiarrhythmic agent. Disopyramide, a I class antiarrhythmic agent, shows significant binding to the plasma proteins, α1– glycoprotein in particular, even at low concentrations. In comparison with the R-(–)-enantiomer of disopyramide, its S-(+)-enantiomer (Fig. 4.78) produces a more prominent antiarrhythmic effect. However, their Mcholinoreceptors blocking effects are virtually the same. The S-enantiomer has a lower renal clearance, its elimination half-life is longer and its apparent volume of distribution is lower than that of the R-enantiomer. This effect is because of its more extensive and stereoselective binding to the plasma proteins (Mehvar et al. 2002; Triggle 1997). Tocainide, a class IB antiarrhythmic agent, also displays stereoselective properties. R-tocainide (Fig. 4.79) is more active as a sodium channel blocker than its S-isomer. The metabolism of tocainide is stereospecific. R-N-carboxytocainide

4.3 Drugs Regulating the Functions of Major Organs and Systems

133

H3C O

CH3

NH2 N

N

CH3

(S)

H3C

(S)-4-(Diisopropylamino)-2-phenyl-2-(pyridin-2-yl)butanamide

Fig. 4.78 Structural formula of S-(+)-disopyramide

CH3

NH2 H N

H (R)

CH3

O CH3

(R)-2-Amino-N-(2,6-dimethylphenyl)propanamide Fig. 4.79 Structural formula of R-tocainide

undergoes glucuronide conjugation more extensively than the S-form (Mehvar et al. 2002). Faster metabolism as well as stereoselectivity of glomerular filtration allows R-tocainide to be excreted faster than its S-enantiomer (Wiela-Hojeñska and Orzechowska-Juzwenko 1995). Propafenon, a class IC antiarrhythmic, is a chiral compound and in clinical practice it is administered in the form of a racemic mixture. Although both enantiomers are equally effective sodium channel blockers, the S-(+)-enantiomer (Fig. 4.80) displays a hundred times greater β-adrenoblocking effect (Antiarrhythmic Drugs 1989). It was demonstrated that the R-enantiomer is excreted faster than the S-enantiomer, resulting in a higher plasma concentration of the S-form upon administration of racemic propafenone. Besides, in vitro studies on microsomal human liver preparations established that enantiomer-enantiomer interactions of S- and R-propafenone were suppressed upon administration of R-propafenone (Chen and Cai 2003; Chen et al. 2000; Zhou et al. 2001a, b). These metabolic differences are only seen at sufficiently high concentration of drugs. They are probably associated with differences in the enzymatic catalysis and/or substrate affinity between the enantiomers (Mehvar and Brocks 2001; Zhou et al. 2003).

134

4 The Significance of Chirality in Pharmacological and Toxicological... O

O H CH3 N H

(S)

OH (S)-1-[2-[2-Oxy-3-(propylamino)propoxy]phenyl]-3-phenylpropan-1-one

Fig. 4.80 Structural formula of S-(+)-propafenone

Fig. 4.81 Structural formula of S-(–)-cibenzoline

H (S)

N

N H

(S)-2-(2,2-Diphenylcyclopropyl)-4,5-dihydro-1H-imidazole

Cibenzoline is a sodium channel blocker that blocks calcium channels as well. Its racemic mixture is used in clinical practice but research showed that the S-(–)enantiomer (Fig. 4.81) is about two times more effective than the R-(+)-enantiomer. Besides, the R-isomer is metabolized approximately 23 times faster, (Niwa et al. 2000). Cardiometabolic Drugs L-Carnitine L-Carnitine (levocarnitine, L-β-hydroxy-γ-N,N,N-trimethylaminobutyric acid, vitamin BT , Fig. 4.82) (Monograph 2005; Spasov and Iezhitsa 2005) is an example of stereopharmacological effectiveness. It takes part in the transport of long-chain fatty acids into the mitochondrial matrix, in the regulation of metabolism involving medium-chain acyl-CoA and branched chain acyl-CoA, as well as in conjugation reactions with xenobiotics (Bohmer and Bremer 1968; Kimura and Yamaguchi 2002; Monograph 2005; Rebouche 1992; Rebouche and Seim 1998). L-carnitine has been used as a component of therapeutic regimen for a wide range of conditions (Ames and Liu 2004; Czeczot et al. 2005; Day et al. 2004a; Famularo et al. 2004; Ferrari et al. 2004; Filipek et al. 2004; Hiatt 2004; Mingrone 2004; Ng et al. 2004; Stanley

4.3 Drugs Regulating the Functions of Major Organs and Systems

H

H3C H3 C

ON

+

CH3

(R)

O

OH

H

H3C H3 C

ON

+

(S)

CH3

CH2COOHO

*

H

CH2N+(CH3)3

135

OH

O

CH2COOH

*

OH

CH2N+(CH3)3

Fig. 4.82 Structural formulas of D- and L-stereoisomers of carnitine (the chiral center is shown with an *)

2004). Most clinical studies have included L-carnitine in the treatment of cardiovascular diseases (Ferrari et al. 2004; Filipek et al. 2004; Hiatt, 2004), for enhancing exercise tolerance (Brass 2004; Karlic and Lohninger 2004), for the treatment of Alzheimer’s disease, age-related senile dementia (Czeczot and Scibior et al. 2005; Monograph 2005; Rebouche 1992), renal diseases and patients undergoing hemodialysis (Calvani et al. 2004; Eknoyan et al. 2003; Monograph 2005; Rebouche 1992). L-carnitine is also indicated in so called mitochondrial diseases (Monograph 2005; Rebouche 1992). Like many other biomolecules, carnitine consists of an asymmetrical C-atom at β-position and therefore, can exist in the form of two isomers. L-carnitine (levo form) and D-carnitine (dextro form). The two isomers of carnitine have identical chemical composition but differ in spatial configurations where each one is a mirror image of the other. This difference results in vastly unequal activity of carnitine stereoisomers (Janiri and Tempesta 1983). Only L-carnitine is present in the body of humans and animals, and this is the form that is biologically active. In the scientific literature of the early 1970s, a potentially harmful effect of Dand DL-carnitine was discussed (Wolff et al. 1971; Rebouche 1977; Seim and Strack 1977; Paulson and Shug 1981). Moreover, when effective methods of obtaining Lcarnitine were developed, the harmful effect of D-carnitine in human was confirmed. Subsequently, the Federal Food and Drug Administration banned the circulation of D-carnitine and DL-carnitine in the USA in 1984. Pharmacological doses of L-carnitine are less effective than its small doses in a normal carnitine-balanced diet (Evans and Fornasini 2003). A high concentration of carnitine D-stereoisomer (in the form of admixture in medications or biologically active nutritional supplements) reduces the already low bioavailability of synthetic L-carnitine by competing for the same transport systems in the intestine thus causing L-carnitine deficiency (Rebouche 1983). This interaction was further confirmed by Duran et al. (Durán et al. 2002). Using isolated chicken enterocytes, they showed that the uptake of L-carnitine by enterocytes was inhibited by some structural analogs or

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4 The Significance of Chirality in Pharmacological and Toxicological...

derivatives of carnitine or its D-isomer. Moreover, these substances could be placed in the order of their inhibiting effect on the transport of L-carnitine in the intestine in the following way: D-carnitine = acetyl-L-carnitine = γ-butyrobetaine > palmitoylL-carnitine > betaine (Durán et al. 2002). These findings agree with the evidence from an earlier work by Gross and Henderson (Gross and Henderson 1984) that utilized labeled D- and L-carnitine. It was shown that the intestinal absorption of L-[14 C]carnitine and D-[3 H]carnitine was of saturable nature, and the high D-isomer concentrations blocked the transport of L-carnitine in the intestine. Distribution of L-carnitine in the body is described with the help of a twocompartment pharmacokinetic model (Rebouche 2004). The first compartment is represented by the liver, kidneys, and other tissues, which are highly vascularized. The other compartment consisted of relatively less vascularized muscular tissue (Rebouche 2004). Upon intake of L-carnitine with normal diet, the average time of its full turnover in the body amounts to 38–119 h (about 5 days). L-carnitine and its short-chain ether do not bind to the plasma proteins. A certain amount of Lcarnitine is also contained in the corpuscular elements of the blood, but the exchange of L-carnitine between erythrocytes and the plasma occurs relatively slowly (Evans and Fornasini 2003). The cardiac and skeletal muscles represent the main reservoir of L-carnitine in the body (Rebouche 2004). Transport proteins intended specifically for L-carnitine have been identified in the cardiac muscle, skeletal muscles, testes, liver, and kidneys (Tein 2003). Transport proteins promote the transfer of L-carnitine from the plasma to cells enabling the cells to accumulate L-carnitine at concentrations that are about ten times higher than in the plasma. Several studies have shown that D-carnitine, acetyl-L-carnitine and γ-butyrobetaine, compete with extracellular L-carnitine for the same transport systems (Chu and Hegsted 1976; Molstad 1980; Molstad et al. 1977; Mehvar et al. 2002). A reduced L-carnitine concentration in the myocardium and skeletal muscle was observed as early as on the fourth day following administration of D-carnitine (Paulson and Shug 1981). Moreover, administration of D-carnitine for 32 days in sexually immature juvenile rats was shown to cause a significant reduction of Lcarnitine levels in the serum and heart. The effect was less significant in skeletal muscles (Rebouche 1983). Later the works by Arancio et al. (1989) provided confirmation for the fact that D-carnitine caused a considerable reduction of L-carnitine content in skeletal muscle in both adult and immature juvenile rats. A study by Cooper et al. (1986) performed on isolated rat epididymis provided additional evidence to confirm that L- and D-carnitine compete for the same transport systems. These authors showed that the transport of L-[3 H] carnitine (50 mkM) through the epididymis epithelium was blocked by D-carnitine (500 mkM) as well as by palmitoyl L-carnitine and desoxycarnitine (Cooper et al. 1986). L-carnitine is excreted mainly by the kidneys (Rebouche 2004). L-Carnitine is excreted in urine either unmetabolized or as acetyl carnitine (Rebouche 2004). Experimental studies on mice (Gross and Henderson 1984; Seim and Strack 1977) have shown that acetyl carnitine comprises of as much as 40% of its total excretion in urine. D-Carnitine is either excreted unmetabolized or is transformed into toxic acetonyltrimethylammonium (Gross and Henderson 1984; Seim and Strack 1977). At normal

4.3 Drugs Regulating the Functions of Major Organs and Systems

137

concentrations renal reabsorption of L-carnitine can be as high as 90–99% (clearance of 1–3 ml/min) (Rebouche 2004). If the concentration of L-carnitine circulating in the blood decreases, the extent of renal reabsorption increases, and clearance reduces accordingly resulting in the restoration of L-carnitine concentration. Gross et al. (1986), Gross and Henderson (1984) showed that D-carnitine also competes with L-carnitine for renal tubular reabsorption. This finding was further supported by Stieger et al. (1995) who showed that the transport of L-carnitine in the vesicles of renal brush border in rats was blocked by both structural analogs or derivatives of L-carnitine (butyrobetaine, acetyl-L-carnitine, trimethyl-lysine), and its D-isomer. L- and D-carnitine not only interact at the level of gastrointestinal absorption, redistribution, transformation, and excretion from the body; there are also differences in their action at the cellular and subcellular level. L-carnitine is an essential nutrient as it plays a key role in the transport of fatty acids to mitochondria (Monograph 2005). Since acyl-CoA, another major fatty acid transporter, cannot penetrate the cellular membrane, L-carnitine deficiency can result in reduced intramitochondrial concentration of fatty acids leading to reduced energy production (Carter et al. 1995). Intracellular acyltransferase catalyzing the conjugation of an activated long-chain fatty acid with L-carnitine, and mitochondrial carnitine translocase catalyzing the transport of acyl-Lcarnitine in mitochondria, are stereospecific for L-carnitine and acyl-L-carnitine (Fritz and Schultz 1965; Pande 1975). This explains the fact that D-carnitine does not affect the intramitochondrial oxidation of long-chain fatty acids (Farinella et al. 1984). Besides, as described above, both isomers utilize the same transport mechanisms in the skeletal muscle fibers. Rebouche (1977, 600). This means that intracellular transport of D-carnitine results in L-carnitine loss. In its turn, intracellular L-carnitine deficiency together with inhibition of fatty acid β-oxidation in mitochondria, results in functional disturbance such as reduced myocardial contractility (Paulson and Shug 1981). In the presence of balanced carnitine homeostasis, the toxicity of D-carnitine per se is not much different from L-carnitine. Experiments on rats have demonstrated that LD50 for L-carnitine is 9.0 g/kg and for D-carnitine it is10.3 g/kg (Seim and Strack 1977; Wolff et al. 1971). Therefore, the total toxicity of both isomers is not high and is within the toxicity range of amino acids. The work by Kargas et al. (1985) on chicken embryos showed that L- and D-carnitine produced a congenital cardiac defects action at doses close to LD50 . Arancio et al. (1989) cite evidence in their work that D-carnitine causes diarrhea in rats (Rebouche 1983) and human (Chapoy et al. 1980; Waber et al. 1982). In clinical practice the toxic effect of D-carnitine was observed in patients with renal insufficiency receiving dialysis (Bazzato et al. 1979; Blum et al. 1971; De Grandis et al. 1980), in patients receiving adriomycin (doxorubicin cardiomyopathy) (Maccari and Ramacci 1981; Strohm et al. 1982) and in patients with ischemic myocardial lesion (Garzya and Amico 1980; Kosolcharoen et al. 1981; Shug et al. 1978; Thomsen et al. 1979; Whitmer et al. 1978). Bazzato et al. (1979) demonstrated that intravenous administration of DL-carnitine at a rate of 2 g three times a week, immediately following hemodialysis in patients

138

4 The Significance of Chirality in Pharmacological and Toxicological...

with progressive renal insufficiency, resulted in pronounced weakness of the muscles of mastication and deglutition. In all the observed patients the Tensilon test proved to be positive demonstrating myasthenic attenuation of action potential (Bazzato et al. 1979). Myasthenic symptoms were resolved after additional hemodialysis and elimination of DL-carnitine. A similar therapy with the L-isomer of carnitine did not produce myasthenic symptoms at all (Bazzato et al. 1979). According to many authors, this observation permits a conclusion that in patients with progressive renal insufficiency, D-carnitine is stored in the striated muscles and probably blocks neuromuscular conduction (Blum et al. 1971; De Grandis et al. 1980). The pharmacodynamic differences between the L- and D-forms of carnitine were demonstrated in vitro on isolated rat hearts with adriamycin cardiomyopathy (Maccari and Ramacci 1981; Strohm et al. 1982). In this study, the cardiotoxic effects of adriamycin were characterized by attenuated cardiac rhythm, diminished coronary blood flow, reduced strength of myocardial contraction, and arrhythmias (Maccari and Ramacci 1981). All these functional disturbances of the myocardium resolved upon administration of L-carnitine but were aggravated by administration of D-carnitine (Maccari and Ramacci 1981). In ischemic heart disease, activated long-chain fatty acids accumulate in the myocardium (Shug et al. 1978; Whitmer et al. 1978). The conjugates of fatty acids interfere with additional myocardial oxygenation and intracellular ATP production. As was shown in some studies, the delay in ATP synthesis as well as intracellular accumulation of long-chain fatty acids can be counteracted by L-carnitine (Shug and Shrago 1973; Suzuki et al. 1983). Liedtke et al. (1981) established in their study on isolated porcine heart that in hypoxic lesion of the myocardium, carnitine blocked the toxic effect of long-chain fatty acids. Moreover, improvement in various biochemical and physiological myocardial parameters by L-isomer of carnitine was significantly higher than that produced by DL-carnitine (Liedtke and Nellis 1979; Liedtke et al. 1981). The findings of these studies were later confirmed by various clinical studies involving patients with coronary artery disease (Garzya and Amico 1980; Kosolcharoen et al. 1981; Thomsen et al. 1979). In these studies the authors evaluated the effect of isosorbide nitrate, DL-carnitine and L-carnitine on the number of anginal episodes and nitroglycerine consumption in patients with ischemic heart disease. Patients of the first group received isosorbide nitrate during a seven-day therapy with nitroglycerine (Garzya and Amico 1980). The other two groups received DL-carnitine or L-carnitine in addition to isosorbide nitrate. As was expected, daily consumption of isosorbide nitrate reduced the number of anginal episodes and nitroglycerine consumption overall. Addition of L-carnitine prolonged the achieved symptomatic improvement. The racemic mixture of carnitine, on the contrary, increased the number of anginal episodes and nitroglycerin consumption in comparison with L-carnitine. Later Watanable et al. (1995) confirmed that Lcarnitine promoted a considerable increase in effort tolerance while DL-carnitine reduced effort tolerance in patients with angina. Pharmacological correction of carnitine deficiency with L-carnitine prevented the development of liver fatty dystrophy to a greater degree, than the administration of other carnitine stereoisomers and promoted the restoration of muscular

4.3 Drugs Regulating the Functions of Major Organs and Systems

139

fiber thickness of skeletal muscles. DL-carnitine administration was accompanied by a moderate correction of fatty dystrophy and did not prevent the development of skeletal muscle atrophy. D-carnitine stereoisomer did not prevent liver fatty dystrophy. Correction of carnitine deficiency with D- stereoisomer was not accompanied by essential morphological and morphometric differences in degree of skeletal muscle atrophy (Spasov et al. 2006b). Additionally, L-Carnitine, but not racemate and D-stereoisomer, improved exercise performance of rats in the forced swimming test (Spasov et al. 2006a). L-carnitine administration to carnitine-deficient rats led to normalization in myocardial function including indices of contractility and relaxation, systolic and diastolic blood pressure in response to volume loading test, adrenoreactivity test, and maximal isometric loading test. Chronic administration of D- and DL-carnitine did not change an altered cardio- and hemodynamics of carnitine-deficient rats compared with L-carnitine (Spasov et al. 2006c). Nephrology offers another field of application for L-carnitine (Calvani et al. 2004; Eknoyan et al. 2003). L-carnitine produced a nephroprotective effect in a model of cisplatin nephrotoxicity in carnitine-deficient rats while D-carnitine, on the contrary, potentiated the nephrotoxic action of cisplatin (Sayed-Ahmed et al. 2004). L-carnitine may also have benefits in sports and rehabilitation medicine (Brass 2004; Karlic and Lohninger 2004). It has been used as an anabolic remedy in anorexia following physical and nervous prostration, after disease or surgery. In an in situ model of physical exhaustion of isolated canine latissimus dorsi muscle, L-carnitine increased the force of contraction by 34% on an average, while D-carnitine produced no effect (Dubelaar et al. 1991). Magnesium L-Asparaginate The stereospecific differences in the physiological actions of different isomers of the same drug are often attributed to the specific mode of their delivery to the site of action. The specific structural and physiological properties of biological membranes may be the cause of stereospecific drug permeation. Moreover, membrane-associated transport systems may stereospecifically transport the isomers and their metabolites through the membranes (Lam 1988; Levy and Boddy 1991). For example, specific amino acid transporters can transport and accumulate L-aminoacids, intracellularly, to a concentration 500 times higher than that in the surrounding medium. These transporters do not transport D-amino acids similarly (Alekseev 1998). Clinical importance of selective enantiomer metabolism cannot be emphasized much unless there are differences in their effect and toxicity. If two enantiomers of a drug in a racemic mixture display the same effect, then it does not matter much which of them is metabolized faster. However, if the enantiomers differ in their efficacy or adverse effects, it is important to take into consideration the differences in the rate of their metabolism (Alekseev 1998). Magnesium aspartate serves as an example in this case (Fig. 4.83). It is well known that only the L-isomers of amino acids are involved in the biochemical processes in the human body. Since they are present endogenously,

140

4 The Significance of Chirality in Pharmacological and Toxicological...

Fig. 4.83 Structural formula of magnesium L-asparaginate

NH3+

O

O

(S)

-

O

NH3+ (S)

-

O H O

+2

Mg

O-

-

O H O

they serve as an acid forming residue and are widely distributed. In the body, Damino acids are metabolized into α-oxoacids by D-amino acid oxidase (DAO) or by D-asparaginic acid oxidase (D’Aniello et al. 1993). The complexes of L-aminoacids with magnesium are expected to possess a higher bioavailability than the magnesium complexes with D-stereoisomers. Therefore, using L-asparaginic acid rather than its racemate, as a magnesium ion chelator, the intracellular magnesium concentration can be increased. This approach can be helpful in the correction of intracellular magnesium deficiency and its pathological consequences. In our study it was shown that the complex of magnesium with the L-stereoisomer of asparaginic acid has a higher bioavailability, in comparison with the complexes of its DL- and D-forms (Iezhitsa 2008). In the calcium chloride-induced arrhythmia model, magnesium L-asparaginate (Spasov et al. 2006a) showed significantly higher efficacy as compared to DL- and D- magnesium asparaginate. It reliably prolonged the time before the onset of rhythm disturbance, reduced the rate of fibrillation episodes, increased survival rates and prolonged the life span of animals. Additionally, in the aconitine-induced arrhythmia model, magnesium L-asparaginate demonstrated a greater antiarrhythmic effect as compared to DL- and D-forms of magnesium asparaginate both in terms of the time of onset of the arrhythmic episode, rate of fibrillation development, the life span of animals, and the absolute value of ED50 (Spasov et al. 2006a). Potassium, Magnesium L-Aspartate (Asparkam-L) Widely used K- and Mg- aspartate preparations (asparkam, panangin, pamaton) are synthesized from aspartic acid representing a racemic mixture of L- and Dstereoisomers. Differences in metabolism and utilization of D- and L-amino acids influence the pharmacological properties of K and Mg L- and D-aspartates. Moreover, the pharmacologically effective doses of Mg and K salts can induce toxicity, which depends on the nature of anions. In a few studies pharmacological and toxicological activities of potassium, magnesium salts of two stereoisomers (D-, L-) and the racemate (DL-) of aspartate acid were compared to identify the most active isomer of aspartate acid (Fig. 4.84). It was shown that administration of potassium, magnesium L- aspartate promoted a faster correction of magnesium deficiency in dietary (Spasov et al. 2010) and druginduced hypomagnesaemia (Iezhitsa et al. 2004) as compared to its DL- and D-forms. Depending on the rate of hypomagnesaemia correction rate, potassium, magnesium salts were ranged in the following order: potassium, magnesium L-aspartate > potassium, magnesium DL-aspartate > potassium, magnesium D-aspartate.

4.3 Drugs Regulating the Functions of Major Organs and Systems NH3+ -

NH3+

O

(S)

O

-

O

K

141

O

NH3+

O

(S)

O

(S)

+

-

O

Mg

+2

-

H

H

H

O

O

O

O-

O

Fig. 4.84 Structural formula of potassium, magnesium L-aspartate (Asparkam-L)

In one of the study, antiarrhythmic effect of potassium, magnesium L-, D-, and DLaspartates was compared using calcium chloride and aconitine induced arrhythmia models in rats and strophanthin-K induced arrhythmia model in guinea pigs. It was found that intravenously administered potassium, magnesium L-aspartate exhibited higher antiarrhythmic effect compared to potassium, magnesium L-aspartate on the strophanthin-K, calcium chloride, and aconitine induced arrhythmia models. Potassium, magnesium L-aspartate more effectively decreased the incidence of arrhythmias, increased the time to onset of the first arrhythmia, and increased the survival rate of the animals after the first arrhythmia onset. At the same time potassium, magnesium L-aspartate demonstrated better acute toxicity profile than D- and DL- aspartate with respect to acute toxicity (LD50 ), effective dose (ED50 ) and antiarrhythmic (therapeutic) ratio (LD50 /ED50 ). (Spasov et al. 2007; Iezhitsa and Spasov 2008). Antisecretory Drugs Omeprazole is a racemic mixture of two optical isomers, S-(–)-omeprazole (esomeprazole, Fig. 4.85) and R-(+)-omeprazole. It was readily demonstrated in clinical conditions that esomeprazole was not subject to racemization and thus did not convert to another stereoisomer (Andersson et al. 2001). Furthermore, in the parietal cells of stomach both the R-and S-omeprazole are converted to the same

H N

O N

S

(S)

H3C O

N

CH3

H3C

O H3 C

(S)-5-Methoxy-2-[(4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl]-1H-benzo[d]imidazole Fig. 4.85 Structural formula of S-(–)-omeprazole (esomeprazole)

142

4 The Significance of Chirality in Pharmacological and Toxicological...

achiral sulfonamide which acts as an inhibitor of the proton pump (H+ /K+ -ATPase) (Lindberg et al. 1990). This means that pharmacokinetic index such as area under the curve (AUC) should correlate with the extent of the inhibition of gastric secretion for both isomers (Lind et al. 1983; Junghard et al. 2002). However, some authors report (Abelö et al. 2000) that the two isomers differ in their systemic effects and pharmacodynamic profile. These differences between the two isomers of omeprazole are attributed to the stereospecific differences in their metabolism. Omeprazole is mostly metabolized by cytochrome P450 CYP2C19 (Andersson 1996; Andersson et al. 1990; Andersson et al. 1993). The gene coding for this enzyme is polymorphic, so in some people the functionally active enzyme is not expressed. In this population, another isoform of omeprazole CYP, CYP3A4 plays the key role. CYP3A4 is usually of less significance in biotransformation of the drug in population with normal enzyme expression. CYP3A4 metabolizes omeprazole at a slow rate and therefore, this population, can be regarded as “poor metabolizers” (3% Caucasian and 15–20% Asians). In vitro studies have shown that hepatic CYP-enzymes metabolizing the drug display stereoselectivity for omeprazole isomers (Abelö et al. 2000). Although both forms of the drug produce the same metabolites, kinetics of metabolic transformation of its enantiomers differ leading to 300 times faster hepatic microsomal clearance of esomeprazole as compared to R-omeprazole. Numerous studies have shown that efficacy of esomeprazole in inhibiting the secretion of hydrochloric acid is greater than that of its isomer. Single dose administration of esomeprazole produces a higher AUC value as compared to the single dose administration of the same omeprazole dose. Besides, with multiple dose administration, the more pronounced nonlinearity of esomeprazole pharmacokinetics yields an almost twice the higher value of AUC for esomeprazole at doses of 15 and 20 mg (1.36 and 4.2 μmol × hr/L) in comparison with the AUC for omeprazole at the same dose (0.67 and 2.3 μmol × hr/L) (Lind et al. 2000; Andersson et al. 2000). The difference in the pharmacological characteristics of esomeprazole and omeprazole results in differences in their effect on hydrochloric acid secretion. The extent of inhibition of the acid secretion is estimated by the percentage of inhibition of pangastrin-stimulated secretion and the temporal interval of 24 h during which the pH value of gastric medium is higher than four. Esomeprazole at a dose of 15 mg was shown to reduce stimulated secretion by 91%, while omeprazole at the same dose reduces it only by 65% (Andersson et al. 2000). The period during which the pH value of gastric medium was higher than four, was 10.8 h for omeprazole at a dose of 20 mg and 12.8 h for omeprazole at a dose of 20 mg, on average (Lind et al. 2000). The results of experimental research that demonstrated considerable pharmacokinetic and pharmacodynamic differences between omeprazole and esomeprazole clearly correlate with the results of clinical research. Two comparative, randomized, multicenter, double-blind clinical studies involving 1960 and 2425 patients demonstrated a higher effectiveness of esomeprazole in 8-week therapy of erosive esophagitis in comparison with omeprazole. The proportion of cured patients in different treatment groups was as follows: in esomeprazole therapy group at a dose of 20 mg–89.9%, at a dose of 40 mg–94.1%; in omeprazole therapy at a dose of 20 mg–84.2%. After 4 weeks of treatment, diminution of symptoms was noted by

4.3 Drugs Regulating the Functions of Major Organs and Systems H3C

O H3C

N

143 O

CH3

CH3

N N

S

(S)

N H

O

(S)-5-Methoxy-2-[(4-methoxy-3,5-dimethylpyridin-2-yl)methylsulfinyl]-1H-imidazo[4,5-b]pyridine

Fig. 4.86 Structural formula of S-(–)-tenatoprazole

81.7% patients receiving esomeprazole at a dose of 40 mg, and by only 68.7% of patients receiving omeprazole at a dose of 20 mg (Kahrilas et al. 2000; Richter et al. 2001; Nexium® (esomeprazole magnesium) 2010). At present the new generation of H+ /K+ -ATPase inhibitors such as S-(–)tenatoprazole, are synthesized in the form of the levorotatory isomer, as a rule, as their apparent advantages are now well established (Fig. 4.86). Anticoagulants Warfarin is a racemic mixture of R- and S-stereoisomers. The S-enantiomer (Fig. 4.87) is 2–5 times more active than the R-isomer, however, its duration of action is shorter than R-isomer. Both isomers are metabolized by CYP2C9, although the S-isomer undergoes faster metabolism. Warfarin metabolism yields inactive and poorly active metabolites that are reabsorbed and excreted in bile. Only the racemate is used in clinical practice (Yamazaki and Shimada 1997; Brocks 2006). O

OH

CH3 (S)

H O

O

(S)-4-Oxy-3-(3-oxo-1-phenylbutyl)-2H-1-benzopyran-2-one

Fig. 4.87 Structural formula of S-warfarin

144

4 The Significance of Chirality in Pharmacological and Toxicological...

4.4 Drugs Affecting the Immunity Processes Antihistamine Drugs Histamine H 1 -Receptor Blockers Levocetirizine is an active R-enantiomer of cetirizine (Fig. 4.88). The S-enantiomer, dextrocetirizine is a distomer without antihistaminic properties (Day et al. 2004a). Levocetirizine is twice as effective as cetirizine and about thirty times more effective than dextrocetirizine in its ability to bind to H1 -receptors and the duration of this receptor interaction (Devalia et al. 2001). In several randomized, double blind, placebo-controlled studies, it was shown that dextrocetirizine lacks the antihistaminic effect. Administration of levocetirizine (2.5 mg) and cetirizine (5 mg) was shown to effectively block nasal (Wang et al. 2001) and cutaneous (Devalia et al. 2001) histamine challenge tests, but the effect of dextrocetirizine was equivalent to that of placebo. Levocetirizine does not undergo chiral inversion to form dextrocetirizine in the human body and is therefore stable (Baltes et al. 2001; Benedetti et al. 2001). Levocetirizine possesses a 600 times greater selectivity for H1 -histamine receptors than for other receptors and ion channels that are similar in structure (Gillard et al. 2002). Phylogenetically, M-cholinoreceptor is the closest to H1 -receptor in the family of G-protein-coupled receptors. The extent of M-cholinoreceptor binding determines the presence of undesirable effects for several antihistamine drugs (dry mouth, tachycardia). Studies using isolated animal organs and in vitro experiments (Gillard et al. 2003), have demonstrated that cetirizine, levocetirizine and fexofenadine (Fig. 4.90) neither interact with muscarinic receptors nor produce an anticholinergic effect (they do not block cholinergic effects of carbachol). Terfenadine, loratadine and desloratadine (Fig. 4.89) bind to all five subtypes of muscarinic receptors. The relative selectivity of these substances for H1 -receptor as compared to muscarinic receptors (“affinity for H1 -receptor / affinity for muscarinic receptors”) is as follows: cetirizine > 20 000;

O

H (R)

N

N O

OH

Cl

(R)-2-[2-[4-(4-Chlorophenyl)(phenyl)methyl)piperazin-1-yl]ethoxy]acetic acid

Fig. 4.88 Structural formula of levocetirizine

4.4 Drugs Affecting the Immunity Processes

145

OH Cl

Cl

N

*

N

N OH

N H

N H3C

I

H3 C

O

CH3

CH3

O

II

1-(4-Tert-butylphenyl)4-[4-(oxydiphenylmethyl)piperidin-1-yl]butan-1-ol

III

Ethyl-4-(8-chloro-5,6-dihydro11H-benzo[5,6]cyclohepta[1,2-b]pyridin-11-ylidene)1-piperidinecarboxylate

8-Chloro-6,11-dihydro-11(4-piperidinylidene)5H-benzo[5,6]cyclohepta[1,2-b]pyridine

Fig. 4.89 Structural formulas of terfenadine (I), loratadine (II), and desloratadine (III) (the chiral center is shown with an *)

OH

HO N

CH3

O

CH3

OH

* H

2-[4-[1-Oxy-4-[4-(oxydiphenylmethyl)piperidin-1-yl]butyl]phenyl]-2-methylpropanoic acid

Fig. 4.90 Structural formula of fexofenadine (* stands for the chiral center)

levocetirizine > 20 000; fexofenadine > 10 000; terfenadine 500–3 000; loratadine 100–500; desloratadine 50–125 (Gillard et al. 2003). In vivo, studies have demonstrated that levocetirizine has higher efficacy in preventing development of hyperemia, urticaria and pruritis following subcutaneous administration of a histaminic agents as compared to other antihistaminic drugs such as desloratadine, ebastine, fexofenadine, mizolastine, loratadine (Clough et al. 2001). Only levocetirizine administration provided a “complete” block of urticaria (in up to 95% of the patients). The urticaria was relieved in 70% patients for 21.4 h on average, upon a single administration of levocetirizine (Purohit et al. 2003). The antihistaminic effect of this drug was also studied in patients with allergic rhinitis. In a double blind, cross-over study, 39 patients allergic to house dust mite were given an allergen challenge test and subsequently were treated with 5 mg of levocetirizine or 10 mg of loratadine or placebo. Considerable relief of symptoms was noted in 83.8% of patients receiving cetirizine whereas loratadine provided relief in 66.7% patients (Horak and Stubner 2001). Another clinical study using controlled

146

4 The Significance of Chirality in Pharmacological and Toxicological...

allergen contact (meadow grass pollen) demonstrated that levocetirizine and fexofenadine required same time for the onset of effect i.e., one hour after administration and both drugs were considerably more effective than placebo. However, 24 h after administration, it was noted that cetirizine had a longer duration of action than fexofenadine (Horak et al. 2005). In another double-blind, placebo-controlled study, the rate of onset of action and duration of effect of levocetirizine were compared with those of desloratadine. Levocetirizine or desloratadine were administered at a dose of 5 mg a day to 373 patients allergic to ambrosia pollen. The study also included a placebo treated group. Observations were made during 2 days of controlled contact with ambrosia pollen. The mean time of onset of the effect was 3 h for desloratadine while for levocetirizine it was 1 h. Both agents were more effective than placebo and levocetirizine was more effective than desloratadine in relieving the symptoms of allergic rhinoconjunctivitis. Both drugs reliably reduced nasal congestion, and on the first day of administration this effect was more pronounced for levocetirizine than for desloratadine. Even 24 h after administration of the first dose, the manifestation of symptoms was significantly lower in the levocetirizine group, indicating its longer duration of its action as compared to loratadine (Day et al. 2004b, c). Like other old-generation antihistaminic agents, levocetirizine also displays some anti-inflammatory properties at therapeutic doses. In vitro studies revealed that levocetirizine inhibits several components of the inflammatory cascade such as chemotaxis and migration of eosinophils, release of soluble molecules like ICAM-1, and increased vascular permeability (Michel et al. 2001; Thomson et al. 2002). The extent to which the anti-inflammatory effect of antihistaminic agents is associated with their direct histamine receptor blocking effect, is a disputable issue (Marone 2005). The H1 -receptor on cellular membranes is present in two conformations, active and inactive ones and a balance exists between these two conformations. Even in the absence of histamine the receptor displays basal activity. Histamine binding with the receptor, “fixes” it in active conformation as a result the active conformation predominates over the inactive one. All sedative and non-sedative histamine blockers fix the receptor in the inactive conformational form, thus reducing the basal activity of the receptors irrespective of the quantity of histamine present in the intercellular space. Besides, basal and histamine-stimulated activity of H1 -receptors, also stimulates the activity of intracellular transcription factor for several anti-inflammatory cytokines and adhesion molecules (NF-kB). Thus, interaction of histamine with H1 receptors increases the activity of NF-kB by eight folds whereas the antihistaminic agents reduce NF-kB and block the response to histamine stimulation (Leurs et al. 2002). Another H1 -receptor blocker, fexofenadine, is (Fig. 4.90), an active terfenadine metabolite. It is a highly effective non-sedative antihistaminic antiallergic agent and is used in clinical practice for the treatment of seasonal allergic rhinitis and bronchitis. It is a racemic mixture of two enantiomers of equal activity but with different pharmacokinetic parameters. In plasma, the drug has a S:R enantiomer ratio of 1:2 (Robbins et al. 1998). Levorotatory cabastine (levocabastine, Fig. 4.91), a selective H1 -histamine receptor blocker, is a second-generation drug and is used topically in the treatment

4.4 Drugs Affecting the Immunity Processes

147

OH

H (s)

F

(S)

N

(R)

(R)

O

N (3S,4R)-1-[(1S,4R)-4-Cyano-4-(4-fluorophenyl)cyclohexyl]-3-methyl-4-phenylpiperidine-4-carboxylic acid Fig. 4.91 Structural formula of levocabastine

of allergic conjunctivitis. A study on guinea pigs showed that levocabastine was 4–90 times more effective than its antipode over the period of 24 h after its peroral administration (Leonov and Bielory 2007). Chlorpheniramine is an antihistamine agent of the first generation. It is manufactured in the form of a racemic mixture (Bui et al. 2000). In vitro studies using isolated guinea pig intestine showed that the R-(–)-isomer of D-chlorpheniramine, (Fig. 4.92) was about twice as active as the racemate. The activity of the L-isomer amounted to one hundredth of the activity of DL-chlorpheniramine. Pharmacokinetic studies have shown that S-(+)-isomer chlorpheniramine binds to plasma albumins and alpha-glycoproteins to a significantly greater extent than the R-(–)-isomer. However, no difference in clinical efficacy could be demonstrated between the chlorpheniramine enantiomers. Pyranenamine is a multifunctional antiallergic agent consisting of two chiral centers. Its mechanism of action can possibly be associated with stabilization of mast cell membrane. Using a model of cutaneous anaphylaxis in rats, it was established

N H CH3

(R)

N CH3 Cl (R)-N,N-Dimethyl-3-(4-chlorophenyl)-3-(pyridin-2-yl)-1-aminopropane

Fig. 4.92 Structural formula of R-(–)-chlorpheniramine

148

4 The Significance of Chirality in Pharmacological and Toxicological...

that the drug was almost 1000 times more active than cromolyn (Leonov and Bielory 2007). Its efficacy was also studied using a model of ocular anaphylaxis in rats and it was concluded that the S,S-stereoisomer (Fig. 4.93) suppressed the allergic response more effectively as compared to R,R-isomer. 5-Lipooxygenase Inhibitors and Leukotriene Receptor Antagonists Montelukast is a chiral antagonist of leukotriene D4 receptors. It is used in clinical practice in the form of the R-enantiomer and does not undergo chiral inversion to S-isomer in the body (Liu et al. 1997). (Fig. 4.94). There is no evidence in literature concerning the effect of S-montelukast on leukotriene receptors. Zileuton is an antiasthmatic agent that inhibits leukotriene production. The drug binds stereoselectively to plasma proteins. The R-(+)-enantiomer (Fig. 4.95) is 96% protein bound, while the S-(–)-isomer is only 88% protein bound (Machinist et al. O CH3 O

HN

OH

OH H

HN

H

O O

CH3

(S)

(S)

HN

O

OH

OH

OH

O

(2S,2'S)-N,N'-[5-[(E)-1-(5-Acetyl-4-oxy-2,6-dioxo-2H-pyran-3(6H)-ylidene)ethylamino]-1,3-phenylene]bis(2,3-dihydroxypropanamide) Fig. 4.93 Structural formula of S,S-E-pyranenamine

O

CH3

HO S

H3C

(R)

Cl

N H

(R,E)-2-[1-[[1-[3-[2-(7-Chloroquinolin-2-yl)vinyl]phenyl]-3-[2-(2-oxypropyl-2)phenyl]propylthio]methyl]cyclopropyl]acetic acid

Fig. 4.94 Structural formula of R-E-montelukast

OH

4.5 Drugs Used in Endocrine Disorders

149 O

Fig. 4.95 Structural formula of R-(+)-zileuton

H2N OH

N H

(R)

S

CH3

(R)-1-(1-(Benzo[b]thiophen-2-yl)ethyl)-1-oxyurea

1995). This difference in pharmacokinetics of two isomers can result in the pharmacodynamic differences, however, no such differences between zileuton isomers are reported in literature.

4.5 Drugs Used in Endocrine Disorders S-(–)-Thyroxin (L-isomer of thyroxin, Fig. 4.96) is a naturally occurring thyroid gland hormone. The dextrorotatory R-(+)-thyroxin displays no hormonal activity but decreases the cholesterol content in the blood. Steroid Hormones Steroid drugs include adrenal cortex hormones (glucocorticoids and mineralocorticoids) as well as steroids of vegetable origin (Fig. 4.97 and Table 4.10). According to the chemical structure and biological effects, steroidal hormones are divided into C21 -steroids with a pregnane structure (gestagens and corticoids), C19 -steroids with an androstane structure (androgens), and C18 -steroids with an estrane structure (estrogens). There is also a group of C27 -steroids with a structure of cholestane (ecdysones and phytoecdysones). C27 -steroids are seen in insects and plants. A specific characteristic of naturally occurring steroids is the presence of a conjugated 4-ene-3-one group (C21 - and C19 -steroids), aromatic 3-hydroxycycle (C18 -steroids) and a conjugated 7-ene-6-one group (C27 -steroids). The naturally occurring steroid hormones have multifaceted action resulting from their ability to Fig. 4.96 Structural formula of naturally occurring S-(–)-thyroxin

I

O H

HO

I (S)

OH

NH2 I

O I

(S)-2-Amino-3-[4-(4-oxy-3,5-diiodophenoxy)-3,5-diiodophenyl]propanoic acid

150

4 The Significance of Chirality in Pharmacological and Toxicological... 29 28 22

21

24

20

CH3

10 3

H (S)

(S)

(S)

H

H

I

7

5 4

(R)

8

B

H

(S)

15

14

2

A

(S)

D

C

9

27

16

13

19

CH3

17

11

O

H3C

23

18 12

1

26 25

O

6

Basic structure of steroids

Hestagens

OH

OH O

O O

CH3 O (S)

CH3

(S)

(R)

H

OH

HO

(R)

(R)

CH3

(S)

(S)

H

H

(S)

(S)

H

(R)

H

(S)

(S)

H

II

O

(S)

H

III

O

Glucocorticoids

Mineralocorticoids OH

CH3 (S)

CH3

(S)

H

OH CH3

H (S)

(S)

(S)

(S)

H

(R)

(R)

H O

H (S)

(S)

(R)

H

H

IV

H

V

HO

Androgens

Estrogens

Fig. 4.97 Chemical structure of steroid hormones: progesterone (I), cortisone (II), aldosterone (III), testosterone (IV), estradiol (V)

Steroid nomenclature

pregn-4-ene-3,20-dione

17,21-dioxy-pregn-4-ene-3,11,20-trione

11β,21-dioxy-pregn-4-ene-3,18,20-trione

17β-oxy-androst-4-ene-3-one

estra-1,3,5(10)-triene-3,17β-diol

Trivial

Progesterone

Cortisone

Aldosterone

Testosterone

Estradiol

Table 4.10 Systematic names of some steroid hormones IUPAC name

(8R,9S,13S,14S,17S)-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenantrene-3,17-diol

(8R,9S,10R,13S,14S,17S)-17-oxy-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenantrene-3(2H)-one

(8S,9S,10R,11S,13R,14S,17S)-11-oxy-17-(2-oxyacetyl)-10-methyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenantrene-13-carbaldehyde

(8S,9S,10R,13S,14S,17R)-17-oxy-17-(2-oxyacetyl)-10,13-dimethyl-7,8,9,10,12,13,14,15,16,17-decahydro-1H-cyclopenta[a]phenantrene-3,11(2H,6H)-dione

(8S,9S,10R,13S,14S,17S)-17-acetyl-10,13-dimethyl-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenantrene-3(2H)-one

4.5 Drugs Used in Endocrine Disorders 151

152

4 The Significance of Chirality in Pharmacological and Toxicological...

interact with several receptors of different specificities. This also explains the high incidence of side effects and limitations for the clinical use of naturally occurring steroid hormones and similar drugs. Attempts have been made to achieve a controlled chemical modification of steroids to overcome these limitations and provide for better pharmacodynamic profile. Due to their rigid chiral structure, steroids have been subjected to numerous stereochemical studies. As early as in the 1970s scientists, by modifying steroid hormones, were able to develop compounds that did not display the typical hormonal effects but produced an immunostimulant, cardiotonic, psychotropic and other effects (Akhrem et al. 1969, 1971). Synthetic analogs of steroid hormones were the source of discovery of several new drug classes such as anabolic steroids, oral contraceptives, general anesthetics, etc. Steroids display three types of isomerism: conformational, geometric, and optical. Pharmacological properties of steroid molecules are to a great extent determined by their shape and the stability of the rings in their structure. It is well known that for cyclohexane derivatives, the enthalpy of chair conformation is lower than the enthalpy of bathtub conformation by 5–6 kcal/mol (Eliel and Wilen 1994). As indicated by the enthalpy of two forms, it is more advantageous when as many as possible rings in the structure of steroids are in the chair conformation because this affects the biological activity. At the same time, the rigid nonplanar cyclic system of steroids allows the existence of geometric isomers. As a rule, cis- and trans- forms of steroids differ very much in their pharmacological effects (Seifulla et al. 1975). It was shown (Caillet and Pullman 1970) that for steroids the most energy-wise advantageous conformations are trans–trans-trans- like in androstane, and cis–trans-trans- like in testane (Fig. 4.98). All naturally occurring saturated sterols are in the trans– trans-trans- configuration, while the cis–trans-trans- variety is typical of biliary acids. The conformation and geometric isomerism of steroids determine the shape, stability, and reactivity of their molecules. As it is the strict complementarity between the steroid structure and its receptor that underlies the biological activity of steroid hormones, one can assume that stable conformations of steroids retain their shape upon interaction with a receptor which in turn adopts a corresponding conformation by altering its active surface. D

D

C

C

B

B A

A

I

II

trans-trans-trans-configuration

cis-trans-trans-configuration

Fig. 4.98 Main conformations of steroids: androstane (I) type and testane (II) type

4.6 Antibacterial Drugs

153

O CH3 OH CH3 O

N

CH3

(S)

H

N H

O

(S)-2-Ethoxy-4-[2-[3-methyl-1-[2-(piperidin-1-yl)phenyl]butylamino]-2-oxoethyl]benzoic acid Fig. 4.99 Structural formula of S-(+)-repaglinide

Optical isomerism of steroid compounds is associated with the presence of asymmetrical carbon atoms in their structure. Keeping in view the relative rigidity of cellular receptor structure, differences in the biological activity of isomers are expected. Accordingly, in a study by Seifulla et al. (1975) it was shown that only D-isomers of cortisone and aldosterone displayed biological activity while their Lisomers were inactive. It is believed (Bush 1962) that only the members of the optical D-isomer family of nonbenzoid steroid hormones can produce biological effects. Synthetic Antidiabetic Drugs Repaglinide is characterized by stereoselectivity in its pharmacological effect. Consequently, only the active S-(+)-stereoisomer is used in the treatment of type II diabetes (Fig. 4.99). The drug is a member of the family of carbamoyl-methyl-benzoic acid. Studies evaluating structure–activity relationship showed that the in vivo hypoglycemic effect of repaglinide was stereoselective, and the activity of the S-stereoisomer was more than a hundred times the activity of the R-(–)-stereoisomer (Mark and Grell 1997).

4.6 Antibacterial Drugs Several new generations of antibacterial agents were developed over the past century and the new discoveries are still ongoing. The most important characteristic of this class of drugs (except for antiseptics) is that they display a selective toxic effect on microorganisms (Albert 1979; Gale et al. 1972; Egorov 1986). The understanding of the mechanism of antibacterial action of these drugs has progressed tremendously. The antibacterial agents have been shown to act by disrupting the integrity of the bacterial cell wall or the cytoplasmic membrane, by suppressing protein synthesis

154

4 The Significance of Chirality in Pharmacological and Toxicological... HO H

O

O

N

O

O (S)

CH3

S

CH3

(R)

N H

(R)

H

H

(2S,5R,6R)-3,3-Dimethyl-7-oxo-6-(2-phenoxyacetamido)-4-thia1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Fig. 4.100 Structural formula of 2S,5R,6R-phenoxymethylpenicillin

after altered tertiary ribosomal structure, or by blocking nucleic acid synthesis (Gale et al. 1972). Some of the protein molecules targeted by antibacterial drugs display enzymatic activity and some do not. It was shown that interaction with these protein molecules is a stereoselective process (Hutt and O’Grady 1996) and determines the selective effect of antibiotics and synthetic antibacterial agents on microorganisms. The first study that evaluated the effect of chirality of phenoxymethylpenicillin, isolated from Penicillium mould, established that the 2S,5R,6R-isomer was the active form (Fig. 4.100) (Naylor 1973). Any chemical modification that altered its stereoform resulted in disappearance of the antimicrobial effect (Naylor 1973). Chloramphenicol isolated from Streptomyces venezuelae contains two chiral centers and so comprises of four stereoisomers (Fig. 4.101). Its antibacterial action is attributed to its ability to cause inhibition of bacterial protein synthesis. It turned out that only the D-(–)-treo-isomer (R,R-configuration) of chloramphenicol produces an antibacterial effect (Gale et al. 1972). At present this isomer can be synthesized (Albert 1979). The antibiotic latamoxef (moxalactam, Fig. 4.102) belongs to the family of cephalosporins and consists of two and more diastereoisomers. The drug used clinically is a mixture of two epimers each of which is levorotatory and is designated as R-(–)- and S-(–)-latamoxef (Wise et al. 1981). Cl

OH (R)

H

H N Cl

(R)

H O

O N+

OH

O2,2-Dichloro-N-[(1R,2R)-1,3-dioxy-1-(4-nitrophenyl)propyl-2]acetamide

Fig. 4.101 Structural formula of R,R-laevomycetin

4.6 Antibacterial Drugs

155 H3C

O H

H

O

O HO

N H

(R)

(R)

(R)

CH3 S

N HO

O

N

O

N N HO

N

O

H 3C

O H

H

O

O HO

N H

(S)

(R)

(R)

CH3 S

N HO

O

N

O

N N HO

N

O

(6R,7R)-7-[(R/S)-2-Carboxy-2-(4-oxyphenyl)acetamido]-7-methoxy-3-[(1-methyl-1H-tetrazol-5-ylthio)methyl]-8-oxo-5-oxa-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid

Fig. 4.102 Structural formulas of the R-(–)- and S-(–)-epimers of latamoxef (arrows point to the epimerization center)

R-S nomenclature is commonly used for the purpose of designating the spatial configuration of naturally occurring derivatives of 6-aminopenicillanic acid and of 7-aminocephalosporic acid (Fig. 4.103). O

O H (R)

R

N H

H S

(R)

7

O

R1

2

5

N

(R)

CH3

1

6

H

CH3

3 (S)

4

N H

7 8

O

H S

(R)

1

2

6

N

3

5 4

R2

O

H OH

I (2S,5R,6R)-6-Acylamino-3,3-dimethyl7-oxo-4-thia-1-azabicyclo[3.2.0]heptane2-carboxylic acid

II

HO

O

(6R,7R)-7-Acylamino-3-methyl-8-oxo-5thia-1-azabicyclo[4.2.0]oct2-ene-2-carboxylic acid

Fig. 4.103 Structural formulas of derivatives of 3S,5R,6R-6-acylamino-penicillanic acid (I) and of 6R,7R-7-acylamino-cephalosporanic acid (II)

156

4 The Significance of Chirality in Pharmacological and Toxicological...

For some drugs, addition of a lateral substituent to their basic structure leads to appearance of an additional chiral center. The stereoconfiguration of these compounds such as ampicillin (Fig. 4.104) and cephalexin (Fig. 4.105) are often characterized according to the D-L-system. Thus, in the British Pharmacopoeia 2005, ampicillin is defined as 6R-6-(αD-phenylglycylamino)-penicillanic acid (Fig. 4.104), and cephalexin—as 7-α-Dphenylglycylamino-3-methyl-3-cephem-4-carboxylic acid (Fig. 4.105). In these names the stereocondition of the lateral substituent is designated according to the D-L-system. The stereochemistry of the ring system is only stated for ampicillin and for only one of the three chiral centers. Two possible diastereoisomers for each one of these two compounds have been described in literature and named according to D-L-nomenclature (Tamai et al. 1988). Many naturally occurring or semisynthetic antibacterial agents are often used in the form of single stereoisomers, but occasionally their racemates or mixtures

O H

H

H (R)

N H

(R)

CH3 S

(R)

NH2

CH3 (S)

N O

O

H OH

(2S,5R,6R)-6-[(R)-2-Amino-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]pentane-2-carboxylic acid Fig. 4.104 Structural formula of the D-epimer of ampicillin

O H (R)

H

H (R)

N H

NH2

S

(R)

N CH3

O HO

O

(6R,7R)-7-[(R)-2-Amino-2-phenylacetamido]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid Fig. 4.105 Structural formula of the D-epimer of cephalexin

4.6 Antibacterial Drugs

157

of diastereoisomers are also used. The stereochemical properties of drugs not only affect the selectivity of their antibacterial action but can also affect some of their pharmacokinetic parameters such as binding to transport proteins in the intestinal wall, interaction with hepatic microsomal enzymes and cotransporters in renal tubules. These aspects can be considered in more detail in the example of β-lactam antibiotics and quinolone derivatives. β-Lactam Antibiotics The presence of the ‘frames’ of 6-aminopenicillanic acid (6-APA) and of 7aminocephalosporanic acid (7-ACA) in the native configuration (Fig. 4.103) of the structure of a compound is considered a prerequisite for the antibacterial properties in the β-lactam group. For example, a change in the stereocondition of any chiral atom in 6-APA results in partial or complete loss of the antibacterial effect (Naylor 1973). Introduction of an α-substituent causes appearance of an additional chiral center in the side acyloamine chain, which results in formation of two epimer diastereoisomers. In case of ampicillin, both epimers possess antibacterial properties, but the D-epimer (R-ampicillin, Fig. 4.104) is more active. The D/L eudysmic index, therefore, varies from two to five, depending on the microorganism (Naylor 1973). Introduction of a carboxyl group to α-position produces carbenicillin (Fig. 4.106). It was shown that single epimers of this compound display a identical effect. Furthermore, it was observed that the two epimers were chemically unstable and undergo fast epimerization in solution (Naylor 1973; Hoover and Dunn 1979), thus, making it impossible to obtain pure carbenicillin epimers. Absorption of antibiotics can be stereoselective as for some drugs it is mediated by the intestinal dipeptide transporters. The alteration in cephalexin epimer absorption due to α-substituent configuration in the side branch (Fig. 4.105) was studied in rats (Tamai et al. 1988). After oral administration of L-cephalexin (the S-isomer), the unaltered drug was undetectable in the serum or urine. On the contrary, D-cephalexin (the R-isomer) turned out to be well absorbed. In vitro experiments showed that both epimers were substrates for one of the transporters in dipeptide transport system.

O H

H (R)

N H

*

H

CH3 S

(R)

CH3 (S)

N HO

O

O

O

H OH

(2S,5R,6R)-6-[(R/S)-2-Carboxy-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptan-2-carboxylic acid

Fig. 4.106 Structural formula of carbenicillin (the chiral center at which epimerization proceeds is shown with an *)

158

4 The Significance of Chirality in Pharmacological and Toxicological...

L- Cephalexin had a higher affinity for this transporter and served as a competitive inhibitor of the D-epimer binding. L-cephalexin also turned out to be more susceptible to the action of hydrolytic enzymes present in tissues. Latamoxef (moxalactam) is a mixture of two epimer forms (Fig. 4.102) that are conventionally designated as R- and S-forms (Yamada et al. 1981). The antimicrobial activity of the R-epimer is about two times higher than that of the S-epimer. The relative activity of the two epimers also depends on the strain that is being used in the bacterial test system (Wise et al. 1981). Both isomers are stereochemically unstable and undergo epimerization yielding an equilibrium mixture in which the R:S ratio of epimers is 50:50 (in the buffer solution) or 45:55 (in the serum). The rate of epimerization of R- and S-isomers may differ depending upon the conditions. In the serum, however, at a temperature of 37 °C the period during which 50% of the stereoisomer undergoes epimerization is the same for both forms amounting to an hour and a half (Wise et al. 1981). Following intravenous administration of latamoxef epimer mixture to humans, its serum concentration was shown to decrease by half in 2.3 h. However, the serum concentration of the less active S-epimer was about two times higher than that of the R-epimer at the end of 4 h, and the R:S ratio for renal clearance was 1.5 (Lüthy et al. 1981). Apart from easy epimerization, the pharmacokinetics of latamoxef is further complicated by stereoselective binding to the plasma proteins. The proportion of unbound fractions of the R- and S-latamoxef amounts to 0.47 and 0.33. Accordingly, the renal clearance values for unbound fractions amount to 140 and 132 ml/min/m2 for the R- and S-epimer respectively (Yamada et al. 1981). Apparently, the proportion of different latamoxef forms in the plasma is determined by both the epimerization and stereoselective binding to the plasma proteins and this results in preferential renal clearance of the R-isomer (Yamada et al. 1981). Interestingly, Luthy et al. (1981) studied the pharmacokinetics of latamoxef and determined its content in the serum both by stereospecific high performance liquid chromatography and bioassays. The concentrations obtained by the bioassay method were always lower than those obtained by HPLC, and the difference in results grew progressively as the samples taken beyond two hours after drug administration were analyzed. It can be proposed that this difference reflects the faster excretion of the more active epimer from plasma. The findings of this study also throw light on the potential problems associated with the use of bioassay for stereoisomer mixtures analysis (Hutt 1990). Stereoselectivity in the antibacterial action is also typical of relatively new subgroups of β-lactam antibiotics, carbapenems and penems (Figs. 4.107 and 4.108). Thienamycin was the first carbapenem derivative to be isolated and described. It is a highly effective antibiotic agent with a wide antibacterial spectrum (Kahan et al. 1983). Naturally occurring thienamycin is a 5R,6S,8R-isomer (Fig. 4.107). In contrast to classic β-lactam antibiotics, the β-lactam ring in this case is in transconfiguration as two hydrogen atoms at positions 5 and 6 are placed on different sides of the ring plane (Albers-Schoenberg et al. 1978). This fact is of significant importance as it proves that the cis- configuration of the beta-lactam ring in penicillins

4.6 Antibacterial Drugs

159

H

H

H

8

H3 C

(R)

OH

NH2

OH H

(R) (S) 6 7

N

3

4

(R)

H3 C

2

5

H

8

S

1

(R)

2

5

7

O

S

1

(S) 6

O

NH

HN H

N

3

4

O

O OH

OH

Thienamycin (5R,6S)-3-(2-Aminoethylthio)-6-[(R)1-oxyethyl]-7-oxo1-azabicyclo[3.2.0]hept-2-ene2-carboxylic acid

Imipenem (5R,6S)-3-(2-Formimidamidoethylthio)6-[(R)-1-oxyethyl]-7-oxo1-azabicyclo[3.2.0]hept-2-ene2-carboxylic acid

Fig. 4.107 Structural formulas of β-lactam antibiotics of carbapenem subgroup

H

H S

(R) 6 7

1

5

N

4

S

(R) 2

6

3

7

O

O

CH3

1

5

N

4

2 3

O

O OH

5R-Penem-3-carboxylic acid (R)-7-Oxo-4-thia1-azabicyclo[3.2.0]hept-2-ene2-carboxylic acid

OH

5R-2-Methylpenem-3-carboxylic acid (R)-3-Methyl-7-oxo-4-thia1-azabicyclo[3.2.0]hept-2-ene2-carboxylic acid

Fig. 4.108 Structural formulas of β-lactam antibiotics of penem subgroup

and cephalosporins is not the only characteristic imparting antibacterial properties. Thienamycin was also found to be resistant to the effect of beta-lactamase and this property of the drug could be attributed to the trans- configuration of its beta-lactam ring. It is known, however, that thienamycin is unstable in solid form, and in a solution, it undergoes dimerization forming an inactive product (Birnbaum et al. 1985). Modification of thioethylamine side chain of thienamycin yielded imipenem, (Fig. 4.107) which preserved the wide antibacterial spectrum and beta-lactamase resistance but had the advantage of a greater chemical stability (Kahan et al. 1983; Barza 1985). After thienamycin was discovered, a number of similar compounds were isolated. They differed from thienamycin in the stereochemistry of the beta-lactam ring and/or α-hydroxyethyl lateral substituent. Substances in which the side chain configuration is opposite to the configuration of thienamycin (S-instead of R-), are referred to as epithienamycines (Birnbaum et al. 1985). Most of these substances are also antibiotics with a wide antibacterial spectrum. However, a change in the stereochemical

160

4 The Significance of Chirality in Pharmacological and Toxicological...

position of both the lateral substituent and the beta-lactam ring results in reduced antibacterial efficacy and increased penicillinase sensitivity (Kahan et al. 1983; Barza 1985). Thienamycin and imipenem have shown acceptable plasma pharmacokinetics but a low renal excretion in animals and human. Both the drugs are metabolized by dihydropeptidase-I (DHP-I) localized on the brush border of microvilli in proximal renal tubules (Kim and Campbell 1982; Parsons et al. 1991). Penems are a group of synthetic beta-lactam antibiotics having a structure that combines the characteristics of chemical structure of penicillins and cephalosporins (Fig. 4.108). Both the enantiomers and racemic penem-3-carboxylic acid have been synthesized and tested for biological activity. The 5R-enantiomer turned out to be two to four times more active as an antibacterial agent than the racemate, and the 5S-enantiomer was inactive (Pfaendle et al. 1979). Likewise, the 5R-enanitomer of 2-methylpenem3-carboxylic acid was twice as active as the racemate (Ernest et al. 1979). Thus the R-configuration of the carbon atom C5 common for two condensed rings apparently provides an important stereochemical condition for antibacterial activity of this group of compounds. Several penem compounds have been synthesized so far and many of them contain a substituent at position 6 of the two-ring structure. This substitution results in an additional chiral center and provides a possibility of cis–trans-isomerism of the beta-lactam ring (McCombie and Ganguly 1988). Besides, a number of penems have a chiral α-hydroxyethyl substituent at position 6, like carbapenems (Zak et al. 1988). Consequently, two series of penem derivatives are possible with stereochemical properties like thienamycin and epithienamycin. Stereochemical properties as indicators of the presence of antimicrobial activity are similar in both series. Sensitivity of penem derivatives to DHP-I and beta-lactamase is the same as in the series of carbapenem antibiotics. The derivatives of both penems and carbapenems are resistant to microbial beta-lactamase and sensitive to mammalian dihydropeptidase-I. Quinolone Derivatives As it was pointed out above, the problem of chirality is of greater importance for synthetic substances than for naturally occurring or semisynthetic ones. Quinolone derivatives are synthetic antibacterial agents and are one of the classes for which the effect of stereochemistry on antibacterial properties has widely been researched (Fig. 4.109). The conjugate system of 4-oxopyridin-carboxylic acid is considered the most important structural fragment in quinolone derivatives (Shen 1994; Chu et al. 1989; Mitscher et al. 1989). Although antibacterial activity has also been observed when the 3-carboxyl group was replaced by other substituents. A ring condensed on that side can be carbo- or heteroaromatic and contain substituents at positions 6 and 7. Chiral substituents in most compounds of this series are at positions 1 and 7.

4.6 Antibacterial Drugs

161 O 4

5 7 8

1

OH

OH

3

6

2

N

N

R2

O

O

O

R1

R1

I

R2

4-Oxo-1,4-dihydroquinolin3-carboxylic acid

2-Oxo-6,7-dihydro-2H-pyrido[2,1-a]isoquinolin-3-carboxylic acid

Fig. 4.109 Structural formulas of antibacterial derivatives of quinolone

Quinolone derivatives exhibit greater antibacterial activity upon introduction of fluorine atoms at positions 6, 8 and to the R1 substituents of structure I (Fig. 4.109). Common formulas of other fluoroquinolones are shown in Fig. 4.110. One of these fluorinated compounds, ofloxacin is commonly used in clinical practice (Fig. 4.111). Due to its pharmacokinetic properties, it has considerable advantages over other antibiotic agents. Stereoselectivity of antimicrobial action of ofloxacin enantiomers was established in vitro long ago (Hayakawa and Atarashi 1991). With regards to the sensitivity for Gram positive and Gram-negative bacteria, the S-enantiomer is 8 to 128 times more active than the R-enantiomer (Hayakawa and Atarashi 1991; Atarashi et al. 1987). The target enzyme of quinolone derivatives is DNA gyrase (Sato et al. 1993). Quinolones demonstrate a clear correlation between 50% inhibitory concentration and the extent of inhibition of DNA gyrase. Inhibition of DNA gyrase is stereoselective. In case of ofloxacin, the IC50 of S-(–)-enantiomer (levofloxacin) is 9/3 times O

O

O

F

O

F OH

R3

OH

N

N

N R1

N

R1

O

H3 C R2

9-Fluoro-1-oxo-1,5,6,7tetrahydropyrido[3,2,1-ij]quinolin-2-carboxylic acid

R2

9-Fluoro-10-(4-methylpiperazin-1-yl)-7-oxo3,7-dihydro-2H-[1,4]oxazino-[2,3,4-ij]-quinolin6-carboxylic acid

Fig. 4.110 Structural formulas of some antibacterial fluoroquinolones

162

4 The Significance of Chirality in Pharmacological and Toxicological... O

O

O

O

F

F OH

N N

OH

N O

N

N CH3

3

N

(S)

H3C

H3 C H

O

H

3 (R)

CH3

9-Fluoro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-3,7-dihydro2H-[1,4]oxazino[2,3,4-ij]quinolin-6-carboxylic acid

Fig. 4.111 Structural formulas of ofloxacin enantiomers

more than the R-(+)-enantiomer and it is 1.3 times more effective than the racemate (Hutt and O’Grady 1996). There is a similarity between DNA gyrase and topoisomerase II of mammalians, so the ability of quinolones to inhibit topoisomerase II and thus to affect mammalian cells attracted the interest of pharmacologists (Zschiesche et al. 2002). For inhibition of topoisomerase II in calf embryo thymus, the S-isomer showed higher activity than the R-isomer and this was also true for bacterial DNA gyrase. However, the S:R enantiomer ratios in terms of effectiveness in inhibiting DNA gyrase and topoisomerase II were 12.4 and 1.8 respectively. Therefore, (S)-(–)-ofloxacin in its relative inhibiting effect “DNA gyrase / topoisomerase II” is about 6.7 times more selective than its R-enantiomer. It is also of interest to note that the achiral ofloxacin analog (3-CH3 -group is missing in the structure shown in Fig. 4.111) is a much less active substance. Thus, the presence and orientation of a methyl group at the chiral carbon atom C3 determines both the extent of activity and the selectivity of the compound (Hoshino and Hayakawa 1991; Sato et al. 1993). The pharmacokinetics of ofloxacin enantiomers were studied following its administration in the form of single enantiomers and as racemic and nonracemic mixtures in rats, dogs, and monkeys (Okazaki et al. 1989, 1991 1992). After single enantiomers administration of the drug to rats, the concentration of R-ofloxacin in the serum was much higher, the AUC was greater, and the apparent elimination half-life was longer as compared to that achieved with the S-enanitomer. These pharmacokinetic differences arise as a result of a combination of three factors: stereoselective conjugation of S-ofloxacin with glucuronic acid, preferential excretion of S-ofloxacin and its glucuronide with bile and excretion of R-ofloxacin with urine. In vitro studies of hepatic microsomal preparations of rats demonstrated a relatively small difference in the rate of glucuronide formation for both enantiomers (the Km constant amounting to 1.43 and 1.14 mM for R- and S-ofloxacin, correspondingly), but the difference in Vmax values was 6.5 times, and the S:R ratio for Vmax /Km (the index of renal clearance) was 8.1. Later, it was established that the R-enantiomer was a competitive inhibitor of S-ofloxacin glucuronidation with an inhibition constant Ki of 2.92 mM. As a result of competitive interaction of enantiomers during metabolism, the concentration of S-ofloxacin in the serum increased after administration of racemic drug

4.7 Antitumor Drugs

163

(in comparison with administration of an equivalent dose of the pure S-enantiomer) resulting in 1.7 times increase in the area under the concentration time curve. Upon peroral administration of racemic ofloxacin to healthy volunteers the plasma concentration versus time curves for single enantiomers were similar in their shape with the curves for the total concentration of the drug (Okazaki et al. 1991). A small but statistically significant difference between enantiomers was noted in the area under the concentration time curve (S > R), the mean residence time (S > R), total and renal clearance (R > S). However, no difference was noted between two enantiomers with regards to plasma protein binding and distribution volume. Since ofloxacin undergoes minimal metabolism in the body, the difference in pharmacokinetic profiles of two enantiomers can be explained by the stereoselectivity of renal clearance (Okazaki et al. 1991). At present the S-form of ofloxacin referred to as levofloxacin is used in clinical practice.

4.7 Antitumor Drugs Oxaliplatin is a chemical analog of cisplatin but it is different in its spectrum of action (Fig. 4.112). Oxaliplatin exists in the form of trans-isomer and consists of a central atom of platinum surrounded by two ligands. One of flat oxalate-anion and the other one is 1,2-diaminocyclohexane with R,R-configuration (Misset et al. 1991, 1997). Like many other platinum derivatives, oxaliplatin blocks DNA synthesis by interacting with its intra- and interstrand cross links. Formation of bonds between oxaliplatin and the DNA is rapid taking about 15 min, whereas in case of cisplatin this process is diphasic and includes a slow stage of four to eight hours duration. Disruption of DNA synthesis results in inhibition of RNA transcription and suppression of cellular protein synthesis. Oxaliplatin is effective for some tumor lines resistant to cisplatin and carboplatin. Melphalan. a derivative of chloroethylamine, is an alkylating agent that inhibits cell growth (Fig. 4.113). The drug is a levorotatory isomer of sarcolysin. The process of alkylation consists of the formation of covalent bonds between two DNA helices at the expense of two chloroethyl groups and nitrogen guanine atoms (at position 7), which results in disturbance of cell replication. Melphalan blocks normal mitosis in rapidly proliferating tissues. The extent of the cytotoxic effect is related to the administered dose. The difference in the pharmacological activity of melphalan Fig. 4.112 Structural formula of oxaliplatin

O

O-

H2 N Pt++

O

O-

N H2

H (R) (R)

H

Oxalato[(1R,2R)-1,2-cyclohexadiamine]platinum (II)

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4 The Significance of Chirality in Pharmacological and Toxicological...

Cl

H

N

O

(S)

Cl

H2N

OH

(S)-2-Amino-3-[4-[bis(2-chloroethyl)amino]phenyl]propanoic acid Fig. 4.113 Structural formula of melphalan

and sarcolysin is because of the differences in their ability to penetrate the cell membrane. The differences between the two isomers in their ability to penetrate the cell membrane can be attributed to stereoselective functions of membrane transporter molecules. The transport of melphalan across the cell membrane has been shown to involve L-amino acid transporters.

4.8 Pros and Cons of Administering Drugs in the Form of Racemic Mixtures or Single Stereoisomers in Clinical Practice Although, the drugs in pure enantiomer forms are preferred over racemic drugs, most chiral drugs have been developed as racemates. Modern technologies of asymmetrical synthesis and chiral separation have made it possible to develop drugs in their pure enantiomer forms. A substantial number of such drugs have been developed in recent years and are approved for use in clinical practice. This trend is becoming widespread in the sphere of chiral drug development and their utilization. The utilization of pure optical isomers has increased from 27% in the year 1996 to 39% in 2002. The pace of this trend continues to grow (Rouhi 2003). It should be considered that working with a mixture of optical isomers means working with a complex mixture of chemical compounds in which each component might have different pharmacological properties and technological tools are required to separately analyze the properties of each component. Since optical isomers possess different biological activity, racemic mixtures can be considered as a combination of drugs. The more the number of chiral centers in the structure of the compound, more complex is the pharmacological and analytical situation. The main advantage of performing a study on a single optical isomer is that there is no need to take the distomer effects into consideration, which has its own pharmacokinetics and pharmacodynamics. Besides, a distomer can affect the pharmacokinetic and/or pharmacodynamic profiles of the eutomer. Pharmacokinetic difficulties can be settled with the help of stereospecific analytical methods that allow assessment of the content of single isomers in physiological media. Pharmacodynamic profile can be easily evaluated if only one isomer is active, which is not usually the case. Hence,

4.8 Pros and Cons of Administering Drugs in the Form of Racemic Mixtures … Fig. 4.114 Structural formula of razoxane (the chiral center is shown with an *)

165

O O N

H HN

N

NH

* CH3

O

O

4,4'-(Propan-1,2-diyl)dipiperazine-2,6-dione

working with an individual isomer only, makes it possible to clearly determine the pharmacokinetic versus pharmacodynamic profiles. Moreover, eutomer may have better efficacy and safety profile as compared to racemate. The investigations into the above-mentioned problems are important for addressing the issues of drug safety and effectiveness. The properties of pure enantiomers can be used for developing most suitable drug formulations and razoxane, a cytostatic drug, serves a good example in this regard (Repta et al. 1976). Pure enantiomers of razoxane are more soluble in water than the racemate and, therefore, are suitable for formulating the drug for intravenous injections. Development of a drug based on a single isomer can be more advantageous in terms of cost-effectiveness, besides being more useful therapeutically due to higher efficacy and safety. Due to these reasons, currently, numerous governmental agencies are often unwilling to consider registration of racemates. Moreover, production of a drug in the form of a pure stereoisomer prevents development of analogous products and/or generics as development of an off patent alternative new method of asymmetrical synthesis would be an extremely challenging task (Fig. 4.114). Although there are several reasons for the preferred use of enantiomers as discussed above, racemates are preferred choice in many cases, for example, in case of no pharmacokinetic and pharmacodynamic differences between isomers, which is rarely the case. Unless the enantiomers display the same activity, the chiral center of a molecule can only be placed in the part which is not responsible for interaction with the biotarget. In this case administration of achiral compounds should be regarded as a more optimal solution. Secondly, the racemates are used when the effect is stereospecific, and the useful action is 100% associated with the eutomer while the distomer displays no (side) effects. If such is the case, administration of a racemate would be acceptable. The conclusion that the distomer has no undesirable effects is made on the basis of relevant pharmacological studies. Generally, a distomer usually displays some pharmacological activity of its own and, frequently, its side effects are only manifest after a prolonged clinical use. On the other hand, even if a distomer is pharmacologically inactive, it is likely to “burden” the metabolic and excretory pathways. This by itself, especially upon a prolonged administration, can lead to unpredictable and undesirable effects.

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4 The Significance of Chirality in Pharmacological and Toxicological...

Sometimes there is pharmacological interaction between enantiomers. The total effect of these interactions, whether positive or negative in relation to the desirable effects, tips the balance either in favor of the enantiomer mixture or the pure isomer. In case, the enantiomers provide synergistic effect, the optimum therapeutic effect can be achieved by using a nonracemic mixture of isomers in optimal non-equimolar concentration. Administration of a racemic mixture, in this case may not produce the synergistic effect as the concentration of two isomers in racemate may not be optimal. It should be borne in mind that the attempt to enhance the therapeutic effect of one drug (one isomer) by administering simultaneously another drug (the other isomer) should be done very accurately considering the patient’s individual characteristics. If inversion of an isomer occurs in the process of drug manufacturing, the same principles of standardization are applicable as used for determining the drug’s stability during storage period. The contents of the active optical isomer should be within the range of 95–100% of the stated value, and the amount of inactive isomer should not exceed the prescribed limit. Inversion in chiral drugs is seldom noted in the solid phase, but in an aqueous medium inversion can occur very quickly. For example, hyoscyamine that was isolated as a pure optical isomer in the beginning of the twentieth century, in aqueous solution rapidly transforms to the racemate, atropine. Consequently, in case of cholinesterase inhibitor poisoning it is appropriate to use atropine in the form of a racemic mixture. When a drug undergoes inversion in the body, the resulting isomer should be regarded as a metabolite. If a eutomer is formed in the body by distomer inversion, the distomer should be regarded as a prodrug. Drugs undergoing isomer inversion in the body require specialized analytical methods that can perform a precise estimation of single isomer concentration in various physiological media. At the same time, the isomer ratio should not change during the storage period of such drugs.

4.9 Rules of Developing and Administering Chiral Drugs Despite the widespread knowledge and availability of extensive data revealing the stereochemical properties of drugs and the specifics of pharmacological action of racemic mixtures and their components (Lien et al. 2006; Shimazowa et al. 2008), governmental agencies responsible for registration and marketing of drugs ignored the issue well into the 1980-s. The Food and Drug Administration (USA) started demanding information about stereochemical aspects as late as in 1987. The information required thereafter for registration of new drugs included several aspects of stereochemical properties of drugs such as the stereochemical position of chiral centers in structural formulas of compounds, the relative content of optical isomers in nonracemic mixtures and data on the study of various properties of single optical isomers. If a drug is introduced in practice in the form of a single isomer which was earlier a component of another registered drug based on a racemic mixture, there was a strict requirement for additional clinical testing. In 1989, the European community implemented recommendations for chiral drug registration.

4.9 Rules of Developing and Administering Chiral Drugs

167

At present regional regulatory documentation is in force in the USA and Europe with regards to the technology of chiral drug production, the procedures of their quality control and methods of investigating their pharmacological effects, pharmacokinetics, toxicology, etc. (Development of new stereoisomeric drugs 1992; Stereochemical issues in chiral drug development 2000; Investigation of chiral active substances 1993). Besides, the quality of chiral drugs is specified in the recommendations by the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) (Q6A: Specifications 1999). This document consists of a Q6A coding and was entitled “Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances”. According to this document, when developing a drug in the form of a single optical isomer, the other isomer is regarded as an impurity. A special test should be designed which can help in distinguishing both optical isomers and the racemate and methods should also be in place for estimating the content of each stereoisomer separately. If the active substance in the drug is a chiral compound, a method for determining the concentration of all its stereoisomers and their ratio in all parts of the body should be designed and tested comprehensively. In its guidelines for preclinical pharmacokinetic investigation, the FDA stated that when the substance under study was a racemate, the developers should be able to analyze the content of each optical isomer in the body separately so as to evaluate the pharmacokinetic profile of the compound with an utmost precision (Guidelines on Non-clinical Pharmacokinetic Studies 1998). In other countries the regulations for chiral drug development correspond with those developed in the USA and Europe, as a rule. The above-mentioned guidelines do not specify the preference as to develop a new pharmaceutical form in the form of a pure optical isomer or as a racemate. However, when applying for registration of a racemic drug the developers are required to submit a scientific substantiation of the advantages of the racemate over single optical isomers. The ICH-Q6A guidelines state that when developing a pure optical isomer, the required test should not only allow for identfication of the isomers but also an analysis of both single optical isomers and the racemic mixture. As for stereochemical parameters, it is advisable to perform stereoselective chromatography for mixtures of optical isomers, in addition to the optical rotation test. When studying pharmacological properties of a racemic drug, it is necessary to perform a study of pharmacodynamics and pharmacokinetics of both the racemate and the isomers composing it. When a pure stereoisomer is tested, it is necessary to prove that it does not undergo chiral inversion. When studying toxicological properties, an investigation of acute toxicity of a racemate usually suffices. At relatively low multiple doses planned for clinical trials the toxic effect may be different from the effect predicted on the basis of the pharmacological properties of the drug. In this case an investigation of toxicity at the dose at which the unexpected toxic effect was revealed should be repeated with single isomers so as to establish whether or not the toxicity is caused by one single isomer.

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4 The Significance of Chirality in Pharmacological and Toxicological...

If administration of a pure isomer eliminates an important toxic effect and at the same time preserves the desired pharmacological action, one should consider the possibility of developing a drug based on this single isomer. It is important to estimate the content of each isomer and to set a limitation on all isomer components, admixtures and impurities in a pharmaceutical form undergoing preclinical investigation and headed for clinical trials. The maximum permissible level of impurities in chiral drugs undergoing clinical trials must not exceed the level of impurities in substances that have gone through preclinical toxicity studies. When developing a drug based on a stereoisomer that is a component of a mixture already tested in preclinical studies, it is advisable, as a rule, to conduct an abridged study of pharmacological and toxicological properties. Experiments should be designed in such a way that the available data about the racemate can be used for developing the toxicological profile of the stereoisomer under study. For a single optical isomer, a toxicity study at a maximum dose (up to three months) is usually performed in addition to a study of reproductive toxicity in the most sensitive species (only of phase two). These investigations should include an additional control group consisting of animals receiving the racemate. If there is no difference between toxicological profiles of the single isomer and that of the racemate, further study would be inadvisable. If the pure isomer is more toxic than the racemate, the causes of this phenomenon should be looked into, and one should consider possible consequences when calculating the clinical dose. When only a small difference in the activity of optical isomers is noted, it is advisable to develop a racemate-based drug. In some cases developing a single isomer drug may be desirable, for instance, when one isomer displays a toxic or undesirable pharmacological effect, and the other one does not. Further study of the properties of single isomers and their active metabolites is justified when the racemate is found to be toxic at clinical doses and this toxic effect is not the extension of the pharmacological effects of the drug, or if there are other unexpected effects. In such situations additional experimental investigations are necessary. It should be borne in mind that toxicity or an unusual pharmacological effect sometimes is not associated with the eutomer (or distomer) itself but with metabolites formed from it. If both optical isomers are pharmacologically active, but differ significantly in their effectiveness, specificity or maximum effect, a more correct methodological approach would be to conduct a clinical study of both isomers rather than testing one isomer with assumption that the other one is inactive. When a racemate is studied, pharmacokinetics of the enantiomers composing it must also be included, with consideration to their possible chiral inversion. Based on the pharmacokinetic findings it should be determined whether conventional nonselective methods of analysis can be used for pharmacokinetic investigations (in case if the ratio of the isomers in the body does not change), or the content of each isomer in the body should be traced separately. If the racemate has already been approved as a drug, and the developer wishes to design a single optical isomer as a new drug, the investigations should include an

4.10 Conclusion

169

assessment of whether any significant transformation of this isomer to another can occur in the body and whether the pharmacokinetics of this pure isomer resemble the pharmacokinetics of the same isomer incorporated into the racemate.

4.10 Conclusion Chiral separation of racemic pharmaceuticals is an indispensable procedure both for the pharmaceutical industry and for clinical practice. In this regard, development of modern technologies for chiral separation remains a top-priority area of theoretical research and industrial developments. However, the choice of using pure isomer should always be based on meticulous studies and comparison of the properties of the racemate and pure enantiomers as in some cases the racemate is a preferred choice in clinical practice. Over the past decade, due to wide-spread use of asymmetrical synthesis and chiral compounds separation methods in pharmaceutical industry, there is steady increase in the number of enantiomer-based drugs submitted for registration and approval. This phenomenon is not entirely for the purpose of developing a safe a effective drug from an approved racemate, but also due to the possibility of renewing a patent for existing approved medicinal substances. Lately, when the 20-year lifetime of patent for an approved drug expires, the drug manufacturers have launched a pure stereoisomer of an approved racemic drug as a new drug, often with assurances about its greater effectiveness and lesser toxicity. Many drugs that have been recently launched are a mixture of stereoisomers. These can be enantiomers, whose molecules are non-superimposable mirror images of each other, or geometric isomers whose molecules are not mirror images. In any case, stereoisomers may display dramatic differences in bioactivity and pharmacokinetics. Federal Food and Drug Administration (USA) now demands the identification and characterization of each isomer in a mixture of chiral compounds. The difference between stereoisomers may or may not be of consequence in clinical practice. For instance, in the case of levofloxacin, an antibacterial agent of fluoroquinolone group, it was proved that the S-(–)-isomer had an important clinical advantage over the racemate. However, in other cases when some stereoisomeric drugs were introduced upon expiry of the patent for racemic mixture, no clinical advantages were detected (Lien et al. 2006). In this case, it should be noted that the administration of an individual stereoisomer drug may relieve the xenobiotic load on the body processes responsible for drug excretion. This fact can be used to substantiate a decision to obtain a pure isomer from the racemic mixture. As the number of racemic and pure enantiomer chiral drugs that are being developed is growing steadily worldwide, the introduction of the method of chiral separation is of key importance both for the pharmaceutical industry and for clinical practice. Nowadays many drugs are used are racemates inspite of their side effects. The reason for this is both the complexity of chiral separation technology and the costs that it entails. If new, simpler and cheaper technologies of chiral separation

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are developed, the number of racemic drugs may decline. Direct manufacturing of a pure enantiomer by method of asymmetrical synthesis makes sense when the antipode enantiomer proves to be toxic or completely ineffective. Obtaining a racemate, however, can provide information about the racemate as well as two pure enantiomers. Theoretically speaking, the pure enantiomer should be used, but in practice this decision should be only taken after a meticulous analysis of the effects of the racemate and its pure enantiomers. In some clinical situations it is more advisable to use the racemate as pure enantiomers often complement the effect of each other. Another problem is the issue of informing doctors and pharmaceutical experts about the new generations of enantiomer drugs, their difference from racemic mixtures and the specifics of their administration in clinical practice. It is important to include the information about the pharmacology of drug stereoisomers into the educational objectives. Besides, the chiral form of the drug should be stated in drug compendia (whether it is a racemate or an enantiomer), along with the pharmacological, pharmacokinetic and toxicological properties of each isomer.

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Chapter 5

In Silico Search for Chiral Drug Compounds

All optical isomers of an optically active medicinal substance require experimental and clinical testing (Calcaterra and D’Acquarica 2018). In this process, as a rule, in silico methods are used in the first stage of screening for pharmacologically active substances. These methods predict the activity of chiral structures. Many computerized systems like Schrödinger (Schrödinger 2021), MOE (Chemical 2021), AutoDock (Goodsell et al. 2021), HIT QSAR (Kuz’min et al. 2021) have been used for investigation of biologically active compounds. However, the accuracy of their prediction is often unsatisfactory. Therefore, at present in silico methods for the search of pharmacologically active chiral compounds are being intensively developed and perfected.

5.1 Ways of Describing Chirality of Compounds for in Silico Analysis A key factor decisive for the ability of any computerized system to predict biological activity of chiral compounds is the ability to utilize the chiral descriptors. The chiral descriptors are the variables that take into account the asymmetry of predicted structures, when describing the chemical structure (Chemoinformatics 2018; Fragmentbased 2016). In this case the prediction method is of secondary importance as a rule. The most pertinent way of representing the structure of optically active compounds is the so-called absolute configuration, a three-dimensional model of a molecule that takes into account stereochemical specifics and indicates geometric coordinates of the atoms composing it (Karnik and Hasan 2021). Such a model permits an answer whether there are symmetry elements of any order (points, axes, planes) in the chemical structure and thus makes it possible to take into consideration any chiral forms present in an organic molecule. However, determining the absolute configuration of a particular chiral molecule presents difficulties from the methodological point of view. That is why in silico systems are used for analyzing the structure–activity © Springer Nature Singapore Pte Ltd. 2022 A. A. Spasov et al., Pharmacology of Drug Stereoisomers, Progress in Drug Research 76, https://doi.org/10.1007/978-981-19-2320-3_5

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relationship as they usually employ formal approaches to description of chirality, which can be conventionally divided into 2D and 3D methods. 2D methods of describing chirality are based on certain ways of coding the stereochemical position of atoms. The overwhelming majority of these methods are only good for analyzing stereoisomers resulting from the presence of asymmetrical atoms in the structure of the compound. As a rule, 2D approaches are not applicable for the description of higher-order chirality (axial, planar, spiral or topological). 2D chiral descriptors can be called qualitative as they do not indicate the extent of chirality in a compound. 2D methods of in silico analysis of “structure—activity” relationships belong to the class of LBDD (Ligand Based Drug Design) methods, since they do not require a 3D model of the target protein. Therefore, the widespread use of these approaches began before the use of three dimensional modeling methods (Usha et al. 2017). 3D methods of description operate with spatial models of molecules, which allow for a quantitative estimation of chirality of any type. Practically, all 3D chiral descriptors are “quantitative”. 3D methods of in silico analysis of “structure—activity” relationships belong to the class of SBDD (Structure Based Drug Design) methods and are based on modeling the interaction of the 3D structure of a low molecular weight ligand with a 3D model of target protein (Usha et al. 2017). At present, the “structure—activity” relationships are studied in the overwhelming majority of works within the framework of 3D approaches. However, the main method for modeling ligand–protein interactions is molecular mechanics, which stipulates the insufficiently high accuracy of 3D in silico prediction of pharmacological activity. In this connection, in terms of predictive ability, 3D methods are often inferior to 2D methods, and therefore, at the moment, the most optimal should be recognized as a combination of 2D and 3D approaches in the search for new drug substances including chiral ones (Yu and MacKerell 2017).

5.2 2D in Silico Analysis of Chiral Compounds A classical 2D structural formula of an organic compound permits reflection of the three-dimensional configuration of an optically active substance with symbolic representation of the bonds “above” and “below” the plane of molecule (Fig. 5.1). Analysis of the well-known MDDR database containing information about over 150 thousand biologically active substances (MDL 2020; Altalib and Salim 2021) showed that in medicinal compounds 81% of all chiral centers were carbon atoms, 17%—nitrogen atoms, 0.93%—sulfur atoms, and 0.3%—phosphorus atoms. Thus the easiest way of describing an enantiomer is using a counter variable equal to the number of asymmetrical atoms of a particular element in the structure of the compound, in R- and S-configurations separately. Chiral indexing of atoms is used in the well-known HQSAR approach (Joshi et al. 2021) to form “molecular holograms”. Accordingly, the stereoindex takes on values (0), (+1), (−1)—for an achiral atom, and for atoms in R- and S-configurations, correspondingly. Similar

5.2 2D in Silico Analysis of Chiral Compounds

OH

O

H3C

(R)

H

195

OH

O CH3

NH

H3C

(S)

H

CH3 NH

1-(Isopropylamino)-3-(1-naphthyloxy)propan-2-ol

Fig. 5.1 Structural formulas of R-(+)- and S-(–)-enantiomers of propranolol

indexing is also used when forming various molecular fingerprints (Chemoinformatics 2018; Fragment-based 2016). The following predictions can serve as examples of successful implementation of this approach: – Prediction of affinity to 5-HT1A -serotonin receptors of arylpiperazine chiral derivatives; accuracy in leave-one-out cross validation (LOOCV), correlation coefficient Q2 amounts to 81% (Weber et al. 2008); – Prediction of the antagonist effect on CCK2 -cholecystokinine receptors of 1,3,4benzotriazepine chiral derivatives; the accuracy in LOOCV Q2 amounted to 74% (Kaur and Talele 2008); – Prediction of the inhibiting effect of 4-pyrrolidylquinazoline chiral derivatives on phosphodiesterase PDE10A ; the accuracy in LOOCV Q2 amounted to 70% (Kulkarni et al. 2008). When fragment-based description is used, chiral substructural descriptors may be formed. For example, the structure of chiral compounds was represented on the basis of four-atom simplex representation in HIT QSAR system (Kuz’min et al. 2021). The following predictions were made using this approach: – Prediction of SIRT1 activity of structurally dissimilar compounds; the accuracy of R2 amounted to 83% (Chauhan and Kumar 2018); – Repositioning of known drugs as inhibitors of Mpro SARS-CoV-2, 42 active compounds found (Alves et al. 2021). When coding the structure of optical isomers with asymmetrical atoms, chiral topological descriptors are widely used. For instance, when calculating Wiener or Randi˙c indices (Devillers and Balaban 2000), in the case of an enantiomer the values of increments of these indices are first calculated for the groups adjoining the atom. Then, according to the Cahn-IngoldPrelog rule, the correction factor is calculated as a product of increment difference of group pairs; the obtained factor value is subtracted from the value of topological index for a planar structure. This approach was successfully employed for QSAR analysis of selectivity of chromatographic separation of naturally occurring oxyand amino acid enantiomers. The accuracy of R2 auto prediction amounted to 90% (Lukovits and Linert 2001).

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Modification of classical topological descriptors is also possible by way of “chiral” correction. It consists in adding a “chirality factor” to all diagonal elements of the connection matrix. In the simplest case the factor equals (0), (+1) or (−1)—for an achiral atom and for atoms in R- or S-configurations, correspondingly (Golbraikh et al. 2001). The final value of chiral topological index is calculated from the modified matrix in a regular manner. This approach was used in predicting H1 -histaminergic effect of structurally heterogeneous chiral compounds (the accuracy in LOOCV Q2 amounting to 69%), and the inhibiting effect on HIV-I protease (the accuracy in LOOCV Q2 amounted to 79%) (Golbraikh and Tropsha 2003). Of interest are indicial R/S-similar physicochemical stereodescriptors. They are based on a certain configuration of chiral atoms in accordance with the Cahn-IngoldPrelog rule but in this case priority is not assigned according to the atomic number. Instead, the values of physicochemical (electronic, steric and lipophilic) parameters of the fragments attached to the atom are considered, from the first layer to the third. The asymmetrical atom in this case is described with a set of binary indices taking on the value of (+1) for the R-, or (−1) for the S-configuration. On the basis of these descriptors and using the random forest analysis, lipase enantioselectivity in relation to primary alcohols can be successfully predicted. General accuracy of classification (ACC) in LOOCV amounts to 88% (Zhang and Aires-de-Sousa 2006). An example of the combined use of chiral atomic indices, chiral topological indices, and physicochemical stereodescriptors in 2D QSAR is the work on predicting the inhibitory activity of pIC50 of chiral structurally dissimilar compounds (amides, N-acylhydrazones, thiazolylhydrazones, thiosemicarbazones, triazine nitriles) against Cruzain (a cysteine protease of Trypanosoma cruzi); the accuracy in LOOCV Q2 amounted to 66% and external R2 amounted to 77% (Rosas-Jimenez et al. 2021). Thus 2D approaches are widely and successfully implemented in prediction of pharmacological effects of stereoisomeric compounds containing asymmetrical atoms. But, as it was pointed out above, these approaches are not applicable for the description of chirality of a higher order.

5.3 3D in Silico Analysis of Chiral Compounds A wider range of possibilities for prediction of biological effects of chiral compounds are offered by 3D modelling. Application of these methods makes it possible to define spatial geometry of the molecules to be analyzed, which allows a quantitative estimation of chirality. A study by Crippen (2008) has described the three main groups of quantitative 3D chiral descriptors that can be calculated for any type of chiral structure with any kind of asymmetry:

5.3 3D in Silico Analysis of Chiral Compounds

1. 2. 3.

197

The overlap volume for common atoms upon superimposition of two stereoisomers; Distortion indices in a transition from a planar structure to the intended stereoconfiguration; Energy parameters of inversion of one stereoisomer to another along the given chirality element.

VolSurf descriptors (Molecular Discovery 2021; Huang et al. 2020) permit a description of chiral molecules by a set of parameters that reflect their ability to interact preferentially with particular functional groups. These functional groups can be placed at the binding site of the given biotarget. The following predictions were made using this approach: – The ability of structurally heterogeneous compounds to penetrate the blood–brain barrier; general prediction accuracy ACC in LOOCV exceeded 90% (Crivori et al. 2000); – Affinity of structurally heterogeneous chiral ligands to proton-coupled transporter of hPepT1 oligopeptides; the accuracy in LOOCV Q2 exceeding 75% (Larsen et al. 2008). The well-known grid-based CoMFA (Silakari and Singh 2020; Joshi et al. 2021) and CoMSIA (Shaker et al. 2021; Joshi et al. 2021) methods also allow a prediction of pharmacological effects of chiral compounds. The CoMFA method was instrumental in obtaining significant 3D QSAR dependencies for D3 -agonist activity of chiral derivatives of tropane, the accuracy of LOOCV Q2 equaling 75% (Robarge et al. 2000). CoMSIA method made a prediction of CYP2D6 inhibiting activity for the stereoisomers of quinidine and quinine derivatives; the accuracy in LOOCV Q2 amounting to 72% (Ai et al. 2009). The same method found 3D QSAR dependencies between the inhibiting effect on NS5B-polymerase of hepatitis C virus and the structure of benzimidazol chiral derivatives; general prediction accuracy ACC in LOOCV achieving 67% (Patel et al. 2008). As an example of sharing of CoMFA and CoMSIA, aldo–keto reductase activity for chiral alcohols was predicted successfully; the accuracy in LOOCV Q2 amounting to 63% and 61%, for CoMFA and CoMSIA respectively (Cheng et al. 2018). Currently, 3D QSAR analysis linked with docking has been developing intensively. As docking is stereoselective in the first place, it is helpful in calculating 3D chiral descriptors. For instance, AutoDock Vina software (Eberhardt et al. 2021) was used in docking analyses of chiral cholesterol isomers in regasrd to five ion channels Kir2.2, KirBac1.1, TRPV1, GABAA and BK. It was shown that specific orientations of the chiral sterols within the binding sites of the channels are distinct, so that a subset of the interacting amino acids is unique to each sterol (Barbera et al. 2019). Using models complementary to target site, 3D QSAR dependencies were calculated using CoMFA, CoMSIA, HQSAR methods and molecular docking on a series of chiral coumarin-based benzamides as histone deacetylases HDAC inhibitors. The

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5 In Silico Search for Chiral Drug Compounds

accuracy of prediction of the inhibiting effect in LOOCV Q2 amounted to 73%, 67% and 81% for CoMFA, CoMSIA and HQSAR, correspondingly (Abdizadeh et al. 2020).

5.4 Molecular Docking of Chiral Compounds In its very essence, docking automatically takes into consideration the difference in interaction of any optical isomers of the same ligand with the binding site of the biotarget (Fig. 5.2). According to the Pfeiffer’s rule (The Practice 2015; Chemoinformatics 2018), the stereoisomer complementary to the target site should form a more stable ligand– protein intermolecular complex than other stereoisomers of the same substance. This hypothesis allows an estimation of the pharmacological effects of any chiral structure when there is no related data available based on previously done studies. This can be done by using a 3D model of the target protein (computer-derived model). This is the major advantages offered by docking and, therefore, the method is widely used for in silico estimation of pharmacological activity of all sorts of chiral compounds. With the help of AutoDock Vina program, docking of R- and S- enantiomers of antifungal new compounds with triazole scaffold into the human and fungal lanosterol-14 α-demethylase binding sites was performed. As a result of docking and experimental studies, the selective substances 4-256 times more potent than fluconazole against Candida species were identified (Irannejad et al. 2020). Docking of piperazine chiral derivatives (Fig. 5.3) was performed by means of AutoDock 4.2.6 software into the binding sites of histone deacetylase 8 HDAC8 (anticancer drug target) using 3D model 1T64 from PDB database. Two highly active HDAC8 inhibitors in R-(+)-form with IC50 = 21.54 nM and IC50 = 10.81 nM were found (Garrido González and Mancilla Percino 2020). A search for tumor cell growth inhibitors was conducted among R-(−)-, S-(+)-, R,R-(−)-, S,S-(+)-enantiomers of derivatives of xanthones and benzophenones (Fig. 5.4) using docking simulations between the (S,S)-Whelk-O1 selector and

Fig. 5.2 Interaction of two enantiomers with biotarget binding site (Patrick 2017)

5.4 Molecular Docking of Chiral Compounds

199

H N

O

O

R1 R2

N H

3,3-Disubstituted-piperazine-2,6-diones Fig. 5.3 General formula of piperazine derivatives, histone deacetylase 8 inhibitors

O

O

R

R

R

R

O

a

b

O

O

H N

H

OH

O

H N O

H

O

c

O OH

2,2'-(9-oxo-9H-xanthene-1,3-diyl)bis(oxy)bis(N-((S)-2-hydroxy-1-phenylethyl)acetamide) (C)

Fig. 5.4 General formulas of xanthone (a) and benzophenone (b) derivatives and the most active tumor cell growth inhibitor (c)

quantum-chemical 3D models of these structures. (S,S)-(+)-enantiomer was found, the most potent against all tested human tumor cell lines, with GI50 = 12.83 μM, GI50 = 12.40 μM and GI50 = 13.06 μM for NCI-H460 non-small cell lung cancer, respectively (Phyo et al. 2021). Docking of enantiomers of the heteroaryl-phenoxyethylamines was performed using homologically derived 5-HT1A -serotonin receptor and LeadIT 2.1.8 software suite. Subsequent to the results of an in silico study, an experimental investigation of

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5 In Silico Search for Chiral Drug Compounds CH3 N N O H N O

O

N-(((S)-2,2-diphenyl-1,3-dioxolan-4-yl)methyl)-2-(2-(1-methyl-1H-imidazol-5yl)phenoxy)ethanamine

Fig. 5.5 Structural formula of the heteroaryl-phenoxyethylamine with a high 5-HT1A -agonist activity

the most promising substances was conducted that resulted in finding the S-isomer of a compound with 5-HT1A -agonist effect pKi = 8.39 (Fig. 5.5) (Linciano et al. 2020). One of the studies (Papke et al. 2020) describes comparative modeling using a flexible docking procedure molecular binding mechanism of two chiral allosteric modulators of alpha7 nicotinic acetylcholine receptors α7 nAChR (Fig. 5.6). A 3D model of the extracellular domain of α7 nAChR was homologically derived with consideration to the aqueous medium. It was shown that allosteric activation binding site suggested a specific interaction of the active enantiomer with threonine 106. It is this interaction that determines the high effect of the most active of the studied compounds GAT1676 in comparison with the reference GAT107 (Fig. 5.7). Comparisons between GAT1676 isomers and active and inactive enantiomers of other related compounds identify the orientation of the cyclopentenyl ring to the plane of the core quinoline to be a crucial determinate of level of positive allosteric modulation. Affinity indicators for 10 chiral polyphenols from Opuntia ficus-indica for SARS-CoV-2 proteins were calculated using web platform COVID-19 Docking Server (Kong et al. 2020). Among these compounds, astragalin showed in silico the highest binding energy for Mpro ΔE = −8.7 kcal/mol, optimal interaction profile for its site (Fig. 5.8), as well as low toxicity. According to obtained data, astragalin is a prospective substance for COVID-19 treatment as Mpro protease inhibitor (Vicidomini et al. 2021).

5.5 Prediction of Pharmacological Activity of Chiral Compounds Using IT Microcosm IT Microcosm, the information technology of computerized prediction of biological activity of chemical compounds is authoring software (Vassiliev et al. 2014). On the basis of 2D stuctural formulas it permits a complex analysis of structure/activity

5.5 Prediction of Pharmacological Activity of Chiral Compounds …

NH2 O

S

201

NH2 O

O

HN

S

O

HN

CH3

CH3

CH3

CH3

Br

GAT107

GAT1676

(3aR,4S,9bS)-4-(4-bromophenyl)-

(3aS,4S,9bR)-4-(2,3,5,6-

3a,4,5,9b-tetrahydro-3H-

tetramethylphenyl)-3a,4,5,9b-tetrahydro-

cyclopenta[c]quinoline-8-sulfonamide

3H-cyclopenta[c]quinoline-8-sulfonamide

Fig. 5.6 Structure formulas of α7 nAChR allosteric modulators

Fig. 5.7 Comparison of docking into a homology model of the extracellular domain of α7 nAChR for GAT107 (A) and GAT1676 (B) compounds (Papke et al. 2020)

relationships both for structurally heterogeneous compounds and for analogs with a common formula. In particular, this technology allows prediction of the presence and extent of pharmacological effect of unstudied compounds (including unsynthesized ones) on the basis of obtained QSAR dependencies. The basis of the prediction method consists of regularities obtained by pattern recognition theory methods (Zhang et al. 2020). These regularities link semiquantitative gradations of biological activity and the structure of compounds represented

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5 In Silico Search for Chiral Drug Compounds

5,7-dihydroxy-2-(4-hydroxyphenyl)-3-((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6(hydroxymethyl)-tetrahydro-2H-pyran-2-yloxy)-4H-chromen-4-one Fig. 5.8 Binding features and pose of astragalin into Mpro site

in the form of a set of substructural QL language descriptors (Vassiliev et al. 2014). During prediction the pattern of the investigational compound (in the form of QLrepresentation) is compared with the models of generalized patterns of active/inactive classes (represented in the form of QL-matrix), and the membership function of the predicted structure to each class are calculated. In IT Microcosm an original complex approach to prediction is employed. It uses three predicting strategies (conservative, normal and risk) (Vassiliev et al. 2014) for generalization of the range of primary prognostic estimates obtained by 4 methods differing mathematically at 11 different levels of structure description. In the process of developing substantiated final conclusions for a particular type of biological activity in a range of 44 presumptive assessments, as a result of prediction by three strategies, the most probable level of this activity is calculated for each one of the tested compounds. QSAR Language (QL), a multidescriptor, hierarchical multilevel language with fragmental substructural notation is used in IT Microcosm for description of chemical structure. The QL version for prediction of the activity of common compounds has been described in detail in the book (Vassiliev et al. 2014). An extended version of the QL language was developed especially for prediction of pharmacological activity of chiral structures. It provides notation of optically active centers. In the process of translating a compound into QL-descriptors, the presence of asymmetrical atoms in its structure is first determined and then, the spatial configuration of these atoms is determined (R- or S-) according to the Cahn-Ingold-Prelog rule. The corresponding structural descriptor is marked as optically active, for instance, >C( 8.5), “moderate” (9 compounds 7.5 < lg(1/EC50 ) ≤ 8.5), and “low” (11 compounds, lg(1/EC50 ) ≤ 7.5). A combined class with “high or moderate” level of activity (15 compounds, lg(1/EC50 ) > 7.5) was added. The results of testing the accuracy of prediction of these four levels of D2 -dopaminergic activity in IT Microcosm are presented in Table 5.1. Clearly, the normal strategy turned out to be most effective in this study. Another research was performed based on the data from a study (Mehvar and Brocks 2001) that featured parameters of adrenoblocking activity and pharmacokinetics of S- and R-isomers of six known structurally similar β-adrenoblockers belonging to the same chemical class of 1-aryloxy-3-alkylamino-propan-2-ol: acebutolol, atenolol, carvediol, metoprolol, pindolol, and sotalol. The information gathered about the pharmacological activity of enantiomers of these compounds included the extent of S-isomer adrenoblocking effect exceeding the effect of the R-isomer in rodents using different models. The pharmacokinetic parameters in humans included Table 5.1 Testing the accuracy of prediction of D2 -dopaminagonistic activity of structurally heterogeneous chiral compounds Activity level

Accuracy of prediction in LOOCV, %a Conservative ACCb

SENS

Normal SPC

ACC

Risk SENS

SPC

ACC

SENS

SPC

High

80

50

90

89

83

90

89

83

90

Moderate

67

57

71

77

67

83

69

89

59

Low

68

50

79

73

73

73

77

55

93

High or moderate

65

79

44

73

73

73

77

93

55

Adequate models numberc

0

a

4

1

In three strategies; the data about inadequate models are shown in italics ACC is general accuracy; SENS is sensitivity or true positive rate; SPC is specificity or true negative rate c With the accuracy value of prediction ACC, SENS, and SPC over 60% b

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5 In Silico Search for Chiral Drug Compounds

the maximum drug concentration in the blood plasma (Cmax ), area under the plasma concentration time curve (AUC), renal clearance (ClR ) and apparent elimination half-life (t1/2 ). In accordance with the value of each of these five parameters, βadrenoblocker enantiomers were divided into two classes: (1) the enantiomer activity higher than that of the racemate; (2) the enantiomer activity lower than that of the racemate. The testing results showed that the conservative and risk strategies yielded no adequate prediction dependences. The normal strategy yielded highly accurate decision rules for all five activities. All the parameters of prediction accuracy ACC, SENS, and SPC equaled 100%. The third research was performed based on the data from an article (Siebert et al. 2008) that cited IC50 values of β 1 -adrenoblocking activity of ten optical isomers of the same drug with four chiral centers, nebivolol. According to the IC50 values, these ten diastereomers were divided into two classes: highly active (IC50 < 30 nM, 5 compounds) and compounds of low activity (IC50 ≥ 30 nM, 5 compounds). The testing results showed that the conservative strategy yielded a highly accurate decision rule (ACC, SENS, and SPC amounting to 100%); the risk strategy also yielded an adequate model (ACC = 70%, SENS = 80% and SPC = 60%). The results of all three studies made it possible to establish that the conservative strategy was not good for prediction of pharmacological activity of chiral compounds. LOOCV testing showed that not a single decision rule had been found. The risk strategy is only partially applicable for this purpose as out of seven obtained QSAR models only two showed satisfactory prediction characteristics. The normal strategy proved to be the most effective in prediction of chiral structure activity relationship as in all seven cases highly accurate prediction dependencies were obtained. This should be due to the fact that on one hand, “standard” regularities determined by the basic chemical structure of compounds play the key role and on the other hand, a considerable contribution is made by specific, subtle relationships associated with the way the asymmetric center interacts with the binding site of the biotarget. Thus IT Microcosm allows a highly accurate prediction of pharmacological activity of chiral compounds both in structurally heterogeneous and in a number of structurally similar substances including the series of optical isomers of one and the same drug.

5.6 Conclusion The analysis of data from literature and the results of our own research demonstrate that using modern computerized QSAR systems and molecular modeling systems allows an effective in silico search for chiral compounds with a high pharmacological activity. 2D and 3D methods of chiral in silico analysis have been utilized widely with the same degree of success. IT Microcosm software package based on 2D substructural description, in particular, permits a sufficiently accurate prediction of pharmacological activity of chiral compounds.

References

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