Journal of Antimicrobial Chemotherapy (1996) 37, 7-32

Review Drug chirality: a consideration of the significance of the stereochemistry of antimicrobial agents
A. J. Hutf and J. O'Grady*
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"Department of Pharmacy, King's College London, Manresa Road, London SW3 6LX; h Daiichi Pharmaceuticals UK Ltd, 76 Shoe Lane, London EC4A 3JB, UK Approximately 25% of drugs are marketed as either racemates or mixtures of diastereoisomers. Such stereoisomers frequently differ in terms of their biological activity and pharmacokinetic profiles and the use of such mixtures may contribute to the adverse effects of the drug particularly if they are associated with the inactive or less active isomer. In recent years drug stereochemistry has become a significant issue for both the pharmaceutical industry and the regulatory authorities. The significance of stereoisomerism in antimicrobial agents is addressed in this review using examples drawn from the /?-lactams, as being representative of semisynthetic agents, and the quinolones, as examples of synthetic agents. Within these two groups of compounds it is clear that stereochemical considerations are of significance for an understanding of concentration effect relationships, selectivity in both action and inactivation and for an appreciation of the mode of action at a molecular level. In the case of some agents the use of a single isomer is precluded due to their facile epimerization, e.g. carbenicillin, in the case of others there are potential advantages with the use of single isomers, e.g. ofloxacin. However, in the case of latamoxef, a compound which undergoes in-vivo epimerization with a half-life similar to its apparent serum elimination half-life the situation is by-no-means clear cut. These agents emphasise the importance of considering each compound individually, i.e. on a case-by-case basis, before deciding to use a single isomer or stereoisomeric mixture.

Introduction Over the last ten years drug chirality has become a 'big issue', not only within the scientific and medical communities but also in the 'quality' lay press (Hawkes, 1993; Moran, 1993) and popular scientific press (Mason, 1984; Matteson, 1991). This interest in chirality has arisen as a result of recent advances in the areas of stereoselective synthesis and stereospecific analysis of chiral drug molecules. As a result of these advances, and the increasing realisation of the significance of the pharmacodynamic and pharmacokinetic differences between the enantiomers of chiral drugs, there has been increasing concern over the use of racemates, and other stereoisomeric mixtures, in therapeutics. The use of such mixtures may present problems, particularly if the adverse affects, or toxicity, of the administered agent is associated with the less active, or inactive, isomer or does not show stereoselectivity.
0305-7453/96/010007 + 26 $12.00/0 7 % 1996 The British Society for Antimicrobial Chemotherapy

A. J. Hntt and J. O'Grady

A survey of 1675 drugs carried out in the early 1980s, indicated that 1200 (71.6%) could be classified as synthetic and 475 (28.4%) as natural products or semisynthetic agents. Four hundred and eighty (28.7%) of the synthetic compounds were chiral and of these 58 (3.5%) were marketed as single isomers, the remainder (25.2%) were marketed as racemates. In contrast 469 (28%) of the natural or semisynthetic products were chiral and of these 98.3% (461) were marketed as single isomers (Aliens, Wuis & Veringa, 1988). More recent surveys have indicated that the position with respect to natural/ semisynthetic products has not changed greatly but that the proportion of synthetic single isomer drugs increased considerably up to 1991 (Millership & Fitzpatrick, 1993). It should be obvious from the above figures that drug chirality is not a problem restricted to a single therapeutic group of agents but an 'across-the-board' problem; mixtures of stereoisomers are found in the majority of therapeutic groups. As many of the agents used in antimicrobial chemotherapy are natural, or semisynthetic, the reader may wonder why this issue is being addressed in this journal. The problems associated with drug stereochemistry are complex, many of the semisynthetic agents are marketed as mixtures of diastereoisomers and a number of the synthetic agents are used as racemates. Such mixtures are regarded by some as 'compounds containing 50% impurity' and their use is essentially 'polypharmacy' with the proportions in the mixture being determined by chemical properties rather than therapeutic need. As a result of this increased concern, drug stereochemistry has become an issue for both the pharmaceutical industry and all the major regulatory authorities (De Camp, 1989; Cayen, 1991; Nation, 1994; Rauws & Groen, 1994). At present there is no absolute requirement from any authority for the development of drugs as single isomers but in the future the introduction of mixtures will require scientific justification. Indeed, several compounds currently marketed as racemates are undergoing re-evaluation as single isomer products and while relatively few, e.g. dexfenfluramine, have been remarketed to date, several such compounds are in an advanced stage of development. A compound frequently cited, particularly in the popular press, to support arguments for the development of single isomer drugs is the teratogen thalidomide. Recent investigations have indicated that the /?-enantiomer of thalidomide has hypnotic properties while (iS)-thalidomide is both an hypnotic and a teratogen in SWS mice (Blaschke et al., 1979). Thus, if the drug had been used as a single isomer then the tragedy of the early 1960s could have been avoided. However, some older data obtained with a more sensitive test species, New Zealand White rabbits, indicates that both enantiomers of the drug are teratogenic (Fabro, Smith & Williams, 1967). An additional problem with the compound is its facile racemization in biological media (Testa, Carrupt & Gal, 1993 and references therein). Taken together these data indicate that the situation with thalidomide is by no means as clear as some of the secondary literature implies. In this review when the structure of a molecule is specifically referred to the name of the molecule is followed by a number in parentheses. These structures can be located within the figures using this numerical identification.
Definitions and nomenclature

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Stereoisomers are compounds which differ only in the spatial arrangement of their constituent atoms or groups and may be classified into two groups, namely enantiomers and diastereoisomers.

Chirality of antimicrobial agents

Enantiomers are stereoisomers which are non-superimposable mirror images of one another and therefore by definition are pairs of compounds related as an object to its mirror image. Such isomers are said to be chiral (Greek chiros meaning handed) and are referred to as optical isomers, due to their ability to rotate the plane of plane polarized light, which is equal in magnitude but opposite in direction. The term diastereoisomers refers to all other stereoisomeric compounds, regardless of their ability to rotate plane polarized light, and the definition therefore includes both optical and geometrical isomers. The fundamental distinction between enantiomerism and diastereoisomerism is that in a pair of enantiomers the intramolecular distances between non-bonded atoms are identical, whereas in diastereoisomers they are not. Thus, the energy content of a pair of enantiomers is essentially identical and therefore their physicochemical properties, e.g. lipid solubility, melting points etc., are also identical and the separation, or resolution, of a racemic mixture (a 1:1 mixture of enantiomers) was, until relatively recently, fairly difficult. Diastereoisomers differ in energy, and therefore in physicochemical properties, and may be relatively readily separable by standard chemical techniques. In terms of compounds of interest in medicinal chemistry the most frequent cause of chirality results from the presence of a tetracoordinate carbon centre in a molecule to which four different groups are attached. The presence of one such centre in a molecule gives rise to a pair of enantiomers, the presence of n such centres gives rise to 2" stereoisomers and half that number of pairs of enantiomers. Those isomers which are not enantiomeric are diastereomeric. Diastereoisomers which differ in configuration about one chiral centre only are termed epimers (see Figure 1). As pointed out above, in physicochemical terms enantiomers differ only in the direction of rotation of the plane of plane polarized light and this property is frequently used in their designation. Those isomers which rotate light to the right are termed dextrorotatory, indicated by a ( + )-sign, while those which rotate light to the left are
HO

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H
"NHCOCHClj O-.N'' (la)

HO
C12CHCOHN

y\%
H ^" (lb) 'NO,

NHCOCHC12

HO CljCHCOHN

(lc) (Id) Figure 1. Stereoisomers of chloramphenicol. The active stercoisomer of chloramphenicol (la) has the R,/{-absolute configuration. The British Pharmacopoeia (1993) designates I a as 2,2-dichloro-A4(ot-/?, /)-/?)/J-hydroxy-/?-hydroxymethyl-4-nitrophenethyl]acetamide. Using this nomenclature the remaining three stereoisomers may be designated as follows: lb, (a-S, /J-S); lc, (a-R,fi-S); Id, (a-S, fl-R). Thus, in this diagram those compounds which are related horizontally (i.e. la with lb; lc with Id) are enantiomeric, while those which are related vertically (i.e. la with both lc and Id; lb with both lc and Id) are diastereomenc. As there are only two chiral centres in the molecule the diasteroisomers are also epimeric to one another, but at different centres. The active isomer la being an a-epimer of Id and a /)-epimer of lc.

10

A. J. Hutt and J. O'Grady

termed laevorotatory indicated by a ( )-sign. A racemic mixture of the two is indicated by () before the name of the compound. It is important to appreciate that this designation yields information concerning the physical property of the material but does not give information concerning the three dimensional spatial arrangement, or absolute configuration, of the molecule. Some care is also required when using the direction of rotation as a stereochemical descriptor as both the magnitude and direction of the rotation may vary with the experimental conditions used to make the determination. For example chloramphenicol (Figure 1) contains two chiral centres and therefore four stereoisomeric forms are possible. The active isomer (la) has the R,^-absolute configuration (see below). However, this compound is dextrorotatory when the determination is made in ethanol and laevorotatory in ethyl acetate (Controulis, Rebstock & Crooks, 1949; Rebstock et ah, 1949). Additional complications arise if the drug material is a mixture of two diastereoisomers, e.g. latamoxef (moxalactam) (2) consists of a mixture of two epimers both of which are laevorotatory and are designated as (-)-(R)- and (-)-(S)-latamoxef (moxalactam) (Wise, Wills & Bedford, 1981). In this case the designation of the material by optical rotation is meaningless and provides no information concerning the stereochemical composition of the material, i.e. single isomer or mixture. Once the structure of a stereoisomer has been determined by, for example, X-ray crystallography then the configuration of the molecule may be indicated by the use of a prefix letter to the name of the compound. Two systems are frequently used, the R/S Cahn-Ingold-Prelog system, or the older D/L system. The D/L system relates the absolute stereochemistry of a molecule to that of the enantiomers of either the carbohydrate D-glyceraldehyde or the amino acid L-serine. The use of this system has lead to ambiguities and is now usually restricted in use to carbohydrates and amino acids. In the R/S system once the structure of the molecule has been determined the substituent atoms attached to the chiral centre are ranked in order of priority based upon their atomic numbers. The higher the atomic number the greater the priority. The molecule is then viewed from the side of the molecule opposite the group of lowest priority and if the remaining highest to lowest priority atoms are in a clockwise direction, i.e. to the right, the chiral centre is of the rectus or ^-absolute configuration and if to the left the isomer is of the sinister or 5-absolute configuration. A particular problem in the nomenclature and stereochemical designation of semisynthetic compounds occurs as both the above systems may be used to define the structure of a single molecule. For example, the absolute stereochemistry of the 6-aminopenicillanic acid (3) and 7-aminocephalosporanic acid (4) nucleii have been determined and defined using the R/S system but the addition of a side chain, e.g. ampicillin (5), cephalexin (6) may result in the introduction of an additional chiral centre, which in the case of these two compounds is frequently defined in terms of the D/L system. Thus, the British Pharmacopoeia (1993) defines ampicillin (5) as (6/?)-6-(a-D-phenylglycylamino)penicillanic acid and cephalexin (6) as 7-a-D-phenylglycylamino-3-methyl-3-cephem-4-carboxylic acid (see Figure 3 for structures). The side chain chiral centre being denoted by the D/L system and only in the case of ampicillin is the stereochemistry of the ring system indicated and then for only one of the three centres. Within the literature the two possible diastereoisomers arising from the introduction of the side chain in these two compounds are frequently referred to in terms of D and L (e.g. Tamai et al., 1988).

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Cbirality of antimicrobial agents

11

*,

I)' Receptor

I)

(a) (b) Figure 2. Biological discrimination between a pair of enantiomers. The enantiomer on the left (a) takes part in three complementary interactions with the active site, whereas that on the right (b) interacts at two sites only. Alternative orientations of the enantiomer (right) to the active site are possible, but only two interactions may take place at any one time. The vertical line represents the mirror plane where the centre structure is the reflection of that on the left.

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Chirality and biological activity As pointed out above differences between enantiomers are under normal circumstances difficult to detect. However, when placed in a chiral environment these differences become much more marked. Biological systems, at a molecular level, are intensely chiral environments being composed, in mammals at least, of macromolecules, e.g. proteins, glycolipids and polynucleotides, from the chiral building blocks of L-amino acids and D-carbohydrates. As many of the processes of drug action and disposition involve an interaction between the enantiomers of a drug molecule and a chiral biological macromolecule it is hardly surprising that stereoselectivity, or specificity, is observed in biological systems. The interaction between a drug molecule and a receptor surface or enzyme active site is associated with bonding interactions between the functionalities of the drug and complementary sites on the receptor surface. Such interactions may have considerable steric constraints, for example in terms of interatomic distance and steric bulk, between such functionalities. In the case of stereoisomers the three dimensional spatial arrangement of the groups is also of considerable significance. This situation is illustrated with respect to a pair of enantiomers in Figure 2. In the case of the 'active' enantiomer three simultaneous bonding interactions between the drug and the biological surface take place, whereas the 'inactive' isomer may take part in two such interactions. Thus the 'fit' of the two enantiomers to the receptor surface are different and the binding energies of the interaction also differ. The differential pharmacological activity of drug enantiomers has also given rise to additional terminology. Thus the isomer with the higher receptor affinity, or activity, is termed the eutomer, and that with the lower affinity, or activity, the distomer. The ratio of activities, a measure of the stereoselectivity, is termed the Eudismic Ratio (Lehmann, DeMiranda & Ariens, 1976). The above designations, and the Eudismic Ratio, refer to one biological action only and for a dual action drug the eutomer for one activity may be the distomer for the other. Examples are known in which the differential biological properties of a pair of enantiomers results in the marketing of

12

A. J. Hutt and J. O'Grady

both isomers with different therapeutic indications. Both enantiomers of the drug propoxyphene are available, one as the analgesic dextropropoxyphene, with the (2R, 3S)-configuration, and the other laevopropoxyphene ((2S, 3^)-configuration) as an antitussive. In the case of this example not only are the molecules mirror image related but so are their trade names (Darvon/Novrad). Stereoselectivity is also observed in drug disposition particularly for those processes which depend on an interaction with a chiral biological macromolecule, e.g. active transport processes, binding to plasma proteins, and drug metabolism (Williams & Lee, 1985; Caldwell, Winter & Hutt, 1988; Tucker & Lennard, 1990). The passage of the majority of drugs through biological membranes depends on their physicochemical properties, e.g. lipid solubility, pKa, size. In such cases differences between enantiomers would not be expected but differences between diastereoisomers may well occur as a result of differences in their solubility. For example the water solubility of the D-diastereoisomer of ampicillin (5, Figure 3) is greater than that of the L-diastereoisomer (Doyle et ah, 1962). However, if a chiral drug molecule is a substrate for an active transport process then differences between both enantiomers and diastereoisomers would be expected with preferential absorption of the stereoisomer with a spatial arrangement similar to that of the natural substrate. In theory such processes may be expected to increase the rate rather than the extent of absorption. In fact the bioavailability of D-methotrexate is only 2.5% that of the L-isomer (Hendel & Brodthagen, 1984). Similarly, selective transport processes may influence drug distribution by selective tissue uptake and renal excretion, as a result of active secretion and/or reabsorption. Plasma protein binding may also influence drug stereoisomer distribution and renal excretion. In metabolism, a process resulting from a direct interaction between a drug and a chiral macromolecule, stereodifferentiation is the rule rather than the exception and stereoselective metabolism is probably responsible for the majority of the differences observed in enantioselective drug disposition (Caldwell et al., 1988). As a result of the above processes the pharmacokinetic profiles of the enantiomers of a drug administered as a racemate may differ markedly. Pharmacokinetic parameters, e.g. clearance, volume of distribution, half-life etc., based on the determination of'total' drug substance present in biological samples is essentially meaningless data and potentially highly misleading or "sophisticated nonsense" (Ariens, 1984). As pointed out above many of the agents used in antimicrobial chemotherapy are natural or semisynthetic products and frequently single isomers are used. However, mixtures of diastereoisomers and enantiomers do occur and the remainder of this article will examine such cases using the /J-lactams and quinolone derivatives as representative examples. /J-Lactams Within the /J-lactam group of compounds the stereochemistry of the 6-aminopenicillanic acid (6-APA) and 7-aminocephalosporanic acid nucleii are thought to be absolute requirements. Alteration of, for example, the configuration of any of the chiral centres in 6-APA results in a marked or total loss of activity (Naylor, 1973). This perhaps should not be surprising considering the mode of action of these compounds. The introduction of an ot-substituent and thus an additional chiral centre in the side chain however, results in the formation of two epimeric diastereoisomers. In the case of

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, H3CO H CH CONH.. I ! ^O COOH COOH Latanioxef (moxalactam) epimers (2) (* indicates the chiral centre which undergoes epimerization)

H H RCONH^l |

(3S, 5fi, 6fi )-6-acylsubstituted-6aminopenicillanic acid (3)

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COOH

4
COOH
to
3

CHjR' COOH (6fl, 7fl)-7-acylsubstituted-7aminocephalosporanic acid (4)

Ampicillin (5)

H w +

NH2 H H

CONH^J

|^S

// //

s\ \N

H * CH C O N H ^ !

CH3 CH3 COOH

COOH O COOH Cephalexin (6) H

Carbenicillin epimers (7) (' indicates the chiral centre which undergoes epimerization) Figure 3. /i-Lactam agents.

14

A. J. Hurt and J. O'Grady

ampicillin (5, Figure 3) the two epimers differ in aqueous solubility (see above) and activity, the ratio (D/L) in activity varying between 2 to 5-fold depending on the test microorganism (Naylor, 1973). In the case of ampicillin the official preparation is the epimer of the D-absolute configuration (which corresponds to the R configuration using the Cahn-Ingold-Prelog system). The introduction of a carboxyl group in the a-position yields carbenicillin (7, Figure 3) a compound used as a mixture of epimers. The individual epimers of this compound reportedly display only slight differences in activity and are stereochemically unstable undergoing rapid epimerization in solution (Naylor, 1973; Hoover & Dunn, 1979). In the case of this compound, separation of the individual epimers for therapeutic use would appear to be a futile exercise. The absorption of a number of /?-lactam antibiotics is mediated by the intestinal dipeptide transport system and as such their absorption would be expected to be stereoselective. The influence of configuration of the a-substituent in the side chain on the absorption of the epimers of cephalexin has been investigated in the rat (Tamai et al., 1988). Following the administration of L-cephalexin the unchanged drug could not be detected in either serum or urine. In contrast the D-isomer was found to be well absorbed. In-vitro studies indicated that both epimers were substrates for a carrier-mediated transport system with the L-epimer showing a higher affinity than, and acting as a competitive inhibitor for, D-cephalexin (6, Figure 3) transport. The L-epimer was also more susceptible to the hydrolytic enzymes present in the tissues and the unchanged drug could not be detected in the analytical samples (Tamai et al., 1988). Latamoxef (moxalactam) (2, Figure 3) is a mixture of two epimeric forms, designated as R and S (see above; Yamada et al., 1981), the antimicrobial activity of the ./?-epimer being ca twice that of the S depending on the test system used (Wise et al., 1981). The two isomers are stereochemically unstable undergoing epimerization to yield equilibrium mixtures in the ratio R:S of 50:50 and 45:55 in buffer and serum respectively. The rates of epimerization depending on the environment and epimeric form (Wise et al., 1981). However, at 37C in serum the half-life of epimerization is the same for both compounds at 1.5 h, compared to a pharmacokinetic apparent serum elimination half-life of 2.3 h for 'total drug' following intravenous infusion of the epimeric mixture to man (Liithy et al., 1981; Wise et al., 1981). In man the serum concentrations of the less active S-epimer are approximately twice those of the /?-epimer within 4 h and the ratio (R:S) in renal clearance is 1.5 (Liithy et al., 1981; Yamada et al., 1981). In addition to the facile epimerization in serum the pharmacokinetics of latamoxef are complicated by stereoselectivity in protein binding, the fractions unbound being 0.47 and 0.33 for (R)- and (S)-latamoxef respectively, resulting in similar unbound renal clearances of 140 and 132 mL/min/1.48 m2 for the R and S-epimers respectively (Yamada et al., 1981). It would, therefore, appear that the epimeric composition of latamoxef in plasma may be explained by a combination of epimerization and stereoselectivity in plasma protein binding resulting in preferential renal clearance of the /?-epimer (Yamada et al., 1981). It is of interest to note that in the pharmacokinetic study by Luthy et al. (1981) the serum concentrations of latamoxef were determined by both stereospecific high-performance liquid chromatography (HPLC) and a bioassay method. The serum concentrations determined using the bioassay methodology were consistently lower than those obtained using the HPLC method, the difference in values increasing progressively in samples obtained up to 2 h post drug administration. This difference

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Chirality of antimicrobial agents

15

presumably reflects the more rapid excretion of the more active epimer and illustrates the potential problems involved using bioassay methodology for the determination of isomeric mixtures (Hutt, 1990). Carbapenems and penems Thienamycin (8, Figure 4), a highly active broad spectrum antibiotic, was the first of the carbapenem derivatives to be isolated and characterised (Kahan et al., 1983; Birnbaum et al., 1985; Moellering, Eliopoulos & Sentochnik, 1989). The absolute stereochemistry of thienamycin has been determined to be 5R, 6S, SR (structure 8) and thus, unlike the classical /Mactam antibiotics, the /Mactam ring has the trans configuration, the two hydrogen atoms at positions 5 and 6 projecting in opposite directions from the plane of the ring (Albers-Schonberg et al., 1978). This observation is of considerable significance as it indicates that the cis ring stereochemistry of the penicillins and cephalosporins is not an absolute requirement for biological activity. In
OH SCH2CH2NHR

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Thienamycin (8) Imipenem (9)

R H CH=NH

CO2H H. HO2C
tCH2S(CH2)4

NHCCK

H i

NH2 Me Me Cilastatin CIO)

COOH (5fl)-Penem-3-carboxylic acid (R=H; 11) (5fl)-2-Methylpenem-3-carboxylic acid rR=CH3; 12) Figure 4. Carbapenems and penems.

16

A. J. Hutt and J. O'Grady

SCH2CH2NHCH=NH2

SCH2CH2NHCH=NH2 HOOC HN
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coo-

OH H H SCH2CH2NHCH=NH2 HOOC N

Flgure 5. Hydrolysis of imipenem (9) by dehydropeptidase-I (DHP-I) to yield a pair of diastereoisomeric 1-pyrroline derivatives. Adapted from RatclifTe el al. (1989).

coo-

addition, thienamycin was found to be remarkably stable to /Mactamases which is presumably related to the trans configuration of the /Mactam ring (Birnbaum et al., 1985). Thienamycin is chemically unstable in the solid state and in concentrated solutions undergoes dimerization to yield an inactive product (Kahan et al., 1983; Birnbaum et al., 1985). The chemical modification of the nucleophilic thioethylamino side chain of thienamycin resulted in the synthesis of imipenem (9, Figure 4) which retains the broad spectrum of activity, shows resistance to /Mactamases and increased chemical stability (Kahan et al., 1983; Barza, 1985; Birnbaum et al., 1985; Kropp et al., 1985). Since the discovery of thienamycin a number of related compounds have been isolated which vary in terms of the stereochemistry of the /Mactam ring and/or the configuration of the hydroxyethyl side chain (Figure 6). The agents which have the opposite stereochemistry to thienamycin in the side chain, i.e. the S- rather than the /^-absolute configuration, are known as epithienamycins (Birnbaum et al., 1985). The majority of these agents are broad spectrum antibiotics, however the alteration of the stereochemistry of both the side chain and the ring system results in a decrease in potency relative to thienamycin and an increased susceptibility to penicillinase (see Figure 6). An examination of the pharmacokinetic and metabolic properties of thienamycin (8) and imipenem (9) (see Figures 4 and 5) in both animals and man have indicated acceptable plasma pharmacokinetics but low urinary drug recoveries (Kropp et al., 1982; Kahan et al., 1983; Birnbaum et al., 1985; Moellering et al., 1989). These agents

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K1 S-R2
CH, 6

COOH

Compound Tliienamycin (8) Iniipencm (9) A'-Acetylthionamycin A/-Pheiiylaoetyltliieiiamycin Epithienamycin A Desacetylcpithicnamycin A Kpithienainyciu B Kpithienamycin C Kpithicnamycin D Kpithicnamyciii E Kpithienamycin F Oil OH OH 011 OH OH OH OH OH OSO3H OSO3H CH2CH2NH2 CHaCHsNHCH-NH CH2CH2NHCOCH3 CI12CH2NHCOCH2C6HB CHaCH2NHCOCH3 CH2CH2NH2 CH=CHNHCOCH3 CH2CH2NHCOCH3 CII=CHNHCOCH3 CH=CHNHCOCH3 CH2CH2NHCOCH3

Stereochemistry
(5-lacLani CS

trans trans trans trans

cis cis cis

R R R R S S

Penicillinase Relative Relative potency susceptibility resistance to DHP-l 1 1 High 0.9 High 4.2 0.53 High
Low Low

g
n

0.29 0.63 0.034 0.023 0.34 0.29

6.4 12 1.3 20 51 30 8.3

trans trans
cis cis

s s s s s

Moderate Moderate Moderate Moderate

Figure 6. Influence of stereochemistry on the penicillinase resistance, relative potency and susceptibility to porcine dehydropeptidase-1 of oirbapenem antibiotics (adapted from Birnbaum et al (1985) and Kropp et al. (1982)); DHP-I, porcine dehydropeptidase-I, penicillinase resistance, hydrolysis by Bacillus ceretis penicillinase, relative potency was determined against a panel of 35 bacterial species.

18

A. J. Hutt and J. O'Grady

have been found to undergo metabolism in the kidney mediated by dehydropeptidase-I (DHP-I; EC 3.4.13.11) a zinc containing metallopeptidase located in the brush border microvilli of the renal proximal tubules the function of which is to scavenge dipeptides found in the glomerular filtrate (Kim & Campbell, 1982; Kropp et al., 1982; Parsons et al., 1991). DHP-I has no activity against penicillins and cephalosporins but is active against most carbapenems (see Figure 6; Kropp et al., 1982). In the case of imipenem (9) the drug undergoes hydrolysis of the /Mactam ring system to yield an approximately 1:1 mixture of diastereoisomeric 1-pyrroline derivatives (Figure 5; Ratcliffe et al., 1989). Investigations on the metabolism of imipenem by DHP-I demonstrated for the first time /Mactamase activity exhibited by a specific mammalian enzyme (Kim & Campbell, 1982). Kropp et al. (1982) have investigated the metabolism of a number of thienamycin and epithienamycin derivatives by DHP-I isolated from porcine kidney preparations. Imipenem showed slightly improved susceptibility to DHP-I compared with thienamycin, but in all other cases the compounds were found to be more sensitive to the enzyme (Figure 6). From the data available it would appear that an alteration in the stereochemistry of the compounds relative to thienamycin, results in a decrease in terms of /Mactamase resistance and relative antibiotic potency but an increase in susceptibility to DHP-I. Trans ring stereochemistry appears to be preferred for penicillinase resistance and the ^-absolute configuration in the side chain apparently reduces the susceptibility to hydrolysis by DHP-I. The concept that co-administration of an inhibitor of DHP-I with imipenem would result in an improved urinary antibiotic profile resulted in the synthesis of cilastatin (10, Figure 4; Graham et al., 1987). The stereochemistry of the agents evaluated during the development of cilastatin was a significant consideration and the activity of a number of enantiomeric and diastereoisomeric derivatives was examined (Graham et al., 1987). Cilastatin, a highly specific reversible competitive inhibitor of DHP-I, was selected for development on the basis of its appropriate pharmacokinetic properties for combination with imipenem (Kahan et al., 1983; Graham et al., 1987). The combination of imipenem and cilastatin, in a ratio of 1:1, known as Primaxim (Clissold, Todd & Campoli-Richards, 1987) results in high urinary concentrations and recovery of imipenem and in addition cilastatin prevents entry of imipenem into the proximal tubular epithelium (Kahan et al., 1983). The penems are a group of synthetic /Mactam antibiotics which, in terms of chemical structure, combine features of both the penicillins and cephalosporins. The synthesis and biological activity of both enantiomers and racemic penem-3-carboxylic acid (11, Figure 4) have been reported and the 5/?-enantiomer is between two to four fold more active than the racemate, the 55-enantiomer being inactive (Pfaendler, Gosteli & Woodward, 1979). Similarly, the 5/J-enantiomer of the 3-methyl derivative (12) is twice as active as the racemate (Ernest, Gosteli & Woodward, 1979). Thus, the ^-absolute configuration at the ring junction appears to be an essential stereochemical requirement for activity within this group of compounds. A considerable number of derivatives of the penem nucleus have been synthesized, many of which involve substitution at position 6 of the bicyclic ring system resulting in the introduction of an additional chiral centre with the possibility of cis or trans stereochemistry in the /Mactam ring (McCrombie & Ganguly, 1988; Zak et al., 1988). In addition, a number of compounds, by analogy with the carbapenems, have a chiral

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Chirality of antimicrobial agents

19

hydroxyethyl substituent at position 6. Therefore two series of penem derivatives corresponding, in stereochemical terms, to thienamycin and the epithienamycin derivatives are possible. In general terms it would appear that the stereochemical requirements for antimicrobial activity are similar within both series (McCrombie & Ganguly, 1988). However, unfortunately the susceptibility of the penem derivatives to both DHP-I and 0-lactamase also appears to correspond to that observed in the carbapenem series (Zak et al., 1988). It is unfortunate that both the penems and the carbapenems, compounds which are resistant to microbial /Mactamases should be susceptible to a mammalian enzyme. Prodrugs Esterification of the carboxyl group, to yield lipophilic ester prodrugs, has been used extensively within the /Mactams in order to improve their absorption following oral administration. A number of these derivatives involve the formation of either an acyloxymethyl or acyloxyethyl function which undergo rapid enzymatic hydrolysis in vivo to yield the corresponding hydroxymethyl or hydroxyethyl esters which, being hemiacetal derivatives, spontaneously cleave with liberation of the active /J-lactam and the corresponding aldehyde. The introduction of the hydroxyethyl function into the promoiety results in the introduction of an additional chiral centre into the molecule and therefore the possibility of a pair of diastereoisomeric compounds, e.g. for cefuroxime axetil (14) and cefdaloxime pentexil (16). As pointed out above diastereoisomers may differ in their physicochemical properties, e.g. solubility, and also in their susceptibility with respect to in-vivo enzymatic hydrolysis (for structures see Figure 7). Cefuroxime axetil (14) is the 1-acetoxyethyl ester prodrug of cefuroxime (13) and undergoes hydrolysis in vivo to yield cefuroxime, acetaldehyde and acetic acid. The drug material consists of an equal parts mixture of the two possible diastereoisomers of the \'S,6R,7R (14a) and l'R,6R,7R (14b) absolute configurations. Following administration to man the prodrug undergoes rapid hydrolysis and cannot be detected in the systemic circulation (Harding, Williams & Ayrton, 1984) and shows a bioavailability with respect to cefuroxime (13) of between 30 to 50% in the fasted and fed states (Harding et al., 1984; Finn et al., 1987). Similar values for bioavailability have been reported following administration of the prodrug to the rat and may be due to an esterase, isolated from intestinal washings, which converts the ester to the unabsorbed drug (Campbell, Chantrell & Eastmond, 1987). A more recent investigation has examined the stereoselectivity of the hydrolysis using both serum and intestinal mucosal esterases isolated from both rat and dog tissue preparations (Mosher, McBee & Shaw, 1992). These workers found that the hydrolysis was stereoselective for the l'S,6^,7./?-diastereoisomer (14a) but that the stereoselectivity varied with both tissue source and species, the ratio I'S/l'R (14a/14b) being 14 and 2.5 for dog serum and intestinal esterases respectively. The corresponding values for rat tissue preparations being 13 and 3.4, the rat tissue esterases being faster in both cases (Mosher et al., 1992). The possible contribution of stereoselectivity in the intestinal enzymatic hydrolysis of the prodrug in man is not known, but such selectivity may contribute to the observed bioavailability of between 30-50%. In human blood the mixture of diastereoisomers has a half-life of 3.5 min (Harding et al., 1984), i.e. is rapid, and thus stereoselectivity in hydrolysis is not a problem (for structures, see Figure 7).
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20

A. J. Hutt and J. O'Grady OCH 3 NT

II

/O

Ji

\J

C0NH

H H

v
CH2OCONH2
COOR

Cefuroxime (13) (l'S, 6R, 7)-Cefuroxime axetil (14a)

R H ^>< / ^' OCOCH3 H ^ fMe > < *OCOCH3

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(l'fl, 6R, 7fl)-Cefuroxime axetil (14 b)

H,;\L

//

CONH^J

J^
CH2OCH3 COOR
R H Me

Cfdaloxime (15) (l'S, 6R, 7fl )-Cefdaloxime pentexil, HR916 K(16a)

OCOCMe3 H (I'R, 6R, 7fl)-Cefdaloximepentexil; HR916 J (16b) Figure 7. Cefuroxime axetil and cefdaloxime pentexil. Me
OCOCMe3

Differential chemical hydrolysis and photochemical stability of cefuroxime axetil diastereoisomers has also been observed (Fabre, Ibork & Lerner, 1994). However, the absolute configurations of the two compounds was not reported. Cefdaloxime (RU 29246; 15) is a third generation cephalosponn with high antibacterial activity against both Gram-positive and Gram-negative pathogens (Bauernfeind el al., 1992; Markus et al., 1992). The drug is poorly absorbed from the gastrointestinal tract and has been esterified to yield the pivaloyloxyethyl prodrug (Defossa et al., 1992). Similarly to cefuroxime axetil (14) the formation of the prodrug results in the introduction of an additional chiral centre and two diastereoisomers of absolute configurations l'S, 6R, 1R for HR 916 K (16a) and VR, 6R, 1R for HR 916 J (16b) (Defossa et al., 1992). Examination of the in-vivo activity of the diastereoisomers, following their individual and mixed administration, in a mouse protection assay indicated similar activity profiles

Chirality of antimicrobial agents

21

for all three stereoisomeric forms of the prodrug (Defossa et al., 1992). A more extensive pharmacokinetic study in mice, rats and dogs, however, yielded some species differences (Isert et al., 1992). In the mouse all three forms of the prodrug showed rapid and essentially complete absorption; in the rat bioavailability of the drug was reduced but no differences were observed between the individual diastereoisomers. However, in the dog the r5,6^,77?-diastereoisomer (HR 916 K; 16a) showed approximately three times the bioavailability of the r^,6^,77?-diastereoisomer (HR 916 J; 16b) as determined by comparison of the areas under the cefdaloxime serum concentration time curves and urinary drug recovery (Isert et al., 1992). Defossa et al. (1992) have also stated that the absorption of the l'S,6.fl,77?-diastereoisomer (16a) was significantly higher in man, but no experimental data were presented. This diastereoisomer, HR 916 K, has been selected for evaluation and the pharmacokinetic properties of cefdaloxime following administration of the prodrug to man have been reported (Mendes et al., 1992). Stereoselectivity in the absorption of diastereoisomeric prodrugs, together with the subsequent availability of the drug, may arise as a result of differential solubility at the absorption site, rates of diffusion through the gut wall and enzymatic activity in the gut contents, mucosa, liver and blood, and as such the potential problems associated with the introduction of a chiral promoiety into a molecule need to be taken into consideration at the compound design stage. Quinolone derivatives As pointed out above the problem of chirality is of greater significance for synthetic agents than with natural or semisynthetic agents. One group of compounds where the significance of stereochemistry in relation to activity has been addressed in some detail are the substituted l,4-dihydro-4-oxopyridine-3-carboxylic acid derivatives (17), collectively known as the quinolones. In terms of structure activity relationships within this series the substituted oxopyridine ring system with the carboxyl group at the 3-position and the 4-carbonyl group being coplaner, is regarded by some as being essential for activity (Shen, 1994) although useful activity has been observed with alternative functionalities in the 3-position (Chu el al., 1989). The fused ring system may be either aromatic or heteroaromatic with substituents at positions 6 and 7. In the majority of compounds in this series the elements of chirality have been introduced at positions 1 and 7 of structure 17 (Mitscher, Sharma & Zavod, 1989) (for structures, see Figure 8).

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Substituents at Nl An important subgroup of the quinolones are those with a fused tricyclic ring system involving attachment at positions 1 and 8 on the bicyclic ring structure (17). This ring fusion imparts a degree of rigidity to the substituent and several of this series possess a chiral centre in the third ring adjacent to the nitrogen atom at position 1 (e.g. structures 18 to 20). The antibacterial activity of several members of this group has been shown to reside in the enantiomers of the 5-absolute configuration (18a, 18c, 19a, 20a), the 7?-enantiomers being considerably less active than the racemic mixture and the S-enantiomers having ca twice the activity of the racemate (Hayakawa et al., 1986; Atarashi et al., 1987; Gerster et al., 1987, 1989; Mitscher et al., 1987; Une et al., 1988). The difference in the in-vitro enantiomeric activity ranges from 4- to 250-fold against

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COOH C0011

COOH

(17) S-(21a) R-(2\b)

(21) R1
CH 3 H

R2 H
CH,

Ciprofloxacin (22) Methyl analogue (23) Phenyl analogue (24)

R1 H CH3 C6Hfi

R2 H CH3 CH3

S q 6

COO11

COOH

COOH

C2"S 9H

HN

(25)

(27)
R2 H

K-(25b)

K1 Call5NHCH2 H

(S (-Tfemafloxacin (26a) (fi)-Temarioxacin(26b)

R1 H CH3

R2 CH3 H

R1 R2 S-(27a) CH2OH H -(27b) H CH2OH

Figure 8. Quinolone derivatives

Chirality of antimicrobial agents O COOH F O

23

R2
(18) (19) (20)

Compound (S)-Flumequine(18a) (fl )-Flumequme (18b) (S)-Methylflumequine (18c) (R )-Methylflumequine (18d) (S (-Ofloxacin (19a) (R )-Ofloxacin (19b) (S)-S-12681 (20a) (tf )-S-12681 (20b)

R1 CH3 H
CH 3

R2
H CH 3 H CH 3

H H
CH 3 CH 3 Cl Cl

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H CH3 H

H
CH 3

H CH3 CH 3 H Figure 9. Quinolone derivatives continued.

both Gram-positive and Gram-negative bacteria depending on both the compound under examination and the test system used (Hayakawa et al., 1986; Atarashi et al., 1987; Gerster et al., 1987, 1989; Mitscher et al., 1987; Une et al., 1988). Also, in the case of methylflumequine (18c, 18d) and ofloxacin (19a, 19b) the corresponding non-chiral analogues (i.e. structures 18 and 19, R1 = R2 = H) are more active than the /?-enantiomers but less active than the racemates (Gerster et al., 1987; Hoshino et al., 1991ft). Such data imply steric constraints at the site of action with the orientation of the methyl group in the /?-enantiomers hindering and that in the S-isomers enhancing the interaction (for structures see Figure 9). Alternative tricyclic ring systems have been examined in which the aromatic, or heteroaromatic ring, fused with the oxopyridine system (see structure 17 Figure 8) has been removed, e.g. 21 (Mitscher et al., 1989). In the case of this compound, in contrast to 18-20, antibacterial activity was found to reside predominantly in the enantiomer of the 7?-absolute configuration (21b) with R/S potency ratios varying between 0.8 and greater than 64 against strains of Escherichia coli and Staphylococcus aureus (Georgopapadakou et al., 1987). This reversal in enantioselectivity is of interest and may imply an alternative binding mode between the two structural series at the site of action. The replacement of the N-ethyl group in norfloxacin with a cyclopropyl ring system results in ciprofloxacin (22) a derivative of greater potency and broader spectrum of activity. The introduction of a second substituent into the cyclopropyl ring results in the formation of two chiral centres and the methyl (23) and phenyl (24) analogues therefore exist in four stereoisomeric forms, i.e. two pairs of enantiomers. Examination

24

A. J. Hutt and J. O'Grady O O COOH

(28) R1 NH 2 H R2 H NH 2

DU-6859 (29)

Amfonelic acid (30)

S-(28a) R-(28b)

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Figure 10. Quinolone derivatives continued.

of the antimicrobial activity of all four stereoisomers, of both 23 and 24, indicated stereoselectivity in action but in all cases the activity was less than that of ciprofloxacin (22) (Mitscher et al., 1989). It is of interest to note that the most potent isomer of the phenyl series, the \'S, 2'/?-stereoisomer, was about four-fold more potent in a DNA gyrase assay, derived from Micrococcus luleus, than its enantiomer and ciprofloxacin, whereas against an E. coli enzyme system the above stereoisomer was equiactive with its enantiomer and about 12-fold less active than ciprofloxacin (Mitscher et al., 1989) (for structures see Figure 8).

Subslituenls at C7 A number of quinolones have been developed substituted at position 7 of the bicyclic nucleus (17) with a heterocyclic ring system containing a chiral centre (structures 25-28, Figures 8 and 10). In comparison to the tricyclic systems (18-20, Figure 9) differences in enantiomeric activity appear to be of relatively minor significance. This is presumably due to the centre of chirality being in a position remote from the critical binding region of the molecules. However, stereoselectivity is observed in this series and appears to vary with the position of substitution on the attached heterocylic ring. Thus in the case of compound 27, the chiral centre being adjacent to the heterocyclic nitrogen attached to the bicyclic nucleus, the /?-enantiomer (27b) is ca. 50 and 30 times more potent against E. coli and S. aureus than its 5-antipode (27a) (Mitscher et al., 1989). In the case of compounds 25, 28 and temafloxacin (26), compounds substituted fi to the nitrogen atom, the in-vitro activities of the individual enantiomers are either similar or show only relatively minor differences (Mitscher et al., 1989; Rosen et al., 1988; Chu et al., 1991). In the case of temafloxacin (26) the enantiomers were found, within experimental error, to possess similar activities against DNA gyrase but (S)-temafloxacin (26a) showed slightly greater in-vivo potency in a mouse protection test (Chu et al., 1991), which may be a result of a better pharmacokinetic profile compared with the .R-enantiomer (26b). A number of novel compounds substituted with chiral functionalities at both positions 1 and 7 of the bicyclic ring system are currently under development. One such

Chirality of antimicrobial agents Table. Inhibitory activity and selectivity of ofloxacin (19) stereoisomers Compound (,S)-ofloxacin (19) (#)-ofloxacin (19b) (5>ofloxacin (19a) Nonchiral analogue (structure 19, R1 = R2 = H) DNA gyrase 0.76 4.7 0.38 3.1 IC (mg/L) topoisomerase II 1870 2550 1380 178 selectivity" 2461 543 3632 57

25

'Ratio IC topoisomerase II/ICM DNA gyrase.

agent DU-6859 (29), a compound with three chiral centres, is ca. 8-64 times more active than ofloxacin against Gram-positive and Gram-negative bacteria (Sato et al., 19926). DU-6859 shows the highest potency and high selectivity (~9000) for DNA gyrase compared to topoisomerase II of the four stereoisomers examined (Hayakawa et al., 1991; Hoshino et al., 1991a). Adverse effects of quinolones Between 1-4% of patients treated with quinolones suffer adverse central nervous system (CNS) effects, e.g. dizziness, insomnia, headache, anxiety etc. (Kitzes-Cohen, 1989). CNS stimulation is a recognised problem with some of these agents, the most potent being amfonelic acid (30, see Figure 10) (Gerster et al., 1989; Chu et al., 1991). The relationship between stereochemistry and pharmacological activity has been investigated for a number of agents including flumequine (18a, 18b), methylflumequine (18c, 18d), S-12681 (20a, 20b) (for structures see Figure 9) and temafloxacin (26 see Figure 8) using locomotor activity and, in the case of S-12681 (20a, 20b), inhibition of dopamine and noradrenaline uptake into synaptosomes (Gerster et al., 1989; Chu et al., 1991). No locomotor stimulation was observed for either flumequine or methylflumequine (Gerster et al., 1989) and neither enantiomer of temafloxacin produced marked stimulant or depressant activity (Chu et al., 1991). The enantiomers of S-12681 produced either a slight, y?-enantiomer (20b), or marked, iS-enantiomer (20a), increase in locomotor activity in mice. In the case of the S-enantiomer (20a) the activity was similar to that observed with amfonelic acid (30). The S-enantiomer was also 6.6 and 5.3 times more potent than the tf-isomer as an inhibitor of dopamine and noradrenaline uptake respectively into rat synaptosomes (Gerster et al., 1989). This observation is unfortunate as the stereoselectivity with respect to the adverse reaction parallels that observed for the antimicrobial activity.

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Ofloxacin The stereoselectivity of the in-vitro antimicrobial action of the enantiomers of ofloxacin (19) has been referred to above. The S-enantiomer (19a) being between 8- to 128-fold more active against both Gram-positive and Gram-negative bacteria than the fl-antipode (19b) (Hayakawa et al., 1986; Atarashi et al., 1987) (for structures, see Figure 9). The target enzyme of the quinolone derivatives is believed to be DNA gyrase (bacterial topoisomerase II) (Sato et al., 1993) and a good correlation between

26

A. J. Hutt and J. O'Grady

antimicrobial activity, as determined by MIC concentrations and IC50 concentrations for inhibition of DNA gyrase have been obtained for the quinolones (Hoshino et al., 19916). In the case of ofloxacin the ( )-5-enantiomer (19a) is 9.3 and 1.3 times more active than the ( + )-/?-enantiomer (19b) and the racemate in terms of enzyme inhibition (Imamura et al., 1987). The rank order of potencies is identical to that observed for MIC activity. As there are similarities between DNA gyrase and mammalian topoisomerase II it is of interest to examine the activity of the quinolones on topoisomerase II and hence their effects on mammalian cells. In the case of ofloxacin (19) the rank order of potency against topoisomerase II, obtained from fetal calf thymus, is the same as that for DNA gyrase inhibition, i.e. S > R,S > R (Table; Hoshino et al., 1991/?; Sato et al., 1993). However, the relative activity (R/S) of the two enantiomers decreases from 12.4 with DNA gyrase to 1.8 against topoisomerase II, but more importantly, ( H^-ofloxacin (19a) is about 6.7 fold more selective than the fl-enantiomer (19b) (Table). It is also of interest to note that the non chiral analogue (structure 19, R' = R2 = H) is the least selective compound of the four (Table). Thus, the presence and orientation of the methyl group at the chiral centre not only determines the potency of the compound but also increases the selectivity. The interaction between the quinolones and both DNA gyrase and DNA has been the subject of extensive investigation (see for example Sato, Hoshino & Mitsuhashi, 1992a; Shen et al., 1989, 1990; Shen, 1993, 1994). Based on these investigations Shen and coworkers have proposed a cooperative quinolone-DNA binding model for the inhibition of DNA gyrase (Shen et al., 1989, 1990). The proposed model requires self-association of the drug molecules via both n-n stacking interactions between the bicyclic ring system, together with hydrophobic interactions involving the substituents on the ring nitrogen. The final complex has been depicted as involving at least four drug molecules such that the hydrophilic groups are projected 'outside' the complex, the 'core' being hydrophobic (Shen et al., 1989, 1990). In terms of substituents on the quinolone nucleus, hydrophobic groups are required at Nl, to enhance interactions between individual molecules, and the steric bulk of substituents at C7 does not appear to be a critical feature for useful activity (Shen et al., 1989). This latter point agrees with the observations presented above regarding the lack of significant differences between the activity of enantiomers on the introduction of a centre of chirality in the 7-position substituent. With the use of molecular graphics techniques Shen et al. (1990) have attempted to rationalise the differential activity of the enantiomers of ofloxacin (19, see Figure 9) in terms of their interaction model. The oxazine fused ring in ofloxacin (19) is partially saturated and is therefore nonplanar with a degree of conformational flexibility. Such conformational flexibility will also influence the orientation of the methyl group at the chiral centre to the ring, which may take up either equatorial or axial positions, and thus influence the structure of the complex formed by molecular self-association. The data obtained by molecular modelling indicated that the most stable molecular complexes for the two enantiomers were also mirror images of one another and that the enantiomers cannot stack in the same way to the asymmetric DNA binding site (Shen et al., 1990; Shen, 1994). Sato et al. (1992a) have reported that the specific binding of both enantiomers of ofloxacin (19) to supercoiled DNA are essentially the same, between 5 and 6 M, but that the apparent number of bound drug molecules varied with configuration, being four and two for (5)- and (W)-ofloxacin, respectively. In terms of

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Chirality of antimicrobial agents

27

the model proposed by Shen et al. (1990) this difference may be due to an unfavourable binding orientation of the /?-enantiomer (19b) such that the molecular self-association cannot take place. The metabolism and pharmacokinetics of the enantiomers of ofloxacin (19 see Figure 9) have been investigated following their administration as such and as both racemic and non-racemic mixtures, to the rat, dog and cynomolgus monkey. Following their individual administration to rats the serum concentrations of (,/?)-ofloxacin (19b) were significantly higher than those of the S-enantiomer (19a) with a corresponding greater area under the serum concentration time curve (AUC) and a longer apparent serum elimination half-life. These pharmacokinetic differences arise due to stereoselective conjugation of (S')-ofloxacin with glucuronic acid, together with preferential biliary excretion of (S^ofloxacin, and its glucuronide, and urinary excretion of (7?)-ofloxacin (Okazaki, Kurata & Tachizawa, 1989). In addition, in-vitro studies, using rat hepatic microsomal preparations have indicated relatively minor differences in the apparent Km for glucuronide formation of the two enantiomers (1.43 and 1.14 mM for (R)- and (S)-ofloxacin, respectively) but a 6.5-fold difference in V^, the ratio of VmMx/Km, an index of intrinsic hepatic clearance, S/R being 8.1 (Okazaki et al., 19916). Further studies indicated that the ./?-enantiomer is a competitive inhibitor of the glucuronidation of (S')-ofloxacin with a A", value of 2.92 mM. As a result of this enantiomeric interaction in metabolism the serum concentrations of (5^-ofloxacin are markedly increased following administration of the racemic drug compared with those observed following an equivalent dose of the single enantiomer, resulting in a 1.7-fold increase in the AUC (Okazaki et al., 19916). Following administration of racemic ofloxacin (19, see Figure 9) to cynomolgus monkeys significant differences were observed between the two enantiomers in AUC (S > R), mean residence time (5 > R) and total clearance (R > S) (Okazaki et al., 1992). Interestingly, administration of the 5-enantiomer with increasing amounts of the /?-isomer resulted in an increase in AUC, a decrease in volume of distribution and a decrease in both total and renal clearance of (S)-ofloxacin (19a). As the drug undergoes minimal metabolism in this species these differences cannot be rationalised by metabolic interactions. The renal excretion of ofloxacin is believed to involve both glomerular filtration and tubular secretion, mediated by the organic cation transport system. Thus the enantiomer-enantiomer interaction in the case of the monkey may be explained by competition for the secretion or reabsorption process (Okazaki et al., 1992). In contrast to the above two species, no differences were observed in the pharmacokinetic parameters of the two enantiomers in the dog (Okazaki et al., 1992). The dispositional properties of the enantiomers of ofloxacin illustrate the potential problems which may arise when dealing with racemic mixtures, i.e. stereoselectivity in metabolism resulting in stereoselectivity in routes of excretion; enantiomeric interactions in both metabolism and active transport processes; species variability in enantiomeric metabolism and excretion together with the associated difficulty of species selection for toxicological evaluation. Following the oral administration of racemic ofloxacin to healthy volunteers the serum concentration time profiles of the individual enantiomers are similar to those obtained following determination of total drug concentrations (Okazaki et al., 1991a). Small, but statistically significant, differences were observed between the enantiomers in AUC (S > R), mean residence time (S > R) and both total and renal clearance (R > S) but not in plasma protein binding or volume of distribution. As the drug

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28

A. J. Hutt and J. O'Grady

undergoes minimal metabolism in man the differences in the pharmacokinetic profiles of the two enantiomers may be accounted for by stereoselectivity in renal clearance (Okazaki et al., 1991a). If a similar situation with respect to the reduced renal clearance of (S')-ofloxacin in the presence of the ^-enantiomer observed in the monkey occurs in man, it could be argued that it may be advantageous to administer the racemate rather than the single active enantiomer and thus increase the serum concentrations of the active isomer. However, it is of interest to note that the single active S-enantiomer of ofloxacin, levofloxacin, has been recently marketed in Japan and is currently undergoing Phase III clinical trials in Europe and the USA (Davis & Bryson, 1994). Concluding comment The above discussion has attempted to highlight the significance of stereochemical considerations in the area of antimicrobial agents. In the current regulatory climate all the components present in a medicinal product require justification and as has been observed in other therapeutic areas, the introduction of single stereoisomers of both new and existing chiral drugs is likely to increase (the so-called racemic switches). However, such introductions are not without problems and may provide unexpected results. Labetalol, an established combined a- and j3-blocking drug used in the treatment of cardiovascular disease, contains two chiral centres and the marketed material is a mixture of all four stereoisomeric forms. Of these stereoisomers the ^-blocking activity resides in the R,R-isomer, the a-blocking activity in the S,/?-isomer and the remaining pair are essentially inactive. Clinical trials with the single ^-blocking, R,R-\somtr, named dilevalol, resulted in elevated liver function tests in a small number of patients. This toxicity had not been observed with labetalol and resulted in the withdrawal of the single isomer. Why such toxicity is not observed with the isomeric mixture is not clear, but this example does illustrate that removal of the isomeric 'impurity' may not be a trivial matter. The decision to market a racemate, nonracemic isomeric mixture or single stereoisomer depends on a number of factors, including technical feasibility, i.e. production on an industrial scale, stereochemical stability, toxicological profile and the clinical significance of the agent, i.e. the risk-benefit ratio. There are no simple answers to the single stereoisomer versus isomeric mixture debate and each example must be examined on a case-by-case basis. Several of the compounds cited above indicate the potential problems that may arise during drug development. For example, the epimerization of carbenicillin appears to be so rapid as to preclude the use of a single isomer. Whereas, in the case of latamoxef, a compound with a half-life of epimerization under physiological conditions only slightly shorter than the apparent serum elimination half-life, the single isomer or mixture question is more difficult to answer. In the case of the quinolones, particularly with respect to ofloxacin and its derivatives, there can be little doubt of the significance of stereochemical considerations, particularly in terms of providing an insight into the mechanism of action at a molecular level, potency and selectivity. References Albers-Schonberg, G., Anson, B. H., Hensens, O. D., Hirschfield, J., Hoogsteen, K.., Kaczka, E. A. et al. (1978). Structure and absolute configuration of thienamycin. Journal of the
American Chemical Society 100, 6491-9.
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