Review Drug chirality: a consideration of the significance

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

Review

Drug chirality: a consideration of the significance of thestereochemistry of antimicrobial agents

A. J. Hutf and J. O'Grady*

"Department of Pharmacy, King's College London, Manresa Road, London SW3 6LX;hDaiichi Pharmaceuticals UK Ltd, 76 Shoe Lane, London EC4A 3JB, UK

Approximately 25% of drugs are marketed as either racemates or mixtures ofdiastereoisomers. Such stereoisomers frequently differ in terms of their biologicalactivity and pharmacokinetic profiles and the use of such mixtures may contributeto the adverse effects of the drug particularly if they are associated with the inactiveor less active isomer. In recent years drug stereochemistry has become a significantissue for both the pharmaceutical industry and the regulatory authorities. Thesignificance of stereoisomerism in antimicrobial agents is addressed in this reviewusing examples drawn from the /?-lactams, as being representative of semisyntheticagents, and the quinolones, as examples of synthetic agents. Within these two groupsof compounds it is clear that stereochemical considerations are of significance for anunderstanding of concentration effect relationships, selectivity in both action andinactivation and for an appreciation of the mode of action at a molecular level. Inthe case of some agents the use of a single isomer is precluded due to their facileepimerization, e.g. carbenicillin, in the case of others there are potential advantageswith the use of single isomers, e.g. ofloxacin. However, in the case of latamoxef, acompound which undergoes in-vivo epimerization with a half-life similar to itsapparent serum elimination half-life the situation is by-no-means clear cut. Theseagents emphasise the importance of considering each compound individually, i.e. ona 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 thescientific and medical communities but also in the 'quality' lay press (Hawkes, 1993;Moran, 1993) and popular scientific press (Mason, 1984; Matteson, 1991). This interestin chirality has arisen as a result of recent advances in the areas of stereoselectivesynthesis and stereospecific analysis of chiral drug molecules. As a result of theseadvances, and the increasing realisation of the significance of the pharmacodynamic andpharmacokinetic differences between the enantiomers of chiral drugs, there has beenincreasing concern over the use of racemates, and other stereoisomeric mixtures, intherapeutics. The use of such mixtures may present problems, particularly if the adverseaffects, or toxicity, of the administered agent is associated with the less active, orinactive, isomer or does not show stereoselectivity.

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0305-7453/96/010007 + 26 $12.00/0 % 1996 The British Society for Antimicrobial Chemotherapy

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8 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 semisyntheticagents. Four hundred and eighty (28.7%) of the synthetic compounds were chiral and ofthese 58 (3.5%) were marketed as single isomers, the remainder (25.2%) were marketedas racemates. In contrast 469 (28%) of the natural or semisynthetic products were chiraland 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 syntheticsingle isomer drugs increased considerably up to 1991 (Millership & Fitzpatrick, 1993).

It should be obvious from the above figures that drug chirality is not a problemrestricted 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 ofthe agents used in antimicrobial chemotherapy are natural, or semisynthetic, the readermay wonder why this issue is being addressed in this journal. The problems associatedwith drug stereochemistry are complex, many of the semisynthetic agents are marketedas mixtures of diastereoisomers and a number of the synthetic agents are used asracemates. Such mixtures are regarded by some as 'compounds containing 50%impurity' and their use is essentially 'polypharmacy' with the proportions in the mixturebeing determined by chemical properties rather than therapeutic need. As a result ofthis increased concern, drug stereochemistry has become an issue for both thepharmaceutical industry and all the major regulatory authorities (De Camp, 1989;Cayen, 1991; Nation, 1994; Rauws & Groen, 1994). At present there is no absoluterequirement from any authority for the development of drugs as single isomers but inthe future the introduction of mixtures will require scientific justification. Indeed, severalcompounds currently marketed as racemates are undergoing re-evaluation as singleisomer products and while relatively few, e.g. dexfenfluramine, have been remarketedto date, several such compounds are in an advanced stage of development.

A compound frequently cited, particularly in the popular press, to support argumentsfor the development of single isomer drugs is the teratogen thalidomide. Recentinvestigations have indicated that the /?-enantiomer of thalidomide has hypnoticproperties 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 thetragedy of the early 1960s could have been avoided. However, some older data obtainedwith a more sensitive test species, New Zealand White rabbits, indicates that bothenantiomers of the drug are teratogenic (Fabro, Smith & Williams, 1967). An additionalproblem with the compound is its facile racemization in biological media (Testa,Carrupt & Gal, 1993 and references therein). Taken together these data indicate thatthe situation with thalidomide is by no means as clear as some of the secondaryliterature implies.

In this review when the structure of a molecule is specifically referred to the nameof the molecule is followed by a number in parentheses. These structures can be locatedwithin the figures using this numerical identification.

Definitions and nomenclature

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

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Chirality of antimicrobial agents 9

Enantiomers are stereoisomers which are non-superimposable mirror images of oneanother and therefore by definition are pairs of compounds related as an object to itsmirror image. Such isomers are said to be chiral (Greek chiros meaning handed) andare referred to as optical isomers, due to their ability to rotate the plane of planepolarized light, which is equal in magnitude but opposite in direction. The termdiastereoisomers refers to all other stereoisomeric compounds, regardless of their abilityto rotate plane polarized light, and the definition therefore includes both optical andgeometrical isomers. The fundamental distinction between enantiomerism anddiastereoisomerism is that in a pair of enantiomers the intramolecular distances betweennon-bonded atoms are identical, whereas in diastereoisomers they are not. Thus, theenergy content of a pair of enantiomers is essentially identical and therefore theirphysicochemical properties, e.g. lipid solubility, melting points etc., are also identicaland the separation, or resolution, of a racemic mixture (a 1:1 mixture of enantiomers)was, until relatively recently, fairly difficult. Diastereoisomers differ in energy, andtherefore in physicochemical properties, and may be relatively readily separable bystandard chemical techniques.

In terms of compounds of interest in medicinal chemistry the most frequent causeof chirality results from the presence of a tetracoordinate carbon centre in a moleculeto which four different groups are attached. The presence of one such centre in amolecule gives rise to a pair of enantiomers, the presence of n such centres gives riseto 2" stereoisomers and half that number of pairs of enantiomers. Those isomers whichare not enantiomeric are diastereomeric. Diastereoisomers which differ in configurationabout one chiral centre only are termed epimers (see Figure 1).

As pointed out above, in physicochemical terms enantiomers differ only in thedirection of rotation of the plane of plane polarized light and this property is frequentlyused in their designation. Those isomers which rotate light to the right are termeddextrorotatory, indicated by a ( + )-sign, while those which rotate light to the left are

HO

H HO y\%"NHCOCHClj C12CHCOHN H

O-.N'' ^ " 'NO,

(la) (lb)

HONHCOCHC12 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 threestereoisomers may be designated as follows: lb, (a-S, /J-S); lc, (a-R,fi-S); Id, (a-S, fl-R). Thus, in thisdiagram those compounds which are related horizontally (i.e. la with lb; lc with Id) are enantiomeric, whilethose 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.

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

termed laevorotatory indicated by a ( —)-sign. A racemic mixture of the two is indicatedby (±) before the name of the compound. It is important to appreciate that thisdesignation yields information concerning the physical property of the material but doesnot give information concerning the three dimensional spatial arrangement, or absoluteconfiguration, of the molecule. Some care is also required when using the direction ofrotation as a stereochemical descriptor as both the magnitude and direction of therotation may vary with the experimental conditions used to make the determination.For example chloramphenicol (Figure 1) contains two chiral centres and thereforefour stereoisomeric forms are possible. The active isomer (la) has the R,^-absoluteconfiguration (see below). However, this compound is dextrorotatory when thedetermination is made in ethanol and laevorotatory in ethyl acetate (Controulis,Rebstock & Crooks, 1949; Rebstock et ah, 1949). Additional complications arise if thedrug 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 designatedas (-)-(R)- and (-)-(S)-latamoxef (moxalactam) (Wise, Wills & Bedford, 1981). In thiscase the designation of the material by optical rotation is meaningless and provides noinformation concerning the stereochemical composition of the material, i.e. singleisomer or mixture.

Once the structure of a stereoisomer has been determined by, for example, X-raycrystallography then the configuration of the molecule may be indicated by the use ofa prefix letter to the name of the compound. Two systems are frequently used, the R/SCahn-Ingold-Prelog system, or the older D/L system. The D/L system relates theabsolute stereochemistry of a molecule to that of the enantiomers of either thecarbohydrate D-glyceraldehyde or the amino acid L-serine. The use of this system haslead to ambiguities and is now usually restricted in use to carbohydrates and aminoacids.

In the R/S system once the structure of the molecule has been determined thesubstituent atoms attached to the chiral centre are ranked in order of priority basedupon their atomic numbers. The higher the atomic number the greater the priority. Themolecule is then viewed from the side of the molecule opposite the group of lowestpriority and if the remaining highest to lowest priority atoms are in a clockwisedirection, i.e. to the right, the chiral centre is of the rectus or ^-absolute configurationand if to the left the isomer is of the sinister or 5-absolute configuration.

A particular problem in the nomenclature and stereochemical designation ofsemisynthetic compounds occurs as both the above systems may be used to define thestructure of a single molecule. For example, the absolute stereochemistry of the6-aminopenicillanic acid (3) and 7-aminocephalosporanic acid (4) nucleii have beendetermined 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 chiralcentre, which in the case of these two compounds is frequently defined in terms of theD/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-phenyl-glycylamino-3-methyl-3-cephem-4-carboxylic acid (see Figure 3 for structures). The sidechain chiral centre being denoted by the D/L system and only in the case of ampicillinis the stereochemistry of the ring system indicated and then for only one of the threecentres. Within the literature the two possible diastereoisomers arising from theintroduction of the side chain in these two compounds are frequently referred to interms of D and L (e.g. Tamai et al., 1988).

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Cbirality of antimicrobial agents 11

B I D

I)'

K B A *, I

I)

Receptor

(a) (b)

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

Chirality and biological activity

As pointed out above differences between enantiomers are under normal circumstancesdifficult to detect. However, when placed in a chiral environment these differencesbecome much more marked. Biological systems, at a molecular level, are intensely chiralenvironments being composed, in mammals at least, of macromolecules, e.g. proteins,glycolipids and polynucleotides, from the chiral building blocks of L-amino acids andD-carbohydrates. As many of the processes of drug action and disposition involve aninteraction between the enantiomers of a drug molecule and a chiral biologicalmacromolecule it is hardly surprising that stereoselectivity, or specificity, is observed inbiological systems.

The interaction between a drug molecule and a receptor surface or enzyme active siteis associated with bonding interactions between the functionalities of the drug andcomplementary sites on the receptor surface. Such interactions may have considerablesteric constraints, for example in terms of interatomic distance and steric bulk, betweensuch functionalities. In the case of stereoisomers the three dimensional spatialarrangement of the groups is also of considerable significance. This situation isillustrated 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 thebiological surface take place, whereas the 'inactive' isomer may take part in two suchinteractions. Thus the 'fit' of the two enantiomers to the receptor surface are differentand the binding energies of the interaction also differ.

The differential pharmacological activity of drug enantiomers has also given rise toadditional 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. Theratio of activities, a measure of the stereoselectivity, is termed the Eudismic Ratio(Lehmann, DeMiranda & Ariens, 1976). The above designations, and the EudismicRatio, refer to one biological action only and for a dual action drug the eutomer forone activity may be the distomer for the other. Examples are known in which thedifferential biological properties of a pair of enantiomers results in the marketing of

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both isomers with different therapeutic indications. Both enantiomers of the drugpropoxyphene are available, one as the analgesic dextropropoxyphene, with the(2R, 3S)-configuration, and the other laevopropoxyphene ((2S, 3^)-configuration) asan antitussive. In the case of this example not only are the molecules mirror imagerelated but so are their trade names (Darvon/Novrad).

Stereoselectivity is also observed in drug disposition particularly for those pro-cesses 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 passageof the majority of drugs through biological membranes depends on their physico-chemical properties, e.g. lipid solubility, pKa, size. In such cases differences betweenenantiomers would not be expected but differences between diastereoisomers may welloccur as a result of differences in their solubility. For example the water solubility ofthe D-diastereoisomer of ampicillin (5, Figure 3) is greater than that of theL-diastereoisomer (Doyle et ah, 1962). However, if a chiral drug molecule is a substratefor an active transport process then differences between both enantiomers anddiastereoisomers would be expected with preferential absorption of the stereoisomerwith a spatial arrangement similar to that of the natural substrate. In theory suchprocesses may be expected to increase the rate rather than the extent of absorption. Infact the bioavailability of D-methotrexate is only 2.5% that of the L-isomer (Hendel& Brodthagen, 1984). Similarly, selective transport processes may influence drugdistribution by selective tissue uptake and renal excretion, as a result of active secretionand/or reabsorption. Plasma protein binding may also influence drug stereoisomerdistribution and renal excretion. In metabolism, a process resulting from a directinteraction between a drug and a chiral macromolecule, stereodifferentiation is the rulerather than the exception and stereoselective metabolism is probably responsible for themajority of the differences observed in enantioselective drug disposition (Caldwell et al.,1988).

As a result of the above processes the pharmacokinetic profiles of the enantiomersof 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 andpotentially highly misleading or "sophisticated nonsense" (Ariens, 1984).

As pointed out above many of the agents used in antimicrobial chemotherapy arenatural or semisynthetic products and frequently single isomers are used. However,mixtures of diastereoisomers and enantiomers do occur and the remainder of this articlewill examine such cases using the /J-lactams and quinolone derivatives as representativeexamples.

/J-Lactams

Within the /J-lactam group of compounds the stereochemistry of the 6-aminopenicil-lanic acid (6-APA) and 7-aminocephalosporanic acid nucleii are thought to be absoluterequirements. Alteration of, for example, the configuration of any of the chiral centresin 6-APA results in a marked or total loss of activity (Naylor, 1973). This perhapsshould not be surprising considering the mode of action of these compounds. Theintroduction of an ot-substituent and thus an additional chiral centre in the side chainhowever, results in the formation of two epimeric diastereoisomers. In the case of

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, H3CO HCH— CONH.. I ! ^O

COOH

COOH

Latanioxef (moxalactam) epimers (2)(* indicates the chiral centre which undergoes

epimerization)

H HR C O N H ^ l |

COOH

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

CHjR'

COOH

(6fl, 7fl)-7-acylsubstituted-7-aminocephalosporanic acid (4)

H NH2

«w + H HCONH^J | ^ S

COOH

Cephalexin (6)

Ampicillin (5)

// s\ * H H

/ / \N CH — C O N H ^ ! 1

COOHO

COOH

H

CH3

CH3

COOH

Carbenicillin epimers (7)(' indicates the chiral centre which undergoes

epimerization)

4to3

Figure 3. /i-Lactam agents.

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14 A. J. Hurt and J. O'Grady

ampicillin (5, Figure 3) the two epimers differ in aqueous solubility (see above) andactivity, the ratio (D/L) in activity varying between 2 to 5-fold depending on the testmicroorganism (Naylor, 1973). In the case of ampicillin the official preparation is theepimer of the D-absolute configuration (which corresponds to the R configuration usingthe 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 thiscompound reportedly display only slight differences in activity and are stereochem-ically unstable undergoing rapid epimerization in solution (Naylor, 1973; Hoover &Dunn, 1979). In the case of this compound, separation of the individual epimers fortherapeutic use would appear to be a futile exercise.

The absorption of a number of /?-lactam antibiotics is mediated by the intestinaldipeptide transport system and as such their absorption would be expected to bestereoselective. The influence of configuration of the a-substituent in the side chain onthe absorption of the epimers of cephalexin has been investigated in the rat (Tamaiet al., 1988). Following the administration of L-cephalexin the unchanged drug couldnot be detected in either serum or urine. In contrast the D-isomer was found to be wellabsorbed. In-vitro studies indicated that both epimers were substrates for acarrier-mediated transport system with the L-epimer showing a higher affinity than, andacting as a competitive inhibitor for, D-cephalexin (6, Figure 3) transport. The L-epimerwas also more susceptible to the hydrolytic enzymes present in the tissues and theunchanged 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, designatedas R and S (see above; Yamada et al., 1981), the antimicrobial activity of the ./?-epimerbeing ca twice that of the S depending on the test system used (Wise et al., 1981). Thetwo isomers are stereochemically unstable undergoing epimerization to yieldequilibrium mixtures in the ratio R:S of 50:50 and 45:55 in buffer and serumrespectively. The rates of epimerization depending on the environment and epimericform (Wise et al., 1981). However, at 37°C in serum the half-life of epimerization is thesame for both compounds at 1.5 h, compared to a pharmacokinetic apparent serumelimination half-life of 2.3 h for 'total drug' following intravenous infusion of theepimeric mixture to man (Liithy et al., 1981; Wise et al., 1981). In man the serumconcentrations of the less active S-epimer are approximately twice those of the /?-epimerwithin 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 oflatamoxef are complicated by stereoselectivity in protein binding, the fractions unboundbeing 0.47 and 0.33 for (R)- and (S)-latamoxef respectively, resulting in similar unboundrenal 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 oflatamoxef in plasma may be explained by a combination of epimerization andstereoselectivity 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) theserum concentrations of latamoxef were determined by both stereospecifichigh-performance liquid chromatography (HPLC) and a bioassay method. The serumconcentrations determined using the bioassay methodology were consistently lowerthan those obtained using the HPLC method, the difference in values increasingprogressively in samples obtained up to 2 h post drug administration. This difference

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presumably reflects the more rapid excretion of the more active epimer and illustratesthe potential problems involved using bioassay methodology for the determination ofisomeric mixtures (Hutt, 1990).

Carbapenems and penems

Thienamycin (8, Figure 4), a highly active broad spectrum antibiotic, was the first ofthe carbapenem derivatives to be isolated and characterised (Kahan et al., 1983;Birnbaum et al., 1985; Moellering, Eliopoulos & Sentochnik, 1989). The absolutestereochemistry of thienamycin has been determined to be 5R, 6S, SR (structure 8) andthus, unlike the classical /Mactam antibiotics, the /Mactam ring has the transconfiguration, the two hydrogen atoms at positions 5 and 6 projecting in oppositedirections from the plane of the ring (Albers-Schonberg et al., 1978). This observationis of considerable significance as it indicates that the cis ring stereochemistry of thepenicillins and cephalosporins is not an absolute requirement for biological activity. In

OH

SCH2CH2NHR

Thienamycin (8)Imipenem (9)

RH

CH=NH

H. tCH2S(CH2)4

HO2C NH2

Cilastatin CIO)

CO2H

HNHCCK i

MeMe

COOH

(5fl)-Penem-3-carboxylic acid (R=H; 11)(5fl)-2-Methylpenem-3-carboxylic acid rR=CH3; 12)

Figure 4. Carbapenems and penems.

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HOOC HN

OHH H

HOOC N

SCH2CH2NHCH=NH2

SCH2CH2NHCH=NH2

coo-

SCH2CH2NHCH=NH2

coo-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).

addition, thienamycin was found to be remarkably stable to /Mactamases which ispresumably related to the trans configuration of the /Mactam ring (Birnbaum et al.,1985).

Thienamycin is chemically unstable in the solid state and in concentrated solutionsundergoes dimerization to yield an inactive product (Kahan et al., 1983; Birnbaumet al., 1985). The chemical modification of the nucleophilic thioethylamino side chainof thienamycin resulted in the synthesis of imipenem (9, Figure 4) which retains thebroad spectrum of activity, shows resistance to /Mactamases and increased chemicalstability (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 isolatedwhich vary in terms of the stereochemistry of the /Mactam ring and/or the configurationof the hydroxyethyl side chain (Figure 6). The agents which have the oppositestereochemistry to thienamycin in the side chain, i.e. the S- rather than the /^-absoluteconfiguration, are known as epithienamycins (Birnbaum et al., 1985). The majority ofthese agents are broad spectrum antibiotics, however the alteration of thestereochemistry of both the side chain and the ring system results in a decrease inpotency relative to thienamycin and an increased susceptibility to penicillinase (seeFigure 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 indicatedacceptable 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

C H , 6

S-R2

COOH

Compound

Tliienamycin (8) OilIniipencm (9) OHA'-Acetylthionamycin OHA/-Pheiiylaoetyltliieiiamycin 011Epithienamycin A OHDesacetylcpithicnamycin A OHKpithienainyciu B OHKpithienamycin C OHKpithicnamycin D OHKpithicnamyciii E OSO3HKpithienamycin F OSO3H

CH2CH2NH2CHaCHsNHCH-NHCH2CH2NHCOCH3

CI12CH2NHCOCH2C6HB

CHaCH2NHCOCH3

CH2CH2NH2CH=CHNHCOCH3

CH2CH2NHCOCH3

CII=CHNHCOCH3

CH=CHNHCOCH3CH2CH2NHCOCH3

Stereochemistry(5-lacLani

transtranstranstranscisciscistranstransciscis

CS

RRRRSS

sssss

Penicillinaseresistance

HighHighHigh

Low

LowModerateModerateModerateModerate

Relativepotency

1

0.53

0.29

0.630.0340.0230.340.29

Relativesusceptibilityto DHP-l10.94.26.4121.3

2051308.3

og

n

3

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

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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 bordermicrovilli of the renal proximal tubules the function of which is to scavenge dipeptidesfound in the glomerular filtrate (Kim & Campbell, 1982; Kropp et al., 1982; Parsonset al., 1991).

DHP-I has no activity against penicillins and cephalosporins but is active againstmost carbapenems (see Figure 6; Kropp et al., 1982). In the case of imipenem (9) thedrug undergoes hydrolysis of the /Mactam ring system to yield an approximately 1:1mixture 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 thienamycinand epithienamycin derivatives by DHP-I isolated from porcine kidney preparations.Imipenem showed slightly improved susceptibility to DHP-I compared withthienamycin, but in all other cases the compounds were found to be more sensitive tothe enzyme (Figure 6). From the data available it would appear that an alteration inthe stereochemistry of the compounds relative to thienamycin, results in a decrease interms of /Mactamase resistance and relative antibiotic potency but an increase insusceptibility to DHP-I. Trans ring stereochemistry appears to be preferred forpenicillinase resistance and the ^-absolute configuration in the side chain apparentlyreduces the susceptibility to hydrolysis by DHP-I.

The concept that co-administration of an inhibitor of DHP-I with imipenem wouldresult 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 duringthe development of cilastatin was a significant consideration and the activity of anumber of enantiomeric and diastereoisomeric derivatives was examined (Grahamet al., 1987). Cilastatin, a highly specific reversible competitive inhibitor of DHP-I, wasselected for development on the basis of its appropriate pharmacokinetic properties forcombination with imipenem (Kahan et al., 1983; Graham et al., 1987). The combinationof imipenem and cilastatin, in a ratio of 1:1, known as Primaxim (Clissold, Todd &Campoli-Richards, 1987) results in high urinary concentrations and recovery ofimipenem and in addition cilastatin prevents entry of imipenem into the proximaltubular epithelium (Kahan et al., 1983).

The penems are a group of synthetic /Mactam antibiotics which, in terms of chemicalstructure, combine features of both the penicillins and cephalosporins. The synthesisand 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 foldmore 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 twiceas active as the racemate (Ernest, Gosteli & Woodward, 1979). Thus, the ^-absoluteconfiguration at the ring junction appears to be an essential stereochemical requirementfor 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 resultingin the introduction of an additional chiral centre with the possibility of cis or transstereochemistry 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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hydroxyethyl substituent at position 6. Therefore two series of penem derivativescorresponding, in stereochemical terms, to thienamycin and the epithienamycinderivatives are possible. In general terms it would appear that the stereochemicalrequirements for antimicrobial activity are similar within both series (McCrombie &Ganguly, 1988). However, unfortunately the susceptibility of the penem derivatives toboth DHP-I and 0-lactamase also appears to correspond to that observed in thecarbapenem series (Zak et al., 1988). It is unfortunate that both the penems and thecarbapenems, compounds which are resistant to microbial /Mactamases should besusceptible to a mammalian enzyme.

Prodrugs

Esterification of the carboxyl group, to yield lipophilic ester prodrugs, has been usedextensively within the /Mactams in order to improve their absorption following oraladministration. A number of these derivatives involve the formation of either anacyloxymethyl or acyloxyethyl function which undergo rapid enzymatic hydrolysisin vivo to yield the corresponding hydroxymethyl or hydroxyethyl esters which, beinghemiacetal derivatives, spontaneously cleave with liberation of the active /J-lactam andthe corresponding aldehyde.

The introduction of the hydroxyethyl function into the promoiety results in theintroduction of an additional chiral centre into the molecule and therefore the possibilityof a pair of diastereoisomeric compounds, e.g. for cefuroxime axetil (14) andcefdaloxime pentexil (16). As pointed out above diastereoisomers may differ in theirphysicochemical properties, e.g. solubility, and also in their susceptibility with respectto in-vivo enzymatic hydrolysis (for structures see Figure 7).

Cefuroxime axetil (14) is the 1-acetoxyethyl ester prodrug of cefuroxime (13) andundergoes hydrolysis in vivo to yield cefuroxime, acetaldehyde and acetic acid. The drugmaterial 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 adminis-tration to man the prodrug undergoes rapid hydrolysis and cannot be detected in thesystemic circulation (Harding, Williams & Ayrton, 1984) and shows a bioavailabilitywith 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 beenreported following administration of the prodrug to the rat and may be due to anesterase, isolated from intestinal washings, which converts the ester to the unabsorbeddrug (Campbell, Chantrell & Eastmond, 1987). A more recent investigation hasexamined the stereoselectivity of the hydrolysis using both serum and intestinal mucosalesterases isolated from both rat and dog tissue preparations (Mosher, McBee &Shaw, 1992). These workers found that the hydrolysis was stereoselective for thel'S,6^,7./?-diastereoisomer (14a) but that the stereoselectivity varied with both tissuesource and species, the ratio I'S/l'R (14a/14b) being 14 and 2.5 for dog serum andintestinal esterases respectively. The corresponding values for rat tissue preparationsbeing 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 ofthe prodrug in man is not known, but such selectivity may contribute to the observedbioavailability of between 30-50%. In human blood the mixture of diastereoisomers hasa half-life of 3.5 min (Harding et al., 1984), i.e. is rapid, and thus stereoselectivity inhydrolysis is not a problem (for structures, see Figure 7).

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20 A. J. Hutt and J.

OCH3

NTII

/ O Ji H H

\J C0NHv

O'Grady

CH2OCONH2

COOR

Cefuroxime (13)

(l'S, 6R, 7«)-Cefuroxime axetil (14a)

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

RH

^ > </ ^

H ^> <

' OCOCH3

f M e

*OCOCH3

H,;\L N

// CONH^J J ^

CH2OCH3

COOR

C«fdaloxime (15)

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

RH

(I'R, 6R, 7fl)-Cefdaloximepentexil; HR916 J (16b)

Figure 7. Cefuroxime axetil and cefdaloxime pentexil.

OCOCMe3

H Me

OCOCMe3

Differential chemical hydrolysis and photochemical stability of cefuroxime axetildiastereoisomers has also been observed (Fabre, Ibork & Lerner, 1994). However, theabsolute configurations of the two compounds was not reported.

Cefdaloxime (RU 29246; 15) is a third generation cephalosponn with highantibacterial activity against both Gram-positive and Gram-negative pathogens(Bauernfeind el al., 1992; Markus et al., 1992). The drug is poorly absorbed from thegastrointestinal tract and has been esterified to yield the pivaloyloxyethyl prodrug(Defossa et al., 1992). Similarly to cefuroxime axetil (14) the formation of the prodrugresults in the introduction of an additional chiral centre and two diastereoisomers ofabsolute 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 individualand mixed administration, in a mouse protection assay indicated similar activity profiles

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for all three stereoisomeric forms of the prodrug (Defossa et al., 1992). A more extensivepharmacokinetic 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 andessentially complete absorption; in the rat bioavailability of the drug was reduced butno differences were observed between the individual diastereoisomers. However, in thedog the r5,6^,77?-diastereoisomer (HR 916 K; 16a) showed approximately three timesthe bioavailability of the r^,6^,77?-diastereoisomer (HR 916 J; 16b) as determined bycomparison of the areas under the cefdaloxime serum concentration time curves andurinary drug recovery (Isert et al., 1992). Defossa et al. (1992) have also stated that theabsorption 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 beenselected for evaluation and the pharmacokinetic properties of cefdaloxime followingadministration of the prodrug to man have been reported (Mendes et al., 1992).

Stereoselectivity in the absorption of diastereoisomeric prodrugs, together with thesubsequent availability of the drug, may arise as a result of differential solubility at theabsorption site, rates of diffusion through the gut wall and enzymatic activity in the gutcontents, mucosa, liver and blood, and as such the potential problems associated withthe introduction of a chiral promoiety into a molecule need to be taken intoconsideration at the compound design stage.

Quinolone derivatives

As pointed out above the problem of chirality is of greater significance for syntheticagents than with natural or semisynthetic agents. One group of compounds where thesignificance of stereochemistry in relation to activity has been addressed in some detailare the substituted l,4-dihydro-4-oxopyridine-3-carboxylic acid derivatives (17),collectively known as the quinolones. In terms of structure activity relationships withinthis series the substituted oxopyridine ring system with the carboxyl group at the3-position and the 4-carbonyl group being coplaner, is regarded by some as beingessential for activity (Shen, 1994) although useful activity has been observed withalternative functionalities in the 3-position (Chu el al., 1989). The fused ring system maybe either aromatic or heteroaromatic with substituents at positions 6 and 7. In themajority of compounds in this series the elements of chirality have been introduced atpositions 1 and 7 of structure 17 (Mitscher, Sharma & Zavod, 1989) (for structures, seeFigure 8).

Substituents at Nl

An important subgroup of the quinolones are those with a fused tricyclic ring systeminvolving attachment at positions 1 and 8 on the bicyclic ring structure (17). This ringfusion imparts a degree of rigidity to the substituent and several of this series possessa 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 beenshown 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 theS-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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C0011

(17)

F C9H2"S

(25)

COO11

K1

Call5NHCH2

K-(25b) H

R2

H

HN

COOH

(21)

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

R1

CH3

H

R2

HCH,

R1

COOH

R2

(S (-Tfemafloxacin (26a) H CH3

(fi)-Temarioxacin(26b) CH3 H

COOH

R1 R2

Ciprofloxacin (22) H HMethyl analogue (23) CH3 CH3

Phenyl analogue (24) C6Hfi CH3

COOH

(27)

R1 R2

S-(27a) CH2OH H«-(27b) H CH2OH

X

S

q6

Figure 8. Quinolone derivatives

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Chirality of antimicrobial agents

O O

COOH F

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

CH3

H

CH3

H

CH3

H

R2

H

CH3

H

CH3

HCH3

HCH3

R°

HH

CH3

CH3

-

-

Cl

Cl

Figure 9. Quinolone derivatives continued.

both Gram-positive and Gram-negative bacteria depending on both the compoundunder 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 thecase of methylflumequine (18c, 18d) and ofloxacin (19a, 19b) the correspondingnon-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 ofthe methyl group in the /?-enantiomers hindering and that in the S-isomers enhancingthe interaction (for structures see Figure 9).

Alternative tricyclic ring systems have been examined in which the aromatic, orheteroaromatic ring, fused with the oxopyridine system (see structure 17 Figure 8) hasbeen removed, e.g. 21 (Mitscher et al., 1989). In the case of this compound, in contrastto 18-20, antibacterial activity was found to reside predominantly in the enantiomerof the 7?-absolute configuration (21b) with R/S potency ratios varying between 0.8 andgreater than 64 against strains of Escherichia coli and Staphylococcus aureus(Georgopapadakou et al., 1987). This reversal in enantioselectivity is of interest andmay imply an alternative binding mode between the two structural series at the site ofaction.

The replacement of the N-ethyl group in norfloxacin with a cyclopropyl ring systemresults in ciprofloxacin (22) a derivative of greater potency and broader spectrum ofactivity. The introduction of a second substituent into the cyclopropyl ring results inthe formation of two chiral centres and the methyl (23) and phenyl (24) analoguestherefore exist in four stereoisomeric forms, i.e. two pairs of enantiomers. Examination

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O

O

COOH

(28)

R1 R2

S-(28a) NH2 HR-(28b) H NH2

DU-6859 (29) Amfonelic acid (30)

Figure 10. Quinolone derivatives continued.

of the antimicrobial activity of all four stereoisomers, of both 23 and 24, indicatedstereoselectivity 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 thephenyl series, the \'S, 2'/?-stereoisomer, was about four-fold more potent in a DNAgyrase assay, derived from Micrococcus luleus, than its enantiomer and ciprofloxacin,whereas against an E. coli enzyme system the above stereoisomer was equiactive withits 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 bicyclicnucleus (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) differencesin enantiomeric activity appear to be of relatively minor significance. This is presumablydue to the centre of chirality being in a position remote from the critical binding regionof the molecules. However, stereoselectivity is observed in this series and appears to varywith the position of substitution on the attached heterocylic ring. Thus in the case ofcompound 27, the chiral centre being adjacent to the heterocyclic nitrogen attached tothe bicyclic nucleus, the /?-enantiomer (27b) is ca. 50 and 30 times more potent againstE. coli and S. aureus than its 5-antipode (27a) (Mitscher et al., 1989). In the case ofcompounds 25, 28 and temafloxacin (26), compounds substituted fi to the nitrogenatom, the in-vitro activities of the individual enantiomers are either similar or show onlyrelatively 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 experimentalerror, 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 bothpositions 1 and 7 of the bicyclic ring system are currently under development. One such

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Table. Inhibitory activity and selectivity of ofloxacin (19) stereoisomers

IC» (mg/L)Compound DNA gyrase topoisomerase II selectivity"

(£,S)-ofloxacin (19)(#)-ofloxacin (19b)(5>ofloxacin (19a)Nonchiral analogue(structure 19, R1 = R2 = H)

0.764.70.383.1

187025501380

178

2461543

363257

'Ratio IC» topoisomerase II/ICM DNA gyrase.

agent DU-6859 (29), a compound with three chiral centres, is ca. 8-64 times more activethan ofloxacin against Gram-positive and Gram-negative bacteria (Sato et al., 19926).DU-6859 shows the highest potency and high selectivity (~9000) for DNA gyrasecompared 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 potentbeing amfonelic acid (30, see Figure 10) (Gerster et al., 1989; Chu et al., 1991).

The relationship between stereochemistry and pharmacological activity has beeninvestigated for a number of agents including flumequine (18a, 18b), methylflumequine(18c, 18d), S-12681 (20a, 20b) (for structures see Figure 9) and temafloxacin (26 seeFigure 8) using locomotor activity and, in the case of S-12681 (20a, 20b), inhibition ofdopamine and noradrenaline uptake into synaptosomes (Gerster et al., 1989; Chu et al.,1991). No locomotor stimulation was observed for either flumequine or methyl-flumequine (Gerster et al., 1989) and neither enantiomer of temafloxacin producedmarked stimulant or depressant activity (Chu et al., 1991). The enantiomers of S-12681produced either a slight, y?-enantiomer (20b), or marked, iS-enantiomer (20a), increasein locomotor activity in mice. In the case of the S-enantiomer (20a) the activity wassimilar to that observed with amfonelic acid (30). The S-enantiomer was also 6.6 and5.3 times more potent than the tf-isomer as an inhibitor of dopamine and noradrenalineuptake respectively into rat synaptosomes (Gerster et al., 1989). This observation isunfortunate as the stereoselectivity with respect to the adverse reaction parallels thatobserved for the antimicrobial activity.

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-foldmore active against both Gram-positive and Gram-negative bacteria than thefl-antipode (19b) (Hayakawa et al., 1986; Atarashi et al., 1987) (for structures, seeFigure 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

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antimicrobial activity, as determined by MIC concentrations and IC50 concentrationsfor 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 moreactive 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 forMIC activity.

As there are similarities between DNA gyrase and mammalian topoisomerase II itis of interest to examine the activity of the quinolones on topoisomerase II and hencetheir effects on mammalian cells. In the case of ofloxacin (19) the rank order of potencyagainst topoisomerase II, obtained from fetal calf thymus, is the same as that for DNAgyrase 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 withDNA 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 alsoof interest to note that the non chiral analogue (structure 19, R' = R2 = H) is the leastselective compound of the four (Table). Thus, the presence and orientation of the methylgroup at the chiral centre not only determines the potency of the compound but alsoincreases the selectivity.

The interaction between the quinolones and both DNA gyrase and DNA has beenthe subject of extensive investigation (see for example Sato, Hoshino & Mitsuhashi,1992a; Shen et al., 1989, 1990; Shen, 1993, 1994). Based on these investigations Shenand coworkers have proposed a cooperative quinolone-DNA binding model for theinhibition of DNA gyrase (Shen et al., 1989, 1990). The proposed model requiresself-association of the drug molecules via both n-n stacking interactions between thebicyclic ring system, together with hydrophobic interactions involving the substituentson the ring nitrogen. The final complex has been depicted as involving at least four drugmolecules such that the hydrophilic groups are projected 'outside' the complex, the'core' being hydrophobic (Shen et al., 1989, 1990). In terms of substituents on thequinolone nucleus, hydrophobic groups are required at Nl , to enhance interactionsbetween individual molecules, and the steric bulk of substituents at C7 does not appearto be a critical feature for useful activity (Shen et al., 1989). This latter point agreeswith the observations presented above regarding the lack of significant differencesbetween the activity of enantiomers on the introduction of a centre of chirality in the7-position substituent.

With the use of molecular graphics techniques Shen et al. (1990) have attempted torationalise the differential activity of the enantiomers of ofloxacin (19, see Figure 9) interms of their interaction model. The oxazine fused ring in ofloxacin (19) is partiallysaturated and is therefore nonplanar with a degree of conformational flexibility. Suchconformational flexibility will also influence the orientation of the methyl group at thechiral centre to the ring, which may take up either equatorial or axial positions, andthus influence the structure of the complex formed by molecular self-association. Thedata obtained by molecular modelling indicated that the most stable molecularcomplexes for the two enantiomers were also mirror images of one another and thatthe 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 bindingof 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 withconfiguration, being four and two for (5)- and (W)-ofloxacin, respectively. In terms of

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the model proposed by Shen et al. (1990) this difference may be due to an unfavourablebinding orientation of the /?-enantiomer (19b) such that the molecular self-associationcannot take place.

The metabolism and pharmacokinetics of the enantiomers of ofloxacin (19 seeFigure 9) have been investigated following their administration as such and as bothracemic and non-racemic mixtures, to the rat, dog and cynomolgus monkey. Followingtheir individual administration to rats the serum concentrations of (,/?)-ofloxacin (19b)were significantly higher than those of the S-enantiomer (19a) with a correspondinggreater area under the serum concentration time curve (AUC) and a longer apparentserum elimination half-life. These pharmacokinetic differences arise due tostereoselective conjugation of (S')-ofloxacin with glucuronic acid, together withpreferential biliary excretion of (S^ofloxacin, and its glucuronide, and urinary excretionof (7?)-ofloxacin (Okazaki, Kurata & Tachizawa, 1989). In addition, in-vitro studies,using rat hepatic microsomal preparations have indicated relatively minor differencesin the apparent Km for glucuronide formation of the two enantiomers (1.43 and 1.14 mMfor (R)- and (S)-ofloxacin, respectively) but a 6.5-fold difference in V^, the ratio ofVmMx/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 theglucuronidation of (S')-ofloxacin with a A", value of 2.92 mM. As a result of thisenantiomeric interaction in metabolism the serum concentrations of (5^-ofloxacin aremarkedly increased following administration of the racemic drug compared with thoseobserved following an equivalent dose of the single enantiomer, resulting in a 1.7-foldincrease in the AUC (Okazaki et al., 19916).

Following administration of racemic ofloxacin (19, see Figure 9) to cynomolgusmonkeys 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 adecrease in both total and renal clearance of (S)-ofloxacin (19a). As the drug undergoesminimal metabolism in this species these differences cannot be rationalised by metabolicinteractions. The renal excretion of ofloxacin is believed to involve both glomerularfiltration and tubular secretion, mediated by the organic cation transport system. Thusthe enantiomer-enantiomer interaction in the case of the monkey may be explained bycompetition for the secretion or reabsorption process (Okazaki et al., 1992). In contrastto the above two species, no differences were observed in the pharmacokineticparameters of the two enantiomers in the dog (Okazaki et al., 1992).

The dispositional properties of the enantiomers of ofloxacin illustrate the potentialproblems which may arise when dealing with racemic mixtures, i.e. stereoselectivity inmetabolism resulting in stereoselectivity in routes of excretion; enantiomericinteractions in both metabolism and active transport processes; species variability inenantiomeric metabolism and excretion together with the associated difficulty of speciesselection for toxicological evaluation.

Following the oral administration of racemic ofloxacin to healthy volunteers theserum concentration time profiles of the individual enantiomers are similar to thoseobtained following determination of total drug concentrations (Okazaki et al., 1991a).Small, but statistically significant, differences were observed between the enantiomersin 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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undergoes minimal metabolism in man the differences in the pharmacokinetic profilesof 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 clearanceof (S')-ofloxacin in the presence of the ^-enantiomer observed in the monkey occurs inman, it could be argued that it may be advantageous to administer the racemate ratherthan the single active enantiomer and thus increase the serum concentrations of theactive isomer. However, it is of interest to note that the single active S-enantiomer ofofloxacin, levofloxacin, has been recently marketed in Japan and is currently undergoingPhase III clinical trials in Europe and the USA (Davis & Bryson, 1994).

Concluding comment

The above discussion has attempted to highlight the significance of stereochemicalconsiderations in the area of antimicrobial agents. In the current regulatory climate allthe components present in a medicinal product require justification and as has beenobserved in other therapeutic areas, the introduction of single stereoisomers of both newand 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 ofcardiovascular disease, contains two chiral centres and the marketed material is amixture of all four stereoisomeric forms. Of these stereoisomers the ^-blocking activityresides in the R,R-isomer, the a-blocking activity in the S,/?-isomer and the remainingpair 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 ofthe single isomer. Why such toxicity is not observed with the isomeric mixture is notclear, but this example does illustrate that removal of the isomeric 'impurity' may notbe a trivial matter.

The decision to market a racemate, nonracemic isomeric mixture or singlestereoisomer depends on a number of factors, including technical feasibility, i.e.production on an industrial scale, stereochemical stability, toxicological profile and theclinical significance of the agent, i.e. the risk-benefit ratio. There are no simple answersto the single stereoisomer versus isomeric mixture debate and each example must beexamined on a case-by-case basis. Several of the compounds cited above indicate thepotential problems that may arise during drug development. For example, theepimerization of carbenicillin appears to be so rapid as to preclude the use of a singleisomer. Whereas, in the case of latamoxef, a compound with a half-life of epimerizationunder physiological conditions only slightly shorter than the apparent serumelimination 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, particularlyin terms of providing an insight into the mechanism of action at a molecular level,potency and selectivity.

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E. A. et al. (1978). Structure and absolute configuration of thienamycin. Journal of theAmerican Chemical Society 100, 6491-9.

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Ariens, E. J. (1984). Stereochemistry, a basis for sophisticated nonsense in pharmacokinetics andclinical pharmacology. European Journal of Clinical Pharmacology 26, 663-8.

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Atarashi, S., Yokohama, S., Yamazaki, K..-I., Sakano, K.-I., Imamura, M. & Hayakawa, I.(1987). Synthesis and antibacterial activities of optically active ofloxacin and its fluoromethylderivative. Chemical and Pharmaceutical Bulletin 35, 1896-902.

Barza, M. (1985). Imipenem: first of a new class of beta-lactam antibiotics. Annals of InternalMedicine 103, 552-60.

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