J3 L iiiniiioerJiylphosphojiic

acid

bacteria, possesses significant activity against P-lactamase staphylococcal producers. Its intrinsic potency is, however, less than that of benzylpenicillin against non-P-lactamase staphylococci, and acid instability precludes the oral usage of methicillin.

Methicillin was soon followed by ampicillin (11.36), and this was another significant advance, because ampicillin was the first semisynthetic penicillin to possess marked activity against Gram-negative organisms (although Ps. aeruginosa is resistant). Ampicillin is stable to acid and is administered orally or by injection, but is susceptible to the p-lactamases produced by S. aureus and most Gram-negative bacteria.

There then followed many other important new semisynthetic penicillins, such as cloxacillin (11.37) (the first oral, p-lactamase stable penicillin, but again without significant action on Gram-negative cells); its derivative flucloxacillin (11.38) claimed to give higher blood levels; carbenicillin (11.39) the first penicillin with activity against Ps. aeruginosa and its derivative ticarcillin (11.40); amoxycillin (11.41) with a similar spectrum to ampicillin, but which is much better absorbed; temocillin (11.42) with a longer half-life allowing twice-daily dosage; and several others.

The design of all the semisynthetic penicillins has had one common goal: to achieve, by the introduction of a different R group (11.2), a new antibiotic with an improved spectrum of activity and/or enhanced stability to p-lactamases. This deliberate design concept, therefore, has achieved some notable successes.

The above examples all illustrate development of new penicillins by substitution at the 6-position in the molecule. Position 3 is also uniquely important, since the introduction of various groups here has led to the design of new esters ('pro-drugs': see Chapter 7)

which are hydrolyzed by enzyme action after absorption from the gut mucosa to give the active antibiotic. Esters (pivampicillin, talampicillin and bacampicillin: see (11.43), (11.44) and (11.45), respectively) at position 3 of the ampicillin molecule break down in vivo to produce higher blood levels of ampicillin than would be obtained if ampicillin itself had been given at an equivalent concentration. Carbenicillin is not absorbed when given orally but esters (carfecillin (11.46), carindacillin (11.47)) in the side-chain at position-6, when given orally will hydrolyse in vivo to give a similar blood level to that obtained with an equivalent dose of carbenicillin given intramuscularly. Thus, 'prodrugs' form a useful development in P-lactam design.

Substituted penicillins, such as piperacillin (11.48), azlocillin (11.49) and mezlocillin (11.50), appear to combine the spectra and degree of activity of ampicillin and carbenicillin. Mecillinam (11.51) a 6p-amidinopenicillin, has limited activity against Gram-positive bacteria but is active against Gram-negative organisms. It binds preferentially to PBP2.

Thus, alterations in the molecule (and especially at positions 6 and 3) can produce penicillins with changes in microbiological and/or pharmacological properties.

Substitution at other sites has also been examined in the quest for improved design: removal of the sulphur atom of the thiazolidine ring usually leads to a reduction of activity, although the oxapenicillin (clavam) clavulanic acid (Section 11.5.4.2) is an important P-lactamase inhibitor. Additionally, the carbapenems (Section 11.5.5.2), in which sulphur is isosterically replaced by a methylene group but which have a double bond in the 5-membered ring, may possess significant activity. Substitution at the C-5 position reduces antibacterial activity, and substitution at the C-2 locus produces penicillins with activity against Gram-positive but not Gram-negative bacteria.

11.5.2 Cephalosporins

Current research on cephalosporins and cephamycins (methoxycephalosporins) is proceeding at a bewildering pace. The cephalosporins may be considered as semisynthetic derivatives of 7-ACA (11.3). Several of the early (first generation) cephalosporins differed more in their pharmacokinetic than in their antibacterial properties. Subsequent developments have been to improve:

(i) antibacterial activity, especially in the context of increasing resistance to P-lacta-mases produced by Gram-negative bacteria, although it must be added that decreased enzyme lability is sometimes paralleled by a reduction in antibacterial potency;

(ii) pharmacokinetic properties by making appropriate substitutions in the molecule, especially at positions 3 and 7.

11.5.2.1 Structure-activity relationships

The cephalosporins (A3-cephalosporins) and penicillins are structurally related in that the p-lactam ring is fused to different rings. The position of the double bond in A3-cephalosporins is very important, since A2-cephalosporins (11.52), irrespective of the composition of the side-chains, are not significantly antibacterial. In contrast, A2-penicillins are highly active against Gram-positive and Gram-negative bacteria (Sections 11.5.4.2 and 11.5.5.2).

The 7a-methyl cephalosporin derivatives have a greatly reduced antibacterial activity, whereas the introduction of a 7a-methoxy group gives compounds (cephamycins) with high antibacterial activity, and possessing considerable stability to most p-lactamases. There is, however, a rapid decrease in activity as the size of the ether group is increased.

Oxacephems (oxacephamycins; see Section 11.5.5.1) have been produced by synthetic means and may have high antibacterial activity, including p-lactamase stability.

An example of the interplay of various factors in antibacterial activity is demonstrated by the following findings. 7a-Methoxy substitution of cefuroxime, cefamandole and cephapirin gives reduced activity agaist E. coli because of a lower affinity for PBPs and not because of reduced permeability. In contrast, similar substitution in cefoxitin enhances activity because of greater penetration through the OM barrier rather than an increased affinity for PBPs.

Cephalosporins are generally less sensitive than penicillins to inactivation by staphylococcal p-lactamase, but may be susceptible to p-lactamases produced by some Gram-negative bacteria. Additional information is provided in Sections 11.4.1.1, including Table 11.4, and 11.5.3.

Various characteristics involved in the activity of ceftazidime are depicted in (11.53).

The 3-acetoxymethyl compounds cephalothin (11.54), cephacetrile (11.55) and cephapirin (11.56) have different 7-acyl groups, which are monosubstituted acetamido groups, and have similar antibacterial activity. They are active against Gram-positive bacteria and against p-lactamase-negative Gram-negative organisms.

11.5.2.2 Pharmacokinetic properties

The 3-acetoxymethyl compounds are converted in vivo by esterases to the antbacterially less active 3-hydroxymethyl derivatives and are excreted partly as such. The rapid excretion means that such cephalosporins have a short half-life in the body.

Replacement of the 3-acetoxymethyl group by a wide variety of groups has rendered other cephalosporins much less prone to esterase attack. For example, cephaloridine (11.57) has an internally compensated betaine group at position 3 and is metabolically stable. It gives higher and more prolonged blood levels than cephalothin.

Cephalosporins such as 3-acetoxymethyl derivatives (11.54), (11.55) and (11.56), cephaloridine (11.57) and cefazolin (11.58) are inactive when given orally. For good oral absorption, the 7-acyl group must be based on phenylglycine and the amino group must remain unsubstituted. At position 3, the substituent must be small, non-polar and stable, with a methyl substituent considered desirable (although this can decrease antibacterial activity). Earlier examples of oral cephalosporins are cephalexin (11.59), cefaclor (11.60) and cephradine (11.61). Although cephalexin has some degree of resistance to p-lactamases produced by Gram-negative bacteria, none of these oral cephalosporins possess a significant degree of resistance to these enzymes. More recent oral cephalosporins such as loracarbef (11.62), cefixime (11.63), cefpodoxime (11.64) and ceftibuten (11.65) show increased stability to Gram-negative p-lactamases. Cefpodoxime is an absorbable ester (see Chapter 7; 'pro-drugs'). During absorption, esterases remove the ester side-chain, liberating the active substance into the blood. Cefixime and

ceftibuten are non-ester drugs and loracarbef is a new oral carbacephem (sulphur at position 1 replaced by carbon). Loracarbef is highly active against Gram-positive organisms, including staphylococci. The others are characterized by activity against Gram-negative and -positive organisms. None of the drugs is active against Ps. aeruginosa.

Parenterally administered cephalosporins which are metabolically stable and which are resistant to many types of P-lactamases include cefamandole (11.66), cefotaxime (11.67), cefoxitin ((11.68) (which has a 7a-methoxy group) and cefuroxime (11.69). An esterified derivative of the latter, cefuroxime axetil, retaining all the proerties of the parent molecule, is also now available for oral administration.

Other cephalosporins include ceftazidime (see (11.53) where the roles played by various components are indicated), ceftriaxone (11.70), ceftizoxime (11.71), cefpirome (11.72), cefepime (11.73) and cefsulodin (11.74). Cefsulodin is a narrowspectrum drug only used for pseudomonal infections. The development of new cephalosporins continues apace and those listed are meant to serve as examples since space precludes an exhaustive list.

Side-chains containing a 2-aimnothiazolyl group at R' (e.g. cefotaxime, ceftriaxone, ceftizoxime and ceftazidime) yield cephalosporins with enhanced affinity for PBPs of Enterobacteriaceae and streptococci.

11.5.3 P-lactamase stability

This short section attempts to summarize the facts presented in Sections 11.5.1 and 11.5.2.

P-lactam antibiotics, both penicillins and cephalosporins, with simple side-chains (Ar-aCH2-CO-NH-) are usually sensitive to P-lactamases, whereas the incorporation of the a-carbon atom into an aromatic ring (e.g. methicillin) increases resistance.

The presence of an additional substituent, e.g. methoxy, in the 6-(penicillins) or 7a(cephalosporins) position greatly increases P-lactamase resistance, although intrinsic antibacterial activity may be decreased. Cefoxitin (11.68), however, has a broad spectrum of activity and is very resistant to P-lactamases. Newer 7a-methoxycephalosporins have been described that have the same spectrum of activity as cefoxitin but are more active. The 1-oxacephem antibiotic latamoxef (moxalactam: (11.75) see Section 11.5.5.1) has oxygen instead of sulphur at position 1, which would tend to make it less chemically stable and more enzyme-labile; however, the presence of the 7a-methoxy group, as in cefoxitin (11.68) stabilizes the molecule.

Absence of sulphur or oxygen in the fused ring results in increased resistance to P-lactamases (see Section 11.5.4; loracarbef (11.62)). Generally, changes at C2 and C3 in a penicillin or cephalosporin do not affect resistance to a P-lactamase.

11.5.4 P-lactamase inhibitors

An exciting concept in antibiotic therapy is the possibility of using P-lactam inhibitors clinically. This is not a revolutionary idea since many of the older penicillins were able to inhibit the enzyme (see Section 11.5.4.1). The development of clavulanic acid (see Section 11.5.4.2) bridged the gap between theoretical desirability and actual practice, and the introduction in 1981 of an antibiotic mixture (amoxyclav; Augmentin®, Beecham Research Laboratories) consisting of clavulanic acid with the P-lactamase sensitive penicillin, amoxycillin, provided the clinician with a new weapon in his armoury against microbial infection. A combination of clavulanic acid with the broad-spectrum penicillin ticarcillin has also been introduced. Other combinations are listed below (Section 11.5.4.3).

11.5.4.1 f--Lactams as inhibitors

Some of the earlier penicillins (e.g. cloxacillin and methicillin) and cephalosporins (such as cephalosporin C) were found to inhibit Bacillus cereus P-lactamase and later were shown to be active against some P-lactamases elaborated by Gram-negative bacteria. This inhibition was competitive in nature, and marked potentiation of a P-lactamase-sensitive P-lactam antibiotic could be achieved in vitro. The problem, nevertheless, was two-fold: (a) high concentrations of inhibitor were necessary, (b) no single antibiotic then available was able to inhibit a wide range of P-lactamases.

Thus, it was then not possible to design for clinical use an antibiotic mixture consisting of a P-lactam and a P-lactamase inhibitor.

11.5.4.2 Naturally occurring fi-lactamase inhibitors

The P-lactamase-inhibitory properties of cephalosporin C (described above, Section 11.5.4.1), itself produced by a micro-organism, stimulated a search for other naturally occurring P-lactamase inhibitors. In principle, this technique has involved testing culture fluids in which Streptomyces species have been growing for their ability to inhibit the P-lactamase produced by a specific strain of Klebsiella aerogenes. Research investigations at Beecham Research Laboratories in the UK and studies elsewhere, notably in the United States and Japan, have shown the production of P-lactamase inhibitors in the culture fluids of Streptomyces olivaceus and Streptomyces clavuligerus.

Three P-lactamase-inhibiting acidic substances, termed the olivanic acids (general structure (11.76)) have, with some difficulty, been isolated from the culture fluids of Streptomyces olivaceus. These possess potent activity against various types of P-lactamases, when they act as inhibitors, and are also broad-spectrum antibiotics in their own right. The olivanic acids, characterized as closely related members of a new class (1-carbapenems (11.77)) of fused P-lactams, are analogues of penicillins or clavulanic acid (11.78) where sulphur or oxygen, respectively, has been replaced by a methylene group. An antibiotic thienamycin (11.79) with a similar structure to the olivanic acids has been isolated from Streptomyces cattleya. It is of interest to note that thienamycin, a very broad-spectrum antibiotic, is often a poor P-lactamase inhibitor (see Section 11.5.5.2).

It would thus seem logical for a member of the olivanic acid group to be utilized as a P-lactamase inhibitor in combination with a P-lactamase-sensitive antibiotic in the design of a new antibiotic mixture. Unfortunately, the olivanic acids are produced in low yields and, as mentioned above, there have been problems with their isolation.

Attention has thus been focused on other types of P-lactamase inhibitors. One of these, clavulanic acid, was sufficiently promising for a comprehensive investigation to be undertaken. Clavulanic acid (a clavam) (11.78) is a fused bicyclic compound containing a P-lactam ring; it is similar in structure to the penicillins except that it contains oxygen in place of sulphur, i.e. an oxazolidine, instead of a thiazolidine, ring. It is produced in higher yields than the olivanic acids, but has a poor antibacterial action; it is, however, a potent inhibitor of staphylococcal P-lactamase and of P-lactamases produced by Gram-negative bacteria, in particular those with a 'penicillinase' rather than a 'cephalosporinase' type of action. Clavulanic acid inhibits P-lactamases of Group 2 (Table 11.4) but is a poor inhibitor of Groups 1, 3 and 4. Clavulanic acid effects a progressive inhibition of P-lactamase, the initial effect probably being competitive in nature, this being followed by a phase of rapid inactivation. Studies with different types of p-lactamases have demonstrated that this inhibitor is a kcat inhibitor (see Chapter 8) acting in a competitive and irreversible manner.

11.5.4.3 Synthetic fi-lactamase inhibitors

Penicillanic acid derivatives are also known to inhibit P-lactamases. Penicillanic acid sulphone (11.80) is a P-lactamase inhibitor that protects ampicillin from hydrolysis by staphylococcal P-lactamase and some, but not all, of the Gram-negative types depicted in Table 11.4. It is however, a less active inhibitor than clavulanic acid. Tazobactam (11.81) is a penicillinic acid sulphone derivative marketed as a combination with piperacillin (11.48). Alone, it has poor intrinsic antibacterial activity but is comparable to clavulanic acid in inhibiting P-lactamase activity. Sulbactam (11.82) is a semisynthetic 6-desaminopenicillin sulphone structurally related to tazobactam. It is an effective inhibitor of many P-lactamases and is also active alone against certain Gram-negative bacteria. It is combined with ampicillin (11.36) for clinical use. 6-Bromopenicillanic acid (11.83) inhibits some types of P-lactamases.

It is essential that the P-lactamase inhibitor and the penicillin have similar pharmacokinetics, i.e. absorption rates, to arrive together at the site of action in the body.

11.5.4.4 Structure-activity relationships in fi-lactamase inhibitors

The p-lactam ring of p-lactamase inhibitors appears to mimic the p-lactam ring of substrate molecules, fitting closely into the catalytic centre of the enzyme. Amino derivatives of clavulanic acid have potent inhibitory activity. A variety of p-lactam molecular types show high activity against cell-free p-lactamases, but not when used against whole cells of Gram-negative bacteria. The reason for this poor effect against periplasmic p-lactamase is their low OM penetrability (see Section 11.4.2). Clavulanic acid is a small hydrophobic molecule. Strongly acidic olivanic acids penetrate the outer membrane less well than clavulanic acid.

11.5.4.5 fi-lactamase inducers

In some bacterial species, exposure to certain P-lactams (e.g. cefoxitin, imipenem) may induce the formation of large amounts of chromosomally mediated Class I

'cephalosporinase' type P-lactamases (see Table 11.4). These may antagonize the activity of a second P-lactam used simultaneously. Continued production of the enzyme is necessary for a continued decrease in susceptibility to the second P-lactam.

11.5.4.6 Mutual pro-drugs

A pro-drug (see Section 11.5.1 and Chapter 7) is an inactive compound that is converted in vivo to an active form. Pro-drugs of P-lactams are usually esters which are broken down by mammalian esterases. A potentially exciting development has been the synthesis of linked esters of penicillins and P-lactamase inhibitors to produce what are termed mutual pro-drugs. These must be well absorbed and the two active constituents released in equal amounts. One problem is that the maximum antibacterial activity is not necessarily achieved at a 1:1 ratio. It has been suggested that it might even be possible to develop a mutual pro-drug (an ester of a penicillin with a P-lactamase inhibitor) to be given in combination with the pro-drug of the non P-lactamase inhibitor moiety. This is an interesting theoretical possibility, but it might itself pose many practical problems in formulation.

11.5.5 Other P-lactam ring systems

For several years, investigations have been carried out on modifications of P-lactam drugs in order to improve and extend their antibacterial activity or to alter their pharmacokinetic properties. The result has been the development of an impressive array of P-lactam antibiotics with new ring systems which can be of value clinically in their own right, or may serve as starting points for the design of still more important antibiotics, or, again, may be potent P-lactamase inhibitors (see Section 11.5.4).

11.5.5.11-Oxacephems

A highly active 1-oxacephem (latamoxef (11.75)) has been obtained semisynthetically from penicillin. The sulphur atom in the cephalosporin dihydrothiazone ring is isosterically replaced by an oxygen atom. Latamoxef shows similarities to other P-lactam antibiotics, e.g. a 7a-methoxygroup (as in cefoxitin), a p-hydroxybenzyl group (amoxycillin), an a-carboxylic acid group (carbenicillin) and a 3-(1-methyltetrazol-5-ylthiomethyl) substituent (cefamandole). Latamoxef is an effective kcat inhibitor of some P-lactamases and a competitive inhibitor of others.

11.5.5.2 Penems

In the penems, the double bond in the dihydrothiazine ring of the cephalosporins has been replaced in the corresponding (thiazolidine) ring of the penicillins. In the carbapenems (11.77), a methylene group has replaced the -S- atom at position 1 in the penicillin molecule. Examples have already been dealt with (olivanic acids, thienamycin) although it must be noted that, depite its high activity and P-lactamase resistance, thienamycin (11.79) suffers the disadvantage of being chemically unstable.

An N-formimidoyl derivative, imipenem (11.84), overcomes this problem. Imipenem has a broad spectrum, covering most Gram-positive and -negative aerobic and anaerobic bacteria and is highly resistant to P-lactamases (although it may act as an inducer: see Section 11.5.4.5). Imipenem is administered intravenously with cilastatin, which acts as a specific, competitive inhibitor of the enzyme, dehydropeptidase-I, that metabolizes imipenem in the kidney.

A new derivative, meropenem (11.85) containing a dimethylcarbamoylpyrrolidine ring, has been shown in vitro to be more active than imipenem (11.84). Of particular note is its activity against Ps. aeruginosa.

11.5.5.3 Nocarcidins

A novel group of P-lactam antibiotics, the nocarcidins (11.86) have been isolated from a strain of nocardia. This group has been characterized into seven closely related compounds (viz. nocarcidins A-G), with nocarcidin A (11.86) the most active. In vitro, nocarcidin A is less active than carbenicillin against Gram-negative bacteria and has no effect on Gram-positive organisms. In vivo, however, nocarcidin A is more active than carbenicillin because the potency of the former is increased in the presence of neutrophils, one type of phagocytic cell.

11.5.5.4 Monobactams

The monobactams are monocyclic P-lactam antibiotics produced by bacteria. They have been isolated from bacteria using as test organism a strains of Bacillus licheniformis which is specific for, and highly sensitive (100 ng ml-1) to, molecules containing a P-lactam ring.

On the basis of the novel nucleus (3-aminomonobactamic acid, 3-AMA (11.87) possessed by these antibiotics a potent monobactam has been synthesized. This is known as aztreonam (11.88). It is highly active against most Gram-negative bacteria and is very stable to most types of P-lactamases, including staphylococcal, although -interestingly- it is without effect on the growth or viability of S. aureus strains. Its lack of effect on staphylococci is believed to result from its predominant effect on PBP3 in Gram-negative organisms since this PBP is absent from staphylococci.

The newest class of P-lactam antibiotics comprises the carbacephems. An oral highly active compound (loracarbef (11.62)) has been described already. The sulphur in the

11.5.5.5 Carbacephems

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(11*84) imipenem

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(11.86) nocardicin A

(11.86) nocardicin A

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