LiLiirioniiit

cephalosporin dihydrothiazine ring is replaced by a methylene group (cf. carbapenems, Section 11.5.5.2). Loracarbef is highly active against Gram-positive bacteria, including staphylococci, but shows poor activity against Gram-negative bacteria.

11.6 DESIGN OF OTHER ANTIBACTERIAL AGENTS

Whilst it is probably true to state that P-lactam antibiotics occupy the greatest attention in the treatment of bacterial infecctions, it would be incorrect to imply that other antimicrobial agents do not have an important role to play. Several other groups of antibiotics exist, notably the aminoglycosides, tetracyclines, macrolides, polymyxins, lincomycins, rifamycins, quinolones together with chloramphenicol, glycopeptides and bacitracin. In recent years, advances have also been demonstrated in several of these groups, resulting in the production of new antibiotics and these are considered in this section.

11.6.1 Aminoglycoside-aminocyclitol antibiotics

The general structure of the 2-deoxystreptamine-containing antibiotics (the aminoglycoside antibiotics) is shown in (11.89), together with examples of the different drugs. Streptomycin (11.90) does not contain 2-deoxystreptamine. The more important aminoglycoside antibiotics are kanamycin (11.27), gentamicin (11.91), tobramycin (11.92), amikacin (11.28), sisomicin (sissomicin) (11.93) and netilmicin (11.94). As a group, the aminoglycoside antibiotics are bactericidal to Gram-negative bacteria and to staphylococci. Two of the major problems, however, have been the development of resistance of Gram-negative bacteria, often by virtue of drug-modifying enzymes (acetyltransferases, adenylylating enzymes and phosphotransferases: see Section 11.4.1.2) and the toxicity associated with a most important member, gentamicin, which has necessitated careful monitoring of blood and body fluid levels. Desirable properties of the newer (post-gentamicin) types have included increased antimicrobial activity, including improved activity against resistant strains, enhanced pharmacokinetic properties or a reduction in, or freedom from, toxicity.

Aminoglycoside antibiotics have several sites at which chemical substitution can be made. Alteration in the 3' position of kanamycin B to give 3'-deoxykanamycin B (tobramycin) (11.92) changes the activity spectrum. Amikacin has a 4-amino-2-hydroxybutanoyl group on the amino group at position 1 in the 2-deoxystreptamine ring,

H

CH-NHj

H CI

CH-NHj

H CI

NHCjH,

CHjMH oh

{11.94) netilmicin and this enhances the resistance of the molecule to modification by some, but not all, types of aminoglycoside-modifying enzymes. Amikacin is thus effective against several resistant strains because fewer sites on the molecule are modified. Netilmicin (A-ethylsisomycin (11.94)) is a semisynthetic derivative of ssisomicin that is less susceptible to some types of bacterial enzymes.

The tetracyclines are no longer used to the same extent as they were in the past. The most imortant members of this group are oxytetracycline (11.95), tetracycline (11.96), doxycycline (11.97), clomocycline (11.98), chlortetracycline (11.99), demethyltetracycline (11.100), methacycline (11.101), and minocycline (11.102). The microbiological spectrum tends to be similar, with cross-resistance between the individual compounds, except for minocycline. Minocycline is active against some tetracycline-resistant strains of Gram-negative bacteria and against tetracycline-resistant staphylococci. Against the former, it appears to enter the cells more readily and may be excreted less rapidly.

An isosterically related, chemically synthesized derivative, thiacycline (11.103), is more active than minocycline against tetracycline-resistant strains. This agent is thought not to inhibit protein synthesis, but exerts a bactericidal effect causing nonspecific damage to the bacterial cytoplasmic membrane. Although some toxicity problems have become apparent in the context of its possible clinical use (due to its non-selectivity), thiacycline could form a useful fore-runner for a new group of highly active tetracycline antibiotics.

Structure-activity studies in the tetracyclines have shown that inhibitory activity is increased significantly by chlorination at position 7 (e.g. (11.99)). Conversely, decreased potency occurs with epimerization of the 4-dimethylamino group (as in 4-epitetracycline) or with ring opening, e.g. in isotetracycline or apo-oxytetracycline. The carboxamide group at the C-2 position of the tetracycline molecule appears to be essential for transport into E. coli cells.

In acid conditions, tetracycline hydrochloride is converted to the inactive 5a,6-anhydro and epi-anhydro compounds. Chlortetracycline (with a 7-chloro substituent)

11.6.2 Tetracyclines is more resistant to acid-catalysed decomposition through 5a,6-anhydro compound formation, as

are (11.100), (11.101) and (11.102) where the tertiary hydroxyl group has been modified or is absent.

Recently, a new group of tetracycline analogues, the glycylcyclines, have been discovered. The glycylcyclines are novel tetracyclines substituted at the C9 position of the molecule with a dimethylglycylamido side-chain (11.104), (11.105).

The glycylcyclines possess activity against organisms expressing resistance to the older tetracyclines mediated by the determinants that encode tetracycline efflux proteins. The glycylcyclines therefore represent a significant advance within the tetracycline group of antibiotics because they are not recognised by the efflux proteins that recognise older members of this class.

These drugs also possess activity against organisms expressing the tetM and tetO determinants which probably mediate resistance to the older tetracyclines by modifying the tetracycline binding site on the bacterial 30S ribosomal subunit. Therefore the affinity, or mechanism of binding of the glycylcyclines to ribosomes modified by the TetM or TetO proteins, is presumably sufficient to prevent aminoacyl-tRNA binding despite expression of the resistance determinant in the cell.

(J H04) N,[sJ-dirncthylgiyL;ylamiti-ii-ij-deTiiicihy!-6'dei)Kytetracy<;Eirie

( 11.105 ) N, N-d i mulhy Ig] y£y larriido- m inucyCä i nu

11.6.3 Macrolides

The most important of the antibacterial macrolide antibiotics is erythromycin (11.106). This is active generally against Gram-positive bacteria and some Gram-negative ones, including Legionella pneumophila. A major effort has been made to synthesize macrolides derived from erythromycin or other naturally occurring compounds. The new macrolides are semisynthetic molecules that differ from the original compounds in the substitution pattern of the lactone ring system. The macrolides consist of a large lactone ring containing 12-16 atoms to which are attached one or more sugars. The most recent clinically useful compounds include roxithromycin (11.107), the methoxy-ethoxy-methyloxime derivative of erythromycin), clarithromycin (11.108), (the methyl derivative of erythromycin), azithromycin (11.109), deoxo-aza-methyl-homo-erythromycin, (the only 15-membered macrolide) and dirithromycin (11.110), a new derivative of

NiCH,)a

NiCH,)a

CH>COCH>CH,-OCH

HO OH I

CHyOOH

CH*CHj

Or CH

(11.106) erythromycin

(11.106) erythromycin

erythromycin. Azithromycin shows good activity against Gram-negative bacteria. The macrolides tend to be unstable and inactived by acidic media and are thus administered in enteric-coated tablets or as the more acid-stable esters and ester salts, e.g. ethyl estolate and succinate. Efforts have also been made to overcome these problems, resulting in new molecules such as miocamycin (11.111), the diacetyl derivative of midecamycin (11.112) and rokitamycin (11.113), the butyryl ester of leucomycin A5 (11.114).

11.6.4 Chloramphenicol

Chloramphenicol (11.29) possesses a broad spectrum of activity against Gram-positive and Gram-negative bacteria, and acts by inhibiting the peptidyl transferase reaction in protein synthesis. The active form is the D-threo isomer; the L-erythro, D-erythro and L-threo isomers do not inhibit protein synthesis and are all inactive as antibacterial agents. Some bacteria possess an enzyme, chloramphenicol acetyltransferase, that can inactivate the antibiotic (Section 11.4.1.3). This can pose a clinical problem, and the practical use of

chloramphenicol for systemic infections is reduced because of its tendency to cause blood dyscrasias, notably aplastic anaemia. Nevertheless, it remains an important drug, e.g. in the treatment of H. influenzae meningitis.

Chloramphenicol is administered orally as the tasteless palmitate, which is hydrolysed to chloramphenicol in the gastrointestinal tract. The highly water-soluble chloramphenicol sodium succinate comprises the injectable form; this acts as a pro-drug, and chloramphenicol is rapidly liberated, although it has been stated that only ca. one-half of the antibiotic in blood is in an active form and that plasma concentrations are lower than those achieved with a comparable oral dose of chloramphenicol.

11.6.5 Folate inhibitors

It was found in 1948 that alkyl- or phenyl- substituted diaminopyrimidines had an antifolate action. 'Small molecule' inhibitors (collectively, diaminopyrimidine derivatives) were thus studied extensively, leading to the development of compounds that were highly active against bacteria or protozoa and also to agents that were selectively toxic for the parasite rather than the host (mammalian) cells. In 1965 it was demonstrated that trimethoprim (11.22) inhibits dihydrofolate reductase (DHFR) and that its specificity of action is at the molecular level (Table 11.6). Unsubstituted diaminobenzylpyrimidines ((11.115) R1=R2=R3=H) bind poorly to E. coli DHFR; the introduction of a single methoxy group (R1 (11.115)) improves binding to some extent, whereas two methoxy groups (R1 and R2 (11.115)) improve it still further, and three methoxy groups (R1, R2 and R3, as in trimethoprim) produce a highly selective potent antibacterial agent, which binds much less strongly to human DHFR than to the bacterial enzyme. Diaminobenzylpyrimidines with good antibacterial activity can be obtained if a methoxy group is retained at R1 and R3 and a methoxyethoxy or methoxymethoxy group introduced at R2. One of the most active compounds is 2,4-diamino-5-(3',5'-dimethoxy-4'-methoxyethoxybenzyl) pyrimidine, known as tetroxoprim (11.24).

Table 11.6 Inhibitors of dihydrofolate reductase (DHFR) in clinical use. Concentrations1 binding to DHFR in: Type of compound Compound__E. coli Rat Specific use

Benzylpyrimidine Benzylpyrimidine Benzylpyrimidine Benzylpyrimidine Diaminopteridine Phenylpyrimidine Conjugated pteridine

1As 50% inhibitory concentration (I50x10-8 M), the concentration that affects 50% binding to DHFR.

Trimethoprim

Tetroxoprim

Ormetroprim

Diaveridine

Triamterene

Pyrimethamine

Methotrexate

0.50 40000 Antibacterial 8.00 40000 Antibacterial 5.00 34000 Antiprotozoal 10.00 7000 Antiprotozoal 170.00 150 Diuretic 250.00 70 Antimalarial Anticancer

The rationale for combining trimethoprim with a sulphonamide (as in co-trimoxazole) is based upon the in vitro assertion that the mixture has a markedly increased activity in comparison to either drug alone, i.e. a 'sequential blockade' in which a sulphonamide acts as a competitive inhibitor of dihydropteroate synthetase and trimethoprim inhibits DHFR. It has, moreover, often been stated that the use of such a combination reduces the risk of the emergence of resistance. Arguments about the clinical use of co-trimoxazole, however, continue and it has also been shown that the two components bind simultaneously to DHFR rather than causing the sequential blockade in the metabolic pathway referred to above. Sulphamethoxazole has been found to bind to purified E. coli DHFR, thereby preventing the conversion of dihydrofolate to tetrahydrofolate. Furthermore, synergism between trimethoprim and sulphamethoxazole is not demonstrated against sulphonamide-resistant bacteria and it is uncommon for an organism with acquired trimethoprim resistance to show sulphonamide sensitivity.

Resistance to trimethoprim, by plasmid mediation, results from an insusceptible target site, viz. an altered DHFR. About 20000 times as much trimethoprim is needed to inhibit the plasmid-encoded enzyme. Future design of diaminopyrimidines as drugs may well have to concentrate on this aspect.

11.6.6 Quinolones

The quinolone antimicrobial agents have been one of the fastest growing group of drugs in recent times. To date, more than 10000 different analogues have been synthesized. They are unusual in being totally synthesized chemically. This means that various side-chains can be altered and the resulting analogues tested for their antibacterial properties. Nalidixic acid (11.13) was derived as a by-product of chloroquine synthesis and is regarded as the progenitor of the newer quinolones. The wealth of compounds and information on structure-activity relationships precludes extensive coverage in the space allowed here but some examples will be highlighted to emphasise the importance of design on microbiological and pharmacokinetic properties.

The most important develpment was the introduction of a fluorine substituent at C-6 (see (11.7)) which led to a great increase in potency and spectrum of antibacterial activity compared to nalidixic acid (11.13). Ciprofloxacin (11.12) and norfloxacin (11.116) are examples of compounds with a fluorine at C-6, often termed collectively fluoroquinolones. These agents show excellent activity against Gram-negative bacteria, good activity against some Gram-positive bacteria but no action against anaerobes. Subsequent design of new quinolones has largely retained the C-6 fluorine moiety, with changes at other sites focusing on increasing activity against Grampositive cocci and anaerobes. An important early discovery was that the side-chain at position N-1 of the basic quinolone ring had a substantial effect on potency. Ciprofloxacin (11.12) possess a cyclopropyl moiety at N-1. Norfloxacin (11.116) is identical to ciprofloxacin in all respects except for an ethyl rather than cyclopropyl side-chain at N-1, yet ciprofloxacin is more active against Gram-negative and -positive bacteria. Ciprofloxacin and norfloxacin belong to the second generation of quinolones.

The third and most recently developed generation of quinolones has maintained many of the properties of the second generation. Some compounds (e.g. lomefloxacin (11.117)) differ in having sufficiently long half-lives to allow once daily dosing. Lomefloxacin has a second fluorine atom at C-8, an ethyl group at N-1 (cf norfloxacin (11.116)) and a methyl group on the piperazinyl group at C-7. There are, however, adverse photosensitivity reactions now being recognised with this compound.

Two other third generation quinolones are worthy of note. Sparfloxacin (11.118), a difluorinated quinolone, shows enhanced activity against Gram-positive cocci and anaerobes, whilst retaining high activity against Gram-negative bacteria. Sparfloxacin possesses a cyclopropyl side-chain at N-1 (cf. ciprofloxacin (11.12), a second fluorine at C-8 (cf. lomefloxacin (11.117)), an amino group at C-5 and a substituted piperazinyl ring. Temafloxacin (11.119) differs from ciprofloxacin in possessing a difluorophenyl side-chain at N-1 (a trifluorinated quinolone) and a methyl substituent on the piperazinyl side-chain at C-7. Unfortunately, severe haemolytic and nephrotoxic reactions occurred unexpectedly after the marketing of this drug, leading to its subsequent withdrawal.

A novel avenue of antibiotic research involves the synthesis of dual action quinolone-cephalosporin hybrids. Simultaneous administration of drugs is used occasionally to broaden the spectrum of activity or to counteract resistance. If two such drugs could be combined in one molecule (prodrug), with release of the active constituents after

metabolism, problems associated with absorption could be reduced. Though experimental quinolone-cephalosporin hybrids have been synthesized, the data available at the time of writing is inconclusive and the clinical applicability of such hybrids is not yet clear.

11.7 DESIGN OF ANTIFUNGAL AGENTS

The design of antifungal agent poses problems different from those associated with the design of antibacterial drugs. Whereas lack of human toxicity with both types of drug is a factor of paramount importance, the differences in structure of, and some biosynthetic processes in, the fungal cell mean that antibacterial antibiotics are usually without action against fungi. Fungal infections are normally less virulent in nature than are bacterial or viral infections. Furthermore, the fungal cells are eucaryotic, as are human cells, and consequently difficulties arise in designing appropriate chemotherapeutic drugs. One possible target is the fungal cell wall, and considerable advances have been made in understanding its structure and biosynthesis. Another is the cytoplasmic membrane.

The ideal antifungal agent should be fungicidal, have a broad spectrum, be in a suitable form for oral and intravenous administration and should have adequate penetration of body fluids; in addition, no resistance should develop during therapy. No drug currently in clinical use satisfies all of these criteria.

The most important chemotherapeutic antifungal agents are the macrolide polyenic antibiotics, imidazole derivatives, flucytosine and griseofulvin, a very small number of useful agents in comparison to the very large number of antibacterial antibiotics.

11.7.1 Polyene antibiotics

The great majority of polyene antibiotics are produced by Streptomyces species, with nystatin (11.120) the first to be isolated. The macrolide ring of the polyenes is larger than that of the other macrolide group (exemplified by erythromycin (11.106)) and contains a series of conjugated double bonds. An ultraviolet absorption spectrum enables a polyenic antibiotic to be classified, on the basis of the number of olefinic (alkenylic) bonds present, into trienes, tetraenes, pentaenes, hexaenes and heptaenes. Generally, antifungal activity increases with the number of conjugated olefinic bonds, although solubility decreases from the tetraenes to the heptaenes.

The polyenes act by combining with the cytoplasmic membrane; this is achieved by an interaction between the antibiotic and membrane sterol, disrupting membrane integrity. Consequently, only those organisms containing sterol in the membrane are sensitive. The

NH
0 0

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