Some Active Derivatives of Penicillin

Transcription

1 APPuED MICOBIOLOGY, Mar. 1969, p Vol. 17, No. 3 Copyright 1969 American Society for Microbiology Printed in U.S.A. Some Active Derivatives of Penicillin LOMAN D. BOWN,1 WALTE A. ZYGMUNT, AND HOME E. STAVELY2 Mead Johnson esearch Center, Evansville, Indiana eceived for publication 9 December 1968 The antibacterial activities of a number of amide derivatives of penicillin against both penicillin-sensitive and penicillin-resistant cultures were determined. Several of them were found to possess significant inhibitory activity against certain grampositive bacteria. The amides, although resistant to the destructive action of f- lactamase, did not protect penicillin G in competitive experiments. One derivative, the O-benzylhydroxamide of penicillin G, was active against six or eight penicillinresistant strains of Staphylococcus aureus (minimal inhibitory concentration, 0.2,ug/ml or less), but was found to have only a minimal in vivo activity against mouse Streptococcus infections. Although several clinically useful synthetic penicillins have been prepared by the acylation of 6-amino-penicillanic acid, chemical modification of the natural penicillins has been unproductive. Several carboxyl derivatives which were microbiologically inactive or possessed only a low order of activity have been prepared (8, 10). It has been generally assumed that they are inactive because they lack the electron-negative carboxyl group necessary for attachment to some electron-deficient active site. A number of semisynthetic penicillins have been developed (methicillin, oxacillin, etc.) which are markedly resistant to destruction by,3- lactamase. Many investigators think that the bulky acyl group adjacent to the,3-lactam ring present in these derivatives contributes to this stability. However, penicillin amide derivatives are also resistant to f3-lactamase destruction (2, 3). Such derivatives may owe their resistance to the fact that, lacking the electron-negative carboxyl group, they are incapable of forming a substrate-enzyme complex. On the other hand, if these penicillin amides can form stable enzyme complexes, they might act as competitive inhibitors and thus prevent the destruction of penicillin G when the two compounds are present simultaneously. The original reports (3, 8) concerning the low activity of the amides of penicillin G referred only to activity against a few microorganisms. Inview of the stability of these compounds toward 3-lactamase, it was decided to test them against a variety of bacteria in the hope that useful activity I Present address: The J. M. Smucker Co., P. 0. Box 280, Orrville, Ohio Present address: Medical College of Ohio at Toledo, Toledo, Ohio would be uncovered against one or more penicillin G-resistant organisms. It occurred to us that a penicillin hydroxamic acid might be a uniquely active compound, impervious to f3-lactamase by virtue of the amide linkage, yet active because of the acidic character of the group. We found that the hydroxamic acids of several penicillins have been disclosed in the patent literature (4, 5). In our experiments, the synthetic methods given in the patents were not productive, so an alternate synthetic method for penicillin G hydroxamic acid was developed. An intermediate in our synthesis was O-benzylhydroxamide of penicillin G (compound 5) from which the hydroxamic acid was obtained by catalytic hydrogenation (Fig. 1). A well-supported mechanism of penicillin action (9, 11) envisions a penicillin acting as a peptide, competing with the cell wall peptide for an enzyme site. It is, therefore, conceivable that the addition of amino acid residues to the penicillin carboxyl group might result in a peptide having a different spectrum of activity. This idea was explored by the preparation of several such peptides, compounds 7 to 10, 13, and 19. MATEIALS AND METHODS Penicillin derivatives. The penicillin amides (compounds 2 to 5, 15, 17, and 18) were all prepared by treating a penicillin-isobutyl carbonic mixed anhydride intermediate with ammonia or a suitable substituted ammonia. A typical synthesis is as follows. Benzyl penicillinamide-benzyl penicillanic acid (3.51 g) was dissolved in 50 ml of methylene chloride containing 1.39 ml of triethylamine and was cooled to -5 C. With vigorous stirring, 1.32 ml of isobutylchloroformate was added at 0 C. After 30 min, 100 ml of 15% dibasic ammonium phosphate solution was added. The reaction mixture was stirred vigor- 339

2 340 BOWN, ZYGMUNT, AND STAVELY APPL. MICOBIOL. CHC2- Co NH -CH3 2 ~~~~~~~~~~CH3 H o,, N s CON-OCH2C6H5 FIG. 1. Alternate synthetic methodfor penicillin G hydroxamic acid. PENICILLIN G DEIVATIVES MISCELLANEOUS DEIVATIVES CHC-N 0 TO N C~ -N CO '-NH S 1 NH2 NHC, H, NHCH2C6 H, NHOCH, C6 H, NH NHCH2CO N CO ' C6H5CHOCO CH3CO CH3CO Cephalothin Cephalothin amide NHqH-CONH-qHCO CH3 CH3 (D-ala-D-ala ) FIG. 3. Miscellaneous derivatives prepared and tested 8 NHCH2CONH-CH-CO C4 H, NHCOOCH2 C, H, (gly-nc-cbz-l-lys) AMPICILLIN DEIVATIVES 9 NHCH2CONHCHCONHCH2CO (gly-gly-gly) 10 NH-CH-CO (CH,),NHCOOCHCH, FIG. 2. Penicillin G derivatives prepared and tested. ously for 3 hr at room temperature, and the layers were separated. The mixture was then treated with 25 ml of a freshly prepared 15% phosphate solution. The two-phase solution was chilled and poured into three volumes of cyclohexane. The product precipitated as a white gum. It was washed with K2C03 solution, crystallized with methylene chloride-cyclohexane, and then dried in a dessicator. Product was obtained as the trihydrate. The yield was 2.76 g, and the melting point was 65 to 66 C. Analysis: C16H19N303S *3H20; calculated: C, 49.59; H, 6.50; N, 10.84; found: C, 49.41; H, 6.50; N, s with a free carboxylic acid group (compounds 7 to 13, 19, and 20) were prepared by the mixed anhydride method and were precipitated as triethylamine salts. A list of the penicillin derivatives prepared and tested is given in Fig. 2 to 4.,#-Lactamase susceptibility. The sensitivity to filactamase was determined by the method of Baudet and Cherbuliez (1), which depends upon the difference in the intensity of ultraviolet (UV) absorption at 240 and nm. Test compounds, 2 jmmoles/ml, were exposed to 3.5 units of penicillinase (iker Laboratories, Inc., Northridge, Calif.) per ml for 16 hr at room temperature CH-CO-NH 'oṉ H ' COOCH2 C6 H, COOCH2C6 H5 COOCH, C6 H, COOCH2C6 H, CO NH2 NHC2Hs NHCH2CO FIG. 4. Ampicillin derivatives prepared and tested. Sensitivity tests. Minimal inhibitory concentrations (MIC) for each test compound were determined by conventional twofold serial dilution techniques in Penassay Broth (Difco). An inoculum level of approximately 105 viable cells per ml of medium was used. All compounds were dissolved in 0.1 M phosphate buffer (ph 6.0) or in N, N-dimethylformamide. Stock solutions of the compounds were sterilized by Seitz filtration and were further diluted aspetically in phosphate buffer to equimolar concentrations (5.37 X 10-4 M). This concentration is equivalent to 200 jug of penicillin G potassium salt per ml. The

3 VOL. 17, 1969 ACTIVE DEIVATIVES OF PENICILLIN 341 TABLE 1. In vitro susceptibility of various bacteria to penicillin derivatives" CdihereCS aureusbs. aureus Corn- E. M. co lyso- M. roes Spygn s lea S pound deikticus Mospus pyogenhagenb 4180C 1 >25d > 25 2 > >25 3 > >25 > >25 >25 4 >25 > > >25 >25 5 >25 > >25 6 >25 > > >25 8 > >25 9 > >25 10 >25 > >25 >25 >25 11 > >25 12 > > >25 13 > >25 14 > >25 15 >25 > >25 > >25 17 > > > > >25 19 > >25 20 > >25 a These compounds were also tested against the following gram-negative organisms: E. coli, (ampicillin-resistant strain), Salmonella choleraesuis, S. typhimurium, Shigella paradysteneriae, Proteus vulgaris, P. morganii, Enterobacter aerogenes, Klebsiella pneumoniae, and Pseudomonas aeruginosa (MIC values all > 100 jig/ml). b Penicillin G-sensitive strain. c Penicillin G-resistant strain. d MIC, in micrograms per milliliter. TABLE 2. Microbiological activity of various penicillins against penicillin-sensitive and penicillin-resistant strains of S. aureus ' Copen- 209P hagen Penicillin G (1) >50 >50 >50 >50 >50 >50 >50 Ampicillin (16) Oxacillin Methicillin Penicillin G benzylhydroxamide (5) Penicillin G hydroxamic acid (6) a in parentheses following name of compound indicates compound number. b MIC, in micrograms per milliliter. antimicrobial activities are expressed in terms of an MIC relative to this stock solution of penicillin G. The in vivo activity of one of the compounds, penicillin G benzylhydroxamide (compound 5), was compared to penicillin G and cloxacillin in mice infected with Streptococcus pyogenes (strain C-203). Groups of 10 mice were infected intraperitoneally, and medication was given parenterally (subcutaneous) at 2, 6, and postinfection. The mean survival time for each group of animals and the ED66 were calculated. ESULTS AND DISCUSSION Initially, all compounds were tested against an ampicillin-sensitive strain of Escherichia coli and a miscellaneous group of seven gram-positive bacteria (Table 1). In this general screen, all amide derivatives possessed high activity against Micrococcus roseus and Corynebacterium diphtheriae, but against the other gram-positive bacteria only some of the derivatives were ac-

4 342 BOWN, ZYGMUNT, AND STAVELY APPL. MICOBIOL. tive. Activity could be a function of the presence of an extracellular amidase which converts the amide derivatives back to penicillin G. This possibility is being investigated. All derivatives which possessed significant activity against the Copenhagen strain of Staphylococcus aureus were retested with a penicillin G-resistant strain (4180) of S. aureus (Table 1). In every case (except compounds 5, 6, and 15), there was significantly less activity against the resistant strain, although compounds 5, 6, 15, and 17 were more active than penicillin G. The O-benzyl-hydroxamide of penicillin V and several other 0-substituted hydroxamides were highly active against two resistant strains of S. aureus (4). s 5 (the O-benzyl derivative) and 6 (penicillin G hydroxamic acid) were then assayed against an additional group of 7- * A A- A A 6- an PENICILLIN /CLOXACILLIN G / (ED5o 3.7 glkg) (ED50 = 0.16 mg/kg) / >~~~~~~ CS >~~~~~~~ // 52-/ I PENICILLINGBENZYLHYDOXAMIDE 1-0/w, (ED50 = 32 mg/kg) 0- i i 4 h i CONTOL O.; Mg/kg (3 doses s. c.) FIG. 5. Chemotherapeutic effect ofpenicillins on streptococcal infections in mice. seven penicillin G-resistant clinical isolates of S. aureus (Table 2). It is apparent that compound 5 has a greater activity against five of these strains than either oxacillin or methicillin. The resistance of this compound to f-lactamase was also determined by the manometric method of Henry and Housewright (7). In this assay, its susceptibility to fl-lactamase approximated that of cloxacillin. Contrary to expectations, penicillin G hydroxamic acid (compound 6) was a disappointing compound and was consistently less active than the neutral O-benzylhydroxamide derivative. The derivatives of penicillin G in which one or more amino acids are linked to the molecule through the carboxyl group (compounds 7 to 10, 13, and 19) were not outstanding in their antibacterial activity, although they were more active against M. roseus, S. pyogenes, Sarcina lutea, and C. diphtheriae than those amide derivatives (compounds 2 to 4) which did not possess a terminal carboxyl group. Cephalothinamide (compound 15) appears to be less active than cephalothin (compound 14) per se (Table 1). (Benzyloxy)carbonylampicillin (compound 20) retained full activity against gram-positive bacteria but was much less active against an ampicillin-sensitive strain of E. coli than ampicillin (compound 16). The stability of several of the penicillin amides toward f-lactamase was confirmed. Attempts to prevent inactivation of penicillin G by competition with penicillin G amide were unsuccessful. Combinations of penicillin G and the amide in TABLE 3. Effect of bovine serum on the antistaphylococcal activity of oxacillin and penicillin G benzylhydroxamide S. aureus strain hr 48 hr 48 hr 48 hr Sodium oxacillin Bovine seruma mg/ml mg/ml... Penicillin G benzylhydroxamide Bovine serum mg/ml mg/ml... b 3.12 >25 < a Bovine serum = lyophilized (Pentex). b MIC, in micrograms per milliliter.

5 VOL. 17, 1969 ACITIVE DEIVATIVES OF PENICILLIN 343 various ratios were incubated with f3-lactamase. There was a suggestion of penicillin G protection by the amide, but no significant differences were found. Similarly, eight different penicillinaseproducing strains of S. aureus were found to be equally refractory (MIC values all greater than 100 mg/ml) to penicillin G, penicillin G amide, and various combinations of the two agents. In view of these results, it is unlikely that amide derivatives can be expected to protect penicillin G against /3-lactamase by a competitive inhibition mechanism. It appears that the amide is able to form only a loose enzyme complex, if at all. Of the 17 compounds synthesized, penicillin G benzyl-hydroxamide (compound 5) demonstrated the greatest degree of antistaphylococcal activity (Table 2). Since the magnitude of the in vivo activity of this compound was unknown, it was of interest to determine its activity in mice infected with streptococci. A low order of chemotherapeutic effectiveness was obtained with this derivative (Fig. 5). The poor in vivo activity of the compound may be attributable to its high degree of binding to serum proteins (Table 3). ACKNOWLEDGMENTS We thank Edward F. Harrison and Peter Zwadyk, Jr., for the mouse protection and manometric studies. We are also indebted to E. Townsend for technical assistance. LITEATUE CITED 1. Baudet, P., and E. Cherbuliez echerches sur la penicillinase. I. Dosage spectrophotometrique de la penicillinase. Helv. Chim. Acta 36: Cooper, D. E Penicillin amides. U.S. Patent no. 2,593,852. B. 3. Cooper, D. E., and S. Am. Chem. Soc. 70: Binkley Penicillin amide. J. 4. Coronelli, C., G. C. Lancini,. Pallanza, G. Tamoni, and P. Sensi Idrossilamidi della penicillina V. Farmaco (Pavia) Ed. Sci. 21: Godfrey, J. C N-hydroxypenicillin amides. U.S. Patent no. 3,301, Goldberg, M. W., and S. Teitel Penicillin derivatives. U.S. Patent no. 3,177, Henry,. J., and. D. Housewright Studies on penicillinase. II. Manometric method of assaying penicillinase and penicillin, kinetics of the penicillin-penicillinase reaction, and the effects of inhibitors on penicillinase. J. Biol. Chem. 167: Holysz,. P., and H. E. Stavely Carboxy derivatives of benzylpenicillin. J. Am. Chem. Soc. 72: Izaki, K., M. Matsuhashi, and J. L. Strominger Glycopeptide transpeptidase and D-alanine carboxypeptidase: penicillin-sensitive enzymatic reactions. Proc. Natl. Acad. Sci. U.S. 55: Johnson, D. A Carboxy derivatives of benzylpenicillin. J. Am. Chem. Soc. 75: Wise, E. M., and J. T. Park Penicillin: its basic site of action as an inhibitor of a peptide cross-linking reaction in cell wall mucopeptide synthesis. Proc. Natd. Acad. Sci. U.S. 54:75-81.