Development of the semi-synthetic penicillins and cephalosporins

International Journal of Antimicrobial Agents 31 (2008) 189–192

Commentary

Development of the semi-synthetic penicillins and cephalosporins J.M.T. Hamilton-Miller ∗ Department of Medical Microbiology, Royal Free and University College Medical School, London, UK Received 24 November 2007; accepted 26 November 2007

Abstract Semi-synthetic penicillins and cephalosporins both derive from their respective chemical nuclei, 6-aminopenicillanic acid (6-APA) and 7-aminocephalosporanic acid (7-ACA). Work leading to their isolation was being carried out in parallel, but following very different pathways, during the last half of the 1950s. The development of 6-APA was reviewed recently in this journal, and in the present article I take a closer look at early work on ‘penicillin amidase’ and revisit the steps that led to 7-ACA. © 2007 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: 6-Aminopenicillanic acid; 7-Aminocephalosporanic acid; ␤-Lactam antibiotics; History of medicine

1. Introduction

2. Work in Japan during the 1950s

Living history, as illustrated in the recent review in this journal by Rolinson and Geddes [1] on the discovery of 6-aminopenicillanic acid (6-APA) 50 years ago, is inspirational. Thus, memories are turned into history. The modern generation of microbiologists and medical practitioners take antibiotics for granted, and it is salutary for them to be able to read at first hand of the ingenuity, insight and hard work that went into the birth of the semi-synthetic penicillins. I am grateful for the opportunity to be able to add some further memories to this story, in which I have personal interests. Some 40 years ago I undertook a comprehensive review of penicillin acylase [2], having been initially confused by the fact that this enzyme was also being called penicillin amidase in the early literature. (I subsequently discovered that there was indeed a genuine penicillin amidase, whose substrate was penicillinamide.) Shortly afterwards I started a postdoctoral fellowship at The Sir William Dunn School of Pathology with Edward Abraham. The latter, with Guy Newton, had isolated 7-aminocephalosporanic acid (7-ACA) and thus followed a path parallel to that of the semi-synthetic penicillins, but, as will be shown below, going by an entirely different route.

Rolinson & Geddes [1] in section 4 of their review discuss some of the early reports from Japan suggesting that “penicillin nucleus” or “penicin” could possibly have been 6APA. This discussion can be amplified somewhat by considering some other papers by the same authors published in the Japanese literature (with complete or partial translation) before the definitive Beechams paper on 6APA in 1959 [12]. These early papers were concerned solely with aspects of the industrial production of penicillin, or biosynthetic pathways. Sakaguchi and Murao [3] investigated a decrease in penicillin titre after peak levels had been obtained. They showed ‘penicin’, represented structurally in their paper by a formula we recognise as 6-APA, to be formed together with phenylacetic acid when mycelium of Penicillium chrysogenum Q167 was incubated with benzylpenicillin. They followed this process by converting penicin to its penicilloate, which they called ‘penicic acid’, and assaying with ninhydrin. Kato [4,5] described what he called ‘penicillin nucleus’ to account for discrepancies in iodometric assays of penicillin production by P. chrysogenum Q167. Crucially, he reported that ‘penicillin nucleus’ was a substrate for, and inducer of, penicillinase from Bacillus mycoides (thus showing that the ␤-lactam ring was present), and was only produced when phenylacetate was absent from the fermentation broth. He was, however, reluctant to confirm that ‘penicillin nucleus’ was identical to the previously described penicin. Murao pub-

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lished two more papers in 1955 [6,7] confirming his previous findings in more detail, and also showing that amidase activity was increased in the presence of sulfathiazole. Murao produced two more papers on penicillin amidase in 1961, but these are entirely in Japanese. Many of the findings reported in the early Japanese work were confirmed later [8–10]. Whilst there were some inconsistencies, in particular the fact that the reported melting point of penicin differed from that of authentic 6-APA, there seems little doubt that both Murao’s ‘penicin’ and Kato’s ‘penicillin nucleus’ were 6-APA. Sir Edward Abraham seemed convinced, as he wrote in 1977 [11], “There can be no serious doubt that 6APA was responsible for these observations”; and in 1990 [12], “Convincing evidence for the existence of the penicillin nucleus in fermentation fluids was first obtained in Japan by Sakaguchi and Murao in 1950. They did not pursue these findings. . .”. However, it must be stressed that the importance of 6APA as a precursor of semi-synthetic penicillins was not appreciated until the ground-breaking studies at Beecham Laboratories, described by Rolinson and Geddes [1].

3. Work at Oxford towards 7-ACA The isolation of 6-APA by scientists at Beecham Laboratories, which actually occurred in the summer of 1957 [1] but was not reported in the literature until 2 years later [13], preceded 7-ACA, isolated in 1959 by Abraham at Oxford [14], reported 2 years later [15]. Abraham was first and foremost a peptide chemist, and an extraordinarily skilled one. He showed his prowess on two separate spectacular but largely unreported occasions, by being right when two of the greatest organic chemists of the 20th century were wrong. In the early 1940s Abraham correctly suggested the fused ␤lactam/thiazolidine structure for penicillin, whilst Sir Robert Robinson strongly favoured an alternative answer, and in the mid 1950s Woodward disagreed with Abraham’s (correct) assignation of a ␤-lactam/dihydrothiazine configuration for cephalosporins [16]. I worked closely with Abraham for 3.5 years and found him always to be at least one step ahead of the current project; he surely would have realised the potential for side-chain replacement as soon as he and Newton noticed in 1955 the resemblance of cephalosporin C to penicillins [17]. Abraham was also keenly aware of clinical needs, following the dramas during the war years involved in penicillin production, and then the rise of penicillinase-producing staphylococci. Abraham reported to Florey, and the latter retained his interest in antibiotics after research on penicillin had ended: Florey investigated several antibiotics during the decade 1945–1955, notably micrococcin against Mycobacterium tuberculosis [18], before reverting to research on the vascular system and mucus. Florey was very interested in the results obtained by Abraham and Newton on the antibiotic properties of Brotzu’s Cephalosporium acremonium cul-

ture; thus, in 1953 when Abraham realised the potential of cephalosporin C, he telephoned Florey in Australia to obtain his permission to continue working on it. Permission was gladly given, provided work on penicillin N continued (this compound seemed at the time very promising and in due course a successful trial was carried out in the USA on typhoid fever), and Florey went on to carry out toxicity and efficacy studies in animals on cephalosporin C. Another incentive to pursue studies at that time on potentially useful antibiotics was undoubtedly a financial one. Contrary to common perception, it was not Florey himself who was against taking out patents on penicillin in the early 1940s; it was others who objected (notably Mellanby) and also there was simply no mechanism at that time for either Oxford University or the Medical Research Council to handle patents [18]. However, there was clear change of attitude during the 1950s, and patents on the cephalosporin antibiotics were taken out by the Oxford workers and assigned to the National Research and Development Corporation (NRDC), to the great benefit of, amongst others, Florey’s department. Abraham was very aware of the possibilities of altering the properties of penicillins and cephalosporins by chemical manipulation. He had seen at first hand in the early 1940s the subtly different properties of the biosynthetic products of P. chrysogenum (e.g. penicillins G, F, K and later V) as well as the marked changes found when an amino group was present in the side chain, as in penicillin N, which he and Newton isolated and identified from C. acremonium in 1954 [19]. He wrote of cephalosporin C in one of his narrative accounts [20], “There was a great incentive to modify the molecule chemically with a view to increasing its intrinsic activity without affecting its resistance to staphylococcal penicillinase”. Further, before any work had been done on cephalosporin C, the Oxford team was alive to the possibility of chemical modification of an existing antibiotic. Dr John Jones tells me [personal communication] he has found a letter dated 25 May 1953 from Florey to Abraham: “It will be very interesting if you can really manipulate the molecule, as seems likely. At any rate it would mean much employment for chemists in the drug firms”. The molecule referred here was presumably penicillin N. Abraham then set up a systematic programme of modifying the existing side chains of cephalosporin C and observing the effects on microbiological activity. Thus, activity against Gram-positive species was increased by substituting a nitrogenous base at C3 (making the cephalosporin CA family—this type of chemistry is present in, e.g., ceftazidime and cefpirome) and by acetylating the amino group in the ␣-aminoadipyl side chain at C7 [21]. Reduced activity was found when the acetyl group at C3 was diminished or removed, giving cephalosporin CC , and also in desacetyland desacetoxy-cephalosporin C (the latter chemistry being found in some orally absorbed cephalosporins, e.g. cefalexin and cefradine) [22,23]. These studies were taking place at the same time as the penicillinase-producing staphylococci were taking a firm hold in hospitals. It was indeed considered a distinct

J.M.T. Hamilton-Miller / International Journal of Antimicrobial Agents 31 (2008) 189–192

possibility that cephalosporin C (or a more active derivative, such as described in the preceding paragraph) would be used clinically, as large amounts of pure substance were available due to the discovery of a mutant of Brotzu’s fungus that produced much higher yields [24]. However, the introduction of methicillin in 1960 immediately halted this line of thought, although there is one report of a small uncontrolled trial of cephalosporin C in urinary infections caused by Klebsiella spp. [25]. It will never be known precisely at what stage Abraham decided to try to isolate 7-ACA. An exact timeline is not available, but we know that he had succeeded by August 1959, as that is when a patent application was made [14]. Abraham certainly knew that isolation of 6-APA was to be of the greatest importance, as in 1958 he was involved in negotiations between NRDC and Eli Lilly; workers in Lilly were at that time endeavouring to isolate 6-APA [24]. They would have been unaware at that time of the success of the Beecham workers in 1957, as the latter’s patent application would have been kept a closely guarded secret. Sheehan reported at a CIBA Foundation Symposium held in March 1958 that his group had produced 6-APA by total chemical synthesis, but he had been very doubtful about its inherent stability. His remarks [26] indicate that he considered 6-APA of interest in the study of the biosynthesis of penicillins rather than the production of novel compounds. The work on isolating 7-ACA had undoubtedly started prior to Beecham’s announcement; Abraham wrote, “After this work was projected, Batchelor, Doyle, Nayler and Rolinson reported the isolation of 6APA” [15]. It seems, therefore, that he had foreseen the potential importance of isolating 7-ACA even before he was certain of the structure of cephalosporin C. The latter had not been firmly established until April 1959 [24], and published 2 years later [27]. Obtaining 7-ACA from cephalosporin C proved to be a very difficult task, and even for such a skilled chemist as Abraham the process must have taken a great deal of time. The routes available for 6-APA production, namely fermentation in the absence of a side chain and enzymatic removal of a side chain, are not applicable for the cephalosporin molecule. The latter was unexpected: “To our considerable surprise, no enzyme able to remove the d-␣-aminoadipyl sidechain could be found” [20]; and obviously much time and effort had gone into looking for such an acylase. On the other hand, the methods used to obtain 7-ACA are not applicable to 6-APA, as the latter is not acid-stable. It should not be forgotten that Abraham’s laboratory was very small: Newton was the only other (semi-permanent) member of staff, and there was usually one DPhil student. Nonetheless, in Abraham’s own words [24]: “by July 1959, small amounts of 7ACA had been isolated in relatively pure state and its N-phenylacetyl derivative had been shown to be much more active than cephalosporin C against a penicillinase-producing strain of Staphylococcus aureus” (the derivative mentioned, later called cephaloram, is the cephalosporin analogue of benzylpenicillin). Guy Newton

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told me that he had used some of that precious supply on the same day that the structure of methicillin was published in 1960 to make the latter’s cephalosporin analogue, so he and Abraham were obviously keeping a very close watch on the penicillin situation. 7-ACA produced in Oxford (by acid hydrolysis of cephalosporin C) was at a yield of <1%, much too low to be of commercial value; it was the discovery at the laboratories of Eli Lilly of a novel chemical method for cleaving the ␣-aminoadipoyl side chain of cephalosporin C that produced 7-ACA in sufficient quantities to make the first of the semi-synthetic cephalosporins [28]. 4. Conclusion As with all great scientific discoveries, there are not only stars that shine brightly in the firmament but also a host of background lights. The production of 6-APA and 7-ACA required great scientific ingenuity and much hard work; there is sufficient reward to be shared by all—as Sir Almroth Wright commented [29] about the discovery of penicillin: Palmam qui meruit ferat [‘Let the credit be given to those who deserve it’]. Acknowledgments I am very grateful to Dr John Jones, Balliol College, Oxford University, for sight of reference [16] prior to publication as well as other helpful communications; to David Evans, Senior Librarian at Hampstead Campus, The Royal Free & University College Medical School, for his help in obtaining various papers; and to Graham Arnot for a patent search. Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required. References [1] Rolinson GN, Geddes AM. The 50th anniversary of the discovery of 6aminopenicillanic acid (6-APA). Int J Antimicrob Agents 2007;29:3–8. [2] Hamilton-Miller JMT. Penicillinacylase. Bacteriol Rev 1966;30:761–71. [3] Sakaguchi K, Murao S. A preliminary report on a new enzyme ‘penicillin-amidase’. J Agric Chem Soc Jpn 1950;23:411. [4] Kato K. Occurrence of penicillin-nucleus in culture broths. J Antibiot Ser A 1953;6:130–6. [5] Kato K. Further notes on penicillin-nucleus. J Antibiot Ser A 1953;6:184–5. [6] Murao S. Studies on penicillin-amidase. Part 2. Research on conditions of producing penicillin-amidase. J Agric Chem Soc Jpn 1955;29:400–3. [7] Murao S. Studies on penicillin-amidase. Part 3. Researches of penicillin-amidase. Mechanism on Na-penicillin G. J Agric Chem Soc Jpn 1955;29:404–7. [8] Erickson RC, Bennett RE. Degradation of penicillins to 6aminopenicillanic acid by Penicillium chrysogenum. Bact Proc 1961:65.

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[9] Claridge CA, Luttinger JR, Lein J. Specificity of penicillin amidases. Proc Soc Exp Biol Med 1963;113:1008–12. [10] Erickson RC, Bennett RE. Penicillin acylase activity of Penicillium chrysogenum. Appl Microbiol 1965;13:738–42. [11] Abraham EP. ␤-Lactam antibiotics and related substances. Jpn J Antibiot 1977;30(Suppl):S1–26. [12] Abraham EP. Oxford, Howard Florey and World War II. In: Moberg CL, Cohn ZA, editors. Launching the antibiotic era. New York, NY: Rockefeller Press; 1990. p. 19–30. [13] Batchelor FR, Doyle FP, Nayler JHC, Rolinson GN. Synthesis of penicillin: 6-aminopenicillanic acid in penicillin fermentations. Nature 1959;183:257–8. [14] Abraham EP, Newton GGF, Boothroyd B. Derivatives of cephalosporin C. British Patent Application no. 26569/59 (Patent 953,695) application date August 4, 1959. [15] Loder B, Newton GGF, Abraham EP. The cephalosporin C nucleus (7amino-cephalosporanic acid) and some of its derivatives. Biochem J 1961;79:408–16. [16] Curtis R, Jones J. Robert Robinson and penicillin: an unnoticed document in the saga of its structure. J Pept Sci 2007;13:769–75. [17] Newton GGF, Abraham EP. Cephalosporin C, a new antibiotic containing sulphur and d-␣-aminoadipic acid. Nature 1955;175:548. [18] Abraham EP. Howard Walter Florey Baron Florey of Adelaide and Marston. Biogr Mem Fellows R Soc 1971;17:255–301. [19] Newton GGF, Abraham EP. Degradation, structure and some derivatives of cephalosporin N. Biochem J 1954;58:103–11.

[20] Abraham EP. Reflections on the development of the cephalosporins. G Ital Chemioter 1970;17:4–13. [21] Hale CW, Newton GGF, Abraham EP. Derivatives of cephalosporin C formed with certain heterocyclic tertiary bases: the cephalosporin CA family. Biochem J 1961;79:403–8. [22] Jeffrey JD, Abraham EP, Newton GGF. Deacetylcephalosporin C. Biochem J 1961;81:591–6. [23] Abraham EP, Newton GGF. Experiments on the degradation of cephalosporin C. Biochem J 1956;62:658–66. [24] Abraham EP, Loder PB. Cephalosporin C. In: Flynn EH, editor. Cephalosporins and penicillins: chemistry and biology. New York/London: Academic Press; 1972. p. 1–26. [25] Fleming PC. Cephalosporin C and cephalosporinase—some laboratory and clinical considerations. Can J Public Health 1963;54:47. [26] Sheehan JC. Discussion following paper by Birch & Smith. In: Wolstenholme GEW, O’Connor CM, editors. CIBA Foundation Symposium on Peptides with Antimetabolic Activity. London, UK: Churchill; 1958. p. 247–60. [27] Abraham EP, Newton GGF. The structure of cephalosporin C. Biochem J 1961;79:377–93. [28] Morin RB, Jackson BG, Flynn EH, Roeske RW. Chemistry of cephalosporin antibiotics I. 7-aminocephalosporanic acid from cephalosporin C. J Am Chem Soc 1962;84:3400–1. [29] Wright AE. Letter to the Editor of The Times, August 31, 1942.