Methyls and Me

Like Proust’s famous encounter with a tasty madeleine, my encounter with the recent paper by Gerstenberger et al. (1) triggered memories of a longstanding romance with methyl moieties, my professional career having been flanked by projects focusing on methyl groups. My master’s studies at the University of Illinois at Urbana–Champaign dealt with the methanogenic dissimilation of methanol (and methylamine) by the archaeon Methanosarcina barkeri, (2) and my doctoral studies at the Worcester Foundation for Experimental Biology, Inc. (Shrewsbury, MA) centered on biohydroxylation reactions at chiral methyl moieties. (3) Some four decades later my encounter with a strategically placed methyl group led to the creation of a pharmaceutical startup and invention of a new and exciting antibacterial drug. Most of my working life has been dedicated to applied microbiological research. Gerstenberger et al.’s publication on the nearly 400-fold improvement in potency observed for a cis-2-methylcyclohexanamine derivative compared to that of the unmethylated congener reminded me of some examples of the value of methylation on the potency of antibacterial products, a subject barely addressed in reviews of methylation effects in medicinal chemistry published during the past 15 years. (4) Coumarins are a class of antibacterial compounds biosynthesized by streptomycetes. The only coumarin antibiotic to have been commercialized is novobiocin, which was used to treat staphylococcal infections until its withdrawal from the market during the 1980s due to safety and efficacy concerns. (5) Nonetheless, interest in antibacterial coumarins persists. (6) In their study of 3-aryl-6-nitrocoumarins, Matos et al. (7) reported that replacement of a hydrogen atom by a methyl group at the meta position of the aryl moiety produced an increase (≥2 log₂ dilution steps) in in vitro activity toward Staphylococcus aureus, though the reason for this enhanced antibacterial activity by meta-methylation was not explored. Quinolones are synthetic broad-spectrum antibiotics suitable for outpatient use due to their high bioavailability. Most members of this antibiotic family contain a fluorine atom at position 6, following Koga et al.’s report (8) that the presence of a fluorine atom at C-6 of the quinolone pharmacophore improved antimicrobial activity and oral bioavailability. One such fluoroquinolone, ciprofloxacin, featured prominently as a treatment for exposure to Bacillus anthracis spores during the anthrax scare following the 9/11 terrorist attacks on the New York City World Trade Center towers. (9) For several decades invention of new (fluoro)quinolones was actively pursued by diverse pharmaceutical houses, especially Bayer AG (Wuppertal, Germany) and Wakunaga Pharmaceutical Co. (Hiroshima, Japan). Chemists at the latter firm invented delafloxacin (Baxdela), the last fluoroquinolone to have been approved by the United States Food and Drug Administration (2017). Though quinolones are still widely used, the pace of development of new derivatives has declined due to the availability of alternative drugs with lower toxicities. In 2008 the FDA advised fluoroquinolone manufacturers to add a “black box” warning to drug labels and prescribing information in response to reports of serious side effects, including (but not restricted to) elevated risk of tendinopathy. (10) But during an earlier period of active development innumerable quinolone variants were synthesized and quantitative structure–activity relationships for antibacterial activity determined. Hagen et al. (11) observed a methyl enhancement effect for variously substituted 4-oxo-3-quinolinecarboxylates. For example, when the 6-fluoroquinolone nucleus was substituted with a cyclopropyl moiety at N-1 and a 3-(aminomethyl)-3-methylpyrrolidinyl moiety at C-7, replacement of C-5 hydrogen by C-5 methyl improved potencies toward Gram-positive cocci. Placement of methyl moieties at discrete sites in an antibiotic molecule can affect parameters other than in vitro potency. The scaffold of the carbapenem family of β-lactam antibiotics derives from thienamycin, a chemically unstable compound recovered from spent medium of Streptomyces cattleya fermentations. The first carbapenem to reach the market, imipenem (FDA approved, 1985), is an N-formimidoyl-stabilized derivative of thienamycin, coadministered with cilastatin (1:1 ^w/w) to prevent its hydrolysis by renal dehydropeptidase I (thereby compromising its use for urinary tract infections) and to reduce imipenem-induced nephrotoxicity. (12) Later generation carbapenems (meropenem, ertapenem, doripenem) incorporate a 1β-methyl group in the fused pyrroline ring, which stabilizes them in the presence of the renal enzyme. (13) Erythromycin A (“erythromycin”), marketed in the United States since 1952, was the first nonpolyene macrolide antibiotic to achieve widespread clinical success. Related macrolide antibiotics have since been commercialized, including clarithromycin, azithromycin, dirithromycin, roxithromycin, spiramycin, josamycin, and rokitamycin. Forty years after the clinical introduction of erythromycin, clarithromycin received FDA marketing approval. The structures of these two macrolides differ by the presence of a methyl moiety on the C-6 hydroxy group of erythromycin (clarithromycin = 6-O-methylerythromycin). Their pharmacokinetic/pharmacodynamic parameters are broadly similar, though the longer serum half-life and better tissue penetration of clarithromycin permits less frequent dosing (typically q12h) than for erythromycin (q8h-q6h). (14a−c) Antibacterial activity of clarithromycin and erythromycin toward Gram-negative bacilli and Gram-positive cocci tend to be similar, but clarithromycin is notably more active toward atypical respiratory pathogens, corynebacteria, Helicobacter pylori, and mycobacteria. (14c) Clarithromycin constitutes part of a first-line triple combination therapy for peptic ulcer disease, (14d) and is used to prevent or treat Mycobacterium avium complex infections in patients carrying the human immunodeficiency virus (HIV). (14e) Enmetazobactam is a penicillanic acid sulfone β-lactamase inhibitor that differs from tazobactam by the presence a methyl group at N-3 of the 1,2,3-triazolyl ring. Invented at Orchid Chemicals and Pharmaceuticals, Ltd. (Chennai, India) by a team of chemists led by Senthilkumar Udayampalayam Palanisamy (15a) and designated “OCID 5090”, the compound languished for years in Orchid’s patent vault before coming to the attention of myself and Dott. Stefano Biondi. I reviewed the available biological data and decided that the compound had clinical potential against clinically relevant Gram-negative pathogens, and proposed pairing it with the fourth-generation cephalosporin cefepime. Biondi, a gifted medicinal chemist with expertise in process chemistry, opined that the structure was compatible with industrial scale manufacture. In 2013 Allecra Therapeutics was founded for the purpose of inlicensing OCID 5090 (renamed “AAI101”, later “enmetazobactam”) and taking the combination of cefepime plus enmetazobactam through the preclinical and clinical steps required for regulatory agency approval. The Orchid synthesis involved a large excess of methyl iodide at a temperature near its boiling point, so Biondi and colleagues (15b) invented an alternative procedure, compatible for preparing sterile product, using a stoichiometric amount of methyl triflate at a much lower temperature, from which enmetazobactam could be isolated at higher yield and purity, with fewer toxic wastes (Dott. S. Biondi, personal communication). Cefepime/enmetazobactam, under the trade name Exblifep, received marketing approval from the FDA, EMA, and CHMP (UK) during the first half of 2024. Addition to tazobactam of a strategically placed methyl group renders the molecule zwitterionic. Compared to tazobactam, enmetazobactam has 4- to 10-fold lower IC₅₀s toward multiple serine β-lactamases. Crystal structures of tazobactam and of enmetazobactam with GES-1, an extended-spectrum β-lactamase (ESBL), indicate an electrostatic interaction between a glutamyl active site residue and the positive charge on the triazolyl ring of enmetazobactam, likely accounting for the increased potency of enmetazobactam vis-à-vis tazobactam toward this enzyme (Dr. P. Hinchliffe, personal communication). Moreover, the zwitterionicity of enmetazobactam is presumed to enhance its penetration through the outer membrane of Gram-negative bacteria, where it accumulates in the periplasm and inhibits β-lactamases localized between the inner and outer membranes. Exblifep was developed as a safe and effective carbapenem-sparing alternative to combat infections by Enterobacterales expressing ESBLs, by far the most prevalent β-lactam resistance mechanism among this order of bacteria. (16a) As such, Exblifep ought to find a place as a first-line empiric treatment, particularly in clinics where ESBL-producing enteric bacteria are of special concern. (16b) (The drug is active toward AmpC and OXA-48 producers, too, as well as toward the nonfermentative pathogen Pseudomonas aeruginosa, though these activities are attributable to the intrinsic properties of the cefepime component. (16c,d)) Exblifep is not intended as a replacement for β-lactam/β-lactamase inhibitor combinations specifically addressing Klebsiella pneumoniae carbapenemase (KPC) resistance [e.g. ceftazidime/avibactam (Avycaz), aztreonam/avibactam (Emblaveo), imipenem/relebactam/cilastatin (Recarbrio), meropenem/vaborbactam (Vabomere)], though Exblifep has shown unexpected activity toward a high proportion of enteric bacteria expressing KPCs, (16e) which may be due to periplasmic accumulation of enmetazobactam overwhelming even some β-lactamases for which the inhibitor does not have a high affinity. In this context, it would be of interest to know what future surveillance and epidemiological studies reveal about the proportion of pathogens successfully treated with Exblifep that turned out to be KPC⁺. The arc of my career has intersected repeatedly with methyl groups. From the bacterial formation of methane and its impact on agriculture and climate change, to the stereochemistry of monooxygenase-catalyzed reactions and its mechanistic implications, and culminating in the design and commercialization of a drug with lifesaving efficacy, the methyl group has been a modest but faithful companion. This humble moiety, the first organic functionality presented to students in their introductory organic chemistry course, is a seemingly jejune entity acting pedagogically as a springboard for more advanced concepts and more interesting chemical structures. But, as demonstrated by Gerstenberger et al. and many others, introduction of a single methyl group at just the right position can have a profound effect on molecular properties, be it for steric/conformational reasons, electronic/electrostatic reasons, hydrophobicity/solubility reasons, and/or any other reasons contributing to the overall physicochemical and pharmacological profile of a molecule. So Gerstenberg et al. were not being facetious by referring to their methyl moiety as “supermethyl”; rather, to put it in contemporary parlance, they were just telling it like it is. Stuart Shapiro is a consultant at the Harry Lime Institute for Penicillin Research (Basel, Switzerland) to companies pursuing the discovery and development of novel antibacterial drugs. He received a B.S. in Biology/Chemistry from New York University (1971), an M.S. in Microbiology from the University of Illinois (1976), and a Ph.D. in Biomedical Sciences (specialization in bioorganic chemistry) from Worcester Polytechnic Institute on behalf of the Worcester Foundation for Experimental Biology, Inc. (1981), followed by a postdoctoral position with Prof. Dr. Leo Vining (Dalhousie University, Halifax, Nova Scotia, Canada). He has worked at Sigma-Tau Industrie Farmaceutiche Riunite S.p.A.; Abteilung für orale Mikrobiologie and allgemeine Immunologie, Zahnärztliches Institut der Universität Zürich; Basilea Pharmaceutica International AG; and Allecra Therapeutics GmbH. He retired from Allecra in 2017. Ambler class C β-lactamase (“Ampicillin class C”) Committee for Medicinal Products for Human Use European Medicines Agency extended-spectrum β-lactamase Food and Drug Administration human immunodeficiency virus Klebsiella pneumoniae carbapenemase This article references 16 other publications. This article has not yet been cited by other publications.