Synthesis and antibacterial action of 3’,6’-disubstituted spectinomycins

The Journal of Antibiotics https://doi.org/10.1038/s41429-024-00750-2 ARTICLE Synthesis and antibacterial action of 3’,6’-disubstituted spectinomycins Suresh Dharuman 1 Gregory A. Phelps1,2 Christine M. Dunn1,2 Laura A. Wilt 1 Patricia A. Murphy1 Robin B. Lee1 Hannah E. Snoke1,2 Petra Selchow3 Klara Haldimann3 Erik C. Böttger 3,4 Sven N. Hobbie Peter Sander3,5 Richard E. Lee 1 ● ● ● ● ● ● ● ● ● ● 3 ● ● 1234567890();,: 1234567890();,: Received: 1 April 2024 / Revised: 22 May 2024 / Accepted: 24 May 2024 © The Author(s) 2024. This article is published with open access Abstract Spectinomycin is an aminocyclitol antibiotic with a unique ribosomal binding site. Prior synthetic modifications of spectinomycin have enhanced potency and antibacterial spectrum through addition at the 6’-position to produce trospectomycin and to the 3’-position to produce spectinamides and aminomethyl spectinomycins. This study focused on the design, synthesis, and evaluation of three 3’,6’-disubstituted spectinomycin analogs: trospectinamide, N-benzyl linked aminomethyl, and Nethylene linked aminomethyl trospectomycins. Computational experiments predicted that these disubstituted analogs would be capable of binding within the SPC ribosomal binding site. The new analogs were synthesized from trospectomycin, adapting the previously established routes for the spectinamide and aminomethyl spectinomycin series. In a cell-free translation assay, the disubstituted analogs showed ribosomal inhibition similar to spectinomycin or trospectomycin. These disubstituted analogs demonstrated inhibitory MIC activity against various bacterial species with the 3’-modification dictating spectrum of activity, leading to improved activity against mycobacterium species. Notably, N-ethylene linked aminomethyl trospectomycins exhibited increased potency against Mycobacterium abscessus and trospectinamide displayed robust activity against M. tuberculosis, aligning with the selective efficacy of spectinamides. The study also found that trospectomycin is susceptible to efflux in M. tuberculosis and M. abscessus. These findings contribute to the understanding of the structure-activity relationship of spectinomycin analogs and can guide the design and synthesis of more effective spectinomycin compounds. Introduction These authors contributed equally: Suresh Dharuman, Gregory A. Phelps * Richard E. Lee 1 Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS#1000, Memphis, TN 38105, USA 2 Graduate School of Biomedical Sciences, St. Jude Children’s Research Hospital, Memphis, TN 38103, USA 3 Institute of Medical Microbiology, University of Zurich, Gloriastrasse 28/30, CH-8006 Zurich, Switzerland 4 Division of Clinical Bacteriology and Mycology, University Hospital Basel, Petersgraben 4, CH-4031 Basel, Switzerland 5 National Reference Center for Mycobacteria, Gloriastrasse 28/30, CH-8006 Zurich, Switzerland Many of our essential classes of antibiotics, such as betalactams, macrolides, and aminoglycosides, trace their origins back to natural products [1]. Through careful modifications, researchers have harnessed the power of semisynthetic chemistry to enhance the effectiveness, broaden the spectrum of activity, and overcome resistance mechanisms exhibited by drug-resistant pathogens [2]. An illustration of this concept can be seen in the strategic modifications applied to the natural product spectinomycin (SPC, 1 in Fig. 1), an aminocyclitol antibiotic produced by Streptomyces spectabilis historically used to treat Neisseria gonorrhoeae infections [3, 4]. SPC exerts its antimicrobial action by binding helix 34 of the 16S rRNA of the bacterial 30S ribosomal subunit thereby impeding bacterial translation [5]. SPC has a limited spectrum of activity due to innate resistance mechanisms in most organisms, largely mediated by efflux, and plasmidencoded modifying enzymes prevalent in Gram-negative bacteria [6–9]. Semisynthetic modifications utilizing the SPC S. Dharuman et al. Fig. 1 Analogs of spectinomycin and trospectomycin. Blue arrows highlight addition of 6’-propyl and red arrows highlight addition to 3’-modifications to the spectinomycin analogs scaffold have successfully expanded the spectrum activity and improved antimicrobial activity for SPC analogs. The most clinically advanced SPC modified analog, trospectomycin (TroSPC, 2 in Fig. 1), was discovered in the 1980’s and developed by the Upjohn company [10]. This 6’-propyl SPC analog displayed markedly increased activity, ranging from 2 to 50 times greater than SPC, against a diverse panel of microorganisms, including atypical bacteria, Gram-positive, Gram-negative, and non-tuberculous mycobacterium (NTM) species [11–13]. TroSPC showed favorable results in early clinical trials for sexually transmitted bacterial infections, but further trials were halted due to intense market competition. Previous work by our group led to the development of three lead series of 3’-SPC analogs that overcome innate resistance to SPC in their relevant pathogens [14–19]. The spectinamides (SPA, 3 in Fig. 1) were developed first as a class of narrow-spectrum Mycobacterium tuberculosis agents that exert their improved action through avoidance of Tapmediated (Rv1258c) efflux [14, 16]. N-benzyl substituted AmSPC (bAmSPC, 5 in Fig. 1) were developed against pathogens causing respiratory tract, biothreat, and sexually transmitted bacterial infections [15, 17, 18]. N-ethylene linked AmSPC (eAmSPC, 7 in Fig. 1), which differ from bAmSPC by an additional linker carbon between the aminomethyl and the terminal substitution, expanded the antimicrobial spectrum of the SPC class to broadly cover mycobacterial species, including the emerging NTM pathogen M. abscessus [20]. In M. abscessus, the increase in potency of eAmSPC is the result of greater accumulation, principally by avoiding high-level, TetV (Mab2780c) mediated efflux [20, 21]. Given TroSPC generally has improved antimicrobial activity compared to SPC [11–13], we hypothesized that combining the 6’-propyl side chain from TroSPC with the 3’-modifications from our lead spectinomycin analogs (3, 5, and 7) might result in improved antimicrobial activity. In this study, we report the synthesis and evaluation of three 3’,6’-disubstituted spectinomycin analogs: trospectinamide (TroSPA, 4 in Fig. 1), N-benzyl linked aminomethyl TroSPC (bAmTroSPC, 6 in Fig. 1) and N-ethylene linked aminomethyl TroSPC (eAmTroSPC, 8 in Fig. 1), as well as amino TroSPC (11 in Scheme 1) and aminomethyl TroSPC (13 in Scheme 2). These analogs exhibit comparable translational inhibitory properties against mycobacterial ribosomes, while closely retaining the antimicrobial characteristics of their corresponding 3’-SPC analogs. Our investigation also demonstrated that TroSPC is subject to efflux in M. tuberculosis and M. abscessus by Rv1258c and Mab2780c, respectively. This is consistent with SPC resistance mec (...truncated)