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  • br Chemistry A total of new ThDP analogs


    Chemistry A total of 35 new ThDP analogs in the series of 7, 12, 15, and 19 were designed and synthesized (Scheme 1). The synthetic route is simple and convenient. In this synthetic route, 4-amino-2-methylpyrimidine-5-carbaldehyde 3 is a key intermediate for the synthesis of title compounds 7, 12, 15, and 19. 3 could be prepared by a catalytic hydrogenation reaction of raney nickel with 4-amino-2-methylpyrimidine-5-carbonitrile 2, which was prepared by the reaction of acetamidine hydrochloride 1 and ethoxymethylene malononitrile. The title compounds 7 could be synthesized by the acid-catalyzed nucleophilic addition of 3 and hydrazides 6, which was prepared by the reaction of ethyl benzoates 5 and hydrazine hydrate.5 was achieved starting from substituted phenols 4 by reacting with ethyl 4-fluorobenzoate in DMF with K2CO3 as base. Title compounds 12 or 15 could be also synthesized by the nucleophilic addition reaction of 3 and hydrazides 11 or 14 in the presence acetic acid, respectively. 11 or 14 were prepared via the reaction of hydrazine hydrate with ethyl benzoates 10 or 13, which was prepared from corresponding acyl chlorides 9 by reacting with 3-aminobenzoic BIBR 1532 ethyl ester or 4-aminobenzoic acid ethyl ester, respectively.9 were obtained starting from acids 8 by chloridization using oxalyl chloride. The title compounds 19 were further synthesized by 3 reacting with corresponding hydrazides 18 via nucleophilic addition in the presence of acetic acid. 18 could be prepared from ethyl benzoates 17, which were prepared starting from isocyanate 16 by reacting with 4-aminobenzoic acid ethyl ester. 35 new title compounds were synthesized smoothly by above synthetic method which showed the advantages of mild and efficient. All title compounds were fully characterized with 1H NMR, 13C NMR, 19F NMR, IR, and ESI-MS spectra and confirmed by elementary analysis.
    Results and discussion
    Conclusion Four series of novel 4-aminopyrimidine derivatives 7, 12, 15, and 19 including 35 compounds were designed and synthesized. As novel ThDP analogs, 12, 15, and 19 were designed by replacing the pyrophosphate moiety of ThDP with N-substituted-benzamide or diphenylurea moiety, were confirmed to have potent inhibition against E. coli PDHc-E1. 15h with IC50 value of 0.29 μM was found to be most effective E. coli PDHc-E1 inhibitor. Molecular docking analysis revealed Pleiotropic gene V192, M194, E571, H640, K392, and H142 (or L264) at active site of E. coli PDHc-E1 played an important role in the binding of 12i, 15h, or 19a with E. coli PDHc-E1. The binding mode of novel inhibitor 12i, 15h, or 19a with E. coli PDHc-E1 was further confirmed by site-directed mutagenesis and enzymatic assays. The study of enzyme-selective inhibition showed 12g, 12i, 15f, and 19a with 100% inhibition against E. coli PDHc-E1 (IC50 = 2.85–0.54 μM) had only 0–18% inhibition against porcine PDHc-E1. The interaction of inhibitor 12i and E. coli or porcine PDHc-E1 was further studied by molecular docking to understand their selectivity. The acylhydrazone and N-phenylbenzamide moieties could form hydrogen bonds with His640 and Leu264 at the active site of E. coli PDHc-E1, but no such hydrogen bonds were formed at the active site of porcine PDHc-E1. Therefore, these title compounds could selectively inhibit E. coli PDHc-E1 because of their stronger interaction with E. coli PDHc-E1 and weak interaction with porcine PDHc-E1. A part of compounds as potent PDHc-E1 inhibitors also exhibited notable antibacterial activity. In particular, 12e, 12f, 12g, 12o, and 19a exhibited 72.0–88.8% inhibition against Xanthomonas oryzae pv. Oryzae and 75.2–92.1% inhibition against Ralstonia solanacearum at 100 μg/mL, respectively. Their effect was much better than thiodiazole-copper (34.3 and 29.1%, respectively) and bismerthiazol (56.4 and 54.5%, respectively) as positive controls. The results proved that we could obtain effective bactericidal as highly selective PDHc inhibitors by rational molecular design utilizing the binding model of active site of E. coli PDHc-E1.