Imidazo[4,5-B] Pyridines As Lumazine Synthase Inhibitors for Their Effective Antimicrobial Activity

Original Article In-silico docking based design and synthesis of [1H,3H] imidazo[4,5-b] pyridines as lumazine synthase inhibitors for their effective antimicrobial activity Sunil L. Harer, Manish S. Bhatia Department of ABSTRACT Pharmaceutical Chemistry, Purpose: The imidazopyridine moiety is important pharmacophore that has proven to be useful for a number Bharati Vidyapeeth College of Pharmacy, of biologically relevant targets, also reported to display antibacterial, antifungal, antiviral properties. Riboflavin Kolhapur, Maharashtra, biosynthesis involving catalytic step of Lumazine synthase is absent in animals and human, but present in India microorganism, one of marked advantage of this study. Still, this path is not exploited as antiinfective target. Here, we proposed different interactions between [1H,3H] imidazo[4,5‑b] pyridine test ligands and target protein Address for correspondence: Lumazine synthase (protein Data Bank 2C92), one‑step synthesis of title compounds and further evaluation of Prof. Sunil L. Harer, them for in vitro antimicrobial activity. Materials and Methods: Active pocket of the target protein involved in E-mail: sunil.harer5@gmail. com the interaction with the test ligands molecules was found using Biopredicta tools in VLifeMDS 4.3 Suite. In‑silico docking suggests H‑bonding, hydrophobic interaction, charge interaction, aromatic interaction, and Vanderwaal forces responsible for stabilizing enzyme‑inhibitor complex. Disc diffusion assay method was used for in vitro antimicrobial screening. Results and Discussion: Investigation of possible interaction between test ligands and target lumazine synthase of Mycobacterium tuberculosis suggested 1i and 2f as best fit candidates showing hydrogen bonding, hydrophobic, aromatic and Vanderwaal’s forces. Among all derivatives 1g, 1j, 1k, 1l, 2a, 2c, 2d, 2e, 2h, and 2j exhibited potent activities against bacteria and fungi compared to the standard Ciprofloxacin and Fluconazole, respectively. The superiority of 1H imidazo [4,5‑b] pyridine compounds having R’ = Cl ˃No2 ˃ NH2 at the phenyl/aliphatic moiety resident on the imidazopyridine, whereas leading 3H imidazo[4,5‑b] pyridine compounds nd containing R/Ar = Cl ˃ No2 ˃ NH2˃ OCH3 substituents on the 2 position of imidazole. Received : 28-12-13 Review completed : 22-03-14 KEY WORDS: Antimicrobial, docking score, imidazopyridine, lumazine synthase, minimum inhibitory Accepted : 23-05-14 concentration itamin B2, commonly called riboflavin, is one of eight biosynthesized by plants and numerous microorganisms, V water-soluble B vitamins. Like its close relative, vitamin but not by animals, whereas animals obtain riboflavin from B1 (thiamine), riboflavin plays a crucial role in certain metabolic dietary sources. A rational approach to therapeutically reactions, for example, in the final metabolic conversion of useful antibiotics would be to selectively inhibit an enzyme monosaccharides, where reduction-equivalents and chemical present in a parasite, but absent in the host. Inhibition of energy in the form of adenosine triphosphate are produced the bio-synthesis of riboflavin provides such a strategy, since through the Embden-Meyerhoff pathway.[1] Riboflavin is pathogenic microorganisms synthesize their own riboflavin, whereas mammals obtain this vitamin through dietary sources. Access this article online In particular, enterobacteria such as Salmonella and Escherichia Quick Response Code: species lack riboflavin due to the apparent absence of transport Website: systems for riboflavin or flavocoenzymes, and are therefore www.jpbsonline.org absolutely dependent on endogenous riboflavin biosynthesis.[2-5] Riboflavin biosynthesis is therefore an attractive target for the DOI: design and synthesis of new antibiotics, which are urgently 10.4103/0975-7406.142962 needed because pathogens are becoming drug resistant at an alarming rate.[6] How to cite this article: Harer SL, Bhatia MS. In-silico docking based design and synthesis of [1H,3H] imidazo[4,5-b] pyridines as lumazine synthase inhibitors for their effective antimicrobial activity. J Pharm Bioall Sci 2014;6:285-96. Journal of Pharmacy and Bioallied Sciences October-December 2014 Vol 6 Issue 4 285 Harer and Bhatia: [1H,3H] imidazo[4,5‑b] pyridines: Antimicrobial agents Lumazine synthase and riboflavin synthase catalyze the last be nonsteroidal antiinflammatory and analgesic agents,[38-41] two steps in the biosynthesis of riboflavin (4) [Figure 1]. and to possess antidepressant,[39-42] antiphlogistic,[41-43] The biosynthetic pathway starts off from one molecule cardiotonic,[41-44] hypotensive and antiarrhythmic activity.[42-45] of guanosine triphosphate,[5-7] which is converted to In addition, certain members of this class had been reported 5-amino-6-ribitylamino-2,4 (1H,3H)-pyrimidinedione (1) by a sequence of ring opening, deamination, reduction, and dephosphorylation.[8,9] Lumazine synthase catalyzes the condensation of 3,4-dihydroxy-2-butanone 4-phosphate (2) with 5-amino-6-ribitylamino-2,4-(1H,3H) pyrimidinedione (1) yielding 6,7-dimethyl-8-D-ribityllumazine (3).[10-19] The final process in the biosynthesis of riboflavin (4) involves a mechanistically unusual dismutation of two molecules of 6,7-dimethyl-8-D-ribityllumazine (3) that results in the formation of one molecule of riboflavin and one molecule of the pyrimidinedione derivative (1).[20-24] Although the precise details remain to be established, the lumazine synthase-catalyzed reaction most likely proceeds along a mechanistic pathway involving the formation of the Schiff base (5), phosphate elimination affording (6), tautomerization to (7), ring closure, and dehydration to yield the lumazine (3) [Figure 2].[9] Variations of this mechanism are possible depending on the Schiff base geometry and possible isomerization, conformational changes, Figure 1: Riboflavin biosynthesis pathway and the timing of phosphate elimination. Until now, the riboflavin pathway has not been exploited as an antiinfective target. In the design and development of inhibitors, there would be no requirement for selective inhibition of the pathogen enzymes as opposed to (nonexistent) homologous enzymes of the human host. This is a marked advantage of biosynthesis pathway with regard to drug development. [7] We report herein an attempt for in-silico design of title compounds for inhibition of target lumazine synthase from Mycobacterium tuberculosis (Protein Data Bank [PDB] 2C92, Figure 3). Possible binding interactions were studied involving types of forces responsible for stabilizing the drug-receptor complex. Among all test ligands docked with receptor, we found 1f and 2j compounds as the best fit ligands. Study of docking is performed with the aim to design [1H,3H] Imidazo[4,5-b] pyridine analogues and to explore further as novel antibiotics against drug resistant pathogenic microbial Figure 2: Catalytic mechanism of lumazine synthase strains. Recent studies from many laboratories, implicate the role of these imidazopyridine scaffolds in the treatment of many of the most common human diseases, including diabetes,[25] cancers,[26] infection by microorganisms,[27] and an array of neurological syndromes.[28] Furthermore, a literature search indicated that benzimidazoles,[29-31] oxadiazoles,[32-34] and phenyl imidazoles[35,36] with different substitution patterns possess a wide range of antimicrobial properties. The imidazopyridine moiety is an important pharmacophore that has proven to be useful for a number of biologically relevant targets.[37] Imidazo[4,5-b] pyridine, known as 1-desasapurine, is a common structural motif found in numerous molecules that display antiviral, antifungal, antibacterial, and antiproliferative activities. The potent biological activity and the prevalence of 1-desazapurines in both natural products and pharmaceuticals has inspired significant interest in the synthesis of these heterocycles. Compounds that belong to Figure 3: Lumazine synthase from Mycobacterium tuberculosis the imidazo[4,5-b] pyridin-2-one class have been shown to (PDB 2C92) 286 Journal of Pharmacy and Bioallied Sciences October-December 2014 Vol 6 Issue 4 Harer and Bhatia: [1H,3H] imidazo[4,5‑b] pyridines: Antimicrobial agents [46] to be a potent inhibitor of Aurora-A, adenosine deaminase aqueous Na2S2O4 (3.0 mmol, 3 mL) [Figure 8, Scheme 1]. After inhibitors,[47] potent inhibitors of inosine 5’-monophosphate heating the reaction mixture at 60°C for 24 h, reaction mixture [48] dehydrogenase. was filtered to remove unreacted Na2S2O4. The clear filtrate was cooled to room tampere and excess solvent was removed Materials and Methods by high vacuum distillation. The concentrated residue formed in the distillating flask was washed with water (2 ml × 15 ml) Melting points (°C) were determined using a Fischer-Jones and dried under reduced pressure to afford the desired product melting point apparatus and were uncorrected. in satisfactory purity. Further recrystallization was carried using Microanalyses (CHN) were performed at the microanalytical ethanol. Purified compounds were subjected for melting point center, University of Pune using Rapid analyzer. Fourier and reaction progress was monitored with TLC and respective Transform Infrared spectra (FT-IR, KBr cm−1) were run on chemical test. JASCO 401 FT-IR spectrometer. 1H NMR and 13C NMR spectra were recorded on BRUKER AVANCE II FT-NMR 1‑(3H‑Imidazo[4,5‑b] pyridin‑2‑yl)‑butane‑1,2,3,4‑tetraol (1a) (400 MHz) using TMS as an internal standard (chemical shifts in δ, ppm), s = singlet, d = doublet,

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