![Imidazo[4,5-B] Pyridines As Lumazine Synthase Inhibitors for Their Effective Antimicrobial Activity](http://data.docslib.org/img/2aa2b1b5610232a420ff4901fb15d4bd-1.webp)
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.
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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)
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[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, m = multiplet, This derivative was synthesized according to the general bs = broad singlet. The relative integrals of peak areas agreed procedure A. Yield 79.65%, as pale yellow solid, m.p. 160-165°C. with those expected for the assigned structures. Mass spectra −1 IR (KBr, cm ): 3600 (O‑Haliph), 3350 (NH), 3250 (CHarom), were recorded on WATERS, Q‑TOF MICROMASS (LC‑MS), 2900 (CHaliph), 1650 (C = Narom), 1500 (C = Carom), 1350 (C‑Caliph), performed at SAIF, Punjab University, Chandigarh. Thin layer 1275 (C‑Narom), 1100 (C‑Oaliph alco), 800 (bend CHaliph), 770 (bend chromatography (TLC) analysis was carried out on silica gel 1 CHarom). HNMR (400 MHz, CDCl3, in ppm): 13.5 (s, 1H), precoated aluminum sheets (Type 60 F 254, Merck) and the 7.89 (t, 3H), 3.78 (s, 4H), 2.0 (s, 4H). 13C NMR (400 MHz, spots were detected under ultraviolet‑lamp at short wavelength CDCl3, in ppm): 154, 149, 138, 128, 116, 33. HRMS (ESI): calcd. λ 254 nm. + for C10H13N3O4 [M + H] : 240.097; found 240.227. Analysis
Calcd. for C10H13N3O4 (239.23); C, 49.06; H, 5.57; N, 15.59; O, In‑silico docking experiment 29.71%. Found: C, 49.06; H, 5.55; N, 15.59; O, 29.79%.
The crystal structures of the target protein was obtained from 1‑(3H‑imidazo[4,5‑b] pyridin‑2‑yl) pentane‑1,2,3,4,5‑pentol (1b) PDB and saved in standard three‑dimensional coordinate format. The PDB is a repository for three‑dimensional This derivative was synthesized according to the general structural data of proteins and nucleic acids. This procedure A. Yield 89.58%, as gray solid, m.p. 145-150°C. database provides the three‑dimensional structure of all −1 IR (KBr, cm ): 3600 (O‑Haliph), 3350 (N‑H), 3250 (CHarom), the proteins by NMR or by X‑ray crystallography. After 2900 (CH ), 1650 (C = N ), 1500 (C = C ), conducting adequate literature review lumazine synthase aliph arom arom 1350 (C‑Caliph), 1275 (C‑Narom), 1100 (C‑Oaliph alcoholic), 800(bend of M. tuberculosis (PDB entry code 2C92) was selected 1 CHaliph), 770 (bend CHarom). H NMR (400 MHz, CDCl3, as the target for the present study. Preparation of ligands in ppm): 13.5 (s, 1H), 7.87 (s, 3H), 3.79 (t, 5H), 2 (t, 5H). was done by drawing the structures using ChemSketch HRMS (ESI): calcd. for C H N O [M + H]+: 270.108447; 12 (ACD) Advanced Chemistry Development (USA) and 11 15 3 5 found 270.118349. Analysis Calcd. for C11H15N3O5 (269.253); Chem Draw Ultra 7 Cambridge Soft Chem Draw Ultra in C, 50.20; H, 5.43; N, 17.55; O, 26.75%. Found: C, 50.22; H, two‑dimensional and saved as MDL Molfile format. 5.33; N, 17.54; O, 26.65%. Further conversion of ligands to three‑dimensional format using VLife Engine tools of VlifeMDS 4.3 VLifesciences, A Division of NovaLead Pharma Pvt Ltd, Pune. Protein visualization was done by loading the structure in SWISS PDB viewer. Further the Energy Minimization was performed by VlifeMDS 4.3 docking suite. Docking simulations were performed with Biopredicta tool using grip docking mode. The number of docking runs was set to 10. Different types of binding interactions were studied between docked three‑dimensional test ligands and three‑dimensional macromolecule target [Figures 4‑7].
Chemistry
General experimental procedure for the synthesis of 3H Imidazo[4,5‑b] pyridine (1a‑1l) (procedure A)
Solution of 2 nitro 3 aminopyridine (1.0 mmol) and substituted Figure 4: Hydrogen bond Interaction of 2-(2-nitrophenyl)-3H- aldehyde (1.0 mmol) in DMF (4 mL) was treated with 1 M imidazo[4,5-b]pyridine(1i) with GLU122C (2.348 A°, 2.528 A°)
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in ppm): 13.5 (s, 1H), 7.89 (t, 3H), 3.78 (s, 4H), 2.0 (s, 4H). 13 C NMR (400 MHz, CDCl3, in ppm): 154, 149, 138, 128, 116, + 33. HRMS (ESI): calcd. for C10H13N3O4 [M + H] : 240.097;
found 239.227. Analysis Calcd. for C10H13N3O4 (239.23); C, 49.06; H, 5.57; N, 15.59; O, 29.71%. Found: C, 49.06; H, 5.55; N, 15.59; O, 29.79%.
2‑(3H‑imidazo[4,5‑b] pyridin‑2‑yl) phenol (1d)
This derivative was synthesized according to the general procedure A. Yield 68.00%, as faint yellow solid, m.p. 135-140°C. −1 IR (KBr, cm ): 3600 (OH), 3350 (NH), 3250 (CHarom),
1650 (C = Narom), 1500 (C = Carom), 1275 (C‑Narom), Figure 5: Aromatic Interaction of 2-(2-nitrophenyl)-3H-imidazo[4,5-b] 1 1250 (C‑Ophenolic), 770 (bend CHarom). HNMR (400 MHz, pyridine (1i) with HIS28C CDCl3, in ppm): 13.4 (s, 1H), 7.8 (s, 3H), 7.3 (s, 4H), 5.1 (s, 1H). 13 C NMR (400 MHz, CDCl3, in ppm): 154, 149, 138, 128, 116, + 33. HRMS (ESI): calcd. for C12H9N3O [M + H] : 212.08183;
found 212.07346. Analysis Calcd. for C12H9N3O (211.21); C, 68.17; H, 4.26; N, 19.89; O, 5.57%. Found: C, 68.17; H, 4.24; N, 19.89; O, 5.56%.
4‑(3H‑imidazo[4,5‑b] pyridin‑2‑yl) phenol (1e)
This derivative was synthesized according to the general procedure A. Yield 78.00%, as faint yellow solid, m. p. 150-155°C. −1 IR (KBr, cm ): 3600 (OH), 3350 (NH), 3250 (CHarom),
1650 (C = Narom), 1500 (C = Carom), 1275 (C‑Narom), 1 1250 (C‑Ophenolic), 770 (bend CHarom). HNMR (400 MHz,
CDCl3, in ppm): 13.4 (s, 1H), 7.8 (s, 3H), 7.3 (s, 4H), 5.1 (s, 1H). 13 Figure 6: Hydrogen bond Interaction of 4-(2-methyl-2,3-dihydro-1H- C NMR (400 MHz, CDCl3, in ppm): 154, 149, 138, 128, 116, + imidazo[4,5-b]pyridin-2-yl)benzene-1,3-diol (2f) with GLU122C 33. HRMS (ESI): calcd. for C12H9N3O [M + H] : 212.08183;
found 212.07346. Analysis Calcd. for C12H9N3O (211.21); C, 68.17; H, 4.26; N, 19.89; O, 5.57%. Found: C, 68.17; H, 4.24; N, 19.89; O, 5.56%.
4‑(3H‑imidazo[4,5‑b] pyridin‑2‑yl)‑2‑methoxyphenol (1f)
This derivative was synthesized according to the general procedure A. Yield 69.06%, as orange solid, m.p. 148-150°C. −1 IR (KBr, cm ): 3600 (OH), 3350 (NH), 3250 (CHarom),
1650 (C = Narom), 1500 (C = Carom), 1275 (C‑Narom), 1 1250 (C‑Ophenolic), 770 (bend CHarom). HNMR (400 MHz,
CDCl3, in ppm): 13.4 (s, 1H), 7.8 (s, 3H), 7.3 (s, 4H), 5.1 (s, 1H). 13 C NMR (400 MHz, CDCl3, in ppm): 154, 149, 138, 128, 116, + 33. HRMS (ESI): Calcd. for C12H9N3O [M + H] : 212.08183;
found 212.07346. Analysis Calcd. for C12H9N3O (211.21); C, 68.17; H, 4.26; N, 19.89; O, 5.57%. Found: C, 68.17; H, 4.24; Figure 7: Hydrophobic interaction of 4-(2-methyl-2,3-dihydro-1H- N, 19.89; O, 5.56%. imidazo[4,5-b]pyridin-2-yl)benzene-1,3-diol (2f), with GLU122C 4‑(3H‑imidazo[4,5‑b] pyridin‑2‑yl)‑N, N‑dimethylaniline (1g) 1‑(3H‑imidazo[4,5‑b] pyridin‑2‑yl) butane‑1,2,3,4‑tetrol (1c) This derivative was synthesized according to the general This derivative was synthesized according to the general procedure A. Yield 75.03%, as yellow solid, m.p. 150-153°C. −1 procedure A. Yield 79.65%, as orange solid, m.p. 150-154°C. IR (KBr, cm ): 3600 (OH), 3350 (NH), 3250 (CHarom), −1 IR (KBr, cm ): 3600 (O‑Haliph), 3350 (NH), 3250 (CHarom), 1650 (C = Narom), 1500 (C = Carom), 1275 (C‑Narom), 1 2900 (CHaliph), 1650 (C = Narom), 1500 (C = Carom), 1250 (C‑Ophenolic), 770 (bend CHarom). HNMR (400 MHz,
1350 (C‑Caliph), 1275 (C‑Narom), 1100 (C‑Oaliph alcoholic), 800 (bend CDCl3, in ppm): 13.4 (s, 1H), 7.8 (s, 3H), 7.3 (s, 4H), 5.1 (s, 1H). 1 13 CHaliph), 770 (bend CHarom). HNMR (400 MHz, CDCl3, C NMR (400 MHz, CDCl3, in ppm): 154, 149, 138, 128, 116,
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+ 33. HRMS (ESI): calcd. for C12H9N3O [M + H] : 212.08183; Analysis Calcd. for C13H11N3O (225.24); C, 69.20; H, 4.9; N, 18.64; found 212.07346. Analysis Calcd. for C12H9N3O (211.21); C, O, 7.10%. Found: C, 69.20; H, 4.91; N, 18.59; O, 7.11%. 68.17; H, 4.26; N, 19.89; O, 5.57%. Found: C, 68.17; H, 4.24; N, 19.89; O, 5.56%. 2‑(2‑nitrophenyl)‑3H‑imidazo[4,5‑b] pyridine (1i)
2‑(4‑methoxyphenyl)‑3H‑imidazo[4,5‑b] pyridine (1h) This derivative was synthesized according to the general procedure A. Yield 63.02%, as dark yellow solid, m.p. 175- −1 This derivative was synthesized according to the general 180°C. IR (KBr, cm ):OH 3600 ( arom), 3350 (NH), procedure A. Yield 82.5%, as faint yellow‑orange solid, m.p. 189- 3250 (CHarom), 2850 (CH3 ), 1650 (C = Narom) , −1 193°C. IR (KBr, cm ): 3350 (NH), 3250 (CHarom), 2850 (CH3), 1500 (C = Carom), 1475 (N = O), 1375 (bend CH3),
1500 (C = Carom), 1650 (C = Narom), 1375 (bend CH3), 1275 (C‑Narom), 1250 (C‑Ophenolic), 770 (bend CHarom). 1 1 1275 (C‑Narom), 770 (bend CHarom). HNMR (400 MHz, CDCl3, in HNMR (400 MHz, CDCl3, in ppm): 13.4 (s, 1H) 7.7(s, 13 13 ppm): 13.4 (s, 1H). 7.8 (s, 3H), 7.5 (s, 4H). C NMR (400 MHz, 3H), 7.3 (s, 4H). C NMR (400 MHz, CDCl3, in ppm):
CDCl3, in ppm): 149.8, 123.6, 122.3, 138, 128, 56. HRMS (ESI): 149, 145, 135, 130, 117, 121.3. HRMS (ESI): Calcd. for + + Calcd. for C13H11N3O [M + H] : 226.097488; found 226.096565. C13H11N3O [M + H] : 226.097488; found 226.096565.
Figure 8: Scheme of synthesis of title compounds
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Analysis Calcd. for C13H11N3O (225.24); C, 69.20; H, 4.9; N, crystalline product of expected purity. Reaction progress was 18.64; O, 7.10%. Found: C, 69.20; H, 4.91; N, 18.59; O, 7.11%. monitored with TLC on precoated plates. Dry crystals were subjected for m.p and respective chemical tests [Figure 8, 4‑(3H‑imidazo[4,5‑b] pyridin‑2‑yl) benzene‑1,2‑diol (1j) Scheme 2]. This type of product has been reported previously by Scheuerman and Tumelty.[49] and is presumably formed This derivative was synthesized according to the general through formylation of the aniline nitrogen, nitro reduction procedure A. Yield 85.55%, as greenish brown solid, m.p. and cyclization. −1 180-185°C. IR (KBr, cm ): 3600 (O‑Hphenol), 3350 (N‑H),
3250 (CHarom), 1650 (C = Narom), 1500 (C = Carom), Formylation of the aniline nitrogen is believed to assist
1350 (C‑Caliph), 1275 (C‑Narom), 1250 (C‑Ophenol), 770 (bend nitro reduction. In summary, we have demonstrated 1 CHarom). HNMR (400 MHz, CDCl3, in ppm): 13.4 (s, that imidazopyridines can be efficiently prepared from 1H), 7.5 (t, 3H), 6.5 (t, 3H), 5.0 (s, 2H). 13C NMR (400 a support‑bound nitro aminopyridine using a “one‑pot”
MHz, CDCl3, in ppm): 149.8, 123.6, 122.3, 138, 128, 56. reduction–cyclisation method. This approach has provided + HRMS (ESI): Calcd. for C12H9N3O2 [M + H] : 228.076753; the shortest solid phase synthesis of imidazopyridines to date. found 228.108912. Analysis Calcd. for C12H9N3O2 (227.21); Since a wide range of amines and aldehydes are commercially C, 63.37; H, 3.96; N, 18.47; O, 14.08%. Found: C, 63.37; available, a large number of pyridoimidazoles and respective H, 3.96; N, 18.47; O, 14.08%. benzimidazoles can be easily prepared using this method.
2‑(2‑chlorophenyl)‑3H‑imidazo[4,5‑b] pyridine (1k) 4‑chloro‑2‑(2‑methyl‑2,3‑dihydro‑1H‑imidazo[4,5‑b] pyridin‑2‑yl) phenol (2a) This derivative was synthesized according to the general procedure A. Yield 98.22%, as off white solid, m.p. 169-172°C. This derivative was synthesized according to the general −1 IR (KBr, cm ): 3350 (N‑H), 3250 (CHarom), 1650 (C = Narom), procedure B. Yield 78.11%, as faint white solid, m.p. 219- −1 1500 (C = Carom), C‑N Ar 1275, 750 (C‑Cl), 770 (bend CHarom). 223°C. IR (KBr, cm ): 3500 (O‑Hphenolic), 3350 (N‑H), 3250 1 HNMR (400 MHz, CDCl3, in ppm): 13.4 (s, 1H), 7.7 (t, 3H), (CHarom), 2850 (CH3), 1650 (C = Narom), 1456 (C = Carom), 13 7.2 (m, 4H). C NMR (400 MHz, CDCl3, in ppm): 149.8, 1275 (C‑Narom), 1250 (C‑Ophenol), 800 (bend CHaliph), 770 (bend 1 136.3, 135.7, 129.6, 128.3, 122.4. HRMS (ESI): Calcd. for CHarom), 750 (C‑Cl). H NMR (400 MHz, CDCl3, in ppm): 7.7 (m, + C12H8ClN3 [M + H] : 230.66502; found 230.57092. Analysis 4H), 7.0 (m, 3H), 5 (s, 1H, OH), 4 (s, 2H, NH), 1.5 (s, 3H, CH3), 13 Calcd. for C12H8ClN3 (229.66); C, 62.88; H, 3.49; N, 18.34; O, C NMR (400 MHz, CDCl3, in ppm): 150, 135, 130, 123, 65, 35. + 15.28%. Found: C, 62.88; H, 3.49; N, 18.34; O, 15.28%. HRMS (ESI): calcd. for C13H12ClN3O [M + H] : 262.074166;
found 262.04697. Analysis Calcd. for C13H12ClN3O (261.70); C, 2‑(furan‑2‑yl)‑3H‑imidazo[4,5‑b] pyridine (1l) 59.61; H, 4.58; N, 16.04; O, 6.11; Cl, 13.37%. Found: C, 59.61; H, 4.58; N, 16.04; O, 6.11; Cl, 13.37%. This derivative was synthesized according to the general procedure A. Yield 89.34%, as purple solid, m.p. 89.6-94.8°C. 4‑(2‑methyl‑2,3‑dihydro‑1H‑imidazo[4,5‑b] pyridin‑2‑yl) −1 IR (KBr, cm ): 3350 (N‑H), 3250(s CHarom), 1650 (C = Narom), benzene‑1,2,3‑triol (2b)
1500 (C = Carom), 1275 (C‑Narom), 1100 (C‑O), 770 (s 1 bend CHarom). H NMR (400 MHz, CDCl3, in ppm): This derivative was synthesized according to the general 13.4 (s, 1H), 7.8 (s, 3H), 6.5 (s, 3H). 13C NMR (400 MHz, procedure B. Yield 67.89%, as white solid, m.p. 167-170°C. −1 CDCl3, in ppm): 154, 135, 140, 123, 111. HRMS (ESI): calcd. for IR (KBr, cm ): 3600 (O‑Haliph), 3350 (N‑H), 3250 (CHarom), + C10H7N3O [M + H] : 186.066188; found 186.044543. Analysis 2900 (CHaliph), 1650 (C = Narom), 1500 (C = Carom),
Calcd. for C10H7N3O (185.18); C, 64.80; H, 3.78; N, 22.68; O, 1350 (C‑Caliph), 1275 (C‑Narom), 1100 (C‑Oaliph alcoholic), 800 (bend 1 8.64%. Found: C, 64.80; H, 3.78; N, 22.68; O, 8.64%. CHaliph), 770 (bend CHarom). H NMR (400 MHz, CDCl3, in ppm): 13.5 (s, 1H), 7.87 (s, 3H), 3.79 (t, 5H), 2 (t, 5H). 13C NMR (400
General experimental procedure for the synthesis of 1H MHz, CDCl3, in ppm): 149, 144, 139, 130, 123, 113, 65, 33. + Imidazo[4,5‑b] pyridine (2a‑2j) (procedure B) HRMS (ESI): calcd. for C13H13N3O3 [M + H] : 260.102968;
found 262.14397. Analysis Calcd. for C13H13N3O3 (259.26); C, In the present synthesis, mixture of 2 nitro 3 aminopyridine 60.17; H, 6.17; N, 16.19; O, 18.51%. Found: C, 60.17; H, 6.18; and substituted acetophenones were refluxed in EtOH along N, 16.20; O, 18.52%. with SnCl2 H2O and formic acid as a catalyst at 60°C for overnight time period. After the reaction is over, the reaction 3‑(aminomethyl)‑4‑(2‑methyl‑2,3‑dihydro‑1H‑imidazo[4,5‑b] mixture was cooled at room temperature and filtered at normal pyridin‑2‑l) benzene‑1,2‑diol (2c) temperature. Clear filtrate was subjected for distillation under reduced pressure (in vacuo). Concentrate in the distillating This derivative was synthesized according to the general flask is kept open at room temperature for air oxidation procedure B. Yield 73.00%, as pale yellow solid, m.p. −1 and evaporation of trapped solvent. Crude solid mass was 211-214°C. IR (KBr, cm ): 3500 (O‑Hphenolic), 3350 (N‑Harom), subjected further for washing with water (2 ml × 30 ml). Next 3300 (N‑Haliph), 3250 (CHarom), 2850 (CH3), 2700 (CH2), recrystallization was performed with solvent benzene to obtain 1650 (C = Narom), 1456 (C = Carom), 1275 (C‑Narom), 1250
290 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
(C‑Ophenolic), 1100 (C‑Naliph), 800 (bend CHaliph), 770 (bend 3500 (O‑Hphenolic), 3350 (N‑H), 3250 (CHarom), 1650 (C = Narom), 1 CHarom). H NMR (400 MHz, CDCl3, in ppm): 7.5 (m, 3H), 1456 (C = Carom), 1275 (C‑Narom), 1250 (C‑Ophenol), 800 (bend 13 1 6.5 (m, 4H), 4.0 (s, 2H, NH), 1.5 (s, 3H, CH3). C NMR CHaliph), 770 (bend CHarom), H NMR (400 MHz, CDCl3, in
(400 MHz, CDsCl3, in ppm): 149, 142, 138, 135, 130, 123, 114, 33, ppm): 7.5(m, 3H), 6.9 (m, 4H), 5.0 (s, 1H, OH), 4.0 (s, 2H, + 13 31. HRMS (ESI): calcd. for C14H16N4O2 [M + H] : 273.134602; NH), 1.5(s, 3H, CH3). C NMR (400 MHz, CDCl3, in ppm): found 273.144312. Analysis Calcd. for C14H16N4O2 (272.30); C, 155, 149, 138, 135, 130, 123, 115, 74, 32. HRMS (ESI): calcd. for + 61.69; H, 5.87; N, 20.56; O, 11.75%. Found: C, 61.69; H, 5.87; C13H13N3O [M + H] : 228.113138; found 228.113458. Analysis
N, 20.55; O, 11.72%. Calcd. for C13H13N3O (227.26); C, 68.64; H, 5.72; N, 18.48; O, 7.04%. Found: C, 68.64; H, 5.72; N, 18.48; O, 7.04%. 3‑(2‑methyl‑2,3‑dihydro‑1H‑imidazo[4,5‑b] pyridin‑2‑yl) aniline (2d) 2‑(4‑chlorophenyl)‑2‑methyl‑2,3‑dihydro‑1H‑imidazo[4,5‑b] pyridine (2h) This derivative was synthesized according to the general procedure B. Yield 77.00%, as yellow solid, m.p. 224-228°C. This derivative was synthesized according to the general −1 IR (KBr, cm ): 3450 (N‑H), 3350 (N‑Harom), 3250 (CHarom), procedure B. Yield 65.23%, as yellow solid, m.p. 215-218°C. −1 2850 (CH3), 1650 C = Narom), 1456 (C = Carom), 1275 (C‑Narom), IR (KBr, cm ): 3350 (N‑H), 3250 (CHarom), 2850 (CH3), 1 770 (bend CHarom). H NMR (400 MHz, CDCl3, in ppm): 7.5 (m, 1650 (C = Narom), 1456 (C = Carom), 1275 (C‑N arom), 800 (bend, 13 1 3H), 6.5 (m, 4H), 4.0 (s, 2H, NH), 1.5 (s, 3H, CH3). C NMR CHaliph), 770 (bend CHarom), 750 (C‑Cl). H NMR (400 MHz,
(400 MHz, CDCl3, in ppm): 149, 143, 138, 135, 123, 113, 117, CDCl3, in ppm): 7.25 (m, 3H), 6.9 (m, 3H), 4.0 (s, 2H, NH), + 13 74, 32. HRMS (ESI): calcd. for C13H14N4 [M + H] : 227.129123; 1.5 (s, 3H, CH3). C NMR (400 MHz, CDCl3, in ppm): 149, found 227.11813. Analysis Calcd. for C13H14N42 (226.27); C, 140, 138, 130, 123, 128, 113, 74, 32. HRMS (ESI): calcd. for + 68.4; H, 6.18; N, 24.74%. Found: C, 68.4; H, 6.18; N, 24.74%. C13H12ClN3 [M + H] : 246.079252; found 246.085552. Analysis
Calcd. for C13H12ClN3 (245.70); C, 63.49; H, 4.88; N, 17.09; Cl, 4‑(2‑methyl‑2,3‑dihydro‑1H‑imidazo[4,5‑b] pyridin‑2‑yl) 14.24%. Found: C, 63.49; H, 4.88; N, 17.09; Cl, 14.24%. aniline (2e) 2‑methyl‑2‑phenyl‑2,3‑dihydro‑1H‑imidazo[4,5‑b] pyridine (2i) This derivative was synthesized according to the general procedure B. Yield 80.11%, as pale yellow solid, m.p. 220-223°C. This derivative was synthesized according to the general −1 IR (KBr, cm ): 3450 (N‑H), 3350 (N‑Harom), 3250 (CHarom), procedure B. Yield 75.99%, as yellow solid, m.p. 119-124°C. −1 2850 (CH3), 1650 C = Narom), 1456 (C = Carom), 1275 (C‑Narom), IR (KBr, cm ): 3350 (N‑H), 3250 (CHarom), 2850 (CH3), 1 770 (bend CH arom). H NMR (400 MHz, CDCl3, in ppm): 1650 (C = Narom), 1456 (C = Carom), 1275 (C‑Narom), 800 (bend 1 7.5 (m, 3H), 6.5 (m, 4H), 4.0 (s, 2H, NH), 1.5 (s, 3H, CHaliph), 770 (bend CHarom). H NMR (400 MHz, CDCl3, in ppm): 13 13 CH3). C NMR (400 MHz, CDCl3, in ppm): 149, 143, 7.2 (m, 5H), 7.0 (m, 3H), 4.0 (s, 2H, NH), 1.5 (s, 3H, CH3). C 138, 135, 123, 113, 117, 74, 32. HRMS (ESI): Calcd. for NMR (400 MHz, CDCl3, in ppm): 149, 142, 138, 130, 128, 123, + + C13H14N4 [M + H] : 227.129123; found 227.11813. Analysis 74, 32. HRMS (ESI): calcd. for C13H13N3 [M + H] : 212.118224; found 212.109232. Analysis Calcd. for C H N (211.26); C, Calcd. for C13H14N42 (226.27); C, 68.4; H, 6.18; N, 24.74%. 13 13 3 Found: C, 68.4; H, 6.18; N, 24.74%. 73.84; H, 6.15; N, 19.88%. Found: C, 73.84; H, 6.15; N, 19.88%.
4‑(2‑methyl‑2,3‑dihydro‑1H‑imidazo[4,5‑b] pyridin‑2‑yl) 4‑methoxyphenyl)[2‑(4‑methoxyphenyl)‑2,3‑dihydro‑1H‑imidaz benzene‑1,3‑diol (2f) o[4,5‑b] pyridine‑2‑yl] methanol (2j)
This derivative was synthesized according to the general This derivative was synthesized according to the general procedure B. Yield 76.89%, as pale yellow solid, m.p. 238-240°C. procedure B. Yield 62.11%, as white solid, m.p. 250-254°C. −1 IR (KBr, cm−1): 3600 (O‑H), 3350 (N‑H), 3250 (CH ), IR (KBr, cm ): 3500 (OHphenolic), 3350 (N‑H), 3250 (CHarom), arom 2850 (CH ), 1650 (C = N ), 1456 (C = C ), 1290 (C‑O), 2850 (CH3),C 1650 ( = Narom),C 1456 ( = Carom),C 1275 ( ‑Narom), 3 arom arom 1275 (C‑Narom), 800 (bend CH ), 770 (bend CH ). 1H 1250 (C‑Ophenol), 800 (bend CHaliph), 770 (bend CHarom). aliph arom 1 NMR (400 MHz, CDCl , in ppm): 7.2 (m, 3H), 6.9 (m, 4H), H NMR (400 MHz, CDCl3, in ppm): 7.5 (m, 3H), 6.9 (m, 3H), 3 13 5.3 (s, 1H, CH ), 4.0 (s, 2H, NH), 2.0 (s, 1H, OH), 3.5 (s, 6H). 5.0 (s, 1H, OH), 4.0 (s, 2H, NH), 1.5 (s, 3H, CH3). C NMR aliph 13C NMR (400 MHz, CDCl , in ppm): 160, 128, 123, 115, 90, 87, (400 MHz, CDCl3, in ppm): 157, 149, 138, 130, 122, 108, 102, 64, 3 + 57. HRMS (ESI): calcd. for C H N O [M + H]+: 364.165568; 31. HRMS (ESI): calcd. for C13H13N3O2 [M + H] : 244.108053; 21 21 3 3 found 364.155549. Analysis Calcd. for C H N O (363.40); C, found 244.117458. Analysis Calcd. for C13H13N3O2 (243.26); C, 21 21 3 3 64.12; H, 5.34; N, 17.26; O, 13.15%. Found: C, 64.12; H, 5.33; 69.34; H, 5.77; N, 11.55; O, 13.20%. Found: C, 69.34; H, 5.77; N, 17.24; O, 13.16%. N, 11.55; O, 13.20%.
4‑(2‑methyl‑2,3‑dihydro‑1H‑imidazo[4,5‑b] pyridin‑2‑yl) In vitro anti‑bacterial procedure phenol (2g) In vitro antimicrobial activity was carried out using disc diffusion This derivative was synthesized according to the general procedure assay.[22,23] Whatman No. 1 filter paper discs of 5 mm diameter B. Yield 82.56%, as yellow solid, m.p. 230-234°C. IR (KBr, cm−1): were sterilized by autoclaving for 15 min at 121°C. The sterile discs
Journal of Pharmacy and Bioallied Sciences October-December 2014 Vol 6 Issue 4 291 Harer and Bhatia: [1H,3H] imidazo[4,5‑b] pyridines: Antimicrobial agents were impregnated with the test compounds (500 µg/disc). The motifs studied using crystal protein lumazine synthase from agar plates were inoculated with standard inoculum (10 cells/ml M. tuberculosis (PDB 2C92).[1] With the aim of rationalizing broth) of the quality control test organism namely Gram‑positive the antimicrobial activity data obtained, docking study was M. tuberculosis, Staphylococcus aureus (ATCC 25923) and performed for the imidazo [4,5‑b] pyridine derivatives 1a‑1l Gram‑negative Escherichia coli (ATCC 25922), Brucella abortus. and 2a‑2j in order to investigate the possible interactions with The impregnated discs were placed on the agar plate medium, lumazine synthase. Minimum docking score for test ligands and the plates were incubated at 5-6°C for 1 h to permit good was showed in Table 3 in comparison with the reference ligand diffusion and then transferred to an incubator at 37°C for 24 h. Trimethoprim. The diameter of inhibition zone was measured using a caliber, to the nearest millimeter. Among the tested compounds, those Reference ligand Trimethoprim binding to the active site exhibiting moderate activity (inhibition zone >15 mm) were of the enzyme showed in Figures 9 and 10 for validation of subjected to a quantitative assay in order to determine their docking protocol and confirmation of the biological data. minimum inhibitory concentrations (MICs) using the 2‑fold Complex between the enzyme’s active site and compounds serial broth dilution assay.[24,7] 2‑(2‑nitrophenyl)‑3H‑imidazo[4,5‑b] pyridine (1i), 4‑(2‑methyl‑2,3‑dihydro‑1H‑imidazo[4,5‑b] pyridin‑2‑yl) Standardized bacterial inoculum were prepared by touching the benzene‑1,3‑diol (2f) showed in Figures 4‑7. Different types top of four or five colonies of a single type and inoculating them of interactions studied provides some important SAR points into a tube containing 5 mL of Mueller‑Hinton broth (Difco) at supporting possible interaction. pH 7.3. Incubations of these microorganism suspensions were carried out at 35°C until a visible turbidity was obtained. Finally, Chemistry the culture was diluted so that, after inoculation, each microplate well had an inoculum size of 5 × 105 colony forming units (CFU)/ Previously one‑step method was reported for benzimidazole ml. Antibacterial assays were performed in Mueller‑Hinton synthesis from o‑nitro aniline and aldehydes.[50] In the present broth (Difco) at pH 7.3. The dilutions in the test medium were one‑step synthesis of 3H imidazo [4,5‑b] pyridines (1a‑1j) successful prepared at the required concentration of 100–1 µg/ml, and for attempt was made from aldehydes and 2‑nitro‑3‑amino pyridine standard compound ciprofloxacin at 40-0.015 µg/ml. through reductive cyclisation using Na2S2O4. Aqueous paste of
Na2S2O4 was prepared as 1M in H2O and added in 3 equivalent After inclusion of 100 µg/ml of the broth containing the standard proportions to the reaction mixture [Figure 8, Scheme 1]. drug or the test compound, 100 µg/ml of bacterial suspensions were inoculated into microplate wells. After incubation for 16-20 h Second reaction for one‑step synthesis of 1H imidazo [4,5‑b] at 35°C, the well‑containing the lowest concentration of the pyridine (2a‑2l) was obtained from ketones and 2‑nitro‑3‑amino standard drug or the test compound that inhibit microorganism pyridine through reductive cyclization using SnCl2.2H2O as growth as detected by the unaided eye, was recorded to represent reductive catalyst [Figure 8, Scheme 2]. This type of approach the MIC expressed in µg/ml [Tables 1 and 2]. The MIC was has been reported previously for obtaining benzimidazole defined as the lowest concentration of the antibiotic or test motifs.[51] Imidazopyridine scaffolds were visited after treatment sample allowing no visible growth. of the substituted acetophenones and 2‑nitro‑3‑amino pyridine
with the addition of SnCl2.H2O in the presence of formic acid. In vitro anti‑fungal procedure It is presumably formed through formylation of the aniline nitrogen, nitro reduction and cyclization. Formylation of the Fungal quality control strains used were Schizosaccharomyces aniline nitrogen is believed to assist nitro reduction, In summary, pombe, Candida albicans (ATCC 90018). Disc diffusion assay[22,23] we have demonstrated that imidazo pyridines can be efficiently method was used as described above. All fungi used were prepared from a support‑bound 2 nitro 3 amino pyridine as cultivated in Sabouraud’s Dextrose Agar medium (Merck) with like the same way benzimidazoles reported using a ‘one‑pot’ L‑glutamine, buffered with 3‑(N‑morpholino) propane‑sulfonic reduction-cyclisation method. Both reactions were clear and acid at pH 7.4. The culture was further diluted after inoculation without any form of side products or byproducts as impurities. with each microplate well had final inoculum density as 0.5- 2.5 × 103 CFU/ml. Standard drug used was fluconazole. The In vitro antimicrobial screening stock solutions of the test compounds and standard compound were prepared in dimethyl sulfoxide (DMSO). The microtiter The antimicrobial activities of the compounds 1a‑1j and 2a‑2l plates were incubated at 35°C and evaluated visually after 48 h. were tested against Gram‑positive bacteria M. tuberculosis, It was established that dilution of DMSO lacked antimicrobial S. aureus, Gram‑negative bacteria E. coli, B. abortus and fungi activity against any of the test microorganisms. S. pombe, C. albicans. All selected isolates were reported to express protein lumazine synthase involved in Riboflavin Results and Discussion path. MIC was measured as described in the experimental section. Ciprofloxacin and Fluconazole were used as positive In‑silico docking controls for antibacterial and antifungal activity, respectively. Solutions of different concentrations of Ciprofloxacin, An attempt tried for in‑silico design of title compounds aiming Fluconazole and test compounds (1a‑1l, 2a‑2j) were prepared to inhibit lumazine synthase. Binding interaction of test by dissolving them in DMSO. Bacterial suspensions at
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Table 1: MIC (µg/ml)a for 3H Imidazo[4,5‑b] pyridine (1a‑1l) Compound Substituent R/Ar Molecular Microorganismb code formula Gram‑positive bacteria Gram‑negative bacteria Fungi (molecular weight) Mycobacterium Staphylococcus Escherichia Brucella Schizosaccharomyces Candida tuberculosis aereus coli abortus pombe albicans
1a C10H13N3O4 25 28 30 29 28 27 (239.23)
1b 2+ + + +2 C H N O 24 27 25 23 27 24 1 2+ 11 15 3 5 (269.253)
1 1 + + 2+ 2+ + 1c + C H N O 20 22 24 21 23 25 1 2+ 2+ 2+ 10 13 3 4 (239.227) 1 1 ++ + 2+ 1d +2 C H N O 27 30 32 36 30 28 + 12 9 3 1 1 (211.219)
1 1 1e C12H9N3O 26 29 23 26 29 30 2+ (211.219) 1 1 + 1 1f C13H11N3O2 19 18 22 29 20 22 2+ (241.243) 1 1 + 2&+
1 &+ 1 g C14H14N4 18 20 25 28 19 20 1 (238.287) &+ 1 1 + 1 1 h C13H11N3O 16 18 23 26 17 19
2&+ (225.245) 1 1 + 2 1 1i C12H8N4O2 18 22 28 26 20 19 1 (240.217)
1 1 + 1 1j C12H9N3O2 22 26 24 22 24 24 2+ (227.218) 1 1 + 2+
1k &O C12H8N3Cl 19 24 28 29 21 23 1 (229.66)
1 1 +
1l 1 2 C10H7N3O 24 29 32 34 28 29 (185.18) 1 1 + G Ciprofloxacin ‑ 25 26 23 24 ‑ ‑ H Fluconazole ‑ ‑ ‑ ‑ ‑ 25 28 aThe lowest concentration of compound needed for prevention of visible growth of microorganism. MIC: Minimum inhibitory concentration
5 × 105 CFU and fungal suspension 2.5 × 103 (CFU/mL) concentration of drug at which no visible growth is observed from were inoculated into the wells. Antibacterial plates were 100 to 1.0 µg/ml [Tables 1 and 2] with the most active compounds incubated at 37°C for 16-20 h and antifungal plates were belonging to those having chloro‑ and nitro‑substituents. evaluated after incubation at 35°C for 48 h. The MIC (µg/ml) Derivatives 2‑(2‑chlorophenyl)‑3H‑imidazo[4,5‑b] pyridine (1k), values were determined using a disc diffusion method. The 2‑(2‑nitrophenyl)‑3H‑imidazo[4,5‑b] pyridine (1i), results of in vitro antimicrobial activities of the synthesized 4‑chloro‑2‑(2‑methyl‑2,3‑dihydro‑1H‑imidazo[4,5‑b] compounds are listed in Tables 1 and 2. pyridin‑2‑yl) phenol (2a), 3‑(aminomethyl)‑4‑(2‑methyl‑2,3‑dihy dro‑1H‑imidazo[4,5‑b] pyridin‑2‑l) benzene‑1,2‑diol (2c), 3‑(2‑me The results showed a wide range of antimicrobial activities among thyl‑2,3‑dihydro‑1H‑imidazo[4,5‑b] pyridin‑2‑yl) aniline (2d), the different derivatives tested with MIC values, defined as the lowest 4‑(2‑methyl‑2,3‑dihydro‑1H‑imidazo[4,5‑b] pyridin‑2‑yl)
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Table 2: MIC (µg/ml)a of 1H imidazo[4,5‑b] pyridine (2a‑2j) Compound Substituent Ar Molecular The tested microorganismb code formula Gram‑positive bacteria Gram‑negative bacteria Fungi (molecular weight) Mycobacterium Staphylococcus Escherichia Brucella Schizosaccharomyces Candida tuberculosis aereus coli abortus pombe albicans &O 2a C13H12ClN3O 18 22 27 28 21 22 + &+ 1 (261.706)
1+ +2 1 2b + &+ C H N O 26 25 30 26 33 34 1 13 13 3 3 2+ (259.260) 1+ +2 2+ 1 2+ 2c C14H16N4O2 16 19 22 20 18 14 + (272.302) 1 2+ &+ 1 1 + 1+
2d + C13H14N4 15 17 24 23 15 17 1 (226.277) 1+ &+ 1 1 + 1+ 2e C13H14N4 17 20 25 26 20 20 + 1 (226.277)
&+ 1 1 + +2 2f C13H13N3O2 27 30 35 30 30 30 + 2+ 1 (243.261) 1 1 + &+ 2+ 2g C13H13N3O 30 31 38 35 31 34 + 1 (227.261)
&+ 1 1 +
2h &O C13H12ClN3 20 23 28 25 26 28 + 1 (245.707)
&+ 1 1 +
2i + C13H13N3 30 25 24 22 26 28 1 (211.262)
&+ 1 1 +
&+ 2j C21H21N3O3 14 16 17 18 16 18 2 (363.409) 2+ + 1 2
1+ &+
1 2k Ciprofloxacin ‑ 25 26 23 24 ‑ ‑ 2l Fluconazole ‑ ‑ ‑ ‑ ‑ 25 28 aThe lowest concentration of compound needed for prevention of visible growth of microorganism. MIC: Minimum inhibitory concentration aniline (2e), 2‑(4‑chlorophenyl)‑2‑methyl‑2,3‑dihydro‑1H‑imid pyridin‑2‑yl) benzene‑1,2,3‑triol (2b), 4‑(2‑methyl‑2,3‑dihydro‑ azo[4,5‑b] pyridine (2h) exhibited potent activities. Compounds 1H‑imidazo[4,5‑b] pyridin‑2‑yl) benzene‑1,3‑diol (2f), 4‑(2‑me 4‑(3H‑imidazo[4,5‑b] pyridin‑2‑yl)‑2‑methoxyphenol (1f), thyl‑2,3‑dihydro‑1H‑imidazo[4,5‑b] pyridin‑2‑yl) phenol (2g), 4‑(3H‑imidazo[4,5‑b] pyridin‑2‑yl)‑N, N‑dimethylaniline (1g), 2‑methyl‑2‑phenyl‑2,3‑dihydro‑1H‑imidazo[4,5‑b] pyridine (2i) 2‑(4‑methoxyphenyl)‑3H‑imidazo[4,5‑b] pyridine (1h), 2‑(furan‑ behave with poor antimicrobial activity against bacteria and 2‑yl)‑3H‑imidazo[4,5‑b] pyridine (1l), 4‑methoxyphenyl)[2‑(4‑me fungi compared to standard Ciprofloxacin and Fluconazole thoxyphenyl)‑2,3‑dihydro‑1H‑imidazo[4,5‑b] pyridine‑2‑yl] respectively. Some qualitative structure activity relationships methanol (2j) showed moderate anti‑microbial activity. Whereas could be concluded from Tables 1 to 2. The superiority of the compounds 4‑(2‑methyl‑2,3‑dihydro‑1H‑imidazo[4,5‑b] compounds having R’ = Cl ˃ No2 ˃ NH2 at the phenyl moiety
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Figure 10: Hydrophobic binding of Trimethoprim to the active pocket ILE31C, GLU122C, GLU122C, LEU126C, LEU126C Figure 9: Aromatic binding of Trimethoprim to the active site HIS28C
Table 3: MolDock scores of various (3H) imidazo[4,5‑b] fluconazole. Characterization of the antimicrobial spectrum pyridines (1a‑1l) and (1H) imidazo[4,5‑b] pyridines (2a‑2j) of the synthesized compounds as shown in Tables 1 and 2, indicating a broad spectrum of antimicrobial activity against Test Minimum docking Test Minimum docking ligands score (1a‑1l) ligands score (2a‑2j) the tested strains. Docking interaction study of lumazine synthase revealed perfect binding of test ligands in comparison 1a −36.184956 2a −39.835743 1b −39.087022 2b −37.596247 with reported ligands. Minimum docking score of best fit ligands 1c −39.279918 2c −40.475413 confirm their usefulness as Lumazine synthase inhibitors for 1d −37.647217 2d −36.600264 in vitro tested pathogenic microorganism causing deadliest 1e −39.388745 2e −37.390861 infection and further to arrest spread of infectious disease. 1f −42.341387 2f −38.798321 These results form the foundation for further investigations 1g −35.661825 2g −38.955099 in our laboratories. 1h −39.206669 2h −40.782873 1i −39.647875 2i −35.520673 1j −40.199156 2j −55.287018 Acknowledgments 1k −39.554708 ‑ ‑ 1l −35.667895 ‑ ‑ Authors are grateful toward SAIF, Punjab University, Chandigarh for performing spectral studies of synthesized compounds. Also sincere thanks to Microanalytical Department, University of Pune for resident on the imidazopyridine ring (1H imidazo 4,5‑b pyridine) performing CHNO analysis. and R/Ar = Cl ˃ No2 ˃ NH2 containing substituent on the nd 2 position of the imidazole ring (3H imidazo 4,5‑b pyridine) References is obviously evident from Tables 1 and 2. Those compounds having to possess alkoxy (OCH3), alkylamine (dimethyl amine) 1. Morgunova E, Illarionov B, Sambaiah T, Haase I, Bacher A, and furan heterocycle were observed with moderate activity, Cushman M, et al. Structural and thermodynamic insights into the whereas compounds with more polar substituents (polyhydroxy) binding mode of five novel inhibitors of lumazine synthase from were observed with poor antimicrobial activity. We next strove Mycobacterium tuberculosis. FEBS J 2006;273:4790‑804. 2. Nakajima H. Tuberculosis: A global emergency. World Health to arrive at the title motifs and study their growth inhibitory 1993;46:3. activity (MIC) against Gram‑positive bacteria M. tuberculosis, 3. Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, et al. S. aureus, Gram‑negative bacteria E. coli, B. abortus and fungi Deciphering the biology of Mycobacterium tuberculosis from the S. pombe, C. albicans. complete genome sequence. Nature 1998;393:537‑44. 4. Sassetti CM, Boyd DH, Rubin EJ. Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol Conclusion 2003;48:77‑84. 5. Cushman M, Mavandadi F, Kugelbrey K, Bacher A. Synthesis of 2,6‑dioxo‑(1H,3H)‑9‑N‑ribitylpurine and In summary, we have performed in‑silico docking with the aim to 2,6‑dioxo‑(1H,3H)‑8‑aza‑9‑N‑ribitylpurine as inhibitors of lumazine study possible interactions between title compounds [1H,3H] synthase and riboflavin synthase. Bioorg Med Chem 1998;6:409‑15. 4,5‑b imidazopyridine and macromolecule lumazine synthase. 6. Talukdar A, Morgunova E, Duan J, Meining W, Foloppe N, Nilsson L, Further comparison of types of interaction with the reference et al. Virtual screening, selection and development of a benzindolone structural scaffold for inhibition of lumazine synthase. Bioorg Med ligand trimethoprim. On the basis of docking experiment Chem 2010;18:3518‑34. next we synthesized and evaluated new imidazo [4,5‑b] 7. Kaiser J, Illarionov B, Rohdich F, Eisenreich W, Saller S, den Brulle JV, pyridines against various Gram‑positive and Gram‑negative et al. A high‑throughput screening platform for inhibitors of the riboflavin biosynthesis pathway. Anal Biochem 2007;365:52‑61. bacteria and fungi expressing lumazine synthase. Many of 8. Zhang Y, Jin G, Illarionov B, Bacher A, Fischer M, Cushman M. the synthesized motifs showed potent antimicrobial activity Structure‑based model of the reaction catalyzed by Lumazine compared to the control antibiotics ciprofloxacin and Synthase from Aquifex aeolicus. J Mol Biol 2003;328:167‑82.
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