untitled European Journal of Chemistry 8 (1) (2017) 25‐32 European Journal of Chemistry ISSN 2153‐2249 (Print) / ISSN 2153‐2257 (Online)  2017 Atlanta Publishing House LLC ‐ All rights reserved ‐ Printed in the USA http://dx.doi.org/10.5155/eurjchem.8.1.25-32.1522 European Journal of Chemistry Journal webpage: www.eurjchem.com Synthesis of 3H‐imidazo[4,5‐b] pyridine with evaluation of their anticancer and antimicrobial activity Rohini Narayan Shelke 1, Dattatraya Navnath Pansare 2, Chandrakant Dhondiram Pawar 2, Arun Khandu Deshmukh 3, Rajendra Pandu Pawar 1 and Saroj Ram Bembalkar 1,* 1 Department of Chemistry, Deogiri College, Aurangabad, 431005, India 2 Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, 431004, India 3 Rao Bahadhur Narayanrao Borawake College Shrirampur, Ahmednagar, 413709, India * Corresponding author at: Department of Chemistry, Deogiri College, Aurangabad, 431005, India. Tel.: +91.0240.2403308. Fax: +91.0240.2400413. E‐mail address: dbsdattatraya10@rediffmail.com (S.R. Bembalkar). ARTICLE INFORMATION ABSTRACT DOI: 10.5155/eurjchem.8.1.25-32.1522 Received: 20 December 2016 Received in revised form: 14 January 2017 Accepted: 15 January 2015 Published online: 31 March 2017 Printed: 31 March 2017   Microwave assisted and conventional synthetic methods of new 6‐bromo‐2‐(substituted)‐ 3H‐imidazo[4,5‐b]pyridine and its derivatives are described, which were obtained in reduced reaction times, higher yields, cleaner reactions than previously described methods. All the synthesized compounds were characterized, and screened for their anticancer and antimicrobial activity. Among synthesized compounds 3b and 3k shows prominent antibacterial activity and compound 3f shows both antibacterial and antifungal activity. Compounds 3h and 3j shows prominent anticancer activity against the both breast cancer cell lines, MCF‐7 and BT‐474. These results suggest that the imidazo[4,5‐b]pyridine moiety may serve as a new promising template for synthesis of anticancer and antimicrobial agents and further study is required for evaluation of their mechanism of action. KEYWORDS Anticancer activity Substituted aldehydes Antimicrobial activity Conventional synthesis Microwave‐assisted synthesis 5‐Bromopyridine‐2,3‐diamine Cite this: Eur. J. Chem. 2017, 8(1), 25‐32 1. Introduction Infections caused by multi‐drug resistant bacteria are of major health concern worldwide. The imidazole and benzimidazole nucleus are important building blocks in the drug discovery. The imidazole ring system is considered to be one of the most imperative heterocyclic substructures found in a large number of natural products and pharmacologically active compounds. An examination of literature revealed that imidazole and their analogues usually possess diverse biological activities like antimicrobial, antioxidant, antihe‐ molytic, cytotoxic [1] and antimycobacterial [2]. Almost all of the major classes of antibiotics have encountered resistance in clinical applications [3]. Because of the versatile core contained in several substances of imidazole derivatives are possess a broad spectrum of pharmacological activities [4,5]. Recently imidazole and its derivatives has been the subject of extensive study of the antimicrobial activity for their potential as effective therapeutic agents. Based on several literature surveys, imidazole derivatives show a range of pharma‐ cological activities, such as antimicrobial, anti‐tubercular, antiviral [6‐11], antioxidant and antifungal [12], antidepres‐ sant [13], anti‐inflammatory and analgesic [14], anti‐tubercu‐ losis [15] anticancer [16,17]. The imidazole scaffold is present in many natural products and is a bioactive substance in human metabolism [18]. There are many clinical drugs being used in the different therapeutic areas based on the imidazole structure, such as antihistaminic (cimetidine), anti‐cancer (dacarbazine), anti‐parasitic (metronidazole), and antihyper‐ tensive (losartan) (Figure 1). Many heterocyclic cores have played significant role in the pharmacological activities, like pyridine core, the high thera‐ peutic properties of pyridine related drugs have encouraged medicinal chemists to the synthesized large number of chemo‐ therapeutic agents. The medicinal properties of pyridine include a variety of pharmacological applications, such as antibacterial [19], antiparacite [20], antitumor [21], anticancer [22,23], antiviral [24] and anti‐inflammatory [25]. In addition, they can act as antagonists of various biological receptors [26]. Various methods for the synthesis of multi‐substituted imidazole derivatives involving different catalysts including AlCl3, FeCl3, Yb(OTf)3, NdCl3, LaCl3 [27], ZrOCl2.8H2O [28], BF3.SiO2, zeolite [29], NaH2PO4 [30], cyclic phosphoric acid [31] and SiO2 [32] etc. 26 Shelke et al. / European Journal of Chemistry 8 (1) (2017) 25‐32 Figure 1. Examples of significant imidazole containing pharmaceuticals. The application of microwave irradiation is the use of catalysts or mineral supported reagents, under solvent‐free conditions, enables organic reactions to occur expeditiously in ambient pressure, thus providing unique chemical processes with special attributes such as enhanced reaction rates and higher product yields [33,34]. 2. Experimental 2.1. Materials and instrumentations 5‐Bromopyridine‐2, 3‐diamine, substituted aldehydes, N,N‐dimethylformamide, glacial acetic acid and various solvents were commercially available. The major chemicals were purchased from Sigma Aldrich and Avra labs. Reaction progress was monitored by TLC on silica gel precoated F254 Merck plates. Developed plates were examined with ultraviolet lamps (254 nm). IR spectra were recorded on a FT‐ IR (Bruker). Melting points were recorded on SRS Optimelt, melting point apparatus and are uncorrected. 1H NMR spectra were recorded on a 400 MHz Bruker spectrometer and 13C NMR spectra were recorded on a 100 MHz Bruker spectrometer are reported as parts per million (ppm) downfield from a tetramethylsilane internal standard. The following abbreviations are used; singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m) and broad (br). Mass spectra were taken with Micromass‐QUATTRO‐II of water mass spectrometer. Microwave reactions were carried out in MicroSYNTH Lab station of Ethusi Milestone. 2.2. General procedure for the synthesis of compounds (3a‐ l) 2.2.1. Microwave‐assisted synthesis of of 6‐bromo‐2‐ (substituted) ‐3H‐imidazo [4,5‐b] pyridine, Method A An equimolar amounts of 5‐bromopyridine‐2,3‐diamine (1) (1 mmol), substituted aldehydes (2a‐l) (1 mmol) was added in DMF. This mixture was taken in a 100 mL round bottom flask subjected to MW irradiation (900 W), at 110 °C temperature for 5‐6 min. The completion of the reaction progress was monitored by using TLC (10%, ethyl acetate: n‐ hexane). The product obtained was poured into water and ethyl acetate (2:8, v:v) (3×10 mL). The combined solvent extracts were concentrated in vacuo. The compounds were recrystallized from ethanol to give a pure product (3a‐l). 2.2.2. Conventional synthesis of 6‐bromo‐2‐(substituted) ‐ 3H‐imidazo [4,5‐b] pyridine, Method B An equimolar amounts of 5‐bromopyridine‐2, 3‐diamine (1) (1 mmol), substituted aldehydes (2a‐l) (1 mmol) were added in DMF. This mixture was taken in a 100 mL round bottom flask and stirring on reflux up to 3‐4 h. The completion of the reaction progress was monitored by using TLC (10%, ethyl acetate: n‐hexane). The product obtained was poured into water and ethyl acetate (2:8, v:v) (3×10 mL). The combined solvent extracts were concentrated in vacuo. The compounds were recrystallized from ethanol to give a pure product (3a‐l) (Scheme 1). 6‐Bromo‐2‐phenyl‐3H‐imidazo[4,5‐b]pyridine (3a): Color: Yellow solid. Yield: 99%. M.p.: 165‐167 °C. FT‐IR (KBr, , cm‐1): 3059 (NH), 2843 (aromatic CH), 1569 (C=N), 1447 (C=C), 702 (C‐Br). 1H NMR (400 MHz, CDCl3, δ, ppm): 6.60‐6.95 (m, 5H, H‐ Aromatic), 7.62 (s, 1H, pyridine H), 9.40 (s, 1H, pyridine H), 10.10 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 119.2, 127.5, 129.3, 131.2, 131.8, 134.7, 138.8, 148.4, 150.2, 161.8. MS (EI, m/z (%)): 276.18 (M+2). 4‐(6‐Bromo‐3H‐imidazo[4,5‐b]pyridin‐2‐yl)phenol (3b): Color: Yellow solid. Yield: 96%. M.p.: 192‐194 °C. FT‐IR (KBr, , cm‐1): 3375 (OH), 3119 (NH), 3013 (aromatic CH), 1612 (C=N), 1468 (C=C), 825 (C‐Br). 1H NMR (400 MHz, CDCl3, δ, ppm): 5.50 (s, 1H, OH), 6.85‐6.87 (d, 2H, H‐Aromatic), 6.96‐6.98 (d, 2H, H‐Aromatic), 8.45 (s, 1H, pyridine), 9.40 (s, 1H, pyridine), 10.10 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 116.4, 119.1, 127.4, 130.5, 131.6, 138.9, 148.2, 150.3, 158.8, 161.1. MS (EI, m/z (%)): 290.25. 6‐Bromo‐2‐(2,4‐dichlorophenyl)‐3H‐imidazo[4,5‐b]pyridine (3c): Color: Yellow solid. Yield: 95%. M.p.: 214‐216 °C. FT‐IR (KBr, , cm‐1): 3027 (NH), 3008 (aromatic CH), 1578 (C=N), 1461 (C=C), 790 (C‐Cl), 688 (C‐Br). 1H NMR (400 MHz, CDCl3, δ, ppm): 7.70‐8.00 (m, 3H, H‐Aromatic), 8.10 (s, 1H, pyridine), 8.20 (s, 1H, pyridine), 8.80 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 119.0, 127.5, 130.6, 130.9, 131.7, 133.5, 133.7, 135.5, 136.5, 148.1, 150.4, 158.8. MS (EI, m/z (%)): 343.00. 6‐Bromo‐2‐(4‐chlorophenyl)‐3H‐imidazo[4, 5‐b]pyridine (3d): Color: Yellow solid. Yield: 96%. M.p.: 137‐139 °C. FT‐IR (KBr, , cm‐1): 3137 (NH), 1653 (C=N), 1464 (C=C), 760 (C‐Cl), 631 (C‐Br). 1H NMR (400 MHz, CDCl3, δ, ppm): 7.50‐7.52 (d, 2H, H‐Aromatic), 7.68‐7.70 (d, 2H, H‐Aromatic), 7.95 (s, 1H, pyridine), 8.15 (s, 1H, pyridine), 8.80 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 119.0, 128.5, 129.6, 131.8, 132.5, 134.7, 138.5, 148.1, 150.4, 161.7. MS (EI, m/z (%)): 308.50. Shelke et al. / European Journal of Chemistry 8 (1) (2017) 25‐32 27 Reaction condition: 5‐bromopyridine‐2,3‐diamine (1) (1 mmol), benzaldehyde (2) (1 mmol), solvent 1 mL, reflux 3‐12 h. Scheme 1 6‐Bromo‐2‐(2‐chlorophenyl)‐3H‐imidazo[4, 5‐b]pyridine (3e): Color: Yellow solid. Yield: 94%. M.p.: 157‐159 °C. FT‐IR (KBr, , cm‐1): 3065 (NH), 1653 (C=N), 1457 (C=C), 702 (C‐Cl), 637 (C‐Br). 1H NMR (400 MHz, CDCl3, δ, ppm): 7.60‐7.86 (m, 4H, H‐Aromatic), 8.20 (s, 1H, pyridine), 8.50 (s, 1H, pyridine), 9.15 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 119.1, 127.5, 128.4, 129.5, 130.1, 131.7, 132.4, 134.7, 138.5, 147.5, 148.0, 150.5. MS (EI, m/z (%)): 308.85. 6‐Bromo‐2‐(4‐methoxyphenyl)‐3H‐imidazo[4, 5‐b]pyridine (3f): Color: Yellow solid. Yield: 95%. M.p.: 191‐193 °C. FT‐IR (KBr, , cm‐1): 3198 (NH), 3006 (aromatic CH), 1553 (C=N), 1461 (C=C), 634 (C‐Br). 1H NMR (400 MHz, CDCl3, δ, ppm): 3.75 (s, 3H, OCH3), 7.05‐7.26 (m, 4H, H‐Aromatic), 8.54 (s, 1H, pyridine), 9.14 (s, 1H, pyridine), 10.76 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 55.9, 114.8, 119.1, 127.4, 130.3, 131.9, 138.8, 148.0, 150.5, 160.6, 161.5. MS (EI, m/z (%)): 304.23. 6‐Bromo‐2‐(2,4‐dimethoxyphenyl)‐3H‐imidazo[4, 5‐b]pyri dine (3g): Color: Yellow solid. Yield: 93%. M.p.: 196‐198 °C. FT‐ IR (KBr, , cm‐1): 3199 (NH), 3016 (aromatic CH), 1563 (C=N), 1451 (C=C), 638 (C‐Br). 1H NMR (400 MHz, CDCl3, δ, ppm): 3.76 (s, 6H, OCH3), 7.15‐7.46 (m, 3H, H‐Aromatic), 8.52 (s, 1H, pyridine), 9.11 (s, 1H, pyridine), 10.71 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 55.8, 56.8, 98.8, 107.6, 110.1, 119.3, 129.4, 131.9, 138.8, 148.0, 150.4, 151.6, 158.3, 161.6. MS (EI, m/z (%)): 334.25. 6‐Bromo‐2‐(4‐fluorophenyl)‐3H‐imidazo[4, 5‐b]pyridine (3h): Color: Yellow solid. Yield: 93%. M.p.: 270‐272 °C. FT‐IR (KBr, , cm‐1): 3219 (NH), 3051 (aromatic CH), 1590 (C=N), 1463 (C=C), 904 (C‐F), 640 (C‐Br). 1H NMR (400 MHz, CDCl3, δ, ppm): 7.43‐7.78 (m, 4H, H‐Aromatic), 8.51 (s, 1H, pyridine), 9.10 (s, 1H, pyridine), 10.75 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 116.0, 119.5, 129.6, 130.8, 131.5, 138.5, 148.1, 150.4, 161.7, 162.9. MS (EI, m/z (%)): 292.11. 6‐Bromo‐2‐(3‐fluorophenyl)‐3H‐imidazo[4, 5‐b]pyridine (3i): Color: Yellow solid. Yield: 94%. M.p.:147‐149 °C. FT‐IR (KBr, , cm‐1): 3218 (NH), 3053 (aromatic CH), 1577 (C=N), 1457 (C=C), 965 (C‐F), 674 (C‐Br). 1H NMR (400 MHz, CDCl3, δ, ppm): 7.33‐7.79 (m, 4H, H‐Aromatic), 8.55 (s, 1H, pyridine), 9.16 (s, 1H, pyridine), 10.78 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 115.5, 116.1, 119.5, 123.2, 127.8, 131.5, 132.4, 138.7, 148.2, 150.6, 161.7, 162.5. MS (EI, m/z (%)): 292.10. 6‐Bromo‐2‐(4‐nitrophenyl)‐3H‐imidazo[4, 5‐b]pyridine (3j): Color: Yellow solid. Yield: 94%. M.p.: 185‐187 °C. FT‐IR (KBr, , cm‐1): 3228 (NH), 3033 (aromatic CH), 1513 (C=N), 1469 (C=C), 1339 (NO2), 686 (C‐Br). 1H NMR (400 MHz, CDCl3, δ, ppm): 8.03 (m, 4H, H‐Aromatic), 8.58 (s, 1H, pyridine), 9.06 (s, 1H, pyridine), 10.79 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 119.5, 124.2, 127.2, 131.5, 138.7, 140.3, 147.6, 148.2, 150.6, 161.7. MS (EI, m/z (%)): 319.20. 4‐(6‐Bromo‐3H‐imidazo[4, 5‐b]pyridin‐2‐yl)benzonitrile (3k): Color: Yellow solid. Yield: 92%. M.p.: 260‐262 °C. FT‐IR (KBr, , cm‐1): 3226 (NH), 3022 (aromatic CH), 2224 (CN), 1569 (C=N), 1464 (C=C), 624 (C‐Br). 1H NMR (400 MHz, CDCl3, δ, ppm): 7.53‐7.79 (m, 4H, H‐Aromatic), 8.54 (s, 1H, pyridine), 9.09 (s, 1H, pyridine), 10.76 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 112.3, 118.6, 119.2, 127.2, 131.5, 132.4, 138.7, 139.3, 148.2, 150.6, 161.7. MS (EI, m/z (%)): 299.10. 6‐Bromo‐2‐(thiophen‐2‐yl)‐3H‐imidazo[4, 5‐b]pyridine (3l): Color: Yellow solid. Yield: 96%. M.p.: 160‐162 °C. FT‐IR (KBr, , cm‐1): 3224 (NH), 3065 (aromatic CH), 1594 (C=N), 1457 (C=C), 702 (C‐Br). 1H NMR (400 MHz, CDCl3, δ, ppm): 7.35‐7.72 (m, 3H, H‐Aromatic), 8.53 (s, 1H, pyridine), 9.12 (s, 1H, pyridine), 10.86 (s, 1H, NH). 13C NMR (100 MHz, CDCl3, δ, ppm): 119.2, 128.2, 129.1, 131.6, 131.8, 132.4, 138.7, 143.2, 148.2, 150.6. MS (EI, m/z (%)): 280.20. 2.3. Biological evaluation 2.3.1. Antibacterial activity All the synthesized compounds (3a‐l) were screened for their in vitro antibacterial activity; three Gram positive bacteria; Bacillus subtilis (NCIM‐2063), Enterococcus faecalis (NCIM‐5443) and Staphylococcus aureus (NCIM‐2901), three Gram negative bacteria; Escherichia coli (NCIM‐2256), Pseudomonas aeruginosa (NCIM‐2037) and Salmonella typhimurium (NCIM‐2501). The organisms were obtained from the National Chemical Laboratory, Pune, MS, India. The antibacterial assay was carried out by a microdilution method [35‐37] in order to determine the antibacterial activity of compounds tested against the human pathogenic bacteria. The bacterial suspensions were adjusted with sterile saline to a concentration of 1.0 × 105 CFU/mL. The inocula were prepared daily and stored at +4 °C until use. The dilutions of the inocula were cultured on solid medium to verify the absence of contamination and to check the validity of the inoculums. All experiments were performed in duplicate and repeated three times. 2.3.2. Antifungal activity All the synthesized compounds (3a‐l) were screened for their in vitro antifungal activity. Six fungal strains; Candida albicans (NCIM‐3471), Aspergillus flavus (NCIM‐539), Aspergillus oryzae (NCIM‐570), Aspergillus Niger (NCIM‐1196), Penicillium chrysogenum (NCIM‐707) and Fusarium oxysporum (NCIM‐1282). The organisms were obtained from the National Chemical Laboratory, Pune, MS, India. The micromycetes were maintained on malt agar and the cultures stored at +4 °C and sub‐cultured once a month. In order to investigate the antifungal activity of the compounds, a modified microdilution technique was used [38]. The fungal spores were washed from the surface of agar plates with sterile 0.85% saline containing 0.1% Tween 80 (v:v). The spore suspension was adjusted with sterile saline to a concentration of approximately 1.0 × 105 in a final volume of 100 µL per well. The inocula were stored at +4 °C for further use. Dilutions of the inocula were cultured on solid malt agar to verify the absence of contamination and to check the validity of the inoculum. The Minimum inhibitory concentration (MIC) determina‐ tions were performed by a serial dilution technique using 96‐ well microtiter plates. The compounds investigated were dissolved in 5% DMSO solution containing 0.1% Tween 80 (v:v) (1 mg/mL) and added in broth Malt medium with inoculum. 28 Shelke et al. / European Journal of Chemistry 8 (1) (2017) 25‐32 Scheme 2 The microplates were incubated at Rotary shaker (160 rpm) for 72 h at 28 °C. The lowest concentrations without visible growth (at the binocular microscope) were defined as MICs. The fungicidal concentrations (MFCs) were determined by serial sub‐cultivation of a 2 µL of tested compounds dissolved in medium and inoculated for 72 h, into microtiter plates containing 100 µL of broth per well and further incubation 72 h at 28 °C. The lowest concentration with no visible growth was defined as MFC indicating 99.5% killing of the original inoculum. The DMSO was used as a negative control, commercial fungicides; fluconazole and miconazole were used as positive controls (1‐3000 µg/mL). All experi‐ ments were performed in duplicate and repeated three times. 2.3.3. Microdilution test The minimum inhibitory and bactericidal concentrations (MICs and MBCs) were determined using 96‐well microtiter plates. The bacterial suspension was adjusted with sterile saline to a concentration of 1.0 × 105 CFU/mL. The compounds to be investigated were dissolved in 5% DMSO solution containing 0.1% Tween 80 (v:v) (1 mg/mL) and added in broth LB medium (100 µL) with bacterial inoculum (1.0 × 104 CFU per well) to achieve the wanted concentrations. The micro‐plates were incubated at Rotary shaker (160 rpm) for 24 h at 48 °C. The lowest concentrations without visible growth (at the binocular microscope) were defined as concentrations that completely inhibited bacterial growth (MICs). The MBCs were determined by serial sub‐cultivation of 2 µL into microtiter plates containing 100 µL of broth per well and further incubation for 24 h. The lowest concentration with no visible growth was defined as the MBC, indicating 99.5% killing of the original inoculum. The optical density of each well was measured at a wavelength of 655 nm by Microplate manager 4.0 and compared with a blank and the positive control. The ciprofloxacin and ampicillin were used as a positive control (1 mg/mL DMSO). The DMSO was used as a negative control. All experiments were performed in duplicate and repeated three times. 2.3.4. Anticancer activity All the synthesized compounds were also tested for their in vitro anticancer activity against two human breast cancer cell lines MCF‐7 and BT‐474. The anticancer activity test is performed according to the procedure developed by the National Cancer Institute (NCI, USA) in the ‘In vitro Anticancer Drug Discovery Screen’ that uses the protein‐binding dye Sulforhodamine B (SRB) to assess cell growth [39,40]. Briefly, cells are grown in 96‐well plates in suspension and then were exposed for 48 hours to four serial concentrations of 10‐7 Molar (M), 10‐6 M, 10‐5 M and 10‐4 M of each compound. Following this, cells were fixed and stained with protein binding SRB stain. Excess stain is washed and bound stain was solubilized, and the absorbance was measured at 492 nm in a plate reader. Concentration of the compounds that inhibited 50% of the net cell growth, growth inhibition of 50% (GI50), was calculated from the dose response curve obtained for each test compound and cell line. GI50 values were presented in micro molar (μM) concentration. Adriamycin (Doxorubicin) was used as positive control for the comparison of cytotoxicity of synthesized compounds. Assays were performed in triplicate on three independent experiments and their mean values are taken as a final reading. 3. Results and discussion 3.1. Chemistry We have developed the protocol for the synthesis of 6‐ bromo‐2‐phenyl‐3H‐imidazo[4,5‐b]pyridine (3) (Scheme 1) by condensation between 5‐bromopyridine‐2,3‐diamine and benzaldehyde. In this reaction, various solvents were selected as a model reaction to optimize the reaction conditions. In terms of the effect of solvent on the condensation reaction, DMF was found to be the best solvent for the reaction (Table 1, entry 4); other solvents, including ethanol, methanol, benzene, toluene and tetrahydrofuran were less efficient (Table 1, entries 1, 2, 3, 5 and 6). Ethanol, methanol, benzene, toluene and tetrahydrofuran gave the corresponding product yield 30, 45, 50, 40 and 45%, respectively, which were the worst among these solvents. Nevertheless, all of these yields were generally low before further optimizations. All the reactions were carried out with equimolar amounts of each compound in 1 mL of solvent. Among these reactions same amounts of the solvent, DMF turned out to be the best choice with yields of 98% (Table 1, entries 4). We would like to mention here that DMF as a solvent was the best choice with a yield of 98% and less time required for the completion of the reaction (Table 1, entry 4). Thus, we decided to carry out the reactions in DMF solvent. The plausible reaction mechanism is shown in Scheme 2. The initial reaction of 5‐bromopyridine‐2,3‐diamine with benzaldehyde produces imine, which at the reflux temperature is dehydrated or loss of water. Then the cyclise the ring, product are formed by the loss of one proton. In continuation of our work [41‐48], on the synthesis of bioactive compounds, we have synthesized some imidazole analogues. In view of the facts mentioned above, imidazole derivatives were synthesized. Shelke et al. / European Journal of Chemistry 8 (1) (2017) 25‐32 29 Table 1. Screening of solvents, reaction time, and yield for the synthesis (3) a. Entry Solvent Time (h) Yield b (%) 1 Ethanol 11 30 2 Methanol 12 45 3 Benzene 10 50 4 DMF 3 98 5 Toluene 7 40 6 Tetrahydrofuran 11 45 a All the reaction was carried out in equimolar amounts of each compound in 1 mL of solvent. b Isolated yield. Table 2. Synthesis of 6‐bromo‐2‐(substituted)‐3H‐imidazo [4, 5‐b] pyridine (3a‐l) a. Compound Aldehydes (2a‐l) Time (min) Yield b % M.p (°C) MW c (min) Conv. d (h) MW c Conv. d 3a Benzaldehyde 5 3 99 98 165‐167 3b 4‐Hydroxybenzaldehyde 5 3 96 88 192‐194 3c 2,4‐Dichlorobenzaldehyde 6 4 95 86 214‐216 3d 4‐Chlorobenzaldehyde 6 3 96 90 137‐139 3e 2‐Chlorobenzaldehyde 6 4 94 90 157‐159 3f 4‐Methoxybenzaldehyde 5 4 95 86 191‐193 3g 2,4‐Dimethoxybenzaldehyde 5 3 93 85 196‐198 3h 4‐Fluorobenzaldehyde 6 4 93 90 270‐272 3i 3‐Fluorobenzaldehyde 6 4 94 86 147‐149 3j 4‐Nitrobenzaldehyde 5 3 94 88 185‐187 3k 4‐Formylbenzonitrile 5 3 92 82 260‐262 3l Thiophene‐2‐carbaldehyde 6 4 96 90 160‐162 a Reaction condition (3a‐l), Method A: Microwave‐assisted synthesis: N,N‐dimethylformamide, 110 °C, 5‐6 min; Method B: Conventional synthesis: N,N‐ dimethylformamide, reflux 3‐4 h. b Isolated yields. c Microwave. d Conventional. Scheme 3 With this in mind, we initiated a program to synthesize a series of new 6‐bromo‐2‐(substitu‐ted)‐3H‐imidazo[4,5‐ b]pyridine derivatives, which have different pharmacological active groups, which can exhibit anticancer and antimicrobial activity. We have successfully synthesized series of new 6‐ bromo‐2‐(substituted)‐3H‐imidazo[4,5‐b]pyridine (3a‐l) (Scheme 3) by using N,N‐dimethylformamide (DMF) as a solvent with glacial acetic acid in catalytic amounts, under microwave‐assisted technique as well as conventional method. All the synthesized compounds are tested for their in vitro antimicrobial activities against selected bacterial and fungal strains as well as for anticancer against MCF‐7 and BT‐474 human breast cancer cell line. Our current investigation describes the convenient synthesis of new 6‐bromo‐2‐(substituted)‐3H‐imidazo[4,5‐ b]pyridine (3a‐l) (Scheme 3) using DMF as a solvent under microwave assisted as well as conventional synthesis with high yield in a short time period. This method is unique, rapid and convenient for the synthesis 3H‐imidazo[4,5‐b]pyridine derivatives. We herein report, the synthesis and screening of anticancer and antimicrobial activity of this series. Microwave used in the study was MicroSYNTH Lab station of Ethusi Milestone with temperature control (Scheme 3, method A). A one‐pot, microwave assisted as well as conventional synthesis by using a 5‐bromopyridine‐2,3‐diamine, substituted alde‐ hydes and DMF as a solvent to give the compounds (3a‐l) (Table 2). These compounds were characterized on the basis of spectral analysis. The IR spectrum of representative compound 6‐bromo‐2‐phenyl‐3H‐imidazo[4,5‐b]pyridine (3a), IR absorption bands in the wave numbers show 3065 cm‐1 that is due to a secondary amine group, 702 cm‐1 that is due to a bromine atom. The mass spectrum revealed a molecular ion peak at m/z was 276.18 (M+2) corresponding to a molecular formula C12H8BrN3. The 1H NMR spectrum of the compound 3a has been shown multiplate of benzyl proton in the range of δ ppm 6.60‐6.95 (m, 5H) and 7.62 (s, 1H, pyridine H), 9.40 (s, 1H, pyridine H), 10.10 (s, 1H, NH). The synthesized com‐ pounds were characterized on the basis of IR, 1H NMR, 13C NMR, Mass spectral analysis. 3.2. Biological evaluation 3.2.1. Antimicrobial activity From the antimicrobial data it is clearly observed that many of the synthesized compounds were shows prominent antimicrobial activity (Table 3 and 4). Antimicrobial data indicate that among the twelve synthesis compounds of present series many shows promising good to moderate level of antimicrobial activity. Some compounds were narrow spectrum, active against only one fungal or bacterial strain while some of them were found to be broad spectrum molecules, active against both one and more fungal and bacterial strains. 30 Shelke et al. / European Journal of Chemistry 8 (1) (2017) 25‐32 Table 3. Antibacterial activities of title compounds (3a‐l). Compounds MIC Values (μg/mL) a B. subtilis S. aureus E. coli E. faecalis P. aeruginosa S. typhimurium 3a MIC 85 95 80 80 90 75 MBC 100 115 100 95 110 95 3b MIC 70 55 3.25 60 75 70 MBC 92 85 55 90 95 100 3c MIC 30 60 55 70 85 90 MBC 60 85 70 95 100 115 3d MIC 15 55 10 20 10 60 MBC 45 80 35 55 30 100 3e MIC 20 65 15 70 80 75 MBC 60 90 30 100 110 100 3f MIC 25 95 20 20 90 25 MBC 60 115 70 45 120 60 3g MIC 90 100 75 100 95 85 MBC 110 120 95 120 110 100 3h MIC 55 70 80 65 75 60 MBC 70 100 110 90 95 100 3i MIC 80 70 90 95 85 90 MBC 100 90 120 120 100 115 3j MIC 70 50 60 65 70 65 MBC 95 85 90 90 95 100 3k MIC 6.0 60 75 95 80 70 MBC 50 85 95 90 100 110 3l MIC 90 100 75 80 85 90 MBC 120 120 100 115 115 110 Ciprofloxacin MIC 6.25 6.25 4.0 6.25 6.25 4.0 MBC 20 20 20 20 20 20 Ampicillin MIC 12.5 12.5 12.5 12.5 12.5 12.5 MBC 25 25 25 25 25 25 a Values are the average of three readings. Table 4. Antifungal activity of title compounds (3a‐l). Compounds MIC Values (μg/mL) a A. oryzae P. chrysogenum F. oxysporum C. albicans A. flavus A. niger 3a MIC 95 80 85 95 100 90 MFC 130 105 110 100 115 120 3b MIC 65 85 70 60 85 90 MFC 90 115 100 95 100 130 3c MIC 75 100 90 60 90 80 MFC 95 130 125 100 115 95 3d MIC 90 70 85 70 80 95 MFC 115 100 115 105 110 115 3e MIC 85 90 100 20 85 90 MFC 100 115 130 60 110 120 3f MIC 30 100 95 90 30 35 MFC 60 125 125 110 70 65 3g MIC 100 80 85 90 85 90 MFC 120 115 105 130 100 115 3h MIC 95 85 90 65 85 90 MFC 115 120 105 95 100 125 3i MIC 95 80 90 85 95 100 MFC 130 100 115 100 130 115 3j MIC 95 90 100 70 80 75 MFC 120 105 130 95 100 95 3k MIC 70 65 75 70 90 80 MFC 115 90 100 95 130 125 3l MIC 95 90 100 80 85 90 MFC 115 130 125 105 100 120 Fluconazole MIC 6.25 10 6.25 10 6.25 6.25 MFC 55 50 45 40 50 40 Miconazole MIC 3.25 3.25 3.25 3.25 3.25 3.25 MFC 40 35 45 40 35 40 a Values are the average of three readings. Among the series compounds 3b and 3k were found to be the narrow spectrum molecule as they were specifically active against the bacterium E. coli and B. subtilis, respectively. They are also most active molecules among the series, more potent than standard antibacterial drugs Ciprofloxacin and Ampicillin, with a MIC of 3.25 μg/mL for compound 3b and 6.0 μg/mL for compound 3k. On the other hand, compounds 3f, 3f and 3c are found to be quite broad spectrum molecules as they are active against one or more bacterial as well as fungal strains. Compounds 3f were found active against the majority of testing strains in the present studies, bacteria B. subtilis (MIC 25) and E. coli (MIC 20), E. faecalis and S. typhimurium, and fungus A. flavus (MIC 30) and A. niger (MIC 35) and A. oryzae (MIC 30). While the compound 3e is active against the fungus C. albicans (MIC 20), in addition to bacterial strains B. subtilis (MIC 20) and E. coli (MIC 15). The compound 3c has the same activity profile of the compound 3e but with somewhat high MIC values. However, the compound 3d is bacterial specific, active agaist the four bacterial strains, B. subtilis (MIC 15), E. faecalis (MIC 20), E. Coli (MIC 10) and P. aeruginosa (MIC 10). Remaining compounds of the series 3a, 3g, 3i and 3l are found to be biologically inactive in nature owing to their high MIC value ranges from 75 to 100 μg/mL and their neutral nature towards the both cancer cell lines. Shelke et al. / European Journal of Chemistry 8 (1) (2017) 25‐32 31 Table 5. Anticancer activity of the compounds (3a‐l). Compounds (IC50) a µMolar b Compounds (IC50) a µMolar b MCF‐7 c BT‐474 d MCF‐7 c BT‐474 d 3a 68.8 56.8 3g 88.4 68.5 3b 65.7 41.0 3h 1.7 0.9 3c 64.8 48.4 3i 82.2 87.1 3d 59.5 56.2 3j 1.3 1.1 3e 59.7 57.6 3k 75.7 66.5 3f 80.5 48.6 3l 81.1 81.2 Adriamycin e <0.1 a GI50 (Growth inhibition of 50) : Concentration of drug that decreases the growth of the cells by 50% compared to non‐treated control cell. b Values are the average of three readings. c MCF‐7: Human Breast cancer cell line. d BT474: Human Breast cancer cell line. e Adriamycin : Positive control compound. 3.2.2. Anticancer activity The result of this study (Table 5) indicates that compound 3h and 3j shows prominent anticancer activity against both cell lines, having Growth inhibition of 50 (GI50) values of 0.9 to 1.7 µM. All experiments were performed in duplicate and repeated three times. The structure activity relationship of the series can be explained as, Effect of phenyl ring: Presence of only phenyl ring, without any substitution, at C2 position on imidazole ring (3a), in 6‐ bromo‐2‐(substituted)‐3H‐imidazo[4,5‐b]pyridine moiety is inactive in nature, do not show any antimicrobial and anticancer activity. It indicates that, to attain the antimicrobial activity, substituted groups may present on the phenyl ring. Effect of hydroxyl group: Substitution of the hydroxyl group at the para position on the phenyl ring (3b), in 6‐bromo‐2‐ (substituted) ‐3H‐imidazo [4,5‐b] pyridine moiety, make the molecule narrow spectrum and active only against bacterium E. coli. The effect of nitrile group: New 6‐bromo‐2‐(substituted)‐ 3H‐imidazo[4,5‐b]pyridine moiety containing nitrile groups at the para position on the phenyl ring (3k) shows strain specific activity, active only against bacterium B. subtilis. Effect of chloro group: Introduction of the chloro group in the para position (3d) on the phenyl ring make a molecule bacterial specific; active towards B. subtilis E. faecalis, P. aeruginosa and E. coli. On the other hand, changing the position of this group from para to ortho (3e) on the phenyl ring make the molecule also active towards the fungus C. albicans along with its antibacterial properties. This indicates that changing the position of the chloro group on phenyl ring, from para to ortho, also changes its antimicrobial activity profile, and this change is favorable for the development of antimicrobial active molecules by incorporating bromo‐2‐ (substituted)‐3H‐imidazo[4,5‐b]pyridine moiety. However, interestingly simultaneous presents of the chloro group in the ortho and para position on the phenyl ring (3c) make the molecule inactive in nature. This may happen due to increase the size of molecules as well as the steric crowding effect imposed by two chloro groups, inhibiting the binding of molecules to its target in biological system. The effect of methoxy group: Introduction of methoxy group on the phenyl ring at the para position (3f) makes the molecule broad spectrum; active against most of the bacterial and fungal strains tested. On the other hand simultaneously substitution of methoxy group at ortho and para position (3g) result in inactivation of the molecule against all bacteria and fungus. It may be due to increase the electron donation effect of dimethoxy group on the phenyl ring. Effect of fluoro group: Presence of fluoro group at the meta position on the phenyl ring (3i) make a molecule biologically inactive in nature. While fluoro group substitution at the para position on the phenyl ring (3h) gives the potent anticancer molecule, inhibiting the growth of breast cancer cell lines, MCF7 and BT‐474, in‐vitro. Effect of nitro group: The substitution of the Nitro group at the para position on the phenyl ring (3j) shows promising anticancer activity. It may be due to the electron withdrawing nature of nitro group. Effect of thiophen‐2‐yl group: The substitution of thiophen‐ 2‐yl group at 2‐position on imidazole moiety (3l), shows inactive in nature. It may be due to the small size of five member sulfur containing ring. 4. Conclusions In conclusion, the compounds were synthesized by microwave‐irradiations as well as conventional methods with reduced reaction times and increased yields of the products. Among the synthesized compounds, 3h and 3j shows prominent anticancer activity against both breast cancer cell lines, MCF‐7 and BT‐474. While the compounds 3b and 3k are specifically active towards bacterium E. coli and B. subtilis, respectively; more potent than standard drugs used. 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