Synthesis and Evaluation of 2, 3, 4, 6-Tetra-Substituted-2, 3- Dihydropyridine Derivatives as Novel Antimicrobial Agents

4268 Int J Pharm Sci Nanotech Vol 11; Issue 5 September October 2018 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 11 Issue 5 September October 2018 Research Paper MS ID: IJPSN-7-19-18-BHAGAVAAN Synthesis and Evaluation of 2, 3, 4, 6-Tetra-Substituted-2, 3- Dihydropyridine Derivatives as Novel Antimicrobial Agents A. Ramesh A 1, M. Bhagavaan Raju 2* and A. Raghuram Rao 3 1 Department of Pharmaceutical Chemistry, Vaagdevi Institute of Pharmaceutical Sciences, Warangal. 2 Department of Pharmaceutical Chemistry, Sree Venkateshwara College of Pharmacy, Hyderabad, India; 3 National Institute of Pharmaceutical Education & Research, Mohali, Punjab, India; and 3 Department of Pharmaceutical Chemistry, UCPSc, Kakatiya University, Warangal, Telangana, India. Received July 19, 2018; accepted August 12, 2018 ABSTRACT The present study has been carried out to synthesize and screen certain heterocyclic antimicrobial compounds with clues from the biologically potent activities of heterocyclic compounds containing pyridine. With a view to synthesize some biologically active compounds, it has been felt worthwhile to study the synthesis of 2, 3, 4, 6-tetrasubstituted-2,3-dihydro pyridine derivatives (7a-t). They were synthesized by the condensation of respective 2-amino-2,3- dihydropyridines with different aromatic aldehydes. 7a-t were purified and characterized by physical and spectral methods (IR, 1HNMR & MS). All the compounds were evaluated for their KEYWORDS: Pyridine; Condensation; Antimicrobial; Antibacterial; Antifungal activity. antimicrobial activity by the agar diffusion method against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Candida albicans and Saccharomyces cerevisiae. All the compounds exhibited mild to moderate antibacterial & antifungal activity. Among these compounds, compound 7h (R= Cl, R1=Cl) showing greater inhibitory activity against all tested organisms employed with zones inhibition of 20 to 15 mm at a concentration of 150 g/ml. Compounds 7g, 7c, 7i and 7j have been found as the next in the order of its antimicrobial potency. These compounds have the potential as novel antimicrobial agents. Introduction It has been observed that during the last 10 years many major pathogenic bacteria and parasites have acquired resistance towards chemotherapeutic agents in market. Concern has been echoed in World Health Assembly (1998) with the adoption of a resolution on antimicrobial resistance (WHO Workshop, 1999). This has raised fears that infectious diseases may once again become major cause of death in developing/developed countries. Now, there is a need to give serious consideration towards development of novel chemotherapeutic agents to combat Multi-Drug Resistant (MDR) strains. Pyridine moieties are common sub-structures in numerous natural products, pharmaceuticals, and functional materials. Polysubstituted pyridines possess important biological & pharmacological activities and mainly anti-microbial agents. Multi-component reactions (MCRs) are powerful tools in modern synthetic organic chemistry, enabling straightforward access to large libraries of structurally related compounds and there by facilitating lead generation. Hence, combined with the use of combinatorial chemistry and high-through put parallel synthesis, such reactions have constituted an increasingly valuable approach to drug discovery efforts in recent years (Hulme et al., 2003). The synthesis of functionalized organic compounds via one-pot reaction may be convenient to perform without the isolation of the intermediates, without discharging any functional groups in short reaction time (Boulard et al., 2004). One of the newly developed combination therapies is multi-target approach which is a promising tool for identification of lead molecule (Zimmermann et al., 2007). Several efficient drugs, such as non-steroidal antiinflammatory drugs (NSAIDs) (Atla et al., 2014), antidepressants (Abdel-Latif et al., 2005), antineurodegenerative agents (Maqbool et al., 2017), multitarget kinase inhibitors (Kalash L et al., 2017) affect many targets simultaneously. And also, multi-target antibodies are increasingly used in cancer therapy to delay the development of resistance (Frantz et al., 2005). The compounds containing an azomethine group ( CH=N ) are important in determines the mechanism of transamination and racemization reactions in biological systems. Due to the great flexibility and diverse structural aspects, a wide range of Schiff bases have been synthesized and their complexation behaviors have been studied. Antibacterial, antifungal, and anticancer activities of Schiff bases have been reported and they are active against a wide range of organisms. Antibacterial activity has been studied more than antifungal activity, because bacteria can achieve resistance to antibiotics through biochemical and morphological modifications. Some Schiff bases bearing aryl groups possess excellent 4268

Ramesh et al: Synthesis and Evaluation of 2, 3, 4, 6 Tetra-Substituted-2, 3-Dihydropyridine Derivatives 4269 biological activities have attracted the attention of many researchers in recent years. The Schiff bases formed from aromatic aldehydes and their derivatives are quite stable. Antimicrobial activity of various Schiff bases has also been reported. Many Schiff bases are known to be medicinally important and are used to design medicinal compounds (Khaksar et al., 2012). In the present study, we attempted to synthesize a new series of 2, 3, 4, 6-tetra-substituted-2,3-dihydro pyridine derivatives (7a-t), which were synthesized by the condensation of respective 2-amino-2,3-dihydropyridines with different aromatic aldehydes. Further, these synthesized Schiff bases were screened for their antimicrobial activity. Materials and Methods Materials Melting points were determined using Boethius Apparatus by capillary method and are uncorrected. FT- IR spectra were taken on a Bruker FT-IR Opus Spectroscopic Software Version 2.0 (Bruker Instruments Inc., USA) from 4000-400 cm-1 using KBr discs. 1 H-NMR spectra were recorded at 400 MHz in DMSO-d6 using a Bruker Avance 400 instrument (Bruker Instruments Inc., USA). Chemical shifts were measured in δ (ppm) unit relative to tetramethylsilane (TMS). FAB-MS spectra were recorded on a Jeol SX 102/DA-6000 Mass Spectrometer (Jeol Ltd. Akishima, Tokyo, Japan) using argon/xenon (6 kv, 10 ma) as FAB gas, m-nitrobenzyl alcohol as matrix, and 10 kv as accelerating voltage at room temperature. Elemental analysis was performed on Vario EL III Elemental Analyser (Elementar, Germany) using Sulfanilamide as standard. All chemicals were purchased from Aldrich, E Merck, Spectrochem, CDH, Himedia, Finar or Avra India. Solvents were of reagent grade and were purified and dried by standard procedure. Reactions were monitored using Thin-layer chromatography on Silica Gel F 254 plates (Merck) with visualization by UV (254 nm) chamber. All the cyanopyridines have been purified by column chromatography performed on silica gel (100-200 mesh, Merck). Final compounds were characterized by 1 H- NMR, FAB-MS and elemental analysis. In the elemental analysis, the observed values were within ±0.4% variation of the calculated values. Experimental Synthesis of 2-amino-3-cyano-4-(substituted phenyl)- 6-phenyl pyridines (5a-d): Substituted aromatic aldehydes 1 (0.005 mol), acetophenone 2 (0.005 mol), malononitrile 3 (0.005 mol), ammonium acetate 4 (0.02 mol) and absolute alcohol (15 ml) in a 100 ml round bottom flask and was refluxed under heat for about 2-3 hrs. Progress of the reaction was monitored by the TLC. After completion of the reaction, the reaction mixture was poured into crushed ice with constant stirring. The precipitated solid was separated and dried. It was purified by column chromatography using ethyl acetate and hexane mixture (Gupta et al., 2010). The synthesized compounds 5a-d were confirmed by Mass spectrum, M/Z (M+1) values were observed at 297 (5a), 304 (5c and 5d) and (M+2) values was observed at 307 (5b). The structure, physico-chemical characterization of compounds 5a-d were presented in Table 1. TABLE 1 Physical data of intermediates (5a-d & 6a-d) and 2, 3, 4, 6-tetra substituted-2,3-dihydropyridine derivatives. S. No. Comp Code Substituents R R 1 Molecular Formula Mol. wt M.P. ( o C) Yield (%) Rf 1. 5a 4-CN NA C19H12N4 296.11 144-146 65 0.36 2. 5b 4-Cl NA C18H12ClN3 305.07 148-150 72 0.41 3. 5c 2,3-OH NA C18H13N3O2 303.10 152-154 59 0.32 4. 5d 2,4-OH NA C18H13N3O2 303.10 150-152 62 0.33 5. 6a 4-CN NA C20H15ClN4O 362.09 162-164 68 0.38 6. 6b 4-Cl NA C19H15Cl2N3O 371.06 170-172 70 0.31 7. 6c 2,3-OH NA C19H16ClN3O3 369.09 198-200 62 0.39 8. 6d 2,4-OH NA C19H16ClN3O3 369.09 194-196 65 0.37 9. 7a 4-CN H C27H19N4OCl 450.91 192-194 78 0.41 10. 7b 4-CN 4-CN C28H18N5OCl 475.92 196-198 72 0.28 11. 7c 4-CN 4-Cl C27H18N4OCl2 485.36 202-204 77 0.29 12. 7d 4-CN 2,3-OH C27H19N4O3Cl 482.91 198-200 72 0.27 13. 7e 4-CN 2,4-OH C27H19N4O3Cl 482.91 202-204 69 0.29 14. 7f 4-Cl H C26H19N3OCl2 460.35 194-196 75 0.38 15. 7g 4-Cl 4-CN C27H18N4OCl2 485.36 208-210 66 0.31 16. 7h 4-Cl 4-Cl C26H18N3OCl3 494.79 228-230 78 0.39 17. 7i 4-Cl 2,3-OH C26H19N3O3Cl2 492.35 216-218 73 0.37 18. 7j 4-Cl 2,4-OH C26H19N3O3Cl2 492.35 214-216 67 0.29 19. 7k 2,3-OH H C26H20N3O3Cl 457.90 196-198 74 0.35 20. 7l 2,3-OH 4-CN C27H19N4O3Cl 482.91 196-198 72 0.26 21. 7m 2,3-OH 4-Cl C26H19N3O3Cl2 492.35 228-230 65 0.36 22. 7n 2,3-OH 2,3-OH C26H20N3O5Cl 489.90 224-226 71 0.41 TABLE 1 Contd

4270 Int J Pharm Sci Nanotech Vol 11; Issue 5 September October 2018 S. No. Comp Code Substituents R R 1 Molecular Formula Molecular Formula Mol. wt M.P. ( o C) Yield (%) Rf 23. 7o 2,3-OH 2,4-OH C26H20N3O5Cl 489.90 220-222 73 0.32 24. 7p 2,4-OH H C26H20N3O3Cl 457.90 202-204 63 0.33 25. 7q 2,4-OH 4-CN C27H19N4O3Cl 482.91 212-214 68 0.24 26. 7r 2,4-OH 4-Cl C26H19N3O3Cl2 492.35 218-220 70 0.28 27. 7s 2,4-OH 2,3-OH C26H20N3O5Cl 489.90 220-222 77 0.39 28. 7t 2,4-OH 2,4-OH C26H20N3O5Cl 489.90 218-220 64 0.32 Synthesis of 2-amino-3-chloro-3-cyano-2-methoxy--4- (substituted phenyl)-6-phenyl-2,3-dihydropyridines (6a-d): 5a-d (0.005 mol), N-Chlorosuccinimide (0.0055 mol) and Methanol (10 ml) in a 50 ml round bottom flask and stirred for 3 hrs at room temperature. Progress of the reaction was monitored by the TLC. After completion of the reaction, the reaction mixture was poured into crushed ice with constant stirring. The precipitated solid was separated and dried. The synthesized compounds 6ad were confirmed by Mass spectrum, M/Z (M+2) values were observed at 364 (6a), 373 (6b), 371 (6c & 6d). The structure, physico-chemical characterization of compounds 6a-d were presented in Table 1. Synthesis of 2-substituted benzylidineamino-3-chloro- 3-cyano-2-methoxy-4-(substituted phenyl)-6-phenyl-2,3- dihydropyridines (7a-t): 6a-d (0.005 mol), respective benzaldehyde (0.005 mol) dissolved in minimum volume of ethanol and added catalytical amount of concentrated sulphuric acid was added then stirred for 4-5 hrs at room temperature. Progress of the reaction was monitored by the TLC. It was cooled to 0 o C, the formed precipitate was filtered, washed with ethanol, and recrystallized from methanol (Tacconi et al., 1980). It was purified by column chromatography using ethyl acetate and hexane mixture. The structure, physico-chemical characterization was presented in Table 1. 2-Benzylidineamino-3-chloro-3-cyano-2-methoxy-4-(4- cyanophenyl)-6-phenyl-2,3-dihydro pyridine. (7a): IR (KBr, cm -1 ): 3497, 3354, 2214, 1632, 1542, 1361, 835; 1 H NMR (400MHz, CDCl3, δ, ppm): 3.29 (s, 3H, -OCH3), 6.38 (s, 1H, Pyridine), 7.18-7.64 (m, 14H, Ar), 8.28 (s, 1H, - CH=N-); MS m/z: 452 (M+2). Analysis Calculated for C27H19N4OCl: C, 71.92; H, 4.25; N, 12.43. Found: C, 71.83; H, 4.36; N, 12.85. methoxy-4-(4-cyanophenyl)-6-phenyl-2,3 -dihydropyridine. (7b): IR (KBr, cm -1 ): 3461, 3356, 2215, 1638, 1571, 1283, 838; 1 H NMR (400MHz, CDCl3, δ, ppm): 3.29 (s, 3H, - OCH3), 6.39 (s, 1H, Pyridine), 7.18-7.64 (m, 13H, Ar), 8.26 (s, 1H, -CH=N-); MS m/z: 477 (M+2). Analysis Calculated for C28H18N5OCl: C, 70.66; H, 3.81; N, 14.72. Found: C, 70.83; H, 3.95; N, 14.96. methoxy-4-(4-cyanophenyl)-6-phenyl-2,3 -dihydropyridine. (7c): IR (KBr, cm -1 ): 3461, 3357, 2216, 1637, 1573, 1284, 838: 1 H NMR (400MHz, CDCl3, δ, ppm): 3.28 (s, 3H, - OCH3), 6.60 (s, 1H, Pyridine), 7.18-7.63 (m, 13H, Ar), 8.26 (s, 1H, -CH=N-); MS m/z: 487 (M+2), 489 (M+4). Analysis Calculated for C27H18N4OCl2: C, 66.81; H, 3.74; N, 11.54. Found: C, 66.95; H, 3.93; N, 11.87. 2-methoxy-4-(cyanophenyl)-6-phenyl -2,3-dihydropyridine. (7d): IR (KBr, cm -1 ): 3737, 3358, 2216, 1638, 1544, 1284, 840; 1 H NMR (400MHz, CDCl3, δ, ppm) 3.28 (s, 3H, - OCH3), 5.20-5.30 (s, 2H, -OH), 6.62 (s, 1H, Pyridine), 7.28-7.65 (m, 12H, Ar), 8.26 (s, 1H, -CH=N-); MS m/z: 484 (M+2). Analysis Calculated for C27H19N4O3Cl: C, 67.15; H, 3.97; N, 11.60. Found: C, 67.64; H, 3.47; N, 11.98. 2-(2,4-Dihydroxybenzylidine) amino-3-chloro-3-cyano- 2-methoxy-4-(cyanophenyl)-6-phenyl -2,3-dihydropyridine. (7e): IR (KBr, cm -1 ): 3461, 3357, 2216, 1638, 1572, 1284, 838; 1 H NMR (400MHz, CDCl3, δ, ppm): 3.29 (s, 3H, - OCH3),5.30-5.40 (s, 2H, -OH), 6.79 (s, 1H, Pyridine),7.28-7.65 (m, 12H, Ar), 8.29 (s, 1H, -CH=N-); MS m/z: 484.91 (M+2). Analysis Calculated for C27H19N4O3Cl: C, 67.15; H, 3.97; N, 11.60. Found: C, 67.72; H, 3.42; N, 11.89. 2-Benzylidineamino-3-chloro-3-cyano-2-methoxy-4-(4- chlorophenyl)-6-phenyl-2,3-dihydro pyridine. (7f): IR (KBr, cm -1 ): 3461, 3356, 2211, 1632, 1573, 1284, 838; 1 H NMR (400MHz, CDCl3, δ, ppm): 3.29(s, 3H, -OCH3), 6.62 (s, 1H, Pyridine), 7.19-7.63 (m, 14H, Ar), 8.29 (s, 1H, - CH=N-); MS m/z: 462.35 (M+2). Analysis Calculated for C26H19N3OCl2: C, 67.83; H, 4.16; N, 9.13. Found: C, 67.34; H, 4.58; N, 9.87. methoxy-4-(4-chlorophenyl)-6-phenyl-2,3 -dihydropyridine. (7g): IR (KBr, cm -1 ): 3669, 3299, 2213, 1637, 1542, 1241, 828. 1 H NMR (400MHz, CDCl3, δ, ppm): 3.20 (s, 3H, - OCH3), 6.37 (s, 1H, Pyridine), 7.18-7.64 (m, 13H, Ar), 8.28 (s, 1H, -CH=N-). MS m/z: 487.35 (M+2). Analysis Calculated for C27H18N4OCl2: C, 66.81; H, 3.74; N, 11.54. Found: C, 66.43; H, 3.98; N, 11.87. methoxy-4-(4-chlorophenyl)-6-phenyl-2,3-dihydropyridine. (7h): IR (KBr, cm -1 ): 3744, 3297, 2213, 1636, 1542, 1241, 828. 1 H NMR (400MHz, CDCl3, δ, ppm): 3.29 (s, 3H, - OCH3), 6.59 (s, 1H, Pyridine),7.28-7.68 (m, 13H, Ar), 8.27 (s, 1H, -CH=N-). MS m/z: 496.79 (M+2). Analysis Calculated for C26H18N3OCl3: C, 63.11; H, 3.67; N, 8.49. Found: C, 63.41; H, 3.92; N, 8.87. 2-methoxy-4-(4-chlorophenyl)-6-phenyl-2,3-dihydropyridine. (7i): IR (KBr, cm -1 ): 3739, 3358, 2211, 1639, 1547, 1282, 827. 1 H NMR (400MHz, CDCl3, δ, ppm): 3.45 (s, 3H, -OCH3), 5.43(s, 2H, -OH), 6.88 (s, 1H, Pyridine), 7.49-7.58 (m, 12H, Ar), 8.72 (s, 1H, -CH=N-). MS m/z: 494.35 (M+2). Analysis Calculated for C26H19N3O3Cl2: C, 63.43; H, 3.89; N, 8.53. Found: C, 63.97; H, 3.58; N, 8.98.

Ramesh et al: Synthesis and Evaluation of 2, 3, 4, 6 Tetra-Substituted-2, 3-Dihydropyridine Derivatives 4271 2-(2,4-Dihydroxybenzylidine) amino-3-chloro-3-cyano- 2-methoxy-4-(4-chlorophenyl)-6-phenyl-2,3-dihydropyridine. (7j): IR (KBr, cm -1 ): 3739, 3358, 2211, 1639, 1547, 1282, 827. 1 H NMR (400MHz, CDCl3, δ, ppm): 3.51 (s, 3H, - OCH3), 5.41 (s, 2H, -OH), 6.41 (s, 1H, Pyridine), 6.89 (s, 1H, Ar),7.48-7.59 (m, 11H, Ar), 8.72 (s, 1H, -CH=N-). MS m/z: 494.35 (M+2). Analysis Calculated for C26H19N3O3Cl2: C, 63.43; H, 3.89; N, 8.53. Found: C, 63.86; H, 3.26; N, 8.87. 2-Benzylidineamino-3-chloro-3-cyano-2-methoxy-4- (2,3-dihydroxyphenyl)-6-phenyl-2,3-dihydropyridine. (7k): IR (KBr, cm -1 ): 3719, 3348, 2216, 1624, 1507, 1280, 822. 1 H NMR (400MHz, CDCl3, δ, ppm): 3.49 (s, 3H, - OCH3), 5.39 (s, 2H, -OH), 6.41 (s, 1H, Pyridine),7.27-7.86 (m, 13H, Ar), 8.30 (s, 1H, -CH=N-). MS m/z: 459.90 (M+2). Analysis Calculated for C26H20N3O3Cl: C, 68.20; H, 4.40; N, 9.18. Found: C, 68.63; H, 4.78; N, 9.67. methoxy-4-(2,3-dihydroxyphenyl)-6-phenyl-2,3-dihydropyridine. (7l): IR (KBr, cm -1 ): 3358, 2216, 1638, 1544, 1284, 840. 1 H NMR (400MHz, CDCl3, δ, ppm): 3.29 (s, 3H, -OCH3), 5.30-5.40 (s, 2H, -OH), 6.30 (s, 1H, Pyridine), 7.22-7.88 (m, 12H, Ar), 8.50 (s, 1H, -CH=N-). MS m/z: 484.91 (M+2). Analysis Calculated for C27H19N4O3Cl: C, 67.15; H, 3.97; N, 11.62. Found: C, 67.43; H, 3.58; N, 11.98. methoxy-4-(2,3-dihydroxyphenyl)-6-phenyl-2,3-dihydropyridine. (7m): IR (KBr, cm -1 ): 3459, 3177, 2208, 1579, 1508, 1244, 849. 1 H NMR (400MHz, CDCl3, δ, ppm): 3.28 (s, 3H, -OCH3), 5.25-5.40 (s, 2H, -OH), 6.38 (s, 1H, Pyridine), 7.18-7.64 (m, 12H, Ar), 8.27 (s, 1H, -CH=N-). MS m/z: 494.35 (M+2). Analysis Calculated for C26H19N3O3Cl2: C, 63.43; H, 3.89; N, 8.53. Found: C, 63.87; H, 3.66; N, 8.88. 2-methoxy-4-(2,3-dihydroxyphenyl)-6-phenyl-2,3-dihydropyridine. (7n): IR (KBr, cm -1 ): 3736, 3265, 2208, 1632, 1557, 1259, 774; 1 H NMR (400MHz, CDCl3, δ, ppm): 3.24 (s, 3H, -OCH3), 5.31 (m, 4H, -OH), 6.21 (s, 1H, Pyridine), 7.29-7.38 (m, 10H, Ar), 7.68 (s, 1H, Ar), 8.42 (s, 1H, - CH=N-); MS m/z: 491.90 (M+2). Analysis Calculated for C26H20N3O5Cl: C, 63.43; H, 3.89; N, 8.53. Found: C, 63.98; H, 3.65; N, 8.89. 2-(2,4-Dihydroxybenzylidine) amino-3-chloro-3-cyano- 2-methoxy-4-(2,3-dihydroxyphenyl)-6-phenyl-2,3-dihydropyridine. (7o): IR (KBr, cm -1 ): 3478, 3178, 2210, 1638, 1579, 1238, 851. 1 H NMR (400MHz, CDCl3, δ, ppm): 3.52 (s, 3H, -OCH3), 5.43 (m, 4H, -OH), 6.32 (s, 1H, Pyridine), 7.49-7.58 (m, 11H, Ar), 8.72 (s, 1H, -CH=N-); MS m/z: 491.90 (M+2). Analysis Calculated for C26H20N3O5Cl: C, 63.74; H, 4.11; N, 8.58. Found: C, 63.97; H, 4.58; N, 8.87. 2-Benzylidineamino-3-chloro-3-cyano-2-methoxy-4- (2,4-dihydroxyphenyl)-6-phenyl-2,3-dihydropyridine. (7p): IR (KBr, cm -1 ): 3728, 3348, 2216, 1612, 1574, 1280, 825. 1 H NMR (400MHz, CDCl3, δ, ppm): 3.29 (s, 3H, - OCH3), 5.30-5.40 (s, 2H, -OH), 6.39 (s, 1H, Pyridine),7.19-7.89 (m, 13H, Ar), 8.37 (s, 1H, -CH=N-), MS m/z: 459.90 (M+2). Analysis Calculated for C26H20N3O3Cl: C, 68.20; H, 4.40; N, 9.18. Found: C, 68.63; H, 4.76; N, 9.87. methoxy-4-(2,4-dihydroxyphenyl)-6-phenyl-2,3-dihydropyridine. (7q): IR (KBr, cm -1 ): 3363, 3177, 2210, 1612, 1574, 1228, 849. 1 H NMR (400MHz, CDCl3, δ, ppm): 3.18 (s, 3H, -OCH3), 5.30-5.40 (s, 2H, -OH), 6.20 (s, 1H, Pyridine), 7.28-7.74 (m, 12H, Ar), 8.57 (s, 1H, -CH=N-), MS m/z: 484.91 (M+2). Analysis Calculated for C27H19N4O3Cl: C, 67.15; H, 3.97; N, 11.60. Found: C, 67.43; H, 3.68; N, 11.87. methoxy-4-(2,4-dihydroxyphenyl)-6-phenyl-2,3-dihydropyridine. (7r): IR (KBr, cm -1 ): 3365, 3178, 2209, 1580, 1506, 1238, 850. 1 H NMR (400MHz, CDCl3, δ, ppm): 3.29 (s, 3H, -OCH3), 5.30-5.40 (s, 2H, -OH), 6.39 (s, 1H, Pyridine), 7.16-7.64 (m, 12H, Ar), 8.29 (s, 1H, -CH=N-). MS m/z: 494.35 (M+2). Analysis Calculated for C26H19N3O3Cl2: C, 63.43; H, 3.89; N, 8.53. Found: C, 63.89; H, 3.57; N, 8.92. 2-methoxy-4-(2,4-dihydroxyphenyl)-6-phenyl-2,3-dihydropyridine. (7s): IR (KBr, cm -1 ): 3364, 3178, 2210, 1579, 1510, 1238, 851. 1 H NMR (400MHz, CDCl3, δ, ppm): 3.28 (s, 3H, -OCH3), 5.48-5.49 (m, 4H, -OH), 6.24 (s, 1H, Pyridine), 7.28-7.50 (m, 11H, Ar), 8.42 (s, 1H, -CH=N-). MS m/z: 491.90 (M+2). Analysis Calculated for C26H20N3O5Cl: C, 63.74; H, 4.11; N, 8.58. Found: C, 63.98; H, 4.38; N, 8.87. 2-(2,4-Dihydroxybenzylidine)amino-3-chloro-3-cyano- 2-methoxy-4-(2,4-dihydroxyphenyl)-6-phenyl-2,3-dihydropyridine. (7t): IR (KBr, cm -1 ): 3744, 3358, 2208, 1622, 1577, 1240, 850. 1 H NMR (400MHz, CDCl3, δ, ppm): 3.28 (s, 3H, -OCH3), 5.43 (s, 4H, -OH), 6.88 (s, 1H, Pyridine), 7.49-7.52 (m, 11H, Ar), 8.72 (s, 1H, -CH=N-). MS m/z: 491.90 (M+2). Analysis Calculated for C26H20N3O5Cl: C, 63.74; H, 4.11; N, 8.58. Found: C, 63.93; H, 4.28; N, 8.87. Antimicrobial Activity Antibacterial Studies The antibacterial activity of the test compounds was assayed using serial double dilution method against nonpathogenic strains of Gram positive bacteria (Bacillus subtilis & Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli & Proteus vulgaris) in nutrient agar medium by the Cup-plate method (Nagashree et al., 2013). Sterilized media was cooled to 40 C and 0.5 ml of inoculum for 100 ml of media was added. The flasks were shaken gently to avoid formation of air bubbles. 25 ml portions of this media were transferred to Petri dishes of 9 cm diameter so as to obtain 4 5 mm thickness of the media layer. The plates were left at room temperature to allow solidification of the media. In each Petri plate, 4 cups of suitable diameter were made with a sterile borer. All these procedures were conducted aseptically under laminar air flow workstation. The test compounds and standard were dissolved in DMSO (0.5%) and solution ranging between 0.1 and 100 mm were prepared. DMSO control was also maintained. 40 ml of the test compounds and standard were added into each cup with the help of a micropipette. Plates were kept undisturbed for at least 2 h at room temperature to allow

4272 Int J Pharm Sci Nanotech Vol 11; Issue 5 September October 2018 for proper diffusion. Petri plates were then incubated at 37 ± 1 C for 24 h. Zone inhibitions (in mm) were measured after incubation. All the studies were performed in triplicate and results were presented in Table 2. Antifungal Studies The antibacterial activity of the test compounds was assayed using serial double dilution method against Saccharomyces cerevisiae & Candida albicans in Sabouraud dextrose agar medium by cup plate method. The sterile medium was inoculated using 24 h slant cultures of test organisms and transferred into sterile Petri dishes and allowed to solidify. 4 cups of suitable diameter were made on the solidified media. The test compounds and standard were dissolved in DMSO (0.5%) and solution ranging between 0.1 and 100 mm were prepared. DMSO control was also maintained. 40 ml of test compounds and standard were added into each cup with the help of a micropipette. Zones of inhibition (in mm) were measured after 24 h of incubation. All the studies were performed in triplicate and results were presented in Table 2. Results and Discussion Chemistry Target compounds, Compounds 7a-t were synthesized following the reaction sequence outlined in Scheme 1. By the condensation reaction of 2-amino-3-chloro-3-cyano-2- methoxy-4-(substitutedphenyl)-6-phenyl-2,3-dihydropyridines with different aromatic aldehydes. The structure of the products, 7a-t was established by physico-chemical and spectroscopic analysis. The IR spectra of 7a-t showed bands at 3400-3200 cm -1 (NH2), 3450-3425 (OH) cm -1, 2220-2210 cm -1 (C-N) and 1640-1550 cm -1 (C=N). The 1 H- NMR spectra of the synthesized compounds gave further support for the cyanopyridines structure, showed a singlet at δ 8.20-8.40 & 5.30-5.50 ppm attributed to the CH=N & -OH protons respectively. The characteristic singlet peak was observed at δ 6.40 ppm indicates the presence of single proton at C-4 position of pyridine ring, the above statement confirms the formation of 2- Substituted benzylidineamino-3-chloro-3-cyano-2- methoxy-4-(substituted phenyl)-6-phenyl-2,3-dihydropyridine. Other aromatic proton signals were appeared at δ 6.0-8.0 ppm. The parent ion peak appeared on the positive mode in the mass spectrum of all the compounds further confirms the structure of cyanopyridines. R = 4-CN, 4-Cl, 2,3-OH, 2,4-OH; R 1 = H, 4-CN, 4-Cl, 2,3-OH, 2,4-OH; Scheme 1 Antimicrobial Activity All twenty derivatives (7a 7t) were evaluated for their in vitro antimicrobial activity, in antibacterial activity adopted serial double dilution method against non-pathogenic strains of (Bacillus subtilis & Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli & Proteus vulgaris). Similarly, they were also evaluated for their antifungal activity against Saccharomyces cerevisiae & Candida albicans (Nagashree et al., 2013). The results are showed in Table 2. From the results, the data reveals that amongst all the synthesized compounds (7a-t), compounds 7h and 7c were exhibited good activity against Gram positive bacteria (Bacillus subtilis & Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli & Proteus vulgaris) when compared to standard (ampicillin), which was statistically significant. Antifungal study revealed that compounds 7h, 7c, 7i, 7m and 7r show more potent than the standard fungicidal Ketoconazole against Saccharomyces cerevisiae & Candida albicans. TABLE 2 Antimicrobial activity of 2-substituted benzylidine amino-3-chloro-3-cyano-2-methoxy-4-substituted phenyl-6-phenyl-2,3-dihydropyridines. Compound Bacillus Subtilis Streptococcus pneumonia Zone of Inhibition (at ug/ml; mm) Escherichia coli Proteus vulgaris Saccharomyces cerevisiae Candida albicans 50 100 150 50 100 150 50 100 150 50 100 150 50 100 150 50 100 150 7a 08 10 12 10 11 13 09 11 13 08 10 12 09 11 13 08 10 12 7b 10 11 13 11 13 14 10 12 14 09 11 13 12 13 15 11 12 15 7c 13 15 17 12 14 16 13 14 16 12 13 15 14 16 18 15 17 19 7d 11 12 14 09 11 13 08 10 12 08 09 11 11 13 14 11 13 15 7e 09 11 13 10 12 13 10 12 13 09 10 12 10 11 13 12 14 15 7f 11 13 15 11 12 14 11 12 14 12 13 15 13 15 17 14 16 18 7g 14 16 18 13 15 17 13 14 17 12 14 16 16 18 20 18 20 22 7h 16 18 20 16 17 19 15 18 20 14 16 18 18 20 22 20 22 24 TABLE 2 Contd

Ramesh et al: Synthesis and Evaluation of 2, 3, 4, 6 Tetra-Substituted-2, 3-Dihydropyridine Derivatives 4273 Compound Bacillus Subtilis Streptococcus pneumonia Zone of Inhibition (at ug/ml; mm) Escherichia coli Proteus vulgaris Saccharomyces cerevisiae Candida albicans 50 100 150 50 100 150 50 100 150 50 100 150 50 100 150 50 100 150 7i 12 14 16 12 14 15 12 13 15 10 12 14 16 18 19 14 16 18 7j 11 13 15 13 15 16 11 14 15 11 13 15 15 17 18 14 16 18 7k 06 07 09 05 06 08 05 06 08 05 06 08 06 07 09 06 07 08 7l 09 11 12 10 11 12 08 09 11 08 10 12 09 11 12 07 09 11 7m 11 13 14 11 12 14 10 12 13 09 11 12 12 14 16 11 13 15 7n 08 09 11 07 09 10 08 09 10 07 09 11 08 09 10 07 09 11 7o 07 08 10 08 10 11 07 09 10 06 08 10 06 08 09 07 09 11 7p 05 07 08 05 06 08 04 05 07 04 05 06 06 07 08 05 06 07 7q 08 09 11 09 10 12 09 10 11 06 08 10 08 10 11 06 08 10 7r 11 12 14 09 11 13 11 12 13 10 12 13 12 14 15 12 14 16 7s 06 07 09 07 08 10 06 07 09 06 07 09 06 07 08 06 08 10 7t 06 08 10 06 07 09 06 08 09 05 06 08 06 08 09 05 07 08 AMP 18 20 22 20 23 24 20 21 23 18 20 22 - - - - - - KTZ - - - - - - - - - - - - 22 24 26 24 26 28 AMP: Ampicillin: KTZ: Ketoconazol Conclusions The 2-amino-3-chloro-3-cyano-2-methoxy-4-(substitutedphenyl)-6-phenyl-2,3-dihydropyridines with different aromatic aldehydes were synthesized and characterized by spectral methods (IR, NMR & MS). They were subjected to evaluation for antimicrobial activity. From these results, the compound (7h) exhibited significant biological activity with reference to standard drugs. It may have potential as novel antimicrobial agent. Acknowledgements One of the authors acknowledge to the Director Synapse Life Sciences, Warangal and the Management and Director, Vaagdevi Institute of Pharmaceutical Sciences, Bollikunta, Warangal for providing necessary facilities to carry out research work, and also thankful to Mrs. Sravanthi Siliveri, Pullareddy College of Pharmacy, Hyderabad. References Abdel-Latif NA (2005). Synthesis and antidepressant activity of some new coumarin derivatives. Scientia Pharmaceutica. 73(4): 15. Atla SR, Nagireddy NR, and Yejella RP (2014). Anti-inflammatory, analgesic and antimicrobial activity studies of novel 4, 6- disubstituted-2-amino-3-cyanopyridines. International Journal of Pharmaceutical Chemistry and Analysis. 1(1): 47-57. Boulard L, BouzBouz S, Cossy J, Franck X, and Figadere B (2004). Two successive one-pot reactions leading to the expeditious synthesis of ( )-centrolobine. Tetrahedron letters. 45(35): 6603-5. Frantz S (2005). Drug discovery: playing dirty. 437: 942-943. Gupta R, Jain A, Jain M, and Joshi R (2010). 'One Pot' Synthesis of 2-Amino-3-cyano-4, 6-diarylpyridines under Ultrasonic Irradiation and Grindstone Technology. Bulletin of the Korean Chemical Society. 31(11): 3180-2. Hulme C and Gore V (2003). Multi-component Reactions: Emerging Chemistry in Drug Discovery: From Xylocain to Crixivan. Current Medicinal Chemistry. 10(1): 51-80. Kalash L, Val C, Azuaje J, Loza MI, Svensson F, Zoufir A, Mervin L, Ladds G, Brea J, Glen R, and Sotelo E (2017). Computer-aided design of multi-target ligands at A 1 R, A 2A R and PDE10A, key proteins in neurodegenerative diseases. Journal of Cheminformatics. 9(1): 67. Khaksar S and Yaghoobi M (2012). A concise and versatile synthesis of 2-amino-3-cyanopyridine derivatives in 2, 2, 2- trifluoroethanol. Journal of Fluorine Chemistry. 142: 41-4. Maqbool M (2017). Rational design, synthesis and biological screening of cyanopyridine-triazine hybrids as lead multitarget anti-alzheimer agents. Alzheimer's & Dementia: The Journal of the Alzheimer's Association. 13(7): 627. Nagashree S, Mallu P, Mallesha L, and Bindya S (2012). Synthesis, Characterization, and Antimicrobial Activity of Methyl-2- aminopyridine-4-carboxylate Derivatives. Journal of Chemistry. 2013. Tacconi G, Gatti G, Desimoni G, and Messori V (1980). A New Route to 4H Pyrano (2, 3 c) pyrazoles. Journal für Praktische Chemie. 322(5): 831-4. World Health Organization (1999). Review of the Literature and Report of a WHO Workshop on the Development of a Global Strategy for the Containment of Antimicrobial Resistance. Communicable Disease Surveillance and Response-Geneva, Switzerland, 4-5 February. Zimmermann GR, Lehar J, and Keith CT (2007). Multi-target therapeutics: when the whole is greater than the sum of the parts. Drug Discovery Today. 12(1-2): 34-42. Address correspondence to: Dr. M. Bhagavaan Raju, Department of Pharmaceutical Chemistry, Sree Venkateshwara College of Pharmacy, Hyderabad. Mob: +91 8790792161. E-mail: mbhagavanraju@gmail.com