HETEROCYCLES

Short Paper | Regular issue | Vol. 85, No. 9, 2012, pp. 2259-2268
Received, 4th June, 2012, Accepted, 6th July, 2012, Published online, 19th July, 2012.
DOI: 10.3987/COM-12-12520

■ A Facile and Convenient Synthesis of Novel Pyridine Derivatives Incorporating Antipyrine Moiety and Investigation of Their Antimicrobial Activities

Nabila A. Kheder, Sayed M. Riyadh,* and Ahlam M. Asiry

Department of Chemistry, Faculty of Science, Cairo University, Giza 12013, Egypt

Abstract

A series of potentially bioactive piperidines and pyridines, pendant to antipyrine moiety, were synthesized and evaluated in vitro for their antimicrobial potential. Condensation of acetamide 1 with arylaldehydes in (2:1 molar ratio) gave hitherto unreported novel piperidine-3-carboxamide derivatives 4a-c. Also, reaction of acetamide 1 with ethyl 2-cyano-3-(4-nitrophenyl)acrylate afforded unexpected piperidine-3-carboxamide 4c. On the other hands, treatment of acetamide 1 with (arylmethylene)malononitriles 7a,b afforded pyridine-3,5-dicarbonitrile derivatives 10a,b. The structures of the synthesized products were confirmed by IR, 1H NMR, 13C NMR and mass spectral techniques.

The antipyrine derivatives have been reported to possess diverse pharmacological activities such as antimicrobial,1 antiviral,2 antioxidant,3 anticancer,4 antipyretic,5 analgesic and anti-inflammatory6 activities. Also, several pyridine derivatives have been developed as pharmaceuticals compounds. They are reported as Calpains inhibitors,7 Glycogen phosphorylase inhibitors,8 cyclooxygenase (COX-2/COX-1) inhibitors,9 HMG-CoA reductase inhibitors10 and as anticancer agents.11 Recently, it was reported that, piperidine linked to aminopyrimidines and aminotriazines have anti-HIV activities.12 In view of the above mentioned findings, and in continuation of our previous work aimed at the synthesis of a variety of heterocyclic systems for biological and pharmacological evaluation,13-20 we report in the present work an efficient method for the synthesis of a series of pyridines and piperidines pendant to antipyrine moiety.
It was previously reported that 2-cyano-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4- yl)acetamide (1),21 condensed with aromatic aldehydes in (1:1 molar ratio) to give arylmethylene derivatives.21 On the other hand, in our study, acetamide 1 condensed with benzaldehyde in (2:1 molar ratio) to give the corresponding piperidine-3-carboxamide derivative 4a via non-isolable intermediates 2a and 3a (Scheme 1). To account for the formation of this product 4a we assumed that the reaction initially proceeds via elimination of water molecule to afford the non-isolable intermediate 2a which reacted with another molecule of the acetamide 1 to give 3a. Intramolecular cyclization of 3a afforded the final product 4a (Scheme 1).

The structure of the isolated product was inferred from its elemental analysis and spectral data [IR, 1H NMR and 13C NMR]. Its IR spectrum showed absorption bands at v = 1670, 1701 (2 CO), 2251 (C≡N), 3323 and 3363 cm-1 (2 NH). Also, its 1H NMR spectrum revealed doublet of doublet signal at δ 3.88 ppm (J = 10, 8 Hz) and another pair of doublets at 4.15 (J = 10 Hz), 5.0 ppm (J = 8 Hz) assignable to (3CH of piperidine ring). Also, two singlet signals (D2O-exchangeable) were shown at 9.4 and 13.7 ppm assignable to (2NH) groups. Four singlet signals at 1.42, 2.33, 2.94 and 3.20 ppm due to four methyl protons, in addition to aromatic multiplet in the region δ 7.30-7.57 ppm were also revealed. The molecular ion peak at m/z = 628 confirmed the bis addition of acetamide 1. Prompted by the foregoing results and to generalize this finding we also studied the reaction of the acetamide 1 with the aromatic aldehydes 2b,c under the same experimental conditions and obtained the respective piperidine-3-carboxamide derivatives 4b,c.
The structure of the isolated products 4b,c was established on the basis of their elemental analyses and spectral data (see experimental part).
It was reported that, reaction of acetamide with ethyl 2-cyano-3-arylacrylate22,23 afforded either aminopyridines24,25 or pyridine-dicarbonitriles.26 In our study, refluxing of an equimolar amounts of acetamide 1 with ethyl 2-cyano-3-(4-nitrophenyl)acrylate (5),22,23 in EtOH, in the presence of catalytic amount of piperidine, gave astonishing the corresponding piperidine-3-carboxamide derivative 4c (identical in all respects to sample obtained from the reaction between acetamide 1 and 4-nitrobenzaldehyde shown in Scheme 1). To account for the formation of this product we suggested that the acetamide 1 reacted with compound 5 to afford non-isolable intermediate 6. Elimination of one molecule of ethyl cyanoacetate from intermediate 6 gave intermediate 2c. Further reaction of the latter intermediate 2c with another molecule of acetamide 1 gave the respective final product 4c via intermediate 3c (Scheme 2).

On the other hand, the behavior of (arylmethylene)malononitriles27 towards acetamides was investigated and the reaction products were 2-oxopyridine-3,5-dicarbonitriles21,28 or 6-imino-2-oxopyridine- 3-carbonitriles.29 Thus, refluxing of an equimolar amounts of acetamide 1 with (arylmethylene)malononitriles27 7a,b, under the same experimental conditions, afforded, in each case (1:1) cycloadduct (Scheme 3). The structure of the isolated cycloadducts was identified as 6-amino-4-aryl-2-oxo-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)pyridine-3,5-dicarbonitriles 10a,b (Scheme 3).

The structure of compound 10 was established for the reaction product based on analytical and spectral data (see Experimental). The 1H NMR spectra exhibited signal at δ 7.9-8.6 ppm (exchangeable with D2O) due to NH2 protons and did not revealed any signal assignable to NH protons. The pyridine structure 10 is assumed to be formed via an initial Micheal addition30 adduct 8 followed by an intramolecular cyclization and subsequent oxidation of 9 to the final product 10 (Scheme 3).

ANTIMICROBIAL EVALUATION
The newly synthesized target compounds (4a-c and 10a,b) were evaluated for their in vitro antibacterial activity against Staphylococcus aureus (SA) and Bacillis subtilis (BS) as examples of Gram-positive bacteria and Pseudomonas aeruginosa (PA) and Escherichia coli (EC) as examples of Gram-negative bacteria. They were also evaluated for their in vitro antifungal potential against Aspergillus fumigatus (AF), Geotrichum candidum (GC), Candida albicans (CA) and Syncephalastrum racemosum (SR) fungal strains. The organisms were tested against the activity of solutions of concentrations (5 mg/mL) and using inhibition zone diameter (IZD) in mm as criterion for the antimicrobial activity (agar diffusion well method). The fungicides Itraconazole, Clotrimazole and the bactericides Penicillin G, Streptomycin were used as references to evaluate the potency of the tested compounds under the same conditions. The results are depicted in Table 1.

The results revealed that most of the tested compounds displayed variable inhibitory effects on the growth of the tested Gram-positive bacteria and Gram-negative bacteria strains and also against fungal strains. In general, most of the tested compounds revealed better activity against the Gram-positive bacteria rather than the Gram-negative bacteria and all compounds exhibited almost no activity against Syncephalastrum racemosum. Compounds 4a-c and 10a,b were found to have moderate inhibition activity, relative to the standard drugs Itraconazole and Clatrimazole, against Aspergillus fumigates, Geotrichum candidum and Candida albicans. Also, compounds 4a and 10a were found to be the most potent relative to the standard drugs, Penicillin G and Streptomycin against Staphylococcus aureus, Bacillis subtilis, Pseudomonas aeruginosa and Escherichia coli. The structure antimicrobial activity relationship of the synthesized compounds revealed that the maximum activity was attained with compound 10a, having pyridine nucleus with chloro substituent in the phenyl group. The results suggest that the new skeleton possessing pyridine and antipyrine may provide valuable leads for synthesis and development of novel antibacterial agents.
Novel series of piperidines and pyridines bearing antipyrine moiety were synthesized and evaluated for their antimicrobial activities. Most of the newly synthesized products have promising activity against Gram-positive and Gram-positive bacteria.

EXPERIMENTAL
All melting points were measured on a Gallenkamp melting point apparatus. The infrared spectra were recorded in potassium bromide disks on a Pye Unicam SP 3300 and Shimadzu FT IR 8101 PC infrared spectrophotometers. The NMR spectra were recorded on a BRUKER VX-500 NMR spectrometer (Varian, Palo Alto, CA, USA). 1H spectra were run at 500 MHz and 13C spectra were run at 125 MHz in deuterated dimethylsulphoxide (DMSO-d6). Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCMS-QP 1000 EX mass spectrometer at 70 e.V. Elemental analyses were carried out at the Microanalytical Center of Cairo University, Giza, Egypt. The biological evaluation of the products 4a-c and 10a,b were carried out in the Medical Mycology Laboratory of the Regional Center for Mycology and Biotechnology of Al-Azhar University, Cairo, Egypt. 2-cyano-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)acetamide (1),21 ethyl 2-cyano-3- (4-nitrophenyl)acrylate (5)22,23 and (arylmethylene)malononitriles 7a,b,27 were prepared as described in literature.

Reaction of acetamide 1 with aromatic aldehydes.
General Procedure:
To an ethanolic solution (10 mL) of acetamide 1 (0.54 g, 2 mmol) and the appropriate aromatic aldehydes (1 mmol) was added few drops of piperidine and the reaction mixture was refluxed for 4 h. The solvent was evaporated under reduced pressure, and the residue was cooled, filtered off, washed with EtOH and purified by recrystallization from DMF/EtOH to afford 4a-c.

5-Cyano-N,1-bis(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-imino-6-oxo-4-phenylpiperidine-3-carboxamide (4a).
White powder, (0.28 g, 44%), mp 245 oC; IR (KBr) ν 3363, 3323 (2NH), 2251 (C≡N), 1701-1670 (4 C=O) cm1; 1H NMR (DMSO-d6) δ 1.42 (s, 3H, CH3), 2.33 (s, 3H, CH3), 2.94 (s, 3H, N-CH3), 3.20 (s, 3H, N-CH3), 3.88 (dd, 1H, J = 10.0, 8.0 Hz, CH-piperidine), 4.15 (d, 1H, J = 10.0 Hz, CH-piperidine), 5.0 (d, 1H, J = 8.0 Hz, CH-piperidine), 7.30-7.57 (m, 15H, Ar-H), 9.40 (s, 1H, D2O-exchangeable, NH), 13.7 (s, 1H, D2O-exchangeable, NH); 13C NMR (DMSO-d6) δ 9.8 (CH3), 10.4 (CH3). 34.6 (N-CH3), 35.8 ( N-CH3), 41.4, 42.9, 52.6, 107.1, 113.5, 123.3, 123.5, 126.1, 126.2, 128.3, 128.4, 128.6, 129.1, 129.3, 133.5, 135.1, 137.9, 142.3, 151.9, 152.1, 159.1, 161.4, 162.7, 166.9, 167.9; MS m/z (%) 628 (M+, 10), 230 (10), 202 (14), 187 (18), 77 (100). Anal. Calcd for C35H32N8O4 (628.25): C, 66.87; H, 5.13; N, 17.82. Found: C, 66.77; H, 5.08; N, 16.93%.

4-(4-Chlorophenyl)-5-cyano-N,1-bis(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-imino-6-oxopiperidine-3-carboxamide (4b).
Yellow powder, (0.30 g, 46%), mp 230 oC; IR (KBr) ν 3287, 3198 (2NH), 2255 (C≡N), 1700-1670 (4 C=O) cm1; 1H NMR (DMSO-d6) δ 1.50 (s, 3H, CH3), 2.33 (s, 3H, CH3), 2.97 (s, 3H, N-CH3), 3.21 (s, 3H, N-CH3), 3.95 (dd, 1H, J = 10.0, 8.0 Hz, CH-piperidine), 4.10 (d, 1H, J = 10.0 Hz, CH-piperidine), 5.03 (d, 1H, J = 8.0 Hz, CH-piperidine), 7.30-7.60 (m, 14H, Ar-H), 9.40 (s, 1H, D2O-exchangeable, NH), 13.65 (s, 1H, D2O-exchangeable, NH); MS m/z (%): 665 (M++2, 4), 663 (M+, 10), 624 (18), 460 (12), 202 (16), 187 (14), 111 (20), 77 (100). Anal. Calcd for C35H31ClN8O4 (663.22): C, 63.39; H, 4.71; N, 16.90. Found: C, 63.47; H, 4.78; N, 16.83%.

5-Cyano-N,1-bis(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-imino-4-(4-nitrophenyl)-6-oxopiperidine-3-carboxamide (4c).
Yellow powder, (0.32 g, 48%); mp 220 oC; IR (KBr) ν 3192, 3109 (2NH), 2255 (C≡N), 1690-1668 (4 C=O) cm1; 1H NMR (DMSO-d6) δ 1.49 (s, 3H, CH3), 2.33 (s, 3H, CH3), 2.94 (s, 3H, N-CH3), 3.21 (s, 3H, N-CH3), 4.19 (dd, 1H, J = 10.0, 8.0 Hz, CH-piperidine), 5.03 (d, 1H, J = 10.0 Hz, CH-piperidine), 5.15 (d, 1H, J = 8.0 Hz, CH-piperidine), 7.29-7.59 (m, 10H, Ar-H), 7.77 (d, 2H, J = 8.0 Hz, Ar-H), 8.31 (d, 2H, J = 8.0 Hz, Ar-H), 9.49 (s, 1H, D2O-exchangeable, NH), 13.71 (s, 1H, D2O-exchangeable, NH); MS m/z (%) 673 (M+, 10), 230 (16), 202 (21), 187 (12), 134 (10), 122 (14), 77 (100). Anal. Calcd for C35H31N9O6 (673.24): C, 62.40; H, 4.64; N, 18.71. Found: C, 62.47; H, 4.68; N, 18.63%.

Reactions of acetamide 1 with ethyl 2-cyano-3-(4-nitrophenyl)acrylate (5).
To a solution of ethyl 2-cyano-3-(4-nitrophenyl)acrylate (5) (1 mmol) in EtOH (10 mL) was added an eqimolar amount of the acetamide 1 (0.27 g, 1 mmol), and few drops of piperidine and the reaction mixture was heated for 3 h. The solid product that obtained was collected by filtration, washed with EtOH and then crystallized from DMF to afford product 4c (50%) which was identical in all respects to sample obtained from the reaction between acetamide 1 and 4-nitrobenzaldehyde.

Reaction of acetamide 1 with (arylmethylene)malononitriles 7a,b.
General procedure.
To a solution of the appropriate (arylmethylene)malononitriles 7a,b (1 mmol) in EtOH (10 mL) was added an equimolar amount of the acetamide 1 (0.27 g, 1 mmol), and few drops of piperidine and the reaction mixture was heated under reflux for 2 h. The solid product that formed was collected by filtration, washed with EtOH and then crystallized from DMF/EtOH to give 10a,b.

6-Amino-4-(4-chlorophenyl)-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (10a).
White powder, (0.32 g, 70%), mp > 300 oC; IR (KBr) ν 3280, 3194 (NH2), 2240, 2213 (2 C≡N), 1667, 1672 (2 C=O) cm1; 1H NMR (DMSO-d6) δ 2.16 (s, 3H, CH3), 3.28 (s, 3H, N-CH3), 7.41-7.69 (m, 9H, Ar-H), 8.6 (s, 2H, D2O-exchangeable, NH2); 13C NMR (DMSO-d6) δ 10.2 (CH3), 34.9 (N-CH3), 74.7, 87.4, 100.4, 115.5, 116.1, 124.8, 127.1, 128.8, 129.2, 129.9, 133.4, 134.3, 135.2, 153.8, 157.7, 158.6, 159.5, 160.5; MS m/z (%) 458 (M++2, 4), 456 (M+, 10), 269 (40), 111 (30), 77 (100). Anal. Calcd for C24H17ClN6O2 (456.11): C, 63.09; H, 3.75; N, 18.39. Found: C, 63.16; H, 3.88; N, 18.19%.

6-Amino-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-4-(4-nitrophenyl)-2-oxo-1,2-dihydropyridine-3,5-dicarbonitrile (10b).
Yellow powder, (0.35 g, 75%), mp 265 oC; IR (KBr) ν 3383, 3161 (NH2), 2240, 2215 (2 C≡N), 1663, 1676 (2 C=O) cm1; 1H NMR (DMSO-d6) δ 2.18 (s, 3H, CH3), 3.28 (s, 3H, N-CH3), 7.40-8.50 (m, 9H, Ar-H), 7.9 (s, 2H, D2O-exchangeable, NH2); 13C NMR (DMSO-d6) δ 10.2 (CH3), 34.9 (N-CH3), 74.7, 87.3, 100.4, 115.4, 115.8, 123.8, 124.8, 127.2, 129.2, 129.7, 134.3, 140.9, 148.5, 153.8, 157.7, 158.4, 159.4, 162.3; MS m/z (%) 467 (M+, 10), 281 (10), 122 (14), 77 (100). Anal. Calcd for C24H17N7O4 (467.13): C, 61.67; H, 3.67; N, 20.98. Found: C, 61.76; H, 3.58; N, 21.09%.

ANTIMICROBIAL ACTIVITY TEST
Agar diffusion well method to determine the antimicrobial activity
The microorganism inoculums were uniformly spread using sterile cotton swab on a sterile Petri dish Malt extract agar (for fungi) and nutrient agar (for bacteria). One hundred µL of each sample was added to each well (10 mm diameter holes cut in the agar gel, 20 mm apart from one another). The systems were incubated for 24-48 h at 37 °C (for bacteria) and at 28 °C (for fungi). After incubation, the microorganism's growth was observed. Inhibition of the bacterial and fungal growth were measured as IZD in mm. Tests were performed in triplicate.31

References

1. H. Bayrak, A. Demirbas, N. Demirbas, and S. A. Karaoglu, Eur. J. Med. Chem., 2010, 45, 4726. CrossRef
2. A. N. Evstropov, V. E. Yavorovskaya, E. S. Vorob'ev, Z. P. Khudonogova, L. N. Gritsenko, E. V. Shmidt, S. G. Medvedeva, V. D. Filimonov, T. P. Prishchep, and A. S. Saratikov, Pharm. Chem. J., 1992, 26, 426. CrossRef
3. N. V. Bashkatova, E. I. Korotkova, Y. A. Karbainov, A. Y. Yagovkin, and A. A. Bakibaev, J. Pharm. Biomed. Anal., 2005, 37, 1143. CrossRef
4. T. Rosu, M. Negoiu, S. Pasculescu, E. Pahontu, D. Poirier, and A. Gulea, Eur. J. Med. Chem., 2010, 45, 774. CrossRef
5. E. V. Shchegol’kov, O. G. Khudina, L. V. Anikina, Y. V. Burgart, and V. I. Saloutin, Pharm. Chem. J., 2006, 40, 373. CrossRef
6. A. E. Rubtsov, R. R. Makhmudov, N. V. Kovylyaeva, N. I. Prosyanik, A. V. Bobrov, and V. V. Zalesov, Pharm. Chem. J., 2002, 36, 608. CrossRef
7. K. Y. Lee, K. S. Lee, C. Jin, and Y. S. Lee, Eur. J. Med. Chem., 2009, 44, 1331. CrossRef
8. N. D. Karis, W. A. Loughlin, I. D. Jenkins, and P. C. Healy, Bioorg. Med. Chem., 2009, 17, 4724. CrossRef
9. J. F. Renard, D. Arslan, N. Garbacki, B. Pirotte, and X. De Leval, J. Med. Chem., 2009, 52, 5864. CrossRef
10. M. Suzuki, H. Iwasaki, Y. Fujikawa, M. Sakashita, M. Kitahara, and R. Sakoda, Bioorg. Med. Chem. Lett., 2001, 11, 1285. CrossRef
11. A. H. Abadi, T. M. Ibrahim, K. M. Abouzid, J. Lehmann, H. N. Tinsley, B. D. Gary, and G. A. Piazza, Bioorg. Med. Chem., 2009, 17, 5974. CrossRef
12. X. Chen, C. Pannecouque, J. Balzarini, E. De Clercq, and X. Liu, Eur. J. Med. Chem., 2012, 51, 60. CrossRef
13. N. A. Kheder and Y. N. Mabkhoot, Int. J. Mol. Sci., 2012, 13, 3661. CrossRef
14. Y. N. Mabkhot, N. A. Kheder, and A. M. Farag, Heterocycles, 2011, 83, 609. CrossRef
15. E. S. Darwish, N. A. Kheder, and A. M. Farag, Heterocycles, 2010, 81, 2247. CrossRef
16. Y. N. Mabkhot, N. A. Kheder, and A. M. Farag, Heterocycles, 2010, 81, 2369. CrossRef
17. S. M. Riyadh, Molecules, 2011, 16, 1834. CrossRef
18. S. M. Riyadh, T. A. Farghaly, M. A. Abdallah, M. M. Abdalla, and M. R. Abd El-Aziz, Eur. J. Med. Chem., 2010, 45, 1042. CrossRef
19. S. M. Riyadh, I. A. Abdelhamid, H. M. Al-Matar, N. M. Hilmy, and M. H. Elnagdi, Heterocycles, 2008, 75, 1849. CrossRef
20. T. A. Farghaly, S. M. Riyadh, M. A. Abdallah, and M. R. Abd El-Aziz, Acta Chim. Solvenica, 2011, 58, 87.
21. A. M. Farag, K. M. Dawood, and H. A. Elmenoufy, Heteroatom Chem., 2004, 15, 508. CrossRef
22. J. A. Cabello, J. M. Campelo, A. Garcia, D. Luna, and J. M. Marinas, J. Org. Chem., 1984, 49, 5195. CrossRef
23. G. Kaupp, M. R. Naimi-Jamal, and J. Schmeyers, Tetrahedron, 2003, 59, 3753. CrossRef
24. W. M. Basyouni, Acta Chem. Solv., 2003, 223.
25. A. A. Al-Najjar, S. A. Amer, M. Riad, I. Elghamry, and M. H. Elnagdi, J. Chem. Res. (S), 1996, 296.
26. A. H. Mandour, O. A. Fathalla, and W. M. Basyouni, Biomed. Prob., 2000, 60, 53.
27. P. R. Rao and R. V. Venkataratnam, Tetrahedron Lett., 1991, 32, 5821. CrossRef
28. K. A. M. El-Bayouki, M. M. Aly, Y. A. Mohamed, W. M. Basyouni, and S. Y. Abbas, World J. Chem., 2009, 4, 161.
29. E. M. Zayed, E. A. Hafez, S. A. S. Ghozlan, and A. A. H. Ibrahim, Heterocycles, 1984, 22, 2553. CrossRef
30. B. C. Ranu and S. Banerjee, Org. Lett., 2005, 7, 3049. CrossRef
31. A. Smania, F. D. Monache, E. F. A. Smania, and R. S. Cuneo, Int. J. Med. Mushrooms, 1999, 1, 325.