Note. Synthesis of some novel pyrido[2,3- d]pyrimidine derivatives and their antimicrobial investigations

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1 Indian Journal of Chemistry Vol. 52B, March 2013, pp ote Synthesis of some novel pyrido[2,3- d]pyrimidine derivatives and their antimicrobial investigations Sangeeta Bhargava* & Lokesh K Rajwanshi Department of Chemistry, University of Rajasthan, Jaipur , India drsbhargava1@gmail.com Received 24 August 2011; accepted (received) 19 December 2012 A series of pyrido[2,3-d]pyrimidine derivatives viz 4-amino- 5,7-disubstituted pyrido[2,3-d]pyrimidines 3a-d, 4-amino-5,7- disubstituted pyrido[2,3-d]pyrimidin-2(1h)-ones 4a-d and 4- amino-5,7-disubstituted pyrido[2,3-d]pyrimidin-2(1h)-thione 5ad have been synthesized by the condensation reaction of 2-amino- 3-cyano-4,6-disubstituted pyridines 2a-d with formamide, urea and thiourea, respectively. The newly synthesized compounds have been established by elemental analysis, IR, 1 H and 13 C MR. All the synthesized compounds have been screened for their antibacterial and antifungal activity. Keywords: Chalcones, 2-amino-3-cyano-4,6-disubstituted pyridines, pyrido [2,3-d]pyrimidine, antimicrobial investigations The synthesis of pyrido[2,3-d]pyrimidine derivatives is of great interest in organic chemistry, because manifold implications viz, antibacterial 1,2, antifungal 3,4, anticancer 5, antitumor 6, antiherpes 7, antiallergic 8, analgesic 9, anti-inflammatory 10, antineuplastic 11, etc. are well proved by a large number of publication and patents on it. Martin et al. 12, have demonstrated that pyrido[2,3-d]pyrimidines are active against P 38 kinase, Reinhard et al. 13, have synthesized pyrido[2,3- d]pyrimidine derivatives as analogs of the antifolates methotrexate. In our previous work we have synthesized some new pyrido[2,3-d]pyrimidines 14,15 and their ribofuranosides have been synthesized which showed varying degree of antimicrobial activity 16. The manifold applications of pyrido[2,3- d]pyrimidines have prompted to target the synthesis of some novel 4-amino-5,7-disubstituted pyrido[2,3- d] pyrimidine derivatives with the goal to achieve some novel biologically effective compounds. All the synthesized compounds have been screened for their antimicrobial investigation against E. coli, S. Aureus, A. flavus and A. niger and are found to be better antimicrobial agents than their precursors pyrido[2,3- d]pyrimidine derivatives. Results and Discussion Chalcones 1a-d, on treatment with malanonitrile and ammonium acetate in ethanol via Micheal-type condensation, gave 2-amino-3-cyano-4,6-disubstituted pyridines 2a-d. Compound 2 on treatment with formamide, urea and thiourea afforded 4-amino-5,7- disubstituted pyrido[2,3-d]pyrimidines 3a-d, 4-amino- 5,7-disubstituted pyrido[2,3-d]pyrimidin-2-(1h)-ones 4a-d and 4-amino-5,7-disubstituted pyrido[2,3- d]pyrimidin-2-(1h)-thiones 5a-d, respectively (Scheme I). The proposed structures of all the synthesized compounds are well supported by elemental analysis, IR, 1 H and 13 C MR data. In the IR spectra of compound 2, absorption bands due to C and H 2 groups appeared in the region and cm -1, respectively. The disappearance of absorption band due to C groups confirmed the formation of heterocyclic ring in compounds 3, 4 and 5. Absorption band due to >H group appeared in the region and cm -1 in compounds 4 and 5, respectively. Absorption band due to >C=O and >C=S group appeared in the region and cm -1, respectively in compounds 4 and 5. The 1 H MR spectra of synthesized compounds 3, 4, 5 revealed a multiplet of aromatic protons in the region δ Signals due to -H 2 protons were found to be merged with aromatic protons and has been confirmed by integration of the multiplet. The >H protons in the compounds 4 and 5 appeared in the region δ Antimicrobial activity All the synthesized compounds 3, 4, 5 were evaluated for their antimicrobial activity following the paper disc method of Verma et al. 17 The concentration applied was 100 µg/disc. Streptomycin and mycostain were used as reference compounds while testing antibacterial and antifungal activity, respectively. All compounds were found to be moderately active against various bacteria, such as Escherichia coli (gram-negative bacterium) and Staphylococcus aureus (gram-positive bacterium) and fungi (Aspergillus niger and Aspergillus flavus).

2 OTES 449 CH CH CH 2 C C 1 a-d + CH 3 COOH 4 EtOH Reflux C 2 a-d H 2 HCOH 2 H 2 CSH 2 H 2 H 2 COH 2 H 2 3 a-d 5 a-d H 2 H S H 4 a-d = a-d: 2,4-dichlorophenyl. = a: phenyl, b: 4-Bromophenyl, c: 4-Hydroxyphenyl, d: 4-Fluorophenyl. O Scheme I Synthesis of pyrido[2,3-d]pyridimine derivatives The results obtained are tabulated in the form of inhibition zone and activity indices (Table I). Experimental Section Melting points of all the synthesized compounds were determined in open capillary tube and are uncorrected. The homogeneity of synthesized compounds was checked by TLC using silica gel G as adsorbent and visualization was accomplished by UV light or iodine vapours. Infrared spectra were recorded on a Shimadzu FT-IR spectrophotometer using KBr pellets. 1 H and 13 C MR spectra were recorded on JEOL-AL spectrometer at 300 and 75 MHz using CDCl 3 /DMSO-d 6 as solvent and TMS as an internal standard. Elemental analyses were carried out on a Elemental Bario EL-III Carlo Erba 1108 CH analyzer. Chalcones were synthesized according to the reported method 18. Synthesis of 2-amino-3-cyano-4,6-disubstituted pyridines, 2a-d A mixture of the appropriate chalcone 1 (0.05 mol), malononitrile (0.05 mol) and ammonium acetate (0.4 mol) in ethanol (80 ml) was refluxed on a water bath for hr. After cooling, the contents of the flask were poured onto crushed ice with constant stirring. The solid mass, thus obtained, was washed with water followed by cold ethanol. The residue was recrystallized from ethanol.

3 450 IDIA J. CHEM., SEC B, MARCH 2013 Table I Antimicrobial activity of the synthesized compounds 3a-d to 5a-d Compd Zone of inhibition in (mm) (Activity Index) Antibacterial activity Antifungal activity E. coli S. aureus A. niger A. flavus 3a 7.8(0.65) 7.4 (0.61) 8.8 (0.74) 8.4 (0.71) 3b 8.2 (0.78) 8.7 (0.70) 9.2 (0.75) 9.4 (0.76) 3c 12.2 (0.08) 12.0 (0.99) 7.8 (0.64) 8.5 (0.69) 3d 11.9 (1.35) 11.7 (1.33) 12.1 (1.40) 11.9 (1.38) 4a 7.6 (0.63) 8.1 (0.96) 9.7 (0.81) 9.0 (0.75) 4b 9.8 (1.08) 7.2 (1.01) 6.1 (0.75) 9.3 (1.0) 4c 12.0 (1.0) 11.8 (0.98) 7.5 (0.63) 7.9 (0.70) 4d 11.7 (1.30) 11.4 (1.28) 11.6 (1.33) 11.5 (1.31) 5a 8.0 (0.65) 7.6 (0.61) 9.1 (0.73) 8.6 (0.70) 5b 10.0 (1.12) 8.3 (1.14) 8.5 (1.03) 9.6 (1.02) 5c 12.4 (0.99) 12.6 (1.02) 7.7 (0.64) 7.4 (0.62) 5d 12.2 (0.36) 12.0 (1.34) 12.6 (1.42) 12.3 (1.40) Activity index = Inhibition area of the sample/inhibition area of the standard Synthesis of 4-amino-5,7-disubstituted pyrido[2,3- d]pyrimidines, 3a-d A mixture of compound 2 (0.01 mol) and formamide (0.04 mol) was refluxed on an oil bath for hr. After cooling, the reaction mixture was poured onto the crushed ice. The solid mass, thus obtained, was washed with water, dried and recrystallized from ethanol. The characteristic data of compound 3a-d are presented below. 4-Amino-5-(2,4 -dichlorophenyl)-7-phenyl pyrido- [2,3-d]pyrimidine, 3a Brown solid, yield 80%, m.p C; IR (KBr): (-H 2 ), 757 cm -1 (-C-Cl); 1 H MR: δ 6.93 (s, 2H, -H 2 ), (m, 10H, ArH); 13 C MR: δ , , , 157.0, , , , , , , , , 127.0, Anal. Calcd for C 19 H 12 Cl 2 4 : C, 62.14; H, 3.29;, Found: C, 62.10; H, 3.27;, 15.28%. pyrimidine, 3b Yellow solid, yield 82%, m.p C; IR (KBr): (-H 2 ), 763 (-C-Cl), 721 cm -1 (-C-Br); 1 H MR: δ 7.01 (s, 2H, -H 2 ), (m, 9H, ArH); 13 C MR: δ , , 158.0, , , , , , , , , , , , , Anal. Calcd for C 19 H 11 BrCl 2 4 : C, 51.15; H, 2.48;, Found: C, 51.12; H, 2.52;, 12.54%. pyrimidine, 3c Brown solid, yield 67%, m.p C; IR (KBr): (-H 2 ), 3315(-OH), 768 cm -1 (-C-Cl); 1 H MR: δ 6.90 (s, 2H, -H 2 ), (m, 9H, Ar- H), 9.82 (1H, s, -OH); 13 C MR: δ , , , , , 150.0, , , , , , 130.0, , , , , Anal. Calcd for C 19 H 12 Cl 2 4 O: C, 59.55; H, 3.16;, Found: C, 59.48; H, 3.12;, 14.74%. pyrimidine, 3d Brown solid, yield 79%, m.p C; IR (KBr): (-H 2 ), (-C-F), 775 cm -1 (C- Cl); 1 H MR: δ 7.05 (s, 2H, -H 2 ), (m, 9H, Ar-H); 13 C MR: δ , , , , , , , , , , , 129.0, , Anal. Calcd for C 19 H 11 Cl 2 F 4 : C, 59.24; H, 2.88;, Found: C, 59.38; H, 2.82;, 14.52%. Synthesis of 4-amnio-5,7-disubstituted pyrido[2,3- d]pyrimidin-2(1h)-ones, 4a-d A mixture of 2-amino-4,6-disubstituted pyridine 2 (0.01 mol) and urea (0.02 mol) was heated on an oil bath at C for 2 hr with constant stirring. The temperature of the reaction was raised gradually to 180 C for 2 hr and finally the reaction mixture was

4 OTES 451 heated at 220 C for 2 hr. The solid mass, thus obtained was filtered, washed with water, saturated sodium bicarbonate solution and finally with cold ethanol. The crude product was recrystallized from DMF:ethanol (1:2). 4-Amino-5-(2,4 -dichlorophenyl)-7-phenylpyrido- [2,3-d]pyrimidin-2(1H)-one, 4a Brown solid, yield 74%, m.p C; IR(KBr): 3150 (>H), (-H 2 ), 1710 (>C=O), 1340, 1445, 1470 (>HCO), 770 cm -1 (-C-Cl); 1 H MR: δ 8.02 (s, 1H, -H), 6.95 (s, 2H, -H 2 ), (m, 9H, Ar-H); 13 C MR: δ , , , , , , , , , , , , , 127.0, , Anal. Calcd for C 19 H 12 Cl 2 4 O: C, 59.55; H, 3.16;, Found: C, 59.40; H, 3.21;, 14.64%. pyrimidin-2(1h)-one, 4b Brown solid, yield 70%, m.p C; IR (KBr): 3270 (>H), (-H 2 ), 1715 (>C=O), 1365, 1455, 1480 (>HCO), 773 (-C-Cl), 724 cm -1 (-C-Br); 1 H MR: δ 7.89 (s, 1H, -H), 6.92 (s, 2H, -H 2 ), (m, 8H, Ar-H); 13 C MR: δ , , , , , , , , , , , , , , , Anal. Calcd for C 19 H 11 BrCl 2 4 O: C, 49.48; H, 2.40;, Found: C, 49.42; H, 2.36;, 12.32%. pyrimidin-2(1h)-one, 4c Brown solid, yield 69%, m.p C; IR (KBr): 3140 (>H), (-H 2 ), 1725 (>C=O), 1375, 1460, 1490 (>HCO), 782 cm -1 (-C-Cl); 1 H MR: δ 7.82 (s, 1H, -H), 7.10 (s, 2H, -H 2 ), (m, 8H, Ar-H), 9.95 (1H, s, -OH); 13 C MR: δ , , , , 156.0, , , , , , 130.0,128.70, , , , Anal. Calcd for C 19 H 12 Cl 2 4 O 2 : C, 57.16; H, 3.03;, Found: C, 57.08; H, 3.14;, 14.08%. pyrimidin-2(1h)-one, 4d Brown solid, yield 80%, m.p C; IR (KBr): 3185 (>H), (-H 2 ), 1740 (>C=O), 1370, 1410, 1480 (>HCO), 1025 (-C-F), 793 cm -1 (-C-Cl); 1 H MR: δ 8.15 (s, 1H, -H), 7.20 (s, 2H, -H 2 ), (m, 8H, Ar-H); 13 C MR: δ , 161.0, , , , , , , , , , , 128.0, , , Anal. Calcd for C 19 H 11 Cl 2 F 4 O: C, 56.88; H, 2.78;, Found: C, 56.93; H, 2.82;, 13.82%. Synthesis of 4-amnio-5,7-disubstituted pyrido[2,3- d]pyrimidin-2(1h)-thiones, 5a-d A mixture of 2-amino-4,6-disubstituted pyridine 2 (0.01 mol) and thiourea (0.02 mol) was heated on an oil bath at C for 2 hr with constant stirring. The temperature of the reaction was raised gradually to 180 C for 2 hr and finally the reaction mixture was heated at 220 C for 2 hr. The solid mass, thus obtained was filtered, washed with water, saturated sodium bicarbonate solution and finally with cold ethanol. The crude product was recrystallized from DMF:ethanol (1:2). 4-Amino-5-(2,4 -dichlorophenyl)-7-phenylpyrido- [2,3-d]pyrimidin-2 (1H)-thione, 5a Brown solid, yield 76%, m.p C; IR (KBr): 3240 (>H), (-H 2 ), 1205 (>C=S), 1345, 1440, 1475 (>HCS), 780 cm -1 (-C-Cl); 1 H MR: δ 7.98 (s, 1H, -H), 6.94 (s, 2H, -H 2 ), (m, 9H, Ar-H); 13 C MR: δ , , , , , , , 136.0, , , , , , , , Anal. Calcd for C 19 H 12 Cl 2 4 S: C, 57.15; H, 3.04;, Found: C, 57.08; H, 3.12;, 14.06%. pyrimidin-2(1h)-thione, 5b Brown solid, yield 71%, m.p C, IR (KBr): 3180 (>H), (-H 2 ), 1230 (>C=S), 1375, 1435, 1480 (>HCS), 786 (-C-Cl), 728 cm -1 (-C-Br); 1 H MR: δ 7.85 (s, 1H, -H), 6.88 (s, 2H, -H 2 ), (m, 8H, Ar-H); 13 C MR: δ , , 160.0, , , , , , , , , , , , , Anal. Calcd for C 19 H 11 BrCl 2 4 S: C, 47.72; H, 2.34;, Found: C, 47.78; H, 2.36;, 11.62%. pyrimidin-2(1h)-thione, 5c Yellow solid, yield 63%, m.p C, IR (KBr): 3165 (>H), (-H 2 ), 1215 (>C=S), 1385, 1455, 1490 (>HCS), 789 cm -1 (-C-Cl); 1 H MR: δ 7.92 (s, 1H, -H), 7.03 (s, 2H, -H 2 ), (m, 8H, Ar-H), 9.98 (1H, s, -OH); 13 C MR: δ ,

5 452 IDIA J. CHEM., SEC B, MARCH , , , , , 137.0, 135.8, 134.0, , , ,128.80, , , , Anal. Calcd for C 19 H 12 Cl 2 4 OS: C, 54.96; H, 2.91;, Found: C, 55.08; H, 2.84;, 13.48%. pyrimidin-2(1h)-thione, 5d Yellow solid, yield 81%, m.p C, IR (KBr): 3150 (>H), (-H 2 ), 1245 (>C=S), 1390, 1465, 1495 (>HCS), 1030 (-C-F), 796 cm -1 (-C-Cl); 1 H MR: δ 8.12 (s, 1H, -H), 7.15 (s, 2H, -H 2 ), (m, 8H, Ar-H); 13 C MR: δ , , , , , , , 136.0, , , , , , , , , Anal. Calcd for C 19 H 11 Cl 2 F 4 S: C, 54.70; H, 2.64;, Found: C, 54.62; H, 2.78;, 13.37%. Acknowledgement One of the authors (LKR) is grateful to the CSIR, ew Delhi for providing financial assistance. The authors are thankful to the Head, Department of Chemistry, University of Rajasthan, Jaipur, for providing necessary facilities during the experimental work. References 1 Singh G, Singh G, Yadav A K & Mishra A K, Indian J Chem, 41B, 2002, Kanth S R, Reddy G V, Rao P S, arsalah B & Murthy V S, Eur J Chem, 41, 2006, Swati, Goyal R D & Prakash L, Indian J Chem, 31B, 1992, Singh G, Singh G, Yadav A K & Mishra A K, Phosphorus, Sulfur, Silicon and Related Elements, 165, 2000, Bar T, Zimmermann P, Boer R, Gekeler V, Isc W, Boss H & Ulrich W R, PCT Int Appl WO 97,19,946 (Cl. CO 7 D 471/04) Jun 5, 1997, CH Appl. 95/3, 322, ov 24, 1995; Chem Abstr, 127, 1997, Tomita K, Chiba K, Kashimoto S, Shibamori K & Tsuzuki Y, PCT Int Appl WO 95,35,559 (Cl CO 7D 417/04) 21 Dec 1995, Jpn Patent Appl. 94/156, 578, 14 Jun 1994; Chem Abstr, 2124, 1996, Verheggen I, Aerschot A V, Toppet S, Snoeck R, Janssen G, Balzarini J, Clercq E D & Herdewijn P, J Med Chem, 36, 1993, ishikawa Y & Matsumoto J, Chem Pharm Bull, 37(5), 1989, Rahamana A & Hafez H, Bioorg Med Chem, 19, 2009, Huang H, Hutta D A, Desfarlais R L, Schuberh C, Ptrounia L P, Chaikin M A, Manthey C L & Player M R, Bioorg Med Chem Lett, 18, 2008, Whi-Gun C, Thomas C K C & Ching-jer C, Tetrahedron, 54, 1998, Chen J J, Dunn J P, Goldstein D M & Stahl C M, (F. Hoffmann-La Roche Ag Switz) PCT Int Appl WO 2002, , A2, 2002, Schonfeld F & Troschutz R, Heterocycles, 55, 2001, Verma P, Kumar, Bhargava S & Yadav A K, Indian J Heterocycl Chem, 16, 2007, Tiwari S, Yadav A K & Mishra A K, E J Chem, 7 S1, 2010, S Kumar, Tiwari S & Yadav A K, Indian J Chem, 46B, 2007, Verma R S & obles W L, J Pharm Sci, 61, 1972, Furniss B S, Hannaford A J, Rogers V, Smith P W G & Tatchell A R, Vogel s Textbook of Practical Organic Chemistry (Longman Group Ltd, London), 1980, 796.