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Microwave Assisted Synthesis of New N1-Substituted 5-Cyano-pyrimidine Derivatives as Potent Antimicrobial Agents
Microwave Assisted Synthesis of New N1-Substituted 5-Cyano-pyrimidine Derivatives as Potent Antimicrobial Agents
Journal of the Korean Chemical Society. 2008. Feb, 52(1): 30-35
Copyright © 2008, The Korean Chemical Society
  • Received : April 09, 2007
  • Published : February 20, 2008
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About the Authors
Yogesh Pore
Gaurav Patil
Ijaj Tamboli
Vaibhav Chavan
Kirti Kamble
Shital Nikam
Bhanudas Kuchekar

Abstract
The purpose of the study was to synthesize new series of 5-cyano substituted pyrimidine analogues with different substitutions at N1 and 6 positions and to evaluate them for antibacterial and antifungal activities. The desired compounds were synthesized by tertiary condensation of ethylcyanoacetate, substituted thioureas and suitable aldehyde in presence of potassium carbonate using MORE technique. The antibacterial and antifungal activities were evaluated by cup plate method in the concentration of 25 μg. The zone of inhibition was measured in mm. All the compounds have shown significant antibacterial and antifungal activities. The maximum activity was shown by P1 and P5 against S.aureus and E.coli respectively, while P6 has shown significant activity against all types of microorganisms. The compound P8 has been found to be significantly effective against C. albicans . Norfloxacin and griseofulvin were used as standards to compare the activites of synthesized compounds. It is concluded that analogues containing p-hydroxy, p-methoxy substituted phenyl moiety at 6 position have been found to be more potent against gram-positive microorganisms, while analogues lacking these substituents on phenyl moiety possessed gram-negative activity. The compounds having pdimethylamino substituent on phenyl moiety at 6 positions have shown moderate activity. Further, only fluorine containing analogue at N1 position was found to possess appreciable antifungal activity. This suggests that electron donating substituent on aryl moiety as well as electron withdrawing substituent at N1 plays important role in determining potency of the compounds.
Keywords
INTRODUCTION
Uracil derivatives play an important role in the field of medicine, as antiretroviral, agents 1 , 2 , 3 . They have also been reported as potent enzyme inhibitors 4 , 5 , 6 . In last few years uracil derivatives substituted either at the C-5 or C-6 position have emerged in chemotherapy as several pyrimidine derivatives have been found to possess antileishmanial and antimicrobial activity. 7 , 8 In case of pyrimidine nucleus structure activity relationship studies have shown that C-6 position is important determinant for the activity. The most general route for the synthesis of pyrimidines is a reagent containing N-C-N and C-C-C skeleton. The N-C-N skeleton containing reagents are urea, thiourea or guanidine whereas C-C-C skeleton can be obtained from 1,3-diketones, diesters and dinitriles. 8 A facile synthesis of 2-thiocytosines has been reported using piperidine in place of potassium carbonate. 9 Pyrimidines have been also synthesized by regiospecific cyclization of 1,3-dicarbonitriles, 10 from ketene dithioacetals 11 and from alkyl N-cyanoimidates or cyanoacetamides. 12 However these classical methods resulted in poor yield of pyrimidine compounds. Therefore, in order to obtain new potent therapeutic agents, various 5-cyano, 6-substituted (alkyl/aryl) pyrimidine derivatives were synthesized using one- pot reaction of aryl substituted thiourea, aldehyde and ethylcyanoacetate utilizing microwave irradiation to get various N1-substituted and 6-aryl/substituted aryl pyrimidines in better yield. These compounds were evaluated for their antibacterial and antifungal activities.
RESULTS
- Chemistry
Pyrimidines have been synthesized by microwave irradiation and found to give higher yields of compounds 13 . The synthesis of substituted 5-cyano, 2-thiouracil derivatives ( P1-P10 ) was accomplished by reacting various aldehydes with equimolar amount of appropriate substituted thioureas, ethylcyanoacetate and potassium carbonate in small amount of ethanol utilizing microwave irradiation ( 1 ). Unlike the conventional methods 9 - 12 where the reaction time required was 5-7 hrs and with yields of 26-75%, microwave-assisted reactions were very facile (4-8 min. with few minutes of interval) and provided very good yields (65-83%). The purity of the compounds was checked by TLC and elemental analysis. Melting points and % yields of synthesized compounds are reported in 1 . Both analytical and spectral data of all the synthesized compounds were in full agreement with the proposed structures.
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Physical data of synthesized compounds
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Physical data of synthesized compounds
- Antibacterial and antifungal activities
The synthesized compounds were evaluated for their antibacterial and antifungal activities by cupplate method against S. aureus, B. subtilis, E. coli, P. aeruginosa, and C. albicans in the concentration of 25 μg. The activites were compared with standard antibiotics; norfloxacin and griseofulvin. The synthesized compounds exhibited zone of inhibition of 09-22 mm in diameter whereas standard norfloxacin and griseofulvin exhibited zone of 25-28 mm in diameter. All the compounds have shown significant antibacterial and antifungal activities. The maximum activity was shown by P1 and P5 against S.aureus and E. coli respectively, while P6 has shown significant activity against all types of microorganisms. The compound P8 has been found to be significantly effective against C. albicans . All other compounds have shown moderate antimicrobial activities. The results of antimicrobial activities are expressed in 2 .
Antibacterial and antifungal activities
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Zone of inhibition: + = 5-10 mm, ++ = 11-15 mm, +++ = 16-20 mm, ++++ = More than 20 mm.
DISCUSSION
In the present investigation new series of 5-cyano, N1 substituted pyrimidine analogues were synthesized by microwave irradiation technique. Unlike the conventional methods where more reaction time is required, with yields of 26-75%, microwave- assisted reactions resulted in higher yields (65-83%) of products. These synthesized compounds were also screened for antibacterial and antifungal activities. Present findings revealed that analogues containing p-hydroxy, p-methoxy substituted phenyl moiety at 6 position have been found to be more potent against gram-positive microorganisms, while analogues lacking these substituents on phenyl moiety possessed gram-negative activity. The compounds having p-dimethylamino substituent on phenyl moiety at 6 positions have shown moderate activity. Further, only fluorine containing analogue at N1 position was found to possess appreciable antifungal activity. The compound P7 has shown least activity among all the synthesized compounds indicating that meta methyl group in phenyl moiety at N1 position is not well tolerated. Further, as the bulkiness on phenyl moiety at N1 increases, antimicrobial activity decreases. This suggests that electron donating substituent on aryl moiety as well as electron withdrawing substituent at N1 plays important role in determining potency of the compounds. The compounds P1, P5 and P6 have shown good antibacterial activities against S.aureus and E.coli whereas compound P8 has shown excellent antifungal activity against C.albicans . It is concluded that N1 and C-6 position are important determinants for the activity. Also electron-withdrawing substituent at C-5 position in pyrimidine increases the acidic dissociation constant of nucleus and enhances the receptor binding. Further studies are needed to explore the differences in the efficacy and safety of synthesized compounds.
EXPERIMENTAL
- Materials
The melting points were determined in open capillary tubes and are uncorrected. The purity of compounds was checked by TLC on silica gel G plates using acetonitrile: chloroform (60:40) as mobile phase. IR spectra were recorded on Digilab FT-IR spectrophotometer. 1 HNMR and 13 CNMR spectra were recorded in DMSO (d6) and CDCI 3 on Varian Mercury YH-300 NMR spectrophotometer. Microwave assisted reactions were carried out in a Catalysts Microwave synthesizer.
- Methods
- Synthesis of aryl-substituted thioureas
These were obtained by reacting the various substituted aromatic amines with ammonium thiocyanate in presence of conc. HCL. The compounds were recrystallized from hot water or ethanol and used for synthesis of pyrimidine derivatives. 14
- Synthesis of N1- substituted, 5-cyano, 6-arylsubstituted thiouracil derivatives (P1-P10)
The titled compounds were synthesized using onepot reaction of aryl-substituted thiourea (0.01 mol), aldehyde (0.01 mol), ethylcyanoacetate (0.01 mol) and potassium carbonate in small amount of ethanol to obtain various N1 substituted pyrimidines. Six different aryl substituted thioureas like phenyl thiourea, p-tolyl thiourea, m-tolyl thiourea, p-anisidine thiourea, 2,4-xylidyl thiourea, p-fluoro phenyl thiourea and four aldehydes like benzaldehyde, 4-hydroxy benzaldehyde, p-methoxybenzaldehyde and p-dimethylaminobenzaldehyde were used to obtain ten different 2-thio, 4-one derivatives. The desired compounds were synthesized by the tertiary condensation of ethylcyanoacetate, aryl substituted thiourea and suitable aldehyde in presence of potassium carbonate and ethanol using MORE (Microwave organic reaction enhancement) technique. The reaction mixture was subjected to microwave pulse for 120 s (240 w), 60 s (350 w) and 60 s (450 w). Between each irradiation an interval of 3-10 minutes was kept. After the completion of reaction, the precipitate obtained was dried, which was potassium salt of nucleobase. The salt was dissolved in warm water and the solution was acidified by acetic acid to precipitate pure nucleobase. The crude product was recrystallised from acetic acid. All the compounds were obtained in good yield.
- P1: 5-Cyano-1-phenyl-6-(4-hydroxyphenyl)-2-thiouracil
I.R.: ν cm -1 3363 (N-H), 2210 (C≡N), 1622 (C=O), 1305 (C=S), 3150 (O-H), 1 HNMR: δ (ppm): 8.17 (s, 1H, NH), 7.24-7.95 (m, 9H, ArH), 4.33-4.40 (s, 1H, OH), 13 CNMR: 72.67 (C=C), 115.81-134.02 (11C), 158.10 (phenyl C 5 ), 115.85 (C≡N), 167.09 (-CONH-), 171.79 (C=C), 174.03 (-CSNH-), Anal: Found: C, 63.52; H, 3.39; N, 13.05; S, 9.93. Calcd for C 17 H 11 N 3 SO 2 : C, 63.54; H, 3.45; N, 13.08; O, 9.96; S, 9.98%.
- P2: 5-Cyano-1-(4-methyl-phenyl)-6-(4-hydroxyphenyl)-2-thiouracil
I.R.: ν cm -1 3010 (C-H), 3288(N-H), 2260 (C≡N), 1610 (C=O), 1320 (C=S), 1 HNMR: δ (ppm): 8.19 (s, 1H, NH), 7.38-7.90 (m, 8H, ArH), 4.31-4.37 (s, 1H, OH), 2.354 (s, 3H, CH 3 ), 13 CNMR: 24.35 (CH 3 ), 72.67 (C=C), 115.82-128.31 (10C), 133.81 (N-phenyl C 4 ), 157.55 (phenyl C 4 ), 115.75 (C≡N), 167.21 (-CONH-), 172.10 (C=C), 174.12 (-CSNH-), Anal: Found: C, 64.41; H, 3.84; N, 12.51; S, 9.52. Calcd for C 18 H 13 N 3 SO 2 : C, 64.46; H, 3.91; N, 12.53; O, 9.54; S, 9.56%.
- P3: 5-Cyano-1,6 bis-(4-methoxy-phenyl)-2-thiouracil
I.R.: ν cm -1 3005 (C-H), 3102 (N-H), 2250 (C≡N), 1590 (C=O), 1280 (C=S), 3175 (O-H), 1275 (O-CH 3 ), 1 HNMR: δ (ppm): 8.12 (s, 1H, NH), 7.31-7.97 (m, 8H, ArH), 3.78-3.91 (s, 6H, OCH 3 ), 13 CNMR: 55.85 (OCH 3 ), 72.61 (C=C), 114.11-127.41 (10C), 159.80 (phenyl C 4 ), 156.64 (N-phenyl C 4 ), 115.75 (C≡N), 167.21 (-CONH-), 171.68 (C=C), 173.93 (-CSNH-), Anal: Found: C, 62.43; H, 4.08; N, 11.46; S, 8.73. Calcd for C 19 H 15 N 3 SO 3 : C, 62.45; H, 4.14; N, 11.50; O, 13.14; S, 8.78%.
- P4: 5-Cyano-1-(4-methyl-phenyl)-6-phenyl-2-thiouracil
I.R.: ν cm -1 2995 (C-H), 3270 (N-H), 2240 (C≡N), 1620 (C=O), 1200 (C=S), 1 HNMR: δ (ppm): 8.00-8.84 (s, 1H, NH), 7.18-7.98 (m, 9H, ArH), 2.35-2.60 (d, 3H, CH 3 ), 13 CNMR: 24.25 (CH 3 ), 72.81 (C=C), 126.51-134.35 (11C), 134.42 (N-phenyl C 4 ), 115.72 (C≡N), 167.15 (-CONH-), 171.95 (C=C), 173.98 (-CSNH-), Anal: Found: C, 67.69; H, 4.05; N, 13.13; S, 10.01. Calcd for C 18 H 13 N 3 SO: C, 67.69; H, 4.10; N, 13.16; O, 5.01; S, 10.04%.
- P5: 5-Cyano-1,6-diphenyl-2-thiouracil
I.R.: ν cm -1 3005 (C-H), 3280 (N-H), 2270 (C≡N), 1610 (C=O), 1265 (C=S), 1 HNMR: d (ppm): δ 9.28(s, 1H, NH), δ 7.17-7.40 (m, 10H, ArH), 13 CNMR: 72.68 (C=C), 126.33-133.90 (12C), 115.95 (C≡N), 167.12 (-CONH-), 171.67 (C=C), 174.23 (-CSNH-), Anal: Found: C, 66.85; H, 3.55; N, 13.75; S, 10.47. Calcd for C 17 H 11 N 3 SO: C, 66.87; H, 3.63; N, 13.76; O, 5.24; S, 10.50%.
- P6: 5-Cyano-1-(2,4-dimethyl-phenyl)-6-(4-methoxyphenyl)-2-thiouracil
I.R.: ν cm -1 3010 (C-H), 3261 (N-H), 2245 (C≡N), 1605 (C=O), 1275 (C=S), 1175 (O-CH 3 ), 1 HNMR: δ (ppm): 8.13-8.15 (d, 1H, NH), 7.32-7.98 (m, 7H, ArH), 2.16-2.17 (s, 6H, CH 3 ), 3.88 (s, 3H, OCH 3 ), 13 CNMR: 16.45 (CH 3 ), 24.55 (CH 3 ), 55.81 (OCH 3 ), 72.69 (C=C), 114.29-133.68 (9C), 134.23 (N-phenyl C 4 ), 139.21 (N-phenyl C 2 ), 160.10 (phenyl C 4 ), 115.78 (C≡N), 167.59 (-CONH-), 171.99 (C=C), 174.33 (-CSNH-), Anal: Found: C, 66.09; H, 4.65; N, 11.53; S, 8.79. Calcd for C 20 H 17 N 3 SO 2 : C, 66.10; H, 4.71; N, 11.56; O, 8.80; S, 8.82 %.
- P7: 5-Cyano-1-(3-methyl-phenyl)-6-(4-methoxyphenyl)-2-thiouracil
I.R.: ν cm -1 3000 (C-H), 3280 (N-H), 2205 (C≡N), 1605 (C=O), 1250 (C=S), 1170 (O-CH 3 ), 1 HNMR: δ (ppm): 8.13 (s, 1H, NH), 7.30-7.98 (m, 8H, ArH), 3.88 (s, 3H, OCH 3 ), 2.17 (s, 3H, CH 3 ), 13 CNMR: 24.13 (CH 3 ), 55.95 (OCH 3 ), 72.67 (C=C), 114.29-134.08 (10C), 138.66 (N-phenyl C 3 ), 159.79 (phenyl C 4 ), 115.71 (C≡N), 167.12 (-CONH-), 172.01 (C=C), 173.96 (-CSNH-), Anal: Found: C, 65.29; H, 4.27; N, 12.01; S, 9.13. Calcd for C 19 H 15 N 3 SO 2 : C, 65.31; H, 4.33; N, 12.03; O, 9.16; S, 9.18%.
- P8: 5-Cyano-1-(4-fluoro-phenyl)-6-(4-methoxyphenyl)-2-thiouracil
I.R.: ν cm -1 2995 (C-H), 3280 (N-H), 2240 (C≡N), 1590 (C=O), 1262 (C=S), 1280 (O-CH 3 ), 1 HNMR: δ (ppm): 8.13-8.15 (s, 1H, NH), 7.33-7.98 (m, 8H, ArH), 3.85-3.90 (s, 3H, OCH 3 ), 13 CNMR: 55.88 (OCH 3 ), 72.55 (C=C), 114.32-129.56 (10C), 159.10 (N-phenyl C 4 ), 159.79 (phenyl C 4 ), 115.68 (C≡N), 167.22 (-CONH-), 171.57 (C=C), 174.13 (-CSNH-), Anal: Found: C, 61.33; H, 3.36; N, 11.90; S, 9.06. Calcd for C 18 H 12 FN 3 SO 2 : C, 61.18; H, 3.42; F, 5.38; N, 11.89; O, 9.06; S, 9.07%.
- P9: 5-Cyano-1-(2,4-dimethyl-phenyl)-6-(4-dimethylamino-phenyl)-2-thiouracil
I.R.: ν cm -1 3002 (C-H), 3300 (N-H), 2245 (C≡N), 1590 (C=O), 1250 (C=S), 1 HNMR: δ (ppm): 8.02 (s, 1H, NH), 7.36-7.90 (m, 7H, ArH), 2.25-2.30 (d, 12H, CH 3 ), 13 CNMR: 16.35 (CH 3 ), 24.65 (CH 3 ), 40.23 (-N(CH 3 ) 2 ), 72.78 (C=C), 114.12-133.98 (9C), 134.43(N-phenyl C 4 ), 139.15 (N-phenyl C 2 ), 148.78 (phenyl C 4 ), 115.68 (C≡N), 167.12 (-CONH-), 171.57 (C=C), 174.11 (-CSNH-), Anal: Found: C, 67.01; H, 5.28; N, 14.86; S, 8.49. Calcd for C 21 H 20 N 4 SO: C, 67.00; H, 5.35; N, 14.88; O, 4.25; S, 8.52%.
- P10: 5-Cyano-1-phenyl-6-(4-dimethylamino-phenyl)-2-thiouracil
I.R.: ν cm -1 3012 (C-H), 3275 (N-H), 2260 (C≡N), 1595 (C=O), 1270 (C=S), 1 HNMR: δ (ppm): 8.03 (s, 1H, NH), 7.34-7.91 (m, 9H, ArH), 3.10-3.79 (s, 6H, CH 3 ), 13 CNMR: 40.31 (-N(CH 3 ) 2 ), 72.87 (C=C), 114.22-133.95 (11C), 148.78 (phenyl C 4 ), 115.60 (C≡N), 167.02 (-CONH-), 171.79 (C=C), 174.13 (-CSNH-), Anal: Found: C, 65.48; H, 4.56; N, 16.03; S, 9.16. Calcd for C 19 H 16 N 4 SO: C, 65.50; H, 4.63; N, 16.08; O, 4.59; S, 9.20%.
- Antimicrobial activity
All synthesized compounds were screened for antibacterial activity by cup- plate method against gram-positive species S. aureus and B. subtilis and gram-negative species E. coli and P.aeruginosa in the concentration of 25 μg. These compounds were also screened for antifungal activity against fungi C. albicans in the same concentration. The activities were compared with standard antibiotics; norfloxacin and grisievofulvin. All the synthesized compounds were dissolved in dimethyl sulphoxide, which was used as a control. The plates were incubated at 37 ℃ for 24 hours and the zone of inhibition was measured in mm.
Acknowledgements
The authors are thankful to Nulife Pharmaceuticals Ltd., Pimpri, Pune and Pune University, Pune, Maharashtra, India for spectroscopy facilities.
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