Synthesis of novel and highly functionalized pyrimidine-5-carboxylate derivatives and their antimicrobial evaluation

Article abstract of DOI:10.2174/1570180811666140401182419

In the present article, the antimicrobial properties of ethyl 4-amino-2-mercapto-6-arylpyrimidine-5-carboxylate (2a-h) and ethyl 2-(2,4-dinitrophenylthio)-4-amino-6-arylpyrimidine-5-carboxylate (3a-h) were studied. Biginelli typed three component reaction between an aldehyde, ethyl cyanoacetate and a thiourea constituent which gives a rapid facile 3,4-dihydropyrimidine ring (1a-h), which on aromatization using SeO2 and followed by reaction with 1-chloro-2,4-dinitrobenzene giving final product (3a-h). The structures of all synthesized compounds have been established by extensive IR, NMR and Mass studies and were assayed for their antibacterial activity against S. aureus MTCC-96 and E. coli MTCC-443 and antifungal activity against A. niger MTCC-282 and C. albicans MTCC-227 at different concentrations compared with Ampicillin, Chloramphenicol, Ciprofloxacin and Griseofulvin as standard drugs. The activity was measured in the zone of inhibition. Among the all synthesized compounds, 2a, 2d, 3a, 3d and 3g are significantly active against bacterial strain, while other compounds are weakly active. All the compounds are moderate to low active against fungal strain.

Full text of DOI:10.2174/1570180811666140401182419

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930
Letters in Drug Design & Discovery, 2014, 11, 930-936
Synthesis of Novel and Highly Functionalized Pyrimidine-5-carboxylate
Derivatives and their Antimicrobial Evaluation
Hardik B. Ghodasara, Rajesh G. Vaghasiya, Bharat G. Patel and Viresh H. Shah*
Department of Chemistry, Saurashtra University, Rajkot 360 005, India
Abstract: In the present article, the antimicrobial properties of ethyl 4-amino-2-mercapto-6-arylpyrimidine-5-carboxylate
(2a-h) and ethyl 2-(2,4-dinitrophenylthio)-4-amino-6-arylpyrimidine-5-carboxylate (3a-h) were studied. Biginelli typed
three component reaction between an aldehyde, ethyl cyanoacetate and a thiourea constituent which gives a rapid facile
3,4-dihydropyrimidine ring (1a-h), which on aromatization using SeO2 and followed by reaction with 1-chloro-2,4-
dinitrobenzene giving final product (3a-h). The structures of all synthesized compounds have been established by exten-
sive IR, NMR and Mass studies and were assayed for their antibacterial activity against S. aureus MTCC-96 and E. coli
MTCC-443 and antifungal activity against A. niger MTCC-282 and C. albicans MTCC-227 at different concentrations
compared with Ampicillin, Chloramphenicol, Ciprofloxacin and Griseofulvin as standard drugs. The activity was meas-
ured in the zone of inhibition. Among the all synthesized compounds, 2a, 2d, 3a, 3d and 3g are significantly active
against bacterial strain, while other compounds are weakly active. All the compounds are moderate to low active against
fungal strain.
Keywords: Antimicrobial assay, Biginelli reaction, Pyrimidine-5-carboxylate, Standard drugs, Zone of inhibition.
INTRODUCTION
cal potential of this class of compounds, we have synthesized
series of compounds using Biginelli typed three-
a
Heterocyclic compounds have considerably a lot of inter-
est due to wide application of pharmacological activity. Dur-
ing last few years, interest in this area was focused on the
evaluation of biological activities of various heterocyclic
compounds with chemical moieties such as Pyridine,
Pyrimidines, Pyrazoline, Thiazolidine, Azetidines, Imida-
zole, Thiazole, etc. Pyrimidine and their derivatives play a
vital role in the field of drugs and agricultural chemicals.
Pyrimidine could be a basic nucleus in DNA & RNA.
Pyrimidine derivatives are well-known for their pharmacol-
ogical properties [1, 2]. These compounds, structurally re-
lated to nucleic acids, have been reported to be anticancer
[3–7], interferon inducer [8], antiviral [9, 10], anti-
hypertensive [11], antitubercular [12-13], hypoglycemic
[14], analgesic and anti-inflammatory drugs [15].
component reaction and evaluated the antibacterial and anti-
fungal activity of ethyl 2-(2,4-dinitrophenylthio)-4-amino-6-
arylpyrimidine-5-carboxylate against various strains of mi-
crobes at different concentrations compared with Ampicillin,
Chloramphenicol, Ciprofloxacin, Norfloxacin and Griseo-
fulvin as standard drugs.
MATERIALS AND METHODS
All research chemicals were purchased from Sigma–
Aldrich and used as it is for the reactions. Reactions were
monitored by thin-layer chromatography (TLC) on pre-
coated silica gel GF254 plates from E-Merck Co and com-
pounds visualized either by exposure to UV. Melting points
were determined in open capillaries and are uncorrected. The
IR spectra were recorded on SHIMADZU- FTIR-8400 spec-
Several methodologies are available for synthesizing this
pharmacologically interesting class of heterocycles. Most of
them make use of a condensation reaction between a Michael
intermediate (arylidine) and amidines [16], guanidine [17],
urea [18], thiourea [19], methylisourea and methyliso-
thiourea [20] in the presence of organic bases as catalysts.
The most common path for the synthesis of pyrimidine is a
reagent containing C-C-C and N-C-N skeleton. The C-C-C
skeleton was obtained from the reaction of reagents with
active methelene group and aryl aldehyde whereas N-C-N
skeleton can be obtained from urea, thiourea or guanidine.
1
trophotometer using KBr pellet method. H and 13C NMR
spectra were recorded on Bruker 300-MHz NMR spectrome-
ter in CDCl3 with TMS as internal standard. Mass spectrum
was recorded on JOEL SX 102/DA-600-Mass spectrometer
and elemental analysis was carried out using Heraus C,H,N
rapid analyzer.
General Procedure for the Synthesis of Ethyl 6-amino-
1,2,3,4-tetrahydro-4-aryl-2-thioxopyrimidine-5-carboxy-
late (1a-h)
As the pyrimidine scaffold is present in many antimicro-
bial agents [21] and keeping in mind various pharmacologi-
A mixture of equimolar quantities of appropriate aryl al-
dehyde (0.01 mmol) ethyl cyanoacetate (0.01 mmol) and
thiourea (0.01 mmol) was refluxed in dry ethanol for 3
hours. The reaction mixture was kept at room temperature
for 2 hours. The yellow crystalline product was obtained.
The product was isolated and recrystallized from ethanol to
give the desired product (1a-h).
*Address correspondence to this author at the Department of Chemistry,
Saurashtra University, Rajkot 360 005, India; Tel/Fax: +91 281 2571989;
E-mail: shah_v_h@yahoo.com
1875-628X/14 $58.00+.00
©2014 Bentham Science Publishers

Synthesis of Novel and Highly Functionalized
Letters in Drug Design & Discovery, 2014, Vol. 11, No. 7 931
General Procedure for Synthesis of Ethyl 4-amino-2-
mercapto-6-arylpyrimidine-5-carboxylate (2a-h)
Ethyl 4-amino-6-(4-chlorophenyl)-2-mercaptopyrimidi-
ne-5-carboxylate (2e)
In a typical procedure, a Compound (1a-h) (0.01 mmol),
selenium dioxide (0.01 mmol), and HOAc (4 mL) was stirred
at room temperature for 30 min. Reaction was monitored by
TLC. After completion of reaction, the reaction mixture was
quenched with a satd. aqueous NaHCO3 solution and the red
solid selenium was filtered off. The filtrate was extracted three
times with dichloromethane. The combined organic layer was
washed with water, dried over anhydr. NaSO4, and concen-
trated to give the pure product (2a-h).
Yield = 55%, m.p. 123-125 ºC; IR (KBr) cm-1: 3414,
3089, 2942, 2790, 2514, 1741, 1645, 1542, 1518, 1475,
1236, 845, 740; 1H NMR (300 MHz, CDCl3) ꢀ ppm : 1.07 (t,
3H, –CH3), 2.34 (q, 2H, –CH2), 3.10 (s, 1H, –SH), 4.12 (s,
2H, -NH2), 6.61-6.90 (m, 4H, Ar-H); 13C NMR
(CDCl3,ꢀppm): 15.4, 44.2, 61.4, 102.7, 126.1, 126.8, 129.1,
144.1, 156.1, 162.5, 166.2; MS (m/z): 309; Anal. Calcd. for
C13H12N3O2SCl: C, 50.40; H, 3.90; N, 13.56. Found: C,
50.39; H, 3.92; N, 13.53.
Ethyl 4-amino-2-mercapto-6-phenylpyrimidine-5-carbox-
ylate (2a)
Ethyl 4-amino-2-mercapto-6-(2-nitrophenyl)pyrimidine-
5-carboxylate (2f)
Yield = 68%, m.p. 150-152 ºC; IR (KBr) cm-1: 3413,
3075, 2945, 2833, 2521, 1750, 1645, 1565, 1530, 1444,
1255, 1141, 941, 830; 1H NMR (300 MHz, CDCl3) ꢀ ppm:
1.20 (t, 3H, –CH3), 2.27 (q, 2H, –CH2), 3.11 (s, 1H, –SH),
4.12 (br s, 2H, -NH2), 7.06-7.14 (m, 5H, Ar-H); 13C NMR
(CDCl3,ꢀppm): 14.2, 44.0, 61.7, 102.3, 126.8, 127.0, 128.6,
143.2, 156.1, 163.0, 167.2; MS (m/z): 275; Anal. Calcd. for
C13H13N3O2S: C, 56.71; H, 4.76; N, 15.26. Found: C, 56.72;
H, 4.77; N, 15.28.
Yield = 49%, m.p. 170-172 ºC; IR (KBr) cm-1: 3402,
3053, 2954, 2825, 2532, 1743, 1640, 1539, 1525, 1508,
1478, 1251, 1143, 845, 723; 1H NMR (300 MHz, CDCl3) ꢀ
ppm: 1.08 (t, 3H, –CH3), 2.18 (q, 2H, –CH2), 3.51 (s, 1H, –
SH), 4.32 (s, 2H, -NH2), 7.32-8.07 (m, 4H, Ar-H); 13C NMR
(CDCl3,ꢀppm): 14.4, 44.1, 62.2, 103.0, 126.7, 128.1, 128.8,
144.2, 155.1, 162.8, 166.8; MS (m/z): 320; Anal. Calcd. for
C13H12N4O4S: C, 48.74; H, 3.78; N, 17.49. Found: C, 48.76;
H, 3.80; N, 17.48.
Ethyl 4-amino-6-(2-hydroxyphenyl)-2-mercaptopyrimid-
ine-5-carboxylate (2b)
Ethyl 4-amino-2-mercapto-6-(3-nitrophenyl)pyrimidine-
5-carboxylate (2g)
Yield = 74%, m.p. 188-190ºC; IR (KBr) cm-1: 3445,
3140, 3108, 2925, 2874, 2541, 1742, 1645, 1525, 1525,
1454, 1366, 1235, 1125, 826; 1H NMR (300 MHz, CDCl3) ꢀ
ppm: 1.17 (t, 3H, –CH3), 2.40 (q, 2H, –CH2), 3.21 (s, 1H, –
SH), 4.22 (s, 2H, -NH2), 5.10 (s, 1H, -OH), 6.61-6.90 (m,
4H, Ar-H); 13C NMR (CDCl3,ꢀppm): 15.0, 43.7, 62.2, 104.1,
125.9, 126.8, 127.6, 141.9, 155.2, 162.8, 166.8; MS (m/z):
291; Anal. Calcd. for C13H13N3O3S: C, 53.60; H, 4.50; N,
14.42. Found: C, 53.62; H, 4.51; N, 14.44.
Yield = 43%, m.p. 131-133 ºC; IR (KBr) cm-1: 3412,
3080, 2938, 2845, 2512, 1751, 1655, 1535, 1525, 1515,
1470, 1256, 1123, 829;; 1H NMR (300 MHz, CDCl3) ꢀ ppm:
1.12 (t, 3H, –CH3), 2.11 (q, 2H, –CH2), 3.23 (s, 1H, –SH),
4.23 (s, 2H, -NH2), 7.40-8.01 (m, 4H, Ar-H); 13C NMR
(CDCl3,ꢀppm): 14.8, 44.5, 62.5, 102.1, 126.4, 126.8, 128.5,
142.1, 154.1, 162.7, 165.1; MS (m/z): 320; Anal. Calcd. for
C13H12N4O4S: C, 48.74; H, 3.78; N, 17.49. Found: C, 48.75;
H, 3.80; N, 17.47.
Ethyl 4-amino-6-(4-hydroxyphenyl)-2-mercaptopyrimidi-
ne-5-carboxylate (2c)
Ethyl 4-amino-2-mercapto-6-(4-methoxyphenyl)pyrimid-
ine-5-carboxylate (2h)
Yield = 56%, m.p141-143ºC; IR (KBr) cm-1: 3422, 3177,
3045, 2924, 2864, 2512, 1747, 1640, 1522, 1509, 1446,
1356, 1244, 830; H NMR (300 MHz, CDCl3) ꢀ ppm: 1.07
Yield = 51%, m.p. 118-120 ºC; IR (KBr) cm-1: 3405,
3075, 2925, 2865, 2531, 1745, 1646, 1540, 1513, 1472,
1250, 1133, 822; 1H NMR (300 MHz, CDCl3) ꢀ ppm: 1.11 (t,
3H, –CH3), 2.22 (q, 2H, –CH2), 3.42 (s, 1H, –SH), 3.73 (s,
3H, -OCH3), 4.33 (s, 2H, -NH2, 6.65-6.95 (m, 4H, Ar-H);
13C NMR (CDCl3,ꢀppm): 15.1, 43.8, 55.4, 60.4, 103.3,
127.1, 127.6, 128.8, 143.5, 156.2, 163.4, 166.8; MS (m/z):
305; Anal. Calcd. for C14H15N3O3S: C, 55.07; H, 4.95; N,
13.76. Found: C, 55.09; H, 4.93; N, 13.76.
1
(t, 3H, –CH3), 2.23 (q, 2H, –CH2), 3.43 (s, 1H, –SH), 4.11 (s,
2H, -NH2), 5.22 (s, 1H, -OH), 6.42-6.72 (m, 4H, Ar-H); 13
C
NMR (CDCl3,ꢀppm): 16.1, 45.3, 62.1, 102.8, 126.1, 126.9,
127.6, 143.8, 156.4, 162.6, 165.2; MS (m/z): 291; Anal.
Calcd. for C13H13N3O3S: C, 53.60; H, 4.50; N, 14.42. Found:
C, 53.59; H, 4.49; N, 14.40.
Ethyl 4-amino-6-(2-chlorophenyl)-2-mercaptopyrimide-
carboxylate (2d)
General Procedure for the Synthesis of ethyl 2-(2,4-
dinitrophenylthio)-4-amino-6-arylpyrimidine-5-carboxy-
late (3a-h)
Yield = 72%, m.p. 148-150ºC; IR (KBr) cm-1: 3400,
3082, 2925, 2854, 2545, 1750, 1651, 1541, 1512, 1477,
1241, 829, 773, 735; H NMR (300 MHz, CDCl3) ꢀ ppm:
Equimoler quantities of ethyl 4-amino-2-mercapto-6-
arylpyrimidine-5-carboxylate 2 (0.01 mmol) and 1-Chloro-2,
4-dinitrobenzene (0.01 mmol) was stirred in dry ethanol in
the presence of 10% alcoholic sodium hydroxide as a cata-
lyst and heated for 3 hours at reflux temperature. After com-
pletion of reaction the content was poured into ice cold water
and filtered. The desired product was isolated and recrystal-
lized from ethanol.
1
1.05 (t, 3H, –CH3), 2.12 (q, 2H, –CH2), 3.45 (s, 1H, –SH),
4.15 (s, 2H, -NH2), 7.00-7.15 (m, 4H, Ar-H); 13C NMR
(CDCl3,ꢀppm): 16.0, 44.1, 61.1, 103.7, 126.4, 127.0, 127.9,
142.9, 155.1, 162.8, 166.4; MS (m/z): 309; Anal. Calcd. for
C13H12N3O2SCl: C, 50.40; H, 3.90; N, 13.56. Found: C,
50.42; H, 3.91; N, 13.55.

932 Letters in Drug Design & Discovery, 2014, Vol. 11, No. 7
Ghodasara et al.
R
C2H5OH
O
H
O
R
O
R
ꢀ
SeO2
O
O
NH
O
N
-H2O
+
O
NH2
HOAc, RT
H
H2N
N
H
S
H2N
N
S
H2N
S
N
1a-h
2a-h
O
R
Cl
NO2
NO2
NaOH
O
N
2a-h
+
ꢀ
H2N
N
S
NO2
NO2
3a-h
Where R= H, 2-OH, 4-OH, 2-Cl, 4-Cl, 2-NO2, 3-NO2, 4-OCH3
Scheme (1). Synthesis of pyrimidine-5-carboxylate derivatives.
457; Anal. Calcd. for C19H15N5O7S: C, 49.89; H, 3.31; N,
15.31;. Found: C, 49.91; H, 3.30; N, 15.32.
Ethyl 2-(2,4-dinitrophenylthio)-4-amino-6-phenylpyrimi-
dine-5-carboxylate (3a)
Yield = 67%, m.p. 146-148 ºC; IR (KBr) cm-1: 3414,
3075, 2912, 2821, 1641, 1554, 1523, 1442, 1374, 924, 842;
1H NMR (300 MHz, CDCl3) ꢀ ppm: 1.15 (t, 3H, –CH3), 2.13
(q, 2H, –CH2), 4.22 (s, 2H, -NH2), 6.32-6.65 (m, 5H, Ar-H),
7.70 (d, 1H, Ar-H), 8.42 (dd, 1H, Ar-H), 8.96 (d, 1H, Ar-H);
13C NMR (CDCl3,ꢀppm): 15.2, 45.4, 61.5, 103.3, 119.1,
126.8, 127.1, 127.5, 128.6, 131.2, 133.7, 143.2, 145.2, 150.7,
155.1, 162.8, 166.9; MS (m/z): 441; Anal. Calcd. for
C19H15N5O6S: C, 51.70; H, 3.43; N, 15.87. Found: C, 51.71;
H, 3.44; N, 15.86.
Ethyl 2-(2,4-dinitrophenylthio)-4-amino-6-(2-chlorophen-
yl)pyrimidine-5-carboxylate (3d)
Yield = 58%, m.p. 162-164 ºC; IR (KBr) cm-1: 3400,
3082, 2925, 2854, 1651, 1541, 1512, 1477, 829, 773, 735; 1H
NMR (300 MHz, CDCl3) ꢀ ppm: 1.15 (t, 3H, –CH3), 2.12 (q,
2H, –CH2), 4.25 (s, 2H, -NH2), 7.04-7.27 (m, 4H, Ar-H),
7.56 (d, 1H, Ar-H), 7.88 (dd, 1H, Ar-H), 8.12 (d, 1H, Ar-H);
13C NMR (CDCl3,ꢀppm): 16.2, 44.2, 61.2, 103.5, 119.4,
126.4, 127.1, 127.5, 127.9, 131.2, 133.7, 142.7, 146.1, 150.7,
155.5, 162.5, 166.1; MS (m/z): 475; Anal. Calcd. for
C19H14ClN5O6S: C, 47.96; H, 2.97; N, 14.72. Found: C,
47.95; H, 2.94; N, 14.73.
Ethyl 2-(2,4-dinitrophenylthio)-4-amino-6-(2-hydroxyph-
enyl)pyrimidine-5-carboxylate (3b)
Yield = 72%, m.p. 115-117 ºC; IR (KBr) cm-1: 3410,
3170, 3040, 2944, 2854, 1640, 1530, 1512, 1454, 1344,
1320, 826; H NMR (300 MHz, CDCl3) ꢀ ppm: 1.17 (t, 3H,
–CH3), 2.40 (q, 2H, –CH2), 4.18 (s, 2H, -NH2), 5.12 (s, 1H, -
OH), 6.45-7.12 (m, 4H, Ar-H), 7.67 (d, 1H, Ar-H), 8.44 (dd,
1H, Ar-H), 8.71 (d, 1H, Ar-H); 13C NMR (CDCl3,ꢀppm):
15.1, 43.5, 62.2, 103.1, 119.4, 124.9, 126.4, 127.2, 127.8,
131.4, 133.7, 141.4, 146.4, 150.4, 155.5, 162.1, 166.3; MS
(m/z): 457; Anal. Calcd. for C19H15N5O7S: C, 49.89; H, 3.31;
N, 15.31;. Found: C, 49.90; H, 3.33; N, 15.30.
Ethyl 2-(2,4-dinitrophenylthio)-4-amino-6-(4-chlorophen-
yl)pyrimidine-5-carboxylate (3e)
1
Yield = 56%, m.p. 233-235 ºC; IR (KBr) cm-1: 3411,
3101, 2932, 2850, 1645, 1542, 1518, 1475, 845, 740; H
1
NMR (300 MHz, CDCl3) ꢀ ppm: 1.23 (t, 3H, –CH3), 2.37 (q,
2H, –CH2), 4.41 (s, 2H, -NH2), 7.00 (d, 2H, Ar-H), 7.15 (d,
2H, Ar-H), 7.45 (d, 1H, Ar-H), 7.92 (dd, 1H, Ar-H), 8.27 (d,
1H, Ar-H); 13C NMR (CDCl3,ꢀppm): 15.2, 44.1, 61.1, 102.8,
119.4, 126.5, 126.8, 127.4, 129.1, 131.4, 133.7, 144.4, 156.1,
162.7, 166.3; MS (m/z): 475; Anal. Calcd. for
C19H14ClN5O6S: C, 47.96; H, 2.97; N, 14.72. Found: C,
47.97; H, 2.99; N, 14.70.
Ethyl 2-(2,4-dinitrophenylthio)-4-amino-6-(4-hydroxyph-
enyl)pyrimidine-5-carboxylate (3c)
Yield = 64%, m.p. 183-185 ºC; IR (KBr) cm-1: 3422,
3177, 3045, 2924, 2864, 1640, 1522, 1509, 1446, 1356, 830;
1H NMR (300 MHz, CDCl3) ꢀ ppm: 1.19 (t, 3H, –CH3), 2.27
(q, 2H, –CH2), 4.24 (s, 2H, -NH2), 5.14 (s, 1H, -OH), 6.45
(d, 2H, Ar-H), 6.89 (d, 2H, Ar-H), 7.72 (d, 1H, Ar-H), 8.12
(dd, 1H, Ar-H), 8.42 (d, 1H, Ar-H); 13C NMR
(CDCl3,ꢀppm): 16.7, 45.2, 62.7, 102.2, 119.1, 126.2, 126.7,
127.5, 127.8, 143.4, 146.1, 156.8, 163.6, 166.2; MS (m/z):
Ethyl 2-(2,4-dinitrophenylthio)-4-amino-6-(2-nitrophen-
yl)pyrimidine-5-carboxylate (3f)
Yield = 59%, m.p. 154-156 ºC; IR (KBr) cm-1: 3402,
3053, 2954, 2825, 1645, 1539, 1525, 1508, 1478, 845, 723;
1H NMR (300 MHz, CDCl3) ꢀ ppm: 1.16 (t, 3H, –CH3), 2.33
(q, 2H, –CH2), 4.41 (s, 2H, -NH2), 7.12-7.89 (m, 4H, Ar-H),
7.71 (d, 1H, Ar-H), 8.12 (dd, 1H, Ar-H), 8.77 (d, 1H, Ar-H);

Synthesis of Novel and Highly Functionalized
Letters in Drug Design & Discovery, 2014, Vol. 11, No. 7 933
Table 1. Molecular Formula and physical parameter of synthesized compounds.
No.
M.F.
R
M.W.
M.P. (°C)
% Yield
2a
2b
2c
2d
2e
2f
C13H13N3O2S
C13H13N3O3S
C13H13N3O3S
C13H12N3O2SCl
C13H12N3O2SCl
C13H12N4O4S
C13H12N4O4S
C14H15N3O3S
C19H15N5O6S
C19H15N5O7S
C19H15N5O7S
C19H14ClN5O6S
C19H14ClN5O6S
C19H14N6O8S
C19H14N6O8S
C20H17N5O7S
C6H5
275
291
291
309
309
320
320
305
441
457
457
475
475
486
486
471
150-152
188-190
141-143
148-150
123-125
170-172
131-133
118-120
146-148
115-117
183-185
162-164
233-235
154-156
165-167
124-126
68
74
56
72
55
49
43
51
67
72
64
58
56
59
53
65
2-OH- C6H4
4-OH- C6H4
2-Cl- C6H4
4-Cl- C6H4
2-NO2- C6H4
3-NO2- C6H4
4-OCH3- C6H4
C6H5
2g
2h
3a
3b
3c
3d
3e
3f
2-OH- C6H4
4-OH- C6H4
2-Cl- C6H4
4-Cl- C6H4
2-NO2- C6H4
3-NO2- C6H4
4-OCH3- C6H4
3g
3h
13C NMR (CDCl3,ꢁppm): 14.6, 44.4, 62.1, 103.2, 119.2,
126.1, 127.5, 128.1, 128.6, 131.4, 133.2, 144.1, 156.1, 163.8,
166.1; MS (m/z): 486; Anal. Calcd. for C19H14N6O8S: C,
46.92; H, 2.90; N, 17.28. Found: C, 46.91; H, 2.93; N, 17.26.
RESULT AND DISCUSSION
Chemistry
Various ethyl 4-amino-2-mercapto-6-arylpyrimidine-5-
carboxylate (2a-h) and ethyl 2-(2,4-dinitrophenylthio)-4-
Ethyl 2-(2,4-dinitrophenylthio)-4-amino-6-(3-nitrophen-
yl)pyrimidine-5-carboxylate (3g)
amino-6-arylpyrimidine-5-carboxylate (3a-h) bearing
a
range of electron withdrawing and electron releasing sub-
stituents, viz., 2-OH; 4-OH; 2-Cl; 4-Cl; 2-NO2; 3-NO2; 4-
OCH3 were synthesized. Biginelli typed three-component
reaction between an aldehyde, ethyl cyanoacetate and a
thiourea constituent gives a rapid facile 3,4-dihydropyr-
imidine ring (1a-h), which on aromatization using SeO2 and
followed by reaction with 1-chloro-2,4-dinitrobenzene gives
final product (3a-h). Designed series of molecules (2a-h to
Yield = 53%, m.p. 165-167 ºC; IR (KBr) cm-1: 3412,
3080, 2938, 2845, 1655, 1535, 1525, 1515, 1470, 829; H
1
NMR (300 MHz, CDCl3) ꢁ ppm: 1.21 (t, 3H, –CH3), 2.30 (q,
2H, –CH2), 4.44 (s, 2H, -NH2), 7.45-8.10 (m, 4H, Ar-H),
7.72 (d, 1H, Ar-H), 8.07 (dd, 1H, Ar-H), 8.52 (d, 1H, Ar-H);
13C NMR (CDCl3,ꢁppm): 15.8, 43.5, 63.5, 102.7, 119.5,
126.4, 126.8, 127.4, 128.5, 131.2, 133.4, 142.2, 146.2, 151.1,
153.1, 163.7, 166.1; MS (m/z): 486; Anal. Calcd. for
C19H14N6O8S: C, 46.92; H, 2.90; N, 17.28. Found: C, 46.90;
H, 2.91; N, 17.27.
1
3a–h) (Table 1) were characterized by H NMR, 13C NMR,
and Mass spectrometry techniques, and elemental analysis.
Antimicrobial Screening
Ethyl 2-(2,4-dinitrophenylthio)-4-amino-6-(4-methoxyph-
enyl)pyrimidine-5-carboxylate (3h)
The miscellaneous biological activity of pyrimidine
and its fused derivatives inspired us to screen the newly syn-
thesized compounds. Nowadays many antimicrobial agents
have been applied for treatment; still the medical field needs
extensive efforts for the development of new antimicrobial
agents to overcome the highly resistant species of microbes.
The newly synthesized compounds 2-3(a-h) were tested for
anti bacterial screening using agar well diffusion method
against S. aurues MTCC-96, B. subtillitis MTCC-441 bacte-
rial strain and antifungal screening against A. niger MTCC-
282 and C. albicans MTCC-227 at different concentrations
(50,100,250 ꢀg/mL), which were compared with Ampicillin,
Chloramphenicol, Ciprofloxacin, Norfloxacin and Griseo-
Yield =65%, m.p. 124-126 ºC; IR (KBr) cm-1: 3405,
3075, 2925, 2865, 1646, 1540, 1513, 1472, 822; H NMR
1
(300 MHz, CDCl3) ꢁ ppm: 1.15 (t, 3H, –CH3), 2.31 (q, 2H, –
CH2), 2.42 (s, 3H, -OCH3), 4.42 (s, 2H, -NH2), 6.65 (d, 2H,
Ar-H), 6.95 (d, 2H, Ar-H), 7.61 (d, 1H, Ar-H), 8.11 (dd, 1H,
Ar-H), 8.43 (d, 1H, Ar-H); 13C NMR (CDCl3,ꢁppm): 15.2,
44.2, 55.1, 60.2, 102.3, 119.4, 126.1, 127.2, 127.8, 128.9,
131.2, 133.4, 143.5, 146.2, 150.4, 156.2, 163.4, 166.4; MS
(m/z): 471; Anal. Calcd. for C20H17N5O7S: C, 50.95; H, 3.63;
N, 14.86. Found: C, 50.97; H, 3.65; N, 14.84.

934 Letters in Drug Design & Discovery, 2014, Vol. 11, No. 7
Table 2. In vitro antimicrobial screening data of 2-3a-h.
Ghodasara et al.
Antibacterial Screening
Antifungal Screening
C. albicans A. niger
S. aureus
MTCC-96
E. Coli
No
R
MTCC-443
MTCC-227
MTCC-282
Conc. In ꢀg/mL
Conc. In ꢀg/mL
Conc. In ꢀg/mL
Conc. In ꢀg/mL
25
50
100
25
50
100
25
50
100
25
50
100
2a
2b
2c
2d
2e
2f
C6H5
2-OH- C6H4
4-OH- C6H4
2-Cl- C6H4
11
12
11
11
10
11
10
12
14
12
12
14
12
11
14
13
13
14
19
-
12
13
14
14
11
12
11
12
14
12
13
15
12
13
14
13
14
19
21
-
14
15
15
16
15
13
15
11
16
14
14
15
14
13
15
14
16
20
22
-
15
14
14
13
14
13
12
13
15
13
14
15
13
12
15
14
15
17
23
-
16
15
15
15
15
14
14
14
16
14
14
17
14
14
16
15
16
23
28
-
18
16
16
18
17
15
16
15
20
15
16
17
16
15
16
15
19
23
28
-
16
17
17
17
14
15
17
17
17
17
17
17
17
17
17
17
-
17
18
18
18
18
18
17
18
18
17
17
18
17
17
17
18
-
20
20
20
20
20
19
20
20
19
19
19
19
19
19
19
19
-
18
18
16
18
18
18
19
18
18
18
18
18
18
18
18
18
-
18
18
19
20
19
19
18
19
19
19
19
19
19
19
19
19
-
21
22
22
23
21
21
22
20
20
20
20
21
20
20
20
20
-
4-Cl- C6H4
2-NO2- C6H4
3-NO2- C6H4
4-OCH3- C6H4
C6H5
2g
2h
3a
3b
3c
3d
3e
3f
2-OH- C6H4
4-OH- C6H4
2-Cl- C6H4
4-Cl- C6H4
2-NO2- C6H4
3-NO2- C6H4
4-OCH3- C6H4
Ampicillin
3g
3h
S1
S2
Chloramphenicol
Ciprofloxacin
Griseofulvin
-
-
-
-
-
-
S3
-
-
-
-
-
-
S4
23
25
26
21
22
23
Bold letters indicate that synthesized compounds are comparatively active as standard drugs
DMF used as control and its antibacterial activity is nil or zero.
Where Standards are S1= Ampicillin, S2= Chloramphenicol
S3= Ciprofloxacin, S4= Norfloxacin, S5= Griseofulvin
The linear growth of the fungus was obtained by measuring
the diameter of the fungal colony after 24h. After 24h
incubation at 37 ºC, the zone of inhibition was measured in
mm. The results are depicted in (Table 2).
fulvin as standard drugs. S. aurues MTCC-96 (Gram positive
bacteria) were grown in nutrient broth and B. subtillitis
MTCC-441 (Gram negative bacteria) in peptone water (PW,
1% bacteriological peptone and 0.5% NaCl) for 24h; this
gave an optimum growth of the best bacteria. Each purified
compound was dissolved in DMF sterilized using filtration
(sintered glass filter) and stored at 4ºC. Each agent was then
added to molten nutrient agar in the 0(control), 50,100,250
ꢀg/mL concentration and poured into sterile petri dish. The
pH of the media was maintained at 7.2-7.4. These were then
spot inoculated on nutrient agar plates containing increasing
amount of a compound, incubated at 37 ºC up to 24h for
determination of the minimum inhibitory concentration
(MIC). DMF was used as a positive control for the experi-
ments, but DMF did not show inhibition of microbes.
Antifungal activity was carried out using cup-plate method.
All the synthesized compounds were tested for their anti-
bacterial and antifungal activity (MIC) in vitro by broth dilu-
tion method with two bacterial strains S. aurues MTCC-96
and B. subtillitis MTCC-441 and two fungal strains A. niger
MTCC-282 and C. albicans MTCC-227 taking Ampicillin,
Chloramphenicol, Ciprofloxacin, Norfloxacin and Griseo-
fulvin as standard drugs. To ensure that the solvent had no
effect on the bacterial growth, a control was performed with
the test medium supplemented with DMF at the same dilu-
tions as used in the experiments and it was observed that
DMF had no effect on the microorganisms in the concentra-
tions studied. The results are depicted in (Table 3).

Synthesis of Novel and Highly Functionalized
Letters in Drug Design & Discovery, 2014, Vol. 11, No. 7 935
Table 3. In vitro antimicrobial screening (MIC in μg mL-1) data of 2-3a-h.
Minimal Inhibition Concentration (μg mL-1)
Antibacterial Screening
Antifungal Screening
No
R
S. aureus
E. Coli
C. albicans
A. niger
MTCC-96
MTCC-443
MTCC-227
MTCC-282
2a
2b
2c
2d
2e
2f
C6H5
2-OH- C6H4
4-OH- C6H4
2-Cl- C6H4
500
250
500
250
500
500
500
500
250
>1000
1000
62.5
250
1000
125
500
250
50
100
1000
250
250
125
500
>1000
250
100
>1000
250
62.5
250
500
62.5
1000
100
50
1000
500
500
500
1000
500
1000
500
100
1000
500
1000
100
250
500
1000
500
100
---
>1000
1000
100
4-Cl- C6H4
2-NO2- C6H4
3-NO2- C6H4
4-OCH3- C6H4
C6H5
500
2g
2h
3a
3b
3c
3d
3e
3f
500
>1000
>1000
500
2-OH- C6H4
4-OH- C6H4
2-Cl- C6H4
250
500
4-Cl- C6H4
500
2-NO2- C6H4
3-NO2- C6H4
4-OCH3- C6H4
Ampicillin
500
3g
3h
S1
1000
500
---
S2
Chloramphenicol
Ciprofloxacin
Griseofulvin
---
---
S3
50
25
---
---
S4
---
---
250
100
Bold letters indicate that synthesized compounds are comparatively active as standard drugs
DMF used as control and its antibacterial activity is nil or zero.
Where Standards are S1= Ampicillin, S2= Chloramphenicol
S3= Ciprofloxacin, S4= Norfloxacin, S5= Griseofulvin
CONCLUSION
ACKNOWLEDGEMENTS
The objective of the present study was to synthesize and
investigate the anti-microbial activity of new pyrimidine-5-
carboxylate derivatives. Results have suggested that pyrim-
idine-5-carboxylate derivatives emerge as valuable com-
pounds with great potential to be used as antibacterial agents,
and as promising candidates for further efficiency evalua-
tion. All of the compounds were found to be inactive against
fungi. Further investigations in the area of pyrimidine-5-
carboxylate derivatives incorporated with different substi-
tuents are in progress in our laboratory.
This work was funded by UGC through grant no. 41-
198/2012(SR). The authors wish to thank UGC for its
financial support. I also wish to acknowledge Professor and
head, Department of Chemistry, Saurashtra University,
Rajkot for providing research facility.
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CONFLICT OF INTEREST
The authors confirm that this article content has no con-
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Received: January 08, 2014
Revised: March 28, 2014
Accepted: March 30, 2014