Synthesis and biological evaluation of novel thio-1,4-dihydropyrimidine-5- carboxylate derivatives

Article abstract of DOI:10.14233/ajchem.2013.13093

We have synthesized ten novel isopropyl 2-(4-substituted benzylthio)-6-methyl-4-phenyl-1,4-dihydropyrimidine-5-carboxylate derivatives (5a-j). All the synthesized compounds were characterized by 1H NMR, 13C NMR, IR, MS and elemental analysis data. Newly synthesized compounds were screened for antiinflammatory activity on acetic acid induced writhing in mice and carrageenan induced paw oedema in rats. Compounds (5g) and (5b) showed a potent antiinflammatory activity (100 % at 100 mg/kg b.w) compared to reference standard drug, nimesulide (100% at 50 mg/kg b.w). The other compounds showed good antiinflammatory activity. All the synthesized compounds were also screened for antioxidant activity, among those three compounds were shown good antioxidant activity by using the in vitro method.

Full text of DOI:10.14233/ajchem.2013.13093

Asian Journal of Chemistry; Vol. 25, No. 1 (2013), 385-389
http://dx.doi.org/10.14233/ajchem.2013.13093
Synthesis and Biological Evaluation of Novel Thio-1,4-dihydropyrimidine-5-carboxylate Derivatives
1,*
2
2
1
P. LAVANYA , S. MADDILA , S.B. JONNALAGADDA and C. VENKATA RAO
1Department of Chemistry, S.V. University, Tirupati-517 502, India
2School of Chemistry, University of KwaZulu-Natal, West Ville Campus, Chilten Hills, Private Bag 54001, Durban-4000, South Africa
*Corresponding author: Fax: +91 877 2248499; E-mail: gajulapallilavanya@gmail.com
(Received: 24 November 2011;
Accepted: 28 July 2012)
AJC-11884
We have synthesized ten novel isopropyl 2-(4-substituted benzylthio)-6-methyl-4-phenyl-1,4-dihydropyrimidine-5-carboxylate derivatives
(5a-j). All the synthesized compounds were characterized by 1H NMR, 13C NMR, IR, MS and elemental analysis data. Newly synthesized
compounds were screened for antiinflammatory activity on acetic acid induced writhing in mice and carrageenan induced paw oedema in
rats. Compounds (5g) and (5b) showed a potent antiinflammatory activity (100 % at 100 mg/kg b.w) compared to reference standard
drug, nimesulide (100% at 50 mg/kg b.w). The other compounds showed good antiinflammatory activity. All the synthesized compounds
were also screened for antioxidant activity, among those three compounds were shown good antioxidant activity by using the in vitro
method.
Key Words: Thio-1,4-dihydropyrimidine-5-carboxylate, Antiinflammatory, Antioxidant activity.
and urea in alcohol using strong mineral acid was first reported
by Biginelli22. These products, popularly known as Biginelli
compounds possess several pharmaceutical properties like
antibacterial, antiviral, antiinflammatory, antihypertensive and
antitumor agents23. Based on the literature reports and recog-
nizing the prospective of substituted thiopyrimidenes as anti-
inflammatory and antimicrobial agents, in a continued quest
for new potential molecules, we designed and synthesized
novel isopropyl 2-(4-substituted benzylthio)-6-methyl-4-
phenyl-1,4-dihydropyrimidine-5-carboxylate derivatives (5a-j)
having substituted benzylthio groups. Structures of the
products were characterized by IR, 1H NMR, 13C NMR, LC-
MS and elemental analysis.
INTRODUCTION
Heterocycles are ubiquitous to among pharmaceutical
compounds1. Pyrimidine moiety is an important class of
N-containing heterocycles widely used as key building blocks
for pharmaceutical agents. These compounds exhibit a wide
spectrum of pharmacophore, as they act as bactericidal,
fungicidal2-5, analgesic6, antioxidant7, antihypertensive8,
antifilarial9 and antitumor agents10. Preclinical data from
literature indicates the continuing research in polysubstituted
pyrimidine as potential antitumor agents11-13. Among these,
thiouracils in particular are used as antiinflammatory and
virucidal agents14. The biological and synthetic significance
places this scaffold at a prestigious position in medicinal
chemistry research.
EXPERIMENTAL
The key role pyrimidines play in cellular processes has
made them valuable leads for drug discovery. One important
class of pyrimidines is 2-thiopyrimidine (2-TP) and its deri-
vatives, which are also recognized as 2-mercaptopyrimidine
compounds15. In 2-thiopyrimidine ring, sulfur atom serves as
an interesting replacement for the existing oxygen atom bonded
to C-2 in uridine base16,17. Based on this approach, 2-thiopyri-
midines have attracted significant attention of synthetic
chemists and biochemists18-20. Pathak et al.21 have reported
potential activity of 2-thiopyrimidine derivatives against
Mycobacterium tuberculosis (Mtb).
All reagents and solvents (Aldrich or Merck) were
purchased and used without further purification. Melting points
were determined on a Fisher-Johns melting point apparatus
were uncorrected. Crude products were purified by column
chromatography on silica gel of 60-120 mesh. IR spectra were
obtained on a Perkin Elmer BX serried FT-IR 5000 spectro-
meter using KBr pellet. NMR spectra were recorded on a
1
Bruker 400 MHz spectrometer for H NMR. The 13C NMR
spectra were recorded on JEOL. The chemical shifts were
reported as ppm down field using TMS as an internal standard.
LC-MS spectra were recorded on a MASPEC low resolution
mass spectrometer operating at 70 eV.
One-step synthesis of 3,4-dihydropyrimidin-2(1H)-one by
three-component condensation of aldehydes, ethyl acetoacetate

386 Lavanya et al.
Asian J. Chem.
General procedure for the synthesis of 5-isopropoxy-
Isopropyl 2-(benzylthio)-6-methyl-4-phenyl-1,4-
dihydropyrimidine-5-carboxylate (5a): Pale yellow solid;Yield
69 %; m.p. 196-198 ºC; IR (KBr, νmax, cm-1): 3313 (NH), 1709
(CO), 1625 (C-N), 1559 (C-C), 641 (C-S); 1H NMR (δ, DMSO-
d6): 1.24 (d, 6H, OCH-(CH3)2), 2.10 (s, 1H, NH), 2.35 (s, 3H,
Ar-CH3), 4.21 (s, 1H, -CH), 4.91 (m, 1H, OCH-(CH3)2), 5.10 (s,
2H, -SCH2), 7.20-7.52 (m, 10H,Ar-H); 13C NMR (δ, DMSO-d6):
170.10, 162.63, 152.96, 142.95, 136.89, 129.63, 129.34, 128.42,
128.14, 127.37, 127.15, 120.22, 66.23, 48.19, 38.25, 23.53, 21.44;
LCMS: (m/z) 381 [M+H].Anal. calcd. for C22H24N2O2S: C, 69.42;
H, 6.35; N, 7.33. Found: C, 69.58; H, 6.47; N, 7.29.
carbonyl-6-methyl-4-phenyl-3,4-dihydropyrimidin-2(1H)-
thione (4)24: To a solution of isopropyl acetoacetate (1 mmol),
aldehyde (1.1 mmol), thiourea (1.5 mmol), SrCl2·6H2O
(1 mmol, 10 mol %) and EtOH (20 mL). The mixture was
heated at 40 ºC and the progress of the reaction was monitored
by TLC. After completion of the reaction (ca. 3-5 h) the
solution was cooled to room temperature and poured into
crushed ice. The resultant solid product was collected and
purified by column chromatography (Scheme-I).
General procedure for the synthesis of isopropyl 2-(4-
substitutedbenzylthio)-6-methyl-4-phenyl-1,4-dihydro-
pyrimidine-5-carboxylate (5a-j): An ice cold solution of 5-
isopropoxycarbonyl-6-methyl-4-phenyl-3,4-dihydropyrimidin-
2(1H)-thione (1 mmol) in DMF (4 vol), potassium carbonate
(1.5 mmol) and substituted benzyl bromides (1.3 mmol) was
taken in a 1 L round bottomed flask equipped with magnetic
stirrer and stirred for 3 h. The residual portion was poured on
to crushed ice, neutralized with dilute acid and the product
obtained isopropyl 2-(4-substituted benzylthio)-6-methyl-4-
phenyl-1,4-dihydropyrimidine-5-carboxylate (5a-j). It was fil-
tered, washed with water, dried and recrystallized from ethanol
(Scheme-I).
Isopropyl 2-(4-bromobenzylthio)-6-methyl-4-phenyl-
1,4-dihydropyrimidine-5-carboxylate (5b): Yellow solid;
Yield 63 %; m.p. 215-216 ºC; IR (KBr, νmax, cm-1): 3315 (NH),
1716 (CO), 1613 (C-N), 1528 (C-C), 638 (C-S); 1H NMR (δ,
DMSO-d6): 1.30 (d, 6H, OCH-(CH3)2), 2.15 (s, 1H, NH), 2.39
(s, 3H, Ar-CH3), 4.27 (s, 1H, -CH), 4.93 (m, 1H, OCH-(CH3)2),
5.24 (s, 2H, -SCH2), 7.08-7.91 (m, 9H, Ar-H); 13C NMR (δ,
DMSO-d6): 170.59, 163.89, 152.71, 143.48, 136.76, 132.66,
132.18, 129.70, 127.63, 127.41, 120.88, 119.93, 67.66, 48.39,
39.42, 23.78, 21.53; LCMS: (m/z) 460 [M + 2H]. Anal. calcd.
for C22H23N2O2SBr: C, 57.50; H, 5.03; N, 6.13. Found: C,
57.71; H, 4.88; N, 6.21.
CHO
S
CH3
O
O
CH3
O
i
+
+
H2N
CH3
NH2
H3C
O
H3C
O
NH
4
1
2
3
H3C
N
S
H
Compound
R
Compound
R
ii
5a
5b
5c
5d
5e
4-H
4-Br
4-C2H5
4-CH3
4-OCH3
5f
5g
5h
5i
4-N(CH3)2
4-Cl
4-CH(CH3)2
4-C(CH3)3
4-C4H7
5j
CH3
O
H3C
O
N
H3C
N
H
S
5a-j
R
(i) SrCl2·6H2O, EtOH, 2 h; (ii) Substituted benzaldehydes, K2CO3, DMF, 3 h
Scheme-I: Synthetic pathway for the compounds 5a-j

Vol. 25, No. 1 (2013)
Synthesis and Biological Evaluation of Novel Thio-1,4-dihydropyrimidine-5-carboxylate Derivatives 387
Isopropyl 2-(4-ethylbenzylthio)-6-methyl-4-phenyl-
1,4-dihydropyrimidine-5-carboxylate (5c): Yellow solid;
Yield 75 %; m.p. 174-175 ºC; IR (KBr, νmax, cm-1): 3310 (NH),
1711 (CO), 1609 (C-N), 1546 (C-C), 654 (C-S); 1H NMR (δ,
DMSO-d6): 1.18 (t, 3H, -ArCH2-CH3), 1.26 (d, 6H, OCH-
(CH3)2), 2.10 (s, 1H, NH), 2.34 (s, 3H, Ar-CH3), 2.47 (q, 2H,
-ArCH2-CH3), 4.23 (s, 1H, -CH), 4.90 (m, 1H, OCH-(CH3)2),
5.15 (s, 2H, -SCH2), 6.91-7.40 (m, 9H, Ar-H); 13C NMR (δ,
DMSO-d6): 169.92, 162.53, 152.98, 142.74, 142.13, 134.12,
129.44, 129.19, 128.48, 127.50, 127.21, 120.38, 66.35, 48.22,
38.44, 27.19, 23.51, 21.47, 17.62; LCMS: (m/z) 409 [M + H].
Anal. calcd. for C24H28N2O2S: C, 70.57; H, 6.91; N, 6.86.
Found: C, 70.78; H, 7.01; N, 6.77.
DMSO-d6): 170.65, 163.58, 153.60, 143.51, 135.48, 132.13,
130.77, 129.95, 129.69, 127.71, 127.41, 120.67, 67.58, 48.40,
39.25, 23.83, 21.54; LCMS: (m/z) 415 [M + H]. Anal. calcd.
for C22H23N2O2SCl: C, 63.67; H, 5.61; N, 6.77. Found: C,
63.78; H, 5.69; N, 6.93.
Isopropyl 2-(4-isopropylbenzylthio)-6-methyl-4-
phenyl-1,4-dihydropyrimidine-5-carboxylate (5h):Yellow
solid;Yield 78 %; m.p. 185-187 ºC; IR (KBr, νmax, cm-1): 3316
1
(NH), 1714 (CO), 1618 (C-N), 1528 (C-C), 652 (C-S); H
NMR (δ, DMSO-d6): 0.98 (d, 6H, -ArCH(CH3)2), 1.26 (d, 6H,
OCH-(CH3)2), 2.12 (s, 1H, NH), 2.33 (s, 3H, Ar-CH3), 2.64
(m, 1H, -ArCH(CH3)2), 4.20 (s, 1H, -CH), 4.89 (m, 1H, OCH-
(CH3)2), 5.10 (s, 2H, -SCH2), 7.12-7.38 (m, 9H, Ar-H); 13C
NMR (δ, DMSO-d6): 169.37, 162.45, 153.08, 146.81, 142.98,
133.77, 129.12, 128.83, 127.47, 127.35, 127.14, 120.42, 66.52,
48.19, 38.83, 34.67, 24.18, 23.53, 21.42; LCMS: (m/z) 423
[M + H]. Anal. calcd. for C25H30N2O2S: C, 71.04; H, 7.18; N,
6.65. Found: C, 70.89; H, 7.23; N, 6.74.
Isopropyl 6-methyl-2-(4-methylbenzylthio)-4-phenyl-
1,4-dihydropyrimidine-5-carboxylate (5d): Pale yellow
solid;Yield 65 %; m.p. 223-224 ºC; IR (KBr, νmax, cm-1): 3312
1
(NH), 1710 (CO), 1617 (C-N), 1544 (C-C), 648 (C-S); H
NMR (δ, DMSO-d6): 1.24 (d, 6H, OCH-(CH3)2), 2.10 (s, 1H,
NH), 2.28-2.33 (s, 6H, -CH3), 4.21 (s, 1H, -CH), 4.89 (m, 1H,
OCH-(CH3)2), 5.10 (s, 2H, -SCH2), 7.08-7.35 (m, 9H, Ar-H);
13C NMR (δ, DMSO-d6): 169.73, 162.51, 152.88, 142.77,
135.67, 132.84, 129.95, 129.31, 129.23, 127.35, 127.09,
120.43, 66.43, 48.10, 38.57, 23.49, 21.85, 21.40; LCMS: (m/
z) 395 [M + H]. Anal. calcd. for C23H26N2O2S: C, 70.05; H,
6.69; N, 7.16. Found: C, 70.17; H, 6.53; N, 7.23.
Isopropyl 2-(4-tert-butylbenzylthio)-6-methyl-4-
phenyl-1,4-dihydropyrimidine-5-carboxylate (5i): Pale
yellow solid;Yield 71 %; m.p. 201-202 ºC; IR (KBr, νmax, cm-1):
3318 (NH), 1713 (CO), 1616 (C-N), 1538 (C-C), 647 (C-S);
1H NMR (δ, DMSO-d6): 1.28 (d, 6H, OCH-(CH3)2), 1.36 (s,
9H, -ArC(CH3)3), 2.12 (s, 1H, NH), 2.35 (s, 3H,Ar-CH3), 4.22
(s, 1H, -CH), 4.89 (m, 1H, OCH-(CH3)2), 5.10 (s, 2H, -SCH2),
7.12-7.36 (m, 9H, Ar-H); 13C NMR (δ, DMSO-d6): 169.94,
162.47, 152.54, 149.12, 143.28, 133.17, 129.63, 129.47,
127.55, 127.13, 123.18, 119.34, 66.79, 48.24, 38.63, 33.72,
30.65, 23.40, 21.39; LCMS: (m/z) 437 [M + H]. Anal. calcd.
for C26H32N2O2S: C, 71.51; H, 7.40; N, 6.43. Found: C, 71.57;
H, 7.49; N, 6.36.
Isopropyl 2-(4-methoxylbenzylthio)-6-methyl-4-
phenyl-1,4-dihydro-pyrimidine-5-carboxylate (5e):Yellow
solid;Yield 76 %; m.p. 170-171 ºC; IR (KBr, νmax, cm-1): 3310
1
(NH), 1706 (CO), 1606 (C-N), 1568 (C-C), 645 (C-S); H
NMR (δ, DMSO-d6): 1.30 (d, 6H, OCH-(CH3)2), 2.15 (s, 1H,
NH), 2.38 (s, 3H, Ar-CH3), 3.76 (s, 3H, -ArOCH3), 4.26 (s,
1H, -CH), 4.92 (m, 1H, OCH-(CH3)2), 5.20 (s, 2H, -SCH2),
6.84-7.36 (m, 9H, Ar-H); 13C NMR (δ, DMSO-d6): 170.51,
163.19, 153.97, 153.24, 143.58, 129.80, 129.23, 128.10,
127.91, 127.44, 120.94, 67.51, 54.11, 48.43, 39.34, 23.80,
21.69; LCMS: (m/z) 411 [M + H]. Anal. calcd. for
C23H26N2O3S: C, 67.29; H, 6.38; N, 6.82. Found: C, 67.18; H,
6.29; N, 6.93.
Isopropyl 6-methyl-4-phenyl-2-((1,2,3,4-tetrahydro-
naphthalen-1-yl)methylthio)-1,4-dihydropyrimidine-5-
carboxylate (5j): Pale yellow solid; Yield 69 %; m.p. 221-
223 ºC; IR (KBr, νmax, cm-1): 3315 (NH), 1720 (CO), 1632 (C-
N), 1555 (C-C), 650 (C-S); 1H NMR (δ, DMSO-d6): 1.28 (d,
6H, OCH-(CH3)2), 1.48-1.52 (m, 4H, -2CH2), 2.12 (s, 1H, NH),
2.36 (s, 3H, Ar-CH3), 2.78 (t, 2H, -CH2), 3.20 (m, 1H, -CH),
3.48 (d, 2H, -SCH2), 4.24 (s, 1H, -CH), 4.90 (m, 1H, OCH-
(CH3)2), 6.84-7.38 (m, 9H, Ar-H); 13C NMR (δ, DMSO-d6):
169.78, 163.10, 153.19, 143.06, 135.83, 135.17, 129.39,
128.64, 127.49, 127.21, 126.30, 124.27, 120.48, 66.74, 48.28,
39.31, 32.46, 30.29, 28.62, 23.58, 21.44, 18.73; LCMS: (m/z)
435 [M + H]. Anal. calcd. for C26H30N2O2S: C, 71.84; H, 6.73;
N, 6.58. Found: C, 71.97; H, 6.73; N, 6.58.
Isopropyl 2-(4-(dimethylamino)benzylthio)-6-methyl-
4-phenyl-1,4-dihydropyrimidine-5-carboxylate (5f):Yellow
solid;Yield 82 %; m.p. 210-212 ºC; IR (KBr, νmax, cm-1): 3314
1
(NH), 1718 (CO), 1610 (C-N), 1538 (C-C), 642 (C-S); H
NMR (δ, DMSO-d6): 1.30 (d, 6H, OCH-(CH3)2), 2.18 (s, 1H,
NH), 2.39 (s, 3H, Ar-CH3), 2.94 (s, 6H, -ArN(CH3)2), 4.28 (s,
1H, -CH), 4.92 (m, 1H, OCH-(CH3)2), 5.25 (s, 2H, -SCH2),
6.65-7.38 (m, 9H, Ar-H); 13C NMR (δ, DMSO-d6): 170.48,
163.62, 153.97, 143.63, 137.18, 129.97, 129.63, 127.80,
127.64, 125.60, 120.96, 114.43, 67.50, 48.49, 40.78, 39.51,
23.87, 21.49; LCMS: (m/z) 424 [M + H]. Anal. calcd. for
C24H29N3O2S: C, 68.05; H, 6.90; N, 9.92. Found: C, 67.94; H,
6.97; N, 9.79.
Pharmacological screening
Antiinflammatory activity: All the synthesized comp-
ounds were tested for their antiinflammatory activity using
Carrageenan induced rat hind paw oedema method of Winter
et al.8. The oedema hind paw was induced by injection of 0.1
mL of 1 % Carrageenan solution into subplanter region of
right hind paw. The volume of the paw was measured plethys-
mographically immediately and after 120 min the injection of
the irritant. The difference in volume gave the amount of
oedema developed. Percentage inhibition of the oedema
between control group and the compound treated group was
calculated and compared with the group receiving standard
drug at 50 mg/kg b.w. The results are tabulated in Table-1.
Isopropyl 2-(4-chlorobenzylthio)-6-methyl-4-phenyl-
1,4-dihydropyrimidine-5-carboxylate (5g): Yellow solid;
Yield 80 %; m.p. 252-253 ºC; IR (KBr, νmax, cm-1): 3312 (NH),
1721 (CO), 1628 (C-N), 1552 (C-C), 648 (C-S); 1H NMR (δ,
DMSO-d6): 1.30 (d, 6H, OCH-(CH3)2), 2.16 (s, 1H, NH), 2.38
(s, 3H, Ar-CH3), 4.27 (s, 1H, -CH), 4.91 (m, 1H, OCH-(CH3)2),
5.22 (s, 2H, -SCH2), 7.18-7.42 (m, 9H, Ar-H); 13C NMR (δ,

388 Lavanya et al.
Asian J. Chem.
TABLE-1
ANTIINFLAMMATORY ACTIVITY OF (5a-j) (100 mg/kg b.w) AND NIMESULIDE (50 mg/kg b.w).
Paw oedema thickness (mm)
Compound
30 m (X SE) Oedema inhibition (%) 60 m (X SE) Oedema inhibition (%) 120 m (X SE) Oedema inhibition (%)
Control
1.3 0.05
1.3 0.05
1.1 0.03
1.2 0.05
1.2 0.06
1.2 0.03
1.2 0.03
1.2 0.03
1.4 0.00
1.2 0.05
1.1 0.03
1.1 0.05
–
1.5 0.03
–
1.7 0.03
1.3 0.08**
1.1 0.03**
1.3 0.03**
1.2 0.03**
1.2 0.05**
1.4 0.03**
1.1 0.03**
1.3 0.10**
1.3 0.06**
1.3 0.05**
1.0 0.00**
–
5a
7.6
1.2 0.05**
1.1 0.00**
1.3 0.03**
1.1 0.05**
1.1 0.03**
1.1 0.06**
1.1 0.00**
1.2 0.03**
1.2 0.05**
1.2 0.03**
1.1 0.00**
20.0
26.6
13.3
26.6
26.6
26.6
26.6
20.0
20.0
20.0
26.6
23.5
41.1
23.5
29.4
29.4
17.6
41.1
23.5
23.5
23.5
41.1
5b
15.3
7.6
5c
5d
7.6
5e
7.6
5f
7.6
5g
15.3
7.6
5h
5i
5j
7.6
15.3
15.3
Nimesulide
Data represent mean values SE of six mice per group and the percent changes versus 30, 60 and 120 m post-carrageenan injection.
Data were analyzed using one-way ANOVA followed by Turkey–Krammer Multiple comparison test **p < 0.01.
Per cent oedema inhibition was calculated as regards saline control group.
**Significant difference from the control value at p < 0.01.
SE = Standard error. The active compounds are marked in bold letters.
Antioxidant screening (in vitro)
% Inhibition - [1-(Vt/Vc)] × 100
Hydrogen peroxide scavenging activity: A solution of
hydrogen peroxide (20 mM) was prepared in phosphate buffer
saline (pH 7.4). Various concentrations (12.5, 25, 50, 100 mg/
mL) of 1 mL of the test samples or standard, ascorbic acid25 in
methanol were added to 2 mL of hydrogen peroxide solution
in phosphate buffer saline. The absorbance was measured at
230 nm after 10 min26.
where Vt - mean absorption of test compound and Vc - mean
absorption of control.
The IC50 value was derived from the % inhibition at diffe-
rent concentration.
RESULTS AND DISCUSSION
The synthesis of thio-1,4-dihydropyrimidine-5-carboxylate
derivatives (5a-j) was carried out according to Scheme-I.
Benzaldehyde (1) and isopropyl acetoacetate (2) in ethanol
was refluxed with thiourea (3) using ethanol as solvent in basic
conditions to yield 5-isopropoxy-carbonyl-6-methyl-4-phenyl-
3,4-dihydropyrimidin-2(1H)-thione. It was synthesized by the
multicomponent Biginelli reaction. The Biginilli compound 4
was treated with substituted benzaldehydes in presence of
potassium carbonate and to afford the titled compounds (5a-j).
The reaction sequences are outlined in Scheme-I.
Nitric oxide scavenging activity: The reaction mixture
(6 mL) containing sodium nitroprusside (10 mM, 4 mL),
phosphate buffer saline (pH 7.4, 1 mL) and test samples or
standard, ascorbic acid solution in dimethyl sulphoxide (1 mL)
at various concentrations (12.5, 25, 50, 100 mg/mL) was
incubated at 25 ºC for 150 min. After incubation, 0.5 mL of
reaction mixture containing nitrite ion was removed, 1 mL of
sulphanilic acid reagent was added to this, mixed well and
allowed to stand for 5 min for completion of diazotization.
Then, 1 mL of naphthyl ethylene diamine dihydrochloride was
added, mixed and allowed to stand for 0.5 h in diffused light.
A pink coloured chromophore was formed. The absorbance
was measured at 640 nm27.
All the synthesized compounds were obtained in good to
high yields. Products were purified and characterized by various
spectroscopic techniques. The IR spectra of compounds
(5a-j) showed characteristic absorption bands at 3318-3310,
1721-1706, 1632-1606, 1568-1525 and 652-647 cm-1 corres-
ponding to the N-H str, ester group of C-O str, C-N str, C-C str
and C-S str functions in the structures. Similarly the 1H NMR
spectra showed peaks due to in the range of δ 1.24-1.30 for
OCH-(CH3)2, δ 2.10-2.18 for NH, δ 2.28-2.39 for Ar-CH3, δ
4.20-4.28 for -CH, δ 4.89-4.92 for OCH-(CH3)2, δ 3.48-5.25
for -SCH2 and δ 6.65-7.91 forAr-H. The mass spectrum of all
the compounds showed molecular ion peak at M + H, at M +
2H corresponding to its molecular formula, which confirmed
its chemical structure. The IR, 1H NMR, 13C NMR, mass spectra
and elemental analysis showed the structure of various thio-
1,4-dihydropyrimidine-5-carboxylate derivatives (5a-j).
Antiinflammatory screening: The results of tested
compounds as well as reference standard were measured
before administration of Carrageenan inflammation.After the
Carrageenan inflammation was administered on rats, the effect
was measured in the intervals of 30, 60 and 120 min. The
Lipid peroxidation inhibitory activity: Egg lecithin
(3 mg/mL phosphate buffer, pH 7.4) was sonicated in an
ultrasonic sonicator for 10 min to ensure proper liposome
formation. Test samples or standard, ascorbic acid (100 mL)
of different concentrations (12.5, 25, 50, 100 mg/mL) were
added to liposome mixture (1 mL); the control was without
test sample. Lipid peroxidation was induced by adding ferric
chloride (10 mL, 400 mM) and L-ascorbic acid (10 mL, 200
mM).After incubation for 1 h at 37 ºC the reaction was stopped
by adding hydrochloric acid (2 mL, 0.25 N) containing trichlo-
roacetic acid (150 mg/mL), thiobarbituric acid (3.75 mg/mL)
and butylated hydroxy anisole (0.50 mg/mL). The reaction
mixture was subsequently boiled for 15 min, cooled, centri-
fuged at 1000 rpm for 15 min and the absorbance of the
supernatant was measured at 532 nm28. For all the above anti-
oxidant methods, experiments were done in triplicate and
average is taken, the % inhibition at different concentration
was calculated by the following formula:

Vol. 25, No. 1 (2013)
Synthesis and Biological Evaluation of Novel Thio-1,4-dihydropyrimidine-5-carboxylate Derivatives 389
per cent oedema inhibition was calculated reference to saline
control group, as depicted in Table-1. All the newly obtained
compounds (5a-j) were tested for antiinflammatory activity.
Compared to the standard, Nimesulide, bulk of the compounds
exhibited moderate to good anti-inflammatory activity. The
results revealed that, while 5b and 5g have shown potent anti-
inflammatory activity, compounds 5a, 5c, 5d, 5e, 5f, 5h, 5i
and 5j exhibited good antiinflammatory activities.
laboratory emerge as a valuable lead series with great potential
to be used as antiinflammatory and antioxidant activity agents
and as promising candidates for further efficacy evaluation.
ACKNOWLEDGEMENTS
The authors are thankful to the authorities of the School of
Chemistry, University of KwaZulu-Natal, Westville campus,
Durban, South Africa for the facilities and encouragement.
Antioxidant activity: All the synthesized compounds 5a-j
were screened for their in vitro antioxidant activity by various
methods such as scavenging of hydrogen peroxide, scavenging
of nitric oxide radical and lipid peroxidation inhibitory activity.
In vitro antioxidant activity of synthesized compound is
summarized in Table-2. The investigation of antioxidant
screening revealed that some of the tested compounds showed
moderate to good antioxidant activity. Particularly, benzylthiol
derivatives (5a-j) showed more promising antioxidant activity
as compared to that of standard, ascorbic acid. This could be
due the availability of free thiol group. In scavenging of nitric
oxide radical techniques compounds 5j and 5i shown low IC50
value than the standard. While, 5j, 5i and 5a has shown more
potent activity by scavenging of hydrogen peroxide. All the
compounds showed higher IC50 value than the standard by
lipid peroxidation inhibitory activity. Derivatives with aliphatic
group on benzene ring having good antioxidant activity
compared with the other compounds in their series. 5h, 5a,
5f, 5b, 5g and 5d having moderate to good antioxidant activity.
REFERENCES
1. T. Eicher and S. Hauptmann, The Chemistry of Heterocycles, Wiley-VCH,
Weinheim, edn. 2 (2003).
2. N.G. Pershin, L.I. Sherbakova, T.N. Zykova and V.N. Sakolova, World
Rev. Pest. Contr., 35, 466 (1972).
3. M. Suresh, P. Lavanya, K.N. Raju, S.B. Jonnalagadda and C.V. Rao,
Org. Commun., 4, 33 (2011).
4. S. Maddila and S.B. Jonnalagadda, Arch. Pharm, 345, 163 (2011).
5. S. Maddila and S.B. Jonnalagadda, Bull. Chem. Soc. Ethiopia, 26, 121
(2012).
6. G. Regnier, L. Canevar, R.J. Le, J.C. Douarec, S. Halstop and J. Daussy,
J. Med. Chem., 15, 295 (1972).
7. H.A. Stefani, C.B. Oliveira, R.B. Almeida, C.M.P. Pereira, R.C. Braga,
R. Cella, V.C. Borges, L. Savegnago and C.W. Nogueira, Eur. J. Med.
Chem., 41, 513 ( 2006).
8. C.A. Winter, E.A.R. Fisley and G.W. Nuss, Proc. Soc. Exp. Biol. Med.,
111, 544 (1962).
9. B.K. Singh, M. Mishra, N. Saxena, G.P. Yadav, P.R. Maulik, M.K.
Sahoo, R.L. Gaur, P.K. Murthy and R.P. Tripathi, Eur. J. Med. Chem.,
43, 2717 (2008).
10. K. Suguira, A.F. Schmid, M.M. Schmid and F.G. Brown, Cancer
Chemother. Rep. Part., 23, 231 (1973).
11. E. Maquoi, N.E. Sounni, L. Devy, F. Olivier, F. Frankenne, H.-W. Krell,
F. Grams, J.-M. Foidart andA. Noel, Clin. Cancer Res., 10, 4038 (2004).
12. M. Huang, Y. Wang, M. Collins, B.S. Mitchell and L.M. Graves, Mol.
Pharmacol., 62, 463 (2002).
TABLE-2
ANTIOXIDANT ACTIVITY (IC50 VALUES)
OF COMPOUNDS (5a–j)
13. N.Von Bubnoff, R. Darren,W.Veach, T. Miller,W. Li, J. Sanger, C. Peschel,
W.G. Bornmann, B. Clarkson and J. Duyster, Cancer Res., 63, 6395
(2003).
IC50 (Mean SD) µg/mL
Scavenging of Scavenging of Lipid peroxidation
Compounds
NO radical
56 0.052
60 0.066
65 0.453
63 0.183
69 0.318
59 0.453
62 0.045
54 0.024
46 0.333
44 0.279
47 0.087
H2O2
inhibitory activity
39 0.066
43 0.318
78 0.045
68 0.087
63 0.162
46 0.453
57 0.279
37 0.318
34 0.087
31 0.121
26 0.333
14. M.M. Mojtahedi, M.R. Saidi, J.S. Shirzi and M. Bolourtchian, Syn.
Commun., 32, 851 (2002).
5a
5b
32 0.318
49 0.121
58 0.318
53 0.066
66 0.162
41 0.087
46 0.024
36 0.121
30 0.183
29 0.087
33 0.121
15. S.M. Sondhi, R.N. Goyal, A.M. Lahoti, N. Singh, R. Shukla and R.
Raghubi, Bioorg. Med. Chem., 13, 3185 (2005).
5c
5d
16. H. Sierzputowska-Gracz, E. Sochacka, A. Malkiewicz, K. Kuo, C.
Gehrke and P.F. Agris, J. Am. Chem. Soc., 109, 7171 (1987).
17. E. Sochacka and I. Fratczak, Tetrahedron Lett., 45, 6729 (2004).
18. S. Stoyanov, I. Petkov, L. Antonov, T. Stoyanova, P. Karagiannides
and P. Aslanidis, Can. J. Chem., 68, 1482 (1990).
5e
5f
5g
5h
19. J.C. Hazelton, B. Iddon, H. Suschitzky and L.H. Woolley, J. Chem.
Soc. Perkin Trans., 685 (1992).
5i
5j
20. Z.G. Hajos and R.M. Kanojia, Eur. Pat. Appl. EP 458459 A2 (1991);
Chem. Abstr., 116, 83701 (1991).
Standard
S.D. = Standard deviation (average of three determination); Standard =
Ascorbic acid.
21. A.K. Pathak, V. Pathak, V.E. Seit, W.J. Sulng and R.C.J. Reynolds, J.
Med. Chem., 47, 273 (2004).
22. P. Biginelli, Gazz. Chim. Ital., 23, 360 (1893).
23. B. Schnell, W. Krenn, K. Faber and C.O. Kappe, J. Chem. Soc. Perkin
Trans., 4382 (2000).
Conclusion
In conclusion, we have described simple and efficient
protocol for the synthesis of novel thio-1,4-dihydropyrimidine-
5-carboxylate derivatives (5a-j) with good yields. All the
synthesized compounds have been investigated for their anti-
inflammatory, antibacterial and antifungal activities. With our
newly synthesized compounds, it is evident that 5b and 5g
have highest antiinflammatory activity; 5a, 5i and 5j have anti-
oxidant activity. Accordingly, these novel class of thio-1,4-
dihydropyrimidine-5-carboxylate derivatives reported from our
24. S. Chitra, D. Devanathan and K. Pandiarajan, Eur. J. Med. Chem., 45,
367 (2010).
25. L. Ismaili, A. Nadaradjane, L. Nicod, C. Guyon, A. Xicluna, J. Robert
and B. Refouvelet, Eur. J. Med. Chem., 43, 1270 (2008).
26. G.K. Jayaprakasha, R.L. Jaganmohan and K.K. Sakariah, Bioorg. Med.
Chem., 12, 5141 (2004).
27. L. Marcocci, L. Packer, M.T. Droy-lefaix,A. Sekaki and M. Gondes-Albert,
Methods Enzymol., 234, 462 (1994).
28. P.D. Duh and G.H. Yen, Food. Chem., 60, 639 (1997).