Synthesis and pharmacological evaluation of newer thiazolo [3,2-a] pyrimidines for anti-inflammatory and antinociceptive activity

Article abstract of DOI:10.1007/s00044-009-9267-8

A new series of thiazolo [3,2-a] pyrimidine derivatives was designed and synthesized using 4-fluoroaniline and ethylacetoacetate as starting material. Anti-inflammatory activity was assessed by the rat paw edema method and antinociceptive activity was evaluated by thermal stimulus technique. The compounds 5-(4-chlorophenyl)-2-(4-fluorobenzylidene)-7-methyl-3-oxo-2,3-dihydro- 5H-thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3l) and 2-(4-chlorobenzylidene)-5-(4-fluorophenyl)-7-methyl-3-oxo-2,3-dihydro-5H- thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3q) were found to possess significant anti-inflammatory and antinociceptive activities. These compounds also showed lower ulcerogenic activity and higher ALD ₅₀ values. Compounds with an aryl ring substituted with a smaller electron withdrawing group at the fourth position displayed better activity than the other derivatives.

Full text of DOI:10.1007/s00044-009-9267-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:1245–1258
DOI 10.1007/s00044-009-9267-8
ORIGINAL RESEARCH
Synthesis and pharmacological evaluation of newer
thiazolo [3,2-a] pyrimidines for anti-inflammatory
and antinociceptive activity
•
•
•
Ozair Alam Suroor A. Khan Nadeem Siddiqui
Waquar Ahsan
Received: 27 April 2009 / Accepted: 15 September 2009 / Published online: 14 October 2009
¨
Ó Birkhauser Boston 2009
Abstract A new series of thiazolo [3,2-a] pyrimidine derivatives was designed
and synthesized using 4-fluoroaniline and ethylacetoacetate as starting material.
Anti-inflammatory activity was assessed by the rat paw edema method and antin-
ociceptive activity was evaluated by thermal stimulus technique. The compounds
5-(4-chlorophenyl)-2-(4-fluorobenzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo
[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3l) and 2-(4-chlo-
robenzylidene)-5-(4-fluorophenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]
pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3q) were found to possess
significant anti-inflammatory and antinociceptive activities. These compounds also
showed lower ulcerogenic activity and higher ALD50 values. Compounds with an
aryl ring substituted with a smaller electron withdrawing group at the fourth position
displayed better activity than the other derivatives.
Keywords Thiazolopyrimidine ꢀ Anti-inflammatory ꢀ Antinociceptive ꢀ
Acute toxicity
Introduction
Acute and chronic inflammation and different types of arthritis are inflammatory
disorders that deal a big blow to humanity, and continual search for newer
nonsteroidal anti-inflammatory agents is the only way to protect against this threat.
In the past few decades the literature has been enriched with newer agents that are
structurally different from the presently known anti-inflammatory drugs, but the
O. Alam (&) ꢀ S. A. Khan ꢀ N. Siddiqui ꢀ W. Ahsan
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University,
Hamdard Nagar, New Delhi 110062, India
e-mail: oalam@jamiahamdard.ac.in

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search for better candidates in terms of higher efficacy and selectivity with lesser
side effects continues to be an area of research for scientists worldwide.
A number of heterocyclic moieties are reported to possess anti-inflammatory
activity, among which pyrimidine derivatives constitute an important class that
displays a number of diverse biological activities including anti-inflammatory and
antinociceptive activity (Kowaluk et al., 2000; Orjales et al., 2008; Alagarsamy
et al., 2007; Falca˜o et al., 2006; Jakubkien et al., 2003; Bruno et al., 2002; Jarvis
et al., 2002). In addition, thiazole derivatives have acquired conspicuous signif-
icance due to their use in inflammation (Bender et al., 1985; Holla et al., 2003).
Since the two heterocyclic moieties constitute two active pharmacophores that
are highly active against inflammation and pain, combining the two is expected to
have a synergistic effect in dealing with the diseases. This idea has been utilized by
some scientists to prepare thiazolopyrimidine derivatives, and their anti-inflamma-
tory activity has been reported (Bekhit et al., 2003; Tozkoparan et al., 1999).
Thiazolopyrimidine derivatives are the biosteric analogues of purines and are
potentially bioactive molecules that have shown anti-inflammatory activity
comparable to that of some standard drugs in vivo, with no or minimal ulcerogenic
effects (Bekhit et al., 2003).
In continuing the search for better anti-inflammatory agents of the thiazolopyrim-
idine class that can also be used as analgesics and that have minimal side effects, we
report here the synthesis of 5-(4-substituted phenyl)-2-(substituted benzylidene)-7-
methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluo-
rophenyl)amides (3a–3t). All the synthesized compounds were tested for anti-
inflammatory and antinociceptive activities. Ulcerogenic activity and acute toxicity
studies of the most active compounds were also performed.
Materials and methods
Chemistry
Melting points were determined in open capillary tubes and are uncorrected. IR
spectra were recorded in KBr pellets on a FT-IR (Bio-Rad FTS) spectrophotometer.
Elemental analyses (C, H, N) of all compounds were performed on the CHNS
Elimentar (Vario EL III; Analysen systime, GmbH, Germany). Proton magnetic
resonance spectra (1H-NMR) were recorded on a DRX-300 NMR spectrometer and
Bruker 400 Ultra Shield, and chemical shifts (d) are expressed as parts per million
(ppm) using TMS (tetramethylsilane; Me4Si) as an internal reference. All TLC was
carried out using silica gel.
General procedure for synthesis of the title compounds (3a–3t)
N-(4-Fluoro-phenyl)-3-oxo-butyramide (1)
A mixture of ethyl acetatoacetate (0.01 mol) and 4-fluoroaniline (0.01 mol) in
20 ml of ethanol containing 0.3 g of sodium hydroxide was refluxed for 5 h. The

Med Chem Res (2010) 19:1245–1258
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reaction mixture was concentrated and the solid product obtained was filtered off
and recrystallized with ethyl acetate. Yield, 76%; m.p., 136°C. IR (KBr, cm-1):
1
3315, 3070, 1672, 1329. H NMR (DMSO-d6): d 2.15 (s, 3H, CH3), 3.78 (s, 2H,
CH2), 7.12–7.83 (m, 4H, ArH), 8.52 (bs, 1H, NH, D2O exchangeable).
6-Methyl-4-(substituted phenyl)-2-thioxo-1,2,3,4-tetrahydro-pyrimidine-5-
carboxylic acid (4-fluorophenyl)amides (2a–2e)
A mixture of N-(4-fluoro-phenyl)-3-oxo-butyramide (1), substituted aryl aldehydes
(0.01 mol), and thiourea (0.015 mol) in 20 ml of ethanol was refluxed for 7–9 h in
the presence of a catalytic amount of concentrated hydrochloric acid. The reaction
mixture was kept overnight and the precipitate obtained was filtered and
recrystallized with ethanol. Yield, 63%; m.p., 179°C. IR (KBr, cm-1): 3416,
1
3358, 3340, 3019, 1658, 1304, 1092. H NMR (DMSO-d6): d 2.21 (s, 3H, CH3),
5.11 (s, 1H, CH), 7.09–7.73 (m, 8H, ArH), 9.20 (bs, 1H, NH, D2O exchangeable),
9.65 (bs, 1H, NH, D2O exchangeable), 9.86 (bs, 1H, NH, D2O exchangeable).
5-(4-Substituted phenyl)-2-(substituted benzylidene)-7-methyl-3-oxo-2,3-dihydro-
5H-thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amides (3a–3t)
A mixture of compounds 2a–2e (0.01 mol), chloroacetic acid (0.01 mol), substi-
tuted aryl aldehydes (0.01 mol), and sodium acetate (1.5 g) in a mixture of acetic
acid and acetic anhydride (25 ml; 1:1) was refluxed for 8–10 h. The reaction
mixture was concentrated and the solid thus obtained was filtered and recrystallized
with ethyl acetate to get the titled compounds.
5-(4-Bromophenyl)-2-(3,4-dimethoxybenzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3a)
Yield, 56%. %CHN found (calc): C, 57.20 (57.24); H, 3.80 (3.81); H, 7.07 (6.91).
IR (KBr, cm-1): 3458, 3034, 2930, 1713, 1658, 1304. 1H NMR (DMSO-d6): d 2.46
(s, 3H, CH3), 3.95 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 5.44 (s, 1H, CH-pyr.), 6.58–
6.84 (m, 7H, ArH), 6.92–7.21 (m, 4H, ArH), 7.68 (s, 1H, CH), 9.36 (bs, 1H, NH,
D2O exchangeable).
5-(4-Bromophenyl)-2-(3,4-dichlorobenzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3b)
Yield, 61%. %CHN found (calc): C,52.52 (52.53); H, 2.75 (2.78); N, 6.79 (6.81). IR
1
(KBr, cm-1): 3404, 3015, 2865, 1704, 1646, 1328, 804. H NMR (DMSO-d6): d
2.38 (s, 3H, CH3), 5.49 (s, 1H, CH-pyr.), 6.66–6.98 (m, 7H, ArH), 7.04–7.34 (m,
4H, ArH), 7.71 (s, 1H, CH), 9.41 (bs, 1H, NH, D2O exchangeable).

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2-Benzylidene-5-(4-bromophenyl)-7-methyl-3-oxo-2,3-dihydro-5H-thiazolo[3,2-
a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3c)
Yield, 59%. %CHN found (calc): C, 59.03 (59.13); H, 3.42 (3.49); N, 7.61 (7.66).
IR (KBr, cm-1): 3416, 2958, 2715, 1714, 1679, 1315, 545. 1H NMR (DMSO-d6): d
2.46 (s, 3H, CH3), 5.51 (s, 1H, CH-pyr.), 6.49–7.32 (m, 9H, ArH), 7.48–7.67 (m,
4H, ArH), 7.84 (s, 1H, CH), 9.64 (bs, 1H, NH, D2O exchangeable).
5-(4-Bromophenyl)-2-(4-chlorobenzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3d)
Yield, 47%. %CHN found (calc): C, 55.61 (55.64); H, 3.15 (3.11); N, 7.27 (7.21).
1
IR (KBr, cm-1): 3455, 2916, 2812, 1694, 1663, 1318, 818, 545. H NMR (DMSO-
d6): d 2.33 (s, 3H, CH3), 5.45 (s, 1H, CH-pyr.), 6.52–7.16 (m, 8H, ArH), 7.39–7.59
(m, 4H, ArH), 7.78 (s, 1H, CH), 9.59 (bs, 1H, NH, D2O exchangeable).
5-(4-Bromophenyl)-7-methyl-2-(4-methylbenzylidene)-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3e)
Yield, 51%. %CHN found. Calc: C, 59.76 (59.79); H, 3.72 (3.76); N, 7.41 (7.47). IR
1
(KBr, cm-1): 3438, 3078, 2768, 1716, 1689, 1352, 598. H NMR (DMSO-d6): d
2.41 (s, 3H, CH3), 2.62 (s, 3H, CH3), 5.48 (s, 1H, CH-pyr.), 6.41–7.28 (m, 8H,
ArH), 7.41–7.63 (m, 4H, ArH), 7.69 (s, 1H, CH), 9.46 (bs, 1H, NH, D2O
exchangeable).
2-(2,4-Dichlorobenzylidene)-7-methyl-5-(4-nitrophenyl)-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3f)
Yield, 59%. %CHN found (calc): C, 55.59 (55.58); H, 2.93 (2.94); N, 9.61 (9.60).
IR (KBr, cm-1): 3418, 3006, 2732, 1684, 1646, 1356, 1338, 813. 1H NMR (DMSO-
d6): d 2.44 (s, 3H, CH3), 5.46 (s, 1H, CH-pyr.), 6.42–7.31 (m, 7H, ArH), 7.51–7.84
(m, 4H, ArH), 7.89 (s, 1H, CH), 9.64 (bs, 1H, NH, D2O exchangeable).
2-(4-Chlorobenzylidene)-7-methyl-5-(4-nitrophenyl)-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3g)
Yield, 61%. %CHN found (calc): C, 59.10 (59.07); H, 3.34 (3.30); N, 10.17 (10.21).
IR (KBr, cm-1): 3464, 2956, 2768, 1724, 1664, 1378, 834.; 1H NMR (DMSO-d6): d
2.42 (s, 3H, CH3), 5.51 (s, 1H, CH-pyr.), 6.47–7.04 (m, 8H, ArH), 7.29–7.48 (m,
4H, ArH), 7.67 (s, 1H, CH), 9.67 (bs, 1H, NH, D2O exchangeable).
2-(4-Methoxybenzylidene)-7-methyl-5-(4-nitrophenyl)-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3h)
Yield, 58%. %CHN found (calc): C, 61.71 (61.76); H, 3.91 (3.89); N, 10.26 (10.29).
IR (KBr, cm-1): 3458, 3016, 2805, 1716, 1654, 1356. 1H NMR (DMSO-d6): d 2.47

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(s, 3H, CH3), 3.23 (s, 3H, OCH3), 5.48 (s, 1H, CH-pyr.), 6.39–7.16 (m, 8H, ArH),
7.31–7.51 (m, 4H, ArH), 7.64 (s, 1H, CH), 9.58 (bs, 1H, NH, D2O exchangeable).
2-(2-Chlorobenzylidene)-7-methyl-5-(4-nitrophenyl)-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3i)
Yield, 68%. %CHN found (calc): C, 59.01 (59.07); H, 3.32 (3.30); N, 10.23 (10.21).
IR (KBr, cm-1): 3426, 3019, 2889, 1741, 1651, 1245, 854. 1H NMR (DMSO-d6): d
2.51 (s, 3H, CH3), 5.59 (s, 1H, CH-pyr.), 6.41–7.04 (m, 8H, ArH), 7.42–7.64 (m,
4H, ArH), 7.71 (s, 1H, CH), 9.61 (bs, 1H, NH, D2O exchangeable).
2-Benzylidene-7-methyl-5-(4-nitrophenyl)-3-oxo-2,3-dihydro-5H-thiazolo[3,2-
a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3j)
Yield, 56%. %CHN found (calc): C, 62.95 (63.03); H, 3.74 (3.72); N, 10.85 (10.89).
IR (KBr, cm-1): 3415, 3056, 2858, 1721, 1668, 1349. 1H NMR (DMSO-d6): d 2.49
(s, 3H, CH3), 5.63 (s, 1H, CH-pyr.), 6.37–7.46 (m, 9H, ArH), 7.78–8.14 (m, 4H,
ArH), 8.23 (s, 1H, CH), 9.78 (bs, 1H, NH, D2O exchangeable).
2-(4-Bromobenzylidene)-5-(4-chlorophenyl)-7-methyl-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3k)
Yield, 61%. %CHN found (calc): C, 55.67 (55.64); H, 3.16 (3.11); N, 7.19 (7.21).
1
IR (KBr, cm-1): 3415, 3047, 2804, 1707, 1678, 1317, 854, 563. H NMR (DMSO-
d6): d 2.41 (s, 3H, CH3), 5.61 (s, 1H, CH-pyr.), 6.56–7.26 (m, 8H, ArH), 7.41–7.61
(m, 4H, ArH), 7.84 (s, 1H, CH), 9.74 (bs, 1H, NH, D2O exchangeable).
5-(4-Chlorophenyl)-2-(4-fluorobenzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3l)
Yield, 43%. %CHN found (calc): C, 62.14 (62.13); H, 3.49 (3.48); N, 8.01 (8.05).
IR (KBr, cm-1): 3398, 3041, 2848, 1709, 1678, 1304, 1294, 849. 1H NMR (DMSO-
d6): d 2.37 (s, 3H, CH3), 5.54 (s, 1H, CH-pyr.), 6.37–7.21 (m, 8H, ArH), 7.51–7.70
(m, 4H, ArH), 7.84 (s, 1H, CH), 9.78 (bs, 1H, NH, D2O exchangeable).
2-(2-Chlorobenzylidene)-5-(4-chlorophenyl)-7-methyl-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3m)
Yield, 59%. %CHN found (calc): C, 60.21 (60.23); H, 3.39 (3.37); N,: 7.79 (7.80).
IR (KBr, cm-1): 3314, 3009, 2856, 1745, 1681, 1325, 849, 816.; 1H NMR (DMSO-
d6): d 2.41 (s, 3H, CH3), 5.51 (s, 1H, CH-pyr.), 6.28–7.31 (m, 8H, ArH), 7.47–7.69
(m, 4H, ArH), 7.91 (s, 1H, CH), 9.75 (bs, 1H, NH, D2O exchangeable).

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5-(4-Chlorophenyl)-2-(2,4-dichlorobenzylidene)-7-methyl-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3n)
Yield, 64%. %CHN found (cal): C, 56.64 (56.61); H, 2.96 (2.99); N, 7.37 (7.34). IR
(KBr, cm-1): 3334, 3037, 2816, 1731, 1694, 1341, 849, 831, 828. 1H NMR (DMSO-
d6): d 2.45 (s, 3H, CH3), 5.51 (s, 1H, CH-pyr.), 6.34-7.24 (m, 7H, ArH), 7.48-7.64
(m, 4H, ArH), 7.91 (s, 1H, CH), 9.46 (bs, 1H, NH, D2O exchangeable).
5-(4-Chlorophenyl)-7-methyl-2-(2-nitrobenzylidene)-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluoro-phenyl)-amide (3o)
Yield, 51%. %CHN found (calc): C, 58.99 (59.07); H, 3.32 (3.30); N, 10.23 (10.21).
IR (KBr, cm-1): 3318, 3015, 2876, 1745, 1658, 1349, 1315, 849.; 1H NMR
(DMSO-d6): d 2.46 (s, 3H, CH3), 5.61 (s, 1H, CH-pyr.), 6.37–7.30 (m, 8H, ArH),
7.44–7.68 (m, 4H, ArH), 7.79 (s, 1H, CH), 9.64 (bs, 1H, NH, D2O exchangeable).
5-(4-Fluorophenyl)-7-methyl-2-(4-nitrobenzylidene)-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3p)
Yield, 56%. %CHN found (calc): C, 60.93 (60.90); H, 3.46 (3.41); N, 10.54 (10.52).
IR (KBr, cm-1): 3345, 3038, 2887, 1741, 1689, 1338, 1298, 1264. 1H NMR
(DMSO-d6): d 2.43 (s, 3H, CH3), 5.59 (s, 1H, CH-pyr.), 6.41–7.09 (m, 8H, ArH),
7.35–7.61 (m, 4H, ArH), 7.78 (s, 1H, CH), 9.56 (bs, 1H, NH, D2O exchangeable).
2-(4-Chlorobenzylidene)-5-(4-fluorophenyl)-7-methyl-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3q)
Yield, 67%. %CHN found (calc): C, 62.19 (62.13); H, 3.46 (3.48); N, 8.01 (8.05).
IR (KBr, cm-1): 3364, 3015, 2879, 1745, 1667, 1264, 1254, 851. 1H NMR (DMSO-
d6): d 2.54 (s, 3H, CH3), 5.51 (s, 1H, CH-pyr.), 6.31–7.04 (m, 8H, ArH), 7.34–7.59
(m, 4H, ArH), 7.97 (s, 1H, CH), 9.64 (bs, 1H, NH, D2O exchangeable).
2-(2-Chlorobenzylidene)-7-methyl-5-(2-nitrophenyl)-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3r)
Yield, 46%. %CHN found (calc): C, 59.10 (59.07); H, 3.31 (3.30); N, 10.25 (10.21).
IR (KBr, cm-1): 3374, 3019, 2816, 1716, 1645, 1347, 1284, 897. 1H NMR (DMSO-
d6): d 2.45 (s, 3H, CH3), 5.61 (s, 1H, CH-pyr.), 6.24–7.11 (m, 8H, ArH), 7.48–7.67
(m, 4H, ArH), 7.67 (s, 1H, CH), 9.69 (bs, 1H, NH, D2O exchangeable).
2-(4-Bromobenzylidene)-7-methyl-5-(2-nitrophenyl)-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3s)
Yield, 49%. %CHN found (calc): C, 54.70 (54.65); H, 3.09 (3.06); N, 9.41
1
(9.44). IR (KBr, cm-1): 3384, 3037, 2816, 1715, 1656, 1345, 1284, 549. H NMR

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(DMSO-d6): d 2.46 (s, 3H, CH3), 5.51 (s, 1H, CH-pyr.), 6.43–7.16 (m, 8H, ArH),
7.47–7.81 (m, 4H, ArH), 7.91 (s, 1H, CH), 9.64 (bs, 1H, NH, D2O exchangeable).
2-(2-Chlorobenzylidene)-5-(4-fluorophenyl)-7-methyl-3-oxo-2,3-dihydro-5H-
thiazolo[3,2-a]pyrimidine-6-carboxylic acid (4-fluorophenyl)amide (3t)
Yield, 51%. %CHN found (calc): C, 62.07 (62.13); H, 3.51 (3.48); N, 8.06 (8.05).
IR (KBr, cm-1): 3304, 3009, 2856, 1718, 1659, 1289, 1274, 864. 1H NMR (DMSO-
d6): d 2.51 (s, 3H, CH3), 5.51 (s, 1H, CH-pyr.), 6.41–7.11 (m, 8H, ArH), 7.47–7.75
(m, 4H, ArH), 7.98 (s, 1H, CH), 9.61 (bs, 1H, NH, D2O exchangeable).
Pharmacology
Anti-inflammatory activity
The investigations were conducted on albino rats of either gender (75–125 g).
Animals were kept under standard laboratory conditions at an ambient temperature
of 25 2°C and allowed free access to food and water except at the time they were
brought out of the cage. All experimental protocols were carried out with
permission from the Institutional Animal Ethics Committee (IAEC), form no. 502.
Animals were obtained from the Central Animal House Facility, Hamdard
University, New Delhi-62. Registration number is 173/CPCSEA; registration date,
28 January 2000.
Preliminary anti-inflammatory activities of the synthesized compounds were
evaluated using the Winter et al., (1962) method described previously. It was
performed at three graded doses (25, 50, and 100 mg/kg) and compared with the
standard drug phenyl butazone. Animals were divided into groups (control, test
compounds, and standard) of six animals each. A freshly prepared suspension of
carrageenin (1.0% in 0.9% saline), 0.05 ml, was injected under the plantar
aponeurosis of the rats’ right paw. One group was kept as controls and the animals
in other groups were pretreated with the test drugs suspended in a methyl cellulose/
water (0.5%) mixture, given orally 1 h before the carrageenin injection. The volume
of the foot was measured before and 1 and 3 h after carrageenin treatment by
mercury displacement in a plethysmograph. The mean increase in paw volume in
each group was measured and the percentage anti-inflammatory activity was
calculated according to the formula
Percentage anti ꢁ inflammatory activity ¼ ½1 ꢁ ðVt=VcÞꢂ ꢃ 100
where Vt and Vc are the volumes of edema in the drug-treated and control groups.
Phenyl butazone was used as the standard drug for comparison.
Antinociceptive activity
Antinociceptive activity was assessed using the thermal stimulus technique (Kendall
et al., 1982). Investigations were performed on albino mice in groups of six each,
which were kept under standard laboratory conditions. Test compounds were

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suspended in a methyl cellulose/water (0.5%) mixture. Each compound was
administered orally at a dose of 20 mg/kg. Antinociceptive activity was assessed 4 h
after administration. The tail of each mouse was gently immersed in thermostat-
ically controlled water at 55°C. The parameter measured in test samples was time
that elapsed between immersion and the attempt to withdraw the tail from hot water
for control as well as treated animals. Diclofenac (20 mg/kg) was used as the
standard drug for comparison.
Ulcerogenic activity
Ulcerogenic activity was measured using Djahanguiri’s (1969) method. Adult
albino rats of either gender were divided into groups of ten animals each. Animals
were kept under standard laboratory conditions and fasted 24 h prior to adminis-
tration of drugs. Water was allowed ad libitum. The most active compounds and the
standard drug (phenyl butazone) were given intraperitoneally and the animals
sacrificed 8 h after drug treatment. The stomach, duodenum, and jejunum were
removed and examined with a hand lens for any evidence of shedding of epithelium,
red spots below the skin, bleeding, and erosion or discrete ulceration with or without
perforation. The presence of any one of these criteria was considered to be evidence
of ulcerogenic activity.
Acute toxicity study
The approximate 50% lethal dose (ALD50) of the promising compounds was
determined in albino mice. Animals of either gender weighing between 20 and 25 g
were chosen for the study. Drugs were injected intraperitoneally (i.p.) at different
dose levels in separate groups of animals. After 24 h of drug administration,
percentage mortality in each group was observed. From the data obtained, ALD50
was calculated by the method of Smith (1960).
Statistical analyses
All statistical analyses of the data were performed using SigmaStat version 7.0 and
ANOVA followed by Dunnett’s multiple-comparison test.
Results and discussion
Chemistry
Synthesis of the title compounds (3a–3t) was carried out according to Scheme 1.
Ethylacetoacetate was refluxed with 4-fluoroaniline in ethanol in the presence of
sodium hydroxide to yield oxobutyramide (1). It was then condensed with thiourea
and substituted benzaldehydes in the presence of concentrated hydrochloric acid to
get pyrimidine carboxamides (2a–2e). In the final step, pyrimidine carboxamides
were treated with chloroacetic acid and substituted benzaldehydes in a mixture of

Med Chem Res (2010) 19:1245–1258
1253
Scheme 1 Reagents and
conditions: (i) NaOH, ethanol,
reflux, 5 h; (ii) thiourea,
aromatic aldehydes; (iii)
aromatic aldehydes, chloroacetic
acid, acetic anhydride, acetic
acid
O
O
CH2CH3
NH2
F
+
H3C
O
(i)
F
NH
O
CH3
(1)
O
(ii)
R
O
NH
NH
S
F
NH
H3C
(2a-e)
(iii)
R'
R
O
H
O
N
S
N
F
NH
H3C
(3a-t)
acetic acid and acetic anhydride to afford the title compounds (3a–3t). All
synthesized compounds were characterized by different spectroscopic techniques.
Physicochemical properties of compounds are presented in Table 1.
Pharmacology
Anti-inflammatory activity
The anti-inflammatory activity of all the title compounds (3a–3t) is presented in
Table 2. All compounds were tested at a dose of 25 mg/kg p.o and have shown
considerable anti-inflammatory activity in the range of 5%–60%. Compounds 3l and

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Med Chem Res (2010) 19:1245–1258
Table 1 Physicochemical parameters of synthesized compounds (3a–3t)
Compound
R
R0
Mol. formula (MW)a
Rf valueb
m.p. (°C)
3a
3b
3c
3d
3e
3f
4-Br
3,4-OCH3
2,4-Cl
H
C29H23BrFN3O4S (608.48)
0.69
0.78
0.54
0.61
0.68
0.74
0.79
0.72
0.64
0.71
0.50
0.66
0.70
0.77
0.63
0.49
0.52
0.57
0.64
0.53
178–181
192–194
185–187
177–179
191–193
197–199
211–213
226–228
213–215
228–230
205–207
185–187
213–215
230–232
176–178
163–165
168–170
233–235
215–217
158–160
4-Br
C27H17BrCl2FN3O2S (617.32)
C27H19BrFN3O2S (548.43)
C27H18BrClFN3O2S (582.87)
4-Br
4-Br
4-Cl
4-Br
4-CH3
2,4-Cl
4-Cl
C28H21BrFN3O2S (562.45)
C27H17Cl2FN4O4S (583.42)
C27H18ClFN4O4S (548.97)
C28H21FN4O5S (544.55)
C27H18ClFN4O4S (548.97)
C27H19FN4O4S (514.53)
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-Cl
3g
3h
3i
4-OCH3
2-Cl
3j
H
3k
3l
4-Br
C27H18BrClFN3O2S (582.87)
C27H18ClF2N3O2S (521.97)
C27H18Cl2FN3O2S (538.42)
C27H17Cl3FN3O2S (572.87)
4-Cl
4-F
3m
3n
3o
3p
3q
3r
3s
3t
4-Cl
2-Cl
4-Cl
2,4-Cl
2-NO2
4-NO2
4-Cl
4-Cl
C27H18ClFN4O4S (548.97)
C27H18F2N4O4S (532.52)
4-F
4-F
C27H18ClF2N3O2S (521.97)
2-NO2
2-NO2
4-F
2-Cl
C27H18ClFN4O4S (548.97)
C27H18BrFN4O4S (593.42)
C27H18ClF2N3O2S (521.97)
4-Br
2-Cl
a
Solvent of crystallization: ethanol
b
Solvent system: toluene/ethyl acetate/formic acid (5:4:1)
3q displayed significant results, comparable to those for the standard drug
phenylbutazone (Fig. 1).
Compounds that showed significant anti-inflammatory activity were 3d, 3g, 3k, 3l,
3o, and 3q. These compounds showed [20% protection from inflammation in
animals. Among these compounds 3l and 3q were found to be the most potent, with
44% and 47% anti-inflammatory activity at 25 mg/kg p.o (P \ 0.001). These
compounds were also tested at 50 and 100 mg/kg p.o. (P \ 0.001) to determine the
potentiality along with the reference drug phenylbutazone. Interestingly, compound
3l showed comparable activity to phenylbutazone at 25 mg/kg p.o. (44%; P \ 0.001),
better activity at 50 mg/kg p.o. (60%; P \ 0.001), and even better activity at 100 mg/
kg p.o. (80%; P \ 0.001). Compound 3q also showed comparable anti-inflammatory
activity, at all three graded doses, to the reference drug.
The structure–activity relationship of the compounds was studied and it was
found that the halo-substituted compounds were the most active in the series.
Substitutions with a halogen atom at both the aryl rings resulted in increased anti-
inflammatory activity. Among the halo derivatives, substitutions at the para position
were more active than the halogen at other positions. The size of the halogen atom
also seemed to play an important role in determining the activity. Compounds with
smaller halogen atoms (F, Cl) were more potent than the larger bromo derivatives.

Med Chem Res (2010) 19:1245–1258
1255
Table 2 Pharmacological data on the title compounds (3a–3t)
Compound
Anti-inflammatory activitya
Antinociceptive activityb
Mean increase in paw
volume SE
% Activity
Mean average reaction
time SE (s)
% Activity
Control
3a
0.78 0.12
0.71 0.11
0.67 0.13
0.74 0.10
0.58 0.08
0.69 0.11
0.64 0.12
0.60 0.07*
0.68 0.10
0.63 0.14
0.72 0.09
0.50 0.13**
0.43 0.15***
0.31 0.10***
0.15 0.18***
0.65 0.11
0.67 0.09
0.65 0.17
0.54 0.06**
0.41 0.04***
0.39 0.08***
0.17 0.12***
0.68 0.09
0.65 0.07
0.63 0.10
0.44 0.08***
0.35 0.13***
0.23 0.14***
–
–
8
4.7 0.44
5.8 0.27
–
23
3b
3c
14
5
6.2 0.31
31
8.3 0.34
76
3d
3e
25
11
17
23
12
19
7
9.6 0.33*
8.1 0.28
104
72
3f
10.4 0.16**
9.6 0.34
121
104
46
3g
3h
3i
6.9 0.25
11.3 0.16**
9.1 0.17
140
93
3j
3k
3l
35
44
60c
80d
16
14
16
30
47
50c
78d
12
16
19
43
55c
70d
–
8.3 0.24
76
12.8 0.34***
172
3m
3n
3o
3p
3q
7.7 0.38
6.9 0.39
63
46
8.6 0.41
82
10.4 0.21*
13.6 0.15***
121
189
3r
8.1 0.17
9.4 0.31
10.3 0.35*
–
72
100
119
–
3s
3t
Phenyl butazone
Diclofenac
17.6 0.25***
274
Note: Number of animals used: six. – Not tested. Solvent used: methyl cellulose/water (0.5%) mixture.
* P \ 0.05; ** P \ 0.01; *** P \ 0.001
a
Tested at a dose of 25 mg/kg
b
Tested at a dose of 20 mg/kg
c
Tested at a dose of 50 mg/kg
d
Tested at a dose of 100 mg/kg
Antinociceptive activity
The antinociceptive activity of all compounds was assessed at 25 mg/kg p.o. and the
results are presented in Table 2. Results are expressed as mean average reaction

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Med Chem Res (2010) 19:1245–1258
Fig. 1 Anti-inflammatory
activity of compounds 3l, 3q,
and phenylbutazone
3l
3q
Phenylbutazone
90
80
70
60
50
40
30
20
10
0
25
50
100
Dose (mg/kg)
time (seconds) SE, and the change from control was noted to calculate the
percentage analgesia. All compounds showed antinociceptive activity in the range
of 23%–189% and were compared to the standard drug diclofenac. The two
compounds, 3l and 3q, that showed significant anti-inflammatory activity were also
found to possess considerable antinociceptive activity (Fig. 2). Compounds that
showed significant antinociceptive activity ([ 100%) include 3d, 3f, 3 g, 3i, 3l, 3p,
3q, and 3t. Among these compounds, 3l and 3q were found to be the most potent,
with 172% and 189% antinociceptive activity (P \ 0.001).
All compounds were found to possess lesser activity than the standard drug
diclofenac, which was found to show 274% antinociceptive activity. Substitutions
with electron-withdrawing functional groups resulted in a significant increase in the
activity. Most of the nitro-substituted derivatives were active in the screen.
Substitutions at the para position increased the activity significantly.
Ulcerogenic activity
The ulcerogenic activity of the most active compounds, 3l and 3q, was assessed in
albino rats 8 h after compound administration intraperitoneally and the results are
Fig. 2 Antinociceptive activity
of compounds 3l, 3q, and
diclofenac
300
250
200
150
100
50
0
3l
3q
Diclofenac
Compounds

Med Chem Res (2010) 19:1245–1258
1257
Table 3 Toxicity studies of the selected compounds
Compound
Ulcerogenic activity
(UD50; mg/kg)a
ALD50 (mg/kg)b
3l
240.36
174.63
70.25
[1000
[1000
[1000
3q
Phenyl butazone
Note: Number of animals used: ten. Solvent used: polyethylene glycol (PEG)
a
Compounds were administered intraperitoneally and animals were assessed 8 h after drug treatment
Approximate lethal dose; percentage mortality was observed 24 h after drug administration
b
reported in Table 3. Interestingly, both the compounds displayed a better safety
profile than the standard drug phenylbutazone. The UD50 values of compounds 3l
and 3q were found to be 240.36 and 174.63 mg/kg, much higher than that of the
standard drug (UD50 = 70.25 mg/kg). This shows that these compounds are less
prone to cause ulcers and are relatively safer agents.
Acute toxicity studies
Acute toxicity studies were performed in albino mice and the approximate lethal
dose (ALD50) was determined 24 h after drug administration. Results are reported
in Table 3. Both compounds showed ALD50 values ([1000 mg/kg) comparable to
that of the standard drug phenylbutazone. This further supported the wide safety
margin of the two compounds.
Conclusion
A new series of thiazolopyrimidines has been identified as anti-inflammatory and
anti-nociceptive agents. Two compounds showed significant activities in both
screens, comparable to those of the standard drugs phenylbutazone and diclofenac.
Interestingly, these compounds displayed higher UD50 and ALD50 values, indicating
the wide safety profile of the compounds. These agents have strong future potential
and have proven to be good candidates for further investigations.
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