Synthesis and pharmacological evaluation of novel thienopyrimidine and triazolothienopyrimidine derivatives

Article abstract of DOI:10.1007/s00044-013-0900-1

Novel tricyclic thienopyrimidines (2, 3, 5, 8) and triazole-fused tetracyclic thienopyrimidines (6a-c and 9a-c) were synthesized from the precursor 2-Amino-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile (1). The structures of newly synthesized compounds were established by spectral and analytical data. The title compounds were screened for analgesic, anti-inflammatory, ulcerogenicity index, and antibacterial activities. Test compounds exhibited significant activity, the compounds (6a-c) and (9a-c) showed more potent analgesic activity, and the compounds (6c) and (9c) showed more potent anti-inflammatory activity than the reference standard Diclofenac Sodium. All the synthesized compounds exhibited remarkable antibacterial activity.

Full text of DOI:10.1007/s00044-013-0900-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0900-1
ORIGINAL RESEARCH
Synthesis and pharmacological evaluation of novel
thienopyrimidine and triazolothienopyrimidine derivatives
•
Jameel Ahmed S. Mulla Mohammed Iqbal A. Khazi
•
•
Shridhar I. Panchamukhi Young-Dae Gong
•
Imtiyaz Ahmed M. Khazi
Received: 28 March 2013 / Accepted: 18 December 2013
Ó Springer Science+Business Media New York 2014
Abstract Novel tricyclic thienopyrimidines (2, 3, 5, 8) and
triazole-fused tetracyclic thienopyrimidines (6a–c and 9a–c)
were synthesized from the precursor 2-amino-4,5,6,7-tetra-
hydro-1-benzothiophene-3-carbonitrile (1). The structures of
newly synthesized compounds were established by spectral
and analytical data. The title compounds were screened for
analgesic, anti-inflammatory, ulcerogenicity index, and anti-
bacterial activities. Test compounds exhibited significant
activity, the compounds (6a–c) and (9a–c) showed more
potent analgesic activity, and the compounds (6c) and (9c)
showed more potent anti-inflammatory activity than the ref-
erence standard Diclofenac Sodium. All the synthesized
compounds exhibited remarkable antibacterial activity.
membered heteroaromatic ring. These are the structural
analogs of the biogenic purines and can be considered as
potential nucleic acid antimetabolites. The derivatives of
thienopyrimidines still continue to attract considerable
attention because of their great practical usefulness, pri-
marily, due to a very wide spectrum of biological activities.
Certain thienopyrimidine derivatives are reported to exhibit
antiallergic (Quintela et al., 1998; Madding and Thompson,
´
1987; Temple, 1978), antiproliferative (Pedeboscq et al.,
2010), antibacterial (Ashalatha et al., 2007; Alagarsamy
et al., 2006; Hozien et al., 1997), antifungal (Ashalatha
et al., 2007), analgesic (Alagarsamy et al., 2006), anti-
inflammatory (Ashalatha et al., 2007; Alagarsamy et al.,
2006; Nabil and Abd El-Galil, 2008), anticonvulsant
(Ashalatha et al., 2007; Cannito et al., 1990; Santagati
et al., 1994), antianaphylactic (Wagner et al., 1993),
Immunostimulant (Guetschow et al., 1995), antiviral
(Nasr and Gineinah, 2002), antihypertensive (Russell
et al., 1988), antidepressant (Wardakhan et al., 2008;
Saito et al., 2011), antidiabetic (Deng et al., 2011), and
antihistaminic (Shishoo et al., 2000; Shishoo et al., 2001)
activities. Bacterial infections often produce pain and
inflammation. In normal practice, two groups of agents
(chemotherapeutic, analgesic, and anti-inflammatory) are
prescribed simultaneously. Unfortunately, none of drugs
possesses both these activities in a single component. The
rapid growth in the literature dealing with the synthesis
and biological activities of the thienopyrimidine deriva-
tives prompted us to undertake the synthesis of novel-
fused thienopyridine derivatives with a hope to get a
single molecular framework which can exhibit both
analgesic and anti-inflammatory activities.
Keywords Thienopyrimidine ꢀ
Triazolothienopyrimidine ꢀ Gewald reaction ꢀ Analgesic ꢀ
Anti-inflammatory ꢀ Antibacterial activity
Introduction
Thienopyrimidines occupy a special position among the
fused heterocyclic systems containing an annulated five-
J. A. S. Mulla ꢀ M. I. A. Khazi ꢀ S. I. Panchamukhi ꢀ
I. A. M. Khazi (&)
Department of Chemistry, Karnatak University,
Dharwad 580003, India
e-mail: drimkorgchem@gmail.com
J. A. S. Mulla
Department of Pharmaceutics, K.L.E. University’s College of
Pharmacy, Hubli, India
In the present study, we have reported the synthesis of
some novel tricyclic thienopyrimidines and triazole-fused
tetracyclic thienopyrimidines and screened them for their
Y.-D. Gong ꢀ I. A. M. Khazi
Center for Innovative Drug Library Research, Science College,
Dongguk University, Seoul, Republic of Korea
123

Med Chem Res
Scheme 1 Syntheses of
thienopyrimidine and
triazolothienopyrimidine
derivatives
H2N
N
O
N
NH
i
ii
NH2
CH3
N
S
S
N
S
1
3
2
iv
iii
N
N
N
N
C
O
C
O
S
7
S
4
C2H5
CH3
v
v
HN
HN
NH2
NH2
N
N
C2H5
CH3
N
N
S
8
S
5
vi
vi
R
N
R
N
N
N
N
N
C2H5
CH3
N
N
S
S
9a-c
6a-c
R = H , CH3, C2H5
R = H , CH3, C2H5
Reagents and conditions:
i) CH3COOH, reflux; (ii), HCONH2, reflux; (iii) Triethylorthoacetate, reflux;
iv) Triethylorthopropionate, reflux; (v) NH2NH2·H2O, room temp; (vi) RC(OEt)3,
DMF, 90°C;
analgesic, anti-inflammatory, ulcerogenicity index, and
antibacterial activities.
Shah, 1998; Sabnis et al., 1999). Formation of 2-amino-
4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile (1) was
characterized by the presence of a band at 2,210 cm-1 due
to a cyano group and mN–H-stretching bands at 3,325 and
3,209 cm-1 in their IR spectra. Furthermore, it was also
supported by the presence of D2O exchangeable broad
Results and discussion
1
Chemistry
singlet at d 4.72 in the H-NMR spectrum due to a NH2
group.
The required 2-amino-4,5,6,7-tetrahydrobenzo[b]thio-
phene-3-carbonitrile (1) was prepared by means of Ge-
wald’s reaction as reported in the literature (Dave and
Imidoformates (4, 7) were prepared in excellent yields
by treating (1) with triethylorthoesters in the refluxing
temperature. The structures of imidoformate (4, 7) were
123

Med Chem Res
Table 1 Analgesic activity
Compound code
Dose (mg/kg)
Percent analgesic activity
30 min
1 h
2 h
3 h
2
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
28 1.12*
31 1.23*
29 1.14*
37 1.32**
29 1.24
31 1.22*
35 1.23*
33 1.46*
41 1.68*
35 1.76**
41 1.46*
36 1.53*
42 1.42**
37 1.32*
52 1.11**
43 1.59*
51 1.75*
55 1.65*
64 1.04*
37 1.32*
48 1.34*
49 1.21***
63 1.33**
61 1.20***
67 1.32**
34 1.08***
39 1.13*
35 1.22***
43 1.21*
39 1.34**
43 1.43*
38 1.21**
44 1.25**
41 1.07**
54 1.10*
45 1.12*
53 1.32**
58 1.21***
67 1.33*
44 1.17***
51 1.62*
53 1.46*
65 1.34**
63 1.42*
69 1.31***
27 1.43*
30 1.21***
26 1.22*
27 1.34**
27 1.46*
32 1.21**
29 1.20*
33 1.54*
26 1.34***
31 1.12*
32 1.33*
33 1.13*
48 1.17*
52 1.43**
30 1.23*
37 1.45*
38 1.55*
41 1.23***
52 1.43**
56 1.12**
3
5
36 1.25***
31 1.54*
37 1.43*
33 1.65*
46 1.34**
38 1.54***
46 1.56*
50 1.23**
57 1.11***
35 1.25*
42 1.46*
43 1.67**
56 1.65***
55 1.45**
62 1.44***
8
6a
6b
6c
9a
9b
9c
Control
3
0.76
7
0.54
4
0.34
4
0.14
Diclofenac
10
20
41 0.54
51 1.34
45 1.32
58 0.79
48 1.67
66 1.54
34 0.89
41 1.23
Each value represents the mean SD (n = 6)
Significance levels * P \ 0.5, ** P \ 0.01, and *** P \ 0.001 as compared with the respective control
Pharmacology
established by the absence of mN–H in IR and the presence
of a triplet at d 1.42 and a quartet at d 4.44 corresponding
to protons of the ethyl group in the 1H-NMR spectrum. The
reaction of imidoformate ester (4, 7) with hydrazine
hydrate afforded the thienopyrimidine (5, 8). Formation of
the products was confirmed by the presence of bands at
3,362, 3,372, 3,283, and 3,250 cm-1 in IR spectrum, due to
amino and imino functional groups. 1HNMR spectrum
displayed D2O exchangeable singlets at d 4.72 and 5.54
due to amino and imino groups, respectively. The com-
pound (2) and (3) were prepared by refluxing 2-amino-
Analgesic activity
The analgesic activity test was performed on the Wistar
Albino mice using the tail flick technique (Kulkarni, 1980).
The results showed that all the test compounds exhibited
significant activity (Table 1). Compounds (6a) and (9a)
showed good activity with an increase in lipophilicity,
Further when lipophilicity was increased (methyl group)
compounds (6b, 9b) registered even more increase in
activity. Compounds (6c, 9c) were found more potent than
Diclofenac Sodium. Other compounds showed moderate
activity.
4,5,6,7-tetrahydrobenzo[b]thiophene-3-carbonitrile
with acetic acid and formamide, respectively.
(1)
The compounds (5, 8) were further converted into
triazolopyrimidine derivatives (6a–c) and (9a–c) by treat-
ment with triethylorthoesters in dimethylformamide. The
formation of triazole ring involving both amino and imino
groups was evident by the absence of absorption bands due
to either of these groups in the IR spectrum of (6a–c) and
(9a–c). Further, 1H NMR and Mass spectrum supported the
formation of triazole-fused tetracyclic thienopyrimidines
derivatives (Scheme 1).
Anti-inflammatory activity
Carrageenan-induced paw edema test was used for the
evaluation of the anti-inflammatory activity in rats (Winter
et al., 1962). The data indicate that the rats were protected
by all the test compounds from Carrageenan-induced
inflammation and have been found to be more potent.
123

Med Chem Res
Table 2 Anti-inflammatory activity
Compound code
Dose (mg/kg)
Percent protection
30 min
1 h
2 h
3 h
2
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
10
20
22 1.32**
35 1.21*
23 1.11*
36 1.34*
26 1.54*
38 1.45*
27 1.42**
37 1.11*
28 1.24***
39 1.34**
31 1.53**
42 1.23*
35 1.43***
45 1.24**
29 1.54*
40 1.21***
32 1.34*
44 1.21***
39 1.34**
47 1.56***
5.4 0.67
28 1.32*
48 1.13**
29 1.43*
49 1.23**
31 1.42*
49 1.13*
33 1.34**
51 1.12*
34 1.33**
52 1.12***
32 1.43*
51 1.54***
39 1.34**
58 1.55***
33 1.33*
53 1.24*
36 1.65***
57 1.34**
43 1.56***
61 1.43***
6.7 0.73
32 1.54**
51 1.32*
34 1.23*
53 1.12*
34 1.43**
52 1.53*
38 1.34*
54 1.44**
37 1.13*
55 1.32*
37 1.12***
55 1.21*
44 1.43*
59 1.22*
38 1.43***
56 1.12***
39 1.43*
59 1.54**
48 1.32***
63 1.57**
6.1 0.54
27 1.54*
33 1.43**
26 1.32*
32 1.40**
28 1.12*
31 1.74**
30 1.32*
35 1.43**
31 1.23**
38 1.54*
30 1.12*
32 1.11*
30 1.34**
41 1.43*
32 1.54**
39 1.26*
32 1.13**
39 1.21*
36 1.12*
45 1.49*
3.5 0.41
34 0.84
3
5
8
6a
6b
6c
9a
9b
9c
Control
Diclofenac
10
20
33 1.23
40 1.41
41 1.57
44 1.32
51 1.26
59 0.65
40 1.72
Each value represents the mean SD (n = 6)
Significance levels * P \ 0.5, ** P \ 0.01, and *** P \ 0.001 as compared with the respective control
Table 3 Evaluation of ulcerogenicity index
Compounds (6c, 9c) showed more anti-inflammatory
activity than Diclofenac Sodium (Table 2).
Compound code
Ulcer index
2
1.05 0.32*
1.12 0.15*
0.96 0.27*
0.97 0.22*
0.89 0.17*
0.79 023*
0.86 0.18**
0.92 0.21**
0.87 0.32*
0.90 0.29**
1.02 0.15
Evaluation of Ulcerogenicity index
3
5
The ulcer index of the test compounds reveals that all
compounds showed a mild ulcer index as compared to
aspirin (Table 3).
8
6a
6b
6c
Antibacterial activity
9a
9b
The Agar dilution method (Barry, 1991) was used to evaluate
the antibacterial activity. Results showed that all the test
compounds exhibited moderate activity against the tested
bacteria (Table 4). Compound (6b) registered good activity
against Staphylococcus aureus and Bacillus subtilis; how-
ever, compound (9c) exhibited good activity against Esch-
erichia coli and Pseudomonas aeruginosa. Compound (2)
displayed good activity against Staphylococcus aureus and
9c
Control
Aspirin
1.71 0.23**
Dose 20 mg for test compounds and 200 mg/ for aspirin. Each value
represents the mean SEM (n = 6)
Significance levels * P \ 0.05 and ** P \ 0.01 as compared with the
respective control
123

Med Chem Res
Table 4 Antibacterial activity
as MIC values (lg/mL)
Compound code
Staphylococcus
aureus
ATCC 25923
Bacillus
subtilis
ATCC 6633
Escherichia
coli
ATCC 25922
Pseudomonas
aeruginosa
ATCC 27853
2
8
32
8
8
64
64
64
32
32
32
64
64
16
8
3
32
32
64
16
8
8
5
32
64
32
8
64
32
32
64
32
64
16
4
8
6a
6b
6c
16
32
64
32
0.5
32
32
32
16
0.5
9a
9b
9c
Ampicillin
2
2
Escherichia coli while the compound (3) exhibited good
activity against Bacillus subtilis and Escherichia coli.
formamide (15 mL) was heated under reflux for 5 h. The
solution was concentrated under reduced pressure. The
resulting residue was crystallized from ethanol.
Conclusion
2-methyl, 5,6,7,8-tetrahydro[1]benzothieno[2,3-
d]pyrimidin-4[3H]-one (2)
We have synthesized a series of novel thienopyrimidine
and triazolothienopyrimidine derivatives and established
their structures with the help of analytical and spectral data.
Some of the synthesized compounds exhibited significant
activity, the compounds (6a–c) and (9a–c) showed more
potent analgesic activity and the compounds (6c) and (9c)
showed more potent anti-inflammatory activity than the
reference standard Diclofenac sodium. All the synthesized
compounds exhibit remarkable antibacterial activity.
Yield: 80%; melting range: 154–156°C; IR (KBr, v, cm-1):
3308 (N–H str), 3056 (C–H str of Ar), 2927 (C–H str of
Aliphatic), 1662 (C=O str), 1590 (C=N str), 1282 (C–N
1
str). H NMR (CDCl3, 300 MHz, TMS) dH (ppm): 11.80
(br s, 1H, NH, D2O exchangeable), 3.12 (s, 3H, CH3), 2.75
(t, J = 8 Hz, 2H, CH2), 2.62(t, J = 8 Hz, 2H, CH2), 1.80
(m, 4H, CH2); 13C NMR (75 MHz, CDCl3) dC (ppm):
158.2 (C-4), 153.2 (C-11), 152.1 (C-2), 136.1 (C-9), 124.2
(C-10), 116.2 (C-12), 25.2 (2-CH3), 24.2 (C-8), 24.1 (C-7),
23.1 (C-6), 22.8 (C-5). Anal. calcd. for C11H12N2OS (%):
Calcd. C, 59.97; H, 5.49; N, 12.72; S,14.56. Found: C,
59.89; H, 5.69; N. 12.37; S,14.63.
Experimental
Chemistry
5,6,7,8,-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4-
General details
ylamine (3)
Melting points were determined in open capillaries. The IR
spectra were recorded on Nicolet Impact 410 FT IR spectro-
Yield: 87%; melting range: 134–136°C; IR (KBr, v,
cm-1): 3318 (N–H str), 3122 (C–H str of Ar), 1562 (C=N),
1
photometer (Model-410, USA) using KBr pellets. H NMR
1
1239 (C–N str). H NMR (DMSO d6, 300 MHz, TMS) dH
spectra were recorded on Bruker 300 MHz NMR spectrometer
(Model RX-300, Switzerland) in CDCl3 and DMSO-d6 with
TMS as internal standard. Mass spectra were recorded on
GCMS Schimadzu (QP-2010S Japan), and elemental analyses
were carried out using Heraeus CHN rapid analyzer.
(ppm): 8.17 (s, 1H, C2–H, pyrimidine), 6.78 (br s, 2H,
NH2, D2O exchangeable), 2.91 (t, J = 8 Hz, 2H, CH2),
2.74 (t, J = 8 Hz, 2H, CH2), 1.81 (m, 4H, CH2); 13C NMR
(75 MHz, DMSO d6) dC (ppm): 155.3 (C-2), 154.3 (C-4),
144.6 (C-11), 136.2 (C-9), 126.5 (C-10), 112.1 (C-12), 25.1
(C-8), 23.2 (C-7), 23.1 (C-6); Mass Calcd. 205.07 Found
m/z: 205.24 (m?). Anal. calcd. for C10H11N3S: C, 58.51; H,
5.40; N, 20.47; S, 15.62 Found: C, 57.68; H, 7.62; N,
20.38; S, 15.74.
General procedure for synthesis of compounds 2–3
A
mixture of 2-amino-4,5,6,7-tetrahydro-1-benzothio-
phene-3-carbonitrile 1 (1 g, 52 mmole) and acetic acid/
123

Med Chem Res
General procedure for synthesis of compounds 4 and 7
solution of 2-amino-4,5,6,7-tetrahydro-1-benzothio-
4-Imino-5,6,7,8-tetrahydro[1]benzothieno[2,3-
d]pyrimidin-3[4H]-amine (5)
A
phene-3-carbonitrile 1 (1.33 g, 5 mmole) in triethylort-
hoacetate / triethylorthopropionate (12 mL) was heated
under reflux for 18 h, and excess of triethylorthoacetate /
triethylorthopropionate was removed under vaccum. The
residue was treated with petroleum ether. Solid that sepa-
rated was filtered and recrystallized with petroleum ether to
afford light brown crystals.
Yield: 77%; melting range: 152–154°C; IR (KBr, v, cm-1):
3362 (N–H str), 3027 (C–H str of Ar), 2936 (C–H str of
Aliphatic), 1552 (C=N str), 1165 (C–N str). 1H NMR
(CDCl3, 300 MHz, TMS) dH (ppm): 5.54 (br s, 1H, NH,
D2O exchangeable), 4.72 (br s, 2H, NH2, D2O exchange-
able), 3.02 (s, 3H, CH3), 2.85 (t, J = 8 Hz, 2H, CH2), 2.65
(t, J = 8 Hz, 2H, CH2), 1.83 (m, 4H, CH2); 13C NMR
(75 MHz, CDCl3) dC (ppm): 160.2 (C-4), 158.3 (C-2),
142.5 (C-12), 136.4 (C-9), 128.1 (C-11), 127.2 (C-10), 25.2
(C-8), 24.2 (C-7), 22.9 (C-6), 22.6 (C-5), 19.7 (2-CH3);
Mass Calcd. 234.07 Found m/z : 220.10 (m?).. Anal. calcd.
For C11H14N4S: C, 56.38; H, 6.02; N, 23.91; S, 13.68.
Found: C, 56.23; H, 6.12; N, 23.99; S, 13.59.
N-(3-Cyano-4,5,6,7-tetrahydro-benzo[b]thiophen-2-yl)-
acetimidic acid ethyl ester (4)
Yield: 89%; melting range: 122–124°C; IR (KBr, v, cm-1):
3089 (C–H str of Ar), 2950 (C–H str of Aliphatic), 2212
(C:N str), 1625 (C=N str), 1329 (C–N str), 1290 (C–O–C
str). 1H NMR (CDCl3, 300 MHz, TMS) dH (ppm): 4.44 (q,
2H, CH2), 3.10 (s, 3H, CH3), 2.78 (t, J = 8 Hz, 2H, CH2),
2.58 (t, J = 8 Hz, 2H, CH2), 1.84 (m, 4H, CH2), 1.42 (t,
3H, CH3); 13C NMR (75 MHz, CDCl3) dC (ppm): 160.1
(N = C–O–), 155.3 (C-2), 136.5 (C-8), 127.5 (C-9), 114.2
(CN), 98.3 (C-3), 56.1 (O–CH2–CH3), 25.2 (C-7), 24.1 (C-
6), 22.9 (C-5), 22.8 (C-4), 20.3 (O–CH2–CH3), 14.7 (N=C–
CH3); Mass Calcd. 248.1 Found m/z : 248.10 (m?). Anal.
calcd. for C13H16N2OS; C, 62.87; H, 6.49; N, 11.28; S,
12.91.Found: C, 62.65; H, 6.23; N, 11.67; S, 13.02.
2-ethyl-4-Imino–5,6,7,8-tetrahydro[1]benzothieno[2,3-
d]pyrimidin-3[4H]-amine (8)
Yield: 65%; melting range: 115–117°C; IR (KBr, v, cm-1):
3372 (N–H str), 3030 (C–H str of Ar), 2933 (C–H str of
Aliphatic), 1547 (C=N str), 1155 (C–N str). 1H NMR
(CDCl3, 300 MHz, TMS)) dH (ppm): 5.52 (br s, 1H, NH,
D2O exchangeable), 4.70 (br s, 2H, NH2, D2O exchange-
able), 4.41 (q, 2H, CH2), 2.78 (t, J = 8 Hz, 2H, CH2), 2.58
(t, J = 8 Hz, 2H, CH2), 1.85 (m, 4H, CH2), 1.32 (t, 3H,
CH3); 13C NMR (75 MHz, CDCl3) dC (ppm): 160.2 (C-4),
153.1 (C-2), 142.3 (C-12), 136.5 (C-9), 128.2 (C-11), 127.1
(C-10), 25.2 (C-8), 24.3 (C-7), 22.9 (C-6), 22.7 (2-CH2–
CH3), 20.9 (C-5), 5.1 (2-CH2–CH3); Mass Calcd. 248.11
Found m/z: 248.30 (m?). Anal. calcd. For C12H16N4S: C,
58.04; H, 6.49; N, 22.56; S, 12.91. Found: C, 58.23; H,
6.12; N, 22.99; S, 12.82.
(Z)-ethyl N-3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-
2-ylpropionimidate (7)
Yield: 76%; melting range: 134–136°C; IR (KBr, v, cm-1):
3078 (C–H str of Ar), 2962 (C–H str of Aliphatic), 2218
(C:N str), 1615 (C=N str), 1324 (C–N str), 1288 (C–O–C
str). 1H NMR (CDCl3, 300 MHz, TMS) dH (ppm): 4.44 (m,
4H, CH2), 2.91 (t, J = 8 Hz, 2H, CH2), 2.74 (t, J = 8 Hz,
2H, CH2), 1.84 (m, 4H, CH2), 1.46 (m, 6H, CH3); 13C NMR
(75 MHz, CDCl3) dC (ppm): 162.2 (N=C–O–), 155.1 (C-2),
136.7 (C-8), 127.1 (C-9), 114.2 (CN), 98.2 (C-3), 58.2 (O-
CH2–CH3), 26.1 (C-7), 25.1 (C-6), 24.1 (C-5), 22.9
(N=CH2–CH3), 22.8 (C-4), 14.8 (–O–CH2–CH3), 4.1
(N=CH2–CH3); Mass Calcd. 262.11 Found m/z: 262.89
(m?). Anal. calcd. for C14H18N2OS; C, 64.09; H, 6.91; N,
10.68; S, 12.22. Found: C, 64.65; H, 6.63; N, 10.67; S, 12.52.
General procedure for synthesis of compounds 6a–c
A mixture of (5) (53 mmole), triethylorthoformate / trie-
thylorthoacetate / triethylorthopropionate (0.6 mL), and
dimethylformamide (0.6 mL) was heated on water bath
(90°C) for 4 h. The reaction mixture was cooled to room
temperature and diluted with petroleum ether (2 mL). The
solid that separated was washed with benzene and petro-
leum ether and dried. The crude product was purified by
means of column chromatography (EtOAc:Hexane:: 6:4) to
furnish yellow granules.
General procedure for synthesis of compounds 5 and 8
A mixture of (4) (55 mmole) and hydrazine hydrate (7 mL)
was stirred at room temperature for 2 h and then diluted
with ethanol (20 mL). The resulting fine solid suspension
was filtered, washed with ethanol, and purified by recrys-
tallisation from ethanol-dioxane mixture to afford analyti-
cally pure pale yellow granules.
5-Methyl-8,9,10,11-tetrahydro[1]benzothieno[3,2-
e][1,2,4]triazolo[1,5-c] pyrimidine (6a)
Yield: 70%; melting range: 118–120°C; IR (KBr, v, cm-1):
3054 (C–H str of Ar), 2963 (C–H str of Aliphatic), 1622
1
(C=N str), 1297 (C–N str). H NMR (CDCl3, 300 MHz,
123

Med Chem Res
TMS) dH (ppm): 9.20 (s, 1H, triazole), 3.10 (s, 3H, CH3),
2.91 (t, J = 8 Hz, 2H, CH2), 2.74 (t, J = 8 Hz, 2H, CH2),
1.81 (m, 4H, CH2); 13C NMR (75 MHz, CDCl3) dC (ppm):
165.9 (C-5), 150.1 (C-2), 147.4 (C-16), 144.5 (C-15), 136.4
(C-13), 128.1 (C-14), 126.8 (C-12), 25.8 (C-8), 24.2
(C-11), 23.7 (C-10), 23.2 (C-9), 19.8 (5-CH3); Mass
Calcd. 244.08 Found m/z: 244.32 (m?). Anal. calcd. for
C12H12N4S: C, 58.99; H, 4.95; N, 22.93; S, 13.12. Found:
C, 59.08; H, 4.88; N, 23.03; S, 13.08.
means of column chromatography (EtOAc:Hexane:: 6:4) to
furnish yellow granules.
5-Ethyl-8,9,10,11-tetrahydro[1]benzothieno[3,2-
e][1,2,4]triazolo[1,5-c] pyrimidine (9a)
Yield: 78%; melting range: 125–127°C; IR (KBr, v, cm-1):
3030 (C–H str of Ar), 2983 (C–H str of Aliphatic), 1554
1
(C=N str), 1181 (C–N str). H NMR (CDCl3, 300 MHz,
TMS) dH (ppm): 9.20 (s, 1H, triazole), 4.34 (q, 2H, CH2),
2.91 (t, J = 8 Hz, 2H, CH2), 2.74 (t, J = 8 Hz, 2H, CH2),
1.81 (m, 4H, CH2), 1.34 (t, 3H, CH3); 13C NMR (75 MHz,
CDCl3) dC (ppm): 170.8 (C-5), 151.2 (C-2), 146.8 (C-16),
145.5 (C-15), 136.5 (C-13), 127.6 (C-14), 126.9 (C-12),
26.8 (5-CH2–CH3), 25.1 (C-8), 24.2 (C-11), 23.3 (C-10),
23.2 (C-9), 14.2 (2-CH2–CH3); Mass Calcd. 258.09 Found
m/z : 258.13 (m?). Anal. calcd. for C13H14N4S: C, 60.44;
H, 5.46; N, 21.69; S, 12.41. Found C,60.24; H, 5.25; N,
21.75; S, 12.76.
2,5-Dimethyl-8,9,10,11-tetrahydro[1]benzothieno[3,2-
e][1,2,4]triazolo[1,5-c]pyrimidine (6b)
Yield: 86%; melting range: 165–167°C; IR (KBr, v, cm-1):
3044 (C–H str of Ar), 2953 (C–H str of Aliphatic), 1616
1
(C=N str), 1291 (C–N str). H NMR (CDCl3, 300 MHz,
TMS) dH (ppm): 3.18 (s, 3H, CH3), 3.08 (s, 3H, CH3), 2.88
(t, J = 8 Hz, 2H, CH2), 2.68 (t, J = 8 Hz, 2H, CH2), 1.79
(m, 4H, CH2); 13C NMR (75 MHz, CDCl3) dC (ppm):
166.3 (C-5), 159.2 (C-2), 147.5 (C-16), 144.1 (C-15), 136.3
(C-13), 128.2 (C-14), 126.6 (C-12), 25.5 (C-8), 24.1 (C-
11), 23.8 (C-10), 23.4 (C-9), 19.5 (5-CH3), 18.1 (2-CH3);
Mass Calcd. 258.09 Found m/z: 258.21 (m?). Anal. calcd.
for C13H14N4S: C, 60.44; H, 5.46; N, 21.69; S, 12.41.
Found: C, 60.24; H, 5.26; N, 21.19; S, 12.11.
5-Ethyl-2-methyl-8,9,10,11-tetrahydro[1]benzothieno[3,2-
e][1,2,4]triazolo[1,5-c]pyrimi dine (9b)
Yield: 74%; melting range: 145–147°C; IR (KBr, v, cm-1):
3056 (C–H str of Ar), 2980 (C–H str of Aliphatic), 1560
1
(C=N str), 1139 (C–N str). H NMR (CDCl3, 300 MHz,
2-Ethyl-5-methyl-8,9,10,11-tetrahydro[1]benzothieno[3,2-
TMS) dH (ppm): 4.35 (q, 2H, CH2), 3.08 (s, 3H, CH3), 2.88
(t, J = 8 Hz, 2H, CH2), 2.68 (t, J = 8 Hz, 2H, CH2), 1.79
(m, 4H, CH2), 1.36 (t, 3H, CH3); 13C NMR (75 MHz,
CDCl3) dC (ppm): 170.8 (C-5), 158.1 (C-2), 147.3 (C-16),
145.2 (C-15), 137.2 (C-13), 128.2 (C-14), 127.1 (C-12),
26.8 (5-CH2–CH3), 25.1 (C-8), 23.9 (C-11), 23.4 (C-10),
23.1 (C-9), 17.9 (2-CH2), 14.2 (2-CH2–CH3); Anal. calcd.
for C14H16N4S: C, 61.74; H, 5.92; N, 20.57; S, 11.77.
Found: C, 61.24; H, 5.26; N, 20.45; S, 11.51.
e][1,2,4]triazolo[1,5-c]pyramidine (6c)
Yield: 66%; melting range: 125–127°C; IR (KBr, v, cm-1):
3050 (C-H str of Ar), 2926 (C–H str of Aliphatic), 1552
1
(C=N str), 1284 (C–N str). H NMR (CDCl3, 300 MHz,
TMS) dH (ppm): 4.16 (q, 2H, CH2), 3.20 (s, 3H, CH3), 2.99
(t, J = 8 Hz, 2H, CH2), 2.74 (t, J = 8 Hz, 2H, CH2), 1.43
(t, 3H CH3), 1.81 (m, 4H, CH2); 13C NMR (75 MHz,
CDCl3) dC (ppm): 167.2 (C-5), 165.1 (C-2), 146.9 (C-16),
145.2 (C-15), 136.4 (C-13), 127.9 (C-14), 126.3 (C-12),
26.9 (2-CH2–CH3), 25.1 (C-8), 24.2 (C-11), 23.6 (C-10),
23.2 (C-9), 19.2 (5-CH3), 14.1 (2-CH2–CH3); Mass Calcd.
272.11 Found m/z: 272.15 (m?). Anal. calcd. for
C14H16N4S: C, 61.74; H, 5.92; N, 20.57; S, 11.77. Found:
C, 61.24; H, 5.44; N, 20.10; S, 11.15
2,5-Diethyl-8,9,10,11-tetrahydro[1]benzothieno[3,2-
e][1,2,4]triazolo[1,5-c]pyrimidine (9c)
Yield: 72%; melting range: 134–136°C; IR (KBr, v, cm-1):
3046 (C–H str of Ar), 2989 (C–H str of Aliphatic), 1562
1
(C=N str), 1152 (C–N str). H NMR (CDCl3, 300 MHz,
TMS) dH (ppm): 4.30 (m, 4H, CH2), 2.84 (t, J = 8 Hz, 2H,
CH2), 2.65 (t, J = 8 Hz, 2H, CH2), 1.81 (m, 4H, CH2), 1.36
(m, 6H CH3); 13C NMR (75 MHz, CDCl3) dC (ppm): 170.3
(C-5), 163.1 (C-2), 147.2 (C-16), 145.1 (C-15), 137.2 (C-
13), 127.8 (C-14), 126.1 (C-12), 26.9 (5-CH2–CH3), 26.8
(2-CH2–CH3), 25.1 (C-8), 23.9 (C-11), 23.3 (C-10), 23.2
(C-9), 14.2 (5-CH2–CH3), 14.1 (2-CH2–CH3); Mass Calcd.
286.13 Found m/z : 286.08 (m?). Anal. calcd. for
C15H18N4S: C, 62.91; H, 6.33; N, 19.56; S, 11.20. Found:
C, 62.71; H, 6.53; N, 19.06; S, 11.45.
General procedure for synthesis of compounds 9a–c
A mixture of (8) (53 mmole), triethylorthoformate / trie-
thylorthoacetate / triethylorthopropionate (0.6 mL), and
dimethylformamide (0.6 mL) was heated on water bath
(90°C) for 4 h. The reaction mixture was cooled to room
temperature and diluted with petroleum ether (2 mL). The
solid that separated was washed with benzene and petro-
leum ether and dried. The crude product was purified by
123

Med Chem Res
volume of rats after the administration of Carrageenan in
the test group (drug treated), ‘‘b’’ is the mean paw volume
of rats after the administration of Carrageenan in the con-
trol group, and ‘‘y’’ is the mean paw volume of rats before
the administration of Carrageenan in the control group.
Pharmacology
The synthesized compounds were also evaluated for anal-
gesic, anti-inflammatory, ulcerogenic index, and antimi-
crobial activities. To ascertain the significance of all the
exhibited activities Student’s t test was carried out. The test
compounds and the standard drugs were administered in
the form of a suspension (1% carboxy methyl cellulose as a
vehicle) by oral route for analgesic and anti-inflammatory
activities, but for the ulcerogenicity studies, the drugs were
administered intraperitoneally as suspension in 10% v/v
Tween. Six animals each made up a group were kept in
colony cages at 25 2°C at a relative humidity of 45–55%
under a light and dark cycle of 12 h each. They were fed
with standard animal feed and were acclimatized for a
week before the tests.
Evaluation of ulcerogenicity index
Albino rats of Wistar strain weighing 150–200 g of either
sex were divided into various groups each of six animals
and ulceration in rats was induced by following the liter-
ature method (Goel et al., 1985). Control group of animals
were administered only with 10% v/v Tween 80 suspension
intraperitoneally. One group was administered with Aspirin
intraperitoneally in a dose of 200 mg/kg once daily for
3 days, and the remaining groups of animals were admin-
istered with test compounds intraperitoneally in a dose of
20 mg/kg. On the fourth day, Pylorus was ligated as per the
method of Shay et al. (1945). Animals were put on a fast
for 36 h before the pylorus ligation procedure. Four hours
after the ligation, animals were sacrificed. The stomach
was removed and opened along with the greater curvature.
Data show the Ulcer index which was determined by the
method of Ganguly and Bhatnagar (1973).
Analgesic activity
The analgesic activity tests were performed using the tail
flick technique on the Wistar Albino mice (25–35 g) of
either sex selected using random sampling technique. As a
reference drug for comparison, Diclofenac sodium at dose
levels of 10 and 20 mg/kg was administered orally. Two
dose levels (10, 20 mg/kg) of the test compounds were
orally administered. The reaction time was recorded at
30 min, 1–3 h after the treatment, and cut-off time was
10 s. The percent analgesic activity (PAA) was calculated
by the following Eq. (1),
Antibacterial activity
The Agar dilution method (Barry, 1991) was used to
evaluate the antibacterial activity. The antibacterial activity
of the synthesized compounds was screened against the
following bacterial strains: Staphylococcus aureus (ATCC
25923), Bacillus subtilis (ATCC 6633), Escherichia coli
(ATCC 25922), and Pseudomonas aeruginosa (ATCC
27853). All bacteria were grown on Muller–Hinton Agar
(Hi-media) plates (37°C, 24 h) and the minimum inhibitory
concentration (MIC) was considered to be the lowest
concentration that completely inhibited the growth on agar
plates, barring a single colony or faint haze caused by the
inoculums. The MIC of the test compounds was compared
with the reference drug ampicillin.
ꢀ
ꢁ
T2 ꢁ T1
10 ꢁ T1
where T1 is the reaction time (s) before treatment, and T2 is
PAA ¼
ꢂ 100
ð1Þ
the reaction time (s) after treatment.
Anti-inflammatory activity
The anti-inflammatory activity was evaluated by the Car-
rageenan-induced paw edema test in rats. As a reference
drug for comparison, Diclofenac sodium at dose levels of
10 and 20 mg/ kg was administered orally. The data indi-
cate that the rats were protected by all the test compounds
from Carrageenan-induced inflammation and have been
found to be more potent. The paw volumes were measured
using the mercury displacement technique with the help of
a Plethysmograph immediately before and 30 min, 1–3 h
after Carrageenan injection. The percent inhibition of paw
edema was calculated using the following Eq. (2).
Acknowledgments The authors are thankful to Karnatak Univer-
sity, Dharwad, University Grants Commission, New Delhi, India, for
financial support and to the University Science Instruments Centre,
Karnatak University, Dharwad, for providing the spectral data.
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