Syntheses of new tetrasubstituted thiophenes as novel anti-inflammatory agents

Article abstract of DOI:10.1016/j.ejmech.2007.01.007

A series of new tetrasubstituted thiophenes (4a-4i, 5a-5i and 6a-6f) have been synthesized as novel anti-inflammatory agents and were evaluated for their anti-inflammatory activity in carrageenin-induced rat hind paw oedema model at the doses of 10, 20 an

Full text of DOI:10.1016/j.ejmech.2007.01.007

European Journal of Medicinal Chemistry 42 (2007) 1049e1058
http://www.elsevier.com/locate/ejmech
Original article
Syntheses of new tetrasubstituted thiophenes as
novel anti-inflammatory agents
b
b
Khurshid I. Molvi a, , Kamala K. Vasu , Swapnil G. Yerande ,
*
Vasudevan Sudarsanam b, Navedul Haque c
a School of Pharmacy, Faculty of Medical Sciences, Jimma University, P.O. Box 378, Jimma, Oromia, Ethiopia
b Department of Medicinal Chemistry, B.V. Patel Pharmaceutical Education and Research Development Centre,
ThaltejeGandhinagar Highway, Thaltej, Ahmedabad 380 054, Gujarat, India
c Department of Chemistry, Faculty of Education, Jimma University, P.O. Box 378, Jimma, Oromia, Ethiopia
Received 21 July 2006; received in revised form 28 December 2006; accepted 9 January 2007
Available online 24 January 2007
Abstract
A series of new tetrasubstituted thiophenes (4ae4i, 5ae5i and 6ae6f) have been synthesized as novel anti-inflammatory agents and were
evaluated for their anti-inflammatory activity in carrageenin-induced rat hind paw oedema model at the doses of 10, 20 and 40 mg/kg body
weight. Among ester series, the best compound 4c showed 71% protection at 10 mg/kg, 72% at 20 mg/kg, and 76% at 40 mg/kg to inflamed
paw; while in acid series 5a showed 79% protection at 10 mg/kg, 80% at 20 mg/kg, and 70% at 40 mg/kg, and 5c showed 72% protection
at 10 mg/kg, 75% at 20 mg/kg, and 69% at 40 mg/kg, to inflamed paw. In case of oxime series 6ae6f, the anti-inflammatory activities of
the candidates were found to be poor as compared to acid and ester series. It was found on the basis of SAR studies of target compounds,
that the presence of OCH3 at R2 position and H, OCH3 at R1 are one of the requirements for eliciting comparable anti-inflammatory activity
in both tetrasubstituted thiophenes’ ester and acid series. Compounds 4ae4i, 5ae5i were investigated for their analgesic activity in acetic
acid induced writhing response model at 10 mg/kg dose. Among the ester series compound 4e showed maximum protection of 60%, while
4a, 4b, and 4i exhibited 55%, 45%, and 43% protection, respectively. The result showed that presence of H, Cl at R1 and OCH3, CH3 at R2
in tetrasubstituted thiophene ester series enhances their analgesic activity. The candidates of acid series 5ae5i showed poor analgesic activity
as compared to the standard drug ibuprofen. Compounds 4ae4i, 5ae5i were evaluated for their in vitro antioxidant nitric oxide radical scav-
enging assay. Among the ester series 4a showed maximum in vitro nitric oxide radical scavenging activity having IC50 value 30.08 mg/ml while
in acid series 5a has IC50 value 25.20 mg/ml. The results showed that the presence of R1 ¼ H, R2 ¼ OCH3 and R1 ¼ R2 ¼ OCH3 enhances nitric
oxide radical scavenging property in tetrasubstituted thiophenes’ acid series.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Tetrasubstituted thiophenes; COX-inhibitors; Anti-inflammatory activity; Analgesic activity; Antioxidant activity
1. Introduction
neutralize, or to destroy an injurious agent in preparation for
the process of healing. The cardinal signs of inflammation
are rubor (redness), calor (heat), dolor (pain), tumor (swell-
ing), and functio laesa (loss of function). Cause of inflamma-
tion includes physical agents, chemical agents, immunological
reactions, and infection by pathogenic organism [1]. Inflam-
mation is divided into acute and chronic patterns. The charac-
teristics of acute inflammation are the exudation of fluid and
plasma proteins (oedema) and the emigration of leuko-
cytes, predominantly neutrophils. Chronic inflammation is
Inflammation is a local reaction of the vascular and sup-
porting elements of a tissue to injury resulting in the forma-
tion of a protein-rich exudates; it is a protective response of
the nonspecific immune system that serves to localize,
* Corresponding author. Tel.: þ251 471 114201; fax: þ251 471 111450.
E-mail address: khurshidmolvi@yahoo.co.in (K.I. Molvi).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.01.007

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K.I. Molvi et al. / European Journal of Medicinal Chemistry 42 (2007) 1049e1058
considered to be inflammation of prolonged duration (weeks
or months) in which active inflammation, tissue destruction,
and attempts at repair are proceeding simultaneously. Chronic
inflammation includes some of the most common and dis-
abling human diseases, such as rheumatoid arthritis, athero-
sclerosis, tuberculosis, and chronic lung diseases [2].
activity. An attempt to generate a three point pharmacophore
for designing better anti-inflammatory agents has been reported
[23]. In continuation of our search for new tetrasubstituted thio-
phene as potent anti-inflammatory agents, we have reported op-
timization of electron withdrawing groups at the para position
in anilino (second position) and benzoyl moiety (fifth position)
in the bioisosteric tetrasubstituted thiophenes, falling in þs, ꢀp
quadrant of the Craig plot and its effect on their anti-inflamma-
tory activity profile [24]. In present study we report synthesis,
anti-inflammatory, analgesic and in vitro antioxidant nitric ox-
ide radical scavenging activity of designed tetrasubstituted thio-
phene acid/ester molecules having the features of (a) COX-1
inhibitor and 5-LOX inhibitor (acid/ester) of the anthranilic
acid type (fenamates), (b) p38 MAP kinase inhibitor, and (c)
the selection of substituents at R1 (both electron releasing and
electron withdrawing) in anilino moiety and R2 (electron releas-
ing only) in benzoyl moiety. The selection of substituents at R1
and R2 was mainly guided by lipophilicity and electronic con-
siderations as defined by sep Craig plot chosen in such
a way that they fall within the ꢁs, ꢀp quadrant of the Craig
plot [25].
For optimization of the presence of electron releasing groups
on anti-inflammatory activity of tetrasubstituted thiophenes we
introduced, electron releasing groups such as p-OCH3
(s ¼ ꢁ0.27; p ¼ ꢁ0.04) and p-CH3 (s ¼ ꢁ0.17; p ¼ þ0.56)
in benzoyl moiety (R2) and keeping them constant, electron re-
leasing groups p-OCH3 and p-CH3 in anilino moiety (R1) were
introduced. Further keeping electron releasing groups p-OCH3
and p-CH3 at R2 in benzoyl moiety as constant, electron with-
drawing substituents p-F (s ¼ þ0.06; p ¼ þ0.14) and p-Cl
(s ¼ þ0.24; p ¼ þ0.71) were introduced in anilino moiety
(R1), to study their effect on anti-inflammatory and analgesic
activity profile of the designed compounds. Oximes of tetrasub-
stituted thiophene esters were synthesized to explore the intro-
duction of oximino feature on the anti-inflammatory activity
profile of the designed compounds. The role of the free radical
(NO) in inflammatory processes is well known [26]. Free radi-
cals liberated from phagocyte cells are important in inflamma-
tory processes because they are implicated in the activation of
nuclear factor kB (NF-kB), which induces the transcription of
inflammatory cytokines and COX-2. Furthermore, antioxidants
have been shown to be able to effectively block the activation of
NF-kB through the stabilization of NF-kB/IkB-a complex [27].
In order to arrive at possible mechanism for anti-inflammatory
activity of synthesized tetrasubstituted thiophene esters 4ae4i
and acids 5ae5i, their in vitro antioxidant nitric oxide radical
scavenging assay was performed.
Nonsteroidal anti-inflammatory drugs (NSAIDs) are widely
used for the choice treatment in various inflammatory diseases
such as arthritis, rheumatisms as well as to relieve the aches and
pain of everyday life [3]. Classical NSAIDs exhibit their action
by restricting the biosynthesis of prostaglandin, some of which
are pro-inflammatory. This is essentially brought about by
inhibiting the rate limiting cyclooxygenase (COX) enzyme in-
volved in the inflammatory cascade [4]. The acid group present
in the classical NSAIDs binds to arginine, the 120th amino acid
residue in the COX, and causes the inactivation of the enzyme
[5]. Therefore classical NSAIDs act as preferential COX-1 in-
hibitors. NSAIDs cause side effects, namely, dyspepsia, gastro-
intestinal ulceration, bleeding, nephrotoxicity [6] and even
death [7]. The discovery of the isoform of constitutive COX-1,
namely, COX-2 (inducible) and its inhibition has proved to be
beneficial in clinical situations had led to the introduction of
many potent anti-inflammatory agents commonly referred to
as COX-2 inhibitors [8]. COX-2 inhibitors, which were once
thought to be a panacea for the treatment of inflammatory dis-
ease, have recently met with a measure of disrepute due to the
emergence of serious side effects in long-term clinical use
[9]. COX-3, a COX-1 variant and a new isozyme and its relation
to analgesic/antipyretic drug has also been reported [10].
MAP kinases are signaling molecules that are activated by
a number of extracellular stress stimuli. p38 MAP kinase is
a member of a family of serineethreonine kinases that are
activated by dual phosphorylation of a TGY motif [11]. The
events that are regulated by p38 MAP kinase lead to the produc-
tion of pro-inflammatory cytokines such as tumor necrosis fac-
tor-a (TNF-a) and interleukin-1b (IL-1b) [12]. Tumor necrosis
factor-a (TNF-a) and interleukin-1b (IL-1b), accumulation ini-
tiates a cascade of events leading to inflammation and tissue de-
struction in diseases such as rheumatoid arthritis [13], Crohn’s
disease [14], inflammatory bowel syndrome, and psoriasis.
Agents having beneficial effects for the treatment of the inflam-
matory diseases and which inhibit the production of TNF-a
antibody and IL-1b are also known [15]. Chimeric TNF-a anti-
body, infliximab [16], has been approved for the treatment of
rheumatoid arthritis and Crohn’s disease, while the TNF-a re-
ceptor fusion protein, etanercept [17] and IL-1 receptor antago-
nist, anakinra [18], have been approved for the treatment of
rheumatoid arthritis. As such, p38 MAP kinase mediated regu-
lation of TNF-a and IL-1b serves as an attractive target for the
treatment of inflammatory diseases [19]. The p38 MAP kinase
inhibitors such as SB203580 [20], BIRB-796, and SCIO-469
[21] are under clinical trial for the treatment of the rheumatoid
arthritis. Aminobenzophenone derivatives have also been re-
ported as a novel class of p38 MAP kinase inhibitors with
high anti-inflammatory activity [22]. Recent years have wit-
nessed the emergence of many candidates incorporating the
oximino feature in their structure having anti-inflammatory
2. Chemistry
The syntheses of tetrasubstituted thiophenes were carried
out as shown in Scheme 1. The intermediate compound (3)
was synthesized by the nucleophilic addition of arylisothiocya-
nate and enamine (2) in ether at 0 ꢂC. Enamine (2) was synthe-
sized by reacting ammonia with methyl acetoacetate (1) as per
reported procedure [28e30]. Tetrasubstituted thiophene esters
(4ae4i) were prepared by reaction of intermediate (3) with

K.I. Molvi et al. / European Journal of Medicinal Chemistry 42 (2007) 1049e1058
1051
O
O
NH2
O
Gujarat, India, and kept under standard laboratory condition at
25 ꢀ 2 ꢂC. The animals were provided with pellet diet (Lipton,
India) and water ad libitum. The institutional ethics committee
constituted by the Ministry of Social Justice and Empowerment,
Government of India approved the experimental protocol. All
experiments were carried out at K.B. Institute of Pharmaceutical
Education and Research, Gandhinagar, Gujarat, India.
(a)
O
O
2
1
(b)
O
O
R1
HN
S
H2N
3
(c)
3.1. Anti-inflammatory activity
(e)
O
R2
O
R1
O
R2
3.1.1. Carrageenin-induced rat hind paw oedema model
The method adopted resembles essentially that described by
Winter et al. [32]. The animals were studied for toxicity of
DMSO up to 10% v/v in saline, and 5% DMSO was selected
as a vehicle to suspend the standard drugs and the test com-
pounds. Albino rats weighing between 150 and 250 g of either
sex were starved for 18 h prior to the experiment. The animals
were weighed, marked for identification and divided into groups
of six. The standard drugs, ibuprofen (20 mg/kg body weight),
mefenamic acid (100 mg/kg body weight) and three graded
doses (10, 20 and 40 mg/kg body weight) of the test compounds
were given orally as a suspension using 5% DMSO as a vehicle.
One hour later foot paw oedema was induced by injecting 0.1 ml
of 1% carrageenin subcutaneously into the planter portion of the
right hind paw of each rat. Initial foot paw volumewas measured
immediately by mercury plethysmometer. Oedema was mea-
sured 3 h after carrageenin administration. The swelling in test
group animals was used to calculate the % inhibition ꢀ SEM
of oedema achieved by the compound at the test dose compared
with the vehicle control group. The % protection of oedema was
calculated according to the formula, % anti-inflammatory acti-
vity ¼ 100 ꢃ (1 ꢁ Vt/Vc) where Vt and Vc are the volume of
oedema in test compounds and control groups, respectively.
R1
O
N
H
S
4a-4i
N
H
S
O
HON
6a-6e(E); 6f(Z)
(d)
R2
R1
COOH
N
H
S
O
5a-5i
Scheme 1. Synthesis of tetrasubstituted thiophenes. Reagents and conditions:
(a) ammonia (25%), diethyl ether, 0e15 ꢂC, 1 h, 62%; (b) ArNCS, diethyl
ether, 0 ꢂCert, 5 h, 50e55%; (c) 4-substituted phenacyl bromides, acetonitrile,
rt, until no more starting material could be detected on TLC; (d) KOH, MeOH,
rt, 1 h, then removed in vacuo, extracted with diethyl ether, aqueous layer acid-
ified with dilute HCl; (e) oxime of 4-substituted phenacyl bromides, acetoni-
trile, rt, until no more starting material could be detected on TLC.
4-substituted phenacyl bromide in acetonitrile without adding
base at room temperature. The corresponding acids (5ae5i)
of tetrasubstituted thiophene esters (4ae4i) were prepared by
hydrolyzing them in methanol with 1 eq of potassium hydrox-
ide solution at room temperature. The intermediate (3) was sep-
arately treated with oxime of 4-substituted phenacyl bromide in
acetonitrile at room temperature to obtained oximino com-
pounds (6ae6f). All the synthesized compounds were charac-
3.2. Analgesic activity
3.2.1. Acetic acid induced writhing response model
1
terized by spectroscopic data such as mass, IR, H NMR and
The compounds of the ester series 4ae4i and their counter
partsinacidseries5ae5i wereselectedforinvestigatingtheiran-
algesicactivityin acetic acidinducedwrithingresponse in albino
mice. Following the method of Siegmund et al. [33], 10 mg/kg of
the selected compounds was administered intra-peritoneally to
six groups of mice (six in each group) starved for 16 h. The first
four groups received the test compounds while the fifth and sixth
groups, which served as positive and negative controls, respec-
tively, received 10 mg/kg ibuprofen and 0.5 ml/100 g body
weight of 1% DMSO solution. One hour after treatment, the an-
imals in each group received 0.1 ml of 3% acetic acid to induce
the characteristic writhing response. The number of writhing oc-
curring within 30 min was recorded and the mean was compared
with that of the control and converted into % inhibition.
elemental analysis.
1H NMR spectroscopy was used to assign E/Z configuration
of oxime compounds 6ae6f. The oxime compounds 6ae6e
were found to have E configuration while 6f has Z configura-
tion. E configuration of oxime compounds (6ae6e) was as-
signed on the basis of the absence of unshielding effect of
oxime oxygen on the CeCH3 singlet of the fourth position of
thiophene ring. For compounds 6a, 6b, 6c, 6d, and 6e shielded
singlet values are Dd ¼ ꢁ0.01, ꢁ0.05, ꢁ0.21, ꢁ0.86 and
ꢁ0.06, respectively. In case of 6f due to unshielding effect of
oxime oxygen on the CeCH3 singlet of the fourth position
thiophene ring, shielded singlet value is Dd ¼ þ0.23 which in-
dicates its Z configuration. These data agrees with previously
published data for E/Z configuration of oximes [31].
3.3. Antioxidant activity
3. Pharmacology
3.3.1. Nitric oxide radical scavenging assay
Albino rats (150e250 g) and mice (20e25 g) of either sex
were supplied by Cadilla Pharmaceuticals, Ahmedabad,
Nitric oxide generated from sodium nitroprusside in aqueous
solution at physiological pH interacts with oxygen to produce

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K.I. Molvi et al. / European Journal of Medicinal Chemistry 42 (2007) 1049e1058
nitrite ions, which can be measured by Griess reagent [34]. The
reaction mixture (3 ml) containing sodium nitroprusside
(10 mmol) in phosphate buffered saline (PBS) and test com-
pounds (4ae4i and 5ae5i) and reference compound at different
concentrations (5, 10, 15, 20, 25, 30, and 35 mg/ml) were incu-
bated at 25 ꢂC for 150 min. Each 30 min, 0.5 ml of the incubated
sample was removed. Griess reagent of 0.5 ml (1% sulphanila-
mide, 0.1% naphthylethylene diamine dihydrochloride in 2%
H3PO4) was added to the 0.5 ml aliquot of the sample removed.
The absorbance of the chromophore formed was measured at
546 nm. The experiment was performed (in triplicate) and %
scavenging activity was calculated using the formula 100 ꢁ
[100/blank absorbance ꢃ sample absorbance]. The activity
was compared with ascorbic acid at concentrations 0.1, 0.2,
0.4, 0.6, 0.8, 1.0 and 1.2 mg/ml, which was used as a standard
antioxidant.
(s ¼ ꢁ0.02; p ¼ ꢁ0.27) and CH3 (s ¼ ꢁ0.17; p ¼ þ0.56) as
hydrophilic and electron releasing groups 4c, 4g, 5c, and 5g,
were synthesized. Now keeping R2 ¼ OCH3 and CH3 as hy-
drophilic and electron releasing groups in benzoyl moiety
we introduced at R1 of the anilino moiety the representative
lipophilic and electron withdrawing substituents p-Cl
(s ¼ þ0.24; p ¼ þ0.71), p-F (s ¼ þ0.06; p ¼ þ0.14) 4d, 4e,
4h, and 4i and corresponding acids 5d, 5e, 5h, and 5i were
synthesized to gain more insight in to the structural require-
ments for the anti-inflammatory activity. In order to study
the anti-inflammatory effect of aryl oxime directly attached
to the fifth position of thiophene, oximino compounds 6ae6f
were synthesized.
In the ester series, 4c showed maximum anti-inflammatory
activity at all the three graded doses employed, 71% at 10 mg/kg,
72% at 20 mg/kg and 76% at 40 mg/kg. For 4a the % pro-
tection was found to be 69% at 10 mg/kg, 70% at 20 mg/kg
and 73% at 40 mg/kg. Similarly, for 4b the % protection
was found to be 50% at 10 mg/kg, 63% at 20 mg/kg, and
70% at 40 mg/kg. Compounds 4de4g showed moderate to
less activity as compared to 4ae4c. In the case of 4h and 4i
the increase in % protection was up to a dose of 20 mg/kg
but % protection decreases at 40 mg/kg dose. To summarize
these findings, the presence of an electron releasing substitu-
ents OCH3 at R2 and OCH3, CH3 at R1 was the necessary
condition to enhance the anti-inflammatory potential of
the candidate. However, the presence of R1 ¼ R2 ¼ CH3
4. Results and discussion
All the synthesized compounds were tested in the carra-
geenin-induced 3 h rat paw oedema model, to determine their
anti-inflammatory potential; the results are shown in Table 1.
The selection of substituents on R1 and R2 in tetrasubstituted
thiophene was mainly guided by lipophilicity and electronic
considerations as defined by sep Craig plot. All the selected
substituents are spread over the three quadrants ꢁs, ꢀp of the
Craig plot [25]. Initially keeping both R1 and R2 ¼ OCH3
Table 1
Compound
R1
R2
Anti-inflammatory activitya carrageenin-induced
rat hind paw oedema % protection
Analgesic activityb acetic acid induced
writhing test % protection
10 mg/kg
20 mg/kg
40 mg/kg
10 mg/kg
4a
4b
4c
4d
4e
4f
H
OCH3
OCH3
OCH3
OCH3
OCH3
CH3
69
50
71
36
42
43
32
38
34
79
56
72
57
49
45
35
38
63
05
22
20
03
13
13
70
63
72
53
40
49
36
46
66
80
63
75
57
49
47
38
44
68
38
27
25
12
21
29
73
70
76
58
39
53
37
23
26
70
62
69
54
43
42
38
42
59
34
30
30
30
30
42
55
45
40
40
60
27
22
37
43
30
26
22
28
30
17
18
22
24
e
CH3
OCH3
F
Cl
H
4g
4h
4i
CH3
F
CH3
CH3
Cl
H
CH3
5a
5b
5c
5d
5e
5f
OCH3
OCH3
OCH3
OCH3
OCH3
CH3
CH3
OCH3
F
Cl
H
5g
5h
5i
CH3
F
CH3
CH3
Cl
H
CH3
6a
6b
6c
6d
6e
6f
OCH3
OCH3
OCH3
OCH3
CH3
CH3
F
e
e
Cl
e
H
Cl
e
e
CH3
a
Oral administration for all test compounds, P < 0.05, Student’s t-test versus controls, the standard drugs (dose and % protection) were ibuprofen (20 mg/kg,
33%) and mefenamic acid (100 mg/kg, 39%).
b
Intra-peritoneal administration for all test compounds, P < 0.05, Student’s t-test versus controls, the standard drug (dose and % protection) was ibuprofen
(10 mg/kg, 60%).

K.I. Molvi et al. / European Journal of Medicinal Chemistry 42 (2007) 1049e1058
1053
remarkably decreased the anti-inflammatory potential of the
candidate 4g. Keeping the electron donating groups OCH3
and CH3 at R2 in benzoyl moiety as constant, introduction
of the representative lipophilic and electron withdrawing sub-
stituents F and Cl at R1 in anilino moiety leads to decrease in
anti-inflammatory activity of the candidates 4d, 4e, 4h, and 4i
at all the three graded doses as compared to 4ae4c. However,
these compounds showed comparable better activity than 4g.
To explore the effect of the acid function at C-3 of thiophene
nucleus as compared to ester we have synthesized the acid an-
alogues 5ae5i. In acid series, there is a general trend of dis-
playing increasing anti-inflammatory activity of compounds
up to 20 mg/kg but decreasing % protection at 40 mg/kg
dose. Compound 5a was found to be most potent compound
having % protection 79% at 10 mg/kg, 80% at 20 mg/kg and
70% at 40 mg/kg dose. Compound 5c showed 72% protection
at 10 mg/kg, 75% at 20 mg/kg and 69% at 40 mg/kg dose. In
acid series also the presence of eCH3 at both R1 and R2 (5g)
remarkably decrease anti-inflammatory activity. Also when
R1 ¼ CH3 and R2 ¼ OCH3 (5b) the activity was less as com-
pared to 5a and 5c. Similar to ester series when R1 ¼ F, Cl
and R2 ¼ OCH3, CH3 (5d, 5e, 5h, and 5i) the anti-inflamma-
tory activity was found to be moderate as compared to 5a
and 5c. In case of oxime series 6ae6f, the anti-inflammatory
activities of the candidates were found to be poor as compared
to acid and ester series. The best candidate among whole se-
ries was 4c, which showed anti-inflammatory activity at
a very low dose as compared to both of the standard drug me-
fenamic acid and ibuprofen.
test in albino mice at 10 mg/kg dose and results are shown
in Table 1. Among the ester series compound 4e showed max-
imum protection of 60%, while 4a, 4b, and 4i exhibited 55%,
45%, and 43% protection, respectively, at 10 mg/kg dose. The
result showed that presence of H, Cl at R1 and OCH3, CH3 at
R2 in tetrasubstituted thiophene ester series enhances their an-
algesic activity. The candidates of acid series 5ae5i showed
poor analgesic activity as compared to the standard drug
ibuprofen.
Taking into account, in vivo comparable anti-inflammatory
activity results of esters 4ae4i and acid compounds 5ae5i, in
vitro antioxidant nitric oxide radical scavenging assay of these
compounds was performed at concentrations 5, 10, 15, 20, 25,
30 and 35 mg/ml and results are shown in Table 2. In the ester se-
ries, 4a showed maximum in vitro nitric oxide radical scaveng-
ing activity having IC50 value 30.08 mg/ml. Compounds 4c and
4d showed nearly comparable activity having IC50 values 72.21
and 74.17 mg/ml, respectively. Compound 4f showed very poor
antioxidant activity. Compounds 4b, 4e, 4g, 4h and 4i have
showed no scavenging activity. In acid series, 5a showed maxi-
mum invitro nitric oxide radical scavenging activity having IC50
value 25.20 mg/ml. Compound 5c was found to have comparable
activity having IC50 value 36.57 mg/ml. Compounds 5d and 5f
showed very poor antioxidant activity.
Compounds 5b, 5e, 5g, 5h and 5i have showed no scaveng-
ing activity. The best candidate among whole series was 5a;
however, it was found to have poor in vitro antioxidant nitric
oxide radical scavenging activity as compared to standard drug
ascorbic acid which showed IC50 value 00.52 mg/ml at concen-
trations 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 and 1.2 mg/ml. From the IC50
values of 4ae4i and 5ae5i, it can be concluded that the
The analgesic activity of ester and acid compound series
was investigated using acetic acid induced writhing response
Table 2
Compound % Scavenging (mean ꢀ SEM) of triplicates
5 mg/ml
10 mg/ml
15 mg/ml
20 mg/ml
25 mg/ml
30 mg/ml
35 mg/ml
aIC50 mg/ml rc
30.08 0.95
4a
13.33 ꢀ 0.002* 18.01 ꢀ 0.001* 28.62 ꢀ 0.003* 32.44 ꢀ 0.001* 36.86 ꢀ 0.002* 48.08 ꢀ 0.003* 63.04 ꢀ 0.002*
4b
4c
e
e
e
e
e
e
e
e
e
4.29 ꢀ 0.001*
5.90 ꢀ 0.002*
7.80 ꢀ 0.002* 11.98 ꢀ 0.003* 15.45 ꢀ 0.001* 20.54 ꢀ 0.002* 25.05 ꢀ 0.001*
72.21
74.17
0.97
0.93
e
4d
4e
5.28 ꢀ 0.002* 14.03 ꢀ 0.002* 15.24 ꢀ 0.001* 18.15 ꢀ 0.003* 20.18 ꢀ 0.001* 23.25 ꢀ 0.001* 25.58 ꢀ 0.001*
e
e
e
e
e
e
e
e
4f
4g
2.08 ꢀ 0.001*
3.16 ꢀ 0.002*
4.59 ꢀ 0.001*
6.06 ꢀ 0.002*
8.16 ꢀ 0.002*
9.35 ꢀ 0.002* 10.25 ꢀ 0.002* 171.43
0.99
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
4h
4i
e
5a
19.60 ꢀ 0.002* 24.71 ꢀ 0.001* 36.47 ꢀ 0.001* 41.17 ꢀ 0.003* 52.58 ꢀ 0.001* 59.45 ꢀ 0.003* 61.22 ꢀ 0.002*
25.20
0.97
e
0.97
0.94
e
5b
5c
e
e
e
e
e
e
e
e
20.15 ꢀ 0.003* 23.47 ꢀ 0.001* 30.63 ꢀ 0.002* 32.47 ꢀ 0.001* 38.47 ꢀ 0.001* 41.23 ꢀ 0.002* 50.98 ꢀ 0.003*
36.57
00.10 ꢀ 0.003* 00.98 ꢀ 0.002* 01.98 ꢀ 0.003* 03.58 ꢀ 0.003* 08.36 ꢀ 0.002* 10.52 ꢀ 0.001* 12.75 ꢀ 0.001* 118.32
5d
5e
e
e
e
e
e
e
e
e
5f
5g
00.26 ꢀ 0.003* 00.85 ꢀ 0.001* 01.25 ꢀ 0.002*
2.68 ꢀ 0.001* 04.35 ꢀ 0.002* 05.13 ꢀ 0.003* 05.55 ꢀ 0.003* 259.47
0.96
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
5h
5i
e
e
0.98
Ascorbic
acidb
02.98 ꢀ 0.001* 11.54 ꢀ 0.002* 29.51 ꢀ 0.001* 39.33 ꢀ 0.003* 41.62 ꢀ 0.003* 58.34 ꢀ 0.001* 70.24 ꢀ 0.002*
0.52
*P < 0.001 compared to reagent blank.
e Showed no scavenging activity.
a
IC50 ¼ 50% Inhibitory concentration.
b
Ascorbic acid tested at 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 mg/ml.
Regration analysis.
c

1054
K.I. Molvi et al. / European Journal of Medicinal Chemistry 42 (2007) 1049e1058
presence of R1 ¼ H and R2 ¼ OCH3 enhances nitric oxide
radical scavenging property. The presence of R1 ¼ R2 ¼ OCH3
also enhances nitric oxide radical scavenging ability in acid
derivative 5c (IC50 value 36.57 mg/ml); while it enhances mod-
erately the nitric oxide radical scavenging ability in ester
derivative 4c (IC50 value 72.21 mg/ml). The presence of CH3
either at R1 and R2 leads to near or total loss of antioxidant
activity of tested compounds.
FT-NMR (Advance DPX200) spectrometer using tetramethylsi-
lane as internal standard and the chemical shifts (d) are reported
in parts per million, coupling constants (J ) are given in hertz.
Masses of all the compounds were recorded on PerkineElmer
Sciex atmospheric pressure ionization liquid chromatography
mass instrument (LCMS). In this technique some of the com-
pounds gave a molecular ion and an additional peak at
(M þ 23), which was due to (M þ Na). Elemental analyses
were carried out on Carlo-Erba 1108 instrument or Elementar’s
Vario EL III micro-analyzer. UV spectra were recorded in Shi-
madzu 1601 UVevis spectrophotometer. All chromatographic
purification was performed with silica gel 60 (100e200 or
200e400 mesh), whereas all TLC (silica gel) development
was performed on silica gel coated (Merck Kiesel 60 GF-254,
0.2 mm thickness) sheets. Ibuprofen, mefenamic acid and ascor-
bic acid drugs were obtained from Baroda Chemist Association
(Baroda, India). Griess reagent was procured from Aldrich
Sigma Chemical Co. Ltd., USA. The chemicals used, namely,
methyl acetoacetate, p-chloroaniline, aniline, carbon disulfide,
triethylamine, acetonitrile, methanol, DMSO, DMF, dichloro-
methane, toluene, chloroform, sodium nitroprusside (Ranbaxy
Fine Chemicals, Punjab, India), phenyl isothiocyanate (Lances-
ter U.K.), substituted acetophenones, carrageenin, ethyl chloro-
formate and 4-fluoroaniline, (Spectrochem Mumbai, India)
were used without further purification unless otherwise stated.
5. Conclusion
A new series of tetrasubstituted thiophene analogues were
designed, synthesized and characterized. The synthesized com-
pounds 4ae4i, 5ae5i, and 6ae6f were evaluated for their anti-
inflammatory activity in carrageenin-induced rat hind paw oe-
dema model; while 4ae4i and 5ae5i were investigated
for their analgesic activity in acetic acid induced writhing re-
sponse model at 10 mg/kg dose. As the synthesized compounds
have the structural features of COX-1, 5-LOX (acid/ester, as in
mefenamic acid) and the p38 MAP kinase inhibitor; the anti-in-
flammatory activity exhibited by the experimental candidates
may be due to blocking of more than one rate limiting steps in
the inflammatory cascade. In search of the optimum structural
requirements for the anti-inflammatory activity around the thio-
phene scaffold it was possible to find, on the basis of SAR stud-
ies of target compounds, that the presence of OCH3 at R2
position and H, OCH3 at R1 in target compounds are the require-
ments for eliciting comparable anti-inflammatory activity in
both the ester and acid series. The best candidate among whole
series was 4c, which showed anti-inflammatory activity at a very
low dose as compared to both of the standard drug mefenamic
acid and ibuprofen. It is, therefore, suggested that the compound
to be studied further to explore its full potential particularly in
the treatment of chronic inflammatory diseases. The best com-
pound of the series was an ester derivative 4e that showed max-
imum analgesic activity of 60% protection in acetic acid
induced writhing response test in albino mice at a dose of
10 mg/kg dose. The result showed that presence of H, Cl at R1
and OCH3, CH3 at R2 in tetrasubstituted thiophene ester series
enhances their analgesic activity. The best candidate among
whole series was 5a which was found to have maximum in vitro
nitric oxide radical scavenging activity having IC50 value
25.20 mg/ml. The results showed the presence of R1 ¼ H,
R2 ¼ OCH3 and also R1 ¼ R2 ¼ OCH3 enhances nitric oxide
radical scavenging property in tetrasubstituted thiophene acid
series. In vitro antioxidant nitric oxide radical scavenging assay
of 4ae4i and 5ae5i compounds showed that the mechanism of
anti-inflammatory activity of potent candidates in ester and acid
series could be mediated through inhibition of nitric oxide burst
in inflammatory situation.
6.1. General procedure for the synthesis
of compounds 4ae4i
As shown in Scheme 1, 1-(a-Carbomethoxy-b-aminothioc-
rotonoyl)-4-substituted aniline 3 was synthesized by nucleo-
philic addition of arylisothiocyanate and enamine 2, which in
turn was synthesized by reacting ammonia with methyl ace-
toacetate. Compounds 4ae4i were synthesized by adding
0.001 moloftherespectivesubstitutedphenacylbromidetoaso-
lution of 3 (0.001 mol) in 2 ml of acetonitrile without adding
base at room temperature. The solution was stirred until the solid
was separated from the reaction mixture or until no more starting
materials could be detected on TLC. The solid that separated
was filtered off, washed with chilled acetonitrile, dried, and
yielded yellow coloured product corresponding to 4ae4i char-
acterized as per the analytical data.
6.1.1. Methyl-2-anilino-5-(4-methoxybenzoyl)-
4-methylthiophene-3-carboxylate (4a)
Yield: 84%; m.p. 120e122 ꢂC; MS: m/z 382 (M þ 1); IR
(KBr): 3250, 3050, 1666 (C]O stretching), 1600, 1445,
1364, 1199e1238, 1170, 996, 697e755 (strongest of thio-
phene bands, CeS stretching) cmꢁ1 1H NMR (300 MHz,
.
CDCl3): d 2.46 (s, 3H, CH3 at fourth position), 3.87 (s, 3H,
OCH3 at fifth position), 3.91 (s, 3H, CH3 of ester at third po-
sition), 6.92e6.95 (d, 2H, J ¼ 5.44, aromatic protons ortho to
OCH3 at fifth position), 7.16e7.41 (m, 5H, aromatic protons
of second position), 7.72e7.75 (d, 2H, aromatic protons
meta to OCH3 at fifth position), 10.59 (s, 1H, NH at second
position). Anal. calcd. for C21H19NO4S: C 66.15, H 4.98, N
3.67. Found: C 66.16, H 5.14, N 3.67%.
6. Experimental
Melting points were recorded on capillary melting point ap-
paratus and are uncorrected. The infrared spectra in KBr were
recorded, on Buck Scientific M-500 Infrared spectrophotome-
1
ter. H NMR spectra were recorded on 300/200 MHz Bruker

K.I. Molvi et al. / European Journal of Medicinal Chemistry 42 (2007) 1049e1058
1055
6.1.2. Methyl-2-(4-methylanilino)-5-(4-methoxybenzoyl)-
4-methylthiophene-3-carboxylate (4b)
Anal. calcd. for C21H18ClNO4S: C 60.67, H 4.33, N 3.36.
Found: C 60.91, H 4.16, N 3.47%.
Yield: 65%; m.p. 247e249 ꢂC; MS: m/z 395 (Mþ); IR
(KBr): 3452, 1615 (C]O stretching), 1577, 1428, 1339,
1191, 680e774 (strongest of thiophene bands, CeS stretch-
ing). 1H NMR (300 MHz, CDCl3): d 2.39 (s, 6H, CH3 at fourth
position and CH3 at second position), 3.86 (s, 6H, CH3 of ester
at third position and OCH3 at fifth position), 7.00e7.02 (d, 2H,
J ¼ 5.44, aromatic protons ortho to OCH3 at fifth position),
7.26e7.28 (m, 4H, aromatic protons of second position),
7.72e7.75 (d, 2H, aromatic protons meta to OCH3 at fifth po-
sition), 10.51 (s, 1H, NH at second position). Anal. calcd. for
C22H21NO4S: C 66.85, H 5.31, N 3.54. Found: C 66.54, H
5.65, N 3.42%.
6.1.6. Methyl-2-anilino-5-(4-methylbenzoyl)-
4-methylthiophene-3-carboxylate (4f)
Yield: 35%; m.p. 138e140 ꢂC; MS: m/z 388 (M þ 23); IR
(KBr): 3550, 2450, 1678 (C]O stretching), 1615, 1368, 1180,
987, 685e745 (strongest of thiophene bands, CeS stretching)
1
cmꢁ1. H NMR (300 MHz, CDCl3): d 2.42 (s, 3H, CH3 at
fourth position), 2.46 (s, 3H, CH3 at fifth position), 3.91 (s,
3H, CH3 of ester at third position), 7.23e7.26 (d, 2H,
J ¼ 5.61, aromatic protons ortho to CH3 at fifth position),
7.33e7.41 (m, 5H, aromatic protons of second position),
7.61e7.64 (d, 2H, aromatic protons meta to CH3 at fifth posi-
tion), 10.61 (s, 1H, NH at second position). Anal. calcd. for
C21H19NO3S: C 69.02, H 5.20, N 3.83. Found: C 68.70, H
5.35, N 4.01%.
6.1.3. Methyl-2-(4-methoxyanilino)-5-(4-methoxybenzoyl)-
4-methylthiophene-3-carboxylate (4c)
Yield: 30%; m.p. 139e141 ꢂC; MS: m/z 412 (M þ 1); IR
(KBr): 3250, 2890, 1667 (C]O stretching), 1552, 1517,
1364, 1263, 1138, 996, 697e755 (strongest of thiophene
6.1.7. Methyl-2-(4-methylanilino)-5-(4-methylbenzoyl)-
4-methylthiophene-3-carboxylate (4g)
1
bands, CeS stretching) cmꢁ1. H NMR (300 MHz, CDCl3):
Yield: 43%; m.p. 204e206 ꢂC; MS: m/z 379 (Mþ); IR
(KBr): 3450, 2370, 1627 (C]O stretching), 1602, 1363,
1142, 940, 685e706 (strongest of thiophene bands, CeS
d 2.48 (s, 3H, CH3 at fourth position), 3.88 (s, 3H, CH3 of ester
at third position), 3.92 (s, 6H, OCH3 at second position and
fifth position), 6.91e6.94 (d, 4H, aromatic protons ortho to
OCH3 at second position and fifth position), 7.72e7.75 (d,
4H, aromatic protons meta to OCH3 at second position and
fifth position), 10.51 (s, 1H, NH at second position). Anal.
calcd. for C22H21NO5S: C 64.23, H 5.10, N 3.40. Found: C
63.59, H 4.92, N 3.71%.
stretching) cmꢁ1 1H NMR (300 MHz, CDCl3): d 2.28 (s,
.
3H, CH3 at fourth position), 2.30 (s, 6H, CH3 at second posi-
tion and fifth position), 3.56 (s, 3H, CH3 of ester at third po-
sition), 6.88e6.91 (d, 4H, J ¼ 6.03, aromatic protons ortho
to CH3 at second position and fifth position), 7.09e7.12 (d,
4H, aromatic protons meta to CH3 at second position and fifth
position), 10.27 (s, 1H, NH at second position). Anal. calcd.
for C22H21NO3S: C 69.64, H 5.53, N 3.69. Found: C 69.78,
H 5.15, N 3.81%.
6.1.4. Methyl-2-(4-fluoroanilino)-5-(4-methoxybenzoyl)-
4-methylthiophene-3-carboxylate (4d)
Yield: 75%; m.p. 146e148 ꢂC; MS: m/z 400 (M þ 1); IR
(KBr): 3100, 2363, 1660 (C]O stretching), 1607, 1431,
1363, 1238, 1174, 721 (strongest of thiophene bands, CeS
6.1.8. Methyl-2-(4-fluoroanilino)-5-(4-methylbenzoyl)-
4-methylthiophene-3-carboxylate (4h)
stretching) cmꢁ1
.
1H NMR (200 MHz, CDCl3): d 2.57 (s,
Yield: 35%; m.p. 129e131 ꢂC; MS: m/z 383 (Mþ); IR
(KBr): 3359, 2365, 1670 (C]O stretching), 1612, 1491,
1363, 1180, 905, 680e744 (strongest of thiophene bands,
3H, CH3 at fourth position), 3.01 (s, 3H, CH3 of ester at third
position), 3.93 (s, 3H, OCH3 at fifth position), 7.05e7.09 (d,
2H, J ¼ 5.28, aromatic protons ortho to OCH3 at fifth posi-
tion), 7.26 (s, 4H, aromatic protons at second position),
7.49e7.53 (d, 2H, aromatic protons meta to OCH3 at fifth po-
sition), 10.34 (s, 1H, NH at second position). Anal. calcd. for
C21H18FNO4S: C 63.17, H 4.50, N 3.50. Found: C 63.67, H
4.92, N 3.71%.
1
CeS stretching) cmꢁ1. H NMR (300 MHz, CDCl3): d 2.35
(s, 3H, CH3 at fourth position), 2.48 (s, 3H, CH3 of ester at
third position), 3.92 (s, 3H, CH3 at fifth position), 7.18e7.21
(d, 2H, J ¼ 6.23, aromatic protons ortho to CH3 at fifth posi-
tion), 7.25 (s, 4H, aromatic protons of second position),
7.62e7.65 (d, 2H, aromatic protons meta to CH3 at fifth posi-
tion), 10.48 (s, 1H, NH at second position). Anal. calcd. for
C21H18FNO3S: C 65.80, H 4.59, N 3.65. Found: C 66.01, H
4.92, N 4.01%.
6.1.5. Methyl-2-(4-chloroanilino)-5-(4-methoxybenzoyl)-
4-methylthiophene-3-carboxylate (4e)
Yield: 90%; m.p. 132e134 ꢂC; MS: m/z 416 (M þ 1); IR
(KBr): 3120, 2365, 1670 (C]O stretching), 1593, 1441,
1368, 1245, 1169, 926, 692e780 (strongest of thiophene
6.1.9. Methyl-2-(4-chloroanilino)-5-(4-methylbenzoyl)-
4-methylthiophene-3-carboxylate (4i)
1
bands, CeS stretching) cmꢁ1. H NMR (300 MHz, CDCl3):
Yield: 25%; m.p. 128e130 ꢂC; MS: m/z 400 (M þ 1); IR
(KBr): 3540, 2455, 1670 (C]O stretching), 1592, 1441,
1368, 1242, 926, 683e780 (strongest of thiophene bands,
d 2.46 (s, 3H, CH3 at fourth position), 3.88 (s, 3H, OCH3 at
fifth position), 3.91 (s, 3H, CH3 of ester at third position),
6.92e6.95 (d, 2H, J ¼ 5.65, aromatic protons ortho to OCH3
at fifth position), 7.26e7.38 (m, 4H, aromatic protons at sec-
ond position), 7.72e7.75 (d, 2H, aromatic protons meta to
OCH3 at fifth position), 10.59 (s, 1H, NH at second position).
1
CeS stretching) cmꢁ1. H NMR (300 MHz, CDCl3): d 2.13
(s, 3H, CH3 at fourth position), 2.30 (s, 3H, CH3 at fifth posi-
tion), 3.88 (s, 3H, CH3 of ester at third position), 7.15e7.17
(d, 2H, aromatic protons ortho to CH3 at fifth position),

1056
K.I. Molvi et al. / European Journal of Medicinal Chemistry 42 (2007) 1049e1058
7.22e7.30 (m, 4H, aromatic protons of second position),
7.41e7.44 (d, 2H, J ¼ 5.26, aromatic protons meta to CH3 at
fifth position), 10.41 (s, 1H, NH at second position). Anal.
calcd. for C21H18ClNO3S: C 63.07, H 4.50, N 3.50. Found:
C 63.09, H 4.35, N 3.73%.
7.85e7.89 (d, 4H, J ¼ 5.72, aromatic protons ortho to OCH3
at second position and fifth position), 10.89 (s, 1H, NH at sec-
ond position). Anal. calcd. for C21H19NO5S: C 63.48, H 4.78,
N 3.52. Found: C 63.89, H 4.65, N 3.32%.
6.2.4. 2-(4-Fluoroanilino)-5-(4-methoxybenzoyl)-
6.2. General procedure for the synthesis
of compounds 5ae5i
4-methylthiophene-3-carboxylic acid (5d)
Yield: 56%; m.p. 202e204 ꢂC; MS: m/z 385 (Mþ); IR
(KBr): 2949, 2405, 1699 (C]O stretching), 1594, 1441,
1391, 1242, 1142, 672e723 (strongest of thiophene bands,
The corresponding acids (5ae5i) of the tetrasubstituted
thiophene esters were synthesized by treating 4ae4i in meth-
anol with 1 eq of potassium hydroxide solution and stirring at
room temperature until TLC showed the complete disappear-
ance of the respective ester. The solvent was then removed
in vacuo, treated with a small amount of distilled water and
extracted with ether. The ether layer was discarded while the
aqueous layer was cooled and treated with 1 eq of dilute hy-
drochloric acid to liberate the acids (5ae5i).
CeS stretching) cmꢁ1 1H NMR (200 MHz, DMSO-d6):
.
d 2.57 (s, 3H, CH3 at fourth position), 3.93 (s, 3H, OCH3 at
fifth position), 7.05e7.09 (d, 2H, aromatic protons ortho to
OCH3 at fifth position), 7.26 (s, 4H, aromatic protons at sec-
ond position), 7.49e7.53 (d, 2H, J ¼ 5.29, aromatic protons
meta to OCH3 at fifth position), 10.28 (s, 1H, NH at second
position). Anal. calcd. for C20H16FNO4S: C 62.33, H 4.15,
N 3.63. Found: C 61.78, H 4.54, N 3.88%.
6.2.1. 2-Anilino-5-(4-methoxybenzoyl)-4-
6.2.5. 2-(4-Chloroanilino)-5-(4-methoxybenzoyl)-
methylthiophene-3-carboxylic acid (5a)
4-methylthiophene-3-carboxylic acid (5e)
Yield: 72%; m.p. 206e208 ꢂC; MS: m/z 367 (Mþ); IR
(KBr): 3212, 2911, 1635 (C]O stretching), 1597, 1450,
1356, 1211, 1172, 1111, 868, 698e756 (strongest of thiophene
Yield: 78%; m.p. 165e167 ꢂC; MS: m/z 403 (M þ 2); IR
(KBr): 3747, 2364, 1670 (C]O stretching), 1590, 1431,
1256, 1174, 931, 691e775 (strongest of thiophene bands,
1
bands, CeS stretching) cmꢁ1. H NMR (200 MHz, DMSO-
CeS stretching) cmꢁ1 1H NMR (200 MHz, DMSO-d6):
.
d6): d 2.40 (s, 3H, CH3 at fourth position), 3.82 (s, 3H,
OCH3 at fifth position), 6.99e7.03 (d, 2H, J ¼ 4.89, aromatic
protons ortho to OCH3 at fifth position), 7.19e7.42 (m, 5H,
aromatic protons of second position), 7.62e7.66 (d, 2H, aro-
matic protons meta to OCH3 at fifth position), 10.60 (s, 1H,
NH at second position). Anal. calcd. for C20H17NO4S: C
65.40, H 4.62, N 3.81. Found: C 65.01, H 4.92, N 4.01%.
d 2.40 (s, 3H, CH3 at fourth position), 3.82 (s, 3H, OCH3 at
fifth position), 6.99e7.03 (d, 2H, J ¼ 5.10, aromatic protons
ortho to OCH3 at fifth position), 7.28e7.48 (m, 4H, aromatic
protons of second position), 7.62e7.66 (d, 2H, aromatic pro-
tons meta to OCH3 at fifth position), 10.75 (s, 1H, NH at sec-
ond position). Anal. calcd. for C20H16ClNO4S: C 59.80, H
3.98, N 3.48. Found: C 59.85, H 4.01, N 3.63%.
6.2.2. 2-(4-Methylanilino)-5-(4-methoxybenzoyl)-
4-methylthiophene-3-carboxylic acid (5b)
6.2.6. 2-Anilino-5-(4-methylbenzoyl)-4-methylthiophene-
3-carboxylic acid (5f)
Yield: 49%; m.p. > 250 ꢂC; MS: m/z 382 (M þ 1); IR
(KBr): 3037, 2360, 1692 (C]O stretching), 1555, 1454,
1240, 1169, 975, 674e714 (strongest of thiophene bands,
Yield: 55%; m.p. 194e196 ꢂC; MS: m/z 351 (Mþ); IR
(KBr): 3124, 2501, 1663 (C]O stretching), 1601, 1358,
1188, 974, 673e760 (strongest of thiophene bands, CeS
CeS stretching) cmꢁ1
.
1H NMR (200 MHz, DMSO-d6):
stretching) cmꢁ1. H NMR (200 MHz, DMSO-d6): d 2.39 (s,
1
d 2.47 (s, 3H, CH3 at fourth position), 2.48 (s, 3H, CH3 at sec-
ond position), 3.32 (s, 3H, OCH3 at fifth position), 6.99e7.03
(d, 2H, J ¼ 4.97, aromatic protons ortho to OCH3 at fifth po-
sition), 7.33e7.48 (m, 4H, aromatic protons of second posi-
tion), 7.62e7.66 (d, 2H, aromatic protons meta to OCH3 at
fifth position), 10.47 (s, 1H, NH at second position). Anal.
calcd. for C21H19NO4S: C 66.15, H 4.98, N 3.67. Found: C
66.16, H 4.80, N 3.97%.
3H, CH3 at fourth position), 3.82 (s, 3H, CH3 at fifth position),
6.70e6.74 (d, 2H, aromatic protons ortho to CH3 at fifth posi-
tion), 7.37e7.43 (m, 5H, aromatic protons of second position),
7.70e7.74 (d, 2H, J ¼ 4.74, aromatic protons meta to CH3 at
fifth position), 10.78 (s, 1H, NH at second position). Anal.
calcd. for C20H17NO3S: C 68.38, H 4.83, N 3.98. Found: C
68.54, H 4.78, N 3.61%.
6.2.7. 2-(4-Methylanilino)-5-(4-methylbenzoyl)-
6.2.3. 2-(4-Methoxyanilino)-5-(4-methoxybenzoyl)-
4-methylthiophene-3-carboxylicacid (5c)
4-methylthiophene-3-carboxylic acid (5g)
Yield: 38%; m.p. 296e298 ꢂC; MS: m/z 365 (Mþ); IR
(KBr): 3350, 2338, 1661 (C]O stretching), 1607, 1542,
1378, 1143, 958, 680e767 (strongest of thiophene bands,
Yield: 40%; m.p. 210e212 ꢂC; MS: m/z 397 (Mþ); IR
(KBr): 3180, 1661 (C]O stretching), 1589, 1495, 1351,
1237, 1122, 1002, 680e785 (strongest of thiophene bands,
1
CeS stretching) cmꢁ1. H NMR (300 MHz, CDCl3): d 2.28
CeS stretching) cmꢁ1
.
1H NMR (200 MHz, DMSO-d6):
(s, 3H, CH3 at fourth position), 2.30 (s, 6H, CH3 at second po-
sition and fifth position), 6.88e6.91 (d, 4H, J ¼ 6.03, aromatic
protons ortho to CH3 at second position and fifth position),
7.09e7.12 (d, 4H, aromatic protons meta to CH3 at second
d 2.48 (s, 3H, CH3 at fourth position), 3.92 (s, 6H, OCH3 at
second position and fifth position), 7.17e7.21 (d, 4H, aromatic
protons meta to OCH3 at second position and fifth position),

K.I. Molvi et al. / European Journal of Medicinal Chemistry 42 (2007) 1049e1058
1057
position and fifth position), 10.40 (s, 1H, NH at second posi-
tion). Anal. calcd. for C21H19NO3S: C 69.05, H 5.20, N
3.83. Found: C 69.25, H 4.80, N 3.84%.
6.3.2. E-4-Methoxyphenyl-[2-(4-methylanilino)-
3-methoxycarbonyl-4-methyl-5-thienyl]ketoxime (6b)
Yield: 40%; m.p. 168e170 ꢂC; MS: m/z 410 (Mþ); IR
(KBr): 3022, 2959, 1660 (C]O stretching), 1596, 1504,
1433, 1360, 1168, 688e780 (strongest of thiophene bands,
6.2.8. 2-(4-Fluoroanilino)-5-(4-methylbenzoyl)-
4-methylthiophene-3-carboxylic acid (5h)
1
CeS stretching) cmꢁ1. H NMR (300 MHz, CDCl3): d 2.34
Yield: 30%; m.p. 185e187 ꢂC; MS: m/z 369 (Mþ); IR
(KBr): 3360, 2400, 1672 (C]O stretching), 1603, 1490,
1360, 1180, 910, 675e740 (strongest of thiophene bands,
(s, 3H, CH3 at fourth position), 2.45 (s, 3H, CH3 at second po-
sition), 3.87(s, 3H, OCH3 at fifth position), 3.90 (s, 3H, CH3 of
ester at third position), 6.91e6.94 (d, 2H, J ¼ 6.08, aromatic
protons ortho to OCH3 at fifth position), 7.17e7.26 (m, 4H,
aromatic protons of second position), 7.71e7.74 (d, 2H, aro-
matic protons meta to OCH3 at fifth position), 10.44 (s, 1H,
NH at second position). Anal. calcd. for C22H22N2O4S: C
64.39, H 5.36, N 6.82. Found: C 64.65, H 5.78, N 6.73%.
1
CeS stretching) cmꢁ1. H NMR (200 MHz, CDCl3): d 2.28
(s, 3H, CH3 at fourth position), 3.45 (s, 3H, CH3 at fifth posi-
tion), 7.05e7.26 (m, 8H, aromatic protons of second position
and fifth position), 10.09 (s, 1H, NH at second position). Anal.
calcd. for C20H16FNO3S: C 65.04, H 4.33, N 3.79. Found: C
65.67, H 3.97, N 3.73%.
6.3.3. E-4-Methoxyphenyl-[2-(4-fluoroanilino)-
6.2.9. 2-(4-Chloroanilino)-5-(4-methylbenzoyl)-
4-methylthiophene-3-carboxylic acid (5i)
3-methoxycarbonyl-4-methyl-5-thienyl]ketoxime (6c)
Yield: 45%; m.p. 130e132 ꢂC; MS: m/z 414 (Mþ); IR
(KBr): 3159, 1660 (C]O stretching), 1608, 1548, 1430,
1280, 1184, 825, 668e721 (strongest of thiophene bands,
Yield: 35%; m.p. 179e181 ꢂC; MS: m/z 386 (M þ 1); IR
(KBr): 3280, 2363, 1700 (C]O stretching), 1588, 1438,
1352, 1246, 945, 680e740 (strongest of thiophene bands,
1
CeS stretching) cmꢁ1. H NMR (200 MHz, CDCl3): d 2.36
CeS stretching) cmꢁ1
.
1H NMR (200 MHz, DMSO-d6):
(s, 3H, CH3 at fourth position), 2.42 (s, 3H, OCH3 at fifth po-
sition), 3.89 (s, 3H, CH3 of ester at third position), 6.90e6.93
(d, 2H, J ¼ 5.86, aromatic protons ortho to OCH3 at fifth po-
sition), 7.17e7.26 (m, 4H, aromatic protons of second posi-
tion), 7.72e7.75 (d, 2H, aromatic protons meta to OCH3 at
fifth position), 10.44 (s, 1H, NH at second position). Anal.
calcd. for C21H19FN2O4S: C 60.88, H 4.58, N 6.75. Found:
C 60.89, H 4.54, N 6.85%.
d 2.42 (s, 3H, CH3 at fourth position), 2.66 (s, 3H, CH3 at fifth
position), 7.03e7.08 (d, 2H, aromatic protons ortho to CH3 at
fifth position), 7.28e7.48 (m, 4H, aromatic protons of second
position), 7.63e7.68 (d, 2H, J ¼ 5.73, aromatic protons meta
to CH3 at fifth position), 10.65 (s, 1H, NH at second position).
Anal. calcd. for C20H16ClNO3S: C 62.25, H 4.14, N 3.62.
Found: C 62.65, H 4.01, N 3.63%.
6.3. General procedure for the synthesis
of compounds 6ae6f
6.3.4. E-4-Methoxyphenyl-[2-(4-chloroanilino)-
3-methoxycarbonyl-4-methyl-5-thienyl]ketoxime (6d)
Yield: 49%; m.p. 136e138 ꢂC; MS: m/z 431 (M þ 1); IR
(KBr): 3242, 1669 (C]O stretching), 1602, 1593, 1552,
1398, 1368, 1288, 1169, 926, 690e786 (strongest of thiophene
Compounds 6ae6f were synthesized by adding 0.001 mol
of the respective substituted oxime of 4-substituted phenacyl
bromides to a solution 0.001 mol of 3 in 2 ml of acetonitrile
without adding base at room temperature. The solution was
stirred until the solid was separated from the reaction mixture
or until no more starting materials could be detected on TLC.
The solid that separated was filtered off, washed with chilled
acetonitrile, and dried, yielded product corresponding to
6ae6f characterized as per the analytical data.
1
bands, CeS stretching) cmꢁ1. H NMR (300 MHz, CDCl3):
d 1.60 (s, 3H, CH3 at fourth position), 2.40 (s, 3H, OCH3 at
fifth position), 3.85 (s, 3H, CH3 of ester at third position),
6.92e6.95 (d, 2H, aromatic protons ortho to OCH3 at fifth po-
sition), 7.26e7.40 (m, 4H, aromatic protons of second posi-
tion), 7.76e7.79 (d, 2H, aromatic protons meta to OCH3 at
fifth position), 10.46 (s, 1H, NH at second position). Anal.
calcd. for C21H19ClN2O4S: C 58.53, H 4.40, N 6.49. Found:
C 58.53, H 4.07, N 6.17%.
6.3.1. E-4-Methoxyphenyl-(2-anilino-3-methoxycarbonyl-
4-methyl-5-thienyl)ketoxime (6a)
Yield: 45%; m.p. 140e142 ꢂC; MS: m/z 396 (Mþ); IR
(KBr): 3241, 3192, 1666 (C]O stretching), 1666, 1552,
1446, 1365, 1238, 1199, 1171, 1024, 697e756 (strongest of
6.3.5. E-4-Methylphenyl-(2-anilino-3-methoxycarbonyl-
4-methyl-5-thienyl)ketoxime (6e)
Yield: 80%; m.p. 125e127 ꢂC; MS: m/z 380 (Mþ); IR
(KBr): 3021, 2959, 2450, 1661 (C]O stretching), 1615,
1595, 1370, 1188, 985, 684e740 (strongest of thiophene
thiophene bands, CeS stretching) cmꢁ1
.
1H NMR
(300 MHz, CDCl3): d 2.47 (s, 3H, CH3 at fourth position),
2.68 (s, 3H, OCH3 at fifth position), 4.00 (s, 3H, CH3 of ester
at third position), 7.19e7.22 (d, 2H, aromatic protons ortho to
OCH3 at fifth position), 7.24e7.38 (m, 5H, aromatic protons
of second position), 7.97e8.00 (d, 2H, J ¼ 4.56, aromatic pro-
tons meta to OCH3 at fifth position), 10.40 (s, 1H, NH at sec-
ond position). Anal. calcd. for C21H20N2O4S: C 63.63, H 5.04,
N 7.06. Found: C 63.16, H 5.14, N 7.01%.
1
bands, CeS stretching) cmꢁ1. H NMR (300 MHz, CDCl3):
d 2.36 (s, 3H, CH3 at fourth position), 2.42 (s, 3H, CH3 at fifth
position), 3.88 (s, 3H, CH3 of ester at third position), 7.11e
7.14 (d, 2H, J ¼ 5.66, aromatic protons ortho to CH3 at fifth
position), 7.23e7.39 (m, 5H, aromatic protons of second posi-
tion), 7.66e7.69 (d, 2H, aromatic protons meta to CH3 at fifth
position), 10.41 (s, 1H, NH at second position). Anal. calcd.

1058
K.I. Molvi et al. / European Journal of Medicinal Chemistry 42 (2007) 1049e1058
[10] N.V. Chandrasekharan, H. Dai, K.L. Roos, N.K. Evanson, J. Tomsik,
T.S. Elton, D.L. Simmons, Proc. Natl. Acad. Sci. U.S.A. 99 (2002)
13926e13931.
for C21H20N3O3S: C 66.30, H 5.25, N 7.36. Found: C 66.70, H
5.35, N 7.42%.
[11] A. Paul, S. Wilson, C.M. Belham, C.J.M. Robinsson, P.H. Scott,
G.W. Gould, R. Plevin, Cell. Signal. 9 (1997) 403e410.
[12] J.C. Lee, J.T. Laydon, P.C. McDonnell, T.F. Gallagher, S. Kumar,
D. Green, D. McNulty, M.J. Blumenthal, J.R. Heys, S.W. Landvatter,
J.E. Strickler, M.M. McLaughlin, I.R. Siemens, S.M. Fisher, G.P. Livi,
J.R. White, J.L. Adams, P.R. Young, Nature 372 (1994) 739e746.
[13] M.L. Foster, F. Halley, J.E. Souness, Drug News Perspect. 13 (2000)
488e497.
6.3.6. Z-4-Methylphenyl-[2-(4-chloroanilino)-
3-methoxycarbonyl-4-methyl-5-thienyl]ketoxime (6f)
Yield: 75%; m.p. 143e145 ꢂC; MS: m/z 415 (M þ 1); IR
(KBr): 3183, 2364, 1662 (C]O stretching), 1591, 1443,
1374, 1246, 936, 673e780 (strongest of thiophene bands,
1
CeS stretching) cmꢁ1. H NMR (300 MHz, CDCl3): d 2.36
(s, 3H, CH3 at fourth position), 2.40 (s, 3H, CH3 at fifth posi-
tion), 3.83 (s, 3H, CH3 of ester at third position), 7.22e7.25
(d, 2H, aromatic protons ortho to CH3 at fifth position),
7.26e7.30 (m, 4H, aromatic protons of second position),
7.41e7.44 (d, 2H, aromatic protons meta to CH3 at fifth posi-
tion), 10.41 (s, 1H, NH at second position). Anal. calcd. for
C21H19ClN3O3S: C 60.80, H 4.58, N 6.75. Found: C 60.98,
H 4.78, N 3.48%.
[14] P. Rutgeerts, D.G. Haens, S. Targan, E. Vasiliauskas, S.B. Hanauer,
D.H. Present, L. Mayer, R.A. Van Hogezand, T. Braakman,
K.L. DeWoody, T.F. Schaible, S.J.H. Van Deventer, Gastroenterology
117 (1999) 761e769.
[15] A.J. Lewis, A.M. Manning, Curr. Opin. Chem. Biol. 3 (1999) 489e494.
[16] D.H. Present, P. Rutgeerts, S. Targan, S.B. Hanauer, L. Mayer, R.A. van
Hogezand, D.K. Podolsky, B.E. Sands, T. Braakman, K.L. DeWoody,
T.F. Schaible, S.J. van Deventer, N. Engl. J. Med. 340 (1999) 1398e1405.
[17] B. Jarvis, D. Faulds, Drugs 57 (1999) 945e966.
[18] B. Bresnihan, BioDrugs 15 (2001) 87e97.
[19] J.C. Lee, S. Kumar, D.E. Griswold, D.C. Underwood, B.J. Votta,
J.L. Adams, Immunopharmacol. 47 (2000) 185e201.
Acknowledgements
[20] J.R. Jackson, B. Bolognese, L. Hillegass, S. Kassis, J. Adams,
D.E. Griswold, J.D. Winkler, J. Pharmacol. Exp. Ther. 284 (1998) 687e692.
[21] C. Pargellis, J. Regan, Curr. Opin. Investig. Drugs 4 (2003) 566e571.
[22] E.R. Ottosen, M.D. Sorensen, F. Bjorkling, T. Skak-Nielsen, M.S. Fjording,
H. Aaes, L. Binderup, J. Med. Chem. 46 (2003) 5651e5662.
[23] A.D. Pillai, S. Rani, P.D. Rathod, F.P. Xavier, K.K. Vasu, H. Padh,
V. Sudarsanam, Bioorg. Med. Chem. 13 (2005) 1275e1283.
[24] K.I. Molvi, K.K. Vasu, M.M. Patel, V. Sudarsanam, N. Haque, Ethiop.
Pharm. J. 24 (2006) 1e12.
The authors are thankful to Prof. Gaurang B. Shah, Head,
Department of Pharmacology, K.B.I.P.E.R., Gandhinagar, In-
dia, for the facilities and encouragement. We also wish to
thank RSIC of CDRI Lucknow, Bombay Tablets Mfg. Co.
Pvt. Ltd., Gandhinagar and Cadilla Pharmaceuticals, Ahmeda-
1
bad, India for providing the H NMR, C, H, N analysis, I.R.
data and animals, respectively.
[25] P.N. Craig, in: M.E. Wolff (Ed.), Burger’s Medicinal Chemistry, Part I,
4th ed., Wiley, New York, 1980, pp. 332e348.
[26] G.R. Schinella, H.A. Tournier, J.M. Prieto, M. Mordujovich, J.L. Rios,
Life Sci. 70 (2002) 1023e1033.
References
[27] Y.C. Huang, J.H. Guh, Z.J. Cheng, Y.L. Chang, T.L. Hwang, C.N. Lin, et
al., Life Sci. 68 (2001) 2435e2447.
[1] L.M. Wilson, in: S.A. Price, L.M. Wilson (Eds.), Pathophysiology: Clin-
ical Concepts of Disease Processes, Mosby St. Louis, Missouri, 2003, pp.
44e61.
[28] S. Rajappa, B.G. Advani, Indian J. Chem. 12 (1974) 4e7.
[29] A.D. Pillai, P.D. Rathod, P.X. Franklin, H. Padh, K.K. Vasu, V. Sudarsanam,
Biochem. Biophys. Res. Commun. 317 (2004) 1067e1074.
[30] A.D. Pillai, P.D. Rathod, F.P. Xavier, M. Patel, M. Nivsarkar, K.K. Vasu,
H. Padh, V. Sudarsanam, Biochem. Biophys. Res. Commun. 301 (2003)
183e186.
[2] T. Collins, in: R.S. Cotran, V. Kumar, T. Collins (Eds.), Robbins Patho-
logic Basis of Disease, Harcourt India Pvt. Ltd., New Delhi, 2001, pp.
50e88.
[3] R.M. Garavito, Nat. Struct. Biol. 3 (11) (1996) 897e901.
[4] J.R. Vane, Nature 231 (1971) 232e235.
[31] H. Baji, M. Flammang, T. Kimny, F. Gasquez, P.L. Compagnon,
A. Delcourt, Eur. J. Med. Chem. 30 (1995) 617e626.
[5] C.J. Hawkey, The Lancet 353 (1999) 307e314.
[6] A. Robert, Adv. Prostaglandin Thromboxane Res. 2 (1976) 507e520.
[7] E. Fosslien, Ann. Clin. Lab. Sci. 28 (2) (1998) 67e81.
[8] K. Seibert, Y. Zhang, K. Leahy, S. Hauser, J. Masferrer, W. Perkins,
L. Lee, P. Isakson, Proc. Natl. Acad. Sci. U.S.A. 91 (1994) 12013e
12017.
[32] C.A. Winter, E.A. Risley, G.W. Nuss, Proc. Soc. Exp. Biol. Med. 111
(1962) 544e547.
[33] E.A. Siegmund, R.A. Cadmus, G. Lu, Proc. Soc. Exp. Biol. Med. 95
(1957) 729e731.
[34] N. Sreejayan, M.N.A. Rao, J. Pharm. Pharmacol. 49 (1997) 105e107.
[9] R.N. DuBois, Gastroenterology 130 (2006) 6e15.