102 Send Orders for Reprints to reprints@benthamscience.

ae
Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2018, 17, 102-114

RESEARCH ARTICLE
ISSN: 1871-5230
eISSN: 1875-614X

Synthesis and Biological Evaluation of Novel 2-(4-acetyl-3-methyl-

5-(arylamino) thiophen-2-yl)-3-arylquinazolin-4(3H)-one Deriva-
tives as Potential Anti-inflammatory and Antioxidant Agents

Priyanka S. Chaudhari*, Sohan S. Chitlange and Rabindra K. Nanda

Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry

Department of Pharmaceutical Chemistry, Dr. D.Y. Patil Institute of Pharmaceutical Sciences and
Research, Pimpri, Pune 411018, Maharashtra, India
Abstract: Background: A new series of 2-(4-acetyl-3-methyl-5-(arylamino) thiophen-2-
yl)-3-arylquinazolin-4(3H)-one derivatives (11a-11j) were synthesized from acetyl acetone,
phenyl isothiocyanate and 2-chloromethyl quinazolinone.
Objective: Due to side effects of Non Steroidal Anti-Inflammatory Drugs (NSAID), an
attempt was made to identify the novel tetrasubstituted thiophene lead compound as
potential anti-inflammatory and antioxidant agent.
Methods: Then newly synthesized compounds were characterized by IR spectroscopy, 1H
ARTICLE HISTORY NMR and mass spectrometry. The synthesized compounds were screened for their in vivo
anti-inflammatory activity in carrageenan-induced rat hind paw edema model at dose
Received: May 03, 2018 20mg/kg body weight using diclofenac sodium as a standard drug. The compounds were al-
Revised: September 01, 2018
Accepted: September 03, 2018 so evaluated for their in vitro DPPH free radical-scavenging activity and nitric oxide radical
scavenging activity at the concentrations of 10, 20, 40, 60, 80 and 100 µg/mL using ascorbic
DOI:
10.2174/1871523017666180910105609 acid as standard drug.
Results: The results from carrageenan-induced rat hind paw edema showed that compounds
11e, 11f and 11b show a significant anti-inflammatory activity of 46.61%, 48.94% and
47.04 % protection respectively to inflamed paw but less than diclofenac sodium. Com-
pounds 11h and 11e show good DPPH free radical scavenging and nitric oxide radical scav-
enging activity, respectively.
Conclusion: From results, it was observed that highly substituted thiophene scaffold exhib-
its anti-inflammatory and antioxidant activity.

Keywords: Anti-inflammatory activity, antioxidant activity, tetrasubstituted thiophene, inflammation,

COX, PGs.

1. INTRODUCTION removal of the necroses cells and tissues. The

Inflammation is defined as the local response of main mediator of inflammation and pain is the key
living mammalian tissues to injury due to physical enzyme Cyclooxygenase (COX), that catalyzes the
agents, chemical agents, immunological reactions conversion of arachidonic acid into Prostaglandins
and infection by pathogenic organism [1]. It is a (PGs). The two isoforms of this enzyme are COX1
body defence mechanism in order to eliminate or and COX2. COX1 is a constitutive enzyme and
limit the spread of injurious agent, followed by responsible for the production of PGs which main-
tain the integrity of gastric mucosa and provide
adequate vascular homeostasis, whereas COX2 is
*Address correspondence to this author at the Department of an inducible enzyme and produces PGs that induce
Pharmaceutical Chemistry, Dr. D.Y. Patil Institute of Phar- undesirable inflammation, fever and pain. Thus,
maceutical Sciences and Research, Pimpri, Pune 411018, pharmacological inhibition of COX provides relief
Maharashtra, India; Mobile: +91 8983438029;
from the symptoms of inflammation and pain.
E-mail: [email protected]
Classical NSAIDs are COX inhibitors and restrict
1875-614X/18 $58.00+.00 © 2018 Bentham Science Publishers
Synthesis and Biological Evaluation Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2018, Vol. 17, No. 2 103

the biosynthesis of PGs. Although NSAIDs thera- 2.1.1. General Procedure for Synthesis of 2-(4-
py effectively reduces symptoms of many painful acetyl-3-methyl-5-(arylamino) thiophen-2-yl)-3-
arthritic syndromes but causes side effects namely arylquinazolin-4(3H)-one Compounds
gastrointestinal ulceration, damage of gastric mu-
To a solution of acetylacetone (1.0 mmol, 1
cosa, bleeding, nephrotoxicity and even death [2-
equiv.) in dimethylformamide (3 mL), dried potas-
9]. NSAIDs like diclofenac or ibuprofen show a
sium carbonate (1.0 mmol,1 equiv.) was added and
high risk of vascular events, similar to patients
the mixture was stirred for 1 hour at room temper-
treated with coxibs [10, 11]. These deleterious ef-
ature. Aryl isothiocyanate [18] (1.0 mmol, 1
fects of NSAIDs seem to be associated with the
equiv.) was then added dropwise and the mixture
inhibition of prostacyclin synthesis, increased
was stirred for 1 hour at room temperature. Then
blood pressure and oxidative stress-induced endo-
quinazolinone (1.0 mmol, 1 equiv.) [19, 20] was
thelial dysfunction [12]. Free radicals are types of
added, the reaction mixture was heated for 1 hour.
reactive oxygen species (ROS), which cause cell
The reaction was quenched with 10 mL water. The
membrane disintegration, membrane protein dam-
crude product tetrasubstituted thiophene [21-23]
age and DNA mutation upon oxidation. According
precipitated and was purified by filtration followed
to the reported literature, reactive oxygen species
by crystallization from methanol. Purity was
or oxidative stress components are involved in the
checked by thin layer chromatography using n-
pathophysiology of NSAIDs-induced ulcerations
hexane: ethyl acetate (3:2) as a solvent.
[13-15].
2.1.2. Characterization of Synthesized Compounds
Thiophene and its derivatives were reported for
their antifungal, antibacterial [16], anti-HIV, anti- 2.1.2.1. 2-(4-acetyl-3-methyl-5-(phenylamino)
depressant, antitubercular, antiarthritic, antitussive, thiophen-2-yl)-3-phenylquinazolin-4(3H)-one
antihistamine, local anesthetic, analgesic, anti- (11a)
inflammatory activities [1-9] and so on [17]. The Yield: 41.62 %; m.p. : 220-222°C; I.R. (KBr,
synthesis of highly substituted thiophenes has at- cm-1): 3400 (N-H), 3051 (C-H s, aromatic), 2850
tracted a great deal of interest over the years due to (CH3-CH), 1678 (C=O), 748 (C-H b, aromatic); 1H
their biological importance. Encouraged by the NMR (300 MHz, CDCl3)δ(ppm): 2.32(s,3H, CH3),
aforementioned findings the aim of present work is 2.44(s,3H, CH3), 7.1-8.3 (m,14H aromatic), 11.80
the synthesis and evaluation of some novel (s,1H,NH); HR-MS, m/z (M+1): 452.14.
tetrasubstituted thiophene derivatives for their an-
ti-inflammatory and antioxidant activity in order to 2.1.2.2. 2-(4-acetyl-3-methyl-5-(phenylamino)
find a lead compound with minimal side effects thiophen-2-yl)-3-p-tolylquinazolin-4(3H)-one
[18-20]. (11b)

2. EXPERIMENTAL Yield: 38.11 %; m.p. : 167-169°C; I.R. (KBr,

cm-1): 3431 (N-H), 3028 (C-H s, aromatic), 2926
2.1. Chemistry (CH3-CH), 2864 (CH3-CH), 1674 (C=O), 777 (C-
All chemicals were purchased from Sigma Al- H b, aromatic); 1H NMR (300 MHz, CDCl3)
drich (Steinheim, Germany), SD Fine, Spectrochem, δ(ppm): 2.32(s,3H, CH3), 2.44(s,3H, CH3),
Merck (Darmstadt, Germany). Melting points were 2.35(s,3H, CH3),7.0-8.4 (m,13H, aromatic), 11.80
determined by open tube capillary method in (s,1H, NH); HR-MS, m/z (M+1): 466.15.
Hicon melting point apparatus (Hicon, India) and 2.1.2.3. 2-(4-acetyl-3-methyl-5-(phenylamino) th-
are uncorrected. IR spectra (KBr) were recorded iophen-2-yl)-3-(4-methoxyphenyl) quinazolin-
on Shimadzu 8400S FTIR. 1H NMR spectra were 4(3H)-one (11c)
recorded on Bruker 300 MHz spectrometer and
chemical shifts (δ) were reported in parts per mil- Yield: 40.45 %; m.p. : 204-206°C; I.R. (KBr,
lion (ppm) with CDCl3 as the solvent. TMS was cm-1): 3053 (N-H), 3003 (C-H s aromatic), 2937
used as the internal standard for NMR. Mass spec- (CH3-CH), 2835 (CH3-CH), 1681 (C=O), 777 (C-
tra were recorded on Bruker Daltonik GmbH, H b, aromatic); 1H NMR (300 MHz, CDCl3)
Germany. The progress of reactions was moni- δ(ppm): 2.47 (s,3H, CH3), 2.36(s,3H, CH3), 3.83
tored by thin layer chromatography using silica gel (s,3H, OCH3),6.90-7.83 (m,13H, aromatic), 11.80
60 F254 pre-coated aluminum plates (Merck). (s,1H, NH); HR-MS, m/z (M+1) : 482.10.
104 Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2018, Vol. 17, No. 2 Chaudhari et al.

2.1.2.4. 2-(4-acetyl-3-methyl-5(phenylamino)thio- 2.1.2.9. 2-(5-(4-methoxyphenylamino)-4-acetyl-3-

phen-2-yl)-3-(2-chlorophenyl) quinazolin-4(3H)- methylthiophen-2-yl)-3-(4-bromophenyl)quinazo-
one (11d) lin-4(3H)-one (11i)
Yield: 39.88 %; m.p. : 192-194°C;); I.R. (KBr, Yield 41.09 %; m.p. : 176-178°C; I.R. (KBr,
cm-1): 3344 (N-H), 3057 (C-H s, aromatic), 2960 cm-1): 3470 (N-H), 3066 (CH s, aromatic), 2866
(CH3-CH), 2926 (CH3-CH), 1683 (C=O), 765 (C- (CH3-CH), 1680 (C=O), 738, (CH b, aromatic),
H b, aromatic); 1H NMR (300 MHz, CDCl3) 630 (C-Br); 1H NMR (300 MHz, CDCl3)δ(ppm):
δ(ppm): 2.47 (s,3H, CH3), 2.49(s,3H, CH3) 7.08- 2.21 (s,3H, CH3), 3.73 (s,3H, OCH3), 2.53(s,3H,
7.86(m,13H, aromatic), 11.80 (s,1H,NH); HR-MS, CH3), 6.36-8.36 (m,13H); HR-MS, m/z (M+1) :
m/z (M+1): 486.10. 561.10.
2.1.2.5. 2-(4-acetyl-5-(4-chlorophenylamino)-3- 2.1.2.10. 2-(5-(4-methoxyphenylamino)-4-acetyl-
methylthiophen-2-yl)-3-(4-chlorophenyl)quinazo- 3-methylthiophen-2-yl)-3-(2-
lin-4(3H)-one (11e) chlorophenyl)quinazolin-4(3H)-one (11j)
Yield: 66.12 %; m.p. : 206-208°C; I.R. (KBr, Yield 41.09 %; m.p. : 176-178°C; I.R. (KBr,
cm-1): 3236 (N-H), 3076 (C-H s, aromatic), 2895 cm-1): 3329 (N-H), 3047 (CH s, aromatic), 2933
(CH), 1676 (C=O), 881 (C-H s, aromatic); 1H NMR (CH3-CH), 1685 (C=O), 742, (CH b, aromatic); 1H
(300 MHz, CDCl3)δ(ppm): 2.20(s, 3H, CH3), 2.32 NMR (300 MHz, CDCl3)δ(ppm): 2.45 (s,3H, CH3),
(s,3H, CH3) 7.2-8.3(m,12H), 11.79 (s,1H, NH), 3.50 (s,3H,OCH3), 7.09-8.36 (m,15H), 11.80
HR-MS, m/z (M+1): 520.06. (s,1H,NH) ; HR-MS, m/z (M+1): 517.17.
2.1.2.6. 2-(4-acetyl-5-(4-chlorophenylamino)-3- 2.2. Pharmacology
methylthiophen-2-yl)-3-o-tolylquinazolin-4(3H)-
one (11f) 2.2.1. Oral Acute Toxicity Study
Yield: 50.93 %; m.p. : 196-198°C; I.R. (KBr, The acute toxicity study of synthesized com-
cm-1): 3028 (N-H), 2922 (CH), 1676 (C=O), 773 pounds was carried out in Swiss albino mice (20-
(C-H s aromatic); 1H NMR (300 MHz, CDCl3) 25g) as per the OECD guidelines no 423. Different
δ(ppm): 2.17 (s,3H, CH3), 2.5-2.6(s,6H, CH3), doses of synthesized compounds were adminis-
7.13-8.3(m,12H),11.74 (s,1H,NH); HR-MS, m/z tered to groups (n=3) up to 2000 mg/kg orally and
(M+): 500.12. animals were observed for a period of 72 h for be-
havioral changes, toxic reactions and mortality if
2.1.2.7. 2-(4-acetyl-3-methyl-5-(p-tolylamino) thi- any.
ophen-2-yl)-3-phenylquinazolin-4(3H)-one (11g)
2.2.2. Anti-inflammatory Activity
Yield: 44.76 %; m.p. : 248-250°C; I.R. (KBr,
cm-1): 3375 (N-H), 3034 (C-H s, aromatic), 2933 All the above-synthesized compounds were
(CH), 2833(CH), 1672 (C=O), 759 (C-H b, aro- screened for their in vivo anti-inflammatory activi-
matic); 1H NMR (300 MHz, CDCl3)δ(ppm): 2.308 ty against carrageenan induced rat paw edema
(s,3H,CH3), 2.32 (s,3H, CH3), 2.41, (s,3H, CH3) model [24]. Male Wistar rats (180-250gm) were
7.06-8.32 (m,13H), 11.7 (s,1H, NH); HR-MS, m/z used for the edema test. The animals were fed with
(M+1): 466.15. commercial feed pellets and were given water ad
libitum. Experimental protocols were approved by
2.1.2.8. 2-(5-(4-methoxyphenylamino)-4-acetyl-3- the Institutional Animal Ethical Committee
methylthiophen-2-yl)-3-(4-methoxyphenyl)quina- (DYPIPSR/IAEC/17-18/P-27) prior to initiation of
zolin-4(3H)-one (11h) the experiment. Animals were divided into 12
Yield 41.09 %; m.p. : 176-178°C; I.R. (KBr, groups consisting of six rats per group and were
cm-1): 3321 (N-H), 3050 (CH s, aromatic), 2926 kept fastened overnight. One group served as the
(CH3-CH), 2830 (CH3-CH), 1681 (C=O), 777, 883 standard, another group served as the control and
(CH b, aromatic); 1H NMR (300 MHz, CDCl3) the remaining groups were used to test the synthe-
δ(ppm): 2.21(s,3H, CH3), 2.55(s,3H, CH3), sized compounds. The rats were received standard
3.73(s,6H, OCH3), 7.08-8.34 (m,13H); HR-MS, drug Diclofenac Sodium and test compounds at a
m/z (M+1): 512.30. dose of 20mg/kg body wt orally suspended in
0.5% sodiumcarboxy methyl cellulose. Control
Synthesis and Biological Evaluation Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2018, Vol. 17, No. 2 105

group administered with vehicle 0.5 % sodium of colored DPPH radical was used. Here, 2.0 ml of
carboxymethyl cellulose (10ml/kg b.w p.o). One methanol solution of different test compounds at
hour post dosing 0.1 ml of 1 % carrageenan solu- various concentration (10, 20, 40, 60, 80, 100
tion in saline was injected subcutaneously in the μg/ml) were mixed with 3.0 ml of DPPH methanol
sub-plantar region of the right hind paw of each solution (0.135 mM). Similar mixtures were pre-
rat. The volume of paw edema was measured at 1, pared for ascorbic acid as standard antioxidant and
2, 3, 4, 5 and 6h after carrageenan injection using control (mix reagent with methanol except for test
plethysmometer (UGO Basile, 7140, Italy). Aver- compounds). The solution was kept for 20 min at
age edema volumes for test compound treated and room temperature in a dark place. After 20 min
positive control rats were compared statistically absorbances of all concentrations were recorded at
with those of the vehicle control animals [25, 26]. 517 nm against blank by UV spectrophotometer
The percentage inhibition of edema was calculated [27-30]. At high concentration of the test
according to the following formula: compounds, the absorbance decreases that is the
evidence of an increase in free radical scavenging.
% Inhibition = (Vc-Vt/Vc) ×100 Inhibition of free radical DPPH in percent (I%)
where Vc paw volume of control, Vt paw volume was calculated as follows:
of test compound. I % = [(A control – A sample) / A control] × 100
2.2.3. Histopathology whereas Acontrol is the absorbance of control re-
At termination, all the rats were euthanized by action (containing all reagent except test com-
CO2 asphyxiation. Necropsy examination was pounds) and Asample is the absorbance of the test
conducted externally and internally. Rats were ex- compound.
amined carefully for external abnormalities. The 2.2.4.2. Nitric Oxide Scavenging Activity
thoracic and abdominal cavities were cut opened
to record the gross lesions in different organs if The procedure is based on the principle that so-
any. Organs viz., site of injection (paw) and stom- dium nitroprusside generates nitric oxide radicals
ach of normal control, disease control, standard at physiological pH, which interact with oxygen to
drug and test compound 1e treated were collected produce nitrite ions. These can be measured using
at 10% Neutral Buffered Formalin for further his- the Griess reagent (0.1% N-(1-naphthyl) ethylene-
topathological examination. Formalin-fixed tissues diamine dihydrochloride, 2% phosphoric acid and
were trimmed and routinely processed. Tissue pro- 1% sulfanilamide). For the assessment, reaction
cessing was done to dehydrate in ascending grades mixture (3 ml) containing sodium nitroprusside
of alcohol, clearing in xylene and embedded in (10 mM) in Phosphate Buffer Saline (PBS) and
paraffin wax. Paraffin wax embedded tissue blocks test compounds at different concentrations (10, 20,
were sectioned at 3 µm thickness with the Rotary 40, 60, 80, 100 μg/ml) were incubated at 25°C for
Microtome (Leica RM2125). All the slides were 150 min and after that, 0.5 mL of test sample was
stained with Hematoxylin & Eosin (H & E) stain. removed and mixed with Griess reagent (0.5 ml).
The prepared slides were examined under a micro- The absorbance of the pink color formed during
scope to note histopathological lesions if any. Se- the diazotization of nitrite with sulphanilamide and
verity of the observed lesions were recorded as 0= subsequent coupling with naphthylethylenedia-
Not Present, 1= Minimal (<1%), 2= Mild (1-25%), mine was read at 546 nm. Ascorbic acid was used
3= Moderate (26-50%), 4= Moderately Severe as a standard antioxidant [28-31]. The experiment
(51-75%), 5= Severe (76-100%) and distribution was performed in triplicates and nitric oxide scav-
was recorded as focal, multifocal and diffuse. enging activity was calculated by the following
equation:
2.2.4. Antioxidant Activity
% Nitric oxide scavenging activity = [(A control –
2.2.4.1. DPPH Free Radical-scavenging Activity A sample) / A control] × 100
2,2-diphenyl-1-picrylhydrazyl (DPPH) radical 2.2.4.3. Statistical Analysis
was used for the determination of free radical
scavenging activity of synthesized derivatives. To Results were analyzed using one-way Analysis
determine free radical scavenging activity a meth- of Variance (ANOVA) using graph pad prism 7
od based on a reduction of a methanolic solution software and expressed as Mean ± SEM. Data was
106 Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2018, Vol. 17, No. 2 Chaudhari et al.

O
O
O
OH
TEA,rt,4 h,DCM OH
Cl
Cl
NH
NH2
Cl
O

(1) (2) (3)

O O
R R
O
PCl3,Toluene H2 N
OH reflux 3 h Cl
N

NH NH rt Cl
N
Cl Cl
O O

(4) (5) (6) (7)

Scheme 1. Synthesis of 2-chloro methyl-3- aryl-3H-quinazolinone-4-ones.

further subjected to Dunnett’s test and differences pure product to get our targeted molecule decreas-
between means were regarded as highly significant es the purity of the final compound. So there was a
at P<0.01 when compared with control. need to develop such method which will give an
intermediate compound in pure form so that final
3. RESULTS AND DISCUSSION compound will be pure and have a good yield. In
3.1. Chemistry the reported method, some modification has been
carried out i.e use of POCl3 and SOCl2 instead of
In designing the synthetic protocol for tetra PCl3 but it gave poor yield as compared to yield
substituted thiophene analogues, it was envisioned obtained by using PCl3. Some modifications have
that 2-chloromethyl-3- aryl-3H-quinazolinone-4- carried out by changing the reaction condition
one (7) and aryl isothiocyanate intermediate repre- while adding aniline. Firstly, 2-(2-chloroacetyl-
sent the interesting starting materials for the syn- amino) benzoic acid and PCl3 were refluxed in tol-
thesis of designed compounds. The synthetic strat- uene and after formation of the acid chloride reac-
egies adopted for the synthesis of intermediates tion mixture was cooled to room temperature and
and target compounds are depicted in Scheme 1 aniline was added dropwise. The reaction was
and 2 respectively. stirred continuously at room temperature and mon-
The synthesis of intermediate compound 2- itored by TLC using n-hexane:ethyl acetate (5:3).
chloromethyl-3- aryl-3H-quinazolinone-4-one was Following this method pure intermediate was ob-
well documented in the literature. The synthesis of tained. Synthesis of tetrasubstituted thiophene was
(7) was furnished by using modified Niemento- carried out as shown in Scheme 2.
wski synthesis, which involves chloroacetylation N,S ketene acetal are key intermediate for the
of anthranillic acid (1) by chloroacetyl chloride (2) preparation of a large number of heterocyclic
to get corresponding 2-(2-chloroacetylamino) ben- compounds. This intermediate was synthesized as
zoic acid (3). This 2-(2-chloroacetylamino) benzo- per method reported by Gilbert et al. [22]. The de-
ic acid was then treated with trichlorophosphate signed targeted compound has been synthesized by
(PCl3) to convert into an acid chloride (5) which condensation of activated methylene compounds
was immediately treated with substituted aniline with aryl isothiocyanates in a basic medium. As
in situ to replace the chloro group of the acid chlo- per literature, 1,3-dicarbonyl compounds was cho-
ride with substituted aniline. This was then re- sen as a substrate, K2CO3 as base and N, N-
fluxed in toluene to get cyclised product [20, 21]. Dimethylformamide (DMF) as the reaction medi-
The product obtained by this method showed two um. From experiment, it was discovered that
major spots on TLC. Further reaction of this im- acetyl acetone reacts with phenyl isothiocyanate in
Synthesis and Biological Evaluation Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2018, Vol. 17, No. 2 107

O
O O O
R2
O O K2CO3, Ar NCS, N
Cl N
DMF N HN
HN SH S
R1 N
rt, 2 h Heat 1h
O
R2
R1

(8) (9) (7) (11)

Scheme 2. Synthetic route of compounds 11a to 11j.

K2CO3, Ar NCS, O O
R2
O O DMF O
Heat 1h
rt, 2 h N
Cl
HN SH N
(8)
(7)

R1
(9) O
O O O
OH
N
N HN
N S
N S N S R1 N
H H H
R1 N R1 N
O
O
O
R2
R2 R2
(11)

Scheme 3. Possible reaction Mechanism for Synthesis of targeted compounds (11a-11j).

presence of K2CO3 in DMF at room temperature to In all mice, neither toxic reactions nor mortality
give nonisolable intermediate N,S-ketene acetals were observed.
to which acetyl acetone was added to give cyclised
Further, all the synthesized compounds were
product, tetrasubstituted thiophene [21]. On the
screened for their in vivo anti-inflammatory activi-
basis of results, mechanism for the reaction of
ty in carrageenan-induced rat paw edema model.
acetyl acetone has been depicted in Scheme 3.
Percent inhibition of standard drug diclofenac so-
dium and tested compounds were calculated in
In presence of K2CO3, deprotonation of active
comparison with vehicle control (Table 2). Com-
methylene acetyl acetone starts to afford enolate. pounds 11e, 11b, 11d, 11f, and 11h possess signif-
Nucleophilic attacks of enolate on phenyl isothio- icant anti-inflammatory activity at but less than
cyanate form N, S-ketene acetal. Further addition diclofenac sodium. Compounds 11a, 11c, 11g, 11i
of intermediate (7) to (9) undergoes intramolecular and 11j show moderate anti-inflammatory activity.
cyclisation and gives rise to the targeted compound, These results suggest that Cl group at the para po-
tetrasubstituted thiophene (11). Physical data of sition of phenyl ring (11a) is essential for activity.
synthesized compounds are given in Table 1. Group OCH3 at the fourth position in 11h and CH3
in 11b is required for anti-inflammatory activity.
3.2. Pharmacology From results, it was observed that possible mecha-
3.2.1. Anti-inflammatory Activity nism for inhibition of paw edema formation was
due to inhibition of prostaglandins, which is re-
The acute oral toxicity study of synthesized leased in second phase (after 3 h) after carragee-
compounds was carried out in Swiss albino mice. nan injection.
108 Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2018, Vol. 17, No. 2 Chaudhari et al.

Table 1. Physical properties of tetrasubstituted thiophene compounds.

O

N
HN S
R1 N

R2

(11)

Molecular Molecular wt Rf
Compounds R1 R2
Formula (M.W.) Value

11a H H C27H21N3O2S 451.54 0.64

11b H 4-CH3 C28H23N3O2S 465.57 0.52
11c H 4-OCH3 C28H23N3O3S 481.57 0.65
11d H 2-Cl C27H20ClN3O2S 485.98 0.51
11e 4-Cl 4-Cl C27H19Cl2N3OS 520.43 0.45
11f 4-Cl 2-CH3 C28H22ClN3O2S 500.01 0.68
11g 4-CH3 H C28H23N3O2S 465.57 0.71
11h 4-OCH3 4-OCH3 C29H25N3O4S 511.59 0.68
11i 4-OCH3 4-Br C28H22BrN3O3S 560.46 0.62
11j 4-OCH3 2-Cl C28H22ClN3O3S 516.01 0.59

Table 2. Effect of 2-(4-acetyl-3-methyl-5-(arylamino) thiophen-2-yl)-3-arylquinazolin-4(3H)-one compounds

on Carrageenan induced paw edema.

Edema Volume (ml) Mean ± SEM (% Inhibition)

Compound
1h 2h 3h 4h 5h 6h

Control 1.125 ± 0.010 1.24 ± 0.01 1.48 ± 0.018 1.57 ± 0.031 1.38 ± 0.016 1.20 ± 0.015
0.94± 0.012* 0.77 ± 0.021** 0.68 ± 0.023** 0.59± 0.009** 0.54 ± 0.013** 0.53 ± 0.012**
DS
(15.85) (37.76) (53.64) (62.15) (60.60) (55.58)
1.10 ± 0.006ns 1.18 ± 0.008ns 1.19 ± 0.008* 0.95 ± 0.006* 0.94 ± 0.012* 0.92 ± 0.012*
11a
(2.07) (4.30) (19.64) (39.42) (31.80) (23.17)
1.06 ±0.006 1.11 ± 0.003 1.18 ± 0.012* 0.84 ± 0.011 0.83 ± 0.013* 0.80 ± 0.019*
11b
(5.48) (10.08) (20.42) (**46.61) (39.79) (33.37)
1.08 ± 0.007ns 1.18 ± 0.007ns 1.19 ± 0.009* 0.93 ± 0.01** 0.91 ± 0.013* 0.86 ± 0.012*
11c
(3.25) (4.70) (19.52) (41.01) (33.78) (28.05)
1.07 ± 0.004 1.13 ± 0.019ns 1.15 ± 0.011** 0.85 ± 0.012** 0.82 ± 0.010** 0.80 ± 0.010*
11d
(4.29) (8.87) (22.22) (45.77) (40.36) (33.51)
1.04 ± 0.009 1.08 ± 0.007* 1.10 ± 0.006** 0.80 ± 0.005** 0.81 ± 0.016** 0.74 ± 0.010*
11e
(7.11) (12.90) (25.36) (48.94) (40.84) (38.20)
(Table 2) contd….
Synthesis and Biological Evaluation Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2018, Vol. 17, No. 2 109

Edema Volume (ml) Mean ± SEM (% Inhibition)

Compound
1h 2h 3h 4h 5h 6h

1.05 ± 0.011 1.10 ± 0.009* 1.11 ± 0.006** 0.83 ± 0.008** 0.80 ± 0.010** 0.77 ± 0.012*
11f
(6.07) (11.15) (24.69) (47.04) (41.68) (35.86)
1.08 ± 0.009ns 1.17 ± 0.008* 1.18 ± 0.016* 0.98 ± 0.023* 0.94 ± 0.009* 0.91 ± 0.004*
11g
(3.40) (4.97) (19.97) (37.73) (32.04) (24.27)
1.05 ± 0.012* 1.16 ± 0.009* 1.17 ± 0.007* 0.88 ± 0.009** 0.86 ± 0.008* 0.83 ± 0.012*
11h
(5.92) (6.04) (21.21) (43.86) (37.71) (30.62)
1.10 ± 0.004ns 1.15 ± 0.007* 1.16 ± 0.020* 0.99 ± 0.034* 0.98 ± 0.010* 0.95 ± 0.012*
11i
(2.07) (6.72) (21.88) (36.78) (29.03) (20.96)
1.07 ± 0.007 1.14 ± 0.008* 1.16 ± 0.020* 1.04 ± 0.037* 1.02 ± 0.011* 0.98 ± 0.023*
11j
(4.44) (7.52) (21.32) (33.82) (26.14) (18.34)
Each value is the mean ± SEM for six rats. *P < 0.05, **P < 0.01, vs control group ns: non significant.
Control: 0.5 % sodium CMC.
DS: Standard Diclofenac Sodium.

Fig. (1). Paw Normal Control: Showing normal histology, Articular cartilage (Ac), Synovial membrane (Sm),
Bone(Bo), Joint cavity (Jc).

3.2.2. Histopathology crosis, neutrophils, macrophages, and lymphocytes

3.2.2.1. Stomach is observed at a synovial area in rats of disease
control group. Focal mild infiltration of lympho-
Rats of normal control, disease control and test cytes is observed at a synovial area in rats of
drug (11e) treated groups did not reveal any lesion standard and test drug treated group (Figs. 1-8).
of pathological significance. However, multifocal
moderate ulceration at the mucosa of the glandular 3.2.3. Antioxidant Activity
stomach is observed in diclofenac sodium. All synthesized compounds in the concentration
3.2.2.2. The Site of Injection (Paw) range from 10, 20, 40, 60, 80 and 100µg/ml were
screened for DPPH free radical scavenging and
No abnormality of pathological significance at nitric oxide scavenging activity. It was found that
the site of injection is observed in the normal con- the percentage of scavenging effect on DPPH radi-
trol group. Multifocal moderate infiltration of ne- cal was increased with an increase in concentration
110 Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2018, Vol. 17, No. 2 Chaudhari et al.

Fig. (2). Paw Disease Control: Showing Macrophage Infiltration (Mc) and Lymphocytic Infiltration (Li) and
Necrosis (Ne) at muscle fibers of synovial area.

Fig. (3). Paw Standard Drug Treated: Showing Lymphocytic Infiltration (Li) at muscle fibers of peri-synovial area.

Fig. (4). Paw Test compound 1e Treated: Showing Lymphocytic Infiltration (Li) at muscle fibers of peri-synovial area.
Synthesis and Biological Evaluation Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2018, Vol. 17, No. 2 111

Fig. (5). Normal Control Stomach: Glandular stomach showing intact Mucosa (Mu) and Submucosa (Sm).

Fig. (6). Disease Control Stomach: Glandular stomach showing intact Mucosa (Mu) and Submucosa (Sm).

Fig. (7). Standard Drug Treated Stomach: Showing area of ulceration at mucosa (Ul).
112 Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2018, Vol. 17, No. 2 Chaudhari et al.

Table 3. % DPPH radical scavenging activity for compounds 11a to 11j.

% DPPH Radical Scavenging Activity ± SEM

Compounds
10 μg/ml 20 μg/ml 40 μg/ml 60 μg/ml 80 μg/ml 100 μg/ml

11a 03.08 ± 0.033 05.18 ± 0.240 06.21 ± 0.474 07.10 ± 0.106 09.12 ± 0.462 12.16 ± 0.526
11b 19.67 ± 0.616 25.93 ± 0.501* 33.88 ± 0.520* 41.49 ± 0.780** 46.82 ± 0.535** 51.77 ± 0.560**
11c 04.07 ± 0.159 07.15 ± 0.499 11.18 ± 0.598 16.82 ± 0.344 20.38 ± 0.398 25.23 ± 0.465*
11d 26.30 ± 0.352 31.12 ± 0.684* 37.34 ± 0.482* 43.75 ± 0.482** 52.40 ± 0.527** 67.50 ± 0.545**
11e 37.30 ± 0.276* 43.03 ± 0.494** 52.76 ± 0.397** 63.56 ± 0.462** 70.54 ± 0.485** 81.68 ± 0.599**
11f 18.53 ± 0.336 24.72 ± 0.433* 30.67 ± 0.762 37.32 ± 0.616* 43.89 ± 0.580** 47.43 ± 0.721**
11g 20.56 ± 0.560 28.36 ± 0.585* 36.23 ± 0.449* 45.78 ± 0.609** 50.23 ± 0.565** 55.21 ± 0.516**
11h 20.93 ± 0.241 32.61 ± 0.554* 37.43 ± 0.587* 55.96 ± 0.627** 66.11 ± 0.546** 86.04 ± 0.673**
11i 25.67 ± 0.320 33.56 ± 0.592* 41.12 ± 0.524** 49.89 ± 0.640** 56.65 ± 0.446** 62.48 ± 0.722**
11j 02.09 ± 0.290 03.17 ± 0.418 04.20 ± 0.085 06.07 ± 0.661 08.12 ± 0.534 10.26 ± 0.597
Ascorbic acid 94.54 ± 0.451** 95.17 ± 0.089** 95.68 ± 0.289** 96.06 ± 0.160** 96.19 ± 0.139** 97.20 ± 0.313**

Table 4. Nitric oxide radical scavenging activity for compounds 11a to 11j.

% Nitric Oxide Scavenging Activity

Compounds
10 μg/ml 20 μg/ml 40 μg/ml 60 μg/ml 80 μg/ml 100 μg/ml

11a 04.65 ± 0.612 09.16 ± 0.536 13.30 ± 0.600 18.15 ± 0.609 26.23 ± 0.520* 30.50 ± 0.447**
11b 29.67 ± 0.513* 33.93 ± 0.661* 38.39 ± 0.482** 43.43 ± 0.638** 48.32 ± 0.689** 52.60 ± 0.640**
11c 12.90 ± 0.447 24.05 ± 0.583* 30.13 ± 0.609** 36.28 ± 0.600 40.38 ± 0.516** 44.52 ± 0.672**
11d 14.60 ± 0.366 20.34 ± 0.586* 28.43 ± 0.256** 34.67 ± 0.612 45.46 ± 0.661** 52.87 ± 0.583**
11e 40.54 ± 0.603* 54.12 ± 0.545* 61.87 ± 0.566** 69.65 ± 0.643** 73.89 ± 0.588** 80.23 ± 0.537**
11f 20.54 ± 0.765 27.80 ± 0.614* 34.16 ± 0.338* 41.64 ± 0.637** 47.87 ± 0.681** 65.25 ± 0.583**
11g 22.56 ± 0.571 28.97 ± 0.575* 35.54 ± 0.482* 39.21 ± 0.579* 43.69 ± 0.641** 52.46 ± 0.577**
11h 25.33 ± 0.574 32.21 ± 0.762* 48.63 ± 0.534** 55.86 ± 0.586** 67.23 ± 0.493** 79.10 ± 0.580**
11i 05.67 ± 0.444 10.56 ± 0.491 15.12 ± 0.587* 20.89 ± 0.606* 26.65 ± 0.621** 32.48 ± 0.782**
11j 22.9 ± 0591 29.34 ± 0.121* 36.76 ± 0.514* 43.98 ± 0.540** 49.65 ± 0.366** 57.45 ± 0.542**
Ascorbic acid 93.54 ± 0.997** 96.17 ± 0.614** 96.68 ± 0.496** 97.06 ± 0.681** 98.19 ± 0.349** 98.87 ± 0.161**

(Table 3). Compound 11h and 11e exhibit the CONCLUSION

highest scavenging effect among all. 11b, 11d, Different tetrasubstituted thiophene compounds
11f, 11g, and 11i possess moderate antioxidant (11a-11j) were synthesized from acetyl acetone,
activity whereas 11a and 11j have very low anti- aryl isothiocyanates and 2-2-chloromethyl-3- aryl-
oxidant activity. The percentage of inhibition in 3H-quinazolinone-4-one. All the compounds were
nitric oxide scavenging assay was maximum for structurally characterized and evaluated for their
compounds 11h and 11e but less than ascorbic ac- in-vivo anti-inflammatory and in-vitro antioxidant
id (Table 4). activity. From results, it was observed that com-
Synthesis and Biological Evaluation Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2018, Vol. 17, No. 2 113

Fig. (8). Test compound 1e treated stomach: Stomach showing Mucosa (Mu) and Submucosa (Sm).

pounds 11h and 11e show comparable anti- ACKNOWLEDGEMENTS

inflammatory and antioxidant activity as compared The authors are thankful to Central Instrumen-
to standard. The observed variations microscopic tation Laboratory, Savitribai Phule Pune Universi-
evaluations revealed that disease control animals ty for providing NMR & Mass facility. The au-
showed necrotic and inflammatory lesions at the thors are also grateful to Dr. D.Y. Patil Institute of
site of injection. Treatment of standard diclofenac Pharmaceutical Sciences and Research, Pimpri,
sodium and test compound 11e mitigates the ef- Pune for providing necessary facility to carry out
fect. Observed lesion in the stomach in standard all research work and animal studies.
drug suggest its adverse effect. So the test com-
pound was found to be safe and show no any gas- SUPPLEMENTARY MATERIAL
tric ulceration. It was concluded that newly syn-
thesized tetrasubstituted thiophene compounds Supplementary material is available on the pub-
show promising anti-inflammatory and antioxidant lisher’s website along with the published article.
activity with fewer side effects.
REFERENCES
ETHICS APPROVAL AND CONSENT TO [1] Molvi, K.I.; Vasu, K.K.; Yerande, S.G.; Sudarsanam,
PARTICIPATE V.; Haque, N. Syntheses of new tetrasubstituted thio-
phenes as novel anti-inflammatory agents. Eur. J.
Experimental protocols were approved by the Med. Chem., 2007, 42, 1049-1058.
Institutional Animal Ethical Committee [2] Sanda, P.D.; Silva, L.J.D.; Zorica, D.J.; Bojan, D.M.;
(DYPIPSR/IAEC/17-18/P-27) prior to initiation of Sote, M.V.; Ivan, O. Docking studies and anti-
the experiment. inflammatory activity of β-hydroxy-β-arylpropanoic
acids juranic 4. Molecules, 2008, 13, 603-615.
HUMAN AND ANIMAL RIGHTS [3] Pillai, A.D.; Rathod, P.D.; Xavier, F.P.; Kamala, K.;
Vasu, K.K.; Padhc, H.; Sudarsanam V. Design,
No humans were used for studies that are the synthesis, and pharmacological evaluation of some 2-
basis of this research. All the reported experiments [4-morpholino]-3-aryl-5-substituted thiophenes as
on mice were carried out as per the OECD guide- novel anti-inflammatory agents: generation of a novel
anti-inflammatory. Pharma. Bio. & Med. Chem.,
lines no 423.
2004, 12, 4667-4671.
CONSENT FOR PUBLICATION [4] Molvi, K.I.; Mansuri, M.; Sudarsanam, V.; Khurshid,
M.I.; Molvi, M.M.; Sudarsanam, V.; Patel, M.M.;
Not applicable. Syed Muzaffar, A.A.; Haque, N. Synthesis, anti-
inflammatory, analgesic and antioxidant activities of
CONFLICT OF INTEREST some tetrasubstituted Thiophenes. J. Enzy. Inh. Med.
Chem., 2008, 23(6), 829-838.
The authors declare no conflict of interest, [5] Radwan, M.A.A.; Fakhr, I.M.I.; Seham, E.B.; Omar,
financial or otherwise. M.E.; El-Salam, A. Synthesis and pharmacological
114 Anti-Inflammatory & Anti-Allergy Agents in Medicinal Chemistry, 2018, Vol. 17, No. 2 Chaudhari et al.

evaluation of 2- substituted benzo[b]thiophenes as [19] Giri, R.S.; Thaker, H.M.; Giordano, T.; Williams, J.;
anti-inflammatory and analgesic agents. Eur. J. Med. Rogers, D.; Sudarsanam, V.; Vasu, K.K. Design, syn-
Chem., 2009, 44, 1718-1725. thesis and characterization of novel 2-(2,4-
[6] Singh, D.; Mohan, S.; Sharma, P.C.; Sarvanan, J. disubstituted-thiazole-5-yl)-3-aryl-3H-quinazoline-4-
Synthesis and evaluation of some novel piperidino one derivatives as inhibitors of NF-kB and AP-1 me-
thiophenes as potential antioxidant and anti-inflam- diated transcription activation and as potential anti-
matory agents. Act. Pharm. Sci., 2007, 49, 29-38. inflammatory agents. Eur. J. Med. Chem., 2009, 44,
[7] Rajender Kumar, P.; Raju, S.; Goud, S.P.; Sailaja, M.; 2184-2189.
Sarma, M.R.; Om Reddy, G.; Prem Kumar, M.; [20] Giri, R.S.; Thaker, H.M.; Giordano, T.; Williams, J.;
Krishna Reddy, V.V.R.M.; Suresh, T.; Hegde, P. Rogers, D.; Vasu, K.K.; Sudarsanam, V. Design,
Synthesis and biological evaluation of thiophene [3,2- synthesis and evaluation of novel 2-thiophen-5-yl-
b] pyrrole derivatives as potential anti-inflammatory 3H-quinazolin-4-one analogues as inhibitors of
agents. Bio. Med. Chem., 2004, 12, 1221-1230. transcription factors NF-kB and AP-1 mediated
[8] Deka, S.; Mohan, S.; Saravanan, J.; Kakati, M.; transcriptional activation: Their possible utilization as
Talukdar, A.; Sahariah, B.J.; Kumar, D.B.; Sarma, R. anti-inflammatory and anti-cancer agents. Bio. Med.
Syntheses, characterization and in-vitro anti- Chem., 2010, 18, 2796-2808.
inflammatory activity of some novel thiophenes. [21] Wang, Y.; Huang, J.; Chai, Y.; Liu, Q.; Liang, Y.;
Maced. J. Med. Sci., 2012, 5(2), 159-163. Dong, D. Efficient one-pot synthesis of highly
[9] Srivastava, S.; Das, B.; Prasad, R. Antimicrobial and substituted thiophene library from 1, 3- dicarbonyl
anti-inflammatory effect of some novel tetra compounds. J. Comb. Chem., 2008, 10, 511-516.
substituted Thiophenes. Der. Pharmacia. Lettre, [22] Sommen, G.; Comel, A.; Kirsch, G. Preparation of
2012, 4, 1214-1220. thieno [2,3-b] pyrroles starting from ketene-N, S-
[10] Ferreira de Melo, T.; Chelucci, R.C.; Pires, M. Phar- acetals. Tetrahedron., 2003, 59, 1557-1564.
macological evaluation and preparation of nonsteroi- [23] Moghaddam, F.M.; Boinee, H.Z. An efficient and
dal anti-inflammatory drugs containing an n-acyl hy- facile one-step synthesis of highly substituted
drazone subunit. Int. J. Mol. Sci., 2014, 15, 5821-5837. thiophenes. Tetrahedron, 2004, 60, 6085-6089.
[11] Bhala, N.; Emberson, J.; Merhi, A.; Abramson, S.; [24] Winter, C.A.; Risley, E.A.; Nuss, G.W. Carrageenin-
Arber, N.; Baron, J.A. Vascular and upper gastroin- induced edema in hind paw of the rat as an assay for
testinal effects of non-steroidal anti-inflammatory anti-inflammatory drugs. Proc. Soc. Exp. Biol. Med.,
drugs: Meta-analyses of individual participant data 1962, 111, 544-547.
from randomized trials. Coxib and traditional NSAID [25] Poojaria, P.; Naik, P.; Krishnamurthy, G.; Kumara, J.;
Trialists’ (CNT) Collaboration. Lancet, 2013, 382, Sunil Kumar, N.; Naik, S. Anti-inflammatory, anti-
769-779. bacterial and molecular docking studies of novel spi-
[12] Fanelli, A.; Ghisi, D.; Fanelli, G. Nonsteroidal anti- ro-piperidine quinazolinone derivatives. J. Taibah
inflammatory drugs (NSAIDS) in clinical practice: Uni. Sci., 2017, 11, 497-511.
Managing gastric and cardiovascular risks. Acta Bio- [26] Banu, H.; Bharathi, K.; Prasad, K.V.S.R.G. Synthesis,
med., 2013, 84, 98-101. characterization and evaluation of in vitro antioxidant
[13] Madhavi, K.; Sree Ramya, G. Synthesis, antioxidant and anti-inflammatory activity of 2-(4-oxo-2-
and anti-inflammatory activities of ethyl 2-(2-cyano- phenylquinazolin-3(4H)-yl) substituted acetic acids.
3- (substituted phenyl) acrylamido)-4,5- IOSR J. Pharm., 2012, 2, 97-104.
dimethylthiophene-3-carboxylates. Asian J. Pharm. [27] Chen, Y., Wang, M., Rosen, R.T., Ho, C.T. 2,2-
Clin. Res., 2017, 10(7), 95-100. Diphenyl-1-picrylhydrazyl radical-scavenging active
[14] Awah, F.M.; Verla, A.W. Antioxidant activity, nitric components from Polygonum multiflorum thunb. J.
oxide scavenging activity and phenolic contents of Agri. Food Chem., 1999, 47, 2226-2228.
Ocimum gratissimum leaf extract. J. Med. Plant. Res., [28] Duh, P.D.; Tu, Y.Y.; Yen, G.C. Antioxidant activity
2010, 4, 2479-2487. of water extract of harng jyur (Chrysanthemum mori-
[15] Mohammad, A.E.Z.; Seyed, M.N.; Seyed, F.N.; folium ramat). LWT-Food Sci. Tech., 1999, 32, 269-
Fatemeh, B.; Ahmad, R.B. Antioxidant and free radi- 277.
cal scavenging activity of H. Officinalis L. Var. An- [29] Kumar, S.; Kumar, V.; Chandrashekhar, M.S. In-vitro
gustifolius, V. Odorata, B. Hyrcana and C. Specio- anti-oxidant and alpha-amylase inhibitory activity of
sum. Pak. J. Pharm. Sci., 2010, 23, 29-34. isolated fractions from methanolic extract of asystasia
[16] Wedad, M.; Al-Adiwish, W.A.; Yaacob, D.; Adan, I.; dalzelliana leaves. Inter. J. Pharm. Tech. Res., 2011,
Nazlina. Synthesis and antibacterial activity of thio- 3, 889-894.
phenes. Inter. J. Adv. Sci. Eng. Inform. Technol., [30] Sharma, A.; Bhardwaj, S.; Mann, A.S.; Jain, A.;
2012, 2, 27-30. Kharya, M.D. Screening methods of antioxidant
[17] Mishra, R.; Jha, K.K.; Kumar, S.; Tomer, I. Synthe- activity: An overview. Pharmacog. Rev., 2007, 2,
sis, properties and biological activity of thiophene: A 232-238.
review. Der Pharma. Chemica., 2011, 3, 38-54. [31] Garg, S.; Garg, A.; Shukla, A.; Dev, S.; Bishnoi, R.;
[18] Hodgkins, J.E.; Preston Reeves, W. The modified Kumar, M. Review on antioxidant evaluation
kaluza synthesis. III. The synthesis of some aromatic methods: In- vitro and in-vivo models. Asian J.
isothiocyanates. J. Org. Chem., 1964, 29, 3098-3099. Pharm. Pharma., 2018, 4, 147-154.