2
C.K. Khatri et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 1. Structures of selective COX-2 inhibitors and designed molecule.
A
literature survey revealed that the substituted 4,5,6,7-
tetrahydro[b]benzothiophene scaffold possess various biological
activities such as anti-inflammatory,12 analgesic,13 antiarrhyth-
mic,14 antibacterial15, and anti-tumor.16 Due to the potent anti-
inflammatory activity, it was chosen as a central heterocyclic motif
for the present study and a phenyl ring attached at position 2 to
maintain its common structural integrity. However, another phe-
nyl ring at position 3 connected via ester linker gives different
insight with new molecular scaffold which results in a stronger
interaction with key role amino acid residues and also favorable
for the COX-2 active site. The objective of making these hybrids
is an attempt to reach novel anti-inflammatory agents with potent
activity and COX-2 selectivity. We herein report the efficient syn-
thesis, anti-inflammatory evaluation and docking studies of 2-phe-
nyl-4,5,6,7-tetrahydro[b]benzothiophene derivatives.
In an attempt to design and develop novel potential anti-
inflammatory agents, we synthesized target compounds starting
with Gewald reaction of cyclohexanone, ethyl cyanoacetate and
sulfur in the presence of diethylamine in ethanol to afford ethyl
2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate 1,17
followed by arylation via modified Gomberg–Buchmann–Hey reac-
tion using p-TSA, triethyl orthoformate (TEOF), CuCl and sodium
nitrite in benzene to get ethyl 2-phenyl-4,5,6,7-tetrahydrobenzo
[b]thiophene-3-carboxylate 2.18
Scheme 1. Reagent and conditions: (i) diethylamine, EtOH, reflux, 2 h, 74%; (ii) p-
TSA, TEOF, CuCl, NaNO2, C6H6, 50 °C, 1 h, 71%; (iii) 10 M KOH, EtOH, reflux, 2 h, 96%;
(iv) phenols, DCC, DMAP, CHCl3, rt, 12 h, 75–85%.
Hydrolysis of compound 2 afforded the common molecular
scaffold
2-phenyl-4,5,6,7-tetrahydrobenzo[b]thiophene-3-car-
were advanced for evaluation of IC50 values on COX-2 and COX-1
enzyme. The most potent COX-2 inhibitor in this series were com-
pounds 4a, 4j, 4k and 4q having IC50 values 0.33 mM, 0.31 mM,
0.67 mM and 1.40 mM respectively on COX-2 enzyme along with
the COX-2 SI values ranging from 48.8–183.8 (Table 1, entry 1,
10, 11, 17) (Fig. 2). Structure-activity relationship showed that
the nature of substituents was the major determinant for activity.
No substitution or presence of substituents like 4-SO2NH2, 4-
NHCOCH3 and 3-COCH3 showed potent activity whereas other sub-
stituents showed less to the moderate activity which is attributed
to H-bonding capacity of these substituents (Table 1, Entry 2–9 and
12–16).
Compounds 4a, 4j, 4k and 4q were further investigated for the
anti-inflammatory via PGE-2 inhibitory properties using RAW
264.7 cells, which produces PGE-2 when stimulated by LPS.21,22
PGE-2 levels in culture well supernatants were analyzed using
PGE-2 assay kits according to the manufacturer’s instructions
(High Sensitivity Prostaglandin E2 Enzyme Immunoassay Kit, Cat-
alog number: KO18-HX1, ARBOR Assays, USA). The cells stimulated
with LPS showed the highest release of PGE-2 as compared with
the cells with no stimulation. The LPS stimulated cells when incu-
bated with celecoxib and the test compounds, celecoxib showed
the lowest release of PGE-2 whereas, compounds 4a, 4j, 4k and
4q showed reduced PGE-2 production as compared to LPS control
cells (Fig. 3).
boxylic acid 3,19 which was esterified via Steglich esterification
with substituted phenols using DCC and DMAP as coupling
reagents in chloroform/acetonitrile to produce the title 2-phenyl-
4,5,6,7-tetrahydro[b]benzothiophene analogues 4a–4q.20 The route
of synthesis of intermediates and target compounds is depicted in
the Scheme 1. All the compounds were characterized by FTIR, 1H
NMR, 13C NMR and mass spectroscopy.
The potency of all the synthesized compounds on COX-1 and
COX-2 were evaluated using COX fluorescent inhibitor screening
assay kit (Catalogue No. 700100, Cayman Chemicals Inc., Ann
Arbor, MI, USA) according to manufacturer’s assay protocol. The
compounds 4a-4q were screened at 60 mM concentration to deter-
mine the different steric and electronic effects upon COX-1 and
COX-2 inhibitory potency and selectivity. The molecules showed
inhibition greater than 75% of COX-2 enzyme were advanced for
determination of IC50 values. Celecoxib and DuP-697 as COX-2
selective and SC-560 as COX-1 selective inhibitors were used as
reference compounds. The enzyme inhibitory data, the respective
COX-2 selectivity index (COX-2 SI) are summarized in Table 1.
In COX enzyme screening assay, celecoxib showed 99.5% inhibi-
tion on COX-2 and 68.8% inhibition on COX-1 at 60 mM concentra-
tion. However, IC50 values were found as 0.03 mM for COX-2 and
18.14 mM for COX-1, which is in the same range as reported in
the literature.4 Most compounds of this series, displayed very
low inhibitory potency toward the COX-1 and COX-2 enzymes.
Compounds 4b-4e, 4g-4i and 4l-4p showed less than 75% inhibi-
tion on both COX-2 and COX-1 at 60 mM concentration. Whereas
compounds 4a, 4f, 4j, 4k, and 4q showed greater than 75% inhibi-
tion of the COX-2 enzyme (Table 1, entry 1, 6, 10, 11, 17), which
Compounds 4a, 4f, 4i, 4j, 4k, 4p and 4q which showed diverse
in vitro activity were evaluated for in vivo anti-inflammatory
potential in carrageenan-induced rat paw edema dose of 150 lg/
kg and results obtained are summarized in Table 2. The observed
anti-inflammatory activity of all the compounds was less than
Khatri, Chetan K.
