Rationally designed hybrid molecules with appreciable COX-2 inhibitory and anti-nociceptive activities

Article abstract of DOI:10.1016/j.bmcl.2013.11.080

Six molecules were obtained by the combination of three biologically and medicinally significant moieties-indole, chrysin and pyrazole. Bio-evaluation of these hybrid molecules showed significant inhibition of COX-2 enzymatic activity over that of COX-1 a

Full text of DOI:10.1016/j.bmcl.2013.11.080

Bioorganic & Medicinal Chemistry Letters 24 (2014) 77–82
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Rationally designed hybrid molecules with appreciable COX-2
inhibitory and anti-nociceptive activities
a
b
b
Palwinder Singh a, , Shaveta , Surbhi Sharma , Rajbir Bhatti
⇑
a Department of Chemistry, Guru Nanak Dev University, Amritsar 143005, India
b Department of Pharmaceutical Sciences, Guru Nanak Dev University, Amritsar 143005, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Six molecules were obtained by the combination of three biologically and medicinally significant moie-
ties—indole, chrysin and pyrazole. Bio-evaluation of these hybrid molecules showed significant inhibition
of COX-2 enzymatic activity over that of COX-1 and appreciable anti-nociceptive activity, checked at
swiss albino mice.
Received 17 September 2013
Revised 8 November 2013
Accepted 28 November 2013
Available online 4 December 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Chrysin
Indole
Anti-inflammatory
Anti-nociceptive
Molecular modeling
Inflammation is an innate immunity response against harmful
stimulus which leads to the activation of arachidonic acid (AA)
metabolism and hence more production of prostaglandins and leu-
kotrienes.1 These metabolites trigger the signals for induction of
inflammation-symptomised as pain, redness, fever, etc. COX-2 is
the enzyme associated with production of inflammatory metabo-
lites during arachidonic acid metabolism2 and hence a primary tar-
get of anti-inflammatory drugs.3 However, the process of blockage
of COX-2 enzyme through the use of various inhibitors suffers from
inhibition of COX-1 which is structurally an isoform of COX-2 but
associated with production of desirable prostaglandins in arachi-
donic acid pathway.4 Starting with the use of aspirin as COX-1/2
nonselective drug,5 treatment of inflammation travelled a long
journey up to the use of selective COX-2 inhibitors, the COXIBS.6
However, the side effects associated with the use of COXIBS and
withdrawal of some of them from the market7 necessitated the
search for their substitutes.
The hybrid molecules, obtained by the combination of two bio-
active molecules, exhibit significantly higher biological activity in
comparison to their individual fragments,8,9 a number of such mol-
ecules containing indole–pyrazole, indole–pyrimidine, indole–
chromone are reported by our group for anti-cancer and COX-2
inhibitory activities.10–12 For the present studies, another biologi-
cally significant moiety chrysin (1, Chart 1) was combined with
indole (2, Chart 1) and pyrazole (3, Chart 1) and through appropri-
ate combination of these three moieties, compounds 4–9 were pro-
cured. While chrysin is an important member of flavone class of
natural products identified with anti-oxidant,13,14 anti-bacterial,15
anti-inflammatory,16,17 anti-cancer,18 antiestrogenic,19 anxiolytic20
activities and a number of its analogues like apigenin,21 tectorige-
nin22 and wogonin23 show significant anti-inflammatory activities,
the biological potential of indole and pyrazole is also well estab-
lished.24–29 Therefore, it is envisaged that the combination of three
biologically significant heterocycles may result in the development
of more efficacious molecules.
N-alkylation of 3-formyl indole (1 mmol) with 1,3-dibromopro-
pane (1 mmol) and 1,4-dibromobutane (1 mmol) provided com-
pounds 10 and 11 (Scheme 1).
In the next step, chrysin, (1, 1 mmol) was refluxed with com-
pounds 10 and 11 in presence of potassium carbonate (1 mmol)
in acetone. The reaction was performed under N2 atmosphere to
procure compounds 12 and 13 (Scheme 2).
Compound 12 (100 mg, 1 mmol) on treatment with 1-(3-chlo-
rophenyl)-3-methyl-2-pyrazolin-5-one (1 mmol) at 160–180 °C
for 10–20 min provided compound 4. Similarly, reaction of com-
pound 13 (100 mg, 1 mmol) with 1-(3-chlorophenyl)-3-methyl-2-
pyrazolin-5-one gave product 5. Under same reaction conditions,
treatment of compounds 12 and 13 with 1,3-dihydro-indol-2-one
(oxindole) and 1-(2,6-dichlorophenyl)-1,3-dihydro-indol-2-one
(indolinone) provided products 6–9, respectively (Scheme 3).30
Compounds 4–9 were screened for inhibition of catalytic activ-
ities of COX-2 and COX-1, the enzymes associated with arachidonic
⇑
Corresponding author. Tel.: +91 183 2258802x3495; fax: +91 183 2258819.
E-mail address: palwinder_singh_2000@yahoo.com (P. Singh).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.11.080

78
P. Singh et al. / Bioorg. Med. Chem. Lett. 24 (2014) 77–82
CH3
N
inhibition of COX-1/2 at four molar concentrations used here. All
the compounds showed insignificant COX-1 inhibition which is
the housekeeping enzyme for the production of useful
prostaglandins.
O
O
HO
O
N
N
H
IC50 of compound 4 for COX-2 is better than that of chrysin.
Although IC50 of compound 4 for COX-2 is comparable to indo-
methacin but its selectivity for COX-2 over COX-1 is better than
that of indomethacin. While IC50 of compound 4 for COX-2 is high-
er than that of diclofenac and celecoxib but its selectivity for COX-2
over COX-1 is better than diclofenac and poorer than celecoxib.
This may be a desirable feature of compound 4 because too poor
and too higher selectivity of a drug for COX-2 over COX-1 leads
to side effects (as it happens in case of aspirin and was a major lim-
itation of rofecoxib). Therefore, better/comparable inhibition and
desirable selectivity index of compound 4 for COX-2 than the drugs
based on individual components of this compound justify the de-
sign of the molecules. Compound 4 may act as a lead molecule
for further refinement to increase its efficacy for COX-2 inhibition.
In order to come across the rationale behind the type of interac-
tions and their extent between the compounds and the enzymes,
molecular docking of compounds 4–9 in the active sites of COX-1
and COX-2 was performed. Compounds were built using the
builder tool kit of the software package Argus Lab 4.0.132 and en-
ergy minimized with semi-empirical quantum mechanical method
PM3. Crystal co-ordinates of COX-1 (PDB ID 1EQG and 3KK6)33,34
and COX-2 (PDB ID 6COX and 3MQE)35,36 were downloaded from
protein data bank (www.rcsb.org) and in the molecule tree view
of the software, the monomeric structures of the crystal coordinate
was selected and the active site was defined as 15 Å around the
ligand.
OH
Cl
2, Indole
3, pyrazole
1, Chrysin
indole/
pyrazole
O
O
O
N
n
OH
4-9
Hybrid molecules
Chart 1.
CHO
CHO
CHO
Br
Br
NaH, DMF
Br
Br
N
N
H
NaH, DMF
N
Br
10 (70%)
Br
11 (65%)
Scheme 1.
The molecule to be docked in the active site of the enzyme was
pasted in the work space carrying the structure of the enzyme. The
docking programme implements an efficient grid based docking
algorithm which approximates an exhaustive search within the
free volume of the binding site cavity. The conformational space
was explored by the geometry optimization of the flexible ligand
(rings were treated as rigid) in combination with the incremental
construction of the ligand torsions. Thus, docking occurs between
the flexible ligand parts of the compound and enzyme. Docking
was repeated several times (approx. 10,000 iterations) until no
change in position of the ligand and a constant value of binding en-
ergy was observed. The ligand orientation was determined by a
shape scoring function based on Ascore and final positions were
ranked by lowest interaction energy values. H-bonds and hydro-
phobic interactions between the respective compound and enzyme
were explored.
acid metabolism producing prostaglandins. All the enzyme inhibi-
tion assays were performed in duplicate as per the manufacturer’s
protocol available along with the enzyme immunoassay kits31 and
the difference between two readings was 3–5%. Compounds were
tested at 10ꢀ5 M, 10ꢀ6 M, 10ꢀ7 M and 10ꢀ8 M concentrations for
COX-1/2 inhibitory activities and based on these results, com-
pounds were further screened at 10ꢀ3 M, 10ꢀ4 M, 10ꢀ5 M and
10ꢀ6 M concentrations for COX-1 inhibition. Catalytic activities of
COX-1/2 enzymes were quantified by measuring the amount of
prostaglandins produced by each enzyme in presence of various
concentrations of the test compounds and comparing with control
experiments.
In general, inhibition of enzymatic activity by test compound 1
1/conc of prostaglandins produced during enzymatic reaction.
Compound 4 showed considerable inhibition of COX-2 enzy-
matic activity with IC50 (50% inhibitory conc) 0.7
IC50 for COX-1 was found to be 118 M. Compound 5, 6 and 8 also
showed good inhibition of COX-2 with IC50 7.9, 7.3 and 6.2 M for
l
M while its
Compound 4 was well docked in the active site of both the crys-
tal coordinates of COX-2 viz. 6COX and 3MQE (Fig. 1a and b). It
showed H-bond interactions of 1.59, 2.04, 2.35 and 2.92 Å between
its pyrazolic nitrogens and T118 amino acid of the enzyme active
l
l
COX-2 enzyme (Table 1). Compounds 7 and 9 did not exhibit
OHC
i) K2CO3, Acetone,
N2 atm.
N
O
O
O
ii) 10
HO
O
O
OH
12 (65%)
i) K2CO3, Acetone,
N2 atm.
OH
O
O
N
1
OHC
ii) 11
OH
O
13 (68%)
Scheme 2.

P. Singh et al. / Bioorg. Med. Chem. Lett. 24 (2014) 77–82
79
N
Cl
H3C
N
OHC
N
H3C
O
N
O
O
N
O
n
O
O
O
N
O
160-180 o
C
n
OH
12, n=3
13, n=4
OH
O
Cl
N
160-180 oC
O
4, n=3 (87%)
5, n=4 (60%)
Cl
160-180 oC
N
H
Cl
N
NH
O
Cl
O
O
O
N
O
N
O
n
n
O
OH
O
OH
8, n=3 (69%)
9, n=4 (67%)
6, n=3 (60%)
7, n=4 (78%)
Scheme 3.
site. It also showed a strong H-bond interaction of 1.66 Å through
its indole nitrogen towards R120 (Fig. 1a), the amino acid residue
forming salt bridge with AA during the turn over phase of the en-
zyme. Similarly, compound 4 fit in the hydrophobic cavity of an-
other crystal structure of COX-2 (Fig. 1b and c).
Compound 4 was also tried to dock in the active site of the
housekeeping enzyme COX-1 (pdb ID 1EQG and 3KK6) but it did
not enter into the active site of COX-1 (Supporting information).
Hence, the molecular docking results, in harmony with the enzyme
inhibition assay results give evidence about the interaction as well
as inhibition of COX-2 with compound 4. Molecular docking of
compounds 5–9 was also performed in the active site of enzymes
COX-1/COX-2. Similar results were obtained on docking com-
pounds 5–9 in the active site of COX-2 and their compatibility in
the active site varies from compound to compound. However, com-
pounds 5–9 did not enter the active site of COX-1 enzyme (Sup-
porting information).
Guru Nanak Dev University, Amritsar, Punjab. Animals were accli-
matized to the laboratory for at least 1 h before testing and were
used only once throughout the experiments. All the protocols have
been duly approved by the Institutional Ethics Committee and are
in accordance with guidelines of Committee for the purpose of
Control and Supervision of Experiments on Animals (CPSCEA), Min-
istry of Environment and Forests, India.
The method used for capsaicin-induced licking was similar to
that described by Sakurada et al.37 with a few modifications.
Briefly, 20 lL of capsaicin (1.6 lg) was injected into the plantar
surface of the right hand paw. Animals were observed individually
for 10 min after capsaicin administration and the time spent paw
licking and twitching was recorded as an indicator of nociception.
Animals were divided into 10 groups of 6 each for studying the
anti-nociceptive activity of compounds 4–6 and 8 using capsaicin
induced paw licking. All the treatments were administered intra-
peretoneally 30 min before capsaicin injection. Group I was control
wherein the animals were injected normal saline in a volume of
0.5 mL. In group II, animals were injected diclofenac at a dose of
25 mg Kgꢀ1; in group III and IV, animals were injected compound
4 at a dose of 5 and 10 mg Kgꢀ1, respectively. Similarly, in group
V and VI, animals were injected compound 5 at a dose of 5 and
10 mg Kgꢀ1, respectively; in group VII and VIII, with compound 6
and in group IX and X, with compound 8, respectively.
Based on the results of enzyme immunoassays, compounds 4–6
and 8 were checked for their anti-nociceptive activities. Swiss albi-
no mice (25–35 g) of either sex were used in the present study. The
animals were housed at 25 2 °C under 12 h light/12 h dark cycle
and free access to food and water in the central animal house at
Table 1
The treatment with diclofenac was found to produce a marked
decrease in the number of paw lickings after capsaicin injection.
Compounds 4 and 6 did not produce any decrease in capsaicin in-
duced pain at a dose of 5 mg Kgꢀ1. However, compound 4 was
found to produce a significant analgesic effect at a dose of
10 mg Kgꢀ1 whereas compound 6 did not attenuate the capsaicin
induced algesia even at a dose of 10 mg Kgꢀ1. The effect of com-
pound 4 at a dose of 10 mg Kgꢀ1 was comparable to the standard
drug diclofenac with 25 mg Kgꢀ1 dose. On the other hand, both
the compounds 5 and 8, showed a remarkable analgesic effect at
a dose of 5 mg Kgꢀ1 while not much improvement was observed
at increasing the dose to 10 mg Kgꢀ1 (Fig. 2). However, compound
5 at 5 mg Kgꢀ1 dose was found to show analgesic activity compa-
rable to the standard drug diclofenac (25 mg Kgꢀ1 dose). Overall,
IC50 (lM) of compounds 4–9 against COX-1, COX-2 and their selectivity index
Compound
IC50
Selectivity index*
(COX-2)
(COX-1)
4
5
6
7
0.7
6.2
7.9
118
90
102
—
168.5
14.5
12.9
—
—
8
9
7.3
—
115
—
15.7
—
Indomethacin
Diclofenac
Celecoxib
Chrysin
0.96
0.02
0.04
25.5
0.08
0.07
15
0.08
3.5
375
1.5
39.3
*
IC50 (COX-1)/IC50 (COX-2).

80
P. Singh et al. / Bioorg. Med. Chem. Lett. 24 (2014) 77–82
Figure 2. Effect of compounds 4–6 and 8 on capsaicin induced pain in mice. All
values are expressed as mean SD (n = 6 per group). Statistical significance has
been calculated using one way ANOVA followed by Tukey’s multiple range
comparison test. ⁄p < 0.001 versus control group.
based on these three individual moieties. As per the results of en-
zyme immunoassays, compounds 4, 5 and 8 exhibit significant
activity for inhibition of COX-2. Molecular modeling studies indi-
cating considerable interactions of these compounds with COX-2
active site amino acids also favor the enzyme immunoassay re-
sults. Compounds 4, 5 and 8 are identified with significant anti-
nociceptive potential comparable with the standard drug
diclofenac.
Acknowledgments
Financial assistance from CSIR, New Delhi and DST, New Delhi is
gratefully acknowledged. Shaveta thanks UGC for fellowship.
Supplementary data
Supplementary data (docking structures, NMR and mass spec-
tra) associated with this article can be found, in the online version,
at http://dx.doi.org/10.1016/j.bmcl.2013.11.080.
References and notes
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6COX). Green lines indicate H-bonds formed between the compound and the
enzyme active site residues. Hs0 are omitted for clarity; (b) compound 4 docked in
the active site of enzyme COX-2 (pdb ID 3MQE); (c) CPK model of the active site of
COX-2 (pdb ID 3MQE) showing fitting of compound 4 in the active site.
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compounds 4, 5 and 8 were identified for promising analgesic
potential.
In conclusion, the results of present studies indicate that the
compounds obtained by the combination of chrysin, indole and
pyrazole are more potent for COX-2 inhibition than the drugs
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7.59 (m, 7H, ArH), 7.77–7.93 (m, 5H, ArH), 8.11 (d, J = 9.3 Hz, 1H, ArH), 9.48 (s,
1H, ArH), 12.67 (s, 1H, OH); 13C NMR (75 MHz, CDCl3) d: 26.40 (CH2), 46.80
(CH2), 67.23 (CH2), 93.23, 98.76, 105.98, 108.87, 110.44, 118.40, 121.66,
121.76, 122.38, 123.13, 126.44, 126.99, 128.08, 129.13, 129.21, 130.63, 131.48,
131.93, 136.11, 136.19, 137.20, 139.07, 157.90, 162.31, 164.10, 164.16, 164.90,
26. Goodman, L. S.; Gilman, A. The Pharmacological Basis of Therapeutics; Mc Graw-
Hill: New Delhi, 1991. pp 358–360.
182.58; HRMS Calcd for
C41H28Cl2O5N2: 699.1448. Found: m/z 699.1421
(M+H)+.
27. Dewick, P. M. Medicinal Natural Products. A Biosynthetic Approach; Wiley: India,
2012. Chapter 6.
1-(2,6-Dichloro-phenyl)-3-{1-[4-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)-butyl]-1H-indol-3-ylmethylene}-1,3-dihydro-indol-2-one (9): Compound
9 was synthesized according to the synthetic procedure given above as a
yellow solid, in a yield of 67%, mp 120 °C; 1H NMR (300 MHz, CDCl3) d: 1.62–
1.89 (m, 2H, CH2), 2.09–2.16 (m, 2H, CH2), 3.98–4.06 (dt, 2H, CH2), 4.26–4.36
(dt, 2H, CH2), 6.29–6.39 (m, 1H, ArH), 6.41–6.46 (m, 2H, ArH), 6.64 (d, J =
3.3 Hz, 1H, ArH), 7.14–7.53 (m, 9H, ArH), 7.83–7.97 (m, 5H, ArH), 8.09 (s, 1H,
ArH), 8.24 (s, 1H, ArH), 9.50 (s, 1H, ArH), 12.66 (s, 1H, OH); 13C NMR (75 MHz,
CDCl3) d: 26.83 (CH2), 27.11 (CH2), 47.34 (CH2), 68.18 (CH2), 93.44, 98.97,
106.19, 109.08, 110.65, 111.72, 117.87, 118.49, 118.61, 121.97, 122.60, 123.01,
123.35, 126.61, 126.65, 127.20, 128.29, 129.34, 129.42, 129.50, 130.85, 131.70,
132.14, 136.32, 136.40, 137.41, 139.28, 157.52, 162.52, 163.71, 163.87, 165.12,
28. Rauf, A.; Farshori, N. N. Microwave-Induced Synthesis of Aromatic Heterocycles-
Springerbriefs in Molecular Science; Springer, 2012. Chapter 6.
29. Dewangan, D.; Kumar, T.; Alexander, A.; Nagori, K.; Tripathi, D. K. Curr. Pharm.
Res. 2011, 1, 369.
30. Melting points were determined in capillaries and are uncorrected. 1H and 13
C
NMR spectra were recorded on JEOL 300 and 75 MHz NMR spectrometer,
respectively, using CDCl3 and/or DMSO-d6 as solvent. Chemical shifts are given
in ppm with TMS as an internal reference. J values are given in Hertz. Signals
are abbreviated as singlet, s; doublet, d; double-doublet, dd; triplet, t;
multiplet, m. Mass spectra were recorded on Bruker micrOTOF Q II Mass
spectrometer.
Compounds 4–9 were prepared through knoevenagel condensation of
compounds 12 and 13 (1 mmol) with active methylene compounds including
1-(3-chlorophenyl)-3-methyl-2-pyrazolin-5-one, oxindole and indolinone
(1 mmol). The reactants were heated at 160–180 °C for about 10–20 min in
oil bath and the completion of the reaction was monitored using TLC. The crude
products were solidified by triturating with diethyl ether and purified by
recrystallization and column chromatography.
182.78; HRMS Calcd for
C42H30Cl2O5N2: 735.1424. Found: m/z 735.1408
(M+Na)+.
31. COX inhibitor screening assay kit, Item No. 560131. Cayman Chemical Co.
General procedure for COX-1/2 inhibitory immunoassay: For studying the COX-1,
COX-2 inhibitory activities of the compounds, various reagents were prepared
as per the protocol of the assays. The background samples were prepared for
both COX-1 (ovine) and COX-2 (human recombinant) by taking 20 ll of each
enzyme in separate test tubes and keeping them in boiling water for 3 min. The
inactivated enzymes were used to generate background values. In two test
4-{1-[3-(5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)-propyl]-1H-indol-3-
ylmethylene}-5-methyl-2-phenyl-2,4-dihydro-pyrazol-3-one (4): Compound
was synthesized according to the synthetic procedure given above as
4
a
tubes named background COX-1 and background COX-2, 970
buffer, 10 l of heme and 10 l of inactive COX-1 or COX-2 were added. 100%
Initial activity tubes were prepared for both COX-1 and COX-2 by adding
950 l of reaction buffer, 10 l of heme and 10 l of COX-1 or COX-2. Inhibitor
tubes were prepared for compounds 4–9. In each sample tube, 950 l reaction
buffer, 10 l of heme, 10 l of COX-1 or COX-2 enzyme and 20 l of the
inhibitor solution was added. All the solutions were incubated for 10 min at
37 °C. After incubation, 10 l of AA was added to all the test tubes and vortex.
They were again incubated for another 2 min. Afterwards, 50 l of 1 M HCl was
added to each test tube to stop the reaction. Then 100 l of stannous chloride
ll of reaction
l
l
yellow solid, in a yield of 87%, mp 202 °C; 1H NMR (300 MHz, CDCl3) d: 2.38 (s,
3H, CH3), 2.46 (t, J = 6 Hz, 2H, CH2), 4.05 (t, J = 5.4 Hz, 2H, CH2), 4.51 (t, J =
6.3 Hz, 2H, CH2), 6.28 (s, 1H, ArH), 6.44 (s, 1H, ArH), 6.60 (s, 1H, ArH), 7.07 (d, J
= 7.8 Hz, 1H, ArH), 7.14–7.36 (m, 4H, ArH), 7.46–7.53 (m, 4H, ArH), 7.70–7.94
(m, 4H, ArH), 8.05 (s, 1H, ArH), 9.86 (s, 1H, ArH), 12.69 (s, 1H, OH); 13C NMR
(75 MHz, CDCl3) d: 13.10 (CH3), 29.33 (CH2), 44.24 (CH2), 64.99 (CH2), 93.12,
98.62, 105.82, 110.67, 112.26, 116.60, 118.21, 118.72, 119.60, 122.83, 123.95,
124.11, 126.24, 129.02, 129.17, 129.68, 131.79, 134.31, 135.27, 136.74, 140.39,
143.93, 150.82, 157.78, 162.19, 163.34, 163.96, 164.22, 182.37; HRMS Calcd for
l
l
l
l
l
l
l
l
l
l
solution was added to each test tube and vortex. Incubated for another 5 min
and kept at 0–4 °C.
C
37H28ClO5N3: 652.1610. Found: m/z 652.1615 (M+Na)+.
4-{1-[4-(5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)-butyl]-1H-indol-3-
ylmethylene}-5-methyl-2-phenyl-2,4-dihydro-pyrazol-3-one (5): Compound
was synthesized according to the synthetic procedure given above as
Prostaglandin screening standards were prepared as test tubes S1–S8. 800
Of EIA buffer was added to S1 and 500 l of the same was added to S2–S7. Then
200 of bulk standard (10 ng/ml) was added to tube S1 and mixed
thoroughly. The standards were diluted serially by removing 500 l from
tube S1 and placing it in tube S2 and mixed thoroughly. Same process was
repeated from S2–S3, S3–S4 up to S7–S8.
ll
5
a
l
ll
yellow solid, in a yield of 60%, mp 200 °C; 1H NMR (300 MHz, CDCl3) d: 1.89–
1.94 (m, 2H, CH2), 2.18–2.23 (m, 2H, CH2), 2.40 (s, 3H, CH3), 4.04 (t, J = 6 Hz, 2H,
CH2), 4.39 (t, J = 6 Hz, 2H, CH2), 6.25 (s, 1H, ArH), 6.45 (s, 1H, ArH), 6.65 (s, 1H,
ArH), 7.23 (d, J = 7.2 Hz, 1H, ArH), 7.24–7.51 (m, 8H, ArH), 7.79-7.85 (m, 5H,
ArH), 8.09 (s, 1H, ArH), 9.89 (s, 1H, ArH), 12.69 (s, 1H, OH); 13C NMR (75 MHz,
CDCl3) d: 13.00 (CH3), 29.10 (CH2), 29.23 (CH2), 44.14 (CH2), 64.69 (CH2), 93.02,
98.52, 105.72, 110.57, 112.16, 116.50, 118.11, 118.62, 119.50, 122.73, 123.85,
124.01, 126.14, 128.92, 129.07, 129.58, 131.14, 131.69, 135.17, 136.64, 140.29,
150.72, 157.66, 162.09, 163.19, 163.92, 164.12, 182.41; HRMS Calcd for
l
To make dilutions for COX reactions, two test tubes named BC1 and BC2 were
prepared. To each test tube was added, 990
buffer and 10 l of background COX-1 or COX-2 and mixed thoroughly. COX
100% initial activity samples were prepared as three test tubes for COX-1 and
COX-2 both and numbered as IA1–IA3. For each sample, aliquot 990 l of EIA
buffer to IA1, 950 l of EIA buffer to IA2 and 500 l of EIA buffer to IA3. 10 l of
COX-1 or COX-2 100% initial activity sample was added to IA1 and mixed
thoroughly. Aliquot, 50 l of tube IA1 and added to tube IA2 and mixed
thoroughly. Again aliquot 500 l from test tube IA2 and added to test tube IA3
ll of EIA (enzyme immunoassay)
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l
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l
C
38H30ClO5N3: 644.1947. Found: m/z 644.1932 (M+H)+.
3-{1-[3-(5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)-propyl]-1H-indol-3-
ylmethylene}-1,3-dihydro-indol-2-one (6): Compound was synthesized
l
6
l
according to the synthetic procedure given above as a yellow solid, in a yield
of 60%, mp 225 °C; 1H NMR (300 MHz, DMSO-d6) d: 2.31-2.48 (m, 2H, CH2),
4.11 (t, J = 5.1 Hz, 2H, CH2), 4.52 (t, J = 7.2 Hz, 2H, CH2), 6.35–6.39 (m, 1H, ArH),
6.71 (d, J = 6 Hz, 1H, ArH), 6.81–6.84 (m, 1H, ArH), 6.96 (s, 1H, ArH), 7.06–7.13
(q, 1H, ArH), 7.19–7.30 (m, 2H, ArH), 7.53–7.74 (m, 6H, ArH), 7.84 (d, J = 6 Hz,
1H, ArH), 8.01–8.29 (m, 4H, ArH), 9.47 (s, 1H, ArH), 10.46 (d, J = 11.4 Hz, 1H,
ArH); 13C NMR (75 MHz, DMSO-d6) d ppm: 26.10 (CH2), 46.97 (CH2), 67.12
(CH2), 92.77, 93.35, 98.78, 105.84, 108.68, 111.64, 118.22, 118.37, 121.72,
122.26, 122.41, 123.18, 126.26, 126.97, 127.93, 129.02, 129.15, 129.48, 135.93,
136.00, 136.25, 157.75, 162.31, 164.05, 164.14, 164.47, 182.26; HRMS Calcd for
and mixed well. In the same manner, COX inhibitor samples were prepared by
further dilutions and named C1–C3 for each concentration.
After preparing all the dilutions, theywere introduced on 96 well plate. The
wells were distributed as blank-1A, NSB (nonspecific binding)-1B and Bo
(maximum binding)-1C. Well 1H was named as TA (Total activity well).Wells
2A–2H were used for S1–S8 and 3A–3H were used for S1–S8 duplicate. Wells
4A and 5A were prepared as BC1 and its duplicate. Similarly, for BC2 wells 4B
and 5B were prepared. Remaining wells were used for inhibitor samples for
COX-1 and COX-2.
The addition of the reagents on 96-well plate was performed as follows: 100
EIA buffer was added to NSB well and 50 l of EIA buffer was added to Bo well.
50 l of prostaglandin screening standard was added to the respective wells
S1–S8 from their respective test tubes S1–S8 and duplicated. 50 l of BC1 and
BC2 were added per well and in duplicate. 50 l of 100% initial activity samples
were added per well and only IA2 and IA3 were assayed in duplicate for both
COX-1 and COX-2. 50 l of COX inhibitor sample was added per well from their
respective dilutions (only C2 and C3 were assayed). 50 l of PG screening AChE
tracer was added to each well except TA and Blank well. At last, 50 l of PG
ll
C
35H26O5N2: 555.1914. Found: m/z 555.1918 (M+H)+.
3-{1-[4-(5-Hydroxy-4-oxo-2-phenyl-4H-chromen-7-yloxy)-butyl]-1H-indol-3-
ylmethylene}-1,3-dihydro-indol-2-one (7): Compound was synthesized
l
l
7
l
according to the synthetic procedure given above as a yellow solid, in a yield
of 78%, mp 156 °C; 1H NMR (300 MHz, CDCl3) d: 1.86–1.89 (m, 2H, CH2), 2.15–
2.18 (m, 2H, CH2), 4.02 (t, J = 5.7 Hz, 2H, CH2), 4.33 (t, J = 6.3 Hz, 2H, CH2), 6.28–
6.33 (m, 1H, ArH), 6.42 (s, 1H, ArH), 6.61–6.65 (m, 1H, ArH), 6.84–6.91 (m, 2H,
ArH), 7.05 (t, J = 7.2 Hz, 1H, ArH), 7.16 (t, J = 6.6 Hz, 1H, ArH), 7.29–7.60 (m, 6H,
ArH), 7.80–7.98 (m, 4H, ArH), 8.10 (s, 1H, ArH), 9.49 (s, 1H, ArH), 12.68 (s, 1H,
OH); 13C NMR (75 MHz, CDCl3) d: 29.30 (CH2), 29.58 (CH2), 43.62 (CH2), 65.82
(CH2), 92.85, 93.03, 98.56, 105.82, 108.66, 111.52, 118.10, 118.15, 121.60,
122.14, 122.19, 123.06, 126.24, 126.85, 127.81, 128.20, 128.90, 129.03, 129.16,
135.81, 135.88, 136.23, 157.63, 162.19, 163.93, 164.24, 164.35, 182.43; HRMS
Calcd for C36H28O5N2: 591.1890. Found: m/z 598.1880 (M+Na)+.
1-(2,6-Dichloro-phenyl)-3-{1-[3-(5-hydroxy-4-oxo-2-phenyl-4H-chromen-7-
yloxy)-propyl]-1H-indol-3-ylmethylene}-1,3-dihydro-indol-2-one (8): Compound
8 was synthesized according to the synthetic procedure given above as a
yellow solid, in a yield of 69%, mp 146 °C; 1H NMR (300 MHz, CDCl3) d: 2.12–
2.17 (m, 2H, CH2), 3.98 (t, J = 6 Hz, 2H, CH2), 4.31 (t, J = 6.6 Hz, 2H, CH2), 6.30 (d,
J = 15 Hz, 1H, ArH), 6.40–6.42 (m, 1H, ArH), 6.62 (d, J = 15 Hz, 1H, ArH), 6.84–
6.91 (m, 2H, ArH), 7.04 (t, J = 7.2 Hz, 1H, ArH), 7.15 (t, J = 7.5 Hz, 1H, ArH), 7.28–
l
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l
screening EIA antiserum was added to each well except TA, NSB and blank
wells. The plate was then covered with plastic film and was incubated for 18 h
at room temperature. After incubation, the plate was developed by emptying
the wells and rinsing the wells with wash buffer for five times. After washing
the wells, 200 ll of Ellman’s reagent was added to each well and 5 ll of tracer
was added to Total activity well. The plate was covered with plastic film and it
was kept for 60–90 min. Before reading the plate, it was wiped from bottom to
remove any fingerprints and finally read at 420 nm.
Calculation of % inhibition and IC50 values: %B/Bo value for each sample (all
compounds 4–9 at 10ꢀ3 M, 10ꢀ4 M, 10ꢀ5 M, 10ꢀ6 M, 10ꢀ7 M and 10ꢀ8 M conc
each) was determined from the absorbance values attained after reading the
96 well plate at 420 nm according to the calculation strategy provided in the
manufacturer’s protocol for inhibition assay.31

82
P. Singh et al. / Bioorg. Med. Chem. Lett. 24 (2014) 77–82
From the values of %B/Bo, conc of prostaglandins formed during the enzymatic
34. Rimon, G.; Sidhu, R. S.; Lauver, D. A.; Lee, J. Y.; Sharma, N. P.; Yuan, C.; Frieler, R.
A.; Trievel, R. C.; Lucchesi, B. R.; Smith, W. L. Proc. Natl. Acad. Sci. U.S.A. 2010,
107, 28.
35. Kurumbail, R. G.; Stevens, A. M.; Gierse, J. K.; McDonald, J. J.; Stegeman, R. A.;
Pak, J. Y.; Gildehaus, J. L.; Miyashiro, J. M.; Penning, T. D.; Seibert, K.; Isakson, P.
C.; Stallings, W. C. Nature 1996, 384, 644.
36. Wang, J. L.; Limburg, D.; Graneto, M. J.; Springer, J.; Hamper, J. R.; Liao, S.;
Pawlitz, J. L.; Kurumbil, R. G.; Maziasz, T.; Talley, J. J.; Kiefer, J. R.; Carter, J.
Bioorg. Med. Chem. Lett. 2010, 20, 7159.
37. Sakurada, T.; Matsumura, T.; Moriyama, T.; Sakurada, C.; Ueno, S.; Sakurada, S.
Pharmacol. Biochem. Behav. 2003, 75, 115.
reaction were calculated for all the compounds at all conc with the help of the
standard curve. After having the prostaglandin conc at each tested conc for all
the compounds, percentage inhibition values were calculated at all the tested
conc. according to the prescribed protocol.
Finally, the percentage inhibitions at all the conc were plotted against the
respective tested conc for each compound using GraphPad Prism version 6.01
for calculating IC50 (50% inhibitory conc).
32. ArgusLab 4.0.1, M. A. Thompson, Planaria Software LLC, Seattle, WA 98155.
33. Selinsky, B. S.; Gupta, K.; Sharkey, C. T.; Loll, P. J. Biochemistry 2011, 40,
5172.