Rational design, synthesis and evaluation of chromone-indole and chromone-pyrazole based conjugates: Identification of a lead for anti-inflammatory drug

Article abstract of DOI:10.1016/j.ejmech.2014.03.003

Conjugates of chromone-indole and chromone-pyrazole were screened for cyclooxygenase-2 (COX-2), cyclooxygenase-1 (COX-1) and 5-lipoxygenase (5-LOX) inhibitory activities. Compounds 8 and 9 were identified as preferred inhibitors of COX-2 over the other two enzymes. Their IC₅₀ for COX-2 was 29 nM and 20 nM, respectively and selectivity indices (SI) for COX-2 over COX-1 was 46 and 337. NMR, mass spectral studies and molecular modelling also indicated preferential interactions of compounds 8 and 9 with COX-2. Tested on albino mice against capsaicin induced algesia, compound 8 exhibited analgesic potential comparable to diclofenac. In addition to the biological profile, the desirable physico-chemical properties of these compounds make them promising leads for anti-inflammatory drugs.

Full text of DOI:10.1016/j.ejmech.2014.03.003

European Journal of Medicinal Chemistry 77 (2014) 185e192
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Rational design, synthesis and evaluation of chromone-indole and
chromone-pyrazole based conjugates: Identification of a lead for anti-
inflammatory drug
Shaveta a, Amrinder Singh a, Matinder Kaur a, Surbhi Sharma b, Rajbir Bhatti b,
,
Palwinder Singh a
*
a Department of Chemistry, UGC Sponsored Centre for Advanced Studies, Guru Nanak Dev University, Amritsar, Punjab 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:
Conjugates of chromone-indole and chromone-pyrazole were screened for cyclooxygenase-2 (COX-2),
cyclooxygenase-1 (COX-1) and 5-lipoxygenase (5-LOX) inhibitory activities. Compounds 8 and 9 were
identified as preferred inhibitors of COX-2 over the other two enzymes. Their IC50 for COX-2 was 29 nM
and 20 nM, respectively and selectivity indices (SI) for COX-2 over COX-1 was 46 and 337. NMR, mass
spectral studies and molecular modelling also indicated preferential interactions of compounds 8 and 9
with COX-2. Tested on albino mice against capsaicin induced algesia, compound 8 exhibited analgesic
potential comparable to diclofenac. In addition to the biological profile, the desirable physico-chemical
properties of these compounds make them promising leads for anti-inflammatory drugs.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Received 14 August 2013
Received in revised form
9 February 2014
Accepted 2 March 2014
Available online 3 March 2014
Keywords:
Chromone
Indole
Pyrazole
Interactions
Cyclooxygenase-2
Inhibitors
1. Introduction
formation of inflammatory prostaglandins through COX-2 enzy-
matic activity are well documented [7]. Hence, succeeding COX-1/2
Being a part of the complex vascular response or an innate-
immunity response, inflammation is a protective attempt [1] by
the body against harmful pathogens and stimuli. It is not consid-
ered as a disease in its initial stage but under chronic conditions
may lead to a number of diseases including rheumatoid arthritis,
asthma and even cancer [2]. Identification of metabolites [3] and
corresponding enzymes of arachidonic acid (AA) metabolism led to
systematic treatment of inflammatory diseases by controlling the
enzymatic activities of phospholipase, lipoxygenase and/or cyclo-
oxygenase. Moreover, differentiation of cyclooxygenase into two
structural and functional isoforms [4,5] viz. cyclooxygenase-1
(COX-1) and cyclooxygenase-2 (COX-2) and disclosure of reasons
for side effects of aspirin, ibuprofen (drugs put to clinical use
without knowing their cellular target) [6] incited the scientific
community to choose COX-2 as the cellular target of anti-
inflammatory drugs. House-keeping functions of COX-1 and
non-selective aspirin, ibuprofen, indomethacin; COXIBS were
developed as selective COX-2 inhibitors [8]. However, revelation of
cardiovascular side effects of rofecoxib and valdecoxib and their
withdrawal from the market in 2004 and 2005, respectively [9,10]
put a question mark on the safety of other similar drugs like cele-
coxib and left the space open for developing more safe and effica-
cious anti-inflammatory drugs.
A wide variety of natural products are being used in the form of
drugs [11]. Several compounds from terpenoid, alkaloid and
flavonoid categories of natural products have been isolated [12]
and evaluated for their COX inhibitory activities amongst which
chromone based compounds [12] (1, 2; Chart 1) have shown
promising inhibition of COX-2 enzymatic activity. Besides chro-
mone; indole and pyrazole are the other naturally occurring
moieties which are part of a number of clinically used drugs like
indomethacin and celecoxib (3, 4; Chart 1) [13,14]. Therefore,
keeping in mind the higher efficacy of hybrid molecules [15,16], it
was envisaged that the compounds obtained by the combination
of chromone-indole and chromone-pyrazole may prove as better
COX-2 inhibitors in comparison to their individual parent
* Corresponding author.
E-mail address: palwinder_singh_2000@yahoo.com (P. Singh).
http://dx.doi.org/10.1016/j.ejmech.2014.03.003
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

186
Shaveta et al. / European Journal of Medicinal Chemistry 77 (2014) 185e192
Chart 1. Design of new molecules.
molecules. As this hypothesis worked well in the development of
cytotoxic agents [17], chromone-indole based compound 5 (Chart
1) with appreciable anti-cancer activity (average GI50 over 60
2. Results and discussion
2.1. Chemistry
human tumour cell lines 3.2
mM) was taken for determining its
COX-2 inhibitory activity. Similar to compound 5, compounds 6e
10 (Chart 1) were synthesized and screened for the inhibition of
enzymatic activity of COX-2, COX-1 and 5-LOX. Results of enzyme
immunoassays were supported with NMR, mass spectral studies
and molecular modelling. Compound 8 was investigated for
analgesic activity on albino mice.
A simple experimental protocol was employed for the synthesis
of compounds. Reaction of chromone-3-carboxaldehyde with
indolinone (1-(2,6-dichlorophenyl)-2-indolinone) under micro-
wave irradiations gave compound 5. Similar reactions of 6-bromo/
isopropyl-chromone-3-carboxaldehyde provided compounds
6
and 7 (Scheme 1). Using the same synthetic protocol, a finely
Scheme 1. Synthesis of compounds 5e10.

Shaveta et al. / European Journal of Medicinal Chemistry 77 (2014) 185e192
187
ground mixture of chromone-3-carboxaldehyde (1 mmol) and
oxindole (1.2 mmol) was irradiated under microwaves to obtain
compound 8 and reaction of 6-bromochromone-3-carboxaldehyde
with oxindole provided compound 9 (Scheme 1). With the limited
activities of these hybrid compounds and chromone, indole, pyr-
azole based drugs; IC50 of wogonin (1, Chart 1) [21], indomethacin
(3, Chart 1) [22] and celecoxib (4, Chart 1) [23] were taken into
consideration. Compounds 8 and 9 showed better COX-2 inhibitory
activity and selectivity for COX-2 over COX-1 than that of wogonin
and indomethacin which justifies the design of the molecules. IC50
of these two compounds for COX-2 was also comparable to that of
celecoxib.
availability
of
6-fluoro-8-nitrochromone-3-carboxaldehyde
(SigmaeAldrich stopped the supply), compound 10 was prepared
by its combination with 1-(3-chlorophenyl)-3-methyl-2-pyrazolin-
5-one (Scheme 1). Therefore, as per the design of the molecules and
in accordance with the fact “the most fruitful basis for the discovery
of a new drug is to start with an old one”, the new compounds were
made, retaining as much as possible the structural features of some
old drugs.
2.3. NMR spectral studies and molecular modelling
1H NMR spectrum of compound 8 (0.35 mM in DMSO-d6) was
recorded (Figs. S1, S2) and the signals for various hydrogens were
identified. D2O exchange experiment identified NH of indole moi-
2.2. Enzyme inhibition assays
ety at
d 10.7. 12 ml (0.08 mM) of COX-2 (low concentration for
Compounds 5e10 were screened for inhibition of catalytic ac-
tivities of COX-2, COX-1 and 5-lipoxygenase (5-LOX); the enzymes
participating in AA metabolism. All the enzyme inhibition assays
were performed in triplicate as per the manufacturer’s protocol
available with the enzyme immunoassay kits. Compounds were
tested at 10ꢀ5 M, 10ꢀ6 M, 10ꢀ7 M and 10ꢀ8 M concentrations. The
enzyme inhibition assay [18,19] used here is based on COX-1/2 and
5-LOX catalysed production of prostaglandins and leukotrienes,
respectively during AA metabolism. Catalytic activities of the three
enzymes were quantified by measuring the amount of prosta-
glandins/leukotrienes produced by each enzyme in the presence of
various concentrations of the test compounds and comparing with
control experiments.
observing signals of the compound only) was added to the solution
of compound 8 in DMSO-d6 (no D2O). NMR spectra were recorded
after every 30 min until no change was observed (Figs. S1, S2). NH
signal was shifted to
d 10.85 and its intensity was considerably
reduced which indicated that the enzyme COX-2 might be inter-
acting with NH group of compound 8.
Looking for the possibility of interactions of compound 8 with
COX-2 through its carbonyl oxygens, 13C NMR spectra of compound
8 alone as well as in the presence of COX-2 (same conc and solvent
as for 1H NMR spectra) were recorded. Comparison of two 13C NMR
spectra (Fig. S3) clearly showed the downfield shift of some of the
signals especially those belonging to carbonyl carbons of chromone
and oxindole moieties at
d 174.9 and 167.5, respectively (Fig. S4).
Enzyme inhibition N 1/conc of prostaglandin or leukotriene in
each enzymatic reaction.
Compounds 5, 7, 8 and 9 showed considerable inhibition of COX-
2 with IC50 (50% inhibitory concentration) 1.3 nM, 5.8 nM, 29 nM
and 20 nM, respectively while their respective IC50 for COX-1 was
Hence, there seems to be the possibility of interaction between
carbonyl oxygen of the compound and amino acid residue of the
enzyme (Fig. S5).
In order to support NMR spectral data and pin-point the in-
teractions of NH and C]O group with amino acid residues, mo-
lecular docking [24] of compound 8 in the active site of COX-2 [25]
0.0047 mM, 0.65 mM, 1.35 mM and 6.75 mM. Since COX-1 and COX-2
ꢀ
ꢀ
are responsible for the synthesis of different prostaglandins in AA
pathway, preferred inhibition of catalytic activity of COX-2 is
desirable for controlling inflammation [20]. Compound 5 showed
considerable inhibition of catalytic activities of both COX-2 and
COX-1, exhibiting negligible selectivity for COX-2 over COX-1
(Table 1) and hence cannot be recognized as a proficient candi-
was performed. H-Bonds of 2.88 A and 2.90 A between NH group
of compound 5 and Arg120 (Arg120 forms salt bridge with AA
during the metabolic phase of the enzyme) and chromone
carbonyl oxygen and Ser530, respectively were observed.
Carbonyl oxygen of indole moiety also showed interaction with
Tyr355 (Fig. 1). Therefore, supporting the results of NMR spectral
investigations, molecular docking studies also showed the in-
teractions of NH and carbonyl oxygens of compound 8 with the
enzyme.
date for its use as COX-2 inhibitor. Compound 6 with IC50 0.63
and 7.6 M for COX-1 and COX-2, respectively also exhibited poor
selectivity for COX-2 over COX-1. Compound 10 exhibited
IC50 > 10 M for both COX-1 and COX-2. As per the results of pre-
mM
m
m
Similarly, interactions of compound 8 with COX-1 were studied.
1H NMR spectrum of compound 8 (DMSO-d6) after the addition of
COX-1 (0.08 mM) resulted into small shift and decrease in intensity
of the NH signal of compound 8 (Fig. S5). 13C NMR spectrum of
compound 8 in the presence of COX-1 showed the shifting of
sent experiments, compounds 8 and 9 are identified with sub-
stantial inhibition of COX-2 as well as desirable selectivity for COX-
2 over COX-1. Only compound 7 showed appreciable inhibition of
5-LOX with IC50 20 nM. For making comparison of COX-2 inhibitory
Table 1
Inhibition of catalytic activity of COX-1, COX-2 and 5-LOX by compounds 5e10.
Compd
COX-1
COX-2
SIa
5-LOX
% Inhibition (Molar conc)
IC50
(
m
M)
% Inhibition (Molar conc)
IC50
(
mM)
% Inhibition (Molar conc)
IC50 (mM)
10ꢀ5
10ꢀ6
10ꢀ7
10ꢀ5
10ꢀ6
10ꢀ7
10ꢀ8
10ꢀ5
10ꢀ6
10ꢀ7
10ꢀ8
5
6
7
8
9
10
80.3
80.3
64.6
51.6
40.5
27.9
e
75
60
56
43
30.4
18
e
63
44.4
47
35
20
15
e
0.0047
0.63
0.65
1.35
6.75
>10
e
78.1
e
11.6
46.5
e
64.9
34.9
67.2
57
61.7
36.0
e
51
23.5
54
52
57.4
15
e
0.0013
7.6
0.0058
0.029
0.020
>10
46
0.96
0.04
4
8.62
50
e
15.5
e
20
37
44.8
22.4
e
e
e
0.08
112
46
337
e
25
30.1
e
10
0.02
e
72.8
96.7
70.4
18.6
e
81
e
73.3
66.3
e
74.1
e
77.6
46.5
e
68.1
8.62
e
e
e
22.4
e
7.4
e
Wogonin
Indomethacin
Celecoxib
e
e
e
e
e
e
0.08
15
e
e
e
e
0.08
375
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
a
Selectivity index ¼ IC50(COX-1)/IC50(COX-2).

188
Shaveta et al. / European Journal of Medicinal Chemistry 77 (2014) 185e192
COX-1/2 interactions with AA e COX-1/2 interactions, association
constants of AA with COX-2, COX-1 were also calculated. Mass
spectra of COX-1 and COX-2 alone as well as in combination with
AA, compound 8 and compound 9 were recorded (Figs. S34, S35,
Supporting information). Association constants indicated better
interaction of compounds 8 and 9 with COX-2 in comparison to
their interactions with COX-1. Moreover, Ka of compounds 8 and 9
for COX-2 were higher than those exhibited by AA with this
enzyme. On the other hand, their Ka for COX-1 was smaller than
that of COX-1 e AA (Table 2). Normally, an effective COX-2 inhibitor
occupies the active site of the enzyme in preference to AA (the
substrate) and provides relief from inflammation by blocking the
production of inflammatory prostaglandins. Since the association
constants of compounds 8 and 9 for COX-2 are better than that of
AA, they may bind with COX-2 in preference to AA and hence may
be developed as highly efficacious COX-2 inhibitors.
Moreover, comparing with aspirin, compounds 8 and 9 exhibi-
ted larger association constants for COX-2 and smaller association
constants with COX-1 which indicated that compounds 8 and 9
may be better and safe COX-2 inhibitors than aspirin. However, Ka
of compound 8 e COX-2 was comparable to Ka of indomethacin e
COX-2 while Ka of compounds 8/9 for COX-1 were less than that of
indomethacin.
Fig. 1. Compound 8 docked in the active site of COX-2 (PDB ID 6COX). Yellow lines
represent H-bonds with bond length in A. Purple amino acids are positively charged,
grey amino acids are hydrophobic while blue amino acids are hydrophilic in nature.
(For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
ꢀ
2.5. Analgesic activity of compound 8
chromone carbonyl carbon from
d 175.03 to d 175.20 (Fig. S6).
Docking of compound 8 in the active site of COX-1 [26] indicated
presence of H-bond interactions between NH and oxindole
carbonyl groups of compound 8 with R120 (Fig. 2). However,
comparing the results of NMR studies and molecular modelling, it
seems that compound 8 exhibited better interactions with COX-2
than that of COX-1.
Based upon the results of enzyme immunoassay, NMR, mass
spectrometric and molecular docking studies, compound 8 was
checked for its analgesic activity (algesia being the major symptom
of inflammation). Swiss albino mice (25e35 g) of either sex were
used in the present study. The animals were housed at 25 ꢁ 2 ꢂC
under 12 h light/12 h dark cycle and free access to food and water in
the central animal house at Guru Nanak Dev University, Amritsar,
Punjab. Animals were acclimatized to the laboratory for at least 1 h
before testing and were used only once throughout the experi-
ments. All the protocols have been duly approved by the Institu-
tional Ethics Committee and are in accordance with guidelines of
Committee for the purpose of Control and Supervision of Experi-
ments on Animals (CPSCEA), Ministry of Environment and Forests,
India.
2.4. Mass spectral studies
In continuation with the results of foregoing experiments,
binding of compounds 8 and 9 with COX-2 and COX-1 was also
evaluated in terms of association constants (Ka). Since electrospray
ionization (ESI) mode of mass spectrometry is a prompt and ac-
curate technique to study non-covalent interactions [27e33], as-
sociation constants of compounds 8 and 9 with COX-1 and COX-2
were calculated from mass spectral data. In order to compare 8/9 e
With a few modifications, the method used for capsaicin-
induced licking was similar to that described by Sakurada et al.
[34]. Capsaicin (1.6 mg/20 m) 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 on paw
licking and twitching was recorded as an indicator of algesia.
Animals were divided into 3 groups of 6 each. All the treatments
were administered intraperitoneally 30 min before capsaicin in-
jection. 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 animals
Table 2
Association constants of compounds 8, 9, AA and drugs for COX-1 and COX-2.
Entry
Ligand
aKa (Mꢀ1) ¼ [EL]/[E]free*[L]free
COX-1
COX-2
1
2
3
4
5
6
Arachidonic acid
8
9
Aspirin
Indomethacin
Celecoxib
0.39 ꢃ 104
0.29 ꢃ 104
0.35 ꢃ 104
0.93 ꢃ 104
0.54 ꢃ 104
e
0.18 ꢃ 104
0.75 ꢃ 104
0.46 ꢃ 104
0.22 ꢃ 104
0.74 ꢃ 104
2.15 ꢃ 104
a
Fig. 2. Compound 8 docked in the active site of COX-1 (PDB ID 1EQG) showing H-
bonds with Arg120.
[EL] ¼ conc of enzymeeligand complex, [E]free ¼ conc of free enzyme, [L]
free ¼ conc of free ligand.

Shaveta et al. / European Journal of Medicinal Chemistry 77 (2014) 185e192
189
Moreover, scanning electron microscope (SEM) images (recor-
ded on Field Emission Scanning Electron Microscope, Zeiss) of
compounds 8 and 9 showed molecular arrangement of the two
compounds in detail. Molecules of compound 8 are arranged in the
form of rods. The diameter and the length of the rods is in the range
of
Compound 9 showed sheet like structures with breadth of the
sheet in the range of 1 m and length of the sheets goes to 10
mm and mm, respectively (Supporting information, Fig. S38).
m
mm
(Supporting information, Fig. S39). Supporting the naked eye
observation; the crystalline, shining surface of compound 8 was
quite evident from its XRD pattern and SEM image. Although
compound 9 was not as crystalline as compound 8 but XRD pattern
and SEM image indicated that it also exists in a quite organized
form.
Fig. 3. Effect of compound
8 on capsaicin induced pain in mice. All values are
expressed as mean ꢁ SEM (n ¼ 6 per group). Statistical significance has been calculated
using one way ANOVA followed by Tukey’s multiple range comparison test.
***p < 0.001 vs. control group.
3. Conclusions
The enzyme specificity of naturally occurring compounds along
with their medicinal potential was utilized for the development of a
lead for anti-inflammatory drug. In comparison to chromone and
indole based drugs, combination of chromone and oxindole in the
form of compounds 8 and 9, resulted into considerable inhibition
and selectivity for COX-2 over COX-1. These two compounds
exhibited IC50 for COX-2 in the nM range and as observed during
NMR, mass spectral studies and molecular modelling, they showed
preferential interactions with COX-2 over COX-1. Interestingly, the
association constants of compounds 8, 9 with COX-2 were higher
than the association constants of AA and aspirin with COX-2.
However, their association constants with COX-1 were smaller
than those of AA, aspirin and indomethacin. Compound 8 at
5 mg kgꢀ1 concentration reduced the number of paw lickings due
to capsaicin induced pain in albino mice by 76% in comparison to
68% reduction on using diclofenac at concentration 25 mg kgꢀ1
which was an indicative of better analgesic activity of compound 8
than that of diclofenac. Adding to all, the desirable physico-
chemical properties and morphology was shown by compounds 8
and 9.
were injected compound 8 at a dose of 5 mg kgꢀ1 and 10 mg kgꢀ1
respectively.
Treatment with diclofenac was found to produce a marked
,
decrease in the number of paw lickings after capsaicin injection. A
decrease of 68% in paw lickings was observed on using 25 mg kgꢀ1
diclofenac. Surprisingly, compound 8 produced far better analgesic
activity than diclofenac and it reduced the paw lickings by 76% at a
dose of 5 mg kgꢀ1. On increasing the dose to 10 mg kgꢀ1, further
enhancement in the analgesic activity was observed (Fig. 3).
Moreover, compound 8, showing considerable analgesic activity is
under further refinements for developing into an anti-
inflammatory drug.
It is worth to make a comparison with an earlier report where
oxindole and indolinone (1-(2,6-dichlorophenyl)-2-indolinone) in
combination with syringaldehyde showed inhibition of dihy-
drofolate reductase (DHFR) [35]. Here, oxindole in combination
with chromone moiety in the form of compounds 8 and 9 was
found to inhibit the enzymatic activity of COX-2. As indole and
chromone based compounds are known for moderate COX-2 inhi-
bition [13,36], it may be the combined inherent selectivity of both
the fragments which make compounds 8 and 9 more selective for
COX-2. Hence, the remarkable specificity and selectivity exhibited
by the natural systems could be a roadmap for developing more
such compounds showing selective inhibition of a particular
enzyme.
4. Experimental protocols
4.1. General methods
Melting points were determined in capillaries and were un-
corrected. 1H and 13C NMR spectra were recorded on JEOL 300 MHz
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. Elemental analysis was performed on Thermoelectron
FLASH EA1112 CHN analyzer. Reactions under microwaves were
performed using microwave oven (INALSA model 1MW17EG) with
microwave power 700 W and operating frequency 2450 MHz. Mass
spectra were recorded on Bruker micrOTOF Q II Mass spectrometer.
SEM images were recorded on Zeiss Supra 55 Scanning Electron
Microscope.
Further to check the physico-chemical properties of compounds
8 and 9, Lipinski parameters and solid state morphology of the
compounds were determined. Both the compounds obeyed Lip-
inski’s rule of ‘5’ (Table 3) [37].
Since solid state morphology of the drug has significant impact
on its bioavailability and stability, the diffraction spectra of com-
pounds 8 and 9 were recorded on Rigaku Miniflex II desktop X-ray
diffractometer (XRD). Appearance of sharp peaks at 2q equal to
11.96ꢂ, 15.04ꢂ, 16.84ꢂ, 17.32ꢂ, 18.08ꢂ, 23.62ꢂ, 24.26ꢂ, 28.04ꢂ and
14.18ꢂ, 14.6ꢂ, 19.78ꢂ, 21.78ꢂ, 22.52ꢂ, 26.6ꢂ, 27.4ꢂ, 31.22ꢂ for com-
pounds 8 and 9, respectively indicated that compounds 8 and 9
are highly crystalline powders (Supporting information, Figs. S36,
S37).
4.2. General procedure
A
finely ground mixture of chromone-3-carboxaldehyde
Table 3
(1 mmol) and oxindole/indolinone (1-(2,6-dichlorophenyl)-2-
indolinone)/1-(3-chlorophenyl)-3-methyl-2-pyrazolin-5-one
(1.2 mmol) was irradiated in microwave oven for 1 min and
completion of the reaction was monitored with TLC. Reaction
mixture was washed with diethyl ether to get pure compounds
5e10.
Lipinski values for compounds 8 and 9.
Compound
Mol wt.
NH, OH
donors
NH, OH
acceptors
Total polar
surface area
Log P
8
9
291
370
1
1
4
4
59.16
59.16
2.92
3.71

190
Shaveta et al. / European Journal of Medicinal Chemistry 77 (2014) 185e192
4.2.1. 1-(2,6-Dichlorophenyl)-3-(4-oxo-4H-chromen-3-
ylmethylene)-1,3-dihydroindol-2-one (5)
(t, J ¼ 7.5 Hz, 1H, ArH), 7.23 (t, J ¼ 7.8 Hz, 1H, ArH), 7.64 (d, J ¼ 7.5 Hz,
1H, ArH), 7.70 (s, 1H, ]H), 7.73 (d, J ¼ 9.0 Hz, 1H, ArH), 7.99e8.03
(dd, J ¼ 2.7 Hz, J ¼ 9.0 Hz, 1H, ArH), 8.20 (d, J ¼ 2.4 Hz, 1H, ArH), 9.91
(s, 1H, 2-H), 10.71 (s, 1H, NH, D2O exchange); 13C NMR (75 MHz,
Compound 5 was synthesized according to the synthetic pro-
cedure given above as yellow solid, in yield of 87%, mp 242 ꢂC; 1H
NMR (300 MHz, CDCl3)
d
ppm: 6.44 (d, J ¼ 7.8 Hz, 1H, ArH), 7.16 (t,
DMSO-d6) d ppm: 109.7, 117.5, 118.5, 120.1, 121.5, 123.9, 124.6, 125.5,
J ¼ 7.5 Hz, 1H, ArH), 7.21e7.26 (m, 1H, ArH), 7.37e7.42 (m, 1H, ArH),
7.54 (d, J ¼ 8.4 Hz, 3H, ArH), 7.67e7.77 (m, J ¼ 7.8 Hz, 3H, ArH), 8.17
(s, 1H, ]H), 8.30e8.33 (dd, J ¼ 1.5 Hz, J ¼ 8.1 Hz, 1H, ArH), 10.27 (s,
127.5, 128.1, 129.6, 130.3, 137.2, 140.8, 154.5, 160.1, 164.7 (C]O),
173.9 (C]O); HRMS (ESI) calcd for [C18H10BrO3N þ H]þ: 367.9917,
369.9897, Found: 367.9905, 369.9889; Anal. For C18H10BrO3N calcd
%: C 58.72, H 2.74, N 3.80; Found%: C 58.70, H 2.79, N 3.55.
1H, 2-H); 13C NMR (75 MHz, CDCl3 þ DMSO-d6)
d ppm: 108.6, 117.3,
118.4, 120.0, 122.8, 125.0, 125.5, 125.9, 127.2, 129.0, 129.3, 129.5,
131.6, 134.4, 134.6, 139.7, 155.4, 160.1, 164.5 (C]O), 174.6 (C]O);
HRMS (ESI) calcd for [C24H13Cl2O3N þ Na]þ: 456.0165, Found:
456.0169; Anal. For C24H13Cl2O3N calcd%: C 66.38, H 3.02, N 3.23;
Found%: C 63.73, H 3.24, N 3.75.
4.2.6. 2-(3-Chlorophenyl)-4-(6-fluoro-8-nitro-4-oxo-4H-chromen-
3-ylmethylene)-5-methyl-2,4-dihydropyrazol-3-one (10)
Compound 10 was synthesized according to the synthetic pro-
cedure given above as orange solid, in yield of 89%, mp 230 ꢂC; 1H
NMR (300 MHz, DMSO-d6)
d ppm: 2.35 (s, 3H, CH3), 7.26 (d,
4.2.2. 3-(6-Bromo-4-oxo-4H-chromen-3-ylmethylene)-1-(2,6-
dichlorophenyl)-1,3-dihydroindol-2-one (6)
J ¼ 7.8 Hz, 1H, ArH), 7.39e7.49 (m, 1H, ArH), 7.79e7.96 (m, 2H, ArH),
8.07 (s, 1H, ]H), 8.24e8.27 (dd, J ¼ 3.0 Hz, J ¼ 7.5 Hz, 1H, ArH),
Compound 6 was synthesized according to the synthetic pro-
8.62e8.65 (dd, J ¼ 3.0 Hz, J ¼ 7.5 Hz, 1H, ArH), 10.30 (s, 1H, 2-H); 13
C
cedure given above as yellow solid, in yield of 87%, mp 229 ꢂC; 1H
NMR (75 MHz, CDCl3 þ DMSO-d6)
d ppm: 12.7 (CH3), 116. 2, 116.9,
NMR (300 MHz, CDCl3)
d
ppm: 6.43 (d, 1H, J ¼ 7.8 Hz, ArH), 7.16 (t,
117.1, 117.4, 118.6, 119.8, 124.4, 125.2, 127.9, 130.5, 133.3, 135.4, 147.0,
151.6, 161.7, 162.3, 172.3 (C]O), 177.7 (C]O); HRMS (ESI) calcd for
[C20H11ClFO5N3 þ H]þ: 428.0444, 430.0416, Found: 428.0450,
430.0450; Anal. For C20H12ClFO5N3 calcd%: C 56.15, H 2.59, N 9.82,
Found%: C 56.14, H 2.60, N 9.83.
J ¼ 7.5 Hz, 1H, ArH), 7.22e7.25 (m, 1H, ArH), 7.37e7.43 (m, 2H, ArH),
7.52e7.55 (m, 2H, ArH), 7.75e7.80 (m, 2H, ArH), 8.10 (s, 1H, ]H),
8.44 (s, 1H, ArH), 10.25 (s, 1H, 2-H); 13C NMR (75 MHz,
CDCl3 þ DMSO-d6)
d ppm: 108.5, 117.6, 118.5, 119.6, 120.3, 122.5,
124.3, 125.3, 125.9, 127.8, 128.5, 129.0, 130.7, 134.7, 136.6, 139.7,
154.1, 160.1, 164.6 (C]O), 173.6 (C]O); HRMS (ESI) calcd for
[C24H12BrCl2O3N þ Na]þ: 533.9270, 535.9249, 537.9221, Found:
533.9205, 535.9185, 537.9165; Anal. For C24H12BrCl2O3N Calcd% C
56.17, H 2.36, N 2.73; Found%: C 56.17, H 2.34, N 2.75.
4.3. Docking procedure
Compounds were built using the builder tool kit of the software
package Argus Lab 4.0.1 [24] and energy minimized with semi-
empirical quantum mechanical method PM3. Crystal co-ordinates
of COX-1 (PDB ID 1EQG) and COX-2 (PDB ID 6COX) were down-
loaded from protein data bank and in the molecule tree view of the
software, the monomeric structures of the crystal co-ordinate was
4.2.3. 1-(2,6-Dichlorophenyl)-3-(6-isopropyl-4-oxo-4H-chromen-
3-ylmethylene)-1,3-dihydro-indol-2-one (7)
Compound 7 was synthesized according to the synthetic pro-
cedure given above as yellow solid, in yield of 91%, mp 232 ꢂC; 1H
selected and the active site was defined as 15 A around the ligand.
ꢀ
NMR (300 MHz, CDCl3)
d
ppm: 1.32 (d, J ¼ 6.6 Hz, 6H, 2 ꢃ CH3),
Validation of the docking programme was checked by docking
celecoxib in the binding site of COX-2 (Supporting information,
Fig. S40).
3.02e3.11 (m, 1H, CH), 6.44 (d, J ¼ 7.5 Hz, 1H, ArH), 7.14e7.24 (m,
2H, ArH), 7.43 (t, J ¼ 7.5 Hz, 2H, ArH), 7.52e7.59 (m, 3H, ArH), 7.78
(d, J ¼ 7.5 Hz, 1H, ArH), 8.15 (s, 1H, ]H), 8.19 (s, 1H, ArH), 10.28 (s,
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 con-
struction of the ligand torsions. Thus, docking occurs between the
flexible ligand parts of the compound and enzyme. The docking
was repeated several times (approx. 10,000 iterations) until no
change in the position of the ligand and a constant value of the
binding energy was observed. The ligand orientation was deter-
mined by a shape scoring function based on Ascore and the final
positions were ranked by lowest interaction energy values. H-
bonds and hydrophobic interactions between the respective com-
pound and enzyme were explored.
1H, 2-H); 13C NMR (75 MHz, CDCl3)
d ppm: 24.2 (CH3), 28.8 (CH3),
116.6, 117.2, 118.2, 118.7, 118.8, 119.2, 123.6, 124.9, 126.4, 126.5, 127.3,
129.8, 130.4, 134.5, 136.6, 139.2, 151.5, 156.0, 162.5, 164.1 (C]O),
175.3 (C]O); HRMS (ESI) calcd for [C27H19Cl2O3N
498.0634, Found: 498.0647; Anal. For C27H19Cl2O3N calcd%: C
69.08, H 4.02, N 2.94, Found%: C 69.11, H 4.03, N 2.91.
þ
Na]þ:
4.2.4. 3-(4-Oxo-4H-chromen-3-ylmethylene)-1,3-dihydroindol-2-
one (8)
Compound 8 was synthesized according to the synthetic pro-
cedure given above as yellow solid, in yield of 78%, mp 230 ꢂC; 1H
NMR (300 MHz, DMSO-d6)
d
ppm: 6.79 (d, J ¼ 7.8 Hz, 1H, ArH), 6.90
(t, J ¼ 7.5 Hz, 1H, ArH), 7.11 (t, J ¼ 7.5 Hz, 1H, ArH), 7.33e7.48 (m, 1H,
ArH), 7.56 (d, J ¼ 7.5 Hz,1H, ArH), 7.70e7.74 (m, 2H, ArH), 7.91 (s,1H,
]H), 8.01e8.10 (dd, J ¼ 1.2 Hz, J ¼ 7.2 Hz, 1H, ArH), 10.03 (s, 1H, 2-
H), 10.48 (s, 1H, NH, D2O exchange); 13C NMR (75 MHz, DMSO-d6)
d
ppm: 109.6, 117.3, 118.6, 119.8, 121.4, 123.1, 124.0, 124.9, 125.4,
4.4. Procedure for COX-1/2 inhibitory immunoassay
126.0, 127.6, 129.4, 134.6, 140.7, 155.4, 159.9, 167.5 (C]O), 174.9 (C]
O); HRMS (ESI) calcd for [C18H11O3N þ Na]þ: 312.0631, Found:
312.0626; Anal. For C18H11O3N calcd%: C 74.73, H 3.83, N 4.84;
Found%: C 74.65, H 3.64, N 4.98.
For studying the COX-1, COX-2 inhibitory activities of the com-
pounds, various reagents were prepared as per the protocol of the
assay. The background samples were prepared for both COX-1
(ovine) and COX-2 (human recombinant) by taking 20 ml of each
4.2.5. 3-(6-Bromo-4-oxo-4H-chromen-3-ylmethylene)-1,3-
dihydroindol-2-one (9)
enzyme in separate test tubes and keeping them in boiling water
for 3 min. The inactivated enzymes were used to generate back-
ground values. In two test tubes named background COX-1 and
Compound 9 was synthesized according to the synthetic pro-
cedure given above as yellow solid, in yield of 75%, mp > 300 ꢂC; 1H
background COX-2, 970
ml of reaction buffer, 10 ml of haem and 10 ml
NMR (300 MHz, DMSO-d6)
d
ppm: 6.84 (d, J ¼ 7.8 Hz, 1H, ArH), 6.99
of inactive COX-1 or COX-2 were added. 100% initial activity tubes

Shaveta et al. / European Journal of Medicinal Chemistry 77 (2014) 185e192
191
were prepared for both COX-1 and COX-2 by adding 950
action buffer, 10 l of haem and 10 l of COX-1 or COX-2. Inhibitor
tubes were prepared for compounds 5-10. In each sample tube,
950 l reaction buffer, 10 l of haem, 10 l of COX-1 or COX-2
enzyme and 20 l of the inhibitor solution was added. All the so-
lutions were incubated for 10 min at 37 ꢂC. 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
m
l of re-
Cayman Chemicals Co). Stock solutions of compounds 5e10 were
prepared so as to obtain conc of 10ꢀ5 M, 10ꢀ6 M, 10ꢀ7 M and 10ꢀ8 M
in the respective wells. The prepared solutions were then intro-
duced on 96 well plate where the cells were distributed as blank-
1A-2A-1D (triplicate), positive control- 1B-2B (duplicate), 100%
initial activity wells- 1C-2C-2D (triplicate). Remaining wells were
designated to inhibitor (compounds 5e10) solutions in duplicate.
The addition of the reagents was done according to the standard
m
m
m
m
m
m
m
m
m
protocol, according to which,100
blank wells, 90 l of lipoxygenase (5-LOX, potato) enzyme and 10
of assay buffer was added to þve control wells. To 100% initial ac-
tivity wells, 90 l of lipoxygenase enzyme and 10 l of solvent
(DMSO) was added. All the inhibitor (compound) wells were
charged with 90 l of lipoxygenase enzyme and 10 l of respective
stock (compound) solution. The reaction was initiated by adding
10 l of the substrate (AA) to all the wells. The plate was then
shaken for five minutes on an orbital shaker. Ultimately, 100 l of
ml of assay buffer was added to the
chloride solution was added to each test tube and vortex. Incubated
m
ml
for another 5 min and kept at 0e4 ꢂC.
Prostaglandin screening standards were prepared as test tubes
m
m
S1eS8. 800
was added to S2eS7. Then 200
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
m
l of EIA buffer was added to S1 and 500
ml of the same
ml of bulk standard (10 ng/ml) was
m
m
m
m
and mixed thoroughly. Same process was repeated from S2eS3,
S3eS4 upto S7eS8.
m
chromogen solution (prepared according to standard protocol) was
added to each well to stop enzyme catalysis. The plate was incu-
bated for half an hour and was read at 500 nm.
To make dilutions for COX reactions, two test tubes named BC1
and BC2 were prepared. To each test tube was added, 990
ml of EIA
(enzyme immunoassay) buffer and 10 l of background COX-1 or
m
COX-2 and mixed thoroughly. COX 100% initial activity samples
were prepared as three test tubes for COX-1 and COX-2 both and
4.6. Procedure for recording mass spectra
numbered as IA1eIA3. For each sample, aliquot 990
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
m
l of EIA buffer
All the mass spectra were recorded on Bruker MicroTOF QII
machine. Solutions of 11 mM (conc calculated from UV spectra) of
COX-1 and COX-2 in ACN-H2O (acetonitrileewater) (7:3) in the
presence of 10 mM ammonium acetate buffer were prepared. So-
lutions of AA and compounds with the enzyme were prepared with
m
m
m
m
m
IA2 and added to test tube IA3 and mixed well. In the same manner,
COX inhibitor samples were prepared by further dilutions and
named C1eC3 for each concentration.
four different concentrations of the ligand viz. 20
mM, 40 mM, 60 mM
and 80 M. The mass spectra of these solutions were recorded
m
in þve and ꢀve modes at different source temperatures (100 ꢂCe
180 ꢂC) and collision energies (2 eVe12 eV). Most consistent results
were obtained in þve mode at source temperature 120 ꢂC and
collision energy 5e7 eV. Therefore, all the mass spectra were
recorded under these conditions and deconvoluted to get m/z.
The association constants were calculated using Equation (1).
After preparing all the dilutions, they were introduced on 96
well plate. The wells were distributed as blank-1A, NSB (Non-spe-
cific binding)-1B and Bo (Maximum binding)-1C. Well 1H was
named as TA (Total activity well). Wells 2Ae2H were used for S1e
S8 and 3Ae3H were used for S1eS8 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.
Ka ¼ ½ELꢄ=½Eꢄfree*½Lꢄfree
(1)
Addition of the reagents on 96-well plate was performed as
follows:
½ELꢄ ¼ ½Eꢄtotal ꢃ IEL=IEL þ IE
where IEL and IE are the intensities of mass peaks of EL complex and
enzyme, respectively. The conc of free enzyme ([E]free) and free
ligand ([L]free) were calculated by subtracting the conc of EL com-
plex ([EL]) from the total conc of enzyme and ligand, respectively.
As apparent from Equation (1), conc of free enzyme and free ligand
were taken into account after the complete complexation occurred
and hence instead of stepwise complexation, overall binding con-
stant, Ka were calculated from this equation irrespective of the
stoichiometry of enzymeeligand complex.
100
was added to Bo well. 50
added to the respective wells S1eS8 from their respective test
tubes S1eS8 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 screening EIA antiserum was
m
l EIA buffer was added to NSB well and 50
ml of EIA buffer
ml of Prostaglandin screening standard was
m
m
m
m
m
4.7. Procedure for recording NMR spectra
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
1H NMR spectra of pure compounds (0.35 mM) were recorded in
DMSO-d6 and deuterium exchange (using D2O) was performed to
identify the NH signals. 12 ml (0.08 mM) of COX-2/COX-1 was added
times. After washing the wells, 200
ml of Ellman’s reagent was
to the compound solutions in DMSO-d6 and the contents of NMR
tube were incubated at 37 ꢂC and NMR spectra were recorded after
each 30 min.
added to each well and 5 l of tracer was added to Total activity
m
well. The plate was covered with plastic film and it was kept for
60e90 min. Before reading the plate, it was wiped from bottom to
remove any fingerprints and finally read at 420 nm.
Acknowledgements
4.5. Procedure for 5-LOX inhibitory immunoassay
Financial assistance by Department of Science & Technology,
New Delhi and Council of Scientific & Industrial Research, New
Delhi is gratefully acknowledged. Shaveta thanks UGC, New Delhi
The reagents were prepared according to the standard protocol
(Lipoxygenase inhibitor screening assay kit, Item No. 760700.

192
Shaveta et al. / European Journal of Medicinal Chemistry 77 (2014) 185e192
W.E. Perkins, K. Seibert, A.W. Veenhuizen, Y.Y. Zhang, P.C. Isakson, Journal of
Medicinal Chemistry 40 (1997) 1347e1365.
[15] B. Meunier, Accounts of Chemical Research 41 (2008) 69e77.
[16] G. Mehta, V. Singh, Chemical Society Reviews 31 (2002) 324e334.
[17] P. Singh, M. Kaur, W. Holzer, European Journal of Medicinal Chemistry 45
(2010) 4968e4982.
and AS, MK thank DST, New Delhi for fellowships. Sincere efforts of
Prof. A. S. Brar, worthy vice-chancellor, GNDU in building up state of
the art research facilities under the UGC-UPE programme are
respectfully acknowledged.
[18] COX Inhibitor Screening Assay kit, Item No. 560131. Cayman Chemical Co.
[19] Lipoxygenase Inhibitor Screening Assay Kit, Item No. 760700. Cayman
Chemicals Co.
Appendix A. Supplementary data
[20] C.R. Rodrigues, M.P. Veloso, H. Verli, C.A.M. Fragal, A.L.P. Miranda, E.J. Barreiro,
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[22] E.A. Meade, W.L. Smith, D.L. DeWitt, Journal of Biological Chemistry 268
(1993) 6610e6614.
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