Dual evaluation of some novel 2-amino-substituted coumarinylthiazoles as anti-inflammatory-antimicrobial agents and their docking studies with COX-1/COX-2 active sites

Article abstract of DOI:10.3109/14756366.2013.805755

Synthesis of total eighteen 2-amino-substituted 4-coumarinylthiazoles including sixteen new compounds (3a-o and 5b) bearing the benzenesulfonamide moiety is described in the present report. All the synthesized target compounds were examined for their in vivo anti-inflammatory (AI) activity and in vitro antimicrobial activity. Results revealed that six compounds (3d, 3f, 3g, 3h, 3j and 3n) exhibited pronounced anti-inflammatory activity comparable to the standard drug indomethacin. AI results were further confirmed by the docking studies of the most active (3n) and the least active compound (3a) with COX-1 and COX-2 active sites. In addition, most of the compounds exhibited moderate antimicrobial activity against Gram-positive bacteria as well as fungal yeast, S. cervisiae. Comparison between 3 and 5 indicated that incorporation of additional substituted pyrazole nucleus into the scaffold significantly enhanced AI activity.

Full text of DOI:10.3109/14756366.2013.805755

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, 2014; 29(4): 476–484
2014 Informa UK Ltd. DOI: 10.3109/14756366.2013.805755
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ORIGINAL ARTICLE
Dual evaluation of some novel 2-amino-substituted coumarinylthiazoles
as anti-inflammatory–antimicrobial agents and their docking studies
with COX-1/COX-2 active sites
Navneet Chandak1, Pawan Kumar1, Pawan Kaushik2, Parul Varshney3, Chetan Sharma4, Dhirender Kaushik2,
Sudha Jain3, Kamal R. Aneja4, and Pawan K. Sharma1
2
1Department of Chemistry, Kurukshetra University, Kurukshetra, Haryana, India, Institute of Pharmaceutical Sciences, Kurukshetra University,
3
4
Kurukshetra, Haryana, India, Department of Chemistry, Lucknow University, Lucknow, Uttar Pradesh, India, and Department of Microbiology,
Kurukshetra University, Kurukshetra, Haryana, India
Abstract
Keywords
Synthesis of total eighteen 2-amino-substituted 4-coumarinylthiazoles including sixteen new
compounds (3a–o and 5b) bearing the benzenesulfonamide moiety is described in the present
report. All the synthesized target compounds were examined for their in vivo anti-inflammatory
(AI) activity and in vitro antimicrobial activity. Results revealed that six compounds (3d, 3f, 3g,
3h, 3j and 3n) exhibited pronounced anti-inflammatory activity comparable to the standard
drug indomethacin. AI results were further confirmed by the docking studies of the most active
(3n) and the least active compound (3a) with COX-1 and COX-2 active sites. In addition, most of
the compounds exhibited moderate antimicrobial activity against Gram-positive bacteria as
well as fungal yeast, S. cervisiae. Comparison between 3 and 5 indicated that incorporation of
additional substituted pyrazole nucleus into the scaffold significantly enhanced AI activity.
2-Amino-substituted coumarinylthiazoles,
anti-inflammatory activity, antimicrobial
activity, benzenesulfonamide, docking,
thiazolylhydrazinomethylidenepyrazoles
History
Received 4 February 2013
Revised 24 April 2013
Accepted 26 April 2013
Published online 18 June 2013
Introduction
such as treatment of alzheimer’s disease3, huntington’s disease4,
prion disease5 and tuberculosis6. They reduce the production of
prostaglandin E2 (PGE2) which is a key mediator of pain and
inflammation7. Their enzymatic inhibitory activities have also
The development of effective non-steroidal anti-inflammatory
drugs (NSAIDs) for the management of inflammation with
minimal gastrointestinal mucosal damage as well as renal toxicity
has undergone continual evolution leading to the emergence of
more efficacious class of drugs, COX-2 inhibitors, better known
as ‘‘coxibs’’. Most of the coxibs have a central heterocyclic
moiety which is appropriately substituted. Many of the sulfona-
mide bearing COX-2 inhibitors have also shown promising
carbonic anhydrase inhibition activities stimulating further inter-
est in these molecules as potential anticancer/antitumour
agents1,2. Thiazole ring system is an important member of the
heterocyclic class and its presence in numerous biologically active
molecules of natural as well synthetic origin have found extensive
applications in the drug development. Tiazofurin (antineoplastic
agent), ritanovir (anti-HIV drug), fanetizole (immunoregulatory
agent), meloxicam (anti-inflammatory agent), nizatidine (anti-
ulcer agent), sulfathiazole (antimicrobial drug) and abafungin
(antifungal drug) are some of the prominent examples of thiazole
bearing marketed drugs. Amongst various thiazole derivatives, 2-
aminothiazole has emerged as a useful structural motif due to
various biological activities associated with this pharmacophore
been well recognized by the chemists as well as biologists8,9
.
2-Aminothiazole derivatives have shown broad spectrum of
antibacterial activity against both Gram-positive and Gram-
negative bacteria by inhibiting Mur1 and Mur2 enzymes which
are responsible for cell wall biosynthesis10.
Coumarins, nowadays, have become indispensable units in
medicinal chemistry applications due to their diverse biological
profiles, e.g. many coumarin derivatives are known for their
special property of scavenging reactive oxygen species (ROS) and
have been found to be inhibitors of cyclooxygenase and
lipooxygenase in the arachidonic acid pathway of inflammation11.
In the development of newer antimicrobials, coumarins have been
identified as target specific plant anti-bacterial agents with growth
inhibitory potential particularly against Gram-positive species12.
Kalakhambkar et al.13 recently synthesized a series of new
fluorinated coumarins and 1-azacoumarins which displayed
excellent anti-inflammatory and moderate analgesic activities.
In addition, most of the compounds were found to be potent
antibacterial as well as antifungal agents.
Appreciation of these findings coupled with the fact that a
generic scaffold consisting of thiazole and coumarin rings14 or in
combination with pyrazole15 is mostly studied for either anti-
inflammatory or antimicrobial activity, prompted us to investigate
dual anti-inflammatory–antimicrobial potential of both these
Address for correspondence: Pawan K. Sharma, Department of
Chemistry, Kurukshetra University, Kurukshetra, Haryana, India.
Tel: +91 9416457355. Fax: +91 1744 238277. E-mail: pksharma@
kuk.ac.in

DOI: 10.3109/14756366.2013.805755
Dual evaluation of some novel 2-amino-substituted coumarinylthiazoles 477
Figure 1. General structures of compounds 3 and 5.
scaffolds by synthesizing two novel series of 2-amino-substituted 4-[4-({2-[4-(2-Oxo-2H-chromen-3-yl)-1,3-thiazol-2-yl]hydrazo-
coumarinylthiazoles bearing the benzenesulfonamide moiety no}methyl)-3-phenyl-1H-pyrazol-1-yl]benzenesulfonamide 3a
(3 and 5) (Figure 1).
Yield: 88%; m.p. 299-302 ꢀC; IR (KBr) cmꢁ1: 3248 (N–H
stretch), 1705 (lactone C¼O stretch), 1566 (C¼N stretch), 1504
Materials and methods
1
(N–H bend), 1335 and 1157 (SO2 stretch); H NMR (300 MHz,
Starting materials were prepared according to literature proced- DMSO-d6): ꢀ 12.04 (s, ex, 1H, NH), 9.00 (s, 1H, CH¼N), 8.52
ures. All solvents (EtOH, THF, DMF, CHCl3) were distilled (s, 1H, coumarin C4-H), 8.19–8.22 (m, 3H, pyrazole C5-H,
before use. All reactions were carried out under atmospheric Ar), 7.98 (d, 2H, J ¼ 8.1 Hz, Ar), 7.76–7.85 (m, 4H, thiazole C5-
pressure. Melting points of all compounds were determined H, coumarin, Ar), 7.35–7.63 (m, 8H, SO2NH2 (ex), coumarin,
putting sample on glass slide using MR-VIS LABINDIA VISUAL Ar); 13C NMR (75.5 MHz, DMSO-d6): ꢀ 167.7, 159.2, 152.7,
Melting Range Apparatus and are uncorrected. The infrared (IR) 152.1, 144.3, 142.3, 141.5, 138.5, 135.1, 132.3, 132.0,
spectra were recorded on ABB MB 3000 DTGS FT-IR 129.2, 129.09, 129.00, 128.3, 127.7, 125.1, 120.9, 119.6, 119.1,
Spectrophotometer using the KBr pellet technique. 1H NMR 118.1, 116.2, 110.9; ES-MS m/z 569.0 (M þ H)þ, 494.1, 493.3,
and 13C NMR spectra were recorded either in pure DMSO-d6 or 492.5, 405.8, 403.8, 401.8, 274.2, 145.4; C28H20N6O4S2Hþ Calcd
in CDCl3/DMSO-d6 mixtures on Bruker NMR spectrometers 569.1.
at 300/400 MHz and 75.5/100 MHz respectively using tetra-
methylsilane (TMS) as internal standard. Chemical shifts are 4-[4-({2-[4-(6-Chloro-2-oxo-2H-chromen-3-yl)-1,3-thiazol-2-
expressed in ꢀ, ppm. The electrospray mass spectra (ES-MS) were yl]hydrazono}methyl)-3-(4-chlorophenyl)-1H-pyrazol-1-yl]benze-
recorded on a THERMO Finnigan LCQ Advantage max ion nesulfonamide 3n
trap mass spectrometer having an ESI source through
Yield: 74%; m.p. 329–332 ꢀC; IR (KBr) cmꢁ1: 3271 (N–H
Finnigan surveyor autosampler. The purity of the compounds
stretch), 1713 (lactone C¼O stretch), 1597 (C¼N stretch), 1566
1
was checked by H NMR and thin layer chromatography (TLC)
(C¼N stretch), 1504 (N–H bend), 1335 and 1157 (SO2 stretch);
on silica gel plates using a mixture of chloroform and methanol as
1H NMR (300 MHz, DMSO-d6): ꢀ 12.11 (s, ex, 1H, NH), 9.03 (s,
eluent. Iodine or UV lamp was used as
a visualizing
1H, CH¼N), 8.46 (s, 1H, coumarin C4-H), 8.18 (d, 3H,
J ¼ 8.7 Hz, pyrazole C5-H, Ar), 7.96–7.99 (m, 3H, coumarin C5-
H, Ar), 7.87 (d, 2H, J ¼ 8.4 Hz, Ar), 7.80 (s, 1H, thiazole C5-H),
7.60–7.62 (m, 3H, coumarin, Ar), 7.48 (d, 3H, J ¼ 8.4 Hz,
SO2NH2 (ex), coumarin); 13C NMR (75.5 MHz, DMSO-d6): ꢀ
167.8, 158.7, 151.2, 150.6, 144.2, 142.4, 141.3, 137.1, 135.0,
133.9, 131.4, 131.2, 130.7, 129.2, 129.0, 128.9, 128.0, 127.8,
121.8, 121.0, 119.1, 118.2, 118.1, 111.8; ES-MS m/z 636.9
(M þ H)þ, 586.2, 585.3, 567.2, 494.0, 493.3, 405.8, 403.8, 401.8,
274.2, 145.4; C28H18Cl2N6O4S2Hþ Calcd 637.0.
agent. Abbreviations ‘‘s’’ for singlet, ‘‘d’’ for doublet, ‘‘m’’ for
multiplet, ‘‘ex’’ for exchangeable proton are used for NMR
assignments. Anti-inflammatory and antimicrobial activity of the
synthesized compounds were determined by carrageenan-induced
rat paw edema method and agar well diffusion method,
respectively.
Synthesis
General procedure for the synthesis of thiazolylhydrazinomethy-
lidenepyrazoles, 3a–o and N-substituted anilinothiazoles, 5a–c
4-{[4-(6-Chloro-2-oxo-2H-chromen-3-yl)-1,3-thiazol-2-yl]ami-
no}benzenesulfonamide 5b
To a solution of appropriate pyrazole-4-carbaldehyde thiosemi-
carbazone (1, 2.0 mmol) or 4-thioureido-benzenesulfonamide
(4, 2.0 mmol) in EtOH : THF (100 mL, 3:2 v/v), was added Yield: 85%; m.p. 330–332 ꢀC; IR (KBr) cmꢁ1: 3364, 3286 and
appropriate 3-bromoacetylcoumarin (2, 2.2 mmol) followed by 3194 (N–H stretch), 1705 (lactone C¼O stretch), 1605 (C¼N
sodium acetate (2.2 mmol) and then refluxed the reaction mixture. stretch), 1535 (C¼C stretch), 1504 (N–H bend), 1327 and 1149
Color of the solution changed yellow or orange within 5 min and (SO2 stretch); 1H NMR (300 MHz, DMSO-d6): ꢀ 10.80 (s, ex, 1H,
solid started separating out within half an hour. Refluxed it for 2 h NH), 8.73 (s, 1H, coumarin C4-H), 8.17 (d, 1H, Jmeta ¼ 2.1 Hz,
for reaction completion, cooled to room temperature for complete coumarin C5-H), 7.94 (d, 2H, J ¼ 8.7 Hz, Ar), 7.88 (s, 1H, thiazole
precipitation, filtered it, washed with water (50 mL) followed by C5-H), 7.84 (d, 2H, J ¼ 8.7 Hz, Ar), 7.65 (dd, 1H, Jortho ¼ 9.0 Hz,
EtOH (20 mL), dried and crystallized using EtOH-DMF mixture Jmeta ¼ 2.1 Hz, coumarin C7-H), 7.49 (d, 1H, Jortho ¼ 9.0 Hz,
to afford the target thiazolylhydrazinomethylidenepyrazoles 3 or coumarin C8-H), 7.24 (s, ex, 2H, SO2NH2); 13C NMR (75.5 MHz,
N-substituted anilinothiazoles 5 as yellow or orange solid DMSO-d6): ꢀ 162.2, 158.8, 151.2, 144.0, 143.7, 138.0, 136.3,
material.
131.5, 129.0, 128.2, 127.69, 127.68, 121.3, 121.0, 116.9, 112.0;

478 N. Chandak et al.
J Enzyme Inhib Med Chem, 2014; 29(4): 476–484
ES-MS m/z 434.0 (M þ H)þ, 403.7, 274.2, 130.1, 109.9; In vitro antimicrobial assay
C18H12ClN3O4S2Hþ Calcd 434.0.
Characterization data of the remaining compounds along with
The antimicrobial activity of 18 synthesized compounds was
evaluated in vitro by agar well diffusion method17. Total six
microbial strains were selected on the basis of their clinical
importance in causing diseases in humans. Two Gram-positive
bacteria (Staphylococcus aureus and Bacillus subtilis), two Gram-
negative bacteria (Escherichia coli and Pseudomonas aeruginosa)
and two fungal yeasts (Candida albicans and Saccharomyces
cerevisiae) were screened for evaluation of antibacterial and
antifungal activity of the synthesized compounds. All the
microbial cultures were adjusted to 0.5 McFarland standard,
which is visually comparable to a microbial suspension of
approximately 1.5 ꢂ 108 cfu/mL18. 20 mL of Mueller Hinton agar
medium was poured into each Petri plate and the agar plates were
swabbed with 100 mL inocula of each test bacterium and kept for
15 min for adsorption. Using sterile cork borer of 8 mm diameter,
wells were bored into seeded agar plates and these were loaded
with a 100 mL volume with concentration of 4.0 mg/mL of each
compound reconstituted in dimethylsulfoxide (DMSO). All the
plates were incubated at 37 ꢀC for 14 h. Antimicrobial activity of
18 synthetic compounds was evaluated by measuring the zone of
growth inhibition against the test microorganisms with zone
reader (HiAntibiotic zone scale). DMSO was used as a negative
control whereas ciprofloxacin was used as positive control for
bacteria and amphotericin-B for fungal yeasts. The experiments
were performed in triplicates. The antibacterial activity of the
compounds was compared with ciprofloxacin as standard and
antifungal activity with amphotericin-B as standard. MIC of
newly synthesized compounds against tested bacteria as well as
yeasts was determined using macrodilution tube method as
recommended by NCCLS18. MIC is the lowest concentration of
an antimicrobial compound that will inhibit the visible growth of
a microorganism after overnight incubation. In this method,
various test concentrations of newly synthesized compounds were
prepared from 128 to 0.25 mg/mL in sterile tubes No. 1 to 10.
100 mL sterile Mueller Hinton Broth (MHB) was poured in each
sterile tube followed by addition of 200 mL test compound in tube
1. Two fold serial dilutions were carried out from tube 1 to tube
10 and excess broth (100 mL) was discarded from the last tube No.
10. To each tube, 100 mL of standard inoculum (1.5 ꢂ 108 cfu/mL)
was added. Ciprofloxacin was used as control against bacteria
and amphotericin-B against yeasts. Turbidity was observed
after incubating the inoculated tubes at 37 ꢀC for 24 h. The
bacteria were subcultured on Nutrient agar whereas yeasts on
Malt yeast agar.
1
all original H and 13C NMR spectra are given in supplementary
information.
Anti-inflammatory testing
Carrageenan-induced rat paw edema assay
Male Wistar albino rats weighing 200–250 g were used
throughout the study16. They were kept in the animal
house under standard conditions of light and temperature with
free access to food and water. Food was withdrawn 12 h before
and during experimental hours. The animals were randomly
divided into groups each consisting of six rats. One group of
six rats was kept as control and received tween 80 (95:5).
Another group received the standard drug indomethacin
i.p. while rest ones were administered the test compounds at a
dose of 50 mg/kg body weight orally. A mark was made on the
left hind paw just beyond the tibiotarsal articulation, so that
every time the paw was dipped up to the fixed mark and
constant paw volume was ensured. Paw volumes were measured
using a plethysmometer (model 7140, Ugo Basile, Italy).
Thirty minutes after administration of test compounds and
standard drug, 0.1 mL of 1% w/v of carrageenan suspension
in normal saline was injected into subplanter region of the
left hind paw of all the animals. The initial paw volume was
measured within 30 s of the injection and remeasured again 1, 2,
3, and 4 h after administration of carrageenan. The edema was
expressed as an increase in the volume of paw and the percentage
of edema inhibition for each rat and each group was obtained as
follows:
ꢀ
ꢁ
ðVt ꢁ V0Þcontrol ꢁ ðVt ꢁ V0Þtest
ðVt ꢁ V0Þcontrol
%inhibition ¼
where
ꢂ 100
Vt ¼ volume of edema at specific time interval and
V0 ¼ volume of edema at zero time interval.
Docking assay
The docking experiments were carried our using the GOLD
docking program. For docking studies, the atomic co-ordinates of
COX-1 (PDB-ID: 2OYE), and COX-2 (PDB-ID: 4COX) were
downloaded from the Brookhaven Protein Data Bank
(www.rcsb.org) and prepared using in Discovery Studio 2.1
(DS2.1) software package where bond orders were assigned, water
and other residues except bound ligands were deleted and finally Results and discussion
the protein was backbone constrained minimized using Charmm
Chemistry
force field implemented in DS2.1. The bound conformation of co-
crystallized ligands were used as controls in order to define the The synthetic routes used to synthesize the target thiazolylhy-
active site in COX-1, and COX-2 respectively, and optimized 3D- drazinomethylidenepyrazoles 3 and N-substituted anilinothiazoles
˚
structures of new ligands were docked within 10 A radius by 5 are outlined in Scheme 1 and 2, respectively. Using Hantzsch
running 20 genetic algorithm (GA) steps for each. The docked thiazole synthesis strategy, the present synthesis of thiazolylhy-
poses of geometry optimized ligands were ranked using the drazinomethylidenepyrazoles (3a–o) consists of the condensation
GoldScore (GS), to find the most optimal binding pose of of appropriate 6-substituted-3-bromoacetylcoumarin (2) with
each ligand. In the GOLD program, the default parameters: appropriate pyrazole-4-carbaldehyde thiosemicarbazone (1) in
population size (100); selection-pressure (1.1); number of refluxing EtOH:THF in the presence of sodium acetate. Likewise
operations (10 000); number of islands (1); niche size (2); in Scheme 2, 4-thioureido-benzenesulfonamide 4 was reacted
and operator weights for migrate (0), mutate (100) and crossover with various 6-substituted-3-bromoacetylcoumarins (2) in reflux-
(100) were applied. Finally, the top five binding poses of each ing EtOH:THF in the presence of sodium acetate to afford
ligand were analyzed to select the best binding pose of N-substituted anilinothiazoles (5a–c). Starting materials, pyra-
each ligand in order to untangle the essential parameters zole-4-carbaldehyde thiosemicarbazones (1)19, 4-thioureido-ben-
in terms of direct- (H-bonds) and indirect (hydrophobic) zenesulfonamide 420 and 6-substituted-3-bromoacetylcoumarins
interactions governing binding disparities among the series of (2)21 were synthesized following established literature procedures.
these compounds.
Spectral data (1H NMR, 13C NMR, IR and mass) of the newly

DOI: 10.3109/14756366.2013.805755
Dual evaluation of some novel 2-amino-substituted coumarinylthiazoles 479
Scheme 1. Synthesis of thiazolylhydrazinomethylidenepyrazoles (3).
Scheme 2. Synthesis of N-substituted anilinothiazoles (5).
synthesized compounds were in full agreement with the proposed research article22 described the synthesis of two of the compounds
1
structures. In general, the H NMR spectra of thiazolylhydrazi- 5a and 5c; however, lack of dependable characterization data
nomethylidenepyrazoles (3a–o) displayed an exchangeable singlet prompted us to report their full spectral characterization. A singlet
1
in the range of ꢀ 12.04–12.12 corresponding to NH proton while in the range of ꢀ 7.73–7.85 in H NMR spectra of thiazolylhy-
N-substituted anilinothiazoles (5a–c) displayed the same in the drazinomethylidenepyrazoles 3 was attributed to C5-proton of
range of ꢀ 10.62–10.80. It is pertinent to mention here that a thiazole ring while N-substituted anilinothiazoles 5 displayed the

480 N. Chandak et al.
J Enzyme Inhib Med Chem, 2014; 29(4): 476–484
same in the narrow range ꢀ 7.85–7.88. In some of the compounds, well as 4 h after carrageenan injection. Four of these compounds
this C5-proton of thiazole is merged with other aromatic protons. (3d, 3f, 3h and 3n) possessed AI activity (490.39% inhibition)
All the target compounds, 3 and 5 were showing characteristic surpassing that of indomethacin after 4 h. Three other compounds
C¼O stretch of lactone nature at 1705–1713 cmꢁ1 in FT-IR due to (3c, 5a and 5c) showed good AI activity with 74–89% inhibition
the presence of coumarin moiety.
after 4 h. Amongst rest of the compounds, many showed moderate
AI activity upto 2 h but failed to maintain AI activity after 3 h.
Considering the fact that carrageenan-induced paw edema assay is
a biphasic event involving the release of histamine and serotonin
as the mediators of inflammation in the first phase which
normally lasts for about 2 h after the carrageenan injection, and
second phase which generally operates 2 h after the carrageenan
injection involving prostaglandins as the mediators of inflamma-
tion23, any anti-inflammatory activity shown by the compounds
after 2 h can be attributed to the inhibition of prostaglandin
synthesis. Excellent anti-inflammatory activity in the second
phase of the biphasic carrageenan induced edema assay indicates
the capability of the tested compounds to inhibit prostaglandin
synthesis. This can be attributed to their ability to bind
cyclooxygenase (COX), the enzyme responsible for the synthesis
of prostaglandins.
In vivo anti-inflammatory activity
All the synthesized thiazolylhydrazinomethylidenepyrazoles (3a–
o) and N-substituted anilinothiazoles (5a–c) were evaluated for
their in vivo anti-inflammatory activity by carrageenan-induced
paw edema assay16. The protocol of animal experiments has been
approved by the Institutional Animal Ethics Committee (IAEC).
Each test compound was dosed orally (50 mg/kg body weight)
30 min prior to induction of inflammation by carrageenan
injection while indomethacin was utilized as a reference anti-
inflammatory drug. The anti-inflammatory activity was then
calculated 1–4 h after induction and presented in Table 1 as the
mean paw volume (mL) in addition to the percentage anti-
inflammatory activity (AI%).
Amongst eighteen compounds (3a–o and 5a–c) tested, six
compounds (3d, 3f, 3g, 3h, 3j and 3n) showed excellent AI
activity comparable to the standard drug indomethacin 3 h as
A comparison between two series of compounds (3 and 5)
indicates that in general, thiazolylhydrazinomethylidenepyrazoles
Table 1. In vivo anti-inflammatory activity of compounds 3 and 5 by carrageenan-induced rat paw edema assay.
Volume of edema (mL)b and %AIc
Compounda
1h
2h
3h
4h
Control
Indomethacin
0.99 ꢃ 0.12
0.04 ꢃ 0.02*
(95.95)
1.98 ꢃ 0.23
0.19 ꢃ 0.03*
(90.40)
1.73 ꢃ 0.22
0.26 ꢃ 0.02*
(84.97)
1.77 ꢃ 0.33
0.17 ꢃ 0.03*
(90.39)
3a
3b
3c
3d
3e
3f
0.23 ꢃ 0.08*
1.44 ꢃ 0.12
1.210 ꢃ 0.19
1.90 ꢃ 0.18
(76.76)
0.12 ꢃ 0.02*
(87.87)
(27.27)
1.37 ꢃ 0.24
(30.80)
(30.05)
1.378 ꢃ 0.16
(20.35)
(ꢁ7.34)
1.29 ꢃ 0.03
(27.11)
0.74 ꢃ 0.2*
0.67 ꢃ 0.14*
0.79 ꢃ 0.16*
0.45 ꢃ 0.18*
(25.25)
0.28 ꢃ 0.07*
(71.71)
(66.16)
0.24 ꢃ 0.09*
(87.87)
(54.33)
0.20 ꢃ 0.15*
(88.43)
(74.57)
0.16 ꢃ 0.08*
(90.96)
0.16 ꢃ 0.04*
1.04 ꢃ 0.1*
1.09 ꢃ 0.32
0.67 ꢃ 0.10*
(83.83)
0.31 ꢃ 0.03*
(68.68)
(47.47)
0.39 ꢃ 0.05*
(80.30)
(36.99)
0.29 ꢃ 0.07*
(83.23)
(62.14)
0.15 ꢃ 0.04*
(91.52)
3g
3h
3i
0.28 ꢃ 0.03*
0.41 ꢃ 0.05*
0.37 ꢃ 0.04*
0.28 ꢃ 0.02*
(71.71)
0.21 ꢃ 0.07*
(78.78)
(79.29)
0.27 ꢃ 0.06*
(86.36)
(78.61)
0.32 ꢃ 0.07*
(81.50)
(84.18)
0.12 ꢃ 0.04*
(93.22)
0.26 ꢃ 0.12*
1.03 ꢃ 0.18*
0.48 ꢃ 0.04*
1.4 ꢃ 0.25
(73.73)
0.30 ꢃ 0.04*
(69.69)
(47.97)
0.32 ꢃ 0.03*
(84.84)
(72.25)
0.28 ꢃ 0.08*
(83.81)
(20.90)
0.19 ꢃ 0.06*
(89.26)
3j
3k
3l
0.32 ꢃ 0.07*
0.28 ꢃ 0.04*
1.56 ꢃ 0.18
1.70 ꢃ 0.27
(67.67)
0.36 ꢃ 0.08*
(63.63)
(85.85)
1.55 ꢃ 0.16
(43)
(9.82)
1.44 ꢃ 0.14
(16.76)
(3.95)
1.46 ꢃ 0.13
(17.51)
3m
3n
3o
5a
5b
5c
0.24 ꢃ 0.08*
1.03 ꢃ 0.23*
1.24 ꢃ 0.23
1.26 ꢃ 0.02
(75.75)
0.21 ꢃ 0.02*
(78.78)
(47.97)
0.22 ꢃ 0.05*
(88.88)
(28.32)
0.22 ꢃ 0.06*
(87.28)
(28.81)
0.11 ꢃ 0.16*
(95.95)
0.98 ꢃ 0.1
1.38 ꢃ 0.25
1.414 ꢃ 0.10
1.19 ꢃ 0.22
(1.01)
0.45 ꢃ 0.15*
(54.54)
(30.30)
0.74 ꢃ 0.20*
(62.62)
(18.26)
0.75 ꢃ 0.25*
(56.64)
(32.76)
0.42 ꢃ 0.17*
(76.27)
0.51 ꢃ 0.15*
0.81 ꢃ 0.09*
1.11 ꢃ 0.19*
0.79 ꢃ 0.03*
(48)
0.21 ꢃ 0.07*
(78.78)
(59.09)
0.40 ꢃ 0.18*
(79.79)
(35.83)
0.58 ꢃ 0.10*
(66.47)
(55.36)
0.19 ꢃ 0.04*
(89.26)
*Significantly different compared to respective control values, p50.01.
aDose levels: 50 mg/kg body wt.
bValues are expressed as mean ꢃ SEM (no. of animals ¼ 6) and analyzed by ANOVA.
cValues in parentheses (percentage anti-inflammatory activity, AI%).

DOI: 10.3109/14756366.2013.805755
Dual evaluation of some novel 2-amino-substituted coumarinylthiazoles 481
3 showed better AI activity as compared to N-substituted revealed that there is no significant change in the binding mode
anilinothiazoles 5 signifying that incorporation of pyrazole of indomethacin both in terms of indomethacin conformation or
moiety into the scaffold enhances AI activity. Within the in the binding amino acid residues, thus suggesting the correct
individual series, it was found that compounds containing binding prediction of docking program (Figure S1 in supplemen-
methyl, methoxy, fluoro or chloro substituent on pyrazole tary information).
3 displayed better AI activity as compared to unsubstituted
The binding site analysis of indomethacin showed that it
pyrazole but no correlation could be drawn with respect to the penetrates deep into the cycloxygenase active site (Figure 2A).
substituent on coumarin ring. However, a close look indicated that The benzoyl oxygen interacts with Ser530 amino acid residue
a combination of methoxy or chloro substituent on pyrazole ring which is the acetylation site of aspirin in both COX-1 and COX-
and chloro substituent on coumarin ring, i.e. 3h and 3n exhibited 225. The carboxylate group of indomethacin is positioned towards
pronounced AI activity.
the mouth of the active site near Arg120 and Tyr355 and the
When we compared the AI results of these compounds methoxy group of indole points towards the secondary pocket of
with phenyl analogues reported earlier by us19, we found that the COX-2 active site. Additional close contacts with Ser353,
in general, coumarin analogues containing methyl, methoxy, fluoro Ala527 and Val523 were also seen (Figure 2A). Figure S2 in
or chloro substituent on pyrazole (3d, 3e, 3g, 3h, 3j, 3k, 3m, 3n) supplementary information represents 2D binding pose view of
displayed better AI activity as compared to phenyl analogues but indomethacin at COX-2 active site.
loss of AI activity was found in coumarin analogues with
The binding site analysis of the most active compound
unsubstituted pyrazole (3a, 3b). The comparison of AI results 3n showed that, the benzenesulfonamide group is positioned
with starting materials, pyrazole-4-carbaldehyde thiosemicarba- in the vicinity of secondary pocket of COX-2 isozyme,
zones displayed an increase in activity by construction of thiazole surrounded by His90, Tyr355, Arg513, Ser353, Gln192
ring in compounds containing methyl, methoxy or fluoro substitu- and Leu352 (Figure 2A and B). This secondary pocket is
ent on pyrazole (3d, 3g, 3j) while decrease in activity in those the major difference in the active site of COX-1 and COX-2
containing no or chloro substituent on pyrazole (3a, 3m).
isoforms as similar pocket is found in COX-1 but is inaccessible
due to the presence of bulkier Ile523 in COX-1 which is Val523
in COX-224,26. The two oxygen atoms of the SO2NH2 group
are inserted into the secondary pocket region, showing
close contact with the residue His90 and Gln192, respectively,
in the COX-2 active site. Moreover, the NH2 of SO2NH2
group showed hydrogen bond with Ser353 and also showed
Van der Waals interaction with His90. The nitrogen atom of
thiazole shows interaction with Arg120, near the entrance to
the COX-2 binding site. However, in case of the least active
compound 3a, the docking results showed that this compound
failed to show its interaction with the secondary pocket residues
due to the flipping of the benzenesulfonamide group but showed
Docking studies
Molecular docking studies were performed using the automated
GOLD docking program on the most active (3n) and the least
active (3a) compound of the series in the anti-inflammatory
studies along with the control drug indomethacin to gain an
insight about the probable nature of their binding at the COX-1
and COX-2 active site using crystal structure data obtained from
the RCSB Protein Data Bank.
Docking to COX-2 active site
Initially, indomethacin was docked at the active site of the COX-2 interaction of the thiazole nitrogen and carbonyl oxygen of
protein co-crystallized with the indomethacin (PDB ID: 4COX)24 coumarin with the Arg120 and Tyr355 at the entrance of COX-2
to ensure the correct binding mode prediction of the docking active site (Figure 2B). Figure S3 and S4 in supplementary
program. The comparative binding site analysis of COX-2 protein information represent 2D binding pose views of compounds 3n
ligand docked complex and COX-2 X-ray crystal structure and 3a, respectively.
Figure 2. Comparative 3D binding pose view of (A) standard drug indomethacin (green colored carbon) along with the most active compound of the
series 3n (pink colored carbon), (B) the most active compound 3n (pink colored carbon) along with the least active compound 3a (blue colored carbon)
of the series at the active site of COX-2.

482 N. Chandak et al.
J Enzyme Inhib Med Chem, 2014; 29(4): 476–484
A
comparison of the position of indomethacin and The benzoyl group of indomethacin and benzenesulfonamide
compound 3n within the COX-2 active site as illustrated group of 3n assumes similar positions. However, the methoxy
in Figure 2(A) showed that methoxy substituent of the indole group of indomethacin and 4-chlorophenyl group of compound 3n
moiety in indomethacin and benzenesulfonamide moiety protrudes a little towards relatively large cavity of COX-1,
in compound 3n assumes a position near the secondary resulting in higher activity of compound 3n than indomethacin.
pocket of COX-2 active site, but the extra bulk of benzenesulfo- Similarly, the comparison of the binding site analysis of the least
namide is well accommodated by the secondary pocket and active compound 3a with the most active compound 3n showed
shows electrostatic interaction with COX-2 active site residues, that although the SO2NH2 group of the compound 3a showed
plausibly explaining better activity profile of 3n in comparison H-bond interaction with Leu384 but failed to interact with
to indomethacin. Likewise, reduced activity of 3a in compari- Tyr348. Moreover, the coumarin ring and thiazole ring also lost
son to 3n can be explained by the incapability of 3a to interact its interaction with Arg120 and Tyr355 respectively presumably
with or accommodate within the secondary pocket of COX-2 contributing to the loss of activity (Figure 3B). Figure S7 in
(Figure 2B).
supplementary information represents 2D binding pose view of
compound 3a (the least active) at COX-1 active site.
Docking to COX-1 active site
In vitro antimicrobial activity
The docking studies has further been performed on the most
active (3n) and the least active (3a) compound of the series along All the synthesized target compounds (3a–o and 5a–c) were
with control drug indomethacin with the COX-1 enzyme (PDB assayed for their in vitro antimicrobial activity against S. aureus
ID: 2OYE)26.
(MTCC 96) and B. subtilis (MTCC 121) representing Gram-
The binding site analysis of indomethacin showed that its positive bacteria, E. coli (MTCC 1652) and P. aeruginosa (MTCC
carboxylate group interacts with Arg120 at the mouth of the COX- 741) representing Gram-negative bacteria, and S. cerevisiae
1 active site (Figure 3A). The benzoyl group showed interactions (MTCC 170) and C. albicans (MTCC 227) representing fungal
with Val346 and Leu352 while the phenyl ring of indole of the yeasts (Table S1 in supplementary information) by agar well
molecule showed hydrophobic interactions with Tyr355, Ile523, diffusion method17 using ciprofloxacin against bacteria and
Ala527 and Ser353. The chloro group showed its orientation amphotericin-B against fungi as the reference drugs. The results
towards Leu384. Similarly, binding site analysis of the most active were recorded for each tested compound as the average diameter
compound 3n revealed that this molecule well occupied the of inhibition zones of microbial growth surrounding the well in
entrance of the COX-1 active site which is evident through mm. The minimum inhibitory concentration (MIC) measurements
H-bond interaction of the sulfur of thiazole and carbonyl oxygen were performed using a macrodilution tube method18 (Table S2 in
of coumarin ring of the molecule with Arg120. The nitrogen of supplementary information).
the thiazole ring showed H-bond interaction with Tyr355 whereas
Results revealed that in general, all the tested compounds
the pyrazole ring of the compound showed sigma pi interaction possessed moderate antibacterial activity against both the Gram-
with Ile523 (Val523 in COX-2). At the same time the NH2 group positive bacteria (S. aureus, B. subtilis) and moderate antifungal
in SO2NH2 showed hydrogen bonding with Leu384 as well as activity against one of the yeasts, S. cerevisiae. However, none of
close contact with Tyr348 (Figure 3A and B). Figure S5 and S6 in them was found to be effective against any Gram-negative
supplementary information represent 2D binding pose views of bacteria (E. coli, P. aeruginosa) and other tested yeast,
indomethacin and compound 3n (the most active), respectively at C. albicans. On the basis of zone of inhibition against the test
COX-1 active site.
bacterium, compounds 3g and 5a were found to be the most
A comparison of the position of indomethacin and compound effective against S. aureus showing zone of inhibition 418.0 mm
3n within the COX-1 active site as illustrated in Figure 3(A) and compounds 3b, 3d, 3e, 3f, 3g, 3o, 5a and 5c producing
showed that the carboxylate group in indomethacin and coumarin ꢄ18.0 mm zone of inhibition against B. subtilis (Table S1) when
ring in 3n are oriented towards the mouth of the active site. compared with standard drug ciprofloxacin which showed the
Figure 3. Comparative 3D binding pose view of (A) standard drug indomethacin (green colored carbon) along with the most active compound of the
series 3n (pink colored carbon), (B) the most active compound 3n (pink colored carbon) along with the least active compound 3a (blue colored carbon)
of the series at the active site of COX-1.

DOI: 10.3109/14756366.2013.805755
Dual evaluation of some novel 2-amino-substituted coumarinylthiazoles 483
zone of inhibition 26.6 mm against S. aureus and 24.0 mm against Acknowledgements
B. subtilis. Rest of the compounds didn’t show significant
One of the authors (NC) is grateful to the Council of Scientific and
Industrial Research (CSIR), New Delhi for the award of senior research
fellowship. The authors are thankful to Sophisticated Analytical
antibacterial activity against any of the Gram-positive bacteria.
However, in terms of MIC, none of them was found to possess
appreciable antibacterial activity. Amongst all the compounds, the Instrument Facility, Central Drug Research Institute, Lucknow for Mass
spectra.
MIC ranged between 32 and 256 mg/mL against Gram-positive
bacteria as compared to standard drug ciprofloxacin having MIC
of 5 mg/mL (Table S2).
In case of fungal yeasts, compounds 3f, 3g, 3o and 5a
Declaration of interest
Defence Research and Development Organization (DRDO), New Delhi is
thankfully acknowledged for financial support in the form of a research
project.
were found to be the most effective against S. cervisiae
showing zone of inhibition ꢄ13.0 mm (Table S1) when
compared with standard drug amphotericin-B producing zone of
The authors report no conflicts of interest.
inhibition of 19.3 mm. However, in terms of MIC, most of
the compounds failed to possess appreciable antifungal
activity. Amongst all the compounds, the MIC ranged between
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