Design and Amberlyst-15 mediated synthesis of novel thienyl-pyrazole carboxamides that potently inhibit Phospholipase A2 by binding to an allosteric site on the enzyme

Article abstract of DOI:10.1016/j.bioorg.2018.06.023

Inflammation-mediated disorders are on the rise and hence, there is an urgent need for the design and synthesis of new anti-inflammatory drugs with higher affinity and specificity for their potential targets. The current study presents an effective and new protocol for the synthesis of thienyl-pyrazoles through 3 + 2 annulations using a recyclable heterogeneous catalyst Amberlyst-15. Chalcones 3(a-g) prepared from 3-methylthiophene-2-carbaldehyde and acetophenones by Claisen-Schmidt approach reacted with semicarbazide hydrochloride 4 in the presence of Amberlyst-15 in acetonitrile at room temperature producing thienyl-pyrazole carboxamides 5(a-h) in good yields. Alternatively, the compounds 5(a-h) were prepared by conventional method using acetic acid (30%) medium. Structures of synthesized new pyrazoles were confirmed by spectral and crystallographic studies. All the new compounds were evaluated for their in vitro inhibition of Phospholipase A2 from Vipera russelli and preliminary studies revealed that, amongst the designed series, compounds 5b, 5c and 5h showed promising inhibition. Further, the compounds exhibited linear mixed-type inhibition behavior for the sPLA₂ enzyme indicating that they bind to an allosteric site distinct from either the calcium or substrate binding site on the enzyme. These kinetic conclusions were further validated by macromolecular rigid-body docking whereby compounds 5c and 5h showed binding to distinct pockets on the protein. These findings present a promising series of lead molecules that can serve as prototypes for the treatment of inflammatory related disorders.

Full text of DOI:10.1016/j.bioorg.2018.06.023

Accepted Manuscript
Design and Amberlyst-15 mediated synthesis of novel thienyl-pyrazole carbox-
amides that potently inhibit Phospholipase A2 by binding to an allosteric site on
the enzyme
Achutha Dileep Kumar, Malledevarapura Gurumurthy Prabhudeva, Srinivasan
Bharath, Karthik Kumara, Neratur Krishnappagowda Lokanath, Kariyappa
Ajay Kumar
PII:
S0045-2068(18)30378-X
https://doi.org/10.1016/j.bioorg.2018.06.023
YBIOO 2403
DOI:
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 April 2018
14 June 2018
18 June 2018
Please cite this article as: A. Dileep Kumar, M. Gurumurthy Prabhudeva, S. Bharath, K. Kumara, N.
Krishnappagowda Lokanath, K. Ajay Kumar, Design and Amberlyst-15 mediated synthesis of novel thienyl-
pyrazole carboxamides that potently inhibit Phospholipase A2 by binding to an allosteric site on the enzyme,
Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.06.023
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Design and Amberlyst-15 mediated synthesis of novel thienyl-pyrazole carboxamides that
potently inhibit Phospholipase A2 by binding to an allosteric site on the enzyme
Achutha Dileep Kumar,a Malledevarapura Gurumurthy Prabhudeva,a Srinivasan Bharath,b
Karthik Kumara,c Neratur Krishnappagowda Lokanath,c Kariyappa Ajay Kumara*
aDepartment of Chemistry, Yuvaraja College, University of Mysore, Mysuru-570005, India.
bMarie Skłodowska-Curie Actions Fellow, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal.
cDepartment of Studies in Physics, University of Mysore, Mysuru-570006, India.
*Corresponding author, E-mail: ajaykumar@ycm.uni-mysore.ac.in
Abstract
Inflammation-mediated disorders are on the rise and hence, there is an urgent need for the
design and synthesis of new anti-inflammatory drugs with higher affinity and specificity for their
potential targets. The current study presents an effective and new protocol for the synthesis of
thienyl-pyrazoles through 3+2 annulations using a recyclable heterogeneous catalyst Amberlyst-
15. Chalcones 3(a-g) prepared from 3-methylthiophene-2-carbaldehyde and acetophenones by
Claisen-Schmidt approach reacted with semicarbazide hydrochloride 4 in the presence of
Amberlyst-15 in acetonitrile at room temperature producing thienyl-pyrazole carboxamides 5(a-
h) in good yields. Alternatively, the compounds 5(a-h) were prepared by conventional method
using acetic acid (30%) medium. Structures of synthesized new pyrazoles were confirmed by
spectral and crystallographic studies. All the new compounds were evaluated for their in vitro
inhibition of Phospholipase A2 from Vipera russelli and preliminary studies revealed that,
amongst the designed series, compounds 5b, 5c and 5h showed promising inhibition. Further, the
compounds exhibited linear mixed-type inhibition behavior for the sPLA2 enzyme indicating that
1

they bind to an allosteric site distinct from either the calcium or substrate binding site on the
enzyme. These kinetic conclusions were further validated by macromolecular rigid-body docking
whereby compounds 5c and 5h showed binding to distinct pockets on the protein. These findings
present a promising series of lead molecules that can serve as prototypes for the treatment of
inflammatory related disorders.
Keywords: anti-inflammatory, chalcone, condensation, pyrazole, thiophene.
1.
Introduction
Phospholipases A2 (PLA2) are enzymes producing arachidonic acid and lysophospholipids
as byproducts of hydrolyzing membrane phospholipids. Subsequently, arachidonic acid and
lysophospholipids are metabolized to eicosanoids and platelet-activating factors which play
critical roles in the initiation and regulation of inflammation and oxidative stress. Several
aberrant neurological conditions are ascribed to inflammation caused by phospholipid
metabolism alterations leading to lipid peroxides accumulation and increased phospholipase
A2 activities in the brain. Hence, it is essential to design and synthesize novel small-molecule
inhibitors for this class of enzymes that can serve as core structures to elaborate for species
specific or context-specific inhibition and physiological outcome. Extracts containing natural
plant metabolites from curcumin plants, Centella asiatica and Ginkgo Biloba, have also been
extensively assessed as potential inhibitors of phospholipase A2 in cell culture.1
Chalcones are the initiators for the biosynthesis of flavonoids and isoflavonoids. Recent
reviews on the chalcone derivatives highlights the anti-inflammatory activities, effect of these
compounds on lipid peroxidation, heme oxygenase 1(HO-1), cyclooxygenase (COX), and
interleukin 5 (IL-5) etc.2,3 A synthesized series of α,β-unsaturated carbonyl compounds have
2

showed potent inhibitory activity on sPLA2, cyclooxygenases (COX), soybean lipoxygenase
(LOX) in addition to pro-inflammatory cytokines comprising IL-6 and TNF-α.4,5
Pyrazoles are an important class of compounds with demonstrated pharmacological
properties. A few examples of such properties would include antimicrobial,6 antioxidant,7 and
anti-inflammatory8 activities. For instance, synthesized series of pyrazolines have reported to
exhibit potent anti‐ inflammatory related activities such as inhibition of phospholipase
A2 (PLA2), cyclooxygenases (COX‐ 1 and COX‐ 2), IL‐ 6, and TNF‐ α.9 A recent study has
also demonstrated by fragment based virtual ligand screening and subsequent experiments that a
novel series of pyrazoles can inhibit phospholipase A2, and the most potent inhibitor of the series
being 5-(5-isopropyl-2-methylthiophen-3-yl)-1H pyrazole 3-carboxamide with IC50 of 7.29
µM.10
Design and development of an easy procedure for the synthesis of heterocycles with various
functionalities that possess wide range of bioactivities with fewer/no side effects is a worthwhile
contribution in organic synthesis. A number of synthetic protocols have been reported for the
synthesis of pyrazole derivatives in the literature11-15 etc. However, many of these methods suffer
from drawbacks, such as low yields, longer reaction time etc. Thus, development of a facile and
eco-friendly method is highly desirable. In recent times, heterogeneous catalysis have played a
pivotal role in organic transformations,16 for example, Amberlyst-15 was found efficient
heterogeneous catalyst in the synthesis of quinazolinones.17 In the present work, we report the
use of an inexpensive and reusable catalyst (Amberlyst-15) mediated synthesis of thienyl-
pyrazoline carboxamides by the reaction of chalcones with semicarbazide hydrochloride. In
order to check the efficiency of the newly employed catalyst, the reaction was carried out by
conventional method in acetic acid medium. The synthesized compounds were characterized by
3

spectral and crystallographic studies, and were evaluated in vitro for their inhibition of
Phospholipase A2 activities from Vipera russelli. The demonstrated synthesis paves the way for
future efforts at synthesizing thienyl-pyrazoles that could find widespread applications in
bioorganic chemistry.
2.
Materials and Methods
Melting points were determined by an open capillary tube method and are uncorrected.
Progress of the reactions was checked on thin layer chromatography (TLC) plates pre-coated
with silica gel using solvent system hexane: ethyl acetate (1:4). The spots were visualized under
1
UV light. H NMR and 13C NMR spectra were recorded on Agilent-NMR 400 MHz and 100
MHz spectrometer respectively. Mass spectra were obtained on ESI/APCI-Hybrid Quadrupole,
Synapt G2 HDMS ACQUITY UPLC model spectrometer. Elemental analysis was obtained on a
Thermo Finnigan Flash EA 1112 CHN analyzer.
2.1
X-Ray diffraction analysis
The compound 3-(2-methoxyphenyl)-5-(3-methylthiophen-2-yl)-4,5-dihydro-1H-
pyrazole-1-carboxamide 5f was recrystallized from methanol to give pale yellow crystals
suitable for single crystal X-ray diffraction (crystal size 0.23 × 0.34 × 0.27 mm) with the
crystallographic parameters: a = 7.681(3) Å, b = 8.346(3) Å, c = 13.149(5) Å, α = 103.609(7)°, β
= 103.170(12)°, γ = 97.531(13)°, V = 782.4(5) Å3, Z = 2, Goodness-of-fit on F2 = 0.944, R indices
(all data) = R1 = 0.1090, wR2 = 0.2150. The complete intensity data sets were processed using
CRYSTAL CLEAR.18 The crystal structure was solved by direct method and refined by full-
matrix least squares method on F2 using SHELXS and SHELXL programs,19 respectively. The
geometrical calculations were performed using PLATON.20 The molecular and packing diagrams
were generated using the software MERCURY.21
4

2.3
Anti-inflammatory activity
Inflammation is a complex immunological cascade driven by several different factors and
can be initiated by manifold cues including, but not limited to, pathogen invasion, tissue damage
due to oxidative challenge etc. COX-2 is an important player in bringing about inflammation.
Primarily during tissue damage, the first enzyme to get activated is sPLA2, which drives the
substrate for COX-2. Inhibition of sPLA2 will result in substrate depletion for COX-2, thereby
bringing down the inflammation, as there will be no pro-inflammatory Prostaglandins (PG). In
this context, we assessed the inhibitory potential of the synthesized thienyl-pyrazole derivatives,
5(a-h) to inhibit sPLA2, rather than COX-2,22 This is to ensure that multiple enzyme targets are
available for combinatorial drug administration to increase potency and efficacy of blocking the
pathway.
2.3.1 Purification of sPLA2 (VRV-PL-8a) from V. russelli venom: sPLA2 (VRV-PL-8a)
from Vipera russelli venom was purified to homogeneity by reported procedure23 and the protein
was estimated by Lowry's method.24 Briefly, V. russelli venom (80 mg) was fractionated on pre-
equilibrated Sephadex G-75 column (1.5 × 160 cm) using 50 mM phosphate buffer pH 7.0. The
protein was resolved into major three peaks. The second peak, constitute about 30% of the total
protein, which showed major sPLA2 activity. This sPLA2 peak fraction was lyophilized and
further subjected to pre-equilibrated CM-Sephadex C-25 column (1.5 × 45 cm) chromatography.
The fractions were eluted stepwise using phosphate buffers of varied ionic strength (50mM –
200mM) and pH (7.0-8.0). They were resolved into two fractions labeled as V and VIII
respectively. The above eluted two fractions were similar to V and VIII protein profiles. The
lyophilized fraction VIII was next subjected to Sephadex G-50 column (0.75 ×40 cm)
5

chromatography and eluted using 50mM phosphate buffer pH 7.0 and obtained peak was
checked for sPLA2 activity. Homogeneity was checked by SDS-PAGE and RP-HPLC.
2.3.2 In vitro inhibition of VRV-PL-8a by thienyl-pyrazole derivatives, 5(a-h): In vitro
inhibition of sPLA2 (VRV-PL-8a) by the synthesized pyrazole derivatives, 5(a-h), was assessed
by the known procedure.24 Briefly, a 50µl activity buffer containing 50mM Tris-HCl buffer pH
7.5, 10mM CaCl2 and 100µM substrate stock (1mM DMPC in methanol containing 2mM Triton
X-100 in Milli-Q water) were added and incubated for 5min at 37ºC. Activity was initiated by
adding 10ng of sPLA2 alone or pre incubated with different concentration of thiophene-pyrazole
conjugates 5(a-h) ranging from 0-100µM for 5min at 37°C. Reaction mixtures were incubated
for 45min at 37ºC. 50µl of quenching solution was added at a final concentration of 2mM NaN3,
50µM ANS and 50mM EGTA, vortexed for 30sec and incubated for 5min at rt. 2µL of this
solution was pipetted to measure RFU in a Nanodrop ND3300 Ver 2.8 using an excitation UV-
LED (370±10nm) and emission was recorded at 480nm in dark condition. Enzyme activity was
calculated using the equation
∆RFU = RFU (Control) ─RFU (Test)
Where, ∆RFU is the change in RFU of test (with sPLA2) with respect to control (without
sPLA2) in the presence of inhibitors. The resultant RFU was compared with the standard LPC
curve to determine the sPLA2 activity in the presence of inhibitors.
2.3.3 Effect of substrate and calcium concentration on VRV-PL-8a: The effect of substrate
and calcium concentration on VRV-PL-8a was estimated by known method.24 The reaction
mixture 0.1ml containing 10mg of sPLA2 alone or with IC50 concentration (11.01µM) of
inhibitor (test compounds) in 50mM Tris-HCl buffer pH 7.5, 10mM CaCl2 and 10µl of varied
substrate stock (0-400µM) was used for sPLA2 assay to check the effect of substrate in presence
6

of test compounds. 50µl of quenching solution was added at final concentration of 50µM ANS,
2mM NaN3 and 50mM EGTA, vortexed for 30 sec and incubated for 5 min at RT. 2µl of this
solution was pipetted to measure RFU. Similar set of experiment was conducted, where in
reaction mixture containing IC50 concentration of inhibitor (test compounds) in 50mM Tris-HCl
buffer, pH 7.5, 10ng of sPLA2, 100µM substrate and varied concentration of CaCl2 (0-12mM).
Reaction was terminated and RFU was measured as described above.
2.3.4 Protein structure quality assessment: The quality of the predicted structure was
assessed. Ramachandran plots were generated to understand whether the phi-psi dihedrals are
within the allowed regions.25 Below is the summary of the analysis. As can be seen, the statistics
indicates that the structure is reasonably good.
Number of residues in favoured region (~98.0% expected) : 112 (93.3%)
Number of residues in allowed region (~2.0% expected) : 8 (6.7%)
Number of residues in outlier region : 0 (0.0%)
Residue [A 32 :PHE] ( -61.55, 105.65) in Allowed region
Residue [A 33 :GLY] ( -33.23, -68.80) in Allowed region
Residue [A 39 :TYR] (-143.95, 28.16) in Allowed region
Residue [A 41 :GLY] ( 47.13,-163.40) in Allowed region
Residue [A 92 :GLY] (-114.18, 59.19) in Allowed region
Residue [A 97 :CYS] ( -66.37, -64.58) in Allowed region
Residue [A 129 :PHE] ( -74.72, 64.44) in Allowed region
Residue [A 132 :LYS] ( -55.19, 174.67) in Allowed region
3.
Results
3.1
Synthesis of chalcones, 3(a-h). To a solution mixture of 3-methylthiophene-2-
carbaldehyde, 1 (10 mmole) and acetophenones, 2(a-h) (10 mmole) in methyl alcohol, potassium
hydroxide solution (40%, 2 mL) was added. Then the solution mixture was stirred at room
7

temperature for 2-3 h. The progress of the reaction was monitored by TLC. After the completion,
the mixture was cooled to room temperature and poured into ice cold water. The solids separated
were filtered, washed successively with cold hydrochloric acid (5%) and cold water. The crude
solids were recrystallized from methyl alcohol to obtain compounds 3(a-h).26
3.2
Synthesis of pyrazole carboxamides, 5(a-h).
Method A: A mixture of chalcones 5(a-h) (5.0 mmol), semicarbazide hydrochloride 4 (5.0
mmol) and Amberlyst-15 (10%, w/w) in acetonitrile (25 mL) was stirred at room temperature for
30-60 minutes. The progress of the reaction was monitored by TLC. After completion of the
reaction, the solid separated was filtered, washed by diethyl ether (2 x 20 mL), dried and treated
with EtOAc (20 mL). After stirring for 10 min the mixture was filtered to remove the insoluble
catalyst. The filtrate was collected and concentrated under vacuum. The solid isolated was
triturated in diethyl ether, filtered and dried to obtain the desired product 5(a-h). The recovered
catalyst was washed with EtOAc, dried and reused effectively for four times for a reaction.
Method B: A solution mixture of thiophene based chalcones, 3(a-f) (5 mmole) and
semicarbazide hydrochloride, 4 (10 mmole) in acetic acid (30%) was refluxed on a water bath for
1-2 h. The progress of the reaction was monitored by TLC. After the completion, the mixture
was filtered and the filtrate was poured into crushed ice. The separated solids were filtered and
washed successively with 5% NaHCO3 and water. The crude solids were recrystallized from
methyl alcohol to get target molecules 5(a-f) in good yields.
3.2.1 5-(3-Methylthiophen-2-yl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carboxamide, 5a:
Obtained from 3-(3-methylthiophen-2-yl)-1-phenylprop-2-en-1-one, 3a (1.14g, 5 mmole) and
1
semicarbazide hydrochloride, 4 (1.11g, 10 mmole) in 72% yield, m.p. 112-114 °C; H NMR
(CDCl3, δ ppm): 2.271 (s, 3H, CH3), 3.196 (dd, 1H, J=6.9, 16.0Hz, C4-Ha), 3.726 (dd, 1H,
8

J=7.1, 12.6Hz, C4-Hb), 5.560 (s, 2H, NH2), 5.745 (dd, 1H, J=6.9, 12.1Hz, C5-H), 6.744 (d, 1H,
Ar-H), 7.240-7.641 (m, 6H, Ar-H); 13C NMR (CDCl3, δ ppm): 13.26 (1C, CH3), 42.60 (1C, C-4),
54.56 (1C, C-5), 120.22 (1C), 122.33 (1C), 124.36 (1C), 128.10 (1C), 128.14 (1C), 128.84 (1C),
128.86 (1C), 133.52 (1C), 130.90 (1C), 135.24 (1C), 151.80 (1C, C-3), 159.87 (1C, C=O). MS
m/z: 285.09 (M+, 100); Anal. Calcd. for C15H15N3OS (%): C, 63.13; H, 5.30; N, 14.73; Found:
C, 63.01; H, 5.26; N, 14.69.
3.2.2 3-(4-Fluorophenyl)-5-(3-methylthiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-
carboxamide, 5b: Obtained from 1-(4-fluorophenyl)-3-(3-methylthiophen-2-yl)prop-2-en-1-one,
3b (1.23g, 5 mmole) and semicarbazide hydrochloride, 4 (1.11g, 10 mmole) in 70% yield, m.p.
1
104-106 °C; H NMR (CDCl3, δ ppm): 2.265 (s, 3H, CH3), 3.181 (dd, 1H, J=7.1, 16.6Hz, C4-
Ha), 3.722 (dd, 1H, J=7.4, 12.3Hz, C4-Hb), 5.554 (s, 2H, NH2), 5.738 (dd, 1H, J=6.8, 12.6Hz,
13
C5-H), 6.786 (d, 1H, Ar-H), 7.235-7.686 (m, 5H, Ar-H); C NMR (CDCl3, δ ppm): 13.35 (1C,
CH3), 45.42 (1C, C-4), 54.40 (1C, C-5), 113.50 (1C), 120.87 (1C), 120.98 (1C), 122.44 (1C),
128.36 (1C), 129.72 (1C), 131.10 (1C), 133.22 (1C), 137.60 (1C), 151.33 (1C, C-3), 153.080
(1C), 159.27 (1C, C=O). MS m/z: 303.08 (M+, 100); Anal. Calcd. for C15H14FN3OS (%): C,
59.39; H, 4.65; N, 13.85; Found: C, 59.30; H, 4.61; N, 13.78.
3.2.3 3-(4-Chlorophenyl)-5-(3-methylthiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-
carboxamide, 5c: Obtained from 1-(4-chlorophenyl)-3-(3-methylthiophen-2-yl)prop-2-en-1-one,
3c (1.31g, 5 mmole) and semicarbazide hydrochloride, 4 (1.11g, 10 mmole) in 69% yield, m.p.
1
114-116 °C; H NMR (CDCl3, δ ppm): 2.294 (s, 3H, CH3), 3.360 (dd, 1H, J=6.5, 17.0Hz, C4-
Ha), 3.912 (dd, 1H, J=6.0, 13.8Hz, C4-Hb), 5.687 (s, 2H, NH2), 5.732 (dd, 1H, J=6.9, 12.4Hz,
C5-H), 6.744 (d, 1H, J=4.0Hz, Ar-H), 6.970-7.352 (m, 4H, Ar-H), 7.760 (d, 1H, J=7.3Hz, Ar-H);
13C NMR (CDCl3, δ ppm): 13.65 (1C, CH3), 45.50 (1C, C-4), 54.36 (1C, C-5), 120.22 (1C),
9

121.14 (1C), 121.85 (1C), 128.18 (1C), 128.26 (1C), 129.34 (1C), 129.40 (1C), 133.16 (1C),
134.74 (1C), 137.88 (1C), 151.54 (1C, C-3), 158.27 (1C, C=O). MS m/z: 321.05 (M+2, 36.5%),
319.05 (M+, 100); Anal. Calcd. for C15H14ClN3OS (%): C, 56.33; H, 4.41; N, 13.14; Found: C,
56.21; H, 4.11; N, 13.07.
3.2.4 3-(4-Methoxyphenyl)-5-(3-methylthiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-
carboxamide, 5d: Obtained from 1-(4-methoxyphenyl)-3-(3-methylthiophen-2-yl)prop-2-en-1-
one, 3d (1.27g, 5 mmole) and semicarbazide hydrochloride, 4 (1.11g, 10 mmole) in 77% yield,
1
m.p. 162-163 °C. H NMR (CDCl3, δ ppm): 2.289 (s, 3H, CH3), 3.356 (dd, 1H, J=6.9, 17.1Hz,
C4-Ha), 3.848 (s, 3H, OCH3), 3.900 (dd, 1H, J=6.2, 13.2Hz, C4-Hb), 5.696 (s, 2H, NH2), 5.721
(dd, 1H, J=6.8, 11.9Hz, C5-H), 6.731 (d, 1H, J=4.0Hz, Ar-H), 6.929-7.365 (m, 4H, Ar-H),
13
7.853 (d, 1H, J=7.6Hz, Ar-H); C NMR (CDCl3, δ ppm): 13.90 (1C, CH3), 45.82 (1C, C-4),
54.45 (1C, C-5), 55.43 (1C, OCH3), 111.55 (1C), 120.60 (1C), 120.80 (1C), 122.22 (1C), 129.06
(1C), 130.12 (1C), 131.33 (1C), 133.34 (1C), 139.46 (1C), 151.99 (1C, C-3), 155.40 (1C),
158.10 (1C, C=O). MS m/z: 315.12 (M+, 100); Anal. Calcd. for C16H17N3O2S (%):C, 60.93; H,
5.43; N, 13.32; Found: C, 60.79; H, 5.38; N, 13.22.
3.2.5 3-(3-Methoxyphenyl)-5-(3-methylthiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-
carboxamide, 5e: The synthesis and characterization was reported earlier.27
3.2.6 3-(2-Methoxyphenyl)-5-(3-methylthiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-
carboxamide, 5f: Obtained from 1-(2-methoxyphenyl)-3-(3-methylthiophen-2-yl)prop-2-en-1-
one, 3f (1.27g, 5 mmole) and semicarbazide hydrochloride, 4 (1.11g, 10 mmole) in 71% yield,
1
m.p. 124-126 °C; H NMR (CDCl3, δ ppm): 2.243 (s, 3H, CH3), 3.189 (dd, 1H, J=4.8, 17.6Hz,
C4-Ha), 3.712 (dd, 1H, J=5.2, 11.2Hz, C4-Hb), 3.827 (s, 3H, OCH3), 5.535 (s, 2H, NH2), 5.765
(dd, 1H, J=4.8, 11.6Hz, C5-H), 6.730-7.030 (m, 3H, Ar-H), 7.206-7.322 (m, 3H, Ar-H); 13C
10

NMR (CDCl3, δ ppm): 13.91 (1C, CH3), 42.71 (1C, C-4), 54.50 (1C, C-5), 55.36 (1C, OCH3),
111.39 (1C), 116.07 (1C), 119.13 (1C), 122.46 (1C), 129.74 (1C), 130.20 (1C), 132.63 (1C),
133.49 (1C), 139.10 (1C), 151.68 (1C, C-3), 155.26 (1C), 159.76 (1C, C=O). MS m/z: 315.10
(M+, 100); Anal. Calcd. for C16H17N3O2S (%): C, 60.93; H, 5.43; N, 13.32; Found: C, 60.81; H,
5.40; N, 13.25.
3.2.7 3-(3,4-Dimethoxyphenyl)-5-(3-methylthiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-
carboxamide, 5g: Obtained from 1-(3,4-dimethoxyphenyl)-3-(3-methylthiophen-2-yl)prop-2-en-
1-one, 3g (1.44g, 5 mmole) and semicarbazide hydrochloride, 4 (1.11g, 10 mmole) in 65%
1
yield, m.p. 130-133 °C. H NMR (CDCl3, δ ppm): 2.280 (s, 3H, CH3), 3.191 (dd, 1H, J=6.7,
16.4Hz, C4-Ha), 3.731 (dd, 1H, J=6.6, 12.9Hz, C4-Hb), 3.848 (s, 6H, OCH3), 5.556 (s, 2H, NH2),
5.784 (dd, 1H, J=6.6, 12.7Hz, C5-H), 6.740 (d, 1H, Ar-H), 6.954-6.977 (m, 1H, Ar-H), 7.040-
13
7.346 (m, 3H, Ar-H); C NMR (CDCl3, δ ppm): 14.30 (1C, CH3), 42.82 (1C, C-4), 54.45 (1C,
C-5), 55.45 (2C, OCH3), 111.46 (1C), 114.36 (1C), 121.06 (1C), 121.50 (1C), 122.14 (1C),
125.15 (1C), 127.33 (1C), 133.44 (1C), 150.09 (1C), 151.77 (1C, C-3), 153.26 (1C), 159.90 (1C,
C=O). MS m/z: 345.11 (M+, 100); Anal. Calcd. for C17H19N3O3S (%): C, 59.11; H, 5.54; N,
12.17; Found: C, 59.00; H, 5.49; N, 12.08.
3.2.8 3-(Benzo[d][1,3]dioxol-5-yl)-5-(3-methylthiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-
carboxamide, 5h: The synthesis and characterization was reported earlier.28
4.
Discussion
4.1
Chemistry
Initially, the intermediate 1-(aryl)-3-(3-methylthiophen-2-yl) prop-2-en-1-ones, 3(a-h), were
synthesized by base catalyzed reaction of 3-methylthiophene-2-carbaldehyde 1, with substituted
acetophenones, 2(a-h) in methyl alcohol.26 Then the reaction of 3(a-h) and semicarbazide
11

hydrochloride 4, in the presence of heterogeneous catalyst Amberlyst-15 (10%, w/w) in
acetonitrile under stirring conditions at room temperature produced thienyl-pyrazole
carboxamides, 5(a-h) in good yields. The target compounds, 5(a-h) were also synthesized by
conventional method in acetic acid (30%) under reflux conditions, which paves way for
understanding the catalytic activity of Amberlyst-15 (Fig. 1).
Fig. 1: Synthesis of thienyl-pyrazoline carboxamides, 5(a-h)
The base catalyzed reaction of 3-methylthiophene-2-carbaldehyde 1, and acetophenones,
2(a-h) in methyl alcohol produced 1-(aryl)-3-(3-methylthiophen-2-yl) prop-2-en-1-one, 3(a-h) in
good yields.26 In search of new potent anti-inflammatory agents, our strategic aim was to
synthesize novel thienyl-pyrazole carboxamides. In this context, we carried out the reaction of
chalcones 3(a-h) with semicarbazide hydrochloride 4 using recyclable heterogeneous catalyst
Amberlyst-15 in acetonitrile at room temperature under stirring conditions to obtain the target
compounds thienyl-pyrazole carboxamides, 5(a-h) in good yields. In order to check the
efficiency of the catalyst, alternatively the reaction was carried out by conventional method in
12

acetic acid medium. We observed that, Amberlyst-15 mediated synthesis requires lesser reaction
time, and needs no thermal energy in comparison to conventional method in acetic acid; however
there was no major difference (±5%) in terms of product yields between the Amberlyte-15
method and the classical method. The Amberlyst-15 catalyst, recovered using the solvent ethyl
acetate, was found efficient for four consecutive similar experiments.
1H NMR spectra of compounds 5(a-h) did not show the signals appearing as doublet for
each of the alkenyl protons of chalcones 3(a-h), confirming the (3+2) annulations between
chalcones and semicarbazide hydrochloride to form the target compounds. Further, the
methylene protons of C-4 atom of newly formed pyrazole ring in compounds 5(a-h) exhibited
typical ABX spin and were of diastereotopic nature. For instance, in 1H NMR spectrum, the 4-Ha
proton of 5f appeared as doublet of doublet at δ 3.361 (J=4.8, 17.6Hz) ppm; whereas, 4-Hb
proton appeared as doublet of doublet at δ 3.712 (J=5.2, 11.2Hz) ppm, respectively. Instead of
appearing as a triplet, 5-H resonates with both 4-Ha and 4-Hb and appears as doublet of doublet
at δ 5.765 (J=4.8, 11.6Hz) ppm. The singlets appearing at δ 2.243, 3.827 and 5.535 ppm were
due to CH3, OCH3 and NH2 protons. Array of signals appearing as multiplets in the region δ
6.730-7.030 and δ 7.206-7.322 ppm were unambiguously assigned to thiophene and aromatic
ring protons.
In the 13C NMR spectrum, compound 5f showed signals at δ 13.91, 55.36 and 159.76
ppm due to CH3, OCH3 and C=O carbons. The C-4, C-5 and C-3 carbons of newly formed
pyrazole ring showed signals correspondingly at δ 42.71, 54.50 and 151.68 ppm. The appearance
of signals for C-4 and C-5 in this region confirms the formation of pyrazoline ring. Aromatic
carbons showed the signals in the region δ 111.39-155.26 ppm. In mass spectrum, compound 5f
showed m/z peak at 315.10 corresponding to its molecular mass. All compounds of the
13

synthesized series 5(a-h) showed similar and consistent pattern signals in their respective spectra
and showed satisfactory elemental analyses compared with theoretical values, which strongly
favours the formation of the designed products. Further, amongst the series, the structure of 5f
was confirmed by single crystal x-ray diffraction studies. Earlier, we had reported the synthesis,
spectral and crystallographic studies of compounds 5e,27 and 5h.28
4.2
Crystallography
The molecular structure of the compound 3-(2-methoxyphenyl)-5-(3-methylthiophen-2-
yl)-4,5-dihydro-1H-pyrazole-1-carboxamide 5f (CCDC#: 1556966) was confirmed by single
crystal X-ray diffraction analysis. The crystal structure of 5f is triclinic with a space group of P-1
and contains pyrazole ring as a central core with three substitutions. Complete crystallographic
data refinement details of compound 5f are given in Table 1. The ORTEP of the molecule with
thermal ellipsoids drawn at 50% probability is shown in Fig. 2.
Table 1: Crystal data and structure refinement details of compound 5f.
Parameter
CCDC deposit No.
Empirical formula
Formula weight
Value
CCDC 1556966
C16H17N3O2S
315.40
Temperature
293 (2) K
Wavelength
0.71073Å
Crystal system, space group
Unit cell dimensions
Triclinic, P-1
a = 7.681(3) Å; b = 8.346(3) Å ; c = 13.149(5) Å
α = 103.609(7)o ; β = 103.170(12)o ; γ = 97.531(13)o
Volume
782.4(5) Å3
Z
2
Density(calculated)
Absorption coefficient
F000
1.339 Mg m −3
0.217 mm −1
332
Crystal size
0.23 × 0.34 × 0.27 mm
14

θ range for data collection
3.31o to 27.47o
Index ranges
−9 ≤ h ≤ 6 ; −10 ≤ k ≤ 10 ; −17 ≤ l ≤ 16
Reflections collected
Independent reflections
Absorption correction
Refinement method
4565
3476 [R int = 0.0664]
multi-scan
Full matrix least-squares on F2
Data / restraints / parameters 3476 / 0 / 201
Goodness-of-fit on F 2
Final [I > 2σ(I)]
0.944
R1 = 0.0630, wR2 = 0.1668
R1 = 0.1090, wR2 = 0.2150
0.290 and −0.371 e Å-3
R indices (all data)
Largest diff. peak and hole
Insert Fig.2 here
Fig. 2: ORTEP of the molecule 5f with thermal ellipsoids drawn at 50% probability.
4.3
Anti-inflammatory activity
The results of sPLA2 inhibition studies of synthesized compounds 5(a-h) are tabulated in
Table 2. The tested thienyl-pyrazoline carboxamides 5(a-h), inhibited sPLA2 in dose depended
manner with an IC50 value ranging from 9.88 to 36.10 µM. Amongst series, 5c and 5h showed
significant inhibition against sPLA2 (VRV-PL-8a) with IC50 values of 9.88µM and 10.48 µM,
respectively, when compared to other structurally related molecules.
Table 2: Neutralization of VRV-PL-8a (sPLA2) by compounds 5(a-h)#
sPLA2
Compound
IC50 (μM) ± SEM
14.550 ± 0.132
13.961 ± 0.104
9.880 ± 0.089
36.108 ± 0.253
25.112 ± 0.220
5a
5b
5c*
5d
5e
15

24.072 ± 0.198
18.446 ± 0.135
10.482 ± 0.179
5f
5g
5h*
*denotes the potent ligand; #Results are expressed as mean ± SEM (n=3).
It has been shown that calcium ions are mandatory for the hydrolysis reaction carried out
by sPLA2 by stabilizing the transition state mediated by coordinating the carbonyl group and by
shielding the phosphate oxygen’s negative charge. Enthalpic interactions contributing to calcium
ion binding include conserved aspartic acid residue and carbonyl oxygens of the Tyr and Gly.
Thus, to understand the mechanism of action of inhibition and to gain further insight into the
sight of binding of the inhibitor vis-à-vis substrate and cofactor, detailed kinetic analysis was
undertaken. We carried out the experiments to assess the effect of cofactor calcium ion
concentrations (0-12 mM) (Table 3 and Fig. 3A-B) and substrate-1,2-dimyristoyl-sn-glycero-3-
phosphocholine (DMPC) concentrations (0-400
PL-8a) enzyme inhibition by the compounds 5c and 5h. To understand this, compound 5c and
5h were kept fixed at their respective IC50 concentrations (9.880 M and 10.482 M)
µM) (Table 4 and Fig. 4C-D) on sPLA2 (VRV-
µ
µ
and the calcium ion was varied from 0-12 mM. Fig 3A shows the primary experimental
curves fit to Michaelis-Menten equation at no inhibitor and in the presence of 5c and 5h
respectively.
Table 3: Effect of calcium concentration on sPLA2 (VRV-PL-8a) enzyme inhibition by
compounds 5c (IC50 -9.88 M) and 5h (IC50 -10.48
µ µM) with varying calcium concentrations (0-
12 mM). The results are expressed as mean ± SEM (n=4).
CaCl2
sPLA2
sPLA2-5c sPLA2-5h
(mM)
0
1
0
0
0
0.122
0.061
0.060
16

2
4
0.165
0.212
0.245
0.290
0.324
0.340
0.064
0.098
0.108
0.132
0.150
0.170
0.061
0.101
0.124
0.151
0.165
0.190
6
8
10
12
Table 4: Effect of DMPC concentration (0-400µM) for sPLA2 (VRV-PL-8a) activity in presence
of compounds 5c (IC50 -9.88 M) and 5h (IC50 -10.48
µ µM). The results are expressed as mean ±
SEM (n=4)
sPLA2
0
sPLA2-5c sPLA2-5h
DMPC (µ
0
0
0
60
0.720
0.986
1.140
1.244
1.422
1.582
1.652
0.533
0.660
0.703
0.748
0.789
0.810
0.839
0.449
0.495
0.580
0.655
0.757
0.828
0.866
120
180
240
300
360
400
As can be seen from the set of hyperbolic curves, both 5c and 5h inhibit the enzyme
activity. The nature of inhibition becomes more evident in the double-reciprocal Lineweaver-
Burk plots (Fig. 3B) whereby both the plots show linear mixed-type inhibition. The Linear
regression for 5c intersects with no inhibitor line in the IIIrd quadrant (indicating increased
apparent affinity for calcium of the enzyme leaning towards uncompetitive inhibition) while that
for %h intersects in the IInd quadrant (indicating decreased apparent affinity for calcium of the
enzyme leaning towards competitive inhibition). However, in both the cases Vma x of the enzyme
decreases indicating unambiguous inhibition. This indicated that the inhibitors bind to allosteric
17

site different than the calcium binding site and yet modulating calcium binding. However, the
experiments would have to be performed at more number of inhibitor concentrations and effects
rising due to possible chelation of calcium ions can also be not excluded.
Insert Fig.3 here
Fig. 3: Kinetic analysis of inhibition by 5c and 5h on sPLA2 activity (A) Non-linear curve fitting
of primary calcium titration data (B) Double reciprocal Lineaweaver-Burk Plots for calcium
titration data. (C) Non-linear curve fitting of primary DMPC substrate titration data (D) Double
reciprocal Lineaweaver-Burk Plots for DMPC substrate titration data
The results of substrate DMPC titrations indicated the same behavior as seen for calcium
ion ( Fig 3C and Fig 3D) possibly suggesting that the binding site of these compounds are distal
than the cofactor and substrate binding site and inhibitor binding can either facilitate substrate
binding ( for 5c) or inhibit substrate binding (for 5h) to sPLA2 (VRV-PL-8a) However, as has
been pointed out above, the experiments would have to be performed at more number of
inhibitor concentrations to enable global nonlinear model fifing and rigorous assessment
mechanism.
4.4
Protein modelling, Ligand-similarity metrics and Docking studies
There are several structures available for Basic phospholipase A2 VRV-PL-VIIIa from
Vipera russelii in the PDB database (UNIPROT ID: P59071). As a first step, we used these
structures for docking studies. However, the results from the above docking analysis prevented
us from assessing the translational potential of these small-molecules as potential intervention
agents for human anti-inflammatory purposes and to compare and contrast the results with the
pyrazole inhibitor reported for human phospholipase A2 (see below for detailed). The aim of the
current exercise was to model a soluble phospholipase A2 with closest similarity to the human
18

homologue. Thus, the protein modelling was performed as follows. The sequence of the human
soluble phospholipase A2 (PDB id: 5G3M) was queried to search for similar sequences in the D.
russelii genome that resulted in acidic phospholipase A2 (A8CG87.1) from D. russelii as the
closest hit (43 % sequence identity and e-value of 3e-26).29 This protein sequence was modelled
on the template 1oz6 with 68.33 % sequence coverage (Global model quality estimate of 0.76).
The quality control parameters of the predicted structure were assessed25 and are as specified in
Fig. 4.
Insert Fig.4 here
Fig. 4: Ramachandran plot of all the residues (left panel) and of general, glycine, proline and
preproline-proline residues (right panel).
Before carrying out detailed docking analysis of the small-molecule with modelled
structure, we wanted to understand possible pharmacophore similarity across the small-
molecules synthesized and their similarity with the closest reported small-molecule inhibitor of
soluble phospholipase A2 reported in literature 5-(5-isopropyl-2-methylthiophen-3-yl)-1H
pyrazole 3-carboxamide.9
Table 5: Comparison of the synthesized molecules by rigid-body and flexible superposition done
using LS-align.30
Mol Ref
Rigid superposition
Flexible superposition
PC Score1 PC Score2 Jaccard RMSDLS PC Score1 PC Score2 Jaccard RMSDLS
Ratio
Ratio
0.9991 (2.7 0.9515 (5.7 0.9524
0.1800
0.0000
0.0000
0.1759
19
0.9991 (2.7 0.9515 (5.7
× 10-5) × 10-5)
0.9965 (2.9 0.9965 (2.9
× 10-5) × 10-5)
1.0000 (2.8 1.0000 (2.8
× 10-5) × 10-5)
0.9407 (7.6 0.9855 (3.7
× 10-5) × 10-5)
5a
(20)
5b
(21)
5c
(21)
5d
5c
(21)
5c
(21)
5c
(21)
5c
0.9524
0.1800
0.0000
0.0000
0.1759
× 10-5)
× 10-5)
0.9965 (2.9 0.9965
(2.9 × 10-5)
1.0000
1.0000
1.0000
0.9545
× 10-5)
1.0000 (2.8 1.0000 (2.8 1.0000
× 10-5) × 10-5)
0.9407 (7.6 0.9855 (3.7 0.9545
× 10-5) × 10-5)

(22)
5e
(22)
(21)
5c
(21)
0.8872
0.9295 (9.2 0.7917
× 10-5)
0.4122
0.3885
0.8879 (1.8 0.9301 (9.1
0.7917
0.7200
0.5377
0.5063
(1.82 × 10-
× 10-4)
× 10-5)
4
)
0.8637
0.9048
0.6538
0.8646
0.9058
(1.36× 10-4)
5f
(22)
5c
(21)
(2.67 × 10-
(1.38 × 10-
(2.63 × 10-
4
4
4
)
)
)
0.8616 (3.1 0.9846 (4.2 0.8750
0.3766
0.2717
0.8616 (3.1 0.9846 (4.2
5g
(24)
5h
5c
(21)
5c
0.8750
0.9130
0.3766
0.2717
× 10-4)
× 10-5)
× 10-4)
× 10-5)
0.8758
0.9592 (6.0 0.9130
× 10-5)
0.8758
0.9592 (6.0
× 10-5)
(2.32 × 10-
(2.32 × 10-
(23)
(21)
4
4
)
)
Ref3
(17)
5c
(21)
0.3637
(0.399462) (0.775864)
0.2944
0.0000
102.5398 0.3637
0.2944
0.0000
102.5398
(0.399462) (0.775864)
1is if normalized by atom number of Query Ligand, 2 is if normalized by atom number of
template Ligand
As can be understood from Table 5, the best hit 5c resembles 5b the closest and distantly
resembles 5e among the ensemble of synthesized compounds. However, compound 5c shares no
similarity, whatsoever, with the only know pyrazole compound 5-(5-isopropyl-2-
methylthiophen-3-yl)-1H pyrazole 3-carboxamide that has been shown to possess activity against
sPLA2 enzyme.9 This observation enhances the importance of the current work and demonstrates
its novelty.
The obtained high quality model was used to perform in silico docking experiments with
the small-molecules 5c and 5h that showed the most potent IC-50 values for phospholipase A2
inhibition. Docking analysis with 5b was not performed because of its high similarity to 5c.
SWISS-DOCK was used to perform the docking with the calcium bound structure. Explicit
hydrogens were added to the ligands employing OpenBabel,31 and the structures were minimized
with Chimera. Table 6 summarizes the docking parameters of the top hits for molecule 5c and
5h.
Table 6: Summary of the docking parameters for the top pose of small molecules 5c and 5h
20

Ligand
Receptor
Full
Fitness Estimated ∆G
(kcal/mol)
(kcal/mol)
Model 1
Model 1
-737.55
-744.66
-8.34
-8.28
5c
5h
Perusal of Fig. 5(A-D) for the top binding clusters of 5c and 5h point to the fact that they
bind to two distinct allosteric pockets. Fig. 5 A-B shows the top ten poses of the molecule 5c and
5h and Fig. 5C-D shows the surface representation depicting the depth of the pocket
accommodating 5c and 5h, respectively. As is observed from the figure, the top poses of the
small-molecules 5c and 5h with low binding energies (-8 to -7 kcal/mol) all cluster compactly (<
1 Å root mean square deviation) within the predicted docking pocket. It has to be pointed out
that the two distinct pockets to which 5c and 5h bind, are different than either the substrate
binding or the cofactor binding pockets.
Insert Fig.5 here
Fig. 5: Determination of the site of binding for 5c and 5h. Ribbon representation of the protein
with top ten docked poses of the small-molecules in ball and stick representation for (A) 5c and
(B) 5h. To emphasize the binding pocket, surface representation of the protein with small-
molecules in ball and stick representation for (C) 5c and (D) 5h
Careful analysis of the top most poses belonging to cluster 1 for 5h indicates that all of
them bind to a highly conserved pocket on the protein’s surface that corresponds exactly to the
pocket in which the only know pyrazole compound 5-(5-isopropyl-2-methylthiophen-3-yl)-1H
pyrazole 3-carboxamide, which has inhibitory activity on sPLA2 enzyme, binds (PDB ID:
4UY1)9 (Fig. 6A). This could explain the distinct inhibition kinetics observed for this compound
vis-à-vis calcium and substrate DMPC. Just to emphasize, compound 5h shows an inhibition
21

pattern with both calcium and DMPC which resembles a linear mixed-type inhibition whereby
binding of inhibitor makes the enzyme lose both its activity and affinity for the
substrate/cofactor.
Insert Fig.6 here
Fig. 6: Superposition of 4uy1 (purple) with pyrazole ligand TJM (red) (5-(2,5-dimethyl-3-
thienyl)-1h-pyrazole-3-carboxamide) with (A) the modeled structure docked with 5c and the (B)
modeled structure docked with 5h. Calcium ion is shown as sphere (orange).
However, the top poses belonging to cluster 1 for 5c binds to a completely novel, yet
unreported, pocket (Fig. 6B). To the best of our knowledge, this is the first report demonstrating
the utilization of this allosteric pocket by a novel pyrazole family of compounds to inhibit the
enzyme. To reiterate the inhibition mechanism shown by this compound, and to find plausible
rationalization from the docking pose, it would have to be pointed out that compound 5c shows
an inhibition pattern with both calcium and DMPC which resembles a linear mixed-type
inhibition whereby binding of inhibitor makes the enzyme lose its activity but gain increased
affinity for the substrate/cofactor.
The structural insights into the site of binding and the nature of interactions for 5c and 5h
shows that the latter binds to a conserved pocket that is shared by an already reported pyrazole
compound. However, the allosteric site to which 5c binds is very novel and unique and paves the
way for a lot of future investigation that would enable the design of better inhibitors for
phospholipase A2 with better geometrical and charge complementarities with the pocket and
resultant increase in its affinity and potency.
Conclusions
22

In the present work, we developed a new procedure for the synthesis of thiophene-
pyrazole conjugates from various thienyl chalcones using a recyclable Amberlyst-15 catalyst.
The method is a simple and reliable approach towards the synthesis of pyrazoles, Amberlyst-15
was developed as new efficient reusable heterogeneous catalyst for the synthesis of pyrazole
derivatives. Synthesized compounds were characterized by spectral and crystallographic studies.
In vitro studies on anti-inflammatory activity of the synthesized compounds, 5(a-h) shows that;
amongst the series, compounds 5c, and 5h exhibit excellent VRV-PL-8a inhibition activities.
The thienyl, chlorophenyl and methylenedioxyphenyl substitution on pyrazole ring is posited to
be the key feature for the biological potency of the synthesized compounds. The results of the
study pave way for the development of new anti-inflammatory drugs.
Conflict of Interest: All authors no conflict of interest including financial, personal or other
relationships with other people or organizations for this article.
Acknowledgments
The authors are grateful to the IOE Instrumentation Facility, Vijnana Bhavana,
University of Mysore, for recording spectra and x-ray diffraction studies.
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