Amberlyst-15 catalyzed synthesis of novel thiophene–pyrazoline derivatives: spectral and crystallographic characterization and anti-inflammatory and antimicrobial evaluation

Article abstract of DOI:10.1007/s11164-018-3501-2

Increasing instances of antimicrobial drug resistance and Inflammation-mediated disorders requires the design and synthesis of new small-molecules with higher affinity and specificity for their potential targets to serve as antibiotics or anti-inflammatory drugs, respectively. The current study presents the synthesis of a series of chalcones, 3(a–h) by the reaction of 3-methylthiophene-2-carbaldehyde, 1 and acetophenones, 2(a–h) by Claisen–Schmidt approach. The chalcones were efficiently transformed into thienyl-pyrazolines, 5(a–h) by their reaction with thiosemicarbazide hydrochloride, 4 in the presence of Amberlyst-15 as a catalyst in acetonitrile at room temperature. Alternatively, the compounds 5(a–h) were prepared by conventional method using acetic acid (40%) medium. Structures were characterized by spectral and single crystal X-ray diffraction studies. Preliminary assessment of the anti-inflammatory properties of the compounds showed that, amongst the series, compounds 5b and 5c have excellent anti-inflammatory activities. Further, compound 5c showed excellent activity against Escherichia coli (MIC, 15?μg/mL), Bacillus subtilis (MIC, 20?μg/mL), Aspergillus niger (MIC, 20?μg/mL), and Aspergillus flavus (MIC 15?μg/mL), respectively. Compounds 5a and 5b were also found to be active against the tested microorganisms.

Full text of DOI:10.1007/s11164-018-3501-2

Res Chem Intermed
https://doi.org/10.1007/s11164-018-3501-2
Amberlyst-15 catalyzed synthesis of novel thiophene–
pyrazoline derivatives: spectral and crystallographic
characterization and anti-inflammatory
and antimicrobial evaluation
Malledevarapura Gurumurthy Prabhudeva1 Karthik Kumara2
•
•
Achutha Dileep Kumar1 Mylarappa B. Ningappa3
Neratur K. Lokanath2 Kariyappa Ajay Kumar1
•
•
•
Received: 21 March 2018 / Accepted: 4 June 2018
Ó Springer Nature B.V. 2018
Abstract Increasing instances of antimicrobial drug resistance and Inflammation-
mediated disorders requires the design and synthesis of new small-molecules with
higher affinity and specificity for their potential targets to serve as antibiotics or
anti-inflammatory drugs, respectively. The current study presents the synthesis of a
series of chalcones, 3(a–h) by the reaction of 3-methylthiophene-2-carbaldehyde, 1
and acetophenones, 2(a–h) by Claisen–Schmidt approach. The chalcones were
efficiently transformed into thienyl-pyrazolines, 5(a–h) by their reaction with
thiosemicarbazide hydrochloride, 4 in the presence of Amberlyst-15 as a catalyst in
acetonitrile at room temperature. Alternatively, the compounds 5(a–h) were pre-
pared by conventional method using acetic acid (40%) medium. Structures were
characterized by spectral and single crystal X-ray diffraction studies. Preliminary
assessment of the anti-inflammatory properties of the compounds showed that,
amongst the series, compounds 5b and 5c have excellent anti-inflammatory activ-
ities. Further, compound 5c showed excellent activity against Escherichia coli
(MIC, 15 lg/mL), Bacillus subtilis (MIC, 20 lg/mL), Aspergillus niger (MIC,
20 lg/mL), and Aspergillus flavus (MIC 15 lg/mL), respectively. Compounds 5a
and 5b were also found to be active against the tested microorganisms.
Keywords Anti-inflammatory Á Antimicrobial Á Chalcone Á Condensation Á
Carbothioamide
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11164-
018-3501-2) contains supplementary material, which is available to authorized users.
&
Kariyappa Ajay Kumar
ajaykumar@ycm.uni-mysore.ac.in
1
2
3
Department of Chemistry, Yuvaraja’s College, University of Mysore, Mysore 570005, India
Department of Studies in Physics, Manasagangotri, University of Mysore, Mysore, India
Rangos Research Center, University of Pittsburgh, Pittsburgh, PA 15201, USA
123

M. G. Prabhudeva et al.
Abbreviations
TLC
Thin layer chromatography
Charge couple device
CCD
CIF
Crystallographic information file
Cambridge Crystallographic Data Centre
Nuclear magnetic resonance
CCDC
NMR
UPLC
ESI
APCI
Ultimate performance liquid chromatography
Electrospray
Atmospheric pressure chemical ionization
Microbial type culture collection
VRV-PL-8a Vipera russelli (Russell’s viper) snake venom phospholipase A2
MTCC
RFU
Relative Fluorescence Unit
Introduction
Inflammation is a normal response of both the innate and adaptive immune systems
whenever the host faces challenge in terms of infection [1]. However, when
uncontrolled, inflammation can cause autoimmune/autoinflammatory disorders
leading to yet greater conditions such as neurodegenerative disease and cancer.
Microbial infections are implicated in inflammation whereby they trigger a
persistent and aggressive immune response to antigens/adjuvants secreted by them
and against normal human microbiota. In spite of the availability of effective anti-
inflammatory agents there is a constant need for newer and safer small-molecules
with inflammation countering properties. Despite numerous attempts to search for
more effective antimicrobial and anti-inflammatory agents, pyrazoles still remain as
the scaffold of choice in bioorganic chemistry. Amongst, the various routes
available in the literature, most commonly employed are: highly regioselective
synthesis of phosphonyl/sulphonyl pyrazoles by the reaction of chalcones with an
Bestmann Ohira Reagent [2], 1.3-dipolar cycloaddition reaction of hydrazones to
alkenes/alkynes [3], base catalyzed reaction of N-arylbenzamidrazones with
2-(ethoxymethylene)malononitriles [4], and the cyclocondensation reaction of
phenylhydrazine hydrochlorides to chalcones [5].
In recent years, heterogeneous catalysis has played a central role in various
organic transformations [6, 7]. Amberlyst-15, a macro reticular polystyrene based
ion exchange resin with strongly acidic sulfonic group that facilitates its utility as an
excellent source of strong acid, has been used in various acid catalyzed reactions in
organic synthesis. The growing interest in the use of Amberlyst-15, a heterogeneous
catalyst, is mainly due to its role in mild and highly selective transformations/
synthesis, and the fact that the catalyst can be regenerated and used several times
[8]. For instance, recently it was successfully employed for the synthesis of
quinazolinones [9].
Further, it is emphasized here that pyrazoles are ubiquitous scaffolds and are
regarded as promising molecules with potential applications in medicinal chemistry.
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Amberlyst-15 catalyzed synthesis of novel thiophene…
Pyrazoles were known to exhibit anti-inflammatory [10], antimicrobial and
antioxidant [11], anesthetic [12], analgesic [13], antioxidant [14], antipsychotic
[15], antidepressant and anticonvulsant [16], activities. In view of wide range of
synthetic and biological applications of pyrazoles, we herein report the synthesis of
thienyl-pyrazole derivatives and the results of their in vitro evaluation for anti-
inflammatory and antimicrobial activities. The demonstrated protocol paves the way
for future efforts at synthesizing derivatives of pyrazoles that could find widespread
applications in medicinal chemistry.
Experimental
Materials and methods
Melting points were determined by an open capillary tube method and are
1
uncorrected. H NMR and 13C NMR spectra were recorded on Agilent-NMR 400
and 125 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 elemental
analyzer.
Synthesis of chalcones, 3(a–h)
To a solution mixture of 3-methylthiophene-2-carbaldehyde, 1 (5 mmol) and
acetophenones, 2(a–h) (5 mmol) in methyl alcohol, potassium hydroxide solution
(40%, 2 mL) was added. Then the solution mixture was stirred at room temperature
for 2–3 h. The progress of the reaction was monitored by TLC. After the
completion, the reaction 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 the compounds 3(a–h).
Synthesis of 5-aryl-3-(3-methylthiophen-2-yl)-4,5-dihydro-1H-pyrazole-
carbothioamides, 5(a–h)
Method A Mixture of chalcones 3(a–h) (5 mmol), thiosemicarbazide hydrochloride
4 (10 mmol) and Amberlyst-15 (10%, w/w) in acetonitrile (25 mL) was stirred at
room temperature for 30–60 min. The progress of the reaction was monitored by
TLC. After completion of the reaction, the separated solid was filtered, washed by
diethyl ether (2 9 20 mL), dried and treated with ethyl acetate (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 ethyl acetate, dried and reused effectively
for four subsequent reactions.
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M. G. Prabhudeva et al.
Method B A solution mixture of chalcones, 3(a–h) (5 mmol) and thiosemicar-
bazide hydrochloride, 4 (10 mmol) in acetic acid (40%) was refluxed on a water
bath for 3–4 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–h) in good yields.
X-ray crystallography
A defect free single crystal of approximate dimension 0.25 9 0.27 9 0.30 mm3
was chosen for X-ray diffraction studies. X-ray intensity data for the title compound
were collected at temperature 293 K on a Bruker Proteum2 CCD diffractometer
with X-ray generator operating at 45 kV and 10 mA, using CuKa radiation of
˚
wavelength 1.54178 A. Data were collected for 24 frames per set with different
settings of u (0° and 90°), keeping the scan width of 0.5°, exposure time of 5 s, the
sample to detector distance of 45.10 mm. Parameters in CIF format are available as
Electronic Supplementary Publication from Cambridge Crystallographic Data
Centre.
Anti-inflammatory activity
Inflammation is initiated by manifold cues such as microbial or viral pathogens,
autoimmune defects, tissue damage due to oxidative challenge etc. COX-2 has been
the main target till date for which anti-inflammatory agents have been designed.
However, inhibiting the enzyme soluble phospholipase A2 (sPLA2) which is an
upstreame target in the inflammation pathway, will result in substrate depletion for
COX-2, thereby bringing down the inflammation, as there will be no pro-
inflammatory and inflammatory Prostaglandins (PG) [17]. In this context, we
assessed the inhibitory potential of the newly synthesized thiophene–pyrazoline
derivatives 5(a–h) to block the upstream enzyme sPLA2, rather than LOX and
COX-2. This approach will result in multiple enzyme targets within a pathway
resulting in combinatorial administration of drugs in order to increase the potency
and efficacy of blocking the pathway.
Purification of sPLA2 (VRV-PL-8a) from V. russelli venom
sPLA2 (VRV-PL-8a) from Vipera russelli venom was purified to homogeneity by
reported procedure [18], and the protein was estimated by Lowry’s method [19].
Briefly, V. russelli venom (80 mg) was fractionated on pre-equilibrated Sephadex
G-75 column (1.5 9 160 cm) using 50 mM phosphate buffer pH 7.0. The protein
was resolved into major three peaks. The second peak, constituting about 30% of the
total protein, showed major sPLA2 activity. This sPLA2 peak fraction was
lyophilized and subjected further to cation exchange chromatographic fractionation
employing
(1.5 9 45 cm). The fractions were eluted stepwise using phosphate buffers of
a
pre-equilibrated Carboxymethyl-Sephadex C-25 column
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Amberlyst-15 catalyzed synthesis of novel thiophene…
varied ionic strength (50–200 mM) and pH (7.0–8.0). They were resolved into two
fractions labeled as V and VIII respectively. The lyophilized fraction VIII was next
subjected to Sephadex G-50 column (0.75 9 40 cm) chromatography and eluted
using 50 mM phosphate buffer pH 7.0 and obtained peak was checked for sPLA2
activity.
In vitro inhibition of VRV-PL-8a by thiophene-pyrazole conjugates, 5(a–h)
In vitro inhibition of sPLA2 (VRV-PL-8a) by the synthesized pyrazole derivatives,
5(a–h), was assessed by known procedure [20]. Briefly, a 50 lL activity buffer
containing 50 mM Tris–HCl buffer pH 7.5, 10 mM CaCl2 and 100 lM substrate
stock (1 mM DMPC in methanol containing 2 mM Triton X-100 in Milli-Q water)
were added and incubated for 5 min at 37 °C. Activity was initiated by adding
10 ng of sPLA2 alone or pre incubated with different concentration of thiophene-
pyrazole conjugates 5(a–h) ranging from 0 to 100 lM for 5 min at 37 °C. Reaction
mixtures were incubated for 45 min at 37 °C. 50 lL of quenching solution was
added at a final concentration of 2 mM NaN3, 50 lM ANS and 50 mM EGTA,
vortexed for 30 s and incubated for 5 min at RT. 2 lL of this solution was pipetted
to measure RFU in a Nanodrop ND3300 Ver 2.8 using an excitation UV-LED
(370 10 nm) and emission was recorded at 480 nm in dark condition. Enzyme
activity was calculated using the equation;
DRFU ¼ RFU ðcontrolÞ ¼ RFU ðtestÞ;
where DRFU 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 curve to determine the sPLA2 activity in the presence of
inhibitors.
Effect of substrate and calcium concentration on VRV-PL-8a
0.1 mL of the reaction mixture containing 10 mg of sPLA2 alone or the inhibitor
(test compounds) concentration at their respective IC50 in 50 mM Tris–HCl buffer
pH 7.5, 10 mM CaCl2 and 10 lL of varied substrate stock (0–400 lM) was used for
sPLA2 assay to check the effect of substrate in presence of test compounds. 50 lL
of quenching solution was added at final concentration of 50 lM ANS, 2 mM
sodium azide (NaN3) and 50 mM (ethylene glycol-bis(b-aminoethyl ether)-
N,N,N0,N0-tetraacetic acid) (EGTA) vortexed for 30 s and incubated for 5 min at
RT. 2 lL of this solution was pipetted to measure Relative Fluorescence Unit
(RFU). Similar set of experiment was conducted, where in reaction mixture
containing IC50 concentration of inhibitor (test samples) in 50 mM Tris–HCl buffer,
pH 7.5, 10 ng of sPLA2, 100 lM substrate and varied concentration of CaCl2
(0–12 mM). Reaction was terminated and RFU was measured as described above.
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M. G. Prabhudeva et al.
Antimicrobial activity
The antimicrobial activities of the compounds 5(a–h) were determined as minimum
inhibitory concentrations (MIC) by serial dilution method [21, 22]. The antibacterial
tests were conducted against bacterial pathogens such as Escherichia coli (MTCC
1687), Bacillus subtilis (MTCC 441) and Staphylococcus aureus (MTCC 737). The
antifungal activity was evaluated against fungal strains Aspergillus niger,
Aspergillus flavus and Candila albicans (MTCC 227). The antibiotics ciprofloxacin
and nystatin were used as positive control against bacterial and fungal species,
respectively. Dimethyl sulfoxide was used as solvent control. The experiments were
carried out in triplicate; the results were taken as a mean standard deviation (SD).
Results and discussion
Synthesis and characterization of compounds, 3(a–h) and 5(a–h)
In search of new anti-inflammatory and antimicrobial agents, our strategic aim was
to synthesize thienyl-pyrazole carbothioamides. In this context, initially, the
intermediate 1-(aryl)-3-(3-methylthiophen-2-yl)prop-2-en-1-ones, 3(a–h) were syn-
thesized by base catalyzed reaction of 3-methylthiophene-2-carbaldehyde 1, with
substituted acetophenones, 2(a–h) in methyl alcohol according to our earlier
procedure [23]. Then the reaction of chalcones, 3(a–h) and thiosemicarbazide
hydrochloride 4, in the presence of catalyst Amberlyst-15 (10%, w/w) in acetonitrile
under stirring conditions at room temperature produced thienyl-pyrazole carboth-
ioamides, 5(a–h) in good yields. The target compounds, 5(a–h) were also
synthesized by conventional method in acetic acid (40%) under reflux conditions,
which paves way for understanding the catalytic activity of Amberlyst-15
(Scheme 1).
It was observed that, Amberlyst-15 mediated synthesis requires lesser reaction
time, and occurs at room temperature in comparison to conventional method which
required thermal energy and more reaction time. Interestingly, yields of the products
obtained were almost same and/or up to ? 4% more in comparison with the
conventional heating. More importantly, Amberlyst-15 was recoverable with the
1
solvent ethyl acetate, and is efficiently reused for four consecutive experiments. H
NMR, 13C NMR, MS studies and elemental analysis provided the structural proof
for the compounds 5(a–h). Further, amongst the series, the structure of compound
5h was confirmed by single crystal X-ray diffraction studies.
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
cycloaddition between chalcones and thiosemicarbazide hydrochloride to form
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
1
diastereotopic nature. For instance, in H NMR spectra, the 4-Ha proton of 5h
appears as doublet of doublet at d 3.110 (J = 3.2, 17.6 Hz) ppm; whereas, 4-Hb
proton appears as doublet of doublet at d 3.792 (J = 11.2, 17.6 Hz) ppm,
123

Amberlyst-15 catalyzed synthesis of novel thiophene…
Scheme 1 Schematic diagram for the synthesis of thienyl-pyrazoline carbothioamides, 5(a–h)
respectively. Instead of appearing as a triplet, 5-H resonates with both 4-Ha and
4-Hb and appears as doublet of doublet at d 6.099 (J = 3.6, 11.2 Hz) ppm. The
singlets appearing at d 2.223, 6.069, and 7.980 ppm were due to CH3, OCH2O and
NH2 protons, respectively. Array of signals appearing as multiplet in the region d
7.160–7.246 and doublets at d 6.732 ppm and d 7.608 ppm were due to thiophene
and aromatic ring protons.
In the 13C NMR spectrum, compound 5h showed signals at d 18.63, 106.56 and
160.13 ppm due to CH3, OCH2O and C=S carbons. The C-4, C-5 and C-3 carbons
of newly formed pyrazole ring showed signals correspondingly at d 47.30, 59.35
and 155.48 ppm. The appearance of signals for C-4 and C-5 in this region confirms
the formation of non-aromatic pyrazoline ring. The signals due to other carbons
appear in the aromatic region. Furthermore, in mass spectrum, compound 5h
showed m/z peaks at 345.07 corresponding to its molecular mass. All compounds of
the series 5(a–h) showed signals with similar patterns. All showed satisfactory
elemental analyses compared with theoretical values, which strongly favour the
formation of the products.
Further, amongst the series, the structure of the compound 3-(benzo[d][1,3]-
dioxol-5-yl)-5-(3-methylthiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carbothioamide
5h was confirmed by single crystal x-ray diffraction studies. The crystal data and the
structure refinement details are given in Table 1. There were two molecules (A and
B) in the asymmetric unit of the crystal. The ORTEP of the molecule A and
molecule B with displacement ellipsoids drawn at 50% probability level is shown in
Figs. 1 and 2 respectively. The hydrogen bonding geometry is listed in Table S1,
and the packing of molecules when viewed down along c axis is shown in Fig. S1.
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M. G. Prabhudeva et al.
Table 1 Crystal data and structure refinement details of the molecule, 5h
CCDC Number
1557004
C16H15N3O2S2
Empirical formula
Formula weight
345.45
296 K
Temperature
˚
1.54178 A
Wavelength
Crystal system, space group
Unit cell dimensions
Triclinic, P - 1
˚
a = 9.6550(3) A; b = 13.2331(4) A; c = 14.0232(4) A
˚
˚
a = 66.164(2)°; b = 89.317(2)°; c = 81.675(2)°
3
1619.44(9) A /4
˚
Volume/Z
Density (calculated)
Absorption coefficient
F000
1.417 Mg/m3
3.091 mm-1
720
Crystal size
0.25 9 0.30 9 0.27 mm
h range for data collection
Index ranges
5.64°–64.53°
- 11 B h B 10; - 15 B k B 15; - 16 B l B 16
22,251
Reflections collected
Independent reflections
Absorption correction
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
Final [I [ 2r(I)]
5372 [R int = 0.0662]
Multi-scan
Full matrix least-squares on F2
5372/0/417
1.027
R1 = 0.0602, wR2 = 0.1706
R1 = 0.0777, wR2 = 0.1887
R indices (all data)
Largest diff. peak and hole
3
˚
0.375 and - 0.346 e/A
The dotted line represents hydrogen bond interactions. The bridging of molecules
through N–HÁÁÁS hydrogen bond interactions between amidogen and sulphur
elements results in an R22 (8) ring motif is shown in Fig. S2. The molecular structure
is comparable with the molecule reported earlier [24, 25].
Analytical data of compounds, 3(a–h), 5(a–h)
3-(3-Methylthiophen-2-yl)-1-phenylprop-2-en-1-one, 3a Obtained from 1 (1.26 g,
1
10 mol) and 2a (1.20 g, 10 mmol) in 76% yield (brownish semisolid); H NMR
(CDCl3, d ppm): 7.32 (d, 1H, J = 15.5 MHz, CH=), 7.74–7.85 (m, 7H, Ar–H), 7.98
(d, 1H, J = 16.4 MHz, =CH); 13C NMR (CDCl3, d ppm): 125.6 (1C), 130.1 (1C),
131.6 (1C), 128.6 (2C), 129.8 (2C), 133.5 (1C), 134.3 (1C), 135.0 (1C), 136.7 (1C),
150.2 (1C), 188.5 (1C, C=O). MS m/z: 228.02 (M?, 100); Anal. Calcd. for
C14H12OS (%): C, 73.65; H, 5.30. Found: C, 73.52; H, 5.27.
1-(4-Fluorophenyl)-3-(3-methylthiophen-2-yl)prop-2-en-1-one, 3b Obtained from
1 (1.26 g, 10 mol) and 2b (1.38 g, 10 mmol) in 89% yield, (brackish semisolid); 1H
NMR (CDCl3, d ppm): 6.98 (dd, 2H, J = 12.0, 3.1 MHz, Ar–H), 7.31 (d, 1H,
J = 15.9 MHz, CH=), 7.46 (dd, 2H, J = 12.3, 2.4 MHz, Ar–H), 7.73–7.92 (m, 2H,
123

Amberlyst-15 catalyzed synthesis of novel thiophene…
Fig. 1 ORTEP of the molecule A with thermal ellipsoids drawn at 50% probability
Fig. 2 ORTEP of the molecule B with thermal ellipsoids drawn at 50% probability
Ar–H), 8.09 (d, 1H, J = 16.0 MHz, =CH); 13C NMR (CDCl3, d ppm): 115.6 (1C),
115.6 (1C), 123.8 (1C), 128.9 (1C), 129.8 (1C), 129.9 (1C), 131.4 (1C), 134.9 (1C),
139.8 (1C), 136.6 (1C), 140.2 (1C), 160.6 (1C), 188.4 (1C, C=O). MS m/z: 246.05
(M?, 100); Anal. Calcd. for C14H11FOS (%): C, 68.27; H, 4.50. Found: C, 68.20; H,
4.47.
1-(4-Chlorophenyl)-3-(3-methylthiophen-2-yl)prop-2-en-1-one, 3c Obtained from
1
1 (1.26 g, 10 mmol) and 2c (1.54 g, 10 mmol) in 86% yield, m.p. 89–91 °C; H
NMR (CDCl3, d ppm): 2.35 (s, 3H, CH3), 6.87 (d, 1H, J = 2.8 MHz, CH=),
123

M. G. Prabhudeva et al.
7.11–7.27 (m, 2H, Ar–H), 7.42 (d, 2H, J = 7.2 MHz, Ar–H), 7.92 (d, 2H,
J = 7.2 MHz, Ar–H), 8.01 (d, 1H, J = 15.2 MHz, =CH); 13C NMR (CDCl3, d ppm):
14.2 (1C), 119.0 (1C), 126.4 (1C), 127.6 (1C), 128.3 (1C), 128.8 (1C), 129.7 (1C),
131.4 (1C), 134.3 (1C), 136.0 (1C), 136.5 (1C), 139.0 (1C), 143.1 (1C), 188.3 (1C,
C=O). MS m/z: 265.07 (MH?, 37Cl, 41), 263.07 (MH?, 35Cl, 100); Anal. Calcd. for
C14H11ClOS (%): C, 64.00; H, 4.50. Found: C, 59.83; H, 4.47.
1-(4-Methoxyphenyl)-3-(3-methylthiophen-2-yl)prop-2-en-1-one,
3d Obtained
from 1 (1.26 g, 10 mmol) and 2d (1.50 g, 10 mmol) in 86% yield, pale yellow
oily mass; 1H NMR (CDCl3, d ppm): 2.33 (s, 3H, CH3), 3.80 (s, 3H, OCH3), 6.84 (d,
1H, J = 5.2 MHz, CH=), 6.92 (d, 2H, J = 8.8 MHz, Ar–H), 7.22–7.27 (m, 2H, Ar–
H), 7.96–7.97 (m, 2H, Ar–H), 8.00 (d, 1H, J = 3.2 MHz, =CH); 13C NMR (CDCl3,
d ppm): 14.0 (1C), 55.4 (1C), 112.7 (1C), 119.0 (1C), 119.7 (1C), 120.8 (1C), 127.3
(1C), 129.5 (1C), 131.4 (1C), 135.5 (1C), 139.6 (1C), 142.7 (1C), 159.8 (1C), 189.4
(1C, C=O). MS m/z: 259.12 (MH?, 100); Anal. Calcd. for C15H14O2S (%): C,
69.74; H, 5.46. Found: C, 69.63; H, 5.43.
1-(3-Methoxyphenyl)-3-(3-methylthiophen-2-yl)prop-2-en-1-one, 3e Obtained
from 1 (1.26 g, 10 mmol) and 2e (1.50 g, 10 mmol) in 87% yield, (light brown
1
semisolid); H NMR (CDCl3, d ppm): 2.36 (s, 3H, CH3), 3.87 (s, 3H, OCH3), 6.89
(d, 1H, J = 4.6 MHz, CH=), 7.10–7.41 (m, 4H, Ar–H), 7.52–7.59 (m, 2H, Ar–H),
8.03 (d, 1H, J = 14.7 MHz, =CH). MS m/z: 259.07 (M?, 100); Anal. Calcd. for
C15H14O2S (%): C, 69.74; H, 5.46. Found: C, 69.65; H, 5.42.
1-(2-Methoxyphenyl)-3-(3-methylthiophen-2-yl)prop-2-en-1-one, 3f Obtained
from 1 (1.26 g, 10 mmol) and 2f (1.50 g, 10 mmol) in 86% yield, m.p.
1
113–115 °C; H NMR (CDCl3, d ppm): 2.38 (s, 3H, CH3), 3.87 (s, 3H, OCH3),
6.89 (d, 1H, J = 4.8 MHz, CH=), 7.10–7.42 (m, 4H, Ar–H), 7.53–7.59 (m, 2H, Ar–
H), 8.03 (d, 1H, J = 14.8 MHz, =CH); 13C NMR (CDCl3, d ppm): 14.2 (1C), 55.4
(1C), 113.6 (1C), 113.7 (1C), 119.5 (1C), 126.9 (1C), 130.6 (2C), 131.1 (1C), 131.3
(1C), 134.6 (1C), 134.7 (1C), 142.3 (1C), 163.3 (1C), 187.9 (1C, C=O). MS m/z:
259.12 (MH?, 100); Anal. Calcd. for C15H14O2S (%): C, 69.74; H, 5.46. Found: C,
69.60; H, 5.43.
1-(3,4-Dimethoxyphenyl)-3-(3-methylthiophen-2-yl)prop-2-en-1-one, 3g Ob-
tained from 1 (1.26 g, 10 mmol) and 2g (1.80 g, 10 mmol) in 90% yield, m.p.
1
93–95 °C; H NMR (CDCl3, d ppm): 2.34 (s, 3H, CH3), 3.85 (s, 6H, OCH3), 6.88
(d, 1H, J = 4.8 MHz, CH=), 7.08–7.42 (m, 3H, Ar–H), 7.55–7.59 (m, 2H, Ar–H),
8.04 (d, 1H, J = 14.2 MHz, =CH). MS m/z: 288.08 (M?, 100); Anal. Calcd. for
C16H16O3S (%): C, 66.64; H, 5.59. Found: C, 66.49; H, 5.54.
1-(Benzo[d][1,3]dioxol-5-yl)-3-(3-methylthiophen-2-yl)prop-2-en-1-one, 3h Ob-
tained from 1 (1.26 g, 10 mmol) and 2h (1.64 g, 10 mmol) in 80% yield, m.p.
1
111–113 °C; H NMR (CDCl3, d ppm): 2.38 (s, 3H, CH3), 6.05 (s, 2H, OCH2O),
6.87–6.89 (m, 2H, Ar–H), 7.20–7.28 (m, 2H, Ar–H), 7.51 (s, 1H, Ar–H), 7.62 (d,
1H, J = 8.0 MHz, CH=), 8.00 (d, 1H, J = 14.2 MHz, =CH); 13C NMR (CDCl3, d
ppm): 13.6 (1C), 101.2 (1C), 107.4 (1C), 118.7 (1C), 123.7 (1C), 126.4 (1C), 130.7
123

Amberlyst-15 catalyzed synthesis of novel thiophene…
(1C), 132.4 (1C), 133.3 (1C), 133.9 (1C), 134.4 (1C), 141.9 (1C), 147.6 (1C), 150.9
(1C), 186.9 (1C, C=O). MS m/z: 273.11 (MH?, 100); Anal. Calcd. for C15H12O3S
(%): C, 66.16; H, 4.44. Found: C, 66.04; H, 4.40.
5-(3-Methylthiophen-2-yl)-3-phenyl-4,5-dihydro-1H-pyrazole-1-carbothioamide,
5a Obtained from 3-(3-methylthiophen-2-yl)-1-phenylprop-2-en-1-one, 3a
(1.14 g, 5 mmol) and thiosemicarbazide hydrochloride, 4 (1.11 g, 10 mmol) in
72% yield, m.p. 151–153 °C; 1H NMR (CDCl3, d ppm): 2.26 (s, 3H, CH3), 3.37 (dd,
1H, J = 4.9, 6.8 Hz, C4-Ha), 3.84 (s, 3H, OCH3), 3.89 (dd, 1H, J = 11.2, 17.7 Hz,
C4-Hb), 5.44 (s, 2H, NH2), 5.73 (dd, 1H, J = 4.9, 8.6 Hz, C5-H), 6.78–7.01 (m, 2H,
Ar–H), 7.44–7.68 (m, 5H, Ar–H); 13C NMR (CDCl3, d ppm): 13.2 (1C, CH3), 44.8
(1C, C-4), 54.4 (1C, C-5), 121.5 (1C), 122.2 (1C), 123.9 (1C), 128.6 (1C), 128.6
(1C), 129.0 (1C), 129.1 (1C), 131.3 (1C), 133.3 (1C), 135.5 (1C), 151.7 (1C, C-3),
164.1 (1C, C=S). MS m/z: 301.02 (M?, 100); Anal. Calcd. for C15H15N3S2 (%): C,
59.77; H, 5.02; N, 13.94; Found: C, 59.62; H, 4.98; N, 13.88.
3-(4-Fluorophenyl)-5-(3-methylthiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carboth-
ioamide, 5b Obtained from 1-(4-fluorophenyl)-3-(3-methylthiophen-2-yl)prop-2-
en-1-one, 3b (1.23 g, 5 mmol) and thiosemicarbazide hydrochloride, 4 (1.27 g,
1
10 mmol) in 81% yield, m.p. 146–148 °C; H NMR (CDCl3, d ppm): 2.25 (s, 3H,
CH3), 3.37 (dd, 1H, J = 4.6, 5.9 Hz, C4-Ha), 3.87 (dd, 1H, J = 11.0, 17.5 Hz, C4-
Hb), 5.46 (s, 2H, NH2), 5.73 (dd, 1H, J = 4.7, 9.0 Hz, C5-H), 6.78 (d, 1H, Ar–H),
7.10 (d, 1H, Ar–H), 7.32 (d, 2H, J = 7.8 Hz, Ar–H), 7.74 (d, 2H, J = 7.1 Hz, Ar–
H); 13C NMR (CDCl3, d ppm): 13.8 (1C, CH3), 44.7 (1C, C-4), 53.9 (1C, C-5),
114.5 (2C), 120.1 (1C), 120.8 (1C), 123.3 (1C), 129.3 (2C), 131.1 (1C), 132.7 (1C),
151.7 (1C, C-3), 163.3 (1C), 162.9 (1C, C=S). MS m/z: 319.06 (M?, 100); Anal.
Calcd. for C15H14FN3S2 (%): C, 56.40; H, 4.42; N, 13.16; Found: C, 56.27; H, 4.40;
N, 13.14.
3-(4-Chlorophenyl)-5-(3-methylthiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-carboth-
ioamide, 5c Obtained from 1-(4-chlorophenyl)-3-(3-methylthiophen-2-yl)prop-2-
en-1-one, 3c (1.31 g, 5 mmol) and thiosemicarbazide hydrochloride, 4 (1.27 g,
1
10 mmol) in 88% yield, m.p. 56–57 °C; H NMR (CDCl3, d ppm): 2.25 (s, 3H,
CH3), 3.35 (dd, 1H, J = 4.5, 5.4 Hz, C4-Ha), 3.87 (dd, 1H, J = 11.1, 18.1 Hz, C4-
Hb), 5.43 (s, 2H, NH2), 5.73 (dd, 1H, J = 4.7, 8.8 Hz, C5-H), 6.73 (d, 1H, Ar–H),
7.31 (d, 1H, Ar–H), 7.48 (d, 2H, J = 7.4 Hz, Ar–H), 7.92 (d, 2H, J = 7.1 Hz, Ar–
H); 13C NMR (CDCl3, d ppm): 13.5 (1C, CH3), 45.3 (1C, C-4), 54.3 (1C, C-5),
119.5 (1C), 120.4 (1C), 121.9 (1C), 128.2 (2C), 128.9 (2C), 132.3 (1C), 134.1 (1C),
135.7 (1C), 151.8 (1C, C-3), 164.1 (1C, C=S). MS m/z: 337.05 (M?2, 36.5%),
335.06 (M?, 100); Anal. Calcd. for C15H14ClN3S2 (%): C, 53.64; H, 4.20; N, 12.51;
Found: C, 53.51; H, 4.16; N, 12.49.
3-(4-Methoxyphenyl)-5-(3-methylthiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-car-
bothioamide, 5d Obtained from 1-(4-methoxyphenyl)-3-(3-methylthiophen-2-
yl)prop-2-en-1-one, 3d (1.27 g, 5 mmol) and thiosemicarbazide hydrochloride, 4
(1.27 g, 10 mmol) in 80% yield, m.p. 162–164 °C; 1H NMR (CDCl3, d ppm): 2.26
(s, 3H, CH3), 3.37 (dd, 1H, J = 4.7, 5.5 Hz, C4-Ha), 3.83 (s, 3H, OCH3), 3.92 (dd,
123

M. G. Prabhudeva et al.
1H, J = 11.3, 17.0 Hz, C4-Hb), 5.45 (s, 2H, NH2), 5.72 (dd, 1H, J = 5.0, 8.2 Hz, C5-
H), 6.73 (d, 1H, Ar–H), 7.32–7.78 (m, 5H, Ar–H); 13C NMR (CDCl3, d ppm): 13.6
(1C, CH3), 44.6 (1C, C-4), 54.4 (1C, C-5), 55.5 (1C, OCH3), 113.4 (2C), 120.8 (1C),
121.6 (1C), 123.1 (1C), 128.0 (1C), 128.1 (2C), 133.2 (1C), 151.8 (1C, C-3), 159.3
(1C), 163.1 (1C, C=S). MS m/z: 331.08 (M?, 100); Anal. Calcd. for C16H17N3OS2
(%): C, 57.98; H, 5.17; N, 12.68; Found: C, 57.85; H, 5.14; N, 12.65.
3-(3-Methoxyphenyl)-5-(3-methylthiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-car-
bothioamide, 5e Obtained from 1-(3-methoxyphenyl)-3-(3-methylthiophen-2-
yl)prop-2-en-1-one, 3e (1.27 g, 5 mmol) and thiosemicarbazide hydrochloride, 4
(1.27 g, 10 mmol) in 77% yield, m.p. 118–120 °C; 1H NMR (CDCl3, d ppm): 2.28
(s, 3H, CH3), 3.35 (dd, 1H, J = 6.9, 17.1 Hz, C4-Ha), 3.84 (s, 3H, OCH3), 3.90 (dd,
1H, J = 6.2, 13.2 Hz, C4-Hb), 5.69 (s, 2H, NH2), 5.72 (dd, 1H, J = 6.8, 11.9 Hz, C5-
H), 6.73 (d, 1H, J = 4.0 Hz, Ar–H), 6.92–7.36 (m, 4H, Ar–H), 7.85 (d, 1H,
J = 7.6 Hz, Ar–H); 13C NMR (CDCl3, d ppm): 13.9 (1C, CH3), 45.8 (1C, C-4), 54.4
(1C, C-5), 55.4 (1C, OCH3), 111.5 (1C), 120.6 (1C), 120.8 (1C), 122.2 (1C), 129.0
(1C), 130.1 (1C), 131.3 (1C), 133.3 (1C), 139.4 (1C), 151.9 (1C, C-3), 155.4 (1C),
158.1 (1C, C=S). MS m/z: 331.08 (M?, 100); Anal. Calcd. for C16H17N3OS2 (%):
C, 57.98; H, 5.17; N, 12.68; Found: C, 57.82; H, 5.13; N, 12.66.
3-(2-Methoxyphenyl)-5-(3-methylthiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-car-
bothioamide, 5f Obtained from 1-(2-methoxyphenyl)-3-(3-methylthiophen-2-
yl)prop-2-en-1-one, 3f (1.27 g, 5 mmol) and thiosemicarbazide hydrochloride, 4
(1.27 g, 10 mmol) in 84% yield, m.p. 132–135 °C; 1H NMR (CDCl3, d ppm): 2.28
(s, 3H, CH3), 3.36 (dd, 1H, J = 4.4, 5.2 Hz, C4-Ha), 3.81 (s, 3H, OCH3), 3.88 (dd,
1H, J = 11.6, 17.2 Hz, C4-Hb), 5.40 (s, 2H, NH2), 5.71 (dd, 1H, J = 4.8, 8.0 Hz, C5-
H), 6.73–7.02 (m, 4H, Ar–H), 7.34–7.38 (m, 1H, Ar–H), 7.85 (d, 1H, Ar–H); 13C
NMR (CDCl3, d ppm): 13.9 (1C, CH3), 45.8 (1C, C-4), 54.4 (1C, C-5), 55.4 (1C,
OCH3), 111.5 (1C), 120.6 (1C), 120.8 (1C), 122.2 (1C), 129.0 (1C), 130.1 (1C),
131.3 (1C), 133.3 (1C), 139.4 (1C), 151.9 (1C, C-3), 155.4 (1C), 158.1 (1C, C=S).
MS m/z: 331.08 (M?, 100); Anal. Calcd. for C16H17N3OS2 (%): C, 57.98; H, 5.17;
N, 12.68; Found: C, 57.80; H, 5.12; N, 12.64.
3-(3,4-Dimethoxyphenyl)-5-(3-methylthiophen-2-yl)-4,5-dihydro-1H-pyrazole-1-
carbothioamide, 5g Obtained from 1-(3,4-dimethoxyphenyl)-3-(3-methylthio-
phen-2-yl)prop-2-en-1-one, 3g (1.44 g, 5 mmol) and thiosemicarbazide hydrochlo-
1
ride, 4 (1.27 g, 10 mmol) in 89% yield, m.p. 185–187 °C; H NMR (CDCl3, d
ppm): 2.25 (s, 3H, CH3), 3.35 (dd, 1H, J = 5.4, 6.2 Hz, C4-Ha), 3.82 (s, 6H, OCH3),
3.87 (dd, 1H, J = 11.2, 17.1 Hz, C4-Hb), 5.45 (s, 2H, NH2), 5.72 (dd, 1H, J = 4.9,
8.4 Hz, C5-H), 6.75–6.92 (m, 2H, Ar–H), 7.34–7.48 (m, 3H, Ar–H); 13C NMR
(CDCl3, d ppm): 13.3 (1C, CH3), 43.8 (1C, C-4), 54.5 (1C, C-5), 55.8 (2C, OCH3),
113.6 (1C), 118.6 (1C), 120.1 (1C), 121.1 (1C), 121.3 (1C), 123.7 (1C), 126.8 (1C),
133.1 (1C), 149.4 (1C), 151.8 (1C, C-3), 152.4 (1C), 159.1 (1C, C=S). MS m/z:
361.09 (M?, 100); Anal. Calcd. for C17H19N3O2S2 (%): C, 56.48; H, 5.30; N,
11.62; Found: C, 56.41; H, 5.25; N, 11.57.
123

Amberlyst-15 catalyzed synthesis of novel thiophene…
3-(Benzo[d][1,3]dioxol-5-yl)-5-(3-methylthiophen-2-yl)-4,5-dihydro-1H-pyrazole-
1-carbothioamide, 5h Obtained from 1-(benzo[d][1,3]dioxol-5-yl)-3-(3-methylth-
iophen-2-yl)prop-2-en-1-one, 3h (1.36 g, 5 mmol) and thiosemicarbazide
hydrochloride, 4 (1.27 g, 10 mmol) in 75% yield, m.p. 115–117 °C; 1H NMR
(C6D6, d ppm): 2.22 (s, 3H, CH3), 3.11 (dd, 1H, J = 3.2, 17.6 Hz, 4-Ha), 3.79 (dd,
1H, J = 11.2, 17.6 Hz, 4-Hb), 6.06 (s, 2H, OCH2O), 6.09 (dd, 1H, J = 3.6, 11.2 Hz,
5-H), 6.73 (d, 1H, Ar–H), 7.16–7.24 (m, 2H, Ar–H); 7.60 (d, 1H, Ar–H), 7.98 (s,
2H, NH2); 13C NMR (CDCl3, d ppm): 18.6 (1C, CH3), 47.3 (1C, C-4), 59.3 (1C,
C-5), 106.5 (1C, OCH2O), 111.0 (1C), 113.3 (1C), 126.5 (1C), 127.4 (1C), 130.8
(1C), 135.0 (1C), 137.5 (1C), 145.5 (1C), 152.8 (1C), 153.8 (1C), 155.4 (1C, C-5),
160.1 (1C, C=S). MS m/z: 345.07 (M?, 100); Anal. Calcd. for C16H15N3O2S2 (%):
C, 55.63; H, 4.38; N, 12.16; Found: C, 55.51; H, 4.35; N, 12.10.
Anti-inflammatory activity of compounds, 5(a–h)
The results of sPLA2 inhibition studies of synthesized compounds 5(a–h) are
tabulated in Table 2. The tested 2-pyrazoline derivatives, 5(a–h) inhibited sPLA2 in
dose dependent manner with an IC50 value ranging from 9.110 to 26.220 lM which
are computed and analyzed using sigmoidal Four Parameter Logistic (4PL) curve fit
using the equation; y = min ? (max - min)/(1 ? (x/EC50)(-Hillslope) in which
y = % effect, min = minimum effect, max = maximum effect, x = concentration.
Two constraints were used for fitting the 4PL function: (a) 0.1 \ min \ 1;
(b) max \ 100. Amongst the series, 5b, and 5c showed significant inhibition against
VRV-PL-8a with IC50 value of 10.76 and 9.11 lM respectively (Table 2),
compared to other structurally related molecules.
To understand the mechanism of action of inhibition, we studied the effect of
substrate and calcium concentrations on VRV-PL-8a enzyme inhibition by 5b, and
5c. We examined its inhibition as a function of calcium and substrate concentra-
tions. Compounds 5b at its IC50 concentration (10.76 lM) and 5c (9.11 lM) with
varying calcium (0–12 mM) concentrations, showed no change in sPLA2 activity
(Table 3).
Preliminary study revealed that compounds 5b and 5c were not competing to the
calcium binding site or active site on the VRV-PL-8a enzyme. Inhibition of sPLA2
Table 2 In vitro anti-
Compound
sPLA2
IC50 (lM) SEM
inflammatory activity of thienyl-
pyrazoline carbothioamides,
5(a–h)
5a
5b
5c
5d
5e
5f
14.660 0.132
10.762 0.102
9.110 0.090
18.010 0.165
19.210 0.186
19.488 0.179
26.220 0.224
25.100 0.240
Results are expressed as
mean standard error of
measurement (SEM) (n = 3)
5g
5h
123

M. G. Prabhudeva et al.
Table 3 Effect of substrate and calcium concentrations on VRV-PL-8a enzyme inhibition by compounds
5b (IC50 - 10.76 lM), 5c (IC50 - 9.11 lM) with varying calcium concentrations (0–12 mM)
CaCl2 (mM)
sPLA2
sPLA2-5b
sPLA2-5c
0
0
0
0
1
0.112
0.173
0.210
0.250
0.295
0.325
0.335
0.060
0.085
0.105
0.132
0.165
0.180
0.198
0.066
0.090
0.122
0.147
0.178
0.185
0.204
2
4
6
8
10
12
The results are expressed as mean SEM (n = 3)
by these compounds is independent of calcium concentration and do not chelate
calcium ions required for the enzyme activity.
Antimicrobial activity of compounds, 5(a–h)
The results of antimicrobial activities of the synthesized compounds 5(a–h) are
summarized in Table 4.
All the synthesized series of 2-pyrazoline carbothioamides, 5(a–h) exerted a
wide range of modest to good in vitro antimicrobial activities having MIC’ values
ranging from 10.0 to 120 lg/mL against bacteria and 15.0–110 lg/mL against
fungal species; in particular, they showed lesser inhibitory effect on S. aureus
(90.0–120.0 lg/mL) and C. albicans (80.0–110.0 lg/mL) organisms. Amongst the
series of 2-pyrazolines, 5g having dimethoxy substitutions, and 5h having
Table 4 Minimum inhibitory concentrations (expressed as lg/mL)a of thienyl-pyrazoline carboth-
ioamides 5(a–h) against bacteria and fungi species by serial dilution method
Sample S. aureus
E. coli
B. subtilis
A. niger
A. flavus
C. albicans
5a
110.0 1.00 25.0 0.50 25.0 0.75
120.0 1.50 20.0 0.50 20.0 0.60
25.0 0.50 25.0 0.60
25.0 0.75 25.0 0.75
20.0 0.60 15.0 0.70
70.0 0.75 75.0 0.60
55.0 0.50 50.0 0.40
40.0 0.50 45.0 0.60
90.0 0.50
95.0 0.75
80.0 0.55
110.0 1.25
90.0 1.00
100.0 1.50
5b
5c
90.0 0.75
15.0 0.50 20.0 0.75
5d
120.0 0.50 60.0 1.00 75.0 1.00
110.0 1.00 50.0 0.70 55.0 1.00
5e
5f
90.0 1.25
55.0 0.50 45.0 0.40
5g
120.0 1.50 90.0 1.00 110.0 1.50 90.0 1.50 100.0 1.25 100.0 2.50
5h
100.0 1.50 90.0 1.50 100.0 1.50 90.0 1.25 90.0 1.00
90.0 1.25
–
CYPb
NYSc
20.0 0.50
–
20.0 0.75 30.0 1.00
–
–
–
–
25.0 0.75 25.0 0.25
20.0 0.50
aValues are mean SD of three replicates
bCiprofloxacin—positive control against bacteria species
cNystatin—positive control against fungi species
123

Amberlyst-15 catalyzed synthesis of novel thiophene…
(benzo[d][1,3]dioxol-5-yl) substitution, exhibited lesser activity against all the
organisms tested. Compound 5c showed excellent activity by inhibiting spore
germination of E. coli (MIC:15 lg/mL), B. subtilis (MIC: 20 lg/mL), A. niger
(MIC: 20 lg/mL), and A. flavus (MIC: 15 lg/mL), respectively.
Promising activities were showed by the compounds 5a against E. coli (25 lg/
mL), B. subtilis (25 lg/mL). A. niger (25 lg/mL) A. flavus (25 lg/mL); 5b against
E. coli (20 lg/mL), B. subtilis (20 lg/mL). A. niger (25 lg/mL) A. flavus (25 lg/
mL), which were incidentally similar to those demonstrated by the respective
standard drugs used in the experiment. 2-Pyrazolines 5d, 5e, and 5f having
methoxyphenyl substitutions, showed moderate activity against bacterial pathogens
E. coli (50.0–80.0 lg/mL), B. subtilis (45.0–75.0 lg/mL); and against fungal stains
A. niger (40.0–70.0 lg/mL), A. flavus (40.0–75.0 lg/mL).
Though this study has not attempted understanding the mode of action for this
family of compounds in antimicrobial and antifungal functions, previous reports
with compounds belonging to the same family have speculated that the mode of
antimicrobial and antifungal activities of these compounds could be mediated
through an elevated production of cytotoxic hydroxyl radicals (due to the molecular
properties of the small-molecules) or through the possible inhibition of oxidative
stress protection function(s) more critically needed in rich media capable of constant
synthesis of free oxide radical [26].
Further, the authors have also raised the possibility whereby the small-molecules
bring about their antimicrobial effects by interfering with iron acquisition in a
nutrient limiting environment. These compounds can bind to iron more tightly than
the corresponding bacterial or fungal siderophores, hence sequestering iron under
limiting conditions of this micronutrient. This, in turn, has the potential to reduce
iron bioavailability thus showing potent antimicrobial activity under the iron-
scarcity condition [26].
Conclusions
To sum up, in the present work, we report a new procedure for the synthesis of
thiophene-pyrazole hybrids from chalcones using a recyclable Amberlyst-15
catalyst. The method is an efficient and reliable approach towards the synthesis
of pyrazoles. Synthesized compounds were characterized by spectral and crystal-
lographic studies. Preliminary studies on anti-inflammatory activity of the
synthesized compounds 5(a–h) show that the compounds 5b and 5c exhibit
excellent anti-inflammatory activities. Further, compound 5c showed excellent
activity by inhibiting E. coli (15, 10 lg/mL), B. subtilis (20, 15 lg/mL), A. niger
(20, 20 lg/mL), and A. flavus (15, 15 lg/mL), respectively. Compounds 5a and 5b
were also found to be active against the tested microorganisms. The demonstrated
synthesis paves the way for future efforts at synthesizing novel derivatives of
pyrazoles that could find widespread applications in medicinal chemistry and
helping the efforts aimed at alleviating dreadful diseases caused by microorganism
induced inflammation.
123

M. G. Prabhudeva et al.
Acknowledgements The authors are grateful to the IOE Instrumentation Facility, Vijnana Bhavana,
University of Mysore, for recording spectra and X-ray diffraction studies.
Compliance with ethical standards
Conflict of interest All authors declare no conflict of interest including financial, personal or other
relationships with other people or organizations for this article.
References
1. C.A. Dinarello, Cell 140, 935 (2010)
2. K. Mohanan, A.R. Martin, L. Toupet, M. Smietana, J.-J. Vasseur, Angew. Chem. Int. Ed. 49, 3196
(2010)
3. G.V. Kumar, M. Govindaraju, N. Renuka, B.B.A. Khatoon, B.N. Mylarappa, K.A. Kumar, Rasayan J.
Chem. 5, 338 (2012)
4. S.R. Stauffer, C.J. Coletta, R. Tedesco, G. Nishiguchi, K. Carlson, J. Sun, B.S. Katzenellenbogen,
J.A. Katzenellenbogen, J. Med. Chem. 43, 4934 (2000)
5. P. Jayaroopa, K. Ajay Kumar, Int. J. Pharm. Pharm. Sci. 5(4), 431 (2013)
6. S.H. Lee, D.R. Park, H. Kim, J. Lee, J.C. Jung, S.Y. Woo, W.S. Song, M.S. Kwon, I.K. Song, Catal.
Commun. 2008, 9 (1920)
7. S. Park, K.M. Cho, M.H. Youn, J.G. Seo, S.H. Baeck, T.J. Kim, Y.M. Chung, S.H. Oh, I.K. Song,
Catal. Lett. 122, 349 (2008)
8. R. Pal, T. Sarkar, S. Khasnobis, Arkivoc 1, 570 (2012)
9. P.V.N.S. Murthy, D. Rambabu, G.R. Krishna, C.M. Reddy, K.R.S. Prasad, M.V.B. Rao, M. Pal,
Tetrahedron Lett. 53, 863 (2012)
10. A.K. Tewari, P. Srivastava, V.P. Singh, A. Singh, R.K. Goel, C.G. Mohan, Chem. Pharm. Bull. 58,
634 (2010)
11. L.D. Mahadevaswamy, A.K. Kariyappa, Pharm. Chem. J. 51, 670 (2017)
12. Z. Christina, D. Florea, D. Constantin, I. Mircea, M. Maria, T. Isabela, M.N. George, Ind. J. Chem.
53B, 733 (2014)
13. R. Katoch-Rouse, O.A. Pavlova, T. Caulder, A.F. Hoffmann, A.G. Mukhin, A.G. Horti, J. Med.
Chem. 46, 642 (2003)
14. N. Renuka, H.K. Vivek, G. Pavithra, K. Ajay Kumar, Russ. J. Bioorg. Chem. 43, 197 (2017)
15. G. Pinna, G. Loriga, G.A. Pinna, G. Chelucci, Molecules 18, 8147 (2013)
16. M. Abdel-Aziz, E.D.A. Abuo-Rahma, A.A. Hassan, Eur. J. Med. Chem. 44, 3480 (2009)
17. V.H. Kameshwar, J.R. Kumar, B.S. Priya, S.N. Swamy, Mol. Cell. Biochem. 426, 161 (2016)
18. S. Kasturi, T.V. Gowda, Toxicon 27, 229 (1989)
19. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193, 265 (1951)
20. D.M. Lokeshwari, N.D. Rekha, B. Srinivasan, H.K. Vivek, A.K. Kariyappa, Bioorg. Med. Chem.
Lett. 27, 3048 (2017)
21. K.R. Raghavendra, N. Renuka, K.A. Kumar, S. Shashikanth, Pharm. Chem. J. 51, 661 (2017)
22. M. Govindaraju, G. Vasanth Kumar, G. Pavithra, M.A. Harish Nayaka, B.N. Mylarappa, K. Ajay
Kumar, IOSR J. Pharm. Biol. Sci. 2(6), 30 (2012)
23. M.G. Prabhudeva, S. Bharath, A.D. Kumar, S. Naveen, N.K. Lokanath, B.N. Mylarappa, K. Ajay
Kumar, Bioorg. Chem. 73, 109 (2017)
24. K. Kumara, N. Shivalingegowda, L.D. Mahadevaswamy, A.K. Kariyappa, N.K. Lokanath, Chem.
Data Coll. 9, 251 (2017)
25. K. Kumara, A.D. Kumar, S. Naveen, K.A. Kumar, N.K. Lokanath, J. Mol. Struct. 1161, 285 (2018)
26. J.A. Ferrera, A. Gupt, N.D. Amin, A. Basu, B.N. Sinha, S. Worgall, V. Jayaprakash, L.E.N. Quadri,
Bioorg. Med. Chem. Lett. 21, 6533 (2011)
123