Synthesis, biological evaluation, and in silico study of pyrazoline-conjugated 2,4-dimethyl-1H-pyrrole-3-carboxylic acid derivatives

Article abstract of DOI:10.1002/ardp.202000267

A potential molecular hybridization strategy was used to develop 24 novel pyrazoline-conjugated 2,4-dimethyl-1H-pyrrole-3-carboxylic acid and amide derivatives. The preliminary in vitro antimicrobial assay delivered four potential derivatives with growth inhibition in the range of 50.87–56.60% at the concentration of 32 μg/ml. In the search of an anticancer candidate, all derivatives were screened by NCI-60 at 10 μM concentration, revealing that 12 derivatives were potential agents against the various types of cancer cell lines, with growth inhibition in the range of 50.21–108.37%. The in vitro cytotoxicity assay against the cell line HEK293 (human embryonic kidney cells) and the hemolysis assay of the representative potent compounds propose their potential for a good therapeutic index. In silico studies of the most potent derivatives qualified their significant pharmacokinetic properties with good predicted oral bioavailability and their adherence to Lipinski's rule of five for druglikeness. A molecular docking study against VEGFR-2 with the best-scored conformations reinforced their anticancer potency. The docking study of the most potent compound against VEGFR-2 with the best-scored conformations displayed a binding affinity (?9.5 kcal/mol) comparable with the drug sunitinib (?9.9 kcal/mol) and exhibited that tighter interactions at the active adenosine triphosphate site might be responsible for anticancer potency.

Full text of DOI:10.1002/ardp.202000267

Received: 26 July 2020
Revised: 16 September 2020
Accepted: 23 September 2020
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DOI: 10.1002/ardp.202000267
F U L L P A P E R
Synthesis, biological evaluation, and in silico study of
pyrazoline‐conjugated 2,4‐dimethyl‐1H‐pyrrole‐3‐carboxylic
acid derivatives
Nishant K. Rasal | Rahul B. Sonawane | Sangeeta V. Jagtap
Department of Chemistry, Baburaoji Gholap
College, Affiliated to Savitribai Phule Pune
Abstract
University, Pune, India
A potential molecular hybridization strategy was used to develop 24 novel
Correspondence
pyrazoline‐conjugated 2,4‐dimethyl‐1H‐pyrrole‐3‐carboxylic acid and amide deriva-
Sangeeta V. Jagtap, Department of Chemistry,
tives. The preliminary in vitro antimicrobial assay delivered four potential deriva-
tives with growth inhibition in the range of 50.87–56.60% at the concentration of
32 µg/ml. In the search of an anticancer candidate, all derivatives were screened by
NCI‐60 at 10 µM concentration, revealing that 12 derivatives were potential agents
against the various types of cancer cell lines, with growth inhibition in the range of
50.21–108.37%. The in vitro cytotoxicity assay against the cell line HEK293 (human
embryonic kidney cells) and the hemolysis assay of the representative potent
compounds propose their potential for a good therapeutic index. In silico studies of
the most potent derivatives qualified their significant pharmacokinetic properties
with good predicted oral bioavailability and their adherence to Lipinski's rule of five
for druglikeness. A molecular docking study against VEGFR‐2 with the best‐scored
conformations reinforced their anticancer potency. The docking study of the most
potent compound against VEGFR‐2 with the best‐scored conformations displayed a
binding affinity (−9.5 kcal/mol) comparable with the drug sunitinib (−9.9 kcal/mol)
and exhibited that tighter interactions at the active adenosine triphosphate site
might be responsible for anticancer potency.
Baburaoji Gholap College, Affiliated to
Savitribai Phule Pune University, Pune
411017, India.
Email: sangeetajagtap@rediffmail.com
K E Y W O R D S
2,4‐dimethyl‐1H‐pyrrole, antimicrobial, antiproliferation, drug discovery, molecular docking
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1
INTRODUCTION
2018, 18.1 million cancer patients were found as new cases and
almost 9.6 million died from the disease. By 2040, the figure for new
Under the healthy condition of the body, the number and growth of
each type of cell follow a highly controlled mechanism. However, any
wrong signal through the alteration of genes directly affects the cell
life cycle and it leads to continuous uncontrolled multiplication,
which results in the accumulation of abnormal cells in the form of a
mass of malignant cells called “cancer.”[1] Worldwide, at present,
cancer ranks first for the mortality and morbidity, and it is estimated
that almost 22 million people may be affected from it by 2030.[2]
According to the World Health Organization (WHO), in the year
cancer patients will rise to 29.4 million.[3] To get relief from cancer at
an early stage, the application of chemotherapy is the most popular
approach, but it has several unbearable side effects on bone marrow,
the gastrointestinal tract, hair, and so forth, and it directly affects an
individual's physical health and quality of life.[4] Hence, myriad efforts
have been made by researchers to develop a new anticancer agent
with minimum side effects.
The topmost potential anticancer molecules semaxanib, obatoclax,
sunitinib, toceranib, and so on, exhibited 2,4‐dimethyl‐1H‐pyrrole
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Arch Pharm. 2020;e2000267.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.202000267

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moiety as a key building block along with the 2‐indoline ring. Further
exploration of this skeleton delivered various significant derivatives, 1,[5]
2,[6] 3,[7] and so on, which exhibited the antiproliferative activity
comparable with sunitinib against various cancer cell lines. Many re-
searchers have endeavored to find various amide functionalities at
the 3‐position of pyrrole along with structural modification on the
2‐indoline ring (Figure 1).
In medicinal chemistry, 2‐pyrazoline acts as a privileged scaf-
fold due to its presence in numerous important medical and bio-
chemical agents that have been effectively utilized as antibacterial,
antifungal, anti‐inflammatory, analgesic, antiparasitic, antiviral, an-
titubercular, anticancer, anesthetic, and insecticidal agents.[8–22]
Recently, a 4,5‐unsaturated pyrazoline ring called pyrazole has
marked its versatile use in the drug design, and it has been found to
have a remarkable presence in various potential anticancer mole-
cules such as celecoxib, SC5584. Liu et al.[23] have confirmed that
celecoxib induces apoptosis in the human osteosarcoma cell line
and enhances the cytotoxic effect of cisplatin.[23] Celecoxib reduces
the risk of manifestation and growth of the various types of cancers,
especially breast cancer.[24] Besides, pyrazoline motifs were re-
ported as potent EGFR inhibitors 4 and 5,[25,26] aurora kinase in-
hibitors,[27] telomerase inhibitors,[28] tubulin assembly inhibitors,[29]
and so forth (Figure 1).
SCHEME 1 The synthesis of scaffold 11. Reagents and
conditions: (a) NaNO2, AcOH, RT, 3 hr; (b) ethyl acetoacetate, Zn
powder, AcOH, 65°C, 3 hr; (c) HCl, EtOH, 70°C, 3 hr; (d)
dimethylformamide, POCl3, dichloromethane, reflux, 2 hr; (e) KOH,
H2O, MeOH, reflux, 5 hr
|
2
RESULTS AND DISCUSSION
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2.1
Chemistry
The scaffold 5‐formyl‐2,4‐dimethyl‐1H‐pyrrole‐3‐carboxylic acid (11) was
synthesized according to Scheme 1 adopted from the literature.[32] The
chalcone derivatives 12a–c were prepared by the Knoevenagel con-
densation and subjected to acid–amine coupling using TBTU as a coupling
reagent to get the desired derivatives 13a–c as per the previously re-
ported synthetic procedure.[30] The targeted alkyl‐ or aryl‐substituted
pyrazoline–pyrrole derivatives 14a–i, 15a–c, 15f–h, and 15k–m were
prepared by treatment of respective chalcones with hydrazine HCl de-
rivatives in the presence of NaOH and ethanol under reflux conditions
(Scheme 2). For the remaining acyl‐substituted pyrazoline–pyrrole deri-
vatives (compounds 15d, 15e, 15i, 15j, 15n, and 15o), chalcone was in-
teracted with the hydrazine hydrate to get a basic pyrazoline compound,
which was further acylated using the respective acyl chloride.
In our earlier work, 2,4‐dimethylpyrrole‐conjugated chalcone
derivatives were delivered as the potential scaffold for further
development.[30] Hence, as an extension of the previous work and
in the continuous search of novel antimicrobial and anticancer
templates,[31] this study presents the new hybrid skeleton
consisting of 2,4‐dimethylpyrrole moiety clubbed with the
2‐pyrazoline ring. The hybrid skeleton was further explored by
the screening of a series of derivatives for in vitro antimicrobial
and anticancer activity.
FIGURE 1 Reported 2,4‐dimethyl‐1H‐
pyrrole and pyrazoline containing anticancer
compounds

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RASAL ET AL.
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SCHEME 2 The synthesis of target compounds 13a–c, 14a–i, and 15a–o. Reagents and conditions: (a) ArCOCH3, KOH, MeOH, RT, 48 hr; (b)
CF3CH2NH2 HCl, DIPEA, TBTU, DMF, RT, 24 hr; (c) R1NHNH2 HCl, NaOH, EtOH, reflux, 2 hr; (d) (i) N2H4, EtOH, H2O, reflux, 2 hr, (ii) R1COCl,
TEA, CHCl3, RT, 2 hr. DIPEA, N,N‐diisopropylethylamine; DMF, dimethylformamide; TEA, triethylamine
The formation of the pyrazoline ring was confirmed by 1H nuclear
magnetic resonance (NMR) spectra, where peaks of hydrogen from al-
kene conjugated to the ketone of chalcone at δ 7.0–7.8 ppm disappeared
and new aliphatic protons appeared in the expected region between δ 2.5
and 5.5 ppm as ABX spin system along with one –NH proton at δ
11.5 ppm. The two magnetically nonequivalent protons of the methylene
group of the pyrazoline ring appeared distinctly at δ 2.5 and 3.5 ppm with
J values ranging from 10 to 20 Hz. Additional peaks due to the alkyl, aryl,
or acyl substitution on the pyrazoline ring confirmed the formation of the
respective desired derivative. Representative derivatives were char-
acterized by 13C NMR, and each spectrum showed the carbon values in
the predictable regions. Also, every newly synthesized derivative was
analyzed by high‐resolution mass spectroscopy (HRMS) and the mass
was confirmed with the calculated values of the target compounds.
bacterial strains, Gram‐positive Staphylococcus aureus and Gram‐negative
Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseu-
domonas aeruginosa using ciprofloxacin as a positive control. At the same
concentration, the antifungal activity was also tested against two fungal
species, that is, Candida albicans and Cryptococcus neoformans var. grubii,
using fluconazole as a positive control. The obtained in vitro assay results
are described as % growth inhibition (GI) in Table 1. This preliminary in
vitro antimicrobial evaluation revealed that ~50% GI was displayed by
compounds 14c, 14f, and 14i against A. baumannii and compound 15o
against C. albicans.
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2.2.2
SAR exploration as an antibacterial study
From the obtained results, it was observed that all acid compounds
showed a significant activity against Gram‐negative A. baumannii and
each structurally comparable hydrazine derivative also displayed
equivalent results. The % GI was found to be increased by the in-
troduction of groups into each chalcone‐acid derivative in the following
|
2.2
Biology activity
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2.2.1
In vitro antimicrobial activity
order:
4‐chlorophenylhydrazine > phenylhydrazine > methylhydrazine.
The antimicrobial activity evaluation of all pyrazoline derivatives was
performed at The Community for Antimicrobial Drug Discovery
(CO‐ADD),[33] funded by the Wellcome Trust (UK) and The University of
Queensland (Australia). The screening was done at 32 µg/ml against five
Notably, derivatives using 4‐chlorophenylhydrazine, that is, 14c, 14f,
and 14i, exhibited GI >50% at 32 µg/ml concentration, whereas com-
pound 14c revealed the highest activity, 56.60%, against Gram‐negative
A. baumannii. Hence, based on the structure–activity relationships, it can

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TABLE 1 In vitro antimicrobial activities (% growth inhibition) of tested compounds at 32 µg/ml
Klebsiella
Pseudomonas
aeruginosa
Acinetobacter
baumannii
Candida
albicans
Cryptococcus
Compound
14a
14b
14c
14d
14e
14f
Staphylococcus aureus Escherichia coli
pneumoniae
neoformans var. grubii
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
11.3
23.9
–
15.1
–
13.2
48.1
56.6
10.4
37.5
50.9
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
10.6
–
–
–
–
–
–
–
–
17.3
35.6
14.0
13.8
18.7
12.6
–
–
–
–
–
–
14g
14h
14i
35.6
13.8
–
–
–
11.1
–
12.3
51.1
19.6
17.0
13.8
20.2
26.3
11.7
34.4
19.2
13.3
23.5
40.2
14.3
15.7
14.5
12.7
–
–
15a
15b
15c
15d
15e
15f
13.5
14.1
15.9
19.0
21.5
20.7
10.9
–
14.1
15.5
10.3
17.5
–
–
–
–
–
12.1
–
–
–
14.4
33.6
26.0
16.8
17.7
25.5
–
14.1
18.1
10.2
15.3
10.8
–
–
15g
15h
15i
–
14.4
17.0
–
17.0
11.1
–
15j
15k
15l
12.1
21.3
–
12.9
12.5
–
–
15m
15n
15o
–
17.1
11.7
10.4
–
10.1
55.7
13.8
–
Note: “–” not active (% GI < 10). Bold faced numerical values highlight the most potent compounds.
be concluded that, in the case of acid derivatives, the electron‐donating
group on the aromatic ring and, in contrast, the electron‐withdrawing
group on phenylhydrazine would result in a more potent compound as an
antibacterial agent. None of the acid derivatives showed any potency
against both fungal species.
against the NCI‐60 cell lines is calculated by dividing the summation of %
GI values over the number of tested cell lines and is illustrated in
Figure 2. This summarized data revealed that compound 14h from the
acid series and compound 15n from amide series were the most potent
derivatives.
The % GI values were calculated from percent of growth of the
treated cells that were determined in comparison to the untreated con-
trol cells,[34–37] and conclusive results of each tested compound are
presented in Tables 2 and 3. Among all the nine acid derivatives, five
derivatives, 14b, 14c, 14f, 14g, and 14h, showed >50% GI against the
A498 cell line from renal cancer even at such a small dose. Notably, all
acid compounds displayed the maximum sensitivity against the A498 cell
line from renal cancer, whereas the most significant compound, 5‐(1,3‐
diphenyl‐4,5‐dihydro‐1H‐pyrazol‐5‐yl)‐2,4‐dimethyl‐1H‐pyrrole‐3‐
carboxylic acid 14b, displayed 70.2% GI. Among the amide derivatives,
most active compound 5‐(1‐acetyl‐3‐(3‐fluoro‐4‐(trifluoromethyl)phenyl)‐
4,5‐dihydro‐1H‐pyrazol‐5‐yl)‐2,4‐dimethyl‐N‐(2,2,2‐trifluoroethyl)‐1H‐
pyrrole‐3‐carboxamide 15n displayed a significant anticancer activity
|
2.2.3
In vitro antiproliferative activity
All the structures of novel derivatives were submitted to the National
Cancer Institute (NCI) and further selected by the Developmental
Therapeutics Program (www.dtp.nci.nih.gov) for in vitro anticancer
screening. As per standard protocol of NCI, all compounds were eval-
uated for their antiproliferative activity at single‐dose assay (10 µM
concentration in dimethyl sulfoxide [DMSO]) on a panel of 60 cancer cell
lines derived from leukemia, non‐small‐cell lung, colon, central nervous
system (CNS), melanoma, ovarian, renal, prostate, and breast cancer as
per protocol. The mean percentage inhibition of each tested compound

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RASAL ET AL.
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FIGURE 2 Mean percentage inhibition values
of all compounds against the NCI‐60 cell line
panel at 10 µM concentration
against almost all cancer cell lines, with few exceptions. It displayed the
highest efficiency against the SNB‐75 cell line from CNS cancer with
8.37% cytotoxicity along with 100% GI (Table 4). Also, most of the amide
compounds were highly sensitive against the A498 cell line from renal
cancer. Hence from the obtained results, it can be concluded that com-
pounds 14b and 15n exhibited a significant anticancer potency equivalent
to that of sunitinib (A498, IC50: 5.2 µM; SNB‐75, IC50: 5.3 µM; source:
NCI database).
cases of acid and amide derivatives, compounds comprising
3‐fluoro‐4‐trifluoromethylphenyl ring were more potent than the
related phenyl and thiophene derivatives. In detail, the
structure–reactivity relationship of tested compounds for leu-
kemia, colon, and CNS cancer cell lines reveals that, for the
aromatic substituent, 3‐fluoro‐4‐trifluoromethyl ring (compounds
14g–i) was more favorable than the phenyl and thiophene ring
(compounds 14a–f), whereas 4‐chlorophenyl and phenylpyrazo-
line rings (compounds 14h and 14i) were more significant than
the methyl pyrazoline moiety (compound 14g). Contradictorily,
phenyl ring derivatives 14a–c were more active against the A498
cell line from renal cancer than the compounds with thiophene
and 3‐fluoro‐4‐trifluoromethyl rings (compounds 14d–i); how-
ever, the introduction of phenyl and 4‐chlorophenylpyrazoline
moieties leads to the most active compounds 14b and 14c with
70.20% and 60.72% GI, respectively.
|
2.2.4
SAR exploration as an antiproliferative study
of acid derivatives 14a–i
The structural comparison concerning mean percentage inhibi-
tions reveals that all amide derivatives demonstrated higher
inhibition than the corresponding acid compounds. In both
TABLE 2 In vitro anticancer activities of
tested acid derivatives at a concentration of
10 µMa
% Growth inhibition (GI)
Colon
Breast
cancer
Leukemia cancer
CNS cancer
Renal cancer
cancer
RPMI‐
MOLT‐4 8226
HCT‐15 SF‐268 SNB‐75 A498 UO‐31 MCF7
Compound NSC No.
14a
14b
14c
14d
14e
14f
14g
14h
14i
821320
821319
821321
821328
821322
821329
821326
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
14.22
13.14
14.74
–
45.26 16.34
–
–
–
70.20 11.03 11.92
60.72 11.26 17.73
–
–
–
–
–
20.22
–
–
10.44
–
–
45.99 10.70 18.32
–
11.76
–
57.36 17.09
53.42 12.00
–
–
–
–
821325 31.36
821327 35.74
23.26
23.80
24.38
25.43
18.63 22.57
20.53 27.59
57.16 27.74 32.14
32.51 28.18 35.62
Note: “–” not active (% GI < 10). Bold faced numerical values highlight the most potent compounds.
Abbreviation: CNS, central nervous system.
aSupporting Information includes growth percentage data for each NCI‐60 cell line.

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TABLE 3 In vitro anticancer activities of tested amide derivatives at a concentration of 10 µMa
% Growth inhibition (GI)
Leukemia cancer
Renal cancer
Non‐small‐cell lung cancer
MOLT‐4 RPMI‐8226 A549/ATCC
CNS cancer
Breast cancer
A498 CAKI‐1 RXF 393 UO‐31 MCF7
SF‐295
Compound NSC No.
13a
13b
13c
15a
15b
15c
15d
15e
15f
821323
821324
–
–
–
–
–
35.86 14.33
18.97 13.26
23.81 16.76
21.77
24.27
15.81
21.20
28.80
17.77
–
12.62 13.64
10.77
–
–
–
–
819928 26.10
821331
37.93
16.90
18.85
27.72 21.81
17.37 10.93
25.33 20.50
26.92 29.52
13.46 14.91
–
–
–
–
–
–
821330 23.79
821332 28.79
–
–
–
53.20 19.85
16.39
11.32
–
20.21 26.88
821333
821334
821336
–
–
–
–
–
–
39.18
–
–
–
–
48.68 14.53
25.93
–
17.92
14.65
–
–
–
–
–
–
–
15g
15h
15i
821335 19.31
821337 11.62
11.20
–
–
79.06 23.59
25.45
22.83
–
28.84 16.31
22.77 10.01
13.14 12.32
–
–
–
39.51 20.80
821338
821339
–
–
–
–
–
46.75 –
15j
–
–
–
29.11 14.10
69.21 18.90
51.81 41.04
25.34 14.86
45.07 41.26
26.40 33.77
16.86
–
25.13
–
15k
15l
821341 27.13
821340 41.67
821342 31.97
821343 17.41
821344 16.55
–
–
–
28.03 19.35
41.23 43.47
39.77 32.13
73.97 26.40
29.34
22.02
–
15.22
21.41
44.25
40.36
12.86
–
19.23
15.92
69.05
42.07
15m
15n
15o
29.12
50.76
12.17
–
17.79
Note: “–” not active (% GI < 10). Bold faced numerical values highlight the most potent compounds.
Abbreviation: CNS, central nervous system.
aSupporting Information includes growth percentage data for each NCI‐60 cell line.
|
2.2.5
SAR exploration as an antiproliferative study
active compounds. There was no selective difference between the
phenyl and thiophene derivatives, but 3‐fluoro‐4‐trifluorophenyl
ring was favorable to boost activity against almost all cancer cell
lines. Within the 3‐fluoro‐4‐trifluorophenyl derivatives, acetyl
pyrazoline derivative 15n was the most potent compound against
almost all cancer cell lines and displayed the highest 100% GI
with 8.37% cytotoxicity for SNB‐75 cell line from CNS cancer.
Hence, the SAR for anticancer activity can be concluded as the
electron‐withdrawing group on the aromatic ring and carbonyl
substitution on the pyrazoline ring would deliver a more potent
compound as an anticancer agent.
of amide derivatives 15a–o
Structural examination of all compounds with respect to the an-
tiproliferative activity showed that almost all the pyrazoline
compounds were more potent than the parent chalcone deriva-
tives (compounds 13a–c). In the case of phenyl and thiophene
rings, phenylpyrazoline derivatives (compounds 15b and 15g) and
4‐chlorophenylpyrazoline derivatives (compounds 15c and 15h)
displayed higher potency than the rest of pyrazoline derivatives,
whereas the carbonyl hydrazine derivatization led to the less
TABLE 4 In vitro anticancer activities of compound 15n at a concentration of 10 µM
% Growth inhibition (GI)
Melanoma
CNS cancer
Ovarian cancer
Renal cancer
Breast cancer
Colon cancer
cancer
MDA‐
HCT‐116
SNB‐19 SNB‐75 MALME‐3M OVCAR‐4 OVCAR‐8 786‐0 ACHN RXF 393 SN12C UO‐31 MB‐231 HS 578T T‐47D
65.98
52.13 108.37 56.53 86.44 58.13 86.94 81.31 69.05 49.94 73.97 61.17 68.19 56.50

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TABLE 5 Calculated rule‐of‐five
parameters by SwissADME
Number of H
bond donors
Number of H bond Molecular
Compound
14b
mlogP
3.14
3.65
3.25
3.22
4.33
3.50
3.97
3.12
3.57
4.63
3.30
3.91
acceptors
weight
359.42
393.87
399.89
383.34
445.41
440.46
474.91
446.49
464.38
526.45
492.39
532.45
C druglikeness
2
2
2
2
2
2
2
2
2
2
2
2
3
3
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
14c
14f
3
14g
7
14h
7
15b
5
15c
5
15g
5
15k
9
15l
9
15n
10
10
Yes
No
15o
|
2.2.6
Cytotoxicity and hemolysis
14b and 15n exhibited a good lipophilic property with consensus
logPo/w value of 3.64 and 4.62, respectively, together with moderate
water solubility and high gastrointestinal absorption. The pink area of
the bioavailability radar chart has signified the advantageous values
of saturation (INSATU), size (SIZE), polarity (POLAR), solubility
(INSOLU), lipophilicity (LIPO), and flexibility (FLEX) about oral bioa-
vailability.[39,40] Thus, compound 15n is absolutely under the pink
area, which signifies its good predicted oral bioavailability, whereas
compound 14b is found to be slightly deviating from the required
solubility value, which could be rectified in the further derivatization.
The BOILED‐Egg graph (Figure 3) identifies that compounds 14b and
15n were predicted to be effluated from the CNS by the P‐glycoprotein,
which clearly reduced the possibility of their resistance by tumor cell lines
through efflux. Compound 14b (molecule 1 in Figure 3) is predicted to
passively permeate through the blood–brain barrier, whereas compound
The toxicity study was performed at CO‐ADD, Australia.[33] The
study was carried out on two random representative potent com-
pounds 14c and 14f for their in vitro cytotoxic activity against human
embryonic kidney (HEK293) cell line, which demonstrated that both
tested compounds possessed no toxic effect (CC50 > 80 µM). Fur-
thermore, the hemolysis assay of the same derivatives displayed
HC10/HC50 values >80 µM and confirmed the nontoxic property of
the tested compounds against human red blood cells.
|
2.3
In silico study
|
2.3.1
Prediction of druglikeness by Lipinski's rule
of five (RO5)
Lipinski's RO5[38] (MW ≤ 500, mlogP ≤ 4.15, N or O ≤ 10, NH, or
OH ≤ 5) is the well‐known thumb rule to predict the druglikeness
properties of biologically potent compounds. Hence, RO5 parameters
of the most potent derivatives were assessed by the SwissADME
(http://www.swissadme.ch/). The calculated values (Table 5) ap-
proved that all the potent derivatives obeyed the RO5 and presented
the significant druglikeness values, except 15l and 15o, which vio-
lated only the molecular weight parameter.
|
2.3.2
ADME prediction
In silico ADME parameters and pharmacokinetic profile of com-
pounds 14b and 15n were assessed by the freely offered web server
SwissADME (http://www.swissadme.ch/). The obtained results in the
form of figures are provided in Supporting Information that revealed
the substantial ADME properties of studied derivatives. Compounds
FIGURE 3 BOILED‐Egg graph of compounds 14b and 15n from
SwissADME web server

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FIGURE 4 Docking images of compounds 14b and 15n and sunitinib. (a) A 2D diagram of bonding interaction between compound 14b and
VEGFR‐2. (b) A 2D diagram of bonding interaction between sunitinib and VEGFR‐2. (c) A 2D diagram of bonding interaction between compound
15n and VEGFR‐2. (d) Alignment of sunitinib (carbons are colored as blue), compound 14b (carbons are colored as purple), and 15n (carbons are
colored as brown) in the active adenosine triphosphate site of protein
15n (molecule 2 in Figure 3) is predicted to be passively absorbed by the
gastrointestinal tract.[41] Overall, compounds 14b and 15n have promis-
ing in vitro anticancer activity along with the essential in silico ADME
properties for druglikeness, hence signifying that both derivatives would
be noble drug candidates.
molecular docking study was performed using the most potent
derivatives 14b and 15n at the active adenosine triphosphate
(ATP)‐binding site of VEGFR‐2 with PDB ID: 4AGD (resolution:
2.81 Å) along with sunitinib. Results revealed that compounds 14b
and 15n along with sunitinib as a co‐crystallized ligand inside the
active ATP‐binding site of VEGFR‐2 adopted a similar molecular or-
ientation, which might be responsible for the observed affinity of the
compounds 14b and 15n (Figure 4).
|
2.3.3
Molecular docking study
Sunitinib displayed
a strong binding interaction between
Molecular docking is an effective and reliable tool to understand the
plausible orientation along with binding affinity between ligand and
target protein, which induces the biological behavior of drug mole-
cules. The drug candidate sunitinib displays the anticancer activity
via inhibition of receptor tyrosine kinases like VEGFR‐2.[42] Hence, a
(2‐oxoindolin‐3‐ylidene)methylpyrrole moiety and ATP‐binding site
of VEGFR‐2 through three hydrogen bonds at Glu917, Cys919, and
Lys868. In the case of compound 14b, oxygen of the carbonyl group
acted as an H‐acceptor and formed two conventional hydrogen
bonding with the Thr926 and Asn923 with distances 1.92 and 2.43 Å,

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EXPERIMENTAL
respectively. The phenyl and pyrazoline rings get accommodated in
the hydrophobic pocket made of Leu840, Val848, Ala866, and
Cys919. The binding pose of 14b was stabilized through exposure to
the wide network of favorable van der Waals interactions with
Phe1047, Phe918, Lys920, Lys838, Phe921, and Gly922. Similar to
14b, carbonyl oxygen of compound 15n acted as an H‐acceptor, re-
sulting in the formation of two conventional hydrogen bonds with
Thr926 and Asn923 with distances 1.97 and 2.34 Å, respectively. The
substituted phenyl and pyrazoline rings were overlapped by the hy-
drophobic pocket consisting of Leu1035, Val848, Ala866, Val916,
and Leu840. The binding pose of 15n was stabilized through ex-
posure to the wide network of favorable van der Waals interactions
with Val899, Cys1045, Phe1047, Cys919, Phe918, Lys920, and
Gly922. The docking study for the best‐scored conformations ex-
hibited a binding affinity for compound 14b (−7.9 kcal/mol) and 15n
(−9.5 kcal/mol), which was comparable with the ligand sunitinib
(−9.9 kcal/mol). Hence, the described docking score data suggested
that the potent derivatives 14b and 15n have good potential to in-
hibit VEGFR‐2 as well as clarified the experimental anticancer
results.
4
|
4.1
Chemistry
|
4.1.1
General
All the reagents and solvents were procured from Spectrochem,
Sigma‐Aldrich, S D Fine‐Chem, and Finar Chemicals and used without
any further purification. The progress of reactions was monitored by
analytical thin‐layer chromatography (TLC; silica gel 60 F254 plates
from Merck, Darmstadt, Germany). The TLC plates were visualized
under short‐ and long‐wavelength UV light. Melting points were
uncorrected and confirmed in an open capillary tube. HRMS spectra
were recorded on a Waters SYNAPT G2 HDMS system with triple
quadrupole mass spectrometer (ESI mode) and the values were ex-
pressed in units of m/z. All the 1H and 13C NMR spectra were re-
corded in DMSO‐d6 on a Bruker Avance‐III 500 MHz spectrometer
using tetramethylsilane as an internal solvent. Chemical shift and
coupling constant values were expressed in ppm and Hz, respectively.
The abbreviations used to describe the peak patterns are singlet (s),
doublet (d), triplet (t), multiplet (m), double doublet (dd), and so on.
All the compounds, 12a–c and 13a–c, were synthesized as per the
reported procedure.[30]
|
3
CONCLUSION
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
This study offered a bunch of potent compounds against various
microbial strains and cancer cell lines like 14f (50.87% GI), 14c
(56.60% GI), and 14i (51.14% GI) against Gram‐negative A. baumannii,
15o (55.74% GI) against fungal strain C. albicans at 32 µg/ml, as well
as 14b (70.20% GI), 14c (60.72% GI), 14f (57.36% GI), 14g (53.42%
GI), 14h (57.16% GI), 15b (53.20% GI), 15c (50.21% GI), 15g (79.06%
GI), 15k (69.21% GI), and 15l (51.81% GI) against A498 cell line from
renal cancer and 15o (50.76% GI) against SF‐295 from CNS cancer at
10 µM concentration. Among all acid and amide derivatives, com-
pound 15n is the most active compound against numerous cancer cell
lines and it displayed 100.0% GI with 8.37% cytotoxicity for SNB‐75
cell line from CNS cancer. Overall, the antiproliferative assay of all
newly demonstrated pyrazoline–pyrrole derivatives revealed that
the several compounds exhibited anticancer potency analogous to
sunitinib (IC50s: 4.0–8.0 µM). SAR study revealed that the dramatic
improvement in the bioactivity of the amide derivatives may be due
to the conjugative effect of the electron‐withdrawing group on the
aryl ring, that is, trifluoromethyl at para‐position and carbonyl pyr-
azoline. In vitro toxicity study of the representative potent com-
pounds signified a potential for a good therapeutic index. In silico
ADME study of most active compounds, 14b and 15n, comply with
the desired ADME parameters and also obey Lipinski's rule of five for
druglikeness. The molecular docking study concluded that the tighter
interactions of 14b and 15n at the active ATP site of VEGFR‐2 might
be responsible for anticancer potency. Hence, the proposed
pyrazoline–pyrrole template can be a potential scaffold to develop
the new multitargeted anticancer drug as well as antimicrobial drugs,
and additionally, compounds 14b and 15n would be noble drug
candidates for the clinical treatment of numerous cancers.
|
4.1.2
General procedure for the synthesis of
compounds 14a–i, 15a–c, 15f–h, 15k–m
To a solution of chalcone derivative (1.0 g, 1.0 equiv) in ethanol (25 ml,
5.0 volumes), hydrazine derivative (1.5 equiv) and NaOH (2.0 equiv)
were added at 20–25°C. The mixture was refluxed for 2 hr and the
progress of the reaction was monitored by TLC. After the complete
conversion, the mixture was poured over an ice–water mixture (50 ml,
50.0 volumes). For acid derivatives, the mass was acidified to pH ~5
with acetic acid. The precipitate was filtered and washed with water
under vacuum. The solid product was dried under vacuum and purified
in ethyl tert‐butyl ether (MTBE) by the method of trituration.
|
4.1.3
General procedure for the synthesis of
compounds 15d, 15e, 15i, 15j, 15n, and 15o
To a solution of chalcone derivative (1.0 g, 1.0 equiv) in ethanol
(10 ml, 10.0 volumes), 99% hydrazine hydrate (2.0 volumes) was
added, and the mixture was refluxed for 2 hr. The progress of the
reaction was monitored by TLC. After the complete conversion, the
mixture was poured over an ice–water mixture (50 ml, 50.0 volumes)
and it was extracted with chloroform (2 × 5.0 volumes) twice. The
organic layer was washed with brine solution (5 ml, 5 volumes) and
dried over sodium sulfate. The dried organic layer was cooled at
~10°C and was charged with triethylamine (2.5 equiv), followed by

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carbonyl chloride derivative (1.1 equiv). The mixture was stirred at
room temperature for 2 hr and the progress of the reaction was
monitored by TLC. After the complete conversion, the mixture was
poured over an ice–water mixture (50 ml, 50.0 volumes). The organic
layer was separated and concentrated till slurry mass. Hexane (5 ml,
5.0 volumes) was added and the precipitate was filtered. The solid
product was dried under vacuum to get the pure compound.
0.9 Hz, 1H, thiophene‐H), 7.09 (dd, J = 5.0, 3.6 Hz, 1H, thiophene‐H),
4.15 (dd, J = 15.0, 9.9 Hz, 1H, –CHX), 3.33 (d, J = 10.0 Hz, 1H, –CHAH),
3.06 (t, J = 15.5 Hz, 1H, –CHBH), 2.62 (s, 3H, –NCH3), 2.36 (s, 3H,
pyrrole –CH3), 2.14 (s, 3H, pyrrole –CH3); HRMS (ESI) calcd. for [M
+H]+ C15H18N3O2S+: 304.1119, found: 304.1117.
2,4‐Dimethyl‐5‐[1‐phenyl‐3‐(thiophen‐2‐yl)‐4,5‐dihydro‐1H‐pyrazol‐
5‐yl]‐1H‐pyrrole‐3‐carboxylic acid (14e)
2,4‐Dimethyl‐5‐(1‐methyl‐3‐phenyl‐4,5‐dihydro‐1H‐pyrazol‐5‐yl)‐
1H‐pyrrole‐3‐carboxylic acid (14a)
The product was obtained as a brown solid; yield: 0.52 g, 78.4%; RF
(mobile phase hexanes/EtOAc 40:60): 0.60; m.p.: 180–182°C; 1H
NMR (500 MHz, DMSO‐d6): δ 11.50 (br s, 1H, –COOH), 11.07 (s, 1H,
–NH), 7.60 (d, J = 6.0 Hz, 1H, Ar‐H), 7.27 (s, 1H, Ar‐H), 7.15 (m, 3H,
Ar‐H), 6.96 (d, J = 10.0 Hz, 2H, Ar‐H), 6.75 (t, J = 8.5 Hz, 1H, Ar‐H),
5.29 (t, J = 13.0 Hz, 1H, –CHX), 3.79 (dd, J = 21.5, 16.0 Hz, 1H,
–CHAH), 3.12 (dd, J = 21.5, 12.0 Hz, 1H, –CHBH), 2.30 (s, 3H, pyrrole
–CH3), 2.21 (s, 3H, pyrrole –CH3); HRMS (ESI) calcd. for [M+H]+
C20H20N3O2S+: 366.1276, found: 366.1272.
The product was obtained as an off‐white solid; yield: 0.41 g, 74.3%; RF
(mobile phase hexanes/EtOAc 50:50): 0.45; m.p.: 171–173°C; 1H NMR
(500 MHz, DMSO‐d6): δ 11.49 (br s, 1H, –COOH), 11.08 (s, 1H, –NH),
7.64 (d, J = 9.0 Hz, 2H, Ar‐H), 7.37 (m, 3H, Ar‐H), 4.15 (dd, J = 18.5,
12.5 Hz, 1H, –CHX), 3.36 (t, J = 12.5 Hz, 1H, –CHAH), 3.02 (t, J = 19.5 Hz,
1H, –CHBH), 2.66 (s, 3H, –NCH3), 2.36 (s, 3H, pyrrole –CH3), 2.14 (s, 3H,
pyrrole –CH3); HRMS (ESI) calcd. for [M+H]+ C17H20N3O2+: 298.1555,
found: 298.1554.
5‐[1‐(4‐Chlorophenyl)‐3‐(thiophen‐2‐yl)‐4,5‐dihydro‐1H‐pyrazol‐5‐
yl]‐2,4‐dimethyl‐1H‐pyrrole‐3‐carboxylic acid (14f)
5‐(1,3‐Diphenyl‐4,5‐dihydro‐1H‐pyrazol‐5‐yl)‐2,4‐dimethyl‐1H‐
pyrrole‐3‐carboxylic acid (14b)
The product was obtained as an off‐white solid; yield: 0.58 g, 79.9%;
RF (mobile phase hexanes/EtOAc 50:50): 0.60; m.p.: 189–191°C; 1H
NMR (500 MHz, DMSO‐d6): δ 11.54 (br s, 1H, –COOH), 11.09 (s, 1H,
–NH), 7.62 (d, J = 6.0 Hz, 1H, thiophene‐H), 7.29 (d, 1H, J = 4.0 Hz,
thiophene‐H), 7.22 (d, J = 11.5 Hz, 2H, Ar‐H), 7.12 (t, J = 5.5 Hz, 2H,
thiophene‐H), 6.93 (d, J = 13.7 Hz, 2H, Ar‐H), 5.32 (dd, J = 15.0, 12.0,
Hz, 1H, –CHX), 3.81 (dd, J = 21.5, 15.5, Hz, 1H, –CHAH), 3.14 (dd,
J = 21.5, 13.0 Hz, 1H, –CHBH), 2.29 (s, 3H, pyrrole –CH3), 2.19 (s, 3H,
pyrrole –CH3); 13C NMR (126 MHz, DMSO‐d6): δ 167.34, 145.16,
144.50, 136.20, 136.10, 129.11, 128.29, 128.13, 128.05, 125.64,
123.12, 117.32, 114.98, 110.81, 56.93, 42.17, 13.92, 11.21; HRMS
(ESI) calcd. for [M+H]+ C20H19ClN3O2S+: 400.0886, found: 400.0872.
The product was obtained as an off‐white solid; yield: 0.52 g, 77.9%;
RF (mobile phase hexanes/EtOAc 50:50): 0.70; m.p.: 195–197°C; 1H
NMR (500 MHz, DMSO‐d6): δ 11.53 (br s, 1H, –COOH), 11.06 (s, 1H,
–NH), 7.76 (d, J = 9.5 Hz, 1H, Ar‐H), 7.44 (t, J = 8.5 Hz, 2H, Ar‐H), 7.38
(t, J = 9.0 Hz, 1H, Ar‐H), 7.18 (t, J = 10.0 Hz, 1H, Ar‐H), 7.03 (d,
J = 10.0 Hz, 1H, Ar‐H), 7.75 (t, J = 9.0 Hz, 1H, Ar‐H), 5.30 (dd, J = 15.0,
12.5 Hz, 1H, –CHX), 3.80 (dd, J = 22.0, 15.5 Hz, 1H, –CHAH), 3.11 (dd,
J = 22.0, 12.0 Hz, 1H, –CHBH), 2.30 (s, 3H, pyrrole –CH3), 2.21 (s, 3H,
pyrrole –CH3); 13C NMR (126 MHz, DMSO‐d6): δ 145.76, 143.85,
133.69, 130.85, 127.15, 126.98, 124.17, 124.01, 117.38, 114.86,
111.54, 54.78, 39.12, 37.85, 11.80, 9.16; HRMS (ESI) calcd. for [M
+H]+ C22H22N3O2+: 360.1712, found: 360.1713.
5‐{3‐[3‐Fluoro‐4‐(trifluoromethyl)phenyl]‐1‐methyl‐4,5‐dihydro‐1H‐
pyrazol‐5‐yl}‐2,4‐dimethyl‐1H‐pyrrole‐3‐carboxylic acid (14g)
The product was obtained as an off‐white solid; yield: 0.44 g, 81.6%;
RF (mobile phase hexanes/EtOAc 50:50): 0.60; m.p.: 200–202°C; 1H
NMR (500 MHz, DMSO‐d6): δ 11.53 (br s, 1H, –COOH), 11.11 (s, 1H,
–NH), 7.79 (t, J = 7.9 Hz, 1H, Ar‐H), 7.64 (t, J = 9.9 Hz, 2H, Ar‐H), 4.34
(dd, J = 14.9, 10.6 Hz, 1H, –CHX), 3.41 (dd, J = 16.4, 10.5 Hz, 1H,
–CHAH), 3.04 (t, J = 15.7 Hz, 1H, –CHBH), 2.72 (s, 3H, –NCH3), 2.37
(s, 3H, pyrrole –CH3), 2.15 (s, 3H, pyrrole –CH3); 13C NMR (126 MHz,
DMSO‐d6): δ 167.36, 160.61, 158.60, 146.87, 140.24, 135.95, 128.10,
126.42, 124.27, 123.06, 122.11, 121.90, 119.95, 119.37, 113.51,
110.75, 63.91, 39.97, 38.77, 13.94, 11.17; HRMS (ESI) calcd. for [M
+H]+ C18H18F4N3O2+: 384.1335, found: 384.1334.
5‐[1‐(4‐Chlorophenyl)‐3‐phenyl‐4,5‐dihydro‐1H‐pyrazol‐5‐yl]‐2,4‐
dimethyl‐1H‐pyrrole‐3‐carboxylic acid (14c)
The product was obtained as off‐white solid; yield: 0.61 g, 83.4%; RF
(mobile phase hexanes/EtOAc 50:50): 0.70; m.p.: 159–161°C; 1H NMR
(500 MHz, DMSO‐d6): δ 11.04 (s, 1H, –NH), 7.76 (d, J = 7.2 Hz, 2H, Ar‐H),
7.42 (m, 3H, Ar‐H), 7.22 (d, J = 8.9 Hz, 2H, Ar‐H), 7.02 (d, J = 9.0 Hz, 2H,
Ar‐H), 5.32 (dd, J = 12.3, 9.6 Hz, 1H, –CHX), 3.80 (dd, J = 17.4, 12.5 Hz, 1H,
–CHAH), 3.14 (dd, J = 17.5, 9.4 Hz, 1H, –CHBH), 2.31 (s, 3H, pyrrole
–CH3), 2.20 (s, 3H, pyrrole –CH3); 13C NMR (126 MHz, DMSO‐d6): δ
148.65, 144.66, 135.60, 132.75, 129.30, 129.11, 129.06, 126.24, 125.67,
123.00, 117.18, 114.98, 56.84, 41.38, 13.91, 11.26; HRMS (ESI) calcd. for
[M+H]+ C22H21ClN3O2+: 394.1322, found: 394.1309.
2,4‐Dimethyl‐5‐[1‐methyl‐3‐(thiophen‐2‐yl)‐4,5‐dihydro‐1H‐
pyrazol‐5‐yl]‐1H‐pyrrole‐3‐carboxylic acid (14d)
5‐{3‐[3‐Fluoro‐4‐(trifluoromethyl)phenyl]‐1‐phenyl‐4,5‐dihydro‐1H‐
pyrazol‐5‐yl}‐2,4‐dimethyl‐1H‐pyrrole‐3‐carboxylic acid (14h)
The product was obtained as an off‐white solid; yield: 0.52 g, 83.0%;
RF (mobile phase hexanes/EtOAc 40:60): 0.70; m.p.: 127–129°C; 1H
NMR (500 MHz, DMSO‐d6): δ 11.54 (s, 1H, –COOH), 11.07 (s, 1H,
–NH), 7.89–7.71 (m, 3H, Ar‐H), 7.21 (t, J = 9.9 Hz, 2H, Ar‐H), 7.09 (d,
The product was obtained as an off‐white solid; yield: 0.42 g, 76.2%;
RF (mobile phase hexanes/EtOAc 50:50): 0.70; m.p.: 225–227°C; 1H
NMR (500 MHz, DMSO‐d6): δ 11.49 (br s, 1H, –COOH), 11.09 (s, 1H,
–NH), 7.54 (dd, J = 5.1, 0.9 Hz, 1H, thiophene‐H), 7.22 (dd, J = 3.5,

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J = 10.0 Hz, 2H, Ar‐H), 6.81 (t, J = 9.1 Hz, 1H, Ar‐H), 5.48 (dd, J = 12.7,
9.4 Hz, –CHX), 3.80 (dd, J = 17.6, 13.0 Hz, 1H, –CHAH), 3.16 (dd,
J = 8.4 Hz, 2H, Ar‐H), 7.03 (d, J = 8.4 Hz, 2H, Ar‐H), 5.32 (t, J = 10.5 Hz,
1H, –CHX), 4.07–3.89 (m, 2H, –NCH2), 3.82 (dd, J = 17.2, 12.7 Hz, 1H,
–CHAH), 3.12 (dd, J = 17.3, 9.1 Hz, 1H, –CHBH), 2.22 (s, 3H, pyrrole
–CH3), 2.13 (s, 3H, pyrrole –CH3); 13C NMR (126 MHz, DMSO‐d6): δ
166.61, 148.62, 144.59, 132.72, 130.73, 129.33, 129.12, 129.08,
126.61, 126.24, 125.87, 124.39, 122.99, 115.52, 114.98, 114.80,
56.82, 41.49, 12.83, 10.35; HRMS (ESI) calcd. for [M+H]+
C24H23ClF3N4O+: 475.1512, found: 475.1504.
J = 17.6, 9.3 Hz, 1H, –CHBH), 2.30 (s, 3H, pyrrole –CH3), 2.21 (s, 3H,
+
pyrrole –CH3); HRMS (ESI) calcd. for [M+H]+ C23H20F4N3O2
:
446.1491, found: 446.1495.
5‐{1‐(4‐Chlorophenyl)‐3‐[3‐fluoro‐4‐(trifluoromethyl)phenyl]‐4,5‐
dihydro‐1H‐pyrazol‐5‐yl}‐2,4‐dimethyl‐1H‐pyrrole‐3‐carboxylic
acid (14i)
The product was obtained as an off‐white solid; yield: 0.56 g, 82.9%; RF
(mobile phase hexanes/EtOAc 50:50): 0.70; m.p.: 151–153°C; 1H NMR
(500 MHz, DMSO‐d6): δ 11.56 (br s, 1H, –COOH), 11.07 (s, 1H, –NH),
7.80 (dt, J = 24.9, 8.1 Hz, 3H, Ar‐H), 7.26 (d, J = 8.9 Hz, 2H, Ar‐H), 7.06 (d,
J = 8.9 Hz, 2H, Ar‐H), 5.49 (dd, J = 12.7, 9.2 Hz, 1H, –CHX), 3.82 (dd,
J = 17.7, 13.0 Hz, 1H, –CHAH), 3.18 (dd, J = 17.6, 9.0 Hz, 1H, –CHBH), 2.30
(s, 3H, pyrrole –CH3), 2.20 (s, 3H, pyrrole –CH3); 13C NMR (126 MHz,
DMSO‐d6): δ 167.33, 160.63, 158.61, 146.03, 143.49, 139.76, 139.69,
136.24, 129.19, 128.11, 125.45, 124.27, 123.89, 122.27, 122.11, 117.61,
115.34, 114.03, 113.85, 110.87, 57.00, 40.72, 13.92, 11.20; HRMS (ESI)
calcd. for [M+H]+ C23H18ClF4N3O2+: 480.1102, found: 480.1093.
5‐(1‐Acetyl‐3‐phenyl‐4,5‐dihydro‐1H‐pyrazol‐5‐yl)‐2,4‐dimethyl‐N‐
(2,2,2‐trifluoroethyl)‐1H‐pyrrole‐3‐carboxamide (15d)
The product was obtained as an off‐white solid; yield: 0.48 g, 82.8%; RF
(mobile phase hexanes/EtOAc 50:50): 0.25; m.p.: 280–282°C; 1H NMR
(500 MHz, DMSO‐d6): δ 10.73 (s, 1H, –NH), 7.88–7.67 (m, 3H, Ar‐H,
–CONH), 7.48 (d, J = 3.4 Hz, 3H, Ar‐H), 5.47 (dd, J = 6.0 Hz, 1H, –CHX),
4.05–3.87 (m, 2H, –NCH2), 3.78 (dd, J = 18.0, 12.6 Hz, 1H, –CHAH), 3.15
(dd, J = 18.2, 6.0 Hz, 1H, –CHBH), 2.23 (d, J = 8.5 Hz, 6H, –NCOCH3,
pyrrole –CH3), 2.03 (s, 3H, pyrrole –CH3); HRMS (ESI) calcd. for [M+H]+
C20H22F3N4O2+: 407.1695, found: 407.1696.
5‐[1‐(Cyclobutanecarbonyl)‐3‐phenyl‐4,5‐dihydro‐1H‐pyrazol‐5‐yl]‐
2,4‐dimethyl‐N‐(2,2,2‐trifluoroethyl)‐1H‐pyrrole‐3‐
2,4‐Dimethyl‐5‐(1‐methyl‐3‐phenyl‐4,5‐dihydro‐1H‐pyrazol‐5‐yl)‐N‐
(2,2,2‐trifluoroethyl)‐1H‐pyrrole‐3‐carboxamide (15a)
carboxamide (15e)
The product was obtained as an off‐white solid; yield: 0.45 g, 83.3%; RF
(mobile phase hexanes/EtOAc 50:50): 0.60; m.p.: 117–119°C; 1H NMR
(400 MHz, DMSO‐d6): δ 10.91 (s, 1H, –NH), 7.74 (t, 1H, –CONH), 7.64
(d, 2H, J = 6.8 Hz, Ar‐H), 7.38 (t, J = 6.8 Hz, 2H, Ar‐H), 7.33 (t, J = 6.8 Hz,
1H, Ar‐H), 4.13 (m, J = 10.0 Hz, 1H, –CHX), 4.00 (m, 2H, –NCH2), 3.33
(m, 1H, –CHAH), 3.01 (t, J = 15.6 Hz, 1H, –CHBH), 2.66 (s, 1H, –NCH3),
2.27 (s, 3H, pyrrole –CH3), 2.07 (s, 3H, pyrrole –CH3); HRMS (ESI)
calcd. for [M+H]+ C19H22F3N4O+: 379.1745, found: 379.1749.
The product was obtained as an off‐white solid; yield: 0.54 g, 84.7%;
RF (mobile phase hexanes/EtOAc 50:50): 0.50; m.p.: 259–261°C; 1H
NMR (500 MHz, DMSO‐d6): δ 10.75 (s, 1H, –NH), 7.82–7.75 (m, 2H,
Ar‐H), 7.73 (t, J = 6.2 Hz, 1H, –CONH), 7.47 (d, J = 4.8 Hz, 3H, Ar‐H),
5.44 (dd, J = 12.4, 6.3 Hz, 1H, –CHX), 4.06–3.87 (m, 2H, –NCH2), 3.76
(m, 2H, –COCH, –CHAH), 3.12 (dd, J = 18.1, 6.3 Hz, 1H, –CHBH), 2.22
(s, 3H, pyrrole –CH3), 2.14 (m, 4H, –CHaH(CH2)CHaH), 2.03 (s, 3H,
pyrrole –CH3), 1.95 (dq, J = 18.3, 9.3 Hz, 1H, –COCCHbH), 1.74 (dt,
J = 16.2, 8.0 Hz, 1H, –COCCHbH); HRMS (ESI) calcd. for [M+H]+
C23H26F3N4O2+: 447.2008, found: 447.0765.
5‐(1,3‐Diphenyl‐4,5‐dihydro‐1H‐pyrazol‐5‐yl)‐2,4‐dimethyl‐N‐
(2,2,2‐trifluoroethyl)‐1H‐pyrrole‐3‐carboxamide (15b)
The product was obtained as an off‐white solid; yield: 0.50 g, 79.5%; RF
(mobile phase hexanes/EtOAc 40:60): 0.70; m.p.: 227–229°C; 1H NMR
(500 MHz, DMSO‐d6): δ 10.91 (s, 1H, –NH), 7.76 (d, J = 7.2 Hz, 3H,
–CONH), 7.44 (t, J = 7.5 Hz, 2H, Ar‐H), 7.38 (t, J = 7.1 Hz, 1H, Ar‐H), 7.18
(t, J = 7.8 Hz, 2H, Ar‐H), 7.06 (d, J = 8.0 Hz, 2H, Ar‐H), 6.76 (t, J = 7.2 Hz,
1H, Ar‐H), 5.29 (dd, J = 12.2, 9.7 Hz, 1H, –CHX), 4.08–3.87 (m, 2H,
–NCH2), 3.80 (dd, J = 17.3, 12.6 Hz, 1H, –CHAH), 3.10 (dd, J = 17.3, 9.5 Hz,
1H, –CHBH), 2.23 (s, 3H, pyrrole –CH3), 2.14 (s, 3H, pyrrole –CH3); 13C
NMR (126 MHz, DMSO‐d6): δ 166.67, 147.80, 145.91, 132.96, 130.58,
129.27, 129.10, 128.84, 126.62, 126.35, 126.12, 124.40, 122.18, 119.46,
115.47, 114.56, 113.65, 56.93, 41.35, 12.84, 10.38; HRMS (ESI) calcd. for
[M+H]+ C24H24F3N4O+: 441.1902, found: 441.1897.
2,4‐Dimethyl‐5‐[1‐methyl‐3‐(thiophen‐2‐yl)‐4,5‐dihydro‐1H‐
pyrazol‐5‐yl]‐N‐(2,2,2‐trifluoroethyl)‐1H‐pyrrole‐3‐
carboxamide (15f)
The product was obtained as an off‐white solid; yield: 0.45 g, 83.4%;
RF (mobile phase hexanes/EtOAc 50:50): 0.50; m.p.: 177–179°C; 1H
NMR (500 MHz, DMSO‐d6): δ 10.92 (s, 1H, –NH), 7.75 (t, J = 6.4 Hz,
1H, –CONH), 7.54 (d, J = 4.5 Hz, 1H, thiophene‐H), 7.22 (d, J = 2.8 Hz,
1H, thiophene‐H), 7.09 (dd, J = 4.9, 3.7 Hz, 1H, thiophene‐H), 4.13 (dd,
J = 14.9, 9.9 Hz, 1H, –CHX), 4.06–3.89 (m, 2H, –NCH2), 3.31 (s, 1H,
–CHAH), 3.06 (dd, J = 22.2, 8.7 Hz, 1H, –CHBH), 2.63 (s, 3H, –NCH3),
2.28 (s, 3H, pyrrole –CH3), 2.07 (s, 3H, pyrrole –CH3); HRMS (ESI)
calcd. for [M+H]+ C17H20F3N4OS+: 385.1310, found: 385.1309.
5‐[1‐(4‐Chlorophenyl)‐3‐phenyl‐4,5‐dihydro‐1H‐pyrazol‐5‐yl]‐2,4‐
dimethyl‐N‐(2,2,2‐trifluoroethyl)‐1H‐pyrrole‐3‐carboxamide (15c)
The product was obtained as an off‐white solid; yield: 0.53 g, 78.2%;
RF (mobile phase hexanes/EtOAc 50:50): 0.60; m.p.: 249–251°C; 1H
NMR (500 MHz, DMSO‐d6): δ 10.90 (s, 1H, –NH), 7.76 (d, J = 7.1 Hz,
3H, Ar‐H, –CONH), 7.42 (dt, J = 27.5, 7.1 Hz, 3H, Ar‐H), 7.23 (d,
2,4‐Dimethyl‐5‐[1‐phenyl‐3‐(thiophen‐2‐yl)‐4,5‐dihydro‐1H‐pyrazol‐
5‐yl]‐N‐(2,2,2‐trifluoroethyl)‐1H‐pyrrole‐3‐carboxamide (15g)
The product was obtained as an off‐white solid; yield: 0.49 g, 78.2%;
RF (mobile phase hexanes/EtOAc 50:50): 0.60; m.p.: 191–193°C; 1H
NMR (500 MHz, DMSO‐d6): δ 10.92 (s, 1H, –NH), 7.76 (t, J = 6.3 Hz,
1H, –CONH), 7.60 (d, J = 5.0 Hz, 1H, thiophene‐H), 7.27 (d, J = 3.3 Hz,

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1H, thiophene‐H), 7.18 (t, J = 7.5 Hz, 2H, Ar‐H), 7.12 (t, J = 4.1 Hz, 1H,
thiophene‐H), 6.99 (d, J = 8.1 Hz, 2H, Ar‐H), 6.76 (t, J = 7.2 Hz, 1H, Ar‐
H), 5.28 (t, J = 105 Hz, 1H, –CHX), 4.06–3.88 (m, 2H, –NCH2), 3.80
(dd, J = 17.1, 12.5 Hz, 1H, –CHAH), 3.10 (dd, J = 17.2, 9.6 Hz, 1H,
–CHBH), 2.23 (s, 3H, pyrrole –CH3), 2.14 (s, 3H, pyrrole –CH3);
13C NMR (126 MHz, DMSO‐d6) δ 166.64, 145.77, 144.35, 136.40,
130.63, 129.29, 128.84, 128.24, 127.74, 127.71, 126.62, 126.06,
124.39, 122.17, 119.55, 115.46, 114.65, 113.67, 57.10, 42.15, 12.83,
10.36; HRMS (ESI) calcd. for [M+H]+ C22H22F3N4OS+: 447.1466,
found: 447.1470.
5‐{3‐[3‐Fluoro‐4‐(trifluoromethyl)phenyl]‐1‐methyl‐4,5‐dihydro‐1H‐
pyrazol‐5‐yl}‐2,4‐dimethyl‐N‐(2,2,2‐trifluoroethyl)‐1H‐pyrrole‐3‐
carboxamide (15k)
The product was obtained as an off‐white solid; yield: 0.47 g, 88.3%;
RF (mobile phase hexanes/EtOAc 50:50): 0.70; m.p.: 167–169°C; 1H
NMR (500 MHz, DMSO‐d6): δ 10.94 (s, 1H, –NH), 7.89–7.71 (m, 2H,
Ar‐H, –CONH), 7.64 (t, J = 9.6 Hz, 2H, Ar‐H), 4.32 (dd, J = 14.9,
10.5 Hz, 1H, –CHX), 4.10–3.88 (m, 2H, –NCH2), 3.41 (dd, J = 16.4,
10.5 Hz, 1H, –CHAH), 3.03 (t, J = 15.7 Hz, 1H, –CHBH), 2.73 (s, 3H,
–NCH3), 2.28 (s, 3H, pyrrole –CH3), 2.08 (s, 3H, pyrrole –CH3); 13C
NMR (126 MHz, DMSO‐d6):
δ 166.68, 160.60, 158.61, 146.85,
5‐[1‐(4‐Chlorophenyl)‐3‐(thiophen‐2‐yl)‐4,5‐dihydro‐1H‐pyrazol‐5‐
yl]‐2,4‐dimethyl‐N‐(2,2,2‐trifluoroethyl)‐1H‐pyrrole‐3‐
140.24, 130.68, 128.84, 128.08, 126.62, 126.43, 124.40, 124.27,
122.94, 122.18, 122.11, 121.90, 119.94, 116.88, 115.45, 113.60,
113.43, 64.08, 40.62, 38.83, 12.83, 10.26; HRMS (ESI) calcd. for [M
+H]+ C20H20F7N4O+: 465.1525, found: 465.1528.
carboxamide (15h)
The product was obtained as an off‐white solid; yield: 0.54 g, 80.0%;
RF (mobile phase hexanes/EtOAc 50:50): 0.75; m.p.: 227–229°C; 1H
NMR (500 MHz, DMSO‐d6): δ 10.92 (s, 1H, –NH), 7.76 (t, J = 5.8 Hz,
1H, –CONH), 7.63 (d, J = 4.7 Hz, 1H, thiophene‐H), 7.30 (d, J = 2.6 Hz,
1H, thiophene‐H), 7.22 (d, J = 8.7 Hz, 2H, Ar‐H), 7.12 (t, J = 3.8 Hz, 1H,
thiophene‐H), 6.95 (d, J = 8.7 Hz, 2H, Ar‐H), 5.31 (t, J = 9.7 Hz, 1H,
–CHX), 4.07–3.89 (m, 2H, –NCH2), 3.82 (dd, J = 17.0, 12.6 Hz, 1H,
–CHAH), 3.12 (dd, J = 17.2, 9.3 Hz, 1H, –CHBH), 2.22 (s, 3H, pyrrole
–CH3), 2.13 (s, 3H, pyrrole –CH3); HRMS (ESI) calcd. for [M+H]+
C22H21ClF3N4OS+: 481.1076, found: 481.1076.
5‐{3‐[3‐Fluoro‐4‐(trifluoromethyl)phenyl]‐1‐phenyl‐4,5‐dihydro‐1H‐
pyrazol‐5‐yl}‐2,4‐dimethyl‐N‐(2,2,2‐trifluoroethyl)‐1H‐pyrrole‐3‐
carboxamide (15l)
The product was obtained as an off‐white solid; yield: 0.49 g, 81.2%;
RF (mobile phase hexanes/EtOAc 50:50): 0.70; m.p.: 191–193°C; 1H
NMR (500 MHz, DMSO‐d6): δ 10.89 (s, 1H, –NH), 8.10–7.56 (m, 4H,
Ar‐H, –CONH), 7.34–7.00 (m, 4H, Ar‐H), 6.81 (t, J = 6.7 Hz, 1H, Ar‐H),
5.45 (t, J = 10.3 Hz, 1H, –CHX), 3.97 (dd, J = 15.1, 10.1 Hz, 2H,
–NCH2), 3.81 (dd, J = 16.8, 13.4 Hz, 1H, –CHAH), 3.13 (dd, J = 17.3,
8.9 Hz, 1H, –CHBH), 2.22 (s, 3H, pyrrole –CH3), 2.13 (s, 3H, pyrrole
–CH3); HRMS (ESI) calcd. for [M+H]+ C25H22F7N4O+: 527.1682,
found: 527.1686.
5‐[1‐Acetyl‐3‐(thiophen‐2‐yl)‐4,5‐dihydro‐1H‐pyrazol‐5‐yl]‐2,4‐
dimethyl‐N‐(2,2,2‐trifluoroethyl)‐1H‐pyrrole‐3‐carboxamide (15i)
The product was obtained as an off‐white solid; yield: 0.52 g,
89.9%; RF (mobile phase hexanes/EtOAc 50:50): 0.30; m.p.:
221–223°C; 1H NMR (500 MHz, DMSO‐d6): δ 10.74 (s, 1H, –NH),
7.73 (t, J = 4.7 Hz, 2H, thiophene‐H, –CONH), 7.44 (d, J = 3.1 Hz, 1H,
thiophene‐H), 7.16 (t, J = 4.2 Hz, 1H, thiophene‐H), 5.47 (dd,
J = 12.1, 6.0 Hz, 1H, –CHX), 4.05–3.87 (m, 2H, –NCH2), 3.78 (dd,
J = 17.7, 12.5 Hz, 1H, –CHAH), 3.13 (dd, J = 17.8, 6.1 Hz, 1H,
–CHBH), 2.22 (s, 3H, –NCOCH3), 2.18 (s, 3H, pyrrole –CH3), 2.03 (s,
3H, pyrrole –CH3); HRMS (ESI) calcd. for [M+H]+ C18H20F3N4O2S+:
413.1259, found: 413.1252.
5‐{1‐(4‐Chlorophenyl)‐3‐[3‐fluoro‐4‐(trifluoromethyl)phenyl]‐4,5‐
dihydro‐1H‐pyrazol‐5‐yl}‐2,4‐dimethyl‐N‐(2,2,2‐trifluoroethyl)‐1H‐
pyrrole‐3‐carboxamide (15m)
The product was obtained as an off‐white solid; yield: 0.49 g, 76.2%;
RF (mobile phase hexanes/EtOAc 60:40): 0.50; m.p.: 139–143°C; 1H
NMR (500 MHz, DMSO‐d6): δ 10.88 (s, 1H, –NH), 7.79 (m, 4H,
–CONH, Ar‐H), 7.24 (dd, J = 8.5, 3.0 Hz, 2H, Ar‐H), 7.08 (dd, J = 8.5,
3.0 Hz, 2H, Ar‐H), 5.48 (dd, J = 16.0, 10.5 Hz, 1H, –CHX), 4.02–3.91
(m, 2H, –NCH2), 3.82 (dd, J = 22.0, 16.0 Hz, 1H, –CHAH), 3.13 (dd,
J = 22.5, 11.0 Hz, 1H, –CHBH), 2.21 (s, 3H, pyrrole –CH3), 2.11 (s, 3H,
pyrrole –CH3); HRMS (ESI) calcd. for [M+H]+ C25H21ClF7N4O+:
561.1292, found: 561.1294.
5‐[1‐(Cyclobutanecarbonyl)‐3‐(thiophen‐2‐yl)‐4,5‐dihydro‐1H‐
pyrazol‐5‐yl]‐2,4‐dimethyl‐N‐(2,2,2‐trifluoroethyl)‐1H‐pyrrole‐3‐
carboxamide (15j)
The product was obtained as an off‐white solid; yield: 0.56 g,
88.2%; RF (mobile phase hexanes/EtOAc 50:50): 0.40; m.p.:
231–233°C; 1H NMR (400 MHz, DMSO‐d6): δ 10.75 (s, 1H, –NH),
7.82–7.71 (m, 2H, –CONH, thiophene‐H), 7.42 (t, J = 3.5 Hz, 1H,
thiophene‐H), 7.15 (d, J = 5.0 Hz, 1H, thiophene‐H), 5.44 (dd,
J = 15.0, 7.5 Hz, 1H, –CHX), 4.00–3.92 (m, 2H, –NCH2), 3.73 (m, 2H,
–COCH, –CHAH), 3.11 (dd, J = 22.5, 9.3 Hz, 1H, –CHBH), 2.19 (s,
3H, pyrrole –CH3), 2.14 (m, 4H, –CHAH(CH2)CHAH), 2.02 (s, 3H,
pyrrole –CH3), 1.93 (dd, J = 18.3, 9.3 Hz, 1H, –COCCHBH), 1.73 (m,
1H, –COCCHBH); HRMS (ESI) calcd. for [M+H]+ C21H24F3N4O2S+:
453.1572, found: 453.1578.
5‐{1‐Acetyl‐3‐[3‐fluoro‐4‐(trifluoromethyl)phenyl]‐4,5‐dihydro‐1H‐
pyrazol‐5‐yl}‐2,4‐dimethyl‐N‐(2,2,2‐trifluoroethyl)‐1H‐pyrrole‐3‐
carboxamide (15n)
The product was obtained as an off‐white solid; yield: 0.48 g, 85.1%;
RF (mobile phase hexanes/EtOAc 50:50): 0.20; m.p.: 285–287°C; 1H
NMR (500 MHz, DMSO‐d6): δ 10.72 (s, 1H, –NH), 7.92–7.72 (m, 4H,
Ar‐H, –CONH), 5.52 (dd, J = 15.5, 7.5 Hz, 1H, –CHX), 4.05–3.92 (m,
2H, –NCH2), 3.78 (dd, J = 22.5, 15.5 Hz, 1H, –CHAH), 3.16 (dd,
J = 23.0, 7.5 Hz, 1H, –CHBH), 2.22 (s, 6H, –NCOCH3, pyrrole –CH3),
2.03 (s, 3H, pyrrole –CH3); 13C NMR (126 MHz, DMSO‐d6): δ 168.28,

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166.70, 160.52, 158.50, 152.09, 138.92, 129.75, 128.83, 128.27,
126.61, 125.95, 124.39, 124.09, 123.30, 122.16, 121.93, 115.45,
115.31, 115.13, 52.82, 40.48, 22.34, 12.76, 10.30; HRMS (ESI) calcd.
for [M+H]+ C21H20F7N4O2+: 493.1474, found: 493.1473.
Synergy HTX plate reader. The percentage of GI was calculated for each
well, using negative control (media only) and positive control (fungi
without inhibitors) on the same plate.
|
In vitro anticancer assay
5‐{1‐Acetyl‐3‐[3‐fluoro‐4‐(trifluoromethyl)phenyl]‐4,5‐dihydro‐1H‐
pyrazol‐5‐yl}‐2,4‐dimethyl‐N‐(2,2,2‐trifluoroethyl)‐1H‐pyrrole‐3‐
carboxamide (15o)
4.2.3
All demonstrated compounds were submitted to National Cancer In-
stitute (NCI), Bethesda, MD, and they were further evaluated for their
antiproliferative activity at the concentration of 10 µM in DMSO on a
panel of 60 cancer cell lines as per the standard protocol of NCI. The
solution of each compound was added to the microtiter culture plates
and kept for the incubation for 48 hr at 37°C. The endpoint of testing was
determined using a protein‐binding dye, that is, sulforhodamine B. The
growth percentage of each treated cell was determined by comparison
with the untreated control cells and the collective result was reported at
the NCI website.
The product was obtained as an off‐white solid; yield: 0.51 g, 83.6%; RF
(mobile phase hexanes/EtOAc 50:50): 0.60; m.p.: 259–261°C; 1H NMR
(400 MHz, DMSO‐d6): δ 10.73 (s, 1H, –NH), 7.92–7.72 (m, 4H, –CONH,
Ar‐H), 5.50 (dd, J = 16.0, 8.0 Hz, 1H, –CHX), 4.02–3.80 (m, 2H, –NCH2),
3.76 (m, 2H, –COCH, –CHAH), 3.13 (dd, J = 23.0, 10.5 Hz, 1H, –CHBH),
2.20 (s, 3H, pyrrole –CH3), 2.15 (m, 4H, –CHAH(CH2)CHAH), 2.03
(s, 3H, pyrrole –CH3), 1.94 (dq, J = 18.3, 9.6 Hz, 1H, –COCCHBH), 1.74
(m, 1H, –COCCHBH); 13C NMR (126 MHz, DMSO‐d6): δ 172.29,
166.70, 160.53, 158.51, 151.87, 139.03, 129.80, 128.83, 128.27,
126.61, 126.19, 126.06, 124.39, 124.10, 123.23, 122.17, 121.94,
119.78, 115.43, 115.25, 115.14, 53.07, 37.92, 25.22, 24.75, 18.16,
+
12.78, 10.21; HRMS (ESI) calcd. for [M+H]+ C24H24F7N4O2
:
4.2.4
In vitro cytotoxicity assay
|
533.1787, found: 533.1787.
The counted HEK293 cells in a Neubauer hemocytometer were plated in
the 384‐well plates containing the compounds to give a density of
5,000 cells/well in a final volume of 50 µl. Dulbecco's modified Eagle's
medium supplemented with 10% fetal bovine serum (FBS) was used as
growth media. Along with the compounds, HEK293 cells were incubated
for 20 hr at 37°C in 5% CO2. Cytotoxicity was measured by fluorescence,
ex: 560/10 nm, em: 590/10 nm (F560/590), after the addition of 5 µl of
25 µg/ml resazurin (2.3 µg/ml final concentration) and after incubation for
further 3 hr at 37°C in 5% CO2. The fluorescence intensity was measured
using a Tecan M1000 Pro monochromator plate reader, using automatic
gain calculation. CC50 values were calculated by curve fitting the inhibi-
tion values versus log (concentration) using a sigmoidal dose–response
function, with variable fitting values for the bottom, top, and slope. Cy-
totoxic samples were analyzed in duplicate.
|
4.2
Biological assays
|
4.2.1
In vitro antibacterial assay
A sample of each cultured bacterium was diluted 40‐fold in fresh broth
and incubated at 37°C for 1.5–3 hr. The resultant mid‐log phase cultures
were diluted (CFU/ml measured by OD600) and then added to each well
of the compound containing plates, giving a cell density of 5 × 105 CFU/ml
and a total volume of 50 µl. Covered plates were incubated at 37°C for
18 hr without shaking. The inhibition of bacterial growth was determined
by measuring absorbance at 600 nm (OD600), using a Tecan M1000 Pro
monochromator plate reader. The % GI was calculated for each well,
using the negative control (media only) and positive control (bacteria
without inhibitors) on the same plate as references.
|
4.2.5
Hemolysis assay
|
4.2.2
In vitro antifungal assay
The human whole blood was washed three times by three volumes of
0.9% sodium chloride solution and then suspended in the same to a
concentration of 0.5 × 108 cells/ml by calculating in a Neubauer he-
mocytometer. The washed cells were then added to 384‐well plates
containing the compound for a final volume of 50 µl. First, the plates
were shaken on a plate shaker for 10 min and then incubated for 1 hr
at 37°C. After incubation, the plates were centrifuged at 1,000 g for
10 min to pellet cells and debris, and 25 µl of the supernatant was
then transferred to a polystyrene 384‐well assay plate. HC10 and
HC50 (concentration at 10% and 50% hemolysis, respectively) were
calculated by curve fitting the inhibition values versus log (con-
centration) using a sigmoidal dose–response function with variable
fitting values for top, bottom, and slope. Hemolysis samples were
analyzed in duplicate.
Fungi strains were cultured for 3 days on yeast extract, peptone, and
dextrose (YPD) agar at 30°C.
yeast suspension of 1 × 106 to
A
5 × 106 CFU/ml (as determined by OD530) was prepared from five co-
lonies. The suspension was subsequently diluted and added to each well
of the compound‐containing plates giving a final cell density of fungi
suspension of 2.5 × 103 CFU/ml and a total volume of 50 µl. All covered
plates were incubated at 35°C for 24 hr without shaking. The GI of C.
albicans was determined by measuring absorbance at 530 nm (OD530),
whereas the GI of C. neoformans was determined by measuring the dif-
ference in absorbance between 600 and 570 nm (OD600–570), after the
addition of resazurin (0.001% final concentration) and incubation at 35°C
for an additional 2 hr. The absorbance was measured using a Biotek

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|
4.3
Molecular docking
[17] M. S. Muneera, J. Joseph, J. Photochem. Photobiol., B 2016, 163, 57.
[18] M. Amir, H. Kumar, S. A. Khan, Bioorg. Med. Chem. Lett. 2008,
18, 918.
A molecular docking study was performed by downloading Protein
Data Bank file 4AGD (resolution: 2.81 Å). Docking simulation was
employed using AutoDock Vina on PyRx,[43] whereas visualization of
the docking results was performed by the Discovery Studio program
following the standard procedure.
[19] Y. Lee, B. S. Kim, S. Ahn, D. Koh, Y. H. Lee, S. Y. Shin, Y. Lim, Bioorg.
Chem. 2016, 68, 166.
[20] S. Y. Shin, S. Ahn, H. Yoon, H. Jung, Y. Jung, D. Koh, Y. H. Lee, Y. Lim,
Bioorg. Med. Chem. Lett. 2016, 26, 4301.
[21] R. S. Joshi, P. G. Mandhane, S. D. Diwakar, S. K. Dabhade, C. H. Gill,
Bioorg. Med. Chem. Lett. 2010, 20, 3721.
[22] P. L. Zhao, W. Fu, M. Z. Zhang, Z. M. Liu, H. Wei, G. F. Yang, J. Agric.
Food Chem. 2008, 56, 10767.
ACKNOWLEDGMENTS
[23] B. Liu, S. Yan, L. Qu, J. Zhu, Cancer Cell. Int. 2017, 17, 1.
[24] J. Li, Q. Hao, W. Cao, J. V. Vadgama, Y. Wu, Cancer Manag. Res. 2018,
10, 4653.
The authors thank the Baburaoji Gholap Research Center for its support,
the Central Instrumental Facility (CIF) at Savitribai Phule Pune University
and the Indian Institute of Science Education and Research (IISER), Pune
for its analytical support. They are also thankful to the Developmental
Therapeutics Program, NCI, for in vitro anticancer evaluation and
CO‐ADD, which was funded by the Wellcome Trust (UK), and The Uni-
versity of Queensland (Australia) for cytotoxicity and hemolysis assay.
[25] R. F. George, M. Kandeel, D. Y. El‐Ansary, A. M. El Kerdawy, Bioorg.
Chem. 2020, 99, 103780.
[26] X. X. Tao, Y. T. Duan, L. W. Chen, D. J. Tang, M. R. Yang, P. F. Wang, C. Xu,
H. L. Zhu, Bioorg. Med. Chem. Lett. 2016, 26, 677.
[27] S. Y. Shin, H. Yoon, D. Hwang, S. Ahn, D. W. Kim, D. Koh, Y. H. Lee, Y.
Lim, Bioorg. Med. Chem. 2013, 21, 7018.
[28] K. M. Amin, S. M. Abou‐Seri, F. M. Awadallah, A. A. M. Eissa, G. S.
Hassan, M. M. Abdulla, Eur. J. Med. Chem. 2015, 90, 221.
[29] Y. J. Qin, Y. jing Li, A. Q. Jiang, M. R. Yang, Q. Z. Zhu, H. Dong, H. L.
Zhu, Eur. J. Med. Chem. 2015, 94, 447.
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
[30] N. K. Rasal, R. B. Sonawane, A. S. Choudhari, S. S. Chakraborty, D. D.
Sarkar, S. V. Jagtap, ChemistrySelect 2018, 3, 9571.
ORCID
Sangeeta V. Jagtap
http://orcid.org/0000-0002-5947-1013
[31] N. K. Rasal, R. B. Sonawane, S. V. Jagtap, Bioorg. Chem. 2020, 97, 103660.
[32] L. Sun, C. Liang, S. Shirazian, Y. Zhou, T. Miller, J. Cui, J. Y. Fukuda, J.
Y. Chu, A. Nematalla, X. Wang, H. Chen, A. Sistla, T. C. Luu, F. Tang, J.
Wei, C. Tang, J. Med. Chem. 2003, 46, 1116.
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https://doi.org/10.1002/ardp.202000267