Design, synthesis, in silico, and in vitro evaluation of 3-phenylpyrazole acetamide derivatives as antimycobacterial agents

Article abstract of DOI:10.1002/ardp.202000349

Mycobacterium tuberculosis (Mtb) is one of the most dangerous pathogens affecting immunocompetent and immunocompromised patients worldwide. Novel molecules, which are efficient and can reduce the duration of therapy against drug-resistant strains, are an

Full text of DOI:10.1002/ardp.202000349

Received: 18 September 2020
DOI: 10.1002/ardp.202000349
Revised: 12 November 2020
Accepted: 21 November 2020
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F U L L P A P E R
Design, synthesis, in silico, and in vitro evaluation of
3‐phenylpyrazole acetamide derivatives as
antimycobacterial agents
Nikhil B. Gaikwad1
Mohammad N. Ahmad2,3 | Grace Kaul2 | Manjulika Shukla2
Srinivas Nanduri1 | Arunava Das Gupta2 | Sidharth Chopra2
| Krishna Nirmale1 | Santosh K. Sahoo1
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Madhavi V. Yaddanapudi1
1Department of Pharmaceutical Technology
and Process Chemistry, National Institute of
Pharmaceutical Education and Research
(NIPER), Hyderabad, Telangana, India
Abstract
Mycobacterium tuberculosis (Mtb) is one of the most dangerous pathogens affecting
immunocompetent and immunocompromised patients worldwide. Novel molecules,
which are efficient and can reduce the duration of therapy against drug‐resistant
strains, are an urgent unmet need of the hour. In our current study, a series of new
2‐(3‐phenyl‐1H‐pyrazol‐1‐yl)acetamide and N′‐benzylidene‐2‐(3‐phenyl‐1H‐pyrazol‐1‐
yl)acetohydrazide derivatives were designed, synthesized, and evaluated for their
antimycobacterial potential. The biological evaluation revealed that 6b, 6m, 6l, 7a, and
7k exhibited selective and potent inhibitory activity against Mtb. Furthermore, com-
pounds 6m and 7h were found to be nontoxic to Vero cells with CC50 of greater
than 20 and 80 mg/ml, respectively, and exhibited promising selectivity indices (SI) of
greater than 666 and 320, respectively. All derivatives exhibited excellent ADME
(absorption, distribution, metabolism, and excretion) properties in silico. Also, all the
derivatives were found compliant with Lipinski's rule of five, showing their druggability
profile. Molecular docking insights of these derivatives have shown outstanding binding
energies on the mycobacterial membrane protein large transporters. These results
indicate that this scaffold may lead to a potential antimycobacterial drug candidate in
the discovery of antitubercular agents.
2Division of Microbiology, CSIR‐Central Drug
Research Institute, Lucknow, Uttar Pradesh,
India
3Academy of Scientific and Innovative
Research (AcSIR), Ghaziabad, Uttar Pradesh,
India
Correspondence
Madhavi V. Yaddanapudi, Department of
Pharmaceutical Technology and Process
Chemistry, National Institute of
Pharmaceutical Education and Research
(NIPER), Balanagar, Hyderabad,
Telangana 500037, India.
Email: yssmadhavi@gmail.com
K E Y W O R D S
2‐(3‐phenyl‐1H‐pyrazol‐1‐yl)acetamide, acetamide, ADMET studies, antitubercular,
Mycobacterium tuberculosis, pyrazole
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1
INTRODUCTION
as soon as the patient's immunity gets weaker. However, people with
LTBI remain a large human pool for TB, which implies that for the
Tuberculosis (TB), caused due to Mycobacterium tuberculosis (Mtb), is
primarily a lung disease but can attack any part of the body such
as the spine, brain, and kidney, hence causing extra‐pulmonary
tuberculosis.[1] In the majority of the patients, Mtb is present in a
latent form (LTBI), which, in turn, transmutes into an active disease
decline in the global TB burden, all forms of TB should be eradi-
cated.[2] This is exemplified by the fact that big eight countries tally
67% of the total patients: India (27%), China (9%), Indonesia (8%),
Pakistan (6%), Philippines (6%), Bangladesh (4%), Nigeria (4%), and
South Africa (3%). WHO announced “The End TB strategy” to
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Arch Pharm. 2020;e2000349.
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eradicate TB from the world by 2035.[3] India's ambitious goal of
ending TB by 2025 is larger and bigger, with India accounting for the
majority of TB patients.[4] Among many factors, drug resistance is a
big impediment in achieving the global targets in due time; thus there
is an urgent unmet demand for newer, better, and novel anti-
mycobacterials to overcome resistance and help eradicate TB.
Over the years, many scaffolds have been explored and developed
against Mtb, that is, pyridine (isoniazid, ethionamide, and prothionamide),
pyrazine (pyrazinamide and clofazimine), quinoline[5] (ofloxacin, levo-
floxacin, moxifloxacin and bedaquiline), isoxazolinone (linezolid, sutezolid,
teriziidone, cycloserine, tedizolid, and LCB01–0371 [delpazolid]).[6,7]
Many of these are given as first‐line and second‐line drugs for the
treatment of TB but increasingly are facing resistance, due to which
there is a negative impact on mortality and morbidity. Thus, the identi-
fication of new scaffolds is an urgent unmet requirement to combat drug
resistance. One such scaffold is pyrazole, which in recent years has
gained attention with its antitubercular potential.[8–13] Various anti-
mycobacterial agents based on C3 and C5 substituted phenylpyrazole
(Figure 1) was reported earlier with antimycobacterial activity.[19]
Also, another interesting amide‐based pyrazole derivative called
rimonabant (Figure 1), a CB1 receptor modulator was found to have
antimycobacterial activity, acting as an MmpL3 inhibitor. Further, the
derivatives of rimonabant were found to have a better potency than
rimonabant.[20] Antitubercular agents based on cyclohexyl‐indole‐2‐
carboxamide were reported in 2013.[21] These derivatives were proved
to be the primary hits, which led to more potent derivatives reported
later.[22,23] With regard to the biological significance of an acetamide
bridge between pyrazole and amine in antimycobacterial activity
and also as
a
part of our ongoing research on pyrazole‐based
new composite
antimycobacterial agents, we have proposed
a
structure of 2‐(3‐phenyl‐1H‐pyrazol‐1‐yl)acetamide followed by its anti-
mycobacterial evaluation. The rationale behind the design was based on
the exceptional activity reported for the repurposed drug rimonabant on
the MmpL3 receptor. But molecules with a low selectivity index (SI) may
be harmful to the central nervous system (CNS). The diaryl moieties of
rimonabant made the molecule hydrophobic because of which it passes
the blood–brain barrier (BBB). Hence, we have modified the scaffold
with a single aromatic ring, and acetohydrazone was chosen as the linker
bridge, which rightfully fits between an amine and pyrazole moiety with
moderate hydrophilicity along with good binding interactions of the
molecule. Hence, substituted phenyl pyrazolyl acetohydrazones were
designed and synthesized (Figure 2). But the synthesized molecules
were found inactive against the ESKAP panel and Mtb H37Rv. Hence,
the scaffold was further modified and the acetohydrazone linker was
replaced with acetamide (Figure 2). The synthesized pyrazole‐based
acetamides were tested against the ESKAP pathogen panel and Mtb
H37Rv. Many of the compounds showed excellent activity, which helped
derivatives have been reported previously.[10,12–14] Compound
I
(Figure 1) with InhA inhibition activity was found active against both
sensitive as well as drug‐resistant Mtb strains.[15] 3‐(4‐Chlorophenyl)
pyrazole derivative II (Figure 1) carrying a substituted azetidinone moi-
ety demonstrated submicromolar activity.[14] Quinolinyl pyrazole hy-
brids, such as III, illustrated excellent antitubercular activity[16,17] and
quinoline‐based hybrid derivatives with pyrazole have also shown
promising antitubercular activity.[18] Hybrid molecules based on
pyrazole and triazole moiety linked with acetamide (IV) (Figure 1)
were reported to inhibit nonpathogenic Mycobacterium smegmatis.[9]
Quinazoline‐ and benzimidazole‐based acetamide derivative
V
FIGURE 1 Pyrazole and acetamide scaffolds with antimycobacterial activity along with the proposed hybrid pyrazole acetamide scaffold

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FIGURE 2 The rationale for the design of
2‐(3‐phenyl‐1H‐pyrazol‐1‐yl)acetamide
derivatives as a new antitubercular agent
to evaluate the primary structural prerequisite for the potency of
compounds (structure–activity relationship [SAR]). The most active mo-
lecules were additionally tested against a panel of drug‐resistant strains
of Mtb H37Rv and cytotoxicity against Vero cells to determine their
drug‐like potential. Further literature scouting on the proposed structure
illustrated that there are no reports available on 2‐(3‐phenyl‐1H‐pyrazol‐
1‐yl)acetamide framework with anti‐Mtb properties.
coupling to get acetamide derivatives (Scheme 1), although genera-
tion of the acid group involves four substeps. In the first step, re-
spective nitroacetophenones were converted to dimethylamino
enaminones (1) by using N,N‐dimethylformamide dimethyl acetal by
the reported procedure.[24] Pyrazole derivatives (2) were ob-
tained from substituted enaminones when treated with 35% hy-
drazine hydrate in the presence of ethyl alcohol in good yields.[25]
The obtained pyrazole 2 was alkylated with ethyl bromoacetate in
the presence of potassium carbonate in N,N‐dimethylformamide, and
stirring for 72 h at 70°C as per the reported protocols, which gave a
white solid product with 90–92% yields.[26,27] The acetate obtained
was hydrolyzed by using lithium hydroxide in tetrahydrofuran and
water mixture in a 1:1 ratio at room temperature for 20 h to yield the
desired 2‐(3‐nitrophenyl‐1H‐pyrazol‐1‐yl)acetic acid with 98%
yield.[27] Lastly, 2‐(3‐phenyl‐1H‐pyrazol‐1‐yl)acetic acid was coupled
with different amine groups to yield acetamide derivatives as a final
product. 1‐Ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC.HCl)
along with 4‐dimethylaminopyridine (DMAP) was used as a coupling
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
Designed molecules were synthesized, as shown in Scheme 1.
All schemes have common starting material, which is substituted
ethyl 2‐(3‐phenyl‐1H‐pyrazol‐1‐yl)acetate. The series
6 and 7
were synthesized in two important steps, mainly generation of
2‐(3‐phenyl‐1H‐pyrazol‐1‐yl)acetic acid and finally acid‐amine
SCHEME 1 Synthetic route to
compounds of series 6 and 7

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agent, leading to products with 61–87% yields (Figure 3). In series
9, 10, and 11, ester 4 was converted to respective pyrazolyl acet-
ohydrazides (Scheme 2). The final step involves the conversion of
acetohydrazides to hydrazones by the reaction of substituted alde-
hydes (Figure 4) in the presence of catalytic acetic acid in ethanol. It
is significant to refer that amine substituents and aldehyde for each
series were selected based on excellent antimycobacterial results
reported in the literature.[28,29] Spectroscopic and spectrometric
data were acquired for all the synthesized compounds (presented in
Supporting Information).
To exemplify, one of the potent derivatives 6m in 1H NMR
(nuclear magnetic resonance) has shown distinctive sharp
singlet peaks of the methylene proton and amide proton at 5.15
and 10.57 ppm, respectively, which ensured the formation of
amide –NH– bond. The proton of pyrazole C‐5 –CH‐ appeared as a
doublet at 7.93 ppm and also pyrazole C‐4 –CH– proton appeared as
a doublet at 6.99 ppm. Protons on phenyl ring attached to pyrazole
appeared as a doublet at 8.26 and 8.08 ppm belonging to meta and
ortho to pyrazole substitution, respectively; similarly, phenyl ring
protons ortho and meta to amine also appeared as doublets at
7.65 and 7.41 ppm, respectively. Likewise, 13C NMR interpretation of
derivative 6m designates the existence of carbonyl carbon of the
amide group at 165.8 ppm. The methylene (–CH2–) carbon bridging
amide and pyrazole was observed at 79.6 ppm. C‐3, C‐4, and C‐5
pyrazole carbon appeared correspondingly at 148.9, 104.8, and
127.7 ppm. Remaining carbons at the ipso, ortho, meta, and para po-
sitions of the phenyl ring attached to pyrazole appeared at 146.8,
126.3, 124.6, and 140.1 ppm, respectively. Lastly, the ipso, ortho,
meta, and para carbons of the phenyl ring attached to amine were
observed at 137.9, 121.2, 129.3, and 134.7 ppm, respectively.
FIGURE 3 Synthesized molecules of series 6 and 7 with their respective yields

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SCHEME 2 Synthetic route to compounds of
series 9, 10, and 11
1H NMR for the compound 9c from the hydrazone series showed
two singlets of the characteristic methyl of the dimethoxy group at
3.78 and 3.81 ppm. The singlet of the –NH– of the hydrazide group
was present at 11.93 ppm along with –CH– of the amine group at
8.09 ppm, which confirmed the synthesis of the hydrazone moiety.
The two hydrogens from –CH2– of the acetamide were observed at
5.46 ppm as a singlet. The rest of the aromatic protons of pyrazole
appeared as doublets at 6.70 and 7.77 ppm, which correspond to
hydrogens at fourth and fifth position of pyrazole, respectively, and
the aromatic protons were present as doublets at 6.97 and 7.31 ppm,
depicting fifth and sixth position, respectively, and a singlet at
7.33 ppm depicting second position of the phenyl ring attached to
pyrazole. Two doublets at 7.91 and 7.95 ppm correspond to meta and
ortho protons of the hydrazine phenyl ring. Finally, all the acetamide
derivatives were characterized by 1H, 13C NMR, high‐resolution
mass spectrometry (HRMS), and Fourier transform infrared spec-
trometry (FTIR) and are in accordance with the pertinent structures
as presented in the experimental section. The HRMS (electrospray
ionization) data of each compound represented as an [M+H]+ or
[M+Na]+ peaks comply with their precise molecular formula.
levofloxacin (LEVO) as reference. All the molecules of the five series
were found inactive on the ESKAP pathogen panel, except compound
11c, which showed a minimum inhibitory concentration (MIC) of
32 μg/ml on Staphylococcus aureus; due to less potency, this molecule
was not considered for further studies. Further antimycobacterial
screening displayed that all molecules of the series 6 and 7 demon-
strated good to excellent activity against Mtb and moderate activity
against NTM and were ineffective against ESKAP pathogen panel,
suggesting the selective inhibition of designed molecules toward slow‐
growing mycobacteria (Table 1). Upon analysis, cycloalkyl amide de-
rivatives were found to have excellent activity than corresponding
aromatic amide derivatives. The antimycobacterial activity was found
to be dependent on the substituents on the amine group and position
of nitro group on phenyl ring attached to the pyrazole acetic acid.
Cyclopropyl, cyclohexyl, and cycloheptyl amides were studied and all
three were found to have excellent activity against Mtb but their
activities against NTM pathogens varied with ring size. Cyclohexyl and
cyclopropyl amide derivatives of respective series exhibited activity
against Mtb, M. fortuitum, and M. chelonae with MIC comparable to the
reference compound (isoniazid, levofloxacin, and rifampicin) used for
testing (Table 2). Although cycloheptyl amide derivatives 6c and 7c of
both series were active on Mtb but found inactive against NTM pa-
thogens. This suggested that the ring size was affecting the selectivity
against these strains. Moreover, in aromatic amide derivatives, sub-
stituents on the phenyl ring were crucial for activity. Variations in
aromatic acetamide derivatives were studied by using electron‐
withdrawing, electron‐donating, or aliphatic group substitutions at
different positions of a phenyl group attached to amine, which led to
molecules with good to excellent anti‐TB activity and the positions of
these substituents on the phenyl ring were found to be crucial for
activity. Para‐substituted derivatives 6a, 6e, 6f, 6h, 6k, 6m, 7f, and 7h
|
2.2
Pharmacology/biology
|
2.2.1
M. tuberculosis H37Rv inhibition studies
All synthesized derivatives 6a–n, 7a–l, 9a–j, 10a–c, and 11a–c were
assessed for their antibacterial activity against ESKAP pathogen panel
by using broth microdilution assay as well as against Mtb H37Rv and
other mycobacterial pathogens (nontuberculous mycobacteria [NTM])
by using a whole‐cell assay with isoniazid (INH), rifampicin (RIF), and

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FIGURE 4 Synthesized molecules of series 9, 10, and 11 with their respective yields
were well tolerated for activity compared to ortho‐ and meta‐sub-
stituted derivatives. It is noteworthy that any substitution on
meta position reduced antimycobacterial potential and ortho sub-
stitution leads to complete loss of activity. In summary, aromatic
derivatives displayed lesser potency as compared to aliphatic deriva-
tives, disubstituted derivatives exhibited similar results to those of
monosubstituted derivatives. Despite having identical substitutions,
6e and 7e displayed disparate activity, showing that the position of
nitro group on pyrazole ring had significant influence in determining
activity. Meta nitro derivative 7e had excellent activity against Mtb
H37Rv compared to 6e but was inactive against NTM strains. Unlike
6e, when the chloro group of 7e was replaced by the NO2 group in 7k,
it was found to elevate potency. To study the significance of hydro-
phobic moieties, naphthylamine, aniline, and trifluoromethylaniline
acetamides were evaluated (compounds 6i, 6l, 6o, and 7i), which
displayed MIC > 64 μg/ml, which indicates that cycloalkyl amines are
more effective than alkyl and aromatic amines. The correlation of
ClogP and MIC values are given in Figure 5. The position of the nitro
group also affects the activity, which was studied by shifting the po-
sition of the nitro group from para to meta in 6c and 6f compounds,
which, in turn, led to partial to complete loss of activity, indicating the
importance of the group and its position. The SAR parameters dis-
cussed are summarized below (in Figure 6).
|
2.2.2
Cytotoxicity against Vero cells
A cytotoxicity assay was performed against Vero cells (African green
monkey kidney cell line; ATCC CCL‐81) using MTT assay (3‐(4,5‐
dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) to assess the

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TABLE 1 Minimum inhibitory concentration (mg/ml) of 2‐[3‐(4‐nitrophenyl)‐1H‐pyrazol‐1‐yl]acetamides 6a–n and 2‐[3‐(3‐nitrophenyl)‐1H‐
pyrazol‐1‐yl]acetamides 7a–l against ESKAP pathogen panel and Mycobacterium tuberculosis
Gram −ve
Staphylococcus aureus Escherichia coli Klebsiella pneumoniae Acinetobacter baumannii Pseudomonas aeruginosa tuberculosis
Gram +ve
Mycobacterium
Entry
6a
6b
6c
6d
6e
6f
ATCC 29213
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
ATCC 25922
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
BAA 1705
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
BAA 1605
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
ATCC 27853
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
H37Rv
0.06
0.06
0.06
>64
16
0.12
>64
0.12
4
6g
6h
6i
6j
>64
>64
0.06
0.06
>64
0.12
0.06
>64
>64
0.06
>64
>64
0.25
>64
>64
0.12
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
6k
6l
6m
6n
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
10a
(Continues)

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TABLE 1 (Continued)
Gram −ve
Gram +ve
Mycobacterium
Staphylococcus aureus Escherichia coli Klebsiella pneumoniae Acinetobacter baumannii Pseudomonas aeruginosa tuberculosis
Entry
10b
10c
ATCC 29213
ATCC 25922
>64
BAA 1705
>64
BAA 1605
>64
>64
>64
>64
>64
8
ATCC 27853
H37Rv
>64
>64
>64
>64
>64
NT
>64
>64
>64
>64
32
>64
>64
>64
>64
>64
1
>64
>64
11a
11b
11c
>64
>64
>64
>64
>64
>64
Levofloxacin <0.5
<0.5
NT
64
Isoniazid
NT
NT
NT
NT
NT
0.06
0.03
Rifampicin
NT
NT
NT
NT
Abbreviation: NT, not tested.
effect of active derivatives 6b, 6f, 6h, 6l, 6m, 7a, 7b, 7h, and 7k on
mammalian cells. The results were denoted as CC50, that is, the
lowest concentration of test compound which results in a 50% re-
duction in cell viability. All the test samples were tested in triplicate
and doxorubicin was used as a reference. Cytotoxicity examination
revealed that 6b, 6f, 6h, 6l, 6m, 7a, 7h, and 7k were found to be
nontoxic to Vero cells (CC50 were ≥05 to ≥10 mg/ml) and exhibited a
promising SI of ≥10 to ≥666. These observed results are presented in
Table 3.
proteins, the antimycobacterial potencies of respective molecules
were assessed. The MmpL3 docking scores were found to be in line
with the biological activity. The molecular insights of acetamide
derivatives revealed that the pyrazole ring has hydrophobic
π–π stacking interaction with TYR646 of MmpL3 protein. The
amide –NH– group has a hydrogen bond with the ASH645; along
with it, the phenyl ring on amine has π–π stacking and π–cation
interactions with PHE649 and PHE260. The nitrophenyl ring on
the pyrazole is found to enhance the binding of the molecules to
the receptor pocket by forming a hydrogen bond with ARG653 and
THR289 amino acids of backbone protein. The phenyl ring at-
tached to the pyrazole ring was found to form the π–π stacking
interactions with TYR257, and the nitro group had hydrogen
bonding with THR289 through oxygen and π–cation interaction
with PHE260 through nitrogen. Also, the majority of molecules
have charged negative interactions with ARG653 and charged
positive interactions with ASH256 and ASH645 (few molecules
with GLU647), also glycine interactions with GLY641, GLY252,
GLY258. The compounds were found to establish hydrophobic
(nonpolar) interactions with PHE260, TYR257, ILE253, LEU686,
LEU708, ILE249, ALA637, VAL638, LEU642, TYR646, ILE297,
VAL648, PHE649, VAL290, and hydrophilic polar interactions with
SER293, SER300, SER301, THR289, THR644, SER643, SER263,
and SER286 amino acid residues of the protein (Figure 7a–c). To
comprehend the detailed outcomes, the most active ligand of each
series 6a and 7a were superimposed and the resultant poses
disclosed that the structural features of both series were super-
imposed similarly to each other, signifying the probable binding
modes of these molecules. The nitro group position on both these
series was different, which led to a different interaction of the
nitro group of meta derivative, that is, series 7 molecules. Other
interactions were found correlating to each other in both the
series. The consequent superimposed poses of the potent ligand
6a and 7a suggested that acetamide functionality of 7h engaged
identically to 6m acetamide group (Figure 7e). The docking scores,
along with their bonding aspects, are presented in Table 4.
|
2.3
In silico studies
|
2.3.1
Molecular docking studies
Exemplary mycobacterial inhibition inspired us to investigate the
binding pattern of these molecules by using molecular docking
studies to understand the molecular requirements for the biolo-
gical activity. Thus, the structural features and previous reports
on pyrazoles[30–33] and especially amide derivatives[34,35] like
rimonabant formed the basis for the design of these molecules.
Rimonabant is a repurposed drug for tuberculosis, which is a pyr-
azole derivative with amide functionality inhibiting the MmpL3
receptor. All designed derivatives that were found active on Mtb
H37Rv were screened on all the transmembrane receptors of the
M. tuberculosis (KasA, DprE1, InhA, and MmpL3). These proteins
play a crucial role in the biosynthesis as well as transportation of
mycolic acid in Mtb. Docking studies were performed on four PDB
IDs of each receptor protein, KasA (PDB ID: 5W2Q), DprE1 (PDB
ID: 4P8H), InhA (PDB ID: 4TZK), and MmpL3 (PDB ID: 6ajj)
(docking results included in the Supporting Information). The
molecular docking results on all these receptor proteins revealed
that the acetamide derivatives have a lesser binding affinity
toward KasA and DprE1 proteins and moderate binding affinity
toward InhA protein and best affinity toward MmpL3 receptor.
After the results of molecular docking on InhA and MmpL3

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TABLE 2 In vitro activities of 2‐[3‐(4‐
nitrophenyl)‐1H‐pyrazol‐1‐yl]acetamides
6a–n and 2‐[3‐(3‐nitrophenyl)‐1H‐pyrazol‐
1‐yl]acetamides 7a–l against
Mycobacterium tuberculosis H37Rv,
M. abscessus, M. fortuitum, M. chelonae,
and ClogP values correlation
Minimum inhibitory concentration
Entry
ClogPa
4.43
3.27
3.83
4.07
4.30
3.17
3.50
3.50
4.27
4.22
3.09
2.55
4.07
3.29
1.83
3.27
3.83
4.07
4.30
3.17
3.50
3.50
4.27
4.22
3.33
3.93
−0.51
‐
H37Rv
0.06
0.06
0.06
>64
16
M. abscessus
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
1
M. fortuitum
>64
1
M. chelonae
>64
1
6a
6b
6c
>64
>64
16
>64
>64
32
6d
6e
6f
0.12
>64
0.12
>64
>64
>64
0.06
0.06
>64
0.12
0.06
>64
>64
0.06
>64
>64
0.25
>64
>64
0.12
>64
NT
2
4
6g
>64
2
>64
2
6h
6i
>64
>64
>64
1
>64
>64
>64
1
6j
6k
6l
6m
1
2
6n
>64
2
>64
2
7a
7b
1
1
7c
>64
>64
>64
2
>64
>64
>64
2
7d
7e
7f
7g
>64
8
>64
8
7h
7i
>64
>64
2
>64
>64
2
7j
7k
7l
>64
0.03
NT
0.5
>64
0.03
NT
0.5
Levofloxacin
Rifampicin
Isoniazid
0.06
0.03
NT
−0.67
NT
Abbreviation: NT, not tested.
aClogP were calculated by ChemBioDraw Ultra, version 14.0.0.117.
|
2.3.2
Prediction of ADME (absorption,
the derivatives were found to be within the mentioned standard
parameters. Lipinski's rule is the most frequently used thumb rule
to decide the drug likeness of the molecules. The rule illustrates
molecular features that are significant for pharmacokinetics of
desired molecules in the human body, based on their absorption,
distribution, metabolism, and excretion (ADME). Most recently, T is
inserted at the end and that becomes ADMET in which T denotes
the toxicity profile of the drug molecule. According to Lipinski's
rule, physicochemical descriptors should be within the range, like
logP values should be less than or equal to 5, molecular weight less
distribution, metabolism, and excretion) properties
To establish the drug likeliness topographies, ADME parameters of
designed pyrazole acetamides were determined by using the
QikProp module of the Schrödinger suite. A large margin of drugs
have the physicochemical properties in a certain range, such as
hydrogen‐bond acceptor (accept HB) 2–20, hydrogen‐bond donor
(donors HB) 0–6, molecular weight 130–725, and predicted QP
logPo/w −2 to 6.5.[36] The projected drug likeness properties of all

10 of 20
GAIKWAD ET AL.
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FIGURE 5 Correlation between minimum inhibitory concentration (MIC) and ClogP values of pyrazole series
than or equal to 500, H‐bond acceptor ≤ 10, and H‐bond donors ≤ 5.
Thus, the molecule not obeying two or more of these parameters
will lead to poor absorption or permeation.[37,38] When all the
26 compounds were studied for the Lipinski rule of five, it was
observed that all 26 compounds did not violate any parameters.
QPPCaco is a parameter to predict Caco‐2 cell permeability of
molecule in nm/s (>500 is great absorption, <25 is poor absorption).
It is the human absorption process by the nonactive transport
mechanism across the gut–blood barrier.[39] The predicted
QPPCaco value for acetamide derivatives illustrates that designed
compounds certainly absorb through the gut layer. For the target
molecule to become CNS active, it must have the CNS value more
than or equal to 2 (–2 [inactive] to +2 [active]).[39,40] The predicted
value for the CNS was in the range of −1 to −2, thus indicating that
these molecules would be inactive on CNS. The logBB values help to
determine the distribution of the compound in the brain. If the
compound is having a log BB greater than 0.3 it will cross the BBB,
and if it is less than −1.0 those compounds will be weakly dis-
tributed to the brain. The Qikprop determined logBB value of the
selected molecules was in the range of −1 to −2, which suggested
that these compounds will not cross the BBB; thus, no central
nervous system activity will be observed. Furthermore, the QPlog
HERG value denotes the predicted IC50 value for the blockage of
HERG K+ channels. Designed compounds demonstrated good
FIGURE 6 Structure–activity relationship of acetamide derivatives of pyrazole

GAIKWAD ET AL.
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|
FIGURE 7 (a) Compound 6a molecular binding interactions with the binding site pocket. The pink solid arrow represents the H‐Bond
interaction with the receptor protein backbone. (b) Compound 6m interactions with the binding site pocket. The green solid line represents π–π
stacking interactions. (c) Compound 7a molecular binding interactions with the binding site pocket. The pink solid arrow represents the H‐bond
interaction with the receptor protein backbone. (d) Compound 7b in the binding pocket of MmpL3 protein. (e) Molecular docking
representation showing the superimposition of 6a (blue) and 7a (red) ligand interactions in receptor binding pocket of MmpL3 protein
(PDB ID: 6ajj). Dashed lines represent hydrogen bonds. (f) Interactions of reference ligand in the binding pocket, the hydrogen bond
between amide –NH– and ASH645 and C═O and TYR646
predicted pharmacological properties like HERG K+ channel (HERG
K+), QPlogKhsa, which predicts binding to human serum albumin,
QPPMDCK predicts MDCK cell permeability. MDCK cells are good
mimic for the BBB, and QP logKp predicts the skin permeability of
the molecule. Also, the percent absorption values of the majority of
the compounds were found to be in the range of 85–100. Only one
compound had a % absorption of 78% (7a), which is because of the
aliphatic ring present in the molecule. Values of predicted de-
scriptors are presented in Table S2 in ESI. The predicted values of
the descriptors suggest that all the compounds were following good
ADME properties. The active molecules of both series were
showing a good ADME profile, which suggests that these molecules
can be taken for further biological evaluation to develop potential
antitubercular agents.
TABLE 3 Cytotoxicity profile against
Vero cells and selectivity index (SI) of
Mycobacterium
Mycobacterium
Mycobacterium tuberculosis
fortuitum
chelonae
CC50
selected compounds on various
Entry
6b
6f
(µg/ml)
MIC (µg/ml)
0.06
SI (CC50/MIC)
MIC
SI
MIC
1
SI
mycobacterial strains
>10
20
>166
160
1
2
2
1
1
2
1
8
2
>10
10
>10
5
0.125
0.125
0.06
4
6h
6l
>20
>10
>20
>20
>6.25
>80
>20
>160
>166
>666
>166
>104
>320
>166
>10
>10
>20
>10
>6.25
>10
>10
2
>10
>10
>10
>10
>6.25
>10
>10
1
6m
7a
0.06
2
0.12
2
7b
7h
7k
0.06
1
0.25
8
0.12
2
Abbreviation: MIC, minimum inhibitory concentration.

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GAIKWAD ET AL.
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TABLE 4 Docking scores, binding
energy, H‐bond interactions for few potent
acetamide derivatives
H‐Bonding
Entry
6l
Docking score
−10.569
−10.372
−11.915
−10.569
−10.972
−8.497
Binding energy
−60.422
−73.17
Ligand
Amino acid
Amide –N–H
Amide –N–H
Amide –N–H
Amide –N–H
Amide –N–H
Amide –N–H
Nitro –N═O
Amide –N–H
Nitro –N═O
Amide –N–H
Amide –N–H
Nitro –N═O
Amide –N–H
ASH645
6h
6i
ASH645
−83.349
−60.422
−73.161
−56.445
ASH645
6l
ASH645
6m
7a
ASH645
ASH645
THR289, ARG653
ASH645
7h
−10.445
−81.294
THR289, GLY192
ASH645
7i
7l
−11.915
−10.538
−83.349
−78.289
ASH645
ARG653, THR289
ASH645
Cocrystal ligand
−11.980
−43.067
|
|
3
CONCLUSION
4
EXPERIMENTAL
Chemistry
|
In summary, a new series of compounds based on 2‐[3‐(4‐nitrophenyl)‐
1H‐pyrazol‐1‐yl]acetamides 6a–n, 2‐[3‐(3‐nitrophenyl)‐1H‐pyrazol‐1‐yl]
acetamides 7a–l, N′‐benzylidene‐2‐[3‐(3,4‐dimethoxyphenyl)‐1H‐pyra-
zol‐1‐yl]acetohydrazides 9a–j, N′‐benzylidene‐2‐[3‐(4‐nitrophenyl)‐1H‐
pyrazol‐1‐yl]acetohydrazides 10a–c, N′‐benzylidene‐2‐[3‐(3‐nitrophenyl)‐
1H‐pyrazol‐1‐yl]acetohydrazides 11a–c were designed, synthesized, and
their in vitro antimycobacterial activity and the cytotoxicity in Vero cells
was evaluated. The synthesis of these molecules is simple and effortless
with readily available reactants, a painless method of purification, and a
satisfying amount of yields, which makes the scheme attractive. Fur-
thermore, many of the synthesized derivatives exhibited potent and se-
lective activity against various Mtb strains on par with the first‐line drugs
INH and RIF and were nontoxic to mammalian cells. Only compound 11c
showed weak antibacterial activity against S. aureus, while the rest of all
molecules were found to be inactive on the ESKAP pathogen panel.
Compounds 6a, 6b, 6c, 6l, 6m, and 7b exhibited excellent anti-
mycobacterial activity with MIC of 0.06 μg/ml and can be considered the
lead structures among the synthesized derivatives. Also, the compounds
6m and 7h exhibited excellent SI of >666 and >320, respectively. In silico
studies have demonstrated that the synthesized derivatives also dis-
played lower binding free energy than cocrystal ligand. Also, compounds
6c, 6g, 6i, 6m, 7g, and 7i exhibited better interaction profiles and almost
similar binding energy to the cocrystal ligand; also the ADME evaluation
by the QikProp module depicted that all the molecules followed all the
druggability parameters. The most potent molecules in this class may
further lead to the novel antitubercular drug candidate.
4.1
|
4.1.1
General
The commercially available chemicals, such as reactants, reagents,
solvents, and starting materials, were acquired from commercial
providers and were used as such without further purification. The
monitoring of reactions was performed by thin‐layer chromato-
graphy (TLC)—MERCK precoated silica gel 60‐F254 (0.5 mm) alu-
minum plates under UV light. Proton (1H) and carbon (13C) NMR
spectra were acquired on Bruker's Avance 500 MHz spectrometer
by using tetramethylsilane (TMS) as the internal standard and che-
mical shifts are reported in ppm. Chemical shifts are mentioned with
reference to TMS (d 0.00 for 1H NMR and 13C NMR), deuterated
dimethyl sulfoxide (DMSO‐d6) (d 2.50 for 1H NMR, and 39.7 for 13C
NMR) or CDCl3 (d 7.267 for 1H NMR and 77.00 or 77.16 for 13C
NMR). Spinning multiplicities are denoted as s for singlet, brs for
broad singlet, d for doublet, dd for double doublet, t for triplet, and m
for multiplet. Coupling constant value (J) is indicated in hertz (Hz).
HRMS data were carried out on Agilent QTOF mass spectrometer
6540 series instrument. Purification of final compounds by column
chromatography was performed using silica gel 60–120 mesh.
Melting points (MP) were noted using the Stuart® SMP30 apparatus.
The InChI codes of the investigated compounds, together with
observed biological activity data, are provided as Supporting
Information Data.

GAIKWAD ET AL.
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|
4.1.2
General procedure for the synthesis of
1.35–1.23 (m, 3H). 13C NMR (126 MHz, CDCl3) δ 167.50, 149.80,
147.19, 139.39, 132.70, 126.19, 124.07, 104.92, 77.28, 77.03, 76.78,
62.12, and 53.49; FTIR (cm−1): 3120 (N–H), 2978 (C–H), and 1733
(C═O); HRMS–QTOF MS/MS: m/z [M+H]+ calcd for C13H13N3O4:
276.0986; found 276.0985.
3‐dimethylamino‐1‐(nitrophenyl)prop‐2‐en‐1‐one (2)
To the stirred solution of nitroacetophenone (24.7 mmol) in dry to-
luene (20 ml) in a round bottom flask, dimethylformamide‐dimethyl
acetal (34.6 mmol) was added. The reaction mixture was continued
to stir at 110°C until TLC showed the completion of the reaction
(48 h). The reaction mixture was allowed to cool to room tempera-
ture. After cooling, the solvent was evaporated under reduced
pressure, and to the residue, cold brine solution 30 ml was added; the
obtained precipitate was filtered and purified by recrystallization
from ethanol to give the yellow solid; MP: 160–170°C. FTIR (cm−1):
2919 (C–H) and 1639 (C═O). 1H NMR (500 MHz, CDCl3) δ 8.24 (d,
J = 8.8 Hz, 2H), 8.01 (d, J = 8.8 Hz, 2H), 7.86 (d, J = 12.2 Hz, 1H), 5.68
(d, J = 12.2 Hz, 1H), 3.20 (s, 3H), and 2.97 (s, 3H); 13C NMR (126 MHz,
DMSO‐d6) δ 184.07, 155.70, 149.03, 146.32, 128.86, 123.84, 91.66,
and 45.17; HRMS–QTOF MS/MS: m/z [M+H]+ calcd for C11H13N2O3:
221.092; found 221.092.
|
4.1.5
General procedure for the synthesis of
2‐[3‐(nitrophenyl)‐1H‐pyrazol‐1‐yl]acetic acid (5)
2‐[3‐(Nitrophenyl)‐1H‐pyrazol‐1‐yl] acetic acid was obtained by hy-
drolysis of ethyl 2‐(3‐phenyl‐1H‐pyrazol‐1‐yl)acetate. Pyrazole acet-
ate (4) (1 mmol) was added to a solution of sodium hydroxide
(2 mmol) in water under stirring; after addition, the reaction mixture
was stirred at room temperature for 12 h. After completion of the
reaction, the resulting mixture was acidified with 3 N hydrochloric
acid. The precipitate obtained was filtered and dried in a hot air oven
and recrystallized from ethanol to yield the white solid; MP:
206–208°C; FTIR (cm−1): 2928, 1730, and 1403; 1H NMR (500 MHz,
DMSO‐d6) δ 13.16 (s, 1H), 8.27 (d, J = 8.9 Hz, 2H), 8.07 (d, J = 8.9 Hz,
2H), 7.88 (d, J = 2.4 Hz, 1H), and 6.96 (s, 1H), 5.07 (s, 2H). 13C NMR
(126 MHz, CDCl3) δ 174.68, 153.53, 151.63, 144.85, 139.22, 139.03,
131.08, 129.36, 109.76, 109.60, and 58.25; HRMS–QTOF MS/MS:
m/z [M+H]+ calcd for C11H9N3O4: 248.0671; found 248.0668.
|
4.1.3
General procedure for the synthesis of
3‐(nitrophenyl)‐1H‐pyrazole (3)
3‐Dimethylamino‐1‐(nitrophenyl)prop‐2‐en‐1‐one (5.9 mmol) was
dissolved in ethanol and water mixture (2:1). To this stirred solution
hydrazine hydrate (6.5 mmol) was added dropwise at room tem-
perature. The reaction mixture was stirred at 80°C (oil bath) for 4 h.
Then, the mixture was poured on the crushed ice and the pre-
cipitated solid was filtered off, washed with brine, and dried. The
dried solid was recrystallized from ethanol to give the white solid;
MP: 123–125°C; 1H NMR (500 MHz, DMSO‐d6) δ 13.25 (s, 1H), 8.25
(d, J = 8.5 Hz, 2H), 8.08 (d, J = 7.9 Hz, 2H), 7.88 (s, 1H), and 6.92
(d, J = 2.2 Hz, 1H); 13C NMR (126 MHz, DMSO‐d6) δ 148.65, 146.74,
140.76, 131.04, 126.30, 124.52, and 103.77; FTIR (cm−1): 3284
(N–H) and 3106 (C–H); HRMS–QTOF MS/MS: m/z [M+H]+ calcd for
C9H7N3O2: 190.0617; found 190.0612.
|
4.1.6
General procedure for the synthesis of
2‐[3‐(nitrophenyl)‐1H‐pyrazol‐1‐yl]acetamides (6a–o)
To
a stirred solution of 2‐[3‐(nitrophenyl)‐1H‐pyrazol‐1‐yl]acetic
acid (5), (0.40 mmol) in anhydrous DMF (10 ml) was added 1‐(3‐
dimethylaminopropyl)‐3‐ethylcarbodiimide hydrochloride (0.56 mmol),
DMAP (0.56 mmol) and corresponding amine (0.40 mmol) at room
temperature and stirred at room temperature for 12–14 h until com-
pletion of the reaction. Brine solution (30 ml) was added into the re-
action mixture; the resulting solid precipitate was collected by filtration,
the filtered solid was washed with cold water (50 ml) and followed by
cold methanol (10 ml) and dried to afford crude product. The resulting
crude compound was purified by using column chromatography
(n‐hexane/EtOAc 4:1) or by recrystallization from methanol to give a
pure target compound.
|
4.1.4
General procedure for the synthesis of
ethyl 2‐[3‐(nitrophenyl)‐1H‐pyrazol‐1‐yl]acetate (4)
The synthesis of ethyl 2‐[3‐(nitrophenyl)‐1H‐pyrazol‐1‐yl]acetate was
done using the reported methodology.[19] The appropriately sub-
stituted ethyl bromoacetate (1.2 mmol) was added to a mixture of
pyrazole (3) (1 mmol) and potassium carbonate (2 mmol) in acetoni-
trile (10 ml). The reaction mixture was stirred at 65°C (oil bath) for
15 h. After completion of the reaction indicated by TLC, the reaction
mixture was poured onto crushed ice. Precipitated solid was col-
lected by filtration, washed with water, and dried to afford the
products in good purity. The obtained product was recrystallized
from ethanol to give the white solid MP: 170–172°C. 1H NMR
(500 MHz, CDCl3) δ 8.24 (d, J = 8.8 Hz, 2H), 7.96 (d, J = 8.8 Hz, 2H),
7.57 (s, 1H), 6.72 (s, 1H), 4.99 (s, 2H), 4.27 (q, J = 7.1 Hz, 2H), and
2‐[3‐(4‐Nitrophenyl)‐1H‐pyrazol‐1‐yl]‐N‐[4‐(trifluoromethyl)phenyl]-
acetamide (6a)
Pale yellow solid; MP: 220–222°C; 1H NMR (500 MHz, DMSO‐d6) δ
(ppm): 10.86 (s, 1H), 8.27 (d, J = 9.0 Hz, 2H), 8.08 (d, J = 9.0 Hz, 2H),
7.95 (d, J = 2.4 Hz, 1H), 7.83 (d, J = 8.5 Hz, 2H), 7.71 (d, J = 8.7 Hz, 2H),
6.99 (d, J = 2.4 Hz, 1H), and 5.20 (s, 2H); 13C NMR (126 MHz, DMSO‐
d6) δ (ppm): 166.44, 148.95, 146.89, 144.74, 142.59, 140.08, 134.83,
126.72, 126.70, 126.36, 124.62, 119.63, 104.88, and 55.36; FTIR
(cm−1): 3068 (C–H), 1699 (C═O), and 1319 (C–F); HRMS–QTOF
MS/MS: m/z [M+H]+ calcd for C18H13F3N4O3: 391.1018; found
391.1014.

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GAIKWAD ET AL.
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N‐Cyclohexyl‐2‐[3‐(4‐nitrophenyl)‐1H‐pyrazol‐1‐yl]acetamide (6b)
Cream solid; MP: 180–182°C; 1H NMR (500 MHz, DMSO‐d6)
δ (ppm): 8.27 (d, J = 9.0 Hz, 2H), 8.06 (d, J = 9.0 Hz, 2H), 7.84
(d, J = 2.4 Hz, 1H), 6.94 (d, J = 2.4 Hz, 1H), 4.83 (s, 2H), 2.67
(ddd, J = 11.4, 7.6, 4.0 Hz, 1H), 1.24 (s, 1H), 0.66 (q, J = 7.0 Hz, 2H),
and 0.52–0.42 (m, 2H); 13C NMR (126 MHz, CDCl3) δ (ppm): 167.80,
148.67, 146.82, 140.19, 134.52, 126.29, 124.60, 104.68, 54.68,
48.19, 32.70, 25.69, and 24.89; FTIR (cm−1): 3217 (C–H), 2937
(C–H), and 1648 (C═O); HRMS–QTOF MS/MS: m/z [M+H]+ calcd for
(d, J = 2.4 Hz, 1H), 7.52 (d, J = 9.1 Hz, 2H), 6.98 (d, J = 2.4 Hz, 1H), 6.91
(d, J = 9.1 Hz, 2H), 5.10 (s, 2H), and 3.73 (s, 3H); 13C NMR (126 MHz,
DMSO‐d6) δ (ppm): 165.13, 155.94, 148.84, 143.32, 134.76, 132.67,
132.15, 126.33, 124.63, 121.19, 114.47, 104.78, 55.64, and 55.30;
HRMS–QTOF MS/MS: m/z [M+H]+ calcd for C18H16N4O4: 353.1250;
found, 353.1266.
N‐2‐Fluorophenyl‐2‐[3‐(4‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (6g)
C
17H20N4O3: 329.1614; found 329.1616.
Light yellow solid; MP: 216–218°C; 1H NMR (500 MHz, DMSO‐d6)
δ (ppm): 10.20 (s, 1H), 8.28 (d, J = 8.6 Hz, 2H), 8.09 (d, J = 8.6 Hz,
2H), 7.93 (s, 2H), 7.29 (s, 1H), 7.20–7.15 (m, 2H), 6.98 (d, J = 1.5 Hz,
1H), and 5.24 (s, 2H); 13C NMR (126 MHz, DMSO‐d6) δ (ppm):
166.28, 148.94, 146.90, 140.11, 134.80, 126.36, 124.97, 124.61,
116.12, 115.97, 104.84, and 55.07; FTIR (cm−1): 2923 (C–H), 1673
(C═O), and 1353 (C–F); HRMS–QTOF MS/MS: m/z [M+H]+ calcd
for C17H13FN4O3: 341.1050; found, 341.1058.
N‐Cycloheptyl‐2‐[3‐(4‐nitrophenyl)‐1H‐pyrazol‐1‐yl]acetamide (6c)
Cream solid; MP: 145–147°C; 1H NMR (500 MHz, DMSO‐d6)
δ
(ppm): 8.59–8.55 (m, 1H), 8.23 (d, J = 8.0 Hz, 1H), 8.19
(d, J = 7.8 Hz, 1H), 8.15 (d, J = 9.6 Hz, 1H), 7.83 (d, J = 2.3 Hz, 1H),
7.71 (t, J = 8.0 Hz, 1H), 6.93 (d, J = 2.3 Hz, 1H), 4.85 (s, 2H),
3.80–3.72 (m, 1H), 1.83–1.76 (m, 2H), and 1.59–1.39 (m, 10H); 13
C
NMR (126 MHz, DMSO‐d6) δ (ppm): 165.46, 148.80, 148.56,
134.37, 131.83, 122.42, 119.55, 103.87, 54.79, 50.32, 34.68,
28.27, and 24.12. FTIR (cm−1): 3279 (C–H), 2930 (C–H), and 1655
(C═O); HRMS–QTOF MS/MS: m/z [M+H]+ calcd for C18H22N4O3:
343.1770; found, 343.1774.
N‐4‐Fluorophenyl‐2‐[3‐(4‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (6h)
Pale yellow solid; MP: 161–163°C 1H NMR (500 MHz, DMSO‐d6) δ
(ppm): 10.57 (s, 1H), 8.27 (d, J = 8.8 Hz, 2H), 8.08 (d, J = 8.8 Hz, 2H),
7.93 (d, J = 2.3 Hz, 1H), 7.64 (d, J = 8.9 Hz, 2H), 7.40 (d, J = 8.8 Hz, 2H),
6.99 (d, J = 2.3 Hz, 1H), and 5.15 (s, 2H); 13C NMR (126 MHz, DMSO‐
d6) δ (ppm): 165.63, 159.60, 148.88, 146.87, 140.13, 135.49, 134.75,
126.34, 124.60, 121.52, 121.46, 116.01, 115.83, 104.80, and 55.30.
HRMS–QTOF MS/MS: m/z [M+H]+ calcd for C17H13FN4O3:
341.1050; found, 341.1054.
N‐3‐Chlorophenyl‐2‐[3‐(4‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (6d)
Yellow solid; MP: 232–234°C; 1H NMR (500 MHz, DMSO‐d6) δ
(ppm): 10.64 (s, 1H), 8.27 (d, J = 8.9 Hz, 2H), 8.08 (d, J = 8.9 Hz, 2H),
7.93 (d, J = 2.4 Hz, 1H), 7.81 (s, 1H), 7.48 (d, J = 7.3 Hz, 1H), 7.38
(t, J = 8.1 Hz, 1H), 7.16 (d, J = 8.0 Hz, 1H), 6.99 (d, J = 2.4 Hz, 1H), and
5.16 (s, 2H); 13C NMR (126 MHz, DMSO‐d6) δ (ppm): 166.15, 148.94,
146.89, 140.45, 140.10, 134.80, 133.65, 131.09, 126.35, 124.61,
123.89, 119.20, 118.10, 104.85, and 55.34; FTIR (cm−1): 3354, 2925
(C–H), 1683 (C═O), and 775 (C–Cl); HRMS–QTOF MS/MS: m/z
[M+H]+ calcd for C17H13ClN4O3: 357.0754; found, 357.0771.
N‐Naphthalene‐1‐yl‐2‐[3‐(4‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (6i)
Brown solid; MP: 179–181°C; 1H NMR (500 MHz, DMSO‐d6) δ
(ppm): 10.42 (s, 1H), 8.30 (d, J = 8.9 Hz, 2H), 8.19 (d, J = 8.0 Hz, 1H),
8.12 (d, J = 8.8 Hz, 2H), 8.00–7.95 (m, 2H), 7.80 (d, J = 8.2 Hz, 1H),
7.74 (d, J = 7.3 Hz, 1H), 7.58 (t, J = 7.4 Hz, 2H), 7.51 (t, J = 7.8 Hz, 1H),
7.01 (d, J = 2.2 Hz, 1H), and 5.34 (s, 2H); 13C NMR (126 MHz, DMSO‐
d6) δ (ppm): 166.63, 148.90, 148.83, 134.71, 134.71, 131.90, 130.86,
128.68, 126.63, 126.07, 123.07, 122.53, 119.61, 104.12, and 55.23;
FTIR (cm−1): 3249, 3027, 2924 (C–H), and 1664 (C═O);
HRMS–QTOF MS/MS: m/z [M+H]+ calcd for C21H16N4O3: 373.1301;
found, 373.1303.
N‐3‐Chloro‐4‐fluorophenyl‐2‐[3‐(4‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (6e)
Light yellow solid; MP: 249–251°C; 1H NMR (500 MHz, DMSO‐d6) δ
(ppm): 10.65 (s, 1H), 8.27 (d, J = 9.0 Hz, 2H), 8.08 (d, J = 9.0 Hz, 2H),
7.92 (d, J = 7.2 Hz, 2H), 7.52–7.48 (m, 1H), 7.41 (t, J = 9.1 Hz, 1H),
6.99 (d, J = 2.3 Hz, 1H), and 5.15 (s, 2H); 13C NMR (126 MHz, DMSO‐
d6) δ (ppm): 166.05, 154.78, 152.84, 148.97, 146.91, 140.09, 134.29,
126.36, 124.60, 121.20, 120.11, 119.69, 117.51, 104.86, and 55.28;
FTIR (cm−1): 3352, 2954 (C‐H), 1687 (C═O), 754 (C═Cl), and 1324
(C–F); HRMS–QTOF MS/MS: m/z [M+H]+ calcd for C17H12ClFN4O3:
375.0660; found, 375.0663.
N‐4‐Bromophenyl‐2‐[3‐(4‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (6j)
Dark brown solid; MP: 201–203°C; 1H NMR (500 MHz, DMSO‐d6) δ
(ppm): 10.47 (s, 1H), 8.21–8.18 (m, 2H), 8.00 (d, J = 9.0 Hz, 2H), 7.85
(d, J = 2.4 Hz, 1H), 7.51 (d, J = 8.9 Hz, 2H), 7.45 (d, J = 8.9 Hz, 2H), 6.91
(d, J = 2.4 Hz, 1H), and 5.07 (s, 2H); 13C NMR (126 MHz, DMSO‐D6) δ
(ppm): 165.89, 148.91, 146.88, 140.10, 138.40, 134.78, 132.19, 126.35,
124.61, 121.62, 115.77, 104.84, and 55.36; FTIR (cm−1): 3357, 2923
(C–H), 1689 (C═O), and 745 (C–Br); HRMS–QTOF MS/MS: m/z
[M+H]+ calcd for C17H13BrN4O3: 401.0249; found, 401.0249.
N‐4‐Methoxyphenyl‐2‐[3‐(4‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (6f)
Yellow solid; MP: 200–202°C; FTIR (cm−1): 3265, 2923 (C–H), 1680
(C═O), and 1242 (C–O); 1H NMR (500 MHz, DMSO‐d6) δ (ppm):
10.25 (s, 1H), 8.27 (d, J = 9.0 Hz, 2H), 8.08 (d, J = 9.0 Hz, 2H), 7.92

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2‐[3‐(4‐Nitrophenyl)‐1H‐pyrazol‐1‐yl]‐N‐phenylacetamide (6k)
Whitish yellow solid; MP: 188–190°C; 1H NMR (500 MHz, DMSO‐d6)
δ (ppm): 10.65 (s, 1H), 8.27 (d, J = 9.0 Hz, 2H), 8.08 (d, J = 9.0 Hz, 2H),
7.92 (d, J = 7.2 Hz, 2H), 7.52–7.48 (m, 1H), 7.41 (t, J = 9.1 Hz, 1H),
6.99 (d, J = 2.3 Hz, 1H), and 5.15 (s, 2H); 13C NMR (126 MHz, DMSO‐
d6) δ (ppm): 165.74, 148.80, 135.43, 134.65,131.86, 130.83, 129.35,
124.12, 122.49, 119.69, 119.60, 104.05, and 55.30; FTIR (cm−1):
3278, 2924 (C–H), and 1866 (C═O); HRMS–QTOF MS/MS: m/z
[M+H]+ calcd for C17H14N4O3: 323.1144; found, 323.1145.
N‐Cyclohexyl‐2‐[3‐(3‐nitrophenyl)‐1H‐pyrazol‐1‐yl]acetamide (7b)
Cream‐colored solid; MP: 190–192°C; 1H NMR (500 MHz, DMSO‐
d6) δ (ppm): 8.27 (d, J = 8.9 Hz, 2H), 8.13 (d, J = 7.7 Hz, 1H), 8.06
(d, J = 9.0 Hz, 2H), 7.84 (d, J = 2.3 Hz, 1H), 6.94 (d, J = 2.4 Hz, 1H), 4.87
(s, 2H), 3.60–3.52 (m, 1H), 1.77 (d, J = 12.2 Hz, 2H), 1.69 (d,
J = 13.0 Hz, 2H), and 1.31–1.13 (m, 6H); 13C NMR (126 MHz, DMSO‐
d6) δ (ppm): 165.65, 148.62, 146.83, 140.22, 134.48, 126.28, 124.60,
104.64, 54.83, 48.22, 32.77, 25.62, and 24.86; FTIR (cm−1): 3271,
3087 (C–H), and 1638 (C═O); HRMS–QTOF MS/MS: m/z [M
+H]+ calcd for C17H20N4O3: 329.1614; found, 329.1624.
N‐4‐Morpholinophenyl‐2‐[3‐(4‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (6l)
N‐Cycloheptyl‐2‐[3‐(3‐nitrophenyl)‐1H‐pyrazol‐1‐yl]acetamide (7c)
Cream solid; MP: 171–173°C; 1H NMR (500 MHz, DMSO‐d6) δ (ppm):
8.27 (d, J = 8.5 Hz, 2H), 8.17 (d, J = 7.7 Hz, 1H), 8.06 (d, J = 8.5 Hz, 2H),
7.84 (d, J = 2.3 Hz, 1H), 6.94 (d, J = 2.3 Hz, 1H), 4.85 (s, 2H), 3.83–3.71
(m, 1H), 1.83–1.76 (m, 2H), and 1.60–1.38 (m, 10H); 13C NMR (126 MHz,
DMSO‐d6) δ (ppm): 165.38, 148.63, 146.84, 140.22, 134.48, 126.28,
124.60, 104.64, 54.86, 50.33, 34.68, 28.25, and 24.11; FTIR (cm−1): 3290
and 2925 (C–H); HRMS–QTOF MS/MS: m/z [M+H]+ calcd for
Light yellow solid; MP: 241–243°C; 1H NMR (500 MHz, DMSO‐d6) δ
(ppm): 10.25 (s, 1H), 8.27 (d, J = 8.93358, 2958, 1682, 1327; Hz, 2H),
8.08 (d, J = 8.9 Hz, 2H), 7.92 (d, J = 2.3 Hz, 1H), 7.48 (d, J = 9.0 Hz, 2H),
6.98 (d, J = 2.3 Hz, 1H), 6.92 (d, J = 9.1 Hz, 2H), 5.10 (s, 2H), 3.74–3.71
(m, 4H), and 3.07–3.03 (m, 4H); 13C NMR (126 MHz, DMSO‐d6) δ (ppm):
164.96, 148.82, 147.95, 146.85, 140.17, 134.72, 131.25, 126.32,
124.60, 120.77, 115.91, 104.74, 66.57, 55.34, and 49.27; FTIR (cm−1):
2958 (C–H), 1682 (C═O), and 1327 (C–O); HRMS–QTOF MS/MS: m/z
[M+H]+ calcd for C21H21N5O4: 408.1672; found, 408.1692.
C18H22N4O3: 343.1770; found, 343.1776.
N‐3‐Chlorophenyl‐2‐[3‐(3‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (7d)
N‐4‐Chlorophenyl‐2‐[3‐(4‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (6m)
Yellow solid; MP: 217–219°C; 1H NMR (500 MHz, DMSO‐d6) δ (ppm):
10.76 (s, 1H), 8.59 (s, 1H), 8.26 (d, J = 7.7 Hz, 1H), 8.16 (d, J = 9.7 Hz, 1H),
7.93 (d, J = 2.2 Hz, 1H), 7.83 (d, J = 8.5 Hz, 2H), 7.74–7.69 (m, 3H), 6.99 (d,
J = 2.2 Hz, 1H), and 5.19 (s, 2H); 13C NMR (126 MHz, DMSO‐d6) δ (ppm):
166.49, 148.90, 148.81, 142.60, 135.39, 134.70, 131.88, 130.8, 126.70,
122.51, 119.64, 104.13, and 55.31; FTIR (cm−1): 3027, 2924 (C–H), 755
(C–Cl), and 1681 (C═O); HRMS–QTOF MS/MS: m/z [M+H]+ calcd for
Cream solid; MP: 140–142°C; 1H NMR (500 MHz, DMSO‐d6) δ
(ppm): 10.57 (s, 1H), 8.27 (d, J = 8.8 Hz, 2H), 8.08 (d, J = 8.8 Hz, 2H),
7.93 (d, J = 2.3 Hz, 1H), 7.64 (d, J = 8.9 Hz, 2H), 7.40 (d, J = 8.8 Hz, 2H),
6.99 (d, J = 2.3 Hz, 1H), and 5.15 (s, 2H); 13C NMR (126 MHz, DMSO‐
d6) δ (ppm): 165.87, 148.91, 146.88, 140.11, 137.99, 134.78, 129.28,
126.35, 124.61, 121.25, 104.83, and 79.65; FTIR (cm−1): 3058, 2923
(C–H), 1690 (C═O), and 745 (C–Cl); HRMS–QTOF MS/MS: m/z
[M+H]+ calcd for C17H13ClN4O3: 357.0754; found, 357.0771.
C17H13ClN4O3: 357.0754; found, 357.0758.
N‐3‐Chloro‐4‐fluorophenyl‐2‐[3‐(3‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (7e)
N‐Mesityl‐2‐[3‐(4‐nitrophenyl)‐1H‐pyrazol‐1‐yl]acetamide (6n)
Light brown solid; MP: 215–217°C; 1H NMR (500 MHz, DMSO‐d6) δ
(ppm): 10.36 (s, 1H), 8.28 (d, J = 8.8 Hz, 2H), 8.08 (d, J = 8.8 Hz, 2H),
7.92 (d, J = 2.3 Hz, 1H), 6.99 (s, 3H), 5.11 (s, 2H), 3.74 (s, 6H), and 3.63
(s, 3H); 13C NMR (126 MHz, DMSO‐d6) δ (ppm): 165.54, 153.27,
148.91, 146.88, 140.13, 135.15, 134.79, 134.13, 126.33, 124.62,
104.77, 97.39, 60.58, 56.16, and 55.37; FTIR (cm−1): 3058 (C–H) and
1660 (C═O); HRMS–QTOF MS/MS: m/z [M+H]+ calcd for
White brown solid; MP: 248–250°C; 1H NMR (500 MHz, DMSO‐d6) δ
(ppm): 10.67 (s, 1H), 8.27 (d, J = 8.9 Hz, 2H), 8.08 (d, J = 8.9 Hz, 2H),
7.92 (dd, J = 7.7, 3.4 Hz, 2H), 7.52–7.48 (m, 1H), 7.41 (t, J = 9.1 Hz,
1H), 6.99 (d, J = 2.4 Hz, 1H), and 5.15 (s, 2H); 13C NMR (126 MHz,
DMSO‐d6) δ (ppm): 166.05, 148.96, 146.91, 140.09, 136.25, 134.79,
126.36, 124.61, 121.19, 120.05, 117.69, 117.52, 104.86, and 55.27;
FTIR (cm−1): 3352, 2924 (C–H), 1687 (C═O), 1324 (C–F), and 754
(C–Cl); HRMS–QTOF MS/MS: m/z [M+H]+ calcd for C17H12ClFN4O3:
375.0660; found, 375.0656.
C
20H20N4O3: 365.1614; found, 365.1590.
N‐Cyclopropyl‐2‐[3‐(3‐nitrophenyl)‐1H‐pyrazol‐1‐yl]acetamide (7a)
Cream solid; MP: 205–507°C; 1H NMR (500 MHz, DMSO‐d6) δ
(ppm): 8.34 (d, J = 3.5 Hz, 1H), 8.27 (d, J = 8.6 Hz, 2H), 8.06
(d, J = 8.6 Hz, 2H), 7.84 (d, J = 2.1 Hz, 1H), 6.94 (d, J = 2.2 Hz, 1H), 4.83
(s, 2H), 2.67 (td, J = 7.2, 3.7 Hz, 1H), 0.66 (q, J = 6.8 Hz, 2H), and 0.45
(q, J = 4.6 Hz, 2H); 13C NMR (126 MHz, DMSO‐d6) δ (ppm): 167.78,
148.68, 146.84, 140.19, 134.51, 126.30, 124.59, 104.68, 54.70,
22.84, and 6.08; FTIR (cm−1): 3254, 2924 (C–H), and 1664 (C═O);
HRMS–QTOF MS/MS: m/z [M+H]+ calcd for C14H14N4O3: 287.1144;
found, 287.1146.
N‐4‐Methoxyphenyl‐2‐[3‐(3‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (7f)
White brown solid; MP: 185–187°C; 1H NMR (500 MHz, DMSO‐d6) δ
(ppm): 10.26 (s, 1H), 8.59 (s, 1H), 8.25 (d, J = 7.8 Hz, 1H), 8.16
(d, J = 8.2 Hz, 1H), 7.91 (d, J = 2.2 Hz, 1H), 7.72 (t, J = 8.0 Hz, 1H), 7.52
(d, J = 8.9 Hz, 2H), 6.97 (d, J = 2.2 Hz, 1H), 6.91 (d, J = 9.0 Hz, 2H), 5.09
(s, 2H), and 3.73 (s, 3H); 13C NMR (126 MHz, DMSO‐d6) δ (ppm):
165.19, 155.96, 148.79, 135.45, 134.61, 132.17, 131.86, 130.82,
122.47, 121.24, 119.60, 114.47, 104.02, 55.65, and 55.25; FTIR (cm−1):

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3254, 2920 (C–H), 1682 (C═O), and 1244 (C–O); HRMS–QTOF MS/
MS: m/z [M+H]+ calcd for C18H16N4O4: 353.1250; found, 353.1254.
(t, J = 8.9 Hz, 2H), 6.98 (d, J = 2.3 Hz, 1H), and 5.17 (s, 2H); 13C NMR
(126 MHz, DMSO‐d6) δ (ppm): 166.04, 148.95, 146.89, 140.08,
134.80, 126.35, 124.61, 118.13, 116.06, 108.86, 108.68, 104.86, and
55.27; FTIR (cm−1): 3324, 2920 (C–H), 1693 (C═O), and 1512 (N–O);
HRMS–QTOF MS/MS: m/z [M+H]+ calcd for C17H12FN5O5:
386.0901; found, 386.0901.
N‐2‐Fluorophenyl‐2‐[3‐(3‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (7g)
Light brown solid; MP: 206–208°C; 1H NMR (500 MHz, DMSO‐d6) δ
(ppm): 10.20 (s, 1H), 8.28 (d, J = 8.9 Hz, 2H), 8.09 (d, J = 8.9 Hz, 2H), 7.94
(d, J = 2.3 Hz, 2H), 7.32–7.27 (m, 1H), 7.20–7.16 (m, 2H), 6.99
(d, J = 2.4 Hz, 1H), and 5.24 (s, 2H); 13C NMR (126 MHz, DMSO‐d6) δ
(ppm): 166.27, 154.92, 148.95, 146.90, 140.10, 134.81, 126.36, 126.05,
124.96, 124.61, 124.24, 116.05, 104.85, and 55.06; FTIR (cm−1): 3320,
2924 (C–H), 1672 (C═O), and 1333 (C–F); HRMS–QTOF MS/MS: m/z
[M+H]+ calcd for C17H13FN4O3: 341.1050; found, 341.1054.
N‐[Benzo(d)thiazol‐2‐yl]‐2‐[3‐(3‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (7l)
Gray solid; MP: 228–230°C; 1H NMR (500 MHz, DMSO‐D6) δ (ppm):
8.61–8.58 (m, 1H), 8.27 (d, J = 7.8 Hz, 1H), 8.16 (d, J = 8.2 Hz, 1H),
7.97 (dd, J = 10.4, 5.0 Hz, 2H), 7.78 (d, J = 8.0 Hz, 1H), 7.72 (s, 1H),
7.45 (t, J = 7.7 Hz, 1H), 7.32 (t, J = 8.1 Hz, 1H), 7.01 (d, J = 2.4 Hz, 1H),
and 5.34 (s, 2H); 13C NMR (126 MHz, DMSO‐D6) δ (ppm): 167.53,
158.46, 149.04, 148.80, 134.80, 131.92, 130.85, 126.63, 124.10,
122.57, 122.24, 121.07, 119.66, 104.29, and 54.71; FTIR (cm−1):
2922, 2852 (C–H), and 1688 (C═O); HRMS–QTOF MS/MS: m/z
[M+H]+ calcd for C18H13N5O3S: 380.0817; found, 380.0814.
N‐4‐Fluorophenyl‐2‐[3‐(3‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (7h)
Brown solid; MP: 271–273°C; 1H NMR (500 MHz, CDCl3) δ (ppm):
10.47 (s, 1H), 8.39 (s, 1H), 7.75 (dd, J = 11.4, 8.1 Hz, 2H), 7.57
(d, J = 8.5 Hz, 1H), 7.34 (t, J = 7.5 Hz, 1H), 5.15 (d, J = 2.5 Hz, 2H), and
2.29 (t, J = 2.5 Hz, 1H); 13C NMR (126 MHz, DMSO‐d6) δ (ppm):
165.70, 148.83, 146.85, 140.15, 135.66, 134.76, 126.34, 124.59,
121.49, 115.94, 115.84, 104.77, 55.29, and 25.34; FTIR (cm−1): 3392,
3076 (C–H), 1663 (C═O), and 1327 (C–F); HRMS–QTOF MS/MS:
m/z [M+H]+ calcd for C17H13FN4O3: 341.1050; found, 341.1056.
|
4.1.7
General procedure for acetohydrazide
synthesis (8)
To the stirred solution of 4 (1 eqv) in ethanol, hydrazine hydrate
(1.5 eqv) was added dropwise. After the addition is over, the reaction
mixture was stirred at 50°C for 6 h. The reaction was monitored by
TLC; after completion of the reaction the ethanol was evaporated on
the rota‐evaporator. The semisolid residue obtained was poured on
crushed ice. Solid obtained was filtered and used without purification
for further reactions.
N‐Naphthalen‐1‐yl‐2‐[3‐(3‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (7i)
Cream solid; MP: 254–256°C; 1H NMR (500 MHz, DMSO‐d6) δ
(ppm): 10.37 (s, 1H), 8.69 (s, 1H), 8.34 (d, J = 7.8 Hz, 1H), 8.23
(t, J = 8.0 Hz, 2H), 8.02 (d, J = 8.7 Hz, 2H), 7.85 (d, J = 8.2 Hz, 1H), 7.78
(t, J = 8.0 Hz, 2H), 7.63 (dd, J = 12.2, 6.8 Hz, 2H), 7.56 (t, J = 7.8 Hz,
1H), 7.05 (d, J = 2.1 Hz, 1H), and 5.37 (s, 2H); 13C NMR (126 MHz,
DMSO‐d6) δ (ppm): 166.63, 148.91, 148.83, 134.71, 131.90, 130.85,
128.68, 126.53, 126.06, 123.06, 122.52, 119.62, 104.11, and 55.22;
FTIR (cm−1): 3209, 3083 (C–H), and 1681 (C═O); HRMS–QTOF MS/
MS: m/z [M+H]+ calcd for C21H16N4O3: 373.1301; found, 373.1307.
|
4.1.8
General procedure for the synthesis of
benzylidine acetohydrazide
To the stirred solution of 8 (1 eqv) and substituted aldehyde (1 eqv) in
ethanol at room temperature, a catalytic amount of acetic acid (1 mol%)
was added and the reaction mixture was stirred at 50°C till completion of
the reaction (5–6 h). After completion of the reaction indicated by TLC,
ethanol was evaporated under reduced pressure, and to the residue
15 ml n‐hexane was added and filtered. The solid obtained was purified
by crystallization to obtain 9a as white solid; thus remaining molecules of
hydrazide series were synthesized following a similar procedure.
N‐4‐Bromophenyl‐2‐[3‐(3‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (7j)
Gray solid; MP: 230–230°C; 1H NMR (500 MHz, DMSO‐d6) δ (ppm):
10.54 (s, 1H), 8.27 (d, J = 9.0 Hz, 2H), 8.07 (d, J = 9.0 Hz, 2H), 7.93
(d, J = 2.4 Hz, 1H), 7.58 (d, J = 8.9 Hz, 2H), 7.52 (d, J = 8.9 Hz, 2H), 6.98
(d, J = 2.4 Hz, 1H), and 5.14 (s, 2H); 13C NMR (126 MHz, DMSO‐d6) δ
(ppm): 165.89, 148.91, 146.89, 140.10, 138.40, 134.78, 132.19, 126.35,
124.61, 121.63, 115.76, 104.84, and 55.36; FTIR (cm−1): 3357, 3065
(C–H), 1688 (C═O), and 746 (C–Br); HRMS–QTOF MS/MS: m/z
[M+H]+ calcd for C17H13BrN4O3: 403.0229; found, 403.0229.
N′‐4‐Chlorobenzylidene‐2‐[3‐(3,4‐dimethoxyphenyl)‐1H‐pyrazol‐1‐
yl]acetohydrazide (9a)
White solid; MP: 147–149°C, FTIR, (cm−1): 2987 (C–H), 1677 (C═O),
and 762 (C–Cl). 1H NMR (500 MHz, DMSO‐d6) δ 11.75 (s, 1H), 8.04
(s, 1H), 7.80–7.76 (m, 3H), 7.52 (d, J = 8.5 Hz, 2H), 7.33 (s, 2H), 6.98
(d, J = 8.4 Hz, 1H), 6.69 (s, 1H), 5.43 (s, 2H), 3.81 (s, 3H), and 3.78
(s, 3H). 13C NMR (126 MHz, DMSO‐d6) δ 169.23, 150.74–150.58
(m), 149.32, 148.85, 143.17, 134.91, 133.86, 133.42, 129.34 (d,
J = 11.7 Hz), 129.11, 126.91, 118.06, 112.37, 109.15, 102.81, 55.97
N‐4‐Fluoro‐3‐nitrophenyl‐2‐[3‐(3‐nitrophenyl)‐1H‐pyrazol‐1‐yl]-
acetamide (7k)
Dark brown solid; MP: 251–253°C; 1H NMR (500 MHz, DMSO‐d6) δ
(ppm): 11.05 (s, 1H), 8.27 (d, J = 8.9 Hz, 2H), 8.08 (d, J = 8.9 Hz, 2H),
7.94 (d, J = 2.2 Hz, 1H), 7.68 (dd, J = 9.0, 5.0 Hz, 2H), 7.17

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(d, J = 5.0 Hz), and 52.92. HRMS–QTOF MS/MS: m/z [M+H]+ calcd for
N′‐2,3,4‐Trimethoxybenzylidene‐2‐[3‐(3,4‐dimethoxyphenyl)‐1H‐
pyrazol‐1‐yl]acetohydrazide (9f)
C
20H20ClN4O3: 399.1224; found, 399.1220.
White solid; MP: 126–128°C; FTIR (cm−1): 2940, 2836 (C–H), and
1259 (C–O); 1H NMR (500 MHz, DMSO‐d6) δ 11.56 (s, 1H), 8.23
(s, 1H), 7.76 (d, J = 2.3 Hz, 1H), 7.64 (d, J = 8.9 Hz, 1H), 7.36 (s, 1H),
7.32 (dd, J = 8.3, 1.9 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.92
(d, J = 8.9 Hz, 1H), 6.68 (d, J = 2.3 Hz, 1H), 5.40 (s, 2H), 3.84 (s, 3H),
3.81 (s, 3H), and 3.78 (d, J = 1.7 Hz, 8H); 13C NMR (126 MHz,
DMSO‐d6) δ 168.50, 153.01, 148.81, 142.04, 140.49, 135.57, 134.69,
131.82, 130.81, 122.40, 121.14, 120.64,120.48, 119.55, 109.21,
104.01, 62.24, 60.97, and 56.52; HRMS–QTOF MS/MS: m/z [M
+H]+ calcd for C23H26N4O6: 455.1931; found, 455.1935.
N′‐2‐Chlorobenzylidene‐2‐[3‐(3,4‐dimethoxyphenyl)‐1H‐pyrazol‐1‐
yl]acetohydrazide (9b)
White solid; MP: 162–164°C; FTIR (cm−1): 3197, 2941 (C–H), 1674
(C═O), and 756 (C–Cl); 1H NMR (500 MHz, DMSO‐d6) δ 11.88 (s, 1H),
8.44 (s, 1H), 8.08 (dd, J = 7.5, 1.9 Hz, 1H), 7.77 (d, J = 2.2 Hz, 1H), 7.54
(dd, J = 7.9, 1.2 Hz, 1H), 7.45 (dt, J = 9.6, 6.5 Hz, 2H), 7.37 (d, J = 1.8 Hz,
1H), 7.32 (dd, J = 8.3, 1.8 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.70
(dd, J = 5.6, 2.2 Hz, 1H), 5.46 (s, 2H), 3.81 (s, 3H), and 3.78 (s, 3H); 13
C
NMR (126 MHz, DMSO‐d6) δ 169.30, 150.73, 149.39, 148.92, 140.53,
133.83, 133.49, 131.80, 130.38, 128.07, 127.53, 126.96, 118.12,
112.47, 109.29, 102.85, 56.05, and 52.97; HRMS–QTOF MS/MS: m/z
[M+H]+ calcd for C20H19ClN4O3: 399.1224; found, 399.1220.
2‐[3‐(3,4‐Dimethoxyphenyl)‐1H‐pyrazol‐1‐yl]‐N′‐(3,4,5‐
trimethoxybenzylidene)acetohydrazide (9g)
Cream solid; MP: 145–147°C; FTIR (cm−1): 2908 (C–H), 1697 (C═O),
and 1260 (C–O); 1H NMR (500 MHz, DMSO‐d6) δ 11.72 (s, 3H), 7.96
(s, 3H), 7.78 (s, 2H), 7.78 (s, 1H), 7.37 (d, J = 1.8 Hz, 6H), 7.07 (s, 6H),
6.98 (d, J = 8.4 Hz, 4H), 6.70 (d, J = 2.3 Hz, 3H), 5.46 (s, 6H), 3.83
(s, 9H), 3.80 (d, J = 14.8 Hz, 27H), and 3.71 (s, 10H); 13C NMR
(126 MHz, DMSO‐d6) δ 169.15, 153.67, 150.67, 149.34, 148.85,
148.13, 144.27, 139.63, 133.90, 129.97, 126.94, 118.05, 112.39,
109.14, 104.95, 104.73, 102.75, 60.59, 56.45, 55.99, 55.95, 53.81,
and 53.07; HRMS–QTOF MS/MS: m/z [M+H]+ calcd for C23H26N4O6:
455.1931; found, 455.1942.
N′‐4‐Cyanobenzylidene‐2‐[3‐(3,4‐dimethoxyphenyl)‐1H‐pyrazol‐1‐
yl]acetohydrazide (9c)
White solid; MP: 155–157°C; FTIR (cm−1): 2924, 2864 (C–H), 1682
(C═O), and 2223 (C≡N); 1H NMR (500 MHz, DMSO‐d6) δ 11.93 (s, 1H),
8.09 (s, 1H), 7.96 (d, J = 8.4 Hz, 2H), 7.92 (d, J = 7.2 Hz, 2H), 7.77
(d, J = 2.3 Hz, 1H), 7.36 (d, J = 1.9 Hz, 1H), 7.32 (dd, J = 8.3, 1.9 Hz, 1H),
6.98 (d, J = 8.4 Hz, 1H), 6.69 (t, J = 2.7 Hz, 1H), 5.46 (s, 2H), 3.81 (s, 3H),
and 3.78 (s, 3H); 13C NMR (126 MHz, DMSO‐D6) δ 169.54, 150.72,
149.33, 148.86, 142.54, 138.92, 133.86, 133.19, 128.21, 128.06, 126.90,
118.08, 112.39, 112.32, 109.16, 102.85, 55.99, 55.95, and 52.96;
HRMS–QTOF MS/MS: m/z [M+H]+ calcd for C21H19N5O3: 390.1566;
found, 390.1565.
N′‐4‐Hydroxy‐3‐methoxybenzylidene‐2‐[3‐(3,4‐dimethoxyphenyl)‐
1H‐pyrazol‐1‐yl]acetohydrazide (9h)
White solid; MP: 160–162°C; FTIR (cm−1): 3048, 2909 (C–H), 1638
(C═O), and 1216 (C–O); 1H NMR (500 MHz, DMSO‐d6) δ 11.52
(s, 1H), 9.51 (s, 1H), 7.93 (s, 1H), 7.77 (s, 1H), 7.35 (d, J = 9.2 Hz, 2H),
7.31 (s, 1H), 7.11 (d, J = 8.2 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 6.84
(d, J = 8.1 Hz, 1H), 6.69 (d, J = 2.3 Hz, 1H), 5.42 (s, 2H), 3.84 (s, 3H),
and 3.81 (s, 6H); 13C NMR (126 MHz, DMSO‐d6) δ 172.41, 169.12,
164.53, 163.81, 162.56, 151.11,150.69, 149.38, 148.90, 147.04,
143.33, 133.83, 131.12, 129.72, 126.98, 118.10, 116.42, 116.24,
112.46, 109.28, 102.80, 56.00, 53.80, 52.93, and 21.48;
HRMS–QTOF MS/MS: m/z [M+H]+ calcd for C21H22N4O5: 411.1668;
found, 411.1666.
N′‐3‐Nitrobenzylidene‐2‐[3‐(3,4‐dimethoxyphenyl)‐1H‐pyrazol‐1‐
yl]acetohydrazide (9d)
White solid; MP: 196–198°C; FTIR (cm−1): 2924, 2854 (C–H), 1681
(C═O), and 1515 (N–O); 1H NMR (500 MHz, DMSO‐d6) δ 11.93
(s, 1H), 8.56 (s, 1H), 8.28–8.22 (m, 2H), 8.18 (s, 1H), 7.77 (dd, J = 9.7,
5.1 Hz, 2H), 7.36 (d, J = 1.8 Hz, 1H), 7.32 (dd, J = 8.3, 1.9 Hz, 1H), 6.98
(d, J = 8.4 Hz, 1H), 6.70 (d, J = 2.3 Hz, 1H), 5.49 (s, 2H), 3.81 (s, 3H),
and 3.78 (s, 3H); 13C NMR (126 MHz, DMSO‐d6) δ 169.46, 150.73,
149.34, 148.86, 148.78, 142.22, 136.34, 133.86, 133.49, 130.87,
126.92, 124.68, 121.69, 118.08, 112.39, 109.17, 102.85, 55.99,
55.95, and 52.99; HRMS–QTOF MS/MS: m/z [M+H]+ calcd for
C
20H19N5O5: 410.1464; found, 410.1462.
N′‐[4‐(Benzyloxy)benzylidene]‐2‐[3‐(3,4‐dimethoxyphenyl)‐1H‐
pyrazol‐1‐yl]acetohydrazide (9i)
N′‐4‐Nitrobenzylidene‐2‐[3‐(3,4‐dimethoxyphenyl)‐1H‐pyrazol‐1‐
yl]acetohydrazide (9e)
Orange solid; MP: 171–173°C; FTIR (cm−1): 3093 (C–H), 1684
(C═O), and 1509 (N–O); 1H NMR (500 MHz, DMSO‐d6) δ 11.99
(s, 1H), 8.29 (d, J = 8.6 Hz, 2H), 8.15 (s, 1H), 8.04 (d, J = 8.7 Hz, 2H),
7.78 (d, J = 2.1 Hz, 1H), 7.37–7.32 (m, 2H), 6.98 (d, J = 8.3 Hz, 1H),
White solid; MP: 178–180°C; FTIR (cm−1): 3213, 2934 (C–H), 1671
(C═O), and 1243 (C–O); 1H NMR (500 MHz, DMSO‐d6) δ 11.57 (s, 1H),
7.99 (s, 1H), 7.77 (d, J = 2.2 Hz, 1H), 7.71–7.67 (m, 2H), 7.47
(d, J = 7.3 Hz, 2H), 7.41 (dd, J = 10.1, 4.7 Hz, 2H), 7.38–7.30 (m, 4H), 7.09
(s, 1H), 6.97 (d, J = 8.4 Hz, 1H), 6.69 (dd, J = 5.7, 2.3 Hz, 1H), 5.41 (s, 2H),
5.17 (d, J = 3.4 Hz, 2H), 3.81 (s, 3H), and 3.78 (s, 3H); 13C NMR
(126 MHz, DMSO‐d6) δ 169.17, 159.17, 150.68, 149.38, 148.90, 144.21,
137.46, 135.91, 133.85, 130.44, 128.90, 128.34, 128.23, 128.14,
126.98, 120.59, 118.10, 117.45, 112.48, 109.28, 102.80, 69.86, 56.03,
55.99, and 52.98; HRMS–QTOF MS/MS: m/z [M+H]+ calcd for
6.70 (d, J = 2.2 Hz, 1H), 5.49 (s, 2H), 3.81 (s, 3H), and 3.78 (s, 3H); 13
C
NMR (126 MHz, DMSO‐d6) 169.60, 150.75, 149.34, 148.92,
δ
148.30, 142.12, 140.75, 133.87, 128.61, 128.43, 126.90, 124.49,
118.08, 112.39, 109.18, 102.88, 55.98, and 52.53; HRMS–QTOF MS/
MS: m/z [M+H]+ calcd for C20H19N5O5: 410.1464; found, 410.1462.
C27H26N4O4: 471.2032; found, 471.2027.

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2‐[3‐(3,4‐Dimethoxyphenyl)‐1H‐pyrazol‐1‐yl]‐N′‐[(2‐oxo‐1,2‐
dihydroquinolin‐3‐yl)methylene]acetohydrazide (9j)
4.1.10
Synthesis of 2‐[3‐(3‐nitrophenyl)‐1H‐
pyrazol‐1‐yl]acetohydrazide (11)
Brown solid; MP: 264–266°C; FTIR (cm−1): 2970 (C–H), 1678 (C═O),
and 1508 (C–O); 1H NMR (500 MHz, DMSO‐d6) δ 12.06 (s, 1H),
11.84 (s, 1H), 8.57 (s, 1H), 8.30 (s, 1H), 7.81–7.75 (m, 2H), 7.55
(t, J = 7.7 Hz, 1H), 7.37–7.32 (m, 3H), 7.23 (t, J = 7.8 Hz, 1H), 6.98
(d, J = 8.4 Hz, 1H), 6.70 (d, J = 2.2 Hz, 1H), 5.50 (s, 2H), 3.81 (s, 3H),
and 3.78 (s, 3H); 13C NMR (126 MHz, DMSO‐d6) δ 169.26, 161.40,
150.69, 149.34, 148.85, 139.48, 135.34, 133.90, 131.64, 129.57,
129.34, 126.93, 125.58, 122.87, 119.55, 118.06, 115.64, 112.39,
109.13, 102.83, 55.99, 55.95, and 53.04.S; HRMS–QTOF MS/MS:
m/z [M+H]+ calcd for C23H21N5O4: 432.1672; found, 432.1029.
Synthesized as per general procedure 4.1.7.
N′‐Benzylidene‐2‐[3‐(3‐nitrophenyl)‐1H‐pyrazol‐1‐yl]
acetohydrazide (11a)
White solid; MP: 230–232°C; FTIR (cm−1): 3091, 2971 (C–H), and
1674 (C═O); 1H NMR (500 MHz, DMSO‐d6) δ 11.74 (s, 1H), 8.59
(s, 1H), 8.26 (d, J = 8.0 Hz, 1H), 8.16 (d, J = 8.2 Hz, 1H), 8.06 (s, 1H),
7.90 (d, J = 1.4 Hz, 1H), 7.76 (d, J = 6.5 Hz, 2H), 7.73 (d, J = 7.9 Hz, 2H),
7.46 (s, 2H), 6.97 (d, J = 1.4 Hz, 1H), and 5.51 (s, 2H); 13C NMR
(126 MHz, DMSO‐d6) δ 168.87, 148.80, 148.54, 144.60, 134.70,
134.42, 131.82, 130.82, 130.52, 129.31, 127.66, 127.46, 122.41,
119.56, 104.03, and 53.27; HRMS–QTOF MS/MS: m/z [M+H]+ calcd
for C18H15N5O3: 350.1253; found, 350.1257.
|
4.1.9
Synthesis of 2‐[3‐(4‐nitrophenyl)‐1H‐
pyrazol‐1‐yl]acetohydrazide (10)
Synthesized by general procedure 4.1.7.
N′‐3,4‐Dimethoxybenzylidene‐2‐[3‐(3‐nitrophenyl)‐1H‐pyrazol‐1‐
yl]acetohydrazide (11b)
N′‐4‐Chlorobenzylidene‐2‐[3‐(4‐nitrophenyl)‐1H‐pyrazol‐1‐
yl]‐acetohydrazide (10a)
White solid; MP: 115–117°C; FTIR (cm−1): 2949 (C–H) and 1683
(C═O); 1H NMR (500 MHz, DMSO‐d6) δ 11.63 (s, 1H), 8.60–8.59
(m, 1H), 8.26 (d, J = 7.8 Hz, 1H), 8.16 (d, J = 8.2 Hz, 1H), 7.97 (s, 1H),
7.90 (d, J = 2.3 Hz, 1H), 7.72 (t, J = 8.0 Hz, 1H), 7.39 (s, 1H), 7.22
(dd, J = 8.2, 1.7 Hz, 1H), 7.02 (d, J = 8.3 Hz, 1H), 6.97 (d, J = 2.4 Hz,
1H), 5.51 (s, 2H), and 3.82 (d, J = 10.3 Hz, 6H); 13C NMR (126 MHz,
DMSO‐d6) δ 168.65, 151.15, 149.56, 148.81, 148.50, 144.64, 135.58,
134.64, 131.80, 130.82, 127.18, 122.40, 121.97, 119.54, 111.97,
108.97, 103.97, 56.02, and 53.36; HRMS–QTOF MS/MS: m/z
[M+H]+ calcd for C20H19N5O5: 410.1464; found, 410.1468.
White solid; MP: 238–240°C; 1H NMR (500 MHz, CDCl3) δ 11.34
(s, 1H), 7.97 (d, J = 8.9 Hz, 2H), 7.73 (d, J = 8.9 Hz, 3H), 7.40
(dd, J = 20.1, 5.4 Hz, 3H), 7.12 (d, J = 8.5 Hz, 2H), 6.52 (d, J = 2.4 Hz,
1H), and 5.22 (s, 2H); 13C NMR (126 MHz, DMSO‐d6) δ 168.46,
149.11, 146.85, 143.73, 140.00, 135.70, 133.33, 132.64, 128.94,
128.30, 126.01, 123.91, 104.53, and 53.31; HRMS–QTOF MS/MS:
m/z [M+H]+ calcd for C22H25N4O5Cl: 384.0863; found, 384.0861.
N′‐3,4‐Dimethoxybenzylidene‐2‐[3‐(4‐nitrophenyl)‐1H‐pyrazol‐1‐
yl]acetohydrazide (10b)
N′‐2,3,4‐Trimethoxybenzylidene‐2‐[3‐(3‐nitrophenyl)‐1H‐pyrazol‐1‐
yl]acetohydrazide (11c)
White solid; MP: 175–179°C; FTIR (cm−1): 3095, 2941 (C–H), and
White solid; MP: 225–227°C; 1H NMR (500 MHz, DMSO‐d6) δ
11.64 (s, 1H), 8.27 (d, J = 8.8 Hz, 2H), 8.08 (d, J = 8.7 Hz, 2H), 7.97
(s, 1H), 7.92 (d, J = 2.1 Hz, 1H), 7.38 (s, 1H), 7.21 (d, J = 8.3 Hz, 1H),
7.02 (d, J = 8.3 Hz, 1H), 6.98 (s, 1H), 5.52 (s, 2H), 3.83 (s, 3H), and
3.81 (s, 3H); 13C NMR (126 MHz, DMSO‐d6) δ 168.55, 151.18,
149.57, 148.62, 146.83, 144.79, 140.25, 134.81, 127.17, 126.35,
126.27, 124.58, 121.97, 112.03, 109.06, 104.84, 104.74, 56.07,
56.00, 53.42, 40.44, 40.27, 40.10, 39.94, 39.77, 39.60, and 39.43;
HRMS–QTOF MS/MS: m/z [M+H]+ calcd for C20H19N5O5: 410.1
464; found, 410.1460.
1677 (C═O); 1H NMR (500 MHz, DMSO‐d6)
δ 11.60 (s, 1H),
8.60–8.58 (m, 1H), 8.25 (d, J = 10.8 Hz, 2H), 8.16 (d, J = 8.2 Hz, 1H),
7.89 (d, J = 2.3 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 8.9 Hz, 1H),
6.97 (d, J = 2.3 Hz, 1H), 6.93 (d, J = 8.9 Hz, 1H), 5.47 (s, 2H), 3.86 (d,
J = 2.2 Hz, 3H), 3.85 (s, 3H), and 3.79 (s, 3H); 13C NMR (126 MHz,
DMSO‐d6) δ 168.52, 155.60, 153.01, 148.81, 148.50, 142.04, 140.49,
135.57, 134.69, 131.82, 130.81, 122.40, 121.14, 120.58, 119.55,
109.21, 104.01, 62.24, 60.97, 56.52, and 53.28; HRMS–QTOF MS/
MS: m/z [M+H]+ calcd for C21H21N5O6: 440.1571; found, 440.1573.
N′‐4‐Methylbenzylidene‐2‐[3‐(4‐nitrophenyl)‐1H‐pyrazol‐1‐
yl]‐acetohydrazide (10c)
White solid; MP: 217–219°C; 1H NMR (500 MHz, DMSO‐d6) δ 11.67
(s, 1H), 8.27 (d, J = 9.0 Hz, 2H), 8.08 (d, J = 9.0 Hz, 2H), 8.02 (s, 1H),
7.91 (d, J = 2.4 Hz, 1H), 7.65 (d, J = 8.1 Hz, 2H), 7.28 (d, J = 8.0 Hz, 2H),
6.98 (d, J = 2.4 Hz, 1H), 5.50 (s, 2H), and 2.36 (s, 3H); 13C NMR
(126 MHz, DMSO‐d6) δ 168.66, 148.60, 146.82, 144.73, 140.35,
140.28, 134.80, 131.71, 129.91, 127.44, 126.28, 124.60, 104.77,
53.35, and 21.50; HRMS–QTOF MS/MS: m/z [M+H]+ calcd for
4.2
Pharmacological/biological assays
|
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4.2.1
Antibiotic susceptibility testing against
mycobacteria
Antimycobacterial susceptibility testing was carried out on newly
synthesized compounds by using broth microdilution assay.[41,42]
Stock solutions (10 mg/ml) of test and control compounds were
C
19H17N5O3: 364.1410; found, 364.1409.

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prepared in DMSO and stored at −20°C. Mycobacterial cultures
were inoculated in Middlebrook 7H9 enriched (Difco; Becton) media
supplemented with 10% ADC‐Tween‐80 (bovine serum albumin,
dextrose, 0.2% glycerol, and 0.05% Tween‐80) and OD600 of the
cultures was measured, followed by dilution to achieve ~106 CFU/
ml.[35] The newly synthesized compounds were tested from 64 to
0.5 mg/l in a two‐fold serial diluted fashion with 2.5 µl of each con-
centration added per well of a 96‐well round‐bottom microtitre
plate. Later, 97.5 µl of bacterial suspension was added to each well
containing the test compound along with appropriate controls. Pre-
sto blue (Thermo Fisher Scientific) resazurin‐based dye was used for
the visualized identification of active compounds. MIC of active
compound was determined as the lowest concentration of compound
that inhibited visible growth after the incubation period. For each
compound, MIC determinations were replicated three times using
duplicate samples. The MIC plates were incubated at 37°C for 7 days
for Mtb and 48 h for other mycobacterial pathogens.
4.3
Molecular docking
The structures of ligands were drawn in ChemBioDraw Ultra 14.0. and
prepared for docking by use of the Ligprep module of Schrödinger Suite
19.4. The required target protein crystal structure was accrued from
the RSCB PDB site. Molecular docking studies were performed against
Mtb enzyme various surface proteins KasA (PDB ID: 5W2Q), DprE1
(PDB ID: 4P8H), InhA (PDB ID: 4TZK), and MmpL3 (PDB ID: 6ajj). With
the aid of Protein Preparation Wizard of Schrödinger Suite 19.4, the
target protein was prepared. The prepared protein was optimized and
minimized using the algorithm OPLS3 (optimized potential for liquid
simulations) force field and followed by the Glide Grid Generation panel
in which the Glide receptor grid was generated. Finally, all the prepared
test ligands were docked by using Glide's extra precision (XP) mode of
docking calculations. On the basis of the XP Glide scoring function and
root mean square deviation, parameters were implemented for getting
the best‐ranked compounds and the specific ligand–protein binding
interactions. Similarly, the in silico ADME properties were calculated by
the use of the Qikprop module of the Schrödinger Suite 19.
|
4.2.2
Antibiotic susceptibility testing against
ESKAP pathogen panel
ACKNOWLEDGMENTS
Nikhil B. Gaikwad conveys his cordial thanks to DoP, Ministry of
Chemicals and Fertilizers, Govt. of India, for the award of NIPER
fellowship. Grace Kaul thanks DST‐INSPIRE for her fellowship
and Manjulika Shukla thanks the University Grants Commission
for her fellowship. The manuscript has a NIPER‐H acknowledgment
(no. NIPER‐H/2020/M079).
Antibiotic susceptibility testing was carried out on the newly synthesized
compounds by determining the MIC with reference to the standard CLSI
guidelines.[43,44] MIC is defined as the minimum concentration of com-
pound at which visible bacterial growth is inhibited. Bacterial cultures
were grown in Mueller–Hinton cation supplemented broth. Optical
density (OD600) of the cultures was measured, followed by dilution for
~106 CFU/ml. This inoculum was added into a series of test wells in a
microtiter plate that contained various concentrations of the compound
under test ranging from 64 to 0.03 mg/ml. Controls, that is, cells alone
and media alone (without compound + cells) and levofloxacin used as a
reference standard. Plates were incubated at 37°C for 16–18 h, followed
by observations of MIC values by the absence or presence of visible
growth. For each compound, MIC determinations were performed in-
dependently three times using duplicate samples each time.
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
ORCID
Nikhil B. Gaikwad
Srinivas Nanduri
Arunava Das Gupta
Sidharth Chopra
https://orcid.org/0000-0002-1103-3786
http://orcid.org/0000-0002-6671-2022
https://orcid.org/0000-0001-9014-1904
https://orcid.org/0000-0001-8823-6074
Madhavi V. Yaddanapudi
https://orcid.org/0000-0002-9813-8694
|
4.2.3
Cell cytotoxicity assay
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How to cite this article: Gaikwad NB, Nirmale K, Sahoo SK,
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https://doi.org/10.1002/ardp.202000349