Synthesis, molecular docking, and biological evaluation of nitroimidazole derivatives as potent urease inhibitors

Article abstract of DOI:10.1007/s00044-021-02727-4

The objective of this study was to design new nitroimidazole-based derivatives as strong urease inhibitors for the treatment of H. pylori infections. New series of nitroimidazole derivatives, 4a–k, were synthesized by using TBTU as the catalyst and assaye

Full text of DOI:10.1007/s00044-021-02727-4

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-021-02727-4
ORIGINAL RESEARCH
Synthesis, molecular docking, and biological evaluation of
nitroimidazole derivatives as potent urease inhibitors
Meysam Talebi1 Elham Hamidian1 Fatemeh Niasari-Naslaji1 Sogand Rahmani1 Faezeh Sadat Hosseini1
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Shahin Boumi1 Mohammad Nazari Montazer1 Mehdi Asadi1 Massoud Amanlou
1,2
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Received: 5 January 2021 / Accepted: 4 April 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
The objective of this study was to design new nitroimidazole-based derivatives as strong urease inhibitors for the treatment
of H. pylori infections. New series of nitroimidazole derivatives, 4a–k, were synthesized by using TBTU as the catalyst and
assayed as Jack bean urease inhibitors. The facile synthetic approach was employed for the preparation of targeted molecular
designs in good to excellent yields, ranged from 65 to 92%. Accordingly, all the synthesized compounds, 4a–k (IC50
=
1.43–7.72 μM), were more potent than the standard urease inhibitors, thiourea, and hydroxyurea. Among the derivatives, 4d
had the most urease inhibitor activity (IC50 = 1.43 μM), over 15-fold more potent than thiourea and 70-fold than
hydroxyurea. True to form, the result of molecular docking studies was in good congruence with those obtained from
in vitro tests.
Graphical Abstract
Supplementary information The online version contains
supplementary material available at https://doi.org/10.1007/s00044-
021-02727-4.
* Massoud Amanlou
University of Medical Sciences, Tehran, Iran
amanlou@tums.ac.ir
2
Experimental Medicine Research Center, Tehran University of
Medical Sciences, Tehran, Iran
1
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran

Medicinal Chemistry Research
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Keywords Urease inhibitor Molecular docking Nitroimidazole TBTU
Introduction
[15]. The focus is now on alternative paths, such as building an
unstable acidic environment for the pathogen. Therefore,
therapeutic achievements depend on abolishing the H. pylori
urease enzyme.
A typical treatment for H. pylori infection involves the
co-administration of a proton pump inhibitor and antibiotics
[16–18]. Drug-resistant strains have been reported after the
frequent use of macrolides (clarithromycin or azi-
thromycin), tetracycline, amoxicillin, and imidazole
(metronidazole or tinidazole) [19–21]. Moreover, the higher
dose of these antibiotics could result in adverse effects such
as nausea, vomiting, abdominal pain, diarrhea, and bloating
[22–24].
Lately, the in vitro efficacy assessment of urease enzyme
inhibitors using phosphoramidites has been conducted
successfully [25, 26]. However, the in-vivo tests and clin-
ical trials were not promising due to problems such as
hydrolytic instability and toxicity [27, 28]. Acetohy-
droxamic acid is one of the few urease inhibitors that has
gained FDA approval [29–31]. Nitroimidazole derivatives
are commonly used in antibacterial and antiprotozoal
therapies [32].
According to previous studies, a considerable number of
H. pylori strains (6–27%) are resistant to metronidazole, a
common nitroimidazole derivative [33], due to frequent
medications with nitroimidazoles [34]. Then, the main
objective of this study was to design new metronidazole-
based derivatives with remarkable inhibitory activity that
could preserve metronidazole’s role as an acceptable anti-
biotic and could be used as strong urease inhibitors in the
treatment of H. pylori infections.
Helicobacter pylori (H. pylori) is one of the most wide-
spread bacterial infections in the human population. How-
ever, a large number of patients do not reveal clinical
pathogenic symptoms. The pathogen is transmitted orally
during childhood through feces, vomitus, saliva, and water
[1, 2]. H. pylori are well-known for triggering gastritis,
gastric ulcers, and gastric adenocarcinoma [3]. Although
both innate and adaptive immunity cooperate to restrain H.
pylori from colonization, the pathogen applies various
strategies to run away from the immune system [4].H.
pylori can exert pathogenicity by overwhelming the acidic
pH of the stomach through its vital enzyme, urease, a
nickel-containing enzyme that catalyzes the hydrolysis of
urea to two molecules of ammonia and carbon dioxide
[5–7]. It utilizes two types of urease; a surface urease, which
works in neutral pH, and a cytoplasmic urease, which is
activated in acidic pH (2.5–6.5). The cytoplasmic urease is
responsible for bacteria’s pH homeostasis. It is regulated by
a Hþ- gated transporter facilitates urea permeability through
the membrane and thus plays an essential role in bacterial
acid resistance [8, 9].
Crystallographic analysis of H. pylori urease illustrates the
spatial complexity of the enzyme that protects it against
environmental catalytic change [10, 11]. Reports indicate that
the active site of this particular enzyme in H. pylori is different
from other bacterial ureases, which leads to a high affinity to
the substrate [12]. The ammonia produced by the hydrolysis
activity of urease neutralizes gastric hydrochloric acid, hence,
eases bacterial colonization in epithelial cells [13].
Due to the growing concern of drug-resistant strains of H.
pylori to antibiotics, the application of antibiotics to eliminate
H. pylori is becoming less frequent and new methods for
eradication are considered. Resistant bacteria survive from the
treatment regimen, propagate, and settle in the mucosal layer of
the stomach [14]. Consequently, the resistant population trig-
gers the inoculum effect, which means a higher “minimum
inhibitory concentration” is required to eradicate the pathogen
Results and discussion
Chemistry
Eleven novel compounds have been synthesized as urease
inhibitors according to the pathway shown in Scheme 1.
Scheme 1 Schematic
representation of the synthesis of
2-(2-methyl-5-nitro-1H-
imidazol-1-yl)acetohydrazides

Medicinal Chemistry Research
In this current work 2-(2-methyl-5-nitro-1H-imidazol-1-yl)
acetohydrazide (3) was synthesized according to the pre-
viously mentioned method [35].
most potent derivative of the series. The desirable activity
of this compound might be as a result of the appropriate
binding interaction of the indole group with the active site
of the enzyme. Subsequently, compound 4d exhibited a
great inhibitory activity against Jack bean urease (Table 1),
which fascinated our interest in more structure modification
of them as lead compounds for new urease inhibitors or
anti-H. pylori agents.
At the first step, ethyl 2-(2-methyl-5-nitro-1H-imidazol-
1-yl)acetate (2) was synthesized from a reaction between 2-
methyl-5-nitro-1H-imidazole (1) and ethyl bromoacetate in
dry acetone as a solvent, then the excess amount of
hydrazine hydrate was added dropwise to compound 2 in
ethanol solvent to produce 2-(2-methyl-5-nitro-1H-imida-
zol-1-yl)acetohydrazide (3) [35]. In the third step, our final
compounds (4a–k) were synthesized through a condensa-
tion reaction between 2-(2-methyl-5-nitro-1H-imidazol-1-
yl)acetohydrazide (3) and different aromatic acid deriva-
tives in the presence of 2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium tetrafluoroborate (TBTU) as a coupling
reagent while there exist triethylamine (TEA) as base and
dimethylformamide (DMF) as a solvent in our reaction
medium at 0 °C. Our final compounds yield ranged from 65
to 92%. Final compound structures were confirmed by
using IR and NMR spectra.
Docking study
To predict possible interactions between the synthesized
compounds and the active site of the Jack bean urease
enzyme, molecular docking simulations were performed.
Docking results indicate that all of the compounds interact
well with the active site of the enzyme with a binding
energy range of −9.54 to −6.84 kcal/mol, which are
stronger than the binding energy of the standard compounds
thiourea and hydroxyurea, with a binding energy of −3.45
and −5.57 kcal/mol, respectively (Table 1). The super-
imposed position of docking poses of all compounds in the
active site of the enzyme was shown in Fig. 1a. The highest
binding energy, −9.54 kcal/mol, is related to compound 4d,
representing the highest urease inhibitory activity with an
IC50 of 1.432 µM in comparison to other compounds. The
placement of the compound 4d in the active site suggests
that the inhibitory effect is probably related to the con-
formational changes that occur at the entrance of the tunnel
leading to the nickel atoms, and therefore, it could prevent
the substrate from entering the tunnel (Fig. 1b). Compound
4d, with an indole ring, forms three hydrogen bonds with
Gln 635, Gly 638, and Gly 641 residues and interacts
hydrophobically with Glu 418, Arg 639, Val 640, and Glu
642 (Fig. 1c). The SwissADME webserver was used to
determine the ADME properties [36]. So, compound 4d,
with a molecular weight of 356.34 g/mol, log p −0.14,
forms 3 and 5 hydrogen bonds, donor and acceptor,
respectively, fulfill all the criteria declared in the Lipinski
rule of five (Fig. 1d) and preset its potential drug ability
[37]. It is expected that compound 4d, with the highest
urease inhibitory activity, and also the highest binding
interactions, and proper ADME properties, could be a
promising candidate for further evaluation.
Biological activity
All eleven synthesized compounds (4a–k) were evaluated
for their inhibitory activity against Jack bean urease.
Thiourea and hydroxyurea were taken as the reference drug
(Table 1). All of the tested compounds presented acceptable
inhibitory activity mainly compounds 4d (IC50 = 1.43 µM)
and 4k (IC50 = 1.70 µM). These two derivatives demon-
strated potent in vitro inhibitory activity, which was com-
parable to standard inhibitor thiourea (IC50 = 22.01 µM) and
hydroxyurea (IC50 = 100.00 µM) (Table 1). The comparison
of the inhibitory activity of the synthesized compounds and
metronidazole defined that the introduction of a hydrazine
moiety instead of 2-hydroxy, and substitution on the ben-
zene ring, play an important role in the urease inhibitory
activity of synthetic compounds. Interestingly, methoxy-
substituted compounds showed a different pattern.
Among them, compound 4k (IC50 = 1.70 µM), which
bears a methoxy group at ortho-position, displayed an
increased inhibitory action in comparison to compounds 4c
(IC50 = 4.18 μM), with a methoxy group at para-position,
and compound 4 h (IC50 = 2.74 μM), that bears both of
them simultaneously.
Order of inhibitory activity stated the respective potency
of 4k>4h > 4c . This outcome indicates that possibly the
methoxy substitution at para-position leads to an increase in
the steric hindrance or the ortho substitution interacts
greatly with the active site of the enzyme. However, all of
these compounds were more active as compared to the
standard. Compound 4d (IC50 = 1.43 µM), holding an
indole-3-acetic acid moiety, displayed fifteen-fold more
activity in comparison to the standard thiourea and was the
Conclusion
In this current work, eleven new nitroimidazole derivatives
were synthesized and their inhibitory activity against the
Jack bean urease enzyme was determined. The obtained
results demonstrated that all the synthesized compounds
were more potent than the standard inhibitors. The docking
study showed that all of the compounds successfully

Medicinal Chemistry Research
Table 1 The urease inhibitory activity and docking results of compounds 4a–k
Entry
4a
R
IC50 (µM)
2.075
ΔG binding (kcal/mol)
MW
337
−7.67
4b
4c
4d
4e
4.926
4.189
1.432
7.12
−8.17
317
333
358
371
−7.73
−9.54
−6.99
4f
2.86
−8.03
−8.56
303
348
4g
3.211

Medicinal Chemistry Research
Table 1 (continued)
Entry
4h
R
IC50 (µM)
2.747
ΔG binding (kcal/mol)
−8.06
MW
363
4i
3.786
7.724
1.702
22.01
100.00
−8.81
−6.84
−7.75
−3.45
−5.57
395
304
333
76
4j
4k
Thiourea
Hydroxyurea
76
Thiourea and hydroxyurea were used as standards urease inhibitors
occupied and interacted with the active site of the urease
enzyme. Eventually, among all compounds, compound 4d
indicates the lowest IC50 value of urease inhibitory activity
and lowest docking binding energy and is known as the
potent compound of this series. According to the urease
inhibitory activity of our final compounds, it seems

Medicinal Chemistry Research
Fig. 1 a Superimposed of docking position of all compounds in the active site of the urease enzyme, atom Ni are in red sphere. b, c 3D and 2D
display of interactions of compound 4d in the active site of urease enzyme, d structure of compound 4d and its ADME property
beneficial to consider nitroimidazole as an important moiety
for design and synthesize potent urease inhibitors.
In brief, the modified Berthelot spectrophotometric
method was used to evaluate urease inhibitory of the syn-
thesized derivatives at the concentration range of 0–10 mg/
ml and measuring the absorbance at 625 nm. The jack bean
urease enzyme was activated after 30 min incubation at
37 °C, and then, the absorbance of the testing solutions was
measured after adding a combination of phenol and alkali
reagents. The thiourea and hydroxyurea were used as the
reference standard inhibitors, and all the experiments were
performed in triplicate.
Experimental/material and methods
General
All chemicals used in this study were obtained from Merck
(Germany) and used without further purification. Melting
points were determined with a Kofler hot stage apparatus
and were uncorrected. 1H and 13C NMR spectra were
recorded with a Bruker FT-500, using TMS as an internal
standard. Coupling constant (J) values are presented in
Hertz (Hz), and spin multiples are given as s (singlet), d
(doublet), t (triple), and m (multiple). Infrared spectra were
acquired on a Nicolet Magna 550-FT spectrometer. IR
spectra of solid were recorded in KBr, and the absorption
Finally, the percentage of urease inhibition activity was
calculated as
Inhibition ð%Þ ¼ ½1 À ðT=Cފ  100
T is the absorbance of the test samples (synthesized
compounds (4a–k) or positive control) in the presence of
the enzyme, and C (control) is the absorbance of the
reagents in the presence of the enzyme. The IC50 values
were calculated using GraphPad Prism 5 software (Graph-
Pad Software Inc., San Diego, CA) (Table 1).
band was given in wavenumbers in cm−1
.
Urease inhibitory activity
Docking method
Sodium nitroprusside and Jack bean urease (EC 3.5.1.5)
were purchased from Sigma (St. Louis, MO, USA). Ultra-
pure water (HPLC grade, Duksan, Korea) was utilized in all
experiments. The compounds including, 4a–k and inhibi-
tors, were dissolved in deionized water. The urease inhibi-
tory activity of the synthesized compounds was assessed by
a procedure explained previously [38, 39].
Preparation of the input files of the ligands and receptor was
done by AutoDockTools 1.5.6 (ADT) [40]. Jack bean
urease enzyme with PDB ID: 3LA4 was downloaded from
the protein data bank. MarvinSketche version 15.2.2 was
used to draw the structure of the ligands in 2D format.
Chem3D ultra version 8.0 was used to minimized energy

Medicinal Chemistry Research
and converting the 2D structure to PDB format. Receptor
preparation was performed as follows, all water molecules
were deleted, and then polar hydrogens were added and,
Kollman charges were assigned, and non-polar hydrogens
were merged. Grid box with the size of 60 × 60 × 60 Å with
grid center −43.931, −43.497, 77.423 (X, Y, and Z) and
grid-point spacing of 0.375 Å was located near the Ni atom
in the active site of the enzyme. The grid maps of each atom
type were calculated by AutoGrid 4.2 [40]. The following
parameter was set to calculate docking simulations by
AutoDock 4.2: 2.5 × 106 maximum number of energy eva-
luations with 30 run jobs and Lamarckian genetic algorithm
(LGA) with the population size of 150, and the default
value for the rest of the parameters [39–41]. The docking
poses with the lowest binding energy value of each com-
pound were used to illustrate by LIGPLOT+ version v.2.2
[42] and PyMol version 1. Level [43].
bicarbonate solution (2 × 10 mL), and water. The crude
product was recrystallized from dichloromethane to obtain
pure final compounds (4a–k).
4-chloro-N′-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetyl)
benzohydrazide (4a)
White solid; yield: 78% (263.4 mg), mp = 231–233 °C. IR
(KBr): νmax 3318 (NH), 3055 (C–H aromatic), 2986 (C–H
1
aliphatic), 1688 (C=O), 1563–1358 (NO2) cm−1. H NMR
(DMSO-d6, 500 MHz): δ = 2.34 (s, 3H, CH3), 4.97 (s, 2H,
CH2), 7.58–7.60 (d, J = 8.1 Hz, 2H, H-3, H-5), 7.88–7.90 (d,
J = 8.1 Hz, 2H, H-2, H-6), 8.35 (s, 1H, CH, Imidazole),
10.55–10.67 (m, 2H, 2×NH) ppm. 13C NMR (DMSO-d6,
500 MHz): δ = 13.1(CH3), 48.2 (CH2), 129.3 (C-1, Ph), 129.9
(CH, Ph), 130.2 (CH, Ph), 131.4 (CH, Ph), 137.5 (C-4, Ph),
137.8 (C-4, imidazole), 145.7 (C-5, imidazole), 146.5 (C-2,
imidazole), 165.1 (C=O), 165.9 (C=O) ppm. HRMS: m/z [M
+H]+ calcd for C13H12ClN5O4: 338.0650, found: 337.0578.
General procedure for the synthesis of compounds
4a–k
4-methyl-N′-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetyl)
benzohydrazide (4b)
Ethyl (2-methyl-5-nitro-1H-imidazol-1-yl)acetate (2)
A mixture of 2-methyl-5-nitro-1H-imidazole 1 (1 mmol),
ethyl bromoacetate (1 mmol), and potassium carbonate
(1.5 mmol) (5–10 mL) was refluxed in dry acetone for 50 h.
The reaction mixture was cooled to room temperature, and
the solvent was distilled off from the filtrate. The crude ester
thus obtained was purified by recrystallization from ethanol.
Yield 85% (213.07 mg); mp 96 °C [35].
White solid; yield: 78% (247.5 mg), mp = 228–230 °C. IR
(KBr): νmax 3375 (NH), 3089 (C–H aromatic), 2979 (C–H
aliphatic), 1680 (C=O), 1558–1346 (NO2) cm−1. 1Hs NMR
(DMSO-d6, 500 MHz): δ = 2.25 (s, 3H, CH3), 2.36 (s, 3H,
CH3), 4.96 (s, 2H, CH2), 7.21–7.23 (d, J = 8.25 Hz, 2H, H-
3, H-5), 7.86–7.88 (d, J = 8.25 Hz, 2H, H-2, H-6), 8.35 (s,
1H, CH, Imidazole’), 10.43 (s, 2H, 2×NH) ppm. 13C NMR
(DMSO-d6, 500 MHz): δ = 13.1 (CH3), 21.6 (CH3), 48.2
(CH2), 123.9 (C-4, Ph), 128.0 (CH, Ph), 129.6 (CH, Ph),
129.7 (C–H, Ph), 129.9 (C-1, Ph), 142.6 (C-4, Imidazole),
145.7 (C-5, Imidazole), 146.5 (C-2, Imidazole), 165.9
(C=O), 166.0 (C=O) ppm. HRMS: m/z [M+H]+ calcd for
C14H15N5O4: 317.1124, found: 318.1199.
2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetohydrazide (3)
Ethyl (2-methyl-5-nitro-1H-imidazol-1-yl)acetate 2 (2 mmol)
was dissolved in ethanol as a solvent and, then, an excessive
amount of hydrazine hydrate (3 mmol) was added dropwise to
the mixture. The reaction mixture was refluxed for 10 h and
then cooled to room temperature. The solid formed, filtered,
dried, and recrystallized from ethanol to get pure compound
3. Yield 60% (238.8 mg); mp 189 °C [35].
4-methoxy-N′-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetyl)
benzohydrazide (4c)
White solid; yield: 72% (239.9 mg), mp = 249–251 °C. IR
(KBr): νmax 3335 (NH), 3026 (C–H aromatic), 2991 (C–H
Experimental procedure for the synthesis of nitroimidazole
derivatives (4a–k)
1
aliphatic), 1680 (C=O), 1558–1354 (NO2) cm−1. H NMR
(DMSO-d6, 500 MHz): δ = 2.36 (s, 3H, CH3), 4.96 (s, 2H,
CH2), 7.02–7.04 (d, J = 8.35 Hz, 2H, H-3, H-5), 7.86–7.88
(d, J = 8.35 Hz, 2H, H-2, H-6), 8.35 (s, 1H, CH, Imida-
zole’), 10.43 (s, 2H, 2×NH) ppm. 13C NMR (DMSO-d6,
500 MHz): δ = 13.1(CH3), 48.2 (CH2), 56.0 (O–CH3),
114.3 (CH-2, Ph), 114.4 (C-6, Ph), 124.0 (CH-3, Ph), 124.3
(CH-5, Ph), 124.8, 130.0 (C-1, Ph), 141.3 (C-4, Imidazole)
145.7 (C-5, Imidazole), 146.5 (C-2, Imidazole), 163.0 (C-4,
Ph), 165.6 (C=O), 165.9 (C=O) ppm. HRMS: m/z [M+H]
A mixture of benzoic acid derivatives (1 mmol), 0.32 g
(1 mmol) of TBTU, and 0.28 mL (2 mmol) of triethylamine
(TEA) in 2 mL dimethylformamide (DMF) was stirred at
0 °C for 3 min. Then 2-(2-methyl-5-nitro-1H-imidazol-1-yl)
acetohydrazide 3 (1 mmol) was added to the reaction
medium. The reaction mixture was stirred at 0 °C for 1 h
and left overnight at room temperature. The reaction mix-
ture was poured into ice water, filtered, washed with 5%
aqueous citric acid solution (2 × 10 mL), saturated sodium
+
calcd for C14H15N5O5: 333.1073, found: 332.1003.

Medicinal Chemistry Research
(1H-Indol-3-yl)-acetic acid N′-[2-(2-methyl-5-nitro-imidazol-
1-yl)-acetyl]-hydrazide (4d)
m/z [M+H]+ calcd for C13H13N5O4: 303.0968, found:
304.1042.
White solid; yield: 65% (231.6 mg), mp = 234–236 °C. IR
(KBr): νmax 3318 (NH), 3055 (C–H aromatic), 2986 (C–H
N′-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetyl)-4-
nitrobenzohydrazide (4g)
1
aliphatic), 1688 (C=O), 1563–1358 (NO2) cm−1. H NMR
(DMSO-d6, 500 MHz): δ = 2.29 (s, 3H, CH3), 3.59 (s, 2H,
CH2), 4.87 (s, 2H, CH2), 6.96–6.99 (t, J = 7.75 Hz, 1H, H-
4), 7.05–7.08 (t, J = 7.75 Hz, 1H, H-5), 7.24 (d, J = 2.5 Hz,
1H, H-2), 7.33–7.35 (d, J = 8.3 Hz, 1H, H-7), 7.57–7.59 (d,
J = 8.3 Hz, 1H, H-4), 8.31 (s, 1H, CH, Imidazole’), 10.20
White solid; yield: 92% (320.4 mg), mp = 272–274 °C. IR
(KBr): νmax 3358 (NH), 3055 (C–H aromatic), 2983 (C–H
1
aliphatic), 1692 (C=O), 1568–1360 (NO2) cm−1. H NMR
(DMSO-d6, 500 MHz): δ = 2.35 (s, 3H, CH3), 4.99 (s, 2H,
CH2), 8.09–8.11 (d, J = 8.25 Hz, 2H, H-2, H-6), 8.35 (s,
1H, CH, Imidazole), 8.36–8.38 (d, J = 8.25 Hz, 2H, H-3, H-
5), 10.80 (s, 2H, 2×NH) ppm. 13C NMR (DMSO-d6,
500 MHz): δ = 13.1(CH3), 48.2 (CH2), 124.0 (C-1,
Ph),124.3 (CH-3, Ph), 124.4 (CH-2, Ph), 138.3 (C-4, Imi-
dazole), 145.7 (C-5, Imidazole), 146.5 (C-2, Imidazole),
150.1(C-4, Ph), 164.6 (C=O), 165.8 (C=O) ppm. HRMS:
m/z [M+H]+ calcd for C13H12N6O6: 348.0818, found:
347.0743.
(s, 1H, NH), 10.37 (s, 1H, NH), 10.89 (s, 1H, NH) ppm. 13
C
NMR (DMSO-d6, 500 MHz): δ = 13.1 (CH3), 31.1 (CH2),
48.1 (CH2), 108.6 (C-3-indole), 111.9 (CH-7-indole), 118.9
(CH-4-indole), 119.3 (CH-5-indole), 121.6 (CH-6-indole),
124.0, (CH-2-indole), 124.5 (C-3a-indole), 127.7 (C-7a-
indole), 136.6 (C-4, Imidazole), 145.7 (C-5, Imidazole),
146.4 (C-2, Imidazole), 165.4 (C=O), 170.2 (C=O) ppm.
HRMS: m/z [M+H]+ calcd for C16H16N6O4: 356.1233,
found: 357.1307.
2,4-dimethoxy-N′-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)
acetyl)benzohydrazide (4 h)
2,6-dichloro-N′-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)
acetyl)benzohydrazide (4e)
White solid; yield: 75% (272.4 mg), mp = 237–239 °C. IR
(KBr): νmax 3328 (NH), 3040 (C–H aromatic), 2975 (C–H
White solid; yield: 65% (241.9 mg), mp = 221–223 °C. IR
(KBr): 3348 (NH), 3029 (C–H aromatic), 2964 (C–H ali-
1
aliphatic), 1680 (C=O), 1555–1355 (NO2) cm−1. H NMR
1
phatic), 1672 (C=O), 1545–1359 (NO2) cm−1. H NMR
(DMSO-d6, 500 MHz): δ = 2.35 (s, 3H, CH3), 3.83 (s, 3H,
O–CH3), 3.90 (s, 3H, O–CH3), 4.92 (s, 2H, CH2), 6.79–6.80
(d, J = 1.5 Hz, 2H, H-3, H-5), 7.79 (s, 1H, H-6), 8.32 (s,
1H, CH, Imidazole’), 9.88 (s, 1H, NH), 10.69 (s, 1H, NH)
ppm. 13C NMR (DMSO-d6, 500 MHz): δ = 13.1 (CH3),
48.1 (CH2), 56.1 (O–CH3), 56.7 (O– CH3), 99.1 (CH-2, Ph),
106.5 (CH-5, Ph), 124.0 (C-1, Ph), 133.1(C-4, Imidazole),
145.7 (C-5, Imidazole), 146.5 (C-2, Imidazole), 159.4 (C-2,
Ph), 163.8 (C-4, Ph), 164.2 (C=O), 164.8 (C=O) ppm.
HRMS: m/z [M+H]+ calcd for C15H17N5O6: 363.1179,
found: 364.1256.
(DMSO-d6, 500 MHz): δ = 2.34 (s, 3H, CH3), 4.46 (s, 2H,
CH2), 7.59–7.62 (t, J = 7.6 Hz, 1H, H-4), 7.79–7.81 (d, J =
7.65 Hz, 2H, H-3, H-5), 8.25 (s, 1H, CH, Imidazole’), 10.21
(s, 2H, 2×NH) ppm. 13C NMR (DMSO-d6, 500 MHz): δ =
13.4(CH3), 48.8 (CH2), 126.4 (CH-4, Ph), 129.1 (CH-3,
Ph), 129.6 (CH-5, Ph), 131.8 (C-2, Ph), 132.3 (C-6, Ph),
133.7 (C-4, Imidazole), 135.1 (C-5, Imidazole), 141.3 (C-2,
Imidazole), 164.0 (C=O), 165.4 (C=O) ppm. HRMS: m/z
[M+H]+ calcd for C13H12FN5O4: 321.0873, found:
322.0943.
N′-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetyl)
benzohydrazide (4f)
N′-(2-(2-methyl-5-nitro-1H-imidazol-1-yl)acetyl)-2,
Phenoxybenzohydrazide (4i)
White solid; yield: 84% (254.7 mg), mp = 234–236 °C. IR
(KBr): νmax 3340 (NH), 3063 (C–H aromatic), 2978 (C–H
White solid; yield: 68% (268.8 mg), mp = 195–197 °C. IR
(KBr): νmax 3346 (NH), 3052 (C–H aromatic), 2984 (C–H
1
1
aliphatic), 1679 (C=O), 1550–1352 (NO2) cm−1. H NMR
aliphatic), 1685 (C=O), 1548–1339 (NO2) cm−1. H NMR
(DMSO-d6, 500 MHz): δ = 2.35 (s, 3H, CH3), 4.97 (s, 2H,
CH2), 7.49–7.52 (t, J = 7.6 Hz, 2H, H-3, H-5), 7.56–7.58 (t,
J = 7.65 Hz, 1H, H-4), 7.87 (d, J = 7.6 Hz, 2H, H-2, H-6),
8.34–8.36 (s, 1H, CH, Imidazole’), 10.53 (s, 2H, 2×NH)
ppm. 13C NMR (DMSO-d6, 500 MHz): δ = 13.1 (CH3),
48.2 (CH2), 124.0 (CH-4, Ph), 128.1(CH-3, Ph), 128.2 (CH-
5, Ph), 129.1 (CH-2, Ph), 129.2 (CH-6, Ph), 132.6 (C-1,
Ph), 136.7 (C-4, Imidazole), 145.7 (C-5, Imidazole), 146.5
(C-2, Imidazole), 165.9 (C=O), 166.1 (C=O) ppm. HRMS:
(DMSO-d6, 500 MHz): δ = 2.32 (s, 3H, CH3), 4.91 (s, 2H,
CH2), 6.87–6.89 (d, J = 8.35 Hz, 1H, H3), 7.05–7.07 (d,
J = 7.9 Hz, 2H, H-2, H-6), 7.16-7.19 (t, J = 8.3 Hz, 1H, H-
4), 7.64–7.66 (d, J = 7.6 Hz, 1H, H-6), 8.32 (s, 1H, CH,
Imidazole’), 10.33 (s, 1H, NH), 10.65 (s, 1H, NH) ppm. 13
C
NMR (DMSO-d6, 500 MHz): δ = 13.1 (CH3), 48.1 (CH2),
119.1 (CH-2ʹ,6ʹ, Ph), 119.2 (CH-4ʹ, Ph), 119.8 (CH-3, Ph),
123.9 (CH-5, Ph), 124.5 (C-1, Ph), 126.1 (CH-6, Ph),
130.6 (CH-3ʹ,5ʹ, Ph), 132.9 (C-4, Imidazole), 145.7 (C-5,

Medicinal Chemistry Research
Imidazole), 146.5 (C-2, Imidazole), 154.9 (C-2, Ph), 156.7
(CH-1ʹ, Ph), 164.9 (C=O), 165.3 (C=O) ppm. HRMS: m/z
[M+H]+ calcd for C19H17N5O5: 395.1229, found:
396.1302.
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benzohydrazide (4k)
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Compliance with ethical standards
Conflict of interest The authors declare no competing interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.

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