Synthesis and antibacterial activity of new azole, diazole and triazole derivatives based on p-aminobenzoic acid

Article abstract of DOI:10.3390/molecules26092597

The p-aminobenzoic acid was applied for the synthesis of substituted 1-phenyl-5-oxopyrrolidine derivatives containing benzimidazole, azole, oxadiazole, triazole, dihydrazone, and dithiosemi-carbazide moieties in the structure. All the obtained compounds w

Full text of DOI:10.3390/molecules26092597

molecules
Article
Synthesis and Antibacterial Activity of New Azole, Diazole and
Triazole Derivatives Based on p-Aminobenzoic Acid
1
3
Birute˙ Sapijanskaite˙-Banevicˇ , Vykintas Palskys 2, Rita Vaickelioniene˙ 1,*, Ju¯rate˙ Šiugždaite˙ ,
Povilas Kavaliauskas 4,5,6 , Birute˙ Grybaite˙ 1 and Vytautas Mickevicˇius 1
1
Department of Organic Chemistry, Kaunas University of Technology, Radvile˙nu˛ Rd. 19,
LT-50254 Kaunas, Lithuania; birute.sapijanskaite@ktu.lt (B.S.-B.); birute.grybaite@ktu.lt (B.G.);
vytautas.mickevicius@ktu.lt (V.M.)
Thermo Fisher Scientific, V. A. Graicˇiu¯no st. 8, LT-02241 Vilnius, Lithuania;
2
vykintas.palskys@themofisher.com
Department of Veterinary Pathobiology, Veterinary Academy, Lithuanian University of Health Sciences,
3
Tilže˙s Str. 18, LT-47181 Kaunas, Lithuania; siujur@gmail.com
Weill Cornell Medicine of Cornell University, 527 East 68th Street, New York, NY 10065, USA;
4
pok4001@med.cornell.edu
Institute for Genome Sciences, School of Medicine, University of Maryland, 655 W. Baltimore Street,
5
Baltimore, MD 21201, USA
Biological Research Center, Veterinary Academy, Lithuanian University of Health Sciences, Tilže˙s Str. 18,
6
LT-47181 Kaunas, Lithuania
*
Correspondence: rita.vaickelioniene@ktu.lt; Tel.: +370-600-16-958
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The p-aminobenzoic acid was applied for the synthesis of substituted 1-phenyl-5-oxopyrrolidine
derivatives containing benzimidazole, azole, oxadiazole, triazole, dihydrazone, and dithiosemi-
carbazide moieties in the structure. All the obtained compounds were evaluated for their in vitro
antimicrobial activity against Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Salmonella
enteritidis, Escherichia coli, and Pseudomonas aeruginosa by using MIC and MBC assays. This study
Citation: Sapijanskaite˙-Banevicˇ, B.;
Palskys, V.; Vaickelioniene˙, R.;
Šiugždaite˙, J.; Kavaliauskas, P.;
Grybaite˙, B.; Mickevicˇius, V.
Synthesis and Antibacterial Activity
of New Azole, Diazole and Triazole
Derivatives Based on
showed a good bactericidal activity of
bial activity of the most promising compounds was higher than ampicillin. Furthermore, two benz-
imidazoles demonstrated good antimicrobial activity against L. monocytogenes (MIC 15.62 g/mL)
that was four times more potent than ampicillin (MIC 65 g/mL). Further studies are needed to
γ-amino acid and benzimidazoles derivatives. The antimicro-
µ
p-Aminobenzoic Acid. Molecules 2021,
26, 2597. https://doi.org/10.3390/
molecules26092597
µ
better understand the mechanism of the antimicrobial activity as well as to generate antimicrobial
compounds based on the 1-phenyl-5-oxopyrrolidine scaffold.
Academic Editors: Scott K. Bur and
Qiu Wang
Keywords: hydrazides; 2-pyrrolidinone; azoles; benzimidazole; antimicrobial activity
Received: 19 March 2021
Accepted: 27 April 2021
Published: 29 April 2021
1. Introduction
Rapidly growing antimicrobial resistance (AMR) has become a major source of mor-
bidity and mortality worldwide [1]. Increasing AMR among various pathogens has led
to fewer treatment options for patients suffering from severe infections caused by drug-
resistant (DR) pathogens. Moreover, infections caused by DR microorganisms require
more extensive treatment, therefore resulting in a longer course of illness and prolonged
hospitalization duration [2,3].
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with regard to jurisdictional claims in
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iations.
The extensive use of various antimicrobials in agriculture and veterinary sectors
played a pivotal role in the development of AMR and the selection of highly virulent
Copyright:
© 2021 by the authors.
bacterial strains [
environment and can be transferred to humans [
encoding AMR phenotypes can be further disseminated via horizontal gene transfer and
accumulate in various bacterial species [10 11]. These processes created a vicious cycle that
4–
7]. The DR pathogens of veterinary origin can further colonize the
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This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
8,9]. In addition, the genetic determinants
,
gave rise to multidrug-resistant (MDR) pathogens harboring multiple resistance mecha-
nisms, resulting in bacterial resistance to two and more antimicrobial drugs [12]. To over-
come this problem, it is important to develop novel compounds targeting MDR pathogens.
Molecules 2021, 26, 2597. https://doi.org/10.3390/molecules26092597
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2597
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The ESKAPE group of pathogens (Enterococcus faecium, Staphylococcus aureus, Kleb-
siella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species
complex) is the leading cause of hospital-acquired infections worldwide [13–15]. This
group of Gram-positive and Gram-negative bacteria causes life-threatening infections
amongst critically and chronically ill or immunocompromised individuals [15]. The grow-
ing antimicrobial resistance among ESKAPE pathogens creates a significant burden on
healthcare systems and has important global economic costs. Therefore, it is important to
develop novel compounds targeting clinically relevant multidrug-resistant ESKAPE group
of pathogens.
p-Aminobenzoic acid (pABA) and its derivatives are well-known for their chemical
properties and the broad spectrum of biological activity, and they have attracted consider-
able pharmacological and industrial interest. pABA is widely distributed in nature and
is abundant in various plant and animal tissues. Moreover, pABA is found in various
animal and plant-based sources such as grains, eggs, milk, and meat. Furthermore, pABA
is frequently found as a structural moiety in drugs and plays an important role as a phar-
macophore. In a voluminous database of commercial pharmaceuticals, 1.5% were found to
contain the pABA moiety [16].
Various compounds bearing a pABA nucleus exert strong antimicrobial [17
timutagenic [19], antioxidant, cytoprotective [20], and immunomodulatory [21 22] proper-
ties. Moreover, various pABA derivatives have antineoplastic, anesthetic, antiarrhythmic,
anticonvulsant, antiemetic, and gastrokinetic [16 23] properties. pABA is also involved
in the biosynthesis of coenzyme Q [24 25]; it also increases the thermotolerance [26] in
,18], an-
,
,
,
plants and plays an important role as a signaling molecule in the recognition of the plant
pathogens [27]. Various pABA derivatives were previously explored as promising im-
munomodulatory agents. pABA was shown to induce the transcriptional activation of
interferons in various cell types [22]. In addition to this, compounds bearing the pABA nu-
cleus were found to be a promising cyclophilin inhibitors [28]. Furthermore, pABA deriva-
tives were shown to prevent steel corrosion [29] and increase dye adsorption during textile
dyeing [30]. The wide spectrum of biological activity of pABA derivatives could be poten-
tially exploited for the development of novel antimicrobial and immunomodulatory drugs.
The 5-oxopyrrolidine or γ-lactam moiety is a constituent of many natural and non-
natural biologically active compounds. The broad range of biological activity displayed
by functionalized 2-pyrrolidinones makes them an attractive group with profound thera-
peutic [31] and antioxidant [32] use. Moreover, compounds bearing γ-lactam moiety were
previously demonstrated to bind the CCR4 chemokine receptor, making these compounds
potential therapeutics in treating T-cell neoplasms [33]. Besides immunomodulatory and
antiviral activity, 5-oxopyrrolidine derivatives were shown to have promising antimi-
crobial [34,35], antioxidative [36], antitumor [37], and anti-inflammatory [38] properties,
showing high binding affinity towards carbonic anhydrase isoforms [39,40].
The benzimidazole scaffold attracts considerable attention due to its numerous biolog-
ical properties. One of the most known structures containing the benzimidazole scaffold
is cyanocobalamin, also known as cobalamin (vitamin B12), which is involved in cellular
metabolism processes.
With a great affinity displayed towards a numerous enzymes and receptors, the ben-
zimidazoles could be potentially explored for the development of new pharmaceuticals.
Numerous studies revealed that compounds derived from a benzimidazole nucleus ex-
hibit analgesic and anti-inflammatory activity [41], as well as antiulcerative [42], anti-
cancer [43], antiparasitic [44], antimicrobial [45
anticancer/antiestrogenic [49 50], antihypertensive [51], antifouling [52], and many oth-
ers [53 60] activities. Therefore, the strategies of developing compounds bearing the benzim-
–47], antioxidant [46], anticonvulsant [48],
,
–
idazole scaffold should be further explored to generate pharmacologically active molecules.
1,2,4-Triazole derivatives have been reported to possess a wide range of bioactivities
such as neuroprotective [61], antifungal [62], anticancer [63], antibacterial [64], antihy-
pertensive, and cardioprotective [65], antiviral [66], and anticonvulsant [67]. The diverse

Molecules 2021, 26, 2597
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pharmaceutical properties of triazoles induced a deep interest in discovering new entities
for their broader applications. This fragment is a constituent of a variety of pharmaceuti-
cals (etizolam, estazolam, trazodone, ribavirin, trapidil, rizatriptan, anastrozole) that are
available in a clinical setting for the treatment of patients suffering from various diseases,
including muscle tension, suppression of seizures, depression, viral diseases, antiplatelet
action, migraine pains and breast cancer [68,69]. The introduction of the thione group,
either in 3- or 5-position, leads to an enhancement of biological activities related to triazole
moiety [70]. The triazolethione system is a cyclic analog of very important components
(such as thiosemicarbazides) that have effective biological applications [71].
The profound biological activity of pABA and various azoles makes them attractive
building blocks for the development of novel antimicrobial compounds targeting clinically
important pathogens. With this notion, in this paper we aim to synthesize a series of new
azole, diazole, and triazole derivatives based on p-aminobenzoic acid and evaluate their
in vitro antimicrobial properties against clinically important bacterial pathogens.
2. Results
2.1. Synthesis
Considering the wide structural and biological diversity of p-aminobenzoic acid and
its derivatives, herein we present the synthesis and antibacterial evaluation of a series of
1-(4-carboxyphenyl)-5-oxopyrrolidine-3-carboxylic acid derivatives using p-aminobenzoic
acid as the starting compound (Scheme 1). Compound
aminobenzoic and itaconic acids by the method described in [72]. The esterification
of compound with methanol afforded methyl ester , which then under the action of
2 was prepared from the 4-
2
3
hydrazine monohydrate in 2-propanol was converted into hydrazide
one hydrazinocarbonyl moiety.
4, containing only
Scheme 1. Synthesis of 5-oxopyrrolidine derivatives
2–13. 7a, Ar = C6H5; 7b, Ar = 4-MeO-C6H4; 7c,
Ar = 4-Me2N-C6H4; 11a, Ar1 = 4-O2N-C6H4; 11b, Ar1 = 4-Cl-C6H4.
The hydrazide functional group can undergo various chemical transformations; using
this ability, we thus performed a series of chemical reactions, applying different carbonyl
compounds. The reaction of hydrazide
ethanol produced pyrazole derivative , and the Paal–Knorr pyrrole synthesis using hexane-
2,5-dione (2,5-HD) afforded pyrrole . A catalytic amount of glacial acetic acid was used
in the reaction. In the 1H NMR spectrum of
, the intense singlets at 2.0 and 5.65 ppm
4 with pentane-2,4-dione (2,4-PD) in refluxing
5
6
6

Molecules 2021, 26, 2597
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were assigned to the protons of two methyl (2- and 5-positions) and two C=CH groups of
the pyrrole ring, respectively. The resonances at 103.10 and 126.74 ppm in the 13C NMR
spectrum of compound
NMR spectra of the synthesized compounds are given in the Supplementary Materials.
Hydrazones 7a were prepared by the condensation of acid hydrazide with ben-
zaldehyde, 4-methoxybenzaldehyde, and 4-dimethylaminobenzaldehyde in refluxing
ethanol ( ) or a mixture of ethanol and 1,4-dioxane (1:2). In the reaction with itaconic acid,
6 finally approved the formed pyrrole cycle in the molecule. All
–
c
4
a,c
the hydrazide that has the amine group can readily undergo autocatalyzed intramolecular
amidation–cyclization reaction to yield a stable 5-membered N-substituted pyrrolidinone
cycle. The reaction was carried out in water at reflux for 15 h. Multiplets in the ranges
of 2.50–2.90 (COCH2), 3.20–3.40 (CH) 3.56–3.72 (NCH2), and 3.87–4.18 (NCH2) ppm, in-
1
tegrated for 10 protons in total in the H NMR spectrum as well as double sets of the
resonances of carbons of the COCH2, CH, NCH2 fragments in the 13C NMR spectra of
compound 8 approve the presence of two pyrrolidinone rings.
For the synthesis of the target benzoylhydrazine derivative
with benzoyl chloride in dichloromethane at reflux for 10 min. The product
9
, hydrazide
4
was reacted
from the
9
reaction mixture was isolated in 54% yield. The formation of the –CONHNHCOPh–
fragment was approved by the presence of two singlets at 10.29 (NH) and 10.47 (NH) ppm,
and the multiplet was integrated for nine protons of the two aromatic rings in the interval
of 7.35–8.08 ppm.
To obtain a compound containing two hydrazinocarbonyl fragments, the reaction was
carried out in hydrazine monohydrate using a 17 excess. The target product was obtained
in a 54% yield.
The comparison of the spectra of compounds
that led to the easy identification of their specific structures. In the 1H NMR spectrum of
compound
, the singlet at 3.82 ppm (13C, 51.99 ppm), integrated for three protons, shows
4 and 10 demonstrated some differences
4
the presence of the methoxy group, and the singlet at 4.35 ppm integrated for two protons
proves the presence of the amino group, while in the 1H NMR of dihydrazide 10, the signal
of methoxy group is absent, and the broad singlet at 4.39 ppm is integrated for four protons,
which proves the presence of two amino groups in the molecule.
Hydrazones 11a
aldehydes. The reaction was carried out in the mixture of 2-propanol and 1,4-dioxane
(ratio of 1:1.7) at reflux for 12 ( ) or 11 ( ) h, and products from the reaction mixtures were
separated in 77% and 88% yields, respectively.
The synthesized hydrazones and 11 possess amide and azomethine groups in their
,b were obtained by the condensation of dihydrazide 10 with aromatic
a
b
7
structures. Based on the experimental and theoretical studies presented in literature [73], it
can be stated that due to the presence of the amide fragment and the restricted rotation
around the CO NH bond, the hydrazones exist in DMSO solutions as a mixture of Z/E
−
rotamers in which the Z rotamer predominates. The clearest proof of the existence of
conformers produced due to the presence of the CONH fragment was the discovery of
the two sets of resonances of the NH group in the low-field region of the 1H NMR spectra
recorded in DMSO-d6, where a stronger-field side signal was related to the resonance of
the rotamer with the Z structure.
The existence of the mixtures of stereoisomers relative to the CH=N structural frag-
1
ment of the molecules was also found in the H NMR spectra of the monosubstituted
hydrazones 7 and 11. Based on the studies described in the academic literature [73], as well
as the data of the spectra of these compounds, we can conclude that the produced mixtures
of stereoisomers with the Z-isomer predominated.
The interaction of dihydrazide 10 with phenyl isothiocyanate in refluxing methanol
led to the formation of thiosemicarbazide 12, which then under the action of 4% sodium hy-
droxide at reflux for 6 h and subsequent acidification of the mixture with dilute hydrochloric
acid (1:1) to pH 2 afforded heterocyclic compound 13 with two 4-phenyl-5-thioxo-1,2,4-
triazole moieties in the structure. Resonances in the interval of 9.39–9.75 (4H, 2NHNHCO)
as well as 10.12 and 10.40 (2H, 2NH) ppm in the 1H NMR spectrum of compound 12 is clear

Molecules 2021, 26, 2597
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evidence for the formation of the –CONHNHCSNH– moiety. Cyclodehydration of this
fragment in the presence of a strong base led to the 1,2,4-triazole 13 formation, which was
confirmed by the absence of thiosemicarbazide-specific spectral lines and the observation
of a decrease and downfield shift of the NH resonances (1H, 13.87 ppm).
Knowing the wide range of applications of the biological properties of benzimidazole
derivatives in various fields, including medicine, pharmacy, optics, and others, we decided
to synthesize compound 14 to have two benzimidazole moieties in its structure (Scheme 2).
Five methods to achieve this goal were used. The reaction conditions and yields of the
obtained product are given in Table 1.
Scheme 2. Synthesis of benzimidazole derivatives 14–18. 14, R = H; 15, R = CH3.
Table 1. Reaction conditions for the synthesis of benzimidazoles 14 and 15 and their corresponding
product yields.
Solvent/
Catalyst
Temperature,
◦C
Entry
Reagent
Time, h
Yield, %
1
2
3
4
5
-
170; 230
Reflux
2; 0.5
96
51
15% HCl
8
Benzene-1,2-
diamine
-
MW, 140 W
Reflux
0.25
25
40
12
2-PrOH/NH4Cl
PPA
120
6
97 (14); 91 (15)
◦
◦
The melting of carboxylic acid
2
with benzene-1,2-diamine at 170 C and then at 230 C
gave the target compound a 51% yield. The condensation of benzene-1,2-diamine with acid
in 15% hydrochloric acid (by the Phillips method), at reflux yielded the desired product 14
2
,
but the process took 96 h, and the product obtained in only a 8% yield. For this reason, we
tried a more modern method using microwaves, where the reaction mixture was exposed
to microwave irradiation (140 W) for 15 min under solvent-free conditions. Benzimidazole
14 was obtained in a 40% yield. The reaction in 2-propanol with ammonium chloride as
a catalyst did not produce the expected yield of the benzimidazole. The yield of bisbenz-
imidazole 14 was found to be only 12%. The most efficient method for the preparation of
compound 14 appeared to be condensation of dicarboxylic acid 2 with o-phenylenediamine

Molecules 2021, 26, 2597
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in polyphosphoric acid (PPA) at 120 ◦C for 6 h. The product was separated in 97% yield.
The last-mentioned method was used to obtain methylbenzimidazole derivative 15.
The pyrrolidinone ring of compound 14 was readily decyclized under alkaline hy-
drolysis conditions by refluxing it in an aqueous 20% sodium hydroxide solution for 2 h.
The NMR spectra of the obtained product 16 showed chemical shifts characteristic to the
open-chain structure in comparison with the initial compound 14.
The esterification of amino acid 16 with methanol in the presence of a catalytic amount
of sulfuric acid was unsuccessful when the action of a strong acid led to the cyclization
of the butanoic fragment to the initial pyrrolidinone ring. Therefore, to prepare acid
hydrazide, we had to choose another synthesis route. Butanoic acid hydrazide 17 was
obtained directly from pyrrolidinone derivatives 14 and its decyclized product—butanoic
acid 16. The interaction of butanoic acid 16 with hydrazine monohydrate proceeded
successfully under mild conditions, i.e., heating the reaction mixture in 2-propanol at reflux
for 20 h afforded acid hydrazide 17. However, to obtain it from the pyrrolidinone derivative
14, tightened reaction conditions were needed. Therefore, the reaction was carried out
in excess hydrazine monohydrate at reflux for 6 h. The 1H and 13C NMR spectra of 17
confirmed the open-chain compound. In the NMR spectra of 17, the triplet at 6.34 ppm
was ascribed to the NH proton, the singlets at 4.43 (NH2, 1H) and 9.12 (NHNH2, 1H), and
the spectral line at 169.90 (C=O, 13C) approved the formed acid hydrazide moiety.
The condensation of hydrazide 17 with hexane-2,5-dione was investigated. As ex-
pected, the reaction in 2-propanol at reflux, with the presence of a catalytic amount of
hydrochloric acid, afforded 2,5-dimethylpyrrole derivative 18. The singlets at 1.62 and 1.95
(CH3) and the doublets at 5.52, 5.57 (C=CH) ppm in the 1H NMR spectrum, in addition to
resonances at 10.50, 11.00, and 102.76, 102.81 ppm of the corresponding groups in the 13
NMR spectrum, prove the presence of the 2,5-dimethylpyrrole fragment.
C
Benzimidazoles play an important role in modern medicinal chemistry by being an
important pharmacophore. With this notion, it is important to develop a large amount of
structurally diverse benzimidazoles with potential medicinal properties.
For this purpose, the functionalization of benzimidazole 14 was performed (Scheme 3).
The functionalization at nitrogen is perhaps the most common, and therefore it was chosen
for the investigation. Initially, N-substituted benzimidazole derivative 19 was synthesized
by the alkylation of bisbenzimidazole 14 with ethyl chloroacetate in acetone in the presence
of potassium carbonate and a catalytic amount of TBAI (tetrabutylammonium iodide). The
reaction was carried out at reflux for 20 h. The obtained ethyl ester 19 further was applied
for the preparation of hydrazide 20. The structures of the synthesized compounds 19 and
20 were determined by spectral methods and chemical transformations.
The refluxing of 19 with hydrazine monohydrate in 1,4-dioxane for 18 h led to the for-
mation of compound 20 containing two 2-hydrazinyl-2-oxoethyl moieties, whose presence
is confirmed by the singlets at 4.49 (2NH2, 1H), 4.87, 4.95, 5.24, and 5.32 (2NCH2CO, 1H),
8.83, 8.88, 9.60, and 9.65 (2NH, 1H), as well as by the resonance lines at 166.01 and 166.34
(2CONH, 13C) ppm.
Carbonyl compounds are frequently used as derivatization agents. The condensation
of acid hydrazide with diketone pentane-2,4-dione gave 3,5-dimethylpyrazole derivatives
21 a 82% yield. The reaction of 21 was performed for 5 h in refluxing 2-propanol and in
the presence of a catalytic amount of hydrochloric acid. The reaction product was isolated
1
from the reaction mixture by diluting it with water. The data of the H and 13C NMR
spectroscopic techniques and elemental analysis confirmed the proposed structures of the
synthesized pyrazole 21.
Oxadiazole derivative 22 was obtained by the ring closure reaction of the hydrazide 20
with carbon disulfide in alkaline medium obtained by using potassium hydroxide, which
was dissolved in methanol, and then CS2 was added dropwise to the cooled solution. After
a thorough stirring for 15 min, the required amount of hydrazide was added, and the
obtained reaction mixture was refluxed for 12 h. The acidifying of the aqueous solution
of the reaction mixture with hydrochloric acid to pH 1 afforded the desired derivative 22

Molecules 2021, 26, 2597
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with two 1,3,4-oxadiazole moieties in the molecule. The signals in the NMR spectra of the
compound were an exact match of the protons and carbon atoms of the obtained structure.
Scheme 3. Synthesis of bisbenzimidazole derivatives 19–24. 24 a, Ar = C6H5; b, Ar = 4-O2N-C6H4; c,
Ar = 4-F-C6H4; d, Ar = 3-Cl-C6H4; e, Ar = 2,3-di(H3CO)-C6H3.
Derivative 23 with two thiosemicarbazide moieties in the molecule was obtained in
the reaction of hydrazide 20 with phenyl isothiocyanate. The reaction was performed
in methanol at reflux for 6 h, and the obtained hydrazinecarbothioamine 23 was then
applied for the synthesis of triazolethione using a method of cyclization in basic conditions.
Cyclization was performed in an aqueous 4% sodium hydroxide solution in order to
prevent 5-oxopyrrolidine ring breakage with the subsequent acidification of the reaction
mixture with dilute hydrochloric acid (1:1) to pH 2. However, the process failed, and
efforts to separate the cyclized product were unsuccessful. The spectral data (NMR, IR,
and elemental analysis) of thiosemicarbazide 23 were in full agreement with the proposed
structure. The multiplet in the range of 9.48–10.04 (4NH) ppm and the singlets at 10.51 and
10.58 (2NH) ppm in the 1H NMR spectrum for 23 as well as additional peaks in the interval
of 7.08–7.95 ppm prove the presence of the CONHNHCSNHPh fragment.
The hydrazones 24 were prepared by the condensation of hydrazide 20 with the
corresponding aromatic aldehyde (benzaldehyde (
and 2,3-dimethoxybenzaldehyde ( )) in a molar ration of 1:7.5. The reactions were carried
out by heating the mixtures at reflux for 5 h, except in case when the reaction proceeded
a), 4-nitro- (b), 4-fluoro- (c), 3-chloro- (d)
e
b
for 8 h. The products were separated in good to excellent yields in the range of 77%–94%.
When 1H-NMR spectra of compounds 24 were observed, two sets of signals of the
protons at different ppm were seen. This is because of the compounds, which have an
arylidene hydrazide structure. The restricted rotation around the CO NH bond causes the
−
formation of a mixture of Z/E rotamers, whereas the presence of a double bond of CH=N
influences the formation of geometric isomers, which are clearly visible in the spectra of
these compounds [74]. The ratio in each case was calculated by using 1H-NMR data, and
they are as follows: 0.75:0.25 for a–c, e and 0.8:0.2 for d.

Molecules 2021, 26, 2597
8 of 23
2.2. The Antimicrobial Activity of the Synthesized Compounds
In this study, we aimed to synthesize a series of novel benzimidazole derivates bearing
ethyl ester, hydrazide, 3,5-dimethylpyrazole, 2,5-dimethylpyrrole, oxadiazole, thiosemicar-
bazide, and hydrazone moieties and to investigate their in vitro antimicrobial properties
against a series of Gram-positive and Gram-negative bacterial pathogens.
The antimicrobial properties of synthesized compounds 3–24 were investigated against
Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Escherichia coli, Pseudomonas
aeruginosa, and Salmonella enteritidis (Supplementary Table S1). The minimal inhibitory
concentration (MIC) was evaluated by the broth dilution method, while the minimal
bactericidal concentration (MBC) was determined by plating.
Compounds
microorganisms, suggesting the possible existence of Gram-positive bacteria-selective tar-
gets for compounds 14 17 19, and 20, which bear dimethyl ester, 3,5-dimethylpyrazole,
bisbenzimidazole, bis 5(6)-methylbenzimidazole, -amino acid and its hydrazide, diethyl
3–24 demonstrated acceptable antimicrobial activity against Gram-positive
3
,
5,
–
,
γ
ester, and dihydrazide fragments, respectively. The antimicrobial activity was highly
structure-depended on and was mostly bactericidal at near-MIC concentration (Table 2).
The most promising compounds demonstrated near-MIC bactericidal activity, therefore fur-
ther showing importance of the abovementioned scaffold as novel antimicrobials targeting
Gram-positive bacterial pathogens.
The results obtained in this study showed that all tested methyl derivatives
hydrazide 10, and its derivatives 12 and 13 demonstrated moderate antimicrobial activity
on all tested bacteria strains (Table 2). Moreover, hydrazones 11a did not demonstrate
3–9, di-
,
b
antimicrobial activity on neither Gram-positive nor Gram-negative bacteria. Hydrazone
11b harboring 4-chlorobenzylidene fragment demonstrated weak but selective bacterici-
dal activity on S. aureus (250 µg/mL) but not on other Gram-positive or Gram-negative
organisms (Table 2).
Diester
MBC of 31.25
3
µ
and pyrazole derivative
g/mL) against B. cereus. Interestingly, compounds
5
exhibited good bactericidal activity (MIC and
and did not show a
3
5
good antimicrobial activity against other Gram-positive organisms, suggesting the possible
presence of B. cereus-specific targets of compound 5 (Table 2).
Benzimidazole 14, methylbenzimidazole 15, γ-amino acid 16, and oxadiazole 22
demonstrated broad-spectrum antimicrobial activity that targets both Gram-negative and
Gram-positive microorganisms (Table 2). Compound 16 demonstrated the highest antimi-
crobial activity against all tested strains, suggesting the important role of
moiety for biological activity.
γ-amino acid
Furthermore, in this study, the bactericidal activity of hydrazide 17 and diester 19
bactericidal activity on S. aureus was comparable to ampicillin (62.5 g/mL). The incor-
poration of -amino acid moiety in compound 16 and 4-(nitrobenzylidene)hydrazinyl
fragment in 24b resulted in a compound with good activity against Gram-negative organ-
isms (MIC and MBC at 31.25 g/mL). In this study, compound 16 bearing -amino acid
µ
γ
µ
γ
moiety demonstrated the most potent antimicrobial activity against a broad spectrum of
microorganisms, demonstrating the importance of the abovementioned fragment as an
antibacterial pharmacophore.
Interestingly, benzimidazoles 14 and 15 were an exception in this assay and had
an exclusive activity against L. monocytogenes, although no activity was observed when
tested against S. aureus or B. cereus. Compound 14 demonstrated slightly better, near-MIC
bactericidal activity (MBC 15.62
µg/mL) (Table 2). Benzimidazole 15 bearing 5(6)-methyl
moiety showed one dilution higher MBC (31.25
µg/mL), suggesting that the 5(6)-methyl
moiety is important for bactericidal activity against L. monocytogenes.
The investigations of structure-activity based relationships revealed some evident facts
that changes in the 1-phenyl-5-oxopyrrolidine backbone by an incorporation of benzimida-
zole moieties greatly affect the biological properties of the compounds. The data presented
in Table 2 demonstrate that benzimidazole 14 shows broad-spectrum antimicrobial activity,
which was most evident when tested against L. monocytogenes. The antimicrobial activity

Molecules 2021, 26, 2597
9 of 23
of compound bearing a nitro group (24b) in the benzene ring was confirmed in this study
when stronger antibacterial properties against the E. coli and P. aeruginosa strains were seen
in comparison to other hydrazones 24.
Table 2. Minimal inhibitory concentrations (MIC) as well as minimal bactericidal concentrations (MBC) of 5-oxopyrrolidine
derivatives 3–24 against various bacterial strains.
Gram-Positive Bacteria
Gram-Negative Bacteria
S. aureus ATCC
B. cereus ATCC
L. monocytogenes
S. enteritidis
ATCC 13076
P. aeruginosa
NCTC 6750
E. coli ATCC 8739
9144
11778
ATCC 7644
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
µg/mL
3
4
250
125
250
125
250
250
250
250
250
250
250
125
>250
250
250
125
62.5
125
31.25
62.5
31.25
62.5
125
62.5
125
62.5
125
125
125
31.25
125
31.25
250
125
250
125
250
250
250
250
250
250
250
250
250
250
250
250
125
250
125
5
250
31.25
125
31.25
125
125
125
125
125
125
125
6
250
250
250
125
125
125
125
125
125
7a
7b
7c
250
250
250
125
125
125
125
250
250
125
125
250
250
250
250
250
125
125
250
250
125
125
250
250
250
125
125
125
125
250
250
125
125
8
250
125
125
125
125
62.5
125
125
125
125
62.5
125
125
9
125
250
250
250
250
125
250
250
125
10
11a
11b
12
13
14
15
16
17
19
20
21
22
24a
24b
24c
24d
24e
125
125
125
125
125
125
125
125
125
125
125
>250
250
>250
>250
125
>250
>250
250
>250
>250
125
>250
>250
250
>250
>250
125
>250
>250
250
>250
>250
125
>250
>250
250
>250
>250
125
>250
>250
250
125
125
125
250
62.5
15.62
15.62
31.25
62.5
62.5
62.5
62.5
62.5
125
62.5
15.62
31.25
31.25
62.5
62.5
62.5
62.5
62.5
125
125
125
125
250
62.5
62.5
62.5
31.25
62.5
62.5
62.5
125
62.5
62.5
62.5
31.25
62.5
62.5
62.5
125
62.5
62.5
31.25
31.25
31.25
62.5
125
62.5
62.5
31.25
62.5
62.5
31.25
62.5
62.5
125
62.5
125
62.5
62.5
31.25
62.5
31.25
62.5
62.5
31.25
125
62.5
62.5
31.25
62.5
62.5
62.5
62.5
31.25
125
62.5
62.5
31.25
62.5
62.5
62.5
62.5
62.5
125
62.5
125
31.25
62.5
62.5
31.25
62.5
62.5
125
31.25
62.5
62.5
62.5
62.5
62.5
125
62.5
125
62.5
62.5
31.25
125
62.5
125
62.5
125
62.5
125
62.5
125
62.5
125
62.5
125
62.5
62.5
125
62.5
62.5
125
31.25
125
31.25
125
31.25
62.5
125
125
125
125
62.5
62.5
62.5
62.5
125
125
125
62.5
62.5
62.5
62.5
62.5
62.5
62.5
125
62.5
62.5
Ampicillin
The results generated during this study are expected to be a foundation for the
development of novel 1-phenyl-5-oxopyrrolidine-based antimicrobials. Further studies are
needed to better understand the mechanism of antimicrobial activity as well as to generate
more potent antimicrobial compounds based on the 1-phenyl-5-oxopyrrolidine nucleus.
3. Materials and Methods
3.1. Synthesis
Reagents and solvents were obtained from Sigma-Aldrich (St. Louis, MO, USA) and
used without further purification. The reaction course and purity of the synthesized

Molecules 2021, 26, 2597
10 of 23
compounds were monitored by TLC using aluminum plates precoated with Silica gel
with F254 nm (Merck KGaA, Darmstadt, Germany). Melting points were determined
with a B-540 melting point analyzer (Büchi Corporation, New Castle, DE, USA) and
were uncorrected. NMR spectra were recorded on a Brucker Avance III (400, 101 MHz)
spectrometer. Chemical shifts were reported in (
(TMS) with the residual solvent as internal reference ([D6]DMSO,
δ = 39.5 ppm for 13C). Data were reported as follows: chemical shift, multiplicity, coupling
constant (Hz), integration, and assignment. IR spectra (
, cm−1) were recorded on a Perkin–
δ
) ppm relative to tetramethylsilane
δ
= 2.50 ppm for 1H and
ν
Elmer Spectrum BX FT–IR spectrometer using KBr pellets. Mass spectra were obtained on
a Bruker maXis UHRTOF mass spectrometer with ESI ionization. Elemental analyses (C, H,
N) were conducted using the Elemental Analyzer CE-440; their results were found to be in
good agreement (±0.3%) with the calculated values.
1-(4-Carboxyphenyl)-5-oxopyrrolidine-3-carboxylic acid (2): A mixture of itaconic acid
(65.05 g, 0.5 mol) and p-aminobenzoic acid
1 (34.3 g, 0.25 mol) was refluxed in water (150 mL)
for 6 h, then cooled down; the formed crystalline precipitate was filtered off, washed with
water, 2-propanol, and recrystallized from 2-propanol to give the title compound
solid, yield 45 g, 72%, m. p. 290 ◦C (decomp.)).
2 (white
1H-NMR (400 MHz, DMSO-d6):
δ = 2.69–2.87 (m, 2H, COCH2), 3.30–3.44 (m, 1H, CH),
3.96–4.11 (m, 2H, NCH2), 7.78 (d, J = 8.7 Hz, 2H, Har), 7.94 (d, J = 8.7 Hz, 2Har), 12.83 (br s,
2H, 2OH) ppm.
13C-NMR (101 MHz, DMSO-d6):
δ
= 35.08 (COCH2), 35.38 (CH), 49.85 (NCH2), 118.49,
125.76, 130.18, 142.91 (Car), 166.89, 172.54, 174.10 (3C=O) ppm.
IR (KBr): ν
= 3121 (OH), 1706, 1671 (3C=O) cm−1
.
max
MS (APCI+, 25 V) m/z, %: 272 (100) [M + Na]+.
Calcd. for C12H11O5N, %: C 57.83; H 4.45; N 5.62. Found, %: C 57.98; H 4.52; N 5.68.
Methyl 1-[4-(methoxycarbonyl)phenyl]-5-oxopyrrolidine-3-carboxylate ( To a solution
of carboxylic acid (31.13 g, 0.125 mol) in methanol (350 mL), concentrated sulfuric acid
3):
2
(12.5 mL) was added dropwise, and the mixture was heated at reflux for 4 h. The solvent
was then evaporated under reduced pressure, and the residue neutralized with 10% sodium
carbonate solution to pH 6–7. After cooling, the obtained solid was filtered off, washed
with plenty of water and 2-propanol, and recrystallized from 2-propanol to give the title
compound 3 (white solid, yield 23.5 g, 68%, m. p. 142–143 ◦C).
1H-NMR (400 MHz, DMSO-d6):
δ = 2.72–2.91 (m, 2H, COCH2), 3.42–3.55 (m, 1H, CH),
3.69 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 3.99–4.16 (m, 2H, NCH2), 7.82 (d, J = 8.8 Hz, 2H,
Har), 7.97 (d, J = 8.8 Hz, 2H, Har) ppm.
13C-NMR (101 MHz, DMSO-d6):
δ = 34.80 (COCH2), 35.14 (CH), 49.56 (NCH2),
51.96 (OCH3), 52.19 (OCH3), 118.52, 124.54, 130.00, 143.11 (Car), 165.72, 172.32, 172.91
(3C=O) ppm.
IR (KBr): ν
= 1735, 1707 (3C=O) cm−1
.
max
MS (APCI+, 25 V) m/z, %: 300 (100) [M + Na]+.
Calcd. for C14H15O5N, %: C 60.64; H 5.45; N 5.05. Found, %: C 60.88; H 5.50; N 5.14.
Methyl 4-[3-(hydrazinocarbonyl)-5-oxopyrrolidin-1-yl]benzoate (
4): To a boiling mixture of
methyl ester (13.86 g, 0.05 mol) and 2-propanol (50 mL), hydrazine monohydrate (7.5 g,
3
0.15 mol) was added, and the mixture was heated at reflux for 1 h. After completion of the
reaction (TLC), the mixture was cooled to room temperature; the formed precipitate was
filtered off, washed with water, 2-propanol, and hexane, and recrystallized from 2-propanol
to give the title compound 4 (white solid, yield 12.06 g, 87%, m. p. 198–199 ◦C).
1H-NMR (400 MHz, DMSO-d6):
δ = 2.61–2.82 (m, 2H, COCH2), 3.09–3.26 (m, 1H, CH),
3.82 (s, 3H, OCH3), 3.84–3.91 (m, 1H, NCH2), 3.98–4.07 (m, 1H, NCH2), 4.35 (s, 2H, NH2),
7.81 (d, J = 8.4 Hz, 2H, Har), 7.95 (d, J = 8.4 Hz, 2H, Har), 9.30 (s, 1H, NH) ppm.
13C-NMR (101 MHz, DMSO-d6): δ = 33.94 (COCH2), 35.93 (CH), 50.58 (NCH2), 51.99
(OCH3), 118.41, 124.40, 130.02, 143.25 (Car), 165.77, 171.44, 172.91 (3C=O) ppm.
IR (KBr): ν
= 3280, 3308 (NH, NH ), 1724, 1685, 1637 (3C=O), 1282 (OCH ) cm−1
.
2 3
max
MS (APCI+, 25 V) m/z, %: 300 (100) [M + Na]+.

Molecules 2021, 26, 2597
11 of 23
Calcd. for C13H15O4N3, %: C 56.32; H 5.42; N 15.16. Found, %: C 56.48; H 5.52;
N 15.22.
Methyl 4-(4-(3,5-dimethyl-1H-pyrazole-1-carbonyl)-2-oxopyrrolidin-1-yl)benzoate (
solution of hydrazide (1.11 g, 4 mmol) in ethanol (8 mL), 2,4-pentanedione (1 g, 10 mmol)
5): To a
4
was added, and the mixture was heated at reflux for 2 h; it then cooled down, and the
formed precipitate was filtered off, washed with ethanol, and recrystallized from methanol
to give the title compound 5 (white solid, yield 0.56 g, 41%, m. p. 161–162 ◦C).
1H-NMR (400 MHz, DMSO-d6):
δ = 2.21, 2.48 (2s, 6H, 2CH3), 2.84–2.99 (m, 2H,
COCH2), 3.82 (s, 3H, OCH3), 4.04–4.28 (m, 2H, NCH2), 4.44–4.54 (m, 1H, CH), 6.23 (s, 1H,
C=CH−C), 7.82 (d, J = 8.7 Hz, 2H, Har), 7.96 (d, J = 8.7 Hz, 2H, Har) ppm.
13C-NMR (101 MHz, DMSO-d6):
δ = 13.58, 14.03 (2CH3), 35.27 (CH + COCH2), 49.96
(NCH2), 51.97 (OCH3), 111.61, 118.58, 124.53, 130.01, 143.12, 143.91, 152.19 (Car), 165.73,
172.39, 172.43 (3C=O) ppm.
IR (KBr): ν
= 1729 (3C=O), 1276 (OCH ) cm−1
.
3
max
MS (APCI+, 25 V) m/z, %: 364 (100) [M + Na]+.
Calcd. for C18H19N3O4, %: C 63.33; H 5.61; 12.31. Found, %: C 63.49; H 5.75; N 12.47.
Methyl 4-(4-((2,5-dimethyl-1H-pyrrol-1-yl)carbamoyl)-2-oxopyrrolidin-1-yl)benzoate ( To
a solution of hydrazide (3,48 g, 12.5 mmol) in ethanol (15 mL), 2,5-hexanedione (1.85
6):
4
mL, 15.8 mmol) and glacial acetic acid (2 mL) were added by stirring, and the mixture
was heated at reflux for 1,5 h. Then the reaction mixture was cooled down, and the
formed crystalline solid was filtered off, washed with ethanol and ether, and recrystallized
from methanol to give the title compound
6 (yellowish solid, yield 2.11 g, 47.5%, m. p.
147–148 ◦C).
1H-NMR (400 MHz, DMSO-d6):
δ = 2.00 (s, 6H, 2CH3), 2.74–3.01 (m, 2H, COCH2),
3.46–3.54 (m, 1H, CH), 3.84 (s, 3H, OCH3), 3.98–4.22 (m, 2H, NCH2), 5.65 (s, 2H, 2C=CH),
7.85 (d, J = 8.8 Hz, 2H, Har), 7.97 (d, J = 8.8 Hz, 2H, Har), 10.94 (s, 1H, NH) ppm.
13C-NMR (101 MHz, DMSO-d6):
δ = 10.94, 10.96 (2CH3), 33.98 (COCH2), 35.87 (CH),
50.21 (NCH2), 51.99 (OCH3), 103.10, 118.53, 124.55, 126.74, 130.05, 143.16 (Car), 165.75,
171.72, 172.41 (3C=O) ppm.
IR (KBr): ν
= 3264 (NH), 1714, 1702, 1671 (3C=O), 1286 (OCH ) cm−1
.
3
max
MS (APCI+, 25 V) m/z, %: 378 (100) [M + Na]+.
Calcd. for C19H21N3O4, %: C 64.21; H 5.96; N 11.82. Found, %: C 64.32; H 6.03;
N 11.76.
General procedure for the preparation of hydrazones 7a–c:
To the boiling solution of hydrazide (1.11 g, 4 mmol) in ethanol (50 mL) (
ethanol:1,4-dioxane (5:10 mL) ( ) the corresponding aromatic aldehyde [benzaldehyde
(6 mmol), 4-methoxybenzaldehyde (5 mmol), 4-dimethylaminobenzaldehyde (6 mmol)]
was added, and the reaction mixture was heated at reflux for 3.5 ( ), 2.5 ( ) h. Then the
4
a,c) or
b
a,c
b
mixture was cooled down; the formed solid was filtered off, washed with 2-propanol, and
recrystallized from 2-propanol to give the title compound 7a (white solid, yield 0.79 g, 54%,
m. p. 201–203 ◦C), 7b (white solid, yield 1.33 g, 84%, m. p. 230–231 ◦C), and 7c (yellowish
solid, yield 1.22 g, 70%, m. p. 199–201 ◦C).
Methyl 4-(4-(2-benzylidenehydrazine-1-carbonyl)-2-oxopyrrolidin-1-yl)benzoate (7a
NMR (400 MHz, DMSO-d6): = Z/E 2.78–2.95 (m, 2H, COCH2), 3.37–3.43 (m, 0.3H, CH,
):
1H-
δ
overlaps with the water peak), 3.83 (s, 3H, OCH3), 4.01–4.24 (m, 0.7H, CH + 2H, NCH2),
7.40–7.47 (m, 3H, Har), 7.69–7.74 (m, 2H, Har), 7.85 (d, J = 8.7 Hz, 2H, Har), 7.93–8.00 (m,
2H, Har), 8.05 (s, 0.7H, CH=N), 8.24 (s, 0.3H, CH=N), 11.62, 11.69 (2s, 1H, NH) ppm.
IR (KBr): ν
= 3254 (NH), 1719, 1677 1658 (3C=O), 1289 (OCH ) cm−1
.
3
max
MS (APCI+, 25 V) m/z, %: 366 (100) [M + H]+.
Calcd. for C20H19N3O4, %: C 65.74; H 5.24; N 11.50. Found, %: C 65.65; H 5.22;
N 11.46.
Methyl 4-(4-(2-(4-methoxybenzylidene)hydrazine-1-carbonyl)-2-oxopyrrolidin-1-yl)benzoate
1H-NMR (400 MHz, DMSO-d6):
= Z/E 2.77–2.93 (m, 2H, COCH2), 3.78 (s, 3H, OCH3),
3.82 (s, 3H, OCH3), 3.79–4.26 (m, 1H, CH + 2H, NCH2), 6.96–7.01 (m, 2H, Har), 7.64 (d,
(7b
):
δ

Molecules 2021, 26, 2597
12 of 23
J = 8.5 Hz, 2H, Har), 7.84 (d, J = 8.7 Hz, 2H, Har), 7.93–8.03 (m, 2H, Har + 0.6H, N=CH), 8.41
(s, 0.4H, N=CH), 11.47, 11.54 (2s, 1H, NH) ppm.
IR (KBr): ν
= 3240 (2NH), 1717, 1680, 1653 (3C=O) cm−1
.
max
MS (APCI+, 25 V) m/z, %: 396 (100) [M + H]+.
Calcd. for C21H21N3O5, %: C 63.79; H 5.35; N 10.63. Found, %: C 63.69; H 5.44;
N 10.67.
Methyl 4-(4-(2-(4-(diethylamino)benzylidene)hydrazine-1-carbonyl)-2-oxopyrrolidin-1-yl)
benzoate (7c = Z/E 1.08 1.10 (2t, J = 7.0 Hz, 6H, 2CH3),
1H-NMR (400 MHz, DMSO-d6):
):
δ
2.72–2.93 (m, 2H, COCH2), 3.34–3.41 (m, 4H, 2CH2CH3 + 0.4H, CH), 3.83 (s, 3H, OCH3),
3.96–4.22 (m, 2H, NCH2 + 0.6H, CH), 6.55–6.80 (m, 2H, Har), 7.48 (d, J = 8.7 Hz, 2H, Har),
7.80–8.01 (m, 4H, Har + 0.6H, N=CH), 8.05 (s, 0.4H, N=CH), 11.28, 11.32 (2s, 1H, NH) ppm.
IR (KBr): ν
= 3248 (2NH), 1718, 1702, 1663 (3C=O) cm−1
.
max
MS (APCI+, 25 V) m/z, %: 437 (100) [M + H]+.
Calcd. for C24H28N4O4, %: C 66.04; H 6.47; N 12.84. Found, %: C 66.01; H 6.48;
N 12.74.
1-(1-(4-(Methoxycarbonyl)phenyl)-5-oxopyrrolidine-3-carboxamido)-5-oxopyrrolidine-3- car-
boxylic acid ( A mixture of hydrazide (1.11 g, 4 mmol), itaconic acid (0.62 g, 4.8 mmol),
8
):
4
and water (15 mL) was refluxed for 15 h and then cooled down; the formed precipitate was
filtered off, washed with plenty of water and hexane, and recrystallized from 2-propanol to
give the title compound 8 (yellowish solid, yield 1.23 g, 79%, m. p. 249–251 ◦C).
1H-NMR (400 MHz, DMSO-d6):
δ = 2.50–2.90 (m, 4H, 2COCH2), 3.20–3.40 (m, 2H, 2CH),
3.56–3.72 (m, 2H, NCH2), 3.83 (s, 3H, OCH3), 3.87–4.18 (m, 2H, NCH2), 7.82 (d, J = 8.8 Hz, 2H,
Har), 7.97 (d, J = 8.8 Hz, 2H, Har), 10.40 (s, 1H, NH), 12.82 (br. s, 1H, OH) ppm.
13C-NMR (101 MHz, DMSO-d6):
δ = 31.26, 33.56, 34.13, 35.62, 49.64, 50.19, 50.21
(2COCH2, 2CH, 2NCH2), 52.01 (OCH3), 118.51, 124.52, 130.06, 143.16 (Car), 165.76, 170.90,
171.29, 172.52 (4C=O), 174.02 (COOH) ppm.
IR (KBr):
Calcd. for C18H19N3O7, %: C 55.53; H 4.92; N 10.79. Found, %: C 55.47; H 4.99;
N 10.92.
Methyl 4-(4-(2-benzoylhydrazine-1-carbonyl)-2-oxopyrrolidin-1-yl)benzoate (
ing solution of hydrazide (5 g, 1.8 mmol) in dichloromethane (100 mL), benzoyl chloride
ν .
max = 3410 (OH), 3275 (NH), 1727, 1702, 1684, 1666 (5C=O), 1286 (OCH3) cm−1
9): To the boil-
4
was added dropwise and the reaction mixture was heated at reflux for 10 min. It was then
cooled down, and the formed precipitate was filtered off, washed with dichloromethane
and ethanol, and recrystallized from methanol to give the title compound
9 (yellowish
solid, yield 3.7 g, 54%, m. p. 239–241 ◦C).
1H-NMR (400 MHz, DMSO-d6):
δ = 2.66–2.94 (m, 2H, COCH2), 3.41–3.49 (m, 1H, CH),
3.83 (s, 3H, OCH3), 3.91–4.22 (m, 2H, NCH2), 7.35–8.08 (m, 9H, Har), 10.29, 10.47 (2s, 2H,
2NH) ppm.
13C-NMR (101 MHz, DMSO-d6):
δ = 33.71, 33.81, 35.51, 35.80, 50.07, 50.46 (COCH2, CH,
NCH2), 52.02 (OCH3), 118.48, 118.52, 124.49, 124.57, 127.44, 128.50, 130.07, 131.90, 132.31,
143.10, 143.22 (Car), 165.55, 165.74, 165.77, 171.62, 171.91, 172.38, 172.71 (4C=O) ppm.
IR (KBr): ν
= 3206 (2NH), 1718, 1682, (4C=O), 1281 (OCH ) cm−1
.
3
max
MS (APCI+, 25 V) m/z, %: 382 (100) [M + H]+.
Calcd. for C20H19N3O5, %: C 62.99; H 5.02; N 11.02. Found, %: C 62.87; H 5.16;
N 11.12.
1-(4-(Hydrazinecarbonyl)phenyl)-5-oxopyrrolidine-3-carbohydrazide (10
methyl ester (13.85 g, 0.05 mol) and hydrazine monohydrate (42.6 g, 0.85 mol) was heated
): A mixture of
3
at reflux for 2 h. After completion of the reaction (TLC), the mixture was cooled to room
temperature, and the formed precipitate filtered off, washed with plenty of water and
2-propanol, and recrystallized from water to give the title compound 10 (white solid, yield
7.5 g, 54%, m. p. 225–226 ◦C).
1H-NMR (400 MHz, DMSO-d6):
δ = 2.57–2.82 (m, 2H, COCH2), 3.03–3.32 (m, 1H,
CH), 3.82–4.10 (m, 2H, NCH2), 4.39 (br. s, 4H, NH2), 7.73 (d, J = 8.7 Hz, 2H, Har), 7.85 (d,
J = 8.7 Hz, 2H, Har), 9.31 (s, 1H, CONH), 9.72 (s, 1H, CONH) ppm.

Molecules 2021, 26, 2597
13 of 23
13C-NMR (101 MHz, DMSO-d6):
δ
= 34.04 (COCH2), 35.90 (CH), 50.61 (NCH2), 118.40,
127.63, 128.32, 141.47 (Car), 165.41, 171.53, 172.60 (3C=O) ppm.
IR (KBr): ν
= 3307, 3279, 3193, 3167 (2NH , 2NH), 1687, 1635 (3C=O) cm−1
.
max
2
MS (APCI+, 25 V) m/z, %: 300 (100) [M + Na]+.
Calcd. for C12H15O3N5, %: C 51.98; H 5.45; N 25.26. Found, %: C 52.04; H 5.53;
N 25.29.
General procedure for the preparation of hydrazones 11a, b:
A mixture of dihydrazide 10 (0.5 g, 1.8 mmol), the corresponding aromatic aldehyde
(10.8 mmol) and 2-propanol/1,4-dioxane (1:1.7) was heated at reflux for 12 (a) or 11 (b) h.
Then the mixture was cooled down, and the formed solid filtered off and washed with
2-propanol, and recrystallized from 1,4-dioxane to give the title compound 11a (yellow
solid, yield 0.76 g, 77%, m. p. 295–296 ◦C and 11b (white solid, yield 0.83 g, 88%, m. p.
298–300 ◦C).
N-(4-nitrobenzylidene)-1-[4-[2-(4-nitrobenzylidenehydrazinocarbonyl)]phenyl]-5-oxopyrrolidine-
3-carbohydrazide (11a): δ = Z/E 2.75–2.98 (m, 2H, COCH2),
1H-NMR (400 MHz, DMSO-d6):
3.38–3.48 (m, 0.35H, CH), 4.01–4.27 (m, 0.65H, CH + 2H, NCH2), 7.76–8.09 (m, 8H, Har),
8.14 (s, 0.65H, CH=N), 8.16–8.32 (m, 4H, Har), 8.33 (s, 0.35H, CH=N), 8.54 (s, 1H, CH=N),
11.89, 11.96 (2s, 1H, NH), 12.10 (s, 1H, NH) ppm.
IR (KBr): ν
= 3433 (2NH), 1667, 1603 (3C=O) cm−1
.
max
MS (APCI+, 25 V) m/z, %: 544(100) [M + H]+.
Calcd. for C26H21N7O7, %: C 57.46; H 3.89; N 18.04. Found, %: C 57.57; H 3.97;
N 17.98.
N-(4-chlorobenzylidene)-1-[4-[2-(4-chlorobenzylidenehydrazinocarbonyl)]phenyl]-5-oxopyrrolidine-
3-carbohydrazide (11b = Z/E 2.73–2.97 (m, 2H, COCH2),
1H-NMR (400 MHz, DMSO-d6):
)
:
δ
3.36–3.44 (m, 0.35H, CH), 3.98–4.26 (m, 0.65H, CH + 2H, NCH2), 7.38–7.62 (m, 4H, Har),
7.63–8.20 (m, 8H, Har + 0.65H, CH=N), 8.23 (s, 0.35H, CH=N), 8.45 (s, 1H, CH=N), 11.66,
11.74 (2s, 1H, NH), 11.88 (s, 1H, NH) ppm.
IR (KBr): ν
= 3431, 3235 (2NH), 1679, 1655 (3C=O) cm−1
.
max
MS (APCI+, 25 V) m/z, %: 523 (100) [M + H]+.
Calcd. for C26H21Cl2N5O3, %: C 59.78; H 4.05; N 13.41. Found, %: C 59.68; H 4.04;
N 13.48.
1-[[5-Oxo-1-(4-[(phenylcarbamoylthioamino)carbamoyl]pyrrolidin-3-carbonyl]-3- phenylthio-
carbamide (12 To a solution of dihydrazide 10 (5 g, 18 mmol) in methanol (50 mL), phenyl
):
isothiocyanate (9.73 g, 72 mmol) was added, and the mixture was heated at reflux for 5 h.
After completion of the reaction, the mixture was cooled down, and the obtained crystalline
solid was filtered off, washed with methanol and boiling water, and recrystallized from
methanol to give the title compound 12 (white solid, yield 8.97 g, 97% m. p. 235–236 ◦C).
1H-NMR (400 MHz, DMSO-d6):
δ = 2.56–2.76 (m, 2H, COCH2), 2.89–3.41 (m, 1H, CH
+ NCH2), 6.28–6.74 (m, 4H, Har), 7.09–7.77 (m, 10H, Har), 9.39–9.75 (m, 4H, 4NH), 10.12,
10.40 (2s, 2H, 2NH) ppm.
13C-NMR (101 MHz, DMSO-d6):
δ = 33.39 (CH, COCH2), 44.37 (NCH2), 111.02, 113.98,
115.79, 116.81, 119.46, 123.68, 124.50, 124.84, 124.88, 124.92, 125.13, 125.23, 125.87, 125. 91,
128.10, 128.84, 129.58, 139.01, 139.34, 151.41, 166.04, 171.80, 173.00, 180.06, 180.98 (Car, 3C=O,
2C=S) ppm.
IR (KBr): ν
= 3312 (NH), 1611 (3C=O), 1332 (C=S) cm−1
.
max
MS (APCI+, 25 V) m/z, %: 548 (100) [M + H]+.
Calcd. for C26H25N7O3S2, %: C 57.02; H 4.60; N 17.90. Found, %: C 57.10; H 4.56;
N 17.93.
4-(4-Phenyl-5-thioxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)-1-(4-(4-phenyl-5-thioxo-4,5-dihydro-
1H-1,2,4-triazol-3-yl)phenyl)pyrrolidin-2-one (13 A mixture of thiosemicarbazide 12 (1.03 g,
):
2 mmol) and 4% sodium hydroxide solution (20 mL) was refluxed for 6 h. The reaction
mixture was then cooled down, acidified, with diluted hydrochloric acid (1:1) to pH 2. The
formed solid was filtered off, washed with water, and recrystallized from methanol to give
the title compound 13 (white solid, yield 0.61 g, 60%, m. p. 320–322 ◦C).

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1H-NMR (400 MHz, DMSO-d6):
δ
= 2.67–2.88 (m, 2H, COCH2), 2.89–3.25 (m, 3H,
NCH2 + CH), 6.81 (d, J = 8.4 Hz, 2H, Har), 7.11–7.22 (m, 4H, Har), 7.31–7.50 (m, 6H, Har),
7.58–7.62 (m, 2H, Har), 13.87 (s, 2H, 2NH) ppm.
13C-NMR (101 MHz, DMSO-d6):
δ = 26.83 (CH), 32.48 (COCH2), 45.39 (NCH2), 110.84,
112.53, 127.94, 128.90, 129.33, 129.39, 129.44, 133.10, 122.14, 135.16, 148.80, 150.05, 150.05,
150.93, 152.46, 167.59, 167.73, 168.16 (Car, C=O, 2C=S) ppm.
Calcd. for C26H21N7OS2, %: C 61.04; H 4.14; N 19.16. Found, %: C 61.13; H 4.10;
N 19.12.
General procedure for the preparation of benzimidazoles 14 and 15.
To a mixture of dicarboxylic acid
2 (5 g, 20 mmol) and benzene-1,2-diamine (14)
or 4-methylbenzene-1,2-diamine (15) (52 mmo◦l), polyphosphoric acid (15 g) was added
dropwise, and the mixture was heated at 120 C for 6 h. It was then cooled down and
neutralized with 7% Na2CO3 to pH 9. The formed precipitate was filtered off, washed with
plenty of water, and recrystallized from methanol to give the title compound 14 (white
solid, yield 7.6 g, 97%, m. p. 215–216 ◦C) or compound 15 (light yellow solid, yield 7.67 g,
91%, m. p. 257–258 ◦C).
3-(1H-benzimidazol-2-yl)-1-[4-(1H-benzimidazol-2-yl)phenyl]pyrrolidin-5-one (14
(400 MHz, DMSO-d6): = 3.01–3.20 (m, 2H, COCH2), 4.01–4.11 (m, 1H, CH), 4.24–4.42 (m,
):
1H-NMR
δ
2H, NCH2), 7.10–7.24 (m, 4H, Har), 7.48–7.69 (m, 4H, Har), 7.91 (d, J = 8.9 Hz, 2H, Har), 8.20
(d, J = 8.8 Hz, 2H, Har), 12.50, 12.87 (2s, 2H, 2NH) ppm.
13C-NMR (101 MHz, DMSO-d6):
δ = 30.61 (COCH2), 37.70 (CH), 52.08 (NCH2), 111.08,
111.19, 118.52, 118.70, 119.29, 119.46, 121.19, 121.61, 122.02, 122.37, 125.54, 126.93, 128.70,
134.57, 135.00, 140.52, 142.82, 143.87 (Car), 150.93, 154.95 (N=C), 172.42 (C=O) ppm.
IR (KBr): ν
= 3197 (2NH), 1680 (C=O) cm−1
.
max
MS (APCI+, 25 V) m/z, %: 394 (100) [M + H]+.
Calcd. for C24H19N5O, %: C 73.27; H 4.87; N 17.80. Found, %: C 73.20; H 4.82; N 17.76.
3-(6-Methyl-1H-benzimidazol-2-yl)-1-[4-(6-methyl-1H-benzimidazol-2-yl)phenyl]pyrrolidin-
5-one (15): δ = 2.39 (s, 3H, CH3), 2.42 (s, 3H, CH3), 2.99–3.12
1H-NMR (400 MHz, DMSO-d6):
(m, 2H, COCH2), 3.98–4.05 (m, 1H, CH), 4.24–4.37 (m, 2H, NCH2), 6.97–7.05 (m, 2H, Har),
7.29–7.48 (m, 4H, Har), 7.88 (d, J = 8.9 Hz, 2H, Har), 8.17 (d, J = 8.8 Hz, 2H, Har), 12.51 (br. s,
2H, 2NH) ppm.
13C-NMR (101 MHz, DMSO-d6):
δ = 21.28 (CH3), 21.36 (CH3), 30.63 (COCH2), 37.72
(CH), 52.12 (NCH2), 113.54, 117.43, 117.54, 119.26, 119.47, 119.92, 122.58, 122.97, 123.45,
125.73, 126.79, 127.62, 128.70, 130.70, 131.16, 140.37 (Car), 150.41, 150.64, 154.56 (N=C),
172.42 (C=O) ppm.
IR (KBr): ν
= 3411, 3243 (2NH), 1684 (C=O) cm−1
.
max
MS (APCI+, 25 V) m/z, %: 422 (100) [M + H]+.
Calcd. for C26H23N5O, %: C 74.09; H 5.50; N 16.62. Found, %: C 74.17; H 5.44; N 16.54.
3-(1H-benzimidazol-2-yl)-4-[4-(1H-benzimidazol-2-yl)anilino]butanoic acid (16 A mixture
):
of benzimidazole 14 (1.97 g, 5 mmol) and aqueous 20% sodium hydroxide (30 mL) solution
was heated at reflux for 2 h, and then was cooled down, diluted with water (50 mL) and
filtered off. The filtrate was acidified with 10% acetic acid to pH 6. The formed solid was
filtered off, washed with water, and purified by dissolving it in 5% sodium hydroxide
solution, filtering and acidifying the filtrate with 10% acetic acid to pH 6 (procedure was
performed twice) to give the title compound 16 (light brown solid, yield 1.03 g, 50%, m. p.
204–205 ◦C).
1H-NMR (400 MHz, DMSO-d6):
δ = 2.76–2.96 (m, 2H, COCH2), 3.29–3.75 (m, 3H,
NHCH2+CH), 6.40 (s, 1H, NHCH2), 6.78 (d, J = 8.5 Hz, 2H, Har), 7.02–7.24 (m, 4H, Har),
7.38–7.62 (m, 4H, Har), 7.92 (d, J = 8.6 Hz, 2H, Har), 12.51 (br. s, 3H, OH + 2NH) ppm.
13C-NMR (101 MHz, DMSO-d6):
δ = 35.64 (COCH2), 36.18 (CH), 46.49 (NHCH2),
111.90, 117.52, 121.24, 121.29, 127.76, 149.92 (Car), 152.47, 155.98 (2N=C), 173.44 (C=O) ppm.
MS (APCI+, 25 V) m/z, %: 412 (100) [M + H]+.
Calcd. for C24H21N5O2, %: C 70.06; H 5.14; N 17.02. Found, %: C 70.17; H 5.07;
N 17.08.

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4-((4-(1H-benzimidazol-2-yl)phenyl)amino)-3-(1H-benzimidazol-2-yl)butanehydrazide (17).
Method A: A mixture of butanoic acid 16 (2.06 g, 5 mmol), hydrazine monohydrate (2.10 g,
42 mmol), and 2-propanol (20 mL) was heated at reflux for 20 h and then cooled to room
temperature. The obtained solid was filtered off, washed with water, and recrystallized
from water to give the title compound 17 (light brown solid, yield 1.08 g, 51%, m. p.
166–167 ◦C).
Method B: A mixture of pyrrolidinone 14 (1.97 g, 5 mmol) and hydrazine monohydrate
(20 g, 400 mmol) was heated at reflux for 6 h and then was cooled to room temperature,
diluted with 2-propanol (30 mL), and filtered off. The filtrate was evaporated under
reduced pressure; the residue was poured with water and stirred for 10 min. The obtained
solid was filtered off, washed with plenty of water, and recrystallized from water to give
the title compound 14 (light brown solid, yield 1.34 g, 63%, m. p. 166–167 ◦C).
1H-NMR (400 MHz, DMSO-d6):
δ = 2.56–2.75 (m, 2H, COCH2), 3.40–3.59 (m, 2H,
NHCH2), 3.72–3.78 (m, 1H, CH), 4.43 (s, 2H, NH2), 6.34 (t, J = 5.9 Hz, 1H, NHCH2), 6.76 (d,
J = 8.6 Hz, 2H, Har), 7.09–7.15 (m, 4H, Har), 7.42–7.58 (m, 4H, Har), 7.91 (d, J = 8.4 Hz, 2H,
Har), 9.12 (s, 1H, CONH), 12.31 (s, 1H, NH), 12.47 (s, 1H, NH) ppm.
13C-NMR (101 MHz, DMSO-d6):
δ = 35.54, (CH), 35.72 (COCH2), 46.46 (NHCH2),
110.88, 111.87, 117.46, 118.18, 121.26, 127.71, 134.89, 143.21, 149.95 (Car), 152.47, 156.12
(N=C), 169.90 (C=O) ppm.
IR (KBr): ν
= 3410 (NHNH ), 1611 (C=O) cm−1
.
2
max
MS (APCI+, 25 V) m/z, %: 426 (100) [M + H]+.
Calcd. for C24H23N7O, %: C 67.75; H 5.45; N 23.04. Found, %: C 67.84; H 5.36; N 23.12.
4-((4-(1H-benzimidazol-2-yl)phenyl)amino)-3-(1H-benzimidazol-2-yl)-N-(2,5-dimethyl-1H-
pyrrol-2-yl)butanamide (18): To a mixture of hydrazide 17 (2.13 g, 5 mmol) and hexane-
2,5-dione (3.42 g, 30 mmol) in 2-propanol (50 mL), conc. hydrochloric acid (2.5 mL) was
added dropwise; the mixture was heated at reflux for 4 h, then cooled to room temperature,
and the solvent was evaporated under reduced pressure. The residue was poured with
water (30 mL) and stirred for 10 min. The obtained solid was filtered off, washed with
water, and recrystallized from methanol to give the title compound 18 (white solid, yield
1.79 g, 71%, m. p. 227–228 ◦C).
1H-NMR (400 MHz, DMSO-d6):
δ = 1.62 (s, 3H, CH3), 1.95 (s, 3H, CH3), 2.93–3.09
(m, 2H, COCH2), 3.54–3.84 (m, 3H, CH + NHCH2), 5.52 (d, J = 3.1 Hz, 1H, C=CH), 5.57
(d, J = 3.1 Hz, 1H, C=CH), 6.90 (d, J = 8.7 Hz, 2H, Har), 7.02 (s, 1H, NHCH2), 7.19–7.24 (m,
2H, Har), 7.34–7.39 (m, 2H, Har), 7.53–7.59 (m, 2H, Har), 7.64–7.68 (m, 2H, Har), 8.06 (d,
J = 8.7 Hz, 2H, Har), 10.86 (s, 1H, CONH), 13.68 (br. s, 2H, 2NH) ppm.
13C-NMR (101 MHz, DMSO-d6):
δ = 10.50 (CH3), 11.00 (CH3), 34.97 (COCH2), 35.55
(CH), 46.03 (NHCH2), 102.76, 102.81 (2C=CH), 112.12, 112.13, 113.52, 113.54, 122.08, 122.12,
123.87, 126.56, 126.79, 129.08, 134.17, 134.22, 139.09, 150.54 (Car), 151.79, 154.99 (2N=C),
169.97 (C=O) ppm.
IR (KBr): ν
= 3050 (NH), 1608 (C=O) cm−1
.
max
Calcd. for C30H29N7O, %: C 71.55; H 5.80; N 19.47. Found, %: C 71.78; H 5.72; N 19.33.
Ethyl 2-(2-(4-(4-(1-(2-ethoxy-2-oxoethyl)-1H-benzimidazol-2-yl)-2-oxopyrrolidin-1-yl)-phenyl)-
1H-benzimidazol-1-yl)acetate (19): A mixture of benzimidazole 14 (3 g, 7.6 mmol), potassium
carbonate (2.1 g, 15.2 mmol), TBAI (0.02 g), and acetone (50 mL) was boiled, and ethyl
chloroacetate (9.9 mL, 91.4 mmol) was slowly added dropwise. The mixture was refluxed
for 20 h and filtered while hot; the filtrate was cooled to room temperature and diluted
with water (100 mL). The formed solid was filtered off, washed with plenty of water, and
recrystallized from methanol to give the title compound 19 (light brown solid, yield 3.78 g,
88%, m. p. 106–107 ◦C).
1H-NMR (400 MHz, DMSO-d6):
δ = 1.15 (t, J = 7.1 Hz, 3H, CH2CH3), 1.25 (t, J = 7.1 Hz,
3H, CH2CH3), 2.94–3.11 (m, 2H, COCH2), 4.10–4.38 (m, 7H, 2CH2CH3 + CH + NCH2), 5.23
(s, 2H, NCH2CO), 5.32 (s, 2H, NCH2CO), 7.20–7.30 (m, 4H, Har), 7.51–7.72 (m, 4H, Har),
7.75 (d, J = 8.7 Hz, 2H, Har), 7.90 (d, J = 8.8 Hz, 2H, Har).

Molecules 2021, 26, 2597
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13C-NMR (101 MHz, DMSO-d6):
δ
= 13.94 (CH3), 14.04 (CH3), 28.41 (CH), 37.76 (COCH2),
44.41 (NCH2CO), 46.06 (NCH2CO), 51.90 (NCH2), 61.40 (CH2CH3), 61.49 (CH2CH3), 110.12,
110.59, 118.85, 119.06, 119.15, 121.92, 122.37, 125.11, 129.42, 135.76, 136.33, 140.46, 141.75,
142.40 (Car), 152.87, 155.42 (2N=C), 168.24, 168.39 (2C=O), 172.33 (NC=O) ppm.
IR (KBr): ν
= 1732, 1708, 1611 (3C=O) cm−1
.
max
MS (APCI+, 25 V) m/z, %: [M + H]+ = 566 (100).
Calcd. for C32H31N5O5, %: C 67.95; H 5.52; N 12.38. Found, %: C 67.86; H 5.54;
N 12.28.
2-(2-(4-(4-(1-(2-Hydrazinyl-2-oxoethyl)-1H-benzimidazol-2-yl)-2-oxopyrrolidin-1-yl)phenyl)-
1H-benzimidazol-1-yl)acetohydrazide (20 A mixture of diester 19 (1.98 g, 3.5 mmol), hy-
):
drazine monohydrate (1.42 g, 28.3 mmol) and 1,4-dioxane (20 mL) was refluxed for 18 h
and cooled to room temperature; the formed solid was filtered off, washed with water and
2-propanol, and recrystallized from methanol to give the title compound 20 (yellowish
solid, yield 1.60 g, 85%, m. p. 206–207 ◦C).
1H-NMR (400 MHz, DMSO-d6):
δ = 2.99–3.15 (m, 2H, COCH2), 4.00–4.74 (m, 7H, CH
+ NCH2 + 2NH2), 4.87, 4.95, 5.24, 5.32 (4s, 2H, NCH2CO), 7.00–7.57 (m, 6H, Har), 7.58–8.00
(m, 6H, Har), 8.83, 8.88, 9.60, 9.65 (4s, 2H, 2NH).
13C-NMR (101 MHz, DMSO-d6):
δ = 28.55 (CH), 37.81 (COCH2), 44.47 (NCH2CO),
45.78 (NCH2CO), 52.09 (NCH2), 110.02, 110.54, 118.82, 118.99, 119.03, 121.75, 122.17, 125.28,
129.78, 135.79, 136.38, 140.46, 141.79, 142.49 (Car), 153.20, 155.77 (2N=C), 166.01, 166.34
(2C=O), 172.54 (NC=O) ppm.
IR (KBr): ν
= 1729, 1674 (3C=O) cm−1
.
max
MS (APCI+, 25 V) m/z, %: [M + H]+ = 566 (100).
Calcd. for C38H30N9O3, %: C 68.56; H 5.30; N 18.94. Found, %: C 68.62; H 5.25;
N 18.87.
4-(1-(2-(3,5-Dimethyl-1H-pyrazol-1-yl)-2-oxoethyl)-1H-benzimidazol-2-yl)-1-(4-(1-(3,5-dimethyl-
1H-pyrazol-1-yl)-2-oxoethyl)-1H-benzimidazol-2-yl)phenyl)pyrrolidin-2-one (21 A mixture of
):
hydrazide 20 (2.15 g, 4 mmol), 2,4-pentanedione (4.4 g, 44 mmol), 2-propanol and conc.
hydrochloric acid (0.25 mL) was refluxed for 5 h and then cooled down and diluted with
water. The formed precipitate was filtered off, washed with water, and recrystallized
from methanol to give the title compound 21 (light yellow solid, yield 2.17 g, 82%, m. p.
178–179 ◦C).
1H-NMR (400 MHz, DMSO-d6):
δ = 1.14 (s, 3H, CH3), 1.16 (s, 3H, CH3), 1.24 (s, 3H,
CH3), 1.25 (s, 3H, CH3), 2.91–3.11 (m, 2H, COCH2), 4.11–4.19 (m, 1H, CH), 4.25–4.37 (m, 2H,
NCH2), 4.89–4.96 (m, 1H, C=CH), 4.97–5.06 (m, 1H, C=CH), 5.20, 5.30 (4s, 4H, 2NCH2CO),
7.20–7.30 (m, 4H, Har), 7.47–7.72 (m, 4H, Har), 7.76 (d, J = 8.6 Hz, 2H, Har), 7.90 (d, J = 8.8 Hz
,
2H, Har).
13C-NMR (101 MHz, DMSO-d6):
δ = 21.37 (CH3), 21.53 (CH3), 28.43, 37.72, 44.60, 46.25,
51.90 (CH, COCH2, NCH2CO, NCH2), 69.19, 69.32 (C-CH-C), 110.04, 110.51, 118.86, 119.06,
119.14, 121.90, 122.25, 122.37, 125.19, 129.37, 135.79, 136.36, 140.45, 141.74, 142.39 (Car),
152.85, 155.37 (2N=C), 167.75, 167.89 (2C=O), 172.31 (NC=O) ppm.
IR (KBr): ν
= 3279, 3203 (2NHNH ), 1689, 1610 (3C=O) cm−1
.
max
2
Calcd. for C28H27N9O35, %: C 62.56; H 5.06; N 23.45. Found, %: C 62.31; H 4.99;
N 23.27.
4-(1-((5-Thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)methyl)-1H-benzo[d]imidazol-2-yl)-1-(4-(1-
((5-thioxo-4,5-dihydro-1,3,4-oxadiazol-2-yl)methyl)-1H-benzo[d]imidazol-2-yl)phenyl)pyrrolidin-2-
one (22): To a cooled solution of potassium hydroxide (1 g, 18 mmol) in methanol (80 mL),
carbon disulfide (0.7 mL, 11 mmol) was added dropwise and the obtained mixture was
stirred at room temperature for 15 min. Then hydrazide 20 (2.15 g, 4 mmol) was added
slowly, and the mixture was heated at reflux for 12 h. After completion of the reaction
(TLC), the volatile fraction was evaporated under reduced pressure; the residue was dis-
solved in water (100 mL) and boiled for 3 min with activated carbon. The mixture was
filtered off, and the filtrate was acidified with diluted hydrochloric acid to pH 1. The

Molecules 2021, 26, 2597
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formed precipitate was filtered off, washed with water, and recrystallized from methanol
to give the title compound 22 (white/yellow solid, yield 2.02 g, 81%, m. p. 262–263 ◦C).
1H-NMR (400 MHz, DMSO-d6):
δ = 2.90–3.17 (m, 2H, COCH2), 4.06–4.53 (m, 2H,
NCH2 + CH), 5.72, 5.81, 5.90, 6.01 (4s, 4H, 2NCH2CO), 7.19–8.04 (m, 12H, Har), 12.41 (br. s,
2H, 2NH) ppm.
13C-NMR (101 MHz, DMSO-d6):
δ = 28.45, 37.82, 38.08, 44.35, 51.94 (CH, COCH2,
NCH2CO, NCH2), 110.40, 110.87, 119.03, 119.13, 119.24, 122.29, 122.73, 124.48, 129.78,
135.35, 135.76, 140.67, 141.76, 142.42, 155.20, 164.35, 165.63, 165.93 (Car), 172.36, 172.42 (C=O,
2C=S) ppm.
IR (KBr): ν
= 3425 (2NH), 1609 (C=O), 1327 (C=S) cm−1
.
max
Calcd. for C30H26N9O3S2, %: C 57.96; H 3.73; N 20.28. Found, %: C 57.87; H 3.80;
N 20.21.
2-(2-(-2-(5-Oxo-1-(4-(1-(2-oxo-2-(2-(phenylcarbamothioyl)hydrazinyl)ethyl)-1H-benzimidazol-
2-yl)phenyl)pyrrolidin-3-yl)-1H-benzimidazol-1-yl)acetyl)-N-phenylhydrazin-1-carbothioamide (23
):
To a boiled mixture hydrazide 20 (2.15 g, 4 mmol) and methanol (100 mL), phenyl isothio-
cyanate (2.03 g, 15 mmol) was added dropwise, and the obtained mixture was heated at
reflux for 6 h and then cooled down; the obtained solid was filtered off, washed with water
and cold methanol, and recrystallized from methanol to give the title compound 23 (light
◦
yellow solid, yield 2.91 g, 90%, m. p. 240 (decomp.) C).
1H-NMR (400 MHz, DMSO-d6):
δ = 3.01–3.17 (m, 2H, COCH2), 4.11–4.37 (m, 3H, CH
+ NCH2), 4.89, 4.97, 5.06, 5.15 (4s, 4H, 2NCH2CO), 7.08–7.95 (m, 22H, Har), 9.48–10.04 (m,
4H, 4NH), 10.51, 10.58 (2s, 2H, 2NH) ppm.
IR (KBr): ν
= 3206 (NH), 1694, 1608 (3C=O) cm−1
.
max
Calcd. for C42H37N11O3S2, %: C 62.44; H 4.62; N 19.07. Found, %: C 62.57; H 4.69;
N.1916
General procedure for the preparation of hydrazones 24a
20 (2.15 g, 4 mmol) in DMF (100 mL), the corresponding aromatic aldehyde (30 mmol) was
added, and the mixture was heated at reflux for 5 ( ) or 8 ( ) h. After completion of
the reaction, the mixture was cooled to room temperature and diluted with water ( ).
Upon cooling the reaction mixture , a solid formed in the DMF. The obtained crystalline
–e: To a solution of hydrazide
a,c
–
e
b
a,c–e
b
substance was filtered off, washed with water, 2-propanol, and diethyl ether, and recrystal-
lized from the mixture of 1,4-dioxane and 2-propanol (1:2) to give the corresponding title
compound 24.
N’-benzylidene-2-(2-(4-(4-(1-(2-(2-(benzylidene)hydrazinyl)-2-oxoethyl)-1H-benzo[d]imidazol-
2-yl)-2-oxopyrrolidin-1-yl)phenyl)-1H-benzo[d]imidazol-1-yl)acetohydrazide (24a
)
:
Light brown
◦
solid, yield 2.34 g, 82%, m. p. 254–255 C. 1H-NMR (400 MHz, DMSO-d6):
δ = Z/E 2.95–3.13
(m, 2H, COCH2), 4.09–4.41 (m, 3H, CH + NCH2), 5.08, 5.14, 5.54, 5.61 (4s, 4H, 2NCH2CO),
7.20–8.00 (m, 22H, Har), 8.06, 8.11 (2s, 0.75(2H), 2N=CH), 8.26, 8.30 (2s, 0.25(2H), 2N=CH),
11.82, 11.89, 12.00, 12.04 (4s, 2H, 2NH) ppm.
IR (KBr): ν
= 3200 (2NH), 1693, 1610 (3C=O) cm−1
.
max
Calcd. for C42H35N9O3, %: C 70.67; H 4.94; N 17.66. Found, %: C 70.62; H 4.98; N.1758
N’-4-nitrobenzylidene-2-(2-(4-(4-(1-(2-(2-(4-nitrobenzylidene)hydrazinyl)-2-oxoethyl)-1H-
benzo[d]imidazol-2-yl)-2-oxopyrrolidin-1-yl)phenyl)-1H-benzo[d]imidazol-1-yl)acetohydrazide (24b):
◦
Light yellow solid, yield 2.48 g, 77%, m. p. 213–214 C. 1H-NMR (400 MHz, DMSO-d6):
δ =
Z/E 2.90–3.11 (m, 2H, COCH2), 4.02–4.43 (m, 3H, CH + NCH2), 5.12, 5.18, 5.60, 5.66 (4s, 4H,
2NCH2CO), 7.09–7.43 (m, 4H, Har), 7.46–8.46 (m, 18H, Har + 2N=CH), 12.08, 12.14,12.24,
12.26 (4s, 2H, 2NH) ppm.
IR (KBr): ν
= 3438 (2NH), 1694, 1610 (3C=O) cm−1
.
max
Calcd. for C42H33N11O5, %: C 62.76; H 4.14; N 19.17. Found, %: C 62.81; H 4.20;
N 19.14.
N’-(4-fluorobenzylidene)-2-(2-(4-(4-(1-(2-(2-(4-fluorobenzylidene)hydrazinyl)-2-oxoethyl)-1H-
benzo[d]imidazol-2-yl)-2-oxopyrrolidin-1-yl)phenyl)-1H-benzo[d]imidazol-1-yl)acetohydrazide (24c
)
:
◦
1
White solid, yield 2.40 g, 80%, m. p. 268–269 C. H-NMR (400 MHz, DMSO-d6):
δ =
Z/E 2.93–3.13 (m, 2H, COCH2), 4.08–4.20 (m, 1H, CH), 4.21–4.39 (m, 2H, NCH2), 5.08,

Molecules 2021, 26, 2597
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5.14,5.54, 5.61 (4s, 4H, 2NCH2CO), 7.05–7.46 (m, 8H, Har), 7.47–7.96 (m, 12H, Har), 8.05,
8.10 (2s, 0.75(2H), 2N=CH), 8.26, 8.30 (2s, 0.25(2H), 2N=CH), 11.82, 11.88, 12.00, 12.03 (4s,
2H, 2NH) ppm.
IR (KBr): ν
= 3206 (2NH), 1694, 1608 (3C=O) cm−1
.
max
Calcd. for C42H33F2N9O3, %: C 62.44; H 4.62; N 19.07. Found, %: C 62.47; H 4.69;
N 19.16.
N’-3-chlorobenzylidene)-2-(2-(4-(4-(1-(2-(2-(3-chlorobenzylidene)hydrazinyl)-2-oxoethyl)-1H-
benzo[d]imidazol-2-yl)-2-oxopyrrolidin-1-yl)phenyl)-1H-benzo[d]imidazol-1-yl)acetohydrazide (24d
Light brown solid, yield 2.47 g, 79%, m. p. 256–257 ◦C. 1H-NMR (400 MHz, DMSO-d6):
= Z/E 2.95–3.09 (m, 2H, COCH2), 3.99–4.52 (m, 3H, CH + NCH2), 5.09, 5.15, 5.56, 5.63
)
:
δ
(4s, 4H, 2NCH2CO), 7.08–7.96 (m, 20H, Har), 8.03, 8.08 (2s, 0.8(2H), 2N=CH), 8.23, 8.27 (2s,
0.2(2H), 2N=CH), 11.89, 11.95, 12.11, 12.14 (4s, 2H, 2NH) ppm.
IR (KBr): ν
= 3393, 3207 (2NH), 1685, 1611 (3C=O) cm−1
.
max
Calcd. for C42H33Cl2N9O3, %C 64.45; H 4.25; N 16.11. Found, %: C 64.36; H 4.20;
N 16.20.
N’-(2,3-dimethoxybenzylidene)-2-(2-(4-(4-(1-(2-(2-(2,3-dimethoxybenzylidene)hydrazinyl)-2-
oxoethyl)-1H-benzo[d]imidazol-2-yl)-2-oxopyrrolidin-1-yl)phenyl)-1H-benzo[d]imidazol-1-yl) ace-
◦
1
tohydrazide (24e
)
:
Light yellow solid, yield 3.14 g, 94%, m. p. 194–195 C. H-NMR
(400 MHz, DMSO-d6):
δ
= Z/E 2.94–3.11 (m, 2H, COCH2), 3.70, 3.76, 3.79, 3.81 (4s, 12H,
4CH3O), 4.10–4.44 (m, 1H, CH+NCH2), 5.05, 5.11, 5.54, 5.61 (4s, 4H, 2NCH2CO), 6.96–7.91
(m, 18H, Har), 8.36, 8.40 (2s, 0.75(2H), 2N=CH), 8.57, 8.60 (2s, 0.25(2H), 2N=CH), 11.77,
11.83, 12.00, 12.01 (4s, 2H, 2NH) ppm. IR (KBr): νmax = 3200, 3054 (2NH), 1682, 1610 (3C=O)
cm−1. Calcd. for C46H43N9O7, %: C, 66.26; H, 5.20; N, 15.12. Found, %: C 66.19; H 5.26;
N 15.21.
3.2. Determination of Antimicrobial Activity
3.2.1. Preparation of Bacterial Inoculum
A panel of clinically important reference bacterial pathogens were obtained from
the American Type Culture Collection (ATCC) and the National Type Culture Collection
(NTCT) (Supplementary Table S1). Each test organism was subcultured on Tryptic Soy Agar
(TSA) at 37 ◦C, for 24 h. After incubation, the representative colonies were suspended in
◦
5 mL of Tryptic Soy Broth (TBS) and further incubated at 37 C for 24 h to initiate the liquid
culture. The bacterial cultures were normalized using a spectrophotometer (OD600 nm), and
the final inoculum (1
×
107 CFU/mL) was achieved by diluting the culture with fresh TSB.
3.2.2. Preparation of the Test Compounds
The test compounds 24 were dissolved in hybridoma grade DMSO to achieve a
3
–
50 mg/mL stock solution. The stock solution was further diluted in TSB supplemented
with 1% of DMSO to produce the series of dilutions (0, 15.62, 31.25, 62.5, 125, 250, 500, and
1000 µg/mL). Ampicillin was dissolved in sterile deionized water and the series of dilutions
(0, 15.62, 31.25, 62.5, 125, 250, 500, and 1000 µg/mL) were prepared as described above.
3.2.3. Evaluation of Minimal Inhibitory Concentration
The minimal inhibitory concentration (MIC) of the compounds 3–24 and ampicillin
were determined by the broth dilution method as described by Balouiri et al. [75] with
brief modifications. The tubes containing diluted compounds in TSB were inoculated
with normalized bacterial inoculum (100
µ
L) to achieve a final bacterial concentration of
1 × 106 CFU/mL. The inoculated tubes were incubated at 37 C for 24 h. After incubation,
the turbidity was evaluated visually and MIC was estimated. The MIC was defined
as the lowest concentration of the test compound that inhibits the visual growth of the
test organism.
◦

Molecules 2021, 26, 2597
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3.2.4. Determination of Minimal Bactericidal Concentration
The minimal bactericidal concentration (MBC) was determined as described by
Parvekar et al. [76]. After a MIC evaluation of the novel compounds and ampicillin,
aliquots of 100
µ
L were taken from tubes without growth and plated on TSA. The plates
◦
were incubated at 37 C for 48 h. After incubation, the plates were evaluated, and the mini-
mal bactericidal concentration (MBC) was estimated. The MBC was defined as the lowest
concentration of the test compound that fully suppresses the growth of the test organism.
4. Conclusions
In this study, the chemical transformations of p-aminobenzoic acid were carried
out, and a series of 1-phenyl-5-oxopyrrolidine derivatives with hydrazone, pyrazole,
thiosemicarbazide, triazole, oxadiazole fragments were obtained. A convenient and effi-
cient method for the synthesis of benzimidazoles by heating reagents in polyphosphoric
acid was proposed.
The synthesized compounds were evaluated for their antibacterial activity against a
panel of clinically relevant Gram-positive and Gram-negative pathogens. The antimicrobial
activity evaluation revealed that the γ-amino acid derivative 16 bearing two benzimidazole
fragments demonstrated the strongest broad-spectrum bactericidal activity on both Gram-
positive and Gram-negative organisms. The antimicrobial activity of compound 16 was
notably greater than that of ampicillin. Furthermore, benzimidazoles 14 and 15 showed
promising, broad-spectrum antibacterial activity against tested pathogens, with notably
good bactericidal activity against L. monocytogenes. Collectively, these results demonstrated
that the 5-oxopyrrolidine 14 could be further explored as a potential pharmacophore in the
development of novel antimicrobials targeting clinically significant bacterial pathogens.
Further studies are needed to better understand the safety, tolerability, and in vivo activity
of the most promising compounds.
1
Supplementary Materials: The following are available online, Figure S1: H-NMR of compound
2
,
1
Figure S2: 13C-NMR of compound
2
, Figure S3: H-NMR of compound
3
, Figure S4: 13C-NMR of
1
compound
3
, Figure S5: H-NMR of compound
4
, Figure S6: 13C-NMR of compound
4, Figure S7:
1
1H-NMR of compound
5
, Figure S8: 13C-NMR of compound
5
, Figure S9: H-NMR of compound
6
,
Figure S10: 13C-NMR of compound
6
, Figure S11: H-NMR of compound 7a, Figure S12: H-NMR
1
1
of compound 7b, Figure S13: 1H-NMR of compound 7c, Figure S14: 1H-NMR of compound
8,
1
Figure S15: 13C-NMR of compound
8
, Figure S16: H-NMR of compound
9
, Figure S17: 13C-NMR
of compound 9, Figure S18: 1H-NMR of compound 10, Figure S19: 13C-NMR of compound 10
,
Figure S20: 1H-NMR of compound 11a, Figure S21: 1H-NMR of compound 11b, Figure S22: 1H
1
NMR of compound 12, Figure S23: 13C-NMR of compound 12, Figure S24: H-NMR of compound 13
,
Figure S25: 13C-NMR of compound 13, Figure S26: H-NMR of compound 14, Figure S27: 13C-NMR
1
of compound 14, Figure S28: 1H-NMR of compound 15, Figure S29: 13C-NMR of compound 15
,
Figure S30: H-NMR of compound 16, Figure S31: 13C-NMR of compound 16, Figure S32: H-NMR
1
1
of compound 17, Figure S33: 13C-NMR of compound 17, Figure S34: 1H-NMR of compound 18
,
Figure S35: 13C-NMR of compound 18, Figure S36: H-NMR of compound 19, Figure S37: 13C-NMR
1
of compound 19, Figure S38: 1H-NMR of compound 20, Figure S39: 13C-NMR of compound 20
,
Figure S40: H-NMR of compound 21, Figure S41: 13C-NMR of compound 21, Figure S42: H-NMR
1
1
of compound 22, Figure S43: 13C-NMR of compound 22, Figure S44: 1H-NMR of compound 23
,
1
1
1
Figure S45: H-NMR of compound 24a, Figure S46: H-NMR of compound 24b, Figure S47: H-NMR
of compound 24c, Figure S48: H-NMR of compound 24d, Figure S49: H-NMR of compound 24e
1
1
.
Table S1: Bacteria strains used in the biological evaluation.
Author Contributions: Conceptualization, B.S.-B. and V.M.; methodology, B.S.-B. and R.V.; software,
V.P.; validation, B.S.-B., V.P., R.V., B.G., P.K. and J.Š.; formal analysis, B.S.-B. and V.M.; investigation,
B.S.-B., V.P., R.V., B.G., P.K. and J.Š.; resources, V.P.; data curation, B.S.-B.; writing—original draft
preparation, R.V., P.K. and J.Š.; writing—review and editing, B.S.-B.; visualization, V.P. and P.K.;
supervision and project administration, B.S.-B. and V.M. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.

Molecules 2021, 26, 2597
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Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from the authors.
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