Synthesis and evaluation of 2-(4-[4-acetylpiperazine-1-carbonyl] phenyl)-1H-benzo[d]imidazole-4-carboxamide derivatives as potential PARP-1 inhibitors and preliminary study on structure-activity relationship

Article abstract of DOI:10.1002/ddr.21843

Although 1H-benzo[d]imidazole-4-carboxamide derivatives have been explored for a long time, the structure–activity relationship of the substituents in the hydrophobic pocket (AD binding sites) has not thoroughly discovered. Here in, a series of 2-(4-[4-acetylpiperazine-1-carbonyl]phenyl)-1H-benzo[d]imidazole-4-carboxamide derivatives have been designed, synthesized, and successful characterization as novel and effective poly ADP-ribose polymerases (PARP)-1 inhibitors to improve the structure–activity relationships about the substituents in the hydrophobic pocket. These derivatives were evaluated for their PARP-1 inhibitory activity and cellular inhibitory against BRCA-1 deficient cells (MDA-MB-436) and wild cells (MCF-7) using PARP kit assay and MTT method. The results indicated that compared with other heterocyclic compounds, furan ring-substituted derivatives 14n-14q showed better PARP-1 inhibitory activity. Among this derivatives, compound 14p displayed the strongest inhibitory effects on PARP-1 enzyme (IC₅₀?=?0.023 μM), which was close to that of Olaparib. 14p (IC₅₀?=?43.56 ± 0.69 μM) and 14q (IC₅₀?=?36.69 ± 0.83 μM) displayed good antiproliferation activity on MDA-MB-436 cells and inactivity on MCF-7 cells, indicating that 14p and 14q have high selectivity and targeting. The molecular docking method was used to explore the binding mode of compound 14p and PARP-1, and implied that the formation of hydrogen bond was essential for PARP-1 inhibition activities. This study also showed that in the hydrophobic pocket (AD binding sites), the introduction of strong electronegative groups (furan ring, e.g.) or halogen atoms in the side chain of benzimidazole might improve its inhibitory activity and this strategy could be applied in further research.

Full text of DOI:10.1002/ddr.21843

Received: 20 April 2021
DOI: 10.1002/ddr.21843
Revised: 22 May 2021
Accepted: 27 May 2021
R E S E A R C H A R T I C L E
Synthesis and evaluation of 2-(4-[4-acetylpiperazine-
1-carbonyl] phenyl)-1H-benzo[d]imidazole-4-carboxamide
derivatives as potential PARP-1 inhibitors and preliminary
study on structure-activity relationship
Miaojia Chen | Honglin Huang | Kaiyue Wu | Yunfan Liu | Lizhi Jiang |
Yang Li | Guotao Tang
| Junmei Peng
| Xuan Cao
Institute of Pharmacy and Pharmacology,
Hunan Province Cooperative Innovation
Center for Molecular Target New Drug Study,
University of South China, Changsheng West
Road 28#, Hengyang, 421001, China
Abstract
Although 1H-benzo[d]imidazole-4-carboxamide derivatives have been explored
for a long time, the structure–activity relationship of the substituents in the
hydrophobic pocket (AD binding sites) has not thoroughly discovered. Here in, a
series of 2-(4-[4-acetylpiperazine-1-carbonyl]phenyl)-1H-benzo[d]imidazole-
4-carboxamide derivatives have been designed, synthesized, and successful char-
acterization as novel and effective poly ADP-ribose polymerases (PARP)-1 inhibi-
tors to improve the structure–activity relationships about the substituents in the
hydrophobic pocket. These derivatives were evaluated for their PARP-1 inhibitory
activity and cellular inhibitory against BRCA-1 deficient cells (MDA-MB-436) and
wild cells (MCF-7) using PARP kit assay and MTT method. The results indicated
that compared with other heterocyclic compounds, furan ring-substituted deriva-
tives 14n-14q showed better PARP-1 inhibitory activity. Among this derivatives,
compound 14p displayed the strongest inhibitory effects on PARP-1 enzyme
(IC50 = 0.023 μM), which was close to that of Olaparib. 14p (IC50 = 43.56
0.69 μM) and 14q (IC50 = 36.69 0.83 μM) displayed good antiproliferation
activity on MDA-MB-436 cells and inactivity on MCF-7 cells, indicating that 14p
and 14q have high selectivity and targeting. The molecular docking method was
used to explore the binding mode of compound 14p and PARP-1, and implied that
the formation of hydrogen bond was essential for PARP-1 inhibition activities.
This study also showed that in the hydrophobic pocket (AD binding sites), the
introduction of strong electronegative groups (furan ring, e.g.) or halogen atoms in
the side chain of benzimidazole might improve its inhibitory activity and this strat-
egy could be applied in further research.
Correspondence
Dr. Xuan Cao PhD, Institute of Pharmacy and
Pharmacology, Hunan Province Cooperative
Innovation Center for Molecular Target New
Drug Study, University of South China, 28#
Chang-Sheng Road, Hengyang 421001,
Hunan, China.
Email: caoxuancx@gmail.com
Funding information
financial support from China Scholarship
Council, Grant/Award Number:
201908430069; Hunan Province Cooperative
Innovation Center for Molecular Target New
Drug Study, Grant/Award Number:
0223-0002-0002000-44; University of South
China startup fundation for advanced talent,
Grant/Award Number: 2018XQD14
K E Y W O R D S
benzimidazole, furan ring, halogen, PARP-1 inhibitors, structure-activity relationship
Miaojia Chen, Honglin Huang, and Kaiyue Wu contributed equally to this work.
Drug Dev Res. 2021;1–9.
wileyonlinelibrary.com/journal/ddr
© 2021 Wiley Periodicals LLC.
1

2
CHEN ET AL.
1
|
INTRODUCTION
to treat BRCA-1/2 deficient cancers. Up to now, several PARP inhibi-
tors were available on the market, such as olaparib (Lynparza),
rucaparib (Rubraca), niraparib (Zejula) and talazoparib (Talzenna), ect,
and many inhibitors were candidate in clinical trials (Figure 1), such as
fuzuopali and veliparib (ABT-888). Although tremendous progress has
been made in the research of PARP-1 inhibitors, there were still many
shortcomings such as serious toxicity in combination with chemother-
apy drugs, in vivo side effects or poor pharmacodynamics and phar-
macokinetic properties (Tao et al., 2006; Zhu et al., 2013). Therefore,
it was crucial to develop more effective and safe PARP-1 inhibitors.
Currently, all PARP-1 inhibitors were mimicked the structure of
NAD+, competing for the combination of NAD+ and PARP catalytic
active sites to inhibit PARP enzymes. Those inhibitor's chemical struc-
ture were shown in Figure 1. The catalytic domain of PARP-1 was
generally characterized as two sub-pockets, nicotinamide-ribose bind-
ing sites (NI sites), and adenine-ribose binding sites (AD sites), respec-
tively. The amide functional group of the known PARP-1 inhibitor
occupy to the Ni site by forming key hydrogen bonds with Ser904
and Gly863, and the aromatic parent nucleus produces π ꢀ π interac-
tion with Tyr907 and Tyr896. Compared with the NI site, the AD site
was a large hydrophobic pocket that can accommodate the ADP-
ribose portion of NAD+. Different groups interacted with this pocket
could increase inhibitory activity and physicochemical properties of
these compounds (Farmer et al., 2005; Griffin et al., 1998; Zaremba
et al., 2007). Penning et al. were introduce basic amine solubilizing
groups of derivatives in which the nitrogen atom interacts with
Asp766 in the active site of PARP with water molecules so as to
increase it's cell permeability (Penning et al., 2010). Jeffrey
W. Johannes et al. modified a cyano-pyridine moiety at the end of the
side chain, and the nitrogen of the nitrile forms a hydrogen bond with
the ─NH of Asp1198, result shows the was prompt (Johannes
et al., 2015). Yi Zhong et al. Using the thiohydantoin ring as the linking
group to forms additional hydrogen bonds with Tyr896 and Ile895,
In 1963, Chambon et al. discovered the poly ADP-ribose polymerase
(PARP) enzyme for the first time (Kim et al., 2005). PARPs, a protein
family consisting of 18 subtypes, were significant enzymes, which role
in DNA damage repair process. It also plays an important role in a
wide range of biological processes such as apoptosis and transcrip-
tional regulation, maintaining genome stability, cell cycle progression
and chromatin dynamics regulation (De Vos et al., 2012; Hassa
et al., 2008; Yi et al., 2019). PARP-1, the most abundant and well-
characterized member of the PARP family, was involved in the base
excision repair (BER) pathway of eukaryotic cells to repair DNA
single-strand breaks (Shen et al., 2015). Once the DNA damaged
PARP-1 would be activated to catalyze the conversion of nicotin-
amide adenine dinucleotide (NAD+) to nicotinamide and ADP-ribose,
transfer the ADP-ribose unit to the nuclear receptor protein and
formed poly(ADP-ribose) on the substrate protein, which was essen-
tial for the transferring process of DNA repair and maintaining
genome stability (Ferraris (2010); Jagtap et al., 2005; Virag
et al., 2002). In 1922, the concept of synthetic lethality was first pro-
posed by Dobzhansky in Drosophila research. The two nonlethal
genes mutations together were lethal for the cell, whereas cells with
one or the other mutations do not undergo apoptosis (Lee
et al., 2014). The researchers found that BRCA-1 and BRCA-2
germline mutations can increase the risk of breast and ovarian cancer.
It was not confirmed until 2005 that the absence of BRCA-1 and
BRCA-2 played an important role in PARP inhibition sensitivity.
Recently, it has been proved that PARP-1 and BRCA-1/2 were syn-
thetically lethal. The PARP-1 inhibitors can selectively target to tumor
cells with defective BRCA-1/2 genes which can express to two essen-
tial proteins in homologous recombination (HR) mediated repair of
double-stranded DNA breaks (Farmer et al., 2005; Turner
et al., 2005). Therefore, PARP-1 inhibitors can be used as single drugs
FIGURE 1 Examples of PARP-1 inhibitors

CHEN ET AL.
3
result shows the is prompt (Zhong et al., 2019). The first PARP-1
inhibitor, approved for the treatment of advanced ovarian cancer with
BRCA gene defects, Olaparib, is also modified the fluorine atom on
the benzyl group to interact with the N-terminus of Gly894 and the
nitrogen on piperazine forms a favorable charge interaction with
Asp766 (Menear et al., 2008). A. Selen Gurkan-Alp et al. synthesized
three compounds with (piperazine-1-carbonyl)phenyl)-1H-benzo[d]
imidazole-4-carboxamide as the skeleton. But little useful information
about structure–activity relationship could be obtained because of
the minority amount (Gurkan-Alp et al., 2020). Herein, 1H-benzo[d]
imidazole-4-carboxamide was used as the model skeleton to improve
the structure–activity relationships about the substituents in the
hydrophobic pocket. A series of 2-aryl-substituted benzo[d]imidazole-
4-carboxamide derivatives were designed as new PARP-1 inhibitors,
in which the heterocyclic isostere such as piperazine, benzimidazole,
and thiazole were arranged on the 40 position of 2-aryl. The results
showed that compared with other heterocyclic compounds, furan
ring-substituted derivatives 14n-14q displayed better PARP-1 inhibi-
tory activity. Among this derivatives, compound 14p performed the
strongest inhibitory effects on PARP-1 enzyme (IC50 = 0.023 μM)
which was close to that of Olaparib. 14p (IC50 = 43.56 0.69 μM)
and 14q (IC50 = 36.69 0.83 μM) displayed good antiproliferation
activity on MDA-MB-436 cells and inactivity on MCF-7 cells, indicat-
ing that 14p and 14q have high selectivity to PARP-1 enzyme.
stirring at room temperature for 4 h. The suspension was filtered and
dried to obtain 1.9654 gm of bright yellow solid with a yield of 96.0%.
The compound is listed in Scheme 1.
2.1.3
|
Synthesis methods of 2-amino-
3-nitrobenzoic acid (3)
The 35 ml NaClO solution was drop-wised to
a mixture of
2-carbamoylꢀ3-nitrobenzoic acid (2) 1.9654 gm (0.009 mol), NaOH
4.0013 gm (0.1 mol), stirring vigorous for 2 h under ice bath. The reac-
tion was then heated to 65ꢁC. After about 3 h, the reaction was com-
pleted and an orange-red solid was produced. The solid was dissolved in
10 ml distilled water and then the pH of the solution was adjusted to
4 by concentrated sulfuric acid. The suspended solid was filtered and
dried to obtain 1.3691 gm of bright yellow solid with a yield of 80.8%.
The compound is listed in Scheme 1.
2.1.4
|
Synthesis methods of 2-amino-3-nitro
benzamide (5)
The 2-amino-3-nitrobenzoic acid (3) 1.3691 gm (0.0075 mol) was added in
3 ml SOCl2 and stirred at 60ꢁC for 2 h. The excess SOCl2 was evaporated
under high vacuum. The residue was dissolved in 10 ml Tetrahydrofuran
(THF) and then dropwise added to 6–8 ml ammonia water, strirring for 1–
2 h under ice bath. The suspension was filtered and washed by MeOH
(10 ml ꢂ 3) to obtain 1.2803 gm of yellow solid with a yield of 94.2%.
The compound is listed in Scheme 1.
2
|
MATERIALS AND METHODS
| Chemicals
2.1
2.1.1
|
Materials and reagents
2.1.5
|
Synthesis methods of
Unless otherwise noted, all chemicals and reagents (reagent grade) were
obtained from standard commercial suppliers and used without further
purification. Melting points (uncorrected) were determined on ZRD-1 full-
automatic melting point apparatus. Matrix assisted laser desorption ioniza-
tion coupled to time-of-flight mass spectrometry (MALDI-TOF MS) were
measured on a Bruker ultraflextreme mass spectrometer, and 1H NMR
spectra were recorded on Bruker AV-400 model spectrometer using indi-
cated solvent and internal standard Tetramethylsilane (TMS). Elemental
analysis was achieved on Perkin Elmer 2400 CHN. The values of the chem-
ical shifts were shown in delta values (ppm) and the coupling constants
(J) in Hertz (Hz). Peak multiplicities were generally described as follows: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; and br, broadened.
2, 3-diaminobenzamide (6)
The 2-amino-3-nitro benzamide (5) 1.2803 gm (0.007 mol), add half a
spoon of Raney Ni and 2 ml 80% hydrazine hydrate were added to
40 ml absolute ethanol solution, reaction at 60ꢁC for 2 h. After filtra-
tion and concentration, the crude product was purified by silicon
chromatography (methylene chloride: methanol
=
30:1) and
0.7920 gm of light yellow solid was obtained with a yield of 74.8%.
The compound is listed in Scheme 1.
2.1.6
|
Synthesis methods of 4-(4-carbamoyl-1H-
benzo[d]imidazol-2-yl)benzoic acid methyl ester (8)
2.1.2
|
Synthesis methods of 2-carbamoyl-
The 3-diaminobenzamide (6) 0.7920 gm (0.005 mol), methyl
4-formylbenzoate (7) 0.8346 gm (0.005 mol), NaHSO3 1.0231 gm
(0.01 mol), and 2 ml of distilled water were added to 40 ml absolute
ethanol solution, reaction at 60ꢁC for 1–2 h. The suspension was fil-
tered and washed with methanol (20 ml ꢂ 3) to obtain 0.9583 gm
white solid with a yield of 65.0%.
3-nitrobenzoic acid (2)
The 3-nitrophthalic anhydride 2.0065 gm (0.01 mol) was dissolved in
30 ml dry tetrahydrofuran at room temperature stirring for 3–4 h. 2 ml
of ammonia water was slowly added dropwise until the solution changed
from clear and transparent to white suspended solution, then continue
The compound is listed in Scheme 1.

4
CHEN ET AL.
SCHEME 1 Synthetic route of 14a-q
TABLE 1 Structure of compound 13a-q
overnight under 40ꢁC water bath. The solvent was removed
using high vacuum. The solid was dissolved in distilled water
and then the pH of solution was adjusted to 7 by citric acid
aqueous solution to obtain 0.8260 gm of white solid with a yield
of 92.0%.
The compound is listed in Scheme 1.
Compound
13a
Substituents (R)
Compound
13j
Substituents (R)
-Boc
2.1.8
|
Synthesis methods of compound 13a-q
13b
13c
─CH3
13k
13l
Carboxylic acid with different substituents 11a-q (0.005 mol), HBTU
1.8239 gm (0.005 mol), and anhydrous potassium carbonate
1.3672 gm (0.01 mol) were added to 40 ml acetone solution, reaction
at room temperature for 0.5 h. Then N-Boc-piperazine 0.9328 gm
(0.005 mol) was added to the solution and the mixture was stirred at
room temperature overnight. The solid was dissolved in 30 ml of dic-
hloromethane and 2 ml of 5 M HCl solution, then 1 M NaOH solution
was added to adjust the pH to 3. After filtration and concentration,
the crude product was purified by silicon chromatography (methylene
chloride: methanol = 20:1).
13d
13m
13e
13f
13n
13o
The compounds are listed in Table 1.
13g
13h
13i
13p
13q
2.1.9
|
Synthesis methods of compound 14a-q
The
(4-carbamoyl-1H-benzo[d]imidazol-2-yl)benzoic
acid
(9)
0.1438 gm (0.0005 mol), HBTU 0.1886 gm (0.0005 mol), and anhy-
drous potassium carbonate 0.1346 gm (0.001 mol) were resolved in
40 ml acetone solution, reaction at room temperature for 0.5 h. Then
13a (0.005 mol) was added and the mixture stirred at ambient temper-
ature overnight. After filtration and concentration, the crude product
was purified by silicon chromatography (methylene chloride: metha-
nol = 40:1) .
2.1.7
|
Synthesis methods of 4-(4-carbamoyl-1H-
benzo[d]imidazol-2-yl)benzoic acid (9)
The 4-(4-carbamoyl-1H-benzo[d]imidazol-2-yl)benzoic acid methyl
ester (8) was added to 40 ml methanol solution. Then the pH of
solution was adjusted to 12 with LiOH aqueous solution, stirring
The preparation method of compound 14b-q was similar to the
above method.

CHEN ET AL.
5
The compounds are listed in Table 2.
The antiproliferative evaluation of 14a-q was performed by using
Thiazolyl blue tetrazolium bromide (MTT) method. About 8 ꢂ 103 cells
per well were placed into well of 96-well microculture plates (Costar) and
grown for 24 h. After incubated with half diluted inhibitors (128, 64, 32,
16, 8, 4, 2, and 1 umol/L) for 48 h, cell cultures were incubated with 20 μl
MTT solution (5 mg/ml) each well for 4 h in dark at 37ꢁC. Then carefully
removed supernatant and 150 μl of Dimethyl sulfoxide (DMSO) was
added to dissolve the formazan crystals by shaking for 10 min. Absor-
bance at 490 nm was measured with a WellscanMK-2 microplate reader.
The assays were independently performed three times.
The 1H NMR, melting point, MALDI-TOF MS, and elemental anal-
ysis spectra data of compounds 14a-m and 14q were supplied as
experiment data.
The 1H NMR, 13C NMR, melting point, MALDI-TOF MS, and ele-
mental analysis spectra data of compounds 14n-p were supplied as
experiment data.
2.2
|
Cell culture
The human breast cancer cells (MCF-7), HA-VSMC cells, and human
breast cancer cells with a BRCA-1 mutation (MDA-MB-436) were
obtained from the Type Culture Collection of the Chinese Academy
of Sciences, Shanghai, China. MCF-7 cells and HA-VSMC cells were
grown in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, NY,
USA) containing 10% (v/v) fetal bovine serum (FBS, Gibco) with 1%
antibiotic-antimycotic solution (Gibco). MDA-MB-436 cells were
grown in L-15 containing 10% (v/v) FBS (science ll), 1% antibiotic-
antimycotic solution (Gibco) and 0.5% insulin (Becton Dickinson and
company). All of the cells were maintained 95% relative humidity.
2.3
|
PARP-1 inhibition assay
We use a commercially available PARP-1 Chemiluminescent Assay Kit
(BPS Bioscience, catalog#80551, San Diego, CA, USA) to measure the
inhibitory activity of the compounds (14a-q). The operation process is
the same as the kit instructions. First, 5ꢂ histone mixture was added
to 384-well plates and incubated overnight under 4ꢁC. Then, a
ribosylation reaction occurs between various concentration of inhibi-
tors and the PARP buffer which contains the PARP-1 enzyme. The
plate was treated with streptavidin-HRP followed by the addition of
the ELISA mixed solution. The content of biotinylated substrates
attached to histones was determined using the color reaction cata-
lyzed by streptavidin-HR, which can indirectly reflect the activity of
PARP-1 under different intervention conditions.
TABLE 2 Structure of compound 14a-q
2.4
|
Molecular docking
The molecular docking experiment was executed by SYBYL-X 2.0. The
3D structure of the PARP-1 receptor in complex with 2-substituted
benzimidazole-4- carboxamide inhibitor (PDB ID: 5WS1), retrieved
from the RCSB Protein Data Bank (PDB), was used in this study. The
ligand chosen in this experiment was compounds 14p. The protein
was optimized for docking by eliminating water molecules and optimiz-
ing the energy of receptors and ligands.
Compound
14a
Substituents (R)
Compound
14j
Substituents (R)
-Boc
14b
14c
─CH3
14k
14l
14d
14m
2.5
|
Drug likeness
This study was conducted using the descriptor computation module
of MOE 2019 (Aziz et al., 2021; Aziz et al., 2020). The molecular
descriptors of all compounds were calculated using the ligand prop-
erty calculation function of MOE 2019. (Table 4).
14e
14f
14n
14o
14g
14h
14i
14p
14q
3
|
RESULTS AND DISCUSSION
| Chemistry
3.1
According to the synthetic route outlined in Scheme 1 1H-benzo[d]-
imidazole-4-carboxamide derivatives were synthesized by 12 step

6
CHEN ET AL.
TABLE 3 The inhibitory activity of
IC50 SD (μM)a
compounds against PARP-1 enzyme and
antitumor activity against MDA-MB-436
cells and MCF-7 cells
Compound
14a
PARP-1 IC50 (μM)
0.195
>0.5
MDA-MB-436
55.09 0.19
>100
MCF-7
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
HA-VSMC
35.17 0.85
>100
14b
14c
>0.5
>100
>100
14d
>0.5
>100
>100
14e
>0.5
>100
>100
14f
0.274
>0.5
>100
>100
14g
>100
>100
14h
0.113
>0.5
54.30 1.60
>100
>100
14i
>100
14j
0.025
>0.5
>100
>100
14k
>100
>100
14l
0.247
0.478
0.293
0.332
0.023
0.043
0.061
0.015
86.01 0.17
45.07 1.18
95.83 1.35
64.66 1.19
43.56 0.69
36.69 0.83
>100
>100
14m
14n
32.21 1.80
>100
>100
>100
>100
>100
>100
>100
>100
14o
>100
14p
>100
14q
>100
Veliparib
Olaparib
>100
19.50 0.39
>100
aData are the average of three independent assays.
TABLE 4 Physicochemical
descriptors of compounds 14a-q
Compound
14a
LogP
2.64
2.04
3.61
1.82
2.45
2.96
2.92
2.91
2.80
3.80
2.82
3.12
3.23
2.98
3.21
4.13
3.83
Lip_acc
Lip_don
LogD (pH = 7)
LogS
TPSA
Weight
449.51
391.43
453.50
443.47
457.49
454.49
454.49
454.49
475.53
539.41
460.53
456.52
520.39
443.48
457.51
522.36
477.92
4
4
4
5
5
5
5
5
6
5
5
4
4
4
4
4
4
2
2
2
3
2
2
2
2
2
2
2
2
3
2
2
2
2
2.63
2.03
3.60
1.81
2.44
2.96
2.91
2.90
2.79
3.79
2.54
2.79
2.91
2.70
2.93
4.12
3.53
ꢀ5.20
ꢀ4.50
ꢀ6.13
ꢀ5.05
ꢀ5.49
ꢀ.5.85
ꢀ5.80
ꢀ5.80
ꢀ6.17
ꢀ6.90
ꢀ5.80
ꢀ5.61
ꢀ5.95
ꢀ5.45
ꢀ5.69
ꢀ6.67
ꢀ6.26
121.62
112.39
112.39
141.07
130.21
125.28
125.28
125.28
138.17
125.28
108.21
100.25
111.11
108.46
108.46
125.53
108.46
14b
14c
14d
14e
14f
14g
14h
14i
14j
14k
14l
14m
14n
14o
14p
14q
chemical reactions, and their chemical structures were shown in
Scheme 1. The key intermediate o-diaminobenzamide (6) was composed
of commercially available 3-nitrophthalic anhydride (1). At first,
3-nitrophthalic anhydride (1) was converted into 2-amino-
3-nitrobenzamide (5) by amino ring-opening, Hofmann rearrangement
reaction, and amidation reaction. After that, using methanol as a solvent,
under Raney Ni catalysis, it was reduced to o-diaminobenzamide (6) with
80% hydrazine hydrates. The intermediate (6) undergoes a cyclization
reaction with the aldehyde group of methyl 4-formylbenzoate (7) and
then hydrolyzed to the corresponding carboxylic acid at a high yield

CHEN ET AL.
7
TABLE 5 Docking score of compound 14a-q
Compound
14a
Scores
6.26
5.61
4.67
3.26
4.40
4.89
4.18
4.54
4.01
4.12
Compound
14k
Scores
4.90
4.50
4.33
3.07
5.48
6.81
5.85
7.06
4.30
14b
14l
14c
14 m
14n
14d
14e
14o
14f
14p
14 g
14 h
14i
14q
Veliparib
Olaparib
14j
under basic conditions (9). Besides, N-Boc-piperazine was condensed
with carboxylic acids (11a-q) containing different groups and then unpro-
tected by BOC to obtain intermediates (13a-q). Finally, the intermediate
(9) and the intermediate (13a-q) were condensed again to obtain a series
of target compounds (14a-q) in reasonable yield. A total of 17 target
compounds were synthesized in this paper, and their structures were
supported by 1H NMR, melting point, and MALDI-TOF mass spectrome-
try. The specific synthetic route and key points of the compound were
also shown in Scheme 1.
FIGURE 2 Molecular docking of compound 14p with PARP-1
(PDB ID: 5WS1) (Zhou et al., 2017). Protein and ligand structures
were colored in green and wheat, respectively. Nitrogen atoms are
shown in blue, whereas oxygen atoms were shown in red. Key
hydrogen bonding interactions were indicated by yellow dotted
lines
3.2
|
In vitro antitumor cell proliferation
experiment
unsaturated heterocycles, such as imidazole ring, pyridine ring,
thiazole ring, pyrrole ring or furan ring. Compounds 14a-q were
screened in vitro for their PARP-1 enzyme inhibitory activity at the
fixed concentration of 0.5 μM, and then the ones with PARP-1 inhibi-
tory rates >50% were selected to further determine the IC50 values.
The results were shown in Table 3, among this compounds, 14j
(IC50 = 0.025 μM), 14p (IC50 = 0.023 μM), and 14q (IC50 = 0.043 μM)
showed excellent PARP-1 enzyme activity, which was comparable
The MTT method was used to evaluate the proliferation inhibitory
effects of all target compounds on human breast cancer MDA-MB-
436 cells with natural BRCA-1 mutations, wild-type human breast
cancer MCF-7 cells and human aortic vascular smooth muscle cells
HA-VSMC, the results were listed in Table 3. Except for compound
14 m, it has no growth inhibitory effect on wild-type MCF-7 cells.
Furan derivatives 14n-q with IC50 values ranging between 36.69–
95.83 μM showed similar effects to the control Olaparib
(IC50 = 19.50 0.39 μM), and its cytostatic activity against BRCA-1
mutation was significantly stronger than that of wild-type cells, indi-
cating that these compounds can selectively kill BRCA-1 mutation
cells. In addition, except for compound 14a, other compounds have
no obvious toxicity to normal cells HA-VSMC. We found that com-
pound 14j showed similar enzymatic activity compared with
compound 14p. But, it had no appreciable proliferation inhibition
against on BRCA-1 mutant MDA-MB-436 cells. Therefore, compound
14p may be a promising lead compound with high activity on PARP-1
enzyme and BRCA-1 deficient cells.
with the control Veliparib (IC50
= 0.061 μM) and Olaparib
(IC50 = 0.015 μM). Compared with 14 g, both 14f and 14 h have
superior PARP-1 enzyme inhibitory activity, which may due to the rel-
atively low electron cloud density in the adjacent-positions and para-
positions. In addition, compared with other heterocyclic compounds,
furan ring-substituted derivatives 14n-q showed better PARP-1 inhib-
itory activity, especially 14p and 14q containing halogen atoms (Br,
Cl). The possible reason to promote the inhibition effect was that the
furan ring has the greatest electronegativity, and the smaller delocali-
zation degree of π electrons which can interact with the amino groups
in hydrophobic pocket by electrostatic force. Furthermore, we noticed
that every compounds substituted of halogen atoms, such as 14j,
14 m, 14p and 14q shows reasonable inhibitory activities. The possi-
ble reason was that the hydrophobic force might increase when
substituted halogen atoms binding with the AD site. It hints that the
introduction of strong electronegative groups or halogen atoms in the
side chain of benzimidazole might improve its inhibitory activity in fur-
ther research.
3.3
|
PARP-1 inhibition assay
In this report, we describe a series of benzimidazole-4-carboxamides
PARP-1 inhibitors in which a side chain was introduced some

8
CHEN ET AL.
3.4
|
Molecular docking
14p (MW 522.36) might have problem with bioavailability and needs
to be modified in further studies.
The molecular docking software SYBYL-2.0 was used to simulate the
binding mode of the designed compound and PARP-1 ligand (PDB
code: 5WS1) to guide and verify the rationality of the designed
PARP-1 inhibitor molecular structure. It can be seen that most of the
compounds and PARP-1 ligands have good strength of action scores
compare to the control Veliparib (7.06) and Olaparib (4.30) (Tables 4
and 5). Furan ring derivatives 14o, 14p and 14q apparently showed
higher scores than other heterocyclic derivatives. The reason could be
that the furan ring has the largest electronegativity and the π electron
has a small delocalization, so it can interact with the amino group in
the hydrophobic pocket through the electrostatic force. Compared
with compound 14n and 14o, compounds 14p and 14q have better
interaction strength with PARP-1 ligand when halogen atoms are
substituted.
4
|
CONCLUSION
In summary, a series of 2-(4-[4-acetylpiperazine-1carbonyl]phenyl)
-1H-benzo[d]imidazole-4-carboxamide derivatives (14a-q) were
successful synthesis and characterization to improve the
structure–activity relationships about the substituents in the
hydrophobic pocket. These compounds were evaluated for their
PARP-1 inhibitory activity. Compared with unsaturated heterocy-
clic compounds such as substituted imidazole ring and pyridine
ring, furan ring substituted derivatives 14n-q showed better
PARP-1 inhibitory activity. Compound 14p displayed the strongest
inhibitory effects on PARP-1 enzyme (IC50 = 0.023 μM). From the
MTT results, 14p and 14q displayed better antiproliferation activ-
ity on MDA-MB-436 cells and inactivity on MCF-7 cells, indicating
that 14p and 14q have high selectivity and targeting. The com-
pound exhibiting most prominent PARP-1 enzyme inhibitory activ-
ity in vitro was subjected to molecular docking analysis on 14p.
The molecular docking analysis showed that the formation of
hydrogen bond was essential for PARP-1 inhibition activities. Fur-
thermore, in the hydrophobic pocket (AD binding sites), it might
improve the inhibitory activity of designed compounds when
strong electronegative groups or halogen atoms were introduced
to the side chain of benzimidazole.
Compared with the their PARP-1 inhibitory activity and cellular
inhibitory against MDA-MB-436 and MCF-7 (Table 3), the data we
obtained was found approximate consistent with the predicted scores.
Therefore, it was inferred that when a strong electronegative group or
a halogen atom was introduced into the side chain of the benzimid-
azole ring, the PARP-1 inhibitory activity of the designed compound
could be improved.
To better understand the interaction forces between the com-
pound and the PARP-1 enzyme ligand, the compound 14p, which
exhibiting the best potency, was chosen for molecular docking analy-
sis with PARP-1 protein. Consistent with previous reports in the liter-
ature, the core of benzimidazole was the active site of PARP-1. The
carboxamide was located at the Ni site which was shown in Figure 2.
The amide structure can restrict its conformation through intramolec-
ular hydrogen bonding or ring formation and has a key hydrogen bond
with Gly863 and Ser904. There was a π-π conjugate between the
electron-rich benzimidazole ring and Tyr907 effect. When the side
chain of 14p extends into the hydrophobic pocket, on the one hand,
the Nitrogen atom on piperazine could forms hydrogen-bond interac-
tion with Asp-766, on the other hand, the bromine atom on furan ring
might has a certain hydrophobic force with this hydrophobic pocket.
This two factors might plays the main role for the promoted inhibitory
activity of PARP-1.
ACKNOWLEDGMENTS
This work was supported by Hunan Province Cooperative Innovation
Center for Molecular Target New Drug Study (0223-0002-0002000-44)
and University of South China startup foundation for advanced talent
(2018XQD14). The authors also gratefully acknowledge financial support
from China Scholarship Council No.201908430069.
CONFLICT OF INTEREST
The authors declare no potential conflict of interests to this work.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are openly available in
[repository name e.g “figshare”] at [doi], reference number [reference
number].
3.5
|
Drug likeness
The Lipin-ski's acceptors (lip-acc), Lipinski's donors (lip-don), log P, log
D (pH = 7), molecular weight, and total polar specific surface area
(TPSA) were calculated to test the pharmaceutical properties of the
synthesized 1H-benzo[d]imidazole-4-carboxamide derivatives (Aziz
et al., 2021). The results revealed that the synthesized 1H-benzo[d]
imidazole-4-carboxamide derivatives (14a-q) showed compliance to
the Lipinski's Ro5. Thus this compounds according with the drug-like
principle have the potential for further studies. The drug likeness of
the 17 compounds were shown in Table 6. According to Lipinski's rule
that molecular weight should be fewer or equal to 500, compound
ORCID
Guotao Tang
Junmei Peng
Xuan Cao
https://orcid.org/0000-0001-9673-4727
https://orcid.org/0000-0002-6887-9184
https://orcid.org/0000-0001-8293-7283
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