Design, synthesis and evaluation of phthalazinone thiohydantoin-based derivative as potent PARP-1 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103181

Two new series of compounds were designed and synthesized as potent PARP-1 inhibitors. These compounds were evaluated for PARP-1 enzyme and cellular inhibitory activities. All efforts lead to the identification of 9k (named as LG-12) with efficient potency both for PARP-1 and BRCA1 deficient MDA-MB-436 cells. Additionally, the novel PARP-1 inhibitor LG-12 is an efficient chemosensitizer, which could potentiate the anti-cancer effect of TMZ. Our data presented herein provide a comprehensive preclinical in vitro evaluation of the potential therapeutic efficacy and potency of chemotherapeutic agent-PARP-1 inhibitor combinations for LG-12. The combined results indicated that LG-12 could be a promising candidate for further study.

Full text of DOI:10.1016/j.bioorg.2019.103181

Journal Pre-Proof
Design, synthesis and evaluation of phthalazinone thiohydantoin-based deriva-
tive as potent PARP-1 inhibitors
Yi Zhong, Ying Meng, Xi Xu, Lulu Zhao, Zhiyu Li, Qidong You, Jinlei Bian
PII:
S0045-2068(19)30065-3
DOI:
https://doi.org/10.1016/j.bioorg.2019.103181
YBIOO 103181
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 January 2019
28 June 2019
1 August 2019
Please cite this article as: Y. Zhong, Y. Meng, X. Xu, L. Zhao, Z. Li, Q. You, J. Bian, Design, synthesis and evaluation
of phthalazinone thiohydantoin-based derivative as potent PARP-1 inhibitors, Bioorganic Chemistry (2019), doi:
https://doi.org/10.1016/j.bioorg.2019.103181
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will
undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing
this version to give early visibility of the article. Please note that, during the production process, errors may be
discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Inc.

JOURNAL PRE-PROOF
Design, synthesis and evaluation of phthalazinone thiohydantoin-based
derivative as potent PARP-1 inhibitors
Yi Zhong a,b,1, Ying Meng a,b,1, Xi Xua,b, Lulu Zhaoa,b, Zhiyu Li a,b, Qidong Youa,b,* and
Jinlei Bian a,b, *
aState Key Laboratory of Natural Medicines and Jiangsu Key Laboratory of Drug Design and
Optimization, China Pharmaceutical University, Nanjing 210009, China
bDepartment of Medicinal Chemistry, School of Medicinal Chemistry, China Pharmaceutical University,
Nanjing 210009, China
1 These two authors contributed equally to this work.
Keywords: PARP-1, antitumor, chemosensitizer, inhibitor
Abstract
Two new series of compounds were designed and synthesized as potent PARP-1 inhibitors. These
compounds were evaluated for PARP-1 enzyme and cellular inhibitory activities. All efforts lead to the
identification of 9k (named as LG-12) with efficient potency both for PARP-1 and BRCA1 deficient MDA-
MB-436 cells. Additionally, the novel PARP-1 inhibitor LG-12 is an efficient chemosensitizer, which
could potentiate the anti-cancer effect of TMZ. Our data presented herein provide a comprehensive
preclinical in vitro evaluation of the potential therapeutic efficacy and potency of chemotherapeutic agent-
PARP-1 inhibitor combinations for LG-12. The combined results indicated that LG-12 could be a
promising candidate for further study.

JOURNAL PRE-PROOF
1. Introduction
Poly (ADP-ribose) polymerases (PARPs) are composed of 18 members, which regulate a number of
cellular processes including surveillance of genome integrity, cell cycle progression, initiation of the DNA
damage response, apoptosis, and regulation of transcription.1 PARP-1, the most abundant and well-
characterized member among PARP family, is a critical component of base excision repair (BER) that
provides repairment of DNA single-strand breaks in eukaryotic cells. Impaired DNA activates PARP-1 to
cleave its substrate nicotinamide adenine dinucleotide (NAD+) and transfer ADP-ribose units to nuclear
target proteins to recruit BER components to facilitate DNA repair subsequently.2-6
It has been demonstrated that inhibition of PARP1/2 is synthetically lethal with the loss of function of
either the BRCA1 or BRCA2 (two essential proteins in homologous recombination (HR) of DNA double-
strand breaks) tumor suppressor gene, a number of PARP-1 inhibitors that were originally developed as
chemo- and radiopotentiators have been used as monotherapy for treating genetically DNA repair-defective
cancers. Indeed, seven PARP-1 inhibitors have undergone clinical evaluation in 2019 and six of them are
entering pre-registraon Phase III clinical trials including AG014699 (Rucaparib), AZD2281(Olaparibe),
MK4827(Niraparib), BMN673(Talazoparib).7-10 It is inspiring that FDA granted an accelerated approval to
olaparib (AZD2281) as a single agent for ovarian cancer treatment on December 19, 2014.11 So far, four
PARP-1 inhibitors have been approved.
O
H2N
O
NH
N
H2N
O
NH
N
O
F
N
N
N
N
H
N
N
H
O
Veliparib (ABT-888)
Olaparib (AZD2281)
Niraparib (MK4728)
CONH2
H
H
H
O
N
N
O
N
N
N
N
Linker
N
N
R
HN
O
F
N
H
N
H
F
O
O
N
S
Linker =
N
F
N
H
Rucaparib (AG014699)
BMN-673
S
Fig. 1. The structures of representative PARP-1 inhibitors and the designed compounds.

JOURNAL PRE-PROOF
Since 1990s multiple companies and academic groups developed PARP-1 inhibitors as chemo- and
radio potentiators and progressed molecules into early stage clinical trials.12 Despite these agents were
efficacious in initial clinical trials, they had not advanced into late stage clinical testing because of the
serious toxicity in combination with chemo- and radiotherapy. Therefore, we have been focusing our
efforts on discovering PARP-1 inhibitors that would significantly potentiate tumor-killing effect of
chemotherapy agents with low toxicity in recent years.
The catalytic pocket of PARP-1 was usually characterized as two sub-pockets which occupied by the
substrate NAD+. One of the binding sites is occupied by the nicotinamide-ribose (NI site) and the other is
the adenine-ribose binding site (AD site). The reported PARP-1 inhibitors can bind the NI site through
hydrogen bonds with residues Ser904 and Gly863, and additional π-π stacking interaction with Tyr907.
Compared with the NI site, the AD site is large enough to accommodate diverse structure motifs. Many
known potent inhibitors, such as AZD-2281, utilized this pocket to improve their potency.13 Herein, we
employed benzo[d]immidazole-4-carboxamide as a core structure to occupy the NI site which has been
proved to be efficient scaffold according to the potent PARP-1 inhibitors including ABT-888 and
NU1085.14-15 Additionally, various substituted heterocycles were selected and introduced on the 2-position
of benzo[d]imidazole ring using sulfonamide or thiohydantoin as the linkers to explore additional
interactions with AD site. In this work, we designed 2-substituted benzo[d]imidazole derivatives as PARP-
1 inhibitors and evaluated their anti-proliferative activity against MDA-MB-436 and MCF-7 cell lines.
Especially for compound LG-12, it exhibited greatly inhibitory activities against PARP-1 enzyme and in
vitro antitumor activity. The cell cycle analysis and Annexin V/PI double staining assay further indicated
that the LG-12 was a chemosensitizer and could be a feasible candidate in combination anti-cancer therapy.
2. Result and Discussion
2.1 Chemistry
The designed target compounds benzimidazole carboxamide of 9a-9k were synthesized according to
the protocol outlined in Scheme 1. Our synthetic work was started from the commercially available
materials 2,3-diaminobenzamide dihydrochloride (1), 2-(((benzyloxy)carbonyl)amino)propanoic acid (2)
and N,N'-Carbonyldiimidazole (CDI). Firstly, 1 and 2 were subjected to condensation reaction to give

JOURNAL PRE-PROOF
product 3. Then the compound 3 was reacted with acetic acid to form 4. Then, the amide group of the
intermediate 4 is hydrolyzed to obtain the intermediate 5. Further treating compound 5 with methyl
chloroacetate to get 6 or with 7 through nucleophilic substitution reaction to give the final products 9a-9c.
Intermediate 6 was treated with 8 to obtain final products 9d-9k in moderate yields. Typically, the crude
products were filtered with 15% HCl, crude products is recrystallized from methanol to increase purity. All
of the compounds were confirmed by 1H NMR and HRMS (ESI) spectra.
H2NOC
NH2
NH
CONH2
NH2
O
O
H
H
N
N
O
HN
i
ii
iii
HCl
O
O
O
N
NH2
O
OH
O
HN
3
1
2
CONH2
4
NH2
O
9a,
9b,
9c,
R1=H, R2=H, R3=NO2
R1=CH3, R2=Cl, R3=H
R1=H, R2=H, R3=CH3
O
H
O
H
N
O
S
R1
R3
N
Cl
N
v
S
H
O
N
R2
N
NH2
R
CONH2
9a-9c
7
5
9d,
9e,
9f,
9g,
R1=H, R2=H, R3=H
R
O
R1=H, R2=H, R3=CH3
R1=H, R2=H, R3=OCH3
R1=H, R2=H, R3=OCF3
NCS
R1
H
N
N
S
N
NH
2 HN
iv
8
R2
R3
9h,
9i,
R1=H, R2=OCH3, R3=H
R1=H, R2=H, R3=F
O
N
N
HN
vi
OCH3
CONH2
9j,
9k,
R1=H, R2=H, R3=Cl
R1=H, R2=H, R3=CF3
O
9d-9k
6
Scheme 1. Reagents and conditions: (i) CDI, DMF/pyridine (1/1), 45 ℃, 3 h, 80%; (ii) CH3COOH, 100 ℃,
18 h, 15.7%; (iii) H2, Pd-C, CH3OH, 4 h, 81%; (iv) Et3N, Methyl chloroacetate, CH3OH, 80 ℃, 40 h, 37%;
(v) Et3N, MeOH, 0 ℃, 6 h, 70-90%; (vi) dimethylformamide, Et3N, 5 ℃, 1 h, 16-50%.
2.2. Biological evaluation
2.2.1
In vitro PARP-1 inhibitory activity assay
The first series of these synthesized compounds were evaluated for their PARP-1 inhibitory activity.
As shown in Table 1, most of the synthesized compounds displayed excellent inhibitory activities against
PARP-1 with IC50 values ranging from 19 nM to 550 nM. Generally, the second series with thiohydantoin
as linkers (compounds 9d-9k) were more potent than sulfonamide (9a-9c). In the first series, compound 9b
with 2-methyl, 3-chloro substitutions showed the most inhibitory activity with IC50 110 nM. As for the
second series, compound 9d bearing a benzothiohydantoin demonstrated reasonable PARP-1 inhibitory

JOURNAL PRE-PROOF
activity with IC50 98 nM. Introducing the methyl or methoxy substituents (9e-9h) on the phenyl ring
indistinguishably affected the inhibitory activity. When the halogen group or trifluoromethyl (9i-9j or 9k)
were introduced at the benzene ring, all of these compounds showed improved activities. Among these
compounds, 9j and 9k showed appreciable enzymatic activity with the IC50 values of 19 and 25 nM,
respectively, which was comparable with the control Rucaparib and Olaparib. The representative
compound 9k was further selected for exploring the enzyme selectivity between PARP-1 and PARP-2. As
shown in Table 2, 9k was equipotent against the two isoforms due the close homology of PARP1 and
PARP-2.
Table 1
The inhibitory activity of compounds against PARP-1 enzyme and antitumor activity against MCF-7 cells
Enzyme
Enzyme
inhibition
(pIC50)
IC50
(MDA-MB-
436, μM)
pIC50
(MDA-MB-
436)
IC50
(MCF-7,
μM)
inhibition
IC50
(nM)
pIC50
(MCF-7)
Compd
R
NH2
O
H
N
O
O
HN
S
N
R
4-NO2
2-CH3, 3-Cl
550 ± 50.0
110 ± 20.3
348 ± 40.1
6.25 ± 0.03
6.96 ± 0.07
6.46 ± 0.05
19.3 ± 3.3
11.2 ± 1.6
32.8 ± 4.0
7.71 ± 0.07
7.95 ± 0.06
7.48 ± 0.05
>100
48.1 ± 10.7
>100
9a
9b
9c
—
7.31 ± 0.09
—
4-CH3
O
N
N
NH
2 HN
O
S
N
R
H
98 ± 8.9
102 ± 10.7
76 ± 5.1
74 ± 4.6
89 ± 9.7
37 ± 7.9
19 ± 4.3
25 ± 5.3
22 ± 8.4
7.00 ± 0.04
6.99 ± 0.04
7.12 ± 0.03
7.13 ± 0.03
7.05 ± 0.04
7.43 ± 0.08
7.72 ± 0.09
7.60 ± 0.08
7.66 ± 0.14
6.1 ± 3.4
1.3 ± 0.4
5.4 ± 1.2
2.9 ± 0.6
6.6 ± 2.5
4.6 ± 1.8
1.7 ± 0.4
1.0 ± 0.2
5.8 ± 1.3
8.21 ± 0.2
8.89 ± 0.1
8.27 ± 0.09
8.54 ± 0.08
8.18 ± 0.1
8.34 ± 0.1
8.77 ± 0.09
9.00 ± 0.08
8.24 ± 0.09
>100
>100
29.2 ± 4.8
>100
>100
>100
15.8 ± 3.2
>100
18.5 ± 5.1
9d
9e
9f
9g
9h
9i
—
4-CH3
4-OCH3
4-OCF3
3-OCH3
4-F
4-Cl
4-CF3
---
—
7.53 ± 0.07
—
—
—
9j
9k
7.80 ± 0.08
—
7.73 ± 0.1
Rucaparib
Olaparib
---
25 ± 5.6
7.60 ± 0.09
20.2 ± 5.3
7.69 ± 0.1
93.1 ± 10.9
7.03 ± 0.05
a Values are means from three independent dose–response curves.

JOURNAL PRE-PROOF
Table 2
The IC50 values of 9k against PARP-1/2 enzyme.
pIC50
IC50 (nM)
Compound
PARP-1
25 ± 5.3
22 ± 8.4
PARP-2
29 ± 2.1
15 ± 2.6
PARP-1
7.60 ± 0.08
7.66 ± 0.1
PARP-2
9k
7.54 ± 0.03
7.75 ± 0.07
Rucaparib
a Values are means from three independent dose–response curves.
2.2.2. Cell proliferation inhibition of potent analogues
All of the synthesized compounds were further evaluated for their inhibition of cell proliferation with
human breast cancer MDA-MB-436 cells carrying natural BRCA1 deficient and wild-type MCF-7 cells. All
of these compounds displayed moderate to good inhibition against MDA-MB-436 cells with the IC50 values
less than 50 μM and were almost inactive in wild-type MCF-7 cells, indicating that these compounds could
selectively kill the BRCA1 deficient cells. The high potency of 9j and 9k against the BRCA1 deficient cells
was consistent with their PARP-1 enzymatic activity. However, compound 9j displayed lower selectivity
between MDA-MB-436 cells and MCF-7 cells than compound 9k. The result indicated that the compound
9j might kill the cancer cells through other pathway in addition to the inhibition of PARP-1. Fortunately,
compound 9k shewed excellent potency in MDA-MB-436 cells with IC50 value of 1.0 μM and good
selectivity toward MCF-7 cells. Based on the results, compound 9k (named as LG-12) could be a
promising lead compound for further study with high activity against PARP-1 enzyme and the BRCA1
deficient cells.
2.2.3. Compound LG-12 caused the accumulation of the DNA double-strand breaks in MDA-MB-436 cells.
The production of DNA single-strand breaks (SSB) is common phenomenon in cancer cells because of
high frequency of replicative stress, and the double-strand breaks would occur and accumulate in cells if
the single-strand break repair by PARP-1 was not timely or the repair pathways are deficient, especially
BRCA-deficient. The frequency of stalled and damaged replication forks in MDA-MB-436 (BRCA1
deficient) caused by PARP-1 inhibitors can be monitored by estimating the formation of nuclear γ-H2AX
foci. To further confirm the PARP-1 enzyme inhibitory activities of LG-12, we assessed the ability of LG-

JOURNAL PRE-PROOF
12 to induce nuclear γ-H2AX foci formation in MDA-MB-436 by immunofluorescence and confocal
microscopic imaging. As shown in Fig. 2A and 2B, treatments with LG-12 at 1 μM and 1.5 μM led to
significantly enhanced levels of γ-H2AX in MDA-MB-436, which was equivalent efficacious as Rucaparib.
These results demonstrated that LG-12 was a potent PARP-1 inhibitor with IC50 at low nanomolar
concentrations.
Fig. 2. LG-12 increases formation of γ-H2AX in response to DNA damage. Nuclei stained with DAPI indicated the cell density; the
red luminescence images revealed the levels of cellular γ-H2AX. (A) Cells were pre-treated with 10 μM H2O2 or 1.5 μM AG014699
(Rucaparib) as positive control for 12 h at 37 °C. Cells untreated with H2O2 or PARP-1 inhibitors were used as negative control. (B)
Cells were pre-treated with serial dilutions of LG-12 for 12 h. LG-12 inhibits the viability of various breast cell lines and sensitizes
breast cells to TMZ in vitro.
2.2.4. Compound LG-12 potentiated cytotoxicity of the DNA damaging agent TMZ in MCF-7 cells.
After obtaining the potent PARP-1 small molecule inhibitor LG-12, we then tested whether it can
enhance the sensitivity of breast cancer cells to a methylation agent TMZ in vitro. TMZ is an oral
alkylating chemotherapeutic agent and the cytotoxicity of TMZ is based on guanine methylation and O6-
methylguanine formation in DNA. The molecular mechanism of PARP inhibitors is to trap PARP enzyme
at damaged DNA. We employ MTT assay to investigate whether LG-12 could enhance the anti-
proliferative effect of TMZ in wild-type MCF-7 cells. The results demonstrated that growth inhibition of
0.5 μM LG-12 combined with TMZ is about 3-fold higher than treatment with TMZ alone (Table 3), in
other words, LG-12 can effectively sensitize MCF-7 to TMZ in vitro.

JOURNAL PRE-PROOF
Table 3
Compound LG-12 potentiated cytotoxicity of TMZ in MCF-7 cells
Compound
IC50/nM
pIC50
Increase fold a
---
TMZ
610.7 ± 20
6.21 ± 0.014
TMZ + 0.5 μM LG-12
217.2 ± 12.4
6.66 ± 0.024
2.81
1.55
TMZ + 0.5 μM Rucaparib
394.3 ±16.7
6.40 ± 0.018
aThe increase fold a was calculated as IC50 (TMZ alone)/IC50 (TMZ in combination with compound).
2.2.5.
Treatment with LG-12 in combination with TMZ arrested cells in G2/M phase in MCF-7 cells
The synergy of TMZ and PARP-1 inhibitors is associated with DNA single-strand damage and can be
reflected in dramatic cell cycle arrest in cancer cells. TMZ methylate DNA at the O6 and N7 position of
guanine and the N3-position of adenine. The excision of these N-methylpurines (N7-MeG and N3-MeA)
generates a DNA single strand break (SSB).16 Since SSB repair must be completed before DNA replication
to prevent the formation of DSBs, the cell cycle progression is forced to delay until the repair is
accomplished successfully. However, in the case of intensive DNA damage, for example downregulation of
PARP-1, impaired DNA generally leads to permanent cell cycle arrest and the accumulation of the p53-
inducible proapoptotic products, which can lead to apoptosis.17-19 To understand and confirm the
mechanism underlying the combination effects on breast cancer cells, cell cycle analysis was determined
(Fig. 3). The treatment of MCF-7 with 125 and 250 μM of TMZ for 48 h, the arrest of cell cycle in G2/M
phase was increased (from 17.5% to 25.8%, 17.5% to 63.2% respectively) which was accompanied by a
concomitant decrease in the proportion of cells in G0/G1 phases. Furthermore, in comparison to the
combinations of TMZ (125 μM and 250 μM) with LG-12 (0.5 μM), addition of LG-12 significantly
arrested cells in G2/M phase (125 μM: 25.8% vs 66.3%, 250 μM: 63.2% vs 80.9%).

JOURNAL PRE-PROOF
Fig. 3. Cell cycle analysis of MCF-7 48 h post treatment with TMZ alone or in combination with 0.5 μM LG-12. Few changes in cell
cycle progression were observed for MCF-7 treated with 0.5 μM LG-12 alone (15%) compared to control group (17.5%). G2/M arrest
observed with TMZ treatment alone (125 μM: 25.8%; 250 μM: 63.2%) was enhanced prominently by the addition of LG-12 (125 μM:
66.3%; 250 μM: 80.9%) in MCF-7.
2.2.6.
LG-12 increases the apoptotic events of TMZ in MCF-7 cells
Since severe cell cycle arrest can lead to the accumulation of the p53-inducible proapoptotic products,
which can result in apoptosis, Annexin V/PI double staining assay was performed to completely evaluate
potentiation mechanism of TMZ combined with LG-12 in MCF-7. We analyzed Annexin V/PI expression
at 24, 48 and 72 h, the results indicated a slight tend of apoptosis increase which presented a dependence on
concentration with presence of TMZ or TMZ with LG-12 in MCF-7 from 24 to 72 h (Fig. 4). We
speculated that distinct capacity of inducing cell cycle arrest might explain the apoptosis events of different

JOURNAL PRE-PROOF
dose. Moreover, as shown in Fig. 4C, treatment with TMZ in combination with LG-12 (14.1% and 22.6%)
may increase late apoptotic events compared to treatment with TMZ alone (11% and 14.4%).
Fig. 4. Annexin V/PI apoptosis assay of MCF-7 treated with TMZ (125 μM and 250 μM) or in combination with LG-12 (0.5 μM) for
24 (A), 48 (B) or 72 h (C). The apoptotic cells include the early apoptotic cells (Annexin V+ /PI-) and late apoptotic cells (Annexin V+
/PI+).
2.3. Computational studies of compound LG-12.
2.3.1. Molecular docking.
Molecular docking of compound LG-12 binding with PARP-1 were performed by GOLD 5.1 (PDB
ID: 4R6E). The docking results were shown in Fig. 5. The benzimidazole carboxamide core provides the
conserved hydrogen-bonding interactions with residues Ser904, Gly863 and His862, and π-π stacking
interactions with residue Tyr907. The thiohydantoin ring of the compound formed a hydrogen-bonding
interaction with Tyr896 and Ile895. In addition, the methyl could form additional H…π interaction with

JOURNAL PRE-PROOF
Tyr896. The preliminary docking result may support the fact that the compound LG-12 was a good
inhibitor of PARP-1.
Fig. 5. (A) Docked conformation of compound LG-12 with PARP-1. (B) The 2D ligand interactions of compound LG-12 with key
residues of PARP-1. The interaction mode was obtained through molecular docking (PDB ID: 4R6E) and depicted using MOE
2018.01. The H-bonds were shown as black dot lines.
2.3.2. In silico toxicity and metabolism of LG-12
To study the potential therapeutic potential of compound LG-12, we performed an in silico prediction
of its toxicity and metabolism in humans using DEREK and StarDrop software, respectively. Based on the
comparison of the structural features of a given compound with one or more toxicophore patterns
(structural alerts) in the Lhasa's knowledge base 2018 (species: human), the toxicity predictions were
obtained with the DEREK Nexus program. There were 50 toxicity endpoints for compound LG-12,
analyzed at the minimum reasoning level of “impossible” (Chart S1). Three alerts are plausible fired for
LG-12, including pulmonary toxicity in mammal due to aromatic thiourea, HERG channel inhibition in
vitro in mammal, and hepatotoxicity in mammal due to benzimidazole group. We would determine these
potential toxicities in our next work for further biological test and pay attention on these predicted plausible
toxicities.
We further evaluated the potential metabolites for CYP3A4 by using the StarDrop software. The
software predicted four metabolites, with the most favored product being the sulfur atom (S9) with 69%
probability. The potential metabolites were shown in Fig. S1. Based on the result of predicted toxicity and

JOURNAL PRE-PROOF
metabolism, our group would design and synthesis several novel PARP-1 inhibitors based on the scaffold
of compound LG-12 with improved drug-like properties.
3. Conclusion
In conclusion, a series of novel 2-substituted benzo[d]imidazole-4-carboxamide derivatives were
designed based on the characteristics of the catalytic domain in PARP-1. These compounds were evaluated
for their PARP-1 enzyme inhibitory activity and cellular inhibitory against wild MCF-7 cells and MDA-
MB-436 BRCA1 deficient cells. All efforts lead to the identification of 9k (named as LG-12) with efficient
potency both for PARP-1 and BRCA1 deficient MDA-MB-436 cells. In addition, the novel PARP-1
inhibitor LG-12 is an efficient chemosensitizer, which could potentiate the anti-cancer effect of TMZ. Our
data presented herein provide a comprehensive preclinical in vitro evaluation of the potential therapeutic
efficacy and potency of chemotherapeutic agent-PARP-1 inhibitor combinations for LG-12. Therefore, we
believed that LG-12 supported to be an efficient instrumental for improving the efficacy for
chemotherapies of cancer and expanding the application of clinical PARP-1 inhibitors.
4. Experimental section
4.1. Chemistry
All reagents were used as obtained from commercial sources unless otherwise indicated. With
1
tetramethylsilane (TMS) as internal standard, the H NMR spectra were recorded on Bruker AV-300 (300
o
MHz) apparatus at 25 C. Samples were prepared as solutions in deuterated solvent. EI-MS was collected
on shimadzu GCMS-2010 instruments. HR-MS spectral data was obtained on Agilent technologies 6520
Accurate-Mass Q-TOF LC/MS instruments. All target compounds were purified via silica gel (60 Å, 70-
230 mesh) column chromatography. All target compounds were found to have > 95% purity.
4.1.1. Synthesis of benzyl (1-((2-amino-3-carbamoylphenyl)amino)-1-oxopropan-2-yl)carbamate (3)
2, 3-Diaminobenzamide dihydrochloride (1, 2 g, 9 mmol) and N, N'-carbonyldiimidazole (CDI) were
dissolved in the mixture solution of DMF (8 mL) and pyridine (8 mL). The reaction mixture was stirred at

JOURNAL PRE-PROOF
room temperature for 3 h. After which, 2-(((benzyloxy)carbonyl)amino)propanoic acid (2) was added
dropwise, then the mixture keep 45 ℃ for 3 h, followed by filtration and concentration in vacuo gave the
crude product in 80%.
4.1.2. Synthesis of benzyl (1-(4-carbamoyl-1H-benzo[d]imidazol-2-yl)ethyl)carbamate (4)
Compound 3 was dissolved in CH3COOH and maintained 100 ℃ for 18 h, then the reaction mixture
was diluted with water and extracted with ethyl acetate. Drying over MgSO4, followed by filtration and
concentration in reducing pressure gave the crude product. The product was ready for the next step without
the further purification.
4.1.3. Synthesis of 2-(1-aminoethyl)-1H-benzo[d]imidazole-4-carboxamide (5)
Intermediate 4 was dissolved in CH3OH and the mixture was stirred at room temperature for 4 h, after
the Pd-C was removed from mixture through filtration. Then CH3OH was evaporated under reduced
pressure, giving a white precipitate.
4.1.4. Synthesis of methyl 2-((1-(4-carbamoyl-1H-benzo[d]imidazol-2-yl)ethyl)amino)acetate (6)
Intermediate 5 (1.6 g, 7.8 mmol) was added to 100 mL of MeOH, then EtN3 (1.8 mL) and methyl
chloroacetate (0.8 mL) drop in the solution and heated to refluxed at 80 °C for 40 h. The solvent was
removed under reduced pressure, and then ethyl acetate (60 mL) was added to afford intermediate 6 at
yellow oil in 37.0%.
4.1.5. Synthesis of General procedure for the synthesis of target compounds (9a-9c)
Compound 6 (200 mg) was dissolved in MeOH (30 mL), after which a solution of compound 7 in
MeOH (4 mL) was added dropwise. The reaction mixture was stirred at 0 ℃ for 6 h. After completion the
reaction mixture was diluted with water and extracted with diethyl ether. The solvent was removed under
reduced pressure to give solid. Then the solid product was washed with MeOH to give final products 9a-9c
in 80-90%.

JOURNAL PRE-PROOF
4.1.5.1. Synthesis of
2-(1-(4-Nitrophenylsulfonamido)ethyl)-1H-benzo[d]imidazole-7-carboxamide (9a)
The title compound was prepared using intermediate 7a and following the general method. Yield: 90.5%.
1
Mp = 232-235 ℃. H NMR (300 MHz, DMSO-d6, ppm) δ: 12.78 (s, 1H), 9.06-8.75 (m, 2H), 8.75 (s, brd,
1H), 8.18 (d, J = 8.4 Hz, 2H), 7.99 (d, J = 8.7 Hz, 2H), 7.83-7.66 (m, 2H), 7.34 (t, J = 7.8 Hz, 1H), 4.95-
4.83 (m, 1H), 1.43 (d, J = 6.9 Hz, 3H). ESI-HRMS m/z [M + H]+ calculated for C16H15N5O5S:390.0872,
found: 390.0864.
4.1.5.2. Synthesis of
2-(1-(3-Chloro-2-methylphenylsulfonamido)ethyl)-1H-benzo[d]imidazole-7-carboxamide(9b)
The title compound was prepared using intermediate 7b and following the general method. Yield:
83.5%. Mp = 228-230 ℃. 1H NMR (300 MHz, DMSO-d6, ppm) δ: 12.49 (s, 1H), 9.03 (s, 1H), 8.70 (d, J =
6 Hz, 1H) , 7.70-7.60 (m, 3H), 7.46 (d, J = 8.1 Hz, 1H), 7.28 (d, J = 7.5 Hz, 1H), 7.18 (t, J = 7.8 Hz, 1H),
7.07 (t, J = 7.8 Hz, 1H), 4.58-4.50 (m, 1H), 2.42 (s, 3H), 1.43 (d, J = 6.9 Hz, 1H). ESI-HRMS m/z [M +
H]+ calculated for C17H17ClN4O3S:393.0788, found: 393.0778.
4.1.5.3. Synthesis of
2-(1-(4-Methylphenylsulfonamido)ethyl)-1H-benzo[d]imidazole-7-carboxamide(9c)
The title compound was prepared using intermediate 7c and following the general method. Yield: 85.0%.
Mp = 220-224 ℃.1H NMR (300 MHz, DMSO-d6, ppm) δ: 12.63 (s, brd, 1 H), 9.01 (s, brd, 2 H) , 8.37 (d, J
= 8.4 Hz, 1H) , 7.78 (d, J = 8.4 Hz, 1H), 7.63-7.57 (m, 3H), 7.27 (t, J = 7.2 Hz, 1H), 7.15 (d, J = 7.8 Hz,
2H), 7.07 (t, J = 7.8 Hz, 1H), 4.62 (s, brd, 1H), 1.39 (d, J = 6.9 Hz, 3H). ESI-HRMS m/z [M + H]+
calculated for C17H18N4O3S: 359.1118, found: 359.1117.
4.1.6. Synthesis of General procedure for the synthesis of target compounds (9d-9k)
Compound 6 (0.1 g) was dissolved in DMF (3 mL), then dimethylformamide (4 mL) and TEA (0.1 mL)
were drop to this solution, and keep cool for 10 minutes. After 8 (0.1 mL) was added into the mixture and
keep 5 ℃ for 1 hour. The solution was treated with water and the mixture was adjusted pH being 1 with 2
M HCl to give crude products. Column chromatography to obtain the final product, as yellow solid in 16-
50 %.

JOURNAL PRE-PROOF
4.1.6.1. Synthesis of 2-(1-(4-oxo-3-phenyl-2-thioxoimidazolidin-1-yl)ethyl)-1H-benzo[d]imidazole-7-
carboxamide (9d)
The title compound was prepared using intermediate 9d and following the general method. Yield: 43.6%.
Mp = 168-172 ℃. 1H NMR (300 MHz, DMSO-d6, ppm) δ: 12.89 (s, 1H), 9.05 (s, 1H), 7.79-7.64 (m, 3H),
7.46-7.37 (m, 3H), 7.29 (d, J = 7.2 Hz, 3H), 6.24 (br., 1H), 4.57 (d, J = 19.2 Hz, 1H), 4.41 (d, J = 19.2 Hz,
1H), 1.74 (d, J = 5.4 Hz, 3H). ESI-HRMS m/z [M + H]+ calculated for C19H17N5O2S: 380.1181, found:
380.1168.
4.1.6.2.
Synthesis
of
2-(1-(4-oxo-2-thioxo-3-(4-methylphenyl)imidazolidin-1-yl)ethyl)-1H-
benzo[d]imidazole-7-carboxamide (9e)
The title compound was prepared using intermediate 9e and following the general method. Yield:
35.3%. Mp = 176-180 ℃. 1H NMR (300 MHz, DMSO-d6, ppm) δ: 12.97 (s, 1H), 9.15 (s, 1H), 7.88-7.71 (m,
3H), 7.37-7.18 (m, 5H), 6.38-6.28 (m, 1H), 4.64 (d, J =19.8 Hz, 1H), 4.47 (d, J =19.8 Hz, 1H), 2.37 (s, 3H),
1.82-1.71 (d, 3H). ESI-HRMS m/z [M + H]+ calculated for C20H19N5O2S: 394.1338, found: 394.1327.
4.1.6.3. Synthesis of 2-(1-(3-(4-methoxyphenyl)-4-oxo-2-thioxoimidazolidin-1-yl)ethyl)-1H-
benzo[d]imidazole-7-carboxamide (9f)
The title compound was prepared using intermediate 9f and following the general method. Yield: 16.9%,
Mp = 158-161 ℃. 1H NMR (300 MHz, DMSO-d6, ppm) δ: 12.83 (s, 1H), 9.01 (s, 1H), 7.73 (d, J = 8.1Hz,
1H), 7.67 (s, 1H), 7.59 (d, J = 8.1Hz, 1H), 7.24 (t, J = 8.1 Hz, 1H),7.14 (d, J = 8.7 Hz, 2H), 6.93 (d, J = 8.7
Hz, 2H), 6.15 (m, J = 6 Hz, 1H), 4.47 (d, J =18.9 Hz, 1H), 4.31 (d, J =18.9 Hz, 1H), 3.67 (s, 3H), 1.68 (d, J
= 6.6 Hz, 3H). ESI-HRMS m/z [M + H]+ calculated for C20H19N5O3S: 410.1278, found: 410.1286.
4.1.6.4. Synthesis of 2-(1-(4-oxo-2-thioxo-3-(4-(trifluoromethoxy)phenyl)imidazolidin-1-yl)ethyl)-1H-
benzo[d]imidazole-7-carboxamide (9g)
The title compound was prepared using intermediate 9g and following the general method. Yield: 25.0%,
Mp = 160-164 ℃.1H NMR (300 MHz, DMSO-d6, ppm) δ: 13.48 (s, 1H), 9.16 (s, 1H), 7.86-7.81 (m, 2H),
7.74 (d, J = 7.8 Hz, 1H), 7.55 (s, 4H), 7.36 (t, J = 7.8 Hz, 1H), 6.29 (m, J = 7.5 Hz, 1H), 4.53 (d, J = 19.2
Hz, 1H), 4.38 (d, J = 19.2 Hz, 1H), 1.84(d, J = 7.8 Hz, 3H). ESI-HRMS m/z [M + H]+ calculated for
C20H16F3N5O2S:464.1004, found: 464.0992.

JOURNAL PRE-PROOF
4.1.6.5. Synthesis of 2-(1-(3-(3-methoxyphenyl)-4-oxo-2-thioxoimidazolidin-1-yl)ethyl)-1H-
benzo[d]imidazole-7-carboxamide (9h)
The title compound was prepared using intermediate 9h and following the general method. Yield: 40.7%.
Mp = 155-158 ℃. 1H NMR (300 MHz, DMSO-d6, ppm) δ: 12.84 (s, 1H), 9.01 (s, 1H), 7.78-7.60 (m, 3H),
7.28-7.11 (m, 2H), 6.89-6.28 (m, 3H), 6.16 (br., 1H), 4.50 (d, J = 18.9 Hz, 1H), 4.33 (d, J = 18.9 Hz, 1H),
3.63 (s, 3H), 1.65 (br., 3H). ESI-HRMS m/z [M + H]+ calculated for C20H16F3N5O3S: 410.1287, found:
410.1283.
4.1.6.6. Synthesis of 2-(1-(3-(4-fluorophenyl)-4-oxo-2-thioxoimidazolidin-1-yl)ethyl)-1H-
benzo[d]imidazole-7-carboxamide (9i)
The title compound was prepared using intermediate 9i and following the general method. Yield: 50.9%.
Mp = 163-165 ℃. 1H NMR (300 MHz, DMSO-d6, ppm) δ: 12.96 (s, 1H), 9.15 (s, 1H), 7.88 (d, J = 7.5 Hz,
1H), 7.79 (s, 1H), 7.74 (d, J = 7.5 Hz, 1H), 7.41-7.36 (m, 5H), 6.30 (br., 1H), 4.64 (d, J = 18.9 Hz, 1H),
4.49 (d, J =18.9 Hz, 1H), 1.83 (d, J = 6.3 Hz, 3H). ESI-HRMS m/z [M + H]+ calculated for C19H16FN5O2S:
398.1087, found: 398.1083.
4.1.6.7. Synthesis of 2-(1-(3-(4-chlorophenyl)-4-oxo-2-thioxoimidazolidin-1-yl)ethyl)-1H-
benzo[d]imidazole-7-carboxamide (9j)
The title compound was prepared using intermediate 9j and following the general method. Yield: 40.9%.
Mp = 168-170 ℃. 1H NMR (300 MHz, DMSO-d6, ppm) δ: 12.83 (s, 1H), 8.99 (s, 1H) , 7.73 (d, J = 7.5 Hz,
1H), 7.65 (s, 1H), 7.60 (d, J = 7.5 Hz, 1H), 7.48 (d, J = 7.5 Hz, 2H), 7.28-7.18 (m, 3H), 6.16 (br., 1H), 4.50
(d, J = 18.9 Hz, 1H), 4.35 (d, J = 18.9 Hz, 1H), 1.68 (d, J = 7.5 Hz, 3H). ESI-HRMS m/z [M + H]+
calculated for C19H16ClN5O2S: 414.0786, 416.0756, found: 414.0787, 416.0768.
4.1.6.8. Synthesis of 2-(1-(4-oxo-2-thioxo-3-(4-(trifluoromethyl)phenyl)imidazolidin-1-yl)ethyl)-1H-
benzo[d]imidazole-7-carboxamide (9k)
The title compound was prepared using intermediate 9k and following the general method. Yield: 33.1%.
Mp = 165-168 ℃. 1H NMR (300 MHz, DMSO-d6, ppm) δ: 12.99 (s, 1H), 9.15 (s, 1H), 7.95 (d, J = 8.1Hz,
2H), 7.89 (d, J = 7.2 Hz, 1H), 7.81 (s, 1H), 7.75 (d, J = 7.2 Hz, 1H), 7.67 (d, J = 8.1 Hz, 2H), 7.38 (t, J =
6.9 Hz, 1H, 6.34-6.27 (q, J = 6.9 Hz, 1H), 4.66 (d, J = 19.5 Hz, 1H), 4.53 (d, J = 19.5 Hz, 1H), 1.84 (d, J =
6.3 Hz, 3H). ESI-HRMS m/z [M + H]+ calculated for C20H16F3N5O2S: 448.1055, found: 448.1049.

JOURNAL PRE-PROOF
4.2. Bioassay
4.2.1. PARP inhibition assay
We used a commercially available 96-well assay kit (Cat# 4690-096-K, Homogeneous PARP
Inhibition Assay Kit from Trevigen) to evaluate the inhibitory activity of the synthesized compounds. The
stock solutions of the various test compounds were dissolved in DMSO and then serially diluted with
distilled water to the required concentrations. In the first step, adding 25 μL 1 × Buffer to each well, 25 μL
2 × NAD+ standard solution was added to the 1C to 1H and 2C to 2H well of 96-well plate for drawing
experimental standard curve. Add 25 μL 2 μM NAD+ to the PARP control group wells. As for test
compound wells, 25 μL 2 μM NAD+ was added to all wells, then 1 μL serially diluted inhibitors was added
with two duplications. And 1 μL of DMSO was added to the standard wells. The 25 μL PARP Mix minus
Enzyme was added to standard wells mixed with pipette. The 25 μL PARP Mix plus Enzyme PARP was
added to the control wells and the test compound wells. Incubate the 96-well plate for 30 minutes at 25 °C.
After 30 minutes, 50 μL Cycling Mix was added to all wells and mixed with pipette. Incubate the 96-well
plate in the dark for 40 minutes at 25 °C. Then 50 μL stop solution was added to each well, and mixed with
pipette. The fluorescence values under λex 544 nm and λem 590 nm were measured (Molecular Devices,
SpectraMax® Paradigm®). Then draw the standard curve and calculate the inhibition rate of each test
compound. IC50 value of each compound was calculated according to the above results.
4.2.2. MTT assays
We used the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazoliumbromide] (Biomatik,
Wilmington, DE) assay to measure cell growth inhibition, which is based on the conversion of MTT to
formazan crystals by mitochondrial dehydrogenases.20 Only in live cells, mitochondrial enzymes can
transform MTT into insoluble formazan. After incubated with serially diluted inhibitors for 96 h, cell
cultures were incubated with MTT solution (5 mg/mL) for 4 h at 37 °C. Then discard the medium and
DMSO was added to solubilize the reaction product formazan by shaking for 10 min. Absorbance at 492
nm was measured with a microplate reader (Thermo, MK3). Cell viability was expressed as an IC50 value.
4.2.3. Immunofluorescence analysis

JOURNAL PRE-PROOF
Within 1-3 minutes of the formation of a double-strand break, serine-139 in the unique carboxyl-terminal
tail of histone H2AX is phosphorylated to generate γ-H2AX. Several γ-H2AX histone molecules then
localize to the site of a double-strand break to form a focus.21 We choose γ-H2AX to measure DNA double-
strand breaks. Cells were cultured on glass coverslips in 6-well plate for 24 h, and then were exposed to
medium containing no drug or serially diluted inhibitors for 24 h at 37 °C before fixation in 4%
paraformaldehyde for 15 minutes at room temperature. As a positive control for DNA damage, cells were
cultured on coverslips and then exposed to 1 mM H2O2 for 10 minutes at 37 °C before fixation. After
fixation, cell cultures were permeabilized in 0.3% Triton X-100 for 15 minutes, and blocked in 10% goat
serum and then incubated overnight at 4 °C in primary anti-γ-H2AX histone IgG mouse monoclonal
antibody (abcam, CN). Cells were next incubated with secondary Alexa Fluor 586-conjugated goat anti-
mouse IgG fluorescent antibody (Invitrogen, MD) for 1 h at room temperature in the dark. Nuclei were
stained with DAPI. The γ-H2AX foci were obtained with a Leica microscope.
4.2.4. Cell cycle analysis
MCF-7 cells were seeded into 6-well plates (5 × 105 cells/well), incubated overnight and then treated with
medium containing no drug, serially diluted TMZ (125 μM, 250 μM), LG-12 (0.5 μM) or serially diluted
TMZ plus LG-12 (0.5 μM) for 48 h. Cells were harvested after trypsinization, washed with PBS, and
stained with DNA staining solution DAPI (containing 0.1 Triton X-100). Cell cycle phase distributions
were determined using FlowSight flow cytometer. The data were analyzed by Flow Jo software.
4.2.5. Annexin V/PI double staining assays
Apoptosis-mediated cell death of tumor cell was examined using a double staining method with FITC-
labeled Annexin V/propidium iodide (PI) Apoptosis Detection kit (Beyotime, CN) according to the
manufacturer's instructions. MCF-7 were seeded into 6-well plates (5 × 105 cells/well), incubated overnight
and then treated with medium containing no drug, serially diluted TMZ (125 μM, 250 μM), LG-12 (0.5 μM)
or serially diluted TMZ plus LG-12 (0.5 μM) for 24, 48 and 72 h. Flow cytometric analysis was performed
immediately after supravital staining. Cells in early stages of apoptosis were Annexin V positive; whereas,

JOURNAL PRE-PROOF
cells that were both Annexin V and PI positive were in the late stage of apoptosis. The data were analyzed
by Flow Jo software.
4.2.6. In silico toxicity and metabolism predictions
To assess toxicity prediction, the Derek Nexus v 6.0.1 software was employed to evaluate alerts and
estimate the likelihood toxicity. The likelihood levels in Derex Nexus in highest to lowest order are: certain,
probable, plausible, equivocal, doubted, improbable, and impossible. The metabolites were predicted by
StarDrop model runner algorithms.
Acknowledgements
We are thankful for the financial support of the National Natural Science Foundation of China (no.
21672260, and 21372260), the Natural Science Foundation of Jiangsu Province of China (no.
BK20170743), the Fundamental Research Funds for the Central Universities, China (2632017PY13).
College Students Innovation Project for the R&D of Novel Drugs (J1310032). National Found for
Fostering Talents of Basic Science (NFFTBS).
Reference
1. P.O. Hassa, M.O. Hottiger, The diverse biological roles of mammalian PARPS, a small but po
werful family of poly-ADP-ribose polymerases, Front. Biosci. 3 (2008) 3046-3082.
2. M. Buege, P.B. Mahajan, Clinical Trials of Poly(ADP-Ribose) Polymerase Inhibitors for Cancer
Therapy: A Review, Reviews on Recent Clinical Trials. 10 (2015) 326-339.
3. Y. Shen, M. Aoyagi-Scharber, B. Wang, J. Pharmacol, Trapping Poly(ADP-Ribose) Polymerase,
Exp. Ther. 353 (2015) 446-457.
4. P. Jones, K. Wilcoxen, M. Rowley, C. Toniatti, Niraparib: A Poly(ADP-ribose) Polymerase (PA
RP) Inhibitor for the Treatment of Tumors with Defective Homologous Recombination, J. Med. Ch
em. 58 (2015) 3302-3314.

JOURNAL PRE-PROOF
5. Y.S. Tong, J.J. Bouska, P.A. Ellis, E.F. Johnson, J. Leverson, X.S. Liu, P.A. Marcotte, A.M. O
lson, D.J. Osterling, M. Przytulinska, L.E. Rodriguez, Y. Shi, N. Soni, J. Stavropoulos, S. Thomas,
C.K. Donawho, D.J. Frost, Y. Luo, V.L. Giranda, T.D. Penning, Synthesis and Evaluation of a Ne
w Generation of Orally Efficacious Benzimidazole-Based Poly(ADP-ribose) Polymerase-1 (PARP-1)
Inhibitors as Anticancer Agents, J. Med. Chem. 52 (2009) 6803-6813.
6. H. Almahli, E. Hadchity, M.Y. Jaballah, R. Daher, H.A. Ghabbour, M.M. Kabil, N.S. Al-shaklia
h, W.M. Eldehnah, Development of novel synthesized phthalazinone-based PARP-1 inhibitors with
apoptosis inducing mechanism in lung cancer, Bioorg. Chem. 77 (2018) 443-456.
7. N. Curtin, Biochem. PARP inhibitors for anticancer therapy. Soc. Trans. 42 (2014) 82-88.
8. J.F. Liu, P.A. Konstantinopoulos, U.A. Matulonis, PARP inhibitors in ovarian cancer: Current st
atus and future promise, Gynecol. Oncol. 133 (2014) 362-369.
9. Y. Shen, F.L. Rehman, Y. Feng, J. Boshuizen, I. Bajrami, R. Elliott, B. Wang, C.J. Lord, L.E.
Post, A. Ashworth, BMN 673, a Novel and Highly Potent PARP1/2 Inhibitor for the Treatment of
Human Cancers with DNA Repair Deficiency, Clin. Cancer. Res. 19 (2013) 5003-5015.
10. M.W. Audeh, J. Carmichael, R.T. Penson, M. Friedlander, B. Powell, K.M. BellMcGuinn, C. S
cott, J.N. Weitzel, A. Oaknin, N. Loman, K. Lu, R.K. Schmutzler, U. Matulonis, M. Wickens, A.
Tutt, Synergistic antitumor effects of S-1 with eribulin in vitro and in vivo for triple-negative brea
st cancer cell lines, Lancet. 376 (2010) 245-251.
11. S. Tangutoori, P. Baldwin, S. Sridhar, PARP inhibitors: A new era of targeted therapy. Maturi
tas. 81 (2015) 5-9.
12. D.V. Ferraris, Evolution of Poly(ADP-ribose) Polymerase-1 (PARP-1) Inhibitors. From Concept
to Clinic, J. Med. Chem. 53 (2010) 4561-4584.
13. L. Lehtio, A.S. Jemth, R. Collins, O. Loseva, A. Johansson, N. Markova, M. Hammarstrom, A.
Flores, L. Holmberg-Schiavone, J. Weigelt, T. Helleday, H. Schuler, T. Karlberg, Structural basis f

JOURNAL PRE-PROOF
or inhibitor specificity in human poly(ADP-ribose) polymerase-3, J. Med. Chem. 52 (2009) 3108-31
11.
14. T.D. Penning, G.D. Zhu, V.B. Gandhi, J. Gong, X. Liu, Y. Shi, V. Klinghofer, E.F. Johnson,
C.K. Donawho, D.J. Frost, V. Bontcheva-Diaz, J.J. Bouska, D.J. Osterling, A.M. Olson, K.C. Mars
h, Y. Luo, V.L. Giranda, Discovery of the Poly(ADP-ribose) polymerase (PARP) inhibitor 2-[(R)-2
-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide (ABT-888) for the treatment of cancer, J.
Med. Chem. 52 (2009) 514-523.
15. A.W. White, R. Almassy, A.H. Calvert, N.J. Curtin, R.J. Griffin, Z. Hostomsky, K. Maegley,
D.R. Newell, S. Srinivasan, B.T. Golding, Resistance-Modifying Agents. 9.1 Synthesis and Biologic
al Properties of Benzimidazole Inhibitors of the DNA Repair Enzyme Poly(ADP-ribose) Polymerase,
J. Med. Chem. 3 (2004) 4084-4097.
16. Z. Tatar, E. Thivat, E. Planchat, P. Gimbergues, E. Gadea, C. Abrial, X. Durando, Temozolom
ide and unusual indications: Review of literature, Cancer Treat Rev. 39 (2013) 125-135.
17. E.A. Prokhorova, A.V. Zamaraev, G.S. Kopeina, B. Zhivotovsky, I.N. Lavrik, Role of the nucl
eus in apoptosis: signaling and execution, Cell Mol. Life Sci. 72 (2015) 4593-4612.
18. H. Tian, Z. Gao, H. Li, B. Zhang, G. Wang, Q. Zhang, D. Pei, J. Zheng, DNA damage respo
nse – A double-edged sword in cancer prevention and cancer therapy, Cancer Lett. 358 (2015) 8-1
6.
19. A. Malki, R.Y. Elbayaa, O. Ali, A. Sultand, A.M. Youssef, Novel Quinuclidinone Derivatives
Induced Apoptosis in Human Breast Cancer via Targeting P53, Bioorg. Chem. 50 (2014) 16-17.
20. W.T. Zhang, J.L. Ruan, P.F. Wu, F.C. Jiang, L.N. Zhang, W. Fang, X.L. Chen, Y. Wang, B.
S. Cao, G.Y. Chen, Y.J. Zhu, J. Gu, J.G. Chen, Design, Synthesis, and Cytoprotective Effect of 2-
Aminothiazole Analogues as Potent Poly(ADP-Ribose) Polymerase-1 Inhibitors, J. Med. Chem. 52 (
2009) 718-725.

JOURNAL PRE-PROOF
21. Y. Drew, E.A. Mulligan, W.T. Vong, H.D. Thomas, S. Kahn, S. Kyle, A. Mukhopadhyay, G.
Los, Z. Hostomsky, E.R. Plummer, R.J. Edmondson, N.J. Curtin, Therapeutic Potential of Poly(AD
P-ribose) Polymerase Inhibitor AG014699 in Human Cancers With Mutated or Methylated BRCA1
or BRCA2, J. Natl. Cancer Inst. 103 (2011) 334-346.
22. C.A. Marchant, K.A. Briggs, A. Long, In Silico Tools for Sharing Data and Knowledge on Toxicity and
Metabolism: Derek for Windows, Meteor, and Vitic, Toxicol Mech. Method. 18(2-3) (2008) 177-187.
23. SeeSARv.8.1, BioSolveIT GmbH, Sankt Augustin, Germany (2018).
24. P.N. Judson, S.A. Stalford, J. Vessey, Assessing confidence in predictions made by knowledge-based
systems, Toxicology Research. 2(1) (2013) 70-79.

JOURNAL PRE-PROOF

JOURNAL PRE-PROOF
Two series compounds (9a-9c, 9d-9k) were evaluated for PARP-1 enzyme
and cellular inhibitory activity in BRCA1 deficient MDA-MB-436 cells.
The novel PARP-1 inhibitor LG-12 (9k) could be an efficient
chemosensitizer, which could potentiate the anti-cancer effect of TMZ.
Compound LG-12 caused the accumulation of the DNA double-strand
breaks in MDA-MB-436 cells.
Compound LG-12 potentiated cytotoxicity of the DNA damaging agent
TMZ in MCF-7 cells.
Treatment with LG-12 in combination with TMZ arrested cells in G2/M
phase in MCF-7 cells and increases the apoptotic events of TMZ in MCF-7
cells