Discovery of 2-substituted 1H-benzo[d]immidazole-4-carboxamide derivatives as novel poly(ADP-ribose)polymerase-1 inhibitors with in?vivo anti-tumor activity

Article abstract of DOI:10.1016/j.ejmech.2017.03.013

Novel 1H-benzo[d]immidazole-4-carboxamide derivatives bearing five-membered or six-membered N-heterocyclic moieties at the 2-position were designed and synthesized as PARP-1 inhibitors. Structure-activity relationships were conducted and led to a number of potent PARP-1 inhibitors having IC₅₀ values in the single or double digit nanomolar level. Some potent PARP-1 inhibitors also had similar inhibitory activities against PARP-2. Among all the synthesized compounds, compound 10a and 11e displayed strong potentiation effects on temozolomide (TMZ) in MX-1?cells (PF₅₀?=?7.10, PF₅₀?=?4.17). In?vivo tumor growth inhibition was investigated using compound 10a in combination with TMZ, and it was demonstrated that compound 10a could strongly potentiate the cytotoxicity of TMZ in MX-1 xenograft tumor model. Two co-crystal structures of compounds 11b and 15e complexed with PARP-1 were achieved and demonstrated a unique binding mode of these benzo-imidazole derivatives.

Full text of DOI:10.1016/j.ejmech.2017.03.013

European Journal of Medicinal Chemistry 132 (2017) 26e41
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of 2-substituted 1H-benzo[d]immidazole-4-carboxamide
derivatives as novel poly(ADP-ribose)polymerase-1 inhibitors with
in vivo anti-tumor activity
,
,
,
, *
Jie Zhou a 1, Ming Ji b 1, Zhixiang Zhu b, Ran Cao a, Xiaoguang Chen b **, Bailing Xu a
a Beijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences &
Peking Union Medical College, Beijing, 100050, China
b State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences &
Peking Union Medical College, Beijing, 100050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 1H-benzo[d]immidazole-4-carboxamide derivatives bearing five-membered or six-membered N-
heterocyclic moieties at the 2-position were designed and synthesized as PARP-1 inhibitors. Structure-
activity relationships were conducted and led to a number of potent PARP-1 inhibitors having IC50
values in the single or double digit nanomolar level. Some potent PARP-1 inhibitors also had similar
inhibitory activities against PARP-2. Among all the synthesized compounds, compound 10a and 11e
displayed strong potentiation effects on temozolomide (TMZ) in MX-1 cells (PF50 ¼ 7.10, PF50 ¼ 4.17).
In vivo tumor growth inhibition was investigated using compound 10a in combination with TMZ, and it
was demonstrated that compound 10a could strongly potentiate the cytotoxicity of TMZ in MX-1
xenograft tumor model. Two co-crystal structures of compounds 11b and 15e complexed with PARP-1
were achieved and demonstrated a unique binding mode of these benzo-imidazole derivatives.
© 2017 Elsevier Masson SAS. All rights reserved.
Received 3 January 2017
Received in revised form
6 March 2017
Accepted 8 March 2017
Available online 18 March 2017
Keywords:
PARP-1 inhibitor
PARP-2 inhibitor
Benzo[d]immidazole-4-carboxamide
Anti-tumor agents
1. Introduction
cells when DNA damaging agents were used as anti-cancer thera-
peutics, and resulted in drug resistance. Therefore, inhibiting PARP-
Poly (ADP-ribose) polymerase-1 (PARP-1) is an abundant nu-
clear enzyme, discovered five decades ago, and is involved in the
repair process of DNA damage [1,2]. As a molecular sensor of DNA
strand breaks, PARP-1 can detect the DNA damage and bind to the
nicks through its N-terminal domain, and its catalytic domain on C-
terminal can locate the nicotinamide adenine dinucleotide (NADþ)
as substrate and catalyze the conversion of NADþ into nicotinamide
and ADP-ribose units. The ADP-ribose units were transferred to
proteins participating in the repair pathway including histone and
PARP-1 itself and formed ADP-ribose polymers on the substrate
proteins. This post-transcriptional poly-ADP-ribosylation has a
pivotal role in the repair of DNA damage, in particular for the single
strand breaks repair through base excursion repair pathway [3e7].
It has been suggested that PARP-1 was strongly activated in tumor
1 could potentiate the toxicity of chemotherapeutics such as
temozolomide (TMZ) and cisplatin in the combination therapy
[8e12]. Even more interestingly, it was found that PARP-1 was
synthetic lethal with BRCA1/2, which is involved in DNA double
strand breaks repair through homologous recombination pathway.
This synthetic lethality conferred tumor cells with BRCA1/2 muta-
tions more susceptible to PARP-1 inhibitors, which could be used as
a single agent in the treatment of BRCA defective cancers [13e16].
In fact, PARP-1 inhibitors were mainly exploited as a treatment
modality for patients with BRCA dysfunctions in clinical trials for
the present.
As targeting the repair pathway of DNA damage has been
recognizing as an intriguing manoeuvre for coping with cancers,
PARP-1 inhibitors have aroused a great deal of interests among
academics and pharmaceutical industries. A significant number of
structurally diversified PARP-1 inhibitors were developed and a
dozen of inhibitors have been advanced into clinical trials,
including ABT-888, AZD2281, AG014699, MK-4827 and BMN673
[17e23]. Among them, Olaparib (AZD-2281) was the first PARP-1
inhibitor approved by FDA in 2014 for the treatment of high-
* Corresponding author.
** Corresponding author.
E-mail addresses: chxg@imm.ac.cn (X. Chen), xubl@imm.ac.cn (B. Xu).
The first two authors contributed equally.
1
http://dx.doi.org/10.1016/j.ejmech.2017.03.013
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

J. Zhou et al. / European Journal of Medicinal Chemistry 132 (2017) 26e41
27
grade serous ovarian cancer. Although tremendous progress has
been made in researches on PARP-1 inhibitors, it is still an exciting
and imperative for the development of novel chemical entities with
varied pharmacodynamic and pharmacokinetic properties, so as to
PARP-1 inhibitors would be able to become effective treatments for
multiple cancers.
derivatives (7a-7c, 8a-8f) were prepared and their inhibitory ac-
tivities were shown in Table 1. Generally, these compounds
moderately inhibited PARP-1 enzymatic activity and had IC50 values
ranging from 199 nM to 1810 nM. And in both series compounds,
introduction of bulky substituents on the nitrogen would lead to
decrease in potency, when compared with non-substituted com-
pounds 7c and 8e.
The substrate NADþ binds in the catalytic domain of PARP-1 and
the binding region was usually characterized as two sub-pockets.
One is the nicotinamide-ribose binding site (NI site) and the
other is the adenine-ribose binding site (AD site). The known PARP-
1 inhibitors take full advantage of the NI site through hydrogen
We also synthesized 4-amino-piperidine derivatives (9a-9o and
10a-10b in Table 2) in order to probe the impact of the nitrogen
position in the piperidine ring on the inhibitory activity. Compound
9a with a Boc group showed moderate inhibitory activity against
PARP-1 with an IC50 value of 644 nM, while removal of the Boc
(compound 9o) resulted in 6-fold enhancement on potency, sug-
gesting that the Boc is somewhat larger for this position. Satisfy-
ingly, it was found that incorporation of a small to mediate size
alkyl group on the piperidine ring (compounds 9b-9g) produced
marked inhibition effect on PARP-1 with IC50 values in the double
digit nanomolar level. In contrast, when a bulkier substituent was
placed on the nitrogen, compounds 9j-9n exhibited decreased ac-
tivity. It was demonstrated that larger moieties with carbon atoms
ꢀ5 were not tolerated on the nitrogen, while smaller hydrophobic
groups would be beneficial for the binding affinity. Notably, com-
pound 9i with a neo-pentyl group on the nitrogen had IC50 value of
41 nM and it was more potent than isomers 9j and 9k, presumably
because the shape of neo-pentyl substituent was well matched
with that of the sub-pocket.
bonds with residues Ser904 and Gly863, and
p-p stacking inter-
action with Tyr907. In comparison with NI site, the AD site has more
space to accommodate diverse structural motifs, and some of
known PARP-1 inhibitors including AZD-2281 utilized this pocket
to improve their potency and PK properties [24,25]. In this work,
we took benzo[d]immidazole-4-carboxamide as a core structure to
occupy the NI site since it was successfully employed in potent
PARP-1 inhibitors such as ABT-888, and NU1085 [17,26]. Further-
more, various substituted cyclic amines were tentatively selected
and introduced on the 2-position of benzo[d]imidazole ring to
explore additional bindings within AD site. Herein, we described
the chemical synthesis and biological evaluation of the designed 2-
subsituted benzo[d]imidazole derivatives as PARP-1 inhibitors. The
biological evaluations were performed with respect to enzymatic
inhibition, potentiation effects on DNA damaging agent and anti-
tumor activity in MX-1 xenograft tumor model. Two co-crystal
structures were also presented to elicit the action mode of the
designed molecules.
Compounds 10a and 10b with a fluoro atom on the 6-position of
benzo-imidazole ring were highly active against PARP-1 (IC50
:
17 nM and 30 nM), and their inhibition was stronger than that of
corresponding non-fluoro-substituted counterparts (compounds 9i
and 9k), respectively. It was demonstrated that grafting a fluoro
atom on the 6-position would be favorable for the binding affinity,
and this result was consistent with the known SARs. Additionally, in
comparison with 3-amino-piperidine derivatives, 4-amino-piperi-
dine moiety seems to be a proper choice for generating potent
PARP-1 inhibitors, since compounds 9a and 9o are somewhat more
active than compounds 8a and 8e.
2. Chemical synthesis
The 1H-benzo[d]immidazole-4-carboxamide derivatives were
prepared according to the synthetic route as outlined in Scheme 1
and their chemical structures were presented in Tables 1e4. Upon
treatment with methyl 2,2,2-trichloroacetimidate and TFA, 2,3-
diamino-benzamides (1 and 2) were converted into 2-(tri-
chloromethyl)-1H-benzo[d]imidazole-4-carboxamides (3 and 4) in
61% and 92% yields, respectively. Under the basic reaction condi-
tions, compounds 3 and 4 were hydrolyzed into the corresponding
carboxylic acids 5 and 6 in high yields, which were the key in-
termediates for preparation of various designed target molecules.
Compounds 5 and 6 were coupled with 1-substituted pyrrolidin-3-
amine, 1-substituted piperidin-3-amine or 1-substituted piperidin-
4-amine generating series of compounds 7e10 (Tables 1 and 2) in
moderate to good yields. And compounds 5 and 6 were subjected to
condensation reaction with N-Boc pyrrolidin-3-amine or N-Boc
piperidin-4-amine, followed by Boc deprotection, reductive ami-
nation or acylation giving rise to series of compounds 11e14
(Table 3) in reasonable yields. Regarding series of compounds 15
and 16 (Table 4) bearing piperazine, hydropyrrolo[3,4-b]pyridine,
or quinuclidin-3-amine motif, they were readily prepared by
condensation of compounds 5 and 6 with the corresponding
amines followed by Boc deprotection or acylation reaction.
When 3-amino-pyrrolidine and 4-amino-piperidine fragments
were linked to the benzo-imidazole scaffold through the ring ni-
trogen atom resulted in series of compounds 11e14 (Table 3). These
compounds showed a wide range of inhibitory activities against
PARP-1 with IC50 values varying from 2.76 nM to 1190 nM. In
general, a bulky group, such as Boc or N-Boc-piperidine-4-yl sub-
stituent taken as the R group would deteriorate the potency, as
exemplified by compounds 11a, 11f, 13a and 13d. Removal of the
Boc group had a significant positive impact on the inhibition and
led to the potency increasing more than 10-fold (11a vs 11b, 12a vs
12b, 13a vs 13b, 14a vs 14b), suggesting a basic amine group was
desirable in this position, presumably a strong hydrogen bond or
electrostatic interaction formed between the amine group and an
acidic residue within the PARP-1 catalytic site. Moreover, com-
pounds (11c-11e, 12c, 13c, 14c) with an alkyl hydrophobic group,
such as a propyl or cyclohexyl moiety on the nitrogen had com-
parable potency with non-substituted derivatives, demonstrating
that variations on the nitrogen by hydrophobic groups were
allowed and this provided an opportunity for improving structural
diversity and physicochemical properties. Compared with 3-
amino-pyrrolidine derivatives (11b vs 12b, 11e vs 12c), grafting a
fluoro atom on the 6-position had a more favorable effect on the
inhibition in 4-amino-piperidine derivatives (13b vs 14b, 13c vs
3. Results and discussion
3.1. Enzymatic activity against PARP-1
The inhibitory activities against PARP-1 of all target compounds
(7a-7c, 8a-8f, 9a-9o, 10a, 10b, 11a-11g, 12a-12c, 13a-13e, 14a-14c,
15a-15k, 16a, 16b) were evaluated. ABT888 and AZD2281 were
used as reference molecules. The corresponding results were
expressed as IC50 values and presented in Tables 1e4
14c). In fact, compounds 14b (IC50
¼
2.76 nM) and 14c
(IC50 ¼ 7.54 nM) are the most potent PARP-1 inhibitors in this series
of benzo-imidazole derivatives, they had single digit nanomolar
activities.
Initially,
3-amino-pyrrolidine
and
3-amino-piperidine
We also incorporated the piperazine, piperazin-2-one and other

28
J. Zhou et al. / European Journal of Medicinal Chemistry 132 (2017) 26e41
Scheme 1. Reagents and conditions: a) methyl 2,2,2-trichloroacetimidate, TFA, DCM, ether, rt; b) NaOH (aq, 1 N), H2O, rt; c) EDC, HOBt, Et3N, DMF, rt or HBTU, HOBt, DIEA, DMF, rt;
d) TFA, DCM, rt; e) acyl chloride, DCM, Et3N, or aldehyde or ketone, DIEA, THF, then NaBH(OAc)3; (f) propionyl chloride, DCM, Et3N, 0 ꢁC.

J. Zhou et al. / European Journal of Medicinal Chemistry 132 (2017) 26e41
29
bulky N-heterocyclic fragments into the 2-position of benzo-
imidazole core to further expand the SARs. As shown in Table 4,
similar to previous derivatives (Tables 1e3), Boc derivatives had
low potency in comparison with non-substituted compounds (15a
vs 15e, 15b vs 15f). Regarding piperazine series, alkylation on the
nitrogen with iso-butyl group (compound 15d, IC50 ¼ 81 nM) was
tolerated, while acylation with propionyl moiety (compound 15k,
IC50 > 100 nM) produced reduced activity, indicating that a basic
amine group was necessary for the binding, probably functioning as
an H-bond donor or an acceptor. In addition, comparing compound
15f bearing an ethyl group on the piperizine ring with 15g, the (S)-
enantiomer (15f, IC50 ¼ 19 nM) was >5 times more potent than the
(R)-enantiomer (15g, IC50 > 100 nM). Thus we considered com-
pound 15f was deserved to be further optimized in terms of its
structural novelty and potent enzymatic activity. As to the piper-
azin-2-one derivatives (15j, 16a and 16b), these compounds dis-
played strong inhibition effect on PARP-1, suggesting that
piperazin-2-one could be exploited as an alternative motif for
achieving potent PARP-1 inhibitors. In addition, compounds 15h
and 15i showed low inhibitory activity and their IC50 values of
>100 nM. Therefore, bulky heterocyclic amines were not preferred
as the structural motifs to be incorporated into the 2-position of
benzo-imidazole scaffold.
TMZ in combination, both dosing at 50 mg/kg, the tumors shrunk
markedly and 88% tumor growth inhibition was achieved. When
TMZ treated group was used as the control, the combination dosing
still produced 68% inhibition effect. It was demonstrated that
compound 10a was a strong chemo-sensitizing agent. Additionally,
it was observed that the combination treatment did not lead to
pronounced body weight changes, suggesting that compound 10a
had low toxicity at this dose.
4. Crystal structures of compounds 11b and 15e complexed
with PARP-1
The co-crystal structures of compounds 11b and 15e located in
the catalytic site of PARP-1 were obtained with an aim to investi-
gate the binding features of the designed benzo-imidazole-based
PARP-1 inhibitors. As shown in Fig. 1, as we expected, the benzo-
imidazole scaffold was nicely situated in NI site and formed 3 key
hydrogen bonds with the backbone of Gly863 and the side chain of
Ser904. It also formed characterized
pep stacking with Tyr907.
These binding features were consistent with that of the known
PARP-1 inhibitors [4,28]. The 3-amino-pyrrolindine ring extended
into AD site, and especially the amino group interacted with
Asp766 via a favorable charge-charge interaction, which was
considered to have great positive contributions to the binding af-
finity of this series of PARP-1 inhibitors. In fact, this indeed gave a
good explanation for previous SAR. Removal of the Boc group
resulted in dramatic enhancement in potency.
As shown in Fig. 2, the binding mode of compound 15e was the
same as that of compound 11b. In this co-crystal structure, the
nitrogen on the piperazine ring formed the key charge-charge
interaction with Asp766. It has been reported that Asp766 could
be exploited to construct novel PARP-1 inhibitors. However, few
known PARP-1 inhibitors presented this interaction in their bind-
ing, except for ABT-888 [29]. Therefore, this unique binding inter-
action in this class of PARP-1 inhibitors deserved to be further
explored, and it might provide a chance to build up more potent
PARP-1 inhibitors with distinct binding features by modifications
on the amine group.
3.2. Enzymatic activity against PARP-2 and cellular potency
PARP-2 is the closest homolog of PARP-1 in the PARP super-
family and its catalytic domain shares 69% similarity to that of
PARP-1. Furthermore, both enzymes are involved in DNA breaks
repair and PARP-1 contributes >90% PARP activity [27]. It has been
well known that most of PARP-1 inhibitors were also recognized as
PARP-2 inhibitors with comparable potency, although these in-
hibitors were initially designed to target PARP-1. In this work, we
selected some potent PARP-1 inhibitors to test their inhibition ef-
fects on PARP-2. As shown in Table 5, these benzo-imidazole de-
rivatives presented somewhat stronger inhibition on PARP-1 over
PARP-2, and none of them showed selectivity more than 10 fold
though.
The cellular potency of twelve PARP-1 inhibitors was evaluated
by measuring their potentiation effects on TMZ-induced cytotox-
icity in MX-1 cells. As presented in Table 5, among the tested de-
rivatives, compounds 10a and 11e could strongly sensitize the
cytotoxicity of TMZ in MX-1 breast cancer cells. The cytotoxicity of
TMZ was increased to 7.1 fold and 4.17 fold, respectively, when TMZ
5. Conclusion
In summary, novel 2-substituted benzo[d]imidazole-4-
carboxamide derivatives were designed based on the characteris-
tics of the catalytic domain in PARP-1. The enzymatic inhibition
results demonstrated that incorporation of a 3-aminopyrrolidine
fragment on the 2-position via the ring nitrogen can give rise to
potent PARP-1 inhibitors with IC50 values in the double digit
nanomolar level (compounds 11b and 12b). The co-crystal struc-
ture of 11b located in PARP-1 proved that the basic 3-amino group
on the pyrrolidine ring was critical for the binding since it created a
key electrostatic interaction with Asp766. More importantly,
various six-membered N-heterocyclic moieties, such as 4-amino
piperidine, piperizine, (S)-2-ethyl piperizine and piperizin-2-one
were allowed to be integrated into the 2-position of benzo-
imidazole scaffold. These derivatives produced strong inhibitory
effects on PARP-1, and the most potent inhibitors (14b and 14c) had
IC50 value of single digit nanomolar level. These diversified struc-
ture motifs provided numerous opportunities to further develop
potent PARP-1 inhibitors with unique structure properties and
physiochemical properties. Furthermore, compound 10a demon-
strated high efficacy as a sensitizer of TMZ in MX-1 xenograft tumor
model, suggesting that these benzo-imidazole-based PARP-1 in-
hibitors could be developed into useful anti-tumor agents.
was used in combination with 10 mM concentration of compounds
10a and 11e. Of note, some compounds had remarkable enzymatic
potency, however, they could not potentiate the cytotoxicity of TMZ
in MX-1 cells, e.g. compounds 12b, 14b and 14c, presumably due to
the poor permeability. Therefore, it is worthwhile to note that
improving both PARP-1 inhibitory activity and cellular potency
could be achieved by variations of the R group on the benzo-
imidazole ring.
3.3. In vivo antitumor activity of compound 10a
As compound 10a showed remarkable potentiation effects on
TMZ-induced cytotoxicity in MX-1 cells, we further tested its anti-
cancer activity as a sensitizer of TMZ using MX-1 xenograft tumor
model. The results were outlined in Table 6. Orally dosing TMZ
(50 mg/kg) to the BalB/c nude mice bearing MX-1 xenograft tumors
obviously resulted in tumor growth inhibition in 62%, while com-
pound 10a was administered at a dose of 50 mg/kg did not inhibit
the tumor growth. However, when compound 10a were used with

30
J. Zhou et al. / European Journal of Medicinal Chemistry 132 (2017) 26e41
Table 1
The chemical structures and PARP-1 inhibitory activities of compounds 7a-7c, 8a-8fa.
Compd.
R
PARP-1 IC50b(nM) SDc
Compd.
R
PARP-1 IC50b(nM) SDc
382
818 75
199 24
793 37
7a
7b
7c
8a
8b
Boc
cyclohexyl
H
Boc
iso-pentyl
1810 152
>1000
343 14
1150 87
708 10
8c
8d
8e
8f
cyclohexyl
N-Boc-piperidine-4-yl
H
8
piperdine-4-yl
a
The ability of compounds to inhibit PARP-1 enzyme activity were tested using NADþ method and ABT888 was used as a positive control. The measured IC50 for ABT-888
was132 nM.
b
c
Concentrations for 50% inhibition in PARP-1 enzyme assay (IC50).
Standard Deviation.
Table 2
The chemical structures and PARP-1 inhibitory activities of compounds 9a-9o, 10a-10ba.
Compd.
R
PARP-1 IC50b(nM) SDc
644 21
Compd.
R
PARP-1 IC50b(nM) SDc
111
302 12
311 24
868 45
316 23
101 11
9a
9b
9c
9d
9e
9f
9g
9h
9i
Boc
ethyl
9j
9k
9l
9m
9n
9o
10a
10b
iso-pentyl
pentan-3-yl
cyclohexyl
N-Boc-piperidine-4-yl
piperidine-4-yl
H
neo-pentyl
pentan-3-yl
7
40
34
83
49
64
92
5
4
7
5
4
8
trifluoroethyl
trifluoroacetyl
propyl
iso-propyl
iso-butyl
17
30
3
5
cyclopropylmethyl
neo-pentyl
108 11
41
5
a
The ability of compounds to inhibit PARP-1 enzyme activity were tested using ELISA method and ABT888 or AZD2281 was used as a positive control. The measured IC50 for
ABT-888 was 5.18 nM, and IC50 for AZD-2281 was 2.09 nM.
b
Concentrations for 50% inhibition in PARP-1 enzyme assay (IC50).
Standard Deviation.
c
6. Experimental section
was recorded in DMSO-d6 or CDCl3. Chemical shifts are reported in
(ppm) units relative to the internal standard tetramethylsilane
d
6.1. Chemical synthesis general
(TMS). High resolution mass spectra (HRMS) were obtained on an
Agilent Technologies LC/MSD TOF spectrometer. All chemicals and
solvents used were of reagent grade without purified or dried
before use. All the reactions were monitored by thin-layer chro-
matography (TLC) on pre-coated silica gel G plates at 254 nm under
a UV lamp. Column chromatography separations were performed
with silica gel (200e300 mesh).
Unless otherwise noted, all materials were obtained from
commercial suppliers and used without further purification. All
reactions involving air- or moisture-sensitive reagents were per-
formed under an argon atmosphere. Melting points were measured
on a Yanaco micro melting point apparatus and are uncorrected. 1H
NMR (300 MHz and 400 MHz) on a Varian Mercury spectrometer

J. Zhou et al. / European Journal of Medicinal Chemistry 132 (2017) 26e41
31
Table 3
The chemical structures and PARP-1 inhibitory activities of compounds 11a-11g, 12a-12c, 13a-13e, 14a-14ca.
Compd.
X
R
PARP-1 IC50b(nM) SDc
857 99
Compd.
X
R
PARP-1 IC50b(nM) SDc
37
11a
11b
11c
11d
11e
11f
11g
12a
12b
H
H
H
H
H
H
H
F
Boc
H
propyl
propionyl
cyclohexyl
N-Boc-piperidine-4-yl
piperidine-4-yl
Boc
H
12c
13a
13b
13c
13d
13e
14a
14b
14c
F
cyclohexyl
Boc
H
cyclohexyl
N-Boc-piperidine-4-yl
piperidine-4-yl
Boc
3
15
79
83
75
3
8
6
7
H
H
H
H
H
F
968 45
29
70
3
6
250 27
316 13
80 14
2.76 0.47
7.54 0.85
1190 68
60
5
>100
12 3.46
F
F
H
F
cyclohexyl
a
The ability of compounds to inhibit PARP-1 enzyme activity were tested using ELISA method and ABT888 or AZD2281 was used as a positive control. IC50 for ABT-888 was
5.18 nM and for AZD-2281 was 2.09 nM.
b
Concentrations for 50% inhibition in PARP-1 enzyme assay (IC50).
Standard Deviation.
c
6.2. Synthesis of compounds
(m, 5H), 2.26 (brs, 1H), 1.95 (brs, 2H), 1.72 (brs, 2H), 1.56 (d,
J ¼ 9.3 Hz, 1H), 1.16e1.40 (m, 6H); 13C NMR (100 MHz, DMSO- d6)
6.2.1. tert-Butyl 3-(4-carbamoyl-1H-benzo[d]imidazole-2-
d (ppm): 165.60, 158.07, 145.25, 139.98, 134.78, 124.02, 123.94,
carboxamido)pyrrolidine-1-carboxylate (7a)
116.13, 62.77, 54.85, 49.48, 47.32, 29.62, 28.63, 24.78, 24.10; HRMS
(ESI): m/z, calcd. For C19H26N5O2 [M þ H]þ 356.2081, found
356.2075.
A
mixture
of
2-carboxyl-1H-benzo[d]immidazole-4-
carboxamide (75 mg, 0.37 mmol), EDC (210 mg, 1.09 mmol), HOBt
(147 mg, 1.09 mmol), Et3N (0.4 mL, 2.93 mmol) and N-Boc-ami-
nopyrrolidine (202 mg, 1.09 mmol) in DMF (5 mL) was stirred at
room temperature for 20 h and then H2O was added to the mixture.
The solution was extracted with the solvent mixture (ethyl acetate/
methanol ¼ 10:1) (30 mL ꢂ 3) and then the organic layer was
washed with brine (20 mL ꢂ 2). The combined organic layer was
dried over anhydrous MgSO4. After filtration and concentration, the
crude product was obtained and purified with column chroma-
tography (methylene chloride/methanol ¼ 50:1 to 40:1) to give
compound 7a as a white solid (74 mg, 54.4%); mp 231e233 ꢁC;
Compound 7a exists as a mixture of two conformers; 1H NMR
6.2.3. N2-(Pyrrolidin-3-yl)-1H-benzo[d]imidazole-2,4-
dicarboxamide 2,2,2-trifluoroacetate (7c)
To a solution of compound 7a in DCM (5.0 mL), TFA (0.27 mL,
4.23 mmol) was added and then the reaction mixture was stirred at
room temperature for 2 h. Ether (20 mL) was added into the
mixture. After filtration, the title compound 7c was afforded as a
white solid (35 mg, 58%); mp 233e235 ꢁC; 1H NMR (300 MHz,
DMSO-d6)
d
(ppm): 13.74 (brs, 1H), 9.29 (d, J ¼ 5.7 Hz, 1H), 9.11 (s,
1H), 9.00 (brs, 2H), 7.94 (d, J ¼ 7.5 Hz, 1H), 7.88 (brs, 1H), 7.72 (d,
J ¼ 7.5 Hz, 1H), 7.43 (t, J ¼ 7.8 Hz, 1H), 4.55e4.70 (m, 1H), 3.23e3.52
(m, 4H), 2.24e2.29 (m, 1H), 2.09e2.14 (m, 1H); HRMS (ESI): m/z,
calcd. For C13H16N5O2 [M þ H]þ 274.1299, found 274.1293.
(300 MHz, DMSO-d6)
d
(ppm): 13.68 (s, 1H), 9.27 (d, J ¼ 7.2 Hz, 1H),
9.16 (brs, 1H), 7.93 (d, J ¼ 7.5 Hz, 1H), 7.83 (brs, 1H), 7.70 (d,
J ¼ 8.1 Hz, 1H), 7.42 (t, J ¼ 7.5 Hz, 1H), 4.50e4.60 (m, 1H), 3.55e3.65
(m, 1H), 3.38e3.47 (m, 2H), 3.24e3.31 (m,1H), 2.12e2.23 (m, 1H),
1.94e2.08 (m, 1H), 1.40 (s, 9H); 13C NMR (100 MHz, DMSO-d6)
6.2.4. tert-Butyl 3-(4-carbamoyl-1H-benzo[d]imidazole-2-
carboxamido)piperidine-1-carboxylate (8a)
d
(ppm): 165.72, 158.30, 153.48, 145.43, 139.85, 134.89, 123.96,
Compound 8a was obtained as a white solid (90 mg, 15.9%); mp
226e228 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d (ppm): 13.70 (s, 1H),
123.86, 123.56, 116.39, 78.43, 50.14 (49.91), 49.09 (48.42), 44.26
(44.01), 30.83 (30.01), 28.17; HRMS (ESI): m/z, calcd. For
9.15 (s, 1H), 9.04 (d, J ¼ 6.8 Hz, 1H), 7.93 (d, J ¼ 6.8 Hz, 1H), 7.85 (s,
1H), 7.71 (d, J ¼ 7.6 Hz, 1H), 7.43 (t, J ¼ 7.2 Hz, 1H), 4.10 (brs, 1H),
3.80e3.88 (m, 2H), 2.72e2.82 (m, 1H), 1.92e1.98 (m, 1H), 1.68e1.78
C
18H24N5O4 [MþH]þ 374.1823, found 374.1812.
This method was used to prepare compounds 7b, 8a-8d, 9a-9m,
(m, 2H),1.39e1.50 (m,11H); 13C NMR (100 MHz, DMSO-d6)
d (ppm):
11a, 13a, 15a-15d.
166.18, 158.23, 154.35, 145.98, 140.27, 135.35, 124.41, 124.28, 123.99,
116.83, 79.30, 47.80, 46.43, 43.65, 30.06, 28.49, 24.34; HRMS (ESI):
m/z, calcd. For C19H26N5O4 [M þ H]þ 388.1979, found 388.1969.
6.2.2. N2-(1-cyclohexylpyrrolidin-3-yl)-1H-benzo[d]imidazole-2,4-
dicarboxamide (7b)
Compound 7b was obtained as a white solid (70 mg, 33%); mp
229e232 ꢁC; 1H NMR (300 MHz, DMSO- d6)
d
(ppm): 13.68 (brs,
6.2.5. N2-(1-iso-Pentylpiperidin-3-yl)-1H-benzo[d]imidazole-2,4-
dicarboxamide (8b)
1H), 9.33 (s, 1H), 9.15 (s, 1H), 7.93 (d, J ¼ 6.9 Hz,1H), 7.79 (s, 1H), 7.71
(d, J ¼ 7.2 Hz, 1H), 7.42 (t, J ¼ 7.5 Hz, 1H), 4.57 (brs, 1H), 2.80e3.50
Compound 8b was obtained as a white solid (75 mg, 33.6%); mp

32
J. Zhou et al. / European Journal of Medicinal Chemistry 132 (2017) 26e41
Table 4
The chemical structures and PARP-1 inhibitory activities of compounds 15a-15k, 16a, 16ba.
Compd.
R
PARP-1 IC50b(nM) SDc
152 11
Compd.
R
PARP-1 IC50b(nM) SDc
15a
15h
>100
15b
100 13
15i
116 10
15c
15d
>100
15j
29
2
81
7
15k
>100
15e
42
19
3
2
16a
16b
H
15
5
(S)-15f
pentan-3-yl
88 31
(R)-15g
>100
a
The ability of compounds to inhibit PARP-1 enzyme activity were tested using ELISA method and ABT888 or AZD2281 was used as a positive control. The measured IC50 for
ABT-888 was 5.18 nM, IC50 for AZD-2281 was 2.09 nM.
b
Concentrations for 50% inhibition in PARP-1 enzyme assay (IC50).
Standard Deviation.
c
216e218 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 13.65 (brs, 1H),
1H), 9.12 (s, 1H), 8.90 (d, J ¼ 8.4 Hz, 1H), 7.93 (d, J ¼ 7.6 Hz, 1H), 7.82
(s, 1H), 7.69 (d, J ¼ 8.0 Hz, 1H), 7.41 (t, J ¼ 8.0 Hz, 1H), 3.97 (brs, 3H),
2.86 (d, J ¼ 9.6 Hz, 1H), 2.63e2.72 (m, 3H), 2.45 (brs, 1H), 2.27 (t,
J ¼ 9.6 Hz, 1H), 2.19 (brs, 1H), 1.81 (brs, 1H), 1.66e1.69 (m, 3H),
1.48e1.50 (m, 2H), 1.37 (s, 9H), 1.27e1.31 (m, 2H); 13C NMR
9.12 (s, 1H), 8.91 (d, J ¼ 7.2 Hz, 1H), 7.93 (d, J ¼ 7.2 Hz, 1H), 7.82 (s,
1H), 7.70 (d, J ¼ 7.6 Hz, 1H), 7.41 (t, J ¼ 8.0 Hz, 1H), 3.99 (brs, 1H),
2.86 (brs, 1H), 2.71 (brs, 1H), 2.32 (brs, 2H), 2.03 (brs, 1H), 1.93 (brs,
1H), 1.83 (brs, 1H), 1.68 (brs, 1H), 1.49e1.60 (m, 3H), 1.31e1.33 (m,
2H), 0.86 (d, J ¼ 6.4 Hz, 6H); HRMS (ESI): m/z, calcd. For C19H28N5O2
[M þ H]þ 358.2238, found 358.2231.
(100 MHz, DMSO-d6) d (ppm): 165.59, 157.53, 153.67, 145.47, 140.02,
134.70, 123.96, 123.87, 123.71, 116.14, 78.71, 61.73, 48.38, 42.57,
28.03, 26.96; HRMS (ESI): m/z, calcd. For C24H35N6O4 [M þ H]þ
471.2714, found 471.2708.
6.2.6. N2-(1-cyclohexylpiperidin-3-yl)-1H-benzo[d]imidazole-2,4-
dicarboxamide (8c)
Compound 8c was obtained as a white solid (90 mg, 35.8%); mp
247e248 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d (ppm): 13.65 (brs, 1H),
6.2.8. N2-(Piperidin-3-yl)-1H-benzo[d]imidazole-2,4-
9.11 (s, 1H), 8.88 (d, J ¼ 6.8 Hz, 1H), 7.93 (d, J ¼ 7.2 Hz, 1H), 7.82 (brs,
1H), 7.70 (d, J ¼ 7.6 Hz, 1H), 7.41 (t, J ¼ 7.6 Hz, 1H), 3.97 (brs, 1H),
2.84 (brs, 1H), 2.69 (brs, 1H), 2.22e2.31 (m, 3H), 1.71e1.80 (m, 6H),
1.48e1.56 (m, 3H), 1.13e1.28 (m, 4H), 0.99e1.08 (m, 1H); 13C NMR
dicarboxamide 2,2,2-trifluoroacetate (8e)
The title compound was synthesized from 8a using the same
method as the preparation of compound 7c: Compound 8e was
obtained as a white solid (48 mg, 71.6%); mp 156e158 ꢁC; 1H NMR
(100 MHz, DMSO-d6) d (ppm): 165.61,157.53,145.50,140.00,134.72,
(400 MHz, DMSO-d6)
d
(ppm): 13.72 (brs, 1H), 9.18 (d, J ¼ 8.0 Hz,
123.94, 123.82, 116.13, 63.69, 52.46, 48.41, 45.53, 29.25, 27.31, 25.40,
25.12; HRMS (ESI): m/z, calcd. For C20H28N5O2 [M þ H]þ 370.2238,
found 370.2232.
1H), 9.08 (brs, 1H), 8.88 (brs, 1H), 8.72 (brs, 1H), 7.94 (d, J ¼ 7.6 Hz,
1H), 7.87 (s, 1H), 7.72 (d, J ¼ 8.0 Hz, 1H), 7.43 (d, J ¼ 8.0 Hz, 1H), 4.25
(brs, 1H), 3.37e3.39 (m, 1H), 3.26 (d, J ¼ 11.6 Hz, 1H), 2.94 (q,
J ¼ 10.4 Hz,1H), 2.75e2.89 (m,1H),1.90e1.96 (m, 2H),1.68e1.74 (m,
6.2.7. tert-Butyl 3-(4-carbamoyl-1H-benzo[d]imidazole-2-
carboxamido)-1,4’-bipiperidine-1’- carboxylate (8d)
2H); 13C NMR (100 MHz, DMSO-d6)
d (ppm): 165.69, 157.84, 145.24,
139.66, 135.14, 124.02, 123.92, 123.49, 116.52, 45.90, 43.38, 42.91,
28.07, 20.78; HRMS (ESI): m/z, calcd. For C14H18N5O2 [M þ H]þ
288.1455, found 288.1451.
Compound 8d was obtained as a white solid (200 mg, 43.6%);
mp 238e240 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d (ppm): 13.65 (s,

J. Zhou et al. / European Journal of Medicinal Chemistry 132 (2017) 26e41
33
Table 5
The enzymatic activities against PARP-1 and PARP-2 and potentiation effects on TMZ in MX-1 cells of selected compounds.
PARP-1 IC50a(nM) SDc
34
PARP-2 IC50a(nM) SDc
PF50
b
Compd
X
R
9c
H
4
100
9
0.93
9h
H
108 11
101 11
130
9
3.53
9o
H
F
ND
74
1.75
7.10
10a
17
3
6
11b
11c
11e
H
H
H
15
79
75
3
8
7
48
75
91
3
5
2
1.98
1.54
4.17
12b
12c
F
F
12 3.46
19
38
1
4
1.07
1.71
37
3
13b
14b
14c
H
F
29
3
73
24
34
3
2
3
1.99
0.98
0.71
2.76 0.47
7.54 0.85
F
a
The ability of compounds to inhibit PARP-1 and PARP-2 enzyme activity were tested using ELISA method and AZD2281 was used as a positive control. The measured IC50
against PARP-1 was 2.09 nM, the measured IC50 against PARP-2 was 2.26 nM.
b
Potentiation factor (PF50): The fold of potentiation was calculated as the ratio of the IC50 for TMZ divided by the IC50 of TMZ þ PARP-1 inhibitor, the test compounds were
used at a fixed concentration of 10
m
M.
c
Standard Deviation.
Table 6
In vivo tumor growth inhibition of compound 10a.
Group
Number start/end
Tumor weights(g SE)
%T/Cveh(%TGI)
Weight change(g)
%T/CTMZ(%TGI)
Vehicle
6/6
6/6
6/6
6/6
6/6
4.09 0.64
1.57 0.64
1.47 0.68
0.50 0.10
3.73 0.64
NA
5.32
0.02
0.23
ꢃ0.79
5
NA
NA
94(6)
32(68)
NA
TMZ(50 mg/kg)
TMZþ10a (25 mg/kg)
TMZþ10a (50 mg/kg)
10a (50 mg/kg)
38 (62)***
36 (64)***
12 (88)***
91 (9)
NA: Not Applicable, ANOVA, ***p < 0.001 vs Vehicle.
6.2.9. N2-(1,40-Bipiperidine-3-yl) 1H-benzo[d]imidazole-2,4-
dicarboxmide 2,2,2-trifluoroacetate (8f)
The title compound was synthesized from 8d using the same
method as the preparation of compound 7c: Compound 8f was
obtained as a white solid (100 mg, 80%); mp 171e173 ꢁC; 1H NMR
1H), 4.33 (brs, 1H), 3.47 (brs, 7H), 2.92 (brs, 3H), 2.22 (brs, 2H), 2.00
(brs, 2H),1.60e1.90 (m, 3H); HRMS (ESI): m/z, calcd. For C19H27N6O2
[M þ H]þ 371.2190, found 371.2183.
6.2.10. tert-Butyl 4-(4-carbamoyl-1H-benzo[d]imidazole-2-
carboxamido)piperidine-1-carboxylate (9a)
(400 MHz, DMSO-d6)
d (ppm): 13.68 (brs, 1H), 10.20 (brs, 1H), 9.26
(brs, 1H), 9.10 (brs, 1H), 8.92 (brs, 1H), 8.61 (brs, 1H), 7.95 (d,
J ¼ 7.2 Hz, 1H), 7.88 (s, 1H), 7.71 (d, J ¼ 7.2 Hz, 1H), 7.44 (t, J ¼ 7.6 Hz,
Compound 9a was obtained as a white solid (130 mg, 46%); mp.
251e253 ꢁC; 1H NMR (300 MHz, DMSO- d6)
d (ppm): 13.62 (s, 1H),

34
J. Zhou et al. / European Journal of Medicinal Chemistry 132 (2017) 26e41
Fig. 1. Co-crystal structure of PARP-1 in complex with 11b. Protein and ligand structures are colored in tan and sky blue, respectively. Hydrogen bonds are represented with cyan
lines. The 2FoeFc electron density map (contoured at 1 ) around the inhibitor is shown as pink mesh (PDB: 5WS1). (For interpretation of the references to colour in this figure
s
legend, the reader is referred to the web version of this article.)
Fig. 2. Co-crystal structure of PARP-1 in complex with 15e. Protein and ligand structures are colored in tan and sky blue, respectively. Hydrogen bonds are represented with cyan
lines. The 2FoeFc electron density map (contoured at 1s) around the inhibitor is shown as pink mesh (PDB: 5WS0). (For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this article.)
8.81 (s, 1H), 7.92 (d, J ¼ 6.9 Hz, 1H), 7.81 (s, 1H), 7.72 (d, J ¼ 7.8 Hz,
1H), 7.43 (t, J ¼ 8.1 Hz, 1H), 6.94 (d, J ¼ 7.5 Hz, 1H), 4.85 (d,
J ¼ 12.3 Hz, 1H), 4.38 (d, J ¼ 12.9 Hz, 1H), 3.61 (brs, 1H), 3.43 (t,
J ¼ 12.3 Hz,1H), 3.05 (t, J ¼ 12.6 Hz,1H),1.83 (brs, 2H),1.38e1.48 (m,
11H); HRMS (ESI): m/z, calcd. For C19H25N5O4 [M þ H]þ 388.1979,
found 388.1971.
1H), 7.71 (d, J ¼ 8.0 Hz, 1H), 7.42 (t, J ¼ 8.0 Hz, 1H), 3.87 (brs, 1H),
3.19 (q, J ¼ 10.0 Hz, 2H), 2.98 (d, J ¼ 11.2 Hz, 2H), 2.47 (t, J ¼ 10.4 Hz,
2H), 1.74e1.79 (m, 4H); 13C NMR (400 MHz, DMSO-d6)
d (ppm):
165.64, 157.42, 145.74, 140.07, 134.67, 126.08 (q, JCF ¼ 278.5), 123.89,
123.76, 123.68, 116.08, 56.67 (q, JCF ¼ 29.0), 52.82, 46.19, 31.18;
HRMS (ESI): m/z, calcd. For C16H19N5O2F3 [M þ H]þ 370.1485, found
370.1480.
6.2.11. N2-(1-Ethylpiperidin-4-yl)-1H-benzo[d]imidazole-2,4-
dicarboxamide (9b)
6.2.13. N2-(1-(2,2,2,-Trifluoroacetyl) piperidin-4-yl)-1H-benzo[d]
imidazole-2,4-dicarboxamide (9d)
Compound 9b was obtained as a white solid (70 mg, 30.4%); mp
272e274 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 13.60 (brs, 1H),
Compound 9d was obtained as a white solid (50 mg, 17.9%);
9.14 (brs, 1H), 9.07 (d, J ¼ 8.0 Hz, 1H), 7.92 (d, J ¼ 7.2 Hz, 1H), 7.80 (s,
1H), 7.70 (d, J ¼ 8.0 Hz, 1H), 7.40 (t, J ¼ 7.6 Hz, 1H), 3.85 (brs, 1H),
2.98 (d, J ¼ 10.8 Hz, 2H), 2.42 (q, J ¼ 10.8 Hz, 2H), 2.09 (t, J ¼ 10.8 Hz,
2H), 1.65e1.85 (m, 4H), 1.02 (t, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz,
mp > 300 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d (ppm): 13.70 (s, 1H),
9.13 (s, 2H), 9.09 (d, J ¼ 8.0 Hz, 1H), 7.92 (d, J ¼ 8.0 Hz, 1H), 7.84 (s,
1H), 7.69 (d, J ¼ 8.0 Hz,1H), 7.41 (t, J ¼ 8.0 Hz,1H), 4.36 (d, J ¼ 9.2 Hz,
1H), 4.20e4.29 (m, 1H), 3.92 (d, J ¼ 9.2 Hz, 1H), 3.41 (t, J ¼ 9.2 Hz,
1H), 3.08 (t, J ¼ 9.2 Hz,1H),1.99 (t, J ¼ 9.2 Hz, 2H),1.51e1.52 (m, 2H);
DMSO-d6)
d (ppm): 165.71, 157.31, 145.62, 139.93, 134.83, 123.88,
123.76, 116.28, 50.95, 50.72, 45.70, 29.46, 10.36; HRMS (ESI): m/z,
13C NMR (100 MHz, DMSO-d6)
d (ppm): 165.74, 157.48, 154.09 (q,
calcd. For C16H22N5O2 [M þ H]þ 316.1768, found 316.1764.
JCF ¼ 34.6), 145.58, 140.04, 134.77, 123.97, 123.84, 123.60, 116.47 (q,
JCF ¼ 286.8), 116.19, 45.71, 44.44, 42.46, 31.68, 30.77; HRMS (ESI): m/
z, calcd. For C16H17N5O3F3 [M þ H]þ 384.1278, found 384.1276.
6.2.12. N2-(1-(2,2,2,-Trifluoroethyl) piperidin-4-yl)-1H-benzo[d]
imidazole-2,4-dicarboxamide (9c)
Compound 9c was obtained as a white solid (49 mg, 26.5%); mp
6.2.14. N2-(1-Propylpiperidin-4-yl)-1H-benzo[d]imidazole-2,4-
dicarboxamide (9e)
276e278 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 13.65 (s, 1H),
9.17 (s, 1H), 9.05 (d, J ¼ 7.6 Hz, 1H), 7.95 (d, J ¼ 7.6 Hz, 1H), 7.83 (s,
Compound 9e was obtained as a white solid (70 mg, 29.2%); mp

J. Zhou et al. / European Journal of Medicinal Chemistry 132 (2017) 26e41
35
259e261 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 13.67 (s, 1H),
6.2.20. N2-(1-(Pentan-3-yl) piperidin-4-yl)-1H-benzo[d]imidazole-
9.17 (brs, 2H), 7.93 (d, J ¼ 7.2 Hz, 1H), 7.82 (s, 1H), 7.68 (d, J ¼ 7.2 Hz,
1H), 7.41 (t, J ¼ 8.0 Hz, 1H), 4.00e4.09 (m, 2H), 2.70e3.40 (m, 5H),
1.96 (brs, 4H), 1.64 (brs, 2H), 0.88 (t, J ¼ 7.2 Hz, 3H); 13C NMR
2,4-dicarboxamide (9k)
Compound 9k was obtained as a white solid (60 mg, 28.7%); mp
240e242 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 13.62 (s, 1H),
(100 MHz, DMSO-d6)
d
(ppm): 165.66,157.89,145.44,139.73,134.97,
9.16 (s, 1H), 9.06 (brs, 1H), 7.92 (d, J ¼ 7.6 Hz, 1H), 7.78 (s, 1H), 7.68
(d, J ¼ 7.6 Hz, 1H), 7.40 (t, J ¼ 7.6 Hz, 1H), 3.82 (brs, 1H), 2.72 (brs,
2H), 2.34 (brs, 2H), 2.18 (brs, 1H), 1.78 (brs, 2H), 1.67 (brs, 2H), 1.44
(brs, 2H), 1.25 (brs, 2H), 0.87 (s, 6H); HRMS (ESI): m/z, calcd. For
123.90, 123.82, 123.51, 116.42, 57.33, 50.62, 44.92, 28.43, 16.70,
11.01; HRMS (ESI): m/z, calcd. For C17H24N5O2 [M þ H]þ 330.1925,
found 330.1921.
C
19H28N5O2 [M þ H]þ 358.2238, found 358.2232.
6.2.15. N2-(1-iso-Propylpiperidin-4-yl)-1H-benzo[d]imidazole-2,4-
dicarboxamide (9f)
6.2.21. N2-(1-cyclohexylpiperidin-4-yl)-1H-benzo[d]imidazole-2,4-
dicarboxamide (9l)
Compound 9f was obtained as a white solid (80 mg, 29.3%); mp
295e297 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 13.70 (s, 1H),
Compound 9l was obtained as a white solid (90 mg, 29.4%); mp
9.36 (brs, 1H), 9.20 (s, 1H), 7.93 (d, J ¼ 7.6 Hz, 1H), 7.80 (s, 1H), 7.70
(d, J ¼ 8.0 Hz, 1H), 7.41 (t, J ¼ 8.0 Hz, 1H), 4.15 (brs, 1H), 3.30e3.50
(m, 3H), 3.05e3.23 (m, 2H), 2.10e2.24 (m, 2H), 2.01e2.09 (m, 2H),
1.28 (d, J ¼ 6.4 Hz, 6H); HRMS (ESI): m/z, calcd. For C17H24N5O24
[M þ H]þ330.1925, found 330.1918.
271e273 ꢁC; 1H NMR (400 MHz, DMSO- d6)
d (ppm): 13.61 (brs,1H),
9.14 (brs, 1H), 9.03 (d, J ¼ 8.4 Hz, 1H), 7.92 (d, J ¼ 7.6 Hz, 1H), 7.79 (s,
1H), 7.69 (d, J ¼ 8.0 Hz, 1H), 7.40 (t, J ¼ 7.6 Hz, 1H), 3.81 (brs, 1H),
2.89 (d, J ¼ 9.6 Hz, 2H), 2.33 (bs, 3H), 1.67e1.82 (m, 8H), 1.56 (d,
J ¼ 9.6 Hz, 1H), 1.06e1.19 (m, 5H); HRMS (ESI): m/z, calcd. For
C
20H28N5O2 [M þ H]þ 370.2238, found 370.2233.
6.2.16. N2-(1-iso-Butylpiperidin-4-yl)-1H-benzo[d]imidazole-2,4-
dicarboxamide (9g)
6.2.22. tert-Butyl 4-(4-carbamoyl-1H-benzo[d]imidazole-2-
carboxamido)1,4’-bipiperidine-1’-carboxylate (9m)
Compound 9g was obtained as a white solid (40 mg, 24.3%); mp
266e268 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d
(ppm): 13.63 (s, 1H),
Compound 9m was obtained as a white solid (70 mg, 23.8%); mp
9.15 (s, 1H), 9.04 (d, J ¼ 6.8 Hz, 1H), 7.92 (d, J ¼ 7.6 Hz, 1H), 7.79 (s,
1H), 7.68 (d, J ¼ 8.0 Hz, 1H), 7.40 (t, J ¼ 7.6 Hz, 1H), 3.85 (brs, 1H),
2.86 (brs, 2H), 2.04 (brs, 2H), 1.94 (brs, 2H), 1.77 (brs, 5H), 0.85 (d,
239e242 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d (ppm): 13.62 (s, 1H),
9.15 (s, 1H), 9.03 (d, J ¼ 8.4 Hz, 1H), 7.92 (d, J ¼ 7.6 Hz, 1H), 7.79 (s,
1H), 7.68 (d, J ¼ 7.6 Hz, 1H), 7.40 (t, J ¼ 7.6 Hz, 1H), 3.95 (d,
J ¼ 11.2 Hz, 2H), 3.82 (brs, 1H), 2.89 (d, J ¼ 10.0 Hz, 2H), 2.69 (brs,
2H), 2.44e2.49 (m, 1H), 2.24 (t, J ¼ 10.4 Hz, 2H), 1.79e1.82 (m, 2H),
1.60e1.71 (m, 4H), 1.38 (s, 9H), 1.22e1.31 (m, 2H); 13C NMR
J ¼ 6.4 Hz, 6H); 13C NMR (100 MHz, DMSO-d6)
d (ppm): 165.65,
157.704, 145.63, 140.09, 134.68, 123.92, 123.84, 123.72, 116.13, 52.04,
45.80, 29.03, 24.16, 20.75; HRMS (ESI): m/z, calcd. For C18H26N5O2
[M þ H]þ 344.2081, found 344.2078.
(100 MHz, DMSO-d6)
d (ppm): 165.62, 157.69, 153.68, 145.63,
140.08, 134.66, 123.88, 116.09, 78.77, 61.56, 47.71, 29.94, 28.04,
26.83; HRMS (ESI): m/z, calcd. For C24H35N6O4 [M þ H]þ 471.2714,
found 471.2712.
6.2.17. N2-(1-(cyclopropylmethyl) piperidin-4-yl)-1H-benzo[d]
imidazole-2,4-dicarboxamide (9h)
Compound 9h was obtained as a white solid (70 mg, 35.1%); mp
262e264 ꢁC; 1H NMR (300 MHz, DMSO-d6)
d
(ppm): 13.63 (brs,1H),
6.2.23. N2-(1,40-Bipiperidine-4-yl) 1H-benzo[d]imidazole-2,4-
dicarboxmide 2,2,2-trifluoroacetate (9n)
The title compound was synthesized from 9m using the same
method as the preparation of compound 7c: Compound 9n was
obtained as a white solid (137 mg, 82%); mp 206e208 ꢁC; 1H NMR
9.13 (brs, 2H), 7.94 (d, J ¼ 7.5 Hz, 1H), 7.82 (s, 1H), 7.71 (d, J ¼ 7.5 Hz,
1H), 7.42 (t, J ¼ 7.5 Hz, 1H), 4.36 (brs, 1H), 3.15 (brs, 3H), 2.37 (brs,
3H), 1.86 (brs, 4H), 0.90 (brs, 1H), 0.51 (d, J ¼ 6.0 Hz, 2H), 0.16 (brs,
2H); 13C NMR (100 MHz, DMSO-d6)
d (ppm): 165.67, 157.90, 145.46,
139.75, 134.80, 123.90, 123.82, 123.51, 116.43, 60.00, 50.38, 44.97,
28.44, 5.29, 4.17; HRMS (ESI): m/z, calcd. For C18H24N5O2 [M þ H]þ
342.1925, found 342.1920.
(400 MHz, DMSO-d6)
d
(ppm): 10.16 (brs, 1H), 9.30 (d, J ¼ 7.6 Hz,
1H), 9.10 (brs, 1H), 8.95 (brs, 1H), 8.69 (brs, 1H), 7.94 (d, J ¼ 7.2 Hz,
1H), 7.84 (s, 1H), 7.72 (d, J ¼ 8.0 Hz, 1H), 7.42 (t, J ¼ 8.0 Hz, 1H),
4.10e4.17 (m, 2H), 3.46e3.55 (m, 4H), 3.16e3.21 (m, 2H), 2.95 (q,
J ¼ 11.2 Hz, 2H), 2.11e2.24 (m, 4H), 1.97 (q, J ¼ 12.4 Hz, 2H), 1.85 (q,
J ¼ 11.2 Hz, 2H); HRMS (ESI): m/z, calcd. For C19H27N6O2 [M þ H]þ
371.2190, found 371.2181.
6.2.18. N2-(1-neo-Pentylpiperidin-4-yl)-1H-benzo[d]imidazole-2,4-
dicarboxamide (9i)
Compound 9i was obtained as a white solid (100 mg, 44.4%); mp
264e266 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d (ppm): 13.64 (s, 1H),
9.17 (s, 1H), 9.04 (d, J ¼ 7.6 Hz, 1H), 7.94 (d, J ¼ 7.2 Hz, 1H), 7.80 (s,
1H), 7.70 (d, J ¼ 8.0 Hz, 1H), 7.42 (t, J ¼ 7.6 Hz, 1H), 3.86 (brs, 1H),
2.85 (brs, 2H), 2.34 (brs, 2H), 2.10 (brs, 2H), 1.78 (brs, 4H), 0.88 (s,
9H); HRMS (ESI): m/z, calcd. For C19H28N5O2 [M þ H]þ 358.2238,
found 358.2226.
6.2.24. N2-(Piperidin-4-yl)-1H-benzo[d]imidazole-2,4-
dicarboxamide 2,2,2-trifluoroacetate (9o)
The title compound was synthesized from 9a using the same
method as the preparation of compound 7c: Compound 9o was
obtained as a white solid (50 mg, 56.8%); mp 252e254; 1H NMR
(300 MHz, DMSO-d6)
d
(ppm): 13.69 (s, 1H), 9.24 (d, J ¼ 7.5 Hz, 1H),
6.2.19. N2-(1-iso-Pentylpiperidin-4-yl)-1H-benzo[d]imidazole-2,4-
dicarboxamide (9j)
9.16 (s, 1H), 8.85 (brs, 1H), 8.40 (brs, 1H), 7.93 (d, J ¼ 7.2 Hz, 1H), 7.84
(brs, 1H), 7.70 (d, J ¼ 7.5 Hz, 1H), 7.42 (t, J ¼ 7.8 Hz, 1H), 4.08e4.20
(m, 1H), 3.34e3.44 (m, 2H), 2.98e3.10 (m, 2H), 1.99e2.18 (m, 2H),
1.73e1.95 (m, 2H); HRMS (ESI): m/z, calcd. For C14H18N5O2 [M þ H]þ
288.1455, found 288.1451.
Compound 9j was obtained as a white solid (70 mg, 23.8%); mp
264e266 ꢁC; 1H NMR (400 MHz, DMSO- d6)
d (ppm): 13.65 (brs,
1H), 9.14 (brs, 1H), 9.03 (d, J ¼ 8.0 Hz, 1H), 7.92 (d, J ¼ 7.6 Hz, 1H),
7.79 (s, 1H), 7.69 (d, J ¼ 8.0 Hz, 1H), 7.40 (t, J ¼ 8.0 Hz, 1H), 3.83 (brs,
1H), 2.91 (d, J ¼ 10.0 Hz, 2H), 2.29 (brs, 2H), 1.97 (brs, 2H), 1.69e1.78
(m, 4H), 1.53e1.60 (m,1H), 1.30e1.34 (m, 2H), 0.86 (d, J ¼ 6.4 Hz,
6.2.25. 6-Fluoro-N2-(1-neo-pentylpiperidin-4-yl)-1H-benzo[d]
imidazole-2,4-dicarboxamide (10a)
A mixture of 6-fluoro-2-carboxyl-1H-benzo[d]immidazole-4-
carboxamide (113 mg, 0.50 mmol), HBTU (379 mg, 1.0 mmol),
HOBt (135 mg, 1.0 mmol), DIEA (0.35 mL, 2.0 mmol) and 1-
neopentylpiperidin-4-amine (170 mg, 1.0 mmol) in DMF (10 mL)
6H); 13C NMR (100 MHz, DMSO- d6)
d
(ppm): 165.64, 157.47, 145.69,
123.83,123.65, 55.54, 52.04, 46.47, 34.95, 30.74, 25.82, 22.50; HRMS
(ESI): m/z, calcd. For C19H28N5O2 [M þ H]þ 358.2238, found
358.2231.

36
J. Zhou et al. / European Journal of Medicinal Chemistry 132 (2017) 26e41
was stirred at room temperature for 20 h and then H2O was added
to the mixture. The solution was extracted with the solvent mixture
(ethyl acetate/methanol ¼ 10:1) (30 mL ꢂ 3) and then the organic
layer was washed with brine (20 mL ꢂ 2). The combined organic
layer was dried over anhydrous MgSO4. After filtration and con-
centration, the crude product was obtained and purified with col-
umn chromatography (methylene chloride/methanol ¼ 60:1 to
20:1) to give compound 10a as a white solid (70 mg, 37.2%); mp
to 5:1) to give compound 11c as a white solid (23 mg, 56.1%); mp
242e244 ꢁC; Compound 11c exists as a mixture of two conformers;
1H NMR (300 MHz, DMSO-d6)
d (ppm): 8.82 (brs, 1H), 7.94 (d,
J ¼ 6.9 Hz, 1H), 7.84 (d, J ¼ 7.5 Hz, 1H), 7.76 (d, J ¼ 7.8 Hz, 1H), 7.44 (t,
J ¼ 7.5 Hz, 1H), 4.18e4.38 (m, 2H), 3.67e3.84 (m, 3H), 2.66e2.80 (m,
2H), 1.93e2.30 (m, 2H), 1.47e1.59 (m, 2H), 0.90 (q, J ¼ 6.6 Hz, 3H);
13C NMR (100 MHz, DMSO-d6)
d (ppm): 165.83, 157.21 (157.10),
145.87 (145.75), 124.27, 124.04, 123.95, 116.60, 57.05 (54.49), 50.58
(49.22), 47.29, 46.62 (45.11), 28.53 (25.59),19.19,10.99; HRMS (ESI):
m/z, calcd. For C16H22N5O2 [M þ H]þ 316.1768, found 316.1765.
261e263 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d (ppm): 13.76 (s, 1H),
9.15 (s, 1H), 9.03 (d, J ¼ 8.4 Hz, 1H), 7.99 (s, 1H), 7.65 (d, J ¼ 10.4 Hz,
1H), 7.42 (d, J ¼ 8.0 Hz, 1H), 3.83 (brs, 1H), 2.78 (brs, 2H), 2.30 (brs,
2H), 2.06 (brs, 2H), 1.74 (brs, 4H), 0.84 (s, 9H); HRMS (ESI): m/z,
calcd. For C19H27N5O2F [M þ H]þ 376.2143, found 376.2142.
6.2.30. 2-(3-Propionamidopyrrolidine-1-carbonyl)-1H-benzol[d]
imidazole-4-carboxamide (11d)
To a solution of 11b (65 mg, 0.13 mmol) in DCM (5 mL), DIEA
(0.15 mL, 0.84 mmol) and propionyl chloride (0.02 mL, 0.2 mmol)
were added at 0 ꢁC. The reaction mixture was stirred at 0 ꢁC for 3 h
and then was evaporated. The residue was dissolved with the sol-
vent mixture (ethyl acetate/methanol ¼ 10:1) (40 mL). The organic
layer was washed with brine (15 mL ꢂ 2) and then the organic layer
dried over anhydrous MgSO4. After filtration and concentration, the
crude product was obtained and purified with column chroma-
tography (methylene chloride/methanol ¼ 30:1) to give compound
11d as a white solid (28 mg, 65%); mp 282e284 ꢁC; Compound 11d
exists as a mixture of two conformers. 1H NMR (400 MHz, DMSO-
6.2.26. 6-Fluoro-N2-(1-(pentan-3-yl) piperidin-4-yl)-1H-benzo[d]
imidazole-2,4-dicarboxamide (10b)
The title compound was synthesized from 6-fluoro-2-carboxyl-
1H-benzo[d]immidazole-4- carboxamide using the same method
as the preparation of compound 10a: Compound 10b was obtained
as a white solid (45 mg, 27.2%); mp 268e270 ꢁC; 1H NMR (400 MHz,
DMSO-d6) d (ppm): 13.80 (s, 1H), 10.02 (brs, 1H), 9.32 (brs, 1H), 9.18
(s, 1H), 8.04 (s, 1H), 7.68 (d, J ¼ 8.0 Hz, 1H), 7.46 (d, J ¼ 7.2 Hz, 1H),
4.18 (brs,1H), 3.40 (brs, 2H), 3.20 (brs, 2H), 2.98 (brs, 1H), 2.03e2.20
(m, 4H), 1.90 (brs, 2H), 1.60 (brs, 2H), 0.98 (brs, 6H); HRMS (ESI): m/
z, calcd. For C19H27N5O2F [M þ H]þ 376.2143, found 376.2142.
d6)
d (ppm): 13.63 (s, 1H), 8.94 (s, 0.5 H), 8.87 (s, 0.5H), 8.09 (t,
J ¼ 8.4 Hz, 1H), 7.93 (d, J ¼ 7.2 Hz, 1H), 7.83 (s, 1H), 7.73 (d, J ¼ 8.0 Hz,
1H), 7.44 (t, J ¼ 7.6 Hz, 1H), 4.05e4.46 (m, 3H), 3.49e3.79 (m, 2H),
2.03e2.16 (m, 3H), 1.91e2.00 (m, 0.5H), 1.79e1.90 (m, 0.5H),
6.2.27. tert-Butyl (1-(4-carbomoyl-1H-benzo[d]imidazole-2-
carbonyl)pyrrolidin-3-yl)carbamate (11a)
Compound 11a was obtained as a white solid (505 mg, 39.49%);
0.94e1.00 (m, 3H); 13C NMR (100 MHz, DMSO-d6)
d (ppm): 172.97
mp. 246e248 ꢁC; Compound 11a exists as a mixture of two con-
(172.88), 165.81, 157.19 (157.09), 146.16 (146.07), 123.95, 123.80,
116.40, 53.79 (52.58), 49.49 (46.96), 46.79 (45.35), 31.73 (28.95),
28.30, 9.81; HRMS (ESI): m/z, calcd. For C16H20N5O3 [M þ H]þ
330.1561, found 330.1556.
formers; 1H NMR (400 MHz, DMSO-d6)
d (ppm): 13.61 (s, 1H), 8.93
(s, 0.5H), 8.88 (s, 0.5H),7.93 (d, J ¼ 7.6 Hz,1H), 7.82 (d, J ¼ 7.6 Hz,1H),
7.72 (d, J ¼ 8.0 Hz, 1H), 7.44 (t, J ¼ 7.6 Hz, 1H), 7.26 (brs, 1H),
4.04e4.24 (m, 3H), 3.49e3.75 (m, 2H), 2.13e2.18 (m, 0.5 H),
2.03e2.08 (m, 0.5H), 1.93e1.95 (m, 0.5H), 1.83e1.85 (m, 0.5H), 1.38
6.2.31. 2-(3-(Cyclohexylamino)pyrrolidine-1-carbonyl)-1H-benzol
[d]imidazole-4-carboxamide (11e)
(d, J ¼ 11.2 Hz, 9H); 13C NMR (100 MHz, DMSO-d6)
d (ppm): 165.75,
157.26 (157.18), 155.32 (155.19), 146.25 (146.10), 140.23, 133.75,
124.07, 123.82, 116.15, 77.99, 53.71 (52.65), 50.82 (48.22), 46.98
(45.33), 31.65 (29.06), 28.21; HRMS (ESI): m/z, calcd. For
To a solution of 11b (100 mg, 0.20 mmol) in THF (5 mL), DIEA
and cyclohexanone were added. The reaction mixture was stirred at
room temperature for 4 d. Sodium triacetoxyborohyride (NaB-
H(OAc)3, 274 mg, 1.29 mmol) was added to the mixture and stirred
at room temperature for 2 h, then H2O (15 mL) was added to the
mixture. The solution was extracted with the solvent mixture (ethyl
acetate/methanol ¼ 10:1) (30 mL ꢂ 6) and then the organic layer
was dried over anhydrous MgSO4. After filtration and concentra-
tion, the crude product was obtained and purified with column
chromatography (methylene chloride/methanol ¼ 15:1 to 10:1) to
C
18H24N5O4 [M þ H]þ 374.1823, found 374.1816.
6.2.28. 2-(3-Aminopyrrolidine-1-carbonyl)-1H-benzol[d]
imidazole-4-carboxamide 2,2,2-trifluoroacetate (11b)
The title compound was synthesized from 11a using the same
method as the preparation of compound 7c: Compound 11b was
obtained as a white solid (415 mg, 75.3%); mp 226e228 ꢁC; 1H NMR
(400 MHz, DMSO-d6)
d
(ppm): 13.71 (brs, 1H), 8.78 (brs, 0.5H), 8.68
give compound 11e as
a
white solid (30 mg, 32.7%); mp
(ppm): 8.89 (brs, 1H),
(brs, 0.5H), 8.17 (brs, 2H), 7.93e7.97 (m, 1H), 7.75e7.88 (m, 2H), 7.45
(t, J ¼ 7.6 Hz, 1H), 4.31e4.37 (m, 2H), 3.99 (brs, 0.5H), 3.93 (brs,
0.5H), 3.71e3.83 (m, 2H), 2.31e2.40 (m, 0.5H), 2.20e2.30 (m, 0.5H),
2.11e2.19 (m, 0.5H), 2.00e2.10 (m, 0.5H); HRMS (ESI): m/z, calcd.
For C13H16N5O2 [M þ H]þ 274.1299, found 274.1294.
233e235 ꢁC;1H NMR (400 MHz, DMSO-d6)
d
7.94 (d, J ¼ 7.2 Hz, 1H), 7.86 (s, 0.5H), 7.83 (s, 0.5H), 7.75 (brs, 1H),
7.44 (t, J ¼ 7.6 Hz, 1H), 4.34e4.43 (m, 1H), 4.15e4.18 (m, 1H),
3.80e3.87 (m, 2H), 3.58e3.64 (m,1H), 3.41e3.43 (m, 1H), 2.19e2.28
(m, 2H), 1.99 (brs, 2H), 1.73 (brs, 2H), 1.58 (brs, 2H), 1.04e1.14 (m,
5H); HRMS (ESI): m/z, calcd. For C19H26N5O2 [M þ H]þ 356.2081,
found 356.2075.
6.2.29. 2-(3-(Propylamino)pyrrolidine-1-carbonyl)-1H-benzol[d]
imidazole-4-carboxamide (11c)
To a solution of 11b (65 mg, 0.13 mmol) in THF (5 mL), DIEA and
propionaldehyde (0.03 mL, 0.42 mmol) were added at ꢃ40 ꢁC. The
reaction mixture was stirred at ꢃ40 ꢁC for 30 min. Sodium tri-
acetoxyborohyride (NaBH(OAc)3, 178 mg, 0.84 mmol) was added to
the mixture and stirred at ꢃ30 ꢁC for 1.5 h, then H2O (15 mL) was
added to the mixture. The solution was extracted with the solvent
mixture (ethyl acetate/methanol ¼ 10:1) (15 mL ꢂ 6) and then the
organic layer dried over anhydrous MgSO4. After filtration and
concentration, the crude product was obtained and purified with
column chromatography (methylene chloride/methanol ¼ 12.5:1
6.2.32. tert-Butyl 4-(1-(4-carbomoyl-1H-benzo[d]imidazole-2-
carbonyl)pyrrolidin-3-ylamino)piperidine-1-carboxylate (11f)
To a solution of 11b (150 mg, 0.30 mmol) in THF (10 mL), DIEA
and N-(tert-butoxycarbonyl)-4-piperidone (386 mg, 1.94 mmol)
were added. The reaction mixture was stirred at room temperature
for
4 d. Sodium triacetoxyborohyride (NaBH(OAc)3, 419 mg,
1.94 mmol) was added to the mixture and stirred at room tem-
perature for 2 h, then H2O (15 mL) was added to the mixture. The
solution was extracted with the solvent mixture (ethyl acetate/
methanol ¼ 10:1) (30 mL ꢂ 4) and then the organic layer was dried

J. Zhou et al. / European Journal of Medicinal Chemistry 132 (2017) 26e41
37
over anhydrous MgSO4. After filtration and concentration, the
crude product was obtained and purified with column chroma-
tography (methylene chloride/methanol ¼ 15:1 to 12.5:1) to give
compound 11f as a white solid (120 mg, 87.7%); mp 209e211 ꢁC; 1H
mp 278e280 ꢁC; 1H NMR (300 MHz, DMSO-d6)
d
(ppm): 13.66 (s,
1H), 9.14 (s, 1H), 9.08 (s, 0.5H), 9.05 (s, 0.5H), 7.93 (d, J ¼ 6.9 Hz, 1H),
7.82 (s, 1H), 7.69 (d, J ¼ 7.8 Hz, 1H), 7.41 (t, J ¼ 7.5 Hz, 1H), 3.97e4.10
(m, 3H), 2.84 (bs, 2H), 1.79e1.82 (m, 2H), 1.49e1.61 (m, 2H), 1.41 (s,
NMR (400 MHz, DMSO-d6)
d
(ppm): 9.55 (brs, 2H), 8.89 (s, 0.5H),
9H); 13C NMR (100 MHz, DMSO-d6)
d (ppm): 165.63, 157.34, 153.81,
8.84 (s, 0.5H), 7.94 (d, J ¼ 7.2 Hz, 1H), 7.85 (s, 1H), 7.74 (d, J ¼ 8.0 Hz,
1H), 7.45 (t, J ¼ 7.6 Hz, 1H), 4.21e4.55 (m, 2H), 3.88e3.99 (m, 4H),
3.60e3.65 (m, 1H), 3.10e3.40 (m, 1H), 2.78 (brs, 2H), 2.22e2.48 (m,
2H), 2.06 (brs, 2H), 1.40e1.52 (m, 2H), 1.39 (brs, 9H); HRMS (ESI): m/
z, calcd. For C23H33N6O4 [M þ H]þ 457.2558, found 457.2546.
145.67, 140.07, 134.65, 123.88, 123.75, 116.07, 78.64, 46.52, 43.15,
31.18, 28.08; HRMS (ESI): m/z, calcd. For C19H26N5O4 [M þ H]þ
388.1979, found 388.1971.
6.2.38. 2-(4-Aminopiperidine-1-carbonyl)-1H-benzol[d]imidazole-
4-carboxamide 2,2,2-trifluoroacetate (13b)
6.2.33. 2-(3-(Piperidin-4-ylamino)pyyrrolidine-1-carbonyl)-1H-
benzo[d]imidazole-4-carboxamide 2,2,2-trifluoroacetate (11g)
The title compound was synthesized from 11f using the same
method as the preparation of compound 7c: Compound 11g was
obtained as a white solid (40 mg, 72.7%); mp 199e201 ꢁC; 1H NMR
The title compound was synthesized from 13a using the same
method as the preparation of compound 7c: Compound 13b was
obtained as a white solid (60 mg, 70.5%); mp 275e277 ꢁC; 1H NMR
(300 MHz, DMSO-d6)
d
(ppm): 13.65 (s, 1H), 9.24 (d, J ¼ 7.5 Hz, 1H),
9.17 (s, 1H), 8.64 (brs, 2H), 7.93 (d, J ¼ 7.2 Hz, 1H), 7.84 (s, 1H), 7.70
(d, J ¼ 8.1 Hz, 1H), 7.42 (t, J ¼ 7.5 Hz, 1H), 4.20e4.42 (m, 1H),
3.33e3.38 (m, 2H), 3.06 (t, J ¼ 13.2 Hz, 2H), 1.99e2.09 (m, 2H),
(400 MHz, DMSO-d6)
d (ppm): 13.70 (brs, 1H), 9.54 (brs, 2H), 8.95
(brs, 1H), 8.83 (brs, 2H), 7.94 (d, J ¼ 7.6 Hz, 1H), 7.85 (brs, 1H), 7.75
(brs, 1H), 7.46 (t, J ¼ 8.0 Hz, 1H), 4.35e4.60 (m, 2H), 3.85e4.25 (m,
4H), 3.62e3.69 (m, 1H), 3.50 (brs, 1H), 2.94 (brs, 2H), 2.15e2.48 (m,
4H), 1.75e1.90 (m, 2H); HRMS (ESI): m/z, calcd. For C18H25N6O4
[M þ H]þ 357.2034, found 357.2027.
1.82e1.92 (m, 2H); 13C NMR (100 MHz, DMSO-d6)
d (ppm): 165.69,
157.80, 145.52, 139.99, 134.69, 123.92, 116.18, 109.30, 44.62, 42.45,
28.24; HRMS (ESI): m/z, calcd. For C14H18N5O2 [M þ H]þ 288.1455,
found 288.1450.
6.2.34. tert-Butyl (1-(4-carbomoyl-6-fluoro-1H-benzo[d]
imidazole-2-carbonyl)pyrrolidin-3-yl)carbamate (12a)
6.2.39. 2-(4-(Cyclohexylamino)piperidine-1-carbonyl)-1H-benzol
[d]imidazole-4-carboxamide (13c)
The title compound was synthesized from 6-fluoro-2-carboxyl-
1H-benzo[d]immidazole-4-carboxamide using the same method as
the preparation of compound 10a: Compound 12a was obtained as
a white solid (120 mg, 18%); mp 252e254 ꢁC; 1H NMR (400 MHz,
The title compound was synthesized from 13b using the same
method as the preparation of compound 11e: Compound 13c was
obtained as a white solid (45 mg, 42%); mp 228e230 ꢁC;1H NMR
(400 MHz, DMSO-d6)
d
(ppm): 8.80 (s, 1H), 7.92 (d, J ¼ 7.6 Hz, 1H),
DMSO-d6)
d
(ppm): 13.70 (s, 1H), 8.84 (d, J ¼ 8.8 Hz, 1H), 8.25 (d,
7.78 (s, 1H), 7.76 (d, J ¼ 8.4 Hz, 1H), 7.42 (t, J ¼ 7.6 Hz, 1H), 4.80 (d,
J ¼ 13.2 Hz, 1H), 4.39 (d, J ¼ 12.8 Hz, 1H), 3.41 (t, J ¼ 11.6 Hz, 1H),
3.05 (t, J ¼ 11.6 Hz, 1H), 2.97e2.99 (m, 1H), 2.59 (t, J ¼ 10.0 Hz, 1H),
1.89e1.95 (m, 2H), 1.83 (d, J ¼ 11.2 Hz, 2H), 1.67 (d, J ¼ 12.8 Hz, 2H),
1.56 (d, J ¼ 12.0 Hz, 1H), 0.97e1.38 (m, 7H); 13C NMR (100 MHz,
J ¼ 7.2 Hz, 1H), 7.68 (d, J ¼ 7.2 Hz, 1H), 7.50 (d, J ¼ 7.2 Hz, 1H), 7.26
(brs, 1H), 4.02e4.30 (m, 3H), 3.58e3.78 (m, 1.5H), 3.48e3.54 (m,
0.5H), 2.12e2.22 (m, 0.5H), 2.02e2.10 (m, 0.5H), 1.90e1.98 (m,
0.5H), 1.82e1.89 (m, 0.5H), 1.39 (d, J ¼ 10.4 Hz, 9H); HRMS (ESI): m/
z, calcd. For C18H23N5O4F [M þ H]þ 392.1729, found 392.1723.
DMSO-d6)
d (ppm): 165.98, 158.14, 146.21, 123.70, 123.60, 123.44,
122.81, 117.07, 52.14, 49.99, 44.92, 41.12, 32.20, 32.07, 31.28, 25.59,
24.38; HRMS (ESI): m/z, calcd. For C20H28N5O2 [M þ H]þ 370.2238,
found 370.2235.
6.2.35. 2-(3-Aminopyrrolidine-1-carbonyl)-6-fluoro-1H-benzol[d]
imidazole-4-carboxamide 2,2,2-trifluoroacetate (12b)
The title compound was synthesized from 12a using the same
method as the preparation of compound 7c: Compound 12b was
obtained as a white solid (180 mg, 70.5%); mp 244e246 ꢁC; 1H NMR
6.2.40. tert-Butyl 4-(1-(4-carbomoyl-1H-benzo[d]imidazole-2-
carbonyl)piperidin-4-ylamino)piperidine-1-carboxylate (13d)
The title compound was synthesized from 13b using the same
method as the preparation of compound 11f: Compound 13d was
obtained as a white solid (100 mg, 72.9%); mp 221e223 ꢁC; 1H
(400 MHz, DMSO-d6)
d (ppm): 13.80 (s, 1H), 8.84 (s, 0.5H), 8.74 (s,
0.5H), 8.05e8.16 (m, 4H), 7.68 (d, J ¼ 7.6 Hz, 1H), 7.50 (d, J ¼ 8.0 Hz,
1H), 4.25e4.40 (m, 2H), 3.90e4.15 (m, 1H), 3.65e3.86 (m, 2H),
2.30e2.40 (m, 0.5H), 2.20e2.29 (m, 0.5H), 2.15 (brs, 0.5H), 2.05 (brs,
0.5H); HRMS (ESI): m/z, calcd. For C13H14N5O2F [M þ H]þ 292.1204,
found 292.1201.
-NMR (400 MHz, DMSO-d6)
d (ppm): 8.76 (brs. 1H), 7.91 (d,
J ¼ 7.6 Hz, 1H), 7.78 (s, 1H), 7.74 (d, J ¼ 8.0 Hz, 1H), 7.41 (t, J ¼ 7.6 Hz,
1H), 4.78 (d, J ¼ 12.8 Hz, 1H), 4.36 (d, J ¼ 12.8 Hz, 1H), 3.83 (d,
J ¼ 12.8 Hz, 2H), 3.42 (t, J ¼ 11.6 Hz,1H), 3.06 (t, J ¼ 11.6 Hz,1H), 2.94
(brs, 1H), 2.76 (brs, 3H), 1.91 (t, J ¼ 10.4 Hz, 2H), 1.77 (d, J ¼ 12.0 Hz,
2H), 1.22e1.38 (m, 11H), 1.05e1.58 (m, 2H); HRMS (ESI): m/z, calcd.
For C24H35N6O4 [M þ H]þ 471.2714, found 471.2708.
6.2.36. 2-(3-(Cyclohexylamino)pyrrolidine-1-carbonyl)-6-fluoro-
1H-benzol[d]imidazole-4-carboxamide (12c)
The title compound was synthesized from 12b using the same
method as the preparation of compound 11e: Compound 12c was
obtained as a white solid (87 mg, 79%); mp 256e258 ꢁC; 1H NMR
6.2.41. 2-(4-(Piperidin-4-ylamino)piperidine-1-carbonyl)-1H-
benzo[d]imidazole-4-carboxamide 2,2,2-trifluoroacetate (13e)
The title compound was synthesized from 13d using the same
method as the preparation of compound 7c: Compound 13e was
obtained as a white solid (88 mg, 77.8%); mp 171e173 ꢁC; 1H NMR
(400 MHz, DMSO-d6)
d (ppm): 8.82 (s, 0.5H), 8.76 (s, 0.5H), 8.15 (s,
0.5H), 8.09 (s, 0.5H), 7.68 (d, J ¼ 10.0 Hz, 1H), 7.52 (d, J ¼ 5.2 Hz, 1H),
4.40e4.45 (m, 0.5H), 4.32e4.36 (m, 0.5H), 4.15e4.17 (m, 1H),
3.76e3.91 (m, 2H), 3.67e3.71 (m, 0.5H), 3.57e3.64 (m, 0.5H), 3.01
(brs, 0.5H), 2.91 (brs, 0.5H), 1.99e2.31 (m, 4H), 1.73 (brs, 2H), 1.58
(brs, 1H), 1.08e1.47 (m, 5H); HRMS (ESI): m/z, calcd. For
(400 MHz, DMSO-d6)
d (ppm): 8.98 (brs, 1H), 8.83 (brs, 1H), 8.66
(brs, 1H), 7.93 (d, J ¼ 7.2 Hz, 1H), 7.85 (brs, 1H), 7.61 (brs, 1H), 7.44 (t,
J ¼ 7.6 Hz, 1H), 5.18 (brs, 1H), 4.64 (d, J ¼ 13.2 Hz, 1H), 3.61 (brs, 1H),
3.51 (brs, 1H), 3.41 (d, J ¼ 12.0 Hz, 2H), 3.32 (t, J ¼ 12.8 Hz, 1H),
2.92e2.99 (m, 3H), 2.10e2.20 (m, 4H), 1.52e1.74 (m, 4H); HRMS
(ESI): m/z, calcd. For C19H27N6O2 [M þ H]þ 371.2190, found
371.2184.
C
19H25N5O2F [M þ H]þ 374.1987, found 374.1987.
6.2.37. tert-Butyl (1-(4-carbomyl-1H-benzo[d]imidazole-2-
carbonyl)piperidin-4-yl)carbamate (13a)
Compound 13a was obtained as a white solid (140 mg, 37.1%);

38
J. Zhou et al. / European Journal of Medicinal Chemistry 132 (2017) 26e41
6.2.42. tert-Butyl (1-(4-carbomyl-6-fluoro-1H-benzo[d]imidazole-
2-carbonyl)piperidin-4-yl)carbamate (14a)
6.2.47. (R)-tert-Butyl 4-(4-carbamoyl-1H-benzo[d]imidazole-2-
carbonyl)-2-ethylpiperazine-1-carboxylate (15c)
The title compound was synthesized from 6-fluoro-2-carboxyl-
1H-benzo[d]immidazole-4-carboxamide using the same method as
the preparation of compound 10a: Compound 14a was obtained as
a white solid (177 mg, 44.5%); mp 262e264 ꢁC; 1H NMR (400 MHz,
Compound 15c was obtained as a white solid (190 mg, 60.7%);
mp 208e210 ꢁC; Compound 15c exists as a mixture of two con-
formers; 1H NMR (300 MHz, DMSO-d6)
d (ppm): 13.70 (s, 1H), 8.78
(s, 0.5H), 8.72 (s, 0.5H), 7.94 (d, J ¼ 7.5 Hz, 1H), 7.86 (brs, 0.5H), 7.80
(brs, 0.5H), 7.75 (d, J ¼ 8.4 Hz, 1H), 7.46 (t, J ¼ 7.5 Hz, 1H), 5.04e5.15
(m, 1H), 4.40e4.50 (m, 1H), 4.05e4.14 (m, 1H), 3.84e3.89 (m, 1H),
3.54e3.62 (m, 0.5H), 3.38e3.49 (m, 0.5H), 2.96e3.12 (m, 2H),
DMSO-d6)
d (ppm): 13.73 (s, 1H), 8.77 (s, 1H), 8.02 (s, 1H), 7.66 (d,
J ¼ 8.0 Hz, 1H), 7.52 (d, J ¼ 8.0 Hz, 1H), 6.95 (d, J ¼ 7.6 Hz, 1H), 4.85
(d, J ¼ 11.2 Hz, 1H), 4.40 (d, J ¼ 11.2 Hz, 1H), 3.63 (brs, 1H), 3.46 (t,
J ¼ 11.2 Hz, 1H), 3.08 (t, J ¼ 11.2 Hz, 1H), 1.78 (brs, 2H), 1.39e1.50 (m,
1.40e1.60 (m,11H), 0.86 (t, J ¼ 7.2 Hz, 2H), 0.74 (t, J ¼ 7.2 Hz,1H); 13
C
11H); 13C NMR (100 MHz, DMSO-d6)
d
(ppm): 165.35, 159.11 (d,
NMR (100 MHz, DMSO-d6) d (ppm): 165.87, 158.83 (158.67), 153.94
JCF ¼ 237.7), 157.56, 154.79, 146.78, 136.40, 134.02, 124.99, 111.65 (d,
JCF ¼ 27.4), 101.96 (d, JCF ¼ 27.6), 77.65, 46.92, 45.13, 41.42, 32.38,
31.57, 28.23; HRMS (ESI): m/z, calcd. For C19H25N5O4F [M þ H]þ
406.1885, found 406.1880.
(153.89), 145.61 (145.56), 139.24, 133.94, 123.93, 123.84, 116.43,
79.11, 52.43, 48.25, 46.22, 44.39, 42.37, 28.01, 21.78 (21.63), 10.37
(10.12); HRMS (ESI): m/z, calcd. For C20H28N5O4 [M þ H]þ 402.2136,
found 402.2130.
6.2.43. 2-(4-Aminopiperidine-1-carbonyl)-6-fluoro-1H-benzol[d]
imidazole-4-carboxamide 2,2,2-trifluoroacetate (14b)
The title compound was synthesized from 14a using the same
method as the preparation of compound 7c: Compound 14b was
obtained as a white solid (150 mg, 78.9%); mp 231e233 ꢁC; 1H NMR
6.2.48. 2-(4-iso-Butylpiperazine-1-carbonyl)-1H-benzo[d]
imidazole-4-carboxamide (15d)
Compound 15d was obtained as a white solid (30 mg,18.7%); mp
228e230 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d (ppm): 13.61 (s, 1H),
8.83 (s, 1H), 7.92 (d, J ¼ 8.4 Hz, 1H), 7.78 (s, 1H), 7.73 (d, J ¼ 7.6 Hz,
1H), 7.44 (t, J ¼ 7.6 Hz, 1H), 4.23 (brs, 2H), 3.76 (brs, 2H), 2.48 (brs,
4H), 2.12 (d, J ¼ 7.2 Hz, 1H), 1.80e1.85 (m, 1H), 0.87 (d, J ¼ 6.4 Hz,
6H); HRMS (ESI): m/z, calcd. For C17H24N5O2 [M þ H]þ 330.1925,
found 330.1921.
(400 MHz, DMSO-d6)
d (ppm): 13.80 (s, 1H), 8.69 (s, 1H), 8.01 (brs,
4H), 7.66 (d, J ¼ 8.0 Hz, 1H), 7.55 (brs, 1H), 5.01 (brs, 1H), 4.54 (d,
J ¼ 13.2 Hz, 1H), 3.30e3.48 (m, 2H), 2.98 (t, J ¼ 12.4 Hz, 1H), 2.03 (t,
J ¼ 12.4 Hz, 2H), 1.45e1.48 (m, 2H); HRMS (ESI): m/z, calcd. For
C
14H17N5O2F [M þ H]þ 306.1361, found 306.1358.
6.2.44. 2-(4-(Cyclohexylamino)piperidine-1-carbonyl)-6-fluoro-
1H-benzol[d]imidazole-4-carboxamide (14c)
6.2.49. 2-(Piperazine-1-carbonyl)-1H-benzo[d]imidazole-4-
carboxamide 2,2,2-trifluoroacetate (15e)
The title compound was synthesized from 14b using the same
method as the preparation of compound 11e: Compound 14c was
obtained as a white solid (37 mg, 42.5%); mp 260e262 ꢁC; 1H NMR
The title compound was synthesized from 15a using the same
method as the preparation of compound 7c: Compound 15e was
obtained as a white solid (90 mg, 75%); mp 235e237 ꢁC; 1H NMR
(400 MHz, DMSO-d6)
d
(ppm): 8.91 (s, 1H), 7.93 (s, 1H), 7.60 (d,
(400 MHz, DMSO-d6) d (ppm): 13.71 (s,1H), 8.96 (brs, 2H), 8.64 (brs,
J ¼ 10.0 Hz,1H), 7.51 (d, J ¼ 8.0 Hz,1H), 4.70 (d, J ¼ 12.8 Hz, 1H), 4.41
(d, J ¼ 12.4 Hz, 1H), 3.31 (t, J ¼ 12.4 Hz, 1H), 2.95e3.04 (m, 2H), 2.66
(brs, 1H), 1.83e1.96 (m, 5H), 1.66 (d, J ¼ 11.2 Hz, 2H), 1.55 (d,
J ¼ 11.2 Hz, 1H), 1.02e1.36 (m, 6H); HRMS (ESI): m/z, calcd. For
1H), 7.93 (d, J ¼ 7.6 Hz, 1H), 7.79 (brs, 2H), 7.45 (t, J ¼ 7.6 Hz, 1H),
4.51 (brs, 2H), 3.91 (brs, 2H), 3.27 (brs, 4H); 13C NMR (100 MHz,
DMSO-d6)
d (ppm): 165.99, 158.19, 145.25, 123.96, 43.42, 42.86,
42.51; HRMS (ESI): m/z, calcd. For C13H16N5O2 [M þ H]þ 274.1299,
C
20H27N5O2F [M þ H]þ 388.2143, found 388.2142.
found 274.1296.
6.2.45. tert-Butyl 4-(4-carbamoyl-1H-benzo[d]imidazole-2-
carbonyl)piperazine-1-carboxylate (15a)
6.2.50. (S)-2-(3-Ethylpiperazine-1-carbonyl)-1H-benzo[d]
imidazole-4-carboxamide 2,2,2-trifluoroacetate (15f)
The title compound was synthesized from 15b using the same
method as the preparation of compound 7c: Compound 15f was
obtained as a white solid (88 mg, 69.2%); mp 250e252 ꢁC; 1H NMR
Compound 15a was obtained as a white solid (180 mg, 48.2%);
mp 232e233 ꢁC; 1H NMR (400 MHz, DMSO-d6)
d (ppm): 13.65 (s,
1H), 8.75 (s, 1H), 7.93 (d, J ¼ 7.2 Hz, 1H), 7.77 (s, 1H), 7.73 (d,
J ¼ 8.0 Hz, 1H), 7.44 (t, J ¼ 8.0 Hz, 1H), 4.23 (brs, 2H), 3.71 (brs, 2H),
(400 MHz, DMSO-d6)
d (ppm): 13.68 (s, 1H), 9.00e9.32 (m, 2H),
3.47 (brs, 4H), 1.42 (s, 9H); 13C NMR (100 MHz, DMSO-d6)
d (ppm):
8.65 (s, 1H), 7.94 (d, J ¼ 7.2 Hz, 1H), 7.74e7.82 (m, 1H), 7.75 (d,
J ¼ 8.0 Hz,1H), 7.46 (t, J ¼ 7.2 Hz,1H), 5.59 (d, J ¼ 10.8 Hz, 0.5H), 5.23
(d, J ¼ 12.0 Hz, 0.5H), 4.50e4.60 (m, 1H), 3.82e3.88 (m, 0.5H),
3.06e3.48 (m, 4.5H), 1.50e1.72 (m, 2H), 0.93e1.03 (m, 3H); HRMS
(ESI): m/z, calcd. For C15H20N5O2 [M þ H]þ 302.1612, found
302.1608.
165.65, 158.17.153.81, 145.59, 139.74, 133.68, 123.94, 116.05, 79.28,
46.26, 43.27, 28.04; HRMS (ESI): m/z, calcd. For C18H24N5O4
[M þ H]þ 374.1823, found 374.1814.
6.2.46. (S)-tert-Butyl 4-(4-carbamoyl-1H-benzo[d]imidazole-2-
carbonyl)-2-ethylpiperazine-1-carboxylate (15b)
Compound 15b was obtained as a white solid (210 mg, 50.6%);
mp 219e221 ꢁC; Compound 15b exists as a mixture of two con-
6.2.51. (R)-2-(3-Ethylpiperazine-1-carbonyl)-1H-benzo[d]
imidazole-4-carboxamide 2,2,2-trifluoroacetate (15g)
formers; 1H NMR (400 MHz, DMSO-d6)
d (ppm): 13.67 (s, 1H), 8.76
(s, 0.5H), 8.72 (s, 0.5H), 7.93 (d, J ¼ 7.2 Hz, 1H), 7.84 (s, 0.5H),
7.72e7.78 (m, 1.5H), 7.44 (t, J ¼ 8.0 Hz, 1H), 5.07 (t, J ¼ 11.2 Hz, 1H),
4.38e4.50 (m, 1H), 3.85e4.15 (m, 2H), 3.52e3.60 (m, 0.5H),
3.30e3.40 (m, 0.5H), 2.95e3.10 (m, 2H), 1.41e1.52 (m, 11H), 0.85 (t,
J ¼ 7.2 Hz, 2H), 0.72 (t, J ¼ 7.2 Hz,1H); 13C NMR (100 MHz, DMSO-d6)
The title compound was synthesized from 15c using the same
method as the preparation of compound 7c: Compound 15g was
obtained as a white solid (90 mg, 70.8%); mp 253e255 ꢁC; 1H NMR
(300 MHz, DMSO-d6)
d (ppm): 13.65 (s, 1H), 9.20 (s, 1H), 9.00 (s,
1H), 8.65 (s, 1H), 7.94 (d, J ¼ 7.2 Hz, 1H), 7.75 (s, 1H), 7.46 (t,
J ¼ 7.2 Hz, 1H), 5.58 (brs, 0.5H), 5.20 (brs, 0.5H), 4.50e4.60 (m, 1H),
3.32e3.80 (m, 3H), 3.05e3.30 (m, 2H), 1.50e1.72 (m, 2H), 0.90e1.05
(m, 3H); HRMS (ESI): m/z, calcd. For C15H20N5O2 [M þ H]þ 302.1612,
found 302.1608.
d
(ppm): 165.86, 158.80 (158.65), 153.91 (153.87), 145.58, 123.90,
123.81, 116.92, 79.08, 52.26, 48.23, 46.21, 44.37, 42.36, 27.99, 21.77
(21.64), 10.35 (10.10); HRMS (ESI): m/z, calcd. For C20H28N5O4
[M þ H]þ 402.2136, found 402.2128.

J. Zhou et al. / European Journal of Medicinal Chemistry 132 (2017) 26e41
39
6.2.52. N2-(Quinuclidin-3-yl) 1H-benzo[d]imidazole-2,4-
dicarboxmide (15h)
J ¼ 7.2 Hz, 1H), 7.75 (brs, 2H), 7.44 (brs, 1H), 4.22e4.29 (m, 2H),
3.69e3.75 (m, 2H), 3.59 (brs, 4H), 2.30e2.52 (m, 2H), 0.99 (brs, 3H);
HRMS (ESI): m/z, calcd. For C16H20N5O3 [M þ H]þ 330.1561, found
330.1558.
The
mixture
of
2-carboxyl-1H-benzo[d]immidazole-4-
carboxamide (150 mg, 0.73 mmol), EDC (280 mg, 1.46 mmol),
HOBt (1973 mg, 1.46 mmol), DMAP (35 mg, 0.3 mmol), Et3N
(0.63 mL, 4.38 mmol) and 3-aminoquinuclidine dihydrochloride
(218 mg, 1.09 mmol) in DMF (10 mL) was stirred at room temper-
ature for 48 h, and then the mixture was evaporated. The crude
product was obtained and purified with column chromatography
(methylene chloride/methanol/Et3N ¼ 20:1:0.2 to 8:1:0.08) to give
compound 15h as a white solid (135 mg, 59%); mp 201e203 ꢁC; 1H
6.2.56. 6-Fluoro-2-(3-oxopiperazine-1-carbonyl)-1H-benzo[d]
imidazole-4-carboxamide (16a)
The mixture of 6-fluoro-2-carboxyl-1H-benzo[d]immidazole-4-
carboxamide (180 mg, 0.8 mmol), HBTU (606 mg, 1.6 mmol), HOBt
(216 mg, 1.6 mmol), DIEA (0.56 mL, 3.2 mmol) and piperazin-2-one
(160 mg, 1.6 mmol) in DMF (20.0 mL) was stirred at room tem-
perature for 24 h then the mixture was evaporated. The crude
product was obtained and purified with column chromatography
(methylene chloride/methanol/Et3N ¼ 20:1:0.2 to 6:1:0.06) to give
compound 16a as a white solid (180 mg, 70%); mp 195e197 ꢁC; 1H
NMR (400 MHz, DMSO-d6)
d
(ppm): 9.16 (s, 1H), 8.94 (d, J ¼ 6.8 Hz,
1H), 7.94 (d, J ¼ 7.6 Hz, 1H), 7.81 (s, 1H), 7.72 (d, J ¼ 8.0 Hz, 1H), 7.42
(t, J ¼ 8.0 Hz, 1H), 4.04e4.08 (m, 1H), 3.17 (t, J ¼ 12.0 Hz, 1H), 2.95 (t,
J ¼ 10.4 Hz, 1H), 2.85 (dd, J1 ¼ 14.0 Hz, J2 ¼ 5.6 Hz, 1H), 2.66e2.78
(m, 3H), 1.95 (brs, 1H), 1.80e1.90 (m, 1H), 1.63 (t, J ¼ 6.0 Hz, 1H), 1.36
NMR (400 MHz, DMSO-d6) d (ppm): 13.58 (brs, 1H), 8.68 (brs, 0.5H),
(t, J ¼ 11.6 Hz, 1H); 13C NMR (100 MHz, DMSO-d6)
d
(ppm): 165.77,
8.62 (brs, 0.5H), 8.18 (d, J ¼ 14.4 Hz,1H), 8.05 (s, 0.5H), 7.95 (s, 0.5H),
7.69 (d, J ¼ 9.6 Hz, 1H), 7.54 (s, 1H), 4.82 (s, 1H), 4.38 (s, 1H), 4.19 (s,
1H), 3.87 (s, 1H), 3.32 (s, 2H); HRMS (ESI): m/z, calcd. For
158.58, 145.72, 139.68, 135.36, 123.87, 123.70, 123.39, 116.66, 52.30,
46.64, 46.39, 45.98, 25.54, 24.51, 19.20; HRMS (ESI): m/z, calcd. For
C
16H20N5O2 [M þ H]þ 314.1611, found 314.1605.
C
13H13N5O3F [M þ H]þ 306.0997, found 306.0996.
6.2.53. 2-((4aR,7aR)-Octahydro-1H-pyrrolo[3,4-b]pyridine-6-
carbonyl)-1H-benzo[d]imidazole-4-carboxamide (15i)
6.2.57. 6-Fluoro-2-(3-oxo-4-(pentan-3-yl)piperazine-1-carbonyl)-
1H-benzo[d]imidazole-4-carboxamide (16b)
The mixture of 2- carboxyl-1H-benzo[d]immidazole-4-
carboxamide (60 mg, 0.29 mmol), HBTU (222 mg, 0.58 mmol),
HOBt (79 mg, 0.58 mmol), Et3N (0.17 mL, 1.17 mmol) and (S,S)-2,8-
Diazabicyclo[4.3.0]nonane (73 mg, 0.58 mmol) in DMF (5.0 mL) was
stirred at room temperature for 48 h, and then the mixture was
evaporated. The crude product was obtained and purified with
column chromatography (methylene chloride/methanol/acetic
acid ¼ 4:1:0.05) to give the corresponding product 15i as a white
solid (30 mg, 32.9%); mp 201e203 ꢁC; 1H NMR (400 MHz, DMSO-
The title compound was synthesized from 6-fluoro-2-carboxyl-
1H-benzo[d]immidazole-4-carboxamide using the same method as
the preparation of compound 16a: Compound 16b was obtained as
a white solid (60 mg, 30.7%); mp 275e277 ꢁC; 1H NMR (300 MHz,
DMSO-d6)
d (ppm): 13.80 (s, 1H), 8.78 (s, 0.5H), 8.68 (s, 0.5H), 8.12
(s, 0.5H), 8.00 (s, 0.5H), 7.64e7.74 (m,1H), 7.52 (brs,1H), 4.96 (s,1H),
4.50 (s, 1H), 4.20e4.31 (m, 2H), 3.91 (s, 1H), 3.20e3.30 (m, 2H),
1.40e1.52 (m, 4H), 0.79 (t, J ¼ 5.7 Hz, 6H); HRMS (ESI): m/z, calcd.
For C18H23N5O3F [M þ H]þ 376.1779, found 376.1777.
d6)
d
(ppm): 8.94 (brs, 0.5H), 8.89 (brs, 0.5H), 7.91 (d, J ¼ 7.2 Hz, 1H),
Note: Except for target compounds, the synthesis and charac-
terization of all other compounds mentioned in Scheme 1 were
described in the supporting information.
7.74e7.78 (m, 2H), 7.41 (t, J ¼ 7.6 Hz, 1H), 4.01e4.23 (m, 2H),
3.61e3.67 (m, 1H), 3.28 (brs, 0.5H), 3.22 (brs, 0.5H), 2.79e2.91 (m,
2H), 2.44 (brs, 1H), 2.26e2.31 (m, 1H), 1.51e1.74 (m, 3H), 1.38 (brs,
1H); HRMS (ESI): m/z, calcd. For C16H20N5O2 [M þ H]þ 314.1611,
found 314.1604.
6.3. Biological evaluation
6.3.1. The assay for PARP-1 and PARP-2 inhibition
6.2.54. 2-(3-Oxopiperazine-1-carbonyl)-1H-benzo[d]imidazole-4-
carboxamide (15j)
Plasmid pET32a-PARP1 was a gift from Prof. Satoh (Canada).
Human recombinant PARP1/2 were expressed and purified as
described [30]. The ability of compounds to inhibit PARP1/2
enzyme activity were tested using ELISA method and the chemical
quantitation of NADþ method as described [30,31]. IC50 values were
calculated using GraphPad Prism 5 software.
The
mixture
of
2-carboxyl-1H-benzo[d]immidazole-4-
carboxamide (100 mg, 0.49 mmol), HATU (371 mg, 0.98 mmol),
HOAt (133 mg, 0.98 mmol), DIEA (0.34 mL, 1.95 mmol) and piper-
azin-2-one (98 mg, 0.98 mmol) in DMF(8.0 mL) was stirred at room
temperature for 24 h and then the mixture was evaporated. The
crude product was obtained and purified with column chroma-
6.3.2. The PF50 assay in MX-1 cells
tography (methylene chloride/methanol/Et3N
¼
20:1:0.2 to
In chemosensitization assays, MX-1 cells were seeded in a
density (2000 cells/well) in 96-well plates. Cells were treated with
PARP inhibitors at a fixed concentration of 10 mmol/L and temo-
zolomide (TMZ) at different concentrations (0e0.5 mmol/L). After
72 h treatment, cell survival was determined by MTT assay. IC50
values were calculated using GraphPad Prism5 software. Potenti-
ation factor (PF50) was calculated as the ratio of the IC50 for TMZ
divided by the IC50 of the combination (TMZ þ PARP inhibitor).
8:1:0.08) to give compound 15j as a white solid (80 mg, 57.2%); mp
266e268 ꢁC; Compound 15j exists as a mixture of two conformers;
1H NMR (400 MHz, DMSO-d6)
d
(ppm): 13.69 (s, 1H), 8.80 (brs,
0.5H), 8.74 (brs, 0.5H), 8.22 (s, 0.5H), 8.18 (s, 0.5H), 7.90e7.98 (m,
1.5H), 7.73e7.82 (m, 1.5H), 7.47 (t, J ¼ 8.4 Hz, 1H), 4.89 (s, 1H), 4.45
(brs, 1H), 4.22 (s, 1H), 3.90 (brs, 1H), 3.40 (s, 2H); 13C NMR
(100 MHz, DMSO-d6)
d (ppm): 165.92, 165.72, 157.90 (157.68),
145.47 (145.40), 123.95, 116.93, 49.90 (46.51), 43.38 (40.32), 39.17;
HRMS (ESI): m/z, calcd. For C13H14N5O3 [M þ H]þ 288.1091, found
288.1090.
6.3.3. Xenograft experiment
Female athymic BALB/c nude mice (8e10 week old) were pur-
chased from Vital River Laboratories, Beijing, China. A total of
1 ꢂ 106 MX-1 cells in 0.1 mL sterilized PBS were implanted sub-
cutaneously in mice. After 6 days, the mice were grouped (6 mice/
group) with tumor volumes at 100e300 mm3 and received treat-
ment. TMZ was given by oral gavage once daily for five days at the
dose of 50 mg/kg/day and compound 10a was administered orally
once daily at the dose of 50 mg/kg/day for ten days. For
6.2.55. 2-(4-Propionylpiperazine-1-carbonyl)-1H-benzo[d]
imidazole-4-carboxamide (15k)
The title compound was synthesized from 15e using the same
method as the preparation of compound 11d: Compound 15k was
obtained as a white solid (37 mg, 90.2%); mp 251e253 ꢁC; 1H NMR
(400 MHz, DMSO-d6)
d (ppm): 13.65 (s, 1H), 8.76 (s, 1H), 7.93 (d,

40
J. Zhou et al. / European Journal of Medicinal Chemistry 132 (2017) 26e41
combination groups, both TMZ and compound 10a were adminis-
trated orally once daily for five days. Body weights and tumor
volumes were measured twice every week. The mice were eutha-
nized at Day 11. %T/C is calculated as (tumor weight of treatment
group/Vehicle or TMZ treatment group) ꢂ 100, while %TGI is
calculated as (100-%T/C). Statistical analyses were performed by
GraphPad Prism5 software and the significance levels were evalu-
ated using one-way ANOVA model.
References
[1] J.C. Ame, C. Spenlehauer, G. de Murcia, The PARP superfamily, Bioessays 26
(2004) 882e893.
[2] T. Helleday, Putting poly (ADP-ribose) polymerase and other DNA repair in-
hibitors into clinical practice, Curr. Opin. Oncol. 25 (2013) 609e614.
[3] M.F. Langelier, J.L. Planck, S. Roy, J.M. Pascal, Structural basis for DNA damage-
dependent poly(ADP-ribosyl)ation by human PARP-1, Science 336 (2012)
728e732.
[4] D.V. Ferraris, Evolution of Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors.
From concept to clinic, J. Med. Chem. 53 (2010) 4561e4584.
ꢀ
ꢀ
[5] L. Virag, C. Szabo, The therapeutic potential of poly(ADPribose)polymerase
inhibitors, Pharmacol. Rev. 54 (2002) 375e429.
6.4. The X-ray crystallographic experiment
[6] H. Florian, H. Philipp, E.R. Nina, P. Michael, K. Stefan, B. Andreas du, Poly(ADP-
ribosyl)ation polymerases: mechanism and new target of anticancer therapy,
Expert Rev. Anticancer Ther. 10 (2010) 1125e1136.
[7] M.O. Hottiger, P.O. Hassa, B. Lüscher, H. Schüler, F. Koch-Nolte, Toward a
unified nomenclature for mammalian ADP-ribosyltransferases, Trends bio-
chem. Sci. 35 (2010) 208e219.
6.4.1. Protein expression and purification
Human PARP-1 catalytic domain (residues 663e1011, catPARP-
1) was cloned to pET-28a (Novagen) to construct the recombinant
expression vector. The V762A mutagenesis was carried out on
pET28a-PARP-1 with Site-Directed Mutagenesis Kit from Transgen
Biotech. The inserted genes were sequenced to ensure the sequence
was correct. The expression and purification of wild type and
mutant PARP-1 was carried out as previously reported. Generally
speaking, upon reaching an optical density (OD600) of 0.5e0.8, the
expression of catPARP-1 protein was induced by adding 0.4 mM
[8] C.A. Delaney, L.Z. Wang, S. Kyle, A.W. White, A.H. Calvert, N.J. Curtin,
B.W. Durkacz, Z. Hostomsky, D.R. Newell, Potentiation of temozolomide and
topotecan growth inhibition and cytotoxicity by novel poly(adenosine
diphosphoribose) polymerase inhibitors in a panel of human tumor cell lines,
Clin. Cancer Res. 6 (2000) 2860e2867.
[9] R.A. Daniel, A.L. Rozanska, E.A. Mulligan, Y. Drew, H.D. Thomas,
D.J. Castelbuono, Z. Hostomsky, E.R. Plummer, D.A. Tweddle, A.V. Boddy,
S.C. Clifford, N.J. Curtin, Central nervous system penetration and enhancement
of temozolomide activity in childhood medulloblastoma models by poly(ADP-
ribose) polymerase inhibitor AG-014699, Br. J. Cancer 103 (2010) 1588e1596.
[10] S. Kummar, J. Ji, R. Morgan, H.J. Lenz, S.L. Puhalla, C.P. Belani, D.R. Gandara,
D. Allen, B. Kiesel, J.H. Beumer, E.M. Newman, L. Rubinstein, A. Chen, Y. Zhang,
L. Wang, R.J. Kinders, R.E. Parchment, J.E. Tomaszewski, J.H. Doroshow,
A phase I study of veliparib in combination with metronomic cyclophospha-
mide in adults with refractory solid tumors and lymphomas, Clin. Cancer Res.
18 (2012) 1726e1734.
isopropyl b-D
-1-thiogalactopyranoside (IPTG) at 18 ꢁC. Following
cell lysis by sonication in lysis buffer (50 mM Tris (pH7.4), 300 mM
NaCl and 15 mM imidazole) with EDTA-free protease-inhibitor
cocktail (Thermo Scientific), the catPARP-1 protein was first puri-
fied using Ni2þ-chelating column, followed by ion-exchange
(HITRAP SP HP, GE Healthcare) and gel-filtration (SUPERDEX 200,
GE Healthcare) purifications. The purified protein was concentrated
to 80 mg/mL in stock buffer (50 mM Tris-HCl (pH 7.4),150 mM NaCl,
5 mM DTT) and stored at ꢃ80 ꢁC.
[11] B. Evers, R. Drost, E. Schut, M. de Bruin, E. van der Burg, P.W. Derksen,
H. Holstege, X. Liu, E. van Drunen, H.B. Beverloo, G.C. Smith, N.M. Martin,
A. Lau, M.J. O'Connor, J. Jonkers, Selective inhibition of BRCA2-deficient
mammary tumor cell growth by AZD2281 and cisplatin, Clin. Cancer Res. 14
(2008) 3916e3925.
[12] C.K. Donawho, Y. Luo, Y.P. Luo, T.D. Penning, J.L. Bauch, J.J. Bouska,
V.D. Bontcheva-Diaz, B.F. Cox, T.L. DeWeese, L.E. Dillehay, D.C. Ferguson,
N.S. Ghoreishi-Haack, D.R. Grimm, R. Guan, E.K. Han, R.R. Holley-Shanks,
B. Hristov, K.B. Idler, K. Jarvis, E.F. Johnson, L.R. Kleinberg, V. Klinghofer,
L.M. Lasko, X. Liu, K.C. Marsh, T.P. McGonigal, J.A. Meulbroek, A.M. Olson,
J.P. Palma, L.E. Rodriguez, Y. Shi, J.A. Stavropoulos, A.C. Tsurutani, G.D. Zhu,
S.H. Rosenberg, V.L. Giranda, D.J. Frost, ABT-888, an orally active poly(ADP-
ribose) polymerase inhibitor that potentiates DNA-damaging agents in pre-
clinical tumor models, Clin. Cancer Res. 13 (2007) 2728e2737.
[13] H. Farmer, N. McCabe, C.J. Lord, A.N. Tutt, D.A. Johnson, T.B. Richardson,
M. Santarosa, K.J. Dillon, I. Hickson, C. Knights, N.M. Martin, S.P. Jackson,
G.C. Smith, A. Ashworth, Targeting the DNA repair defect in BRCA mutant cells
as a therapeutic strategy, Nature 434 (2005) 917e921.
6.4.2. Crystallography
The purified V762A mutant PARP-1 (17 mg/mL) was crystallized
by hanging drop vapor diffusion method. The crystals were grown
at 18 ꢁC after 5 days. The protein-ligand complex crystals were
obtained by soaking apo crystals in reservoir solution (2.45 M
(NH4)2SO4, 0.1 M Tris, pH 7.5) containing 2 mM of tested com-
pounds. Then crystals were immediately cryoprotected by
immersing in the well solution containing 20% v/v glycerol before
being flash-frozen in liquid nitrogen. Diffraction data were
collected at Shanghai Synchrotron Research Facility (SSRF) using
the beam-line BL17U. The data were processed with HKL-3000
software [32]. The PARP-1 structures were primarily determined
by molecular replacement using the published PDB structure (PDB
code: 4L6S) as the searching model. After initial refinement, the
specific compound was modeled into the electron density with
COOT software [33]. The structures of the PARP-1 complexed with
small molecules were finally refined with restrains using REFMAC5
[34] in CCP4 suite. The structures have been deposited to the Pro-
tein Data Bank (PDB) with accession codes: 5WS1 (compound 11b),
5WS0 (compound 15e).
[14] N. Curtin, PARP inhibitors for anticancer therapy, Biochem. Soc. Trans. 42
(2014) 82e88.
[15] P.C. Fong, D.S. Boss, T.A. Yap, A. Tutt, P. Wu, M. Mergui-Roelvink, P. Mortimer,
H. Swaisland, A. Lau, M.J. O'Connor, A. Ashworth, J. Carmichael, S.B. Kaye,
J.H. Schellens, J.S. de Bono, Inhibition of poly(ADP-ribose) polymerase in tu-
mors from BRCA mutation carriers, N. Engl. J. Med. 361 (2009) 123e134.
[16] M. Anwar, H.M. Aslam, S. Anwar, PARP inhibitors, Hered. Cancer Clin. Pract. 13
(2015) 1e4.
[17] T.D. Penning, G.D. Zhu, J. Gong, V.B. Gandhi, Y. Luo, X. Liu, Y. Shi, V. Klinghofer,
E.F. Johnson, D. Frost, C. Donawho, L. Rodriguez, G. Bukofzer, K. Jarvis,
J. Bouska, D.J. Osterling, A. Olson, K.C. Marsh, S.H. Rosenberg, V. 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) 514e523.
[18] K.A. Menear, C. Adcock, R. Boulter, X. Cockcroft, L. Copsey, A. Cranston,
K.J. Dillon, J. Drzewiecki, S. Garman, S. Gomez, H. Javaid, F. Kerrigan,
C. Knights, A. Lau, V.M.L Jr., I.T.W. Matthews, S. Moore, M.J. O'Connor,
G.C.M. Smith, N.M.B. Martin, 4-[3-(4-Cyclopropanecarbonylpiperazine-1-
carbonyl)-4-fluorobenzyl]-2H-phthalazin-1-one: a novel bioavailable inhibi-
tor of poly(ADP-ribose) polymerase-1, J. Med. Chem. 51 (2008) 6581e6591.
[19] H.D. Thomas, C.R. Calabrese, M.A. Batey, S. Canan, Z. Hostomsky, S. Kyle,
K.A. Maegley, D.R. Newell, D. Skalitzky, L.Z. Wang, S.E. Webber, N.J. Curtin,
Preclinical selection of a novel poly(ADP-ribose) polymerase inhibitor for
clinical trial, Mol. Cancer Ther. 6 (2007) 945e956.
[20] P. Jones, S. Altamura, J. Boueres, F. Ferrigno, M. Fonsi, C. Giomini, S. Lamartina,
E. Monteagudo, J.M. Ontoria, M.V. Orsale, M.C. Palumbi, S. Pesci, G. Roscilli,
R. Scarpelli, C. Schultz-Fademrecht, C. Toniatti, M. Rowley, Discovery of 2-{4-
[(3S)-piperidin-3-yl]phenyl}-2H-indazole-7-carboxamide (MK-4827): a novel
oral poly(ADP-ribose)polymerase (PARP) inhibitor efficacious in BRCA-1 and
-2 mutant tumors, J. Med. Chem. 52 (2009) 7170e7185.
Acknowledgements
This work is supported by National Natural Science Foundation
of China (No. 81673300) and Special Funds for National Public
Benefit Research Institutes (2016ZX350039). We greatly appreciate
Dr. Niu Huang and Yanli Wang (National Institute of Biological
Sciences, Beijing) for their supports on X-ray crystallographic
experiment.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.03.013.
[21] G. Papeo, H. Posteri, D. Borghi, A.A. Busel, F. Caprera, E. Casale, M. Ciomei,

J. Zhou et al. / European Journal of Medicinal Chemistry 132 (2017) 26e41
A. Cirla, E. Corti, M. D'Anello, M. Fasolini, B. Forte, A. Galvani, A. Isacchi, (2000) 4084e4097.
41
A. Khvat, M.Y. Krasavin, R. Lupi, P. Orsini, R. Perego, E. Pesenti, D. Pezzetta,
S. Rainoldi, F. Riccardi-Sirtori, A. Scolaro, F. Sola, F. Zuccotto, E.R. Felder,
D. Donati, A. Montagnoli, Discovery of 2-[1-(4,4-Difluorocyclohexyl)piperidin-
4-yl]-6-fluoro-3-oxo-2,3-dihydro-1H-isoindole-4-carboxamide (NMSP118):a
potent, orally available, and highly selective PARP-1 inhibitor for cancer
therapy, J. Med. Chem. 58 (2015) 6875e6898.
[27] J. Yelamos, J. Farres, L. Llacuna, C. Ampurdanes, J. Martin-Caballero, PARP-1
and PARP-2: new players in tumour development, Am. J. Cancer Res. 1 (2011)
328e346.
[28] Y.Q. Wang, P.Y. Wang, Y.T. Wang, G.F. Yang, A. Zhang, Z.H. Miao, An update on
poly(ADP-ribose)polymerase-1 (PARP-1) inhibitors: opportunities and chal-
lenges in cancer therapy, J. Med. Chem. 59 (2016) 9575e9598.
€
[22] 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) 5003e5015.
[23] B. Wang, D. Chu, Y. Feng, Y.Q. Shen, M. Aoyagi-Scharber, L.E. Post, Discovery
and Characterization of (8S,9R)-5-Fluoro-8-(4-fluorophenyl)-9-(1-methyl-1H-
1,2,4-triazol-5-yl)-2,7,8,9-tetrahydro-3H-pyrido[4,3,2-de]phthalazin-3-one
[29] T. Karlberg, M. Hammarstrom, P. Schütz, L. Svensson, H. Schüler, Crystal
structure of the catalytic domain of human PARP2 in complex with PARP
inhibitor ABT-888, Biochemistry 49 (2010) 1056e1058.
[30] Z.X. Zhu, J. Jin, N. Xue, X. Song, X.G. Chen, Development and validation of high-
throughput screening assays for poly(ADP-ribose) polymerase-2 inhibitors,
Anal. Biochem. 449 (2014) 188e194.
[31] K.S. Putta, P.J. Hergenrother, An enzymatic assay for poly(ADP-ribose) poly-
merase-1 (PARP-1) via the chemical quantitation of NADt: application to the
high-throughput screening of small molecules as potential inhibitors, Anal.
Biochem. 326 (2004) 78e86.
[32] W. Minor, M. Cymborowski, Z. Otwinowski, M. Chruszcz, HKL-3000: the
integration of data reduction and structure solution e from diffraction images
to an initial model in minutes, Acta Crystallogr. Sect. D. Biol. Crystallogr. 62
(2006) 859e866.
[33] P. Emsley, B. Lohkamp, W.G. Scott, K. Cowtan, Features and development of
coot, acta crystallographica, section d, Biol. Crystallogr. 66 (2010) 486e501.
[34] G.N. Murshudov, P. Skubak, A.A. Lebedev, N.S. Pannu, R.A. Steiner,
R.A. Nicholls, M.D. Winn, F. Long, A.A. Vagin, REFMAC5 for the refinement of
macromolecular crystal structures, Acta Crystallographica, Section D, Biol.
Crystallogr. 67 (2011) 355e367.
(BMN 673, Talazoparib),
a Novel, Highly Potent, and Orally Efficacious
Poly(ADP-ribose) Polymerase-1/2 Inhibitor, as an Anticancer Agent, J. Med.
Chem. 59 (2016) 335e357.
[24] A. Ruf, G. de Murcia, G.E. Schulz, Inhibitor and NADþ binding to poly(ADP-
ribose) polymerase as derived from crystal structures and homology
modeling, Biochemistry 37 (1998) 3893e3900.
€
[25] 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. Schüler, T. Karlberg, Structural basis for inhibitor specificity in human
poly(ADP-ribose) Polymerase-3, J. Med. Chem. 52 (2009) 3108e3111.
[26] 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 biological properties of benzimidazole inhibitors of
the DNA repair enzyme poly(ADP-ribose) polymerase, J. Med. Chem. 43