Discovery of the PARP (poly ADP-ribose polymerase) inhibitor 2-(1-(4,4-difluorocyclohexyl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide for the treatment of cancer

Article abstract of DOI:10.1016/j.bioorg.2021.105026

In this work, two series of cyclic amine-containing benzimidazole carboxamide derivatives were designed and synthesized as potent anticancer agents. PARP1/2 inhibitory activity assays indicated that most of the compounds showed significant activity. The in vitro antiproliferative activity of these compounds was investigated against four human cancer cell lines (MDA-MB-436, MDA-MB-231, MCF-7 and CAPAN-1), and several compounds exhibited strong cytotoxicity to tumor cells. Among them, 2-(1-(4,4-difluorocyclohexyl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide (17d) was found to be effective PARP1/2 inhibitors (IC₅₀ = 4.30 and 1.58 nM, respectively). In addition, 17d possessed obvious selective antineoplastic activity and noteworthy microsomal metabolic stability. What's more, further studies revealed that 17d was endowed with an excellent ADME profile. These combined results indicated that 17d could be a promising candidate for the treatment of cancer.

Full text of DOI:10.1016/j.bioorg.2021.105026

Journal Pre-proofs
Discovery of the PARP (poly ADP-ribose polymerase) Inhibitor 2- (1- (4,4-
difluorocyclohexyl) piperidin-4-yl) -1H-benzo [d] imidazole-4-carboxamide
for the Treatment of Cancer
Lin Tang, Weibin Wu, Cunlong Zhang, Zhichao Shi, Dawei Chen, Xin Zhai,
Yuyang Jiang
PII:
S0045-2068(21)00403-X
DOI:
https://doi.org/10.1016/j.bioorg.2021.105026
YBIOO 105026
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
9 October 2020
6 May 2021
24 May 2021
Please cite this article as: L. Tang, W. Wu, C. Zhang, Z. Shi, D. Chen, X. Zhai, Y. Jiang, Discovery of the PARP
poly ADP-ribose polymerase) Inhibitor 2- (1- (4,4-difluorocyclohexyl) piperidin-4-yl) -1H-benzo [d]
imidazole-4-carboxamide for the Treatment of Cancer, Bioorganic Chemistry (2021), doi: https://doi.org/
0.1016/j.bioorg.2021.105026
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Discovery of the PARP (poly ADP-ribose polymerase) Inhibitor 2- (1-
4,4-difluorocyclohexyl) piperidin-4-yl) -1H-benzo [d] imidazole-4-carboxamide for the
(
Treatment of Cancer
Lin Tanga,b,†, Weibin Wub,c,†, Cunlong Zhang , Zhichao Shi , Dawei Chen , Xin Zhai *,
Yuyang Jianga,e,f,
b,c
d
b
a,
*
a Department of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, Liaoning 110016,
PR China
b Shenzhen Kivita Innovative Drug Discovery Institute, Shenzhen 518057, PR China
c National & Local United Engineering Lab for Personalized Anti-tumor Drugs, The Graduate School at Shenzhen,
Tsinghua University, Shenzhen 518055, PR China
d Department of Chemistry, Tsinghua University, Beijing 100084, PR China
e Joint Key State Laboratory of Tumor Chemogenomics, Tsinghua Shenzhen International Graduate School,
Tsinghua University, Shenzhen 518055, PR China
f School of pharmaceutical sciences, Tsinghua University, Beijing 100084, PR China
†These authors contributed equally to this work.
*
Corresponding author.
E-mail addresses: jiangyy@sz.tsinghua.edu.cn (Yuyang Jiang); zhaixin_syphu@126.com (Xin Zhai).
Abstract ： In this work, two series of cyclic amine-containing benzimidazole
carboxamide derivatives were designed and synthesized as potent anticancer agents. PARP1/2
inhibitory activity assays indicated that most of the compounds showed significant activity.
The in vitro antiproliferative activity of these compounds was investigated against four human
cancer cell lines (MDA-MB-436, MDA-MB-231, MCF-7 and CAPAN-1), and several
compounds exhibited strong cytotoxicity to tumor cells. Among them, 2- (1- (4,
4
-difluorocyclohexyl) piperidin-4-yl) -1H-benzo [d] imidazole-4- carboxamide (17d) was
found to be effective PARP1/2 inhibitors (IC = 4.30 and 1.58nM, respectively). In addition，
5
0
1
7d possessed obvious selective antineoplastic activity and noteworthy microsomal metabolic
stability. What’s more, further studies revealed that 17d was endowed with an excellent ADME
profile. These combined results indicated that 17d could be a promising candidate for the
treatment of cancer.
Keywords: Drug design; PARP inhibitors; Anti-cancer; ADME properties.
1
. Introduction
Poly ADP-ribose polymerases (PARPs) are a family of enzymes related to DNA damage
repair process. The formation of ADP-ribose polymers is catalyzed using Nicotinamide
1

+
Adenine Dinucleotide (NAD ) as a substrate by activated PARP enzymes. [1,2] Nowadays, at
least 17 PARP enzymes are known to be involved in this mechanism. Among all proteins
belonging to PARP family, only PARP1 and PARP2 carry DNA binding domains which
facilitates the recognition and localization of the DNA damage sites. Both PARP1 and
PARP2 can repair single-strand DNA (ssDNA) breaks. But PARP1 can also repair
double-strand DNA (dsDNA) breaks and stalled replication forks. PARP1 was firstly
identified and is the best studied PARP enzyme, along with PARP2, was found in the nucleus.
PARP1 is acting as a “molecular nick sensor” to ssDNA breaks and assists in their repairs.
PARP1 is mostly correlated with the progress of DNA damage repair which generates nearly
9
0% of poly ADP-ribose chains after the occurrence of DNA damage events. [3-7]
It has been demonstrated that inhibition of PARP1/2 accelerates the damage of injured
DNA, which is synthetically lethal to DNA-repairing-deficient cancer cells, such as
+
BRCA1/2-deficient cells.[4,8-9] PARP inhibitors are small molecule NAD mimetics with
+
different specificities and potencies, which can bind to the NAD sites in the catalytic domain
of PARP proteins. Due to the influence of PARP inhibitors, PARP enzymes are unable to use
+
NAD to catalyze the transfer of ADP-ribose units to nuclear target proteins upon oxidative
stress and DNA injury.[10-12] A large number of heterocyclic derivatives have been
developed as scaffolds of PARP inhibitors, like benzimidazole, quinazoline, phthalazine and
phenanthridone derivatives, because their structures are similar to the natural substrate of
+
NAD .[13-15] Several PARP1 inhibitors entered the arena as promising chemo- and
radiotherapy potentiators, and they have been used as monotherapy in breast and ovarian
cancers with mutant BRCA.[16-17] Olaparib (Fig. 1) is the first PARP inhibitor approved by
the FDA for marketing in 2014.[18] A total of four PARP inhibitors have been approved so
far, and the other three are Rucaparib, Niraparib and Talazoparib (Fig. 1).
O
H
H
O
N
NH
N
N
N
N
O
NH2
N
O
O
F
N
HN
N
NH
N
N
F
N
H
N
H
N
F
F
Olaparib
O
Rucaparib
Niraparib
Talazoparib
Fig. 1. Structures of PARP inhibitors approved by FDA.
According to the position and function of the catalytic sites, the catalytic pockets of
2

PARP1 were characterized as three sub-pockets (Fig. 2) which occupied by the substrate
+
NAD . One is the nicotinamide-ribose binding site (NI site), and the other two are the
phosphate binding site (PH site) and the adenine-ribose binding site (AD site). The PARP1
inhibitors described in the literature are able to bind the NI site through hydrogen bonds with
the Gly863 and Ser904 residues, and additional π-π stacking interaction with the Tyr907
residue. The AD site, unlike the NI site, contains a large hydrophobic pocket to accommodate
diverse molecular structures.[19]
Fig. 2. Three catalytic sub-pockets of PARP1.
Plenty of researches on reported PARP1 inhibitors revealed that limitation of free
rotation of the amide group on the aromatic ring could greatly improve the PARP1 inhibitory
activity. [20-21] Because the hydrogen atom on the amide bond forms an intramolecular
hydrogen bond with the nitrogen atom on the imidazole ring, which limits the freedom of the
amide bond and enables it to bind to the NI site better. Among them, veliparib (Fig. 3) is
considered as one of the most competitive members to obtain approval in the future.
In 2001, hundreds of 2-alkylamine substitutes were synthesized by Abbott Labs and a
part of compounds were screened out with both PARP1 and cellular IC values under 10nM.
5
0
After further optimization, the final structure of veliparib was achieved. Preclinical research
results showed that Veliparib had potent anti-tumor activity and good bioavailability. At
present, Veliparib has entered phase III clinical studies.[22-28] In the process of structural
optimization to veliparib, A-620223 (Fig. 3) was found to have good potency against both
PARP1 and PARP2, along with effective oral efficacy in vivo studies. Although A-620223
was preclinically abandoned afterwards since the Abbott Labs had found the more ideal
3

candidate veliparib, the excellent biological activity and the potential druggability of
A-620223 raised our great interests.[23-24] Besides, due to its comparatively low molecular
weight and high intrinsic potency, the benzimidazole carboxamide scaffold contained in
A-620223 and Veliparib is considered to be an essential basic structure in PARP1
inhibitors.[22-24] Consequently, A-620223 and Veliparib (Fig. 3) were chosen to be the lead
compounds in our study. Two series of cyclic amine-containing benzimidazole carboxamide
derivatives (I and II in Fig. 3) based on A-620223 and Veliparib were designed rationally and
synthesized as PARP inhibitors in this work.
O
NH2
O
NH2
O
NH2
O
NH2
N
N
R1
N
N
N
N R3
N
N
H
N
N
N
N
R2
H
H
H
H
Veliparib
A-620223
I
II
Fig. 3. Structures of lead compounds and designed derivatives.
The basic cyclic amine-containing (the pyrrolidinyl and piperidyl) benzimidazole
carboxamide scaffolds of lead compounds were reserved. However, the lead compounds do
not form distinct interactions with the residues in the hydrophobic pocket (AD site) of PARP1
because of their short molecular structures. Consequently, in order to develop more promising
drug candidates, various substituted short straight chains, rings, heterocycles and aromatic
nucleus were selected and introduced to the nitrogen atom on the five/six-membered cyclic
amine to explore additional interactions with the AD site. Four human cancer cell lines, one
with mutant BRCA1 (MDA-MB-436, breastꢀcancer), two with non-mutant BRCA1/2
(MDA-MB-231 and MCF-7, breastꢀcancer), and one with mutant BRCA2ꢀ (CAPAN-1,
pancreaticꢀcancer), were chosen to evaluate the in vitro antiproliferative activity of these
compounds. Among them, 17d exhibited potent inhibitory activity against both PARP1 and
PARP2 enzyme, significant in vitro antitumor activity and noteworthy microsomal metabolic
stability. Furthermore, 17d also possessed excellent ADME properties, which indicated that
1
2
2
7d could be a potential candidate for treatment of cancer.
. Results and discussion
.1. Chemistry
The target compounds were synthesized via a synthetic route from benzylamine (1) and
4

isonipecotic acid (12), as shown in Scheme 1 and Scheme 2. The benzimidazole ring system
was constructed as described in the literature. [23-24, 29] Saponification of the
CBZ-protected cyclic amine carboxylic esters (6a, 6b) gave the acids (7a, 7b). 7a, 7b and 13
were
,1-carbonyldiimidazole (CDI) conditions to selectively give amides (8a, 8b, 14). The amides
8a, 8b, 14) were refluxed in acetic acid to provide benzimidazoles (9a, 9b, 15). The CBZ
protecting groups were removed under hydrogenolysis conditions to give secondary amines
10a, 10b, 16). Finally, compounds 11a–11p, 17a–11o were synthesized from the reaction of
0a–10b, 16 with the corresponding side chain reactant under appropriate alkaline condition.
coupled
to
a
2,3-diaminobenzamide
hydrochloride
under
standard
1
(
(
1
All the synthesized compounds were purified using recrystallization or silica gel column
1
chromatography. The structures of the target compounds were characterized using H-NMR,
13C-NMR, and HRMS spectral analyses.
5

Si
Si
R1
O
H N
2
N
R1 O
O
a
HN
b
O
N
3
c
d
O
HN
1
2
4a,4b
5a,5b
CONH2
NH2
R O
R O
e
Cbz
1
f
Cbz
1
g
h
N
N
NH
O
OH
R1
O
N Cbz
6
a,6b
7a,7b
8a,8b
O
NH2
O
NH2
O
NH2
j
i
R1
R1
N
R1
N
N
N
NH
N
N
H
N
N
H
Cbz
R2
H
4
a - 10a : R = H
1
9
a,9b
10a,10b
11a-11p
4b - 10b : R = F
1
R = H
R = F
1
1
F
F
N
F
F
R =
F
R =
2
F
2
F
O
O
F
1
1a
11b
11c
11d
11e
11n
11o
H
N
O
O
11g
1
1f
11h
Br
N
N
O
N
O
O
H
1
1i
11j
11k
11l
11m
11p
o
o
Scheme 1. Reagents and conditions: (a) CMM3, CH
3
CN, 90 C; (b) formaldehyde, MeOH, K
2
CO
3
, 0 C~RT; (c) R
1
=H,
o
o
methyl acrylate, TFA, DCM, 0 C; R
1
=F, methyl 2-fluoroacrylate, TFA, DCM, 0 C; (d) HCl in dioxane, H
2
, 10% Pd/C, MeOH;
o
o
(
e) NaHCO
3
, CbzCl, toluene, 0 C; (f) LiOH, THF, H
2
O, 50 C; (g) R
1
=H, CDI, 2,3-Diaminobenzamide dihydrochloride, pyridine,
=H, AcOH, reflux; R =F, AcOH,
o
DMF, 0 C; R
1
=F, 2,3-Diaminobenzamide dihydrochloride, TBTU, DIPEA, AcOH; (h) R
1
1
0oC; (i) H
, 10% Pd/C, MeOH, 50 C; (j) the corresponding side chain reactant, appropriate alkaline condition.
o
5
2
6

CONH2
COOH
NH2
O
NH2
COOH
k
l
m
N
NH
N Cbz
N
N
H
N
O
Cbz
H
N
Cbz
1
2
13
14
15
O
NH2
O
NH2
n
o
N
N
NH
N R3
N
N
H
H
16
17a-17o
F
F
N
F
F
R =
F
3
F
F
F
17a
17b
17c
17d
17e
H
N
N
O
O
17f
17g
17h
17i
N
O
N
NH
O
H
17j
17k
17l
17m
17n
17o
Scheme 2. Reagents and conditions: (k) CBzCl, NaOH, THF, H
2
O; (l) CDI, 2,3-Diaminobenzamide dihydrochloride,
o
o
pyridine, DMF, 45 C; (m) AcOH, reflux; (n) H
2
, 10% Pd/C, MeOH, 50 C; (o) the corresponding side chain reactant, appropriate
alkaline condition.
2
2
.2. Biological activity
.2.1. Evaluation of PARP1/2 inhibitory activity of target compounds
The 31 designed compounds were first evaluated for the PARP1/2 inhibitory activity
using ELISA assay. Veliparib was used as a positive control. The inhibition rates at 10nM of
3
1 compounds and the IC values of 9 more effective compounds are summarized in Table
50
1
. Apparently, most of the target compounds showed significant PARP1/2 inhibitory activity
at 10nM. 9 Compounds (11f, 11g, 11h, 11i, 11j, 17d, 17f, 17g, 17h) exhibited potent activity
against both PARP1 and PARP2 enzyme, with IC50 values near or lower than 10nM. The
PARP1/2 inhibitory activity of 11f, 17d and17h were similar to Veliparib.
7

Table 1. The PARP1/2 inhibitory activity of compounds 11a-11p, 17a-17o.
%
Inhibition rate at 10nM
IC50(nM)
%Inhibition rate at 10nM
IC50(nM)
Comp.
Comp.
PARP1
PARP2
PARP1
PARP2
PARP1
PARP2
PARP1
PARP2
1
1a
1b
1c
1d
1e
1f
1g
1h
27.21
10.83
3.40
47.24
38.62
37.44
72.90
37.40
86.39
76.76
81.34
75.20
82.51
71.26
61.75
59.04
65.03
76.73
77.65
/
/
17a
17b
12.96
7.16
77.58
72.07
72.31
90.82
13.31
56.28
53.98
71.86
50.95
68.66
37.06
59.19
83.60
85.57
59.53
86.94
/
/
1
/
/
/
/
1
/
/
17c
14.14
47.9
/
/
1
27.06
22.07
66.15
68.02
41.42
47.17
65.51
37.59
37.92
6.38
/
/
17d
4.30
1.58
1
/
/
17e
16.31
38.57
70.19
60.13
31.79
31.62
19.06
57.80
50.18
61.25
21.89
72.65
/
/
1
2.26
2.50
17f
10.41
3.22
1
3.64
3.15
17g
4.36
2.57
1
11.22
1.94
17h
4.60
1.57
1
1i
6.89
3.65
17i
/
/
/
/
1
1j
5.46
2.50
17j
/
1
1k
1l
1m
1n
/
/
/
/
/
/
/
/
/
/
/
/
17k
/
1
17l
/
1
17m
17n
/
/
1
4.70
/
/
/
/
1
1o
18.84
24.39
17o
1
1p
Veliparib
3.30
1.51
2
.2.2. Evaluation of cytotoxicity of target compounds
To investigate the relationship between anticancer activity and PARP inhibitory activity,
7
more effective compounds were further evaluated for the in vitro cytotoxicity against four
human cancer cell lines, one with mutant BRCA1 (MDA-MB-436, breastꢀcancer), two with
nonmutant BRCA1/2 (MDA-MB-231 and MCF-7, breastꢀcancer), and one with
mutantꢀBRCA2ꢀ(CAPAN-1, pancreaticꢀcancer) using MTS assay.[30] Veliparib was also used
as a positive control. The IC values for each compound are summarized in Table 2.
5
0
8

Table 2. The anti-cancer activity of compounds 11f, 11g, 11i, 11j, 17d, 17g, 17h.
MDA-MB-436
MDA-MB-231
MCF-7
CAPAN-1
Comp.
IC50(μM)
IC50(μM)
IC50(μM)
IC50(μM)
1
1f
1g
1i
1j
7d
7g
7h
Veliparib
27.81
11.97
14.61
40.55
28.33
15.82
59.10
15.96
33.67
27.29
33.40
49.02
96.83
41.88
> 100
42.08
60.95
7.22
> 100
29.90
> 100
> 100
> 100
28.78
> 100
> 100
1
1
55.12
74.60
60.81
7.53
1
1
1
1
70.17
65.37
In the cytotoxic assay, 11g and 17g showed the best antineoplastic activity against all
four cell lines. On the contrary, 11i and 17h exhibited the weakest antitumor activity against
four cell lines. However, compared with the other three cell lines, 11f, 11i and 17d showed
considerable selective cytotoxic activity against MDA-MB-436 cell line. In general, most of
the compounds with good PARP1/2 inhibitory activity also showed significant anticancer
activity. This finding implied a significant correlation between PARP1/2 inhibitory activity
and anticancer activity.
2
.3. Evaluation of microsomal stability of target compounds
The liver is the main organ of drug metabolism in the body. Subcellular fractions such as
liver microsomes are useful in vitro models of hepatic clearance as they contain many of the
drug metabolising enzymes found in the liver. Microsomal stability assay was used to
determine the in vitro intrinsic clearance of 11f, 11g, 11i, 11j, 17d, 17g and 17h by
monitoring the rate of disappearance of parent compounds following incubation with human
and rat liver microsomes. The results revealed that 17d possessed the most high T1/2 and low
CL in both HLM and RLM (Table 3). It demonstrated that 17d was most stable in human and
rat liver microsomes and more likely to be made into an anti-tumor drug.
9

Table 3. Metabolic stability of 11f, 11g, 11i, 11j, 17d, 17g, 17h in pooled human/rat liver microsomes.
Human liver microsomes (HLM) Rat liver microsomes (RLM)
Comp.
T
1/2 (min)
30.4
CL ( μL/min/mg)
T
1/2 (min)
31.5
CL ( μL/min/mg)
1
1f
1g
1i
1j
7d
7g
7h
45.6
56.2
26.7
17.1
5.3
44.0
55.2
80.1
12.2
＜1
1
24.7
25.1
1
51.9
17.3
1
81.0
114.0
1
262.6
39.9
＞1000
43.5
1
34.9
173.8
31.9
11.9
1
8.0
116.2
2
.4. Study on the early ADME properties of 17d
Further ADME researches were conducted to examine the pharmacological performance
of compound 17d as a drug.
2
.4.1. Evaluation of kinetic/thermodynamic solubility of 17d
Solubility properties of a compound is one of the most important considerations in drug
design and development. A chemical’s solubility or lack thereof has far reaching implications
throughout the development process, potentially impacting dosing route, formulation
strategies, bioavailability and the design of in vitro assays. Kinetic and Thermodynamic
solubility Assays were used to determine the apparent kinetic and thermodynamic solubility
in PBS buffer (pH 7.4) of 17d. The result showed that 17d had good kinetic and
thermodynamic solubility (98.33 μg/mL and 63.99 μg/mL, respectively, Table 4).
2
.4.2. Evaluation of permeability of 17d and whether 17d is a P-gp substrate
In addition to solubility, oral bioavailability is largely dependent on a drug’s
permeability. Moreover, P-glycoprotein (P-gp) is an ATP-binding cassette drug efflux
transporter which is apically expressed in the gastrointestinal tract, liver, kidney and brain
endothelium. Consequently, P-gp plays an important role in the oral bioavailability, CNS
distribution and biliary and renal elimination of drugs which are substrates of this transporter.
Permeability assay in hMDR1-MDCK Ⅱ was used to decide whether 17d was a P-gp
substrate or not and its permeability through hMDR1-MDCK Ⅱ cell monolayers. The results
1
0

demonstrated that 17d was a P-gp substrate with high permeability (Table 4).
2
.4.3. Evaluation of red blood cell (RBC) to plasma ratio of 17d
Calculation of pharmacokinetic parameters is typically performed by the analysis of drug
concentrations in plasma rather than whole blood. Therefore, pharmacokinetic parameters
calculated from the plasma data may be misleading if differences exist between
concentrations of the drug in the plasma and the red blood cells due to differential binding to
a specific component in the blood. The blood to plasma ratio determines the concentration of
the drug in whole blood compared to plasma and provides an indication of drug binding to
erythrocytes. At blood to plasma ratios of greater than 1 (usually as a consequence of the drug
distributing into the erythrocyte), the plasma clearance significantly overestimates blood
clearance and could exceed hepatic blood flow. Blood to plasma ratio can also be used to
understand potential haemotoxicity. The distribution of 17d between red blood cells and
plasma was determined by using RBC to plasma ratio assay, and the KRBC/PL is 1.58 (Table 4).
Table 4. Solubility, Permeability and RBC to plasma ratio results of 17d.
Thermodynamic
solubility
Efflux ratiob
(without P-gp
inhibitor)
RBC to plasma
ratio
Kinetic Solubility
Permeability
Comp.
a
-6
(μg/mL)
Papp (×10 cm/s)
(μg/mL)
K
RBC/PL
1
7d
98.33
63.99
27.30
20.80
1.58
-6
a Papp＞25×10 cm/s means the permeability is high.
b Efflux ratio＞2.0 indicates the test compound is a P-gp substrate.
2
.4.4. Evaluation of plasma protein binding of 17d
The extent of binding to plasma influences the way in which a drug distributes into
tissues in the body. If a compound is highly bound, then it is retained in the plasma, which
results in a low volume of distribution. This may impact on the therapeutic effects of the
compound by limiting the amount of free compound which is available to act at the target
molecule. Extensive plasma protein binding also limits the amount of free compound
available to be metabolised which can, in turn, reduce the clearance of the compound. Plasma
Protein Binding Assay was used to determine the plasma protein binding of 17d by using
Rapid Equilibrium Dialysis (RED) method. As listed in Table 5, the PPB rates varied greatly
among different species, and 17d bound considerable human plasma proteins.
1
1

Table 5. Plasma Protein Binding results of 17d.
Plasma Protein Binding (PPB)
Comp.
Human
Rat
Mouse
Dog
1
7d
56.3%
42.9%
24.3%
11.0%
2
.4.5. Pharmacokinetic evaluation of 17d metabolites in SD rats after i.v. and p.o.
administration
Pharmacokinetics is the study of the concentration of compound in the body over time,
and is related to the absorption, distribution, metabolism, excretion (ADME) of a compound.
In the pharmacokinetic rat model, we used i.v. and intragastric administration of the
metabolite of 17d and measured its pharmacokinetic parameters. As shown in Table 6, the
metabolite of 17d possessed good pharmacokinetic parameters and high oral bioavailability.
Table 6. Plasma concentrations (mg/mL) and PK parameters of 17d in male SD rats.
1
7d
i.v. (1 mg/kg)
p.o. (5 mg/kg)
T
1/2 (h)
max (h)
max (ng/mL)
2.50±1.70
-
4.76±0.48
0.67±0.29
T
C
251.7±64.0
398.9±108.8
410.8±115.6
2.23±0.92
8.37±3.73
2.59±0.82
-
193.3±42.8
999.0±248.3
1450.8±439.0
3.31±0.05
AUC0-t (h·ng/mL)
AUC0-∞ (h·ng/mL)
MRTlast (h)
Vd/F (L/kg)
Cl/F (L/h/kg)
F(%)
24.53±4.46
3.64±0.95
49.64±12.45
2
2
.5. Molecular modeling
.5.1 The binding mode of 17d with PARP1
7d was modified from A-620223, so it was docked into the active site of PARP1
1
complexed with A-620223 (PDB ID: 2RCW) using the SYBYL-X 2.0 protocol to elucidate its
interaction mode. As shown in Fig. 4, the amide group on the aromatic ring of 17d bound to
the NI site of PARP1 through hydrogen bonds with the Gly202 and Ser243 residues, and the
benzene ring of the benzimidazole formed additional π-π stacking interaction with the Tyr246
residue. Moreover, the fluorine atom on the six-membered ring of the side chain interacted
1
2

with the Ala209 residue, suggesting that 17d extended out of the nicotinamide pocket and
made further interaction with the amino acid residue in the ADP-ribose pocket.
Fig. 4. The binding mode of 17d with PARP1(PDB ID: 2RCW).
2
.5.2 Molecular dynamics simulations
In order to prove the reliability of the docking result, the binding mode between
compound 17d and PARP1 protein need to be further investigated, and molecular dynamics
MD) simulation of 50ns was carried out using AMBER14 software package. The RMSD plot
(
of Cα for the complex was shown in Fig. 5. After a few times, the RSMD fluctuation of the
complex was in a very small range between 0.5 Å and 1.5 Å, which indicated that the system
had reached a state of stability.
Fig. 5. RMSD values of the complex during 50 ns MD simulations. ( 17d colored red and PARP1 colored black)
The average MD structure of the complex was shown in Fig. 6, and the binding mode
of 17d had changed a little. The amide group on the aromatic ring of 17d bound to the NI site
of PARP1 through hydrogen bonds with the Lys242 and Ser243 residues, and the benzene
ring of the benzimidazole still formed additional π-π stacking interaction with the Tyr246
1
3

residue. Although 17d had no interaction with the Ala209 residue, it formed two new
hydrogen bonds with the Asp105 residue. These results validated the reliability of the docking
result.
Fig. 6. The binding mode of the MD average structure of 17d with PARP1(PDB ID: 2RCW).
2
.5.3 Validation of docking reliability
MD was based on the result of molecular docking, so it was necessary to verify the
reliability of the docking result. The structure of A-620223 was redocked into the binding site
of PARP1 (PDB ID: 2RCW) using the SYBYL-X 2.0 protocol to compare the docking pose
with that of its original crystal structure. Its redocking pose and original docking pose were
superimposed. As shown in Fig. 7, the redocking pose and the original docking pose were in
similar spatial orientations with the similarity being 0.60. The numerical value of similarity is
closer to 1, the docking result is more reliable. The result suggested that the docking result
was reasonable and could be used for further simulation and analysis.
Fig. 7. Superimposing of redocking pose (green) of the ligand (A-620223) and its original docking pose (blue).
3
. Conclusions
1
4

A structure activity relationship study revealed that the introduction of short straight
chains or unsaturated five/six-membered heterocycles to the nitrogen atom on the
five/six-membered cyclic amine had no evident effect on activity, whereas an increase in
activity was observed with aromatic nucleus or saturated six-membered rings/heterocycles
substitution. When a carbonyl group existed between the five/six-membered cyclic amine and
the benzene ring, the activity was significantly increased.
In summary, two series of cyclic amine-containing benzimidazole carboxamide
derivatives have been synthesized and evaluated in vitro for PARP1/2 inhibitory activity and
potential cytotoxic activity against four types of cancer cell lines. Most of the compounds
under investigation exhibited significant PARP1/2 inhibitory activity. Among them, 11g and
1
1
7g showed the best antineoplastic activity against all four cancer cell lines. And 11f, 11i and
7d not only showed significant PARP1/2 inhibitory activity, but also exhibited obvious
selective anti-proliferative activity against the MDA-MB-436 cancer cell line. Early in vitro
ADME studies revealed that 17d possessed good solubility and permeability, moderate
binding rate with human plasma proteins, and good stability in human and rat liver
microsomes. Further in vivo pharmacokinetic experiment on SD rats demonstrated that 17d
was endowed with favorable ADME properties. The findings highlighted the potential of
these derivatives as new anticancer agents and 17d as a candidate for the treatment of cancer.
Further detailed research will be conducted to evaluate the molecular mechanism underlying
the anticancer activity of these compounds.
4
. Experimental Section
4
.1 Chemistry
All chemicals and reagents were purchased from commercial suppliers were of reagents
grade and used without further purification. Reactions were monitored by TLC, performed on
silica gel glass plates containing 60 GF-254, and visualization was achieved by UV light
(λmax = 254 or 365nm). Purification of compounds was done through silica gel (200-300
1
13
mesh) column chromatography. H-NMR and C-NMR spectra were recorded with Bruker
AV-400 NMR spectrometers using TMS as internal standard. Mass spectral data were
obtained by electron spray ionization on a Micromass ZabSpec high-resolution mass
spectrometer.
1
5

Note: Only the synthesis and characterization of target compounds are presented in this
article. The intermediates mentioned in Scheme 1 and 2 are described in Supplementary
Materials.
4
.1.1. General synthetic procedures for the synthesis of compounds (11a–11c, 11g)
To a solution of 10a (1.0mmol, 1eq.) in 12mL DMF were added the corresponding side
chain reactant (1.2mmol, 1.2eq.) and K CO (2.0mmol, 2eq.). The reaction mixture was
2
3
stirred at 50℃ for about 3h. After completion of reaction (monitored by TLC), the mixture
was poured into ice water, the formed precipitate was filtered, washed with water and dried
under vacuum. The crude product was purified by column chromatography on silica gel to get
the target compounds (11a–11c , 11g).
2
-(1-(2-fluoroethyl)pyrrolidin-3-yl)-1H-benzo[d]imidazole-4-carboxamide
(11a).
1
Obtained in 46.7% yield. H-NMR (400MHz , CDCl ) δ (ppm): 7.99 (s, 1H), 7.63 (d, J =
3
5
1
.7 Hz, 1H), 7.31 (t, J = 7.8 Hz, 1H), 5.99 (s, 1H), 4.73 (t, J = 4.8 Hz, 1H), 4.61 (t, J = 4.8 Hz,
H), 3.77 (td, J = 6.6, 3.4 Hz, 1H), 3.23 (d, J = 9.8 Hz, 2H), 3.10 – 2.88 (m, 2H), 2.86 – 2.73
1
3
(
m, 1H), 2.63 – 2.39 (m, 2H), 2.16 – 2.04 (m, 1H); C-NMR (100MHz, MeOD) δ (ppm):
1
8
69.29 (s), 158.31 (s), 139.51 (s), 136.90 (s), 122.25 (s), 121.46 (s), 120.16 (s), 116.37 (s),
2.20 (d, J = 167.1 Hz), 58.88 (s), 55.27 (d, J = 19.7 Hz), 53.85 (s), 37.03 (s), 29.82 (s);
+
HRMS-ESI (m/z) Calcd. for C H FN O [M + H] : 277.1465, Found: 277.1472.
1
4
18
4
2
-(1-(2,2-difluoroethyl)pyrrolidin-3-yl)-1H-benzo[d]imidazole-4-carboxamide
(11b).
1
Obtained in 23.3% yield. H-NMR (400MHz , MeOD) δ (ppm): δ 7.85 (d, J = 7.4 Hz, 1H),
7
.66 (d, J = 7.9 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 6.02 (tt, J = 55.9, 4.3 Hz, 1H), 3.80 – 3.62
(m, 1H), 3.25 – 3.16 (m, 1H), 3.13 – 2.79 (m, 5H), 2.48 – 2.32 (m, 1H), 2.25 (td, J = 13.6, 6.9
1
3
Hz, 1H); C-NMR (100MHz, MeOD) δ (ppm): 169.32 (s), 158.38 (s), 139.39 (s), 136.56 (s),
1
2
2
22.22 (s), 121.46 (s), 120.11 (s), 116.45 (s), 115.80 (t, J = 240.2 Hz), 59.23 (s), 57.05 (t, J =
+
5.0 Hz), 54.17 (s), 37.18 (s), 29.91 (s); HRMS-ESI (m/z) Calcd. for C H F N O [M + H] :
1
4
17
2
4
95.1370, Found: 295.1370.
2
-(1-(2,2,2-trifluoroethyl)pyrrolidin-3-yl)-1H-benzo[d]imidazole-4-carboxamide (11c).
1
Obtained in 37.1% yield. H-NMR (400MHz , CDCl ) δ (ppm): 7.96 (s, 1H), 7.66 (d, J =
3
7
3
.6 Hz, 1H), 7.33 (t, J = 7.8 Hz, 1H), 6.00 (s, 1H), 3.88 – 3.74 (m, 1H), 3.37 – 3.18 (m, 4H),
.05 – 2.92 (m, 1H), 2.75 (dd, J = 16.5, 8.7 Hz, 1H), 2.57 – 2.40 (m, 1H), 2.22 – 2.06 (m,
1
6

1
3
1
1
3
3
H); C-NMR (100MHz, MeOD) δ (ppm): 169.32 (s), 158.08 (s), 139.67 (s), 136.87 (s),
22.26 (q, J = 278.7 Hz), 122.25 (s), 121.47 (s), 120.11 (s), 116.39 (s), 59.08 (s), 55.47 (q, J =
+
1.5 Hz), 54.10 (s), 37.37 (s), 30.06 (s); HRMS-ESI (m/z) Calcd. for C H F N O [M + H] :
1
4
16
3
4
13.1276, Found: 313.1261.
2
-(1-(3-oxo-3-phenylpropyl)pyrrolidin-3-yl)-1H-benzo
[d]imidazole-4-carboxamide
1
(
11g). Obtained in 69.0% yield. H-NMR (400MHz , DMSO-d ) δ (ppm): 9.10 (s, 1H), 8.05
6
–
7
4
1
1
7.94 (m, 2H), 7.80 (d, J = 7.5 Hz, 1H), 7.77 – 7.60 (m, 3H), 7.60 – 7.48 (m, 2H), 7.34 –
.19 (m, 1H), 4.08 – 3.96 (m, 1H), 3.93 – 3.79 (m, 1H), 3.72 – 3.60 (m, 3H), 3.60 – 3.43 (m,
1
3
H), 2.62 – 2.49 (m, 1H), 2.35 (s, 1H); C-NMR (100MHz, DMSO-d ) δ (ppm): 197.43 (s),
6
66.96 (s), 155.69 (s), 136.48 (s), 134.16 (s), 129.38 (s), 129.31 (s), 129.03 (s), 128.70 (s),
28.53 (s), 122.69 (s), 122.36 (s), 122.13 (s), 56.90 (s), 53.56 (s), 49.87 (s), 36.59 (s), 35.11
+
(
s), 29.83 (s); HRMS-ESI (m/z) Calcd. for C H N O [M + H] : 363.1821, Found: 363.1815.
2
1
23
4
2
4
.1.2. General synthetic procedures for the synthesis of compounds (11d–11f, 11h–11m)
To a solution of 10a (1.0mmol, 1eq.) in 12mL CH OH were added the corresponding
3
side chain reactant (1.2mmol, 1.2eq.) and NaBH CN (2.0mmol, 2eq.). The reaction mixture
3
was stirred at RT for about 7h. After completion of reaction (monitored by TLC), the mixture
was poured into ice water, the formed precipitate was filtered, washed with water and dried
under vacuum. The crude product was purified by column chromatography on silica gel to get
the target compounds (11d–11f , 11h-11m).
2
-(1-(4,4-difluorocyclohexyl)pyrrolidin-3-yl)-1H-benzo[d]imidazole-4-carboxamide
1
(
11d). Obtained in 78.4% yield. H-NMR (400MHz , MeOD) δ (ppm): 7.87 (d, J = 7.6 Hz,
1
3
2
H), 7.68 (d, J = 8.0 Hz, 1H), 7.30 (t, J = 7.8 Hz, 1H), 3.74 (td, J = 15.3, 7.4 Hz, 1H), 3.31 –
.25 (m, 1H), 3.08 – 2.78 (m, 3H), 2.52 – 2.34 (m, 2H), 2.28 (dt, J = 20.9, 6.7 Hz, 1H), 2.15 –
1
3
.03 (m, 3H), 1.95 – 1.50 (m, 5H); C-NMR (100MHz, MeOD) δ (ppm): 169.28 (s), 158.31
(s), 139.50 (s), 137.01 (s), 122.82 (t, J = 241.0 Hz), 122.24 (s), 121.46 (s), 120.13 (s), 116.49
(s), 60.64 (s), 56.35 (s), 51.17 (s), 36.95 (s), 31.26 (t, J = 24.6 Hz), 29.66 (s), 27.19 (d, J = 7.1
+
Hz); HRMS-ESI (m/z) Calcd. for C H F N O [M + H] : 349.1840, Found: 349.1845.
1
8
23
2
4
2
-(1-(pyridin-2-ylmethyl)pyrrolidin-3-yl)-1H-benzo[d]imidazole-4-carboxamide(11e).
1
Obtained in 81.1% yield. H-NMR (400MHz , DMSO-d ) δ (ppm): 9.10 (s br, 1H), 8.51 (d,
6
J = 4.2 Hz, 1H), 7.90 – 7.74 (m, 2H), 7.75 – 7.56 (m, 2H), 7.46 (d, J = 7.8 Hz, 1H), 7.34 –
1
7

7
2
.15 (m, 2H), 3.97 (s, 2H), 3.75 – 3.72 (m, 1H), 3.29 – 3.14 (m, 1H), 3.13 – 3.01 (m, 1H),
.99 – 2.79 (m, 2H), 2.44 – 2.27 (m, 1H), 2.29 – 2.09 (m, 1H); 13C-NMR (100MHz,
DMSO-d ) δ (ppm): 167.34 (s), 158.52 (s), 157.81 (s), 149.27 (s), 139.86 (s), 137.31 (s),
6
1
5
3
36.60 (s), 123.34 (s), 122.99 (s), 122.47 (s), 121.86 (s), 120.68 (s), 116.38 (s), 60.74 (s),
+
8.66 (s), 53.80 (s), 37.06 (s), 30.03 (s); HRMS-ESI (m/z) Calcd. for C H N O [M + H] :
1
8
20
5
22.1668, Found: 322.1664.
2
-(1-((1H-Indol-5-yl)methyl)pyrrolidin-3-yl)-1H-benzo[d]imidazole-4-carboxamide(11f)
1
.
Obtained in 87.6% yield. H-NMR (400MHz , MeOD) δ (ppm): 7.91 (d, J = 7.0 Hz, 1H),
.77 (d, J = 1.1 Hz, 1H), 7.72 (dd, J = 8.0, 0.7 Hz, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.40 – 7.31
7
(m, 2H), 7.29 (dd, J = 8.4, 1.7 Hz, 1H), 6.55 (dd, J = 3.1, 0.7 Hz, 1H), 4.56 – 4.44 (m, 2H),
4
2
.07 (dt, J = 15.5, 7.6 Hz, 1H), 3.93 – 3.75 (m, 2H), 3.64 – 3.45 (m, 2H), 2.75 – 2.61 (m, 1H),
1
3
.50 (dt, J = 20.8, 7.1 Hz, 1H); C-NMR (100MHz, DMSO-d ) δ (ppm): 167.27 (s), 156.18
6
(s), 136.47 (s), 128.20 (s), 126.89 (s), 123.45 (s), 123.20 – 123.12 (m), 122.54 (dd, J = 62.0,
3
1
5.1 Hz), 122.74 – 120.75 (m), 122.24 (d, J = 36.1 Hz), 120.70 (s), 115.37 (s), 112.22 (s),
01.83 (s), 59.14 (s), 56.81 (s), 53.37 (s), 36.33 (s), 29.45 (s); HRMS-ESI (m/z) Calcd. for
+
C H N O [M + H] : 360.1824, Found: 360.1814.
2
1
22
5
2
-(1-(1-(4-methoxyphenyl)propan-2-yl)pyrrolidin-3-yl)-1H-benzo[d]imidazole-4-carbox
1
amide(11h). Obtained in 70.4% yield. H-NMR (400MHz , MeOD) δ (ppm): 7.91 (d, J =
7
.5 Hz, 1H), 7.73 (d, J = 7.9 Hz, 1H), 7.41 – 7.32 (m, 1H), 7.20 (d, J = 8.3 Hz, 2H), 6.92 (d, J
7.1 Hz, 2H), 4.00 – 3.89 (m, 1H), 3.86 – 3.75 (m, 4H), 3.74 – 3.55 (m, 2H), 3.44 – 3.36 (m,
=
1
3
2
H), 3.31 – 3.18 (m, 2H), 2.68 – 2.54 (m, 2H), 2.48 – 2.36 (m, 1H), 1.21 (s, 3H); C-NMR
100MHz, DMSO-d ) δ (ppm): 167.21 (s), 158.32 (s), 157.95 (s), 131.93 (s), 131.05 (s),
(
6
1
5
30.81 (s), 130.79 (s), 130.71 (s), 122.51 (s), 121.91 (s), 114.29 (s), 113.92 (s), 67.91 (s),
5.48 (s), 55.43 (s), 50.97 (s), 44.99 (s), 36.82 (s), 29.90 (s), 23.46 (s); HRMS-ESI (m/z)
+
Calcd. for C H N O [M + H] : 379.2134, Found: 302.2135.
2
2
27
4
2
2
-(1-(4-(dimethylamino)benzyl)pyrrolidin-3-yl)-1H-benzo[d]imidazole-4-carboxamide
1
(
11i). Obtained in 62.5% yield. H-NMR (400MHz , DMSO-d ) δ (ppm): 9.03 (s, 1H), 7.79
6
(d, J = 7.5 Hz, 1H), 7.71 (s, 1H), 7.66 (d, J = 7.9 Hz, 1H), 7.31 – 7.16 (m, 3H), 6.69 (d, J =
8
1
.6 Hz, 2H), 4.06 – 3.86 (m, 2H), 3.87 – 3.74 (m, 1H), 3.45 – 3.28 (m, 1H), 3.28 – 3.13 (m,
1
3
H), 3.13 – 2.95 (m, 2H), 2.86 (s, 6H), 2.46 – 2.33 (m, 1H), 2.34 – 2.20 (m, 1H); C-NMR
1
8

(
100MHz, DMSO-d ) δ (ppm): 167.21 (s), 157.31 (s), 150.66 (s), 139.22 (s), 130.81 (s),
6
1
22.48 (s), 121.87 (s), 121.52 (s), 116.75 (s), 113.21 (s), 112.58 (s), 58.47 (s), 57.46 (s), 53.24
+
(
s), 40.48 (s), 36.63 (s), 29.58 (s); HRMS-ESI (m/z) Calcd. for C H N O [M + H] :
2
1
26
5
3
64.2137, Found: 364.2132.
2
-(1-((1H-pyrrol-2-yl)methyl)pyrrolidin-3-yl)-1H-benzo[d]imidazole-4-carboxamide
1
(
11j). Obtained in 60.3% yield. H-NMR (400MHz , MeOD) δ (ppm): 7.91 (dd, J = 7.6, 0.8
Hz, 1H), 7.73 (dd, J = 8.0, 0.8 Hz, 1H), 7.36 (t, J = 7.8 Hz, 1H), 6.92 (dd, J = 2.7, 1.5 Hz,
1
6
1
1
1
H), 6.42 (dd, J = 3.4, 1.3 Hz, 1H), 6.27 – 6.14 (m, 1H), 4.48 (s, 2H), 4.06 (ddd, J = 15.2, 8.4,
.7 Hz, 1H), 3.99 – 3.89 (m, 1H), 3.89 – 3.76 (m, 1H), 3.66 – 3.43 (m, 2H), 2.79 – 2.58 (m,
1
3
H), 2.55 – 2.37 (m, 1H); C-NMR (100MHz, DMSO-d ) δ (ppm): 167.22 (s), 155.82 (s),
6
38.97 (s), 122.33 (d, J = 55.8 Hz), 121.99 – 121.95 (m), 121.83 (s), 121.45 (s), 120.13 (s),
17.35 (s), 114.09 (s), 111.24 (s), 108.81 (s), 56.32 (s), 52.96 (s), 50.81 (s), 36.28 (s), 29.54
+
(
s); HRMS-ESI (m/z) Calcd. for C H N O [M + H] : 310.1668, Found: 310.1666.
1
7
20
5
2
-(1-(furan-2-ylmethyl)pyrrolidin-3-yl)-1H-benzo[d]imidazole-4-carboxamide
(11k).
1
Obtained in 48.0% yield. H-NMR (400MHz , MeOD) δ (ppm): 7.91 (dd, J = 7.6, 0.8 Hz,
1
6
H), 7.72 (dd, J = 8.0, 0.7 Hz, 1H), 7.61 (dd, J = 1.7, 0.6 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H),
.56 (d, J = 3.1 Hz, 1H), 6.53 – 6.41 (m, 1H), 4.21 (s, 2H), 4.03 – 3.82 (m, 1H), 3.66 – 3.44
1
3
(
m, 2H), 3.27 (t, J = 7.2 Hz, 2H), 2.70 – 2.48 (m, 1H), 2.48 – 2.32 (m, 1H); C-NMR
(
100MHz, DMSO-d ) δ (ppm): 167.21 (s), 157.76 (s), 150.59 (s), 143.54 (s), 139.67 (s),
6
1
5
3
36.96 (s), 122.51 (s), 121.91 (s), 121.50 (s), 116.80 (s), 111.13 (s), 110.21 (s), 57.95 (s),
+
3.38 (s), 50.77 (s), 36.91 (s), 29.85 (s); HRMS-ESI (m/z) Calcd. for C H N O [M + H] :
1
7
19
4
2
11.1508, Found: 311.1493.
2
-(1-(tetrahydro-2H-pyran-4-yl)pyrrolidin-3-yl)-1H-benzo[d]imidazole-4-carboxamide
1
(
11l). Obtained in 83.0% yield. H-NMR (400MHz , MeOD) δ (ppm): 7.91 (dd, J = 7.6, 0.9
Hz, 1H), 7.72 (dd, J = 8.0, 0.9 Hz, 1H), 7.35 (t, J = 7.8 Hz, 1H), 4.17 – 3.94 (m, 3H), 3.89 –
3
1
1
6
.70 (m, 2H), 3.58 – 3.41 (m, 4H), 3.32 – 3.22 (m, 1H), 2.73 – 2.55 (m, 1H), 2.54 – 2.35 (m,
1
3
H), 2.20 – 2.04 (m, 2H), 1.87 – 1.64 (m, 2H); C-NMR (100MHz, DMSO-d ) δ (ppm):
6
67.37 (s), 156.42 (s), 139.23 (s), 137.39 (s), 122.64 (s), 122.07 (s), 121.36 (s), 117.18 (s),
5.80 (s), 60.73 (s), 54.93 (s), 51.00 (s), 36.30 (s), 30.31 (s), 29.58 (s); HRMS-ESI (m/z)
+
Calcd. for C H N O [M + H] : 315.1821, Found: 315.1836.
1
7
23
4
2
1
9

2
-(1-(1-methylpiperidin-4-yl)pyrrolidin-3-yl)-1H-benzo[d]imidazole-4-carboxamide
1
(
11m). Obtained in 34.0% yield. H-NMR (400MHz , MeOD) δ (ppm): 7.88 (d, J = 7.6 Hz,
1
2
2
H), 7.71 (d, J = 8.0 Hz, 1H), 7.32 (t, J = 7.8 Hz, 1H), 3.82 – 3.72 (m, 1H), 3.31 – 3.21 (m,
H), 3.20 – 3.11 (m, 2H), 3.11 – 3.04 (m, 1H), 2.95 (t, J = 6.9 Hz, 2H), 2.50 (s, 3H), 2.48 –
1
3
.41 (m, 2H), 2.36 – 2.21 (m, 2H), 2.14 – 2.02 (m, 2H), 1.85 – 1.66 (m, 2H); C-NMR
(
100MHz, DMSO-d ) δ (ppm): 167.10 (s), 158.83 (s), 141.10 (s), 135.20 (s), 122.33 (s),
6
1
3
3
4
21.69 (s), 116.51 (s), 115.11 (s), 59.66 (s), 56.39 (s), 53.53 (s), 50.99 (s), 45.15 (s), 36.98 (s),
+
0.18 (s), 29.86 (s); HRMS-ESI (m/z) Calcd. for C H N O [M + H] : 328.2137, Found:
1
8
26
5
28.2142.
.1.3. Synthetic procedure for 2- (1-butyryl-3-fluoropyrrolidin-3-yl) -1H-benzo [d]
imidazole-4-carboxamide (11n)
To a solution of 10b (1.0mmol, 1eq.) in 10mL DCM were added DIPEA (1.5mmol,
o
o
1
.5eq.) and butyryl chloride (1.2mmol, 1.2eq.) at 0 C. The mixture was stirred at 0 C for
another 1h and at RT overnight. The formed precipitate was filtered, washed with DCM and
1
dried under vacuum. Obtained in 54.6% yield. H-NMR (400MHz , DMSO-d ) δ (ppm):
6
1
1
2
3.49 (s, 1H), 9.00 (s, 1H), 7.88 (d, J = 7.3 Hz, 1H), 7.85 – 7.77 (m, 1H), 7.73 (d, J = 7.8 Hz,
H), 7.38 (t, J = 7.8 Hz, 1H), 4.34 – 3.90 (m, 2H), 3.88 – 3.44 (m, 2H), 2.88 – 2.52 (m, 2H),
.40 – 2.13 (m, 2H), 1.62 – 1.43 (m, 2H), 0.97 – 0.78 (m, 3H); 13C-NMR (100MHz,
DMSO-d ) δ (ppm): 171.45 (s), 171.19 (s), 166.33 (s), 151.43 (d, J = 26.5 Hz), 140.54 (s),
6
1
5
5
3
4
35.19 (s), 123.69 (s), 123.56 (d, J = 24.8 Hz), 116.08 (s), 100.83 (s), 99.29 (s), 97.54 (s),
5.82 (s), 55.57 (s), 44.93 (s), 44.32 (s), 37.05 (s), 36.82 (s), 36.05 (s), 35.64 (s), 18.15 (d, J =
+
.3 Hz), 14.28 (s); HRMS-ESI (m/z) Calcd. for C H FN O [M + Na] : 341.1390, Found:
1
6
20
4
2
41.1580.
.1.4. Synthetic procedure for 2- (1- (cyclopropanecarbonyl) -3-fluoropyrrolidin-3-yl)
-1H-benzo [d] imidazole-4-carboxamide (11o)
To a solution of 10b (1.0mmol, 1eq.) in 10mL DCM were added DIPEA (1.5mmol,
o
1
.5eq.) and cyclopropanecarbonyl chloride (1.2mmol, 1.2eq.) at 0 C. The mixture was stirred
o
at 0 C for another 1h and at RT overnight. The mixture was poured into ice water and
extracted with DCM. The organic phases were combined, dried with MgSO and concentrated
4
in vacuo. The crude product was purified by column chromatography on silica gel to get 11o.
2
0

1
Obtained in 66.9% yield. H-NMR (400MHz , DMSO-d ) δ (ppm): 13.50 (s, 1H), 9.01 (s,
6
1
3
H), 7.88 (d, J = 6.5 Hz, 1H), 7.83 – 7.61 (m, 2H), 7.48 – 7.19 (m, 1H), 4.57 – 3.97 (m, 2H),
1
3
.97 – 3.43 (m, 2H), 2.94 – 2.54 (m, 2H), 1.89 – 1.70 (m, 1H), 0.87 – 0.66 (m, 4H); C-NMR
(
100MHz, DMSO-d ) δ (ppm): 171.98 (s), 171.69 (s), 166.40 (s), 151.34 (dd, J = 26.4, 8.6
6
Hz), 140.53 (s), 135.25 (s), 124.53 – 123.77 (m), 123.56 (d, J = 20.2 Hz), 116.09 (s), 100.73
(
s), 99.19 (s), 98.97 (s), 97.45 (s), 56.68 – 55.81 (m), 56.00 (s), 55.88 (d, J = 24.3 Hz), 55.39
s), 45.06 (s), 44.67 (s), 36.74 (d, J = 22.2 Hz), 36.62 – 35.87 (m), 29.35 (d, J = 30.9 Hz),
(
1
2.57 (s), 12.18 (s), 7.73 (dd, J = 17.9, 4.4 Hz); HRMS-ESI (m/z) Calcd. for C H FN O [M
16 18 4 2
+
+
H] : 317.1414, Found: 317.1407.
4
.1.5. Synthetic procedure for 2- (1- (2- (3-bromophenyl) acetyl) -3-fluoropyrrolidin-3-yl)
-1H-benzo [d] imidazole -4-carboxamide (11p)
To a solution of 10b (1mmol, 1.0eq.) in 10mL CH CN were added 2-(3-bromophenyl)
3
acetic acid (1.2mmol, 1.2eq.) , TBTU (1.5 mmol, 1.5eq.) in CH CN (8mL) and DIPEA
3
(2.0mmol, 2.0eq.). The mixture was stirred at RT for 4.5 h. The mixture was poured into ice
water and extracted with DCM. The organic phases were combined, dried with MgSO and
4
concentrated in vacuo. The crude product was purified by column chromatography on silica
1
gel to get 11p. Obtained in 53.2% yield. H-NMR (400MHz , DMSO-d ) δ (ppm): 13.51 (s,
6
1
H), 9.00 (s, 1H), 7.95 – 7.87 (m, 1H), 7.87 – 7.77 (m, 1H), 7.77 – 7.69 (m, 1H), 7.50 – 7.45
(m, 1H), 7.45 – 7.34 (m, 2H), 7.29 – 7.21 (m, 2H), 4.33 – 4.09 (m, 1H), 4.09 – 3.91 (m, 1H),
3
.91 – 3.65 (m, 3H), 3.64 – 3.47 (m, 1H), 2.90 – 2.55 (m, 2H); 13C-NMR (100MHz,
DMSO-d ) δ (ppm): 169.26 (s), 168.98 (s), 166.35 (s), 151.89 – 151.09 (m), 151.07 (s),
6
1
1
1
4
1
4
40.57 (d, J = 4.4 Hz), 138.76 (s), 135.16 (s), 132.81 (d, J = 2.8 Hz), 130.75 (d, J = 2.3 Hz),
29.79 (d, J = 1.7 Hz), 129.24 (s), 123.74 (d, J = 6.3 Hz), 123.47 (s), 121.87 (d, J = 0.9 Hz),
16.11 (s), 100.97 (s), 99.26 (s), 97.50 (s), 56.66 (s), 56.43 (s), 56.06 (s), 55.86 (s), 45.28 (s),
4.73 (s), 42.35 (s), 37.00 (s), 36.76 (s), 35.61 (d, J = 22.0 Hz), 35.47 – 35.23 (m), 18.58 (s),
+
7.23 (s); HRMS-ESI (m/z) Calcd. for C H BrFN O [M + H] : 445.0675, Found: 445.0695.
2
0
19
4
2
.1.5. General synthetic procedure for the synthesis of compounds (17a–17c, 17g)
To a solution of 16 (1.0mmol, 1eq.) in 12mL DMF were added the corresponding side
chain reactant (1.2mmol, 1.2eq.) and K CO (2.0mmol, 2eq.). The reaction mixture was
2
3
stirred at 50℃ for about 3h. After completion of reaction (monitored by TLC), the mixture
2
1

was poured into ice water, the formed precipitate was filtered, washed with water and dried
under vacuum. The crude product was purified by column chromatography on silica gel to get
the target compounds (17a–17c , 17g).
2
-(1-(2-fluoroethyl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide
(17a).
1
Obtained in 49.5% yield. H-NMR (400MHz , MeOD) δ (ppm): 7.90 (s, 1H), 7.65 (s, 1H),
7
3
2
1
8
.30 (t, J = 7.8 Hz, 1H), 4.73 – 4.63 (m, 1H), 4.61 – 4.49 (m, 1H), 3.13 (d, J = 11.9 Hz, 2H),
.02 (t, J = 11.3 Hz, 1H), 2.87 – 2.79 (m, 1H), 2.79 – 2.68 (m, 1H), 2.35 (td, J = 11.7, 2.3 Hz,
1
3
H), 2.15 (d, J = 11.6 Hz, 2H), 2.10 – 1.99 (m, 2H); C-NMR (100MHz, MeOD) δ (ppm):
69.31 (s), 158.97 (s), 141.35 (s), 134.79 (s), 122.29 (s), 121.37(s), 120.85 (s), 114.91 (s),
1.19 (d, J = 167.1 Hz), 58.09 (d, J = 19.9 Hz), 53.24 (s), 35.80 (s), 29.95 (s); HRMS-ESI
+
(
m/z) Calcd. for C H FN O [M + H] : 291.1621, Found: 291.1628.
1
5
20
4
2
-(1-(2,2-difluoroethyl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide
(17b).
1
Obtained in 12.8% yield. H-NMR (400MHz , MeOD) δ (ppm): 7.87 (s, 1H), 7.67 (d, J =
7
–
4
1
3
.4 Hz, 1H), 7.29 (t, J = 7.8 Hz, 1H), 6.02 (tt, J = 55.9, 4.3 Hz, 1H), 3.19 – 3.06 (m, 2H), 3.06
2.91 (m, 1H), 2.82 (td, J = 15.3, 4.3 Hz, 2H), 2.44 (td, J = 11.6, 2.5 Hz, 2H), 2.28 – 1.89 (m,
1
3
H); C-NMR (100MHz, MeOD) δ (ppm): 169.35 (s), 159.02 (s), 122.21 (s), 121.36 (s),
20.30 (s), 115.83 (s), 115.65 (t, J = 240.5 Hz), 59.88 (t, J = 24.9 Hz), 53.72 (s), 35.68 (s),
+
0.16 (s); HRMS-ESI (m/z) Calcd. for C H F N O [M + H] : 309.1527, Found: 309.1521.
1
5
19
2
4
2
-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide (17c).
1
Obtained in 23.7% yield. H-NMR (400MHz , MeOD) δ (ppm): 7.91 (s, 1H), 7.70 (d, J =
7
–
.7 Hz, 1H), 7.35 – 7.28 (m, 1H), 6.16 (s, 1H), 3.18 – 3.09 (m, 2H), 3.08 – 2.98 (m, 3H), 2.62
1
3
2.51 (m, 2H), 2.19 – 2.07 (m, 4H); C-NMR (100MHz, MeOD) δ (ppm): 169.36 (s), 159.06
(s), 135.03 (s), 125.84 (q, J = 280.3 Hz), 122.21 (s) , 121.35 (s), 120.59 (s), 115.14 (s), 57.17
(
q, J = 30.5 Hz), 53.57 (s), 35.65 (s), 30.38 (s); HRMS-ESI (m/z) Calcd. for C H F N O [M
1
5
18
3
4
+
+
H] : 327.1433, Found: 327.1448.
2
-(1-(3-oxo-3-phenylpropyl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide (17g).
1
Obtained in 87.2% yield. H-NMR (400MHz , DMSO-d ) δ (ppm): 13.01 (s, 1H), 9.30 (d, J
6
=
2.4 Hz, 1H), 8.00 (d, J = 7.4 Hz, 2H), 7.80 (d, J = 7.5 Hz, 1H), 7.74 (d, J = 2.7 Hz, 1H),
7
3
.69 – 7.61 (m, 2H), 7.57 – 7.50 (m, 2H), 7.31 – 7.19 (m, 1H), 3.60 – 3.47 (m, 4H), 3.23 –
1
3
.08 (m, 3H), 2.95 – 2.75 (m, 2H), 2.30 – 2.18 (m, 2H), 2.16 – 2.03 (m, 2H); C-NMR
2
2

(
100MHz, DMSO-d ) δ (ppm): 198.15 (s), 166.83 (s), 158.17 (s), 141.15 (s), 136.75 (s),
6
1
5
35.06 (s), 134.01 (s), 129.29 (s), 128.51 (s), 122.71 (s), 122.52 (s), 122.09 (s), 115.27 (s),
2.17 (s), 34.55 (s), 34.20 (s), 29.51 (s), 28.65 (s); HRMS-ESI (m/z) Calcd. for C H N O
2
2
2
25
4
+
[
M + H] : 377.1978, Found: 377.1974.
4
.1.6. General synthetic procedure for the synthesis of compounds (17d–17f, 17h–17o)
To a solution of 16 (1.0mmol, 1eq.) in 12mL CH OH were added the corresponding side
3
chain reactant (1.2mmol, 1.2eq.) and NaBH CN (2.0mmol, 2eq.). The reaction mixture was
3
stirred at RT for about 7h. After completion of reaction (monitored by TLC), the mixture was
poured into ice water, the formed precipitate was filtered, washed with water and dried under
vacuum. The crude product was purified by column chromatography on silica gel to get the
target compounds (17d–17f , 17h-17o).
2
-(1-(4,4-difluorocyclohexyl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide(17d)
1
.
Obtained in 26.3% yield. H-NMR (400MHz , MeOD) δ (ppm): 7.89 (s, 1H), 7.65 (s, 1H),
.30 (t, J = 7.8 Hz, 1H), 3.16 – 3.05 (m, 2H), 3.05 – 2.91 (m, 1H), 2.55 (t, J = 11.0 Hz, 1H),
.47 (td, J = 11.6, 2.1 Hz, 2H), 2.22 – 2.04 (m, 4H), 2.04 – 1.89 (m, 4H), 1.89 – 1.73 (m, 2H),
7
2
1
1
3
1
3
.73 – 1.57 (m, 2H); C-NMR (100MHz, MeOD) δ (ppm): 170.65 (s), 160.43 (s), 142.68 (s),
36.08 (s), 124.14 (t, J = 239.8 Hz), 124.12 (s), 122.72 (s), 116.26 (s), 62.73 (s), 50.20 (s),
7.62 (s), 33.67 (t, J = 25.4 Hz), 31.78 (s), 25.28 (d, J = 9.6 Hz); HRMS-ESI (m/z) Calcd. for
+
C H F N O [M + H] : 363.1996, Found: 363.2001.
1
9
25
2
4
2
-(1-(Pyridin-2-ylmethyl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide
(17e).
1
Obtained in 22.7% yield. H-NMR (400MHz , MeOD) δ (ppm): 8.57 (d, J = 4.2 Hz, 1H),
7
7
2
1
1
.93 – 7.80 (m, 2H), 7.67 (d, J = 7.9 Hz, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.43 – 7.34 (m, 1H),
.32 – 7.24 (m, 1H), 4.05 (s, 2H), 3.35 – 3.28 (m, 2H), 3.21 – 3.12 (m, 1H), 2.84 – 2.66 (m,
H), 2.27 – 2.10 (m, 4H); 13C-NMR (100MHz, MeOD) δ (ppm): 169.27 (s), 158.16 (s),
54.57 (s), 148.82 (s), 139.57 (s), 137.50 (s), 136.37 (s), 124.10 (s), 123.22 (s), 122.33 (s),
21.55 (s), 120.34 (s), 116.17 (s), 62.22 (s), 52.70 (s), 34.71 (s), 29.02 (s); HRMS-ESI (m/z)
+
Calcd. for C H N O [M + H] : 336.1824, Found: 336.1823.
1
9
22
5
2
-(1-((1H-Indol-5-yl)methyl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide (17f).
1
Obtained in 61.6% yield. H-NMR (400MHz , DMSO-d ) δ (ppm): 12.81 (s, 1H), 11.29 (s,
6
1
H), 9.26 (s, 1H), 7.86 – 7.60 (m, 4H), 7.48 (d, J = 8.3 Hz, 1H), 7.45 – 7.39 (m, 1H), 7.28 (t,
2
3

J = 7.7 Hz, 1H), 7.21 (d, J = 8.3 Hz, 1H), 6.49 (s, 1H), 4.36 (s, 2H), 3.57 – 3.35 (m, 4H), 3.29
1
3
–
3.20 (m, 1H), 2.39 – 2.18 (m, 2H), 2.18 – 1.89 (m, 2H); C-NMR (100MHz, DMSO-d ) δ
6
(
(
(
ppm): 166.80 (s), 157.62 (s), 141.14 (s), 136.72 (s), 134.91 (s), 128.22 (s), 127.12 (s), 124.10
d, J = 38.4 Hz), 123.53 (s), 122.72 (s), 122.46 (s), 122.28 (s), 120.39 (s), 115.35 (s), 112.25
s), 101.88 (s), 61.03 (s), 51.30 (s), 33.55 (s), 28.30 (s); HRMS-ESI (m/z) Calcd. for
+
C H N O [M + H] : 374.1981, Found: 374.1985.
2
2
24
5
2
-(1-(1-(4-Methoxyphenyl)propan-2-yl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxa
1
mide (17h). Obtained in 36.7% yield. H-NMR (400MHz , DMSO-d ) δ (ppm): 12.70 (s,
6
1
7
2
–
1
1
1
H), 9.36 (s, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.70 (d, J = 2.9 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H),
.27 (t, J = 7.8 Hz, 1H), 7.11 (d, J = 8.5 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 3.71 (s, 3H), 3.08 –
.75 (m, 5H), 2.59 – 2.51 (m, 1H), 2.40 (dd, J = 13.9, 10.6 Hz, 2H), 2.20 – 1.95 (m, 2H), 1.95
1
3
1.72 (m, 2H), 0.90 (d, J = 6.3 Hz, 3H); C-NMR (100MHz, DMSO-d ) δ (ppm): 166.32 (s),
6
58.87 (s), 157.42 (s), 140.74 (s), 134.47 (s), 131.98 (s), 129.97 (s), 122.01 (d, J = 11.5 Hz),
21.36 (s), 114.52 (s), 113.57 (s), 61.20 (s), 54.91 (s), 47.17 (s), 37.42 (s), 35.89 (s), 30.46 (s),
+
3.82 (s); HRMS-ESI (m/z) Calcd. for C H N O [M + H] : 393.2291, Found: 393.2288.
2
3
29
4
2
2
-(1-(4-(Dimethylamino)benzyl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide
1
(
17i). Obtained in 61.6% yield. H-NMR (400MHz , DMSO-d ) δ (ppm): 12.81 (s, 1H),
6
9
7
2
.27 (s, 1H), 7.82 (d, J = 7.5 Hz, 1H), 7.78 – 7.70 (m, 1H), 7.67 (d, J = 7.9 Hz, 1H), 7.35 –
.20 (m, 3H), 6.76 (d, J = 8.7 Hz, 2H), 4.10 (s, 2H), 3.34 – 3.13 (m, 3H), 3.11 – 2.94 (m, 2H),
1
3
.92 (s, 6H), 2.32 – 2.18 (m, 2H), 2.18 – 1.97 (m, 2H); C-NMR (100MHz, DMSO-d ) δ
6
(ppm): 166.80 (s), 157.68 (s), 151.32 (s), 141.14 (s), 134.93 (s), 132.37 (s), 122.70 (d, J =
2
5.9 Hz), 122.23 (s), 117.56 (s), 115.31 (s), 112.50 (s), 59.93 (s), 50.95 (s), 40.38 (s), 33.43
+
(
s), 28.04 (s); HRMS-ESI (m/z) Calcd. for C H N O [M + H] : 378.2294, Found: 378.2307.
2
2
28
5
2
-(1-((1H-Pyrrol-2-yl)methyl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide
1
(
17j). Obtained in 43.8% yield. H-NMR (400MHz , DMSO-d ) δ (ppm): 12.79 (s, 1H),
6
1
7
3
0.97 (s, 1H), 9.25 (s, 1H), 7.82 (d, J = 7.3 Hz, 1H), 7.73 (s, 1H), 7.67 (d, J = 7.8 Hz, 1H),
.29 (t, J = 7.6 Hz, 1H), 6.89 (s, 1H), 6.25 (s, 1H), 6.11 (s, 1H), 4.19 (s, 2H), 3.54 – 3.36 (m,
1
3
H), 3.10 – 2.90 (m, 2H), 2.39 – 2.19 (m, 2H), 2.19 – 1.95 (m, 2H); C-NMR (100MHz,
DMSO-d ) δ (ppm): 166.78 (s), 161.33 (s), 157.57 (s), 141.12 (s), 134.94 (s), 123.36 – 122.07
6
(m), 122.42 (d, J = 35.6 Hz), 122.42 (d, J = 35.6 Hz), 120.34 (s), 115.30 (s), 111.96 (s),
2
4

1
08.91 (s), 52.87 (s), 50.92 (s), 33.30 (s), 28.01 (s); HRMS-ESI (m/z) Calcd. for C H N O
18 22 5
+
[
M + H] : 324.1824, Found: 324.1828.
2
-(1-(Furan-2-ylmethyl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide
(17k).
1
Obtained in 38.3% yield. H-NMR (400MHz , DMSO-d ) δ (ppm): 12.68 (s, 1H), 9.34 (d, J
6
=
3.0 Hz, 1H), 7.79 (d, J = 7.1 Hz, 1H), 7.70 (d, J = 2.9 Hz, 1H), 7.60 (d, J = 7.5 Hz, 1H),
7
3
–
1
1
.58 (d, J = 1.1 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 6.39 (dd, J = 3.1, 1.9 Hz, 1H), 6.27 (d, J =
.0 Hz, 1H), 3.51 (s, 2H), 2.96 – 2.79 (m, 3H), 2.21 – 2.06 (m, 2H), 2.05 – 1.94 (m, 2H), 1.90
1
3
1.72 (m, 2H); C-NMR (100MHz, DMSO-d ) δ (ppm): 166.85 (s), 159.45 (s), 152.39 (s),
6
42.81 (s), 141.25 (s), 135.00 (s), 122.53 (d, J = 13.6 Hz), 121.90 (s), 115.05 (s), 110.80 (s),
09.11 (s), 54.74 (s), 52.78 (s), 36.08 (s), 30.76 (s); HRMS-ESI (m/z) Calcd. for C H N O
2
1
8
21
4
+
[
M + H] : 325.1665, Found: 325.1668.
2
-(1-(Tetrahydro-2H-pyran-4-yl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide
1
(
17l). Obtained in 74.4% yield. H-NMR (400MHz , MeOD) δ (ppm): 7.88 (d, J = 6.5 Hz,
1
3
2
H), 7.68 (d, J = 7.7 Hz, 1H), 7.41 – 7.23 (m, 1H), 4.05 (dd, J = 11.4, 4.0 Hz, 2H), 3.55 –
.36 (m, 4H), 3.20 (t, J = 11.1 Hz, 1H), 3.06 (t, J = 11.4 Hz, 1H), 2.86 (t, J = 11.4 Hz, 2H),
1
3
.40 – 2.23 (m, 2H), 2.23 – 2.03 (m, 2H), 2.03 – 1.86 (m, 2H), 1.82 – 1.53 (m, 2H); C-NMR
(100MHz, MeOD) δ (ppm): 169.24 (s), 157.77 (s), 143.29 (s), 122.40 (s), 121.63 (s), 120.63
(s), 115.89 (s), 66.43 (s), 61.82 (s), 48.53 (s), 34.51 (s), 28.88 (s), 28.17 (s); HRMS-ESI (m/z)
+
Calcd. for C H N O [M + H] : 329.1978, Found: 302.1967.
1
8
25
4
2
2
-(1'-Methyl-[1,4'-bipiperidin]-4-yl)-1H-benzo[d]imidazole-4-carboxamide
(17m).
1
Obtained in 62.0% yield. H-NMR (400MHz , MeOD) δ (ppm): 7.88 (d, J = 7.0 Hz, 1H),
7
2
2
1
.66 (d, J = 7.8 Hz, 1H), 7.37 – 7.15 (m, 1H), 3.08 (d, J = 11.8 Hz, 2H), 3.02 – 2.92 (m, 3H),
.46 – 2.33 (m, 3H), 2.31 (s, 3H), 2.17 – 2.06 (m, 4H), 2.04 – 1.79 (m, 5H), 1.68 – 1.56 (m,
1
3
H); C-NMR (100MHz, MeOD) δ (ppm): 169.24 (s), 158.96 (s), 140.95 (s), 134.81 (s),
22.21 (s), 121.34 (s), 120.42 (s), 115.26 (s), 61.17 (s), 54.62 (s), 48.75 (s), 44.42 (s), 36.12
+
(
s), 30.20 (s), 26.94 (s); HRMS-ESI (m/z) Calcd. for C H N O [M + H] : 342.2294, Found:
1
9
28
5
3
42.2309.
2
-([1,4'-Bipiperidin]-4-yl)-1H-benzo[d]imidazole-4-carboxamide (17n). Obtained in
1
5
6.5% yield. H-NMR (400MHz , DMSO-d ) δ (ppm): 9.29 (s, 1H), 7.75 (dd, J = 7.6, 0.8
6
Hz, 1H), 7.66 (d, J = 2.4 Hz, 1H), 7.63 – 7.54 (m, 1H), 7.20 (t, J = 7.8 Hz, 1H), 4.59 (s, 1H),
2
5

4
2
.21 (d, J = 9.8 Hz, 1H), 3.00 – 2.78 (m, 5H), 2.36 (t, J = 11.3 Hz, 2H), 2.30 – 2.16 (m, 3H),
1
3
.05 – 1.91 (m, 2H), 1.87 – 1.69 (m, 2H), 1.68 – 1.53 (m, 2H), 1.33 – 1.16 (m, 2H); C-NMR
(
100MHz, DMSO-d ) δ (ppm): 167.16 (s), 160.07 (s), 140.82 (s), 136.29 (s), 122.07 (d, J =
6
3
0.4 Hz), 121.47 (s), 115.81 (s), 62.70 (s), 49.01 (s), 46.42 (s), 36.86 (s), 31.52 (s), 29.62 (s);
+
HRMS-ESI (m/z) Calcd. for C H N O [M + H] : 328.2137, Found: 328.2153.
1
8
26
5
2
-(1-(4-Methylbenzyl)piperidin-4-yl)-1H-benzo[d]imidazole-4-carboxamide
(17o).
1
Obtained in 52.1% yield. H-NMR (400MHz , MeOD) δ (ppm): 7.87 (s, 1H), 7.62 (s, 1H),
7
2
.30 – 7.22 (m, 1H), 7.20 (d, J = 7.9 Hz, 2H), 7.13 (d, J = 7.9 Hz, 2H), 3.50 (s, 2H), 3.02 –
1
3
.86 (m, 3H), 2.31 (s, 3H), 2.20 – 2.10 (m, 2H), 2.08 – 1.91 (m, 4H); C-NMR (100MHz,
MeOD) δ (ppm): 169.32 (s), 159.07 (s), 141.31 (s), 136.86 (s), 134.70 (s), 133.72 (s), 129.43
(s), 128.57 (s), 122.28 (s), 121.35 (s), 120.90 (s), 114.84 (s), 62.52 (s), 52.70 (s), 36.01 (s),
+
2
3
4
4
9.98 (s), 19.81 (s); HRMS-ESI (m/z) Calcd. for C H N O [M + H] : 349.2028, Found:
2
1
25
4
49.2035.
.2. Biological Evaluation
.2.1. PARP1/2 Inhibitory Activity Assay
The PARP1 and PARP2 inhibition assays were performed by a CRO company, Shanghai
Medicilon Inc. (Shanghai, China). The PARP1 and PARP2 inhibitory activity of the test
compounds were measured using PARP1 Chemiluminescent Assay Kit (BPS Bioscience,
catalog 80569, San Diego, CA, USA) and PARP2 Chemiluminescent Assay Kit (BPS
Bioscience, catalog 80552), respectively, according to the manufacturer’s instructions.
Briefly, PARP1 or PARP2 biotinylated substrate was incubated with test compounds or
solvent control at various concentrations and an assay buffer containing the PARP1 or PARP2
enzyme. After incubation, the plate was treated with streptavidin-HRP followed by addition
of the HRP substrate and the luminescent signal was measured using a chemiluminescence
reader (Perkin Elmer Envision 2104 Multi Label Microplate Reader, Waltham, MA, USA).
The IC values were calculated using GraphPad Prism Software (GraphPad Prism 5, La Jolla,
5
0
CA, USA). For each concentration, at least three wells were performed to calculate the
average parameter.
4
4
.2.2. Cytotoxic activity assays
.2.2.1. Cell culture
2
6

The four types of human cancer cell lines (MDA-MB-436, MDA-MB-231, MCF-7 and
CAPAN-1) were cultured aseptically using Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% (v/v) fetal bovine serum (FBS) and penicillin (100
−
1
−1
units·mL )/streptomycin (100 mg·mL ), at pH7.2 and 5% CO humidified atmosphere at
2
3
7°C. After attaining 80% confluence, the cells were trypsinized with 0.25 trypsin–EDTA and
diluted with media to a fixed number of cells.
4
.2.2.2. MTS assay
Cytotoxic activity was assessed using the standard MTS method by using triplicate
assay. The cells were seeded into 96-well plates containing the medium at the density of
000–6000 cells/mL (100μL/well). The compounds were dissolved in DMSO to the
concentration of 100mM and diluted in a culture medium to the concentrations needed. After
4
2
4 h, the cultured cells were treated with concentrations of test compounds (3.125μM to
00μM for tumor cells) for 48h. After 48h of incubation, the supernatant was replaced by
1
fresh medium(100μL/well), and 10μL MTS reagent ([3- (4,5-dimethylthiazol-2-yl) -5-
3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H -tetrazolium, inner salt]) was added to
(
each well. The plate was further incubated for 3h at 37°C in 5% CO . The optical absorbance
2
in individual well was determined at 492nm using a microplate reader. The inhibition rates
were calculated using the following formula:
Inhibition rate (%) = (ODnegative control– ODsample) / (ODnegative control– ODblank) × 100%.
The IC values were calculated using GraphPad Prism Software (GraphPad Prism 5, La
5
0
Jolla, CA, USA). For each concentration, at least three wells were performed to calculate the
average parameter.
4
.3. Microsomal Stability Assay
The positive control and test compounds were diluted to working concentrations at
.25mM with 70% acetonitrile. The cofactor used in this study was NADPH regenerating
0
system, that was composed of 6.5mM NADP, 16.5mM G-6-P, 3 U/mL G-6-PDH. The
quenching agent was consisted of acetonitrile containing internal standards (tolbutamide and
propanolol). The buffer used in this study was 100mM phosphate buffer with 3.3mM MgCl2.
0
.5 mg/mL liver microsomal protein and 1μM test compounds/positive control were dissolved
in 100mM potassium phosphate buffer to prepare the incubation mixtures.
2
7

The 0-minute samples were prepared by addition of an 80µL aliquot of each incubation
mixture to 300µL quenching agent to precipitate proteins. The samples were vortexed, and
then 20µL aliquot of the NADPH regenerating system were added in. The reaction was
initiated by addition of 80µL of the NADPH regenerating system to 320µL of each incubation
mixture. The final incubation conditions achieved in 400µL (0.5 mg/mL microsomal protein,
1
µM test compounds/positive control, 1.3mM NADP, 3.3mM G-6-P, 0.6 U/mL G-6-PDH).
The mixtures were incubated in a 37°C water bath with gentle shaking. A 100µL aliquot of
each mixture was removed at 10, 30, 90 min to a clean 96-well plate which contained 300µL
quenching agent to precipitate proteins, and centrifuged (4000 ×g, 15 min). 80µL of
supernatant were taken into 96-well assay plates pre-added with 160µL ultrapure water, and
then analyzed by LC-MS/MS.
4
.4. Study on the early ADME properties
The studies of early ADME properties were all performed by a CRO company, Sandia
Medical Technology (Shanghai) Co., Ltd. The test compound (17d) was dissolved in DMSO
to the concentration of 10mM as a stock solution.
4
.4.1. Kinetic/ Thermodynamic solubility Assays
The PBS (pH7.4) used in the assays contained 3.3mM MgCl2.
.4.1.1. Kinetic Thermodynamic solubility Assay
4
The stock solution of 17d was diluted in PBS and acetonitrile to concentration at 100
µg/mL respectively. The PBS sample was incubated at 37°C in water bath for 120 min, while
the acetonitrile sample was incubated at the ambient temperature for the same time. The
supernatant of PBS sample was separate by centrifugation (4000×g, 15min). 20µL
supernatant (or the acetonitrile sample) was added into 96-well plate pre-added with 380μL
7
0% acetonitrile, and then diluted 6-fold with an internal standard (200 ng/mL Tolbutamide)
as a quenching agent. 50μL quenching agent was diluted and well mixed with 300μL
ultrapure water to obtain the sample of injection. Finally, 10μL sample was injected to
LC-MS/MS for sample analysis.
4
.4.1.2 Thermodynamic solubility Assay
The stock solution of 17d was diluted in PBS to obtain a supersaturated solution (~5
mg/mL). The supersaturated solution was short-vortexed and sonicated for 5 min. Then, the
2
8

solution was incubated by being vortexed at 25℃ for 24h. 200μL of the supersaturated
solution was pipetted and filtered with a 0.4µm filter plate to get the filtrate (Solution A).
Another 5µL stock solution of 17d was first diluted in 495μL 100% acetonitrile to 100 μg/mL,
and 20μL of the above solution was second diluted in 380μL 70% acetonitrile to 5 μg/mL
(Solution B). The Solution A (or the Solution B) was mixed with triple amount of the
acetonitrile solution containing internal standards (200 ng/mL of Tolbutamide and 50 ng/mL
Propranolol) to obtain a pre-injection solution. 100µL of the pre-injection solution was
subsequently diluted in 200µL ultrapure water to generate the injection solution, which was
injected to LC-MS/MS with an appropriate volume for analysis.
4
.4.2. Permeability Assay in hMDR1-MDCK Ⅱ
The cell culture and incubation conditions were the same as described above. The
hMDR1-MDCK Ⅱ cells were seeded into 24-multiwell insert systems with PET
2
(
polyethylene terephthalate) membranes (1 micron pore size and 0.3 cm surface area) at an
5
optimized density of 2×10 cells/mL (1 mL/well) in cell culture medium. Before the
experiment, all the apical sides and basolateral sides was washed and incubated by 0.3mL and
1
mL PBS buffer (pH7.4) for 30 min. The hMDR1-MDCK Ⅱ cell monolayers were
preincubated in transport media, all the apical sides and basolateral sides were preincubated
by 0.2mL and 0.7mL transport media with or without specific P-gp inhibitor (cyclosporin A)
for 40 min. The test compound (17d) was diluted in DMEM to the final concentration of
1
0µM. For A to B directional transport, 0.2mL donor working solution with 17d was added to
the A compartment and 0.7mL transport media as receiver working solution was added to the
B compartment. For B to A directional transport, 0.7mL donor working solution with 17d or
cyclosporin A was added to the B compartment and 0.2mL transport media as receiver
working solution was added to the A compartment. The cells were incubated for 90 min.
8
0µL samples were taken from both donor and receiver compartments into 96-well assay
plates, which pre-added with 320µL internal standard solution of acetonitrile in each well,
and centrifuged (4000×g, 10min). 80µL of the supernatant were added into 96-well assay
plates pre-added with 160µL ultrapure water and then analyzed by LC-MS/MS.
4
.4.3. Red Blood Cell (RBC) to Plasma Ratio Assay
The positive control Chloroquine and 17d were diluted to working concentrations at
2
9