Design and synthesis of 2-(4,5,6,7-tetrahydrothienopyridin-2-yl)-benzoimidazole carboxamides as novel orally efficacious Poly(ADP-ribose)polymerase (PARP) inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.01.018

The nuclear protein poly(ADP-ribose) polymerases-1/2 (PARP-1/2) are involved in DNA repair damaged by endogenous or exogenous process. And PARP-1/2 inhibitors have been proved to be clinically efficacious for DNA repair deficient tumors in the past decade

Full text of DOI:10.1016/j.ejmech.2018.01.018

Accepted Manuscript
Design and synthesis of 2-(4,5,6,7-tetrahydrothienopyridin-2-yl)-benzoimidazole
carboxamides as novel orally efficacious Poly(ADP-ribose)polymerase (PARP)
inhibitors
Xuxing Chen, Xiajuan Huan, Qiufeng Liu, Yuqin Wang, Qian He, Cun Tan, Yi Chen,
Jian Ding, Yechun Xu, Zehong Miao, Chunhao Yang
PII:
S0223-5234(18)30018-7
10.1016/j.ejmech.2018.01.018
EJMECH 10095
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 October 2017
Revised Date: 5 January 2018
Accepted Date: 5 January 2018
Please cite this article as: X. Chen, X. Huan, Q. Liu, Y. Wang, Q. He, C. Tan, Y. Chen, J. Ding, Y. Xu,
Z. Miao, C. Yang, Design and synthesis of 2-(4,5,6,7-tetrahydrothienopyridin-2-yl)-benzoimidazole
carboxamides as novel orally efficacious Poly(ADP-ribose)polymerase (PARP) inhibitors, European
Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.01.018.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
1
2
3
Design and Synthesis of
2-(4,5,6,7-Tetrahydrothienopyridin-2-yl)-benzoimidazole Carboxamides as
Novel Orally Efficacious Poly(ADP-ribose)polymerase (PARP) Inhibitors
4
5
Xuxing Chen,a,b,ǀǀ Xiajuan Huan,c,ǀǀ Qiufeng Liu,d,b,ǀǀ YuqinWang,a Qian He,a Cun Tan,a Yi Chen,c Jian
Ding,c YechunXu,*,d Zehong Miao,*,c and ChunhaoYang*,a
6
7
aSynthetic Organic & Medicinal Chemistry Laboratory, State Key Laboratory of Drug Research,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
8
bUniversity of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China
9
cDivision of Anti-tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
10
11
12
dDrug Discovery and Design Center, CAS Key Laboratory of Receptor Research, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
13
14
15
ǀǀThese three authors contributed equally to this work.
16
17
18
*Corresponding Authors. For C. Yang: phone, 86-21-50806770; E-mail, chyang@simm.ac.cn. For Z.
Miao: phone, 86-21-50806820; E-mail, zhmiao@simm.ac.cn. For Y. Xu: phone, 86-21-50801267;
E-mail, ycxu@simm.ac.cn.
19
20
Abstract
1

ACCEPTED MANUSCRIPT
1
2
The nuclear protein poly(ADP-ribose) polymerases-1/2 (PARP-1/2) are involved in DNA repair
damaged by endogenous or exogenous process. And PARP-1/2 inhibitors have been proved to be
clinically efficacious for DNA repair deficient tumors in the past decade. We have developed a series of
4,5,6,7-tetrahydrothienopyridin-2-yl benzimidazole carboxamides as novel and potent PARP-1/2
inhibitors. The best compound resulted from this series is compound 27 which displays excellent
PARP-1 and PARP-2 inhibitory activity with IC50 of 18 nM and 42 nM, respectively. Furthermore, it
can selectively kill BRCA2 deficient V-C8 cells with a CC50 of 920 nM. In the MDA-MB-436
(BRCA-1 mutant) xenograft model, this compound was well tolerated and showed single-agent activity.
Based on the results above, compound 27 has been selected as a lead candidate targeting PARP-1/2 and
its preclinical characterization is also underway.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
1. Introduction
Poly(ADP-ribose) polymerases (PARPs), a family of 17 protein members, conduct protein
post-translational modification using nicotinamide adenine dinucleotide (NAD+) to add ADP-ribose
units onto acceptor proteins1-2. The PARP protein superfamily plays wide-ranging roles in cellular
processes such as proliferation3, apoptosis4-5, transcriptional regulation6-7, DNA methylation8-9, and
WNT signalling10. All of the PARP family enzymes share a conserved catalytic domain, but only
PARP-1 and PARP-2 contain a DNA-binding domain which facilitates localization to the site of DNA
damage. PARP-1 is more active than PARP-2, contributing 90% to 95% of the total PARP activity in
response to DNA damage11-12. Once the damaged DNA was recognized by PARP-1 DNA-binding
domain, the catalytic activity of PARP-1 is stimulated dramatically13-15 followed by
poly(ADP-ribosyl)ation of a variety of nuclear proteins (histones, transcription factors and PARP itself)
and repair of DNA damage through the base excision repair (BER) pathway16-18
.
Repair of DNA damage induced by chemotherapeutics or radiation during cancer therapy usually
leads to drug resistance and continued tumor growth19-20. Since PARP-1 is involved in maintaining the
genomic stability, particularly in the repair of single-strand DNA lesions, PARP-1 inhibitors have been
2

ACCEPTED MANUSCRIPT
1
2
used as potentiators in combination with DNA damaging cytotoxic agents or radiation to compromise
the cancer cell DNA repair mechanism, resulting in genomic dysfunction and cell death16, 20. Moreover,
inhibition of PARP-1 can induce synthetic lethality in tumor cells defective in homologous
recombination (HR) repair, such as cancer cells carrying at least one of BRCA1 and BRCA2 mutant
genes21-22. Therefore, PARP-1 inhibitors can be used as single-agent therapy to kill BRCA1/2-deficient
tumor cells selectively and efficiently.
3
4
5
6
7
In the past three decades, a large number of PARP-1 inhibitors have been investigated23, including
AZD2281 (olaparib, 1)24, ABT-888 (veliparib, 2)25, AG014699 (rucaparib, 3)26, MK-4827 (niraparib,
4)27, and BMN-673 (talazoparib, 5)28 (Figure 1). In December 2014, olaparib was approved by EMA
and FDA as monotherapy for the maintenance treatment of women with relapsed BRCA-mutated
ovarian cancer. Up to now, three PARP-1 inhibitors have been approved. All inhibitors developed to
date share a nicotinamide-based structure competing with NAD+ in PARP-1 catalytic site29-30. Hence, all
of the known PARP-1 inhibitors have the similar binding mode with PARP-1. Hydrogen bonds formed
between the lactam or the carboxamide group of the small molecules and residues Ser904 and Gly863 of
8
9
10
11
12
13
14
15
16
17
18
PARP-1, and a π−
π stacking between the aromatic ring and side chain of Tyr90729 are crucial to
interactions between these inhibitors and PARP-1. And due to the high homology of the catalytic
domain between PARP-1 and PARP-2, PARP-1 inhibitors usually display inhibitory activities against
PARP-2.
3

ACCEPTED MANUSCRIPT
O
H
N
O
NH2
O
NH
N
HN
N
O
F
N
N
HN
F
N
H
H
N
O
Olaparib (KU 59436, AZD2281)
1
Veliparib ABT-888
Rucaparib (AG 014699, PF01367338)
3
(
)
2
O
6
NH2
H
O
N
O
NH2
N
N
7'
4'
N
N
S
6'
5'
X
Y
NH
N
N
N
N
R1
3'
H
F
N
H
F
X = NR2, Y = CH2
or X = CH2, Y = NR2
Niraparib (MK-4827)
4
Talazoparib (BMN-673)
5
1
2
Figure 1. Approved or PARP-1 inhibitors undergoing clinical trials (compound 1-5) and our proposal.
Benzimidazole carboxamide, one class of PARP-1 inhibitors, was first developed by researchers at the
University of Newcastle31-32. This series of compounds displayed relatively high intrinsic potency
against PARP-1, but suffered from poor activity in the whole cell assay. To improve the cellular activity
of benzimidazole carboxamide scaffold inhibitors, a series of unsaturated basic amino-containing
substituents have been introduced to the C-2 position of the pharmacophore by Abbott, leading to the
identification of ABT-88825. However, ABT-888 has a lesser cytotoxicity than other clinical PARP
inhibitors28 and it failed in two phase III combination therapy trials in patients with squamous
non-small-cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC). In this report, we
incorporated a tetrahydrothienopyridin-2-yl substituent to this scaffold as an alternative optimization
strategy (Figure 1), and discovered a novel benzimidazole compound 27 as a potent PARP inhibitor
with both in vitro and in vivo activity.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
2. Chemistry
Boc-protected 4,5,6,7-tetrahydrothienopyridine-2-carbaldehydes were the first intermediates needed
for preparing the desired sulfur-containing benzimidazole carboxamides. Preparation of
5-Boc-4,5,6,7-tetrahydrothieno[3,2-c]pyridine-2-carbaldehyde
(42)
and
6-Boc-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-2-carbaldehyde (43) is shown in Scheme 1. After
4

ACCEPTED MANUSCRIPT
1
2
bromination
of
5-Boc-4,5,6,7-tetrahydrothieno[3,2-c]pyridine
(38)
and
6-Boc-4,5,6,7-tetrahydrothieno[3,2-c]pyridine (39), the carboxaldehyde functionality was then
introduced at the C-2 position of the tetrahydrothienopyridine via metallation with n-BuLi and
subsequent formylation with dry DMF.
3
4
5
7-alkyl-6-Boc-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-2-carbaldehydes were prepared using the
method described in Scheme 2. 2-(Thiophen-3-yl)ethanamine hydrochloride (44) was condensed with
various acyl chlorides and the resulting amides were treated with POCl3 at 60 °C. After reduction by the
6
7
8
NaBH4
and
protection
with
di-tert-butyl
dicarbonate,
9
7-alkyl-6-Boc-4,5,6,7-tetrahydrothieno[2,3-c]pyridines 45-52 were given33. The Boc-protected
derivatives were then transformed into the aldehydes 61-68 in two steps: bromination with NBS to
obtain the bromides 53-60 and then treatment with t-BuLi and dry DMF.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
5-Methyl-6-Boc-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-2-carbaldehyde (64) was prepared using the
method described in Scheme 3. Nitroethane aldol reaction with thiophene-3-carbaldehyde (69) gave
nitro olefin 70. After reduction with LiAlH4, the resulting (2-amino)ethylthiophene was protected with
tert-butyl dicarbonate and then cyclized with paraformaldehyde and p-TsOH34. The desired compound
74 was obtained from 5-methyl-6-Boc-4,5,6,7-tetrahydrothieno[2,3-c]pyridine (72) following a
procedure similar to that of preparation of 61-68.
Starting from the known 6-Boc-2-amino-3-ethoxylcarbonyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine
(75)35, a synthetic route forward to 6-Boc-3-methyl-2-formyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine
(81) was developed (Scheme 4). Removal of amino group with isopentyl nitrate at 60 °C for 2 h and
reduction of ester functionality with LiBH4 gave the alcohol 77. The hydroxyl group was easily
converted to chloride with MeSO2Cl and NEt3, followed by dechlorination to furnish
6-Boc-3-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridine (79). After two steps described before, the
aldehyde 81 was obtained.
5

ACCEPTED MANUSCRIPT
1
2
Aldehydes 83, 85 and 87 were synthesized in an effort to further understand the SAR of the
tetrahydrothienopyridinyl moiety as shown in Scheme 5. Reaction of 82, 84 and 86 with n-BuLi and dry
DMF gave compounds 83, 85 and 87, respectively.
3
4
Some of dianiline analogues 97-99 were prepared as outlined in Scheme 6. Nitration of isatins 88-90
with fuming HNO3 and concentrated H2SO4 gave the corresponding nitroisatins 91-93, which were
subjected to Baeyer-Villiger rearrangement conditions followed by hydrolysis under alkali conditions
and esterification under acid conditions to afford the intermediates 94-96. The nitro group could be
converted to amino group by hydrogenation catalyzed by Pd/C.
5
6
7
8
9
With the aldehydes and diamines in hand, the target benzimidazoles 6-33 were synthesized as shown
in Schemes 7 and 8. Dianiline analogues 97-100 reacted with various aldehydes in the presence of
sodium bisulfite at 60 °C to give the corresponding compounds. Deprotection of Boc derivatives 101,
105, 108, 110, 112, 115, 118-127, 138, 140 and 142 gave the corresponding compounds 102, 106, 109,
111, 113, 116, 128-137, 139, 141, and 143. Then alkylation by reductive methylation with
formaldehyde or ethylation with bromoethane gave the corresponding compounds 103, 104, 107, 114,
117. Aminolysis of 102-104, 106, 107, 109, 111, 113, 114, 116, 117, 128-137, 139, 142 and 143 with
ammonia in methanol gave 6-26 and 31-33, respectively.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Alkylation of 112, 122 and 123 with alkyl halides afforded 1-alkylbenzimidazole compounds 144-147,
following by deprotection and aminolysis gave the target compounds 27-30 (Scheme 9). The synthesis
of tricyclic benzimidazoles 34 and 35 was described in Scheme 10. Sandmeyer reaction of compound 94
provided aryl bromide 152 in high yield, which was then reacted with ethylenediamine to give a
seven-membered amide 15332. Under hydrogen atmosphere and in the presence of a catalytic amount of
Pd/C, 153 in methanol was transformed into diamine 154, which was further reacted with 42 or 43 in the
presence of sodium bisulfite at 60 °C. The generated 155 and 156 were easily converted to the desired
compounds 33 and 34 by removal of Boc. S-23 and R-23 were prepared from S-134 and R-134 with >
99% ee and > 98% ee, respectively. The S-134 and R-134 were separeted from racemic 134 by chiral
HPLC. And the absolute configuration of S-134 was assigned by X-ray crystallographic analysis36.
6

ACCEPTED MANUSCRIPT
1
Scheme 1. Synthesis of intermediates 42 and 43 a
2
3
aReagents and conditions: (i) NBS, MeCN, rt, 1 h; (ii) n-BuLi, THF, DMF, –78 °C, 2 h.
68 a
4
Scheme 2. Synthesis of intermediates 61
−
5
6
aReagents and conditions: (i) RCOCl, Et3N, DCM, rt, 1 h; (ii) POCl3, 60 °C, 2 h; (iii) MeOH,NaBH4,
rt, 1 h; (iv) (Boc)2O, DCM, rt, 1 h; (v) NBS, MeCN, rt, 1 h; (vi) t-BuLi, THF, DMF, –78 °C, 2 h.
Scheme 3. Synthesis of intermediate 74 a
7
8
S
S
S
i
ii, iii
O
NHBoc
85%
55%
NO2
69
70
71
72: R1 = H, 49%
S
iv
NBoc
v
R
: R1 = Br, 81%
73
vi
1
74
: R = CHO, 64%
9
10
aReagents and conditions: (i) EtNO2, AcONH4, reflux, 2 h; (ii) LiAlH4, THF, 0~60 °C, 1 h; (iii)
(Boc)2O, DCM, rt, 1 h; (iv) paraformaldehyde, TsOH·H2O, toluene, reflux, overnight; (v) NBS, MeCN,
rt, 1 h; (vi) t-BuLi, THF, DMF, –78 °C, 2 h.
11
12
13
Scheme 4. Synthesis of intermediate 81 a
7

ACCEPTED MANUSCRIPT
1
2
aReagents and conditions: (i) isopentyl nitrate, THF, rt to 60 °C, 2 h; (ii) LiBH4, THF/toluene, rt~100
°C, 5 h; (iii) Et3N, MeSO2Cl, DCM, 5 h; (iv) Raney Ni, H2(1 atm), reflux, 4 h; (v) NBS, MeCN, rt, 1 h;
(vi) n-BuLi, THF, DMF, –78 °C, 2 h.
3
4
5
Scheme 5. Synthesis of intermediates 83, 85 and 87 a
6
7
aReagents and conditions: (i) n-BuLi, THF, DMF, –78 °C, 2 h for 82 and 84, 5 min for 86.
Scheme 6. Synthesis of intermediates 97-99 a
8
9
10
aReagents and conditions: (i) fuming HNO3, H2SO4(conc.), –5 °C, 1 h; (ii) NaOH (aq, 5 N), H2O2
(30%), 0 °C, 5 h, then HCl (aq, 2 N); (iii) MeOH,TsOH·H2O, reflux, 36 h; (iv) Pd/C, H2(1 atm), rt, 5 h.
Scheme 7. Synthesis of 6-16 a
11
12
8

ACCEPTED MANUSCRIPT
1
2
aReagents and conditions: (i) NaHSO3, EtOH, 60 °C, overnight; (ii) TFA, DCM, 1 h; (iii)
NH3/MeOH, 80 °C; 20 h; (iv) CH2O (37% aq), CH3CO2H, 95 °C, 4 h; (v) EtBr, Cs2CO3, 80°C, 1 h.
Scheme 8. Synthesis of 21-26 and 31-33 a
3
4
9

ACCEPTED MANUSCRIPT
1
2
aReagents and conditions: (i) NaHSO3, EtOH, 60 °C, overnight; (ii) TFA, DCM, 1 h; (iii)
3
4
NH3/MeOH, 80 °C; 20 h.
Scheme 9. Synthesis of 27-30 a
5
10

ACCEPTED MANUSCRIPT
1
2
aReagents and conditions: (i) MeI, K2CO3, acetone, rt, overnight; (ii) EtBr, Cs2CO3, DMF, 90 °C,
overnight; (iii) TFA, DCM, 1 h; (iv) NH3/MeOH, 80 °C; 20 h.
3
Scheme 10. Synthesis of 34 and 35 a
4
5
6
7
aReagents and conditions: (i) isopentyl nitrite, CuBr2, MeCN, 0 to 60 °C, overnight; (ii)
1,2-ethanediamine, DMA, 100 °C, 4 h; (iii) Pd/C, H2(1 atm), rt, 5 h; (iv) NaHSO3, EtOH, 60 °C,
overnight; (v) TFA, DCM, 1 h.
8
Scheme 11. Synthesis of S-23 and R-23 a
9
10
aReagents and conditions: (i) NH3/MeOH, 80 °C; 20 h.
11
12
13
14
3. Results and discussion
3.1 PARP-1/2 Inhibition and Cellular Potency.
To assess the potency of our newly synthesized compounds, we tested them against PARP-1/2 activity
at the enzymatic level and in cells of Chinese hamster lung fibroblast V79 (wild-type) and V-C8
11

ACCEPTED MANUSCRIPT
1
2
3
(BRCA2 deficient). AZD2281 and ABT-888 were used as reference compounds. The results were
shown in Tables 1-3.
Table 1. Enzymatic and Cellular Assays of Compounds 6-16 a.
4
PARP1
PARP2
V-C8 (BRCA2-/-)
CC50 (nM)
V79 (BRCA2+/+
CC50 (nM)
)
Compd
R
X
IC50 (nM)
IC50 (nM)
H
H
H
H
H
Me
Cl
F
6
7
58
123
90
113
131
140
53
NA
NA
NA
NA
NA
NA
NA
2364
9832
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
8
9
14
10
11
12
13
14
15
55
247
281
382
124
109
73
345
51
17
F
22
F
27
F
16
58
83
11
55
3
456
NA
NA
AZD2281
70
2344
ABT-888
a
5
6
7
IC50 values were calculated by Logit method from the results of at least three independent tests with six
concentrations each (standard deviations were within 25% of the mean values); Cytotoxic effect (CC50) means the
concentration required to reduce cell proliferation and growth by 50%. Values were calculated by Logit method from
12

ACCEPTED MANUSCRIPT
1
2
3
the results of at least three independent tests with six concentrations each (standard deviations were within 25% of the
mean values); NA: not active at 10 µM.
Initially, 4,5,6,7-tetrahydrothieno[3,2-c]pyridines and 4,5,6,7-tetrahydrothieno[2,3-c]pyridines were
4
5
introduced onto 2-position of the benzimidazoles. As shown in Table 1, all the synthesized compounds
displayed excellent inhibitory activities against PARP-1 with IC50 values ranging from 14 nM to 345
nM. In generally, 4,5,6,7-tetrahydrothieno[2,3-c]pyridine analogues (compounds 9, 10, 13, and 14) are
more potent than 4,5,6,7-tetrahydrothieno[3,2-c]pyridine analogues (compounds 6-8, 15 and 16),
indicating that the nitrogen atom preferred at the 6-position. According to previous literature, small
hydrophobic substituents on the aromatic ring, adjacent to the amide, contribute to the inhibitory
potency29. Therefore, anti-PARP activities of compounds having Me, Cl or F on the benzimidazole ring
were investigated (compounds 11-16). Fluorine-containing compounds 13 and 14 have high PARP-1
potency with IC50 values of 17 nM and 22 nM, respectively. Even though possessing high enzymatic
potency, the cellular activities of these two compounds significantly drops. They have CC50 values
of 2364 nM and 9832 nM against BRCA2-deficient V-C8 cells, respectively. And all the new
benzimidazole compounds together with reference compounds showed no proliferation inhibition to the
wild-type V79 cells, validating that the tested compounds 13 and 14 can selectively kill
BRCA-deficient cells by inhibition of PARP-1 activity. Due to the high homology of the catalytic
domain between PARP-1 and PARP-2, PARP-1 inhibitors usually also bind PARP-2. As expected, all
of these compounds displayed comparable inhibitory activities against PARP-2.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Table 2. Enzymatic and Cellular Assays of Compounds 17-30 a.
21
PARP1
PARP2
V-C8 (BRCA2-/-)
CC50 (nM)
V79 (BRCA2+/+
CC50 (nM)
)
Compd
R1
R2
R3
R4
IC50 (nM)
IC50 (nM)
H
H
H
H
H
H
H
13
17
17
40
124
87
2364
8623
NA
NA
Me
13

ACCEPTED MANUSCRIPT
Et
n-Pr
n-Bu
i-Pr
H
H
H
H
H
18
19
20
21
22
23
24
25
26
27
28
29
30
25
86
130
27
14
25
36
16
8
145
324
348
196
239
86
5333
5203
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
H
H
H
H
H
H
H
2846
2285
1624
6320
6515
4880
920
cyclopropyl
t-Bu
i-Bu
H
H
H
H
H
H
H
H
H
H
219
14
Me
H
H
H
H
H
Me
H
30
H
H
Me
Et
Me
Me
18
250
153
20
42
H
H
H
359
328
117
NA
t-Bu
i-Bu
H
H
2063
2320
H
H
1
2
3
a Values were calculated by Logit method from the results of at least three independent tests with six concentrations
each (standard deviations were within 25% of the mean values); NA: not active at 10 µM.
Next, we conducted optimizations based on compound 13. A variety of analogues with hydrophobic
4
5
substituents on the 4,5,6,7-tetrahydrothieno[2,3-c]pyridine and benzimidazole moieties were prepared
and the results are summarized in Table 2. Alkyl substituents at the 7-position of the
4,5,6,7-tetrahydrothieno[2,3-c]pyridine were well tolerated, having inhibitory activities against PARP-1
with IC50 values ranging from 14 to 130 nM (compounds 17-24). Compound 23 bearing a bulky
tert-butyl group at this position showed an IC50 value of 25 nM on PARP-1 enzyme and a CC50 value of
1624 nM against V-C8 cells. The introduction of a methyl group at 5- or 3-position of
4,5,6,7-tetrahydrothieno[2,3-c]pyridine appendages led to compounds 25 and 26. Compared to
compound 13, compound 25 has similar inhibitory activity against PARP-1. But it is 9-fold more potent
than 13 with an IC50 value of 14 nM on PARP-2 enzyme. The 3-methyl analogue 26 displayed the best
enzymatic potency against PARP-1 (IC50 = 8 nM). However, both compounds 25 and 26 showed a
lower potency than the lead 13 in cellular assay. N-methylated derivative 27 maintained PARP-1
potency with an IC50 value of 18 nM and showed increased potency in V-C8 cells (CC50 = 920 nM).
6
7
8
9
10
11
12
13
14
15
14

ACCEPTED MANUSCRIPT
1
2
3
4
5
Replacing the methyl group with a longer ethyl group at the same position results in compound 28,
which has a sharp drop in inhibitory activity both in vitro and in vivo. In addition, potency improvement
was not successful by adding a methyl group to compounds 23 and 24 to obtain compounds 29 and 30,
respectively.
Table 3. Enzymatic and Cellular Assays of Compounds 31-35, S-23, R-23 a
PARP1
PARP2
V-C8 (BRCA2-/-)
CC50 (nM)
V79 (BRCA2+/+)
CC50 (nM)
Compd
Structure
IC50 (nM)
IC50 (nM)
31
244
42
37
25
25
15
92
144
64
NA
NA
NA
NA
NA
864
NA
NA
NA
NA
NA
NA
NA
NA
32
33
149
49
34
35
99
S-23
R-23
70
356
6
7
8
a Values were calculated by Logit method from the results of at least three independent tests with six concentrations
each (standard deviations were within 25% of the mean values); NA: not active at 10 µM.
Changing the tetrahydrothienopyridinyl in compound 13 to tetrahydrofuropyridinyl in 31 and
tetrahydrothiazolopyridinyl in 33 resulted in a 14- and 2-fold drop of inhibitory potency against PARP-1,
respectively (Table 3). To examine whether the tetrahydrothienopyridinyl ring could be opened,
compound 32 was prepared and assayed. As a result, compound 32 displayed an IC50= 42 nM to
9
10
11
15

ACCEPTED MANUSCRIPT
1
2
PARP-1 and an IC50 = 64 nM to PARP-2. There has been a reported benefit by locking the carboxamide
in the favored conformation of benzimidazole carboxamides through a seven-membered ring32, here we
found that compounds 34 and 35 demonstrated a similar enzymatic potency compared to compounds 13
and 15. Unfortunately, neither of them had appreciable proliferation inhibition against BRCA2-deficient
V-C8 cells. As described above, compound 23 is racemic. The two enantiomers of 23 were prepared to
evaluate the relative PARP activity. The S enantiomer (S-23) was modestly preferred and provide a
compound with a CC50 value of 864 nM in the V-C8 cytotoxity assay.
3
4
5
6
7
8
3.2 Co-crystal Structures of Compounds 13, 15 and 31 Bound to the PARP-1 Catalytic Domain.
In order to gain insight into the structure basis for differential inhibitory potencies among these
aforementioned compounds, co-crystal structures of compounds 13, 15 and 31 in complex with PARP-1
catalytic domain were determined (Figure 2). These three compounds are structural similar with a subtle
difference in side-chain. Benzimidazole carboxamide core provides the conserved hydrogen-bonding
interactions with residues Ser 904 and Gly863, and π-π stacking interactions with residue Tyr907. The
tetrahydropyridine ring of the compounds formed a hydrophobic interaction with residue Tyr889. The
nitrogen atom on the tetrahydropyridine functionality in 13 and 15 formed hydrogen bonds with residue
Glu763. The distance between this nitrogen atom and the carboxyl oxygen of residue Glu763 was closer
in 13 than 15. A closer distance of the sulfur atom in 13 to the aromatic ring of Tyr889 than that in 15
resulted in an additional hydrophobic interaction, contributing to greater affinity of 13 binding to
PAPR-1. Compound 31 bearing a tetrahydrofuropyridinyl group instead of a tetrahydrothienopyridinyl
group in compound 13 led to a significant loss of potency. The smaller furan ring together with an
intramolecular hydrogen bond formed between oxygen atom of the furan ring and the NH group of the
imidazole ring increase the distance of 31 to Glu763 and thus break the hydrogen bond between 31 and
Glu763. This makes the tetrahydrofuropyridinyl group of 31 more flexible in the complex structure and
the resultant inhibitory activity of 31 to PARP-1 is decreased to 14-fold of 13.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
16

ACCEPTED MANUSCRIPT
1
2
Figure 2. Co-crystal structures of PARP-1 catalytic domain bound with compounds 13 (A), 15 (B) and
31 (C). The PDB codes for three complex structures are 5XSR (PRAR-1/13), 5XST (PRAR-1/15) and
5XSU (PRAR-1/31), respectively. Distances of H-bonds between the side chain of E763 and NH in the
tetrahydropyridine ring are labeled in Å.
3
4
5
6
3.3 Thermodynamic Behavior of Compounds 13 and 31 Binding to PARP-1.
7
To further elucidate the binding modes between inhibitors and PARP-1, the thermodynamic behavior
of compounds 13 and 31 binding to the catalytic domain of PARP-1 in solution were investigated by
Isothermal Titration Calorimetry (ITC). The averaged dissociation constant (Kd), binding free energy
(∆G), enthalpy (∆H), and entropy (-T∆S) resulted from three independent ITC measurements are listed
in Table 4. It was revealed that compound 13 possesses stronger binding capability to PARP-1 than 31
8
9
10
11
17

ACCEPTED MANUSCRIPT
1
2
as its ∆G and ∆H are stronger than those of 31. Although the binding of two compounds to the catalytic
domain of PARP-1 is mainly enthalpy-driven, the entropic effect to the binding free energy is positive
in 13 but negative in the case of 31. The difference between two compounds is that 31 bears a
tetrahydrofuropyridinyl group while it is a tetrahydrothienopyridinyl group instead in 13. The
thermodynamic data combined with the structure information shown in Figure 2 leads us to conjecture
that the hydrogen bond of 13 to Glu763 reduced the flexibility of the tetrahydrothienopyridinyl group,
resulting in the negative contribution of the entropy to the binding free energy. In contrast, such a
hydrogen bond to Glu763 is missed in the complex structure of 31 and the intramolecular hydrogen
bond is formed instead, so that the flexibility of tetrahydrofuropyridinyl group is somehow maintained
and thus the entropy effect to the ∆G is positive. However, the entropy effect is not complement to
enthalpy loss due to deficiency of hydrogen bond to Glu763, the resulted ∆G is weaker in 31 compared
to 13. With one atom changed from sulfur to oxygen, two compounds have different thermodynamic
binding mechanism.
3
4
5
6
7
8
9
10
11
12
13
14
Table 4. Thermodynamic Properties of Compounds 13 and 31 Binding to PARP-1
Compd
13
Kd(M)
∆G (kJ/mol) ∆H (kJ/mol) -T∆S (kJ/mol)
(2.03 ± 0.30)× 10-8
(1.80± 0.44)× 10-7
-44.79
-39.14
-54.83 ± 0.59
-34.74 ± 0.67
10.04
-4.40
31
15
16
17
18
19
20
21
22
23
3.4 Pharmacokinetic Properties.
Compounds 13 and 27 were further evaluated for their physicochemical properties and in vitro
ADME parameters (Table 5). Compound 13 has a poor permeability in Caco-2 cells with A→B = 0.20
× 10-6 cm/s, with a high efflux ratio at 74.8. While compound 27 has a moderate permeability as
measured in Caco-2 cells at A→B = 2.65 × 10-6 cm/s, with an efflux ratio at 4.5. The improved
permeability may contribute to the higher cellular potency of 27 than 13. Intrinsic clearance of
compounds 13 and 27 was measured by in vitro incubation experiments with liver microsome. There
were significant interspecies differences observed with regards to the in vitro microsomal clearances.
The highest in vitro microsomal clearance (> 100 µL/min/mg protein) was observed in mice for these
18

ACCEPTED MANUSCRIPT
1
2
3
4
5
two compounds. And the in vitro clearance measured from human-derived microsomes for compounds
13 (23 µL/min/mg protein) and 27 (28 µL/min/mg protein) was low. When primary iv/oral PK studies
were performed in rats (see Supplementary Material), compound 27 displayed a high clearance of 11.61
L/h/kg, which was well predicted by in vitro clearance.
Table 5. Characterization of Compounds 13 and 27.
Solubility (µM)
in vitro ADME
Compd MW
mp (°C)
Caco-2 (A → B, 10-6cm/s efflux ratio)
pH 7.0 pH 6.8 pH 7.4 Clint (µL/min/mg protein, M/R/H)a
316.4
330.4
> 300
0.20/74.8
2.65/4.5
> 50
> 50
> 50
> 50
> 50
> 50
123/340/23
75/196/28
13
27
265-267
6
7
a Intrinsic clearance (Clint) of compounds in mouse (M)/rat (R)/human (H) liver microsomes (expressed in µL/min/mg
protein).
8
9
3.5 In vivo Antitumor Studies of Compound 27.
Consideration of the excellent in vitro potency and relatively good DMPK properties, xenograft
studies were performed on compound 27 hydrochloride (Figure 3 and Supplementary Material Table
S3/S4). In the in vivo BRCA-1-mutated MDA-MB-436 xenograft model, a significant inhibition of
tumor growth was observed at daily oral dose of 100 mg/kg after 3 weeks of treatment. At the lower
dose of 20 mg/kg, 27 was still more effective than AZD-2281 dosed orally at 30 mg/kg QD. Compound
27 hydrochloride was well-tolerated, as indicated by the less than 10% reduction in body weight at these
doses after 21 consecutive days of treatment.
10
11
12
13
14
15
16
19

ACCEPTED MANUSCRIPT
1
2
Figure 3. Activity of 27 in MDA-MB-436 (BRCA-1 mutated) xenograft bearing mice when dosed orally
at 100 mg/kg q.d. or 20 mg/kg q.d. for 21 days.
3
4. Conclusions
4
In summary, the discovery and characterization of a novel, potent, orally efficacious PARP-1/2
inhibitor 27, has been described. Commencing with 1H-benzo[d]imidazole-4-carboxamide, the
introduction of the 4,5,6,7-tetrahydrothienopyridin-2-yl moiety improved potency against PARP-1, and
further substitution with fluorine atom at C6 of benzimidazole ring yielded compounds 13 and 15 with
good enzymatic inhibitory activity. Structural analysis together with thermodynamic studies elucidated
the intrinsic binding mechanisms of these inhibitors to PARP-1. The addition of a methyl group at N1 of
5
6
7
8
9
10
11
12
13
14
15
16
benzimidazole
core
resulted
in
compound
27,
6-fluoro-2-(4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carboxamide,
with
excellent inhibitory potency against the PARP-1/2 enzymes and a CC50 of 920 nM against BRCA2
deficient V-C8 cells. In the MDA-MB-436 (BRCA-1 mutated) xenograft model, this compound was
well tolerated and showed significant single-agent activity. Compound 27 has been selected as a lead
candidate and is currently undergoing further characterization of the preclinical profile.
5. Experimental Section
17
18
19
20
21
22
23
24
25
26
27
28
5.1 Chemical Synthesis. Analytical thin layer chromatography (TLC) was HSGF 254 (0.15-0.2 mm
thickness, Yantai Huiyou Company, China). Column chromatography was carried out on silica gel
(200-300 mesh). NMR spectra were recorded on Varian Mercury-300, -400, -500 or -600 spectrometers.
Tetramethylsilane (TMS) was used as internal standard. Chemical shifts were reported in parts per
million (ppm, δ). Proton coupling patterns are described as singlet (s), doublet (d), triplet (t), quartet (q),
heptet (hept), mutipet (m) and broad (br). Low- and high-resolution mass spectra (LRMS and HRMS)
were recorded on a Finnigan/MAT-95 (EI), FinniganLCQ/DECA and Micromass Ultra Q-TOF (ESI)
spectrometer. Melting points (m.p.) were measured by Büchi 510 melting point apparatus and were
uncorrected. The purity of all final was over 95% determined by an Agilent 1100 series LC system
(PLATISIL ODS 5 µL 250 mm × 4.6 mm) with two solvent systems (acetonitrile/water or
acetonitrile/buffer (0.1% CF3COOH in water)). Reverse phase HPLC purifications were performed on a
Gilson preparative HPLC system controlled by Unipoint software, using a Chiralpak As 4.6 mm × 250
20

ACCEPTED MANUSCRIPT
mm column. Chiral LC analysis was performed on an Shimadzu LC-10AT VP LC system using a Chiral
AS-H 5 µL 250 mm × 4.6 mm column.
1
2
3
4
2-(4,5,6,7-Tetrahydrothieno[3,2-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carboxamide (6): In a
hydrothermal synthesis reactor, were added 102 (110 mg, 0.35 mmol) and saturated ammonia solution
in methanol (10.0 mL). The reactor was capped and was heated at 80 °C for 20 h. After cooling and
removal of the solvent, the residue was chromatographed on silica gel using DCM/MeOH (10:1, v/v) as
5
6
1
7
the eluent to give a white solid (77 mg, 74%); H NMR (300 MHz, DMSO-d6) δ 9.09 (brs, 1 H), 7.83
8
(dd, J = 7.6, 1.1 Hz, 1 H), 7.73 (brs, 1 H), 7.68 (dd, J = 8.0, 1.1 Hz, 1 H), 7.63 (s, 1 H), 7.31 (t, J = 7.8
Hz, 1 H), 3.81 (s, 2 H), 3.00 (t, J = 5.6 Hz, 2 H), 2.76 (t, J = 5.6 Hz, 2 H); 13C NMR (125 MHz,
DMSO-d6) δ 166.75, 148.54, 138.68, 136.96, 135.53, 128.84, 126.66, 126.66, 123.10, 122.49, 122.04,
115.62, 45.30, 43.40, 26.09; HRMS (ESI): m/z Calcd. For C15H15N4OS [M+H]+: 299.0967; Found:
299.0959.
9
10
11
12
13
14
15
16
17
18
19
20
2-(5-Methyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carboxamide
(7): Starting from 103 (128 mg, 0.39 mmol), and following the procedure similar to that of preparation
of 6 to give a white solid (94 mg, 77%); 1H NMR (300 MHz, DMSO-d6) δ 9.49 (brs, 1 H), 7.74 (dd, J =
7.5, 1.2 Hz, 1 H), 7.65 (dd, J = 7.9, 1.2 Hz, 1 H), 7.59 (brs, 1 H), 7.57 (s, 1 H), 7.18 (t, J = 7.7 Hz, 1 H),
13
3.47 (s, 2 H), 2.90 (t, J = 5.6 Hz, 2 H), 2.71 (t, J = 5.6 Hz, 2 H), 2.41 (s, 3 H); C NMR (125 MHz,
DMSO-d6) δ 167.58, 151.40, 143.15, 140.55, 135.75, 135.59, 133.04, 125.06, 121.69, 121.20, 120.59,
116.90, 54.71, 52.43, 45.66, 25.74; HRMS (ESI): m/z Calcd. For C16H17N4OS [M+H]+: 313.1123;
Found: 313.1114.
21
22
23
24
25
26
27
28
2-(5-Ethyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carboxamide
(8): Starting from 104 (150 mg, 0.44 mmol), and following the procedure similar to that of preparation
of 6 to give a pale yellow solid (87 mg, 61%); 1H NMR (500 MHz, DMSO-d6) δ 13.44 (brs, 1 H), 9.14
(brs, 1 H), 7.84 (d, J = 7.6 Hz, 1 H), 7.78 (brs, 1 H), 7.68 (d, J = 8.0 Hz, 1 H), 7.64 (s, 1 H), 7.32 (t, J =
13
7.7 Hz, 1 H), 3.66 (s, 2 H), 2.92 (s, 2 H), 2.89 (brs, 2 H), 2.67 (brs, 2 H), 1.14 (t, J = 7.2 Hz, 3 H); C
NMR (125 MHz, DMSO-d6) δ 166.02, 147.64, 141.29, 137.31, 135.04, 129.40, 126.13, 122.83, 122.23,
121.93, 114.62, 51.36, 50.76, 49.46, 24.89, 11.86; HRMS (ESI): m/z Calcd. For C17H19N4OS [M+H]+:
327.1280; Found: 327.1273.
29
30
31
32
2-(4,5,6,7-Tetrahydrothieno[2,3-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carboxamide
(9):
Starting from 106 (158 mg, 0.50 mmol), and following the procedure similar to that of preparation of 6
to give a white solid (130 mg, 87%); 1H NMR (500 MHz, DMSO-d6) δ 9.12 (brs, 1 H), 7.82 (d, J = 7.5
Hz, 1 H), 7.75 (s, 1 H), 7.69 (dd, J = 7.9, 0.9 Hz, 1 H), 7.66 (brs, 1 H), 7.31 (t, J = 7.8 Hz, 1 H), 3.92 (s,
21

ACCEPTED MANUSCRIPT
1
2
3
2 H), 2.96 (t, J = 5.7 Hz, 2 H), 2.62 (t, J = 5.4 Hz, 2 H); 13C NMR (125 MHz, DMSO-d6) δ 166.71,
148.52, 141.84, 139.75, 136.01, 128.92, 123.15, 122.53, 115.26, 44.83, 42.98, 26.57; HRMS (ESI): m/z
Calcd. For C15H15N4OS [M+H]+: 299.0967; Found: 299.0959.
4
5
2-(6-Methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carboxamide
(10): Starting from 107 (134 mg, 0.41 mmol), and following the procedure similar to that of preparation
of 6 to give a grey solid (102 mg, 80%); 1H NMR (500 MHz, DMSO-d6) δ 9.15 (brs, 1H), 7.84 (d, J =
6.3 Hz, 1 H), 7.77 (brs, 1 H), 7.69 (d, J = 7.9 Hz, 1 H), 7.65 (brs, 1 H), 7.32 (t, J = 7.4 Hz, 1 H), 3.60 (s,
2 H), 2.73 (t, J = 5.0 Hz, 2 H), 2.67 (t, J = 5.5 Hz, 2 H), 2.39 (s, 3 H); 13C NMR (125 MHz, DMSO-d6)
δ 166.54, 148.28, 141.82, 137.62, 135.55, 135.14, 129.45, 128.26, 123.31, 122.71, 122.43, 115.12,
53.74, 52.01, 45.42, 25.67; HRMS (ESI): m/z Calcd. For C16H17N4OS [M+H]+: 313.1123; Found:
313.1118.
6
7
8
9
10
11
12
13
14
15
16
17
18
6-Methyl-2-(4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carboxamide
(11): Starting from 109 (59 mg, 0.15 mmol), and following the procedure similar to that of preparation
1
of 6 to give a pale brown solid (31 mg, 67%); M.p. > 300 °C; H NMR (500 MHz, DMSO-d6) δ 9.09
(brs, 1 H), 7.79 (s, 1 H), 7.75 (brs, 1 H), 7.64 (s, 1 H), 7.49 (s, 1 H), 4.22 (s, 2 H), 3.22 (t, J = 5.8 Hz, 2
13
H), 2.84 ((t, J = 5.8 Hz, 2 H), 2.44 (s, 3 H); C NMR (125 MHz, DMSO-d6) δ 166.75, 147.45, 139.87,
135.96, 134.71, 133.37, 132.29, 130.91, 128.18, 124.58, 121.66, 115.19, 42.77, 41.46, 23.69, 21.67;
HRMS (ESI): m/z Calcd. For C16H17N4OS [M+H]+: 313.1123; Found: 313.1119.
19
20
21
22
23
24
25
6-Chloro-2-(4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carboxamide
(12): Starting from 111 (48 mg, 0.14 mmol), and following the procedure similar to that of preparation
1
of 6 to give a pale brown solid (30 mg, 65%); M.p. > 300 °C; H NMR (500 MHz, DMSO-d6) δ 8.96
(brs, 1 H), 7.94 (brs, 1 H), 7.83 (s, 1 H), 7.74 (d, J = 2.2 Hz, 1 H), 7.73 (d, J = 2.2 Hz, 1 H), 4.13 (s, 2
H), 3.15 (t, J = 6.2 Hz, 2 H), 2.76 (t, J = 6.2 Hz, 2 H); 13C NMR (125 MHz, DMSO-d6) δ 165.49,
149.53, 136.28, 135.26, 129.80, 129.21, 126.88, 122.73, 115.09, 43.52, 41.95, 29.47; HRMS (ESI): m/z
Calcd. For C15H14ClN4OS [M+H]+: 333.0577; Found: 333.0570.
26
27
28
29
30
31
32
6-Fluoro-2-(4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carboxamide
(13): Starting from 113 (110 mg, 0.35 mmol), and following the procedure similar to that of preparation
of 6 to give a white solid (72 mg, 76%); M.p. > 300 °C; 1H NMR (500 MHz, DMSO-d6) δ 9.05 (brs, 1
H), 7.94 (s, 1 H), 7.72 (brs, 1 H), 7.61–7.54 (m, 2 H), 3.96 (s, 2 H), 2.99 (brs, 2 H), 2.65 (brs, 2 H); 13C
NMR (125 MHz, DMSO-d6) δ 165.62, 158.60 (d, J = 234.0 Hz, C-F), 149.79, 139.68, 135.98, 129.11,
128.95 (d, J = 26.0 Hz, CH-CF), 110.12 (d, J = 25.8 Hz, CH-CF), 44.77, 42.92, 26.47; HRMS (ESI):
m/z Calcd. For C15H14FN4OS [M+H]+: 317.0872; Found: 317.0861.
22

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
6-Fluoro-2-(6-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carb
oxamide (14): Starting from 114 (77 mg, 0.22 mmol), and following the procedure similar to that of
1
preparation of 6 to give a grey solid (36 mg, 55%); H NMR (500 MHz, DMSO-d6) δ 9.08 (brs, 1 H),
7.94 (s, 1 H), 7.68 (brs, 1 H), 7.59–7.52 (m, 2 H), 3.60 (s, 2 H), 2.73 (t, J = 5.3 Hz, 2 H), 2.67 (t, J = 5.6
Hz, 2 H), 2.39 (s, 3 H); 13C NMR (125 MHz, DMSO-d6) δ 165.49, 158.72 (d, J = 235.6 Hz, C-F ),
149.43, 137.74, 136.05, 135.16, 129.23, 128.59, 122.98, 110.32 (d, J = 26.5 Hz, CH-CF), 101.83, 53.72,
51.99, 45.40, 25.65; HRMS (ESI): m/z Calcd. For C16H16FN4OS [M+H]+: 331.1029; Found: 331.1020.
8
9
6-Fluoro-2-(4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carboxamide
(15): Starting from 116 (100 mg, 0.30 mmol), and following the procedure similar to that of preparation
of 6 to give a brown solid (78 mg, 82%); M.p. 288–290 °C; 1H NMR (500 MHz, DMSO-d6) δ 9.02 ( brs,
1 H), 7.88 (brs, 1 H), 7.61 (s, 1 H), 7.52 (dd, J = 10.0, 2.5 Hz, 1 H), 7.50 (dd, J = 8.0, 2.5 Hz, 1 H), 3.79
(s, 2 H), 2.99 (t, J = 5.2 Hz, 2 H), 2.74 (t, J = 5.2 Hz, 2 H); 13C NMR (125 MHz, DMSO-d6) δ 165.69,
158.58 (d, J = 236.0 Hz, C-F), 149.91, 138.63, 137.79,136.69, 128.92, 126.80, 122.55,110.05 (d, J =
26.5 Hz, CH-CF), 102.57 (d, J = 25.2 Hz, CH-CF), 79.63, 45.19, 43.30, 25.93; HRMS (ESI): m/z Calcd.
For C15H14FN4OS [M+H]+: 317.0872; Found: 317.0867.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
6-Fluoro-2-(5-methyl-4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carb
oxamide (16): Starting from 117 (100 mg, 0.29 mmol), and following the procedure similar to that of
1
preparation of 6 to give a pale yellow solid (58 mg, 61%); M.p.> 300 °C; H NMR (400 MHz,
DMSO-d6) δ 9.08 (brs, 1 H), 7.95 (s, 1 H), 7.65 (s, 1 H), 7.58–7.50 (m, 2 H), 3.57 (s, 1 H), 2.92 (t, J =
5.1 Hz, 2 H), 2.79 (t, J = 5.0 Hz, 2 H), 2.45 (s, 3 H); 13C NMR (125 MHz, DMSO-d6) δ 165.35, 158.78
(d, J = 237.0 Hz, C-F), 149.18, 138.48, 137.68, 136.04, 135.24, 129.62, 126.82, 123.20, 110.38 (d, J =
24.0 Hz, CH-CF), 101.78 (d, J = 25.0 Hz, CH-CF), 54.18, 52.00, 45.11, 25.31; HRMS (ESI): m/z Calcd.
For C16H16FN4OS [M+H]+: 331.1029; Found: 331.1022.
24
25
26
27
28
29
30
31
32
6-Fluoro-2-(7-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carb
oxamide (17): Starting from 128 (123 mg, 0.35 mmol), and following the procedure similar to that of
1
preparation of 6 to give a pale yellow solid (102 mg, 87%); M.p. > 300 °C; H NMR (300 MHz,
DMSO-d6) δ 8.96 (brs, 1 H), 7.90 (brs, 1 H), 7.68 (s, 1 H), 7.54 (dd, J = 6.3, 3.0 Hz, 1 H), 7.51 (dd, J =
4.4, 3.0 Hz, 1 H), 4.17 (q, J = 6.6 Hz, 2 H), 3.28-3.17 (m, 1 H), 2.99-2.86 (m, 1 H), 2.73-2.58 (m, 2 H),
13
1.41 (d, J = 6.6 Hz, 3 H); C NMR (125 MHz, DMSO-d6) δ 165.88, 158.92 (d, J = 237.4 Hz, C-F),
149.34, 143.87, 135.76, 129.20, 129.02, 122.86, 110.32 (d, J = 26.9 Hz, CH-CF), 102.04 (d, J = 25.6
Hz, CH-CF), 50.37, 41.89, 25.74, 22.74; HRMS (ESI): m/z Calcd. For C16H16FN4OS [M+H]+:
331.1029; Found: 331.1023.
23

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
2-(7-Ethyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-6-fluoro-1H-benzo[d]imidazole-4-carbox
amide (18): Starting from 129 (144 mg, 0.40 mmol), and following the procedure similar to that of
1
preparation of 6 to give a pale yellow solid (119 mg, 86%); M.p. 295–297 °C; H NMR (500 MHz,
DMSO-d6) δ 9.00 (brs, 1 H), 7.97 (brs, 1 H), 7.78 (s, 1 H), 7.56 (dd, J = 10.7, 2.7 Hz, 1 H), 7.53 (dd, J =
8.3, 2.7 Hz, 1 H), 4.23 (t, J = 6.0 Hz, 1 H), 3.34 (dt, J = 12.4, 4.1 Hz, 1 H), 3.06 (dt, J = 12.4, 4.1 Hz, 1
H), 2.87–2.71 (m, 2 H), 1.99-1.90 (m, 1 H), 1.89–1.76 (m, 1 H), 1.08 (t, J = 7.3 Hz, 3 H); 13C NMR
(125 MHz, DMSO-d6) δ 165.56, 158.71 (d, J = 238.1 Hz, C-F), 149.09, 140.19, 135.63, 130.15, 128.89,
122.95, 110.39 (d, J = 26.3 Hz, CH-CF), 102.48 (d, J = 27.1 Hz, CH-CF), 55.63, 41.39, 28.94, 24.58,
10.69; HRMS (ESI): m/z Calcd. For C17H18FN4OS [M]+: 345.1185; Found: 345.1178.
10
11
12
13
14
15
16
17
18
19
6-Fluoro-2-(7-propyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carb
oxamide (19): Starting from 130 (144 mg, 0.40 mmol), and following the procedure similar to that of
1
preparation of 6 to give a white solid (137 mg, 61%); M.p. 210–213 °C; H NMR (400 MHz,
DMSO-d6) δ 8.99 (brs, 1 H), 7.93 (brs, 1 H), 7.69 (s, 1 H), 7.54 (dd, J = 10.9, 2.5 Hz, 1 H), 7.51 (dd, J =
8.6, 2.6 Hz, 1 H), 4.12 (dd, J = 8.1, 4.6 Hz, 1 H), 3.23 (dt, J = 12.6, 4.8 Hz, 1 H), 2.94 (ddd, J = 13.2,
8.9, 5.4 Hz, 1 H), 2.78–2.57 (m, 2 H), 1.84-1.62 (m, 2 H), 1.59–1.41 (m, 2 H), 0.93 (t, J = 7.3 Hz, 3 H);
13C NMR (100 MHz, DMSO-d6) δ 165.47, 158.64 (d, J = 236.1 Hz, C-F), 149.21, 142.22, 135.84,
129.42, 128.92, 122.91, 110.29 (d, J = 25.9 Hz, CH-CF), 102.02 (d, J = 26.3 Hz, CH-CF), 54.22, 41.62,
38.79, 25.44, 19.09, 14.31; HRMS (ESI): m/z Calcd. For C18H20FN4OS [M+H]+: 359.1342; Found:
359.1334.
20
21
22
23
24
25
26
27
28
29
2-(7-Butyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-6-fluoro-1H-benzo[d]imidazole-4-carbox
amide (20): Starting from 131 (240 mg, 0.62 mmol), and following the procedure similar to that of
1
preparation of 6 to give a pale yellow solid (178 mg, 77%); M.p. 245–247 °C; H NMR (500 MHz,
DMSO-d6) δ 9.03 (brs, 1 H), 7.93 (brs, 1 H), 7.67 (s, 1 H), 7.55 (dd, J = 14.8, 2.4, 1 H), 7.53 (dd, J =
12.8, 2.4, 1 H), 7.52 (d, J = 2.4 Hz, 1H), 3.98–3.89 (m, 1 H), 3.15 (dt, J = 12.4, 4.4 Hz, 1 H), 2.83 (ddd,
J = 13.1, 8.8, 5.0 Hz, 1 H), 2.67-2.54 (m, 2 H), 1.82–1.71 (m, 1 H), 1.69–1.60 (m, 1 H), 1.52–1.41 (m, 2
H), 1.41–1.27 (m, 2 H), 0.90 (t, J = 7.3 Hz, 3 H); 13C NMR (125 MHz, DMSO-d6) δ 165.57, 158.66 (d,
J = 237.0 Hz, C-F), 149.55, 144.43, 136.38, 129.17, 128.78, 122.97, 110.24 (d, J = 26.2 Hz, CH-CF),
102.18 (d, J = 27.9 Hz, CH-CF), 54.61, 42.05, 37.09, 28.12, 26.55, 22.66, 14.45; HRMS (ESI): m/z
Calcd. For C19H22FN4OS [M+H]+: 373.1498; Found: 373.1490.
30
31
32
33
6-Fluoro-2-(7-isopropyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-ca
rboxamide (21): Starting from 132 (248 mg, 0.66 mmol), and following the procedure similar to that of
1
preparation of 6 to give a pale brown solid (166 mg, 70%); M.p. > 300 °C; H NMR (500 MHz,
DMSO-d6) δ 9.08 (brs, 1 H), 7.97 (brs, 1 H), 7.76 (s, 1 H), 7.55 (t, J = 10.8 Hz, 2 H), 4.24–4.17 (m, 1
24

ACCEPTED MANUSCRIPT
1
2
3
4
5
H), 3.35–3.28 (m, 1 H), 3.03–2.95 (m, 1 H), 2.85–2.75 (m, 1 H), 2.74–2.65 (m, 1 H), 2.23–2.15 (m, 1
H), 1.11 (d, J = 6.9 Hz, 3 H), 0.95 (d, J = 6.9 Hz, 3 H); 13C NMR (125 MHz, DMSO-d6) δ 165.33,
158.77 (d, J = 236.9 Hz), 149.04, 138.46, 136.85, 136.09, 129.99, 129.05, 123.34, 110.44 (d, J = 24.4
Hz), 101.82 (d, J = 26.9 Hz), 59.80, 42.35, 33.49, 29.49, 19.62, 17.32. HRMS (ESI): m/z Calcd. For
C18H20FN4OS [M+H]+: 359.1342; Found: 359.1336.
6
7
2-(7-Cyclopropyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-6-fluoro-1H-benzo[d]imidazole-4-
carboxamide (22): Starting from 133 (298 mg, 0.80 mmol), and following the procedure similar to that
1
8
of preparation of 6 to give a pale brown solid (260 mg, 91%); M.p. 258–261 °C; H NMR (300 MHz,
9
DMSO-d6) δ 9.00 (brs, 1 H), 7.88 (brs, 1 H), 7.68 (s, 1 H), 7.53 (dd, J = 11.0, 2.6 Hz, 1 H), 7.49 (dd, J =
9.6, 2.6 Hz, 1 H), 3.28-3.17 (m, 2 H), 2.92–2.76 (m, 1 H), 2.76–2.54 (m, 2 H), 1.18–0.98 (m, 2 H),
0.75–0.62 (m, 1 H), 0.6–0.40 (m, 2 H); 13C NMR (125 MHz, DMSO-d6) δ 165.56, 158.67 (d, J = 236.6
Hz, C-F), 149.54, 143.14, 135.92, 129.37, 128.80, 122.83, 110.24 (d, J = 26.2 Hz, CH-CF), 102.16 (d, J
= 24.6 Hz, CH-CF), 60.43, 42.54, 29.50, 25.92, 18.06, 4.30, 3.84; HRMS (ESI): m/z Calcd. For
C18H18FN4OS [M+H]+: 357.1185; Found: 357.1179.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
2-(7-(tert-Butyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-6-fluoro-1H-benzo[d]imidazole-4-c
arboxamide (23): Starting from 134 (234 mg, 0.60 mmol), and following the procedure similar to that
1
of preparation of 6 to give a brown solid (144 mg, 65%); M.p. 295–296 °C; H NMR (300 MHz,
DMSO-d6) δ 9.08 (brs, 1 H), 7.88 (brs, 1 H), 7.67 (brs, 1 H), 7.56 (dd, J = 8.4, 2.5 Hz, 1 H), 7.52 (dd, J
= 8.0, 2.5 Hz, 1 H), 3.73 (s, 1 H), 3.21 (t, J = 4.0 Hz, 1 H), 2.84–2.70 (m, 1 H), 2.66–2.54 (m, 2 H), 1.07
(s, 9 H); 13C NMR (125 MHz, DMSO-d6) δ 165.40, 158.71 (d, J = 235.0 Hz, C-F), 149.29, 140.61,
139.06, 138.61, 136.02, 128.94, 123.26, 110.34 (d, J = 30.3 Hz, CH-CF), 101.80 (d, J = 25.4 Hz,
CH-CF), 64.77, 43.02, 35.89, 27.42, 27.35; HRMS (ESI): m/z Calcd. For C19H22FN4OS [M+H]+:
373.1498; Found: 373.1490.
24
25
26
27
28
29
30
31
32
33
6-Fluoro-2-(7-isobutyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-car
boxamide (24): Starting from 135 (374 mg, 0.97 mmol), and following the procedure similar to that of
1
preparation of 6 to give a pale yellow solid (241 mg, 67%); M.p. 173–175 °C; H NMR (500 MHz,
DMSO-d6) δ 9.01 (brs, 1 H), 7.92 (brs, 1 H), 7.62 (s, 1 H), 7.53 (dd, J = 10.7, 2.6 Hz, 1 H), 7.50 (dd, J =
8.5, 2.6 Hz, 1 H), 3.94 (dd, J = 10.1, 4.3 Hz, 1 H), 3.11 (dt, J = 13.1, 5.4 Hz, 1 H), 2.81 (ddd, J = 13.4,
8.7, 5.4 Hz, 1 H), 2.63-2.52 (m, 2 H), 1.99–1.89 (m, 1 H), 1.58 (ddd, J = 14.2, 9.7, 4.7 Hz, 1 H), 1.48
13
(ddd, J = 13.6, 9.2, 4.3 Hz, 1 H), 0.93 (d, J = 6.6 Hz, 3 H), 0.92 (d, J = 6.6 Hz, 3 H); C NMR (125
MHz, DMSO-d6) δ 165.46, 158.6 (d, J = 237.5 Hz, C-F), 149.45, 145.03, 136.18, 129.01, 128.49,
122.76, 110.11 (d, J = 25.8 Hz, CH-CF), 102.11 (d, J = 23.4 Hz, CH-CF), 52.54, 46.85, 41.76, 26.57,
24.56, 23.87, 21.91; HRMS (ESI): m/z Calcd. For C19H22FN4OS [M+H]+: 373.1498; Found: 373.1490.
25

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
6-Fluoro-2-(5-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carb
oxamide (25): Starting from 136 (119 mg, 0.34 mmol), and following the procedure similar to that of
preparation of 6 to give a brown solid (82 mg, 73%); M.p. 285–288 °C; 1H NMR (300 MHz, DMSO-d6)
δ 8.96 (brs, 1 H), 7.89 (brs, 1 H), 7.67 (s, 1 H), 7.57–7.45 (m, 2 H), 4.06 (d, J = 28.9, 1 H), 4.01 (d, J =
29.1, 1 H), 2.99 (brs, 1 H), 2.74 (dd, J = 16.3, 3.9 Hz, 1 H), 2.41–2.25 (m, 1 H), 1.19 (d, J = 6.3 Hz, 3
H); HRMS (ESI): m/z Calcd. For C16H16FN4OS [M+H]+: 331.1029; Found: 311.1023.
7
8
6-Fluoro-2-(3-methyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carb
oxamide (26): Starting from 137 (226 mg, 0.65 mmol), and following the procedure similar to that of
1
9
preparation of 6 to give a pale yellow solid (151 mg, 70%); M.p. 286–288 °C; H NMR (300 MHz,
10
11
12
13
14
DMSO-d6) δ 9.14 (brs, 1 H), 7.92 (brs, 1 H), 7.55 (dd, J = 10.7, 2.6 Hz, 1 H), 7.47 (dd, J = 8.5, 2.6 Hz,
1 H), 3.92 (s, 2 H), 3.17 (s, 2 H), 3.00 (t, J = 5.7 Hz, 2 H), 2.46 (s, 3 H); 13C NMR (100 MHz,
DMSO-d6) δ 165.65, 158.43 (d, J = 234.4 Hz, C-F), 149.83, 138.36, 138.23, 137.78, 136.51, 122.68,
110.12 (d, J = 26.1 Hz, CH-CF), 102.30 (d, J = 27.0 Hz, CH-CF), 44.56, 42.63, 25.27, 13.76; HRMS
(ESI): m/z Calcd. For C16H16FN4OS [M+H]+: 331.1029; Found: 331.1023.
15
16
17
18
19
20
21
6-Fluoro-2-(5-((methylamino)methyl)thiophen-2-yl)-1H-benzo[d]imidazole-4-carboxamide (32):
Starting from 141 (216 mg, 0.68 mmol), and following the procedure similar to that of preparation of 6
to give a white solid (144 mg, 70%); M.p. 197–200 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.00 (brs, 1
H), 7.90 (brs, 1 H), 7.79 (brs, 1 H), 7.56–7.50 (m, 2 H), 7.08 (d, J = 3.9 Hz, 1 H), 3.90 (s, 2 H), 2.33 (s,
3 H); 13C NMR (125 MHz, DMSO-d6) δ 165.09, 158.17 (d, J = 236.6 Hz, C-F), 149.06, 148.66, 130.63,
127.95, 126.09, 122.22, 109.76 (d, J = 26.2 Hz, CH-CF), 101.94 (d, J = 27.1 Hz, CH-CF), 49.43, 35.13;
HRMS (ESI): m/z Calcd. For C14H14FN4OS [M+H]+: 305.0872; Found: 305.0863.
22
23
24
25
26
27
28
29
6-Fluoro-2-(4,5,6,7-tetrahydrothiazolo[5,4-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carboxamid
e (33): Starting from 143 (103 mg, 0.31 mmol), and following the procedure similar to that of
1
preparation of 6 to give a pale brown solid (65 mg, 66%); M.p. > 300 °C; H NMR (300 MHz,
DMSO-d6) δ 8.86 (brs, 1 H), 8.04 (brs, 1 H), 7.64 (dd, J = 10.6, 2.6 Hz, 1 H), 7.49 (dd, J = 8.4, 2.6 Hz,
1 H), 4.39 (brs, 2 H), 3.41 (t, J = 6.2 Hz, 2 H), 3.06 (t, J = 5.9 Hz, 2 H); 13C NMR (101 MHz, TFA-d) δ
167.73, 161.60 (d, J = 253.6 Hz, C-F), 150.70, 147.41, 142.78, 132.11(d, J = 12.8 Hz, C-CH-CF),
129.52, 126.60, 119.47 (d, J = 10.8 Hz, C-CH-CF), 116.01, 105.65 (d, J = 28.4 Hz, CH-CF), 42.37,
28.98, 22.73; HRMS (ESI): m/z Calcd. For C14H13FN5OS [M+H]+: 318.0825; Found: 318.0820.
30
31
32
6-Fluoro-1-methyl-2-(4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carb
oxamide (27): Starting from 148 (154 mg, 0.45 mmol), and following the procedure similar to that of
preparation of 6 to give a white solid (116 mg, 79%); Rf value = 0.1, DCM: MeOH = 8: 1, v/v; M.p.
26

ACCEPTED MANUSCRIPT
1
1
2
3
4
5
6
7
265–267 °C; H NMR (500 MHz, DMSO-d6) δ 9.05 (brs, 1 H), 8.00 (brs, 1 H), 7.81 (d, J = 8.6 Hz, 1
13
H), 7.60 (s, 1 H), 7.58 (d, J = 12.9 Hz, 1 H), 4.01 (brs, 5 H), 3.03 (brs, 2 H), 2.68 (brs, 2 H); C NMR
(125 MHz, DMSO-d6) δ 165.22, 158.77 (d, J = 237.0 Hz, C-F), 149.48, 138.10 (d, J = 12.9 Hz,
C-CH-CF), 136.97, 136.02, 130.17, 127.91, 123.36 (d, J = 8.6 Hz, C-CH-CF), 110.93 (d, J = 26.3 Hz,
CH-CF), 101.49 (d, J = 28.6 Hz, CH-CF), 44.28, 42.66, 32.80, 26.01; 19F NMR (471 MHz, DMSO) δ
-117.97 (t, J = 9.5 Hz, CH-CF); HRMS (ESI): m/z Calcd. For C16H16FN4OS [M]+: 331.1029; Found:
331.1024.
8
9
1-Ethyl-6-fluoro-2-(4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carbox
amide (28): Starting from 149 (156 mg, 0.43 mmol), and following the procedure similar to that of
1
10
11
12
13
14
15
16
preparation of 6 to give a brown solid (120 mg, 81%); M.p. 217–220 °C; H NMR (500 MHz,
DMSO-d6) δ 9.04 (brs, 1 H), 8.03 (brs, 1 H), 7.88 (dd, J = 8.9, 2.6 Hz, 1 H), 7.60 (dd, J = 9.2, 2.4 Hz, 1
H), 7.59 (s, 1 H), 4.54 (q, J = 7.2 Hz, 2 H), 4.10 (s, 2 H), 3.11 (t, J = 5.7 Hz, 2 H), 2.76 (t, J = 5.7 Hz, 2
H), 1.40 (t, J = 7.1 Hz, 3 H); 13C NMR (125 MHz, DMSO-d6) δ 165.11, 158.87 (d, J = 237.1 Hz, C-F),
148.49, 137.47, 137.08 (d, J = 8.8 Hz, C-CH-CF), 137.07, 135.83, 129.43, 128.15, 123.60 (d, J = 8.4
Hz, C-CH-CF), 111.16 (d, J = 26.6 Hz, CH-CF), 101.56 (d, J = 28.2 Hz, CH-CF), 43.70, 42.21, 29.44,
25.22, 15.17; HRMS (ESI): m/z Calcd. For C17H18FN4OS [M+H]+: 345.1185; Found: 345.1177.
17
18
19
20
21
22
23
24
2-(7-(tert-Butyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-6-fluoro-1-methyl-1H-benzo[d]imi
dazole-4-carboxamide (29): Starting from 150 (196 mg, 0.49 mmol), and following the procedure
similar to that of preparation of 6 to give a white solid (132 mg, 70%); M.p. 290–293 °C; 1H NMR (500
MHz, TFA-d) δ 7.90 (d, J = 8.2 Hz, 1 H), 7.85 (s, 1 H), 7.71 (d, J = 5.3 Hz, 1 H), 4.71 (s, 1 H), 4.17 (s,
3 H), 3.97–3.88 (m, 1 H), 3.61–3.48 (m, 1 H), 3.27– 3.08 (m, 2 H), 1.26 (s, 9 H); 13C NMR (125 MHz,
TFA-d) δ 169.79, 161.95, 147.83, 139.38, 138.84, 137.26, 136.51 (d, J = 12.4 Hz, C-CH-CF), 127.63,
121.64, 105.90 (d, J = 28.9 Hz, CH-CF), 67.26, 44.61, 36.93, 34.18, 26.61, 23.50; HRMS (ESI): m/z
Calcd. For C20H24FN4OS [M+H]+: 387.1655; Found: 387.1648.
25
26
27
28
29
30
31
32
6-Fluoro-2-(7-isobutyl-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-1-methyl-1H-benzo[d]imida
zole-4-carboxamide (30): Starting from 150 (192 mg, 0.48 mmol), and following the procedure similar
to that of preparation of 6 to give a white solid (128 mg, 69%); M.p. 257–260 °C; 1H NMR (400 MHz,
DMSO-d6) δ 9.04 (brs, 1 H), 7.96 (brs, 1 H), 7.79 (dd, J = 8.8, 2.6 Hz, 1 H), 7.56 (s, 1 H), 7.55 (d, J =
10.4, 2.5 Hz, 1 H), 4.00 (s, 3 H), 3.13 (brs, 1 H), 2.81 (brs, 1 H), 2.59 (brs, 2 H), 1.94 (s, 1 H), 1.66–1.54
(m, 1 H), 1.53-1.48 (m, 1 H), 0.94 (d, J = 6.1 Hz, 3 H), 0.92 (d, J = 6.1 Hz, 3 H); 13C NMR (100 MHz,
DMSO-d6) δ 165.15 , 158.70 (d, J = 236.9 Hz, C-F), 149.56 , 138.07 (d, J = 13.4 Hz, C-CH-CF),
136.97, 136.64, 130.38, 127.38, 123.30 (d, J = 8.5 Hz, C-CH-CF), 110.84 (d, J = 26.3 Hz, CH-CF),
27

ACCEPTED MANUSCRIPT
101.43 (d, J = 28.2 Hz, CH-CF), 52.48, 47.06, 32.77, 24.59, 24.04, 21.86; HRMS (ESI): m/z Calcd. For
C20H24FN4OS [M+H]+: 387.1655; Found: 387.1649.
1
2
3
4
6-Fluoro-2-(4,5,6,7-tetrahydrofuro[2,3-c]pyridin-2-yl)-1H-benzo[d]imidazole-4-carboxamide
(31): Starting from 139 (28 mg, 0.09 mmol), and following the procedure similar to that of preparation
of 6 to give a brown solid (20 mg, 74%); M.p. 220–223 °C; 1H NMR (300 MHz, DMSO-d6) δ 9.09 (brs,
1 H), 7.96 (brs, 1 H), 7.56 (dd, J = 10.7, 2.5 Hz, 1 H), 7.51 (dd, J = 8.4, 2.4 Hz, 1 H), 7.34 (s, 1 H), 4.16
(s, 2 H), 3.17 (t, J = 5.0 Hz, 2 H), 2.68 (t, J = 5.0 Hz, 2 H); 13C NMR (151 MHz, DMSO-d6) δ 172.01,
165.47, 158.78 (d, J = 237.0 Hz, C-F), 157.99, 149.26, 145.61, 143.50, 118.56, 113.29, 110.51 (d, J =
27.0 Hz, CH-CF), 102.11, 49.05, 42.21, 41.57; HRMS (ESI): m/z Calcd. For C15H14FN4O2 [M+H]+:
301.1101; Found: 301.1093.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
4-Fluoro-1-(4,5,6,7-tetrahydrothieno[3,2-c]pyridin-2-yl)-8,9-dihydro-2,7,9a-triazabenzo[cd]azul
en-6(7H)-one (34): Starting from 155 (90 mg, 0.20 mmol), and following the procedure similar to that
1
of preparation of 102 to give a white solid (94 mg, 67%); M.p. 240–243 °C; H NMR (400 MHz,
DMSO-d6) δ 8.65 (t, J = 5.8 Hz, 1 H), 7.72 (dd, J = 8.9, 2.6 Hz, 1 H), 7.56 (dd, J = 10.1, 2.6 Hz, 1 H),
7.55 (s, 1 H), 4.53 (brs, 2 H), 4.24 (s, 2 H), 3.61 (dd, J = 9.7, 5.6 Hz, 2 H), 3.44 (t, J = 6.1 Hz, 2 H), 3.10
(t, J = 6.1 Hz, 2 H); 13C NMR (101 MHz, DMSO-d6) δ 166.52 (d, J = 1.2 Hz, C-C-CH-CF), 158.58 (d, J
= 235.6 Hz, C-F), 149.68, 144.41 (d, J = 12.7 Hz, C-CH-CF), 136.74, 130.33, 130.03 (d, J = 2.4 Hz,
C-C-CH-CF), 128.29, 118.94 (d, J = 8.2 Hz, C-CH-CF), 112.82 (d, J = 27.0 Hz, CH-CF), 108.86 (d, J =
24.0 Hz, CH-CF), 50.74, 42.62, 41.23, 40.45, 22.24; HRMS (EI): m/z Calcd. For C17H16FN4OS [M]+:
343.1023; Found: 343.1021.
21
22
23
24
25
26
27
28
29
30
4-Fluoro-1-(4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-8,9-dihydro-2,7,9a-triazabenzo[cd]azul
en-6(7H)-one (35): Starting from 156 (81 mg, 0.18 mmol), and following the procedure similar to that
1
of preparation of 102 to give a pale green solid (94 mg, 67%);M.p. 280–284 °C; H NMR (400 MHz,
DMSO-d6) δ 8.63 (t, J = 5.7 Hz, 1 H), 7.70 (dd, J = 9.0, 2.6 Hz, 1 H), 7.55 (dd, J = 10.6, 2.6 Hz, 1 H),
7.47 (s, 1 H), 4.55 (brs, 2 H), 4.05 (s, 2 H), 3.61 (dd, J = 9.4, 5.4 Hz, 2 H), 3.07 (t, J = 5.8 Hz, 2 H), 2.69
(t, J = 5.8 Hz, 2 H); 13C NMR (125 MHz, DMSO-d6) δ 166.60 (d, J = 1.3 Hz, C-C-CH-CF), 158.52 (d, J
= 235.4 Hz, C-F), 150.29, 144.51 (d, J = 12.1 Hz, C-CH-CF), 138.15, 135.61, 130.56, 130.03, 128.39,
118.77 (d, J = 9.0 Hz, C-CH-CF), 112.51 (d, J = 26.8 Hz, CH-CF), 108.72 (d, J = 23.8 Hz, CH-CF),
50.75, 44.11, 42.52, 40.08, 25.65; HRMS (EI): m/z Calcd. For C17H15FN4OS [M]+: 343.1023; Found:
343.1019.
31
32
(S)-Methyl
2-(7-(tert-butyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-6-fluoro-1H-benzo[d]imidazole-4-car
28

ACCEPTED MANUSCRIPT
1
2
boxylate (S-134): Separeted from racemic 134 by chiral HPLC (Chiralpak As, 0.46 cm I.D. × 25 cm L;
5 µL; CO2 /IPA/DEA = 60/40/0.1 (v/v/v); 2.5 mL/min; UV λ = 254 nm; 35 °C) to give a white solid; 1H
NMR (300 MHz, Chloroform-d) δ 10.40 (s, 1 H), 7.63 (dd, J = 15.5, 1.8 Hz, 1 H), 7.60 (dd, J = 15.8,
1.7 Hz, 1 H), 7.39 (s, 1 H), 4.02 (s, 3 H), 3.84 (s, 1 H), 3.43–3.31 (m, 1 H), 2.98–2.85 (m, 1 H), 2.80–
2.58 (m, 2 H), 1.13 (s, 9 H); MS (ESI) 388 [M+H]+; [α]D20 –169 (c, 0.2, MeOH); tR = 3.60 min.
(S)-2-(7-(tert-Butyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-6-fluoro-1H-benzo[d]imidazole-
4-carboxamide (S-23): Starting from S-134 (200 mg, 0.52 mmol), and following the procedure similar
3
4
5
6
7
1
8
to that of preparation of 6 to give a pale brown solid (125 mg, 65%); M.p. 290–292 °C; H NMR (300
9
MHz, DMSO-d6) δ 9.08 (brs, 1 H), 7.88 (brs, 1 H), 7.67 (brs, 1 H), 7.56 (dd, J = 8.4, 2.5 Hz, 1 H), 7.52
(dd, J = 8.0, 2.5 Hz, 1 H), 3.73 (s, 1 H), 3.21 (t, J = 4.0 Hz, 1 H), 2.84–2.70 (m, 1 H), 2.66–2.54 (m, 2
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
H), 1.07 (s, 9 H); HRMS (ESI): m/z Calcd. For C19H22FN4OS [M+H]+: 373.1498; Found: 373.1490; [α]
20
D
–191 (c, 0.08, MeOH); ee > 99%; tR = 12.69 min (Chiralpak AD-H;0.40 cm I.D. × 25 cm L; 5 µL;
Hexane/IPA/DEA = 70/30/0.05 (v/v/v); 0.40 mL/min; UV λ = 254 nm; 20 °C).
(R)-methyl
2-(7-(tert-butyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-6-fluoro-1H-benzo[d]imidazole-4-car
boxylate (R-134): Separeted from racemic 134 by chiral HPLC (Chiralpak As, 0.46 cm I.D. × 25 cm L;
5 µL; CO2 /IPA/DEA = 60/40/0.1 (v/v/v); 2.5 mL/min; UV λ = 254 nm; 35 °C) to give a white solid; 1H
NMR (300 MHz, Chloroform-d) δ 10.40 (s, 1 H), 7.63 (dd, J = 15.5, 1.8 Hz, 1 H), 7.60 (dd, J = 15.8,
1.7 Hz, 1 H), 7.39 (s, 1 H), 4.02 (s, 3 H), 3.84 (s, 1 H), 3.43–3.31 (m, 1 H), 2.98–2.85 (m, 1 H), 2.80–
2.58 (m, 2 H), 1.13 (s, 9 H); MS (ESI) 388 [M+H]+; [α]D20 141 (c, 0.2, MeOH); tR = 1.93 min.
(R)-2-(7-(tert-butyl)-4,5,6,7-tetrahydrothieno[2,3-c]pyridin-2-yl)-6-fluoro-1H-benzo[d]imidazole-
4-carboxamide (R-23): Starting from R-134 (200 mg, 0.52 mmol), and following the procedure similar
1
to that of preparation of 6 to give a pale brown solid (120 mg, 62%); M.p. 295–296 °C; H NMR (300
MHz, DMSO-d6) δ 9.08 (brs, 1 H), 7.88 (brs, 1 H), 7.67 (brs, 1 H), 7.56 (dd, J = 8.4, 2.5 Hz, 1 H), 7.52
(dd, J = 8.0, 2.5 Hz, 1 H), 3.73 (s, 1 H), 3.21 (t, J = 4.0 Hz, 1 H), 2.84–2.70 (m, 1 H), 2.66–2.54 (m, 2
H), 1.07 (s, 9 H); HRMS (ESI): m/z Calcd. For C19H22FN4OS [M+H]+: 373.1498; Found: 373.1490; [α]
29