Development of novel benzimidazole-derived neddylation inhibitors for suppressing tumor growth in vitro and in vivo

Article abstract of DOI:10.1016/j.ejmech.2020.112964

Ubiquitin-like protein neddylation is overactivated in various human cancers and correlates with disease progression, and targeting this pathway represents a valuable therapeutic strategy. Our previous work disclosed an antihypertensive agent, candesartan cilexetic (CDC), serves as a novel neddylation inhibitor for suppressing tumor growth by targeting Nedd8-activating enzyme (NAE). In this study, 42 benzimidazole derivatives were designed and synthesized based on lead compound CDC to improve the neddylation inhibition and anticancer efficacy. Optimal benzimidazole-derived 35 displayed superior neddylation inhibition in enzyme assay compared to CDC (IC₅₀ = 5.51 μM vs 16.43 μM), along with promising target inhibitory activity and killing selectivity in cancer cell. The results of cellular mechanism research combined with tumor growth suppression in human lung cancer cell A549 in vivo, accompanied with docking model, revealed that 35 has the potential to be developed as a promising neddylation inhibitor for anticancer therapy.

Full text of DOI:10.1016/j.ejmech.2020.112964

European Journal of Medicinal Chemistry xxx (xxxx) xxx
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Development of novel benzimidazole-derived neddylation inhibitors
for suppressing tumor growth in vitro and in vivo
Xin Chen a , Xi Yang , Fei Mao , Jinlian Wei , Yixiang Xu , Baoli Li , Jin Zhu ,
,
1
b
,
1
a
,
1
a
,
1
a
a
a
b,
**
, Lijun Jia b , Jian Li
,
***
a, c, d, *
Shuaishuai Ni
a
State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of New Drug Design, East China University of Science and Technology, 130 Mei
Long Road, Shanghai, 200237, China
Cancer Institute, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai, 200032, China
College of Pharmacy and Chemistry, Dali University, 5 Xue Ren Road, Dali, Yunnan, 671000, China
Frontiers Science Center for Materiobiology and Dynamic Chemistry, East China University of Science and Technology, 130 Mei Long Road, Shanghai,
b
c
d
2
00237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 June 2020
Received in revised form
Ubiquitin-like protein neddylation is overactivated in various human cancers and correlates with disease
progression, and targeting this pathway represents a valuable therapeutic strategy. Our previous work
disclosed an antihypertensive agent, candesartan cilexetic (CDC), serves as a novel neddylation inhibitor
for suppressing tumor growth by targeting Nedd8-activating enzyme (NAE). In this study, 42 benz-
imidazole derivatives were designed and synthesized based on lead compound CDC to improve the
neddylation inhibition and anticancer efficacy. Optimal benzimidazole-derived 35 displayed superior
21 September 2020
Accepted 19 October 2020
Available online xxx
neddylation inhibition in enzyme assay compared to CDC (IC50 ¼ 5.51
mM vs 16.43 mM), along with
Keywords:
Neddylation
cullin1-Nedd8
Benzimidazole-derived inhibitor
Anticancer
promising target inhibitory activity and killing selectivity in cancer cell. The results of cellular mecha-
nism research combined with tumor growth suppression in human lung cancer cell A549 in vivo,
accompanied with docking model, revealed that 35 has the potential to be developed as a promising
neddylation inhibitor for anticancer therapy.
©
2020 Elsevier Masson SAS. All rights reserved.
1
. Introduction
methods that still faces plenty of challenges, such as therapeutic
resistance, monotonous research approaches, system toxicities and
side effects. To overcome the defects and decelerate the spread of
drug resistance, it is urgent to develop the next class of chemo-
therapeutics with novel mechanisms of action.
The ubiquitin proteasome system (UPS) acts as one of the most
important protein degradation pathways that regulates the
degradation of intracellular proteins, and it has important roles in a
variety of cellular and organismal processes [4]. In the UPS, cullin-
RING ubiquitin E3 ligases (CRLs) are the largest family of multiunit
E3 ubiquitin ligases [5,6], which are assembled with the best-
known cullin family members and activated by cullin neddylation
Cancer has become one of the most serious health issues for
global public health [1]. According to a report from the World
Health Organization (WHO) in 2018, cancer is the first or second
leading cause of death before seventy years of age in 91 of 172
countries [2]. The GLOBOCAN 2018 database provides estimates of
18.1 million new cancer cases and 9.6 million cancer deaths
worldwide [3]. Although the improvement of diagnostic methods
has made significant progress in decreasing the tumor incidence
and mortality, chemotherapy acts as one of the main anticancer
[
7,8]. Neddylation is a posttranslational modification of conjugating
the neuronal precursor cell-expressed developmentally down-
regulated protein 8 (Nedd8) to cullin family members and other
noncullin substrates.
*
Corresponding author. State Key Laboratory of Bioreactor Engineering, Shanghai
Key Laboratory of New Drug Design, East China University of Science and Tech-
nology, 130 Mei Long Road, Shanghai, 200237, China.
*
*
* Corresponding author.
** Corresponding author.
During the process of neddylation (Fig. 1A), Nedd8 first binds to
2þ
Nedd8-activating enzyme (NAE) in an Mg /ATP-dependent
E-mail addresses: nss1106@126.com (S. Ni), ljjia@shutcm.edu.cn (L. Jia), jianli@
manner [9,10] and then reacts to yield an acyl adenylate between
the carboxyl terminus of Nedd8 and AMP (Nedd8-AMP) coupled
ecust.edu.cn (J. Li).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2020.112964
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.
Please cite this article as: X. Chen, X. Yang, F. Mao et al., Development of novel benzimidazole-derived neddylation inhibitors for suppressing
tumor growth in vitro and in vivo, European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112964

X. Chen, X. Yang, F. Mao et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 1. The process of neddylation (A) and reported NAE inhibitors (B).
with the release of inorganic pyrophosphate. Nedd8-AMP reacts
with the thiol of the active site cysteine residue in the NAE subunit
to form a NAE-Nedd8 thioester and release AMP. Subsequently,
Nedd8 is transferred to one of the two Nedd8 conjugating enzymes
conferred significant antitumor activity in phase II/III clinical tri-
als [24]. However, recent preclinical studies identified the resis-
tance to MLN4924 for heterozygous mutations nearby its
covalently binding site with NAE, suggesting the development of
covalent NAE inhibitors is possibly limited in practice [25,26]. For
the R&D of noncovalent neddylation inhibitors, Lu et al. devel-
oped a series of piperidin-4-amine scaffold neddylation inhibitors
that presented submicromolar NAE inhibitory activities in
enzyme-based assays [27,28]. Leung et al. and partners reported
serial studies on the discovery of neddylation inhibitors by virtual
screening [29e33]. In addition, several series of DCN1 inhibitors
have been discovered by high throughput screening (HTS) in
recent years [34e39]. However, the small molecule inhibitors
mentioned above (except MLN4924) are in preclinical research;
thus, there is an urgent need to develop novel inhibitors with
better druggability.
CDC (01), as an FDA-approved antihypertensive agent, is con-
verted into candesartan via the first-pass effect of the gastroin-
testinal tract in the body to play an antihypertensive effect, but we
have revealed in a previous study that candesartan has no ned-
dylation inhibitory activity, and CDC is capable of blocking the
neddylation pathway of various types of cancers by inhibiting NAE
[40]. Although it has moderate neddylation inhibition and anti-
cancer activity, nonoral administration due to its prodrug struc-
ture has limited the further development of CDC. In this study,
novel CDC-derived neddylation inhibitors were designed and
synthesized to promote the neddylation inhibition and overcome
the disadvantages of anticancer activities and oral administration
of CDC.
(
UBE2M/Ubc12 [11] or UBE2F [12], E2s) through a transthiolation
reaction and forms E2-Nedd8. In the presence of Nedd8 ligases
E3s), Nedd8 is ultimately transferred from E2-Nedd8 to specific
(
substrates. The best-known substrates for neddylation are mem-
bers of the cullin family, which are involved in the assembly of
cullin-RING E3 ubiquitin ligases (CRLs). CRLs act as a large family of
ubiquitin ligases that regulate cellular protein substrates for pro-
teasomal degradation. The inhibition of the neddylation pathway
inactivates CRLs and decreases the protein levels of ubiquitination
and subsequent degradation of substrates that are regulated by
CRLs substrates, such as Wee1 [13], p27 [14], and Nrf2 [15], leading
to the suppression of tumor growth. In recent years, many studies,
including our studies, have shown that the level of neddylation
enzymes are higher in various types of human cancers compared to
adjacent normal tissues [16e20], providing a sound rationale as an
attractive cancer treatment strategy. Accumulated experimental
data have clearly demonstrated that the overexpression of neddy-
lation enzymes is associated with disease progression, conferring a
worse overall patient survival [13,16,17]. Thus, targeting the ned-
dylation pathway is an effective approach to suppressing tumor
growth [21e23].
In fact, the potential of NAE as a target in the development of
novel anti-cancer therapeutics recently received attention
(
(
Fig. 1B). A terminal sulfonamide-based compound, pevonedistat
MLN4924), represents a potently covalent NAE inhibitor that
2

X. Chen, X. Yang, F. Mao et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
2
. Results and discussion
Derivatives 9e46 were prepared as described in Scheme 2.
Derivatives 9 and 10 were prepared by using similar synthetic
routes as those of 7 or 8. Analog 11 was achieved by methylation of
CDC. Candesartan reacts with triphenylmethyl bromide to obtain
intermediate 12a, which was then condensed with various
substituted amines; finally, trifluoroacetic acid was hydrolyzed to
produce derivatives 12e46.
2.1. Design and synthesis
To improve the neddylation inhibitory activity and anticancer
efficacy of the lead compound CDC and obtain a novel structural
scaffold, chemical modifications were performed in three cycles in
this study (Fig. 2). We divided the structure of CDC into three
functional regions, namely, regions A, B and C. In our previous
work, we showed that region C is a key pharmacophore domain for
regulating the neddylation inhibitory activity by comparing with
other FDA-approved A2T1R antagonists (structural analogs of CDC)
2
2
.2. In vitro biological evaluations
.2.1. Preliminary investigation of neddylation inhibitory activities
and anti-proliferation abilities of benzimidazole derivatives
[
40]. Hence, based on the strategy of scaffold hopping, we first
Cullins act as one of the most important neddylation substrates
that can accurately reflect the level of Nedd8 modification in cancer
cells, while the formation of cullin1-Nedd8 adduct was explored as
a critical indicator for investigating the inhibitory activities of de-
rivatives rationally [5,6]. Initial studies involved in the investigation
of structurally modified derivatives in region A and region B were
performed by exploring the levels of cullin1-Nedd8 adduct at the
enzyme-based assay, along with anti-proliferation activities on
A549 human lung cancer cells. As shown in Table 1, we first tried to
replace benzimidazole (region A) with ring-opening (6), resulting
in the reduced neddylation inhibitory activity of the derivatives.
Next, we replaced, removed or methylated the tetrazolium group to
obtain derivatives 9e11. Unfortunately, they have poorer neddyla-
tion inhibitory activity compared to CDC.
opened the ring of the benzimidazole in region A and changed the
substitution position of the cilexetil group to generate derivatives
5
e8 (Table 1). The tetrazole group of CDC was then replaced with
various steric substituents in region B to generate derivatives 9e11.
Subsequently, comprehensive modifications of the side linker (cil-
exetil group) in region C aimed to elevate the inhibitory activities
and reverse the easy hydrolysis by oral administration of CDC.
The synthetic routes for the preparation of derivatives 5e8 are
outlined in Scheme 1. Intermediate 5a was first obtained by a
cyclization reaction of 2,3-diaminobenzoic acid and tetraethyl
orthocarbonate (commercially available), followed by a nucleo-
0
philic substitution reaction with N-(triphenylmethyl)-5-(4 -bro-
momethylbiphenyl-2-yl)tetrazole to yield intermediate 5b. Finally,
intermediate 5b was hydrolyzed with 20% trifluoroacetic acid to
give target derivative 5. Methyl anthranilate (commercially avail-
able) was reacted with butyryl chloride to obtain a mono-
substituted amide, which was directly input into the next step
without purification to generate intermediate 6a, followed by the
nucleophilic substitution of 1-chloroethylcyclohexyl carbonate
under the conditions of NaHCO /DMF to generate intermediate 6b;
3
we finally achieved 6 by 20% trifluoroacetic acid hydrolysis. Inter-
mediate 7a was first obtained by a cyclization reaction of methyl
In comparison with lead compound CDC, removal of the cilexetil
group (5) in region C and replacement of the cilexetil group with 5
(
8) and 6 (7) substitutions leads to a decrease in neddylation
inhibitory activity. Perhaps the presence of cilexetil groups and the
-position substitution are important for maintaining the neddy-
7
lation inhibition of the derivatives. Combined with the results of
molecular docking in our previous study, we preliminarily specu-
late that regions A and B are groups that maintain neddylation
inhibitory activity. Thus, to keep it unchanged, we turned our
attention to region C. As is known, the cilexetil group has emerged
as a prodrug group that is vulnerable upon oral administration. In
addition, CDC has poor water solubility, and its water solubility can
be improved by replacing ester bonds with amide bonds to increase
stability.
Next, we focused on the systemic variation in region C for sta-
bilizing the structure and elevating the inhibitory activities and
water solubility of the new derivatives. As shown in Table 2, the
cilexetil group was replaced with an aliphatic chain or an aliphatic
ring to obtain a series of derivatives, 12e20, but the neddylation
inhibitory activity was reduced to varying degrees compared to that
3
,4-diaminobenzoate and tetraethyl orthocarbonate. Next, the key
intermediate 7b (6-substituted) and its isomer 8b (5-substituted)
were achieved by 7a reacting with N-(triphenylmethyl)-5-(4 -bro-
momethylbiphenyl-2-yl)tetrazole under the conditions of K CO /
2 3
DMF separation and purification by silica gel column chromatog-
raphy. Intermediates 7b and 8b were hydrolyzed under lithium
hydroxide to obtain their carboxylates, and the crude products
were directly reacted with 1-chloroethylcyclohexyl carbonate to
obtain intermediates 7c and 8c. Intermediate 7c and 8c were
deprotected under 20% trifluoroacetic acid conditions to obtain the
target compounds 7 and 8.
0
Fig. 2. Chemical optimization strategies and purposes for lead compound CDC.
3

X. Chen, X. Yang, F. Mao et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Table 1
Neddylation inhibition in enzyme-based assay and the antitumor activities against A549 human lung cancer cells of derivatives 5e11.
Compd.
Structure
Cullin1-Nedd8 inhibition % @ 25
18.9%
m
Ma
A549 inhibition% @ 100
9.5%
m
Mb
5
6
32.6%
15.2%
7
8
43.2%
44.7%
56.9%
33.3%
9
29.5%
34.2%
34.2%
14.6%
1
1
0
1
41.2%
87.8%
90.5%
16.8%
69.2%
91.4%
CDC (01)
MLN4924
a
The neddylation rate of cullin1-Nedd8 in enzyme-based assay and are reported as the average of three experiments.
The anti-profileration rate in A549 and are reported as the average of three experiments.
b
of CDC. Considering that the NAE pocket is relatively flat, a series of
electron-donating ones (22, 24, 33e35 vs 23, 27, 31). (4) Sub-
stitutions at the phenyl ring by chlorine atoms presented superior
efficacy over other groups, while the substitution at the para-
position provided significant improvements in activities (35, 39,
46 vs. 38, 40e45).
aromatic ring-substituted derivatives were synthesized (21e37).
The replacement of the cilexetil group with phenylethylamine led
to the superior inhibition of derivatives, such as 33 and 35. Ac-
cording to the results, more terminal phenethylamine derivatives
were designed and synthesized to evaluate the neddylation and
proliferation inhibitory activities (Table 3, Fig. 2), some of which
2.2.3. Optimal compounds suppress the growth of cancer cell by
(
39, 46) displayed superior efficacy than the lead compound CDC.
inhibiting the neddylation pathway
Next, derivatives 35, 39 and 46 as the optimal compounds were
selected for investigating neddylation inhibitory activities and
cellular anti-proliferation abilities at various concentrations with
either two human lung cancer cell lines (A549 and H1299) or two
human normal cell lines (16H6E and BEAS-2E), as shown in Table 4.
The results showed that three derivatives displayed excellent
inhibitory activities on the formation of cullin1-Nedd8 adducts at
the micromolar concentration in the enzyme-based assay, along
with selectively inhibiting the proliferation of cancer cells without
affecting the normal ones. In general, derivative 35 presented su-
perior neddylation inhibitory activities and anticancer selectivity.
In light of the inhibitory activity of the cullin1-Nedd8 adduct
and selective anti-proliferation activities in cancer cells, derivative
35 was chosen to further investigate the neddylation inhibition in
2
.2.2. Structure-activity relationships (SARs)
According to the structural features and the preliminary results
of the neddylation inhibitory abilities and anti-proliferation activ-
ities of the derivatives, the SARs are summarized in detail. (1) The
scaffolds of benzimidazole and tetrazole groups are important for
improving the neddylation inhibitory activities of the derivatives (1
vs. 5e10). (2) The replacements of the substituents of (cyclo) alkyl,
benzoalicyclic and heterocyclic groups into region C are unfavor-
able for the neddylation inhibitory activities of the derivatives (24,
3
3 vs. 12e20, 25e27), while the replacement of phenyl groups is
beneficial for those of the derivatives. (3) The introduction of
electron-withdrawing groups or halogen atoms into the phenyl
rings increased more neddylation inhibition than that of the
4

X. Chen, X. Yang, F. Mao et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Scheme 1. Synthesis of Compounds 5-8a
a
ꢀ
0
ꢀ
Reagents and conditions: (a) C(OEt)
4
, AcOH, 0 C to rt, 4 h, 90%; (b) N-(triphenylmethyl)-5-(4 -bromomethylbiphenyl-2-yl)tetrazole, K
2
CO
3
, DMF, 60 C, overnight, 45%; (c) 20%
trifluoroacetic acid in dichloromethane, rt, 4 h, 90%; (d) Butyryl chloride, DIPEA, dichloromethane, reflux, overnight, 90%; (e) LiOH$H
2
O, 5eq, MeOH: H
2
O ¼ 1: 1, reflux, 6 h; (f) 1-
ꢀ
chloroethyl cyclohexyl carbonate, NaHCO
3
, NaI, DMF, 60 C, overnight, 40%.
the cell-based assay. Cullin family members act as the best-known
substrates of neddylation that are involved in the assembly of CRLs,
which also represent a significant index for evaluating the neddy-
lation inhibitory activities of inhibitors. Furthermore, in the process
of cullin neddylation, individual cullins 1, 2, 3, 4a/b are specially
regulated by Nedd8 conjugating enzyme Ubc12, while cullin 5 is
controlled by another Nedd8 conjugating enzyme UBE2F. In addi-
tion, when the upstream Nedd8 activating enzyme, NAE, is blocked,
the entire cullin-Nedd8 adduct will be inhibited (Fig. 3A). Accord-
ing to the principle described above, our previous work demon-
strated that lead compound CDC can inhibit the formation of the
cullin-Nedd8 adduct by targeting NAE. We continuously verified
the specific target of representative 35 by utilizing these methods.
Herein, the levels of various cullin-Nedd8 (cullins 1, 2, 4a, 5)
adducts were first examined by immunoblotting after the treat-
ment of 35 in A549 (Fig. 3B). In general, 35 acted as an efficient
neddylation inhibitor for suppressing the entire cullins neddylation
apoptosis. As shown in Fig. 4A, an annexin-V/PI double staining
assay was performed to further assess the ability of derivative 35 to
induce apoptosis. The results showed that the total ratio of early
and late apoptotic A549 cells increased from 2.6% to 35.81% with
increasing concentrations of 35, suggesting that 35 induced cancer
cell apoptosis. Besides, considering that targeting the neddylation
pathway usually induces cellular senescence. As shown in Fig. 4B,
an SA-b-gal staining assay was performed to assess the ability of
derivative 35 to induce senescence. The results demonstrated 35
treatment promoted the senescence of lung cancer cell at the
concentration of 10
cellar senescence.
mM, indicating that compound 35 can induce
2.3. Molecular modeling studies
Our previous work confirmed that lead compound CDC as a
novel neddylation inhibitor can bind with the ATP-binding pocket
of NAE, as shown by molecular modeling. Similarly, molecular
modeling was established to investigate the binding mode of 35
with NAE using Autodock 4.0. As shown in Figs. 4Ce1 and 2, 35 was
found to form various interactions in the binding pocket, including
several H-bonds with the amino acid residues of K124 (2.2 Å), N168
(2.7 Å), Q112 (1.9 Å) and G76 (2.4 Å). Furthermore, the side chain
guanidine groups of R22 (3.1 Å) and R111 (3.3 Å) formed two
significantly at 12.5e25 mM, which also displayed significant effects
compared with lead compound CDC upon comparison with
MLN4924. Furthermore, the results of cullins and enzyme UBE2M
(
E2) neddylation inhibition suggested that 35 inhibited neddylation
by targeting the NAE enzyme in the mechanism, similar to CDC. The
level of global protein neddylation was explored in parallel with the
displayed significant decline with the increase in concentration of
3
5, which further indicated that 35 inhibited NAE. Meanwhile, two
representative CRL substrates, Wee1 and p27, accumulated obvi-
ously after treatment with 35 at the concentration of 50
Fig. 3C), validating that 35 stabilized the CRL substrates by sup-
pressing the neddylation-CRL pathway.
additional p-p conjugations with structural benzimidazole and
phenyl groups of derivative 35, respectively, suggesting that the
affinity of 35 with NAE was higher than that of CDC (Figs. 4Ce2 and
3).
mM
(
Accumulated studies showed that blocking the neddylation
pathway induces cell death via apoptosis. The result in Fig. 3B
showed that the apoptosis-related marker cleaved-PARP was
2.4. Neddylation inhibitor 35 suppresses the growth of A549
xenograft tumors in mice
accumulated by the treatment of 35 at the concentration of 12.5
in A549, which preliminarily indicates that 35 induced cellular
m
M
To investigate the anticancer activity of 35 in vivo, we admin-
istered 35 to nude mice bearing human tumor cell xenografts and
5

X. Chen, X. Yang, F. Mao et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
b
Scheme 2. Synthesis of Compounds 9e46
b
ꢀ
ꢀ
Reagents and conditions: (a) C(OEt)
4
, AcOH, 0 C to rt, 4 h, 90%; (b) 4-bromomethylbiphenyl-2-yl, K
, DMF, 60 C, overnight, 76%; (d) Triphenylmethyl bromide, NEt
2
CO
3
, dichloromethane, 60 C, overnight, 84%; (c) 1-chloroethyl cyclohexyl
ꢀ
carbonate, NaHCO
3
3
, dichloromethane, rt, overnight, 85%; (e) 20% trifluoroacetic acid in DCM, rt, 4 h, 90%; (f) CH
3
I,
dichloromethane, reflux, overnight, 52%; (g) ReNH
2
, EDCI, HOBT, DIPEA, DMF, overnight, 28e78%.
monitored the tumor growth rate. First, 2 ꢁ 10 A549 cells in 0.1 mL
6
house “old drug bank”, and CDC was discovered as a potential
neddylation inhibitor [40]. To overcome the defect of structurally
easy hydrolysis and promote the anticancer ability of lead com-
pound CDC, 42 derivatives with a novel benzimidazole skeleton
were designed and synthesized through a rational structure-based
drug design strategy in this study. Derivative 35 displayed both
superior neddylation inhibitory activity and cellular anti-
proliferation ability of those of other compounds. Additional
of PBS were subcutaneously inoculated into female nude mice.
After the tumor size reached the requirements (tumor volume of
0e100 mm , approximately 8 days), the tumor-bearing mice were
randomly divided into 5 groups (6 mice per group): a mock group, a
CDC-treated group as the positive control administered by intra-
peritoneal injection (30 mg/kg qd), a 35-treated group adminis-
tered by intraperitoneal injection (30 mg/kg qd) and two 35-
treated groups by oral administration (60 mg/kg qd or 30 mg/kg
qd), (Fig. 5).
3
7
annexin-V/PI double staining assays and SA-b-gal staining assay
showed that 35 induces apoptosis and promotes senescence in the
A549 cancer cells. Antitumor activity in vivo indicated that 35 not
only suppressed the growth of A549 xenograft tumors in the mice
upon administration of intraperitoneal injection (30 mg/kg) but
also presented promising efficacy through oral administration
(60 mg/kg). In summary, we have discovered a new class of ned-
dylation inhibitors of the benzimidazole scaffold, which can be
used as candidate compounds for the development of new anti-
cancer drugs.
As shown in Fig. 5A and B, in contrast to the mock group, three
5-treated groups showed a significant decrease in tumor volume
3
and tumor weight except for that with oral administration at the
dosage of 30 mg/kg qd, while all treated groups showed no effect on
body weight after 24 days (Fig. 5A and B). The 35-treated group
with the high dosage (60 mg/kg qd) by oral administration pre-
sented significant tumor inhibitory activity, suggesting that the
strategy of elevating the stability of derivatives in vivo is feasible by
modifying the structure of CDC. In addition, the 35-treated group
by intraperitoneal injection (30 mg/kg) displayed superior anti-
cancer activity in comparison with the tumor weight and size of the
other treated groups (Fig. 5C and D).
4. Experimental sections
4.1. General methods
3
. Conclusion
The synthetic starting materials, reagents, and solvents were
obtained from commercial supplier, such as Alfa Aesar, Ada-
Our previous work screened 1331 approved drugs from an in-
masbeta, Energy Chemical, J&K, and TCI at the highest commercial
6

X. Chen, X. Yang, F. Mao et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Table 2
Neddylation inhibition in enzyme-based assay and the antitumor activities against A549 human lung cancer cells of derivatives12e37.
quality and used without further purification. Reaction progress
was monitored using analytical thin layer chromatography (TLC),
standard. Chemical shifts were reported in parts per million (ppm,
d) downfield from tetramethylsilane. Proton coupling patterns
HSGF 254 (150e200
light (254 nm) and I
Melting points were measured in capillary tubes on a SGWX-4
m
m thickness; Yantai Huiyou Co.; China), UV
were used to visualize the components.
were described as singlet (s), doublet (d), triplet (t), quartet (q), and
multiplet (m). High-resolution mass spectra (HRMS) were obtained
by electric ionization (EI) and electrospray ionization (ESI) using a
2
1
13
1
melting point apparatus without correction. H NMR and
NMR spectra were recorded on a Bruker AMX 400 and AMX 500
spectrometer in DMSO‑d , MeOD-d or CDCl with TMS as internal
C
Waters GCT Premie and XEVO-G2TOF NotSet. All target com-
pounds were purified to ꢂ95% purity as determined by an Agilent
6
4
3
1100 with a quaternary pump and diode array detector (DAD).
7

X. Chen, X. Yang, F. Mao et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Table 3
Neddylation inhibition in enzyme-based assay and the antitumor activities against A549 human lung cancer cells of derivatives 38e46.
Table 4
The inhibitory activities of selected compounds 35, 39 and 46 on the formation of cullin1-Nedd8 adducts, the proliferation of Human lung cancer cell lines (A549, H1299) and
a
human normal cell lines (16H6E, BEAS-2B).
Compd.
Cullin1-Nedd8 inhibition (IC50
,
m
M)
Anti-proliferative activity (IC50, mM)
A549
H1299
16H6E
BEAS-2B
3
3
4
5
9
6
5.51 ± 0.33
6.67 ± 0.32
9.94 ± 0.41
16.43 ± 0.12
0.18 ± 0.02
20.39 ± 1.47
39.29 ± 1.37
19.12 ± 1.09
65.92 ± 1.63
0.63 ± 0.06
21.76 ± 2.7
36.74 ± 1.01
18.25 ± 1.53
45.02 ± 1.55
0.49 ± 0.04
76.06 ± 2.43
64.91 ± 3.82
44.7 ± 1.21
82.67 ± 2.22
4.03 ± 0.07
79.35 ± 1.89
80.84 ± 3.10
37.91 ± 2.59
79.76 ± 1.90
1.22 ± 0.1
CDC
MLN4924
a
The values are reported as the average ± SD of three experiments.
0
4
.2. Synthesis of intermediates and target compounds 5e11
.2.1. 2-Ethoxy-1H-benzo[d]imidazole (5a)
4.2.2. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-2-
ethoxy-1H benzo[d]imidazole (5)
4
In a 50 mL round-bottomed flask, add intermediates 5a (0.13 g,
The compound benzene-1,2-diamine (0.56 g, 3.68 mmol) and
0.5 mmol), K
2
CO
3
(0.1 g, 0.75 mmol) in DMF (4 mL). Then N-(tri-
0
acetic acid (20 mL) were added to a round bottom flask. After
mixing, tetraethyl orthocarbonate (0.9 mL, 4.30 mmol) was slowly
added dropwise to the system under ice bath conditions, stirring
was continued under ice bath conditions, and reacted at room
temperature for 4 h. After the reaction was completed, the reaction
phenylmethyl)-5-(4 -bromomethylbiphenyl-2-yl-)terazole (0.28 g,
0.5 mmol) was added after stirring at room temperature for 10 min.
ꢀ
After 60 C overnight reaction, when the reaction was completed,
the reaction solution was poured into an ice water bath, diluted
with EtOAc and washed with distilled water, followed by brine. The
solution was poured into an ice water bath. Brown solid, yield 88%.
4
organic layer was dried (MgSO ), filtered and concentrated in vacuo
1
H NMR (400 MHz, CDCl
3
)
d
9.56 (s, 1H), 7.51e7.32 (m, 2H),
to give the desired intermediates without further purification.
Intermediate was added to the round bottom flask, then a DCM
solution (4 mL) containing 20% trifluoroacetic acid was added
7.23e7.00 (m, 2H), 4.58 (q, J ¼ 7.1 Hz, 2H), 1.44 (t, J ¼ 7.1 Hz, 3H).
8

X. Chen, X. Yang, F. Mao et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 3. Analog 35 is an NAE inhibitor. (A) The cullin1-Nedd8 inhibitory activities of optimal 35, 39, 46 and CDC. (B) 35 inhibited global neddylation and induced the accumulation of
apoptosis marker cleaved-parp in human lung A549 cancer cell line. (C) 35 inhibited cullins neddylation and CRL substrates degradation in A549 cancer cell line.
dropwise under ice bath conditions. After 4 h of reaction at room
temperature, the solvent was removed by rotary evaporation,
4.2.4. 1-(((cyclohexyloxy)carbonyl)oxy)ethyl 2-(N-((2’-(1-trityl-
1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)butyramido)benzoate
0
neutralized to neutral in NaHCO
3
solution, EtOAc was extracted
3 ꢁ 50 mL), followed by brine. The organic layer was dried
MgSO ), filtered and concentrated in vacuo to give the desired
(6b)
(
(
The intermediate 6a (0.80 g, 1.16 mmol) was added to a solution
of the LiOH$H
4
2
O (5 mL) in MeOH/H
2
O mixture (4 mL, MeOH:
ꢀ
crude product. After separation and purification by silica gel col-
umn chromatography to give the white solid compound 5. White
H
2
O ¼ 1:1) at room temperature and stirred at 80 C in air. After 6 h,
the reaction was cooled to room temperature, diluted with EtOAc
and washed with distilled water, followed by brine. The organic
ꢀ
1
solid, yield 35%, mp ¼ 170e172 C. H NMR (400 MHz, DMSO‑d
12.35 (s, 1H), 7.68e7.58 (m, 2H), 7.58e7.52 (m, 2H), 7.32 (t,
J ¼ 7.7 Hz, 2H), 7.27 (d, J ¼ 7.2 Hz, 2H), 7.01 (d, J ¼ 8.5 Hz, 1H), 6.90
6
)
d
4
layer was dried (MgSO ), filtered and concentrated in vacuo to give
the desired carboxylate intermediates without further purification.
The carboxylate intermediates (0.51 g, 0.6 mmol), 1-chloroethyl
cyclohexyl carbonate (0.15 g, 0.72 mmol), NaHCO
(
1
d, J ¼ 8.1 Hz,1H), 6.80 (d, J ¼ 8.1 Hz, 1H), 5.35 (s, 2H), 4.68e4.54 (m,
H), 1.40 (dd, J ¼ 13.6, 6.6 Hz, 3H).
3
(0.08 g,
0.9 mmol), NaI (0.01 g, 0.09 mmol), DMF (5 mL) were added to the
0
ꢀ
4
4
.2.3. Methyl 2-(N-((2’-(1-trityl-1H-tetrazol-5-yl)-[1,1 -biphenyl]-
-yl)methyl)butyramido)benzoate (6a)
round-bottom flask. After 60 C reaction overnight, the reaction
liquid was poured into ice water bath, diluted with EtOAc and
washed with distilled water, followed by brine. The organic layer
In a round bottom flask was added the compound methyl
anthranilate (0.75 g, 5 mmol), DIPEA (2.6 mL, 15 mmol) was dis-
solved in DCM (20 mL), and stirred at room temperature for 10 min,
then add butyryl chloride (1.5 mL, 15 mmol). The reaction mixture
was stirred and refluxed overnight. After the reaction was
completed, the crude product is obtained by water quenching,
dichloromethane extraction, vacuum evaporation and solvent
removal. The crude product was directly subjected to the next re-
action without purification.
4
was dried (MgSO ), filtered and decompressed to remove solvents.
The crude products were separated and purified by silica gel col-
umn chromatography, and the corresponding intermediates 6b
were obtained.
4.2.5. 1-(((cyclohexyloxy)carbonyl)oxy)ethyl 2-(N-((2’-(1H-
0
tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)butyramido)benzoate (6)
Compound 6 was synthesized according to a similar protocol as
ꢀ
1
To a round bottom flask was added corresponding intermediate
that described for 5. White solid, yield 52%, mp ¼ 172e175 C. H
(
0.08 g, 0.5 mmol), K
2
CO
3
(0.07 g, 0.6 mmol), DMF (2 mL), and
NMR (400 MHz, MeOD-d
4
)
d
8.04 (t, J ¼ 7.3 Hz, 1H), 7.74e7.66 (m,
stirred at room temperature for 10 min, then add N-(triphe-
2H), 7.65e7.47 (m, 4H), 7.12 (d, J ¼ 7.8 Hz, 2H), 7.04 (d, J ¼ 7.9 Hz,
2H), 6.98e6.88 (m, 1H), 6.82 (d, J ¼ 7.8 Hz, 1H), 5.61 (dd, J ¼ 14.5,
9.5 Hz, 1H), 4.72e4.59 (m, 1H), 4.07e3.95 (m, 1H), 2.03e1.96 (m,
2H), 1.92 (s, 2H), 1.75 (s, 2H), 1.64e1.38 (m, 11H), 0.84 (t, J ¼ 7.4 Hz,
0
nylmethyl)-5-(4 -bromomethylbiphenyl-2-yl)terazole (0.28 g,
ꢀ
0
.5 mmol), then 60 C overnight reaction. After the reaction was
completed, the reaction mixture was poured into an ice water bath,
3H). HRMS (ESI) m/z calcd C34
634.2640.
H
37
N
5
O
6
[MþNa]þ 634.2642, found
EtOAc was extracted (3 ꢁ 50 mL), followed by brine. The organic
layer was dried (MgSO
4
), filtered and concentrated in vacuo to give
the desired crude product, which was purified by silica gel column
chromatography to obtain the corresponding intermediate 6a
4.2.6. Methyl 2-ethoxy-1H-benzo[d]imidazole-6-carboxylate (7a)
Compound 7a was synthesized according to a similar protocol as
(
0.19 g, 62%).
9

X. Chen, X. Yang, F. Mao et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 4. 35 induces the apoptpsis and promotes senescence of lung cancer cell A549 by the target of enzyme NAE. (A) Evaluation of apoptosis on A549 cell line by Annexin V/PI
staining and flow cytometry detecting. 0.1% DMSO treated A549 cells in 48 h; 35 treated A549 cells in 48 h at the concentration of 10, 20, or 40 M. (B) 35 treatment promotes the
senescence of lung cancer cell lines A549 by SA- -gal staining; scale bar: 50 m. (C) Molecular modeling results (PDB ID: 3GZN): (1) Low-energy binding conformations of 35
shown in blue) bound to NAE heterodimer generated by virtual ligand docking; (2) The hydrogen-bonding interaction between the structure of 35 with NAE; (3) Merging between
m
b
m
(
35 pose (shown in blue) and CDC conformation (shown in yellow) from NAE crystal. (4) Merging between 35 pose (shown in blue) and MLN4924 conformation (shown in green)
from NAE crystal. Statistical significance was determined by the Mann-Whitney test (two-tailed): ***p < 0.001. (For interpretation of the references to colour in this figure legend,
the reader is referred to the Web version of this article.)
1
that described for 5a. White solid, yield 87%, H NMR (400 MHz,
4.2.8. 1-(((cyclohexyloxy)carbonyl)oxy)ethyl 1-((2’-(1H-tetrazol-5-
yl)-[1,1 -biphenyl]-4-yl)methyl)-2-ethoxy-1H-benzo[d]imidazole-6-
carboxylate (7)
0
CDCl
3
) 7.53e7.30 (m, 2H), 7.27e7.05 (m, 2H), 4.68 (q, J ¼ 7.1 Hz, 2H),
3
.89 (s, 3H), 1.29 (t, J ¼ 7.1 Hz, 3H).
Compound 7 was synthesized according to a similar protocol as
ꢀ
1
that described for 5. White solid, yield 42%, mp ¼ 171e175 C. H
NMR (400 MHz, DMSO‑d
7.99 (s, 1H), 7.73 (d, J ¼ 8.2 Hz, 2H),
.65e7.57 (m, 3H), 7.51 (d, J ¼ 8.3 Hz, 1H), 7.20 (d, J ¼ 8.2 Hz, 2H),
.03 (d, J ¼ 8.2 Hz, 2H), 6.89e6.84 (m, 1H), 6.08 (s, 1H), 5.29 (s, 2H),
.60e4.55 (m, 2H), 4.10 (dd, J ¼ 10.5, 5.2 Hz, 1H), 1.81 (s, 3H),
0
.2.7. 2-Ethoxy-1-((2’-(1-trityl-1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-
yl)methyl)-1H-benzo[d]imidazole-5-carboxylic acid (7b)
The intermediate 7b (0.13 g, 0.5 mmol) was added to a solution
(0.1 g, 0.75 mmol) in DMF (4 mL) at room temperature,
then N-(triphenylmethyl)-5-(4 -bromomethylbiphenyl-2-yl-)ter-
4
6
) d
7
7
4
of the K
2
CO
3
0
1
C
.70e1.45 (m, 10H), 1.38 (s, J ¼ 7.1 Hz, 3H). HRMS (ESI) m/z calcd
azole (0.28 g, 0.50 mmol) was added after stirring at room tem-
perature for 10 min. Finally, stirred at 60 C overnight reaction in
H
33 34
N
6
O
6
[MþH]þ 611.2618, found 611.2619.
ꢀ
air. After 6 h, the reaction was cooled to room temperature, diluted
with EtOAc and washed with distilled water, followed by brine. The
4.2.9. 1-(((cyclohexyloxy)carbonyl)oxy)ethyl 2-ethoxy-1-((2’-(1-
trityl-1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-1H-benzo[d]
imidazole-5-carboxylate (8)
0
organic layer was dried (MgSO
in vacuo, the residue was purified by flash column chromatography
CHCl
-EtOAc ¼ 20:1). The first eluate was concentrated in vacuo to
give 7b. White solid, yield 37%. Intermediate 7b: H NMR (400 MHz,
DMSO‑d
12.74 (s, 1H), 7.77 (dd, J ¼ 7.6, 1.3 Hz, 1H), 7.68 (dd,
J ¼ 7.9, 1.0 Hz, 1H), 7.55e7.46 (m, 3H), 7.38 (dd, J ¼ 12.6, 6.7 Hz, 5H),
.31 (t, J ¼ 7.4 Hz, 5H), 7.31e7.08 (m, 1H), 6.97 (d, J ¼ 8.2 Hz, 2H),
4
), after the solvent was evaporated
(
3
Compound 8 was synthesized according to a similar protocol as
1
ꢀ
1
that described for 5. White solid, yield 35%, mp ¼ 172e175 C. H
6
)
d
NMR (400 MHz, DMSO‑d
6
)
d
7.99 (s, 1H), 7.73 (d, J ¼ 8.2 Hz, 2H),
7.65e7.57 (m, 3H), 7.51 (d, J ¼ 8.3 Hz, 1H), 7.20 (d, J ¼ 8.2 Hz, 2H),
7.03 (d, J ¼ 8.2 Hz, 2H), 6.89e6.84 (m, 1H), 6.08 (s, 1H), 5.29 (s, 2H),
4.60e4.55 (m, 2H), 4.10 (dd, J ¼ 10.5, 5.2 Hz, 1H), 1.81 (s, 3H),
1.70e1.45 (m, 10H), 1.38 (s, J ¼ 7.1 Hz, 3H). HRMS (ESI) m/z calcd
7
6
.96e6.64 (m, 8H), 5.34 (s, 2H), 4.43 (q, J ¼ 7.1 Hz, 2H), 1.35 (t,
J ¼ 7.1 Hz, 3H). and the second eluate was concentrated in vacuo to
give 8b [41].
C
H
33 34
N
6
O
6
[MþH]þ 611.2618, found 611.2615.
10

X. Chen, X. Yang, F. Mao et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
Fig. 5. 35 inhibited A549 xenograft growth in nude mice (n ¼ 6). After intraperitoneally administering vehicle (black), CDC (30 mg/kg I.P.; red), 35 (30 mg/kg I.P.; yellow), 35 (60 mg/
kg P$O.; brown), 35 (30 mg/kg P$O.; blue) for three weeks, the mice were sacrificed, and the tumors were weighed. (A) Tumor volume changed during treatment; (B) Body weight
changed of mice during treatment. (C) The images of tumors from mice at 24 days after initiation of treatment; (D) The tumorous weight of each group. Statistical significance was
determined by the Student’s t-test (two-tailed): *p < 0.05, **p < 0.01, ***p < 0.001, n. s. indicates no significant difference. (For interpretation of the references to colour in this
figure legend, the reader is referred to the Web version of this article.)
0
4
.2.10. 1-(((cyclohexyloxy)carbonyl)oxy)ethyl 1-([1,1 -biphenyl]-4-
DMSO‑d
6
)
d
7.76e7.68 (m, 2H), 7.57 (dt, J ¼ 7.6, 3.8 Hz, 1H),
ylmethyl)-2-ethoxy-1H-benzo[d]imidazole-7-carboxylate (9)
7.54e7.49 (m, 1H), 7.46 (d, J ¼ 7.8 Hz, 1H), 7.39 (d, J ¼ 7.7 Hz, 1H),
7.22 (t, J ¼ 7.9 Hz,1H), 7.02 (d, J ¼ 8.2 Hz, 2H), 6.88 (d, J ¼ 8.1 Hz, 2H),
6.81 (q, J ¼ 5.3 Hz, 1H), 5.51 (d, J ¼ 6.8 Hz, 2H), 4.63 (q, J ¼ 7.0 Hz,
2H), 4.59e4.53 (m, 1H), 4.21 (s, 3H), 1.81 (s, 2H), 1.61 (s, 2H),
1.45e1.33 (m, 9H), 1.23 (t, J ¼ 12.1 Hz, 3H). HRMS (ESI) m/z calcd
ꢀ
1
White solid, yield 35%, mp ¼ 178e181 C. H NMR (400 MHz,
DMSO‑d
7.74e7.66 (m, 2H), 7.54 (dt, J ¼ 7.6, 3.8 Hz, 1H),
.67e7.53 (m, 1H), 7.46 (d, J ¼ 7.8 Hz, 1H), 7.45 (d, J ¼ 7.7 Hz, 1H),
6
) d
7
7
6
2
.34 (t, J ¼ 7.9 Hz,1H), 7.12 (d, J ¼ 8.2 Hz, 2H), 6.79 (d, J ¼ 8.1 Hz, 2H),
.76 (q, J ¼ 5.3 Hz, 2H), 5.51 (d, J ¼ 6.8 Hz, 2H), 4.63 (q, J ¼ 7.0 Hz,
C
H
34 36
N
6
O
6
[MþH]þ 625.2775, found 625.2773.
H), 4.59e4.53 (m, 1H), 4.21 (s, 3H), 1.83 (s, 2H), 1.61 (s, 2H),
1
C
.56e1.43 (m, 9H), 1.31 (t, J ¼ 12.1 Hz, 3H). HRMS (ESI) m/z calcd
4.3. General procedure for compounds 12e46
þ
34
36
H N
6
O
6
[MþH] 543.2495, found 543.2497.
In round bottom flask, add candesartan (1 g, 2.27 mmol), tri-
methylamine (0.34 g, 3.40 mmol), 4-dimethylaminopyridine
(DMAP) (0.04 g, 0.34 mmol), and trityl bromide (0.73 g,
2.27 mmol) was dissolved in dichloromethane (20 mL), and the
reaction mixture was stirred at room temperature for 18 h. On
completion of the reaction, 100 mL water was added to the reaction
mixture at room temperature and EtOAc was extracted (3 ꢁ 50 mL),
4
.2.11. 1-(((cyclohexyloxy)carbonyl)oxy)ethyl 2-ethoxy-1-((2’-(5-
0
oxo-2,5-dihydro-1,2,4-oxadiazol-3-yl)-[1,1 -biphenyl]-4-yl)methyl)-
1
H-benzo[d]imidazole-7-carboxylate (10)
White solid, yield 67%, mp ¼ 182e184 C. H NMR (400 MHz,
DMSO‑d
7.81e7.67 (m, 2H), 7.57 (dt, J ¼ 7.6, 3.8 Hz, 1H),
.54e7.48 (m, 1H), 7.46 (d, J ¼ 7.8 Hz, 1H), 7.39 (d, J ¼ 7.7 Hz, 1H),
.20 (dd, J ¼ 20.1, 12.2 Hz, 1H), 7.00 (t, J ¼ 12.3 Hz, 2H), 6.88 (d,
J ¼ 8.1 Hz, 2H), 6.84e6.72 (m, 1H), 5.51 (q, J ¼ 16.2 Hz, 2H),
ꢀ
1
6
) d
7
7
4
followed by brine. The organic layer was dried (MgSO ), filtered and
concentrated in vacuo to give the desired crude product. Purifica-
tion of the crude was done by column chromatography using
dichloromethane: methanol (20:1) as solvent system, to yield the
desired product 12a. Add HOBT (103 mg, 0.76 mmol), EDCI (192 mg,
4
.70e4.48 (m, 3H), 1.81 (s, 2H), 1.61 (s, 2H), 1.47e1.35 (m, 9H), 1.23
(
6
t, J ¼ 11.1 Hz, 3H). HRMS (ESI) m/z calcd C34
34
H N
4
O
8
[MþH]þ
27.2455, found 627.2452.
1.0 mmol), triethylamine (0.21 mL, 1.5 mmol) and 12a (520 mg,
4
.2.12. 1-(((cyclohexyloxy)carbonyl)oxy)ethyl 2-ethoxy-1-((2’-(1-
0.76 mmol) to round bottom flask, and dissolved in 10 mL DMF. The
solution (10 mL) was stirred at room temperature for 30 min. Then,
amine (0.76 mmol) was added to the reaction mixture, and the
reaction was carried out at room temperature overnight. After the
0
methyl-1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-1H-benzo[d]
imidazole-7-carboxylate (11)
White solid, yield 79%, mp ¼ 187e190 ꢀC. H NMR (400 MHz,
1
11

X. Chen, X. Yang, F. Mao et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
0
reaction was completed, it was quenched with water and extracted
4.3.5. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-(3-
with EtOAc (3 ꢁ 50 mL). The organic phase was collected, dried over
(dimethylamino)propyl)-2-ethoxy-1H-benzo[d]imidazole-7-
carboxamide (16)
4
anhydrous MgSO , and finally the solvent was removed under
Gray solid, yield 67%, mp ¼ 198e200 C. 1H NMR (400 MHz,
ꢀ
reduced pressure. The crude product was used directly in the next
step without further purification. The corresponding amide com-
pound was dissolved in dichloromethane, and trifluoroacetic acid
was added dropwise under ice bath, the reaction was carried out at
room temperature for 3 h. After the reaction was completed, it was
quenched with water and extracted with EtOAc (3 ꢁ 50 mL). The
DMSO‑d
6
)
d
8.52 (t, J ¼ 5.8 Hz, 1H), 7.60e7.53 (m, 2H), 7.46e7.37 (m,
2H), 7.38e7.31 (m, 1H), 7.19e7.10 (m, 2H), 6.99 (d, J ¼ 8.2 Hz, 2H),
6.78 (d, J ¼ 8.2 Hz, 2H), 5.45 (s, 2H), 4.55 (q, J ¼ 7.0 Hz, 2H),
3.22e3.18 (m, 2H), 2.95e2.86 (m, 2H), 2.63 (s, 6H),1.60 (dd, J ¼ 14.6,
7.1 Hz, 2H), 1.36 (t, J ¼ 7.0 Hz, 3H). HRMS (ESI) m/z calcd C29
32 8 2
H N O
[MþH]þ 525.2726, found 525.2727.
4
organic phase was collected, dried over anhydrous MgSO , and
finally the solvent was removed under reduced pressure to obtain
the crude product. The crude product was purified by silica gel
0
4.3.6. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-2-
column chromatography (CH
pounds 12e46.
2
Cl
2
: MeOH ¼ 10: 1) to afford com-
ethoxy-N-(3-methoxypropyl)-1H-benzo[d]imidazole-7-
carboxamide (17)
Gray solid, yield 87%, mp ¼ 176e180 C. 1H NMR (400 MHz,
ꢀ
0
4.3.1. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-2-
DMSO‑d
6
)
d
8.35 (t, J ¼ 5.6 Hz, 1H), 7.70e7.61 (m, 2H), 7.60e7.51 (m,
ethoxy-N-phenyl-1H-benzo[d]imidazole-7-carboxamide (12)
2H), 7.47 (d, J ¼ 7.5 Hz, 1H), 7.16e7.10 (m, 2H), 7.02e6.91 (m, 4H),
5.76 (s, 1H), 5.41 (s, 2H), 4.61e4.53 (m, 2H), 3.27 (t, J ¼ 6.2 Hz, 2H),
3.21e3.12 (m, 5H), 1.62e1.53 (m, 2H), 1.38 (t, J ¼ 8.7, 5.4 Hz, 3H).
ꢀ
1
White solid, yield 77%, mp ¼ 182e185 C. H NMR (400 MHz,
DMSO‑d
7.76 (t, J ¼ 5.5 Hz, 1H), 7.70 (dd, J ¼ 7.5, 1.4 Hz, 1H),
.50e7.46 (m, 1H), 7.38e7.31 (m, 2H), 7.31e7.28 (m, 1H), 7.17 (dt,
J ¼ 3.2, 1.6 Hz, 1H), 7.09e7.04 (m, 1H), 7.00 (d, J ¼ 8.3 Hz, 2H), 6.82
d, J ¼ 8.3 Hz, 2H), 5.44 (s, 2H), 4.64 (q, J ¼ 7.1 Hz, 2H), 3.18e3.15 (m,
H), 1.52 (dd, J ¼ 14.5, 7.3 Hz, 2H), 1.46 (dd, J ¼ 9.5, 4.7 Hz, 3H), 0.88
6
) d
7
HRMS (ESI) m/z calcd
C
H
28 29
N
7
O
3
[MþH]þ 512.2410, found
512.2411.
(
0
2
4.3.7. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-(3-
(cyclohexylamino)propyl)-2-ethoxy-1H-benzo[d]imidazole-7-
carboxamide (18)
13
(
t, J ¼ 7.4 Hz, 3H). C NMR (151 MHz, DMSO‑d
40.42, 140.35, 134.38, 131.86, 129.83, 129.31, 128.93, 128.55, 126.60,
26.05, 125.30, 121.20, 120.12, 119.80, 118.29, 69.17, 65.68, 44.84,
6
) d 166.36, 160.01,
1
1
ꢀ
1
White solid, yield 87%, mp ¼ 191e195 C. H NMR (400 MHz,
DMSO‑d
8.50 (t, J ¼ 5.7 Hz, 1H), 7.61e7.49 (m, 2H), 7.46e7.33 (m,
3H), 7.21e7.11 (m, 2H), 6.97 (d, J ¼ 8.2 Hz, 2H), 6.77 (d, J ¼ 8.2 Hz,
4
4.49, 40.36, 33.43, 28.40, 21.40, 15.10, 13.86, 13.33, 10.87. HRMS
6
) d
(
ESI) m/z calcd C27
H
27
N
7
O
2
[MþH]þ 482.2304, found 482.2306.
2
H), 5.46 (s, 2H), 4.53 (q, J ¼ 7.0 Hz, 2H), 3.22e3.14 (m, 2H), 2.71 (d,
0
4.3.2. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-
J ¼ 33.9 Hz, 3H), 1.87 (s, 2H), 1.70e1.46 (m, 5H), 1.36 (t, J ¼ 7.0 Hz,
3H), 1.16 (dd, J ¼ 19.2, 12.1 Hz, 4H), 0.94 (s, 2H). HRMS (ESI) m/z
cyclopropyl-2-ethoxy-1H-benzo[d]imidazole-7-carboxamide (13)
ꢀ
1
[MþH]þ 579.3196, found 579.3198.
White solid, yield 83%, mp ¼ 195e198 C. H NMR (400 MHz,
DMSO‑d
8.40 (d, J ¼ 4.5 Hz, 1H), 7.99 (d, J ¼ 8.4 Hz, 1H), 7.73 (d,
38 8 2
calcd C33H N O
6
) d
0
J ¼ 8.4 Hz, 1H), 7.68e7.62 (m, 2H), 7.60e7.50 (m, 3H), 7.49e7.39 (m,
4.3.8. Ethyl (1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-
2-ethoxy-1H-benzo[d]imidazole-7-carbonyl)glycinate (19)
2
2
5
H), 7.15e7.05 (m, 2H), 5.76 (s, 1H), 5.42 (s, 2H), 4.57 (q, J ¼ 7.0 Hz,
ꢀ
1
H), 2.80e2.70 (m, 1H), 1.38 (t, J ¼ 7.1 Hz, 3H), 0.59 (tt, J ¼ 10.1,
White solid, yield 62%, mp ¼ 190e192 C. H NMR (400 MHz,
DMSO‑d
8.73 (t, J ¼ 5.9 Hz, 1H), 7.94e7.83 (m, 4H), 7.59e7.54 (m,
2H), 7.47 (ddd, J ¼ 14.8, 7.7, 1.2 Hz, 2H), 7.42e7.39 (m, 1H), 7.18e7.13
m,1H), 7.11e7.06 (m,1H), 5.31 (s, 2H), 4.50 (dq, J ¼ 14.2, 7.1 Hz, 2H),
.2 Hz, 2H), 0.38e0.31 (m, 2H). HRMS (ESI) m/z calcd C27
25
H N
7
O
2
6
) d
þ
[MþH] 480.2148, found 480.2149.
(
0
4.3.3. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-
4.07e3.98 (m, 2H), 3.85 (t, J ¼ 9.1 Hz, 2H),1.29 (t, J ¼ 7.0 Hz, 3H),1.11
cyclopentyl-2-ethoxy-1H-benzo[d]imidazole-7-carboxamide (14)
(t, J ¼ 7.1 Hz, 3H). HRMS (ESI) m/z calcd C28
27
H N
7
O
4
[MþH]þ
ꢀ
1
White solid, yield 75%, mp ¼ 173e175 C. H NMR (400 MHz,
DMSO‑d
8.37 (d, J ¼ 7.3 Hz, 1H), 7.66 (dd, J ¼ 16.4, 7.7 Hz, 2H),
.60e7.50 (m, 2H), 7.45 (d, J ¼ 7.7 Hz, 1H), 7.16e7.07 (m, 2H),
.04e6.92 (m, 4H), 5.42 (s, 2H), 4.56 (q, J ¼ 7.0 Hz, 2H), 4.11 (dq,
J ¼ 14.0, 7.0 Hz, 1H), 1.72 (dt, J ¼ 12.4, 6.2 Hz, 2H), 1.56 (dd, J ¼ 14.1,
.3 Hz, 2H), 1.47 (dd, J ¼ 14.3, 7.5 Hz, 2H), 1.38 (t, J ¼ 7.1 Hz, 3H),
.36e1.29 (m, 2H). 13C NMR (101 MHz, DMSO‑d
166.94, 158.15,
41.47, 141.40, 138.69, 137.08, 131.49, 131.12, 130.99, 130.03, 129.39,
28.24, 127.18, 123.94, 122.07, 121.42, 120.92, 119.36, 66.76, 51.26,
526.2203, found 526.2204.
6
) d
0
7
7
4.3.9. Methyl (1r,4r)-4-(1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-
yl)methyl)-2-ethoxy-1H-benzo[d]imidazole-7-carboxamido)
cyclohexane-1-carboxylate (20)
ꢀ
1
7
1
1
1
White solid, yield 68%, mp ¼ 182e185 C$ H NMR (400 MHz,
6
)
d
DMSO‑d
6
)
d
8.33 (d, J ¼ 7.7 Hz, 1H), 7.96 (s, 1H), 7.72e7.61 (m, 2H),
7.60e7.51 (m, 2H), 7.45 (d, J ¼ 7.7 Hz, 1H), 7.18e7.08 (m, 2H),
7.05e6.94 (m, 4H), 5.47 (d, J ¼ 32.9 Hz, 2H), 4.58 (dq, J ¼ 14.2, 7.1 Hz,
2H), 3.48e3.41 (m, 1H), 3.18 (s, 3H), 2.29e2.14 (m, 1H), 1.90 (dd,
J ¼ 14.2, 3.6 Hz, 2H), 1.70 (dd, J ¼ 12.9, 3.9 Hz, 2H), 1.44e1.35 (m,
4
5.92, 32.37, 23.94, 14.91. HRMS (ESI) m/z calcd C29
29
H N
7
O
2
þ
[MþH] 508.2461, found 508.2460.
4H), 1.26e1.04 (t, 3H). HRMS (ESI) m/z calcd C32
33
H N
7
O
4
[MþH]þ
0
4.3.4. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-
580.2672, found 580.2673.
cyclohexyl-2-ethoxy-1H-benzo[d]imidazole-7-carboxamide (15)
ꢀ
1
0
White solid, yield 66%, mp ¼ 191e195 C. H NMR (400 MHz,
DMSO‑d
8.28 (t, J ¼ 12.7 Hz, 1H), 7.65 (dd, J ¼ 15.6, 7.8 Hz, 2H),
.59e7.49 (m, 2H), 7.44 (d, J ¼ 7.5 Hz, 1H), 7.12 (q, J ¼ 7.2 Hz, 2H),
.02e6.87 (m, 4H), 5.41 (d, J ¼ 7.9 Hz, 2H), 4.55 (q, J ¼ 7.1 Hz, 2H),
.67e3.57 (m, 1H), 1.70e1.49 (m, 6H), 1.37 (dd, J ¼ 12.7, 5.7 Hz, 3H),
4.3.10. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-2-
ethoxy-N-propyl-1H-benzo[d]imidazole-7-carboxamide (21)
6
) d
ꢀ
1
7
7
White solid, yield 72%, mp ¼ 202e205 C. H NMR (400 MHz,
DMSO‑d 10.35 (s, 1H), 7.68e7.58 (m, 5H), 7.58e7.52 (m, 1H), 7.32
6
) d
3
(t, J ¼ 7.7 Hz, 3H), 7.27 (d, J ¼ 7.2 Hz, 1H), 7.20 (t, J ¼ 7.7 Hz, 1H), 7.10
(t, J ¼ 7.4 Hz,1H), 7.01 (d, J ¼ 8.5 Hz,1H), 6.90 (d, J ¼ 8.1 Hz, 2H), 6.80
(d, J ¼ 8.1 Hz, 2H), 5.35 (s, 2H), 4.68e4.54 (q, 2H), 1.40 (t, J ¼ 13.6,
13
1
.23 (t, J ¼ 14.8, 9.2 Hz, 3H), 1.11 (d, J ¼ 7.6 Hz, 1H). C NMR
151 MHz, DMSO‑d 165.45, 157.08, 140.40, 140.34, 136.07, 130.50,
30.06, 129.89, 128.96, 128.27, 127.19, 126.11, 121.06, 120.32, 119.86,
(
1
6
) d
13
6
6.6 Hz, 3H). C NMR (151 MHz, DMSO‑d ) d 166.10, 158.28, 141.63,
1
18.29, 65.67, 47.59, 44.87, 31.37, 24.54, 24.06,13.82. HRMS (ESI) m/z
141.40, 139.57, 136.59, 131.46, 131.03, 129.90, 129.31, 129.04, 128.25,
127.36, 124.19, 121.88, 121.29, 121.13, 120.38, 119.86, 66.92, 46.03,
calcd C30
31
H N
7
O
2
[MþH]þ 522.2617, found 522.2618.
12

X. Chen, X. Yang, F. Mao et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
1
4.92. HRMS (ESI) m/z calcd C30
H
25
N
7
O
2
[MþH]þ 516.2148, found
126.36, 120.31, 120.21, 119.93, 118.71, 109.86, 106.30, 65.72, 44.91,
516.2147.
35.38, 13.82. HRMS (ESI) m/z calcd C29
H
25
N
7
O
3
[MþH]þ 520.2097,
found 520.2098.
0
4.3.11. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-2-
0
ethoxy-N-(4-fluorophenyl)-1H-benzo[d]imidazole-7-carboxamide
22)
White solid, yield 82%, mp ¼ 187e190 C. H NMR (400 MHz,
DMSO‑d
10.39 (s, 1H), 7.71e7.59 (m, 5H), 7.54 (dd, J ¼ 10.7,
.3 Hz, 1H), 7.32 (d, J ¼ 7.7 Hz, 1H), 7.26 (d, J ¼ 6.6 Hz, 1H), 7.22e7.11
4.3.16. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-
(benzo[d] [1,3]dioxol-5-yl)-2-ethoxy-1H-benzo[d]imidazole-7-
carboxamide (27)
(
ꢀ
1
ꢀ
1
6
)
d
White solid, yield 78%, mp ¼ 192e195 C. H NMR (400 MHz,
4
DMSO‑d
6
)
d
10.25 (s, 1H), 7.59 (dtd, J ¼ 21.9,14.5, 7.4 Hz, 4H), 7.35 (d,
(
5
(
1
m, 3H), 6.87 (d, J ¼ 8.2 Hz, 2H), 6.81 (d, J ¼ 8.2 Hz, 2H), 5.76 (s, 1H),
J ¼ 7.7 Hz, 1H), 7.28 (d, J ¼ 1.5 Hz, 1H), 7.25e7.15 (m, 2H), 7.05 (d,
13
.34 (s, 2H), 4.60 (q, J ¼ 7.1 Hz, 2H), 1.41 (t, J ¼ 7.1 Hz, 3H). C NMR
165.39, 158.97, 157.72, 157.38, 141.06, 140.80,
38.19, 135.97, 135.35, 130.81, 130.46, 130.41, 129.33, 128.73, 127.66,
J ¼ 8.3 Hz, 1H), 6.95e6.78 (m, 5H), 5.99 (s, 2H), 5.34 (s, 2H), 4.60 (q,
1
3
151 MHz, DMSO‑d
6
)
d
J ¼ 7.0 Hz, 2H), 1.40 (t, J ¼ 7.0 Hz, 3H). C NMR (151 MHz, DMSO‑d
6
)
d 164.67, 157.20, 146.31, 142.64, 140.53, 140.34, 137.66, 135.54,
1
6
5
26.65, 121.58, 121.52, 121.13, 120.67, 120.57, 119.35, 115.11, 114.97,
132.84, 130.36, 129.97, 129.94, 128.82, 128.24, 127.17, 126.21, 120.81,
120.14, 120.06, 118.73, 112.26, 107.29, 101.35, 100.39, 65.84, 44.94,
6.36, 45.45, 14.34. HRMS (ESI) m/z calcd C30
H24FN
7
O
2
[MþH]þ
34.2054, found 534.2053.
13.83. HRMS (ESI) m/z calcd C31
H
25
N
7
O
4
[MþH]þ 560.2046, found
560.2045.
0
4.3.12. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-2-
0
ethoxy-N-(4-methoxyphenyl)-1H-benzo[d]imidazole-7-
carboxamide (23)
4.3.17. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-
benzyl-2-ethoxy-1H-benzo[d]imidazole-7-carboxamide (28)
ꢀ
1
ꢀ
1
White solid, yield 59%, mp ¼ 185e188 C. H NMR (400 MHz,
White solid, yield 63%, mp ¼ 181e184 C. H NMR (400 MHz,
DMSO‑d
6
)
d
10.23 (s, 1H), 7.68e7.50 (m, 6H), 7.35 (d, J ¼ 7.7 Hz, 1H),
DMSO‑d
6
)
d
8.91 (t, J ¼ 6.0 Hz, 1H), 7.57e7.51 (m, 2H), 7.49e7.44 (m,
7
6
2
.25 (d, J ¼ 6.6 Hz, 1H), 7.18 (t, J ¼ 7.7 Hz, 1H), 6.90 (dd, J ¼ 8.5,
2H), 7.38 (dt, J ¼ 4.9, 3.2 Hz, 4H), 7.28 (dd, J ¼ 7.3, 5.8, 4.1 Hz, 4H),
7.20 (dd, J ¼ 5.2, 4.2 Hz, 1H), 7.12 (t, J ¼ 7.7 Hz, 1H), 6.97 (t, J ¼ 7.7 Hz,
2H), 6.79 (d, J ¼ 8.2 Hz, 2H), 5.37 (s, 2H), 4.60 (q, J ¼ 7.0 Hz, 2H), 4.00
.7 Hz, 4H), 6.83 (d, J ¼ 8.2 Hz, 2H), 5.35 (s, 2H), 4.59 (q, J ¼ 7.0 Hz,
13
H), 3.74 (s, 3H), 1.40 (t, J ¼ 7.0 Hz, 3H). C NMR (151 MHz,
165.13, 157.68, 155.46, 141.02, 140.78, 138.27, 136.03,
32.09, 130.72, 130.47, 130.45, 129.35, 128.75, 127.63, 126.80, 121.40,
1
3
DMSO‑d
1
6
)
d
(s, 2H), 1.41 (t, J ¼ 7.0 Hz, 3H). C NMR (151 MHz, DMSO‑d
6
)
d 166.39, 159.22, 157.25, 140.49, 139.87, 139.50, 138.78, 134.56,
1
20.68, 120.54, 119.15, 113.61, 66.31, 55.07, 53.34, 14.34. HRMS (ESI)
133.53, 130.26, 129.85, 129.48, 129.02, 128.54, 128.26, 128.00,
127.83, 127.63, 127.30, 126.71, 126.29, 126.08, 125.49, 120.77, 120.17,
m/z calcd C31
H
27
N
7
O
3
[MþH]þ 546.2254, found 546.2255.
1
19.91, 118.56, 65.72, 41.93, 41.65, 13.85. HRMS (ESI) m/z calcd
0
[MþH]þ 530.2304, found 530.2303.
4.3.13. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-2-
31 27 7 2
C H N O
ethoxy-N-(4-sulfamoylphenyl)-1H-benzo[d]imidazole-7-
carboxamide (24)
0
4.3.18. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-2-
ethoxy-N-(4-fluorobenzyl)-1H-benzo[d]imidazole-7-carboxamide
(29)
ꢀ
1
Brown solid, yield 35%, mp ¼ 201e204 C. H NMR (400 MHz,
DMSO‑d
10.39 (s, 1H), 7.69e7.56 (m, 5H), 7.54 (dd, J ¼ 10.7,
.3 Hz, 1H), 7.32 (d, J ¼ 7.7 Hz, 1H), 7.26 (d, J ¼ 6.6 Hz, 1H), 7.31e7.15
m, 3H), 6.98 (s, 2H), 6.87 (d, J ¼ 8.2 Hz, 2H), 6.81 (d, J ¼ 8.2 Hz, 2H),
6
) d
ꢀ
1
4
(
White solid, yield 77%, mp ¼ 178e190 C. H NMR (400 MHz,
DMSO‑d
6
)
d
8.91 (t, J ¼ 6.0 Hz, 1H), 7.60 (dd, J ¼ 7.5, 1.1 Hz, 1H),
5
3
5
.68 (s, 1H), 5.24 (s, 2H), 4.60 (q, J ¼ 7.1 Hz, 2H), 1.41 (t, J ¼ 7.1 Hz,
7.55e7.52 (m, 1H), 7.45 (td, J ¼ 7.5, 1.2 Hz, 1H), 7.40e7.22 (m, 4H),
7.21e7.05 (m, 4H), 7.00e6.92 (m, 2H), 6.83 (d, J ¼ 8.2 Hz, 2H), 5.38
(s, 2H), 4.58 (q, J ¼ 7.0 Hz, 2H), 4.32 (d, J ¼ 5.9 Hz, 2H), 1.39 (t,
H). HRMS (ESI) m/z calcd C30
H
26
N
8
O
4
S [MþH]þ 595.1876, found
95.1831.
13
J ¼ 7.0 Hz, 3H). C NMR (151 MHz, DMSO‑d
6
) d 166.35, 161.31,
0
4.3.14. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-2-
159.70, 157.24, 155.85, 140.50, 140.12, 138.27, 135.51, 134.86, 130.71,
130.66, 129.98, 129.83, 129.49, 129.07, 128.72, 128.67, 128.38,
126.92, 125.86, 125.01, 120.52, 120.17, 119.95, 118.65, 114.94, 114.80,
114.42, 114.28, 65.73, 44.91, 41.26, 13.82. HRMS (ESI) m/z calcd
ethoxy-N-(pyridin-4-yl)-1H-benzo[d]imidazole-7-carboxamide
25)
White solid, yield 90%, mp ¼ 197e199 C. H NMR (400 MHz,
DMSO‑d
11.12 (s, 1H), 8.62e8.51 (m, 2H), 7.87 (d, J ¼ 6.5 Hz, 2H),
.69 (dd, J ¼ 7.8, 1.0 Hz, 1H), 7.66e7.58 (m, 2H), 7.57e7.49 (m, 1H),
.31 (d, J ¼ 6.6 Hz, 1H), 7.25 (t, J ¼ 7.7 Hz, 2H), 6.84e6.74 (m, 4H),
(
ꢀ
1
)
d
C
31
H26FN
7
O
2
[MþH]þ 548.2210, found 548.2211.
6
7
7
0
4.3.19. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-
benzhydryl-2-ethoxy-1H-benzo[d]imidazole-7-carboxamide (30)
5
3
1
.76 (s, 1H), 5.33 (s, 2H), 4.63 (q, J ¼ 7.1 Hz, 2H), 1.42 (t, J ¼ 7.1 Hz,
13
ꢀ
1
H). C NMR (151 MHz, DMSO‑d
49.09, 145.25, 140.74, 140.25, 137.67, 135.09, 130.40, 129.95, 129.87,
28.84, 128.31, 128.27, 127.24, 125.84, 122.62, 120.29, 120.20, 120.16,
6
)
d
166.15, 157.44, 154.33, 150.55,
White solid, yield 76%, mp ¼ 168e170 C. H NMR (400 MHz,
DMSO‑d
6
)
d
9.57 (d, J ¼ 9.0 Hz, 1H), 7.67e7.60 (m, 2H), 7.54 (dd,
1
J ¼ 12.7, 7.8 Hz, 2H), 7.49e7.42 (m, 1H), 7.38 (d, J ¼ 7.3 Hz, 4H), 7.28
(t, J ¼ 7.2 Hz, 5H), 7.24e7.12 (m, 3H), 6.83 (d, J ¼ 8.0 Hz, 2H), 6.67 (d,
J ¼ 8.1 Hz, 2H), 6.44 (d, J ¼ 9.1 Hz, 1H), 5.34 (s, 2H), 4.58 (q,
119.84, 119.33, 113.69, 66.11, 45.19, 13.83. HRMS (ESI) m/z calcd
þ
C
29
24
H N
8
O
2
[MþH] 517.2100, found 517.2101.
13
J ¼ 7.0 Hz, 2H), 3.17 (s, 1H), 1.39 (t, J ¼ 7.0 Hz, 3H). C NMR
0
4.3.15. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-2-
6
(151 MHz, DMSO‑d ) d 165.62, 157.19, 141.74, 140.50, 140.05, 137.93,
ethoxy-N-(furan-2-ylmethyl)-1H-benzo[d]imidazole-7-
135.46, 130.04, 129.91, 129.84, 129.22, 128.33, 128.30, 127.85, 127.79,
127.02, 126.81, 126.59, 126.36, 120.99, 119.95, 119.81, 118.86, 65.73,
55.73, 44.76, 13.83. HRMS (ESI) m/z calcd C37H N O
31 7 2
carboxamide (26)
ꢀ
1
[MþH]þ
White solid, yield 91%, mp ¼ 202e204 C. H NMR (400 MHz,
DMSO‑d
8.92 (t, J ¼ 5.8 Hz, 1H), 7.69e7.61 (m, 2H), 7.60e7.50 (m,
H), 7.46 (d, J ¼ 7.6 Hz, 1H), 7.14 (dt, J ¼ 15.1, 6.9 Hz, 2H), 7.00e6.89
6
)
d
606.2617, found 606.2618.
3
0
(
2
3
m, 4H), 6.34 (dd, J ¼ 3.1, 1.9 Hz, 1H), 6.21 (d, J ¼ 3.0 Hz, 1H), 5.38 (s,
4.3.20. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-
(benzo[d] [1,3]dioxol-5-ylmethyl)-2-ethoxy-1H-benzo[d]imidazole-
7-carboxamide (31)
H), 4.57 (q, J ¼ 7.1 Hz, 2H), 4.36 (d, J ¼ 5.7 Hz, 2H),1.39 (t, J ¼ 7.0 Hz,
13
6
H). C NMR (151 MHz, DMSO‑d ) d 166.25, 157.21, 151.45, 141.42,
White solid, yield 78%, mp ¼ 193e195 ꢀC. H NMR (400 MHz,
1
140.49, 140.39, 137.61, 135.85, 130.44, 130.02, 129.08, 128.32, 127.17,
13

X. Chen, X. Yang, F. Mao et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
DMSO‑d
2
6
6
)
d
8.89 (t, J ¼ 5.9 Hz, 1H), 7.70e7.62 (m, 2H), 7.60e7.52 (m,
H), 7.45 (d, J ¼ 7.6 Hz, 1H), 7.22e7.10 (m, 2H), 6.97e6.93 (m, 2H),
.88 (dd, J ¼ 9.3, 4.8 Hz, 3H), 6.78 (dt, J ¼ 7.9, 4.7 Hz, 2H), 5.94 (d,
127.76, 127.04, 121.77, 121.10, 121.01, 119.63, 66.81, 46.03, 38.97,
32.53, 14.89. HRMS (ESI) m/z calcd
C
32
H27Cl
2
N
7
O
2
[MþH]þ
612.1682, found 612.1680.
J ¼ 4.0 Hz, 2H), 5.39 (s, 2H), 4.58 (q, J ¼ 7.1 Hz, 2H), 4.26 (d,
13
0
J ¼ 6.1 Hz, 2H), 1.39 (t, J ¼ 7.1 Hz, 3H). C NMR (151 MHz, DMSO‑d
166.71, 157.72, 147.07, 145.92, 140.99, 140.92, 138.14, 136.35,
33.00, 130.89, 130.54, 130.48, 129.57, 128.80, 127.66, 126.67, 121.01,
6
)
4.3.25. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-
(benzyloxy)-2-ethoxy-1H-benzo[d]imidazole-7-carboxamide (36)
d
ꢀ
1
1
White solid, yield 87%, mp ¼ 178e181 C. H NMR (400 MHz,
DMSO‑d
11.66 (s, 1H), 7.59 (dd, J ¼ 18.3, 15.4, 7.5 Hz, 4H),
7.43e7.31 (m, 6H), 7.12 (dt, J ¼ 7.6, 7.0 Hz, 2H), 6.98 (s, 4H), 5.42 (s,
120.71, 120.54, 120.46, 119.13, 108.02, 107.88, 100.67, 66.23, 45.41,
6
) d
4
2.28, 13.83. HRMS (ESI) m/z calcd C32
H N
27 7
O
4
[MþH]þ 574.2203,
1
3
found 574.2204.
2H), 4.66 (s, 2H), 4.56 (q, J ¼ 7.0 Hz, 2H), 1.36 (t, J ¼ 7.1 Hz, 3H).
C
NMR (151 MHz, DMSO‑d 163.46, 157.22, 140.51, 140.31, 135.94,
6
) d
0
4.3.21. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-2-
135.11, 130.41, 130.01, 129.97, 129.49, 128.40, 128.20, 127.70, 127.18,
125.91, 120.54, 119.99, 119.25, 117.23, 76.22, 65.82, 45.04, 13.78.
ethoxy-N-phenethyl-1H-benzo[d]imidazole-7-carboxamide (32)
ꢀ
1
[MþH]þ 546.2254, found
White solid, yield 92%, mp ¼ 192e195 C. H NMR (400 MHz,
DMSO‑d
8.41 (t, J ¼ 5.5 Hz, 1H), 7.63 (t, J ¼ 6.4 Hz, 2H), 7.54 (t,
J ¼ 7.5 Hz, 2H), 7.42 (d, J ¼ 7.5 Hz, 1H), 7.31e7.23 (m, 2H), 7.23e7.15
m, 3H), 7.09 (dt, J ¼ 7.5, 7.1 Hz, 2H), 6.96 (q, J ¼ 8.3 Hz, 4H), 5.76 (s,
H), 5.39 (s, 2H), 4.56 (q, J ¼ 7.1 Hz, 2H), 3.39e3.34 (m, 2H), 2.67 (t,
HRMS (ESI) m/z calcd C31
546.2253.
H
27
N
7
O
3
6
)
d
0
(
1
4.3.26. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-2-
ethoxy-N-(4-phenylbutyl)-1H-benzo[d]imidazole-7-carboxamide
(37)
13
J ¼ 7.4 Hz, 2H), 1.37 (t, J ¼ 7.0 Hz, 3H). C NMR (151 MHz, DMSO‑d
166.28, 157.19, 140.45, 140.42, 138.80, 136.05, 130.42, 130.01,
29.98, 129.01, 128.31, 128.03, 127.71, 127.18, 126.03, 125.49, 120.85,
6
)
ꢀ
1
d
White solid, yield 53%, mp ¼ 186e190 C. H NMR (400 MHz,
1
DMSO‑d
6
)
d
8.37 (t, J ¼ 5.6 Hz, 1H), 7.66e7.56 (m, 2H), 7.55e7.48 (m,
120.03, 119.92, 118.47, 65.71, 54.31, 40.08, 34.14, 13.82. HRMS (ESI)
2H), 7.39 (d, J ¼ 6.9 Hz, 1H), 7.30e7.18 (m, 3H), 7.12 (dt, J ¼ 7.5,
6.2 Hz, 5H), 6.96 (d, J ¼ 8.2 Hz, 2H), 6.91 (d, J ¼ 8.2 Hz, 2H), 5.76 (s,
m/z calcd C32
H
29
N
7
O
2
[MþH]þ 544.2461, found 544.2462.
1
H), 5.39 (s, 2H), 4.57 (q, J ¼ 7.0 Hz, 2H), 3.19e3.06 (m, 2H), 2.56 (dd,
0
13
4.3.22. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-(4-
J ¼ 18.3, 7.5 Hz, 2H),1.38 (t, J ¼ 7.0 Hz, 4H),1.25 (t, J ¼ 7.4 Hz, 3H).
C
bromophenethyl)-2-ethoxy-1H-benzo[d]imidazole-7-carboxamide
33)
White solid, yield 89%, mp ¼ 205e207 C. H NMR (400 MHz,
DMSO‑d
8.42 (t, J ¼ 5.6 Hz, 1H), 7.67e7.59 (m, 2H), 7.54 (td,
J ¼ 7.7, 1.2 Hz, 2H), 7.50e7.38 (m, 4H), 7.15 (s, 1H), 7.12 (dd, J ¼ 10.6,
.8 Hz, 2H), 7.05 (dd, J ¼ 7.6, 1.1 Hz, 1H), 6.98 (d, J ¼ 8.3 Hz, 2H), 6.92
d, J ¼ 8.3 Hz, 2H), 5.37 (s, 2H), 4.56 (q, J ¼ 7.0 Hz, 2H), 2.64 (t,
NMR (151 MHz, DMSO‑d 166.31, 157.18, 155.62, 141.52, 140.43,
6
) d
(
140.18, 138.16, 135.66, 129.99, 129.85, 129.65, 128.97, 128.35, 127.70,
127.66, 127.59, 126.95, 125.95, 125.00, 121.00, 120.11, 119.91, 118.38,
65.70, 52.78, 38.31, 34.14, 27.84, 27.79, 13.83. HRMS (ESI) m/z calcd
ꢀ
1
6
) d
C
H N
34 33 7
O
2
[MþH]þ 572.2774, found 572.2775.
4
(
0
4.3.27. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-
(2,6-dichlorophenethyl)-2-ethoxy-1H-benzo[d]imidazole-7-
carboxamide (38)
13
J ¼ 7.2 Hz, 2H), 1.37 (t, J ¼ 7.1 Hz, 3H). C NMR (151 MHz, DMSO‑d
166.79, 157.70, 140.89, 138.80, 138.11, 136.51, 131.01, 130.87, 130.51,
30.47, 129.50, 128.81, 127.73, 127.66, 127.23, 126.45, 124.41, 121.26,
6
)
d
ꢀ
1
1
White solid, yield 77%, mp ¼ 188e190 C. H NMR (400 MHz,
120.50, 120.44, 119.03, 119.00, 109.54, 66.21, 45.43, 40.22, 33.97,
DMSO‑d
6
)
d
8.52 (t, J ¼ 5.7 Hz, 1H), 7.62 (t, J ¼ 7.1 Hz, 2H), 7.57e7.50
14.31. HRMS (ESI) m/z calcd C32
H28BrN
7
O
2
[MþH]þ 622.1566, found
(m, 2H), 7.43 (dd, J ¼ 15.9, 7.8 Hz, 3H), 7.31e7.24 (m, 1H), 7.18e7.09
6
22.1565.
(m, 2H), 6.96 (q, J ¼ 8.3 Hz, 4H), 5.42 (s, 2H), 4.57 (q, J ¼ 7.0 Hz, 2H),
1
3
3
.38e3.33 (m, 2H), 3.04e2.95 (m, 2H), 1.38 (t, J ¼ 7.0 Hz, 3H).
C
0
4.3.23. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-2-
6
NMR (151 MHz, DMSO‑d ) d 166.25, 157.19, 140.48, 140.41, 137.61,
ethoxy-N-(4-nitrophenethyl)-1H-benzo[d]imidazole-7-
136.04, 134.31, 134.09, 130.33, 129.99, 129.95, 129.03, 128.49,
128.32, 127.90, 127.89, 127.16, 126.03, 120.75, 120.12, 119.91, 118.56,
65.73, 44.98, 36.69, 30.15, 13.83, 13.82. HRMS (ESI) m/z calcd
carboxamide (34)
ꢀ
1
White solid, yield 56%, mp ¼ 184e185 C. H NMR (400 MHz,
DMSO‑d
6
)
d
8.44 (t, J ¼ 5.4 Hz, 1H), 8.13 (d, J ¼ 8.6 Hz, 2H), 7.62 (t,
C
32
H
27Cl
2
N
7
O
2
[MþH]þ 612.1682, found 612.1683.
J ¼ 5.5 Hz, 2H), 7.54 (t, J ¼ 7.2 Hz, 2H), 7.47 (d, J ¼ 8.6 Hz, 2H), 7.42 (d,
J ¼ 7.4 Hz, 1H), 7.12 (t, J ¼ 7.7 Hz, 1H), 7.05 (d, J ¼ 7.0 Hz, 1H), 6.98 (d,
J ¼ 8.2 Hz, 2H), 6.92 (d, J ¼ 8.2 Hz, 2H), 5.75 (s, 1H), 5.37 (s, 2H), 4.56
0
4.3.28. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-
(2,5-dichlorophenethyl)-2-ethoxy-1H-benzo[d]imidazole-7-
carboxamide (39)
(
2
1
q, J ¼ 7.0 Hz, 2H), 3.39 (dt, J ¼ 16.2, 8.1 Hz, 2H), 2.82 (t, J ¼ 7.0 Hz,
13
ꢀ
1
H), 1.37 (t, J ¼ 7.0 Hz, 3H). C NMR (151 MHz, DMSO‑d
6
)
d
166.87,
White solid, yield 89%, mp ¼ 203e205 C. H NMR (400 MHz,
57.73, 147.82, 145.98, 140.97, 140.89, 138.08, 136.53, 130.91, 130.52,
DMSO‑d
6
)
d
8.43 (t, J ¼ 5.5 Hz, 1H), 7.65e7.57 (m, 2H), 7.52 (t,
130.47, 129.94, 129.52, 128.83, 127.69, 126.43, 123.28, 121.16, 120.52,
J ¼ 8.0 Hz, 2H), 7.47e7.36 (m, 3H), 7.31 (dd, J ¼ 8.5, 2.6 Hz, 1H), 7.11
(t, J ¼ 7.7 Hz, 1H), 7.08e7.02 (m, 1H), 6.97 (d, J ¼ 8.2 Hz, 2H), 6.91 (d,
J ¼ 8.2 Hz, 2H), 5.37 (s, 2H), 4.57 (q, J ¼ 7.0 Hz, 2H), 3.38 (dd, J ¼ 12.8,
1
20.46, 119.07, 66.23, 54.82, 45.46, 34.49, 14.32. HRMS (ESI) m/z
calcd C32
H
28
N
8
O
4
[MþH]þ 589.2312, found 589.2313.
13
6
.8 Hz, 2H), 2.83 (t, J ¼ 6.9 Hz, 2H), 1.38 (t, J ¼ 7.1 Hz, 3H). C NMR
0
4
.3.24. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-
6
(151 MHz, DMSO‑d ) d 166.86,157.72,155.56, 140.98, 140.80, 138.94,
(
2,4-dichlorophenethyl)-2-ethoxy-1H-benzo[d]imidazole-7-
carboxamide (35)
White solid, yield 88%, mp ¼ 172e175 C. H NMR (400 MHz,
DMSO‑d
8.43 (t, J ¼ 5.6 Hz, 1H), 7.62 (t, J ¼ 6.3 Hz, 2H), 7.58e7.50
m, 3H), 7.42 (d, J ¼ 7.3 Hz, 1H), 7.32 (dt, J ¼ 14.8, 5.2 Hz, 2H), 7.10
dt, J ¼ 7.6, 7.1 Hz, 2H), 6.98 (d, J ¼ 8.2 Hz, 2H), 6.92 (d, J ¼ 8.2 Hz,
H), 5.39 (s, 2H), 4.57 (q, J ¼ 7.0 Hz, 2H), 3.40e3.35 (m, 2H), 2.80 (t,
138.41, 136.30, 131.82, 131.49, 130.70, 130.67, 130.48, 130.41, 129.48,
128.85, 127.90, 127.56, 126.46, 124.25, 121.24, 120.47, 120.40, 119.04,
66.24, 45.45, 38.23, 32.28, 14.33. HRMS (ESI) m/z calcd
ꢀ
1
[MþH]þ 612.1682, found 612.1684.
6
)
d
C
32
H
27Cl
2
N
7
O
2
(
(
2
0
4.3.29. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-2-
ethoxy-N-(3-methoxyphenethyl)-1H-benzo[d]imidazole-7-
carboxamide (40)
13
J ¼ 7.0 Hz, 2H), 1.38 (t, J ¼ 7.0 Hz, 3H). C NMR (151 MHz, DMSO‑d
167.40, 158.28, 141.55, 138.71, 137.07, 136.45, 134.56, 132.84,
32.19, 131.39, 131.08, 131.03, 130.08, 129.41, 129.11, 128.23, 127.80,
6
)
ꢀ
1
d
White solid, yield 66%, mp ¼ 185e188 C. H NMR (400 MHz,
1
6
DMSO‑d ) d
8.40 (t, J ¼ 5.5 Hz, 1H), 7.61 (dd, J ¼ 11.8, 4.5 Hz, 2H),
14

X. Chen, X. Yang, F. Mao et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
7
.58e7.49 (m, 2H), 7.42 (d, J ¼ 7.7 Hz, 1H), 7.22e7.15 (m, 1H),
4.54 (q, J ¼ 7.1 Hz, 2H), 3.43 (dd, J ¼ 13.2, 6.8 Hz, 2H), 2.76 (t,
13
7
.15e7.04 (m, 2H), 6.98 (d, J ¼ 8.3 Hz, 2H), 6.93 (d, J ¼ 8.3 Hz, 2H),
J ¼ 7.0 Hz, 2H), 1.46 (t, J ¼ 7.1 Hz, 3H). C NMR (151 MHz, DMSO‑d
6
)
6
3
3
.76 (d, J ¼ 6.6 Hz, 3H), 5.38 (s, 2H), 4.57 (q, J ¼ 7.0 Hz, 2H), 3.71 (s,
d 166.31, 157.21, 140.45, 139.87, 138.92, 137.93, 135.21, 130.08,
130.04, 129.97, 129.93, 129.89, 129.64, 128.96, 128.54, 128.43,
127.94, 127.62, 127.58, 126.64, 125.58, 120.92, 119.97, 119.89, 118.44,
H), 3.34 (d, J ¼ 4.9 Hz, 2H), 2.64 (t, J ¼ 7.4 Hz, 2H), 1.38 (t, J ¼ 7.1 Hz,
13
6
H). C NMR (151 MHz, DMSO‑d ) d 166.27, 158.66, 157.19, 140.45,
1
40.39, 137.68, 135.96, 130.25, 129.99, 129.95, 128.99, 128.71, 128.31,
65.71, 44.91, 39.81, 33.40, 13.83. HRMS (ESI) m/z calcd C32
7 2
H28ClN O
127.13, 125.99, 120.88, 120.27, 120.01, 119.91, 118.46, 113.51, 111.09,
[MþH]þ 578.2071, found 578.2020.
6
C
5.71, 54.25, 44.92, 39.96, 34.15, 13.82. HRMS (ESI) m/z calcd
þ
0
H
33 31
N
7
O
3
[MþH] 574.2567, found 574.2568.
4.3.34. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-
(
2,4-difluorophenethyl)-2-ethoxy-1H-benzo[d]imidazole-7-
carboxamide (45)
Brown solid, yield 87%, mp ¼ 199e201 C. H NMR (400 MHz,
CDCl
0
4.3.30. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-2-
ꢀ
1
ethoxy-N-(4-methylphenethyl)-1H-benzo[d]imidazole-7-
carboxamide (41)
3
)
d
8.00 (d, J ¼ 7.0 Hz, 1H), 7.57e7.49 (m, 2H), 7.33 (d,
ꢀ
1
White solid, yield 79%, mp ¼ 192e195 C. H NMR (400 MHz,
J ¼ 6.0 Hz, 2H), 7.15 (dd, J ¼ 15.0, 8.3 Hz,1H), 7.04 (dd, J ¼ 8.6, 5.2 Hz,
2H), 6.98 (d, J ¼ 8.1 Hz, 2H), 6.87e6.72 (m, 5H), 6.02 (s, 1H), 5.39 (s,
2H), 4.53 (q, J ¼ 7.0 Hz, 2H), 3.43 (dd, J ¼ 13.0, 6.6 Hz, 2H), 2.79 (t,
DMSO‑d
6
) d
8.40 (t, J ¼ 5.5 Hz,1H), 7.61 (dd, J ¼ 11.9, 4.4 Hz, 2H), 7.53
(
7
5
dd, J ¼ 7.5, 4.3,1.6 Hz, 2H), 7.41 (d, J ¼ 7.8 Hz,1H), 7.16e7.09 (m,1H),
13
.09e7.05 (m, 5H), 6.97 (d, J ¼ 8.4 Hz, 2H), 6.93 (d, J ¼ 8.4 Hz, 2H),
J ¼ 6.8 Hz, 2H), 1.47 (t, J ¼ 6.7 Hz, 3H). C NMR (151 MHz, DMSO‑d
6
)
.39 (s, 2H), 4.57 (q, J ¼ 7.0 Hz, 2H), 3.29 (d, J ¼ 7.8 Hz, 2H), 2.61 (t,
d 166.34, 161.24, 161.16, 160.88, 160.80, 159.63, 159.54, 159.25,
13
J ¼ 7.5 Hz, 2H), 2.24 (s, 3H), 1.38 (t, J ¼ 7.1 Hz, 3H). C NMR
151 MHz, DMSO‑d 166.25, 157.19, 140.45, 140.37, 137.66, 135.98,
35.68, 134.37, 130.26, 130.00, 129.95, 129.00, 128.31, 128.28, 127.89,
27.13, 126.00, 123.09, 120.88, 120.04, 119.92, 118.46, 65.71, 44.93,
159.17, 157.22, 155.33, 140.47, 140.26, 138.04, 135.77, 131.59, 131.54,
131.48, 129.97, 129.86, 129.80, 129.00, 128.35, 127.01, 125.90, 124.19,
121.78, 121.76, 121.68, 120.79, 119.98, 119.93, 118.52, 110.70, 110.67,
110.56, 110.53, 103.18, 103.00, 102.83, 65.73, 44.95, 27.15, 13.82.
(
1
1
6
) d
4
0.20, 33.73, 20.02, 13.82. HRMS (ESI) m/z calcd C33
31
H N
7
O
2
HRMS (ESI) m/z calcd C32
580.2272.
27 2
H F N
7
O
2
[MþH]þ 580.2273, found
þ
[MþH] 558.2617, found 558.2583.
0
0
4
(
7
.3.31. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-(2-
4.3.35. N-(2-(1H-indol-3-yl)ethyl)-1-((2’-(1H-tetrazol-5-yl)-[1,1 -
biphenyl]-4-yl)methyl)-2-ethoxy-1H-benzo[d]imidazole-7-
carboxamide (46)
benzo[d] [1,3]dioxol-5-yl)ethyl)-2-ethoxy-1H-benzo[d]imidazole-
-carboxamide (42)
White solid, yield 87%, mp ¼ 189e191 C. H NMR (400 MHz,
CDCl
ꢀ
1
ꢀ
1
Brown solid, yield 78%, mp ¼ 189e192 C. H NMR (400 MHz,
3
)
d
7.88 (d, J ¼ 7.4 Hz,1H), 7.55 (d, J ¼ 6.4 Hz,1H), 7.49e7.37 (m,
DMSO‑d
6
)
d
10.82 (s, 1H), 8.45 (t, J ¼ 5.5 Hz, 1H), 7.62 (d, J ¼ 7.3 Hz,
2
2
(
4
H), 7.34 (d, J ¼ 7.6 Hz, 1H), 7.09e6.96 (m, 4H), 6.82 (d, J ¼ 8.0 Hz,
1H), 7.58e7.47 (m, 4H), 7.37 (d, J ¼ 7.0 Hz, 2H), 7.33 (d, J ¼ 8.0 Hz,
1H), 7.14 (s, 1H), 7.13e7.08 (m, 2H), 7.09e7.03 (m, 1H), 6.94 (q,
J ¼ 8.6 Hz, 5H), 5.41 (s, 2H), 4.57 (q, J ¼ 7.0 Hz, 2H), 3.39 (d,
H), 6.68 (d, J ¼ 7.8 Hz, 1H), 6.58 (d, J ¼ 8.0 Hz, 2H), 6.44 (s, 1H), 6.30
t, J ¼ 5.7 Hz, 1H), 5.85 (t, J ¼ 5.0 Hz, 2H), 5.27 (d, J ¼ 21.7 Hz, 2H),
13
.59 (q, J ¼ 7.0 Hz, 2H), 3.43 (dd, J ¼ 12.8, 6.7 Hz, 2H), 2.68 (t,
J ¼ 11.0 Hz, 2H), 2.83e2.73 (m, 2H), 1.38 (t, J ¼ 7.1 Hz, 3H). C NMR
13
J ¼ 6.8 Hz, 2H), 1.42 (t, J ¼ 7.1 Hz, 3H). C NMR (151 MHz, DMSO‑d
166.32, 159.96, 157.22, 146.79, 146.54, 145.40, 144.86, 140.44,
40.20, 139.39, 134.47, 132.71, 131.35, 130.44, 129.78, 129.39, 128.93,
28.54, 126.82, 126.17, 125.29, 121.13, 121.10, 120.94, 119.98, 119.85,
6
)
6
(151 MHz, DMSO‑d ) d 166.29, 157.19, 140.44, 140.27, 137.88, 135.85,
d
135.63,133.89, 129.96,129.91, 128.99,128.32,127.03,126.92, 126.60,
125.95, 122.01, 120.30, 120.08, 119.91, 118.42, 117.63, 117.60, 111.13,
110.77, 65.70, 44.92, 28.40, 24.17, 13.83. HRMS (ESI) m/z calcd
1
1
1
3
5
18.37, 108.47, 108.43, 107.72, 107.45, 100.24, 100.01, 65.71, 40.30,
3.81, 32.23, 13.85. HRMS (ESI) m/z calcd C33
C
H
34 30
N
8
O
2
[MþH]þ 583.2570, found 583.2571.
[MþH]þ
29
H N
7
O
4
88.2359, found 588.2358.
4.4. Cell culture and reagents
0
4
.3.32. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-(2-
A549, H1299 human lung cancer cells, 16H6E and BEAS-2B hu-
man normal lung epithelial cells were obtained from the American
Type Culture Collection (Manassas, Virginia) and passaged six times
before use. Cultured in Dulbecco’s modified Eagle’s medium
chlorophenethyl)-2-ethoxy-1H-benzo[d]imidazole-7-carboxamide
43)
Brown solid, yield 75%, mp ¼ 187e190 C. H NMR (400 MHz,
CDCl
8.03e7.95 (m, 1H), 7.54 (p, J ¼ 7.3 Hz, 2H), 7.38e7.28 (m,
H), 7.16 (dd, J ¼ 6.9, 3.7 Hz, 4H), 7.06e6.96 (m, 2H), 6.93 (d,
J ¼ 8.1 Hz, 2H), 6.78 (d, J ¼ 7.9 Hz, 2H), 5.93 (s, 1H), 5.38 (s, 2H), 4.44
q, J ¼ 7.0 Hz, 2H), 3.50e3.38 (m, 2H), 2.87 (t, J ¼ 7.0 Hz, 2H),
(
ꢀ
1
3
)
d
(Hyclone, Logan, UT), which contains 10% fetal bovine serum
ꢃ1
3
(Biochrom AG, Berlin, Germany) and 100 units ml
penicillin/
streptomycin solution, and store in a humidified atmosphere at
ꢀ
(
1
1
1
37 C, and 5% CO
2
(standard culture conditions). MLN4924, CDC and
13
.48e1.41 (t, J ¼ 7.1 Hz, 3H). C NMR (151 MHz, DMSO‑d
57.20, 140.47, 140.35, 137.76, 136.16, 135.93, 132.53, 130.49, 130.15,
29.99, 129.93, 129.01, 128.64, 128.34, 127.60, 127.11, 126.62, 125.99,
6
)
d
166.31,
derivatives 2e48 were dissolved in dimethyl sulfoxide (DMSO) and
ꢀ
maintained at ꢃ20 C for in vitro studies. In vivo studies MLN4924,
CDC and derivative 2e47 were dissolved in 5% 2-hydroxypropyl-b-
1
23.32, 120.77, 120.07, 119.91, 118.53, 65.72, 44.95, 38.18, 31.95,
cyclodextrin and 5% castor oil (Macklin reagent, Shanghai, China)
for in vivo study. MLN4924, CDC and derivatives 2e47 are all freshly
prepared when used.
13.86. HRMS (EI) m/z calcd C32
H28ClN
7
O
2
[MþH]þ 578.2071, found
578.2004.
0
4
.3.33. 1-((2’-(1H-tetrazol-5-yl)-[1,1 -biphenyl]-4-yl)methyl)-N-(4-
4.5. Enzyme-based neddylation activity assay
chlorophenethyl)-2-ethoxy-1H-benzo[d]imidazole-7-carboxamide
44)
White solid, yield 68%, mp ¼ 197e199 C. H NMR (400 MHz,
CDCl
7.95 (d, J ¼ 7.0 Hz, 1H), 7.51 (dtd, J ¼ 13.7, 7.5, 6.1 Hz, 2H),
.40e7.36 (m, 1H), 7.34e7.30 (m, 1H), 7.24 (d, J ¼ 8.3 Hz, 2H),
.20e7.12 (m, 1H), 7.09 (d, J ¼ 8.3 Hz, 2H), 7.06e6.98 (m, 2H), 6.95
d, J ¼ 8.2 Hz, 2H), 6.81 (d, J ¼ 8.0 Hz, 2H), 6.08 (s, 1H), 5.37 (s, 2H),
(
The protein Nedd8, NAE, Ubc12, Rbx1/Cullin1CTD were prepared
and gifted by Prof. Jin Huang, Shanghai Jiao Tong University School
of Medicine, China. The procedure of enzyme-based assay detailed
ꢀ
1
3
) d
7
7
(
as below: 1
m
L UBA3-NAE (final concentration, 0.025
M), 1 L Ubc12 (final concentration,
(final concentration, 1 M), 1 L Tris-
mM), 1 mL
Nedd8 (final concentration, 10
m
m
CDT
1
mM), 1
mL RBX1/cullin1
m
m
15

X. Chen, X. Yang, F. Mao et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
HCl (1 M, pH ¼ 7.4; final concentration, 50 mM), 1
m
L MgCl2 (0.1 M),
O, were added
L water so-
M) or test drugs (final
L (final concentration, 20 M) ATP was
female nude mice were subcutaneously injected with 100
m
L of PBS
9 cells. After tumor induction
(approximately 7e9 days), nude mice were randomly divided into 5
groups: solvent group (10% 2-hydroxypropyl- -cyclodextrin/water
6
54
1
mL DTT (10 mM), 0.2
m
L 0.1 mg/mL BSA, 2.8
m
L ddH
2
buffer containing 2 ꢁ 10
A
into in each well of 96-well plates, followed with 2.0
lution of MLN4924 (final concentration, 1
concentration, 50 M). 4.0
m
m
b
m
m
m
solution) and positive group (CDC-30 mg/kg), The other three
groups were administered compound 39 in different ways and
doses: 35 (30 mg/kg) was administered intraperitoneally, and 35
(30 mg/kg, 60 mg/kg) was administered by oral gavage. All groups
were treated daily once that give it five days and off two days. Mice
weight and tumor volume were measured every two days. On day
32 after vaccination, all mice were sacrificed. The tumor volume
and mass of each group were measured.
added to reaction after the mixture was incubated for 10 min at
room temperature. The mixture was incubated at 37 C for 30 min.
ꢀ
The reaction was quenched by 10% loading buffer, and heated for
ꢀ
5
min at 95 C by a real-time PCR. Protein samples were electro-
phoresed under non-reducing conditions on a 10e15% SDS-PAGE
gel, and cullin1-Nedd8 levels were determined by immunoblot-
ting analysis. To determine the neddylation inhibitory activities of
3
5, 39, 46 and CDC, various concentrations of groups were used by
two-thirds decrease.
Statistical analysis
4
.6. Cell-based neddylation activity assay
All data were reported only if at least three independent ex-
periments showed consistent results. The data were analyzed by
one-way ANOVA followed by Dunnett’s test for multiple compari-
sons or by Student’s t-test for single comparison. Statistical signif-
icance was established at p value < 0.05.
A549 cells were exposed to the specified concentrations of
MLN4924 (1
2
mM) or CDC, 35, 39 and 46 (75
m
M, 50
M) or 0.1% (v/v)
mM, 32 mM,
5
m
M, 18 M, 12.5
m
m
M, 9 M, 6.25 M and 4.5 m
m
m
DMSO was performed for 6 h or 12 h, respectively. Cells were
washed 3 times with ice-cold PBS, resuspended in RIPA lysis buffer,
Supporting information
and incubated on ice for 30 min. Centrifuge at 15,000 rpm for
ꢀ
1
0 min at 4 C to remove cell debris. The protein concentration of
HPLC reports for the purity check of compounds 2e46, the
cullin1-Nedd8 inhibitory activities of analogs 2e46 by the enzyme-
based assay, survival curve of the cullin1-Nedd8 adduction after
incubating with 35, 39, 46 and CDC, H NMR, MASS spectra and
HPLC Traces of compounds 14, 22, 23, 25, 26 and 28e33 and the
the supernatant was determined using a Thermo Fisher protein
assay dye reagent (Thermo Fisher). The same amount of protein
was electrophoresed on SDS-PAGE under non-reducing (cullin1-
Nedd8, cullin2-Nedd8, cullin3-Nedd8, cullin4a-Nedd8 and
cullin5-Nedd8) or reduced (Wee1 or p27) conditions and execute
Western blot analysis.
1
intermediates.
Declaration of competing interest
4.7. Cell apoptosis assays and cell proliferation
The author declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
The FITC Annexin V Apoptosis detection kit was used to evaluate
the apoptosis of A549 cells. A549 cells were seeded at
5
2
.5 ꢁ 10 cells per well in 6-well plates and allowed to attach
overnight. Cells were treated with successively increasing con-
centrations of 35 (10 M, 20 M, 40 M) for 48 h. Cells were
dissociated using trypsin, washed 3 times with ice-cold PBS buffer,
and resuspended in 100 L of 1x binding buffer. Add 5 L FITC
Annexin V staining solution and 5 L PI staining solution. After
5 min of incubation at room temperature in the dark, another
00 L of 1x binding buffer was added. Stained cells were imme-
Acknowledgments
m
m
m
This work was supported by the Chinese Minister of Science and
Technology grant (2016YFA0501800), the National Natural Science
Foundation of China (Grants 81872747, 21702061, 81625018,
81820108022), the Innovative Research Team of High-level Local
Universities in Shanghai, the National Special Fund for State Key
Laboratory of Bioreactor Engineering (2060204), and the National
Key R&D Program of China (2017YFB0202600). Innovation Program
of Shanghai Municipal Education Commission (2019-01-07-00-10-
E00056), Program of Shanghai Academic/Technology Research
Leader (18XD1403800).
m
m
m
1
4
m
diately analyzed by fluorescence activated cell sorting (FACS).
To assess the proliferation of cultured cells, cells (A549, H1299,
6H6E, and BEAS-6B) were seeded into 96-well plates with
500 cells per well and cultured overnight in triplicate, treated
with CDC, 39, 41, and 47 for 48 h, and then perform Cell Counting
Kit 8 (CCK-8) analysis.
1
2
Appendix A. Supplementary data
4.8. Molecular modeling
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112964.
The X-ray structure of NAE protein was downloaded from the
protein database (PDB ID: 3GZN). Removal of crystal water, ligands
and other conformations through the protein preparation protocol
in Autodock 4.0. The docking process between small molecule and
NAE is performed through the DOCK program in Autodock 4.0, and
finally the structure image is obtained using PyMOL software.
Abbreviations
CRLs
cullin-RING ligases
Nedd8
neuronal precursor cell-expressed developmentally
down-regulated protein 8
Nedd8-activating enzyme E1
adenosine triphosphate
4.9. Tumor xenograft growth inhibition
NAE
ATP
The animal handling and experiment protocols were approved
by the Institutional Animal Care and Use Committee of Longhua
Hospital Shanghai University of Traditional Chinese Medicine
A2T1R
I$P.
DMF
HOBT
Angiotensin II type 1 receptor
intraperitoneal injection
N,N-Dimethylformamide
(
2019-N038). According to the tumor transplantation protocol,
1-Hydroxybenzotriazole
16

X. Chen, X. Yang, F. Mao et al.
European Journal of Medicinal Chemistry xxx (xxxx) xxx
EDCI
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride
Ethyldiisopropylamine
structure-activity relationships
maturation through repression of Hippo signaling, Proc. Natl. Acad. Sci. Unit.
States
Am.
115
(2018)
E4101eE4110,
https://doi.org/10.1073/
pnas.1719309115.
DIPEA
SARs
[
22] W. Zhou, J. Xu, H. Li, M. Xu, Z.J. Chen, W. Wei, Z. Pan, Y. Sun, Neddylation E2
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cullin-RING ligases for anticancer therapy, Antioxidants Redox Signal. 21
(2014) 2383e2400, https://doi.org/10.1089/ars.2013.5795.
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