Design, synthesis and biological evaluation of novel FXIa inhibitors with 2-phenyl-1H-imidazole-5-carboxamide moiety as P1 fragment

Article abstract of DOI:10.1016/j.ejmech.2021.113437

Factor XIa, as a blood coagulation enzyme, amplifies the generation of the last enzyme thrombin in the blood coagulation cascade. It was proved that direct inhibition of factor XIa could reduce pathologic thrombus formation without an enhanced risk of bleeding. WSJ-557, a nonpurine imidazole-based xanthine oxidase inhibitor in our previous reports, could delay blood coagulation during its animal experiments, which prompted us to investigate its action mechanism. Subsequently, during the exploration of the action mechanism, it was found that WSJ-557 exhibited weak in vitro factor XIa binding affinity. Under the guide of molecular modeling, we adopted molecular hybridization strategy to develop novel factor XIa inhibitors with WSJ-557 as an initial compound. This led to the identification of the most potent compound 44g with a Ki value of 0.009 μM, which was close to that of BMS-724296 (Ki = 0.0015 μM). Additionally, serine protease selectivity study indicated that compound 44g display a desired selectivity, more 400-fold than those of thrombin, factor VIIa and factor Xa in coagulation cascade. Moreover, enzyme kinetics studies suggested that the representative compound 44g acted as a competitive-type inhibitor for FXIa, and molecular modeling revealed that it could tightly bind to the S1, S1′ and S2′ pockets of factor XIa. Furthermore, in vivo efficacy in the rabbit arteriovenous shunt model suggested that compound 44g demonstrated dose-dependent antithrombotic efficacy. Therefore, these results supported that compound 44g could be a potential and efficacious agent for the treatment of thrombotic diseases.

Full text of DOI:10.1016/j.ejmech.2021.113437

European Journal of Medicinal Chemistry 220 (2021) 113437
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis and biological evaluation of novel FXIa inhibitors
with 2-phenyl-1H-imidazole-5-carboxamide moiety as P1 fragment
,
,
Yu Lei a 1, Bing Zhang a 1, Yan Zhang b, Xiwen Dai a, Yulin Duan a, Qing Mao a, Jun Gao a,
Yuwei Yang a, Ziyang Bao a, Xuefeng Fu a, Kunqi Ping a, Chengda Yan c, Yanhua Mou b
,
,
**
Shaojie Wang a
,
*
a Key Laboratory of Structure-Based Drugs Design & Discovery of Ministry of Education, School of Pharmaceutical Engineering, Shenyang Pharmaceutical
University, 103 Culture Road, Shenhe District, Shenyang, 110016, China
b Department of Pharmacology, Shenyang Pharmaceutical University, 103 Culture Road, Shenhe District, Shenyang, 110016, China
c Department of Pharmacy, The First Hospital of China Medical University, 155 Nanjing North Street, Shenyang, 110001, Liaoning, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Factor XIa, as a blood coagulation enzyme, amplifies the generation of the last enzyme thrombin in the
blood coagulation cascade. It was proved that direct inhibition of factor XIa could reduce pathologic
thrombus formation without an enhanced risk of bleeding. WSJ-557, a nonpurine imidazole-based
xanthine oxidase inhibitor in our previous reports, could delay blood coagulation during its animal
experiments, which prompted us to investigate its action mechanism. Subsequently, during the explo-
ration of the action mechanism, it was found that WSJ-557 exhibited weak in vitro factor XIa binding
affinity. Under the guide of molecular modeling, we adopted molecular hybridization strategy to develop
novel factor XIa inhibitors with WSJ-557 as an initial compound. This led to the identification of the most
Received 29 November 2020
Received in revised form
5 March 2021
Accepted 30 March 2021
Available online 20 April 2021
Keywords:
Biological evaluation
2-Phenyl-1H-imidazole
FXIa inhibitors
potent compound 44g with a Ki value of 0.009
mM, which was close to that of BMS-724296
(Ki ¼ 0.0015 M). Additionally, serine protease selectivity study indicated that compound 44g display a
m
desired selectivity, more 400-fold than those of thrombin, factor VIIa and factor Xa in coagulation
cascade. Moreover, enzyme kinetics studies suggested that the representative compound 44g acted as a
competitive-type inhibitor for FXIa, and molecular modeling revealed that it could tightly bind to the S1,
S10 and S20 pockets of factor XIa. Furthermore, in vivo efficacy in the rabbit arteriovenous shunt model
suggested that compound 44g demonstrated dose-dependent antithrombotic efficacy. Therefore, these
results supported that compound 44g could be a potential and efficacious agent for the treatment of
thrombotic diseases.
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
novel anticoagulants targeting two key serine proteases in the
blood coagulation cascade: thrombin and factor Xa (FXa), are
Thrombotic diseases which include stroke, myocardial infarc-
tion and deep vein thrombosis, continue to be major causes of
death worldwide [1e3]. For decades, warfarin and heparin have
been the mainstay in the treatment and prevention of these
thrombotic diseases [4e7]. However, they both cause enhanced risk
of bleeding and have a narrow therapeutic index [7e9]. As a result,
popular in treating thrombotic diseases [1,10,11]. The approved
drugs such as direct FXa inhibitors (rivaroxaban [11], apixaban [12],
and edoxaban [13]) and direct thrombin inhibitor (dabigatran [14])
are highly effective for the prevention and treatment of these dis-
eases. Although they have offered patients an alternative to
warfarin and heparin [4,15,16], there is still a risk of major bleeding
as high as 2e3% per year in patients with thrombotic diseases
which limit their widespread use [1,4,15,16]. Consequently, novel
anticoagulants that can effectively reduce thrombosis without
enhanced risk of bleeding are urgently needed [1,4,5,15,16].
* Corresponding author.
** Corresponding author.
Factor XIa (FXIa) as a potential feedback activator of FXI [4]
amplifies the generation of the last enzyme thrombin in the blood
E-mail addresses: mu_hua_jj@sina.com (Y. Mou), wangshaojie@syphu.edu.cn
(S. Wang).
1
These two authors contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2021.113437
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
coagulation cascade [17,18], which further leads to fibrin clot for-
mation [17,18]. Several preclinical studies have demonstrated that
inhibition of FXI (antisense oligonucleotides or neutralizing anti-
bodies [19e21]) or FXIa (small-molecule inhibitors) could provide
antithrombotic efficacy with a low risk of bleeding in animal
thrombosis models [17,19e21]. In addition, it is shown that FXI
gene disruption in mice does not lead to an increase in bleeding
times [10,22], and human subjects with FXI deficiency such as
hemophilia C [1,4] do not suffer spontaneous bleeding [1,4].
Furthermore, a recent phase II clinical trial with an antisense
oligonucleotide targeting FXI proved efficacious in preventing
venous thrombosis in patients undergoing total knee arthroplasty,
and also appeared to be safe with respect to bleeding [17,20]. Taken
together, these cases suggest that FXIa may serve as a powerful
route to achieve an antithrombotic effect safer than that of FXa or
thrombin [1,17e21].
have entered clinical trials. In addition, other FXIa inhibitors, such
as 4 (BMS-724296) [2,28], 5 [17], 6 [29], 7 [30], 8 [20], 9 [4], 10 [16]
and 11 [17] shown in Fig. 1, are in preclinical research. These cases
inspire us to explore novel FXIa inhibitors to fulfill the urgent needs
in clinical practice [1,17e21].
Human FXIa is a serine protease disulfide homodimer belonging
to the intrinsic coagulation pathway [5,6]. The active site of FXIa
contains several subsites with characteristic features for substrate
selectivity.
Substrate
amino
acid
nomenclatures
are
P4eP3eP2eP1eP1ʹeP2ʹeP3ʹeP4ʹ, respectively, and the substrate
binding pockets are labeled in a corresponding manner to the
substrate: S4, S3, S2, S1, S1ʹ, S2ʹ, S3ʹ and S4ʹ, respectively [5,6].
Among them, P1eP1ʹ denotes the cleaved peptide bond, which
implies that the corresponding S1 and S1ʹ pockets are crucial for
catalyzing reaction of substrates [5,6]. It is reported that binding in
multiple pockets in FXIa can lead to improve affinity and selectivity,
and the active site of FXIa contains a deep S1 pocket accompanied
by open and shallow prime binding regions S10 and S20 (Fig. 2)
[1,5,6]. Therefore, For the S1 pocket that catalyzes the cleavage of
the substrate peptide, S1eS1ʹeS2ʹ mode in FXIa is often used to
Recently, several monoclonal antibodies targeting FXI (e.g., BAY
1213790 [23], Xisomab 3G3 [24] and MAA-868 [25]) and small-
molecule FXIa inhibitors including 1 (Milvexian [26], phase II), 2
(BMS-962212 [21], phase I), and 3 (EP-7041 [27], phase I) in Fig. 1
Fig. 1. Structures of the representative FXIa inhibitors: 1 (Milvexian, phase II), 2 (BMS-962212, phase I), 3 (EP-7041, phase I),4 (BMS-724296), 5, 6, 7, 8, 9, 10 and 11.
2

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
implied that WSJ-557 could be used as a P1 fragment to design
novel FXIa inhibitors, and it was hoped that hydroxyl and carbonyl
groups on imidazole moiety could interact with key amino acid
residues (Gly193, Asp194 and Ser195) of oxyanion hole. Therefore,
we attempted to introduce P10 and P20 fragments of FXIa inhibitor 7
into the carboxyl group of WSJ-557 to design a series of novel FXIa
inhibitors to investigate the possibility of WSJ-557 as a P1 fragment
(Fig. 3).
In this paper, we describe the procedure in discovery of a series
of novel FXIa inhibitors. These FXIa inhibitors were evaluated on
FXIa binding affinity in vitro, and the structure-activity relation-
ships (SAR) were further explored. Moreover, serine protease
selectivity profile, including thrombin, FVIIa, FXa and plasma
kallikrein, was performed to investigate the selectivity of the most
potent compound 44g. Additionally, to determine the inhibitory
behavior of these target compounds, the representative compound
44g was further evaluated by molecular modeling studies and
steady-state kinetic analysis. Furthermore, in vivo efficacy in the
rabbit arteriovenous shunt model was carried out to explore the
antithrombotic effect of 44g.
Fig. 2. Crystal structure of inhibitor 7 with FXIa (PDB: 5E2O) [30], and the surfaces of
S1, S10 and S20 pockets are shown as green, yellow and cyan, respectively.
2. Results and discussion
design novel FXIa inhibitors [1,5,6]. In addition, the oxyanion hole
completes the perimeter of the S1 pocket and is formed by the
backbone nitrogen atoms of residues Gly193, Asp194 and Ser195,
which stabilizes the negative charge formed during proteolysis, and
it is indicated that Gly193, Asp194 and Ser195 of oxyanion hole may
be key amino acid residues for catalyzing the substrate peptide
[1,5,6]. Moreover, the above reported compounds 1e11 (Fig. 1) all
could contact with Ser195 of oxyanion hole through hydrogen
bonds or covalent bonds, displaying excellent FXIa binding affinity,
which further suggests that the interaction between P1 group and
oxyanion hole is essential for design of FXIa inhibitors [16,17,20,21].
2.1. Discovery of lead compound 28a
Molecular modeling study was employed to guide the structural
optimization of imidazole derivatives. The result of molecular
modeling showed that isobutoxy group of WSJ-557 was inserted
into the deep part of FXIa S1 pocket (Fig. 4), and its carboxyl group
formed two electrostatic interactions with Arg39 in the FXIa active
site. However, this bulky isobutoxy group pushed the hydroxyl
group of imidazole moiety so far away from key residue Cys191 that
it could not form hydrogen bond interaction, which reminded of us
that decreasing the size of 40-substitutions on phenyl moiety could
induce phenyl moiety into the S1 pocket tightly in the hope of
improving the FXIa affinity. Subsequently, the isobutoxy group on
phenyl moiety was changed into methoxy, hydroxy groups or
removed to give the imidazole derivatives 16a, 17 and 16e (Table 1),
which all improved the FXIa affinity by more than 2-fold (WSJ-
In
our
previous
reports,
WSJ-557
(2-(3-cyano-4-
isobutoxyphenyl)-1-hydroxy-4-methyl-1H-imidazole-5-carboxylic
acid, a nonpurine XO inhibitor, Fig. 3) demonstrated stronger XO
inhibitory potency than that of febuxostat (WSJ-557 vs febuxostat,
IC50 ¼ 0.003 and 0.01
mM for XO) [31e33]. Interestingly, during its
animal experiments, it was found that WSJ-557 could delay blood
coagulation [33], which encouraged us to investigate its action
mechanism. Subsequently, molecular modeling study suggested
that WSJ-557 was accommodated in the FXIa S1 pocket, which
557 < 16a < 17 < 16e, Ki ¼ >500, 243, 228 and 120
mM, respectively,
Table 1), thus verifying the above envisages. Among them, com-
pound 16e displayed more potent than those of others. In addition,
the results of molecular modeling suggested that imidazole
Fig. 3. Design of FXIa inhibitors based on WSJ-557.
3

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
Fig. 4. Binding modes of compounds 16a, 16e, 17 and WSJ-557 within the FXIa binding pocket. (A) The surface of the protein (PDB code 4TY7) [2] is shown in rainbow, and small
molecules as well as residues (green) are shown as sticks. 16a (yellow), 16e (magenta), 17 (grey) and WSJ-557 (cyan) occupy the same S1 binding pocket in FXIa. Electrostatic
interactions of WSJ-557 are shown as black dashed lines, and hydrogen bonds of 16e are shown as red dashed lines, respectively. (B) Docking pose of 16e (magenta) with FXIa.
Table 2
Table 1
The effect of substituents around the 2-phenyl-imidazole moiety on FXIa binding
affinity.
Structural modification of imidazole derivatives.
Compounds
R
Ki (m
M)a
WSJ-557
>500b
16a
16e
-OCH3
243
120
Compounds
R1
R2
R3
R4
Ki (m
M)a
e
17
-OH
228
28a
28b
28c
28d
28e
28f
28g
28h
28i
-H
-H
-CN
-H
-Cl
-H
-H
-H
-H
-H
-H
-CN
-H
-H
-H
-H
-Cl
-H
-Cl
-Cl
-Cl
-Cl
-H
-CN
-H
-H
-H
-H
-Cl
-F
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-H
23.88
18.86
>100b
>100b
>100b
14.50
>100b
11.76
26.38
22.37
>100b
a
Ki values were derived from IC50 values using Cheng Prussof transformation
[37,38].
b
Inhibition in 500 mM: 29.83%.
derivative 16e could fit more tightly into S1 pocket than others.
Specifically, carboxyl group of 16e pointed into the oxyanion hole
and formed two hydrogen bonds with key residues Gly193 and
Ser195, in addition, its hydroxyl group could engage an extra
hydrogen bond with Cys191 (Fig. 4B). Meanwhile, this optimization
also made imidazole derivative 16e almost closer to comply with
the “rule of three” of the hit (see Table S1) [34e36]. Therefore,
compound 16e was selected as a hit for further optimization of
novel FXIa inhibitors.
-F
-F
-F
28j
28k
-CH3
-OCH3
a
Ki values were derived from IC50 values using Cheng Prussof transformation
[37,38].
b
Inhibition rates of compounds 28b, 28c, 28e, 28g and 28k at concentration of
M were 16.4%, 0%, 22.5%, 34.1% and 0%, respectively.
100
m
It was reported that binding in multiple pockets in FXIa could
improve affinity and selectivity [5,6]. As a result, with using 2-
phenyl-1H-imidazole fragment of 16e as a P1 group, we intro-
duced P10 phenylalanine and P20 p-aminobenzoic acid fragments
into the carbonyl group on imidazole ring to afford the lead com-
pound 28a, which displayed a 5.03-fold increase for FXIa affinity in
ortho-, para- and no cyano substituted derivatives 28b-d were
prepared. Among them, the ortho-cyano substituted derivative 28b
retained a similar FXIa binding affinity as that of compound 28a
(28b vs 28a, Ki ¼ 18.86 and 23.88
mM, respectively), whereas para-
and no cyano substituted derivatives 28c and 28d led to the loss of
the FXIa affinity (Ki values of 28c and 28d were both more than
comparison to 16e (Table 2, 28a vs 16e, Ki ¼ 23.88 and 120
mM,
respectively). This suggested that linking P10 and P20 fragments to
fit into active pockets (S10 and S20) of FXIa was necessary for
improving FXIa affinity. To investigate the effect of cyano group
positions for P1 groups binding to S1 pocket, the corresponding
100 mM). According to the current case, the introduction of halogen
atoms on P1 phenyl moiety could enhance the affinity [4,17,21].
Then, substituting the cyano groups on phenyl moiety with halogen
atoms gave compounds 28e-h. The compounds 28f, h exhibited
4

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
Table 3
moiety was performed to occupy this space to investigate the FXIa
binding affinity. Initially, the addition of halogen atoms (fluorine,
chlorine and bromine) at para-position of P10 phenyl moiety of
compound 34b offered compounds 39a-c (Table 4) with Ki values of
The FXIa binding affinity of 2-phenyl-1H-imidazole derivatives with various R5
moieties.
0.486, 0.356 and 0.962
mM, respectively, which showed a
2.93e7.92-fold improvement for FXIa binding affinity compared
with compound 34b. But changing these halogen atoms to a nitro
or amine group could damage the FXIa binding affinity
(39e < 39d < 34b, Ki ¼ 7.32, 3.08 and 2.82
mM, respectively).
Then, with carboxamide as a linker, insertion of various sub-
stitutions at the carbonyl carbon atom of the carboxamide moiety
to fill the S10 active pocket was further explored. Specifically,
increasing the size of the substituent at carbonyl carbon of the
carboxamide moiety from a methyl to an isobutyl group could
steadily strengthen the FXIa binding potency (44a < 44b < 44c,
Ki ¼ 0.392, 0.156 and 0.026
mM, respectively). Moreover, the
replacement of saturated alkyl groups with an ethynyl or prop-1-
yn-1-yl group at carbonyl carbon of the carboxamide moiety pro-
Compounds
R5
Ki (m
M)a
34a
6.86
vided compounds 44d-e with Ki values of 0.053 and 0.031
mM,
retaining desired FXIa binding potency, nevertheless, they did not
show apparent increase on FXIa binding potency compared with
that of 44c. This finding indicated that increasing the size of satu-
rated alkyl and alkynyl groups at carbonyl carbon of the carbox-
amide moiety could maintain a satisfactory FXIa binding potency,
which might be due to the improved hydrophobic interactions with
residues at the S10 active pocket.
34b
2.82
a
Ki values were derived from IC50 values using Cheng Prussof transformation
To continue to investigate the size effect of the R6 substituents
on binding potency, the conversion of saturated alkyl groups into
various cycloalkyl substitutions led to compounds 44f-j (Table 4).
Interestingly, compound 44g showed the most potent FXIa binding
[37,38].
slightly stronger than that of 28a (28f > 28h > 28a, Ki ¼ 11.76, 14.50
and 23.88 M, respectively), while there was no improvement in
m
affinity with a Ki value of 0.009
mM, which was close to that of BMS-
FXIa binding affinity through inserting chlorine atom to ortho- and
para-positions of phenyl moiety (Ki values of 28e and 28g were
both more than 100 mM). As we continued to explore modifications
724296 (Ki ¼ 0.0015 M). Presumably, the 2-methylcyclopropane-
m
1-carboxamido substitution on P10 phenyl moiety kept the P10
group in a more favorable position so that it could fill better in FXIa
S10 active pocket. Following this discovery, cycloalkyl substitutions
were changed into various aromatic rings, and compounds 44k-m
were prepared, yet they resulted in a 9.22e40.67-fold decrease in
FXIa binding affinity in comparison to 44g (44k, 44l, 44m < 44g,
of P1 group, compounds 28i and 28k were prepared through
changing hydroxyl group on imidazole moiety into hydrogen,
methyl or methoxy group, and they showed no increase in FXIa
binding affinity (28i, 28j and 28k vs 28h, Ki ¼ 26.38, 22.37, >100
and 11.76 mM, respectively). These results indicated that the meta-
Ki ¼ 0.366, 0.155, 0.083 and 0.009
mM, respectively). This implied
unsubstituted aromatic groups on P10 phenylalanine linker was not
fit to fill in S10 active pocket, likely owing to the steric hindrance of
these aromatic substituents with the amino acids at the entrance of
the S10active pocket.
chlorine and hydroxyl groups on 2-phenyl-1H-imidazole moiety
were critical for improving FXIa binding affinity. Hence, compound
28h was selected for further optimization by exploring structural
modifications of P10 and P20 groups.
2.2. Discovery of compound 44g
2.3. Serine protease selectivity
We next explored the modification of P20 groups based on
compound 28h. Inserting a carbon atom between the carboxyl
group and phenyl ring gave compound 34a, which was 1.71-fold
more potent than 28h against FXIa (34a > 28h, Ki ¼ 6.86 and
To investigate the selectivity of the most potent compound 44g,
serine protease selectivity was further evaluated against other
serine proteases including factor VIIa (FVIIa), factor Xa (FXa),
thrombin and plasma kallikrein in the coagulation cascade. In
Table 5, compound 44g exhibited >400-fold selectivity against
FVIIa, FXa and thrombin, except for plasma kallikrein (21.3-fold).
11.76
mM, respectively, Table 3), suggesting that carboxyl group
closer to the S20 active pocket might be beneficial for FXIa binding
affinity. Subsequently, in order to decrease the distance between
the carboxyl group and key residues of S20 pocket, the phenyl ring
of P20 group was replaced with an indole ring to afford compounds
34b (Table 3), and results revealed that the addition of a carboxyl
group to 2-position on the indole ring provided a 4.17-fold
improvement in FXIa binding affinity in comparison with com-
2.4. Molecular modeling
To explore a probable binding model of inhibitors and FXIa
active site, molecular docking of the representative compound 44g
in the substrate binding pocket of FXIa was performed using the
€
pound 28h (34b > 28h, Ki ¼ 2.82 and 11.76
m
M, respectively).
Glide XP docking protocol (2016, Schrodinger Suite) [39], and BMS-
Therefore, compound 34b was selected for further optimization to
investigate the FXIa binding affinity.
724296 was used as a reference [2]. Co-crystallized ligand BMS-
724296 was redocked using the Glide XP docking protocol. The
results suggested that the key interactions between ligand and key
residues of active pocket were consistent with that of crystallo-
graphic bound ligand (Fig. 5A and B), which verified that the
docking model was reliable.
Based on the crystal structure of inhibitor 7, it was found that
there was a large space in P10 prime region to accommodate sub-
stitutions at para-position of P10 phenyl moiety. Therefore, intro-
duction of various substitutions at the para-position of P10 phenyl
5

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
Table 4
The effect of para-substituents at P10 phenyl moiety on FXIa binding affinity.
a
Compounds
R6
-F
Ki (
m
M)
Compounds
R6
Ki (m
M) a
39a
0.486
0.356
0.962
7.32
44f
0.259
39b
39c
39d
-Cl
-Br
44g
44h
44i
0.009
0.0225
0.0339
-NO2
39e
44a
44b
44c
44d
44e
-NH2
3.08
44j
44k
0.0563
0.366
0.392
0.156
0.026
0.053
0.031
44l
0.155
44m
0.0813
0.0015
BMS-724296
-
a
Ki values were derived from IC50 values using Cheng Prussof transformation [37,38].
6

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
Table 5
Human serine protease selectivity profile for compound 44g.
Human enzyme (Ki)
44g (nM)a
FXIa
9.0
FVIIa
4193
FXa
>8000
>12,500
191.8
Thrombin
Plasma kallikrein
a
Ki values were derived from IC50 values using Cheng
Prussof transformation [37,38].
In the P1 prime region (Fig. 5A, C), 2-phenyl-1H-imidazole
scaffold as a P1 group could bind deep into the S1 active pocket.
Specifically, the carbonyl oxygen of 2-phenyl-1H-imidazole-5-
amide moiety was positioned over the oxyanion hole formed by
Gly193, Asp194 and Ser195 to participate in two key hydrogen
bonds with the backbone amino groups of Gly193 and Ser195,
which was consistent with that of the carbonyl oxygen atom in
BMS-724296 [2]. Moreover, the hydroxyl group of imidazole moi-
ety could engage in a hydrogen bond with backbone carbonyl of
Cys191. Interestingly, two halogen interactions were observed be-
tween the fluorine atom of P1 phenyl moiety and Ser214 and
Trp215 [40]. Consequently, the above results suggested that 2-
phenyl-1H-imidazole scaffold of WSJ-557 could be used as P1
fragment to design novel FXIa inhibitor. In the P10 prime region
(Fig. 5C), phenyl ring of P10 phenylalanine linker occupied on the
entrance of S10 active pocket and interacted with Cys42 through a
Fig. 6. Lineweaver-Burk plots of FXIa inhibition by compound 44g.
0.005e0.04
mM of compound 44 g were 45.54, 60.28, 89.85 and
128.25 M, respectively, and Vmax was a constant value of 0.17
m
mM/
min. This behavior revealed that 44g acted as a competitive-type
inhibitor with respect to FXIa. In addition, dose-dependent inhi-
bition of FXIa by 44g was exhibited (Fig. 7) [41].
2.6. In vivo efficacy in the rabbit arteriovenous shunt model
To determine its antithrombotic effect, compound 44g was next
performed in the rabbit arteriovenous shunt (AV shunt) thrombosis
p
-sulfur interaction. In the deepest part of the P10 active pocket, the
amide carbonyl group on the phenyl ring of P10 formed an addi-
tional hydrogen bond with the guanidine group of Arg39. In the P20
prime region (Fig. 5C), the nitrogen atom of the indole moiety
formed a hydrogen bond with the side chain of Tyr143. Further-
more, carboxyl group interacted with the side chain of Lys192 via a
strong electrostatic interaction.
2.5. Steady-state kinetic analysis
To investigate the action mode of the target compounds with
FXIa, enzyme kinetics studies of the representative compound 44g
were performed. As shown in Fig. 6, the analysis on the data of
compound 44g indicated that Vmax remained unchanged with
changing slope (Km/Vmax) in the presence of increasing concen-
trations of the inhibitor, where the Km values in the presence of
Fig. 7. The inhibition of FXIa by compound 44g. Values are means SDs, n ¼ 3.
Fig. 5. Binding modes of compounds 44g and BMS-724296 within the FXIa binding pocket. (A) Superposition of compound 44g (orange), re-docked BMS-724296 (magenta) and
crystallographic ligand BMS-724296 (cyan) occupy the same binding site in FXIa. The surface of the protein (PDB code 4TY7) is shown in grey, and small molecules are shown as a
line. (B) FXIa residues interacting with re-docked BMS-724296 are depicted by green lines. Hydrogen bonds and
p-
p
stacking interactions are shown as red and magentas dashed
lines, respectively. (C) FXIa residues interacting with compound 44g are depicted by green lines. Hydrogen bonds, p-p stacking interactions, electrostatic interactions and halogen
interactions are shown as red, magenta, black and cyan dashed lines, respectively.
7

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
8.0 mg kgꢀ1$ hꢀ1, and inhibitions of thrombus weight were 26.80%,
42.67% and 51.04%, respectively, which suggested that compound
44g caused a dose-dependent reduction of thrombus weight with
an ED50 of 4.7 mg/kg þ 7.5 mg kgꢀ1$ hꢀ1. Specifically, at the highest
dose (5.0 mg/kg þ 8.0 mg kgꢀ1$ hꢀ1), antithrombotic effect of
compound 44g was compared to that of BMS-724296 (0.5 mg/
kg þ0.8 mg kgꢀ1$ hꢀ1). The results of in vivo efficacy in the rabbit
arteriovenous shunt model suggested that compound 44 g was a
potential and efficacious agent in the treatment of thrombotic
diseases.
Fig. 8. Antithrombotic effect of compounds 44g and BMS-724296 in the rabbit AV-
shunt model. Data are mean values SD (n ¼ 3 per group).
2.7. Chemistry
model (Fig. 8), and BMS-724296 was used as a positive control. At a
The syntheses of compounds 16a-j and 17 were shown in
Scheme 1. The key intermediate ethyl 2-hydroxyimino-3-
oxobutanoate 13 was obtained by nitrosation of the commercially
available ethyl 3-oxobutanoate 12 with sodium nitrite in acetic acid
[31], which was cyclized with the corresponding benzaldehyde
derivatives 14a-j to give the intermediates 15a-j [31]. Then, com-
pounds 16a-j were prepared through hydrolysis of intermediates
single intravenous (IV) dose of 0.5 mg/kg þ 0.8 mg kgꢀ1$ hꢀ1
,
positive control BMS-724296 could markedly reduce thrombus
weight, suggesting that the model was successfully established.
Moreover, compound 44g was given as a single loading dose fol-
lowed by continuous IV infusion at three doses: 0.5 mg/kg þ
0.8 mg kgꢀ1$ hꢀ1, 2.5 mg/kg þ 4.0 mg kgꢀ1$ hꢀ1, 5.0 mg/kg þ
Scheme 1. Reagents and conditions: (a) NaNO2, AcOH, 0ꢁС; (b) 13, CH3COOH/CH3COONH4, 50ꢁС; (c) LiOH, THF, H2O, 50ꢁС; (d) H2, Pd/C, Methanol, 25ꢁС.
Scheme 2. Reagents and conditions: (a) Me3SiCl, NaI, CH3CN, reflux; (b) MeI, K2CO3, N2, DMF, 25ꢁС; (c) LiOH, THF, H2O, 50ꢁС; (d) Me2SO4, K2CO3, DMF, 0ꢁС.
8

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
Scheme 3. Reagents and conditions: (a) HATU, DMF, DIPEA, methyl 4-aminobenzoate, 25ꢁС; (b) 4 M HCl in ethyl acetate; (c) HATU, DMF, DIPEA, 16a-j, 20, 22 or 23, 25ꢁС; (d) LiOH,
THF, H2O, 50ꢁС.
Scheme 4. Reagents and conditions: (a) THF, 10% Pd/C, HCOONH4, 60ꢁС; (b) HATU, DMF, DIPEA, amines, 25ꢁС; (c) 4 M HCl in ethyl acetate; (d) HATU, DMF, DIPEA, 16j, 25ꢁС; (e)
LiOH, THF, H2O, 50ꢁС.
15a-j using an aqueous solution of lithium hydroxide, among them,
the benzyl group of compound 16b was removed in H2 and Pd/C to
provide compound 17.
hydrolysis of the esters 27a-k offered target compounds 28a-k in
an aqueous solution of lithium hydroxide with a good yield.
The syntheses of compounds 34a-b were described in Scheme 4.
Ethyl 5-amino-1H-indole-2-carboxylate 30 was prepared by
reducing the nitro group of ethyl 5-nitro-1H-indole-2-carboxylate
29 catalyzed by Pd/C in HCOONH4 and THF. Following the same
procedure as described in Scheme 3, condensation of (tert-butox-
The syntheses of compounds 20, 22 and 23 were shown in
Scheme 2. With compound 15j as a starting material, removal of a
hydroxyl group in chlorotrimethylsilane and sodium iodide affor-
ded intermediate 18 [31], and insertion of a methyl group offered
intermediate 21 by alkylation reaction with Me2SO4 in K2CO3 and
DMF. Then, intermediate 19 was prepared through the alkylation
reaction of the resulting intermediate 18 with iodomethane in DMF
in the presence of K2CO3. Finally, compounds 20, 22 and 23 were
obtained by hydrolyzing intermediates 18, 19 and 21 in an aqueous
solution of lithium hydroxide.
Syntheses of compounds 28a-k were shown in Scheme 3.
Commercially available starting material 24 was activated with
HATU as a coupling reagent and then reacted with methyl 4-
aminobenzoate to yield intermediate 25 [42], which further pro-
vided intermediate 26 through removing the Boc protected group
[2]. Then, the resulting intermediate 26 was treated with carboxylic
acids 16a-j, 20, 22 and 23 to give intermediates 27a-k. Subsequent
ycarbonyl)- -phenylalanine 24 with 30, ethyl 2-(4-aminophenyl)
L
acetate or methyl 5-amino-1H-indole-3-carboxylate gave in-
termediates 31a-b in the presence of HATU and DIPEA. Then,
removal of Boc protected groups in aqueous hydrochloric acid
afforded 32a-b, and amino groups of 32a-b followed by conden-
sation with intermediate 16j provided intermediates 33a-b, which
were followed by hydrolysis reactions using an aqueous solution of
lithium hydroxide to give compounds 34a-b.
The compounds 39a-e were prepared as shown in Scheme 5.
Reaction of commercially available starting materials 35a-d with 30
afforded compounds 36a-d in the same manner as Schemes 3 and
4. Then, deprotection of Boc group in aqueous 4 M HCl in ethyl
acetate yielded compounds 37a-d, which further reacted with
9

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
Scheme 5. Reagents and conditions: (a) HATU, DMF, DIPEA, 30, 25ꢁС; (b) 4 M HCl in ethyl acetate; (c) HATU, DMF, DIPEA, 16j, 25ꢁС; (d) LiOH, THF, H2O, 50ꢁС; (e) 5% Pt/C, Ethanol,
H2, 25ꢁС.
Scheme 6. Reagents and conditions: (a) 5% Pt/C, EtOH, H2, 25ꢁС; (b) HATU, DMF, DIPEA, carboxylic acid derivatives, 25ꢁС; (c) HCl in ethyl acetate; (d) HATU, DMF, DIPEA, 16j, 25ꢁС;
(e) LiOH, THF, H2O, 50ꢁС.
intermediate 16j by condensation reaction to give compounds 38a-
d. Among them, compound 38d was converted into 38e by
reducing the nitro group under a hydrogen atmosphere at room
temperature catalyzed by Pd/C in ethanol. The resulting com-
pounds 38a-e were subsequently hydrolyzed to provide com-
pounds 39a-e.
The compounds 44a-m were prepared in five steps from inter-
mediate 36d as shown in Scheme 6. Reduction of intermediate 36d
gave compound 40, followed by condensation with carboxylic acid
derivatives which afforded compounds 41a-m in the presence of
HATU and DIPEA. Deprotection of compounds 41a-m with an
aqueous 4 M HCl provided compounds 42a-m, followed by
condensation with intermediate 16j to offer compounds 43a-m.
Subsequent hydrolysis of the resulting compounds 43a-m afforded
compounds 44a-m in the same method as Schemes 1e5.
3. Conclusion
Based on our previously reported nonpurine XO inhibitor WSJ-
557 with an apparent anticoagulation potency, structural modifi-
cation was employed, which resulted in the discovery of hit 16e
(Ki ¼ 120
mM) under the guide of molecular modeling. Subse-
quently, we adopted molecular hybridization strategy to introduce
P10 phenylalanine and P20 p-aminobenzoic acid fragments into the
carbonyl group of hit 16e to afford lead compound 28a
(Ki ¼ 23.88
mM), which was further structurally optimized to lead to
10

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
identification of the most potent compound 44g (Ki ¼ 0.009
m
M),
99.8e101.2ꢁС. ESI-MS (m/z) ¼ 281.07 [M þH] þ; 1H NMR (400 MHz,
close to that of BMS-724296 (Ki ¼ 0.0015
m
M). Moreover, molecular
DMSO‑d6)
d
11.75 (s, 1H), 7.60 (dt, J ¼ 9.2, 1.3 Hz, 1H), 7.57e7.49 (m,
modeling provided the molecular basis for rationalizing the bind-
ing affinity of the designed compounds and suggested that 2-
phenyl-1H-imidazole-5-carboxamide moiety could be used as a
P1 fragment. In addition, the result of serine protease selectivity
indicated that the representative compound 44g displayed a
desired selectivity against thrombin, FVIIa, FXa and plasma kalli-
krein in the coagulation cascade. Furthermore, in vivo efficacy in the
rabbit AV shunt model suggested that compound 44g demon-
strated dose-dependent antithrombotic efficacy. Therefore, these
results supported that compound 44g could be a potential and
efficacious agent for treatment of thrombotic diseases. The in-
vestigations performed in this research confirmed the possibility of
nonpurine imidazole XO inhibitor WSJ-557 as a P1 fragment to
design novel FXIa inhibitors.
2H), 7.46 (dd, J ¼ 8.0, 1.6 Hz, 1H), 4.29 (q, J ¼ 7.1 Hz, 2H), 2.39 (s, 3H),
1.32 (t, J ¼ 7.1 Hz, 3H).
4.1.4. Synthesis of ethyl 2-(3-chloro-2-fluorophenyl)-1-hydroxy-4-
methyl-1H-imidazole-5-carboxylate (15j)
Compound 15j was prepared from the compound 14j in the
same manner as described for 15g. A white solid, yield: 80.6%. Mp
þ
137.8e139.1 ꢁC. ESI-MS (m/z) ¼ 299.09 [M þ H]
;
1H NMR
(400 MHz, DMSO‑d6)
d 12.01 (s, 1H), 7.79e7.69 (m, 1H), 7.62 (dd,
J ¼ 10.2, 3.8 Hz, 1H), 7.36 (td, J ¼ 8.0, 0.7 Hz, 1H), 4.30 (q, J ¼ 7.1 Hz,
2H), 2.39 (s, 3H), 1.32 (t, J ¼ 7.1 Hz, 3H).
4.1.5. Synthesis of 2-(4-cyanophenyl)-1-hydroxy-4-methyl-1H-
imidazole-5-carboxylic acid (16c)
A mixture of ethyl 2-(4-cyanophenyl)-1-hydroxy-4-methyl-1H-
4. Experimental protocols
imidazole-5-carboxylate 15c (2.9 mmol), 1 M LiOH aqueous
(11 mL), THF (5 mL), and ethanol (5 mL) was stirred at 50 ꢁC for 6 h.
The solvent was concentrated in a vacuum, and the residue was
acidified with dilute hydrochloric acid to pH 1. The resulting pre-
cipitate was filtered, dried, and recrystallized with a mixture of
methanol and ethyl acetate (2:1) to yield the corresponding 2-(4-
cyanophenyl)-1-hydroxy-4-methyl-1H-imidazole-5-carboxylic
acid 16c as a white solid, yield: 82.3%. Mp 221.3e222.8ꢁС. ESI-MS
4.1. Chemistry
Compounds WSJ-557, 13, 15a-f, 16a-b and 17 were prepared
from our previous report [31]. In addition, positive control BMS-
724296 was synthesized by the reported literature [2], and com-
pounds 12, 14a-j, 24, 29 and 35a were commercially available. Re-
agents and solvents were purchased from commercial sources and
used without further purification. All reactions were monitored by
TLC using silica gel aluminum cards (0.2 mm thickness) with
254 nm and 365 nm fluorescent indicator. Melting points were
obtained using a YRT-3 melting apparatus and were uncorrected.
1H NMR spectra were recorded on a Bruker 400 or 600 MHz
spectrometer, and 13C NMR spectra were recorded on a Bruker 400
or 600 MHz spectrometer. Chemical shifts were expressed in parts
per million using tetramethylsilane as an internal reference and
DMSO‑d6 as the solvent. ESI-MS data were gathered using an Agi-
lent 1100 instrument and ESI-HRMS data were recorded in the
Agilent 6540 Series Q-TOF-MS system.
(m/z) ¼ 244.06 [M þ H] þ;1H NMR (400 MHz, DMSO‑d6)
(d, J ¼ 8.6 Hz, 2H), 7.95 (d, J ¼ 8.5 Hz, 2H), 2.39 (s, 3H).
d 8.24
4.1.6. Synthesis of 1-hydroxy-4-methyl-2-phenyl-1H-imidazole-5-
carboxylic acid (16d)
Compound 16d was prepared from the compound 15d in the
same manner as described for 16c. A white solid, yield: 89.2%. Mp
196.6e198.1ꢁС. ESI-MS (m/z) ¼ 217.11 [M ꢀ H] -; 1H NMR (400 MHz,
DMSO‑d6)
2.1 Hz, 1H), 2.52 (s, 3H).
d
8.30e8.24 (m, 2H), 7.62e7.56 (m, 2H), 7.54 (dt, J ¼ 5.2,
4.1.7. Synthesis of 2-(3-cyanophenyl)-1-hydroxy-4-methyl-1H-
imidazole-5-carboxylic acid(16e)
4.1.1. Synthesis of ethyl 2-(4-chlorophenyl)-1-hydroxy-4-methyl-
1H-imidazole-5-carboxylate (15g)
Compound 16e was prepared from the compound 15e in the
same manner as described for 16c. A white solid, yield: 69.7%. Mp
þ
A mixture of 4-chlorobenzaldehyde 14g (32.1 g, 0.229 mol),
ethyl 2-hydroxyimino-3-oxobutanoate 13 (43.7 g, 0.285 mol),
ammonium acetate (176.3 g, 2.29 mol), and acetic acid (600 mL)
was stirred at 50 ꢁC under nitrogen atmosphere for 24 h. The re-
action mixture was cooled to room temperature and then slowly
poured into cold water (2000 mL). The resulting precipitate was
filtered, dried, and washed with ethyl acetate to obtain the com-
pound 15g as a white solid, yield: 87.1%. Mp 147.5e148.7 ꢁC. ESI-MS
227.5e228.8ꢁС. ESI-MS (m/z) ¼ 244.07 [M þH]
;
1H NMR
(400 MHz, DMSO‑d6)
d
8.84 (s, 1H), 8.57 (d, J ¼ 8.1 Hz, 1H), 7.97 (t,
J ¼ 7.8 Hz, 1H), 7.78 (t, J ¼ 7.9 Hz, 1H), 2.53 (s, 3H).
4.1.8. Synthesis of 2-(2-cyanophenyl)-1-hydroxy-4-methyl-1H-
imidazole-5-carboxylic acid(16f)
Compound 16f was prepared from the compound 15f in the
same manner as described for 16c. A white solid, yielþd: 77.8%. Mp
;
1H NMR
(m/z) ¼ 281.11 [M þH] þ; 1H NMR (400 MHz, DMSO‑d6)
d
8.35e7.89
181.2e182.7ꢁС. ESI-MS (m/z)
¼
244.05 [M þH]
(m, 2H), 7.72e7.23 (m, 2H), 4.31e4.26 (m, 2H), 2.37 (s, 3H), 1.31 (t,
J ¼ 7.1 Hz, 3H).
(400 MHz, DMSO‑d6)
0.9 Hz, 1H), 7.64 (td, J ¼ 7.8, 1.7 Hz, 1H), 7.57e7.52 (m, 1H), 2.52 (s,
d
7.77 (dd, J ¼ 7.6, 1.4 Hz, 1H), 7.70 (dd, J ¼ 8.0,
3H).
4.1.2. Synthesis of ethyl 2-(3-chlorophenyl)-1-hydroxy-4-methyl-
1H-imidazole-5-carboxylate (15h)
4.1.9. Synthesis of 2-(4-chlorophenyl)-1-hydroxy-4-methyl-1H-
imidazole-5-carboxylic acid (16g)
Compound 15h was prepared from the compound 14h in the
same manner as described for 15g. A wihte sþolid, yield: 81.8%. Mp
144.2e145.4ꢁС. ESI-MS (m/z) ¼ 281.07 [MþH] ; 1H NMR (400 MHz,
Compound 16g was prepared from the compound 15g in the
same manner as described for 16c. A white solid, yield: 88.3%. Mp
þ
DMSO‑d6)
d
12.32 (s, 1H), 8.08 (s, 1H), 8.05e7.98 (m, 1H), 7.51 (dd,
219.7e221.2ꢁС. ESI-MS (m/z) ¼ 253.05 [M þH]
;
1H NMR
J ¼ 3.7, 2.0 Hz, 2H), 4.28 (q, J ¼ 7.1 Hz, 2H), 2.21 (s, 3H), 1.31 (t,
(400 MHz, DMSO‑d6)
d
8.32 (d, J ¼ 8.6 Hz, 2H), 7.66 (d, J ¼ 8.6 Hz,
J ¼ 7.1 Hz, 3H).
2H), 2.52 (s, 3H).
4.1.3. Synthesis of ethyl 2-(2-chlorophenyl)-1-hydroxy-4-methyl-
1H-imidazole-5-carboxylate (15i)
4.1.10. Synthesis of 2-(3-chlorophenyl)-1-hydroxy-4-methyl-1H-
imidazole-5-carboxylic acid (16h)
Compound 15i was prepared from the compound 14i in the
same manner as described for 15g. A yellow solid, yield: 77.6%. Mp
Compound 16h was prepared from the compound 15h in the
same manner as described for 16c. A white solid, yield: 72.4%. Mp
11

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
þ
229.4e231.3ꢁС. ESI-MS (m/z) ¼ 253.05 [M þH]
;
1H NMR
4.1.18. Synthesis of 2-(3-chloro-2-fluorophenyl)-4-methyl-1H-
imidazole-5-carboxylic acid (23)
Compound 23 was prepared from the compound 18 in the same
manner as described for 16c, and it was used for the next reaction
without further purification.
(400 MHz, DMSO‑d6)
d
8.41 (d, J ¼ 1.8 Hz, 1H), 8.26 (dt, J ¼ 7.3,
1.7 Hz, 1H), 7.76e7.50 (m, 2H), 2.53 (s, 3H).
4.1.11. Synthesis of 2-(2-chlorophenyl)-1-hydroxy-4-methyl-1H-
imidazole-5-carboxylic acid (16i)
4.1.19. Synthesis of methyl (S)-4-{2-[(tert-butoxycarbonyl)amino]-
3-phenylpropanamido}benzoate (25)
Compound 16i was prepared from the compound 15i in the
same manner as described for 16c. A white solid, yield: 73.6%. Mp
243.2e244.6ꢁС. ESI-MS (m/z) ¼ 251.12 [M ꢀ H] þ;1H NMR
A solution of (tert-butoxycarbonyl)- -phenylalanine 24 (10.0 g,
L
37.7 mmol), methyl 4-aminobenzoate (5.7 g, 37.7 mmol), DIPEA
(5.85 g, 45.24 mmol) and HATU (17.2 g, 45.24 mmol) in DMF
(100 mL) was stirred at 25ꢁС for 4 h. The reaction mixture was
poured into 150 mL ethyl acetate and 100 mL water. The organics
were washed with 1 M NaOH (100 mL), 1 M HCl (100 mL) and brine
(100 mL), dried (Na2SO4), and concentrated to afford 25 as a yellow
solid, yield: 77.3%. Mp 114.6e116.3ꢁС. ESI-MS (m/z) ¼ 421.29 [M þ
(400 MHz, DMSO‑d6)
d
7.77 (dd, J ¼ 7.6, 1.5 Hz, 1H), 7.70 (dd, J ¼ 8.0,
1.0 Hz, 1H), 7.64 (td, J ¼ 7.8, 1.7 Hz, 1H), 7.55 (td, J ¼ 7.5, 1.1 Hz, 1H),
2.52 (s, 3H).
4.1.12. Synthesis of 2-(3-chloro-2-fluorophenyl)-1-hydroxy-4-
methyl-1H-imidazole-5-carboxylic acid (16j)
Compound 16j was prepared from the compound 15j in the
þ
Na]
; d 10.39 (s, 1H), 7.93 (d,
1H NMR (400 MHz, DMSO‑d6)
same manner as described for 16c. A white solid, yield: 87.6%. Mp
J ¼ 8.6 Hz, 2H), 7.74 (d, J ¼ 8.7 Hz, 2H), 7.45e7.07 (m, 6H), 4.36 (d,
J ¼ 9.4 Hz, 1H), 3.83 (s, 3H), 2.98 (d, J ¼ 4.2 Hz, 1H), 2.87 (d,
J ¼ 10.2 Hz, 1H), 1.32 (s, 9H).
þ
233.0e234.8ꢁС. ESI-MS (m/z) ¼ 271.04 [M þH]
;
1H NMR
(400 MHz, DMSO‑d6)
d
8.09 (t, J ¼ 6.8 Hz, 1H), 7.92e7.66 (m, 1H),
7.45 (t, J ¼ 8.0 Hz, 1H), 2.53 (s, 3H).
4.1.20. Synthesis of methyl (S)-4-(2-amino-3-phenylpropanamido)
benzoate (26)
4.1.13. Synthesis of ethyl 2-(3-chloro-2-fluorophenyl)-4-methyl-
1H-imidazole-5-carboxylate (18)
Methyl
(S)-4-{2-[(tert-butoxycarbonyl)amino]-3-
A suspension of compound 15j (2.98 g, 10 mmol), potassium
iodide (1.16 g, 10 mmol), chlorotrimethylsilane (1.63 g, 15 mmol)
and acetonitrile (30 mL) was stirred at 60ꢁС for 6 h. The reaction
mixture was poured into a solution of 1 M sodium hydroxide
aqueous and was stirred for 1 h. The precipitate was filtered,
washed with ethyl acetate to obtain compound 18 as a white solid.
phenylpropanamido}benzoate 25 was added into a solution of
4 M HCl in ethyl acetate, and stirred in 25ꢁС. The resulting mixture
was filtered and precipitate was washed with 20 mL ethyl acetate,
dried to afford 26 as a white solid, yield: 84.3%. Mp 127.4e128.8ꢁС.
ESI-MS (m/z) ¼ 299.20 [M þ H]þ; 1H NMR (400 MHz, DMSO‑d6)
d
11.27 (s, 1H), 8.45 (s, 3H), 7.93e7.91 (d, J ¼ 8.4 Hz, 2H), 7.73e7.71
Yield: 67.8%. 1H NMR (400 MHz, DMSO‑d6)
d
7.91 (t, J ¼ 6.8 Hz, 1H),
(d, J ¼ 8.8 Hz, 2H), 7.32e7.23 (m, 5H), 4.34 (m, 1H), 3.81 (s, 3H),
7.57 (t, J ¼ 6.9 Hz, 1H), 7.29 (t, J ¼ 7.9 Hz, 1H), 4.24 (q, J ¼ 7.1 Hz, 2H),
3.24e3.12 (m, 2H).
2.49 (s, 3H), 1.29 (t, J ¼ 7.1 Hz, 3H). ESI-MS (m/z) ¼ 281.11[M ꢀ H]-.
4.1.21. General procedure for synthesis of methyl (S)-4-[2-(2-
phenyl-1-hydroxy-4-methyl-1H-imidazole-5-carboxamido)-3-
phenylpropanamido]benzoate derivatives (27a-27k)
4.1.14. Synthesis of ethyl 2-(3-chloro-2-fluorophenyl)-1,4-dimethyl-
1H-imidazole-5-carboxylate (19)
A 100 mL flask was charged with 5 mL of DMF, 10 mmol of the
compounds 16a-j, 20, 22 and 23, DIPEA (0.15 g, 12 mmol) and HATU
(0.46 g, 12 mmol). The complex was stirred for 24 h, and the re-
action mixture was poured into 20 mL ethyl acetate and 20 mL
water. Then, the organics were washed with 1 M NaOH (20 mL),1 M
HCl (20 mL) and brine (20 mL), dried (Na2SO4), and concentrated to
give the compounds 27a-27k, which were used for the next reac-
tion without further purification.
A solution of compound 18 (0.56 g, 2.0 mmol), anhydrous po-
tassium carbonate (0.33 g, 2.40 mmol) and iodomethane (0.33 g,
2.4 mmol) in DMF (5.4 mL) was stirred at 35 ꢁC under nitrogen
atmosphere for 1 h. After the completion of the reaction, the re-
action mixture was poured into 11 mL water and stirred for 10 min.
The precipitate was filtered and washed with water, then ethyl
acetate (5.0 mL) was added to wash residue to yield compound 19
as a white solid, which was used for the next reaction without
further purification.
4.1.22. General procedure for synthesis of (S)-4-[2-(2-phenyl-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido)-3-
phenylpropanamido]benzoic acids derivatives (28a-28k)
4.1.15. Synthesis of 2-(3-chloro-2-fluorophenyl)-1,4-dimethyl-1H-
imidazole-5-carboxylic acid (20)
A 100 mL flask was charged with 5 mL of DMF, 10 mmol of the
compounds 16a-j, 20, 22 and 23, DIPEA (0.15 g, 12 mmol) and HATU
(0.46 g, 12 mmol). The complex was stirred for 24 h, and the reaction
mixture was poured into 20 mL ethyl acetate and 20 mL water. Then,
the organics were washed with 1 M NaOH (20 mL), 1 M HCl (20 mL)
and brine (20 mL), dried (Na2SO4), and concentrated to give the
compounds 27a-27k, which were used for the next reaction without
further purification. A mixture of the crude materials 27a-27k, 1 M
LiOH aqueous (10 mL) and methanol (10 mL) was stirred at 40 ꢁC
until the material spot disappeared by TLC. The mixture was washed
with tert-butyl methyl ether (10 mL), and water phase was acidified
with dilute hydrochloric acid to pH 3e4, filtered dried and purified
by silica gel chromatography with dichloromethane/methanol (10:
1) to afford the corresponding 2-[4-alkoxy-3-(1H-tetrazol-1-yl)
phenyl]-6-oxo-1, 6-dihydropyrimidine-5-carboxylic acids (S)-4-[2-
(2-phenyl-1-hydroxy-4-methyl-1H-imidazole-5-carboxamido)-3-
phenyl propanamido] benzoic acids derivatives 28a-28k.
Compound 20 was prepared from the compound 19 in the same
manner as described for 16c, and it was used for the next reaction
without further purification.
4.1.16. Synthesis of ethyl 2-(3-chloro-2-fluorophenyl)-1-methoxy-
4-methyl-1H-imidazole-5-carboxylate (21)
Compound 21 was prepared from the compound 15j in the same
manner as described for 19, and it was used for the next reaction
without further purification.
4.1.17. Synthesis of 2-(3-chloro-2-fluorophenyl)-1-methoxy-4-
methyl-1H-imidazole-5-carboxylic acid (22)
Compound 22 was prepared from the compound 21 in the same
manner as described for 16c. Yield: 91.2%. 1H NMR (400 MHz,
DMSO‑d6)
d
8.09 (t, J ¼ 6.8 Hz, 1H), 7.92e7.66 (m, 1H), 7.45 (t,
J ¼ 8.0 Hz, 1H), 2.53 (s, 3H). ESI-MS (m/z) ¼ 285.09[M þ H]þ.
12

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
4.1.23. Synthesis of (S)-4-{2-[2-(3-cyanophenyl)-1-hydroxy-4-
methyl-1H-imidazole-5-carboxamido]-3-phenylpropanamido}
benzoic acid (28a)
2.21 (s, 3H). 13C NMR (100 MHz, DMSO)
d171.15, 166.28, 159.33,
143.69, 140.50, 138.17, 137.83, 134.44, 132.66, 130.82, 130.74, 129.69,
129.18, 128.73, 127.08, 124.68, 120.73, 119.23, 55.98, 37.52, 15.10.
A white solid, yield: 15.45%. Mp 152.1e153.8 ꢁC. ESI-HRMS calcd
for C28H23N5O5 [M ꢀ OH]- 492.1677 found: 492.1671; 1H NMR
4.1.28. Synthesis of (S)-4-{2-[2-(3-chlorophenyl)-1-hydroxy-4-
methyl-1H-imidazole-5-carboxamido]-3-phenylpropanamido}
benzoic acid (28f)
(400 MHz, DMSO‑d6)
d
10.68 (s, 1H), 8.45 (d, J ¼ 7.7 Hz, 1H), 8.06
(dd, J ¼ 3.5, 1.8 Hz, 2H), 7.96 (d, J ¼ 8.7 Hz, 2H), 7.87 (d, J ¼ 7.8 Hz,
1H), 7.79 (d, J ¼ 8.8 Hz, 2H), 7.64 (t, J ¼ 8.1 Hz, 1H), 7.29 (dd, J ¼ 13.5,
7.2 Hz, 3H), 7.16 (t, J ¼ 7.6 Hz, 2H), 4.94 (dd, J ¼ 14.6, 9.8 Hz,1H), 3.24
A white solid, yield: 24.46%. Mp135.8e136.9 ꢁC. ESI-HRMS calcd
for C27H23ClN4O5 [M þ Na]þ 541.1249 found: 541.1240; 1H NMR
(d, J ¼ 4.5 Hz, 1H), 3.11 (dd, J ¼ 13.8, 9.9 Hz, 1H), 2.23 (s, 3H). 13
C
(400 MHz, DMSO‑d6)
d
10.67 (s, 1H), 8.42 (d, J ¼ 7.3 Hz, 1H), 7.96 (d,
NMR (150 MHz, DMSO‑d6)
d
171.11, 166.28, 159.21, 143.67, 138.40,
J ¼ 8.8 Hz, 2H), 7.80 (dd, J ¼ 8.2, 5.5 Hz, 4H), 7.50 (d, J ¼ 4.5 Hz, 2H),
7.20 (t, J ¼ 7.6 Hz, 3H), 7.38 (d, J ¼ 7.2 Hz, 2H), 4.98 (d, J ¼ 9.9 Hz,1H),
3.26 (s, 1H), 3.10 (s, 1H), 2.22 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
137.84, 133.00, 132.53, 132.10, 130.84, 130.81, 130.34, 129.97, 129.71,
129.47, 128.72, 127.08, 124.71, 121.03, 119.24, 112.17, 55.93, 37.55,
15.07.
d171.12,166.28,159.27,143.68,140.00,138.22,137.83,133.75,132.61,
131.02, 130.76, 130.23, 129.93, 129.70, 128.71, 127.03, 126.15, 124.70,
120.92, 119.24, 55.96, 37.53, 15.08.
4.1.24. Synthesis of (S)-4-{2-[2-(2-cyanophenyl)-1-hydroxy-4-
methyl-1H-imidazole-5-carboxamido]-3-phenylpropanamido}
benzoic acid (28b)
4.1.29. Synthesis of (S)-4-{2-[2-(2-chlorophenyl)-1-hydroxy-4-
methyl-1H-imidazole-5-carboxamido]-3-phenylpropanamido}
benzoic acid (28g)
A white solid, yield: 18.18%. Mp 100.4e101.3 ꢁC. ESI-HRMS calcd
for C28H23N5O5 [M ꢀ OH]- 492.1677 found: 492.1685; 1H NMR
(400 MHz, DMSO‑d6)
d
10.70 (s, 1H), 8.21 (d, J ¼ 3.9 Hz, 2H), 7.92 (d,
A white solid, yield: 17.44%. Mp.98.1e99.2 ꢁC. ESI-HRMS calcd
for C27H23ClN4O5 [M þ Na]þ 541.1249 found: 541.1253; 1H NMR
J ¼ 8.6 Hz, 2H), 7.77 (d, J ¼ 8.7 Hz, 2H), 7.69e7.53 (m, 3H), 7.45 (d,
J ¼ 7.4 Hz, 2H), 7.30 (t, J ¼ 7.5 Hz, 2H), 7.21 (t, J ¼ 7.3 Hz, 1H),
4.97e4.83 (m, 1H), 3.24 (dd, J ¼ 13.7, 4.8 Hz, 1H), 3.15 (dd, J ¼ 13.6,
(400 MHz, DMSO‑d6)
d
10.66 (s, 1H), 8.29 (d, J ¼ 7.6 Hz, 1H),
8.01e7.83 (m, 2H), 7.82e7.73 (m, 2H), 7.58 (dt, J ¼ 6.6, 5.0 Hz,2H),
7.47e7.39 (m, 2H), 7.31e7.23 (m, 3H), 7.16 (t, J ¼ 7.6 Hz, 2H),
5.03e4.92 (m, 1H), 3.28e3.16 (m, 1H), 3.09 (dd, J ¼ 13.8, 9.5 Hz, 1H),
9.7 Hz, 1H), 2.49 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
d170.82,
167.35, 164.09, 159.65, 143.45, 143.31, 137.93, 135.13, 134.78, 132.01,
131.93, 131.89, 130.85, 129.80, 129.76, 128.72, 128.67, 127.27, 127.02,
126.96, 125.89, 124.77, 119.15, 56.22, 37.65, 11.04.
2.24 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
d171.08, 166.27, 159.30,
143.65, 140.49, 137.95, 137.72, 133.60, 132.97, 132.77, 132.05, 130.83,
130.12, 129.75, 128.79, 127.59, 127.09, 124.70, 119.84, 119.24, 55.79,
37.56, 15.24.
4.1.25. Synthesis of (S)-4-{2-[2-(4-cyanophenyl)-1-hydroxy-4-
methyl-1H-imidazole-5-carboxamido]-3-phenylpropanamido}
benzoic acid (28c)
4.1.30. Synthesis of (S)-4-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-
A white solid, yield: 21.86%. Mp 161.3e162.6ꢁС. ESI-HRMS calcd
for C28H23N5O5 [M ꢀ OH]- 492.1677 found: 492.1664; 1H NMR
phenylpropanamido}benzoic acid (28h)
(400 MHz, DMSO‑d6)
d
11.25 (s, 1H), 8.58 (d, J ¼ 7.8 Hz, 1H), 7.91
A white solid, yield: 15.23%. Mp 181.3e183.1 ꢁC. ESI-HRMS calcd
for C27H22ClFN4O5 [M ꢀ H]- 535.1190 found: 535.1197; 1H NMR
(ddd, J ¼ 12.8, 8.6, 3.2 Hz, 8H), 7.28 (dd, J ¼ 9.4, 5.4 Hz, 3H), 7.15 (d,
J ¼ 7.5 Hz, 2H), 5.04e4.96 (m, 1H), 3.31 (d, J ¼ 4.2 Hz, 1H), 3.11 (dd,
J ¼ 13.5, 10.5 Hz, 1H), 2.20 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
(400 MHz, DMSO‑d6)
d
10.54 (s, 1H), 8.31 (s, 1H), 7.91 (d, J ¼ 8.5 Hz,
2H), 7.78e7.66 (m, 3H), 7.42 (t, J ¼ 8.0 Hz, 1H), 7.34e7.12 (m, 5H),
4.95 (dd, J ¼ 13.6, 7.8 Hz, 1H), 3.19 (dd, J ¼ 13.8, 5.3 Hz, 1H), 3.02 (dd,
J ¼ 13.7, 8.7 Hz, 1H), 2.49 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
d170.12,165.20,158.07,142.80,138.73,137.24,136.88,131.36,129.74,
129.62, 128.70, 127.75, 127.36, 126.94, 125.91, 123.45, 120.47, 118.10,
110.57, 55.14, 36.31, 15.23.
d
170.63, 167.34, 159.45, 155.45, 152.94, 143.26, 137.49, 132.44,
130.85, 129.64, 129.42, 128.74, 127.05, 126.06, 126.02, 125.88, 121.14,
120.89, 120.72, 119.14, 54.88, 38.68, 12.16.
4.1.26. Synthesis of (S)-4-[2-(1-hydroxy-4-methyl-2-phenyl-1H-
imidazole-5-carboxamido)-3-phenylpropanamido]benzoic acid
(28d)
4.1.31. Synthesis of (S)-4-{2-[2-(3-chloro-2-fluorophenyl)-4-
methyl-1H-imidazole-5-carboxamido]-3-phenylpropanamido}
benzoic acid (28i)
A white solid, yield: 17.76%. Mp 136.8ꢁС-137.6ꢁС. ESI-HRMS
calcd for C27H24N4O5 [M ꢀ OH]- 467.1725 found: 467.1738; 1H
NMR (400 MHz, DMSO‑d6)
d12.70 (s, 1H), 10.54 (s, 1H), 7.94 (dd,
A white solid, yield: 14.72%. Mp 181.3e183.0ꢁС. ESI-HRMS calcd
for C27H22ClFN4O4 [M ꢀ H]- 519.1241 found: 519.1244; 1H NMR
J ¼ 7.8, 5.5 Hz, 4H), 7.84 (d, J ¼ 8.0 Hz, 1H), 7.74 (d, J ¼ 8.7 Hz, 2H),
7.48 (t, J ¼ 7.6 Hz, 2H), 7.39 (t, J ¼ 7.4 Hz, 1H), 7.28 (q, J ¼ 7.6 Hz, 4H),
7.20 (dt, J ¼ 9.2, 4.2 Hz,1H), 4.90 (dd, J ¼ 14.0, 7.8 Hz, 1H), 3.24e3.05
(400 MHz, DMSO‑d6)
d
10.55 (s, 1H), 8.31 (t, J ¼ 6.3 Hz, 1H), 7.92 (d,
J ¼ 8.6 Hz, 2H), 7.74 (dd, J ¼ 16.1, 7.9 Hz, 3H), 7.42 (t, J ¼ 8.0 Hz, 1H),
7.29 (q, J ¼ 8.0 Hz, 4H), 7.20 (t, J ¼ 6.7 Hz, 1H), 4.96 (dd, J ¼ 13.6,
7.8 Hz, 1H), 3.20 (dd, J ¼ 13.8, 5.3 Hz, 1H), 3.03 (dd, J ¼ 13.7, 8.7 Hz,
(m, 2H), 2.48 (s, 3H). 13C NMR (150 MHz, DMSO‑d6)
d 171.15, 166.28,
159.30, 143.69, 141.43, 137.81, 132.74, 130.82, 130.62, 129.88, 129.69,
129.15, 128.77, 128.72, 127.09, 124.69, 120.53, 119.24, 55.97, 37.55,
15.00.
1H), 2.49 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
d 172.52, 171.17,
167.50, 159.99, 158.34, 143.31, 137.75, 131.65, 130.77, 129.76, 128.65,
126.97, 126.09, 125.34, 119.12, 118.36, 118.28, 116.80, 116.65, 113.03,
54.61, 38.35, 21.61.
4.1.27. Synthesis of (S)-4-{2-[2-(4-chlorophenyl)-1-hydroxy-4-
methyl-1H-imidazole-5-carboxamido]-3-phenylpropanamido}
benzoic acid (28e)
4.1.32. Synthesis of (S)-4-{2-[2-(3-chloro-2-fluorophenyl)-1,4-
dimethyl-1H-imidazole-5-carboxamido]-3-phenylpropanamido}
benzoic acid (28j)
A white solid, yield: 22.36%. Mp 178.7e179.9ꢁС. ESI-HRMS calcd
for C27H23ClN4O5 [M ꢀ H]- 517.1284 found: 517.1265; 1H NMR
(400 MHz, DMSO‑d6)
d
10.66 (s, 1H), 8.38 (d, J ¼ 7.6 Hz, 1H), 7.96 (d,
A white solid, yield: 18.39%. Mp 162.3e163.7ꢁС. ESI-HRMS calcd
for C28H24ClFN4O4 [M ꢀ H]- 533.1397 found: 533.1422; 1H NMR
J ¼ 8.8 Hz, 2H), 7.89e7.81 (m, 2H), 7.79 (d, J ¼ 8.8 Hz, 2H), 7.58e7.48
(m, 2H), 7.30 (q, J ¼ 7.7 Hz, 3H), 7.22e7.19 (m, 2H), 5.17e4.54 (m,
1H), 3.24 (dd, J ¼ 13.8, 4.8 Hz, 1H), 3.08 (dd, J ¼ 13.8, 9.9 Hz, 1H),
(400 MHz, DMSO‑d6)
d 13.15 (s, 1H), 11.24 (s, 1H), 10.56 (s, 1H), 8.39
(s, 1H), 7.92 (t, J ¼ 8.3 Hz, 2H), 7.81e7.60 (m, 3H), 7.42 (t, J ¼ 8.0 Hz,
13

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
1H), 7.34e7.24 (m, 4H), 7.20 (dt, J ¼ 9.3, 5.0 Hz, 1H), 4.93 (dd,
J ¼ 13.5, 8.0 Hz, 1H), 3.83 (s, 3H), 3.18 (dd, J ¼ 13.8, 5.4 Hz, 2H), 3.01
(dd, J ¼ 13.7, 8.7 Hz, 1H), 2.49 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
4.1.39. Synthesis of ethyl (S)-2-{4-{2-[2-(3-chloro-2-fluorophenyl)-
1-hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-
phenylpropanamido}phenyl}acetate (33a)
Compound 33a was prepared from 32a in the same manner as
described for 27a-27k, and it was used for the next reaction
without further purification.
d
170.74, 166.26, 159.55, 152.85, 143.62, 137.48, 132.36, 130.75,
129.62, 129.33, 128.75, 127.06, 126.05, 126.00, 124.68, 121.10, 120.87,
120.70, 119.24, 54.89, 52.36, 38.65, 11.87.
4.1.40. Synthesis of ethyl (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-
phenylpropanamido}-1H-indole-2-carboxylate (33b)
Compound 33b was prepared from 32b in the same manner as
described for 27a-27k, and it was used for the next reaction
without further purification.
4.1.33. Synthesis of (S)-4-{2-[2-(3-chloro-2-fluorophenyl)-1-
methoxy-4-methyl-1H-imidazole-5-carboxamido]-3-
phenylpropanamido}benzoic acid (28k)
A white solid, yield: 38.11%. Mp 118.4e120.1 ꢁC. ESI-HRMS calcd
for C28H24ClFN4O5 [M ꢀ H]- 549.1341 found: 549.1372; 1H NMR
(400 MHz, DMSO‑d6) d 10.71 (s, 1H), 8.50 (s, 1H), 8.34e8.24 (m, 1H),
8.12 (d, J ¼ 8.1 Hz, 1H), 7.95 (d, J ¼ 8.7 Hz, 2H), 7.78 (d, J ¼ 8.6 Hz,
2H), 7.63 (t, J ¼ 7.8 Hz, 1H), 7.40 (d, J ¼ 7.3 Hz, 2H), 7.32 (t, J ¼ 7.5 Hz,
2H), 7.23 (t, J ¼ 7.3 Hz, 1H), 5.12e4.80 (m, 1H), 3.74 (s, 1H), 3.28 (dd,
J ¼ 13.6, 3.8 Hz, 1H), 3.18e3.02 (m, 1H), 2.26 (s, 3H). 13C NMR
4.1.41. Synthesis of (S)-2-{4-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-
phenylpropanamido}phenyl}acetic acid (34a)
Compound 34a was prepared from 33a in the same manner as
described for 28a-28k.A yellow solid, yield: 12.33%. Mp
187.1e189.0ꢁС. ESI-HRMS calcd for C28H24ClFN4O5 [M þ Na]þ
(100 MHz, DMSO‑d6) d 170.91, 167.79, 167.36, 158.70, 158.60, 143.32,
139.91, 137.77, 135.57, 130.92, 130.86, 129.96, 129.73, 129.47, 129.13,
128.73, 127.07, 125.91, 120.01, 119.15, 67.90, 55.61, 37.65, 14.76.
573.1311 found: 573.1309; 1H NMR (600 MHz, DMSO‑d6)
d 10.36 (s,
1H), 10.13 (d, J ¼ 3.6 Hz, 1H), 8.34 (dd, J ¼ 15.8, 7.8 Hz,1H), 7.71e7.62
(m, 1H), 7.58 (d, J ¼ 8.4 Hz,1H), 7.50 (dd, J ¼ 14.5, 8.3 Hz, 2H), 7.37 (t,
J ¼ 6.6 Hz, 2H), 7.19 (d, J ¼ 8.0 Hz, 3H), 7.14e7.05 (m, 2H), 4.95 (dd,
J ¼ 13.5, 8.6 Hz, 1H), 3.62 (s, 2H), 3.30e3.18 (m, 1H), 3.11 (dd,
J ¼ 13.7, 9.8 Hz, 1H), 2.23 (s, 3H). 13C NMR (150 MHz, DMSO‑d6)
4.1.34. Synthesis of ethyl 5-amino-1H-indole-2-carboxylate (30)
A mixture of ethyl 5-nitro-1H-indole-2-carboxylate 29 (17.0 g,
77.0 mmol) and 5% Pd/C (3.4 g) in ethanol was stirred at room
temperature for 12 h under hydrogen atmosphere. After the
completion of the reaction, the Pd/C was filtered out and the filtrate
was evaporated to give a grey soliþd, yield: 87.9%. Mp 126.1e127.2ꢁС.
d
170.60, 169.51, 157.97, 154.66, 152.98, 137.55, 136.82, 131.62, 131.17,
129.28, 128.96, 128.62, 127.59, 127.56, 125.93, 124.60, 119.25, 118.77,
118.59, 54.47, 40.81, 36.71, 13.46.
ESI-MS (m/z) ¼ 205.09 [M þH]
;
1H NMR (400 MHz, DMSO‑d6)
d
11.40 (s, 1H), 7.17 (d, J ¼ 9.0 Hz, 1H), 6.78 (d, J ¼ 53.9 Hz, 3H), 4.42
(t, J ¼ 73.8 Hz, 4H), 1.32 (t, J ¼ 7.1 Hz, 3H).
4.1.42. Synthesis of (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-
4.1.35. Synthesis of ethyl (S)-2-{4-{2-[(tert-butoxycarbonyl)
amino]-3-phenylpropanamido}phenyl}acetate (31a)
Compound 31a was prepared from the commercially available
ethyl 2-(4-aminophenyl)acetate in the same manner as described
for 25, and it was used for the next reaction without further
purification.
phenylpropanamido}-1H-indole-2-carboxylic acid (34b)
Compound 34b was prepared from 33b in the same manner as
described for 28a-28k.
A white solid, yield: 21.23%. Mp
186.2e187.6 ꢁC. ESI-HRMS calcd for C29H23ClFN6O6 [M ꢀ H]-
574.1299 found: 574.1276; 1H NMR (400 MHz, DMSO‑d6)
d 11.54 (s,
1H), 10.08 (s, 1H), 8.26 (s, 1H), 7.97 (d, J ¼ 17.6 Hz, 1H), 7.78e7.51 (m,
1H), 7.36 (dt, J ¼ 4.9, 3.3 Hz, 3H), 7.29 (d, J ¼ 6.3 Hz, 2H), 7.27e7.22
(m, 2H), 7.21e7.17 (m, 1H), 7.15 (d, J ¼ 8.6 Hz, 1H), 6.80 (d, J ¼ 8.6 Hz,
1H), 5.10e4.72 (m, 1H), 3.16 (dd, J ¼ 13.5, 5.8 Hz, 1H), 3.06e2.95 (m,
4.1.36. Synthesis of ethyl (S)-5-{2-[(tert-butoxycarbonyl)amino]-3-
phenylpropanamido}-1H-indole-2-carboxylate (31b)
Compound 31b was prepared from the commercially available
ethyl 5-amino-1H-indole-2-carboxylate in the same manner as
described for 25. A grey solid, yield: 77.6%. Mp 192.5e193.7ꢁС. 1H
1H), 2.45 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
d174.26, 169.86,
163.46, 158.36, 155.62, 138.08, 134.47, 133.62, 132.09, 129.81, 129.72,
128.63, 127.48, 126.84, 125.54, 120.72, 120.60, 118.79, 118.13, 116.80,
112.81, 112.33, 109.83, 107.02, 54.41, 38.03, 15.19.
NMR (400 MHz, DMSO‑d6)
d 11.79 (s, 1H), 9.83 (s, 1H), 7.99 (s, 1H),
7.36 (dt, J ¼ 9.0, 5.3 Hz, 2H), 7.11 (s, 1H), 6.95 (d, J ¼ 8.0 Hz, 2H), 6.85
(d, J ¼ 8.1 Hz, 1H), 6.47 (d, J ¼ 8.1 Hz, 2H), 4.34 (q, J ¼ 7.1 Hz, 2H),
4.22 (dd, J ¼ 13.3, 8.3 Hz,1H), 2.83 (dd, J ¼ 13.7, 4.7 Hz,1H), 2.69 (dd,
J ¼ 13.3, 9.7 Hz, 1H), 1.42e1.21 (m, 12H).
4.1.43. Synthesis of ethyl (S)-5-{2-[(tert-butoxycarbonyl)amino]-3-
(4-fluorophenyl)propanamido}-1H-indole-2-carboxylate (36a)
Compound 36a was prepared from commercially available (S)-
2-[(tert-butoxycarbonyl)amino]-3-(4-fluorophenyl)propanoic acid
35a in the same manner as described for 25. A grey solid, yield:
71.9%. Mp 173.5e175.0ꢁС. ESI-MS (m/z) ¼ 492.30 [M þ Na]
;
1H
4.1.37. Synthesis of ethyl (S)-2-[4-(2-amino-3-phenylpropanamido)
phenyl]acetate (32a)
þ
NMR (400 MHz, DMSO‑d6)
d 11.82 (s, 1H), 9.94 (s, 1H), 8.01 (s, 1H),
Compound 32a was prepared from 31a in the same manner as
described for 26. A grey solid, yield: 85.4%. ESI-MS (m/z) ¼ 325.29
7.68e7.24 (m, 4H), 7.24e6.89 (m, 4H), 4.34 (q, J ¼ 7.1 Hz, 3H),
3.26e2.88 (m, 1H), 2.94e2.68 (m, 1H), 1.71e0.94 (m, 12H).
[M ꢀ H]-; 1H NMR (400 MHz, DMSO‑d6)
d
7.55 (d, J ¼ 8.5 Hz, 2H),
7.26 (t, J ¼ 4.1 Hz, 5H), 7.18 (d, J ¼ 8.5 Hz, 3H), 4.07 (q, J ¼ 7.1 Hz, 2H),
3.64e3.53 (m, 4H), 3.00 (dd, J ¼ 13.4, 5.5 Hz, 1H), 2.72 (dd, J ¼ 13.4,
8.0 Hz, 1H), 1.18 (t, J ¼ 7.1 Hz, 3H).
4.1.44. Synthesis of ethyl (S)-5-{2-[(tert-butoxycarbonyl)amino]-3-
(4-chlorophenyl)propanamido}-1H-indole-2-carboxylate (36b)
Compound 36b was prepared from commercially available (S)-2-
[(tert-butoxycarbonyl)amino]-3-(4-chlorophenyl)propanoicacid35b
in the same manner as described for 25. A grey sþolid, yield: 69.6%. Mp
213.7e215.2ꢁС. ESI-MS(m/z)¼ 508.30[Mþ Na] ;1HNMR(400 MHz,
4.1.38. Synthesis of ethyl (S)-5-(2-amino-3-phenylpropanamido)-
1H-indole-2-carboxylate (32b)
Compound 32b was prepared from 31b in the same manner as
DMSO‑d6) d 11.83 (s, 1H), 9.95 (s, 1H), 8.01 (s, 1H), 7.68e7.24 (m, 6H),
described for 26. A white solid, yield: 87.7%. Mp 135.2e136.7ꢁС. ESI-
7.12(t, J¼ 5.5Hz, 2H), 4.38e4.03(m, 3H), 3.02(dd, J ¼ 13.6, 4.5 Hz,1H),
þ
MS (m/z) ¼ 352.10 [M þ H]
.
2.85 (dd, J ¼ 13.6, 10.4 Hz, 1H), 1.45e1.16 (m, 12H).
14

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
4.1.45. Synthesis of ethyl (S)-5-{3-[4-bromophenyl]-2-[(tert-
butoxycarbonyl)amino]propanamido}-1H-indole-2-carboxylate
(36c)
Compound 36c was prepared from commercially available (S)-
3-(4-bromophenyl)-2-[(tert-butoxycarbonyl)amino]propanoic acid
4.1.51. Synthesis of ethyl (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-(4-
fluorophenyl)propanamido}-1H-indole-2-carboxylate (38a)
Compound 38a was prepared from 37a in the same manner as
described for 27a-27k, and it was used for the next reaction
without further purification.
35c in the same manner as described for 25. A grey solid, yield:
þ
66.3%. Mp 218.0e219.6ꢁС. ESI-MS (m/z) ¼ 552.28 [M þ Na]
;
1H
NMR (400 MHz, DMSO‑d6)
d 11.83 (s, 1H), 9.97 (s, 1H), 8.01 (s, 1H),
4.1.52. Synthesis of ethyl (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-(4-
chlorophenyl)propanamido}-1H-indole-2-carboxylate (38b)
Compound 38b was prepared from 37b in the same manner as
described for 27a-27k, and it was used for the next reaction
without further purification.
7.49 (d, J ¼ 8.2 Hz, 2H), 7.40 (d, J ¼ 8.8 Hz, 1H), 7.39e7.28 (m, 3H),
7.11 (t, J ¼ 5.0 Hz, 2H), 4.33 (t, J ¼ 7.1 Hz, 3H), 2.98 (dt, J ¼ 23.1,
11.6 Hz, 1H), 2.82 (dt, J ¼ 25.1, 12.5 Hz, 1H), 1.58e0.83 (m, 12H).
4.1.46. Synthesis of ethyl (S)-5-{2-[(tert-butoxycarbonyl)amino]-3-
(4-nitrophenyl)propanamido}-1H-indole-2-carboxylate (36d)
Compound 36d was prepared from commercially available (S)-
2-[(tert-butoxycarbonyl)amino]-3-(4-nitrophenyl)propanoic acid
4.1.53. Synthesis of ethyl (S)-5-{3-(4-bromophenyl)-2-[2-(3-chloro-
2-fluorophenyl)-1-hydroxy-4-methyl-1H-imidazole-5-
carboxamido]propanamido}-1H-indole-2-carboxylate (38c)
Compound 38c was prepared from 37c in the same manner as
described for 27a-27k, and it was used for the next reaction
without further purification.
35d in the same manner as described for 25. A grey solid, yield:
þ
;
1H
72.7%. Mp 200.3e201.8ꢁС. ESI-MS (m/z) ¼ 519.24 [M þNa]
NMR (400 MHz, DMSO‑d6)
d 11.83 (s, 1H), 10.01 (s, 1H), 8.19 (d,
J ¼ 8.6 Hz, 2H), 8.01 (s, 1H), 7.63 (d, J ¼ 8.5 Hz, 2H), 7.40 (s, 1H), 7.36
(d, J ¼ 1.9 Hz,1H), 7.21 (d, J ¼ 8.5 Hz,1H), 7.13 (d, J ¼ 1.5 Hz,1H), 4.34
(q, J ¼ 7.1 Hz, 3H), 3.17 (dd, J ¼ 13.4, 4.2 Hz, 1H), 3.00 (dd, J ¼ 13.2,
10.7 Hz, 1H), 1.33 (dd, J ¼ 14.5, 7.4 Hz, 12H).
4.1.54. Synthesis of ethyl (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-(4-
nitrophenyl)propanamido}-1H-indole-2-carboxylate (38d)
Compound 38d was prepared from 37d in the same manner as
described for 27a-27k, and it was used for the next reaction
without further purification.
4.1.47. Synthesis of ethyl (S)-5-[2-amino-3-(4-fluorophenyl)
propanamido]-1H-indole-2-carboxylate (37a)
Compound 37a was prepared from 36a in the same manner as
described for 26. A white solid, yield: 88.2%. Mp 268.2e269.8ꢁС.
4.1.55. Synthesis of ethyl (S)-5-{3-(4-aminophenyl)-2-[2-(3-chloro-
2-fluorophenyl)-1-hydroxy-4-methyl-1H-imidazole-5-
carboxamido]propanamido}-1H-indole-2-carboxylate (38e)
Compound 38e was prepared from 37d in the same manner as
described for 30, and it was used for the next reaction without
further purification.
þ
ESI-MS (m/z) ¼ 370.28 [M þ H]
;
1H NMR (400 MHz, DMSO‑d6)
d
11.79 (s, 1H), 7.41e7.34 (m, 2H), 7.29 (dd, J ¼ 8.6, 5.7 Hz, 2H), 7.10
(dd, J ¼ 10.5, 7.3 Hz, 3H), 4.34 (d, J ¼ 7.1 Hz, 2H), 3.55 (dd, J ¼ 7.8,
5.6 Hz, 1H), 3.00 (dd, J ¼ 13.4, 5.4 Hz, 1H), 2.74 (dd, J ¼ 13.5, 8.0 Hz,
1H), 1.34 (s, 3H).
4.1.48. Synthesis of ethyl (S)-5-[2-amino-3-(4-chlorophenyl)
propanamido]-1H-indole-2-carboxylate (37b)
4.1.56. Synthesis of (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-(4-
Compound 37b was prepared from 36b in the same manner as
fluorophenyl)propanamido}-1H-indole-2-carboxylic acid (39a)
Compound 39a was prepared from 38a in the same manner as
described for 26. A white solid, yield: 78.4%. Mp 277.2e278.5ꢁС.
þ
ESI-MS (m/z) ¼ 386.27 [M þ H]
;
1H NMR (400 MHz, DMSO‑d6)
described for 28a-28k.
A white solid, yield: 19.35%. Mp
148.6e150.1ꢁС. ESI-HRMS calcd for C29H22ClF2N5O5 [M ꢀ H]-
d
11.78 (s, 2H), 8.00 (s, 1H), 7.41e7.21 (m, 4H), 7.09 (dd, J ¼ 10.2,
592.1205 found: 592.1210; 1H NMR (400 MHz, DMSO‑d6)
d 11.83 (s,
7.5 Hz, 3H), 4.33 (q, J ¼ 7.1 Hz, 2H), 3.65e3.44 (m, 1H), 2.99 (dd,
J ¼ 13.4, 5.5 Hz, 1H), 2.79e2.67 (m, 1H), 1.34 (t, J ¼ 7.1 Hz,3H).
1H), 10.23 (s, 1H), 8.34 (d, J ¼ 7.9 Hz, 1H), 8.14e7.98 (m, 1H),
7.47e7.39 (m, 4H), 7.36e7.31 (m, 1H), 7.14e7.06 (m, 5H), 4.97 (td,
J ¼ 8.6, 5.1 Hz, 1H), 3.25 (dd, J ¼ 13.9, 4.8 Hz, 1H), 3.12 (dd, J ¼ 13.8,
4.1.49. Synthesis of ethyl (S)-5-[2-amino-3-(4-bromophenyl)
propanamido]-1H-indole-2-carboxylate (37c)
9.4 Hz, 1H), 2.26 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
d 170.81,
167.36, 160.51, 159.45, 158.02, 143.41, 138.62, 137.95, 137.78,
134.91134.60, 133.08, 132.24, 130.84, 130.39, 129.81, 129.76, 128.66,
127.37, 127.01, 125.89, 125.62, 119.16, 116.85, 56.31, 37.59, 11.02.
Compound 37c was prepared from 36c in the same manner as
described for 26. A white solid, yield: 75.5%. Mp 257.4e259.1ꢁС. ESI-
MS (m/z) ¼ 430.23 [M þ H] þ; 1H NMR (400 MHz, DMSO‑d6)
d 11.85
(s, 1H), 10.34 (s, 1H), 7.97 (s, 1H), 7.51 (d, J ¼ 8.1 Hz, 2H), 7.40 (s, 1H),
7.37e7.31 (m, 1H), 7.27 (d, J ¼ 8.2 Hz, 2H), 7.13 (s, 1H), 4.34 (d,
J ¼ 7.1 Hz, 2H), 4.03 (d, J ¼ 14.0 Hz,1H), 3.44 (dt, J ¼ 13.9, 6.9 Hz,1H),
3.14 (dd, J ¼ 13.5, 5.8 Hz, 1H), 2.99 (d, J ¼ 7.3 Hz, 1H), 1.34 (s, 3H).
4.1.57. Synthesis of (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-(4-
chlorophenyl)propanamido}-1H-indole-2-carboxylic acid (39b)
Compound 39b was prepared from 38b in the same manner as
described for 28a-28k.
167.2e168.6ꢁС. ESI-MS (m/z)
(400 MHz, DMSO‑d6)
A
white solid, yield: 18.47%. Mp
4.1.50. Synthesis of ethyl (S)-5-[2-amino-3-(4-nitrophenyl)
propanamido]-1H-indole-2-carboxylate (37d)
¼
610.31 [M
þ
H]þ; 1H NMR
d
11.83 (s, 1H), 10.24 (s, 1H), 8.34 (d, J ¼ 7.9 Hz,
Compound 37d was prepared from 36d in the same manner as
described for 26. A yellow solid, yield: 69.4%. Mp 168.0e169.4ꢁС.
ESI-MS (m/z) ¼ 397.26 [M þ H]þ; 1H NMR (400 MHz, DMSO‑d6)
1H), 8.04 (s, 1H), 7.75e7.62 (m, 1H), 7.45e7.38 (m, 4H), 7.37e7.30
(m, 3H), 7.26 (d, J ¼ 2.9 Hz, 1H), 7.13 (d, J ¼ 2.2 Hz, 1H), 5.00e4.95
(m, 1H), 3.28e3.20 (m, 1H), 3.13 (dd, J ¼ 13.9, 9.4 Hz, 1H), 2.26 (s,
d
11.79 (s, 1H), 9.82 (s, 1H), 8.15 (d, J ¼ 7.4 Hz, 2H), 8.01 (s, 1H), 7.56
3H). 13C NMR (100 MHz, DMSO‑d6)
d169.69, 161.69, 159.03, 155.77,
(d, J ¼ 7.4 Hz, 2H), 7.36 (d, J ¼ 6.6 Hz, 2H), 7.10 (s, 1H), 4.33 (d,
J ¼ 6.6 Hz, 2H), 3.64 (s,1H), 3.14 (d, J ¼ 9.2 Hz,1H), 2.90 (d, J ¼ 7.7 Hz,
1H), 1.34 (s, 3H).
154.09, 138.65, 137.17, 137.10, 134.85, 132.71, 132.28, 132.20, 131.73,
130.60, 130.35, 129.73, 128.65, 128.43, 127.07, 120.36, 119.46, 113.17,
112.52, 108.13, 55.34, 37.36, 14.78.
15

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
4.1.58. Synthesis of (S)-5-{3-(4-bromophenyl)-2-[2-(3-chloro-2-
fluorophenyl)-1-hydroxy-4-methyl-1H-imidazole-5-carboxamido]
propanamido}-1H-indole-2-carboxylic acid (39c)
butoxycarbonyl)amino]propanamido}-1H-indole-2-carboxylate 40
(0.47 g, 1.0 mmol), acetic acid (0.77 g, 1.28 mmol), DIPEA (0.17 g,
1.32 mmol) and HATU (0.49 g,1.3 mmol) in DMF (10 mL) was stirred
at 25ꢁС for 4 h. The reaction mixture was poured into 15 mL ethyl
acetate and 10 mL water. The organics were washed with 1 M NaOH
(10 mL), 1 M HCl (10 mL) and brine (10 mL), dried (Na2SO4), and
concentrated to afford 41a, which was used for the next reaction
without further purification.
Compound 39c was prepared from 38c in the same manner as
described for 28a-28k.
A white solid, yield: 21.05%. Mp
152.3e153.6ꢁС. ESI-HRMS calcd for C29H22BrClFN5O5 [M ꢀ H]-
652.0404 found: 652.0438; 1H NMR (400 MHz, DMSO‑d6)
d 10.67
(s, 1H), 8.40 (d, J ¼ 7.6 Hz, 1H), 7.96 (d, J ¼ 8.8 Hz, 2H), 7.79 (d,
J ¼ 8.8 Hz, 2H), 7.68 (t, J ¼ 6.6 Hz, 1H), 7.39 (d, J ¼ 7.1 Hz, 2H),
7.29e7.23 (m, 2H), 7.19 (t, J ¼ 7.4 Hz, 1H), 7.11 (t, J ¼ 7.7 Hz, 2H), 4.98
4.1.63. Synthesis of ethyl (S)-5-{2-[(tert-butoxycarbonyl)amino]-3-
{4-[(isopropylcarbonyl)amino]phenyl}propanamido}-1H-indole-2-
carboxylate (41b)
(s, 1H), 3.25 (d, J ¼ 4.5 Hz, 1H), 3.18e2.90 (m, 1H), 2.23 (s, 3H). 13
C
NMR (100 MHz, DMSO‑d6)
d 169.29, 163.17, 161.38, 159.37, 155.42,
153.73, 137.22, 134.70, 132.73, 131.95, 131.56, 129.81, 129.46, 127.14,
126.07, 126.04, 121.05, 120.93, 119.13, 117.89, 115.68, 112.97, 107.78,
54.34, 38.41, 12.67.
Compound 41b was prepared from commercially available iso-
butyric acid in the same manner as described for 41a. An off-white
solid, yield: 69.7%. Mp 223.1e224.8ꢁС. ESI-MS (m/z) ¼ 559.49
[M þNa]þ; 1H NMR (400 MHz, DMSO‑d6)
d 11.79 (s, 1H), 9.87 (s, 1H),
4.1.59. Synthesis of (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-(4-
nitrophenyl)propanamido}-1H-indole-2-carboxylic acid (39d)
Compound 39d was prepared from 38d in the same manner as
9.71 (s, 1H), 7.98 (s, 1H), 7.50 (d, J ¼ 8.3 Hz, 2H), 7.35 (dt, J ¼ 8.9,
5.3 Hz, 2H), 7.22 (d, J ¼ 8.2 Hz, 2H), 7.11 (d, J ¼ 1.5 Hz, 1H), 6.99 (d,
J ¼ 8.1 Hz, 1H), 4.34 (d, J ¼ 7.1 Hz, 3H), 2.99e2.87 (m, 1H), 2.79 (dd,
J ¼ 26.6, 13.1 Hz, 1H), 2.56 (dt, J ¼ 13.8, 6.9 Hz, 1H), 1.33 (s, 12H), 1.08
(d, J ¼ 6.8 Hz, 6H).
described for 28a-28k.
A white solid, yield: 15.34%. Mp
178.2e179.6ꢁС. ESI-HRMS calcd for C29H22ClFN6O7 [M ꢀ H]-
619.1150 found: 619.1165; 1H NMR (400 MHz, DMSO‑d6)
d
13.90 (s,
4.1.64. Synthesis of ethyl (S)-5-{2-[(tert-butoxycarbonyl)amino]-3-
{4-[(isobutylcarbonyl)amino]phenyl}propanamido}-1H-indole-2-
carboxylate (41c)
Compound 41c was prepared from commercially available 3-
methylbutanoic acid in the same manner as described for 41a. An
off-white solid, yield: 70.1%. Mp 163.2e164.9ꢁС. 1H NMR (400 MHz,
1H), 11.81 (s, 1H), 11.45 (s, 1H), 8.32 (s, 1H), 7.64e7.45 (m, 2H), 7.40
(d, J ¼ 8.6 Hz, 1H), 7.25 (t, J ¼ 7.8 Hz, 1H), 7.13 (d, J ¼ 8.5 Hz, 1H), 7.04
(s, 1H), 6.92 (d, J ¼ 10.0 Hz, 1H), 6.72 (s, 1H), 6.33 (d, J ¼ 9.9 Hz, 1H),
6.19 (d, J ¼ 10.1 Hz, 1H), 6.04 (d, J ¼ 9.4 Hz, 1H), 3.44 (dd, J ¼ 13.8,
6.9 Hz, 2H), 3.17 (s, 1H), 2.43 (s, 3H). 13C NMR (100 MHz, DMSO‑d6)
d
165.52, 162.13, 159.13, 154.20, 152.51, 145.46, 135.56, 135.46,
DMSO‑d6) d 11.80 (s, 1H), 9.95 (s, 1H), 9.81 (s, 1H), 7.99 (s, 1H), 7.50
131.38, 130.64, 128.77, 128.31, 127.76, 126.14, 125.32, 124.10, 123.96,
120.78, 119.53, 119.41, 118.52, 116.43, 111.93, 106.44, 55.39, 39.43,
17.93.
(d, J ¼ 8.2 Hz, 2H), 7.47e7.30 (m, 2H), 7.23 (d, J ¼ 8.1 Hz,2H), 7.11 (d,
J ¼ 1.5 Hz, 1H), 6.99 (d, J ¼ 8.2 Hz, 1H), 4.34 (q, J ¼ 7.1 Hz, 3H), 2.97
(dd, J ¼ 13.5, 4.4 Hz, 1H), 2.89e2.71 (m, 1H), 2.17 (d, J ¼ 7.0 Hz, 2H),
2.06 (dt, J ¼ 13.8, 6.7 Hz, 1H), 1.46e1.19 (m, 12H), 0.92 (d, J ¼ 6.5 Hz,
6H).
4.1.60. Synthesis of (S)-5-{3-(4-aminophenyl)-2-[2-(3-chloro-2-
fluorophenyl)-1-hydroxy-4-methyl-1H-imidazole-5-carboxamido]
propanamido}-1H-indole-2-carboxylic acid (39e)
4.1.65. Synthesis of ethyl (S)-5-{2-[(tert-butoxycarbonyl)amino]-3-
{4-[(ethynylcarbonyl)amino]phenyl}propanamido}-1H-indole-2-
carboxylate (41d)
Compound 41d was prepared from commercially available
propiolic acid in the same manner as described for 41a. An off-
white solid, yield: 65.3%. Mp 162.4e163.9ꢁС.
Compound 39e was prepared from 38e in the same manner as
described for 28a-28k.
A white solid, yield: 13.22%. Mp
167.3e168.9ꢁС. ESI-HRMS calcd for C29H24ClFN6O5 [M ꢀ H]-
589.1408 found: 589.1613; 1H NMR (400 MHz, DMSO‑d6)
d 10.72 (d,
J ¼ 8.0 Hz, 1H), 8.25 (dd, J ¼ 6.6, 2.8 Hz, 1H), 7.93 (d, J ¼ 8.0 Hz, 2H),
7.78 (dd, J ¼ 8.6, 7.6 Hz, 2H), 7.70e7.56 (m, 2H), 7.52e7.36 (m, 3H),
7.30 (td, J ¼ 7.4, 3.4 Hz, 2H), 7.22 (dd, J ¼ 10.5, 4.1 Hz, 1H), 5.03e4.81
(m, 1H), 3.29e3.20 (m, 1H), 3.19e3.09 (m, 1H), 2.50 (s, 3H). 13C NMR
4.1.66. Synthesis of ethyl (S)-5-{3-{4-{[(prop-1-yn-1-yl)carbonyl]
amino}phenyl}-2-[(tert-butoxycarbonyl) amino]propanamido}-1H-
indole-2-carboxylate (41e)
(100 MHz, DMSO‑d6)
d 170.06, 165.38, 164.03, 162.82, 156.45,
155.52, 153.84, 137.88, 134.14, 131.95, 131.39, 120.12, 129.92, 129.58,
127.31, 125.28, 125.25, 120.63, 120.51, 119.31, 117.41, 112.59, 112.24,
109.72, 54.81, 39.16, 15.20.
Compound 41e was prepared from commercially available but-
2-ynoic acid in the same manner as described for 41a. An off-white
solid, yield: 66.4%. Mp 149.6e151.3ꢁС. 1H NMR (400 MHz,
DMSO‑d6) d 11.80 (s, 1H), 10.51 (s, 1H), 9.93 (s, 1H), 7.99 (s, 1H), 7.49
4.1.61. Synthesis of ethyl (S)-5-{3-(4-aminophenyl)-2-[(tert-
butoxycarbonyl)amino]propanamido}-1H-indole-2-carboxylate
(40)
(d, J ¼ 8.3 Hz, 2H), 7.36 (dt, J ¼ 9.0, 5.3 Hz, 2H), 7.25 (d, J ¼ 8.2 Hz,
2H), 7.11 (d, J ¼ 1.6 Hz, 1H), 7.00 (d, J ¼ 8.2 Hz, 1H), 4.79e3.94 (m,
3H), 2.97 (dd, J ¼ 13.6, 4.4 Hz, 1H), 2.87e2.73 (m, 1H), 2.03 (s, 3H),
1.55e1.14 (m, 12H).
Compound 40 was prepared from 36d in the same manner as
described for 30. An off-white solid, yield: 82.6%. Mp
-
200.8e201.6ꢁС. ESI-MS (m/z) ¼ 465.45 [M ꢀ H]
;
1H NMR
4.1.67. Synthesis of ethyl (S)-5-{2-[(tert-butoxycarbonyl)amino]-3-
{4-[(cyclopropylcarbonyl)amino]phenyl}propanamido}-1H-indole-
2-carboxylate (41f)
Compound 41f was prepared from commercially available
cyclopropanecarboxylic acid in the same manner as described for
41a. A white solid, yield: 77.0%. Mp 152.4e153.8ꢁС. ESI-MS (m/
(400 MHz, DMSO‑d6)
d 11.79 (s, 1H), 9.83 (s, 1H), 7.99 (s, 1H), 7.36
(dt, J ¼ 9.0, 5.3 Hz, 2H), 7.11 (s, 1H), 6.95 (d, J ¼ 8.0 Hz, 2H), 6.85 (d,
J ¼ 8.1 Hz, 1H), 6.47 (d, J ¼ 8.1 Hz, 2H), 4.34 (q, J ¼ 7.1 Hz, 2H), 4.22
(dd, J ¼ 13.3, 8.3 Hz, 1H), 2.83 (dd, J ¼ 13.7, 4.7 Hz, 1H), 2.69 (dd,
J ¼ 13.3, 9.7 Hz, 1H), 1.73e0.70 (m, 12H).
z) ¼ 557.25 [M þNa] þ; 1H NMR (400 MHz, DMSO‑d6)
d 11.80 (s, 1H),
4.1.62. Synthesis of ethyl (S)-5-{3-[4-(acetylamino)phenyl]-2-
[(tert-butoxycarbonyl)amino]propanamido}-1H-indole-2-
carboxylate (41a)
10.08 (s, 1H), 9.88 (s, 1H), 7.99 (s, 1H), 7.48 (d, J ¼ 8.1 Hz, 2H), 7.37
(dd, J ¼ 14.4, 5.2 Hz, 2H), 7.23 (d, J ¼ 8.1 Hz, 2H), 7.12 (d, J ¼ 1.2 Hz,
1H), 6.97 (dd, J ¼ 15.6, 7.2 Hz, 1H), 4.34 (d, J ¼ 7.1 Hz, 3H), 2.96 (dd,
J ¼ 13.6, 4.4 Hz,1H), 2.88e2.77 (m, 1H), 1.86e1.66 (m, 1H),1.44e1.18
A
solution of ethyl (S)-5-{3-(4-aminophenyl)-2-[(tert-
16

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
(m, 12H), 0.77 (t, J ¼ 6.0 Hz, 4H).
J ¼ 8.3 Hz, 1H), 4.34 (d, J ¼ 7.1 Hz, 3H), 3.00 (dd, J ¼ 13.8, 4.6 Hz, 1H),
2.91e2.72 (m, 1H), 1.52e1.14 (m, 12H).
4.1.68. Synthesis of ethyl 5-{(2S)-2-[(tert-butoxycarbonyl)amino]-
3-{4-[(2-methylcyclopropylcarbonyl)amino]phenyl}propanamido}-
1H-indole-2-carboxylate(41g)
4.1.73. Synthesis of ethyl (S)-5-{2-[(tert-butoxycarbonyl)amino]-3-
{4-{[(pyridin-2-yl)carbonyl]amino}phenyl}propanamido}-1H-
indole-2-carboxylate (41l)
Compound 41g was prepared from commercially available 2-
methylcyclopropane-1-carboxylic acid in the same manner as
described for 41a. An off-white solid, yield: 69.7%. Mp
221.4e222.8ꢁС. ESI-MS (m/z) ¼ 547.55[M ꢀ H] -; 1H NMR (400 MHz,
Compound 41l was prepared from commercially available
picolinic acid in the same manner as described for 41a. An off-white
solid, yield: 74.4%. Mp 147.4e148.7ꢁС. ESI-MS (m/z) ¼ 594.45[M þ
þ
Na] ; d 11.80 (s, 1H), 10.55 (s, 1H),
1H NMR (400 MHz, DMSO‑d6)
DMSO‑d6)
d
11.79 (s, 1H), 10.02 (s, 1H), 9.88 (s, 1H), 7.98 (s, 1H), 7.47
(d, J ¼ 8.3 Hz, 2H), 7.35 (dt, J ¼ 9.0, 5.2 Hz, 2H), 7.21 (d, J ¼ 8.2 Hz,
2H), 7.11 (d, J ¼ 1.4 Hz, 1H), 6.99 (d, J ¼ 8.0 Hz, 1H), 4.45e4.21 (m,
3H), 3.02e2.87 (m, 1H), 2.87e2.70 (m, 1H), 1.49 (dt, J ¼ 7.9, 4.0 Hz,
1H), 1.41e1.13 (m, 13H), 1.09 (t, J ¼ 6.3 Hz, 3H), 1.02e0.91 (m, 1H),
0.67e0.47 (m, 1H).
9.92 (s, 1H), 8.73 (d, J ¼ 4.5 Hz, 1H), 8.16 (d, J ¼ 7.8 Hz, 1H), 8.07 (td,
J ¼ 7.7, 1.6 Hz, 1H), 8.01 (s, 1H), 7.82 (d, J ¼ 8.3 Hz, 2H), 7.72e7.60 (m,
1H), 7.39 (dd, J ¼ 14.6, 5.3 Hz, 2H), 7.33 (dd, J ¼ 10.5, 5.0 Hz, 2H), 7.12
(d, J ¼ 1.3 Hz, 1H), 7.04 (d, J ¼ 8.2 Hz, 1H), 4.48e4.06 (m, 3H), 3.01
(dd, J ¼ 13.6, 4.3 Hz, 1H), 2.92e2.77 (m, 1H), 1.65e1.13 (m, 12H).
4.1.69. Synthesis of ethyl (S)-5-{2-[(tert-butoxycarbonyl)amino]-3-
{4-[(cyclobutylcarbonyl)amino]phenyl}propanamido)-1H-indole-2-
carboxylate (41h)
4.1.74. Synthesis of ethyl (S)-5-{2-[(tert-butoxycarbonyl) amino]-3-
{4-{[(pyrimidine-2-yl)carbonyl]amino}phenyl}propanamido}-1H-
indole-2-carboxylate (41m)
Compound 41h was prepared from commercially available
cyclobutanecarboxylic acid in the same manner as described for
41a. A yellow solid, yield: 73.7%. Mp 189.2e190.9ꢁС. ESI-MS (m/
Compound 41m was prepared from commercially available
pyrimidine-2-carboxylic acid in the same manner as described for
41a. An off-white solid, yield: 65.8%. Mp 236.3e238.0ꢁС. 1H NMR
z) ¼ 571.44 [M þ Na] þ; 1H NMR (400 MHz, DMSO‑d6)
d
11.82 (s,1H),
(400 MHz, DMSO‑d6) d 11.80 (s, 1H), 10.66 (s, 1H), 9.92 (s, 1H), 9.03
9.98 (d, J ¼ 14.9 Hz, 1H), 9.68 (s, 1H), 8.08e7.89 (m, 1H), 7.60e7.44
(m, 2H), 7.44e7.31 (m, 2H), 7.23 (d, J ¼ 8.4 Hz, 2H), 7.15e7.07 (m,
1H), 6.99 (t, J ¼ 9.0 Hz, 1H), 4.40e4.23 (m, 3H), 3.31e3.10 (m, 1H),
3.04e2.92 (m, 1H), 2.86e2.76 (m, 1H), 2.28e1.71 (m, 6H), 1.50e1.13
(m, 12H).
(d, J ¼ 4.9 Hz, 2H), 8.01 (s, 1H), 7.79 (d, J ¼ 8.3 Hz, 2H), 7.73 (t,
J ¼ 4.9 Hz, 1H), 7.45e7.27 (m, 4H), 7.12 (d, J ¼ 1.5 Hz, 1H), 7.05 (d,
J ¼ 8.2 Hz, 1H), 4.34 (q, J ¼ 7.0 Hz, 3H), 3.02 (dd, J ¼ 13.6, 4.4 Hz, 1H),
2.91e2.81 (m, 1H), 1.45e1.08 (m, 12H).
4.1.75. Synthesis of ethyl (S)-5-{3-[(4-acetylamino)phenyl]-2-
aminopropanamido}-1H-indole-2-carboxylate (42a)
Compound 42a was prepared from 41a in the same manner as
described for 26, and it was used for the next reaction without
further purification.
4.1.70. Synthesis of ethyl (S)-5-{2-[(tert-butoxycarbonyl)amino]-3-
{4-[(cyclopentylcarbonyl)amino]phenyl}propanamido}-1H-indole-
2-carboxylate (41i)
Compound 41i was prepared from commercially available
cyclopentanecarboxylic acid in the same manner as described for
41a. An off-yellow solid, yield: 77.3%. Mp 237.9e239.2ꢁС. ESI-MS
4.1.76. Synthesis of ethyl (S)-5-{2-amino-3-{4-[(isopropylcarbonyl)
amino]phenyl}propanamido}-1H-indole-2-carboxylate (42b)
Compound 42b was prepared from 41b in the same manner as
þ
(m/z) ¼ 585.48[M þ Na]
; d 11.80
1H NMR (400 MHz, DMSO‑d6)
(d, J ¼ 1.4 Hz, 1H), 9.88 (s, 1H), 9.76 (s, 1H), 7.99 (s, 1H), 7.50 (d,
J ¼ 8.4 Hz, 2H), 7.43e7.31 (m, 2H), 7.22 (d, J ¼ 8.3 Hz, 2H), 7.12 (d,
J ¼ 1.5 Hz, 1H), 6.99 (d, J ¼ 8.2 Hz, 1H), 4.34 (d, J ¼ 7.1 Hz, 3H), 2.94
(d, J ¼ 4.6 Hz, 1H), 2.86e2.67 (m, 2H), 1.83 (d, J ¼ 8.0 Hz, 2H),
1.76e1.62 (m, 4H), 1.55 (d, J ¼ 2.6 Hz, 2H), 1.35 (s, 12H).
described for 26. An off-white solid, yield: 82.4%. Mp
þ
275.3e276.8ꢁС. ESI-MS (m/z) ¼ 459.33[M þ Na]
;
1H NMR
(400 MHz, DMSO‑d6) d 11.79 (s, 1H), 9.76 (s, 2H), 8.01 (s, 1H), 7.50 (d,
J ¼ 7.8 Hz, 2H), 7.36 (s, 2H), 7.16 (d, J ¼ 7.7 Hz, 2H), 7.09 (s, 1H),
4.85e4.02 (m, 2H), 3.54 (s, 1H), 2.96 (d, J ¼ 8.9 Hz, 1H), 2.75e2.63
(m, 1H), 2.61e2.54 (m, 1H), 1.34 (t, J ¼ 6.8 Hz, 3H), 1.08 (d, J ¼ 6.5 Hz,
6H).
4.1.71. Synthesis of ethyl (S)-5-{2-[(tert-butoxycarbonyl)amino]-3-
{4-[(cyclohexylcarbonyl)amino]phenyl}propanamido}-1H-indole-2-
carboxylate (41j)
Compound 41j was prepared from commercially available
cyclohexanecarboxylic acid in the same manner as described for
41a. An off-white solid, yield: 81.2%. Mp 252.3e253.7ꢁС. ESI-MS (m/
4.1.77. Synthesis of ethyl (S)-5-{2-amino-3-{4-[(isobutylcarbonyl)
amino]phenyl}propanamido}-1H-indole-2-carboxylate (42c)
Compound 42c was prepared from 41c in the same manner as
z) ¼ 599.49[M þ Na] þ; 1H NMR (400 MHz, DMSO‑d6)
d
11.80 (s, 1H),
described for 26. An off-white solid, yield: 83.6%. Mp
-
9.87 (s, 1H), 9.69 (s, 1H), 7.98 (s, 1H), 7.50 (d, J ¼ 8.3 Hz, 2H), 7.36 (dt,
J ¼ 10.3, 5.2 Hz, 2H), 7.22 (d, J ¼ 8.2 Hz, 2H), 7.12 (s, 1H), 6.98 (d,
J ¼ 8.1 Hz, 1H), 4.34 (d, J ¼ 7.1 Hz, 3H), 2.95 (dd, J ¼ 13.5, 4.5 Hz, 1H),
2.87e2.71 (m, 1H), 2.30 (dd, J ¼ 15.5, 7.3 Hz, 1H), 1.76 (t, J ¼ 11.5 Hz,
4H), 1.65 (d, J ¼ 10.6 Hz, 1H), 1.51e1.07 (m, 17H).
237.1e238.6ꢁС. ESI-MS (m/z)
¼
449.45[M
ꢀ
H]
;
1H NMR
(400 MHz, DMSO‑d6) d 11.79 (s,1H), 9.75 (s, 2H), 8.00 (s,1H), 7.49 (d,
J ¼ 7.7 Hz, 2H), 7.37 (t, J ¼ 6.9 Hz, 2H), 7.16 (d, J ¼ 7.6 Hz, 2H), 7.10 (s,
1H), 4.34 (d, J ¼ 6.8 Hz, 2H), 3.58 (s, 1H), 2.97 (d, J ¼ 8.3 Hz, 1H),
2.78e2.66 (m, 1H), 2.15 (d, J ¼ 6.6 Hz, 2H), 2.04 (dd, J ¼ 10.7, 8.0 Hz,
1H), 1.34 (t, J ¼ 6.7 Hz, 3H), 0.92 (d, J ¼ 6.0 Hz, 6H).
4.1.72. Synthesis of ethyl (S)-5-{3-{4-[(phenylcarbonyl)amino]
phenyl}-2-[(tert-butoxycarbonyl)amino]propanamido}-1H-indole-
2-carboxylate (41k)
4.1.78. Synthesis of ethyl (S)-5-{2-amino-3-{4-[(ethynylcarbonyl)
amino]phenyl}propanamido}-1H-indole-2-carboxylate (42d)
Compound 42d was prepared from 41d in the same manner as
described for 26. An off-white solid, yield: 72.3%. Mp
Compound 41k was prepared from commercially available
benzoic acid in the same manner as described for 41a. An off-white
solid, yield: 72.7%. Mp 225.4e226.9ꢁС. ESI-MS (m/z) ¼ 593.47[M þ
217.6e219.3ꢁС. 1H NMR (400 MHz, DMSO‑d6)
d 11.90 (s, 1H), 10.73
þ
Na]
;
1H NMR (400 MHz, DMSO‑d6)
d
11.80 (s, 1H), 10.17 (s, 1H),
(s, 1H), 7.99 (s, 1H), 7.64 (dd, J ¼ 16.3, 9.1 Hz, 2H), 7.41 (dd, J ¼ 14.2,
7.7 Hz, 3H), 7.32e7.23 (m, 1H), 7.13 (s, 1H), 4.56e4.08 (m, 3H), 3.87
(s, 1H), 3.20 (dd, J ¼ 13.5, 6.2 Hz, 1H), 3.15e2.96 (m, 1H), 1.34 (t,
J ¼ 6.7 Hz, 3H).
9.91 (s, 1H), 8.00 (s, 1H), 7.97e7.90 (m, 2H), 7.69 (d, J ¼ 8.4 Hz, 2H),
7.58 (d, J ¼ 7.2 Hz, 1H), 7.53 (t, J ¼ 7.3 Hz, 2H), 7.39 (s, 1H), 7.37 (d,
J ¼ 1.8 Hz,1H), 7.30 (d, J ¼ 8.3 Hz, 2H), 7.12 (d, J ¼ 1.6 Hz,1H), 7.03 (d,
17

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
4.1.79. Synthesis of ethyl (S)-5-{2-amino-3-{4-{[(prop-1-yn-1-yl)
carbonyl]amino}phenyl}propanamido}-1H-indole-2-carboxylate
(42e)
4.1.84. Synthesis of ethyl (S)-5-{2-amino-3-{4-
[(cyclohexylcarbonyl)amino]phenyl}propanamido}-1H-indole-2-
carboxylate (42j)
Compound 42e was prepared from 41e in the same manner as
described for 26. An off-white solid, yield: 78.3%. Mp
Compound 42j was prepared from 41j in the same manner as
described for 26. A white solid, yield: 82.5%. Mp > 280.0ꢁС. ESI-MS
209.6e210.9ꢁС. ESI-MS (m/z)
¼
455.31[M
þ
Na]þ; 1H NMR
(m/z) ¼ 477.41 [M þ H] þ; 1H NMR (400 MHz, DMSO‑d6)
d 11.78 (s,
(400 MHz, DMSO‑d6)
d
11.88 (s, 1H), 10.79 (s, 1H), 10.61 (s, 1H), 8.45
1H), 9.69 (s, 2H), 8.01 (d, J ¼ 6.8 Hz, 1H), 7.49 (d, J ¼ 8.4 Hz, 2H), 7.37
(t, J ¼ 6.2 Hz, 2H), 7.21e7.04 (m, 3H), 4.34 (q, J ¼ 7.1 Hz, 2H), 3.53 (s,
1H), 2.95 (dd, J ¼ 13.5, 5.5 Hz, 1H), 2.79e2.60 (m, 1H), 2.31 (d,
J ¼ 14.9 Hz, 1H), 1.76 (s, 4H), 1.42e1.15 (m, 9H).
(s, 3H), 7.97 (s, 1H), 7.54 (d, J ¼ 8.4 Hz, 2H), 7.40 (dt, J ¼ 9.0, 5.3 Hz,
2H), 7.29 (d, J ¼ 8.4 Hz, 2H), 7.13 (d, J ¼ 1.5 Hz, 1H), 4.47e4.22 (m,
3H), 3.20 (dd, J ¼ 13.8, 6.6 Hz,1H), 3.12 (dd, J ¼ 13.8, 7.0 Hz,1H), 2.03
(s, 3H), 1.34 (t, J ¼ 7.1 Hz, 3H).
4.1.85. Synthesis of ethyl (S)-5-{2-amino-3-{4-[(phenylcarbonyl)
amino]phenyl}propanamido}-1H-indole-2-carboxylate (42k)
Compound 42k was prepared from 41k in the same manner as
4.1.80. Synthesis of ethyl (S)-5-{2-amino-3-{4-
[(cyclopropylcarbonyl)amino]phenyl}propanamido}-1H-indole-2-
carboxylate (42f)
described for 26. An off-white solid, yield: 84.5%. Mp
þ
278.4e280.1ꢁС. ESI-MS (m/z) ¼ 471.35 [M þ H]
;
1H NMR
Compound 42f was prepared from 41f in the same manner as
described for 26. An off-white solid, yield: þ81.9%. Mp
(400 MHz, DMSO‑d6) d 11.87 (s, 1H), 10.77 (s, 1H), 10.28 (s, 1H), 8.48
(s, 3H), 8.01e7.90 (m, 3H), 7.75 (d, J ¼ 8.5 Hz, 2H), 7.58 (dd, J ¼ 8.4,
6.0 Hz, 1H), 7.52 (t, J ¼ 7.4 Hz, 2H), 7.46e7.35 (m, 2H), 7.32 (d,
J ¼ 8.5 Hz, 2H), 7.14 (d, J ¼ 2.0 Hz, 1H), 4.34 (dd, J ¼ 14.2, 7.1 Hz, 3H),
3.23 (dd, J ¼ 13.8, 6.8 Hz, 1H), 3.15 (dd, J ¼ 13.8, 7.0 Hz, 1H), 1.34 (t,
J ¼ 7.1 Hz, 3H).
212.3e214.0ꢁС. ESI-MS (m/z) ¼ 435.34[M þ H]
;
1H NMR
d 11.82 (s, 1H), 10.24 (s, 1H), 8.01 (s, 1H), 7.50
(400 MHz, DMSO‑d6)
(d, J ¼ 8.3 Hz, 2H), 7.37 (s, 2H), 7.16 (d, J ¼ 8.4 Hz, 2H), 7.09 (s, 1H),
4.34 (q, J ¼ 7.1 Hz, 2H), 3.61e3.47 (m, 1H), 2.96 (dd, J ¼ 13.4, 5.3 Hz,
1H), 2.68 (dd, J ¼ 13.3, 8.1 Hz, 1H), 1.80 (m, 1H), 1.34 (t, J ¼ 7.1 Hz,
4H), 0.76 (t, J ¼ 6.8 Hz, 4H).
4.1.86. Synthesis of ethyl (S)-5-{2-amino-3-{4-{[(pyridin-2-yl)
carbonyl]amino}phenyl}propanamido}-1H-indole-2-carboxylate
(42l)
4.1.81. Synthesis of ethyl 5-{(2S)-2-amino-3-{4-{[(2-
methylcyclopropyl)carbonyl]amino}phenyl}propanamido}-1H-
indole-2-carboxylate (42g)
Compound 42l was prepared from 41l in the same manner as
described for 26. An off-white solid, yield: 86.3%. Mp
þ
200.9e202.2ꢁС. ESI-MS (m/z) ¼ 472.36 [M þ H]
;
1H NMR
Compound 42g was prepared from 41g in the same manner as
(400 MHz, DMSO‑d6) d 11.82 (s, 1H), 10.54 (s, 1H), 9.87 (s, 1H), 8.73
described for 26. A white solid, yield: 76.1%. Mp 271.7e273.0ꢁС. ESI-
(d, J ¼ 4.4 Hz, 1H), 8.15 (d, J ¼ 7.7 Hz, 1H), 8.06 (dd, J ¼ 15.1, 6.2 Hz,
2H), 7.81 (d, J ¼ 8.3 Hz, 2H), 7.73e7.60 (m, 1H), 7.38 (s, 2H), 7.26 (d,
J ¼ 8.3 Hz, 2H), 7.10 (s, 1H), 4.33 (q, J ¼ 7.0 Hz, 2H), 3.59 (s, 1H), 3.01
(dd, J ¼ 13.3, 5.3 Hz, 1H), 2.73 (dd, J ¼ 13.3, 7.8 Hz, 1H), 1.34 (t,
J ¼ 7.1 Hz, 3H).
MS (m/z) ¼ 449.38 [M þ H]þ; 1H NMR (400 MHz, DMSO‑d6)
d 11.79
(s, 1H), 10.04 (s, 1H), 8.01 (s, 1H), 7.47 (d, J ¼ 8.3 Hz, 2H), 7.41e7.27
(m, 2H), 7.18 (t, J ¼ 14.6 Hz, 2H), 7.10 (s, 1H), 4.34 (q, J ¼ 7.1 Hz, 2H),
3.69e3.44 (m, 1H), 2.96 (dd, J ¼ 13.4, 5.3 Hz, 1H), 2.68 (dd, J ¼ 13.5,
7.8 Hz, 1H), 1.50 (dt, J ¼ 8.1, 4.1 Hz, 1H), 1.34 (t, J ¼ 7.1 Hz, 3H), 1.10 (t,
J ¼ 6.2 Hz, 4H), 0.98 (dt, J ¼ 8.1, 3.9 Hz, 1H), 0.60 (dd, J ¼ 11.4, 5.8 Hz,
1H).
4.1.87. Synthesis of ethyl (S)-5-{2-amino-3-{4-{[(pyrimidin-2-yl)
carbonyl]amino}phenyl}propanamido}-1H-indole-2-carboxylate
(42m)
Compound 42m was prepared from 41m in the same manner as
4.1.82. Synthesis of ethyl (S)-5-{2-amino-3-{4-
described for 26. An off-white solid, yield: 81.3%. Mp > 280ꢁС. 1H
[(cyclobutylcarbonyl)amino]phenyl}propanamido}-1H-indole-2-
carboxylate (42h)
NMR (400 MHz, DMSO‑d6) d 11.79 (s, 1H), 10.66 (s, 1H), 9.78 (s, 1H),
9.03 (d, J ¼ 4.9 Hz, 2H), 8.03 (s, 1H), 7.90e7.59 (m, 3H), 7.37 (s, 2H),
7.26 (d, J ¼ 8.3 Hz, 2H), 7.10 (s,1H), 4.34 (q, J ¼ 7.1 Hz, 2H), 3.66e3.50
(m, 1H), 3.02 (dd, J ¼ 13.4, 5.4 Hz, 1H), 2.74 (dd, J ¼ 13.4, 7.8 Hz, 1H),
1.34 (t, J ¼ 7.1 Hz, 3H).
Compound 42h was prepared from 41h in the same manner as
described for 26. A white solid, yield: 75.8%. Mp 251.3e252.7ꢁС. ESI-
MS (m/z) ¼ 449.36 [M þ H] þ; 1H NMR (400 MHz, DMSO‑d6)
d 11.81
(s, 1H), 9.65 (s, 1H), 8.00 (t, J ¼ 9.0 Hz, 1H), 7.49 (t, J ¼ 8.1 Hz, 2H),
7.43e7.28 (m, 2H), 7.16 (d, J ¼ 8.4 Hz, 1H), 7.10 (d, J ¼ 6.1 Hz, 1H),
6.90 (d, J ¼ 8.2 Hz,1H), 6.47 (t, J ¼ 7.5 Hz,1H), 4.34 (q, J ¼ 7.1 Hz, 2H),
3.25e3.16 (m, 1H), 2.96 (dd, J ¼ 13.4, 5.5 Hz, 1H), 2.69 (dd, J ¼ 13.5,
7.9 Hz, 1H), 2.60e2.54 (m, 1H), 2.29e1.69 (m, 6H), 1.34 (t, J ¼ 7.1 Hz,
3H).
4.1.88. Synthesis of ethyl (S)-5-{3-phenyl-2-[2-(3-chloro-2-
fluorophenyl)-1-hydroxy-4-methyl-1H-imidazole-5-carboxamido]
propanamido}-1H-indole-2-carboxylate (43a-m)
Compound 43a was prepared from 42a in the same manner as
described for 27a-27k, and they were used for the next reaction
without further purification.
4.1.83. Synthesis of ethyl (S)-5-{2-amino-3-{4-
[(cyclopentylcarbonyl)amino]phenyl}propanamido}-1H-indole-2-
carboxylate (42i)
4.1.89. Synthesis of (S)-5-{3-(4-acetylaminophenyl)-2-[2-(3-
chloro-2-fluorophenyl)-1-hydroxy-4-methyl-1H-imidazole-5-
carboxamido]propanamido}-1H-indole-2-carboxylic acid (44a)
Compound 44a was prepared from 43a in the same manner as
described for 28a-28k. An off-white solid, yield: 17.33%. Mp
173.6e174.7ꢁС. ESI-HRMS calcd for C31H27ClFN6O6 [M þ H]þ
Compound 42i was prepared from 41i in the same manner as
described for 26. A white solid, yield: 74.3%. Mp 277.2e278.9ꢁС.
þ
ESI-MS (m/z) ¼ 463.38 [M þ H]
;
1H NMR (400 MHz, DMSO‑d6)
d
11.78 (s, 1H), 9.77 (s, 2H), 8.00 (s, 1H), 7.50 (d, J ¼ 8.3 Hz, 2H),
633.1659 found: 633.1524; 1H NMR (600 MHz, DMSO‑d6)
d 11.15 (s,
7.41e7.28 (m, 2H), 7.16 (d, J ¼ 8.4 Hz, 2H), 7.09 (s, 1H), 4.33 (q,
J ¼ 7.1 Hz, 2H), 3.54 (dd, J ¼ 7.5, 5.7 Hz,1H), 2.95 (dd, J ¼ 13.4, 5.3 Hz,
1H), 2.83e2.54 (m, 2H), 1.95e1.76 (m, 2H), 1.69 (dd, J ¼ 13.3, 5.8 Hz,
4H), 1.59e1.45 (m, 2H), 1.34 (t, J ¼ 7.1 Hz, 3H).
1H), 10.04 (d, J ¼ 10.4 Hz, 2H), 8.33 (d, J ¼ 8.0 Hz, 1H), 7.96 (s, 1H),
7.88 (s, 1H), 7.55 (s, 1H), 7.49 (d, J ¼ 7.4 Hz, 2H), 7.38e7.09 (m, 5H),
6.73 (s, 1H), 4.62 (d, J ¼ 4.3 Hz, 1H), 3.00 (d, J ¼ 13.3 Hz, 1H),
2.86e2.74 (m, 1H), 2.32 (s, 3H), 2.02 (s, 3H). 13C NMR (100 MHz,
18

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
DMSO‑d6)
d
176.67, 170.37, 161.68, 155.26, 153.57, 138.24, 138.04,
(s, 1H), 7.87 (s, 1H), 7.61e7.40 (m, 3H), 7.29 (d, J ¼ 8.5 Hz, 1H), 7.20
(dd, J ¼ 13.3, 8.0 Hz, 4H), 6.76 (s, 1H), 4.81 (d, J ¼ 6.8 Hz, 1H),
136.05, 133.88, 133.11, 131.78, 130.03, 129.61, 129.38, 128.85, 127.50,
125.23, 124.68, 120.44, 120.37, 119.96, 117.82, 112.44, 112.32, 112.20,
112.11, 105.47, 54.89, 38.55, 15.88.
3.10e2.99 (m, 1H), 2.94e2.85 (m, 1H), 2.31 (s, 3H), 1.76 (s, 3H). 13
C
NMR (100 MHz, DMSO‑d6)
d 170.42, 169.67, 165.58, 163.17, 161.18,
155.42, 153.73, 134.64, 132.93, 131.99, 129.94, 129.86, 129.39, 127.12,
126.15, 126.12, 121.06, 120.95, 119.30, 119.21, 117.93, 115.27, 112.89,
112.45, 107.78, 95.20, 63.47, 52.78, 37.89, 14.69, 12.50.
4.1.90. Synthesis of (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-{4-
[(isopropylcarbonyl)amino]phenyl}propanamido}-1H-indole-2-
carboxylic acid (44b)
Compound 44b was prepared from 43b in the same manner as
4.1.94. Synthesis of (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-{4-
[(cyclopropylcarbonyl)amino]phenyl}propanamido}-1H-indole-2-
carboxylic acid (44f)
described for 28a-28k.
A
white solid, yield: 18.14%.
Mp173.1e174.5 ꢁC. ESI-HRMS calcd for C33H30ClFN6O6 [M þ Na]þ
683.1792 found: 683.1797; 1H NMR (600 MHz, DMSO‑d6)
d 10.93 (s,
1H), 9.96 (d, J ¼ 8.6 Hz, 1H), 7.92 (d, J ¼ 8.7 Hz, 2H), 7.84 (d,
J ¼ 8.7 Hz, 2H), 7.68 (dd, J ¼ 14.4 Hz, 1H), 7.52 (t, J ¼ 7.9 Hz, 1H), 7.39
(d, J ¼ 7.5 Hz, 2H), 7.26 (t, J ¼ 7.5 Hz, 3H), 7.19 (d, J ¼ 7.8 Hz, 2H), 5.32
(t, J ¼ 4.7 Hz, 1H), 4.37 (d, J ¼ 11.5 Hz, 1H), 3.16 (d, J ¼ 7.0 Hz, 1H),
3.06 (d, J ¼ 9.1 Hz, 1H), 2.39 (s, 3H), 1.23 (s, 6H). 13C NMR (150 MHz,
Compound 44f was prepared from 43f in the same manner as
described for 28a-28k.
A yellow solid, yield: 14.83%. Mp
200.2e201.9ꢁС. ESI-HRMS calcd for C33H28ClFN6O6 [M ꢀ H]-
657.1670 found: 657.1672; 1H NMR (600 MHz, DMSO‑d6)
d 11.41 (s,
1H), 10.12e9.79 (m, 2H), 8.28 (s, 1H), 7.95 (d, J ¼ 14.8 Hz, 2H),
7.60e7.45 (m, 2H), 7.27 (tdd, J ¼ 15.0, 14.7, 8.1 Hz, 4H), 6.88 (s, 1H),
4.92e4.80 (m, 1H), 3.15e3.04 (m, 1H), 2.96 (dd, J ¼ 15.5, 8.2 Hz, 1H),
2.35 (d, J ¼ 14.5 Hz, 3H), 1.23 (s, 3H), 0.85 (t, J ¼ 6.8 Hz, 1H). 13C NMR
DMSO‑d6)
d 175.55, 169.98, 163.48, 160.23, 155.20, 153.52, 138.30,
134.48, 132.41, 132.03, 131.07, 130.30, 129.83, 129.51, 127.18, 125.50,
125.48, 120.70, 120.63, 120.58, 119.41, 118.92, 112.78, 112.41, 107.27,
54.86, 38.48, 35.30, 19.96, 14.41.
(100 MHz, DMSO‑d6) d 171.93,169.68,168.49,167.44,163.80,162.80,
157.77, 138.34, 133.72, 133.57, 132.16, 132.01, 130.74, 129.87, 129.42,
129.13, 127.31, 125.92, 121.59, 120.80, 120.69, 119.26, 114.11, 112.86,
110.85, 55.40, 38.52, 14.91, 14.02, 7.49.
4.1.91. Synthesis of (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-{4-
[(isobutylcarbonyl)amino]phenyl}propanamido}-1H-indole-2-
carboxylic acid (44c)
Compound 44c was prepared from 43c in the same manner as
4.1.95. Synthesis of (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-{4-[(2-
methylcyclopropylcarbonyl)amino]phenyl}propanamido}-1H-
indole-2-carboxylic acid (44g)
described for 28a-28k.
177.7e179.2ꢁС. ESI-HRMS calcd for C27H23ClN4O5 [M
673.1983 found: 673.1973; 1H NMR (400 MHz, DMSO‑d6)
A
white solid, yield: 10.69%. Mp
H]-
11.68 (s,
ꢀ
d
1H), 10.10 (s, 1H), 8.16 (s, 1H), 8.11e7.92 (m, 2H), 7.77e7.68 (m, 2H),
7.48 (d, J ¼ 8.6 Hz, 2H), 7.40e7.31 (m, 3H), 7.21 (d, J ¼ 8.2 Hz, 2H),
7.04 (s, 1H), 4.88 (t, J ¼ 5.7 Hz,1H), 3.14 (dd, J ¼ 11.0, 7.1 Hz, 1H), 2.98
(d, J ¼ 10.9 Hz, 1H), 2.42 (s,3H), 1.68e1.55 (m, 4H), 0.91 (t, J ¼ 7.4 Hz,
Compound 44g was prepared from 43g in the same manner as
described for 28a-28k. An off yellow solid, yield: 15.32%. Mp
203.9e205.4ꢁС. ESI-HRMS calcd for C34H30ClFN6O6 [M þ Na]þ
695.1792 found: 695.1809; 1H NMR (400 MHz, DMSO‑d6)
d 11.50 (s,
6H). 13C NMR (100 MHz, DMSO‑d6)
d 169.61, 167.80, 163.15, 161.69,
1H),10.1 (d, J ¼ 7.1 Hz,1H) 9.99 (s,1H), 8.36 (d, J ¼ 7.1 Hz,1H) 8.26 (s,
1H), 8.14 (d, J ¼ 7.3 Hz, 1H) 7.95 (d, J ¼ 7.6 Hz, 1H) 7.55 (t, J ¼ 8.1 Hz,
1H) 7.32 (d, J ¼ 8.3 Hz, 2H), 7.27e7.21 (m, 3H), 7.18 (t, J ¼ 14.6 Hz,
3H), 6.92 (s, 1H), 4.83 (q, J ¼ 7.1 Hz, 1H), 3.08 (m, 1H), 2.92 (dd,
J ¼ 13.4, 5.3 Hz, 1H), 2.37 (s, 1H), 1.50 (dt, J ¼ 8.1, 4.1 Hz, 1H), 1.34 (d,
J ¼ 7.1 Hz, 1H), 1.10 (t, J ¼ 6.2 Hz, 3H), 0.98 (dt, J ¼ 8.1, 3.9 Hz, 1H),
157.53, 155.48, 153.80, 138.05, 134.73, 132.17, 131.67, 131.11, 129.76,
129.13, 127.11, 126.04, 125.74, 125.71, 120.96, 120.85, 119.38, 117.69,
113.37, 112.31, 118.11, 55.48, 46.06, 38.55, 27.02, 22.77, 14.78.
4.1.92. Synthesis of (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-{4-
[(ethynylcarbonyl)amino]phenyl}propanamido}-1H-indole-2-
carboxylic acid (44d)
0.60 (d, J ¼ 11.4, 5.8 Hz, 1H). 13C NMR (100 MHz, DMSO‑d6)
d 170.40,
169.02, 162.75, 161.70, 154.14, 152.46, 145.72, 138.70, 137.17, 133.78,
133.19, 131.38, 130.88, 128.78, 128.36, 126.44, 126.18, 124.06, 119.50,
119.39, 119.28, 118.04, 117.48, 111.56, 111.20, 53.73, 37.45, 22.33,
16.93, 14.74, 14.47, 11.19.
Compound 44d was prepared from 43d in the same manner as
described for 28a-28k.
A white solid, yield: 14.01%. Mp
183.6e184.7ꢁС. ESI-HRMS calcd for C32H24ClFN6O6 [M ꢀ H]-
641.1357 found: 641.5392; 1H NMR (400 MHz, DMSO‑d6)
d 10.80 (s,
1H), 8.51 (d, J ¼ 7.8 Hz, 1H), 8.28 (d, J ¼ 11.6 Hz, 2H), 7.91 (dd,
J ¼ 13.7, 7.7 Hz, 3H), 7.74 (t, J ¼ 7.8 Hz, 1H), 7.65 (d, J ¼ 8.2 Hz, 2H),
7.40 (dd, J ¼ 13.3, 8.3 Hz, 4H), 4.94 (dd, J ¼ 12.4, 9.3 Hz, 1H), 3.91 (s,
1H), 3.26 (dd, J ¼ 13.7, 3.8 Hz, 1H), 3.19e3.04 (m, 1H), 2.23 (s, 3H).
4.1.96. Synthesis of (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-{4-
[(cyclobutylcarbonyl)amino]phenyl}propanamido}-1H-indole-2-
carboxylic acid (44h)
13C NMR (150 MHz, DMSO‑d6)
d173.42, 170.33, 169.71, 166.25,
158.77, 140.89, 139.31, 138.91, 138.73, 138.35, 137.17, 133.36, 131.72,
131.67, 130.84, 130.49, 130.36, 129.33, 128.59, 127.97, 120.67, 120.22,
118.77, 118.66, 112.67, 68.02, 67.82, 55.68, 37.07, 15.14.
Compound 44h was prepared from 43h in the same manner as
described for 28a-28k. An off yellow solid, yield: 17.77%. Mp
192.2e194.0ꢁС. ESI-HRMS calcd for C34H30ClFN6O6 [M þ Na]þ
695.1792 found: 695.1788; 1H NMR (600 MHz, DMSO‑d6)
d 13.15 (s,
4.1.93. Synthesis of (S)-5-{3-{4-{[(prop-1-yn-1-yl)carbonyl]amino}
phenyl}-2-[2-(3-chloro-2-fluorophenyl)-1-hydroxy-4-methyl-1H-
imidazole-5-carboxamido]propanamido}-1H-indole-2-carboxylic
acid (44e)
1H), 11.70 (s, 1H), 10.07 (s, 1H), 9.63 (s, 1H), 8.40 (s, 1H), 8.02 (d,
J ¼ 16.6 Hz, 2H), 7.76 (s, 2H), 7.40 (m, 7H), 7.20 (s, 2H), 7.05 (s, 1H),
4.90 (s, 1H), 3.15 (d, J ¼ 11.1 Hz, 4H), 2.96 (s, 2H), 2.19 (s, 2H), 2.07 (s,
2H), 1.92 (s, 1H), 1.79 (s, 1H). 13C NMR (150 MHz, DMSO‑d6)
d173.16,
Compound 44e was prepared from 43e in the same manner as
169.58, 163.17, 161.13, 159.36, 155.41, 153.73, 138.29, 134.66, 132.97,
132.35, 132.14, 132.06, 129.85, 129.42, 127.12, 126.16, 126.03, 122.37,
120.83, 119.42, 119.15, 117.93, 112.91, 112.47, 107.77, 54.71, 38.54,
25.04, 18.18, 12.46, 11.92.
described for 28a-28k.
A white solid, yield: 12.38%. Mp
181.1e182.8ꢁС. ESI-MS (m/z) ¼ 655.36 [M ꢀ H]-; 1H NMR (600 MHz,
DMSO‑d6) d 11.71 (s, 1H), 11.19 (s, 1H), 10.16 (s, 1H), 9.87 (s, 1H), 8.35
19

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
4.1.97. Synthesis of (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-{4-
[(isopentylcarbonyl)amino]phenyl}propanamido}-1H-indole-2-
carboxylic acid (44i)
134.71, 133.36, 132.53, 132.45, 132.08, 130.64, 129.94, 129.45, 127.30,
127.16, 125.02, 122.76, 121.04, 120.53, 119.23, 116.55, 116.34, 112.94,
112.57, 107.80, 54.73, 38.59, 12.07.
Compound 44i was prepared from 43i in the same manner as
4.1.101. Synthesis of (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-{4-
{[(pyrimidin-2-yl)carbonyl]amino}phenyl}propanamido}-1H-
indole-2-carboxylic acid (44m)
described for 28a-28k.
A white solid, yield: 16.21%. Mp
186.3e187.8ꢁС. ESI-HRMS calcd for C35H32ClFN6O6 [M ꢀ H]-
685.1983 found: 685.2048; 1H NMR (400 MHz, DMSO‑d6)
d 11.09 (s,
1H), 10.05 (s, 1H), 9.92 (s, 1H), 8.34 (d, J ¼ 8.3 Hz, 1H), 7.92 (d,
J ¼ 10.9 Hz, 2H), 7.53 (d, J ¼ 7.9 Hz, 3H), 7.38e7.07 (m, 4H), 6.73 (s,
1H), 4.62 (t, J ¼ 14.3, 8.8 Hz, 1H), 3.01 (dd, J ¼ 13.9, 4.1 Hz, 1H), 2.81
(dd, J ¼ 14.6, 9.8 Hz, 1H), 2.47 (s, 3H), 2.41e2.24 (m, 1H), 1.76 (m,
Compound 44m was prepared from 43m in the same manner as
described for 28a-28k. An off yellow solid, yield: 18.14%. Mp
181.1e182.6ꢁС. ESI-HRMS calcd for C34H26ClFN6O6 [M þ H]þ
697.1721 found: 697.1958; 1H NMR (600 MHz, DMSO‑d6)
d 11.85 (d,
4H), 1.22 (m, 4H). 13C NMR (150 MHz, DMSO‑d6)
d
173.52, 169.13,
J ¼ 1.3 Hz, 1H), 10.64 (s, 1H), 10.26 (s, 1H), 9.02 (d, J ¼ 4.9 Hz, 2H),
8.32 (d, J ¼ 7.8 Hz, 1H), 8.06 (s, 1H), 7.80 (d, J ¼ 8.5 Hz, 2H), 7.73 (t,
J ¼ 4.9 Hz, 1H), 7.72e7.64 (m, 1H), 7.36e7.32 (m, 1H), 7.27e7.18 (m,
2H), 7.08 (t, J ¼ 7.7 Hz, 2H), 6.86 (d, J ¼ 7.1 Hz, 2H), 4.98 (td, J ¼ 8.2,
5.3 Hz, 1H), 3.25 (dd, J ¼ 13.9, 4.9 Hz, 1H), 3.12 (dd, J ¼ 13.8, 9.3 Hz,
168.45, 164.06, 161.64, 160.10, 137.14, 132.34, 131.56, 130.29, 128.93,
128.66, 128.58, 127.60, 126.47, 118.95, 118.10, 118.03, 117.55, 117.24,
116.02, 115.99, 111.07, 110.88, 103.08, 54.41, 43.85, 36.67, 28.52,
24.50, 13.19.
1H), 2.29 (s, 3H). 13C NMR (150 MHz, DMSO‑d6)
d169.90, 161.69,
4.1.98. Synthesis of (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-{4-
[(isohexylcarbonyl)amino]phenyl}propanamido}-1H-indole-2-
carboxylic acid (44j)
158.97, 158.29, 155.76, 154.08, 138.75, 137.38, 137.16, 134.84, 133.71,
132.71, 132.26, 130.37, 130.20, 130.05, 129.69, 128.72, 128.63, 128.40,
127.07, 125.67, 123.60, 119.52, 113.15, 112.55, 108.14, 55.53, 37.63,
14.78.
Compound 44j was prepared from 43j in the same manner as
described for 28a-28k.
A
white solid, yield: 18.96%. Mp
4.1.102. Synthesis of trans-N-{(S)-1-[4-(3-amino-1H-indazol-6-yl)-
5-chloro-1H-imidazole-2-yl]-2-phenylethyl}-4-(aminomethyl)
cyclohexanecarboxamide, bis-hydrochloric acid salt (BMS-724296)
Compound BMS-724296 was synthesized by the reported
171.5e172.9 ꢁC. ESI-HRMS calcd for C36H34ClFN6O6 [M þ Na]þ
723.2105 found: 723.2103; 1H NMR (400 MHz, DMSO‑d6)
d 11.49 (s,
1H), 9.91 (s, 1H), 9.69 (s, 1H), 8.32 (s, 1H), 7.92 (s, 1H), 7.50 (m, 3H),
7.27 (m, 5H), 6.93 (s, 1H), 4.82 (dd, J ¼ 14.3, 7.2 Hz, 1H), 3.06 (dd,
J ¼ 13.4, 6.0 Hz, 1H), 2.91 (dd, J ¼ 12.5, 7.1 Hz, 1H), 2.39e2.17 (m,
4H), 1.83e1.67 (m, 4H), 1.50e1.05 (m, 6H). 13C NMR (100 MHz,
literature [2],1H NMR (400 MHz, DMSO‑d6)
d 12.70 (s, 1H), 10.57 (s,
1H), 8.39 (d, J ¼ 7.6 Hz, 1H), 8.18e8.04 (m, 2H), 7.70e7.59 (m, 1H),
7.31 (td, J ¼ 6.5, 3.3 Hz, 1H), 7.27e7.09 (m, 4H), 5.28e5.03 (m, 2H),
4.73 (dd, J ¼ 12.7, 9.6 Hz, 1H), 1.88e1.72 (m, 2H), 1.69e1.55 (m, 2H),
1.49e1.30 (m, 1H), 1.24 (s, 2H), 1.06e0.72 (m, 8H). ESI-MS (m/
DMSO‑d6)
d 174.54, 170.21, 163.77, 161.09, 155.22, 153.54, 138.30,
134.32, 132.61, 132.03, 130.02, 129.81, 129.65, 129.40, 127.26, 124.97,
120.56, 120.44, 120.22, 119.27, 118.65, 112.94, 112.67, 112.28, 106.81,
54.86, 45.28, 38.52, 29.59, 25.87, 25.70, 14.41.
þ
z) ¼ 492.31[M þ H]
4.2. Enzyme affinity assays
4.1.99. Synthesis of (S)-5-{3-{4-[(phenylcarbonyl)amino]phenyl}-2-
[2-(3-chloro-2-fluorophenyl)-1-hydroxy-4-methyl-1H-imidazole-5-
carboxamido]propanamido}-1H-indole-2-carboxylic acid (44k)
Compound 44k was prepared from 43k in the same manner as
described for 28a-28k. An off yellow solid, yield: 26.28%. Mp
163.4e165.1 ꢁC. ESI-HRMS calcd for C36H28ClFN6O6 [M ꢀ H]-
FXa, FXIa and substrate of FXIa (p-Glu-Pro-Arg-pNa, HCl) were
purchased from Haematologic Technologies [43]. The substrates
(CH3SO2-D-CHA-But-Arg-pNA, AcOH and CH3OCO-D-CHA-Gly-
Arg-pNA, AcOH) of FVIIa and FXa were purchased from Pentapharm
(Basel, Switzerland). The substrates (H-D-Phe-Pip-Arg-pNa, 2HCl
and D-Pro-Phe-Arg-pNa, 2HCl) of thrombin and plasma kallikrein
as well as thrombin were purchased from Hyphen BioMed. FVIIa
and plasma kallikrein were purchased from Enzyme Research
Laboratories.
According to manufacturer’s manual, FXIa was suspended in
0.9% NaCl for a concentration of 31.5 pM (stored at ꢀ20ꢁС).
Thrombin, FVIIa, FXa, plasma kallikrein and their corresponding
substrates were diluted with water to final concentrations of 4NIH/
mL, 0.025 mg/mL, 10 nM, 33.3 nM, 4 mM, 4 mM, 4 mM and
0.475 mM, respectively. The substrate of FXIa was dissolved and
693.1670 found: 693.1710; 1H NMR (600 MHz, DMSO‑d6)
d13.08 (s,
1H), 11.70 (s, 1H), 11.21 (s, 1H), 10.15 (d, J ¼ 13.8 Hz, 2H), 8.40 (s, 1H),
7.99 (s, 1H), 7.92 (d, J ¼ 6.7 Hz, 2H), 7.75 (s, 1H), 7.68 (d, J ¼ 7.3 Hz,
2H), 7.55 (m, 4H), 7.45e7.32 (m, 3H), 7.28 (d, J ¼ 7.4 Hz, 2H), 7.06 (s,
1H), 4.95 (d, J ¼ 6.0 Hz, 1H), 3.22e3.10 (m, 1H), 3.01 (s, 1H), 2.51 (s,
3H). 13C NMR (150 MHz, DMSO‑d6)
d 169.61, 165.88, 163.19, 159.41,
154.97, 153.31, 138.09, 135.48, 134.68, 132.99, 132.30, 132.07, 131.94,
129.84, 129.46, 128.82, 128.05, 127.14, 126.02, 121.20, 120.83, 120.72,
120.66, 119.17, 115.13, 112.94, 112.50, 107.78, 54.71, 38.59, 11.96.
4.1.100. Synthesis of (S)-5-{2-[2-(3-chloro-2-fluorophenyl)-1-
hydroxy-4-methyl-1H-imidazole-5-carboxamido]-3-{4-{[(pyridin-
2-yl)carbonyl]amino}phenyl}propanamido}-1H-indole-2-carboxylic
acid (44l)
diluted with 0.9% NaCl to provide the substrate solution (425 mM).
The buffers used by these enzymes: thrombin, FVIIa, FXa, FXIa and
plasma kallikrein, included a (50 mM Tris and 0.3 M NaCl, pH 8.4); b
(0.1 M Tris, 0.1 M NaCl, 5 mM CaCl2 and 0.1%BSA, pH 8.4); c (0.1 M
Tris, 0.2 M NaCl and 0.1%BSA, pH 7.4); d (50 mM Tris and 0.3 M NaCl,
Compound 44l was prepared from 43l in the same manner as
described for 28a-28k. An off yellow solid, yield: 17.32%. Mp
177.2e178.8 ꢁC. ESI-HRMS calcd for C35H27ClFN6O6 [M þ Na]þ
pH 7.4); e (50 mM Tris-HCl, 0.25 M NaCl and 1.5 mM BSA, pH 7.5). All
target compounds and positive control BMS-724296 were dis-
solved in DMSO (initial concentration: 10 mM), which were then
further diluted with the corresponding buffers to obtain the
required concentrations. The test protocols for serine proteases
718.1588 found: 718.1587; 1H NMR (400 MHz, DMSO‑d6)
d11.82 (d,
J ¼ 5.2 Hz, 1H),10.66 (d, J ¼ 3.9 Hz, 1H),10.08 (d, J ¼ 4.6 Hz,1H), 9.02
(dd, J ¼ 7.2, 4.9 Hz, 3H), 7.89e7.82 (m, 1H), 7.81e7.70 (m, 4H), 7.66
(d, J ¼ 8.4 Hz, 1H), 7.47e7.28 (m, 6H), 7.24 (d, J ¼ 8.4 Hz, 1H), 4.70
(dd, J ¼ 13.5, 7.8 Hz, 1H), 3.12 (dd, J ¼ 12.1, 8.9 Hz, 1H), 3.03 (d,
were different, as follows: 1. the buffer (100
sample (15 L) or blank solution (the buffer) were added to 96-well
plates (COSTAR 3599) and incubated at 37ꢁС for 5 min. Then, the
mixture was added with substrate (30 L) to the plates, which was
mL), FXIa (15 mL) and
m
J ¼ 5.9 Hz, 1H), 2.46 (s, 3H). 13C NMR (150 MHz, DMSO‑d6)
d169.66,
163.17, 162.77, 160.16, 159.47, 157.67, 150.37, 148.84, 138.55, 137.27,
m
20

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
further measured at 405 nm [20,43]; 2. the buffer (70
m
L), FVIIa
Studio Visualizer 2017 and Pymol [48] were used for graphic
display.
(10
added to 96-well plates (COSTAR 3599) and incubated at 37ꢁС for
10 min. Then, the mixture was added with substrate (10 L) to the
plates, which was further measured at 405 nm [44]; 3. the buffer
(70 L), plasma kallikrein (10 L) and sample (10 L) or blank so-
mL) and sample (10 mL) or blank solution (the buffer) were
m
4.4. Steady-state kinetic analysis
m
m
m
An enzyme kinetics study of the most potent compound 44g
was carried out to further evaluate and identify the type of inhi-
bition, and enzyme kinetic assays were performed in the same way
as the in vitro FXIa inhibitory activity but with varying concentra-
lution (the buffer) were added to 96-well plates (COSTAR 3599) and
incubated at 37ꢁС for 15 min. Then, the mixture was added with
substrate (10
mL) to the plates, which was further measured at
405 nm [45]; 4. the buffer (35
m
L), FXa (5 L) and sample (5 L) or
m
m
tions of the substrate at 212.5, 425, 850 and 1700
centrations of the substrate were 42.5, 85, 170 and 340
respectively). The Lineweaver-Burk plot was established from
which we could calculate the Km and Vmax of the inhibitor [17,41].
m
M (final con-
M,
blank solution (the buffer) were added to 96-well plates (COSTAR
m
3599) and incubated at 25ꢁС for 10 min. Then, the mixture was
added with substrate (5
measured at 405 nm [46]; 5. the buffer (70
and sample (10 L) or blank solution (the buffer) were added to 96-
well plates (COSTAR 3599) and incubated at 25ꢁС for 10 min. Then,
the mixture was added with substrate (10 L) to the plates, which
was further measured at 405 nm [44]. The test compounds that
presented binding effects over 50% at a concentration of 30
mL) to the plates, which was further
m
L), thrombin (10 L)
m
m
4.5. In vivo efficacy in the rabbit arteriovenous shunt model
m
The AV shunt model was initiated according to the previously
described method with minor modifications [2,21]. Male New
Zealand White rabbits (2e2.5 kg) were obtained from the Animal
Center of Shenyang Pharmaceutical University (Shenyang, China).
Animal maintenance and treatment met the protocols approved by
the Ethics Review Committee for Animal Experimentation of She-
nyang Pharmaceutical University. The rabbits had free access to
food and water and were maintained on a 12-h light/dark cycle in a
temperature- and humidity-controlled room for one week.
mM
were further tested at a wide range of concentrations to calculate
their IC50 values using SPSS 20.0 software. Then, The
MichaelisꢀMenten constant, Km, for substrate hydrolysis was
determined by the following relationship: MichaelisꢀMenten
equation,
Vmax
Km þ S
S
Briefly, male New Zealand White rabbits were anesthetized
urethane (1.25 g/kg, i. v.) and then placed on a heating pad to
maintain body temperature. Then, their femoral artery, jugular vein
and femoral vein were catheterized to an AV-shunt device through
a PE60 cannula, and significant thrombosis formation was induced
through contact with a 4.8 cm long 2-0 silk thread. After 40 min, the
AV-shunt was disconnected from the arterial and venous catheters,
and a mixture of silk thread and thrombosis were removed and
weighed. The wet weight of thrombosis was obtained by subtrac-
tion, which was used as the control [2,21]. Subsequently, after the
compound or saline vehicle was given as a bolus intravenously
followed by a continuous IV infusion via the jugular vein, the
thrombosis formation was induced by a new AV-shunt for 40 min
immediately, and the measurement method was as described
above. Data were reported as % inhibition of thrombosis formation
in the treatment period versus in control period. Compound 44g
was tested at three doses: 0.5 mg/kg þ 0.8 mg kgꢀ1 hꢀ1, 2.5 mg/kg þ
v ¼
where v represents velocity of the reaction, Vmax is the maximal
velocity, S is the final concentration of substrate and Km is the
MichaelisꢀMenten constant for the substrate [20].
Competitive inhibition was assumed for all proteases. The Ki
values were further derived from IC50 values by the following
relationship [20]:
IC50
Ki ¼
½Sꢂ
1 þ K
m
4.3. Molecular modeling
Molecular modeling studies were progressed using GLIDE (2016,
4.0 mg kgꢀ1
h
ꢀ1, 5.0 mg/kg þ 8.0 mg kgꢀ1$ hꢀ1using a dosing
€
Schrodinger Suite) [39]. The crystal structures (PDB code: 4TY7 and
vehicle of saline, and BMS-724296 was used as a positive control
with a dose of 0.5 mg/kg þ 0.8 mg kgꢀ1$ hꢀ1. The saline vehicle did
not significantly inhibit the AV-shunt-induced thrombosis forma-
tion. A total of three rabbits were used for each group. The ED50
represented the dose that produced 50% inhibition of thrombus
formation and was estimated by linear regression.
5E2O) of FXIa were retrieved from the RCSB Protein Data Bank,
which was further prepared using the Protein Preparation Wizard
€
tool (2016, Schrodinger Suite) [39] by adding all hydrogen atoms as
well as the missed side chains of residues and deleting all bound
water. The ligands were built within Maestro BUILD (2016,
€
Schrodinger Suite) [39] and prepared by the LIGPREP module (2016,
€
Schrodinger Suite) [39]. The tautomeric forms of ligands, which
Declaration of competing interest
included the keto and enol forms of ligands, were generated at a
physiological pH (7.0 2.0). The crystal structure 4TY7 was selected
for molecular model, since its cocrystallized ligand BMS-724296
was used for comparison in biological evaluation. In addition, the
crystal structure 5E2O with cocrystallized ligand FXIa inhibitor 7
was used to display the active pockets of FXIa. The Glide Grid was
built using an inner box of dimensions 24 ꢃ 24 ꢃ 24 Å3 around the
centroid of the ligand, assuming that the ligands to be docked were
of a size similar to the cocrystallized ligand. This docking meth-
odology has been validated by extracting the crystallographic
bound BMS-724296 and redocking it with the Glide module using
extra precision (XP) [39]. Different docking poses of ligands were
generated and analyzed by Accelrys Discovery Studio Visualizer
2017 [47] for interpretation of the final results. Accelrys Discovery
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
Authors are grateful to Jiyang Guo for his support in graphic
display.
Appendix ASupplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113437.
21

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
based factor XIa selective inhibitors as leads for potential new anticoagulants,
Bioorg. Med. Chem. Lett 30 (2020) 127072.
References
[19] P.C. Wong, M.L. Quan, C.A. Watson, E.J. Crain, M.R. Harpel, A.R. Rendina,
J.M. Luettgen, R.R. Wexler, W.A. Schumacher, D.A. Seiffert, In vitro, antith-
[1] M.L. Quan, D.J.P. Pinto, J.M. Smallheer, W.R. Ewing, K.A. Rossi, J.M. Luettgen,
D.A. Seiffert, R.R. Wexler, Factor XIa inhibitors as new anticoagulants, J. Med.
Chem. 61 (2018) 7425e7447.
[2] J.J. Hangeland, T.J. Friends, K.A. Rossi, J.M. Smallheer, C. Wang, Z. Sun,
J.R. Corte, T. Fang, P.C. Wong, A.R. Rendina, F.A. Barbera, J.M. Bozarth,
J.M. Luettgen, C.A. Watson, G. Zhang, A. Wei, V. Ramamurthy, P.E. Morin,
G.S. Bisacchi, S. Subramaniam, P. Arunachalam, A. Mathur, D.A. Seiffert,
R.R. Wexler, M.L. Quan, Phenylimidazoles as potent and selective inhibitors of
coagulation factor XIa with in vivo antithrombotic activity, J. Med. Chem. 57
(2014) 9915e9932.
[3] Z. Chen, D. Seiffert, B. Hawes, Inhibition of factor XI activity as a promising
antithrombotic strategy, Drug Discov. Today 19 (2014) 1435e1439.
[4] W. Yang, Y. Wang, A. Lai, C.G. Clark, J.R. Corte, T. Fang, P.J. Gilligan, Y. Jeon,
K.B. Pabbisetty, R.A. Rampulla, A. Mathur, M. Kaspady, P.R. Neithnadka,
A. Arumugam, S. Raju, K.A. Rossi, J.E. Myers, S. Sheriff, Z. Lou, J.J. Zheng,
S.A. Chacko, J.M. Bozarth, Y. Wu, E.J. Crain, P.C. Wong, D.A. Seiffert,
J.M. Luettgen, P.Y.S. Lam, R.R. Wexler, W.R. Ewing, Discovery of a high affinity,
orally bioavailable macrocyclic FXIa inhibitor with antithrombotic activity in
preclinical species, J. Med. Chem. 63 (2020) 7226e7242.
rombotic and bleeding time studies of BMS-654457,
a small-molecule,
reversible and direct inhibitor of factor XIa, J. Thromb. Thrombolysis 40
(2015) 416e423.
[20] J.R. Corte, T. Fang, H. Osuna, D.J.P. Pinto, K.A. Rossi, J.E. Myers, S. Sheriff, Z. Lou,
J.J. Zheng, T.W. Harper, J.M. Bozarth, Y. Wu, J.M. Luettgen, D.A. Seiffert,
C.P. Decicco, R.R. Wexler, M.L. Quan, Structure-based design of macrocyclic
factor XIa inhibitors: discovery of the macrocyclic amide linker, J. Med. Chem.
60 (2017) 1060e1075.
[21] D.J.P. Pinto, M.J. Orwat, L.M. Smith, M.L. Quan, P.Y.S. Lam, K.A. Rossi, A. Apedo,
J.M. Bozarth, Y. Wu, J.J. Zheng, B. Xin, N. Toussaint, P. Stetsko,
O. Gudmundsson, B. Maxwell, E.J. Crain, P.C. Wong, Z. Lou, T.W. Harper,
S.A. Chacko, J.E. Myers, S. Sheriff, H. Zhang, X. Hou, A. Mathur, D.A. Seiffert,
R.R. Wexler, J.M. Luettgen, W.R. Ewing, Discovery of
a parenteral small
molecule coagulation factor XIa inhibitor clinical candidate (BMS-962212),
J. Med. Chem. 60 (2017) 9703e9723.
[22] X. Wang, Q. Cheng, L. Xu, G.Z. Feuerstein, M.-Y. Hsu, P.L. Smith, D.A. Seiffert,
W.A. Schumacher, M.L. Ogletree, D. Gailani, Effects of factor IX or factor XI
deficiency on ferric chloride-induced carotid artery occlusion in mice,
J. Thromb. Haemostasis 3 (2010) 695e702.
[23] M. Schaefer, A. Buchmueller, F. Dittmer, J. Straßburger, A. Wilmen, Allosteric
inhibition as a new mode of action for BAY 1213790, a neutralizing antibody
targeting the activated form of coagulation factor XI, J. Mol. Biol. 431 (2019)
4817e4833.
[5] R.A. Al-Horani, U.R. Desai, Factor XIa inhibitors: a review of the patent liter-
ature, Expert Opin. Ther. Pat. 26 (2016) 323e345.
[6] R.A. Al-Horani, D.K. Afosah, Recent advances in the discovery and develop-
ment of factor xi/xia inhibitors, Med. Res. Rev. 28 (2018) 1974e2023.
[7] P.C. Wong, E.J. Crain, C.A. Watson, W.A. Schumacher, A small-molecule factor
XIa inhibitor produces antithrombotic efficacy with minimal bleeding time
prolongation in rabbits, J. Thromb. Thrombolysis 32 (2011) 129e137.
[8] N.R. Patel, D.V. Patel, P.R. Murumkar, M.R. Yadav, Contemporary de-
velopments in the discovery of selective factor Xa inhibitors: a review, Eur. J.
Med. Chem. 121 (2016) 671e698.
[9] W.A. Schumacher, J.M. Luettgen, M.L. Quan, D.A. Seiffert, Inhibition of factor
XIa as a new approach to anticoagulation, Arterioscler. Thromb. Vasc. Biol. 30
(2010) 388e392.
[10] Z. Hu, P.C. Wong, P.J. Gilligan, W. Han, K.B. Pabbisetty, J.M. Bozarth, E.J. Crain,
T. Harper, J.M. Luettgen, J.E. Myers, V. Ramamurthy, K.A. Rossi, S. Sheriff,
C.A. Watson, A. Wei, J.J. Zheng, D.A. Seiffert, R.R. Wexler, M.L. Quan, Discovery
[24] Q. Cheng, E.I. Tucker, M.S. Pine, I. Sisler, A. Matafonov, M.-f. Sun, T.C. White-
ꢀ
Adams, S.A. Smith, S.R. Hanson, O.J.T. McCarty, T. Renne, A. Gruber, D. Gailani,
A role for factor XIIaemediated factor XI activation in thrombus formation
in vivo, Blood 116 (2010) 3981e3989.
[25] A.W. Koch, N. Schiering, S. Melkko, S. Ewert, J. Salter, Y. Zhang, P. McCormack,
J. Yu, X. Huang, Y.-H. Chiu, Z. Chen, S. Schleeger, G. Horny, K. DiPetrillo,
^
L. Muller, A. Hein, F. Villard, M. Scharenberg, P. Ramage, U. Hassiepen, S. Cotẻ,
J. DeGagne, C. Krantz, J. Eder, B. Stoll, K. Kulmatycki, D.L. Feldman,
P. Hoffmann, C.T. Basson, R.J.A. Frost, Y. Khder, MAA868da novel FXI antibody
with a unique binding modedshows durable effects on markers of anti-
coagulation in humans, Blood 133 (2019) 1507e1516.
of
a potent parenterally administered factor XIa inhibitor with hydrox-
ꢀ
ꢀ
[26] A. Gomez-Outes, M. García-Fuentes, M.L. Suarez-Gea, Discovery methods of
coagulation-inhibiting drugs, Expert Opin. Drug Discov. 12 (2017)
1195e1205.
yquinolin-2(1H)-one as the P20 moiety, ACS Med. Chem. Lett.
590e595.
6 (2015)
[11] T. Xue, S. Ding, B. Guo, Y. Zhou, P. Sun, H. Wang, W. Chu, G. Gong, Y. Wang,
X. Chen, Y. Yang, Design, synthesis, and structureeactivity and
structureepharmacokinetic relationship studies of novel [6,6,5] tricyclic fused
oxazolidinones leading to the discovery of a potent, selective, and orally
bioavailable FXa inhibitor, J. Med. Chem. 57 (2014) 7770e7791.
[12] D.J.P. Pinto, M.J. Orwat, S. Koch, K.A. Rossi, R.S. Alexander, A. Smallwood,
P.C. Wong, A.R. Rendina, J.M. Luettgen, R.M. Knabb, K. He, B. Xin, R.R. Wexler,
P.Y.S. Lam, Discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-
yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide
(apixaban, BMS-562247), a highly potent, selective, efficacious, and orally
bioavailable inhibitor of blood coagulation factor Xa, J. Med. Chem. 50 (2013)
5339e5356.
[13] M. Poulakos, J.N. Walker, U. Baig, T. David, Edoxaban: a direct oral anticoag-
ulant, Am. J. Health Syst. Pharm. 74 (2017) 117e129.
[14] H. Yang, Q. Liu, X. Gao, Y. Ren, Y. Gao, Novel dabigatran derivatives with a
fluorine atom at the C-2 position of the terminal benzene ring: design, syn-
thesis and anticoagulant activity evaluation, Eur. J. Med. Chem. 126 (2017)
799e809.
[27] C.V. Pollack, M.A. Kurz, N.J. Hayward, EP-7041, a factor XIa inhibitor as a
potential antithrombotic strategy in extracorporeal membrane oxygenation: a
brief report, Crit. Care Explor. 2 (2020), e0196.
ꢀ
[28] O. Szekely, M. Borgi, G.Y.H. Lip, Factor XI inhibition fulfilling the optimal ex-
pectations for ideal anticoagulation, Expert Opin. Emerg. Drugs 24 (2019)
55e61.
[29] M.L. Quan, P.C. Wong, C. Wang, F. Woerner, J.M. Smallheer, F.A. Barbera,
J.M. Bozarth, R.L. Brown, M.R. Harpel, J.M. Luettgen, P.E. Morin, T. Peterson,
V. Ramamurthy, A.R. Rendina, K.A. Rossi, C.A. Watson, A. Wei, G. Zhang,
D. Seiffert, R.R. Wexler, Tetrahydroquinoline derivatives as potent and selec-
tive factor XIa inhibitors, J. Med. Chem. 57 (2014) 955e969.
[30] L.M. Smith, M.J. Orwat, Z. Hu, W. Han, C. Wang, K.A. Rossi, P.J. Gilligan,
K.B. Pabbisetty, H. Osuna, J.R. Corte, A.R. Rendina, J.M. Luettgen, P.C. Wong,
R. Narayanan, T.W. Harper, J.M. Bozarth, E.J. Crain, A. Wei, V. Ramamurthy,
P.E. Morin, B. Xin, J. Zheng, D.A. Seiffert, M.L. Quan, P.Y.S. Lam, R.R. Wexler,
D.J.P. Pinto, Novel phenylalanine derived diamides as factor XIa inhibitors,
Bioorg. Med. Chem. Lett 26 (2016) 472e478.
[31] Y. Lei, B. Zhang, D. Liu, J. Zhao, X.W. Dai, J. Gao, Q. Mao, Y. Feng, J.X. Zhao,
F.W. Lin, Y.L. Duan, Y. Zhang, Z.Y. Bao, Y.W. Yang, Y.H. Mou, S.J. Wang,
Switching a xanthine oxidase inhibitor to a dual-target antagonist of P2Y1 and
P2Y12 as an oral antiplatelet agent with a wider therapeutic window in rats
than ticagrelor, J. Med. Chem. 63 (2020) 15752e15772.
[15] Z. Xie, Y. Tian, X. Lv, X. Xiao, M. Zhan, K. Cheng, S. Li, C. Liao, The selectivity and
bioavailability improvement of novel oral anticoagulants: an overview, Eur. J.
Med. Chem. 146 (2018) 299e317.
[16] E. Lorthiois, J. Roache, D. Barnes-Seeman, E. Altmann, U. Hassiepen, G. Turner,
R. Duvadie, V. Hornak, R.G. Karki, N. Schiering, W.A. Weihofen, F. Perruccio,
A. Calhoun, T. Fazal, D. Dedic, C. Durand, S. Dussauge, K. Fettis, F. Tritsch,
C. Dentel, A. Druet, D. Liu, L. Kirman, J. Lachal, K. Namoto, D. Bevan, R. Mo,
G. Monnet, L. Muller, R. Zessis, X. Huang, L. Lindsley, T. Currie, Y.-H. Chiu,
C. Fridrich, P. Delgado, S. Wang, M. Hollis-Symynkywicz, J. Berghausen,
E. Williams, H. Liu, G. Liang, H. Kim, P. Hoffmann, A. Hein, P. Ramage,
A. D’Arcy, S. Harlfinger, M. Renatus, S. Ruedisser, D. Feldman, J. Elliott,
R. Sedrani, J. Maibaum, C.M. Adams, Structure-based design and preclinical
characterization of selective and orally bioavailable factor XIa inhibitors:
demonstrating the power of an integrated S1 protease family approach,
J. Med. Chem. 63 (2020) 8088e8113.
[17] J.R. Corte, D.J.P. Pinto, T. Fang, H. Osuna, W. Yang, Y. Wang, A. Lai, C.G. Clark, J.-
H. Sun, R. Rampulla, A. Mathur, M. Kaspady, P.R. Neithnadka, Y.-X.C. Li,
K.A. Rossi, J.E. Myers, S. Sheriff, Z. Lou, T.W. Harper, C. Huang, J.J. Zheng,
J.M. Bozarth, Y. Wu, P.C. Wong, E.J. Crain, D.A. Seiffert, J.M. Luettgen,
P.Y.S. Lam, R.R. Wexler, W.R. Ewing, Potent, orally bioavailable, and efficacious
macrocyclic inhibitors of factor XIa. Discovery of pyridine-based macrocycles
possessing phenylazole carboxamide P1 groups, J. Med. Chem. 63 (2019)
784e803.
[32] S. Chen, T. Zhang, J. Wang, F. Wang, H. Niu, C. Wu, S. Wang, Synthesis and
evaluation
of
1-hydroxy/methoxy-4-methyl-2-phenyl-1H-imidazole-5-
carboxylic acid derivatives as non-purine xanthine oxidase inhibitors, Eur. J.
Med. Chem. 103 (2015) 343e353.
[33] D. Zhang, T. Yang, J. Lin, A novel xanthine oxidase inhibitor WSJ-557 study on
pharmacokinetics and tissue distribution in rats by UPLCeMS/MS,
J. Chromatogr. B 1113 (2019) 77e83.
[34] D.C. Rees, M. Congreve, C.W. Murray, R. Carr, Fragment-based lead discovery,
Nat. Rev. Drug Discov. 3 (2004) 660e672.
€
[35] S. Schultes, C.D. Graaf, E.E.J. Haaksma, I.J.P.D. Esch, R. Leurs, O. Kramer, Ligand
efficiency as a guide in fragment hit selection and optimization, Drug Discov.
Today Technol. 7 (2010) e147ee202.
[36] A. Daina, O. Michielin, V. Zoete, SwissADME: a free web tool to evaluate
pharmacokinetics, drug-likeness and medicinal chemistry friendliness of
small molecules, Sci. Rep. 7 (2017) 42717.
[37] B.F. Krippendorff, R. Neuhaus, P. Lienau, A. Reichel, W. Huisinga, Mechanism-
based inhibition: deriving ki and kinact directly from time-dependent ic50
values, J. Biomol. Screen 14 (2010) 913e923.
[38] M. Zeilinger, F. Pichler, L. Nics, W. Wadsak, H. Spreitzer, M. Hacker,
M. Mitterhauser, New approaches for the reliable in vitro assessment of
binding affinity based on high-resolution real-time data acquisition of
radioligand-receptor binding kinetics, EJNMMI Res. 7 (2017) 1e13.
[18] Z. Hussain, A.J. Cooke, S. Neelamkavil, L. Brown, E. Carswell, W.M. Geissler,
Z. Guo, B. Hawes, T.M. Kelly, Y. Kiyoi, K. Lai, C. Lesburg, E. Pow, Y. Zang,
H.B. Wood, S.D. Edmondson, W. Liu, Design and synthesis of novel proline
22

Y. Lei, B. Zhang, Y. Zhang et al.
European Journal of Medicinal Chemistry 220 (2021) 113437
[39] Glide, Schrodinger, LLC, New York, NY, 2016.
[40] P. Politzer, J.S. Murray, T. Clark, Halogen bonding and other
teractions: a perspective, Phys. Chem. Chem. Phys. 15 (2013) 11145e11588.
[41] P.C. Wong, M.L. Quan, C.A. Watson, E.J. Crain, M.R. Harpel, A.R. Rendina,
J.M. Luettgen, R.R. Wexler, W.A. Schumacher, D.A. Seiffert, In vitro, antith-
Y. Sakai-Moritani, T. Kawasaki, S. Sakamoto, M. Okada, M. Ohta, S.-
i. Tsukamoto, Potent and selective TF/FVIIa inhibitors containing a neutral P1
ligand, Bioorg. Med. Chem. 14 (2006) 7688e7705.
s-hole in-
[45] D.P. Teufel, G. Bennett, H. Harrison, K.V. Rietschoten, S. Pavan, C. Stace,
F.L. Floch, T.V. Bergen, E. Vermassen, P. Barbeaux, T.T. Hu, J.H.M. Feyen,
M. Vanhove, Stable and long-lasting, novel bicyclic peptide plasma kallikrein
inhibitors for the treatment of diabetic macular edema, J. Med. Chem. 61
(2018) 2823e2836.
rombotic and bleeding time studies of BMS-654457,
reversible and direct inhibitor of factor XIa, J. Thromb. Thrombolysis 40
(2015) 416e423.
a small-molecule,
[42] J. Nicolette, R.E. Neft, J. Vanosdol, J. Murray, Peptide bond-forming reagents
HOAt and HATU are not mutagenic in the bacterial reverse mutation test,
Environ. Mol. Mutagen. 57 (2016) 236e240.
[43] Q. Wei, Z. Zheng, S. Zhang, X. Zheng, F. Meng, J. Yuan, Y. Xu, C. Huang, Frag-
ment-based lead generation of 5-phenyl-1H-pyrazole-3-carboxamide de-
rivatives as leads for potent factor Xia inhibitors, Molecules 23 (2018) 23.
[44] M. Miura, N. Seki, T. Koike, T. Ishihara, T. Niimi, F. Hirayama, T. Shigenaga,
[46] X. Sun, Z. Hong, M. Liu, S. Guo, D. Yang, Y. Wang, T. Lan, L. Gao, H. Qi, P. Gong,
Y. Liu, Design, synthesis, and biological activity of novel tetrahydropyr-
azolopyridone derivatives as FXa inhibitors with potent anticoagulant activity,
Bioorg. Med. Chem. 25 (2017) 2800e2810.
[47] A.D.S. Visualizer, Version 4. 5, Softw. Vis. Anal. Protein Struct, 2017.
€
[48] The PyMOL Molecular Graphics System, version 1.5.0.3; Schrodinger, LLC:
New York.
23