N1-Substituted benzimidazole scaffold for farnesoid X receptor (FXR) agonists accompanying prominent selectivity against vitamin D receptor (VDR)

Article abstract of DOI:10.1016/j.bmc.2020.115512

As a cellular bile acid sensor, farnesoid X receptor (FXR) participates in regulation of bile acid, lipid and glucose homeostasis, and liver protection. With respect to the bone metabolism, FXR positively regulates bone metabolism through both bone format

Full text of DOI:10.1016/j.bmc.2020.115512

Journal Pre-proofs
N1-Substituted benzimidazole scaffold for farnesoid X receptor (FXR) ago-
nists accompanying prominent selectivity against vitamin D receptor (VDR)
Arisa Masuda, Keigo Gohda, Yusuke Iguchi, Ko Fujimori, Yukiko
Yamashita, Keisuke Oda, Mizuho Une, Naoki Teno
PII:
S0968-0896(20)30338-2
https://doi.org/10.1016/j.bmc.2020.115512
BMC 115512
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
26 March 2020
11 April 2020
13 April 2020
Please cite this article as: A. Masuda, K. Gohda, Y. Iguchi, K. Fujimori, Y. Yamashita, K. Oda, M. Une, N. Teno,
N1-Substituted benzimidazole scaffold for farnesoid X receptor (FXR) agonists accompanying prominent
selectivity against vitamin D receptor (VDR), Bioorganic & Medicinal Chemistry (2020), doi: https://doi.org/
10.1016/j.bmc.2020.115512
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N1-Substituted benzimidazole scaffold for farnesoid X receptor (FXR) agonists
accompanying prominent selectivity against vitamin D receptor (VDR)
Arisa Masuda,a Keigo Gohda,b Yusuke Iguchi,c Ko Fujimori,d Yukiko Yamashita,c
Keisuke Oda,c Mizuho Une,a,c Naoki Teno,a,e,*
aGraduate School of Pharmaceutical Sciences, Hiroshima International University,
5-1-1 Hirokoshingai, Kure, Hiroshima 737-0112, Japan
bComputer-aided Molecular Modeling Research Center, Kansai (CAMM-Kansai),
3-32-302, Tsuto-Otsuka, Nishinomiya 663-8241, Japan
cFaculty of Pharmaceutical Sciences, Hiroshima International University, 5-1-1
Hirokoshingai, Kure, Hiroshima 737-0112, Japan
dDepartment of Pathobiochemistry, Osaka University of Pharmaceutical Sciences,
4-20-1 Nasahara, Takatsuki, Osaka 569-1094, Japan
eFaculty of Clinical Nutrition, Hiroshima International University, 5-1-1 Hirokoshingai,
Kure, Hiroshima 737-0112, Japan
*Corresponding author:
Prof. Naoki Teno
Hiroshima International University,
Graduate School of Pharmaceutical Sciences,
5-1-1, Hirokoshingai, Kure, Hiroshima 737-0112, Japan
Tel,: +81 823 73 8593; Fax: +81 823 73 8981
e-mail: n-teno@hirokoku-u.ac.jp
Abbreviations:
FXR, Farnesoid X receptor; CDCA, Chenodeoxycholic acid; CYP7A1, Cholesterol
7α-hydroxylase; BSEP, Bile salt export pump; SHP, Small heterodimer partner; OSTα,
Organic solute transporter α; CAR, Constitute androstane receptor; PXR, Pregnane X
receptor; RXR, Retinoid X receptor ; 9-cis-RA, 9-cis-Retinoic acid; 1,25-OH-VD3,
1α,25-Dihydroxy-vitamin D3; TGR5, Transmembrane G protein-coupled receptor 5;
FGF15, Fibroblast growth factor 15; NASH, Non-alcoholic steatohepatitis; LBD,
Ligand binding domain; TR-FRET, Time-resolved fluorescence resonance energy
transfer; RT-PCR, Reverse transcription polymerase chain reaction; BMP-2, Bone
morphogenetic protein-2; ST-2 MSCs, Mouse bone marrow-derived mesenchymal stem
1

cell (MSC)-like ST2 cells; GS, Guggulsterone; ALP, Alkaline phosphatase; SAR,
Structure-activity relationship; Et3N, Triethylamine; DMF, N,N-Dimethylformamide;
THF,
Tetrahydrofuran;
Boc,
tert-Butoxycarbonyl;
HOAt,
WSCI.HCl,
TBAF,
1-Hydroxy-7-azabenzotriazole;
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride;
tetra-n-Butylammonium fluoride;
2

Abstract
As a cellular bile acid sensor, farnesoid X receptor (FXR) participates in regulation of
bile acid, lipid and glucose homeostasis, and liver protection. With respect to the bone
metabolism, FXR positively regulates bone metabolism through both bone formation
and resorption of the bone remodeling pathways. Some of FXR agonists possessing
isoxazole moiety are undergoing clinical trials for the treatment of non-alcoholic
steatohepatitis. To date, therefore, the activation of FXR leads to considerable interest in
FXR as potential therapeutic targets. We have identified a series of nonsteroidal FXR
agonists bearing N1-methyl benzimidazole and isoxazole moieties that are bridged with
aromatic derivatives. They showed affinity to FXR, but also weak affinity toward the
vitamin D receptor (VDR) that involves regulation of calcium and phosphate
homeostasis and is activated by bile acids. The deployment of FXR agonists without
activity against VDR as off-target is therefore crucial in the development of FXR
ligands. Our efforts focusing on increasing the agonist properties towards FXR led to
the discovery of 19, which activates FXR at and below nanomolar levels (EC50 = 26.5 ±
10.5 nM TR-FRET and 0.8 ± 0.2 nM luciferase, respectively) and functions as a FXR
agonist: the affinity toward FXR over eight nuclear receptors, including VDR [IC50
(VDR) / EC50 (FXR) > 5000] and TGR5, effects FXR target genes, and activates bone
morphogenetic protein-2-induced differentiation of mouse bone marrow-derived
mesenchymal stem cell-like ST2 cells into osteoblast.
Key words
FXR agonist, Benzimidazole, Osteoblast differentiation.
3

1. Introduction
Nuclear hormone receptors (NHRs) are transcription factors that work in concert with
coactivators and co-repressors to regulate gene expression.1 Bile acid synthesis is under
negative feedback control through activation of the nuclear receptor farnesoid X
receptor (FXR, NR1H4) in the ileum and liver.2,3 FXR and the cholesterol-sensing liver
X receptor (LXR, NR1H2/3) form a complicated control network with other members
of NHRs, the constitute androstane receptor (CAR, NR1I3) and the pregnane X receptor
(PXR, NR1I2)4 that function as the xenobiotic-sensing nuclear receptors in liver
diseases.5 Chenodeoxycholic acid (CDCA),2 as an endogenous ligand of FXR,
transcriptionally regulates the expression of cholesterol 7α-hydroxylase (CYP7A1)
through the nuclear receptors small heterodimer partner (SHP) and the liver receptor
homolog-1 (LRH-1).6 Recent data suggest that intestinal FXR regulates hepatic
CYP7A1 through a fibroblast growth factor 15 (FGF15)-dependent mechanism.7 FXR
regulates transport of proteins, the bile salt export pump (BSEP) and the organic solute
transporter α (OSTα) that are involved in hepatocellular excretion.8 With respect to the
bone metabolism, bile acids enhance the differentiation of mesenchymal stem cells
(MSCs) into osteoblasts though FXR.9,10 FXR deficiency reveals a decrease in bone
mineral density11 and deletion of FXR-regulated SHP decreased bone mass through
repression of osteoblast differentiation.12 FXR agonists could activate osteoblast
differentiation,13 indicating that osteoblast differentiation is available to evaluate the
activity of FXR agonist.
To date, many FXR agonists have been reported13-30 and the most representative
non-steroidal FXR agonist is GW4064 (1), which is composed of isoxazole and stilbene
moieties.30 (Figure 1) These portions of GW4064 (1) lie co-planar to each other,
allowing the agonist to fit into a narrow region of the ligand binding domain (LBD) in
FXR.31 Some FXR agonists (2-12) possess an alternative structure to the stilbene
substituent which might elicit a potentially toxic15-17,32-37 while preserving isoxazole
derivatives.13-24 (Figure 1) From these analogs, tropifexor (2), a highly potent
nonsteroidal FXR agonist, is undergoing clinical trials for the treatment of
non-alcoholic steatohepatitis (NASH).14,38 A partial FXR agonist (9) was identified by
Eli Lilly.21 Terns Pharmaceuticals licensed this compound in 2018 and TERN-101 (9) is
in Phase 1.38,39 Cilofexor (10),40 which is analogous to 3,22 is also currently undergoing
clinical evaluation.38
We have previously reported the chemotype for FXR agonists in which an isoxazole
moiety30 and N1-substituted benzimidazole are bridged with aromatic derivatives
4

(12-15).13 (Table 1) These analogs revealed the agonistic activity against FXR and a
significant affinity with FXR toward eight nuclear receptors and transmembrane G
protein-coupled receptor 5 (TGR5). In addition, we have identified that 13 and 14
activated bone morphogenetic protein-2 (BMP-2)-induced differentiation of mouse
bone marrow-derived MSC-like ST2 cells (ST-2 MSCs) into osteoblasts.13 The
modeling study implied that 14 occupied the same binding pocket as GW4064; there is
the possibility that LBD is not occupied by GW4064.13 In further profiling of the
activities of 12-15, they retained a weak antagonism against the vitamin D receptor
(VDR, NR1I1) at micromolar levels even though GW4064 (1) had no affinity with
VDR. (Table 1)
Figure 1. Representative FXR agonists with isoxazole derivatives
N
O
O H
Cl
O H
Cl
N
O H
Cl
Cl
O
O
F
S
Cl
O H
F
Cl
O
O
O
N
N
F
O
F
N
N
O
O
N
O
O
1: GW4064
2: Tropifexor
3
N
Cl
H
N
O H
O H
Cl
Cl
Cl
O
Cl
O
O
N
O
N
O
O H
N
O
O
O
4
5
6
O H
O
O
O H
Cl
Cl
O H
HN
Cl
N
Cl
N
Cl
N
O
N
Cl
O
Cl
O
O
N
O
O
N
O
O
7
8
9: TERN-101
O
O H
N
O H
Cl
O
N
N
N
O
O
N
Cl
O
O
Cl
O H
Cl
Cl
Cl
Cl
O H
O
O
N
N
O
O
10: Cilofexor
11
12
Table 1. Effects of reported analogs, 12-15 on FXR and VDR
Cl
Cl
R1
R2
N
O
R3
R4
N
N
O
Cpds.
R1
R2
R3
R4
FXR
EC50 (nM)
TR-FRET
Relative
potency (%)
FXR
EC50 (nM)
Luciferase
Relative
potency
(%)
VDR
IC50 (nM)
Luciferase
Relative
potency
(%)
1
-
-
-
-
96.2±62.7*
43.7±54.2*
64.4±135.1*
13.4±7.9*
135.8±42.6*
58.2±26.2*
58.3±20.4*
51.1±27.6*
76.4±23.4*
13.2±13.9
32.0±38.1
25.8±26.0
19.0±17.4
3.66±4.66
102.4±8.9
101±31.1
65.6±12.2
64.6±8.1
>10000
47.5±7.6
55.2±4.5
62.9±4.8
74.2±13.6
81.1±0.7
12*
13*
14*
15*
H
H
Cl
H
H
H
H
Cl
COOH
H
H
COOH
H
7151.3±3853.3
4407.7±284.0
2454.3±2006.5
3519.0±716.9
COOH
COOH
H
31.6±26.9*
70.1±15.1
*: see reference No. 22
5

VDR is also known as a member of the NHR1 subfamily of the NHR superfamily.41
VDR binds 1α,25-dihydroxy-vitamin D3 (1,25-OH-VD3, calcitriol) with high affinity
and mediates regulation of calcium and phosphate homeostasis.42 Common feature of
FXR and VDR is that both receptors are activated by bile acids as cholesterol
products.43 Indeed, docking studies suggested a common key interaction for bile acid
with FXR and VDR.43 Although the majority of the VDR ligand was based on the
secosteroidal scaffold of calcitriol and recently several other non-secosteroidal VDR
ligands have been reported.44-48
The deployment of FXR agonists without activity against VDR as off-target is crucial
for future design of FXR ligands. Toward the development of the selective FXR
ligands, our efforts have focused on increasing the agonistic potency against FXR based
on the structures of 12-15. The primary modeling study data13 enabled further insight
that the region not occupied by GW4064 (1) in LBD could be used to increase the
interaction with FXR. Considering this observation, substituents extending from
nitrogen atom (N1) in benzimidazole of 12 could be appropriate for filling the vacant
space in LBD of FXR. We describe herein that a structure-activity relationship (SAR)
on the N1-substituted benzimidazole of 12 was explored to identify the potent and
selective FXR agonists and examine the agonism by the activation of ST-2-MCSs
differentiation into osteoblasts.
2. Chemistry
Preparation of 19 is outlined in Scheme 1 as the representative example. Synthetic
protocol (Schemes S1-S7) and characterizations of other analogs are available in
Supplementary
Data.
The
starting
material,
methyl
4-amino-3-(1-ethylpropylamino)benzoate49 was coupled with 4-benzyloxybenzoic acid
using Et3N, HOAt and WSCI.HCl in DMF was performed to yield 19a. Immediate ring
closure of 19a in CH3COOH gave 19b. After removal of benzyl group of 19b, followed
by coupling with 4-(bromomethyl)-3-(2,6-dichlorophenyl)-5-isopropyl-isoxazole9 using
K2CO3 in DMF afforded 19d. The methyl ester of 19d was hydrolyzed by 1M NaOH in
MeOH to yield 19.
3. Biology
The binding activity of the analogs to LBD of FXR was evaluated by TR-FRET-based
coactivator recruitment assay.13 Evaluation of FXR agonist activity and ligand activity
for other nuclear receptors and TGR5 by these compounds in living cells was carried
out by a luciferase assay as previously published.50 Analysis of ligand-induced
6

regulation of FXR target genes was conducted using real time RT-PCR according to a
previous report.50 Values of P used to compare the statistical significance were
determined using Student's t-test in Excel. Validation of activating differentiation of
ST-2 MSCs into osteoblasts was conducted according to the literature.13 Statistical
analysis was performed using One-way ANOVA, followed by Tukey’s post hoc test
(Statcel 3 software; OMS Publishing, Saitama, Japan). The experimental protocols are
available in Supplementary Data.
O
NH2
H
N
NH
H
N
O
a
b
O
N
N
O
O
O
O
O
O
19a
19b
Cl
Cl
Cl
O
O
O
Cl
e
c
d
O
N
N
N
N
N
N
O H
O
N
N
O
O
O
HO
O
19c
19d
19
Scheme 1. Reagents and conditions:(a) 4-Benzyloxybenzoic acid, Et3N, HOAt, WSCI.HCl,DMF, 15 h,
0 oC→ rt; (b) CH3COOH, 2 h, 80 oC; (c) 10 % Pd/C, H2, MeOH/THF, 15 h, rt; (d) 4-(Bromomethyl)-3-
(2,6-dichlorophenyl)-5-isopropyl-isoxazole,K2CO3,DMF, 15 h, rt; (e) 1M NaOH, MeOH/THF, 15 h, rt.
4. Results and Discussion
Our efforts for the development of potent and selective FXR agonists began with a
SAR exploration starting with the replacement of the N1-methyl group on
benzimidazole of 12. The synthesized analogs (16-39) were assessed by an FXR
time-resolved fluorescence resonance energy transfer (TR-FRET) binding assay and a
luciferase reporter assay according to a previous publication.13 An antagonistic activity
against VDR as off-target was simultaneously evaluated, and the target selectivity was
calculated from EC50 and IC50 measured by a luciferase assay and shown as the ratio of
IC50 (VDR) / EC50 (FXR). The modification on the nitrogen atom in benzimidazole
made a difference in the agonistic activity for FXR.
Replacing N1-methyl group of 12 with various size substituents was carried out.
(Table 2) The methyl moiety of 12 was replaced by linear alkyl groups, ethyl (16) and
propyl (17). The former appeared to bind nearly 2- and 15-fold greater than 12 in
TR-FRET binding and luciferase assays, respectively, and the inhibition against VDR
was equivalent to that of 12, resulting that a ratio of IC50 (VDR) / EC50 (FXR) exceeded
2000 times. Although the latter modification was inferior to 16 in either assay for FXR,
it showed a single-digit nanomolar activity against FXR in the luciferase assay and a
slight increase in the affinity toward VDR. In addition to the size (length) effect as seen
7

Table 2. Effects of 16-33 on FXR and VDR
Cl
Cl
O
R
N
O
N
O H
O
N
Cpds.
R
FXR
EC50 (nM)
TR-FRET
Relative
potency
(%)
FXR
EC50 (nM)
Luciferase
Relative
potency
(%)
VDR
IC50 (nM)
Luciferase
Relative
potency (%)
VDR (IC50) /
FXR (EC50
Luciferase
)
12*
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
CH3
43.7±54.2
19.3±9.8
96.4±60.4
56.7±63.6
26.5±10.5
30.3±10.8
116.8±84.4
-
58.2±26.2
61.2±14.2
14.5±12.4
27.6±20.2
68.7±10.9
70.1±10.8
26.4±13.9
-
32.0±38.1
2.31±1.82
9.3±0.7
101.0±31.1
76.4±7.8
50.9±9.2
56.3±3.7
70.4±2.5
68.9±7.4
59.1±5.2
50.8±3.9
36.8±8.7
61.1±9.4
55.6±6.9
3.9±3.1
7151.3±3853.3
5760.9±748.3
1401.3±290.2
3551.9±459.4
5688.0±1431.8
>10000
55.2±4.5
66.0±8.6
86.2±1.5
85.8±4.2
51.7±0.7
20.1±12.5
78.1±2.0
89.3±1.9
83.1±1.1
68.9±5.4
76.8±2.9
0
223
2439
150
1047
7110
-
3.39±1.61
0.8±0.2
1.8±1.2
16.5±0.3
3197.7±714.1
1104.0±179.5
928.8±95.7
193
76
14.4±1.5
-
-
13.6±0.8
68
-
-
4.21±0.54
14.8±2.4
1448.6±201.3
921.1±55.3
344
62
113.4±93.0
-
31.0±9.1
-
O
N
1432.0±1672.3
5.26±4.58
13.0±5.2
>10000
-
24.7±14.0
24.8±22.7
29.1±22.9
19.9±10.7
-
23.5±11.2
20.1±11.9
26.5±7.5
51.4±10.5
-
66.6±7.42
55.0±11.7
61.4±17.0
70.9±0.5
11.1±7.4
90.2±7.85
70.9±6.9
1183.4±828.3
861.3±21.8
82.1±8.6
86.0±4.2
74.9±1.8
82.7±4.9
24.3±20.9
76.1±7.7
76.2±3.4
224
66
19.4±13.7
0.52±0.14
1166.1±1144.3
32.2±18.6
3.31±0.63
2652.5±1520.1
346.0±144.9
>10000
136
665
-
O
N
F
15.1±11.4
105.3±135.2
9.1±10.0
30.2±31.6
2413.8±2160.3
1141.6±430.3
74
F
344
-: Not determined
in 16 and 17, we successively investigated the size (balkiness) of N1--branched alkyl
groups (18-20). The isopropyl group (18) had almost the same activity against FXR as
the ethyl substituent (16) and a slight increase in the inhibitory activity against VDR,
with a decrease in the selectivity ratio. The linear extension by branching (19) indicated
that the agonistic activity against FXR was at and below nanomolar level (EC50 = 26.5 ±
10.5 nM TR-FRET, 0.8 ± 0.2 nM luciferase, respectively). Although 19 still retained
weak antagonism against VDR (IC50 = 5688.0 ± 1431.8 nM), the ratio of IC50 (VDR) /
EC50 (FXR) was more than 7000-fold. Further extension (20) of 19 showed a slight
attenuation of the agonistic effect on FXR and complete loss of the antagonism against
VDR relative to 19. Since the N1--branched analogs shared promising results in both
assays for FXR, cyclic (21-26) and N1--branched (27-31) analogs were evaluated.
8

Analogs 21-26 bearing cyclic substituents on benzimidazole revealed less activity than
19 in the luciferase assay for FXR but had a slight increase in the affinity with VDR
compared to 19. The selective ratios of 21-25 were, therefore, reduced as compared to
19. An acylated piperidine moiety (26) showed no significant effect on FXR.
N1--Branched derivatives (27 and 28) preserved the agonistic activity against FXR in
the luciferase assay at almost the same level as 21-25 and had an upward trend in the
inhibitory activity against VDR. Extension of the chain length of the cyclopropyl ring
showed no significant difference in the activity in the luciferase receptor assay (21 vs
29). Notably, robust potency was observed for 30 (EC50 = 19.9 ± 10.7 nM TR-FRET,
0.52 ± 0.14 nM luciferase), being nearly equipotent with 19; besides, the substituent of
30 (IC50=346.0±144.9 nM) facilitated the interaction with VDR. Like analog 26, the
substituent of 31 was not tolerated in either receptor. The aromatic substituents (32 and
33) were less favorable in the ratio of IC50 (VDR) / EC50 (FXR). It turns out that
N1-substituents of 19 and 20 represent key building blocks to achieve nanomolar level
potency against on-target and lead to the selectivity ratio of IC50 (VDR) / EC50 (FXR)
greater than 5000-fold.
We next attempted to determine whether the analogs (34-37) derived from 19 and 30
substantially cause the changes in agonism against FXR (Table 3) since 15 substantially
changed it in the luciferase assay (12 vs 15).13 Even if fluoride (34, 36) and chloride
(35, 37) were introduced into 19 and 30, respectively, there were no substantial changes
in agonism (34, 35) and the EC50 values in the luciferase assay of 36 and 37 increased
about 10-fold compared to that of 30. Introduction of fluoride (34, 36) and chloride (35,
37) revealed a counter trend on the antagonism against VDR relative to 19 and 30,
respectively. This observation led to the discovery of 34, which exhibited an EC50
=
69.9 ± 22.0 nM in TR-FRET and EC50 = 1.3 ± 0.2 nM in the luciferase assay and loss of
antagonism against VDR.
9

Table 3. Effects of 34-37 on FXR and VDR
Cl
Cl
O
R2
R1
N
O
N
O H
O
N
Cpds.
R1
R2
FXR
EC50 (nM)
TR-FRET
Relative
potency
(%)
FXR
EC50 (nM)
Luciferase
Relative
potency (%)
VDR
IC50 (nM)
Luciferase
Relative
potency (%)
VDR(IC50)/
FXR(EC50
Luciferase
)
19
34
H
F
26.5±10.5
69.3±22.0
68.7±10.9
48.7±12.9
0.8±0.2
1.3±0.2
70.4±2.5
43.1±6.9
5688.0±1431.8
>10000
51.7±0.7
7110
54.7±11.5
-
35
30
36
37
Cl
H
21.6±12.7
19.9±10.7
-
95.8±12.9
51.4±10.5
-
1.6±0.03
0.52±0.14
7.8±2.9
54.5±11.7
70.9±0.5
58.0±6.5
53.9±8.8
1012.6±43.9
346.0±144.9
839.5±86.9
160.3±8.9
74.0±2.8
82.7±4.9
78.8±3.0
76.5±5.9
632
665
49
F
Cl
66.1±53.8
18.7±5.5
7.6±1.9
21
-: Not determined
Our computer-assisted modeling studies suggested that 19 (white) and 30 (white) (left
and right panels in Figure 2, respectively) occupy the same binding site of LBD in FXR
as GW4064 (yellow) and exhibit interactions between the acidic moieties of each
agonist and Arg331. (Figure 2) The N1--branched alkyl group of 19 and
N1--branched cyclohexyl group of 30 are similarly placed in the vacant space of LBD
that GW4064 does not fill. Both groups are hydrophobic and therefore may contribute
to enhancing the EC50 activities for FXR because the cholesterol binding-site of
FXR-LBD is known to be hydrophobic in nature.51
10

30: Top-view
19: Top-view
Arg331
Met328
Arg331
Met328
Yellow: GW4064 x-ray
Yellow: GW4064 x-ray
Figure 2. Modeling of FXR complexedwith GW4064,19 and 30.
A complex-modelof human FXRα-LBDmonomer(PDB ID: 3DCT,purple cylinders and
orange sticks) with GW4064 (X-ray; yellow sticks) and 19 (left panel, white sticks) and
30 (right panel, white sticks) was built using AutoDockVina1.1.2.
The acidic moiety of our chemotype interacts with Arg331 in FXR through a
hydrogen bond. (Figure 2) Contribution of this moiety relative to FXR activity was
confirmed by 38 and 39, which lack acidic moiety of 30. (Table S1) The affinity with
FXR and VDR of both analogs dropped sharply. The results revealed that the acidic
moiety on N1-substituted benzimidazole is required for the interaction with LBD in
FXR.
Tetrazolium colorimetric (MTT) assays are the most common employed for the
detection of cytotoxicity or cell viability following exposure to toxic substances.52 As
Huh-7 and HEK293T cells were used for the assay of agonism toward FXR and
antagonism against VDR, respectively, the cytotoxic activities of the synthesized
analogs in both cell lines were evaluated (Table S2). The results obtained in the MTT
assays suggested that cytotoxicity tends to be partially affected by altering
N1-substituents on benzimidazole.
To gain insights into the agonism and target engagement for 19 (Figure 3) and 20
(Figure S1), the effect in modulating FXR target genes, BSEP, SHP and OST, was
assessed in a Huh-7 cell line by real-time PCR, with GW4064 (0.01-1 μM) as the
reference compound. Analogs 19 and 20 at 0.01-1 μM concentrations were as potent as
GW4064 in the induction of mRNA expression.
11

BSEP
SHP
OST
25
20
15
10
5
8
6
4
2
0
160
120
80
40
0
0
1
GW4064
0.01 0.1
1
1
1
(M)
0.01 0.1
0.1
1
0.01 0.1
0.1
1
0.01 0.1
0.01
0.01
(M)
(M)
GW4064
GW4064
19
19
19
Figure 3. Effectof 19 on FXR target genes.Expressionof FXR-induced genes BSEP,SHP and OST was
significantly increased by treatment of GW4064or 19 in Huh-7 cells (n=3, P < 0.01 v.s. N.C.)
Further, in vitro profile of 19 and 20 was expanded to common off-targets for bile
acid receptor ligands. (Figure 4 and Figure S2, respectively) Both analogs were unable
to induce RXRα, RARα, VDR, PPARα, γ and δ, LXRα and β, TGR5 transactivation on
Huh-7 cell lines. In brief, both analogues 19 and 20 (1 μM) had no agonism toward any
of these receptors other than FXR.
FXR
RAR
RXR
VDR
PPAR 
16
14
12
10
8
20
15
10
5
40
30
20
10
0
15
10
5
25
20
15
10
5
6
4
2
0
0
0
GW4064
19
0
GW7647
19
9-cis-RA
19
1,25-OH-VD3
-
-
+
+
+
-
-
-
+
+
+
-
-
-
+
-
+
+
-
+
-
-
+
+
+
-
9-cis-RA
-
-
+
-
+
+
-
+
19
-
+
-
+
19
-
+
PPAR 
LXR 
LXR 
PPAR 
TGR5
15
10
5
25
20
15
10
5
15
10
5
15
10
5
8
6
4
2
0
0
0
0
0
GW501516
19
-
-
+
-
+
+
-
TO901317
19
TO901317
19
GW1929
19
-
-
+
+
+
-
-
-
+
+
+
-
-
-
+
+
+
-
LCA
19
-
-
+
-
+
+
-
+
+
-
+
-
+
-
+
Figure 4 .Selectivity screening of 19 against human nuclear receptors and TGR5.The concentration of
ligands for each nuclear receptorwas as follows;19: 1 μM, GW4064:100 nM, 9-cis-retinoicacid (9-cis-RA):
50 nM, 1,25-hydroxyvitamin D3 (1,25-OH-VD3):100nM, Lithocholic acid (LCA): 200nM, GW7647:50nM,
GW501516:50nM, GW1929:50nM, TO901317:50nM.
We subsequently investigated whether 19 and 30 activate the BMP-2-induced
differentiation of ST-2 MSCs into osteoblasts, like 14 and 15 according to a previous
publication.13 At first, viability of ST-2 MSCs in the presence of 19 or 30 was evaluated
by a WST-8 assay (Figure 5A) Cells were cultured for 12 days in RPMI-1640 medium
containing various concentrations of 19 or 30 (0−10 μM); no significant toxic effects
were observed up to 5 μM in the cells. The ST-2 MSCs differentiated into osteoblasts
after 6 or 12 days in RPMI-1640 medium with BMP-2 in the presence or absence of
12

FXR agonists. The staining and activity of alkaline phosphatase (ALP) that is the
biomarker of bone formation53 were enhanced by treatment with BMP-2 during
osteoblast differentiation of ST-2 MCSs after 6 days and 12 days. (Figure 5B, C)
Enhancing the staining and activity of ALP was clearly observed for FXR agonists
including 19 and 30 and the activity was repressed by the co-treatment with
guggulsterone (GS).54 (Figure 5B, C) Thus, our structure-activity analyses of
N1-substituents on benzimidazole culminated in the identification of a remarkable series
of FXR agonists bearing potency below nanomolar level.
5. Conclusion
We have developed a series of FXR agonists with different N1-substituents on the
benzimidazole pharmacophore as potential modulators of FXR. As exemplified by 19
and 30, the size (length and bulkiness) of N1-substituents greatly affected antagonism
against FXR as on-target and the selective ratio of on-target versus off-target.
Unfortunately, no clear SAR on the analogs reported here against FXR and VDR was
apparent, but 19, 20 and 34, which share N1--branched aliphatic group, led to an
increase in potency for FXR and nearly a complete loss of antagonism against VDR.
Analog 19 was an FXR agonist: the affinity with FXR over eight nuclear receptors and
TGR-5, effects on FXR target genes, and ALP activity BMP-2-induced differentiation
of ST-2 MSCs into osteoblast. Furthermore, molecular modeling implied that 19 fills
the leeway of LBD in FXR that the stilbene of GW4064 (1) cannot cover, resulting that
19 shares agonism against FXR below nanomolar level. In contrast, introduction of
slightly bulkier cyclic group than that of 19, as observed with 30, facilitated the
interaction with VDR and increased the potency for VDR even though 30 showed the
similar agonism against FXR as 19. According to selective optimization of side
activities (SOSA) concept,55 the chemical modification of 30 may serve to develop the
new chemotype for future non-secosteroidal VDR ligands as the modification of
omeprazole led to the discovery of nanomolar bombesin receptor subtype 3 agonists.56
13

A
180
160
140
120
100
80
60
40
20
0
0
0.5
1
5
10
0
0.5
1
5
10
1
19 (μM)
30 (μM)
CDCA
GW4064
19
30
B
(μM)
10
10
+
5
+
-
5
+
+
1
+
-
5
+
-
5
+
+
5
+
-
5
+
+
-
-
BMP-2
GS
+
-
+
-
+
-
+
C
4
3
2
1
0
*
*
*
*
*
#
#
#
#
#
#
#
#
BMP-2
GS
(μM)
+
+
+
+
5
+
+
+
5
+
+
+
5
+
+
+
+
+
5
+
+
+
5
+
+
+
+
+
+
+
+
+
+
-
-
+
+
- - -
-
- -
- -
- - -
-
- -
- -
10 10
5
1
5
1
5
10 10
5
5
1
5
1
5
- -
- -
V
V
CDCA GW4064
6d
19
30
V
V
CDCA GW4064
12d
19
30
Figure 5. Activationof BMP-2-induced osteoblast differentiationby novel synthesized FXR agonist 19 and 30 in
ST-2 MSCs. (A) Cell viability. The ST-2 MSCs(vehicle; white columns) were cultured for 12 daysin the medium
containing0-10 μM of 19 or 30 (blackcolumns). Cell viability wasmeasured by a WST-8 assay. Cell viability at
0 μM was definedas100%. Data are presented asmeans± S.D.(n=5). (B) ALP staining. The ST-2 MSCswere
differentiatedintoosteoblastsfor 12 daysin the medium containing BMP-2 (50 ng/ml) together with 10 μM CDCA,
5 μM GW4064, or 1 or 5 μM of 19 or 30 in the presence or absence of 25 μM GS. ALP staining wasperformedas
described in Materialsand Methods. The resultsare the representative from three experiments. (C) ALP activity. The
ST-2 MSCs(vehicle: V; white columns) were culturedfor 6 or 12 daysin the medium containing BMP-2 (50 ng/ml;
gray columns) together with one of theFXR agonists, 10 μM CDCA, 5 μM GW4064, and 1 or 5 μM of 19 or 30 (black
columns) in the presence or absence of 25 μM GS (hatched columns). Data are shown as means± S.D.(n=3).
*p<0.01, ascompared withthe BMP-2-inducedcells, #p<0.01,ascompared withthe FXR agonist-treated cells.
6. Experimentals
6.1 Materials and Methods
All chemicals were purchased from Tokyo Chemical Industry Co., Ltd., FUJIFILIM
Wako Pure Chemical Industries, Ltd. or Aurora Fine Chemicals LLC and used without
further purification. All protected amino acids and the coupling reagents were purchased
1
from Watanabe Chemical Industries, Ltd. H-NMR experiments were recorded on a
14

JMTC-600 (JEOL Ltd.) 600 NMR spectrometer with CDCl3 or DMSO-d6 as solvents.
Chemical shifts are expressed in parts per million (ppm, ) and referred to the solvent
signal. HRMS spectra were recorded on The AccuTOF (JMS-T100LC) equipped with
an electrospray ion source (JEOL Ltd.). The analytical HPLC system consisted of a
SHINADZU CBM-20A System Controller and a SHINADZU Pump Unit LC-20AT, a
SHIMADZU In-Line Degasser DGU-20A3R, a SHIMADZU SPD-20A Absorbance
Detector, a SHIMADZU FCV-11AL Valve Unit and a SHIMADZU FRC-10A Fraction
Collector (SHIMADZU Corporation). The absorbance detector was operated at 254 nm.
The mobile phase for preparation and analysis was a combinations of water (A) and
acetonitrile (B), both containing 0.1% TFA, and a flow rate of 1 ml/min, respectively.
TSK gel ODS column (4.6 x 150 mm for analysis, TOSOH Corporation) were used.
6.2. Methyl 4-[(4-benzyloxybenzoyl)amino]-3-(1-ethylpropylamino)benzoate (19a) :
Methyl 4-amino-3-(1-ethylpropylamino)benzoate50 (172 mg, 0.73 mmol) in DMF (5 ml)
was added to a solution of 4-benzyloxybenzoic acid (166 mg, 0.73 mmol) in DMF (2
o
ml) at 0 C. Subsequently, HOAt (119 mg, 0.88 mmol), WSCI.HCl (207 mg, 1.09
o
mmol) and Et3N (306 µl, 2.19 mmol) were added to the former solution at 0 C and
stirred for 15 h at room temperature. The reaction mixture was quenched with
sat.NaHCO3 and extracted with EtOAc. The combined extracts were washed with H2O
and brine, dried over MgSO4 and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography to give 19a in 61 % yield. Rf=0.48
(n-Hexane/EtOAc=2/1). 1H NMR (600 MHz, CDCl3): δ 8.11 (brs, 1H), 7.87 (d, J=8.5
Hz, 2H), 7.77 (brs, 1H), 7.56-7.54 (m, 2H), 7.44-7.34 (m, 5H), 7.07 (d, J=8.8 Hz, 2H),
5.15 (s, 2H), 3.91 (s, 3H), 3.40 (brs, 1H), 3.24-3.22 (m, 1H), 1.63-1.48 (m, 4H), 0.94 (t,
J=7.5 Hz, 6H). HRMS (ESI/TOF) m/z: [M+H]+ Calcd for C27H31N2O4 447.22838 ;
Found 447.22575.
6.3. Methyl 2-(4-benzyloxyphenyl)-3-(1-ethylpropyl)benzimidazole-5-carboxylate
(19b) : 19a (197 mg, 0.44 mmol) was dissolved in CH3COOH (5 ml) at room
o
temperature and stirred for 2 h at 80 C. The reaction mixture was evaporated and
quenched by addition of sat.NaHCO3. The mixture was extracted with EtOAc, and
organic layer was washed successively with H2O and brine, dried over MgSO4 and
concentrated under reduced pressure. The residue was purified by silica gel column
1
chromatography to give 19b in 86 % yield. Rf=0.28 (n-Hexane/EtOAc=2/1). H NMR
(600 MHz, CDCl3): δ 8.30 (s, 1H), 8.00 (d, J=8.4 Hz, 1H), 7.81 (d, J=8.5 Hz, 1H), 7.56
(d, J=7.8 Hz, 2H), 7.47 (d, J=7.7 Hz, 2H), 7.42 (t, J=7.6 Hz, 2H), 7.36 (t, J=6.8 Hz, 1H),
15

7.12 (d, J=8.7 Hz, 2H), 5.15 (s, 2H), 4.30-4.26 (m, 1H), 3.97 (s, 3H), 2.23-2.20 (m, 2H),
2.00-1.96 (m, 2H), 0.74 (t, J=7.3, 6H). HRMS (ESI/TOF) m/z: [M+H]+ Calcd for
C27H29N2O3 429.21782 ; Found 429.21805.
6.4. Methyl 3-(1-ethylpropyl)-2-(4-hydroxyphenyl)benzimidazole-5-carboxylate
(19c) : To 19b (161 mg, 0.38 mmol) and 10 % Pd/C (40 mg), was added
dist.MeOH/dist.THF (2.5 ml/2.5 ml) and hydrogenated under a hydrogen atmosphere
for 15 h at room temperature. The solution was filtered and concentrated under reduced
1
pressure to give 19c in quantitative yield. Rf=0.59 (n-Hexane/EtOAc=1/2). H NMR
(600 MHz, CDCl3): δ 9.76 (brs, 1H), 8.31 (s, 1H), 8.02 (dd, J=8.5, 1.5 Hz, 1H), 7.84 (d,
J=8.5 Hz, 1H), 7.36 (d, J=8.6 Hz, 2H), 6.84 (d, J=8.6 Hz, 2H), 4.31-4.26 (m, 1H), 3.97
(s, 3H), 2.23-2.18 (m, 2H), 2.00-1.95 (m, 2H), 0.74 (t, J=7.4 Hz, 6H). HRMS
(ESI/TOF) m/z: [M+H]+ Calcd for C20H23N2O3 339.17087 ; Found 339.17323.
6.5.
Methyl
2-[4-[[3-(2,6-dichlorophenyl)-5-isopropyl-isoxazol-4-yl]methoxy]phenyl]-3-(1-ethyl
propyl)benzimidazole-5-carboxylate (19d) : To a solution of 19c (70 mg, 0.21 mmol)
and 4-(bromomethyl)-3-(2,6-dichlorophenyl)-5-isopropyl-isoxazole30 (72 mg, 0.21
mmol) in DMF (4 ml), K2CO3 (57 mg, 0.41 mmol) was added at room temperature and
stirred for 15 h at room temperature. The reaction mixture was quenched with
sat.NH4Cl and extracted with EtOAc. The combined extracts were washed with H2O
and brine, dried over MgSO4 and concentrated under reduced pressure. The residue was
purified by silica gel column chromatography to give 19d in 95 % yield. Rf=0.65
(n-Hexane/EtOAc=1/1). 1H NMR (600 MHz, CDCl3): δ 8.29 (s, 1H), 7.99 (d, J=8.5 Hz,
1H), 7.79 (d, J=8.5 Hz, 1H), 7.49 (d, J=8.1 Hz, 2H), 7.41 (d, J=8.2 Hz, 2H), 7.34 (t,
J=8.8 Hz, 1H), 6.90 (d, J=8.2 Hz, 2H), 4.82 (s, 2H), 4.25-4.20 (m, 1H), 3.96 (s, 3H),
3.39-3.34 (m, 1H), 2.23-2.18 (m, 2H), 1.99-1.93 (m, 2H), 1.45 (d, J=7.0 Hz, 6H),
0.73-0.70 (m, 6H). HRMS (ESI/TOF) m/z: [M+H]+ Calcd for C33H34Cl2N3O4
606.19264 ; Found 606.19148.
6.6. 2-[4-[[3-(2,6-Dichlorophenyl)-5-isopropyl-isoxazol-4-yl]methoxy]phenyl]-3-
(1-ethylpropyl)benzimidazole-5-carboxylic acid (19) : To a suspension of 19d (118
mg, 0.20 mmol) in MeOH/THF (2 ml/2 ml), 1M NaOH (0.98 ml, 0.98 mmol) was
added at room temperature and the mixture was stirred for 15 h at room temperature.
The reaction mixture was neutralized with 1M HCl and evaporated down. A white solid
product appeared and filtered to give 96 mg of 19 in 51 % yield. Rf=0.63
16

1
(CH2Cl2/MeOH=9/1), H NMR (600 MHz, DMSO-d6): δ 13.2 (brs, 1H), 8.37 (s, 1H),
8.02 (d, J=8.3 Hz, 1H), 7.87 (d, J=8.4 Hz, 1H), 7.71 (d, J=8.1 Hz, 2H), 7.64-7.60 (m,
3H), 7.11 (d, J=8.5 Hz, 2H), 5.03 (s, 2H), 4.28 (m, 1H), 3.58-3.54 (m, 1H), 2.16-2.03
(m, 4H), 1.42 (d, J=7.0 Hz, 6H), 0.74-0.69 (m, 6H). 13C NMR (600 MHz, DMSO-d6): δ
175.7, 167.0, 159.2, 158.5, 155.6, 142.2, 134.3, 132.1, 131.6, 131.1, 128.1, 126.8,
125.3, 124.1, 120.1, 117.5, 114.7, 114.1, 108.9, 61.0, 58.7, 25.7, 25.6, 20.3, 10.4.
HRMS (ESI/TOF) m/z: [M+H]+ Calcd for C32H32Cl2N3O4 592.17699 ; Found
592.17973.
Acknowledgements
We are grateful to Dr. Lawrence H. Lazarus for critically reading the manuscript. We
thank Professor Hidetoshi Tahara for data collection.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at
XXX.
17

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Figure caption
Figure 1. Representative FXR agonists with isoxazole derivatives.
Figure 2. Modeling of FXR complexed with GW4064, 19 and 30. A complex-model of
human FXRα-LBD monomer (PDB ID: 3DCT, purple cylinders and orange sticks) with
GW4064 (X-ray; yellow sticks) and 19 (left panel, white sticks) and 30 (right panel,
white sticks) was built using AutoDock Vina1.1.2.
Figure 3. Effect of 19 on FXR target genes. Expression of FXR-induced genes BSEP,
SHP and OST was significantly increased by treatment of GW4064 or 19 in Huh-7
cells (n=3, P < 0.01 v.s. N.C.)
Figure 4. Selectivity screening of 19 against human nuclear receptors and TGR5. The
concentration of ligands for each receptor was as follows; 19: 1 μM, GW4064: 100 nM,
9-cis-retinoic acid (9-cis-RA): 50 nM, 1α,25-dihydroxyvitamin D3 (1,25-OH-VD3):
100nM, Lithocholic acid (LCA): 200nM, GW7647: 50nM, GW501516: 50nM,
GW1929: 50nM, TO901317: 50nM.
Figure 5. Activation of BMP-2-induced osteoblast differentiation by 19 and 30 in ST-2
MSCs. (A) Cell viability. The ST-2 MSCs (vehicle; white columns) were cultured for
12 days in the medium containing 0-10 μM of 19 or 30 (black columns). Cell viability
was measured by a WST-8 assay. Cell viability at 0 μM was defined as 100%. Data are
presented as means ± S.D.(n=5). (B) ALP staining. The ST-2 MSCs were differentiated
into osteoblasts for 12 days in the medium containing BMP-2 (50 ng/ml) together with
10 μM CDCA, 5 μM GW4064, or 1 or 5 μM of 19 or 30 in the presence or absence of
25 μM GS. ALP staining was performed as described in Materials and Methods. The
results are the representative from three experiments. (C) ALP activity. The ST-2 MSCs
(vehicle: V; white columns) were cultured for 6 or 12 days in the medium containing
BMP-2 (50 ng/ml; gray columns) together with one of the FXR agonists, 10 μM CDCA,
5 μM GW4064, and 1 or 5 μM of 19 or 30 (black columns) in the presence or absence
of 25 μM GS (hatched columns). Data are shown as means ± S.D.(n=3). *p<0.01, as
compared with the BMP-2-induced cells, #p<0.01, as compared with the FXR
agonist-treated cells.
24

Graphical Abstract
N1-Substituted benzimidazole scaffold for farnesoid X receptor (FXR) agonists
accompanying prominent selectivity against vitamin D receptor (VDR)
Arisa Masuda, Keigo Gohda, Yusuke Iguchi, Ko Fujimori, Yukiko Yamashita, Keisuke
Oda, Mizuho Une, Naoki Teno
Cl
Cl
O
O
N
O
N
N
OH
19
Modeling of FXR complexed with GW4064 and 19.
A complex-modelof human FXRα-LBDmonomer(PDB ID: 3DCT,
purple cylinders and orange sticks) with GW4064 (X-ray; yellow
sticks) and 19 (white sticks) and was built using AutoDockVina1.1.2.
25