Investigation around the Oxadiazole Core in the Discovery of a New Chemotype of Potent and Selective FXR Antagonists

Article abstract of DOI:10.1021/acsmedchemlett.8b00534

Recent findings have shown that Farnesoid X Receptor (FXR) antagonists might be useful in the treatment of cholestasis and related metabolic disorders. In this paper, we report the discovery of a new chemotype of FXR antagonists featured by a 3,5-disubsti

Full text of DOI:10.1021/acsmedchemlett.8b00534

Subscriber access provided by TULANE UNIVERSITY
Letter
Investigation around the oxadiazole core in the discovery of
a new chemotype of potent and selective FXR antagonists
Carmen Festa, Claudia Finamore, Silvia Marchianò, Francesco Saverio Di
Leva, Adriana Carino, Maria Chiara Monti, Federica del Gaudio, Sara Ceccacci,
Vittorio Limongelli, Angela Zampella, Stefano Fiorucci, and Simona De Marino
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00534 • Publication Date (Web): 10 Jan 2019
Downloaded from http://pubs.acs.org on January 12, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 8
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Investigation around the oxadiazole core in the discovery of a new
chemotype of potent and selective FXR antagonists
Carmen Festa,1 Claudia Finamore,1 Silvia Marchianò,2 Francesco Saverio Di Leva1, Adriana Carino,2
Maria Chiara Monti,4 Federica del Gaudio,4 Sara Ceccacci,4 Vittorio Limongelli,1,3 Angela Zampella,1
Stefano Fiorucci2 and Simona De Marino1*
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1Department of Pharmacy, University of Naples "Federico II", Via D. Montesano 49, 80131 Naples, Italy.
2Department of Surgery and Biomedical Sciences, Nuova Facoltà di Medicina, Perugia, Italy.
3Università della Svizzera Italiana (USI), Faculty of Biomedical Sciences, Institute of Computational Science - Center for
Computational Medicine in Cardiology, Via G. Buffi 13, CH-6900 Lugano, Switzerland.
4Department of Pharmacy, University of Salerno, Via Giovanni Paolo II, 132, 84084, Fisciano, Salerno, Italy.
KEYWORDS: FXR antagonists, bile acid receptor, 1,2,4-oxadiazole core, piperidine ring and pyrrolidine ring
ABSTRACT: Recent findings have shown that FXR antagonists might be useful in the treatment of cholestasis and related metabolic
disorders. In this paper we report the discovery of a new chemotype of FXR antagonists featured by a 3,5-disubstituted oxadiazole
core. In total, 35 new derivatives were designed and synthesized and notably, compounds 3f and 13, containing a piperidine ring,
displayed the best antagonistic activity against FXR with promising cellular potency (IC50=0.58±0.27 M and 0.127±0.02 M,
respectively). The excellent pharmacokinetic properties make compound 3f as the most promising lead identified in this study.
Farnesoid X Receptor (FXR, also known as BAR, NR1H4), a (NASH).10 Unfortunately, the side effects resulted from the phase
ligand-dependent transcription factor, belongs to the super-family III clinical trials of 6-ECDCA, the most promising steroidal FXR
of metabolic nuclear receptors. FXR is highly expressed in the agonist,10 raised concerns about the use of FXR agonists in the
liver, intestine, kidney, and adrenals, and it responds to bile acids1 treatment of metabolic liver diseases. In addition, the recent
regulating their homeostasis and governing lipid and glucose finding that the FXR gene ablation protects from liver injury
metabolism in the liver.2-6 For all these reasons, FXR represents a caused by bile duct ligation (BDL), has moved the attention
promising pharmacological target in the management of metabolic towards the FXR antagonism in the treatment of obstructive
disorders.
cholestasis.11
The most potent endogenous ligand of FXR is chenodeoxycholic As in the case of the agonists, both steroidal and non-steroidal
acid (CDCA), a primary bile acid found in human bile. FXR compounds12-14 have been identified as FXR antagonists. Among
agonists bind to the FXR’s ligand-binding activation function 2 the steroidal scaffolds, the most promising are natural compounds
(AF-2) domain (LBD) inducing a receptor conformational change with glyco-β-muricholic acid (GβMCA) currently in advanced
responsible for the release of co-repressor molecules (such as N- pre-clinical trials for the treatment of obesity, NAFLD, and insulin
Cor) and the recruitment of co-activators (such as SRC-1). The resistance.15 In this work, starting from the recent discovery of
FXR/co-activator complex assisted by the RXR protein binds to Flesch et al.16 who showed that structural fragmentation on
specific regions of DNA enhancing the expression of target genes. GW4064, a highly potent and selective non-steroidal FXR agonist,
On the other hand, the binding of an antagonist molecule to the can lead to the shift of the receptor modulation from the agonism
FXR LBD induces a shift of AF-2 helix which assumes an to the antagonism, we aimed at developing novel non-steroidal
orientation unable to recruit the co-activators, thus leading to FXR antagonists. In particular, Flesch et al.16 reported that the
transcriptional silencing.7
removal of the bulky 4-substituent from the isoxazole ring of
GW4064 can be a valuable starting point for the synthesis of FXR
antagonists. In detail, the replacement of the isoxazole ring in
GW4064 with a 3,5-disubsituted oxadiazole led to compounds
with different activity profiles, from agonism, partial agonism to
antagonism.
The strong interest towards FXR agonists is linked to the ability
of these compounds in regulating bile acid homeostasis in
cholestasis, lowering plasma lipogenesis, repressing very low
density lipoprotein production, increasing plasma triglyceride
clearance, improving insulin sensitivity, and promoting the
storage of glycogen.8,9
The 1,2,4-oxadiazole ring is interesting from the chemical
synthesis point of view, being an accessible and highly
diversifiable scaffold that can be exploited to improve the
pharmacokinetics, pharmacodynamics and the selectivity profile
of the drug candidates.
In recent years, several FXR agonists, belonging to steroidal and
non-steroidal scaffolds, have been identified as potential leads for
the treatment of metabolic disorders with some of them progressed
in advanced clinical trials for the treatment of patients with
primary biliary cirrhosis (PBC) and non-alcoholic steatohepatitis
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 2 of 8
This is the case of compound 1 in Figure 1, a highly simplified Briefly, treatment of nitriles 1a-f with hydroxylamine
GW4064 derivative endowed with an antagonistic profile toward hydrochloride afforded the intermediate amidoximes 2a-f in
1
2
FXR.
quantitative yield.
3
4
5
6
7
8
9
Figure 1
Reaction with N-Boc-isonipecotic acid (N-Boc-Inp-OH), using
HBTU as coupling agent, followed by deprotection of the Boc
protecting group in acidic conditions, led to compounds 3a-f
(Scheme 1). Compounds 3a-f were evaluated in a luciferase
reporter assay on HepG2 cells transiently transfected with human
FXR (Table 1). None of the above compounds resulted able to
transactivate FXR in agonistic mode at 10 μM concentration
(Figure S3) whereas cell co-stimulation in presence of CDCA
(10ꢀμM) reveals that, among this series, derivative 3f was
relatively effective (80% antagonistic rate) in inhibiting FXR
activation caused by CDCA, and the above efficacy data was
accompanied by a promising potency (IC50 0.58±0.27 M).
Having identified the 3-naphtyl-1,2,4-oxadiazole as a promising
core in FXR antagonism, substitutions preserving the naphtyl at
C-3 and the protonable core at C-5 have been explored. First,
compounds 13 and 23 have been prepared reacting the amidoxima
2f with N-Boc-L-pipecolic acid (N-Boc-L-Pip-OH) and N-Boc-L-
proline (N-Boc-L-Pro-OH) (Scheme 2), in order to explore the
influence of different size and different position of the nitrogen in
the heterocyclic moiety at C-5. Compound 13 was also
demonstrated a high potent FXR antagonist (IC50 0.127±0.02
M), whereas a dramatic loss of potency was observed for the
corresponding proline derivative 23, thus identifying the N-
bearing six membered heterocycle as a key structural element at
C-5 position on the central 1,2,4-oxadiazole moiety. Then, the
effect of the introduction of N-alkyl side chain with differentiated
lengths and functionalization has been focused, using compounds
3f, 13 and 23 as cornerstone intermediates. N-alkylation with , 
or ‐bromo esters, as depicted in the Scheme 2, allowed the
introduction of C-3/C-4 or C-5 side chains with the methyl ester
end-group. Basic hydrolysis with LiOH and reduction with
DIBAL-H afforded the corresponding acids and alcohols (Scheme
2).
OCH3
O
N
O
O
NH
5
O
N
N
3
5
N
Cl
N
3
HOOC
Cl
R
Cl
1
Compound
IC50 16.4 M
GW4064
Despite the promising pharmacological profile of 1, a strong
rationale of its activity, supported by the elucidation of the ligand
binding mode, is lacking. Therefore, we have decided to explore
further this scaffold, modifying both substituents at C-3 and C-5
of the oxadiazole moiety developing a small library of derivatives
listed in Table 1. In particular, the lipophilic p-methoxyphenyl at
C-5 in 1 was replaced by different protonable N-bearing
heterocyclic substituents, such as a piperidine or a pyrrolidine
ring. It is worth noting that a similar substituent has been recently
reported by Teno et al,13 in a new chemotype of FXR antagonists
with the evidence that the replacement of a cyclohexyl component
with a ionizable piperidine ring increases the binding activity to
FXR. The research of Teno et al. led finally to the identification
of N-acylated electrically neutral potent FXR antagonists. At
variance with the Teno’s compounds, in our derivatives the N-
bearing ring is not acetylated, hence protonable, thus endowing
basic properties to our compounds and allowing to explore the
piperidine moiety as available H-bond donor group in the FXR-
LBD.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Here we report the pharmacological evaluation, the assessment of
the in vitro pharmacokinetic properties and the ligand binding
mode to FXR of the newly synthesized compounds that allowed
the identification of novel potent FXR antagonists with promising
pharmacokinetic properties.
The structure of the synthesized compounds was confirmed by
spectroscopic and analytical techniques and the structural
characterization is available in the SI.
Scheme 2. Synthesis of analogues 4-32
Synthesis started with oxadiazole derivatives 3a-f prepared from
the nitriles 1a-1f and N-Boc-isonipecotic acid (N-Boc-Inp-OH), as
starting materials (Scheme 1).
14
15
16
17
18
19
20
21
22
n=2 R1=COOCH3
n=2 R1=COOH
n=2 R1=CH2OH
n=3 R1=COOCH3
n=3 R1=COOH
n=3 R1=CH2OH
n=4 R1=COOCH3
n=4 R1=COOH
n=4 R1=CH2OH
f
g
g
g
R1
n
O
O
N
HN
N
N
f
n
N
Br
N
R1
Scheme 1. General synthetic procedure to prepare 1,2,4-
oxadiazole derivatives 3a-fa
e
f
13
c,b
OH
OH
N
O
N
O
N
O
N
N
3
5
R1
a
b,c
CN
NH
n
Br
N
NH
a,b
R1
R
N
NH2
N
n
R
NH2
2a-f
N
R
e
1a-f
3a-f
3f
2f
4
5
6
7
8
9
10
11
12
n=2 R1=COOCH3
n=2 R1=COOH
n=2 R1=CH2OH
n=3 R1=COOCH3
n=3 R1=COOH
n=3 R1=CH2OH
n=4 R1=COOCH3
n=4 R1=COOH
n=4 R1=CH2OH
f
f
g
g
R1
d,b
n
N
H
N
CF3
CF3
O
O
N
N
N
b
a
n
Br
N
R1
f
g
e
R=
23
d
f
c
e
24
n=2 R1=COOCH3
n=2 R1=COOH
n=2 R1=CH2OH
n=3 R1=COOCH3
n=3 R1=COOH
n=3 R1=CH2OH
n=4 R1=COOCH3
n=4 R1=COOH
n=4 R1=CH2OH
f
g
g
25
26
27
28
29
30
31
32
f
f
g
aReagents and conditions: a) NH2OH HCl, K2CO3 in CH3OH,
reflux, 100-90% yield; b) N-Boc-Inp-OH, DIPEA, HBTU in DMF
dry, 80 °C, 60-80% yield; c) TFA:CH2Cl2 1:1, 2 h, quantitative
yield.
aReagents and conditions a) N-Boc-Inp-OH, DIPEA, HBTU in
DMF, 80 °C, 80% yield; b) TFA:CH2Cl2 1:1, 2 h, quantitative
yield; c) N-Boc-L-Pip-OH, DIPEA, HBTU in DMF dry, 80 °C,
78% yield; d) N-Boc-L-Pro-OH, DIPEA, HBTU in DMF dry, 80
°C, 83% yield; e) methyl 3-bromopropanoate, methyl 4-
bromobutanoate or methyl 5-bromopentanoate, DIPEA, in
ACS Paragon Plus Environment

Page 3 of 8
ACS Medicinal Chemistry Letters
CH3CN dry, 60 °C, over night, 65-92% yield; f) LiOH in Of interest, the replacement of the carboxylic end group with a
THF/H2O (2:1), 0 °C then room temperature, 24 h, 40-60% yield; neutral alcoholic functional group preserves the antagonistic
g) DIBAL-H (1 M in toluene) in THF dry, 0 °C, 48 h, 57-82% activity toward FXR. Furthermore, the biological activity
1
2
3
4
5
6
7
8
yield.
indicates that the efficacy in antagonizing FXR correlates with the
size of the installed side chain, with the best match found for the
C-4 side chain as in compounds 9 and 19 (IC50 value of 1.17 and
7.0 M, respectively). The comparison between the IC50 values of
the N-alkylated piperidine derivatives with those of the N-free
piperidine 3f and 13, shows that the modification at the nitrogen
atom alters the ligand potency, prompting the non-substituted
piperidine ring as a key structural element in the FXR antagonism,
with compounds 3f and 13 the best hits identified in this study.
Alkylation at the nitrogen affects FXR antagonism. As shown in
Table 1, the effects in term of efficacy/potency is highly
dependent on the position of the nitrogen in the cycle, the length
of the alkyl chain and the nature of the terminal functional group
(COOH or CH2OH). Foremost, the introduction of alkyl side
chains bearing a carboxylic group produces substantial changes in
FXR antagonistic activity compared to non-alkylated
counterparts, with dramatic increase in IC50 values. Importantly,
the above loss in potency is independent by the length of the N-
alkyl chain and by the position of the nitrogen on the heterocycle
at C-5.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
All the synthetized compounds showed no agonistic activity
towards FXR (Figure S3).
Table 1. FXR antagonistic activity of substituted 1,2,4-oxadiazole derivatives
O
N
X
N
3
5
nY
R
b
a
Compound
3a
R
n
X
Y
IC50
Efficacy [%]
2
2
CH2
CH2
NH
NH
0
-
-
3b
44
CF3
3c
2
2
CH2
CH2
NH
NH
9
-
-
3d
13
CF3
3e
3f
2
2
CH2
CH2
NH
NH
49
80
-
0.58±0.27 M
Naph
Naph
Naph
Naph
Naph
Naph
Naph
Naph
Naph
Naph
Naph
Naph
Naph
Naph
Naph
Naph
Naph
5
2
2
2
2
2
2
2
2
2
2
2
2
2
1
1
1
CH2
CH2
CH2
CH2
CH2
CH2
NH
N(CH2)2COOH
N(CH2)2CH2OH
N(CH2)3COOH
N(CH2)3CH2OH
N(CH2)4COOH
N(CH2)4CH2OH
CH2
19
67
74
95
2
-
6
40.4±7.66 M
8
25.7±1.69 M
9
1.17±0.41 M
11
12
13
15
16
18
19
21
22
23
25
26
-
48
96
17
59
40
82
49
71
88
70
73
-
0.127±0.02 M
-
N(CH2)2COOH CH2
N(CH2)2CH2OH CH2
N(CH2)3COOH CH2
N(CH2)3CH2OH CH2
N(CH2)4COOH CH2
N(CH2)4CH2OH CH2
27.0±5.7 M
-
7.0±2.18 M
-
10.5±1 M
34.5±2.5 M
28.4±4.7 M
20.5±0.65 M
NH
CH2
N(CH2)2COOH CH2
N(CH2)2CH2OH CH2
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 8
28
29
31
32
Naph
Naph
Naph
Naph
1
1
1
1
N(CH2)3COOH CH2
N(CH2)3CH2OH CH2
N(CH2)4COOH CH2
N(CH2)4CH2OH CH2
44
85
8
-
1
2
3
4
5
6
33.8±0.4 M
-
68
23.0±0.9 M
aEfficacy has been calculated in transactivation assay on HepG2 cells as percent of inhibition of transactivation caused by CDCA at 10
μM, set as 100%. IC50 has been determined for efficacy ≥ 50%. IC50 values (μM) were calculated from at least three experiments.
b
Results are expressed as mean ± SEM.
7
8
9
The efficacy of compounds 3f and 13 on FXR was then evaluated
in a cell-free SRC-1 recruitment Alpha Screen experiment. In this
assay, FXR-SRC-1 recruitment is measured in presence of CDCA
at 500 nM with or without different concentrations of 3f and 13.
As reported in Figure 2A, both compounds show high efficacy in
counteracting SRC-1 peptide recruitment prompted by CDCA
when tested at 500 nM and 1000 nM. Afterwards, we attempted
to see whether 3f and 13 interacts with common off-targets, such
as the nuclear receptors liver X receptor α and β (LXRα and β),
the peroxisome proliferator-activated receptor γ (PPARγ) and
pregnane X receptor (PXR) (Figure 2B-2C). Compounds 3f and
13 showed no agonistic and antagonistic effects toward any of
these receptors.
formation via the amine group. Instead, large differences were
observed in their metabolic stability, as resulted by in vitro
incubation with rat liver microsomes. Compound 13 showed a
very low metabolic stability in vitro, with only 9.4% of
unmodified molecule remaining after 40 min, and these data rule
out its pharmacological potential.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 2. In vitro pharmacokinetics for compounds 3f and 13
b
Compound Solubilitya logD Clint
(µM)
t (min) %c
½
3f
200
0.34
0.58
20.67 111.8
201 11.5
76.2
9.4
13
110.4
b
aAqueous solubility at pH 7.4; Reported as µL/min/mg protein;
cPercentage of compound remaining in solution after 40 min
incubation. Each measurement has been repeated in triplicate and
SD < 5%.
A
140
500 nM
120
1000 nM
100
80
60
40
20
0
Collectively, compound 3f combines the high potency/efficacy in
antagonizing FXR (Table 1) with excellent metabolic stability
(about 76% remaining after 40 min of in vitro incubation, Table
2), thus representing a new promising chemotype of FXR
antagonist. Furthermore, we tested also its behavior in the parallel
artificial membrane permeability assay (PAMPA), to measure its
effective permeability (express as logPe) through an artificial lipid
membrane.17 Propanolol and furosemide at 250 μM were used as
positive and negative control molecules giving a logPe of -4.875
for propanolol and of -6.503 for furosemide. In this assay, 3f
displayed an average propensity to cross the membrane in vitro
(logPe -5.046).
CDCA
3f
13
B
LXRα
LXRβ
PPARγ
PXR
6
4
2
*
*
*
*
To gain insights into the antagonism of compound 3f, we then
investigated the downstream genes of FXR by real-time PCR
(Figure 3). The HepG2 (1ꢀ×ꢀ106) were plated and, after 24ꢀhours
of starvation, were stimulated with CDCA (10ꢀμM) and compound
3f at 0.1 and 1 μM concentration.
0
NT GW
3f
13
NT ROSI 3f
13
NT
RIF
3f
13
NT GW
3f
13
C
10
8
6
4
2
0
8
6
4
2
0
2.5
2.0
1.5
1.0
0.5
0.0
*
NT
*
CDCA
CDCA + 3f
NT
3f
GW
13
NT
3f
GW
13
NT
3f
13
NT
3f
RIF
13
#
ROSI
#
Figure 2. A) The effect of 3f and 13 at 500 nM and 1000 nM in
antagonizing SRC-1 recruitment on FXR by CDCA at 500 nM.
Each measurement has been repeated in triplicate and SD < 5%;
B-C) Off-target activity. B) HepG2 cells were stimulated with
GW3965 (GW, 10 M) or Rosiglitazone (ROSI, 500 nM), or
Rifaximin (RIF, 10 M) as positive controls and compounds 3f
and 13 (10 M); C) Cells were stimulated with 5 M of
compounds 3f and 13 in combination with the relative positive
control. Results are expressed as mean ± SEM. ∗p < 0.05 versus
not treated cells (NT).
5
4
3
2
1
0
1.5
1.0
0.5
0.0
6
4
2
0
*
*
#
#
#
*
Figure 3. Compound 3f modulates FXR target genes. Real-time
PCR analysis of mRNA expression on FXR target genes in HepG2
cells primed with CDCA (10 μM) alone or in combination with
increasing concentrations of 3f (0.1 and 1 µM), or with vehicle
In order to evaluate the physicochemical parameters of the above-
mentioned analogs, LC-MS analysis were assessed (Table 2).
Both compounds exhibited good aqueous solubility offering also
the possibility to improve their solubility and polarity by a salt
ACS Paragon Plus Environment

Page 5 of 8
ACS Medicinal Chemistry Letters
(DMSO) alone. Values are normalized to GAPDH. The relative
mRNA expression is analyzed according to the CT method. Data
were calculated from at least three experiments. Results are
expressed as actual mean between the two concentrations ± SEM
*p < 0.05 versus not treated cells (NT); # p<0.05 versus CDCA
stimulated cells. .
Docking of 3f and 13 (lowest energy conformation, Figure 4) in
the FXR-LBD antagonist conformation shows that the ligand
oxadiazole ring engages both a water-mediated hydrogen bond
and a T-shape interaction with the side chain of His451 on H11.
Additionally, the naphtyl ring of 3f and 13 establishes a number
of hydrophobic contacts with residues such as Ala295, Val299,
Leu455, and Met456. On the other side, the piperidine ring of 3f
and 13 can establish favorable interactions with Ile356 and Ile361,
and might form water-mediated hydrogen bonds with Ser336 and
Tyr373. The overlap in the FXR-LBD of the binding mode of 3f
and 13 with that of the FXR co-crystallized antagonist N-benzyl-
N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-
1
2
3
4
5
6
7
8
As shown in Figure 3, CDCA induced the expression of SHP,
OSTα, PEPCK, GC6P whereas decrease CYP7A1 relative mRNA
expression and 3f reverts the effect of CDCA on the above FXR
targeted genes. These results demonstrate that compound 3f is a
potent, effective and selective FXR antagonist.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
In order to elucidate the binding mode of the newly synthesized
FXR antagonists, we performed docking calculations, which is a
widely used computational technique to generate and rank
(dimethylamino) benzamide (NDB) shows that the ligands occupy
similarly the ligand binding site, although NDB can form
additional interactions with the receptor though its 3-(tert-butyl)-
4-hydroxyphenyl moiety (Figure S1). Interestingly, additional
docking calculations on 9 show that also the binding mode of this
compound is superimposable to that of NBD (Figure S2). This
finding explains why compounds endowed with an alcoholic side
chain on the piperidine nucleus (i.e. 9, 19, 22 and 26) have FXR
antagonistic profile.
ligand/protein complexes based on empirical scoring functions.18-
20
In particular, we performed molecular docking calculations on the
most potent derivatives 3f and 13 (Figure 4) in the crystal structure
of the FXR-ligand binding domain (LBD) in the antagonist
conformation (PDB code: 4OIV).21
In conclusion, we report the discovery of a new chemotype of
FXR antagonists, featuring a central oxadiazole ring with a
protonable piperidine nucleus at C-5 and a naphtyl group at C-3.
We note that the piperidine moiety plays a different role in our
compounds if compared with the compounds reported by Teno et
al.13 In particular, in the Teno’s compounds the piperidine works
as a spacer to place the H-bond acceptor group (the carbonyl CO)
in the proper position to interact with His298, while in our
compounds the piperidine is not acetylated and therefore available
as H-bond donor group in the FXR-LBD. Furthermore, the
presence of a free piperidine makes our compounds basic with the
piperidine protonated and an overall net charge equals to +1.
Our study identified compounds 3f and 13 as potent and selective
FXR antagonists. The ability in reverting the effect of CDCA on
the expression of several FXR targeted genes combined with the
excellent pharmacokinetic properties makes compound 3f the
most promising lead identified in this study and suitable for
further development.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Experimental procedures, docking poses of 3f and 13 with the
crystallographic pose of NBD at the FXR-LBD; 1H NMR of
compounds 3a-3f and 4-32 (PDF).
Figure 4. Docking pose of (A) 3f (cyan sticks) and (B) 13 (yellow
sticks) in the crystal structure of the FXR-LBD (PDB code:
4OIV).21 FXR is shown as grey cartoons, with amino acids
important for ligand binding depicted as sticks. Non-polar
hydrogens are omitted for clarity. Hydrogen bonds are shown as
dashed black lines.
AUTHOR INFORMATION
Corresponding Author
*Prof. Simona De Marino; e-mail: sidemari@unina.it
We note that the structure of the FXR-LBD in this conformation
is rather different if compared with that of the FXR-LBD in the
agonist form. In particular, in the agonist state the ligand binding
site is defined by helices 3, 5, 6, 7, 11, and 12, with the latter
forming the recognition site for the coactivator peptides.22 At
variance with the agonist form, in the antagonist state the
hydrophobic C-terminal part of H11 is bended filling part of the
ligand binding pocket and sending H12 to the binding site of the
coactivators peptides of the other FXR monomer. This
conformational change blocks the receptor in a not activable
state.21
Notes
The authors declare no competing financial interest
Funding Sources
This work was supported by University of Naples Federico II
"Finanziamento della Ricerca in Ateneo (DR/2016/341, February
2016)", by MIUR-ITALY PRIN2015 “Top-down and Bottom-up
approach in the development of new bioactive chemical entities
inspired on natural products scaffolds” (Project N.
2015MSCKCE_003) and by the Swiss National Science
Foundation (Project N. 200021_163281).
ACS Paragon Plus Environment
5

ACS Medicinal Chemistry Letters
Page 6 of 8
based farnesoid X receptor antagonists. Bioorg. Med. Chem. 2018, 26,
4240-4253.
(15) Gonzalez, F. J.; Jiang, C.; Patterson, A. D. An intestinal
microbiota-farnesoid X receptor axis modulates metabolic disease.
Gastroenterology 2016, 151, 845-59.
ACKNOWLEDGMENTS
1
2
3
V.L. also thanks the COST action CA15135 (Multi-target
paradigm for innovative ligand identification in the drug discovery
process MuTaLig) for the support.
4
5
6
7
8
9
(16) Flesch, D.; Gabler, M.; Lill, A.; Carrasco Gomez, R.; Steri, R.;
Schneider, G.; Stark, H.; Schubert-Zsilavecz, M.; Merk, D.
Fragmentation of GW4064 led to a highly potent partial farnesoid X
receptor agonist with improved drug-like properties. Bioorg. Med.
Chem. 2015, 23, 3490-3498.
(17) Miele, E.; Valente, S.; Alfano, V.; Silvano, M.; Mellini, P.;
Borovika, D.; Marrocco, B.; Po, A.; Besharat, Z.; Catanzaro, G.;
Battaglia, G.; Abballe, L.; Zwergel, C.; Stazi, G.; Milite, C.;
Castellano, S.; Tafani, M.; Trapencieris, P.; Mai, A.; Ferretti, E.; The
histone methyltransferase EZH2 as a druggable target in SHH
medulloblastoma cancer stem cells. Oncotarget. 2017, 40, 68557-
68570.
(18) Anzini, M.; Braile, C.; Valenti, S.; Cappelli, A.; Vomero, S.;
Marinelli, L.; Limongelli, V.; Novellino, E.; Betti, L.; Giannaccini, G.;
Lucacchini, A.; Ghelardini, C.; Norcini, M.; Makovec, F.; Giorgi G.;
Ian Fryer, R. Ethyl 8-Fluoro-6-(3-nitrophenyl)-4H-imidazo[1,5-
a][1,4]benzodiazepine-3-carboxylate as novel, highly potent, and safe
antianxiety agent. J. Med. Chem. 2008, 51, 4730-4743.
(19) Famiglini, V.; La Regina, G.; Coluccia, A.; Pelliccia, S.;
Brancale, A.; Maga, G.; Crespan, E.; Badia, R.; Riveira-Muñoz, E.;
Esté, J. A.; Ferretti, R.; Cirilli, R.; Zamperini, C.; Botta, M.; Schols, D.;
Limongelli, V.; Agostino, B.; Novellino, E.; Silvestri, R.
Indolylarylsulfones carrying a heterocyclic tail as very potent and
broad spectrum HIV-1 non-nucleoside reverse transcriptase inhibitors.
J. Med. Chem. 2014, 57, 9945-9957.
(20) Nuti, E.; Casalini, F.; Avramova, S. I.; Santamaria, S.; Fabbi, M.;
Ferrini, S.; Marinelli, L.; La Pietra, V.; Limongelli, V.; Novellino, E.;
Cercignani, G.; Orlandini, E.; Nencetti, S.; Rossello, A. Potent
arylsulfonamide inhibitors of tumor necrosis factor-α converting
enzyme able to reduce activated leukocyte cell adhesion molecule
shedding in cancer cell models. J. Med. Chem. 2010, 53, 2622-2635.
(21) Xu, X.; Xu, X.; Liu, P.; Zhu, Z. Y.; Chen, J.; Fu, H. A.; Chen, L.
L.; Hu, L. H.; Shen, X. Structural basis for small molecule NDB (N-
Benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-4-
(dimethylamino) benzamide) as a selective antagonist of farnesoid X
receptor  (FXR ) in stabilizing the homodimerization of the receptor
J. Biol. Chem. 2015, 290, 19888-19899.
ABBREVIATIONS
AF-2, activation function 2 (domain); Boc, tert-butyloxycarbonyl;
CDCA, chenodeoxycholic acid; Clint, intrinsic clearance; CYP7A1,
cholesterol
7
alpha-hydroxylase;
DIPEA,
N,N-
diisopropylethylamine; DMF, N,N-dimethylformamide; 6-
ECDCA, 6-ethyl chenodeoxycholic acid; FXR-LBD, Farnesoid X
Receptor-Ligand Binding Domain; HBTU, N,N,N′,N′-tetramethyl-
O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate; NAFLD,
non-alcoholic fatty liver disease; GC6P, glucose-6-phosphatase;
NDB, N-benzyl-N-(3-(tert-butyl)-4-hydroxyphenyl)-2,6-dichloro-
4-(dimethylamino) benzamide; OSTα, organic solute transporter
alpha; PAMPA, parallel artificial membrane permeability assay;
PCR, polymerase chain reaction; PDB, protein data bank; PEPCK,
phosphoenolpyruvate carboxykinase; SHP, small heterodimer
partner; SRC-1, steroid receptor coactivator-1; TFA, trifluoroacetic
acid.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
REFERENCES
(1) Forman, B. M.; Goode, E.; Chen, J.; Oro, A. E.; Bradley, D. J.;
Perlmann, T.; Noonan, D. J.; Burka, L. T.; McMorris, T.; Lamph, W.
W.; Evans, R. M.; Weinberger, C. Identification of a nuclear receptor
that is activated by farnesol metabolites. Cell 1995, 81, 687-693.
(2) Makishima, M.; Okamoto, A. Y.; Repa, J. J.; Tu, H.; Learned, R.
M.; Luk, A.; Hull, M. V.; Lustig, K. D.; Mangelsdorf, D. J.; Shan, B.
Identification of a nuclear receptor for bile acids. Science 1999, 284,
1362-1365.
(3) Parks, D. J.; Blanchard, S. G.; Bledsoe, R. K.; Chandra, G.;
Consler, T. G.; Kliewer, S. A.; Stimmel, J. B; Willson, T. M.; Zavacki,
A. M.; Moore, D. D.; Lehmann, J. M. Bile acids: natural ligands for an
orphan nuclear receptor. Science 1999, 284, 1365-1368.
(4) Zhang, Y.; Lee, F. Y.; Barrera, G.; Lee, H.; Vales, C.; Gonzalez,
F. J.; Willson, T. M.; Edwards, P. A. Activation of the nuclear receptor
FXR improves hyperglycemia and hyperlipidemia in diabetic mice.
Proc. Natl. Acad. Sci. USA 2006, 103, 1006-1011.
(5) Claudel, T.; Staels, B.; Kuipers, F. The Farnesoid X receptor: a
molecular link between bile acid and lipid and glucose metabolism.
Arterioscler. Thromb. Vasc. Biol. 2005, 25, 2020-2030.
(6) Cipriani, S.; Mencarelli, A.; Palladino, G.; Fiorucci, S. FXR
activation reverses insulin resistance and lipid abnormalities and
protects against liver steatosis in Zucker (fa/fa) obese rats. J. Lipid Res.
2010, 51, 771-784.
(7) Brzozowski, A. M.; Pike, A. C.; Dauter, Z.; Hubbard, R. E.;
Bonn, T.; Engström, O.; Ohman, L.; Greene, G. L.; Gustafsson, J. A.;
Carlquist, M. Molecular basis of agonism and antagonism in
theoestrogen receptor. Nature 1997, 389, 753-758.
(8) Prawitt, J.; Caron, S.; Staels, B. How to modulate FXR activity
to treat the metabolic syndrome. Drug Discov. Today Dis. Mech. 2009,
6, e55-e64.
(9) Cariou, B.; Staels, B. FXR: a promising target for the metabolic
syndrome? Trends Pharmacol. Sci. 2007, 28, 236-243.
(10) Sepe, V.; Distrutti, E.; Fiorucci, S.; Zampella, A. Farnesoid X
receptor modulators 2014-present: a patent review. Expert Opin. Ther.
Pat. 2018, 28, 351-364.
(22) Mi, L. Z.; Devarakonda, S.; Harp, J. M.; Han, Q.; Pellicciari, R.;
Willson, T. M.; Khorasanizadeh, S.; Rastinejad, F. Structural basis for
bile acid binding and activation of the nuclear receptor FXR. Mol. Cell
2003, 11, 1093-1100.
(11) Stedman, C.; Liddle, C.; Coulter, S.; Sonoda, J.; Alvarez, J. G.;
Evans, R. M.; Downes, M. Benefit of farnesoid X receptor inhibition
in obstructive cholestasis. Proc. Natl. Acad. Sci. USA 2006, 103,
11323-11328.
(12) Xu, Y. Recent progress on bile acid receptor modulators for
treatment of metabolic diseases. J. Med. Chem. 2016, 59, 6553-6579.
(13) Teno, N.; Yamashita, Y.; Iguchi, Y.; Fujimori, K.; Une, M.;
Nishimaki-Mogami, T.; Hiramoto, T.; Gohda, K. Nonacidic
Chemotype Possessing N-Acylated Piperidine Moiety as Potent
Farnesoid X Receptor (FXR) Antagonists. ACS Med. Chem. Lett. 2018,
9, 78-83.
(14) Schmidt, J.; Schierle, S.; Gellrich, L.; Kaiser, A.; Merk, D.
Structural optimization and in vitro profiling of N-phenylbenzamide-
ACS Paragon Plus Environment
6

Page 7 of 8
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment
7

ACS Medicinal Chemistry Letters
Page 8 of 8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
44x23mm (300 x 300 DPI)
ACS Paragon Plus Environment