GPBAR1 activation by C6-substituted hyodeoxycholane analogues protect against colitis

Article abstract of DOI:10.1021/acsmedchemlett.9b00636

GPBAR1 agonists have been identified as potential leads for the treatment of diseases related to colon inflammation such as Crohn's and ulcerative colitis. In this paper, we report the discovery of a small library of hyodeoxycholane analogues, decorated at C-6 with different substituents, as potent and selective GPBAR1 agonists. In vitro pharmacological assays showed that compound 6 selectively activates GPBAR1 (EC₅₀ = 0.3 μM) and reduces the production of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-a) in THP1 cells. The binding mode of compound 6 in GPBAR1 was elucidated by docking calculations. Moreover, compound 6 protects against TNBSinduced colitis in Gpbar1^+/+ rodent model, representing an intriguing lead for the treatment of these inflammatory disorders.

Full text of DOI:10.1021/acsmedchemlett.9b00636

Subscriber access provided by Linköping University Library
Letter
GPBAR1 activation by C6-substituted
hyodeoxycholane analogs protect against colitis
Simona De Marino, Claudia Finamore, Michele Biagioli, Adriana Carino, Silvia Marchianò, Rosalinda
Roselli, Cristina Di Giorgio, Martina Bordoni, Francesco Saverio Di Leva, Ettore Novellino,
Chiara Cassiano, Vittorio Limongelli, Angela Zampella, Carmen Festa, and Stefano Fiorucci
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00636 • Publication Date (Web): 02 Mar 2020
Downloaded from pubs.acs.org on March 4, 2020
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 9
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
GPBAR1 activation by C6-substituted hyodeoxycholane analogs
protect against colitis
Simona De Marino,1 Claudia Finamore,1 Michele Biagioli,2 Adriana Carino,2 Silvia Marchianò,2
Rosalinda Roselli,1 Cristina Di Giorgio,2 Martina Bordoni,2 Francesco Saverio Di Leva,1 Ettore
Novellino,1 Chiara Cassiano,1 Vittorio Limongelli,1,3 Angela Zampella,1 Carmen Festa1,* and Stefano
Fiorucci.2
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.
KEYWORDS. GPBAR1 agonists, bile acid receptor, hyodeoxycholane derivatives, inflammatory bowel disease (IBD), colitis.
ABSTRACT: GPBAR1 agonists have been identified as potential lead for the treatment of diseases related to colon inflammation
such as Crohn’s and ulcerative colitis. In this paper, we report the discovery of a small library of hyodeoxycholane analogs, decorated
at C-6 with different substituents, as potent and selective GPBAR1 agonists. In vitro pharmacological assays showed that compound
6 selectively activates GPBAR1 (EC50 = 0.3 M) and reduces the production of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-
α,) in THP1 cells. The binding mode of compound 6 in GPBAR1 was elucidated by docking calculations. Moreover, compound 6
protects against TNBS-induced colitis in Gpbar1+/+ rodent model representing an intriguing lead for the treatment of these
inflammatory disorders.
In the last decades the onset of several chronic inflammatory
disorders is increased in the worldwide population. Pathologies
such as inflammatory type 2 diabetes mellitus, metabolic
syndrome, inflammatory bowel disease (IBD), atherosclerosis
and rheumatoid arthritis have become more and more frequent.
Inflammatory bowel disease (IBD) is a common disorder that
involves chronic remittent or progressive inflammatory
conditions of gastrointestinal tract. Crohn’s disease (CD), a life-
long disease, and ulcerative colitis (UC), are the most common
forms of IBD. Multiple factors play an important role in the
pathogenesis of IBD. Among these may be included diet,
immunological factors, environmental factors, microbiome,
infectious agents and genetic susceptibility.1
The current treatment of IBD includes anti-inflammatory
aminosalicylates and corticosteroids, antibiotics, biologic,
immunosuppressive and anti-TNF- agents.2
The lack of alternative approaches and the limited efficacy of
this treatment, due to the incomplete remission and the arising
of severe side effects, urge for new pharmacological tools,
including molecules able to regulate the intestinal immune
response. In this scenario, the targeting of GPBAR1 constitutes
a therapeutic option for the treatment of IBDs.
and tauro-DCA (TDCA) as well as many steroids.3-6 Activation
of GPBAR1 results in the increase of intracellular cAMP levels,
which in turn triggers specific intracellular signalling cascades.
In the human body, GPBAR1 is expressed in the gallbladder,
small intestine, and is also located in tissues and cells not
participating in the enterohepatic circulation such as muscle,
brain, adipose tissue, immune system, and endothelial cells.7
Thus in recent years, academic and industrial investigations
have been focused on the development of steroidal and non-
steroidal GPBAR1 modulators useful in the treatment of
enterohepatic and metabolic disorders.8-12
GPBAR1 has an important role in intestinal homeostasis and
inflammation-driven immune disfunction. Previous studies
have shown that Gpbar1-/- mice have an altered intestinal
morphology with increased permeability and higher
susceptibility to develop colitis. In addition, in the immune
system, GPBAR1 reduces phagocytic activity and the
production of pro-inflammatory cytokines (TNF-, IL-1, IL-
1, IL-6, and IL-8).13
Recently, manipulating bile acid scaffolds, we succeeded in the
discovery of potent and selective GPBAR1 agonists and
demonstrated that GPBAR1 agonism shifts macrophagic
population toward an anti-inflammatory M2 phenotype in
mouse models of colitis, thus confirming that targeting
GPBAR1 can be a valid therapeutic option in IBDs.14
In this context, our speculation started from the observations
that the concomitant introduction of an alkyl group at C-6 and
a nitrile end group on C-23 side chain produces a potent and
GPBAR1, cell-surface G-protein coupled bile acid receptor 1
(also known as TGR5 or M-BAR1), belongs to the rhodopsin-
like superfamily of G protein coupled receptors (GPCRs) and
can be activated by a wide range of ligands, including
endogenous BAs primarily lithocholic acid (LCA), deoxycholic
acid (DCA) and their taurine-conjugates, tauro-LCA (TLCA)
1
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
selective GPBAR1 agonist (NorECDCN in Figure 1),15 and that
Page 2 of 9
concomitant epimerization at C-5 affording compound 15 in
quantitative yield. One-carbon degradation at C-24 followed by
Grignard reactions, as previously described, afforded
compounds 17-20. In agreement with the flat shape due to the
trans A/B ring junction and the steric influence played by the
methyl group at C-10, the approach of the nucleophile
proceeded from the  face of the steroidal nucleus producing
exclusively the 6-alkyl epimers (Scheme 1).
hyodeoxycholic acid (HDCA, 1),16,17 a natural secondary bile
acid, is a weak GPBAR1 agonist.18,19 Thus, combining this
information, and considering that chemical modifications on
bile acid scaffold profoundly influence the pharmacokinetic
properties and the activity towards BAs receptors,20 our
medicinal chemistry strategy has consisted in the evaluation of
the effect on GPBAR1 activation, introducing a nitrile group in
the side chain and different alkyl, alkenyl or aromatic
substituents at C-6 on HDCA (Figure 1).
Since GPBAR1 can be activated by many natural steroids and
terpenoids featuring a flat shape,5 we have speculated on the
stereochemistry of A/B ring junction of the steroidal nucleus in
order to investigate the effect of physical shape change on the
affinity towards GPBAR1.
1
2
3
4
5
6
7
8
Scheme 1. Synthetic procedure to prepare derivatives 4-14
9
and 17-20a
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
23
CN
CN
CN
c
d
6
HO
HO
HO
H
H
H
OH
R
OH
O
Figure 1.
2
3
6-analogs
6-analogs
O
23
CH3
CH3
CN
HO
CN
4
R=
R=
R=
R=
R=
R=
R=
5
R=
R=
R=
R=
21
CH2CH3
OH
CH2CH3
a , b
O
6
H
H
7
CH2CH2CH3
CH(CH3)2
CH=CH2
CH2CH=CH2
CH2C6H5
18
8
13
14
24
OH
19
9
6
HO
OH
HO
10
11
12
H
H
H
(CH2)3CH=CH2
C6H5
OH
R
OH
3
6
HO
norECDCN
H
1
HDCA (
)
OH
1
HDCA (
)
The synthetic protocol started with HDCA (1) that was first
formylated at C-3 and C-6 hydroxyl groups and then subjected
to one-carbon degradation at C-24 through the so-called second
order “Beckmann rearrangement”21. Alkaline conditions
(NaOH 6N) used in the treatment of the reaction afforded the
C-3 and C-6 deprotection giving the nornitrile (2) (Scheme 1).
Oxidation with pyridinium dichromate (PDC) afforded a
mixture of C-6 and C-3 mono and di-keto derivatives which
were efficiently separated by flash chromatography to obtain
the desired 6-keto nornitrile (3) in 60% chemical yield.
Treatment with several Grignard reagents gave 6-substituted
compounds 4-14 showed in the Scheme 1.
From a stereochemical point of view, treatment with methyl and
ethyl magnesium bromides produced a mixture of 6α- and 6β-
alkylated derivatives, in a 63/37 ratio for compounds 4 and 5
and in a 81/19 ratio for compounds 6 and 7, efficiently separated
by HPLC, where invariably the 6-alkyl epimers (5 and 7) were
eluted more rapidly than the corresponding 6-alkyl ones (4 and
6).22 Of interest, in agreement with the steric influence played
by the ring A oriented on the α-face of ring B, the reaction with
more hindered/longer Grignard reagents, such as propyl-,
isopropyl-, vinyl-, 4-pentenyl-, phenyl- magnesium bromide,
proceeded in a stereoselective manner, affording the exclusive
formation of the corresponding 6β epimers 8-12, through the
approach of the Grignard reagent from the upper-face of the
steroidal nucleus.
e,c,f
O
CN
CN
OH
a,b
d
6
HO
HO
HO
H
H
H
OH
O
R
O
15
16
17
18
19
20
R= CH3
R=CH2CH3
R=CH2CH2CH3
R=CH2CH=CH2
aReagents and conditions. a) HCOOH, HClO4, 45-50°C; b) TFA,
trifluoroacetic anhydride, NaNO2, 45-50°C and then NaOH, 90%
over two steps; c) PDC, in DCM dry, 60% yield; d) Methyl-, ethyl-,
propyl-, isopropyl-, allyl-, vinyl-, 4-pentenyl-, phenyl- and benzyl-
magnesium bromides, in THF dry, 0°C; e) p-TsOH, MeOH dry; f)
NaOH, MeOH:H2O (1:1), reflux, quantitative yield.
The synthetized compounds were evaluated on GPBAR1 in
transactivation assays. They were also tested on Farnesoid X
Receptor (FXR) in order to determine their activity towards this
related nuclear bile acid receptor. For this purpose, Hek293T
cells transfected with GPBAR1 and HepG2 cells transfected
with FXR, were incubated with 10 µM of compounds 2 and 4-
20. Activities of compounds on GPBAR1 and FXR were
compared to those of the reference compounds TLCA and
CDCA, respectively, that were set as 100% of activity. As
shown in Table 1, all tested compounds, with a cis A/B ring
junction, were effective in transactivating GPBAR1, while they
exerted a minimal effect on FXR, showing a marked selectivity
toward GPBAR1 over this receptor (Table 1). Compounds 17-
20, endowed with a trans junction, showed no agonistic activity
towards GPBAR1 and FXR (Figures S2 and S3).
Results shown in Table 1 demonstrated that the best match in
terms of % efficacy, EC50 and selectivity has been found for
compounds 4-7 and 11.
The physicochemical parameters of the most efficacious
compounds were measured by LC-MS analysis. As shown in
Table S1 all compounds exhibited good aqueous solubility and
a suitable logD.
Finally, the treatment with allyl- and benzyl magnesium
bromides afforded surprisingly the 6-substituted compounds
13 and 14.
Structural and stereochemical characterization was based on
careful analysis of NMR data. According to literature data,23 in
the 1H NMR spectra, the -orientation of the hydroxyl group at
C-6 as in compounds 5 and 7 produces the diagnostic down-
field shifted signal for CH3-19 (i.e. H 1.14 in 5 vs 0.98 in 4; H
1.15 in 7 vs 1.01 in 6). Additionally, NOESY cross peaks H2-
24/H-5, H-8 and H3-19 confirmed the 6-orientation of the
ethyl group at C-6 on compound 6.
To access to compounds 17-20 with the trans A/B ring junction,
HDCA (1) was subjected to methylation on the side chain and
then to C-6 regioselective oxidation as previously described.
Alkaline hydrolysis at the side chain methyl ester produced the
Therefore, we have investigated whether these compounds
could exert an in vitro activity on GPBAR1-target genes. For
this purpose, THP1 monocytic/macrophage cells, were primed
with LPS (lipopolysaccharide), and co-incubated with or
2
ACS Paragon Plus Environment

Page 3 of 9
ACS Medicinal Chemistry Letters
without compounds 4-7 and 11 at 10 µM. As shown in Figure
2, all compounds reduced the production of pro-inflammatory
Table 1. Efficacy, EC50 values and selectivity for compounds 2 and 4-14
cytokines (IL-1β, IL-6) in THP1 cells but only compounds 4
and 6-7 decrease also the production of TNF-
1
2
3
Compound
C-6 substituents
GPBAR1*
FXR**
GPBAR1#
GPBAR1/FXR##
4
5
Eff. (% vs TLCA)
Eff. (% vs CDCA)
EC50 µM
Selectivity
CN
6
7
6
HO
H
OH
R
8
9
2
4
H
118.7 ± 3.16
95.4 ± 0.24
83.4 ± 2.4
3.5 ± 0.34
6.4 ± 0.23
5.5 ± 0.7
2.65
6.9
33.9
14.9
15.1
8.2
6-CH3
6-CH3
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
5
0.57
0.3
6
101.2 ± 4.1
65.9 ± 1.69
67.1 ± 2.1
12.3 ± 0.47
6.6 ± 0.065
3.1 ± 0.29
4.3 ± 0.31
2.2 ± 0.5
6-CH2CH3
6-CH2CH3
7
0.66
1.2
9.9
8
21.6
5.16
28.4
56.31
2.13
15.2
1.88
6-CH2CH2CH3
6-CH(CH3)2
6-CH=CH2
9
22.2 ± 0.13
62.5 ± 2.18
90.1 ± 1.35
38.8 ± 1.79
62.5 ± 2.15
8.3 ± 0.015
2.78
2.26
0.78
4.58
0.1
10
11
12
13
14
1.6 ± 0.05
18.2 ± 1.08
4.1 ± 0.04
4.4 ± 0.49
6-(CH2)3CH=CH2
6-C6H5
6-CH2CH=CH2
6-CH2C6H5
9.33
*Activity toward GPBAR1 in a reporter assay was assessed in HEK-293T cells transfected with a cAMP responsive element (CRE) cloned
upstream to the luciferase gene. For calculation of efficacy data, maximal transactivation of CRE caused by each compound (10ꢀμM) was
compared to maximal transactivation caused by TLCA (10 μM). **Activity toward FXR in a reporter assay was assessed in HepG2 cells
transfected with a FXR responsive element (IR1) cloned upstream to the luciferase gene. For calculation of efficacy data, maximal
transactivation of IR1 caused by each compound (10 μM) was compared to maximal transactivation caused by CDCA (10ꢀμM). #GPBAR1
affinity expressed as EC50 values. ##GPBAR1/FXR selectivity calculated as ratio between % of activity toward GPBAR1 and % of activity
toward FXR. Data are calculated from the same experiment conducted in triplicate.
Additionally, exposure to compound 6 increased the expression
B
A
*
*
*
*
*
for anti-inflammatory gene IL-10, that is a specific target of
GPBAR1.These results prompted us to investigate the effect of
compound 6 in a TNBS-induced colitis model in Gpbar1+/+ and
Gpbar1-/- mice. The development and the severity of colitis in
Gpbar1+/+ mice, assessed by body weight loss, CDAI (Clinical
Disease Activity Index) and histological sections, were
attenuated by treatment with 6. Conversely, this compound did
not produce effects in the Gpbar1-/- mice (Figures 3 A-C).
We also analyzed the expression of pro- and anti-inflammatory
cytokines in the colon of these animals (Figure 4). As expected,
TNBS administration increased the expression of pro-
inflammatory cytokines IL-6, IL-1 and TNF-α both in wild-
type mice and in Gpbar1-/- mice. On the contrary, compound 6
was able to revert this inflammatory pattern by reducing the
expression of pro-inflammatory cytokines and also increasing
the expression of the anti-inflammatory cytokine, IL-10. These
beneficial effects were lost in mice lacking the receptor.
To elucidate the binding mode of the newly developed HDCA
analogues, we performed docking calculations that are widely
used to generate and assess ligand/protein complexes based on
scoring functions.24-26 In particular, we investigated the most
promising compound of the series, 6, into the homology model
of hGPBAR1 that we have previously reported and validated
through drug design studies.17,27,28
200
*
800
*
*
*
*
*
*
150
100
50
600
400
200
0
0
*
D
C
*
*
*
300
60
40
20
0
*
*
*
*
200
100
0
E
*
*
15
NT
LPS
*
*
LPS + 5
LPS + 4
LPS + 7
LPS + 6
LPS + 11
10
5
0
Figure 2. In vitro effects of selected compounds on human
monocytes activation. Relative mRNA expression of pro- and anti-
inflammatory genes was assessed by RT-PCR in THP1 cells: A)
IL-1β; B) IL-6; C) TNF-α; D) IL-10 and E) GPBAR1, relative
mRNA expression. Values are normalized relative to GAPDH
mRNA and are expressed as mean ± SEM (n = 3); *p < 0.05.
In the best scored and most recurrent docking pose, compound
6 is predicted to bind GPBAR1 similar to other BA derivatives
previously reported as agonists of this receptor.17,27-29 The
ligand’s steroidal scaffold is hosted in a wide hydrophobic
pocket where it can form favourable interactions with the side
3
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
chains of residues such as Leu71, Phe96, Leu174, and Trp237
(Figure 5). Additional Van der Waals contacts are formed by
the 6β-ethyl group of 6 which occupies the ancillary pocket
shaped by residues Leu97, Phe138 and Leu173.
Page 4 of 9
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
4
ACS Paragon Plus Environment

Page 5 of 9
ACS Medicinal Chemistry Letters
$
*
* $
A
B
1
100
2
3
4
10
*
*
*
*
*
*
8
90
*
5
6
6
7
8
9
Gpbar1+/+ NT
4
80
70
Gpbar1+/+ TNBS
Gpbar1+/+ TNBS + 6 30 mg/Kg
Gpbar1-/- NT
2
Gpbar1-/- TNBS
0
Gpbar1-/- TNBS + 6 30 mg/Kg
Day 1
Day 2
Day 3
Day 4
0
1
2
3
4
-1
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
NT
TNBS
TNBS + 6
C
Gpbar1+/+
Gpbar1-/-
Figure 3. Compound 6 protects against colitis in a GPBAR1-dependent manner. Gpbar1+/+ and Gpbar1-/- mice were treated with TNBS alone
or in combination with compound 6 (30 mg/kg). A) Changes in body weight. B) CDAI of mice during the course of TNBS-induced colitis.
C) H&E staining of colon sections from control mice, mice treated with TNBS, and mice treated with TNBS and compound 6 (original
magnification 10x). (n = 5);#p < 0.05
Besides this hydrophobic interaction network, the 3α- and 6α-
OHs of the ligand can establish H-bonds with the side chains of
Glu169 on TM5 and Asn93 on TM3, respectively. On the
opposite side of the binding cavity, the ligand side chain points
toward the amphipathic cleft defined by the side chains of
residues of TM helices 1, 2, and 7, which have been described
in previous papers as interacting points for BAs derivatives.27
Here, the ligand’s nitrile group can form direct or water-
mediated H-bond interactions with Ser267 and Ser270, which
might contribute to stabilize the ligand binding pose.
A
B
*
*
20
15
15
*
*
10
5
*
*
*
*
10
5
0
0
D
C
2.0
*
10
8
*
*
1.5
1.0
0.5
0.0
6
4
2
0
*
*
E
1.5
1.0
0.5
0.0
Gpbar1+/+ NT
Gpbar1+/+ TNBS
Gpbar1+/+ TNBS + 6 30 mg/Kg
Gpbar1-/- NT
Gpbar1-/- TNBS
Gpbar1-/- TNBS + 6 30 mg/Kg
Figure 5. Binding mode of 6 in hGPBAR1.27 The ligand is
represented as green sticks, while the receptor is shown as gray
cartoons. Amino acids involved in the ligand binding are depicted
as sticks. Non-polar hydrogens are omitted for clarity, while H-
bonds are shown as black dashed lines.
n.d.n.d. n.d.
Figure 4. Compound 6 reversed TNBS-induced inflammation in a
GPBAR1-dependent manner. Gpbar1+/+ and Gpbar1-/- mice were
treated with TNBS alone or in combination with compound 6 (30
mg/kg). Relative mRNA expression of pro- and anti-inflammatory
genes was assessed by RT-PCR colon samples: A) Il-1β, B) Il-6,
C) Tnf-α, D) Il-10 and E) Gpbar1, relative mRNA expression.
Values are normalized relative to Gapdh mRNA and are expressed
as mean ± SEM (n = 5); *p < 0.05.
We note that the ligand interaction with Glu169 has been
previously identified by us and by other authors as a major
anchor point for BAs to GPBAR1, presumably triggering the
receptor activation.27,30
5
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 9
(1) Knight-Sepulveda, K.; Kais, S.; Santaolalla, R.; Abreu, M. T.
Diet and inflammatory bowel disease. Gastroenterol. Hepatol. 2015,
11, 511-520.
(2) Mosli, M. H.; Rivera-Nieves, J.; Feagan. B. G. T-cell trafficking
and anti-adhesion strategies in inflammatory bowel disease: current
and future prospects. Drugs 2014, 74, 297-311.
(3) Maruyama, T.; Miyamoto, Y.; Nakamura, T.; Tamai, Y.; Okada,
H.; Sugiyama, E.; Nakamura, T.; Itadani, H.; Tanaka, K. Identification
of membrane-type receptor for bile acids (M-BAR). Biochem. Biophys.
Res. Commun. 2002, 298, 714-719.
On the other hand, the interaction with Asn93 was reported to
play a minor role in the binding of BAs to GPBAR1. In fact,
LCA derivatives display good GPBAR1 agonist profiles,
although they do not present alcohol groups at either the C-6 or
C-7 position that could interact with Asn93.28 These data might
explain our findings indicating that compound 7, which
displays an inverted configuration at C-6 and thus cannot
interact with Asn93 (Figure S1), shows however a good EC50
value, comparable to that of 6 (Table 1).
1
2
3
4
5
6
7
8
9
(4) Watanabe, M.; Houten, S. M.; Mataki, C.; Christoffolete, M. A.;
Kim, B. W.; Sato, H.; Messaddeq, N.; Harney, J. W.; Ezaki, O.;
Kodama, T.; Schoonjans, K.; Bianco, A. C.; Auwerx, J. Bile acids
induce energy expenditure by promoting intracellular thyroid hormone
activation. Nature 2006, 439, 484-489.
(5) De Marino, S., Festa, C., Sepe, V., Zampella, A. Chemistry and
pharmacology of GPBAR1 and FXR selective agonists, dual agonists,
and antagonists. Handb. Exp. Pharmacol. 2019, 256, 137-165.
(6) Imperatore, C.; D’Aniello, F.; Aiello, A.; Fiorucci, S.; D’Amore,
C.; Sepe, V.; Menna, M. Phallusiasterols A and B: Two new sulfated
sterols from the mediterranean tunicate Phallusia fumigata and their
effects as modulators of the PXR receptor. Mar. Drugs 2014, 12, 2066–
2078.
(7) Perino, A.; Schoonjans, K. TGR5 and immunometabolism:
Insights from physiology and pharmacology. Trends Pharmacol. Sci.
2015, 36, 847-57.
(8) Gioiello, A.; Rosatelli, E.; Nuti, R.; Macchiarulo, A.; Pellicciari,
R. Patented TGR5 modulators: a review (2006 -- present). Expert Opin.
Ther. Pat. 2012, 22,1399-414.
(9) Xu, Y. Recent progress on bile acid receptor modulators for
treatment of metabolic diseases. J. Med. Chem. 2016, 59, 6553-6579.
(10) Duan, H.; Ning, M.; Zou, Q.; Ye, Y.; Feng, Y.; Zhang, L.; Leng,
Y.; Shen, J. Discovery of intestinal targeted TGR5 agonists for the
treatment of type 2 diabetes. J. Med. Chem. 2015, 58, 8, 3315-3328.
(11) Chen, T.; Reich, N. W.; Bel, N.; Finn, P. D.; Rodriguez, D.;
Kohler, J.; Kozuka, K.; He, L.; Spencer, A. G.; Charmot, D.; Navre,
M.; Carreras, C. W.; Koo-McCoy, S.; Tabora, J.; Caldwell, J. S.;
Jacobs, J. W.; Lewis, J. G. Design of gut-restricted thiazolidine agonists
of G Protein-Coupled Bile Acid Receptor 1 (GPBAR1, TGR5). J. Med.
Chem. 2018, 61, 7589-7613.
(12) Cao, H.; Chen, Z. X.; Wang,K.; Ning, M. M.; Zou, Q. A.; Feng,
Y.; Ye, Y. L.; Leng, Y.; Shen, J. H. Intestinally-targeted TGR5
agonists equipped with quaternary ammonium have an improved
hypoglycemic effect and reduced gallbladder filling effect. Sci. Rep.
2016, 6, 28676.
(13) Cipriani, S.; Mencarelli, A.; Chini, M. G.; Distrutti, E.; Renga,
B.; Bifulco, G.; Baldelli, F.; Donini, A.; Fiorucci, S. The bile acid
receptor GPBAR-1 (TGR5) modulates integrity of intestinal barrier
and immune response to experimental colitis. Plos One 2011, 6,
e25637.
(14) Biagioli, M.; Carino, A.; Cipriani, S.; Francisci, D.; Marchianò,
S.; Scarpelli, P.; Sorcini, D.; Zampella, A.; Fiorucci, S. The bile acid
receptor GPBAR1 regulates the M1/M2 phenotype of intestinal
macrophages and activation of GPBAR1 rescues mice from murine
colitis. J. Immunol. 2017, 199, 718-733.
In conclusion, we report the discovery of a new chemotype of
GPBAR1 agonists designed on hyodeoxycholic acid scaffold.
We have decorated the C-6 position with different substituents
and speculated also on the stereochemistry of A/B ring junction
of the steroidal nucleus obtaining a small library of derivatives.
Interestingly, we note that compounds featured with a cis A/B
ring junction showed a marked selectivity toward GPBAR1
over FXR in transactivation assays, exerting minimal activating
effects on the latter receptor. Our studies identified compound
6 as potent and selective GPBAR1 agonist. The activation of
the receptor, combined with the ability to revert both the
expression of inflammatory genes in vitro and the inflammatory
pattern in TNBS-induced colitis model in a GPBAR1-
dependent manner, indicate compound 6 as a promising lead for
the treatment of GPBAR1-related inflammatory bowel
disorders.
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
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
Experimental procedures; binding mode of compound 7 in
hGPBAR1; transactivation assay on GPBAR1 of compounds 17-
20; AlphaScreen assay on FXR of compounds 17-20;
transactivation assays on LXRα, LXRβ, and PPARγ of compound
1
6; Physiochemical properties of compounds 4-7 and 11; H NMR
of compounds 2, 4-14 and 17-20; NOESY spectrum of compound
6 and ROESY spectrum of compound 8 (PDF).
AUTHOR INFORMATION
Corresponding Author
*Dr. Carmen Festa; e-mail: carmen.festa@unina.it
Notes
Stefano Fiorucci and Angela Zampella have filed the Italian patent
application No. 102019000023403 in the name of PRECISION
BIO-THERAPEUTICS S.R.L., a spinoff of the University of
Perugia, on same the compounds described in this paper.
ABREVIATIONS
(15) Festa, C.; Renga, B.; D’Amore, C.; Sepe, V.; Finamore C.; De
Marino, S.; Carino, A.; Cipriani, S.; Monti, M. C.; Zampella, A.;
Fiorucci,S. Exploitation of cholane scaffold for the discovery of potent
and selective farnesoid X receptor (FXR) and G-protein coupled bile
acid receptor 1 (GP-BAR1) ligands. J. Med. Chem., 2014, 57, 8477-
8495.
(16) De Marino, S.; Carino, A.; Masullo, D.; Finamore, C.; Sepe, V.;
Marchianò, S.; Di Leva, F. S.; Limongelli, V.; Fiorucci, S.; Zampella,
A. Epoxide functionalization on cholane side chains in the
identification of G-protein coupled bile acid receptor (GPBAR1)
selective agonists. RSC Adv. 2017, 7, 32877-32885.
(17) De Marino, S.; Carino, A.; Masullo, D.; Finamore, C.;
Marchianò, S.; Cipriani, S.; Di Leva, F. S.; Novellino, E.; Catalanotti,
B.; Limongelli, V.; Fiorucci, S.; Zampella, A. Hyodeoxycholic acid
derivatives as liver X receptor  and G-protein-coupled bile acid
receptor agonists. Sci. Rep. 2017, 7:43290.
BA, Bile acid; CDAI, colitis disease activity index; CDCA,
chenodeoxycholic acid; DCM, dichloromethane; FXR, farnesoid X
receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
GPBAR1, G protein-coupled bile acid receptor 1; HDCA,
hyodeoxycholic acid; IBD, inflammatory bowel disease; IL-6,
interleukin 6; IL-1β, interleukin 1; IL-10, interleukin 10; LCA,
lithocholic acid; LDB, ligand binding domain; LPS,
lipopolysaccharide; NOESY, Nuclear Overhauser Effect
SpectroscopY; PDC, pyridinium dichromate; TNBS, 2,4,6-
Trinitrobenzenesulfonic acid; TNF-α, Tumor necrosis factor alpha;
TFA, trifluoroacetic acid; TLCA, taurolithocholic acid.
REFERENCES
6
ACS Paragon Plus Environment

Page 7 of 9
ACS Medicinal Chemistry Letters
(18) Song, C.; Hiipakka, R. A.; Liao, S. Selective activation of liver
X receptor alpha by 6-alpha-hydroxy bile acids and analogs. Steroids
2000, 65, 423-427.
a][1,4]benzodiazepine-3-carboxylate as novel, highly potent, and safe
antianxiety agent. J. Med. Chem. 2008, 51, 4730-4743.
1
2
3
4
5
6
7
8
(25) 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. Indolylaryl-
sulfones carrying a heterocyclic tail as very potent and broad spectrum
HIV-1 non-nucleoside reverse transcriptase inhibitors. J. Med. Chem.
2014, 57, 9945-9957.
(26) 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.
(27) D’Amore, C.; Di Leva, F. S.; Sepe, V.; Renga, B.; Del Gaudio,
C.; D’Auria, M. V.; Zampella, A.; Fiorucci, S.; Limongelli, V. Design,
synthesis, and biological evaluation of potent dual agonists of nuclear
and membrane bile acid receptors. J. Med. Chem. 2014, 57, 937-954.
(28) Di Leva, F. S.; Festa, C.; Renga, B.; Sepe, V.; Novellino, E.;
Fiorucci, S.; Zampella, A.; Limongelli, V. Structure-based drug design
targeting the cell membrane receptor GPBAR1: exploiting the bile acid
scaffold towards selective agonism. Sci. Rep. 2015, 5, 16650.
(29) Di Leva, F. S.; Di Marino, D.; Limongelli, V. Structural insight
into the binding mode of FXR and GPBAR1 modulators. Handb. Exp.
Pharmacol. 2019, 256, 111-136.
(19) Sato, H.; Macchiarulo, A.; Thomas, C.; Gioiello, A.; Une, M.;
Hofmann, A. F.; Saladin, R.; Schoonjans, K.; Pellicciari, R.; Auwerx J.
Novel potent and selective bile acid derivatives as TGR5 agonists:
biological screening, structure-activity relationships, and molecular
modeling studies. J. Med. Chem. 2008, 51, 1831-1841.
(20) Roda, A.; Pellicciari, R.; Gioiello, A.; Neri, F.; Camborata, C.;
Passeri, D.; De Franco, F.; Spinozzi, S.; Colliva, C.; Adorini, L.;
Montagnani, M.; Aldini, R. Semisynthetic bile acid FXR and TGR5
agonists: Physicochemical properties, pharmacokinetics, and
metabolism in the rats. J. Pharmacol. Exp. Ther. 2014, 350, 56-68.
(21) Schteingart, C. D.; Hofmann, A. F. Synthesis of 24-nor-5-
cholan-23-oic acid derivatives: a convenient and efficient one-carbon
degradation of the side chain of natural bile acids. J. Lipid Res. 1988,
29, 1387-1395.
(22) Une, M.; Cohen, B. I.; Mosbach, E. H. New bile acid analogs:
3,7-dihydroxy-7-methyl-5-cholanoic acid, 3,7-dihydroxy-7-
methyl-5-cholanoic acid, and 3-hydroxy-7ξ-methyl-5-cholanoic
acid. J. Lipid Research 1984, 25, 407-410.
(23) Matoba, N.; Mosbach, E. H.; Cohen, B. I.; Une, M.; McSherry,
C. K. Synthesis of new bile acid analogues and their metabolism in the
hamster: 3,6-dihydroxy-6methyl-5-cholanoic acid and 3,6-
dihydroxy-6-methyl-5-cholanoic acid, J. Lipid Res. 1989, 30, 1005-
1014.
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
(30) Gertzen C. G. W.; Spomer, L.; Smits, S. H. J.; Häussinger, D.;
Keitel, V.; Gohlke, H. Mutational mapping of the transmembrane
binding site of the G-protein coupled receptor TGR5 and binding mode
prediction of TGR5 agonists. Eur. J. Med. Chem. 2015, 104, 57-72.
(24) 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-
7
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 8 of 9
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
8
ACS Paragon Plus Environment

Page 9 of 9
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
35x21mm (300 x 300 DPI)
ACS Paragon Plus Environment