Discovery of 6α-ethyl-23(S)-methylcholic acid (S-EMCA, INT-777) as a potent and selective agonist for the TGR5 receptor, a novel target for diabesity

Article abstract of DOI:10.1021/jm901390p

In the framework of the design and development of TGR5 agonists, we reported that the introduction of a C23(S)-methyl group in the side chain of bile acids such as chenodeoxycholic acid (CDCA) and 6-ethylchenodeoxycholic acid (6-ECDCA, INT-747) affords selectivity for TGR5. Herein we report further lead optimization efforts that have led to the discovery of 6R-ethyl-23(S)- methylcholic acid (S-EMCA, INT-777) as a novel potent and selective TGR5 agonist with remarkable in vivo activity.

Full text of DOI:10.1021/jm901390p

7958 J. Med. Chem. 2009, 52, 7958–7961
DOI: 10.1021/jm901390p
Chart 1. Natural and Semisynthetic BAs
Discovery of 6r-Ethyl-23(S)-methylcholic Acid
(S-EMCA, INT-777) as a Potent and Selective
Agonist for the TGR5 Receptor, a Novel Target
for Diabesity
Roberto Pellicciari,*,† Antimo Gioiello,†
Antonio Macchiarulo,† Charles Thomas,‡,§
Emiliano Rosatelli,† Benedetto Natalini,†
Roccaldo Sardella,† Mark Pruzanski, Aldo Roda,^
Elisabetta Pastorini,^ Kristina Schoonjans,‡,# and
Johan Auwerx‡,#,¥
†Dipartimento di Chimica e Tecnologia del Farmaco, Universitaꢀ di
Perugia, 06123 Perugia, Italy, ‡Institut de Geꢁneꢁtique et de Biologie
Moleꢁculaire et Cellulaire (IGBMC), CNRS/INSERM/ULP, 67404
Illkirch, France, §Center of Phenogenomics, Ecole Polytechnique
Feꢁdeꢁrale de Lausanne, 1015 Lausanne, Swizerland, Intercept
Pharmaceuticals, New York, New York 10013, ^Dipartimento di
Scienze Farmaceutiche, Alma Mater Studiorum, Universitaꢀ di
Bologna, 40126 Bologna, Italy, #Laboratory of Integrative and
expressed but most importantly in metabolic tissues such as
liver, muscle, intestine, and brown adipose tissue.4 In the
murine enteroendrocrine cell line (STC-1), in vitro BA-
mediated activation of TGR5 leads to a rise in intracellular
levelsofcAMP, triggeringanincreaseinglucagon-likepeptide
1 (GLP-1) release.6 Additionally, in brown adipocytes TGR5-
dependent enhancement of cAMP levels increases type 2
iodothyronine deiodinase-mediated energy expenditure.6,7
TGR5 signaling in the muscle, the liver (especially in the
sinusoidal endothelial cells (SECs) and in the Kuppfer cells),8,9
and the gallbladder10 contributes to the control of energy
expenditure,2c the regulation of gallbladder and liver func-
tions,10 the protection of liver against lipid peroxidation, and
the reduction of proinflammatory cytokine production.8,9
Accordingly, TGR5 may have broad therapeutic application,
ranging from the treatment of metabolic disorders to liver and
inflammatory diseases.1
Starting from the discovery of FXR as a BA nuclear receptor
in 1999,11-13 a finding that heralded the paradigm shift of BAs
from detergent-like molecules to signaling hormones, and
following the disclosure of TGR5 as a BA membrane
receptor,3,4 we have been engaged in the search for potent
and selective modulators of these receptors through a strategic
approach based on the screening of a large number of BA
derivatives prepared by extensive modifications of their core
structure.5,14-19 In the case of FXR, an important break-
through came with the discovery that the agonist activity at
this receptor was dramatically increased when introducing an R
ethyl moiety in the C6 position of chenodeoxycholic acid
(CDCA, 3), a primary BA and FXR endogenous agonist
(FXR-EC50=13 μM). This finding resulted in the disclosure
of 6-ECDCA (INT-747, 5, FXR-EC50 = 0.099 μM), a com-
pound endowed with remarkable properties of potency, selec-
tivity, and metabolic stability.15 Currently, 6-ECDCA (5) is in
phase II clinical studies for primary biliary cirrhosis and is
being advanced in nonalcoholic steatohepatitis (NASH). When
5 was docked into the ligand binding domain of FXR, a nice fit
was observed in a small hydrophobic cavity of the receptor.16
In our search for novel potent and selective TGR5 ago-
nists, we have followed an analogous evaluative strategy
which in this case involved initial screening of natural and
semisynthetic BA libraries for TGR5 activity. As a result, we
discovered that the incorporation of a methyl moiety with
the preference of the S over the R chiral configuration
at the C23 position of the CDCA side chain (3) afforded
selective, albeit not very potent, TGR5 agonist properties
ꢁ ꢁ
Systems Physiology (LISP), Ecole Polytechnique Federale de
Lausanne, 1015 Lausanne, Swizerland, and ¥Institut Clinique de la
Souris (ICS), 67404 Illkirch, France
Received September 18, 2009
Abstract: In the framework of the design and development of
TGR5 agonists, we reported that the introduction of a C23(S)-
methyl group in the side chain of bile acids such as chenodeoxy-
cholic acid (CDCA) and 6-ethylchenodeoxycholic acid (6-ECDCA,
INT-747) affords selectivity for TGR5. Herein we report further
lead optimization efforts that have led to the discovery of 6R-ethyl-
23(S)-methylcholic acid (S-EMCA, INT-777) as a novel potent and
selective TGR5 agonist with remarkable in vivo activity.
In the quest for improved therapies targeting the variety of
pathways involved in obesity, diabetes, and prediabetic in-
sulin resistanceinthe metabolicsyndrome, two bileacid (BAa)
activated receptors, namely, the nuclear farnesoid-X receptor
(FXR) and, more recently, the G protein coupled receptor
(GPCR) TGR5, have emerged as especially attractive targets
for drug discovery efforts.1 Indeed, it is well established that
FXR and TGR5 have key roles in the regulation of the
intricate network governing lipid, cholesterol, and energy
homeostasis, the transport and metabolism of fatty acids
and triglycerides, and the regulation of glucose homeostasis.2
In this paper, we focus on TGR5, a receptor that was
discovered in 2002 and further characterized in 2003 by two
independent Japanese groups as coupled to GRs-proteins and
shown to be mainly activated by lithocholic acid (LCA, 1) and
deoxycholic acid (DCA, 2) (Chart 1).3-5 TGR5 is widely
*To whom correspondence should be addressed. Phone: þ39 075
5855120. Fax: þ39 075 5855124. E-mail: rp@unipg.it.
a Abbreviations: BA, bile acid; FXR, farnesoid-X receptor; GPCR,
G-protein-coupled receptor; GLP-1, glucagon-like peptide 1; cAMP,
cyclic adenosine monophosphate; CA, cholic acid; LCA, lithocholic
acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; 6-ECD-
CA, 6R-ethylchenodeoxycholic acid; EMCA, 6R-ethyl-23-methylcholic
acid; bw, body weight.
r
pubs.acs.org/jmc
Published on Web 11/12/2009
2009 American Chemical Society

Letter
Scheme 1
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7959
a
Table 1. TGR5 and FXR Activities of Cholic Acid and Its Derivativesa
TGR5b
FXRc
d
compd
EC50
efficacy
101
EC50
efficacy
CA (4)
13.6
(6.66 - 22.87)
3.44
>100
0e
49
6-ECA (14)
S-EMCA (8)
R-EMCA (9)
95
166
86
>100
>100
>100
(2.37 - 5)
0.82
18e
114
(0.54 - 1.24)
4.79
(3.44 - 6.66)
a Data represent average values of at least three independent experi-
ments. b Units are μM for EC50 and % of 10 μM LCA (1) value for
efficacy. c Units are μM for EC50 and % of 10 μM 5 value for efficacy.
d 95% confidence intervals in parentheses. e Curve fit, <0.5.
Table 2. Physicochemical Properties of BAsa
a Reagents and conditions: (i) p-TSA, MeOH; (ii) LDA, TMSCl,
Wsb
cmcc
(mM)
STcmc
(dyne/cm)
albumin
binding (%)
d
Et3N, THF, -78 ꢀC; (iii) MeCHO, BF3 OEt2, CH2Cl2, -60 ꢀC; (iv) H2,
3
compd
(μM)
PtO2, AcOH/HCl; (v) NaOH, MeOH; (vi) NaBH4, THF/H2O; (vii) p-
TSA, MeOH; (viii) 3,4-DHP, p-TSA, dioxane, room temp; (ix) LDA,
MeI, THF, -78 ꢀC; (x) HCl, MeOH, 45 ꢀC; (xi) NaOH, MeOH, reflux.
CDCA (3)
CA (4)
S-EMCA (8)
30
270
99
3
10
2
45.5
48.2
50.1
93
54
62
a The methods used for the measurements of the physicochemical
properties are those reported in ref 26. b Ws: water solubility as protonated
species in 0.1 M HCl water solution. c cmc: critical micellar concentration
determined in 0.15 M NaCl water solution. d STcmc: surface tension at cmc in
0.15 M NaCl water solution.
(6, TGR5-EC50 = 3.58 μM, FXR-EC50 > 100 μM).19 When
this additional chemical feature was next introduced in 6-
ECDCA (5), we were pleasantly surprised to discover a remar-
kable reversal of the activity profile. In the resulting derivative
7, the FXR activity was remarkably decreased (FXR-EC50=
11.80 μM) and the TGR5 efficacy boosted to nanomolar con-
centration (TGR5-EC50=0.095 μM).19 Further conclusions on
the structure-activity relationships (SAR) profile of BA-de-
rived TGR5 agonists were also drawn by the results of our
screening.6,19
of the toxic secondary BA DCA (2) via extensive and efficient
intestinal bacteria 7R-dehydroxylation.24
On the basis of the above considerations, we focused our
attention on the incorporation of the crucial 6R-ethyl- and 23-
methyl moieties in the chemical scaffold of CA (4) as key
features to improve potency, selectivity, and metabolic stability
of the resulting compound. The synthesis, physicochemical
properties, and selected biological properties of 6R-ethyl-23(S)-
methylcholic acid (S-EMCA, INT-777, 8) are herein reported.
The synthetic route to S-EMCA (8) and R-EMCA (9) is
illustrated in Scheme 1. Treatment of methyl 7-ketodeoxy-
cholate with LDA in THF at -78 ꢀC followed by reaction of
the enolate with trimethylchlorosilane afforded the corre-
sponding silyl enol ether 11 in nearly quantitative yield. The
intermediate 11 was then reacted with acetaldehyde in the
As a continuation to this work and to find a suitable
candidate for TGR5 clinical studies, we directed our attention
to the optimization of 7 in search for a compound endowed
not only with analogous properties of efficacy and selectivity
but also with improved pharmacokinetic properties and a
more favorable metabolic profile. To this end, we were
attracted by the peculiar biological and physicochemical
properties of CA (4), a primary bile acid in human and many
animal species, also reported as one of the main components
together with bilirubin of Calculus Bovis, a highly valued
traditional Chinese medicine (TCM) remedy.20 CA (4) differs
from CDCA (3) by the presence at C12 of an additional R-
hydroxyl group oriented on the polar side of the molecule.
This “minor” structural difference accounts for the remark-
ably different physicochemical and biological features of these
two BAs. With respect to CDCA (3) protonated CA (4) is
about 10-fold more soluble and relatively less detergent as a
result of its hydrophobic/hydrophilic balance and polarity
(Table 2).21-25 Moreover, CA (4) is devoid of activity
toward FXR receptor (EC50 > 100 μM) while showing a
moderate agonistic activity on TGR5 (EC50 = 13.6 μM).5
As an even more important consideration, it was previously
reported thatthe pharmacologicaladministrationofCA(4) at
0.5% w/w in diet-induced obese mice efficiently prevents and
treats metabolic syndrome.6 While this study provided inter-
esting clues about the endocrine functions of bile acids, the
high dosage required (0.5% w/w) still limited the proof of
concept concerning the therapeutic relevance of TGR5 in the
context of metabolic diseases, since the modulation of other
and unknown targets could not be ruled out at that dose. An
additional issue was also the risk associated with testing
a high dose of CA (4) in clinical trials due to the production
presence of BF3 OEt2 at -60 ꢀC in CH2Cl2 to obtain the
3
methyl 3R,12R-dihydroxy-6-ethylidene-7-keto-5β-cholan-24-
oate (12) in 85% yield. Hydrogenation of 12 with PtO2 in
glacial acetic acid/hydrochloric acid, followed by alkali hydro-
lysis (10% NaOH in methanol) gave selectively the 6R-ethyl
derivative 13 in good yield. Selective reduction of the
C7-ketone with NaBH4 in a mixture of THF/H2O at room
temperature afforded the 6-ECA (14) in 46% overall yield
(from 10). C23-Methylation of the corresponding 3R,7R,12R-
trihydroxy-protected ester 15 and the following acidic and basic
hydrolysis reactions18 gave the desired S- and R-EMCA (8, 9)
in 40% and 22% yield (from 6-ECA), respectively. The abso-
lute configuration assignment to epimers 8 and 9 was based
upon the previously reported single-crystal X-ray analysis of
23(S)-Me-CA18 and 13C NMR comparison.
Compounds 8, 9, and 14 were evaluated for TGR5 and
FXR activities, assessing their abilities to increase cAMP-
responsive element (CRE) driven luciferase reporter activity in
CHO cells transiently transfected with hTGR5 or to activate
FXR on COS1 (ATCC) cells in cell-based bioluminescence
assays, respectively (Table 1). In accordance with the reported
SAR schemes of TGR5 agonists and FXR modulators,18,19

7960 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24
Pellicciari et al.
Table 3. Biliary Lipid Secretion Parameters after iv and id Infusion at a
Dose of 1 (μmol/min)/kg bw over 1 h of BAsa
SV0
id (iv)
SBA
id (iv)
% free
id (iv)
% conjug
id (iv)
compd
CDCA (3)
57 ( 7
(51 ( 9)
64 ( 6
(78 ( 8)
112 ( 12
(131 ( 11)
81 ( 8
(90 ( 5)
46 ( 4
(48 ( 4)
0.7 ( 0.2
(0.8 ( 0.1)
1.0 ( 0.4
(1.3 ( 0.2)
0.5 ( 0.2
(0.7 ( 0.3)
0.4 ( 0.2
(0.5 ( 0.1)
0.4 ( 0.1
(0.4 ( 0.1)
3 ( 1
(4 ( 1)
12 ( 2
(8 ( 3)
94 ( 6
(93 ( 5)
68 ( 8
(65 ( 4)
96 ( 8
(98 ( 5)
90 ( 4
(92 ( 6)
10 ( 5
(7 ( 3)
32 ( 7
(26 ( 6)
CA (4)
S-EMCA (8)
R-EMCA (9)
saline
a Data represent average values and standard deviations of six
independent experiments. The vehicle used for the id administration
was saline solution. The vehicle used for the iv administration was 3%
BSA saline solution, pH 7.2. SV0: maximum bile secretion rate ((μL/
min)/kg bw). SBA: maximum BA secretion rate ((μmol/min)/kg bw). %
free: percentage of the administered dose recovered in bile of the
molecules as such. % conjugate: percentage of the administered dose
recovered as conjugated BA.
Figure 2. Impact of 1 h exposure to indicated concentration of
S-EMCA (8) on GLP-1 release ex vivo in ileal explants isolated from
18-weeks HF-fed TGR5-Tg male mice (n = 4). The data are
represented as mean ( SE: Student’s unpaired t-test, (/) P <
0.05, 8 treated ileal explants vs vehicle treated.
the femoral vein) or oral gavage administration in the duode-
num at a dose of 1 (μmol/min)/kg bw for 1 h (bile fistula rat
model).27,28 The results in Table 3 reveal that S-EMCA (8) has
a potent choleretic effect, with the maximum bile secretion rate
(SV0) being significantly higher than those of CDCA (3) and
CA (4). Accordingly, our results show that S-EMCA (8) is
resistant to conjugation, with more than 90% of the compound
being secreted into the bile in its unconjugated form after
intravenous or intraduodenal infusion (Table 3). In contrast
CDCA and CA cannot be secreted into bile as such, requiring
the conjugation step. In accordance with previous studies,29,30
we can envisage that the C23(S) methyl group of S-EMCA (8)
prevents carboxyl CoA activation and subsequent conjugation,
thereby favoring its cholehepatic shunt pathway with a ductu-
lar absorption and a potent choleretic effect.
To further study the influence of the configuration of the
C23 methyl group on the side chain amidation and choleretic
effect of the compound, similar analyses were also carried out
on the other epimer, namely, 6R-ethyl-23(R)-methylcholic
acid (R-EMCA, 9, Table 3). The inspection of the maximum
bile secretion rate (SV0) shows that the choleretic effect of 9 is
still higher than CA (4), though lower than 8. As a result, these
data suggest that the orientation of the 23 methyl group is
important for the conjugation of the carboxyl group, with the
methyl moiety fitting poorly in the catalytic pocket of the
conjugating enzyme in the case of the C23(S) epimer. Alto-
gether, these results show that S-EMCA (8) is efficiently
absorbed and undergoes enterohepatic cycling albeit with
relatively little liver conjugation. The low rate of conjugation
may also allow S-EMCA (8) to escape hepatic first pass
clearance and reach the systemic blood circulation.
Given the relatively good albumin binding of S-EMCA (8)
(Table 2), circulation of S-EMCA (8) in the blood may be
facilitated, thereby favoring the systemic targeting of TGR5
in peripheral tissues such as muscle and brown adipose
tissue. Supporting this hypothesis, we show in Figure 2 that
S-EMCA (8) dramatically and dose-dependently induces the
release of GLP-1 ex vivo. Along with these effects, we have
recently demonstrated in a joint paper that the pharmacologi-
cal targeting of TGR5 by S-EMCA (8) efficiently increases
GLP-1 secretion in vivo.31 Also consistent with systemic TGR5
activation was an increase in energy expenditure in diet-
induced obese mice that resulted in a significant reduction in
weight gain and adiposity. Very interestingly, these effects are
Figure 1. Stability of S-EMCA (9) (2) and CA (4) (9) in human
stool culture.
S-EMCA (8) was qualified as a novel potent and selective
TGR5 agonist and was next submitted to a preliminary
pharmacokinetic characterization and an assessment of the
compound’s physicochemical properties and metabolic stabi-
lity (Tables 2 and 3). On the basis of the higher surface
tension at the CMC (STCMC), despite a lower CMC value,
S-EMCA (8) shows a detergency power similar to CA (4),
albeit with slightly lower water solubility (Table 2).
It is known that intestinal bacteria hydrolyze the C24 amide
bond of taurine and glycine conjugated BAs and remove the
7R-hydroxyl group of CA, leading to the formation of toxic
lipophilic secondary BAs such as DCA (2).25 To determine
the sensitivity of S-EMCA (8) to intestinal flora-mediated
7-dehydroxylation, its metabolic stability was assessed in hu-
man stool broth culture as previously described.26 S-EMCA (8)
appears not to be sensitive to this process and was shown to be
highly stable with more than 95% of the compound unmodified
after 12 h of incubation. By comparison, more than 50% of
CA (4) was metabolized after 1 h and up to 90% within 8 h
(Figure 1). According to previous studies,26 it is likely that the
extended stability of S-EMCA (8) is related to the alkylation of
the C6 position which provides steric hindrance to the bacterial
7R-dehydroxylation process.
We next evaluated preliminary pharmacokinetic profiles of
S-EMCA (8) with the aim of gaining insights into the efficiency
of its intestinal absorption, hepatic uptake, transport, and
biliary secretion upon intravenous administration (infusion in

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 24 7961
associated with an improvement of liver function in high fat fed
mice with a concomitant reduction of steatosis and fibrosis.31
In conclusion, while our recent studies have univocally
demonstrated the promising therapeutic potential of pharma-
cological activation of TGR5 for the treatment of diabetes,
obesity, and related disorders such as NASH, the present
results call for additional pharmacokinetic evaluation in
support of the selection of S-EMCA (8) as a novel lead
candidate to advance into clinical studies.
(13) Parks, D. J.; Blanchard, S. G.; Bledsoe, R. K.; Chandra, G.;
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Acknowledgment. This work was supported by Intercept
Pharmaceuticals (New York, NY), ANR PHYSIO (BASE),
EU (Eugene2), EFPL, SNF. We thank Luciano Adorini and
Gianni Rizzo of Intercept for helpful discussions. Thanks are
due also to Erregierre (Bergamo, Italy) for the gift of bile acids
as starting materials.
(17) Costantino, G.; Entrena-Guadix, A.; Macchiarulo, A.; Gioiello,
A.; Pellicciari, R. Molecular dynamics simulation of the ligand
binding domain of farnesoid X receptor. Insights into helix-12
stability and coactivator peptide stabilization in response to ago-
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Supporting Information Available: Description of the synthe-
tic procedures, biological methods, and analytical analysis of all
target compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
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