Nongenomic actions of bile acids. Synthesis and preliminary characterization of 23- and 6,23-alkyl-substituted bile acid derivatives as selective modulators for the G-protein coupled receptor TGR5

Article abstract of DOI:10.1021/jm070633p

23-Alkyl-substituted and 6,23-alkyl-disubstituted derivatives of chenodeoxycholic acid are identified as potent and selective agonists of TGR5, a G-protein coupled receptor for bile acids (BAs). In particular, we show that methylation at the C-23(S) position of natural BAs confers a marked selectivity for TGR5 over FXR, while the 6α-alkyl substitution increases the potency at both receptors. The present results allow for the first time a pharmacological differentiation of genomic versus nongenomic effects mediated by BA derivatives.

Full text of DOI:10.1021/jm070633p

J. Med. Chem. 2007, 50, 4265-4268
4265
Nongenomic Actions of Bile Acids. Synthesis and
Preliminary Characterization of 23- and
,23-Alkyl-Substituted Bile Acid Derivatives as
Chart 1. Synthetic and Natural Bile Acids
6
Selective Modulators for the G-Protein Coupled
Receptor TGR5
,
†
‡
†
Roberto Pellicciari,* Hiroyuki Sato, Antimo Gioiello,
Gabriele Costantino,†,§ Antonio Macchiarulo,
†
†
|
Bahman M. Sadeghpour, Gianluca Giorgi,
‡
‡,⊥
Kristina Schoonjans, and Johan Auwerx
Dipartimento di Chimica e Tecnologia del Farmaco, UniVersit a` di
Perugia, Via del Liceo 1, 06123 Perugia, Italy, Institut de
G e´ n e´ tique et de Biologie Mol e´ culaire et Cellulaire (IGBMC),
CNRS/INSERM/ULP, 67404 Illkirch, France, Dipartimento di
Chimica, UniVersit a` degli Studi di Siena, Via Aldo Moro,
I-53100 Siena, Italy, and Institut Clinique de la Souris (ICS),
67404 Illkirch, France
clearly different from that of BA activating FXR. TGR5 is
broadly expressed in human tissues, including those that are
not a target for BAs. In particular, TGR5 is highly expressed
ReceiVed June 4, 2007
Abstract: 23-Alkyl-substituted and 6,23-alkyl-disubstituted derivatives
of chenodeoxycholic acid are identified as potent and selective agonists
of TGR5, a G-protein coupled receptor for bile acids (BAs). In
particular, we show that methylation at the C-23(S) position of natural
BAs confers a marked selectivity for TGR5 over FXR, while the 6R-
alkyl substitution increases the potency at both receptors. The present
results allow for the first time a pharmacological differentiation of
genomic versus nongenomic effects mediated by BA derivatives.
7
in the adipose tissue and in macrophages. While this latter
observation may allow a reconsideration of earlier reports
8-13
occasionally indicating an immunosuppressive effect for BAs,
the former one has been linked to a report stating that cholate
inhibits high-fat diet-induced hyperglycemia and obesity in
1
4
rats. Indeed, administration of BAs to mice increased energy
expenditure in the brown adipose tissue (BAT) and prevented
diet-induced obesity and insulin resistance. This effect was
ascribed to the increased cAMP-dependent induction of the type
2 iodothyronine deiodinase (D2) enzyme, which converts
thyroxine (T4) into 3,5,3′-triiodothyronine (T3), thus giving rise
to increased thyroid hormone activity. The up-regulation of D2
depends on the activation by BAs of membrane-bound TGR5,
Considered for many years as the final products of cholesterol
catabolism and involved in dietary lipid and vitamin adsorption,
bile acids (BAs ) are experiencing a new lease of life, being
now recognized as key elements of paracrine and endocrine
functions related to the homeostasis of cholesterol levels, control
of lipid and carbohydrate metabolism, and regulation of the
a
1
5
which mobilizes cAMP. Consistent with a role for TGR5 in
energy homeostasis, female TGR5 deficient mice, although not
obese under chow fed conditions, are predisposed to accelerated
1
immune system. BAs exert their role as signaling molecules
through interactions with membrane and nuclear receptors, as
well as with components of the mitogen-activated protein kinase
pathway. In 1999, the orphan farnesoid X receptor (FXR) was
adopted as a BA nuclear receptor, which is now recognized
as a master regulator of the pleietropic actions of endogenous
BAs within the systemic endocrine apparatus and the entero-
hepatic circulation. In 2002, two groups independently reported
the identification and the preliminary characterization of a
membrane-type, G-protein coupled receptor for BAs, named
M-BAR or TGR5. Thus, BAs potently induced cyclic ad-
enosine monophosphate (cAMP) formation in CHO cells
transiently expressed with TGR5 in a dose-dependent manner;
the cAMP formation is independent of nuclear receptor activa-
tion and the rank order of potency for BAs activating TGR5 is
1
6
obesity when fed a high fat diet. Although the translation of
this mechanism toward clinically exploitable strategies is still
far, TGR5 is emerging as a very promising target for metabolic
control and energy homeostasis, thus allowing the potential
unlinking of metabolism control from the diet.
2-4
5
The further pharmacological and pathophysiological charac-
terizations of TGR5, as well as its validation as clinically useful
target requires the availability of potent and selective ligands.
While screening of nonsteroidal libraries may provide suitable
and chemically diverse templates for further optimization,17
chemical elaboration around the steroid nucleus of BAs remains
an effective route toward potent BA receptors ligands, although
the selectivity among different receptors (i.e., FXR vs TGR5)
is a relevant issue.
6
,7
*
To whom correspondence should be addressed. Tel.: +39 075
We have previously reported extensive structure activity
relationship studies on BAs derivatives as FXR modulators
5
855120. Fax: +39 075 5855124. E-mail: rp@unipg.it.
18-20
†
Universit a` di Perugia.
Institut de G e´ n e´ tique et de Biologie Mol e´ culaire et Cellulaire, CNRS/
‡
and discovered 6ECDCA (INT747, 1, Chart 1) as the most
1
8
INSERM/ULP.
potent steroid ligand for FXR so far available. We have also
accumulated a considerable experienwhichce in the design,
synthesis and biological evaluation of unnatural BA derivatives
modified in the side chain, reporting for some of them interesting
properties, such as reduced hepatic amidation, alternative
conjugation pathways for sustained biliary secretion, and altered
§
Current address: Dipartimento Farmaceutico, Universit a` di Parma, via
G. Usberti 27A, 43100 Parma.
|
Universit a` degli Studi di Siena.
Institut Clinique de la Souris.
Abbreviations: BA, bile acid; BAT, brown adipose tissue; CA, cholic
⊥
a
acid; cAMP, cyclic adenosine monophosphate; CDCA, chenodeoxycholic
acid; D2, type 2 iodothyronine deiodinase; FXR, farnesoid X receptor; LBD,
ligand binding domain; M-BAR, membrane-bile acid receptor; 6ECDCA,
2
1-27
ability to enter the enterohepatic circulation.
Furthermore,
6
R-ethyl-chenodeoxycholic acid.
we noticed regio- and stereoselective biological properties for
1
0.1021/jm070633p CCC: $37.00 © 2007 American Chemical Society
Published on Web 08/09/2007

4
266 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 18
Letters
Scheme 1a
Figure 1. Drawing of the crystal structure of 23(S)-methyl-3R,7R,12R-
trihydroxy-5â-cholan-24-oic acid (7d) × CH
3
OH. Ellipsoids enclose
0% probability. C(24a), O(4a), and O(5a) have s.o.f. equal to 0.55(1).
5
The solvate methanol molecule has been omitted for clarity.
Table 1. TGR5 and FXR Activities of Bile Acids and Bile Acid
Derivativesa
TGR5b
efficacy
FXRc
efficacy
EC50 ratio
(TGR5/FXR)
index
EC50
EC50
2
1
6.71
0.755
3.58
105
101
110
100
105
102
101
105
13.0
0.361
>100
10.5
11.6
11.8
62
0.52
2.1
<0.036
2.4
100
d
7
8
7
7
3
7
8
a
a
b
c
0
25.5
49
23
73
0.140
0.095
13.6
4.39
>51.9
0.012
0.0081
e
e
e
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.e
n.d.e
n.d.e
n.d.e
e
d
d
d
e
75
a
Data represents average values of at least three independent experiments.
a
Reagent and conditions: (i) 3,4-DHP, p-TSA, dioxane, rt; (ii) (a) LDA,
b
c
Units are µM for EC50 and % of 10 µM LCA value for efficacy. Units
CH3I, THF, -78 °C; (b) HCl, CH3OH, rt; (iii) NaOH, CH3OH, reflux.
d
are µM for EC50 and % of 10 µM 6ECDCA value for efficacy. Plateau
activation level not reached; the maximum concentration tested was 125
µM for 8d and 100 µM for 7a. e n.d. ) not determined.
some of the side-chain modified BA derivatives, thus inferring
the potential for a fine-tuning modulation of their biological
activities. Based on these considerations, and given the potential
differences between TGR5 and FXR in terms of either different
tissue expression or topological requirements for ligand interac-
tion, we deemed appropriate to initially evaluate side chain
modified analogs of BAs in search for TGR5/FXR selective
modulators. Given the order of potency of naturally occurring
BAs at TGR5,6,7 we initially focused our attention on cheno-
deoxycholic acid (CDCA, 2, Chart 1) and cholic acid (CA, 3,
Chart 1) derivatives, which are expected to have a high potency
at TGR5, while keeping a reasonable hydrophobic/hydrophilic
balance. As a result of the screening, we identified a subset of
C-23 alkyl-substituted analogs of CDCA and CA. Their
synthesis and preliminary evaluation as TGR5 ligands is reported
herein.
Chemistry. The synthetic route to the 23(R)- and 23(S)-
methyl-BA derivatives (7a-d and 8a-d) is outlined in Scheme
. Thus, treatment of the methyl cholanoates 4a-d
,4-dihydro-2H-pyran in dioxane in the presence of a catalytic
amount of p-toluenesulfonic acid (p-TSA) afforded the corre-
sponding hydroxy-protected BA derivatives 5a-d in quantitative
yields. Reaction of 5a-d with methyl iodide at -78 °C using
lithium diisopropylamide as a base and tetrahydrofuran (THF)
as solvent, followed by treatment with methanolic HCl afforded
the corresponding 23-methylated BA esters 6a-d. Hydrolysis
of the methyl esters 6a-d with NaOH in methanol at reflux
yielded, respectively, a mixtures of two compounds, which were
separated by flash chromatography into less polar acids, 23(A)-
methyl BA derivatives 7a-d, and more polar acids, 23(B)-
methyl BA derivatives 8a-d.
The absolute configuration assignment to the eight diastereo-
isomeric BAs 7a-d and 8a-d was based upon the single-crystal
X-ray analysis performed on a suitable selected compound
chosen depending on the quality of its crystal. Thus, X-ray
analysis of the 23(A)-methyl-3R,7R,12R-trihydroxy-5â-cholan-
uration of the other BA diastereoisomers by 13C NMR com-
parison.
Biology. The newly synthesized compounds were evaluated,
along with CDCA (2) and CA (3) as reference controls, for
their ability to increase cAMP-responsive element (CRE)-driven
luciferase reporter activity in CHO cells, which transiently
transfected with hTGR5 or to activate FXR on COS1 (ATCC)
cells in cell-based luciferase assay in the case of CDCA
derivatives (Table 1).
Interestingly, a significantly different behavior is observed
between the respective C-23-methyl BA epimers. In particular,
while 8a is moderately active at both FXR and TGR5, with an
TGR5/FXR EC50 ratio of 2.4, the corresponding C-23(S)-epimer
7a is inactive at FXR up to a 100 µM concentration, while
activating TGR5, with an EC50 ) 3.58 µM (TGR5/FXR EC50
ratio < 0.036) and 110% of efficacy with respect to 10 µM
LCA. Thus, 23(S)-methyl-CDCA (7a) resulted to be highly
selective TGR5 agonist. We previously reported that the
insertion of alkyl groups at the 6R-position of CDCA (2), greatly
enhances the potency at FXR and discovered the most potent
2
8
1
8,19
1
3
with
29
18
steroid ligand for FXR, 6ECDCA (1). Analogously, we
wondered whether a similar modification would have improved
the potency and selectivity of 7a. Thus, the 6R-methyl (7b) and
6R-ethyl (7c) derivatives of 7a were synthesized and evaluated
in the above assays. As shown in Table 1, the introduction of
the 6R-methyl or 6R-ethyl group had a considerable impact on
the TGR5 activity, with an increase in potency of 1 or 2 orders
of magnitude, respectively. Thus, 6R-ethyl-23(S)-methyl-3R,7R-
dihydroxy-5â-cholan-24-oic acid (7c) turned out to be the most
potent and selective TGR5 agonist so far reported.
Molecular Modeling. With the aim of gaining insights into
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docked both the 23(S)- and 23(R)-Me-CDCA (7a and 8a,
respectively) epimers into the canonical binding pocket of the
ligand binding domain (LBD) of FXR (pdb code: 1OSV). Both
compounds were superimposed to the experimentally determined
2
2
4-oic acid (7d) confirmed the S-chirality of the carbon at the
3-position (Figure 1) and allowed to fix the absolute config-

Letters
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Supporting Information Available: Description of the syn-
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