6alpha-ethyl-chenodeoxycholic acid (6-ECDCA), a potent and selective FXR agonist endowed with anticholestatic activity.

Article abstract of DOI:10.1021/jm025529g

A series of 6alpha-alkyl-substituted analogues of chenodeoxycholic acid (CDCA) were synthesized and evaluated as potential farnesoid X receptor (FXR) ligands. Among them, 6alpha-ethyl-chenodeoxycholic acid (6-ECDCA) was shown to be a very potent and selec

Full text of DOI:10.1021/jm025529g

© Copyright 2002 by the American Chemical Society
Volume 45, Number 17
August 15, 2002
Letters
Ch a r t 1
6r-Eth yl-Ch en od eoxych olic Acid
(6-ECDCA), a P oten t a n d Selective F XR
Agon ist En d ow ed w ith An tich olesta tic
Activity
Roberto Pellicciari,*,† Stefano Fiorucci,§
Emidio Camaioni,† Carlo Clerici,§
Gabriele Costantino,† Patrick R. Maloney,‡
Antonio Morelli,§ Derek J . Parks,‡ and
Timothy M. Willson‡
Dipartimento di Chimica e Tecnologia del Farmaco,
Universita` di Perugia, Via del Liceo, 06123 Perugia, Italy,
Dipartimento di Medicina Clinica e Sperimentale, Clinica di
Gastroenterologia ed Epatologia, Universita` di Perugia,
06123 Perugia, Italy, and Discovery Research,
GlaxoSmithKline, Research
Triangle Park, North Carolina 27709
Received April 11, 2002
Abstr a ct: A series of 6R-alkyl-substituted analogues of cheno-
deoxycholic acid (CDCA) were synthesized and evaluated as
potential farnesoid X receptor (FXR) ligands. Among them, 6R-
ethyl-chenodeoxycholic acid (6-ECDCA) was shown to be a very
potent and selective FXR agonist (EC50 ) 99 nM) and to be
endowed with anticholeretic activity in an in vivo rat model
of cholestasis.
In tr odu ction . The farnesoid X receptor (FXR, NRIH4)
is an orphan member of the superfamily of nuclear
receptors, a growing ensemble of ligand-dependent
transcription factors that are activated by small, lipo-
philic hormones.1 FXR was originally proposed to be a
receptor for the intermediary metabolite farnesol.2
However, farnesol does not bind to FXR and supra-
physiological concentrations were required to activate
the receptor. In 1999, three groups independently
proposed FXR as a nuclear receptor for bile acids (BA).3
These studies revealed that physiological concentrations
of BA bound and activated FXR, with the primary bile
acid chenodeoxycholic (CDCA, 1, Chart 1) being the
most potent ligand with EC50 values ranging between
10 and 50 µM.3 Lithocholic (LCA, 2) and deoxycholic
(DCA, 3) acid also activate FXR, while ursodeoxycholic
acid (UDCA, 4) is inactive. Nonsteroid ligands for FXR
have also been discovered, of which the combinatorial
chemistry-derived isoxazole GW4064 (5) is the most
potent.4
* To whom correspondence should be addressed. Tel: +39 075 585
5120. Fax: +39 075 585 5124. E-mail: rp@unipg.it.
† Dipartimento di Chimica e Tecnologia del Farmaco, Universita` di
Perugia.
§ Dipartimento di Medicina Clinica e Sperimentale, Universita` di
Perugia.
‡ GlaxoSmithKline.
10.1021/jm025529g CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/20/2002

3570 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Letters
Ta ble 1. Binding Potency of Synthetic Bile Acids to FXR
compd
EC50 (µM)
efficacya
5 (GW4064)
1 (CDCA)
6a
6b (6-ECDCA)
6c
6d
0.037 ( 0.006
8.66 ( 0.45
0.75 ( 0.08
0.099 ( 0.01
1.11 ( 0.13
>30
117 ( 3
100 ( 3
148 ( 3
144 ( 5
156 ( 2
5 ( 2
a
Relative recruitment of the SRC1 peptide to FXR where CDCA
(1) ) 100% (ref 4). All data ( standard error, n ) 4.
Sch em e 1a
F igu r e 1. FXR regulates the expression of target genes
involved in bile acids and cholesterol homeostasis.
FXR shares a common modular architecture with the
other members of the nuclear receptor superfamily,
comprising a highly conserved DNA binding domain
(DBD) and a carboxy-terminal ligand binding domain
(LBD). The DBD recognizes specific DNA sequences,
known as response elements, within the enhancer
region of the receptor’s target genes. FXR binds as a
heterodimer with the 9-cis retinoic acid receptor (RXR)
to an IR-1 response element. The binding of a small,
lipophilic ligand to the LBD results in the recruitment
of transcriptional coactivators (e.g., SRC1), which couple
the receptor to the cellular transcriptional machinery.5
In its capacity as a BA receptor, FXR regulates the
expression of several target genes involved in the BA
homeostasis (Figure 1). In particular, FXR indirectly
represses the expression of the BA biosynthetic enzymes
CYP7A and CYP8B by increasing the levels of the
inhibitory nuclear receptor SHP in the liver and intes-
tine.6 BA-activated FXR also positively regulates the
expression of genes encoding proteins involved in the
transport of BA, including I-BABP, BSEP, and cMOAT.7
Although many of the molecular mechanisms involved
in FXR-mediated gene transcription regulation remain
to be fully understood, a picture has emerged of the role
of this receptor in BA and cholesterol homeostasis.8
Another intriguing therapeutic opportunity associated
with FXR modulation is the control of bile flow. Indeed,
the FXR-mediated activation of SHP causes the negative
feedback regulation of NTCP, the principal BA trans-
porter in the liver. Notably, FXR null mice display
defects in BA homeostasis such as elevated serum BA,
reduced BA pools, and reduced fecal BA secretion.9 So,
FXR may coordinate an integrated pathway aimed at
the down-regulation of BA import and synthesis. The
regulation of this process may protect hepatocytes from
BA toxicity.
a
Reagents and conditions: (1) pTsOH, 3,4-dihydropyrane,
dioxane, 25 °C. (2) (a) LDA, R-Br (or Me-I for compound 9a ),
THF, -78 °C; (b) 10% HCl, MeOH, reflux. (3) NaBH4. (4) 10%
NaOH, MeOH.
to identify potent BA-based ligands. We decided to
investigate a series of CDCA analogues as potential FXR
ligands and explore their in use the treatment of
cholestasis.
Resu lts. Initially, we surveyed a number of com-
pounds previously synthesized in our laboratory12 and
found that 6R-methyl-chenodeoxycholic acid (6-MeCD-
CA, 6a ) was a more potent FXR agonist than CDCA (1)
(Table 1). This result prompted us to synthesize CDCA
analogues 6b-d characterized by increasingly bulkier
6R-substituents. The synthetic route to the 6R-alkyl
CDCA analogues 6a -d is outlined in Scheme 1. Treat-
ment of the 3-tetrahydropyranyloxy derivative 8 of
7-keto-lithocholic acid (7) with methyl-, ethyl-, propyl-,
and benzyl bromide, respectively, at -78 °C using
lithium diisopropylamide as a base and HMPTA/tetra-
hydrofuran (THF) as solvents, followed by treatment
with methanolic HCl, afforded the corresponding methyl
3R-hydroxy-7keto-6R-methyl-5â-cholan-24-oate (9a ),
methyl 3R-hydroxy-7keto-6R-ethyl-5â-cholan-24-oate (9b),
methyl 3R-hydroxy-7keto-6R-propyl-5â-cholan-24-oate
(9c), and methyl 3R-hydroxy-7keto-6R-benzyl-5â-cholan-
24-oate (9d ) in 22, 12, 5, and 13% yield, respectively.
Finally, selective reduction of the 6R-alkyl-7-keto bile
acid methyl esters 9a -d with sodium borohydride13 and
subsequent hydrolysis of the methyl ester with alkali
afforded the desired 3R,7R-dihydroxy-6R-alkyl-5â-cholan-
24-oic acids 6a -d .14 It is worth noting that in each case
only the 6R-alkyl derivative is formed, a selectivity that
Cholestasis is a condition characterized by an impair-
ment or cessation of bile flow that occurs in a variety of
human liver diseases such as primary biliary cirrhosis,
primary sclerosing cholangitis, cystic fibrosis, and in-
trahepatic cholestasis of pregnancy.10 Currently, drug
treatment for cholestasis is limited to the secondary BA
UDCA (4)11 whose long-term efficacy remains unproven.
The molecular mechanism of action of UDCA (4) is
unknown, although its anticholestatic activity may be
due in part to the displacement of more hydrophobic BA
from the hepatocyte. Given the proposed role of FXR in
BA biosynthesis and transport from the liver, we sought

Letters
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17 3571
F igu r e 2. FXR Activity of 6b in HuH7 cells. Data points
represent the mean of assays performed in triplicate.
F igu r e 3. Effect of infusion of LCA alone (open circle) or in
combination with 6b (filled circle) on bile flow. Data are mean
( standard error of 4-6 rats/group.
can be explained by the 1,3-diaxial interaction that
would be experienced by a 6â-alkyl derivative with the
C19 methyl group.
Biology: In Vitr o Ch a r a cter iza tion . All new com-
pounds were tested in an established cell-free ligand
sensing assay, which measured the ligand-dependent
recruitment of an SRC1 peptide to FXR by fluorescence
resonance energy transfer.4 The results, reported in
Table 1, show that 6-ECDCA (6b) is a very potent FXR
agonist with an EC50 of 99 nM. Also, the 6R-MeCDCA
(6a ) and 6R-PrCDCA (6c) derivatives demonstrated
good potency as FXR agonists, while the 6R-BnCDCA
derivative (6d ) was essentially inactive.
In a reporter gene, (hsp70EcRE)2-tk-LUC,6a assay
employing the full length human FXR in HuH7 cells,
6-ECDCA (6b) was a potent full agonist with an EC50
of 85 nM (Figure 2). When tested across a standard
panel (described in ref 6a) of nuclear receptor LBD-
GAL4 chimeric receptors,4 1 µM 6b activated only the
FXR(LBD)-GAL4 chimera (data not shown). No signifi-
cant activation of other receptors was seen at 1 µM.
Thus, 6b is a potent and selective steroidal FXR agonist.
F igu r e 4. Histologic analysis of liver of rats treated with no
agent (a), LCA alone (b), LCA plus 6b (c), and LCA + CDCA
(d). Each compound was infused at the concentration of 3 µmol/
kg/min. Arrows indicate necrotic areas. Haematoxylin and
Eosin staining, original magnification 200×.
Biology: In Vivo Ch a r a cter iza tion in a n An im a l
Mod el of Ch olesta sis. The most potent derivative
6-ECDCA (6b) was selected for further characterization
in an in vivo model of cholestasis. Male Wistar rats
(225-300 g) were catheterized at the right jugular vein
using PE-50 polyethylene tubing, and the abdomen was
opened through a midline incision. The common bile
duct was isolated and cannulated. Saline solution was
infused via the external jugular vein at the same
infusion rate used later for BA, until a steady state in
the bile flow was reached (75 min). BA were then
dissolved in saline solution with 2% bovine serum
albumin, pH 7.4, and infused for 90 min followed by 60
min of saline infusion. During the treatment, bile
samples were collected every 15 min and weighed in
order to determine the bile flow. Two protocols were
used. In the first protocol, rats were randomly assigned
to receive one of the following BA: LCA (2), 6-ECDCA
(6b), or CDCA (1) at 1.0, 1.5, or 3 µmol/kg/min. 6-ECD-
CA (6b) was also infused at a higher dose (7 µmol/kg/
min). In the second protocol, cholestasis was induced
by intravenous infusion of LCA (2). Three groups of
animals were treated as follows: LCA (2) (3 µmol/kg/
min) alone, LCA (2) plus 6-ECDCA (6b) (3 µmol/kg/min),
or LCA (2) plus CDCA (1) (3 µmol/kg/min). The results
are shown in Figure 3. Administration of LCA alone at
a rate of 3 µmol/kg/min caused a dramatic reduction in
bile flow and extensive necrosis of liver cells (Figure 4b,
arrow).
At lower doses, LCA (2) was unable to induce cholesta-
sis. Neither CDCA (1) nor 6-ECDCA (6b) induced
cholestasis when administered alone. In the coinfusion
protocol, 6-ECDCA (6b) dosed at a rate of 3 µmol/kg/
min fully reversed the impairment of bile flow and
protected against liver cell injury induced by 3 µmol/
kg/min of LCA (2) (Figure 4c). Although 6-ECDCA (6b)
was effective in protecting against cholestasis induced
by LCA (2), the effect was lost over time, and a drop in
bile flow was observed as expected when the infusion
of the compound was stopped. In contrast, CDCA (1) at
doses up to 7 µmol/kg/min failed to protect against
colestasis caused by LCA (2) (data not shown), consis-
tent with its low FXR affinity. Higher doses of CDCA
(1) coinfused with LCA (2) were lethal (data not shown).
Discu ssion . The quest for potent and selective FXR
ligands is driven by the need to further clarify the
physiological and pathophysiological role of this nuclear
BA receptor. We have reported here the synthesis and
the preliminary evaluation of a series of potent steroidal

3572 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 17
Letters
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nuclear receptor. Science 1999, 284, 1365-1368. (c) Wang, H.;
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FXR agonists. Compound 6b is almost 2 orders of
magnitude more potent than CDCA (1), the putative
physiological FXR ligand. Moreover, the synthesis of a
series of 6R-substituted CDCA derivatives has allowed
us to delineate a key structure-activity relationship
within the steroidal skeleton. Indeed, our results point
to the existence of a discrete hydrophobic pocket in the
receptor, whose size is particularly suited to small linear
alkyl substituents. Although further experiments are
needed to fully characterize compounds 6a -d , they
appear to be useful chemical tools to probe the biological
function of FXR. Thus, 6b was active in an in vivo model
of cholestasis, and when infused to rats codosed with
LCA (2), it prevented bile flow impairment caused by
this agent. Remarkably, despite its high lipophilicity,
6b had no intrinsic cholestatic activity and did not show
any evidence of acute liver toxicity.
(4) Maloney, P. R.; Parks, D. J .; Haffner, C. D.; Fivush, A. M.;
Chandra, G.; Plunket, K. D.; Creech, K. L.; Moore, L. B.; Wilson,
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In conclusion, we report the identification of a potent
and selective steroidal FXR agonist, 6-ECDCA (6b).
Importantly, not only did 6-ECDCA (6b) promote bile
flow but it also protected hepatocytes against acute
necrosis caused by LCA. Thus, FXR activation not only
regulates endogenous bile acid synthesis but also acti-
vates protective pathways in hepatocytes challenged
with toxic xenobiotics. 6-ECDCA (6b ) and related
analogues may, therefore, offer a rational approach to
the treatment of cholestatic liver disease. Studies aimed
at further elucidating the potential of FXR modulators
in cholestasis as well as in other therapeutic areas are
under way.
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Ack n ow led gm en t. We thank Cristin Galardi and
Kelli Plunket for determination of the activity of 6b in
the cell-based reporter assays.
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(14) Selected analytical data for 6-EDCA (6b). 1H NMR (CDCl3): δ
0.67 (s, 3H, CH3-18); 0.90-0.96 (m, 9H, CH2-CH3-6, CH3-19, and
CH3-21); 2.22-2.46 (2m, 2H, CH2-23); 3.39-3.47 (m, 1H, CH-
3), 3.72 (brs, 1H, CH-7). 13C NMR (CDCl3): δ 11.65; 11.80; 18.25,
20.76; 22.23; 23.14; 23.69; 28.17; 30.53; 30.81; 30.95; 33.23; 33.90;
35.38; 35.52; 35.70; 39.60; 40.03; 41.19; 42.77; 45.19; 50.49; 55.80;
70.97; 72.38; 179.19. GC-MS methyl ester-trimethylsylyl ether
derivative of 6-ECDCA, m/z (relative intensity): 579 (M + H+,
1); 398 (base peak, 100).
J M025529G