Extending SAR of bile acids as FXR ligands: Discovery of 23-N-(carbocinnamyloxy)-3α,7α-dihydroxy-6α-ethyl-24-nor- 5β-cholan-23-amine

Article abstract of DOI:10.1016/j.bmc.2011.03.004

Within our efforts in the discovery of novel potent and selective ligands for the FXR receptor, 23-N-(carbocinnamyloxy)-3α,7α-dihydroxy- 6α-ethyl-24-nor-5β-cholan-23-amine was synthesized and evaluated for its ability to activate and modulate the biological response of the receptor. Alphascreen and RT-PCR revealed that the 6α-ethyl-24-norcholanyl-23-amine derivate behaves as full FXR agonist endowed with high binding affinity and efficacy, representing a promising lead candidate for further optimization. In addition, docking studies provide new insights into the molecular basis governing the partial and full agonist activity at FXR.

Full text of DOI:10.1016/j.bmc.2011.03.004

Bioorganic & Medicinal Chemistry 19 (2011) 2650–2658
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Extending SAR of bile acids as FXR ligands: Discovery
of 23-N-(carbocinnamyloxy)-3a,7a-dihydroxy-6a-ethyl-24-nor-5b-cholan-23-
amine
Antimo Gioiello a, Antonio Macchiarulo a, Andrea Carotti a, Paolo Filipponi a, Gabriele Costantino b,
Giovanni Rizzo c, Luciano Adorini c, Roberto Pellicciari a,
⇑
a Dipartimento di Chimica e Tecnologia del Farmaco, Università degli Studi di Perugia, Via del Liceo 1, 06123 Perugia, Italy
b Dipartimento Farmaceutico, Università degli Studi di Parma, V.le G.P. Usberti 27/A, I-43124 Parma, Italy
c Intercept Pharmaceuticals Italia Srl, Via Togliatti, 06073 Corciano, Perugia, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Within our efforts in the discovery of novel potent and selective ligands for the FXR receptor, 23-N-(car-
Received 3 January 2011
Revised 28 February 2011
Accepted 3 March 2011
Available online 10 March 2011
bocinnamyloxy)-3
for its ability to activate and modulate the biological response of the receptor. Alphascreen and RT-PCR
revealed that the 6 -ethyl-24-norcholanyl-23-amine derivate behaves as full FXR agonist endowed with
high binding affinity and efficacy, representing a promising lead candidate for further optimization. In
addition, docking studies provide new insights into the molecular basis governing the partial and full
agonist activity at FXR.
a,7a-dihydroxy-6a-ethyl-24-nor-5b-cholan-23-amine was synthesized and evaluated
a
Keywords:
FXR
Bile acid
Carbamate
RMDS
Ó 2011 Elsevier Ltd. All rights reserved.
Allosteric site
1. Introduction
CO2H
CO2H
The farnesoid-X receptor (FXR, also known as BAR; NR1H4)
belongs to the superfamily of intracellular ligand-activated
transcription factors that are involved in many physiological,
developmental and toxicological processes.1 Although initially
characterized as a metabolic nuclear receptor activated by farne-
sol,2 since 1999 FXR is considered a key nuclear receptor for bile
acids (BAs), with chenodeoxycholic acid (CDCA, 1, Fig. 1) being
its most potent endogenous agonist.3 FXR is abundantly expressed
in tissues exposed to BAs, such as liver, intestine, kidneys, and in
the ileal epithelium along the intestinal tract, where BAs are
mainly absorbed.2,4
HO
OH
HO
OH
H
H
Chenodeoxycholic Acid
6α-Ethyl-chenodeoxycholic Acid
(6-ECDCA, INT-747, 2)
(CDCA, 1)
Figure 1. CDCA derivatives as FXR agonists.
have shown the key role of FXR in regulating different metabolic
pathways, including the homeostasis of BAs, lipids and glucose,1
as well as in liver regeneration.8
Two FXR genes exist in lower mammalians including rodents,
The regulation of BA metabolism, for instance, occurs at three
complementary levels of action: (i) decreasing the BA synthetic
pathway; (ii) supporting the bile flow in the enterohepatic circula-
tion; (iii) increasing the clearance of toxic biliary constituents. At
the first level, FXR negatively regulates the BA synthetic pathway
through the down-regulation of the rate-limiting enzyme CYP7A1,
and the repression of CYP8B1, another key enzyme in BA synthe-
sis.9 Interestingly, the repression of CYP7A1 occurs through the
activation of FXR in the liver and intestine (Gut-Liver Axis) via
the induction of the small heterodimer partner (SHP) and the acti-
vation of the fibroblast growth factor (mouse FGF15/human
rabbits and dogs, that encode for
a and b types of receptor. While
FXRb is a pseudogene in humans and primates,5 the expression of
FXRa gene further originates four isoforms (FXRa1-4) as a result of
alternative splicing and different promoters.6 Although the physio-
logical importance of each isoform is still unclear, it is known that
the four isoforms of FXRa exhibit diverse DNA binding and coreg-
ulator recruitment.7 Over the past decade, a number of evidences
⇑
Corresponding author. Tel.: +39 075 585 5120; fax: +39 075 585 5124.
E-mail address: rp@unipg.it (R. Pellicciari).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.03.004

A. Gioiello et al. / Bioorg. Med. Chem. 19 (2011) 2650–2658
2651
FGF19) signaling pathway.10 In addition, FXR inhibits HMG-CoA
reductase and down-regulates lanosterol 14 -demethylase which
H
N
a
O
are both important in the synthesis of cholesterol, the endogenous
precursor of BAs.11 At the second level of action, FXR supports bile
flow in the enterohepatic circulation through the induction of
genes involved in BA secretion from liver, such as the bile salt ex-
port pump (BSEP) and the multidrug resistant-associated protein 2
(MRP2); the down-regulation of genes involved in the import of
BAs into the liver, that include the organic anion transporting
polypeptide-2 and -8 (OATP-2 and -8) and the sodium-dependent
taurocholate co-transporting protein (NTCP); the regulation of
genes involved in the transport of BAs in the intestine such as
the intestinal bile acid binding protein (IBABP), the apical
sodium-dependent bile acid cotransporter (ASBT) and the organic
O
R
FXR-EC50: 2.7-0,4 μM
FXR-Efficacy: 47-106%
HO
OH
H
R= Alk, Ar, CH2CHCHPh
24-Norcholanyl-23-amine Derivatives
H
H
N
N
O
O
O
O
6
6
HO
OH
HO
H
OH
H
solute transporters-
a and -b (OST-a
and -b).12 Finally, FXR
increases the clearance of toxic biliary constituents trough the reg-
ulation of additional genes, including the dehydroepiandrosterone
sulfotransferase (SULT2A1), the bile acid N-acetyltransferase (BAT),
and the bile acid CoA synthetase (BACS).1d
23-N-(carbocinnamyloxy)-3α,7α-
23-N-(carbocinnamyloxy)-3α,7α-dihydroxy-
6α-ethyl-24-nor-5β-cholan-23-amine (4)
dihydroxy-24-nor-5β-cholan-23-amine (3)
Figure 2. Structure and FXR activity of 24-norcholanyl-23-amine derivatives.
The unraveling of the physiological functions of FXR has dis-
closed unprecedented opportunities for this receptor as drug target
in different therapeutic areas such as liver diseases, metabolic syn-
drome and, more recently, hepatocarcinogenesis.1 In this scenario,
a number of molecules have been discovered, including both non-
steroidal and steroidal compounds that were able to bind and
interact with the ligand binding domain (LBD) of FXR in different
ways, leading to ligand and promoter selectivity for the FXR-
mediated gene transcription.1e,f While non-steroidal compounds
have been identified using high-throughput screenings of chemical
libraries eventually filtered for lead-like and/or drug-like proper-
ties,1a,b,f,13 the identification of FXR steroidal modulators has
mostly relied on a design strategy based on extensive modifica-
tions of the BA body and side chain.14 Embracing the latter
approach, we have developed thorough structure activity relation-
ships of BA derivatives as FXR ligands. These studies unveiled a
striking increase of agonist activity at the receptor when introduc-
ing an ethyl group in the C6 alpha position of CDCA (1), thereby
disclosing 6-ECDCA (INT-747, 2) as the most potent and orally
bioavailable agonist for FXR.14e 6-ECDCA (2) is being advanced in
clinical studies for non-alcoholic steatohepatitis (NASH), and
recent results of phase II clinical studies for primary biliary
cirrhosis (PBC) show a significant reduction of primary endpoint
biomarkers of the clinical status and progression of the disease.
While significant progress has been made towards the discov-
ery of potent agonists, great efforts are still needed for the develop-
ment of selective FXR modulators (referred to as ‘selective bile acid
receptor modulators’, SBARMs), compounds able to regulate indi-
vidual or a cluster of FXR target genes.1f,g These peculiar FXR mod-
ulators will offer the possibility to reduce the pleiotropism of the
receptor’s action and to explore novel therapeutic opportunities
which cannot be reacted by the use of FXR activators. In pursing
this aim, we have reported that the substitution of the carboxylic
tail of CDCA (1) with diverse substituted carbamate moieties was
able to affect the agonist potency of BAs at FXR, leading to a broad
range of efficacy in both cell-free FRET and cell-based luciferase as-
says (Fig. 2).14b,c Docking experiments suggested for the first time
that the LBD of FXR contains two contiguous pockets: the steroid
binding pocket and the ‘back door’ pocket.14b The former, in partic-
ular, accommodates the BA body and C24 side chain, whereas the
latter lodges the extended side chain substitution. Although the
significance of this observation and its possible extension to other
members of the nuclear receptor superfamily deserves further
investigation, we demonstrated that it is possible to achieve full
agonism, partial agonism, or antagonism by modifying a part of
the molecule that is not directly interacting with any of the LBD
elements known to affect coactivator binding.
With the aim of shedding light on the existence of any cross-
talk between the steroid binding pocket and the ‘back door’ pocket
responsible for the transactivation properties of FXR, and continu-
ing our efforts in the identification of FXR selective modulators, in
this study we report the synthesis, biological evaluations and
molecular modeling studies of 23-N-(carbocinnamyloxy)-3
-dihydroxy-6 -ethyl-24-nor-5b-cholan-23-amine (4) (Fig. 2).
We were, indeed, attracted by the interesting biological properties
of 23-N-(carbocinnamyloxy)-3 ,7 -dihydroxy-24-nor-5b-cholan-
a,-
7a
a
a
a
23-amine (3), a carbamate BA derivative 10-fold more potent than
CDCA (1) but endowed with partial agonism in FRET-assay
(EC50 = 0.62
particular, on the incorporation of the crucial 6
l
M, efficacy = 63%).14b We focused our attention, in
a-ethyl group in
the scaffold of 3 as key structural element to improve the potency
of the resulting compound.
2. Results
2.1. Chemistry
The synthesis of 4 is outlined in Scheme 1.14b,16d Treatment of
methyl 7-keto-deoxycholate 5 with LDA in THF at ꢀ78 °C followed
by reaction of the enolate thus formed with trimethylchlorosilane
(TMSCl) afforded the corresponding silyl enolether 6, in nearly
quantitative yield. Aldol-type addition of intermediate 6 with acet-
aldehyde in the presence of BF3ꢁOEt2 at ꢀ60 °C in CH2Cl2 gave the
desired methyl
3a-hydroxy-6-ethylidene-7-keto-5b-cholan-24-
oate (7) (Z:E, 85:15) in 80% yield. Hydrogenolysis of 7 with plati-
num dioxide (PtO2) in a mixture of glacial acetic acid/hydrochloric
acid, followed by alkali hydrolysis (10% NaOH in refluxing metha-
nol) afforded selectively 3a-hydroxy-6a-ethyl-7-keto-5b-cholan-
24-oic acid in good yield. Selective reduction of the C7-ketone with
sodium borohydride (NaBH4) in a mixture of THF/H2O at room
temperature afforded the 6-ECDCA (2), in 58% overall yield (from
5) (Scheme 1). Protection of the 3,7-hydroxy groups of 6-ECDCA
(2) was achieved by reaction with formic acid in the presence of
catalytic amount of p-toluensulfonic acid (p-TSA) in refluxing ben-
zene (96% yield). 3a,7a-Diformyl CDCA (8) was converted into the
corresponding acyl azides, via acyl chloride intermediate (oxalyl
chloride), followed by treatment with aqueous sodium azide at
0 °C. The crude acyl azide mixture was then refluxed in dry toluene
to give the corresponding isocyanate 9 by Curtius rearrangement.
Addition of t-cinnamyl alcohol (1.2 equiv) and removal of the for-
myl groups by hydrolysis with potassium carbonate in methanol at
room temperature gave the desired 23-N-(carbocinnamyloxy)-

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A. Gioiello et al. / Bioorg. Med. Chem. 19 (2011) 2650–2658
CO2Me
CO2Me
HO
CO2Me
ii
i
HO
O
Me3SiO
OSiMe3
O
H
H
H
7
6
5
CO2H
vii
CO2H
iii-v
vi
OHCO
OCHO
HO
OH
H
H
8
2
H
N
NCO
viii, ix
O
O
OHCO
OCHO
HO
OH
H
H
4
9
Scheme 1. Synthesis of 23-N-(carbocinnamyloxy)-3a,7a-dihydroxy-6a-ethyl-24-nor-5b-cholan-23-amine (4). Reagent and conditions: (i) LDA, TMSCl, Et3N, THF, ꢀ78 °C; (ii)
MeCHO, BF3ꢁOEt2, CH2Cl2, ꢀ60 °C; (iii) H2, PtO2, AcOH/HCl, rt; (iv) NaOH, MeOH, refl.; (v) NaBH4, THF/H2O, rt; (vi) HCO2H, p-TSA, benzene, refl.; (vii) (a) (COCl)2, 45 °C; (b)
NaN3, H2O, acetone, 0 °C; (c) toluene, refl.; (viii) t-cinnamoyl alcohol, toluene, reflux; (ix) K2CO3, MeOH, rt.
107
3a,7a-dihydroxy-6a-ethyl-24-nor-5b-cholan-23-amine (4), in 48%
overall yield (Scheme 1).
2.0
×
*
2.2. Biology
*
1.5
1.0
5.0
×
107
107
106
In order to characterize the binding potency of 3 and 4 as
ligands for FXR, we used
a coactivator recruitment assay
(Alphascreen),15 in which the ligand induces the recruitment of a
*
×
×
coactivator to the LBD of human FXR. In this assay, 4 shows a very
potent agonist activity on FXR, with an EC50 = 0.15
efficacy = 290%, while 3 confirms to be a partial agonist with an
EC50 = 0.9 M and efficacy = 55% (Table 1). The effect on FXR
lM and
l
activation of 3 and 4 was then tested in cell-based transactivation
assay using a human hepatic cell line (HepG2) transfected with the
full-length FXR and FXRE(IR1)3-Luc. As a result, compound 4 shows
**
0
NT
2
4
1
3
full agonistic activity on FXR at 1
CA (2), whereas 3 proves to be only a partial agonist at FXR even at
a concentration of 20 M (Fig. 3).
lM, comparable to that of 6-ECD-
Figure 3. Biological activity of norcholanylamine derivatives
transactivation assay. Cells were exposed to 20 M of 1 and 3 and 1
3
and
4 in FXR
l
l
l
M of 2 and 4.
In addition, since natural BA and BA derivatives are known to
activate the TGR5 receptor,16 we investigated if the 24-norchola-
nyl-23-amine derivatives 3 and 4 could bind also the metabotropic
receptor. By using time-resolved fluorescence resonance energy
transfer (TR-FRET) we have determined the increase of intracellu-
lar levels of cAMP in human enteroendocrine cell line (NCI-H716)
physiologically expressing TGR5. As shown in Table 1, both deriv-
atives 3 and 4 increase cAMP at high dose indicating only a weak or
absent TGR5 agonist activity.
Luciferase activity was normalized using renilla luciferase as the internal control.
Relative luciferase expression in untreated cells (NT) is shown.
Next, we investigated their gene expression profile, using a
quantitative real-time PCR and assessing the mRNA expression of
selected FXR target genes. In particular, we analyzed the expres-
sion of SHP, BSEP, Ostb, and Cyp7a1 in HepG2 cells (Fig. 4).
The results indicate that both derivatives 3 and 4 induce the
expression of SHP, BSEP, and Ostb genes, while down-regulating
the expression of Cyp7A1 (Fig. 4). However, while the effect of 3
in modulating FXR gene expression was less efficient than CDCA
(1) and 6-ECDCA (2), 4 resulted to be more potent than 2 in activat-
ing the receptor and regulating FXR target genes.
Table 1
FXR and TGR5 activities of bile acid derivativesa
Compound
FXR
TGR5
EC50 SD
Efficacyb
EC50 SD
2.3. Molecular modeling
CDCA (1)
6-ECDCA (2)
3
4
21.0 3.5
0.20 0.05
0.90 0.04
0.15 0.05
100
245
55
30
20
5
4
Docking experiments were performed on 3 and 4 in the attempt
to identify the molecular basis responsible of their partial and full
>100
55.0 5.7
290
agonism at FXR. In the case of PPARc modulators, it has been re-
Data represents average values of at least three independent experiments.
a
ported that full agonists stabilize the H12 helix whereas partial
Units are
lM for EC50 and% for efficacy.
agonists mainly act on the H3 stabilization.17 Accordingly, we
b
Units are % of 20 M 1.
l

A. Gioiello et al. / Bioorg. Med. Chem. 19 (2011) 2650–2658
2653
BSEP
OSTβ
40
30
20
10
0
40
30
20
10
0
**
**
**
**
**
**
*
*
CTRL
2
1
3
4
CTRL
2
1
3
4
CYP7α1
SHP
3
2
1
0
1.5
1.0
0.5
0.0
*
**
*
**
**
**
2
**
CTRL
1
3
4
CTRL
2
1
3
4
*p<0.05 and **p<0.001
Figure 4. Quantitative gene expression induced by 3 and 4 measured by using real-time PCR. HepG2 cells were treated with vehicle alone, 3 at 20
M concentration. CDCA (1) and 6-ECDCA (2) were used as reference standard at 20 and 1 M, respectively.
l
M concentration, and 4 at
1
l
l
Table 2
Results of docking experiments of compounds 3 and 4 into the LBD of FXR.
Compound
Glide XP score S2
Backbone RMSD S2 (Å)
Glide XP Score S3
Backbone RMSD S3 (Å)
3
4
ꢀ11.75
ꢀ13.57
1.97 0.13
1.88 0.11
ꢀ11.58
ꢀ13.17
2.44 0.16
2.06 0.16
deemed of interest to investigate the existence of a similar scenario
in the case of FXR to explain the pharmacological profiles of 3 and
4. To this end, we firstly docked each compound into the LBD of
FXR (Table 2).
the steroidal binding site (S1), and the extended side chain fitting
the ‘back door’ site (S2). This binding solution was instrumental
to investigate the stability of helices H3 and H12 as molecular
determinants for full and partial agonism of compounds 3 and 4
at FXR (Fig. 6). In particular, this was achieved by calculating the
mean RMSD values of the backbone atoms constituting the resi-
dues of helices H3 (residues Ser276-Lys300) and H12 (residues
Thr459–Trp466) from the starting agonistic conformation of the
receptor. Thus, it was found that 3 and 4 stabilize helix H12 to
different values of RMSD (3 RMSD = 1.59 0.41 Å; 4 RMSD =
1.46 0.43 Å), albeit the large standard deviations suggest the
presence of high fluctuating residues. In addition, compounds 3
and 4 show a diverse stabilization pattern for helix H3, with the
partial agonist inducing a larger stabilization than the full agonist
(3 RMSD = 1.60 0.22 Å; 4 RMSD = 1.84 0.19 Å), and in both cases
the presence of less fluctuating residues with the respect to helix
H12.
As a result, the two ligands bind to the steroidal binding site
(S1) with the steroid nucleus, while their extended side chain fills
two alternative pockets: the ‘back door’ site (S2), or a solvent ex-
posed site (S3). While the ‘back door’ site is composed of Tyr257,
Phe298 and Thr383, S3 is mostly exposed to the solvent, with only
three residues (Asn268, Lys335 and Pro338) being the possible
anchoring points (Fig. 5). The inspection of the binding energy
scores (Glide XP scores, Table 2) suggests that the occupancy of
the ‘back door’ site (S2) is slightly preferred over the S3 site for
both compounds, with 4 showing higher scores than 3 in agree-
ment to its potency. However, given the almost identical scores
of compounds 3 and 4 in S2 and S3, we deemed advisable to con-
sider as plausible all the four solutions at this stage. Accordingly,
four molecular dynamic experiments were carried out in water sol-
vated boxes with periodic boundary conditions for 10 ns. The anal-
ysis of the mean interaction energies between the ligand and the
protein in each simulation suggests that the occupancy of the ‘back
door’ site (S2) by the extended side chain is more favorable than
that of the S3 site, with the energetic differences being of ꢀ8.55
and ꢀ17.82 kcal/mol in the case of compound 3 and 4, respectively.
The preference of this binding solution is further supported by the
lower root mean square deviation (RMSD) of the backbone atoms
as calculated in the trajectories from the starting docked pose:
RMSD = 1.97 0.13 Å for 3 binding S2 versus RMSD = 2.44 0.16 Å
for 3 binding S3; RMSD = 1.88 0.11 Å for 4 binding S2 versus
RMSD = 2.06 0.16 Å for 4 binding S3.
3. Discussion
While substitution of the carboxylic group of CDCA (1) by an
amino group preserved both the potency and efficacy of the parent
derivative 1, carbamoylation of the distal C24-amino derivative
with a cinnamyl functionality gave the corresponding carbamate
derivative 3, which displayed the profile of a partial FXR agonist
(Table 1).14b,14c Further insertion of an ethyl group in position
C6a resulted in 23-N-(carbocinnamyloxy)-3a,7a-dihydroxy-6a-
ethyl-24-nor-5b-cholan-23-amine (4), which was endowed with
a high FXR binding affinity and, surprisingly, with a better efficacy
when compared to 2. Consistent with this finding, RT-PCR experi-
ments revealed that 4 is a potent FXR agonist, thereby regulating
Thus, based on the above results, we selected as plausible the
binding pose of compounds 3 and 4 with the BA scaffold occupying

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A. Gioiello et al. / Bioorg. Med. Chem. 19 (2011) 2650–2658
Figure 5. (A) Docking poses of compounds 3 (left) and 4 (right). The two ligands display two alternative binding modes: one with the carbamic moiety oriented towards S2 (3,
green; 4, violet) and another pointing to S3 (3, bluet, 4, pink). Hydrogen bonds are displayed in yellow and the key residues are labeled. (B) 2D representations of the
compounds 3 and 4 in the two proposed binding modes (left: S2 oriented; right: S3 oriented). Hydrogen bonds are displayed with green arrows, polar and charged residues
are shown in pink and lipophilic residues are shown in green.
Figure 6. Stability of helices H3 (blue) and H12 (magenta) as expressed by the RMSD (Å) of the relative backbone atoms during the molecular dynamic simulations of
compounds 3 (left) and 4 (right).
FXR target gene expression. At the cellular level, while both norch-
olanylamine derivatives 3 and 4 gave a similar gene expression
profile with respect to the endogenous FXR agonist CDCA (1), 4
was found to be more effective than 6-ECDCA (2), considered so
far the most potent BA-based agonist for FXR. In particular, a
strong induction of BSEP and OSTb expression was observed
indicating that 4 presumably leads to an increase of BA secretion
and efflux in ileum and other ASBT-expressing tissues, while the

A. Gioiello et al. / Bioorg. Med. Chem. 19 (2011) 2650–2658
2655
by standard methods. Melting points were determined with an
electrothermal apparatus and are uncorrected. NMR spectra were
recorded on a Bruker AC 200 or 400 MHz spectrometer and the
chemical shifts are reported in parts per million (ppm). The abbre-
viations used are as follows: s, singlet; br s, broad singlet; d, dou-
blet; dd, double doublet; m, multiplet. IR spectra were recorded
with a Pelkin-Elmer 1320 Spectrometer. Flash column chromatog-
raphy was performed using Merck Silica Gel 60 (0.040–0.063 mm).
Thin-layer chromatography was performed using glass plates
coated with Silica Gel 60 F-254. Spots were visualized by staining
and worming with phosphomolybdate reagent (5% solution in
EtOH). All reactions were carried out under a nitrogen atmosphere.
Purity of final compounds was determined by NMR and HPLC anal-
ysis. All tested compound showed an HPLC purity more than 98% in
two different systems: (a) analytical column: Ultra aqueous RP-18
Restek (Polar Base Deactivated); eluent: H2O/CH3CN: 20:80 (v/v);
flow rate: 1.5 mL/min; detection at 250 nm; (b) column Grace
Smart RP-18; eluent: H2O/CH3CN/CH3OH: 35:35:10 (v/v/v); flow
rate: 1 mL/min; detection at 254 nM. The HPLC analyses were car-
ried out with a EZ Start chromatography data software, a LC-10
ATVP pump, a SCL-10AVP system controller, a FCV-10ALVP low
pressure gradient formation unit, a DGU-14A on-line degasser
and a 7725i injector with a 20 mL stainless steel loop; DAD or
SPD-10A variable-wavelength UV–vis were used as detectors.
Figure 7. Proposed molecular basis for the different stabilization of helices H3
(yellow ribbon) and H12 (cyan ribbon) by compounds
Hydrophobic residues are labeled in yellow.
3 (left) and 4 (right).
repression of CYP7A1 and the slight induction of SHP may result in
a decreased BA synthesis. On the basis of these considerations, it
could be speculated that such compounding effects is potentially
positive to contrast hepatotoxicity induced by an excessive hepatic
accumulation of BAs.
Docking experiments and molecular dynamic simulations pro-
vided the molecular basis for rationalizing the activity of the new
derivatives. In particular, both compounds adopt similar binding
poses into the LBD of FXR (Fig. 5), with the BA scaffold and the ex-
tended side chain occupying the steroid binding site (S1) and the
‘‘back door’’ site (S2) of the receptor, respectively. Furthermore,
in agreement with previous studies reporting a helix H3 dependent
23-N-(carbocinnamyloxy)-3
a,7a-dihydroxy-24-nor-5b-cholan-23-
amine (3) was prepared as previously reported.14b
4.1.2. Methyl 3a,7-dimethylsilyloxy-5b-cholan-6-en-24-oate (6)
To a solution of diisopropylamine (10.4 mL, 0.079 mol) in dry
THF (50 mL) was added dropwise a solution of n-butyllithium
(31.6 mL, 2.5 M in hexane) at ꢀ78 °C under nitrogen atmosphere.
After 30 min, trimethylchlorosilane (18.0 mL, 0.097 mol) was
added, and the resulting mixture was reacted for additional
mechanism for partial agonists at PPARc, we found that the com-
pound 3 promotes a stabilization of helices H3 and H12 whereas
the full agonist 4 mostly stabilizes helix H12 (Fig. 7). While the sta-
bilization of H12 by 4 occurs through an indirect mechanism med-
iated by the extension of the hydrophobic contacts between the
ligand and the steroid binding site (S1) that ensue the insertion
of the ethyl group (Fig. 7, right panel), more challenging is the
explanation of the molecular basis underpinning the stabilization
of H3 by compound 3 (Fig. 7, left panel). Helix H3 is indeed part
of S2 through the residues Ile294, Leu295, Val296, Glu297,
Phe298, Thr299, Lys300 and Arg301. The hypothesis is that, unlike
compound 4 (H3 RMSD = 1.84 0.19 Å), the occupancy of S2 by the
partial agonist 3 may favor the packing of helix H3 to the binding
site (H3 RMSD = 1.60 0.22 Å) through its hydrophobic residues
Ile294, Leu295, Val296 and Phe298. This is not observed with the
full agonist 4 because the insertion of the ethyl group would con-
versely disrupt the packing of H3 to the steroid binding site (S1)
(H3 RMSD = 1.84 0.19 Å), while promoting the packing of H12
(H12 RMSD = 1.46 0.43 Å) and the full activation of the receptor.
In conclusion, our study provides evidence that also in the case
of FXR, partial agonists mainly act trough the stabilization of helix
H3, a finding potentially fruitful to design new FXR modulators
with different biological features and gene expression profiles.
10 min. A solution of methyl 3a-hydroxy-7-keto-5b-cholan-24-
oate (5.0 g, 0.012 mol) in dry THF (18 mL) was added dropwise in
40 min. The system was kept to ꢀ78 °C for an additional 30 min,
and then triethylamine (24.1 mL, 0.18 mol) was added. After 1 h
the reaction mixture was allowed to warm to ꢀ20 °C, treated with
aqueous saturated solution of NaHCO3 (25 mL), and brought up to
room temperature in 3 h. The organic phase was separated, and
aqueous phase was extracted with EtOAc (3 ꢂ 25 mL). The com-
bined organic phases were washed several times with saturated
solution of NaHCO3, water, and brine. After drying over anhydrous
Na2SO4, the residue was evaporated under vacuum affording to a
6.6 g of crude compound: Rf = 0.86 (PET/EtOAc, 9:1); 1H NMR
(400 MHz, CDCl3) d 0.13-0.18 (m, 18H, 3-OSiMe3 and 7-OSiMe3),
0.70 (s, 3H, 18-CH3), 0.84 (s, 3H, 19-CH3), 0.94 (d, J = 6.36 Hz, 3H,
21-CH3), 3.53 (m, 1H, 3-CH), 3.69 (s, 3H, CO2CH3), 4.75 ppm (dd,
J1 = 1.86 Hz, J2 = 5.91 Hz, 1H, 6-CH). HRMS (+ESI) calcd for
C31H57O4Si2 (M+H)+: 549.3795; found: 549.3791.
4.1.3. Methyl-3a-hydroxy-6-ethylidene-7-keto-5b-cholan-24-
oate (7)
To a cooled (ꢀ60 °C) and stirred solution of acetaldehyde
(1.3 mL, 22.2 mmol) and methyl ,7-diitrimethylsyliloxy-5b-
Moreover, 23-N-(carbocinnamyloxy)-3a,7a-dihydroxy-6a-ethyl-
3
a
24-nor-5b-cholan-23-amine (4) revealed here represent a novel
avenue in search of new BA derivatives as FXR ligands, and poten-
tially could serve as a promising new lead for further optimization.
chol-6-en-24-oate (6) (6.6 g) in dry CH2Cl2 (20 mL) was added
dropwise a solution of BF3ꢁOEt2 (0.044 mol) in 15 mL of CH2Cl2.
The reaction mixture was stirred for 2 h at ꢀ60 °C and allowed
to warm to room temperature. The mixture was quenched with
a saturated aqueous solution of NaHCO3 and extracted with
CH2Cl2. The combined organic extracts were washed with brine,
dried (Na2SO4) and concentrated under vacuum. The crude resi-
due was dissolved in CH2Cl2 (45 mL), treated with HCl (3 N,
20 mL), and stirred at 0 °C for 1 h. The reaction mixture was
quenched with a saturated aqueous solution of NaHCO3 (50 mL)
and extracted with CH2Cl2. The combined organic phases were
4. Experimental
4.1. Chemistry
4.1.1. General methods
Chemical reagents and solvents were obtained from commer-
cial sources. When necessary, solvents were dried and purified

2656
A. Gioiello et al. / Bioorg. Med. Chem. 19 (2011) 2650–2658
dried over Na2SO4 anhydrous, filtered and evaporated under re-
duced pressure. The crude residue was purified by flash chroma-
tography on silica gel using PET/EtOAc 7:3 as eluent to afford
4.13 g of 7 (9.6 mmol, 80%). Rf 0.28 (CH2Cl2/MeOH, 97:3); 1H
4.1.6. 23-N-(Carbocinnamyloxy)-3
nor-5b-cholan-23-amine (4)
a,7a-dihydroxy-6a-ethyl-24-
3a,7a-Diformyloxy-6a-ethyl-5b-cholan-24-oic acid (8) (0.82 g,
1.71 mmol) was treated with oxalyl chloride (8.0 mL) and reacted
at 45 °C for 2 h under nitrogen atmosphere. Removal of the excess
of oxalyl chloride by evaporation gave the corresponding acyl chlo-
ride, which was dissolved in dry acetone (8 mL). A solution of NaN3
(0.67 g, 10.26 mmol) in water (4 mL) was added to the acyl chlo-
ride solution at 0–5 °C, and the resulting reaction mixture was stir-
red for an additional 3 h at the same temperature. The solvents
were removed, and the residue was poured into cold water
(25 mL) and extracted with Et2O (3 ꢂ 20 mL). The combined organ-
ic phases were dried over anhydrous Na2SO4 and evaporated to ob-
tain the corresponding acyl azide (IR, 2134 and 2267 cmꢀ1). The
acyl azide intermediate so obtained was then refluxed in dry tolu-
ene (20 mL) for 5 h. To the resulting solution isocyanate derivative
9 (IR 2271 cmꢀ1) t-cinnamyl alcohol (0.30 g, 2.05 mmol) was
added at the same temperature under nitrogen atmosphere. The
end of the reactions was checked by TLC. The reaction mixture
was then poured into water (20 mL), the organic phase was sepa-
rated, and the aqueous phase was extracted with EtOAc
(3 ꢂ 20 mL). The combined organic phases were washed with
brine, dried (Na2SO4), filtered, and concentrated under vacuum.
NMR (400 MHz, CDCl3)
d 0.63 (s, 3H, 18-CH3), 0.92 (d,
J = 6.26 Hz, 3H, 21-CH3), 1.00 (s, 3H, 19-CH3), 1.68 (d,
J = 7.17 Hz, 3H, 20-CH3), 2.55-2.58 (dd, 1H, 5-CH), 3.56-3.69 (m,
4H, 3-CH and CO2CH3), 6.17 ppm (q, 1H, 10-CH); 13C NMR
(100.6 MHz, CDCl3+CD3OD) d 11.9, 12.6, 18.3, 21.2, 22.7, 25.84,
28.30, 29.60, 30.88, 30.92(ꢂ2), 34.34, 34.46, 35.10, 37.43, 38.82,
38.96, 43.45, 45.37, 48.57, 50.54, 51.40, 54.34, 70.38, 129.76,
143.20, 174.61, 204.79 ppm. HRMS (+ESI) calcd for C27H43O4
(M+H)+: 431.3163; found: 431.3165.
4.1.4. 3
(6-ECDCA, 2)
solution of methyl
a
,7
a
-Dihydroxy-6
a
-ethyl-5b-cholan-24-oic acid
A
3a
-hydroxy-6-ethylidene-7-cheto-5b-
cholan-24-oate (7) (4.0 g, 9.3 mmol) in glacial acetic acid/HCl
(200/10 mL, v/v) was hydrogenised in presence of platinum oxide
(0.4 g) at 32 psi for 24 h. The catalyst was filtered off and the
filtrate was concentrated. The residue was taken into a mixture
of water (200 mL) and EtOAc (150 mL) and neutralized with an
aqueous saturated solution of NaHCO3. The separated aqueous
layer was extracted with EtOAc (3 ꢂ 150 mL). The combined
organic phases were washed with brine, dried (Na2SO4) and
evaporated under reduced pressure. The crude product was
The
crude
23-N-(carbocinnamyloxy)-3a,7a-diformyloxy-6a-
ethyl-24-nor-5b-cholan-23-amine was then treated overnight
with a saturated methanolic solution of K2CO3 (20 mL) at room
temperature. After evaporation of the solvent, the residues were
dissolved in water (20 mL), acidified with 2 N HCl, and extracted
with CH2Cl2 (3 ꢂ 20 mL). The combined organic extracts were
dried over anhydrous Na2SO4, filtered, concentrated under vac-
uum. The residual was purified by silica gel flash chromatography
using a mixture of CH2Cl2/MeOH as eluent, affording the desired
compound 4 as a white solid (0.82 g, 1.49 mmol, 87%). Rf 0.18
(CHCl3/MeOH, 96:4); mp: 88-92 °C; 1H NMR (400 MHz, CDCl3) d
0.64 (s, 3H, 18-CH3), 0.91-0.88 (m, 6H, 19-CH3 e 20-CH3), 0.96 (d,
J = 6 Hz, 3H, 21-CH3), 3.13 (m, 1H, 23-CH), 3.26 (m, 1H, 23-CH),
3.40 (m, 1H, 3-CH), 3.68 (s, 1H, 7-CH), 4.71 (d, J = 5.6 Hz, 2H,
CH2–CH@CH–C6H5), 6.29-6.31 (m, 1H, CH2–CH@CH–C6H5), 6.62-
6.66 (d, J = 16 Hz, 1H, CH2–CH@CH–C6H5), 7.25-7.39 ppm (m, 5H,
Ph); 13C NMR (100.6 MHz, CDCl3) d 11.65, 11.72, 18.59, 20.69,
22.19, 23.11, 23.64, 28.34, 30.58, 33.17, 33.76, 33.92, 35.46(ꢂ2),
36.04, 38.63, 39.55, 39.96, 41.13, 42.72, 45.14, 50.46, 55.96,
65.27, 70.83, 72.28, 124.01, 126.56, 127.90, 128.54, 133.52,
hydrolysed overnight using
a methanol solution of sodium
hydroxide (10%, 500 mL). The mixture was then concentrated
under vacuum, diluted with water, acidified with HCl 11 N and
extracted with ethyl acetate (3 ꢂ 150 mL). The collected organic
phases were washed with brine, dried over Na2SO4 anhydrous
and evaporated under reduced pressure. The residual was dis-
solved in a solution of THF/H2O (500 mL, 4:1, v/v) and treated
with NaBH4 overnight at room temperature. After evaporation
of the solvents the residue was diluted with water, acidified with
HCl 3 N and extracted with CH3Cl/MeOH 9:1 (3 ꢂ 150 mL). The
combined organic phases were washed with brine, dried over
Na2SO4 anhydrous and evaporated under reduced pressure. The
crude residue was purified by flash chromatography on silica
gel using dichloromethane/methanol (7:3, v/v) as eluent, to af-
ford 2.8 g (6.7 mmol, 72%) of 2. Rf 0.38 (acetone/CH2Cl2/AcOH,
30:60:10); mp: 108-110 °C; 1H NMR (400 MHz, CDCl3+CD3OD) d
0.67 (s, 3H, 18-CH3), 0.90-0.93 (m, 6H, 20-CH3, 19-CH3), 0.95 (d,
J = 6.43 Hz, 3H, 21-CH3), 3.42 (m, 1H, 3-CH), 3.72 ppm (s, 1H,
136.32, 156.26 ppm; IR (KBr):
m = 3355.05, 2927.90, 1701.39,
1525.90, 1448.28, 1256.88, 1133.46, 1060.17, 967.61 cm-1. HRMS
(+ESI) calcd for C35H53NO4 (M+H)+: 552.4053; found: 552.4051.
7-CH); 13C NMR (100.6 MHz, CDCl3+CD3OD)
d 11.62, 11.76,
18.23, 20.73, 22.20, 23.12, 23.65, 28.15, 30.46(ꢂ2), 30.85, 33.19,
33.79, 35.39(ꢂ2), 39.57, 39.99, 41.15, 42.73, 45.16, 50.44, 55.78,
4.2. Biology
70.90, 72.28, 178.00 ppm. HRMS (+ESI) calcd for
(M+H)+: 421.3318; found: 421.3316.
C26H45O4
4.2.1. FXR Alphascreen assay
Activity on FXR was assayed by using Alphascreen technology
in a coactivator recruitment assay. Anti-GST-coated Acceptor beads
were used to capture the GST-fusion FXR-LBD whereas the biotin-
ylated-SRC-1 peptide was captured by the streptavidin Donor
beads. Upon illumination at 680 nm chemical energy is transferred
from Donor to Acceptor beads across the complex streptavidin-
Donor/Src-1-Biotin/GSTFXR-LBD/Anti-GST-Acceptor and a signal
is produced. The assay was performed in white, low-volume,
4.1.5. 3a,7a-Diformyloxy-6a-ethyl-5b-cholan-24-oic acid (8)
6-ECDCA (2) (0.90 g, 2.14 mmol) was dissolved in 30 mL of
benzene and treated with p-toluenesulfonic acid (0.33 g,
1.73 mmol) and formic acid 99% (5.5 mL). The resulting solution
was azeotropically distilled for 2 h using Dean-Stark apparatus.
After evaporation of the solvent, the residue was taken up into
a mixture of EtOAc/water, extracted with EtOAc (3 ꢂ 30 mL),
dried, filtered and concentrated on vacuum to obtain 0.82 g
(1.71 mmol, 96%) of compound 8 which was used for the next
step without further purifications. Rf 0.51 (CHCl3/MeOH, 9:1);
1H NMR (200 MHz, CDCl3) d 0.65 (s, 3H, 18-CH3), 0.79-0.95
(m, 9H, 19-CH3, 21-CH3 and 20-CH3), 4.61-4.96 (m, 1H, 3-CH),
5.18 (m, 1H, 7-CH), 8.04 (s, 1H, CHO), 8.15 ppm (s, 1H, CHO).
384-well Optiplates (PerkinElmer) using a final volume of 25
containing final concentrations of 10 nM of purified GST-tagged
FXR-LBD protein, 30 nM biotinylated Src-1 peptide, 20 g/ml
anti-GST acceptor beads acceptor beads and 10 g/ml of streptavi-
lL
l
l
din donor bead (PerkinElmer). The assay buffer contained 50 mM
Tris (pH 7.4), 50 mM KCl, 0.1% BSA, and 1 mM DTT. The stimulation
HRMS (+ESI) calcd for
474.3215.
C
28H44NO5 (M+H)+: 474.3219; found:
times with 1
lL of tested compound (solubilized in 100% DMSO)
were fixed to 30 min at room temperature. The concentration of

A. Gioiello et al. / Bioorg. Med. Chem. 19 (2011) 2650–2658
2657
DMSO in each well was maintained at a final concentration of 4%.
After the addition of the detection mix (acceptor and donor beads)
the plates were incubated in the dark for 4 h at room temperature
and then were read in Envision microplate analyzer (PerkinElmer).
Dose response curves were done in triplicate and Z0-factor was
used to validate the assays. Non linear regression curves, without
constraints, were performed by using four parameter equation
and GraphPad Prism Software (GraphPad Inc.), to obtain the EC50
values.
ml Matrigel (BD Biosciences) according to manufacturer’s instruc-
tions just prior to use, at a density of 12 ꢂ 103 cells/well in DMEM
supplemented with 10% (v/v) FBS, 100 units/ml penicillin and
100 lg/mL streptomycin sulfate, and cultured for 24 h, which al-
lowed cell adhesion to the bottom of the plate. The cells were
washed twice with PBS and medium was exchanged for cAMP as-
say medium [OPTIMEM containing 0.1% (w/v) BSA and 1 mM 3-iso-
buty1-1-methylxanthine (IBMX)]. After incubation for 60 min. at
37 °C, the cells were treated with increasing concentrations of
tested compound in stimulation Buffer (5 mM HEPES, 0.1% BSA in
HBSS pH 7.4) containing the europium chelate—Streptavidin and
the ALEXA Fluor 647-conjugated antibody anti-cAMP (PerkinEl-
mer) for 1 h at room temperature. The level of intracellular cAMP
was determined with Lance kit (PerkinElmer). Lithocholic acid
was used as control ligand. Z0-factor was used to validate assays.
Non linear regression curves, without constraints, were performed
by using four parameter equation and GraphPad Prism Software
(GraphPad Inc.), to obtain the EC50 values.
4.2.2. Cell culture and FXR transactivation assay
HepG2 and HEK293T cells were cultured in E-MEM and DMEM
respectively, either supplemented with 1% penicillin/streptomycin,
1% L-glutamine and 10% fetal bovine serum (high glucose) (Invitro-
gen, Carlsbad, CA). Cells were grown at 37 °C in 5% CO2. All the
transfections were made using 5:2 (volume:weight) of Fugene
HD Trasfection reagent (5 lL) to DNA (2 lg), respectively, accord-
ing to the manufacturer’s instructions (Roche). Twenty-four hours
before transfection HEK293T or HepG2 cells were seeded onto a
96-well plate at a density of 10,000 or 15,000 cells/well, respec-
tively. Transient transfections were performed using 100 ng of re-
porter vector pGL4.29[luc2P/CRE/Hygro] (Promega), 40 ng of
pGL4.74 (Renilla), as internal control for transfection efficiency,
and 10 ng of expression plasmid pCMV-SPORT6-hFXR. The pGEM
vector was added to normalize the amounts of DNA transfected
4.3. Molecular modeling
All the calculations were performed with the Schrodinger Suite
2008 (Schrodinger Inc., NY, USA). The docked compounds were
prepared with the default options of the LigPrep module to assign
all the possible protonation states at pH 7.0 2. The crystal struc-
ture of FXR in complex with 6-ECDCA (pdb code = 1OSV) was used
for docking experiments after being prepared with the standard
options of the Protein Preparation Wizard protocol. The docking
grid was calculated placing the coordinates of the center of mass
of the co-crystallized ligand as center of a cubic box, having a side
length of 10 Å. All docking calculations were performed using the
extra precision (XP) mode of Glide 5.0. The best poses were stored
and used for molecular dynamic simulations (MD) that were car-
ried out with Desmond package. In particular, the four resulting
complexes were firstly solvated with water TIP3P and neutralized
with the proper counterions using the system builder module. The
box used was orthorhombic, having each side at a minimum dis-
tance of 10 Å from any atom of the complex. The force field used
was the OPLS 2005 in an NPT (isothermic-isobaric) environment.
The thermostat used was Berendsen type. The dynamic protocol
was set in subsequent steps, with the first being a relaxing minimi-
zation, then a progressive increase of temperature to reach 300 K
in 140 ps, and finally a production run of 10 ns. Frames of the sim-
ulation were recorded every 5 ps. The MD trajectories were ana-
lyzed using either the simulation event analysis tool, as
implemented in Desmond package, and the VMD tools.
in each assay (2 lg). Twenty-four hours post-transfection the cells
were stimulated with increasing concentrations of tested com-
pound for 18 h. Control cultures received vehicle (0.1% DMSO)
alone. The cells were then lysed by adding 75
lL of Dual-Glo Lucif-
erase Reagent (Promega) to 75 L of medium containing cells/well.
l
Renilla luciferase activity was measured by adding volume:volume
of Dual-Glo Stop & Glo reagent and original culture medium. Lucif-
erase activities were expressed as ratio between luciferase unit and
renilla luciferase unit. Each data point is the average of triplicate
assays. Each experiment was repeated almost three times.
4.2.3. FXR target gene expression
The mRNA expression level of FXR target genes was measured
by Real-Time Polymerase Chain Reaction (Q-RTPCR). Total RNA
was isolated (Aurum Total RNA Mini Kit BioRad) from HepG2 cells
stimulated with increasing concentration of tested compound for
18 h. The RNA was random reverse-transcribed with Iscript cDNA
Synthesis kit (BioRad) in 20
was used in 20 L final volume reaction of Real-Time PCR contain-
ing 0.3 M of each primer and 10 L of 2X SYBR Green PCR Master
lL reaction volume. Ten ng template
l
l
l
MIX (Bio-Rad). All reactions were performed in triplicate and the
thermal cycling conditions were: 3 min at 95 °C, followed by 45 cy-
cles of 95 °C for 10 s, and 60 °C for 30 s in iCycler iQ5 instrument
(Biorad, Hercules, CA). The mean value of the replicates for each
sample was calculated and expressed as cycle threshold (CT: cycle
number at which each PCR reaction reaches a predetermined fluo-
rescence threshold, set within the linear range of all reactions). The
amount of gene expression was then calculated as the difference
Acknowledgments
This work was supported by Intercept Pharmaceuticals (New
York, NY). Thanks are due also to Erregierre (Bergamo, Italy) for
the gift of bile acids as starting materials.
Supplementary data
(DCT) between the CT value of the sample for the target gene
and the mean CT value of that sample for the endogenous control.
Relative expression was calculated as the difference (DDCT) be-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.03.004.
tween the
DCT values of the test sample and of the control sample
(WT) for each target gene. The relative quantization value was ex-
pressed and shown as 2-DDCT. All PCR primers were intron span-
ning designed using the software Beacon Designer on published
sequence data from the NCBI database.
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