Bile acid derivatives as ligands of the farnesoid X receptor. Synthesis, evaluation, and structure - Activity relationship of a series of body and side chain modified analogues of chenodeoxycholic acid

Article abstract of DOI:10.1021/jm049904b

The farnesoid X receptor (FXR) is activated by endogenous bile acids (BAs) and plays a variety of physiological roles related to modulation of gene transcription. In particular, FXR positively regulates the cholesterol catabolism while feedback inhibits the BA synthesis by repressing the expression of the CYP7A and CYP8B genes. We have previously shown that 6α-ethyl-CDCA (6ECDCA) is a potent and selective FXR agonist. In this paper we report an extensive structure-activity relationship for a series of synthetic bile acids. Our results indicate that the 6α position plays a fundamental role in determining affinity and that the side chain of BA is amenable to a variety of chemical modification. Although none of the new derivatives is more potent than 6ECDCA, we show here that a wide variability in efficacy, from full agonists to partial antagonists, can be obtained.

Full text of DOI:10.1021/jm049904b

J . Med. Chem. 2004, 47, 4559-4569
4559
Bile Acid Der iva tives a s Liga n d s of th e F a r n esoid X Recep tor . Syn th esis,
Eva lu a tion , a n d Str u ctu r e-Activity Rela tion sh ip of a Ser ies of Bod y a n d Sid e
Ch a in Mod ified An a logu es of Ch en od eoxych olic Acid
Roberto Pellicciari,*,† Gabriele Costantino,† Emidio Camaioni,† Bahman M. Sadeghpour,† Antonio Entrena,†,
Timothy M. Willson,‡ Stefano Fiorucci,§ Carlo Clerici,§ and Antimo Gioiello†
Dipartimento di Chimica e Tecnologia del Farmaco, Universita` di Perugia, Via del Liceo, 1, 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 February 3, 2004
The farnesoid X receptor (FXR) is activated by endogenous bile acids (BAs) and plays a variety
of physiological roles related to modulation of gene transcription. In particular, FXR positively
regulates the cholesterol catabolism while feedback inhibits the BA synthesis by repressing
the expression of the CYP7A and CYP8B genes. We have previously shown that 6R-ethyl-
CDCA (6ECDCA) is a potent and selective FXR agonist. In this paper we report an extensive
structure-activity relationship for a series of synthetic bile acids. Our results indicate that
the 6R position plays a fundamental role in determining affinity and that the side chain of BA
is amenable to a variety of chemical modification. Although none of the new derivatives is
more potent than 6ECDCA, we show here that a wide variability in efficacy, from full agonists
to partial antagonists, can be obtained.
Ch a r t 1. Naturally Occurring BAs
In tr od u ction
The identification of the former orphan nuclear recep-
tor farnesoid X receptor (FXR) as a bile acid (BA)
receptor 1-3 has greatly increased the understanding of
processes related to physiological control of BA homeo-
stasis into the enterohepatic circulation. Indeed, FXR
was shown to respond to physiological concentration of
the primary BA chenodeoxycholic acid (CDCA, 1, Chart
1), as well as to lithocholic acid (LCA, 2) and deoxycholic
acid (DCA, 3), but not to ursodeoxycholic acid (UDCA,
4) and to regulate the expression of several genes
controlling the BA disposal through modulation of
cholesterol metabolism and of the BA transport sys-
tems.4,5
Although many salient traits of the drawing still need
to be painted, quite a clear picture has emerged for FXR
as a BA sensor that feed-forward regulates the choles-
terol catabolism and feedback inhibits the BA synthesis
by repressing the expression of the CYP7A and CYP8B
genes6 through increase in the level of the nuclear
receptor SHP-17,8 and by activating gene expression of
the bile acid transporters I-BABP, BSEP, and MAOT.9
The intimate role of FXR in the transcriptional control
of genes regulating cholesterol and BA biosynthesis and
disposal makes it a particularly attractive target for the
discovery of drugs for the treatment of cholestasis10 or
hyperlipedimia.11 There is therefore pressure for the
development of potent, specific, and pharmacokinetically
suitable ligands to be employed in the validation of the
viability of FXR as a therapeutic target.
A major breakthrough toward a more precise under-
standing of the FXR pathophysiological role was the
discovery, facilitated by the availability of cell-free
ligand sensing assays and reporter gene assays, of
selective, highly potent agonists. Thus, exploitation of
an isoxazole-based combinatorial library allowed GSK
to discover GW4064 (5, Chart 2) as a nanomolar
nonsteroidal activator of FXR.12
In a different approach, rational modification of 6R-
methyl-CDCA (6), identified as an FXR ligand from the
survey of a small libraries of synthetic BA, allowed us
to develop 6R-ethyl-CDCA (6ECDCA, 7), the most potent
steroidal FXR agonist so far reported.10 6ECDCA (7)
turned out to be very efficacious in recruiting the SRC-1
peptide in the cell-free assay, was significantly active
in reverting cholestasis in an in vivo rat model, and was
successfully employed in the determination of the first
crystal structure of the holo conformation of FXR.13
* To whom correspondence should be addressed. Phone: +39 075
585 5120. Fax: +39 075 585 5124. E-mail: rp@unipg.it.
On leave from Departamento de Quimica Farmaceutica y Or-
ganica, Universidad de Granada, Spain.
† Dipartimento di Chimica e Tecnologia del Farmaco, Universita` di
Perugia.
‡ GlaxoSmithKline.
§ Dipartimento di Medicina Clinica
e
Sperimentale, Clinica di
Gastroenterologia ed Epatologia, Universita` di Perugia.
10.1021/jm049904b CCC: $27.50 © 2004 American Chemical Society
Published on Web 07/23/2004

4560 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Ch a r t 2. Synthetic FXR Ligands
Pellicciari et al.
F igu r e 1. Selected modifications on CDCA skeleton.
(Figure 1). We have previously shown that 6R substitu-
tion of CDCA with small linear alkyl substituents
increases the potency with respect to the parent com-
pound, with 6R-ethyl (7) being the optimal one.10 Now,
we engaged ourselves in the synthesis and the evalua-
tion of the effect of additional alkyl substituents, includ-
ing 6R-allyl (9), 6R-(2′-hydroxyethyl) (10), and 6R-
propargyl (11). Furthermore, more hydrophilic sub-
stituents were introduced, such as the 6R-hydroxy (12)
and 6R-methoxy (13) ones. Fluorine was also considered
as a potentially useful probe because of its small size,
relative lipophilicity, and moderate ability to act as
hydrogen acceptor. Thus, the 6R-F-CDCA (14) and 6â-
F-CDCA (15) derivatives were synthesized. Finally, the
effect of the hydroxyl group in the 3 position was
evaluated by the synthesis of 3-deoxy-CDCA (16) (Chart
3).
Finally, optimization of a benzopyrane-based combina-
torial derived libraries led to the identification of
fexaramine (8) as a structurally diverse nonsteroidal
agonist, also endowed with nanomolar potency.14,15
Interestingly, the gene expression array induced by
6ECDCA (7), GW4064 (5), and fexaramine (8) is differ-
ent,15 thus pointing out a different genomic target for
FXR when stimulated by structurally divergent ago-
nists. Apparently consistent with this, the comparison
of the high-resolution crystal structures of the two
complexes fexaramine-FXR and 6ECDCA-FXR, indi-
cated two similar but not overlapping binding sites and
slightly different conformations of the two receptors,
thus inferring that the two ligands may induce different
degrees of receptor activation. Further evidence comes
from the very recent observation that endogenous BAs
may interact with FXR in unique ways leading to ligand
and promoter selectivity for FXR-mediated gene tran-
scription.16
Although combinatorial chemistry may offer the op-
portunity for the detection of structurally novel chemical
entities able to interact with both the hormone binding
site and possible modulatory sites, manipulation of the
steroidal skeleton toward synthetic BA analogues is
endowed with a number of potential advantages. In this
regard, of particular interest is the possibility that a
BA-based FXR ligand may efficiently be taken up into
the enterohepathic circulation, thus preventing the
depletion of the BA pool size secondary to the inhibition
of BA synthesis induced by FXR activation. Further-
more, a BA-based ligand should inherently be targeted
to hepatocytes and endowed with a pharmacokinetic
profile suitable for in vivo administration. With this
consideration in mind and as a continuation of our work
in the field,10,17,18a-l we report here a thorough structure-
activity relationship of a series of synthetic BA ana-
logues as potential FXR modulators. Of these new
derivatives, some were previously synthesized by us in
attempts to optimize the physicochemical properties of
BA; others were derive from a systematic modification
over the CDCA (1) skeleton.
The side chain was also extensively studied (Chart
4). Positions 22 and 23 were explored by introducing a
22-hydroxy group (17), a 23-hydroxy group (18, 19), and
a 23-methyl group (20). Furthermore, the four diastereo-
isomers (21-24) resulting from the 22,23 cyclopropa-
nation were analyzed in terms of the conformational
requirement of the steroid side chain. The role of the
side chain length was proved by degradation of CDCA
(1) to bisnor-CDCA (25). Finally, but not less impor-
tantly, we decided to check the precise role of the acidic
tail of BA. Thus, the distal 23 or 24 position was
systematically scanned by the introduction of groups
with different electrostatic, steric, hydrogen bonding,
and pKa profiles, such as amino (26), ethylcarbamate
(27), oxazolidin-3,5-dione (28), sulfate (29), and sul-
fonate (30).
Ch em istr y
Compounds 1-4 and 12 were commercially available
and purchased from Aldrich and Fluka (St. Louis, MO).
All the other derivatives were synthesized as described
below. Thus, compounds 14 (3R,7R-dihydroxy-6R-fluoro-
5â-cholan-24-oic acid),18a,j,19 15 (3R,7R-dihydroxy-6â-
fluoro-5â-cholan-24-oic acid),18a,j,19 16 (7R-hydroxy-5â-
cholan-24-oic acid),20 17 (3R,7R,22R-trihydroxy-5â-cholan-
24-oic acid),18k,l 18 (3R,7R,23â-trihydroxy-5â-cholan-24-
oic acid),18k,l 19 (3R,7R,23R-trihydroxy-5â-cholan-24-oic
acid,18k,l 25 (3R,7R-dihydroxy-22-bisnor-5â-cholanoic
acid,21 29 (3R,7R,24-trihydroxy-5â-cholane-24-sulfate,22
and 30 (3R,7R-dihydroxy-5â-cholan-24-ol-24-O-sulfonic
acid)23 were prepared as reported elsewhere.
Design Str a tegy
The 6R-alkyl analogues of CDCA (9-11) were pre-
pared according to the procedure previously described
for the synthesis of 6RMeCDCA (6) and 6RECDCA (7).10
In particular, 7-ketolithocholic acid 31 was protected at
Two major patterns of modification were elaborated
around CDCA (1), namely, those relating to the B ring
of the steroid body and those affecting the side chain

Synthetic Bile Acids as Potent FXR Ligands
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4561
Ch a r t 3. Body Modified CDCA Analogues
the 3 position by treatment with 3,4-dihydro-2H-pyran
in dioxane in the presence of a catalytic amount of
p-toluenesulfonic acid (p-TSA) to give the corresponding
3-tetrahydropyranyloxy derivative of 7-ketolithocholic
acid 32. Reaction of 32 with allyl and propargyl bromide
at -78 °C, using lithium diisopropylamide as a base and
HMPTA/tetrahydrofuran (THF) as solvent followed by
treatment with methanolic HCl, afforded the corre-
sponding methyl 3R-hydroxy-7-keto-6R-allyl-5â-cholan-
24-oate (33) and methyl 3R-hydroxy-7-keto-6R-propargyl-
5â-cholan-24-oate (34) in 28% and 22% yield, respectively
(Scheme 1). Selective reduction with sodium borohy-
dride24 and subsequent hydrolysis of the methyl esters
33 and 34 with alkali afforded the desired 3R,7R-
dihydroxy-6â-allyl-5â-cholan-24-oic and 3R,7R-dihydroxy-
6R-propargyl-5â-cholan-24-oic acids (9 and 11, respec-
tively) in about quantitative yields.
45% yield. Selective reduction of 40 with sodium boro-
hydride and basic saponification gave the final com-
pound 3R,7R-dihydroxy-6R-methoxy-5â-cholan-24-oic acid
(13) in 40% yield.
Compound 20 (3R,7R-dihydroxy-6R,23-dimethyl-5â-
cholan-24-oic acid) was obtained as a byproduct in the
synthesis of 6R-methyl-CDCA.10 Briefly, alkylation of
the 3-tetrahydropyranyloxy derivative of 7-ketolitho-
cholic acid 32 with methyl iodide, followed by treatment
with methanolic HCl, afforded the corresponding methyl
3R-hydroxy-7-keto-6R-methyl-5â-cholan-24-oate (42) and
methyl 3R-hydroxy-7-keto-6R,23-dimethyl-5â-cholan-24-
oate (43) in 21% and 4% yield, respectively (Scheme 4).
Selective reduction with sodium borohydride and sub-
sequent hydrolysis of the methyl ester 43 with alkali
afforded the 3R,7R-dihydroxy-6R,23-dimethyl-5â-cholan-
24-oic acid (20) in 52% yield.
Compound 10 (3R,7R-dihydroxy-6R-(2′-hydroxyethyl)-
5â-cholan-24-oic) was obtained by submitting interme-
diate 33 of Scheme 1 to a reductive (sodium borohydride)
ozonolysis to afford the corresponding hydroxymethyl
derivative 37 in 31% yield (Scheme 2). Final basic
saponification with alkali of 37 afforded the desired
3R,7R-hydroxy-6R-(2′-hydroxyethyl)-5â-cholan-24-oic acid
(10) in 89% yield.
3R,7R-Dihydroxy-22,23-methylene-5â-cholan-24-oic acid
isomers 21-24 were prepared following the procedure
reported elsewhere for the 3R,7â-dihydroxy-5â-cholan-
24-oic analogues (UDCA derivatives).18f-i Protection
with acetyl groups of CDCA and degradation of the side
chain to afford the corresponding 21,22 unsaturated
derivative 45 was performed according to a known
procedure.18k,28 As reported in Scheme 5, the derivative
4528 was submitted to a dirhodium(II) tetracetate
catalyzed cyclopropanation with ethyldiazoacetate, thus
obtaining a mixture of diacetyl 22,23-methylenecholan-
oic esters 46. Alkaline hydrolysis followed by medium-
pressure liquid chromatography afforded the (22S,23S)-
3R,7R-dihydroxy-22,23-methylene-5â-cholan-24-oic acid
(21), (22R,23R)-3R,7R-dihydroxy-22,23-methylene-5â-
cholan-24-oic acid (22), (22S,23R)-3R,7R-dihydroxy-22,-
23-methylene-5â-cholan-24-oic acid (23), and (22R, 23S)-
3R,7R-dihydroxy-22,23-methylene-5â-cholan-24-oic acid
(24) in 15%, 12%, 32%, and 27% yield, respectively. The
assignment of absolute stereochemistry was performed
3R,7R-Dihydroxy-6R-methoxy-5â-cholan-24-oic acid (13)
was synthesized as reported in Scheme 3.
Thus, methyl 3R,6R-dihydroxy-7-keto-5â-cholan-24-
oate (38), obtained from commercially available 3R-
hydroxy-7-keto-5â-cholan-24-oic acid (31) according to
a known procedure,25 was selectively protected at the 3
position with the tert-butyldiphenylsilyl group affording
the corresponding derivative 39 in 43% yield. Alkylation
of 39 with methyl iodide, 15-crown-5, and sodium
hydride26 and subsequent removal of the silyl protecting
group with tetrabutylammonium fluoride gave methyl
3R,7R-hydroxy-6R-methoxy-5â-cholan-24-oate (40) in

4562 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Ch a r t 4. Side Chain Modified CDCA Analogues
Pellicciari et al.
by comparison with that previously reported for the
ursoderivatives.18f,i
to the 3,5-dioxo-1,2,4-oxadiazolidine moiety by following
a known procedure.30 Thus, condensation of 50 with
O-benzylhydroxylamine in the presence of sodium cy-
anoborohydrate gave the corresponding N-benzyloxyami-
no derivative 51 in 42% yield. The intermediate 51 was
then reacted with ethoxycarbonyl isocyanide to afford
the derivative 52 in 72% yield. Removal of the benzyl
protecting group by hydrogenolysis gave the corre-
sponding N-hydroxy ester derivative 53 in 64% yield,
which was submitted to the final hydrolysis under basic
conditions leading to the cyclization and the removal of
the acetyl protecting groups, thus obtaining the final
compound 28 in 46% yield.
3R,7R-Dihydroxy-5â-23-norcholanylamine (26) and
23N-carbetoxy-3R,7R-dihydroxy-5â-23-norcholanyla-
mine (27) were prepared as reported in Scheme 6. Thus,
the diacetyl-protected CDCA 47 was converted into the
corresponding acyl azides via the acyl chloride inter-
mediate (thionyl chloride) followed by treatment with
aqueous sodium azide. The crude acyl azide mixture was
then refluxed overnight in EtOH to give the protected
N-carbethoxyamino derivative 48 in 62% yield, which
upon treatment with potassium hydroxide gave a mix-
ture, which was separated by chromatography, of the
corresponding N-carbethoxyamino 27 and norcholanil-
amino derivative 26 in 26% and 23% yield, respectively.
Compound 28 (3R,7R-dihydroxy-5â-23-norcholanyl)-
3,5-dioxo-1,2,4-oxadiazolidine) was obtained in a mul-
tistep synthesis. Derivative 4528 was submitted to
hydroboration and oxidation reactions29 to yield the
corresponding alcohol derivative 49 in 63% yield. Sub-
sequent oxidation by utilizing pyridinium chlorocromate
afforded 3R,7R-diacetoxy-5â-23-norcholanaldehyde (50)
in 61% yield. The aldehyde group was then transformed
Biologica l Resu lts a n d Discu ssion
All the studied compounds were tested in a cell-free
ligand sensing assay that measures the ligand-depend-
ent recruitment of the SRC peptide to FXR by fluores-
cence resonance energy transfer, as previously de-
scribed.12 The results are listed in Table 1.
The 3R-hydroxy group (16) can be eliminated without
significant loss of activity with respect to CDCA (1). This
result could be interpreted on the basis of the crystal

Synthetic Bile Acids as Potent FXR Ligands
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4563
Sch em e 1a
a
(i) DHP, pTSOH, dioxane, room temp; (ii) (a) LDA, THF, R-Br, -78 °C, (b) HCl, MeOH, reflux; (iii) NaBH4, MeOH, room temp; (iv)
NaOH, MeOH, reflux.
Sch em e 2a
effect, according to its peculiar hydrophobic/hydrophilic
balance. Thus, the 6R-fluoro derivative (14) is twice less
potent than CDCA (1) but equally efficacious, while the
6â epimer (15) is about 8 times more potent but less
efficacious (76%).
Modification of the side chain of CDCA (1) resulted
in a complex pattern of results. Introduction of a
hydroxyl group in the 22 or 23R or 23S position (17 or
18 or 19, respectively) had an overall unfavorable effect
on EC50, although it did not abolish the activity. In
detail, functionalization of the 23 position is quite
acceptable because compounds 18-20 showed only a
minimal decrease in activity with respect to the parent
CDCA (1) albeit with a marked reduction in the efficacy.
Less prone to modification is the 22 position because
compound 17 is 3 times less potent than CDCA (1).
Remarkably, all the above side chain substituted CDCA
analogues did not reach the maximum effect and behave
as partial agonists. Cyclopropanation on the 22,23
position yielded very useful information. The two cis
isomers (21, 22) were devoid of any activity, whereas
the two trans ones (23, 24) were able to activate FXR.
In particular, among the two trans derivatives, the 22S,-
23R isomer (23) was about 4 times more potent than
CDCA (1) although it was unable to reach the maximum
efficacy. The 22R,23S isomer (24) was, in contrast, 5-fold
less potent than CDCA (1). These data clearly indicate
a conformational preference for an extended disposition
of the side chain of BA when interacting with the FXR
receptor. This is in agreement with the experimentally
determined conformation of 6ECDCA (7) complexed
with FXR and is indirectly confirmed by the lack of
activity of bisnor-CDCA (25), where the carboxylic
moiety cannot reach the position of that of a conforma-
tionally extended cholanoic acid.
a
(i) (a) O3, CH2Cl2, MeOH, room temp, (b) NaBH4, MeOH, room
temp; (ii) NaOH, MeOH, reflux.
structure of the ligand binding domain of FXR.13 Modi-
fication on ring B of the steroid body profoundly affected
the ability of activating FXR. The key importance of the
6R position, figured out by the excellent potency of
6ECDCA (7),10 is furthermore highlighted by the analy-
sis of the novel 6-substituted CDCA analogues (com-
pounds 9-15). Indeed, while linear alkyl or alkyl-
substituted groups (9-11) induced both high potency
and efficacy consistent with the physicochemical nature
of the pocket, more polar substituents such as the
hydroxy (12) or methoxy (13) groups had a markedly
reduced effect. This loss of potency is clearly not due to
steric reasons and must be ascribed to the excessive
desolvation cost that more hydrophilic molecules must
pay to fit into the hydrophobic pocket. The effect of
desolvation seems to be crucial for understanding the
potency of BA analogues and will be thoroughly ad-
dressed in a following paper. Interestingly, insertion of
the fluorine atom in the 6 position has an intermediate
Of great interest is the analysis of the effect of the
replacement of the 24-carboxylic acid group by structur-

4564 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Pellicciari et al.
Sch em e 3a
a
(i) TBDPSCl, imidazole, DMF, room temp; (ii) CH3I, 15-crown-5, NaH, THF, room temp; (iii) NaBH4, MeOH, room temp; (iv) NaOH,
MeOH, reflux.
Sch em e 4a
a
(i) (a) LDA, THF, MeI, -78 °C, (b) HCl, MeOH, reflux; (ii) NaBH4, MeOH, room temp; (iii) NaOH, MeOH, reflux.
ally diverse moieties. Introduction in the 23 position of
a oxazolidin-3,5-dione moiety (28) turned out to repre-
sent a true bioisosteric substitution because the result-
ing derivative is endowed with both high potency and
high efficacy. Other acidic groups had, however, a less
straightforward behavior. Thus, while the 24-sulfonic
acid derivative (30) kept both the potency and the
efficacy comparable with that of CDCA (1), the 24-
sulfate acid (29) had a slightly better potency than
CDCA but only 27% of efficacy, thus demonstrating it
to be endowed with marked antagonist properties. The
substitution of the distal carboxylic acid group by an
amino group yielded derivative 26, which was surpris-
ingly as equipotent and as efficacious as CDCA (1).
Indeed, the substitution of the carboxylate by an amino
group profoundly affects both the electrostatic profile
and the hydrogen bond acceptor/donor characteristic of
the molecule and the interpretation of the equiactivity
between the two moieties requires extensive modeling
approaches, whose results will be communicated in due
course. Interestingly, the ethylcarbamate derivative (27)
of compound 26 showed a potency 3 times higher than
the parent derivative but only 47% of efficacy, being
therefore a partial antagonist of FXR.
Con clu sion
Taken together, these data offer quite a clear picture
of the structural requirements needed for the modula-
tion of the FXR receptor by steroid derivatives. While
recent reports on the structure of ligand binding domain
of FXR have allowed us to elucidate the main determi-
nants of binding of steroids such as 6ECDCA (7)13,17 and
non-steroids such as fexaramine (8),15 the present
structure-activity relationship on a large series of
CDCA analogues permits a closer examination of the
possibility of fine-tuning the FXR response by selected
chemical manipulations. The main findings can be
grouped as follows. (i) Although none of the here-
reported derivatives is more potent than 6ECDCA (7),10
the beneficial effect of a 6R-alkyl substitution is con-
firmed. In particular, a group larger than an ethyl group
can be fitted into the pocket, thus suggesting a certain

Synthetic Bile Acids as Potent FXR Ligands
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4565
Sch em e 5a
Ta ble 1. Binding Potency and Efficacy of Synthetic
BAs to FXR
a
(i) EDA, Rh2(OAc)4, CH2Cl2, room temp; (ii) (a) NaOH, EtOH,
reflux, (b) MPLC.
Sch em e 6a
a
(i) (a) SOCl2, C6G6, reflux, (b) NaN3, H2O, room temp, (c) EtOH,
reflux; (ii) NaOH, MeOH, reflux.
degree of receptor flexibility or ligand accommodation.
Remarkably, some 6R-alkyl derivatives, such as 9, have
a very high efficacy. More polar and hydrogen-bonding
groups have a negative impact on both potency and
efficacy, consistent with the very hydrophobic nature
of the pocket complementary to the 6R position. (ii) The
side chain can be functionalized, and the resulting
derivatives (17-24), despite a general, moderate loss
of potency, display an interesting partial agonist char-
acter. The four cyclopropane isomers (21-24) provide
stringent clues about the need of an extended disposi-
tion of the side chain of BA analogues because the two
cis isomers 21 and 22, reminiscent of a more folded
conformation, are completely inactive. (iii) The carbox-
ylic group can be substituted. Indeed, intuitive bioiso-
steric replacers such as oxazolidin-3,5-dione (28) kept
the potency of the parent derivative, while others had
an unpredictable effect. In particular, going from an
amino (26), carbamate (27), and sulfate (29) to sulfonate
(30), it is possible to achieve a variety of functional
effects, from full agonism to partial antagonism, without
significant loss of potency.
a
Relative recruitment of the SRC1 peptide to FXR where CDCA
(1) is 100%.
synthetic BA previously identified by us as potent and
selective FXR agonist.10
Exp er im en ta l Section
In conclusion, the present results complete and extend
Ch em istr y. Melting points were determined with a Buchi
the structure-activity relationship for a class of semi-
535 electrothermal apparatus and are uncorrected. NMR

4566 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Pellicciari et al.
spectra were obtained with a Bruker AC 200 or 400 MHz
spectrometer, and the chemical shifts are reported in parts
per million (ppm). The abbreviations used are as follows: s,
singlet; bs, broad singlet; d, doublet; dd, double doublet; m,
multiplet. Specific rotations were recorded on a J asco Dip-360
digital polarimeter. Flash column chromatography was per-
formed using Merck silica gel 60 (0.040-0.063 mm). TLC was
carried out on precoated TLC plates with silica gel 60 F-254
(Merck). Spots were visualized by staining and worming with
phosphomolybdate reagent (5% solution in EtOH). All reac-
tions were carried out under a nitrogen atmosphere.
EtOAc 70/30 afforded to 1.30 g of derivative 33 (27.8% yield).
1H NMR (CDCl3) δ: 0.58 (s, 3H, CH3-18), 0.87 (d, J ) 6.2 Hz,
3H, CH3-21), 1.15 (s, 3H, CH3-19), 3.30-3.55 (m, 1H, CH-3),
3.60 (s, 3H, CO2CH3), 4.90-5.10 (m, 2H, CHdCH2), 5.50-5.70
(m, 1H, CHdCH2).
Meth yl 3r-Hyd r oxy-6r-p r op a r gyl-7-k eto-5â-ch ola n -24-
oa te (34). This compound was prepared using method A
(propargyl bromide 12.5 g, 105 mmol). Elution with light
petroleum/EtOAc 70/30 afforded to 0.76 g of derivative 34 (22%
1
yield). H NMR (CDCl3) δ: 0.58 (s, 3H, CH3-18), 0.84 (d, J )
6.2 Hz, 3H, CH3-21), 1.16 (s, 3H, CH3-19), 3.40-3.59 (m, 1H,
CH-3), 3.60 (s, 3H, CO2CH3).
6r-Alk yla t ion of 7-Ket o-5â-ch ola n oic Der iva t ives.
Meth od A. n-Butyllithium (18.7 mL, 1.8 M solution in hexane)
and then HMPA (5.8 mL. 33.7 mmol) were added dropwise at
-78 °C to a solution of diisopropylamine (4.7 mL, 33.7 mmol)
in dry THF (250 mL). The system was kept to -78 °C for an
additional 30 min, and then 3R-tetrahydropyranyloxy-7-keto-
5â-cholan-24-oic acid (32) (5 g, 10.5 mmol) dissolved in dry
THF (50 mL) was added dropwise to the mixture. After 20 min,
suitable alkyl bromide (105 mmol) dissolved in dry THF (20
mL) was slowly added and the mixture was allowed to warm
overnight to room temperature. The solvents were removed
under vacuum, acidified by 10% HCl, extracted with EtOAc
(5 × 200 mL), washed with 5% Na2S2O3 solution (2 × 300 mL),
dried (over anhydrous Na2SO4), filtered, and evaporated under
vacuum. The crude residue was then refluxed with a solution
of 2 N HCl in MeOH (50 mL) for 12 h. The residue was
evaporated under vacuum and taken up with EtOAc (300 mL),
washed with a saturated NaHCO3 solution (2 × 100 mL), dried
(Na2SO4), and evaporated under vacuum. The residue was
purified by silica gel flash chromatography.
Selective Red u ction of 7-Keto-5â-ch ola n oic Der iva -
tives.24 Meth od B. Methyl 7-keto-5â-cholanoate derivative
(0.85 mmol) was dissolved in MeOH (50 mL), and NaBH4 (96
mg, 2.56 mmol) was added. The mixture was stirred at room
temperature for 3 h. H2O (10 mL) was then added, and the
mixture was partially concentrated under vacuum and ex-
tracted with EtOAc (3 × 20 mL). The combined organic
fractions were washed with a saturated NaCl solution (brine)
(1 × 50 mL), dried (Na2SO4), and evaporated under vacuum
to obtain the crude reduced derivative that was passed to the
next step without further purification.
Sa p on ifica tion of Meth yl 5â-Ch ola n oic Ester s. Meth od
C. Methyl 5â-cholanoate derivative (0.71 mmol) was dissolved
in MeOH (25 mL), and 10% NaOH in MeOH (5.7 mL,
14.2.mmol) was added. The mixture was refluxed for 16 h. The
mixture was acidified with 3 N HCl and extracted with EtOAc
(3 × 20 mL). The combined organic fractions were washed with
brine (1 × 50 mL), dried (Na2SO4), and evaporated under
vacuum. The residue was purified by silica gel flash chroma-
tography.
3r-Tetr ah ydr opyr an yloxy-7-keto-5â-ch olan -24-oic Acid
(32). p-Toluenesulfonic acid (0.12 g, 0.64 mmol) and 3,4-
dihydro-2H-pyrane (1.9 mL, 20.4 mmol) were added to a
solution of 7-ketolithocholic acid 31 (5.0 g, 12.8 mmol) in
dioxane (50 mL). The reaction mixture was stirred at room
temperature for 15 min. H2O (50 mL) was then added, and
the mixture was partially concentrated under vacuum and
extracted with EtOAc (3 × 50 mL). The combined organic
fractions were washed with brine (1 × 50 mL), dried (Na2SO4),
and evaporated under vacuum. The residue was dissolved in
MeOH (50 mL) and treated overnight with 10% NaOH (MeOH
solution) at room temperature. The solvent was removed under
vacuum, and the crude was dissolved in H2O (100 mL),
neutralized with 2 N HCl, and extracted with EtOAc (3 × 100
mL). The organic phases were dried (Na2SO4) and evaporated
under vacuum. The residue was chromatographated on silica
gel column. Elution with light petroleum/EtOAc 80/20 afforded
4.2 g of compound 32 (84% yield). 1H NMR (CDCl3) δ: 0.56 (s,
3H, 18-CH3), 0.79 (d, 3H, J ) 6.7 Hz21-CH3), 1.03 (s, 3H, CH3-
19), 2.70 (m, 1H, CH-8), 3.37 (m, 4H), 3.79 (m, 1H, 3-CH).
Meth yl 3r,7r-Dih ydr oxy-6r-allyl-5â-ch olan -24-oate (35).
Methyl 3R-hydroxy-6R-allyl-7-keto-5â-cholan-24-oate (33) (0.380
g, 0.85 mmol) was converted to 0.30 g of product 35 (81% yield)
following method B. 1H NMR (CDCl3) δ: 0.58 (s, 3H, CH3-18),
0.84-0.87 (d, J ) 6.2 Hz, 3H, CH3-21), 1.15 (s, 3H, CH3-19),
3.30-3.47 (m, 1H, CH-3), 3.60 (m, 2H, CH-7 and CO2CH3),
4.90-5.10 (m, 2H, CHdCH2), 5.60-5.80 (m, 1H, CHdCH2).
Met h yl 3r,7r-Dih yd r oxy-6r-p r op a r gyl-5â-ch ola n -24-
oa te (36). Methyl 3R-hydroxy-6R-propargyl-7-keto-5â-cholan-
24-oate (34) (0.30 g, 0.68 mmol) was converted to 0.23 g of
1
product 36 (76% yield) following method B. H NMR (CDCl3)
δ: 0.64 (s, 3H, CH3-18), 0.84 (s, 3H, CH3-19), 0.89 (d, J ) 6.2
Hz, 3H, CH3-21), 3.40-3.59 (m, 1H, CH-3), 3.59 (s, 3H,
CO2CH3), 3.74 (brs, 1H, CH-7).
Meth yl 3r,7r-Dih ydr oxy-6r-(2-h ydr oxyeth yl)-5â-ch olan -
24-oa te (37). Methyl 3R-hydroxy-6R-allyl-7-keto-5â-cholan-24-
oate (33) (0.380 g, 0.85 mmol) was dissolved in 50 mL of
CH2Cl2/MeOH (1/1) and treated at 0 °C with O3 until a blue
solution was reached. The mixture was stirred at room
temperature for 1 h, NaBH4 (96 mg, 2.56 mmol) was added,
and the mixture was stirred for an additional 3 h. The mixture
was acidified with 3 N HCl and extracted with EtOAc (3 × 20
mL). The combined organic fractions were washed with brine
(1 × 50 mL), dried with Na2SO4, and evaporated under
vacuum. The residue was chromatographated on silica gel
column. Elution with light petroleum/EtOAc 70/30 afforded
1
0.12 g of derivative 37 (31%yield). H NMR (CD3OD) δ: 0.58
(s, 3H, CH3-18), 0.85 (m, 6H, CH3-19 and CH3-21), 3.24-3.30
(m, 1H, CH-3), 3.59 (s, 3H, CO2CH3), 3.64 (brs, 1H, CH-7).
3r,7r-Dih yd r oxy-6r-a llyl-5â-ch ola n -24-oic Acid (9).
Methyl 3R,7R-dihydroxy-6R-allyl-5â-cholan-24-oate (35) (0.1 g,
0.22 mmol) was treated according to method C. Elution with
CHCl3/MeOH 98/2 with 0.1% AcOH afforded 0.08 g of final
compound 9 (86% yield) as a white solid (mp: 99-101 °C). 1H
NMR (CDCl3) δ: 0.59 (s, 3H, CH3-18), 0.83 (s, 3H, CH3-19),
0.86 (d, J ) 6.4 Hz, 3H, CH3-21), 2.20-2.44 (2m, 2H, CH2-
23), 3.20-3.47 (m, 1H, CH-3), 3.60 (brs, 1H, CH-7), 4.90-5.10
(m, 2H, CHdCH2), 5.50-5.80 (m, 1H, CHdCH2). 13C NMR
(CDCl3) δ: 11.79, 18.24, 20.69, 23.06, 23.67, 28.15, 30.39, 30.80,
30.99, 33.14, 33.85, 34.24, 35.39, 35.59, 39.43, 39.57, 40.03,
42.75, 45.42, 50.42, 55.79, 70.84, 72.25, 115.98, 137.32, 179.27.
3r,7r-Dih yd r oxy-6r-(2-h yd r oxye t h yl)-5â-ch ola n -24-
oic Acid (10). Methyl 3R,7R-dihydroxy-6R-(2-hydroxyethyl)-
5â-cholan-24-oate (37) (0.1 g, 0.22 mmol) was treated according
to method C. Elution with CHCl3/MeOH 95/5 with 0.1% AcOH
afforded of 0.07 g of compound 10 (73% yield) as a white solid
(mp: 114-116 °C). 1H NMR (CD3OD) δ: 0.63 (s, 3H, CH3-18),
0.87 (m, 6H, CH3-19 and CH3-21), 2.23-2.30 (m, 2H, CH2-23),
3.22-3.28 (m, 1H, CH-3), 3.54 (3, 3H, CH2-OH and CH-7).
13C NMR (CD3OD) δ: 11.50, 18.00, 20.53, 22.86, 23.35, 27.96,
29.48, 30.00, 30.92, 32.07, 32.92, 33.45, 35.24, 35.30, 35.43,
36.38, 39.42, 39.64, 42.48, 45.83, 49.01, 50.28, 55.69, 60.52,
70.46, 71.91, 177.04.
3r,7r-Dih yd r oxy-6r-p r op a r gyl-5â-ch ola n -24-oic Acid
(11). Methyl 3R,7R-dihydroxy-6R-propargyl-5â-cholan-24-oate
(36) (0.10 g, 0.22 mmol) was treated according to method C.
Elution with CHCl3/MeOH 98/2 with 0.1% AcOH afforded of
0.08 g of compound 11 (89% yield) as a white solid (mp: 108-
110 °C). 1H NMR (CD3OD) δ: 0.64 (s, 3H, CH3-18), 0.87 (s,
3H, CH3-19), 0.90 (d, J ) Hz, 3H, CH3-19), 2.23-2.27 (m, 2H,
CH2-23), 3.24-3.27 (m, 1H, CH-3), 3.65 (brs, 1H, CH-7). 13C
NMR (CD3OD) δ: 11.26, 17.81, 19.35, 22.60, 28.27, 30.16,
Meth yl 3r-Hyd r oxy-6r-a llyl-7-k eto-5â-ch ola n -24-oa te
(33). This compound was prepared using method A (allyl
bromide 12.7 g, 105 mmol). Elution with light petroleum/

Synthetic Bile Acids as Potent FXR Ligands
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18 4567
1
31.13, 31.40, 33.21, 33.30, 35.72, 40.01, 40.13, 40.56, 42.76,
45.38, 50.62, 56.36, 69.13, 69.92, 71.98, 78.48, 83.33, 177.48.
product 44 (80% yield) following method B. H NMR (CDCl3)
δ: 0.62 (s, 3H, CH3-18), 0.87 (s, 3H, CH3-19), 0.92 (d, J ) 6.0
Hz, 3H, CH3-21), 0.97 (d, J ) 7.1 Hz, 3H, CH3-6), 3.27-3.40
(m, 1H, CH-3), 3.55 (brs, 1H, CH-3), 3.63 (s, 3H, CO2CH3).
3r,7r-Dih yd r oxy-6r,23-d im eth yl-5â-ch ola n -24-oic Acid
(20). Methyl 3R,7R-dihydroxy-6R,23-dimethyl-5â-cholan-24-
oate (44) (0.08 g, 0.19 mmol) was treated according to method
C. Elution with CH2Cl2/MeOH 95/5 with 0.1% AcOH afforded
of 0.05 g of product 20 (65% yield) as a white solid (mp: 103-
105 °C). 1H NMR (CDCl3) δ: 0.67 (s, 3H, CH3-18), 0.92 (s, 3H,
CH3-19), 0.95 (m, complex, CH3-21 and CH3-23), 1.01 (d, J )
7.1 Hz, 3H, CH3-6), 2.54 (m, 1H, CH-23), 3.39-3.48 (m, 1H,
CH-3), 3.56 (brs, 1H, CH-7). 13C NMR (CDCl3) δ: 11.77, 15.72,
18.58, 18.88, 20.65, 23.12, 23.66, 25.63, 28.19, 30.51, 32.67,
33.84, 33.99, 34.62, 35.44, 37.03, 38.91, 39.62, 40.04, 40.73,
47.37, 50.46, 56.61, 72.35, 72.87, 179.10.
Meth yl 3r-(ter t-Bu th yld ip h en ylsilyloxy)-6r-h yd r oxy-
7-k et o-5â-ch ola n -24-oa t e (39). Methyl 3R,6R-dihydroxy-7-
keto-5â-cholan-24-oate (38) (4.0 g, mmol) was dissolved in
DMF (80 mL), and tert-butyldiphenylsilyl chloride (5.8 mL,
14.2.mmol) and imidazole (1.5 g, mmol) were added. The
reaction mixture was stirred at 60 °C for 5 h. Cold H2O (250
mL) was then added, and the mixture was extracted with
EtOAc (3 × 150 mL). The organic fraction was washed with
brine (1 × 50 mL), dried (Na2SO4), and evaporated under
vacuum. The residue was chromatographated on silica gel
column. Elution with light petroleum/EtOAc 80/20 afforded 2.7
g of compound 39 (43% yield). 1H NMR (CDCl3) δ: 0.56 (s,
3H, CH3-18), 0.81 (d, 3H J ) 6.85 Hz, CH3-21), 0.95 (s, 9H,
t-Bu), 1.03 (s, 3H, CH3-19), 3.40 (m, 1H, CH-3), 3.59 (s, 3H,
CO2CH3), 3.69 (m, 1H, CH-6), 7.30 (m, 6H, Ph), 7.62 (m, 4H,
Ph).
Meth yl 3r-Hyd r oxy-6r-m eth oxy-7-k eto-5â-ch ola n -24-
oa te (40). Methyl 3R-(tert-buthyldiphenylsilyloxy)-6R-hydroxy-
7-keto-5â-cholan-24-oate (39) (0.5 g, 0.76 mmol) and 15-
crown-5 (0.17 mL, 0.84 mmol) dissolved in dry THF (10 mL)
were slowly added to a suspension of NaH (0.04 g, 1.06 mmol,
60% in oil) in dry ether (40 mL) at 0 °C. The reaction mixture
was then treated dropwise with methyl iodide (0.12 g, 0.84
mmol) dissolved in THF (10 mL) and stirred at room temper-
ature for an additional 4 h. Cold H2O (50 mL) was added, and
the mixture was extracted with EtOAc (3 × 20 mL). The
organic fraction was washed with brine (1 × 50 mL), dried
(Na2SO4), and evaporated under vacuum. The residue was
taken up with THF (40 mL), tetrabutylammonium fluoride
(0.26 g, 0.99 mmol) was added, and the mixture was stirred
at room temperature for 16 h. H2O (150 mL) was added, and
the mixture was extracted with EtOAc (3 × 50 mL). The
organic fraction was washed with brine (1 × 50 mL), dried
(Na2SO4), and evaporated under vacuum. The residue was
chromatographated on a silica gel column. Elution with light
petroleum/EtOAc 50/50 afforded 0.15 g of the derivative 40
(45% yield). 1H NMR (CDCl3) δ: 0.56 (s, 3H, CH3-18), 0.81 (d,
3H J ) 7.06 Hz, CH3-21), 1.04 (s, 3H, CH3-19), 3.18 (s, 3H,
OCH3), 3.41 (m, 1H, CH-3), 3.59 (s, 3H, CO2CH3), 3.95 (m, 1H,
CH-6).
3r,7r-Dih yd r oxy-22,23-m eth ylen e-5â-ch ola n -24-oic Ac-
id s (21-24). Ethyl diazoacetate (0.478 g, 1.19 mmol) in dry
CH2Cl2 (15 mL) was slowly added dropwise to a stirred
suspension of 3R,7R-diacetoxy-5â-norcholan-22,23-ene (45) (0.6
g, 1.39 mmol) in the presence of dirhodium(II) tetraacetate (9
mg, 0.02 mmol) in dry CH2Cl2 (15 mL) under nitrogen at room
temperature. The reaction mixture was filtered and washed
with H2O (20 mL), dried (Na2SO4), and evaporated under
vacuum, thus affording a mixture of the four diastereoisomeric
esters 46. The esters 46 were successively dissolved in EtOH
(15 mL) and treated with a solution of 10 N NaOH (10 mL) at
reflux for 4 h, cooled, poured onto cold H2O (50 mL), acidified
with 2 N HCl, and extracted with EtOAc (3 × 15 mL). The
organic phase was washed with brine (10 mL), dried (Na2SO4),
and concentrated under vacuum. The residue was chroma-
tographated on silica gel. Elution with CH2Cl2/MeOH 96/4 with
AcOH 0.1% afforded 0.087 g (15% yield) of (22S,23S)-3R,7R-
dihydroxy-22,23-methylene-5â-cholan-24-oic acid (21) and 0.065
g (11.5% yield) of (22R,23R)-3R,7R-dihydroxy-22,23-methylene-
5â-cholan-24-oic acid (22). Elution with CH2Cl2/MeOH 95.5/
4.5 with 0.1% AcOH afforded 0.18 g (32% yield) of (22S,23R)-
3R,7R-dihydroxy-22,23-methylene-5â-cholan-24-oic acid (23)
and 0.15 g (26.7% yield) of (22R, 23S)-3R,7R-dihydroxy-22,23-
methylene-5â-cholan-24-oic acid (24) as white solids.
21. Mp: 148-150 °C. [R]20 +5.16 (c 1, EtOH). 1H NMR
D
(CD3OD and CDCl3) δ: 0.67 (s, 3H, 18-CH3), 0.90 (s, 3H, 19-
CH3), 0.96 (d, J ) 6.68 Hz, 3H, 21-CH3), 3.40-3.50 (m, 1H,
3-CH), 3.85 (m, 1H, 7-CH). 13C NMR (CDCl3) δ: 12.20, 16.80,
17.08, 20.80, 21.00, 23.15, 24.10, 28.30, 30.80, 31.30, 33.30,
34.80, 34.90, 35.40, 35.70, 39.80, 39.90, 41.80, 43.40, 50.55,
58.20, 68.90, 72.30, 177.00.
Meth yl 3r,7r-Dih ydr oxy-6r-m eth oxy-5â-ch olan -24-oate
(41). Methyl 3R-hydroxy-6R-methoxy-7-keto-5â-cholan-24-oate
(40) (0.12 g, 0.28 mmol) was converted to 0.085 g of product
1
41 (71% yield) following method B. H NMR (CDCl3) δ: 0.55
(s, 3H, CH3-18), 0.81 (m, 6H, CH3-19 and CH3-21), 3.27-3.37
(m and s, 4H, CH-3 and OCH3), 3.56 (s, 3H, CO2CH3), 3.70
(brs, 1H, CH-7).
22. Mp: >230 °C. [R]20 -38.19 (c 1.1, CH3Cl/MeOH 1:1).
D
1H NMR (CD3OD and CDCl3) δ: 0.50 (s, 3H, 18-CH3), 0.86 (s,
3H, 19-CH3), 0.96 (d, J ) 6.40 Hz, 3H, 21-CH3), 3.40-3.60 (m,
1H, 3-CH), 3.80 (m, 1H, 7-CH). 13C NMR (CDCl3) δ: 12.00,
12.50, 20.90, 21.00, 21.10, 23.00, 23.80, 27.10, 30.50, 31.00,
32.10, 33.10, 34.80, 35.30, 35.60, 39.50, 39.70, 39.85, 41.80,
43.00, 50.40, 58.50, 68.60, 72.00, 176.90.
3r,7r-Dih ydr oxy-6r-m eth oxy-5â-ch olan -24-oic Acid (13).
Methyl 3R,7R-dihydroxy-6R-methoxy-5â-cholan-24-oate (41)
(0.08 g, 0.18 mmol) was treated according to method C. Elution
with CHCl3/MeOH 90/10 and further purification on an RP8
column, eluting with CH3CN/H2O 80/20, afforded of 0.04 g of
compound 13 (56% yield) as a white solid (mp: 105-107 °C).
1H NMR (CDCl3) δ: 0.59 (s, 3H, CH3-18), 0.83 (s, 3H, CH3-
19), 0.87 (d, J ) Hz, 3H, CH3-19), 2.18-2.32 (2m, 2H, CH2-
23), 3.27-3.37 (m, 1H, CH-3), 3.30 (s, 3H, OCH3), 3.88 (brs,
1H, CH-7). 13C NMR (CDCl3) δ: 11.67, 18.24, 20.64, 23.13,
23.55, 28.10, 30.51, 30.79, 30.90, 32.75, 32.85, 35.39, 35.68,
35.76, 38.24, 39.41, 42.66, 44.74, 50.06, 55.65, 55.91, 68.96,
71.90, 78.76, 179.12.
23. Mp: 221-225 °C. [R]20 -40.22 (c 1, EtOH). 1H NMR
D
(CD3OD and CDCl3) δ: 0.56 (s, 3H, 18-CH3), 0.86 (s, 3H, 19-
CH3), 1.16 (d, J ) 6.60 Hz, 3H, 21-CH3), 3.10-3.30 (m, 1H,
3-CH), 3.85 (m, 1H, 7-CH). 13C NMR (CDCl3) δ: 12.10, 18.30,
18.55, 20.00, 20.90, 23.10, 24.00, 28.20, 30.70, 31.70, 33.20,
34.80, 35.40, 35.70, 39.80, 40.10, 41.80, 43.15, 50.40, 57.80,
68.90, 72.20, 178.40.
24. Mp: 136-140 °C. [R]20 +13.66 (c 1, EtOH). 1H NMR
D
Meth yl 3r-Hyd r oxy-6r,23-d im eth yl-7-k eto-5â-ch ola n -
24-oa te (43). This compound was prepared using method A
(methyl iodide 15 g, 105 mmol). Elution with light petroleum/
EtOAc 70/30 afforded 0.92 g of methyl 3R-hydroxy-6R-methyl-
7-keto-5â-cholan-24-oate (42) (21% yield) and 0.18 g of product
(CD3OD and CDCl3) δ: 0.56 (s, 3H, 18-CH3), 0.86 (s, 3H, 19-
CH3), 0.96 (d, J ) 6.66 Hz, 3H, 21-CH3), 3.40-3.60 (m, 1H,
3-CH), 3.80 (m, 1H, 7-CH). 13C NMR (CDCl3) δ: 12.00, 13.50,
19.90, 20.90, 22.50, 23.10, 24.00, 28.00, 30.70, 31.60, 33.20,
34.90, 35.40, 35.65, 39.70, 39.73, 41.80, 43.10, 50.40, 58.00,
68.80, 72.20, 177.60.
1
43 (4% yield). H NMR (CDCl3) δ: 0.60 (s, 3H, CH3-18), 0.87
(d, J ) 6.3 Hz, 3H, CH3-21), 0.88 (d, J ) 6.6 Hz, 3H, CH3-6),
(m, 3H, CH3-23), 1.19 (s, 3H, CH3-19), 3.45-3.53 (m, 1H, CH-
OH), 3.61 (s, 3H, CO2CH3).
Meth yl 3r,7r-Dih yd r oxy-6r,23-d im eth yl-5â-ch ola n -24-
oate (44). Methyl 3R-hydroxy-6R,23-dimethyl-7-keto-5â-cholan-
24-oate (43) (0.10 g, 0.24 mmol) was converted to 0.08 g of
23-N -Ca r b e t h oxy-3r,7r-d ia ce t oxy-5â-n or ch ola n ila -
m in e (48). 3R,7R-Diacetoxy-5â-cholan-24-oic acid (47) (0.3 g,
0.63 mmol) was suspended in dry benzene (30 mL) and treated
with SOCl2 (0.8 mL). The mixture was refluxed for 2 h. After
the mixture was cooled, the solvents were removed under
vacuum and THF (10 mL) was added and the mixture was

4568 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 18
Pellicciari et al.
1
treated at 0 °C with a solution of NaN3 (0.12 g, 1.9 mmol) in
H2O (5 mL). After 1 h, the reaction mixture was poured into
cold H2O (100 mL) and extracted with EtOAc (3 × 100 mL).
The organic phase was dried (Na2SO4) and evaporated to
obtain the acyl azide intermediate (IR: 2134 and 2267 cm-1).
The residue was then dissolved in absolute EtOH (50 mL) and
refluxed overnight. The reaction mixture was concentrated
under vacuum to give 0.21 g of the crude product 48 (65%
51 (42% yield). H NMR (CDCl3) δ: 0.63 (s, 3H, CH3-18), 0.86
(s, 3H, CH3-19), 0.89 (d, J ) 6.1 Hz, 3H, CH3-19), 2.05 and
2.08 (2s, 6H, 2CH3CO), 2.73 (m, 2H, CH2-23), 4.24 (q, J ) 6
Hz, 2 H, CH2CH3), 4.41 (m, 1H, CH-3), 4.56 (m, 1H, CH-7),
4.91 (s, 2 H, CH2Ph), 5.77 (brs, 1 H, HNOBn), 7.35 (brs, 5 H,
Ph).
3r,7r-Diacetoxy-5â-n or ch olan -23-(N-ben zyloxy)-N-(eth -
oxyca r bon ylu r eid o)a m in e (52). 3R,7R-Diacetoxy-5â-nor-
cholan-23-(N-benzyloxy)amine (51) (0.6 g, 1.08 mmol) was
dissolved in dry THF (5 mL), and 90% ethoxycarbonyl isocy-
anate (135 µL, 1.32 mmol) was added after which the reaction
mixture was stirred for 30 min. The solvent was removed
under vacuum, and the residue was chromatographated on
silica gel. Elution with CHCl3 gave 0.52 g of the derivative 52
1
yield). H NMR (CDCl3) δ: 0.63 (s, 3H, CH3-18), 0.91 (s, 3H,
CH3-19), 0.91 (d, J ) 6.07 Hz, 3H, CH3-21), 1.23 (t, 3H J )
4.06 Hz, CH2CH3), 2.01 and 2.03 (2s, 6H, 2CH3CO), 4.90-4.53
(m, 1H, CH-3), 4.74 (bs, 1H, CH-7).
3r,7r-Dih yd r oxy-5â-n or ch ola n ila m in e (26) a n d 23-N-
Ca r beth oxy-3r,7r-d ih yd r oxy-5â-n or ch ola n ila m in e (27).
23-N-Carbethoxy-3R,7R-diacetoxy-5â-norcholanilamine (48) (0.2
g, 0.39 mmol) was treated according to method C. Elution with
CH2Cl2/MeOH 90/10 afforded 0.035 g of the less polar product
27 (26% yield) and 0.028 g of the more polar product 26 (23%
yield) as white solids.
1
(72% yield). H NMR (CDCl3) δ: 0.63 (s, 3H, CH3-18), 0.86 (s,
3H, CH3-19), 0.89 (d, J ) 6.5 Hz, 3H, CH3-19), 1.38 (m, 3H),
2.05 and 2.08 (2s, 6H, 2CH3CO), 2.73 (m, 2H, CH2-23), 4.27
(m, 3H, CH-3 and CH2CH3), 4.58 (m, 1H, CH-7), 4.89 (s, 2H,
CH2Ph), 7.41 (br s, 5 H, Ph), 7.80 (br s, 1 H, CONHCO).
26. Mp: 131-133 °C. 1H NMR (CD3OD) δ: 0.64 (s, 3H, CH3-
18), 0.87 (s, 3H, CH3-19), 0.90 (d, J ) 6.45 Hz, 3H, CH3-21),
2.20-2.40 (m, 2H, CH2-24), 3.27-3.33 (m, 1H, CH-3), 3.74 (bs,
1H, CH-7). 13C NMR (CD3OD) δ: 17.45, 20.33, 22.07, 23.18,
27.79, 29.88, 31.01, 32.56, 34.43, 34.77, 35.11, 35.31, 38.96,
39.26, 39.55, 41.64, 42.23, 48.14, 50.06, 55.83, 67.62, 71.40.
3r,7r-Dia cetoxy-5â-n or ch ola n -23-(N-h yd r oxy)-N-(eth -
oxyca r bon ylu r eid o)a m in e (53). 3R,7R-Diacetoxy-5â-nor-
cholan-23-(N-benzyloxy)-N-(ethoxycarbonylureido)amine (52)
(0.47 g, 0.70 mmol) was dissolved in 20 mL of 96% EtOH, and
this solution was carefully mixed with about 0.1 g of 10% Pd/
C. The solution was hydrogenated at 30 psi for 3 h. The
solution was then filtered through Celite and washed with
EtOH, and the solvent was removed under vacuum. The
residue was chromatographated on silica gel. Elution with
CHCl3/MeOH 95/5 gave 0.26 g of the derivative 53 (64% yield).
1H NMR (CDCl3) δ: 0.66 (s, 3H, CH3-18), 0.95 (s, 3H, CH3-
19), 0.99 (d, J ) 6.7 Hz, 3H, CH3-19), 1.32 (m, 3 H, CH3), 2.05
and 2.08 (2s, 6H, 2CH3CO), 3.61 (m, 1H), 3.72 (m, 1H), 4.27
(m, 2H, CH2), 4.61 (m, 1H, CH-3), 4.90 (bs, 1H, CH-7), 7.29 (s,
1H, NHO), 8.06 (s, 1 H, CONHCO), 8.72 (s, 1 H, NOH).
1
27. Mp: 90-92 °C. H NMR (CD3OD) δ: 0.61 (s, 3H, CH3-
18), 0.85 (s, 3H, CH3-19), 0.90 (d, J ) 6.52 Hz, 3H, CH3-21),
2.95-3.24 (2m, 2H, CH2-24), 3.36-3.44 (m, 1H, CH-3), 3.79
(bs, 1H, CH-7), 4.04 (bq, CH2CH3). 13C NMR (CDCl3) δ: 14.68,
18.61, 20.57, 22.77, 23.71, 28.33, 30.68, 32.84, 33.80, 34.61,
35.05, 35.32, 36.16, 38.56, 39.43, 39.63, 39.92, 41.48, 42.74,
50.47, 56.02, 60.64, 68.53, 72.01, 199.98.
3r,7r-Dia cetoxy-5â-n or ch ola n -23-ole (49). 3R,7R-Diac-
etoxy-5â-norcholan-22,23-ene (45) (6.4 g, 14.8 mmol) was
dissolved in 150 mL of THF, and 1 M solution of the BH3‚
THF complex (30 mL) was added at 0 °C. The mixture was
then stirred at room temperature for 3 h. The reaction mixture
was then cooled (ice bath) and treated with 3 N NaOH (23
mL) and 30% H2O2 (26 mL) and stirred for an additional 16 h
at room temperature. The mixture was acidified with 3 N HCl
and extracted with EtOAc (3 × 300 mL). The combined organic
fractions were washed with brine (1 × 150 mL), dried (Na2-
SO4), and evaporated under vacuum. The residue was chro-
matographated on silica gel. Elution with CH2Cl2/MeOH 95/5
gave 4.2 g of product 49 (63% yield). 1H NMR (CDCl3) δ: 0.69
(s, 3H, CH3-18), 0.97 (m, 6H, CH3-19 and CH3-21), 2.08 and
2.10 (2s, 6H, 2CH3CO), 3.65 (m, 2H, CH2-23), 4.53 (m, 1H, CH-
3), 4.92 (bs, 1H, CH-7).
1-(3r,7r-Dih yd r oxy-5â-23-n or ch ola n yl)-3,5-d ioxo-1,2,4-
oxa d ia zolid in e (28). 3R,7R-Diacetoxy-5â-norcholan-23-(N-
hydroxy)-N-(ethoxycarbonylureido)amine (53) (0.17 g, 0.29
mmol) was dissolved in a mixture of THF and H2O (20 mL,
1/1). Sodium hydroxide (0.12 g, 0.29 mmol) was added, and
the reaction mixture was stirred at room temperature for 1 h.
The reaction then was quenched with 10% citric acid solution.
The residue was extracted with EtOAc (5 × 50 mL). The
organic layer was dried (Na2SO4), and the solvent was then
removed under vacuum. The residue was chromatographated
on silica gel. Elution with CHCl3/MeOH 90/10 afforded 0.06 g
of product 28 (46% yield) as a white solid (mp: 96-98 °C). 1H
NMR (CD3OD) δ: 0.63 (s, 3H, CH3-18), 0.86 (s, 3H, CH3-19),
0.89 (d, J ) Hz, 3H, CH3-19), 2.63-2.73 (m, 2H, CH2-24), 3.26-
3.41 (m, 1H, CH-3), 3.80 (m, 2H, CH2), 3.92 (brs, 1H, CH-7),
7.95 (s, 1H, NH). 13C NMR (CD3OD) δ: 10.57, 17.46, 20.26,
21.87, 23.09, 27.79, 29.85, 32.24, 32.55, 33.29, 34.38, 34.72,
35.05, 38.98, 39.26, 39.50, 41.68, 46.56, 50.03, 55.75, 67.53,
71.35, 77.97, 152.75, 157.99.
3r,7r-Dia cet oxy-5â-n or ch ola n -23-a ld eh yd e (50). PCC
(2.41 g, 11.2 mmol) suspended in dry methylene chloride (70
mL) was added dropwise to 3R,7R-diacetoxy-5â-norcholan-23-
ole (49) (4.1 g, 9.3 mmol) dissolved in dry dichloromethane (70
mL). The mixture was stirred at room temperature for 12 h.
Et2O (25 mL) was then added, and the black solid that
precipitated was filtered and washed with diethyl ether several
times. The filtrate was concentrated under vacuum and the
residue was passed on fluorisil (CH2Cl2), affording 2.6 g of
crude product 50 (61% yield) that was used for the next step
Biology. Compounds were assayed by fluorescence reso-
nance energy transfer for recruitment of the SRC1 peptide to
human FXR using a cell-free LiSA as described elsewere.2,12
Ack n ow led gm en t. A.E. gratefully acknowledges
financial support from the “Secretaria de Estado de
Universidades y Educacion” of Spain. The authors are
grateful to Erregierre (Bergamo, Italy) for the starting
material 7-ketolithocholic acid and to Intercept Phar-
maceuticals (New York) for financial support.
1
without further purification. H NMR (CDCl3) δ: 0.67 (s, 3H,
CH3-18), 0.95 (m, 6H, CH3-19 and CH3-21), 2.05 and 2.08 (2s,
6H, 2CH3CO), 2.38 (m, 2H, CH2-23), 4.51 (m, 1H, CH-3), 4.86
(bs, 1H, CH-7), 9.76 (s, 1H, CHO).
3r,7r-D ia c e t o x y -5â-n o r c h o la n -23-(N -b e n zy lo x y )-
a m in e (51). Benzylhydroxylamine (0.43 g, 2.68 mmol), 3R,7R-
diacetoxy-5â-norcholan-23-aldehyde (50) (1.2 g, 2.68 mmol),
and sodium cyanoborohydride (0.34 g, 5.4 mmol) were dis-
solved in distilled MeOH (50 mL). The pH of the solution was
brought to 5 by addition of 2 N HCl in MeOH, and the reaction
mixture was stirred for 48 h at room temperature. H2O (50
mL) was added, and the solvents were removed under vacuum.
The crude product was extracted with EtOAc (3 × 50 mL) and
H2O (50 mL). The organic layer was washed with brine (1 ×
50 mL) and dried (Na2SO4). The residue was chromatographat-
ed on silica gel. Elution with CHCl3 afforded 0.63 g of product
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