Asymmetric synthesis of the four diastereoisomers of a novel non-steroidal farnesoid X receptor (FXR) agonist: Role of the chirality on the biological activity

Article abstract of DOI:10.1016/j.bmc.2013.04.038

An asymmetric synthetic strategy was designed for the preparation of the four possible diastereoisomers of 3,6-dimethyl-1-(2-methylphenyl)-4-(4- phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7-one, a non-steroidal FXR agonist, we recently discovered following a virtual screening approach. The results obtained from an AlphaScreen assay clearly demonstrated that only the isomer endowed with 4R,6S absolute configuration is responsible for the biological activity. A deep investigation of the different putative binding modes adopted by these enantiomerically pure ligands using computational modeling studies confirmed the enantioselectivity of FXR towards this class of molecules.

Full text of DOI:10.1016/j.bmc.2013.04.038

Bioorganic & Medicinal Chemistry 21 (2013) 3780–3789
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Asymmetric synthesis of the four diastereoisomers of a novel
non-steroidal farnesoid X receptor (FXR) agonist: Role of the chirality
on the biological activity
a
a
a
a
Maura Marinozzi a, , Andrea Carotti , Roccaldo Sardella , Federica Buonerba , Federica Ianni ,
⇑
Benedetto Natalini a, Daniela Passeri b, Giovanni Rizzo c, Roberto Pellicciari a
a Dipartimento di Chimica e Tecnologia del Farmaco, Università di Perugia, Via del Liceo, 1-06123 Perugia, Italy
b TES Pharma S.r.l., Via Palmiro Togliatti 22bis, 06073 Corciano, Italy
c Intercept Pharmaceuticals, 18 Desbrosses Street, New York, NY 10013, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
An asymmetric synthetic strategy was designed for the preparation of the four possible diastereoisomers
of 3,6-dimethyl-1-(2-methylphenyl)-4-(4-phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiaze-
pin-7-one, a non-steroidal FXR agonist, we recently discovered following a virtual screening approach.
The results obtained from an AlphaScreen assay clearly demonstrated that only the isomer endowed with
4R,6S absolute configuration is responsible for the biological activity. A deep investigation of the different
putative binding modes adopted by these enantiomerically pure ligands using computational modeling
studies confirmed the enantioselectivity of FXR towards this class of molecules.
Received 18 March 2013
Revised 9 April 2013
Accepted 10 April 2013
Available online 23 April 2013
Keywords:
Farnesoid X receptor (FXR)
Non-steroidal FXR agonist
Virtual screening
Ó 2013 Elsevier Ltd. All rights reserved.
1H-Pyrazole[3,4-e][1,4]thiazepin-7-one
Heterocyclic chemistry
1. Introduction
protective role in liver carcinogenesis and protects against intesti-
nal infection.9 All these evidences make FXR a promising potential
Starting from 1999 when the orphan nuclear receptor farnesoid
X receptor (FXR) was ‘de-orphanized’ by the demonstration that
primary bile acids (BAs) were its endogenous ligands,1–3 great pro-
gress has been made in the understanding of its physiological roles.
FXR was initially identified as a regulator of the expression of var-
ious transport proteins and biosynthetic enzymes that maintain
cholesterol and bile acid homeostasis.4 Indeed, activation of FXR
by bile acids or synthetic agonists results in (a) transcriptional
target for the treatment of a variety of pathological conditions,
including steatohepatitis, liver fibrosis, cholestasis, liver cancer,
inflammatory bowel disease, cholesterol gallstone disease, athero-
sclerosis, erectile dysfunction, obesity, diabetes, and metabolic
syndrome.10–13
For all these evidences, over the last decade, many efforts have
been dedicated to the search of FXR small molecule modulators
either by the structural modification of the endogenous ligands
or by the screening of non-steroidal compound libraries.14 Follow-
repression of cholesterol 7a-hydroxylase (CYP7A1), the rate-limit-
ing enzyme in the bile acid biosynthesis pathway, (b) induction of
the small heterodimer partner (SHP), a transcriptional repressor
found in the liver and intestine, and (c) induction of genes encod-
ing for some bile acid transport proteins, such as intestinal bile
acid-binding protein (IBABP)5 and bile salt export pump (BSEP).6
Furthermore, bile acid-mediated FXR activation has been recently
recognized as a major underlying pathway for energy homeostasis
and glucose and lipid metabolism.7 More recently, additional func-
tional roles of FXR have been identified; specifically, it has been
shown that FXR regulates normal liver regeneration,8 plays a
ing the first approach, our group reported in 2002, 6a-ethylcheno-
deoxycholic acid (obeticholic acid, INT-747, 1) as a highly potent
and orally available FXR full agonist.15 Positive data from two
phase II clinical trials using INT-747 (1), recently demonstrated
the clinical utility of this compound in the treatment of primary
biliary cirrhosis, a chronic inflammatory cholestatic condition in
the liver, and type 2 diabetes. The first non-steroidal FXR agonist,
namely GW4064 (2), was identified by GlaxoSmithKline from an
iterative combinatorial library synthesis and screening approach.16
A lot of work was then dedicated to further explore structure/activ-
ity relationships (SAR) of 2 and improve its drug developability.17
High-throughput screening combined with optimization of benzo-
pyran-based combinatorial library result in 2003 in the discovery
⇑
Corresponding author. Tel.: +39 075 585 5159; fax: +39 075 585 5161.
E-mail address: maura@chimfarm.unipg.it (M. Marinozzi).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.038

M. Marinozzi et al. / Bioorg. Med. Chem. 21 (2013) 3780–3789
3781
of fexaramine (3), another structurally diverse non-steroidal FXR
agonist.18 Aside from mentioned GW4064 (2) and fexaramine (3),
high-throughput screening campaigns revealed benzimidazole-
(4)19 and azepino(4,5-b)indole (5)20 derivatives as new classes of
FXR agonists, while pyrazoline-3,5-dione- (6)21 and 1-(4-methylpi-
perazin-1-yl)-3-phenoxypropan-2-ol (7)22 derivatives were dis-
closed by virtual screening approaches (Fig. 1).
series of analogues of 9 modified at C-3 and C-6-core positions will
be described.
As a part of a project aimed at finding a novel class of non-ste-
roidal FXR agonists we recently reported the identification of 4-
(2,4-dimethoxyphenyl)-3,6-dimethyl-1-(2-mehylphenyl)-4,8-
dihydro-1H-pyrazole[3,4-e][1,4]thiazepin-7-one (8), as a new scaf-
fold endowed with FXR activity. Our first optimization work fo-
cused to explore substitution patterns of A and B rings of 8,
revealed that, among the two diastereoisomeric couples, the syn
one is responsible for the biological activity. Similar indications ar-
ose also from the evaluation of the binding energies correlated to a
set of docking studies, that confirmed the different putative bind-
ing pose of the two diastereoisomeric couples.23 Having thus be-
come aware that the relative disposition of the substituents is a
crucial parameter for the activity and in consideration of the fact
that the receptor recognition is expected to be enantioselective,
we considered important to evaluate the activity of the single
enantiomers of a target compound. With this aim, herein we report
the synthetic approach allowing us to obtain the four possible dia-
stereoisomers of 3,6-dimethyl-1-(2-methylphenyl)-4-(4-phenoxy-
phenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7-one
(9),23 one of the most potent derivatives obtained from the optimi-
zation of 8. Furthermore, as a continuation of the SAR study of this
class of molecules, the synthesis and the biological evaluation of a
2. Chemistry
Although the preparation of functionalized 1H-pyrazolo[3,4-
e][1,4]thiazepin-7-ones is known,24 there are no examples in the
literature reporting the preparation of these compounds in enanti-
omerically pure form. The general synthetic strategy provides for
the condensation reaction between a 5-aminopyrazole and an
aldehyde, followed by the addition of the appropriate 2-mercapto-
carboxylic acid to the intermediate 2,4-dihydro-3H-pyrazol-5-
imine, first formed (Scheme 1). Since two stereogenic centers are
present in the structure of the final compounds (if R3 – H), they
Figure 1. Structures of selected steroidal- and non-steroidal FXR agonists.

3782
M. Marinozzi et al. / Bioorg. Med. Chem. 21 (2013) 3780–3789
Scheme 1. General synthesis of functionalized 1H-Pyrazolo[3,4-e][1,4]thiazepin-7-ones.
will be obtained as mixture of two racemic anti- and syn-couples,
separable by chromatography.
other diastereoisomeric pair, (4R,6R)-9 and (4S,6R)-9, was achieved
in the same fashion by using (R)-2-mercaptopropanoic acid [(R)-
12] in the condensation step (Scheme 2). After MPC, (4R,6R)-9
and (4S,6R)-9 were obtained in 37% and 36% yield, respectively.
Although (S)- and (R)-2-mercaptopropanoic acids [(S)- and (R)-
12] were synthesized according to the procedure reported by Kel-
logg and co-workers,25 we deemed advisable to determinate their
optical purity, not by the reported NMR methods, but by chiral
HPLC, that is the same methodology we use for checking the enan-
tiomeric purity of the final compounds. A series of unsuccessful
analyses carried out on the racemate of 2-mercaptopropanoic acid
(12) prompted us to attempt the enantioseparation of the corre-
sponding S-trityl derivative considering the aromatic moiety able
to emphasize the enantiodifference into an anisotropic
environment.
By analyzing this reaction we realized that, while the asymmet-
ric center at C-4 is formed during the reaction, the C-6 one derives
from the starting 2-mercaptocarboxylic acid and remains intact
during the reaction. Thus, it could be supposed that the use of a
chiral, non-racemic 2-mercaptocarboxylic acid, in place of the cor-
responding racemate, should afford, after chromatography, the
anti- and syn enantiomerically pure diastereoisomers. By repeating
the reaction using the acid endowed with the opposite absolute
configuration, it would then be obtained the other two
diastereoisomers.
Thus, the addition of (S)-2-mercaptopropanoic acid [(S)-12]
to the adduct derived from the condensation between
3-methyl-1-(2-methylphenyl)-1H-pyrazol-5-amine (10b) and
4-phenoxybenzaldehyde (11) afforded the mixture of the two 6S-
diastereoisomers, which were separated by medium pressure
chromatography (MPC) (Scheme 2). As detailed in our previous pa-
per,23 spectroscopic analysis allowed us to assign the anti disposi-
tion of the substituents to the first eluted compound, thus
endowed with (4S,6S) absolute configuration and the syn (4S,6R)
one to the more polar diastereoisomer. The preparation of the
A profitable enantioresolution (a = 1.33, RS = 2.31) of the race-
mate of S-trityl-2-mercaptopropanoic acid was achieved by run-
ning the analysis in the polar-organic mode of elution, with an
anion-exchange chiral stationary phase based on quinine-carba-
mate chiral selector units (QN-AX CSP).26 The chromatographic
trace of the racemic mixture along with those referring to the
(R)- and (S)-S-trityl-2-mercaptopropanoic acids are shown in
O
N
O
N
S
S
S
R
S
S
CH3
CO2H
N
H
N
H
O
O
N
N
HS
S
S-12
O
b
(4S,6S)-9
(4R,6S)-9
O
N
NH2
N
a
+
N
CHO
b
NH
N
11
10b
O
O
CH3
CO2H
HS
R
S
R
S
R
R
R-12
S
N
N
N
H
O
N
N
O
N
H
(4S,6R)-9
(4R,6R)-9
a (a) i. toluene, reflux; (b) i. reflux; ii. MPC.
Scheme 2. Synthesis of (4S,6S)-, (4R,6S)-, (4R,6R)- and (4S,6R)-3,6-Dimethyl-1-(2-methylphenyl)–4-(4-phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7-
ones [(4S,6S)-9, (4R,6S)-9, (4R,6R)-9 and (4S,6R)-9]. Reagents and conditions: (a) (i) toluene, reflux; (b) (i) reflux; (ii) MPC.

M. Marinozzi et al. / Bioorg. Med. Chem. 21 (2013) 3780–3789
3783
NHNH2
O
N
NH2
a
N
CN
21
20
10c
a (a) i. EtOH abs, reflux, 12 h, 50 %.
Scheme 5. Synthesis of 3-Ethyl-1-(2-methylphenyl)-1H-pyrazol-5-amine (10c).
Reagents and conditions: (a) (i) EtOH abs, reflux, 12 h, 50%.
Figure 2. Chromatographic traces of (A) racemic S-Trityl-2-mercaptopropanoic
acid; (B) S-Trityl-(R)-2-mercaptopropanoic acid and (C) S-Trityl-(S)-2-mercapto-
propanoic acid. Mobile phase composition: MeOH/AcOH—99:1 (v/v).
Figure 3. Chromatographic trace of (A) (4R,6R)-9; (B) (4S,6R)-9; (C) (4S,6S)-9 and (D) (4R,6S)-9. Mobile phase composition: n-hexane/chloroform/EtOH—88:10:2 (v/v/v).
CHO
O
O
O
R
N
a
+
NH2
N
S
S
R
R
R1
R1
N
N
N
N
H
O
O
N
N
H
10a (
10b (
10c (
=
=
=
R
R
R
H)
CH3)
C2H5)
11
a( )13
a( )9
s( )13
(R = H, R1 = CH3)
s( )9 (R = CH3, R1 = CH3)
( )14
15
s( )14
a
(R = C2H5, R1 = CH3)
(R = CH3, R1 = H)
a( )16
+
s( )16 (R = CH3, R1 = C2H5)
( )17
s( )17
(R = CH3, R1 = C3H7)
a
Scheme 3. Synthesis of 1H-Pyrazolo[3,4-e][1,4]thiazepin-7-ones, a( )- and s( )9, 13–17. Reagents and conditions: (a) (i) toluene, reflux; (ii) thioglycolic acid (for 15), 2-
mercaptopropanoic acid (for 9, 13, 14), 2-mercaptobutanoic acid (for 16), 2-mercaptopentanoic acid (for 17), toluene, reflux; (iii) MPC.
CO2Et
NHNH2
CO2Et
a
N
N
b
NH2
NH2
EtO
N
N
CN
19
20
18
10a
a (a) i. EtOH abs, reflux, 24 h, 56 %; (b) 12 N HCl, reflux, 12 h, 75 %.
Scheme 4. Synthesis of 1-(2-Methylphenyl)-1H-pyrazol-5-amine (10a). Reagents and conditions: (a) (i) EtOH abs, reflux, 24 h, 56%; (b) 12 N HCl, reflux, 12 h, 75%.

3784
M. Marinozzi et al. / Bioorg. Med. Chem. 21 (2013) 3780–3789
Table 1
Instead, a cellulose-based CSP in combination with a ‘non-stan-
dard’ mobile phase system,27 given the presence of chloroform,
succeeded in the enantioresolution of the four diastereoisomers,
(4R,6R)-9, (4S,6R)-9, (4R,6S)-9 and (4S,6S)-9, which showed an ee
of 87%, (Fig. 3A), 90% (Fig. 3B), 85% (Fig. 3C) and 80% (Fig. 3D),
FXR functional activity for (4S,6S)-, (4R,6S)-, (4R,6R)- and (4S,6R)-3,6-Dimethyl-1-(2-
methylphenyl)–4-(4-phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-
7(6H)-ones [(4S,6S)-9, (4R,6S)-9, (4R,6R)-9 and (4S,6R)-9]
Efficacyb (%)
a
Compound
EC50
(
lM)
(4S,6S)-9
(4R,6S)-9
(4R,6R)-9
(4S,6R)-9
>150
1.4 0.2
>150
respectively
(a = 1.14, RS = 1.89, for (4R,6R)-9 and (4S,6S)-9;
130
5
a
= 1.07, RS = 1.11, for (4R,6S)-9 and (4S,6R)-9). These values clearly
indicate that the enantiomeric purity of the starting 2-mercapto-
propanoic acid maintained nearly unaltered in the course of the
synthetic procedures.
With the aim to explore the effects of modifications at C-3 and
C-6 positions of 9, following the synthetic protocol above
described, 1H-pyrazolo[3,4-e][1,4]thiazepin-7-one derivatives
13–17 were also prepared (Scheme 3). Accordingly, the condensa-
tion reaction between the appropriate 1-(2-methylphenyl)-1H-
pyrazol-5-amine 10 and 4-phenoxybenzaldehyde (11), carried
out in refluxing toluene with concomitant water removal, and
>150
a
Ligand-dependent recruitment of Src-1 peptide assessed by AlphaScreen assay.
hFXR-LBD-GST was incubated with increasing concentrations of the indicated
ligand in the presence of biotinylated Src-1 peptide. The AlphaScreen signal
increases when the complex receptor-co-activator is formed.31
b
Efficacy: % of compound effect versus 10 lM of CDCA.
Figure 2. Enantiomeric excess (ee) values of 85% and 90% were
computed for the (S)-isomer and the (R)-isomer, respectively.
Figure 4. Ligand interaction diagrams of the best ranked docking poses of (A) the inactive syn enantiomer (4S,6R)-9 in comparison (B) to the active one (4R,6S)-9. The
interacting residues around the 1H-pyrazolo[3,4-e][1,4]thiazepin-7-one scaffold are completely different due to an opposite orientation of the core of these two ligands in the
FXR LBD (1OSV pdb32 residue numbering).

M. Marinozzi et al. / Bioorg. Med. Chem. 21 (2013) 3780–3789
3785
Table 2
FXR functional activity and calculated binding energies for dihydro-1H-Pyrazolo[3,4-e][1,4]thiazepin-7-ones, 9, 13–17
O
R
S
R1
N
N
O
N
H
9, 13-17
Compound
R
R1
EC50
(l
M)c
Efficacy (%)d
DDG Binding solv. (kcal/mol)e
DDG Binding lipo (kcal/mol)e
s( )9a
s( )13a
s( )14a
15
CH3
H
CH2CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
CH2CH3
CH2CH2CH3
3.0 1.0
>150
2.0 0.1
14
3.0 0.6
>150
150 30
Reference
+6.01
Reference
+8.44
+7.10
+6.23
+1.51
140 50
8
ꢀ5.87
+4.01
16b
35
8
+5.95
+6.64
s( )17a
+9.40
a
The symbol s refers to the racemic couple endowed with syn-disposition of the substituents at C-4 and C-6 positions. The anti-couples resulted in any case less active than
the corresponding syn-couples. Data relative to the anti-couples are not shown.
b
Compound tested as mixture of syn- and anti-couples.
Ligand-dependent recruitment of Src-1 peptide assessed by AlphaScreen assay. hFXR-LBD-GST was incubated with increasing concentrations of the indicated ligand in
c
the presence of biotinylated Src-1 peptide. The AlphaScreen signal increases when the complex receptor–coactivator is formed.31
d
Efficacy: % of compound effect versus 10
l
M of CDCA.
Difference of the binding energies estimated by Prime MM-GBSA calculation using the (4R,6S)-9 values as the reference ones. In particular the
(desolvation) and G binding lipo (lipophylic) contributes are reported.
e
D
G binding solv.
D
the subsequent addition of the appropriate 2-mercaptocarboxylic
acid afforded the title compounds 13–17, obtained as mixture of
two racemic couples (with the exception of the derivative 15 pre-
senting only one stereogenic center). MPC of the crude reaction
mixtures allowed us to obtain the less polar racemic couples
a( )13–17, endowed with an anti disposition of the substituents
and the more polar ones, s( )13–17 showing a syn disposition of
the substituents.23 The separation was not possible for the com-
pound 16 which was therefore tested as a mixture of four
diastereoisomers.
The precursor 5-aminopyrazole 10b, characterized by the pres-
ence of the methyl group at C-3 position, was prepared in high
yield following a known procedure involving the reaction of 3-
aminocrotononitrile with 2-tolylhydrazine in concentrated hydro-
chloric acid.28 1-(2-Methylphenyl)-1H-pyrazol-5-amine (10a) was
synthesized (Scheme 4) by acidic hydrolysis and decarboxylation
from ethyl 5-amino-1-(2-methylphenyl)-1H-pyrazole-3-carboxyl-
ate (18), in turn obtained by the condensation reaction between
ethyl 3-ethoxy-2-cyanoacrylate (19) and 2-tolylhydrazine (20).29
Whereas the condensation between the same hydrazine 20 and
3-oxopentanenitrile (21)30 allowed us to obtain 3-ethyl-substi-
tuted pyrazole 10c (Scheme 5). With regard to 2-mercaptocarbox-
ylic acids, whereas thioglycolic and 2-mercaptopropanoic acids are
commercially available, 2-mercaptobutanoic and 2-mercaptopen-
tanoic acids were prepared from the corresponding 2-bromoacids
by treatment with cesium thiobenzoate followed by aminolysis,
according to the procedure followed by us for the preparation of
(S)-and (R)-2-mercaptopropanoic acids [(S)- and (R)-12].25
The data relating to the four diastereoisomers (4S,6S)-, (4R,6S)-,
(4R,6R)- and (4S,6R)-9, reported in Table 1, clearly demonstrated
that the recognition process of the receptor towards our ligands
is highly enantioselective. Only the diastereoisomer endowed with
4R,6S absolute configuration, indeed, was able to activate FXR
receptor with a potency of 1.4 lM and a full efficacy. The absolute
configuration of the active isomer (4R,6S)-9 corresponds to a syn
disposition of the substituents confirming what we have already
observed on all derivatives of this class, with the syn couples
resulting in any case more active than the corresponding anti-
ones.23
With the aim to interpret these biological results, we applied
some computational docking experiments followed by more accu-
rate rescoring estimating the binding energies of the compounds.
As reported in our previous work,23 the pdb used (code: 1OSV)32
in the structure based approach applied to this series of derivatives
was chosen between a set of three published complexes, compris-
ing also the pdb entries 3BEJ33 and 3DCT,17a–c using an ensemble
docking technique. By the docking program present in the Schro-
dinger Suite 2012,34 a Glide 5.5 run in single precision mode (Glide-
Score SP) was instrumental to collect the ten best ranked poses for
each molecule. The docking experiments carried out on the active
derivative (4R,6S)-9, and its enantiomer (4S,6R)-9, highlighted
their different putative binding pose in the LBD of FXR. The best
ranked solutions of each compound are presented in the Ligand
interaction diagrams pictures (Fig. 4) maintaining fixed the orien-
tation of the bicyclic core of the molecules (3D-docking images are
available in the Supplementary data). It’s noteworthy that the
interacting residues around the 1H-pyrazolo[3,4-e][1,4]thiazepin-
7-one region are completely different, thus indicating an opposite
orientation of the core of the two enantiomers in the binding site.
By comparing the binding energies calculated using the Prime MM-
GBSA34 protocol on all the ten collected poses, and analyzing the
energetically best solutions, we estimated a loss of lipophilic
3. Results and discussion
The ability of the synthesized compounds to act as FXR agonists
was evaluated by ligand-dependent recruitment of steroid receptor
coactivator-1 (SRC-1) peptide assessed by AlphaScreen assay.31

3786
M. Marinozzi et al. / Bioorg. Med. Chem. 21 (2013) 3780–3789
contacts (
in terms of desolvation energy (
D
G binding lipo) of 5.20 kcal/mol and of 11.57 kcal/mol
G binding solv)35 for the inactive
tives of 9, modified at C-3 and C-6 positions, has been also pre-
D
pared and evaluated. The presence of a methyl or ethyl group at
C-3 position of the core is essential for the activity of the corre-
sponding compounds, whereas the methyl group is optimal at C-
6 position, as the unsubstituted 15, and bulkier alkyl-substituted
derivatives 16 and 17 resulted less active then the reference com-
pound 9. The computational studies confirmed the observed bio-
logical results, giving an estimation of the binding energies of the
putative binding poses, useful to understand the mechanism of ac-
tion of these compounds in stabilizing the AF-2 helix region in a
bioactive conformation able to recruit the co-activator. The new in-
sights gained in this work will be instrumental to further optimize
this series of non-steroidal FXR agonists.
(4S,6R)-9 in comparison to the active (4R,6S)-9. Moreover, it is
important to note that (4S,6R)-9 was unable to reproduce the ori-
entation of its enantiomer (4R,6S)-9 in all the ten poses analyzed.
These results are in line with the results of the biological evalua-
tions and confirms the crucial role of the hydrophobic interactions
in the LBD of FXR.
As evidenced from the data reported in Table 2, the presence of
a methyl- or an ethyl group [s( )14] at C-3 position of the pyrazole
seems to be essential for the activity of the corresponding com-
pounds as the unsubstituted derivative s( )13 resulted inactive.
An hydrophobic cleft in the ligand binding site, defined by
PHE284, THR288, TRP454 and PHE461 becomes occupied by these
alkyl groups at C-3 position, giving stabilizing Van der Waals inter-
actions to the secondary structures, mainly AF-2 helix (residue
range 461–468) and helix 3, that are directly involved in the co-
activator recruitment process. Moreover, from a conformational
point of view, the substituent at C-3 position seems to be instru-
mental to allow the correct orientation of the phenoxyphenyl moi-
ety inside the receptor. By the aim of the computational studies,
the sum of the estimated binding energies reported a loss of
1.23 kcal/mol for the (4R,6S)-14 and of 14.45 kcal/mol for the
(4R,6S)-13 with respect to the (4R,6S) 9 (Table 2). Also the substi-
tuent at C-6 position of the seven-members ring has a significant
influence on the activity of the corresponding compounds. The
derivative 15, not substituted at this position, indeed, showed an
almost complete loss of potency in comparison to the reference
compound s( )9. The replacement of the methyl group of s( )9
by ethyl one, as in s( )16, was more or less neutral in terms of
EC50, whereas resulted in a substantial decrease in efficacy. Also
in this case the computational studies highlighted an overall loss
of interaction energies of 10.24 kcal/mol and 7.46 kcal/mol for
(4R)-15 and (4R,6S)-16 respectively, using the (4R,6S)-9 as refer-
ence (Table 2). The introduction of bulkier alkyl group, such as n-
propyl, resulted in the inactive derivative s( )17. Also in this case,
we observed from the docking studies, a modification of the orien-
tation of both the aryl moieties of the molecules inside the recep-
tor, changing the interaction networks displayed by the most
active compound s( )9 that are plausible to explain the loss of po-
tency (results shown in the Supplementary data). Indeed the
(4R,6S)-17 showed a gain in the interaction energies of 2.76 kcal/
mol with respect to (4R,6S)-9 compound (Table 2), but displayed
a loss of the key interactions with PHE284 and TRP454, the above
cited two residues localized in the region responsible for the co-
activator recruitment.
5. Experimental section
5.1. Chemistry
Commercially available starting materials, reagents, and sol-
vents were used as supplied. MPC was performed on Merck LiChro-
prep Si 60 Lobar columns. Flash chromatography was performed
on Merck silica gel (0.040–0.063 mm). 1H NMR spectra were re-
corded on a Bruker AC400 as solutions in CDCl3. Chemical shifts
were recorded in ppm (d) downfield of tetramethylsilane. The spin
multiplicities are indicated by the symbols s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), and bs (broad). Melting points
were determined by the capillary method on a Büchi 535 electro-
thermal apparatus and are uncorrected. All target compounds pos-
sessed acceptable purity as verified by HPLC (see Supporting
information).
5.1.1. General procedure for the synthesis of 1H-Pyrazolo[3,4-
e][1,4]thiazepin-7-ones 9, 13–17
The appropriate 1-(2-methylphenyl)-1H-pyrazol-5-amine 10a–
c (1.0 mmol) and 4-phenoxybenzaldehyde (11, 1.0 mmol) were
heated under reflux in toluene (50 mL) for 7–9 h; during this per-
iod produced water was removed with the aid of a Dean–Stark
apparatus. The reaction mixture was cooled, the proper 2-mercap-
tocarboxylic acid (1.0 mmol) was added and the mixture heated
again at reflux for 12 hours. The solvent was removed in vacuo
and the residue submitted to MPC. Elution by light petroleum–
ethyl acetate mixtures afforded the desired compounds.
5.1.2. (4S,6S)-3,6-Dimethyl-1-(2-methylphenyl)-4-(4-
phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-
7-one [(4S,6S)-9]
Light petroleum/ethyl acetate (80:20), yield 32%, white solid,
4. Conclusion
mp 93–95 °C; ½a 2D0
ꢁ
ꢀ18.5° (c 0.50, CH2Cl2); ee = 80% (HPLC); 1H
NMR (CDCl3, 400 MHz) d = 0.96 (d, J = 7.2 Hz, 3H, 6-CH3), 1.97 (s,
3H, 2-CH3), 2.14 (s, 3H, 3-CH3), 3.22 (q, J = 7.2 Hz, 1H, 6-CH), 5.15
(s, 1H, 4-CH), 6.95–7.50 (m, 14H, aromatics and NH); 13C NMR
(CDCl3, 100 MHz) d = 12.72, 16.34, 17.251, 36.23, 42.18, 105.04,
118.39, 119.27, 123.70, 127.51, 128.51, 129.29, 129.82, 130.58,
131.73, 135.10, 136.20, 136.38, 136.96, 147.68, 156.57, 156.86,
172.12; Anal. calcd for C27H25N3O2S: C, 71.18; H, 5.53; N, 9.22; O,
7.02; S, 7.04, found: C, 71.20; H, 5.54; N, 9.21; S, 7.02.
In conclusion, the synthesis and the biological evaluation of the
four possible diastereoisomers of the FXR full agonist 3,6-di-
methyl-1-(2-methylphenyl)-4-(4-phenoxyphenyl)-4,8-dihydro-
1H-pyrazolo[3,4-e][1,4]thiazepin-7-one (9) allowed us to demon-
strate that the receptor recognition is highly enantioselective for
this class of agonists, as only (4R,6S)-9 exhibited low micromolar
range of potency and full efficacy, whereas the other three diaste-
reoisomers resulted completely inactive. (4S,6S)-, (4R,6S)-, (4R,6R)-
and (4S,6R)-9 represent the first examples of enantiomerically pure
4,6-disubstituted 4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7-
ones, here prepared by the asymmetric version of a known multi-
component reaction using both optically pure 2-mercaptopropa-
noic acids. Moreover, a novel HPLC method for determining the
enantiomeric excesses of these acids has been reported as a valid
alternative to the known NMR one. With the aim to enlarge the
SAR of this class of non-steroidal FXR agonists a series of deriva-
5.1.3. (4R,6S)-4-(4-Phenoxyphenyl)-3,6-dimethyl-1-(2-
methylphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7-
one [(4R,6S)-9]
Light petroleum/ethyl acetate (80:20), yield 37%, white solid,
mp 152–155 °C; ½a D20
ꢁ
+43.3° (c 0.65, CH2Cl2); ee = 85% (HPLC); 1H
NMR (CDCl3, 400 MHz) d = 1.46 (d, J = 7.2 Hz, 3H, 6-CH3), 1.90 (s,
3H, 2-CH3), 2.10 (s, 3H, 3-CH3), 3.81 (q, J = 7.2 Hz, 1H, 6-CH), 5.63
(s, 1H, 4-CH), 6.95–7.41 (m, 14H, aromatics and NH); 13C NMR

M. Marinozzi et al. / Bioorg. Med. Chem. 21 (2013) 3780–3789
3787
(CDCl3, 100 MHz) d = 13.34, 15.24, 17.21, 39.00, 44.91, 108.47,
118.76, 119.14, 123.56, 127.29, 128.25, 129.59, 129.78, 130.32,
131.54, 133.72, 135.30, 135.46, 136.60, 148.09, 156.71, 157.08,
172.67; Anal. calcd for C27H25N3O2S: C, 71.18; H, 5.53; N, 9.22; O,
7.02; S, 7.04, found: C, 71.19; H, 5.52; N, 9.20; S, 7.01.
134.21, 135.17, 135.73, 136.69, 152.91, 156.74, 157.00, 172.97;
Anal. calcd for C28H27N3O2S: C, 71.61; H, 5.80; N, 8.95; O, 6.81; S,
6.83, found: C, 71.62; H, 5.81; N, 8.93; S, 6.81.
5.1.10. ( )-3-Methyl-1-(2-methylphenyl)-4-(4-phenoxyphenyl)-
4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7-one [( )15]
Light petroleum/ethyl acetate (80:20), yield 44%, white solid,
mp 172–173 °C; 1H NMR (400 MHz, CDCl3) 2.00 (s, 3H, 2-CH3),
2.20 (s, 3H, 3-CH3), 3.33 (d, J = 15.4 Hz, 1H, 6-CHa), 3.40 (d,
J = 15.4 Hz, 1H, 6-CHb), 5.31 (s, 1H, 4-CH), 7.03–7.45 (m, 14H, aro-
matics and –NH); 13C NMR (100 MHz, CDCl3) 12.77, 17.21, 31.72,
42.47, 105.83, 118.46, 119.34, 132.73, 127.50, 128.44, 129.46,
129.82, 130.59, 131.72, 135.11, 135.28, 135.54, 136.93, 147.86,
156.52, 157.11, 170.10; Anal. calcd for C26H23N3O2S: C, 70.72; H,
5.25; N, 9.52; O, 7.25; S, 7.26, found: C, 70.75; H, 5.33; N, 9.49; S,
7.24.
5.1.4. (4R,6R)-3,6-Dimethyl-1-(2-methylphenyl)-4-(4-
phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-
7-one [(4R,6R)-9]
½
a 2D0
+20.0° (c 0.60, CH2Cl2); ee = 87% (HPLC).
ꢁ
5.1.5. (4S,6R)-3,6-Dimethyl-1-(2-methylphenyl)-4-(4-
phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-
7-one [(4S,6R)-9]
½
a 2D0
ꢁ
ꢀ38.5° (c 0.40, CH2Cl2); ee = 90% (HPLC).
5.1.6. anti-( )-6-Methyl-1-(2-methylphenyl)-4-(4-
phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-
7-one [a( )13]
5.1.11. anti-( )+syn-( )-6-Ethyl-3-methyl-1-(2-methylphenyl)-
4-(4-phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-
Light petroleum/ethyl acetate (80:20), yield 25%, white solid,
mp 189–192 °C; 1H NMR (400 MHz, CDCl3) 1.29 (d, J = 7.2 Hz, 3H,
6-CH3), 2.12 (s, 3H, 2-CH3), 3.50 (q, J = 7.2 Hz,1H, 6-CH), 5.40 (s,
1H, 4-CH), 6.73–7.50 (m, 15H, aromatics and NH); 13C NMR
(100 MHz, CDCl3) 15.61, 17.27, 36.73, 41.98, 108.71, 118.49,
119.27, 123.68, 127.40, 128.12, 129.64, 129.83, 130.56, 131.71,
135.40, 136.46, 136.67, 140.00, 156.65, 157.06, 172.37; Anal. calcd
for C26H23N3O2S: C, 70.72; H, 5.25; N, 9.52; O, 7.25; S, 7.26, found:
C, 70.78; H, 5.28; N, 9.510; S, 7.22.
e][1,4]thiazepin-7-one [a( )16+s( )-16]
Light petroleum/ethyl acetate (80:20), yield 41%, white solid; 1H
NMR (400 MHz, CDCl3) 0.60 (t, J = 7.3 Hz, 3H, CH2CH3, anti), 1.13 (t,
J = 7.3 Hz, 3H, CH2CH3, syn), 1.46–1.52 (m, 2H, CH2CH3, syn), 1.66–
1.74 (m, 2H, CH2CH3, anti), 1.91 (s, 3H, 2-CH3, anti), 1.98 (s, 3H, 3-
CH3, anti), 2.10 (s, 3H, 2-CH3, syn), 2.15 (s, 3H, 3-CH3, syn), 2.92 (m,
1H, 6-CH, anti), 3.56 (t, J = 7.3 Hz, 1H, 6-CH, syn), 5.23 (s, 1H, 4-CH,
anti), 5.78 (s, 1H, 4-CH, syn), 6.70–7.53 (m, 28H, aromatics and –
NH, anti + syn); 13C NMR (100 MHz, CDCl3) 10.37, 11.92, 12.81,
13.34, 14.14, 17.24, 22.17, 22.52, 42.20, 43.50, 45.01, 46.05,
104.94, 108.53, 118.63, 118.74, 118.87, 119.09, 123.50, 127.24,
127.43, 128.23, 128.47, 129.46, 129.59, 129.75, 130.20, 130.42,
131.47, 131.65, 133.96, 135.37, 136.27, 136.48, 136.72, 147.72,
148.08, 156.67, 157.01, 171.97, 172.12; Anal. calcd for
5.1.7. syn-( )-6-Methyl-1-(2-methylphenyl)-4-(4-
phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-
7-one [s( )13]
Light petroleum/ethyl acetate (80:20), yield 10%, white solid,
mp 179–182 °C; 1H NMR (400 MHz, CDCl3) 1.49 (d, J = 7.2 Hz, 3H,
6-CH3), 2.07 (s, 3H, 2-CH3), 3.72 (q, J = 7.2 Hz,1H, 6-CH), 5.58 (s,
1H, 4-CH), 6.73–7.50 (m, 15H, aromatics and NH); 13C NMR
(100 MHz, CDCl3) 15.79, 17.19, 40.43, 45.87, 110.38, 118.69,
119.25, 123.61, 127.34, 128.16, 129.77, 130.51, 131.65, 134.07,
134.77, 135.41, 136.75, 140.26, 156.64, 157.35, 172.04; Anal. calcd
for C26H23N3O2S: C, 70.72; H, 5.25; N, 9.52; O, 7.25; S, 7.26, found:
C, 70.73; H, 5.26; N, 9.53; S, 7.25.
C28H27N3O2S: C, 71.61; H, 5.80; N, 8.95; O, 6.81; S, 6.83, found: C,
71.64; H, 5.83; N, 8.91; S, 6.81.
5.1.12. anti-( )-6-nPropyl-3-methyl-1-(2-methylphenyl)-4-(4-
phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-
7-one [a( )17]
Light petroleum/ethyl acetate (80:20), yield 21%, white solid;
mp 168–170 °C; 1H NMR (400 MHz, CDCl3) 0.99 (t, J = 7.1 Hz, 3H,
CH2CH3), 1.67–1.54 (m, 4H, CH2CH2), 1.91 (s, 3H, 2-CH3), 2.10 (s,
3H, 3-CH3), 3.64 (t, J = 6.8 Hz, 1H, 6-CH), 5.65 (s, 1H, 4-CH), 6.78
(s, 1H, NH), 6.98–7.47 (m, 13H, aromatics); 13C NMR (100 MHz,
CDCl3,) 13.38, 13.87, 17.19, 20.50, 31.17, 44.18, 45.11, 108.56,
118.80, 119.14, 123.55, 127.33, 128.26, 129.62, 129.79, 130.30,
131.55, 133.92, 135.37, 135.62, 136.53, 148.16, 157.08, 172.17;
Anal. calcd for C29H29N3O2S: C, 72.02; H, 6.04; N, 8.69; O, 6.62; S,
6.63, found: C, 72.04; H, 6.06; N, 8.68; S, 6.61.
5.1.8. anti-( )-3-Ethyl-6-methyl-1-(2-methylphenyl)-4-(4-
phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-
7-one [a( )14]
Light petroleum/ethyl acetate (80:20), yield 17%, white solid,
mp 163–175 °C; 1H NMR (400 MHz, CDCl3) 1.16 (t, J = 7.5 Hz, 3H,
CH2CH3), 1.30 (d, J = 7.2 Hz, 3H, 6-CH3), 2.14 (s, 3H, 2-CH3), 2.31
(m, 2H, CH2CH3), 3.23 (q, J = 7.2 Hz, 1H, 6-CH), 5.22 (s, 1H, 4-CH),
6.98–7.47 (m, 14H, aromatics and NH); 13C NMR (100 MHz, CDCl3)
12.09, 16.33, 17.24, 20.38, 36.13, 42.04, 104.29, 118.30, 119.24,
123.66, 127.47, 128.48, 129.30, 129.80, 130.45, 131.74, 135.37,
135.96, 136.78, 137.11, 152.41, 156.58, 156.81, 172.07; Anal. calcd
for C28H27N3O2S: C, 71.61; H, 5.80; N, 8.95; O, 6.81; S, 6.83, found:
C, 71.63; H, 5.81; N, 8.94; S, 6.80.
5.1.13. syn-( )-6-nPropyl-3-methyl-1-(2-methylphenyl)-4-(4-
phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-
7-one [s( )17]
Light petroleum/ethyl acetate (80:20), yield 23%, white solid;
mp 163–165 °C; 1H NMR (400 MHz, CDCl3) 0.66 (t, J = 7.3 Hz, 3H,
CH2CH3), 0.79–0.88 (m, 1H, CH2CHaCH3), 1.21–1.28 (m, 1H,
CH2CHbCH3), 1.46–1.52 (m, 1H, CHaCH2CH3), 1.83–1.93 (m, 1H,
CHbCH2CH3), 1.99 (s, 3H, 2-CH3), 2.16 (s, 3H, 3-CH3), 3.04 (t,
J = 6.8 Hz, 1H, 6-CH), 5.22 (s, 1H, 4-CH), 6.78 (s, 1H, NH), 6.97–
7.49 (m, 13H, aromatics); 13C NMR (100 MHz, CDCl3,) 12.81,
13.36, 17.28, 18.93, 31.01, 41.47, 42.27, 105.04, 118.85, 123.48,
127.46, 128.34, 128.54, 129.51, 129.80, 130.06, 130.49, 131.69,
133.36, 135.36, 136.31, 136.81, 147.77, 157.04, 172.26; Anal. calcd
for C29H29N3O2S: C, 72.02; H, 6.04; N, 8.69; O, 6.62; S, 6.63, found:
C, 72.03; H, 6.06; N, 8.67; S, 6.62.
5.1.9. syn-( )-3-Ethyl-6-methyl-1-(2-methylphenyl)-4-(4-
phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-
7-one [s( )14]
Light petroleum/ethyl acetate (80:20), yield 27%, white solid,
mp 172–175 °C; 1H NMR (400 MHz, CDCl3) 1.12 (t, J = 7.5 Hz, 3H,
CH2CH3), 1.47 (d, J = 7.2 Hz, 3H, 6-CH3), 2.07 (s, 3H, 2-CH3), 2.27
(m, 2H, CH2CH3), 3.79 (q, J = 7.2 Hz, 1H, 6-CH), 5.71 (s, 1H, 4-CH),
6.98–7.47 (m, 14H, aromatics and NH); 13C NMR (100 MHz, CDCl3)
12.20, 15.11, 17.22, 20.82, 38.82, 44.57, 108.05, 118.75, 119.09,
123.51, 127.18, 128.22, 129.53, 129.75, 129.91, 130.14, 131.49,

3788
M. Marinozzi et al. / Bioorg. Med. Chem. 21 (2013) 3780–3789
5.2. HPLC analysis
Acknowledgments
The analytical HPLC measurements were made on a Shimadzu
(Kyoto, Japan) LC-20A Prominence, equipped with a CBM-20A
communication bus module, two LC-20AD dual piston pumps, a
SPD-M20A photodiode array detector, and a Rheodyne 7725i injec-
We gratefully acknowledge the financial support of Intercept
Pharmaceuticals, New York, USA. We thank Professor Wolfgang
Lindner (University of Vienna, Austria) for the generous gift of
the Chiralpak QN-AX column used in this work.
tor (Rheodyne Inc., Cotati, CA, USA) with a 20 lL stainless steel
loop. A LC solution software from Shimadzu allowed the manipula-
tion of the chromatographic data. Column temperature was con-
trolled through a Grace (Sedriano, Italy) heater/chiller (Model
7956R) thermostat.
The chiral stationary phase employed for the enantioseparation
of S-trityl-2-mercaptopropanoic acids was a prototype of the com-
mercially available Chiralpak QN-AX column (from Chiral Technol-
Supplementary data
Supplementary data (experimental procedures and character-
izations for 2-mercaptobutanoic- and 2-mercaptopentanoic acids.
HPLC estimation of the sample purity of 1H-pyrazolo[3,4-
e][1,4]thiazepin-7-ones described in the paper. 3-D Docking
images of the enantiomers (4S,6R)-9 and (4R,6S)-9. Ligand interac-
tion diagrams of (4R,6S)-13, (4R,6S)-14, (4R)-15, (4R,6S)-16 and
(4R,6S)-17) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2013.04.038.
ogies Europe, Illkirch, France) (120 Å pore size, 5 lm particle
diameter and column dimension of 150 ꢂ 4 mm I.D.) which was
kindly provided by Professor Wolfgang Lindner. Instead, the col-
umn employed for the enantioseparation of 9 was the commer-
cially available Chiralpak IB (from Chiral Technologies, West
Chester, PA, USA) (250 mm ꢂ 4.6 mm I.D., containing cellulose
References and notes
1. Wang, W.; Chen, J.; Hollister, K.; Sowers, L. C.; Forman, B. M. Mol. Cell 1999, 3,
543.
2. Parks, D. J.; Blanchard, S. G.; Bledsoe, R. K.; Chandra, G.; Consler, T. G.; Kliewer,
S. A.; Stimmel, J. B.; Willson, T. M.; Zavacki, A. M.; Moore, D. D.; Lehmann, J. M.
Science 1999, 284, 1365.
3. Makashima, M.; Okamoto, A. Y.; Repa, J. J.; Tu, H.; Learned, R. M.; Luk, A.; Hull,
M. V.; Lustig, K. D.; Mangelsdorf, D. J.; Shan, B. Science 1999, 284, 1362.
4. (a) Sinal, C. J.; Tohkin, M.; Miyata, M.; Ward, J. M.; Lambert, G.; Gonzales, F. J.
Cell 2000, 102, 731; (b) Lu, T. T.; Makishima, M.; Repa, J. J.; Schoonjans, K.; Kerr,
T. A.; Auwerx, J.; Mangelsdorf, D. J. Mol. Cell 2000, 6, 507.
5. (a) Landrier, J. F.; Grober, J.; Zaghini, I.; Besnard, P. Mol. Cell. Biochem. 2002, 239,
149; (b) Campana, G.; Pasini, P.; Roda, A.; Spampinato, S. Biochem. Pharmacol.
2005, 69, 1755.
6. Yu, L.; Lo, J. L.; Huang, L.; Zhao, A.; Metzger, E.; Adams, A.; Meinke, P. T.; Wright,
S. D.; Crui, J. J. Biol. Chem. 2002, 277, 31441.
7. Stayrook, K. R.; Bramlett, K. S.; Savkur, R. S.; Ficorilli, J.; Cook, T.; Christe, M. E.;
Michael, L. F.; Burris, T. P. Endocrinology 2006, 146, 984.
8. Huang, W.; Ma, K.; Zhang, J.; Qatanani, M.; Cuvillier, J.; Liu, J.; Dong, B.; Huang,
X.; Moore, D. D. Science 2006, 312, 233.
9. Inagaki, T.; Moschetta, A.; Lee, Y.-K.; Peng, L.; Zhao, G.; Downes, M.; Yu, R. T.;
Shelton, J. M.; Richardson, J. A.; Repa, J. J.; Mangelsdorf, D. J.; Kliewer, S. A. Proc.
Natl. Acad. Sci. U.S.A. 2006, 103, 3920.
10. Wang, Y.-D.; Chen, W.-D.; Huang, W. Histol. Histopathol. 2008, 23, 621.
11. Fiorucci, S.; Rizzo, G.; Donini, A.; Distrutti, E.; Santucci, L. Trends Mol. Med.
2007, 13, 298.
12. (a) Cariou, B. Diabetes Metab. 2008, 34, 685; (b) Zhang, S.; Wang, J.; Liu, Q.;
Harnish, D. C. J. Hepatol. 2009, 51, 380.
13. Mencarelli, A.; Fiorucci, S. J. Cell. Mol. Med. 2010, 14, 79.
14. For a recent review about FXR ligands, see: Merk, D.; Steinhilber, D.; Schubert-
Zsilavecz, M. Future Med. Chem. 2012, 4, 1015.
15. Pellicciari, R.; Fiorucci, S.; Camaioni, E.; Clerici, C.; Costantino, G.; Maloney, P.
R.; Morelli, A.; Parks, D. J.; Willson, T. M. J. Med. Chem. 2002, 45, 3569.
16. Maloney, P. R.; Parks, D. J.; Haffner, C. D.; Fivush, A. M.; Chandra, G.; Plunket, K.
D.; Creech, K. L.; Moore, L. B.; Wilson, J. G.; Lewis, M. C.; Jones, S. A.; Willson, T.
M. J. Med. Chem. 2000, 43, 2971.
tris(3,5-dimethylphenylcarbamate) immobilized onto a 5
gel).
lm silica
Before the use, all the employed mobile phases were degassed
through sonication. Analytes to be injected were solubilized into
the selected mobile phase. The Chiralpak QN-AX column was con-
ditioned with the selected mobile phase at a 1.0 mL minꢀ1 flow
rate for 20 min, before running the analysis; the Chiralpak IB col-
umn for at least 40 min.
The detection wavelength was always set at 254 nm. Unless
otherwise stated, all the analyses were carried out at
a
0.7 mL minꢀ1 eluent flow rate, while the column temperature fixed
at 25 °C.
All the following chromatographic parameters were calculated
according to the German Pharmacopoeia (DAB). The retention fac-
tor (k) values were computed by taking the retention time (tR) at
the peak maximum. Enantioseparation factor (a), resolution factor
(RS) were computed from the following Equations 1 and 2:
k2
k1
a
¼
ð1Þ
ð2Þ
tR ꢀ tRp
RS ¼ 1:18
W0:5 þ Wp0:5
where k1 is the retention factor of the first eluted enantiomer, k2 is
the retention factor of the second eluted enantiomer.
17. (a) Akwabi-Ameyaw, A.; Bass, J. Y.; Caldwell, R. D.; Caravella, J. A.; Chen, L.;
Creech, K. L.; Deaton, D. N.; Jones, S. A.; Kaldor, I.; Liu, Y.; Madauss, K. P.; Marr,
H. B.; McFadyen, R. B.; Miller, A. B.; Navas, F., III; Parks, D. J.; Spearing, P. K.;
Todd, D.; Williams, S. P.; Wisely, G. B. Bioorg. Med. Chem. Lett. 2008, 18, 4339;
(b) Akwabi-Ameyaw, A.; Bass, J. Y.; Caldwell, R. D.; Caravella, J. A.; Chen, L.;
Creech, K. L.; Deaton, D. N.; Madauss, K. P.; Marr, H. B.; McFadyen, R. B.; Miller,
A. B.; Navas, F., III; Parks, D. J.; Spearing, P. K.; Todd, D.; Williams, S. P.; Wisely,
G. B. Bioorg. Med. Chem. Lett. 2009, 19, 4733; (c) Abel, U.; Schlüter, T.; Schulz, A.;
5.3. Docking and modeling studies
A structure based procedure was applied to discover and study
the putative binding mode of the most potent compounds in the
series. The pdb used (code: 1OSV32) was firstly identified in our
previous work23 between a set of three published complexes com-
prising also the pdb entries 3BEJ33 and 3DCT17a–c using an ensem-
ble docking technique. All the PDBs used in this work were
previously submitted to the Protein Preparation Wizard protocol
of the Schrödinger Suite. All the compounds were prepared by the
aim of the LigPrep protocol present in the Schrödinger Suite
2012.34 The grid generation and all the docking runs were per-
formed leaving all the variables at default values in the Glide
5.534 program used in single precision docking mode (SP) and sav-
ing up to 10 best poses per compound. The obtained results were
further submitted to the Prime MM-GBSA34 protocol to estimate
the Free Binding Energy of the ligands using a minimization shell
of 8 Å from the center of mass of the binder.
´
Hambruch, E.; Steeneck, C.; Hornberger, M.; Hoffmann, T.; Perovic-Ottstadt, S.;
Kinzel, O.; Burnet, M.; Deuschle, U.; Kremoser, C. Bioorg. Med. Chem. Lett. 2010,
20, 4911; (d) Feng, S.; Yang, M.; Zhang, Z.; Wang, Z.; Hong, D.; Richter, H.;
Benson, G. M.; Bleicher, K.; Grether, U.; Martin, R. E.; Plancher, J.-M.; Kuhn, B.;
Rudolph, M.; Chen, L. Bioorg. Med. Chem. Lett. 2009, 19, 2595; (e) Bass, J. Y.;
Caldwell, R. D.; Caravella, J. A.; Chen, L.; Creech, K. L.; Deaton, D. N.; Madauss, K.
P.; Marr, H. B.; McFadyen, R. B.; Miller, A. B.; Parks, D. J.; Todd, D.; Williams, S.
P.; Wisely, G. B. Bioorg. Med. Chem. Lett. 2009, 19, 2969; (f) Akwabi-Ameyaw, A.;
Caravella, J. A.; Chen, L.; Creech, K. L.; Deaton, D. N.; Madauss, K. P.; Marr, H. B.;
Miller, A. B.; Navas, F., III; Parks, D. J.; Spearing, P. K.; Todd, D.; Williams, S. P.;
Wisely, G. B. Bioorg. Med. Chem. Lett. 2011, 21, 6154.
18. Nicolaou, K. C.; Evans, R. M.; Roecker, A. J.; Hughes, R.; Downes, M.; Pfefferkorn,
J. A. Org. Biomol. Chem. 2003, 1, 908.
19. Richter, H. G. F.; Benson, G. M.; Blum, D.; Chaput, E.; Feng, S.; Gardes, C.;
Grether, U.; Hartman, P.; Kuhn, B.; Martin, R. E.; Plancher, J. M.; Rudolph, M. G.;
Schuler, F.; Taylor, S.; Bleicher, K. H. Bioorg. Med. Chem. Lett. 2011, 21, 191.

M. Marinozzi et al. / Bioorg. Med. Chem. 21 (2013) 3780–3789
3789
20. (a) Flatt, B.; Martin, R.; Wang, T.-L.; Mahaney, P.; Murphy, B.; Gu, X.-H.; Foster,
P.; Li, J.; Pircher, P.; Petrowski, M.; Schulman, I.; Westin, S.; Wrobel, J.; Yan, G.;
Bischoff, E.; Daige, C.; Mohan, R. J. Med. Chem. 2009, 52, 904; (b) Lundquist, J. T.,
IV; Harnish, D. C.; Kim, C. Y.; Mehlmann, J. F.; Unwalla, R. J.; Phipps, K. M.;
Crawley, M. L.; Commons, T.; Green, D. M.; Xu, W.; Hum, W.-T.; Eta, J. E.;
Feingold, I.; Patel, V.; Evans, M. J.; Lai, K.; Borges-Marcucci, L.; Mahaney, P. E.;
Wrobel, J. E. J. Med. Chem. 2010, 53, 1774.
and quinine carbamate-based anion-exchange type chiral stationary phase to
cope with limited chemoselectivity on the latter: A case study illustrated for
simultaneous T3 and T4 enantiomer separation J. Sep. Sci. 2008, 31, 1702–1711;
(c) Sardella, R.; Marinozzi, M.; Ianni, F.; Lisanti, A.; Natalini, B. Simultaneous
diastereo- and enantioseparation of farnesoid X receptor (FXR) agonists with a
quinine carbamate-based chiral stationary phase Anal. Bioanal. Chem. 2013,
405, 847–862.
21. Deng, G.; Li, W.; Shen, J.; Jiang, H.; Chen, K.; Liu, H. Bioorg. Med. Chem. Lett.
2008, 18, 5497.
22. Shuster, D.; Markt, P.; Grienke, U.; Mihaly-Bison, J.; Binder, M.; Noha, S. M.;
Rollinger, J. M.; Stuppner, H.; Bochkov, V. N.; Wolber, G. Bioorg. Med. Chem.
2011, 19, 7168.
27. (a) Natalini, B.; Sardella, R.; Massari, S.; Ianni, F.; Tabarrini, O.; Cecchetti, V.
Talanta 2011, 85, 1392; (b) Gioiello, A.; Sardella, R.; Rosatelli, E.; Sadeghpour, B.
M.; Natalini, B.; Pellicciari, R. Steroids 2012, 77, 250.
28. Nam, N. L.; Grandberg, I. I.; Sorokin, V. I. Chem. Heterocycl. Compd. 2000, 36,
281.
23. Marinozzi, M.; Carotti, A.; Sansone, E.; Macchiarulo, A.; Rosatelli, E.; Sardella,
R.; Natalini, B.; Rizzo, G.; Adorini, L.; Passeri, D.; De Franco, F.; Pruzanski, M.;
Pellicciari, R. Bioorg. Med. Chem. 2012, 20, 3429.
24. (a) Swett, L. R.; Ratajczyk, J. D.; Nordeen, C. W.; Aynilian, G. H. J. Heterocycl.
Chem. 1975, 12, 1137; (b) Joshi, K. C.; Pathak, V. N.; Garg, U. J. Heterocycl. Chem.
1980, 17, 789.
25. Strijtveen, B.; Kellogg, R. M. J. Org. Chem. 1986, 51, 3664.
26. (a) Sardella, R.; Lämmerhofer, M.; Natalini, B.; Lindner, W. Enantioselective
HPLC of potentially CNS-active acidic amino acids with a cinchona carbamate
based chiral stationary phase Chirality 2008, 20, 571–576; (b) Sardella, R.;
Lämmerhofer, M.; Natalini, B.; Lindner, W. In-line coupling of reversed-phase
29. Schmidt, P.; Druey, J. Helv. Chim. Acta 1956, 39, 986.
30. Ji, Y.; Trenkle, W. C.; Vowles, J. V. Org. Lett. 2006, 8, 1161.
31. Rizzo, G.; Passeri, D.; De Franco, F.; Ciaccioli, G.; Donadio, L.; Rizzo, G.; Orlandi,
S.; Sadeghpour, B.; Wang, X. X.; Jiang, T.; Levi, M.; Pruzanski, M.; Adorini, L. Mol.
Pharmacol. 2010, 78, 617.
32. Mi, L. Z.; Devarakonda, S.; Harp, J. M.; Han, Q.; Pellicciari, R.; Willson, T. M.;
Khorasanizadeh, S.; Rastinejad, F. Mol. Cell 2003, 11, 1093.
33. Soisson, S. M.; Parthasarathy, G.; Adams, A. D.; Sahoo, S.; Sitlani, A.; Sparrow,
C.; Cui, J.; Becker, J. W. PNAS 2008, 105, 5337.
34. Schrödinger, LLC., New York, NY, 2009.
35. Li, J.; Abel, R.; Zhu, K.; Cao, Y.; Zhao, S.; Friesner, R. A. Proteins 2011, 79, 2794.