Identification of trisubstituted-pyrazol carboxamide analogs as novel and potent antagonists of farnesoid X receptor

Article abstract of DOI:10.1016/j.bmc.2014.04.014

Farnesoid X receptor (FXR, NRIH4) plays a major role in the control of cholesterol metabolism. This suggests that antagonizing the transcriptional activity of FXR is a potential means to treat cholestasis and related metabolic disorders. Here we describe the synthesis, biological evaluation, and structure-activity relationship (SAR) studies of trisubstituted-pyrazol carboxamides as novel and potent FXR antagonists. One of these novel FXR antagonists, 4j has an IC₅₀ of 7.5 nM in an FXR binding assay and 468.5 nM in a cell-based FXR antagonistic assay. Compound 4j has no detectable FXR agonistic activity or cytotoxicity. Notably, 4j is the most potent FXR antagonist identified to date; it has a promising in vitro profile and could serve as an excellent chemical tool to elucidate the biological function of FXR.

Full text of DOI:10.1016/j.bmc.2014.04.014

Bioorganic & Medicinal Chemistry 22 (2014) 2919–2938
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Identification of trisubstituted-pyrazol carboxamide analogs as novel
and potent antagonists of farnesoid X receptor
Donna D. Yu a, , , Wenwei Lin b, , Barry M. Forman a, , Taosheng Chen b, ,
⇑
⇑
a Department of Diabetes and Metabolic Diseases Research, Beckman Research Institute, City of Hope National Medical Center, Duarte, CA 91010, USA
b Department of Chemical Biology & Therapeutics, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Farnesoid X receptor (FXR, NRIH4) plays a major role in the control of cholesterol metabolism. This sug-
gests that antagonizing the transcriptional activity of FXR is a potential means to treat cholestasis and
related metabolic disorders. Here we describe the synthesis, biological evaluation, and structure–activity
relationship (SAR) studies of trisubstituted-pyrazol carboxamides as novel and potent FXR antagonists.
One of these novel FXR antagonists, 4j has an IC50 of 7.5 nM in an FXR binding assay and 468.5 nM in
a cell-based FXR antagonistic assay. Compound 4j has no detectable FXR agonistic activity or cytotoxicity.
Notably, 4j is the most potent FXR antagonist identified to date; it has a promising in vitro profile and
could serve as an excellent chemical tool to elucidate the biological function of FXR.
Received 3 February 2014
Revised 29 March 2014
Accepted 7 April 2014
Available online 16 April 2014
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
novel, potent and selective modulators as agonists or antagonists
of FXR, both for potential clinical applications, as well as studies
The activation of farnesoid X receptor (FXR, NRIH4),1 in partic-
ular, gives bile acids the ability to modulate genomic signaling
pathways. FXR is a ligand-dependent transcription factor that reg-
ulates gene networks involved in regulating lipid and cholesterol
homeostasis.2 As such, FXR is expressed primarily in tissues
exposed to high concentrations of bile acids, such as the intestine,
kidney, adrenal gland, and liver.3 Bile acids are the purported
endogenous agonists for FXR.3,4 As the bile acid sensor, FXR regu-
lates the expression of transporters and biosynthetic enzymes cru-
cial for the physiological maintenance of bile acid homeostasis.
Because of FXR’s role in bile acid homeostasis, modulating FXR
may be beneficial for treating all aspects of the metabolic syn-
drome, a complex disease cluster that includes risk factors such
as dyslipidemia, insulin-resistance, increased blood pressure, vis-
ceral obesity and hypercoagubility.5 Recent findings also suggest
that FXR acts as a key metabolic regulator in the liver to maintain
the homeostasis of liver metabolites.6 FXR ligands have been inves-
tigated in preclinical studies for targeted therapy of metabolic dis-
eases, but have shown limitations.7 There is, therefore, a need for
to better understand FXR’s biological functions.
Several potent, selective FXR agonists have been reported
recently, such as GW40648 and 6
a-ethylchenodeoxycholic acid
(6-ECDCA).9 However, preclinical development of FXR agonists is
limited by the complex response, including possible undesirable
effects, triggered by activation of FXR in the liver. For example,
FXR activation inhibits bile acid synthesis and basolateral efflux
of bile through indirect downregulation of cholesterol 7 alpha-
hydroxylase, or cytochrome P450 7A1 (CYP7A1), the rate-limiting
enzyme of the bile acid synthesis pathway.10 In addition, although
activation of FXR would reduce triglyceride levels in hypertriglyc-
eridemic patients, it would also decrease the levels of high density
lipoprotein and lead to accumulation of cholesterol as a conse-
quence of the inhibited bile acid synthesis. Furthermore, FXR ago-
nists interfere with the ability of constitutive androstane receptor
to regulate basolateral transporters in hepatocytes.11 These effects
might worsen liver injury in a subset of patients who have obstruc-
tive cholestasis (a severe liver disease that impairs bile flow and
causes irreversible liver damage), thereby limiting the preclinical
development and possible clinical use of FXR agonists. In contrast,
FXR antagonists might be useful for understanding the function of
FXR and ultimately for treating liver disorders, if they targeted a
specific cluster of genes and thus avoided the detrimental side
effects mediated by FXR agonists. Treatment with FXR antagonists
could potentially upregulate CYP7A1 expression and decrease
overall cholesterol concentrations. This activity would occur
through FXR’s regulation of the expression of small heterodimer
Abbreviations: HTS, high-throughput screen; FXR, farnesoid X receptor; NRs,
nuclear receptors; Cyp7A1, cytochrome 7A1; BAs, bile acids; BSEP, bile salt export
pump; SAR, structure–activity relationship; LBD, ligand-binding domain.
⇑
Corresponding authors. Tel.: +1 (626) 359 8111x65993; fax: +1 (626) 256 8704
(D.D.Y.); tel.: +1 (901) 595 5937 (T.C.).
E-mail addresses: dyu@coh.org (D.D. Yu), taosheng.chen@stjude.org (T. Chen).
These authors contributed equally.
http://dx.doi.org/10.1016/j.bmc.2014.04.014
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

2920
D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
partner.2 In addition, FXR can promote expression of an intestinal
bile acid-binding protein (I-BABP) gene.12 When expression of I-
BABP is repressed by an antagonist of FXR, re-absorption of bile
acids in the ileum is repressed, which may reduce the amount of
bile acid that returns to the liver, and, thereby promote the expres-
sion of CYP7A1. Thus, an FXR antagonist could potentially induce
repression of I-BABP expression in the ileum, leading to reduced
levels of serum cholesterol, and suggesting potential as a therapeu-
tic agent for hypercholesterolemia in humans. Therefore, an FXR-
specific antagonist could be used to validate the clinical relevance
of antagonizing FXR.
Despite the potential of FXR as a therapeutic target for meta-
bolic diseases, few selective FXR antagonists (synthetic small mol-
ecules or natural products) are described in the literature. Recently,
the natural product guggulsterone was identified as the first puta-
tive FXR antagonist.13,14 However, the mechanism by which it
antagonizes FXR is unclear. It appears that rather than being a true
antagonist of FXR, guggulsterone is a unique FXR ligand that has
antagonistic activity in coactivator association assays and can
enhance the action of FXR agonists in vivo.15
compounds.24a,b The pyrazole heterocycle has long been consid-
ered a privileged scaffold in drug discovery on the basis of its
wide-ranging biological activities, including modulating G-protein
coupled receptor modulators, inhibiting cyclooxygenase-2, and
p38 MAP kinase.25 A challenge in investigating pyrazole heterocy-
cles as potential FXR antagonists is selecting a structural motif to
serve as the scaffold of a focused library, given the diversity of
structures that can be optimized to develop more potent and selec-
tive FXR antagonists. Therefore we started by assessing the antag-
onistic effect of
a collection of known substituted pyrazole
carboxamides (Enamine Small Molecule Compound Libraries)
against FXR, and identified E16, a 1,3-disubstituted-pyrazole-4-
carboxamide as a potent FXR antagonist. We then used E16 as
the template for further structural optimization, which led to sev-
eral novel compounds. These compounds were exemplified by 1-
(3-methoxybenzyl)-3-(3-methoxyphenyl)-N-(4-methyl-3-(mor-
pholinosulfonyl) phenyl)-1H-pyrazole-4-carboxamide) 4j, which
had significantly improved FXR antagonistic activities as compared
to E16. To our knowledge, 4j is the most potent FXR antagonist to
date. Our efforts in designing structurally novel, nonsteroidal FXR
antagonists by modifying pyrazole carboxamides may represent
an important direction in developing novel FXR antagonists.
In addition to guggulsterone, several marine products, such as
sulfated polyhydroxy sterols isolated from marine invertebrates,
have been identified as putative FXR modulators.16 This is exempli-
fied by sulfated sterol. Theonellasterol has also been reported as an
FXR antagonist. In rodent models, theonellasterol attenuated both
liver injury and the extent of liver necrosis caused by bile duct liga-
tion.17 In addition, several scalarane sesterterpenes, isolated from a
marine sponge, inhibited FXR with IC50 values between 2.4 and
2. Results and discussion
2.1. Identification of a substituted pyrazole-4-carboxamide E16
as a template for designing novel FXR antagonists
24 l
M.18 Most recently, suvanine, a marine sponge sesterterpene,
We began by evaluating the FXR antagonistic activities of
known or commercially available substituted pyrazole carboxam-
ides. We first evaluated 16 commercially available substituted pyr-
azole carboxamides (Enamine Small Molecule Compound
Libraries) by FXR biochemical TR-FRET binding, FXR cell-based
agonistic and antagonistic, and cytotoxicity assays (Tables 1 and
2). Among the carboxamide analogs, E16 (Table 1), a 1,3,4-trisub-
stituted-pyrazole carboxamide analog, had the highest FXR bind-
ing affinity (IC50 = 47 nM) and was a potent FXR antagonist
was reported by Di Leva et al. as an FXR antagonist.19 Using suva-
nine as a template, Di Leva et al. developed suvanine derivative as a
novel FXR antagonist (Fig. 1).
The nonsteroidal antagonist AGN34 displayed an interesting
profile of antagonist properties of the RXR/FXR heterodimer acting
at RXR. AGN43 antagonizes FXR in vitro reporter assays but acts in
a gene-selective manner in vivo.20 Interestingly, the nonsteroidal
compound1,3,4-trisubstituted-pyrazolone derivative was recently
identified as an FXR antagonist (Fig. 1).21 This compound strongly
suppresses the regulating effects of CDCA on FXR target genes.
These findings further suggest the feasibility of identifying FXR
antagonists that regulate a specific subset of FXR responsive genes
in a gene-specific fashion.22
(IC50 = 2.62 lM) (Table 2 and Fig. 2). None of the substituted pyra-
zole carboxamides tested displayed FXR agonistic activity (data not
shown) or significant cytotoxicity (<5% of control) (Table 2).
2.2. Design
We recently reported the discovery of an FXR antagonist
through the use of a time-resolved fluorescence resonance energy
transfer (TR-FRET) assay,23 as well as several chemotypes identi-
fied as inhibitors of FXR. These chemotypes are represented by het-
erocyclic amide compounds, including substituted pyrazole
With the novel substituted pyrazole-4-carboxamide framework
in hand, we started to design a series of new trisubstituted-pyra-
zole carboxamide analogs to evaluate their receptor binding affin-
ity and antagonistic potency in modulating the transcriptional
HO
OH
O
NO2
O
H3CO
O
N
N
Scalarane Sesterterpene
1,3,4-Trisubstituted-pyrazolone
Derivative
O
N
Cl
OH
O
O
O
Cl
OH
Cl
Suvanine
GW4064
Figure 1. Chemical structures of examples of FXR ligands.

D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
2921
Table 1
Structures of the 16 substituted pyrazole-4-carboxamides (from Enamine Small Molecule Compound Libraries)
O
O
O
S
NH
O
NH
O
F
NH
O
N
N
N
N
N
N
N
N
O
S
O
S
NH
O
N
N
O
N
O
N O
O
O
O
S
S
S
S
O
O
N
N
N
N
O
O
O
O
F
O
O
E1
E2
E3
E4
O
S
F3C
O
NH
O
NH
O
NH
O
NH
O
N
N
N
N
N
N
N
N
N
O
O
O
S
S
S
O
S
N O
N
N
N
O
O
O
O
O
O
O
F
E5
E6
E7
E8
O
S
O
NH
O
NH
O
NH
O
NH
O
N
N
N
N
N
N
N
N
N
O
S
N
O
O
N
O
O
O
Cl
Cl
E9
E10
E11
E12
H
N
N
N
NH
O
NH
O
NH
O
N
N
N
N
N
N
N
O
S
O
N
O
S
O
O
N
N
O
O
O
O
E13
E14
E15
E16
activity of FXR. Recently, several crystal structures of the FXR-
ligand binding domain (LBD) in complex with agonists were
reported.26 However, no structural information is available on
the binding mode of these FXR antagonists nor are co-crystal struc-
tures of a substituted pyrazole carboxamide framework in complex
with the LBD of FXR available. Therefore, we could not directly
identify the structural feature of the substituted pyrazole carbox-
amide framework that interacts with the ligand-dependent activa-
tion function 2 motif of the FXR LBD.27 However, the flexible nature
of the FXR side chains suggests the FXR LBD may have considerable
flexibility to accommodate ligands of various shapes by changing
its conformation in response to ligand binding.28 Huang et al. pre-
dicted the binding poses of the 1,3,4-substituted-pyrazolone ana-
logs in the FXR ligand binding pocket by means of molecular
docking to address the flexibility of the FXR LBD.21 It is recognized
that the chemical structure of 1,3,4-substituted-pyrazolone is the
most similar to the substituted 1,3,4-substituted-pyrazole, even
though their functions are diverse.
between steric/hydrophobic effects of the 1,3-substituent of the
pyrazolone core seemed to be critical for high FXR antagonistic
potency. These observations suggest that varying the 1,3-substitu-
ents of the pyrazole core could be critical for high antagonistic
potency.
On the basis of the detailed SAR studies for the 16 substituted
pyrazole carboxamides (Tables 1 and 2), the substituted morphol-
inosulfonyl moiety of aniline on the 4-position appeared to be nec-
essary to improve the antagonistic activity against FXR (E16 in
Table 1). To increase the diversity of substituted moieties (R1, R2,
and R3 in Fig. 3) and optimize the 1,3,4-trisubstituted pyrazole-
based molecules to improve their antagonistic activity against
FXR, we investigated the alternative deconstructive hypothesis
for fragment binding.29 Retrospectively, 1,3,4-trisubstituted-pyra-
zole carboxamides can be considered as being formed by two frag-
ments: the substituted-N1-C3-pyrazole core (F1) and the
substituted morpholinosulfonyl aniline moiety chain (F2) (Fig. 3).
On the basis of our preliminary results (Tables 1 and 2) suggest-
ing the substituted N1-C3-pyrazole moiety is sensitive to activity,
various hydrophobic residues, such as alkyl or methoxy residues,
substituted in the R1 or R2 positions of the phenyl-ring might sig-
nificantly improve the antagonistic activity (Tables 1 and 2). We
also determined whether the substituted N1-C3-pyrazole core
(F1) and morpholinosulfonyl aniline (F2) could bind separately to
FXR. We selected three commercially available products, 2-or 4-
methyl-3-(morpholinosulfonyl)aniline and 3-(morpholinosulfo-
nyl)aniline as F2 functionalities for this evaluation. A series of
To understand the structural basis for the SAR, we analyzed the
published binding poses of the 1,3,4-substituted-pyrazolone deriv-
ative (Fig. 1). The binding model showed that the 1,3-substituted
alkyl rings of the pyrazolone core are crucial for the hydrophobic
interactions between the two phenyl rings and the two aromatic
residues (Phe329 and His294), even though the antagonistic
potency of the pyrazolone derivative for FXR is still relatively low
(IC50 = 8.96 3.62
tuted-pyrazolone derivative demonstrated that a subtle interplay
l
M).21 The SAR analysis of the 1,3,4-substi-

2922
D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
Table 2
Biological activities of the 16 substituted pyrazole-4-carboxamides as determined by FXR TR-FRET binding, cell-based FXR antagonistic and cytotoxic assays
Compound
FXR TR-FRET Binding
aIC50
GB FXR Cell Anta.
aIC50
GB FXR Cell Cyto
%Inh.
%Inh.
%Inh.
NT
IC50
NA
E1
21.2 0.6
(164 nM)
70.6 4.2
NA
34.7 1.2
NA
(40
99.0 3.7
(40 M)
27.0 0.3
(40 M)
80.3 0.1
(40 M)
101.1 2.7
(40 M)
18.6 0.4
(40 M)
54.9 2.1
(40 M)
42.6 0.9
(40 M)
82.3 2.6
(40 M)
66.9 3.0
(40 M)
38.8 1.7
(40 M)
20.6 0.3
(40 M)
32.8 1.1
(40 M)
16.1 0.5
(40 M)
24.0 0.9
(40 M)
77.2 2.2
(40 M)
lM)
E2
3.3 0.6
NA
l
l
M
M
11.8 0.6
NA
l
M
47.4 1.4
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
(13.3
l
M)
l
(40
NT
lM)
E3
11.6 0.2
(54.8 nM)
83.8 1.4
l
E4
8.3 0.9
9.4 0.9
11.8 1.1
NA
lM
27.4 1.2
(40 M)
51.3 1.7
(40 M)
7.4 0.3
(40 M)
7.1 0.3
(40 M)
16.3 0.2
(13.3
99.8 6.1
(13.3 M)
l
M)
l
l
E5
996 67 nM
l
M
l
l
l
E6
12.7 0.9
(493 nM)
44.4 1.9
(493 nM)
53.3 0.6
(493 nM)
90.4 4.0
NA
NA
NA
l
l
E7
NA
l
l
E8
NA
l
(40
NT
lM)
E9
1.3 0.2
6.6 0.7
NA
l
l
M
M
10.8 0.4
29.3 1.7
NA
l
M
M
(4.44
85.1 0.9
(13.3 M)
l
M)
l
E10
E11
E12
E13
E14
E15
E16
l
NT
NT
NT
l
l
36.9 1.5
(54.8 nM)
17.8 0.2
(54.8 nM)
27.5 0.9
(493 nM)
18.8 1.2
l
NA
NA
l
NA
NA
5.4 0.2
(40 M)
27.9 0.7
(40 M)
10.3 0.3
(40 M)
17.9 0.5
(40 M)
l
l
NA
NA
(13.3
23.0 0.5
(1.48 M)
l
M)
l
l
NA
NA
l
l
l
100.2 5.2
(164 nM)
47.0 5.1 nM
2.62 0.13 lM
l
l
None of the 16 compounds showed FXR agonistic activity (data not shown).
NA: not applicable.
NT: Not toxic (toxicity less than 5% of control).
GB FXR Cell Anta: GeneBLAzer FXR cell-based antagonistic assay.
GB FXR Cell Cyto: Cytotoxicity assay performed in the GeneBLAzer FXR cells.
%Inh: %Inhibition is presented as the means SE of at least three independent experiments calculated as described in the Section 4. The value shown in the table represents
the maximum percent inhibition observed, and its lowest corresponding compound concentration is noted in the parentheses.
a
IC50 values represent the means SE of at least three independent experiments derived as described in the Section 4.
substituted F1 fragments was prepared (described in Section 4;
120
A
B
compounds 3a–3e). These fragments failed to show an antagonistic
effect against FXR. However, by connecting F1 moieties to F2 moi-
eties through a linker of a single covalent bond by an amide bridge,
the resulting carboxamide compounds had dramatically improved
antagonistic activity against FXR, exemplified by compounds in
series 4 (Fig. 4). This suggested that 1,3,4-trisubstituted-pyrazole
carboxamides can be created as potent antagonists of FXR by link-
ing the F1 and F2 fragments.
Because the F2 fragments have a hydrophobic region centered
on the double hydrogen bond acceptor (sulfone) and had very
weak antagonistic activity against FXR, it may provide considerable
conformational rigidity to the receptor recognition after joining
with F1 fragments.30 Detailed SAR studies indicated that the posi-
tion of the morpholinosulfonyl moiety of the F2 phenyl ring is
important to modifications (Tables 1 and 2). Further analysis of
the essential structural requirements for effective FXR antagonists
showed that the meta-position-substituted morpholinosulfonyl
moiety on the aniline amide moiety would influence the antago-
nistic activity. Therefore, we hypothesized that an appropriate 3-
(morpholinosulfonyl)aniline moiety of the amide moiety needed
to be fixed to act as a specific anchor during FXR binding
recognition.
GW4064
E16
Lithocholic acid
100
80
60
40
20
0
-12 -11 -10 -9
-8
-7
-6
-5
-4
Log [M]
4
3
2
1
0
GW4064
E16
E16 + 400 nM GW4064
Lithocholic acid
Lithocholic acid + 400 nM GW4064
-10
-9
-8
-7
-6
-5
-4
Log [M]
Figure 2. (A) FXR binding activities of GW4064, E16, and lithocholic acid in an FXR
TR-FRET binding assay. (B) FXR agonistic activities (GW4064, E16, or lithocholic
acid alone) and antagonistic activities (E16 and lithocholic acid in the presence of
400 nM GW4064) in a cell-based FXR transactivation assay. GW4064 and litho-
cholic acid were used as agonistic and antagonistic controls, respectively.
Variations of R3 on the F2 fragments could also affect the antag-
onistic potency. For example, introduction of a 4-methyl group on
the F2 fragment resulted in greater antagonistic activity (Tables 1

D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
2923
R3
O
H
N
1
R1
R3
S
4
3
R1
N
HO
NH2
N
N
S
N
O
O
N
O
O
N
+
O
R2
R2
O
O
F2
1,3,4-trisubstituted pyrazole
carboxamides
F1
Figure 3. The retro-analysis of 1,3,4-trisubstituted pyrazole carboxamides.
R1
NH
O
which could act as a template to put substituents into the correct
positions to fit the receptor. A scaffold expansion based on two
diverse points given by the substituents and their positions on
the pyrazole ring, as well as the substituents (R1 and R2) on the
N1,C3-pyrazole rings, was therefore designed (Figs. 5 and 6).
On the basis of the above design, two protocols for preparing
the 1,3,4-trisubstituted-pyrazole carboxamides were developed
to optimize the synthesis of compounds 4. These protocols
included preparing various structural isomers to determine
whether the positions of the R1 and R2 could affect compound
activity. The key intermediates 3 and 7 (Figs. 5 and 6) could be pre-
pared by two possible routes to allow access to final functional
analogs. In the first route, lead optimization was initiated by
exploring the phenyl functionality located on the N1-position. A
series of para- and meta-substitutions on the N1-pyrazole ring
was investigated (Fig. 5). In general procedures for preparing
phenyl hydrazones, synthesis often begins with commercially
available substituted acetophenone 12 and substituted phen-
ylhydrazines 11. Condensation between acetophenone and substi-
tuted phenylhydrazines provides hydrazones 1. A Vilsmeier–Haack
reaction, which is the key reaction for the cyclization of hydra-
zones to pyrazoles, assembles the pyrazole nucleus scaffolds.31
However, these conditions either do not form key aldehydes or
have poor yields. We therefore modified the Vilsmeier–Haack reac-
tion by adding the corresponding phenylhydrazones to an excess
equivalence of POCl3 and DMF at 0 °C, then heated the reaction
mixture to 60–70 °C with stirring over 18 h under N2 to afford
the desired key cyclized aldehydes 2 at a 70–90% yield after hydro-
lysis. Oxidation of the substituted pyrazole-4-carboxaldehydes 2
with a Jones’ reagent32 yielded the corresponding substituted pyr-
azole-4-carboxylic acids 3. Finally, the corresponding acids 3 were
coupled with commercially available 4-methyl-3-(morphol-
inosulfonyl)aniline 13 through a Mitsunobu reaction33 in the pres-
ence of cross-linking reagents (i.e., HOBt and EDCI) to produce the
desired 4. The synthetic pathways used to obtain the 5 analogs of
1,3,4-trisubstituted-1H-pyrazoles 4 are illustrated in Figure 5.
An alternative strategy to explore the SAR by introducing
selected R2 functional groups to investigate the structure require-
ments for FXR antagonistic activity is shown in Figure 6. The intro-
duction of R2 groups was carried out by treating commercially
available b-keto esters 14 with triethyl orthoformate in the pres-
ence of Ac2O as a catalyst to provide the corresponding ethoxym-
ethylene intermediate 5. Cyclization of 5 with hydrazine in dry
ethanol afforded the pyrazole core 6. The N1-alkylation reaction
of 6 with excess appropriate bromoalkyl and bromobenzyl was
performed in the presence of cesium carbonate as the base and
acetonitrile as solvent. The ethyl esters 6 were hydrolyzed under
basic conditions to afford the corresponding carboxylic acid 7. This
was followed by a Mitsunobu coupling with 4-methyl-3-(morphol-
inosulfonyl)aniline in the presence of EDCI/DMAP as a fixed cou-
pling reagent to produce the desired 14 products 4f–4s. Notably,
in this alternative pathway, the combination of an activating cou-
pling agent (i.e., HOBt and EDCI) did not work well, so the combi-
nation of EDCI/DMAP as an alternative coupling reagent was used.
N
N
O
S
O
N
R2
O
Figure 4. A focused two-component small array of pyrazoles designed for
optimization.
and 2). Additionally, replacing the 4-methyl substituent on the
aniline ring with a 2-methyl substituent conjugated with the
same F1 resulted in lower antagonistic activities. Further, inclu-
sion of an unmethylated 3-morpholinosulfonyl aniline substan-
tially decreased the antagonistic potency (Tables 1 and 2). Taken
together, this preliminary SAR analysis of a set of analogs provided
important insight into the essential structural requirements that
indicated 4-methyl-3-(morpholinosulfonyl)aniline in the F2 func-
tionality would be an appropriate factor for effective FXR
antagonists.
Guided by the SAR insights for the F2 fragments described
above, we envisioned that F2 fragments might be a key pharmaco-
phore region where an appropriate substituent, such as 4-methyl
substituted aniline, would be crucial for hydrophobic interactions
with FXR. We chose the 4-methyl-3-(morpholinosulfonyl)aniline
as a fixed essential fragment and modified two regions of F1 frag-
ments. We hypothesized that by subtly optimizing the steric/
hydrophobic effects of the R1 substituents and fine-tuning the
hydrogen-bonding capability of the R2 substituents, we could
establish a focused library to develop novel FXR antagonists that
could display high FXR binding affinity and potent cellular antago-
nistic effects against FXR.
2.3. Chemistry
2.3.1. Synthesis of 1,3,4-trisubstituted pyrazole nonsteroidal
FXR antagonists
As indicated in Figure 4, the N1-and C3-pyrazole moieties on the
pyrazole core could be explored with various electronic and hydro-
phobic substituted alkyls and phenyls. We hypothesized that
potent analogs of 1,3-disubstituted-pyrazole-4-carboxamides
may have structural features in common. From a parallel library
synthesis perspective, compounds of this type are attractive
because they contain only two vectors (denoted R1 and R2) that
allow the introduction of considerable structural diversity. To
explore novel carboxamide analogs of 1,3-disubstituted-pyrazole-
4-carboxamides that could have high binding affinity and antago-
nistic effects against FXR, an efficient synthetic strategy was devel-
oped to optimize the N1-C3-pyrazole core structure through
substitutions at R1 and R2 as a focused two-component small array
of pyrazole. Combining structural considerations and chemical fea-
sibility, our initial strategy comprised traditional medicinal chem-
istry involving variations in both the electronic and steric
properties of the appendages of the central pyrazole nucleus,

2924
D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
O
O
NH2
OH
H
HN
R1
EtOH/acetic acid
N
POCl3/DMF
N
+
Jones' reagent
N
N
N
rt
R1
HN
R1
O
11
R1
12
2
1
3
2a: R1 = 4-F-Ph
1a: R1 = 4-F-Ph
3a: R1 = 4-F-Ph
3b: R1 = 3-Cl-Ph
3c: R1 = 2,4-di-Cl-Ph
3d: R1 = 3-CF3-Ph
2b: R1 = 3-Cl-Ph
2c: R1 = 2,4-di-Cl-Ph
2d: R1 = 3-CF3-Ph
1b: R1 = 3-Cl-Ph
1c: R1 = 2,4-di-Cl-Ph
1d: R1 = 3-CF3-Ph
N
N
N
2e: R1
=
1e: R1
=
3e: R1 =
S
S
S
NH2
R1
O
N
NH
3
N
N
S
+
O
N
O
EDCl/HOBt
S
O
O
O
O
4
13
4a: R1 = 4-F-Ph
4b: R1 = 3-Cl-Ph
4c: R1 = 2,4-di-Cl-Ph
4d: R1 = 3-CF3-Ph
N
4e: R1
=
S
Figure 5. Initial exploration of pyrazole core via Vilsmeier-Haack reaction by the introduction of the first diversity element (R1).
O
O
O
O
O
R2
NH2NH2/EtOH
(EtO)3CH/Ac O
2
OCH2CH3
OCH2CH3
OEt
R2
OCH2CH3
R2
N
N
H
14
6
5
5a: R2 = 4-CH3-Ph
5b: R2 = CH(CH3)2
5c: R2 = 3-OCH3-Ph
5d: R2 = 4-CF3-Ph
5c: R2 = (CH2)2-OCH3
6a: R2 = 4-CH3-Ph
6b: R2 = CH(CH3)2
6c: R2 = 3-OCH3-Ph
6d: R2 = 4-CF3-Ph
6c: R2 = (CH2)2-OCH3
O
R2
OH
R1
NH2
EDCl/DMAP
1. R1-X/Cs2CO3
2. KOH/EtOH
NH
N
+
N
O
O
N
N
O
S
S
O
O
N
R1
7
N
R2
O
O
4
7a: R1 = CH(CH3)2, R2 = 4-CH3-Ph
7b: R1 = (CH2)3CH3, R2 = 4-CH3-Ph
7c: R1 R2 = 4-CH3-Ph
4f: R1 = CH(CH3)2, R2 = 4-CH3-Ph
4g: R1 = (CH2)3CH3, R2 = 4-CH3-Ph
4h: R1 R2 = 4-CH3-Ph
=
=
7d: R1 = CH2CH(CH2)2, R2 = 4-CH3-Ph
4i: R1 = CH2CH(CH2)2, R2 = 4-CH3-Ph
4j: 1 =CH2-3-OCH3-Ph, R2 = 4-CH3-Ph
4k: R1 = CH2-3-OCF3-Ph, R2 = 4-CH3-Ph
4l: 1 = (CH2)4OH, R2 = 4-CH3-Ph
7e: R1 =CH2-3-OCH3-Ph, R2 = 4-CH3-Ph
7f: R1 = CH2-3-OCF3-Ph, R2 = 4-CH3-Ph
7g: R1 = (CH2)4OH, R2 = 4-CH3-Ph
R
R
7h: R1 = CH2-3-OCH3-Ph, R2 = CH(CH3)2
7i: R1 = CH(CH3)2, R2 = 3-OCH3-Ph
7j: R1 = CH2-3-OCH3-Ph, R2 = 3-OCH3-Ph
7k: R1 = CH(CH3)2, R2 = 4-CF3-Ph
4m: R1 = CH2-3-OCH3-Ph, R2 = CH(CH3)2
4n: R1 = CH(CH3)2, R2 = 3-OCH3-Ph
4o: R1 = CH2-3-OCH3-Ph, R2 = 3-OCH3-Ph
4p: R1 = CH(CH3)2, R2 = 4-CF3-Ph
7l: R1 = CH2-3-OCH3-Ph, R2 = 4-CF3-Ph
7m: R1 = CH(CH3)2, R2 = (CH2)2-OCH3
7n: R1 = CH2-3-OCH3-Ph, R2 = (CH2)2-OCH3
4q: R1 = CH2-3-OCH3-Ph, R2 = 4-CF3-Ph
4r: R1 = CH(CH3)2, R2 = (CH2)2-OCH3
4s: R1 = CH2-3-OCH3-Ph, R2 = (CH2)2-OCH3
Figure 6. Alternative exploration of the pyrazole core to introduce R1 and R2 as substituted N1-C3-substituted pyrazoles of 4f-4s.

D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
2925
To explore the linker between the amide bridge and the aro-
matic core, we focused our efforts on modifying the chemical space
in the amide moiety to examine if the amide bridge was sensitive
to activity. Three amides of series 4 were altered to generate com-
pounds 8. We introduced a methyl moiety to assess the SAR. Com-
pounds 4f, 4m, and 4o were chosen to react with iodomethane in
the presence of sodium hydride to give N-methylation compounds
8 (Fig. 7). Because the amide position was hypothesized to be a site
of potential metabolism,34 further optimization involved reduction
of amides. The amide groups in 4f, 4m, and 4o were reduced to cor-
responding amines with 1N borane–THF complex to yield com-
pounds 9 (Fig. 7).
Noticeably, 4j had an IC50 of 7.5 nM in the FXR TR-FRET binding
assay, which was 5.26-fold more potent than E16 (IC50 of 47 nM)
(Tables 3–5; Fig. 8A).
2.4.2. Cell-based FXR transactivation assay
To investigate the cellular activities of the novel 1,3-substi-
tuted-pyrazol-4-carboxamide analogs, we used
a GeneBLAzer
FXR cell-based assay, which detects the levels of a b-lactamase
reporter controlled by a promoter containing the binding site of
the DNA binding domain (DBD) of the GAL4 transcription factor;
the Gal4 DBD was fused to the LBD of FXR in this assay.36 In typical
test for agonists, DMSO (vehicle and negative control), 10 lM
GW4064 (positive control), and titrations of GW4064, LCA or the
23 newly synthesized novel 1,3-trisubstituted-pyrazol-4-carbox-
amide analogs were mixed with GeneBLAzer FXR-UAS-bla HEK
293T cells. Reporter assays were performed after a16 h incubation.
No agonistic activities were observed for any of the newly synthe-
sized compounds (data not shown).
For the experiments to evaluate the antagonistic activity, titra-
tions of compounds were tested in the presence of 400 nM
GW4064. Assay controls included: DMSO alone (100% inhibition),
400 nM GW4064 alone (0% inhibition), and titrations of LCA in
the presence of 400 nM GW4064 (as an assay reference). Activities
of compounds that displayed activating or inhibitory activities in a
dose-dependent manner were fit into a sigmoidal dose-response
equation to derive individual EC50 or IC50 values. The tested chem-
icals’ FXR activities are summarized in Tables 3–5. Many of these
compounds had significantly improved antagonistic activity when
compared to E16. Noticeably, in the FXR antagonistic assay, 4j had
an IC50 of 468.5 nM, which was 4.59-fold more potent than that of
2.4. Biological evaluation
2.4.1. FXR TR-FRET binding assay
The FXR binding affinities of E16, the 23 newly synthesized
novel 1,3-trisubstituted-pyrazol-4-carboxamide analogs shown in
Figures 5–7, and control compounds GW4064 and lithocholic acid
(LCA) were tested through an in vitro FXR TR-FRET binding assay
using DY246 as the fluorescent probe.23 We chose TR-FRET assays
because of their advantages of low background fluorescence inter-
ference and increased sensitivity as compared to other fluores-
cence-based assays, such as FI, FP and FRET assays.35 We chose
DY246 because it is a derivative of GW4064, which is a potent
FXR agonist. In addition, DY246 acts as a potent FXR agonist
(EC50 of 550 nM) and has successfully been used as a fluorescent
probe in a high throughput screening campaign to identify FXR
antagonists.23 In a representative assay, DMSO (vehicle and nega-
tive control), 10 lM GW4064 (positive control), and titrations of
GW4064, LCA or other chemicals were incubated for 20 min with
an appropriate mixture of GST-FXR-LBD, terbium (Tb)-anti-GST
and DY246, after which the TR-FRET signals were collected and
activity for each chemical was normalized to DMSO (negative con-
E16 (IC50 of 2.62 lM) (Tables 3–5; Fig. 8B).
2.4.3. Cytotoxicity assay
For the FXR cell-based antagonistic assays, if a tested compound
caused a signal reduction, it was usually considered an antagonist.
However, signal reduction could also be caused non-specifically if a
chemical was cytotoxic, although GeneBLAzer assays are relatively
insensitive to cytotoxicity because of their use of ratiometric read-
ing. To examine if certain chemical’s antagonistic effect was caused
by cytotoxicity, we used the same GeneBLAzer FXR cells in
trol; 0% inhibition) and 10 lM GW4064 (positive control; 100%
inhibition). The activities of compounds that displayed dose-
dependent inhibitory effects were fit into a one-site competitive
binding equation to derive IC50 values. The TR-FRET binding activ-
ities are summarized in Tables 3–5. Many of these compounds had
significantly improved FXR binding affinity when compared to E16.
R1
NH
R1
N
N
N
CH3I/NaH/DMF
N
N
O
O
O
S
O
S
O
N
O
R2
N
R2
O
O
4
8
8a: R1 = CH(CH3)2, R2 = p-CH3-Ph
4f: R1 = CH(CH3)2, R2 = p-CH3-Ph
8b: R1 = CH2-3-OCH 3-Ph, R2 = CH(CH3)2
8c: R1 = CH2-3-OCH3-Ph, R2 = 3-OCH3-Ph
4m: R1 = CH2-3-OCH3-Ph, R2 = CH(CH3)2
4o: R1 = CH2-3-OCH3-Ph, R2 = 3-OCH3-Ph
BH3-THF
reflux
R1
N
N
NH
O
N
R2
S
O
O
9
9a: R1 = CH(CH3)2, R2 = p-CH3-Ph
9b: R1 = CH2-3-OCH3-Ph, R2 = CH(CH3)2
9c: R1 = CH2-3-OCH3-Ph, R2 = 3-OCH3-Ph
Figure 7. N-methylation and amide reduction of 4f, 4m, and 4o gave corresponding compounds 8 and 9.

2926
D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
Table 3
The effect of varying the substitution pattern of the 1,3,4-substituted-pyrazole-4-carboxamide derivatives on their biological activities
R1
NH
O
N
N
O
S
N
O
R2
O
Compds.
R1
R2
FXR TR-FRET Binding
GB FXR Cell Anta.
GB FXR Cell Cyto
%Inh.
a
a
%Inh.
IC50
%Inh.
NA
IC50
IC50
NA
GW4064
100 4.3
19.8 2.5 nM
NA
NT
(10
82.3 0.9
(40 M)
lM)
Lithocholic acid
18.3 1.2
(40 lM)
16.1 0.5
NA
NT
NA
NA
l
E16
4a
Ph
4-CH3-Ph
4-CH3-Ph
4-CH3-Ph
4-CH3-Ph
4-CH3-Ph
100.2 5.2
(164 nM)
95.0 1.2
(493 nM)
99.3 1.1
(164 nM)
119.8 7.8
(164 nM)
77.5 4.3
(164 nM)
47.0 5.1 nM
24.3 0.6 nM
10.4 0.9 nM
14.1 0.3 nM
23.9 2.1 nM
77.2 2.2
2.62 0.13
l
M
17.9 0.5
(40
18.5 1.0
(40
NT
(40
72.0 2.2
(40 M)
60.6 0.9
(40 M)
89.9 1.9
(40 M)
54.8 1.3
(40 M)
79.1 2.4
(40 M)
lM)
lM)
4-F-Ph
3-Cl-Ph
2,4-Di-Cl-Ph
3-CF3-Ph
N
2.49 0.0.06
lM
l
lM)
4b
4c
5.91 0.15
1.24 0.12
22.29 1.14
l
M
M
NA
NA
NA
l
l
NT
l
4d
l
M
7.3 0.2
(40 M)
11.5 0.2
l
l
101.8 2.8
(164 nM)
l
(13.3
lM)
4e
4f
4-CH3-Ph
4-CH3-Ph
17.7 0.4 nM
49.1 9.9 nM
7.91 1.16
l
M
NA
NA
S
(CH3)2CH
130.6 3.8
99.1 4.4
(40 M)
97.9 8.1
(20 M)
84.4 4.1
(40 M)
1.69 0.18 l
M
NT
(4.4
130.1 0.7
(1.5 M)
lM)
l
4g
CH3(CH2)3
4-CH3-Ph
52.5 4.1 nM
11.51 0.07
l
M
10.3 0.3
(4.4 M)
7.9 0.3
NA
l
l
l
97.8 1.2
(164 nM)
4h
4i
4-CH3-Ph
4-CH3-Ph
4-CH3-Ph
10.2 1.6 nM
76.9 2.7 nM
7.5 0.8 nM
844.1 10.3 nM
848.8 69.0 nM
468.5 8.4 nM
NA
NA
NA
l
(4.4
lM)
105.0 4.9
100.1 3.2
(40 M)
NT
(1.5
lM)
l
OCH3
130.1 5.4
(164 nM)
98.9 4.3
(40 M)
ꢀ4j
NT
NT
l
CF3
99.2 6.4
(164 nM)
93.4 3.9
(40 M)
4k
4l
4-CH3-Ph
4-CH3-Ph
18.4 1.2 nM
130.3 16 nM
515.0 25.4 nM
NA
NA
l
HO(CH2)4
(CH3)2CH
(CH3)2CH
(CH3)2CH
89.3 0.1
(493 nM)
97.6 3.7
(40 M)
99.2 2.7
(40 M)
2.61 0.29
l
M
5.9 0.2(40 lM)
l
OCH3
OCH3
OCH3
OCH3
100.6 5.6
4m
4n
CH(CH3)2
199.4 21.2 nM
159.7 22.6 nM
3.11 0.58
3.49 0.64
l
M
M
NT
NA
(4.4
lM)
l
3-CH3O-Ph
149.8 6.9
97.8 1.5
(40 M)
89.4 4.2
(40 M)
l
10.8 0.2
(4.4
(13.3 M)
l
l
lM)
114.1 3.3
(493 nM)
4o
4p
3-CH3O-Ph
4-CF3-Ph
42.8 0.5 nM
77.5 3.7 nM
1.24 0.09
1.95 0.28
l
M
M
NT
NA
NA
l
133.2 5.3
100.0 3.7
(40 M)
97.6 0.9
(40 M)
l
19.5 1.1
(40 lM)
(4.4
lM)
l
109.1 3.6
(164 nM)
4q
4r
4-CF3-Ph
30.9 3.6 nM
592.7 3.0 nM
29.58 1.75 lM
NT
NT
NA
NA
l
CH3O(CH2)2
98.2 3.8
3.42 0.19
lM
62.5 0.8
(40 M)
95.9 2.4
(40 M)
(40
113.5 0.8
(13.3 M)
lM)
l
4s
CH3O(CH2)2
389.5 9.4 nM
11.1 0.64
l
M
NT
NA
l
l
NA: not applicable.
NT: Not toxic (toxicity less than 5%).
GB FXR Cell Anta: GeneBLAzer FXR cell-based antagonistic assay.
GB FXR Cell Cyto: Cytotoxicity assay performed in the GeneBLAzer FXR cells.
%Inh: %Inhibition is presented as the means SE of at least three independent experiments calculated as described in the Section 4. The value shown in the table represents
the maximum percent inhibition observed, and its lowest corresponding compound concentration is noted in the parentheses.
a
IC50 values represent the means SE of at least three independent experiments derived as described in the Section 4.
cytotoxicity assays. These assays were set up the same as for an
FXR agonistic assay, with the exception of signal reading. Instead
of detecting reporter activities, the CellTiter-GloÒ assay was used
to detect cell viability. Conditions including DMSO with or without
cells were used as negative (0% inhibition) or positive (100% inhi-
bition) controls, respectively. Cytotoxic activities are summarized
in Tables 3–5. All chemicals tested showed marginal toxicity (%
inhibition <5%), therefore the antagonistic effect of the compounds
was not caused by non-specific cytotoxicity.
2.5. SAR
We investigated the effect of introducing electron withdrawing
groups, such as fluorine, chlorine, dichlorines, and trifluoromethyl

D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
2927
Table 4
The effect of varying the substitution pattern of the 1,3,4-substituted-pyrazole-4-carboxamide derivatives on their biological activities (N-methylation)
R1
N
O
N
N
O
S
N
O
R2
O
Compd.
R1
R2
FXR TR-FRET binding
IC50
GB FXR Cell Anta.
IC50
GB FXR Cell Cyto
%Inh. IC50
15.0 0.7
(40 M)
13.5 0.4
%Inh.
%Inh.
86.4 2.8
(40 M)
2.4 0.1
8a
(CH3)2CH
4-CH3-Ph
132.9 6.2
489.4 21.2 nM
16.16 0.28
lM
NA
(40
80.3 5.2
(40 M)
l
M)
l
l
OCH3
OCH3
8b
8c
(CH3)2CH
5.33 0.24
3.81 0.20
l
M
M
NA
NA
NA
l
(20 M)
l
(40 M)
l
69.7 3.5
(40 M)
23.8 0.4
(40 M)
19.7 0.4
(40 M)
3-CH3O-Ph
l
NA
l
l
l
Table 5
The effect of varying the substitution pattern of the 1,3,4-substituted-pyrazole-4-carboxamide derivatives on their biological activities (amide reduction)
R1
NH
N
N
O
S
N
O
R2
O
Compd.
R1
R2
FXR TR-FRET Binding
IC50
GB FXR Cell Anta.
IC50
GB FXR Cell Cyto
%Inh.
%Inh.
36.8 0.3
(40 M)
64.8 1.9
%Inh.
10.5 0.3
(40 M)
13.9 0.6
IC50
NA
9a
(CH3)2CH
4-CH3-Ph
99.5 2.6
(13.3 M)
72.5 2.7
(4.4 M)
2.04 0.02
l
M
NA
l
l
l
OCH3
OCH3
9b
9c
(CH3)2CH
2.31 0.32
NA
l
M
27.83 1.54
NA
l
M
NA
NA
l
(40 M)
l
(40
lM)
39.8 1.8
(1.48 M)
3.1 0.1
(40 M)
3-CH3O-Ph
NT
l
l
atoms, at ortho, meta, or para positions of the N1-phenyl.37 All of
these derivatives (4a, 4b, 4c, 4d, and 4e) improved FXR antagonis-
tic activity by about 3- to 4-fold as compared to E16 (Table 3), indi-
cating that the electron withdrawing groups were well tolerated
for antagonistic activity. This observation can be attributed to the
higher interaction strength of fluorine or chlorine atoms compared
to hydrogen, which enables a tighter fit.
N1-position and the positive influence of the m-methoxybenzyl
group may be generally preferred at the N1-position of the pyrazole
ring.
Replacing the original C3-p-methyl phenyl of 4j with the stron-
ger electron-donating m-methoxy phenyl group resulted in analog
4o, which had slightly decreased antagonistic activity (Table 3).
This indicated that the binding pocket of FXR that hosted the m-
methoxy phenyl group on the C3 position of pyrazole is not suffi-
ciently wide to accept larger substituents for hydrogen bond inter-
actions with FXR. Notably, replacing the original of C3-p-methyl
phenyl (R2) of 4j with a smaller isopropyl group (analog 4m) signif-
icantly decreased the potency and affinity, demonstrating that the
C3 moiety is very sensitive in terms of steric bulkiness and/or
hydrophobicity. For instance, introducing a p-trifluoromethyl phe-
nyl with electron withdrawing properties at the C3 position of the
pyrazole ring gave products 4p and 4q, which had nanomolar IC50
values for in vitro potency. 4p and 4q appeared to have similar
antagonistic activities as their parent compounds 4f and 4o. In con-
trast, replacing the p-trifluoromethyl phenyl at the C3 position of
the pyrazole ring with a linear 2-methoxy-ethyl group to obtain
compounds 4r and 4s, resulted in severe loss of potency relative
to that of 4f and 4o, indicating that an alkyl methoxy in the C3
position of pyrazole ring is not beneficial. Therefore, we speculate
that an aromatic phenyl ring at the C3-position of the pyrazole ring
is required not only to optimally bind to a hydrophobic area of FXR,
Based on this initial SAR, we next introduced longer benzyl
chains or small alkyl groups to replace the N1-phenyl of the pyra-
zole moiety to see if it was a suitable position to find more potent
antagonists for FXR. The alternative synthetic methodology for
constructing the corresponding pyrazole-4-carboxylic acids
worked well. Replacing the phenyl ring with small hydrophobic
substituents such as isopropyl (4f) or n-butyl (4g) allowed slight
retention of the antagonistic activity toward FXR. Substituting with
a methylcyclopropane (4i) decreased the activity by 2-fold for FXR
as compared to E16. Interestingly, incorporating a bulkier group,
such as benzo[d]thiazole (4e), led to 3-fold higher potency for
FXR. Other substitutions, including longer and bulkier benzyl aro-
matic substituents such as 2-naphthyl (4h) and benzyl with an
electron donating group m-methoxy (4j), enhanced antagonistic
activity. Among the N1-alkylated analogs, 4j exhibited the most
potent FXR-antagonistic activity, having IC50 values of 7.5 nM
and 468.5 nM in a FXR TR-FRET binding assay and a FXR cell-based
antagonistic assay, respectively. In addition, 4j had no FXR
agonistic activity and no cytotoxicity. These results suggest
that an expanded hydrophobic moiety is necessary at the
but is also critical for
p–p interaction with the receptor to increase
FXR antagonistic activity. These examples demonstrate the

2928
D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
140
120
100
80
ther profiling 4j in vitro and expanding the SAR of these analogs
with the goal of developing FXR antagonists that can be delivered
orally and are active in vivo are under way.
A
B
E16
4j
4. Experimental section
4.1. Chemistry
60
40
20
0
General procedures: Organic reagents were purchased from com-
mercial suppliers unless otherwise noted and were used without
further purification. All solvents were analytical or reagent grade.
All reactions were carried out in flame-dried glassware under
argon or nitrogen. Melting points were determined and reported
automatically by an optoelectronic sensor in open capillary tubes
and were uncorrected. 1H NMR and 13C NMR spectra were mea-
sured at 500 MHz and 125 MHz, respectively, using CDCl3 or CD3-
OD as solvents and tetramethylsilane (Me4Si) as the internal
standard. Flash column chromatography was performed using
Sigma-Aldrich silica gel 60 (200–400 mesh), carried out under
moderate pressure with columns of an appropriate size packed
and eluted with appropriate eluents. All reactions were monitored
by thin layer chromatography (TLC) on precoated plates (silica gel
HLF). TLC spots were visualized either by exposure to iodine vapor
or by irradiation with UV light. Organic solvents were removed
under vacuum by a rotary evaporator. Elemental analyses were
performed by Columbia Analytical Services Inc, Tucson, Arizona.
-11
-10
-9
-8
-7
-6
-5
Log [M]
4
3
2
1
0
E16
E16+400 nM GW4064
4j
4j +400 nM GW4064
-10
-9
-8
-7
Log [M]
-6
-5
-4
Figure 8. (A) FXR binding activities of E16 and 4j in a FXR TR-FRET binding assay.
(B) Agonistic (E16 and 4j) and antagonistic activities (E16 and 4j in an FXR cell-
based transactivation assay.
favorable interaction that can be gained from fine-tuning the
hydrophobic shape complementarily.
We also modified compounds 4f, 4m, and 4o with amide moie-
ties. The carboxamide nitrogens of 4f, 4m, and 4o were methylated
to form N-methyl analogs 8a, 8b, and 8c (Fig. 7). Unfortunately, N-
4.1.1. (E)-1-(4-Fluorophenyl)-2-(1-p-tolylethylidene)hydrazine
(1a)
To a solution of 40-methylacetophenone (0.5 g, 3.73 mmol) in
8 mL of acetic acid and 5 mL of MeOH, 4-fluorophenylhydrazine
hydrochloride (0.6 g, 3.73 mmol) was added. The reaction mixture
was stirred at room temperature for 18 h. The reaction mixture
was diluted with an additional 30 mL of water and subsequently
filtered. The solid was dried in vacuo to afford 0.63 g of beige solid
1a (70% yield): mp 101.8 °C. 1H NMR (CDCl3) d 7.68 (d, 2H), 7.19 (d,
3H), 7.07 (t, 2H), 6.98 (d, 2H), 2.37 (s, 3H), 2.23 (s, 3H).
methylation of the CONH function of amide compounds
8
decreased the antagonistic activity (Table 4), as compared to the
activity of the original amides, 8a, 8b, and 8c were essentially inac-
tive at 1
lM against FXR (data not shown). Similarly, compounds 9
had weak activity at 1
lM, at least on FXR (data not shown). Com-
pound 9 had more than 300-fold less affinity and potency as com-
pared with 4j. The discrepancies in activity of these analogs
between in vitro and in vivo might be due to their stability, solubil-
ity and /or permeability.
4.1.2. (E)-1-(3-Chlorophenyl)-2-(1-p-tolylethylidene)hydrazine
(1b)
3. Conclusions
To a solution of 40-methylacetophenone (0.5 g, 3.73 mmol) in
8 mL of acetic acid and 5 mL of water/MeOH, 3-chlorophenylhydra-
zine hydrochloride (0.67 g, 3.73 mmol) was added. The reaction mix-
ture was stirred at room temperature for 18 h. The reaction mixture
was diluted with an additional 30 mL of water and subsequently fil-
tered. The solid was dried in vacuo to afford 0.58 g of beige solid 1b
(60% yield): mp 100.5 °C. 1H NMR (CDCl3) d 7.68 (t, 2H), 7.20 (t, 5H),
6.98 (s, 1H), 6.83 (d, 1H), 2.38 (s, 3H), 2.28 (s, 3H).
FXR is a nuclear receptor that functions as a physiological
receptor for bile acids. A highly potent and selective antagonist
of FXR would represent a novel option that could prove useful
for treating dyslipidemia and cholestasis. Thus, there is consider-
able need to identify lead structures and pharmacophore in this
relatively unexplored area. We investigated the preliminary SAR-
based optimization of 1,3,4-trisubstituted-pyrazole carboxamides
as FXR antagonists. In an effort to develop specific FXR antagonists,
a traditional medicinal chemistry strategy was used. A series of
1,3,4-trisubstituted-pyrazole carboxamide analogues were devel-
oped and analyzed for physicochemical properties and SAR based
on in vitro data. All of these modified compounds were evaluated
for FXR binding affinities in biochemical assays and antagonistic
activity in cell-based transactivation assays. All amide compounds
reported here behaved as potent FXR antagonists, and did not have
FXR agonistic activity (data not shown). Of interest, compound 4j
had IC50 values of 7.5 nM and 468.5 nM in FXR TR-FRET binding
assays and FXR cell-based antagonistic assays, respectively, sug-
gesting it is a promising candidate FXR antagonist. In addition,
compound 4j does not have any FXR agonistic activity and cytotox-
icity, suggesting that expansion of R1 and R2 on the pyrazole scaf-
fold increases hydrogen bond acceptor interactions with FXR. This
work has resulted in the most potent antagonist to date for FXR, 4j,
which may emerge as novel chemical tool to explore the effect of
FXR inhibition in pharmacological settings. Studies aimed at fur-
4.1.3. (E)-1-(2,4-Dichlorophenyl)-2-(1-p-
tolylethylidene)hydrazine (1c)
To a solution of 40-methylacetophenone (0.5 g, 3.73 mmol) in
8 mL of acetic acid and 5 mL of water/MeOH, 2,4-dichlorophenylhy-
drazine hydrochloride (0.8 g, 3.73 mmol) was added. The reaction
mixture was stirred at room temperature for 18 h. The reaction mix-
ture was diluted with an additional 30 mL of water and subsequently
filtered. The solid was dried in vacuo to afford 0.98 g of beige solid 1c
(90% yield): mp 122.7 °C. 1H NMR (CDCl3) d 7.71 (t, 2H), 7.61 (d, 2H),
7.29 (s, 1H), 7.021(d, 3H), 2.38 (s, 3H), 2.28 (s, 3H).
4.1.4. (E)-1-(3-Trifluromethylphenyl)-2-(1-p-
tolylethylidene)hydrazine (1d)
To a solution of 40-methylacetophenone (0.5 g, 3.73 mmol) in
8 mL of acetic acid and 5 mL of water/MeOH, 3-trif-
luoromethylphenylhydrazine hydrochloride (0.66 g, 3.73 mmol)
was added. The reaction mixture was stirred at room temperature

D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
2929
for 18 h. The reaction mixture was diluted with an additional
30 mL of water and subsequently filtered. The solid was dried in
vacuo to afford 1.01 g of beige solid 1d (93% yield): mp 103.7 °C.
1H NMR (CDCl3) d 7.72 (d, 2H), 7.44 (m, 4H), 7.28 (d, 2H), 7.14 (s,
1H), 2.41 (s, 3H), 2.27 (s, 3H).
purification. 1H NMR (CDCl3) d 10.05 (s, 1H), 8.48 (s, 1H), 7.66
(m, 3H), 7.54 (s, 1H), 7.42 (d, 1H), 7.37 (d, 2H), 2.38 (s, 3H). 13C
NMR (CDCl3) d 186.6, 156.59 140.9, 137.4, 136.8, 134.1, 132.0,
131.7, 130.8, 129.4, 127.2, 105.7, 22.7.
4.1.9. 3-p-Tolyl-1-(3-(trifluoromethyl)phenyl)-1H-pyrazole-4-
carbaldehyde (2d)
4.1.5. (E)-2-(2-(1-(p-
Tolyl)ethylidene)hydrazinyl)benzo[d]thiazole (1e)
A solution of DMF (3 mL) and POCl3 (1.5 mL, 16.4 mmol) was
made in an ice bath under argon and stirred for 10 min. 1d
(0.75 g, 2.56 mmol) dissolved in dichloroethane (7 mL) was added
to this solution; the mixture was heated to 70 °C and stirred for an
additional 18 h. The reaction mixture was then cooled to room
temperature and slowly poured into saturated aqueous NaHCO3
(200 mL) under ice cold conditions and then warmed to room tem-
perature and stirred for 3 h. The aqueous layer was extracted with
EtOAc. The organic layers were washed with water and brine, dried
over Na2SO4 and concentrated in vacuo to yield 0.79 g (93%) of the
title compound (mp 155.1 °C), which was used without further
purification. 1H NMR (CDCl3) d 10.08 (s, 1H), 8.60 (s, 1H), 8.13 (s,
1H), 7.99 (q, 1H), 7.74 (d, 2H), 7.67 (d, 2H), 7.35 (d, 2H), 2.45 (s,
3H). 13C NMR (CDCl3) d 186.6, 157.3, 141.7, 140.8, 132.3, 131.8,
130.9, 130.3, 126.6, 126.2, 123.8, 22.8.
To a solution of 40-methylacetophenone (0.5 g, 3.73 mmol) in
8 mL of acetic acid and 1 mL of water, 2-hydrazinobenthiazole
(0.62 g, 3.73 mmol) was added. The reaction mixture was stirred
at room temperature for 18 h. The reaction mixture was diluted
with an additional 20 mL of water and subsequently filtered. The
solid was dried in vacuo to afford 1 g of beige solid 1e (99% yield):
mp 112.6 °C. 1H NMR (CDCl3) d 7.74 (d, 2H), 7.68 (d, 1H), 7.53 (d,
1H), 7.36 (d, 1H), 7.25 (d, 2H), 7.16 (d, 1H), 2.41 (s, 3H), 2.17 (s, 3H).
4.1.6. 1-(4-Fluorophenyl)-3-p-tolyl-1H-pyrazole-4-
carbaldehyde (2a)
A solution of DMF (3 mL) and POCl3 (1.5 mL, 16.4 mmol) was
made in an ice bath under argon and stirred for 10 min. 1a
(0.21 g, 0.87 mmol) dissolved in dichloroethane (5 mL) was added
to this solution; the mixture was warmed to 70 °C and stirred for
an additional 18 h. The reaction mixture was then cooled to room
temperature, slowly poured into saturated aqueous NaHCO3
(200 mL) under ice cold conditions, and then warmed to room tem-
perature and stirred for 3 h. The aqueous layer was extracted from
the reaction mixture with EtOAc. The organic layers were washed
with water and brine, dried over Na2SO4 and concentrated in vacuo
to yield 0.21 g (88%) of the title compound: mp 143.8 °C, which
was used without further purification. 1H NMR (CDCl3) d 10.05
(s, 1H), 8.47 (s, 1H), 7.78 (m, 1H), 7.71 (d, 2H), 7.33 (d, 2H), 7.19
(m, 3H), 2.44 (s, 3H). 13C NMR (CDCl3) d 187.0, 156.5, 132.7,
130.9, 130.2, 129.8, 123.9, 123.1, 118.1, 117.8, 23.3.
4.1.10. 1-(2,3-Dihydro-1H-inden-1-yl)-3-(p-tolyl)-1H-pyrazole-
4-carbaldehyde (2f)
A solution of DMF (3 mL) and POCl3 (1.5 mL, 16.4 mmol) was
made in an ice bath under argon and stirred for 10 min. 1f (1.0 g,
3.55 mmol) dissolved in dichloroethane (5 mL) was added to the
solution; the mixture was heated to 60 °C and stirred for an addi-
tional 18 h. The reaction mixture was then cooled to room temper-
ature, slowly poured into saturated aqueous NaHCO3 (200 mL)
under ice cold conditions and then warmed to room temperature
and stirred for an additional 5 h. The aqueous layer was extracted
with EtOAc. The organic layers were washed with water, brine,
dried over Na2SO4 and concentrated in vacuo to yield 0.74 g
(70%) of the title compound: mp 145.3 °C, which was used without
further purification. 1H NMR (CDCl3) d 10.11 (s, 1H), 9.08 (s, 1H),
7.98 (d, 1H), 7.89 (d, 1H), 7.79 (d, 2H), 7.55 (t, 1H), 7.44 (t, 1H),
7.35 (d, 2H), 2.41 (s, 3H). 13C NMR (CDCl3) d 186.0, 157.1, 152.0,
141.5, 134.0, 130.9, 130.3, 128.3, 124.4, 123.2, 23.4.
4.1.7. 1-(3-Chlorophenyl)-3-p-tolyl-1H-pyrazole-4-
carbaldehyde (2b)
A solution of DMF (3 mL) and POCl3 (1.5 mL, 16.4 mmol) was
made in an ice bath under argon and stirred for 10 min. 1b
(0.58 g, 2.24 mmol) dissolved in dichloroethane (5 mL) was added
to this solution; the mixture was heated to 70 °C and stirred for an
additional 18 h. The reaction mixture was then cooled to room
temperature and slowly poured into saturated aqueous NaHCO3
(200 mL) under ice cold conditions, then warmed to room temper-
ature and stirred for 3 h. The aqueous layer was extracted with
EtOAc. The organic layers were washed with water, brine, dried
over Na2SO4 and concentrated in vacuo to yield 0.64 g (88%) of
the title compound: mp 118.7 °C, which was used without further
purification. 1H NMR (CDCl3) d 10.05 (s, 1H), 8.53 (s, 1H), 7.88 (s,
1H), 7.67 (d, 3H), 7.46 (t, 1H), 7.31 (d, 3H), 2.44 (s, 3H). 13C NMR
(CDCl3) d 186.5, 159.3, 141.1, 132.1, 130.9, 130.2, 129.3, 121.6,
118.8, 23.0.
4.1.11. 1-(4-Fluorophenyl)-3-p-tolyl-1H-pyrazole-4-carboxylic
acid (3a)
To a solution of 2a (0.21 g, 0.75 mmol) dissolved in 16 mL of
acetone, 5 mL of Jones’ reagent was added. The reaction mixture
was stirred at room temperature and monitored by TLC. The reac-
tion mixture was poured into water and extracted with EtOAc. The
combined organic layers were washed with water and brine, dried
over Na2SO4, and concentrated in vacuo to afford 0.16 g (72%) of
the title acid: mp 202.0 °C. 1H NMR (CDCl3) d 8.51 (s, 1H), 7.75
(m, 3H), 7.32 (d, 2H), 7.18 (t, 4H), 2.41 (s, 3H). 13C NMR (CDCl3) d
170.7, 164.5, 155.9, 140.6, 135.1, 132.6, 130.9, 130.6, 130.2,
130.1, 122.8, 118.2, 22.7. Anal. Calcd for C17H13FN2O2: C, 68.91;
H, 4.41; N, 9.45. Found: C, 68.13; H, 4.43; N, 8.94.
4.1.8. 1-(2,4-Dichlorophenyl)-3-p-tolyl-1H-pyrazole-4-
carbaldehyde (2c)
A solution of DMF (3 mL) and POCl3 (1.5 mL, 16.4 mmol) was
made in an ice bath under argon and stirred for 10 min. 1c
(0.61 g, 2.08 mmol) dissolved in dichloroethane (5 mL) was added
to this solution; the mixture was heated to 70 °C and stirred for an
additional 18 h. The reaction mixture was then cooled to room
temperature and slowly poured into saturated aqueous NaHCO3
(200 mL) under ice cold conditions and then warmed to room tem-
perature and stirred for 3 h. The aqueous layer was extracted with
EtOAc. The organic layers were washed with water, brine, dried
over Na2SO4 and concentrated in vacuo to yield 0.53 g (78%) of
the title compound: mp 85.8 °C, which was used without further
4.1.12. 1-(3-Chlorophenyl)-3-p-tolyl-1H-pyrazole-4-carboxylic
acid (3b)
To a solution of 2b (0.51 g, 1.72 mmol) dissolved in 16 mL of
acetone, 5 mL of Jones’ reagent was added. The reaction mixture
was stirred at room temperature and monitored by TLC. The reac-
tion mixture was poured into water and extracted with EtOAc. The
combined organic layers were washed with water and brine, dried
over Na2SO4, and concentrated in vacuo to afford 0.51 g (95%) of
the title acid: mp 194.4 °C. 1H NMR (CDCl3) d 8.57 (s, 1H), 7.86
(d, 1H), 7.77 (t, 2H), 7.66 (d, 1H), 7.44 (t, 1H), 7.34 (d, 1H), 7.28
(m, 2H), 2.41 (s, 3H). 13C NMR (CDCl3) d 168.8, 156.9, 147.7,

2930
D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
141.6, 140.3, 137.2, 134.7, 132.1, 130.6, 129.1, 121.3, 118.7, 22.8.
Anal. Calcd for C17H13FClN2O2: C, 65.29; H, 4.19; N, 8.96. Found:
C, 64.65; H, 3.37; N, 8.80.
resulting yellow solid was purified by flash column chromatogra-
phy (Hexanes/EtOAc 1:1) to afford 4a as a white solid (0.09 g,
26% yield), mp 95.9 °C. 1H NMR (CDCl3) d 8.52 (s, 1H), 7.73 (s,
1H), 7.72 (d, 2H), 7.64 (t, 2H), 7.54 (d, 2H), 7.36 (d, 2H), 7.27 (d,
3H), 3.72 (d, 4H), 3.17 (d, 4H), 2.57 (s, 3H), 2.47 (s, 3H). 13C NMR
(CDCl3) d 162.1, 152.6, 141.4, 137.5, 137.4, 134.8, 134.6, 132.7,
131.2, 130.7, 130.2, 125.2, 122.7, 122.3, 119.5, 117.9, 117.7, 67.7,
46.8, 23.9, 22.6. Anal. Calcd for C28H27FN4O4S: C, 62.91; H, 5.09;
N, 10.48; F, 3.55. Found: C, 62.75; H, 4.57; N, 10.46; F, 3.4.
4.1.13. 1-(2,4-Dichlorophenyl)-3-p-tolyl-1H-pyrazole-4-
carboxylic acid (3c)
To a solution of 2c (0.53 g, 1.6 mmol) dissolved in 16 mL of ace-
tone, 5 mL of Jones’ reagent was added. The reaction mixture was
stirred at room temperature and monitored by TLC. The reaction
mixture was poured into water and extracted with EtOAc. The
combined organic layers were washed with water and brine, dried
over Na2SO4, and concentrated in vacuo to afford 0.51 g (91%) of
the title acid: mp 211.7 °C. 1H NMR (CDCl3) d 8.52 (s, 1H), 7.75
(d, 2H), 7.66 (d, 1H), 7.58 (s, 1H), 7.41 (d, 1H), 7.24 (d, 2H), 2.43
(s, 3H). 13C NMR (CDCl3) d 171.6, 155.7, 139.3, 131.9, 130.6,
130.2, 134.7, 132.1, 130.6, 129.8, 22.7. Anal. Calcd for C17H12Cl2N2-
O2: C, 58.81; H, 3.48; N, 8.07. Found: C, 58.46; H, 3.40; N, 7.68.
4.1.17. 1-(3-Chlorophenyl)-N-(4-methyl-3-
(morpholinosulfonyl)phenyl)-3-(p-tolyl)-1H-pyrazole-4-
carboxamide (4b)
To a solution of 3b (0.2 g, 0.67 mmol) in CH2Cl2 (10 mL) was
added EDCI (0.22 g, 1.16 mmol) and HOBT (0.15 g, 1.16 mmol).
The reaction mixture was stirred for 1 h. To the reaction was added
4-methyl-3-(morpholinosulfonyl)aniline (0.17 g, 0.68 mmol). The
reaction mixture was stirred at room temperature for 12 h and
quenched with water. The organic layer was collected and the
aqueous layer was extracted with EtOAc. The combined organic
solution was dried over Na2SO4, evaporated under a vacuum. The
resulting yellow solid was purified by flash column chromatogra-
phy (H/EtOAc 1:1) to afford 4b as a white solid (0.07 g, 19% yield),
mp 204.9 °C. 1H NMR (CDCl3) d 8.60 (s, 1H), 7.88 (s, 1H), 7.72 (d,
1H), 7.62(m, 3H), 7.45 (d, 2H), 7.41 (m, 3H), 7.24 (d, 2H), 3.75 (d,
4H), 3.21 (d, 4H), 2.59 (s, 3H), 2.50 (s, 3H). 13C NMR (CDCl3) d
162.1, 152.7, 147.6, 141.7, 137.9, 134.8, 132.8, 132.0, 131.3,
130.7, 128.9, 125.1, 122.3, 121.3, 118.5, 67.7, 64.9, 23.0, 21.5, Anal.
Calcd for C28H27ClN4O4S: C, 61.03; H, 4.94; N, 10.17; Cl, 6.43.
Found: C, 60.78; H, 4.61; N, 9.94; Cl, 6.7.
4.1.14. 3-(p-Tolyl)-1-(3-(trifluoromethyl)phenyl)-1H-pyrazole-
4-carboxylic acid (3d)
To a solution of 2d (0.79 g, 2.39 mmol) dissolved in 16 mL of
acetone, 5 mL of Jones’ reagent was added. The reaction mixture
was stirred at room temperature and monitored by TLC. The reac-
tion mixture was poured into water and extracted with EtOAc. The
combined organic layers were washed with water and brine, dried
over Na2SO4, and concentrated in vacuo to afford 0.81 g (98%) of
the title acid: mp 181.8 °C. 1H NMR (CDCl3) d 8.64 (s, 1H), 8.09
(s, 1H), 7.98 (d, 1H), 7.79 (d, 2H), 7.63 (d, 2H), 7.31 (t, 2H), 2.45
(s, 3H). 13C NMR (CDCl3) d 168.2, 155.7, 140.8, 140.5, 134.8,
131.7, 130.7, 130.2, 129.8, 125.5, 123.7, 117.9, 114.5, 22.8. Anal.
Calcd for C18H13F3N2O2: C, 62.43; H, 3.78; N, 8.09. Found: C,
61.95; H, 3.74; N, 7.62.
4.1.18. 1-(2,4-Dichlorophenyl)-N-(4-methyl-3-
(morpholinosulfonyl)phenyl)-3-(p-tolyl)-1H-pyrazole-4-
carboxamide (4c)
4.1.15. 1-(Benzo[d]thiazol-2-yl)-3-(p-tolyl)-1H-pyrazole-4-
carboxylic acid (3e)
To a solution of 3c (0.2 g, 0.58 mmol) in CH2Cl2 (5 mL) was
added EDCI (0.22 g, 1.16 mmol) and HOBT (0.16 g, 1.16 mmol).
The reaction mixture was stirred for 1 h. To the reaction was added
4-methyl-3-(morpholinosulfonyl)aniline (0.15 g, 0.58 mmol). The
reaction mixture was stirred at room temperature for 12 h and
quenched with water. The organic layer was collected and the
aqueous layer was extracted with EtOAc. The combined organic
solution was dried over Na2SO4, evaporated under a vacuum. The
resulting yellow solid was purified by flash column chromatogra-
phy (H/EtOAc 1:1) to afford 4c as a white solid (0.06 g, 20% yield),
mp 99.8 °C. 1H NMR (CDCl3) d 8.52 (s, 1H), 7.73 (s, 1H), 7.62 (m,
3H), 7.45 (d, 2H), 7.38 (m, 3H), 7.24 (d, 2H), 3.73 (d, 4H), 3.19 (d,
4H), 2.57 (s, 3H), 2.46 (s, 3H). 13C NMR (CDCl3) d 162.0, 152.6,
141.9, 139.9, 137.7, 134.7, 134.3, 132.0, 131.3, 130.8, 129.5,
127.4, 125.2, 122.0, 118.3, 116.6, 67.7, 46.8, 22.9, 21.8. Anal. Calcd
for C28H26Cl2N4O4S: C, 57.44; H, 4.48; N, 9.57; Cl, 12.22. Found: C,
57.11; H, 4.27; N, 8.91; Cl, 12.2.
To a solution of 2e (0.71 g, 2.31 mmol) dissolved in 16 mL of
acetone, 5 mL of Jones’ reagent was added. The reaction mixture
was stirred at room temperature and monitored by TLC. The reac-
tion mixture was poured into water and extracted with EtOAc. The
combined organic layers were washed with water and brine, dried
over Na2SO4, and concentrated in vacuo to afford 0.41 g (57%) of
the title acid: mp 208.0 °C. 1H NMR (CDCl3) d 9.09 (s, 1H), 7.98
(d, 1H), 7.90 (d, 1H), 7.81 (d, 2H), 7.54 (d, 1H), 7.43 (d, 1H), 7.35
(d, 2H), 2.45 (s, 3H). 13C NMR (CDCl3) d 171.2, 160.1, 151.6,
141.3, 134.0, 131.3, 130.3, 129.0, 126.9, 124.2, 123.5, 23.0.
General procedure of Mitsunobu reaction: EDCI and HOBT were
added to a solution of 3 in CH2Cl2. The reaction mixture was stirred
for 1–2 h. 4-Methyl-3-(morpholinosulfonyl)aniline was added to
the reaction and the reaction mixture was stirred at room temper-
ature overnight and quenched with water. Layers were separated
and the aqueous layer was extracted with EtOAc. The combined
organic solution was dried over Na2SO4 and then evaporated under
a vacuum. The resulting yellow solids were purified by flash col-
umn chromatography to afford 4a–4e.
4.1.19. N-(4-Methyl-3-(morpholinosulfonyl)phenyl)-3-(p-tolyl)-
1-(3-(trifluoromethyl) phenyl)-1H-pyrazole-4-carboxamide (4d)
To a solution of 3d (0.2 g, 0.58 mmol) in CH2Cl2 (10 mL) was
added EDCI (0.34 g, 1.76 mmol) and HOBT (0.24 g, 1.76 mmol).
The reaction mixture was stirred for 1 h. To the reaction was added
4-methyl-3-(morpholinosulfonyl)aniline (0.2 g, 0.78 mmol). The
reaction mixture was stirred at room temperature for 16 h and
quenched with water. The organic layer was collected and the
aqueous layer was extracted with EtOAc. The combined organic
solution was dried over Na2SO4, evaporated under a vacuum. The
resulting yellow solid was purified by flash column chromatogra-
phy (H/EtOAc 1:1) to afford 4d as a white solid (0.1 g, 31% yield),
mp 192.7 °C. 1H NMR (CDCl3) d 8.67 (s, 1H), 8.12 (s, 1H), 7.92 (d,
1H), 7.72 (s, 1H), 7.64 (d, 4H), 7.51 (t, 3H), 7.41 (d, 2H), 3.74 (d,
4.1.16. 1-(4-Fluorophenyl)-N-(4-methyl-3-
(morpholinosulfonyl)phenyl)-3-p-tolyl-1H-pyrazole-4-
carboxamide (4a)
To a solution of 3a (0.2 g, 0.67 mmol) in CH2Cl2 (10 mL) was
added EDCI (0.22 g, 1.16 mmol) and HOBT (0.16 g, 1.16 mmol).
The reaction mixture was stirred for 1 h. To the reaction was added
4-methyl-3-(morpholinosulfonyl)aniline (0.17 g, 0.68 mmol). The
reaction mixture was stirred at room temperature for 12 h and
quenched with water. Layers were separated, and the aqueous
layer was extracted with EtOAc. The combined organic solution
was dried over Na2SO4 and then evaporated under a vacuum. The

D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
2931
4H), 3.19 (d, 4H), 2.58 (s, 3H), 2.50 (s, 3H). 13C NMR (CDCl3) d 165.0,
151.6, 141.9, 139.9, 137.7, 134.7, 134.3, 132.0, 131.3, 130.8, 129.5,
127.4, 125.2, 124.1, 118.3, 116.6, 67.8, 46.9, 23.9, 23.6. Anal. Calcd
for C29H27F3N4O4S: C, 59.58; H, 4.66; N, 9.58; F, 9.75. Found: C,
60.07; H, 4.38; N, 9.53; F, 10.
aqueous layer was extracted with EtOAc. The combined organic
solution was dried over Na2SO4, evaporated under a vacuum. The
resulting yellow oil was purified by flash column chromatography
(H/EtOAc 1:1) to afford 4g as a white solid (0.2 g, 53% yield), mp
177.4 °C. 1H NMR (CDCl3) d 8.07 (s, 1H), 7.69 (s, 1H), 7.54 (d, 2H),
7.44 (d, 2H), 7.35 (d, 2H), 7.23 (s, 1H), 4.18 (m, 2H), 3.74 (d, 4H),
3.17 (d, 4H), 2.56 (s, 3H), 2.46 (s, 3H), 1.92 (t, 2H), 1.41 (t, 2H),
0.98 (t, 3H). 13C NMR (CDCl3) d 162.7, 151.3, 140.9, 137.6, 134.8,
131.7, 131.5, 130.7, 125.2, 122.1, 120.9, 116.9, 67.7, 53.9, 46.8,
33.5, 22.8, 21.4, 21.1, 14.9. Anal. Calcd for C26H32N4O4S: C, 62.88;
H, 6.49; N, 11.28. Found: C, 62.65; H, 6.27; N, 11.08.
4.1.20. 1-(Benzo[d]thiazol-2-yl)-N-(4-methyl-3-
(morpholinosulfonyl)phenyl)-3-(p-tolyl)-1H-pyrazole-4-
carboxamide (4e)
To a solution of 3e (0.2 g, 0.59 mmol) in CH2Cl2 (5 mL) was
added EDCI (0.14 g, 0.71 mmol) and HOBT (0.1 g, 0.7 mmol). The
reaction mixture was stirred for 1 h. To the reaction was added
4-methyl-3-(morpholinosulfonyl)aniline (0.15 g, 0.59 mmol). The
reaction mixture was stirred at room temperature for 12 h and
quenched with water. The organic layer was collected and the
aqueous layer was extracted with EtOAc. The combined organic
solution was dried over Na2SO4, evaporated under a vacuum. The
resulting yellow oil was purified by flash column chromatography
(H/EtOAc 1:1) to afford 4e as a pale-yellow solid (0.07 g, 21% yield),
mp 153.1 °C. 1H NMR (CDCl3) d 8.51 (s, 1H), 8.02 (s, 1H), 7.97 (d,
2H), 7.79 (d, 1H), 7.71 (t, 2H), 7.55 (t, 1H), 7.40 (dd, 4H), 3.74 (d,
4H), 3.19 (d, 4H), 2.63 (s, 3H), 2.44 (s, 3H). 13C NMR (CDCl3) d
165.0, 159.6, 144.5, 135.4, 137.7, 131.3, 131.0, 130.7, 130.2,
128.5, 126.1, 123.1, 123.0, 67.7, 47.7, 23.0, 21.7. Anal. Calcd for C29-
H27N5O4S2: C, 60.71; H, 4.74; N, 12.21. Found: C, 60.57; H, 4.2; N,
12.6.
General procedure of Mitsunobu reaction for producing 4f–4s:
EDCI and DMAP were added to a solution of 7 in CH2Cl2. To the
reaction was added 4-methyl-3-(morpholinosulfonyl)aniline. The
reaction mixture was stirred at room temperature for 6 h and
quenched with water and then treated with 10% HCl. The organic
layer was collected, and the aqueous layer was extracted with
EtOAc. The combined organic solution was dried over Na2SO4,
evaporated under a vacuum. The resulting yellow oil was purified
by flash column chromatography to afford 4f–4s.
4.1.23. N-(4-Methyl-3-(morpholinosulfonyl)phenyl)-1-
(naphthalen-2-ylmethyl)-3-(p-tolyl)-1H-pyrazole-4-
carboxamide (4h)
To a solution of 7c (0.2 g, 0.78 mmol) in CH2Cl2 (10 mL) was
added EDCI (0.18 g, 0.94 mmol) and DMAP (0.11 g, 0.94 mmol).
To the reaction was added 4-methyl-3-(morpholinosulfonyl)ani-
line (0.2 g, 0.78 mmol). The reaction mixture was stirred at room
temperature for 6 h and then treated with 10% HCl. The organic
layer was collected, and the aqueous layer was extracted with
EtOAc. The combined organic solution was dried over Na2SO4,
evaporated under a vacuum. The resulting yellow oil was purified
by flash column chromatography (H/EtOAc 1:1) to afford 4h as a
white solid (0.2 g, 50% yield), mp 144.9 °C. 1H NMR (CDCl3) d
8.13 (s, 1H), 7.85 (m, 4H), 7.68 (s, 1H), 7.53 (m, 4H), 7.45 (m,
2H), 7.35 (d, 2H), 7.20 (d, 2H), 5.51 (s, 2H), 3.72 (t, 4H), 3.15 (t,
4H), 2.51 (s, 3H), 2.46 (s, 3H). 13C NMR (CDCl3) d 162.2, 151.2,
141.2, 137.4, 136.9, 135.6, 134.8, 133.7, 131.3, 130.8, 130.4,
129.4, 129.2, 129.1, 128.1, 127.1, 125.1, 122.1, 67.8, 58.4, 46.9,
22.9, 21.6. Anal. Calcd for C33H32N4O4S: C, 68.25; H, 5.55; N, 9.65.
Found: C, 68.00; H, 6.04; N, 9.48.
4.1.24. 1-(Cyclopropylmethyl)-N-(4-methyl-3-
(morpholinosulfonyl)phenyl)-3-(p-tolyl)-1H-pyrazole-4-
carboxamide (4i)
Selected examples are given below:
To a solution of 7d (0.2 g, 0.78 mmol) in CH2Cl2 (10 mL) was
added EDCI (0.18 g, 0.94 mmol) and DMAP (0.11 g, 0.94 mmol).
To the reaction was added 4-methyl-3-(morpholinosulfonyl)ani-
line (0.2 g, 0.78 mmol). The reaction mixture was stirred at room
temperature for 6 h and then treated with 10% HCl. The organic
layer was collected, and the aqueous layer was extracted with
EtOAc. The combined organic solution was dried over Na2SO4,
evaporated under a vacuum. The resulting yellow solid was puri-
fied by crystallization from EtOAc gave 4i as a white solid (0.3 g,
78% yield), mp 210.0 °C. 1H NMR (CDCl3) d 8.23 (s, 1H), 7.71 (d,
1H), 7.54 (d, 2H), 7.46 (d, 2H), 7.35 (d, 2H), 7.21 (d, 1H), 4.05 (d,
2H), 3.74 (t, 4H), 3.17 (t, 4H), 2.51 (s, 3H), 1.36 (br s, 1H), 0.74 (q,
2H), 0.45 (t, 2H). 13C NMR (CDCl3) d 162.5, 150.8, 141.0, 137.6,
137.1, 131.3, 130.8, 125.0, 122.1, 67.8, 58.9, 46.9, 23.2, 21.9, 12.0,
4.1.21. 1-Isopropyl-N-(4-methyl-3-
(morpholinosulfonyl)phenyl)-3-(p-tolyl)-1H-pyrazole-4-
carboxamide (4f)
To a solution of 7a (0.11 g, 0.43 mmol) in CH2Cl2 (5 mL) was
added EDCI (0.14 g, 0.73 mmol) and DMAP (0.09 g, 0.73 mmol).
To the reaction was added 4-methyl-3-(morpholinosulfonyl)ani-
line (0.11 g, 0.43 mmol). The reaction mixture was stirred at room
temperature for 6 h, quenched with water and then treated with
10% HCl. The organic layer was collected, and the aqueous layer
was extracted with EtOAc. The combined organic solution was
dried over Na2SO4, evaporated under a vacuum. The resulting yel-
low oil was purified by flash column chromatography (H/EtOAc
1:1) to afford 4f as a white solid (0.11 g, 50% yield), mp 198.2 °C.
1H NMR (CDCl3) d 8.13 (s, 1H), 7.69 (s, 1H), 7.54 (d, 2H), 7.44 (d,
2H), 7.35 (d, 2H), 7.26 (d, 1H), 4.55 (m, 1H), 3.74 (d, 4H), 3.17 (d,
4H), 2.56 (s, 3H), 2.46 (s, 3H), 1.59 (d, 6H). 13C NMR (CDCl3) d
163.2, 145.3, 140.8, 135.2, 134.7, 130.3, 130.1, 126.1, 121.8,
120.9, 117.9, 109.9, 67.7, 56.5, 46.7, 24.1, 22.9, 21.1. Anal. Calcd
for C25H30N4O4S. ½H2O: C, 61.07; H, 6.15; N, 11.39. Found: C,
61.26; H, 6.31; N, 11.29.
1
5.6. Anal. Calcd for C26H30N4O4S. 4H2O: C, 61.98; H, 6.00; N,
11.02. Found: C, 61.63; H, 5.82; N, 10.84.
4.1.25. 1-(3-Methoxybenzyl)-N-(4-methyl-3-
(morpholinosulfonyl)phenyl)-3-(p-tolyl)-1H-pyrazole-4-
carboxamide (4j, DY268)
To a solution of 7e (0.1 g, 0.31 mmol) in CH2Cl2 (8 mL) was
added EDCI (0.071 g, 0.37 mmol) and DMAP (0.045 g, 0.37 mmol).
To the reaction was added 4-methyl-3-(morpholinosulfonyl)ani-
line (0.08 g, 0.31 mmol). The reaction mixture was stirred at room
temperature for 6 h and then treated with 10% HCl. The organic
layer was collected, and the aqueous layer was extracted with
EtOAc. The combined organic solution was dried over Na2SO4,
evaporated under a vacuum. The resulting yellow oil was purified
by flash column chromatography (H/EtOAc 1:1) to afford 4j as a
white solid (0.08 g, 47% yield), mp 183.3 °C. 1H NMR (CDCl3) d
4.1.22. 1-Butyl-N-(4-methyl-3-(morpholinosulfonyl)phenyl)-3-
(p-tolyl)-1H-pyrazole-4-carboxamide (4g)
To a solution of 7b (0.2 g, 0.77 mmol) in CH2Cl2 (10 mL) was
added EDCI (0.18 g, 0.92 mmol) and HOBT (0.12 g, 0.92 mmol).
The reaction mixture was stirred for 1 h. To the reaction was added
4-methyl-3-(morpholinosulfonyl)aniline (0.2 g, 0.77 mmol). The
reaction mixture was stirred at room temperature for 12 h and
quenched with water. The organic layer was collected, and the

2932
D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
8.01 (s, 1H), 7.69 (s, 1H), 7.55 (d, 2H), 7.44 (d, 2H), 7.34 (m, 3H),
7.22 (d, 2H), 6.93 (d, 1H), 6.87 (s, 1H), 5.32 (s, 2H), 3.85 (s, 3H),
3.74 (t, 4H), 3.17 (t, 4H), 2.56 (s, 3H), 2.46 (s, 3H). 13C NMR (CDCl3)
d 162.6, 160.0, 141.4, 137.1, 135.5, 134.8, 131.5, 131.3, 130.8,
125.1, 122.1, 115.5, 115.4, 67.7, 58.0, 56.7, 46.8, 22.8, 21.6. Anal.
Calcd for C30H32N4O5S: C, 64.27; H, 5.75; N, 9.99. Found: C,
64.01; H, 5.77; N, 9.63.
Acetic anhydride (3.8 g, 37.92 mmol) was then added, and reflux-
ing continued for about 12 h. The reaction mixture was cooled
and diluted with EtOAc, and water was added to the solution,
which was then stirred for 10 min to decompose excess triethylor-
thoformate. The aqueous layer was extracted with EtOAc. The
organic layers were washed with water and brine, dried over Na2-
SO4 and concentrated in vacuo to yield 2.49 g (92%) of the title
compound, which was used without further purification. 1H NMR
(CDCl3) d 7.52 (s, 1H), 4.29 (q, 2H), 4.19 (q, 2H), 3.14 (m, 1H),
1.37 (t, 3H), 1.32 (t, 3H), 1.16 (s, 3H), 1.11 (s, 3H). 13C NMR (CDCl3)
d 203.4, 165.8, 163.1, 114.7, 73.4, 62.2, 42.1, 20.2, 19.3, 16.6, 15.6.
4.1.26. N-(4-Methyl-3-(morpholinosulfonyl)phenyl)-3-(p-tolyl)-
1-(3-(trifluoromethyl)benzyl)-1H-pyrazole-4-carboxamide (4k)
To a solution of 7f (0.11 g, 0.34 mmol) in CH2Cl2 (5 mL) was
added EDCI (0.078 g, 0.41 mmol) and DMAP (0.05 g, 0.41 mmol).
To the reaction was added 4-methyl-3-(morpholinosulfonyl)ani-
line (0.087 g, 0.34 mmol). The reaction mixture was stirred at room
temperature for 6 h and then treated with 10% HCl. The organic
layer was collected and the aqueous layer was extracted with
EtOAc. The combined organic solution was dried over Na2SO4,
evaporated under a vacuum. The resulting yellow solid was puri-
fied by crystallization from EtOAc gave 4k as a white solid (0.1 g,
56% yield), mp 179.3 °C. 1H NMR (CDCl3) d 8.07 (s, 1H), 7.68 (s,
1H), 7.59 (s, 1H), 7.51 (t, 4H), 7.45 (t, 2H), 7.43 (d, 2H), 7.21 (d,
2H), 5.41 (s, 2H), 3.72 (t, 4H), 3.15 (t, 4H), 2.51 (s, 3H), 2.46 (s,
3H). 13C NMR (CDCl3) d 162.4, 151.6, 141.4, 137.3, 135.7, 134.8,
134.6, 132.8, 131.3, 130.8, 126.2, 125.1, 122.2, 118.4, 67.7, 57.4,
46.8, 22.8, 21.6. Anal. Calcd for C30H29F3N4O4S: C, 60.19; H, 4.88;
N, 9.36, F, 9.52. Found: C, 60.45; H, 4.70; N, 9.15, F, 9.20.
4.1.30. (Z)-Ethyl 3-ethoxy-2-(3-methoxybenzoyl)acrylate (5c)
Ethyl (3-methoxybenzoyl) acetate (1.0 g, 4.5 mmol) and tri-
ethylorthoformate (1.07 g, 7.2 mmol) were heated to reflux for
30 min. Acetic anhydride (1.37 g, 13.5 mmol) was then added,
and refluxing continued for about 12 h. The reaction mixture was
cooled and diluted with EtOAc, and water was added to the solu-
tion, which was then stirred for 10 min to decompose excess tri-
ethylorthoformate. The aqueous layer was extracted with EtOAc.
The organic layers were washed with water and brine, dried over
Na2SO4 and concentrated in vacuo to yield 1.1 g (88%) of the title
compound, which was used without further purification. 1H NMR
(CDCl3) d 7.68 (s, 1H), 7.47 (t, 2H), 7.35 (t, 1H), 7.11 (t, 1H), 4.17
(q, 2H), 4.11 (q, 2H), 1.16 (t, 3H), 1.06 (t, 3H). 13C NMR (CDCl3) d
193.3, 167.1, 162.9, 161.2, 140.1, 130.7, 130.5, 123.7, 121.4,
114.1, 73.2, 62.0, 56.8, 16.6, 15.6.
4.1.27. 1-(4-Hydroxybutyl)-N-(4-methyl-3-
(morpholinosulfonyl)phenyl)-3-(p-tolyl)-1H-pyrazole-4-
carboxamide (4l)
4.1.31. (Z)-Ethyl 3-ethoxy-2-(3-
(trifluoromethyl)benzoyl)acrylate (5d)
To a solution of 7g (0.09 g, 0.34 mmol) in CH2Cl2 (5 mL) was
added EDCI (0.078 g, 0.41 mmol) and DMAP (0.05 g, 0.41 mmol).
To the reaction was added 4-methyl-3-(morpholinosulfonyl)aniline
(0.087 g, 0.34 mmol). The reaction mixture was stirred at room tem-
perature for 6 h and then treated with 10% HCl. The organic layer
was collected, and the aqueous layer was extracted with EtOAc.
The combined organic solution was dried over Na2SO4, evaporated
under a vacuum. The resulting yellow oil was purified by flash col-
umn chromatography (CHCl3/MeOH 95:5) to afford 4l as a white
solid (0.05 g, 27% yield), mp 57.8 °C. 1H NMR (CDCl3) d 8.01 (s, 1H),
7.68 (s, 1H), 7.50 (d, 2H), 7.49 (t, 1H), 7.15 (t, 4H), 4.21 (d, 2H),
4.20 (d, 2H), 3.72 (t, 4H), 3.11 (t, 4H), 2.51 (s, 3H), 2.46 (s, 3H),
1.94 (t, 2H), 1.26 (t, 2H). 13C NMR (CDCl3) d 162.2, 154.9, 141.3,
139.8, 137.6, 135.2, 134.8, 131.8, 131.3, 130.6, 129.9, 125.2, 122.3,
67.7, 63.3, 53.8, 46.8, 30.7, 27.2, 21.6. Anal. Calcd for C26H32N4O4S:
C, 60.71; H, 4.74; N, 12.21. Found: C, 60.57; H, 4.2; N, 12.26.
Ethyl (4-trifluoromethylbenzoyl) acetate (1.0 g, 3.84 mmol) and
triethylorthoformate (0.91 g, 6.15 mmol) were heated to reflux for
30 min. Acetic anhydride (1.18 g, 11.53 mmol) was then added,
and refluxing continued for about 12 h. The reaction mixture was
cooled and diluted with EtOAc, and water was added to the solu-
tion, which was then stirred for 10 min to decompose excess tri-
ethylorthoformate. The aqueous layer was extracted with EtOAc.
The organic layers were washed with water and brine, dried over
Na2SO4 and concentrated in vacuo to yield 1.08 g (89%) of the title
compound, which was used without further purification. 1H NMR
(CDCl3) d 8.06 (d, 1H), 7.97 (d, 1H), 7.87 (s, 1H), 7.74 (d, 2H),
4.16 (q, 2H), 4.11 (q, 2H), 1.27 (t, 6H). 13C NMR (CDCl3) d 193.2,
164.1, 130.7, 130.3, 127.8, 127.3, 126.8, 114.1, 73.6, 62.1, 16.6,
15.5.
4.1.32. (Z)-Methyl 2-(ethoxymethylene)-5-methoxy-3-
oxopentanoate (5e)
4.1.28. (Z)-Ethyl 3-ethoxy-2-(4-methylbenzoyl)acrylate (5a)
Ethyl 3-oxo-3-(p-tolyl)propanoate (1.0 g, 4.85 mmol) and tri-
ethylorthoformate (1.15 g, 7.76 mmol) were heated to reflux and
stirred for 30 min. Acetic anhydride (1.5 g, 14.55 mmol) was then
added, and refluxing continued for 12 h. Then the mixture was
cooled and diluted with EtOAc, and water was added to the solution,
which was then stirred for 10 min to decompose excess triethylor-
thoformate. The aqueous layer was extracted with EtOAc. The
organic layers were washed with water and brine, dried over Na2SO4
and concentrated in vacuo to yield 1.21 g (94%) of the title com-
pound, which was used without further purification. 1H NMR (CDCl3)
d 7.82 (d, 2H), 7.69 (s, 1H), 7.27 (d, 2H), 4.17 (q, 2H), 4.11 (q, 2H), 2.40
(s, 3H), 1.28 (t, 3H), 1.17 (t, 3H). 13C NMR (CDCl3) d 192.9, 162.7,
145.5, 136.2, 130.8, 130.5, 130.3, 113.7, 73.1, 61.9, 23.1, 16.6, 15.6.
Methyl 5-methoxy-3-oxovalerate (2.0 g, 12.48 mmol) and tri-
ethylorthoformate (3.0 g, 19.96 mmol) were heated to reflux for
30 min. Acetic anhydride (3.82 g, 37.44 mmol) was then added,
and refluxing continued for about 6 h. The reaction mixture was
cooled and diluted with EtOAc, and water was added to the solu-
tion, which was then stirred for 10 min to decompose excess tri-
ethylorthoformate. The aqueous layer was extracted with EtOAc.
The organic layers were washed with water and brine, dried over
Na2SO4 and concentrated in vacuo to yield 2.27 g (84%) of the title
compound, which was used without further purification. 1H NMR
(CDCl3) d 7.90 (s, 1H), 4.25 (q, 2H), 3.66 (t, 2H), 3.35 (s, 3H), 3.23
(t, 2H), 1.31 (t, 3H). 13C NMR (CDCl3) d 200.5, 185.9, 165.7, 108.1,
67.7, 67.6, 60.3, 51.4, 38.8, 15.2.
4.1.33. Ethyl 3-(p-tolyl)-1H-pyrazole-4-carboxylate (6a)
4.1.29. (Z)-Ethyl 2-(ethoxymethylene)-4-methyl-3-
oxopentanoate (5b)
Ethyl isobutyryl acetate (2.0 g, 12.64 mmol) and triethylortho-
formate (3.0 g, 20.22 mmol) were heated to reflux for 30 min.
A solution of hydrazine monohydrate (0.2 mL, 4.2 mmol) in dry
ethanol (1 mL) was added dropwise at 0 °C to a solution of (Z)-
Ethyl 3-ethoxy-2-(4-methylbenzoyl)acrylate (5a) (1.0 g, 3.81 mmol)
in ethanol (5 mL). The reaction mixture was stirred at room

D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
2933
temperature for 12 h and the resulting solid was filtered, washed
with water and cold ethanol, and dried to afford 0.77 g (88%) of
6a as a white solid, mp 83.5 °C. 1H NMR (CDCl3) d 8.04 (s, 1H),
7.59 (d, 2H), 7.27 (d, 2H), 4.27 (q, 2H), 2.40 (s, 3H), 1.31 (t, 3H).
13C NMR (CDCl3) d 164.8, 149.3, 142.8, 140.8, 130.4, 130.3, 127.9,
113.1, 61.6, 22.8, 15.7.
acetate (3 ꢁ 15 mL). The combined organic layers were washed
with brine (3 ꢁ 10 mL), dried over Na2SO4, filtered and concen-
trated to give crude. 1H NMR (CDCl3) d 8.00 (s, 1H), 7.67 (d, 2H),
7.22 (d, 2H), 4.54 (m, 1H), 4.25 (q, 2H), 2.38 (s, 3H), 1.56 (d, 6H),
1.31 (t, 3H). The crude residue in ethanol (12 mL) was treated with
1 N aqueous potassium hydroxide (7.5 mL) at 75 °C for 2.5 h. The
reaction was cooled, acidified with 5 N HCl and the ethanol
removed in vacuo. The solids were filtered, washed with water,
hexanes and dried in vacuo to afford 0.31 g (58%) of 7a as a beige
powder, mp 129.0 °C. 1H NMR (CDCl3) d 8.06 (s, 1H), 7.66 (d, 2H),
7.21 (d, 2H), 4.53 (m, 1H), 2.38 (s, 3H), 1.56 (d, 6H). 13C NMR
(CDCl3) d 169.8, 154.6, 139.7, 134.6, 131.0, 130.6, 130.1, 55.9,
24.1, 24.0, 22.9.
4.1.34. Ethyl 3-isopropyl-1H-pyrazole-4-carboxylate (6b)
A solution of hydrazine monohydrate (0.67 g, 13.40 mmol) in
dry ethanol (1 mL) was added dropwise at 0 °C to a solution of
(Z)-ethyl 2-(ethoxymethylene)-4-methyl-3-oxopentanoate (5b)
(2.49 g, 11.62 mmol) in ethanol (10 mL). The reaction mixture
was stirred at room temperature for 12 h and the solvent was
removed, solidified by pet. ether to afford 1.28 g (61%) of 6b as a
white solid, mp 71.5 °C. 1H NMR (CDCl3) d 11.46 (br s, 1H), 7.96
(s, 1H), 4.32 (q, 2H), 3.69 (m, 1H), 1.36 (s, 6H), 1.33 (t, 3H). 13C
NMR (CDCl3) d 165.1, 142.1, 111.6, 61.3, 27.1, 22.9, 15.8.
4.1.39. 1-Butyl-3-(p-tolyl)-1H-pyrazole-4-carboxylic acid (7b)
A solution of 6a (0.5 g, 2.17 mmol), 1-bromobutane (0.29 g,
3.25 mmol) and cesium carbonate (2.12 g, 6.51 mmol) in acetoni-
trile (10 mL) was stirred at room temperature for 16 h, and the
reaction was stopped. Water (10 mL) was added into the reaction
mixture and the aqueous layer was extracted with ethyl acetate
(3 ꢁ 15 mL). The combined organic layers were washed with brine
(3 ꢁ 10 mL), dried over Na2SO4, filtered and concentrated. The
crude residue in ethanol (12 mL) was treated with 1 N aqueous
potassium hydroxide (7.5 mL) at 75 °C for 3.5 h. The reaction was
cooled, acidified with 5 N HCl and the ethanol removed in vacuo.
Solvent was removed and the resulting yellow oil was purified
by flash column chromatography (CHCl3/MeOH 9:1) to afford 7b
as pale-yellow oil (0.5 g, 50% yield). 1H NMR (CDCl3) d 8.06 (s,
1H), 7.66 (d, 2H), 7.21 (d, 2H), 4.53 (m, 1H), 2.38 (s, 3H), 1.56 (d,
6H). 13C NMR (CDCl3) d 169.8, 154.6, 139.7, 134.6, 131.0, 130.6,
130.1, 55.9, 24.1, 24.0, 22.9.
4.1.35. Ethyl 3-(3-methoxyphenyl)-1H-pyrazole-4-carboxylate
(6c)
A solution of hydrazine monohydrate (0.22 g, 4.35 mmol) in dry
ethanol (1 mL) was added dropwise at 0 °C to a solution of (Z)-
ethyl
3-ethoxy-2-(3-methoxybenzoyl)acrylate
(5c)
(1.1 g,
3.95 mmol) in ethanol (5 mL). The reaction mixture was stirred
at room temperature for 12 h, and the solvent was removed. The
crude was resolved in EtOAc, washed with water, and concentrated
to afford 0.97 g (98%) of 6c as a red oil, which was used without
further purification. 1H NMR (CDCl3) d 8.05 (s, 1H), 7.36 (t, 1H),
7.29 (t, 3H), 6.98 (d, 1H), 4.28 (q, 2H), 3.81 (s, 3H), 1.30 (t, 3H).
13C NMR (CDCl3) d 164.6, 160.7, 141.6, 132.2, 130.7, 122.8, 116.5,
116.2, 112.8, 61.6, 56.8, 15.7.
4.1.36. Ethyl 3-(4-(trifluoromethyl)phenyl)-1H-pyrazole-4-
carboxylate (6d)
4.1.40. 1-(Naphthalen-2-ylmethyl)-3-(p-tolyl)-1H-pyrazole-4-
carboxylic acid (7c)
A solution of hydrazine monohydrate (0.22 g, 4.35 mmol) in dry
ethanol (1 mL) was added dropwise at 0 °C to a solution of (Z)-
ethyl 3-ethoxy-2-(3-methoxybenzoyl)acrylate (5d) (1.08 g,
3.41 mmol) in ethanol (5 mL). The reaction mixture was stirred
at room temperature for 12 h and the solvent was removed. The
crude was resolved in EtOAc, washed with water, and concentrated
to afford a red oil which was solidified by EtOAc to give 0.79 g
(82%) of 6d as a pale red solid, mp 177.2 °C. 1H NMR (MeOD) d
8.12 (s, 1H), 7.84 (d, 2H), 7.67 (d, 2H), 4.28 (q, 2H), 1.29 (t, 3H).
13C NMR (MeOD) d 164.7, 136.1, 131.2, 131.1, 130.7, 127.4,
126.7, 126.2, 125.7, 112.6, 61.4, 14.4.
A solution of 6a (0.18 g, 0.78 mmol), 2-(bromomethyl)naphtha-
lene (0.26 g, 1.17 mmol) and cesium carbonate (0.76 g, 2.34 mmol)
in acetonitrile (5 mL) was stirred at room temperature for 16 h, and
the reaction was stopped. Water (10 mL) was added into the reac-
tion mixture and the aqueous layer was extracted with ethyl ace-
tate (3 ꢁ 15 mL). The combined organic layers were washed with
brine (3 ꢁ 10 mL), dried over Na2SO4, filtered and concentrated.
The crude residue in ethanol (10 mL) was treated with 1 N aqueous
potassium hydroxide (7.5 mL) at 75 °C for 3.5 h. The reaction was
cooled, acidified with 5 N HCl and the ethanol removed in vacuo
to afford 7c as a pale-yellow solid (0.2 g, 77% yield), mp 151.2 °C.
1H NMR (CDCl3) d 8.06 (s, 1H), 7.75 (m, 3H), 7.71 (d, 2H), 7.53 (d,
2H), 7.48 (m, 1H), 7.20 (m, 3H), 5.45 (s, 2H), 2.40 (s, 3H). 13C
NMR (CDCl3) d 169.3, 155.2, 139.8, 134.7, 131.3, 130.6, 130.5,
130.4, 129.9, 129.7, 129.6, 129.3, 129.2, 129.1, 128.9, 128.0, 59.9,
22.8.
4.1.37. Methyl 3-(2-methoxyethyl)-1H-pyrazole-4-carboxylate
(6e)
A solution of hydrazine monohydrate (0.57 g, 11.54 mmol) in
dry ethanol (1 mL) was added dropwise at 0 °C to a solution of
(Z)-methyl
2-(ethoxymethylene)-5-methoxy-3-oxopentanoate
(5e) (2.27 g, 10.49 mmol) in ethanol (7 mL). The reaction mixture
was stirred at room temperature for 12 h, and the solvent was
removed. The crude was resolved in EtOAc, washed with water,
and concentrated to afford a red oil which was solidified by EtOAc
to give 1.72 g (89%) of 6e as a yellow oil. 1H NMR (CDCl3) d 7.90 (s,
1H), 4.24 (q, 2H), 3.76 (s, 1H), 3.65 (t, 2H), 3.33 (s, 3H), 1.28 (t, 3H).
13C NMR (CDCl3) d 165.2, 148.3, 141.5, 122.4, 72.1, 60.3, 52.5, 27.3.
4.1.41. 1-(Cyclopropylmethyl)-3-(p-tolyl)-1H-pyrazole-4-
carboxylic acid (7d)
A solution of 6a (0.5 g, 2.17 mmol), bromomethylcyclopropane
(0.43 g, 3.25 mmol) and cesium carbonate (2.12 g, 6.51 mmol) in
acetonitrile (10 mL) was stirred at room temperature for 16 h.
Water (10 mL) was added into the reaction mixture and the aque-
ous layer was extracted with ethyl acetate (3 ꢁ 15 mL). The com-
bined organic layers were washed with brine (3 ꢁ 10 mL), dried
over Na2SO4, filtered and concentrated to give the crude. 1H NMR
(CDCl3) d 8.09 (s, 1H), 7.68 (d, 2H), 7.26 (d, 2H), 4.24 (q, 2H), 4.02
(t, 2H), 2.42 (s, 3H), 1.33 (br s, 1H), 1.22 (t, 3H), 0.72 (t, 2H), 0.48
(t, 2H). The crude residue in ethanol (10 mL) was treated with
1 N aqueous potassium hydroxide (7.5 mL) at 75 °C for 3.5 h. The
reaction was cooled, acidified with 5 N HCl and the ethanol
removed in vacuo. The solids were filtered, washed with water
4.1.38. 1-Isopropyl-3-(p-tolyl)-1H-pyrazole-4-carboxylic acid
(7a)
A solution of 6a (0.5 g, 2.17 mmol), 1-Bromo-2-methyl-propane
(0.55 g, 3.25 mmol) and cesium carbonate (2.12 g, 6.51 mmol) in
acetonitrile (10 mL) was stirred at room temperature for 16 h,
and the reaction was stopped. Water (10 mL) was added into the
reaction mixture, and the aqueous layer was extracted with ethyl

2934
D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
hexanes and dried over in vacuo to afford 7d as a pale-yellow solid
(0.3 g, 55% yield), mp 142.6 °C. 1H NMR (CDCl3) d 8.18 (s, 1H), 7.69
(d, 2H), 7.23 (d, 2H), 4.04 (d, 2H), 2.44 (s, 3H), 1.36 (br s, 1H), 0.74
(t, 2H), 0.44 (t, 2H). 13C NMR (CDCl3) d 169.7, 154.9, 143.7, 139.7,
136.6, 130.6, 130.5, 130.1, 112.0, 58.7, 22.8, 12.0, 5.5, 5.3.
2H), 1.55 (t, 2H). 13C NMR (CDCl3) d 168.8, 154.9, 139.7, 137.3,
131.1, 130.6, 130.1, 130.7, 112.0, 63.1, 53.7, 30.5, 27.9, 22.8.
4.1.45. 3-Isopropyl-1-(3-methoxybenzyl)-1H-pyrazole-4-
carboxylic acid (7h)
A solution of 6b (0.5 g, 2.74 mmol), 3-methoxybenzyl bromide
(0.83 g, 4.11 mmol) and cesium carbonate (2.68 g, 8.22 mmol) in
acetonitrile (10 mL) was stirred at room temperature for 16 h
and the reaction was stopped. Water (10 mL) was added into the
reaction mixture and the aqueous layer was extracted with ethyl
acetate (3 ꢁ 15 mL). The combined organic layers were washed
with brine (3 ꢁ 10 mL), dried over Na2SO4, filtered and concen-
trated to give crude. 1H NMR (CDCl3) d 7.73 (s, 1H), 7.29 (d, 1H),
6.87 (t, 1H), 6.83 (d, 1H), 6.77 (s, 1H), 5.22 (s, 2H), 4.26 (q, 2H),
3.82 (s, 3H), 3.54 (m, 1H), 1.35 (d, 6H), 1.26 (t, 3H). The crude res-
idue in ethanol (12 mL) was treated with 1 N aqueous potassium
hydroxide (7.5 mL) at 75 °C for 4.5 h. The reaction was cooled;
the ethanol was removed in vacuo, acidified with 5 N HCl, and
dried in vacuo to afford 7h as an white solid (0.59 g, 78% yield),
mp 111.9 °C. 1H NMR (CDCl3) d 7.79 (s, 1H), 7.29 (t, 1H), 6.88 (d,
1H), 6.84 (d, 1H), 6.78 (s, 1H), 5.23 (s, 2H), 3.79 (s, 3H), 3.55 (m,
1H), 1.32 (d, 6H). 13C NMR (CDCl3) d 169.6, 163.0, 161.5, 138.0,
136.4, 131.4, 121.7, 115.3, 115.1, 111.8, 57.5, 56.7, 28.0, 23.3.
4.1.42. 1-(3-Methoxybenzyl)-3-(p-tolyl)-1H-pyrazole-4-
carboxylic acid (7e)
A solution of 6a (0.1 g, 0.43 mmol), 3-methoxybenzyl bromide
(0.13 g, 0.65 mmol) and cesium carbonate (0.42 g, 1.29 mmol) in
acetonitrile (5 mL) was stirred at room temperature for 16 h. Water
(10 mL) was added to the reaction mixture, and the aqueous layer
was extracted with ethyl acetate (3 ꢁ 15 mL). The combined
organic layers were washed with brine (3 ꢁ 10 mL), dried over Na2-
SO4, filtered and concentrated to give the crude. 1H NMR (CDCl3) d
8.05 (s, 1H), 7.90 (d, 2H), 7.24 (m, 3H), 6.89 (d, 2H). 6.84 (s, 1H),
5.29 (s, 2H), 4.21 (q, 2H), 3.75 (s, 3H), 2.41 (s, 3H), 1.21 (t, 3H).
The crude residue in ethanol (5 mL) was treated with 1 N aqueous
potassium hydroxide (3.5 mL) at 75 °C for 3.5 h. The reaction was
cooled, acidified with 5 N HCl and the solvent removed in vacuo.
The solids were filtered, washed with hexanes and dried over in
vacuo to afford 7e as a white solid (0.1 g, 77% yield), mp
169.6 °C. 1H NMR (CDCl3) d 7.95 (s, 1H), 7.68 (d, 2H), 7.31 (t, 1H),
7.22 (t, 2H), 6.90 (d, 2H), 6.84 (s, 1H), 5.29 (b, 2H), 3.73 (s, 3H),
2.41 (s, 3H). 13C NMR (CDCl3) d 168.9, 155.2, 139.8, 137.3, 131.5,
131.2, 130.6, 130.5, 130.1, 121.8, 115.4, 115.3, 57.8, 56.7, 22.7.
4.1.46. 3-Isopropyl-1-(3-methoxybenzyl)-1H-pyrazole-4-
carboxylic acid (7i)
A solution of 6c (0.3 g, 1.22 mmol), 2-iododpropane (0.31 g,
1.83 mmol) and cesium carbonate (1.19 g, 3.66 mmol) in acetoni-
trile (5 mL) was stirred at room temperature for 16 h, and the reac-
tion was stopped. Water (10 mL) was added to the reaction
mixture, and the aqueous layer was extracted with ethyl acetate
(3 ꢁ 15 mL). The combined organic layers were washed with brine
(3 ꢁ 10 mL), dried over Na2SO4, filtered and concentrated to give
crude. 1H NMR (CDCl3) d 8.01 (s, 1H), 7.39 (d, 1H), 7.32 (t, 2H),
6.92 (d, 1H), 4.26 (q, 2H), 4.14 (m, 1H), 3.85 (s, 3H), 1.43 (d, 6H),
1.17 (t, 3H). 13C NMR (CDCl3) d 164.6, 161.6, 142.5, 133.5, 130.8,
123.5, 117.0, 116.1, 61.4, 56.7, 55.8, 51.9, 24.2, 15.7. The crude res-
idue in ethanol (12 mL) was treated with 1 N aqueous potassium
hydroxide (7.5 mL) at 75 °C for 12 h. The reaction was cooled;
the ethanol was removed in vacuo, acidified with 5 N HCl, and
dried in vacuo to afford 7i as a yellow oil (0.25 g, 78% yield). 1H
NMR (CDCl3) d 8.08 (s, 1H), 7.39 (d, 2H), 7.32 (t, 1H), 6.95 (d,
1H), 4.51 (m, 1H), 3.85 (s, 3H), 1.58 (d, 6H). 13C NMR (CDCl3) d
169.6, 160.6, 154.3, 143.5, 134.9, 134.7, 130.9, 123.3, 116.9,
115.9, 56.7, 55.9, 24.1, 24.0.
4.1.43. 3-(p-Tolyl)-1-(3-(trifluoromethyl)benzyl)-1H-pyrazole-4-
carboxylic acid (7f)
A solution of 6a (0.1 g, 0.43 mmol), 3-(trifluoromethyl)benzyl
bromide (0.15 g, 0.65 mmol) and cesium carbonate (0.42 g,
1.29 mmol) in acetonitrile (5 mL) was stirred at room temperature
for 6 h, and the reaction was stopped. Water (10 mL) was added to
the reaction mixture, and the aqueous layer was extracted with
ethyl acetate (3 ꢁ 15 mL). The combined organic layers were
washed with brine (3 ꢁ 10 mL), dried over Na2SO4, filtered and
concentrated to give the crude. 1H NMR (CDCl3) d 7.97 (s, 1H),
7.68 (d, 2H), 7.58 (s, 1H), 7.51 (m, 3H). 7.23 (d, 2H), 5.39 (s, 2H),
4.26 (q, 2H), 2.41 (s, 3H), 1.26 (t, 3H). The crude residue in ethanol
(5 mL) was treated with 1 N aqueous potassium hydroxide
(3.5 mL) at 75 °C for 5.5 h. The reaction was cooled, acidified with
5 N HCl and the ethanol removed in vacuo. The solids were filtered,
washed with water hexanes and dried over in vacuo to afford 7f as
a white solid (0.11 g, 77% yield), mp 149.9 °C. 1H NMR (CDCl3) d
8.01 (s, 1H), 7.67 (d, 2H), 7.52 (s, 1H), 7.49 (d, 2H), 7.21 (m, 3H),
5.39 (s, 2H), 2.41 (s, 3H). 13C NMR (CDCl3) d 169.2, 156.4, 143.9,
140.1, 137.65 132.7, 131.1, 130.7, 130.6, 130.1, 126.8, 126.2,
119.5, 118.5, 57.2, 22.7.
4.1.47. 1-(3-Methoxybenzyl)-3-(3-methoxyphenyl)-1H-
pyrazole-4-carboxylic acid (7j)
A solution of 6c (0.3 g, 1.22 mmol), benzyl-3-methoxybenzene
(0.37 g, 1.83 mmol) and cesium carbonate (1.19 g, 3.66 mmol) in
acetonitrile (5 mL) was stirred at room temperature for 6 h, and
the reaction was stopped. Water (10 mL) was added to the reaction
mixture and the aqueous layer was extracted with ethyl acetate
(3 ꢁ 15 mL). The combined organic layers were washed with brine
(3 ꢁ 10 mL), dried over Na2SO4, filtered and concentrated to give
crude. 1H NMR (CDCl3) d 7.92 (s, 1H), 7.39 (s, 1H), 7.32 (t, 3H),
6.94 (d, 3H), 6.84 (s, 1H), 5.31 (s, 2H), 4.24 (q, 2H), 3.85 (s, 3H),
3.76 (s, 3H), 1.86 (t, 3H). The crude residue in ethanol (12 mL)
was treated with 1 N aqueous potassium hydroxide (7.5 mL) at
75 °C for 12 h. The reaction was cooled; the ethanol was removed
in vacuo, acidified with 5 N HCl, and dried in vacuo to afford 7j as a
yellow oil (0.27 g, 66% yield). 1H NMR (CDCl3) d 7.96 (s, 1H), 7.38
(d, 2H), 7.32 (t, 3H), 6.92 (d, 2H), 6.84 (s, 1H), 5.29 (s, 2H), 3.83
(s, 3H), 3.73 (s, 3H). 13C NMR (CDCl3) d 168.7, 160.6, 161.1, 143.5,
137.7, 137.4, 134.6, 131.5, 130.4, 123.2, 121.8, 116.2, 115.9,
115.4, 115.3, 57.9, 56.7.
4.1.44. 1-(4-Hydroxybutyl)-3-(p-tolyl)-1H-pyrazole-4-carboxylic
acid (7g)
A solution of 6a (0.1 g, 0.43 mmol), 4-bromo-1-butanol (0.10 g,
0.65 mmol) and cesium carbonate (0.42 g, 1.29 mmol) in acetoni-
trile (5 mL) was stirred at room temperature for 6 h, and the reac-
tion was stopped. Water (10 mL) was added to the reaction
mixture, and the aqueous layer was extracted with ethyl acetate
(3 ꢁ 15 mL). The combined organic layers were washed with brine
(3 ꢁ 10 mL), dried over Na2SO4, filtered and concentrated to give
the crude. 1H NMR (CDCl3) d 7.98 (s, 1H), 7.65 (d, 2H), 7.20 (d,
2H), 4.24 (q, 2H). 4.09 (t, 2H), 3.45 (t, 2H), 2.41 (s, 3H), 1.67 (t,
2H), 1.57 (t, 2H), 1.42 (t, 3H). The crude residue in ethanol (5 mL)
was treated with 1 N aqueous potassium hydroxide (3.5 mL) at
75 °C for 5.5 h. The reaction was cooled; the ethanol was removed
in vacuo, acidified with 5 N HCl, and dried in vacuo to afford 7g as
an oil (0.09 g, 82% yield). 1H NMR (CDCl3) d 7.99 (s, 1H), 7.65 (d,
2H), 7.19 (d, 2H), 4.14 (q, 2H), 3.62 (t, 2H), 2.40 (s, 3H), 1.96 (t,

D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
2935
4.1.48. 1-Isopropyl-3-(4-(trifluoromethyl)phenyl)-1H-pyrazole-
4-carboxylic acid (7k)
To the reaction was added 4-methyl-3-(morpholinosulfonyl)ani-
line (0.25 g, 0.96 mmol). The reaction mixture was stirred at room
temperature for 12 h and then treated with 10% HCl. The organic
layer was collected, and the aqueous layer was extracted with
EtOAc. The combined organic solution was dried over Na2SO4,
evaporated under a vacuum. The resulting yellow oil was purified
by flash column chromatography (H/EtOAc 1:1) to afford 4n as a
white solid (0.26 g, 55% yield), mp 158.0 °C. 1H NMR (CDCl3) d
8.15 (s, 1H), 7.72 (s, 1H), 7.53 (s, 1H), 7.47 (d, 2H), 7.24 (d, 2H),
7.19 (s, 1H), 7.05 (m, 1H), 4.57 (m, 1H), 3.86 (s, 3H), 3.74 (t, 4H),
3.17 (t, 4H), 2.57 (s, 3H), 1.61 (s, 6H). 13C NMR (CDCl3) d 163.2,
161.7, 150.6, 142.2, 137.7, 136.9, 134.8, 134.5, 132.9, 132.6,
131.7, 125.6, 123.6, 123.1, 122.0, 117.6, 67.7, 56.8, 56.0, 46.9,
A solution of 6d (0.3 g, 1.06 mmol), 2-iodopropane (0.36 g,
1.83 mmol) and cesium carbonate (1.19 g, 3.66 mmol) in acetoni-
trile (5 mL) was stirred at room temperature for 6 h, and the reac-
tion was stopped. Water (10 mL) was added to the reaction
mixture, and the aqueous layer was extracted with ethyl acetate
(3 ꢁ 15 mL). The combined organic layers were washed with brine
(3 ꢁ 10 mL), dried over Na2SO4, filtered and concentrated to give
crude. 1H NMR (CDCl3) d 8.00 (s, 1H), 7.92 (s, 1H), 7.93 (d, 2H),
7.64 (d, 2H), 4.25 (q, 2H), 4.10 (m, 1H), 1.61 (d, 6H), 1.28 (t, 3H).
The crude residue in ethanol (10 mL) was treated with 2 N aqueous
potassium hydroxide (5.5 mL) at 75 °C for 12 h. The reaction was
cooled; the ethanol was removed in vacuo. The residue was taken
up with EtOAc and water and acidified with 5 N HCl to pH 3.
Organic phases were dried over Na2SO4, filtered and concentrated
in vacuo to afford 7k as a yellow solid (0.21 g, 67% yield), mp
161.1 °C. 1H NMR (CDCl3) d 10.17 (br s, 1H), 8.10 (s, 1H), 7.93 (d,
2H), 7.62 (d, 2H), 4.51 (br s, 1H), 1.52 (s, 6H). 13C NMR (CDCl3) d
168.1, 151.5, 143.9, 142.1, 135.9, 133.5, 132.7, 130.3, 129.6,
125.3, 54.7, 22.4, 22.3.
1
24.2, 21.7. Anal. Calcd for C25H30N4O5S. 4H2O: C, 59.53; H, 5.92;
N, 11.10. Found: C, 59.76; H, 5.67; N, 11.10.
4.1.52. 1-(3-Methoxybenzyl)-3-(3-methoxyphenyl)-N-(4-
methyl-3-(morpholinosulfonyl)phenyl)-1H-pyrazole-4-
carboxamide (4o)
To a solution of 7j (0.25 g, 0.73 mmol) in CH2Cl2 (5 mL) was
added EDCI (0.17 g, 0.88 mmol) and DMAP (0.11 g, 0.88 mmol).
To the reaction was added 4-methyl-3-(morpholinosulfonyl)ani-
line (0.19 g, 0.73 mmol). The reaction mixture was stirred at room
temperature for 12 h and then treated with 10% HCl. The organic
layer was collected, and the aqueous layer was extracted with
EtOAc. The combined organic solution was dried over Na2SO4,
evaporated under a vacuum. The resulting yellow oil was solidified
by MeOH to afford 4o as a white solid (0.31 g, 74% yield), mp
190.1 °C. 1H NMR (CDCl3) d 8.04 (s, 1H), 7.72 (s, 1H), 7.54 (s, 1H),
7.48 (t, 2H), 7.34 (s, 1H), 7.28 (m, 3H), 7.20 (d, 1H), 6.94 (t, 1H),
6.88 (s, 1H), 3.82 (s, 3H), 3.80 (s, 3H), 3.74 (t, 4H), 3.17 (t, 4H),
2.57 (s, 3H). 13C NMR (CDCl3) d 163.2, 161.6, 160.0, 151.4, 137.7,
137.4, 135.7, 134.9, 134.5, 131.7, 131.6, 125.0, 123.1, 122.1,
122.0, 116.9, 116.2, 115.5, 115.4, 67.8, 58.1, 56.9, 56.7, 46.9, 21.7.
Anal. Calcd for C30H32N4O5S. ½H2O: C, 61.51; H, 5.50; N, 9.56.
Found: C, 61.50; H, 5.28; N, 9.39.
4.1.49. 1-(3-Methoxybenzyl)-3-(4-(trifluoromethyl)phenyl)-1H-
pyrazole-4-carboxylic acid (7l)
A solution of 6d (0.2 g, 0.7 mmol), 3-methoxy benzyl bromide
(0.21 g, 1.05 mmol) and cesium carbonate (0.68 g, 2.1 mmol) in ace-
tonitrile (5 mL) was stirred at room temperature for 12 h. Water
(10 mL) was added to the reaction mixture, and the aqueous layer
was extracted with ethyl acetate (3 ꢁ 15 mL). The combined organic
layers were washed with brine (3 ꢁ 10 mL), dried over Na2SO4, fil-
tered and concentrated to give crude. 1H NMR (CDCl3) d 7.96 (t,
2H), 7.66 (d, 2H), 7.29 (d, 2H), 6.88 (d, 2H), 6.83 (s, 1H), 5.28 (s,
2H), 4.23 (q, 2H), 3.79 (s, 3H), 1.26 (t, 3H). The crude residue in eth-
anol (5 mL) was treated with 2 N aqueous potassium hydroxide
(4 mL) at 75 °C for 12 h. The reaction was cooled; the ethanol was
removed in vacuo. The residue was taken up with EtOAc/water and
acidified with 5 N HCl to pH 3. Organic phases were dried over Na2-
SO4, filtered and concentrated in vacuo to afford 7l as a yellow solid
4.1.53. 1-Isopropyl-N-(4-methyl-3-
(morpholinosulfonyl)phenyl)-3-(4-(trifluoromethyl)phenyl)-
1H-pyrazole-4-carboxamide (4p)
1
(0.21 g, 80% yield), mp 141.7 °C. H NMR (CDCl3) d 7.92 (s, 1H), 7.87
(d, 2H), 7.23 (t, 1H), 6.84 (d, 2H), 6.77 (s, 1H), 5.24 (s, 2H), 3.74 (s, 3H).
13C NMR (CDCl3) d 166.1, 160.8, 159.6, 142.4, 139.2, 135.9, 135.6,
130.0, 129.5, 129.1, 125.4, 119.8, 113.6, 113.0, 112.7, 64.8, 55.0.
To a solution of 7k (0.2 g, 0.67 mmol) in CH2Cl2 (5 mL) was
added EDCI (0.15 g, 0.81 mmol) and DMAP (0.08 g, 0.81 mmol).
To the reaction was added 4-methyl-3-(morpholinosulfonyl)ani-
line (0.17 g, 0.67 mmol). The reaction mixture was stirred at room
temperature for 12 h, quenched with water and then treated with
10% HCl. The organic layer was collected, and the aqueous layer
was extracted with EtOAc. The combined organic solution was
dried over Na2SO4, evaporated under a vacuum. The resulting yel-
low oil was purified by flash column chromatography (H/EtOAc
1:1) to afford 4p as a yellow solid (0.18 g, 51% yield), mp
192.2 °C. 1H NMR (CDCl3) d 8.12 (s, 1H), 8.09 (s, 1H), 7.87 (d, 2H),
7.81 (d, 2H), 7.65 (d, 2H), 7.24 (d, 1H), 4.53 (m, 1H), 3.64 (t, 4H),
3.07 (t, 4H), 2.54 (s, 3H), 1.53 (s, 6H). 13C NMR (CDCl3) d 161.5,
148.8, 138.6, 136.2, 134.9, 133.7, 133.3, 130.4, 129.3, 125.7,
119.4, 66.2, 66.1, 45.3, 45.2, 22.5, 20.1, 20.0. Anal. Calcd for C25H27-
F3N4O4S. ½H2O: C, 55.03; H, 4.98; N, 10.44. Found: C, 55.23; H,
5.03; N, 10.26.
4.1.50. 3-Isopropyl-1-(3-methoxybenzyl)-N-(4-methyl-3-
(morpholinosulfonyl)phenyl)-1H-pyrazole-4-carboxamide (4m)
To a solution of 7h (0.12 g, 0.44 mmol) in CH2Cl2 (5 mL) was
added EDCI (0.1 g, 0.53 mmol) and DMAP (0.07 g, 0.53 mmol). To
the reaction was added 4-methyl-3-(morpholinosulfonyl)aniline
(0.11 g, 0.44 mmol). The reaction mixture was stirred at room tem-
perature for 6 h and then treated with 10% HCl. The organic layer
was collected, and the aqueous layer was extracted with EtOAc.
The combined organic solution was dried over Na2SO4, evaporated
under a vacuum. The resulting yellow oil was purified by flash col-
umn chromatography (H/EtOAc 1:1) to afford 4m as a white solid
(0.18 g, 83% yield), mp 179.7 °C. 1H NMR (CDCl3) d 8.10 (s, 1H), 7.79
(dd, 3H), 7.31 (m, 2H), 6.83 (t, 2H), 6.82 (s, 1H), 5.28 (s, 2H), 3.81 (s,
3H), 3.66 (t, 4H), 3.59 (m, 1H), 3.11 (t, 4H), 2.60 (s, 3H), 1.39 (d, 6H).
13C NMR (CDCl3) d 163.2, 161.2, 160.0, 138.8, 138.5, 136.3, 134.5,
135.1, 131.8, 131.4, 126.1, 122.6, 122.7, 121.7, 115.2, 115.1, 67.6,
57.4, 56.7, 46.8, 28.4, 23.6, 21.5. Anal. Calcd for C26H32N4O5S: C,
60.92; H, 6.29; N, 10.93. Found: C, 60.45; H, 5.97; N, 10.51.
4.1.54. 1-(3-Methoxybenzyl)-N-(4-methyl-3-
(morpholinosulfonyl)phenyl)-3-(4-(trifluoromethyl)phenyl)-
1H-pyrazole-4-carboxamide (4q)
To a solution of 7l (0.06 g, 0.16 mmol) in CH2Cl2 (2 mL) was
added EDCI (0.04 g, 0.019 mmol) and DMAP (0.02 g, 0.019 mmol).
To the reaction was added 4-methyl-3-(morpholinosulfonyl)ani-
line (0.04 g, 0.16 mmol). The reaction mixture was stirred at room
temperature for 12 h, quenched with water and then treated with
4.1.51. 1-Isopropyl-3-(3-methoxyphenyl)-N-(4-methyl-3-
(morpholinosulfonyl)phenyl)-1H-pyrazole-4-carboxamide (4n)
To a solution of 7i (0.25 g, 0.96 mmol) in CH2Cl2 (5 mL) was
added EDCI (0.22 g, 1.15 mmol) and DMAP (1.15 g, 1.15 mmol).

2936
D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
10% HCl. The organic layer was collected and the aqueous layer was
extracted with EtOAc. The combined organic solution was dried
over Na2SO4, evaporated under a vacuum. The resulting yellow
oil was purified by flash column chromatography (H/EtOAc 1:1)
to afford 4q as a yellow solid (0.022 g, 61% yield), mp 208.2 °C.
1H NMR (CDCl3) d 7.98 (s, 1H), 7.89 (d, 2H), 7.81 (s, 1H), 7.74 (d,
2H), 7.62 (d, 1H), 7.47 (s, 1H), 7.27 (t, 1H), 7.26 (t, 1H), 6.94 9 m,
3H), 5.34 (s, 2H), 3.82 (s, 3H), 3.72 (t, 4H), 3.15 (t, 4H), 2.58 (s,
3H). 13C NMR (CDCl3) d 161.5, 148.8, 138.6, 136.2, 134.9, 133.7,
133.3, 130.4, 129.3, 125.7, 119.4, 66.2, 66.1, 45.3, 45.2, 22.5, 20.1,
20.0. Anal. Calcd for C30H29F3N4O5S. ½H2O: C, 57.77; H, 4.68; N,
8.98. Found: C, 58.08; H, 4.64; N, 8.64.
water (50 mL) was added into the reaction mixture and the aqueous
layer was extracted with ethyl acetate (3 ꢁ 25 mL). The combined
organic layers were washed with water and brine (3 ꢁ 10 mL), dried
over Na2SO4, filtered and concentrated. The resulting yellow oil was
purified by flash column chromatography (H/EtOAc 1:1) to afford 8c
as a clear oil (0.08 g, 87% yield). 1H NMR (CDCl3) d 7.45 (s, 1H), 7.21
(t, 2H), 7.14 (t, 1H), 6.85 (d, 2H), 6.80 (t, 3H), 6.72 (d, 2H), 6.70 (s, 1H),
5.12 (s, 2H), 3.72 (s, 6H), 3.58 (t, 4H), 3.26 (s, 3H), 2.84 (t, 4H), 2.41 (s,
3H). 13C NMR (CDCl3) d 164.3, 159.0, 158.6, 148.3, 140.8, 135.5,
134.4, 133.9, 132.6, 131.6, 131.3, 129.0, 128.3, 127.0, 119.3, 118.8,
113.3, 112.8, 111.3, 65.2, 54.3, 54.2, 44.0, 19.0. HRMS (ESI); Calcd
for C31H34N4O6S (M+1): 591.2272. Found: 591.2270.
4.1.55. 1-Isopropyl-N-methyl-N-(4-methyl-3-
(morpholinosulfonyl)phenyl)-3-(p-tolyl)-1H-pyrazole-4-
carboxamide (8a)
4.1.58. N-((1-Isopropyl-3-(p-tolyl)-1H-pyrazol-4-yl)methyl)-4-
methyl-3-(morpholinosulfonyl)aniline (9a)
To a solution of 4f (0.1 g, 0.21 mmol) in anhydrous THF (5 mL),
1 N borane-THF complex (7 mL) was added at 0 °C under N2. The
reaction mixture was heated to reflux for 3 h. After it was cooled
to 0 °C, 1 N methanolic HCl (3 mL) was added dropwise to quench
excess borane reagent and MeOH (5 mL) was added. The reaction
mixture was heated to reflux for 2 h. The solvent was evaporated
at reduced pressure. The oily residue was dissolved in EtOAc and
neutralized with 2 N NaOH. The resulting mixture was extracted
with EtOAc (3 ꢁ 15 mL). The combined extracts were washed with
0.5 N KOH, dried over Na2SO4, and evaporated in vacuo. The result-
ing yellow oil was purified by flash column chromatography (H/
EtOAc 1:1) to afford 9a as colorless oil (0.08 g, 81% yield). 1H
NMR (CDCl3) d 7.55 (d, 2H), 7.47 (s, 1H), 7.20 (d, 2H), 7.14 (s,
1H), 7.10 (d, 1H), 6.72 (d, 1H), 4.52 (m, 1H), 4.27 (s, 2H), 3.71 (t,
4H), 3.12 (t, 4H), 2.51 (s, 3H), 2.36 (s, 3H), 1.53 (d, 6H), 13C NMR
(CDCl3) d 150.5, 147.6, 138.8, 136.7, 135.1, 130.7, 128.8, 128.5,
127.6, 118.7, 115.7, 69.0, 46.7, 33.1, 24.1, 24.0. HRMS (ESI); Calcd
for C25H32N4O3S (M+1): 469.6116. Found: 469.2715.
A mixture of 4f (0.1 g, 0.21 mmol), NaH (60% dispersion in oil)
(0.018 g, 0.46 mmol) and anhydrous DMF (10 mL) was stirred at
room temperature for 30 min. The reaction mixture was cooled
to 0 °C, and iodomethane (0.1 mL, 1.47 mmol) was added to it.
The resulting mixture was stirred at room temperature under an
atmosphere of N2 and monitored by TLC. The reaction was stopped,
water (30 mL) was added to the reaction mixture, and the aqueous
layer was extracted with ethyl acetate (3 ꢁ 15 mL). The combined
organic layers were washed with water and brine (3 ꢁ 10 mL),
dried over Na2SO4, filtered and concentrated. The resulting yellow
oil was purified by flash column chromatography (H/EtOAc 1:1) to
afford 8a as a clear oil (0.07 g, 70% yield). 1H NMR (CDCl3) d 7.34 (s,
1H), 7.15 (d, 2H), 7.09 (t, 2H), 6.94 (d, 2H), 6.87 (d, 1H), 4.26 (m,
1H), 3.67 (t, 4H), 3.27 (s, 3H), 3.05 (t, 4H), 2.55 (s, 3H), 2.41 (s,
3H), 1.31 (d, 6H). 13C NMR (CDCl3) d 166.5, 144.0, 140.8, 136.8,
134.4, 131.6, 130.8, 130.7, 130.4, 129.6, 129.3, 128.5, 127.4, 67.6,
51.5, 46.6, 39.0, 22.8, 22.7, 21.6. HRMS (ESI); Calcd for C26H32N4O4S
(M+1): 497.2217. Found: 497.2215.
4.1.59. N-((3-Isopropyl-1-(3-methoxybenzyl)-1H-pyrazol-4-
yl)methyl)-4-methyl-3-(morpholinosulfonyl)aniline (9b)
4.1.56. 3-Isopropyl-1-(3-methoxybenzyl)-N-methyl-N-(4-
methyl-3-(morpholinosulfonyl)phenyl)-1H-pyrazole-4-
carboxamide (8b)
To a solution of 4m (0.1 g, 0.19 mmol) in anhydrous THF (5 mL),
1 N borane-THF complex (7 mL) was added at 0 °C under N2. The
reaction mixture was heated to reflux for 3 h. After it was cooled
to 0 °C, 1 N methanolic HCl (3 mL) was added dropwise to quench
excess borane reagent and additional MeOH was added. The reac-
tion mixture was heated to reflux for 3 h. The solvent was evapo-
rated at reduced pressure. The oily residue was dissolved in
EtOAc and neutralized with 2 N NaOH. The resulting mixture was
extracted with EtOAc (3 ꢁ 15 mL). The combined extracts were
washed with 0.5 N KOH, dried over Na2SO4, and evaporated in
vacuo. The resulting yellow oil was purified by flash column chro-
matography (H/EtOAc 1:1) to afford 9b as colorless oil (0.07 g, 77%
yield). 1H NMR (CDCl3) d 7.22 (d, 2H), 7.21 (s, 1H), 7.12 (s, 1H), 7.10
(d, 1H), 6.83 (d, 1H), 6.77 (d, 1H), 6.72 (t, 2H), 5.18 (s, 2H), 4.11 (t,
2H), 3.76 (s, 3H), 3.69 (t, 4H), 3.11 (t, 4H), 3.05 (m, 1H), 2.48 (s, 3H),
1.26 (d, 6H). 13C NMR (CDCl3) d 161.3, 157.7, 147.6, 139.6, 136.4,
135.1, 131.2, 130.5, 127.1, 121.3, 118.5, 116.6, 115.6, 114.8,
114.7, 67.7, 57.1, 56.6, 46.7, 39.8, 26.5, 24.0, 23.6, 21.1. HRMS
(ESI); Calcd for C26H34N4O4S (M+1): 499.6376. Found: 499.3676.
A mixture of 4m (0.3 g, 0.58 mmol), NaH (60% dispersion in oil)
(0.046 g, 1.27 mmol) and anhydrous DMF (15 mL) was stirred at
room temperature for 30 min. The reaction mixture was cooled
to 0 °C, and iodomethane (0.25 mL, 4.09 mmol) was added to it.
The resulting mixture was stirred at room temperature under an
atmosphere of N2 and monitored by TLC. The reaction was stopped,
water (50 mL) was added to the reaction mixture and the aqueous
layer was extracted with ethyl acetate (3 ꢁ 25 mL). The combined
organic layers were washed with water and brine (3 ꢁ 10 mL),
dried over Na2SO4, filtered and concentrated. The resulting yellow
oil was purified by flash column chromatography (H/EtOAc 1:1) to
afford 8b as a clear oil (0.27 g, 87% yield). 1H NMR (CDCl3) d 7.45 (s,
1H), 7.18 (dd, 3H), 6.78 (d, 1H), 6.52 (d, 2H), 6.50 (d, 1H), 4.95 (s,
2H), 3.71 (s, 3H), 3.63 (t, 4H), 3.34 (s, 3H), 3.31 (m, 1H), 2.82 (t,
4H), 2.53 (s, 3H), 1.23 (d, 6H). 13C NMR (CDCl3) d 166.5, 144.0,
140.8, 136.8, 134.4, 131.6, 130.8, 130.7, 130.4, 129.6, 129.3,
128.5, 127.4, 67.6, 51.5, 46.6, 39.0, 22.8, 22.7, 21.6. HRMS (ESI);
Calcd for C27H34N4O5S (M+1): 527.2323. Found: 527.2321.
4.1.60. N-((1-(3-Methoxybenzyl)-3-(3-methoxyphenyl)-1H-
pyrazol-4-yl)methyl)-4-methyl-3-(morpholinosulfonyl)aniline
(9c)
To a solution of 4o (0.1 g, 0.17 mmol) in anhydrous THF (5 mL),
1 N borane-THF complex (8 mL) was added at 0 °C under N2. The
reaction mixture was heated to reflux for 3 h. After it was cooled
to 0 °C, 1 N methanolic HCl (3 mL) was added dropwise to quench
excess borane reagent and additional MeOH was added. The
reaction mixture was heated to reflux for 2 h. The solvent was
evaporated at reduced pressure. The oily residue was dissolved in
4.1.57. 1-(3-Methoxybenzyl)-3-(3-methoxyphenyl)-N-methyl-N-
(4-methyl-3-(morpholinosulfonyl)phenyl)-1H-pyrazole-4-
carboxamide (8c)
A mixture of 4o (0.1 g, 0.17 mmol), NaH (60% dispersion in oil)
(0.02 g, 0.51 mmol) and anhydrous DMF (10 mL) was stirred at
room temperature for 30 min. The reaction mixture was cooled
to 0 °C, and iodomethane (0.075 mL, 1.21 mmol) was added to it.
The resulting mixture was stirred at room temperature under an
atmosphere of N2 and monitored by TLC. The reaction was stopped,

D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
2937
EtOAc and neutralized with 2 N NaOH. The resulting mixture was
extracted with EtOAc (3 ꢁ 15 mL). The combined extracts were
washed with 0.5 N KOH, dried over Na2SO4, and evaporated in
vacuo. The resulting yellow oil was purified by flash column chroma-
tography (H/EtOAc 1:1) to afford 9c as colorless oil (0.065 g, 72%
yield). 1H NMR (CDCl3) d 7.38 (s, 1H), 7.32 (m, 5H), 7.18 (d, 1H),
7.11 (s, 1H), 6.89 (t, 2H), 6.81 (d, 2H), 6.69 (t, 1H), 5.28 (s, 2H),
4.26 (s, 2H), 3.81 (s, 3H), 3.76 (s, 3H), 3.71 (t, 4H), 3.11 (t, 4H),
2.49 (s, 3H). 13C NMR (CDCl3) d 161.3, 161.2, 151.1, 147.3, 139.1,
136.4, 135.1, 131.7, 131.3, 127.4, 121.5, 118.6, 117.7, 115.7, 115.3,
115.1, 116.2, 114.8, 114.1, 67.7, 57.5, 56.6, 46.7, 40.5, 21.2. HRMS
(ESI); Calcd for C30H34N4O5S (M+Na): 565.6798. Found: 585.2798.
4.2.3. FXR cell-based transactivation assay
FXR transactivation assays were performed using GeneBLAzer
FXR-UAS-bla HEK 293T cells (Invitrogen). Cells were maintained
according to the manufacturer’s instructions. Briefly, cells were
grown in DMEM supplemented with 10% dialyzed FBS, 0.1 mM
non-essential amino acids, 25 mM HEPES, 100
cin B, 100 g/mL of zeocin, 100 units/mL penicillin and 100
streptomycin in regular tissue culture flasks. Upon transactivation
assay, DMSO, 400 nM GW4064, 10 M GW4064, and titrations of
l
g/mL of hygromy-
l
lg/mL
l
GW4064, LCA or chemicals without 400 nM GW4064 (for FXR ago-
nistic activity determination) or with 400 nM GW4064 (for FXR
antagonistic activity determination) were mixed with cells
(20,000 cells/well in a 30 lL) in assay medium (phenol red-free
DMEM supplemented with 2% charcoal/dextran-treated FBS,
0.1 mM non-essential amino acids, 1 mM sodium pyruvate,
4.2. Biology
100 units/mL penicillin and 100 lg/mL streptomycin) in black
384-well tissue culture-treated clear bottom plates (Corning Incor-
4.2.1. Materials
DMSO and charcoal/dextran-treated FBS were obtained from
Fisher Scientific (Pittsburgh, PA). GW4064 was purchased from
R&D Systems, Inc. (Minneapolis, MN); LCA and rifampicin were
purchased from Sigma-Aldrich (St. Louis, MO); TO901317 was
obtained from Cayman Chemical Company (Ann Arbor, MI);
Z26476908 (E16) was purchased from Enamine LLC (Monmouth
Junction, NJ). Black polypropylene 384-well plates were purchased
from Matrical Bioscience (Spokane, WA). Black 384-well tissue cul-
ture-treated clear bottom plates were obtained from Corning
Incorporated (Corning, NY). White tissue culture-treated 384-well
plates were purchased from PerkinElmer (Waltham, MA). GST-
hFXR-LBD, Tb-anti-GST antibody, coregulator buffer G, 1M DTT,
GeneBLAzer FXR-UAS-bla HEK 293T cells, DMEM, phenol red-free
DMEM, dialyzed FBS, non-essential amino acids, sodium pyruvate,
HEPES, hygromycin B, zeocin, penicillin/ streptomycin, G418 and
CCF4-AM substrate were purchased from Invitrogen (Carlsbad, CA).
All chemicals were initially solubilized in DMSO as 10 mM
stocks and then diluted in corresponding assay buffer or medium
to indicated concentrations. All experiments were repeated in
triplicate.
porated, Corning, NY). As a background control, the wells in col-
umn 24 of each plate were filled with 30 lL assay medium along
with 0.5% DMSO. The final DMSO concentration was 0.5% in each
assay well. Plates were incubated (16 h, cell culture incubator at
37 °C), after which 6 lL/well of loading solution with CCF4-AM
substrate was added. Plates were then incubated in the dark for
90 min at room temperature before measuring the fluorescent
emissions at 460 nm and 535 nm (using excitation at 400 nm) with
an Envision plate reader with bottom read capacity. After back-
ground signal subtraction for individual emission channels, emis-
sion signals at 460 and 535 nm from each well were used to
determine the ratio of 460 nm/535 nm. For agonist assays, the
DMSO group and 10 lM GW4064 group served as negative (0%
activation) and positive (100% activation) controls, respectively.
The% activation was calculated using Eq. 2.
%Activation ¼ 100%
Chemical460 nm=535 nm ꢀ DMSO460 nm=535 nm
ꢁ
:
10
l
MGW 4064460 nm=535 nm ꢀ DMSO460 nm=535 nm
ð2Þ
4.2.2. FXR TR-FRET binding assay
For antagonistic assays in which 400 nM GW4064 was included
in the assay conditions, the DMSO group and 400 nM GW4064
group served as positive (100% inhibition) and negative (0% inhibi-
tion) controls, respectively. The% inhibition was calculated using
Eq. 3.
The FXR TR-FRET binding assay was performed as previously
described23 with modifications. Briefly, in a black polypropylene
384-well plate, DMSO, 10 lM GW4064, and titrations of GW4064,
lithocholic acid or the synthesized chemicals were mixed with 10
nM GST-hFXR-LBD, 1.5 nM Tb-anti-GST antibody, and 10 nM
DY246 in a 20 lL/well of coregulator buffer G supplemented with
10 mM DTT. The final DMSO concentration was 0.4% for all wells.
The plate was spun down after a brief shake and incubated at room
temperature for 20 min. The TR-FRET emission signals at 520 nm
and 490 nm for each well were determined using a PHERAstar plate
reader (BMG LABTECH, Cary, NC) and an excitation wavelength of
%Inhibition ¼ 100% ꢀ 100%
ðChemical þ 400 nM GW 4064Þ460
ꢀ DMSO460
nm=535 nm
nm=535 nm
ꢁ
:
400nMGW 4064460
nm ꢀ DMSO460
nm=535
nm=535 nm
ð3Þ
For those chemicals with a maximal %inhibition >50% and activ-
ities that behaved in a dose-dependent manner, the inhibitory
activities at different concentrations were fit into a sigmoidal
dose-response equation with GraphPad PRISM 6.01 to derive IC50
values.
337 nm, 100
520 nm/490 nm ratio from each well was then calculated. The DMSO
group and 10 M GW4064 group served as negative (0% inhibition)
ls delay time, and 200 ls integration time. The
l
and positive (100% inhibition) controls, respectively. The %inhibition
of tested chemicals at given concentration was calculated based on
negative and positive controls using Eq. 1.
4.2.4. Cell-based cytotoxicity assay
Cytotoxicity assays were performed side-by-side with the FXR
agonistic assay using GeneBLAzer FXR-UAS-bla HEK 293T cells
and the CellTiter-GloÒ Luminescent Cell Viability Assay reagent
(Promega). Briefly, cells were maintained in DMEM supplemented
with 10% dialyzed FBS, 0.1 mM non-essential amino acids, 25 mM
%Inhibition ¼ 100% ꢀ 100%
Chemical520 nm=490 nm ꢀ 10
lMGW 4064520 nm=490 nm
MGW 4064520 nm=490 nm
ð1Þ
ꢁ
:
DMSO520 nm=490 nm ꢀ 10
l
HEPES, 100
lg/mL of hygromycin B, 100
lg/mL of zeocin, 100
For those chemicals with a maximal %inhibition >50% and activ-
ities that behaved in a dose-dependent manner, the inhibitory
activities at different concentrations were fit into a one-site com-
petitive binding equation with GraphPad PRISM 6.01 (GraphPad
Software, Inc., La Jolla, CA) to derive IC50 values.
units/mL penicillin and 100
culture flasks. For the cytotoxicity assay, DMSO, titrations of
GW4064, LCA or of chemicals were mixed with cells
l
g/mL streptomycin in regular tissue
(20,000 cells/well in a 30
lL) in assay medium (phenol red-free
DMEM supplemented with 2% charcoal/dextran-treated FBS,

2938
D. D. Yu et al. / Bioorg. Med. Chem. 22 (2014) 2919–2938
11. Renga, B.; Migliorati, M.; Mencarelli, A.; Cipriani, S.; D’Amore, C.; Distrutti, E.;
Fiorucci, S. Biochim. Biophys. Acta 2011, 1809, 157.
0.1 mM non-essential amino acids, 1 mM sodium pyruvate,
100 units/mL penicillin and 100 g/mL streptomycin) in black
384-well tissue culture-treated clear bottom plates. As a positive
control, the wells in column 24 of each plate were filled with
30 lL assay medium along with 0.5% DMSO. The final DMSO con-
l
12. Makishima, 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.
13. Urizar, N. L.; Liverman, A. B.; Dodds, D. T.; Silva, F. V.; Ordentlich, P.; Yan, Y. Z.;
Gonzalez, F. J.; Heyman, R. A.; Mangelsdorf, D. J.; Moore, D. D. Science 2002, 296,
1703.
14. Urizar, N. L.; Moore, D. D. Annu. Rev. Nutr. 2003, 23, 303.
15. Pellicciari, R.; Costantino, G.; Fiorucci, S. J. Med. Chem. 2005, 17, 1.
16. Sepe, V.; Bifulco, G.; Renga, B.; D’Amore, C.; Fiorucci, S.; Zampella, A. J. Med.
Chem. 2011, 54, 1314.
centration was 0.5% in each assay well. After a 16 h incubation in
a cell culture incubator at 37 °C, the cell plates were cooled at room
temperature for 20 min and then CellTiter-GloÒ reagent 20
lL/well
was added followed by an incubation for 20 min in the dark to
allow the development of optimal luminescence signal. The lumi-
nescence signal from each well was collected with an Envision
plate reader equipped with Ultra-sensitive Luminescence detector.
The DMSO group and no cell group served as negative (0% activa-
tion) and positive (100% activation) controls, respectively. The%
inhibition was calculated using Eq. 4 below.
17. Renga1, B.; Mencarelli, A.; D’Amore, C.; Cipriani, S.; D’Auria, M. V.; Sepe, V.;
Chini, M. G.; Monti, M. C.; Bifulco, G.; Zampella, A.; Fiorucci, S. PLoS One 2012, 7,
e30443.
18. Nam, S. J.; Ko, H.; Shin, M.; Ham, J.; Chin, J.; Kim, Y.; Kim, H.; Shin, K.; Choi, H.;
Kang, H. Bioorg. Med. Chem. Lett. 2006, 16, 5398.
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Novellino, E.; Limongelli, V.; Zampella, A.; Fiorucci, S. J. Med. Chem. 2013, 56,
4701.
20. Dussault, I.; Beard, R.; Lin, M.; Hollister, K.; Chen, J.; Xiao, J. H.; Chandraratna,
R.; Forman, B. M. J. Biol. Chem. 2003, 278, 7027.
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22. Sayin, S. I.; Wahlstroem, A.; Felin, J.; Jaentti, S.; Marschall, H.-U.; Bamberg, K.;
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and Use Thereof, WO/2007/052843, 2007.
Signalcompound ꢀ SignalNo
Cell
%Inhibition ¼ 100% ꢀ 100% ꢁ
ð4Þ
SignalDMSO ꢀ SignalNo
Cell
Acknowledgments
We thank Drs. Arthur D. Riggs and Wendong Huang for support
and encouragement. We also thank Min Lin for technical assistance
and Dr. Keely Walker for editing the manuscript. This work was
supported by the American Lebanese Syrian Associated Charities
(ALSAC), St. Jude Children’s Research Hospital (St. Jude), National
Institute of General Medical Sciences [Grant GM086415] (to T.C.),
and National Cancer Institute [Grant P30-CA21765].
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