Discovery of Tropifexor (LJN452), a Highly Potent Non-bile Acid FXR Agonist for the Treatment of Cholestatic Liver Diseases and Nonalcoholic Steatohepatitis (NASH)

Article abstract of DOI:10.1021/acs.jmedchem.7b00907

The farnesoid X receptor (FXR) is a nuclear receptor that acts as a master regulator of bile acid metabolism and signaling. Activation of FXR inhibits bile acid synthesis and increases bile acid conjugation, transport, and excretion, thereby protecting the liver from the harmful effects of bile accumulation, leading to considerable interest in FXR as a therapeutic target for the treatment of cholestasis and nonalcoholic steatohepatitis. We identified a novel series of highly potent non-bile acid FXR agonists that introduce a bicyclic nortropine-substituted benzothiazole carboxylic acid moiety onto a trisubstituted isoxazole scaffold. Herein, we report the discovery of 1 (tropifexor, LJN452), a novel and highly potent agonist of FXR. Potent in vivo activity was demonstrated in rodent PD models by measuring the induction of FXR target genes in various tissues. Tropifexor has advanced into phase 2 human clinical trials in patients with NASH and PBC.

Full text of DOI:10.1021/acs.jmedchem.7b00907

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Drug Annotation
Discovery of Tropifexor (LJN452), a Highly Potent Non-
Bile Acid FXR Agonist for the Treatment of Cholestatic
Liver Diseases and Nonalcoholic Steatohepatitis (NASH)
David C. Tully, Paul V Rucker, Donatella Chianelli, Jennifer Williams, Agnes Vidal,
Phil B. Alper, Daniel Mutnick, Badry Bursulaya, James Schmeits, Xiangdong Wu,
Dingjiu Bao, Jocelyn Zoll, Young Kim, Todd Groessl, Peter McNamara, H. Martin
Seidel, Valentina Molteni, Bo Liu, Andrew Phimister, Sean B Joseph, and Bryan Laffitte
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 16 Nov 2017
Downloaded from http://pubs.acs.org on November 16, 2017
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Discovery of Tropifexor (LJN452), a Highly Potent Non-Bile
Acid FXR Agonist for the Treatment of Cholestatic Liver
Diseases and Nonalcoholic Steatohepatitis (NASH)
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David C. Tully,* †‡ Paul V. Rucker,† Donatella Chianelli,† Jennifer Williams,† Agnès Vidal,† Phil B.
Alper,† Daniel Mutnick,† Badry Bursulaya,† James Schmeits,† Xiangdong Wu,† Dingjiu Bao,† Jocelyn
Zoll,† Young Kim,† Todd Groessl, † Peter McNamara,† H. Martin Seidel,†◊ Valentina Molteni,† Bo Liu,†
Andrew Phimister,‡ǁ Sean B. Joseph,†§ and Bryan Laffitte†
†Genomics Institute of the Novartis Research Foundation, San Diego, California 92121
‡Novartis Institutes for Biomedical Research, Emeryville, California 94608
KEYWORDS Farnesoid X Receptor, FXR Agonist, cholestasis, dyslipidemia, nonalcoholic
steatohepatitis (NASH)
ABSTRACT: The farnesoid X receptor (FXR) is a nuclear receptor that acts as a master regulator of
bile acid metabolism and signaling. Activation of FXR inhibits bile acid synthesis and increases bile
acid conjugation, transport, and excretion, thereby protecting the liver from the harmful effects of bile
accumulation, leading to considerable interest in FXR as a therapeutic target for the treatment of
cholestasis and non-alcoholic steatohepatitis. We identified a novel series of highly potent non-bile acid
FXR agonists that introduce a bicyclic nortropine-substituted benzothiazole carboxylic acid moiety onto
a trisubstituted isoxazole scaffold. Herein, we report the discovery of 1 (tropifexor, LJN452) a novel
and highly potent agonist of FXR. Potent in vivo activity was demonstrated in rodent PD models
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measuring the induction of FXR target genes in various tissues. Tropifexor has advanced into Phase 2
human clinical trials in patients with NASH and PBC.
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INTRODUCTION AND BIOLOGICAL RATIONALE
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The farnesoid X receptor (FXR) is a ligand-activated nuclear receptor that is highly expressed
in the liver, gall bladder, intestines and kidney, and acts as a key regulator of bile acid
production, conjugation, and transport.1 Bile acids are resorbed in the ileum and activate FXR,
which leads to increased expression of bile acid binding proteins and FGF19, a key metabolic
regulator. In the liver, FXR activation modulates expression of genes involved in bile acid
transport and metabolism, as well as glucose and lipid metabolism. Specifically, activation of
FXR directly increases the expression of enzymes responsible for bile acid detoxification and
canalicular and basolateral bile acid efflux, while also inhibiting bile acid synthesis and
basolateral bile acid uptake by hepatocytes.2 Activation of FXR can repress bile acid synthesis
by either of two complementary mechanisms. In the liver, FXR activation induces SHP
(NR0B2) which is a negative regulator of CYP7A1 expression, an enzyme which catalyzes the
rate limiting step of the neutral bile acid biosynthetic pathway.3 Activation of FXR in the
intestines induces transcription of the endocrine hormone FGF19, which is secreted into portal
circulation and subsequently binds to the FGFR4 receptor in the liver, which in turn activates a
MAPK signalling pathway that results in the downregulation of CYP7A1 expression, thereby
inhibiting bile acid synthesis.4
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Cholestasis is a pathological condition in which bile flow from the liver to the intestine is
reduced or interrupted due either to impaired secretion by hepatocytes, or to obstruction of bile
flow through the bile ducts. The consequence of the accumulation of bile salts is disruption of
cellular membranes, which results in injury to the affected tissues, whereby the liver is typically
most affected. Primary biliary cholangitis (PBC) is a chronic and progressive cholestatic
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disease, presumed to be autoimmune in origin, which results from the progressive destruction of
intrahepatic bile ducts.5 Decreased FXR expression and mutations of the FXR target genes
ABCB11 (encoding BSEP) or ABCB4 (encoding MDR3) are observed in hereditary forms of
human cholestasis and lead to a severe form of cholestasis known as progressive familial
intrahepatic cholestasis (PFIC-2 and -3).6,7 In addition, loss-of-function mutations in the NR1H4
gene that encodes FXR have been determined to be the cause for a neonatal form of PFIC.8
Ursodeoxycholic acid (UDCA) is a naturally occurring primary bile acid, and though not an FXR
agonist, is currently the standard of care for the treatment of PBC. Obeticholic acid (OCA), a
synthetic bile-acid FXR agonist, recently received accelerated approval for the treatment of PBC
in combination with UDCA for patients who do not respond adequately to UDCA therapy
alone.9
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In addition to its role in bile acid metabolism and signaling, FXR is also involved in regulation
of triglyceride metabolism and lipogenesis. FXR activation suppresses the activity of SREBP-
1c, a key transcription factor that regulates triglyceride synthesis by inducing enzymes involved
in lipogenesis. Non-alcoholic steatohepatitis (NASH) is a condition characterized by the
accumulation of fat in the liver (steatosis), inflammation, hepatocyte ballooning, and fibrosis.
NASH is a leading cause of cirrhosis and liver transplantation, and cases are expected to rise due
to the increased prevalence worldwide of obesity and type 2 diabetes. It is hypothesized that
FXR activation can reduce the liver steatosis, inflammation and fibrosis in patients with NASH
by suppressing lipogenesis and promoting triglyceride oxidation and clearance.
In a recent clinical trial in patients with NASH, treatment with OCA led to improvements in
serum transaminases and liver histology,10 however, higher rates of pruritus were experienced by
patients treated with OCA compared to placebo-treated patients.10,11 Pruritus, or severe itch, is a
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common symptom associated with cholestatic disorders, and recent clinical trials have
demonstrated that OCA therapy in patients with PBC further exacerbates pruritus, which may
ultimately limit the clinical utility of OCA in susceptible patient populations.11,12 OCA also
caused adverse effects on serum lipid profiles including elevated LDL cholesterol and total
cholesterol in heathy volunteers and in patients with NASH, which is a concern in a patient
population with an elevated risk for cardiovascular disease.10–13
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Our efforts to discover safe and more effective therapies for the treatment of cholestatic
disorders have focused on the development of novel FXR agonists to improve upon the efficacy,
safety, and tolerability of the current standard of care. To achieve this goal, the intended target
drug candidate profile was for a highly potent and selective, non-bile acid, FXR agonist that
would be effective at low systemic exposures. As FXR is expressed predominantly in the liver
and intestines, the desire to limit the oral bioavailability and avoid high systemic exposures
stemmed from the wish to avoid the prolonged systemic activation of FXR. Herein, we report
our discovery of a novel series of highly potent non-bile acid FXR agonists that features a
bicyclic [3.2.1] tropane linker ring as the key component responsible for the exceptional potency.
This series potently and selectively activates FXR in vitro, and optimized analogs potently
regulate FXR target genes in vivo at very low systemic exposures in preclinical
pharmacodynamic models. The most advanced compound from this series (1) has progressed
into Phase II clinical trials for the treatment of patients with PBC14 and NASH.15
BACKGROUND
Researchers at Glaxo-SmithKline (GSK) previously described the biological and
pharmacological activity of the synthetic FXR agonist GW4064 (2).16 Since this initial report,
there have been extensive optimization efforts by several groups to overcome liabilities of this
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scaffold class such as low aqueous solubility and overall poor ADME and PK properties.17–22
Detailed accounts of much of this research has been thoroughly summarized in recent review
articles.23,24 Researchers at GSK demonstrated that the stilbene moiety of 2 could be replaced
with a fused biaryl ring system such as the isoquinoline in GSK2324 (3, EC50 = 50 nM, 102%
efficacy).18 This additional conformational restraint retained potent in vitro activity with full
efficacy; however such analogs likely lacked the necessary physicochemical properties and PK
to progress into development. More recently, researchers at Eli Lilly reported that the central
phenyl ring could be replaced by a piperidine ring exemplified by 4 (LY2562175, EC50 = 193
nM, 41% efficacy).25 The replacement of the extended conjugated aromatic system with a
piperidine linker resulted in an improvement in overall drug-like properties, despite resulting in a
loss of potency and only partial FXR agonist activity relative to 2 and 3.
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RESULTS AND DISCUSSION
Early in our efforts to identify a novel full FXR agonist with improved potency and PK, we
discovered that replacement of the indole moiety of the partial FXR agonist 4 with a 2-
substituted benzothiazole-6-carboxylic acid resulted in a dramatic improvement in potency and
exhibited full agonist efficacy in a cellular assay.26,27 The ligand-induced interaction with a
coactivator protein is a critical step in transcriptional activation by FXR, and this is determined
in the FXR-HTRF biochemical assay that measures the interaction between FXR and the Steroid
Receptor Coactivator-1 (SRC1). Benzothiazole 5 was determined to have an EC50 = 8.3 nM with
96% efficacy relative to 2, the most commonly used comparator FXR agonist (Table 1). The
potent functional activity of 5 translates well in the FXR BSEP-luc reporter gene cellular assay,
which is designed to measure cellular transcriptional activity of full length FXR on the BSEP
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promoter driving a luciferase reporter gene. Compound 5 has an EC50 = 9.5 nM in this cellular
assay and activates FXR as a full agonist with 118% efficacy relative to 2.
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The pharmacokinetics in rats was determined, and 5 exhibited high plasma clearance and low
oral bioavailability (F = 6%) (Table 2). In vitro metabolic stability in rat and human liver
microsomes (RLM, HLM) showed moderate to high extraction ratios, (Table 1) which was
consistent with the observed in vivo rat clearance, and suggested that oral bioavailability was
limited by the high in vivo clearance. Furthermore, in vitro metabolite ID studies in liver
microsomes suggested oxidative metabolism on the 2,6-dichlorophenyl ring as the primary route
for metabolic clearance, which was not surprising due to the highly lipophilic nature of that
moiety.
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Having identified that benzothiazole moiety provides a significant improvement in potency
versus 4 with full agonist efficacy, this carboxylic acid-bearing pharmacophore was fixed while
exploring the SAR in other regions of the scaffold for further optimization. In order to reduce
address the metabolic liability, replacement of the 2,6-dichlorophenyl group was explored in the
attempt to improve the metabolic clearance and aqueous solubility. Replacement of the 2,6-
dichlorophenyl with either 2,6-difluorophenyl (6) or 2-trifluoromethylphenyl (7) resulted in a
modest loss of FXR agonist activity, and metabolic stability in RLM was not improved, although
the HLM stability showed some improvement compared to 5.
In contrast, the 2-
trifluoromethoxyphenyl group (compound 8) proved to be highly beneficial in several respects.
Compound 8, in addition to improved FXR agonist activity (EC50 = 19 nM) and full efficacy
(100%), showed a notable improvement in metabolic stability, with low extraction ratios in rat
and human liver microsomes. The improvement in the in vitro metabolic stability translated
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nicely in vivo, as 8 had significantly improved rat pharmacokinetics versus the 2,6-dichloro
analog 5, with moderate clearance (24 mL/min/kg) and modest oral bioavailability (F = 37%).
Next, we examined the SAR involving replacements for the piperidine ring which serves as the
framework linking the essential trisubstituted isoxazole pharmacophore to the aryl carboxylic
acid moiety. A decrease in ring size to the azetidine (9) or pyrrolidine (10) was not tolerated,
and resulted in a substantial loss of FXR agonist activity (>100-fold). Conversely, an increase in
ring size to the racemic azepane (11) afforded a modest improvement in potency compared to
piperidine 8. However, despite this improvement in FXR potency versus the piperidine 8, the
azepane analog 11 was undesirable due to its poor metabolic stability in liver microsomes, and
displayed high extraction ratios in both RLM and HLM (0.75, 0.89).
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In the continued effort to optimize the linker moiety, we sought to increase the conformational
restraint of this central scaffolding ring. We therefore installed the 8-azabicyclo[3.2.1]octane ring
derived from nortropine, which resulted in a dramatic improvement in potency. Nortropine 12
had full FXR agonist efficacy with sub-nM potency in both the FXR-HTRF coactivator
interaction assay (EC50 = 0.54 nM) and FXR BSEP-luc cellular assay (EC50 = 0.69 nM). This
significant development represented a 30-fold improvement in potency over piperidine precursor
8, and more than two orders of magnitude greater potency versus 4.
Importantly, the in vitro ADME properties of nortropine 12 were attractive, with decent
solubility (110 µM) in fasted state simulated intestinal fluid (FASSIF, pH = 6.8), and it showed
moderate and low extraction ratios in rat (0.51) and human (<0.20) liver microsomes,
respectively. Nortropine 12 exhibited reduced plasma clearance in rats (CL = 17 mL/min/kg)
versus piperidine 8, although the terminal half-life was short and oral bioavailability was low (F
= 11%) (Table 2). Having achieved a remarkable improvement in potency for this scaffold, we
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focused on further optimization of this promising nortropine lead. The focus of continued
optimization remained to achieve reduced metabolic clearance and longer in vivo half-life in
order to improve in vivo activity in a rodent PD model measuring induction of FXR target genes
in the liver and intestines. Along with preserving the highly potent FXR agonist activity, limited
systemic exposure and oral bioavailability was desired in order to minimize prolonged systemic
activation in FXR, particularly in tissues other than liver and intestines.
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To this end, we examined the influence of various substituents at the synthetically accessible
4-position of the benzothiazole ring (Table 3). While the addition of either a methyl (13) or
methoxy (14) group retained potent FXR activity, the rat PK was not significantly improved, as
both compounds had low oral bioavailability and short T1/2, despite the low clearance (Table 2).
In contrast, the 4-fluoro substituted analog 1 gave further modest improvement in the FXR
agonist activity (EC50 = 0.26 nM) compared to 12 with full agonist activity in the FXR BSEP
cellular assay. Likewise the solubility in FASSIF (600 µM) was improved considerably
compared to close analogs 12, 13, and 14, possibly explained by the lower pKa of the 4-fluoro
substituted benzothiazole carboxylic acid (pKa = 4.0).
Moreover, the rat pharmacokinetics were further improved, as compound 1 had low clearance
(CL = 9 mL/min/kg) and a significantly longer terminal half-life (T1/2 = 3.7 h) than what had
been observed with previous analogs from this scaffold, and was consistent with the low
extraction ratio seen in rat liver microsomes. Oral bioavailability of 1 in the rat was 20% from
an aqueous microemulsion formulation, a modest improvement over other nortropine analogs
(Table 2). With the 4-fluorobenzothiazole in place, further examination of the phenyl ring
substituent demonstrated that the ortho-trifluoromethoxy group could be modified to either
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difluoromethoxy 15 or trifluoromethyl 16 (LJP305) while preserving similarly potent FXR
agonist activity with comparable rat pharmacokinetics.
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To help rationalize the remarkable boost in potency attained through the introduction of the
novel nortropine-benzothiazole pharmacophore, we examined computational molecular
modeling simulations of 1 docked into the X-ray structure of the FXR ligand binding domain
(PDB 3DCT). In good agreement with published X-ray co-crystal structures of related
isoxazoles bound to the FXR ligand binding domain, the docking model in Figure 2 showed the
key H-bond between the isoxazole nitrogen and H447 and hydrophobic interactions with W468
of helix 12.17–19 The cyclopropyl group fills the hydrophobic pocket formed by side chains of
F284, L287, F461, and W454, while the 2-trifluoromethoxyphenyl ring establishes hydrophobic
contacts with M328, M365, and I352. The bridged bicyclic nortropine ring system fills the
central portion of the binding pocket, and, due to its limited flexibility, provides conformational
restraint to the central linker section of the ligand. Furthermore, the ethylene bridge establishes a
favorable intramolecular hydrophobic interaction with the face of the 2-trifluoromethoxyphenyl
ring. Additionally, the benzothiazole ring efficiently fills the narrow portion of the hydrophobic
pocket and delivers the optimal geometry for the carboxylic acid substituent to establish the
essential electrostatic interaction with R331 in helix 5.
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Having achieved the optimization of highly potent FXR agonists with improved rat PK,
compounds 1 and 16 were selected for further evaluation in pre-clinical models of FXR
activation. Compound 1 was examined in rat primary hepatocytes in an assay designed to
measure the induction of FXR target genes BSEP and SHP mRNA by quantitative real-time PCR
(qPCR). Robust induction of both target genes was observed in primary cells by 1 in a
concentration-dependent manner (Figure 3). BSEP induction above vehicle (DMSO) control
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was observed at concentrations as low as 1 nM, while strong induction of SHP (15-fold above
vehicle) was observed at 10 nM, and modest induction of SHP at 1 nM (3-fold).
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Compounds 1 and 16 were evaluated in a PD model designed to measure the induction of FXR
target genes in the rat following a single oral dose. Both test compounds were administered
orally using a range of four doses (0.03, 0.1, 0.3, and 1.0 mg/kg) and compared directly to the
vehicle control group (vehicle: 0.5% methylcellulose, 0.5% Tween80, 99% water, suspension).
Tissues were harvested 7 h post-dose, and mRNA was isolated from the liver and ileum and
analyzed by real-time quantitative PCR using primer and probe sets specific to SHP (NR0B4),
BSEP (ABCB11) and FGF15. Both compounds demonstrated robust induction of FXR target
genes in a dose-dependent fashion with highly potent in vivo activity. Similar levels of induction
of SHP in the liver were observed, with maximal induction of ~6-fold achieved at 1.0 mg/kg for
both compounds 1 and 16, while significant BSEP induction was observed at the lower dose of
0.3 mg/kg (Figure 4). In the ileum, where FXR is also highly expressed, FXR target genes SHP
and FGF15 (rodent ortholog of human FGF19) were potently induced by 16 at 0.3 mg/kg (Figure
4). Even more impressively, compound 1 demonstrated highly potent induction of SHP and
FGF15 in the ileum as doses as low as 0.1 mg/kg, with mRNA levels >20-fold above vehicle
treated animals (Figure 5).
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In order to more extensively evaluate the pharmacological activity and PK/PD relationship of
compound 1 at steady state, an integrated PK/PD study was carried out in naïve rats that were
treated orally with compound 1 once daily for 14 days. Considering the potent activity observed
in the previous single dose PD study, a wider range of six doses were chosen to better determine
the minimally efficacious dose and exposure required for regulation of FXR target genes: 0.003,
0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 mg/kg, all administered orally using a standard suspension
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formulation (0.5% methylcellulose, 0.5% Tween80, 99% water). Multiple plasma samples were
collected from each group on days 1 and 11 for determination of pharmacokinetics. Tissues
(liver and ileum) were harvested on day 14 of the study 7 h after the final dose, and mRNA was
analyzed by real-time qPCR. In addition, the total compound concentrations in the liver were
determined at 1 h and 7 h for each dosing group.
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In the liver, robust induction of SHP was observed at 0.01 mg/kg of compound 1 with maximal
levels of gene induction achieved at 0.3 mg/kg. Initial BSEP induction in the liver appeared at
the lowest dose tested (0.003 mg/kg), and maximal BSEP target gene induction was achieved
above 0.1 mg/kg (Figure 6). Activation of FXR in the liver resulted in the downregulation of
sterol 12-α hydroxylase (CYP8B1) gene transcription, thereby reducing the conversion of
cholesterol to bile acids. Expression of CYP8B1 mRNA following 14 day treatment with 1 was
already apparent at the lowest dose (0.003 mg/kg), and CYP8B1 gene expression was fully
repressed at doses above 0.03 mg/kg. Extrapolation of the data provided a mean ED50 of
approximately 0.02 mg/kg in the liver for SHP and BSEP and 0.01 mg/kg for CYP8B1 (Table 4).
In the ileum, FXR target genes SHP and FGF15 are robustly induced at similarly low doses
down to 0.01 mg/kg as shown in Figure 7. The mean ED50 for induction of SHP in the ileum
was determined to be approximately 0.01 mg/kg, while for FGF15 in the ileum ED50 was around
0.05 mg/kg.
Serum triglycerides and plasma FGF15 protein levels were also analyzed, as these circulating
biomarkers could potentially be utilized as clinically translatable indicators of target engagement.
Activation of FXR in the ileum induces the expression of FGF15/FGF19, which is then secreted
in the portal blood and signals repression of CYP7A1 expression in the liver. In agreement with
the observation of increased FGF15 mRNA expression in the ileum, treatment of rats with
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compound 1 exhibited a clear dose-dependent increase in plasma FGF15 protein, with maximal
levels of FGF15 detected at 7 h post-dose (Figure 8). Importantly, at 24 h following the final
dose, plasma FGF15 protein concentrations had normalized to baseline levels, suggesting that
repeat daily administration of compound 1 does not result in the prolonged activation of FXR.
Serum triglycerides are an indirect biomarker of FXR activation, whereby FXR directly
induces SHP and downregulates the transcription factor SREBP-1c which is a master regulator
of fatty acid and lipid synthesis. Treatment with compound 1 for 14 days produced a robust
dose-dependent reduction in serum triglycerides and reached a maximal response with a 0.3
mg/kg dose, resulting in a decrease of triglyceride levels to approximately 79% below the
vehicle control group (Figure 9).
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Plasma samples were collected at multiple time points (0.5, 1, 3, 7, and 24 h) on days 1 and 11
for determination of the pharmacokinetics of compound 1 in this study following the first dose
and at steady state. The plasma Cmax occurred between 3 h and 7 h, and total concentrations
were generally similar on day 1 as on 11, ranging from 150 pM at the lowest dose to 171 nM at
the highest dose. Compound 1 demonstrated a linear increase in exposure with increasing dose
across the entire range of doses. The plasma exposures were very low at efficacious doses, with
AUC ranging from 2 nM*h at the lowest dose (0.003 mg/kg) up to 1688 nM*h at the highest
dose (3.0 mg/kg) (Table 5 and Figure 10). Importantly, no significant accumulation was
observed at steady state following multiple days of administration compared to the exposures
observed on day 1 following the initial dose.
Liver exposures were determined at the end of study on day 14 at 1 h and 7 h following the
final dose (Table 6). Mean concentrations of 1 in the liver were low single digit nM levels at the
lowest doses, and ranged between 600-900 nM at the highest dose. The liver concentrations
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averaged approximately 9-fold higher than mean plasma concentrations, with the exception of
the two lowest doses, which showed somewhat higher liver:plasma concentration ratios (average
of 18.5). The higher liver:plasma ratios observed at the two lowest doses may have been due to
inherent variability in the quantitation methods as the plasma concentrations were well below 1
nM and near the limit of detection.
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It is noteworthy that efficacy was observed at such low doses (mean ED50 range 0.01–0.05
mg/kg) with plasma Cmax concentrations near 1 nM and below, especially considering that PPB
of compound 1 is approximately 99.9%. This implies that the unbound concentration of 1 in the
plasma is well below the cellular EC50 (0.26 nM) at efficacious doses. Moreover, the minimal
total plasma concentrations of 1 that are sufficient to regulate FXR target gene expression in the
liver and intestines are only marginally higher than the cellular EC50 (day 11 Cmax = 1.9 at 0.03
mg/kg dose). The liver concentrations of 1 at the lowest effective doses (0.01 and 0.03 mg/kg,
nearest to ED50 values in Table 4) ranged from 5 nM to 12 nM, which are roughly 8 to 22-fold
higher than corresponding plasma concentrations at the same doses. Unbound concentrations of
1 in the liver were not determined in this study since drug concentrations were quantified from
tissue homogenates, and therefore a strict PK/PD relationship for the free drug concentrations of
1 required to regulate target gene expression in tissues cannot be explicitly determined.
Moreover, while this PK data suggests that the concentration of compound 1 required for
pharmacological activity is within the vicinity of the cellular EC50, further studies are warranted
to delineate whether the pharmacodynamics of an FXR agonist regulating target gene expression
is driven primarily by Cmax or by AUC.
It is instructive to compare the minimum effective concentrations of 1 required for induction of
BSEP and SHP in the liver in the rat PD model to the concentrations required in primary rat
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hepatocytes (Figure 3). The liver concentrations of 1 (5–12 nM) at the lowest effective doses
(0.01–0.03 mg/kg) are consistent with and in the same range as the lowest effective
concentrations required in vitro to drive expression of FXR target genes BSEP and SHP in
primary hepatocytes (1–10 nM range, Figure 3). Considering these relatively low total drug
concentrations in the liver and very high PPB (~99.9%), it is reasonable to estimate that the
efficacious free drug concentrations in the liver are likely near or possibly even below the
cellular EC50 (0.26 nM). This suggests the notion that the activation, or occupancy, of only a
fraction of the total available receptors is required for a full agonist of FXR to elicit a
pharmacological response, as is often the case for agonists of other nuclear receptors and
GPCRs.
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Extensive preclinical profiling demonstrated that 1 was highly selective for FXR: a broad
panel screen of enzyme, ion channel, nuclear receptor, and GPCR assays showed no significant
off-target activity (>10000-fold selectivity for FXR, data not shown). Importantly, compound 1
displayed no measurable activity on TGR5, the bile acid-activated GPCR (>10 ꢀM) (Table 7).
This is significant as several recent reports have proposed that activation of TGR5 may be
responsible for mediating bile acid-induced itch.28,29 Recent clinical trials in patients with PBC
treated with OCA have established a dose-dependent relationship between OCA and the
incidence and severity of pruritus.11,30 To further highlight the potential advantages of a non-
bile acid derived FXR agonist, we also tested OCA and its primary active metabolites, the
glycine and taurine conjugates of OCA in the same assay measuring TGR5 activation. Whereas
OCA itself is a rather weak agonist of hTGR5 with EC50 = 0.918 µM (83% efficacy), the glycine
and taurine conjugates are considerably more potent hTGR5 agonists, with EC50 values 0.315
µM and 0.205 µM respectively, both with full (100%) efficacy versus control (Table 7).
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Following daily administration of OCA, the glycine and taurine conjugates of OCA undergo
enterohepatic recirculation and eventually incorporate into the bile acid pool, reaching steady
state serum concentrations of 13.8 and 12.3-fold higher than parent OCA itself.31 Moreover, at
therapeutic doses of OCA, the total concentration of OCA and its taurine and glycine conjugates
are near or above the EC50 concentrations for hTGR5 activation, which could help explain the
dose-dependent relationship between OCA and pruritus.11
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The pharmacokinetics of compound 1 was evaluated in the mouse and in the dog, and this data
is shown in Table 8 for comparison to rat PK. In the mouse, following IV administration
compound 1 exhibited low clearance and small volume of distribution, similar to the rat, with a
half-life of 2.6 h. In the dog, following IV injection, the clearance was low and T1/2 was 7.4 h,
also with a small volume of distribution (0.46 L/kg). Plasma samples from the dog PK studies
were also monitored for any conjugated metabolites of compound 1. Only very low levels of the
acylglucuronide metabolite (≤1 nM) could be detected in some animals, however there were no
taurine or glycine conjugated metabolites of 1 observed in any of the animals. Oral
bioavailability in all three species (mouse, rat, and dog) was about 10% following administration
using an aqueous methylcellulose suspension formulation. The pharmacokinetic parameters
from all three species were used for prediction of human PK and estimation of the efficacious
human clinical dose using GastroPlus modeling and simulation software. Simulated human PK
predicted human clearance to be low, with a moderate T1/2, and oral bioavailability projected at
17%. Using the simulated human PK, the clinically efficacious human dose of compound 1 was
projected to be in the range of 30 µg to 110 µg once daily, and thereby supported advancement
to the clinic.
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A first-in-human Phase 1 clinical study was recently completed in which the safety,
tolerability, PK, and PD of compound 1 was assessed in healthy volunteers. Compound 1 was
found to be generally safe and well-tolerated following single oral doses ranging from 10 µg to 3
mg. Notably, dose-dependent, transient increases in circulating levels of FGF19 protein were
observed, demonstrating potent on-target FXR agonist activity. Preliminary data from this
clinical study has recently been disclosed,32a and detailed data and results will be published
elsewhere.32b
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CHEMICAL SYNTHESIS OF COMPOUND 1
The synthesis of compound 1, outlined in Scheme 1, was initiated from the commercially
available 2-(trifluoromethoxy)benzaldehyde (17a). Chloroxime 19a was prepared from aldehyde
17a by condensation with hydroxylamine, followed by chlorination with NCS, with both steps
proceeding in high yield.
Next, cycloaddition of 19a with methyl 3-cyclopropyl-3-
oxopropanoate afforded the desired isoxazole 20a in moderate yield. The methyl ester was then
reduced using LiAlH4 to give the corresponding primary alcohol 21a. Subsequent bromination
of alcohol 21a, followed by alkylation with N-Boc nortropine under basic conditions resulted in
the Boc-protected intermediate 1f, which was then treated with TFA to afford nortropine 1g.
Benzothiazole 25a was prepared in two steps from commercially available aniline 23a which
was treated with NaSCN and bromine using the classical Hugerschoff reaction to generate the
aminobenzothiazole 24a. Next, the aminobenzothiazole 24a was brominated under modified
Sandmeyer reaction conditions using tert-butyl nitrite to yield the desired bromobenzothiazole
25a. N-arylation of amine 1g with 25a was carried out under mild thermal conditions using
Cs2CO3 in DMF. Finally, saponification of the methyl ester generated the carboxylic acid 1 in
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nine total linear steps. The synthesis and characterization of all other analogs is described in
supporting information and in references 24 and 25.
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CONCLUSION
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Lead optimization successfully overcame the ADME and physicochemical liabilities of earlier
phenylisoxazole FXR agonists by significantly improving the potency and modestly improving
the ADME and PK properties. Compound 1 is a highly potent and efficacious non-bile acid
FXR agonist that is effective at inducing FXR target genes in the liver and intestines following
very low doses and systemic exposures. Compound 1 (LJN452, tropifexor) was found to be
generally safe and well-tolerated at pharmacologically active doses in healthy volunteers and has
progressed into clinical development where it is currently under evaluation in several Phase 2
human clinical trials in patients with PBC and NASH.14,15
EXPERIMENTAL SECTION
Materials and Methods
Commercially available starting materials were used as supplied without further purification.
Reactions were carried out using dry organic solvents (DCM, ACN, DMF, etc.) unless otherwise
noted. Reactions were monitored using thin layer chromatography and an Agilent Technologies
1200 series 6140 Quadrupole LC-MS with UV detection at 254 nm in electrospray ionization
(ESI) mode. For LC-MS, all retention times reported are at 254 nm UV channel unless
otherwise noted. All NMR spectra were recorded on a Bruker AVANCE-400 spectrometer
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operating at a frequency of 400.13 MHz for H and 100.61 MHz for C equipped with a 5mm
1
QNP cryoprobe with Z-gradient. Chemical shifts for H and 13C spectra were referenced to
residual solvent. MS were obtained on an Agilent Technologies 1200 series 6140 Quadrupole
LC-MS in electrospray ionization (ESI) mode. HRMS-ESI data were recorded using an Agilent
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6520 Accurate-Mass Q-TOF LC-MS system with HPLC-Chip Cube interface and an Agilent
1200 HPLC. All final compounds were isolated analytically pure, >99% purity by HPLC unless
otherwise indicated. More information is in the Supporting Information.
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N-hydroxy-2-(trifluoromethoxy)benzimidoyl chloride (19a). To a solution of sodium
hydroxide (7.00 g, 175 mmol, 1.19 equiv) in water (120 mL) was added a stirred solution of
NH2OH•HCl (11.8 g, 170 mmol, 1.15 equiv) in water (120 mL) at 0 °C. The resulting solution
was stirred for 10 min at 0 °C. A solution of 2-(trifluoromethoxy)benzaldehyde 17a (28.0 g, 147
mmol, 1.00 equiv) in ethanol (120 mL) was then added. The reaction was allowed to stir for an
additional 1 h at room temperature, at which point it was diluted with water (500 mL). The
aqueous layer was extracted with ethyl acetate (2×700 mL), and the organic layers were
combined, washed with brine (2×300 mL), dried over anhydrous sodium sulfate, and
concentrated under vacuum to give 30.0 g of (E)-2-(trifluoromethoxy)benzaldehyde oxime 18a
(83% yield). The oxime 18a was carried directly into the next step without further purification.
LC-MS m/z 206.0 (M+H); method A; RT = 1.43 min. NCS (22.0 g, 166 mmol, 1.12 equiv) was
slowly added to a stirred solution of (E)-2-(trifluoromethoxy) benzaldehyde oxime 18a (30.0 g,
146 mmol, 1.00 equiv) in DMF (300 mL) keeping the internal temperature below 25 °C. The
reaction mixture was stirred for 1 h at room temperature, and then diluted with water (300 mL)
and extracted with ethyl acetate (2×500 mL). The organic layers were combined, washed with
brine (5×300 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum to give
35.0 g of N-hydroxy-2-(trifluoromethoxy)benzimidoyl chloride 19a as a light yellow residue
1
(99% yield). This material was used directly in the next step without further purification. H
NMR (d4-MeOD, 400 MHz) δ 7.95 – 7.90 (m, 1H), 7.83 (d, J = 7.6, 1H), 7.62– 7.43 (m, 2H).
LC-MS m/z 240.0 (M+H); method A; RT = 2.03 min.
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Methyl
5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazole-4-carboxylate
(20a).
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Potassium carbonate (11.0 g, 79.7 mmol, 1.09 equiv) was suspended in THF (100 mL), and the
mixture was cooled to –10 °C and treated with a solution of methyl 3-cyclopropyl-3-
oxopropanoate (11.0 g, 77.5 mmol, 1.06 equiv) in THF (50 mL) dropwise over 5 minutes. To
this reaction mixture was then added a solution of N-hydroxy-2-(trifluoromethoxy)benzimidoyl
chloride 19a (17.6 g, 73.6 mmol, 1.0 equiv) in THF (50 mL) and monitored to ensure that the
internal temperature did not exceed –5 °C during the time of the addition. Following this
addition, the reaction was warmed to 35 °C. Upon cooling, the reaction mixture was diluted with
water (200 mL) and extracted with ethyl acetate (2×300 mL). The organic layer was washed
with brine (2×200 mL), dried over anhydrous sodium sulfate, concentrated under vacuum, and
then purified by silica gel column chromatography using ethyl acetate/petroleum ether (1:100-
1:20) to afford methyl 5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazole-4-carboxylate 20a
as a white solid, 12.5 g (52% yield), M.P. 145–150 °C (broad). 1H NMR (d4-MeOD, 400 MHz)
δ 7.65–7.60 (m, 1H), 7.53 (dd, J = 7.6, 2Hz, 1H), 7.48–7.43 (m, 2H), 3.68 (s, 3H), 2.89 (pentet, J
= 6.8Hz, 1H), 1.30 (app br d, J = 6.8 Hz, 4H). LC-MS m/z 328.1 (M+H); method A; RT = 2.91
min.
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(5-Cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-yl)-methanol (21a). A 250-mL
round-bottom flask was purged and maintained with an inert atmosphere of nitrogen, and then
charged with a suspension of LiAlH4 (2.50 g, 65.8 mmol, 2.87 equiv) in THF (50 mL). A
solution of methyl 5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl) isoxazole-4-carboxylate 20a
(7.50 g, 22.9 mmol, 1.00 equiv) in tetrahydrofuran (50 mL) was added dropwise at –10 °C. The
resulting solution was stirred for 30 min at –10 °C, and the reaction was quenched by the
sequential addition of ethyl acetate (3 mL), water (3 mL), and 15% aqueous NaOH (10 mL).
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The resulting mixture was filtered through celite, and the filter cake was washed with ethyl
acetate (200 mL). The filtrate was washed with brine (2×100 mL), dried over anhydrous sodium
sulfate, and concentrated under vacuum, affording 7 g of (5-cyclopropyl-3-(2-
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(trifluoromethoxy)phenyl)isoxazol-4-yl)methanol 21a as yellow oil, (93% yield). H NMR
(CDCl3, 400 MHz) δ 7.57–7.55 (m, 2H), 7.41–7.40 (m, 2H), 4.50 (s, 2H), 2.22–2.20 (m, 1H),
1.72 (s, 1H) 1.11–1.28 (m, 4H). LC-MS m/z 300.1 (M+H); method B; RT = 1.69 min.
Tert-Butyl
3-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-yl)methoxy)-8-
azabicyclo[3.2.1]octane-8-carboxylate (1f). Into a 100 mL round bottom flask was placed (5-
cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-yl)-methanol 21a (4.00 g, 13.3 mmol),
triphenylphosphine (5.6 g, 20 mmol, 1.5 equiv), and dichloromethane (40 mL). The mixture was
stirred until completely dissolved and then slowly added dropwise to a solution of carbon
tetrabromide (6.6 g, 20 mmol, 1.5 equiv) in dichloromethane (20 ml). The reaction was stirred
for 1 h, and the solvent was evaporated in vacuo. The crude material was purified by silica gel
chromatography using a gradient of ethyl acetate/hexanes (0–50%). The bromide 22a was
obtained as a clear oil (4.18 g, 87% yield) and then used immediately (owing to stability) in the
1
next step. H NMR (CDCl3, 400 MHz) δ 7.55–7.50 (m, 1H), 7.49–7.45 (m, 1H), 7.40–7.32 (m,
2H), 4.27 (s, 2H), 2.11–1.99 (m, 1H), 1.24–1.17 (m, 2H), 1.17–1.09 (m, 2H). LC-MS m/z
362.0/364.0 (M + H); (Br isotope pattern); method B; RT = 1.62 min. Into a dry 250-mL flask
was placed N-Boc nortropine (2.9 g, 13 mmol), 18-crown-6 (3.4 g, 12 mmol) and anhydrous
THF (80 mL). Potassium tert-butoxide (2.9 g, 26 mmol) was added portionwise, and the mixture
was stirred vigorously under nitrogen for 1h. 4-(Bromomethyl)-5-cyclopropyl-3-(2-
(trifluoromethoxy)-phenyl)isoxazole 22a (4.18 g, 11.6 mmol) was dissolved in anhydrous THF
(20 mL) and added dropwise. The reaction mixture was stirred overnight under a positive
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nitrogen pressure. The solvent was removed in vacuo, and the mixture was diluted with water
(100 mL) and ethyl acetate (100 mL). The organic layer was separated, dried (MgSO4),
evaporated under vacuum, and then purified by silica gel chromatography using a gradient of
ethyl acetate/hexanes (0–100%) to provide the desired product as a yellow oil, 4.07 g (69%
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yield). H NMR (DMSOd6, 400 MHz) δ 7.98–7.64 (m, 3H), 7.64 (app d, J = 7.6, 1H), 4.38 (s,
2H), 3.82 (bs, 2H), 3.56 (t, J = 4.5Hz, 1H), 2.36–1.98 (m, 3H), 1.82–1.70 (m, 6H), 1.15 (s, 9H),
1.14–1.07 (m, 4H). LC-MS m/z 509.2 (M+H); method A; RT = 2.42 min.
4-((8-Azabicyclo[3.2.1]octan-3-yloxy)methyl)-5-cyclopropyl-3-(2-
(trifluoromethoxy)phenyl)isoxazole
(1g).
tert-Butyl-3-((5-cyclopropyl-3-(2-
1f
(trifluoromethoxy)phenyl)isoxazol-4-yl)methoxy)-8-azabicyclo[3.2.1]octane-8-carboxylate
(3.98 g) was dissolved in a solution of 20% TFA in dichloromethane (30 mL). The solution was
stirred for 1 h at room temperature, and then the solvent was evaporated. The residue was
dissolved in ethyl acetate (125 mL) and washed with a saturated solution of sodium bicarbonate
(100 mL). The organic layer was dried with anhydrous sodium sulfate and evaporated in vacuo.
The crude residue was purified by silica gel chromatography using a gradient of 0–20%
ethanol/dichloromethane to afford 3.01 g of the desired product 1g as a colorless oil (94% yield).
1H NMR (DMSOd6, 400 MHz) δ 8.51 (br s, 1H, NH), 7.72–7.68 (m, 1H), 7.64 (dd, J = 7.6,
1.8Hz, 1H), 7.58–7.52 (m, 2H), 4.33 (s, 2H), 3.81 (bs, 2H), 3.55 (t, J = 4.5Hz, 1H), 2.36–2.33
(m, 1H), 1.98 (app dt, J = 14.8, 4.0 Hz, 2H), 1.91–1.76 (m, 6H), 1.14–1.07 (m, 4H). LC-MS m/z
409.2 (M+H); method A; RT = 1.72 min.
2-(3-((5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazol-4-yl)methoxy)-8-
azabicyclo[3.2.1]octan-8-yl)-4-fluorobenzo[d]thiazole-6-carboxylic acid (1). Into a 25-mL
round-bottom flask equipped with a stir bar, the following were added sequentially: 4-((8-
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azabicyclo[3.2.1]octan-3-yloxy)methyl)-5-cyclopropyl-3-(2-(trifluoromethoxy)phenyl)isoxazole
1g (0.525 g, 1.29 mmol), 3.6 mL of DMA, cesium carbonate (1.08 g, 3.31 mmol), and methyl 2-
chloro-4-methoxybenzo[d]thiazole-6-carboxylate 25a (synthesis in the Supporting Information,
1.12 g, 3.87 mmol). After the resulting slurry was stirred at room temperature for 10 minutes,
the mixture was warmed to 60 °C and stirred for 1 h. The reaction was then allowed to cool to
room temperature, and it was diluted with 200 mL of ethyl acetate and washed with water (3×30
mL). The organic solvent extract was concentrated under vacuum and purified using by silica
gel chromatography (40 g silica column) with a gradient of ethyl acetate/hexanes (10–60%, 15
min gradient). Desired fractions were concentrated under vacuum, and the resulting residue
crystallized upon standing to furnish 0.60 g of desired intermediate methyl ester 1h (75% yield).
1H NMR (DMSOd6, 400 MHz) δ 8.13 (d, J =1.6 Hz, 1H), 7.67–7.59 (m, 3H), 7.54–7.50 (m, 2H),
4.41 (s, 2H), 4.31 (bs, 2H), 3.90 (s, 3H), 3.60 (t, J = 4.8 Hz, 1H), 2.31–2.25 (m, 1H), 2.10 (app
dt, J =14.8, 4.0 Hz, 2H), 2.02–1.91 (m, 4H), 1.83 (app d, J =14.8 Hz, 2H), 1.19–1.15 (m, 4H).
LC-MS m/z 618.2 (M+H); method A; RT = 3.26 min. Into a 25-mL round-bottom flask
equipped with a stir bar was added sequentially: the methyl ester 1h (0.55 g, 0.89 mmol), THF
(4.0 mL), MeOH (2.0 mL), and 3 N aqueous KOH solution (1 mL, 3 mmol). The resulting
homogenous solution was stirred for 1 h at 70 °C and then allowed to cool to room temperature.
The reaction was quenched with AcOH (0.2 mL of glacial acetic acid, 3 mmol) until pH=6 was
achieved as monitored by pH-strip paper (Whatman class type CF Cat. No 2613991). The
resulting solution was diluted with ethyl acetate (40 mL) and washed with water (3×5 mL). The
ethyl acetate layer was concentrated down to an oily residue via vacuum in a 40 mL Wheaton
glass vial, and this oil was then dissolved into MeOH (6 mL). The resulting homogenous
solution soon began to crystallize and allowed to stand for 2.5 h. The mother liquor was
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withdrawn, and the crystals were washed with ice cold MeOH (3×2 mL). The crystals were dried
under vacuum (10 mm Hg pressure at 45 °C overnight). A first crop of crystals (516 mg, 91%
yield) were harvested and shown to be the methanol solvate by elemental analysis. Elemental
Analysis as the methanol solvate: (C30H29F4N3O6S): C 56.69, H 4.60, N 6.61; Found: C 56.79, H
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4.61, N 6.65. H NMR (CDCl3, 400 MHz, for the methanol solvate) δ (d, J = 2.0 Hz, 1H), 7.76
(dd, J = 9.0, 1.0 Hz, 1H), 7.57–7.50 (m, 2H), 7.43–7.36 (m, 2H), 4.37 (s, 2H), 4.35 (bs, 2H), 3.60
(t, J = 4.8 Hz, 1H), 3.50 (br s, 3H, peak corresponding to the presence of the methanol solvate),
2.19–2.10 (m, 3H), 2.03–1.90 (m, 4H), 1.83 (app d, J = 12.8 Hz, 2H), 1.22–1.19 (m, 2H), 1.15–
1.09 (m, 2H). LC-MS m/z 604.2 (M + 1).
The methanol solvate form of 1 was converted into a solvate-free crystalline form using the
following procedure: The methanol solvate of 1 (500 mg) was fully dissolved in dry acetonitrile
(50 mL) with gentle heating for 8 minutes (60 °C). This solution was allowed to stand for 4 days
to crystallize. Upon drying, 484 mg of crystals were obtained with two collection crops. High
Resolution Mass Spectroscopic analysis, 604.1514 for the [M+H]+ ion is consistent with the
molecular formula C29 H25 F4 N3O5S, with a deviation of 1.6 ppm from the calculated [M+H]+
ion mass of 604.1530. Purity was found to be >99 % by LC-MS analysis, two separate protocols.
First protocol: [M+H]+ = 604.2 using LC-MS method A, RT = 3.22 min. Second LC-MS method
B, RT = 2.21 min, MS m/z 604.1 (M+H). IR (cm -1): major stretches at 1679 (C=O stretch), 1242
(C-O-C stretch), 1213 (C-N stretch), 1167 (C-F stretch). Elemental Analysis of solvate free form
1
(C29H25F4N3O5S): C 57.71, H 4.18, N 6.96; Found: C: 57.66, H: 4.20, N: 6.94. H NMR
(DMSOd6, 400 MHz) δ 12.96 (br s,1H), 8.20 (d, J = 1.5 Hz, 1H), 7.69–7.66 (m, 1H), 7.64 (dd, J
= 7.6, 1.7 Hz, 1H), 7.59–7.57 (m, 1H), 7.56–7.55 (m, 1H), 7.54 (d, J = 1.1 Hz, 1H), 4.34 (s, 2H),
4.22 (app d, J = 5.7 Hz, 2H), 3.55 (t, J = 4.7 Hz, 1H), 2.34 (tt, J = 8.39, 5.18, 1H), 2.01–1.95
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(m, 2H), 1.86–1.82 (m, 4H), 1.75 (app d, J =14.5 Hz, 2H), 1.16–1.13 (m, 2H), 1.10–1.07 (m,
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2H). F (DMSOd6, 376 MHz) –126.4, –56.3 ppm. C (DMSOd6, 100 MHz) 171.79, 166.77,
166.37, 159.41, 151.70 (d, J = 249 Hz), 146.35, 145.10, 133.65 (d, J = 5.2 Hz), 132.26, 132.11,
128.25, 124.09, 123.22, 121.88, 120.40 (q, J = 258.3 Hz), 119.78, 113.39 (dd, J = 19.2, 2.4 Hz),
112.11, 72.35, 59.68, 57.28, 33.68, 27.91, 8.43, 7.50. M.P. = 221 °C sharp, corrected.
Assignment table for proton and carbon spectra is in the Supporting information.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at
DOI:
Experimental procedures and analytical data for the synthesis of all compounds and Molecular
Formula Strings; FXR-HTRF coactivator interaction biochemical assay; FXR BSEP-luc reporter
gene cellular assay; rat in vitro hepatocyte gene expression assay; PK protocols; in vivo rat PD
study protocols. (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel: (510) 879-9456. E-mail: david.tully@novartis.com
Present Addresses
*(D.C.T) Novartis Institutes for Biomedical Research, Emeryville, California, USA
ǁ(A.P.) WuXi AppTec, San Francisco, California, USA
◊(H.M.S.) IFM Therapeutics, Boston, Massachusetts, USA
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§(S.B.J.) Calibr, La Jolla, California, USA
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Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors thank David Jones for NMR assistance, Vivian Wang for technical assistance,
Wendy Richmond and Perry Gordon for formulation support, Barbara Saechao for bioanalytical
support, and Glen Spraggon for X-ray crystallography.
ABBREVIATIONS
FXR, Farnesoid X Receptor; NASH, nonalcoholic steatohepatitis; PBC, primary biliary
cholangitis; PFIC, progressive familial intrahepatic cholestasis; BSEP, bile salt export pump;
UDCA, ursodeoxycholic acid or ursodiol, SHP, short heterodimer partner; FGF15/FGF19,
fibroblast growth factor 15/19; FGFR4; fibroblast growth factor receptor 4; LDL, low-density
lipoprotein; MAPK, mitogen-activated protein kinase; CYP7A1, cytochrome P450 7A1 or
cholesterol 7 α-hydroxylase, SREPB-1c, Sterol regulatory element-binding protein 1c; TGR5, G
protein-coupled bile acid receptor 1 (GPBAR1); GPCR, G protein–coupled receptor; OCA,
obeticholic acid; HTRF, Homogeneous Time Resolved Fluorescence; MDR3, multidrug
resistance protein 3; ABCB4/ABCB11, ATP-binding cassette, sub-family B member 4/11;
NR1H4, nuclear receptor subfamily 1, group H, member 4; SRC1, Steroid Receptor Coactivator-
1, HLM, human liver microsomes; RLM, rat liver microsomes; FASSIF, fasted state simulated
intestinal fluid; qPCR, quantitative polymerase chain reaction;
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