Discovery of 6-(4-{[5-Cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl]methoxy}piperidin-1-yl)-1-methyl-1H-indole-3-carboxylic Acid: A Novel FXR Agonist for the Treatment of Dyslipidemia

Article abstract of DOI:10.1021/acs.jmedchem.5b01161

The farnesoid X receptor (FXR) is a member of the "metabolic" subfamily of nuclear receptors. Several FXR agonists have been reported in the literature to have profound effects on plasma lipids in animal models. To discover novel and effective therapies for dyslipidemia and atherosclerosis, we have developed a series of potent FXR agonists that robustly lower plasma LDL and vLDL in LDLr-/- mice. To this end the novel piperidinylisoxazole system LY2562175 was discovered. This molecule is a potent and selective FXR agonist in vitro and has robust lipid modulating properties, lowering LDL and triglycerides while raising HDL in preclinical species. The preclinical ADME properties of LY2562175 were consistent with enabling once daily dosing in humans, and it was ultimately advanced to the clinic for evaluation in humans. The synthesis and biological profile of this molecule is discussed.

Full text of DOI:10.1021/acs.jmedchem.5b01161

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Brief Article
Discovery of 6-(4-{[5-Cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-
yl]methoxy}piperidin-1-yl)-1-methyl-1H-indole-3-carboxylic
Acid: A Novel FXR Agonist for the Treatment of Dyslipidemia
Michael J. Genin, Ana B Bueno, Javier Agejas, Peter R. Manninen, Wayne
Bocchinfuso, Chahrzad Montrose-Rafizadeh, Ellen Cannady, Timothy Jones, John
Stille, Eyas Raddad, Charles Reidy, Amy Cox, Mervyn Michael, and Laura Michael
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01161 • Publication Date (Web): 14 Nov 2015
Downloaded from http://pubs.acs.org on November 16, 2015
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Discovery of 6-(4-{[5-Cyclopropyl-3-(2,6-
dichlorophenyl)isoxazol-4-yl]methoxy}piperidin-1-yl)-1-
methyl-1H-indole-3-carboxylic Acid: A Novel FXR Agonist for
the Treatment of Dyslipidemia
Michael J. Genin,*a Ana B. Bueno,a Javier Agejas Francisco,a Peter R. Manninen,a Wayne P. Boc-
chinfuso,a Chahrzad Montrose-Rafizadeh,a Ellen A. Cannady,b Timothy M. Jones,b John R. Stille,e Eyas
Raddad,e Charles Reidy,d Amy Cox,c M. Dodson Michael,c and Laura F. Michaeld
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a
AUTHOR ADDRESS Discovery Chemistry Research and Technology, bDrug Disposition and Metabolism, cDiabetes Ther-
apeutic Area, dCardiovascular and Metabolic Diseases Therapeutic Area, Lilly Research Laboratories, A Division of Eli Lilly
and Company, eChorus, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Indiana 46285, USA.
KEYWORDS. Farnesoid X receptor, FXR, nuclear hormone, agonist, piperidine, indole, dyslipidemia, atherosclerosis, diabe-
tes.
ABSTRACT: The farnesoid X receptor (FXR) is a member of the ‘metabolic’ subfamily of nuclear receptors. Several FXR ago-
nists have been reported in the literature to have profound effects on plasma lipids in animal models. In our efforts to discover nov-
el and effective therapies for dyslipidemia and atherosclerosis we have developed a series of potent FXR agonists that robustly low-
er plasma LDL and vLDL in LDLr-/- mice. To this end the novel piperidinylisoxazole system LY2562175 was discovered. This
molecule is a potent and selective FXR agonist in vitro and has robust lipid modulating properties lowering LDL and triglycerides
while raising HDL in preclinical species. The pre-clinical ADME properties of LY2562175 were consistent with enabling once
daily dosing in humans and it was ultimately advanced to the clinic for evaluation in humans. The synthesis and biological profile
of this molecule is discussed.
INTRODUCTION
Researchers from GlaxoSmithKline were first to report in
vivo activity of a potent, synthetic FXR agonist, GW4064.
When administered to Fischer rats, GW4064 elevated circulat-
ing HDL levels and reduced triglycerides.5 Since this report,
GW4064 has been successfully utilized as a research tool for
exploring FXR mechanism of action and pharmacology.
The farnesoid X receptor (FXR) is a member of the ‘meta-
bolic’ subfamily of nuclear receptors. The discovery of bile
acids as natural physiological ligands for FXR and the devel-
opment of potent FXR-selective synthetic agonists illuminated
the potential use of FXR agonists for treatment of chronic
hepatitis, cholestasis, liver fibrosis and atherosclerosis.1-3 The
first evidence illustrating the importance of FXR in regulation
of lipid homeostasis came from mice harboring a deletion
construct of the FXR/BAR gene. When maintained on a 1%
cholesterol diet, hepatic cholesterol and triglyceride contents
were greater in FXR/BAR null versus wild-type mice. When
maintained on chow diet, serum total cholesterol, phospholip-
id, triglyceride, and cholesterol ester levels for FXR/BAR null
mice were significantly greater than those for wild-type mice.
Thus, disruption of FXR/BAR gene results in a potentially
proatherogenic serum lipoprotein profile that is exacerbated by
increased dietary cholesterol and the evidence from the
FXR/BAR null mice suggests that an FXR agonist may pro-
vide therapeutic benefit for dyslipidemia.4
Improvements in dyslipidemia and glucose homeostasis in
disease-state animal models following FXR agonist treatment
suggest beneficial outcomes of FXR agonists for the indication
of atherosclerosis. The pathological consequences of the loss
of FXR function on the risk and severity of atherosclerosis
was first tested when FXR null mice were crossed onto the
atherogenic ApoE null background.6 Loss of functional FXR
on the ApoE null background led to further increases of total
blood cholesterol and triglyceride levels as well as increased
VLDL and LDL in the ApoE null model. High fat/high cho-
lesterol (HF/HC) feeding in the FXR null x ApoE null mice
caused the most severe effects on hepatic lipid accumulation,
focal necrosis, and inflammatory gene expression. These phe-
notypic outcomes were associated with the most severe degree
of atherosclerotic lesion formation and the lowest survivability
in the HF/HC-fed FXR null x ApoE null mice as compared
with the control genotypes.7 Based on these results, FXR ap-
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pears to play a protective role in managing risk factors that
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contribute to the development of atherosclerosis.
Scheme 1. Synthesis of compound 1 (LY2562175).
In our efforts to discover novel and effective therapies for
dyslipidemia we have developed a series of potent FXR ago-
nists that robustly lower plasma LDL and vLDL in LDLr-/-
mice. To this end the novel piperidinylisoxazole system
LY2562175 was discovered. This molecule is a potent and
selective FXR agonist in vitro and has robust lipid modulating
properties lowering LDL and triglycerides while raising HDL
in preclinical species. LY2562175 also has ADME properties
consistent with enabling once daily dosing and it was ad-
vanced to clinical evaluation in humans.
NH OH-HCl,
2
NCS, DMF
3N NaOH
40°C
EtOH, 60°C
(99%)
4
2
3
NEt3, EtOH
(46%,2 steps)
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DIBAL
(85%)
TPP, CBr4
DCM
(90%)
8
6
7
KO-tBu,
THF
18-c-6
9
1 (LY2562175)
TFA, DCM
(66%, 2 steps)
10
11
12
1. CuI, Proline,
DMSO, 80°C
(30%)
2. 2N KOH
GW4064
Figure 1. Structures of FXR agonists 1 (LY2562175) and
GW4064.
1
RESULTS AND DISCUSSION
Chemistry. The synthesis of compound 1 was achieved in
9-linear steps as the longest sequence and is outlined in
Scheme 1. Initially the oxime 3 was formed by condensing
ammonium hydroxide with the commercially available alde-
hyde 2. Clorination of 3 with NCS provided cloroxime 4
which was subjected to cyclization with the beta-keto ester 5
under mildly basic conditions to yield the desired isoxazole
system 6. Reduction of the ester (DIBAL) was followed by
bromination of the resulting alcohol 7 to form the isoxazolyl-
Scheme 2. Synthesis of indole intermediate 12.
Br , HOAc
2
(67%)
14
bromide system 8. Reaction of
8
with boc-4-
13
hydroxypiperidine was done under basic conditions followed
by deprotection to form the coupling partner 11. Coupling of
11 with the protected indole species 12 under copper/proline
catalyzed cross-coupling conditions provided the penultimate
intermediate as a methyl ester. Hydrolysis of the ester resulted
in formation of the desired product 1 in ~7% overall yield.
MeOH,
AcCl
(62%)
K CO ,
2
3
Dimethylcarbonate
DMF, 130°C
The intermediate indole 12 was prepared as shown in
Scheme 2. Commercially available indole-3-carboxylate 13
was brominated and esterified to form 15 under standard con-
ditions. The indole nitrogen was then methylated to yield the
requisite intermediate 12.
12
(82%)
15
Biology and Pharmacology. The effect of 1 on a function-
al measure of FXR transcription transactivation activity was
measured using co-transfected HEK293 cells, the human FXR
cDNA, and an FXRE-luciferase reporter. The ability of the
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compound to modulate the secondary structure of the FXR lipoprotein subtypes by FPLC. ED50s (dose producing half-
1
2
3
4
5
6
7
8
ligand binding domain to enable recruitment of a transcrip-
tional coactivator was measured using purified recombinant
FXR ligand binding domain protein and the nuclear receptor
interaction domain of the co-activator protein, SRC-1, and the
AlphaScreen technology (Table 1). LY2562175 promotes
transcriptional activation of human FXR in a cell-based co-
transfection assay with an EC50 of 193 nM (geometric mean)
and demonstrates ‘partial agonist-type’ efficacy since the per-
cent activation as compared to a full FXR agonist is approxi-
mately 41.3% efficacy relative to GW4064 a full agonist.
LY2562175 promotes recruitment of a peptide from the nucle-
ar receptor interaction domain of the co-activator SRC-1 with
a relative EC50 of 121 nM and 93.5% efficacy as compared to
GW4064.
maximal effect) for the decrease in serum cholesterol and tri-
glycerides were determined by nonlinear regression analysis.
In the LDLR null mouse dose-response study, LY2562175
caused a dose-dependent decrease in serum cholesterol and
serum triglycerides. At the dose of 10 mg/kg, the decrease in
cholesterol with LY2562175 was 80% below vehicle-treated
animals, and the decrease in serum triglycerides was 76%
from control group. The ED50 for serum cholesterol was de-
termined to be 2 mg/kg and 3.4 mg/kg for serum triglycerides
(Table II, Figure 2).
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Table II. Total cholesterol- and triglyceride-lowering in LDL
Receptor null mice following a 7 day dosing regimen*
Dose
Percent Change
Table I. FXR Agonist LY2562175 in vitro Profile.
(mg/kg)
serum TG
-25
total chol
VLDL
-59
LDL
-1
hFXR FXRE Ag*
hFXR\hSRC1 CoAR 25uM**
1
3
-37
-57
Compound
Relative
EC50 (nM)
% Relative
Efficacy
Relative
EC50 (nM)
% Relative
Efficacy
-46
-78
-15
-50
-62
2562175
GW4064
193
41
121
220
93
10
-76
-80
-96
373
100
100
30
-83
-83
-98
*FXR transcriptional potency and efficacy of LY2562175 in the HEK293
co-transfection assay. HEK293 cells were co-transfected with a human
FXR expression plasmid and 2XIR-1-luciferase reporter plasmid. Deter-
mination of LY2562175 potency and % efficacy was established as com-
pared to the full agonist, GW4064. **Recombinant FXR LBD protein was
co-incubated with the nuclear receptor interaction domain of SRC-1.
Interaction between the proteins was measured using AlphaScreen tech-
nology. Determination of LY2562175 potency and % efficacy was estab-
lished as compared to the full agonist, GW4064.
ED50
3.40 mg/kg
2.00 mg/kg
* Male LDLR null mice were treated with varying doses of LY2562175
for 7 days. Total cholesterol and triglycerides were quantified by enzy-
matic analysis using a Hitachi 912 Clinical Chemistry Analyzer with
Roche reagents, and lipoprotein fractions were quantified by FPLC.
The ZDF is an inbred rat model that through genetic muta-
tion (fa mutation results in shortened leptin receptor protein
that does not effectively interact with leptin) and a managed
diet of Purina 5008 will closely mimic human adult onset dia-
betes (Type 2) and related complications. When fed a diet of
Purina 5008, homozygous recessive males develop obesity,
hyperlipidemia, fasting hyperglycemia and Type 2 diabetes.
The obese female ZDF rat does not typically develop diabetes
on standard rodent diets but does exhibit obesity, insulin re-
sistance, and hyperlipidemia.
Nuclear receptor selectivity was determined using a panel of
nuclear receptor co-transcriptional activation assays and bio-
chemical ligand binding assays. LY2562175 demonstrates
selectivity for FXR since it does not promote transcriptional
activation of other members of the nuclear receptor superfami-
ly, such as glucocorticoid receptor, androgen receptor, miner-
alocorticoid receptor and progesterone receptor (EC50 >
10000 nM). Thus, LY2562175 is a potent agonist of human
FXR both in biochemical assays and in cells and demonstrates
selectivity as compared to other members of the nuclear recep-
tor superfamily, including GR, AR, MR and PR.
Treatment of female ZDF rats with LY2562175 results in a
dose dependent lowering of plasma triglycerides in both the
fasted and non-fasted states (Table III). When administered
as a fixed dose combination with rosiglitazone, LY2562175
further lowers fasted and non-fasted plasma triglycerides.
FPLC fractionation of the lipoproteins reveals that
LY2562175-treatment results in a reduction in VLDL-C and a
dramatic increase in HDL-C in this animal model. In fact the
high dose of 30mpk resulted in a near doubling of HDL rela-
tive to vehicle controls. Thus, treatment of the female ZDF rat
with FXR agonists causes dramatic reduction of circulating
triglycerides and elevates HDL-cholesterol, and it is tempting
to speculate that FXR agonists may be efficacious in treating
components of diabetic dyslipidemia.
To assess the potency and efficacy of the FXR agonist
LY2562175 in vivo, studies were conducted in 8 week old,
male LDLR null mice fed a ‘western’ diet. Animals were
allowed to acclimate to the high fat/high cholesterol chow
TD88137 (containing 0.15% cholesterol and 42% fat) for two
weeks prior to the study. Animals were divided into groups of
six and dosed once daily for one week by gavage with solu-
tions of LY2562175 in situ sodium salt or with vehicle (5 %
Solutol, 5% EtOH, 1% wt/v CMC) at a dose volume of 5
mL/kg. On the seventh day, animals were bled by cardiac
puncture under anesthesia with CO2. Serum was prepared
from individual animals for determination of cholesterol and
triglycerides by enzymatic analysis using a Hitachi 912 Clini-
cal Chemistry Analyzer with Roche reagents. Pooled samples
from each treatment group were used for determination of
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Table III. Evaluation of LY2562175 alone or in combination
with a low, fixed dose (0.3mpk) of rosiglitazone in female ZDF
rats for 9 days*
1
2
3
4
5
6
7
8
800
700
600
500
400
300
200
100
0
Dose (mg/kg)
VLDL
LDL/lgHDL
HDL
Total
ED50=3.4 +/-0.59 mg/kg
Vehicle
35
17.8
10.8
12.5
18.6
20.3
19.0
48.3
62.5
74.4
94.0
95.0
61.3
101.1
95.2
3
10
21.9
18.2
13.2
8.5
105.1
125.9
123.8
106.5
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30
10 + Rosi
Rosiglitazone
26.3
0.1
1
10
100
*Female ZDF rats were treated with varying doses of LY2562175, rosig-
litazone (rosi), or a fixed dose combination for 9 days. Lipoprotein frac-
tions were quantified by FPLC and are expressed as mg/dl.
LY2562175 (mg/kg/d)
1400
Pharmacokinetics in Human. A 2-cohort, 3-period, double-
blind, placebo-controlled, single-dose escalation study was con-
ducted at a single site, to determine the PK parameters of
LY2562175. Two cohorts of 9 healthy subjects were included in
this study. The cohorts were dosed alternatively in the course of a
dose escalation with the following dose escalation scheme: 5 mg,
25 mg, 75mg, 200 mg, 400 mg, and 600 mg of LY2562175. After
treatment with a single oral dose of LY2562175 in the dose range
of 5 to 600 mg, the drug was well tolerated and maximum
LY2562175 plasma concentrations were reached on average 2 to
3 hours post-dose (Figure 3). Concentration area under the curve
and maximal concentrations reached had less than dose-
proportional increases over the dose range studied. The mean
T1/2 was similar across the dose range 75 to 600 mg LY2562175
and varied between 16.9 and 24.2 hours. The LY2562175 Cmax
observed at the ED50 dose for lipid modulation in rodents was
4.62ng/mL (dashed line), and the human plasma concentration at
the lowest dose tested (5mg) peaked at over twice that level and
remained so to approximately 10 hours.
1200
1000
800
600
400
200
0
ED50=2.0+/-0.43 mg/kg
0.1
1
10
100
LY2562175 (mg/kg/d)
Figure 2. Male LDLR null mice were treated with varying doses
of LY2562175 for 7 days. Total cholesterol and triglycerides
were quantified by enzymatic analysis using a Hitachi 912 Clini-
cal Chemistry Analyzer with Roche reagents (Mean ± SD; n=6).
ED50s (dose producing half-maximal effect) for the decrease in
serum cholesterol and triglycerides were determined by nonlinear
regression analysis.
Pharmacokinetics (PK) in Nonclinical Species. Pharma-
cokinetic studies were conducted in Sprague Dawley (SD)
rats, Beagle dogs, and Cynomolgus monkeys to assess the oral
and intravenous exposure for Compound 2562175. Several
PK parameters, including the calculated oral bioavailability
were also determined. IV doses were administered at 1 mg/kg
(rat and dog) or 0.91 mpk (monkey) in a standard 5% Solutol /
5% EtOH in saline formulation. Oral/nasogastric doses were
administered at 3 mpk in a standard vehicle (1.0 % CMC/
0.25% PS80/ 0.05% antifoam in purified water). Samples
were collected, processed to plasma, and analyzed by LC-
MS/MS. The PK data are summarized in Table IV. Follow-
ing IV administration, the mean terminal elimination half-life
(T1/2) of LY2562175 ranged from approximately 2 to 10
hours, depending on the species. Clearance was low to mod-
erate for all species where values were less than hepatic blood
flow in each respective species.8 Oral exposures were higher
in non-rodent compared to rodent species. The Tmax ranged
from approximately 1 to 3 hours for all species. The oral bio-
availability for rats, dogs and monkeys was 21 ± 5%, 82%,
and 24 ± 3%, respectively. The fraction unbound in rat and
human plasma was 0.020 and 0.032 respectively.
Figure 3. Human SAD Total Plasma Concentration vs Time
Profiles for LY2562175.
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Table IV: Summary of PK parameters after administration of LY2562175 to SD rats, Beagle dogs, or Cynomolgus monkeys.
(Mean ± SD, N=2-3).
1
2
3
Species/Study
SD Rat
Beagle Dog
Cynomolgus Monkey
4
5
6
7
8
9
Dose (Route)
AUC0-24hr (ng*h/mL)
AUC Extrap (ng*h/mL)
% AUC Extrap
T1/2 (h)
1 mpk (IV)
577 ± 181
586 ± 179
2 ± 1
3 mpk (PO)
365 ± 178
373 ± 177
2 ± 1
1 mpk (IV)
3 mpk (PO)
0.91 mpk (IV)
3 mpk (PO)
1813 ± 350
1831 ± 346
1 ± 1
1785 ± 90
1855 ± 124
4 ± 4
4686
4707
0.5
7
2302 ± 215
2322 ± 226
1 ± 1
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2 ± 1
2 ± 0
10 ± 4
9 ± 1
5 ± 1
9 ± 2
CL (mL/min/kg)
Vdss (mL/kg)
Co (ng/mL)
30 ± 8
---
---
6 ± 1
---
1235 ± 622
2527 ± 676
---
---
2483 ± 832
1184 ± 191
---
---
597 ± 275
4222 ± 605
---
---
---
---
---
Tmax (h)
1.08 ± 0.88
130 ± 96
21 ± 5
0.75
1243
82
3.33 ± 1.15
221 ± 66
24 ± 3
Cmax (ng/mL)
F%
---
---
---
---
---
---
Conclusion. In summary, LY2562175 is a potent and partial ago-
nist relative to GW4064 for human FXR as determined by cell-
based transcriptional transactivation assays (Avg EC50=193 nM).
Modulation of plasma lipids in LDLr KO mouse models reveals
ABBREVIATIONS
FXR; farnesoid X receptor, NCS; n-chlorosuccinimide, SD; Spra-
gue-Dawley, LDL; low density liproprotein, HDL; high density
liproprotein, Rosi; rosiglitazone.
.
robust triglyceride and cholesterol lowering.
Furthermore,
LY2562175 reduces triglycerides significantly and elevates HDL-
c by up to 95% in the insulin resistant female ZDF rat model.
Due to the excellent lipid modulation profile in rodents as well as
acceptable PK properties, LY2562175 was advanced into human
clinical trial evaluation for dyslipidemia. Results from SAD stud-
ies revealed PK in humans that support once daily dosing.
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ands for an orphan nuclear receptor. Science 1999, 284, 1365-1368
ASSOCIATED CONTENT
Supporting Information. Experimental procedures and analyti-
cal data for Compound 1, in vitro and in vivo pharmacology pro-
cedures. This material is available free of charge via the Internet
at http://pubs.acs.org.
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Makishima, M., Okamoto, A. Y., Repa, J. J., Tu, H.,
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AUTHOR INFORMATION
Goodwin B., Jones, S.A., Price, R.R., Watson, M.A.,
Corresponding Author
*Phone: 317-651-3849. E-mail: geninmi@lilly.com
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Hanniman, E.A., Lambert, G., McCarthy, T.C., Sinal, C.J.
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Frants, R.R., van Tol, A., Hattori, H., Smelt, A.H., van Dijk, K.W.
Plasma apoAV levels are markedly elevated in severe hypertriglycer-
idemia and positively correlated with the APOA5 S19W polymor-
phism. Atherosclerosis 2007, 193, 129-34
Author Contributions
The manuscript was written by M.J.G. and edited by L.F.M. All
authors have given approval to the final version of the manuscript.
Notes
Henneman, P., Schaap, F.G., Havekes, L.M., Rensen, P.C.,
The authors declare no competing financial interest.
ACKNOWLEDGMENT
8.
Davies B. and Morris T. Physiological parameters in labor-
atory animals and humans. Pharm. Res. 1993, 10, 1095-93.
The authors would like to express their sincere gratitude for the
broad support from Chorus, Eli Lilly and Company for the Phase I
study.
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