Discovery of XL335 (WAY-362450), a highly potent, selective, and orally active agonist of the farnesoid X receptor (FXR)

Article abstract of DOI:10.1021/jm8014124

Azepino[4,5-b]indoles have been identified as potent agonists of the farnesoid X receptor (FXR). In vitro and in vivo optimization has led to the discovery of 6m (XL335, WAY-362450) as a potent, selective, and orally bioavailable FXR agonist (EC₅₀ = 4 nM, Eff = 149%). Oral administration of 6m to LDLR^-/- mice results in lowering of cholesterol and triglycerides. Chronic administration in an atherosclerosis model results in significant reduction in aortic arch lesions.

Full text of DOI:10.1021/jm8014124

904
J. Med. Chem. 2009, 52, 904–907
Letters
Discovery of XL335 (WAY-362450), a Highly
Potent, Selective, and Orally Active Agonist
of the Farnesoid X Receptor (FXR)
Brenton Flatt,*,† Richard Martin,† Tie-Lin Wang,†
Paige Mahaney,3 Brett Murphy,† Xiao-Hui Gu,†
Paul Foster,# Jiali Li,‡ Parinaz Pircher,‡ Mary Petrowski,‡
Ira Schulman,‡ Stefan Westin,‡ Jay Wrobel,§ Grace Yan,|
Eric Bischoff, Chris Daige, and Raju Mohan†
Departments of Medicinal Chemistry, Structural Biology, Molecular
Biology, Lead DiscoVery, and Pharmacology, Exelixis Inc.,
4757 Nexus Centre DriVe, San Diego, California 92121, and
169 Harbor Way, South San Francisco, California 94083, and
Chemical and Screening Sciences, Wyeth Research, 500 Arcola
Road, CollegeVille, PennsylVania 19426
Figure 1. FXR agonists.
synthesis, conjugation, secretion, absorption, and refilling of the
gall bladder. Activation of FXR results in increased hepatic
expression of receptors that are involved in lipoprotein clearance
(VLDL receptor and syndecan-1) and increased apoC-II that
coactivates lipoprotein lipase (LPL). In addition FXR activation
results in decreased expression of proteins (apoC-III and
ANGPTL3)5 that normally function as inhibitors of LPL. Studies
with Fxr-/- mice also demonstrate differential roles for FXR
in controlling lipid homeostasis.6 Collectively these data
implicate FXR with lowering plasma triglyceride levels via
multiple mechanisms, repressing hepatic lipogenesis and trig-
lyceride secretion, as well as increasing the clearance of
triglyceride-rich lipoproteins from the blood. A number of recent
reviews have been published that discuss the role of FXR in
regulating cholesterol, lipoprotein, and glucose metabolism and
its implication in liver disease.7-9
Bile acids such as chenodeoxycholic acid (CDCA) have been
accepted as the physiologically relevant ligands for FXR10 with
data available from human and animal studies that are consistent
with a key role for bile acids in controlling plasma lipids.11
However, bile acids activate other nuclear receptors such as
pregnane X receptor (PXR), vitamin D receptor (VDR), and
constitutive androstane receptor (CAR). In addition, bile acids
have also recently been shown to activate TGR5,12 an intestine-
expressed GPCR, further complicating the differentiation of
FXR-dependent and FXR-independent pathways.
ReceiVed NoVember 7, 2008
Abstract: Azepino[4,5-b]indoles have been identified as potent agonists
of the farnesoid X receptor (FXR). In vitro and in vivo optimization
has led to the discovery of 6m (XL335, WAY-362450) as a potent,
selective, and orally bioavailable FXR agonist (EC50 ) 4 nM, Eff )
149%). Oral administration of 6m to LDLR-/- mice results in lowering
of cholesterol and triglycerides. Chronic administration in an athero-
sclerosis model results in significant reduction in aortic arch lesions.
Farnesoid X receptor (FXR,a NR1H4), a member of the
nuclear receptor (NR) subfamily, was originally identified in
1995.1,2 Nuclear receptors are ligand-activated transcription
factors that regulate the expression of specific target genes and
are involved in several physiological functions including
reproduction, development, and metabolism.3 Relative to other
nuclear receptors, FXR expression is relatively restricted,
primarily in tissues exposed to a high concentration of bile acids
such as the intestine, kidney, adrenals, and the liver.4 Similar
to some other nuclear receptors, FXR binds to specific response
elements, as a heterodimer with the retinoid X receptor (RXR).
FXR, as a key sensor for bile acids, regulates multiple aspects
of bile acid homeostasis and metabolism, including bile acid
Several potent and selective FXR agonists have been reported
(Figure 1) such as GW4064,13 fexaramine,14 and 6R-ethyl-
chenodeoxycholic acid (6-ECDCA).15 While these compounds
have served as useful tools to investigate the role of FXR
biology, the nonsteroidal analogues have suboptimal in vitro
and in vivo characteristics, thus limiting their utility as small-
molecule drugs for treating FXR-mediated metabolic diseases.
6-ECDCA is currently reported to be in phase II clinical trials.15b
Herein, we describe the identification and SAR of a novel
series of azepino[4,5-b]indoles as potent FXR agonists leading
up to the identification of the clinical candidate 6m (XL335,
WAY-362450, FXR-450).16
Our efforts on the discovery of FXR agonists were initiated
by the high-throughput screening of the Exelixis compound
library and the identification of the azepino[4,5-b]indole 1
(Figure 2) as a FXR agonist lead compound. This discovery
also represented a novel class of nonsteroidal, selective FXR
agonists.17 Compound 1 was identified by screening our
* To whom correspondence should be addressed. Phone, (858) 458-4549;
Fax, (858) 458-4501; E-mail, bflatt@exelixis.com.
3
For information relating to WAY-362450 contact P.M.: phone, (484)
865-0627; fax, (484) 865-9399; e-mail, mahanep@wyeth.com.
†
Department of Medicinal Chemistry, Exelixis Inc.
Wyeth Research.
Department of Structural Biology, Exelixis Inc.
Department of Molecular Biology, Exelixis Inc.
§
#
‡
|
Department of Lead Discovery, Exelixis Inc.
Department of Pharmacology, Exelixis Inc.
a Abbreviations: FXR, farnesoid X receptor; LDLR, low-density lipo-
protein receptor; NR, nuclear receptor; RXR, retinoid X receptor; VLDL,
very-low-density lipoprotein; apoC, apolipoprotein C; LPL, lipoprotein
lipase; ANGPTL3, angiopoietin-like 3; CDCA, chenodeoxycholic acid;
GPCR, G-protein-coupled receptor; LBD, ligand-binding domain; NHR,
nuclear hormone receptor; LXR, liver X receptor; PPAR, peroxisome
proliferator-activated receptor; RAR, retinoic acid receptor; SXR, steroid
and xenobiotic receptor; ER, estrogen receptor; GR, glucocorticoid receptor;
AR, androgen receptor; MR, mineralocorticoid receptor; PR, progesterone
receptor; CYP7A1, cholesterol 7R-hydroxylase; SHP, small heterodimer
partner; IBABP, intestinal bile acid binding protein; BSEP, bile salt excretory
pump.
10.1021/jm8014124 CCC: $40.75
2009 American Chemical Society
Published on Web 01/21/2009

Letters
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 905
Table 2. SAR of Azepino[4,5-b]indole Analogues 6
a c
R1
R2
R3
EC50 (nM)
Effb c (%)
,
,
compd
6d
6e
6f
Me,H
Me,H
Me,H
Me,H
Et
Et
Et
Et
4-F
4-Cl
4-OMe
3,4-F2
57
73
60
32
160
155
145
170
6g
a Compounds were screened in a transient transfection assay using hFXR
on an EcREx7-TK-Luc construct in CV-1 cells. b Maximum efficacy relative
to 100 µM CDCA. c All values are the mean of n g 3, and standard
deviations are e30% of the mean values.
Figure 2. Azepino[4,5-b]indole 1.
Scheme 1. Synthesis of Azepino[4,5-b]indoles 6a
Table 3. SAR of Azepino[4,5-b]indole Analogues 6
a c
Effb c (%)
,
,
compd
R1
R2
R3
EC50 (nM)
6h
6i
6j
6k
6l
6m
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Me, Me
Et
Et
Et
Et
i-Pr
i-Pr
4-F
3,4-F2
4-Cl
4-OMe
4-F
3,4-F2
22
12
25
29
8
148
144
190
139
146
149
4
a Compounds were screened in a transient transfection assay using hFXR
on an EcREx7-TK-Luc construct in CV-1 cells. b Maximum efficacy relative
to 100 µM CDCA. c All values are the mean of n g 3, and standard
deviations are e30% of the mean values.
a Reagents: (a) di-tert-butyl dicarbonate, Et3N, DMAP, DCM, 84%; (b)
(i) LiHMDS (1.0 equiv), THF, -78 °C; (ii) MeI, -78 to 25 °C, 71%; (c)
MeI (2.5 equiv), LiHMDS (2.5 equiv), THF, -78 to 25 °C, 84%; (d) TFA,
DCM, 99%; (e) LiAlH4, THF, reflux, 82%; (f) HCl, ethyl or isopropyl
bromopyruvate, EtOH, reflux, 16 h; (g) Et3N or pyridine, 100 °C, 40%
(two steps); (h) ArCOCl, DIPEA, DCE, 70 °C, 78-85%.
Table 1. SAR of Azepino[4,5-b]indole Analogues 6
a c
Effb c (%)
,
,
compd
R1
R2
R3
EC50 (nM)
1
H, H
H, H
H, H
H, H
Et
i-Pr
n-Pr
n-Bu
4-F
4-F
4-F
4-F
600
500
500
100
120
130
109
6a
6b
6c
1100
a Compounds were screened in a transient transfection assay using hFXR
on an EcREx7-TK-Luc construct in CV-1 cells. b Maximum efficacy relative
to 100 µM CDCA. c All values are the mean of n g 3, and standard
deviations are e30% of the mean values.
compounds in a 384-well format using a single point cell-based
h-FXR cotransfection luciferase gene reporter assay. Resynthesis
and full dose-response provided an in vitro potency of 0.6 µM.
Azepino[4,5-b]indoles were prepared via the general synthetic
route shown in Scheme 1. 2-(1H-indol-3-yl)acetonitrile was
converted to the tert-butyl carbamate followed by mono- or
dialkylation to afford the respective analogues 2. Preparation
of dialkylated indoles 2 were obtained when excess reagents
are used. Subsequent deprotection of 2 with TFA and reduction
with LiAlH4 in THF at reflux afforded the corresponding
tryptamines 3. Condensation of 3 with a bromopyruvate in the
presence of HCl provided the intermediate carbolines 4 (not
isolated) via the Pictet-Spengler reaction. When the sample
was heated in pyridine, 4 were converted to azepino[4,5-
b]indoles 5, presumably via rearrangement of an intermediate
aziridine.18 Finally azepino[4,5-b]indoles 5 were reacted with
the desired aroyl chlorides to afford the acylated products 6.
Lead optimization was initiated with exploration of the ester
functionality of the HTS lead 1, and the data are shown in Table
1. A large number of alkyl esters (linear and branched) were
explored with only a select set shown in Table 1. Activity was
maintained with the isopropyl and n-propyl esters (6a, 6b), while
reduced activity was observed for longer alkyl chains (6c).
We then proceeded to explore the alkylation of the 1-position
starting initially with the monomethyl analogues (Table 2). The
monoalkylated compound 1-methyl azepino[4,5-b]indole 6d was
found to increase the FXR potency ∼10-fold when compared
to the HTS lead 1. With the monoalkylated azepinoindole, we
explored the SAR around the benzamide functionality, and
Figure 3. X-ray crystal structure of 6m in hFXR LBD.
selected substitution options are presented in Table 2. 4-Sub-
stitution with F, Cl, and OMe was activity neutral (6d-f), while
the 3,4-difluoro analogue 6g showed a nominal 2-fold improve-
ment.
Results of dialkylation of the azepineindole on the 1-position
with the chemistry described in Scheme 1 are shown in Table
3. In general, a 2-3× improvement in potency was observed
for various benzamides explored, with the 3,4-difluoro analogue
6i showing the highest potency within the ethyl esters, similar
to that observed for the monomethyl analogue 6g (Table 2).
Incorporation of an isopropyl ester (6l and 6m) yielded
another 3-fold potency improvement over the ethyl ester
analogues. Overall, 6m was identified as the most potent FXR
agonist within our series (EC50 ) 4 nM, Eff ) 149%) and was
selected for further profiling (in vitro and in vivo).
The crystal structure of the complex of 6m bound to the
ligand-binding domain (LBD) of human FXR was determined
to a resolution of 2.0 Å (Figure 3). The ligand resides in a
predominately hydrophobic pocket with only a few polar atoms
making contact with the ligand. Among these are a 3.1 Å
hydrogen bond from the peptide amide of A295 (R3) to the
carbonyl oxygen of the isopropyl ester, along with a 2.9 Å
hydrogen bond from the hydroxyl of Y373 (R7) to the amide
oxygen attached to the azepine ring. Similar to that observed

906 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Table 4. PK Profile of 6m in Rat at 3 mg/kg
Letters
route
iva
pob
Cmax (µM)
Tmax (h)
AUC0-t (µM h)
Cl ((mL/h)/kg)
Vss (L/kg)
t1/2 (h)
30.5
0.02
19.1
341
3.3
0.5
10.0
7.0
40.1
24.6
37.6
f (%)
a iv: NMP/Solutol/PEG400/H2O (10:10:40:40). b po: corn oil.
Figure 4. Effect of 6m on total cholesterol and plasma triglycerides
in LDLR-/- mice fed a high-fat diet: (*) P < 0.05 versus vehicle
control, one way ANOVA followed by Bonferroni post hoc test.
in the structure of other FXR agonists, the C-terminal AF2 motif
(R12) adopts the “agonist” conformation, although no direct
contact with the ligand is observed. Nevertheless, residues F465
and W478 (R12) are positioned within 4.5 Å of the indole ring
of 6m and form a lid presumably sequestering the ligand.
The selectivity of 6m was evaluated using a panel of nuclear
receptor expression plasmids transiently transfected in CV-1
cells together with NHR responsive luciferase reporters. The
panel tested consisted of LXRR, LXRꢀ, PPARR, PPARγ,
PPARδ, RXRR, RARγ, VDR, SXR, ERR, ERꢀ, GR, AR, MR,
and PR. Compound 6m is highly selective, as no significant
cross-reactivity with these receptors was observed at concentra-
tions up to 10 µM.
FXR has been shown to regulate several target genes
associated with bile acid synthesis and transport.19 Activation
of FXR by bile acids or synthetic agonists results in transcrip-
tional repression of cholesterol 7R-hydroxylase (CYP7A1), the
rate-limiting enzyme in the bile acid biosynthesis pathway,20
induction of the small heterodimer partner (SHP), a transcrip-
tional repressor found in the liver and intestine,21 and induction
of genes encoding for some bile acid transport proteins, such
as intestinal bile acid binding protein (IBABP)22 and bile salt
excretory pump (BSEP).23 In promoter assays, utilizing reporter
constructs under control of the human BSEP, human SHP, and
mouse IBABP genes were up-regulated by 6m with EC50 of
17, 230, and 33 nM, respectively. In addition, 6m significantly
induced mRNAs encoding for BSEP, SHP, and IBABP in
human cell cultures at 1 µM (13-, 2-, and 20-fold, respectively).
As anticipated, the observed induction of BSEP, SHP, and
IBABP promoters by our FXR agonists is consistent with these
genes being direct targets of FXR.
Figure 5. Effect of 6m on reduction of aortic arch lesion by en face
analysis: (*) P < 0.05 versus vehicle control, one way ANOVA
followed by Bonferroni post hoc test.
inhibited lesion formation by 18% and 36% at doses of 1 and
3 mg/kg, respectively (see Figure 5).
In summary, we have identified a series of novel azepino[4,5-
b]indole analogues from HTS that act as novel and selective
FXR agonists. Our lead optimization efforts have led to the
discovery of 6m as a potent, selective, and orally bioavailable
FXR agonist. We have successfully solved the cocrystal structure
of 6m and related analogues, gaining a strong understanding
of the SAR around the azepino[4,5-b]indole core. Compound
6m displays potent agonist activity in the FXR reporter gene
assays and on FXR target genes in cell-based assays. Compound
6m also showed potent effects on cholesterol and triglyceride
lowering in LDLR-/- mice and antiatherogenic activity with
respect to reduction of aortic arch lesions. On the basis of these
data and other in vitro and in vivo characterization, 6m has
been selected for further clinical evaluation.16
Compound 6m was dosed in rat at 3 mg/kg (po and iv) and
shows good oral bioavailability (38%) (Table 4). There is a
protracted half-life of 25 h, modest volume of distribution, and
low clearance (3.3 L/kg, ∼10% of hepatic blood flow).
Additional pharmacokinetic studies in mice and higher species
have been completed, and the data will be reported elsewhere.
To evaluate the pharmacokinetic and pharmacodynamic
effects of 6m on lipid profiles, we administered 6m (10 mg/kg,
po) to normal C57bl/6 mice for a period of 7 days and sampled
plasma 3 h after the final dose. Compound 6m significantly
lowered triglycerides (82.3 ( 2.9 and 62.0 ( 6.4 mg/dL for
vehicle [80% PEG/20% Tween] and 6m, respectively) and total
cholesterol (99.8 ( 4.0 and 78.1 ( 5.0 mg/dL for vehicle and
6m, respectively). To further evaluate the efficacy of 6m, we
evaluated it in LDLR-/- mice that had been fed a Western diet
(Purina 21551) for 8 weeks at the beginning of the study. After
6 weeks of daily oral treatment, 6m lowered both triglycerides
(by 19% and 39% at 1 and 3 mg/kg, respectively) and total
cholesterol (by 23% and 50% at 1 and 3 mg/kg respectively;
see Figure 4).
Acknowledgment. The authors thank the Analytical and
Bioanalytical Chemistry groups at Exelixis for support of the
pharmacokinetic studies.
Note Added after ASAP Publication. This paper was published
ASAP on January 21, 2009 with an error in Figure 1. The revised
version was published on January 30, 2009.
Supporting Information Available: Details of the synthesis and
characterization of azepino[4,5-b]indole 6m and analogues; pro-
tocols for in vitro and in vivo experiments; X-ray crystallographic
data. This material is available free of charge via the Internet at
http://pubs.acs.org.
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