Identification of an N-oxide pyridine GW4064 analog as a potent FXR agonist

Article abstract of DOI:10.1016/j.bmcl.2009.03.008

According to the docking studies and the analysis of a co-crystal structure of GW4064 with FXR, a series of 3-aryl heterocyclic isoxazole analogs were designed and synthesized. N-Oxide pyridine analog (7b) was identified as a promising FXR agonist with po

Full text of DOI:10.1016/j.bmcl.2009.03.008

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2595–2598
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of an N-oxide pyridine GW4064 analog as a potent FXR agonist
a
a
a
a
b
Song Feng a, , Minmin Yang , Zhenshan Zhang , Zhanguo Wang , Di Hong , Hans Richter ,
Gregory Martin Benson b, Konrad Bleicher b, Uwe Grether b, Rainer E. Martin b,
Jean-Marc Plancher b, Bernd Kuhn b, Markus Georg Rudolph b, Li Chen a
*
a Roche R&D Center(China) Ltd, 720 Cai Lun Road, Building 5, Pudong, Shanghai 201203, China
b F. Hoffmann-La Roche Ltd, Grenzacherstrasse, CH-4070 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
According to the docking studies and the analysis of a co-crystal structure of GW4064 with FXR, a series
of 3-aryl heterocyclic isoxazole analogs were designed and synthesized. N-Oxide pyridine analog (7b)
was identified as a promising FXR agonist with potent binding affinity and good efficacy, supporting
our hypothesis that through an additional hydrogen bond interaction between the pyridine substituent
of isoxazole analogs and Tyr373 and Ser336 of FXR, binding affinity and functional activity could be
improved.
Received 16 December 2008
Revised 12 February 2009
Accepted 3 March 2009
Available online 9 March 2009
Keywords:
Farnesoid X receptor (FXR)
Agonist
Ó 2009 Elsevier Ltd. All rights reserved.
N-Oxide pyridine
Isoxazole
The Farnesoid X Receptor (FXR), a member of the nuclear recep-
tor superfamily, has bile acids (e.g., chenodeoxycholic acid (CDCA))
as its natural ligands, and plays a key role in regulating cholesterol
and bile acid homeostasis.1–4 The key factor to this function is the
ability of bile acid-activated FXR to down-regulate the expression
of Cyp7a1,5,6, which is the rate-limiting step in the liver for the con-
version of free cholesterol to bile acids, and the up-regulation of
the bile salt excretion pump (BSEP).7 Treatment of ob/ob and db/
db mice with the synthetic FXR agonist GW4064 (2) significantly
improves hypercholesterolemia8 and lowers free fatty acid9,10
and triglyceride levels in plasma.11 Similarly, treatment with
GW4064 increases insulin sensitivity in ob/ob and db/db mice.8,10
As such, FXR agonists may have utility in treating metabolic syn-
drome, a clustering of cardiovascular risk factors characterized by
dyslipidemia (elevated triglyceride and low HDL levels, also add
high LDL-c levels), insulin resistance, and poor glucose regulation.
Several reviews have summarized the current state of FXR ago-
nists12,13 and the development of FXR modulators for the treat-
ment of diabetes and metabolic syndrome14 remains the current
focus.
compound 5 has recently entered Phase I clinic trials;21,22 and only
few FXR antagonistshave been found so far.2,3
GW4064 (2) is a well-demonstrated, non-steroid agonist to-
wards FXR, and its biological effects on hyperlipidemia in diabetic
mice have been identified and confirmed.1–4 Since the report of
GW4064, a large amount of research work has been directed to-
wards the search replacements of the isoxazole core and the stil-
bene side-chain.23–26 Many publications and patents have
reported on some new modifications and SAR studies related to
GW4064 analogues. So far, most of the modifications around the
isoxazole 3-position were limited to modifications of the aryl sub-
stituents whereas heterocyclic substituents such as pyridines have
been rarely reported.27
We report herein the exploration around the 3-aryl substituent
of GW4064 and the discovery of an N-oxide pyridine analogue
(7b), which exhibited highly potent binding affinity and functional
activity for FXR, as well as improved physicochemical properties
compared with other described GW4064 analogues.
The co-crystal structures of GW4064 and other analogues
with FXR have been resolved in-house28 and reported recently.29
Analysis of the crystal structure of GW4064 suggested that there
are good hydrogen bond interactions between the carbonyl acid
group and isoxazole hetero atoms with Arg335, and His451,
respectively (Fig. 2). These interactions play important roles for
the binding to FXR. In addition to these interactions, some desir-
able substituents especially on the 3-aryl region of isoxazole
could gain more favorable Hydrogen bond interaction with sev-
eral amino acid residues such as Tyr373/Ser336. Therefore some
modifications such as introduction of more polar heterocycles or
After deorphanization of FXR in 19992–4, extensive studies have
been directed to the creation of FXR ligands able to modulate FXR-
mediated specific gene expression, and several steroidal- and non-
steroidal FXR ligands have been described (Fig. 1). Most of the re-
ports deal with FXR agonists (1,11 2,15,16 3,17,18
4
19 and 520), now
* Corresponding author. Tel.: +86 21 38954910 3390; fax: +86 21 50790201.
E-mail addresses: Andrew.feng@roche.com, songfls@hotmail.com (S. Feng).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.008

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S. Feng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2595–2598
O
Cl
OH
H
O
Cl
O
N
H
H
H
OH
O
HO
HO
Cl
H
1
2
O
O
O
O
F
N
OH
N
F
N
H
O
N
CO2Me
4
5
3
Figure 1. Structures of FXR agonists. 1 (6-ECDCA, Intercept), 2 (GW4064, GSK), 3 (Fexaramine, Amgen), 4 (AGN-31, Allergan), and 5 (Exelixis).
The typical synthetic strategy for the preparation of compound
(7b) is outlined in Scheme 1. The key intermediate 8 was pre-
pared mainly according to the reported synthesis procedure of
GW4064 (6a) analogues.11 Pyridine ester 8 was firstly oxidized
with m-CPBA in refluxing DCE, and then followed by the treat-
ment with trifluoro acetic anhydride to furnish another important
intermediate, pyridone ester 9. The following o-methylation of 9
was realized via refluxing with silver carbonate and methyl iodide
in chloroform to afford methyl ester 10. Reduction of the ester
moiety in 10 did not work very well in a number of different sol-
vents, however DCM was found to be effective, providing the de-
sired alcohol 11 in 60% yield. Chlorination of alcohol 11 with
thionyl chloride in chloroform, followed by oxidation with m-
CPBA produced N-oxidated pyridine 13. Deprotonation of phenol
16 with mild base in DMF and subsequent reaction with 4-chlo-
romethyl isoxazole 13 provided coupled intermediate indole alde-
hyde 14. Finally, oxidation of the corresponding aldehyde 14 with
sodium hypochlorite in t-butene/dioxane solution mediated by
monobasic sodium phosphate gave rise to the target compound
7b. Compounds 7a and 7c–h were synthesized following similar
procedures.
The FXR binding and functional activity data of 6a–d and 7a–h
are listed in Table 1. Replacement of the 2,6-dichloro substituent
with either ortho-methoxy (6b) or para-methylsulfonyl (6c) groups
resulted in a 3- to 4-fold decrease in binding affinity concomitant
with a significant loss in functional activity. Most likely the large
methylsulfonyl substituent interferes with the hydrogen bond
interaction with FXR, and weakens its activity. 2,6-Dichloro pyri-
Figure 2. Co-crystal structure of GW4064 bound to FXR ligand binding domain
(LBD). FXR species: human; resolution: 2.3 Å. Residues in H-bonds are Arg335 and
Tyr373, both of which are conserved in FXR (according to PFAM alignment).
dine analogue 6d showed reduced binding affinity (0.69
lM), how-
substituents toward this position could potentially improve the
binding affinity and interaction with the transactivation helix-
12. On the other hand, from the point of drug-like properties,
the envisaged modifications should help to improve the physico-
chemical properties while maintaining a high degree of binding
affinity at least.
ever maintained the weak transactivation activity, of which
permeability was improved slightly compared with GW4064.
Combination of the pyridine head with an optimized stilbene
replacement, N-methyl indole ring afforded a promising compound
7a, which exhibited good activity (0.094 lM) in the human SPA
binding assay. The predicted membrane permeability was im-
proved, too. Screening of additional analogs led to the discovery
of the most potent compound (7b) in this series, with a binding
affinity of 45 nM. The compound has proved to be a partial agonist
(54% and 89% max efficacy relative to GW4064) with an EC50 at
According to the previous hypothesis, more polar moieties such
as methoxy and methylsulfonyl substituents were introduced to
the ortho- and para-positions at the phenyl moiety of isoxazole
(Fig. 3, 6b, 6c), respectively. Dichloro-pyridine also was utilized
to replace the dichloro-phenyl moiety affording compound 6d.
The synthesis of 6b–d is quite similar to the reported method.11,30
Dozens of heterocyclic GW4064 analogues with pyridine, N-oxide
pyridine, and pyridone as 3-aryl substituent replacements have
been designed and synthesized (7a–h). Since the phenyl indole
side-chain of isoxazole has been revealed recently by Eli Lilly’ pat-
ent as a good replacement of stilbene23,25, the phenyl indole car-
boxylic acid was selected to combine into our design.
0.1 lM, 0.22 lM, 7b also revealed the best PAMPA data among
these series. All other analogues (7c–h) exhibited moderate to
weak activity either in binding or functional assay, with the excep-
tion of 7c which showed good transactivation activity. The reduced
activity might result from the minor changes of the head group ori-
entation, leading to less favorable interactions between the ligands
and the binding pocket of FXR.

S. Feng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2595–2598
2597
O
S
O
N
Cl
O
Cl
Cl
O
Cl
Cl
Cl
R
O
O
c
d
N
a
(GW4064)
b
O
6
O-
H
N+
O
N
O
N
N
Cl
Cl
Cl
Cl
Cl
Cl
O
O
N
d
a
O
b
O
c
N
R
7
O
O
O
O
N
NH
N
N
Cl
Cl
Cl
Cl
Cl
g
h
f
e
Figure 3. Designed and synthesized analogues of GW4064.
1
Table 1
R
FXR binding and functional activity and predicted permeability for selected GW4064
analogues
N+
N
R
Entry Compounds32 IC50 SPAa
(affinity,
EC50TA(CH/m-CHb)
(efficacy, M)
PAMPAc
Cl
Cl
Cl
Cl
O
(10ꢀ6 cm/s)
d
a
l
M)
l
N
O
R2
-
N
O
1
2
3
4
5
6
6a (GW4064) 0.064
0.9(100%)/2(100%)
10(54.3%)/10(19.8%)
0(L)
NA
0(L)
0.09(L)
0.19(L)
587(H)
O
6b
6c
6d
7a
7b
0.25
0.17
0.69
0.094
0.045
0(>10
lM)/0(>10
lM)
R1= -- R2=O
R1= -- R2= Cl
R1= O R2= Cl
8
9(59.4%)/11(48%)
0.9(38%)/3(71%)
0.102(53.7%)/
0.2(88.5%)
5(58.5%)/7(63.3%)
7(12%)/6(17%)
12(9%)/11(15%)
4(27%)/4(39%)
2(23%)/2(12%)
R = H
11
b
e
f
R = OH
9
12
13
c
R = OMe
10
7
8
9
10
11
12
7c
7d
7e
7f
7g
7h
1.10/1.13
7.33/1.72
4.17/2.75
0.88/0.235
0.75/0.31
5.16/4.13
NA
NA
NA
NA
0.7(H)
NA
1
R
N+
O
R2
Cl
Cl
Cl
0(>10 lM)/18(8%)
N
O
N
O
a
SPA: scintillation proximity assay, Binding affinity was assessed in a radioligand
displacement assay, according to the method reported by Nichols et al.31
b
TA: transactivation activity; (m)-CH: (mouse)-chimeric receptors, TA activity
was measured by luciferase transcriptional reporter gene assays,31 efficacies are
relative to GW4064 (set at 100%), the value is a mean.
R1= -- R2= H
R1= -- R2= O
14
7b
g
c
-
Kinetic permeability coefficient (Pe). Pe 6 0.2: low (L), Pe > 0.2: medium to high
(M&H).
CHO
h
CHO
HO
N
Br
N
16
15
Scheme 1. The synthesis of N-oxide pyridine and other heterocyclic head GW4064
analogues. Reagents and conditions: (a) DIBAL-H/DCM, rt, 60%; (b) (1) m-CPBA/DCE,
reflux 3 h; (2) (CF3CO)2O/DMF, 8 h, 60%; (c) K2CO3/AgI, CHCl3, 3 h, 80% (d) 10/K2CO3/
DMF, 80°C, 3 h, 70%; (e) Thionyl chloride/CHCl3, reflux, 2 h, 95%; (f) m-CPBA/DCE,
reflux, 2 h, 80%; (g) NaClO2/NaH2PO4/tert-butene,12 h, 65%; (h) 3-methyl-4-
(4,4,5,5-tetramethyl-[1,3,2] dioxaborolan-2-yl)phenol, Pd(PPh3)4/dioxane/K3PO4,
60%.
In conclusion, our exploration on 3-aryl isoxazole moiety of
GW4064 has led to the identification of the novel N-oxide pyridine
analogue (7b) as a potent and efficacious FXR agonist with im-
proved permeability as well. The molecular docking and modelling
studies suggest that the oxygen of the N-oxide of isoxazole ana-
logue most likely acts as an H-bond acceptor to interact with
Tyr373 on Helix 7 and/or Ser336 on Helix 5 (Fig. 4), leading to
Figure 4. Docked N-oxide pyridine compound (7b) in complex with FXR LBD
binding site. The helix are shown in ribbon for clarification. Cyan: N-Oxide pyridine
analog (7b). Left: Helix 12, AF-2 shown in red cartoon; co-activator, SRC-1, shown in
yellow; Upper right: superposition of docked 7b (cyan) and crystallized GW4064
(yellow). Lower right: 7b complexed with FXR LBD. The potential key hydrogen
0
bonds are indicated by black dash line with the same distance of 2.8 AÅ.

2598
S. Feng et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2595–2598
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NMR (400 MHz, DMSO-d6): 8.18 (d, J = 1.2 Hz, 1H), 8.10 (s, 1H), 8.00 (dd,
J = 2.0, 8.0 Hz), 7.85 (m, 2H), 7.78 (t, J = 8.0 Hz, 2H), 7.50 (t, J = 7.6 Hz, 1H),
7.40 (d, J = 16.4 Hz, 1H), 7.27 (d, J = 16.0 Hz, 1H), 6.99 (d, J = 2.4 Hz, 1H),
6.81 (dd, J = 2.0, 8.8 Hz), 5.00 (s, 2H), 2.50 (t, J = 1.6 Hz), 1.35 (d, J = 6.8 Hz,
6H).Compound 7b MS (ESI+), m/z: 566 (M+H)+; Purity: 99.5% (HPLC); 1H
NMR (400 MHz, CDCl3): 8.35 (s, 2H), 8.22 (d, J = 8.0 Hz, 1H), 7.92 (s, 1H),
7.27 (m, 1H), 7.22 (dd, J = 8.4, 2H), 6.69 (m, 2H), 4.83 (s, 1H), 3.89 (s, 2H),
3.38 (m, 1H), 2.27 (s, 3H), 1.48 (d, J = 7.2 Hz, 6H).Compound 7e MS (ESI+),
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3.38 (m, 1H), 2.27 (s, 3H), 1.48 (d, J = 7.2 Hz, 6H).Compound 7f MS (ESI+),
m/z: 546.3 (M+H)+; Purity: 99.6% (HPLC); 1H NMR (400 MHz, CDCl3): 8.23
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3H), 3.38 (m, 4H), 2.27 (s, 3H), 1.49 (m, 6H).
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