Novel heterocyclic scaffolds of GW4064 as farnesoid X receptor agonists

Article abstract of DOI:10.1016/j.bmcl.2014.11.050

The farnesoid X receptor (FXR) may play a crucial role in a number of metabolic diseases and, as such, could potentially serve as a target for the development of therapeutics as a treatment for those diseases. Previous work has described GW4064 as an FXR

Full text of DOI:10.1016/j.bmcl.2014.11.050

Bioorganic & Medicinal Chemistry Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Novel heterocyclic scaffolds of GW4064 as farnesoid X receptor
agonists
a,
Terrence L. Smalley Jr. a, , Sharon Boggs , Justin A. Caravella b,à, Lihong Chen c, Katrina L. Creech d,
⇑
David N. Deaton a, Istvan Kaldor a, Derek J. Parks d
a Muscle Metabolism DPU, Metabolic Pathways & Cardiovascular Discovery Performance Unit, GlaxoSmithKline, Inc., Five Moore Drive, Research Triangle Park, NC 27709, United States
b Computational and Structural Sciences, Platform Technology & Science, GlaxoSmithKline, Inc., Five Moore Drive, Research Triangle Park, NC 27709, United States
c Enteroendocrine DPU, Metabolic Pathways & Cardiovascular Discovery Performance Unit, GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709, United States
d Molecular Discovery Research, Platform Technology & Science, GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The farnesoid X receptor (FXR) may play a crucial role in a number of metabolic diseases and, as such,
could potentially serve as a target for the development of therapeutics as a treatment for those diseases.
Previous work has described GW4064 as an FXR agonist with an interesting activity profile. This
manuscript will describe the synthesis of novel analogs of GW4064 and the activity profile of those
analogs.
Received 14 October 2014
Revised 17 November 2014
Accepted 19 November 2014
Available online xxxx
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
FXR agonist
Heterocycles
Liver
The farnesoid X receptor (FXR) is an orphan nuclear receptor
highly expressed in the liver, kidney and intestines.1,2 Natural bile
acids, such as chenodeoxycholic acid and its conjugates, are the
native ligands for FXR. Upon ligand binding, FXR heterodimerizes
with the retinoid X receptor (RXR), leading to recruitment of
various co-repressors and co-activators, that in turn lead to gene
transcription or repression.3 Recent studies have suggested that
FXR plays a key role in glucose homeostasis,4 liver fibrosis,5–7
inflammatory bowel disease8,9 and cholestasis.10–12 In addition,
FXR agonists may be useful in regenerating damaged liver.
GW4064 was reported nearly 14 years ago.13 While GW4064 is
an important tool compound for determining the in vivo profile
of FXR agonists, its limited pharmacokinetic profile precludes any
further testing in a clinical setting. In addition, the stilbene moiety
is light sensitive and degradation was observed upon standing in
light, which could potentially lead to toxicity issues.14,15 As a
result, several conformationally constrained analogs of GW4064
were synthesized and were shown to be potent FXR agonists.16,17
In this communication we will describe the synthesis and FXR
activity of several isoxazole replacement analogs as novel FXR
agonists.
A previously solved co-crystal structure of GW4064 bound to
the active site of FXR was used in the de novo design of these ana-
logs (Fig. 1).16 The isoxazole ring plays a crucial role in FXR activa-
tion through its interaction with 454Trp located on helix 12, which
is critical for recruiting accessory proteins for modulating gene
transcription. In theory, different heterocyclic replacements of
the isoxazole ring might be expected to alter this interaction with
helix 12, causing association with different modulators which
might display higher affinities/efficacies, act as partial agonists,
antagonists, or even inverse agonists allowing for selective gene
profiles to be assessed. Based on this rationale, a scaffold hopping
approach was pursued. The binding of GW4064 within the LBD of
the FXR receptor contains a number of key interactions. First, the
carboxylic acid group forms an important electrostatic interaction
with 331Arg in helix 5, in similar fashion with the binding mode of
O
N
O
O
Cl
Cl
HO
Cl
GW4064
There has been a long standing research program at GlaxoSmithK-
line targeting FXR agonists. In fact, the discovery of FXR agonist
⇑
Corresponding author Tel.: +1 (919) 483 1054.
E-mail address: terry.l.smalley@gsk.com (T.L. Smalley).
Current address: Cree, 4600 Silicon Drive, Durham, NC 27703, United States.
Current address: FORMA Therapeutics, 500 Arsenal St., Suite 100, Watertown, MA
à
02472, United States.
http://dx.doi.org/10.1016/j.bmcl.2014.11.050
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Smalley Jr., T. L.; et al. Bioorg. Med. Chem. Lett. (2014), http://dx.doi.org/10.1016/j.bmcl.2014.11.050

2
T. L. Smalley Jr. et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Horner–Wadsworth–Emmons reaction with phosphonate 2 and
deprotection of the resulting silyl ether to provide the expected
key intermediate stilbene derivative 3.19
The oxazolidinone 6 was synthesized by adapting a closely
related literature procedure20 as outlined in Scheme 2. Treatment
of isobutyric anhydride with methyl isocyanoacetate followed by
hydrolysis provided the 2-amino-b-keto ester (4). Cyclization using
triphosgene provided the oxazolidinone ester (5), which was
alkylated with 2,6-dichlorobenzyl bromide, followed by reduction
of the ester to provide the desired oxazolidinone (6). Alternatively,
alkylating intermediate 5 with 2-(2,6-dichlorophenyl)-1-iodoeth-
ane followed by reduction gave oxazolidinone 7.
The synthesis of the furan analog is depicted in Scheme 3. The
propargyl b-keto ester was formed from the reaction of isobutyryl
chloride with Meldrum’s acid. Treatment with propargyl alcohol
gave the ester which was further reacted with p-acetamido-
benzenesulfonyl azide to give 8. Rhodium catalyzed cyclization21
provided the bicyclic furan 9 which was treated with methanol22
followed by Mitsunobu alkylation with 2,6-dichlorophenol to
provide the ester 10. Reduction gave the desired furanyl alcohol 11.
The synthesis of regioisomeric trisubstituted triazoles is shown
in Scheme 4. Thermal cyclization of 2,6-dichlorophenyl azide23
with methyl 4-methylpentynoate24 provided the desired triazole
12. Reduction of the ester group to alcohol 13 was performed with
DIBAH. Alternatively, the regioisomeric triazole was more chal-
lenging. 2,6-Dichlorobenzoyl chloride was treated with methyl
2-(triphenylphosphoranylidene)acetate to provide 14, which
underwent thermal rearrangement to provide the desired alkyne
15. Thermal cyclization with isopropyl azide25 provided a mixture
of the two possible regioisomers 16 and 17 which were separated
by silica gel chromatography. Reduction with DIBAH gave the
desired alcohol 18.
Synthesis of the pyrazole analog is depicted in Scheme 5.
3-Methylbutyraldehyde was treated with morpholine to provide
enamine 19.26 Acetylation27 of 19 with acetoxyacetyl chloride, fol-
lowed by cyclization with 2,6-dichlorophenylhydrazine gave the
desired protected pyrazole 20. Removal of the acetate protecting
group was accomplished by treatment with potassium carbonate
in methanol to provide the desired alcohol 21.
The synthesis of the 1-isopropylimidazole derivative is shown
is Scheme 6. Alkylation of methyl 4-(hydroxymethyl)-1H-imidaz-
ole-5-carboxylate with isopropyl bromide provided a mixture of
regioisomers, which were further alkylated using Mitsunobu
conditions to provide 22 following chromatographic separation.
Figure 1. An overlay of oxazolidinone 26 (green) with that of GW4064 (magenta) in
the binding pocket of FXR.
the carboxylic acids of the native ligands.18 The carboxylic acid-
aryl ring-stilbene portion of GW4064 lie co-planar to each other,
allowing the ligand to fit into a narrow portion of the receptor.
Also, an edge-to-face stacking interaction of the isoxazole moiety
with 454Trp on helix 12 that appears to make a hydrogen bond with
447His was observed. The optimized isopropyl moiety in the 5-
position of the isoxazole that occupied a specific, well-defined
hydrophobic area within the binding pocket formed by 284Phe,
287Leu, 454Trp, and 461Phe, was held constant. When a model of
oxazolidine 26 was docked into the binding pocket and compared
with GW4064, many of the same interactions appear possible.
Interestingly, the carbonyl group of the oxazolidinone reaches
deeper into the pocket, potentially allowing for a stronger hydro-
gen bond with 447His.
The strategy employed in the synthesis of these analogs is
depicted in Scheme 1. 2-Chloro-4-hydroxybenzaldehyde was
protected as the tert-butyldimethylsilyl (TBS) ether 1 followed by
OH
Cl
Cl
OHC
a
OHC
MeO2C
b, c
Cl
OH
OTBDMS
1
3
Scheme 1. Reagents and conditions: (a) TBSCl, imidazole, DMF, 99%; (b) diethyl [3-(methoxycarbonyl)phenyl]methylphosphonate (2), NaH, THF, 0 °C to RT, 48%; (c) Bu4NF,
THF, 75%.
O
N
O
O
O
O
c
a, b
d or e, f
O
N
O
HO
O
O
OMe
Cl
NH2
MeO2C
n
H
Cl
4
5
6
7
n=1
n=2
Scheme 2. Reagents and conditions: (a) Methyl isocyanoacetate, DBU, THF, 0 °C, 94%; (b) p-TsOH, MeOH, reflux; (c) triphosgene, Et3N, THF, À50 °C, 100%;
(d) 2,6-dichlorobenzyl bromide, K2CO3, acetone, 20%; (e) 2-(2,6-dichlorophenyl)-1-iodoethane, K2CO3, 20%; (f) LiAlH4, THF, 88%.
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T.L. Smalley Jr. et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
3
O
O
O
O
N
N
d
a, b, c
H
N
N
N
O
g
Cl
HO
a, b
O
c
N
N2
H
O
O
O
O
O
9
O
Cl
Cl
OH
O
8
Cl
Cl
O
22
23
O
e, f
HO
O
N
N
N
Cl
O
Cl
O
O
N
N
d
HO
O
e
N
O
Cl
Cl
O
Cl
Cl
H
O
Cl
Cl
10
11
Scheme 3. Reagents and conditions: (a) Meldrum’s Acid, pyridine, CH2Cl2, 0 °C to
RT, 76%; (b) propargyl alcohol, PhMe, reflux, 53%; (c) p-acetamidobenzenesulfonyl
azide, Et3N, CH3CN, 84%; (d) Rh2(octanoate)4, PhH, 80 °C, quant.; (e) Amberlyst 15,
MeOH, 35%; (f) 2,6-dichlorophenol, DIAD, PPh3, CH2Cl2, 67%; (g) LiAlH4, THF, 94%.
24
25
Scheme 6. Reagents and conditions: (a) Isopropyl bromide, K2CO3, DMF, 90 °C,
separate regioisomers; (b) 2,6-dichlorophenol, DIAD, PPh3, THF, 24% over 2 steps;
(c) DIBAH, toluene; (d) 2,6-dichlorophenethyl bromide, Cs2CO3, DMF, 100 °C; (e)
DIBAH, toluene, RT, 25% over 2 steps.
Reduction of the ester with DIBAH gave the desired alcohol 23.
Alternatively, the 4-isopropyl imidazole was prepared by alkyl-
ation of methyl 4-isopropyl-1H-imidazole-5-carboxylate28 with
2,6-dichlorophenethyl bromide to provide the desired product
24, which was reduced with DIBAH to provide alcohol 25.
The coupling of these novel heterocyclic fragments to the diaryl
stilbene fragment 3 was performed by Mitsunobu alkylation to
provide the desired compounds as the corresponding methyl esters
(see Table 1). Saponification of the esters provided the desired
acids.
The assay results of our novel scaffolds are shown in Table 1.
Oxazolidinones 26 and 27 showed good potency and agonist activ-
ity in both the fluorescence resonance energy transfer (FRET) and
the transient transfection (TT) assays. It is postulated that the
carbonyl oxygen acts as the key H-bonding element in these com-
pounds, occupying a space that is similar to the nitrogen atom of
the isoxazole ring in GW4064. Based on this hypothesis, it is not
surprising that the furan analog (28) lost agonist activity against
Cl
N
N
N
N
N
N
b
N3
Cl
a
HO
CO2Me
MeO2C
Cl
+
Cl
Cl
Cl
12
13
CO2Me
O
Cl
O
Cl
CO2Me
PPh3
Cl
c
d
Cl
Cl
Cl
Cl
14
15
N
N
N
N
N
N
N
N
HO
Cl
f
e
MeO2C
Cl
MeO2C
Cl
N
+
Cl
Cl
Cl
16
17
18
Scheme 4. Reagents and conditions: (a) 100 °C, neat, 30%; (b) DIBAL-H (2.1 equiv), THF, 87%; (c) Ph3P@CHCO2Me, PhMe, 100 °C, 20%; (d) o-dichlorobenzene, 250 °C,
microwave, 66%; (e) iPrN3, PhMe, microwave, 125 °C, 41%; (f) DIBAH, PhMe.
N
N
a
b, c
d
HO
AcO
CHO
N
N
N
Cl
Cl
O
Cl
Cl
19
20
21
Scheme 5. Reagents and conditions: (a) Morpholine, cyclohexane, 50 °C, 59%; (b) acetoxyacetyl chloride, (i-Pr)2NEt, THF, 35%; (c) 2,6-dichlorophenylhydrazineÁHCl, Et3N,
EtOH, 30%; (d) K2CO3, MeOH, 88%.
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T. L. Smalley Jr. et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx
Table 1
FXR agonist activity
OH
O
R
a, b
MeO2C
HO2C
Cl
Cl
FXR FRET29,30
FXR TT31,30
FXR FRET
FXR TT
Cl
Cl
Cl
Cl
Cl
O
N
N
N
110 nM (90%)
54 nM (104%)
617 nM (49%)
676 nM (35%)
O
Cl
Cl
Cl
Cl
30
26
N
N
O
O
N
66 nM (99%)
35 nM (82%)
123 nM (80%)
112 nM (106%)
42 nM (105%)
145 nM (47%)
35 nM (125%)
20 nM (100%)
N
Cl
O
27
28
31
Cl
Cl
O
N
N
1.23
lM (51%)
<10 lM
N
Cl
Cl
32
N
O
N
309 nM (88%)
562 nM (62%)
N
N
Cl 29
Cl
33
Cl
O
59 nM (100%)
65 nM (100%)
N
Cl
GW4064
(a) Heterocyclic alcohol, PS-PPh3, DIAD, CH2Cl2; (b) NaOH, MeOH, 60 °C.
FXR in both assays. Although the 5-hydrogen atom can be accom-
modated by the protein, since the carbonyl oxygen of the oxazolid-
inone fits into the binding pocket, a hydrogen bond acceptor has
been lost. This result provides further support that the isoxazole
nitrogen atom in GW4064 accepts a key H-bonding interaction
with the backbone of FXR. The absence of this key interaction in
28 leads to a significant drop in potency. The 1-isopropyl imidazole
(29) showed a slight, but significant, reduction in affinity, even
though a nitrogen atom is available for hydrogen bonding in what
is being proposed as the optimal position. Also, 4-isopropyl imidaz-
ole analog 30 showed a decline in potency and, perhaps more
significantly, in agonist efficacy. It should be noted that while 30
contains a potential hydrogen bonding nitrogen, the atom does
not appear to be aligned in the optimal position to support a
hydrogen bond. Both triazoles (31, 32) displayed good activity,
with the 4-isopropyl derivative 32 being particularly potent and
efficacious. It is somewhat interesting to observe that the while
the 1-isopropyl analog 31 showed good potency, the efficacy of
the agonist was only approximately half of that of the 32 in the
TT assay. Finally, the pyrazole derivative 33 was very potent in
both assays and also showed excellent agonist activity.
when dosed in rats, giving corresponding low exposures and short
half-lives. Thus, none were suitable for progression into in vivo
pharmacodynamic experiments for further development.
In summary, several novel heterocyclic analogs of GW4064
were designed, synthesized and assayed as FXR agonists. The more
active analogs, such as 26, 27, 32 and 33, maintain a putative
H-bond acceptor, placed strategically in the ring. These analogs
are valuable in designing subsequent analogs, potentially in combi-
nation with novel constrained analogs of GW4064, which may be
presented in future communications.
Acknowledgment
The authors would like to thank Aaron B. Miller for his assis-
tance with crystal structure overlays.
References and notes
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30. The maximum percent efficacy of the test compound is relative to FXR
activation by GW4064.
31. FXR transient transfection assay measuring the ligand-mediated luminescence
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kidney cells, then treated with the test compound. The pEC50 values are the
mean of at least two assays.
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