GW4064 | Inhibitor library

Novel heterocyclic scaffolds of GW4064 as farnesoid X receptor agonists
Terrence L. Smalley Jr. a,⇑, Sharon Boggs a,ti , Justin A. Caravella b,ti , Lihong Chen c, Katrina L. Creech d, David N. Deaton a, Istvan Kaldor a, Derek J. Parks d
aMuscle Metabolism DPU, Metabolic Pathways & Cardiovascular Discovery Performance Unit, GlaxoSmithKline, Inc., Five Moore Drive, Research Triangle Park, NC 27709, United States
bComputational and Structural Sciences, Platform Technology & Science, GlaxoSmithKline, Inc., Five Moore Drive, Research Triangle Park, NC 27709, United States
cEnteroendocrine DPU, Metabolic Pathways & Cardiovascular Discovery Performance Unit, GlaxoSmithKline, Five Moore Drive, Research Triangle Park, NC 27709, United States
dMolecular 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

Article history:
Received 14 October 2014 Revised 17 November 2014 Accepted 19 November 2014 Available online xxxx

Keywords: FXR agonist Heterocycles Liver
a b s t r a c t

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.
ti 2014 Elsevier Ltd. All rights reserved.

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.

Cl
O
N

Cl
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

GW4064
There has been a long standing research program at GlaxoSmithK- line targeting FXR agonists. In fact, the discovery of FXR agonist
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

⇑ Corresponding author Tel.: +1 (919) 483 1054.
E-mail address: [email protected] (T.L. Smalley).
ti Current address: Cree, 4600 Silicon Drive, Durham, NC 27703, United States.
ti Current address: FORMA Therapeutics, 500 Arsenal St., Suite 100, Watertown, MA 02472, United States.

http://dx.doi.org/10.1016/j.bmcl.2014.11.050

0960-894X/ti 2014 Elsevier Ltd. All rights reserved.
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

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

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.

Cl Cl

OHC a OHC b, c MeO2C

OH OTBDMS
Cl

1 3

Scheme 1. Reagents and conditions: (a) TBSCl, imidazole, DMF, 99%; (b) diethyl [3-(methoxycarbonyl)phenyl]methylphosphonate (2), NaH, THF, 0 ti C to RT, 48%; (c) Bu4NF, THF, 75%.

O O
a, b
O O
c O d or e, f
O
O

O OMe O HO N

NH2
MeO2C
N
H
Cl
n

4 5 Cl
6n=1
7n=2

Scheme 2. Reagents and conditions: (a) Methyl isocyanoacetate, DBU, THF, 0 tiC, 94%; (b) p-TsOH, MeOH, reflux; (c) triphosgene, Et3N, THF, ti 50 ti C, 100%; (d) 2,6-dichlorobenzyl bromide, K2CO3, acetone, 20%; (e) 2-(2,6-dichlorophenyl)-1-iodoethane, K2CO3, 20%; (f) LiAlH4, THF, 88%.

T.L. Smalley Jr. et al. / Bioorg. Med. Chem. Lett. xxx (2014) xxx–xxx 3

a, b, c

O
O

N2
O

H
d

O
O

O
9

a, b

O
N

O
Cl

c
N
HO N

e, f

O O

N
N

Cl
10
Cl
11
O

O
N
H
O

Cl
Cl

Scheme 3. Reagents and conditions: (a) Meldrum’s Acid, pyridine, CH2Cl2, 0 tiC to RT, 76%; (b) propargyl alcohol, PhMe, reflux, 53%; (c) p-acetamidobenzenesulfonyl
24
25

azide, Et3N, CH3CN, 84%; (d) Rh2(octanoate)4, PhH, 80 ti C, quant.; (e) Amberlyst 15, MeOH, 35%; (f) 2,6-dichlorophenol, DIAD, PPh3, CH2Cl2, 67%; (g) LiAlH4, THF, 94%.

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.
Scheme 6. Reagents and conditions: (a) Isopropyl bromide, K2CO3, DMF, 90 ti 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 ti C; (e) DIBAH, toluene, RT, 25% over 2 steps.

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

CO2Me
a

MeO2C
N
N

Cl
b
HO
N
N

Cl Cl Cl

12 13

CO2Me

Cl
O Cl
Cl

Cl
O
PPh3 Cl

d
Cl
CO2Me

Cl
14 15

N N N N HO N N

e MeO2C N
+
MeO2C N f N

Cl
Cl
Cl
Cl
Cl
Cl

16 17 18

Scheme 4. Reagents and conditions: (a) 100 ti C, neat, 30%; (b) DIBAL-H (2.1 equiv), THF, 87%; (c) Ph3P@CHCO2Me, PhMe, 100 ti C, 20%; (d) o-dichlorobenzene, 250 ti C, microwave, 66%; (e) iPrN3, PhMe, microwave, 125 ti C, 41%; (f) DIBAH, PhMe.

CHO a

O
b, c
N
AcO N Cl
Cl
d
HO

Cl
N
N

19 20 21

Scheme 5. Reagents and conditions: (a) Morpholine, cyclohexane, 50 ti C, 59%; (b) acetoxyacetyl chloride, (i-Pr)2NEt, THF, 35%; (c) 2,6-dichlorophenylhydrazinetiHCl, Et3N, EtOH, 30%; (d) K2CO3, MeOH, 88%.

Table 1
FXR agonist activity
OH O R

MeO2C
a, b
HO2C

Cl Cl

FXR FRET29,30 FXR TT31,30 FXR FRET FXR TT

Cl Cl

N O

110 nM (90%) 54 nM (104%)
N N

617 nM (49%) 676 nM (35%)

Cl
O
26
Cl

NO
66 nM (99%) 35 nM (82%) N
N
123 nM (80%) 145 nM (47%)

27
O
Cl

1.23 lM (51%) <10 lM
N

N
N

112 nM (106%) 35 nM (125%)

Cl
28
Cl

O
N 309 nM (88%) 562 nM (62%)
N
N 42 nM (105%) 20 nM (100%)

Cl
29
Cl
33

N
O
59 nM (100%) 65 nM (100%)

Cl
GW4064
(a) Heterocyclic alcohol, PS-PPh3, DIAD, CH2Cl2; (b) NaOH, MeOH, 60 ti 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.
Several of the agonists were profiled in pharmacokinetic exper- iments to determine the feasibility of progressing them to in vivo experiments. All compounds profiled showed high clearances
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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29.FXR ligand-seeking assay measuring ligand-mediated interaction of the SRC-1 peptide (B-CPSSHSSLTERHKILHRLLQEGSPS-CONH2) with the FXR237–472 LBD, using 5 nM biotinylated FXR LBD coupled to 5 nM allophycocyanin-labeled streptavidin and 10 nM biotinylated SRC-1 coupled to 5 nM Europium-labeled streptavidin as reagents in 10 mM DTT, 0.1 g/L BSA, 50 mM NaF, 50 mM MOPS, 1 mM EDTA, and 50 lM CHAPS, at pH 7.5. The pEC50 values are the mean of at least two measurements.
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 resulting from FXR-induced transcription of a luciferase reporter. FXR and the luciferase reporter genes are transfected into African green monkey CV-1 kidney cells, then treated with the test compound. The pEC50 values are the mean of at least two assays.