Optimization of GPR40 agonists for type 2 diabetes

Article abstract of DOI:10.1021/ml400501x

GPR40 (FFA1 and FFAR1) has gained significant interest as a target for the treatment of type 2 diabetes. TAK-875 (1), a GPR40 agonist, lowered hemoglobin A1c (HbA1c) and lowered both postprandial and fasting blood glucose levels in type 2 diabetic patient

Full text of DOI:10.1021/ml400501x

Letter
pubs.acs.org/acsmedchemlett
Optimization of GPR40 Agonists for Type 2 Diabetes
Jiwen (Jim) Liu,* Yingcai Wang,* Zhihua Ma, Mike Schmitt, Liusheng Zhu, Sean P. Brown,
Paul J. Dransfield, Ying Sun, Rajiv Sharma, Qi Guo, Run Zhuang, Jane Zhang, Jian Luo, George R. Tonn,
Simon Wong, Gayathri Swaminath, Julio C. Medina, Daniel C.-H. Lin, and Jonathan B. Houze
Department of Therapeutic Discovery, Metabolic Disorders, Translational Sciences, Amgen Inc., 1120 Veterans Boulevard, South San
Francisco, CA 94080, United States
S
* Supporting Information
ABSTRACT: GPR40 (FFA1 and FFAR1) has gained
significant interest as a target for the treatment of type 2
diabetes. TAK-875 (1), a GPR40 agonist, lowered hemoglobin
A1c (HbA1c) and lowered both postprandial and fasting blood
glucose levels in type 2 diabetic patients in phase II clinical
trials. We optimized phenylpropanoic acid derivatives as
GPR40 agonists and identified AMG 837 (2) as a clinical candidate. Here we report our efforts in searching for structurally
distinct back-ups for AMG 837. These efforts led to the identification of more polar GPR40 agonists, such as AM-4668 (10), that
have improved potency, excellent pharmacokinetic properties across species, and minimum central nervous system (CNS)
penetration.
KEYWORDS: GPR40, FFAR1, FFA1, GPCR, agonist, type II diabetes, insulin secretagogue
searching for structurally distinct GPR40 agonists. We
ype 2 diabetes mellitus (T2DM) is a metabolic
Tdysfunction characterized by increased insulin resistance emphasized achieving structural diversity through introducing
greater polarity into the molecule. Because compound 2 is a
lipophilic acid with low polar surface area (46.5, Table 1), it is
likely that 2 has significant central nervous system (CNS)
exposure. A close analogue of 2 showed a rat brain to plasma
ratio of 0.6 three hours after an oral dose of 5 mg/kg. While
GPR40 is expressed in the brain, its function there is unclear.
However, its role in modulating insulin secretion is through
direct activity on the pancreatic beta cell.6,13,18 In order to avoid
both potential target mediated and off-target effects in the CNS,
we aimed to design non-CNS penetrant GPR40 agonists. Since
the range of physicochemical properties that allows ready
access to the CNS is rather limited,19 we took the approach of
introducing greater polar surface area (PSA) into the molecules
to decrease CNS penetration.20
One successful way to introduce greater PSA was by
replacing the propynyl group of AMG 837 (2) with polar
heterocycles. Among the initial heterocycles studied were
imidazole, triazole, and tetrazole (Table 1). We initially set the
biphenyl moiety to that of compound 3 (Figure 1) because it
maximized potency. GPR40 is a Gαq-coupled GPCR, so
activation of GPR40 by the compounds was measured in an
aequorin assay using CHO cells stably transfected with human
GPR40 and in an inositol phosphate accumulation assay using
A9 cells also stably transfected with human GPR40.14,15 As
shown in Table 1, imidazole 4 and triazole 5 showed similar
EC50 to those of compounds 2 and 3. Tetrazole 6 was more
and impaired insulin secretion, resulting in higher blood
glucose levels. Insulin secretagogues, such as sulfonylureas and
glinides, are commonly used to increase insulin levels in
diabetic patients.1 However, these drugs promote insulin
secretion independent of blood glucose levels, thereby leading
to the risk of hypoglycemia.1,2 Novel insulin secretagogues with
a low risk of hypoglycemia are thus desirable.
G-protein coupled receptor 40 (GPR40, IUPHAR recom-
mended name: FFA1) is highly expressed in pancreatic β cells
and responds to endogenous fatty acids resulting in
amplification of insulin secretion only in the presence of
elevated glucose levels.3−6 Therefore, GPR40 agonists may
lower blood glucose levels with decreased risk of hypoglycemia
compared to classic insulin secretagogues. On the basis of these
findings, GPR40 has drawn considerable attention as a potential
therapeutic target for T2DM.7−13 Robust antiglycemic effects
were demonstrated using GPR40 agonists in rodent diabetic
models.7−9,14,15 Positive proof of concept data was obtained in
humans using a GPR40 agonist TAK-875 (1, Figure 1) in phase
II clinical trials: TAK-875 lowered hemoglobin A1c (HbA1c) by
an amount similar to that of glimepiride in type 2 diabetic
patients at week 12, and hypoglycemic incidents for all doses of
TAK-875 were similar to placebo and significantly lower
compared to glimepiride treatment groups.16 TAK-875 is now
being evaluated in phase III clinical trials.17
We reported the identification of AMG 837 (2, Figure 1),
which is a potent and selective GPR40 agonist selected for
evaluation in human clinical trials.14,15 AMG 837 shows similar
PK properties, potency and efficacy in rats to those of 1.7,15
After the discovery of compound 2, we initiated an effort in
Received: December 7, 2013
Accepted: February 6, 2014
Published: February 6, 2014
© 2014 American Chemical Society
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Letter
Figure 1. TAK-875 (1), AMG 837 (2), and a close analogue (3).
Table 1. GPR40 Activity and Rat Pharmacokinetic Properties of Compounds 4−6
a
GPR40 aequorin assay in CHO cells stably transfected with human GPR40. Assay was run in buffer containing 0.01% human serum albumin.
b
Values are reported as the average of at least two determinations. GPR40 IP3 assay using A9 cells stably transfected with human GPR40 in 0.3%
human serum. See refs 14 and 15 for assay protocol. V values are reported as the average of at least two determinations. Topological polar surface
area was calculated using Daylight Toolkit v4.8.1. IV dose at 0.5 mg/kg. Oral dose at 2 mg/kg. Values are from one experiment. Not determined.
c
d
e
f
g
Table 2. GPR40 Activity and Rat Pharmacokinetic Properties of Compounds 7−9
a
GPR40 aequorin assay in CHO cells stably transfected with human GPR40. Assay was run in buffer containing 0.01% human serum albumin.
b
Values are reported as the average of at least two determinations. GPR40 IP3 assay using A9 cells stably transfected with human GPR40 in 0.3%
human serum. See refs 14 and 15 for assay protocol. Values are from one experiment. Topological polar surface area was calculated using Daylight
Toolkit v4.8.1. IV dose at 0.5 mg/kg. Not determined. Oral dose at 2 mg/kg.
c
d
e
f
potent, but its rat clearance was high (CL = 2.8 L/h/kg). It was
interesting to notice that the clearance values trended higher as
the number of nitrogen in the heterocycles increased.
The observations revealed in Table 1 were confirmed in
compounds that have different biaryl tail pieces. The phenyl-
thiazole tail (Table 2) has a good degree of structural diversity
compared to the biphenyl tails and retains potency on the
receptor. With the phenylthiazole tail, tetrazole 9 was also more
potent than imidazole 7 and triazole 8 (Table 2). Once again,
in vivo rat clearance increased with increasing nitrogen count in
the heterocyclic ring β to the carboxylic acid (compounds 7−
9).
It was desired to retain the improved potency of the
tetrazoles without the increased clearance. The microsomal
stability of the tetrazoles was similar to that of the imidazoles
and triazoles. It is possible that the tetrazole head piece is
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Letter
Table 3. GPR40 Activity of Compounds 10−12
a
GPR40 aequorin assay in CHO cells stably transfected with human GPR40. Assay was run in buffer containing 0.01% human serum albumin.
b
Values are reported as the average of at least two determinations. GPR40 IP3 assay using A9 cells stably transfected with human GPR40 in 0.3%
human serum. See refs 14 and 15 for assay protocol. Values are from one experiment. Topological polar surface area was calculated using Daylight
Toolkit v4.8.1. Values are from one experiment. Not determined.
c
d
e
Table 4. Pharmacokinetic Properties of AM-4668
a
b
species clearance (L/h/kg) T1/2 (h) Vdss (L/kg) oral bioavailability
rat
0.09
0.15
0.04
5.3
5.6
14
0.68
0.60
0.35
77%
100%
65%
dog
cyno
c
a
b
c
IV dose at 0.5 mg/kg. Oral dose at 2 mg/kg. Cynomolgus monkey.
recognized by an efflux transporter, a phase II metabolic
enzyme, or both. The recognition seems to be reinforced as the
number of heteroatoms in the heterocycles rises. On the basis
of this observation, we reduced the number of heteroatoms and
evaluated positioning of the remaining ones to maintain the
good potency of the tetrazoles. A number of heterocycles were
evaluated, and isoxazole 10 (designated AM-4668) was as
potent as the corresponding tetrazole (Table 3). Other
oxazoles, such as 11 and 12, were less potent.
With the number of heteroatoms reduced, AM-4668 indeed
displayed excellent pharmacokinetic properties, not only in rat
but also in dog and cynomolgus monkey (Table 4). It had low
clearance, moderate to long half-life, and very good oral
bioavailability across species. Unlike some compounds with the
propynyl headgroup as 2 and 3, CNS penetration of AM-4668
Figure 3. Insulin secretion of AM-4668 (10) in islets isolated from
hGPR40 knock-in mice.
was low, as indicated by a rat brain to plasma ratio of 0.02 three
hours after an oral dose of 5 mg/kg. In addition, this compound
showed no inhibition of the hERG channel and demonstrated
low potential to induce drug−drug interaction by various CYP
inhibition and induction assays.
Since the activity of AM-4668 was weak against rodent
GPR40, it was difficult to study its efficacy in standard rodent
diabetes models. We therefore evaluated it in mice where the
Figure 2. Oral glucose challenge study of AM-4668 (10) in hGPR40 knock-in mice.
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Scheme 1. Synthesis of Isoxazole AM-4668 (10)
Notes
human GPR40 gene has been replaced with the mouse GPR40
gene but is under the control of the endogenous mouse GPR40
promoter.21 Oral administration of AM-4668 at 10 mg/kg dose
1 h before oral glucose challenge significantly reduced the
blood glucose levels (Figure 2A). The glucose AUC in the
compound-treated animals is 19% lower than that in the
vehicle-treated animals (Figure 2B). In a separate mouse oral
PK study, the plasma concentration of AM-4668 at 1 mg/kg
dose was about 2 μM for the first 4 h, which corresponded to
an 8 nM unbound concentration. In addition, AM-4668
induced insulin secretion in pancreatic islets isolated from
human GPR40 knock-in mice with an EC50 of 55 nM (Figure
3).22 Because AM-4668 does not effectively enter the CNS,
these results demonstrate that agonism of GPR40 by this
compound in the CNS is likely not necessary for its glucose
lowering effects in this model.
The synthesis of AM-4668 is shown in Scheme 1. Michael
addition of (S)-3-acetyl-4-benzyl-2-oxazolidinone (14) to trans-
β-nitrostyrene 13 in the presence of titanium chloride and
Hunig’s base produced compound 15.23 Generation of the
nitrile oxide24 from the nitro group in 15 and reaction with
vinyl bromide generated the isoxazole compound 16.
Deprotection of the benzyl group of 16 using boron trichloride
methyl sulfide complex afforded phenol 17, which was alkylated
using commercially available choloromethylthiazole 18 to yield
compound 19. Finally the oxazolidinone in compound 19 was
removed in the presence of lithium hydroxide and hydrogen
peroxide to afford AM-4668 (10).
The authors declare no competing financial interest.
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ASSOCIATED CONTENT
* Supporting Information
■
S
Detailed synthetic experimental procedures and character-
ization for all compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*(J.L.) E-mail: jiwenl@amgen.com.
*(Y.W.) E-mail: yingcai02@gmail.com.
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(21) Human GPR40 knock-in animals were generated at Ozgene
(Perth, Australia) by replacing the mouse GPR40 gene (a single exon
gene) with the human GPR40 gene. Briefly, the targeting vector was
created by PCR amplifying two fragments of genomic DNA (from
C57Bl/6 mice) corresponding to 5637 bp upstream and 5126 bp
downstream of mouse GPR40 and inserting these into the plasmid L-
Sniper (Ozgene). Human GPR40 was PCR amplified and fused in
between these fragments along with the selectable neomycin resistance
marker driven by the PGK promoter. The targeting vector was
electroporated into ES cells, selected for by culturing in neomycin, and
successful insertion of the human GPR40 was verified by PCR of
genomic DNA. Following ES cell injection into blastocysts, generation
of chimeric mice and germline transmission of the knockin allele,
human GPR40 knock-in mice were created and confirmed by PCR of
genomic DNA. mRNA expression of human GPR40 and lack of
expression of mouse GPR40 were confirmed by qPCR using RNA
from islets isolated from human GPR40 knock-in mice. Oral glucose
tolerance tests were performed on female human GPR40 knock-in
mice at 28 weeks of age. Mice were dosed via oral gavage at 10 mg/kg
of compound 10 following a 6 h fast. Compound 10 was formulated in
1% methylcellulose and 1% Tween-80. Glucose was administered by
oral gavage at 2 g/kg 1 h post drug dose. Blood glucose measurements
were taken from tail vein samples at various time points using
AlphaTRAK (Abbott) blood glucose monitoring system.
(22) Insulin secretion was assessed using islets isolated from human
GPR40 knock-in mice and was performed as described in ref 15.
Assays were performed in buffer containing 16.7 mM glucose and 0.1%
human serum albumin.
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