Improving the pharmacokinetics of GPR40/FFA1 full agonists

Article abstract of DOI:10.1021/ml4005123

We recently reported the discovery of a potent GPR40 full agonist AM-1638 (1). Herein, we describe our efforts in improving the drug-like properties of the full agonists through the systematic introduction of polar groups in the C-, D-, and A-rings. This led to the discovery of new GPR40 full agonists with significantly improved pharmacokinetic propeties. Compound 8 and 20 also showed potent in vivo efficacy in oral glucose tolerance tests in mice in addition to the improvement in properties.

Full text of DOI:10.1021/ml4005123

Letter
pubs.acs.org/acsmedchemlett
Improving the Pharmacokinetics of GPR40/FFA1 Full Agonists
Xiaohui Du,* Paul J. Dransfield, Daniel C.-H. Lin, Simon Wong, Yingcai Wang, Zhongyu Wang,
Todd Kohn, Ming Yu, Sean P. Brown, Marc Vimolratana, Liusheng Zhu, An-Rong Li, Yongli Su,
Xianyun Jiao, Jiwen (Jim) Liu, Gayathri Swaminath, Thanhvien Tran, Jian Luo, Run Zhuang, Jane Zhang,
Qi Guo, Frank Li, Richard Connors, Julio C. Medina, and Jonathan B. Houze
Departments of Therapeutic Discovery, Metabolic Disorders, Pharmaceutics, and Pharmacokinetic and Drug Metabolism, Amgen
Inc., 1120 Veterans Boulevard, South San Francisco, California 94080, United States
S
* Supporting Information
ABSTRACT: We recently reported the discovery of a potent
GPR40 full agonist AM-1638 (1). Herein, we describe our
efforts in improving the drug-like properties of the full agonists
through the systematic introduction of polar groups in the C-,
D-, and A-rings. This led to the discovery of new GPR40 full
agonists with significantly improved pharmacokinetic propeties.
Compound 8 and 20 also showed potent in vivo efficacy in oral
glucose tolerance tests in mice in addition to the improvement in properties.
KEYWORDS: GPR40, FFA1, full agonist, insulin secretagoue, type 2 diabetes
ype 2 diabetes is characterized by impaired glucose
hoped to reduce the CLogP and improve the pharmacokinetic
profile of this class of GPR40 agonists by introducing polarity
throughout the structure including the C-ring, D-ring, and the
A-ring.14,15
T
homeostasis due to insulin resistance and insufficient
secretion of insulin from the pancreas.1 Consequently, blood
glucose increases and is a major risk factor for microvascular
and macrovascular complications including retinopathy, nephr-
opathy, neuropathy, and accelerated cardiovascular disease.2−4
Treating type 2 diabetes remains a significant medical need to
this day.
An aequorin assay13 was used to evaluate the potency of
newly synthesized GPR40 agonists. GPR40 couples primarily to
Gq and elicits a calcium response. The assay was performed
with cells stably overexpressing the GPR40 receptor to examine
the extent of calcium release upon activation by the synthetic
GPR40 agonists.
GPR40 (FFA1) belongs to Class A of the G-protein coupled
receptor (GPCR) family. It is primarily expressed in pancreatic
islet β-cells and also in enteroendocrine cells. GPR40 is not
only involved in direct potentiation of glucose-stimulated
insulin secretion in pancreatic islet β-cells but also stimulates
GLP-1 release from enteroendocrine L-cells.5 The dual
mechanism of action provides a compelling rationale to
develop GPR40 agonists as glucose-dependent insulin
secretagogues for the treatment of type 2 diabetes.6−8
Consequently, it has attracted interest from many in the
pharmaceutical community.9−11
We previously described a GPR40 full agonist AM-1638
(1)12,13 (Figure 1), which displayed superior antidiabetic
efficacy in several rodent models. However, AM-1638 (1) is
very lipophilic and has unsatisfactory physicochemical proper-
ties such as a calculated CLogP of 9.3. AM-1638 (1) exhibits
clearance of 0.91 L/(h·kg) and a half-life of 1.8 h in rat. We
To introduce polarity into the C-ring of AM-1638 (1), we
decided to explore the use of pyridine as a replacement for the
phenyl group. This strategy has been applied in a different
GPR40 scaffold by Ulven et al.16 Methoxy-pyridines were
employed as the C-ring with the ring nitrogen at the ortho,
meta, and para positions relative to the B-ring (Table 1).17 The
ortho- and meta-pyridines (compounds 3 and 4, Table 1) were
investigated with the tert-butyl group in place of the D-ring.
Both 3 and 4 were less potent as compared to their nonpyridine
analogue 2. This strategy was revisited after the more potent
dimethyl cyclopentenyl D-ring was discovered. Compound 5
with a 2′-fluoro-5′-methoxy pyridine C-ring maintained both
the potency and efficacy in the aequorin assay compared to
AM-1638 (1), while lowering the CLogP by 0.7 unit.
Subsequently, the 2′-fluoro-5′-methoxy pyridine C-ring was
combined with the best head groups and the 2,2-dimethylcy-
clopentyl D-ring to evaluate its impact on pharmacokinetic
profile (Table 2).
Received: December 12, 2013
Accepted: February 3, 2014
Published: February 3, 2014
Figure 1. GPR40 full agonist AM-1638 (1).
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Table 1. Replacing the C-Ring with Various Methoxy Pyridines
a
b
c
Mean of at least two runs; mean standard deviation is 14%. % Maximal efficacy compared to AM-1638 (1). Calculated from ChemBioDraw
Ultra 12.0 by CambridgeSoft.
Table 2. Methoxy Pyridine Replacement Improved the Pharmacokinetics of the GPR40 Agonists
a
b
c
Mean of at least two runs; mean standard deviation is 14%. % Maximal efficacy compared to AM-1638 (1). Metabolic stability in clearance
intrinsic: CL intrinsic (μL/min/mg) = {ln 100 − ln[(conc. after the metabolic reaction)/(conc. at initial 1 μM) × 100]}/[30 min]/[0.25 (mg/mL)
d
e
human or rat liver microsomes] × 1000 at 37 °C. IV dose: 0.5 mg/kg. n = 2; PO dose: 2.0 mg/kg. n = 3. Calculated from ChemBioDraw Ultra
12.0 by CambridgeSoft.
Compound 7 with the 2′-fluoro-5′-methoxy pyridine C-ring
showed improved metabolic stability, clearance, and half-life
compared to its 2′-fluoro-5′-methoxy phenyl C-ring analogue 6.
The addition of a fluorine atom onto the headgroup resulted in
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ACS Medicinal Chemistry Letters
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compound 8, which further increased the half-life (4.6 h) and
oral bioavailability (98%) compared to those of compound 7
while maintaining good metabolic stability. Both 7 and 8 have
lower CLogP values due to the pyridine C-ring.
Table 3. Polar Group Introduction at the D-Ring
Given the excellent pharmacokinetic profile of compound 8,
it was dosed in high fat fed/STZ type II diabetic mice.5
Compound 8 demonstrated a dose-proportional decrease in
blood glucose levels with comparable efficacy at 30 mg/kg
compared to a maximally efficacious dose of 60 mg/kg AM-
1638 (1) during an oral glucose tolerance test (Figure 2).
Compound 8 also stimulated dose-dependent GLP-1 release,
reconfirming the dual mechanism of action of GPR40.18
Figure 2. In vivo results of dosing compound 8 in a HF/STZ type II
diabetic model in mice. Compound 8 lowered blood glucose in a dose-
dependent manner. Statistical significance compared to vehicle
treatment is denoted by *(p < 0.05), **(p < 0.01), ***(p < 0.001),
and ****(p < 0.0001), as determined by two-way ANOVA, and is
color-coded to the treatment in the figure legends.
a
b
Mean of at least two runs; mean standard deviation is 14%.
%
c
Maximal efficacy compared to AM-1638 (1). IV dose: 0.5 mg/kg. n =
2. Calculated from ChemBioDraw Ultra 12.0 by CambridgeSoft.
d
Polar groups were also evaluated at the D-ring (Table 3).
Compound 9 had a less lipophilic neopentyl group replacing
the 2,2-dimethylcyclopentyl group. However, it displayed
higher clearance in rat. The addition of a fluorine to the
neopentyl group was tolerated in compound 10. A methoxy
replacement of the fluorine (compound 11) further reduced
the CLogP to 7.7 and led to a reduced clearance and a longer in
vivo half-life in rats compared to those of compound 6. Ethoxy
replacement (12) did not significantly change the potency or
DMPK profile compared to that of compound 11. Constraining
the ethoxy dimethylpropyl group into a dimethyltetrahydropyr-
an D-ring (13) led to a decrease in potency. Compound 14, a
diastereomer of 11, suffered a 10-fold loss in potency thus
indicating the importance of the methoxy-bearing stereogenic
center. These agonists suggested that the methoxy dimethyl-
propyl group in compound 11 could be a good functionality to
combine with the best findings in other areas of the GPR40
molecules.
Seeking additional areas to introduce polarity, we also
investigated into the A-ring (Table 4). Among the analogues
with 2,2-dimethylcyclopentyl group as the D-ring, introducing
an additional pyridine as the A-ring significantly lowered the
clearance to 0.086 L/(h·kg) and extended the half-life to 9.7 h
(compound 15). Substitution at the benzylic position of the
pyridine A-ring was explored. Exchanging the cyclopropyl
group to an ethyl group (16) was less advantageous and
resulted in a nearly 1.5-fold increase in clearance. Trimming the
cyclopropyl group down to a methyl (17) led to a 3-fold drop
in potency.
Replacing the D-ring of 15 with the methoxy dimethylpropyl
group led to compound 20 with the lowest clearance of 0.061
L/(h·kg) among the compounds shown in Table 4. In addition,
compound 20 has a long half-life (8.1 h) and good oral
bioavailability of 54%. The improved pharmacokinetics
correlated well with the improved CLogP value (6.0) of
compound 20, which is more than 3 units lower than that of
AM-1638 (1).
To further confirm that these compounds are full agonists
and that the signaling of the receptor is indeed through Gq that
results in accumulation of IP3, an upstream signal preceding
calcium release, we performed an IP3 assay on transfected A9
cells stably overexpressing the GPR40 receptor. While the
aequorin assay was performed on cells that express low levels of
the FFA1 receptor, the IP3 assays were performed on cells
stably expressing the FFA1 receptor at high levels. This
accounts for the increased potency shift in IP3 assay compared
to aequorin assay. The use of different expression level of
receptor helps to delineate if the compound is a true full agonist
or not. All compounds in Table 4 were confirmed to be full
agonists in both the aqeuorin and the IP3 assays. Compound 20
has the same potency (162 nM) as AM-1638 (1) (161 nM) in
aequorin assay, but it is much more potent in IP3 assay (0.6
nM) than AM-1638 (1) (13 nM).
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Table 4. Polar and Constrained Groups Introduction at the Head Group
a
b
c
Mean of at least two runs; mean standard deviation is 14%. % Maximal efficacy compared to AM-1638 (1). Mean of at least two runs; mean
d
e
standard deviation is 10%. IV dose: 0.5 mg/kg. n = 2; PO dose: 2.0 mg/kg. n = 3. Calculated from ChemBioDraw Ultra 12.0 by CambridgeSoft.
A few of the constrained head groups15 were combined with
the findings in the C-ring and D-ring to form compounds 18,
19, 21, and 22. They generally showed improved clearance over
AM-1638 (1) with the exception of compound 22; however,
they did not show superiority over compounds 15 and 20.
Compound 20 had a desirable combination of potency,
CLogP, and pharmacokinetics. It was scaled up for further
studies through the synthesis shown in Scheme 1. Dimethyl 4-
hydroxyisophthalate 23 was converted to triflate 24. Subse-
quent palladium-catalyzed Suzuki coupling with (5-fluoro-2-
methoxypyridin-4-yl) boronic acid yielded biphenyl 25.
Selective hydrolysis of the less-hindered ester in 25 resulted
in compound 26, and subsequent reduction of the carboxylic
acid in 26 led to alcohol 27. tert-Butyl lithium addition to the
ester in 27 generated ketone 28. Then it was converted to diol
29 through reduction of the ketone in 28 followed by chiral
separation. The benzylic alcohol in 29 was selectively protected
as a TBS ether, and then the secondary hydroxy group in 29
was protected as a methyl ether. After deprotection of the TBS
group in 30, the benzylic alcohol in 31 was converted to a
bromide 32, which would be used to couple to headgroup 38.
To synthesize headgroup 38, 2-fluoroisonicotinaldehyde 33
was condensed with Meldrum’s acid to generate compound 34.
1,4-Addition of cyclopropyl magnesium bromide to 34 and
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ACS Medicinal Chemistry Letters
Letter
a
Scheme 1
Figure 3. Compound 20 showed glucose-lowering effect in a BDF-
DIO model in mice. Statistical significance compared to vehicle
treatment is denoted by *(p < 0.05), **(p < 0.01), ***(p < 0.001),
and ****(p < 0.0001), as determined by two-way ANOVA, and is
color-coded to the treatment in the figure legends.
glucose-lowering effect similar to that of compound 8 at 30
mg/kg. Though compound 20 had no further increase in
efficacy from 3 to 10 mg/kg, the blood glucose levels were not
dropped lower into a hypoglycemic range. This result indicates
that targeting GPR40 might provide a potential advantage as
diabetes therapeutics in terms of hypoglycemic control. The
potent in vivo efficacy of compound 20 is consistent with its
potent in vitro activity as well as its superior pharmacokinetics.
In conclusion, lipophilicity oftentimes correlates with high
metabolism and poor oral absorption. Our strategy of
improving the lipophilicity of the GPR40 agonists through
introducing polar groups in the C-ring, D-ring, and A-ring led
to the discovery of a number of compounds with improved
pharmacokinetics compared to AM-1638 (1). The improved
pharmacokinetics generally correlated well with lowered
calculated CLogP values. In addition, compounds 8 and 20
displayed potent glucose-lowering efficacy in mouse models.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures and data for compounds, biological
assay procedures, in vivo procedures, and GLP-1 release data
for compound 8. This material is available free of charge via the
Internet at http://pubs.acs.org.
a
■
Reagents and conditions: (a) PhN(Tf)2, Et3N, CH2Cl2, 88%; (b) (5-
S
fluoro-2-methoxypyridin-4-yl)boronic acid, Pd(PPh3)4, K2CO3, DMF,
90 °C, 90%; (c) KOH, THF, MeOH, H2O, 99%; (d) BH3·THF, 90%;
(e) t-BuLi, THF, 80%; (f) LiAlH4, THF, then chiral separation with
4% IPA in hexanes on Chiracel-OD column, 44%; (g) (i) TBSCl,
DMAP, Et3N, 90%; (ii) MeI, NaH, DMF, 73%; (h) TBAF, THF,
100%; (i) NBS, PPh3, 90%; (j) Meldrum’s acid, HOAc, piperidine,
benzene, reflux, 68%; (k) (i) cyclopropylMgBr, THF; (ii) DMF, H2O,
95 °C, 84%; (l) chiral separation on chiral IC column with 50 g/min
IPA +0.2% DEA and 50 g/min CO2 on Thar 350 SFC, 45%; (m) aq.
HCl, 100 °C, 96%; (n) MeOH, H2SO4, reflux, 89%; (o) 32, Ag2CO3,
toluene, 83%; (p) LiOH, THF, MeOH, H2O, 87%.
AUTHOR INFORMATION
Corresponding Author
*(X.D.) E-mail: xdu@amgen.com.
Notes
■
The authors declare no competing financial interest.
decarboxylation generated acid 35, which was chirally separated
to obtain the desired enantiomer 36. The fluoro-pyridine in 36
was hydrolyzed to form pyridone 37. Esterification of
compound 37 led to headgroup 38. Coupling of 38 with tail
group 32 facilitated by silver carbonate formed the O-alkylated
compound 39, and it was subsequently hydrolyzed with lithium
hydroxide to give compound 20.
The in vivo efficacy of compound 20 was demonstrated in a
BDF-DIO model19 in mice (Figure 3). At a low dose of 0.3
mg/kg, it lowered the blood glucose significantly during an oral
glucose tolerance test. At 3 mg/kg, it showed comparable
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(17) The general syntheses of the agonists including the compounds
in Tables 1−3 and the compounds with constrained head groups in
Table 4 (18, 19, 21, and 22) follow our previous publications in refs
12 and 15.
(18) See Supporting Information for detailed GLP-1 release data for
compound 8.
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