Solid phase synthesis and SAR of small molecule agonists for the GPR40 receptor

Article abstract of DOI:10.1016/j.bmcl.2006.12.084

The discovery, synthesis and structure-activity relationship (SAR) of novel carboxylic acid agonists for GPR40 are described. Aryl propionic acid 1, identified from a high throughput screen, was selected for chemical exploration. Compound 2 was identified as our lead molecule through efficient solid phase combinatorial array chemistry and had an attractive in vitro and in vivo pharmacokinetic profile in rat. These ligands may prove useful in establishing a role for GPR40 in insulin regulation.

Full text of DOI:10.1016/j.bmcl.2006.12.084

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1584–1589
Solid phase synthesis and SAR of small molecule agonists
for the GPR40 receptor
Stephen C. McKeown,a,* David F. Corbett,a Aaron S. Goetz,b Thomas R. Littleton,b
Eric Bigham,b Celia P. Briscoe,c,ꢀ Andrew J. Peat,c
Steve P Watsona and Deirdre M. B. Hickeyd
aMolecular Discovery Research, GlaxoSmithKline, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, UK
bMolecular Discovery Research, GlaxoSmithKline Research & Development, 5 Moore Drive,
Research Triangle Park, NC 27709, USA
cMetabolic Centre of Excellence for Drug Discovery, GlaxoSmithKline Research & Development, 5 Moore Drive,
Research Triangle Park, NC 27709, USA
dInfectious Diseases Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Third Avenue,
Harlow, Essex CM19 5AW, UK
Received 26 October 2006; revised 21 December 2006; accepted 26 December 2006
Available online 4 January 2007
Abstract—The discovery, synthesis and structure–activity relationship (SAR) of novel carboxylic acid agonists for GPR40 are
described. Aryl propionic acid 1, identified from a high throughput screen, was selected for chemical exploration. Compound 2
was identified as our lead molecule through efficient solid phase combinatorial array chemistry and had an attractive in vitro
and in vivo pharmacokinetic profile in rat. These ligands may prove useful in establishing a role for GPR40 in insulin regulation.
Ó 2007 Elsevier Ltd. All rights reserved.
GPR40, GPR41 and GPR43 belong to a family of
G-protein coupled receptors (GPCRs) that have been
shown to be activated by free fatty acids.1–6 GPR40 is
activated by long chain fatty acids (e.g., linoleic and pal-
mitic acids), whereas GPR41 and GPR43 are both acti-
vated by short chain fatty acids (e.g., acetic and
propionic acid). GPR40 receptor mRNA has been
reported to be expressed in human and rodent pancreat-
ic islets2,3,7 and it is well established that free fatty acids
play a role in maintaining basal insulin secretion and to
‘prime’ the islet b-cells to respond to glucose following a
prolonged fast.8–12 A key role for GPR40 in pancreatic
b-cells was suggested based on the siRNA downregula-
tion of GPR40 expression in the mouse insulinoma cell
line MIN6 which resulted in a decrease of the linoleic
acid potentiation of insulin secretion.3,13,14 Further-
more, our group has demonstrated the enhancement of
insulin secretion in MIN6 cells by the selective GPR40
agonist (2) described in this paper.15 Recent evidence
also suggests that voltage-gated K+ channel regulation
of insulin release in rat pancreatic b-cells is mediated
through the GPR40 receptor.16,17 These data suggest
that a small molecule GPR40 ligand could help regulate
insulin secretion and as such present GPR40 as a poten-
tial target for Type II Diabetes. The discovery of selec-
tive tool molecules, both antagonists and agonists, will
help us to further understand the role of this receptor.
We have recently reported the discovery of the aryl pro-
pionic acid 1 and the chemical optimisation of a subse-
quent analogue 2 as potent GPR40 agonists.18 We
describe here the solid phase combinatorial synthesis
and initial SAR of the hit compound 1 that led to the
discovery of compound 2, Figure 1.
A high throughput screen of the GlaxoSmithKline com-
pound collection yielded the aryl propionic acid 1 as a
hit with a potency of pEC50 6.3 in the GPR40 FLIPR
assay.15 Aryl propionic acid 1 produces a similar maxi-
mal response to linoleic acid (pEC50 = 5.6) indicating
that it is a full agonist for the GPR40 receptor (com-
pared to linoleic acid).
Keywords: GPR40 agonist; Diabetes; Solid phase chemistry.
*
Corresponding author. Tel.: +44 1279 627558; fax: +44 1279 87
5414; e-mail: Steve.C.McKeown@gsk.com
ꢀ
Present address: Diabetes Biology Department, Pfizer, La Jolla, San
Diego, CA 92121, USA.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.084

S. C. McKeown et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1584–1589
1585
O
O
OH
OH
O
N
N
H
H
2
1
Cl
Figure 1. Optimisation of GPR40 agonist hit 1.
OH
a)
Br
O
O
O
O
c)
X
CO2H
X
X
b)
O2N
O2N
H2N
10
3-8
9
d)
e)
O
OH
O
f)
X
X
O
R1
N
R1
H
N
H
11
12
Scheme 1. Reagents and conditions: (a) Ph3PBr2, CH2Cl2, 3 h, rt; (b) CsI, DIPEA, DMF, rt, 20 h, 70–88%; (c) SnCl2 Æ 2H2O, NMP, 67 h, rt; (d)
R1CHO, 1% CH3CO2H, DMF, 2 h, rt; (e) NaCNBH3, rt, 20 h and (f) TFA (33%), H2O (2.5%), CH2Cl2, rt, 2 h, 20–75%.
Aryl propionic acid 1 exhibited good aqueous solubility
(0.83 mg/mL), a low molecular weight (289 Da) and a
clogP of 3.6. In addition, 1 was an attractive molecule
for chemical optimisation since it can be assembled with
relatively simple chemistry allowing for rapid explora-
tion of the SAR. A chemical programme was therefore
initiated with the aim of enhancing the potency and
exploring the SAR of 1. To achieve this, we chose to
simultaneously vary two regions of the template exploit-
ing the carboxylic acid as a solid phase attachment
moiety to facilitate combinatorial array chemistry,
Scheme 1 (Table 1).
available). These reagents were selected to explore length
and regiochemistry of the carboxylic acid head group
(Fig. 2) together with varying substituents in the lipophil-
ic aromatic tail whilst producing compounds with a
molecular weight below 400 and a clogP less than 7.
The nitro acids (3–8) were first coupled to the functional-
ised Wang-Br resin by using CsI and DIPEA (Scheme 1).
Reduction of resin bound nitro product 9 was then effect-
ed with SnCl2. The reductive alkylation of 10 was carried
out by first forming the imine with the aldehyde, followed
by the addition of sodium cyanoborohydride. These
reactions were performed in a combinatorial manner
using the IRORI directed sorting process.19 The solid
bound anilines (10) were placed in MicroKansTM (mic-
roreactors from IRORI) (0.04 mmol/reactor) with a
radiofrequency tag, and then reacted with different alde-
hydes in parallel such that each aniline was reacted with
every aldehyde in different microreactors. At each stage
of the synthesis resin washings were carried out using a
variety of solvents such as CH2Cl2, DMF, THF and
H2O. Final compound cleavage from the resin (11) was
achieved using 33% TFA, 2.5% H2O in CH2Cl2. Any
N-Boc or tert-butyl protecting groups present in the
The building blocks for the chemical array consisted of 6
nitro acids and 32 aromatic aldehydes (commercially
Table 1. Profile of HTS hit compound 1
Parameter
Value
GPR40 pEC50–FLIPR assay15
GPR40 pEC50–reporter assay15
Solubility at pH 7.4 (mg/mL)
clogP
6.3 (100%)
6.0 (111%)
0.83
3.6
0.98
logD at pH 7.4
Mw
289
O
OH
OH
OH
OH
O
O2N
O2N
O2N
O2N
3
4
5
O
O
OH
OH
O
O
O2N
O2N
6
7
8
Figure 2. Nitro acid building blocks.

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S. C. McKeown et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1584–1589
molecules (11) were also removed during the cleavage
step. Those compounds that showed <85% purity by
UV (HPLC) were purified by preparative HPLC, other-
wise the cleaved dry products (12) were assayed directly.
Table 3 shows the variation of R1 when partnered with
the optimal phenylpropionic acid head group. In general
it can be seen that a lipophilic group at R1 is preferred
since any polar substituent results in the loss of potency.
The favoured lipophilic groups are 3-phenoxy and
4-phenyl. These groups were consistently more potent
than other lipophilic groups even when partnered with
the alternative non-optimal acidic head groups.
In total 167 compounds were submitted for GPR40 test-
ing (not all data are shown). A significant increase in
GPR40 potency was obtained (e.g., compound 2) and
clear SAR was defined (Table 2).
Compound 2 was found to be inactive against family
members GPR41 and GPR43, Table 4. This result is
not surprising given that GPR40 is activated by long
chain fatty acids such as lineolic acid, and GPR41 and
GPR43 are activated by short chain fatty acids such as
acetic and propionic acid. Furthermore, compound 2
was selective over a range of other fatty acid receptors,
such as S1P1, HM74A, PPARa, d and c, and the pros-
taglandin receptors, EP1, EP3 and EP4. The detailed
pharmacological characterization and broader selectivi-
ty of compound 2 has previously been described by our
group.15
Table 2 shows the variation of the acid head group
when combined with two of the preferred lipophilic tails.
A 2-carbon spacer between the aryl and the carboxylic
acid appeared to be important in maintaining GPR40
potency and efficacy with the original linear propionic
acid (entries 2 and 19) and cyclopropyl chains (entries
14 and 20) being optimal. Homologation or shortening
of the chain (entries 16–18 and 22–24) led to a drop in
potency. Interestingly, the 3-regioisomer 15 and 21
retained potency and appeared to possess higher intrin-
sic activities.
Table 2. GPR40 SAR of the acid head groups
X
O
N
H
N
X
H
X
Compound
GPR40 pEC50 (% Max)c
7.1 0.2 (69 10)b
Compound
GPR40 pEC50 (% Max)c
6.6 0.2 (111 6)a
O
2
19
OH
OH
O
14
15
16
17
18
7.3 0.1 (74 14)b
6.4 0.1 (148 17)a
5.5 0.1 (74 24)a
4.78 0.4 (34 5)a
5.4 0.1 (32 5)a
20
21
22
23
24
7.4 0.1 (80 13)b
6.3 0.1 (160 7)a
5.8 0.1 (62 5)a
<4.5 (21 1)a
OH
O
OH
O
OH
O
OH
4.91 0.6 (69 7)a
O
a pEC50 values are the average of two experiments.
b pEC50 values are the average of four experiments.
c Reporter gene assay expressing GPR40 in a CHO cell line.2

S. C. McKeown et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1584–1589
Table 3. GPR40 SAR of the lipophilic tails R1
1587
Table 3. (continued)
Compound
R1
GPR40 pEC50 (% Max)c
<4.5 (141 23)b
O
OH
O
O
37
R1
N
H
Compound
R1
GPR40 pEC50 (% Max)c
7.1 0.2 (69 10)b
38
39
40
41
<4.5 (9 3)a
HO
PhO
2
O
<4.5 (37 13)a
19
25
6.6 0.2 (111 6)a
HO
Ph
Cl
<4.5 (49 2)a
6.6 0.4 (117 25)b
NC
Cl
5.9 0.1 (159 2)a
MeO
26
27
6.1 0.1 (123 21)a
6.1 0.1 (120 27)a
O
Br
Cl
42
43
<4.5 (18 2)a
H2N
N
<4.5 (4 1)a
S
28
5.7 0.03 (143 34)a
NH
H
N
6.0 0.3 (111 18)b
5.7 0.1 (133 5)a
5.4 0.05 (122 1)a
5.4 (152)d
S
44
<4.5 (9 2)a
1
O
O
Cl
29
30
31
45
6.71 (139)d
F3C
PhO
a pEC50 values are the average of two experiments.
b pEC50 values are the average of at least four experiments.
c Reporter gene assay expressing GPR40 in a CHO cell line.2
d Single experiment.
Cl
Table 4. Selectivity of compound 2
Receptor
pEC50
Receptor
fpKi
F
32
5.3 0.1 (120 1)a
GPR40
GPR41
GPR43
S1P1
7.3
<4.3
<4.3
<4.5
<5.2
PPARa
PPARd
PPARc
EP1
<5.5
<5.5
<5.5
<5.8
6.4
Cl
HM74A
EP3
EP4
N
<5.5
33
34
5.3 0.2 (112 0.4)a
CF3
Table 5 shows the physicochemical properties and
DMPK (in vitro and in vivo) data for compound 2.
Compound 2 had moderate aqueous solubility of
0.29 mg/mL (at pH 7.4), a molecular weight of 347
and a measured logD of 1.95 (at pH 7.4). The
compound showed no inhibition of the CYP450 iso-
forms tested (1A2, 2C9, 2C19, 2D6 and 3A4 all
>33 lM) and had acceptable microsomal stability with
clearances of 2.3 (rat) and 0.74 (human) mL/min/g liv-
er. As is common with lipophilic acids, compound 2
5.1 0.4 (144 11)b
N
35
36
<4.5 (4 1)a
<4.5 (12 2)a
N

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S. C. McKeown et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1584–1589
Table 5. In vitro DMPK and Physicochemical profile of 2
O
OH
O
N
H
2
Parameter
Value
0.29
In vivo rat profileb
Parameter
Value
Solubility at pH 7.4 (mg/mL)
CYP450a, IC50 (lM)
Microsomal clint (mL/min/g liver)
Permeability (nm/sec)c
logD at pH 7.4
Cl (mL/min/kg)
Vdss (L/kg)
t1/2 (po) (h)
24.9
4.7
5.9
5.3
65
1A2, 2C9, 2C19, 2D6, 3A4 all >33
2.3 rat; 0.74 mouse
599
t1/2 (iv) (h)
Bioavailability (%)
1.95
347
Mw
Protein bindingd(%)
>99.6
a In vitro CYP450 assay results using Gentest protocol.
b Average of three experiments. Doses = 3.9 mg/kg po; 1.0 mg/kg iv.
c Permeability measured using MDCK type I (low Pgp expressing) cell line.20
d Ultrafiltration assay using rat plasma.
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