Discovery of diacylphloroglucinols as a new class of GPR40 (FFAR1) agonists

Article abstract of DOI:10.1016/j.bmcl.2008.10.085

In this letter, we report discovery of diacylphloroglucinol compounds as a new class of GPR40 (FFAR1) agonists. Several diacylphloroglucinols with varying length of acyl functionality and substitution on aromatic hydroxyls were synthesized and evaluated for GPR40 agonism using functional calcium-flux assay. Out of 17 compounds evaluated, 14, 17, 19 and 25 exhibited good GPR40 agonistic activity with EC₅₀ values ranging from 0.07 to 8 μM (pEC₅₀ 7.12-5.09), respectively, with maximal agonistic response of 84-102%.

Full text of DOI:10.1016/j.bmcl.2008.10.085

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6357–6361
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of diacylphloroglucinols as a new class of GPR40 (FFAR1) agonists
Sandip B. Bharate a, Atish Rodge a, Rajendra K. Joshi c, Jaspreet Kaur c, Shaila Srinivasan c, S. Senthil Kumar b,
Asha Kulkarni-Almeida b, Sarala Balachandran a, Arun Balakrishnan b, Ram A. Vishwakarma a,
*
a Department of Medicinal Chemistry, Piramal Life Sciences Limited, 1 Nirlon Complex, Off Western Express Highway, Goregaon (E), Mumbai 400063, India
b Department of Screening and Biotechnology, Piramal Life Sciences Limited, 1 Nirlon Complex, Off Western Express Highway, Goregaon (E), Mumbai 400063, India
c Department of Pharmacology, Piramal Life Sciences Limited, 1 Nirlon Complex, Off Western Express Highway, Goregaon (E), Mumbai 400063, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2008
Revised 26 September 2008
Accepted 20 October 2008
Available online 1 November 2008
In this letter, we report discovery of diacylphloroglucinol compounds as a new class of GPR40 (FFAR1)
agonists. Several diacylphloroglucinols with varying length of acyl functionality and substitution on aro-
matic hydroxyls were synthesized and evaluated for GPR40 agonism using functional calcium-flux assay.
Out of 17 compounds evaluated, 14, 17, 19 and 25 exhibited good GPR40 agonistic activity with EC50 val-
ues ranging from 0.07 to 8
102%.
l
M (pEC50 7.12–5.09), respectively, with maximal agonistic response of 84–
Keywords:
Ó 2008 Elsevier Ltd. All rights reserved.
Diacylphloroglucinols
FFAR1 agonist
GPR40 agonist
Type-2 diabetes
The G-protein coupled receptor GPR40 (also known as free fatty
acid receptor 1, FFAR1) is highly expressed in human and rodent
pancreatic islets and is activated by medium and long-chain free
fatty acids (e.g., linoleic and palmitic acids) resulting in stimulation
of downstream signaling pathways which activate protein kinase C
(PKC) and elevate intracellular [Ca2+].1,2 Since both activation of
PKC and increasing intracellular [Ca2+] play a key role in insulin
exocytosis in pancreatic b-cells,3 GPR40 may serve as a signaling
mechanism through which free fatty acids directly affect insulin
secretion. Treatment of MIN6 pancreatic b-cells with small inter-
fering RNA (siRNA) specific for GPR40 prevents fatty acid stimula-
tion of insulin secretion. This study further supported that GPR40
may serve as an attractive target to mediate insulin secretion. Fur-
thermore, agents that serve as GPR40 agonists may be useful for
the treatment of type-2 diabetes.4–7
derivative 7 (EC50 < 0.1
(pEC50 5.1, Heptahelix AB),17 conformationally constrained 3-(4-
hydroxy-phenyl)-substituted propanoic acid (EC50 0.01 M,
Amgen Inc.),18 cyclopropane carboxylic acid compound 10 (119%
activation at 100
M, Takeda Pharmaceuticals)19 and phenylprop-
l
M, Amgen Inc.),16 thiazolidinedione 8
9
l
l
anoic acid derivative 11 (EC50 19 nM, Takeda Pharmaceuticals).20
Besides these reports, Tikhonoca et al. have recently identified
different new scaffolds active at FFAR1 as full agonists, partial ago-
nists or pure antagonists by virtual screening based on two-dimen-
sional (2D) similarity, three-dimensional (3D) pharmacophore
searches and docking studies using structure of known ago-
nists.21,22 Most of these compounds possess terminal carboxylic
acid group with a tether linking to an aromatic nucleus. This letter
describes our research efforts towards identification of diacylphl-
oroglucinols as a new series of GPR40 agonists.
In the literature, different classes of GPR40 agonists are re-
ported (beyond the fatty acids) as shown in Figure 1. These include
aminophenyl propionic acid derivative GW9508 (1, pEC50 7.1,
GlaxoSmithcline),8–10 4-alkoxy phenyl propionic acid derivative 2
Acyl phloroglucinol compounds widely occur in nature in Myrt-
aceae family and are reported to possess wide range of biological
activities viz. antimicrobial, antileishmanial, antimalarial, cancer
chemopreventive, antifouling, anti-HIV and plant growth regula-
tory activity.23–25 Apart from these reports, this class of compounds
are also reported to possess antidiabetic activities viz. macrocarpals
isolated from Eucalyptus grandis inhibit aldose reductase which is
the target enzyme for the control of diabetic complications such
as cataracts, retinopathy, neuropathy and nephropathy26; achy-
rofuran has been isolated from Achyrocline satureioides by bioactiv-
ity-guided fractionation using the db/db mouse model for
type-2 diabetes and it significantly lowered blood glucose levels
in this model when administered orally at 20 mg/kg/day27; and
(EC50 0.3
0.1–100 nM, Merck),12 4,5-diphenyl-pyrimidinylamino substituted
carboxylic acid 4 (104% activation at 100
M, Sanofi-Aventis),13
phenylamino-benzoxazole substituted carboxylic acid 5 (111%
activation at 100
M, Sanofi-Aventis),14 oxadiazolidinedione com-
pound 6 (EC50 0.26
M, Astellas Pharma),15 bicyclic carboxylic acid
l
M, Johnson and Johnson),11 bicyclic compound 3 (EC50
l
l
l
* Corresponding author. Tel.: +91 22 30818302; fax: +91 22 30818334.
E-mail address: ram.vishwakarma@piramal.com (R.A. Vishwakarma).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.085

6358
S. B. Bharate et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6357–6361
O
Cl
O
7
OH
O
H
O
O
N
N
O
OH
N
HO
H
F3C
OH
N
O
O
1
3
2
4
O
O
OH
O
O
O
2
S
O
H3C
O
O
N
7
N-
Na+
Cl
N
O
N
NH
HO
H
O
Cl
O
O
5
6
7
8
O
O
OH
N
O
OH
O
N
S
O
S
O
OH
O
9
10
11
Figure 1. Structures of GPR40 agonists reported in literature.
phlorotannins, such as eckols isolated from Eisenia bicyclis exhib-
ited inhibitory activity on glycation and R-amylase and thus
possess the potential for the effective treatment of diabetic compli-
cations.28 But this class of compounds have never been evaluated
for their GPR40 agonistic potential.23
As a part of our continuing efforts towards discovery of new
class of compounds against different therapeutic areas29 and based
on the literature reports, we designed a dual pharmacophore
which possess a long aliphatic chain of free fatty acids and a phenyl
propanoid part of known GPR40 agonists. Herein, we report syn-
thesis of several diacylphloroglucinol compounds using simple
synthetic strategy and evaluation of these compounds for GPR40
agonistic activity.
Series of diacyl phloroglucinol compounds were synthesized
starting from commercially available phloroglucinol (12) by vary-
ing acyl functionality. Phloroglucinol (12) on treatment with isova-
leric acid in presence of boron trifluoride etherate under reflux for
2 h resulted in formation of phloroisovalerophenone (13) and
2,4-diisovaleryl phloroglucinol (14) in 90% (30:70–mono:di)
yield.30 Compound 14 was further treated with formaldehyde solu-
tion in dichloromethane at 60 °C for 8–10 h resulting in formation
of dimer 15 in 85% yield (Scheme 1).
Phloroglucinol (12) on treatment with chloroacetyl chloride in
presence of AlCl3 in nitrobenzene led to formation of desired 2,4-
di-(chloroacetyl) phloroglucinol (16) in 50% yield. Further, several
diacyl phloroglucinol compounds were synthesized using different
short to long-chain fatty acids. Treatment of phloroglucinol (12)
with isobutyric acid, caprylic acid, lauric acid, stearic acid and
palmitic acid in presence of boron trifluoride etherate under reflux
for 2 h resulted in formation of desired diacyl phloroglucinol
compounds 17–21 in 50–75% yield (Scheme 2). Compound 18
was earlier reported in literature as an antagonist of thromboxane
A2 and Leukotriene D4.31
In order to develop structure–activity relationship, O- and
C-alkylated analogues 22–25 of 2,4-diisovaleryl phloroglucinol
(14) were synthesized (Scheme 3). Treatment of 14 with n-butyl
bromide in presence of potassium carbonate in acetone at room
temperature for 10 h resulted in formation of three products 22–24
in 50% yield (ratio of 22:23:24 = 50:30:20). Compound 25 was syn-
thesized by refluxing 14 with n-butyl bromide in presence of
freshly prepared sodium methoxide for 2 h.32 Further, analogues
with terminal carboxylic acid group on acyl chain (26 and 28) were
synthesized in order to study the effect of negatively charged func-
tionality on GPR40 agonistic activity. Compounds 26 and 28 were
synthesized by treatment of 12 or 13 with glutaric anhydride in
presence of BF3 etherate at room temperature for 2 h in 40–45%
yield. However, under similar reaction conditions, when phloroglu-
cinol (12) was treated with succinic anhydride in presence of BF3
etherate, desired product with terminal carboxylic acid group
was not formed; instead a condensed product 27 was obtained.
Further, a hydroxyethyl substituted analogue 29 was synthesized
by reaction of 14 with bromoethanol in presence of potassium
carbonate in acetone at room temperature for 10 h in 50% yield.
Synthesis of analogues 26–29 is depicted in Scheme 4.
All compounds were evaluated for GPR40 agonistic activity
using a functional calcium-flux assay.1,33 Linoleic acid was used
as a standard GPR40 agonist. The agonist activity was measured
as the fold increase of calcium flux over control in CHO cells
expressing the hGPR40 vector using the Flex Station. The activity
of all compounds was compared with that of linoleic acid. Linoleic
O
O
HO
O
OH
O
HO
OH
HO
O
OH
HO
O
OHHO
OH
O
b
a
+
OH
OH
OH
13
OH
OH
12
14
15
Scheme 1. Reagents and conditions: (a) (CH3)2CHCH2COOH, BF3–Et2O, 100 °C, 2 h, 90% (mono:di = 30:70); (b) HCHO, CH2Cl2, 60 °C, 8–10 h, 85%.

S. B. Bharate et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6357–6361
6359
HO
O
OH
HO
OH
HO
OH
b
a
R
R
Cl
Cl
OH
O
O
OH O
OH
16
12
17: R = -CH(CH3)2
18: R = -(CH2)6CH3
19: R = -(CH2)10CH3
20: R = -(CH2)14CH3
21: R = -(CH2)16CH3
Scheme 2. Reagents and conditions: (a) RCOOH, BF3–Et2O, 100 °C, 2 h, 50–75%; (b) ClCOCH2Cl, AlCl3, PhNO2, 60 °C, 2 h, 50%.
R2O
O
OR1
O
HO
O
OH
O
a
b
14
OR3
22: R1 = R2 = CH2CH2CH2CH3, R3 = H
23: R1 = R3 = CH2CH2CH2CH3, R2 = H
24: R1 = CH2CH2CH2CH3, R2 = R3 = H
OH
25
Scheme 3. Reagents and conditions: (a) K2CO3, acetone, CH3CH2CH2CH2Br (4 equiv), rt, 10 h, 50%; (b) Na/MeOH, CH3CH2CH2CH2Br, 90 °C, 2 h, 60%.
HO
OH
O
HO
OH
O
HO
OH
O
OH
OH
c
d
OH
26
O
OH
OH
12
HO
OH
27
a
+
HO
OH
O
HO
O
O
OH
b
c
OH
14
13
O
OH
O
O
OH
29
28
Scheme 4. Reagents and conditions: (a) (CH3)2CHCH2COOH, BF3–Et2O, 100 °C, 2 h, 90% (mono:di = 30:70); (b) K2CO3/acetone, BrCH2CH2OH, rt, 10 h, 50%; (c) glutaric
anhydride, BF3–Et2O, rt, 2 h, 40–45%; (d) succinic anhydride, BF3–Et2O, rt, 2 h, 60%.
acid exhibited GPR40 agonism with an EC50 of 0.54
Itoh et al. have reported comparable calcium flux response in the
FLIPR for linoleic acid (EC50 = 1.8 M) with similarly hGPR40 trans-
fected CHO cells.2 Out of the 17 compounds tested, compounds 14,
l
M. Previously,
Specificity of compounds for GPR40 was confirmed by running par-
allel Ca2+ flux assay in CHO cells with the vector lacking the
hGPR40 transfection. From results (Table 1), it was noticed that
substitution of phenolic hydroxyls with alkyls (22–24) resulted
in loss of activity while alkylation on aromatic ring (25) did not
alter GPR40 agonistic potential. Dimerization (compound 15) of
active compound 14 resulted in loss of activity. Analogues with
terminal carboxylic acid group on acyl chain (26 and 28) did not
showed GPR40 agonistic activity. As well as hydroxyethyl substi-
tuted analogue 29 was inactive.
l
17, 19 and 25 showed good agonistic activity at 10
l
M (Table 1).
Table 1
GPR40 agonistic activity of diacylphloroglucinols.
Entry
GPR40 agonist activity (at 10
Calcium flux
l
M)
Fold increase
Comments
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
1.116
1.893
1.115
1.211
2.313
1.211
2.326
1.024
1.185
1.347
1.459
1.024
2.121
1.000
1.006
1.003
1.018
2.547
Not observed
Observed
Not observed
Not observed
Observed
Not active
Active
Not active
Not active
Active
Table 2
EC50 and pEC50 values of active compounds.*
Entry
EC50 (pEC50b) (
l
M)
% Maximal responsec
a
Not observed
Observed
Not active
Active
14
17
19
25
6.0 0.1 (5.22 0.01)
8.0 0.2 (5.09 0.01)
0.07 0.004 (7.12 0.03)
0.97 0.06 (6.01 0.03)
0.54 0.02 (6.26 0.02)
102.2 5.3%
88.7 6.2%
100.2 1.9%
84.5 2.3%
—
Not observed
Not observed
Not observed
Not observed
Not observed
Observed
Not observed
Not observed
Not observed
Not observed
Observed
Not active
Not active
Not active
Not active
Not active
Active
Not active
Not active
Not active
Not active
Active
Linoleic acid
a
Concentration of the samples that produce 50% of the maximal response, and
was calculated from dose–response curves.
b
Negative log of the molar concentration required to elicit a half-maximal
response.
c
The maximal agonistic response elicited by the compound as a percentage of
29
the maximal response evoked by Linoleic acid at 10
lM.
Linoleic acid
*
Data represents mean value SEM.

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Basal
LA
19
14
17
Compounds at 10µM
Figure 2. Effect of compounds 14, 17, 19 and linoleic acid on insulin secretion from
the hamster pancreatic insulinoma cell line (HIT-T15).
12. Min, G. E.; He, J.; Lau, F. W. Y.; Liang, G. B.; Lin, S.; Liu, W.; Walsh, S. P.; Yang, L.
World Patent WO2007136572, 2007 (Merck & Co., Inc.).
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Herling, A.; Bartoschek, S. World Patent WO 2007131620, 2007 (Sanofi-
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14. Defossa, E.; Follmann, M.; Klabunde, T.; Drosou, V.; Hessler, G.; Stengelin, S.;
Haschke, G.; Herling, A.; Bartoschek, S. World Patent WO2007131622, 2007
(Sanofi-Aventis).
Active compounds 14, 17, 19 and 25 were further tested and
EC50 and pEC50 values were determined as shown in Table 2. Com-
pound 25 showed EC50 of 0.97 lM (pEC50 6.01) with 84.5% maxi-
mal response, which suggests that introduction of alkyl chain on
aromatic nucleus of 14, resulted in improved GPR40 agonistic
15. Negoro, K.; Iwasaki, F.; Ohnuki, K.; Kurosaki, T.; Yonetoku, Y.; Asai, N.; Yoshida,
S.; Soga, T. World Patent WO2007123225, 2007 (Astellas Pharma).
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World Patent WO2007106469, 2007 (Amgen).
17. Goddard, C.; Owman, C.; Olde, B.; Rome, D.; Sterner, O. World Patent
WO2007049050, 2007 (Heptahelix-AB).
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(Amgen).
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(Takeda Pharmaceuticals).
20. Yasuma, T.; Sasaki, S.; Sakai, N. World Patent WO2005063725, 2005 (Takeda
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Gershengorn, M. C. J. Med. Chem. 2008, 51, 625.
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Singh, I. P. Bioorg. Med. Chem. 2006, 14, 1750.
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activity than that of 14 (EC50
sponse). Compound 19 showed potent activity with EC50 value of
0.07 0.004 M (pEC50 7.12, 100.2% maximal response). The effi-
cacy of the compound is shown as a percentage of the maximal
agonistic response elicited by the test compound with respect to
the maximal response evoked by the internal standard (linoleic
6 lM, pEC50 5.22, 102.2% maximal re-
l
acid) at a dose of 10
pound 19 was also tested for hPPAR-
showed very weak agonistic activity for hPPAR-
l
M. In order to determine the selectivity, com-
agonistic activity and it
. DMPK study on
c
c
one of representative of the scaffold (compound 14) was per-
formed and pharmacokinetic parameters (Tmax 0.25 h, Cmax
0.88 lg/mL, AUClast 3–13 h lg/mL, AUC0-infinity 3.41 h lg/mL
and half-life 6.59 h) showed that this scaffold has good PK
properties.
Activation of the GRP40 receptor is known to play a role in pan-
creatic and neurological function and the receptor is specifically
localized in the brain and pancreas.1 In the pancreas the receptor
expression is restricted to insulin producing b-cells. Since the key
objective of GPR40 agonists is to prime islet b-cells to respond to
glucose by inducing insulin secretion, we further evaluated these
active compounds for their ability to induce insulin secretion in
pancreatic islet cells. Further, the GPR40 mRNA is known to be ex-
pressed significantly in most pancreatic b-cell lines with highest
expression levels in MIN6, followed by b-TC and HIT-T15.2 We
therefore selected the HIT-T15 cell line for insulin secretion stud-
30.
A typical procedure for synthesis of diacylphloroglucinols: A mixture of
phloroglucinol (12, 1 mmol), respective carboxylic acid (2.5 mmol) and boron
trifluoride-diethyl ether (10 mL) was refluxed at 100 °C for 2 h. Reaction
mixture was poured in crushed ice and stirred for 5 min and then extracted
with ethyl acetate. Ethyl acetate layer was washed with brine solution and
finally dried over anhydrous sodium sulphate. Solvent was removed under
reduced pressure and crude product was purified by silica gel column
chromatography (#100–200) using hexane/EtOAc (3:1) as eluent. 1,3-Di-(3-
methyl-butanoyl)-2,4,6-trihydroxy benzene (14): cream colored solid; yield:
67%; 1H NMR (CDCl3, 300 MHz): d 5.84 (s, 1H), 2.98 (d, J = 6.4 Hz, 4H), 2.25 (m,
2H), 0.99 (d, J = 6.6 Hz, 12H); CIMS: m/z 295 [M+1]+. 1,3-Diisobutyryl-2,4,6-
trihydroxy benzene (17): cream colored sticky solid; yield: 55%; 1H NMR
(CDCl3, 300 MHz): d 6.13 (s, 1H), 3.93 (m, 2H), 1.17 (d, J = 6.7 Hz, 12H); CIMS:
m/z 267 [M+1]+. 1,3-Didodecanoyl-2,4,6-trihydroxybenzene (19): cream
colored solid; yield: 60%; 1H NMR (CDCl3, 300 MHz): d 16.27 (s, 1H, OH),
5.79 (s, 1H), 3.10 (t, J = 7.5 Hz, 4H), 1.67 (m, 4H), 1.32 (m, 32H), 0.98 (t,
J = 7.5 Hz, 6H); MS (ESI): m/z 489.4 [MÀ1]+.
ies. HIT-T15 from hamsters were treated with 10 lM of compound
14, 17 and 1934 Each of these induced insulin secretion comparable
to the effect of linoleic acid (Fig. 2). Further, the inactive com-
pounds did not show any antagonist effect for linoleic acid induced
calcium flux (data not included).9
In conclusion, compounds that activate GPR40 at low sub-
micromolar concentrations have been identified, the majority of
which behave as full agonists as compared to the endogenous
long-chain fatty acid ligands. This series of compounds have a po-
tential to emerge as a lead candidate with potent GPR40 agonistic
activity for the treatment of type-2 diabetes.
31. Tada, M.; Chiba, H.; Takakuwa, T.; Kojima, E. J. Med. Chem. 1992, 35, 1209.
32. Procedure for synthesis of 1,3-diisopentanoyl-5-butyl-2,4,6-trihydroxybenzene
(25): To a solution of 14 (1 mmol) in methanol, a freshly prepared solution
of sodium methoxide was added followed by addition of n-butyl bromide
(1.2 mmol). Reaction mixture was then refluxed for 2 h. Solvent was
evaporated on vacuo rotavapor and crude product was purified by silica gel
column chromatography (#100–200) using hexane/EtOAc (4:1) as eluent.
Yield: 60%; yellow oil; 1H NMR (CDCl3, 300 MHz): d 16.18 (s, 1H), 14.96 (s, 1H),
References and notes
1. Briscoe, C. P.; Tadayyon, M.; Andrews, J. L.; Benson, W. G.; Chambers, J. K.;
Eilert, M. M.; Ellis, C.; Elshourbagy, N. A.; Goetz, A. S.; Minnick, D. T.; Murdock,
P. R.; Sauls, H. R., Jr.; Shabon, U.; Spinage, L. D.; Strum, J. C.; Szekeres, P. G.; Tan,
K. B.; Way, J. M.; Ignar, D. M.; Wilson, S.; Muir, A. J. Biol. Chem. 2003, 278, 11303.

S. B. Bharate et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6357–6361
6361
2.99 (d, J = 6.9 Hz, 4H), 2.57 (m, 2H), 2.28 (m, 2H), 1.52–1.46 (m, 4H), 0.99 (m,
15H); MS (APCI): m/z 351.1 [M+1]+.
for 10 min before reading in the FlexStation II. The change in fluorescence over
baseline was used to determine the agonistic response. The agonistic responses
for the screened actives were checked with the mock control (CHO K1 cells
transfected without hGPR40 in the vector pCDN) for its specificity (data not
shown). Statistical analysis was performed with Graphpad prism software,
version 3.03.
33. GPR40 assay1: CHO K1 cells stably transfected with hGPR40 in the vector pCDN
were seeded in the density of 10,000 cells/well into poly
D-lysine coated 96-
well microplate, black/clr bottom. After 48 h, the cells were loaded for 1 h with
2.5
lM Fluo-4AM fluorescent indicator dye (Molecular probes, Inc.,) in assay
buffer (10 mM Hepes, 200
7.5). Cells were washed three times with assay buffer and incubated at 37 °C
l
M Ca2+ and 2.5 mM probenecid in 1Â HBSS, pH
34. Saito, Y.; Kato, M.; Kubohara, Y.; Kobayashi, I.; Tatemoto, K. Biochem. Biophys.
Res. Commun. 1996, 221, 577.