Identification of diarylsulfonamides as agonists of the free fatty acid receptor 4 (FFA4/GPR120)

Article abstract of DOI:10.1016/j.bmcl.2014.05.012

The exploration of a diarylsulfonamide series of free fatty acid receptor 4 (FFA4/GPR120) agonists is described. This work led to the identification of selective FFA4 agonist 8 (GSK137647A) and selective FFA4 antagonist 39. The in vitro profile of compounds 8 and 39 is presented herein.

Full text of DOI:10.1016/j.bmcl.2014.05.012

Accepted Manuscript
Identification of Diaryl Sulfonamides as Agonists of the Free Fatty Acid Re-
ceptor 4 (FFA4/GPR120)
Steven M. Sparks, Grace Chen, Jon L. Collins, Dana Danger, Steven T. Dock,
Channa Jayawickreme, Stephen Jenkinson, Christopher Laudeman, M. Anthony
Leesnitzer, Xi Liang, Patrick Maloney, David C. McCoy, David Moncol,
Vincent Rash, Thomas Rimele, Padmaja Vulimiri, James Way, Sean Ross
PII:
S0960-894X(14)00499-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.012
BMCL 21625
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 February 2014
2 May 2014
5 May 2014
Please cite this article as: Sparks, S.M., Chen, G., Collins, J.L., Danger, D., Dock, S.T., Jayawickreme, C., Jenkinson,
S., Laudeman, C., Anthony Leesnitzer, M., Liang, X., Maloney, P., McCoy, D.C., Moncol, D., Rash, V., Rimele,
T., Vulimiri, P., Way, J., Ross, S., Identification of Diaryl Sulfonamides as Agonists of the Free Fatty Acid Receptor
4 (FFA4/GPR120), Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.
2014.05.012
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Identification of Diaryl Sulfonamides as Agonists of the Free
Fatty Acid Receptor 4 (FFA4/GPR120)
Steven M. Sparksa*, Grace Chenb, Jon L. Collinsa, Dana Dangera, Steven T. Docka,
Channa Jayawickremeb, Stephen Jenkinsonc, Christopher Laudemanc, M. Anthony
Leesnitzerb, Xi Lianga, Patrick Maloneyc, David C. McCoyc, David Moncolb, Vincent
Rashb, Thomas Rimeleb, Padmaja Vulimirib, James Waya, and Sean Rossa.
aEnteroendocrine Discovery Performance Unit, bPlatform Technology & Science, cMetabolic Center for Excellence in Drug
Discovery, GlaxoSmithKline, P.O. Box 13398, Research Triangle Park, North Carolina 27705
This is where the receipt/accepted dates will go; Received Month XX, 2000; Accepted Month XX, 2000 [BMCL RECEIPT]
Abstract— The exploration of a diarylsulfonamide series of free fatty acid receptor 4 (FFA4/GPR120) agonists is described. This work
led to the identification of selective FFA4 agonist 8 (GSK137647A) and selective FFA4 antagonist 39. The in vitro profile of compounds
8 and 39 is presented herein. ©2000 Elsevier Science Ltd. All rights reserved.
Type 2 diabetes is a major worldwide public health
problem with devastating chronic complications such as
cardiovascular disease, retinopathy, neuropathy, and
nephropathy. Type 2 diabetes affects more than 300
million people globally and is predicted to become the
seventh leading cause of death by 2030, with total
deaths from diabetes projected to increase by 50% in the
next 10 years.1 The disease is polygenic in nature and
characterized by hyperglycemia, defects in pancreatic
insulin secretion, and insulin resistance in skeletal
muscle, adipose tissue, and liver. In addition to these
derangements, the rate of endogenous glucose
production is significantly elevated in type 2 diabetics
relative to healthy subjects.2,3
vildagliptin) have successfully validated GLP-1 as an
important target for the development of new antidiabetic
therapies.6
GLP-1 is released in response to ingestion of
nutrients including carbohydrates, protein, and fat.7
Recent studies aimed at identifying the receptors
involved in nutrient stimulated GLP-1 secretion have
identified free fatty acid receptor 4 (FFA4/GPR120) as
a potential 7TM receptor involved in long-chain fatty
acid stimulated GLP-1 secretion.8 FFA4 is highly
expressed in the intestine and the intestinal endocrine
cell line STC-1, additionally FFA4 mRNA has been
detected in murine L-cells.9 Elucidating the mechanism
of FFA4 mediated GLP-1 secretion would be greatly
aided by the identification of selective small molecule
agonists and antagonists.10 With this goal in mind,
efforts directed toward the identification of small
molecule FFA4 modulators culminating in a series of
diaryl sulfonamide agonists are described herein.
A variety of pharmacological strategies have
been successfully employed to improve glycemic
control in Type 2 diabetics which directly target the
secretion and sensitization of insulin. More recently,
approaches targeting the endogenous antidiabetic
hormone glucagon-like peptide-1 (GLP-1) have met
with success.4 GLP-1, secreted from L-cells located in
the ileum and colon, stimulates glucose-dependent
insulin secretion and has been shown to inhibit
glucagon release, gastric emptying, and feeding.5 GLP-1
(T1/2 = 2 min) is rapidly degraded by the enzyme
dipeptidylpeptidase-4 (DPP-4) and agents developed as
long-acting agonists of GLP-1 (exenatide and
liraglutide) and inhibitors of DPP-4 (sitagliptin and
Compound 1 was identified as a starting point
for optimization following a FLIPR based medium
throughput screening campaign conducted in a HEK293
cell line expressing human FFA4. The potency and
efficacy of compound 1 was determined using a ten-
point response curve in mammalian U2OS cells
expressing human FFA4 with a FLIPR readout run in
both agonist and antagonist modes.11 The ease of
synthesis of diaryl sulfonamides combined with the

plethora of commercially available starting materials
made compound 1 an attractive starting point for SAR
development.
With our initial survey of the aniline SAR
completed, we turned our attention toward exploration
of the substitution on the arylsufonyl group (Table 2).
Varying the position of a methoxy substituent revealed
that ortho-substitution (12) was not tolerated while
para-substitution (8) provided the most potent agonists.
With this information in hand, we concentrated on para-
substitution and identified that small nonpolar
substituents were preferred, with the 4-methoxy
substituent (8) providing the best balance of potency
and efficacy. Interestingly, substitution with an electon
withdrawing trifluoromethoxy group (14) provided
nearly equal potency yet reduced efficacy (53% max
response). Disubstitution at the 3- and 4-postions was
well tolerated, with 5-membered fused rings (27 vs 28)
providing optimal activity.
O
H
N
S
O
O
O
1
Human FFA4 pEC50 = 5.4 0.2
% Max Resp = 109 12%
Figure 1. In vitro profile of sulfonamide 1.
The general synthesis of the diarylsulfonamide
agonists is shown in Scheme 1. Sulfonamide bond
formation was typically formed by reaction of the
aniline and arylsulfonyl chloride in pyridine at room
temperature. In cases where electron-deficient anilines
were employed, heating to elevated temperatures was
required.
Table 2. In vitro FFA4 Agonism: phenyl sulfonyl substitution.
H
R
N
S
H
N
R
O
O
a
NH
R
2
Cl
S
R
S
+
R
O
O
O
O
Compound
R
H
FFA4
% Max
Resp (SD)
86 (32)
a
pEC50
4.9 (0.1)
<4.5
I
11
Scheme 1. General syntheses of the diarylsulfonamides. Reagents and
conditions: (a) pyridine, RT or 50 °C
12
2-MeO
3-MeO
4-MeO
4-CF3O
4-Me
13
4.9 (0.1)
6.3 (0.2)
5.9 (0.1)
6.0 (0.1)
<4.5
58 (12)
100b
8 (GSK137647A)
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
53 (8)
83 (19)
--
Our initial studies explored substitution about
the aniline ring of the diarylsulfonamides (Table 1).
Whereas monoakyl-substitution on the aniline ring
resulted in compounds devoid of activity (2-4), dialkyl-
substitution (5-6) provided activity equivalent to hit
compound 1. Larger alkyl groups at positions 2 and 6 of
the aniline ring were not tolerated (7), however addition
of an alkyl group at the 4-position, to provide 2,4,6-
trimethyl analog 812 provided a substantial increase in
potency. N-substitution (9-10) was not tolerated.
4-CO2H
4-Ac
5.1 (0)
90 (5)
113 (33)
104 (17)
17 (3)
--
4-F
5.2 (0.2)
5.5 (0.2)
6.4 (0.1)
<4.5
4-Cl
4-Pr
4-tBu
4-Ph
<4.5
--
4-OiPr
4-OBu
5.7 (0.1)
<4.5
67 (6)
--
3-MeO-4-Cl
3,4-DiMeO
6.1 (0.1)
5.00 (0.1)
6.5 (0.1)
5.7 (0.2)
73 (10)
42 (3)
78 (7)
103 (28)
3,4-Ethylenedioxy
3,4-Propylenedioxy
aValues are means of at least three experiments, standard deviation is
given in parentheses. bGSK137647A (8) was used as a standard in the
U2OS assay with the % max response set to 100.
Table 1. In vitro FFA4 Agonism: aniline substitution
O
R'
N
S
R
O
O
Compound
R
R’
FFA4
% Max Resp
a
pEC50
<4.5
(SD)
--
Finally we explored the SAR at the 4-position
of the aniline (Table 3). Incremental increases in the
length of the alkyl group (29) maintained FFA4 activity,
while further increases in the steric bulk of the alkyl
group eroded activity (30). Consistent with the previous
SAR small lipophilic groups were tolerated (31-33),
however, attempts to introduce more polar functionality
at the 4-position (34-38) were not successful.
2
3
4
5
6
7
2-Me
3-Me
H
H
H
H
H
H
H
<4.5
--
4-Me
<4.5
--
2,4-DiMe
2,6-DiMe
2,6-DiEt
5.6 (0.1)
5.5 (0.1)
<4.5
81 (22)
63 (9)
--
8 (GSK137647A) 2,4,6-TriMe
6.3 (0.2)
<4.5
100b
9
10
2,4,6-TriMe Me
2,4,6-TriMe Et
--
<4.5
--
Linoleic Acid
--
--
6.5
138
aValues are means of at least three experiments, standard deviation is
given in parentheses. bGSK137647A (8) was used as a standard in the
U2OS assay with the % max response set to 100, linoleic acid data is
shown for comparison.
Table 3. In vitro FFA4 Agonism: aniline substitution
O
H
N
S
O
O
R

Compound
R
FFA4
pEC50a (SD)
6.3 (0.2)
6.5 (0.1)
<4.5
% Max
Resp (SD)
expected given the closely related structures of agonist
8 and antagonist 39, further studies would be needed to
confirm the precise nature of antagonism due to the
potential for insurmountable antagonism in these
studies.14
8 (GSK137647A)
Me
100b
29
30
31
32
33
34
35
36
37
38
Allyl
85 (10)
Cyclopropylmethyl
--
F
6.0 (0.1)
6.3 (0.0)
6.4 (0.2)
<4.5
75 (12)
Cl
131 (8)
OMe
OH
54 (11)
--
--
--
--
--
H
N
S
CN
<4.5
O
O
CO2H
CO2Me
CH2OH
<4.5
O
<4.5
<4.5
aValues are means of at least three experiments, standard deviation is
given in parentheses. bGSK137647A (8) was used as a standard in the
U2OS assay with the % max response set to 100.
39
FFA4: Human pIC50 = 7.1 0.35
Human pEC50 < 4.5
Mouse pIC50 = 8.1
Rat pIC50 = 8.1
FFA1: Human pIC50 < 4.6
Human pEC50 < 4.3
Having completed our SAR studies around the
diarylsulfonamide template, we chose agonist 8,
GSK137647A, for further in vitro selectivity profiling
(Figure 2). Compound 8 was evaluated against a panel
of 65 targets in both full curve functional and binding
assays and was shown to provide at least 100-fold
selectivity against the panel which included 41 G-
Figure 3. In vitro profile of FFA4 antagonist 39.
protein-coupled
receptors
(GPCRs),
including
additional members of the free fatty acid receptors
(FFA1, FFA2, & FFA3). Profiling against the rodent
ortholog receptors showed compound 8 maintained
activity against the mouse and rat FFA4 receptors while
maintaining selectivity over the rodent FFA1, FFA2,
and FFA3 receptors. While the selectivity of compound
8 is promising, the poor solubility of the compound
measured in simulated intestinal fluid (FASSIF) greatly
limited the utility of the compound for in vivo
experiments.
O
H
N
S
O
O
GSK137647A (8)
FFA4: Human pEC50 = 6.3 (0.17), 100% Max Response
Mouse pEC50 = 6.2 (0.17), 100% Max Response
Rat pEC50 = 6.1 (0.16), 100% Max Response
Human FFA1, FFA2, & FFA3 pEC50s < 4.5
Mouse FFA1, FFA2, & FFA3 pEC50s < 4.5
Rat FFA1, FFA2, & FFA3 pEC50s < 4.5
FASSIF solubility = 2.9 µg/mL
Figure 2. In vitro profile of FFA4 agonist GSK137647A (8).
During the course of our optimization of the
diarylsulfonamide chemotype as FFA4 agonists, we
identified xanthene 39 as a potent and selective
antagonist of FFA4 (Figure 3).13 Xanthene 39 was able
to block the agonist induced intracellular Ca2+ response
elicited by both linoleic acid and compound 8 in
competition studies run in FFA4 expressing U2OS cells
(Figure 4). While competitive antagonism maybe

Figure 5. Effect of agonist 8 and antagonist 39 on insulin secretion from
the MIN6 mouse insulinoma cell line in the presence of 25 mM glucose.
Finally, effects on GLP-1 secretion utilizing
compounds 8 and 39 were studied in the human
intestinal cell line NCI-H716 (Figure 6).19 While
literature reports vary on the levels of FFA4 expression
in the NCI-H716 cell line,8,20 our in-house data was
supportive of the presence of the receptor.17 Treatment
with agonist agonist 8 provided a modest increase in
GLP-1 secretion which could be blocked with the
addition of the antagonist 39.
Figure 4. Inhibition of linoleic acid and GSK137647A (8) induced
intracellular Ca2+ increase by compound 39, a selective FFA4 receptor
antagonist.
Pharmacological profiling of the responses of
the selective FFA4 agonist and antagonist in both
murine and human cell lines was next explored.
Previous studies have shown that free fatty acids such as
linoleic acid can produce a glucose-dependent increase
in insulin secretion from the mouse insulinoma cell line
MIN6.15 As our data demonstrate the presence of FFA4
gene expression in the MIN6 cell line, consistent with
findings reported in the literature, we chose to examine
the effects of compound 8 on glucose-stimulated insulin
secretion (GSIS) in MIN6 cells. 8,16,17
Compound
8
produced
a
concentration-
dependent increase in glucose stimulated insulin
secretion (Figure 5) under high glucose conditions (25
mM). The magnitude of the potentiation of insulin
secretion observed under conditions of high glucose is
noteworthy and is similar to that demonstrated in our
laboratories with other insulin secretagogues including
glibenclamide, linoleic acid, and FFA1 agonists.18 The
enhancement in glucose-stimulated insulin secretion by
8 was abolished in the presence of the selective FFA4
antagonist 39.
Figure 6. Effect of agonist 8 and antagonist 39 on GLP-1 secretion from
the NCI-H716 cells.
In summary, the SAR of a series of diaryl
sulfonamide FFA4 agonists was explored which
resulted in the identification of a selective FFA4 agonist
8 (GSK137647A) and a selective FFA4 antagonist 39.
While the tool compounds did not have appropriate
properties for utilization in an in vivo setting,
experiments in secretory cell lines detail the utility of
this highly synthetically accessible class of low
molecular weight diaryl sulfonamides for the in vitro
exploration of FFA4-mediated biology. Further

sarcoma ATCC HTB-96; ATCC, USA) cells were
grown in Dulbecco’s modified Eagle’s medium
(DMEM)/F-12 supplemented with 10% fetal
improvements in physiochemical properties for this
series will need to be realized in order to access tool
compounds suitable for in vivo experimentation.
bovine
serum
and
2mM
L-glutamine.
Recombinant FFA4 (NM_001195755) expressing
cells were generated by transducing U2OS cells
with BacMam viruses encoding the respective
receptor and the chimeric G-protein Gα16
according to established protocols. In brief, cells
were plate to a density of 2x105 cells/mL in cell
culture medium containing human FFA4 (0.25%,
v/v), mouse FFA4 (0.5%, v/v) or rat FFA4 (0.8%,
v/v) BacMam virus. Gα16 BacMam virus
(0.12%, v/v) was also transduced in preparation of
recombinant human FFA4 cells to allow efficient
coupling of human FFA4 receptor to
phospholipase C pathway. This solution of
cells/virus mixture was then plated at a density of
104 cells/well and cultured at 37°C, 5% CO2, 95%
humidity for 24h. Functional EC50 Studies cells
were performed in cells incubated with Hank’s
buffered salt solution (HBSS) containing the
cytoplasmic calcium indicator, Fluo-4 dye in the
acetylmethyl form (4mM), 2.5mM probenecid
and 250µM Brilliant Black at 37 °C for 60 min.
Compound plates were generated containing 3%
dimethyl sulfoxide in dye loading buffer.
Compounds (i.e. FFA4 agonist GSK137647A or
Histamine for host untransduced U2OS cells)
were added to the cells at a 1:3 dilution and
calcium mobilization was measured using a
Fluorescence image plate reader (FLIPR,
Molecular Devices). Data were converted into
normalized responses with respect to assay
standards GSK137647A (for FFA4) or Histamine
(for Host U2OS cells). Data were further analyzed
using a 4-parameter fit to calculate EC50 values.
(b) Condreay, J. P.; Witherspoon, S. M.; Clay, W.
C.; Kost. T. A. 1999. Proc. Natl. Acad. Sci. 1999,
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O
H
N
H
S
N
S
O
O
O
O
O
8
40
(GSK137647A)
FFA4: Human pEC50 = 6.3
FFA4: Human pIC50 = 7.1 ± 0.35
Mouse pEC50 = 6.2
Human pEC50 < 4.5
Rat pEC50 = 6.1
Mouse pIC50 = 8.1
Human FFA1, FFA2, & FFA3 pEC50s < 4.5
Mouse FFA1, FFA2, & FFA3 pEC50s < 4.5
Rat FFA1, FFA2, & FFA3 pEC50s < 4.5
Rat pIC50 = 8.1
FFA1: Human pIC50 < 4.6
Human pEC50 < 4.3