Discovery of the first potent and orally efficacious agonist of the orphan G-protein coupled receptor 119

Article abstract of DOI:10.1021/jm8006867

GPR119 is a rhodopsin-like GPCR expressed in pancreatic β-cells and incretin releasing cells in the GI tract. As with incretins, GPR119 increases cAMP levels in these cell types, thus making it a highly attractive potential target for the treatment of dia

Full text of DOI:10.1021/jm8006867

5172
J. Med. Chem. 2008, 51, 5172–5175
Discovery of the First Potent and Orally
Efficacious Agonist of the Orphan G-Protein
Coupled Receptor 119
Graeme Semple,*,† Beatriz Fioravanti,† Guillherme Pereira,†
Imelda Calderon,† Jane Uy,† Karoline Choi,† Yifeng Xiong,†
Albert Ren,† Michael Morgan,‡ Vibha Dave,§
William Thomsen,§ David J. Unett,§ Charles Xing,§
Stuart Bossie,§ Chris Carroll,§ Zhi-Liang Chu,§
Andrew J. Grottick,§ Erin K. Hauser,§ James Leonard,§ and
Robert M. Jones†
Departments of Medicinal Chemistry, DMPK, and DiscoVery
Biology, Arena Pharmaceuticals, 6166 Nancy Ridge DriVe,
San Diego, California 92121
Figure 1. Structure of the inverse agonist screening hit.
endogenously expressed modulators are neither potent nor
selective. GPR119 is activated by phospholipids and lipid
amides, such as oleoyl ethanolamide, in vitro,4,5 but it remains
unclear as to whether these are physiologically relevant ligands.
A possible role for lipid amides in the activation of GPR119 is
supported by its phylogenetic proximity to cannabinoid recep-
tors,6 however the potency of GPR119-interacting lipid amides
is somewhat weak and they are also known to modulate other
biological targets, including PPARR nuclear receptors and
TRPV1 channels.7 Lipid amides might also be expected to
possess poor stability in vivo. Hence, to confirm early data with
molecular biological tools and to enable proof of principle in
appropriate pharmacological models, we required a potent and
selective agonist of the receptor. A preliminary report of the
discovery of weak GPR119 agonists has recently appeared,8 but
we herein describe the identification of the first highly potent
ligands for GPR119 for use in target validation studies.
A high-throughput screen of our compound collection, using a
96-well Flashplate membrane cyclase assay, capable of detecting
both agonist and inverse agonist responses,9 identified 1 (Figure
1) as a single tractable hit. The hit molecule was considered to be
somewhat unpromising as it was an inverse agonist of the receptor
(IC50 ) 84 nM; n ) 17), whereas all the preliminary data described
above suggested that an agonist would be required to have a
positive effect on modulation of insulin secretion and hence in
diabetes models. In addition, 1 contained both an undesirable
aromatic nitro group and a labile ester group. As expected, no
parent compound remained 15 min after intravenous administration
of 1 to rats and the major circulating metabolite was the carboxylic
acid resulting from ethyl ester hydrolysis, which proved to be
inactive at the receptor in our cyclase assay. However, the nitro-
pyrimidine core structure lent itself to parallel synthesis approaches,
and we therefore set out to try and generate agonist tools based on
the premise that, historically, nonpeptide agonists of rhodopsin-
like GPCRs reported in the literature have borne a remarkable
structural similarity to antagonists or inverse agonists of the same
receptor, as has been discussed previously.10 It was clear from the
lack of activity of a number of related compounds in our compound
collection that the piperidine ester function was making a significant
contribution to the observed activity, so despite the obvious
metabolic liability, we initially elected to keep this group in place
while examining the SAR at the opposite end of the molecule in
the search for an agonist starting point.
ReceiVed June 5, 2008
Abstract: GPR119 is a rhodopsin-like GPCR expressed in pancreatic
ꢀ-cells and incretin releasing cells in the GI tract. As with incretins,
GPR119 increases cAMP levels in these cell types, thus making it a
highly attractive potential target for the treatment of diabetes. The
discovery of the first reported potent agonist of GPR119, 2-fluoro-4-
methanesulfonyl-phenyl)-{6-[4-(3-isopropyl-[1,2,4]oxadiazol-5-yl)-pi-
peridin-1-yl]-5-nitro-pyrimidin-4-yl}-amine (8g, AR231453), is de-
scribed starting from an initial inverse agonist screening hit. Compound
8g showed in vivo activity in rodents and was active in an oral glucose
tolerance test in mice following oral administration.
Injectable peptide agonists of the glucagon-like peptide-
1a (GLP-1) receptor, such as exendin-4 (exenatide), amplify glu-
cose-dependent insulin release and preserve pancreatic ꢀ-cell
mass and have shown promise as antidiabetic agents; exenatide
has been approved for this indication.1 These compounds elevate
intracellular cAMP levels in ꢀ-cells via stimulation of the GLP-1
receptor (GLP-1R), which is positively coupled to adenylate
cyclase (GRs). GLP-1R is a class B (secretin-like) G-protein
coupled receptor (GPCR), and it has proven difficult over the
years to identify nonpeptide agonist ligands of this subfamily
that may be suitable for oral administration. As a result, we
were interested in identifying novel, class A (rhodopsin-like)
GRs-coupled GPCRs that would have similar ꢀ-cell expression
and G-protein coupling to GLP-1R. From this effort, we
identified GPR119 (recently termed glucose-dependent insuli-
notropic receptor, or GDIR) as a constitutively active, ꢀ-cell-
expressed receptor capable of elevating intracellular cAMP
levels in both transfected CHO cells or pancreatic ꢀ-cell lines.2
Additionally, we showed that GPR119 could stimulate incretin
hormone release from cells localized in the GI tract, demonstrat-
ing that the receptor can regulate glucose homeostasis by
multiple mechanisms.3 Thus, our early target localization and
validation studies suggested that GPR119 would be an attractive
target for the treatment of type 2 diabetes, and we assumed that
it may also be more amenable to the discovery of orally acting
small molecule agonists as a result of its phylogenicity.
It has proven difficult to investigate functional aspects of
GPR119 with native ligands, in part because the reported
* To whom correspondence should be addressed. Phone: +1 858 453
7200. Fax: + 1 858 453 7210. E-mail: gsemple@arenapharm.com.
Using the simple synthetic procedure outlined in Scheme 1,11
we replaced the trifluoromethyl pyrazole with a series of aryl ethers
(Scheme 1, X ) O). Reasonable inverse agonist activity was
retained in the screening assay with a range of phenyl ethers
substituted with simple halo-, trifluoromethyl-, cyano-, or alkyl ether
groups (e.g., 4a), but no agonists were identified among the first
several dozen analogues prepared. However, when the phenyl group
was substituted in the 4-position with a more complex keto-ester
group, we obtained 4d, our first example of a GPR119 agonist.
†
Department of Medicinal Chemistry, Arena Pharmaceuticals.
Department of DMPK, Arena Pharmaceuticals.
Department of Discovery Biology, Arena Pharmaceuticals.
‡
§
a Abbreviations: GPR119, G-protein coupled receptor 119, also known
as GDIR, Glucose-dependent insulinotropic receptor; GLP-1R, glucagon-
like peptide-1 receptor; cAMP, cAMP, 3′,5′-cyclic adenosine monophos-
phate; t1/2, terminal half-life; Cmax, observed maximal plasma concentration
following oral dosing; tmax, time to reach the Cmax; AUC, total exposure as
measured by the area under the exposure curve; %F, oral bioavailability;
oGTT, oral glucose tolerance test.
10.1021/jm8006867 CCC: $40.75
2008 American Chemical Society
Published on Web 08/13/2008

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5173
Scheme 2. Preparation of Oxadiazole Analoguesa
Scheme 1. Two Step Synthesis of Nitropyrimidine Core
Compoundsa
i
a Reagents and conditions: (a) Ethyl piperidine-4-carboxylate, PrNEt,
DCM, 0 °C, 91%. (b) Ph(R)XH (X ) O or NH) dioxin: DMF (5/1, v/v),
microwave heating, 20-65%.
Table 1. Activity of Compounds 4a-4k in a Membrane cAMP
Flashplate Assay
compd no.
R
X
IC50/EC50 (µM)a
function
4a
4b
4c
4d
4e
4f
4g
4h
4i
4-Br
4-COEt
O
O
O
O
O
O
O
O
O
N
N
0.05
n.e.
n.e.
1.0
1.7
0.5
1.9
3.0
2.5
1.2
0.4
inverse agonist
4-(CH2)2OMe
4-COCH2CO2Me
4-(CH2)2COMe
4-O(CH2)2OMe
4-SO2Me
4-SO2NH2
4-(1-(1,2,4-triazole))
4-SO2Me
agonist
agonist
inverse agonist
agonist
agonist
agonist
agonist
agonist
4j
4k
4-(1-(1,2,4-triazole))
a Data from the flashplate assay were not run with sufficient replicates
to be regarded as truly quantitative (usually n ) 1 or n ) 2), and so standard
error data are not applicable.
a Reagents and conditions: (a) NH2OH.HCl, Na2CO3, H2O/EtOH. (b) 1-Boc-
ethyl piperidine-4-carboxylate, NaH, THF, 4 M HCl/dioxan. (c) 2, PrNEt,
i
DCM, 0 °C, 91%. (d) 4-MeSO2PhNH2, dioxane: DMF (5/1, v/v), microwave,
180 °C, 300s. (e) R′NH2, K2CO3, dioxane, microwave, 250 °C, 300s.
Although we had succeeded in inverting the functional activity in
our favor, we had done so at the expense of incorporating a second
labile ester group into the molecule. Some simple examination of
the SAR from the same early focused library showed that neither
a simple ketone (4b) nor an ether (4c, 4f) were sufficient to provide
agonist activity, but a ketone further displaced from the ring (4e)
also produced the desired functional response. We thus hypoth-
esized that a hydrogen-bond accepting group was required for
measurable agonist activity but that its optimal position was still
undetermined. Hence we focused our further efforts on substituted
phenyl analogues incorporating this type of feature with a view to
more closely defining the H-bonding acceptor requirements and
identifying a more stable agonist trigger group so that we could
return to addressing the stability issues at the other end of the
molecule. Extensive parallel SAR work led to the identification of
multiple agonist triggers, selected examples of which are shown
in Table 1 and include sulfones, sulfonamides, and various
5-membered heterocycles. The best activity was seen when such
a group was attached to the 4-position of the phenyl ring. In
addition, with the appropriate trigger in place, agonist activity was
maintained with either an ether or aniline linker (4, X ) O or NH).
Although our HTS assay had served its purpose of enabling us
to identify agonist molecules, the reproducibility of the assay and
hence the ability to obtain truly quantitative data was relatively
poor as a result of the requirement for transient transfection of the
receptor prior to each preparation of membranes; cells expressing
the receptor at high levels were also prone to apoptosis. This is
believed to be an artifact of receptor overexpression (many times
higher than under physiological conditions) and was cell type
dependent. Clear agonism or inverse agonism could be readily
determined, but it was difficult to rapidly and accurately determine
structural features that would enhance potency as the generation
of high quality data (SD < 0.4 log units) required too many
replicates. Hence we changed our assay platform to a melanophore
dispersion assay. The melanophore assay uses Xenopus cells, which
when transiently transfected with GPCRs can engage their existing
intracellular machinery to respond to receptor activation by changes
in pigment trafficking. This results in an apparent color change
that can be quantified by simple measurement of light trans-
mission in high-throughput format to produce concentra-
tion-response curves. These cells are particularly suited to
measuring Gi or Gs coupled GPCR activation (producing pigment
aggregation and dispersion respectively).12 This assay switch
provided data that proved to be significantly more reproducible
and consequently more suitable for more subtle SAR examination.
With a clear indication that agonist molecules could be identified
within the series and a new assay in hand, we now sought to replace
the ester group to provide more metabolically stable compounds.
As the ester group had proven important for the initial hit
identification, we elected first to replace it with a highly conserva-
tive isosteric replacement. The oxadiazole ring can provide all of
the hydrogen bond donor and acceptor capabilities of the ester itself,
and so we prepared a series of such derivatives as outlined in
Scheme 2. Reaction of a series of alkyl and aryl nitriles with
hydroxylamine provided hydroxyamidine intermediates, which
were condensed directly with 1-Boc-ethyl piperidine-4-carboxylate.
Removal of the Boc-protecting group under acidic conditions
provided the oxadiazole building blocks 5. Reaction of 5 with 2
under similar conditions as used previously to incorporate pip-
eridines provided the intermediates 6, which upon displacement
of the second chloro- group from the pyrimidine with aniline-4-
methyl sulfone under microwave irradiation conditions duly
provided the target compounds 7. As can be seen in Table 2,
keeping constant the aniline sulfone group which we had previously
shown to enable good agonist activity, allowed the identification
of a number of more potent agonist molecules. The SAR of the
alkyl substituent on the oxadiazole portion was particularly
remarkable. Increasing the size of this substituent from methyl to
ethyl provided a more than 25-fold improvement in agonist potency.
The addition of more carbon atoms to the chain in a linear manner
did not provide any further improvements in activity, whereas the
inclusion of R-branching (but not ꢀ-branching) in this group again
gave a significant improvement in potency. In addition to alkyl

5174 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17
Letters
Table 2. Agonist Activity of Selected Oxadiazole Analogues in the
acceptor in this region of the molecule. Addition of a further
fluoro-substituent onto this aryl ring provided two additional
compounds with improved potency, with 8g (AR231453) being
the first subnanomolar agonist compound that we had observed.
Replacement of the sulfone with sulfonamides did not provide
any further improvement, and although a similar SAR was
Melanophore Dispersion Assay12
compd no.
R
EC50 (nM)
log SD (n)a
7a
7b
7c
7d
7e
7f
7g
7h
7i
Me
Et
260
10
430
25
160
5.8
1.5
660
24
0.26 (8)
0.19 (8)
0.28 (4)
0.20 (8)
0.13 (3)
0.12 (5)
0.38 (3)
0.28 (7)
0.27 (3)
nPr
i
cPr
observed with other oxadiazoles in place of the Pr oxadiazole
in this series, once more, no real potency advantage ensued.
c
CH2 Pr
iPr
Further profiling of 8g showed it to be a highly selective ligand
for GPR119 with no off-target activity across a 76 receptor and
enzyme profiling panel (CEREP), which included the known
incretin receptors and no activity against 140 known and orphan
GPCRs in melanophore dispersion assays in-house. In addition,
8g had no activity against dipeptidyl peptidase-4, a well validated
target for the treatment of diabetes. Importantly for a pharmaco-
logical tool, 8g had activity across species, albeit with somewhat
lower potency in rodents (EC50 in melanophore for other species
tested: cynomologus monkey GPR119 ) 0.4 nM; mouse GPR119
) 12 nM; rat GPR119 ) 14 nM; dog GPR119 ) 1.6 nM). Across
all species, 8g displayed a significantly greater level of efficacy in
these assays than the putative endogenous ligand oleoyl ethano-
lamide and has since become our standard compound for compar-
ing the efficacy of other series of compounds at the receptor.
Comparative efficacy studies of a number of naturally occurring
lipid amides will be detailed in further reports from our laboratories.
tBu
3-pyridyl
3-CF3-Ph
a The standard deviation from the mean of the EC50 value. The number
of determinations is shown in parentheses.
Table 3. Agonist Activity of Isopropyl Oxadiazole Analogues in the
Melanophore Dispersion Assay12
compd no.
R′
EC50 (nM)
log SD (n)a
8a
8b
8c
8d
8e
8f
8g
8h
8i
4-(SO2Et)-Ph
4-(SO2nPr)-Ph
4-(SO2 Pr)-Ph
4-(SOEt)-Ph
4-(SEt)-Ph
17
4.7
7.8
17.1
110
3.4
0.68
7.0
0.15 (3)
0.4 (5)
i
0.44 (7)
0.15 (3)
0.17 (2)
0.44 (3)
0.37 (569)
0.14 (2)
0.24 (6)
3-F, 4-(SO2Me)-Ph
2-F, 4-(SO2Me)-Ph
2-F, 4-(SO2Et)-Ph
i
2-F, (4-SO2 Pr)-Ph
7.9
Compound 8g did not inhibit any of the major CYP enzymes
and had no activity up to 10 µM at the hERG channel in a
patch clamp assay. Furthermore, excellent exposure was ob-
served after oral administration to C57/bl6J mice (10 mg/kg po
dose: tmax ) 0.5 h, Cmax ) 4975 ng/mL (9.84 µM), AUC )
19803 h·ng/mL, t1/2 ) 3.4 h; %F was not calculated as the iv
PK profile was not determined in mouse), making this a
promising tool for studying the function of GPR119 in this
species. As has been recently reported,2 8g stimulated cAMP
production via endogenously expressed GPR119 in the ꢀ-cell-
like hamster insulinoma line HIT-T15, with an EC50 of 4.7 nM,
a value comparable to the EC50 observed in cell lines overex-
pressing the cloned rodent receptors.
As can be seen in Figure 2, the in vivo effect of 8g was similar
in terms of % inhibition of glucose AUC in an oral glucose
tolerance test (oGTT) in both C57/bl6J mice and Sprague-
Dawley rats at a dose of 3 mg/kg ip despite the clear species
differences in the magnitude of the glucose excursion. This
similarity may be expected in light of the similar potency and
efficacy of 8g versus both mouse and rat GPR119. In addition, 8g
improved oral glucose tolerance in wild-type C57/bl6J mice after
per oral administration (Figure 3). Importantly, in terms of target
validation, 8g also enhanced glucose-dependent insulin release and
a The standard deviation from the mean of the EC50 value. The number
of determinations is shown in parentheses.
groups in this position, we also examined a range of aryl groups
(two examples of which are shown in Table 2), but no further
compounds were identified that matched the single-digit nanomolar
potency of 7f or 7g. Subsequently, alternative heterocycles were
also examined in place of the oxadiazole in 7 (including the
isomeric oxadiazoles) and the SAR for compounds of this type
will be reported elsewhere.
Having identified a stable replacement for the ester group,
we turned our attention back to the other end of the molecule
to re-examine whether our previous SAR observations still held
true. The compounds 8a-8i were all prepared from the same
i
intermediate (6, R ) Pr) by nucleophilic displacement of the
pyrimidine chloro- group with various anilines, with the
exception of 8d, which was prepared from 8e by oxidation of
the thioether group to the sulfoxide with meta-chloro-perbenzoic
acid. As can be seen from Table 3, no further improvement in
potency was observed by increasing the size of the sulfone alkyl
group. Interestingly, the sulfoxide 8d was comparable in potency
to 7f, whereas the thioether analogue 8e was significantly less
potent, re-emphasizing the preference for a good hydrogen bond
Figure 2. The effect of compound 8g on oral glucose tolerance in mouse and rat following ip administration.

Letters
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 17 5175
References
(1) Gallwitz, B. Exenatide in type 2 diabetes: treatment effects in clinical
studies and animal study data. Int. J. Clin. Pract. 2006, 60, 1654–1661.
(2) Chu, Z.-L.; Jones, R. M.; He, H.; Carroll, C.; Gutierrez, V.; Lucman, A.;
Moloney, M.; Gao, H.; Mondala, H.; Bagnol, D.; Unett, D.; Liang, Y.;
Demarest, K.; Semple, G.; Behan, D. P.; Leonard, J. A role for ꢀ-Cell-
Expressed GPR119/ GDIR in Glycemic Control by Enhancing Glucose-
Dependent Insulin Release. Endocrinology 2007, 148, 2601–2609.
(3) Chu, Z.-L.; Carroll, C.; Alfonso, J.; Gutierrez, V.; He, H.; Lucman,
A.; Pedraza, M.; Mondala, H.; Gao, H.; Bagnol, D.; Chen, R.; Jones,
R. M.; Behan, D. P.; Leonard, J. A Role for Intestinal Endocrine Cell-
Expressed GPR119 in Glycemic Control by Enhancing GLP-1 and
GIP Release. Endocrinology 2008, 149, 20382047.
(4) Soga, T.; Ohishi, T.; Matsui, T.; Saito, T.; Matsumoto, M.; Takasaki,
J.; Matsumoto, S.; Kamohara, M.; Hiyama, H.; Yoshida, S.;.; Momose,
K.; Ueda, Y.; Matsushime, H.; Kobori, M.; Furuichi, K. Lysophos-
phatidylcholine enhances glucose-dependent insulin secretion via an
orphan G-protein-coupled receptor. Biochem. Biophys. Res. Commun.
2005, 326, 744–751.
(5) Overton, H. A.; Babbs, A. J.; Doel, S. M.; Fyfe, M. C.; Gardner, L. S.;
Griffin, G.; Jackson, H. C.; Procter, M. J.; Rasamison, C. M.; Tang-
Christensen, M.; Widdowson, P. S.; Williams, G. M.; Reynet, C.
Deorphanization of a G protein-coupled receptor for oleoylethanola-
mide and its use in the discovery of small-molecule hypophagic agents.
Cell Metab. 2006, 3, 167–175.
Figure 3. The effect of compound 8g on oral glucose tolerance in
mouse after oral administration.
improved oral glucose tolerance in wild-type C57/bl6J mice but
not in GPR119-deficient mice at a dose of 20 mg/kg po, clearly
demonstrating the receptor dependence of the observed effects.2
Diabetic KK/Ay mice were also highly responsive to 8g. It should
be noted, however, that a statistically significant effect on glucose
excursion in an oGTT following oral administration of 8g was not
observed in rats, although a trend toward efficacy was seen at a
dose of 30 mg/kg (data not shown). We believe that this lack of a
consistent effect results from the combination of a narrower window
for measuring the decrease in glucose excursion in rats as well as
the significantly poorer exposure for 8g in rat compared to mouse
following oral administration (10 mg/kg po dose: tmax ) 1 h, Cmax
) 126 ng/mL (0.250 µM), AUC ) 263 h ·ng/mL, t1/2 ) 1.1 h, F
) 12%). Taken together with the molecular biological data, these
pharmacological studies with a selective receptor agonist strongly
suggest that targeting GPR119 will be a useful approach to provide
novel antidiabetic agents acting in a glucose-dependent fashion.
In summary, the manipulation of our initial inverse agonist
screening hit to provide an orally bioavailable pharmacological
tool compound has been outlined. The incorporation of a
hydrogen-bond acceptor group to the southern portion around
the nitro pyrimidine core was required but was not always
sufficient for agonist activity. Replacement of the ester function
in the original hit with an isosteric oxadiazole provided useful
improvements in stability, and the inclusion of small branched
alkyl groups on this oxadiazole gave compounds with increased
activity. Finally fluorination on the southern ring provided a
further enhancement in potency, resulting in the identification
of 8g, which has proven to be an excellent tool compound for
target validation studies in mice. In addition, we have shown
that it is possible to identify orally bioavailable agonists of the
receptor, which may make GPR119 a more attractive target for
future drug development then other GRs-coupled receptors
expressed on pancreatic ꢀ-cells such as GLP-1R. Further SAR
enhancements leading to the identification of a first clinical
candidate for GPR119 will be reported in due course.
(6) Brown, A. J. Novel cannabinoid receptors. Br. J. Pharmacol. 2007,
152, 567–575.
(7) (a) Fu, J.; Gaetani, S.; Oveisi, F.; Verme, J. L.; Serrano, A.; Rodrı´guez
de Fonseca, F.; Rosengarth, A.; Luecke, H.; Di Giacomo, B.; Tarzia,
G.; Piomelli, D. Oleylethanolamide regulates feeding and body weight
through activation of the nuclear receptor PPAR-R. Nature 2003, 425,
90–93. (b) Ahern, G. P. Activation of TRPV1 by the Satiety Factor
Oleoylethanolamide. J. Biol. Chem. 2003, 278, 30429–30234.
(8) Fyfe, M. C. T.; Babbs, A. J.; Bertram, L. S.; Bradley, S. E.; Doel,
S. M.; Gadher, S.; Gattrell, W. T.; Jeevaratnam, R. P.; Keily, J. F.;
McCormack, J. G.; Overton, H. A.; Rasamison, C. M.; Reynet, C.;
Rushworth, P. J.; Sambrook-Smith, C. P.; Shah, V. K.; Stonehouse,
D. F.; Swain, S. A.; White, J. R.; Widdowson, P. S.; Williams, G. M.;
Procter, M. J. Synthesis, SAR, and in vivo efficacy of novel GPR119
agonists with a 4-[3-(4-methanesulfinylphenoxy)propyl]-1-Boc-pip-
eridine core. Abstracts of Papers, 234th ACS National Meeting,
Boston, MA, August 19-23, 2007, MEDI-062. The recent patent
literature has also been reviewed: Fyfe, M. C. T.; McCormack, J. G.;
Overton, H. A.; Procter, M. J.; Reynet, C. GPR119 agonists as potential
new oral agents for the treatment of type 2 diabetes and obesity. Expert
Opin. Drug DiscoVery 2008, 3, 403–413.
(9) HEK293 cells transiently expressing GPR119 were prepared using
standard transfection protocols and crude plasma membranes were
prepared 48 h post-transfection as described by Chu et al.2 Compound
stimulation of membrane adenylyl cyclase activity was measured using
a 96-well FlashPlate kit (NEN Life Sciences, Boston, MA) as described
by Chu et al.3 Briefly, assay buffer contained 20 mM HEPES, pH
7.4, 10 µM GTP, 0.1 mM ATP, 500 µM IBMX, 10 mM phospho-
creatine, and 10 U/50 µL creatine phosphokinase. Test compound
incubations were performed for 60 min at room temperature in the
presence of 15 µg of membrane protein. cAMP measurements were
extrapolate from a standard curve included on each assay plate.
(10) Sugg, E. E. Nonpeptide agonists for peptide receptors: lessons from
ligands. Annu. Rep. Med. Chem. 1997, 32, 277–283.
(11) Jones, R. M.; Semple, G.; Fioravanti, B.; Pereira, G.; Calderon, I.;
Uy, J.; Duvvuri, K.; Choi, K.; Xiong, Y.; DaveV. Preparation of 1,2,3-
trisubstituted aryl and heteroaryl derivatives, in particular pyrimidines,
as modulators, in particular agonists and inverse agonists, of G-coupled
protein receptor and their use in the prophylaxis and treatment of
metabolic disorders such as diabetes and hyperglycemia. V. PCT Int.
Appl. WO 2004065380, 2004.
Supporting Information Available: Synthetic experimental
procedures, overexpression of GPR119 in RIN5F cells can amplify
insulin secretion in the presence of high concentrations of glucose,
examples of dose-response curves for an inverse agonist (4a) and
an agonist (4i) molecule in the cAMP membrane flashplate assay,
data showing correlation of two assay platforms used and reproduc-
ibility of the melanophore assay. This material is available free of
charge via the Internet at http://pubs.acs.org.
(12) Cultured Xenopus dermal melanophores were transiently transfected
with plasmid DNA encoding the GPR119 receptor and plated into
clear 384-well assay plates following standard protocols. At 48 h post-
transfection, the cells were treated with melatonin (10nM, 1 h) to
induce pigment aggregation. Cells were then exposed to test com-
pounds for 1 h, and the resulting GPR119-induced pigment dispersion
was measured using an absorbance microplate reader. For a full
description of standard protocol see: Potenza, M.; Graminski, G.;
Lerner, M. A method for evaluating the effects of ligands upon Gs
protein-coupled receptors using a recombinant melanophore-based
bioassay. Anal. Biochem. 1992, 206, 315–322.
Note Added after ASAP Publication.
This manuscript published ASAP on August 13, 2008 with
an error in Table 3. The revised version was published on
August 19, 2008.
JM8006867