Discovery of fused bicyclic agonists of the orphan G-protein coupled receptor GPR119 with in vivo activity in rodent models of glucose control

Article abstract of DOI:10.1016/j.bmcl.2011.03.007

We herein outline the design of a new series of agonists of the pancreatic and GI-expressed orphan G-protein coupled receptor GPR119, a target that has been of significant recent interest in the field of metabolism, starting from our prototypical agonist

Full text of DOI:10.1016/j.bmcl.2011.03.007

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3134–3141
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of fused bicyclic agonists of the orphan G-protein coupled receptor
GPR119 with in vivo activity in rodent models of glucose control
Graeme Semple a, , Albert Ren a, Beatriz Fioravanti a, Guillherme Pereira a, Imelda Calderon a, Karoline Choi a,
⇑
Yifeng Xiong a, Young-Jun Shin a, Tawfik Gharbaoui a, Carleton R. Sage a, Michael Morgan b, Charles Xing c,
Zhi-Liang Chu c, James N. Leonard c, Andrew J. Grottick c, Hussein Al-Shamma c, Yin Liang d,
Keith T. Demarest d, Robert M. Jones a
a Medicinal Chemistry, Arena Pharmaceuticals, 6166 Nancy Ridge Drive, San Diego, CA 92121, USA
b DMPK, Arena Pharmaceuticals, 6166 Nancy Ridge Drive, San Diego, CA 92121, USA
c Discovery Biology, Arena Pharmaceuticals, 6166 Nancy Ridge Drive, San Diego, CA 92121, USA
d Johnson & Johnson Pharmaceutical Research & Development, LLC. Spring House, PA 19477, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We herein outline the design of a new series of agonists of the pancreatic and GI-expressed orphan G-pro-
tein coupled receptor GPR119, a target that has been of significant recent interest in the field of metab-
olism, starting from our prototypical agonist AR231453. A number of key parameters were improved first
by incorporation of a pyrazolopyrimidine core to create a new structural series and secondly by the intro-
duction of a piperidine ether group capped with a carbamate. Chronic treatment with one compound
from the series, 3k, showed for the first time that blood glucose and glycated hemoglobin (HbA1c) levels
could be significantly reduced in Zucker Diabetic Fatty (ZDF) rats over several weeks of dosing. As a result
of these and other data described here, 3k (APD668, JNJ-28630368) was the first compound with this
mechanism of action to be progressed into clinical development for the treatment of diabetes.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 17 January 2011
Revised 28 February 2011
Accepted 2 March 2011
Available online 13 March 2011
Keywords:
GPR119 agonists
GPCR
Diabetes
GDIS
We have recently described the identification of GPR119 (also
termed glucose-dependent insulinotropic receptor, or GDIR) as a
constitutively active, pancreatic b-cell-expressed G-protein cou-
pled receptor (GPCR) whose activation can elevate intracellular
cAMP levels in both transfected CHO cells and pancreatic b-cell
lines.1 Such activity would be expected to stimulate glucose-
dependent insulin release from b-cells. In addition, we showed that
GPR119 could stimulate incretin hormone release from cells local-
ized in the GI tract and thus the receptor might be capable of reg-
ulating glucose homeostasis by multiple mechanisms.2 These early
target validation studies suggested that GPR119 may be an inter-
esting target for the treatment of type 2 diabetes that may be ame-
nable to the discovery of orally acting agents.
also be expected to possess poor stability in vivo. Hence, the dis-
covery of AR231453 (1a, Fig. 1) was essential for initial investiga-
tions into the role of this target in plasma glucose control.7
However, whereas 1a was an excellent tool for acute studies in
mouse, it had a number of drawbacks. The relatively poor exposure
in rat and the toxicity observed after multiple doses of 1a in mouse
(which may in part have been a consequence of the vehicle mix-
tures it was necessary to use as a result of poor solubility) meant
that it was not suitable for examining the effect of GPR119 modu-
lation in any of the known and well characterized chronic models
of diabetes in rodents. Hence we undertook to look for alternative
structures to demonstrate activity in proof of concept experiments
of a chronic or sub-chronic nature, with the long term goal of iden-
tifying a compound suitable for clinical testing.
Starting from our prototypical GPR119 agonist 1a, we were
keen to investigate the effect of removing both the undesirable ani-
line and nitro portions of the molecule simultaneously by intro-
ducing a simple 6,5-fused ring system. We envisioned that such
a modification would maintain several of the hydrogen bond
acceptor interactions that we believed may be important for activ-
ity, such as the two nitrogens in the pyrimidine ring and the sul-
fone portion, and these would be held in a similar position to
those in 1a by introduction of this constraint to a key dihedral an-
gle.8 The resulting compound (2), which could be prepared in four
GPR119 can be activated by phospholipids and lipid amides in
vitro,3,4 but it remains unclear as to whether these are physiologi-
cally important ligands. A role for lipid amides in the activation of
GPR119 could be implied by its phylogenetic proximity to cannab-
inoid receptors.5 However, the potency and efficacy of these puta-
tive ligands at the GPR119 receptor is modest and some examples
are also known to modulate other biological targets, including
PPARa
nuclear receptors and TRPV1 channels.6 Lipid amides might
⇑
Corresponding author. Tel.: +1 858 453 7200; fax: +1 858 453 7210.
E-mail address: gsemple@arenapharm.com (G. Semple).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.007

G. Semple et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3134–3141
3135
the oxadiazole piperidine portion of the monocyclic pyrimidine
series into this pyrazolopyrimidine scaffold.9 The reversed piperi-
dine carbamate ether modification could be prepared from the
same intermediate used to prepare 2 (Scheme 1).10 Thus, 6 was re-
acted with the tert-butyl carbamate (Boc) derivative of 4-hydroxy
piperidine under Mitsonobu conditions to furnish 3a in reasonable
yield. In contrast to 2, 3a was more similar in activity to 1a
(EC50 = 0.65 nM; n = 304) and the des-fluoro analogue 1b
(EC50 = 5.1 nM; n = 4) in our melanophore assay (Table 1). In addi-
tion, we could discern no significant difference in apparent efficacy
between the compounds in this assay platform. Hence we set
about exploring some of the aspects of the SAR around our new
bicyclic scaffold GPR119 agonist.
N
O
N
O
N
N
R'
N
N
O
O
N
O
N
N
N
NO2
NH
N
N
N
N
N
N
N
R
R
O
S
S
O
Me
O
Me
O
We assumed in the first instance that the new series of com-
pounds would be binding to the receptor in a similar orientation
to the monocyclic series as implied by our scaffold design de-
scribed above. Thus for our first investigation we maintained the
4-methylsulfone or 2-fluoro-4-methylsulfone substitution pattern
on the southern aromatic ring portion and examined the effect of
changes to the carbamate or linker portion (X in compound 8).
Compounds 3b–k (Table 1) were prepared in the same manner
as 3a via Mitsonobu reaction of 6 with the appropriate carbamate
derivative of 4-hydroxy piperidine, or alternatively could be pre-
pared by removal of the Boc group from 3a and reaction of the
3
2
1a, R=F; AR231453
1b, R=H
Figure 1. Design of bicyclic GPR119 analogues starting from AR231453.
steps by the route shown in Scheme 1, did retain significant agonist
activity at the human GPR119 receptor (EC50 = 42 nM; n = 2 in the
melanophore assay7) but was around 50-fold less potent than 1a in
our hands. Encouraged by this, we incorporated a second modifica-
tion which we had previously shown to be a useful replacement for
OR'
O
N
O
O
N
EtO
NC
HN
(i)
(iv)
N
N
+
NHNH2
N
N
N
CN
R
N
4
5
6
3
R
R
(ii)
OR'
O
N
Cl
X
N
N
N
(iii)
(v)
N
N
N
2
N
N
8
[ X = NH2, N(R)CH2, S ]
7
R
R
(vii)
(vi)
OR'
OR'
O
N
O
N
X
N
N
N
N
N
N
N
N
10
9
[X = SO, SO2]
R
R
Scheme 1. Synthesis of pyrazolopyrimidine analogues. Reagents and conditions: (i) (a) MeOH, reflux, (b) H2O, HCO2H, reflux; 70–85%; (ii) POCl3, PhNMe2, reflux, 30–70%; (iii)
4-(3-isopropyl-1,2,4-oxadiazol-5-yl)piperidine, NaH, THF. rt 65%; (iv) 4-hydroxypiperidine carbamates, DEAD, Ph3P, THF rt, reverse addition; 40–60%; (v) 4-X-substituted
piperidine carbamates, NaH, THF, rt; 40–80%; (vi) mCPBA, DCM, 75%; (vii) (a) tert-butyl 4-methylenepiperidine-1-carboxylate (prepared in 60% yield over two-steps from
piperidine-4-one by standard Boc protection followed by Wittig reaction (CH3PPh3Br, nBuLi, 0 °C, 4 h)), 9-BBN, THF reflux; mixture added to 7 then (b) PdCl2(dppf)2, AsPh3,
K2CO3, DMF, H2O, microwave, 150 °C, 20 min, 20%.

3136
G. Semple et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3134–3141
resultant secondary amine with the requisite chloroformate re-
agent to prepare the desired carbamate. Minor changes to the alkyl
group of the carbamate did not result in significant changes in the
agonist activity, and in some cases apparent efficacy, of the result-
ing molecules. However, it should be noted that secondary and pri-
mary alcohol carbamates were significantly more stable under
acidic conditions than the Boc derivatives, making them much
more suitable for dosing via the oral route. When the size of the al-
kyl group was further increased, however, (3f) a 3–10-fold de-
crease in potency was observed. Furthermore, the incorporation
of heteroatoms into this portion of the molecule in an effort to im-
prove water solubility, resulted in a decrease in both activity and
efficacy (e.g., 3g) and all activity was lost when we attempted to
introduce basic groups into this region (e.g., 3i). In contrast to
the monocyclic series described previously, where we had seen a
significant improvement in potency by introducing a fluoro substi-
tuent onto the southern aromatic ring,7 the same modification in
this bicyclic series where X@O were generally of similar potency
and apparent efficacy to the des-fluoro analogue (e.g., 3j and 3k).
We next investigated the result of changing the linker atom or
atoms between the bicyclic scaffold and the appended piperidine
ring. Such compounds could be prepared by nucleophilic displace-
ment of the 4-chloro substituent from the pyrazolopyrmidine 7
with a piperidine derivative containing the appropriate linker por-
tion XH. The sulfoxide and sulfone linker derivatives were in turn
prepared by oxidation of the thioether linked analogues (Scheme
1). The incorporation of a simple secondary amine or aminometh-
ylene linker (8a and 8b) was not well tolerated. However, in the
case of the aminomethylene linker, the presence of an additional
alkyl group on the nitrogen to provide the tertiary amine analogues
(8c–e), again resulted in compounds with single digit nanomolar
EC50 values. However, in contrast to the ether linked series, com-
pounds of this type were subject to rapid metabolism both
in vitro and in vivo and as a result, were not pursued further.
In a somewhat more conservative change, switching from the
ether linker to the thioether had little effect on activity (8f and
8g). However, oxidation of the thioether to either the sulfoxide
(9a) or the sulfone (9b) linked analogues resulted in a significant
decrease in activity.
Finally we examined the effect of replacing the ether linker with
a methylene group. Compounds 10a and 10b were prepared in
moderate yield from the intermediates 7 (R = 4-SO2Me and 2-F,
4-SO2Me, respectively) via a palladium catalyzed cross-coupling
(PdCl2(dppf)2 in the presence of triphenyl arsine ligand) with a
borane reagent prepared from the Wittig product of Boc-piperi-
din-4-one and methyltriphenylphosphonium bromide. Again, little
difference in the activity between 10a and 10b and their ether
linked counterparts was observed and as this series was signifi-
cantly more difficult to prepare, it was not pursued further.
For the next stage of our SAR exploration, we examined a num-
ber of modifications to the bicyclic core portion of the molecule.
Initially, we tried to apply highly conservative changes, such as
the introduction of methyl groups onto the pyrazolopyrimidine
core. Generally, such compounds could be prepared using very
similar routes to those outlined earlier, but in most cases each of
the intermediates was isolated, as shown in Scheme 2. For the
preparation of the 3-methyl derivative 15a, the pyrazole interme-
diate 11 (R1 = Me) was prepared as shown and acylated with for-
mic acid and the intermediate cyclized without isolation to
provide 13a in good yield. Similarly, for the preparation of 15b,
the acylation was carried out with acetyl chloride following which
the intermediate 12b (which was isolated) was subsequently cy-
clized to provide 13b. Each of these intermediates could be con-
verted to the test compounds by the usual sequence via reaction
with phosphoryl chloride to give the aromatic chlorides (14a and
14b) which underwent nucleophilic displacement with Boc-4-hy-
droxy piperidine under microwave conditions to provide 15a and
15b. However, each addition of a methyl group, first to the 3-posi-
tion and then to the 6-position, resulted in an order of magnitude
reduction in the agonist potency compared to 3a.
We next prepared some alternative 6,5-fused bicyclic heterocy-
cles to assess how more extensive changes to the core may affect
activity at the receptor. The preparation of the first three examples
from
a
common intermediate 17 is shown in Scheme 3.
R1
R1
N
N
EtO
R1
CN
CN
(i)
(ii)
MeO2S
N
MeO2S
N
CN
CN
H2N
HN
O
R2
4b; R1 = Me
11
12a; R1=Me, R2 = H
12b; R1=Me, R2 = Me
(iii)
BocN
OH
R1
Cl
N
R1
O
N
R1
N
N
N
N
N
(v)
(iv)
N
N
R2
N
N
R2
N
R2
SO2Me
SO2Me
SO2Me
15a; R1=Me, R2 = H
15b; R1=Me, R2 = Me
14a; R1=Me, R2 = H
14b; R1=Me, R2 = Me
13a; R1=Me, R2 = H
13b; R1=Me, R2 = Me
Scheme 2. Synthesis of substituted pyrazolopyrimidine analogues. Reagents and conditions: (i) (a) 4-MeSO2-PhNHNH2, NaOMe, MeOH, reflux, 70–90% yield; (ii) either AcCl
(R2 = Me) or HCO2H (R2 = H), (intermediate 12a not isolated); (iii) H2O, KOH, reflux; 30–50% yield two-steps; (iv) POCl3, PhNMe2, reflux; 50–60% yield;
(v) 4-hydroxypiperidine carbamates, K2CO3, DMF 100 °C, microwave; 40–60% yield.

G. Semple et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3134–3141
3137
BocN
Cl
N
Cl
O
NO2
Cl
N
N
N
N
N
N
(iv)
N
N
N
(i)
19
(iii)
18
SO2CH3
SO2CH3
Cl
N
BocN
Cl
N
R"
NH
N
N
N
O
N
N
(v)
(iv)
N
N
N
R
N
N
21
20
SO2CH3
SO2CH3
(vi)
SO2CH3
16; R"=NO2
17; R"=NH2
(ii)
BocN
Cl
H
O
N
N
(iv)
N
H
N
O
F
N
N
O
N
N
F
23
22
SO2CH3
SO2CH3
Scheme 3. Preparation of alternative nitrogen containing bicyclic analogues. Reagents and conditions: (i) 4-(methylsulfonyl)aniline (or 2-fluoro analogue), 0 °C, 90%; (ii) EtOAc,
H2, 10% Pd/C, rt, 85%; (iii) C(OEt)3, Ac2O, reflux, 45%; (iv) THF, NaH, 4-hydroxypiperidine carboxylic acid tBu ester, rt 25–60%; (v) AcOH, NaNO2, rt, 25%; (vi) COCl2, 30%.
OEt
O
O
O
OH
O
NO2
N
(i)(ii)
(iii)(iv)
O2N
SO2Me
SO2Me
SO2Me
24
25
26
(v)(vi)
O
R'O
N
OH
N
NH2
O
O
O
N
N
O
O
N
(ix)
(vii)
N
N
H2N
N
SO2Me
SO2Me
SO2Me
29
28
27
Scheme 4. Preparation of isoxazolopyrimidine analogues. Reagents and conditions: (i) (a) COCl2, DCM, DMF 0 °C–rt; (b) phenol, Et3N, 0 °C–rt; (ii) KOtBu, DMSO/CH3NO2,
0 °C–rt; (iii) EtOH, NH2OHꢀHCl, AcOH, reflux; (iv) Et2O, THF; EtOCOCOCl, rt; (v) satd NH4Cl, Zn, rt; (vi) NH4OH (aq), MeOH, THF, rt; (vii) CH(OEt)3, Ac2O, reflux; (viii) POCl3,
i
reflux; (ix) THF, NaH, and either, 4-hydroxypiperidine-1-carboxylic acid tBu ester (to prepare 29a) or 4-hydroxypiperidine-1-carboxylic acid Pr ester (to prepare 29b).
4,6-Dichloro-5-nitro-pyrimidine could be selectively reacted with
4-sulfone substituted anilines to provide 16 in good yield. Reduc-
tion of the nitro group by catalytic hydrogenation provided 17 in
essentially quantitative yield. In practical terms, 17 had to be
reacted immediately in the next step to avoid decomposition but
the construction of three alternative heterocycles was possible
form this starting point. Firstly, reaction with triethyl orthoformate
in the presence of acetic anhydride provided the purine intermedi-

3138
G. Semple et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3134–3141
Table 1
SAR around the pyrazolopyrimidine core
R'
O
O
N
X
N
N
N
N
R
a
b
a
R
X
R0
hGPR119 EC50 (nM)
% Emax
n
rGPR119 EC50 (nM)
n
3a
3b
3c
3d
3e
3f
3g
3h
3i
4-SO2Me
4-SO2Me
4-SO2Me
4-SO2Me
4-SO2Me
4-SO2Me
4-SO2Me
4-SO2Me
4-SO2Me
2-F,4-SO2Me
2-F,4-SO2Me
4-SO2Me
4-SO2Me
4-SO2Me
4-SO2Me
2-F,4-SO2Me
4-SO2Me
2-F,4-SO2Me
4-SO2Me
4-SO2Me
4-SO2Me
2-F,4-SO2Me
O
O
O
O
O
O
O
O
tBu
1.1
3.8
3.5
7.5
96
91
83
79
99
80
71
105
—
79
89
101
87
89
67
82
104
98
—
14
3
6
6
5
13
3
5
3
9
109
3
3
4
7
4
4
7
2
9.8
53
2
3
—
—
—
2
—
3
—
4
18
1
—
—
—
3
Et
iPr
n.d.
n.d.
n.d.
21
n.d.
65
n.d.
19.6
33
>10,000
n.d.
n.d.
n.d.
264
15.5
7.3
nBu
iBu
4.2
cPent
3-THF
CH2tBu
CH2CH2NMe2
tBu
14.6
435
8.1
>10,000
0.9
O
O
O
NH
NHCH2
N(Me)CH2
N(Et)CH2
N(Et)CH2
S
S
SO
SO2
CH2
3j
3k
8a
8b
8c
8d
8e
8f
8g
9a
9b
10a
10b
iPr
2.7
tBu
250
265
11.6
6.3
3.4
5.6
tBu
tBu
tBu
iPr
tBu
3
4
1
2
5
3
tBu
0.5
tBu
2400
>10,000
7.3
>10,000
>10,000
120
88
tBu
—
91
72
6
15
3
tBu
CH2
tBu
3.4
a
Mean EC50 from multiple determinations (n) in melanophores transfected with either the human (hGPR119) or rat (rGPR119) sequence of the receptor.
Emax = apparent maximum efficacy in the melanophore assay when the curve height was normalized to the positive control (AR231453; defined as a full agonist, that is,
b
100% efficacy).
ate 18 which could be reacted with Boc-4-hydroxypiperidine, un-
der similar conditions to those used previously in the pyrazolopyr-
imidine series, to prepare 19. An alternate cyclization of the
diamine 17 could be effected with sodium nitrite under acidic con-
ditions to provide the triazolopyrimidine 20, which again could be
converted to the test compound by reaction with Boc-4-hydroxypi-
peridine. Finally, the ring closure to form a five-membered ring
could also be carried out with phosgene from the fluorinated inter-
mediate (17, R = F) to provide the purinone 23 by way of the inter-
mediate 22 (R = F). Each of these test compounds retained
significant agonist activity at human GPR119 (Table 2), although
19 appeared to be somewhat less potent than either 21 or 23 or in-
deed the parent compound, 3a. We hypothesized at this stage, that
the bicyclic core was essentially interchangeable as long as it could
display the key binding motifs (i.e., the hydrogen bond acceptor of
the sulfone group on the southern aromatic ring and the piperidine
carbamate in the northern portion) in the appropriate positions for
interaction with the receptor. This was later confirmed to some ex-
tent, when it was reported that a very close analogue in which the
bicyclic portion was a dihydropyrrolopyrimidine also retained sig-
nificant activity at the receptor.11 We also suspected however, that
the core itself was making key binding interactions with the recep-
tor. The somewhat reduced activity of the purine 19 suggested to
us that a hydrogen bond acceptor functionality in the 2-position
of the bicyclic ring system, as present in 3a, 21 and 23, was advan-
tageous. This led us to propose the next target for synthesis, the
isoxazolpyrimidine 29a in which such a group was retained. The
synthesis of this core was considerably more complex than for
the other series and is outlined in Scheme 4. The key step in the
preparation of 29a was the acylation and subsequent cyclization
of the oxime, prepared from 25, with ethyl 2-chloro-2-oxoacetate
to provide 26. Reduction of the nitro group and conversion of the
ester to the primary amide provided the intermediate 27 which
could be converted to the bicyclic intermediate 28 with triethyl
orthoformate in a similar manner to the ring closure reaction used
for the pyrazolopyrimidine series. The sequence was completed by
conversion of 28 to the chloro-heterocycle by treatment with POCl3
and finally nucleophilic displacement with the appropriate carba-
mate protected 4-hydroxy piperidine. Again, the new heterocyclic
core template provided compounds that retained significant activ-
ity at the human GPR119 receptor (Table 2).
At this stage, we wanted to show that the non-acid labile com-
pounds (i.e., compounds with carbamates other than Boc on the
piperidine group) that appeared to have activity in the low nano-
molar range or below in our in vitro assay, would show similar
acute activity in the oral glucose tolerance test (oGTT) to the pro-
totypical GPR119 agonist, AR231453. So as to be able to correlate
the in vivo data with receptor activity, we first tested a selected
number of compounds, including several Boc derivatives, using
the rat sequence of GPR119, again using the melanophore plat-
form. In each case there was a significant 3–20-fold rightward shift
in the rat receptor dose–response curves relative to the human
receptor (Tables 1 and 2), in line with what was previously ob-
served for AR231453.7 Indeed, across our whole program in this
area, we have consistently observed such a difference with very
few significant outliers from this trend regardless of the core scaf-
fold. The efficacy of the compounds (measured relative to 1a) was
however, similar between species in this assay platform. Despite

G. Semple et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3134–3141
3139
Table 2
Effect of core changes on GPR119 activity
R'
O
O
N
O
R
SO2Me
a
b
a
Core
R
R0
hGPR119 EC50 (nM)
% Emax
n
rGPR119 EC50 (nM)
n
∗
N
N
3a
H
tBu
1.1
96
14
9.8
42
2
N
N
N
∗
∗
15a
15b
19
H
H
H
H
F
tBu
tBu
tBu
tBu
tBu
tBu
iPr
15
116
33.4
7.4
103
90
5
7
3
4
4
7
7
2
N
N
N
∗
∗
N
n.d.
n.d.
42
—
—
2
N
N
N
∗
∗
N
N
∗
N
N
N
N
N
95
N
∗
N
N
21
103
64
N
N
∗
∗
H
N
23
0.6
n.d.
17
—
5
O
N
∗
N
∗
O
N
29a
29b
H
H
0.7
100
65
N
∗
∗
O
N
14
40
2
N
∗
a
Mean EC50 from multiple determinations (n) in melanophores transfected with either the human (hGPR119) or rat (rGPR119) sequence of the receptor.
Emax = apparent maximum efficacy in the melanophore assay when the curve height was normalized to the positive control (AR231453; defined as a full agonist, that is,
b
100% efficacy).
this apparent species difference in agonist potency, 3k, the most
potent isopropyl carbamate in the pyrazolopyrimidine series and
its isoxazole analogue 29b, were both shown to have in vivo activ-
ity in both the acute mouse and rat oGTT after oral administra-
tion.12 Compound 3k appeared to be somewhat more active in
this assay compared to 29b, with a minimum efficacious dose of
around 3 mg/kg (mouse) or 10 mg/kg (rat, Fig. 2). We speculated
that this difference may be due to differences in compound adsorp-
tion through the gut, based on the observation that in our hands 3k
was more soluble in aqueous solutions then the isoxazolopyrimi-
dine analogue. In any event, this greater in vivo potency coupled
with the significantly simpler route needed to prepare 3k led us
to profile it more extensively to determine whether it could be a
suitable tool for a broader examination of how GPR119 was in-
volved in glucose control both acutely and under more chronic
conditions.
We first confirmed in alternative in vitro platforms that 3k was
a potent and selective agonist of the receptor of interest. Thus, 3k
was shown to increase adenylate cyclase activation in HEK293
cells transfected with human GPR119 (but not in non-transfected

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G. Semple et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3134–3141
Figure 2. Effect of 3k on oral glucose tolerance in c57/bl6 Mouse (Panel A) and Sprague–Dawley rat (Panel B), as well as hyperglycemic clamp in Sprague–Dawley rat (Panel C
& D). Compound 3k markedly reduced blood glucose levels during oral glucose tolerance test in a dose-dependent manner in both mouse and rat. In hyperglycemic clamp, 3k
showed no effect during euglycemic condition, but significantly stimulated insulin release when blood glucose levels were raised to approximately 300 mg/dl.
cells) in a concentration-dependent manner with an EC50 of 23 nM.
Compound 3k also enhanced insulin release from both rat and hu-
man isolated pancreatic islets in a glucose-dependent manner as
previously observed for 1a.1 In a standard panel of around 80
known receptors and ion channels, 3k did not show any binding
in excess of 50% of control to any other proteins at concentrations
mice, rats, dogs, and monkeys (0.8–3.9 h). The data from rat were
used to confirm that good exposure was observed at the doses
shown to have activity in the oGTT. Interestingly, the pharmacoki-
netic profile of 3k in Zucker fa/fa rats was somewhat different from
that in SD rats. After oral administration, the tmax and t1/2 were
longer, and the AUC and the oral bioavailability were greater in
the Zucker compared with the SD rats. Following iv administration,
the Zucker rats also had larger AUC values, longer t1/2 values and
greater Vdss values. In animal models under euglycemic conditions,
3k had no effect on plasma insulin and blood glucose levels at
doses up to 30 mg/kg. However, 3k significantly improved blood
glucose handling during glucose challenge in several diabetic and
non-diabetic rodent models. In addition 3k (0.08 mg/kg/min iv)
showed a clear glucose-dependent effect on insulin release in a
hyperglycemic clamp model in the Sprague–Dawley rat (Fig. 2).13
With these data in hand, we examined the effect of chronic
treatment with 3k, which had not been possible with previous
receptor ligands, in Zucker Diabetic Fatty (ZDF) rats. Compound
3k significantly reduced blood glucose and glycated hemoglobin
(HbA1c) levels over eight weeks of treatment (QD), with no desen-
sitization of the acute drug response over this period at 30 mg/kg
po.14 Taken together, these profiling, pharmacokinetic and phar-
macology data showed for the first time that chronic activation
of the GPR119 receptor with a selective agonist could have a posi-
tive effect on glycemic parameters and at least with 3k, was not
subject to any major concerns regarding tachyphylaxis that has
been associated with prolonged activation of other GPCRs. These
observations, coupled with the acute effect observed in an oGTT
in cynomolgus monkeys (10 mg/kg po),15 led us to progress 3k
(APD668, JNJ-28630368) into clinical studies as a first-in-class
up to 10 lM.
Compound 3k was highly bound to plasma proteins of male and
female cynomolgus monkeys and humans (P99%), but was less
extensively bound to male (93.0%) and female (96.6%) rats. In hu-
man hepatic microsomes, 3k showed no significant inhibition of
any of the five major CYP isoforms with the exception of CYP2C9
(Ki = 0.1 lM). In addition, 3k was a poor substrate of P-glycopro-
tein and was highly permeable across Caco-2 cell monolayers.
Compound 3k was not genotoxic in the in vitro bacterial/micro-
somal activation, mouse lymphoma, or the in vivo mouse micronu-
cleus assays but did show moderate inhibition of the hERG channel
in a patch clamp assay (IC50 = 3 lM).
In pharmacokinetic assessments across multiple species using
single oral doses of 3k, absorption was rapid to moderate
(tmax 62 h) in mice, Sprague–Dawley (SD) rats, and monkeys, but
slower in dogs (tmax = 6 h) and showed a dose-dependent increase
in rats and monkeys. In general, exposure was dose-dependent at
lower doses and appeared to plateau at doses greater than
300 mg/kg. Exposure was greater in female rats compared with
males. Absolute oral bioavailability was moderate to good in mice,
rats, and monkeys (44–79%), but was lower in dogs (22%). The vol-
ume of distribution (Vdss) values were somewhat variable ranging
from 0.1 L/kg in monkey to 2.6 L/kg in rats. Elimination, based on
mean t1/2 after intravenous (iv) dosing, was rapid to moderate in

G. Semple et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3134–3141
3141
120 min, the animals were euthanized.
Rat: Animals are fasted 3–16 h.
GPR119 agonist to examine the effects of modulation of this recep-
tor in humans. The clinical data will be reported elsewhere in due
course.
The oral glucose tolerance test (OGTT) was executed as follows: At time ꢁ30 min,
blood was collected via a tail nick and a glucose test performed with a hand
held glucometer, and compound was then administered. At time 0, blood was
again collected for glucose reading and then a bolus of glucose (3 g/kg po, 6 ml/
kg) administered. Blood glucose levels were further tested at 30, 60, and
120 min post glucose administration. During the entire oGTT, a total volume of
approximately eight drops of blood was collected. Rats were used twice with a
one week lapse between experiments. After the second test, the animals are
euthanized.
References and notes
1. 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. Endocrinology 2007, 148, 2601.
2. 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.
Endocrinology 2008, 149, 2038.
3. 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. Biochem. Biophys. Res. Commun. 2005,
326, 744.
4. 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. Cell Metab. 2006, 3, 167.
5. Brown, A. J. Br. J. Pharm. 2007, 152, 567.
13. Hyperglycemic clamp method: Male SD rats were fasted overnight before the
clamp experiment. After 1 h of baseline blood glucose and plasma insulin level
data collection, vehicle or compound was infused into the jugular vein for 2 h.
GLP-1 or glybenclamide were infused in different groups of rats as the positive
control. Blood glucose levels were maintained at 100 mg/dl for the first and
second hour of clamp study. For the third hour of clamp study, the blood
glucose levels were increased to, and maintained, at 300 mg/dl via a bolus of
30% glucose injection followed by constant glucose infusion with various
infusion rates.
14. Male ZDF rats (6 weeks old, baseline body weight of 200–250 g) were divided
into 3 treatment groups based upon fed body weight and blood glucose levels.
Blood glucose and HbA1c levels were determined on Days 1, 31, and 56 of
vehicle (100% PEG400) or 3k treatment. Blood glucose and HbA1c levels in rats
treated with 3k at 30 mg/kg/day were significantly decreased, thus these rats
did not develop diabetes, whereas, the vehicle treated rats did.
6. (a) Fu, J.; Gaetani, S.; Oveisi, F.; Verme, J. L.; Serrano, A.; Rodríguez de Fonseca,
F.; Rosengarth, A.; Luecke, H.; Di Giacomo, B.; Tarzia, G.; Piomelli, D. Nature
2003, 425, 90; (b) Ahern, G. P. J. Biol. Chem. 2003, 278, 30429.
7. Semple, G.; Fioravanti, B.; Pereira, G.; Calderon, I.; Uy, J.; Choi, K.; Xiong, Y.; Ren,
A.; Morgan, M.; Dave, V.; Thomsen, W.; Unett, D. J.; Xing, C.; Bossie, S.; Carroll,
C.; Chu, Z.; Leonard, J.; Jones, R. M. J. Med. Chem. 2008, 51, 5172.
8. Hodge, C. N.; Aldrich, P. E.; Wasserman, Z. R.; Fernandez, C. H.; Arvanitis, A.;
Chorvat, R. J.; Cheeseman, R. S.; Christos, T. E.; Scholfield, E.; Krenitsky, P.;
Gilligan, P. J.; Ciganek, E.; Strucely, P. J. Med. Chem. 1999, 42, 819.
9. Jones, R. M. Abstracts of Papers, 239th ACS National Meeting, San Francisco, CA,
Mar 21–25, 2010; MEDI-316.
Effect of chronic 56-day administration of 3k on blood glucose and hba1c levels in ZDF
rats
10. Jones, R. M.; Semple, G.; Xiong, Y.; Shin, Y.-J.; Ren, A. S.; Calderon, I.; Fioravanti,
B.; Choi, J. S. K.; Sage, C. R. PCT Int. Appl. WO/2005007658, 2005.
11. Carpenter, A. J.; Katamreddy, S.; Peckham, G. E.; Caldwell, R.; Ammala, C.;
Croom, D.; Bullard, S.; McNulty, J.; Nystrom, C. Abstracts of Papers, 239th ACS
National Meeting, San Francisco, CA, Mar 21–25, 2010; MEDI-32.
12. Animals were grouped (rats 2/cage, mice 4/cage) in regular cages with bedding
under normal light conditions (lights on 6:30 am–6:30 pm). They were given
ad libitum access to food and water. Animals were allowed to acclimate to the
facility for several days before handling.
Blood
HbA1c (%)
Day 1
Body
weight
gain (g)
glucose
(mg/dL,
Day 56)
Day 31
Day 56
Vehicle, (100%
PEG400) (n = 5)
3k, 10 mg/kg (n = 6)
3k, 30 mg/kg (n = 7)
408.0 46.4 3.6 0.2 7.3 0.4 7.4 0.8 206.8 15.4
359.3 49.3 3.5 0.1 7.3 0.6 8.2 1.0 224.9 4.1
105.2 4.9a 3.4 0.1 5.2 0.1a 4.7 0.1a 204.2 3.5
Procedures:
Mouse: Following two handling sessions, animals were fasted for 3–16 h.
The oral glucose tolerance test (OGTT) was executed as follows: Compound was
administered 0–30 min prior to first blood sample. At time 0, a tail nick and
glucose test was performed with a hand held glucometer, then a bolus of
glucose (2 mg/kg, po) was administered. Glucose was again tested at 20, 40, 60,
and 120 min post glucose administration. During the entire oGTT, a total
volume of approximately 10 drops of blood was collected. After the sample at
Data represent the mean standard error of eight values.
HbA1c = glycated hemoglobin; n = number of subjects.
a
Statistically different compared with that in vehicle treated group (T-test, P < 0.05).
15. Liang, Y.; Leonard, J. N. unpublished data.