Optimization of oxadiazole derivatives with a spirocyclic cyclohexane structure as novel GPR119 agonists

Article abstract of DOI:10.1016/j.bmcl.2019.07.004

We describe here a novel GPR119 agonist 24, which showed a potent and long-acting hypoglycemic effect in rats via oral dosing. For the discovery of 24, we chose compound 5, which possessed an oxadiazole linker, as a lead compound among our spirocyclic cyclohexane GPR119 agonist series, taking into account its lower plasma protein binding nature. 3,5-Difluoro and 4-methylsulfonylmethy groups on the left side phenyl group, and a gem-difluoro group on the right side of 24 are important for its agonist potency and metabolic stability, respectively.

Full text of DOI:10.1016/j.bmcl.2019.07.004

Accepted Manuscript
Optimization of oxadiazole derivatives with a spirocyclic cyclohexane structure
as novel GPR119 agonists
Kazuhito Harada, Jun Mizukami, Takashi Watanabe, Genki Mori, Minoru
Ubukata, Katsunori Suwa, Sumiaki Fukuda, Tamotsu Negoro, Motohide Sato,
Takashi Inaba
PII:
S0960-894X(19)30450-0
https://doi.org/10.1016/j.bmcl.2019.07.004
BMCL 26545
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 April 2019
28 June 2019
2 July 2019
Please cite this article as: Harada, K., Mizukami, J., Watanabe, T., Mori, G., Ubukata, M., Suwa, K., Fukuda, S.,
Negoro, T., Sato, M., Inaba, T., Optimization of oxadiazole derivatives with a spirocyclic cyclohexane structure as
novel GPR119 agonists, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.
2019.07.004
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Optimization of oxadiazole derivatives with a spirocyclic cyclohexane structure as novel
GPR119 agonists
Kazuhito Harada, Jun Mizukami, Takashi Watanabe, Genki Mori, Minoru Ubukata, Katsunori Suwa,
Sumiaki Fukuda, Tamotsu Negoro, Motohide Sato, Takashi Inaba
Central Pharmaceutical Research Institute, Japan Tobacco Inc., 1-1, Murasaki-cho, Takatsuki,
Osaka 569-1125, Japan
Abstract:
We describe here a novel GPR119 agonist 24, which showed a potent and long-acting
hypoglycemic effect in rats via oral dosing. For the discovery of 24, we chose compound 5, which
possessed an oxadiazole linker, as a lead compound among our spirocyclic cyclohexane GPR119
agonist series, taking into account its lower plasma protein binding nature. 3,5-Difluoro and 4-
methylsulfonylmethy groups on the left side phenyl group, and a gem-difluoro group on the right
side of 24 are important for its agonist potency and metabolic stability, respectively.
Keywords:
GPR119 agonist
Spirocyclic
GPR119 is a G protein-coupled receptor (GPCR) expressed predominantly in pancreatic beta cells
and enteroendocrine cells.1 Activation of GPR119 promotes glucagon-like peptide-1 (GLP-1)
secretion in the intestinal tract and mediates glucose-stimulated insulin secretion (GSIS) in the
pancreas. Therefore, it is expected that GPR119 agonists have potential for the treatment of type 2
diabetes with low-risk of hypoglycemia.
To date, a number of synthetic GPR119 agonists have been reported, several of which have advanced
into clinical trials.2 Many reported GPR119 agonists are composed of three distinct parts as depicted
in Figure 1: a piperidine or a piperazine ring N-substituted with a carbamate or a heteroaryl group at
the right side; a phenyl group substituted with a methylsulfonyl or a heteroaryl group (X) at the left
side; and a linker part connecting the two.2,3 The N-substituted piperidine or piperazine ring as the
right side moiety is a characteristic structure of many of GPR119 agonists.

Right side
R
Linker
Left side
N
Y
Y= CH or N
X
O
O
S
O
N
O
N
N
N
O
N
N
N
O
O
S
N
H
O
N
O
N
N
N
N
N
O
F
S
O
O
N
F
MBX-2982
PSN119-2
Arena's compound
Figure 1. Typical structure and representatives of GPR119 agonists.
As shown in Figure 2, we have recently found that a spirocyclic cyclohexane structure in
compound 2 is a novel right side part of GPR119 agonist 14 and the structure is a good alternative to
N-carbamate (heteroaryl group) piperidine compatible with various left side-linker structures of
previously reported GPR119 agonists including 4-anilinopyrimidine left side-linker as shown in
compound 3.5 Moreover, optimization of 3 with a pyrimidine linker gave 4, which exhibited
excellent pharmacokinetic profiles and a hypoglycemic effect after 16 hours of oral administration in
rats (at dosing of 10 mg/kg).
O
N
O
O
N
1
O
GPR119 EC50 (IA)
CLogP
10 nM (102%)
3.9
O
S
O
O
O
O
S
O
O
N
N
N
N
O
N
N
N
O
H
H
F
N
F
4
2
4 nM (104%)
5.1
3
O
13 nM (91%)
4.0
19 nM (120%)
3.7
Figure 2. Our reported GPR119 agonists with a spirocyclic cyclohexane structure. The results
expressed the potency as EC50 values and the inherent activity (IA) as percentages which were
compared to the test compound with oleoylethanolamide (OEA), an endogenous ligand of GPR119
(defined as 100% activation). The ClogP value was calculated using software from ChemAxon.

The objective of this work was to develop a new GPR119 agonist showing a long-acting
hypoglycemic effect at a lower dose. Our strategy was to reduce plasma protein binding to gain
higher free drug plasma concentration and thus to increase in vivo potency. We therefore sought a
new lead compound exhibiting low binding to plasma proteins with the anticipation of obtaining a
compound with the desired profiles after optimization efforts.
Binding to plasma proteins by the three lead candidates 3, 5 and 6, which was described in our
previous work,5 were evaluated and compared (Figure 3).
O
O
O
O
O
S
N
N
O
S
O
N
N
N
N
O
N
N
N
O
H
O
S
N
O
F
F
3
5
6
GPR119 EC50 (IA)
CLogP
13 nM (91%)
4.0
89 nM (109%)
3.4
85 nM (79%)
3.7
plasma protein binding
human / rat
98.3% / 98.1%
96.7% / 97.7%
98.6% / 98.5%
Figure 3. Plasma protein binding of derivatives with a spirocyclic cyclohexane structure.
Compound 5 having an oxadiazole linker showed lower binding to both human and rat plasma
proteins compared with pyrimidine derivative 3 and thiazole derivative 6. Despite its lower agonistic
activity (EC50 = 89 nM) compared to the others, we considered that 5 would be worth optimizing and
selected this compound as a new lead compound, appreciating its lower protein binding nature. Our
initial optimization efforts focused on the improvement of agonistic activity of 5 while maintaining or
reducing the plasma protein binding. In general, the binding to plasma proteins has been positively
correlated with lipophilicity, so we took care not to increase values of CLogP in our SAR studies.

F
R
a, b, c
N
X
O
HO
O
N
25 33
–
7 24
–
O
O
O
F
F
HO
HO
HO
HO
HO
HO
25
26
28
31
O
O
O
O
F
F
HO
HO
32
29
F
F
O
O
HO
33
27
30
Scheme 1. Reagents and conditions: (a) bromoacetic acid or 2-bromopropanoic acid, NaH, dioxane,
rt to 90 °C, 30–91%; (b) the corresponding N-hydroxyl amidine, EDC, HOBt, i-Pr2NEt, DMF, rt; (c)
toluene, reflux, 12–69% (2 steps).
The general syntheses of compounds 7–24 are shown in Scheme 1. Reactions of spiro alcohols 25–
33 with bromoacetic acid or 2-bromopropanonic acid, and subsequent condensation of the resulting
carboxylic acids with the corresponding N-hydroxyl amidines yielded the final products 7–24.
O
OH
O
O
O
O
HO
a
b
c, d
e
O
O
O
HO
O
O
O
O
34
35
36
37
26
f, g, h, i, j, k
l, m, n, o
HO
HO
O
O
p
q
Bn
Bn
Bn
O
HO
O
O
39
29
38
40
Scheme 2. Reagents and conditions: (a) methyl isobutyrate, LDA, THF, -10 °C to rt, 97%; (b)
LiAlH4, THF, 0 °C, 97%; (c) MeSO2Cl, pyridine, 0 °C to rt, (d) AcOH, H2O, 100 °C, 90% (2 steps);
(e) NaBH4, MeOH, rt, then separation by column chromatography on silica gel, 36%; (f) AcOH,
H2O, 100 °C, 90%; (g) BnBr, NaH, THF, 0 °C to rt, 56%; (h) LiAlH4, THF, 0 °C; (i) Ac2O, pyridine,
N,N-dimethylaminopyridine, CHCl3, 0 °C; (j) SOCl2, pyridine, rt; (k) 4N NaOH, THF, MeOH, rt,
89% (4 steps); (l) Dess-Martin reagent, CHCl3, 0 °C, 87%; (m)

methoxymethyltriphenylphosphonium chloride, KO-t-Bu, THF, rt; (n) PPTS, THF, rt; (o) NaBH4,
MeOH, rt, 18% (3 steps); (p) Amberlyst-15, toluene, 90 °C, then separation by column
chromatography on silica gel, 32%; (q) H2 (15 psi), Pd(OH)2-C, MeOH, rt, 99%.
Spiro alcohol 25 was obtained by the procedure cited in our previous report.4 As shown in Scheme
2, both spiro[4.5] and [5.5] alcohols (26 and 29) were synthesized via spiro[4.5] lactone 35 which was
obtained from commercially available epoxide 34. For the preparation of 26, reduction of 35 with
LiAlH4 to diol 36, and the following reaction with MeSO2Cl and heating under acidic conditions
yielded spiro[4.5] ketone 37. Reduction of 37 with NaBH4 and subsequent separation of the resulting
mixture of stereoisomers by column chromatography on silica gel gave the desired alcohol 26. For the
preparation of 29, multiple steps from 35 to alcohol 38 and following a one-carbon elongation led to
alcohol 39. Treatment of 39 with Amberlyst-15 and isolation of the desired stereoisomer by column
chromatography on silica gel gave spiro ether 40. The benzyl group of 40 was removed by
hydrogenation in the presence of Pd(OH)2-C to yield alcohol 29.
OH
HO
MeO2C
O
O
c, d, e, f
g
a, b
h
O
O
O
Bn
O
HO
Bn
O
O
43
28
41
42
44
Scheme 3. Reagents and conditions: (a) triphosgene, pyridine, 0 °C; (b) methyl isobutyrate, LDA,
THF, -10 °C to rt, 65% (2 steps); (c) AcOH, H2O, 100 °C; (d) NaBH4, MeOH, rt; (e) BnBr, NaH,
THF, 0 °C to rt; (f) LiAlH4, THF, 0 °C, 27% (4 steps); (g) BF3·OEt2, CHCl3, rt; (h) H2 (15 psi), Pd-C,
MeOH, rt, then separation by column chromatography on silica gel, 30% (2 steps).
Synthesis of spiro[5.5] alcohol 28 is shown in Scheme 3. Chlorination of commercially available
allyl alcohol 41 and coupling reaction of thus obtained allyl chloride with the lithium enolate generated
from methyl isobutyrate afforded ester 42, which was then converted to intermediate 43 via several
steps. Alcohol 43 was treated with BF3·OEt2 to give spiro[5.5] ether 44, whose benzyl protection was
then removed to afford a mixture of stereoisomers containing desired 28. Separation by column
chromatography on silica gel successfully provided the desired alcohol 28.

TBDMS
O
HO
O
O
a
b
c
d
O
Bn
Bn
O
Bn
Bn
O
HO
O
O
45
46
47
48
27
g
HO
EtO2C
O
O
e
f
c
d
Bn
Bn
O
Bn
Bn
O
HO
O
O
52
49
50
51
30
h
OH
O
O
O
O
F
F
i
j, k
l
d
O
F
F
Bn
Bn
Bn
Bn
O
O
O
O
HO
53
54
55
56
31
Scheme 4. Reagents and conditions: (a) 3-((t-butyldimethylsilyl)oxypropyl)triphenylphosphonium
bromide, which was readily prepared from (3-bromopropoxy)-tert-butyldimethylsilane), NaH, DMF,
0 °C, 72%; (b) 1 M TBAF in THF, THF, rt, 88%; (c) Amberlyst-15, toluene, 90 °C, then separation
by column chromatography on silica gel, 40–52%; (d) H2 (15 psi), Pd(OH)2-C, MeOH, rt, 97–99%;
(e) 3-(ethoxycarbonyl)propyltriphenylphosphonium bromide, KO-t-Bu, THF, 0 °C to rt, 74%; (f)
LiAlH4, THF, 0 °C, 95%; (g) methyltriphenylphosphonium bromide, KO-t-Bu, THF, 0 °C, 65%; (h)
m-CPBA, NaHCO3, CHCl3, 0 °C, then separation by column chromatography on silica gel, 24%; (i)
1 M vinylmagnesium bromide in THF, THF, 0 °C to rt, 79%; (j) NaIO4, t-BuOH, H2O, 50 °C, 35%;
(k) Dess-Martin reagent, CHCl3, 0 °C, 95%; (l) bis(2-methoxyethyl)aminosulfur trifluoride, CH2Cl2,
rt, 71%.
Both unsubstituted spiro[4.5] and [5.5] alcohols (27 and 30), and difluorinated spiro[4.5] alcohol 31
were synthesized from commercially available ketone 45 (Scheme 4). For the preparation of 27 and
30, ketone 45 was converted to alcohols 47 and 50 via introduction of three- and four-carbon units
with the corresponding phosphonium ylides, respectively. Treatment of 47 and 50 with Amberlyst-15
furnished ethers of spiro[4.5] and spiro[5.5] systems, respectively. The resulting mixtures of
stereoisomers were separated by column chromatography on silica gel to give the desired spiro ethers
48 and 51. For the preparation of 31, olefination of 45 to 52 and subsequent oxidation with m-CPBA
gave epoxide 53 as a mixture of stereoisomers. Chromatographically purified 53 was treated with vinyl
magnesium bromide to obtain olefin 54, which was converted to spiro[4.5] ketone 55 by stepwise
oxidation via the corresponding hydroxyl spiro ether. Treatment of 55 with bis(2-methoxyethyl)
aminosulfur trifluoride afforded difluoride 56. The benzyl groups of 48, 51 and 56 were removed by

hydrogenation in the presence of Pd(OH)2-C to yield spiro alcohols 27, 30 and 31, respectively.
O
O
O
O
F
F
O
a, b
c
d
e, f
O
F
F
O
O
O
O
O
O
O
O
HO
O
57
58
60
32
59
Scheme 5. Reagents and conditions: (a) trans-3-(tert-butyldimethylsilyloxy)-N,N-dimethyl-1,3-
butadiene-1-amine, 2-BuOH, rt; (b) AcCl, Et2O, -78 °C, 63% (2 steps); (c) H2 (15 psi), Pd-C, MeOH,
rt, 69%; (d) bis(2-methoxyethyl)aminosulfur trifluoride, CHCl3, rt, 69%; (e) AcOH, H2O, 100 °C,
71%; (f) NaBH4, MeOH, 0 °C, then separation by flash chromatography on silica gel, 85%.
Difluorinated spiro[5.5] alcohol 32 was synthesized from commercially available ketone 57 (Scheme
5). Hetero-Diels-Alder reaction of 57 with trans-3-(tert-butyldimethylsilyloxy)-N,N-dimethyl-1,3-
butadiene-1-amine and subsequent treatment with acetyl chloride gave dispiro enone 58.6
Hydrogenation of 58 and subsequent difluorination of thus obtained ketone 59 afforded difluoride 60,
whose ketal protection was removed and the resultant ketone was reduced with NaBH4, to provide a
mixture of stereoisomers containing desired 32. Purification of 32 was achieved by column
chromatography on silica gel.
O
S
O
S
F
F
F
F
O
S
F
F
F
O
O
O
HO
NH
a, b
c
d
CN
Br
Br
HN OH
F
61
62
63
64
Scheme 6. Reagents and conditions: (a) MeSO2Cl, NEt3, EtOAc, 0 °C; (b) LiBr, MeSO2Na, DMF, rt
to 80 °C, 92% (2 steps); (c) K4[Fe(CN)6]·3H2O, Pd(OAc)2, Na2CO3, DMA, 130 °C, 73%; (d)
NH2OH·HCl, K2CO3, EtOH, H2O, 90 °C, 75%.
F
F
F
F
O
Br
O
OH
a, b, c
d, e
f
g, h
Bn
Bn
Bn
O
O
Bn
O
O
45
65
66
67
F
F
O
F
F
O
O
F
F
F
F
O
S
O
O
i
j
k, l
F
N
O
HO
O
O
HO
N
O
O
F
68
33
69
24
Scheme 7. Reagents and conditions: (a) triethyl phosphonoacetate, EtONa, EtOH, THF, 0 °C to rt,

92%; (b) diisobutylaluminum hydride, toluene, -78 °C, 93%; (c) NBS, PPh3, THF, DMF, 0 °C to rt,
25%; (d) ethyl bromodifluoroacetate, Zn, CuCN, THF, rt, 17%; (e) LiAlH4, THF, 0 °C, 92%; (f) p-
TsOH·H2O, toluene, 100 °C, 80%; (g) H2 (15 psi), 20% Pd(OH)2-C, MeOH, rt, 85%; (h) 1-Me-
AZADO, NaClO, KBr, tetrabutylammonium bromide, NaHCO3, CHCl3, H2O, 0 °C, 93%; (i)
LiAlH4, THF, 0 °C, 90%; (j) (S)-(-)-2-bromopropanonic acid, NaH, dioxane, rt, 67%; (k) 64, EDC,
HOBt, i-Pr2NEt, DMF, rt; (l) NMP, reflux, 27%.
Synthesis of compound 24 is shown in Scheme 6 and 7. Commercially available benzyl alcohol 61
via the intermidiates 62 and 63 was converted to the corresponding N-hydroxyl amidine 64 (Scheme
6). For the preparation of the other counterpart difluorinated spiro[5.5] alcohol 33, coupling reaction
of ethyl bromodifluoroacetate with bromide 65 obtained from commercially available ketone 45 in the
presence of zinc powder and copper cyanide, and following reduction of thus obtained ester gave gem-
difluoro alcohol 66. Heating of 66 under acidic conditions afforded spiro[5.5] ether 67, and whose
benzyl protection was removed and the regenerated hydroxyl group was oxidized to give ketone 68.
Stereoselective reduction (98:2 d.r.) of ketone 68 with LiAlH4 gave the desired alcohol 33. Reaction
of 33 with (S)-(-)-2-bromopropanoic acid and following condensation with 64 afforded 24 (Scheme
7).
To investigate the left side and the linker parts starting from the lead compound 5, compounds 7–
12 were initially synthesized and evaluated for their activity on the human GPR119 receptor in a cell-
based cAMP assay (Table 1).7
Table 1. SAR of oxadiazole derivatives with a spirocyclic cyclohexane structure.
O
R
N
X
O
O
N
F
GPR119
EC50 (nM)
89
IAa
(%)
Compound
X
R
CLogPb
5
MeSO2
Me (rac)
H
109
3.4
7
8
MeSO2
261
103
86
2.9
3.8
Me2NCO
Me (rac)
2,457

9
Me2NCOCH2
MeSO2CH2
MeSO2CH2
MeSO2CH2
Me (rac)
Me (rac)
Me (S)
69
44
131
130
106
106
3.9
3.3
3.3
3.3
10
11
12
430
27
Me (R)
a Percent of maximum response of oleoylethanolamide (OEA).
b The ClogP value was calculated using software from ChemAxon.
Removal of the methyl group on the linker of 5 resulted in 3-fold loss of agonistic activity
(compound 7). Conversion of the left side structure to a dimethylcarbamoyl group, known as one of
the alternative structures of the methylsulfonyl group,4,8 resulted in a significant decrease of agonistic
activity (compound 8). On the other hand, the agonistic activity of phenylacetamide 9, being one
carbon unit longer than 8, was dramatically improved compared with 8. This result inspired us to
prepare benzyl methyl sulfone 10 having one more carbon unit than 5. As anticipated, 10 showed an
about 2-fold improvement in agonistic activity compared to 5. These results suggested that the
arrangement of a hydrogen bond acceptor2 at the left side had a great influence on the agonistic activity.
It was also noteworthy that the CLogP value of 10 was comparable to that of 5. The R-isomer 12 was
found to be 15 times more potent (EC50 = 27 nM) than the S-isomer 11.
We next explored the effect of a F substituent at the benzene ring of compound 10 since introduction
of a F atom at this position reportedly boosted agonistic activity in some known GPR119 agonists.2 In
addition, installation of F atoms was considered to have little impact on lipophilicity and was
anticipated to barely influence plasma protein binding. The effects of F atom(s) toward agonistic
activity are shown in Table 2.
Table 2. Effects of F atom(s) toward GPR119 agonistic activity.
O
O
O
F
R
5
S
6
N
O
O
N
3
2
GPR119
IAa
Compound
Position of F
R
CLogPb
EC50 (nM)
(%)

10
13
12
3-F
none
3-F
Me (rac)
Me (rac)
Me (R)
44
426
27
130
120
106
3.3
3.1
3.3
14
15
16
2-F, 3-F
3-F, 6-F
3-F, 5-F
Me (R)
Me (R)
Me (R)
301
64
5
77
118
109
3.4
3.4
3.4
a Percent of maximum response of oleoylethanolamide (OEA).
b The ClogP value was calculated using software from ChemAxon.
The agonistic activity of compound 13, with no F atom at the 3-position, decreased about ten times
compared with 10 suggesting that the F atom at the 3-position in 10 played a vital role for potent
agonistic activity. Addition of F atom to the 2- or 6-position also decreased agonistic activity (14, 15
vs 12). On the other hand, installation of an additional F atom at the 5-position boosted the agonistic
activity significantly and the thus-obtained 16 showed an EC50 value of 5 nM.
The values of lipophilicity and plasma protein binding of 16 were equivalent to those of lead
compound 5 (for 16, CLogP = 3.4, hPPB (plasma protein binding in human) = 96.5%, rPPB (plasma
protein binding in rat) = 94.0%).
In liver microsomal stability studies, 16 showed moderate metabolic stability (percentages of
remaining after 1 hour incubation with human liver microsomes and with rat liver microsomes were
65% and 83%, respectively).
To gain insights toward the improvement of metabolic stability, we next investigated the spirocyclic
cyclohexane structures at the right side of the molecule using the structure of compound 10 as a
platform (Table 3).
Table 3. Exploration of spirocyclic cyclohexane structures.
O
S
O
N
O
O
N
F
Compound
Right side part
GPR119
IAa
CLogPb
hMsc
rMsd

EC50 (nM)
(%)
(%)
(%)
O
10
17
18
19
20
21
44
31
130
115
109
113
114
112
3.3
3.4
3.8
3.7
2.6
3.0
62
15
0
86
55
56
53
54
64
*
O
*
O
25
*
*
O
29
0
O
720
86
49
39
*
O
*
a Percent of maximum response of oleoylethanolamide (OEA).
b The CLogP value was calculated using software from ChemAxon.
c Percent of compound (5 μM) remaining after 1 hour incubation with human liver microsomes
(0.2 mg/mL).
d Percent of compound (5 μM) remaining after 1 hour incubation with rat liver microsomes (0.2
mg/mL).
We synthesized compounds 17–19 to examine the position of the ether oxygen atom and the gem-
dimethyl group, and the ring size in the spirocyclic cyclohexane structure. Whereas agonistic activities
of compounds 17–19 were comparable to 10, the metabolic stabilities of compounds 17–19 were
greatly diminished by these transformations, which was especially remarkable for human liver
microsomes. These results indicated that the exact position of the ether oxygen atom and the gem-
dimethyl group, and the ring size were quite important structural determinants for metabolic stability.
In addition to these factors, removal of the gem-dimethyl group improved metabolic stability for
human liver microsomes in spite of attenuation of agonistic activity (20 vs 17, 21 vs 18, 19). These
results strongly suggested that the gem-dimethyl group might be one of the metabolic sites.
Therefore, our final optimization efforts were focused on improvement of metabolic stability by
tuning the right side of 16 while maintaining its highly potent agonistic activity (Table 4).

Table 4. Final optimization of oxadiazole derivatives with a spirocyclic cyclohexane structure.
O
F
S
O
N
O
O
N
F
GPR119
IAa
CLogPb
hMsc
(%)
rMsd
(%)
Compound
Right side part
EC50 (nM)
(%)
O
16
22
23
24
5
7
109
116
106
112
3.4
3.1
3.3
3.3
65
83
97
98
83
85
91
98
*
O
F
F
*
O
F
F
27
6
*
F
O
F
*
a Percent of maximum response of oleoylethanolamide (OEA).
b The CLogP value was calculated using software from ChemAxon.
c Percent of compound (5 μM) remaining after 1 hour incubation with human liver microsomes
(0.2 mg/mL).
d
Percent of compound (5 μM) remaining after 1 hour incubation with rat liver microsomes (0.2
mg/mL).
We expected conversion of the gem-dimethyl group to the gem-difluoro group on the spirocyclic
cyclohexane structures would improve the metabolic stability.9 Indeed, compound 22, a gem-difluoro
counter pair of 16, showed higher human metabolic stability than 16 while its agonistic activity was
comparable to that of 16. In addition, 22 showed lower lipophilicity and plasma protein binding than
16 (for 22, CLogP = 3.1, hPPB = 93.4%, rPPB = 91.0%). These results inspired us to examine gem-
difluoro derivatives of spiro[5.5] system 23 and 24, and these were found to be fairly stable against
both human and rat microsomes. Especially, 24 was as potent as 16, showing an EC50 value of 6 nM
with lower plasma protein binding (hPPB = 92.5%, rPPB = 91.4%) than 4 (hPPB = 95.2%, rPPB =
96.2%) and was therefore expected to exhibit good in vivo activity.
The pharmacokinetic profiles of 24 were evaluated in Sprague Dawley rats (Table 5). Compound
24 exhibited a durable profile and its oral bioavailability was 43%. In addition, 24 showed no
inhibitory activity (IC50 > 50 μM) against all tested six CYP isoforms (CYP 3A4, 2D6, 1A2, 2C9,
2C8, 2C19).

Table 5. Phamacokinetic profiles of 24 in Sprague-Dawley rats.
Rat IV
Rat PO
(1 mg/kg)
(3 mg/kg)
t1/2β
CL
Vdss
MRT
(h)
BA
(%)
(h)
(L/h/kg)
(L/kg)
5.1
0.5
3.5
6.4
43
We finally performed intraperitoneal glucose tolerance tests (ipGTT) in Sprague-Dawley rats to
investigate in vivo activity of compound 24 (Figure 4).10 Glucose was intraperitoneally administrated
16 hours after the oral administration of 24. After administration of glucose, the concentration of
plasma glucose up to 2 hours and the concentration of plasma insulin up to 1 hour were measured. As
expected, 24 suppressed the increase in plasma glucose levels with insulin secretion after glucose
loading while showing no effect on plasma glucose and insulin levels before glucose loading. The area
under the curve (AUC) of the plasma glucose levels were decreased by 10% and 12% when 24 was
dosed at levels of 1 mg/kg and 3 mg/kg, respectively.
Figure 4. Effect of 24 on plasma glucose and insulin levels in ipGTT in rats.
In summary, we have identified a highly potent GPR119 agonist 24, which possesses an isoxazole
linker and a spirocyclic cyclohexane structure at the right side, from the lead compound 5 and using
plasma protein binding as an index for an in vivo potency predictor. Compound 24 showed an ED50 of

6 nM as well as excellent metabolic stability that was achieved by introduction of a gem-difluoro group
on the right side part of the molecule. Compound 24 also displayed long-lasting and orally active
pharmacokinetics profiles with no CYP inhibitory activity, and exhibited a hypoglycemic effect with
insulin secretion dependent on glucose concentration after 16 hours of oral administration in rats at 1
mg/kg.
Acknowledgments:
We thank Dr. Hisashi Shinkai for helpful discussions. We also appreciate Dr. Hiromasa Hashimoto
for suggestions on writing this manuscript.
References and notes:
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Balanced Salt Solution (Invitrogen), 20 mM HEPES, 0.1% bovine serum albumin, 0.1 mM 3-
isobutyl-1-methylxanthine, and 0.1 mM 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone)
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determined as the concentration of the test compound required to achieve 50% of the maximal
oleoylethanolamide (OEA) stimulated response.

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10. We conducted ipGTT in place of oral glucose tolerance test (oGTT) to evaluate the inherent
glucose lowering effect of the compound without nonspecific effects, such as gastric emptying
inhibitory action. The ipGTT was performed in the rats (7 weeks of age) after overnight fasting.
Compounds were administrated by oral gavage. After 16 hours, glucose (1 g/kg of body weight)
was administrated intraperitoneally to rats and blood samples were collected from the tail vein
prior to and at 10, 30 and 60 minutes after glucose loading. Plasma glucose levels were measured
using an automatic analyzer (Hitachi7170S, Tokyo, Japan). Plasma insulin levels were
measured with a rat-insulin ELISA kit (Morinaga Institute of Biological Science, Yokohama,
Japan).