Design and synthesis of 1H-pyrazolo[3,4-c]pyridine derivatives as a novel structural class of potent GPR119 agonists

Article abstract of DOI:10.1016/j.bmcl.2016.06.050

Design and synthesis of a novel class of 1H-pyrazolo[3,4-c]pyridine GPR119 receptor agonists are described. Lead compound 4 was identified through the ligand-based drug design approach. Modification of the left-hand aryl group (R¹) and right-ha

Full text of DOI:10.1016/j.bmcl.2016.06.050

Accepted Manuscript
Design and synthesis of 1H-pyrazolo[3,4-c]pyridine derivatives as a novel
structural class of potent GPR119 agonists
Daisuke Matsuda, Yohei Kobashi, Ayako Mikami, Madoka Kawamura,
Fumiyasu Shiozawa, Kenichi Kawabe, Makoto Hamada, Koji Oda, Shinichi
Nishimoto, Kayo Kimura, Masako Miyoshi, Noriko Takayama, Hiroyuki
Kakinuma, Norikazu Ohtake
PII:
S0960-894X(16)30661-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.06.050
BMCL 24003
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 March 2016
14 June 2016
18 June 2016
Please cite this article as: Matsuda, D., Kobashi, Y., Mikami, A., Kawamura, M., Shiozawa, F., Kawabe, K., Hamada,
M., Oda, K., Nishimoto, S., Kimura, K., Miyoshi, M., Takayama, N., Kakinuma, H., Ohtake, N., Design and
synthesis of 1H-pyrazolo[3,4-c]pyridine derivatives as a novel structural class of potent GPR119 agonists,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.06.050
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Design and synthesis of 1H-pyrazolo[3,4-c]pyridine derivatives as a novel structural class of
potent GPR119 agonists
a
Daisuke Matsuda a,*, Yohei Kobashi a, Ayako Mikami , Madoka Kawamura a, Fumiyasu Shiozawa a,
a
a
a
b
c
Kenichi Kawabe , Makoto Hamada , Koji Oda , Shinichi Nishimoto , Kayo Kimura , Masako
Miyoshi b, Noriko Takayama b, Hiroyuki Kakinuma a, Norikazu Ohtake a.
a Chemistry Laboratories, Taisho Pharmaceutical Co., Ltd. ; 1–403 Yoshino-cho, Kita-ku, Saitama
331–9530, Japan.
b Pharmacology Laboratories, Taisho Pharmaceutical Co., Ltd. ; 1–403 Yoshino-cho, Kita-ku,
Saitama 331–9530, Japan.
c Research Support, Taisho Pharmaceutical Co., Ltd.; 1–403 Yoshino-cho, Kita-ku, Saitama
331–9530, Japan.
Abstract:
Design and synthesis of a novel class of 1H-pyrazolo[3,4- c]pyridine
GPR119 receptor agonists are described. Lead compound 4 was identified
through the ligand-based drug design approach. Modification of the
left-hand aryl group (R1) and right-hand piperidine N-capping group(R2)
ledtotheidentificationofcompound24asasingle-digitnanomolarGPR119
agonist.
Keywords: Type 2 diabetes mellitus; GPR119 agonist; G-protein coupled receptor; pyrazolopyridine.
Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease characterized by hyperglycemia
due to impaired insulin secretion and insulin resistance. Long-term hyperglycemia is a major risk
factor for diabetic complications such as nephropathy, retinopathy, and neuropathy. The number of
people with T2DM worldwide is more than 300 million, and the prevalence is rapidly increasing.1,2
Despite the availability of multiple antidiabetic agents, a large number of patients fail to achieve the
desired glycemic control level.3 Therefore, there remains a significant need for the development of
new medical treatments for T2DM.
GPR119 is a G-protein coupled receptor (GPCR) that is predominantly expressed in the pancreatic
β-cells and gastrointestinal L-cells. Activation of the GPR119 receptor increases the cellular cAMP
1

levels, leading to glucose-dependent insulin secretion from the pancreatic β-cells.4 In addition,
activation of the GPR119 receptor in the gut results in the release of incretins, such as glucagon-like
peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), from the
enteroendocrine cells.5 GLP-1 and GIP stimulate insulin secretion from the β-cells in a
glucose-dependent manner and protect the β-cells against apoptosis.6,7 This glucose-dependent dual
mechanism of action suggests that GPR119 agonists can improve glycemic control without the risk
of inducing hypoglycemia.
Oleoyl-lysophosphatidylcholine and oleoylethanolamide (OEA) have been identified as the
endogenous agonists for the GPR119 receptor.8,9 Many research groups have investigated
small-molecule GPR119 agonists,10,11 which has led to the development of several clinical
compounds such as APD668,12 GSK1292263,13 MBX-298214 (Figure 1).
Figure 1. Representative GPR119 agonists.
We envisaged that most GPR119 agonists have a common structural feature: the
methylsulfonyl-subsituted-phenyl group linked to a piperidine carbamate group (or its isostere) via a
central spacer (Scheme 1).
In our medicinal chemistry program, one of the objectives was to identify an orally active small
molecule GPR119 agonist with a novel structure. Therefore, we first pursued a highly potent
GPR119 agonist, assuming that the orientation of the methylsulfonylphenyl group and piperidine
carbamate group would have an impact on the agonist potency, and we designed compounds
focusing on conformationally restricted central spacers as the first step.15-18
Evaluation of the designed spacers was conducted based on a ligand-based pharmacophore model.
Pharmacophore generation was carried out with the compounds (1, 2, 3) using the “Common Feature
Pharmacophore Generation” protocol in Discovery Studio (BIOVIA).19 Conformations of the
compounds were generated by the “FAST” method, and pharmacophore hypotheses were created
with the default parameters of the protocol, with the exception of feature selection;
hydrogen-acceptor, hydrophobic, and ring-aromatic features were selected for the generation. As
shown in Figure 2, the compounds (1, 2, 3) overlapped closely and a six-feature hypothesis was
created. Various fused rings, such as indole, tetrahydroquinoline, tetrahydroisoquinoline, isoindoline,
2

were designed as rigid spacers (Scheme 1). Among them, the indole derivative A fit well to the
pharmacophore model (Figure 3). Since the lipophilicity of compound A itself was relatively high
(ClogP = 3.99), we successively designed several more hydrophilic compounds with the same 6-5
fused ring system containing two or more nitrogen atoms based on superposition of the compounds
(1, 2, 3 and A). The designed compounds as shown in Table 1 were synthesized and evaluated for
hGPR119 agonism20, which led to the identification of the novel 1H-pyrazolo[3,4-c]pyridine
derivative 4 having an EC50 value of 42 nM.
Scheme 1. Design of the 1H-pyrazolo[3,4-c]pyridine derivative
Table 1
SAR of the 6-5 fused ring spacers
hGPR119
Compound
R
X
N
Y
ClogPa
EC50 (nM)
42
iPr
CH
2.21
4
tBu
tBu
tBu
N
CH
CH
N
20
31
63
2.60
3.43
2.86
5
6
7
CH
CH
3

a The ClogP value was calculated using a software from Daylight
Chemical Information Systems, Inc.
Figure 2. Superposition of compound 1 (carbon atoms are colored purple), 2 (orange), 3 (yellow) and a
generated pharmacophore model. The pharmacophore model consists of two hydrogen bond acceptors
(green arrows and spheres), two hydrophobic sites (cyan spheres) and two aromatic rings (orange arrows
and spheres).
Figure 3. Mapping of compound A (gray) onto the pharmacophore model.
Herein, we describe the synthesis and structure-activity relationships (SARs) of this new series of
1H-pyrazolo[3,4-c]pyridine derivatives, focusing on improving the potency without increasing the
lipophilicity.
This
series
of
compounds
was
synthesized
from
commercially
available
2-bromo-4-methyl-5-nitropyridine 26 (Schemes 2-5). Reduction of 26 with hydrogen in the presence
of a catalytic amount of Raney-Ni yielded 27 (93% yield). Cyclization of 27 with NaNO2 under
acidic conditions led to the formation of 28 (59% yield). Treatment of 28 with tert-butyl
4-(methylsulfonyloxy)piperidine-1-carboxylate, followed by separation of the mixture of
regioisomers by silica-gel column chromatography provided the key intermediate 29 (36% yield).
Removal of the Boc group from 29 followed by treatment with isopropyl chloroformate or
4

2-chloro-5-ethylpyrimidine yielded 30a and 30b, respectively. Suzuki coupling of 30a or 30b with
an appropriate aryl boronic acid yielded the desired products 10, 11 and 24 (Scheme 3, a-b).
Compounds 4 and 18-23 were prepared from 29 via Suzuki coupling, deprotection and the
N-capping reaction (Scheme 3, b-c). Amide compounds 12-17 and 25 were prepared by Suzuki
coupling of 30a or 30b, followed by condensation with an appropriate amine using
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride
(EDCI·HCl)
and
1,2,3-benzotriazol-1-ol monohydrate (HOBt·H2O) (Scheme 4).
Scheme 2. (a) H2, Raney-Ni, THF, rt; (b) NaNO2, AcOH, rt; (c) Cs2CO3, tert-butyl
4-(methylsulfonyloxy)piperidine-1-carboxylate, DMF, 80 ˚C.
Scheme 3. (a) (i) HCl, MeOH, 1,4-dioxane, rt ; (ii) isopropyl chloroformate, DIPEA, CHCl3, rt or
2-chloro-5-ethylpyrimidine, Cs2CO3, DMSO, 180 ˚C; (b) for 5, 24, ArB(OH)2, PdCl2(dppf)·CH2Cl2,
Na2CO3, H2O, DMF, 100 ˚C; for 10, 11, 31, ArB(OH)2, Pd(PPh3)4, Na2CO3, H2O, EtOH, microwave, 160
˚C; (c) (1) HCl, MeOH, 1,4-dioxane, rt; (2) for 4, 18, alkyl chloroformate, Et3N, CHCl3, rt; for 19, 23,
1-{[(1,1-difluoro-2-methylpropan-2-yl)oxy]carbonyl}-3-methyl-1H-imidazol-3-ium iodide, Et3N, CHCl3,
rt; for 22, iPnBr, K2CO3, CH3CN, reflux; for 20, 2-chloro-5-ethylpyrimidine, Cs2CO3, DMSO, microwave,
180 ˚C; for 21, 2-bromo-5-methylpyridine, Cs2CO3, DMSO, 180 ˚C.
5

Scheme 4. (a) ArB(OH)2, Pd(PPh3)4, Na2CO3, H2O, EtOH, microwave, 160 ˚C; (b) R6R7NH, EDCI·HCl,
HOBt·H2O, Et3N, DMF, rt.
Compounds 8 and 9 were synthesized as shown in Scheme 5. Copper-mediated Ullmann-type
coupling of 29 with 4-(methylsulfonyl)phenol using a catalytic amount of CuI and picolinic acid
yielded 33 (42% yield). Deprotection of the Boc group followed by treatment with isopropyl
chloroformate provided the desired compound 8. Compound 9 was prepared from 30a under similar
conditions using the copper catalyst described above.
Scheme 5. (a) 4-(methylsulfonyl)phenol, CuI, picolinic acid, K3PO4, DMSO, 105 ˚C; (b) (i) HCl, MeOH,
1,4-dioxane, rt; (ii) isopropyl chloroformate, Et3N, CHCl3, rt; (c) 3-(methylsulfonyl)phenol, CuI, picolinic
acid, K3PO4, DMSO, 100 ˚C.
The synthesized compounds were evaluated for their GPR119 agonist potency by using a cAMP
assay in a human GPR119 cell line.20
Initially, we conducted an SAR study focusing on the substituent (R1) at the 5-position of the
pyrazolopyridine ring (Table 2) of the representative compound 4. In this study, we attempted to
improve the potency of the compound. However, a high lipophilicity of the compound may cause
poor solubility and ADME properties. Therefore, we monitored the LipE value (LipE = pEC50
-
ClogP)21,22 to balance the potency and lipophilicity. Insertion of an oxygen atom between the
6

4-(methylsulfonyl)phenyl moiety and pyrazolopyridine ring of 4 led to a decrease in the potency.
This result suggested that an increase in the flexibility of the R1 substituent lowered the potency as
we
expected.
Replacement
of
the
4-(methylsulfonyl)phenyl
moiety
with
a
2-(methylsulfonyl)pyridin-5-yl one (10) resulted in a decrease of the agonist potency. Meanwhile,
installation of a fluorine atom into the 2-position of the 4-(methylsulfonyl)phenyl group yielded
compound 11,23,24 which showed comparable potency while maintaining the LipE value, as
compared to 4. Subsequently, further modifications of this R1 substituent were conducted.
Replacement of the methylsulfonyl group in 11 with a carbamoyl group (12) resulted in a 3-fold
decrease of the potency. Installment of an additional fluorine atom (13) into the R1 moiety of 12 was
carried out, because the meta-fluoro-substituted aryl group is known as a potent left-hand component,
24
which improved the potency by twofold with an improved LipE value (4.89). The carbamoyl
moiety of 13 was replaced with dimethylcarbamoyl (14), ethylcarbamoyl (15), propylcarbamoyl (16),
and 2-hydroxyethylcarbamoyl (17) moieties to evaluate their effect on the agonist potency. The
dimethylcarbamoyl derivative (14) was equipotent to the carbamoyl derivative (13). By contrast,
ethylcarbamoyl (15), and propylcarbamoyl (16) yielded significant improvement in potency,
although the LipE value of 16 was lower than that of the compounds 13 and 15, due to its high
lipophilicity. The 2-hydroxyethylcarbamoyl (17) showed a 9-fold decrease of the potency, while a
high LipE value (4.95) was maintained, due to its low lipophilicity. We selected the
2-fluoro-4-methylsulfonyl-phenyl group (11), which conferred high potency with a good LipE value,
and the 2,5-difluoro-4-((2-hydroxyethyl)carbamoyl)phenyl group (17), which conferred low
lipophilicity with a good LipE value, as promising R1 groups for the combination study described
below (Table 4).
7

Table 2
SAR of the pyrazolopyridine substituents
hGPR119
Compound
R1
ClogPa LipEb
EC50 (nM)
42
2.21
2.12
5.17
4.63
4
8
177
266
197
27
2.12
2.09
2.42
4.46
4.61
5.15
9
10
11
97
52
2.63
2.39
4.39
4.89
12
13
46
2.62
4.71
14
10
14
92
3.18
3.71
2.09
4.82
4.15
4.95
15
16
17
a The ClogP value was calculated using a software from
Daylight Chemical Information Systems, Inc.
b LipE = pEC50 - CLogP
8

Next, we clarified the effect of the piperidine N-substituent (R2) of 4 on the agonist potency (Table
3). In this study, we simultaneously investigated the relationship between the R2 group and the
human microsomal stability of the compounds. Therefore, we took into account not only the potency,
but also the metabolic stability. Replacement of the iso-propylcarbamate of 4 with an iso-pentyl
moiety yielded 22, which was significantly less potent than 4. Replacement of the
iso-propylcarbamate with a tert-butylcarbamate (5) or an iso-butylcarbamate (18) improved the
potency as compared with that of 4. However, compound 18 was unstable in the human liver
microsomal assay, probably due to the lability of the iso-butylcarbamate moiety. To prevent the
metabolism without loss of potency, fluorine atoms were introduced into the alkyl group of the
carbamate. Fluorinated tert-butylcarbamate (19) exhibited an increase in potency as compared to 4
and showed great stability in the human liver microsomes as expected. It is likely that replacement
of the carbamate moiety in 4 with a carbamate isostere such as pyridine or pyrimidine, would
maintain the high agonist potency. In fact, 5-ethylpyrimidine (20) resulted in a 3-fold increase in
potency as compared to 4, and 5-methylpyridine (21) also conferred good potency. However,
improvement of the metabolic stability of these compounds was not obtained. The above rough SAR
study on the agonist potency suggested that a hydrogen bond acceptor at the R2 group was important
for obtaining a high potency. We selected a fluorinated tert-butylcarbamate group (19), which
conferred metabolic stability and 5-ethylpyrimidine (20), which conferred high potency as a
promising R2 group for the combination study described in Table 4.
9

Table 3
SAR of the piperidine substituents
Compound
R2
hGPR119 EC50 (nM)
42
ClogPa
LipEb
5.17
hMS(%)c
24.1
2.21
4
20
11
2.60
2.82
2.65
2.03
2.27
3.10
5.09
5.13
4.99
5.85
5.25
3.55
NTd
79.8
3.9
5
18
19
20
21
22
23
13
20.3
44.1
NTd
30
220
a The ClogP value was calculated using a software from Daylight Chemical Information Systems, Inc.
b LipE = pEC50 - CLogP
c % Metaborized after 15-min incubation with human liver microsomes (1 mg protein/mL).
d Not tested.
Finally, we optimized the 1H-pyrazolo[3,4-c]pyridine derivatives by a combination of the selected
left-hand aryl groups and right-hand piperidine N-capping groups (Table 4). The compound 23
showed a higher potency and better metabolic stability than 4, as expected. However, its aqueous
solubility was extremely poor. The compound (24), containing a combination of a
4-methylsulfonyl-2-fluorophenyl group (a left-hand substituent) with a 5-ethylpyrimidin-2-yl group
(a right-hand substituent), exhibited the highest agonist potency (hGPR119 EC50 = 4 nM) among this
class of compounds. The 2-hydroxyethylcarbamoyl derivative (25) possessed an EC50 value of 15
nM, but was metabolically unstable.
10

Table 4
Combinations of promissing components
hGPR119
EC50 (nM)
Solubility
(μg/ml)b
Compound
Structure
hMS(%)a
24.1
ClogPc
LipEd
5.17
42
10
4
0.67
<0.06
0.34
2.21
4
2.1
2.87
2.25
5.15
6.12
23
24
14.3
15
35.8
0.69
1.91
5.92
25
a % Metabolized after 15-min incubation with human liver microsomes (1 mg protein/mL).
b Thermodynamic solubility in pH6.8 phosphate buffer.
c The ClogP value was calculated using a software from Daylight Chemical Information Systems, Inc.
d LipE = pEC50 - CLogP
Given the promising potency along with metabolic stability, we conducted a pharmacokinetic study
of compound 24 (TP0456330) in SD rats. The PK parameters are shown in Table 5. Unfortunately,
this compound showed poor bioavailability, although it had a low systemic clearance and a low
volume of distribution. It exhibited high membrane permeability (112 × 10-6 cm/s at pH6.2) in the
PAMPA assay. These results suggested that the low bioavailability of 24 may be due to its low
aqueous solubility. To identify compounds for in vivo efficacy studies, further improvement of the
aqueous solubility would be required.
Table 5
Pharmacokinetic parameters of 24 in SD rats
IV (3 mg/kg)
Vdss
PO (10 mg/kg)
AUC0-∞
CL
t1/2
(h)
Compound
%F
(mL/h/kg) (mL/kg)
(ng · h/mL)
759 537
0.617
1930
12.6
24
11

In summary, we identified 1H-pyrazolo[3,4-c]pyridine derivatives as a novel structural class of
GPR 119 agonists using a ligand based pharmacophore model. To identify a highly potent compound
in this series, we conducted an exploratory SAR study, which led to the identification of compound
24 (TP0456330) having the most potent agonism of hGPR119 (EC50 = 4 nM). However, compound
24 exhibited low bioavailability in rat, probably due to its poor solubility. Further optimization of the
1H-pyrazolo[3,4-c]pyridine series will be reported in due course.
Acknowledgements
The authors thank Yuki Shimizu for the contribution to the synthesis of the compounds, Naoto
Ohsaki for collecting the solubility data, and Satoshi Wakabayashi for collecting the ADME data.
The authors also thank Dr. Hiroshi Kawamoto for his help in preparing this manuscript.
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Graphical abstract
14