Synthesis and structure-activity relationship of fused-pyrimidine derivatives as a series of novel GPR119 agonists

Article abstract of DOI:10.1016/j.bmc.2012.08.054

A series of fused-pyrimidine derivatives have been discovered as potent and orally active GPR119 agonists. A combination of the fused-pyrimidine structure and 4-chloro-2,5-difluorophenyl group provided the 5,7-dihydrothieno[3,4-d] pyrimidine 6,6-dioxide derivative 14a as a highly potent GPR119 agonist. Further optimization of the amino group at the 4-position in the pyrimidine ring led to the identification of 2-{1-[2-(4-chloro-2,5-difluorophenyl)-6,6-dioxido-5,7- dihydrothieno[3,4-d]pyrimidin-4-yl]piperidin-4-yl}acetamide (16b) as an advanced analog. Compound 16b was found to have extremely potent agonistic activity and improved glucose tolerance at 0.1 mg/kg po in mice. We consider compound 16b and its analogs to have clear utility in exploring the practicality of GPR119 agonists as potential therapeutic agents for the treatment of type 2 diabetes mellitus.

Full text of DOI:10.1016/j.bmc.2012.08.054

Bioorganic & Medicinal Chemistry 20 (2012) 6442–6451
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and structure–activity relationship of fused-pyrimidine derivatives
as a series of novel GPR119 agonists
⇑
Kenji Negoro , Yasuhiro Yonetoku, Ayako Moritomo, Masahiko Hayakawa, Kazuhiko Iikubo,
Shigeru Yoshida, Makoto Takeuchi, Mitsuaki Ohta
Drug Discovery Research, Astellas Pharma Inc., 21 Miyukigaoka, Tsukuba, Ibaraki 305-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of fused-pyrimidine derivatives have been discovered as potent and orally active GPR119 ago-
nists. A combination of the fused-pyrimidine structure and 4-chloro-2,5-difluorophenyl group provided
the 5,7-dihydrothieno[3,4-d]pyrimidine 6,6-dioxide derivative 14a as a highly potent GPR119 agonist.
Further optimization of the amino group at the 4-position in the pyrimidine ring led to the identification
of 2-{1-[2-(4-chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-dihydrothieno[3,4-d]pyrimidin-4-yl]piperidin-
4-yl}acetamide (16b) as an advanced analog. Compound 16b was found to have extremely potent agonis-
tic activity and improved glucose tolerance at 0.1 mg/kg po in mice. We consider compound 16b and its
analogs to have clear utility in exploring the practicality of GPR119 agonists as potential therapeutic
agents for the treatment of type 2 diabetes mellitus.
Received 23 July 2012
Revised 22 August 2012
Accepted 22 August 2012
Available online 7 September 2012
Keywords:
Type 2 diabetes mellitus
Glucose-dependent insulin secretion
G-protein-coupled receptor 119
GPR119 agonist
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
orphan GPCRs have been paired with lysophospholipids, bile acids,
arachidonic acid metabolites, dioleoyl phosphatidic acid, and
The onset and progression of type 2 diabetes mellitus (T2DM)
are due to the depletion of glucose-dependent insulin secretion
(GDIS) from pancreatic b cells.1 Oral agents that stimulate insulin
secretion, such as sulfonylureas (SUs), reduce blood glucose and
have been used as a first-line T2DM therapy for nearly 30 years.2
However, these agents act to force b cells to secrete insulin contin-
uously regardless of blood glucose levels, and thereby tend to pro-
mote hypoglycemia and accelerate the loss of islet function and,
eventually, diminish their efficacy.3 Despite the availability of a
range of agents for T2DM, many diabetic patients fail to achieve
or to maintain glycemic targets.4 In addition, stricter glycemic
guidelines have been proposed to define a direction for diabetes
prevention through the identification and treatment of the pre-dia-
betes state.5 Agents that can promote GDIS have great potential to
replace SUs as first-line therapy for treating T2DM. The recent
emergence of glucagon-like peptide 1 (GLP-1)-based GDIS agents,6
including inhibitors of dipeptidyl peptidase-47 and peptidase-sta-
ble analogs such as exendin-4,8 is undoubtedly a major advance
in this direction. Nevertheless, it remains to be confirmed whether
the effects of GLP-1-related agents on b cells mass and function are
truly durable and beneficial.
short-, medium-, and long-chain free fatty acids.9 From these dis-
coveries, GPR40, GPR119, and GPR120 have been reported to play
a role in regulating GDIS and to therefore have potential as novel
targets for the treatment of type 2 diabetes.10 GPR119, which is
abundantly expressed in pancreatic b cells and the gastrointestinal
tract, was identified as a receptor for lysophospholipids and certain
ethanolamide derivatives of long-chain fatty acids, such as lyso-
phosphatidylcholine and oleoylethanolamide.11 Activation of
GPR119 directly promotes GDIS and indirectly increases GLP-1 le-
vel through the upregulation of intracellular cAMP followed by a
reduction in blood glucose level,12 suggesting that GPR119 may
present an attractive drug target for treating T2DM, and that its
agonists may represent potential new insulin secretagogues with-
out the risk of hypoglycemia. Thus, considerable effort has been in-
vested in exploring GPR119 agonists as novel antidiabetic drugs,13
such as AR231453 (1; Fig. 1) or PSN632408 (2), which were re-
ported to show both insulinotropic and antiobesity effects. Clinical
trials of several other GPR119 agonists are currently ongoing.13
Our continued interest in GPR119 as a drug target for the treat-
ment of T2DM prompted an investigation into novel GPR119 ago-
nists. In the course of structure–activity relationship (SAR) studies
around our lead compound 5,14,15 we found that introduction of a
2,4,5-trihalogenatedphenyl group at the 2-position in the pyrimi-
dine ring markedly improved GPR119 agonistic activity and dis-
covered the pyrimidine derivative 6 as a potent GPR119 agonist.
In addition, compound 6 was found to improve not only glucose
tolerance in ICR mice but also blood glucose, plasma insulin,
The molecular pharmacology of lipid and lipid-like mediators
that signal through G protein-coupled receptors (GPCRs) has
expanded significantly over the past few years. To date, several
⇑
Corresponding author. Tel.: +81 (0)29 847 6631; fax: +81 (0)29 847 1519.
E-mail address: kenji.negoro@astellas.com (K. Negoro).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.054

K. Negoro et al. / Bioorg. Med. Chem. 20 (2012) 6442–6451
6443
O O
S
Me
N
N
N O
O
Me
Me
O
N
H
N
N
N
N
Me
Me
F
NO2
Me
N
O
O
1, AR231453
2, PSN632408
N
N
Me
O
S
N
O
Me
O
S
N
N
N
N
N
N
N
N
N
O
O
N
Me
Et
3, MBX-2982
4, GSK1292263
Me
Et
N
N
N
F
N
F
OH
N
N
H
N
H
OH
N
N
OH
O
Br
Cl
5
6
7
Figure 1. Structures of representative GPR119 agonists AR231453 (1), PSN632408 (2), MBX-2982 (3), GSK1292263 (4), 5, 6 and 7.
triglyceride levels and pancreatic insulin content in diabetic kk/Ay
Table 1
mice.15 Separately to these findings, we also identified another lead
In vitro SARs for analogs with replacement of the cyclopentane ring in compound 6
compound 7, which had a cyclopentane fused-pyrimidine struc-
ture and showed moderate in vitro GPR119 agonistic activity with
an EC value of 9.1 lM. From these discoveries, we then designed
R
N
novel fused-pyrimidine derivatives using a combination of the
structure of compounds 6 and 7. In this paper, we describe the syn-
thesis and SAR studies of these fused-pyrimidine derivatives as no-
vel GPR119 agonists.
F
N
N
O
Cl
F
GPR119/pCRE
ECa (nM)
R
IAb (%)
Compound
2. Chemistry
Synthesis of the 4-morpholino-fused-pyrimidine derivatives
12a–f listed in Table 1 is shown in Scheme 1. Condensation of
4-chloro-2,5-difluorobenzamidine 815 with the corresponding cyc-
lic b-keto ester 9a–f16 followed by chlorination using phosphorous
oxychloride afforded the 4-chloro-fused-pyrimidines 11a–f. These
compounds 11a–f were then subjected to substitution with mor-
pholine to obtain the 4-morpholino-fused-pyrimidine derivatives
12a–f.
Synthesis of the 5,7-dihyd.rothieno[3,4-d]pyrimidine 6,6-
dioxide derivatives 14a–f, 15a and 16a–d listed in Table 2 is
shown in Scheme 2. Oxidation of the 5,7-dihydrothieno[3,4-d]
pyrimidine derivative 11d with m-chloroperbenzoic acid (m-CPBA),
followed by substitution with the corresponding amines gave the
4-substituted 6,6-dioxide derivatives 14a–j. Hydrolysis of the ethyl
esters 14g–j afforded the carboxylic acid derivatives 15a–d, and
amidation of compound 15a–d with ammonium chloride or
methylamine provided the amide derivatives 16a–d.
12a
12b
350
770
405
313
O
S
12c
12d
4100
150
205
467
S
12e
12f
970
57
258
426
S
O
S
3. Results and discussion
O
14a
28
668
The synthesized compounds were evaluated for their agonistic
activities toward GPR119 using a cAMP reporter assay system in
which HEK293 cells were transfected with human GPR119 and
cAMP-responsive element (pCRE)-luciferase expression plas-
mids.17 In this assay system, we relatively evaluated our com-
pounds by comparing their EC and IA values with those of lead
compound 5. EC value refers to the concentration of a tested
compound in this assay system at which they showed as potent
a
Concentration of tested compounds showing equipotent efficacy to that of 5 at
lM in human recombinant cell-based assay. See Section 5.
10
b
Relative efficacy of tested compounds at 10
lM compared to efficacy of 5 at
10
lM. See Section 5.
activity (%) of the tested compounds compared to the efficacy of
compound 5 at 10
M in the same assay system.15a
efficacy as compound 5 at 10
lM. IA value refers to the relative
l

6444
K. Negoro et al. / Bioorg. Med. Chem. 20 (2012) 6442–6451
NH
NH2
a or b
c
N
F
N
F
F
+
O
F
F
N
Cl
N
H
O
CO2R
9a-f
Cl
F
Cl
Cl
8
10a-f
11a-f
d
N
F
F
N
N
O
Cl
12a-f
Scheme 1. Synthesis of the fused-pyrimidine derivatives 12a–f. Reagents and conditions: (a) MeOH, reflux; (b) NaOMe, MeOH, rt to 50 °C; (c) POCl3, reflux; (d) morpholine,
MeCN, rt.
Table 2
In vitro SARs for analogs with replacement of the 4-morpholino group in compound 14a
O
O
S
N
F
Cl
N
R
F
Compound
–R
GPR119/pCRE
OGTT/ICR mice
% Decreasec (1 mg/kg po)
Solubility
ECa (nM)
IAb (%)
MEDd (mg/kg po)
1
pH 6.8e
(lM)
N
14a
14b
28
668
30
32
<1
O
N
N
14
575
528
1
91
OH
NTf
15a
560
>3
NTf
(15 at 3 mg/kg po)
CO2H
N
14c
16a
14d
21
51
294
457
292
12
3
3
1
40
11
CONH2
N
N
NEg
CONHMe
CONMe2
350
32
29
>100
N
N
14e
16b
3.9
8.3
501
483
0.3
0.1
16
15
OH
35
CONH2
(27 at 0.1 mg/kg po)
N
N
14f
2.8
506
29
1
23
OH
NTf
16c
16d
18
44
627
737
0.3
3
8
(30 at 0.3 mg/kg po)
CONH2
N
17
>100
N
CONH2
a,b
See the corresponding footnotes to Table 1.
c
Antihyperglycemic effects of tested compounds in male ICR mice at 1 mg/kg po. See Section 5.
Minimum effective dose (MED) of tested compounds.
Solubility of tested compounds in the buffer solution of pH 6.8. See Section 5.
Not tested.
d
e
f
g
Not effective at 1 mg/kg po.

K. Negoro et al. / Bioorg. Med. Chem. 20 (2012) 6442–6451
6445
O
O
S
O
S
S
O
a
b
N
F
N
F
N
F
F
N
Cl
F
F
N
Cl
N
N
Q
Cl
Cl
Cl
11d
13
14a-j
O
S
O
S
O
O
c
d
N
N
F
F
(Q = X(CH) CO2Et)
N
N
N
N
O
2 n
X
CO2H
X
R
( )n
( )n
N
H
Cl
F
Cl
F
15a-d
16a-d
Scheme 2. Synthesis of the 5,7-dihydrothieno[3,4-d]pyrimidine 6,6-dioxide derivatives. Reagents and conditions: (a) m-CPBA, CHCl3, rt; (b) amine, i-Pr2NEt, MeCN, 70 °C; (c)
1 M NaOH, EtOH, THF, rt; (d) NH4Cl (R = H) or 1 M MeNH2/THF (R = Me), Et3N, EDACÁHCl, HOBt, DMF, rt.
We first designed and synthesized several fused-pyrimidine
derivatives. As shown in Table 1, the cyclopentane fused-pyrimi-
dine derivative 12a showed agonistic activity comparable to that
of compound 6, indicating that introduction of 4-chloro and 2,5-di-
fluoro substituents into the phenyl moiety of the fused-pyrimidine
derivative 7 was effective in improving GPR119 agonistic activity.
Ring expansion of 12a (compound 12b) resulted in reduced agonis-
tic activity, suggesting that the five-membered ring was preferable
for agonistic activity compared to the six-membered ring. Replace-
ment of the methylene linker at the 6-position in compound 12a
with the ether linker (compound 12c) led to a 10-fold loss of activ-
ity compared to 12a. In contrast, a two-fold increase in activity was
observed for the thioether derivative 12d. These results suggested
that the introduction of a hetero atom on the fused-ring was toler-
ated, and that the hydrophobicity in this region was particularly
favorable for agonistic activity. The 5,6-dihydrothieno[2,3-
d]pyrimidine derivative 12e was found to be less potent than the
5,7-dihydrothieno[3,4-d]pyrimidine derivative 12d; however, the
6,7-dihydrothieno[3,2-d]pyrimidine derivative 12f showed potent
activity, with an EC value in the subnano molar range, indicating
that introduction of a sulfur atom at the 5- or 6-position might
be preferable to that at the 7-position. Oxidation of the thioether
linker in compound 12d (compound 14a; EC = 28 nM) resulted in
a five-fold improvement of GPR119 agonistic activity over com-
pound 12d, suggesting that the 5,7-dihydrothieno[3,4-d]pyrimi-
dine 6,6-dioxide structure might be suitable for showing potent
agonistic activity.18 Meanwhile, the low solubility in water of com-
group. In addition, compound 14b was found to have dramatically
improved water solubility over compound 14a. Replacement of the
hydroxy group in compound 14b with a carboxyl group (com-
pound 15a) led to 40-fold reduction in agonistic activity compared
to compound 14b, indicating that introduction of the highly acidic
group in this region was unfavorable for agonistic activity. The car-
bamoyl derivative 14c was found to be equipotent to the hydroxy
derivative 14b and to have improved water solubility over com-
pound 14a. The N-methylated analog 16a was slightly less potent
than the carbamoyl derivative 14c and the N,N-dimethylated ana-
log 14d showed approximately 15-fold less potent activity com-
pared to compound 14c, suggesting that hydrogen-bond donation
in this region was important for showing potent agonistic activity.
One carbon elongated analogs of 14b and 14c (compound 14e and
16b) showed extremely potent agonistic activity with EC values in
the nano molar range, which were approximately three-fold more
potent than the corresponding non-elongated analogs 14b and 14c,
respectively. Two carbon elongated analogs of 14b (compound 14f)
was found to show slightly more potent agonistic activity than the
corresponding one carbon elongated analog 14e. In contrast, two
carbon elongated analogs of 14c (compound 16c) was found to
show two-fold less potent agonistic activity than the correspond-
ing one carbon elongated analog 16b. These results indicated that
the introduction of a hydrogen-bond donating group to the two-
carbon-chain length away from the 4-position of the piperidine
ring might be most preferable for agonistic activity. Replacement
of the piperidine ring in compound 16b with the piperazine ring
(compound 16d) resulted in an improvement in water solubility,
but also led to a five-fold reduction in agonistic activity compared
to compound 16b. These results revealed that the piperidine ring
was more favorable than the piperazine ring as a cyclic amine at
the 4-position in the pyrimidine ring for GPR119 agonistic activity.
We then evaluated the antihyperglycemic effects of the all com-
pounds listed in Table 2 via the oral glucose tolerance test (OGTT)
in male ICR mice by measuring the blood glucose-lowering ratio
(%) at 30 min after glucose loading in comparison with animals
in the vehicle group. In each evaluation dose of the tested com-
pounds, minimum effective dose (MED) was defined as the lowest
dose showing a greater than 20% decrease in blood glucose-lower-
ing ratio. The morpholino (14a) and hydroxypiperidino (14b, 14e
and 14f) derivatives demonstrated excellent potency in vivo at
1 mg/kg po, whereas the carboxypiperidino derivative 15a did
not show a significant glucose-lowering effect at 3 mg/kg po. The
carbamoyl derivatives 14c, 14d and 16a–d all demonstrated anti-
hyperglycemic effects at a dose of 3 mg/kg po or lower. In particu-
lar, the 4-carbamoylmethylpiperidino derivative 16b was found to
show the lowest MED among the compounds listed in Table 2, with
pound 14a (<1 lM in pH 6.8 buffer solution) highlighted the need
to improve the physicochemical properties of this compound. Nev-
ertheless, we selected the 5,7-dihydrothieno[3,4-d]pyrimidine 6,6-
dioxide derivative 14a as a template for further investigation.
Having identified a favorable scaffold which showed highly po-
tent GPR119 agonistic activity, we then shifted our focus to optimi-
zation of the morpholine moiety of compound 14a to improve
water solubility and agonistic activity. As described in our previous
reports,15 introduction of the oxy functional groups onto the amino
group at the 4-position in the pyrimidine ring was effective in
inducing potent GPR119 agonistic activity. These groups could be
considered to influence the water solubility of the compounds.
Therefore, replacement of the morpholino group in compound
14a with other cyclic amines which have oxy functional groups,
such as alcohols, amides and carboxylic acids, was examined (Ta-
ble 2). Compound 14b, which had a 4-hydroxypiperidino group,
showed a two-fold improvement in in vitro agonistic activity over
the morpholino derivative 14a, implying that introduction of a
hydrogen-bond donating group in this region was favorable for
agonistic activity compared to that of a hydrogen-bond accepting

6446
K. Negoro et al. / Bioorg. Med. Chem. 20 (2012) 6442–6451
a MED of 0.1 mg/kg. We speculated that compound 16b might
show an excellent in vivo antihyperglycemic effect as a result of
both its potent GPR119 agonistic activity and good pharmacoki-
netic profile. In addition, compound 16b was found to have low
clearance in human liver microsomes (58 mL/min/kg) and no
appreciable inhibition of the CYP450 enzyme isoforms tested up
number of different interactions (Fig. 2). In this docking model,
the 4-chloro-2,5-difluorophenyl group extends toward the hydro-
phobic region sandwiched between Pro140 (TM4) and Met178,
Val182 (TM5), and the chloro group at the 4-position of the phenyl
moiety forms a halogen bond20 with Pro144 (TM4). These results
might explain why the 4-chloro-2,5-difluorophenyl group in-
creased activity. The sulfone linker in the fused-ring moiety of
compound 16b forms hydrophobic interactions with Val85
(TM3), Trp265 (TM7) and Phe241 (TM6), suggesting that the sul-
fone linker would be recognized as the hydrophobic element.20
In addition, the amino group at the 4-position in the pyrimidine
ring extends to the narrow space formed by TM5, TM6 and TM3,
and the carbamoyl moiety forms a hydrogen-bond with Cys186
(TM5). This information also afforded the opportunity to investi-
gate the effect of the substituent in the amino group.
to concentrations of 50 lM.
A homology model of hGPR119 based on an agonist-bound
hA2AAR X-ray structure (PDB code 3QAK)19 was used to study the
putative interactions between compound 16b and GPR119. A dock-
ing study of 16b to the GPR119 homology model suggested that
16b exerts its GPR119 agonistic activity by effectively utilizing a
4. Conclusion
A series of fused-pyrimidine derivatives have been prepared
and evaluated in order to create potent and orally active GPR119
agonists. A combination of the cyclopentane ring fused-pyrimidine
structure and 4-chloro-2,5-difluorophenyl group provided a potent
GPR119 agonist 12a with an original scaffold. Optimization of the
fused-pyrimidine moiety in compound 12a led to identification
of the 5,7-dihydrothieno[3,4-d]pyrimidine 6,6-dioxide derivative
14a and an approximately 10-fold improvement in GPR119 agonis-
tic activity. Subsequent replacement of the amino group at the
4-position in the pyrimidine ring to improve activity and water
solubility led to the identification of 2-{1-[2-(4-chloro-2,5-dif-
luorophenyl)-6,6-dioxido-5,7-dihydrothieno[3,4-d]pyrimidin-4-yl]
piperidin-4-yl}acetamide (16b) as an advanced analog. Compound
16b was found to have extremely potent agonistic activity, with an
EC value of 8.3 nM, and to improve glucose tolerance at 0.1 mg/kg
po in mice. In addition, a docking study of 16b to the GPR119
homology model suggested that 16b exerts its GPR119 agonistic
activity by effectively utilizing a number of different interactions.
We consider that compound 16b and its analogs have clear utility
in exploring the practicality of GPR119 agonists as potential ther-
apeutic agents for the treatment of T2DM.
5. Experimental
5.1. Chemistry
1H NMR spectra were recorded on a JEOL JNM-EX400 spectrom-
eter and were referenced to an internal standard, tetramethylsil-
ane. The abbreviations used for the signal patterns are as
follows: s, singlet; br, broad; d, doublet; t, triplet; q, quartet; dd,
double doublet; m, multiplet. Mass spectra were recorded on a
JEOL LX-2000 or Waters ZQ-2000 mass spectrometer. Elemental
analyses were performed with a Yanako MT-5 microanalyzer (C,
H, N) and a Yokogawa IC-7000S ion chromatographic analyzer (hal-
ogens, S). Where analyses are indicated by symbols, the analytical
results are within 0.4% of the theoretical values. Drying of organic
solutions during workup was done over anhydrous MgSO4. Column
chromatography was performed with Wakogel C-200 or Merck sil-
ica gel 60.
5.2. 2-(4-Chloro-2,5-difluorophenyl)-3,5,6,7-tetrahydro-4H-
cyclopenta[d]pyrimidin-4-one (10a)
To a solution of 4-chloro-2,5-difluorobenzamidine (815, 800 mg)
in methanol (10 mL) was added methyl 2-oxocyclopentanecarb-
oxylate (9a, 700 mg), and the reaction mixture was stirred at reflux
temperature for 5 h. To the mixture was added 2-oxocyclopentan-
Figure 2. (A) Binding mode of compound 16b to the hGPR119 homology model.
The template was built based on the crystal structure of an agonist-bound hA2AAR
(PDB code 3QAK); (B) two-dimensional diagram showing the interactions of 16b
with hGPR119 prepared using the ligand interactions application in MOE.

K. Negoro et al. / Bioorg. Med. Chem. 20 (2012) 6442–6451
6447
ecarboxylate (150 mg), and the reaction mixture was stirred at re-
5.8. 4-Chloro-2-(4-chloro-2,5-difluorophenyl)-6,7-dihydro-5H-
flux temperature for 21 h. The mixture was then evaporated in va-
cuo, and the resulting residue was washed with diethyl ether and
dried in vacuo to give 10a (810 mg, 68%) as a white solid: 1H NMR
(DMSO-d6) d 1.92–2.10 (2H, m), 2.69 (2H, t, J = 7.7 Hz), 2.80 (2H, t,
J = 7.7 Hz), 7.77 (1H, dd, J = 6.4, 9.3 Hz), 7.83 (1H, dd, J = 6.1,
9.5 Hz), 10.90–12.10 (1H, br); FAB-MS m/z 283, 285 [(M+H)+].
The following compound (10b) was prepared by a procedure
similar to that described for 10a.
cyclopenta[d]pyrimidine (11a)
A mixture of 2-(4-chloro-2,5-difluorophenyl)-3,5,6,7-tetrahy-
dro-4H-cyclopenta[d]pyrimidin-4-one (10a, 0.80 g) and phospho-
rus oxychloride (10 mL) was stirred at reflux temperature for 3 h.
The mixture was cooled to room temperature, and evaporated in
vacuo. To the resulting residue was added water, and extracted
with ethyl acetate. The organic layer was dried, filtered and evap-
orated in vacuo to give 11a (0.75 g, 88%) as a light brown solid: 1H
NMR (DMSO-d6) d 2.10–2.21 (2H, m), 3.01 (2H, t, J = 7.7 Hz), 3.10
(2H, t, J = 7.7 Hz), 7.81 (1H, dd, J = 6.4, 10.3 Hz), 7.96 (1H, dd, J =
6.4, 9.8 Hz); FAB-MS m/z 301, 303 [(M+H)+].
5.3. 2-(4-Chloro-2,5-difluorophenyl)-5,6,7,8-tetrahydroquinazolin
-4(3H)-one (10b)
Yellow solid (yield 96%); 1H NMR (DMSO-d6) d 1.62–1.80 (4H,
m), 2.39 (2H, t, J = 5.9 Hz), 2.57 (2H, t, J = 6.2 Hz), 7.76 (1H, dd,
J = 6.3, 9.3 Hz), 7.85 (1H, dd, J = 6.2, 9.6 Hz), 12.30–12.80 (1H, br);
FAB-MS m/z 297, 299 [(M+H)+].
The following compounds (11b–f) were prepared by a proce-
dure similar to that described for 11a.
5.9. 4-Chloro-2-(4-chloro-2,5-difluorophenyl)-5,6,7,8-
tetrahydroquinazoline (11b)
5.4. 2-(4-Chloro-2,5-difluorophenyl)-5,7-dihydrofuro[3,4-d]
pyrimidin-4(3H)-one (10c)
White solid (yield 94%); 1H NMR (DMSO-d6) d 1.78–1.90 (4H,
m), 2.70–2.80 (2H, m), 2.85–2.95 (2H, m), 7.81 (1H, dd, J = 6.4,
10.3 Hz), 7.95 (1H, dd, J = 6.6, 9.5 Hz); FAB-MS m/z 315, 317
[(M+H)+].
To a solution of methyl 4-oxotetrahydrofuran-3-carboxylate
(9c16a, 1.36 g) in methanol (15 mL) were added 4-chloro-2,5-diflu-
orobenzamidine (815, 1.50 g) and sodium methoxide (420 mg), and
the reaction mixture was stirred at room temperature overnight.
The mixture was then stirred at 50 °C for 9 h and cooled to room
temperature. To the mixture were added 1 M hydrochloric acid
(10 mL) and water (40 mL) at 5 °C, and the reaction mixture was
stirred at room temperature for 4 h. The resulting precipitates were
collected by filtration, washed with water and dried in vacuo at
50 °C to give 10c (756 mg, 68%) as a brown solid: 1H NMR
(DMSO-d6) d 4.85–4.93 (2H, m), 4.93–5.00 (2H, m), 7.82 (1H, dd,
J = 6.4, 9.3 Hz), 7,89 (1H, d, J = 6.1, 9.5 Hz), 12.50–13.50 (1H, br);
ESI-MS m/z 285, 287 [(M+H)+].
5.10. 4-Chloro-2-(4-chloro-2,5-difluorophenyl)-5,7-
dihydrofuro[3,4-d]pyrimidine (11c)
Brown solid (yield 19%); 1H NMR (DMSO-d6) d 5.13 (2H, t,
J = 2.0 Hz), 5.20 (2H, t, J = 2.0 Hz), 7.87 (1H, dd, J = 5.9, 10.3 Hz),
8.03 (1H, dd, J = 6.9, 9.8 Hz); FAB-MS m/z 303, 305 [(M+H)+].
5.11. 4-Chloro-2-(4-chloro-2,5-difluorophenyl)-5,7-
dihydrothieno[3,4-d]pyrimidine (11d)
The following compound (10d–f) was prepared by a procedure
similar to that described for 10c.
Pale red solid (yield 97%); 1H NMR (DMSO-d6) d 4.33 (2H, t,
J = 2.0 Hz), 4.44 (2H, t, J = 2.0 Hz), 7.85 (1H, dd, J = 6.4, 10.3 Hz),
8.01 (1H, dd, J = 6.4, 9.8 Hz); FAB-MS m/z 319, 321 [(M+H)+].
5.5. 2-(4-Chloro-2,5-difluorophenyl)-5,7-dihydrothieno[3,4-d]
pyrimidin-4(3H)-one (10d)
5.12. 4-Chloro-2-(4-chloro-2,5-difluorophenyl)-5,6-
dihydrothieno[2,3-d]pyrimidine (11e)
Compound 10d was prepared from 8 and methyl 4-oxotetrahy-
drothiophene-3-carboxylate 9d.16a
Brown oil (yield 83%); 1H NMR (DMSO-d6) d 3.35–3.50 (2H, m),
3.50–3.65 (2H, m), 7.82 (1H, dd, J = 5.9, 10.3 Hz), 7.95 (1H, dd,
J = 6.4, 9.8 Hz); FAB-MS m/z 319, 321 [(M+H)+].
Brown solid (yield 69%); 1H NMR (DMSO-d6) d 3.95–4.10 (2H,
m), 4.15–4.25 (2H, m), 7.81 (1H, dd, J = 6.3, 9.2 Hz), 7,89 (1H, d,
J = 5.9, 9.6 Hz), 12.70–13.30 (1H, br); FAB-MS m/z 301, 303
[(M+H)+].
5.13. 4-Chloro-2-(4-chloro-2,5-difluorophenyl)-6,7-
dihydrothieno[3,2-d]pyrimidine (11f)
5.6. 2-(4-Chloro-2,5-difluorophenyl)-5,6-dihydrothieno[2,3-d]
pyrimidin-4(3H)-one (10e)
Brown solid (yield 76%); 1H NMR (DMSO-d6) d 3.52–3.58 (4H,
m), 7.81 (1H, dd, J = 6.4, 10.3 Hz), 7.96 (1H, dd, J = 6.9, 9.8 Hz);
FAB-MS m/z 319, 321 [(M+H)+].
Compound 10e was prepared from 815 and ethyl 2-oxotetrahy-
drothiophene-3-carboxylate 9e.16b
Pale yellow solid (yield 86%); 1H NMR (DMSO-d6) d 2.50–3.05
(4H, m), 3.60–3.75 (1H, m), 7.80 (1H, dd, J = 6.4, 9.3 Hz), 7.86
(1H, dd, J = 6.1, 9.5 Hz), 11.00–12.60 (1H, br); FAB-MS m/z 319,
321 [(M+H2O+H)+].
5.14. 2-(4-Chloro-2,5-difluorophenyl)-4-(morpholin-4-yl)-6,7-
dihydro-5H-cyclopenta[d]pyrimidine hydrochloride (12a)
To a solution of 4-chloro-2-(4-chloro-2,5-difluorophenyl)-6,7-
dihydro-5H-cyclopenta[d]pyrimidine (11a, 730 mg) in THF (5 mL)
was added morpholine (5 mL), and the mixture was stirred at room
temperature for 12 h. The mixture was evaporated in vacuo and to
the resulting residue was added water (30 mL). The solid was col-
lected by filtration, washed with water and then suspended with
methanol (30 mL). To the suspension was added 4 M hydrochloride
solution in ethyl acetate (5 mL), and the mixture was evaporated in
vacuo. The resulting solid was recrystallized from methanol and
diethyl ether to give 12a (581 mg, 62%) as a colorless crystal: 1H
5.7. 2-(4-Chloro-2,5-difluorophenyl)-6,7-dihydrothieno[3,2-d]
pyrimidin-4(3H)-one (10f)
Compound 10f was prepared from 815 and methyl 3-oxotetra-
hydrothiophene-2-carboxylate 9f.16a
Brown solid (yield 18%); 1H NMR (DMSO-d6) d 3.25–3.80 (4H,
m), 7.78 (1H, dd, J = 6.4, 9.3 Hz), 7.86 (1H, dd, J = 5.9, 9.8 Hz),
12.50–13.20 (1H, br); FAB-MS m/z 301, 303 [(M+H)+].

6448
K. Negoro et al. / Bioorg. Med. Chem. 20 (2012) 6442–6451
NMR (DMSO-d6) d 2.00–2.15 (2H, m), 2.95 (2H, t, J = 7.6 Hz), 3.12
(2H, t, J = 7.6 Hz), 3.65–3.78 (4H, m), 3.80–3.90 (4H, m), 3.90–
6.00 (1H, br), 7.89 (1H, dd, J = 6.4, 9.8 Hz), 8.04 (1H, dd, J = 6.3,
9.8 Hz); FAB-MS m/z 352, 354 [(M+H)+]. Anal. (C17H16N3OClF2ÁHCl):
C, H, N, Cl, F.
hexane to give 13 (6.10 g, 69%) as a slightly yellow crystal: 1H
NMR (DMSO-d6) d 4.81 (2H, s), 4.93 (2H, s), 7.88 (1H, dd, J = 6.4,
10.3 Hz), 8.03 (1H, dd, J = 6.6, 9.6 Hz); FAB-MS m/z 351, 353
[(M+H)+].
The following compounds (12b–f) were prepared by a proce-
dure similar to that described for 12a.
5.21. 2-(4-Chloro-2,5-difluorophenyl)-4-(morpholin-4-yl)-5,7-
dihydrothieno[3,4-d]pyrimidine 6,6-dioxide (14a)
5.15. 2-(4-Chloro-2,5-difluorophenyl)-4-(morpholin-4-yl)-
5,6,7,8-tetrahydroquinazoline hydrochloride (12b)
A
mixture of 4-chloro-2-(4-chloro-2,5-difluorophenyl)-5,
7-dihydrothieno[3,4-d]pyrimidine 6,6-dioxide (13, 250 mg),
morpholine (80 L), N,N-diisopropylethylamine (0.37 mL) and ace-
l
White solid (yield 85%); 1H NMR (DMSO-d6) d 1.55–1.75 (2H,
m), 1.75–1.95 (2H, m), 2.60–2.72 (2H, m), 2.80–2.95 (2H, m),
3.50–4.00 (8H, m), 7.92 (1H, dd, J = 6.1, 10.0 Hz), 8.04 (1H, dd, J =
6.4, 9.3 Hz); FAB-MS m/z 366, 368 [(M+H)+]. Anal.
(C18H18N3OClF2ÁHClÁ0.9H2O): C, H, N, Cl, F.
tonitrile (5 mL) was stirred at 70 °C for 15 h and then cooled to
room temperature. To the mixture was added water (20 mL). The
solid was collected by filtration, washed with water and dried in
vacuo at 50 °C to give a pale yellow solid. The obtained solid was
dissolved with THF (10 mL) and methanol (5 mL), and to the solu-
tion was added 4 M hydrochloride solution in dioxane (2 mL). The
mixture was evaporated in vacuo and the resulting residue was
recrystallized from ethanol and ethyl acetate to obtain 14a
(234 mg, 82%) as a colorless crystal: 1H NMR (DMSO-d6) d 3.60–
3.75 (8H, m), 4.54 (2H, s), 4.74 (2H, s), 7.76 (1H, dd, J = 6.4,
10.3 Hz), 7.99 (1H, dd, J = 6.9, 9.8 Hz); FAB-MS m/z 402, 404
[(M+H)+]. Anal. (C16H14N3O3SClF2Á0.08HCl): C, H, N, S, Cl, F.
The following compounds (14b–j) were prepared by a proce-
dure similar to that described for 14a.
5.16. 2-(4-Chloro-2,5-difluorophenyl)-4-(morpholin-4-yl)-5,7-
dihydrofuro[3,4-d]pyrimidine hydrochloride (12c)
Light brown solid (yield 61%); 1H NMR (DMSO-d6) d 3.50–3.75
(8H, m), 4.00–4.80 (1H, br), 4.87 (2H, t, J = 2.4 Hz), 5.26 (2H, t,
J = 2.4 Hz), 7.75 (1H, dd, J = 5.9, 10.3 Hz), 8.00 (2H, dd, J = 6.6,
10.0 Hz);
FAB-MS
m/z
354,
356
[(M+H)+].
Anal.
(C16H14N3O2ClF2Á0.7HClÁ0.5H2O): C, H, N, Cl, F.
5.17. 2-(4-Chloro-2,5-difluorophenyl)-4-(morpholin-4-yl)-5,7-
dihydrothieno[3,4-d]pyrimidine hydrochloride (12d)
5.22. 1-[2-(4-Chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-
dihydrothieno[3,4-d]pyrimidin-4-yl]piperidin-4-ol
hydrochloride (14b)
Pink solid (yield 84%); 1H NMR (DMSO-d6) d 3.65–3.78 (8H, m),
3.80–4.12 (1H, br), 4.15 (2H, t, J = 2.2 Hz), 4.39 (2H, t, J = 2.2 Hz),
7.75 (1H, dd, J = 5.9, 10.3 Hz), 7.99 (2H, dd, J = 6.6, 10.0 Hz); FAB-
MS m/z 370, 372 [(M+H)+]. Anal. (C16H14N3OSClF2ÁHClÁ0.5C2H6O):
C, H, N, S, Cl, F.
Ivory crystal (yield 85%); 1H NMR (DMSO-d6) d 1.35–1.55 (2H,
m), 1.73–1.90 (2H, m), 3.25–3.43 (2H, m), 3.70–3.85 (1H, m),
3.90–4.05 (2H, m), 4.52 (2H, s), 4.69 (2H, s), 5.20–6.10 (1H, br),
7.76 (1H, dd, J = 6.1, 10.0 Hz), 7.97 (1H, dd, J = 6.9, 9.8 Hz); FAB-
MS m/z 416, 418 [(M+H)+]. Anal. (C17H16N3O3SClF2ÁHCl): C, H, N,
S, Cl, F.
5.18. 2-(4-Chloro-2,5-difluorophenyl)-4-(morpholin-4-yl)-5,6-
dihydrothieno[2,3-d]pyrimidine hydrochloride (12e)
5.23. 1-[2-(4-Chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-
dihydrothieno[3,4-d]pyrimidin-4-yl]piperidine-4-carboxamide
(14c)
Brown solid (yield 32%); 1H NMR (DMSO-d6) d 3.30–3.50 (4H,
m), 3.58–3.75 (8H, m), 4.20–4.80 (1H, br), 4.39 (2H, t, J = 2.2 Hz),
7.73 (1H, dd, J = 6.4, 10.3 Hz), 7.94 (2H, dd, J = 6.8, 9.8 Hz); FAB-
MS
m/z
370,
372
[(M+H)+].
Anal.
White solid (yield 78%); 1H NMR (DMSO-d6) d 1.50–1.70 (2H,
m), 1.70–1.88 (2H, m), 2.35–2.50 (1H, m), 3.00–3.17 (2H, m),
3.20–3.50 (2H, m), 4.20–4.35 (2H, m), 4.52 (2H, s), 4.70 (2H, s),
6.81 (1H, s), 7.31 (1H, s), 7.76 (1H, dd, J = 6.4, 10.3 Hz), 7.98 (1H,
dd, J = 6.6, 10.0 Hz); FAB-MS m/z 443, 445 [(M+H)+]. Anal.
(C18H17N4O3SClF2): C, H, N, S, Cl, F.
(C16H14N3OSClF2Á0.95HClÁ0.25H2O): C, H, N, S, Cl, F.
5.19. 2-(4-Chloro-2,5-difluorophenyl)-4-(morpholin-4-yl)-6,7-
dihydrothieno[3,2-d]pyrimidine hydrochloride (12f)
Yellow solid (yield 72%); 1H NMR (DMSO-d6) d 3.22–3.42 (4H,
m), 3.65–3.75 (8H, m), 4.00–5.25 (1H, br), 4.39 (2H, t, J = 2.2 Hz),
7.73 (1H, dd, J = 6.4, 10.3 Hz), 7.98 (2H, dd, J = 6.9, 9.8 Hz); FAB-
MS m/z 370, 372 [(M+H)+]. Anal. (C16H14N3OSClF2ÁHCl): C, H, N, S,
Cl, F.
5.24. 1-[2-(4-Chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-
dihydrothieno[3,4-d]pyrimidin-4-yl]-N,N-dimethylpiperidine-
4-carboxamide hydrochloride (14d)
Pale yellow solid (yield 41%); 1H NMR (DMSO-d6) d 1.50–1.80
(4H, m), 2.81 (3H, s), 2.95–3.25 (6H, m), 4.20–4.40 (2H, m), 4.52
(2H, s), 4.71 (2H, s), 4.75–5.80 (1H, br), 7.76 (1H, dd, J = 5.9,
10.3 Hz), 7.98 (1H, dd, J = 6.4, 9.8 Hz); FAB-MS m/z 471, 473
[(M+H)+]. Anal. (C20H21N4O3SClF2Á0.6HClÁ0.75H2OÁ0.2C4H8O2): C,
H, N, S, Cl, F.
5.20. 4-Chloro-2-(4-chloro-2,5-difluorophenyl)-5,7-
dihydrothieno[3,4-d]pyrimidine 6,6-dioxide (13)
To a mixture of 4-chloro-2-(4-chloro-2,5-difluorophenyl)-5,7-
dihydrothieno[3,4-d]pyrimidine (11d, 8.00 g) and chloroform
(200 mL) was added m-CPBA (20.19 g), and then the mixture was
stirred at room temperature for 1 h. To the mixture was added
sat. aqueous sodium hydrogen carbonate. The mixture was ex-
tracted with chloroform, and the organic layer was dried. The des-
iccant was removed by filtration and the filtrate was evaporated in
vacuo. The resulting residue was purified by silica gel column chro-
matography (hexane–ethyl acetate) to obtain a pale yellow solid.
The obtained solid was recrystallized from ethyl acetate and
5.25. {1-[2-(4-Chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-
dihydrothieno[3,4-d]pyrimidin-4-yl]piperidin-4-yl}methanol
hydrochloride (14e)
Pale yellow crystal (yield 87%); 1H NMR (DMSO-d6) d 1.10–1.30
(2H, m), 1.60–1.82 (3H, m), 2.90–3.12 (2H, m), 3.28 (2H, d,
J = 5.9 Hz), 4.20–4.40 (2H, m), 4.52 (2H, s), 4.69 (2H, s), 6.30–7.40

K. Negoro et al. / Bioorg. Med. Chem. 20 (2012) 6442–6451
6449
(1H, br), 7.76 (1H, dd, J = 5.9, 10.3 Hz), 7.97 (1H, dd, J = 6.9, 9.8 Hz);
FAB-MS m/z 430, 432 [(M+H)+]. Anal. (C18H18N3O3SClF2ÁHCl): C, H,
N, S, Cl, F.
with diethyl ether and the organic layer was washed with water
and water, and then dried. The desiccant was removed by filtration
and the filtrate was evaporated in vacuo to give a solid (500 mg,
44%). The obtained solid (100 mg) was dissolved with THF
(10 mL), and to the solution was added 4 M hydrochloride solution
in dioxane (1 mL). The mixture was evaporated in vacuo and the
resulting residue was washed with diethyl ether to obtain 15a
(95 mg, 39%) as a white solid: 1H NMR (DMSO-d6) d 1.50–1.70
(2H, m), 1.80–2.00 (2H, m), 2.51–2.70 (1H, m), 3.10–3.25 (2H, m),
3.60–5.20 (3H, m), 4.52 (2H, s), 4.70 (2H, s), 7.77 (1H, dd, J = 6.1,
10.0 Hz), 7.98 (1H, dd, J = 6.9, 9.8 Hz); ESI-MS m/z 444 [(M+H)+].
Anal. (C18H16N3O4SClF2Á0.4HClÁ1.25H2OÁ0.2C4H10O): C, H, N, S, Cl, F.
The following compounds (15b–d) were prepared by a proce-
dure similar to that described for 15a.
5.26. 2-{1-[2-(4-Chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-
dihydrothieno[3,4-d]pyrimidin-4-yl]piperidin-4-yl}ethanol
hydrochloride (14f)
Yellow crystal (yield 84%); 1H NMR (DMSO-d6) d 1.09–1.27 (2H,
m), 1.30–1.48 (2H, m), 1.63–1.83 (3H, m), 2.92–3.10 (2H, m), 3.46
(2H, t, J = 6.6 Hz), 4.21–4.35 (2H, m), 4.51 (2H, s), 4.68 (2H, s), 5.10–
6.00 (1H, br), 7.76 (1H, dd, J = 6.4, 10.3 Hz), 7.96 (1H, dd, J = 6.3,
9.8 Hz);
FAB-MS
m/z
444,
446
[(M+H)+].
Anal.
(C19H20N3O3SClF2ÁHCl): C, H, N, S, Cl, F.
5.27. Ethyl 1-[2-(4-chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-
dihydrothieno[3,4-d]pyrimidin-4-yl]piperidine-4-carboxylate
(14g)
5.32. {1-[2-(4-Chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-
dihydrothieno[3,4-d]pyrimidin-4-yl]piperidin-4-yl}aceticacid
hydrochloride (15b)
White solid (yield 83%); 1H NMR (DMSO-d6) d 1.19 (3H, t,
J = 7.1 Hz), 1.55–1.70 (2H, m), 1.85–1.98 (2H, m), 2.63–2.76 (1H,
m), 3.10–3.25 (2H, m), 4.08 (2H, q, J = 7.1 Hz), 4.13–4.25 (2H, m),
4.52 (2H, s), 4.70 (2H, s), 7.77 (1H, dd, J = 6.1, 10.0 Hz), 7.98 (1H,
dd, J = 6.9, 9.8 Hz); FAB-MS m/z 470, 472 [(M+H)+].
Brown crystal (yield 65%); 1H NMR (DMSO-d6) d 1.15–1.34 (2H,
m), 1.70–1.84 (2H, m), 1.90–2.10 (1H, m), 2.19 (2H, d, J = 6.8 Hz),
2.98–3.15 (2H, m), 4.22–4.35 (2H, m), 4.52 (2H, s), 4.68 (2H, s),
7.76 (1H, dd, J = 6.4, 10.3 Hz), 7.97 (1H, dd, J = 6.6, 10.0 Hz); FAB-
MS m/z 458, 460 [(M+H)+]. Anal. (C19H18N3O4SClF2Á0.9HClÁ0.25H2O
Á0.15C4H10OÁ0.1C2H3N): C, H, N, S, Cl, F.
5.28. Ethyl {1-[2-(4-chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-
dihydrothieno[3,4-d]pyrimidin-4-yl]piperidin-4-yl}acetate
(14h)
5.33. 3-{1-[2-(4-Chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-
dihydrothieno[3,4-d]pyrimidin-4-yl]piperidin-4-yl}propanoic
acid (15c)
White solid (yield 98%); 1H NMR (DMSO-d6) d 1.18 (3H, t,
J = 7.1 Hz), 1.20–1.33 (2H, m), 1.68–1.81 (2H, m), 1.95–2.10 (1H,
m), 2.26 (2H, d, J = 7.4 Hz), 2.98–3.13 (2H, m), 4.07 (2H, q,
J = 7.1 Hz), 4.20–4.35 (2H, m), 4.51 (2H, s), 4.67 (2H, s), 7.76 (1H,
dd, J = 6.2, 10.1 Hz), 7.97 (1H, dd, J = 6.9, 9.8 Hz); FAB-MS m/z
486, 488 [(M+H)+].
Pale yellow solid (yield 96%); 1H NMR (DMSO-d6) d 1.08–1.26
(2H, m), 1.40–1.65 (3H, m), 1.66–1.82 (2H, m), 2.25 (2H, t,
J = 7.4 Hz), 2.90–3.10 (2H, m), 4.20–4.40 (2H, m), 4.50 (2H, s),
4.68 (2H, s), 7.76 (1H, dd, J = 6.4, 10.0 Hz), 7.96 (1H, dd, J = 6.6,
10.2 Hz), 11.90–12.20 (1H, br); FAB-MS m/z 472, 474 [(M+H)+].
5.29. Ethyl 3-{1-[2-(4-chloro-2,5-difluorophenyl)-6,6-dioxido-
5,7-dihydrothieno[3,4-d]pyrimidin-4-yl]piperidin-4-
yl}propanoate (14i)
5.34. 4-[2-(4-Chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-
dihydrothieno[3,4-d]pyrimidin-4-yl]piperazin-1-yl}acetic acid
(15d)
Pale yellow solid (yield 96%); 1H NMR (DMSO-d6) d 1.08–1.26
(5H, m), 1.42–1.65 (3H, m), 1.65–1.82 (2H, m), 2.32 (2H, t,
J = 7.6 Hz), 2.90–3.07 (2H, m), 4.05 (2H, q, J = 7.2 Hz), 4.20–4.36
(2H, m), 4.50 (2H, s), 4.67 (2H, s), 7.76 (1H, dd, J = 6.1, 10.0 Hz),
7.96 (1H, dd, J = 6.9, 9.8 Hz); FAB-MS m/z 500, 502 [(M+H)+].
Pale yellow solid (yield 98%); 1H NMR (DMSO-d6) d 2.58–2.70
(4H, m), 3.21 (2H, s), 3.60–3.80 (4H, m), 2.70–4.00 (1H, br), 4.53
(2H, s), 4.71 (2H, s), 7.77 (1H, dd, J = 6.2, 10.2 Hz), 7.99 (1H, dd,
J = 6.8, 10.0 Hz); FAB-MS m/z 459 [(M+H)+].
5.35. 1-[2-(4-Chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-dihy-
drothieno[3,4-d]pyrimidin-4-yl]-N-methylpiperidine-4-carbox-
amide (16a)
5.30. Ethyl {4-[2-(4-chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-
dihydrothieno[3,4-d]pyrimidin-4-yl]piperazin-1-yl}acetate
(14j)
To a solution of 1-[2-(4-chloro-2,5-difluorophenyl)-6,6-dioxido
-5,7-dihydrothieno[3,4-d]pyrimidin-4-yl]piperidine-4-carboxylic
acid (free form of 15a, 256 mg) in DMF (5 mL) were added 1-ethyl-
3-(3-dimethylaminopropyl)carbodiimide (EDAC) hydrochloride
(0.17 g), 1-hydroxybenzotriazole (HOBt, 0.12 g) and 2 M methyl-
amine solution in THF (0.43 mL), and the mixture was stirred at
room temperature for two days. To the mixture was added water
and the solid was collected by filtration, washed with chloroform
and ethyl acetate and dried in vacuo to obtain 16a (205 mg, 78%)
as a white solid: 1H NMR (DMSO-d6) d 1.50–1.90 (2H, m),
2.35–2.55 (2H, m), 2.34–2.48 (1H, m), 2.57 (3H, d, J = 4.4 Hz),
3.00–3.20 (2H, m), 4.20–4.40 (2H, m), 4.52 (2H, s), 4.70 (2H, s),
7.70–7.81 (2H, m), 7.98 (1H, dd, J = 6.6, 10.0 Hz); FAB-MS m/z
457, 459 [(M+H)+]. Anal. (C19H19N4O3SClF2Á0.4H2OÁ0.01CHCl3): C,
H, N, S, Cl, F.
Pale gray solid (yield 99%); 1H NMR (DMSO-d6) d 1.19 (3H, t,
J = 7.1 Hz), 2.57–2.68 (4H, m), 3.29 (2H, s), 3.62–3.75 (4H, m),
4.09 (2H, q, J = 7.1 Hz), 4.53 (2H, s), 4.71 (2H, s), 7.76 (1H, dd,
J = 5.8, 10.2 Hz), 7.98 (1H, dd, J = 6.4, 9.6 Hz); FAB-MS m/z 487,
489 [(M+H)+].
5.31. 1-[2-(4-Chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-
dihydrothieno[3,4-d]pyrimidin-4-yl]piperidine-4-carboxylic
acid hydrochloride (15a)
To a solution of 1-[2-(4-chloro-2,5-difluorophenyl)-6,6-dioxi-
do-5,7-dihydrothieno[3,4-d]pyrimidin-4-yl]piperidine-4-carboxyl-
ate (14g, 1.11 g) in THF (12 mL) and ethanol (12 mL) was added
1 M aqueous sodium hydroxide (5 mL), and the mixture was stir-
red at room temperature for 8 h. To the mixture were added 1 M
hydrochloric acid (5 mL) and water. The mixture was extracted
The following compounds (16b–d) were prepared by a proce-
dure similar to that described for 16a.

6450
K. Negoro et al. / Bioorg. Med. Chem. 20 (2012) 6442–6451
5.36. 2-{1-[2-(4-Chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-
dihydrothieno[3,4-d]pyrimidin-4-yl]piperidin-4-yl}acetamide
(16b)
solution. Separately to this, to 10
of the test compound was added accurately 1 mL of methanol, fol-
lowed by stirring, thereby giving a standard solution. 10 L por-
lL of the 10 mM DMSO solution
l
tions each of the sample solution and standard solution were
tested by liquid chromatography, and the ratio of the peak area
of the sample solution to the peak area of the standard solution
was determined, thereby calculating the solubility.
Colorless crystal (yield 60%); 1H NMR (DMSO-d6) d 1.10–1.30
(2H, m), 1.65–1.80 (2H, m), 1.90–2.05 (3H, m), 2.95–3.10 (2H, m),
4.20–4.35 (2H, m), 4.51 (2H, s), 4.67 (2H, s), 6.76 (1H, s), 7.26
(1H, s), 7.76 (1H, dd, J = 6.0, 10.4 Hz), 7.97 (1H, dd, J = 6.8,
9.6 Hz); FAB-MS m/z 457 [(M+H)+]. Anal. (C19H19N4O3SClF2ÁH2O):
C, H, N, S, Cl, F.
5.42. Homology modeling and ligand docking
Crystal structures of GPCRs are now available for rhodopsin,
adrenergic, and adenosine receptors in both inactive and activated
forms, as well as for chemokine, dopamine, and histamine recep-
tors in inactive conformations.21 Homology between the hGPR119
sequence and transmembrane domain sequence of these seven
families were compared using MOE,22 during which human A2A
adenosine receptor (hA2AAR) was found to be the highest. A homol-
ogy model of hGPR119 was constructed using the crystal structure
of agonist-bound hA2AAR (PDB code 3QAK),19 obtained from the
RCSB Protein Data Bank, as a structural template. Sequence align-
ment between hGPR119 and hA2AAR was created using PRIME,23
with the ICL3 loop of hA2AAR excluded and manual revision.
Homology modeling was performed using PRIME.23
5.37. 3-{1-[2-(4-Chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-
dihydrothieno[3,4-d]pyrimidin-4-yl]piperidin-4-
yl}propanamide hydrochloride (16c)
Pale yellow crystal (yield 94%); 1H NMR (DMSO-d6) d 1.00–1.30
(2H, m), 1.37–1.64 (3H, m), 1.68–1.82 (2H, m), 2.02–2.15 (2H, m),
2.92–3.10 (2H, m), 4.20–4.40 (2H, m), 4.51 (2H, s), 4.68 (2H, s),
4.90–6.00 (1H, br), 6.50–7.00 (1H, br), 7.00–7.60 (1H, br), 7.76
(1H, dd, J = 6.2, 9.8 Hz), 7.97 (1H, dd, J = 6.8, 9.6 Hz); FAB-MS m/z
471, 473 [(M+H)+]. Anal. (C20H21N4O3SClF2ÁHClÁ0.3H2OÁ0.2C4H10O):
C, H, N, S, Cl, F.
The ligand molecule was sketched in Maestro24 and energy-
minimized using Confgen25 with the OPLS_2005 force field.
A docking study was performed using GOLD.26 The ligand bind-
ing pocket was defined using the O from Thr86 as a central atom
with radius of 10 Å. The ligand molecule was docked 10 times.
The top scoring pose, as assessed by goldscore, was employed for
discussions. The two-dimensional diagram was prepared using
the ligand interactions application in MOE.22
5.38. 2-{4-[2-(4-Chloro-2,5-difluorophenyl)-6,6-dioxido-5,7-
dihydrothieno[3,4-d]pyrimidin-4-yl]piperazin-1-yl}acetamide
hydrochloride (16d)
Slightly yellow crystal (yield 91%); 1H NMR (DMSO-d6) d 3.20–
3.80 (6H, m), 3.99 (2H, s), 4.25–4.55 (2H, m), 4.61 (2H, s), 4.79
(2H, s), 5.90–7.00 (1H, br), 7.71 (1H, s), 7.79 (1H, dd, J = 6.2,
10.2 Hz), 8.05 (1H, dd, J = 6.6, 9.8 Hz), 8.13 (1H, s), 10.40–11.30
(1H,
br);
FAB-MS
m/z
458,
460
[(M+H)+].
Anal.
(C18H18N5O3SClF2Á1.6HClÁ0.25H2O): C, H, N, S, Cl, F.
Acknowledgments
5.39. Human GPR119 cAMP reporter assay
We are grateful to Dr. Masakazu Imamura for his useful advice,
and to the staff of the Division of Analysis & Pharmacokinetics Re-
search Laboratories for the elemental analysis, spectral measure-
ments and solubility test.
Human GPR119 agonist activity was evaluated in HEK293 cells
stably expressing human GPR119 and pCRE-Luc. HEK293-hGPR119
cells were seeded in 96-well plates at 2.5 Â 104 cells/well, incu-
bated overnight at 37 °C in 5% CO2, and then exposed to the test
compound dissolved in DMSO at concentrations ranging from
References and notes
0.01 to 10 lM. After 6 h incubation, cells were harvested using
0.2% Triton X-100 in phosphate-buffered saline (pH 7.4). Luciferase
activity was measured using a model ML-3000 Luminometer (Dy-
nex Tech, VA, USA). Three replicates for each concentration were
performed.
1. (a) Leahy, J. L. Arch. Med. Res. 2005, 36, 197; (b) Wajchenberg, B. L. Endocr. Rev.
2007, 28, 187.
2. (a) Doyle, M. F.; Egan, J. M. Pharmacol. Rev. 2003, 55, 105; (b) Rendell, M. Drugs
2004, 64, 1339.
3. (a) Maedler, K.; Carr, R. D.; Bosco, D.; Zuellig, R. A.; Berney, T.; Donath, M. Y. J.
Clin. Endcrinol. Metab. 2005, 90, 501; (b) Del Guerra, S.; Marselli, L.; Lupi, R.;
Boggi, U.; Moska, F.; Benzi, L.; Del Prato, S.; Marchetti, P. J. Diabetes
Complications 2005, 19, 60.
5.40. Oral glucose tolerance test (OGTT)
4. (a) UK Prospective Diabetes Study (UKPDS) Group. Diabetes 1995, 44, 1249.; (b)
Kahn, S. E.; Haffner, S. M.; Heise, M. A.; Herman, W. H.; Holman, R. R.; Jones, N.
P.; Kravitz, B. G.; Lachin, J. M.; O’Neill, M. C.; Zinman, B.; Viberti, G. N. Engl. J.
Med. 2006, 355, 2427; (c) Grant, R. W.; Wexler, D. J.; Watson, A. J.; Lester, W. T.;
Cagliero, E.; Campbell, E. G.; Nathan, D. M. Diabetes Care 2007, 30, 1448.
5. Alberti, K. G.; Zimmet, P.; Shaw, J. Diabet. Med. 2007, 24, 451.
6. (a) Drucker, D. J. Cell Metab. 2006, 3, 153; (b) Drucker, D. J. Diabetes Care 2007,
30, 1335; (c) Brubaker, P. L. Trends Endocrinol. Metab. 2007, 18, 240.
7. Herman, G. A.; Stein, P. P.; Thomberry, N. A.; Wagner, J. A. Clin. Pharmacol. Ther.
2007, 81, 761.
Eight-week-old male ICR mice were fasted overnight and then
orally administered 0.5% methyl-cellulose (vehicle) or 10 mg/kg
test compounds. After 10 min, glucose was given orally at a dose
of 2 g/kg/10 mL, and a blood sample was collected from a tail vein
after 30 min. Blood glucose levels were determined using the Glu-
cose CII test (Wako, Osaka, Japan).
8. DeFrozo, R. A.; Ratner, R. E.; Han, J.; Kim, D. D.; Fineman, M. S.; Baron, A. D.
Diabetes Care 2005, 28, 1092.
9. (a) Im, D. S. J. Lipid Res. 2004, 45, 410; (b) Kostenis, E. Pharmacol. Ther. 2004, 102,
243; (c) Wang, J.; Wu, X.; Simonavicius, N.; Tain, H.; Ling, L. J. Biol. Chem. 2006,
281, 34457.
5.41. Solubility test in pH 6.8 buffer solution with the
precipitation method
To 13 lL of a 10 mM DMSO solution of a test compound that
10. Rayasam, G. V.; Tulasi, V. K.; Davis, J. A.; Bansal, V. S. Expert Opin. Ther. Targets
2007, 11, 661.
had been prepared in advance was added exactly 1 mL of a second
liquid (pH 6.8) for a disintegration test of Japanese Pharmacopoeia,
followed by shaking at 25 °C for 20 h, thereby giving a sample stock
11. (a) 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,
28, 744; (b) 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.
solution. Next, using a filter impregnated with 200
ple stock solution, 200 L of a fresh sample stock solution was
added for filtration to obtain a liquid, which was taken as a sample
lL of the sam-
l

K. Negoro et al. / Bioorg. Med. Chem. 20 (2012) 6442–6451
6451
12. (a) 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 2008, 149, 2038; (b)
Drucker, D. J. Diabetes 1998, 47, 159; (c) Yip, R. G.; Wolfe, M. M. Life Sci. 2000,
66, 91.
13. (a) Jones, R. M.; Leonard, J. N.; Buzard, D. J.; Lehmann, J. Expert Opin. Ther. Pat.
2009, 19, 1339; (b) Jones, R. M.; Leonard, J. N. Annu. Rep. Med. Chem. 2009, 44,
149.
17. Yoshida, S.; Ohishi, T.; Matsui, T.; Tanaka, H.; Oshima, H.; Yonetoku, Y.;
Shibasaki, M. Biochem. Biophys. Res. Commun. 2010, 402, 280.
18. We didn’t consider the oxidation of the 6,7-dihydrothieno[3,2-d]pyrimidine
derivative 12f as effective for improving the activity, because {1-[2-(4-chloro-
2,5-difluorophenyl)-5,5-dioxido-6,7-dihydrothieno[3,2-d]pyrimidin-4-
yl]piperidin-4-yl}methanol was found to show 100-fold less potent activity
with an EC value of 410 nM than the 5,7-dihydrothieno[3,4-d]pyrimidine 6,6-
dioxide derivative 14e.
14. (a) Yonetoku, Y.; Maruyama, T.; Negoro, K.; Moritomo, H.; Imanishi, N.;
Shimada, I.; Moritomo, A.; Hamaguchi, W.; Misawa, H.; Yoshida, S.; Ohishi, T.
Patent WO 03/026661, 2003.; (b) Yonetoku, Y.; Negoro, K.; Misawa, H.;
Maruyama, T.; Harada, H.; Shimada, I.; Yoshida, S.; Ohishi, T. Patent JP 2004/
269468, 2004.; (c) Yonetoku, Y.; Negoro, K.; Misawa, H.; Harada, H.; Shimada,
I.; Takeuchi, M.; Yoshida, S.; Ohishi, T. Patent JP 2004/269469, 2004.
15. (a) Negoro, K.; Yonetoku, Y.; Maruyama, T.; Yoshida, S.; Takeuchi, M.; Ohta, M.
Bioorg. Med. Chem. 2012, 20, 2369; (b) Negoro, K.; Yonetoku, Y.; Misawa-Mukai,
H.; Hamaguchi, W.; Maruyama, T.; Yoshida, S.; Takeuchi, M.; Ohta, M. Bioorg.
Med. Chem. 2012, 20, 5235.
19. Xu, F.; Wu, H.; Katritch, V.; Han, G. W.; Jacobson, K. A.; Gao, Z.-G.; Cherezov, V.;
Stevens, R. C. Science 2011, 332, 322.
20. Bissantz, C.; Kuhn, B.; Stahl, M. J. Med. Chem. 2010, 53, 5061.
21. (a) Katritch, V.; Cherezov, V.; Stevens, R. C. Trends Phamacol. Sci. 2012, 33, 17;
(b) Kontoyianni, M.; Liu, Z. Curr. Med. Chem. 2012, 19, 544.
22. Molecular Operating Environment (MOE), version 2011.10; Chemical
Computing Group Inc.: Montreal, QC, Canada H3A 2R7, 2011.
23. PRIME, version 3.0; Schrödinger LLC: New York, 2011.
24. Maestro, version 9.2; Schrödinger LLC: New York, 2011.
25. Confgen, version 2.3; Schrödinger LLC: New York, 2011.
26. GOLD, version 5.1; CCDC: Cambridge, UK, 2011.
16. (a) Dowd, P.; Choi, S.-C. Tetrahedron 1991, 47, 4847; (b) Taguchi, Y.; Suhara, Y.
Bull. Chem. Soc. Jpn. 1986, 59, 2321.