2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3, 4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (DSR-12727): A potent, orally active dipeptidyl peptidase IV inhibitor without mechanism-based inactivati

Article abstract of DOI:10.1016/j.bmc.2011.07.042

We report on the identification of 2-({6-[(3R)-3-amino-3-methylpiperidine- 1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d] pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (DSR-12727) (7a) as a potent and orally active DPP-4 inhibitor withou

Full text of DOI:10.1016/j.bmc.2011.07.042

Bioorganic & Medicinal Chemistry 19 (2011) 5490–5499
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4
-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile
(DSR-12727): A potent, orally active dipeptidyl peptidase IV inhibitor without
mechanism-based inactivation of CYP3A
⇑
Yukihiro Nishio , Hidenori Kimura, Naoyuki Sawada, Eiji Sugaru, Masakuni Horiguchi, Michiko Ono,
Yudai Furuta, Mutsuko Sakai, Yumi Masui, Misato Otani, Takahiko Hashizuka, Yayoi Honda, Jiro Deguchi,
Tsutomu Nakagawa, Hiroyuki Nakahira
Drug Research Division, Dainippon Sumitomo Pharma Co. Ltd, 3-1-98 Kasugade Naka, Konohana-ku, Osaka 554-0022, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We report on the identification of 2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-
1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (DSR-12727) (7a) as a
potent and orally active DPP-4 inhibitor without mechanism-based inactivation of CYP3A. Compound 7a
showed good DPP-4 inhibitory activity (IC50 = 1.1 nM), excellent selectivity against related peptidases
and other off-targets, good pharmacokinetic and pharmacodynamic profile, great in vivo efficacy in Zuc-
ker-fatty rat, and no safety concerns both in vitro and in vivo.
Received 6 June 2011
Revised 22 July 2011
Accepted 22 July 2011
Available online 4 August 2011
Keywords:
DPP IV inhibitors
Ó 2011 Elsevier Ltd. All rights reserved.
Mechanism-based inactivation
1. Introduction
the body, modulates the activity of a wide variety of regulatory
peptides. Based on the report about DPP-4 knockout mice, the
Type 2 diabetes (T2D) is a major metabolic disorder affecting
approximately 194 million people worldwide. This number is esti-
mated to reach 366 million by 2030.1 The beneficial effects of anti-
diabetic agents currently used, such as sulphonylurea, biguanide,
use of DPP-4 inhibition in T2D therapy seems to have lots of advan-
tages.5–7 It is suggested that DPP-4 inhibitors might be able to
stimulate production of new b-cell in patients with T2D, and thus
prevent deterioration of the disease. In addition, DPP-4 inhibition
is unlikely to cause serious hypoglycemia, because GLP-1 is strictly
glucose-dependent.
and
a-glucosidase inhibitors that can effectively increase insulin
secretion or decrease glucose absorption are known to be associ-
ated with a number of side effects including hypoglycemia, weight
gain, gastrointestinal disorders, and lactic acidosis. Thus, current
treatments for T2D are considered to be unsatisfactory in terms
of prevention of complications and preservation of quality of life.2
Under these circumstances, intensive effort has been made to find
better and safer drugs for T2D.
Glucagon-like peptide 1 (GLP-1), an important incretin hor-
mone that regulates body blood glucose, has recently been receiv-
ing great attention as a new target for the development of novel
therapies for T2D.3 However, the active state of GLP-1 (7–36) is
short-lived as this hormone is quickly cleaved by dipeptidyl pepti-
dase IV (DPP-4).4 This means that DPP-4 inhibition, and conse-
quently increase in GLP-1 level, presents a new approach for the
treatment of T2D. DPP-4, a serine protease distributed throughout
In recent years, a number of DPP-4 inhibitors,8 such as 1 (sitag-
liptin),9 2 (vildagliptin),10 3 (saxagliptin),11 4 (alogliptin),12 and 5
(linagliptin),13 (Fig. 1) have been marketed or are under clinical
development, and have shown great efficacy in patients with T2D.
In our research about DPP-4 inhibitors, we found compound 6,14
which showed excellent inhibitory activity against DPP-4 (0.34 nM),
however, had two pharmacokinetic drawbacks. One is reversible
inhibition of Cytochrome P450 (CYP) 3A4 (IC50 = 1.6
lM), CYP1A2
(IC50 = 14.7 M), and CYP2D6 (IC50 = 7.5 M) caused by the parent
l
l
drug. The other is potent mechanism-based inactivation (MBI)15
which is irreversible inhibition of CYP3A. MBI is time- and concen-
tration-dependent CYP enzymes inhibition, and caused by metabo-
lites of original compounds. Some specific structures, such as nitroso
species, to coordinate with the Fe-center of CYP enzymes or bind
covalently with it have been reported as the cause of MBI, however,
few examples to prevent or alleviate potent MBI have been re-
ported.16 Our investigation about metabolites of compounds having
⇑
Corresponding author. Tel.: +81 6 6466 5223; fax: +81 6 6466 5287.
E-mail address: yukihiro-nishio@ds-pharma.co.jp (Y. Nishio).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.07.042

Y. Nishio et al. / Bioorg. Med. Chem. 19 (2011) 5490–5499
5491
F
F
OH
OH
F
NH2
O
O
O
CN
CN
H
N
N
N
N
N
N
N
NH2
1
2
CF3
CN
3
O
NH2
O
NH2
N
N
N
N
N
N
N
N
O
N
O
N
4
5
Figure 1. Structures of known DPP-4 inhibitors.
the (R)-3-aminopiperidine unit, which includes 6, revealed that
metabolism of these compounds mainly undergo at the uracil struc-
ture to give demethylated compounds, at the piperidine moiety to
give hydroxylated derivatives and at the (R)-3-amino position of
the piperidine unit to give hydroxyl amine. Among them, hydroxyl
amine is supposed to give nitroso species, which is believed to form
a metabolic intermediate complex and cause MBI.16,17
rearrangement using diphenylphosphoric azide (DPPA), the crude
solution was first washed with water to remove by-products, and
then treated with an excess amount of tert-butyl alcohol and a cat-
alytic amount of potassium tert-butoxide to give 12a–b. The Cbz
group at the 1-amino position was removed by hydrogenation to
afford the target amines (R)-3-amino-3-methylpiperidine (13a)
and rac-3-amino-3-ethylpiperidine moiety (13b) (Scheme 1).
Compounds 6, 7a, and 7b were prepared in five steps. The inter-
mediate 15a–c was prepared from starting material 14 as previ-
ously described.20 The pyrrole moiety of 15a–c was treated with
KOCN to give an urea intermediate, which was first cyclized and
then methylated under basic condition to provide 16a–c.
Cyanation of 16a–c gave 17a–c, and the final structures 6, 7a and
rac-7b were obtained by removal of the tert-butoxy carbonyl
(Boc) group and the tert-butyl ester at the C7-position using
benzensulphonic acid. Each enantiomer of 7b, (+)-7b, and (ꢀ)-7b,
were prepared by chiral separation at Daicel Chemical Industries,
Ltd (http://www.daicel.co.jp/) (Scheme 2). Absolute configurations
of following enantiomers, 7a, (+)-7b, and (ꢀ)-7b, have not been
determined, and configurations written in this article are supposed
by the synthetic ways, inhibitory activities, and the information of
other DPP-4 inhibitors, such as 412 and 5.13
We have previously reported new chemotype DPP-4 inhibitors
having (R)-3-amino-3-methylpiperidine as a pharmacophore and
showed that these inhibitors have good in vitro activity and phar-
macokinetic/pharmacodynamic (PK/PD) profiles.18 In addition to
those results, a methyl substitution seemed to have good influence
on CYP enzymes inhibition because the reported compounds
hardly had strong inhibition of CYP enzymes. Thus, it was expected
that compound 6 strong inhibition of CYP3A4 would be alleviated
by it. As for MBI, we assumed that introduction of a small alkyl
substituent at the 3-position of piperidine unit could prevent
metabolism at the amino group and/or coordinate of reactive
metabolites with the Fe-center of CYP3A to avoid MBI. We there-
fore considered that compound 7a, having a methyl instead of a
hydrogen at 3-position of compound 6 piperidine unit, would be-
come an ideal DPP-4 inhibitor. In addition, to compare the effects
of a small alkyl substituent on the inhibitory activity and potent
MBI, the ethyl substituted compound 7b was also prepared and
evaluated (Fig. 2). Here, we disclosed our evaluation of these com-
pounds, and the identification of 2-({6-[(3R)-3-amino-3-methylpi-
peridine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyr-
2.2. Influence on in vitro potency
The in vitro DPP-4 inhibitory activity of the compounds listed in
Table 1 was measured using human plasma DPP-4. To evaluate our
compounds DPP-4 inhibitory activity, some approved drugs (1, 2,
and 4) were used as reference. The parent compound 6 showed a
strong DPP-4 inhibitory activity (IC50 = 0.34 nM), being almost
two-fold that of 2 (IC50 = 0.58 nM). As for compound 7a having
(R)-3-amino-3-methyl piperidine, its DPP-4 inhibitory activity
(IC50 = 1.1 nM) was three-fold less than that of 6, but almost the
same as that of 4 (IC50 = 1.0 nM). The ethyl substituted compound
(+)-7b on the other hand showed a DPP-4 inhibitory activity
(IC50 = 26 nM) 80-fold less than that of the parent compound 6.
The other enantiomer (ꢀ)-7b did not have any inhibition against
DPP-4. Based on these findings, only the methyl substituted com-
pound 7a could retain an acceptable DPP-4 inhibitory activity as
we expected. To maintain strong in vitro DPP-4 inhibitory, the sub-
rolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile
(DSR-
12727) (7a) as a potent and orally active DPP-4 inhibitor without
mechanism-based inactivation of Cytochrome P450 3A.
2. Results and discussion
2.1. Chemistry
The 3-amino-3-alkyl-piperidine structures 13a–b were synthe-
sized in four steps.18 The intermediate 9b was prepared from ethyl
nipecotate 8 same as 9a.19 The nitrogen-atom of 9a–b was
protected by a carbobenzyloxy (Cbz) to give 10a–b, which were
hydrolyzed to afford the carboxylic acid 11a–b. After Curtius
F
CN
N
F
CN
N
F
CN
N
O
N
O
N
O
N
Et
H
Me
NH2
NH2
NH2
N
N
N
N
N
N
O
O
O
(+)-7b
6
7a
Figure 2. Structures of test-compounds.

5492
Y. Nishio et al. / Bioorg. Med. Chem. 19 (2011) 5490–5499
O
R
N
R
1CO2R2
1CO2Et
d
b
O
HN
9a: R1 = (R)-Me
10a: R1 = (R)-Me, R2 = Et
10b: R1 = rac-Et, R2 = Et
8: R1 = H
c
a
11a: R1 = (R)-Me, R2 = H
11b: R1 = rac-Et, R2 = H
9b: R1 = rac-Et
O
R
R
1 NHBoc
1NHBoc
e
HN
O
N
13a: R1 = (R)-Me
13b: R1 = rac-Et
12a: R1 = (R)-Me
12b: R1 = rac-Et
Scheme 1. Synthetic route for tetra-substituted amines. Reagents and conditions: (a) NaHMDS (1.2 equiv), EtI (1.0 equiv), toluene, ꢀ20 °C – rt; (b) CbzCl (1.0 equiv), Et3N
(1.5 equiv), THF, rt, overnight; (c) 3 N NaOH aq, THF, MeOH, reflux, 1 day for 10a, 3 days for 10b; (d) DPPA (1.0 equiv), Et3N, toluene, 100 °C, 1 h, then t-BuOK (0.1 equiv), t-
BuOH, 40 °C, 3 h; (e) Pd/C, H2, MeOH, rt, 3 h.
F
Br
O
MeS
NC
R1
a-c
d, e
NHBoc
SMe
O
N
R2O
N
O
H2N
O
15a: R1 = (R)-Me
15b: R1 = rac-Et
15c: R1 = (R)-H
14
O
R2 = Et or tert-Bu
F
Br
F
CN
O
N
O
R1
R1
g, (h)
f
NHBoc
NHBoc
N
N
N
N
N
O
N
O
O
N
16a: R1 = (R)-Me
16b: R1 = rac-Et
16c: R1 = (R)-H
17a: R1 = (R)-Me
17b: R1 = rac-Et
17c: R1 = (R)-H
O
O
O
F
CN
N
F
CN
N
Chiral HPLC
column separation
O
N
O
N
R1
Et
NH2
NH2
N
N
N
rac-7b
N
O
O
(+)-7b: (R)-Et
(-)-7b : (S)-Et
7a: R1 = (R)-Me
rac-7b: R1 = rac-Et
6: R1 = (R)-H as a HCl salt
Scheme 2. Synthetic route for the pyrrolo[3,2-d]pyrimidine structure. Reagents and conditions: (a) 13a, 13b or tert-butyl [(R)-3-piperidine-3-yl]carbamate (1.0 equiv,
respectively), CH3CN, 50 °C, 1 h, then 2-bromo-5-fluorobenzyl amine (1.2 equiv), DBU (2.0 equiv), CH3CN, 80 °C, 10 h; (b) ethyl bromoacetate (1.3 equiv), K2CO3 (3.0 equiv),
DMF, 50 °C, 2 h; (c) LiNH2 (2.5 equiv), t-BuOH (20 equiv), CH3CN, 30 °C, 2 h; (d) KOCN (1.6 equiv), AcOH, 40 °C, 2 h; (e) K2CO3 (1.6 equiv), DMF, 50 °C, 5 h, then MeI (2.0 equiv),
K2CO3 (1.6 equiv), 30 °C, 2 h; (f) Pd(t-Bu3P)2 (0.1 equiv), Zn(CN)2 (0.6 equiv), NMP, 100 °C, 1 h; (g) PhSO3H monohydrate (2.0 equiv), CH3CN, 80 °C, 2 h; (h) only for 6, 1.0 N HCl
aq, MeOH, H2O, rt.
Table 1
interact with Glu205, Glu206, and Tyr662 of the enzyme, we as-
sumed that an ethyl substituent might influence this interaction,
In vitro inhibitory activity of DPP-4 inhibitors
Compound
IC50 (nM)
or the conformation of piperidine, more than a methyl substituent.
1
3.5
2
4
0.58
1.0
0.34
1.1
26
>1.000
2.3. Estimation of the potency of mechanism-based inactivation
(MBI)
6
7aa
(+)-7ba
(ꢀ)-7ba
MBI for CYP enzymes is considered as a risk of drug–drug-inter-
action (DDI). However, in clinical usage not all compounds show-
ing MBI are subject to DDI.21 In general, compounds potential for
DDI can be evaluated using MBI kinetic parameters.22 The apparent
constant inactivation ratio (kobs) is estimated from the initial slope
of a plot of the natural log of the remaining enzymatic activity
a
Absolute configuration has not been determined.
stituent at the 3-position of the piperidine had to be kept small. As
it has been shown that the (R)-3-aminopiperidine of 412 and 513

Y. Nishio et al. / Bioorg. Med. Chem. 19 (2011) 5490–5499
5493
Table 2
Table 3
Test-compounds MBI kinetic parameters
Pharmacokinetic profile of 7a in the rat, dog, and monkey
Compound
kinact (minꢀ1
)
KI (n
l
)
kinact/KI (minꢀ1/nM)
Species
dose (mg/ CL (mL/min/ Cmax (ng/
Vdss (L/
kg)
t1/2
(h)
B.A.
(%)
kg)
kg)
mL)
b
7a
(+)-7b
0.0391
N.A.a
0.0227
0.026
N.A.a
0.022
1.50
–
1.03
Rat
1a
3b
1a
3b
40
–
29
–
38
–
–
76
–
245
–
77
6.5
–
5.8
–
10
–
4.1
3.3
3.1
3.9
4.8
15
–
52
–
91
–
38
Dog
a
N.A. means not applicable. No clear change was observed
Monkey 1a
3b
a
Test compounds were administered intravenously.
Test compounds were administered orally.
(a)-1
(a)-2
b
0µM
0.08
1000
10µM
100µM
Data
Fitting line
0.06
0.04
was more associated with decreased kinact value than KI value
(Fig. 3).
100
10
At first, we assumed that steric hindrance created by the alkyl
substituent prevented metabolism of the amino group. However,
this hypothesis could not accord with the case of an ethyl group.
This led us to investigate the metabolites of 7a. Like in compound
6, we found that metabolism of 7a occurred at the 3-amino posi-
tion and gave a hydroxyl amine as one of the metabolites. When
a dansyl Glutathione (dGSH) trapping assay to detect reactive
0.02
0
0
20
40
60
80
100
[I] (µM)
0
5
10
15
Pre incubation (min)
(b)-1
(b)-2
0µM
5µM
1000
100
10
metabolites of 7a was carried out, production of 0.14 lM dGSH ad-
0.08
duct was observed. These findings indicate that the methyl substi-
tuent does not prevent metabolism, but only affects coordination
with the Fe-center of CYP3A to give MI complex. The reason why
compound (+)-7b having ethyl substituent did not prevent MBI
completely same as compound 7a is unclear and under investiga-
tion now. However, the drop in kinact of (+)-7b was observed, and
the result means that introduction of steric hindrance at the
metabolism sites supposed to give reactive metabolites might re-
duce or avoid the risk of MBI. Based on these results, for example,
DPP-4 inhibitory activity and potent MBI, compound 7a was se-
lected for further evaluation.
0.06
0.04
100µM
Data
0.02
0
0
20
40
60
80
100
[I] (µM)
0
5
10
15
Pre incubation (min)
(c)-1
(c)-2
0µM
1000
0.08
0.06
10µM
100µM
Data
Fitting line
2.4. Compound 7a selectivity against DPP-4 related peptidases
and off-targets
0.04
0.02
0
100
10
0
20
40
[I] (µM)
60
80
100
Before investigating the pharmacokinetics (PK) of compound
7a, the selectivity of this compound against DPP-4 related pepti-
dases and potential off-targets, for example muscarinic receptor
1,13 2, and 423 was evaluated. Compound 7a showed great selectiv-
ity against DPP-4 related peptidases, such as dipeptidyl peptidase II
(DPP-2)24, dipeptidyl peptidase VIII (DPP-8),25 dipeptidyl peptidase
IX (DPP-9),26,27 prolidase, prolyl oligpeptidase (POP), fibroblast
0
5
10
15
Pre incubation (min)
Figure 3. MBI parameter of each test-compound; (a) 6, (b) 7a, and (c) (+)-7b. [1]
CYP3A remaining activity plotted against pre-incubation-time and [2] relationship
between kobs and inactivator concentration [I].
activation protein alpha (FAPa
),28 and aminopeptidase P (Amino.
P) (IC50 >50 lM). As for compound 7a selectivity against off-tar-
against pre-incubation time. The maximum inactivation rate con-
stant (kinact) and the inactivator concentration at half-maximal rate
of enzyme inactivation (KI) are determined by the non-linear least
squares fitting of Eq. (1). [I] is the inactivator concentration.
gets, a thorough evaluation using Pharma ScreenÒ showed no affin-
ity for a wide variety of receptors, including muscarinic receptor 1,
2, and 4 (IC50 >10 lM).
As compound 6 had concerns about inhibition against CYP en-
kobs ¼ ðkinact ½IꢁÞ=ðKI þ ½IꢁÞ
ð1Þ
zymes and hERG18, these profiles of compound 7a were evaluated.
Compound 7 showed weak inhibition of hERG (IC50 = 30
had no or only weak in vitro CYP enzymes inhibition (CYP 1A2,
2A6, 2C8, 2C9, 2C19, and 3A4; IC50 >50 M, CYP 2D6;
IC50 = 38 M). These result let us to carry out further studies of
lM) and
In our research, kinact/KI ratio has been used to compare a given
compound MBI potency.22 Before measuring our compounds
kinact/KI ratio, other DPP-4 inhibitors were assayed to investigate ef-
fects of pharmacophore differences on MBI. While compounds 1, 2,
and 3 does not show any MBI potency, 4 and 5 having (R)-3-amino
piperidine same as 6, have potent MBI (kinact/KI = 4.1 for 4 and 8.8
for 5, respectively). These results strongly support our assumption
that (R)-3-aminopiperidine is the cause of MBI.
l
l
compound 7a.
2.5. Pharmacological evaluation of 7a
PK profile of compound 7a was assessed in the rat, dog, and
monkey (Table 3). Compound 7a showed great bioavailability
(B.A.) in dogs (91%) and moderate B.A. in rats and monkeys (52%
and 38%, respectively). Compound 7a systemic clearance (CL) and
maximal concentration (Cmax) in rats (40 mL/min/kg and 76 ng/
mL, respectively) were similar to those in monkeys (38 mL/min/
MBI kinetic parameters of compounds 6, 7a, and (+)-7b for
CYP3A are given in Table 2. Compound 6 has kinact/KI ratio less than
those of 4 and 5. As expected, compound 7a with a methyl
substituent showed no change in MBI kinetic parameters, but, con-
trary to our expectations, compound (+)-7b exhibited an MBI
approximately 70% lower than that of 6. This decrease in MBI

5494
Y. Nishio et al. / Bioorg. Med. Chem. 19 (2011) 5490–5499
(a)
(b)
120
1000
p.o. 1 mg/kg
100
p.o. 3 mg/kg
100
10
80
60
40
20
0
1
0.1
-6
0
6
12
18
24
30
0
6
12
18
24
-20
Time (hr)
Time (hr)
Figure 4. PK/PD profile of 7a in ZF rats; (a) time course of plasma concentration and (b) plasma DPP-4 activity after oral administration of compound 7 in rats. Each vertical
bar represents the means SD (n = 4–5).
and its protein binding ratio (78% for rat, 59% for dog, 69% for mon-
key, and 73% for human), compound 7a was expected to show
300
(a)
great in vivo efficacy. Next, we evaluated compound 7a in vivo
inhibitory activity for DPP-4 and examined its effect on blood glu-
250
cose level in oral glucose tolerance test (OGTT) using Zucker-fatty
(ZF) rats.
For DPP-4 inhibition experiment, male ZF rats aged 9 weeks
200
150
100
50
were fasted overnight and dosed orally with compound 7a (1.0
or 3.0 mg/kg). Compound 7a in vivo inhibitory activity for DPP-4
was then measured against time. As shown in Figure 4, compound
7a at 3.0 mg/kg produced maximal inhibition of DPP-4 activity 3 h
after administration, and its Cmax was 309 ng/mL with an area un-
der the curve (AUC) of 2167 ng h/mL.
ZF : Vehicle
0.5%MC
0.03mg/kg
0.1mg/kg
0.3mg/kg
1.0mg/kg
For the OGTT test, male ZF rats aged 10 weeks were fasted over-
night and dosed orally with either the vehicle or compound 7a at
different doses (0.03, 0.1, 0.3, and 1.0 mg/kg). Thirty minutes later
(t = 0), the rats were orally administrated glucose (2 g/kg), and
blood glucose levels were measured against time. As shown in
Figure 5, Compound 7a (0.3 or 1 mg/kg) dose-dependently reduced
blood glucose level in ZF rats. Compound 7a decreased blood glu-
cose level against glucose tolerance and DPP-4 activity in a dose-
dependent manner. These findings suggest that the therapeutic ef-
fect of 7a is mediated via increase in GLP-1 level following inhibi-
tion of DPP-4.
lean : Vehicle 0.5%MC
0
-60
-30
0
30
60
90 120
150
Time (minute)
(b)
30000
25000
20000
15000
10000
2.6. Additional profiling of compound 7a
Based on the results described above, compound 7a was further
investigated in safety battery test. For that, it was necessary to
determine the final form of 7a. First, we considered using 7a as
an HCl salt. However, differential scanning calorimetry (DSC) and
thermogravimetric analysis (TGA) revealed that the HCl salt form
of 7a is unstable at low temperatures. In fact, the HCl salt form
of 7a generated heat in DSC and showed decrease in weight at
around 100 °C in TGA. A wide variety of salts, that is, sulfuric salt,
phosphate salt, and methanesulfonic salt were consequently tried,
but were found difficult to treat. Finally, a free amine as an anhy-
drous crystal form was found to be suitable. The free amine form
of 7a was nonhygroscopic under kinetic measurements and dis-
solved in buffered water (pH 1.2, 5.5, and 6.8) over 1.0 mg/mL.
In toxicology studies, compound 7a showed no genotoxicity in
Ames test and no severe target organ toxicity in 2-week re-
peated-dose toxicity studies in rats (n = 5, each sex) and monkeys
(n = 1, each sex). The No observable adverse effect level (NOAEL)
of compound 7a in these studies was 60 mg/kg/day.
5000
0
ZF : Vehicle
0.5%MC
0.03mg/kg
0.1mg/kg
0.3mg/kg
1.0mg/kg
lean : Vehicle
0.5%MC
Figure 5. OGTT of 7a in ZF rats; (a) time course of blood glucose concentration and
(b) AUC of blood glucose concentration. Reactive plasma glucose AUC was
calculated from
0 to 120 min. All test-compounds in Methyl-Cellulose were
administered to ZF rats as a solution with appropriate concentration. Each vertical
bar represents the means SD (n = 4–9). White circle and yellow bar show the
results in lean rat. ⁄⁄P <0.01, Compound 7a-treated Zucker fatty rats versus vehicle-
treated Zucker fatty rats; Dunnett multiple comparison.
kg and 77 ng/mL, respectively), resulting in a close B.A. in these
two species. On the other hand, compound 7a relatively low sys-
temic CL (29 mL/min/kg) and high Cmax (245 ng/mL) in dogs are be-
lieved to contribute to its great B.A. Compound 7a terminal half-
life (t1/2) in dogs (15 h) was longer than that in rats (3.3 h), and
its distribution volume (Vdss) in dogs (10 L/kg) was 1.5-fold higher
than that in rats (6.5 L/kg). Considering DPP-4 inhibitory activity
2.7. Conclusion
In summary, we identified in this study 2-({6-[(3R)-3-amino-
3-methylpiperidine-1-yl]-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahy-

Y. Nishio et al. / Bioorg. Med. Chem. 19 (2011) 5490–5499
5495
dro-5H-pyrrolo[3,2-d]pyrimidine-5-yl}methyl)-4-fluorobenzoni-
3.2. Benzyl (3R)-3-[(tert-butoxycarbonyl)amino]-3-
trile (DSR-12727) (7a) as a potent and orally active DPP-4 inhib-
itor without mechanism-based inactivation of cytochrome P450
3A which is observed in some of other DPP-4 inhibitors. Com-
pound 7a showed good pharmacokinetics, and dose-dependently
reduced blood glucose level in ZF rats. Moreover, compound 7a
exhibited no safety concerns both in vitro and in vivo. It is
therefore expected that compound 7a, as a novel DPP-4 inhibi-
tor, would be useful in the treatment of type 2 diabetes.
methylpiperidine-1-carboxylate (12a)
To a solution of 9a (45 g; 263 mmol) and TEA (28.0 g) in THF
(270 mL) was slowly added CbzCl (45.2 g; 266 mmol) at 0 °C, and
the resulting mixture was allowed to warm to room temperature
with the ice-bath and then stirred overnight. The reaction mixture
was next filtered through Celite, washed with satd NH4Cl aq and
brine, dried over Na2SO4, and the filtrate was concentrated to give
10a (80.0 g), which was used for the next reaction without purifi-
cation. A mixture of 10a, 3 N NaOH aq (150 mL), and MeOH
(150 mL) was stirred under reflux for 1 day, and the solvent was re-
moved. The mixture was then extracted by EtOAc under acidic con-
dition, and the organic layer was dried over Na2SO4 and
concentrated to give the intermediate carboxylic acid 11a
(63.82 g), which was used for the next reaction without purifica-
tion. To a solution of the carboxylic acid and TEA (24.3 g) in toluene
(100 mL) heated to 100 °C was added slowly dropwise DPPA
(61.5 g), and the resulting mixture was stirred for 2 h. After cooling
to room temperature, the reaction mixture was washed with H2O,
dried over Na2SO4, and the filtrate was concentrated to half-vol-
ume. To the residue were added tert-butyl alcohol (24.4 g) and
potassium tert-butoxide (2.20 g), and the resulting slurry was stir-
red for 1 h at 40 °C. The mixture was finally washed with H2O and
brine, dried over Na2SO4, and the filtrate was concentrated. The
residue was purified by SiO2 column chromatography (Hexane/
EtOAc = 15/1 to 7/1) to give 53.0 g (58%) of 12a as a white solid.
1H NMR (400 MHz, CDCl3) d: 1.31 (3H, s), 1.41 (9H, s), 1.20–1.80
(6H, m), 2.80–3.10 (2H, m), 3.80–4.00 (2H, m), 4.40–4.80 (1H, m),
5.00–5.10 (2H, m), 7.28–7.39 (5H, m). LRMS (ESI+): m/z 349.3.
Chiral analytical conditions: The column was Chiral-PackÒ AD-
H, the eluent volume was 0.8 mL/min with Hexane/2-propanol/
diethyl amine = 1/5/0.2 (v/v), the temperature was 30 °C and the
measuring wavelength was set at 254 nm. Under these conditions
12a was detected at 8.5 min (>95.0% ee), and the other chiral iso-
mer was detected at 6.8 min.
3. Experimental section
All reagents and solvents were obtained from commercial
suppliers and used without further drying or purification. Other
DPP-4 inhibitors were synthesized in our laboratory based on
the reported ways. All reactions were performed under nitrogen
atmosphere. Normal-phase column chromatography was carried
out on a Yamazen W-prep system with pre-packed SiO2 columns
or Amino-SiO2 columns. Visualization was performed under UV
light (254 nm) or ninhydrine. 1H NMR spectra were recorded
on Brucker AVANCE 400, and 13C NMR spectra were recorded
on Brucker AVANCE 400 set at 100 MHz; all chemical shift (d)
values were referenced to CDCl3 or DMSO as an internal stan-
dard and reported as shift (proton count, multiplicity, coupling
constant (J)). IR spectra were recorded on a JEOL JIR-SPX60 spec-
trometer as ATR or KBr Mass spectra (LRMS) were recorded on
API 150EX performed by electron spray ionization (ESI) and
high-resolution mass spectra (HRMS) were recorded on a Ther-
mo Fischer Scientific LTQ orbitrap Discovery MS equipment. All
chemical analyses were performed on a CE Instrument EA1110
and a Yokokawa analytical system IC7000. Test compounds pur-
ity was determined by Ultra Fluent Liquid chromatography
(UFLC) with Shim-pack XR-ODC 75 mm ꢂ 3.0. Purity samples
were dissolved in MeOH/0.1% TFA, and the eluent volume was
1.0 mL/min with 10 min-gradient (from 1.0%B to 95%B). Solvent
A was H2O/0.1% TFA and solvent B was CH3CN/0.1% TFA. Test
compounds purity was determined by UFLC and was P95%.
3.3. Benzyl 3-[(tert-butoxycarbonyl)amino]-3-ethylpiperidine-
1-carboxylate (12b)
3.1. 1-Benzyl 3-ethyl 3-ethylpiperidine-1,3-dicarboxylate (10b)
The synthetic route for 12b was the same as the latter part of
the synthesis of 12a. Starting from 10b (7.28 g; 22.8 mmol), 12b
(4.23 g; 51%) was obtained as oil. As for the hydroxylation step,
the reaction needed 3 days.
The 3-ethyl 3-ethylpiperidin carboxylate 9b was synthesized
as reported in the literature.19 To a solution of ethyl nipecotate
8 (7.00 g; 44.6 mmol) in THF (10 mL), was added dropwise so-
dium hexamethyl disilazane (NaHMDS) in THF (1.05 M, 50 ml)
at ꢀ20 °C to ꢀ10 °C, and the resulting solution was stirred for
1 h. EtI (6.95 g; 44.6 mmol) was then added to the solution while
keeping the temperature at ꢀ10 °C to ꢀ20 °C, and the resulting
mixture was allowed to warm to room temperature without
the cold bath. The reaction mixture was next quenched by H2O
and separated, and the organic layer was dried over Na2SO4
and concentrated to afford the intermediate 9b (8.20 g), which
was used in the next reaction without purification. To a solution
of 9b and triethylamine (TEA) (6.75 g) in THF was slowly added
carbobenzyloxy chloride (CbzCl) (6.4 ml) at 0 °C, and the result-
ing mixture was allowed to warm to room temperature with the
ice-bath. The reaction mixture was then filtered through Celite,
washed with satd NH4Cl aq and brine, dried over Na2SO4, and
the filtrate was concentrated. The residue was purified by SiO2
column chromatography (Hexane/EtOAc = 10/1 to 5/1) to give
7.28 g (51%) of 10b as a white solid.
1H NMR (400 MHz, CDCl3) d: 0.84 (3H, bs), 1.41 (9H, s), 1.20–
1.80 (6H, m), 2.70–3.05 (2H, m), 3.70–4.10 (2H, m), 5.00–5.30
(2H, m), 7.28–7.39 (5H, m). LRMS (ESI+): m/z 363.3.
3.4. tert-Butyl [(3R)-3-methylpiperidin-3-yl]carbamate (13a)
A mixture of 12a (10.4 g; 29.9 mmol), 10% Pd/C (50% wet)
(1.05 g), and MeOH (60 mL) was stirred at room temperature under
H2 atmosphere for 3 h. The slurry was then filtered through Celite
and concentrated, and the residue was purified by Amino-SiO2 col-
umn chromatography (CHCl3/MeOH = 20/1 to 10/1) to give 5.16 g
(81%) of 13a as oil.
1H NMR (400 MHz, CDCl3) d: 1.32 (3H, s,), 1.44 (9H, s), 1.50–
1.60 (1H, m), 1.60–1.75 (1H, m), 2.22 (1H, m), 2.50–2.70 (3H, m),
3.00–3.18 (2H, m), 5.09 (1H, bs). LRMS (ESI+): m/z 215.4.
3.5. tert-Butyl (3-ethylpiperidin-3-yl)carbamate (13b)
1H NMR (400 MHz, CDCl3) d: 0.82 (3H, bs), 1.18 (3H, br s),
1.40–1.70 (5H, m), 2.04–2.10 (1H, m), 3.10–3.25 (2H, m), 3.60–
3.65 (1H, m), 4.00–4.18 (3H, m), 5.12 (2H, s), 7.27–7.40 (5H, m).
LRMS (ESI+): m/z 414.2.
The synthetic route for 13b was the same as that for 13a. Start-
ing from 12b (4.23 g; 11.7 mmol), 13b (2.65 g; 99%) was obtained
as oil.

5496
Y. Nishio et al. / Bioorg. Med. Chem. 19 (2011) 5490–5499
1H NMR (400 MHz, CDCl3) d: 0.81 (3H, t, J = 7.5 Hz), 1.22 (1H, dt,
1H NMR (400 MHz, CDCl3) d: 1.25 (3H, s), 1.40 (9H, s), 1.61 (9H,
s), 1.50–1.65 (3H, m), 2.10 (1H, br s), 2.72 (1H, br s), 2.80–2.92 (1H,
m), 2.99–3.12 (2H, m), 3.36 (3H, s), 3.59 (3H, s), 4.54 (1H, br s), 6.16
(1H, d, J = 7.6 Hz), 6.86 (1H, td, J = 8.4, 3.0 Hz), 7.53 (1H, q, J = 8.7,
5.2 Hz). LRMS (ESI+): m/z 678.5, 680.5.
J = 13.6, 4.2 Hz), 1.43 (9H, s), 1.40–1.71 (2H, m), 1.87 (1H, br s), 2.11
(1H, s), 2.27 (1H, s), 2.41 (1H, d, J = 12.2 Hz), 2.55 (1H, dt. J = 11.6,
3.0 Hz), 3.00 (2H, dd, J = 30, 12.1 Hz), 4.85 (1H, bs). LRMS (ESI+):
m/z 229.4.
3.6. 4-tert-Butyl 2-ethyl 3-amino-1-(2-bromo-5-fluorobenzyl)-
5-{(3R)-3-[(tert-butoxycarbonyl)amino]-3-methylpiperidin-1-
yl}-1H-pyrrole-2,4-dicarboxylate (15a)
3.8. tert-Butyl 6-{(3R)-3-[(tert-butoxycarbonyl)amino]-3-
methylpiperidin-1-yl}-5-(2-cyano-5-fluorobenzyl)-1,3-
dimethyl-2,4-dioxo-2,3,4,5-tetrahydro-1H-pyrrolo[3,2-
d]pyrimidine-7-carboxylate (17a)
A mixture of 14 (5.00 g; 20.4 mmol) and 13a (4.37 g; 20.4 mmol)
in CH3CN (60 ml) was stirred at 50 °C for 1 h. After cooling to room
temperature, 1,8-diazabicyclo[5,4,0]undeca-7-ene (DBU) (6.20 g;
40 mmol) and 2-bromo-5-fluoro-benzylamine (4.99 g; 24.4 mmol)
were added to the mixture, and the resulting mixture was stirred
at 80 °C for 10 h. After removal of the solvent, the residue was di-
luted with EtOAc, and washed with H2O, 10%KHSO4 aq, 10%NaOH
aq and brine. The EtOAc layer was dried over Na2SO4 and the filtrate
was concentrated.
The residue was dissolved in DMF (60 mL), and ethyl bromoace-
tate (2.80 mL; 28 mmol) and K2CO3 (8.5 g) were added. The result-
ing slurry was then stirred at 50 °C for 2 h and cooled to room
temperature. The slurry was diluted with EtOAc, filtered through
Celite, and washed with satd NH4Cl aq and brine. The organic layer
was dried over Na2SO4, and the filtrate was concentrated.
To tert-butyl alcohol (30 mL) was added lithium amide (1.16 g;
48 mmol). After stirring at 80 °C for 1 h, the resulting mixture was
cooled to 30 °C, and CH3CN (40 mL) was added. A solution of the
alkylated product from the third step in toluene (10 mL) was next
added dropwise, and the reaction mixture was stirred for 2 h at
30 °C. The solvent was removed, and to the residue was added
EtOAc. The resulting mixture was finally washed with satd NH4Cl
aq and brine, the organic layer was dried over Na2SO4, and the fil-
trate was concentrated. The residue was purified by SiO2 column
chromatography (Hexane/EtOAc = 6/1 to 3/1) to give 5.80 g (44%
from 14) of 15a as amorphous solid. In addition to the target prod-
uct 15a, the ester exchange product (R2 = tert-butyl) was also ob-
tained as an amorphous (415 mg).
A mixture of 16a (204 mg; 0.3 mmol), Pd(t-Bu3P)2 (15 mg;
0.03 mmol), zinc cyanide (22 mg; 0.18 mmol), and N-methyl pyr-
rolidone (NMP) (3.0 mL) was air-vacuated, flushed with N2, and
then stirred for 1 h at 100 °C. After cooling to room temperature,
the reaction mixture was filtered through Celite, and NH4OH-satd
NH4Cl aq-H2O (4/4/1, (v/v)) was added with vigorous stirring.
The formed light-yellow solid was collected by filtration, and stir-
red in CH3CN at 80 °C for 2 h, then at 0 °C for 1 h, and finally fil-
tered to give a crude compound. The crude compound was
purified by SiO2 column chromatography (CHCl3/CH3CN = 15/1 to
5/1) to give 102 mg (54%) of 17a as a white solid.
1H NMR (400 MHz, CDCl3) d: 1.25 (3H, s), 1.39 (9H, s), 1.62 (9H,
s), 1.50–1.65 (3H, m), 2.10 (1H, br s), 2.72 (1H, br s), 2.80–2.92 (1H,
m), 3.05–3.12 (2H, m), 3.34 (3H, s), 3.58 (3H, s), 4.48 (1H, br s),
6.35(1H, d, J = 7.4 Hz), 7.05 (1H, td, J = 8.3, 2.6 Hz), 7.71 (1H, dd,
J = 8.5, 5.2 Hz). LRMS (ESI+): m/z 625.5.
3.9. 2-({6-[(3R)-3-amino-3-methylpiperidine-1-yl]-1,3-
dimethyl-2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-
d]pyrimidine-5-yl}methyl)-4-fluorobenzonitrile (7a)
To a asolution of intermediate 17a (102 mg; 0.17 mmol) in
CH3CN (20 mL), was added dropwise
a solution of PhSO3H
monohydrate (58 mg; 0.33 mmol) in EtOH (5.0 mL) at 40 °C,
and the resulting mixture was stirred at 80 °C for 2 h. After cool-
ing to room temperature, the reaction mixture was diluted with
EtOAc, and 2 N NaOH aq was added to adjust the pH between 8
and 10. The mixture was then separated, and the H2O layer was
washed twice with EtOAc. The collected organic layer was
washed with brine, dried over Na2SO4, and the filtrate was con-
centrated. The residue was purified by Amino-SiO2 column chro-
matography (CHCl3/MeOH = 20/1 to 10/1) to give 70 mg (94%) of
7a as a white solid.
1H NMR (400 MHz, CDCl3) d: 1.13 (3H, t, J = 7.1 Hz), 1.26 (3H, s),
1.39 (9H, s), 1.40–1.60 (3H, m), 1.63 (9H, s), 2.10–2.40 (1H, m),
2.50–2.9 (2H, m), 3.10 (1H, s), 3.40 (1H, m), 4.17 (2H, q,
J = 7.1 Hz), 5.30–5.60 (2H, m), 6.21 (1H, d, J = 9.0 Hz), 6.86 (1H,
td, J = 8.3, 2.8 Hz), 7.51 (1H, dd, J = 8.7, 3.6 Hz). LRMS (ESI+): m/z
653.5, 655.5.
Chiral analytical conditions: The column was Chiral-PackÒ AD-H.
The eluent volume was 0.8 mL/min with Hexane/2-propanol/
diethyl amine = 80/20/0.2 (v/v), the temperature was 25 °C and
the measured wavelength was at 291 nm. Under these conditions
7a was detected at 24.8 min (>99.0% ee), and the other enantiomer
was detected at 29.5 min (>97% ee).
3.7. tert-Butyl 5-(2-bromo-5-fluorobenzyl)-6-{(3R)-3-[(tert-
butoxycarbonyl)amino]-3-methylpiperidin-1-yl}-1,3-dimethyl-
2,4-dioxo-2,3,4,5-tetrahydro-1H-pyrrolo[3,2-d]pyrimidine-7-
carboxylate (16a)
To a solution of 15a (784 mg; 1.6 mmol) and ester exchange
product (42 mg) in AcOH (15 mL) was added dropwise KOCN
(0.20 g; 2.5 mmol) in H2O (0.40 g) at 40 °C. After stirring for 2 h,
the resulting mixture was diluted with EtOAc and washed with
H2O, 20% NaHCO3 aq and brine. The organic layer was then dried
over Na2SO4, and the filtrate was concentrated. The residue was used
for the next reaction without further purification. A mixture of the
residue, K2CO3 (340 mg; 2.2 mmol), and DMF (10 mL) was stirred
at 50 °C for 5 h, and then cooled to 30 °C. To the slurry was added
additional K2CO3 (340 mg; 2.2 mmol), and then methyl iodide
(0.20 mL; 3.2 mmol) was added dropwise. After stirring for 5 h,
H2O (excess) was added to the mixture, and the precipitate was col-
lected by filtration. The white solid was slurred in 2-propanol at
60 °C for 2 h, filtered, and dried to afford 550 mg (51%) of 16a as a
white solid.
1H NMR (400 MHz, DMSO) d: 0.89 (3H, s), 1.28–1.60 (5H, m),
1.61–1.71 (1H, m), 2.59 (2H, q, J = 11.2, 10.3 Hz), 2.70–2.83 (2H,
m), 3.14 (3H, s), 3.38 (3H, s), 5.65 (2H, s), 6.00 (1H, s), 6.34 (1H,
dd, J = 9.6, 2.5 Hz), 7.33 (1H, td, J = 8.5, 2.6 Hz), 7.99 (1H, q,
J = 8.6, 5.5 Hz). 13C NMR (100 MHz, CDCl3) d: 22.1, 27.2, 27.71,
31.8, 37.9, 45.5, 48.2, 54.0, 65.4, 84.7, 106.05, 106.1
(4J(C,F) = 3.3 Hz), 114 (2J(C,F) = 24.0 Hz), 115.3 (2J(C,F) = 22.7 Hz),
116.4, 135.1 (3J(C,F) = 9.5 Hz), 136.3, 146.3 (3J(C,F) = 8.5 Hz),
151.5, 151.7, 155.2, 165.4 (1J(C,F) = 255 Hz). IR (KBr): 2939,
2223, 1689, 1640 cmꢀ1. HRMS (ESI+): m/z 425.2082 (Calcd m/z
425.2096 for C22H25FN6O2 +H). Anal. Calcd for C22H25FN6O2: C,
62.25; H, 5.94; N, 19.80; F, 4.48. Found: C, 61.94; H, 5.86; N,
19.81; F, 4.34. mp 176–179 °C (IPA/H2O). ½a D25
ꢀ9.5 (c 0.93,
ꢁ
EtOH/H2O (95:5, v/v)).

Y. Nishio et al. / Bioorg. Med. Chem. 19 (2011) 5490–5499
5497
3.10. 4-tert-Butyl 2-ehtyl 3-amino-1-(2-bromo-5-fluorobenzyl)-
5-{3-[(tert-butoxycarbonyl)amino]-3-ethylpiperidin-1-yl}-1H-
pyrrole-2,4-dicarboxylate (15b)
was detected at 9.2 min (>97.0% ee), and (ꢀ)-7b was detected at
7.8 min (>99.0% ee).
½
a 2D5
ꢁ
+6.62 (c 1.27, EtOH/H2O (95:5, v/v)) for (+)-7b, and ½a D25
ꢁ
ꢀ5.85 (c 1.27, EtOH/H2O (95:5, v/v)) for (ꢀ)-7b.
The synthetic route for 15b was the same as that for 15a. The
target compound 15b (2.52 g: 34%) was obtained from 14
(2.69 g; 11 mmol).
1H NMR (400 MHz, DMSO) d: 0.64 (1H, t, J = 7.5 Hz), 1.10–1.20
(1H, m), 1.20–1.40 (5H, m), 1.48–1.59 (1H, m), 1.62–1.73 (1H,
m), 2.57 (2H, t, J = 12.1 Hz), 2.70–2.80 (1H, m), 2.80–2.90 (1H, m),
3.14 (3H, s), 3.38 (3H, s), 5.65 (2H, dd, J = 17.0, 9.8 Hz), 6.00 (1H,
s), 6.40 (1H, dd, J = 9.6, 2.5 Hz), 7.33 (1H, td, J = 8.5, 2.6 Hz), 8.00
(1H, dd, J = 8.6, 5.4 Hz). 13C NMR (100 MHz, CDCl3) d: 6.9, 21.6,
27.8, 31.8, 34.7, 45.6, 51.3, 54.1, 54.3, 62.8, 84.7, 85.5, 106.15,
1H NMR (400 MHz, CDCl3) d: 0.76 (3H, br s), 1.20–1.30 (1H, m),
1.40 (9H, s), 1.40–1.70 (2H, m), 1.62 (9H, s), 1.88 (2H, m), 2.80–3.50
(5H, m), 4.17 (2H, q, J = 7.1 Hz), 5.30–5.60 (2H, m), 6.25 (1H, d,
J = 7.2 Hz), 6.86 (1H, td, J = 8.4, 3.0 Hz), 7.52 (1H, dd, J = 8.7,
5.2 Hz). LRMS (ESI+): m/z 667.5, 669.5.
106.2
(4J(C,F) = 3.3 Hz),
114.0
(2J(C,F) = 23.9 Hz),
115.4
(2J(C,F) = 22.8 Hz), 116.4, 135.2 (3J(C,F) = 9.5 Hz), 136.3, 146.4
(3J(C,F) = 8.5 Hz), 151.5, 151.6, 155.2, 165.5 (1J(C,F) = 255 Hz). IR
(ATR): 2935, 2802, 2223, 1699, 1643 cmꢀ1. HRMS (ESI+): m/z
439.2241 (Calcd m/z 439.2252 for C23H27FN6O2 +H). Anal. Calcd
for C23H27FN6O2: C, 63.00; H, 6.21; N, 19.17. Found: C, 62.68; H,
6.23; N, 18.99.
3.11. tert-Butyl 5-(2-bromo-5-fluorobenzyl)-6-{3-[(tert-
butoxycarbonyl)amino]-3-ethylpiperidin-1-yl}-1,3-dimethyl-
2,4-dioxo-2,3,4,5-tetrahydro-1H-pyrrolo[3,2-d]pyrimidine-7-
carboxylate (16b)
The synthetic route for 16b was almost the same as that for 16a.
The target compound 16b (1.60 g; 68%) was obtained from 15b
(2.30 g; 3.4 mmol). Purification was carried out using SiO2 column
chromatgraphy (Hexane/EtOAc = 3/1 to 1/1).
3.15. 4-tert-Butyl 2-ehtyl 3-amino-1-(2-bromo-5-fluorobenzyl)-
5-{(3R)-3-[(tert-butoxycarbonyl)amino]piperidin-1-yl}-1H-
pyrrole-2,4-dicarboxylate (15c)
1H NMR (400 MHz, CDCl3) d: 0.73 (3H, s), 1.40 (9H, s), 1.61 (9H,
s), 1.40–1.65 (3H, m), 1.73–1.82 (1H, m), 2.60–3.00 (3H, m), 3.10–
3.20 (1H, m), 3.35 (3H, s), 3.59 (3H, s), 4.42 (1H, br s), 5.59 (2H, m),
6.19 (1H, d, J = 7.9 Hz), 6.85 (1H, td, J = 8.3, 3.0 Hz), 7.53 (1H, dd,
J = 8.8, 5.2 Hz). LRMS (ESI+): m/z 692.7, 694.7.
The synthetic route for 15c was the same as that for 15a. The
target compound 15c (279 mg; 44%) was obtained from 14
(245 mg; 1.0 mmol) and tert-butyl [(R)-3-piperidine-3-yl]carba-
mate (200 mg; 1.0 mmol).
1H NMR (400 MHz, CDCl3) d: 1.12 (3H, br s), 1.40 (9H, s), 1.30–
1.41 (1H, m), 1.50–1.61 (2H, m), 1.60 (9H, s), 1.70–1.81 (1H, m),
1.88–1.92 (1H, m), 2.50 (1H, m), 2.90–3.30 (2H, m), 3.51-3.80
(1H, m), 4.11 (2H, q, J = 7.1 Hz), 5.40–5.60 (2H, m), 6.23 (1H, d,
J = 7.2 Hz), 6.81 (1H, m), 7.50 (1H, m). LRMS (ESI+): m/z 639.5,
641.5.
3.12. tert-Butyl 6-{3-[(tert-butoxycarbonyl)amino]-3-
ethylpiperidin-1-yl}-5-(2-cyano-5-fluorobenzyl)-1,3-dimethyl-
2,4-dioxo-2,3,4,5-tetrahydro-1H-pyrrolo[3,2-d]pyrimidine-7-
carboxylate (17b)
The synthetic route for 17b was almost the same as that for 17a.
The target compound 17b (1.00 g; 68%) was obtained from 16b
(1.60 g; 2.3 mmol). Purification was carried out using SiO2 column
chromatography (Hexane/EtOAc = 3/1 to 1/1), and was followed by
recrystallization from CH3CN.
3.16. tert-Butyl 5-(2-bromo-5-fluorobenzyl)-6-{(3R)-3-[(tert-
butoxycarbonyl)amino]piperidin-1-yl}-1,3-dimethyl-2,4-dioxo-
2,3,4,5-tetrahydro-1H-pyrrolo[3,2-d]pyrimidine-7-carboxylate
(16c)
1H NMR (400 MHz, CDCl3) d: 0.74 (3H, s), 1.39 (9H, s), 1.61 (9H,
s), 1.50–1.62 (3H, m), 1.65–1.80 (2H, m), 2.80–2.92 (1H, m), 2.99–
3.12 (2H, m), 3.34 (3H, s), 3.58 (3H, s), 4.34 (1H, br s), 5.79 (2H, s),
6.38 (1H, d, J = 9.1 Hz), 7.06 (1H, td, J = 8.2, 2.5 Hz), 7.71 (1H, dd,
J = 8.6, 5.3 Hz). LRMS (ESI+): m/z 639.8
The synthetic route for 16c was almost the same as that for 16a.
The target compound 16c (175 mg; 66%) was obtained from 15c
(263 mg; 0.40 mmol). Purification was carried out using SiO2 col-
umn chromatgraphy (Hexane/EtOAc = 3/1 to 1/1).
1H NMR (400 MHz, CDCl3) d: 1.39 (9H, s), 1.30–1.40 (2H, m),
1.59 (9H, s), 1.50–1.60 (1H, m), 1.73–1.82 (1H, m), 2.70–3.00 (3H,
m), 3.24 (1H, br s), 3.34 (3H, s), 3.57 (3H, s), 3.50–3.80 (1H, m),
4.66 (1H, br s), 5.50–5.80 (2H, m), 6.14 (1H, d, J = 7.9 Hz), 6.83
(1H, td, J = 8.3, 3.0 Hz), 7.52 (1H, dd, J = 8.8, 5.2 Hz). LRMS (ESI+):
m/z 664.7, 666.7.
3.13. 2-({6-[(3R)-3-Amino-3-ethylpiperidin-1-yl]-1,3-dimethyl-
2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-
yl}methyl)-4-fluorobenzonitrile ((+)-7b)
See Section 3.14
3.14. 2-({6-[(3S)-3-amino-3-ethylpiperidin-1-yl]-1,3-dimethyl-
2,4-dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-
yl}methyl)-4-fluorobenzonitrile ((-)-7b)
3.17. tert-Butyl-6-{(3R)-3-[(tert-
butoxycarbonyl)amino]piperidin-1-yl}-5-(2-cyano-5-
fluorobenzyl)-1,3-dimethyl-2,4-dioxo-2,3,4,5-tetrahydro-1H-
pyrrolo[3,2-d]pyrimidine-7-carboxylate (17c)
The synthetic route for 7b was the same as that for 7a. The tar-
get compound rac-7b (0.70 g; 99%) was obtained from 17b (1.00 g;
1.6 mmol).
The racemic compound rac-7b (563 mg) was separated to each
enantiomer by chiral HPLC column resolution at Daicel Co. Ltd, to
afford (+)-7b (250 mg) and (ꢀ)-7b (220 mg) as amorphous solid.
Chiral analytical conditions: The column was Chiral-PackÒ AD-H.
Theeluentvolumewas1.0 mL/minwithHexane/2-propanol/diethyl
amine = 70/30/0.1 (v/v), the temperature was 40 °C and the mea-
sured wavelength was at 264 nm. Under these conditions (+)-7b
The synthetic route for 17c was almost the same as that for 17a.
The target compound 17c (89 mg; 56%) was obtained from 16c
(175 mg; 0.26 mmol). Purification was carried out using SiO2 col-
umn chromatography (Hexane/EtOAc = 3/1 to 1/1).
1H NMR (400 MHz, CDCl3) d: 1.38 (9H, s), 1.35–1.41 (1H, m),
1.60 (9H, s), 1.50–1.80 (3H, m), 2.70–3.00 (3H, m), 3.16 (1H, br
s), 3.34 (3H, s), 3.55 (3H, s), 3.40–3.70 (1H, m), 4.50 (1H, br s),
5.79 (2H, s), 6.39 (1H, d, J = 9.1 Hz), 7.03 (1H, td, J = 8.2, 2.5 Hz),
7.71 (1H, dd, J = 8.6, 5.3 Hz). LRMS (ESI+): m/z 611.8.

5498
Y. Nishio et al. / Bioorg. Med. Chem. 19 (2011) 5490–5499
3.18. 2-({6-[(3R)-3-Amino-piperidin-1-yl]-1,3-dimethyl-2,4-
dioxo-1,2,3,4-tetrahydro-5H-pyrrolo[3,2-d]pyrimidine-5-
yl}methyl)-4-fluorobenzonitrile HCl salt (6)
pling performed 12 h after dosing. The rats received the test-
compound (substance) solution orally by gavage. Blood samples
were collected 0.5 h before (ꢀ0.5 h), and at 0.25, 0.5, 1, 2, 4, 6,
8, 10, 12, 16, and 24 h after dosing (0 h). The time-course of
plasma concentration and change in plasma DPP-4 activity after
administration of 7a (1.0, 3.0 mg/kg) were determined. DPP-4
activity in plasma samples was measured in the same manner
as that for measurement of DPP-4 inhibition rate. Plasma dilu-
tion buffer (5 lL containing 80 mmol/L MgCl2, 140 mmol/L NaCl,
25 mmol/L HEPES, 1% (w/v%) BSA, pH 7.8) was placed into each
well of a 96-well plate and a 96 well Half Area Black with Clear
Flat Bottom 3881 (Corning Incorporated), and 5 lL of each plas-
The synthetic route for 6 was the same as that for 7a. The free
amine form of compound 6 (58 mg; quant) was obtained from
17c (89 mg; 0.14 mmol).
After purification using Amino-SiO2 column chromatography,
the free amine was dissolved in MeOH (2.0 mL). To the resulting
solution was added 1.0 N HCl aq (0.2 mL), and the mixture was
concentrated, co-evaporated with water three times, and dried to
give 62 mg (quant) of 6 as an amorphous solid.
1H NMR (400 MHz, DMSO) d: 1.50–1.60 (2H, m), 1.75–2.00 (2H,
m), 2.70–2.80 (1H, m), 2.81–2.90 (2H, m), 3.12 (3H, s), 3.20–3.40
(2H, m), 3.39 (3H, s), 5.59 (2H, s), 6.12 (1H, s), 6.40 (1H, dd,
J = 9.4, 2.4 Hz), 7.33 (1H, td, J = 8.5, 2.5 Hz), 7.99 (1H, dd, J = 8.6,
5.5 Hz), 8.05–8.20 (3H, m). 13C NMR (100 MHz, DMSO) d: 22.1,
27.1, 27.5, 31.9, 45.3, 46.5, 53.2, 54.7, 86.0, 105.3, 106.3
(4J(C,F) = 2.8 Hz), 113.8 (2J(C,F) = 23.8 Hz), 115.7 (2J(C,F) = 22.6 Hz),
116.8, 136.1, 136.2, 136.4, 146.5 (3J(C,F) = 8.5 Hz), 150.8, 151.0,
154.6, 164.9 (1J(C,F) = 252 Hz). IR (ATR): 2946, 2225, 1685,
1633 cmꢀ1. HRMS (ESI+): m/z 411.1929 (Calcd m/z 411.1939 for
C21H23FN6O2 +H). Anal. Calcd for C21H23FN6O2 HCl 1.25H2O: C,
53.73; H, 5.69; N, 17.90; Cl, 7.55. Found: C, 53.56; H, 5.63; N,
17.95; Cl, 7.47.
ma sample or distilled water (for measurement of background
reaction) was added to individual wells, mixed and left to stand
for at least 5 min. To initiate the reaction, 10 lL of 100 lmol/L
Gly-Pro-MCA solution diluted with substrate dilution buffer
(140 mmol/L NaCl, 25 mmol/L HEPES, 1% (w/v%) BSA, pH 7.8)
was added to each well containing a plasma sample or distilled
water. Two minutes later, fluorescence intensity in RFUs (excita-
tion wavelength 380 nm/emission wavelength 460 nm) was con-
tinuously monitored for 3 min at 30 s-intervals using
a
fluorescence microplate reader (SpectraMaxÒ Gemini EM). Next,
20
40
l
l
L of standard AMC solution (1.28, 2.52, 5, 10, 20, and
mol/L; diluted with plasma dilution buffer, substrate dilu-
tion buffer, rat plasma and DMSO) was placed into each of the
wells with no plasma samples or distilled water, and fluores-
cence intensity in RFUs (excitation wavelength 380 nm/emission
wavelength 460 nm) of the standard AMC solution was mea-
sured to construct a calibration curve of RFU against AMC per
well. The curve was constructed on an arithmetic scale using un-
weighted linear regression implemented in software, Soft-
MaxÒPro version 4.3.1 (Molecular Devices Corporation), within
the fluorescence microplate reader. The rates of AMC production
in the samples and background wells were calculated using the
calibration curve. The net rate of AMC production (nmol/min)
in a given sample well was determined by subtracting mean
background rate from the rate for the sample well. DPP-4 activ-
ity (mU/mL) per plasma unit was calculated by dividing the rate
of AMC production by the volume of plasma sample used for the
measurement. Duplicate measurements were performed for stan-
dard AMC solution and for plasma samples.
3.19. Measurement of DPP-4 inhibition rate
DPP-4 activity in plasma samples was measured using Glycyl-L-
Proline 4-Methylcoumaryl-7-Amide (Gly-Pro-MCA) as substrate.
Relative fluorescence units (RFUs) of 7-amino-4-methylcoumarin
(AMC) liberated from Gly-Pro-MCA by DPP-4 in plasma were mea-
sured to determine the amount of cleaved substrate, and DPP-4
activity was calculated from this measurement (1 U = 1 lmol sub-
strate cleavage/min). DPP-4 concentration in human blood plasma
was adjusted to 10% and used as source of enzyme with the con-
centration of each compound. The final concentration was adjusted
to 50 lL by addition of the substrate Gly-Pro-MCA. Fluorescence
intensity in RFUs (excitation wavelength 380 nm/emission wave-
length 460 nm) was continuously monitored by a fluorescence
plate reader at room temperature (excitation at 380 nm, measure-
ment at 460 nm) to determine enzymatic activity. The concentra-
tion of each test-compound that inhibited enzymatic activity by
3.22. OGTT test procedure
50% was determined as IC50
.
Zucker fatty rats (male, 10 weeks) and Zucker lean rats (male,
10 weeks old) described as lean in Figure 5 were fasted for 24 h
3.20. Estimation of MBI parameters
starting from the day before each OGTT. Glucose (0.2 g/mL
cose solution) was orally loaded by gavage in a volume of 10 mL/
kg (2 g of -glucose/kg). Each concentration of 7a (0.03, 0.1, 0.3,
and 1.0 mg/kg) suspension or 0.5% Methyl Cellulose (MC) solution
used as a control was administered once 30 min prior to glucose
loading. Blood samples were taken just before administration of
test-substance or 0.5% MC solution (ꢀ30 min relative to the time
of glucose loading), just before glucose loading (taken as 0 min),
and 10, 30, 60, and 120 min after glucose loading for determination
of blood glucose concentration. Blood glucose concentration was
D-glu-
A mixture of human liver microsomes and appropriate dilutions
of test-compound (0, 1, and 10 lL) was pre-incubated for 15 min at
D
37 °C with or without NADPH. The mixture was next diluted 20-
fold with assay buffer containing midazolam, a probe substrate
of CYP3A, and NADPH, incubated for an additional 5 min at 37 °C,
and quenched in three-fold the amount of MeOH to completely
stop the reaction. The amount of midazolam metabolite, which
was in the form of 1-hydroxy midazolam, was measured by LC/
MS/MS to determine the remaining CYP3A activity. The decrease
in activity with and without NADPH was divided by time
(15 min) to give kobs, which in turn provided the concentration of
the inactivator [I] in Eq. (1), kinact and KI, and subsequently allowed
determination of MBI.
measured for all blood collection time points. A 10
taken from the tail vein of each rat was immediately mixed with
100 L of 0.62 mol/L perchloric acid solution. Potassium carbonate
solution (50 L, 0.37 mol/L) was then added and mixed. The depro-
lL blood sample
l
l
teinized samples were stored in a refrigerator set at 4 °C until mea-
surement of blood glucose concentration. Blood glucose
concentration was measured using a commercially available kit,
Glucose CII test Wako (Wako Pure Chemical Industries, Ltd). The
deproteinized samples were centrifuged at 2,000 rpm for 10 min,
3.21. PK/PD test procedure
Zucker fatty rats (male, 9 weeks old) were fasted for 24 h
starting from removal of food until the start of pre-dose blood
sampling (ꢀ0.5 h), and kept fasted until the end of blood sam-
and 15 lL of the supernatant of each test sample and glucose

Y. Nishio et al. / Bioorg. Med. Chem. 19 (2011) 5490–5499
5499
10. Villhauer, E. B.; Brinkman, J. A.; Naderi, G. B.; Burkey, B. F.; Dunning, B. E.;
Prasad, K.; Mangold, B. L.; Russell, M. E.; Hughes, T. E. J. Med. Chem. 2003, 46,
2774.
11. Augeri, D. J.; Robl, J. A.; Betebenner, D. A.; Magnin, D. R.; Khanna, A.; Robertson,
J. G.; Wang, A.; Simpkins, L. M.; Taunk, P.; Huang, Q.; Han, S.-P.; Abboa-Offei, B.;
Cap, M.; Xin, L.; Tao, L.; Tozzo, E.; Welzel, G. E.; Egan, D. M.; Marcinkeviciene, J.;
Chang, S. Y.; Biller, S. A.; Kirby, M. S.; Parker, R. A.; Hamann, L. G. J. Med. Chem.
2005, 48, 5025.
standard solutions (100, 250, 500, and 750 mg/dL) were added to
96-well plates, followed by addition of 200 L of coloring reagent
to each well and mixing. Color reaction was allowed to develop
for 18–35 min at room temperature and absorbance was measured
using a microplate reader (SpectraMaxÒ190) at a main wavelength
of 505 nm and a sub-wavelength of 700 nm. A linear calibration
l
12. (a) Gwaltney, S. L.; Aertgeerts, K.; Feng, J.; Kaldor, S. W.; Kassel, D. B.; Manuel,
M.; Navre, M.; Prasad, G. S.; Shi, L.; Skene, R. J.; Stafford, J. A.; Wallace, M.;
Wu, R.; Ye, S.; Zhang, Z. Abstaracts of Papers, 231st, Meeting of the American
Chemical Society, Atlanta, GA, 2006; American Chemical Society: Washington,
DC, 2006; MEDI-018.; (b) Feng, J.; Zhang, Z.; Wallace, M. B.; Sttaford, J. A.;
Kaldor, S. W.; Kassel, D. B.; Navre, M.; Shi, L.; Skene, R. J.; Asakawa, T.; Takeuchi,
K.; Xu, R.; Webb, D. R.; Gwaltney, S. L. J. Med. Chem. 2007, 50, 2297.
13. Eckhardt, M.; Langkopf, E.; Mark, M.; Tadayyon, M.; Thomas, L.; Nar, H.;
Pfrengle, W.; Guth, B.; Lotz, R.; Sieger, P.; Fuchs, H.; Himmelsbach, F. J. Med.
Chem. 2007, 50, 6450.
curve was constructed from absorbance differences (A505 ꢀ A700
,
Y-axis) of glucose standard solutions (mg/dL, X-axis), using a soft-
ware provided with the microplate reader (SoftMaxÒPro version
4.3.1). Glucose concentration of each test sample was determined
by interpolation of its absorbance difference (A505 ꢀ A700) on the
standard curve. Each sample and standard solutions were deter-
mined in duplicate.
14. (a) Nakahira, H.; Hochigai, H. World Patent 2004048379, 2004; (b)
Nakahira, H.; Hochigai, H; Takeda, T.; Kobayashi, T.; W. Ewan Hume. World
Patent 2005021550, 2005; (c) Nakahira, H.; Hochigai, H.; Uchiyama, K.; World
Patent 2006030847, 2006; (d) Nakahira, H.; Kimura, H.; Hochigai, H. World
Patent 2006068163, 2006; (e) Nakahira, H.; Kimura, H.; Hochigai, H. World
Patent 2006112331, 2006
Acknowledgments
We are thankful to Mr. H. Hochigai for providing compound 6,
Mr. S. Suetsugu for making the metabolite of compounds, K. Bando
for performing the elemental analysis, and Ms. I. Taoka for record-
ing MS spectra.
15. For a review, see: Kalgutkar, A. S.; Obach, R. S.; Maurer, T. S. Curr. Drug Metab.
2007, 8, 407.
16. Tang, W.; Stearns, R. A.; Wang, R. W.; Miller, R. R.; Chen, Q.; Ngui, J.; Bakshi, R.
K.; Nargund, R. P.; Dean, D. C.; Baillie, T. A. Xenobiotica 2008, 38, 1437.
17. Cerny, M. A.; Hanzlik, R. P. Arch. Biochem. Biophys. 2005, 436, 265.
18. (a) Nakahira H.; Nishio, Y.; Kimura, H.; World Patent 2009084497, 2009; (b)
Nishio, Y.; Kimura, H.; Tosaki, S.; Sugaru, E.; Sakai, M.; Horiguchi, M.; Masui, Y.;
Ono, M.; Nakagawa, T.; Nakahira, H. Bioorg. Med. Chem. Lett. 2010, 20, 7246.
19. Nelson, T. D.; Rosen, J. D.; Smitrovich, J. H.; Payack, J.; Craig, B.; Matty, L.;
Huffman, M. A.; McNamara, J. Org. Lett. 2005, 7, 55.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.07.042.
20. Nishio, Y.; Uchiyama, K.; Kito, M.; Nakahira, H. Tetrahedron 2011, 67, 3124.
21. (a) Ma, B.; Prueksaritanont, T.; Lin, J. H. Drug Metab. Dispos. 1999, 28, 125; (b)
Hollenberg, P. F.; Kent, U. M.; Bumpus, N. N. Chem. Res. Toxicol. 2008, 21, 189.
22. Obach, R. S.; Walsky, R. L.; Venkatakrishnan, K. Drug Metab. Dispos. 2006, 35,
246.
23. Kowalchick, J. E.; Leiting, B.; Pryor, K. D.; Marsilio, F.; Wu, J. K.; He, H.; Lyons, K.
A.; Eiermann, G. J.; Petrov, A.; Scapin, G.; Patel, R. A.; Thornberry, N. A.; Weber,
A. E.; Kim, D. Bioorg. Med. Chem. Lett. 2007, 17, 5934.
24. McDonald, J. K.; Leibach, F. H.; Grindeland, R. E.; Ellis, S. J. Biol Chem. 1968, 243,
4143.
25. Abbott, C. A.; Yu, D. M.; Woollatt, E.; Sutherland, G. R.; McCaughan, G. W.;
Gorrell, M. D. Biochem 2000, 267, 6140.
References and notes
1. Colberg, S. R. ACSM’S Health Fitness J. 2008, 12, 16. For latest information, please
see the website of World Health Organization at http://www.who.int/diabetes/
facts/world_figures/en/.
2. For
a review, see: (a) Mizuno, C. S.; Chittiboyina, A. G.; Kurtz, T. W.;
Pershadsingh, H. A.; Avery, M. A. Curr. Med. Chem. 2008, 15, 61; (b) Barnett,
A. H. Br. J. Diab. Vasc. Dis. 2008, 8, 45.
3. For some references, see: (a) Holst, J. J. Diabetologia 2006, 49, 253; (b) Knudsen,
L. B. J. Med. Chem. 2004, 47, 4128.
4. (a) Mentlein, R.; Gallwitz, B.; Schmidt, W. E. J. Eur. Biochem. 1993, 214, 829; (b)
Kieffer, T. J.; McIntosh, C. H.; Pederson, T. A. Endocrinology 1995, 136, 3585; (c)
Holst, J. J.; Deacon, C. F. Diabetologia 2005, 48, 612; For reviews, see: (d) Deacon,
C. F. Horm. Metab. Res. 2004, 36, 761.
5. Farilla, L.; Hui, H.; Bertolotto, C.; Kang, E.; Bulotta, A.; Di Mario, U.; Perfetti, R.
Endocrinology 2002, 143, 4397.
6. Reimer, M. K.; Holst, J. J.; Ahren, B. Eur. J. Endocrinol. 2002, 146, 717.
7. Pospisilik, A.; Martin, J.; Doty, T.; Ehses, J. A.; Pamir, N.; Lynn, F. C.; Piteau, S.;
Demuth, H. –U.; McIntosh, C. H. S.; Pederson, R. A. Diabetes 2003, 52, 741.
8. For a review, see: Havale, S. H.; Manojit, Pal Bioorg. Med. Chem. 2009, 17, 1783.
9. (a) Kim, D.; Wang, L.; Beconi, M.; Eiermann, G. J.; Fisher, M. H.; He, H.; Hickey,
G. J.; Kowalchick, J. E.; Leiting, B.; Lynos, K.; Marsilio, F.; McCann, M. E.; Patel, R.
A.; Petrov, A.; Scapin, G.; Patel, S. B.; Roy, R. S.; Wu, J. K.; Wyvratt, M. J.; Zhang,
B. B.; Zhu, L.; Thronberry, N. A.; Weber, A. E. J. Med. Chem. 2005, 48, 141; (b)
Miller, S. A.; St. Onge, E. L. Ann. Pharmcother. 2006, 40, 1336.
26. Olse, C.; Wagtmann, N. Gene 2002, 299, 185.
27. (a) Lankas, G. R.; Leiting, B.; Sinha Roy, R.; Eiermann, G. J.; Beconi, M. G.; Biftu,
T.; Chan, C.-C.; Edmondson, S.; Feeney, W. P.; He, H.; Ippolito, D. E.; Kim, D.;
Lynos, K. A.; Ok, H. O.; Patel, R. A.; Petrov, A. N.; Pruor, K. A.; Qian, X.; Reigle, L.;
Woods, A.; Wu, J. K.; Zaller, D.; Zhang, X.; Zhu, L.; Weber, A. E.; Thronberry, N.
A. Diabetes 2005, 54, 2988; (b) Burkey, B. F.; Hoffmann, P. K.; Hassiepen, U.;
Trappe, J.; Juedes, M.; Foley, J. E. Diab. Obes. Metab. 2008, 10, 1057; (c) Wu, J.-J.;
Tang, H.-K.; Yeh, T.-K.; Chen, C.-M.; Shy, H.-C.; Chu, Y.-R.; Chien, C.-H.; Tsai, T.-
Y.; Huang, Y.-C.; Huang, Y.-L.; Huang, C.-H.; Tseng, H.-Y.; Jiaang, W.-T.; Chao,
Y.-S.; Chen, X. Biochem. Pharmacol. 2009, 78, 203.
28. Scanlan, M. J.; Raj, B. K. M.; Calvo, B.; Garin-Chesa, P.; Sanz-Moncasi, M. P.;
Healey, J. H.; Old, L. J.; Rettig, W. J. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5657.