Discovery and preclinical profile of teneligliptin (3-[(2S,4S)-4-[4-(3- methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-ylcarbonyl] thiazolidine): A highly potent, selective, long-lasting and orally active dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes

Article abstract of DOI:10.1016/j.bmc.2012.08.012

Dipeptidyl peptidase IV (DPP-4) inhibition is suitable mechanism for once daily oral dosing regimen because of its low risk of hypoglycemia. We explored linked bicyclic heteroarylpiperazines substituted at the γ-position of the proline structure in the course of the investigation of l-prolylthiazolidines. The efforts led to the discovery of a highly potent, selective, long-lasting and orally active DPP-4 inhibitor, 3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5- yl)piperazin-1-yl]pyrrolidin-2-ylcarbonyl]thiazolidine (8g), which has a unique structure characterized by five consecutive rings. An X-ray co-crystal structure of 8g in DPP-4 demonstrated that the key interaction between the phenyl ring on the pyrazole and the S₂ extensive subsite of DPP-4 not only boosted potency, but also increased selectivity. Compound 8g, at 0.03 mg/kg or higher doses, significantly inhibited the increase of plasma glucose levels after an oral glucose load in Zucker fatty rats. Compound 8g (teneligliptin) has been approved for the treatment of type 2 diabetes in Japan.

Full text of DOI:10.1016/j.bmc.2012.08.012

Bioorganic & Medicinal Chemistry 20 (2012) 5705–5719
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and preclinical profile of teneligliptin (3-[(2S,4S)-
4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-ylcarbon-
yl]thiazolidine): A highly potent, selective, long-lasting and orally active
dipeptidyl peptidase IV inhibitor for the treatment of type 2 diabetes
Tomohiro Yoshida a, , Fumihiko Akahoshi a, , Hiroshi Sakashita a,à, Hiroshi Kitajima a,
⇑
Mitsuharu Nakamura a, Shuji Sonda a,§, Masahiro Takeuchi a, Yoshihito Tanaka a, Naoko Ueda a,
Sumie Sekiguchi a,–, Takayuki Ishige a, Kyoko Shima a, Mika Nabeno a, Yuji Abe a, , Jun Anabuki a,
Aki Soejima a, Kumiko Yoshida a, Yoko Takashina a, Shinichi Ishii a, Satoko Kiuchi a, Sayaka Fukuda a,
Reiko Tsutsumiuchi a,§, Keigo Kosaka a, Takahiro Murozono a,k, Yoshinobu Nakamaru a, Hiroyuki Utsumi a,
Naoya Masutomi a, Hiroyuki Kishida b, Ikuko Miyaguchi b, Yoshiharu Hayashi a,
a Research Division, Mitsubishi Tanabe Pharma Corporation, 2-2-50, Kawagishi, Toda-shi, Saitama 335-8505, Japan
b R&D Strategy Dept., Corporate Strategy Division, Mitsubishi Chemical Corporation, 1000, Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Dipeptidyl peptidase IV (DPP-4) inhibition is suitable mechanism for once daily oral dosing regimen
because of its low risk of hypoglycemia. We explored linked bicyclic heteroarylpiperazines substituted
at the c-position of the proline structure in the course of the investigation of L-prolylthiazolidines. The
Received 19 July 2012
Revised 9 August 2012
Accepted 9 August 2012
Available online 17 August 2012
efforts led to the discovery of a highly potent, selective, long-lasting and orally active DPP-4 inhibitor,
3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-ylcarbonyl]thiazol-
idine (8g), which has a unique structure characterized by five consecutive rings. An X-ray co-crystal
structure of 8g in DPP-4 demonstrated that the key interaction between the phenyl ring on the pyrazole
and the S2 extensive subsite of DPP-4 not only boosted potency, but also increased selectivity. Compound
8g, at 0.03 mg/kg or higher doses, significantly inhibited the increase of plasma glucose levels after an
oral glucose load in Zucker fatty rats. Compound 8g (teneligliptin) has been approved for the treatment
of type 2 diabetes in Japan.
Keywords:
DPP-4 inhibitor
Thiazolidine
Bicyclic heteroarylpiperazine
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
elevated hepatic glucose production, and reduced glucose-stimu-
lated insulin secretion. Moreover, long-term lack of glycemic con-
Type 2 diabetes is a rapidly growing metabolic disease that af-
fects millions of people worldwide,1 and is characterized by ele-
vated fasting plasma glucose, insulin resistance, abnormally
trol increases the risk of micro- and macrovascular complications,
such as coronary artery disease, stroke, hypertension, nephropathy,
peripheral vascular disease, neuropathy and retinopathy.2 Current
treatment strategies include reducing insulin resistance, supple-
menting the insulin deficiency with exogenous insulin, enhancing
endogenous insulin secretion, reducing hepatic glucose output,
and limiting glucose absorption.3 Among them, insulin secreta-
gogues such as glinides (e.g., repaglinide), which are widely used,
need to be taken before each meal for avoiding a risk of hypoglyce-
mia. Therefore antihyperglycemic agents which are orally available
in a once-daily regimen without causing hypoglycemia have been
much awaited in medical settings.
⇑
Corresponding author.
E-mail address: Akahoshi.Fumihiko@mk.mt-pharma.co.jp (F. Akahoshi).
Present address: Development Division, Mitsubishi Tanabe Pharma Corporation,
17–10, Nihonbashi-Koamicho, Chuo-ku, Tokyo 103-8405, Japan.
à
Present address: Tanabe R&D Service Corporation, 1000, Kamoshida-cho, Aoba-
ku, Yokohama 227-0033, Japan.
§
Present address: CMC Division, Mitsubishi Tanabe Pharma Corporation, 3-16-89,
Kashima, Yodogawa-ku, Osaka-shi, Osaka 532-8505, Japan.
–
Present address: Pharmacovigilance & Quality Assurance Division, Mitsubishi
Tanabe Pharma Corporation, 17–10, Nihonbashi-Koamicho, Chuo-ku, Tokyo 103-
8405, Japan.
Present address: Pharmacovigilance & Quality Assurance Division, Mitsubishi
Tanabe Pharma Corporation, 2-6-6, Hiranocho, Chuo-ku, Osaka-shi, Osaka 541-0046,
Japan.
An incretin hormone, glucagon-like peptide-1 (GLP-1) stimu-
lates insulin biosynthesis and secretion in response to meal inges-
tion, inhibits glucagon secretion, and promotes proliferation of
pancreatic b cells.4 In contrast to other insulinotropic agents, for
k
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.08.012

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T. Yoshida et al. / Bioorg. Med. Chem. 20 (2012) 5705–5719
example, sulfonylureas, the insulinotropic effect of GLP-1 depends
even more closely on the actual plasma glucose concentration pro-
viding the possibility of glucose normalization with minimizing
the risk of hypoglycemia. Continuous infusion of GLP-1 for 6 weeks
to patients with type 2 diabetes increased insulin secretion and
normalized both fasting and post-prandial blood glucose levels
without serious side-effects.5 On the basis of the above evidence,
GLP-1 itself is one of the logical candidates for therapeutic agent
in the treatment of diabetes.6 However, active GLP-1 (GLP-1[7–
36]amide) is rapidly degraded by dipeptidyl peptidase IV (DPP-
4),7 which is a serine protease to produce a dipeptide and an
inactive GLP-1.8 DPP-4 inhibition increases the plasma concentra-
tion of active GLP-1 and causes the secretion of insulin in response
to increased blood glucose level.9 Therefore, DPP-4 inhibitors are
expected to be glucose-lowering, safer and once daily agents.10
Several DPP-4 inhibitors have already been approved, including
sitagliptin (1), vildagliptin (2), alogliptin (3), saxagliptin (4) and
linagliptin (5) (Fig. 1).11 Several other candidates have been re-
ported to be in an advanced stage in clinical trials for type 2
diabetes.10b
DPP-4 is a dipeptidase that recognizes an amino acid sequence
having proline or alanine at the N-terminal penultimate (P1) posi-
tion,12 and many DPP-4 inhibitors therefore have substituted pyr-
rolidines or thiazolidines as a proline mimetic in the P1 part.13 In
particular, pioneering inhibitors possess an electrophilic trap such
as a nitrile group to form a covalent bond with the Ser630 of the
catalytic triad in the active site. DPP-4 inhibitors possessing the
electrophilic trap have two main problems: (i) general chemical
instability, and (ii) low selectivity against other related prolyl pep-
tidases, DPP-8 and DPP-9.14 Their chemical instability is due to
intramolecular cyclization between the electrophilic nitrile and
the amine of the P2 part,15 and the resulting lack of durability
makes it difficult to control plasma glucose all day long for once-
daily treatment. The low selectivity is probably due to covalent
bond formation with the Ser630 which is a common amino acid
conserved in the S1 subsite of DPP-4, DPP-8 and DPP-9.16 Since
DPP-8 and DPP-9 inhibition are reported to be associated with
multiorgan toxicities in rats and dogs and inhibition of T cell acti-
vation/proliferation,17 DPP-4 selectivity is one of the key issues for
clinical use.
2-quinolylpiperazine substituent in DPP-4 inhibitory activity.19b
Moreover, the X-ray crystal structure determination of compound
7a in complex with human DPP-4 indicated that interactions gen-
erated between the quinolyl ring and the S2 extensive subsite en-
hanced the DPP-4 inhibitory activity and selectivity.19b
Here we addressed the introduction of another nonlinear struc-
ture, a linked bicyclic heteroaryl group on the piperazine or piper-
idine moiety, instead of a fused bicyclic heteroaryl group (Fig. 2),
and discovered highly efficacious long-lasting DPP-4 inhibitors
that have led to the identification of compound 8g (teneligliptin),
which has been approved for the treatment of type 2 diabetes in
Japan.
2. Results and discussion
2.1. Rational design
To explore a new type of substituent that can interact with the
S2 extensive subsite more effectively, we focused on the introduc-
tion of a nonlinear, linked, bicyclic group on the piperazine or
piperidine moiety (Fig. 2). A docking study was carried out to pre-
dict the difference between compounds having a six-membered
ring and those having a five-membered ring as a linker connecting
the terminal phenyl group and the piperazine or piperidine group.
We selected ortho-substituted biphenyl and 1-phenylimidazol-2-yl
groups (8a, 8b) as a nonlinear linked bicycle. Figure 3 shows dock-
ing models of compounds 8a and 8b both of which are superim-
posed on the cocrystal structure of 7a in the DPP-4 active site
(PDB code: 3VJM).19b While the phenyl group on compound 8b
faced Arg358 in the S2 extensive subsite, that of compound 8a
faced the opposite direction of Arg358 presumably due to steric
repulsion. Thus, compound 8b was expected to have similar
potency to compound 7a and we decided to synthesize 1-aryl-
imizaol-2-yl, 1-arylterazol-5-yl, and 1-arylpyrazol-5-yl derivatives
to optimize the substituent for the S2 extensive subsite.
2.2. Synthesis of derivatives having a linked bicyclic group
4-Heterocycle-substituted piperazinyl intermediates 12 were
synthesized following the routes illustrated in Scheme 1. Reaction
of N-Cbz-protected piperazine 10a and phenyl isothiocyanate fol-
lowed by methylation afforded methylthioimidate 11a, which
was cyclized with aminoacetaldehyde dimethyl acetal by heating
and then deprotected by hydrogenolysis to afford phenylimidazole
12a. After treatment of the piperazine 10a with cyclohexyl isocya-
nate, compound 11b was cyclized into the corresponding tetrazole
according to the literature procedure,20 and then the Cbz group
was removed to afford cyclohexyltetrazole 12b. Pyrazole deriva-
tives 12c–12n were synthesized by dehydration of ß-ketoamides
11c–11e with various arylhydrazines followed by cyclodehydra-
tion with phosphorus oxychloride in pyridine21 and subsequent
deprotection. The synthesis of ß-ketoamides 11c-11e is as follows.
Piperazine 10a was condensed with 3,3-diethoxypropionic acid in
the presence of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiim-
ide/3-hydroxybenztriazole followed by treatment with 50% trifluo-
roacetic acid/water to afford formylacetamide 11c, which was used
in the next pyrazole synthesis step without purification. Treatment
of N-Boc-protected piperazine 10b with diketene gave acetoaceta-
mide 11d. Acetylation of piperazine 10a followed by aldol reaction
with trifluoroacetate yielded trifluoroacetoacetamide 11e.
We started our research on novel DPP-4 inhibitors without the
electrophilic nitrile moiety to increase chemical stability and fo-
cused on the substituent at the
c-position of the proline moiety
of the prolylthiazolidine core structure to increase affinity with
the S2 subsite.18 We previously reported that substitution of aryl-
piperazine (6) or fused bicyclic heteroarylpiperazine (7) resulted
in highly potent and long-lasting inhibitors.19 The SAR study of
fused bicyclic heteroarylpiperazine parts revealed that the
nonlinear (L-shaped) structure such as a 4-quinolylpiperazine sub-
stituent was more suitable than the linear (I-shaped) one such as a
F
OH
F
O
CN
NH2
NH2
O
H3C
O
N
N
N
N
N
N
N
H
F
N
CN
O
N
CF3
sitagliptin (1)
vildagliptin (2)
alogliptin (3)
CH3
O
HO
NH2
The procedure for the synthesis of piperidinyl intermediates 15
and 20 is summarized in Scheme 2. Coupling of N-Cbz-protected
isonipecotic acid (13) with aniline followed by tetrazole-forming
reaction using trimethylsilylazide under Mitsunobu conditions22
and deprotection gave phenyltetrazole 15. Aldol reaction of
4-acetylpyridine 16 with ethyl trifluoroacetate and subsequent
N
N
N
N
N
N
H2N
N
O
N
CH3
CN
O
CH3
saxagliptin (4)
linagliptin (5)
Figure 1. DPP-4 inhibitors.

T. Yoshida et al. / Bioorg. Med. Chem. 20 (2012) 5705–5719
5707
DPP-4
S2 subsite
DPP-4
X
S2 subsite
S2 subsite DPP-4
CF3
N
Ar
N
N
N
γ
N γ
N
R
S
S
S
S2 extensive
N
N
N
N
H
N
H
S2 extensive
subsite
N
H
subsite
O
O
O
6: Ar = aryl, heteroaryl
7a, IC50 = 0.37 nmol/L
8: X = N
9: X = CH
7: Ar = fused bicyclic heteroaryl
Figure 2. (S)-
c
-substituted L-prolythiazolidines as DPP-4 inhibitors.
N
N-Boc-trans-4-hydroxy-
21.
D
-proline was used instead of the
L-proline
N
N
N
N
N
2.3. In vitro and in vivo DPP-4 inhibition
S
S
N
N
N
H
N
H
O
O
A series of the prolylthiazolidine analogs 6, 7, 8 and 9 was eval-
uated for DPP-4 inhibitory activity in human and rat plasma
in vitro by fluorescence assay using Gly-Pro-MCA and the results
are listed in Tables 1 and 2. Since evaluation of ex vivo plasma
DPP-4 inhibition can be utilized to predict the efficacy of antihy-
perglycemic activity as a surrogate biomarker and provide infor-
mation of pharmacokinetic–pharmacodynamic relationships, the
plasma DPP-4 activity was measured at 0.5 h and 9 h after oral
8a
8b
dosing (3 lmol/kg) of each compound in rats (Tables 1 and 2).
We previously reported that 2- and 4-pyridyl analogs 6a and 6b
had a moderate DPP-4 inhibitory activity19a and that 4-quinolyl
compound 7c having a nonlinear structure in the piperazine-aryl
part was more potent than 2-quinolyl compound 7b having a lin-
ear one.19b The comparisons of the activity between compounds
6a, 6b, 7b and 7c suggested that the nonlinear structure on the
piperazine such as compound 7c was suitable for potent DPP-4
inhibition. X-ray crystal structure determination of compound 7a
Figure 3. Docking models of linked bicyclic compounds 8a and 8b superimposed
on the X-ray co-crystal structure of compound 7a in the DPP-4 active site.
Compound 7a is shown as orange stick. Compounds 8a and 8b are shown as green
and pink sticks, respectively.
indicated that CH–p interactions between the quinolyl ring and
the guanidinyl group of Arg358 of the S2 extensive subsite en-
hanced the DPP-4 inhibitory activity.19b To explore a linked bicyclic
group on the piperazine or piperidine moiety instead of the fused
bicyclic ones as another nonlinear structure, we attempted the
introduction of 1-arylimizaol-2-yl, 1-arylterazol-5-yl, and 1-aryl-
pyrazol-5-yl groups on the piperazine or piperidine moiety.
While introduction of a 4-phenylthiazol-2-yl group (8c), which
has a linear structure, led to a decrease in DPP-4 inhibitory activity
(4.5 nmol/L), introduction of a nonlinear structure such as a 1-
phenylimidazol-2-yl or 1-phenyltetrazol-5-yl groups (8b, 8d) led
to an increase in activity. On the other hand, substitution of a
cyclohexyl group for the phenyl ring led to a decrease in activity
(8e). The results suggested that the phenyl group of compounds
cyclization with phenylhydrazine afforded pyridylpyrazole 18.
N-benzylation of 18 followed by reduction of pyridine core with so-
dium borohydride gave N-benzyl tetrahydropyridine 19. Simulta-
neous deprotection and saturation of the compound 19 in the
presence of a palladium catalyst and ammonium formate afforded
phenylpyrazole 20.
A series of piperazine- or piperidine-substituted prolylthiazoli-
dines (8, 9) was synthesized as previously reported (Scheme 3).18a
The key intermediate 2218a was prepared by coupling N-Boc-trans-
4-hydroxy-L-proline (21) with thiazolidine followed by oxidation
with dimethyl sulfoxide and sulfur trioxide.23 Reductive amination
of ketone 22 with commercially available amines or the above-de-
scribed intermediates 12, 15, 20 afforded only the cis-isomers. Sub-
sequent removal of the Boc group yielded analogs 8 and 9.
Diastereomer 8r (2S,4R) and enantiomer 8s (2R,4R) of compound
8g (2S,4S) were prepared as follows. Silylation of the hydroxyl
8b and 8d forms CH–p interactions with the S2 extensive subsite.
Although pyrazole compound 8f had less potent in vitro activity
than imidazolyl or tetrazolyl compounds (8b, 8d), these com-
pounds showed similar ex vivo DPP-4 inhibitory activity. The re-
sults suggested that compound 8f had better pharmacokinetic
property than compounds 8b and 8d. Furthermore, introduction
of a methyl group into the 3-position of the pyrazolyl ring led to
a threefold increase in DPP-4 inhibitory activity, while maintaining
duration of ex vivo activity (8g). 3-Trifluoromethylpyrazole analog
8h also exhibited in vitro and ex vivo potency. On the other side,
tetrazolyl and pyrazolyl piperidine analogs (9a, 9b) remarkably
decreased in vitro inhibitory activity compared with the corre-
sponding piperazine analogs (8d, 8h). These findings suggested
that piperidine analogs would not have any interaction with the
S2 extensive subsite.
group on N-Boc-cis-4-hydroxy-L-proline (23) with tert-butyldi-
methylsilyl chloride followed by coupling with thiazolidine and
subsequent removal of the protective group with tetrabutylammo-
nium fluoride gave N-Boc-cis-4-hydroxy-L-prolylthiazolidide (24),
as previously described.18b Mesylation of the intermediate 24
followed by SN2 nucleophilic substitution with phenylpyrazole
12d in N,N-dimethylformamide at high temperature afforded the
diastereomer 8r. The enantiomer 8s was prepared by the same pro-
cedure as that for the synthesis of compound 8g except that

5708
T. Yoshida et al. / Bioorg. Med. Chem. 20 (2012) 5705–5719
SCH3
N
N
a, b
c, d
PG N
NH
PG
N
N
HN
HN
HN
N
N
Ph
e
Ph
11a: PG = Cbz
12a
10a: PG = Cbz
10b: PG = Boc
O
N
N
f - i
N
N
PG N
N
N
HN cHex
11b: PG = Cbz
j, k or
l or m, n
cHex
12b
Y
O
o - q
PG N
N
O
Y
N
N
N
Ar
11c:
12c:
Y = H, PG = Cbz
Y = H, Ar = Ph
11d: Y = CH3, PG = Boc
11e: Y = CF3, PG = Cbz
12d: Y = CH3, Ar = Ph
12e: Y = CF3, Ar = Ph
12f:
Y = CH3, Ar = 4-F-Ph
12g: Y = CH3, Ar = 3-F-Ph
12h: Y = CH3, Ar = 2-F-Ph
12i:
Y = CH3, Ar = 4-Cl-Ph
12j: Y = CH3, Ar = 4-CN-Ph
12k: Y = CH3, Ar = 4-Pyr
12l:
Y = CH3, Ar = 3-Pyr
12m: Y = CH3, Ar = 2-Pyr
12n: Y = CH3, Ar = 5-CN-2-Pyr
Scheme 1. Reagents and conditions: (a) Ph-NCS, acetone, rt; (b) CH3I, CH3OH, CH2C12, rt; (c) N2NCH2CH(OCH3)2, pyridine, 100 °C; (d) 10%Pd/C, H2, CH3OH, r.t; (e) cHex-NCO,
CH2C12, rt; (f) POCI3, THF, 70 °C; (g) triazole, CH3CN, rt; (h) NaN3, H2O, CH3OH, 70 °C; (i) 10%PdVC, H2, EtOH, AcOEt; (j) (EtO)2CHCH2CO2H, EDC, HOBt, DMF, rt; (k) 50%TFA/
H2O, CHC13, 0 °C to rt; (l) diketene, DMF, rt; (m) Ac2O, pyridine, rt; (n) LHMDS, THF, ꢀ78 °C, then CF3CO2Et, ꢀ78 °C to rt; (o) ArNHNH2, MsOH or MS3A, EtOH, rt; (p) POCl3,
pyridine, rt; (q) TFA, CH2C12 rt, or 10%Pd/C, H2, CH3OH, rt.
N
N
N
a
b, c
Cbz
N
CO2H
Cbz
N
CONHPh
HN
N
Ph
13
14
15
CF3
O
O
d
e
N
N
O
N
N
N
CH3
N
Ph
CF3
16
17
CF3
18
CF3
f, g
h
Bn
N
HN
N
N
N
Ph
Ph
19
20
Scheme 2. Reagents and conditions: (a) PhNH2, EDC, HOBt, THF, rt; (b) PPh3, i-PrO2CNNCO2-i-Pr, TMSN3, 0 °C to rt; (c) 10% Pd/C, H2, CH3OH, rt; (d) CF3CO2Et NaOCH3/CH3OH,
MTBE, rt; (e) PhNHNH2, EtOH, rt, (f) BnCl, CH3CN, rt; (g) NaBH4, EtOH, rt; (h) 10%Pd/C, HCO2NH4, MeOH, rt.
HO
HO
O
RR'N
RR'N
We subsequently turned our attention to substitution on the
phenyl ring of compound 8g, which had shown a favorable effect
on in vitro and ex vivo DPP-4 inhibitory activity. The results are
summarized in Table 2. para-Substitution of the phenyl ring
decreased activity (8i, 8l, 8m), suggesting that the S2 extensive
subsite may be spatially rather restricted. Comparison of activity
in terms of the substituted position on the phenyl ring indicated
that 3-fluoro analog (8j) was superior to 2- or 4-fluoro one (8k,
8i), and compound 8j had similar potency and efficacy compared
with non-substituted analog 8g. While 4-pyridyl analog 8n was
also about 13-fold weaker than the phenyl analog 8g, the 2- and
3-pyridyl analogs (8p, 8o) relatively retained the activity. Substitu-
tion of a cyano group at the 5-position of the 2-pyridyl ring (8q),
however, led to decreased activity in vitro. The plasma DPP-4
inhibitory activity of the 2-pyridyl analog 8p in Wister rats was
stronger than that of 3-pyridyl analog 8o. Regarding the
S
S
a, b
c, d
OH
OH
N
N
N
Boc
N
Boc
N
H
O
O
O
O
21
22
8, 9
HO
S
S
e, a, f
g, h, i
N
N
N
Boc
N
Boc
N
H
O
24
O
23
8r
Scheme 3. Reagents and conditions: (a) Thiazolidine, HOBt, EDC, DMF, rt; (b)
DMSO, SO3–pyridine, Et3N, CH2C12, 0 °C; (c) RR0NH, NaBH(OAc)3, AcOH, 1,2-
dichloroethane, rt; (d) HCl, AcOEt, rt; (e) TBDMSCl, imidazole DMF, rt; (f) TBAF, THF,
0 °C; (g) MsCl, Et3N, CH2Cl2, 0 °C; (h) 12d, DMF, 120 °C; (i) TFA, CH2C12, rt.

T. Yoshida et al. / Bioorg. Med. Chem. 20 (2012) 5705–5719
5709
Table 1
Inhibition of derivatives having a linked bicyclic group
Ar
X
N
S
N
N
H
O
Compd
Ar
X
In vitro DPP-4 inhiition IC50 (nmol/L)
Ex vivo DPP-4 inhibition at 3
l
mol/kg po, Wistar ratsa (%)
9 h
Human
Rat
0.5 h
6a
6b
N
N
2.7
3.1
3.5
62.7 2.7
19.7 8.7
N.T.
3.9
3.0
N.T.
N.T.
7b
7c
N
N
2.2
N.T.
0.95
1.0
5.4
29.7 6.1
N.T.
12.9 4.5
8c
N
N
4.5
N.T.
8b
0.26
0.19
59.3 3.1
58.0 2.2
8d
9a
N
CH
0.20
4.0
0.18
2.9
85.1 1.5
N.T.
70.7 1.5
N.T.
8e
8f
N
N
4.3
3.1
25.6 2.9
90.6 1.3
10.5 3.3
50.8 8.2
0.94
0.69
8g
N
0.37
0.29
94.8 0.1
78.3 1.6
8h
9b
N
CH
0.32
5.6
0.31
2.5
87.4 3.2
N.T.
81.8 3.6
N.T.
N.T.: not test.
a
Data are expressed as means SEM (n = 3).
stereochemistry, a diastereomer 8r (2S,4R) of 8g showed decreased
in Table 3. The arylpyrazolyl analogs 8g, 8j and 8p showed more
potent DPP-4 inhibitory activity than phenyl analog 6c and pyrid-
inyl analog 6d, and a marked decrease in inhibitory activities
against DPP-8 and DPP-9. The results indicated that compounds
8g, 8j and 8p showed at least 290-fold selectivity, while com-
pounds 6c and 6d had at most 70-fold selectivity over DPP-8 and
DPP-9. Especially, 8g had about 700-and 1500-fold selectivity over
DPP-8 and DPP-9, respectively. Therefore the interaction of the aryl
group on the pyrazole with the S2 extensive subsite in DPP-4 and
steric crashes with the corresponding site in DPP-8 and DPP-9
would contribute to the high selectivity of 8g as discussed in more
detail in the next section. Compound 8g showed high potency and
off-target selectivity and was selected for further evaluation of
X-ray crystal structure, pharmacokinetic profiles and in vivo
pharmacology.
inhibitory activity, and enantiomer 8s (2R,4R) resulted in
a
complete loss of activity.
2.4. Selectivity against DPP-8 and DPP-9
The enzymes most closely related to DPP-4 are fibroblast activa-
tion protein (FAP), DPP-II, DPP-8 and DPP-9.14 Although the precise
physiological functions of these enzymes have not been elucidated,
DPP-8 and DPP-9 are widely distributed cytosolic enzymes, and
their inhibition would induce the toxicity of DPP-4 inhibitors
identified to date, including alopecia, thrombocytopenia, anemia,
enlarged spleen, multiple histological pathologies, and animal
mortality.17 Thus selectivity of representative compounds against
DPP-8 and DPP-917 was measured and the results are summarized

5710
T. Yoshida et al. / Bioorg. Med. Chem. 20 (2012) 5705–5719
Table 2
Inhibition of 1-arylpyrazolyl derivatives
CH3
N
N
Ar'
N
N
4
S
2
N
N
H
O
Compd
Ar0
In vitro DPP-4 inhibition IC50 (nmol/L)
Human Rat
Ex vivo DPP-4 inhibition at 3
0.5 h
l
mol/kg po, Wistar ratsa (%)
9 h
8g
8i
Ph
4-FPh
0.37
2.9
0.29
2.3
94.8 0.1
N.T.
78.3 1.6
N.T.
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
3-FPh
2-FPh
4-ClPh
4-CNPh
4-Pyr
3-Pyr
2-Pyr
5-CN-2-Pyr
Ph (2S,4R)
Ph (2R,4R)
0.30
0.85
3.4
12
5.0
0.49
0.53
2.9
13
4.7
0.77
0.72
14
5.7
96.1 0.1
75.9 3.1
N.T.
N.T.
N.T.
66.9 8.4
83.0 5.5
N.T.
N.T.
N.T.
75.1 3.9
34.7 2.5
N.T.
N.T.
N.T.
40.8 4.6
53.7 3.1
N.T.
N.T.
N.T.
0.62
0.63
15
6.3
>1000
>1000
N.T.: not test.
a
Data are expressed as means SEM (n = 3)
Table 3
Selectivity of DPP-4 inhibitors against DPP-8 and DPP-9
Ar
N
N
S
N
N
H
O
Compound
Ar
Inhibitory activity IC50 (nmol/L) on human enzymes
DPP-4
1.6
DPP-8
DPP-9
6c
12
30
6d
1.6
40
110
8g
8j
0.37
260
540
330
610
0.30
0.63
86
8p
220
2.5. X-ray crystal structure determination
and Glu206, and the carbonyl oxygen forms a hydrogen bond with
Asn710. The pyrazolyl ring is stacked with the side chain of Phe357
The X-ray crystal structure shows that the characteristic five
rings of 8g fit into the active site of DPP-4 (Fig. 4). The thiazolidine
moiety fully occupies the S1 hydrophobic subsite. The secondary
amino group of the proline moiety forms salt bridges to Glu205
and the piperazinyl ring forms CH–p interaction with Phe357. In
addition, the phenyl substituent on the pyrazolyl ring is oriented
favorably for hydrophobic interactions with the side chains of
Ser209 and Arg358. In this S2 extensive subsite, the 4-position

T. Yoshida et al. / Bioorg. Med. Chem. 20 (2012) 5705–5719
5711
carbon of the phenyl ring of 8g is so close to the carbonyl oxygen of
the main chain of Val207 (distanceC. . .O = 3.3 Å) and a weak hydro-
gen bond could be formed between them. This subsite that accom-
modates the phenyl ring is quite tight, and the fact explains a loss
in activity by replacement of the phenyl with a cyclohexyl (8e) or
introduction of a para-substituent into the phenyl ring (8i, 8l, 8m,
8q), in agreement with the SAR results (Tables 1 and 2). Moreover,
the electric repulsion between the nitrogen atom of 4-pyridyl ana-
log 8n and the carbonyl oxygen of Val207 seems to decline the
inhibitory activity. This interaction with the S2 extensive subsite
not only increased potency, but also improved selectivity against
DPP-8 and DPP-9 as discussed above. It has been reported that
neither DPP-8 nor DPP-9 has a residue corresponding to Ser209
of DPP-4 and that Asp435 of DPP-8 and Asp425 of DPP-9 corre-
spond to Arg358 of DPP-4.16 Homology models show that DPP-8
and DPP-9 have unique P2-loop structures quite different from that
of DPP-4, which comprises 16 amino acids (Ser349-Phe362 in DPP-
4).16 Moreover, Arg358 and Ser209 have been suggested to play a
crucial role for selectivity on the basis of each docking model of
sitagliptin (1) with DPP-8 and DPP-9.16 The phenyl group of com-
pound 8g, as in the case of the trifluoromethyl group on sitagliptin,
would not interact with Asp435 (DPP-8) or Asp425 (DPP-9) by
induced fit because of their low flexibility to form hydrogen bonds
with Arg508 and Arg498 in DPP-8 and DPP-9, respectively.16
Therefore compound 8g showed weak activities in DPP-8 and
DPP-9. Besides, steric clashes between the phenyl group of 8g
and the P2-loops of DPP-8 and DPP-9 caused a drastic reduction
of the activity against them. These findings accentuate that the
S2 extensive subsite is unique to DPP-4.
Figure 5. X-ray structure of compound 9b (cyan carbon atoms) bound to DPP-4
(Arg 358 and key residues are shown as cyan and gray carbon atoms, respectively.
Superimposed is the bound conformation of compound 8g and Arg358 of DPP-4
(purple carbon atoms).
2.6. Pharmacokinetic profile of compound 8g
The plasma concentration profiles and pharmacokinetic param-
eters of compound 8g are shown in Figure 6 and Table 4, respec-
tively. After oral administration of compound 8g (HBr salt) to
rats and monkeys at each dose of 0.1, 0.3, 1.0 mg/kg, unchanged
8g reached maximal concentrations (Cmax) at 0.75–0.88 h in rats,
and 0.50–1.38 h in monkeys. At the same time, long terminal
half-lives (8–16 h in rats, and 15–19 h in monkeys) were observed
after oral dosing, which was suitable for long-lasting plasma DPP-4
inhibitory activity in vivo. In the range of 0.1–1.0 mg/kg, dose-pro-
portionality of area under curve was confirmed in rats. Compound
8g showed excellent bioavailability (BA) ranging from 63% to 86%
in rats, and 44% to 83% in monkeys, and was mainly excreted into
feces via bile in both rats and monkeys. This profile may be useful
for diabetes patients having impaired renal functions.
Figure 5 shows the X-ray crystal structure of piperidinyl analog
9b in complex with human DPP-4. Compound 9b has critical inter-
actions with the S1 hydrophobic subsite and hydrogen bonding
interactions with the secondary amino group in the proline
moiety; however, the orientation of the 1-phenylpyrazol-2-yl moi-
ety is completely different from that of the piperazinyl analog 8g
(inclined at about 45 degrees). This difference may be caused by
the generally planar conformation to conjugate a lone pair of the
2.7. In vivo pharmacological evaluation of compound 8g
nitrogen atom at the piperazinyl ring with
p-electron of pyrazole
in the compound 8g. The phenyl group of 8g pushes the side chain
of Arg358 away, and then the S2 extensive subsite becomes avail-
able. As a result, the piperidinyl analogs (9a, 9b) earn no hydropho-
bic interactions with the S2 extensive subsite, and exhibit almost
20-fold reduction in activity as compared to the corresponding
piperazine analogs 8d and 8h. These results suggested that the
interaction with the S2 extensive subsite played an important role
for DPP-4 inhibition.
On the basis of its excellent in vitro potency, selectivity, and
pharmacokinetic profiles, compound 8g was chosen for in vivo
evaluation. Compound 8g (HBr salt) was administered orally to
Wistar rats at a dose of 0.03, 0.1, 0.3 or 1 mg/kg and the plasma
DPP-4 activity was evaluated ex vivo. As shown in Figure 7, 8g
indicated dose-dependent, fast-onset and long-lasting DPP-4
inhibitory activity. It is most noteworthy that more than 50% inhi-
bition of plasma DPP-4 level was sustained at a single oral dose of
1000.0
rat 0.3 mg/kg p.o.
rat 0.3 mg/kg i.v.
monkey 0.3 mg/kg p.o.
monkey 0.3 mg/kg i.v.
100.0
10.0
1.0
0.1
0
8
16
24
32
Time after administration (h)
Figure 4. Binding interactions in active site. Co-crystallization of compound 8g and
human DPP-4. The surface in pink represents S2 extensive subsite.
Figure 6. Plasma concentration–time profiles of compound 8g (HBr salt) after oral
and intravenous administration to rats and monkeys (Mean S.D., n = 4).

5712
T. Yoshida et al. / Bioorg. Med. Chem. 20 (2012) 5705–5719
Table 4
Pharmacokinetics properties of compound 8g (HBr salt)
Animal
Rat
Dose (mg/kg)
Phamacokinetic parametera
t1/2 (h)
tmax (h)
Cmax (ng/mL)
AUC0–1 (ng h/mL)
BA (%)
0.1
0.3
1.0
0.88 0.25
0.75 0.29
0.75 0.29
5.48 2.94
20.65 6.58
152.41 16.60
15.84 2.32
8.97 1.64
8.43 1.75
91.81 24.07
202.12 29.59
672.94 99.32
85.9b
63.0b
62.9b
Monkey
0.1
0.3
1.0
0.50 0.00
1.38 1.11
0.88 0.25
28.27 5.30
85.13 28.61
273.54 70.41
18.86 2.46
15.24 4.59
16.11 1.77
295.44 66.17
613.16 75.83
1571.64 250.84
83.2 16.7
57.6 5.3
44.1 2.6
a
Data are expressed as the mean S.D. of 4 animals.
BA was calculated using the mean AUC0-1 of each group.
b
120
100
80
levels in an oral glucose tolerance test (Fig. 8). The treatment with
compound 8g significantly lowered the delta AUC(0–60min) and the
maximum increase in glucose levels in Zucker fatty rats. Near nor-
malization of the glucose excursion relative to lean controls was
seen following a 0.03 mg/kg oral dose of compound 8g and the sig-
nificant improvements were observed at more than 35% of DPP-4
inhibition in compared to pre-dosing. These ex vivo and in vivo
studies in rats suggest that once-daily and lower dosing of
compound 8g is expected to keep proper blood glucose without
causing hypoglycemia in humans.
60
Vehicle
40
20
0
0.03 mg/kg
0.1 mg/kg
0.3 mg/kg
1 mg/kg
3. Conclusion
A culmination of work focused on the optimization at the
c-position of the proline moiety of L-prolylthiazolidine series has
led to the discovery of the compound 8g, which proved to be a
highly potent, selective and long-lasting DPP-4 inhibitor possess-
ing a unique structure of five consecutive rings. X-ray crystal struc-
ture determination of 8g demonstrated that the key interaction
between the phenyl ring on the pyrazole and the S2 extensive sub-
site of DPP-4 not only boosted potency but also increased
selectivity against DPP-8 and DPP-9. Compound 8g (HBr salt) at
0.03 mg/kg or higher doses, significantly inhibited the increase of
plasma glucose levels after an oral glucose load in Zucker fatty rats.
Teneligliptin, 3-[(2S,4S)-4-[4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)
piperazin-1-yl]pyrrolidin-2-ylcarbonyl]thiazolidine (8g) has been
approved for the treatment of type 2 diabetes in Japan.
0
6
12
18
24
30
36
42
48
Time after administaration (h)
Figure 7. Effects of compound 8g (HBr salt) on plasma DPP-4 activity (% change of
baseline) in Wistar rat. Compound 8g (HBr salt) was orally administrated at a dose
of 0.03, 0.1, 0.3 or 1 mg/kg at 0 h. Data are expressed as means SEM (n = 3).
1 mg/kg until 24 h. Compound 8g was assessed for its ability to im-
prove glucose tolerance in Zucker fatty rats. Pretreatment with
compound 8g at the doses from 0.03 mg/kg to 1 mg/kg 30 min be-
fore glucose challenge (1 g/kg) inhibited the increase in glucose
a
b
c
100
90
80
70
60
50
40
30
20
10
0
7000
6000
5000
4000
3000
2000
1000
0
150
100
50
Lean Vehicle
Fatty Vehicle
Fatty 8g 0.03 mg/kg
Fatty 8g 0.1 mg/kg
Fatty 8g 0.3 mg/kg
Fatty 8g 1 mg/kg
glucose
0
Vehicle Vehicle
Lean
0.03
0.1
0.3
1
Vehicle
Lean
Vehicle
0.03
0.1
0.3
1
compound 8g, mg/kg
Fatty
compound 8g, mg/kg
Fatty
-50
-30
0
30
60
90 120 150 180 210 240
Time after glucose load (min)
Figure 8. (a) Effects of compound 8g (HBr salt) on delta glucose levels after an oral glucose tolerance test in Zucker lean and fatty rats. Compound 8g (HBr salt) or 0.5% HPMC
solution (vehicle) was administered 30 min prior to an oral glucose challenge (1 g/kg). Values are mean SEM (n = 10) as the delta glucose levels adjusted from the pre-
glucose levels at 0 min. (b) The incremental areas under the curves of glucose levels from 0 min to 60 min after glucose load during an oral glucose tolerance test in Zucker
rats treated with vehicle or 8g (HBr salt). (c) The plasma DPP-4 activities before glucose load in Zucker rats treated with vehicle or compound 8g (HBr salt). Values are
means SEM (n = 10). ##P <0.01 versus ‘Zucker lean + Vehicle’ by Student’s t-test. ⁄⁄P <0.01 versus ‘Zucker fatty + Vehicle’ by Dunnett’s multiple comparison test.

T. Yoshida et al. / Bioorg. Med. Chem. 20 (2012) 5705–5719
5713
Table 5
triacetoxyborohydride (1.27 g, 6.00 mmol) and the mixture stirred
at room temperature for 13 h. The reaction mixture was poured
into a saturated aqueous sodium hydrogen carbonate solution
and extracted with chloroform. The extract was washed with brine,
dried and concentrated under reduced pressure. The residue
purified by silica gel chromatography with chloroform/methanol
(30:1, v/v) to give 3-{(2S,4S)-1-tert-butoxycarbonyl-4-[4-(4-phen-
ylthiazol-2-yl)piperazin-1-yl]pyrrolidin-2-ylcarbonyl}thiazolidine
(1.59 g, 100%) as a pale yellow amorphous. 1H NMR (300 MHz,
CDCl3): d 1.41, 1.46 (9H, s), 1.84–1.97 (1H, m), 2.40–2.73 (4H, m),
2.82–3.21 (4H, m), 3.34 (1H, t, J = 9.9 Hz), 3.47–4.13 (7H, m),
4.38–4.82 (3H, m), 6.78 (1H, s), 7.25 (1H, t, J = 7.1 Hz), 7.37 (2H,
t, J = 7.1 Hz), 7.82 (2H, d, J = 7.1 Hz).
Data collection and refinement statistics
8g
9b
PDB entry code
3VJK
3VJL
Crystal
Space group
Unit cell parameters: a (Å)
b (Å)
c (Å)
P212121
117.96
126.41
138.01
P212121
117.81
125.83
137.13
Data
Resolution (Å)
50.00–2.49 (2.58–
2.49)
72,839 (6849)
3.8 (3.1)
50.00–2.39 (2.48–
2.39)
80,861 (7811)
5.0 (4.7)
Unique reflections
Redundancy
Completeness (%)
Rmerge
I/r (I)
90.2 (94.7)
0.092 (0.491)
15.6
99.0 (97.9)
0.051 (0.173)
20.9
To a solution of the above compound (1.59 g, 3.00 mmol) in
ethyl acetate (6 mL) was added 4 mol/L hydrochloric acid in etha-
nol (7 mL), and the mixture stirred at room temperature for 12 h.
The precipitated solid was collected by filtration to give the title
compound (1.41 g, 83%) as a pale-brown powder. Mp: >135 °C;
1H NMR (300 MHz, DMSO-d6): d 2.27–2.42 (1H, m), 2.95–3.18
(3H, m), 3.37–4.18 (16H, m), 4.47–4.78 (3H, m), 7.30 (1H, t,
J = 7.3 Hz), 7.37–7.45 (3H, s), 7.87 (2H, d, J = 7.1 Hz), 9.17
(1H, br s), 10.93 (1H, br s); Anal. Calcd for C21H27N5OS2ꢁ33/
10HClꢁ2/5C2H6O: C, 46.07; H,5.80; N, 12.32. Found: C, 45.84; H,
6.14; N, 11.93; LC–MS (ESI) m/z 430.4 [M+H]+.
a
Refinement
Resolution (Å)
30.00–2.49 (2.55–
2.49)
61,379 (4707)
88.9 (93.6)
3211 (243)
0.225 (0.281)
0.279 (0.333)
30.00–2.39 (2.46–
2.39)
76,090 (5399)
99.1 (96.4)
4025 (264)
0.184 (0.210)
0.230 (0.291)
Unique reflections
Completeness (%)
Data in the test set
R-work
R-free
Structure
Protein non-H atoms/B (Å2)
Ligand atoms/B (Å2)
Water oxygen atoms/B (Å2)
12,180/37.7
60/32.5
456/32.1
12,166/25.7
66/35.4
894/27.2
4.1.3. 1-Benzyloxycarbonyl-4-[(methylthio)phenyliminom
ethyl]piperazine (11a)
Rmsd
Bond lengths (Å)
Bond angles (°)
0.011
1.435
0.010
1.343
To a solution of 10a (5.00 g, 22.7 mmol) in acetone (50 mL) was
added phenyl isothiocyanate (2.9 mL, 24 mmol). The mixture was
stirred for 1 h at room temperature. The precipitate was colle-
cted by filtration and washed with acetone to give 1-(ani-
linocarbothioyl)-4-(benzyloxycarbonyl)piperazine (5.08 g, 63%) as
a white powder. 1H NMR (300 MHz, DMSO-d6): d 3.52 (4H, br s),
3.93 (4H, br s), 5.12 (2H, s), 7.11 (1H, t, J = 8.5 Hz), 7.27–7.45 (9H,
m), 9.37 (1H, br s).
To a solution of the above compound (5.07 g, 14.3 mmol) in
methanol (100 mL) was added methyl iodide (1.1 mL, 18 mmol)
under ice-cooling. The mixture stirred at room temperature for
17 h, and then concentrated under reduced pressure. The residue
was poured into a saturated aqueous sodium hydrogen carbonate
solution and extracted with dichloromethane. The extract was
washed with brine, dried and concentrated under reduced pressure
to give the title compound (5.71 g, 100%) as a pale yellow oil. 1H
NMR (300 MHz, CDCl3): d 2.03 (3H, s), 3.59 (8H, br s), 5.17 (2H,
s), 6.87 (2H, d, J = 7.3 Hz), 7.00 (1H, t, J = 7.3 Hz), 7.26 (2H, t,
J = 7.3 Hz), 7.32–7.43 (5H, m).
Ramachandran plot
Most favored regions (%)
Additionally allowed regions
(%)
Generously allowed regions
(%)
87.3
12.0
88.5
11.0
0.7
0.5
Values in parentheses are for highest-resolution shell.
a
Rmerge
=
R
|(I ꢀ <I>)|/
R(I), where I is the observed intensity.
4. Experimental
4.1. Chemistry
4.1.1. General
1H NMR spectra were measured on a Bruker DPX-300 instru-
ment or on a Bruker AMX-500 with tetramethylsilane as the inter-
nal standard; chemical shifts are reported in parts per million
(ppm, d units). Splitting patterns are designated as s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets;
br s, broad singlet. Mass spectra (MS) were recorded on a JEOL JMS-
700 instrument operating in the chemical ionization (CI) mode.
Electron analysis for carbon, hydrogen, and nitrogen was per-
formed with a Yanagimoto CHN CORDER MT-6. Chromatography
refers to flash chromatography conducted on silica gel BW-300
(Fuji Silysia). All chemicals and solvents were of reagent grade un-
less otherwise specified. For drying organic solutions in extraction,
anhydrous sodium sulfate or anhydrous magnesium sulfate was
used unless otherwise indicated. The following abbreviations are
used: DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide;
EDC, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydro-
chloride; HOBT, 3-hydroxybenztriazole hydrate.
4.1.4. 1-(1-Phenyl-1H-imidazol-2-yl)piperazine (12a)
A mixture of 11a (3.00 g, 8.12 mmol) and aminoacetaldehyde
dimethyl acetal (1.8 mL, 17 mmol) in pyridine (15 mL) was stirred
at 100 °C for 2 days, and then concentrated under reduced pres-
sure. The residue was poured into a saturated aqueous sodium
hydrogen carbonate solution and extracted with chloroform. The
extract was washed with brine, dried and concentrated under re-
duced pressure. A solution of the residue in 2 mol/L hydrochloric
acid (30 mL) was stirred for 2 h at 100 °C. After cooling to room
temperature, the reaction mixture was poured into a saturated
aqueous sodium hydrogen carbonate solution and extracted with
chloroform. The extract was washed with brine, dried and concen-
trated under reduced pressure. The residue was purified by silica
gel chromatography with chloroform/methanol (30:1, v/v) to give
1-benzyloxycarbonyl-4-(1-phenyl-1H-imidazol-2-yl)piperazine
(1.16 g, 39%) as a brown oil. 1H NMR (300 MHz, CDCl3): d 2.96–3.03
(4H, m), 3.44–3.52 (4H, m), 5.12 (2H, s), 6.86 (1H, d, J = 1.6 Hz),
6.88 (1H, d, J = 1.6 Hz), 7.27–7.54 (10H, m).
4.1.2. 3-{(2S,4S)-4-[4-(4-Phenylthiazol-2-yl) piperazin-1-
yl]pyrrolidin-2-ylcarbonyl}thiazolidine trihydrochloride (8c)
To a solution of 2218a (901 mg, 3.00 mmol), 4-phenyl-2-(pipera-
zin-1-yl)thiazole (810 mg, 3.30 mmol) and acetic acid (0.17 mL,
3.0 mmol) in 1,2-dichloroethane (15 mL) was added sodium

5714
T. Yoshida et al. / Bioorg. Med. Chem. 20 (2012) 5705–5719
To the above compound (1.16 g, 3.20 mmol) in methanol
H,6.91; N, 21.16. Found: C, 44.02; H, 7.29; N, 20.90; LC–MS (ESI)
m/z 421.2 [M+H]+.
(30 mL) was added 10% palladium on carbon (232 mg). The mix-
ture was stirred at room temperature for 20 h under a hydrogen
atmosphere (1 atm). Palladium on carbon was removed by filtra-
tion. The filtrate was concentrated under reduced pressure to give
the title compound (0.742 g, 100%) as a white amorphous. 1H NMR
(300 MHz, CDCl3): d 3.04–3.28 (8H, m), 3.49 (1H, s), 6.85 (1H, d,
J = 1.6 Hz), 6.88 (1H, d, J = 1.6 Hz), 7.34–7.52 (5H, m).
4.1.9. 1-(1-Phenyl-5-pyrazolyl)piperazine (12c)
To solution of ethyl 3,3-diethoxypropionate (5.34 g,
a
28.1 mmol) in tetrahydrofuran (60 mL) was added a 1 mol/L aque-
ous sodium hydroxide solution (29 mL, 29 mmol). The mixture was
stirred for 12 h at room temperature, and then concentrated under
reduced pressure. To a solution of the residue in DMF (60 mL) were
added HOBT (5.16 g, 33.7 mmol), EDC hydrochloride (6.46 g,
33.7 mmol) and 10a (6.20 g, 28.1 mmol) at room temperature.
The mixture was stirred at room temperature for 6 h, and then con-
centrated under reduced pressure. The residue was poured into
water and extracted with ethyl acetate. The extract solution was
washed with a saturated aqueous sodium hydrogen carbonate
solution and brine, dried and concentrated under reduced pres-
sure. The residue was purified by silica gel with n-hexane/ethyl
acetate (3:7, v/v) to give 1-benzyloxycarbonyl-4-(3,3-diethoxypro-
pionyl)piperazine (10.1 g, 98%) as an oil.
4.1.5. 3-{(2S,4S)-4-[4-(1-Phenyl-1H-imidazol-1-yl)piperazin-1-
yl]pyrrolidin-2-ylcarbonyl}thiazolidine trihydrochloride (8b)
The title compound was prepared in 58% yield using 12a in the
procedures outlined for 8c. Mp: >182 °C; 1H NMR (300 MHz,
DMSO-d6): d 2.10–2.26 (1H, m), 2.83–4.05 (16H, m), 4.43–4.77
(3H, m), 7.48 (1H, d, J = 2.3 Hz), 7.53 (1H, d, J = 2.3 Hz), 7.54–7.72
(5H, m), 9.07 (1H, br s), 10.98 (1H, br s); Anal. Calcd for
C
21H28N6OSꢁ3HClꢁ3/5C4H8O2ꢁ5/2H2O: C, 45.34; H,6.63; N, 13.56.
Found: C, 45.16; H, 6.86; N, 13.66; LC–MS (ESI) m/z 413.4 [M+H]+.
4.1.6. 3-{(2S,4S)-4-[4-(1-Phenyl-1H-tetrazol-5-yl)piperazin-1-
yl]pyrrolidin-2-ylcarbonyl}thiazolidine trihydrochloride (8d)
The title compound was prepared in 68% yield using 1-(1-phe-
nyl-1H-tetrazol-5-yl)piperazine in the procedures outlined for 8c.
Mp: 184 °C; 1H NMR (500 MHz, DMSO-d6): d 2.02–2.22 (1H, m),
2.80–3.95 (16H, m), 4.45–4.73 (3H, m), 7.57–7.73 (5H, m), 9.04
(1H, br s), 10.61 (1H, br s); Anal. Calcd for C19H26N8OSꢁ5/2HClꢁ1/
4C4H8O2ꢁ1/2H2O: C, 44.76; H,5.92; N, 20.88. Found: C, 44.82; H,
6.31; N, 20.94; LC–MS (ESI) m/z 415.4 [M+H]+.
To a solution of the above compound (3.28 g, 9.00 mol) in chlo-
roform (30 mL) was added a 50% aqueous trifluoroacetic acid solu-
tion (20 mL) under ice-cooling. The mixture was stirred at room
temperature for 24 h. The reaction solution was extracted with
chloroform. The extract was washed with water and brine, dried
and concentrated under reduced pressure to give a mixture includ-
ing 11c. To a solution of the mixture in ethanol (60 mL) was added
phenylhydrazine (0.89 mL, 9.0 mmol) and methanesulfonic acid
(0.060 mL, 0.90 mmol), and the solution was stirred at room tem-
perature for 3 h. Pyridine (1.0 mL) was added, and then the mixture
was concentrated under reduced pressure. To a solution of the res-
idue in pyridine (50 mL), was added phosphorus oxychloride
(1.68 mL, 18.0 mmol). The mixture was stirred at room tempera-
ture for 18 h, and then concentrated under reduced pressure. The
residue was poured into water and extracted with ethyl acetate.
The extract solution was washed with brine, dried and concen-
trated under reduced pressure. The residue was purified by silica
gel chromatography with n-hexane/ethyl acetate (7:3, v/v) to give
1-benzyloxycarbonyl-4-(1-phenylpyrazol-5-yl)piperazine
4.1.7. 1-(1-Cyclohexyl-1H-tetrazol-5-yl)piperazine (12b)
To a solution of 10a (2.07 g, 9.40 mmol) in dichloromethane
(50 mL) was added cyclohexyl isocyanate (1.2 mL, 9.4 mmol). The
mixture was stirred for 1 h at room temperature, and then concen-
trated under reduced pressure. To a solution of the residue in tet-
rahydrofuran (100 mL) was added phosphorus oxychloride
(8.8 mL, 94 mmol), and the mixture was refluxed for 18 h. The
reaction mixture was concentrated under reduced pressure, and
a 0.5 mol/L triazole in acetonitrile (100 mL, 50 mmol) was added
to the residue. The mixture was stirred at room temperature for
3 h, and then concentrated under reduced pressure. The residue
was poured into a saturated aqueous sodium hydrogen carbonate
solution and extracted with ethyl acetate. The extract was washed
with brine, dried and concentrated under reduced pressure. To the
residue in methanol (100 mL) was added an aqueous solution
(20 mL) of sodium azide (6.50 g, 100 mmol). The mixture was stir-
red at 70 °C for 3 h, and then concentrated under reduced pressure.
Water was added to the residue, and the mixture was extracted
with ethyl acetate. The extract was washed with brine, dried and
concentrated under reduced pressure. The residue was purified
by silica gel chromatography with n-hexane/ethyl acetate
(7:3, v/v) to give 1-benzyloxycarbonyl-4-(1-cyclohexyl-1H-tetra-
zol-5-yl)piperazine (390 mg, 11%) as a white powder.
(0.218 g, 11%) as an oil. 1H NMR (300 MHz, CDCl3): d 3.06–3.18 (8H,
m), 5.93 (1H, d, J = 1.8 Hz), 7.32 (1H, t, J = 7.5 Hz), 7.45 (2H, t,
J = 7.5 Hz), 7.54 (1H, d, J = 1.8 Hz), 7.72 (1H, d, J = 7.5 Hz).
The title compound was prepared quantitatively using the
above compound in the procedures outlined for 12a. 1H NMR
(300 MHz, CDCl3): d 2.83 (4H, br s), 3.49–3.48 (4H, m), 5.12 (2H,
s), 5.85 (1H, d, J = 1.9 Hz), 7.24–7.45 (8H, m), 7.51 (1H, d,
J = 1.9 Hz), 7.78 (2H, d, J = 8.2 Hz).
4.1.10. 3-{(2S,4S)-4-[4-(1-Phenyl-1H-pyrazol-5-yl)piperazin-1-
yl]pyrrolizin-2-ylcarbonyl}thiazolidine trihydrochloride (8f)
The title compound was prepared in 50% yield using 12c in the
procedures outlined for 8c. Mp: 187 °C; 1H NMR (500 MHz, DMSO-
d6): d 2.10–2.30 (1H, m), 2.80–4.10 (16H, m), 4.46–4.74 (3H, m),
6.10 (1H, d, J = 1.7 Hz), 7.34–7.37 (1H, m), 7.49–7.52 (2H, m),
7.56 (1H, d, J = 1.7 Hz), 7.79–7.81 (2H, m), 9.07 (1H, br s), 10.65
(1H, br s); Anal. Calcd for C21H28N6OSꢁ5/2HClꢁ1/2C4H8O2ꢁ3/4H2O:
C, 49.22; H,6.46; N, 14.97. Found: C, 49.25; H, 6.59; N, 15.20; LC–
MS (ESI) m/z 413.4 [M+H]+.
The title compound was prepared quantitatively using the
above compound in the procedures outlined for 12a. 1H NMR
(500 MHz, CDCl3): d 1.28–1.47 (3H, m), 1.68–2.07 (7H, m), 3.04–
3.08 (4H, m), 3.18–3.23 (4H, m), 3.37–3.57 (1H, m), 3.95–4.08
(1H, m).
4.1.8. 3-{(2S,4S)-4-[4-(1-Cyclohexyl-1H-tetrazol-5-yl)piperazin-
1-yl]pyrrolidin-2-ylcarbonyl}thiazolidine trihydrochloride (8e)
The title compound was prepared in 56% yield using 12b in the
procedures outlined for 8c. Mp: 178 °C; 1H NMR (500 MHz, DMSO-
d6): d 1.20–1.34 (1H, m), 1.40–1.50(2H, m), 1.64–1.88 (5H, m),
1.97–2.03 (2H, m), 2.12–2.32 (1H, m), 2.90–4.05 (16H, m), 4.25
(1H, m), 4.48–4.75 (3H, m), 9.10 (1H, br s), 10.67 (1H, br s); Anal.
4.1.11. 1-Acetoacetyl-4-tert-butoxycarbonylpiperazine (11d)
A solution of 10b (5.02 g, 27.0 mmol) and diketene (2.50 mL
32.6 mmol) n DMF (90 mL) was stirred for 1.5 h at room tempera-
ture, and then concentrated under reduced pressure. The residue
was poured into water and extracted with ethyl acetate. The
extract was washed with brine, dried and concentrated under re-
duced pressure to give the title compound (6.26 g, 86%) as a
Calcd for
C19H32N8OSꢁ5/2HClꢁ1/10C4H8O2ꢁ1/2H2O: C, 44.00;

T. Yoshida et al. / Bioorg. Med. Chem. 20 (2012) 5705–5719
5715
pale-brown powder. 1H NMR (500 MHz, DMSO-d6): d 1.41 (9H, s),
2.15 (3H, s), 3.27–3.35 (6H, m), 3.43 (2H, m), 3.66 (2H, s).
The mixture was stirred for 18 h, and then concentrated under re-
duced pressure. The residue was poured into a 10% aqueous citric
acid solution and extracted with ethyl acetate. The extract was
washed with brine, dried and concentrated under reduced pressure
to give 4-acetyl-1-benzyloxycarbonylpiperazine (22.6 g, 100%) as
an oil. 1H NMR (300 MHz, DMSO-d6): d 2.78 (2H, s), 3.35–3.68 (8H,
m), 5.10 (2H, s), 7.28–7.42 (5H, m).
To a solution of the above compound (7.12 g, 27.1 mmol) in
tetrahydrofuran (150 mL) was added a 1 mol/L lithium bis(tri-
methylsilyl)amide in tetrahydrofuran (41 mL, 41 mmol) dropwise
at ꢀ78 °C over 40 min. After stirring at constant temperature for
1 h, a solution of ethyl trifluoroacetate (4.85 mL, 40.1 mmol) in tet-
rahydrofuran (20 mL) was added to the reaction solution. After
warming to room temperature over 18 h, the mixture was poured
into a saturated aqueous ammonium chloride solution and ex-
tracted with ethyl acetate. The extract solution was washed with
brine, dried and concentrated under reduced pressure. The residue
was purified by silica gel chromatography with n-hexane/ethyl
acetate (7:3, v/v) to give 1-benzyloxycarbonyl-4-(trifluoroaceto-
acetyl)piperazine (7.35 g, 76%) as a pale-yellow solid. 1H NMR
(500 MHz, CDCl3): d 2.86 (4H, br s), 3.49–3.55 (4H, m), 5.12 (2H,
s), 6.12 (1H, s), 7.32–7.43 (6H, m), 7.46 (2H, t, J = 7.5 Hz), 7.74
(2H, d, J = 7.5 Hz).
4.1.12. 1-(3-Methyl-1-phenyl-5-pyrazolyl)piperazine (12d)
To a solution of 11d (6.24 g, 23.1 mmol) in ethanol (500 mL) was
added phenylhydrazine (2.27 mL, 23.1 mmol) and methanesulfonic
acid (0.35 mL, 5.4 mmol) at room temperature. The mixture was
stirred for 14 h, and then pyridine (6 mL) was added. The mixture
was concentrated under reduced pressure. A solution of the residue
and phosphorus oxychloride (5.0 mL, 54 mmol) in pyridine
(250 mL) was stirred for 20 h at room temperature, and then concen-
trated under reduced pressure. The residue was acidified with dilute
hydrochloric acid to pH 3. The mixture was extracted with ethyl ace-
tate. The extract was washed with a saturated aqueous sodium
hydrogen carbonate solution and brine, dried and concentrated
under reduced pressure. The residue was purified by silica gel chro-
matography with n-hexane/ethyl acetate (7:3, v/v) to give 1-tert-
butoxycarbonyl-4-(3-methyl-1-phenyl-1H-pyrazol-5-yl)piperazine
(935 mg, 12%) as an oil. 1H NMR (500 MHz, DMSO-d6): d 1.39 (9H, s),
2.15 (3H, s), 2.73 (4H, m), 3.37 (4H, m), 5.83 (1H, s), 7.28 (1H, t,
J = 7.4 Hz), 7.46 (2H, t, J = 7.4 Hz), 7.76 (2H, d, J = 7.4 Hz).
To a solution of the above compound (935 mg, 2.72 mmol) in
dichloromethane (10 mL) was added trifluoroacetic acid (5 mL) at
room temperature. The mixture was stirred for 1.5 h, and then con-
centrated under reduced pressure. The residue was poured into
water (50 mL) and washed with diethyl ether. The aqueous layer
was basified with a aqueous sodium hydrogen carbonate solution,
and the mixture was extracted with chloroform. The extract was
dried and concentrated under reduced pressure to give the title
compound (584 mg, 88%) as a brown powder. 1H NMR (300 MHz,
DMSO-d6): d 2.14 (3H, s), 2.66–2.76 (8H, m), 5.77 (1H, s), 7.26
(1H, t, J = 7.5 Hz), 7.45 (2H, t, J = 7.5 Hz), 7.76 (2H, d, J = 7.5 Hz);
The title compound was prepared in 13% yield using the above
compound in the procedures outlined for 12c. 1H NMR (300 MHz,
CDCl3): d 3.22 (8H, br s), 6.21 (1H, s), 7.42 (1H, t, J = 7.5 Hz), 7.49
(2H, t, J = 7.5 Hz), 7.66 (2H, d, J = 7.5 Hz), 9.96 (1H, br s).
4.1.15. 3-{(2S,4S)-4-[4-(3-Trifluoromethyl-1-phenyl-1H-pyrazol-
5-yl)piperazin-1-yl]pyrrolizin-2-ylcarbonyl}thiazolidine
trihydrochloride (8h)
The title compound was prepared in 52% yield using 12e in the
procedures outlined for 8c. 1H NMR (300 MHz, DMSO-d6): d 2.00–
2.28 (1H, m), 2.80–4.00 (16H, m), 4.44–4.74 (3H, m), 6.64 (1H, s),
7.44–7.49 (1H, m), 7.54–7.59 (2H, m), 7.77–7.79 (2H, m), 9.03
4.1.13. 3-{(2S,4S)-4-[4-(3-Methyl-1-phenyl-1H-pyrazol-5-
yl)piperazin-1-yl]pyrrolidin-2-yl-carbonyl}thiazolidine
hemipentahydrobromide (8g)
3-{(2S,4S)-1-tert-Butoxycarbonyl-4-[4-(3-methyl-1-phenyl-1H-
pyrazol-5-yl)piperazin-1-yl]pyrrolidin-2-yl-carbonyl}thiazolidine
was prepared in 50% yield using 12d in the procedures outlined for
8c.
(1H,
br
s),
10.55
(1H,
br
s);
Anal.
Calcd
for
C
22H27F3N6OSꢁ2.5HClꢁ0.25H2O: C, 45.86; H, 5.25; N, 14.59. Found:
C, 45.89; H, 5.63; N, 14.23; LC–MS (ESI) m/z 481.4 [M+H]+.
4.1.16. 1-[1-(4-Fluorophenyl)-3-methyl-1H-pyrazol-5-
yl]piperazine (12f)
To a solution of the above compound (25.45 g) in dichlorometh-
ane (200 mL) was added trifluoroacetic acid (50 mL) at room tem-
perature. The mixture was stirred for 19 h, and then concentrated
under reduced pressure. The residue was poured into a saturated
aqueous sodium hydrogen carbonate solution and extracted with
chloroform. The extract was washed with, dried and concentrated
under reduced pressure. The residue was purified by silica gel col-
umn chromatography with chloroform/methanol (9:1, v/v) to give
free base of the title compound (19.28 g, 93%) as a solid.
To a solution of the above compound (5.09 g, 11.9 mmol) in eth-
anol (51 mL) was added 48% hydrobromic acid (5.03 g) at the
refluxing temperature, The mixture was cooled to room tempera-
ture over 1 h with stirring, and further stirred at room temperature
for 1 h. The precipitate was collected by filtration, washed with
ethanol (5 mL) to give the title compound as white crystals
(6.76 g, 90%). Mp: 201 °C; 1H NMR (DMSO-d6, 300 MHz): d 2.28–
2.08 (1H, m), 2.17 (3H, s), 2.95–4.22 (16H, m), 4.43–4.80 (3H, m),
5.95 (1H, s), 7.32 (1H, t, J = 7.5 Hz), 7.48 (2H, t, J = 7.5Hz), 7.78
(2H, d, J = 7.5Hz), 9.18 (1H, br s),9.90 (1H, br s); Anal. Calcd. for
The title compound was prepared in 38% yield from 11d using
4-fluorophenylhydrazine hydrochloride in the procedures outlined
for 12d. 1H NMR (300 MHz, CDCl3): d 2.26 (3H, s), 2.75–2.92 (8H,
m), 5.68 (1H, s), 7.10 (2H, ddd, J = 2.3, 4.8, 8.4 Hz), 7.72–7.79 (2H,
m).
4.1.17. 3-((2S,4S)-4-{4-[1-(4-Fluorophenyl)-3-methyl-1H-
pyrazol-5-yl]piperazin-1-yl}pyrrolizin-2-ylcarbonyl)
thiazolidine trihydrochloride (8i)
The title compound was prepared in 41% yield using 12f in the
procedures outlined for 8c. Mp: >145 °C; 1H NMR (300 MHz,
DMSO-d6): d 2.17 (3H, s), 2.20–2.40 (1H, m), 2.90–4.35(16H, m),
4.43–4.82 (3H, m), 5.95 (1H, s), 7.21–7.37 (2H, m), 7.74–7.89
(2H, m), 9.13 (1H, br s), 11.10 (1H, br s); Anal. Calcd for
C
22H29FN6OSꢁ3HClꢁ0.5C4H8O2ꢁ2.2H2O: C, 44.80; H, 6.44; N, 13.63.
Found: C, 44.67; H, 6.38; N, 13.64; LC–MS (ESI) m/z 445.2 [M+H]+.
4.1.18. 1-[1-(3-Fluorophenyl)-3-methyl-1H-pyrazol-5-yl]
piperazine (12g)
C
22H30N6OSꢁ2.5HBrꢁ1.5H2O: C, 40.29; H, 5.46; N, 12.81. Found: C,
40.23; H, 5.34; N, 12.81; LC–MS (ESI) m/z 427.4 [M+H]+.
The title compound was prepared in 23% yield from 11d using
3-fluorophenylhydrazine hydrochloride in the procedures outlined
for 12d. 1H NMR (300 MHz, CDCl3): d 2.27 (3H, s), 2.82–2.97 (8H,
m), 5.69 (1H, s), 6.91–6.98 (1H, m), 7.31–7.40 (1H, m), 7.60–7.67
(2H, m).
4.1.14. (3-Trifluoromethyl-1-phenyl-1H-pyrazol-5-yl)piperazine
(12e)
To a solution of 10a (19.0 g, 86.3 mmol) in pyridine (150 mL) was
added acetic anhydride (9.0 mL, 95 mmol) at room temperature.

5716
T. Yoshida et al. / Bioorg. Med. Chem. 20 (2012) 5705–5719
4.1.19. 3-((2S,4S)-4-{4-[1-(3-Fluorophenyl)-3-methyl-1H-
pyrazol-5-yl]piperazin-1-yl}pyrrolizin-2-ylcarbonyl)
thiazolidine trihydrochloride (8j)
for 12d. 1H NMR (300 MHz, CDCl3): d 2.27 (3H, s), 2.86–2.99 (8H,
m), 5.77 (1H, s), 7.92 (2H, dd, J = 1.6, 4.8 Hz), 8.60 (2H, dd, J = 1.6,
4.8 Hz).
The title compound was prepared in 72% yield using 12g in the
procedures outlined for 8c. Mp: 172 °C; 1H NMR (300 MHz, DMSO-
d6): d 2.10–2.35 (1H, m), 2.17 (3H, s), 2.90–4.15 (16H, m), 4.46–4.76
(3H, m), 5.98 (1H, s), 7.11–7.19 (1H, m), 7.47–7.55 (1H, m), 7.59–
7.64 (1H, m), 7.70–7.73 (1H, m), 9.09 (1H, br s), 10.79 (1H, br s); Anal.
Calcd for C22H29FN6OSꢁ3HClꢁ0.75H2O: C, 46.56; H, 5.95; N, 14.81.
Found: C, 46.63; H, 6.29; N, 14.45; LC–MS (ESI) m/z 445.4 [M+H]+.
4.1.27. 3-((2S,4S)-4-{4-[3-Methyl-1-(pyridin-4-yl)-1H-pyrazol-5-
yl] piperazin-1-yl}pyrrolizin-2-ylcarbonyl)thiazolidine
trimaleate (8n)
A free base of the title compound was prepared in 90% yield
using 12k in the procedures outlined for 8c. To this free base
(3.20 g, 7.49 mmol) in ethanol (200 mL) was added a solution of
maleic acid (3.00 g, 25.8 mmol) in ethanol (20 mL) under ice-cool-
ing. The precipitate was collected by filtration to give the title com-
pound (4.30 g, 74%) as a white powder. Mp: 168–171 °C; 1H NMR
(300 MHz, DMSO-d6): d 1.60–1.80 (1H, m), 2.18 (3H, s), 2.55–3.90
(20H, m), 4.43–4.72 (4H, m), 5.98 (1H, s), 6.18 (6H, s), 7.92–7.94
(2H, m), 8.61–8.63 (2H, m); Anal. Calcd for C21H29N7OSꢁ3C4H4O4:
C, 51.09; H, 5.32; N, 12.63. Found: C, 50.87; H, 5.27; N, 12.58;
LC–MS (ESI) m/z 428.4 [M+H]+.
4.1.20. 1-[1-(2-Fluorophenyl)-3-methyl-1H-pyrazol-5-
yl]piperazine (12h)
The title compound was prepared in 23% yield from 11d using
2-fluorophenylhydrazine hydrochloride in the procedures outlined
for 12d. 1H NMR (300 MHz, CDCl3): d 2.28 (3H, s), 2.79 (8H, br s),
5.67 (1H, s), 7.16–7.55 (4H, m).
4.1.21. 3-((2S,4S)-4-{4-[1-(2-Fluorophenyl)-3-methyl-1H-
pyrazol-5-yl]piperazin-1-yl}pyrrolizin-2-ylcarbonyl)
thiazolidine trihydrochloride (8k)
4.1.28. 1-[3-Methyl-1-(pyridin-3-yl)-1H-pyrazol-5-yl]piperazine
(12l)
The title compound was prepared in 67% yield using 12h in the
procedures outlined for 8c. Mp: 162 °C; 1H NMR (300 MHz, DMSO-
d6): d 2.03–2.25 (1H, m), 2.16 (3H, s), 2.72–4.00 (16H, m), 4.45–
4.71 (3H, m), 5.91 (1H, s), 7.32–7.35 (1H, m), 7.40–7.44 (1H, m),
7.51–7.57 (2H, m), 9.02 (1H, br s), 10.41 (1H, br s); Anal. Calcd
for C22H29FN6OSꢁ3HClꢁ1.5H2O: C, 45.48; H, 6.07; N, 14.43. Found:
C, 45.61; H, 5.96; N, 14.14; LC–MS (ESI) m/z 445.4 [M+H]+.
The title compound was prepared in 19% yield from 11d using
3-hydrazinopyridine dihydrochloride in the procedures outlined
for 12d. 1H NMR (300 MHz, CDCl3): d 2.28 (3H, s), 2.83–2.96 (8H,
m), 5.76 (1H, s), 7.36 (1H, dd, J = 4.7, 8.3 Hz), 8.13 (1H, ddd,
J = 1.4, 2.4, 8.3 Hz), 8.49 (1H, dd, J = 1.4, 4.7 Hz), 9.15 (1H, d,
J = 2.4 Hz).
4.1.22. 1-[1-(4-Chlorophenyl)-3-methyl-1H-pyrazol-5-
yl]piperazine (12i)
The title compound was prepared in 32% yield from 11d using
4-chlorophenylhydrazine hydrochloride in the procedures outlined
for 12d. LC–MS (ESI) m/z 277.4 [M+H]+.
4.1.29. 3-((2S,4S)-4-{4-[3-Methyl-1-(pyridin-3-yl)-1H-pyrazol-5-
yl] piperazin-1-yl}pyrrolizin-2-ylcarbonyl)thiazolidine
trimaleate (8o)
The title compound was prepared in 43% yield using 12l in the
procedures outlined for 8n. Mp: 165–167 °C; 1H NMR (300 MHz,
DMSO-d6): d 1.60–1.78 (1H, s), 2.17 (3H, s), 2.50–3.90 (20H, m),
4.42–4.71 (4H, m), 5.91 (1H, s), 6.19 (6H, s), 7.49–7.53 (1H, m),
8.12–8.16 (1H, m), 8.18–8.50 (1H, m), 8.98–8.99 (1H, m); Anal.
Calcd for C21H29N7OSꢁ3C4H4O4: C, 51.09; H, 5.32; N, 12.63. Found:
C, 50.90; H, 5.37; N, 12.78; LC–MS (ESI) m/z 428.4 [M+H]+.
4.1.23. 3-((2S,4S)-4-{4-[1-(4-Chlorophenyl)-3-methyl-1H-
pyrazol-5-yl]piperazin-1-yl}pyrrolizin-2-ylcarbonyl)
thiazolidine trihydrochloride (8l)
The title compound was prepared in 73% yield using 12i in the
procedures outlined for 8c. Mp: 210 °C; 1H NMR (300 MHz, DMSO-
d6): d 2.17 (3H, s), 2.25–2.40 (1H, m), 2.95–4.15 (17H, m), 4.46–
4.77 (3H, m), 5.97 (1H, s), 7.48–7.53 (2H, m), 9.13 (1H, br s),
11.01 (1H, br s); Anal. Calcd for C22H29ClN6OSꢁ2.5HClꢁ1.5H2O: C,
45.62; H, 6.00; N, 14.51. Found: C, 45.27; H, 6.29; N, 14.19; LC–
MS (ESI) m/z 461.4 [M+H]+.
4.1.30. 1-[3-Methyl-1-(pyridin-2-yl)-1H-pyrazol-5-yl]piperazine
(12m)
The title compound was prepared in 43% yield from 11d using
2-hydrazinopyridine dihydrochloride in the procedures outlined
for 12d. 1H NMR (300 MHz, CDCl3): d 2.29 (3H, s), 3.14 (8H, br s),
5.75 (1H, s), 7.19 (1H, m), 7.74–7.86 (2H, m), 8.50 (1H, m).
4.1.24. 1-[1-(4-Cyanophenyl)-3-methyl-1H-pyrazol-5-
yl]piperazine (12j)
The title compound was prepared in 40% yield from 11d using
4-cyanophenylhydrazine hydrochloride in the procedures outlined
for 12d. LC–MS (ESI) m/z 269.4 [M+H]+.
4.1.31. 3-((2S,4S)-4-{4-[3-Methyl-1-(pyridin-2-yl)-1H-pyrazol-5-
yl] piperazin-1-yl}pyrrolizin-2-ylcarbonyl)thiazolidine
dimaleate (8p)
4.1.25. 3-((2S,4S)-4-{4-[1-(4-Cyanophenyl)-3-methyl-1H-
pyrazol-5-yl]piperazin-1-yl}pyrrolizin-2-ylcarbonyl)
thiazolidine trihydrochloride (8m)
The title compound was prepared in 67% yield using 12j in the
procedures outlined for 8c. Mp: 220 °C; 1H NMR (300 MHz, DMSO-
d6): d 2.19 (3H, s), 2.20–2.40 (1H, m), 2.95–4.15 (17H, m), 4.46–
4.77 (3H, m), 6.05 (1H, s), 7.91 (2H, d, J = 9.0 Hz), 8.08 (2H, d,
J = 9.0Hz), 9.13 (1H, br s), 10.09 (1H, br s); Anal. Calcd for
The title compound was prepared in 33% yield using 12m in the
procedures outlined for 8n. Mp: 120–122 °C; 1H NMR (300 MHz,
DMSO-d6): d 2.19 (3H, s), 2.24–2.44 (1H, m), 2.88–4.20 (16H, m),
4.42–4.80 (3H, m), 5.99 (1H, s), 7.30–7.40 (1H, m), 7.77 (1H, d,
J = 8.3 Hz), 7.92–8.01 (1H, m), 8.46–8.54 (1H, m), 9.14 (1H, br s),
11.05 (1H, br s); Anal. Calcd for C21H29N7OSꢁ2C4H4O4ꢁH2O: C,
51.39; H, 5.80; N, 14.47. Found: C, 51.54; H, 5.52; N, 14.47; LC–
MS (ESI) m/z 428.4 [M+H]+.
C
23H29N7OSꢁ2.5HClꢁ0.05C4H8O2ꢁ1.5H2O: C, 48.53; H, 6.13; N, 17.08.
Found: C, 48.35; H, 6.42; N, 16.71; LC–MS (ESI) m/z 452.4 [M+H]+.
4.1.32. 1-[1-(5-Cyanopyridin-2-yl)-3-methyl-1H-pyrazol-5-
yl]piperazine (12n)
The title compound was prepared in 19% yield from 11d using
2-cyano-5-hydrazinopyridine in the procedures outlined for 12d.
LC–MS (ESI) m/z 269.4 [M+H]+.
4.1.26. 1-[3-Methyl-1-(pyridin-4-yl)-1H-pyrazol-5-yl]piperazine
(12k)
The title compound was prepared in 54% yield from 11d using
4-hydrazinopyridine dihydrochloride in the procedures outlined

T. Yoshida et al. / Bioorg. Med. Chem. 20 (2012) 5705–5719
5717
4.1.33. 3-((2S,4S)-4-{4-[1-(5-Cyanopyridin-2-yl)-3-methyl-1H-
pyrazol-5-yl]piperazin-1-yl}pyrrolizin-2-ylcarbonyl)
thiazolidine trihydrochloride (8q)
The title compound was prepared in 72% yield using 12n in the
procedures outlined for 8c. Mp: 210 °C; 1H NMR (300 MHz, DMSO-
d6): d 2.21 (3H, s), 2.25–2.45 (1H, m), 2.95–4.19 (17H, m), 4.47–
4.77 (3H, m), 6.05 (1H, s), 7.97 (1H, d, J = 8.7 Hz), 8.37 (1H, dd,
J = 2.3, 8.7 Hz), 8.93 (1H, d, J = 2.3 Hz), 9.15 (1H, br s), 10.80 (1H,
1.40, 1.44 (9H, s), 1.94–2.10 (2H, m), 2.27 (3H, s). 2.18–2.57 (4H,
m), 2.82–4.12 (11H, m), 4.41–4.76 (3H, m), 5.67 (1H, s), 7.24 (1H,
t, J = 7.3 Hz), 7.40 (2H, t, J = 7.6 Hz), 7.75 (2H, d, J = 7.7 Hz).
To a solution of the above compound (2.67 g, 5.07 mmol) in
dichloromethane (15 mL) was added trifluoroacetic acid (30 mL)
under ice-cooling. The mixture was stirred for 1 h, and then con-
centrated under reduced pressure. The residue was poured into a
saturated aqueous sodium hydrogen carbonate solution and ex-
tracted with chloroform. The extract was washed with brine, dried
and concentrated under reduced pressure. The residue was puri-
fied by silica gel column chromatography with chloroform/metha-
nol (10:1, v/v) to give free base of the title compound (1.58 g, 73%)
as an amorphous.
br s); Anal. Calcd for
C
22H28N8OSꢁ3HClꢁ0.2C2H4O2ꢁ1.5H2O: C,
44.77; H, 5.84; N, 18.64. Found: C, 44.54; H, 6.12; N, 18.50; LC–
MS (ESI) m/z 430.4 [M+H]+.
4.1.34. 3-[(2S,4S)-1-(tert-Butoxycarbonyl)-4-hydroxypyrrolid
inylcarbonyl]thiazolidine (24)
To a solution of the above compound (1.57 g, 3.68 mmol) in eth-
anol (20 mL) was added a solution of maleic acid (0.854 g,
7.36 mmol) in ethanol (20 mL) under ice-cooling. The precipitate
was collected by filtration to give the title compound (1.77 g, 72%)
as a white powder. Mp: 136 °C; 1H NMR (300 MHz, DMSO-d6): d
2.04–2.15 (1H, m), 2.15 (3H, s), 2.27 (1H, m), 2.43–2.60 (4H, m),
2.73–2.84 (4H, m), 2.95–3.13 (4H, m), 3.42–3.88 (3H, m), 4.40–
4.72 (3H, m), 5.81 (1H, s), 6.15 (4H, s), 7.28 (1H, t, J = 7.5 Hz), 7.45
(2H, t, J = 7.5 Hz), 7.73 (d, J = 7.5 Hz, 2H); Anal. Calcd for
To a solution of N-(tert-Butoxycarbonyl)-cis-4-hydroxy-L-pro-
line (23) (5.00 g, 21.6 mmol) and imidazole (6.48 g, 95.2 mmol)
in DMF (60 mL) was added tert-butyldimethylsilyl chloride
(7.16 g, 47.5 mmol) at room temperature. After stirring at room
temperature for 19 h, to the mixture were added water (60 mL)
and a 10% citric acid aqueous solution (200 mL) under ice-cooling.
The mixture was extracted with ethyl acetate. The extract was
washed with brine, dried and concentrated under reduced pressure
to give (2S,4S)-1-(tert-butoxycarbonyl)-4-(tert-butyldimethylsilyl-
oxy)pyrrolidine -2-carboxylic acid (10.9 g) as a light tan solid.
To a solution of the above compound (10.9 g), thiazolidine
(1.9 mL, 24 mmol) and HOBt (3.67 g, 24.6 mmol) in DMF (70 mL)
was added EDC hydrochloride (4.97 g, 25.9 mmol) under ice-cool-
ing. The reaction mixture was stirred at room temperature for 6 h.
The mixture was poured into a saturated aqueous sodium hydro-
gen carbonate solution and extracted with ethyl acetate. The ex-
tract was washed with brine, dried and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography with n-hexane/ethyl acetate (7:3, v/v) to give
3-[(2S,4S)-1-(tert-butoxycarbonyl)-4-(tert-butyldimethylsilyloxy)
pyrrolidinylcarbonyl]thiazolidine (7.28 g, 81%) as a white solid.
To a solution of the above compound (7.27 g, 17.4 mmol) in tet-
rahydrofuran (110 mL) was added 1 mol/L tetra-n-butylammo-
nium fluoride in tetrahydrofuran (19.2 mL) under ice-cooling.
After stirring at room temperature for 1.5 h, the mixture was con-
centrated under reduced pressure. The residue was poured into
brine and extracted with chloroform. The extract was dried and
concentrated under reduced pressure. The residue was purified
The residue was purified by silica gel column chromatography with
chloroform/methanol (20:1, v/v) to give the title compound
(5.25 g, 100%) as a white solid. 1H NMR (300 MHz, CDCl3): d 1.45,
1.43 (9H, s), 1.95–2.35 (2H, m), 2.95–3.23 (2H, m), 3.45–3.98
(3H, m), 4.30–5.77 (6H, m).
C
22H30N6OSꢁ2C4H4O4ꢁ0.8H2O: C, 53.53; H, 5.92; N, 12.48. Found: C,
53.56; H, 5.74; N, 12.10; LC–MS (ESI) m/z 427.2 [M+H]+.
4.1.36. 3-{(2R,4R)-4-[4-(3-Methyl-1-phenyl-1H-pyrazol-5-
yl)piperazin-1-yl]pyrrolidin-2-yl-carbonyl}thiazolidine
dimaleate (8s)
The title compound was prepared in 49% yield using 12d and 3-
((R)-1-tert-butoxycarbonyl-4-oxopyrrolidin-2-ylcarbonyl)thiazoli-
dine in the procedures outlined for 8n. Mp: 166–167 °C; 1H NMR
(300 MHz, DMSO-d6): d 1.66 (1H, m), 2.15 (3H, s), 2.51–2.54 (5H,
m), 2.64–2.79 (5H, m), 3.02–3.14 (4H, m), 3.41 (1H, m), 3.60–
3.90 (2H, m), 4.42–4.71 (3H, m), 5.80 (1H, s), 6.16 (4H, s), 7.28
(1H, t, J = 7.3 Hz), 7.45 (2H, t, J = 7.3 Hz), 7.73 (2H, d, J = 7.3 Hz);
Anal. Calcd for C22H30N6OSꢁ2C4H4O4: C, 54.70; H, 5.81; N, 12.76.
Found: C, 54.61; H, 5.78; N, 12.68.
4.1.37. 1-Benzyloxycarbonylisonipecotic acid anilide (14)
To a solution of 1-benzyloxycarbonylisonipecotic acid (13,
13.1 g, 49.8 mmol), aniline (5.15 g, 54.7 mmol) and HOBt (11.4 g,
74.4 mmol) in tetrahydrofuran (200 mL) was added EDC hydro-
chloride (11.4 g, 59.5 mmol). The reaction mixture was stirred at
room temperature for 17 h, and then concentrated under reduced
pressure. The residue wad poured into a saturated aqueous sodium
hydrogen carbonate solution and extracted with ethyl acetate. The
extract was washed with brine, dried and concentrated under re-
duced pressure to give the title compound (8.43 g, 50%) as a
pale-yellow oil. 1H NMR (300 MHz, CDCl3): d 1.61–2.06 (4H, m),
2.44–2. 56 (1H, m), 2.77–3.02 (2H, m), 4.03–4.36 (2H, m), 5.14
(2H, s), 7.11 (1H, t, J = 7.4 Hz), 7.20–7.44 (7H, m), 7.50 (2H, d,
J = 7.9 Hz).
4.1.35. 3-{(2S,4R)-4-[4-(3-Methyl-1-phenyl-1H-pyrazol-5-
yl)piperazin-1-yl]pyrrolidin-2-yl-carbonyl}thiazolidine
dimaleate (8r)
To
a solution of 24 (2.88 g, 9.52 mmol) and 2,6-lutidine
(1.22 mL, 10.5 mmol) in dichloromethane (50 mL) was added a
solution of trifluoromethanesulfonic anhydride (2.95 g, 10.5 mmol)
in dichloromethane (5 mL) dropwise under ice-cooling over
10 min. The mixture was stirred for 30 min and 12d (2.10 g,
8.67 mmol) and diisopropylethylamine (3.6 mL, 21 mmol) were
added to the solution. After stirring at room temperature at 21 h,
the reaction mixture was poured into a saturated aqueous sodium
hydrogen carbonate solution and extracted with chloroform. The
extract was washed with brine, dried and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography with chloroform/methanol (30:1, v/v) to give 3-
{(2S,4R)-1-(tert-butoxycarbonyl)-4-[4-(3-methyl-1-phenyl-1H-
pyrazol-5-yl)piperazin-1-yl]-2-pyrrolidinylcarbonyl}-1,3-thiazoli-
dine (2.70 g, 54%) as a amorphous. 1H NMR (300 MHz, CDCl3): d
4.1.38. 4-(1-Phenyl-1H-tetrazol-5-yl)piperidine (15)
To a solution of 14 (2.00 g, 5.91 mmol), triphenylphosphine
(3.10 g, 11.8 mmol) and a 40% diisopropyl azodicarboxylate–tolu-
ene solution (6.00 g, 11.9 mmol) in tetrahydrofuran (50 mL) was
added trimethylsilylazide (1.57 mL, 11.8 mmol) under ice-cooling.
The mixture was stirred at room temperature for 5 days, and then
concentrated under reduced pressure. The residue was purified by
silica gel chromatography with n-hexane/ethyl acetate (1:1, v/v) to
give
1-benzyloxycarbonyl-4-(1-phenyl-1H-tetrazol-5-yl)piperi-
dine (4.09 g) as a brown oil.
To a solution of the above compound (4.09 g) in methanol
(50 mL) was added 10% palladium/carbon (420 mg). The resulting
mixture was stirred at room temperature under a hydrogen

5718
T. Yoshida et al. / Bioorg. Med. Chem. 20 (2012) 5705–5719
atmosphere (1 atm) for 5 days. Palladium/carbon was removed by
filtration. The filtrate was concentrated under reduced pressure to
give the title compound (1.42 g, 55%) as a gray solid. 1H NMR
(300 MHz, CDCl3): d 1.55–2.02 (4H, m), 2.63 (2H, m), 3.00 (1H,
m), 3.16 (2H, m), 7.41–7.68 (5H, m).
hydrogen carbonate solution and extracted with chloroform. The
extract was dried and concentrated under reduced pressure to give
the title compound (90 mg, 82%) as an oil. 1H NMR (300 MHz,
CDCl3): d 1.47–1.93 (4H, m), 2.54 (2H, dt, J = 2.4, 12.3 Hz), 2.76
(1H, m), 3.07 (2H, m), 6.48 (1H, s), 7.36–7.55 (5H, m).
4.1.44. 3-{(2S,4S)-4-[4-(3-Trifluoromethyl-1-phenyl-1H-pyrazol-
5-yl)piperidino]pyrrolizin-2-ylcarbonyl}thiazolidine dihydro
chloride (9b)
The title compound was prepared in 56% yield using 20 in the
procedures outlined for 8a. Mp: 280 °C; 1H NMR (500 MHz,
DMSO-d6): d 1.90–2.30 (5H, m), 2.83–4.00 (13H, m), 4.46–4.71
(3H, m), 6.78 (1H, s), 7.57–7.62 (5H, m), 9.07 (1H, br s), 10.45
(1H, br s), 11.82 (1H, br s); Anal. Calcd for C23H28F3N5OSꢁ2HClꢁH2O:
C, 48.43; H, 5.65; N, 12.28. Found: C, 48.34; H, 5.78; N, 12.03; LC–
MS (ESI) m/z 480.4 [M+H]+.
4.1.39. 3-{(2S,4S)-4-[4-(1-Phenyl-1H-tetrazol-5-yl)piperidino]
pyrrolizin-2-ylcarbonyl}thiazolidine dihydrochloride (9a)
The title compound was prepared in 43% yield using 15 in the
procedures outlined for 8a. Mp: >186 °C; 1H NMR (300 MHz,
DMSO-d6): d 1.93–2.34 (5H, m), 2.85–3.95 (13H, m), 4.43–4.77
(3H, m), 7.69 (5H, s), 9.12 (1H, br s), 10.74 (1H, br s), 12.04 (1H,
br s); Anal. Calcd for C20H27N7OSꢁ2HClꢁ0.4C2H6Oꢁ2H2O: C, 46.18;
H, 6.60; N, 18.13. Found: C, 46.39; H, 6.48; N, 18.26; LC–MS (ESI)
m/z 414.4 [M+H]+.
4.1.40. 4-Trifluoroacetoacetylpyridine (17)
4.2. X-Ray crystallographic studies
To a solution of ethyl trifluoroacetate (6.32 g, 44.5 mmol) and a
28% sodium methoxide–methanol solution (9.4 g, 49 mmol) in tert-
butyl methyl ether (10 mL) was added a solution of 4-acetylpyri-
dine (16, 4.90 g, 40.4 mmol) in tert-butyl methyl ether (20 mL) at
room temperature and the mixture was stirred for 22 h. A 10%
citric acid aqueous solution was added until the reaction solution
became about pH 4. The precipitate was collected by filtration,
washed with water and dried to give the title compound (5.46 g,
62%) as a yellow solid. 1H NMR (500 MHz, DMSO-d6): d 6.60 (1H,
br s), 7.96 (2H, d, J = 5.5 Hz), 8.82 (2H, d, J = 5.5 Hz).
The protein of human DPP-4 (33ꢀ766) secreted from insect cells
was purified and crystallized according to the method reported by
Hiramatsu et al.24 The protein–inhibitor complex was obtained by
soaking a preformed DPP-4 crystal in the presence of compound
8g and preserved in liquid nitrogen for data collection at 100 K. X-
ray diffraction data were collected at the High Energy Accelerator
Research Organization (KEK) beam line BL5 and processed using
the program HKL2000.25 The structure of DPP-4 inhibitor complex
was solved by molecular replacement with the program PHASER,26
utilizing the previously determined coordinates of DPP-4 with Pro-
tein Data Bank accession code 1J2E. Data collection and model
refinement statistics are summarized in Table 5.
4.1.41. 4-(3-Trifluoromethyl-1-phenyl-1H-pyrazol-5-yl)pyridine
(18)
To a suspension of 17 (760 mg, 3.50 mmol) of ethanol (20 mL)
was added phenylhydrazine (0.38 mL, 3.9 mmol) at room temper-
ature. After stirring for 23 h, the reaction mixture was concen-
trated under reduced pressure. The residue was poured into
water and extracted with ethyl acetate. The extract was washed
with brine, dried and concentrated under reduced pressure. The
residue was purified by silica gel chromatography with chloro-
form/methanol (7:3, v/v) to give the title compound (470 mg,
47%) as an oil. 1H NMR (300 MHz, CDCl3): d 6.89 (1H, s), 7.22
(2H, d, J = 5.9 Hz), 7.30–7.47 (5H, m), 8.59 (2H, d, J = 5.9 Hz).
4.3. Docking studies in DPP-4
The X-ray crystal structure of DPP-4 (PDB code: 3VJM) was uti-
lized in the docking calculations. The compounds were docked into
DPP-4 using Glide 5.7.27
4.4. Pharmacokinetic studies on rats and monkeys
4.4.1. Measurement of compound 8g concentration in plasma
Concentrations of compound 8g in plasma were determined by
liquid chromatography with tandem mass spectrometry (LC–MS/
4.1.42. 1-Benzyl-4-(3-trifluoromethyl-1-phenyl-5-pyrazolyl)-
1,2,3,6-tetrahydropyridine (19)
MS). Plasma samples (50 lL) were placed in polypropylene test
To a solution of 18 (470 mg, 1.62 mmol) in acetonitrile (50 mL)
was added benzyl chloride (0.38 mL, 3.3 mmol). After stirring under
reflux for 24 h, the reaction mixture was concentrated under re-
duced pressure, and diethyl ether was added to the residue. The
precipitate was collected by filtration. To a solution of this solid in
ethanol was added sodium borohydride (130 mg, 3.44 mmol) under
ice-cooling. The mixture was stirred at room temperature for 22 h.
The reaction solution was poured into water and extracted with
ethyl acetate. The extract was washed with brine, dried and concen-
trated under reduced pressure. The residue was purified by silica gel
chromatography with n-hexane/ethyl acetate (4:1, v/v) to give the
title compound (142 mg, 23%) as an oil. 1H NMR (300 MHz, CDCl3):
d 2.15 (2H, m), 2.53 (2H, d, J = 5.6 Hz), 3.03 (2H, m), 3.56 (2H, s), 5.78
(1H, m), 6.53 (1H, s), 7.23–7.51 (10H, m).
tubes and the internal standard was added. The solution was
loaded into a solid-phase extraction cartridge (OASIS HLB), pre-
conditioned with acetonitrile (1 mL) and water (1 mL). The car-
tridge was washed twice with water (1 mL), and the analyte and
internal standard were eluted with acetonitrile (1 mL). The eluate
was evaporated to dryness under a nitrogen stream (40 °C) and
the residue was reconstituted with 200 lL of the mixed solvent
(water/acetonitrile/formic acid, 95:5:1, v/v). After the mixture
was mixed by the vortex-mixer, it was centrifuged and a portion
of the supernatant was injected into the LC–MS/MS system. Ana-
lyte concentrations were evaluated using the internal standard
method. Standard curves were calculated from the peak area ratio
(par) of analyte/internal standard. The nominal compound 8g con-
centrations were obtained using linear regression y = a + bx. The
measured peak area ratios of samples were converted into concen-
trations using the equation: concentration = par (analyte/internal
standard) minus intercept of the corresponding standard curve
divided by slope of the standard curve.
4.1.43. 1-(3-Trifluoromethyl-1-phenyl-1H-pyrazol-5-yl)
piperidine (20)
To a solution of 19 (142 mg, 0.37 mmol) and ammonium for-
mate (240 mg, 3.81 mmol) in methanol (20 mL) was added 10%
palladium/carbon (150 mg). The mixture was heated under reflux
under a nitrogen atmosphere for 2 h. After removal of the insoluble
materials by filtration, the filtrate was concentrated under reduced
pressure. The residue was poured into a saturated aqueous sodium
4.4.2. Pharmacokinetic analysis
The compound 8g plasma concentration–time profiles were
analyzed and its pharmacokinetic parameters were calculated

T. Yoshida et al. / Bioorg. Med. Chem. 20 (2012) 5705–5719
5719
using non-compartment analysis in the pharmacokinetic analysis
Acknowledgments
program WinNonlin (Pharsight Corporation, Ver. 4.0.1).
The authors thank Dr. Kunitomo Adachi, Dr. Hitoshi Kubota and
Dr. Kousuke Yasuda for their helpful discussions; Dr. Yoshitaka Ike-
da and Mr. Masami Yamashita for analytical support; and Dr. Tohru
Nakajima and Dr. Takao Kondo for their insight and guidance for
course of this work.
4.5. Biological experiments
4.5.1. DPP-4 inhibitory activity
The DPP-4 inhibitory activity of human and rat plasma was
measured by fluorescence assay using Gly-Pro-MCA (Peptide
Institute Inc.) as a DPP-4-specific fluorescent substrate. Reaction
References and notes
solutions containing 20
luted solution for human plasma and 10-fold diluted solution
for rat plasma), 20 of fluorescent substrate (100 mol/L),
140 L of buffer (0.003% Brij-35 containing PBS), and 20 L of test
substrate (of various concentrations) were incubated at room
temperature for 60 min using a 96-well flat-bottomed microtiter
plate. The measured fluorescent intensity (excitation 360 nm/
emission 465 nm, SPECTRA FLUOR, TECAN) was taken as the
DPP-4 activity. The inhibitory rate relative to the solvent addition
group was calculated and IC50 values were determined by logistic
analysis.
lL of human or rat plasma (20-fold di-
1. http://www.who.int/mediacentre/factsheets/fs312/en/ (accessed February 22,
2012).
2. (a) Kannel, W. B.; McGee, D. L. Circulation 1979, 59, 8; (b) Krolewski, A. S.;
Kosinski, E. J.; Warram, J. H.; Leland, O. S.; Busick, E. J.; Asmal, A. C.; Rand, L. I.;
Christlieb, A. R.; Bradley, R. F.; Kahn, C. R. Am. J. Cardiol. 1987, 59, 750.
3. Bolen, S.; Feldman, L.; Vassy, J.; Wilson, L.; Yeh, H. C.; Marinpoulos, S.; Wiley, C.;
Selvin, E.; Wilson, R.; Bass, E. B.; Brancati, F. L. Ann. Intern. Med. 2007, 147, 386.
4. Kreymann, B.; Williams, G.; Ghatei, M. A.; Bloom, S. R. Lancet 1987, 2, 1300.
5. Zander, M.; Madsbad, S.; Madsen, J. L.; Holst, J. J. Lancet 2002, 359, 824.
6. Nauck, M. A.; Kleine, N.; Orskov, C.; Holst, J. J.; Willms, B.; Creutzfeldt, W.
Diabetologia 1993, 36, 741.
7. (a) Holst, J. J.; Deacon, C. F. Diabetes 1998, 47, 1663; (b) Vilsboll, T.; Agerso, H.;
Krarup, T.; Holst, J. J. J. Clin. Endocrinol. Metab. 2003, 88, 220.
8. (a) Kieffer, T. J.; McIntosh, C. H.; Pederson, R. A. Endocrinology 1995, 136, 3585;
(b) Deacon, C. F.; Nauck, M. A.; Toft-Nielson, M.; Pridal, L.; Willms, B.; Holst, J. J.
Diabetes 1995, 44, 1126.
lL
l
l
l
9. Pospisilik, J. A.; Stafford, S. G.; Demuth, H.-U.; Brownsey, R.; Parkhouse, W.;
Finegood, D. T.; McIntosh, C. H. S.; Pederson, R. A. Diabetes 2002, 51, 943.
10. For comprehensive reviews on DPP-4 inhibitors, see: (a) Idris, I.; Donnelly, R.
Diabetes Obes. Metab. 2007, 9, 153; (b) von Geldern, T. W.; Trevillyan, J. M. Drug
Dev. Res. 2006, 67, 627; (c) Weber, A. E. J. Med. Chem. 2004, 47, 4135.
11. Gallwitz, B. Expert Opin. Investig. Drugs 2011, 20, 723.
4.5.2. DPP-8,9 inhibitory activity
The DPP8 and DPP9 activity were evaluated using the cytoplas-
mic fractions of cells expressing human DPP8 or DPP9 as enzyme
sources. Reaction solutions containing 20
of various concentrations, 20 L of enzyme preparations, 140
of buffer (0.003% Brij-35 containing PBS), and 20 L of Gly-Pro-
MCA (50 mol/L, Peptide Institute Inc.) were incubated at 37 °C
lL of test compounds
l
lL
12. Lambeir, A. M.; Durinx, C.; Scharpe, S.; De Meester, I. Crit. Rev. Clin. Lab. Sci.
2003, 40, 209.
l
l
13. Havale, S. H.; Pal, M. Bioorg. Med. Chem. 2009, 17, 1783.
14. (a) Park, J. E.; Lenter, M. C.; Zimmermann, R. N.; Garin-Chesa, P.; Old, L. J.;
Rettig, W. J. J. Biol. Chem. 1999, 274, 36505; (b) Abbott, C. A.; Yu, D. M.;
Woollatt, E.; Sutherland, G. R.; McCaughan, G. W.; Gorrell, M. D. Eur. J. Biochem.
2000, 267, 6140; (c) Ajami, K.; Abbott, C. A.; McCaughan, G. W.; Gorrell, M. D.
Biochim. Biophys. Acta 2004, 1679, 18.
for 30 min using a 96-well flat-bottomed microtiter plate. The
measured fluorescence intensity of 7-amino-4-methylcoumarin
was taken as the enzyme.
15. Ashworth, D. M.; Atrash, B.; Baker, G. R.; Baxter, A. J.; Jenkins, P. D. Bioorg. Med.
Chem. Lett. 1996, 6, 1163.
16. Rummey, C.; Metz, G. Proteins 2007, 66, 160.
4.5.3. Plasma DPP-4 activity after oral administration of 8g to
Wistar rats
17. Lankas, G. R.; Leiting, B.; Roy, R. S.; Eiermann, G. J.; Beconi, M. G.; Biftu, T.; Chan,
C. C.; Edmondson, S.; Feeney, W. P.; He, H.; Ippolito, D. E.; Kim, D.; Lyons, K. A.;
Ok, H. O.; Patel, R. A.; Petrov, A. N.; Pryor, K. A.; Qian, X.; Reigle, L.; Woods, A.;
Wu, J. K.; Zaller, D.; Zhang, X.; Zhu, L.; Weber, A. E.; Thornberry, N. A. Diabetes
2005, 54, 2988.
18. (a) Sakashita, H.; Akahoshi, F.; Kitajima, H.; Tsutsumiuchi, R.; Hayashi, Y.
Bioorg. Med. Chem. 2006, 14, 3662; (b) Sakashita, H.; Akahoshi, F.; Yoshida, T.;
Kitajima, H.; Hayashi, Y.; Ishii, S.; Takashina, Y.; Tsutsumiuchi, R.; Ono, S.
Bioorg. Med. Chem. 2007, 15, 641.
19. (a) Yoshida, T.; Sakashita, H.; Akahoshi, F.; Hayashi, Y. Bioorg. Med. Chem. Lett.
2007, 17, 2618; (b) Yoshida, T.; Akahoshi, F.; Sakashita, H.; Sonda, S.; Takeuchi,
M.; Tanaka, Y.; Nabeno, M.; Kishida, H.; Miyaguchi, I.; Hayashi, Y. Bioorg. Med.
Chem. 2012, 20, 5033.
20. Konstantinchenko, A. A.; Lyashenko, P. I.; Pozharskii, A. F. Chem. Heterocycl.
Compd. 1981, 17, 832.
Male Wistar rats (7–9 weeks of age) fasted overnight were used.
Compound 8g (HBr salt) was dissolved in 0.5% hydroxypropyl
methylcellulose (HPMC) and administered orally at a dose of
0.03, 0.1, 0.3 and 1 mg/kg. At pre-administration and at 0.5, 1, 2,
3, 6, 9, 12, 24 and 48 h after administration, 0.1 mL of blood was
collected from tail vein. After centrifugation, 10 lL of plasma was
diluted 10-fold using buffer (0.003% Brij-35 containing PBS).
Twenty microliter of the diluted plasma was used instead of
20 lL of test substrate for the determination of DPP-4 inhibitory
activity by fluorescence as described above.
21. Bouillon, J.-P.; Ates, C.; Janousek, Z.; Viehe, H. G. Tetrahedron Lett. 1993, 34,
5075.
4.5.4. Oral glucose tolerance test (OGTT) in Zucker fatty rats
An OGTT in Zucker fatty rats was carried out based on the meth-
od of Balkan et al.28 Male Zucker fatty and lean rats (13 weeks of
age; n = 10) were fasted overnight. Compound 8g (HBr salt) was dis-
solved in 0.5% HPMC solution and administered orally. After
30 min, glucose solution was orally administered at 1 g/kg body
weight. Blood samples were collected from the tail veins at the indi-
cated times and plasma samples were prepared. Plasma glucose
concentrations as well as plasma DPP-4 activity were measured.
22. Duncia, J. V.; Pierce, M. E.; Santella, J. B. J. Org. Chem. 1991, 56, 2395.
23. Parikh, R.; Doering, W. vonE. Arn. Soc. 1967, 89, 5505.
24. Hiramatsu, H.; Kyono, K.; Shima, H.; Fukushima, C.; Sugiyama, S.; Inaka, K.;
Yamamoto, A.; Shimizu, R. Acta Crystallogr. 2003, D59, 595.
25. Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276, 307.
26. McCoy, A. J.; Grosse-Kunstleve, R. W.; Storoni, L. C.; Read, R. J. Acta Crystallogr.
2005, D61, 458.
27. Glide; Schrodinger, LLC, New York, NY. 2011 http://www.schrodinger.com/.
28. Balkan, B.; Kwasnik, L.; Miserendino, R.; Holst, J. J.; Li, X. Diabetologia 1999, 42,
1324.