[(S)-γ-(Arylamino)prolyl]thiazolidine compounds as a novel series of potent and stable DPP-IV inhibitors

Article abstract of DOI:10.1016/j.bmc.2006.01.022

Dipeptidyl peptidase-IV (DPP-IV) inhibitors, or glucagon-like peptide-1 (GLP-1) enhancers, are looked to as a potential new class of antidiabetic agents. In particular, potent and long-acting inhibitors might offer advantages in exploiting DPP-IV inhibition. The series of [(S)-γ-(arylamino)prolyl]-(S)-2-cyanopyrrolidine compounds on which we reported previously has a highly potent inhibitory activity but seemed to be unstable in neutral aqueous solution. Here, we describe [(S)-γ-(arylamino)prolyl]thiazolidine compounds as a novel series of potent and stable DPP-IV inhibitors. They are the thiazolidine analogs of [(S)-γ-(arylamino)prolyl]-(S)-2-cyanopyrrolidine but with the electrophilic nitrile removed to improve chemical stability in aqueous solution. Of the compounds investigated in the present study, the [((S)-γ-3,4-dicyanophenylamino)prolyl]thiazolidine 12m was the most potent. The structure-activity relationship (SAR) of the γ-substituent in the proline moiety of the thiazolidide was similar to that obtained with the (S)-2-cyanopyrrolidide. The γ-substituent in the proline moiety of both the (S)-2-cyanopyrrolidide and the thiazolidide may engage with the S₂ binding pocket of DPP-IV and thereby achieve hydrophobic interaction in the same manner. Based on pharmacokinetic experiments in rats, the representative compound 11, which displayed high oral bioavailability (BA = 83.9%) and long half-life in plasma (t_1/2 = 5.27 h), was found to have an excellent pharmacokinetic profile.

Full text of DOI:10.1016/j.bmc.2006.01.022

Bioorganic & Medicinal Chemistry 14 (2006) 3662–3671
[(S)-c-(Arylamino)prolyl]thiazolidine compounds as a novel series
of potent and stable DPP-IV inhibitors
Hiroshi Sakashita,a Fumihiko Akahoshi,a,* Hiroshi Kitajima,a
Reiko Tsutsumiuchib and Yoshiharu Hayashic
aResearch Laboratory II, Pharmaceuticals Research Unit, Research and Development Division,
Mitsubishi Pharma Corporation, 1000, Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan
bPharmaceutical Development Laboratories, Pharmaceutical Technology Center, Technology and Production Division,
Mitsubishi Pharma Corporation, 14, Sunayama, Kamisu, Ibaraki 314-0255, Japan
cGlobal Projects Coordination Department, Research and Development Division, Mitsubishi Pharma Corporation,
2-2-6, Nihonbashi-Honcho, Chuo-ku, Tokyo 103-8405, Japan
Received 1 December 2005; revised 10 January 2006; accepted 11 January 2006
Available online 7 February 2006
Abstract—Dipeptidyl peptidase-IV (DPP-IV) inhibitors, or glucagon-like peptide-1 (GLP-1) enhancers, are looked to as a potential
new class of antidiabetic agents. In particular, potent and long-acting inhibitors might offer advantages in exploiting DPP-IV inhi-
bition. The series of [(S)-c-(arylamino)prolyl]-(S)-2-cyanopyrrolidine compounds on which we reported previously has a highly
potent inhibitory activity but seemed to be unstable in neutral aqueous solution. Here, we describe [(S)-c-(arylamino)prolyl]thiaz-
olidine compounds as a novel series of potent and stable DPP-IV inhibitors. They are the thiazolidine analogs of [(S)-c-(arylami-
no)prolyl]-(S)-2-cyanopyrrolidine but with the electrophilic nitrile removed to improve chemical stability in aqueous solution. Of the
compounds investigated in the present study, the [((S)-c-3,4-dicyanophenylamino)prolyl]thiazolidine 12m was the most potent. The
structure–activity relationship (SAR) of the c-substituent in the proline moiety of the thiazolidide was similar to that obtained with
the (S)-2-cyanopyrrolidide. The c-substituent in the proline moiety of both the (S)-2-cyanopyrrolidide and the thiazolidide may
engage with the S2 binding pocket of DPP-IV and thereby achieve hydrophobic interaction in the same manner. Based on pharma-
cokinetic experiments in rats, the representative compound 11, which displayed high oral bioavailability (BA = 83.9%) and long
half-life in plasma (t1/2 = 5.27 h), was found to have an excellent pharmacokinetic profile.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
come to be seen as a potential new type of antidiabetic
agent free of side effects such as hypoglycemia and
exhaustion of pancreatic b-cells. In particular, a potent
and long-acting inhibitor might offer advantages in
exploiting DPP-IV inhibition.
Dipeptidyl peptidase-IV (EC 3.4.14.5, DPP-IV) is a
member of the serine protease family that recognizes
an amino acid sequence having proline or alanine at
the second position from the N-terminal and produces
dipeptide.1 DPP-IV is widely distributed in mammalian
tissues and plays several physiological roles; in particu-
lar, its role as a peptidase that rapidly inactivates gluca-
gon-like peptide-1 (GLP-1) has drawn interest.2 GLP-1
is secreted in response to meal ingestion and stimulates
insulin secretion.3 It has been suggested that potentia-
tion and extension of the action of GLP-1 by DPP-IV
inhibition would stimulate insulin secretion after meal
ingestion only,4 and DPP-IV inhibitors have therefore
Although a number of DPP-IV inhibitors have been
reported and classified into structural types, most of
them are substrate analogs composed of the P2–P1 frag-
ment (Fig. 1).5 (S)-2-Cyanopyrrolidine and thiazolidine
structures are used as proline mimic in the P1 part.
Dipeptide-type inhibitors containing an (S)-2-cyano-
pyrrolidine structure, for example, NVP-DPP728 (1)6–8
and NVP-LAF237 (2),8,9 have been reported as potent
inhibitors. The latter is under clinical trials as an antidi-
abetic agent.9
We previously reported that a series of [(S)-c-(arylami-
Keywords: DPP-IV inhibitor; Thiazolidine.
*
no)prolyl]-(S)-2-cyanopyrrolidine compounds had
a
Corresponding author. Tel.: +81 45 963 4293; fax: +81 45 963
4211; e-mail: Akahoshi.Fumihiko@mk.m-pharma.co.jp
potent inhibitory activity (Fig. 2b and Table 1).10 The
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.01.022

H. Sakashita et al. / Bioorg. Med. Chem. 14 (2006) 3662–3671
3663
S2 pocket
S1 pocket
P2
+
H3N
N
O
Ser
Xaa
O
-
COO
O
P1
P1'
P2
OH
H
N
N
N
N
H
N
CN
N
H
O
CN
NC
O
NVP-DPP728 (1)
NVP-LAF237 (2)
Figure 1.
S2 pocket
CN
S2 pocket
DPP-IV
DPP-IV
N
Ar
HN
γ
S1 pocket
X
S1 pocket
HN
S
γ
N
N
N
N
H
N
H
N
H
O
O
CN
O
Ser630
O
N
a
b
c
NVP-DPP728 (1)
(folded conformation)
[(S)-γ-(arylamino)prolyl]-(S)-2-cyanopyrrolidine
(X = ArNH-)
3 (X = (5-cyano-2-pyridyl)amino)
4 (X = H)
[(S)-γ-(arylamino)prolyl]thiazolidine
Figure 2. Design of [(S)-(c-arylamino)prolyl]-(S)-2-cyanopyrrolidine and thiazolidine.
Table 1. DPP-IV inhibition of (S)-2-cyanopyrrolidines and thiazolidines
V
N
U
W
Compound
U
V
W
DPP-IV inhibition,
IC50 (nmol/L)
Human
Rat
H
N
N
N
H
NVP-DPP728 (1)
15
CH2
S
CN
H
1.4
2.3
500
O
270
NC
4
14
CH2
S
CN
H
2.9
2.9
607
N
H
538
O
NC
H
N
N
3
11
CH2
S
CN
H
0.25
25.4
0.27
30.4
N
H
O

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H. Sakashita et al. / Bioorg. Med. Chem. 14 (2006) 3662–3671
representative compound 3 is an analog conformational-
ly restricted using a proline structure and which mimics
the folded conformation of the conformationally flexible
compound 1 to allow interaction of the (5-cyano-2-pyr-
idyl)amino moiety with the S2 binding pocket as shown
in Figure 2a.6 As a result, compound 3 showed 5-fold
more potent inhibitory activity than the flexible com-
pound 1. In addition, compound 3, with an arylamino
group introduced at the c-position, has 10-fold more po-
tent inhibitory activity than the c-unsubstituted com-
pound 4.11
cyclic products (Fig. 3).8 Ashworth and coworkers
reported on the DPP-IV-inhibitory activity of dipep-
tide-type analogs having (S)-2-cyanopyrrolidide and
their chemical stability in aqueous solution (pH
7.4).11 They found that, although most of the tested
dipeptide analogs exhibited excellent half-lives
(T1/2decomp > 48 h or T1/2decomp = 48 h), the prolyl-(S)-
2-cyanopyrrolidine 4 has a relatively short half-life
(T1/2decomp = 7.5 h).
[(S)-c-(Arylamino)prolyl]-(S)-2-
cyanopyrrolidines, bearing the same core structure,
seemed to cyclize relatively rapidly in neutral aqueous
solution in the same way as shown in Figure 3, and
their instability may result in the short half-life in
plasma.
The (S)-2-cyanopyrrolidide class of compounds thus
includes many potent DPP-IV inhibitors. It suffers,
however, from chemical instability whereby the N-ter-
minal amine intramolecularly attacks the electrophilic
nitrile of the (S)-2-cyanopyrrolidine moiety, affording
Meanwhile, dipeptide-type analogs of pyrrolidide (5)
and thiazolidide (6) lacking the electrophilic nitrile are
known as DPP-IV inhibitors; in general, the thiazolidide
6 is more effective than the corresponding pyrrolidide 5
(Fig. 4).12,13 Free from intramolecular cyclization, they
are chemically stable. P32/98 (7), one example of this
compound class, produced improved glucose tolerance
in diabetic patients and healthy volunteers in clinical tri-
al,14,15 but exhibited only modest inhibitory activity
(IC50 = 75 nmol/L for human plasma16). To identify a
DPP-IV inhibitor with more potent and favorable phar-
macokinetic action, we focused on the [(S)-c-(arylami-
no)prolyl]thiazolidine compounds (Fig. 2c). It was
expected that, even in the thiazolidide, the introduction
at the c-position of a proline moiety would bring more
potent inhibitory activity.
R1
R1
R2
H
R2
O
O
N
N
N
N
Y
N
Y = NH, O
Figure 3.
R1
Z
Z
The inhibitory activity of the most potent of the com-
pounds resulting from this approach, 12m, is single
nanomolar, which is ca. 10-fold that of P32/98 (7).
The representative compound 11 was found to have an
excellent pharmacokinetic profile and improved chemi-
cal stability. Herein we report on [(S)-c-(arylamino)pro-
lyl]thiazolidine as a novel series of potent and stable
DPP-IV inhibitors.
N
N
H2N
H2N
O
O
5 (Z = CH2)
6 (Z = S)
P32/98 (7)
Figure 4.
11 (Ar = 5-cyano-2-pyridyl)
12b (Ar = 2-cyanophenyl)
12c (Ar = 4-cyanophenyl)
12d (Ar = 4-nitrophenyl)
H
Ar
HO
H2N
HO
N
S
S
S
ii, iii, iv
v, vi
i
12e (Ar = 4-methanesulfonylphenyl)
12k (Ar = 2-chloro-4-cyanophenyl)
12l (Ar = 3-chloro-4-cyanophenyl)
12m (Ar = 3,4-dicyanophenyl)
N
N
N
N
N
N
N
COOH
H
Boc
Boc
Boc
HCl
O
O
O
10
8
9
vii
viii
H
H2N
H
N
Ar
N
O
S
S
ix, vi
S
N
N
N
N
N
H
H
N
O
HCl
HCl
Boc
O
O
12h
12a (Ar = phenyl)
13
12f (Ar = 4-chlorophenyl)
12g (Ar = 4-methoxyphenyl)
12i (Ar = 3,4-methylenedioxyphenyl)
12j (Ar = 3-chloro-4-methoxyphenyl)
Scheme 1. Reagents and conditions: (i) thiazolidine, HOBT, EDC, DMF, rt; (ii) MsCl, Et3N, CH2Cl2, 0 ꢁC; (iii) NaN3, DMF, 80 ꢁC; (iv) H2, Pd/C,
EtOH, rt; (v) Ar-Cl or Ar-F, i-PrNEt2, N-methyl-2-pyrrolidone, 80–100 ꢁC; (vi) HCl, AcOEt; (vii) H2, Pd/C, HCl, dioxane, AcOEt; (viii) DMSO,
SO3–pyridine complex; (ix) aniline, NaBH3CN, AcOH, MeOH; or Ar-NH2, NaBH(OAc)3, 1,2-dichloroethane.

H. Sakashita et al. / Bioorg. Med. Chem. 14 (2006) 3662–3671
3665
Table 2. DPP-IV inhibition of [(S)-c-(arylamino)prolyl]thiazolidines
2. Chemistry
H
Ar
The synthesis of [(S)-c-(arylamino)prolyl]thiazolidine
compounds is shown in Scheme 1. The trans-hydroxy
group of the amide 9, which was prepared by coupling
the trans-Boc-hydroxyproline 8 with thiazolidine, was
converted to its mesylate. Treatment of the mesylate
with sodium azide, followed by catalytic hydrogenation,
gave the cis-amino compound 10. Reaction of the amine
10 with 2-chloro-5-cyanopyridine or other electron-defi-
cient aromatic halides and subsequent removal of the
Boc group afforded the (S)-c-substituted derivatives 11
and 12b–e and k–m. The aniline derivative 12h was pre-
pared from the nitrophenyl derivative 12d by catalytic
hydrogenation. The non-electron-deficient phenyl deriv-
atives 12a, f, g, i, and j were prepared by reductive ami-
nation of the ketone 13 with anilines and subsequent
removal of the Boc group.
N
S
N
N
H
O
Compound
Ar
DPP-IV inhibition,
IC50 (nmol/L)
Human
Rat
12a
12b
12c
12d
147
190
CN
24.1
25.2
19.2
41.3
NC
33.6
21.0
O2N
3. Results and discussion
The thiazolidide and related compounds were evaluated
for DPP-IV-inhibitory activity in human and rat plasma
by fluorescence assay using Gly-Pro-MCA (Tables 1 and
2).
O O
S
12e
12f
12g
12h
12i
52.3
45.4
51.5
105
43.8
55.1
54.9
Me
Cl
First of all, we examined the effect of the conversion of
the (S)-2-cyanopyrrolidine moiety into a thiazolidine
structure and conformational restriction using a proline
structure on the thiazolidide (Table 1).
MeO
H2N
O
As a result of the conversion, the thiazolidide 1517
showed 200-fold decreased activity compared to the
(S)-2-cyanopyrrolidide 1. In the same way, the prolyl-
thiazolidine 14 was 200-fold less active than the pro-
pyl-(S)-2-cyanopyrrolidine 4.11 These results indicate
that interaction of the nitrile group and Ser630 at the ac-
tive site of the DPP-IV is a dominant factor for exhibi-
tion of inhibitory activity; an (S)-2-cyanopyrrolidine
structure might therefore be widely used for DPP-IV
inhibitors despite decomposition by intramolecular
cyclization.
125
29.0
19.9
18.2
11.1
8.4
34.5
14.8
21.8
13.0
10.8
O
MeO
12j
Cl
NC
Cl
12k
12l
NC
In a previous study of (S)-2-cyanopyrrolidides, we
found that compound 3 has 5-fold more potent inhibito-
ry activity than NVP-DPP728 (1). Compound 3 is an
analog of the flexible compound 1 conformationally
restricted using a proline structure to produce interac-
tion of the (5-cyano-2-pyridyl) moiety with the S2 bind-
ing site as shown in Figure 2. As a result of the present
research on thiazolidides, compound 11 was found to be
10-fold more potent than the flexible compound 15.17
Restriction using a proline structure thus produces more
potent inhibitory activity in the thiazolidides in the same
way as in (S)-2-cyanopyrrolidides.
Cl
NC
12m
NC
step designed to bring about a recovery of the decreased
activity by conversion of the (S)-2-cyanopyrrolidine
moiety into a thiazolidine structure.
In the next step, as the phenylamino-derivatives of (S)-2-
cyanopyrrolidide had shown potent inhibitory activity
comparable to that of (5-cyano-2-pyridyl)amino-deriva-
tive 3,10 we examined the inhibitory activity of (S)-c-
phenylamino-substituted derivatives of thiazolidide
(12a–m). Table 2 summarizes their inhibitory activity.
The non-substituted phenylamino-compound 12a
Moreover, compared to the c-unsubstituted compound
14, compound 11, as a result of the introduction of a
c-substituent, was 20-fold more potent. This result indi-
cates that the c-substituent of compound 11 plays a sig-
nificant part in the binding with DPP-IV. Modification
of the c-substituent was therefore undertaken in the next

3666
H. Sakashita et al. / Bioorg. Med. Chem. 14 (2006) 3662–3671
showed 6-fold less activity than the (5-cyano-2-pyr-
idyl)amino compound 11, but introduction of a substi-
tution on the benzene ring of 12a produced potent
activity (12b–m). Di-substituted phenylamino com-
pounds tended to be more potent than mono-substituted
phenylamino-compounds, for example, 12c versus 12l or
m; or 12g versus 12i or j. However, no clear-cut electron-
ic influence of the substituent emerged in these
compounds; for example, substitution of an electron-do-
nating methoxy group (12g) or an electron-withdrawing
cyano or nitro group (12b–d) both resulted in 3- to 9-
fold more potent activity than the non-substituted
compound 12a. These results of SAR study of the c-sub-
stituent in the thiazolidide are similar to those obtained
in the (S)-2-cyanopyrrolidide. The c-substituent in both
the (S)-2-cyanopyrrolidide and the thiazolidide may
engage with the S2 binding pocket of DPP-IV and there-
by achieve hydrophobic interaction in the same manner.
Of the compounds investigated in the present study,
compound 12m, bearing a 3,4-dicyanophenylamino-
group, showed the highest inhibitory activity, which,
despite the lack of the electrophilic nitrile, was single
nanomolar (IC50 = 8.4 nmol/L for human plasma) or
ca. 10-fold more potent than that of P32/98 (7).
10 µmol/kg p.o.
30 µmol/kg p.o.
100 µmol/kg p.o.
120
100
80
60
40
20
0
0
4
8
12
16
20
24
Time after administration (h)
Figure 6. Plasma DPP-IV activities after oral administration of
thiazolidide 11 to Wistar rats.
100000
i.v.
10000
p.o.
1000
100
10
1
As proof of concept, the solution stability of (S)-2-
cyanopyrrolidide and thiazolidide was examined at
37 ꢁC using a buffer of pH 6.8. The (S)-2-cyanopyrroli-
dide 3 and the thiazolidide 11, both bearing (5-cyano-
2-pyridyl)amino group, the component parts of
NVP-DPP728 (1), at the c-position of the proline moiety
were selected as representative compounds. As shown in
Figure 5, the (S)-2-cyanopyrrolidide 3 decomposed rap-
idly (T1/2decomp = 1.3 h), whereas the thiazolidide 11 was
quite stable. We therefore undertook ex vivo evaluation
of the plasma DPP-IV-inhibitory activity and the phar-
macokinetic study of the thiazolidide 11. The plasma
DPP-IV activity after oral administration of 11 to Wis-
tar rats is shown in Figure 6. Half-inhibition was ob-
served at 9 h after administration of 100 lmol/kg. The
plasma drug concentration profiles and pharmacokinet-
ic parameters after intravenous or oral administration
(10 mg/kg, 19 lmol/kg) of 11 to Sprague–Dawley rats
are shown in Figure 7 and Table 3, respectively. With
high oral bioavailability (BA = 83.9%) and long half-life
in plasma (t1/2 = 5.27 h), the thiazolidide 11 exhibited an
excellent pharmacokinetic profile. The pharmacokinetic
0
4
8
12
16
20
24
Time after administration (h)
Figure 7. Plasma drug concentration profiles after a intravenous or
oral administration of thiazolidide 11 at a dose of 10 mg/kg to
Sprague–Dawley rats.
data support the ex vivo experimental finding of long-
lasting action.
4. Conclusion
We have described here a novel series of potent DPP-IV
inhibitors, the [(S)-c-(arylamino)prolyl]thiazolidine
compounds, which are the thiazolidine analogs of the
previously reported [(S)-c-(arylamino)prolyl]-(S)-2-
cyanopyrrolidine compounds.10 The electrophilic nitrile
has been removed in them to improve chemical stability
in aqueous solution. Of the compounds investigated in
the present study, the (S)-c-3,4-dicyanophenylamino-
substituted compound 12m showed the most potent
inhibitory activity. The SAR of the c-substituent in the
proline moiety of the thiazolidide was similar to that ob-
tained in the (S)-2-cyanopyrrolidide. The c-substituent
in both the (S)-2-cyanopyrrolidide and the thiazolidide
may engage with the S2 binding pocket of DPP-IV and
thereby achieve hydrophobic interaction in the same
manner. Based on ex vivo and pharmacokinetic experi-
ments in rats, the representative compound 11 exhibited
an excellent pharmacokinetic profile. Through conver-
sion of the (S)-2-cyanopyrrolidine moiety into a thiaz-
olidine structure, we consider that removal of the
electrophilic nitrile is one of the most promising
methods for improvement of the chemical stability of
prolyl-(S)-2-cyanopyrrolidine compounds. Further
optimization of these proline-containing inhibitors for
100
10
(S)-2-cyanopyrrolidide 3
thiazolidide 11
1
0
2
4
6
Time (h)
Figure 5. Solution stability of (S)-2-cyanopyrrolidide 3 and thiazoli-
dide 11 at pH 6.8 and 37 ꢁC.

H. Sakashita et al. / Bioorg. Med. Chem. 14 (2006) 3662–3671
3667
Table 3. Pharmacokinetic parameters of drug in plasma after intravenous or oral administration of thiazolidide 11 to Sprague–Dawley rats
na
AUCc (ng h/mL)
BAe (%)
b
t1/2 (h)
d
Cmax (ng/mL)
Dosage
10 mg/kg iv
10 mg/kg po
3
3
5.27 1.55
46,463 5196
39,937 5461
9939.6 2364.5
83.9 11.5
a Number of animals.
b Elimination half-life.
c Area under plasma concentration–time curve from zero to infinity.
d Maximum plasma concentration of unchanged drug.
e Oral bioavailability: (AUCpo · doseiv)/(AUCiv · dosepo) · 100.
more potent activity, focused on the S2 binding pocket,
will be described in due course.
for 1 h, the reaction mixture was washed with water and
dried. The solvent was evaporated under reduced pres-
sure to give 3-((2S,4R)-1-tert-butoxycarbonyl-4-metha-
nesulfonyloxy-2-pyrrolidinylcarbonyl)-1,3-thiazolidine
(70.5 g, 100%) as an oil. (2) The compound thus pre-
pared (70.5 g, 185 mmol) and sodium azide (13.3 g,
204 mmol) were dissolved in DMF (500 mL) and stirred
at 80 ꢁC for 5 h before concentration of the reaction mix-
ture under reduced pressure and addition of water. The
mixture was then extracted with ethyl acetate and the
organic layer dried and concentrated. The residue was
purified by silica gel chromatography to give the com-
pound 3-((2S,4S)-4-azido-1-tert-butoxycarbonyl-2-pyr-
rolidinylcarbonyl)-1,3-thiazolidine (39.5 g, 65%) as a
white solid. (3) The compound thus prepared (26.7 g,
81.6 mmol) was dissolved in ethanol (270 mL) and the
mixture stirred under a hydrogen atmosphere (1 atm)
for 18 h in the presence of 10% palladium carbon cata-
lyst (13.4 g). The reaction mixture was filtered and the fil-
trate concentrated under reduced pressure to give the
title compound (24.5 g, quant) as a black solid. 1H
NMR (CDCl3) d 1.40–1.45 (9H, m), 1.70–1.83 (1H, m),
2.07 (2H, br s), 2.32–2.56 (1H, m), 2.90–3.19 (2H, m),
3.25–3.58 (2H, m), 3.60–4.14 (3H, m), 4.31–4.80 (3H, m).
5. Experimental
5.1. Chemistry
1H NMR spectra were measured on a Bruker DPX-300
instrument with tetramethylsilane as the internal stan-
dard; chemical shifts are reported in parts per million
(ppm, d units). Splitting patterns are designated as s, sin-
glet; 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 electron ionization (EI) or the chemical
ionization (CI) mode. Electron analysis for carbon,
hydrogen, and nitrogen was performed with a Yanagim-
oto 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 unless 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, dimethylsulfoxide;
EDC, 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride; HOBT, 3-hydroxybenztriazole hydrate.
5.1.3. Representative example of preparation of 11, 12b–e,
12k–m: general method A
5.1.3.1. 3-[(2S,4S)-4-(5-Cyano-2-pyridyl)amino-2-pyr-
rolidinylcarbonyl]-1,3-thiazolidine dihydrochloride (11).
(1) To a solution of compound 10 (904 mg, 3 mmol) in
N-methyl-2-pyrrolidone (9 mL) were added 2-chloro-5-
cyanopyridine (416 mg, 3 mmol) and diisopropylethyl-
amine (9 mmol) and the mixture stirred at 80 ꢁC for
18 h. The reaction mixture was added to saturated aque-
ous sodium hydrogen carbonate solution and the mix-
ture extracted with ethyl acetate. The extract was dried
and the solvent evaporated under reduced pressure.
The residue was purified by silica gel chromatography
to give 3-[(2S,4S)-1-tert-butoxycarbonyl-4-(5-cyano-2-
pyridyl)amino-2-pyrrolidinylcarbonyl]-1,3-thiazolidine
(807 mg, 67%) as a white amorphous substance. (2) The
compound thus prepared (711 mg) was dissolved in ethyl
acetate (1.76 mL) and 4 mol/L hydrochloric acid–ethyl
acetate (2.20 mL) added to the solution. The mixture
was stood at room temperature for 18 h and the precip-
itated solid collected by filtration to give the title
compound (709 mg) as a white powder. 1H NMR
(DMSO-d6) d 1.74–1.94 (1H, m), 2.78–2.94 (1H, m),
2.97–3.26 (3H, m), 3.40–3.77 (3H, m), 4.40–4.80
(4H, m), 6.64 (1H, d, J = 9.0 Hz), 7.77 (1H, dd,
J = 8.7, 2.4 Hz), 8.08 (1H, br s), 8.46 (1H, d,
J = 1.8 Hz), 8.86 (1H, br s), 10.37 (1H, br s). Anal. Calcd
5.1.1.
3-[(2S,4R)-1-tert-Butoxycarbonyl-4-hydroxy-2-
pyrrolidinylcarbonyl]-1,3-thiazolidine (9). To a solution
of thiazolidine (29.4 g, 0.33 mmol) in DMF (300 mL)
were added N-tert-butoxycarbonyl-L-trans-hydroxypro-
line (8, 69.4 g, 0.3 mol), HOBT (50.5 g, 0.33 mol), and
EDC (63.3 g, 0.33 mol). The mixture was stirred at room
temperature for 18 h and the reaction mixture concen-
trated. To the concentrate were added brine and saturat-
ed aqueous sodium hydrogen carbonate solution and
the mixture was extracted with ethyl acetate. The extract
was dried and the solvent evaporated under reduced
pressure to give the title compound (56.3 g, 62%) as a
colorless transparent oil. 1H NMR (CDCl3) d 1.41–
1.45 (9H, m), 1.95–2.34 (2H, m), 2.62–3.25 (2H, m),
3.40–3.98 (4H, m), 4.40–4.90 (4H, m).
5.1.2. 3-((2S,4S)-4-Amino-1-tert-butoxycarbonyl-2-pyr-
rolidinylcarbonyl)-1,3-thiazolidine (10). (1) To a solution
of 3-((2S,4R)-1-tert-butoxycarbonyl-4-hydroxy-2-pyrro-
lidinylcarbonyl)-1,3-thiazolidine (9, 56.3 g, 186 mmol)
and triethylamine (28.5 mL) in dichloromethane (1 L)
was added methanesulfonyl chloride (15.1 mL, 195
mmol) under ice cooling. After stirring under ice cooling

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H. Sakashita et al. / Bioorg. Med. Chem. 14 (2006) 3662–3671
for C14H17N5OSÆ2HClÆC4H8O2Æ1/4C4H9ClÆ3/2H2O: C,
44.35; H, 6.32; N, 13.61. Found: C, 44.19; H, 6.36; N,
13.66. HRMS (CI) calcd. for C14H18N5OS (M+H+) m/
e 304.1232, found m/e 304.1231.
5.1.3.5. 3-[(2S,4S)-4-(4-Methanesulfonylphenyl)amino-
2-pyrrolidinylcarbonyl]-1,3-thiazolidine hydrochloride (12e).
(1) From compound 10 (904 mg, 3 mmol) with addition of
diisopropylethylamine (1.57 mL, 9 mmol) and 4-fluorophe-
nyl methyl sulfone (523 mg, 3 mmol), and using a procedure
analogous to general method A (1) (N-methyl-2-pyrroli-
done, 100 ꢁC, 18 h), the compound 3-[(2S,4S)-1-tert-butox-
ycarbonyl-4-(4-methanesulfonylphenyl)amino-2-pyrrolidi-
nylcarbonyl]-1,3-thiazolidine (27 mg, 2.0%) was prepared.
(2) The compound thus prepared (27 mg) was deprotected
using a procedure analogous to general method A (2) to give
the title compound (19.4 mg, 84%). 1H NMR (DMSO-d6) d
1.65–1.82 (1H, m), 2.89–3.23 (7H, m), 3.49–3.98 (3H, m),
4.18–4.78 (4H, m), 6.74 (2H, d, J = 9.0 Hz), 6.80–6.92
(1H, m), 6.63 (2H, d, J = 9.0 Hz), 9.30 (2H, br s). HRMS
(EI) calcd. for C15H21N3O3S2 (M+) m/e 355.1024, found
m/e 355.1025.
5.1.3.2. 3-[(2S,4S)-4-(2-Cyanophenyl)amino-2-pyrro-
lidinylcarbonyl]-1,3-thiazolidine hydrochloride (12b). (1)
From compound 10 (1810 mg, 6 mmol) with addition
of diisopropylethylamine (3.14 mL, 18 mmol) and
2-fluorobenzonitrile (727 mg, 6 mmol), and using a pro-
cedure analogous to general method A (1) (N-methyl-2-
pyrrolidone, 80 ꢁC, 32 h), the compound 3-[(2S,4S)-1-
tert-butoxycarbonyl-4-(2-cyanophenyl)amino-2-pyrro-
lidinylcarbonyl]-1,3-thiazolidine (88 mg, 3.6%) was
prepared. (2) The compound thus prepared (88 mg)
was deprotected using a procedure analogous to gener-
al method A (2) to give the title compound (25 mg,
34%). 1H NMR (DMSO-d6) d 1.74–1.95 (1H, m),
2.85–3.16 (3H, m), 3.30–3.92 (4H, m), 4.27–4.79 (4H,
m), 6.15–6.27 (1H, m), 6.77 (1H, t, J = 7.5Hz), 6.87
(1H, d, J = 8.4 Hz), 7.38–7.59 (2H, m), 8.90 (1H, br
s), 10.80 (1H, br s). HRMS (EI) calcd. for
C15H18N4OS (M+) m/e 302.1201, found m/e 302.1196.
5.1.3.6. 3-[(2S,4S)-4-(2-Chloro-4-cyanophenyl)amino-2-
pyrrolidinylcarbonyl]-1,3-thiazolidine hydrochloride (12k).
(1) From compound 10 (904 mg, 3 mmol) with addition of
diisopropylethylamine (1.57 mL, 9 mmol) and 3-chloro-4-
fluorobenzonitrile (467 mg, 3 mmol), and using a proce-
dure analogous to general method A (1) (N-methyl-2-pyr-
rolidone, 80 ꢁC, 8 h), the compound 3-[(2S,4S)-1-tert-
butoxycarbonyl-4-(2-chloro-4-cyanophenyl)amino-2-pyr-
rolidinylcarbonyl]-1,3-thiazolidine (460 mg, 35%) was
prepared as a white solid. (2) The compound thus prepared
(395 mg) was deprotected using a procedure analogous to
general method A (2) to give the title compound (177 mg,
5.1.3.3. 3-[(2S,4S)-4-(4-Cyanophenyl)amino-2-pyrro-
lidinylcarbonyl]-1,3-thiazolidine hydrochloride (12c). (1)
From compound 10 (1810 mg, 6 mmol) with addition
of diisopropylethylamine (3.14 mL, 18 mmol) and 4-flu-
orobenzonitrile (727 mg, 6 mmol), and using a proce-
dure analogous to general method A (1) (N-methyl-2-
pyrrolidone, 100 ꢁC, 24 h), the compound 3-[(2S,4S)-1-
tert-butoxycarbonyl-4-(4-cyanophenyl)amino-2-pyrro-
lidinylcarbonyl]-1,3-thiazolidine (170 mg, 7.0%) was
prepared. (2) The compound thus prepared (170 mg)
was deprotected using a procedure analogous to gener-
al method A (2) to give the title compound (69.3 mg,
1
53%) as a white powder. H NMR (DMSO-d6) d 1.80–
1.99 (1H, m), 2.82–3.17 (3H, m), 3.25–3.94 (4H, m),
4.36–4.54 (2H, m), 4.54–4.80 (2H, m), 6.42 (1H, d,
J = 7.8 Hz), 6.93 (1H, d, J = 8.7 Hz), 7.64 (1H, dd,
J = 8.4, 1.8 Hz), 7.82 (d, 1H, J = 1.8 Hz). Anal. Calcd for
C15H17ClN4OSÆHClÆ1/5C4H8O2Æ3/5H2O: C, 47.24; H,
5.22; N, 13.95. Found: C, 47.32; H, 5.07; N, 13.89.
1
49%) as a pale-brown powder. H NMR (DMSO-d6)
d 1.64–1.80 (1H, m), 2.84–3.20 (4H, m), 3.45–3.96
(3H, m), 4.15–4.34 (1H, m), 4.39–4.78 (3H, m), 6.70
(2H, d, J = 8.8 Hz), 6.85–7.01 (1H, m), 7.52 (2H, d,
J = 8.7 Hz), 9.45 (2H, br s). Anal. Calcd for
C15H18N4OSÆHClÆ3/10C4H8O2Æ5/4H2O: C, 50.17; H,
6.21; N, 14.45. Found: C, 50.14; H, 6.21; N, 14.14.
5.1.3.7. 3-[(2S,4S)-4-(3-Chloro-4-cyanophenyl)amino-2-
pyrrolidinylcarbonyl]-1,3-thiazolidine hydrochloride (12l).
(1) From compound 10 (0.904 g, 3 mmol) with addition
of diisopropylethylamine (1.57 mL, 9 mmol) and 2-chlo-
ro-4-fluorobenzonitrile (0.467 g, 3 mmol), and using a
procedure analogous to general method A (1) (N-methyl-
2-pyrrolidone, 80 ꢁC, 8 h), the compound 3-[(2S,4S)-1-
tert-butoxycarbonyl-4-(3-chloro-4-cyanophenyl)amino-2-
pyrrolidinylcarbonyl]-1,3-thiazolidine (0.630 g, 48%) was
prepared as a colorless transparent oil. (2) The compound
thus prepared (0.630 g) was deprotected using a procedure
analogous to general method A (2) to give the title com-
pound (0.465 g, 87%) as a white powder. 1H NMR
(DMSO-d6) d 1.65–1.81 (1H, m), 2.84–2.99 (1H, m),
2.99–3.22 (3H, m), 3.48–3.95 (3H, m), 4.16–4.37 (1H, m),
4.39–4.78 (3H, m), 6.68 (1H, dd, J = 8.7, 2.1 Hz), 6.85
(1H, d, J = 1.8 Hz), 7.30–7.45 (1H, m), 7.60 (1H, d, J =
8.7 Hz), 9.60 (2H, br s). Anal. Calcd for C15H17ClN4OSÆ
HClÆ2/5C4H8O2Æ3/5H2O: C, 47.54; H, 5.38; N, 13.36.
Found: C, 47.77; H, 5.27; N, 12.96.
5.1.3.4. 3-[(2S,4S)-4-(4-Nitrophenyl)amino-2-pyrrolid-
inylcarbonyl]-1,3-thiazolidine hydrochloride (12d). (1)
From compound 10 (904 mg, 3 mmol) with addition of
diisopropylethylamine (1.57 mL, 9 mmol) and 4-fluoro-
nitrobenzene (423 mg, 3 mmol), and using a procedure
analogous to general method A (1) (N-methyl-2-pyrrol-
idone, 80 ꢁC, 24 h), the compound 3-[(2S,4S)-1-tert-bu-
toxycarbonyl-4-(4-nitrophenyl)amino-2-pyrrolidinylcar-
bonyl]-1,3-thiazolidine (919 mg, 73%) was prepared as a
yellow amorphous substance. (2) The compound thus
prepared (795 mg) was deprotected using a procedure
analogous to general method A (2) to give the title com-
pound (647 mg, 96%) as a yellow powder. 1H NMR
(DMSO-d6) d 1.68–1.87 (1H, m), 2.88–3.30 (4H, m),
3.48–3.98 (3H, m), 4.24–4.80 (4H, m), 6.72 (2H, d,
J = 9.3 Hz), 7.40–7.56 (1H, m), 8.04 (2H, d, J =
7.5 Hz), 9.51 (2H, br s). Anal. Calcd for C14H18N4O3SÆ
HClÆ2/5C4H8O2Æ3/5H2O: C, 46.28; H, 5.83; N, 13.84.
Found: C, 46.19; H, 5.80; N, 13.56.
5.1.3.8. 3-[(2S,4S)-4-(3,4-Dicyanophenyl)amino-2-pyr-
rolidinylcarbonyl]-1,3-thiazolidine hydrochloride (12m).
(1) From compound 10 (0.904 g, 3 mmol) with addition
of diisopropylethylamine (1.57 mL, 9 mmol) and 4-flu-

H. Sakashita et al. / Bioorg. Med. Chem. 14 (2006) 3662–3671
3669
orophthalonitrile (0.438 g, 3 mmol), and using a proce-
dure analogous to general method A (1) (N-methyl-2-
pyrrolidone, 80 ꢁC, 4 h), the compound 3-[(2S,4S)-1-
tert-butoxycarbonyl-4-(3,4-dicyanophenyl)amino-2-pyr-
rolidinylcarbonyl]-1,3-thiazolidine (1.08 g, 84%) was
prepared as a white amorphous substance. (2) The com-
pound thus prepared (0.924 g) was deprotected using a
procedure analogous to general method A (2) to give
the title compound (0.782 g, 99%) as a yellow powder.
1H NMR (DMSO-d6) d 1.66–1.84 (1H, m), 2.90–3.27
(4H, m), 3.49–3.95 (3H, m), 4.20–4.40 (1H, m), 4.40–
5.79 (3H, m), 7.00 (1H, dd, J = 8.7, 2.4 Hz), 7.22 (1H,
s), 7.52–7.67 (1H, m), 7.76 (1H, d, J = 9.0 Hz). Anal.
Calcd for C16H17N5OSÆHClÆ3/5C4H8O2Æ1/10C4H9ClÆ-
H2O: C, 50.86; H, 5.83; N, 15.77. Found: C, 51.19; H,
5.94; N, 15.57. HRMS (EI) calcd for C16H17N5OS
(M+) m/e 327.1154, found m/e 327.1151.
extracted with ethyl acetate, and the extract washed with
brine and dried. The solvent was evaporated under re-
duced pressure and the residue purified by silica gel
chromatography and crystallized from diethyl ether to
give 3-((2S,4S)-4-anilino-1-tert-butoxycarbonyl-2-pyr-
rolidinylcarbonyl)-1,3-thiazolidine (156 mg, 25%) as
white crystals. (2) The compound thus prepared
(142 mg) was dissolved in ethyl acetate (2 mL), 4 mol/
L hydrochloric acid–ethyl acetate (0.5 mL) added, and
the mixture stirred at room temperature for 12 h. The
precipitated solid was collected by filtration to give the
title compound (89 mg, 68%) as a white powder. 1H
NMR (DMSO-d6) d 1.64–1.78 (1H, m), 2.84–2.97 (1H,
m), 3.00–3.19 (3H, m), 3.43–3.55 (1H, m), 3.60–4.20
(5H, m), 4.41–4.76 (3H, m), 6.56–6.67 (3H, m), 7.13
(2H, t, J = 7.2 Hz), 8.79 (1H, br s), 10.29 (1H, br s).
Anal. Calcd for C14H19N3OSÆ2HCl: C, 48.00; H, 6.04;
N, 12.00. Found: C, 48.35; H, 6.34; N, 11.83.
5.1.4. 3-[(2S,4S)-4-(4-Aminophenyl)amino-2-pyrrolidinyl-
carbonyl]-1,3-thiazolidine hydrochloride (12h). To com-
pound 12d (200 mg, 557 mmol) dissolved in ethanol
(10 mL) were added 4 mol/L hydrochloric acid-1,4-diox-
ane (0.28 mL) and 10% palladium/carbon (100 mg) and
the mixture stirred under a hydrogen atmosphere
(1 atm) at room temperature for 18 h. The reaction mix-
ture was filtered, the filtrate concentrated under reduced
pressure, and the obtained solid washed with ethanol to
give the title compound (13 mg, 6.9%) as a white pow-
5.1.7. Representative example of preparation of 12f, g, i, j:
general method B
5.1.7.1. 3-[(2S,4S)-4-(4-Chlorophenyl)amino-2-pyrro-
lidinylcarbonyl]-1,3-thiazolidine dihydrochloride (12f).
(1) To a solution of compound 13 (450 mg, 1.50 mmol),
p-chloroaniline (230 mg, 1.80 mmol) and acetic acid
(0.09 mL, 1.57 mmol) in 1,2-dichloroethane (8 mL) was
added
sodium
triacetoxyborohydride
(636 mg,
3.00 mmol) and the mixture stirred at room temperature
for 3 h before addition of saturated aqueous sodium
hydrogen carbonate solution to the reaction mixture,
which was then extracted with ethyl acetate. The extract
was washed with brine and dried. The solvent was evap-
orated under reduced pressure and the residue purified
by silica gel chromatography to give the compound 3-
[(2S,4S)-1-tert-butoxycarbonyl-4-(4-chlorophenyl)ami-
1
der. H NMR (DMSO-d6) d 1.58–1.80 (1H, m), 2.83–
3.00 (1H, m), 3.00–3.20 (3H, m), 3.60–3.90 (3H, m),
4.08–4.25 (1H, m), 4.39–4.79 (3H, m), 6.67 (2H, d,
J = 8.7 Hz), 7.15 (2H, d, J = 8.7 Hz), 8.81 (1H, br s),
10.00 (3H, br s), 10.25 (1H, br s). HRMS (EI) calcd.
for C14H20N4OS (M+) m/e 292.1358, found m/e
292.1353.
no-2-pyrrolidinylcarbonyl]-1,3-thiazolidine
(415 mg,
5.1.5. 3-((S)-1-tert-Butoxycarbonyl-4-oxo-2-pyrrolidinyl-
carbonyl)-1,3-thiazolidine (13). To a solution of com-
pound 9 (55.4 g, 183 mmol) and triethylamine (46 mL)
in dichloromethane (350 mL) was added sulfur trioxide
pyridine complex (52.4 g, 329 mmol) in DMSO
(150 mL) under ice cooling and the mixture stirred for
2 h. Saturated aqueous sodium hydrogen carbonate
solution was added to the reaction mixture and the mix-
ture extracted with ethyl acetate. The extract was
washed with brine and dried. The solvent was evaporat-
ed under reduced pressure and the residue purified by
silica gel chromatography to give the title compound
(30.3 g, 55%) as a white solid. 1H NMR (CDCl3) d
1.47 (9H, s), 2.45–2.57 (1H, m), 2.70–2.93 (1H, m),
2.97–3.22 (2H, m), 3.66–3.78 (0.6H, m), 3.80–4.10 (3H,
m), 4.28–4.38 (0.4H, m), 4.45–5.08 (3H, m).
67%) as an oil. (2) The compound thus produced
(412 mg) was dissolved in ethyl acetate (5 mL), 4 mol/
L hydrochloric acid–ethyl acetate (1.25 mL) added,
and the mixture stirred at room temperature for 13 h.
The precipitated solid was collected by filtration to give
the title compound (297 mg, 80%) as a white powder. 1H
NMR (DMSO-d6) d 1.63–1.78 (1H, m), 2.84–2.97 (1H,
m), 3.00–3.17 (3H, m), 3.5–3.92 (3H, m), 4.07–4.18
(1H, m), 4.40–4.73 (3H, m), 6.62 (2H, d, J = 8.8 Hz),
7.15 (2H, d, J = 8.8 Hz), 8.86 (1H, br s), 10.23 (1H, br
s). Anal. Calcd for C14H18N3OSÆ8/5HCl: C, 45.43; H,
5.34; N, 11.35. Found: C, 45.24; H, 5.58; N, 11.10.
5.1.7.2. 3-[(2S,4S)-4-(p-Anisidino)-2-pyrrolidinylcar-
bonyl]-1,3-thiazolidine dihydrochloride (12g). (1) From
compound 13 (450 mg, 1.50 mmol) with addition of p-
anisidine (222 mg, 1.80 mmol), and using a procedure
analogous to general method B (1), the compound 3-
[(2S,4S)-4-(p-anisidino)-1-tert-butoxycarbonyl-2-pyrro-
lidinylcarbonyl]-1,3-thiazolidine (515 mg, 84%) was
prepared as a white solid. (2) The compound thus pre-
pared (448 mg) was deprotected using a procedure anal-
ogous to general method B (2) to give the title
compound (223 mg, 55%) as a white powder. 1H
NMR (DMSO-d6) d 1.77–1.90 (1H, m), 2.77–2.89 (1H,
m), 3.00–3.14 (3H, m), 3.20–4.20 (1H, m), 3.60–4.20
(6H, m), 4.40–4.72 (3H, m), 6.87 (4H, s), 8.84 (1H, br s),
5.1.6. 3-((2S,4S)-4-Anilino-2-pyrrolidinylcarbonyl)-1,3-
thiazolidine dihydrochloride (12a). Compound 13
(501 mg, 1.67 mmol), aniline (0.20 mL, 2.19 mmol),
and acetic acid (0.10 mL, 1.75 mmol) were dissolved in
methanol (10 mL) and stirred at room temperature for
1.5 h before addition of sodium cyanoborohydride
(95%, 145 mg, 2.19 mmol) to the reaction mixture, stir-
ring for a further 2 h, and evaporation under reduced
pressure. Saturated aqueous sodium hydrogen carbon-
ate solution was added to the residue, the mixture

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H. Sakashita et al. / Bioorg. Med. Chem. 14 (2006) 3662–3671
10.33 (1H, br s). Anal. Calcd for C15H21N3O2SÆ2HClÆ-
H2O: C, 46.27; H, 6.21; N, 10.79. Found: C, 45.93; H,
6.43; N, 10.49.
5.2. Biological methods
5.2.1. DPP-IV-inhibitory activity. The DPP-IV-inhibito-
ry activity of human plasma and rat plasma was mea-
sured by fluorescence assay using Gly-Pro-MCA
(Peptide Institute Inc.) as a DPP-IV-specific fluorescent
substrate. Reaction solutions containing 20 lL of human
or rat plasma (10-fold diluted solution), 20 lL of fluores-
cent substrate (100 lmol/L), 140 lL of buffer (0.003%
Brij-35 containing PBS), and 20 lL of test substrate (of
various concentrations) were incubated at room temper-
ature 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-IV activity. The inhibitory rate
relative to the solvent addition group was calculated
and IC50 values determined by logistic analysis.
5.1.7.3. 3-[(2S,4S)-4-(3,4-Methylenedioxyphenyl)ami-
no-2-pyrrolidinylcarbonyl]-1,3-thiazolidine dihydrochlo-
ride (12i). (1) From compound 13 (450 mg, 1.50 mmol)
with addition of 3,4-methylenedioxyaniline (249 mg,
1.80 mmol), and using a procedure analogous to general
method B (1), the compound 3-[(2S,4S)-1-tert-butoxy-
carbonyl-4-(3,4-methylenedioxyphenyl)amino-2-pyrro-
lidinylcarbonyl]-1,3-thiazolidine (553 mg, 87%) was
prepared as a pale-brown-reddish solid. (2) The com-
pound thus prepared (549 mg) was deprotected using a
procedure analogous to general method B (2) to give
the title compound (457 mg, 89%) as a pale-brown-red-
dish powder. 1H NMR (DMSO-d6) d 1.72–1.85 (1H, m),
2.820–2.93 (1H, m), 3.00–3.28 (3H, m), 3.45–3.57 (1H,
m), 3.60–3.95 (2H, m), 4.08–4.20 (1H, m), 4.42–4.75
(3H, m), 5.92 (2H, s), 6.25–6.32 (1H, m), 6.53 (1H, s),
6.76–6.83 (1H, m), 8.89 (1H, br s) 10.36 (1H, br s). Anal.
Calcd for C15H19N3O3SÆ2HClÆ1/5H2O: C, 45.28; H,
5.42; N, 10.56. Found: C, 45.18; H, 5.34; N, 10.28.
5.2.2. Plasma DPP-IV activity after oral administration
of 11 to wistar rats. Male Wistar rats (7–9 weeks of age)
fasted overnight were used. Compound 11 was dissolved
in 0.5% hydroxypropylmethyl-cellulose and adminis-
tered orally at a dose of 10, 30, and 100 lmol/kg. At
pre-administration and at 0.5, 1, 2, 3, 5, 7, 9, and 24 h
after administration, 0.1 mL of blood was collected
from the jugular vein. After centrifugation, 10 lL of
plasma was diluted 10-fold using buffer (0.003% Brij-
35 containing PBS). Twenty microliters of the diluted
plasma was used instead of 20 lL of test substrate for
the determination of DPP-IV-inhibitory activity by fluo-
rescence as described above.
5.1.7.4. 3-[(2S,4S)-4-(3-Chloro-4-methoxyphenyl)ami-
no-2-pyrrolidinylcarbonyl]-1,3-thiazolidine dihydrochlo-
ride (12j). (1) From compound 13 (450 mg, 1.50 mmol)
with addition of 3-chloro-4-methoxyaniline (284 mg,
1.80 mmol), and using a procedure analogous to general
method B (1), the compound 3-[(2S,4S)-1-tert-butoxy-
carbonyl-4-(3-chloro-4-methoxyphenyl)amino-2-pyrro-
lidinylcarbonyl]-1,3-thiazolidine (569 mg, 86%) was
prepared as an oil. (2) The compound thus produced
(561 mg) was deprotected using a procedure analogous
to general method B (2) to give the title compound
(429 mg, 81%) as a pale-brown powder. 1H NMR
(DMSO-d6) d 1.62–1.76 (1H, m), 2.82–2.95 (1H, m),
3.00–3.18 (3H, m), 3.5–3.92 (6H, m), 4.07–4.18 (1H,
m), 4.40–4.73 (3H, m), 6.61 (1H, dd, J = 8.8, 2.7 Hz),
6.75 (1H, d, J = 2.7 Hz), 6.98 (1H, d, J = 8.8 Hz), 8.80
(1H, br s), 10.15 (1H, br s). Anal. Calcd for
C15H20ClN3O2SÆHCl: C, 43.44; H, 5.35; N, 10.13.
Found: C, 43.07; H, 5.41; N, 9.91.
5.2.3. Pharmacokinetic profile of 11 after intravenous or
oral administration to rats. Female Sprague–Dawley rats
(7 weeks of age) were used. A solution of compound 11
in 0.9% sodium chloride (for iv) or 0.5% hydroxypro-
pylmethylcellulose (for po) was administered at a dose
of 10 mg/kg. Rats were fasted overnight before dosing
and for 6 h after dosing. At 0.05 (for iv), 0.25 (for po),
0.5, 1, 2, 4, 6, 8, and 24 h after administration, blood
was collected from the jugular vein. The blood sample
was centrifuged at 10,000 rpm for 5 min and the separat-
ed plasma stored at À20 ꢁC until analysis. For determi-
nation of unchanged compound 11, 0.05 mL of water
and 0.1 mL of 0.1 mol/L sodium hydroxide were added
to 0.1 mL of plasma sample. The mixture was applied
to an OASYS HLB cartridge (30MG/1CC) [which was
preconditioned with 1 mL of methanol followed by
1 mL of water]. After rinsing the cartridge with 1 mL
of water, the targeted compound 11 was eluted from
the cartridge using 1 mL of methanol. The eluate was
evaporated by centrifugal concentration at 40 ꢁC, the
residue reconstituted in 0.2 mL of mobile phase, and
5 lL injected into an LC–MS/MS system. Chromato-
graphic separation was performed using a Develosil
ODS-HG-3 (50 mm · 2 mm id, 3 lm, Nomura Chemical
Co., Japan), with a mixture of 0.05% trifluoroacetic acid
and acetonitrile (85/15) as the mobile phase. The flow
rate was 0.25 mL/min. Protonated analyte ions were
generated using electron spray ionization. For quantita-
tion, the ion signal was recorded by selective reaction
monitoring. Pharmacokinetic parameters were calculat-
ed by a non-compartmental analysis.
5.1.8. Solution stability of 3 and 11 in a neutral buffer at
37 ꢁC. Compounds 3 and 11 were dissolved in a cool
buffer of pH 6.8 (second fluid of Disintegration Test in
Japanese Pharmacopoeia) to produce test solutions of
0.05 mg/mL. A 2 mL sample of each test solution was
incubated at 37 ꢁC with shaking at 100 times/min for
1, 2, and 4 h (for compound 3), or for 2, 4, and 6 h
(for compound 11). After incubation, 0.1 mL of 1 mol/
L hydrochloric acid and 8 mL of cool mobile phase were
added to each test solution and 20 lL was injected into a
HPLC instrument. The drug concentrations were mea-
sured by HPLC analysis. Chromatographic separation
was performed using
a
Develosil ODS-HG-5
(150 mm · 4.6 mm id, 5 lm, Nomura Chemical Co.,
Japan), with a mixture of 0.05 mol/L sodium perchlorate
(pH 2.5, adjusted with 70% perchloric acid) and acetoni-
trile (85/15 for 3, 80/20 for 11) as the mobile phase. The
flow rate was 1 mL/min. The UV detector was set at
270 nm (for 3) or 200 nm (for 11).

H. Sakashita et al. / Bioorg. Med. Chem. 14 (2006) 3662–3671
3671
Weldon, S. C.; Hughes, T. E. J. Med. Chem. 2002, 45,
2362.
Acknowledgments
8. 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.
We wish to thank Dr. Tohru Nakajima and Dr. Takao
Kondo for their insight and guidance during the course
of this work.
´
9. McIntyre, J. A.; Castaner, J. Drugs Future 2004, 29, 887.
10. Sakashita, H.; Kitajima, H.; Nakamura, M.; Akahoshi,
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