Lead optimization of [(S)-γ-(arylamino)prolyl]thiazolidine focused on γ-substituent: Indoline compounds as potent DPP-IV inhibitors

Article abstract of DOI:10.1016/j.bmc.2006.10.059

Dipeptidyl peptidase IV (DPP-IV) inhibitors are looked to as a potential new antidiabetic agent class. A series of [(S)-γ-(arylamino)prolyl]thiazolidine compounds in which the electrophilic nitrile is removed are chemically stable DPP-IV inhibitors. To discover a structure for the γ-substituent of the proline moiety more suitable for interacting with the S₂ pocket of DPP-IV, optimization focused on the γ-substituent was carried out. The indoline compound 22e showed a DPP-IV-inhibitory activity 100-fold more potent than that of the prolylthiazolidine 10 and comparable to that of NVP-DPP728. It also displayed improved inhibitory selectivity for DPP-IV over DPP8 and DPP9 compared to compound 10. Indoline compounds such as 22e have a rigid conformation with double restriction of the aromatic moiety by proline and indoline structures to promote interaction with the binding site in the S₂ pocket of DPP-IV. The double restriction effect provides a potent inhibitory activity which compensates for the decrease in activity caused by removing the electrophilic nitrile.

Full text of DOI:10.1016/j.bmc.2006.10.059

Bioorganic & Medicinal Chemistry 15 (2007) 641–655
Lead optimization of [(S)-c-(arylamino)prolyl]thiazolidine focused
on c-substituent: Indoline compounds as potent DPP-IV inhibitors
Hiroshi Sakashita,a Fumihiko Akahoshi,a,* Tomohiro Yoshida,a Hiroshi Kitajima,a
Yoshiharu Hayashi,b Shinichi Ishii,c Yoko Takashina,c Reiko Tsutsumiuchid
and Satoshi Onoa
aChemistry Laboratory, Pharmaceuticals Research Division, Mitsubishi Pharma Corporation, 1000,
Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan
bGlobal Projects Coordination Department, Pharmaceuticals Development Division, Mitsubishi Pharma Corporation,
2-2-6, Nihonbashi-Honcho, Chuo-ku, Tokyo 103-8405, Japan
cResearch Laboratory II, Pharmaceuticals Research Division, Mitsubishi Pharma Corporation, 1000, Kamoshida-cho,
Aoba-ku, Yokohama 227-0033, Japan
dGMP Compliance Department, Pharmaceutical Technology Center, Technology & Production Division,
Mitsubishi Pharma Corporation, 14, Sunayama, Kamisu, Ibaraki 314-0255, Japan
Received 10 October 2006; revised 27 October 2006; accepted 28 October 2006
Available online 1 November 2006
Abstract—Dipeptidyl peptidase IV (DPP-IV) inhibitors are looked to as a potential new antidiabetic agent class. A series of [(S)-c-
(arylamino)prolyl]thiazolidine compounds in which the electrophilic nitrile is removed are chemically stable DPP-IV inhibitors. To
discover a structure for the c-substituent of the proline moiety more suitable for interacting with the S2 pocket of DPP-IV, optimi-
zation focused on the c-substituent was carried out. The indoline compound 22e showed a DPP-IV-inhibitory activity 100-fold more
potent than that of the prolylthiazolidine 10 and comparable to that of NVP-DPP728. It also displayed improved inhibitory selec-
tivity for DPP-IV over DPP8 and DPP9 compared to compound 10. Indoline compounds such as 22e have a rigid conformation
with double restriction of the aromatic moiety by proline and indoline structures to promote interaction with the binding site in
the S2 pocket of DPP-IV. The double restriction effect provides a potent inhibitory activity which compensates for the decrease
in activity caused by removing the electrophilic nitrile.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
only,4 and DPP-IV inhibitors have therefore come to
be seen as a potential new type of antidiabetic agent
free of side effects such as hypoglycemia and exhaustion
of pancreatic beta-cells. In particular a potent and
long-acting inhibitor might offer advantages in exploit-
ing DPP-IV inhibition. Recent in vivo studies indicate
that inhibitory selectivity for DPP-IV over other related
prolyl dipeptidases, such as DPP8 and DPP9, is one of
the key issues for clinical use since inhibition of DPP8
and/or DPP9 has produced profound toxicity in animal
studies.5
Dipeptidyl peptidase IV (EC 3.4.14.5, DPP-IV) is a ser-
ine protease which 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 sev-
eral physiological roles; in particular its role as a pepti-
dase that rapidly inactivates glucagon-like peptide 1
(GLP-1) has drawn interest.2 GLP-1 is secreted in re-
sponse to meal ingestion and stimulates insulin secre-
tion.3 It has been suggested that potentiation and
extension of the action of GLP-1 by DPP-IV inhibition
would stimulate insulin secretion after meal ingestion
Several DPP-IV inhibitors have been reported (Fig. 1),6
a number of which are substrate analogs of the P2-P1
fragment. As proline mimics at the P1 part, (S)-2-cyano-
pyrrolidine and thiazolidine structures are used. (S)-2-
Cyanopyrrolidine inhibitors, for example NVP-DPP728
(1),7–9 LAF237 (2),9,10 and BMS-477118 (3),11 which
Keywords: DPP-IV inhibitor; Thiazolidine; Indoline.
*
Corresponding author. Tel.: +81 45 963 3436; fax: +81 45 963
4211; e-mail: Akahoshi.Fumihiko@mk.m-pharma.co.jp
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.10.059

642
H. Sakashita et al. / Bioorg. Med. Chem. 15 (2007) 641–655
S2 pocket
S1 pocket
P2
+
H3N
N
O
Ser630
Xaa
O
N
-
COO
O
P1
P1'
P2
OH
H
N
N
N
N
H
CN
N
H
O
CN
NC
O
LAF237 (2)
NVP-DPP728 (1)
HO
S
N
H2N
N
H2N
O
CN
O
BMS-477118 (3)
P32/98 (4)
Figure 1.
contain a nitrile group as an electrophilic trap for the
Ser630 of the catalytic triad, have been reported as potent
inhibitors. Thiazolidine inhibitors, which lack an electro-
philic nitrile group, are generally of only modest potency.
In contrast, P32/98 (4) improved glucose tolerance in pa-
tients with diabetes and in healthy volunteers despite
exhibiting intrinsic moderate inhibitory properties.12,13
compound 5 is an analog conformationally restricted
using a proline structure and which mimics the folded
conformation of the conformationally flexible com-
pound NVP-DPP728 to allow interaction of the (5-cya-
no-2-pyridyl)amino moiety with the S2 pocket as shown
in Figure 2a. As a result, compound 5 has 5-fold more
potent inhibitory activity than NVP-DPP728. In addi-
tion, compound 5 with an arylamino group introduced
at the c-position has over 10-fold more potent inhibitory
activity than the prolyl-(S)-2-cyanopyrrolidine 616 and
the (R)-isomer 7.14
We previously reported that a series of [(S)-c-(arylami-
no)prolyl]-(S)-2-cyanopyrrolidine compounds had a po-
tent inhibitory activity (Fig. 2b).14 The representative
S2 pocket
CN
S2 pocket
S2 pocket
DPP-IV
DPP-IV
DPP-IV
N
X
S1 pocket
S1 pocket
Y
X
γ
S1 pocket
γ
HN
S
N
N
N
N
O
H
N
Ser630
H
N
O
H
O
O
N
(a)
(b)
(c)
(folded conformation)
[(S)-γ-(arylamino)prolyl]-(S)-2-cyanopyrrolidine
(X = ArNH-)
[(S)-γ-(arylamino)prolyl]thiazolidine
(X = ArNH-)
1 NVP-DPP728 (V = CH, W = CN)
IC50 = 1.4 nmol/L
5 (X = 5-cyano-2-pyridylamino, Y = H)
IC50 = 0.25 nmol/L
8 (X = 5-cyano-2-pyridylamino)
IC50 = 25.4 nmol/L
9 (V = S, W = H)
IC50 = 270 nmol/L
6 (X =Y =H)
IC50 = 2.9 nmol/L
10 (X = H)
IC50 = 538 nmol/L
7 (X= H, Y = 5-cyano-2-pyridylamino)
IC50 = 6.6 nmol/L
Figure 2. [(S)-c-(Arylamino)prolyl]-(S)-2-cyanopyrrolidine and [(S)-c-(arylamino)prolyl]thiazolidine. The human DPP-IV-inhibitory activity was
measured by fluorescence assay using Gly-Pro-MCA as described in Section 5.

H. Sakashita et al. / Bioorg. Med. Chem. 15 (2007) 641–655
643
However, the series of [(S)-c-(arylamino)prolyl]-(S)-2-
cyanopyrrolidine compounds seems to be unstable in
neutral aqueous solution (e.g., for compound 5,
T1/2decomp = 1.3 h at pH 6.8 aqueous solution15). The
instability may result from the intra-molecular cycliza-
tion of the nucleophilic amine in the proline moiety
and the electrophilic nitrile in the (S)-2-cyanopyrrolidine
moiety. We previously reported that compound 8, in
which the (S)-2-cyanopyrrolidine moiety is converted
to a thiazolidine structure, has improved chemical
stability and a long half-life in plasma (t1/2 = 5.27 h);
but compound 8 showed 100-fold decreased inhibitory
activity compared to the (S)-2-cyanopyrrolidine com-
pound 5 (Fig. 2b and c). This effect demonstrates that
the nitrile group of (S)-2-cyanopyrrolidine compounds
plays a dominant part in the exhibition of inhibitory
activity by realizing interaction with the Ser630 of the
catalytic triad. On the other hand, the thiazolidine ana-
logs have displayed similar features to the (S)-2-cyano-
pyrrolidine compounds; the restricted compound 8 is
10-fold more potent than the flexible compound 917
and 20-fold more potent than the prolylthiazolidine
10. These results indicate that introducing a substituent
at the c-position of the proline moiety of the prolylthiaz-
olidine core structure provides potent DPP-IV inhibi-
tion with improved chemical stability and a good
pharmacokinetic profile.15
c-position restricted using an indoline structure, is 100-
fold more potent than the prolylthiazolidine 10
(IC50 = 538 nmol/L) and has comparable activity to
NVP-DPP728 (IC50 = 1.4 nmol/L) despite its lack of
an electrophilic nitrile. Its inhibitory selectivity for
DPP-IV over DPP8 and DPP9 is ca. 100-fold. The rigid
conformation realized by the double restriction with
proline and indoline structures is concluded to be effec-
tive for improving DPP-IV-inhibitory activity.
2. Chemistry
Compounds bearing a non-cyclized nitrogen at the c-po-
sition of the proline moiety of the ((S)-c-substituted pro-
lyl)thiazolidine structure (15a–h, 17a–i) were prepared
from ((S)-c-aminoprolyl)thiazolidine 1315 (Scheme 1).
Introduction of an alkyl, acyl, sulfonyl or carbamoyl
group at the c-amino group of 13, followed by removal
of the Boc group with HCl/AcOEt, afforded the N-
mono-introduced compounds 15a–h. The further modi-
fied compounds 17a–i were prepared by alkylation or
acylation of the c-amino-compound 13 (for 17d) or the
c-[(4-cyanobenzyl)amino]-compound 14b (for 17a–c,
17e–i) and subsequent removal of the Boc group.
The ester compounds 15i and j were synthesized from
the ((S)-c-hydroxyprolyl)thiazolidine 20, which was pre-
pared from the cis-Boc-hydroxyproline 18 by protection
of the hydroxyl group as its silylether and amidation
with thiazolidine followed by removal of the silyl group.
The alcohol 20 was converted to its benzoate followed
by deprotection to give the desired esters 15i and j
(Scheme 2).
In the present study, to compensate for the decrease in
activity caused by the conversion of the (S)-2-cyano-
pyrrolidine moiety to a thiazolidine structure, we opti-
mized the arylamino structure at the c-position of the
proline moiety. The resulting compound 22e
(IC50 = 4.8 nmol/L), with the aromatic moiety at the
H
H
R1
N
R1
N
H2N
S
15a, b (R1 = ArCH -)
S
2
S
i
ii
15c, d (R1 = ArCO-)
N
N
15e, f (R1 = ArSO -)
N
H
N
N
2
N
15g, h (R1 = ArNHCO-)
Boc
O
O
14a-h
Boc
O
13
iv
iii
R1 = 4-CN-PhCH -
2
NC
NC
R2
R2
N
ii
N
S
N
S
N
N
N
H
O
Boc
O
17a-d (R2 = Me,i-Pr, Bu, 4-CN-PhCH-)
2
16a-i
17e-h (R2 = EtOCOCH -, H2NCOCH2-, HOCOCH -, HOCH CH2-)
2
2
2
17i (R2 = 4-CN-PhCO-)
Scheme 1. Reagents: (i) ArCHO, NaBH3CN, AcOH (for 14a, b), ArCOCl, Et3N (for 14c, d), ArSO2Cl, N-methylmorpholine (for 14e, f), or ArNCO
(for 14g, h); (ii) H+; (iii) HCHO, acetone or butyraldehyde, NaBH3CN or NaBH(OAc)3, AcOH (for 16a–c); R2–Br, i-Pr2NEt (for 16e–h); or 4-CN–
PhCOCl, Et3N (for 16i); (iv) 4-CN–PhCH2Br, i-Pr2NEt.

644
H. Sakashita et al. / Bioorg. Med. Chem. 15 (2007) 641–655
R3
TBDMSO
HO
HO
S
S
iv, v
i, ii
iii
O
N
N
N
N
O
N
S
COOH
Boc
Boc
Boc
O
O
N
N
H
O
18
19
20
15i (R3 = H)
15j (R3 = CN)
Scheme 2. Reagents: (i) TBDMSCl, imidazole; (ii) thiazolidine, WSCIÆHCl, HOBtÆH2O, Et3N; (iii) TBAF; (iv) 4-R3–PhCOCl, Et3N; (v) H+.
H2N
S
N
i, iii
R4
a
N
Boc
O
13
b
N
S
O
ii, iii
S
N
N
H
N
O
N
Boc
22a-h (a, b = bond, CH2, or (CH2)2
O
R4 = H, NO2, MeO, F, Cl, Br)
21
Scheme 3. Reagents: (i) a,a0-dibromo-ortho-xylene; (ii) isoindoline, tetrahydroquinoline or indoline derivatives, NaBH(OAc)3, AcOH; (iii) H+.
Compounds having an isoindoline, tetrahydroquinoline
or indoline structure at the c-position (22a–h) were pre-
pared by alkylation of the amine 13 with a,a0-dibromo-
ortho-xylene (for 22a) or reductive amination of the ke-
tone 2115 with cyclic amines (for 22b–h) and subsequent
removal of the Boc group (Scheme 3).
reductive alkylation as shown in Scheme 1 and bears a
nucleophilic nitrogen at the c-position of the proline
moiety that allows the introduction of further substitu-
tions. We therefore selected this compound as the tem-
plate for further structure–activity relationship (SAR)
study of the c-substituent of the proline moiety.
We introduced alkyl, aralkyl, or other functionalized
groups onto the nitrogen at the c-position of compound
15b (Table 2). All of the resulting compounds showed
moderately increased activity compared to compound
15b except the N-acyl compound 17i. Introduction of
an alkyl (17a–c) or benzyl (17d) group both resulted in
3- to 5-fold more potent inhibitory activity. Non-polar
groups tended to produce more potency than polar
groups (17a–d vs 17e–h). However, no pronounced
SAR was evident. This finding suggests that the intro-
duced groups do not interact with the S2 binding site
itself, but allow the aromatic moiety of the flexible
4-cyanobenzyl group to locate the appropriate site for
interaction through their steric hindrance effect.
3. Results and discussion
The ((S)-c-substituted prolyl)thiazolidine and related
compounds were evaluated for DPP-IV-inhibitory activ-
ity in human and rat plasma by fluorescence assay using
Gly-Pro-MCA (Tables 1–4).
In a previous study of ((S)-c-substituted prolyl)-(S)-2-
cyanopyrrolidine, we noted the importance of the aro-
matic moiety at the c-position of the proline moiety
for DPP-IV inhibition.14 First, we examined various
spacers between this aromatic moiety and the proline
moiety. In [(S)-c-(arylamino)prolyl]thiazolidine, since
the 4-cyanophenyl compound 12 showed more potent
inhibitory activity than the phenyl compound 11,15 we
selected both phenyl and 4-cyanophenyl groups for the
structure of the aromatic moiety (Table 1). Of the result-
ing compounds, the 4-cyanobenzylamino 15b and the 4-
cyanophenylcarboxamido 15d had comparable activity
to the 4-cyanophenylamino compound 12. The 4-cya-
nobenzylamino compound 15b is prepared easily by
We were therefore interested in the effect of conforma-
tional restriction of the aromatic moiety of the c-substi-
tuent of the proline moiety (Tables 3, 4). The aromatic
moiety of compounds 11 and 15a was restricted by a
carbon chain, formed isoindoline, tetrahydroquinoline
or indoline structure (22a–c, Table 3). All the restricted
analogs 22a–c showed more potent activity than the

H. Sakashita et al. / Bioorg. Med. Chem. 15 (2007) 641–655
645
Table 1. DPP-IV inhibition by ((S)-c-substituted prolyl)thiazolidines
Table 2. DPP-IV inhibition by ((S)-c-substituted prolyl)thiazolidines
X
NC
S
N
N
R2
N
H
0
S
N
N
Compound
X
DPP-IV inhibition,
IC50 (nmol/L)
H
O
Human
Rat
Compound
R2
DPP-IV inhibition,
IC50 (nmol/L)
11
12
147
190
NH
Human
Rat
34.3
15b
17a
17b
17c
H
34.0
9.6
7.1
9.1
NC
Me
i-Pr
n-Bu
13.6
12.5
5.0
25.2
33.6
NH
17d
6.4
9.3
NH
CN
15a
15b
181
171
17e
17f
17g
17h
CH2COOEt
CH2CONH2
CH2COOH
CH2CH2OH
O
13.6
13.9
16.8
17.0
15.3
16.5
16.3
23.0
NH
34.0
34.3
NC
NC
17i
228
168
CN
O
15c
15d
>300
37.2
236
NH
O
Table 3. DPP-IV inhibition by ((S)-c-substituted prolyl)thiazolidines
34.1
NH
X
S
O O
S
N
N
15e
15f
>300
>300
>300
>300
NH
H
O
O O
S
Compound
X
DPP-IV inhibition,
IC50 (nmol/L)
NH
NC
NC
Human
Rat
O
15g
15h
154
151
11
147
190
N
H
NH
NH
NH
O
15a
181
171
85.9
76.2
N
H
NH
O
22a
26.4
39.0
15i
15j
>300
>300
>300
>300
O
N
O
O
22b
22c
112
99.8
19.5
NC
N
N
11.0
phenylamino (11) or benzyl (15a) derivatives, with the
indoline 22c being the most potent. It appeared thus that
an indoline structure is more suited to DPP-IV inhibi-
tion than a phenylamino or benzylamino structure.
Next, compound 22c was modified by introducing a
number of substituents at the 5-position of the indoline
moiety [22d–h, Table 4(A)] and the results were com-
pared with the corresponding phenylamino compounds
[23-25, Table 4(B)]. Of the indoline compounds (A),
although all the compounds with substituents intro-
duced (22d–h) showed increased activity compared to
compound 22c, no influence of electronic properties
was observed. For example, substitution with an elec-
tron-withdrawing nitro group (22d) and an electron-do-
nating methoxy group (22e) both resulted in more
potent activity. This result is similar to that previously
obtained in phenylamino compounds (23–25). On the
other hand, in comparison of compounds bearing the
same substituent on the aromatic moiety, indoline com-

646
H. Sakashita et al. / Bioorg. Med. Chem. 15 (2007) 641–655
Table 4. DPP-IV inhibition by indolinyl compounds (A) and phe-
nylamino compounds (B)
R4
R4
H
N
N
S
S
N
N
N
N
H
H
O
O
(A)
(B)
Compound
R4
DPP-IV inhibition,
IC50 (nmol/L)
Human
Rat
19.5
190
10.3
22c
11
H
A
11.0
147
7.9
H
B
A
B
A
B
A
A
B
A
22d
23
NO2
NO2
19.2
4.8
21.0
6.1
22e
24
MeO
MeO
F
51.5
5.8
54.9
10.1
13.4
55.1
10.7
22f
22g
25
Cl
Cl
7.0
45.4
6.5
22h
Br
pounds (A) showed 2- to 10-fold more potent inhibitory
activity than the corresponding phenylamino com-
pounds (B). This result indicates that, although the aro-
matic moiety of the indoline compounds (A) interacts
with the same binding site as does that of the phenylami-
no compounds (B), conformational restriction of the
aromatic moiety with an indoline structure produces
more potent activity.
To further understand the mechanism of the inhibition,
a modeling study was carried out to investigate the pos-
sible interaction between the (c-substituted prolyl)thiaz-
olidine compounds and DPP-IV. The predicted binding
modes for the 5-methoxyindolinyl compound 22e and
the 4-methoxyphenylamino compound 24 at the active
site of DPP-IV are shown in Figure 3. The models sug-
gest that the aromatic moiety of both the indolinyl
group of 22e and the phenylamino group of 24 achieves
an aromatic p–p interaction with the side chain of the
Phe357 in the S2 pocket. The aromatic moiety of 22e
seems to be potentially more suited to aromatic p–p
interaction with the side chain of Phe357 due to the
restricting effect of the indoline structure, which may
provide potent inhibitory activity.
Figure 3. (a) Compounds 22e and 24 bound to DPP-IV (PDB 1N1M,
Ref. 18) are docked by ASEDock (Refs. 19 and 20) with MOE (MOE,
Chemical Computing Group). Carbon atoms of the enzyme are in gray
and, carbon atoms of ligand compounds 22e and 24 are orange and
green, respectively. Oxygen and nitrogen atoms of both enzyme and
ligands are in red and blue, respectively. (b) Molecular surface
representation showing the interaction of compounds and DPP-IV
(green, hydrophobic, blue, hydrophilic, and red, exposed).
The inhibitory activity of 22e is comparable to that of
NVP-DPP728 (IC50 = 1.4 nmol/L) despite its lack of
an electrophilic nitrile. Of the P2–P1 fragment substrate
analog inhibitors, compounds bearing (S)-c-substituted
proline at the P2 site seem to represent a suitable struc-
ture to produce interaction of their c-substituents with
the side chain of the amino acid residues in the S2 pocket
of DPP-IV.14,15 Compounds optimized by further
restriction with an indoline structure at the c-position
therefore have potent inhibitory activity compensating
for the decrease caused by removing the electrophilic ni-
trile at the P1 site.
The indoline compounds presented in this paper have a
rigid conformation based on the flexible compounds 9
or NVP-DPP728 but doubly restricted by proline and
indoline structures which allows interaction of their aro-
matic moiety with the binding site in the S2 pocket of
DPP-IV. The double restriction produces potent inhibi-
tory activity which compensates for the decreased activ-
ity caused by removing the electrophilic nitrile. For
example, compound 22e (IC50 = 4.8 nmol/L) has 50-fold
more potent activity than the flexible compound 9
(IC50 = 270 nmol/L) and 100-fold more potent activity
than the prolylthiazolidine 10 (IC50 = 538 nmol/L).

H. Sakashita et al. / Bioorg. Med. Chem. 15 (2007) 641–655
647
Table 5. Selectivity of representative DPP-IV inhibitors
moiety doubly restricted by proline and indoline struc-
tures to allow interaction with the binding site in the
S2 pocket of DPP-IV. The double restriction provides
potent inhibitory activity that compensates for the de-
crease in activity caused by removing the electrophilic
nitrile.
X
S
N
N
H
O
Compound
X
IC50 (nmol/L)
DPP8
DPP-IV
558
DPP9
6129
5. Experimental
5.1. Chemistry
10
H
4687
NC
8
25.4
4756
3735
1H NMR spectra were measured on a Bruker DPX-300
instrument or on a Bruker AMX-500 with tetramethylsi-
lane as the internal standard; chemical shifts are report-
ed in parts per million (ppm, d units). Splitting patterns
are designated as s, singlet; d, doublet; t, triplet; q, quar-
tet; 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 ioniza-
tion (CI) mode. Electron analysis for carbon, hydrogen,
and nitrogen was performed with a Yanagimoto CHN
CORDER MT-6. Chromatography refers to flash chro-
matography conducted on silica gel BW-300 (Fuji Sily-
sia). 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-di-
methylformamide; DMSO, dimethylsulfoxide; EDC,
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydro-
chloride; HOBT, 3-hydroxybenztriazole hydrate.
NH
N
NH
15b
22c
22e
22f
34.0
11.0
4.8
678.9
736.5
378.8
562.8
748.7
501.1
710.7
518.9
NC
N
MeO
F
N
5.8
N
Since inhibition of DPP8 and/or DPP9 has shown pro-
found toxicity in in vivo studies,5 it is important for clin-
ical use to develop a selective DPP-IV inhibitor. The
results for selectivity are shown in Table 5. The prolyl-
thiazolidine 10 showed weak selectivity for DPP-IV over
DPP8 and DPP9 (both ca. 10-fold), while the c-substi-
tuted prolyl thiazolidine compounds 8, 15b, 22c, e, f
exhibited improved selectivity for DPP-IV over DPP8
and DPP9 arising from the increased DPP-IV-inhibitory
activity. For example, the indoline compounds 22e, f
had ca. 100-fold selectivity. This finding of improved
selectivity of c-substituted prolylthiazolidine com-
pounds may indicate that a DPP-IV-specific binding site
exists in the S2 pocket, as suggested in the above model-
ing study, and that it was binding with this site that in-
creased the selectivity of DPP-IV inhibition.
5.1.1. 3-((2S,4S)-4-Benzylamino-2-pyrrolidinylcarbonyl)-
1,3-thiazolidine dihydrochloride (15a). (1) To a solution
of 3-((2S,4S)-4-amino-1-tert-butoxycarbonyl-2-pyrro-
lidinylcarbonyl)-1,3-thiazolidine14 (13, 904 mg, 3 mmol)
in methanol (16 mL) was added benzaldehyde (318 mg,
3 mmol), and the mixture was stirred at room tempera-
ture for 30 min. Sodium cyanoborohydride (189 mg,
3 mmol) and acetic acid (5 drops) were added to the
reaction mixture, and the mixture was stirred for 6 h.
The reaction mixture was evaporated under reduced
pressure. Saturated aqueous sodium hydrogencarbonate
solution was added to the residue and the mixture was
extracted with ethyl acetate. The extract was dried and
the solvent was evaporated under reduced pressure.
The residue was purified by silica gel chromatography
to give 3-[(2S,4S)-4-benzylamino-1-tert-butoxycarbon-
yl-2-pyrrolidinylcarbonyl]-1,3-thiazolidine (14a, 742 mg,
63%) as a colorless transparent oil.
4. Conclusion
A series of [(S)-c-(arylamino)prolyl]thiazolidine com-
pounds with the electrophilic nitrile removed are chem-
ically stable DPP-IV inhibitors.15 In the present study,
we carried out optimization focused on the c-substituent
of these compounds. The resulting compounds bearing
an indoline structure at the c-position showed potent
DPP-IV inhibitory activity. The representative com-
pound 22e (IC50 = 4.8 nmol/L) had 50-fold more potent
activity than the flexible compound 9 (IC50 = 270 nmol/
L) and 100-fold more potent activity than the prolyl-
thiazolidine 10 (IC50 = 538 nmol/L). The inhibitory
selectivity of 22e for DPP-IV over DPP8 and DPP9
was ca. 100-fold. The indoline compounds presented in
this paper have a rigid conformation with an aromatic
(2) Compound 14a (742 mg, 1.90 mmol) was dissolved
in ethyl acetate (3.8 mL) and 4 mol/L hydrochloric
acid–ethyl acetate (2.4 mL) was added to the solution.
The mixture was stirred at room temperature for 18 h
and the precipitated solid was collected to give the title
compound (540 mg, 74%) as a white powder.
1H NMR (300 MHz, DMSO-d6) d 2.10–2.32 (1H, m),
2.86–3.20 (3H, m), 3.49–4.03 (5H, m), 4.21 (2H, s),
4.39–4.80 (3H, m), 7.31–7.52 (3H, m), 7.52–7.72 (2H,
m), 10.17 (4H, br s). Anal. Calcd for C15H21N3OSÆ13/

648
H. Sakashita et al. / Bioorg. Med. Chem. 15 (2007) 641–655
5HCl: C, 46.65; H, 6.16; N, 10.88. Found: C, 46.44; H,
6.07; N, 10.89.
5.1.4. 3-[(2S,4S)-4-(4-Cyanobenzoyl)amino-2-pyrrolidi-
nylcarbonyl]-1,3-thiazolidine hydrochloride (15d). (1)
From compound 13 (543 mg, 1.80 mmol), 4-meth-
ylmorpholine (238 lL, 2.16 mmol), and 4-cyanobenzoyl
chloride (313 mg, 1.89 mmol) using a procedure analo-
gous to synthesis of 14c, the compound 3-[(2S,4S)-1-
tert-butoxycarbonyl-4-(4-cyanobenzoyl)amino-2-pyrro-
lidinylcarbonyl]-1,3-thiazolidine (14d, 804 mg) was pre-
pared as a white powder.
5.1.2. 3-[(2S,4S)-4-(4-Cyanophenylmethyl)amino-2-pyr-
rolidinylcarbonyl]-1,3-thiazolidine dihydrochloride (15b).
(1) From compound 13 (904 mg, 3 mmol) and 4-cyano-
benzaldehyde (393 mg, 3 mmol) with addition of sodium
cyanoborohydride (189 mg, 3 mmol) and acetic acid (5
drops) using a procedure analogous to synthesis of
14a, the compound 3-[(2S,4S)-1-tert-butoxycarbonyl-4-
(4-cyanophenylmethyl)amino-2-pyrrolidinylcarbonyl]-
1,3-thiazolidine (14b, 389 mg, 31%) was prepared as a
colorless transparent oil.
(2) Compound 14d (798 mg) was dissolved in ethyl ace-
tate (0.5 mL) and 4 mol/L hydrochloric acid–ethyl ace-
tate (2.3 mL) was added to the solution. The mixture
was stirred at room temperature for 19 h. The precipitat-
ed solid was collected to give the title compound
(513 mg, 67%) as a white powder.
(2) Compound 14b (389 mg, 0.943 mmol) was dissolved
in ethyl acetate (0.9 mL) and 4 mol/L hydrochloric acid–
ethyl acetate (1.2 mL) was added to the solution. The
mixture was stood at room temperature for 18 h. The
precipitated solid was collected to give the title com-
pound (286 mg, 74%) as a white powder.
1H NMR (500 MHz, DMSO-d6) d 2.01–2.06 (1H, m),
2.81–2.86 (1H, m), 3.03–3.14 (2H, m), 3.36–3.50 (2H,
m), 3.65–3.94 (2H, m), 4.45–4.75 (4H, m), 7.98–8.06
(4H, m), 8.86 (1H, br s), 9.07–9.12 (1H, m), 10.49 (1H,
br s).
1H NMR (300 MHz, DMSO-d6) d 2.06–2.34 (1H, m),
2.85–3.01 (1H, m), 3.01–3.20 (2H, m), 3.50–4.06 (5H,
m), 4.30 (2H, s), 4.41–4.79 (3H, m), 7.80 (2H, d,
J = 8.1 Hz), 7.95 (2H, d, J = 8.4 Hz), 9.05 (1H, br s),
10.30 (3H, br s).
Anal. Calcd for C16H18N4 O2SÆHClÆ3/10C4H8O2: C,
50.23; H, 5.73; N, 13.62. Found: C, 50.21; H, 5.75; N,
13.70.
Anal. Calcd for C16H20N4OSÆ2HCl: C, 49.36; H, 5.70;
N, 14.39. Found: C, 49.13; H, 5.89; N, 14.23.
5.1.5. 3-((2S,4S)-4-Phenylsulfonylamino-2-pyrrolidinyl-
carbonyl)-1,3-thiazolidine hydrochloride (15e). (1) From
compound 13 (543 mg, 1.80 mmol), 4-methylmorpho-
line (240 lL, 2.18 mmol), and benzenesulfonyl chloride
(240 lL, 1.88 mmol) using a procedure analogous to
synthesis of 14c, the compound 3-((2S,4S)-1-tert-butoxy-
carbonyl-4-phenylsulfonylamino-2-pyrrolidinylcarbony-
l)-1,3-thiazolidine (14e, 644 mg, 81%) was prepared as a
white powder.
5.1.3. 3-((2S,4S)-4-Benzoylamino-2-pyrrolidinylcarbon-
yl)-1,3-thiazolidine (15c). (1) To a solution of 3-
((2S,4S)-4-amino-1-tert-butoxycarbonyl-2-pyrrolidinyl-
carbonyl)-1,3-thiazolidine15 (13, 499 mg, 1.65 mmol) in
dichloromethane (10 mL) were added 4-methylmorpho-
line (218 lL, 1.98 mmol) and benzoyl chloride (202 lL,
1.74 mmol) and the mixture stirred at room temperature
for 22 h. To the reaction mixture was added 10% citric
acid solution, and the mixture was extracted with ethyl
acetate. The extract was washed successively with satu-
rated aqueous sodium hydrogencarbonate solution and
brine, and dried. The solvent was evaporated under re-
duced pressure to give 3-((2S,4S)-4-benzoylamino-1-
tert-butoxycarbonyl-2-pyrrolidinylcarbonyl)-1,3-thiazoli-
dine (14c, 652 mg, 97%) as a white powder.
(2) Compound 14e (634 mg, 1.44 mmol) was dissolved
in ethyl acetate (4 mL) and 4 mol/L hydrochloric
acid–ethyl acetate (1.8 mL) was added to the solution.
The mixture was stirred at room temperature for
67 h. The precipitate was collected by filtration to
give the title compound (437 mg, 72%) as a pale-yel-
low powder.
1H NMR (500 MHz, DMSO-d6) d 1.68–1.74 (1H, m),
2.50–2.58 (1H, m), 3.00–3.08 (3H, m), 3.17–3.20 (1H,
m), 3.53–3.87 (3H, m), 4.38–4.63 (3H, m), 7.62–7.71
(3H, m), 7.84–7.85 (2H, m), 8.24–8.27 (1H, m) 9.50
(2H, br s).
(2) Compound 14c (648 mg, 1.60 mmol) was dissolved in
ethyl acetate (4 mL) and 4 mol/L hydrochloric acid–eth-
yl acetate (2 mL) was added to the solution. The mixture
was stirred at room temperature for 19 h. The precipitat-
ed solid was collected and added sodium hydrogencar-
bonate solution, and the mixture was extracted with
chloroform. The extract was washed with brine. The sol-
vent was evaporated under reduced pressure to give the
title compound (250 mg, 51%) as a white powder.
Anal. Calcd for C14H19N3O3S2ÆHClÆ3/10C4H8O2ÆH2O:
C, 43.22; H, 5.82; N, 9.95. Found: C, 43.21; H, 5.64;
N, 10.19.
5.1.6. 3-[(2S,4S)-4-(4-Cyanophenylsulfonyl)amino-2-pyr-
rolidinylcarbonyl]-1,3-thiazolidine hydrochloride (15f). (1)
From compound 13 (1.09 g, 3.63 mmol), 4-meth-
ylmorpholine (480 lL, 4.37 mmol), and 4-cya-
nobenzenesulfonyl chloride (0.780 g, 3.87 mmol) using
1H NMR (500 MHz, DMSO-d6) d 1.76–1.81 (1H, m),
2.33–2.39 (1H, m), 2.91–3.09 (5H, m), 3.63–3.95 (3H,
m), 4.34–4.70 (3H, m), 7.44–7.53 (3H, m), 7.80–7.82
(2H, m), 8.38 (1H, br s).
a
procedure analogous to synthesis of 14c, the
compound 3-[(2S,4S)-1-tert-butoxycarbonyl-4-(4-
cyanophenylsulfonyl)amino-2-pyrrolidinylcarbonyl]-1,
Anal. Calcd for C15H19N3O2SÆ1/3H2O: C, 57.85; H,
6.37; N, 13.49. Found: C, 57.75; H, 6.11; N, 13.44.

H. Sakashita et al. / Bioorg. Med. Chem. 15 (2007) 641–655
649
3-thiazolidine (14f, 1.67 g, 99%) was prepared as a white
powder.
(3 mL), and the mixture was stirred at room temperature
for 8 h. The solvent was evaporated under reduced pres-
sure, and to the residue was added saturated aqueous
sodium hydrogencarbonate solution. The mixture was
extracted with chloroform. The extract was concentrat-
ed under reduced pressure to give the title compound
(140 mg, 18%) as a white powder.
(2) Compound 14f (798 mg, 1.71 mmol) was dissolved in
ethyl acetate (5 mL) and 4 mol/L hydrochloric acid–eth-
yl acetate (2.2 mL) was added to the solution. The mix-
ture was stirred at room temperature for 68 h. The
precipitate was collected by filtration to give the title
compound (544 mg, 70%) as a pale-yellow powder.
1H NMR (300 MHz, DMSO-d6) d 1.60–1.68 (1H, m),
2.22–2.32 (1H, m), 2.67–2.72 (1H, m), 2.91–3.11 (4H,
m), 3.65–3.93 (3H, m), 4.13–4.16 (1H, m), 4.43–4.72
(2H, m), 6.47 (1H, d, J = 7.2 Hz), 7.56 (2H, d,
J = 8.7 Hz), 7.65 (2H, d, J = 8.7 Hz), 9.11 (1H, s).
1H NMR (500 MHz, DMSO-d6) d 1.68–1.75 (1H, m),
2.53–2.59 (1H, m), 3.02–3.09 (3H, m), 3.23–3.28 (1H,
m), 3.54–3.90 (3H, m), 4.40–4.64 (3H, m), 8.01 (2H, d,
J = 8.4 Hz), 8.13 (2H, d, J = 8.4 Hz), 8.62–8.65 (1H,
m), 9.93 (2H, br s).
Anal. Calcd for C16H19N5O2SÆ9/13H2O: C, 53.70; H,
5.74; N, 19.57. Found: C, 54.09; H, 5.63; N, 19.17.
Anal. Calcd for C15H18N4O3S2ÆHClÆ2/5C4H8O2ÆH2O:
C, 43.71; H, 5.35; N, 12.28. Found: C, 43.69; H, 5.26;
N, 12.48.
5.1.9. 3-{(2S,4S)-4-[N-(4-Cyanophenylmethyl)-N-meth-
ylamino]-2-pyrrolidinylcarbonyl}-1,3-thiazolidine
dihy-
drochloride (17a). (1) To a solution of 3-[(2S,4S)-1-
tert-butoxycarbonyl-4-(4-cyanophenylmethyl)amino-2-pyr-
rolidinylcarbonyl]-1,3-thiazolidine (14b, 1.35 g) in acetoni-
trile (20 mL) was added 37% formaldehyde solution
(0.788 mL), and the mixture was stirred at room temper-
ature for 30 min. Sodium cyanoborohydride (0.305 g)
and acetic acid (5 drops) were added to the reaction mix-
ture, and the reaction mixture was stirred for 1 h. The
reaction mixture was evaporated under reduced pres-
sure. Saturated aqueous sodium hydrogencarbonate
solution was added to the residue and the mixture was
extracted with ethyl acetate. The extract was dried and
the solvent was evaporated under reduced pressure.
The residue was purified by silica gel chromatography
to give 3-{(2S,4S)-1-tert-butoxycarbonyl-4-[N-(4-cyan-
ophenylmethyl)-N-methylamino]-2-pyrrolidinylcarbon-
yl}-1,3-thiazolidine (16a, 0.953 g, 68%) as white
amorphous.
5.1.7.
3-[(2S,4S)-4-(3-Phenylureido)-2-pyrrolidinylcar-
bonyl]-1,3-thiazolidine trifluoroacetate (15g). (1) To a
solution of 3-((2S,4S)-4-amino-1-tert-butoxycarbonyl-
2-pyrrolidinylcarbonyl)-1,3-thiazolidine15 (13, 401 mg,
1.33 mmol) in tetrahydrofuran (10 mL) was added phen-
yl isocyanate (167 mg, 1.40 mmol) and the mixture stir-
red at room temperature for 18 h. To the reaction
mixture was added 10% citric acid solution and the mix-
ture was extracted with ethyl acetate. The extract was
washed successively with saturated aqueous sodium
hydrogencarbonate solution and brine. The solvent
was evaporated under reduced pressure to give 3-
[(2S,4S)-1-tert-butoxycarbonyl-4-(3-phenylureido)-2-
pyrrolidinylcarbonyl]-1,3-thiazolidine (14g, 560 mg,
quant.) as a white solid.
(2) To compound 14g (532 mg, 1.27 mmol) was added
trifluoroacetic acid (2 mL) and the mixture was stirred
at room temperature for 3 h. The mixture was evaporat-
ed under reduced pressure to give the title compound
(363 mg, 63%) as a brown powder.
(2) Compound 16a (0.818 g, 1.90 mmol) was dissolved in
ethyl acetate (3.8 mL) and 4 mol/L hydrochloric acid–
ethyl acetate (2.4 mL) was added to the solution. The
mixture was stirred at room temperature for 5 h. The
precipitated solid was collected to give the title com-
pound (0.683 g, 77%) as a white powder.
1H NMR (500 MHz, DMSO-d6) d 1.75–1.80 (1H, m),
2.75–2.80 (1H, m), 3.04–3.20 (3H, m), 3.43–3.47 (1H,
m), 3.68–3.89 (2H, m), 4.40–4.71 (4H, m), 6.72–6.75
(1H,m), 6.91 (1H, t, J = 7.4 Hz), 7.21–7.24 (2H, m),
7.39 (2H, d, J = 7.8 Hz), 8.85 (1H, br s), 8.89–8.90
(1H, m), 9.60 (1H, br s).
1H NMR (300 MHz, DMSO-d6) d 2.20–2.48 (1H, m),
2.57 (3H, s), 2.80–3.20 (3H, m), 3.57–4.17 (5H, m),
4.20–4.85 (5H, m), 7.88 (2H, d, J = 7.8 Hz), 7.96 (2H,
d, J = 8.4 Hz), 9.12 (1H, br s).
Anal. Calcd for C15H20N4O2SÆC2HF3O2ÆH2O: C, 45.13;
H, 5.12; N, 12.38. Found: C, 45.24; H, 5.11; N, 12.18.
Anal. Calcd for C17H22N4OSÆ2HClÆ1/2C4H8O2ÆH2O: C,
49.03; H, 6.50; N, 12.04. Found: C, 49.07; H, 6.62; N,
12.08.
5.1.8. 3-{[2S,4S]-4-[3-(4-Cyanophenyl)ureido]-2-pyrrolid-
inylcarbonyl}-1,3-thiazolidine (15h). (1) From compound
13 (640 mg, 2.12 mmol) and 4-cyanophenyl isocyanate
(321 mg, 2.23 mmol) using a procedure analogous to
synthesis of 14g, the compound 3-{(2S,4S)-1-tert-butoxy-
carbonyl-4-[3-(4-cyanophenyl)ureido]-2-pyrrolidinylcar-
bonyl}-1,3-thiazolidine (14h, 992 mg, quant.) was
prepared as a white powder.
5.1.10.
3-{(2S,4S)-4-[N-(4-Cyanophenylmethyl)-N-iso-
propylamino]-2-pyrrolidinylcarbonyl}-1,3-thiazolidine
dihydrochloride (17b). (1) To a solution of 3-[(2S,4S)-1-
tert-butoxycarbonyl-4-(4-cyanophenylmethyl)amino-2-
pyrrolidinylcarbonyl]-1,3-thiazolidine (14b, 833 mg,
2 mmol) in 1,2-dichloroethane (10 mL) were added ace-
tone (5.73 mL), acetic acid (0.229 mL, 4 mmol), and
sodium triacetoxyborohydride (1.70 g, 8 mmol), and
the mixture was stirred at room temperature for 3 days.
(2) To a solution of compound 14h (978 mg, 2.20 mmol)
in chloroform (5 mL) was added trifluoroacetic acid

650
H. Sakashita et al. / Bioorg. Med. Chem. 15 (2007) 641–655
Saturated aqueous sodium hydrogencarbonate solution
was added to the reaction mixture and the mixture
was extracted with ethyl acetate. The extract was evap-
orated under reduced pressure.The residue was purified
by silica gel chromatography to give 3-{(2S,4S)-1-tert-
butoxycarbonyl-4-[N-(4-cyanophenylmethyl)-N-isopro-
pylamino]-2-pyrrolidinylcarbonyl}-1,3-thiazolidine (16b,
818 mg, 90%).
chromatography to give 3-{(2S,4S)-1-tert-butoxycarbon-
yl-4-[N-(4-cyanophenylmethyl)-N-(ethoxycarbonylmeth-
yl)amino]-2-pyrrolidinylcarbonyl}-1,3-thiazolidine (16e,
1.01 g, quant.) as an oil.
(2) Compound 16e (976 mg, 1.94 mmol) was dissolved in
ethyl acetate (3.88 mL) and 4 mol/L hydrochloric acid–
ethyl acetate (2.43 mL) was added to the solution. The
mixture was stirred at room temperature for 18 h and
the precipitated solid was collected to give the title com-
pound (630 mg, 68%) as a white powder.
(2) Compound 16b (792 mg, 1.73 mmol) was dissolved
in ethyl acetate (3.47 mL) and 4 mol/L hydrochloric
acid–ethyl acetate (2.16 mL) was added to the solution.
The mixture was stirred at room temperature for 18 h
and the precipitated solid was collected to give the title
compound (637 mg, 72%) as a white powder.
1H NMR (300 MHz, DMSO-d6) d 1.18 (3H, t,
J = 7.1Hz), 1.67–1.90 (1H, m), 2.56–2.75 (1H, m),
2.94–3.22 (3H, m), 3.25–4.00 (8H, m), 4.07 (2H, q,
J = 7.1 Hz), 4.34–4.78 (3H, m), 7.54 (2H, d,
J = 8.2 Hz), 7.81 (2H, d, J = 8.2 Hz), 8.86 (1H, br s),
10.40 (1H, br s).
1H NMR (300 MHz, DMSO-d6) d 0.90–1.60 (6H, m),
1.95–2.45 (1H, m), 2.65–3.20 (3H, m), 3.40–4.90 (11H,
m), 7.50–8.30 (4H, m).
Anal. Calcd for C20H26N4O3SÆ2HClÆ1/10H2O: C, 50.33;
H, 5.96; N, 11.74. Found: C, 50.72; H, 6.36; N, 11.75.
Anal. Calcd for C19H26N4OSÆ2HClÆ3/5C4H8O2ÆH2O: C,
50.53; H, 6.90; N, 11.01. Found: C, 50.49; H, 7.05; N,
11.26.
5.1.13. 3-{(2S,4S)-4-[N-(Carbamoylmethyl)-N-(4-cyan-
ophenylmethyl) amino]-2-pyrrolidinylcarbonyl}-1,3-thiaz-
olidine dihydrochloride (17f). (1) From compound 14b
(0.833 g, 2 mmol), 2-bromoacetamide (0.276 mL,
2 mmol), and diisopropylethylamine (1.05 mL, 6 mmol)
using a procedure analogous to synthesis of 16e, the
compound 3-{(2S,4S)-1-tert-butoxycarbonyl-4-[N-(car-
bamoylmethyl)-N-(4-cyanophenylmethyl)amino]-2-pyr-
rolidinylcarbonyl}-1,3-thiazolidine (16f, 0.599 g, 63%)
was prepared.
5.1.11.
3-{(2S,4S)-4-[N-Butyl-N-(4-cyanophenylmeth-
yl)amino]-2-pyrrolidinylcarbonyl}-1,3-thiazolidine dihy-
drochloride (17c). (1) From compound 14b (833 mg,
2 mmol), n-butyraldehyde (216 mg, 3 mmol), acetic acid
(0.229 mL, 4 mmol), and sodium triacetoxyborohydride
(0.848 g, 4 mmol) using a procedure analogous to syn-
thesis of 16b, the compound 3-{(2S,4S)-1-tert-butoxy-
carbonyl-4-[N-butyl-N-(4-cyanophenylmethyl)amino]-2-
pyrrolidinylcarbonyl}-1,3-thiazolidine (16c, 837 mg,
89%) was prepared.
(2) Compound 16f (599 mg, 1.27 mmol) was dissolved in
ethyl acetate (2.53 mL) and 4 mol/L hydrochloric acid–
ethyl acetate (1.27 mL) was added to the solution. The
mixture was stirred at room temperature for 6 h and
the precipitated solid was collected to give the title com-
pound (416 mg, 69%) as a white powder.
(2) Compound 16c (830 mg, 1.76 mmol) was dissolved in
ethyl acetate (3.51 mL) and 4 mol/L hydrochloric acid–
ethyl acetate (2.20 mL) was added to the solution. The
mixture was stirred at room temperature for 18 h and
the precipitated solid was collected to give the title com-
pound (607 mg, 72%) as a pale-yellow powder.
1H NMR (300 MHz, DMSO-d6) d 1.76–1.99 (1H, m),
2.62–2.83 (1H, m), 2.90–4.10 (11H, m), 4.25–4.80 (3H,
m), 7.22 (1H, br s), 7.44 (1H, br s), 7.64 (2H, d,
J = 8.1 Hz), 7.84 (2H, d, J = 8.4 Hz), 8.82 (1H, br s),
10.35 (1H, br s).
1H NMR (300 MHz, DMSO-d6) d 0.80 (3H, t,
J = 7.2 Hz), 1.18 (2H, quint, J = 6.9 Hz), 1.30–1.90
(2H, m), 2.10–2.50 (1H, m), 2.60–3.24 (5H, m), 3.54–
4.87 (9H, m), 7.60–8.20 (4H, m).
Anal. Calcd for C18H23N5O2SÆ2HClÆ1/5C4H8O2Æ4/
5H2O: C, 47.20; H, 5.94; N, 14.64. Found: C, 47.49;
H, 6.01; N, 14.27.
Anal. Calcd for C20H28N4OSÆ2HClÆ4/5H2O: C, 52.24;
H, 6.93; N, 12.18. Found: C, 52.31; H, 7.21; N, 11.94.
5.1.12. 3-{(2S,4S)-4-[N-(4-Cyanophenylmethyl)-N-(eth-
oxycarbonylmethyl)amino]-2-pyrrolidinylcarbonyl}-1,3-
thiazolidine dihydrochloride (17e). (1) To a solution of
3-[(2S,4S)-1-tert-butoxycarbonyl-4-(4-cyanophenylmeth-
yl)amino-2-pyrrolidinylcarbonyl]-1,3-thiazolidine (14b,
0.833 g, 2 mmol) in N-methyl-2-pyrrolidone (6 mL) were
added ethyl bromoacetate (0.333 mL, 3 mmol) and diiso-
propylethylamine (1.05 mL, 6 mmol) and the mixture
was stirred at room temperature for 18 h. The reaction
mixture was added to saturated aqueous sodium hydrog-
encarbonate solution, and the mixture was extracted
with ethyl acetate. The extract was evaporated under re-
duced pressure. The residue was purified by silica gel
5.1.14. 3-{(2S,4S)-4-[N-(Carboxymethyl)-N-(4-cyanophe-
nylmethyl)amino]-2-pyrrolidinylcarbonyl}-1,3-thiazolidine
2 trifluoroacetate (17g). (1) From compound 14b
(0.833 g, 2 mmol), tert-butyl bromoacetate (0.443 mL,
3 mmol), and diisopropylethylamine (1.05 mL, 6 mmol)
using a procedure analogous to synthesis of 16e, the
compound 3-{(2S,4S)-1-tert-butoxycarbonyl-4-[N-(tert-
butoxycarbonylmethyl)-N-(4-cyanophenylmethyl)ami-
no]-2-pyrrolidinylcarbonyl}-1,3-thiazolidine (16g, 0.990 g,
93%) was prepared.
(2) Compound 16g (881 mg, 1.53 mmol) was dissolved
in ethyl acetate (3.06 mL) and 4 mol/L hydrochloric

H. Sakashita et al. / Bioorg. Med. Chem. 15 (2007) 641–655
651
acid–ethyl acetate (6.91 mL) was added to the solution.
The mixture was stirred at room temperature for 3 days.
The precipitated solid was purified by HPLC to give the
title compound (141 mg, 17%) as a white powder.
(2) Compound 16i (514 mg, 0.941 mmol) was dissolved
in ethyl acetate (1.88 mL) and 4 mol/L hydrochloric
acid–ethyl acetate (1.18 mL) was added to the solution.
The mixture was stirred at room temperature for 18 h
and the precipitated solid was collected to give the title
compound (320 g, 66%) as a white powder.
1H NMR (300 MHz, DMSO-d6) d 1.65–1.84 (1H, m),
2.57–2.74 (1H, m), 2.96–3.19 (3H, m), 3.22–4.00 (8H,
m), 4.37–4.72 (3H, m), 7.53 (2H, d, J = 8.4 Hz), 7.81
(2H, d, J = 8.1 Hz), 8.77 (1H, br s), 9.63 (1H, br s).
1H NMR (300 MHz, DMSO-d6) d 1.62–2.39 (1H, m),
2.45–2.82 (1H, m), 2.90–3.25 (2H, m), 3.30–3.95 (4H,
m), 4.25–5.00 (6H, m), 7.30–8.20 (8H, m).
Anal. Calcd for C18H22N4O3SÆ8/5C2HF3O2: C, 45.72; H,
4.27; N, 10.06. Found: C, 45.74; H, 4.53; N, 10.07.
Anal. Calcd for C24H23N5O2SÆHClÆ1/5C4H8O2Æ4/5H2O:
C, 57.95; H, 5.33; N, 13.62. Found: C, 57.92; H, 5.25;
N, 13.47.
5.1.15.
3-{(2S,4S)-4-[N-(4-Cyanophenylmethyl)-N-(2-
hydroxyethyl)amino]-2-pyrrolidinylcarbonyl}-1,3-thiazoli-
dine dihydrochloride (17h). (1) To a solution of 3-[(2S,4S)-
1-tert-butoxycarbonyl-4-(4-cyanophenylmethyl)amino-
2-pyrrolidinylcarbonyl]-1,3-thiazolidine (14b, 1.67 g,
4 mmol) in N-methyl-2-pyrrolidone (12 mL) were added
2-bromoethanol (1.42 mL, 20 mmol) and diisopropyleth-
ylamine (2.09 mL, 12 mmol), and the mixture was stirred
at 80 °C for 2 days. The reaction mixture was added to
saturated aqueous sodium hydrogencarbonate solution
and the mixture was extracted with ethyl acetate. The ex-
tract was dried and the solvent was evaporated under re-
duced pressure. The residue was purified by silica gel
chromatography to give 3-{(2S,4S)-1-tert-butoxycarbon-
yl-4-[N-(4-cyanophenylmethyl)-N-(2-hydroxyethyl)amino]-
2-pyrrolidinylcarbonyl}-1,3-thiazolidine (16h, 0.480 g,
26%).
5.1.17. 3-{(2S,4S)-4-[Bis(4-cyanophenylmethyl)]amino-2-
pyrrolidinylcarbonyl}-1,3-thiazolidine
dihydrochloride
(17d). (1) To a solution of 3-((2S,4S)-4-amino-1-tert-bu-
toxycarbonyl-2-pyrrolidinylcarbonyl)-1,3-thiazolidine15
(13, 0.904 g, 3 mmol) in N-methyl-2-pyrrolidone (9 mL)
were added 4-cyanobenzyl bromide (1.29 g, 6.6 mmol)
and diisopropylethylamine (1.57 mL, 9 mmol), and the
mixture was stirred at room temperature for 18 h. The
reaction mixture was added to saturated aqueous sodi-
um hydrogencarbonate solution, and the mixture was
extracted with ethyl acetate. The extract was dried and
the solvent was evaporated under reduced pressure.
The residue was purified by silica gel chromatography
to give 3-{(2S,4S)-1-tert-butoxycarbonyl-4-[bis(4-cyan-
ophenylmethyl)]amino-2-pyrrolidinylcarbonyl}-1,3-thia-
zolidine (16d, 1.27 g, 80%).
(2) Compound 16h (480 mg, 1.04 mmol) was dissolved
in ethyl acetate (2.08 mL) and 4 mol/L hydrochloric
acid–ethyl acetate (1.04 mL) was added to the solution.
The mixture was stirred at room temperature for 8 h and
the precipitated solid was collected to give the title com-
pound (351 mg, 65%) as a brown powder.
(2) Compound 16d (1.13 g, 2.12 mmol) was dissolved in
ethyl acetate (4.24 mL) and 4 mol/L hydrochloric acid–
ethyl acetate (2.65 mL) was added to the solution. The
mixture was stirred at room temperature for 18 h and
the precipitated solid was collected to give the title com-
pound (444 mg, 39%) as a white powder.
1H NMR (300 MHz, DMSO-d6) d 1.85–1.97 (1H, m),
2.02–2.33 (2H, m), 2.70–4.80 (14H, m), 7.60–8.00 (4H,
m), 9.00 (1H, br s), 10.50 (1H, br s).
1H NMR (300 MHz, DMSO-d6) d 1.81–1.94 (1H, m),
2.57–2.79 (1H, m), 3.00–3.95 (11H, m), 4.39–4.75 (3H,
m), 7.55 (4H, d, J = 8.1Hz), 7.79 (4H, d, J = 8.1 Hz),
8.78 (1H, br s), 10.19 (1H, br s).
Anal. Calcd for C18H24N4O2SÆ2HClÆ 7/10C4H8O2Æ7/
5H2O: C, 48.02; H, 6.66; N, 10.77. Found: C, 48.09;
H, 6.81; N, 10.47.
Anal. Calcd for C24H25N5OSÆ2HClÆ1/4C4H8O2Æ2/5H2O:
C, 56.26; H, 5.63; N, 13.12. Found: C, 56.42; H, 5.84;
N, 13.09.
5.1.16.
3-{(2S,4S)-4-[N-(4-Cyanobenzoyl)-N-(4-cyan-
ophenylmethyl)amino]-2-pyrrolidinylcarbonyl}-1,3-thiaz-
olidine hydrochloride (17i). (1) 3-[(2S,4S)-1-tert-
butoxycarbonyl-4-(4-cyanophenylmethyl)amino-2-pyr-
rolidinylcarbonyl]-1,3-thiazolidine (14b, 0.833 g) was
dissolved in dichloromethane (10 mL), and triethyl-
amine (0.418 mL) and 4-cyanobenzoyl chloride
(0.331 g) were added to the solution. The mixture was
stirred at room temperature for 18 h. The reaction mix-
ture was added to saturated aqueous sodium hydrogen-
carbonate solution, and the mixture was extracted with
ethyl acetate. The extract was dried and the solvent
was evaporated under reduced pressure. The residue
was purified by silica gel chromatography to give 3-
{(2S,4S)-1-tert-butoxycarbonyl-4-[N-(4-cyanobenzoyl)-
N-(4-cyanophenylmethyl)amino]-2-pyrrolidinylcarbo-
nyl}-1,3-thiazolidine (16i, 0.956 g, 87%).
5.1.18. 3-((2S,4S)-1-tert-Butoxycarbonyl-4-hydroxy-2-
pyrrolidinylcarbonyl)-1,3-thiazolidine (20). (1) To a solu-
tion of N-tert-butoxycarbonyl-L-cis-4-hydroxyproline
(18, 23.1 g, 100 mmol) and imidazole (30.0 g, 441 mmol)
in DMF (300 mL) was added tert-butyldimethylsilyl
chloride (33.3 g, 221 mmol). After stirring at room tem-
perature for 16 h, water (300 mL) was gradually added
under ice-cooling. The reaction solution was acidified
with 10% aqueous citric acid solution and extracted with
ethyl acetate. The extract was washed 3 times with water
and with brine, and dried. The solvent was evaporated
under reduced pressure. The residue was purified by sil-
ica gel chromatography to give N-tert-butoxycarbonyl-
L-cis-4-tert-butyldimethylsilyloxyproline (27.4 g, 79%)
as a white solid.

652
H. Sakashita et al. / Bioorg. Med. Chem. 15 (2007) 641–655
(2) To a solution of N-tert-butoxycarbonyl-L-cis-4-tert-
butyldimethylsilyloxyproline (5.55 g, 16.1 mmol) and
thiazolidine (1.4 mL, 18.8 mmol) in DMF (55 mL) were
added triethylamine (2.24 mL, 16.1 mmol), HOBT
(2.96 g, 19.3 mmol), and EDC (3.70 g, 19.3 mmol), and
the mixture was stirred at room temperature for 13 h.
The reaction mixture was added to saturated aqueous
sodium hydrogencarbonate solution and extracted with
ethyl acetate. The extract was washed with water and
brine, and dried. The solvent was evaporated under re-
duced pressure and the residue was purified by silica
gel chromatography to give 3-[(2S,4S)-1-tert-butoxycar-
bonyl-4-tert-butyldimethylsilyloxy-2-pyrrolidinylcarbon-
yl]-1,3-thiazolidine (19, 3.41 g, 51%) as a white solid.
(1H, d, J = 12.8 Hz), 3.57–3.77 (1.5H, m), 3.83–3.98
(1.5H, m), 4.42 (0.5H, d, J = 9.5 Hz), 4.48–4.58 (1H,
m), 4.72 (0.5H, d, J = 9.5 Hz), 5.28–5.36 (1H, m), 7.52
(2H, t, J = 7.4 Hz), 7.65 (1H, t, J = 7.4 Hz), 7.93 (2H,
d, J = 7.4 Hz).
HRMS (CI) calcd for C15H19N2O3S (M+H+) m/e
307.1116; found m/e 307.1111.
5.1.20. 3-[(2S,4S)-4-(4-Cyanobenzoyl)oxy-2-pyrrolidinyl-
carbonyl]-1,3-thiazolidine (15j). (1) From compound 20
(445 mg,
1.50 mmol),
triethylamine
(0.63 mL,
4.52 mmol), and 4-cyanobenzoyl chloride (371 mg,
2.24 mmol) using a procedure analogous to step (1) of
synthesis of 15i, the compound 3-[(2S,4S)-1-tert-butoxy-
carbonyl-4-(4-cyanobenzoyl)oxy-2-pyrrolidinylcarbonyl]-
1,3-thiazolidine (519 mg, 80%) was prepared as a pale-
brown solid.
(3) Compound 19 (3.36 g, 8.06 mmol) was dissolved in
tetrahydrofuran (50 mL), and a 1.0 mol/L solution
(9 mL) of tetrabutylammonium fluoride in tetrahydrofu-
ran was added dropwise under ice-cooling. The mixture
was stirred at room temperature for 18 h, and the sol-
vent was evaporated under reduced pressure. The resi-
due was added to brine and extracted with ethyl
acetate. The extract was dried and the solvent was evap-
orated under reduced pressure. The residue was purified
by silica gel chromatography to give the title compound
(2.44 g, 100%) as a white solid.
(2) From the compound thus prepared (386 mg,
0.895 mmol) using a procedure analogous to step (2)
of synthesis of 15i, the title compound (280 mg, 94%)
was prepared as a pale-yellow solid.
1H NMR (300 MHz, DMSO-d6) d 1.88–1.97 (1H, m),
2.46–2.58 (1H, m), 2.88 (1H, dd, J = 12.9, 4.0 Hz),
2.99 (1H, t, J = 6.3 Hz), 3.09 (1H, t, J = 6.3 Hz), 3.23
(1H, d, J = 12.9 Hz), 3.57–3.76 (1.5H, m), 3.84–3.99
(1.5H, m), 4.42 (0.5H, d, J = 9.5 Hz), 4.48–4.57 (1H,
m), 4.72 (0.5H, d, J = 9.5 Hz), 5.33–5.38 (1H, m), 8.01
(2H, d, J = 8.3 Hz), 8.07 (2H, d, J = 8.3 Hz).
1H NMR (300 MHz, DMSO-d6) d 1.25–1.45 (9H, m),
1.52–1.70 (1H, m), 2.35–2.50 (1H, m), 2.95–3.20 (3H,
m), 3.50–3.80 (3H, m), 4.10–4.25 (1H, m), 4.37–4.78
(3H, m), 5.18 (1H, br s).
5.1.19. 3-((2S,4S)-4-Benzoyloxy-2-pyrrolidinylcarbonyl)-
1,3-thiazolidine (15i). (1) To a solution of compound 20
(643 mg, 2.13 mmol) in dichloromethane (10 mL) were
added triethylamine (0.63 mL, 4.52 mmol), benzoyl
chloride (0.44 mL, 3.79 mmol), and 4-(dimethylami-
no)pyridine (23 mg), and the mixture was stirred at
room temperature for 16 h. Saturated aqueous sodium
hydrogencarbonate solution was added to the reaction
mixture and the mixture was extracted with chloroform.
The extract was evaporated under reduced pressure. The
residue was purified by silica gel chromatography to give
3-((2S,4S)-4-benzoyloxy-1-tert-butoxycarbonyl-2-pyrro-
lidinylcarbonyl)-1,3-thiazolidine (515 mg, 60%) as an
oil.
HRMS (CI) calcd for C16H18N3O3S (M+H+) m/e
332.1069; found m/e 332.1071.
5.1.21.
3-[(2S,4S)-4-(2-Isoindolinyl)-2-pyrrolidinylcar-
bonyl]-1,3-thiazolidine 2 trifluoroacetate (22a). (1) To
a solution of 3-((2S,4S)-4-amino-1-tert-butoxycarbon-
yl-2-pyrrolidinylcarbonyl)-1,3-thiazolidine14 (13, 1.49 g)
in DMF (50 mL) were added potassium carbonate
(2.04 g) and a,a0-dibromo-ortho-xylene (1.37 g), and
the mixture was stirred at room temperature for
18 h. To the reaction mixture was added water and
the mixture was extracted with chloroform. The ex-
tract was washed with brine and dried. The solvent
was evaporated under reduced pressure. The residue
was purified by silica gel chromatography to give 3-
[(2S,4S)-1-tert-butoxycarbonyl-4-(2-isoindolinyl)-2-pyr-
rolidinylcarbonyl]-1,3-thiazolidine (1.26 g, 63%) as a
pale-brown solid.
(2) The compound thus prepared (413 mg, 1.02 mmol)
was dissolved in ethyl acetate (4 mL) and 4 mol/L
hydrochloric acid–ethyl acetate (1.3 mL) was added to
the solution. The mixture was stirred at room tempera-
ture for 14 h and the solvent was evaporated under re-
duced pressure. The residue was added to aqueous
sodium hydrogencarbonate solution and extracted with
ethyl acetate. The extract was washed with brine and
dried. The solvent was evaporated under reduced pres-
sure and the residue was purified by silica gel chroma-
tography to give the title compound (315 mg, quant.)
as an oil.
(2) The compound thus prepared (730 mg, 1.81 mmol)
was dissolved in dichloromethane (5 mL) and trifluoro-
acetic acid (2 mL) was added to the solution. The mix-
ture was stirred at room temperature for 15 h and the
solvent was evaporated under reduced pressure. Ether
was added to the residue and the unsolved solid was col-
lected to give the title compound (910 mg, 90%) as a
brown powder.
1H NMR (300 MHz, DMSO-d6) d 1.83–1.93 (1H, m),
2.45–2.57 (1H, m), 2.87 (1H, dd, J = 12.8, 4.2 Hz),
2.99 (1H, t, J = 6.3 Hz), 3.08 (1H, t, J = 6.3 Hz), 3.19
1H NMR (300 MHz, DMSO-d6) d 2.05–2.14 (1H, m),
2.88–2.96 (1H, m), 3.05–3.17 (2H, m), 3.42–4.02 (5H,
m), 4.44–4.75 (7H, m), 7.31–7.37 (4H,m).

H. Sakashita et al. / Bioorg. Med. Chem. 15 (2007) 641–655
653
Anal. Calcd for C16H21N3OSÆ2C2HF3O2Æ3/2H2O: C,
43.01; H, 4.69; N, 7.52. Found: C, 43.07; H, 4.43; N,
7.33.
5.1.24. 3-[(2S,4S)-4-(5-Nitro-1-indolinyl)-2-pyrrolidinyl-
carbonyl]-1,3-thiazolidine hydrochloride (22d). (1) From
compound 21 (450 mg, 1.50 mmol), 5-nitroindoline
(295 mg, 1.80 mmol), acetic acid (0.090 mL, 1.57 mmol),
and sodium triacetoxyborohydride (636 mg, 3.00 mmol)
using a procedure analogous to step (1) of synthesis of
22b, the compound 3-[(2S,4S)-1-tert-butoxycarbonyl-4-
(5-nitro-1-indolinyl)-2-pyrrolidinylcarbonyl]-1,3-thiazoli-
dine (153 mg, 23%) was obtained as an oil.
5.1.22.
3-[(2S,4S)-4-(1,2,3,4-Tetrahydro-1-quinolyl)-2-
pyrrolidinylcarbonyl]-1,3-thiazolidine hydrochloride (22b).
(1) To a solution of 3-((S)-1-tert-butoxycarbonyl-4-oxo-2-
pyrrolidinylcarbonyl)-1,3-thiazolidine15 (21, 450 mg,
1.50 mmol), 1,2,3,4-tetrahydroquinoline (0.23 mL, 1.83
mmol), and acetic acid (0.09 mL, 1.57 mmol) was added
sodium triacetoxyborohydride (0.636 g), and the mixture
was stirred at room temperature for 24 h. To the reac-
tion mixture was added aqueous sodium hydrogencar-
bonate solution and extracted with ethyl acetate. The
extract was washed with brine and dried. The solvent
was evaporated under reduced pressure. The residue
was purified by silica gel chromatography to give 3-
[(2S,4S)-1-tert-butoxycarbonyl-4-(1,2,3,4-tetrahydro-1-quin-
(2) From the compound thus prepared (153 mg,
0.341 mmol) using a procedure analogous to step (2)
of synthesis of 22b, the title compound (116 mg, 88%)
was obtained as a yellow powder.
1H NMR (300 MHz, DMSO-d6) d 1.92–2.06 (1H, m),
2.67–2.80 (1H, m), 3.00–3.17 (4H, m), 3.27–3.94 (6H,
m), 4.42–4.78 (4H, m), 6.62 (1H, d, J = 8.9 Hz), 7.87
(1H, d, J = 2,3 Hz), 8.04 (1H, dd, J = 8.9, 2.3 Hz), 9.1
(1H, br s), 10.2 (1H, br s).
olyl)-2-pyrrolidinylcarbonyl]-1,3-thiazolidine
16%) as an oil.
(100 mg,
(2) The compound thus prepared (100 mg, 0.239 mmol)
was dissolved in ethyl acetate (3 mL) and 4 mol/L
hydrochloric acid–ethyl acetate (0.3 mL) was added to
the solution. The mixture was stirred at room tempera-
ture for 17 h and the precipitated solid was collected to
give the title compound (60 mg, 65%) as a pale-brown-
reddish powder.
Anal. Calcd for C16H20N4O3SÆ11/10HCl: C, 49.46;
H, 5.47; N, 14.42. Found: C, 49.60; H, 5.61; N,
14.03.
5.1.25. 3-[(2S,4S)-4-(5-Methoxy-1-indolinyl)-2-pyrrolidi-
nylcarbonyl]-1,3-thiazolidine dihydrochloride (22e). (1)
From compound 21 (751 mg, 2.50 mmol), 5-methoxy-
indoline (410 mg, 2.75 mmol), acetic acid (0.143 mL,
2.50 mmol), and sodium triacetoxyborohydride (1.06 g,
5.00 mmol) using a procedure analogous to step (1) of
synthesis of 22b, the compound 3-[(2S,4S)-1-tert-butox-
ycarbonyl-4-(5-methoxy-1-indolinyl)-2-pyrrolidinylcar-
bonyl]-1,3-thiazolidine (1010 mg, 93%) was obtained as
a white solid.
1H NMR (300 MHz, DMSO-d6) d 1.76–1.97 (3H, m),
2.59–2.73 (3H, m), 3.02–3.5 (6H, m), 3.62–3.94 (2H,
m), 4.42–4.86 (4H, m), 6.57 (1H, d, J = 7.2 Hz), 6.80
(1H, d, J = 8.3 Hz), 6.92 (1H, d, J = 7.2 Hz), 6.97–7.07
(1H, m), 8.84 (1H, br s), 10.04 (1H, br s).
Anal. Calcd for C17H23N3OSÆ7/5HClÆ1/5C4H8O2: C,
55.37; H, 6.79; N, 10.88. Found: C, 55.36; H, 7.16; N,
10.58.
(2) From the compound thus prepared (326 mg,
0.752 mmol) using a procedure analogous to step (2)
of synthesis of 22b, the title compound (262 mg, 78%)
was obtained as a white powder.
5.1.23. 3-[(2S,4S)-4-(1-Indolinyl)-2-pyrrolidinylcarbonyl]-
1,3-thiazolidine dihydrochloride (22c). (1) From com-
pound 21 (601 mg, 2.00 mmol), indoline (0.27 mL,
2.41 mmol), acetic acid (0.11 mL, 1.92 mmol), and sodi-
um triacetoxyborohydride (850 mg, 4.01 mmol) using a
procedure analogous to step (1) of synthesis of 22b,
the compound 3-[(2S,4S)-1-tert-butoxycarbonyl-4-(1-
indolinyl)-2-pyrrolidinylcarbonyl]-1,3-thiazolidine (460 mg,
57%) was obtained as a white solid.
1H NMR (300 MHz, DMSO-d6) d 1.80–1.95 (1H, m),
2.62–2.75 (1H, m), 2.86 (2H, t, J = 7.9 Hz), 3.04 (1H,
t, J = 7.0 Hz), 3.12 (1H, t, J = 6.2 Hz), 3.17–3.52 (4H,
m), 3.65 (3H, s), 3.66–4.08 (6H, m), 4.28–4.77 (4H,
m), 6.54 (1H, d, J = 8.5 Hz), 6.63 (1H, dd, J = 8.5,
2.4 Hz), 6.75 (1H, d, J = 2.4 Hz), 8.83 (1H, br s),
10.40 (1H, br s).
(2) From the compound thus prepared (436 mg,
1.08 mmol) using a procedure analogous to step (2) of
synthesis of 22b, the title compound (293 mg, 74%)
was obtained as a white powder.
Anal. Calcd for C17H23N3O2SÆ19/10HClÆ1/2C4H8O2: C,
51.08; H, 6.52; N, 9.41. Found: C, 51.46; H, 6.78; N,
9.07.
5.1.26. 3-[(2S,4S)-4-(5-Fluoro-1-indolinyl)-2-pyrrolidinyl-
carbonyl]-1,3-thiazolidine dihydrochloride (22f). (1) From
compound 21 (496 mg, 1.65 mmol), 5-fluoroindoline
(200 mg, 1.46 mmol), acetic acid (0.084 mL, 1.47 mmol),
and sodium triacetoxyborohydride (0.618 g, 2.92 mmol)
using a procedure analogous to step (1) of synthesis of
22b, the compound 3-[(2S,4S)-1-tert-butoxycarbonyl-4-
(5-fluoro-1-indolinyl)-2-pyrrolidinylcarbonyl]-1,3-thiaz-
olidine (492 mg, 80%) was obtained as a pale-brown
solid.
1H NMR (300 MHz, DMSO-d6) d 1.83–1.97 (1H, m),
2.62–2.77 (1H, m), 2.88 (2H, t, J = 8.2 Hz), 3.04 (1H,
t, J = 7.0 Hz), 3.12 (1H, t, J = 6.2 Hz), 3.20–3.95 (6H,
m), 4.40–4.78 (4H, m), 6.55–6.68 (2H, m), 6.98–7.09
(2H, m), 8.84 (1H, br s), 10.31 (1H, br s).
Anal. Calcd for C16H21N3OSÆ17/10HCl: C, 52.59;
H, 6.26; N, 11.50. Found: C, 52.67; H, 6.54; N,
11.45.

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H. Sakashita et al. / Bioorg. Med. Chem. 15 (2007) 641–655
(2) From the compound thus prepared (487 mg,
1.16 mmol) using a procedure analogous to step (2) of
synthesis of 22b, the title compound (357 mg, 79%)
was obtained as a white powder.
Anal. Calcd for C16H20BrN3OSÆHClÆ2/5H2O: C, 45.11;
H, 5.16; N, 9.86. Found: C, 45.04; H, 5.31; N, 9.69.
5.2. Biological methods
1H NMR (300 MHz, DMSO-d6) d 1.80–1.95 (1H, m),
2.62–2.75 (1H, m), 2.88 (2H, t, J = 8.2 Hz), 3.04 (1H,
t, J = 7.0 Hz), 3.11 (1H, t, J = 6.2 Hz), 3.18–3.52 (4H,
m), 3.60–3.94 (2H, m), 4.35–4.78 (4H, m), 6.55 (1H,
dd, J = 8.8, 4.3 Hz), 6.85 (1H, td, J = 8.8, 2.6 Hz), 6.94
(1H, dd, J = 8.5, 2.6 Hz), 8.90 (1H, br s), 10.44 (1H,
br s).
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 hu-
man or rat plasma (10-fold diluted solution), 20 lL of
fluorescent substrate (100 lmol/L), 140 lL of buffer
(0.003% Brij-35 containing PBS), and 20 lL of test sub-
strate (of various concentrations) were incubated at
room temperature for 60 min using a 96-well flat-bot-
tomed microtiter plate. The measured fluorescent inten-
sity (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 were determined by lo-
gistic analysis.
Anal. Calcd for C16H20FN3OSÆ19/10HCl: C, 49.19; H,
5.65; N, 10.76. Found: C, 49.18; H, 5.94; N, 10.54.
5.1.27. 3-[(2S,4S)-4-(5-Chloro-1-indolinyl)-2-pyrrolidinyl-
carbonyl]-1,3-thiazolidine hydrochloride (22g). (1) From
compound 21 (665 mg, 2.21 mmol), 5-chloroindoline
(340 mg, 2.21 mmol), acetic acid (0.127 mL, 2.22 mmol),
and sodium triacetoxyborohydride (0.937 g, 4.42 mmol)
using a procedure analogous to step (1) of synthesis
of 22b, the compound 3-[(2S,4S)-1-tert-butoxycarbon-
yl-4-(5-chloro-1-indolinyl)-2-pyrrolidinylcarbonyl]-1,3-
thiazolidine (393 mg, 41%) was obtained as a white
solid.
5.2.2. DPP8, 9-inhibitory activity. DPP8 and DPP9 en-
zymes were prepared from cytoplasmic fractions of cells
expressing recombinant human DPP8 or DPP9, respec-
tively. Reaction solutions containing 20 lL of test com-
pounds of various concentrations, 20 lL of enzyme
preparations, 140 lL of buffer (0.003% Brij-35 contain-
ing PBS), and 20 lL of Gly-Pro-MCA (50 lmol/L, Pep-
tide Institute Inc.) were incubated at 37 °C for 30 min
using a 96-well flat-bottomed microtiter plate. The mea-
sured fluorescence intensity of AMC was taken as the
enzyme activity. IC50 value was calculated as described
above.
(2) From the compound thus prepared (389 mg,
0.888 mmol) using a procedure analogous to step (2)
of synthesis of 22b, the title compound (242 mg, 72%)
was obtained as a white powder.
1H NMR (300 MHz, DMSO-d6) delta 1.81–1.95 (1H,
m), 2.62–2.74 (1H, m), 2.90 (2H, t, J = 8.3 Hz), 3.04
(1H, t, J = 7.1 Hz), 3.12 (1H, t, J = 6.2 Hz), 3.18-3.52
(4H, m), 3.60–3.94 (2H, m), 4.38–4.77 (4H, m), 6.57
(1H, d, J = 8.3 Hz), 7.03–7.11 (2H, m), 8.86 (1H, br s),
10.38 (1H, br s).
Acknowledgments
We thank Kumiko Yoshida and Tadashi Hatano for
their technical support. We also thank Dr. Tohru Nak-
ajima and Dr. Takao Kondo for their insight and guid-
ance during the course of this work.
Anal. Calcd for C16H20ClN3OSÆHClÆ3/10H2O: C, 50.61;
H, 5.73; N, 11.07. Found: C, 50.65; H, 5.62; N, 11.00.
5.1.28. 3-[(2S,4S)-4-(5-Bromo-1-indolinyl)-2-pyrrolidinyl-
carbonyl]-1,3-thiazolidine hydrochloride (22h). (1) From
compound 21 (0.901 g, 3.00 mmol), 5-bromoindoline
(0.713 g, 3.60 mmol), acetic acid (0.180 mL, 3.14 mmol),
and sodium triacetoxyborohydride (1.27 g, 5.99 mmol)
using a procedure analogous to step (1) of synthesis of
22b, the compound 3-[(2S,4S)-4-(5-bromo-1-indolinyl)-
1-tert-butoxycarbonyl-2-pyrrolidinylcarbonyl]-1,3-thiaz-
olidine (1.31 g, 91%) was obtained as a white solid.
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1H NMR (300 MHz, DMSO-d6) d 1.82–1.96 (1H, m),
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