[(S)-γ-(4-Aryl-1-piperazinyl)-l-prolyl]thiazolidines as a novel series of highly potent and long-lasting DPP-IV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2007.01.110

In the search for an inhibitor of dipeptidyl peptidase IV (DPP-IV) highly potent both in vitro and in vivo, we synthesized a series of l-prolylthiazolidine-based DPP-IV inhibitors having 4-arylpiperazine or 4-arylpiperidine at the γ-position of the proline structure. Of these compounds, the 4-(5-nitro-2-pyridyl)piperazine analog 21e showed a sub-nanomolar (IC₅₀ = 0.92 nmol/L) DPP-IV inhibitory activity and a long-lasting in vivo DPP-IV inhibition profile.

Full text of DOI:10.1016/j.bmcl.2007.01.110

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2618–2621
[(S)-c-(4-Aryl-1-piperazinyl)-L-prolyl]thiazolidines as a novel
series of highly potent and long-lasting DPP-IV inhibitors
Tomohiro Yoshida,a Hiroshi Sakashita,a Fumihiko Akahoshia,* and Yoshiharu Hayashib
aChemistry Laboratory, Pharmaceuticals Research Division, Mitsubishi Pharma Corporation,
1000, Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan
bGlobal Projects Coordination Pharmaceuticals Department, Development Division,
Mitsubishi Pharma Corporation, 2-2-6, Nihonbashi-Honcho, Chuo-ku, Tokyo 103-8405, Japan
Received 12 December 2006; revised 11 January 2007; accepted 31 January 2007
Available online 8 February 2007
Abstract—In the search for an inhibitor of dipeptidyl peptidase IV (DPP-IV) highly potent both in vitro and in vivo, we synthesized
a series of L-prolylthiazolidine-based DPP-IV inhibitors having 4-arylpiperazine or 4-arylpiperidine at the c-position of the proline
structure. Of these compounds, the 4-(5-nitro-2-pyridyl)piperazine analog 21e showed a sub-nanomolar (IC50 = 0.92 nmol/L)
DPP-IV inhibitory activity and a long-lasting in vivo DPP-IV inhibition profile.
Ó 2007 Elsevier Ltd. All rights reserved.
Glucagon-like peptide (GLP)-1 is an incretin hormone
that has multifaceted actions, including glucose-induced
stimulation of insulin biosynthesis and secretion, regula-
tion of gene expression, trophic effects on b cells, inhibi-
tion of food intake, and slowing of gastric emptying.
These effects, which contribute to the normalization of
elevated blood glucose and to the control of body
weight, are tempered by the rapid enzymatic degrada-
tion of the peptide. Inhibition of dipeptidyl peptidase
IV (DPP-IV) suppresses the degradation of many pep-
tides, including GLP-1, thereby extending their bioactiv-
ity. Indeed, many DPP-IV inhibitors have been reported
and several of them are undergoing late-stage clinical
trials.1–5 Recently, sitagliptin (1, MK-0431)6 was ap-
proved as the first-in-class DPP-IV inhibitor for treat-
ment of type 2 diabetes.
F
F
F
NH2
O
H
N
N
N
N
N
H
N
N
CN
O
N
NC
CF3
sitagliptin (1)
NVP-DPP728 (2)
OH
HO
H2N
N
N
N
H
CN
CN
O
O
vildagliptin (3)
Figure 1. DPP-IV inhibitors.
saxagliptin (4)
the Ser630 of the catalytic triad in DPP-IV and have
highly potent inhibitory activities. We previously report-
ed14 that a series of [(S)-c-(alylamino)-L-prolyl]-(S)-2-
cyanopyrrolidine analogs (e.g., 5) showed highly potent
DPP-IV inhibitory activity. These compounds were,
however, chemically unstable due to the intramolecular
cyclization between the secondary amine of the proline
and the electrophilic nitrile of the (S)-2-cyanopyrroli-
dine moiety. We also reported15,16 that thiazolidide
replacement (e.g., 6) in this series conferred chemical
stability, and that efforts to optimize the c-alylamino
moiety led to improved in vitro activity against
DPP-IV (Fig. 2). The in vivo efficacy was nevertheless
DPP-IV is a dipeptidase that recognizes an amino acid
sequence having a proline or alanine residue at the P1
position,7 and many known DPP-IV inhibitors therefore
have substituted pyrrolidines or thiazolidines as a pro-
line mimetic in the P1 part.8 In particular, inhibitors pos-
sessing a (S)-2-cyanopyrrolidine moiety, for example
NVP-DPP728 (2),9–11 vildagliptin (3, LAF237),11,12
and saxagliptin (4, BMS-477118)13 (Fig. 1), interact with
Keywords: DPP-IV inhibitor; Thiazolidine; 4-Arylpiperazine.
*
Corresponding author. Tel.: +81 45 963 3436; fax: +81 45 963
4211; e-mail: Akahoshi.Fumihiko@mk.m-pharma.co.jp
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.01.110

T. Yoshida et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2618–2621
2619
S2 pocket
S2 pocket
S2 pocket
R
DPP-IV
S1 pocket
DPP-IV
CN
CN
DPP-IV
Z
W
S1 pocket
S1 pocket
N
N
N
γ
HN
γ
HN
γ
S
S
N
N
N
N
H
N
H
N
H
O
O
O
Ser630
O
N
unstable (decomp. t1/2 = 1.3h)
stable
6, IC50 = 25 nmol/L
W = N or CH
Z = N or CH
5, IC50 = 0.25 nmol/L
Figure 2. Design of (S)-c-substituted L-prolylthiazolidines as DPP-IV inhibitors.
Table 1. Inhibition of ((S)-c-substituted-prolyl)thiazolidines
still insufficient. We focused further on c-substituents of
the proline moiety at the P2 position in the search for
highly potent DPP-IV inhibitor both in vitro and in vivo.
Here we describe how the 4-aryl-1-piperazine or 4-aryl-
1-piperidine substitution at the c-position of proline
resulted in not only increased potency, but also im-
proved in vivo efficacy.
O
X
N
S
NH
Compound
X
H
DPP-IV inhibition
IC50 (nmol/L)
Human
Rat
607
The synthesis of a series of cyclic amine substituted prol-
ylthiazolidine compounds is shown in Scheme 1. The
key intermediate 8 was prepared by coupling the
N-Boc-trans-4-hydroxy-L-proline 7 with thiazolidine,
followed by DMSO-oxidation, as previously report-
ed.14,15 Reductive amination of the ketone 8 with a vari-
ety of cyclic amines afforded only cis-configuration
intermediates and subsequent removal of the Boc group
yielded analogs 10–21.
9
538
127
10
121
N
N
11
12
13
14
47.6
74.4
13.9
57.5
10.9
18.9
81.1
17.6
N
N
N
O
N
Table 1 summarizes the DPP-IV inhibitory activity17 of
L-prolylthiazolidide DPP-IV inhibitors bearing a repre-
sentative cyclic amino group at their c-position. Substi-
tution of a 5- or 6-membered cyclic amino group
undoubtedly increased the activity compared to the
non-substituted compound 9. Of these compounds, the
4-phenylpiperazine analog 14 exhibited almost 50-fold
more potent activity. We therefore next examined the
substituent of the phenyl ring of compound 14. The
results are shown in Table 2.
N
15
16
30.5
21.0
38.3
31.4
N
N
N
N
4-Methoxy (17a) or 4-fluoro (17b) substituents were sim-
ilar to 14, while 4-chloro (17c) or 4-bromo (17f) substit-
uents led to
a slight improvement in potency.
Comparison of potency according to the substituted po-
sition on the phenyl ring indicated that the 3-chloro
(17c) substituent was superior to the 2-chloro (17e) or
the 4-chloro (17c) analog. A more significant improve-
ment was seen with the 4-nitro (17g) substituent
(IC50 = 1.6 nmol/L), which was 7-fold more potent than
the unsubstituted phenyl analog 14. Similar improve-
ment was found in electron-withdrawing 4-cyano (17h)
and 4-trifluoromethyl (17i) analogs. Moreover, disubsti-
tution of the electron-withdrawing group increased
potency compared with monosubstitution (17c vs 17j,
17h vs 17k). The 4-phenylpiperidine analogs 18 and 19
also had a strong potency and produced similar SAR
results to the piperazine analogs. These results suggested
that the piperazine and the piperidine play linker roles
and arrange the aryl moiety in the appropriate space
in the S2 pocket. Replacement of the piperazine linker
in this series with a homopiperazine led to a slight de-
crease in potency (17g vs 20). In light of the SAR results
O
S
X
HO
S
c, d
a, b
N
OH
N
N
Boc
N
Boc
N
O
H
O
O
10 -21
7
8
Scheme 1. Reagents: (a) thiazolidine, HOBt, EDC, DMF; (b) DMSO,
SO3-pyridine complex; (c) amine X, NaBH(OAc)3, AcOH, 1,2-dichlo-
roethane; (d) H+.

2620
T. Yoshida et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2618–2621
Table 2. Inhibition of ((S)-c-phenylpiperazinylproly1) thiazolidines
Table 3. Inhibition of ((S)-c-pyridylpiperazinylprolyl) thiazolidines
O
O
n
N
N
S
N
S
Ar
N
N
Ar W
NH
NH
Compound
Ar
DPP-IV inhibition,
IC50 (nmol/L)
Compound
Ar
Ph
W
n
DPP-IV inhibition,
IC50 (nmol/L)
Human
Rat
Human
Rat
21a
21b
21c
21d
21e
21f
21g
21h
2-Pyridyl
4-Pyridyl
5-Cl-2-pyridyl
2.7
3.1
2.5
1.6
0.92
1.5
1.0
1.2
3.5
3.9
3.1
1.9
1.1
1.8
1.4
1.2
14
N
N
N
N
N
N
N
N
N
N
N
N
C
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
10.9
13.5
12.2
8.7
17.6
16.9
17.5
10.3
7.2
17a
17b
17c
17d
17e
17f
17g
17h
17i
17j
17k
18
4-MeOPh
4-FPh
4-ClPh
5-CN-2-pyridyl
5-NO2-2-pyridyl
5-CF3-2-pyridyl
4-CN-2-pyridyl
3-CN-2-pyridyl
3-ClPh
2-ClPh
4-BrPh
4.1
10.8
8.2
10.9
9.0
4-NO2Ph
4-CNPh
4-CF3Ph
3,4-Cl2Ph
3,4-CN2Ph
Ph
1.6
4.3
2.2
4.7
6.8
3.0
7.2
4.2
17k
21e
6
1.3
5.6
1.8
9.6
100
80
60
40
20
0
19
20
4-NO2Ph
4-NO2Ph
C
2.3
2.4
3.3
2.9
N
from Table 2, it appears that the introduction of the
electron-withdrawing group to the phenyl ring is desir-
able to improve potency.
These results encouraged us to incorporate a pyridine
substitution as an electron-deficient aromatic ring into
the piperazine scaffold (Table 3). As expected, each of
the pyridine analogs had a highly potent activity. Most
noteworthy among them was the 5-nitro-2-pyridine ana-
log 21e, which provided a sub-nanomolar activity
(IC50 = 0.92 nmol/L) despite the lack of an electrophilic
trap at the P1 position. Comparison of potency accord-
ing to the substituted position on the pyridine ring indi-
cated the superiority of the 4-cyano position (21g vs 21d,
21h). From the many SAR studies of DPP-IV inhibitors
reported by other groups, it seems that the S2 pocket in
DPP-IV is able to accommodate a large substituent.18 It
was hypothesized that the 4-arylpiperazine group in this
series filled the S2 pocket better than our prototype com-
pound 6 (Fig. 1) and as a result compensated for the
potency lost due to the lack of an electrophilic trap at
the P1 position.
0
2
4
6
8
10
Time after administration (hr)
Figure 3. Effects of 17k, 21e, and 6 on plasma DPP-IV activity (%
change of baseline) in Wistar rat. Each compound was orally
administered at a single dose of 10 lmol/kg at 0 h. Data are expressed
as means SEM (n = 3).
and good PK profile of the 4-arylpiperazine analogs
(PK profile not evaluated).
In conclusion, the present report details our program
of optimizing c-substituted L-prolylthiazolidine-based
DPP-IV inhibitors by introducing 4-arylpiperazine,
which resulted in highly potent and long-lasting inhib-
itors. SAR study revealed that the introduction of an
electron-deficient 4-arylpiperazine scaffold was desir-
able for improved potency, as demonstrated by com-
pounds 21a–h. In particular, compound 21e proved
exceptionally potent in vitro (IC50 = 0.92 nmol/L) de-
spite the lack of an electrophilic trap at the P1 position.
Moreover, this compound showed in vivo DPP-IV
inhibition of fast-onset and of longer duration than
that of the prototype compound 6. Further efforts fo-
cused on the aromatic ring in this arylpiperazine scaf-
fold and its predicted interaction mode with DPP-IV
will be reported in due course.
Evaluation of plasma DPP-IV activity after oral dosing
of the compounds can be used to predict the efficacy of
antihyperglycemic activity and the pharmacokinetic
profile. The representative compounds 17k and 21e
and the prototype compound 6 were tested for in vivo
DPP-IV inhibition. Each compound was administered
orally to Wistar rats at a dose of 10 lmol/kg and the
plasma DPP-IV activity was evaluated ex vivo.19 As
shown in Figure 3, compound 6 showed only 70% max-
imum inhibition of plasma DPP-IV activity and its effi-
cacy diminished within 5h, whereas the maximum
inhibition of both compounds 17k and 21e reached
about 95% within 30 min, with more than 60% inhibi-
tion of DPP-IV persisting for 9 h after oral dosing.
These fast-onset and long-lasting DPP-IV inhibitory
activities were most likely due to the increased potency
Acknowledgments
We thank Dr. Hiroshi Kitajima for discussion. We also
thank Dr. Tohru Nakajima and Dr. Takao Kondo for

T. Yoshida et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2618–2621
2621
15. Sakashita, H.; Akahoshi, F.; Kitajima, H.; Tsutsumiuchi,
R.; Hayashi, Y. Bioorg. Med. Chem. 2006, 14, 3662.
16. Sakashita, H.; Akahoshi, F.; Yoshida, T.; Kitajima, H.;
Hayashi, Y.; Ishii, S.; Takashina, Y.; Tsutsumiuchi, R.;
Ono, S. Bioorg. Med. Chem. 2007, 15, 641.
their insight and guidance during the course of this
work.
17. The DPP-IV inhibitory activity of human plasma and rat
plasma was measured 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 fluorescent 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 temperature for 60 min using a 96-well flat-bottom
microtiter plate. The measured fluorescent intensity (exci-
tation 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.
18. (a) Xu, J.; Wei, L.; Mathvink, R. J.; Edmondson, S. D.;
Eiermann, G. J.; He, H.; Leone, J. F.; Leiting, B.; Lyons,
K. A.; Marsilio, F.; Patel, R. A.; Patel, S. B.; Petrov, A.;
Scapin, G.; Wu, J. K.; Thornberry, N. A.; Weber, A. E.
Bioorg. Med. Chem. Lett. 2006, 16, 5373; (b) Edmondson,
S. D.; Mastracchio, A.; Mathvink, R. J.; He, J.; Harper,
B.; Park, Y.-J.; Beconi, M.; Salvo, J. D.; Eiermann, G. J.;
He, H.; Leiting, B.; Leone, J. F.; Levorse, D. A.; Lyons,
K.; Patel, R. A.; Patel, S. B.; Petrov, A.; Scapin, G.;
Shang, J.; Roy, R. S.; Smith, A.; Wu, J. K.; Xu, S.; Zhu,
B.; Thornberry, N. A.; Weber, A. E. J. Med. Chem. 2006,
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