1-((S)-γ-Substituted prolyl)-(S)-2-cyanopyrrolidine as a novel series of highly potent DPP-IV inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.03.077

1-(γ-Substituted prolyl)-(S)-2-cyanopyrrolidines were designed based on the predicted binding mode of the known DPP-IV inhibitor NVP-DPP728 and evaluated for their inhibitory activity. In structure-activity relationship study at the γ-position of proline,

Full text of DOI:10.1016/j.bmcl.2005.03.077

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2441–2445
1-((S)-c-Substituted prolyl)-(S)-2-cyanopyrrolidine as a novel
series of highly potent DPP-IV inhibitors
Hiroshi Sakashita,a Hiroshi Kitajima,a Mitsuharu Nakamura,a
Fumihiko Akahoshia,* and Yoshiharu Hayashib
aPharmaceuticals Research Unit, Research & Development Division, Mitsubishi Pharma Corporation, 1000, Kamoshida-cho,
Aoba-ku, Yokohama 227-0033, Japan
bPharmaceuticals Development Unit, Research & Development Division, Mitsubishi Pharma Corporation, 2-2-6, Nihonbashi-Honcho,
Chuo-ku, Tokyo 103-8405, Japan
Received 23 February 2005; revised 9 March 2005; accepted 22 March 2005
Available online 12 April 2005
Abstract—1-(c-Substituted prolyl)-(S)-2-cyanopyrrolidines were designed based on the predicted binding mode of the known DPP-
IV inhibitor NVP-DPP728 and evaluated for their inhibitory activity. In structure–activity relationship study at the c-position of
proline, it became clear that compounds bearing (S)-stereochemistry were 20-fold more potent than the antipode. Of these com-
pounds, the (3,4-dicyanophenyl)amino- and (3-chloro-4-cyanophenyl)amino-derivatives showed the highest inhibitory activity.
Ó 2005 Elsevier Ltd. All rights reserved.
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 only after
meals,4 and DPP-IV inhibitors have therefore come to
be seen as a potential new type of anti-diabetic agent
free of side effects such as hypoglycemia and exhaustion
of pancreatic beta-cells.
reversible and potent inhibitors.6 Hughes et al. investi-
gated a series of DPP-IV inhibitors, 1-(N-substituted
glycyl)-2-cyanopyrrolidides, NVP-DPP728 (2), and
NVP-LAF237 (3), the latter is under clinical trials as
anti-diabetic agent (Fig. 1).7–9 As the mode of NVP-
DPP728 binding to DPP-IV, they estimated that hydro-
phobic interaction in the S2 pocket stabilizes the P2 site
side-chain binding.7 This description suggests that, when
NVP-DPP728 displays inhibitory activity, the [2-(5-
cyanopyridyl)amino]ethyl moiety assumes not an ex-
tended but a folded conformation, as shown in Figure
2a.
We hypothesized that analogs, which have a rigid con-
formation capable of interacting with the S2 pocket of
DPP-IV show more potent inhibitory activity. As prol-
yl-(S)-2-cyanopyrrolidine6 (4) has a comparable inhibi-
tory activity to NVP-DPP728, and as most of the
proline derivatives are commercially available, we se-
lected a proline structure for the P2 site and introduced
several functional groups at the c-position. In this way
we designed a series of 1-(c-substituted prolyl)-(S)-
2-cyanopyrrolidine compounds (Fig. 2b) as conforma-
tionally constrained analogs of NVP-DPP728 and
evaluated them for inhibitory activity.
Although a number of DPP-IV inhibitors have been re-
ported and classified into structural types, most of them
are substrate analogs of the P2–P1 fragment.5 Dipeptide
inhibitors containing (S)-2-cyanopyrrolidine as a proline
mimic at the P1 site and an aliphatic amino acid at the P2
site, for example, 1 (Fig. 1), have been reported as
Keywords: DPP-IV inhibitor; 2-Cyanopyrrolidine.
*
The (R)-substituted derivative 8 was synthesized from
the cis-hydroxyproline 5 (Scheme 1). Conversion of the
Corresponding author. Tel.: +81 45 963 4569; fax: +81 45 963
4211; e-mail: Akahoshi.Fumihiko@mk.m-pharma.co.jp
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.03.077

2442
H. Sakashita et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2441–2445
S2 pocket
S1 pocket
R
+
H3N
N
N
O
Xaa
H2N
OH
Ser
CN
-
O
O
COO
O
1
P1
P1'
P2
H
N
N
N
N
H
N
N
H
CN
O
CN
NC
O
NVP-DPP728 (2)
NVP-LAF237 (3)
Figure 1.
S2 pocket
S2 pocket
CN
DPP-IV
CN
O
DPP-IV
N
N
S1 pocket
S1 pocket
HN
HN
γ
N
O
N
O
N
H
N
H
Ser
Ser
O
N
N
NVP-DPP728
(folded conformation)
1-(γ-substituted prolyl)-(S)-2-cyanopyrrolidine
(a)
(b)
Figure 2. Design of 1-(c-substituted prolyl)-(S)-2-cyanopyrrolidine.
NC
H
N
NC
H
HO
N
H2N
N
N
i, ii, iii
iv, v
vi, vii
N
N
N
N
Boc
H
N
Boc
COOH
COOMe
COOMe
CN
O
Boc
5
6
7
8
Scheme 1. Reagents: (i) MsCl, Et3N; (ii) NaN3; (iii) H2, Pd/BaSO4; (iv) 2-chloro-5-cyanopyridine, Et3N; (v) NaOH aq; (vi) (S)-2-cyanopyrroli-
dineÆHCl, HOBt, WSCI, Et3N; (vii) H+.
mesylate of 5 to an azide compound and subsequent cata-
lytic hydrogenation gave the trans-c-amino proline
derivative 6. Reaction of 6 with 2-choloro-5-cyanopyri-
dine in the presence of Et3N at room temperature fol-
lowed by alkaline hydrolysis gave the trans-c-
pyridylaminoproline compound 7. The acid 7 was
coupled with (S)-2-cyanopyrrolidine6 and treated with
HCl/AcOEt to afford the desired constrained analog of
NVP-DPP728 (8).
introduce a pyridine moiety under the same conditions
as described above. However, no reaction occurred at
room temperature and intramolecular cyclization at
high temperature resulted in the lactam 11. In order to
avoid this cyclization, a pyridyl group was introduced
into the amide 14. Amidation of the trans-hydroxypro-
line 12 with (S)-2-cyanopyrrolidine,6 followed by trans-
formation of the trans-hydroxy group to a cis-amino
group, afforded the amine 14. Reaction of the latter with
2-chloro-5-cyanopyridine or other electron-deficient
aromatic halides and subsequent removal of the Boc
group afforded the (S)-substituted derivatives 15 and
17d–g. Meanwhile, the non-electron-deficient phenyl
The syntheses of (S)-substituted derivatives are shown in
Scheme 2. The cis-amino proline 10 was prepared from
the trans-hydroxyproline 9 and an attempt was made to

H. Sakashita et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2441–2445
2443
HO
H2N
H
N
iv
i, ii, iii
O
N
Boc
N
Boc
N
Boc
COOMe
COOMe
COOH
9
10
11
H
N
Ar
HO
HO
H2N
v
i, ii, iii
vi, vii
N
N
N
N
N
Boc
N
Boc
N
N
Boc
H
CN
CN
CN
CN
O
O
O
14
12
13
15,17d-g
X
Y
viii
H
N
O
ix, vii
N
N
Boc
N
H
CN
O
16
O
17a: X = Y = H
17b: X = MeO, Y = H
17c: X = Y = MeO
Scheme 2. Reagents: (i) MsCl, Et3N; (ii) NaN3; (iii) H2, Pd/BaSO4; (iv) 2-chloro-5-cyanopyridine, Et3N; (v) (S)-2-cyanopyrrolidineÆHCl, HOBt,
WSCI, Et3N; (vi) Ar–Cl or Ar–F, Et3N or N-methylmorpholine; (vii) H+; (viii) DMSO, SO3–pyridine complex, Et3N; (ix) Ar–NH2, NaBH3CN,
AcOH.
derivatives 17a–c were prepared by reductive amination
of the ketone 16 with anilines and subsequent
deprotection.
adapt to the S2 pocket of DPP-IV, whereas that of the
(R)-isomer 8 cannot achieve interaction at this site. As
expected, conformationally restricted analogs, which
mimic the constrained structure of NVP-DPP728
showed more potent inhibitory effect. These findings
indicate that the 1-((S)-c-substituted prolyl)-(S)-2-
cyanopyrrolidine has a structure suited to DPP-IV
inhibition.
Table 1 summarizes the DPP-IV inhibitory effects of the
conformationally restricted NVP-DPP728 analogs repre-
sented by 1-prolyl-(S)-2-cyanopyrrolidines having a
(5-cyano-2-pyridyl)amino group at the c-position of
the proline moiety.10 Compound 15, bearing (S)-stereo-
chemistry at the c-position, showed 5–10-fold more po-
tent inhibitory activity than NVP-DPP728 or the non-
substituted derivative 4.6 The (R)-isomer 8 was less po-
tent than the (S)-isomer 15. This result indicates that
the pyridylamino group of the (S)-isomer 15 is able to
Next, the effect of substituents at the c-position of
the proline moiety was investigated (Table 2). Introduc-
tion of an amino or cyclohexylamino group (18, 19)
resulted in a decrease in DPP-IV inhibitory activity com-
pared to the (5-cyano-2-pyridyl)amino compound 15.
Table 1.
Y
X
γ
N
N
H
CN
O
Compound no.
Configuration
at c position
X
Y
H
DPP-IV inhibition,
IC50 (nmol/L)
Human
Rat
H
N
N
15
8
S
0.25
6.6
0.27
NC
H
N
N
R
—
H
6.7
NC
4
NVP-DPP728 (2)
H
H
2.9
1.4
2.9
2.3

2444
H. Sakashita et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2441–2445
Table 2.
between the phenyl and the nitrogen atom at the c-posi-
tion (17a, 20–24). Insertion of all the spacers tested re-
sulted in decreased DPP-IV inhibitory activity. We
next examined the influence of substituents on the benz-
ene ring. Substitution of an electron-donating methoxy
group (17b) or an electron-withdrawing cyano or nitro
group (17d, 17e) both resulted in ca. 2–3-fold more
potent activity. In these structure–activity relationship
studies, no correlation was observed between the elec-
tronic effect of the substituents on the benzene ring
and inhibitory activity. It is possible that they have some
hydrophobic interaction with the S2 pocket. In investi-
gation of NVP-DPP728, it has been reported that elec-
tron-withdrawing substituents on the 2-aminopyridine
ring do not appear to be essential.8 Of the compounds
investigated in the present study, compounds 17f and
17g, bearing 3,4-dicyanophenyl and 3-chloro-4-cyano-
phenyl groups, showed the highest inhibitory activity.
H
N
R
N
N
H
CN
O
Compound no.
R
DPP-IV inhibition,
IC50 (nmol/L)
Human
0.25
Rat
NC
15
0.27
N
18
19
H
9.7
3.0
9.1
3.6
Rasmussen et al. reported the crystal structure of DPP-
IV in complex with a valine–pyrrolidide inhibitor and
indicated that the isopropyl side chain of this inhibitor
points into a large cavity.11 Given the presence of a large
cavity in the S2 pocket, it could hold a hydrophobic
binding site interacting with the c-substituent. Further
optimization of these proline-containing inhibitors, fo-
cused on the binding site in the cavity, is in progress.
17a
20
0.53
4.6
0.53
3.0
21
1.9
1.5
O
In conclusion, we designed a series of 1-(c-substituted
prolyl)-(S)-2-cyanopyrrolidine compounds as novel
DPP-IV inhibitors and investigated their inhibitory
activity. In structure–activity relationship study at the
c-position of proline, it became clear that compounds
bearing (S)-stereochemistry were more potent than the
antipode. Of these compounds, 1-[(S)-c-[(3,4-dicyano-
phenyl)amino]prolyl]-(S)-2-cyanopyrrolidine (17f) and
1-[(S)-c-[(3-chloro-4-cyanophenyl)amino]prolyl]-(S)-2-
cyanopyrrolidine (17g) showed the highest inhibitory
activity.
22
5.5
3.8
O
23
24
1.5
6.7
1.1
3.9
N
H
O
O
S
MeO
MeO
17b
17c
17d
17e
17f
0.33
0.28
0.19
0.18
0.13
0.28
0.24
0.26
0.17
0.17
Acknowledgements
We wish to thank Dr. Tohru Nakajima and Dr. Takao
Kondo for their insight and guidance during the course
of this work.
MeO
NC
O2N
NC
References and notes
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NC
NC
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Cl
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2445
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10. 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
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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,
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