3-[2-((2S)-2-Cyano-pyrrolidin-1-yl)-2-oxo-ethylamino]-3-methyl-butyramid e analogues as selective DPP-IV inhibitors for the treatment of type-II diabetes

Article abstract of DOI:10.1016/j.bmcl.2006.12.019

Based on the structures of NVP-DPP728 (1) and NVP-LAF237 (Vildagliptin, 2), three series of DPP-IV inhibitors were synthesized by linking substituted anilines, benzylamines, and phenylethylamines to (2S)-cyanopyrrolidine through a linker. More than 20 compounds were evaluated for their in vitro DPP-IV inhibition and selectivity profile over DPP-II, DPP8, and FAP enzymes. Selected compounds 5f and 7i showed in vivo plasma DPP-IV inhibition and inhibited glucose excursion in OGTT after oral administration in Wistar rats. Compound 5f (DPP-IV IC₅₀ = 116 nM) has the potential for development as antidiabetic agent.

Full text of DOI:10.1016/j.bmcl.2006.12.019

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1274–1279
3-[2-((2S)-2-Cyano-pyrrolidin-1-yl)-2-oxo-ethylamino]-3-methyl-
butyramide analogues as selective DPP-IV inhibitors for the
treatment of type-II diabetes
Mohane Selvaraj Coumar, Chung-Nien Chang, Chiung-Tong Chen, Xin Chen,
Chia-Hui Chien, Ting-Yueh Tsai, Jai-Hong Cheng, Hsin-Yi Wu,
Chia-Hung Han, Ssu-Hui Wu, Yu-Wen Huang, Tsu Hsu,
Li-Jen Hsu, Yu-Sheng Chao, Hsing-Pang Hsieh* and Weir-Torn Jiaang*
Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, No. 35, Keyan Rd.,
Zhunan Town, Miaoli County 350, Taiwan, ROC
Received 22 September 2006; revised 1 December 2006; accepted 4 December 2006
Available online 12 December 2006
Abstract—Based on the structures of NVP-DPP728 (1) and NVP-LAF237 (Vildagliptin, 2), three series of DPP-IV inhibitors were
synthesized by linking substituted anilines, benzylamines, and phenylethylamines to (2S)-cyanopyrrolidine through a linker. More
than 20 compounds were evaluated for their in vitro DPP-IV inhibition and selectivity profile over DPP-II, DPP8, and FAP
enzymes. Selected compounds 5f and 7i showed in vivo plasma DPP-IV inhibition and inhibited glucose excursion in OGTT after
oral administration in Wistar rats. Compound 5f (DPP-IV IC50 = 116 nM) has the potential for development as antidiabetic agent.
Ó 2006 Elsevier Ltd. All rights reserved.
OH
One of the emerging new drug treatments for type-II dia-
betes therapy is dipeptidyl peptidase IV (DPP-IV; CD26;
E.C. 3.4.14.5) inhibition with small molecules.1–3 Several
DPP-IV inhibitors have entered clinical development,
including NVP-DPP728 (1),4,5 NVP-LAF237 (Vildaglip-
tin, 2),6–8 MK-0431 (Sitagliptin, 3),9–11 and BMS-477118
(Saxagliptin, 4)12 (Fig. 1). Vildagliptin and Sitagliptin are
under review by US FDA as new treatment option for
type-II diabetes.
NC
O
O
H
H
N
N
N
N
N
N
H
NC
NC
1 (NVP-DPP728)
2 (NVP-LAF237)
F
F
HO
NH2
O
N
N
Glucagon-like peptide-1 (GLP-1) is an incretin hormone
secreted by intestinal L-cells in response to food in-
take.13 The active form of GLP-1 is a 30-amino acid
peptide, which stimulates insulin release, inhibits gluca-
gons release, and slows gastric emptying, each a benefit
in the control of glucose homeostasis in patients with
type II diabetes. The active form of GLP-1 is rapidly
N
H2N
N
F
N
O
CN
3 (MK-0431)
CF3
4 (BMS-477118)
Figure 1. DPP-IV inhibitors.
inactivated (t1/2 ꢀ 1 min) by the plasma DPP-IV, which
cleaves a dipeptide from the N-terminus.14,15 Thus inhi-
bition of DPP-IV extends the half-life of endogenously
secreted GLP-1, which in turn enhances insulin secretion
and improves the glucose tolerance. DPP-IV inhibitors
offer several potential advantages over existing therapies
including decreased risk of hypoglycemia, potential for
Keywords: DPP-IV; Type-II diabetes.
*
Corresponding authors. Tel.: +886 37 246166x35712 (W.T.J);
tel.: +886 37 246166x35708 (H.P.H); fax: +886 37 586456; e-mail
addresses: hphsieh@nhri.org.tw; wtjiaang@nhri.org.tw
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.019

M. S. Coumar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1274–1279
1275
P2
P1
OH
Glycine
H
N
N
N
R
N
N
N
N
H
H
H
O
O
CN
CN
O
N
CN
NC
1 (NVP-DPP728)
General structure of 1 and 2
2 (NVP-LAF237)
Design & synthesis
R
Y
Y= CH or N
SAR exploration
& optimisation
DPP-IV
IC50 (nM)
n
5a-h n=0
6a-g n=1
7a-i n=2
n
n
NH
NH
298
452
0
1
5
6
7
O
N
O
N
2
564
N
H
N
H
O
O
CN
CN
Leads reported in ref 16
Compounds reported in this paper
Figure 2. Design of DPP-IV inhibitors based on NVP-DPP728 (1) and NVP-LAF237 (2).
weight loss, and the potential for regeneration and dif-
ferentiation of pancreatic b-cells.1
inhibition (IC50 = 298–564 nM) in in vitro assay,
but poor selectivity over related enzymes DPP8 and
DPP-II (see Tables 1–3).16 Selectivity of the DPP-IV
inhibitors for closely related prolyl peptidase such as
DPP8/9, DPP-II, and FAP is an important criteria for
their further development as antidiabetic agents, since
inhibition of DPP-II results in the apoptosis of quiescent
T-cells19 and also recent in vivo studies indicate that
selective inhibition of DPP8/9 may be associated with
profound toxicities.20 Reports suggest that fibroblast
activation protein (FAP) is involved in tumor growth
and invasion.21 Based on these facts we embarked on
lead optimization of compounds 5–7 to improve the
overall potency and selectivity. Herein we report the
We are involved in the design and synthesis of DPP-IV
inhibitors and had reported potent and selective DPP-IV
inhibitors.16–18 Design of DPP-IV inhibitors in one of
our previous publication16 was based on the general
structure of NVP-DPP728 (1) and NVP-LAF237 (2) as
shown in Figure 2, by retaining the (2S)-cyanopyrroli-
dine in the P1 site and modifying the N-substitution of
glycine in the P2 site. Aniline, benzylamine and phenyl-
ethylamine were attached to the N-terminus of glycine
through a linker to give leads 5, 6, and 7, respectively.
All the three compounds showed moderate DPP-IV
Table 1. Inhibition of DPP-IV, DPP8, DPP-II, and FAP by aniline derivatives
O
H
H
N
N
N
R
X1 X2
O
NC
X1 = X2
IC50 (lM)
DPP8
a
Compound
R
DPP-IVb
DPP-II
FAP
5
H
H
0.298c
0.207
0.197
0.253
0.225
0.174
0.116
0.127
0.269
0.855c
>20
>20
>20
>20
>20
>20
>20
>20
5.7176c
>20
>20
NA
>20
>20
>20
>20
>20
>20
>20
>20
5a
5b
5c
5d
5e
5f
5g
5h
H
4-F
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
4-OCH3
3,4-di-F
3,4-di-CH3
3,4-di-OCH3
2,3,4-tri-OCH3
4-Ph
>20
>20
>20
>20
>20
0.800
NA, not available.
a See Ref. 23.
b Values are expressed as the mean of three independent determinations.
c Data from reference 16.

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M. S. Coumar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1274–1279
Table 2. Inhibition of DPP-IV, DPP8, DPP-II, and FAP by benzylamine derivatives
O
H
H
N
R
N
Y
N
X X
X1 X2
O
NC
X1
X2
Y
IC50 (lM)
DPP8
a
Compound
R
X
DPP-IVb
DPP-II
FAP
6
H
H
H
H
CH
CH
CH
N
0.452c
0.239
0.190
0.433
0.119c
1.108c
0.911
0.042
10.744c
>20
>20
17.400c
>20
>20
>20
>20
>20
>20
>20
>20
>20
NA
6a
6b
6c
6d
6e
6f
6g
H
H
CH3
CH3
CH3
H
CH3
CH3
CH3
H
3-CF3
H
H
H
>20
>20
H
CH3
CH3
CH3
CH3
CH
CH
CH
CH
8.338c
>20c
>20
>20c
>20c
>20
H
CH3
CH3
H
CH3
H
H
H
CH3
1.473
>20
NA, not available.
a See Ref. 23.
b Values are expressed as the mean of three independent determinations.
c Data from reference 16.
Table 3. Inhibition of DPP-IV, DPP8, DPP-II, and FAP by phenylethylamine derivatives
O
H
H
N
N
N
R
X X
X1 X2
O
Y
NC
X1 = X2
Y
IC50 (lM)
a
Compound
R
X
DPP-IVb
DPP8
DPP-II
FAP
7
H
H
H
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
0.564c
0.144
1.300
0.252
0.236
0.247
0.320
0.232
0.181
0.118
2.592c
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20c
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
CH3
H
4-CH3
4-CF3
3-CF3
2-CF3
3,4-di-OCH3
3,4-di-Cl
H
H
H
H
H
H
H
a See Ref. 23.
b Values are expressed as the mean of three independent determinations.
c
Data from reference 16.
structural optimization of the three lead compounds 5–7
for DPP-IV activity and selectivity, also in vivo efficacy
of selected compounds is reported.
to the carboxylic acid group using freshly generated
KOCl.22 Compound 13 was synthesized in 2 steps start-
ing from L-prolinamide as reported in literature.4 All the
synthesized compounds were analyzed by LC–MS and
1H NMR to confirm their structures.
The general synthetic route for the preparation of three
series of DPP-IV inhibitors (5a–h, 6a–g, and 7a-i) is
shown in Scheme 1. Commercially available anilines,
phenylethylamines or benzylamines (11) were condensed
with N-Boc protected substituted b-alanine 10 using
DCC. Deprotection of 12 using TFA, followed by
reaction of the free amine with 1-(2-bromo acetyl)-
(2S)-cyanopyrrolidine 13 gave the final products. The
b-alanine derivative 10 could be synthesized starting
from commercially available keto amines (8) in two
steps, first by masking the amino functionality with
Boc group, followed by oxidation of the keto function
Final compounds (5a–h, 6a–g, and 7a–i) were assayed
for their ability to inhibit DPP-IV in in vitro and their
results are reported in Tables 1–3 along with their
DPP-II, DPP8, and FAP inhibitory potentials.23 The
lead aniline derivative 5 (Table 1) had a moderate
DPP-IV inhibition (IC50 = 298 nM) and a poor selectiv-
ity index of 3-fold over DPP8 and 19-fold over DPP-II.
Introduction of gem-dimethyl substituent adjacent to
the P2 site amine in 5 gave 5a, which resulted in a slight
enhancement in DPP-IV activity and more than 97-fold

M. S. Coumar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1274–1279
1277
H
N
H
N
(c)
(a)
(b)
NH2
.HCl
HO
O
O
NH2
X1 X2
X1 X2
X1 X2
O
R
O
Y
O
O
O
O
0-2
8
9
10
R
O
11
Y
N
Br
O
NC
H
N
H
R
H
H
N
N
N
O
N
Y
(d), (e)
0-2
0-2
X1 X2
X1 X2
O
O
Y = CH/N
NC
X1 ; X2 = H/CH3
5a-h, 6a-g, 7a-i
12
Scheme 1. Reagents: (a) Et3N, (BOC)2O, 1,4-dioxane; (b) KOCl, 1,4-dioxane/H2O; (c) HOSu, DCC, 1,4-dioxane; (d) CF3COOH; (e) K2CO3, 13,
THF.
selectivity over DPP8, DPP-II, and FAP. The improve-
ment in the selectivity of 5a by the introduction of
gem-dimethyl group was consistent with our previous
study.16
selectivity profile as 6a. But the replacement of the phen-
yl ring with a 2-pyridyl ring resulted in 2-fold loss of
DPP-IV potency in 6c compared to 6a. Thus, we focused
our attention back to the modification of the linker.
Moving the gem-dimethyl substituent of 6a to a position
adjacent to the aromatic ring gave compound 6d with
2-fold improved DPP-IV potency, but with a slight loss
of selectivity (70-fold) over DPP8 compared to 6a (>84-
fold). This prompted us to introduce another set of gem-
dimethyl group, however compound 6e with two sets of
gem-dimethyl group showed a 4.5-fold loss of DPP-IV
potency compared to 6a. Since steric crowding in 6e
may be the reason for the loss of DPP-IV potency, we
thought to introduce a single methyl group adjacent to
P2 site amine instead of the gem-dimethyl group. A dif-
ferential effect on activity was obtained depending on
the stereochemistry of the methyl group introduced.
When a S-methyl group was introduced, a highly potent
DPP-IV inhibitor 6g (IC50 = 42 nM) was obtained, but
it also possessed DPP8 inhibition (IC50 = 1.47 lM).
While, a R-methyl group introduction (6f) led to a 4-fold
loss of DPP-IV potency compared to 6a.
Having attained one of our objectives of improving the
selectivity of 5, we turned our attention to the effect of
aromatic ring substitution. The ring-substituted com-
pounds 5b–5g retained the high degree of selectivity
(>100-fold) toward DPP-IV over DPP8, DPP-II, and
FAP similar to that of 5a. Monosubstituted compounds
with 4-F (5b) and 4-OCH3 (5c) groups showed very sim-
ilar range of DPP-IV inhibition to that of 5a. The disub-
stituted compounds with 3,4-di-F (5d) and 3,4-di-CH3
(5e) also showed similar level of DPP-IV inhibition.
But the 3,4-di-OCH3 compound 5f showed almost
2-fold improvement in DPP-IV activity (IC50 = 116 nM)
compared to 5a. Three other dimethoxy substituted
compounds (2,4-di-OCH3; 2,5-di-OCH3; 3,5-di-OCH3)
were synthesized and tested for DPP-IV inhibitory activ-
ity (data not shown) and found to be less potent than 5f.
As the dimethoxy compound 5f was twice as potent as
the monomethoxy compound 5c, we synthesized 3,4,5-
tri-OCH3 substituted 5g, but the introduction of the
third methoxy group did not show any further improve-
ment in potency. Having found the electronic effects of
substituents, we looked at the steric effect of aromatic
substitution by introducing a bulky phenyl ring at the
4-position. Compound 5h showed a decrease in DPP-
IV potency and also loss of selectivity over DPP-II com-
pared to the unsubstituted 5a. By chemical modification
of the lead 5, we obtained 5f with an improved DPP-IV
potency (3-fold) and better selectivity (57-fold over
DPP8) in comparison to 5.
The last series of compounds were developed from the
lead 7 (Table 3), which showed a moderate DPP-IV inhi-
bition (IC50 = 564 nM) and poor selectivity (4.5-fold)
over DPP8. Introduction of gem-dimethyl substituent
adjacent to P2 site amine similar to that in the previous
two series of compounds resulted in compound 7a,
which showed a 4-fold improvement in DPP-IV inhibi-
tion and an enhancement in selectivity (>140-fold over
DPP8) in comparison to 7. Introduction of additional
set of gem-dimethyl group in the linker portion however
led to a 9-fold loss of DPP-IV potency in 7b in compar-
ison to 7a. As two sets of gem-dimethyl group in the
linker seems to be deleterious for the DPP-IV activity,
we retained one set of gem-dimethyl group adjacent to
P2 site amine and modified the aromatic portion to opti-
mize the activity and selectivity. Introduction of mono
substituents such as 4-CH3 (7c), 4-CF3 (7d), 3-CF3 (7e)
or 2-CF3 (7f) only led to loss of potency in comparison
to the unsubstituted compound 7a. Even a 3,4-di-OCH3
(7g) and 3,4-di-Cl (7h) substituents led to a loss of DPP-
IV potency. However when we replaced the phenyl ring
(7a) with 2-pyridyl ring system (7i), we were able to
improve the DPP-IV potency (IC50 = 118 nM) slightly
Next, we took up lead 6 for optimization, which showed
moderate DPP-IV inhibition (IC50 = 452 nM) and
24-fold selectivity over DPP8 and 38-fold selectivity
over DPP-II (Table 2). Introduction of gem-dimethyl
substituent adjacent to P2 site amine similar to the ani-
line series resulted in compound 6a, which showed
almost 2-fold improvement in DPP-IV inhibition and
an enhancement in selectivity (>84-fold over DPP8
and DPP-II). Modification in the aromatic portion of
the molecule by the introduction of 3-CF3 group result-
ed in 6b with a similar level of DPP-IV inhibition and

1278
M. S. Coumar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1274–1279
with retention of high selectivity (>167-fold) over other
enzymes.
5f
7i
Potent and selective compound 5f (Table 1) from the
aniline series and 7i (Table 3) from phenylethylamine
series were selected for further evaluation based on their
in vitro DPP-IV potency and selectivity profile. As one
of the limiting factor for the development of DPP-IV
inhibitors containing 2(S)-cyanopyrrolidine portion is
their inherent chemical instability due to the formation
of inactive diketopiperazine,6,12 we analyzed an aqueous
solution of 5f and 7i by LC–MS. Both the compounds
were stable over extended periods of time in aqueous
solutions and no formation of cyclized diketopiperazine
was observed even after 2 days.
Control
10
0
0
10
Time (hr)
20
To access the in vivo efficacy of 5f and 7i, ability to
inhibit glucose excursion after oral glucose load in oral
glucose tolerance test24 (OGTT, Fig. 3) and in vivo plas-
ma DPP-IV inhibition assay25 (Fig. 4) were carried out.
For the OGTT, compounds were administered by oral
route at 10 mpk to Wistar rats 30 min before glucose
administration (1.5 g/kg), and then blood samples
drawn and analyzed for glucose levels. Both compounds
5f and 7i significantly suppressed the glucose excursion
observed after glucose load. For the plasma DPP-IV
inhibition assay, the compounds were administered to
Wistar rats by oral route at 10 mpk, blood samples were
collected and analyzed for plasma DPP-IV activity.
Both compounds showed strong plasma DPP-IV inhibi-
tion after oral administration with similar onset of ac-
tion, but 5f is more potent at inhibiting the plasma
DPP-IV level than 7i. Compound 5f inhibited plasma
DPP-IV activity (>80%) within 30 min, and more than
50% inhibition lasted upto 8 h after drug treatment.
The DPP-IV activity returned back to original values
after around 24 h in both 5f- and 7i-treated rats. Sup-
pression of glucose elevation in OGTT and inhibition
of DPP-IV activity in the plasma of drug treated rats
Figure 4. Effects of 5f and 7i on the plasma DPP-IV activity in Wistar
rats. Each compound was orally administered at a single dose of
10 mg/kg to rats at 0 h. Data are expressed as means SEM (n = 4/
group).
are a proof of the oral absorption and downstream
antidiabetic activity of the compounds. Further pharma-
cological evaluation is required to appreciate the antidi-
abetic effect of the compounds.
In summary, three series of compounds (>20 analogs)
were synthesized by structural modification of the lead
compounds 5–7 with improved DPP-IV inhibition
potency and selectivity profile. Selected compounds 5f
and 7i showed in vivo efficacy in two animal models
after oral administration. Compound 5f has the poten-
tial for further development as antidiabetic agent.
Acknowledgment
The study was financially supported by National Health
Research Institutes, Taiwan.
210
Vehicle control
5f
7i
Supplementary data
180
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.12.019.
150
120
90
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23. DPPII, DPP-IV, and DPP8 IC50 determinations were
performed as discussed in ref. 16. The detailed protocol for
the cloning and expression of FAP will be described
elsewhere. Briefly, cDNA of FAP was cloned into pBac-
PAC8-CD5 vector (ref: Chien et al., J. Biol. Chem. 2004,
279, 52338). The expression and purification of FAP was
carried out essentially as described in ref Chen et al.,
Protein Expr. Purif. 2004, 35, 142. The substrates used for
FAP assay was Gly-Pro-pNA at 6 mM concentration.
24. OGTT was performed on overnight fasted adult male
Wistar rats and the blood glucose was measured with the
Accu-Chek Compact System from Roche (Basel, Switzer-
land). The animals were orally gavaged with the test
compounds dissolved in distilled water at a 10 mpk dose.
Thirty minutes after the oral dosing of test compounds,
the animals were orally gavaged with freshly prepared
glucose solution of 400 mg/ml in distilled water at 1.5 g
glucose/kg. Blood glucose levels of these dosed animals
were monitored at 0, 15, 30, 60, and 120 min after the oral
glucose challenge.
25. The in vivo DPP-IV inhibition assay was performed as
discussed in ref. 16. Adult male rats were orally gavaged
with the test compounds at a single dose of 10 mg/kg. The
plasma DPP-IV activity was determined by cleavage rate of
Gly-Pro-AMC (H-glycyl-prolyl-7-amino-4-methylcouma-
rin; BACHEM).