Novel trans-2-aryl-cyclopropylamine analogues as potent and selective dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.02.020

A series of trans-2-aryl-cyclopropylamine derived compounds were synthesized and evaluated their biological activities against DPP-IV. The structure-activity relationships (SAR) led to the discovery of novel series of DPP-IV inhibitors, having IC₅₀ values of <100 nM with excellent selectivity over the closely related enzymes, DPP8, DPP-II and FAP. The studies identified a potent and selective DPP-IV inhibitor 24b, which exhibited the ability to both significantly inhibit plasma DPP-IV activity in rats and improve glucose tolerance in lean mice and diet induced obese mice.

Full text of DOI:10.1016/j.bmc.2009.02.020

Bioorganic & Medicinal Chemistry 17 (2009) 2388–2399
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel trans-2-aryl-cyclopropylamine analogues as potent and selective
dipeptidyl peptidase IV inhibitors
Ting-Yueh Tsai, Tsu Hsu, Chiung-Tong Chen, Jai-Hong Cheng, Teng-Kuang Yeh, Xin Chen, Chung-Yu Huang,
Chung-Nien Chang, Kai-Chia Yeh, Su-Huei Hsieh, Chia-Hui Chien, Yi-Wei Chang, Chih-Hsiang Huang,
Yu-Wen Huang, Chen-Lung Huang, Ssu-Hui Wu, Min-Hsien Wang, Cheng-Tai Lu, Yu-Sheng Chao,
*
Weir-Torn Jiaang
Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, No. 35, Keyan Rd., Zhunan Town, Miaoli Country 350, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of trans-2-aryl-cyclopropylamine derived compounds were synthesized and evaluated their bio-
logical activities against DPP-IV. The structure-activity relationships (SAR) led to the discovery of novel
series of DPP-IV inhibitors, having IC50 values of <100 nM with excellent selectivity over the closely
related enzymes, DPP8, DPP-II and FAP. The studies identified a potent and selective DPP-IV inhibitor
24b, which exhibited the ability to both significantly inhibit plasma DPP-IV activity in rats and improve
glucose tolerance in lean mice and diet induced obese mice.
Received 27 November 2008
Revised 4 February 2009
Accepted 5 February 2009
Available online 20 February 2009
Keywords:
Dipeptidyl peptidase IV
Inhibitor
Ó 2009 Elsevier Ltd. All rights reserved.
trans-2-Aryl-cyclopropylamine
1. Introduction
F
OH
F
NH2
O
Dipeptidyl peptidase IV (DPP-IV, also known as CD26)
(EC 3.4.14.5) is a drug target for type II diabetes. It is a prolyl dipep-
tidase involved in the in vivo degradation of two insulin-sensing
hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent
insulinotropic polypeptide (GIP), by cleaving at the peptide bond of
the penultimate position.1,2 The incretin hormone GLP-1 stimu-
lates insulin biosynthesis and secretion, inhibits glucagon release
and slows gastric emptying; each a benefit in the control of glucose
homeostasis.3–7 DPP-IV knockout mice and rats have consistently
displayed healthy phenotypes.8–10 When challenged with a high
concentration of glucose, these knockout animals show improved
glucose tolerance, enhanced insulin secretion and increased circu-
lating active GLP-1 peptide.8–10 Small-molecular inhibitors of DPP-
IV have been shown to prolong the beneficial effects of this incretin
hormone in animal models.5 Antidiabetic efficacy has been demon-
strated clinically with DPP-IV inhibitors; sitagliptin 1 (MK-0431)
was approved by the US Food and Drug Administration for the
treatment of type 2 diabetes,11 and vildagliptin 2 (LAF237) was ap-
proved for use in the European market (Fig. 1).12
N
N
N
N
N
H
F
N
O
CN
CF3
Sitagliptin 1
Vildagliptin 2
Figure 1. DPP-IV inhibitors.
antidiabetic agents, since inhibitionof DPP-II results in the apoptosis
of quiescent T-cells13 and also recent in vivo studies indicate that
selective inhibition of DPP8/9 may be associated with profound tox-
icities.14 The toxic effects of DPP8/9 inhibitors identified thus far in
preclinical studies have included alopecia, thrombocytopenia, ane-
mia, enlarged spleen, multiple histological pathologies, and animal
mortality shown in rats.14 Reports suggest that fibroblast activation
protein (FAP) is involved in tumor growth and invasion.15
With few exceptions, most DPP-IV inhibitors resemble the P2-
P1 dipeptidyl substrate cleavage product. As shown in Figure 1,
inhibitor 2 is a covalent inhibitor. Often this electrophilic cyano-
pyrrolidine is able to bind covalently with the serine 630 in the
S1 pocket of DPP-IV,12 whereas the non-covalent inhibitor 1 de-
pends on non-covalent protein–ligand interactions and the substi-
tuted phenyl group occupies very well the hydrophobic S1 pocket
of the enzyme.11 We are involved in the design and synthesis of
Selectivity of the DPP-IV inhibitors for closely related prolyl pep-
tidases such as DPP8/9, DPP-II and FAP (Fibroblast activation pro-
tein) is an important criterion for their further development as
* Corresponding author. Tel.: +886 37 246166x35712; fax: +886 37 586456.
E-mail address: wtjiaang@nhri.org.tw (W.-T. Jiaang).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.02.020

T.-Y. Tsai et al. / Bioorg. Med. Chem. 17 (2009) 2388–2399
2389
1
R
Cl
R
R
2
1
N
NH
NH
O
F
N
O
N
O
N
H
N
O
CN
N
H
N
H
O
CN
O
CN
I
24b
18
Figure 2. Design of phenylcyclopropylamine derivatives as DPP-IV inhibitors.
DPP-IV inhibitors and have reported potent and selective DPP-IV
inhibitors.16–18 Previous report described 2-[3-[[2-[(2S)-2-cyano-
1-pyrrolidinyl]-2-oxoethyl]amino]-1-oxopropyl]-based (structure I)
DPP-IV inhibitors (Fig. 2), the structure–activity relationships
(SAR) of several series of structure I-based DPP-IV inhibitors were
investigated, and these studies successfully identified potent,
selective and orally available DPP-IV inhibitor with excellent
chemical stability.16 However, selected potent compounds in those
series of structure I-based DPP-IV inhibitors suffered from low oral
bioavailability (data not shown). In this paper, we described our
extensive SAR studies regarding structure I-based derivatives to
develop long-acting DPP-IV inhibitors with improved pharmacoki-
netic profile, we here report on the discovery of a novel series of 2-
phenyl-cyclopropylamine derivatives 18 as DPP-IV inhibitors. To
further improve the potency of this series of compounds, introduc-
tion of fluoro substitution at the 4-position of 2-cyanopyrrolidine
led to the discovery of enantiopure compound 24b, which was a
potent DPP-IV inhibitor (IC50 = 15 nM) with excellent selectivity
over DPP-II, DPP8 and FAP (>1300-fold) (Fig. 2). This compound
was selected for extensive in vivo evaluation and demonstrated
in vivo efficacy better than that of DPP-IV inhibitor 1. In addition,
pharmacokinetic studies are also presented.
1. The synthesis commonly began with commercially available
trans arylated acrylic acid 3 as a starting material. Acid 3 was con-
verted to its methyl ester followed by cyclopropanation of E al-
kenes employing diazomethane to afford trans-2-phenyl-
cyclopropanecarboxylic acid methyl ester 4. Ester 4 was saponified
to yield the corresponding racemic acids 5. To further our under-
standing of the SAR for hydrophilic compound, 2-(4-methyl-thia-
zol-5-yl)-cyclopropylamine was prepared. Thermal reaction of 4-
methyl-thiazole 6 and ethyl diazoacetate 7 according to the meth-
od of Ivanskii and Maksimov afforded the racemate 2-(4-methyl-
thiazol-5-yl)-cyclopropanecarboxylic acid ethyl esters trans-8 and
cis-9,19 trans/cis isomers could be separated by flash column in a
modest yield and approximately a 2:1 ratio. Alkaline hydrolysis
gave the desired trans-2-aryl-cyclopropanecarboxylic acid 5. These
racemic acids 5 were transformed to the corresponding Boc-pro-
tected amines by Curtius rearrangement and then removal of the
Boc group yielded racemic 2-aryl-cyclopropylamine 10.
Enantiopure amines (1R,2S)-14 and (1S,2R)-14 were synthesized
following the strategy illustrated in Scheme 2. Activation of the acid
3 with thionyl chloride followed by addition of lithiated (4R)-4-phe-
nyl-2-oxazolidinone chiral auxiliary gave 11. Cyclopropanation of E
alkenes 11 employing diazomethane provided a separable mixture
of diastereomeric cycloadducts trans-12 in a 6:4 ratio. The chiral
auxiliary of separated enantiopure 12 was removed by base hydro-
lysis to yield the corresponding enantiomeric acids 13. To determine
the absolute configuration of two isomers 13, more polar fraction
compound 13a (R1 = H) was converted into the trans-phenyl-cyclo-
2. Chemistry
The general synthetic route for the preparation of racemic trans-
2-aryl-cyclopropylamine derivatives 10 was illustrated in Scheme
X
R1
Ar
Ar
X
R1
b
d, e
a
HCl
NH2
OH
O
OH
O
10 (racemate)
5
O
O
3 (X = C or N)
4
b
N
S
N
S
c
O
+
N
OEt
+
OEt
N2
EtO
S
O
O
8
7
9
6
less polar fraction
Scheme 1. Reagents and conditions: (a) (i) CH2N2, CH2Cl2, (ii) Pd(OAc)2, CH2N2, CH2Cl2/Et2O (3:1), 0 °C, 1 h; (b) NaOH, MeOH/H2O; (c) neat, 135 °C; (d) DPPA, (Et)3N, t-BuOH,
90 °C, 8 h; (e) (i) TFA, rt, 0.5 h, (ii) HCl 1.0 M in diethylether.

2390
T.-Y. Tsai et al. / Bioorg. Med. Chem. 17 (2009) 2388–2399
1
R
1
R
1
R
b
a
O
O
N
N
OH
O
O
O
O
O
12a, b
3
11
Two diastereomers
seperation
c
1
1
R
R
1
1
R
R
R
S
S
d, e
R
S
HCl
2
HCl
OH
OH
NH
NH
2
R
S
R
O
O
(1S,2R)-10a
(1R,2S)-10b
(1R,2R)-13b
(1S,2S)-13a
Less polar
fraction
More polar
fraction
f
1
R
O
O
14
R1 = H
[α]D20 = 264.1 (c 0.46, CHCl3)
Scheme 2. Reagents and conditions: (a) (i) SOCl2, 55 °C, 4 h, (ii) (4R)-4-phenyl-2-oxazolidinone, NaH, THF, 0 °C?rt, 12 h; (b) CH2N2, Pd(OAc)2, CH2Cl2/Et2O (3:1), 0 °C, 1 h; (c)
LiOH, H2O2, THF/H2O (5:1), ꢁ5 °C?0 °C, 2 h; (d) DPPA, (Et)3N, t-BuOH, 90 °C, 8 h; (e) (i) TFA, rt, 0.5 h, (ii) HCl 1.0 M in diethylether; (f) CH2N2, CH2Cl2.
propanecarboxylic acid methyl ester 14 by esterification with diazo-
methane. When a comparative study of the specific rotation of ester
14 was performed, it was observed that specific rotation of com-
pound 14 is 264.1 (c 0.46, CHCl3) as literature value for enantiopure
(1S,2S)-14.20 Enantiopure (1S,2S)-13 and (1R,2R)-13 were trans-
formed to the corresponding Boc-protected amines by Curtius rear-
rangement and then removal of the Boc group yielded primary
amines (1S,2R)-10 and (1R,2S)-10, respectively.
A series of trans-2-aryl-cyclopropylamine derivatives as DPP-IV
inhibitors, compounds 18–24 were prepared as described in
Scheme 3 and are listed in Tables 1–3. 3-Boc-amino-3-methylbu-
tyric acid 1516 was EDC-coupled with various cyclopropyl amines
to give 16. Removal of the N-Boc group of 16 with trifluoroacetic
acid followed by coupling with 1-bromoacetyl-2-cyano-(S)-pyrrol-
idine derivatives 17 to provide the desired 2-cyanopyrrolidine ana-
logues 18–24. The synthesis of bromo compounds 17 were carried
out according to the literature procedure.21
line-specific enzymes DPP8/9, DPP-II and FAP. To speedily under-
stand the effect of substituent at the phenyl ring attached on
cyclopropylamine, easily prepared diastereomeric mixture 18
was firstly evaluated. As shown in Table 1, the lead compound,
the unsubstituted 2-phenyl-cyclopropylamine 18a, is a potent
DPP-IV inhibitor with an IC50 value of 85 nM. However, substitu-
tion at the phenyl ring had little effect on DPP-IV activity and selec-
tivity; compounds 18b-m exhibited high potency (IC50 6 100 nM)
against DPP-IV, with excellent selectivity versus the other pepti-
dases (IC50 > 20
lM) except that 18a had weak inhibition against
DPP-II (IC50 ꢀ 16
l
M). The most potent compound in Table 1 is
3-chloro substituted 18k, which has an IC50 value of 42 nM for
DPP-IV inhibition.
Since diastereomeric mixture 3-chloro 18k is most potent com-
pound in Table 1, we investigated the activity and selectivity of the
two diastereomers (1R,2S)-21 and (1S,2R)-22 of compound 18k
(Table 2). Additionally, to further improve the potency of com-
pounds 21 and 22, introduction of fluoro substituent at the 4-posi-
tion of 2-cyanopyrrolidine (S form) at the P1 site provided the
diastereomers (1R,2S)-23 and (1S,2R)-24,22 and evaluated fluoro ef-
fects on both potency and selectivity. As shown in Table 2, the chi-
ral centers at C1 and C2 of trans-2-phenylcyclopropylamines had
3. Biological evaluation
The DPP-IV inhibitory activity and selectivity of analogues in
the present series were assayed against human DPP-IV and pro-

T.-Y. Tsai et al. / Bioorg. Med. Chem. 17 (2009) 2388–2399
2391
R2
Ar
O
O
a
HO
Ar
+
N
H
N
+
Boc
Br
HCl
N
H
CN
NH
NH2
O
Boc
15
10
17
16
b, c
R1
R1
R1
N
N
S
R
S
S
NH
R
NH
NH
NH
R2
NH
R2
F
F
O
O
O
O
O
N
N
N
N
N
N
H
N
H
N
H
N
H
N
H
O
CN
CN
CN
O
CN
O
O
O
CN
R2 = H
R2 = F
18
19
20
(1R,2S)-21
(1R,2S)-23
(1S,2R)-22
1S,2R -24
Diastereomeric mixture Diastereomeric mixture Diastereomeric mixture
Scheme 3. Reagents and conditions: (a) (i) HOBT, EDC, (Et)3N, CH2Cl2/1,4-dioxane, rt, 18 h; (b) (i) TFA, rt, 0.5 h, (ii) K2CO3, THF, rt, 1 h; (c) THF, rt, 18 h.
Table 1
Effects of substituent at the R1 position
Table 2
Inhibitory properties of 2-phenyl-cyclopropylamine isomers
1
Cl
Cl
R
S
R
S
NH
R
NH
NH
R2
R2
O
O
O
N
N
N
N
N
H
N
H
H
O
CN
O
CN
O
CN
R2 = H
R2 = F
18
(1R,2S)-21
(1R,2S)-23
(1S,2R)-22
Diastereomeric mixture
(1S,2R)-24b
Compd
R
IC50 (lM)
Compd
IC50 (lM)
DPP-IV
DPP8
DPP-II
FAP
DPP-IV
DPP8
DPP-II
FAP
18a
18b
18c
18d
18e
18f
18g
18h
18i
H
4-F
3-F
2,4-diF
3,5-diF
3,4-diF
4-CF3
3-CF3
3-OCF3
4-Cl
0.085
0.054
0.064
0.084
0.075
0.062
0.085
0.060
0.073
0.086
0.042
0.068
0.101
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
16
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
21
22
23
24b
0.056
0.047
0.027
0.015
>20
>20
11
17
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
5.2
>20
>20
dine derivatives 23 and 24b exhibited twofold and threefold better
DPP-IV inhibitory activity than 4-unsubstituted 21 and 22, respec-
tively. In view of these facts, we focused our efforts on studying the
(1S,2R) series of compounds with (4S)-4-fluoro-2-cyanopyrrolidine
in the P1 site.
18j
18k
18l
18m
3-Cl
3,4-Cl
3,4-OCH3
The potent and selective ((1S,2R) isomer 24b was then elabo-
rated further by modifying the substitution at the phenyl ring. As
we can see in Table 1, a similar SAR trend was observed in Table
3. The unsubstituted compound 24a, 3-fluoro substituted 24c
and the alkoxy substituted compounds 24d–g have very closely
inhibitory activities of DPP-IV compared to lead 24b. Compound
24a–g also exhibited similar selectivity profiles except for 24c
and 24f; compound 24c showed moderate inhibition against
little effect on DPP-IV inhibition (21 vs 22 and 23 vs 24b). While
(1R,2S) configuration was found to be detrimental to selectivity;
(1R,2S)-21 had weak inhibition against DPP-II (IC50 > 10
(4S)-4-fluoro-2-cyanopyrrolidine (1R,2S)-23 exhibited low micro-
molar activity against DPP-II (IC50 = 5.2 M) and weak inhibition
against DPP8 (IC50 = 11 M). As expected, 4-fluoro-2-cyanopyrroli-
lM),
l
l

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T.-Y. Tsai et al. / Bioorg. Med. Chem. 17 (2009) 2388–2399
Table 3
Inhibitory properties of selected DPP-IV inhibitors
Cl
R
N
N
R
R
S
S
NH
NH
NH
NH
O
S
F
F
F
F
F
O
O
O
N
N
N
N
N
N
N
N
H
H
H
H
CN
CN
CN
O
CN
O
O
O
19
20
(1S,2R)-25
(1S,2R)-24
Diastereomeric mixture Diastereomeric mixture
Compd
R
IC50 (lM)
DPP-IV
DPP8
DPP-II
FAP
24a
24b
24c
24d
24e
24f
24g
19
20
25
1
2
H
3-Cl
3-F
4-OMe
3-OMe
3-OEt
3-OCF3
0.019
0.015
0.021
0.022
0.026
0.017
0.020
0.024
0.016
0.026
0.030
0.051
>20
>20
8.6
>20
>20
>20
>20
>20
>20
12
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
13
>20
>20
>20
0.59
>20
>20
>20
14
DPP8 (IC50 = 8.6
toward FAP (IC50 = 13
l
M) and compounds 24f showed weak inhibition
ance is very high (189 mL/min/kg). Selected compounds 24b and
25 did not block the hERG channel at a test concentration of 10
lM). Since Gao et al. reported that heteroat-
oms or polar substituents at the P2 site would help to increase the
DPP8 IC50/DPP-IV IC50 ratio, namely, to provide more selective
DPP-IV inhibitors.23 Alternative approach to improve potency and
selectivity is replacement of the phenyl group present in cyclopro-
pylamine with a polar heterocyclic ring. Two analogues with a pyr-
idine moiety 19 and thiazole moiety 20 were prepared and
evaluated their potency and selectivity. Both of them had potency
toward DPP-IV similar to 3-chlorophenyl analogue 24b; they did
exhibit relatively high selectivity for inhibition of DPP-IV over
l
M (Table 4).
Compound 24b was selected for further in vivo evaluation
based on its potency, off-target selectivity, pharmacokinetic profile
and lack of binding affinity to the hERG channel. Although hetero-
cyclic compound 20 is a diastereomeric mixture, it is equipotent
with 24b as DPP-IV inhibitor and shows excellent selectivity over
DPP8; and further, compounds 20 is still very potent DPP-IV inhib-
itor at 50% human and rat serum, IC50 = 10 nM and 15 nM, respec-
tively. For these reasons, compound 20 was chosen for more
extensive studies. Firstly, compounds 20 and 24b were assessed
for their ability to inhibit plasma DPP-IV activity in rats (Fig. 3).
For the plasma DPP-IV inhibition assay, the compounds were
administered to Wistar rats by oral route at 10 mg/kg, blood sam-
ples were collected and analyzed for plasma DPP-IV activity. Com-
pound 20 showed moderate plasma DPP-IV inhibition (60%) within
30 min after oral administration and provided only 30% inhibition
of DPP-IV activity after 8 h. Compound 24b is more potent and
longer action at inhibiting the plasma DPP-IV level than 20, com-
pound 24b inhibited plasma DPP-IV activity (>85%) within
30 min, and more than 70% inhibition of DPP-IV activity lasted
for 8 h after oral dosing. The inhibition achieved by 24b was more
potent than inhibitor 1, which is the first and only DPP-IV inhibitor
available in US for the treatment of type 2 diabetes. The DPP-IV
activity returned back to original values after around 24 h in both
24b- and 1-treated rats. 2-(4-Methyl-thiazol-5-yl)-cyclopropyl-
amine analogue 20 showed unexpectedly less potent plasma
DPP-IV inhibition in rats relative to in vitro potency (IC50 = 15
nM at 50% rat serum). The disconnection between in vivo efficacy
and in vitro potency was most likely due to the decreased oral
absorption for the increase in hydrophilicity.
DPP8 (17% and 23% inhibition at 20
lM for compounds 19 and
20, respectively). Both 19 and 20 represents the most selective
DPP-IV inhibitor among the trans-2-aryl-cyclopropylamine series
of compounds. Interestingly, compound 25 with 3,3-difluoropyrr-
olidine in the P1 site exhibited submicromolar potency against
DPP-II (IC50 = 588 nM). Generally, this series of inhibitors with
3,3-difluoropyrrolidine in the P1 site significantly inhibited DPP-
II activity (data not shown). Each of the analogues shown in Table
3 was comparable to first-generation DPP-IV inhibitors 1 and 2 in
terms of intrinsic potency and off-target selectivity profile.
Inhibitors that possessed excellent potency and selectivity pro-
files were chosen for extensive pharmacokinetic screening in rats,
and the data are summarized in Table 4. 3-Chlorophenyl derivative
24b displayed excellent half-life (4.8 h), moderate oral bioavail-
ability (42%) and acceptable clearance (22 mL/min/kg). Replacing
3-chloro group with 3-fluoro group gave compound 24c, which
showed faster clearance (55 mL/min/kg) and shorter half-life (t1/2
=
1.2 h) compared to 24b. The 4-OMe substituted analogue 24d
exhibited similar clearance and half-life to 24b. Both compounds
24c and 24d displayed moderate oral bioavailability (F = 28% and
29%, respectively). Unlike 4-OMe analogue 24d, 3-OMe analogue
24e had very high clearance (124 mL/min/kg). Nevertheless, 24e
exhibited excellent half-life (t1/2 = 6.8 h) and in excess of theoret-
ical 100% for oral bioavailability. In comparison with 3-chloro-
phenyl 24b, analogue 25 with 3,3-difluoropyrrolidine in the P1
site showed comparable half-life and oral bioavailability, but clear-
Compound 24b was chosen for further evaluation of its ability
to improve glucose tolerance in C57BL/6j mice. In an oral glucose
tolerance test (OGTT),16 when compound 24b was administered
by oral route to C57BL/6j mice 30 min before glucose administra-
tion (3 g/kg), and then blood samples drawn and analyzed for glu-

T.-Y. Tsai et al. / Bioorg. Med. Chem. 17 (2009) 2388–2399
2393
Table 4
Pharmacokinetic properties of selected DPP-IV inhibitors in ratsa and hERG binding
Compd
CLP (mL/min/kg)
PO Cmax (ng/mL)
T1/2 (po, h)
PO AUCnom (ng/mL h)
F (%)
hERGb,c (%)
24b
24c
24d
24e
25
22
55
20
124
189
621
175
347
898
454
4.8
1.2
5.3
6.8
3.2
1905
449
1179
1345
1195
42
29
28
194
53
11
ND
ND
ND
15
a
24b, 24c and 24e dosed at 5/10 mg/kg iv/po. 24d and 25 dosed at 5/25 mg/kg iv/po.
b
c
Inhibition at a test concentration of 10
ND: not determined.
lM.
120
100
80
60
40
20
0
1
20
24b
Control
0
5
10
Time (h)
15
20
25
Figure 3. Effects of 1, 20 and 24b on the plasma DPP-IV activity in rats. Each
compound was orally administered at a single dose of 10 mg/kg to rats at 0 h. Data
are expressed as mean SEM (n = 4–6/group).
cose levels. The glucose AUC was determined from 0 to 120 min,
and OGTT data on 24b were summarized in Figure 4. The results
shown in Figure 4 demonstrated that 24b significantly reduced
the blood glucose excursion in a dose-dependent manner from 1
mg/kg (50% reduction) to 10 mg/kg (73% reduction). The potency
of 24b on reduction of plasma glucose excursion during OGTT
was almost equal to that of compound 1 at the dose of 3 mg/kg
in mice (data not shown). Blood glucose lowering was also demon-
strated in diet-induced obese (DIO) mice. In comparison to the con-
trol, glucose AUC as measured by the area under the curve was
significantly reduced at all three doses from 1 mg/kg (72% reduc-
tion), 3 mg/kg (98% reduction) to 10 mg/kg (83% reduction) (Fig. 5).
Compound 24b is a potent, selective and orally active inhibitor
of DPP-IV; besides, 24b also possesses an excellent safety pharma-
cology profile. Additional in vitro profiling of 24b in an extensive
panel of 167 enzyme assays showed no significant inhibition at a
Figure 4. (a) Effects of 24b (1, 3 and 10 mg/kg) on the glucose excursion levels of
C57BL/6j mice during oral glucose tolerance test. All rats received 3 g/kg glucose
orally at 0 min. Each of compounds was orally administered to mice at ꢁ30 min. (b)
The suppression of glucose AUC was calculated from
0 to 120 min. Data are
test concentration of 10
lM except endothelin converting en-
represented as mean SEM (n = 6/group).
zyme-1 (ECE-1, 53% inhibition at 10
l
M). Based on the data pre-
sented above, compound 24b is a better candidate for further
evaluation.
DPP-IV activity in rats and suppression of blood glucose elevation
in mice, were also demonstrated. The result of in vivo DPP-IV inhi-
bition indicates that 24b offers longer duration of action than the
inhibitor 1.
4. Conclusion
This report describes the SAR studies of optimizing 2-[3-[[2-
[(2S)-2-cyano-1-pyrrolidinyl]-2-oxoethyl]amino]-1-oxopropyl]-
based DPP-IV inhibitors by introducing the trans-2-aryl-cyclopro-
pylamine building blocks. This approach led to develop a series
of potent and selective inhibitors 18–24. Notable among these is
compound 24b, which is a 15 nM DPP-IV inhibitor with high selec-
5. Experimental
5.1. Chemistry
All commercial chemicals and solvents are reagent grade and
were used without further treatment unless otherwise noted. 1H
NMR spectra were obtained with a Varian Mercury-300 or a Varian
Mercury-400 spectrometer. Chemical shifts were recorded in parts
tivity over DPP-II, DPP8 and FAP (>20
lM). Compound 24b also
possessed good pharmacokinetic profiles in rats. In addition, the
in vivo effects of compound 24b, including inhibition of plasma

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T.-Y. Tsai et al. / Bioorg. Med. Chem. 17 (2009) 2388–2399
acetate (0.01 mmol) in CH2Cl2/ether (30/10 mL) kept at 0 °C. After
the addition of diazomethane ethereal solution was complete, the
reaction was stirred at room temperature for 1 h. The excess diazo-
methane was quenched by dropwise addition of acetic acid. The
mixture was diluted with CH2Cl2, washed sequentially with satu-
rated sodium bicarbonate and brine, and then dried over MgSO4.
The organic layer was concentrated and purified by chromatogra-
phy using EtOAc/hexane (1:9) as an eluant to give the desired
product 4 (>70%) as a colorless oil. Only the data of representative
compounds are showed.
5.2.1. Methyl trans-2-(3-chlorophenyl)cyclopropanecarbox-
ylate (4, X = C, R1 = 3-Cl)
1H NMR (300 MHz, CDCl3) d 7.22–7.15 (m, 2H), 7.06 (t, J = 1.8
Hz, 1H), 6.98 (dt, J = 6.3, 1.8 Hz, 1H), 3.71 (s, 3H), 2.49 (ddd, J =
4.5, 6.3, 9.3 Hz, 1H), 1.90 (ddd, J = 4.5, 5.4, 8.4 Hz, 1H), 1.65–1.57
(m, 1H), 1.30 (ddd, J = 4.5, 6.3, 8.4 Hz, 1H). MS (ES+) m/z calcd for
C11H11ClO2: 210.66; found: 211.1 (M+H), 233.0 (M+Na).
5.2.2. Methyl trans-2-(pyridin-3-yl)cyclopropanecarboxylate
(4, X = N and R1 = H)
The title compound was prepared from corresponding com-
pound 3 in 78% yield as a colorless oil and used EtOAc/hexane
(4:6) as an eluant. 1H NMR (300 MHz, CDCl3) d 8.44 (d, J = 1.8
Hz, 0.6H), 8.43 (d, J = 1.8 Hz, 1.4H), 7.35 (t, J = 1.8 Hz, 0.45H),
7.32 (t, J = 1.8 Hz, 0.55H), 7.19 (d, J = 5.1 Hz, 0.55H), 7.16 (d, J =
5.1 Hz, 0.45H), 3.71 (s, 3H), 2.51 (ddd, J = 4.5, 6.6, 9.3 Hz, 1H),
1.91 (ddd, J = 4.5, 5.4, 8.4 Hz, 1H), 1.68 – 1.59 (m, 1H), 1.32 (ddd,
J = 4.5, 6.6, 8.4 Hz, 1H). MS (ES+) m/z calcd for C10H11NO2:
177.20; found: 178.1 (M+H).
5.3. Ethyl trans-2-(4-methyl-1,3-thiazol-5-yl)cyclopro-
panecarboxylate (8) and ethyl cis-2-(4-methyl-1,3-thiazol-5-
yl)cyclopropanecarboxylate (9)
Figure 5. (a) Effects of 24b (1, 3 and 10 mg/kg) on the glucose excursion level of
DIO mice during oral glucose tolerance test. All rats received 3.0 g/kg glucose orally
at 0 min. Each of compounds was orally administered to mice at ꢁ30 min. (b) The
suppression of glucose AUC was calculated from 0 to 120 min. Data are represented
as mean SEM (n = 6/group).
5-Ethenyl-4-methyl-1,3-thiazole 6 (2.5 g, 20 mmol) was stirred
at 135 °C in an oil bath as ethyl diazoacetate 7 (1.14 g, 10 mmol)
was added portionwise over a period of 2 h. After cooling, the mix-
ture was by flash chromatography (silica gel, eluting with 15:85
EtOAc/hexanes) to give the less polar product 8 (1.29 g, 61%) as a
colorless oil. 1H NMR (300 MHz, CDCl3) d 8.49 (s, 1H), 3.96 (q, J =
7.2 Hz, 2H), 2.45–2.37 (m, 4H, overlapped singlet at 2.40), 2.19–
2.11 (m, 1H), 1.61–1.45 (m, 2H), 1.07 (t, J = 7.2 Hz, 3H). MS (ES+)
m/z calcd for C10H13NO2S: 211.07; found: 212.1 (M+H), 234.1
(M+Na). The more polar product 9 (0.55 g, 26%) as a pale yellow
oil. 1H NMR (300 MHz, CDCl3) d 8.42 (s, 1H), 4.12 (q, J = 7.2 Hz,
2H), 2.50 (ddd, J = 4.2, 6.0, 9.0 Hz, 1H), 2.39 (s, 3H), 1.81 (ddd, J =
4.2, 5.1, 8.4 Hz, 1H), 1.64–1.58 (m, 1H), 1.23 (t, J = 7.2 Hz, 3H),
1.13 (ddd, J = 4.2, 6.0, 8.4 Hz, 1H). MS (ES+) m/z calcd for
C10H13NO2S: 211.07; found: 212.1 (M+H), 234.1 (M+Na).
per million (ppm, d) and were reported relative to the solvent peak
or TMS. LC/MS data were measured on an Agilent MSD-1100 ESI-
MS/MS System. Flash column chromatography was done using sil-
ica gel (Merck Kieselgel 60, No. 9385, 230–400 mesh ASTM). Reac-
tions were monitored by TLC using Merck 60 F254 silica gel glass
backed plates (5 ꢂ 10 cm); zones were detected visually under
ultraviolet irradiation (254 nm) or by spraying with phosphomo-
lybdic acid reagent (Aldrich) followed by heating at 80 °C. All start-
ing materials and amines were commercially available unless
otherwise indicated.
5.2. General procedure for the preparation of compound 4
5.4. General procedure for the preparation of compound 5
The ethereal solution of diazomethane (10 mmol, prepared by a
Aldrich Mini Dazald apparatus) was continuously added into a stir-
red solution of 3 (2.0 mmol) in CH2Cl2/ether (10/5 mL) kept at 0 °C.
After the addition of diazomethane ethereal solution was com-
plete, the reaction was stirred at room temperature for 1 h. The ex-
cess diazomethane was quenched by dropwise addition of acetic
acid. The mixture was diluted with CH2Cl2, washed sequentially
with saturated sodium bicarbonate and brine, and then dried over
MgSO4. The organic layer was concentrated and purified by chro-
matography to give the desired ester, which was used without fur-
ther purification. The ethereal solution of diazomethane (ꢀ16
mmol, prepared by a Aldrich Mini Dazald apparatus) was continu-
ously added into a stirred solution of above ester and palladium(II)
A solution of compound 4 or 8 (10 mmol) in methanol (30 mL)
was added 4 N NaOH aqueous solution (5 mL). The reaction was stir-
red at room temperature for 4 h. The resulting solution was neutral-
ized by 1 N HCl aqueous solution and concentrated in vacuo. The
crude product was added ethyl acetate (100 mL) and washed by
1 N HCl aqueous solution (100 mL). The organic layer was dried over
MgSO4 and concentratedin vacuo to give thecrude acid 5, whichwas
used for the next reaction without further purification.
5.5. General procedure for the preparation of compound 11
A solution of cinnamic acid 3 (10.0 mmol) in thionyl chloride
(4.76 g, 40.0 mmol) was stirred at 55 °C under nitrogen for 4 h.

T.-Y. Tsai et al. / Bioorg. Med. Chem. 17 (2009) 2388–2399
2395
The volatiles were removed under reduced pressure to give the de-
sired acid chloride, which was used without further purification. To
a solution of sodium hydride (400 mg, 10.0 mmol, 60% dispersion
in oil) in THF (10 mL) was added a solution of (R)-4-phenyl-2-oxa-
zolidinone (1.63 g, 10.0 mmol) in THF (15 mL) at 0 °C. The mixture
was stirred at room temperature for 1 h and the above acid chlo-
ride was added at 0 °C. The mixture was stirred at 0 °C for 1 h
and then at room temperature for additional 10 h. The reaction
was quenched with 2 N HCl at 0 °C, extracted with CH2Cl2, dried
over MgSO4, and concentrated in vacuo. The residue was purified
by silica gel column chromatography with EtOAc/hexanes (1:9)
to yield 11 (80–90%) as a white solid. Only the data of representa-
tive compound is showed.
aqueous lithium hydroxide (1 N, 4 mL) within a period of 1 h by
a syringe pump. The reaction was stirred at 0 °C for 2 h and a solu-
tion of aqueous sodium sulfite (1.5 N, 10 mL) was added. After THF
was evaporated in vacuo, the residue was partitioned between
CH2Cl2 and 1 N hydrochloric acid. The organic phase was dried over
MgSO4 and concentrated in vacuo to give 13 (67%) as a white solid,
which was used for the next reaction without further purification.
To a solution of (1S,2R)-13a, (1R,2S)-13b or racemic form 5 (5
mmol) in dry tert-butyl alcohol (10 mL) was added diphenylphos-
phoryl azide (1.38 g, 5 mmol) and triethylamine (0.60 g, 6 mmol).
The mixture was stirred at 90 °C for 8 h. After concentration, the
mixture was diluted with CH2Cl2, washed sequentially with satu-
rated sodium bicarbonate and brine, and then dried over MgSO4.
The organic layer was concentrated and purified by flash chroma-
tography (silica gel, eluting with 15:85 EtOAc/hexanes for phenyl
series of analogues, and 4:6 EtOAc/hexanes for heterocyclic series
of analogues) to give N-BOC protected compound 10 (45–60%) as
a white solid. To a stirred solution of above N-BOC protected com-
pound 10 (2.75 mmol) was added 2 mL of trifluoroacetic acid and
the mixture was stirred at room temperature for 10 min. After con-
centration, the residue was diluted with 1N hydrochloric acid (10
mL) followed by lyophilization to give (1S,2R)-10a, (1R,2S)-10b or
racemic form 10 as a white solid (>90%). Only the data of represen-
tative compounds are showed.
5.5.1. (4R)-3-[(2E)-3-(3-Chlorophenyl)prop-2-enoyl]-4-phenyl-
1,3-oxazolidin-2-one (11, R1 = 3-Cl)
Mp 104–106 °C. 1H NMR (300 MHz, CDCl3) d 7.92 (d, J = 15.6 Hz,
1H), 7.69 (d, J = 15.6 Hz, 1H), 7.56–7.31 (m, 9H), 5.55 (dd, J = 3.9, 8.7
Hz, 1H), 4.75 (t, J = 8.7 Hz, 1H), 4.32 (dd, J = 3.9, 8.7 Hz, 1H). MS
(ES+) m/z calcd for C18H14ClNO3: 327.07; found: 328.1 (M+H),
350.1 (M+Na).
5.6. General procedure for the preparation of compounds 12a
and 12b
The ethereal solution of diazomethane (ꢀ16 mmol, prepared by
a Aldrich Mini Dazald apparatus) was continuously added into a
stirred solution of 11 (2.0 mmol) and palladium(II) acetate (2 mg,
0.01 mmol) in CH2Cl2/ether (56/20 mL) kept at 0 °C. After the addi-
tion of diazomethane ethereal solution was complete, the reaction
was stirred at room temperature for 1 h. The excess diazomethane
was quenched by dropwise addition of acetic acid. The mixture
was diluted with CH2Cl2, washed sequentially with saturated so-
dium bicarbonate and brine, and then dried over MgSO4. The or-
ganic layer was concentrated and purified by chromatography by
using a Biotage system (FLASH12i, silica gel, eluting with 1:9
EtOAc/hexane) to give the less polar product 12b (0.23 g, 33%)
and the more polar product 12a (0.46 g, 67%). Only the data of rep-
resentative compounds are showed.
5.7.1. (1S,2R)-2-(3-Chlorophenyl)cyclopropanamine
hydrochloride (10a, R1 = 3-Cl)
Mp 210–212 °C. 1H NMR (400 MHz, D2O) d 7.34–7.28 (m, 2H),
7.23 (t, J = 2.0 Hz, 1H), 7.12 (dt, J = 6.8, 2.0 Hz, 1H), 2.90 (ddd, J =
3.6, 4.4. 8.0 Hz, 1H), 2.44 (ddd, J = 3.6, 6.8, 10.0 Hz, 1H), 1.47
(ddd, J = 4.4. 6.8, 10.0 Hz, 1H), 1.36 (q like, J = 3.8 Hz, 1H). MS
(ES+) m/z calcd for C9H10ClN: 167.05; found: 168.1 (M+H). The spe-
cific rotation of 10a is ½a D25
¼ þ37:6 (c 0.05, MeOH).
ꢃ
5.7.2. (1R,2S)-2-(3-Chlorophenyl)cyclopropanamine
hydrochloride (10b, R1 = 3-Cl)
1H NMR spectrum of 10b is the same as that of 10a. The specific
rotation of 10b is ½a D25
¼ ꢁ38:6 (c 0.05, MeOH).
ꢃ
5.8. General procedure for EDC coupling reaction for
compounds 16
5.6.1. (4R)-3-{[(1S,2S)-2-(3-Chlorophenyl)cyclopropyl]
carbonyl}-4-phenyl-1,3-oxazolidin-2-one (12a, R1 = 3-Cl)
Mp 151–153 °C. 1H NMR (400 MHz, CDCl3) d 7.42–7.29 (m, 5H),
7.21–7.16 (m, 2H), 7.10 (t, J = 1.6 Hz, 1H), 7.00 (dt, J = 6.8, 1.6 Hz,
1H), 5.44 (dd, J = 4.0, 8.0 Hz, 1H), 4.71 (t, J = 8.8 Hz, 1H), 4.27 (dd, J =
4.0, 8.8 Hz, 1H), 3.55 (ddd, J = 4.4, 5.2, 8.4 Hz, 1H), 2.51 (ddd, J = 4.4,
6.4, 9.2 Hz, 1H), 1.67 (ddd, J = 4.4, 5.2, 9.2 Hz, 1H), 1.40 (ddd, J = 4.4,
6.4, 8.4 Hz, 1H). MS (ES+) m/z calcd for C19H16ClNO3: 341.79; found:
342.1 (M+H), 364.1 (M+Na).
A solution of N-BOC-protected carboxyl compound 15 (1.0
equiv) and HOBT (1.2 equiv) in 20 mL CH2Cl2/1,4-dioxane (2/1)
was cooled in an ice-water bath. To this solution was added
EDC (1.2 equiv) and stirred at room temperature for 1 h. To
the resulting solution was added the appropriate aryl cyclopro-
pylamine (10, 1.5 equiv, pretreated with 1.5 equiv Et3N). After
18 h of stirring at room temperature, the precipitate was re-
moved by filtration and washed by ethyl acetate. The filtrate
and washings were combined, washed sequentially with 10%
aqueous citric acid and saturated aqueous NaHCO3. The organic
layer was dried over MgSO4 and concentrated in vacuo. Purifica-
tion by flash column chromatography (eluted with hexane/EA =
3/7 for phenyl series of analogues, hexane/EA = 6/4 for heterocy-
clic series of analogues) yielded the desired compound 16 (>70%)
as a light yellow solid. Only the data of representative compound
is showed.
5.6.2. (4R)-3-{[(1R,2R)-2-(3-Chlorophenyl)cyclopropyl]car-
bonyl}-4-phenyl-1,3-oxazolidin-2-one (12b, R1 = 3-Cl)
Mp 102–104 °C. 1H NMR (300 MHz, CDCl3) d 7.43–7.30 (m, 5H),
7.21–7.14 (m, 3H), 7.02 (dt, J = 6.6, 2.4 Hz, 1H), 5.44 (dd, J = 3.6, 8.7
Hz, 1H), 4.70 (t, J = 8.7 Hz, 1H), 4.29 (dd, J = 3.6, 8.7 Hz, 1H), 3.54
(ddd, J = 4.2, 5.1, 8.7 Hz, 1H), 2.56 (ddd, J = 4.2, 6.9, 9.6 Hz, 1H),
1.64 (ddd, J = 4.5, 5.1, 9.6 Hz, 1H), 1.40 (ddd, J = 4.5, 6.9, 8.7 Hz,
1H). MS (ES+) m/z calcd for C19H16ClNO3: 341.79; found: 342.1
(M+H), 364.1 (M+Na).
5.8.1. tert-Butyl (2-methyl-4-{[(1S,2R)-2-(3-chlorophenyl)-
cyclopropyl]amino}-4-oxobutan-2-yl)carbamate (16b, R1 = 3-Cl)
Mp 118–120 °C. 1H NMR (300 MHz, CDCl3) d 7.21–6.23 (m, 4H),
6.22 (s, 1H), 4.88 (s, 1H), 2.85 (ddd, J = 3.3, 4.6, 7.5 Hz, 1H), 2.57 (q
like, J = 13.2 Hz, 2H), 1.96 (ddd, J = 3.3, 6.3, 9.6 Hz, 1H), 1.42 (s, 9H),
1.37 (s, 6H), 1.25–1.08 (m, 2H). MS (ES+) m/z calcd for
C19H27ClN2O3: 366.17; found: 389.2 (M+Na).
5.7. General procedure for the preparation of (1S,2R)-10a,
(1R,2S)-10b or racemic form 10
To a stirred solution of 12 (2.0 mmol) in THF/H2O (10/2 mL) at
ꢁ5 °C was added hydrogen peroxide solution (35 wt. % in water,
0.9 mL). To the resulting slurry was added dropwise a solution of

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T.-Y. Tsai et al. / Bioorg. Med. Chem. 17 (2009) 2388–2399
5.9. General procedure for the synthesis of compounds 18–25
5.9.6. 3-({2-[(2S)-2-Cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-
3-methyl-N-{2-[4-(trifluoromethyl)phenyl]cyclopropyl}
butanamide (18g)
A solution of compound 16 (1 equiv) in cool TFA was stirred at
room temperature for 30 min and concentrated in vacuo for 3 h.
The resultant yellowish oil was dissolved in THF, added K2CO3 (5
equiv) and stirred for a period of 1 h to liberate free amine. The
solution was diluted with EA and filtered to remove potassium
salts. The filtrate was concentrated in vacuo so as to obtain about
0.5 M free amine solution. To this solution was added bromide
compound 17 (0.5 equiv), the reaction mixture was stirred 18 h
at room temperature. The mixture was diluted with CH2Cl2, and
then washed with saturated aqueous NaHCO3, dried over MgSO4,
and concentrated in vacuo. Purification by flash column chroma-
tography (eluted with CH2Cl2/MeOH = 98/2–95/5) yielded desired
compounds 18–25 (40–70%) as light yellow gums.
1H NMR (300 MHz, CDCl3) (5/1 mixture of trans/cis amide
rotomers) d 8.70 (br s, 5/6H), 8.45–8.40 (m, 1/6H), 7.52 (d, J =
8.1 Hz, 3H), 7.25 (d, J = 8.1 Hz, 2H), 4.81–4.76 (m, 5/6H),
4.69–4.64 (m, 1/6H), 3.64–3.34 (m, 4H), 2.97–2.92 (m, 1H),
2.33–2.03 (m, 7H), 1.32–1.19 (m, 8H, overlapped singlet at
1.19). MS (ES+) m/z calcd for C22H27F3N4O2: 436.21; found:
437.2 (M+H).
5.9.7. 3-({2-[(2S)-2-Cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-
3-methyl-N-{2-[3-(trifluoromethyl)phenyl]cyclopropyl}
butanamide (18h)
1H NMR (300 MHz, CDCl3) (5/1 mixture of trans/cis amide
rotomers) d 8.74 (dd, J = 10.2, 3.3 Hz, 5/6H), 8.50 (br d, J = 10.2
Hz, 1/6H), 7.43–7.26 (m, 4H), 4.79–4.76 (m, 5/6H), 4.68–4.63 (m,
1/6H), 3.65–3.32 (m, 4H), 2.97–2.92 (m, 1H), 2.36–2.08 (m, 7H),
1.27–1.21 (m, 2H), 1.19(s, 6H). MS (ES+) m/z calcd for
C22H27F3N4O2: 436.21; found: 437.2 (M+H).
5.9.1. 3-({2-[(2S)-2-Cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-
3-methyl-N-[trans-2-phenylcyclopropyl]butanamide (18a)
1H NMR (400 MHz, CDCl3) (4/1 mixture of trans/cis amide
rotomers) d 8.57 (br d, J = 10.4 Hz, 4/5H), 8.32–8.24 (m, 1/5H),
7.27–7.12 (m, 5H), 4.81–4.76 (m, 4/5H), 4.71–4.66 (m, 1/5H),
3.64–3.33 (m, 4H), 2.96–2.93 (m, 1H), 2.35–2.03 (m, 7H), 1.35–
1.16 (m, 8H, overlapped singlet at 1.18). MS (ES+) m/z calcd for
C21H28N4O2: 368.22; found: 369.2 (M+H).
5.9.8. 3-({2-[(2S)-2-Cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-
3-methyl-N-[trans-2-(3-trifluoromethoxyphenyl)cyclopropyl]
butanamide (18i)
1H NMR (300 MHz, CDCl3) (5/1 mixture of trans/cis amide
rotomers) d 8.71 (dd, J = 9.6, 3.3 Hz, 5/6H), 8.48 (dd, J = 9.6,
3.3 Hz, 1/6H), 7.27 (t, J = 7.5 Hz, 1H), 7.11–6.99 (m, 3H), 4.79–
4.76 (m, 5/6H), 4.68–4.65 (m, 1/6H), 3.65–3.32 (m, 4H), 2.97–
2.90 (m, 1H), 2.36–1.98 (m, 7H), 1.27–1.17 (m, 8H, overlapped
singlet at 1.18). MS (ES+) m/z calcd for C22H27F3N4O3: 452.20;
found: 453.2 (M+H).
5.9.2. 3-({2-[(2S)-2-Cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-
3-methyl-N-[trans-2-(4-fluorophenyl)cyclopropyl]butanamide
(18b)
1H NMR (300 MHz, CDCl3) (5/1 mixture of trans/cis amide
rotomers) d 8.60 (br s, 5/6H), 8.34–8.28 (m, 1/6H), 7.16–7.10 (m,
2H), 6.94 (t, J = 8.7 Hz, 2H), 4.79–4.76 (m, 5/6H), 4.69–4.64 (m,
1/6H), 3.62–3.36 (m, 4H), 2.88–2.84 (m, 1H), 2.35–2.0 (m, 7H),
1.17–1.13 (m, 8H, overlapped singlet at 1.16). MS (ES+) m/z calcd
for C21H27FN4O2: 386.21; found: 387.2 (M+H).
5.9.9. 3-({2-[(2S)-2-Cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-
3-methyl-N-[trans-2-(4-chlorophenyl)cyclopropyl]butanamide
(18j)
5.9.3. 3-({2-[(2S)-2-Cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-
3-methyl-N-[trans-2-(3-fluorophenyl)cyclopropyl]butanamide
(18c)
1H NMR (300 MHz, CDCl3) (5/1 mixture of trans/cis amide
rotomers) d 8.64 (br s, 5/6H), 8.36 (br d, J = 10.2 Hz, 1/6H),
7.23–7.20 (m, 2H), 7.11–7.06 (m, 2H), 4.80–4.75 (m, 5/6H),
4.69–4.64 (m, 1/6H), 3.66–3.36 (m, 4H), 2.90–2.85 (m, 1H),
2.34–1.99 (m, 7H), 1.25–1.16 (m, 8H, overlapped singlet at
1.16). MS (ES+) m/z calcd for C21H27ClN4O2: 402.18; found:
403.1 (M+H), 405.0 (M+3).
1H NMR (400 MHz, CDCl3) (5/1 mixture of trans/cis amide
rotomers) d 8.67 (br d, J = 7.2 Hz, 5/6H), 8.42 (br d, J = 7.2 Hz, 1/
6H), 7.23–7.17 (m, 1H), 6.93 (t, J = 7.2 Hz, 1H), 6.86–6.82 (m,
2H), 4.78 (d, J = 7.6 Hz, 5/6H), 4.69–4.65 (m, 1/6H), 3.64–3.32 (m,
4H), 2.94–2.91 (m, 1H), 2.36–2.12 (m, 6H), 2.05 (dt, J = 3.2, 7.6
Hz, 1H), 1.25–1.18 (m, 8H, overlapped singlet at 1.18). MS (ES+)
m/z calcd for C21H27FN4O2: 386.21; found: 387.2 (M+H).
5.9.10. 3-({2-[(2S)-2-Cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-
3-methyl-N-[trans-2-(3-chlorophenyl)cyclopropyl]butanamide
(18k)
5.9.4. 3-({2-[(2S)-2-Cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-
3-methyl-N-[trans-2-(3,5-di-fluorophenyl)cyclopropyl]
butanamide (18e)
1H NMR (300 MHz, CDCl3) (5/1 mixture of trans/cis amide
rotomers) d 8.65 (br d, J = 7.2 Hz, 5/6H), 8.42–8.38 (m, 1/6H),
7.20–7.13 (m, 3H), 7.03 (t, J = 6.6 Hz, 1H), 4.78 (d, J = 6.6 Hz,
5/6H), 4.65 (d, J = 7.5 Hz, 1/6H), 3.75–3.30 (m, 4H), 2.92–2.90
(m, 1H), 2.40–2.10 (m, 6H, overlapped singlet at 2.27), 2.05–
1.99 (m, 1H), 1.24–1.16 (m, 8H, overlapped singlet at 1.16). MS
(ES+) m/z calcd for C21H27ClN4O2: 402.18; found: 403.2 (M+H),
425.2 (M+Na).
1H NMR (400 MHz, CDCl3) (6/1 mixture of trans/cis amide
rotomers) d 8.76–8.71 (m, 6/7H), 8.52–8.46 (m, 1/7H), 6.69–6.57
(m, 3H), 4.79–4.77 (m, 6/7H), 4.69–4.65 (m, 1/7H), 3.64–3.34 (m,
4H), 2.92–2.90 (m, 1H), 2.36–2.01 (m, 7H), 1.28–1.18 (m, 8H, over-
lapped singlet at 1.18). MS (ES+) m/z calcd for C21H26F2N4O2:
404.20; found: 405.2 (M+H).
5.9.11. 3-({2-[(2S)-2-Cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-
3-methyl-N-[trans-2-(3,4-di-chlorophenyl)cyclopropyl]
butanamide (18l)
5.9.5. 3-({2-[(2S)-2-Cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-
3-methyl-N-[trans-2-(3,4-di-fluorophenyl)cyclopropyl]
butanamide (18f)
1H NMR (400 MHz, CDCl3) (6/1 mixture of trans/cis amide
rotomers) d 8.74 (br s, 6/7H), 8.51 (br s, 1/7H), 7.31–7.24 (m,
2H), 7.01 (dt, J = 2.0, 8.4 Hz, 1H), 4.77 (br d, J = 6.8 Hz, 6/7H),
4.66 (br d, J = 6.8 Hz, 1/7H), 3.64–3.32 (m, 4H), 2.91–2.84 (m,
1H), 2.36–1.99 (m, 7H), 1.25–1.16 (m, 8H, overlapped singlet at
1.16). MS (ES+) m/z calcd for C21H26Cl2N4O2: 436.14; found: 437.0
(M+H), 439.0 (M+3).
1H NMR (400 MHz, CDCl3) (5/1 mixture of trans/cis amide
rotomers) d 8.69 (br s, 5/6H), 8.43 (br s, 1/6H), 7.26–6.89 (m,
3H), 4.78 (br d, J = 7.2 Hz, 5/6H), 4.64–4.68 (m, 1/6H), 3.62–3.32
(m, 4H), 2.85–2.82 (m, 1H), 2.35–2.19 (m, 6H), 2.04–1.99 (m,
1H), 1.20–1.12 (m, 8H, overlapped singlet at 1.17). MS (ES+) m/z
calcd for C21H26F2N4O2: 404.20; found: 405.2 (M+H).

T.-Y. Tsai et al. / Bioorg. Med. Chem. 17 (2009) 2388–2399
2397
5.9.12. 3-({2-[(2S)-2-Cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-
3-methyl-N-[trans-2-(3,4-di-methoxyphenyl)cyclopropyl]
butanamide (18m)
7.00 (m, 4H), 5.51 (t, J = 3.3 Hz, 2/5H), 5.43 (t, J = 3.3 Hz, 1/10H),
5.34 (t, J = 3.3 Hz, 2/5H), 5.26 (t, J = 3.3 Hz, 1/10H), 4.98 (d, J =
9.3 Hz, 4/5H), 4.94 (d, J = 9.3 Hz, 1/5H), 4.02–3.61 (m, 2H), 3.39
(q like, J = 16.8 Hz, 2H), 2.96–2.87 (m, 1H), 2.74 (t, J = 15.3 Hz, 1/
5H), 2.67 (t, J = 15.3 Hz, 4/5H), 2.51–2.22 (m, 3H, overlapped singlet
at 2.28), 2.05–1.98 (m, 1H), 1.25–1.06 (m, 8H, overlapped singlet at
1.17). MS (ES+) m/z calcd for C21H26ClFN4O2: 420.17; found: 421.2
(M+H).
1H NMR (300 MHz, CDCl3) (5/1 mixture of trans/cis amide
rotomers) d 8.56–8.52 (m, 5/6H), 8.24–8.16 (m, 1/6H), 6.81–6.67
(m, 3H), 4.79–4.64 (m, 1H), 3.87 (s, 3H), 3.84 (s, 3H), 3.64–3.42
(m, 4H), 2.92–2.86 (m, 1H), 2.38–1.99 (m, 7H), 1.30–1.09 (m, 8H,
overlapped singlet at 1.20). MS (ES+) m/z calcd for C23H32N4O4:
428.24; found: 429.2 (M+H).
5.9.18. 3-({2-[(2S,4S)-2-Cyano-4-fluoropyrrolidin-1-yl]-2-
oxoethyl}amino)-3-methyl-N-[(1S,2R)-2-phenylcyclopropyl]
butanamide (24a)
5.9.13. 3-({2-[(2S,4S)-2-Cyano-4-fluoropyrrolidin-1-yl]-2-
oxoethyl}amino)-3-methyl-N-[trans-2-(pyridin-3-yl)
cyclopropyl]butanamide (19)
1H NMR (CDCl3): (4/1 mixture of trans/cis amide rotomers) d
8.35 (br d, J = 3.3 Hz, 4/5H), 8.18 (br d, J = 3.3 Hz, 1/5H), 7.27–
7.10 (m, 5H), 5.46 (t, J = 3.3 Hz, 2/5H), 5.38 (t, J = 3.3 Hz, 1/10H),
5.29 (t, J = 3.3 Hz, 2/5H), 5.21 (t, J = 3.3 Hz, 1/10H), 5.00 (d, J =
9.0 Hz, 1/5H), 4.93 (d, J = 9.0 Hz, 4/5H), 4.03–3.55 (m, 2H), 3.39
(q like, J = 16.5 Hz, 2H), 2.96–2.89 (m, 1H), 2.69 (t, J = 15.3 Hz, 1/
5H), 2.61 (t, J = 15.3 Hz, 4/5H), 2.52–2.19 (m, 3H), 2.08–2.01 (m,
1H), 1.25–0.96 (m, 8H, overlapped singlet at 1.17). MS (ES+) m/z
calcd for C21H27FN4O2: 386.46; found: 387.2 (M+H), 409.2
(M+Na). Anal. (C21H27FN4O2ꢄ0.33H2O) C, H, N.
1H NMR (400 MHz, DMSO-d6) (5/1 mixture of trans/cis amide
rotomers) d 8.56 (dd, J = 4.0, 12.8 Hz, 1H), 8.41 (t, J = 2.8 Hz, 1H),
8.35 (dd, J = 0.8, 4.8 Hz, 1H), 8.35 (ddd, J = 2.0, 4.0 8.0 Hz, 1H),
7.26 (dd, J = 4.8, 8.0 Hz, 1H), 5.55 (t, J = 3.2 Hz, 5/12H), 5.48 (t, J
= 3.2 Hz, 1/12H), 5.42 (t, J = 3.2 Hz, 5/12H), 5.38 (d, J = 8.4 Hz, 1/
6H), 5.35 (t, J = 3.2 Hz, 1/12H), 4.97–4.95 (m, 5/6H), 3.98–3.27
(m, 4H, overlapped singlet at 3.33), 2.85–2.80 (m, 1H), 2.50–2.31
(m, 2H), 2.16 (dd, J = 15.2, 20.4 Hz, 2H), 1.96–1.91 (m, 1H), 1.24–
1.18 (m, 2H), 1.06 (s, 6H). MS (ES+) m/z calcd for C20H26FN5O2:
387.21; found: 388.2 (M+H), 410.2 (M+Na). HRMS (FAB) calcd for
C20H27FN5O2: 388.2149; found: 388.2151.
5.9.19. 3-({2-[(2S,4S)-2-Cyano-4-fluoropyrrolidin-1-yl]-2-
oxoethyl}amino)-3-methyl-N-[(1S,2R)-2-(3-chlorophenyl)
cyclopropyl]butanamide (24b)
5.9.14. 3-({2-[(2S),4-2S-Cyano-4-fluoropyrrolidin-1-yl]-2-oxoethyl}-
amino)-3-methyl-N-[trans-2-(4-methyl-1,3-thiazol-5-yl)cyclo-
propyl]butanamide (20)
1H NMR (CDCl3): (4/1 mixture of trans/cis amide rotomers) d
8.41 (br d, J = 3.0 Hz, 4/5H), 8.15 (br s, 1/5H), 7.17–6.99 (m, 4H),
5.52 (t, J = 3.3 Hz, 2/5H), 5.42 (t, J = 3.3 Hz, 1/10H), 5.35 (t, J =
3.3 Hz, 2/5H), 5.26 (t, J = 3.3 Hz, 1/10H), 4.96 (d, J = 9.3 Hz,
4/5H), 4.92 (d, J = 9.3 Hz, 1/5H), 3.91 (dd, J = 23.4, 23.1 Hz, 4/5H),
3.77 (d, J = 3.6 Hz, 1/5H), 3.73 (d, J = 3.9 Hz, 1/5H), 3.65–3.61 (m,
4/5H), 3.38 (q like, J = 16.5 Hz, 2H), 2.94–2.88 (m, 1H), 2.76 (t, J =
15.3 Hz, 1/5H), 2.69 (t, J = 15.3 Hz, 4/5H), 2.43–2.22 (m, 3H, over-
lapped singlet at 2.27), 2.05–1.99 (m, 1H), 1.24–1.16 (m, 8H, over-
lapped singlet at 1.16). MS (ES+) m/z calcd for C21H26ClFN4O2:
420.17; found: 421.2 (M+H). Anal. (C21H26ClFN4O2ꢄ0.33H2O) C, H,
N.
1H NMR (300 MHz, CDCl3) (4/1 mixture of trans/cis amide
rotomers) d 8.58 (br s, 4/5H), 8.45 (s, 1H), 8.34 (br s, 1/5H), 5.52
(t, J = 3.3 Hz, 2/5H), 5.44 (t, J = 3.3 Hz, 1/10H), 5.35 (t, J = 3.3 Hz,
2/5H), 5.26 (t, J = 3.3 Hz, 1/10H), 4.94 (d, J = 9.3 Hz, 4/5H), 4.93
(d, J = 9.3 Hz, 1/5H), 3.99–3.49 (m, 2H), 3.40 (q like, J = 16.5 Hz,
2H), 2.91–2.89 (m, 1H), 2.76 (t, J = 15.9 Hz, 1/5H), 2.68 (t, J =
15.9 Hz, 4/5H), 2.55–2.22 (m, 6H, overlapped two singlet at 2.48,
2.27), 2.11–2.03 (m, 1H), 1.37–1.21 (m, 1H), 1.21–0.95 (m, 7H,
overlapped singlet at 1.16). MS (ES+) m/z calcd for C19H26FN5O2S:
407.18; found: 408.2 (M+H). Anal. (C19H26FN5O2SꢄH2O) C, H, N.
5.9.15. 3-({2-[(2S)-2-Cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-
3-methyl-N-[(1R,2S)-2-(3-chlorophenyl)cyclopropyl]
butanamide (21)
5.9.20. 3-({2-[(2S,4S)-2-Cyano-4-fluoropyrrolidin-1-yl]-2-
oxoethyl}amino)-3-methyl-N-[(1S,2R)-2-(3-fluorophenyl)
cyclopropyl]butanamide (24c)
1H NMR (300 MHz, CDCl3) (5/1 mixture of trans/cis amide
rotomers) d 8.65 (br s, 5/6H), 8.36 (br s, 1/6H), 7.39–7.11 (m,
3H), 7.02 (br d, J = 7.2 Hz, 1H), 4.78 (d, J = 6.3 Hz, 5/6H), 4.65 (d,
J = 8.4 Hz, 1/6H), 3.73–3.30 (m, 4H), 2.95–2.90 (m, 1H), 2.33–
2.17 (m, 6H, overlapped singlet at 2.27), 2.06–1.99 (m, 1H), 1.25–
1.16 (m, 8H, overlapped singlet at 1.18). MS (ES+) m/z calcd for
C21H27ClN4O2: 402.18; found: 403.2 (M+H), 425.2 (M+Na).
1H NMR (CDCl3): (3/1 mixture of trans/cis amide rotomers) d
8.43 (br d, J = 3.3 Hz, 3/4H), 8.42 (br s, 1/4H), 7.20 (q like, J = 7.2
Hz, 1H), 6.94–6.81 (m, 3H), 5.51 (t, J = 3.3 Hz, 3/8H), 5.43 (t, J =
3.3 Hz, 1/8H), 5.34 (t, J = 3.3 Hz, 3/8H), 5.26 (t, J = 3.3 Hz, 1/8H),
4.95 (d, J = 9.3 Hz, 1H), 3.91 (dd, J = 23.7, 23.4 Hz, 3/4H), 3.78 (d,
J = 3.6 Hz, 1/4H), 3.74 (d, J = 3.9 Hz, 1/4H), 3.66–3.61 (m, 3/4H),
3.39 (q like, J = 16.5 Hz, 2H), 2.95–2.88 (m, 1H), 2.74 (t, J = 15.3
Hz, 1/4H), 2.67 (t, J = 15.3 Hz, 3/4H), 2.45–2.22 (m, 3H, overlapped
singlet at 2.27), 2.10–1.98 (m, 1H), 1.28–1.17 (m, 8H, overlapped
singlet at 1.20). MS (ES+) m/z calcd for C21H26F2N4O2: 404.20;
found: 405.2 (M+H), 427.2 (M+Na). HRMS (FAB) calcd for
C21H27FN4O2: 405.2102; found: 405.2094.
5.9.16. 3-({2-[(2S)-2-Cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-
3-methyl-N-[(1S,2R)-2-(3-chlorophenyl)cyclopropyl]
butanamide (22)
1H NMR (CDCl3): (5/1 mixture of trans/cis amide rotomers) d 8.65
(d, J = 3.3 Hz, 5/6H), 8.45 (d, J = 3.3 Hz, 1/6H), 7.18–7.09 (m, 3H), 7.02–
6.98 (m, 1H), 4.77–4.74(m, 5/6H), 4.71 (d, J =2.4Hz, 1/6H), 3.63–3.38
(m, 4H, overlapped two singlet at 3.46, 3.44), 2.93–2.89 (m, 1H),
2.35–2.15 (m, 6H, overlapped singlet at 2.32), 2.06–1.99 (m, 1H),
1.26–1.43 (m, 8H, overlapped singlet at 1.20). MS (ES+) m/z calcd
for C21H27ClN4O2: 402.18; found: 403.2 (M+H), 425.2 (M+Na).
5.9.21. 3-({2-[(2S,4S)-2-Cyano-4-fluoropyrrolidin-1-yl]-2-
oxoethyl}amino)-3-methyl-N-[(1S,2R)-2-(4-methoxyphenyl)
cyclopropyl]butanamide (24d)
1H NMR (CDCl3): (3/1 mixture of trans/cis amide rotomers) d
8.29 (br d, J = 3.3 Hz, 3/4H), 8.00 (br s, 1/4H), 7.08 (d, J = 8.4 Hz,
2H), 6.80 (d, J = 8.4 Hz, 2H), 5.50 (t, J = 3.0 Hz, 3/8H), 5.42 (t, J =
3.0 Hz, 1/8H), 5.33 (t, J = 3.0 Hz, 3/8H), 5.24 (t, J = 3.0 Hz, 1/8H),
4.97 (d, J = 8.8 Hz, 1/4H), 4.95 (d, J = 8.8 Hz, 3/4H), 3.96–3.54 (m,
5H, overlapped singlet at 3.76), 3.40 (q like, J = 16.5 Hz, 2H),
2.88–2.82 (m, 1H), 2.73 (t, J = 15.6 Hz, 1/4H), 2.66 (t, J = 15.6 Hz,
3/4H), 2.45–2.23 (m, 3H, overlapped singlet at 2.28), 2.0–1.97 (m,
5.9.17. 3-({2-[(2S,4S)-2-Cyano-4-fluoropyrrolidin-1-yl]-2-
oxoethyl}amino)-3-methyl-N-[(1R,2S)-2-(3-chlorophenyl)
cyclopropyl]butanamide (23)
1H NMR (300 MHz, CDCl3) (4/1 mixture of trans/cis amide
rotomers) d 8.45 (br d, J = 3.0 Hz, 4/5H), 8.16 (br s, 1/5H), 7.21–

2398
T.-Y. Tsai et al. / Bioorg. Med. Chem. 17 (2009) 2388–2399
1H), 1.19–1.09 (m, 8H, overlapped singlet at 1.18). MS (ES+) m/z
calcd for C22H29FN4O3: 416.22; found: 417.2 (M+H), 439.2 (M+Na).
in our previously published method.25 The purification of DPP-II
was carried out as described in Ref. 25 with modifications. The
detailed protocol for the cloning and expression of FAP will be de-
scribed elsewhere. Briefly, cDNA of FAP was cloned into pBac-
PAC8-CD5 vector. The buffer for DPP-IV and DPP8 assays are
2 mM Tris–HCl, pH 8.0 and PBS buffer, respectively. The substrate
used is Gly-Pro-pNA from Bachem at the concentrations of 500
5.9.22. 3-({2-[(2S,4S)-2-Cyano-4-fluoropyrrolidin-1-yl]-2-
oxoethyl}amino)-3-methyl-N-[(1S,2R)-2-(3-methoxyphenyl)
cyclopropyl]butanamide (24e)
1H NMR (300 MHz, CDCl3) (3/1 mixture of trans/cis amide
rotomers) d 8.33 (br s, 3/4H), 8.05 (br s, 1/4H), 7.17 (t, J = 7.5 Hz,
1H), 6.74–6.67 (m, 3H), 5.53 (t, J = 3.3 Hz, 3/8H), 5.43 (t, J = 3.3
Hz, 1/8H), 5.36 (t, J = 3.3 Hz, 3/8H), 5.26 (t, J = 3.3 Hz, 1/8H), 4.97
(d, J = 9.3 Hz, 3/4H), 4.93 (d, J = 9.3 Hz, 1/4H), 4.12–3.62 (m, 5H,
overlapped singlet at 3.78), 3.38 (q like, J = 16.5 Hz, 2H), 2.97–
2.91 (m, 1H), 2.76 (t, J = 15.9 Hz, 1/4H), 2.70 (t, J = 15.9 Hz, 3/
4H), 2.45–2.22 (m, 3H, overlapped singlet at 2.28), 2.06–2.00 (m,
1H), 1.33–1.11 (m, 8H, overlapped singlet at 1.17). MS (ES+) m/z
calcd for C22H29FN4O3: 416.22; found: 417.2 (M+H), 439.2
(M+Na). Anal. (C22H29FN4O3ꢄH2O) C, H, N.
lM, 6000 lM and 2500 lM for DPP-IV, FAP and DPP8 assays,
respectively. DPP-II activity was assayed by 1.5 mM Gly-Pro-
pNA in 50 mM potassium phosphate buffer, pH 5.5. The concen-
tration of DPP-II is 10 nM. Reaction developed at 37 °C and OD405
was monitored.
5.10.2. DPP-IV inhibition in Wistar rats
Adult male Wistar rats (n = 4–6/group) were orally gavaged
with the test compounds dissolved in 0.5% methyl cellulose at a
single dose of 10 mg/kg. Blood samples of 25–50 ll were collected
from the tail veins at the time points indicated in Figure 3 and the
plasma fraction was kept frozen until DPP-IV activity measure-
ment. The plasma DPP-IV activity was determined by cleavage rate
of Gly-Pro-AMC (H-glycyl-prolyl-7-amino-4-methylcoumarin; BA-
5.9.23. 3-({2-[(2S,4S)-2-Cyano-4-fluoropyrrolidin-1-yl]-2-
oxoethyl}amino)-3-methyl-N-[(1S,2R)-2-(3-ethoxyphenyl)
cyclopropyl]butanamide (24f)
1H NMR (400 MHz, DMSO-d6) (4/1 mixture of trans/cis amide
rotomers) d 8.47 (br d, J = 4.4 Hz, 4/5H), 8.43 (br d, J = 4.4 Hz, 1/
5H), 7.13 (t, J = 7.6 Hz, 1H), 6.70–6.61 (m, 3H), 5.55 (t, J = 2.8 Hz,
2/5H), 5.47 (t, J = 2.8 Hz, 1/10H), 5.42 (t, J = 2.8 Hz, 2/5H), 5.37
(d, J = 9.2 Hz, 1/5H), 5.34 (t, J = 2.8 Hz, 1/10H), 4.97–4.95 (m, 4/
5H), 3.98 (q, J = 6.8 Hz, 2H), 3.92–3.27 (m, 4H, overlapped singlet
at 3.33), 2.83–2.78 (m, 1H), 2.57–2.31 (m, 2H), 2.15 (dd, J = 14.0,
20.4 Hz, 2H), 1.90–1.85 (m, 1H), 1.30 (t, J = 7.2 Hz, 3H), 1.17–
1.08 (m, 2H), 1.06 (s, 6H). MS (ES+) m/z calcd for C23H31FN4O3:
CHEM). Plasma (10 ll) was mixed with 140 ll of 150 lM Gly-Pro-
AMC in assay buffer that was composed of 25 mM tris(hydroxy-
methyl)-aminomethane HCl (pH 7.4), 140 mM NaCl, 10 mM KCl
and 0.1% bovine serum albumin. The fluorescence was determined
by using Fluoroskan Ascent FL (excitation at 390 nm and emission
at 460 nm) (Thermo LabSystems; Thermo Electron Corporation).
DPP-IV activity in plasma was described as unit per mL (U/mL).
One unit of activity is defined as the amount of enzyme that pro-
duces 1 lM products per minute.
430.24;
found:
431.3
(M+H),
453.3
(M+Na).
Anal.
(C21H31FN4O3ꢄ0.33H2O) C, H, N.
5.10.3. Oral glucose tolerance test in C57BL/6j lean mice and
C57BL/6j DIO mice
5.9.24. 3-({2-[(2S,4S)-2-Cyano-4-fluoropyrrolidin-1-yl]-2-
oxoethyl}amino)-3-methyl-N-[(1S,2R)-2-(3-trifluorometho-
xyphenyl)cyclopropyl]butanamide (24g)
Adult male C57BL/6 mice (20–25 g) (6–8 weeks of age, n = 6/
group) or C57BL/6j DIO mice (40–50 g) (27 weeks of age, n = 6/
group) were fasted overnight. Blood samples were obtained from
the tail veins with needles of 27G and the blood glucose was mea-
sured with the Accu-Chek Compact System from Roche (Basel,
Switzerland) and animals were grouped according to the glucose
levels. The animals were then orally gavaged with the test com-
pounds dissolved in distilled water at a dose indicated in Figure
4. Thirty minutes after the oral dosing of test compounds, the ani-
mals were orally gavaged with freshly prepared glucose solution of
400 mg/mL in distilled water at 3 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.
1H NMR (400 MHz, DMSO-d6) (5/1 mixture of trans/cis amide
rotomers) d 8.54 (br d, J = 4.4 Hz, 5/6H), 8.50 (br d, J = 4.4 Hz, 1/
6H), 7.37 (t, J = 8.0 Hz, 1H), 7.14–7.10 (m, 3H), 5.55 (t, J = 3.2 Hz,
5/12H), 5.47 (t, J = 3.2 Hz, 1/12H), 5.42 (t, J = 3.2 Hz, 5/12H), 5.37
(d, J = 8.4 Hz, 1/6H), 5.34 (t, J = 3.2 Hz, 1/12H), 4.97–4.94 (m, 5/
6H), 3.99–3.51 (m, 2H), 3.35 (q like, J = 16.4 Hz, 2H), 2.87–2.80
(m, 1H), 2.57–2.31 (m, 2H), 2.15 (dd, J = 14.0, 22.8 Hz, 2H), 1.98–
1.89 (m, 1H), 1.24–1.16 (m, 2H), 1.06 (s, 6H).MS (ES+) m/z calcd
for C22H26F4N4O3: 470.19; found: 471.2 (M+H), 493.2 (M+Na).
Anal. (C22H26F4N4O3ꢄ0.33H2O) C, H, N.
5.9.25. 3-({2-[(2S)-2-Cyano-4,4-difluoropyrrolidin-1-yl]-2-
oxoethyl}amino)-3-methyl-N-[(1S,2R)-2-(3-chlorophenyl)
cyclopropyl]butanamide (25)
Acknowledgment
National Health Research Institutes and National Science Coun-
cil, Taiwan, financially supported the study.
1H NMR (300 MHz, CDCl3) (4/1 mixture of trans/cis amide rotom-
ers) d 8.18 (d, J = 2.4 Hz, 4/5H), 7.81 (br s, 1/5H), 7.21–7.12 (m, 3H),
7.02 (d, J = 7.2 Hz, 1H), 5.21 (br d, J = 6.0 Hz, 1/5H), 4.97 (t, J = 606
Hz, 4/5H), 4.03–3.83 (m, 2H), 3.64–3.37 (m, 2H, overlapped singlet
at 3.37), 2.94–2.87 (m, 1H), 2.80–2.70 (m, 2H), 2.26 (s, 2H), 2.02
(dt, J = 7.5, 3.3 Hz, 1H), 1.22–1.16 (m, 8H, overlapped singlet at
1.16). MS (ES+) m/z calcd for C21H25ClF2N4O2: 438.16; found: 439.2
(M+H), 461.2 (M+Na). Anal. Calcd for C21H25ClF2N4O2ꢄ0.5H2O: C,
56.30; H, 5.75; N, 12.51. Found: C, 56.23; H, 6.03; N, 12.23.
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