Design, synthesis and biological evaluation of hetero-aromatic moieties substituted pyrrole-2-carbonitrile derivatives as dipeptidyl peptidase IV inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.01.021

A series of novel hetero-aromatic moieties substituted α-amino pyrrole-2-carbonitrile derivatives was designed and synthesized based on structure-activity relationships (SARs) of pyrrole-2-carbonitrile inhibitors. All compounds demonstrated good dipeptidyl peptidase IV (DPP4) inhibitory activities (IC₅₀ = 0.004-113.6 μM). Moreover, compounds 6h (IC₅₀ = 0.004 μM) and 6n (IC₅₀ = 0.01 μM) showed excellent inhibitory activities against DPP4, good selectivity (compound 6h, selective ratio: DPP8/DPP4 = 450.0; DPP9/DPP4 = 375.0; compound 6n, selective ratio: DPP8/DPP4 = 470.0; DPP9/DPP4 = 750.0) and good efficacy in an oral glucose tolerance test in ICR mice. Furthermore, compounds 6h and 6n demonstrated moderate PK properties (compound 6h, F% = 37.8%, t_1/2 = 1.45 h; compound 6n, F% = 16.8%, t_1/2 = 3.64 h).

Full text of DOI:10.1016/j.ejmech.2014.01.021

European Journal of Medicinal Chemistry 75 (2014) 111e122
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of hetero-aromatic
moieties substituted pyrrole-2-carbonitrile derivatives as dipeptidyl
peptidase IV inhibitors
,
b
,
,
c
,
,
Xun Ji a 1, Mingbo Su b 1, Jiang Wang b 1, Guanghui Deng b, Sisi Deng b, Zeng Li b,
**
Chunlan Tang b, Jingya Li b, Jia Li b , Linxiang Zhao , Hualiang Jiang b, Hong Liu b
,
a
a,
,
*
a School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, 103 Wen Hua Road, Shenyang, Liaoning 110016, PR China
b CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road,
Shanghai 201203, PR China
c East China of Normal University, 3663 Zhongshan Road, Shanghai 200062, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel hetero-aromatic moieties substituted a-amino pyrrole-2-carbonitrile derivatives was
designed and synthesized based on structureeactivity relationships (SARs) of pyrrole-2-carbonitrile
inhibitors. All compounds demonstrated good dipeptidyl peptidase IV (DPP4) inhibitory activities
Received 22 September 2013
Received in revised form
8 January 2014
Accepted 15 January 2014
Available online 23 January 2014
(IC50 ¼ 0.004e113.6
excellent inhibitory activities against DPP4, good selectivity (compound 6h, selective ratio: DPP8/
DPP4 470.0; DPP9/
m
M). Moreover, compounds 6h (IC50 ¼ 0.004
m
M) and 6n (IC50 ¼ 0.01
mM) showed
¼
450.0; DPP9/DPP4 ¼ 375.0; compound 6n, selective ratio: DPP8/DPP4 ¼
DPP4 ¼ 750.0) and good efficacy in an oral glucose tolerance test in ICR mice. Furthermore, compounds
6h and 6n demonstrated moderate PK properties (compound 6h, F% ¼ 37.8%, t1/2 ¼ 1.45 h; compound 6n,
F% ¼ 16.8%, t1/2 ¼ 3.64 h).
Keywords:
DPP4 inhibitors
Selectivity
Reversible kinetic characterization
Oral bioavailability
Oral glucose tolerance test
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
aspects of glucose homeostasis and is important in the normali-
zation of blood glucose levels in diabetic patients. To date, the
Inhibition of dipeptidyl peptidase IV (DPP IV, DPP4, also known
as CD26) has become an attractive and potential therapeutic
strategy for treatment of type 2 diabetes mellitus (T2DM) [1]. DPP4
is a multifunctional type II transmembrane serine protease glyco-
protein [2] which is responsible for the rapid inactivation of
glucagon-like peptide-1 (GLP-1). GLP-1 is an important incretin
therapeutic rationale for DPP4 inhibition has focused on prolonging
the half-life of GLP-1 bioactivity [1].
Recently, several DPP4 inhibitors [7e14] including 1 (Sitagliptin,
MK-0431) [15], 2 (Vildagliptin, LAF237) [16], 3 (Saxagliptin, BMS-
477118) [17], 4 (Alogliptin, SYR-322) [18], and 5 (Linagliptin,
BI1356) [19] (Fig. 1) have been approved for T2DM treatment.
These agents have been demonstrated to be sensitive to lower
glucose and HbA1c levels and have successfully improved
glucose tolerance in T2DM patients [20]. However, the issue of
poor selectivity was still to focus according to peptidomimetic in-
hibitors [21e25]. Compared to peptidomimetic inhibitors, non-
peptidomimetic inhibitors showed good selectivity [26e29]. In
which regulates blood glucose levels [3], and is released from L-cells
and stimulates insulin biosynthesis and secretion [4], inhibits
glucose release [5], and delays gastric emptying resulting in
reduced appetite [6]. The bioactivity of GLP-1 influences multiple
our group, a novel series of 1-(
g-1,2,3-triazol substituted prolyl)-
(S)-3,3-difluoropyrrolidines derivatives [30] had been reported,
Abbreviation: DPP4, dipeptidyl peptidase IV; GLP-1, glucagon-like peptide-1;
T2DM, type 2 diabetes mellitus; SARs, structureeactivity relationships; OGTTs, oral
glucose tolerance tests; PK, pharmacokinetics; AUC, area under curve.
* Corresponding author. Tel./fax: þ86 21 50807042.
which showed good selectivity. However, 1-(
substituted prolyl)-(S)-3,3-difluoropyrrolidines
g
-1,2,3-triazol
derivatives
showed moderate DPP4 inhibitory activities (micro-molar levels).
Aim to discovery of compounds with excellent DPP4 inhibitory
activities and selectivity, we designed and synthesized hetero-
** Corresponding author. Tel.: þ 86 21 50801313; fax: þ 86 21 50801552.
E-mail addresses: jli@mail.shcnc.ac (J. Li), hliu@mail.shcnc.ac (H. Liu).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.021

112
X. Ji et al. / European Journal of Medicinal Chemistry 75 (2014) 111e122
Fig. 1. Representative DPP4 inhibitors.
aromatic moieties substituted
a
-amino pyrrole-2-carbonitrile de-
2.2. Biological evaluation
rivatives as a novel, potent, and selective DPP4 inhibitors for the
treatment of T2DM.
2.2.1. In vitro enzyme inhibition studies
All the synthesized compounds (6aen) were evaluated in vitro
for their capacity to inhibit DPP4 (Table 1). Due to the diversity of
serine proteases, the inhibitory activities of the other members of
the serine protease family (DPP8 and DPP9) were also evaluated.
Because inhibition of DPP8/9 was associated with toxicity in animal
studies [31], selectivity against DPP8/9 was particularly important.
The SARs of synthesized pyrrole-2-carbonitrile derivatives were
discussed as follows. Compound 6a, which was furan-3-yl
substituted, showed moderate inhibitory activity against DPP4
2. Results and discussion
2.1. Chemistry
2.1.1. Design of compounds
Investigation of the SARs of the pyrrole-2-carbonitrile inhibitors
previously reported [31], which showed that the nitrile group
effectively formed a covalent adduct with the catalytic Ser630
residue and bind to DPP4 in the S1 pocket. Therefore, the pyrrole-2-
carbonitrile moiety was retained in our design (Fig. 2). Further-
more, the P2 region has been mainly substituted with aromatic
moieties [9,32,33] in previous studies. Aliphatic moieties with
research involving hetero-aromatic moiety substituted compounds
were limited [7,10,16,17,34,35]. Therefore, a novel series of com-
pounds including penta- and hexa-hetero-aromatic systems (6aee)
was designed. Furthermore, introducing the fluorine atom in
Denagliptin [32] demonstrated that the inhibitory activity was
improvement compared to pyrrole-2-carbonitrile derivatives. The
reason could be the fluorine atom interacted with certain amino
acid residues via hydrogen bond. Accordingly, a series of novel
(IC50 ¼ 113.6
furan group was replaced with thiophene, thiazole, and pyridine
M)
mM). In order to improve DPP4 inhibitory activities, the
group. Compounds 6b (IC50 ¼ 0.17
m
M) and 6c (IC50 ¼ 0.03
m
which were thiophen-3-yl and benzo[b]thiophen-3-yl substituted
demonstrated 668.2-fold and 3786.7-fold improvement in DPP4
inhibitory activities, respectively. While thiazol-4-yl and pyridin-2-
yl group substituted compounds 6d (IC50 ¼ 0.33
mM) and 6e
(IC50 ¼ 0.52 M) showed 344.2-fold and 218.5-fold improvement in
m
inhibitory activities. The discrepancy in inhibitory activities with
compounds could be enhancement the liposolubility of compounds
6b and 6c when the oxygen atom was replaced with a sulfur atom.
However, the pyridin-2-yl group substituted compound 6e
exhibited decreased inhibitory activity when compared to com-
pound 6b, by 3.1-fold. This could be the difference of the electro-
negativity of two compounds.
hetero-aromatic moiety substituted
carbonitrile derivatives (6fen) was designed.
a-amino 4-fluoropyrrole-2-
Investigation of the inhibitory activities and selectivity of the
pyrrolidine-2-carbonitrile derivatives revealed that compound 6c
showed good DPP4 inhibition, however, the selectivity of com-
pound 6c was lower (SR: DPP8/DPP4 ¼ 327.6; DPP9/DPP4 ¼ 425.9)
than Sitagliptin (SR: DPP8/DPP4 ¼ 1680.0; DPP9/DPP4 ¼ 5455.0).
2.1.2. Synthesis of target compounds
Compounds 6aee were synthesized according to Scheme 1. The
important intermediate compound 11 was obtained from
commercially available compound 7 via protection, amidation,
dehydration, and deprotection, followed by coupling reactions with
N-Boc-a-amino acids (12aee, commercially available) to yield the
compounds 13aee. Deprotection of 13aee with trifluoroacetic acid
(TFA) produced the target compounds 6aee.
The synthetic route of compounds 6fen was shown in Scheme
2. Commercially available compound 14 was protected and then
fluorinated with diethylaminosulfur trifluoride (DAST) to generate
compound 16. Compound 16 produced compound 20 [36] via
demethylation, amidation, dehydration, and deprotection, followed
by coupling reactions with N-Boc-a-amino acids (12fen) to yield
the compounds 21fen. Deprotection of 21fen with TFA produced
the target compounds 6fen.
Fig. 2. Design of novel a-amino pyrrole-2-carbonitrile derivatives.

X. Ji et al. / European Journal of Medicinal Chemistry 75 (2014) 111e122
113
Scheme 1. Reagents and conditions: (a) (Boc)2O, NaHCO3, dioxane, 24 h; (b) (Boc)2O, NH4HCO3, pyridine, dioxane, 6 h; (c) cyanuric chiloride, DMF, 1 h; (d) TsOH, CH3CN, rt, 24 h; (e)
HATU, DIPEA, DMF, 5 h; (f) CH2Cl2, TFA, 0 ꢀC to rt, 1 h.
Therefore, to enhance the DPP4 inhibitory activities and selectivity,
the fluorine atom was introduced to 4-position of the pyrrole-2-
carbonitrile moiety, and the 4-fluoropyrrole-2-carbonitrile de-
rivatives were synthesized.
From the SARs of 4-fluoropyrrole-2-carbonitrile derivatives, the
inhibitory activities and selectivity were relatively improved.
Compared to the pyrrole-2-carbonitrile compound 6c
inhibition mode of compounds 6h and 6n with DPP4 were rapid
binding, evidenced by the stable inhibition rates from the first
second incubation by 0.004 mM of compound 6h and 0.01 mM of
compound 6n (Fig. 3A). The reversible dissociation was shown by
recovery activity of DPP4 by the dialysis process (Fig. 3B). The
further kinetic characterization of compounds 6h and 6n were
determined, and shown as the competitive inhibitory mode by the
same Y-intercept but with different slopes and X-intercepts be-
tween different concentration of inhibitor from the double-
reciprocal plotting (1/v vs 1/[S]) (Fig. 3CeD), the Ki values of 6h
(IC50 ¼ 0.03
m
M), compound 6f (IC50 ¼ 0.005
mM, SR: DPP8/
DPP4 ¼ 280.0; DPP9/DPP4 ¼ 520.0) showed 6.0-fold improvement
in DPP4 inhibitory activity. However, introduction of fluorine at the
4-position of pyrrole-2-carbonitrile moiety of compound 6c resul-
ted in a 1.7-fold decrease in inhibitory activity (compound 6g,
and 6n were 0.0035
using the MichaeliseMenten equation of 1/v
[Vmax[S]])(1 þ [I]/Ki)þ1/Vmax
m
M and 0.009
m
M respectively, calculated by
¼
(Km/
IC50 ¼ 0.05
m
M). To our pleasure, compound 6h (IC50 ¼ 0.004
m
M,
.
SR: DPP8/DPP4 ¼ 450.0; DPP9/DPP4 ¼ 375.0), which was thiazole-
4-yl substituted, showed excellent DPP4 inhibitory activity and
more prominent selectivity than compound 6f. The DPP4 inhibitory
activity of compound 6h was 82.5-fold higher than that of com-
To gain structural information for further optimization, the 3D
binding modes of compounds 6d, 6h, and 6n to DPP4 (from 2AJL)
were generated based on docking simulations (Fig. 4). The binding
modes indicated that the pyrrole-2-carbonitrile moiety of com-
pounds 6d, 6h, and 6n occupied the S1 hydrophobic pocket, and the
nitrile group interacted with the side chains of Ser630 and His740.
The carbonyl group formed a hydrogen bond with residue Arg125,
pound 6d (IC50 ¼ 0.33
mM). This suggested that the fluorine atom
was an important influence on improving inhibitory activities and
selectivity. Considering compound 6h was apt to metabolize, some
groups including of methyl, aryl, and thiophen-2-yl substituted at
the 2-position of the thiazole were synthesized and evaluated.
However, these compounds 6iem decreased DPP4 inhibitory ac-
tivities by 10.0- to 97.5-fold when compared to compound 6h.
Replacement of thiophene moiety with 6-bromobenzo[d][1,3]
while the a-amino group formed two hydrogen bonds with two
glutamate residues (Glu205 and Glu206). The simulations further
indicated that the 6-bromobenzo[d][1,3]dioxole moiety of com-
pound 6n could stack against the side chain of Phe357. Introduction
of the fluorine atom at the 4-position of pyrrole-2-carbonitrile,
resulted in 82.5-fold and 33.0-fold improvement in inhibitory ac-
tivity from compounds 6h and 6n, respectively in comparison of
compound 6d. The binding mode indicated that the fluorine atom
may form a hydrogen bond with residue Tyr547, which could be the
main reason for the inhibitory activity enhancement.
dioxol-5-yl yielded compound 6n (IC50 ¼ 0.01
mM, SR: DPP8/
DPP4 ¼ 470.0; DPP9/DPP4 ¼ 750.0), which also possessed excellent
DPP4 inhibitory activity and selectivity against related peptides.
2.2.2. Inhibition mode and binding mode of compounds 6d, 6h, and
6n
From the SARs of pyrrolidine-2-carbonitrile derivatives and 4-
fluoropyrrolidine-2-carbonitrile derivatives, compounds 6h and
6n were found to be potent and selective DPP4 inhibitors. The
2.2.3. In vivo studies
Based on in vitro potency and selectivity analysis, compounds 6h
and 6n were selected for acute efficacy evaluation by the oral
Scheme 2. Reagents and conditions: (a) (Boc)2O, NaHCO3, dioxane, 24 h; (b) DAST, CH2Cl2, ꢁ78 ꢀC to rt, 24 h; (c) LiOH, dioxane, H2O, overnight; (d) (Boc)2O, NH4HCO3, pyridine,
dioxane, 6 h; (e) cyanuric chiloride, DMF, 1 h; (f) TsOH, CH3CN, rt, 24 h; (g) EDCI, HOBt, TEA, DMF, 20 h; (h) CH2Cl2, TFA, 0 ꢀC to rt, 1 h.

114
X. Ji et al. / European Journal of Medicinal Chemistry 75 (2014) 111e122
Table 1
Potency and selectivity of pyrrolidine-2-carbonitrile derivatives.
Compd.
R1
R2
IC50
(m
M)a
SRb
DPP4
DPP8
DPP9
DPP8/DPP4
DPP9/DPP4
>2.5
6a
6b
6c
6d
H
H
H
H
113.6 ꢂ 17.6
0.17 ꢂ 0.02
0.03 ꢂ 0.02
0.33 ꢂ 0.02
>288.2
>288.2
>2.5
327.6
410.0
34.8
55.7 ꢂ 5.7
12.3 ꢂ 1.3
11.5 ꢂ 1.7
72.4 ꢂ 11.8
10.6 ꢂ 1.1
13.9 ꢂ 1.2
425.9
353.3
42.1
6e
H
0.52 ꢂ 0.02
125.1 ꢂ 4.1
143.6 ꢂ 15.1
240.6
276.1
6f
F
F
0.005 ꢂ 0.001
0.05 ꢂ 0.00
1.4 ꢂ 0.4
3.4 ꢂ 0.7
2.6 ꢂ 0.4
3.2 ꢂ 0.5
280.0
68.0
520.0
64.0
6g
6h
6i
F
F
0.004 ꢂ 0.004
0.04 ꢂ 0.00
1.8 ꢂ 0.3
3.3 ꢂ 0.6
1.5 ꢂ 0.3
6.8 ꢂ 0.9
450.0
82.5
375.0
170.0
6j
F
0.39 ꢂ 0.03
0.9 ꢂ 0.2
5.4 ꢂ 0.7
2.3
13.8
6k
6l
F
F
F
0.05 ꢂ 0.01
0.05 ꢂ 0.00
0.12 ꢂ 0.02
1.0 ꢂ 0.3
1.3 ꢂ 0.2
0.7 ꢂ 0.2
2.0 ꢂ 0.2
1.3 ꢂ 0.1
2.7 ꢂ 0.4
20.0
26.0
5.8
40.0
26.0
22.5
6m
6n
F
/
0.01 ꢂ 0.00
0.02 ꢂ 0.06
4.7 ꢂ 0.6
7.5 ꢂ 0.3
470.0
750.0
Sitagliptin
/
33.6 ꢂ 5.1
109.1 ꢂ 12.7
1680.0
5455.0
a
Data represent the mean value ꢂ SD with quadruplicate assay.
b
Selectivity ratio.
glucose tolerance test (OGTT) in ICR mice. A single dose of either
compound 6h or 6n (15 and 50 mg/kg) was administered to ICR
mice. The OGTT produced a significant decrease in glucose level
after 4 h compared with the vehicle group. Vildagliptin (LAF237),
which was used as a positive control, reduced the area under curve
from 0 to 120 min (AUC)0e120 min to 19.9% (LAF237, 2516.4 ꢂ 145.0;
vehicle control, 3140.8 ꢂ 104.5) at a dose of 30 mg/kg. Compound
6h reduced the value to 10.7% (2804.1 ꢂ 119.2) and 12.8%
(2739.8 ꢂ 211.3), respectively. Compound 6n reduced the value to
12.2% (2756.3 ꢂ 149.9) and 23.6% (2398.7 ꢂ 284.6) at dose of 15 mg/
kg and 50 mg/kg in a dose-dependent manner, respectively (see
Fig. 5).
Based on the DPP4 inhibitory activities (compound 6h,
IC50 ¼ 0.004
mM; compound 6n, IC50 ¼ 0.01
mM) and selectivity

X. Ji et al. / European Journal of Medicinal Chemistry 75 (2014) 111e122
115
(compound 6h, selective ratio: DPP8/DPP4
¼
450.0; DPP9/
spectroscopy was performed on a Bruker AMX-400 and AMX-300
NMR (IS as TMS). Chemical shifts were reported in parts per
million (ppm, d) downfield from tetramethylsilane. Proton coupling
patterns were described as singlet (s), doublet (d), triplet (t),
quartet (q), multiplet (m), and broad (br). Low- and high-resolution
mass spectra (LRMS and HRMS) were given with electric, electro-
spray, and matrix-assisted laser desorption ionization (EI, and ESI)
produced by a Finnigan MAT-95, LCQ-DECA spectrometer and
DPP4 ¼ 375.0; compound 6n, selective ratio: DPP8/DPP4 ¼ 470.0;
DPP9/DPP4 ¼ 750.0), compound 6h was selected for the chronic
effects experiment.
The chronic effects of compound 6h were investigated in dia-
betic BKS db/db mice with multiple doses (5 mg/kg/day to 50 mg/
kg/day) for 5 weeks. LAF237 (15 mg/kg/day) was included as a
positive control. Table 2 showed that compound 6h significantly
decreased the fasting blood glucose level compared to vehicle
control group (18.1 ꢂ 1.9 of 50 mg/kg/day v.s. 25.0 ꢂ 1.2, respec-
tively). The OGTT revealed that the glucose tolerance capacity
during 60e120 min was significant improved by treatment with
compound 6h in a dose-dependent manner, as demonstrated by
the reduction of the AUC0e120 (3094.8 ꢂ 261.7 of 15 mg/kg/day,
2958.5 ꢂ 321.9 of 50 mg/kg/day group v.s. 3915.4 ꢂ 166.9 of vehicle
control, see Table 2).
IonSpec 4.7 T. Optical rotations were reported as follows: [a]D [22]
(c ¼ g/100 mL, in solvent).
4.1.1. (2S)-1-(tert-Butoxycarbonyl)pyrrolidine-2-carboxylic acid (8)
A solution of (2S)-pyrrolidine-2-carboxylic acid (7) (0.5 g,
4.34 mmol) in dioxane (15 mL) was added (Boc)2O (1.14 g,
5.21 mmol) and saturated NaHCO3 (10.5 mL). The reaction was
stirred at room temperature for overnight. The solvent was
removed in vacuo and CH2Cl2 was added. The organics were
washed with H2O and saturated NaCl, dried, filtered and
concentrated. The residue was purified by flash chromatography
on silica gel, eluted with a mixture of EA/PE (1:5, v/v) to afford 8
2.3. Pharmacokinetic evaluation of compounds 6h and 6n
The pharmacokinetic (PK) profiles of the selected compounds
6h and 6n were assessed in SpragueeDawley (SD) rats (Table 3).
The Cmax of compound 6h at 0.3 h was 2711 ng/mL with an AUC0eN
2731 ng/mL*h at a dose of 50 mg/kg. Moreover, compound 6h
demonstrated high clearance in rats. The absolute oral bioavail-
ability for compound 6h was moderate (37.8%) and low for com-
pound 6n (16.8%), while compound 6h had a lower half-life than
compound 6n (1.45 h v.s. 3.64 h, respectively).
(0.8 g, 86%) as a white solid. 1H NMR (CDCl3, 300 MHz):
d 4.37e
4.24 (m, 1H), 3.58e3.32 (m, 2H), 2.41e1.85 (m, 4H), 1.46 (s, 9H).
MS (ESI) m/z 216 [M þ H]þ.
4.1.2. tert-Butyl (2S)-2-carbamoylpyrrolidine-1-carboxylate (9)
A mixture of compound 8 (1.00 g, 4.65 mmol), (Boc)2O (1.52 g,
6.97 mmol), NH4HCO3 (0.55 g, 6.97 mmol) and pyridine (1.0 mL) in
dioxane (20 mL) was stirred at room temperature for 6 h the
product was extracted with CH2Cl2, washed with 1 M HCl and
saturated NaCl, dried, filtrated, and concentrated. n-hexane
(100 mL) was added and the product 9 (0.85 g, 85%) began to
precipitate using the ultrasound as a white solid. 1H NMR (CDCl3,
2.4. hERG testing of compounds 6h and 6n
Blockade of the hERG channel is a significant hurdle encoun-
tered in drug discovery [37]. Based on in vivo results; compounds
6h and 6n were chosen for hERG testing (Table 4). The IC50 values of
300 MHz):
d 4.37e4.34 (m, 1H), 3.47e3.36 (m, 2H), 2.07e1.85 (m,
compounds 6h and 6n on hERG were 176.6
FluxORÔ thallium assay, respectively.
mM and 48.0
mM using
4H), 1.49 (s, 9H). MS (ESI) m/z 215 [M þ H]þ.
4.1.3. tert-Butyl (2S)-2-cyanopyrrolidine-1-carboxylate (10)
2.5. Liver metabolic enzymes P450 testing of compounds 6h and 6n
A mixture of compound 9 (5 g, 23.3 mmol) and cyanuric chi-
loride (2.58 g, 14.0 mmol) in DMF (10 mL) was stirred at room
temperature for 1 h (monitored by TLC). After the reaction
completed, the solution was extracted with EtOAc, washed, dried,
concentrated, and purified by flash chromatography on silica gel,
eluted with a mixture of PE/EA (1/1, v/v) to afford 10 (3.48 g, 76%) as
Compound 6h showed no inhibition of liver metabolic enzymes
such as CYP3A4 and CYP2C9 (percentage inhibition, 7.4% and 11.2%,
respectively, in 100
m
M). On the other hand, compound 6n
inhibition of CYP2C9 (percentage
M) while the inhibitory activity of
M (Table 4).
demonstrated no
inhibition, ꢁ20.3% in 100
m
a white solid. 1H NMR (CDCl3, 300 MHz):
d 4.76 (s, 1H), 3.51 (s, 2H),
CYP3A4 was 2.2
m
2.34e2.31 (m, 1H), 2.17e2.10 (m, 2H), 1.90e1.87 (m, 1H), 1.49 (s,
9H). MS (ESI) m/z 197 [M þ H]þ.
3. Conclusion
4.1.4. (2S)-Pyrrolidine-2-carbonitrile 4-methylbenzene-1-sulfonic
acid (11)
On the basis of SARs investigations of pyrrole-2-carbonitrile
inhibitors, we have designed, synthesized, and evaluated a series
A solution of compound 10 (10.0 g, 50.96 mmol) in CH3CN
(50 mL) was added 4-methylbenzenesulfonic acid hydrate
(14.54 g, 76.43 mmol) and stirred at room temperature for 24 h.
After the reaction completed, the solution was removed in vacuo.
The residual white solid was dissolved in EtOAc (100 mL) and
put into fridge overnight, the product 11 (10.3 g, 75%) was
of novel hetero-aromatic moieties substituted
a-amino pyrroli-
dine-2-carbonitrile derivatives as potent and selective DPP4 in-
hibitors. Compounds 6h and 6n possessed an excellent DPP4
inhibitory activities, high selectivity, good pharmacokinetic pro-
files, and good in vivo efficacy in an OGTT in ICR mice. Moreover,
compound 6h showed no hERG binding and no inhibition to liver
metabolic enzymes. Further investigation of hetero-aromatic moi-
precipitated as
300 MHz):
a
white needle crystal. 1H NMR (CD3OD,
7.77 (d, J ¼ 7.8 Hz, 2H), 7.20 (d, J ¼ 8.1 Hz, 2H),
d
eties substituted
a-amino 4-fluoropyrrolidine-2-carbonitrile de-
4.76e4.75 (m, 1H), 3.51e3.50 (m, 2H), 2.38 (s, 3H), 2.34e2.31 (m,
1H), 2.17e2.09 (m, 2H), 1.90e1.87 (m, 1H). MS (ESI) m/z 97
[M þ H]þ.
rivatives is in progress.
4. Experimental section
4.1. Chemistry
4.1.5. tert-Butyl N-[(2S)-1-[(2S)-2-cyanopyrrolidin-1-yl]-3-(furan-
3-yl)-1-oxopropan-2-yl]carbamate (13a)
A solution of (2S)-2-[(tert-butoxycarbonyl)amino]-3-(furan-3-
yl)propanoic acid (compound 12a, 105 mg, 0.410 mmol) in DMF
(5 mL) was added HATU (283 mg, 0.745 mmol) and DIPEA
The reagents (chemicals) were purchased and used without
further purification. Nuclear magnetic resonance (NMR)

116
X. Ji et al. / European Journal of Medicinal Chemistry 75 (2014) 111e122
Fig. 3. Characterization of compounds 6h and 6n inhibitory effect on DPP4. (A) Time-independent inhibition of DPP4 by compounds 6n and 6h. (B) Reversibility of DPP4 inhibition
by compounds 6n and 6h. (C) Kinetics of DPP4 inhibition by compound 6h. (D) Kinetics of DPP4 inhibition by compound 6n.
(96 mg, 0.745 mmol). After stirring for 30 min compound 11
(100 mg, 0.373 mmol) and additional DIPEA were added. This
solution was allowed to stir at room temperature for 20 h and
then the saturated NaHCO3 was added. The mixture was
extracted with EtOAc and washed with saturated NaCl, dried over
Na2SO4 and concentrated. The residue was purified with flash
chromatography on silica gel, eluted with a mixture of PE/EA (4/
1, v/v) to afford 13a (105 mg, 85%) as a white solid. 1H NMR
4.1.8. tert-Butyl N-[(2S)-1-[(2S)-2-cyanopyrrolidin-1-yl]-1-oxo-3-
(thiazol-4-yl)propan-2-yl]carbamate (13d)
In the same manner as described for 13a, 13d was prepared
from (2S)-2-[(tert-butoxycarbonyl)amino]-3-(thiazol-4-yl)prop-
anoic acid (12d). 1H NMR (CDCl3, 400 MHz)
d 8.75e8.74 (m, 1H),
7.12 (s, 1H), 7.28e7.26 (m, 1H), 5.59 (d, J ¼ 8.8 Hz, 1H), 4.77e4.66
(m, 2H), 3.62e3.56 (m, 1H), 3.32e3.27 (m, 1H), 3.19e3.17 (m, 2H),
2.92e2.82 (m, 1H), 2.15e2.07 (m, 2H), 1.34 (s, 9H). MS (ESI) m/z
351 [M þ Na]þ.
(CDCl3, 400 MHz)
d 7.33e7.32 (m, 1H), 6.28e6.27 (m, 1H), 6.14e
6.13 (m, 1H), 5.46 (d, J ¼ 8.0 Hz, 1H), 4.70e4.68 (m, 1H), 4.62e
4.56 (m, 1H), 3.55e3.49 (m, 1H), 3.05e3.00 (m, 3H), 2.04e1.99
(m, 3H), 1.37 (s, 9H). MS (ESI) m/z 334 [M þ H]þ.
4.1.9. tert-Butyl N-[(2S)-1-[(2S)-2-cyanopyrrolidin-1-yl]-1-oxo-3-
(pyridin-2-yl)propan-2-yl]carbamate (13e)
In the same manner as described for 13a, 13e was prepared from
(2S)-2-[(tert-butoxycarbonyl)amino]-3-(pyridin-2-yl)propanoic
4.1.6. tert-Butyl N-[(2S)-1-[(2S)-2-cyanopyrrolidin-1-yl]-1-oxo-3-
(thiophen-3-yl)propan-2-yl]carbamate (13b)
acid (12e). 1H NMR (CDCl3, 400 MHz)
d 8.55e8.53 (m, 2H), 7.59e
7.56 (m, 1H), 7.31e7.26 (m, 1H), 5.29 (d, J ¼ 8.8 Hz, 1H), 4.74e4.71
(m, 1H), 4.61e4.55 (m, 1H), 3.57e3.50 (m, 1H), 3.05e2.94 (m, 3H),
2.19e2.11 (m, 3H), 1.40 (s, 9H). MS (ESI) m/z 345 [M þ H]þ.
In the same manner as described for 13a, 13b was prepared from
(2S)-2-[(tert-butoxycarbonyl)amino]-3-(thiophen-3-yl)propanoic
acid (12b). 1H NMR (CDCl3, 400 MHz)
d 7.27e7.25 (m, 1H), 7.09 (s,
1H), 6.98e6.97 (m, 1H), 5.41 (d, J ¼ 8.8 Hz, 1H), 4.68e4.65 (m, 1H),
4.52e4.46 (m, 1H), 3.44e3.38 (m, 1H), 3.04e2.99 (m, 4H), 2.80e
2.75 (m, 2H), 1.39 (s, 9H). MS (ESI) m/z 350 [M þ H]þ.
4.1.10. 1-tert-Butyl 2-methyl (2S,4R)-4-hydroxypyrrolidine-1,2-
dicarboxylate (15)
In the same manner as described for 8, 15 was prepared from
(2S,4R)-methyl 4-hydroxypyrrolidine-2-carboxylate hydrochloride
4.1.7. tert-Butyl N-[(2S)-1-[(2S)-2-cyanopyrrolidin-1-yl]-1-oxo-3-
(benzo[b]thiophen-3-yl)propan-2-yl]carbamate (13c)
(14). 1H NMR (CDCl3, 300 MHz):
d 4.35e4.48 (m, 2H), 3.72 (s, 3H),
3.46e3.65 (m, 2H), 2.03e2.29 (m, 2H), 1.39 (s, 9H). MS (ESI) m/z 246
In the same manner as described for 13a, 13c was prepared
from (2S)-2-[(tert-butoxycarbonyl)amino]-3-(benzo[b]thiophen-
[M þ H]þ.
3-yl)propanoic acid (12c). 1H NMR (CDCl3, 400 MHz)
d 7.93e7.82
(m, 2H), 7.44e7.32 (m, 2H), 7.28e7.26 (m, 1H), 5.54 (d, J ¼ 8.0 Hz,
1H), 4.71e4.62 (m, 2H), 3.36e3.32 (m, 1H), 3.25e3.19 (m, 2H),
2.41e2.35 (m, 1H), 2.06e1.98 (m, 3H), 1.42 (s, 9H). MS (ESI) m/z
400 [M þ H]þ.
4.1.11. 1-tert-Butyl 2-methyl (2S,4S)-4-fluoropyrrolidine-1,2-
dicarboxylate (16)
Under the Nitrogen protected, a solution of compound 15 (2 g,
8.2 mmol) in dry CH2Cl2 cooled to ꢁ78 ꢀC, was added DAST (1.97 g,

X. Ji et al. / European Journal of Medicinal Chemistry 75 (2014) 111e122
117
Fig. 4. Three-dimensional structural modes of inhibitors 6d (a), 6h (b), and 6n (c) to DPP4 (PDB ID: 2AJL) derived from the docking simulations. These three images were generated
using the Pymol program.
12.2 mmol). After stirring 3 h, the reaction slowly warmed to room
temperature overnight. Then the reaction solution was poured into
200 mL ice and NaHCO3 mixture solution and stirred acutely until
no CO2 evolution. The organic layer was separated and the aqueous
layer extracted with CH2Cl2, dried, filtered and concentrated. The
residue was purified by flash chromatography on silica gel, eluted
with a mixture of EA/PE (1:4, v/v), to afford 16 (1.6 g, 78%) as a
colorless oil. 1H NMR (CDCl3, 300 MHz):
d 5.18e5.34 (m, 2H), 3.72 (s,
Fig. 5. Single dose of compounds 6h and 6n on OGTT in ICR mice. Compounds 6h and 6n were administered to ICR mice after 16-h starvation, then the oral glucose tolerance test
(2.5 g/kg) was conducted after 4 h of the single dose, the blood glucose level at 0, 30, 60, 90 and 120 min were recorded for area under curve calculation. The results are presented as
the mean ꢂ SE.*, p < 0.05 compared to vehicle group; **, p < 0.01 compared to vehicle group (n ¼ 10).

118
X. Ji et al. / European Journal of Medicinal Chemistry 75 (2014) 111e122
Table 2
Chronic effects of compound 6h on OGTT of diabetic BKS db/db mice.
Blood glucose (mmol/L) after orally glucose challenge
AUC 0e120 min
0
30
60
90
120
Veh
6h-5
6h-15
6h-50
LAF237
25.0 ꢂ 1.2
46.6 ꢂ 2.2
45.1 ꢂ 2.4
41.3 ꢂ 2.2
38.0 ꢂ 4.0*
31.3 ꢂ 1.2**
30.2 ꢂ 1.5
27.2 ꢂ 1.3
24.1 ꢂ 2.6
20.8 ꢂ 2.6*
20.5 ꢂ 2.2**
20.7 ꢂ 1.6**
28.1 ꢂ 1.3
3915.4 ꢂ 166.9
3589.4 ꢂ 218.1
3094.8 ꢂ 261.7**
2958.5 ꢂ 321.9**
2778.6 ꢂ 183.2**
21.5 ꢂ 1.7*
18.0 ꢂ 1.7**
18.1 ꢂ 1.9**
17.9 ꢂ 1.6**
27.5 ꢂ 1.4
24.5 ꢂ 2.4*
20.6 ꢂ 2.4**
20.4 ꢂ 2.3**
21.4 ꢂ 2.2**
21.8 ꢂ 2.4**
19.9 ꢂ 2.0**
21.0 ꢂ 1.9**
C57BKS db/db mice with 6h (5 mg/kg/day to 50 mg/kg/day) treatment, oral glucose tolerance test (1.5 g/kg) was carried out after 6-hrs starvation of 5th-week treatment, the
blood glucose level was recorded for the glucose tolerance capacity evaluation. The results are presented as the mean ꢂ SE. *, p < 0.05 compared to vehicle group; **, p < 0.01
compared to vehicle group (n ¼ 8e11).
3H), 3.46e3.65 (m, 2H), 2.03e2.29 (m, 2H), 1.40 (s, 9H). MS (ESI) m/
z 248 [M þ H]þ.
1H), 4.99e5.04 (m, 1H), 3.42e3.67 (m, 2H), 2.30e2.70 (m, 3H), 3.17
(s, 3H). 13C NMR (CD3OD, 100 MHz):
143.3, 142.0, 130.0, 127.0,
116.4, 93.9, 92.5, 53.8 (d, J ¼ 19.0 Hz), 46.8, 38.4 (d, J ¼ 17.0 Hz), 21.4.
MS (EI) m/z 114 [M]þ. HRMS (EI) m/z calcd C5H7 F N2 114.0593 [M]þ,
found 114.0668.
d
4.1.12. (2S,4S)-1-[(tert-Butoxy)carbonyl]-4-fluoropyrrolidine-2-
carboxylic acid (17)
A solution of compound 16 (2.46 g, 9.96 mmol) in dioxane
(20 mL), was added 10 mL of H2O followed by lithium hydroxide
hydrate (2.09 g, 49.8 mmol) at room temperature, the reaction was
stirred for 3 h (monitored by TLC). Then the solution was filtered
removing the excess lithium hydroxide, the filtrate was removed
the solvent in vacuo, the residue was added 10 mL of H2O and
acidified with concentrated HCl to pH 3e4, the product began to
precipitate, filtered and dried to afford 17 (2.2 g, 95%) as a white
4.1.16. tert-Butyl N-[(2S)-1-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-1-oxo-3-(thiophen-2-yl)propan-2-yl]carbamate (21f)
A solution of (2S)-2-[(tert-butoxycarbonyl)amino]-3-(thiophen-
2-yl)propanoic acid (compound 12f, 104.2 mg, 0.384 mmol) in DMF
(5 mL) was added HOBt (141.6 mg, 1.05 mmol) and EDCI (133.9 mg,
0.698 mmol). After stirring for 30 min compound 20 (100 mg,
0.349 mmol) and additional TEA (0.15 mL, 1.05 mmol) were added.
This solution was allowed to stir at room temperature for 20 h and
then the saturated NaHCO3 was added. The mixture was extracted
with EtOAc and washed with saturated NaCl, dried over Na2SO4 and
concentrated. The residue was purified with flash chromatography
solid. 1H NMR (CD3OD, 300 MHz):
d 5.12e5.30 (m, 1H), 4.38e4.43
(m, 1H), 3.59e3.72 (m, 2H), 2.39e2.46 (m, 2H), 1.47 (s, 9H). MS (ESI)
m/z 232 [M e H]ꢁ.
on silica gel, eluted with a mixture of PE/EA (4/1, v/v) to afford 21f
4.1.13. tert-Butyl (2S,4S)-2-carbamoyl-4-fluoropyrrolidine-1-
carboxylate (18)
1
(112 mg, 86%) as a white solid. H NMR (CDCl3, 300 MHz):
d 7.20
(dd, J1 ¼ 6.0 Hz, J2 ¼ 6.0 Hz, 1H), 6.93e6.97 (m, 1H), 6.92 (dd,
J1 ¼ 6.0 Hz, J2 ¼ 6.0 Hz, 1H), 5.13e5.18 (m, 1H), 4.93 (d, J ¼ 9.0 Hz,
1H), 4.45e4.52 (m, 1H), 3.74e3.92 (dd, J1 ¼ 12.0 Hz, J2 ¼ 12.0 Hz,
1H), 3.16e3.35 (m, 3H), 2.56 (t, 1H, J ¼ 15.0 Hz), 2.14e2.36 (m, 1H),
1.41 (s, 9H). MS (ESI) m/z 390 [M þ Na]þ.
In the same manner as described for compound 9, compound
18 was prepared from (2S,4S)-1-[(tert-Butoxy)carbonyl]-4-
fluoropyrrolidine-2-carboxylic acid (17). 1H NMR (CDCl3,
300 MHz): d 5.59 (br, s,1H), 5.13e5.31 (m,1H), 4.36 (br, s,1H), 3.52e
3.81 (m, 2H), 2.31e2.78 (m, 2H), 1.48 (s, 9H). MS (ESI) m/z 233
[M þ H]þ.
4.1.17. tert-Butyl N-[(2S)-3-(benzo[b]thiophen-3-yl)-1-[(2S,4S)-2-
cyano-4-fluoropyrrolidin-1-yl]-1-oxopropan-2-yl]carbamate (21g)
In the same manner as described for compound 21f, compound
21g was prepared from (2S)-3-(benzo[b]thiophen-3-yl)-2-[(tert-
butoxycarbonyl)amino]propanoic acid (12g). 1H NMR (CDCl3,
4.1.14. tert-Butyl (2S,4S)-2-cyano-4-fluoropyrrolidine-1-
carboxylate (19)
In the same manner as described for compound 10, compound
19 was prepared from tert-Butyl (2S,4S)-2-carbamoyl-4-
fluoropyrrolidine-1-carboxylate (18). 1H NMR (CDCl3, 300 MHz):
300 MHz):
d 7.88e7.92 (m, 2H), 7.40e7.45 (m, 2H), 7.32 (s, 1H),
5.09e5.14 (m, 1H), 4.91 (d, J ¼ 9.0 Hz, 1H), 4.60e4.68 (m, 1H), 3.50e
3.67 (dd, J1 ¼ 12.0 Hz, J2 ¼ 12.0 Hz, 1H), 3.24e3.39 (m, 2H), 3.75e
3.87 (m, 1H), 2.51 (t, 1H, J ¼ 15.0 Hz), 2.06e2.29 (m, 1H), 1.43 (s, 9H).
MS (ESI) m/z 440 [M þ Na]þ.
d
5.21e5.41 (m,1H), 4.62e4.76 (m,1H), 3.49e3.93 (m, 2H), 2.63 (dd,
J1 ¼ 15.0 Hz, J2 ¼ 15.3 Hz, 1H), 2.30e2.44 (m, 1H), 1.49 (s, 9H). MS
(ESI) m/z 215 [M þ H]þ.
4.1.15. (2S,4S)-4-Fluoropyrrolidine-2-carbonitrile: 4-
methylbenzene-1-sulfonic acid (20)
4.1.18. tert-Butyl N-[(2S)-1-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-1-oxo-3-(thiazol-4-yl)propan-2-yl]carbamate (21h)
In the same manner as described for compound 21f, compound
21h was prepared from (2S)-2-[(tert-butoxycarbonyl)amino]-3-
(thiazol-4-yl)propanoic acid (12h). 1H NMR (CDCl3, 300 MHz):
In the same manner as described for compound 11, compound
20 was prepared from tert-butyl (2S,4S)-2-cyano-4-fluoropy
rrolidine-1-carboxylate (19). 1H NMR (CD3OD, 300 MHz):
d 7.51
(dd, J1 ¼ 12.0 Hz, J2 ¼ 6.0 Hz, 2H), 7.11e7.16 (m, 2H), 5.45e5.62 (m,
Table 3
Selected PK parameters for compounds 6h and 6n in SD rats.
Compd.
Admini.
Dose mg/kg
Tmax
h
Cmax ng/mL
AUC0et ng/mL*h
AUC0eN ng/mL*h
MRT h
t1/2
h
CLz L/h/kg
F %
6h
p.o.
i.v.
p.o.
i.v.
50
20
20
5
0.3
0.3
0.3
0.3
2711
2957
2204
2027
2731
2889
1291
1918
2838
2896
1581
1918
1.47
1.48
3.15
0.55
1.45
2.27
3.64
0.63
/
37.8
/
16.8
/
6.96
/
2.61
6n
p.o., oral administration; i.v., intravenous injection.

X. Ji et al. / European Journal of Medicinal Chemistry 75 (2014) 111e122
119
Table 4
hERG and liver metabolic enzymes P450 testing of compounds 6h and 6n.
4.1.23. tert-Butyl N-[(2S)-1-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-1-oxo-3-(2-(thiophen-2-yl)thiazol-4-yl)propan-2-yl]carbamate
(21m)
In the same manner as described for compound 21f, compound
21m was prepared from (2S)-2-[(tert-butoxycarbonyl)amino]-3-(2-
(thiophen-2-yl)thiazol-4-yl)propanoic acid (12m). 1H NMR (CDCl3,
Compd.
hERGa (IC50
,
m
M)
CYP450 (IC50
CYP3A4
, mM)
CYP2C9
6h
6n
176.6
48.0
NIb
2.2
NI
NI
300 MHz):
d
7.49 (d, J ¼ 3.0 Hz, 1H), 7.38 (d, J ¼ 6.0 Hz, 1H), 7.04e
a
using FluxORÔ thallium assay.
NI: no inhibition (100 mM).
7.10 (m, 1H), 7.01 (s, 1H), 5.40 (d, J ¼ 9.0 Hz, 1H), 5.20e5.28 (m, 1H),
4.88 (d, J ¼ 9.0 Hz, 1H), 4.75e4.83 (m, 1H), 3.81e3.99 (dd,
J1 ¼12.0 Hz, J2 ¼ 12.0 Hz,1H), 3.75e3.87 (m,1H), 3.09e3.24 (m, 2H),
2.51 (t, 1H, J ¼ 15.0 Hz), 2.11e2.36 (m, 1H), 1.41 (s, 9H). MS (ESI) m/z
409 [M þ Na]þ.
b
d
7.77 (d, J ¼ 3.0 Hz, 1H), 7.15 (d, J ¼ 3.0 Hz, 1H), 5.50 (d, J ¼ 6.0 Hz,
1H), 5.19e5.24 (m, 1H), 4.88 (d, J ¼ 6.0 Hz, 1H), 4.69e4.75 (m, 1H),
3.82e3.95 (dd, J1 ¼ 9.0 Hz, J2 ¼ 9.0 Hz, 1H), 3.63e3.72 (m, 1H), 3.22
(d, J ¼ 6.0 Hz, 2H), 2.53 (t,1H, J ¼ 12.0 Hz), 2.18e2.33 (m,1H),1.38 (s,
9H). MS (ESI) m/z 391 [M þ Na]þ.
4.1.24. tert-Butyl N-[(2S)-3-(6-bromobenzo[d][1,3]dioxol-5-yl)-1-
[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]-1-oxopropan-2-yl]
carbamate (21n)
In the same manner as described for compound 21f, compound
21n was prepared from (2S)-3-(6-bromobenzo[d][1,3]dioxol-5-yl)-
2-[(tert-butoxycarbonyl)amino]propanoic acid (12n). 1H NMR
4.1.19. tert-Butyl N-[(2S)-1-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-3-(2-methylthiazol-4-yl)-1-oxopropan-2-yl]carbamate (21i)
In the same manner as described for compound 21f, compound
21i was prepared from (2S)-2-[(tert-butoxycarbonyl)amino]-3-(2-
methylthiazol-4-yl)propanoic acid (12j). 1H NMR (CDCl3,
(CDCl3, 300 MHz):
d 6.96 (s, 1H), 6.69 (s, 1H), 5.93 (s, 1H), 5.87 (s,
1H), 5.30e5.36 (m, 1H), 4.87 (d, J ¼ 9.0 Hz, 1H), 4.45e4.61 (m, 1H),
3.74e3.90 (dd, J1 ¼ 12.0 Hz, J2 ¼ 12.0 Hz, 1H), 3.31e3.45 (m, 1H),
3.08e3.14 (m,1H), 2.85e2.94 (m, 2H), 2.56 (t,1H, J ¼ 15.0 Hz), 2.21e
2.35 (m, 1H), 1.35 (s, 9H). MS (ESI) m/z 507 [M þ Na]þ.
300 MHz):
d
6.90 (s, 1H), 5.18e5.23 (m, 1H), 4.90 (d, J ¼ 9.0 Hz, 1H),
4.68e4.74 (m, 1H), 3.86e3.99 (dd, J1 ¼ 12.0 Hz, J2 ¼ 12.0 Hz, 1H),
3.73e3.83 (m, 1H), 3.12 (d, J ¼ 6.0 Hz, 2H), 2.68 (s, 3H), 2.55 (t, 1H,
J ¼ 12.0 Hz), 2.17e2.40 (m, 1H), 1.40 (s, 9H). MS (ESI) m/z 405
[M þ Na]þ.
4.1.25. (2S)-1-[(2S)-2-amino-3-(Furan-3-yl)propanoyl]pyrrolidine-
2-carbonitrile (6a)
A solution of 13a (120 mg) in dry CH2Cl2 (2 mL) was added
CF3COOH (1 mL) at ice-bathe and warmed to room temperature.
After 1 h, the mixture was concentrated, and the residue was added
10 mL Et2O. The white solid was precipitated, filtered and to afford
6a as TFA salt (72 mg, 85%). HPLC: 96.12%, tR ¼ 1.32 min 1H NMR
4.1.20. tert-Butyl N-[(2S)-1-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-1-oxo-3-(2-phenylthiazol-4-yl)propan-2-yl]carbamate (21j)
In the same manner as described for compound 21f, compound
21j was prepared from (2S)-2-[(tert-butoxycarbonyl)amino]-3-(2-
phenylthiazol-4-yl)propanoic acid (12j). 1H NMR (CDCl3,
(400 MHz, DMSO-d6):
d 8.19 (br, s, 2H), 7.60 (s, 1H), 6.44e6.43 (m,
300 MHz): d 7.90e7.97 (m, 2H), 7.40e7.45 (m, 3H), 7.08 (s, 1H), 5.46
1H), 6.25 (d, J ¼ 3.2 Hz, 1H), 4.80 (q, J ¼ 4.0 Hz, 1H), 4.33 (t,
J ¼ 6.4 Hz, 1H), 3.53 (q, J ¼ 9.2 Hz, 2H), 3.11e3.09 (m, 1H), 2.93e2.89
(m, 1H), 2.17e2.08 (m, 2H), 1.94e1.83 (m, 2H). 13C NMR (100 MHz,
(d, J ¼ 6.0 Hz, 1H), 5.23e5.28 (m, 1H), 4.90 (d, J ¼ 9.0 Hz, 1H), 4.75e
4.83 (m, 1H), 3.80e3.98 (dd, J1 ¼ 12.0 Hz, J2 ¼ 12.0 Hz, 1H), 3.68e
3.80 (m, 1H), 3.21e3.26 (m, 2H), 2.52 (t, 1H, J ¼ 15.0 Hz), 2.17e2.36
(m, 1H), 1.39 (s, 9H). MS (ESI) m/z 467 [M þ Na]þ.
DMSO-d6):
d 167.9, 134.8, 129.3, 127.1, 124.7, 118.9, 52.5, 51.9, 49.0,
46.7, 29.2, 24.4. MS (ESI) m/z 234 [M þ H]þ. HRMS (ESI) calcd for
C
11H15N4OS 234.1243 [M þ H]þ, found 234.1265.
4.1.21. tert-Butyl N-[(2S)-3-(2-(4-chlorophenyl)thiazol-4-yl)-1-
[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-yl]-1-oxopropan-2-yl]
carbamate (21k)
4.1.26. (2S)-1-[(2S)-2-Amino-3-(thiophen-3-yl)propanoyl]
pyrrolidine-2-carbonitrile (6b)
In the same manner as described for compound 21f, com-
pound 21k was prepared from (2S)-2-[(tert-butoxycarbonyl)
amino]-3-(2-(4-chlorophenyl)thiazol-4-yl)propanoic acid (12l).
In the same manner as described for compound 6a, compound
6b was prepared from compound 13b. HPLC: 100.00%, tR ¼ 1.31 min
1H NMR (400 MHz, DMSO-d6):
d 8.19 (br, s, 2H), 7.50e7.49 (m, 1H),
1H NMR (CDCl3, 300 MHz):
d
7.88 (d, J ¼ 3.0 Hz, 2H), 7.40 (d,
7.03e7.01 (m, 1H), 6.95 (d, J ¼ 3.2 Hz, 1H), 4.80 (q, J ¼ 4.8 Hz, 1H),
4.32 (t, J ¼ 7.6 Hz, 1H), 3.52 (q, J ¼ 7.2 Hz, 1H), 3.34e3.19 (m, 2H),
2.93e2.88 (m, 1H), 2.16e2.05 (m, 2H), 1.90e1.78 (m, 2H). 13C NMR
J ¼ 9.0 Hz, 2H), 7.09 (s, 1H), 5.47 (d, J ¼ 9.0 Hz, 1H), 5.24e5.32 (m,
1H), 4.88 (d, J ¼ 9.0 Hz, 1H), 4.75e4.90 (m, 1H), 3.80e3.97 (dd,
J1 ¼ 12.0 Hz, J2 ¼ 12.0 Hz, 1H), 3.69e3.80 (m, 1H), 3.18e3.24 (m,
2H), 2.52 (t, 1H, J ¼ 15.0 Hz), 2.11e2.36 (m, 1H), 1.40 (s, 9H). MS
(ESI) m/z 501 [M þ Na]þ.
(100 MHz, DMSO-d6):
d 167.5, 159.4, 147.3, 145.4, 142.7, 118.9, 51.8,
46.9, 46.8, 33.3, 29.8, 25.2. MS (ESI) m/z 250 [M þ H]þ. HRMS (ESI)
calcd for C11H15N4OS 250.1014 [M þ H]þ, found 250.1004.
4.1.22. tert-Butyl N-[(2S)-1-[(2S,4S)-2-cyano-4-fluoropyrrolidin-1-
yl]-1-oxo-3-(2-(4-(trifluoromethyl)phenyl)thiazol-4-yl)propan-2-
yl]carbamate (21l)
In the same manner as described for compound 21f, compound
21l was prepared from (2S)-2-[(tert-butoxycarbonyl)amino]-3-(2-
(4-(trifluoromethyl)phenyl)thiazol-4-yl)propanoic acid (12l). 1H
4.1.27. (2S)-1-[(2S)-2-Amino-3-(benzo[b]thiophen-3-yl)propanoyl]
pyrrolidine-2-carbonitrile (6c)
In the same manner as described for compound 6a, compound
6c was prepared from compound 13c. HPLC: 96.16%, tR ¼ 1.36 min
1H NMR (400 MHz, DMSO-d6):
1H), 7.90 (d, J ¼ 7.2 Hz, 1H), 7.50e7.41 (m, 3H), 4.80 (q, J ¼ 4.8 Hz,
1H), 4.38 (t, J ¼ 8.0 Hz, 1H), 3.45e3.36 (m, 2H), 2.68e2.64 (m, 1H),
2.15e2.10 (m, 2H), 2.05e2.00 (m, 1H), 1.83e1.80 (m, 1H). 13C NMR
d
8.28 (br, s, 2H), 8.05 (d, J ¼ 8.0 Hz,
NMR (CDCl3, 300 MHz):
d
8.04 (d, J ¼ 9.0 Hz, 2H), 7.67 (d, J ¼ 9.0 Hz,
2H), 7.17 (s, 1H), 5.50 (d, J ¼ 9.0 Hz, 1H), 5.21e5.26 (m, 1H), 4.84 (d,
J ¼ 9.0 Hz, 1H), 4.77e4.85 (m, 1H), 3.81e3.98 (dd, J1 ¼ 12.0 Hz,
J2 ¼ 12.0 Hz, 1H), 3.69e3.81 (m, 1H), 3.20e3.32 (m, 2H), 2.51 (t, 1H,
J ¼ 15.0 Hz), 2.13e2.34 (m, 1H), 1.40 (s, 9H). MS (ESI) m/z 535
[M þ Na]þ.
(100 MHz, DMSO-d6): d 168.1, 140.0, 138.4,129.2, 126.5, 125.0, 123.6,
122.0, 118.8, 67.4, 50.8, 46.8, 40.2, 29.8, 25.7, 25.2. MS (ESI) m/z 300
[M þ H]þ. HRMS (ESI) calcd for C11H15N4OS 300.1171 [M þ H]þ,
found 300.1176.

120
X. Ji et al. / European Journal of Medicinal Chemistry 75 (2014) 111e122
4.1.28. (2S)-1-[(2S)-2-Amino-3-(thiazol-4-yl)propanoyl]
pyrrolidine-2-carbonitrile (6d)
4.1.33. (2S,4S)-1-[(2S)-2-Amino-3-(2-methylthiazol-4-yl)
propanoyl]-4-fluoropyrrolidine-2-carbonitrile (6i)
In the same manner as described for compound 6a, compound
6d was prepared from compound 13d. HPLC: 98.73%,
In the same manner as described for compound 6a, compound
6i was prepared from compound 21i. HPLC: 95.77%, tR ¼ 1.30 min
tR ¼ 1.31 min 1H NMR (400 MHz, DMSO-d6):
d
8.13 (br, s, 2H),
1H NMR (CD3OD, 400 MHz),
d 7.20 (s, 1H), 5.30e5.44 (m, 1H), 4.99
7.56e7.54 (m, 1H), 7.30e7.29 (m, 1H), 7.03e7.01 (m, 1H), 4.79 (q,
J ¼ 4.8 Hz, 1H), 4.29 (t, J ¼ 8.0 Hz, 1H), 3.48 (q, J ¼ 6.8 Hz, 1H),
3.12e3.05 (m, 2H), 2.88e2.84 (m, 1H), 2.15e2.07 (m, 2H), 1.91e
1.88 (m, 1H), 1.78e1.75 (m, 1H). 13C NMR (100 MHz, DMSO-d6):
(d, J ¼ 8.0 Hz, 1H), 4.43 (t, 1H, J ¼ 6.0 Hz), 3.61e3.80 (m, 2H), 3.34 (s,
1H), 3.26e3.29 (m, 2H), 2.69 (s, 3H), 2.54 (t, 1H, J ¼ 12.0 Hz), 2.31e
2.47 (m, 1H). 13C NMR (CD3OD, 100 MHz):
d 168.1, 167.5, 147.9, 117.2,
117.0, 92.2 (d, J ¼ 142.0 Hz), 52.7 (d, J ¼ 19.0 Hz), 51.3, 44.9, 35.5 (d,
d
171.1, 165.3, 155.0, 150.2, 118.9, 60.2, 56.5, 55.1, 28.9, 25.2, 22.3.
J ¼ 17.0 Hz), 31.9, 17.4. MS (ESI) m/z 283 [M þ H]þ. HRMS (ESI) calcd
MS (ESI) m/z 251 [M þ H]þ. HRMS (ESI) calcd for C11H15N4OS
C
12H15FN4OS 283.1029 [M þ H]þ, found 283.1025.
251.0967 [M þ H]þ, found 251.0943.
4.1.34. (2S,4S)-1-[(2S)-2-amino-3-(2-phenylthiazol-4-yl)
propanoyl]-4-fluoropyrrolidine-2-carbonitrile (6j)
4.1.29. (2S)-1-[(2S)-2-Amino-3-(pyridin-2-yl)propanoyl]
pyrrolidine-2-carbonitrile (6e)
In the same manner as described for compound 6a, compound
6j was prepared from compound 21j. HPLC: 96.34%, tR ¼ 1.32 min
In the same manner as described for compound 6a, compound
6e was prepared from compound 13e. HPLC: 95.91%, tR ¼ 1.30 min
1H NMR (CD3OD, 400 MHz),
d 7.90e7.96 (m, 2H), 7.40e7.50 (m, 3H),
1H NMR (400 MHz, DMSO-d6):
d 8.17 (br, s, 2H), 7.16e7.14 (m, 2H),
7.37 (s, 1H), 5.27e5.42 (m, 1H), 4.58 (t, 1H, J ¼ 6.0 Hz), 4.11 (q,
J ¼ 12.0 Hz, 1H), 3.87e4.01 (m, 1H), 3.61e3.72 (m, 1H), 3.34e3.49
(m, 2H), 2.66 (m, 1H), 2.12e2.29 (m, 1H). 13C NMR (CD3OD,
6.89e6.87 (m, 2H), 4.81e4.78 (m, 1H), 4.26 (t, J ¼ 8.0 Hz, 1H), 3.46e
3.40 (m, 1H), 3.05e2.91 (m, 2H), 2.74e2.67 (m, 1H), 2.17e2.03 (m,
2H), 1.88e1.83 (m, 1H), 1.72e1.67 (m, 1H). 13C NMR (100 MHz,
100 MHz):
d 169.0, 164.3, 164.1, 150.9, 133.0, 130.2, 128.9, 126.0,
117.5, 89.5 (d, J ¼ 175.0 Hz), 56.6, 54.2, 52.1 (d, J ¼ 15.0 Hz), 35.8 (d,
DMSO-d6):
d 167.4, 148.5, 143.2, 118.8, 111.3, 109.3, 52.4, 50.4, 49.1,
J ¼ 21.0 Hz), 33.7. MS (ESI) m/z 345 [M þ H]þ. HRMS (ESI) calcd
46.7, 29.1, 24.3. MS (ESI) m/z 245 [M þ H]þ. HRMS (ESI) calcd
17H17FN4OS 345.1185 [M þ H]þ, found 345.1192.
C
13H17N4O 245.1402 [M þ H]þ, found 245.1412.
C
4.1.35. (2S,4S)-1-[(2S)-2-Amino-3-(2-(4-chlorophenyl)thiazol-4-yl)
propanoyl]-4-fluoropyrrolidine-2-carbonitrile (6k)
4.1.30. (2S,4S)-1-[(2S)-2-Amino-3-(thiophen-2-yl)propanoyl]-4-
fluoropyrrolidine-2-carbonitrile (6f)
In the same manner as described for compound 6a, compound
6k was prepared from compound 21k. HPLC: 100.00%, tR ¼ 1.35 min
In the same manner as described for compound 6a, compound
6f was prepared from compound 21f. HPLC: 100.00%, tR ¼ 1.29 min
1H NMR (CD3OD, 400 MHz),
d
7.98 (d, J ¼ 8.0 Hz, 2H), 7.48 (d,
1H NMR (CD3OD, 400 MHz),
d 7.30e7.38 (m, 1H), 6.99e7.05 (m, 2H),
J ¼ 12.0 Hz, 2H), 7.40 (s, 1H), 5.25e5.39 (m, 1H), 5.02 (d, J ¼ 8.0 Hz,
1H), 4.56 (t, 1H, J ¼ 6.0 Hz), 3.74e3.83 (m, 2H), 3.39 (d, J ¼ 8.0 Hz,
2H), 2.53 (t, 1H, J ¼ 12.0 Hz), 2.34e2.49 (m, 1H). 13C NMR (CD3OD,
5.23e5.26 (m, 1H), 5.04 (d, J ¼ 8.0 Hz,1H), 4.28e4.35 (m, 1H), 3.65e
3.78 (m, 1H), 3.39e3.44 (m, 2H), 3.14e3.29 (m, 1H), 2.53 (t, 1H,
J ¼ 12.0 Hz), 2.29e2.44 (m,1H). 13C NMR (CD3OD,100 MHz):
d 167.6,
100 MHz): d 168.1, 167.5, 161.8, 149.7, 134.0, 131.8, 128.9, 127.5, 118.3,
134.2, 128.1, 127.5, 125.5, 117.0, 92.0 (d, J ¼ 142.0 Hz), 52.7, 52.6 (d,
J ¼ 20.0 Hz), 45.1, 35.5 (d, J ¼ 17.0 Hz), 30.8. MS (ESI) m/z 268
[M þ H]þ. HRMS (ESI) calcd C12H14FN3OS 268.0920 [M þ H]þ, found
268.0909.
117.2, 92.2 (d, J ¼ 177.0 Hz), 52.7 (d, J ¼ 23.0 Hz), 51.1, 45.0, 35.4 (d,
J ¼ 21.0 Hz), 32.1. MS (ESI) m/z 379 [M þ H]þ. HRMS (ESI) calcd
C
17H16ClFN4OS 379.0796 [M þ H]þ, found 379.0801.
4.1.36. (2S,4S)-1-[(2S)-2-Amino-3-(2-(4-(trifluoromethyl)phenyl)
thiazol-4-yl)propanoyl]-4-fluoropyrrolidine-2-carbonitrile (6l)
In the same manner as described for compound 6a, compound
6l was prepared from compound 21l. HPLC: 100.00%, tR ¼ 1.35 min
4.1.31. (2S,4S)-1-[(2S)-2-Amino-3-(benzo[b]thiophen-3-yl)
propanoyl]-4-fluoropyrrolidine-2-carbonitrile (6g)
In the same manner as described for compound 6a, compound
6g was prepared from compound 21g. HPLC: 100.00%, tR ¼ 1.32 min
1H NMR (CD3OD, 400 MHz),
d
8.19 (d, J ¼ 8.0 Hz, 2H), 7.77 (d,
1H NMR (CD3OD, 400 MHz),
d 7.89e7.98 (m, 2H), 7.39e7.50 (m, 3H),
J ¼ 12.0 Hz, 2H), 7.49 (s, 1H), 5.25e5.39 (m, 1H), 5.02 (d, J ¼ 8.0 Hz,
1H), 4.58 (t, 1H, J ¼ 6.0 Hz), 3.74e3.83 (m, 2H), 3.42 (d, J ¼ 8.0 Hz,
2H), 2.53 (t, 1H, J ¼ 12.0 Hz), 2.34e2.48 (m, 1H). 13C NMR (CD3OD，
5.04e5.17 (m, 1H), 5.00 (d, J ¼ 8.0 Hz, 1H), 4.39e4.43 (m, 1H), 3.60
(dd, J1 ¼ 4.0 Hz, J2 ¼ 4.0 Hz, 1H), 3.42e3.54 (m, 2H), 2.67e2.75 (m,
1H), 2.45 (t, 1H, J ¼ 12.0 Hz), 2.21e2.37 (m, 1H). 13C NMR (CD3OD,
100 MHz):
d
167.5, 167.4, 161.5, 150.1, 136.5, 131.4 (q, J1 ¼ 26.0 Hz,
100 MHz):
d 168.0, 140.7, 137.9, 127.4, 126.4, 124.5, 122.8, 120.7,
J2 ¼ 26.0 Hz), 126.8, 125.7, 119.2, 117.2, 92.2 (d, J ¼ 22.0 Hz), 52.7 (d,
J ¼ 19.0 Hz), 51.1, 45.0, 35.4 (d, J ¼ 17.0 Hz), 32.1. MS (ESI) m/z 413
[M þ H]þ. HRMS (ESI) calcd C18H16F4N4OS 413.1059 [M þ H]þ, found
413.1047.
116.9, 91.6 (d, J ¼ 143.0 Hz), 52.5 (d, J ¼ 19.0 Hz), 51.0, 45.0, 35.3 (d,
J ¼ 17.0 Hz), 29.8. MS (ESI) m/z 318 [M þ H]þ. HRMS (ESI) calcd
C
16H16FN3OS 318.1076 [M þ H]þ, found 318.1083.
4.1.32. (2S,4S)-1-[(2S)-2-Amino-3-(thiazol-4-yl)propanoyl]-4-
fluoropyrrolidine-2-carbonitrile (6h)
4.1.37. (2S,4S)-1-[(2S)-2-Amino-3-(2-(thiophen-2-yl)thiazol-4-yl)
propanoyl]-4-fluoropyrrolidine-2-carbonitrile (6m)
In the same manner as described for compound 6a, compound
6h was prepared from compound 21h. HPLC: 100.00%,
In the same manner as described for compound 6a, compound
6m was prepared from compound 21m. HPLC: 100.00%,
tR ¼ 1.29 min 1H NMR (CD3OD, 400 MHz),
d
9.31 (s, 1H), 7.65 (s,
tR ¼ 1.30 min 1H NMR (CD3OD, 400 MHz),
d 7.57e7.63 (m, 2H), 7.29
1H), 5.31-5.45 (m, 1H), 5.03 (d, J ¼ 8.0 Hz, 1H), 4.55 (t, 1H,
J ¼ 12.0 Hz), 3.78e3.91 (m, 1H), 3.61e3.70 (m, 1H), 3.42e3.47 (m,
2H), 2.88 (s, 1H), 2.39e2.53 (m, 3H), 2.04e2.11 (m, 1H). 13C NMR
(s, 1H), 7.09e7.15 (m, 1H), 5.27e5.39 (m, 1H), 5.02 (d, J ¼ 12.0 Hz,
1H), 4.55 (t, 1H, J ¼ 6.0 Hz), 3.75e3.82 (m, 2H), 3.33 (d, J ¼ 8.0 Hz,
2H), 2.57 (t, 1H, J ¼ 12.0 Hz), 2.31e2.49 (m, 1H). 13C NMR (CD3OD,
(CD3OD, 100 MHz):
d
167.0, 156.7, 146.2, 120.4, 117.2, 92.3 (d,
100 MHz): d 167.6, 163.2, 148.9, 136.4, 128.1, 127.8, 127.1, 117.3, 117.2,
J ¼ 141.0 Hz), 53.1 (d, J ¼ 19.0 Hz), 50.9, 45.1, 35.5 (d, J ¼ 17.0 Hz),
30.2. MS (ESI) m/z 269 [M þ H]þ. HRMS (ESI) calcd C11H13FN4OS
269.0872 [M þ H]þ, found 269.0887.
92.2 (d, J ¼ 178.0 Hz), 52.7 (d, J ¼ 24.0 Hz), 51.0, 44.9, 35.4 (d,
J ¼ 21.0 Hz), 32.1. MS (ESI) m/z 351 [M þ H]þ. HRMS (ESI) calcd
C
15H15FN4OS2 351.0750 [M þ H]þ, found 351.0756.

X. Ji et al. / European Journal of Medicinal Chemistry 75 (2014) 111e122
121
4.1.38. (2S,4S)-1-[(2S)-2-Amino-3-(6-bromobenzo[d][1,3]dioxol-5-
yl)propanoyl]-4-fluoropyrrolidine-2-carbonitrile (6n)
Ki)þ1/Vmax, where Km is the Michaelis constant, v is the initial rate,
Vmax is the maximum rate, and [S] is the substrate concentration.
In the same manner as described for compound 6a, compound
6n was prepared from compound 21n. HPLC: 100.00%, tR ¼ 1.30 min
4.2.5. Pharmacokinetic profile in SD rats
1H NMR (CD3OD, 400 MHz),
d
7.10 (s,1H), 6.74 (s,1H), 5.98e6.05 (m,
Compounds 6h and 6n were administered to SD rats. After oral
and intravenous administration, blood samples were collected. The
blood samples were centrifuged to obtain the plasma fraction. The
plasma samples were deproteinized with methanol containing an
internal standard. After centrifugation, the supernatant was diluted
with methanol and centrifuged again. The compound concentra-
tions in the supernatant were measured by LC/MS/MS.
2H), 5.89 (s, 1H), 5.17e5.31 (m, 1H), 4.99 (d, J ¼ 8.0 Hz, 1H), 4.28e
4.32 (m, 1H), 3.61e3.74 (m, 1H), 2.92e3.12 (m, 3H), 2.50 (t, 1H,
J ¼ 12.0 Hz), 2.25e2.43 (m,1H). 13C NMR (CD3OD,100 MHz):
d 167.5,
161.6, 149.2, 148.6, 125.3, 116.9, 115.6, 114.2, 112.2, 111.1, 102.2, 92.0
(d, J ¼ 142.0 Hz), 52.5 (d, J ¼ 19.0 Hz), 50.2, 44.9, 37.5, 35.3 (d,
J ¼ 17.0 Hz). MS (ESI) m/z 384 [M þ H]þ. HRMS (ESI) calcd
C
15H15BrFN3O3 384.0359 [M þ H]þ, found 384.0352.
4.2.6. Oral glucose tolerance test in ICR mice and chronic study in
C57BKS db/db mice
4.2. In vitro DPP4, FAP, DPP7, DPP8, and DPP9 enzyme assay
All animal experiments were approved by the Animal Care and
Use Committee of Shanghai Institute of Materia Medica. For the
acute single dose study, vehicle (0.5% methycellulose, 10 mL/kg),
compounds 6h (15 and 50 mg/kg), 6n (15 and 50 mg/kg) and
LAF237 (30 mg/kg) were administered to ICR mice after 16-hrs
starvation, then the oral glucose tolerance test (2.5 g/kg) was
conducted after 30 min of the single dose, the blood glucose level at
0, 15, 30, 60, 90, and 120 min were recorded for area under curve
calculation. For the chronic study in C57BKS db/db mice with
compound 6h (5, 15 and 50 mg/kg/day) treatment, oral glucose
tolerance test (1.5 g/kg) was carried out after 6 h starvation of 5th-
week treatment, the blood glucose level was recorded for the
glucose tolerance capacity evaluation. Vehicle (0.5% methycellu-
lose, 10 mL/kg/day) and LAF237 (15 mg/kg/day) were included as
negative and positive control, respectively.
4.2.1. Preparation of the DPPs enzyme
The DPP4, FAP, DPP7, DPP8 and DPP9 enzymes were expressed
in high five cells using a baculoviral system (Bac-To-Bac; Life
Technologies) according to the literature [38], and his6-tagged re-
combinant proteins were purified by Ni-NTA resin individually.
4.2.2. Enzyme-based assay of DPP4
To measure the activity of DPP4, a continuous fluorometric assay
was employed using Ala-Pro-AMC, which is cleaved by the enzyme
to release the fluorescent aminomethylcoumarin (AMC). Liberation
of AMC was monitored using an excitation wavelength of 355 nm
and an emission wavelength of 460 nm using Envision microplate
reader (PerkinElmer). A typical reaction contained 50 pmol/L
enzyme, 10
compounds synthesized in this work, and assay buffer (100 mmol/L
HEPES, pH 7.5, 0.1 mg/mL BSA) in a total reaction volume of 50 L.
mmol/L Ala-Pro-AMC, different concentrations of the
The results are presented as the mean ꢂ SE. Differences between
the groups were analyzed with the Student’s t-test. *, p < 0.05 or **,
p < 0.01 was regarded as statistically significant.
m
The dose response of inhibition test was carried out in quadrupli-
cate. And the IC50 data was calculated using the software GraphPad
Prism 5, and chosen the equation “sigmoidal doseeresponse (var-
iable slope)” for curve fitting.
4.2.7. hERG testing using FluxORÔ thallium assay
Step1: Growing cells. CHO-hERG-ZG cells are grown in 75 cm [2]
flask with complete medium with 100 mg/mL G418 and 100 mg/mL
4.2.3. Enzyme-based assay of DPPs inhibition selectivity
The inhibitory effect of each compounds on DPPs were assayed
by continuous fluorometric method. We used Nle-Pro-AMC as
substrate to measure the activities of DPP7 and FAP, and Ala-Pro-
AMC for DPP8 and DPP9 in the optimized pH (5.5 for DPP7 and
8.0 for other members) assay system. The selective dose response of
inhibition test on DPPs and data analysis is the same as DPP4 assay
system in quadruplicate.
HygromycinB until 80e90% confluency. Wash cells with PBS once.
Incubate cells with 1 mL 0.25% Trypsin until all cells are rounded
and can be easily dislodged from the surface. Add 10 mL complete
medium to stop Trypsin activity. Disassociate cells by thoroughly,
repetitively pipetting. Transfer them to 50 mL Falcon tube and spin
down at 1000 rpm for 5 min. Aspirate medium and resuspend cells
using a small volume of complete medium, like 0.5 mL. Count cell
density. Step 2: Cell seeding. CHO-hERG-ZG cells are plated into 96-
well plates and after plating, tap plates on sides to separate cells
and let plates sit in the dark at RT for 30 min before incubation at
37 ꢀC for 16e18 h. Cells will reach 80% confluency. After overnight
incubation the cells are media changed in loading buffer (old media
is tapped out) and incubated in the dark at RT for 90 min. Remove
the loading buffer and replace with assay buffer. Compounds 6h
and 6n were added to the cell plate. The cell plate is incubated with
compounds for 20 min in the dark at RT. Load the cell plates on
FDSS. Fluorescent signals will be recorded every 2 s till 10 s. At 10 s,
stimulus buffer will be added to cells. Then fluorescent signals will
be recorded every second till 180 s, data QC on FDSS.
4.2.4. Characterization of compound 6h and 6n on DPP4
In the time-independent inhibition experiment, 50 pmol/L DPP4
were incubated by 0.004
pound 6n (2% DMSO as blank) for different times, and then add
45 L mixture of enzyme and compounds to a final 50 L assay
mM of compound 6h or 0.01 mM of com-
m
m
system to initiate the enzyme reaction. And reversibility of DPP4
inhibition by compounds was demonstrated by dialysis experi-
ments. In brief, mixture of 50 pmol/L DPP4 and compounds
(0.004
blank) were dialyzed for different times, and then add 5
strate solution to 45 L dialysis samples to start the enzymatic
mM compound 6h, 0.01
m
M compound 6n, and 2% DMSO as
mL sub-
m
assay. The dissociation of the enzymeeinhibitor complex was
monitored by substrate hydrolysis by measuring the fluorescence
using Envision microplate reader.
4.3. Binding studies
The DPP4 protein was extracted from RCSB Protein Data Bank
(PDB ID: 2AJL). Compounds were generated using Sybyl program.
GasteigereHückel charge was used and the conformation of each
compound was minimized using default parameters. Docking
studies were performed using Glide program. The DPP4 protein was
processed by minimal minimization with OPLS2005 force field. The
For DPP4 kinetic study, the assay was carried out in a 50
mL
system containing 100 mmol/L HEPES, pH 7.5, 0.1 mg/mL BSA,
50 pmol/L DPP4, substrate Ala-Pro-AMC in 2-fold dilution from
160 mmol/L to 1.25 mmol/L, and different concentrations of the in-
hibitors. In the presence of the competitive inhibitor, the Michae-
liseMenten equation is described as 1/v ¼ (Km/[Vmax[S]])(1þ[I]/
ꢀ
grid was sized to 15 A in each direction at the center of the binding

122
X. Ji et al. / European Journal of Medicinal Chemistry 75 (2014) 111e122
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We gratefully acknowledge financial support from the National
Natural Science Foundation of China (Grants 21021063, 91229204,
81025017, 81125023), National S&T Major Projects (Grants
2012ZX09103101-072, 2012ZX09301001-005 and 2013ZX09507-
001), Sponsored by Program of Shanghai Subject Chief Scientist
(Grant 12XD1407100).
(c) B. Ahren, L. Landing-Olsson, P.A. Jansson, M. Sevensson, D. Holmes,
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