Synthesis and biological evaluation of novel benzyl-substituted (S)-phenylalanine derivatives as potent dipeptidyl peptidase 4 inhibitors

Article abstract of DOI:10.1016/j.bmc.2013.07.034

A series of novel benzyl-substituted (S)-phenylalanine derivatives were synthesized and evaluated for their dipeptidyl peptidase 4 (DPP-4) inhibitory activity and selectivity. It was found that most synthesized target compounds were potent DPP-4 inhibitors with IC₅₀ values in 3.79-25.52 nM, which were significantly superior to that of the marketed drug sitagliptin. Furthermore, the 4-fluorobenzyl substituted phenylalanine derivative 6g not only displayed the potent DPP-4 inhibition with an IC₅₀ value of 3.79 nM, but also showed better selectivity against DPP-4 over other related enzymes including DPP-7, DPP-8, and DPP-9. In an oral glucose tolerance test (OGTT) in normal Sprague Dawley rats, compound 6g reduced blood glucose excursion in a dose-dependent manner.

Full text of DOI:10.1016/j.bmc.2013.07.034

Bioorganic & Medicinal Chemistry 21 (2013) 5679–5687
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of novel benzyl-substituted
(S)-phenylalanine derivatives as potent dipeptidyl peptidase
4 inhibitors
Yang Liu a, Meimei Si b, Li Tang a, Shihao Shangguan a, Haoshu Wu b, Jia Li c, Peng Wu a, Xiaodong Ma a,
Tao Liu a, , Yongzhou Hu a,
⇑
⇑
a ZJU-ENS Joint Laboratory of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
b Institute of Pharmacology and Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China
c Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, 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 benzyl-substituted (S)-phenylalanine derivatives were synthesized and evaluated for
their dipeptidyl peptidase 4 (DPP-4) inhibitory activity and selectivity. It was found that most synthe-
sized target compounds were potent DPP-4 inhibitors with IC50 values in 3.79–25.52 nM, which were sig-
nificantly superior to that of the marketed drug sitagliptin. Furthermore, the 4-fluorobenzyl substituted
phenylalanine derivative 6g not only displayed the potent DPP-4 inhibition with an IC50 value of 3.79 nM,
but also showed better selectivity against DPP-4 over other related enzymes including DPP-7, DPP-8, and
DPP-9. In an oral glucose tolerance test (OGTT) in normal Sprague Dawley rats, compound 6g reduced
blood glucose excursion in a dose-dependent manner.
Received 23 April 2013
Revised 25 June 2013
Accepted 16 July 2013
Available online 25 July 2013
Keywords:
Dipeptidyl peptidase 4 inhibitors
Type 2 diabetes
Ó 2013 Elsevier Ltd. All rights reserved.
1,2,3-Triazole
(S)-Phenylalanine derivatives
Selectivity
1. Introduction
vildagliptin, saxagliptin, linagliptin, alogliptin and teneligliptin
have already been approved for the treatment of type 2 diabetes
Dipeptidyl peptidase 4 (DPP-4), a ubiquitous transmembrane
serine protease, specifically cleaves active polypeptides with a pro-
line or alanine at the penultimate position of the N-terminus, such
as the incretin hormone glucagon-like peptide 1 (GLP-1).1 GLP-1
has multiple physiological functions including stimulating insulin
secretion, inhibiting glucagon release, and retarding gastric empty-
ing.2 All of these biological effects benefit in the regulation of glu-
cose homeostasis. Unfortunately, GLP-1 is rapidly degraded in vivo
by DPP-4 into an inactive form.3 Thus, inhibition of DPP-4 increases
the level of active GLP-1 in the circulation, which in turn enhances
insulin secretion and improves the glucose tolerance. Therefore,
DPP-4 inhibitors have emerged as a new therapeutic strategy for
the treatment of type 2 diabetes.4–6 Additionally, DPP-4 inhibitors
offer several potential advantages over existing antidiabetic agents
including a lowered risk of hypoglycemia, no weight gain, and the
potential for regeneration and differentiation of pancreatic b-
cells.7,8 Up to now, a variety of potent DPP-4 inhibitors have been
developed, and several advanced compounds including sitagliptin,
(Fig. 1).9,10
Our continuing interest in the design and development of DPP-4
inhibitors led to the discovery of a promising lead (S)-phenylalanine
derivative 1 (Fig. 2),11 which showed moderate DPP-4 inhibitory
activity (IC50 = 265 nM) but poor selectivity (3 -fold over DPP-7,
24-fold over DPP-8, and 28-fold over DPP-9). These preliminary re-
sults prompted us to optimize lead compound 1 to improve the
overall activity and selectivity. Optimization focus was placed on
the substituent at the N-1 position of 1,2,3-triazole ring because,
as revealed in previous study, substituent at this position had a sig-
nificant effect on activity and selectivity. Herein, with the purpose
of improving DPP-4 inhibitory activity through the formation of
additional interaction with Phe357 residues,12 a benzyl group was
introduced into the N-1 position of 1,2,3-triazole ring. To validate
the design concept, docking study of compound 6a with DPP-4
was performed using the FlexiDock module of Sybyl 6.9 version
(PDB code: 2AJL13). As shown in Figure 3, the cyanopyrrolidine moi-
ety fitted into the S1 pocket, the amino formed hydrogen bonds
with Glu205 and Glu206, the 1-position nitrogen of 1,2,3-triazole
ring formed hydrogen bond with Arg358, and the benzyl group
⇑
Corresponding authors. Tel./fax: +86 571 88208458 (T.L.); tel./fax: +86 571
88208460 (Y.H.).
formed hydrophobic and
p–p interaction with Phe357. Based on
these observations, a series of benzyl-substituted (S)-phenylalanine
E-mail addresses: lt601@zju.edu.cn (T. Liu), huyz@zju.edu.cn (Y. Hu).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.07.034

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Y. Liu et al. / Bioorg. Med. Chem. 21 (2013) 5679–5687
Figure 1. Structures of the approved DPP-4 inhibitors.
Figure 2. Optimization of lead compound 1.
TMS-protected group of compound 3 was removed by tetra-n-
butylammonium fluoride (TBAF) in THF at room temperature to af-
ford 4-ethynylphenylalanine derivative 4. Click reaction of 4 with
different azides14 in the presence of CuSO4ꢀ5H2O and sodium
ascorbate yielded 1,2,3-triazole derivatives 5a–p. Deprotection of
the Boc-group of compounds 5a–p using HCl/Et2O solution pro-
vided 6a–p, which were isolated as hydrochloride salts. The struc-
tures of all synthesized compounds were confirmed by 1H NMR,
13C NMR, MS and elemental analysis.
2.2. Biological activity and SAR
The (S)-phenylalanine derivatives 6a–p were evaluated in vitro
for their DPP-4 inhibitory activity and selectivity profiles against
DPP-4 over other related enzymes including DPP-7, DPP-8, and
DPP-9. Inhibition of DPP-7, DPP-8, and DPP-9 was associated with
potential side effects.15,16 Sitagliptin, the first DPP-4 inhibitor ap-
proved by FDA, was used as the positive control. The results are
summarized in Table 1.
As shown in Table 1, most of the tested compounds showed po-
tent DPP-4 inhibitory activity with IC50 values in 3.79–25.52 nM,
which were significantly superior to that of the positive control
sitagliptin (IC50 = 24.83 nM). In addition, most target compounds
showed moderate selectivity over DPP-7 (>100-fold), DPP-8 (>50-
fold), and DPP-9 (>50-fold).
Introduction of a benzyl group into the N-1 position of 1,2,3-tri-
azole ring led to compound 6a, which showed improved DPP-4
inhibitory activity and selectivity in comparison with that of lead
compound 1 (6a, DPP-4, IC50 = 9.39 nM; 68-fold over DPP-7; 72-
fold over DPP-8; 67-fold over DPP-9; vs 1, DPP-4, IC50 = 265 nM;
7-fold over DPP-7; 24-fold over DPP-8; 28-fold over DPP-9). When
the benzyl group of compound 6a was converted into the phen-
ethyl group, compound 6b showed significantly decreased DPP-4
inhibitory activity and selectivity (DPP-4, IC50 = 25.52 nM; 40-fold
over DPP-7; 10-fold over DPP-8; 24-fold over DPP-9). Compound
6c with 4-pyridinemethyl group at the N-1 position of 1,2,3-tria-
zole ring displayed better selectivity against DPP-7, DPP-8 and
DPP-9, but its DPP-4 inhibitory activity was slightly decreased
(IC50 = 17.61 nM) in comparison with that of 6a. Compound 6d
Figure 3. Docking study of (S)-phenylalanine derivative 6a (yellow) with DPP-4
(PDB code: 2AJL). Hydrogen bonds are shown as dashed red lines. For clarity, only
key amino acid residues are shown.
derivatives were designed and synthesized. All synthesized com-
pounds were evaluated in vitro for their DPP-4 inhibitory activity
and selectivity.
2. Results and discussion
2.1. Chemistry
The general synthetic route for (S)-phenylalanine derivatives
6a–p is outlined in Scheme 1. (S)-N-Boc-4-iodophenylalanine
derivative 2 was prepared starting from (S)-phenylalanine accord-
ing to a reported method with minor modifications.11,13 Treatment
of compound 2 with trimethylsilylacetylene (TMSA) under Sono-
gashira coupling condition gave compound 3. Subsequently, the

Y. Liu et al. / Bioorg. Med. Chem. 21 (2013) 5679–5687
5681
Scheme 1. Reagents and conditions: (a) TMSA, Pd(PPh3)2Cl2, CuI, Et3N, rt; (b) TBAF, THF, rt; (c) RN3, CuSO4ꢀ5H2O, sodium ascorbate, EtOH, rt; (d) HCl/Et2O, CH2Cl2, rt.
Table 1
In vitro DPP-4 inhibitory activity and selectivity of compounds 6a–pa
Compounds
R
DPP-4
DPP-7
DPP-8
DPP-9
IC50 (nM)
IC50 (nM)
IC50 (nM)
IC50 (nM)
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
Benzyl
Phenethyl
9.39
640
680
280
1130
510
330
430
440
390
320
340
1210
300
450
690
240
470
1100
114,830
630
620
1610
450
340
360
440
280
350
340
550
310
330
440
260
600
66
25.50
17.61
8.28
19.54
7.56
1030
1880
340
440
1040
930
4-Pyridinemethyl
2-Thiophenemethyl
2-Fluorobenzyl
3-Fluorobenzyl
4-Fluorobenzyl
2-Chlorobenzyl
3-Chlorobenzyl
4-Chlorobenzyl
2-Methoxybenzyl
3-Methoxybenzyl
4-Methoxybenzyl
3,4-Difluorobenzyl
3,4-Dichlorobenzyl
3,4-Dimethoxybenzyl
—
3.79
15.24
10.45
6.05
20.84
7.50
4.35
8.57
10.02
15.70
3.0
520
1460
1200
2520
1100
2090
660
570
1560
>500,000
654,430
b
Vildagliptin
Sitagliptin
—
24.83
130,240
a
IC50 values are an average of three independent.
Values in literature.17
b
with 2-thiophenemethyl group at the N-1 position of 1,2,3-triazole
ring showed similar inhibition to DPP-4 and its selectivity was
worse than that of 6a.
group was more beneficial for the DPP-4 inhibitory activity and
selectivity compared to substitution at the 2-position and 3-posi-
tion. However, the 3,4-difluoro, 3,4-dichloro, and 3,4-dimethoxyl
substituted compounds 6n–p did not show further improvement
in activity and selectivity.
To further investigate the impact of substituents on benzene
ring of benzyl group on activity and selectivity, the fluoro, chloro,
and methoxyl were introduced into the benzyl group of compound
6a, respectively. It was discovered that compound 6g with a 4-flu-
orobenzyl group showed the best DPP-4 inhibitory activity
(IC50 = 3.79 nM) and selectivity, while 3-fluoro substituted 6f
slightly decreased DPP-4 inhibitory (IC50 = 7.56 nM) activity and
selectivity. But 2-fluoro substituted 6e (IC50 = 19.54 nM) showed
the worse activity and selectivity. This trend was also observed
for the chloro and methoxyl substituted compounds 6h–j and
6k–m (6h, IC50 = 15.24 nM; 6i, IC50 = 10.45 nM; 6j, IC50 = 6.05 nM;
6k, IC50 = 20.84 nM; 6l, IC50 = 7.50 nM; 6m, IC50 = 4.35 nM). The re-
sults suggested that substitution pattern had a significant effect on
activity and selectivity, substitution at the 4-position of benzyl
2.3. Oral glucose tolerance test
Compound 6g with the most potency and better selectivity was
selected for further biological evaluation. An oral glucose tolerance
test (OGTT) in normal Sprague Dawley rats was conducted to
determine the effect of 6g in vivo. Following oral administration
of 1, 3, and 10 mg/kg 6g, blood glucose levels of rats were de-
creased and glucose-induced blood glucose excursion was signifi-
cantly inhibited by 20.8%, 21.7%, and 28.8%, respectively, in a
dose-dependent manner. These results suggested that 6g could im-
prove blood glucose tolerance in vivo (Fig. 4).

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Y. Liu et al. / Bioorg. Med. Chem. 21 (2013) 5679–5687
Figure 4. Effects of 6g on blood glucose levels during the oral glucose tolerance test (OGTT) in normal Sprague Dawley rats, the rats fasted for 16 h were received various
doses of 6g (1, 3, and 10 mg/kg) by gastric intubation 30 min prior to an oral glucose challenge (2.5 g/kg). The rats of vehicle control group were given only 0.5% CMC–Na and
glucose. Vildagliptin was used as the positive control drug. Blood glucose levels were monitored at 0 (preintubation), 30, 60, and 120 min after glucose administration. (A)
Change of blood glucose levels; (B) change rate of AUC of blood glucose. Each value represents means SEM, n = 5 rats in each group. ⁄P < 0.01 versus vehicle control.
(20 mg, 0.1 mmol) in EtOH (10 mL) was stirred for 3 h at room
temperature. The solvent was removed under reduced pressure.
The residue was dissolved in CH2Cl2 (30 mL) and washed with
brine (3 ꢁ 10 mL), dried over anhydrous Na2SO4, and concentrated
under vacuum. The crude product was purified by flash column
chromatography using EtOAc/petroleum ether (5:1) as eluent to
yield compounds 5a–p.
3. Conclusions
In summary, optimization of lead compound 1 resulted in the
identification of 6a with improved activity and selectivity. Further
optimization on the benzyl ring of 6a obtained a series of novel
benzyl-substituted (S)-phenylalanine derivatives, which were po-
tent DPP-4 inhibitors with IC50 values in 3.79–25.52 nM and
showed moderate selectivity against DPP-4 over other related en-
zymes including DPP-7, DPP-8, and DPP-9. Preliminary SAR studies
found that 4-substitution at benzyl ring exhibited better activity
and selectivity than 2-substitution. In particular, the 4-fluoroben-
zyl substituted phenylalanine derivative 6g not only displayed
the potent DPP-4 inhibition with an IC50 value of 3.79 nM, but also
showed better selectivity. In addition, compound 6g can reduce
blood glucose excursion in an oral glucose tolerance test in normal
Sprague Dawley rats. These results provided the guidance for fur-
ther optimization towards the discovery of potential DPP-4 inhib-
itors for the treatment of type 2 diabetes.
4.1.1.1. tert-Butyl {(S)-3-[4-(1-benzyl-1H-1,2,3-triazol-4-yl)phe-
nyl]-1-[(S)-2-cyanopyrrolidin-1-yl]-1-oxopropan-2-yl}carba-
mate (5a).
Yield: 87%; colorless oil; 1H NMR (500 MHz,
CDCl3): d 7.77 (d, 2H, J = 8.0 Hz, ArH), 7.66 (s, 1H, @CH), 7.40–
7.36 (m, 3H, ArH), 7.33–7.32 (m, 2H, ArH), 7.30 (d, 2H, J = 8.0 Hz,
ArH), 5.56 (d, 2H, J = 4.5 Hz, CH2Ar), 5.35 (d, 1H, J = 9.0 Hz, NHBoc),
4.69–4.67 (m, 1H, CHCN), 4.55–4.50 (m, 1H, CH), 3.39–3.35 (m, 1H,
CH2), 3.06–3.02 (m, 2H, CH2), 2.70–2.65 (m, 1H, CH2), 2.08–2.04 (m,
2H, CH2), 1.90–1.85 (m, 1H, CH2), 1.78–1.69 (m, 1H, CH2), 1.42 (s,
9H, t-Bu-H) ppm; ESI-MS (m/z): 501 [M+H]+; Anal. Calcd for
C28H32N6O3: C, 67.18; H, 6.44; N, 16.79. Found: C, 67.10; H, 6.52;
N, 16.75.
4. Experimental
4.1. Chemistry
4.1.1.2. tert-Butyl {(S)-1-[(S)-2-cyanopyrrolidin-1-yl]-1-oxo-3-
[4-(1-phenethyl-1H-1,2,3-triazol-4-yl)phenyl]propan-2-yl}car-
bamate (5b).
Yield: 85%; colorless oil; 1H NMR (500 MHz,
Standard Schlenk techniques were employed for air and water
sensitive reaction. THF and Et2O were dried over and distilled from
Na/benzophenone under a dry nitrogen atmosphere. CH2Cl2 and
Et3N were dried over calcium hydride and KOH pellets then dis-
tilled for use, respectively. Other commercially available chemicals
and solvents are reagent grade and were used without further
purification. Melting points were determined on a Büchi B-540
apparatus (Büchi Labortechnik, Flawil, Switzerland) and were
uncorrected. All 1H NMR and 13C NMR spectra were recorded on
Bruker 500 and 400 MHz instruments (Bruker Bioscience, Billerica,
MA, USA) with CDCl3 or DMSO-d6 as solvent, respectively. ESI Mass
spectral data were obtained by Esquire-LC-00075 spectrometer
(Brucker Bioscience). Elemental analysis for carbon, hydrogen,
and nitrogen was performed with an Eager 300 analyzer. Optical
rotation was measured with a JASCO P-1010 polarimeter. Flash col-
umn chromatography was performed using silica gel (200–
300 mesh). All yields are unoptimized and only represent one
experiment’s result. Intermediates 2–4 were synthesized using
previously reported procedures.11
CDCl3): d 7.75 (d, 2H, J = 8.0 Hz, ArH), 7.51 (s, 1H, @CH), 7.32–
7.29 (m, 4H, ArH), 7.26–7.23 (m, 1H, ArH), 7.16–7.14 (d, 2H,
J = 8.0 Hz, ArH), 5.38 (d, 1H, J = 8.5 Hz, NHBoc), 4.71–4.69 (m, 1H,
CHCN), 4.64 (t, 2H, J = 7.0 Hz, CH2), 4.57–4.52 (m, 1H, CH), 3.43–
3.38 (m, 1H, CH2), 3.27 (t, 2H, J = 7.0 Hz, CH2), 3.09–3.03 (m, 2H,
CH2), 2.74–2.69 (m, 1H, CH2), 2.13–2.07 (m, 2H, CH2), 1.91–1.88
(m, 1H, CH2), 1.79–1.73 (m, 1H, CH2), 1.43 (s, 9H, t-Bu-H) ppm;
ESI-MS (m/z): 515 [M+H]+; Anal. Calcd for C29H34N6O3: C, 67.68;
H, 6.66; N, 16.33. Found: C, 67.59; H, 6.77; N, 16.35.
4.1.1.3. tert-Butyl {(S)-1-[(S)-2-cyanopyrrolidin-1-yl]-1-oxo-3-
[4-(1-(pyridin-4-ylmethyl)-1H-1,2,3-triazol-4-yl)phenyl]pro-
pan-2-yl}carbamate (5c).
Yield: 90%; colorless oil; 1H NMR
(500 MHz, CDCl3): d 8.62 (d, 2H, J = 6.0 Hz, pyridine-H), 7.78 (d,
2H, J = 8.0 Hz, ArH), 7.75 (s, 1H, @CH), 7.31 (d, 2H, J = 8.0 Hz, ArH),
7.17 (d, 2H, J = 6.0 Hz, pyridine-H), 5.59 (d, 2H, J = 1.5 Hz, CH2Ar),
5.38 (d, 1H, J = 8.5 Hz, NHBoc), 4.69–4.67 (m, 1H, CHCN), 4.56–
4.51 (m, 1H, CH), 3.43–3.38 (m, 1H, CH2), 3.05–3.03 (m, 2H, CH2),
2.75–2.71 (m, 1H, CH2), 2.11–2.04 (m, 2H, CH2), 1.92–1.86 (m,
1H, CH2), 1.79–1.73 (m, 1H, CH2), 1.41 (s, 9H, t-Bu-H) ppm; ESI-
MS (m/z): 502 [M+H]+; Anal. Calcd for C27H31N7O3: C, 64.65; H,
6.23; N, 19.55. Found: C, 64.51; H, 6.45; N, 19.56.
4.1.1. General procedure for the synthesis of compounds (5a–p)
A suspension of 4 (367 mg, 1.0 mmol), corresponding azides
(1.5 mmol), CuSO4ꢀ5H2O (25 mg, 0.1 mmol) and sodium ascorbate

Y. Liu et al. / Bioorg. Med. Chem. 21 (2013) 5679–5687
5683
4.1.1.4. tert-Butyl {(S)-1-[(S)-2-cyanopyrrolidin-1-yl]-1-oxo-3-
[4-(1-(thiophen-2-ylmethyl)-1H-1,2,3-triazol-4-yl)phenyl]pro-
pan-2-yl}carbamate (5d).
4.1.1.9. tert-Butyl {(S)-3-[4-(1-(3-chlorobenzyl)-1H-1,2,3-tria-
zol-4-yl)phenyl]-1-[(S)-2-cyanopyrrolidin-1-yl]-1-oxopropan-2-
yl}carbamate (5i).
Yield: 88%; colorless oil; 1H NMR
Yield: 86%; colorless oil; 1H NMR
(500 MHz, CDCl3): d 7.78 (d, 2H, J = 8.5 Hz, ArH), 7.72 (s, 1H,
@CH), 7.35 (d, 1H, J = 5.0 Hz, thiophene-H), 7.31 (d, 2H, J = 8.5 Hz,
ArH), 7.16 (d, 1H, J = 3.0 Hz, thiophene-H), 7.04–7.02 (m, 1H, thio-
phene-H), 5.74 (d, 2H, J = 1.5 Hz, CH2Ar), 5.33 (d, 1H, J = 8.5 Hz,
NHBoc), 4.69–4.67 (m, 1H, CHCN), 4.55–4.51 (m, 1H, CH), 3.40–
3.35 (m, 1H, CH2), 3.07–3.02 (m, 2H, CH2), 2.69–2.65 (m, 1H,
CH2), 2.12–2.05 (m, 2H, CH2), 1.90–1.86 (m, 1H, CH2), 1.80–1.72
(m, 1H, CH2), 1.41 (s, 9H, t-Bu-H) ppm; ESI-MS (m/z): 507
[M+H]+; Anal. Calcd for C26H30N6O3S: C, 61.64; H, 5.97; N, 16.59.
Found: C, 61.63; H, 6.12; N, 16.54.
(500 MHz, CDCl3): d 7.77 (d, 2H, J = 8.0 Hz, ArH), 7.70 (s, 1H,
@CH), 7.33–7.28 (m, 5H, ArH), 7.19–7.18 (m, 1H, ArH), 5.52 (d,
2H, J = 2.5 Hz, CH2Ar), 5.38 (d, 1H, J = 8.0 Hz, NHBoc), 4.68–4.66
(m, 1H, CHCN), 4.55–4.50 (m, 1H, CH), 3.41–3.36 (m, 1H, CH2),
3.03–3.01 (m, 2H, CH2), 2.72–2.68 (m, 1H, CH2), 2.10–2.03 (m,
2H, CH2), 1.89–1.85 (m, 1H, CH2), 1.78–1.70 (m, 1H, CH2), 1.40 (s,
9H, t-Bu-H) ppm; ESI-MS (m/z): 535 [M+H]+; Anal. Calcd for C28H31-
ClN6O3: C, 62.86; H, 5.84; N, 15.71. Found: C, 62.80; H, 5.98; N,
15.70.
4.1.1.10. tert-Butyl {(S)-3-[4-(1-(4-chlorobenzyl)-1H-1,2,3-tria-
zol-4-yl)phenyl]-1-[(S)-2-cyanopyrrolidin-1-yl]-1-oxopropan-2-
yl}carbamate (5j).
4.1.1.5. tert-Butyl {(S)-1-[(S)-2-cyanopyrrolidin-1-yl]-3-[4-(1-(2-
fluorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl]-1-oxopropan-2-
yl}carbamate (5e).
Yield: 94%; colorless oil; 1H NMR
Yield: 93%; colorless oil; 1H NMR
(500 MHz, CDCl3): d 7.78 (d, 2H, J = 8.0 Hz, ArH), 7.66 (s, 1H,
@CH), 7.38 (d, 2H, J = 8.0 Hz, ArH), 7.31 (d, 2H, J = 8.0 Hz, ArH),
7.27 (d, 2H, J = 8.0 Hz, ArH), 5.53 (s, 2H, CH2Ar), 5.32 (d, 1H,
J = 8.5 Hz, NHBoc), 4.70–4.68 (m, 1H, CHCN), 4.56–4.51 (m, 1H,
CH), 3.40–3.37 (m, 1H, CH2), 3.08–3.03 (m, 2H, CH2), 2.71–2.67
(m, 1H, CH2), 2.09–2.03 (m, 2H, CH2), 1.90–1.85 (m, 1H, CH2),
1.78–1.72 (m, 1H, CH2), 1.42 (s, 9H, t-Bu-H) ppm; ESI-MS (m/z):
535 [M+H]+; Anal. Calcd for C28H31ClN6O3: C, 62.86; H, 5.84; N,
15.71. Found: C, 62.88; H, 5.95; N, 15.60.
(500 MHz, CDCl3): d 7.79 (d, 2H, J = 8.0 Hz, ArH), 7.75 (s, 1H,
@CH), 7.46–7.45 (m, 1H, ArH), 7.35–7.27 (m, 5H, ArH), 5.71 (s,
2H, CH2Ar), 5.32 (d, 1H, J = 8.5 Hz, NHBoc), 4.70–4.67 (m, 1H,
CHCN), 4.56–4.51 (m, 1H, CH), 3.42–3.37 (m, 1H, CH2), 3.06–3.03
(m, 2H, CH2), 2.72–2.68 (m, 1H, CH2), 2.12–2.04 (m, 2H, CH2),
1.91–1.85 (m, 1H, CH2), 1.82–1.74 (m, 1H, CH2), 1.42 (s, 9H, t-Bu-
H) ppm; ESI-MS (m/z): 519 [M+H]+; Anal. Calcd for C28H31FN6O3:
C, 64.85; H, 6.03; N, 16.21. Found: C, 64.80; H, 6.22; N, 16.10.
4.1.1.6. tert-Butyl {(S)-1-[(S)-2-cyanopyrrolidin-1-yl]-3-[4-(1-(3-
fluorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl]-1-oxopropan-2-
yl}carbamate (5f).
4.1.1.11. tert-Butyl {(S)-1-[(S)-2-cyanopyrrolidin-1-yl]-3-[4-(1-
(2-methoxybenzyl)-1H-1,2,3-triazol-4-yl)phenyl]-1-oxopropan-
2-yl}carbamate (5k).
Yield: 91%; colorless oil; 1H NMR
Yield: 90%; colorless oil; 1H NMR
(500 MHz, CDCl3): d 7.77 (d, 2H, J = 8.0 Hz, ArH), 7.70 (s, 1H,
@CH), 7.37–7.33 (m, 1H, ArH), 7.30 (d, 2H, J = 8.0 Hz, ArH), 7.10
(d, 1H, J = 7.5 Hz, ArH), 7.07–7.03 (m, 1H, ArH), 7.01–6.99 (m, 1H,
ArH), 5.55 (d, 2H, J = 2.5 Hz, CH2Ar), 5.43 (d, 1H, J = 8.5 Hz, NHBoc),
4.69–4.66 (m, 1H, CHCN), 4.55–4.50 (m, 1H, CH), 3.41–3.37 (m, 1H,
CH2), 3.05–3.02 (m, 2H, CH2), 2.73–2.68 (m, 1H, CH2), 2.10–2.03 (m,
2H, CH2), 1.89–1.87 (m, 1H, CH2), 1.78–1.74 (m, 1H, CH2), 1.40 (s,
9H, t-Bu-H) ppm; ESI-MS (m/z): 519 [M+H]+; Anal. Calcd for C28H31-
FN6O3: C, 64.85; H, 6.03; N, 16.21. Found: C, 65.01; H, 6.21; N,
16.17.
(500 MHz, CDCl3): d 7.77 (d, 2H, J = 8.0 Hz, ArH), 7.70 (s, 1H,
@CH), 7.36–7.33 (m, 1H, ArH), 7.29 (d, 2H, J = 8.0 Hz, ArH), 7.24–
7.22 (m, 1H, ArH), 6.97–6.92 (m, 2H, ArH), 5.57 (d, 2H, J = 1.5 Hz,
CH2Ar), 5.33 (d, 1H, J = 8.5 Hz, NHBoc), 4.68–4.66 (m, 1H, CHCN),
4.54–4.50 (m, 1H, CH), 3.88 (s, 3H, OCH3), 3.39–3.37 (m, 1H, CH2),
3.05–3.01 (m, 2H, CH2), 2.68–2.64 (m, 1H, CH2), 2.11–2.05 (m,
2H, CH2), 1.90–1.85 (m, 1H, CH2), 1.77–1.71 (m, 1H, CH2), 1.41 (s,
9H, t-Bu-H) ppm; ESI-MS (m/z): 531 [M+H]+; Anal. Calcd for
C29H34N6O4: C, 65.64; H, 6.46; N, 15.84. Found: C, 65.55; H, 6.74;
N, 15.75.
4.1.1.7. tert-Butyl {(S)-1-[(S)-2-cyanopyrrolidin-1-yl]-3-[4-(1-(4-
fluorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl]-1-oxopropan-2-
yl}carbamate (5g).
4.1.1.12. tert-Butyl {(S)-1-[(S)-2-cyanopyrrolidin-1-yl]-3-[4-(1-
(3-methoxybenzyl)-1H-1,2,3-triazol-4-yl)phenyl]-1-oxopropan-
2-yl}carbamate (5l).
Yield: 95%; colorless oil; 1H NMR
Yield: 84%; colorless oil; 1H NMR
(500 MHz, CDCl3): d 7.76 (d, 2H, J = 8.5 Hz, ArH), 7.66 (s, 1H,
@CH), 7.32–7.28 (m, 4H, ArH), 7.07 (t, 2H, J = 8.5 Hz, ArH), 5.52 (d,
2H, J = 2.5 Hz, CH2Ar), 5.35 (d, 1H, J = 8.5 Hz, NHBoc), 4.69–4.66
(m, 1H, CHCN), 4.54–4.52 (m, 1H, CH), 3.39–3.35 (m, 1H, CH2),
3.05–3.02 (m, 2H, CH2), 2.71–2.67 (m, 1H, CH2), 2.09–2.04 (m,
2H, CH2), 1.89–1.86 (m, 1H, CH2), 1.76–1.70 (m, 1H, CH2), 1.41 (s,
9H, t-Bu-H) ppm; ESI-MS (m/z): 519 [M+H]+; Anal. Calcd for C28H31-
FN6O3: C, 64.85; H, 6.03; N, 16.21. Found: C, 64.76; H, 6.20; N,
16.12.
(500 MHz, CDCl3): d 7.77 (d, 2H, J = 8.0 Hz, ArH), 7.67 (s, 1H,
@CH), 7.30 (d, 3H, J = 7.5 Hz, ArH), 6.90–6.87 (m, 2H, ArH), 6.84
(s, 1H, ArH), 5.52 (d, 2H, J = 4.0 Hz, CH2Ar), 5.35 (d, 1H, J = 8.5 Hz,
NHBoc), 4.68–4.66 (m, 1H, CHCN), 4.55–4.50 (m, 1H, CH), 3.78 (s,
3H, OCH3), 3.40–3.35 (m, 1H, CH2), 3.05–3.01 (m, 2H, CH2), 2.70–
2.66 (m, 1H, CH2), 2.11–2.02 (m, 2H, CH2), 1.90–1.86 (m, 1H,
CH2), 1.78–1.68 (m, 1H, CH2), 1.41 (s, 9H, t-Bu-H) ppm; ESI-MS
(m/z): 531 [M+H]+; Anal. Calcd for C29H34N6O4: C, 65.64; H, 6.46;
N, 15.84. Found: C, 65.69; H, 6.67; N, 15.77.
4.1.1.8. tert-Butyl {(S)-3-[4-(1-(2-chlorobenzyl)-1H-1,2,3-tria-
zol-4-yl)phenyl]-1-[(S)-2-cyanopyrrolidin-1-yl]-1-oxopropan-2-
yl}carbamate (5h).
4.1.1.13. tert-Butyl {(S)-1-[(S)-2-cyanopyrrolidin-1-yl]-3-[4-(1-
(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)phenyl]-1-oxopropan-
2-yl}carbamate (5m).
(500 MHz, CDCl3): d 7.76 (d, 2H, J = 8.0 Hz, ArH), 7.62 (s, 1H,
@CH), 7.31–7.27 (m, 4H, ArH), 6.92 (d, 2H, J = 8.0 Hz, ArH), 5.49
(d, 2H, J = 3.0 Hz, CH2Ar), 5.35 (d, 1H, J = 8.5 Hz, NHBoc), 4.68–
4.64 (m, 1H, CHCN), 4.54–4.50 (m, 1H, CH), 3.80 (s, 3H, OCH3),
3.40–3.35 (m, 1H, CH2), 3.06–3.00 (m, 2H, CH2), 2.68–2.64 (m,
1H, CH2), 2.11–2.03 (m, 2H, CH2), 1.89–1.85 (m, 1H, CH2), 1.81–
1.69 (m, 1H, CH2), 1.41 (s, 9H, t-Bu-H) ppm; ESI-MS (m/z): 531
Yield: 89%; colorless oil; 1H NMR
Yield: 82%; colorless oil; 1H NMR
(500 MHz, CDCl3): d 7.78 (d, 2H, J = 8.5 Hz, ArH), 7.75 (s, 1H,
@CH), 7.45–7.44 (m, 1H, ArH), 7.34–7.21 (m, 5H, ArH), 5.70 (s,
2H, CH2Ar), 5.36 (d, 1H, J = 8.5 Hz, NHBoc), 4.69–4.64 (m, 1H,
CHCN), 4.56–4.51 (m, 1H, CH), 3.42–3.37 (m, 1H, CH2), 3.06–3.02
(m, 2H, CH2), 2.72–2.68 (m, 1H, CH2), 2.11–2.05 (m, 2H, CH2),
1.92–1.85 (m, 1H, CH2), 1.81–1.76 (m, 1H, CH2), 1.41 (s, 9H, t-Bu-
H) ppm; ESI-MS (m/z): 535 [M+H]+; Anal. Calcd for C28H31ClN6O3:
C, 62.86; H, 5.84; N, 15.71. Found: C, 62.83; H, 5.90; N, 15.69.

5684
Y. Liu et al. / Bioorg. Med. Chem. 21 (2013) 5679–5687
[M+H]+; Anal. Calcd for C29H34N6O4: C, 65.64; H, 6.46; N, 15.84.
Found: C, 65.62; H, 6.63; N, 15.81.
5.77; N, 19.23. Found: C, 63.14; H, 5.99; N, 19.09; ½a D25
ꢂ
= 40.52
(c = 0.1, CH3OH).
4.1.1.14. tert-Butyl {(S)-1-[(S)-2-cyanopyrrolidin-1-yl]-3-[4-(1-
(3,4-difluorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl]-1-oxopro-
pan-2-yl}carbamate (5n).
4.1.2.2. (S)-1-{(S)-2-Amino-3-[4-(1-phenethyl-1H-1,2,3-triazol-
4-yl)phenyl]propanoyl}pyrrolidine-2-carbonitrile hydrochlo-
Yield: 92%; colorless oil; 1H NMR
ride (6b).
Yield: 84%; white powder; mp 179–180 °C; 1H
(500 MHz, CDCl3): d 7.78 (d, 2H, J = 8.0 Hz, ArH), 7.69 (s, 1H,
@CH), 7.31 (d, 2H, J = 8.0 Hz, ArH), 7.19–7.13 (m, 2H, ArH), 7.07–
7.06 (m, 1H, ArH), 5.52 (s, 2H, CH2Ar), 5.34 (d, 1H, J = 8.5 Hz,
NHBoc), 4.70–4.67 (m, 1H, CHCN), 4.56–4.51 (m, 1H, CH), 3.40–
3.37 (m, 1H, CH2), 3.05–3.03 (m, 2H, CH2), 2.72–2.68 (m, 1H,
CH2), 2.12–2.05 (m, 2H, CH2), 1.90–1.87 (m, 1H, CH2), 1.78–1.72
(m, 1H, CH2), 1.42 (s, 9H, t-Bu-H) ppm; ESI-MS (m/z): 537
[M+H]+; Anal. Calcd for C28H30F2N6O3: C, 62.68; H, 5.64; N, 15.66.
Found: C, 62.56; H, 5.90; N, 15.58.
NMR (500 MHz, DMSO-d6): d 8.55 (s, 1H, @CH), 8.41 (br, 3H,
NHþ3 ), 7.77 (d, 2H, J = 8.0 Hz, ArH), 7.32 (d, 2H, J = 8.5 Hz, ArH),
7.28 (d, 2H, J = 8.0 Hz, ArH), 7.23–7.21 (m, 3H, ArH), 4.81–4.77
(m, 1H, CHCN), 4.67 (t, 2H, J = 7.0 Hz, CH2), 4.35–4.32 (m, 1H,
CH), 3.51–3.47 (m, 1H, CH2), 3.24 (t, 2H, J = 7.0v, CH2), 3.19–3.15
(m, 1H, CH2), 3.08–3.03 (m, 1H, CH2), 2.81–2.77 (m, 1H, CH2),
2.16–2.11 (m, 1H, CH2), 2.06–1.99 (m, 1H, CH2), 1.87–1.81 (m,
1H, CH2), 1.71–1.64 (m, 1H, CH2) ppm; ESI-MS (m/z): 415 [M+H]+.
13C NMR (100 MHz, DMSO-d6): d 167.3, 145.7, 137.5, 133.7,
130.2, 129.9, 128.6, 128.3, 126.5, 125.1, 121.4, 118.2, 51.9, 50.6,
46.2, 46.1, 36.2, 35.4, 29.2, 24.6 ppm; Anal. Calcd for C24H26N6-
OꢀHCl: C, 63.92; H, 6.03; N, 18.64. Found: C, 63.94; H, 6.24; N,
4.1.1.15. tert-Butyl {(S)-1-[(S)-2-cyanopyrrolidin-1-yl]-3-[4-(1-
(3,4-dichlorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl]-1-oxopro-
pan-2-yl}carbamate (5o).
Yield: 90%; colorless oil; 1H NMR
18.55; ½a 2D5
ꢂ
= 35.24 (c = 0.1, CH3OH).
(500 MHz, CDCl3): d 7.77 (d, 2H, J = 8.0 Hz, ArH), 7.70 (s, 1H,
@CH), 7.46 (d, 1H, J = 8.5 Hz, ArH), 7.43–7.41 (m, 1H, ArH), 7.31
(d, 2H, J = 8.0 Hz, ArH), 7.16–7.14 (m, 1H, ArH), 5.51 (s, 2H, CH2Ar),
5.36 (d, 1H, J = 8.5 Hz, NHBoc), 4.69–4.67 (m, 1H, CHCN), 4.56–4.51
(m, 1H, CH), 3.42–3.37 (m, 1H, CH2), 3.05–3.02 (m, 2H, CH2), 2.74–
2.69 (m, 1H, CH2), 2.10–2.03 (m, 2H, CH2), 1.90–1.85 (m, 1H, CH2),
1.79–1.73 (m, 1H, CH2), 1.41 (s, 9H, t-Bu-H) ppm; ESI-MS (m/z): 569
[M+H]+; Anal. Calcd for C28H30Cl2N6O3: C, 59.05; H, 5.31; N, 14.76.
Found: C, 59.01; H, 5.51; N, 14.52.
4.1.2.3.
(S)-1-{(S)-2-Amino-3-[4-(1-(pyridin-4-ylmethyl)-1H-
1,2,3-triazol-4-yl)phenyl]propanoyl}pyrrolidine-2-carbonitrile
hydrochloride (6c). Yield: 80%; white powder; mp 82–85 °C;
1H NMR (500 MHz, DMSO-d6): d 8.83–8.80 (m, 2H, pyridine-H),
8.76 (s, 1H, @CH), 8.48 (br, 3H, NHþ3 ), 7.82 (d, 2H, J = 8.0 Hz, ArH),
7.69–7.66 (m, 2H, pyridine-H), 7.33 (d, 2H, J = 8.0 Hz, ArH), 5.97
(s, 2H, CH2Ar), 4.79–4.77 (m, 1H, CHCN), 4.33–4.32 (m, 1H, CH),
3.51–3.48 (m, 1H, CH2), 3.22–3.18 (m, 1H, CH2), 3.07–3.02 (m,
1H, CH2), 2.78–2.73 (m, 1H, CH2), 2.14–2.09 (m, 1H, CH2), 2.02–
1.97 (m, 1H, CH2), 1.85–1.80 (m, 1H, CH2), 1.67–1.63 (m, 1H, CH2)
ppm; ESI-MS (m/z): 402 [M+H]+. 13C NMR (100 MHz, DMSO-d6):
d 167.3, 158.5, 158.1, 154.2, 146.4, 142.8, 134.1, 130.3, 129.4,
125.3, 125.1, 122.6, 118.3, 116.7, 113.9, 51.8, 51.5, 46.2, 46.1,
36.3, 29.2, 24.6 ppm; Anal. Calcd for C22H23N7OꢀHCl: C, 60.34; H,
4.1.1.16. tert-Butyl {(S)-1-[(S)-2-cyanopyrrolidin-1-yl]-3-[4-(1-
(3,4-dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)phenyl]-1-oxopro-
pan-2-yl}carbamate (5p).
Yield: 87%; colorless oil; 1H NMR
(500 MHz, CDCl3): d 7.77 (d, 2H, J = 8.0 Hz, ArH), 7.64 (s, 1H,
@CH), 7.30 (d, 2H, J = 8.0 Hz, ArH), 6.94–6.91 (m, 1H, ArH), 6.87–
6.84 (m, 2H, ArH), 5.48 (d, 2H, J = 4.0 Hz, CH2Ar), 5.33 (d, 1H,
J = 8.5 Hz, NHBoc), 4.69–4.67 (m, 1H, CHCN), 4.55–4.50 (m, 1H,
CH), 3.88 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.40–3.35 (m, 1H,
CH2), 3.07–3.01 (m, 2H, CH2), 2.70–2.65 (m, 1H, CH2), 2.11–2.03
(m, 2H, CH2), 1.90–1.84 (m, 1H, CH2), 1.79–1.71 (m, 1H, CH2),
1.42 (s, 9H, t-Bu-H) ppm; ESI-MS (m/z): 561 [M+H]+; Anal. Calcd
for C30H36N6O5: C, 64.27; H, 6.47; N, 14.99. Found: C, 64.19; H,
6.60; N, 15.03.
5.52; N, 22.39. Found: C, 60.30; H, 5.65; N, 22.38; ½a D25
ꢂ
= 40.30
(c = 0.1, CH3OH).
4.1.2.4. (S)-1-{(S)-2-Amino-3-[4-(1-(thiophen-2-ylmethyl)-1H-
1,2,3-triazol-4-yl)phenyl]propanoyl}pyrrolidine-2-carbonitrile
hydrochloride (6d).
Yield: 79%; white powder; mp 147–
149 °C; 1H NMR (500 MHz, DMSO-d6): d 8.65 (s, 1H, @CH), 8.40
(br, 3H, NHþ3 ), 7.81 (d, 2H, J = 8.0 Hz, ArH), 7.54 (d, 1H, J = 5.0 Hz,
thiophene-H), 7.31 (d, 2H, J = 8.0 Hz, ArH), 7.24 (d, 1H, J = 3.5 Hz,
thiophene-H), 7.04 (t, 1H, J = 5.0 Hz, thiophene-H), 5.84 (s, 2H, CH2-
Ar), 4.78–4.76 (m, 1H, CHCN), 4.34–4.32 (m, 1H, CH), 3.50–3.44 (m,
1H, CH2), 3.18–3.15 (m, 1H, CH2), 3.06–3.01 (m, 1H, CH2), 2.76–2.72
(m, 1H, CH2), 2.13–2.08 (m, 1H, CH2), 2.02–1.97 (m, 1H, CH2), 1.83–
1.78 (m, 1H, CH2), 1.66–1.63 (m, 1H, CH2) ppm; ESI-MS (m/z): 407
[M+H]+. 13C NMR (100 MHz, DMSO-d6): d 167.3, 146.2, 137.5,
133.8, 130.2, 129.7, 128.0, 127.1, 127.0, 125.2, 121.1, 118.2, 51.8,
47.5, 46.2, 46.1, 36.3, 29.2, 24.6 ppm; Anal. Calcd for C21H22N6-
OSꢀHCl: C, 56.94; H, 5.23; N, 18.97. Found: C, 57.00; H, 5.39; N,
4.1.2. General procedure for the synthesis of compounds (6a–p)
To a stirred solution of 5 (0.1 mmol) in CH2Cl2 (1.0 mL) was
added HCl/Et2O solution (3.0 mL, 4.0 N). The mixture was stirred
for 2 h at room temperature under a N2 atmosphere. After that,
the reaction was concentrated under vacuum. The residue was dis-
solved in CH2Cl2 and after addition of HCl/Et2O solution with ice-
cooling. The resulting salt was collected by filtration, washed with
anhydrous Et2O to give compounds 6a–p.
4.1.2.1.
(S)-1-{(S)-2-Amino-3-[4-(1-benzyl-1H-1,2,3-triazol-4-
18.91; ½a 2D5
ꢂ
= 42.16 (c = 0.1, CH3OH).
yl)phenyl]propanoyl}pyrrolidine-2-carbonitrile hydrochloride
(6a).
Yield: 81%; white powder; mp 155–156 °C; 1H NMR
4.1.2.5.
(S)-1-{(S)-2-Amino-3-[4-(1-(2-fluorobenzyl)-1H-1,2,3-
(500 MHz, DMSO-d6): d 8.67 (s, 1H, @CH), 8.38 (br, 3H, NHþ),
triazol-4-yl)phenyl]propanoyl}pyrrolidine-2-carbonitrile
hydrochloride (6e). Yield: 78%; white powder; mp 119–
3
7.82 (d, 2H, J = 8.0 Hz, ArH), 7.40–7.33 (m, 5H, ArH), 7.30 (d, 2H,
J = 8.0 Hz, ArH), 5.63 (s, 2H, CH2Ar), 4.79–4.76 (m, 1H, CHCN),
4.33–4.32 (m, 1H, CH), 3.47–3.45 (m, 1H, CH2), 3.16–3.13 (m, 1H,
CH2), 3.05–3.01 (m, 1H, CH2), 2.76–2.74 (m, 1H, CH2), 2.14–2.08
(m, 1H, CH2), 2.03–1.97 (m, 1H, CH2), 1.83–1.78 (m, 1H, CH2),
1.67–1.61 (m, 1H, CH2) ppm; ESI-MS (m/z): 401 [M+H]+; 13C NMR
(100 MHz, DMSO-d6): d 167.3, 146.2, 135.9, 133.8, 130.2, 129.8,
128.7, 128.1, 127.9, 125.2, 121.6, 118.2, 53.0, 51.8, 46.2, 46.1,
36.3, 29.2, 24.6 ppm; Anal. Calcd for C23H24N6OꢀHCl: C, 63.22; H,
121 °C; 1H NMR (500 MHz, DMSO-d6): d 8.63 (s, 1H, @CH), 8.39
(br, 3H, NHþ3 ), 7.82 (d, 2H, J = 8.5 Hz, ArH), 7.54–7.52 (m, 1H,
ArH), 7.43–7.36 (m, 2H, ArH), 7.32–7.27 (m, 3H, ArH), 5.74 (s, 2H,
CH2Ar), 4.79–4.75 (m, 1H, CHCN), 4.36–4.32 (m, 1H, CH), 3.51–
3.45 (m, 1H, CH2), 3.18–3.14 (m, 1H, CH2), 3.07–3.02 (m, 1H,
CH2), 2.79–2.74 (m, 1H, CH2), 2.14–2.08 (m, 1H, CH2), 2.04–1.97
(m, 1H, CH2), 1.84–1.79 (m, 1H, CH2), 1.68–1.62 (m, 1H, CH2)
ppm; ESI-MS (m/z): 419 [M+H]+. 13C NMR (100 MHz, DMSO-d6):

Y. Liu et al. / Bioorg. Med. Chem. 21 (2013) 5679–5687
5685
d 167.3, 146.0, 133.8, 133.0, 132.6, 130.5, 130.2, 130.1, 129.7,
129.6, 127.7, 125.3, 121.9, 118.2, 51.8, 50.7, 46.2, 46.1, 36.3, 29.2,
24.6 ppm; Anal. Calcd for C23H23FN6OꢀHCl: C, 60.72; H, 5.32; N,
[M+H]+. 13C NMR (100 MHz, DMSO-d6): d 167.3, 146.3, 138.2,
133.9, 133.2, 130.7, 130.2, 129.7, 128.1, 127.9, 126.7, 125.2,
121.7, 118.2, 52.2, 51.8, 46.2, 46.1, 36.3, 29.2, 24.6 ppm; Anal. Calcd
for C23H23ClN6OꢀHCl: C, 58.60; H, 5.13; N, 17.83. Found: C, 58.54;
18.47. Found: C, 60.73; H, 5.50; N, 18.41; ½a D25
ꢂ
= 39.20 (c = 0.1,
CH3OH).
H, 5.30; N, 17.81; ½a D25
ꢂ
= 31.04 (c = 0.1, CH3OH).
4.1.2.6.
(S)-1-{(S)-2-Amino-3-[4-(1-(3-fluorobenzyl)-1H-1,2,3-
4.1.2.10. (S)-1-{(S)-2-Amino-3-[4-(1-(4-chlorobenzyl)-1H-1,2,3-
triazol-4-yl)phenyl]propanoyl}pyrrolidine-2-carbonitrile
hydrochloride (6f). Yield: 86%; white powder; mp 130–
triazol-4-yl)phenyl]propanoyl}pyrrolidine-2-carbonitrile
hydrochloride (6j).
Yield: 76%; white powder; mp 130–
132 °C; 1H NMR (500 MHz, DMSO-d6): d 8.69 (s, 1H, @CH), 8.41
(br, 3H, NHþ3 ), 7.81 (d, 2H, J = 8.0 Hz, ArH), 7.46–7.41 (m, 1H,
ArH), 7.32 (d, 2H, J = 8.0 Hz, ArH), 7.23–7.16 (m, 3H, ArH), 5.66 (s,
2H, CH2Ar), 4.79––4.77 (m, 1H, CHCN), 4.35–4.31 (m, 1H, CH),
3.49–3.44 (m, 1H, CH2), 3.16–3.13 (m, 1H, CH2), 3.06–3.01 (m,
1H, CH2), 2.76–2.74 (m, 1H, CH2), 2.14–2.08 (m, 1H, CH2), 2.03–
1.97 (m, 1H, CH2), 1.84–1.79 (m, 1H, CH2), 1.66–1.64 (m, 1H, CH2)
ppm; ESI-MS (m/z): 419 [M+H]+. 13C NMR (100 MHz, DMSO-d6):
d 167.8, 146.7, 134.3, 131.4, 131.3, 130.7, 125.7, 124.7, 124.6,
122.2, 118.7, 52.8, 52.3, 46.7, 46.6, 36.8, 29.7, 25.1 ppm; Anal. Calcd
for C23H23FN6OꢀHCl: C, 60.72; H, 5.32; N, 18.47. Found: C, 60.64; H,
132 °C; 1H NMR (500 MHz, DMSO-d6): d 8.66 (s, 1H, @CH), 8.37
(br, 3H, NHþ3 ), 7.81 (d, 2H, J = 8.5 Hz, ArH), 7.46 (d, 2H, J = 8.5 Hz,
ArH), 7.39 (d, 2H, J = 8.5 Hz, ArH), 7.31 (d, 2H, J = 8.5 Hz, ArH),
5.63 (s, 2H, CH2Ar), 4.79–4.75 (m, 1H, CHCN), 4.34–4.30 (m, 1H,
CH), 3.39–3.31 (m, 1H, CH2), 3.17–3.13 (m, 1H, CH2), 3.07–3.01
(m, 1H, CH2), 2.77–2.72 (m, 1H, CH2), 2.14–2.08 (m, 1H, CH2),
2.02–1.97 (m, 1H, CH2), 1.83–1.78 (m, 1H, CH2), 1.68–1.61 (m,
1H, CH2) ppm; ESI-MS (m/z): 435 [M+H]+. 13C NMR (100 MHz,
DMSO-d6): d 167.3, 158.4, 146.2, 134.9, 133.8, 132.8, 130.2,
129.9, 129.8, 129.7, 128.7, 125.2, 121.6, 118.2, 52.2, 51.8, 46.2,
46.1, 36.3, 29.2, 24.6 ppm; Anal. Calcd for C23H23ClN6OꢀHCl: C,
58.60; H, 5.13; N, 17.83. Found: C, 58.57; H, 5.36; N, 17.75;
5.51; N, 18.36; ½a D25
ꢂ
= 30.96 (c = 0.1, CH3OH).
½
a 2D5
= 34.16 (c = 0.1, CH3OH).
ꢂ
4.1.2.7.
triazol-4-yl)phenyl]propanoyl}pyrrolidine-2-carbonitrile
hydrochloride (6g). Yield: 84%; white powder; mp 138–
(S)-1-{(S)-2-Amino-3-[4-(1-(4-fluorobenzyl)-1H-1,2,3-
4.1.2.11.
1,2,3-triazol-4-yl)phenyl]propanoyl}pyrrolidine-2-carbonitrile
hydrochloride (6k). Yield: 78%; white powder; mp 158–
(S)-1-{(S)-2-Amino-3-[4-(1-(2-methoxybenzyl)-1H-
140 °C; 1H NMR (500 MHz, DMSO-d6): d 8.66 (s, 1H, @CH), 8.41
(br, 3H, NHþ3 ), 7.79 (d, 2H, J = 8.0 Hz, ArH), 7.45–7.42 (m, 2H,
ArH), 7.30 (d, 2H, J = 8.0 Hz, ArH), 7.24–7.20 (m, 2H, ArH), 5.62 (s,
2H, CH2Ar), 4.78–4.74 (m, 1H, CHCN), 4.34–4.29 (m, 1H, CH),
3.40–3.35 (m, 1H, CH2), 3.18–3.14 (m, 1H, CH2), 3.08–3.01 (m,
1H, CH2), 2.76–2.71 (m, 1H, CH2), 2.15–2.08 (m, 1H, CH2), 2.02–
1.97 (m, 1H, CH2), 1.84–1.77 (m, 1H, CH2), 1.67–1.60 (m, 1H, CH2)
ppm; ESI-MS (m/z): 419 [M+H]+. 13C NMR (100 MHz, DMSO-d6):
d 172.5, 168.3, 165.9, 151.5, 139.1, 137.4, 137.3, 135.6, 135.5,
135.4, 135.0, 130.4, 126.8, 123.5, 120.9, 120.7, 57.4, 57.1, 51.5,
51.4, 41.6, 34.5, 29.9 ppm; Anal. Calcd for C23H23FN6OꢀHCl: C,
60.72; H, 5.32; N, 18.47. Found: C, 60.70, H, 5.41; N, 18.38;
160 °C; 1H NMR (500 MHz, DMSO-d6): d 8.52 (s, 1H, @CH), 8.39
(br, 3H, NHþ3 ), 7.81 (d, 2H, J = 8.0 Hz, ArH), 7.38–7.33 (m, 1H,
ArH), 7.30 (d, 2H, J = 8.5 Hz, ArH), 7.17–7.14 (m, 1H, ArH), 7.07 (d,
1H, J = 8.0 Hz, ArH), 6.96 (t, 1H, J = 7.5 Hz, ArH), 5.56 (s, 2H, CH2Ar),
4.79–4.75 (m, 1H, CHCN), 4.37–4.30 (m, 1H, CH), 3.50–3.44 (m, 1H,
CH2), 3.17–3.14 (m, 1H, CH2), 3.07–3.01 (m, 1H, CH2), 2.78–2.73 (m,
1H, CH2), 2.14–2.10 (m, 1H, CH2), 2.03–1.98 (m, 1H, CH2), 1.84–1.79
(m, 1H, CH2), 1.67–1.63 (m, 1H, CH2) ppm; ESI-MS (m/z): 431
[M+H]+. 13C NMR (100 MHz, DMSO-d6): d 167.3, 156.8, 145.8,
133.8, 130.1, 129.9, 129.8, 129.6, 125.2, 123.3, 121.7, 120.5,
118.2, 111.1, 55.5, 51.8, 48.3, 46.2, 46.1, 36.3, 29.2, 24.6 ppm; Anal.
Calcd for C24H26N6O2ꢀHCl: C, 61.73; H, 5.83; N, 18.00. Found: C,
½
a 2D5
= 27.76 (c = 0.1, CH3OH).
ꢂ
61.64; H, 6.06; N, 17.96; ½a D25
ꢂ
= 34.72 (c = 0.1, CH3OH).
4.1.2.8. (S)-1-{(S)-2-Amino-3-[4-(1-(2-chlorobenzyl)-1H-1,2,3-
triazol-4-yl)phenyl]propanoyl}pyrrolidine-2-carbonitrile
4.1.2.12.
1,2,3-triazol-4-yl)phenyl]propanoyl}pyrrolidine-2-carbonitrile
hydrochloride (6l). Yield: 79%; white powder; mp 155–
(S)-1-{(S)-2-Amino-3-[4-(1-(3-methoxybenzyl)-1H-
hydrochloride (6h).
Yield: 80%; white powder; mp 132–
135 °C; 1H NMR (500 MHz, DMSO-d6): d 8.63 (s, 1H, @CH), 8.40
(br, 3H, NHþ3 ), 7.82 (d, 2H, J = 8.0 Hz, ArH), 7.54–7.52 (m, 1H,
ArH), 7.43–7.36 (m, 2H, ArH), 7.32–7.27 (m, 3H, ArH), 5.74 (s, 2H,
CH2Ar), 4.79–4.75 (m, 1H, CHCN), 4.34–4.31 (m, 1H, CH), 3.50–
3.45 (m, 1H, CH2), 3.18–3.15 (m, 1H, CH2), 3.07–3.02 (m, 1H,
CH2), 2.79–2.74 (m, 1H, CH2), 2.14–2.08 (m, 1H, CH2), 2.04–1.97
(m, 1H, CH2), 1.84–1.79 (m, 1H, CH2), 1.68–1.64 (m, 1H, CH2)
ppm; ESI-MS (m/z): 435 [M+H]+. 13C NMR (100 MHz, DMSO-d6):
d 167.3, 146.0, 133.9, 133.0, 132.6, 130.6, 130.2, 130.1, 129.7,
129.6, 127.7, 125.2, 121.9, 118.2, 51.8, 50.7, 46.2, 46.1, 36.2, 29.2,
24.6 ppm; Anal. Calcd for C23H23ClN6OꢀHCl: C, 58.60; H, 5.13; N,
158 °C; 1H NMR (500 MHz, DMSO-d6): d 8.68 (s, 1H, @CH), 8.43
(br, 3H, NHþ3 ), 7.82 (d, 2H, J = 8.5 Hz, ArH), 7.32–7.29 (m, 3H,
ArH), 6.96–6.89 (m, 3H, ArH), 5.60 (s, 2H, CH2Ar), 4.80–4.76 (m,
1H, CHCN), 4.35–4.31 (m, 1H, CH), 3.50–3.46 (m, 1H, CH2), 3.20–
3.16 (m, 1H, CH2), 3.08–3.02 (m, 1H, CH2), 2.77–2.73 (m, 1H,
CH2), 2.15–2.09 (m, 1H, CH2), 2.04–1.99 (m, 1H, CH2), 1.86–1.80
(m, 1H, CH2), 1.69–1.62 (m, 1H, CH2) ppm; ESI-MS (m/z): 431
[M+H]+. 13C NMR (100 MHz, DMSO-d6): d 167.3, 159.4, 146.2,
137.3, 133.8, 130.2, 129.9, 129.8, 125.2, 121.6, 120.0, 118.2,
113.8, 113.4, 55.1, 52.9, 51.8, 46.2, 46.1, 36.3, 29.2, 24.6 ppm; Anal.
Calcd for C24H26N6O2ꢀHCl: C, 61.73; H, 5.83; N, 18.00. Found: C,
17.83. Found: C, 58.63; H, 5.30; N, 17.73; ½a D25
ꢂ
= 49.24 (c = 0.1,
CH3OH).
61.65; H, 6.08; N, 17.98; ½a D25
ꢂ
= 30.92 (c = 0.1, CH3OH).
4.1.2.9. (S)-1-{(S)-2-Amino-3-[4-(1-(3-chlorobenzyl)-1H-1,2,3-
triazol-4-yl)phenyl]propanoyl}pyrrolidine-2-carbonitrile
4.1.2.13.
1,2,3-triazol-4-yl)phenyl]propanoyl}pyrrolidine-2-carbonitrile
hydrochloride (6m). Yield: 82%; white powder; mp 131–
(S)-1-{(S)-2-Amino-3-[4-(1-(4-methoxybenzyl)-1H-
hydrochloride (6i).
Yield: 83%; white powder; mp 154–
157 °C; 1H NMR (500 MHz, DMSO-d6): d 8.71 (s, 1H, @CH), 8.38
(br, 3H, NHþ3 ), 7.83 (d, 2H, J = 8.5 Hz, ArH), 7.47–7.41 (m, 3H,
ArH), 7.34–7.31 (m, 3H, ArH), 5.67 (s, 2H, CH2Ar), 4.81–4.76 (m,
1H, CHCN), 4.37–4.33 (m, 1H, CH), 3.48–3.45 (m, 1H, CH2), 3.18–
3.15 (m, 1H, CH2), 3.07–3.03 (m, 1H, CH2), 2.80–2.75 (m, 1H,
CH2), 2.17–2.10 (m, 1H, CH2), 2.05–2.01 (m, 1H, CH2), 1.86–1.80
(m, 1H, CH2), 1.70–1.64 (m, 1H, CH2) ppm; ESI-MS (m/z): 435
134 °C; 1H NMR (500 MHz, DMSO-d6): d 8.61 (s, 1H, @CH), 8.38
(br, 3H, NHþ3 ), 7.79 (d, 2H, J = 8.0 Hz, ArH), 7.35 (d, 2H, J = 8.0 Hz,
ArH), 7.30 (d, 2H, J = 8.5 Hz, ArH), 6.94 (d, 2H, J = 8.5 Hz, ArH),
5.53 (s, 2H, CH2Ar), 4.79–4.76 (m, 1H, CHCN), 4.35–4.31 (m, 1H,
CH), 3.73 (s, 3H, OCH3), 3.37–3.35 (m, 1H, CH2), 3.15–3.12 (m, 1H,
CH2), 3.05–3.00 (m, 1H, CH2), 2.75–2.73 (m, 1H, CH2), 2.13–2.09
(m, 1H, CH2), 2.02–1.97 (m, 1H, CH2), 1.83–1.78 (m, 1H, CH2),

5686
Y. Liu et al. / Bioorg. Med. Chem. 21 (2013) 5679–5687
1.65–1.62 (m, 1H, CH2) ppm; ESI-MS (m/z): 431 [M+H]+. 13C NMR
(100 MHz, DMSO-d6): d 167.3, 159.1, 146.1, 133.8, 130.1, 129.8,
129.7, 129.6, 127.8, 125.2, 121.3, 118.2, 114.1, 55.1, 52.5, 51.8,
46.2, 46.1, 36.3, 29.2, 24.6 ppm; Anal. Calcd for C24H26N6O2ꢀHCl:
C, 61.73; H, 5.83; N, 18.00. Found: C, 61.70; H, 6.04; N, 18.02;
460 nm with an Envision microplate reader (PerkinElmer, Wal-
tham, MA, USA). Reaction solutions containing 20 L of enzyme
(DPPs), 20 L of fluorescent substrate (100 mol/L), 140 L of assay
buffer (100 mmol/L HEPES, pH 7.5, 0.1 mg/mL BSA) and 20 L of
test substrate (of various concentrations) were incubated for
60 min at room temperature using a 96-well flat-bottomed micro-
titer plate.18–21 The continuous fluorescence intensity changes
were recorded, according to these data, IC50 values were deter-
mined by using the Graphpad Prism software with three indepen-
dent determinations.
l
l
l
l
l
½
a 2D5
= 30.72 (c = 0.1, CH3OH).
ꢂ
4.1.2.14.
1,2,3-triazol-4-yl)phenyl]propanoyl}pyrrolidine-2-carbonitrile
hydrochloride (6n). Yield: 85%; white powder; mp 117–
(S)-1-{(S)-2-Amino-3-[4-(1-(3,4-difluorobenzyl)-1H-
119 °C; 1H NMR (500 MHz, DMSO-d6): d 8.67 (s, 1H, @CH), 8.40
(br, 3H, NHþ3 ), 7.80 (d, 2H, J = 8.5 Hz, ArH), 7.53–7.43 (m, 2H,
ArH), 7.31 (d, 2H, J = 8.5 Hz, ArH), 7.25–7.18 (m, 1H, ArH), 5.63 (s,
2H, CH2Ar), 4.79–4.74 (m, 1H, CHCN), 4.34–4.31 (m, 1H, CH),
3.49–3.44 (m, 1H, CH2), 3.18–3.14 (m, 1H, CH2), 3.06–3.01 (m,
1H, CH2), 2.77–2.72 (m, 1H, CH2), 2.14–2.08 (m, 1H, CH2), 2.03–
1.97 (m, 1H, CH2), 1.83–1.79 (m, 1H, CH2), 1.67–1.61 (m, 1H, CH2)
ppm; ESI-MS (m/z): 437 [M+H]+. 13C NMR (100 MHz, DMSO-d6):
d 167.3, 150.5, 150.4, 148.1, 147.9, 146.2, 133.9, 133.4, 130.2,
129.7, 125.3, 125.2, 121.6, 118.2, 118.0, 117.8, 117.6, 117.4, 51.8,
51.7, 46.2, 46.1, 36.3, 29.2, 24.6 ppm; Anal. Calcd for C23H22F2N6-
OꢀHCl: C, 58.41; H, 4.90; N, 17.77. Found: C, 58.31; H, 5.10; N,
4.3. Molecular docking
The molecular docking study was performed using the Flexi-
Dock module of Sybyl 6.9 version (Sybyl molecular modeling soft-
ware packages. Tripos Associates Inc., St. Louis, MO63144, 2002).
The X-ray co-crystal structure of DPP-4 in complex with its inhib-
itor was obtained from the RCSB Protein Data Bank (PDB code:
2AJL). The ligands were energy-minimized under a Tripos force
field. The protein receptor was preprocessed by removing the ini-
tial ligand, solvent molecules, and adding hydrogen atoms. Koll-
man all-atom charges were assigned to the DPP-4 protein. A
binding pocket was defined to cover all of the amino acid residues
within 4.0 Å radius sphere centered by the docked compound in
the initial compound and DPP-4 complex. All the single bonds of
residues side chains within the defined DPP-4 binding pocket were
considered as rotatable or flexible bonds. The structure optimiza-
tion was performed for 100,000 generation using a genetic algo-
rithm, and the 20 best scoring ligand–protein complexes were
kept for further analyses. The conformation with the lowest energy
was selected as a final model to analyze the interaction of DPP-4
with its inhibitor. The pictures were generated by Pymol (http://
pymol.sourceforge.net/).
17.70; ½a 2D5
ꢂ
= 35.80 (c = 0.1, CH3OH).
4.1.2.15.
(S)-1-{(S)-2-Amino-3-[4-(1-(3,4-dichlorobenzyl)-1H-
1,2,3-triazol-4-yl)phenyl]propanoyl}pyrrolidine-2-carbonitrile
hydrochloride (6o). Yield: 87%; white powder; mp 124–
127 °C; 1H NMR (500 MHz, DMSO-d6): d 8.68 (s, 1H, @CH), 8.36
(br, 3H, NHþ3 ), 7.80 (d, 2H, J = 8.0 Hz, ArH), 7.68–7.65 (m, 2H,
ArH), 7.35–7.33 (m, 1H, ArH), 7.31 (d, 2H, J = 8.0 Hz, ArH), 5.66 (s,
2H, CH2Ar), 4.79–4.75 (m, 1H, CHCN), 4.36–4.30 (m, 1H, CH),
3.49–3.44 (m, 1H, CH2), 3.18–3.13 (m, 1H, CH2), 3.06–3.02 (m,
1H, CH2), 2.80–2.74 (m, 1H, CH2), 2.16–2.09 (m, 1H, CH2), 2.04–
1.97 (m, 1H, CH2), 1.85–1.80 (m, 1H, CH2), 1.69–1.62 (m, 1H, CH2)
ppm; ESI-MS (m/z): 469 [M+H]+. 13C NMR (100 MHz, DMSO-d6):
d 167.3, 146.3, 134.3, 133.2, 131.5, 130.5, 129.8, 129.7, 129.6,
128.0, 127.1, 125.2, 121.6, 118.2, 52.1, 51.8, 46.2, 46.1, 36.2, 29.2,
24.6 ppm; Anal. Calcd for C23H22Cl2N6OꢀHCl: C, 54.61; H, 4.58; N,
4.4. Oral glucose tolerance test in rats
Male Sprague–Dawley rats (200–220 g) were fasted overnight
and randomly assigned into five groups (n = 5, each group). Each
group of rat was respectively administered with vehicle (0.5% car-
boxymethyl cellulose sodium, CMC–Na), vildagliptin (3 mg/kg), or
compound 6g at doses of 1, 3, and 10 mg/kg. After 30 min, all ani-
mals received an oral glucose load (2.5 g/kg), this time was taken
as the zero starting point. Blood samples were collected from the
tail vein before and after glucose administration at the time per-
iod of ꢃ30, 0, 30, 60 and 120 min. Subsequently, blood glucose
levels at these time points were monitored with blood glucose
meter.
16.61. Found: C, 54.55; H, 4.78; N, 16.55; ½a D25
ꢂ
= 29.04 (c = 0.1,
CH3OH).
4.1.2.16. (S)-1-{(S)-2-Amino-3-[4-(1-(3,4-dimethoxybenzyl)-1H-
1,2,3-triazol-4-yl)phenyl]propanoyl}pyrrolidine-2-carbonitrile
hydrochloride (6p).
Yield: 84%; white powder; mp 122–
124 °C; 1H NMR (500 MHz, DMSO-d6): d 8.61 (s, 1H, @CH), 8.34
(br, 3H, NHþ3 ), 7.80 (d, 2H, J = 8.5 Hz, ArH), 7.30 (d, 2H, J = 8.5 Hz,
ArH), 7.05–7.03 (m, 1H, ArH), 6.95–6.90 (m, 2H, ArH), 5.52 (s, 2H,
CH2Ar), 4.79–4.75 (m, 1H, CHCN), 4.35–4.33 (m, 1H, CH), 3.74 (s,
3H, OCH3), 3.72 (s, 3H, OCH3), 3.44–3.39 (m, 1H, CH2), 3.15–3.12
(m, 1H, CH2), 3.05–3.00 (m, 1H, CH2), 2.78–2.74 (m, 1H, CH2),
2.14–2.08 (m, 1H, CH2), 2.03–1.97 (m, 1H, CH2), 1.84–1.78 (m,
1H, CH2), 1.65–1.61 (m, 1H, CH2) ppm; ESI-MS (m/z): 461 [M+H]+.
13C NMR (100 MHz, DMSO-d6): d 167.3, 148.7, 146.3, 133.7,
130.1, 129.8, 128.0, 125.2, 121.2, 120.7, 118.2, 112.0, 111.8, 55.5,
52.9, 51.8, 46.2, 46.1, 36.3, 29.2, 24.6 ppm; Anal. Calcd for C25H28-
N6O3ꢀHCl: C, 60.42; H, 5.88; N, 16.91. Found: C, 60.32; H, 6.09; N,
Acknowledgments
The authors thank Jianyang Pan (Pharmaceutical Informatics
Institute, Zhejiang University) for performing NMR spectrometry
and Mass spectrometry for structure elucidation, the authors are
also grateful to Peng Peng and Liying Zhao for help in molecular
modeling and optical rotation test.
16.86; ½a 2D5
ꢂ
= 36.12 (c = 0.1, CH3OH).
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