Design, Synthesis, Structure-Activity Relationships, and Docking Studies of 1-(γ-1,2,3-Triazol Substituted Prolyl)-(S)-3,3-Difluoropyrrolidines as a Novel Series of Potent and Selective Dipeptidyl Peptidase-4 Inhibitors

Article abstract of DOI:10.1111/cbdd.12058

Dipeptidyl peptidase-4 inhibitors hold great potential for the treatment of type 2 diabetes. A series of 1-(γ-1,2,3-triazol substituted prolyl)-(S)-3,3-difluoropyrrolidines were designed, synthesized, and evaluated as novel dipeptidyl peptidase-4 inhibitors. Most of the compounds exhibited good in vitro potency against dipeptidyl peptidase-4. Among these, compounds 7j, 7q, and 7s displayed good dipeptidyl peptidase-4 activity and excellent selectivity versus other proteases including dipeptidyl peptidase-8, dipeptidyl peptidase-9, and FAP. The possible binding modes of compounds 7j, 7q, and 7s with dipeptidyl peptidase-4 were also explored by molecular docking simulation.

Full text of DOI:10.1111/cbdd.12058

Chem Biol Drug Des 2013; 81: 198–207
Research Article
Design, Synthesis, Structure–Activity Relationships,
and Docking Studies of 1-(c-1,2,3-Triazol Substituted
Prolyl)-(S)-3,3-Difluoropyrrolidines as a Novel Series of
Potent and Selective Dipeptidyl Peptidase-4 Inhibitors
Lei Zhang1, Mingbo Su2, Jingya Li2, Xun Ji1,
as well as stimulating b-cell growth, survival, and differenti-
ation (2–4). However, the active form of GLP-1 is rapidly
metabolized by enzyme dipeptidyl peptidase-4 (DPP-4)
which selectively cleaves the dipeptide from the N-termi-
Jiang Wang1, Zeng Li1, Jia Li2,*, Hong Liu1,*
1State Key Laboratory of Drug Research, Shanghai
Institute of Materia Medica, Shanghai Institutes for
Biological Sciences, Chinese Academy of Sciences, 555
Zu Chong Zhi Road, Shanghai, China
2National Center for Drug Screening, Shanghai Institute of
Materia Medica, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, 189 Guo Shou
Jing Road, Shanghai, China
nus of GLP-1, thereby limiting its duration of action (5,6).
Therefore, inhibition of DPP-4 can extend the duration of
GLP-1 action and consequently improve both b-cell insulin
secretion and glycaemic control. This concept has been
well validated by the approval of DPP-4 inhibitors Sitaglip-
tin 1 (Januviaꢀ; Merck) (7,8), Vildagliptin 2 (Galvusꢀ; Nov-
artis) (9,10), Saxagliptin 3 (Onglyzaꢀ; Bristol-Myers Squibb)
(11), Alogliptin 4 (Nesinaꢀ; Takeda) (12), and Linagliptin 5
(Tradjentaꢀ; Boehringer Ingelheim) (13) for the treatment of
type 2 diabetes (Figure 1). DPP-4 inhibitors have also been
demonstrated to be effective and safe compounds to con-
trol blood glucose with good patient compliance and
reduced risks of hypoglycemia and other side effects.
*Corresponding authors: Jia Li and Hong Liu,
jli@mail.shcnc.ac.cn; hliu@mail.shcnc.ac.cn
Dipeptidyl peptidase-4 inhibitors hold great potential
for the treatment of type 2 diabetes. A series of 1-(c-
1,2,3-triazol substituted prolyl)-(S)-3,3-difluoropyrroli-
dines were designed, synthesized, and evaluated as
novel dipeptidyl peptidase-4 inhibitors. Most of the
compounds exhibited good in vitro potency against
dipeptidyl peptidase-4. Among these, compounds 7j,
7q, and 7s displayed good dipeptidyl peptidase-4
activity and excellent selectivity versus other proteases
including dipeptidyl peptidase-8, dipeptidyl peptidase-
9, and FAP. The possible binding modes of com-
pounds 7j, 7q, and 7s with dipeptidyl peptidase-4 were
also explored by molecular docking simulation.
Structurally, DPP-4 resembles with several other prote-
ases, it is important to consider selectivity of DPP-4 inhibi-
tors over other serine proteases, especially DPP-8 and
DPP-9, to avoid potential side effects associated with
these off-target enzymes (14). Our continued interest in
DPP-4 inhibition as
a therapy for type 2 diabetes
prompted the investigation into other novel DPP-4 inhibi-
tors. Herein, we report our efforts toward the design, syn-
thesis, structure–activity relationships (SAR), and docking
studies of 1-(c-1,2,3-triazol substituted prolyl)-(S)-3,3-diflu-
oropyrrolidines as a novel series of DPP-4 inhibitors.
Key words: dipeptidyl peptidase-4, inhibitor, molecular dock-
ing, triazole, type 2 diabetes
Received 21 June 2012, revised 4 September 2012 and
accepted for publication 8 September 2012
Molecular Design
Although a number of structurally diverse DPP-4 inhibitors
have been reported in the last two decades, most of them
occupy the S1 and S2 pockets at the DPP-4 active site
and share common structural features of the P2 and P1
fragments that resemble the N-terminal dipeptide residues
of the enzymatic substrates (Figure 2) (15). For example,
simple L-prolyl-L-prolinenitrile 6 has been reported as an
effective DPP-4 inhibitor (16). However, DPP-4 inhibitors
possessing prolylprolinenitrile scaffold are well known to
suffer from chemical instability as illustrated in Figure 2A.
One strategy to solve this problem is to replace the pro-
linenitrile as 3,3-difluoropyrrolidine. The 1,2,3-triazole ring
Type 2 diabetes is a chronic disorder characterized by
hyperglycemia coupled with a gradual decline in insulin
sensitivity and insulin secretion. The main goal of the man-
agement of type 2 diabetes is to achieve glucose homeo-
stasis to reduce the risk of late-stage complications (1).
The gut hormone glucagon like peptide-1 (GLP-1) is
released from gastrointestinal tract in response to food
intake. It plays an important role in regulating glucose
homeostasis by increasing insulin secretion, inhibiting glu-
cagon release, retarding gastric emptying, inducing satiety,
198
ª 2012 John Wiley & Sons A/S. doi: 10.1111/cbdd.12058

Design, Synthesis, Structure–Activity Relationships
Figure 1: Approved DPP-4 inhibitors for the treatment of type 2 diabetes.
stability and versatile biological activities of 1,2,3-triazole,
we then introduced this moiety as a novel P2 fragment to
enhance the chemical stability of compounds 7. In this
way, we designed a novel series of 1-(c-1,2,3-triazol
substituted prolyl)-(S)-3,3-difluoropyrrolidine compounds
(Figure 2B) and evaluated them for inhibitory activity
toward DPP-4.
A
B
Methods and Materials
Chemistry
The synthesis of target compounds
7 is shown in
Figure 2: (A) Chemical instability of cynaopyrrolidine 6. (B) Molec-
ular design of novel DPP-4 inhibitors 7.
Scheme 1. Briefly, condensation of commercially available
Boc-L-hydroxyproline 8 and 3,3-difluoropyrrolidine hydro-
chloride 9 easily afforded 10, which was subsequently
transformed to azide 11 using diphenylphosphoryl azide
(DPPA). A click reaction of various terminal alkynes with
azide 11 provided compounds 12. Target compounds 7
were then smoothly generated by deprotection of com-
pounds 12 with trimethylsilyl trifluoromethanesulfonate
(TMSOTf) and 2,6-lutidine.
Biological evaluation
Enzyme-based assay of DPP-4 inhibition
All the synthesized compounds were tested in vitro against
human DPP-4 employing a continuous fluorometric assay
(22). Ala-Pro-amidomethylcoumarin (Ala-Pro-AMC) was
used as the substrate of DPP-4, which is cleaved by the
enzyme to release the fluorescent AMC. Liberation of AMC
was monitored using an excitation wavelength of 355 nm
and an emission wavelength of 460 nm by an Envision mi-
croplate reader (PerkinElmer).a A typical reaction contained
50 pM of enzyme DPP-4, 10 lM of Ala-Pro-AMC, different
concentrations of test compounds, and assay buffer
Figure 3: Representative drugs containing 1,2,3-triazole moiety.
is a quite stable five-membered heterocycle (17–21). It is
also an important bioisostere of imidazole, oxazole, pyraz-
ole, thiazole, amide moiety, etc., in designing pharmaceuti-
cal molecules. Many triazole-based compounds as
medicinal agents have been frequently employed for the
treatment of various types of diseases, such as Tazobac-
tam, Cefatrizine, TSAO, and CAI (Figure 3). In light of the
Chem Biol Drug Des 2013; 81: 198–207
199

Zhang et al.
F
HO
F
F
HO
F
N3
HCl
HN
b
F
a
OH
F
F
N
N
N
N
Boc
N
Boc
O
R
O
Boc
8
O
9
10
11
N
R
Scheme 1: Reagents and condi-
tions: (a) HOBt, EDCI, Et3N, DMF,
rt; (b) DPPA, DEAD, PPh3, THF, 0
ꢁC; (c) CuSO4Æ5H2O, sodium
ascorbate, tert-butyl alcohol ⁄ H2O,
rt; (d) TMSOTf, 2,6-lutidine,
CH2Cl2, rt.
N
R
N
F
N
F
N
F
d
N
c
N
N
N
N
Boc
O
H
12
O
7
(100 mM HEPES, pH 7.5, 0.1 mg ⁄ mL BSA) in a total reac-
tion volume of 50 lL. The DPP-4 enzyme used in these
studies was soluble recombinant human protein produced
by high five cells with baculovirus expression system (Bac-
To-Bac; Life Technologies)b (23) and purified with Ni-NTA
resin (Novagen)c.
characterized by 1H NMR, 13C NMR and LRMS. Nuclear
magnetic resonance spectra were recorded on a Bruc-
ker AMX-300 or 400 MHz instrument (TMS as IS).
Chemical shifts were reported in parts per million (ppm,
d) downfield from tetramethylsilane. Proton coupling pat-
terns were described as singlet (s), doublet (d), triplet (t),
quartet (q), multiplet (m), and broad (br).
Enzyme-based assay of inhibition selectivity
against DPPs
General procedures for the preparation of
compounds 7
Inhibition of the related proteases DPP-8, DPP-9, and
fibroblast activation protein (FAP) was also assessed. All
DPP proteins were expressed in high five cells using a ba-
culovirus system, and the activities of DPPs were assayed
using a continuous fluorometric method. We used Nle-
Pro-AMC as substrate to measure the activities of FAP,
and Ala-Pro-AMC for DPP-8 and DPP-9 at the optimized
assay system (pH = 8.0).
1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-4-phenyl-1H-1,2,3-
triazole (7a)
(i) Triethylamine (2.75 mL, 19.03 mmol) was added to a
suspension of 3,3-difluoropyrrolidine hydrochloride
9
(2.73 g, 19.03 mmol), in 25 mL of DMF. After five min
(4R)-1-(tert-butoxycarbonyl)-4-hydroxy-L-proline
17.30 mmol), hydroxybenzotriazole (HOBt,
8
(4 g,
2.57 g,
19.03 mmol), and 1-ethyl-3-(3-dimethylaminopropyl) car-
bodiimide (EDCI, 3.65 g, 19.03 mmol) were added. After
stirring the reaction overnight, the mixture was extracted
with ethyl acetate, washed sequentially with saturated
sodium bicarbonate and brine, dried over magnesium sul-
fate, filtered, and concentrated. The residue was purified by
chromatography to afford (3R,5S)-5-[(3,3-difluoropyrrolidin-
1-yl)carbonyl]pyrrolidin-3-ol 10 as a white solid (4.43 g,
80%). MS (EI, m ⁄ z) 321 [M+H]+. (ii) Diethyl azodicarboxylate
(DEAD, 1.35 mL, 8.55 mmol) was slowly added dropwise
via syringe to an ice-cooled stirred solution of compound 10
(2.11 g, 6.59 mmol) and triphenylphosphine (Ph3P, 2.16 g,
8.22 mmol) in dry THF (60 mL) under nitrogen. Diphenyl-
phosphoryl azide (DPPA, 1.77 mL, 8.22 mmol) was added
dropwise via syringe to the solution, and the temperature
was allowed to rise to room temperature. After 24 h, the sol-
vent was removed by rotary evaporation. The compound
was purified by chromatography to give compound 11 as
colorless oil (2.02 g, 89%). MS (EI, m ⁄ z) 346 [M+H]+. (iii) To
a solution of azide 11 (400 mg, 1.16 mmol) and ethynylben-
zene (140 lL, 1.27 mmol) in tert-butyl alcohol ⁄ H2O (1 ⁄ 1,
20 mL) were added sodium ascorbate (92 mg, 0.46 mmol)
and CuSO4Æ5H2O (56 mg, 0.23 mmol). After being stirring
overnight at ambient temperature, the reaction mixture was
diluted with ethyl acetate and washed with water. The
organic phase was dried with MgSO4 and concentrated in
Molecular docking
The Glide program from the Schro¨ dinger Suite was
employed for the docking study.d All molecules were
generated and minimized using SYBYL 7.3, Tripos.e The
protein receptor was obtained from the worldwide Protein
Data Bank (PDB ID: 3F8S) (24) and processed by remov-
ing all solvents, adding hydrogens, and carrying out mini-
mal minimization with the OPLS2001 force field using
protein preparation wizard. The grid was sized to 15 A in
f
˚
each direction and centered using the original small-mole-
cule inhibitor. In all cases, all compounds were prepared
for docking using LigPrep under its default parameters.
The maximum number of heavy atoms permitted per com-
pound was 120 with the maximum number of rotatable
bonds set at 30. Docking was performed using Glide (25)
in standard precision mode, with up to 40 conformers
saved per molecule. The top scoring conformer for each
compound, as assessed by its Glide score, was exported
to a Maestro-formatted output file.
Experimental
All commercially available compounds and solvents were
used without further treatment. All target products were
200
Chem Biol Drug Des 2013; 81: 198–207

Design, Synthesis, Structure–Activity Relationships
vacuo. The residue was purified by chromatography to pro-
vide tert-butyl (2S,4S)-2-[(3,3-difluoropyrrolidin-1-yl)car-
bonyl]-4-(4-phenyl-1H-1,2,3-triazol-1-yl)pyrrolidine-1-car-
boxylate 12a as a white solid (476 mg, 92%). MS (EI, m ⁄ z)
448 [M+H]+. (iv) To a stirred solution of compound 12a
(200 mg, 0.45 mmol) and 2,6-lutidine (105 lL, 0.89 mmol)
in dry CH2Cl2 (2 mL) at room temperature was added drop-
wise trimethylsilyl triflate (122 lL, 0.67 mmol). After the
reaction was completed monitored by TLC, the reaction
mixture was quenched with saturated aqueous ammonium
chloride solution and extracted with ethyl acetate. The com-
bined organic phase was washed with water and then brine,
dried with MgSO4. The concentrated residue was purified
by chromatography to give compound 7a as a white solid
1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-4-(4-fluorophenyl)-1H-
1,2,3-triazole (7d)
1H NMR (400 MHz, CD3OD) d 8.43 (s, 1H), 8.08 (t,
J = 7.6 Hz, 1H), 7.39–7.34 (m, 1H), 7.26 (t, J = 7.6 Hz,
1H), 7.23–7.18 (m, 1H), 5.34–5.29 (m, 1H), 4.14–3.67 (m,
5H), 3.54–3.48 (m, 1H), 3.38–3.33 (m, 1H), 2.95–2.86 (m,
1H), 2.54–2.35 (m, 3H). 13C NMR (125 MHz, CD3OD) d
172.85, 160.66 (d, J = 247.7 Hz), 142.43, 130.98,
128.68, 128.03 (t, J = 243.8 Hz), 125.81, 123.80, 119.61,
117.01, 63.01, 59.94, 54.27, 53.69 (t, J = 32.6 Hz),
45.28, 37.51, 35.02 (t, J = 25.0 Hz). LRMS (ESI, m ⁄ z):
366.2 [M+H]+. HRMS (ESI, m ⁄ z): calcd for C17H19N5OF3,
366.1542 [M+H]+; found 366.1534.
1
(147 mg, 94%). H NMR (300 MHz, CD3OD) d 8.48 (s, 1H),
7.81 (d, J = 8.4 Hz, 2H), 7.42 (t, J = 8.1 Hz, 2H), 7.36–7.33
(m, 1H), 5.29 (m, 1H), 4.15–3.67 (m, 5H), 3.52–3.48 (m,
1H), 3.39–3.34 (m, 1H), 2.96–2.85 (m, 1H), 2.58–2.32 (m,
3H). 13C NMR (125 MHz, CD3OD) d 172.85, 149.03,
131.72, 129.97, 129.36, 128.39 (d, J = 93.8 Hz), 126.66,
121.06, 62.97, 59.88, 54.28, 53.65 (t, J = 32.9 Hz), 45.27,
37.48, 35.00 (t, J = 23.8 Hz). LRMS (ESI, m ⁄ z): 348.3
[M+H]+. HRMS (ESI, m ⁄ z): calcd for C17H20N5OF2,
348.1636 [M+H]+; found 348.1651.
4-{1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-1H-1,2,3-triazol-4-
yl}benzonitrile (7e)
1H NMR (300 MHz, CD3OD) d 8.64 (s, 1H), 8.01 (d,
J = 8.4 Hz, 2H), 7.79 (d, J = 8.1 Hz, 2H), 5.34 (m, 1H),
4.28–3.68 (m, 5H), 3.54–3.42 (m, 2H), 3.07–2.97 (m, 1H),
2.59–2.34 (m, 3H). 13C NMR (125 MHz, CD3OD)
d
172.83, 147.14, 136.35, 133.97, 127.21, 122.99, 120.02
(d, J = 110.0 Hz), 112.58, 62.57, 59.86, 53.84, 53.70 (t,
J = 32.8 Hz), 45.36, 37.20, 34.97 (t, J = 23.8 Hz). LRMS
(ESI, m ⁄ z): 373.0 [M+H]+. HRMS (ESI, m ⁄ z): calcd for
1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-4-(2-fluorophenyl)-1H-
1,2,3-triazole (7b)
C
18H19N6OF2, 373.1588 [M+H]+; found 373.1586.
1H NMR (300 MHz, CD3OD) d 8.41 (s, 1H), 8.08 (t,
J = 7.5 Hz, 1H), 7.40–7.33 (m, 1H), 7.26 (t, J = 7.5 Hz,
1H), 7.20–7.16 (m, 1H), 5.38 (m, 1H), 4.34–3.48 (m,
7H), 3.08–2.95 (m, 1H), 2.56–2.37 (m, 3H). 13C NMR
(125 MHz, CD3OD) d 171.61, 160.63 (d, J = 247.8 Hz),
142.46, 131.06, 128.66, 127.95 (t, J = 245.0 Hz),
125.82, 124.22, 119.45, 117.02, 62.02, 59.95, 53.69 (t,
1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-4-[2-
(trifluoromethyl)phenyl]-1H-1,2,3-triazole (7f)
1H NMR (300 MHz, CD3OD) d 8.28 (s, 1H), 7.83 (d,
J = 7.5 Hz, 1H), 7.73–7.71 (m, 2H), 7.64–7.59 (m, 1H), 5.39
(m, 1H), 4.35–4.19 (m, 1H), 3.95–3.72 (m, 4H), 3.54 (m, 2H),
3.12–2.98 (m, 1H), 2.57–2.31 (m, 3H). 13C NMR (125 MHz,
CD3OD) d 170.82, 145.89, 133.40, 133.21, 130.24, 129.18
(d, J = 31.3 Hz), 128.21 (d, J = 87.5 Hz), 127.33 (q,
J = 6.25 Hz), 125.47, 125.09, 117.88, 61.07, 59.95, 53.78
(t, J = 32.5 Hz), 52.66, 45.42, 36.51, 34.93 (t, J = 23.8 Hz).
LRMS (ESI, m ⁄ z): 416.2 [M+H]+. HRMS (ESI, m ⁄ z): calcd for
C18H19N5OF5, 416.1510 [M+H]+; found 416.1517.
J = 32.6 Hz),
53.43,
45.33,
36.92,
34.97
(t,
J = 25.0 Hz). LRMS (ESI, m ⁄ z): 366.2 [M+H]+. HRMS
(ESI, m ⁄ z): calcd for C17H19N5OF3, 366.1542 [M+H]+;
found 366.1534.
1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-4-(3-fluorophenyl)-1H-
1,2,3-triazole (7c)
1H NMR (300 MHz, CD3OD) d 8.52 (s, 1H), 7.66–7.56 (m,
2H), 7.48–7.41 (m, 1H), 7.08 (t, J = 8.1 Hz, 1H), 5.34 (m,
1H), 4.35–3.54 (m, 7H), 3.08–2.98 (m, 1H), 2.60–2.33 (m,
3H). 13C NMR (125 MHz, CD3OD) d 173.52, 164.32 (d,
J = 244.3 Hz), 147.42, 133.44, 131.78, 127.57 (t,
J = 245.0 Hz), 125.15, 122.96, 122.41, 115.89 (d,
J = 21.3 Hz), 113.07 (d, J = 22.5 Hz), 61.08, 59.63, 53.70
(t, J = 32.5 Hz), 52.65, 45.34, 36.44, 34.66 (t,
J = 25.0 Hz). LRMS (ESI, m ⁄ z): 366.3 [M+H]+. HRMS (ESI,
m ⁄ z): calcd for C17H19N5OF3, 366.1542 [M+H]+; found
366.1528.
1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-4-(4-methoxyphenyl)-
1H-1,2,3-triazole (7g)
1H NMR (300 MHz, CD3OD) d 8.32 (s, 1H), 7.71 (d,
J = 9.0 Hz, 2H), 6.95 (d, J = 8.7 Hz, 2H), 5.27 (m, 1H),
4.33–3.86 (m, 3H), 3.79 (s, 3H), 3.75–3.69 (m, 2H), 3.46
(m, 2H), 3.09–2.96 (m, 1H), 2.55–2.24 (m, 3H). 13C NMR
(125 MHz, CD3OD) d 173.45, 161.40, 148.90, 128.02,
124.05, 121.72 (d, J = 318.3 Hz), 120.90, 115.35, 62.31,
59.47, 55.76, 53.84, 53.68 (t, J = 32.3 Hz), 45.37, 37.31,
34.94 (t, J = 25.0 Hz). LRMS (ESI, m ⁄ z): 378.2 [M+H]+.
Chem Biol Drug Des 2013; 81: 198–207
201

Zhang et al.
HRMS (ESI, m ⁄ z): calcd for C18H22N5O2F2, 378.1742
130.54, 130.28, 130.03, 129.84, 129.06, 127.84, 124.35,
63.85, 59.92, 59.69, 53.53 (t, J = 33.2 Hz), 51.42, 45.38,
35.23, 34.93 (t, J = 23.8 Hz). LRMS (ESI, m ⁄ z): 378.1
[M+H]+. HRMS (ESI, m ⁄ z): calcd for C18H22N5O2F2,
378.1742 [M+H]+; found 378.1728.
[M+H]+; found 378.1729.
1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-4-[4-(pentyloxy)phenyl]-
1H-1,2,3-triazole (7h)
1H NMR (300 MHz, CD3OD) d 8.35 (s, 1H), 7.70 (d,
J = 8.4 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H), 5.28 (m, 1H),
4.18–3.67 (m, 5H), 3.98 (t, J = 6.6 Hz, 2H), 3.51–3.33 (m,
2H), 2.98–2.85 (m, 1H), 2.55–2.32 (m, 3H), 1.82–1.73 (m,
2H), 1.51–1.36 (m, 4H), 0.95 (t, J = 7.2 Hz, 3H). 13C NMR
(126 MHz, CD3OD) d 172.86, 160.83, 149.04, 128.36 (d,
J = 91.3 Hz), 128.00, 124.01, 120.34, 115.88, 69.06,
62.71, 59.44, 54.11, 53.65 (t, J = 32.3 Hz), 45.30, 37.40,
34.99 (t, J = 24.2 Hz), 30.11, 29.39, 23.55, 14.40. LRMS
(ESI, m ⁄ z): 434.2 [M+H]+. HRMS (ESI, m ⁄ z): calcd for
C22H30N5O2F2, 434.2368 [M+H]+; found 434.2339.
3-{1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-1H-1,2,3-triazol-4-
yl}phenol (7l)
1H NMR (300 MHz, CD3OD) d 8.41 (s, 1H), 7.25–7.19 (m,
3H), 6.78–6.75 (m, 1H), 5.29 (m, 1H), 4.16–3.67 (m, 5H),
3.51–3.47 (m, 1H), 3.39–3.29 (m, 1H), 2.97–2.85 (m, 1H),
2.58–2.31 (m, 3H). 13C NMR (125 MHz, CD3OD) d 172.86,
159.11, 149.12, 132.88, 131.05, 128.38 (d, J = 93.3 Hz),
121.08, 117.95, 116.34, 113.40, 62.90, 59.88, 54.25, 53.68
(t, J = 32.7 Hz), 45.28, 37.45, 35.00 (t, J = 24.0 Hz). LRMS
(ESI, m ⁄ z): 364.3 [M+H]+. HRMS (ESI, m ⁄ z): calcd for
C
17H20N5O2F2, 364.1585 [M+H]+; found 364.1570.
4-(4-tert-Butylphenyl)-1-[(3S,5S)-5-[(3,3-
difluoropyrrolidin-1-yl)carbonyl]pyrrolidin-3-yl]-1H-
1,2,3-triazole (7i)
2-{1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-1H-1,2,3-triazol-4-
yl}pyridine (7m)
1H NMR (300 MHz, CD3OD) d 8.42 (s, 1H), 7.72 (d,
J = 8.1 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H), 5.31 (m, 1H),
4.24–3.69 (m, 5H), 3.54–3.38 (m, 2H), 3.02–2.92 (m, 1H),
2.58–2.32 (m, 3H), 1.33 (s, 9H). 13C NMR (125 MHz, CD3OD)
d 173.11, 152.68, 148.98, 128.71, 128.32 (d, J = 91.3 Hz),
126.87, 126.43, 121.28, 62.46, 59.89, 53.94, 53.68 (t,
J = 32.7 Hz), 45.33, 37.32, 35.50, 35.15 (t, J = 23.9 Hz),
31.67. LRMS (ESI, m⁄ z): 404.2 [M+H]+. HRMS (ESI, m⁄ z):
calcd for C21H28N5OF2, 404.2262 [M+H]+; found 404.2274.
1H NMR (300 MHz, CD3OD) d 8.58 (s, 2H), 8.02 (d,
J = 7.8 Hz, 1H), 7.91 (t, J = 7.5 Hz, 1H), 7.38 (m, 1H),
5.36 (m, 1H), 4.30–3.43 (m, 7H), 3.05–2.98 (m, 1H), 2.57–
2.36 (m, 3H). 13C NMR (125 MHz, CD3OD) d 172.85,
150.86, 150.53, 148.71, 138.98, 128.35 (d, J = 92.4 Hz),
124.62, 123.55, 121.67, 62.75, 59.93, 53.98, 53.68 (t,
J = 32.5 Hz), 45.33, 37.36, 34.98 (t, J = 25.0 Hz). LRMS
(ESI, m ⁄ z): 349.2 [M+H]+. HRMS (ESI, m ⁄ z): calcd for
C
16H19N6OF2, 349.1588 [M+H]+; found 349.1601.
1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-4-(4-fluoro-3-
methylphenyl)-1H-1,2,3-triazole (7j)
1H NMR (300 MHz, CD3OD) d 8.42 (s, 1H), 7.69 (d,
J = 7.5 Hz, 1H), 7.66–7.62 (m, 1H), 7.08 (t, J = 8.7 Hz,
1H), 5.31 (m, 1H), 4.32–3.71 (m, 5H), 3.54–3.44 (m, 2H),
3.09–2.96 (m, 1H), 2.59–2.34 (m, 3H), 2.30 (s, 3H). 13C
4-Benzyl-1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-1H-1,2,3-triazole (7n)
1H NMR (300 MHz, CD3OD) d 7.83 (s, 1H), 7.30–7.16 (m,
5H), 5.17 (m, 1H), 4.09–3.64 (m, 5H), 4.02 (s, 2H), 3.45–
3.38 (m, 1H), 3.28–3.15 (m, 1H), 2.88–2.76 (m, 1H), 2.56–
2.35 (m, 2H), 2.30–2.22 (m, 1H). 13C NMR (125 MHz,
CD3OD) d 172.78, 148.64, 140.43, 129.63, 128.38 (d,
J = 93.2 Hz), 127.50, 122.62, 62.73, 59.83, 54.08, 53.62
(t, J = 32.6 Hz), 45.22, 37.43, 34.99 (t, J = 25.0 Hz),
32.62. LRMS (ESI, m ⁄ z): 362.4 [M+H]+. HRMS (ESI, m ⁄ z):
calcd for C18H22N5OF2, 362.1792 [M+H]+; found
362.1803.
NMR (125 MHz, CD3OD)
d
173.16, 162.66 (d,
J = 245.2 Hz), 148.19, 129.93, 128.28 (d, J = 91.3 Hz),
127.66, 126.57 (d, J = 17.7 Hz), 126.01, 121.68, 116.50,
62.10, 59.89, 53.69 (t, J = 32.5 Hz), 53.65, 45.40, 37.12,
34.94 (t, J = 24.1 Hz), 14.49. LRMS (ESI, m ⁄ z): 380.2
[M+H]+. HRMS (ESI, m ⁄ z): calcd for C18H21N5OF3,
380.1698 [M+H]+; found 380.1708.
4-({1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-1H-1,2,3-triazol-4-
yl}methoxy)-2,6-difluorobenzonitrile (7o)
(2-{1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-1H-1,2,3-triazol-4-
yl}phenyl)methanol (7k)
(i) To
a solution of 2,6-difluoro-4-hydroxybenzonitrile
1H NMR (300 MHz, CD3OD) d 8.38 (s, 1H), 7.69–7.67 (m,
1H), 7.55–7.53 (m, 1H), 7.41–7.38 (m, 2H), 5.62 (m, 1H),
4.81–4.75 (m, 1H), 4.66 (s, 2H), 4.12–3.72 (m, 6H), 3.30–
3.22 (m, 1H), 2.85–2.74 (m, 1H), 2.61–2.38 (m, 2H). 13C
(500 mg, 3.22 mmol) and potassium carbonate (668 mg,
4.84 mmol) in acetone was added propargylbromide
(364 lL, 4.84 mmol). The reaction mixture was stirred
overnight at room temperature. After the reaction was
completed monitored by TLC, the solvent was evaporated
NMR (125 MHz, CD3OD)
d 166.96, 148.09, 139.92,
202
Chem Biol Drug Des 2013; 81: 198–207

Design, Synthesis, Structure–Activity Relationships
and extracted with ethyl acetate. The organic solvent was
washed with water and then brine, dried with MgSO4, and
concentrated in vacuo to give 2,6-difluoro-4-(prop-2-yn-1-
yloxy)benzonitrile as a white solid (591 mg, 95%). MS (EI,
m ⁄ z) 194 [M+H]+. (ii) To a solution of 2,6-difluoro-4-(prop-
2-yn-1-yloxy)benzonitrile (200 mg, 1.04 mmol) and azide
11 (393 mg, 1.14 mmol) in tert-butyl alcohol ⁄ H2O (1 ⁄ 1,
20 mL) were added sodium ascorbate (82 mg, 0.41 mmol)
and CuSO4Æ5H2O (50 mg, 0.21 mmol). After being stirring
overnight at ambient temperature, the reaction mixture
was diluted with ethyl acetate and washed with water. The
organic phase was dried with MgSO4 and concentrated in
vacuo. The residue was purified by chromatography to
provide tert-butyl (2S,4S)-4-[4-(4-cyano-3,5-difluorophen-
oxymethyl)-1H-1,2,3-triazol-1-yl]-2-[(3,3-difluoropyrrolidin-
1-yl)carbonyl]pyrrolidine-1-carboxylate 12o as a white solid
(500 mg, 90%). MS (EI, m ⁄ z) 539 [M+H]+. (iii) To a stirred
solution of compound 12o (200 mg, 0.37 mmol) and 2,6-
lutidine (87 lL, 0.74 mmol) in dry CH2Cl2 (2 mL) at room
temperature was added dropwise trimethylsilyl triflate
(124 lL, 0.56 mmol). After the reaction was completed
monitored by TLC, the reaction mixture was quenched
with saturated aqueous ammonium chloride solution, and
extracted with ethyl acetate. The combined organic phase
was washed with water and then brine, dried with MgSO4.
The concentrated residue was purified by chromatography
to give compound 7o as a white solid (111 mg, 82%). 1H
NMR (400 MHz, CDCl3) d 8.12 (s, 1H), 6.72 (s, 1H), 6.69
(s, 1H), 5.37–5.32 (m, 1H), 5.22 (s, 2H), 4.09–3.69 (m,
5H), 3.40–3.37 (m, 2H), 2.85–2.75 (m, 1H), 2.53–2.32 (m,
3H), 2.22 (br s, 1H). 13C NMR (100 MHz, CDCl3) d
171.33, 165.65 (d, J = 7.3 Hz), 163.91 (t, J = 13.7 Hz),
163.08 (d, J = 7.4 Hz), 142.27, 126.60 (d, J = 96.5 Hz),
122.37, 109.71, 99.98 (d, J = 2.9 Hz), 99.74 (d,
J = 2.9 Hz), 85.11 (t, J = 20.0 Hz), 62.91, 61.73, 59.17,
54.58, 53.14 (t, J = 32.4 Hz), 44.18, 36.59, 34.50 (t,
J = 24.0 Hz). LRMS (ESI, m ⁄ z): 439.1 [M+H]+. HRMS (ESI,
m ⁄ z): calcd for C19H19N6O2F4, 439.1506 [M+H]+; found
439.1517.
1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-4-(4-phenylphenyl)-1H-
1,2,3-triazole (7q)
1H NMR (300 MHz,CD3OD) d 8.51 (s, 1H), 7.90 (d,
J = 8.7 Hz, 2H), 7.72–7.64 (m, 4H), 7.45 (t, J = 7.2 Hz,
2H), 7.37–7.32 (m, 1H), 5.49 (m, 1H), 4.60–4.44 (m,
1H), 4.10–3.72 (m, 7H), 2.65–2.37 (m, 4H). 13C NMR
(125 MHz, DMSO) d 170.05, 146.09, 139.58, 139.47,
129.03, 128.75 (d, J = 285.0 Hz), 127.20, 126.55,
125.66, 121.99, 120.40, 60.44, 58.17, 52.44, 52.23 (t,
J = 31.4 Hz), 43.62, 35.46, 33.58 (t, J = 23.3 Hz).
LRMS (ESI, m ⁄ z): 424.2 [M+H]+. HRMS (ESI, m ⁄ z):
calcd for C23H24N5OF2, 424.1949 [M+H]+; found
424.1963.
{1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-1H-1,2,3-triazol-4-
yl}diphenylmethanol (7r)
1H NMR (300 MHz, CD3OD) d 7.80 (s, 1H), 7.35–7.22 (m,
10H), 5.30 (m, 1H), 4.38–3.47 (m, 7H), 3.08–2.97 (m, 1H),
2.55–2.30 (m, 3H). 13C NMR (125 MHz, DMSO) d 171.48,
154.07, 147.19, 127.61, 127.12, 126.78, 75.86, 61.82,
59.13, 52.21, 52.10 (t, J = 30.0 Hz), 43.61, 35.20, 33.64
(t, J = 23.8 Hz). LRMS (ESI, m ⁄ z): 454.0 [M+H]+. HRMS
(ESI, m ⁄ z): calcd for C24H25N5O2F2Na, 476.1874 [M+Na]+;
found 476.1872.
Methyl 1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-1H-1,2,3-triazole-4-
carboxylate (7s)
1H NMR (300 MHz, CD3OD) d 8.67 (s, 1H), 5.60 (m, 1H),
4.88–4.75 (m, 1H), 4.06–3.66 (m, 6H), 3.91 (s, 3H), 3.26–
3.23 (m, 1H), 2.81–2.72 (m, 1H), 2.58–2.40 (m, 2H). 13C
NMR (125 MHz, DMSO)
d 165.66, 160.68, 138.97,
128.99, 127.61 (d, J = 86.7 Hz), 58.10, 57.86, 52.45 (t,
J = 32.5 Hz), 52.01, 49.65, 43.86, 33.55 (t, J = 23.8 Hz),
33.48. LRMS (ESI, m ⁄ z): 330.2 [M+H]+. HRMS (ESI, m ⁄ z):
calcd for
C
13H18N5O3F2, 330.1378 [M+H]+; found
330.1386.
1-[(3S,5S)-5-[(3,3-difluoropyrrolidin-1-
yl)carbonyl]pyrrolidin-3-yl]-4-(6-
methoxynaphthalen-2-yl)-1H-1,2,3-triazole (7p)
1H NMR (300 MHz, CD3OD) d 8.56 (s, 1H), 8.24 (s, 1H),
7.86–7.79 (m, 3H), 7.26 (s, 1H), 7.17 (d, J = 9.0 Hz, 1H),
5.62 (m, 1H), 4.85–4.77 (m, 1H), 4.12–3.73 (m, 6H), 3.92
(s, 3H), 2.86–2.75 (m, 1H), 2.62–2.41 (m, 3H). 13C NMR
(100 MHz, DMSO) d 165.70, 157.58, 146.92, 134.02,
129.56, 128.52, 127.56 (d, J = 69.4 Hz), 127.52, 125.66,
123.98, 123.46, 120.96, 119.27, 106.06, 57.79, 57.56,
55.27, 52.36 (t, J = 46.0 Hz), 49.54, 43.84, 33.50 (t,
J = 28.8 Hz), 33.46. LRMS (ESI, m ⁄ z): 428.1 [M+H]+.
HRMS (ESI, m ⁄ z): calcd for C22H24N5O2F2, 428.1898
[M+H]+; found 428.1886.
4-[(tert-Butoxy)methyl]-1-[(3S,5S)-5-[(3,3-
difluoropyrrolidin-1-yl)carbonyl]pyrrolidin-3-yl]-1H-
1,2,3-triazole (7t)
1H NMR (300 MHz, CD3OD) d 8.05 (s, 1H), 5.29–5.20 (m,
1H), 4.53 (s, 2H), 4.13–3.66 (m, 5H), 3.46–3.33 (m, 2H),
2.93–2.81 (m, 1H), 2.58–2.25 (m, 3H), 1.28 (s, 9H). 13C
NMR (125 MHz, CD3OD) d 172.76, 147.57, 128.39 (d,
J = 93.0 Hz), 123.53, 75.23, 62.79, 59.89, 56.75, 54.22,
53.65 (t, J = 32.5 Hz), 45.25, 37.43, 35.00 (t,
J = 23.9 Hz), 27.79. LRMS (ESI, m ⁄ z): 358.2 [M+H]+.
HRMS (ESI, m ⁄ z): calcd for C16H25N5O2F2Na, 380.1874
[M+Na]+; found 380.1861.
Chem Biol Drug Des 2013; 81: 198–207
203

Zhang et al.
Table 1: In vitro DPP-4 inhibitory activity and selectivity
Table 2: In vitro DPP-4 inhibitory activity and selectivity
N
R2
R1
N
N
F
N
F
F
N
F
N
N
N
N
N
H
O
H
O
IC50 (lM) at 20 lg ⁄ mL
DPP-4 DPP-8 DPP-9
IC50 (lM) at 20 lg ⁄ mL
Compound R1
FAP
NI
Compound R2
DPP-4 DPP-8 DPP-9 FAP
7a
H
3.20
2.14
9.26
1.05
2.83
16.07
2.11
NI
NI
NI
NI
58.65
NI
NI
73.50
12.32
NI
68.15
61.03
NI
39.40
48.67
NI
30.79
28.46
NI
7m
6.88
NI
NI
NI
7b
7c
7d
7e
2-F
3-F
4-F
NI
NI
NI
NI
NI
NI
NI
23.35
NI
NI
N
7n
13.67
2.65
NI
50.27
5.12
NI
NI
4-CN
7f
2-CF3
4-OCH3
7g
7h
7i
7j
7k
F
7o
25.32
N
4-O(CH2)4CH3 2.62
4-C(CH3)3
3-CH3-4-F
2-CH2OH
3-OH
2.18
1.32
1.72
0.62
F
O
NI
NI
NI
O
7l
44.76
27.79
NI
7p
1.66
9.90
8.54 106.02
Alogliptin
–
0.03 1703.29 2583.97 1253.97
NI, no inhibition.
7q
7r
0.66
NI
NI
NI
NI
NI
NI
Results and Discussion
NI
OH
O
All the synthesized compounds were evaluated in vitro for
inhibition of human recombinant DPP-4. Inhibitory potency
was measured by monitoring the hydrolytic reaction of
Ala-Pro-AMC by human DPP-4 with alogliptin as the posi-
tive control, and the results are reported as concentrations
for 50% inhibition (IC50). DPP-8, DPP-9, and FAP data are
also presented because the importance of selectivity over
DPP-8 ⁄ 9 has been frequently mentioned in animal toxicity
studies. SAR for these compounds are summarized in
Tables 1 and 2.
7s
0.72
NI
NI
NI
NI
NI
NI
O
7t
12.39
O
Alogliptin
–
0.03 1703.29 2583.97 1253.97
Initially, our SAR started from phenyl-substituted triazole
compounds. The effects of regiochemical substitution and
electronics on the phenyl ring were explored. As shown in
Table 1, most of the compounds exhibited good in vitro
potency against DPP-4. Additionally, all of these inhibitors
occupied a narrow potency range, indicating tolerability for
a wide variety of substituents on the phenyl ring. The
mono-fluoro substituted compounds demonstrated the
NI, no inhibition.
provided potent DPP-4 inhibitory activity. The combination
of the 4-fluoro group with substitution at C-3 of the phenyl
ring (7j) resulted in satisfactory DPP-4 activity with an IC50
value of 1.32 lM and excellent selectivity over DPP-8, DPP-
9, and FAP. In addition, ortho-hydroxymethyl substitution on
the phenyl ring demonstrated good DPP-4 potency
(7k = 1.72 lM) and excellent selectivity. While substitution
with a 3-hydroxy group led to more potent DPP-4 inhibition
(7l = 0.62 lM) but unfavorable selectivity. Eventually, the
SAR from this small group of compounds found compound
7j as a potent and selective inhibitor of DPP-4.
regiochemical
preference
of
para>ortho>meta
(7d = 1.05 lM, 7b = 2.14 lM, 7c = 9.26 lM). Both elec-
tron-donating and electron-withdrawing groups on the
phenyl ring were tolerated for DPP-4 inhibition. For exam-
ple, compounds 7e (4-CN) and 7g (4-OCH3) exhibited
good inhibitory activities against DPP-4 with IC50 values of
2.83 and 2.11 lM, respectively. Compounds containing a
long-chain alkyl substituent (7h) at the para-position of the
phenyl ring displayed good DPP-4 potency. Introducing
steric hindrance at the 4-position of the phenyl ring (7i) also
Next, variation of substituents on the triazole ring was
investigated to understand the effect of substituents
(Table 2). The SAR study reveals that one-carbon linked
204
Chem Biol Drug Des 2013; 81: 198–207

Design, Synthesis, Structure–Activity Relationships
A
B
C
D
Figure 4: (A) Superposition of
compounds 7j, 7q, and 7s with
ligand PF-00734200 from 3F8S.
Docking structures of compounds
7j (B), 7q (C), and 7s (D) in the
active site of DPP-4. Hydrogen
atoms have been omitted for clar-
ity. All figures were prepared using
PyMol (http://www.pymol.org).
aryl 7n provided decreased potency compared with com-
pound 7a. Compound 7o showed good DPP-4 activity
but low selectivity against DPP-8 and DPP-9. It was
observed that pyridine-substitution (7m) was somewhat
less favored than phenyl-substitution on the triazole scaf-
fold. The 6-methoxy-2-naphthoxy analog 7p was slightly
more active than compound 7a but with poor selectivity
against other DPPs. Biphenyl derivative 7q was found to
display good DPP-4 inhibitory activity (IC50 = 0.66 lM) and
excellent selectivity over DPP-8, DPP-9, and FAP. DPP-4
activity diminished when the substituent was changed to
hydroxydiphenylmethyl group (7r). Additionally, alkyl and
ester substitutions on the triazole ring were also tolerated
with excellent selectivity. For example, compound 7s dem-
onstrated good potency to DPP-4 with an IC50 value of
0.72 lM. In this series, compounds 7q and 7s were dem-
onstrated to be potent DPP-4 inhibitors with excellent
selectivity over DPP-8, DPP-9, and FAP.
defined hydrophobic S1 pocket and makes extensive
hydrophobic interactions with the side chains of Trp659,
Val 656, Val711, Tyr631, Ser630, Tyr666, and Tyr662.
One of the pyrrolidide fluorines appears to be within hydro-
gen-bonding distance to form a hydrogen bond with either
the side chain of Ser630 or the main chain amide of
Tyr631. In the S2 pocket, the secondary amine of the pyr-
rolidine participates in a hydrogen-bonding network with
glutamic acids Glu206 and Glu205.
The observable differences in the binding modes originate
from the substituents on the triazole ring. The P2 fragment
of compound 7j (Figure 4B) positions in S2 pocket of
DPP-4 and makes hydrophobic interactions with residues
lining this pocket. Compared to compound 7j, the biphe-
nyl group of compound 7q (Figure 4C) extends to S2
pocket and forms a more favorable pi-pi stacking interac-
tion with Phe357, which may account for the increased
inhibitory activity than that of compound 7j. In terms of
compound 7s (Figure 4D), the carbonyl oxygen on the
triazole ring may interact with residue Ser209 through a
hydrogen bond.
To gain insight into the binding modes of these com-
pounds against DPP-4, molecular modeling experiments
were carried out to investigate the binding interactions
between this series of compounds and the active site of
DPP-4 using Glide program. The crystal structure of DPP-
4 (PDB ID: 3F8S) (24) was used in molecular docking sim-
ulation. Although DPP-4 exists as a dimer in the unit cell
and the protein-inhibitor interactions are much the same
for both molecules in the asymmetric unit, therefore, Fig-
ure 3 only shows the main interactions between com-
pounds 7j, 7q, 7s, and DPP-4 monomer.
Conclusion
In conclusion, we designed, synthesized, and evaluated a
series of 1-(c-1,2,3-triazol substituted prolyl)-(S)-3,3-diflu-
oropyrrolidines as novel DPP-4 inhibitors. In vitro evalua-
tion found that most of the compounds exhibited good in
vitro potency against DPP-4 in low micromolar range.
Among these, compounds 7j (IC50 = 1.32 lM) and 7s
(IC50 = 0.72 lM) showed both good DPP-4 potency and
excellent selectivity against other proteases. Compound
7q was found to display the best DPP-4 inhibitory activity
with an IC50 value of 0.66 lM and excellent selectivity over
Superposition of 7j, 7q, and 7s with the ligand PF-
00734200 from 3F8S (24) showed that these compounds
occupy the S1 and S2 pockets and share similar binding
modes (Figure 4). For example, the difluoropyrrolidide moi-
ety of this series of compounds is buried in the well-
Chem Biol Drug Des 2013; 81: 198–207
205

Zhang et al.
DPP-4-related enzymes including DPP-8, DPP-9, and
FAP. Molecular docking simulation demonstrated that this
series of compounds occupied the S1 and S2 pockets of
DPP-4. These potent compounds represent a novel scaf-
fold for the development of DPP-4 inhibitors. Current
efforts are aimed at further modifying these compounds to
achieve more potent and selective DPP-4 inhibitors.
Hughes
tyl)amino]acetyl]-2-cyano-(S)-pyrrolidine:
selective, and orally bioavailable dipeptidyl peptidase IV
T.E.
(2003)
1-[[(3-hydroxy-1-adaman-
a
potent,
inhibitor with antihyperglycemic properties.
Chem;46:2774–2789.
J Med
11. Augeri D.J., Robl J.A., Betebenner D.A., Magnin D.R.,
Khanna A., Robertson J.G., Wang A. et al. (2005) Dis-
covery and preclinical profile of Saxagliptin (BMS-
477118): a highly potent, long-acting, orally active dip-
eptidyl peptidase IV inhibitor for the treatment of type 2
diabetes. J Med Chem;48:5025–5037.
Acknowledgments
12. Feng J., Zhang Z., Wallace M.B., Stafford J.A., Kaldor
S.W., Kassel D.B., Navre M., Shi L., Skene R.J., Asak-
awa T., Takeuchi K., Xu R., Webb D.R., Gwaltney S.L.
2nd (2007) Discovery of alogliptin: a potent, selective,
bioavailable, and efficacious inhibitor of dipeptidyl pepti-
dase IV. J Med Chem;50:2297–2300.
We gratefully acknowledge financial support from National
Basic Research Program of China (Grants 2009CB940903
and 2009CB918502), the National Natural Science Foun-
dation of China (Grants 20721003 and 81025017),
National S&T Major Projects (2012ZX09103-101-072) and
Silver Project (260644).
13. Eckhardt M., Langkop E., Mark M., Tadayyon M., Tho-
mas L., Nar H., Pfrengle W., Guth B., Lotz R., Sieger
P., Fuchs H., Himmelsbach F. (2007) 8-(3-(R)-aminopi-
peridin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazo-
lin-2-ylmethyl)-3,7-dihydropurine-2,6-dione (BI 1356), a
highly potent, selective, long-acting, and orally bioavail-
able DPP-4 inhibitor for the treatment of type 2 diabe-
tes. J Med Chem;50:6450–6453.
References
1. Tahrani A.A., Bailey C.J., Del Prato S., Barnett A.H.
(2011) Management of type 2 diabetes: new and future
developments in treatment. Lancet;378:182–197.
2. Kim W., Egan J.M. (2008) The role of incretins in glu-
cose homeostasis and diabetes treatment. Pharmacol
Rev;60:470–512.
3. Holst J.J. (2007) The physiology of glucagon-like pep-
tide 1. Physiol Rev;87:1409–1439.
4. Holst J.J., Vilsboll T., Deacon C.F. (2009) The incretin
system and its role in type 2 diabetes mellitus. Mol Cell
Endocrinol;297:127–136.
5. Mentlein R., Gallwitz B., Schmidt W.E. (1993) Dipept-
idyl-peptidase IV hydrolyses gastric inhibitory polypep-
tide, glucagon-like peptide-1(7-36)amide, peptide
histidine methionine and is responsible for their degra-
dation in human serum. Eur J Biochem;214:829–835.
6. Kieffer T.J., McIntosh C.H., Pederson R.A. (1995) Deg-
radation of glucose-dependent insulinotropic polypep-
tide and truncated glucagon-like peptide 1 in vitro and
in vivo by dipeptidyl peptidase IV. Endocrinol-
ogy;136:3585–3596.
7. Biftu T., Scapin G., Singh S., Feng D., Becker J.W.,
Eiermann G., He H. et al. (2007) Rational design of a
novel, potent, and orally bioavailable cyclohexylamine
DPP-4 inhibitor by application of molecular modeling
and X-ray crystallography of sitagliptin. Bioorg Med
Chem Lett;17:3384–3387.
14. Augustyns K., Van der Veken P., Senten K., Haemers
A. (2005) The therapeutic potential of inhibitors of dip-
eptidyl peptidase IV (DPP IV) and related proline-spe-
cific
dipeptidyl
aminopeptidases.
Curr
Med
Chem;12:971–998.
15. Havale S.H., Pal M. (2009) Medicinal chemistry
approaches to the inhibition of dipeptidyl peptidase-4
for the treatment of type 2 diabetes. Bioorg Med
Chem;17:1783–1802.
16. Ashworth D.M., Atrash B., Baker G.R., Baxter A.J.,
Jenkins P.D., Jones D.M., Szelke M. (1996) 2-Cya-
nopyrrolidides as potent, stable inhibitors of dipeptidyl
peptidase IV. Bioorg Med Chem Lett;6:1163–1166.
17. Zhou C.H., Wang Y. (2012) Recent researches in triaz-
ole compounds as medicinal drugs. Curr Med
Chem;19:239–280.
18. Kharb R., Yar M.S., Sharma P.C. (2011) New insights
into chemistry and anti-infective potential of triazole
scaffold. Curr Med Chem;18:3265–3297.
19. Kharb R., Yar M.S., Sharma P.C. (2011) Recent
advances and future perspectives of triazole analogs as
promising antiviral agents. Mini Rev Med Chem;11:84–
96.
20. Kharb R., Sharma P.C., Yar M.S. (2011) Pharmacologi-
cal significance of triazole scaffold. J Enzyme Inhib Med
Chem;26:1–21.
21. Agalave S.G., Maujan S.R., Pore V.S. (2011) Click
chemistry: 1,2,3-triazoles as pharmacophores. Chem
Asian J;6:2696–2718.
22. Lankas G.R., Leiting B., Roy R.S., Eiermann G.J., Be-
coni M.G., Biftu T., Chan C.C. et al. (2005) Dipeptidyl
peptidase IV inhibition for the treatment of type 2 diabe-
8. Karasik A., Aschner P., Katzeff H., Davies M.J., Stein
P.P. (2008) Sitagliptin, a DPP-4 inhibitor for the treat-
ment of patients with type 2 diabetes: a review of
recent clinical trials. Curr Med Res Opin;24:489–496.
9. Stamataros G., Schneider S.H. (2011) Vildagliptin in the
treatment of type 2 diabetes mellitus. Expert Opin Phar-
macother;12:1967–1973.
10. Villhauer E.B., Brinkman J.A., Naderi G.B., Burkey B.F.,
Dunning B.E., Prasad K., Mangold B.L., Russell M.E.,
206
Chem Biol Drug Des 2013; 81: 198–207

Design, Synthesis, Structure–Activity Relationships
tes: potential importance of selectivity over dipeptidyl
peptidases 8 and 9. Diabetes;54:2988–2994.
(2004) Glide: a new approach for rapid, accurate dock-
ing and scoring. 1. Method and assessment of docking
accuracy. J Med Chem;47:1739–1749.
23. Dobers J., Zimmermann-Kordmann M., Leddermann
M., Schewe T., Reutter W., Fan H. (2002) Expression,
purification, and characterization of human dipeptidyl
peptidase IV ⁄ CD26 in Sf9 insect cells. Protein Expr Pu-
rif;25:527–532.
24. Ammirati M.J., Andrews K.M., Boyer D.D., Brodeur
A.M., Danley D.E., Doran S.D., Hulin B. et al. (2009)
(3,3-Difluoro-pyrrolidin-1-yl)-[(2S,4S)-(4-(4-pyrimidin-2-
Notes
aPerkinElmer Inc., Waltham, MA, USA.
bLife Technologies Corp, Grand Island, NY, USA.
cNovagen, Madison, WI, US.
dGlide version 4.0; Schro¨ dinger, LLC, New York, NY, USA,
available at: http://www.schrodinger.com.
eTripos, Inc., St Louis, MO, USA.
yl-piperazin-1-yl)-pyrrolidin-2-yl]-methanone:
a potent,
selective, orally active dipeptidyl peptidase IV inhibitor.
Bioorg Med Chem Lett;19:1991–1995.
fMaestro, Version 8.0; Schro¨ dinger, LLC, New York, NY,
USA.
25. Friesner R.A., Banks J.L., Murphy R.B., Halgren T.A.,
Klicic J.J., Mainz D.T., Repasky M.P., Knoll E.H., Shel-
ley M., Perry J.K., Shaw D.E., Francis P., Shenkin P.S.
Chem Biol Drug Des 2013; 81: 198–207
207