Discovering Novel α-aminoacyl-Containing Proline Derivatives with Potent and Selective Inhibitory Activity Against Dipeptidyl Peptidase IV: Design, Synthesis, Biological Evaluation, and Molecular Modeling

Article abstract of DOI:10.1111/j.1747-0285.2012.01438.x

On the basis of the enzyme-binding features of known potent inhibitors of dipeptidyl peptidase IV, novel α-aminoacyl-containing proline analogs (8Aa-8Ak, 8Ba-8Bj, 8Ca-8Ck, and 8Da-8Di) with the S configuration were designed, synthesized, and their activit

Full text of DOI:10.1111/j.1747-0285.2012.01438.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01438.x
Chem Biol Drug Des 2012; 80: 843–852
Research Article
Discovering Novel a-aminoacyl-Containing Proline
Derivatives with Potent and Selective Inhibitory
Activity Against Dipeptidyl Peptidase IV: Design,
Synthesis, Biological Evaluation, and Molecular
Modeling
Xiaodong Zhang1†, Jiang Wang1†, Mingbo
glucose-dependent insulinotropic polypeptide (GIP), by cleaving the
peptide bond in the penultimate position (1,2). GLP-1 is a potent anti-
hyperglycemic hormone, inducing glucose-dependent stimulation of
insulin secretion while suppressing glucagon secretion (3,4). The
Su2†, Zeng Li1, Jingya Li2, Jia Li2,* and
Hong Liu1,*
1State Key Laboratory of Drug Research, Shanghai Institute of
active form of GLP-1 is rapidly inactivated by plasma DPP IV through
cleavage of the dipeptide from the N-terminus, limiting its duration of
action. Inhibition of DPP IV results in elevated circulating levels of
endogenous GLP-1, which is produced by L-cells in the small intestine
in response to food intake (5–8). Thus, inhibition of DPP IV extends
the half-life of endogenously secreted GLP-1, which in turn enhances
insulin secretion and improves glucose tolerance. Dipeptidyl pepti-
dase IV inhibitors offer several potential advantages over the existing
therapies including decreased risk of hypoglycemia, potential for
weight loss, and the potential for regeneration and differentiation of
pancreatic b-cells. Therefore, DPP IV has become a validated target
for the treatment of type 2 diabetes. Several DPP IV inhibitors are cur-
rently undergoing late-stage clinical trials. The first DPP IV inhibitor,
sitagliptin 1 (Januvia; Merck, Rahway, NJ, USA), was approved by
the Food and Drug Administration (FDA) in October 2006 (Figure 1) (9–
11); vildagliptin 2 (Glavus; Novartis, Basel, Switzerland) was approved
by the European Medicines Agency in September 2007 (12–14); sax-
agliptin 3 (Onglyza; Bristol-Myers Squibb, New York City, NY, USA)
was approved by the FDA as a once-daily diabetes medication for the
treatment of type 2 diabetes in August 2009 (15); alogliptin 4 (Nesina;
Takeda, Osaka, Honshu, Japan) was launched in Japan as a potent
and highly selective inhibitor of DPP IV in May 2010 (16); and linaglip-
tin 5 (Tradjenta; Boehringer Ingelheim, Biberach, Baden-Wꢀrttemberg,
Germany) was approved by the FDA in June 2011 (17) and is indicated
as an adjunct to diet and exercise to improve glycemic control in
adults with type 2 diabetes mellitus.
Materia Medica, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai
201203, 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 201303, China
*Corresponding author: Hong Liu, hliu@mail.shcnc.ac.cn
These authors contributed equally to this work.
On the basis of the enzyme-binding features of
known potent inhibitors of dipeptidyl peptidase
IV, novel a-aminoacyl-containing proline analogs
(8Aa–8Ak, 8Ba–8Bj, 8Ca–8Ck, and 8Da–8Di) with
the S configuration were designed, synthesized,
and their activity profiled. Their structural
features were determined by nuclear magnetic
resonance (NMR) spectroscopy, low- and high-res-
olution mass spectroscopy. Five compounds (8Aa,
8Aj, 8Ch, 8Ck, and 8Dc) were shown to have
promising inhibitory activities against dipeptidyl
peptidase IV. Two of them, compounds 8Aa and
8Aj inhibited dipeptidyl peptidase IV with IC50 val-
ues of 4.56 and 8.4 lM, respectively, and displayed
no inhibition at other dipeptidyl peptidase IV. The
possible binding modes of compounds 6, 7, 8Aa,
and 8Aj with dipeptidyl peptidase IV were also
explored by molecular docking simulation. This
study provides promising new templates for the
further development of antidiabetic agents.
Recently, various classes of structurally different DPP IV inhibitors
have been reported (18–20). Moriarty and colleagues (18) identified
compound 6 (Figure 2) as a potent DPP IV inhibitor with high inhibi-
tory activity (IC50 = 63 nM). Compound 7 was reported by Edmondson
et al. (19) as a potent and highly selective inhibitor of DPP IV
(IC50 = 18 nM). Clearly, inhibitor 6 has structural features similar to
compound 7 because both of them contain a pyrrole scaffold. On the
basis of the above findings, we speculated that a combination of seg-
ment A of compound 6 and segment B of compound 7 may result in
novel a-aminoacyl-containing proline analogs as potential inhibitors
of DPP IV. To verify our hypothesis, we firstly carried out biological
Key words: a-aminoacyl-containing proline derivatives, dipeptidyl
peptidase IV, inhibitor, molecular docking, type 2 diabetes
Received 16 April 2012, revised 28 May 2012 and accepted for publica-
tion 10 June 2012
Dipeptidyl peptidase IV (DPP IV, also known as CD26, EC 3.4.14.5) is
a prolyl dipeptidase involved in the in vivo degradation of two
insulin-sensing hormones, glucagon-like peptide-1 (GLP-1), and
843

Zhang et al.
Figure 1: Approved dipeptidyl
peptidase IV (DPP IV) inhibitors.
Figure 2: Design strategy of
a-aminoacyl-containing proline
derivatives as dipeptidyl peptidase
IV (DPP IV) inhibitors.
evaluation of compound 8Aa against DPP IV. As expected, compound
8Aa showed moderate DPP IV inhibitory efficacy (IC50 = 4.56 lM).
We continued our structure–activity relationship efforts around com-
pound 8Aa. A series of a-aminoacyl-containing proline derivatives
were designed and synthesized, and their biological activities against
DPP IV and other DPPs were evaluated. Among these compounds,
8Aa, 8Aj, 8Ch, 8Ck, and 8Dc showed moderate DPP IV inhibitory
activities. Compounds 8Aa and 8Aj inhibited DPP IV with IC50 values
of 4.56 and 8.4 lM, respectively, and displayed no inhibition against
other DPPs. The possible binding modes of compounds 6, 7, 8Aa,
and 8Aj to DPP IV were also explored using molecular docking simu-
lation. Herein, we report the design, synthesis, and biological activity
of a series of novel a-aminoacyl-containing proline derivatives (8Aa–
8Ak, 8Ba–8Bj, 8Ca–8Ck, and 8Da–8Di).
Table 1) were designed and synthesized. Keeping the R2 as
hydrogen, we obtained compounds 8Ab–8Ak by introducing differ-
ent electronic substituents at various positions of the benzene ring
of compound 8Aa. When R2 is methyl (Me), ethyl (Et), or phenyl
(Ph), further 30 compounds (8Ba–8Bj, 8Ca–8Ck, and 8Da–8Di)
were prepared (Table 1).
Synthetic procedures of target compounds
The general synthetic procedures for target compounds are outlined in
Scheme 1. The straightforward six-step synthetic route enabled us to
vary positions R1 and R2. The initial step in the synthesis of 8Aa–8Di
was the coupling of phenol 9 and a-bromoesters 10 followed by N-de-
protection. Amines 11 were then coupled with N-Boc-proline followed
by deprotection to give compounds 12, which were subsequently cou-
pled with compound 13 and deprotected to afforded analogs 8Aa–8Di.
Methods and Materials
Chemistry
Biological evaluation
Design of analogs
Enzyme-based assay of DPP IV
On the basis of the structural features of compound 8Aa, 41
compounds (8Aa–8Ak, 8Ba–8Bj, 8Ca–8Ck, and 8Da–8Di,
To measure the activity of DPP IV, a continuous fluorometric assay
was employed using Ala-Pro-aminomethylcoumarin (AMC), which is
844
Chem Biol Drug Des 2012; 80: 843–852

Design, Synthesis, Biological Evaluation, and Molecular Modeling
Table 1: Chemical structures of compounds 8Aa–8Di and their
inhibitory activities against DPP IV
concentrations of test compounds, and assay buffer (100 mM HE-
PES, pH 7.5, 0.1 mg ⁄ mL BSA) in a total reaction volume of 50 lL.
The DPP IV enzyme used in these studies was soluble human
recombinant protein produced in a baculovirus expression system
(Bac-To-Bac; Life Technologies, Grand Island, NE, USA).
O
COOEt
H
O
N
N
R2
O
Enzyme-based assay of DPPs inhibition
selectivities
R1
NH2
All DPP proteins were expressed in high five cells using a baculovi-
rus system, and the activities of DPPs were assayed using a contin-
uous fluorometric method. We used Nle-Pro-AMC as the substrate
to measure the activities of DPP 7 and FAP, and Ala-Pro-AMC for
DPP 8 and DPP 9 at the optimized pH (5.5 for DPP 7 and 8.0 for
other members) assay system.
DPP IV inhibition
at 20 lg ⁄ mL (%)
Compound
R1
R2
8Aa
8Ab
8Ac
8Ad
8Ae
8Af
8Ag
8Ah
8Ai
4-F
4-Me
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Me
Me
Me
Me
Me
Me
Me
Et
Et
Et
Et
Et
Et
Et
Et
Et
98.7
25.9
27.7
48.1
29.3
6.0
19.5
9.4
18.1
92.8
18.3
0
3.1
23.9
8.7
14.0
0
14.0
24.1
21.2
8.7
4-OMe
4-Ph
4-CN
4-CF3
H
2-CN
2-CF3
2-F
Molecular docking
The Glide program from the Schrçdinger Suite was employed for
the docking study.a All molecules were generated and minimized
using SYBYL 7.3, Tripos. The protein receptor was obtained from the
worldwide Protein Data Bank (PDB ID: 2BUB) (22) and processed by
removing all solvents, adding hydrogens, and carrying out minimal
minimization with the OPLS2001 force field using protein prepara-
tion wizard. The grid was sized to 15 ꢁ in each direction and cen-
tered using the original small molecule inhibitor. In all cases, all
compounds were prepared for docking using LigPrep under its
default parameters. The maximum number of heavy atoms permit-
ted per compound was 120 with the maximum number of rotatable
bonds set at 30. Docking was performed using Glide (23) in stan-
dard 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.
8Aj
8Ak
8Ba
8Bb
8Bc
8Bd
8Be
8Bf
3-CN
H
4-Me
4-OMe
4-CN
4-CF3
4-F
2-CN
2-F
3-CN
3-F
8Bg
8Bh
8Bi
8Bj
8Ca
8Cb
8Cc
8Cd
8Ce
8Cf
H
0
4-Me
4-OMe
4-Ph
4-CN
4-F
2-CN
2-CF3
2-F
3-CN
3-CF3
H
4-OMe
4-CN
4-CF3
4-F
17.4
40.8
23.6
18.6
27.0
32.6
46.5
31.2
26.8
43.4
32.2
40.7
40.4
26.0
27.5
38.3
40.5
33.6
27.4
101.1
Experimental Section
8Cg
8Ch
8Ci
The reagents (chemicals) were purchased from commercial sources,
and used without further purification. Analytical thin-layer chroma-
tography was HSGF254 (0.15–0.2 mm thickness). Yields were not
optimized. Nuclear magnetic resonance (NMR) spectra were given
on a Brucker AMX-400 and AMX-300 (internal standard as tetra-
methylsilane). Chemical shifts were reported in parts per million
(ppm, d) downfield from tetramethylsilane. Proton-coupling patterns
were described as singlet, doublet, triplet, quartet, multiplet, and
broad. Low- and high-resolution mass spectra were given with an
electric ionization (ESI) and electrospray and a LCQ-DECA spectrom-
eter produced by Finnigan MAT-95.
8Cj
Et
Et
8Ck
8Da
8Db
8Dc
8Dd
8De
8Df
8Dg
8Dh
8Di
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
–
2-CN
2-CF3
3-CN
3-CF3
–
6
General procedures for the preparation of
compounds 8Aa–8Di
DPP IV, dipeptidyl peptidase IV.
A solution of compound 13 (0.10 mmol) in DMF (5 mL) was added
HATU (0.10 mmol) and DIPEA (0.10 mmol). After stirring for 30 min,
compound 12 (0.11 mmol) and additional DIPEA (0.11 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 chro-
cleaved by the enzyme to release the fluorescent AMC (21). Libera-
tion of AMC was monitored using an excitation wavelength of
355 nm and an emission wavelength of 460 nm using an Envision
microplate reader (PerkinElmer, Waltham, MA, USA). A typical reac-
tion contained 50 pM enzyme DPP IV, 10 lM Ala-Pro-AMC, different
Chem Biol Drug Des 2012; 80: 843–852
845

Zhang et al.
OH
O
COOEt
Br
COOEt
a,b
O
c,b
H
N
+
R2
+
R2
10
N
Boc
Boc
OH
NH2
9
11
Scheme 1: Synthetic route to
a-aminoacyl-containing proline
derivatives 8Aa–8Di. Reagents
and conditions: (a) K2CO3, MeOH,
r.t., overnight; (b) TFA, anhydrous
CH2Cl2, r.t., 3 h; (c) TEA, THF,
)15 ꢀC, iso-butyl chloroformate,
r.t., overnight; (d) HATU, DIPEA, r.t.,
overnight.
O
COOEt
H
N
R2
O
O
COOEt
+
O
H
N
NH2
O
R2
d,b
R1
N
OH
NHBoc
R1
NH
O
8A (a-k)
8B (a-j)
8C (a-k)
8D (a-i)
12
13
matography on silica gel, eluted with a mixture of PE ⁄ EA (4 ⁄ 1, v ⁄ v)
to afford target compound as a white solid. A solution of the target
compound (100 mg) in dry CH2Cl2 (2 mL) was added CF3COOH
(0.2 mL) at 0 ꢀC and warmed to room temperature. After 3 h, the
mixture was concentrated. The residue was purified with flash chro-
matography on silica gel, eluted with a mixture of CH2Cl2 ⁄ MeOH
(20 ⁄ 1, v ⁄ v) to afford compounds 8Aa–8Di.
3.50–3.33 (m, 4H), 2.41–2.18 (m, 4H), 1.38 (t, J = 6.9 Hz, 3H) ppm;
ESI-MS m ⁄ z 484, [M + H]+. HRMS (ESI) m ⁄ z calcd C26H33N3O6
[M + H]+ 484.2448, found 484.2449.
Ethyl-2-(4-(((S)-1-((S)-2-amino-3-(4-cyanophenyl)
propanoyl)pyrrolidine-2-carboxamido)-methyl)
phenoxy)acetate (8Ae)
1H NMR (CDCl3, 300 MHz): d 7.41–7.21 (m, 2H), 7.18–7.00 (m, 4H),
6.98–6.77 (m, 2H), 4.89 (s, 2H), 4.56–4.21 (m, 3H), 4.11–3.98 (m,
3H), 3.42–3.15 (m, 4H), 2.32–1.86 (m, 4H), 1.44 (t, J = 4.2 Hz, 3H)
ppm; ESI-MS m ⁄ z 479, [M + H]+. HRMS (ESI) m ⁄ z calcd C26H30N4O5
[M + H]+ 479.2294, found 479.2283.
Ethyl-2-(4-(((S)-1-((S)-2-amino-(4-fluorophenyl)
propanoyl)pyrrolidine-2-carboxamido)-methyl)
phenoxy)acetate (8Aa)
1H NMR (CDCl3, 300 MHz): d 7.46–7.13 (m, 6H), 6.91–6.78 (m, 2H),
4.79 (s, 2H), 4.60–4.39 (m, 3H), 4.17–3.93 (m, 3H), 3.56–3.37 (m,
4H), 2.71–2.43 (m, 4H), 1.53–1.39 (m, 3H) ppm; ESI-MS m ⁄ z 472,
[M + H]+. HRMS (ESI) m ⁄ z calcd C25H30FN3O5 [M + H]+ 472.2248,
found 472.2239.
Ethyl-2-(4-(((S)-1-((S)-2-amino-3-(4-(trifluoromethyl)
phenyl)propanoyl)pyrrolidine-2-carboxamido)-
methyl)phenoxy)acetate (8Af)
1H NMR (CDCl3, 300 MHz): d 7.49–7.34 (m, 2H), 7.21–7.17 (m,
2H), 7.10–7.01 (m, 2H), 6.90–6.81 (m, 2H), 4.73 (s, 2H),
4.49–4.39 (m, 3H), 4.21–3.93 (m, 3H), 3.59–3.37 (m, 4H), 2.89–
2.78 (m, 4H), 1.43–1.21 (m, 3H) ppm; ESI-MS m ⁄ z 522, [M + H]+.
HRMS (ESI) m ⁄ z calcd C26H30F3N3O5 [M + H]+ 522.2216, found
522.2208.
Ethyl-2-(4-(((S)-1-((S)-2-amino-3-3-p-tolylpropanoyl)
pyrrolidine-2-carboxamido)-methyl)phenoxy)
acetate (8Ab)
1H NMR (CDCl3, 300 MHz): d 7.41–7.32 (m, 2H), 7.24–7.13 (m, 4H),
6.75–6.63 (m, 2H), 4.61 (s, 2H), 4.35–4.21 (m, 3H), 4.15–4.01 (m,
3H), 3.42–3.15 (m, 4H), 2.20–1.85 (m, 7H), 1.34 (t, J = 6.9 Hz, 3H)
ppm; ESI-MS m ⁄ z 468, [M + H]+. HRMS (ESI) m ⁄ z calcd C26H33N3O5
[M + H]+ 468.2498, found 468.2487.
Ethyl-2-(4-(((S)-1-((S)-2-amino-3-phenylpropanoyl)
pyrrolidine-2-carboxamido)-methyl)phenoxy)
acetate (8Ag)
Ethyl-2-(4-(((S)-1-((S)-2-amino-3-(4-methoxyphenyl)
pyrrolidine-2-carboxamido)-methyl)phenoxy)
acetate (8Ac)
1H NMR (CDCl3, 300 MHz): d 7.38–7.09 (m, 4H), 6.90–6.73 (m, 4H),
4.69 (s, 2H), 4.49–4.30 (m, 3H), 4.17–3.97 (m, 3H), 3.88 (s, 3H),
3.50–3.33 (m, 4H), 2.41–2.18 (m, 4H), 1.38 (t, J = 6.9 Hz, 3H) ppm;
ESI-MS m ⁄ z 484, [M + H]+. HRMS (ESI) m ⁄ z calcd C26H33N3O6
[M + H]+ 484.2448, found 484.2449.
1H NMR (CDCl3, 300 MHz): d 7.34–7.30 (m, 2H), 7.28–7.11 (m, 5H),
6.87–6.81 (m, 2H), 4.84 (s, 2H), 4.43–4.31 (m, 3H), 4.14–4.01 (m,
3H), 3.32–3.11 (m, 4H), 2.10–1.91 (m, 4H), 1.41 (t, J = 6.3 Hz, 3H)
ppm; ESI-MS m ⁄ z 454, [M + H]+. HRMS (ESI) m ⁄ z calcd C25H29N3O5
[M + H]+ 454.2342, found 454.2338.
Ethyl-2-(4-(((S)-1-((S)-2-amino-3-(2-cyanophenyl)
propanoyl)pyrrolidine-2-carboxamido)-methyl)
phenoxy)acetate (8Ah)
Ethyl-2-(4-(((S)-1-((S)-2-amino-3-(biphenyl-4-yl)
propanoyl)pyrrolidine-2-carboxamido)-methyl)
phenoxy)acetate (8Ad)
1H NMR (CDCl3, 300 MHz): d 7.38–7.09 (m, 4H), 6.90–6.73 (m, 4H),
4.69 (s, 2H), 4.49–4.30 (m, 3H), 4.17–3.97 (m, 3H), 3.88 (s, 3H),
1H NMR (CDCl3, 300 MHz): d 7.67–7.34 (m, 2H), 7.29–7.16 (m, 4H),
6.98–6.81 (m, 2H), 4.97 (s, 2H), 4.64–4.43 (m, 3H), 4.22–3.99 (m,
3H), 3.64–3.33 (m, 4H), 2.65–2.29 (m, 4H), 1.53–1.32 (m, 3H) ppm;
ESI-MS m ⁄ z 479, [M + H]+. HRMS (ESI) m ⁄ z calcd C26H30N4O5
[M + H]+ 479.2294, found 479.2287.
846
Chem Biol Drug Des 2012; 80: 843–852

Design, Synthesis, Biological Evaluation, and Molecular Modeling
Ethyl-2-(4-(((S)-1-((S)-2-amino-3-(2-(trifluoromethyl)
phenyl)propanoyl)pyrrolidine-2-carboxamido)-
methyl)phenoxy)acetate (8Ai)
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(4-cyanophenyl)
propanoyl)pyrrolidine-2-carboxamido)-methyl)
phenoxy)propanoate (8Bd)
1H NMR (CDCl3, 300 MHz): d 7.53–7.27 (m, 2H), 7.19–7.03 (m, 4H),
6.88–6.73 (m, 2H), 4.83 (s, 2H), 4.53–4.43 (m, 3H), 4.24–3.89 (m,
3H), 3.54–3.23 (m, 4H), 2.45–2.09 (m, 4H), 1.45 (t, J = 4.8 Hz, 3H)
ppm; ESI-MS m ⁄ z 522, [M + H]+. HRMS (ESI) m ⁄ z calcd
C26H30F3N3O5 [M + H]+ 522.2216, found 522.2209.
1H NMR (CDCl3, 300 MHz): d 7.60–7.55 (m, 2H), 7.46–7.43 (m,
2H), 7.14–7.11 (m, 2H), 6.78–6.73 (m, 2H), 4.68 (t, J = 13.3 Hz,
1H), 4.36–4.09 (m, 6H), 3.44–3.24 (m, 4H), 2.05–2.02 (m, 4H),
1.60–1.51 (m, 3H), 1.30–1.24 (m, 3H) ppm; ESI-MS m ⁄ z 493,
[M + H]+. HRMS (ESI) m ⁄ z calcd C27H32N4O5 [M + H]+ 493.2451,
found 493.2449.
Ethyl-2-(4-(((S)-1-((S)-2-amino-3-(2-fluorophenyl)
propanoyl)pyrrolidine-2-carboxamido)-methyl)
phenoxy)acetate (8Aj)
1H NMR (CDCl3, 300 MHz): d 7.56–7.34 (m, 2H), 7.21–7.01 (m, 4H),
6.87–6.81 (m, 2H), 4.85 (s, 2H), 4.58–4.42 (m, 3H), 4.20–3.99 (m,
3H), 3.51–3.21 (m, 4H), 2.76–2.43 (m, 4H), 1.34–1.21 (m, 3H) ppm;
ESI-MS m ⁄ z 472, [M + H]+. HRMS (ESI) m ⁄ z calcd C25H30N3O5
[M + H]+ 472.2248, found 472.2241.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(4-(trifluoromethyl)
phenyl)propanoyl)pyrrolidine-2-carboxamido)-
methyl)phenoxy)propanoate (8Be)
1H NMR (CDCl3, 300 MHz): d 7.58–7.53 (m, 2H), 7.41–7.35 (m, 2H),
7.14–7.09 (m, 2H), 6.78–6.70 (m, 2H), 4.67 (dd, J1 = 9.3 Hz,
J2 = 15.6 Hz, 1H), 4.31–4.10 (m, 6H), 3.21–3.04 (m, 4H), 2.03–1.85
(m, 4H), 1.59–1.52 (m, 3H), 1.28 (t, J = 3.0 Hz, 3H) ppm; ESI-MS
m ⁄ z 536, [M + H]+. HRMS (ESI) m ⁄ z calcd C27H32F3N3O5 [M + H]+
536.2372, found 536.2367.
Ethyl-2-(4-(((S)-1-((S)-2-amino-3-(3-cyanophenyl)
propanoyl)pyrrolidine-2-carboxamido)-methyl)
phenoxy)acetate (8Ak)
1H NMR (CDCl3, 300 MHz): d 8.01–7.73 (m, 3H), 7.51–7.26 (m, 3H),
6.88–6.73 (m, 2H), 4.79 (s, 2H), 4.59–4.41 (m, 3H), 4.29–4.07 (m,
3H), 3.59–3.37 (m, 4H), 2.43–2.01 (m, 4H), 1.49–1.28 (m, 3H) ppm;
ESI-MS m ⁄ z 479, [M + H]+. HRMS (ESI) m ⁄ z calcd C26H30N4O5
[M + H]+ 479.2294, found 479.2291.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(4-fluorophenyl)
propanoyl)pyrrolidine-2-carboxamido)-methyl)
phenoxy)propanoate (8Bf)
1H NMR (CDCl3, 300 MHz): d 7.65–7.55 (m, 2H), 7.45–7.42 (m, 2H),
7.16–7.13 (m, 2H), 6.78–6.72 (m, 2H), 4.68–4.63 (m, 1H), 4.38–4.18
(m, 6H), 3.53–3.21 (m, 4H), 1.99 (m, 4H), 1.63–1.53 (m, 3H), 1.29–
1.22 (m, 3H) ppm; ESI-MS m ⁄ z 486, [M + H]+. HRMS (ESI) m ⁄ z
calcd C26H32FN3O5 [M + H]+ 486.2404, found 486.2401.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-phenylpropanoyl)
pyrrolidine-2-carboxamido)-methyl)phenoxy)
propanoate (8Ba)
1H NMR (CDCl3, 300 MHz): d 7.45–7.39 (m, 2H), 7.23–7.11 (m, 5H),
6.98–6.89 (m, 2H), 4.85 (dd, J1 = 4.2 Hz, J2 = 9.6 Hz, 1H), 4.43–4.41
(m, 3H), 4.21–4.16 (m, 3H), 3.32–3.19 (m, 4H), 2.05–1.98 (m, 4H), 1.59–
1.56 (m, 3H), 1.31–1.28 (m, 3H) ppm; ESI-MS m⁄ z 468, [M + H]+.
HRMS (ESI) m ⁄ z calcd C26H33N3O5 [M + H]+ 468.2498, found 468.2496.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(2-cyanophenyl)
propanoyl)pyrrolidine-2-carboxamido)-methyl)
phenoxy)propanoate (8Bg)
1H NMR (CDCl3, 300 MHz): d 7.51–7.43 (m, 2H), 7.41–7.39 (m, 2H),
7.20–7.16 (m, 2H), 6.78–6.74 (m, 2H), 4.71–4.68 (m, 1H), 4.28–4.13
(m, 6H), 3.45–3.28 (m, 4H), 2.09–2.07 (m, 4H), 1.68–1.61 (m, 3H),
1.30–1.22 (m, 3H) ppm; ESI-MS m ⁄ z 493, [M + H]+. HRMS (ESI)
m ⁄ z calcd C27H32N4O5 [M + H]+ 493.2451, found 493.2447.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-p-tolylpropanoyl)
pyrrolidine-2-carboxamido)-methyl)phenoxy)
propanoate (8Bb)
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(2-
1H NMR (CDCl3, 300 MHz): d 7.14–7.01 (m, 6H), 6.80–6.74 (m, 2H),
4.67 (dd, J1 = 9.0 Hz, J2 = 15.0 Hz, 1H), 4.32–4.31 (m, 3H), 4.23–4.14
(m, 3H), 3.21 (m, 2H), 3.09 (m, 2H), 2.27 (s, 3H), 2.00–1.94 (m, 4H),
1.59–1.53 (m, 3H), 1.28–1.22 (m, 3H) ppm; ESI-MS m⁄ z 482, [M + H]+.
HRMS (ESI) m ⁄ z calcd C27H35N3O5 [M + H]+ 482.2655, found 482.2651.
fluorophenyl)propanoyl)pyrrolidine-2-
carboxamido)-methyl)phenoxy)propanoate (8Bh)
1H NMR (CDCl3, 300 MHz): d 7.59–7.48 (m, 2H), 7.39–7.33 (m,
2H), 7.19–7.16 (m, 2H), 6.81–6.74 (m, 2H), 4.85–4.81 (m, 1H),
4.23–4.05 (m, 6H), 3.50–3.38 (m, 4H), 2.13–2.08 (m, 4H), 1.58–
1.47 (m, 3H), 1.31–1.25 (m, 3H) ppm; ESI-MS m ⁄ z 486, [M + H]+.
HRMS (ESI) m ⁄ z calcd C26H32FN3O5 [M + H]+ 486.2404, found
486.2410.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(4-methoxyphenyl)
propanoyl)pyrrolidine-2-carboxamido)-methyl)
phenoxy)propanoate (8Bc)
1H NMR (CDCl3, 300 MHz): d 7.14–7.09 (m, 4H), 6.81–6.74 (m, 2H),
4.69–4.67 (m, 1H), 4.28–4.15 (m, 6H), 3.75 (s, 3H), 3.47–3.18 (m,
4H), 2.00–1.99 (m, 4H), 1.59–1.55 (m, 3H), 1.29–1.23 (m, 3H) ppm;
ESI-MS m ⁄ z 498, [M + H]+. HRMS (ESI) m ⁄ z calcd C27H35N3O6
[M + H]+ 498.2604, found 498.2611.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(3-cyanophenyl)
propanoyl)pyrrolidine-2-carboxamido)-methyl)
phenoxy)propanoate (8Bi)
1H NMR (CDCl3, 300 MHz): d 7.64–7.63 (m, 1H), 7.55–7.44 (m, 3H),
7.15–7.13 (m, 2H), 6.77–6.74 (m, 2H), 4.67 (dd, J1 = 1.8 Hz,
Chem Biol Drug Des 2012; 80: 843–852
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Zhang et al.
J2 = 6.6 Hz, 1H), 4.38–4.15 (m, 6H), 3.48–3.46 (m, 1H), 3.18–3.16
(m, 2H), 3.00–2.99 (m, 1H), 2.00 (m, 4H), 1.58 (d, 3H, J = 3.0), 1.25
(t, 3H, J = 6.9) ppm; ESI-MS m ⁄ z 493, [M + H]+. HRMS (ESI) m ⁄ z
calcd C27H32N4O5 [M + H]+ 493.2451, found 493.2447.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(4-cyanophenyl)
propanoyl)pyrrolidine-2-carboxamido)methyl)
phenoxy)butanoate (8Ce)
1H NMR (CDCl3, 300 MHz): d 7.36–7.21 (m, 4H), 7.10–6.99 (m, 2H),
6.73–6.69 (m, 2H), 4.54–4.47 (m, 2H), 4.22–4.13 (m, 4H), 3.53–3.48
(m, 1H), 3.36–3.21 (m, 4H), 2.01–1.95 (m, 6H), 1.31–1.21 (m, 3H),
1.10–1.05 (m, 3H) ppm; ESI-MS m ⁄ z 507, [M + H]+. HRMS (ESI)
m ⁄ z calcd C28H34N4O5 [M + H]+ 507.2067, found 507.2071.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(3-fluorophenyl)
propanoyl) pyrrolidine-2-carboxamido)-methyl)
phenoxy)propanoate (8Bj)
1H NMR (CDCl3, 300 MHz): d 7.60–7.56 (m, 2H), 7.46–7.41 (m,
2H), 7.14–7.11 (m, 2H), 6.78–6.74 (m, 2H), 4.69–4.67 (m,
1H), 4.21–4.17 (m, 6H), 3.50–3.23 (m, 4H), 2.03 (m, 4H), 1.60–
1.53 (m, 3H), 1.30–1.24 (m, 3H) ppm; ESI-MS m ⁄ z 486, [M + H]+.
HRMS (ESI) m ⁄ z calcd C26H32FN3O5 [M + H]+ 486.2404, found
486.2401.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(4-fluorophenyl)
propanoyl)pyrrolidine-2-carboxamido)methyl)
phenoxy)butanoate (8Cf)
1H NMR (CDCl3, 300 MHz): d 7.26–7.10 (m, 4H), 6.94 (m, 2H), 6.79–
6.74 (m, 2H), 4.51–4.47 (m, 2H), 4.21–4.14 (m, 4H), 3.51–3.48 (m,
1H), 3.40–3.11 (m, 4H), 1.97–1.93 (m, 6H), 1.28–1.19 (m, 3H), 1.07–
1.02 (m, 3H) ppm; ESI-MS m ⁄ z 500, [M + H]+. HRMS (ESI) m ⁄ z
calcd C27H34FN3O5 [M + H]+ 500.2561, found 500.2557.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-phenylpropanoyl
)pyrrolidine-2-carboxamido)methyl)phenoxy)
butanoate (8Ca)
1H NMR (CDCl3, 300 MHz): d 7.29–7.10 (m, 7H), 6.80–6.75 (m, 2H),
4.52–4.14 (m, 7H), 3.14 (m, 1H), 3.09–2.91 (m, 3H), 1.98–1.87 (m,
6H), 1.32–1.23 (m, 3H), 1.07–1.01 (m, 3H) ppm; ESI-MS m ⁄ z 482,
[M + H]+. HRMS (ESI) m ⁄ z calcd C27H35N3O5 [M + H]+ 482.2655,
found 482.2651.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(2-cyanophenyl)
propanoyl)pyrrolidine-2-carboxamido)methyl)
phenoxy)butanoate (8Cg)
1H NMR (CDCl3, 300 MHz): d 7.67–7.63 (m, 2H), 7.60–7.51 (m, 2H),
7.47–7.41 (m, 2H), 6.79–6.74 (m, 2H), 4.48–4.18 (m, 6H), 3.83 (m,
1H), 3.54–3.52 (m, 2H), 3.20–3.16 (m, 2H), 2.06–1.90 (m, 3H), 1.65–
1.63 (m, 1H), 1.38–1.29 (m, 2H), 1.25–1.08 (m, 3H), 1.05–1.00 (m,
3H) ppm; ESI-MS m ⁄ z 507, [M + H]+. HRMS (ESI) m ⁄ z calcd
C28H34N4O5 [M + H]+ 507.2067, found 507.2071.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-p-tolylpropanoyl)
pyrrolidine-2-carboxamido)methyl)phenoxy)
butanoate (8Cb)
1H NMR (CDCl3, 300 MHz): d 7.11–6.98 (m, 6H), 6.80–6.75 (m,
2H), 4.51–4.47 (m, 1H), 4.34–4.23 (m, 3H), 4.22–4.14 (m,
3H), 3.42 (m, 1H), 3.07–2.91 (m, 3H), 2.00–1.80 (m, 6H), 1.28–
1.23 (m, 3H), 1.09–1.02 (m, 3H) ppm; ESI-MS m ⁄ z 496, [M + H]+.
HRMS (ESI) m ⁄ z calcd C28H37N3O5 [M + H]+ 496.2811, found
496.2803.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(2-(trifluoromethyl)
phenyl)propanoyl)pyrrolidine-2-carboxamido)
methyl)phenoxy)butanoate (8Ch)
1H NMR (CDCl3, 300 MHz): d 7.66–7.64 (m, 1H), 7.46–7.39 (m, 3H),
7.15–7.13 (m, 2H), 6.78–6.75 (m, 2H), 4.48–4.15 (m, 6H), 3.54 (dd,
J1 = 6.9 Hz, J2 = 14.1 Hz, 1H), 3.35–3.28 (m, 4H), 1.95–1.91 (m,
6H), 1.22 (dd, 3H, J1 = 6.9 Hz, J2 = 13.8 Hz), 1.02 (t, 3H,
J = 6.9 Hz) ppm; ESI-MS m ⁄ z 550, [M + H]+. HRMS (ESI) m ⁄ z calcd
C28H34F3N3O5 [M + H]+ 550.2529, found 550.2520.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(4-methoxyphenyl)
pyrrolidine-2-carbox amido)methyl)phenoxy)
butanoate (8Cc)
1H NMR (CDCl3, 300 MHz): d 7.13–7.07 (m, 4H), 6.83–6.75 (m, 4H),
4.50–4.47 (m, 1H), 4.23–4.14 (m, 5H), 3.76–3.74 (m, 1H), 3.70 (s,
3H), 3.19 (m, 1H), 3.07 (m, 2H), 2.91 (m, 1H), 2.04–1.89 (m, 6H),
1.27–1.21 (m, 3H), 1.06–1.01 (m, 3H) ppm; ESI-MS m ⁄ z 513,
[M + H]+. HRMS (ESI) m ⁄ z calcd C28H37N3O6 [M + H]+ 513.2761,
found 513.2754.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(2-fluorophenyl)
propanoyl)pyrrolidine-2-carboxamido)methyl)
phenoxy)butanoate (8Ci)
1H NMR (CDCl3, 300 MHz): d 7.63–7.54 (m, 2H), 7.47–7.36 (m, 4H),
7.28–7.11 (m, 2H), 6.83–6.78 (m, 2H), 4.59–4.01 (m, 6H), 3.78 (m,
1H), 3.41–3.30 (m, 2H), 3.20–3.10 (m, 2H), 2.19–1.80 (m, 6H), 1.30–
1.13 (m, 3H), 1.08–0.98 (m, 3H) ppm; ESI-MS m ⁄ z 500, [M + H]+.
HRMS (ESI) m ⁄ z calcd C27H34FN3O5 [M + H]+ 500.2561, found
500.2558.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(biphenyl-4-yl)
propanoyl)pyrrolidine-2-carboxamido)methyl)
phenoxy)butanoate (8Cd)
1H NMR (CDCl3, 300 MHz): d 8.00–7.98 (m, 3H), 7.53–7.49 (m, 2H),
7.37–7.26 (m, 2H), 7.10–7.08 (m, 2H), 6.78–6.72 (m, 4H), 4.50–4.16
(m, 6H), 3.55 (dd, J1 = 7.5 Hz, J2 = 14.4 Hz, 1H), 3.18–2.97 (m, 4H),
2.02–1.81 (m, 6H), 1.27–1.19 (m, 3H), 1.04–1.00 (m, 3H) ppm; ESI-
MS m ⁄ z 558, [M + H]+. HRMS (ESI) m ⁄ z calcd C33H39N3O5 [M + H]+
558.2968, found 558.2960.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(3-cyanophenyl)
propanoyl)pyrrolidine-2-carboxamido)
methyl)phenoxy)butanoate (8Cj)
1H NMR (CDCl3, 300 MHz): d 7.68–7.67 (m, 1H), 7.57–7.44 (m, 3H),
7.19–7.13 (m, 2H), 6.78–6.74 (m, 2H), 4.49–4.20 (m, 6H), 3.83 (m,
848
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Design, Synthesis, Biological Evaluation, and Molecular Modeling
1H), 3.61–3.54 (m, 2H), 3.27–3.13 (m, 2H), 2.31–2.01 (m, 3H), 1.63–
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(4-fluorophenyl)
propanoyl)pyrrolidine-2-carboxamido)methyl)
phenoxy)-2-phenylacetate (8De)
1H NMR (CDCl3, 300 MHz): d 7.53–7.51 (m, 2H), 7.40–7.34 (m, 3H),
7.13–7.06 (m, 4H), 6.95–6.83 (m, 4H), 5.55 (s, 1H), 4.28–4.21 (m,
2H), 4.20–4.08 (m, 2H), 3.38–3.06 (m, 3H), 1.93–1.78 (m, 3H), 1.24–
1.15 (m, 3H) ppm; ESI-MS m ⁄ z 548, [M + H]+. HRMS (ESI) m ⁄ z
calcd C31H34FN3O5 [M + H]+ 548.2561, found 548.2559.
1.59 (m, 1H), 1.33–1.29 (m, 2H), 1.25–1.11 (m, 3H), 1.08–1.03 (m,
3H) ppm; ESI-MS m ⁄ z 507, [M + H]+. HRMS (ESI) m ⁄ z calcd
C28H34N4O5 [M + H]+ 507.2067, found 507.2063.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(3-(trifluoromethyl)
phenyl)propanoyl)pyrrolidine-2-carboxamido)
methyl)phenoxy)butanoate (8Ck)
1H NMR (CDCl3, 300 MHz): d 7.64 (m, 1H), 7.58–7.44 (m, 3H), 7.29–
7.18 (m, 2H), 6.81–6.70 (m, 2H), 4.31–4.17 (m, 6H), 3.81 (m, 1H),
3.61–3.41 (m, 2H), 3.27–3.13 (m, 2H), 2.33–2.13 (m, 3H), 1.61–1.51
(m, 1H), 1.37–1.31 (m, 2H), 1.25–1.20 (m, 3H), 1.08–1.01 (m, 3H)
ppm; ESI-MS m ⁄ z 550, [M + H]+. HRMS (ESI) m ⁄ z calcd
C28H34F3N3O5 [M + H]+ 550.2529, found 550.2521.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(2-cyanophenyl)
propanoyl)pyrrolidine-2-carboxamido)methyl)
phenoxy)-2-phenylacetate (8Df)
1H NMR (CDCl3, 300 MHz): d 7.57–7.52 (m, 4H), 7.42–7.39 (m, 5H),
7.15–7.13 (m, 2H), 6.84–6.81 (m, 2H), 5.53 (s, 1H), 4.28–4.11 (m,
6H), 3.51–3.14 (m, 4H), 1.91–1.80 (m, 3H), 1.57 (m, 1H), 1.18 (t,
J = 2.7 Hz, 3H) ppm; ESI-MS m ⁄ z 555, [M + H]+. HRMS (ESI) m ⁄ z
calcd C32H34N4O5 [M + H]+ 555.2602, found 555.2595.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-phenylpropanoyl)
pyrrolidine-2-carboxamido)methyl)phenoxy)-2-
phenylacetate (8Da)
1H NMR (CDCl3, 300 MHz): d 7.54–7.52 (m, 2H), 7.41–7.34 (m, 3H),
7.19–7.08 (m, 7H), 6.87–6.80 (m, 2H), 5.55 (s, 1H), 4.36–4.20 (m,
3H), 4.19–4.08 (m, 3H), 3.19–3.04 (m, 3H), 2.71 (m, 1H), 1.86–1.73
(m, 4H), 1.17 (t, J = 7.5 Hz, 3H) ppm; ESI-MS m ⁄ z 530, [M + H]+.
HRMS (ESI) m ⁄ z calcd C31H35N3O5 [M + H]+ 530.2655, found
530.2651.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(2-
(trifluoromethyl)phenyl)propanoyl)pyrrolidine-2-
carboxamido)methyl)phenoxy)-2-phenylacetate
(8Dg)
1H NMR (CDCl3, 300 MHz): d 7.68–7.66 (m, 2H), 7.51–7.49 (m, 3H),
7.45–7.38 (m, 4H), 7.15–7.08 (m, 2H), 6.87 (m, 2H), 5.57 (s, 1H),
4.21–4.19 (m, 4H), 3.58–3.39 (m, 2H), 3.10–2.74 (m, 4H), 1.99–1.87
(m, 4H), 1.26–1.19 (m, 3H) ppm; ESI-MS m ⁄ z 598, [M + H]+. HRMS
(ESI) m ⁄ z calcd C32H34F3N3O5 [M + H]+ 598.2529, found 598.2523.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(4-methoxyphenyl)
propanoyl)pyrrolidine-2-carboxamido)methyl)
phenoxy)-2-phenylacetate (8Db)
1H NMR (CDCl3, 300 MHz): d 7.54–7.51 (m, 2H), 7.38–7.36 (m, 3H),
7.14–7.05 (m, 4H), 6.92–6.73 (m, 4H), 5.56 (s, 1H), 4.30–4.10 (m,
6H), 3.72 (s, 3H), 3.14–3.08 (m, 4H), 2.00–1.89 (m, 4H), 1.22–1.16
(m, 3H) ppm; ESI-MS m ⁄ z 560, [M + H]+. HRMS (ESI) m ⁄ z calcd
C32H37N3O6 [M + H]+ 560.2761, found 560.2753.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(3-cyanophenyl)
propanoyl)pyrrolidine-2-carboxamido)methyl)
phenoxy)-2-phenylacetate (8Dh)
1H NMR (CDCl3, 300 MHz): d 7.68–7.61 (m, 4H), 7.54–7.47 (m, 5H),
7.19–7.13 (m, 2H), 6.88–6.83 (m, 2H), 5.61 (s, 1H), 4.43–4.28 (m,
3H), 4.21–4.13 (m, 3H), 3.33–3.15 (m, 3H), 2.10–2.01 (m, 4H), 1.28
(t, J = 6.9 Hz, 3H) ppm; ESI-MS m ⁄ z 555, [M + H]+. HRMS (ESI)
m ⁄ z calcd C32H34N4O5 [M + H]+ 555.2602, found 555.2593.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(4-cyanophenyl)
propanoyl)pyrrolidine-2-carboxamido)methyl)
phenoxy)-2-phenylacetate (8Dc)
1H NMR (CDCl3, 300 MHz): d 7.50–7.47 (m, 4H), 7.34–7.26 (m, 5H),
7.13–7.10 (m, 2H), 6.86–6.83 (m, 2H), 5.56 (s, 1H), 4.30–4.10 (m,
6H), 3.20–2.94 (m, 4H), 1.92–1.39 (m, 4H), 1.17 (t, J = 6.9 Hz, 3H)
ppm; ESI-MS m ⁄ z 555, [M + H]+. HRMS (ESI) m ⁄ z calcd C32H34N4O5
[M + H]+ 555.2602, found 555.2597.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(3-(trifluoromethyl)
phenyl)propanoyl) pyrrolidine-2-carboxamido)
methyl)phenoxy)-2-phenylacetate (8Di)
1H NMR (CDCl3, 300 MHz): d 7.58–7.56 (m, 4H), 7.53–7.49 (m,
5H), 7.13–7.11 (m, 2H), 6.86–6.84 (m, 2H), 5.58 (s, 1H), 4.31–
4.22 (m, 3H), 4.21–4.11 (m, 3H), 3.24–3.01 (m, 3H), 2.02–1.91 (m,
4H), 1.20 (t, J = 4.5 Hz, 3H) ppm; ESI-MS m ⁄ z 598, [M + H]+.
HRMS (ESI) m ⁄ z calcd C32H34F3N3O5 [M + H]+ 598.2529, found
598.2526.
Ethyl 2-(4-(((S)-1-((S)-2-amino-3-(4-(trifluoromethyl)
phenyl)propanoyl)pyrrolidine-2-carboxamido)
methyl)phenoxy)-2-phenylacetate (8Dd)
1H NMR (CDCl3, 300 MHz): d 7.53–7.50 (m, 4H), 7.38–7.36 (m, 5H),
7.13–7.08 (m, 2H), 6.86–6.82 (m, 2H), 5.56 (s, 1H), 4.37–4.40 (m,
3H), 4.21–4.07 (m, 3H), 3.20–3.03 (m, 4H), 2.00 (m, 4H), 1.19 (t,
J = 6.0 Hz, 3H) ppm; ESI-MS m ⁄ z 598, [M + H]+. HRMS (ESI) m ⁄ z
calcd C32H34F3N3O5 [M + H]+ 598.2529, found 598.2521.
Ethyl 2-(4-(aminomethyl)phenoxy)acetate (11a)
1H NMR (CDCl3, 400 MHz): d 7.20 (d, J = 8.4 Hz, 2H), 6.86 (d,
J = 8.4 Hz, 2H), 4.60 (s, 2H), 4.25 (q, J = 7.2 Hz, 2H), 4.15 (s, 2H),
1.30 (t, J = 7.2 Hz, 3H) ppm; ESI-MS m ⁄ z 210, [M + H]+.
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Zhang et al.
(S)-ethyl 2-(4-((pyrrolidine-2-carboxamido)methyl)
phenoxy)acetate (12a)
Table 2: Selectivity profiles of selected DPP IV inhibitors (IC50,
lM)
1H NMR (DMSO-d6, 400 MHz): d 7.19 (d, J = 8.4 Hz, 2H), 6.87 (d,
J = 8.4 Hz, 2H), 4.25–4.14 (m, 4H), 2.50–2.49 (m, 6H), 1.39 (s, 1H),
1.27 (s, 2H), 1.20 (t, J = 7.2 Hz, 3H) ppm; ESI-MS m ⁄ z 307, [M + H]+.
Compound
DPP IV
FAP
DPP 7
DPP 8
DPP 9
8Aa
8Aj
4.56
8.4
NI
NI
NI
NI
NI
NI
NI
NI
DPP IV, dipeptidyl peptidase IV; NI, no inhibition.
Results and Discussion
(4-Me), 8Ac (4-OCH3), 8Ad (4-Ph), 8Ae (4-CN), and 8Af (4-CF3)
exhibited mild inhibition activities against DPP IV, with 25.9%,
27.7%, 48.1%, 29.3%, and 6.0% inhibitions at 20 lg ⁄ mL, respec-
tively. Therefore, for the para-position substitutions, the fluorine
group appeared to be essential for DPP IV inhibition. While elec-
tron-withdrawing groups were introduced at ortho- and meta-posi-
tion of the benzene ring, the DPP IV activities were dramatically
decreased. For instance, compounds 8Ah (2-CN), 8Ai (2-CF3),
and 8Ak (3-CN) showed mild inhibition activity toward DPP IV.
However, compound 8Aj, which was fluorine-substituted at
ortho-position, provided an increase in inhibitory potency with
92.8% inhibition at 20 lg ⁄ mL. When R1 is fluorine, the inhibition
against DPP IV was increased, as illustrated in compounds 8Aa
and 8Aj, with IC50 values of 4.56 and 8.4 lM, respectively. All
these indicated that the fluorine group was very important for
the potency,
Chemistry
In total, 41 compounds (8Aa–8Ak, 8Ba–8Bj, 8Ca–8Ck, and
8Da–8Di) were designed and synthesized based on structural fea-
tures from compounds 6 and 7, and their chemical structures are
shown in Table 1. These compounds were synthesized through the
route outlined in Scheme 1, and the details of the synthetic proce-
dures and structural characterizations are described in the Experi-
mental section.
Biological evaluation
For the primary screening assay, the inhibition percentages of the
compounds (8Aa–8Ak, 8Ba–8Bj, 8Ca–8Ck, and 8Da–8Di) at
20 lg ⁄ mL were measured. The results are summarized in Table 1
and illustrated that most of these compounds exhibited moderate
inhibitory activities.
In the comparison of 8Aa–8Ak to 8Ba–8Bj, 8Ca–8Ck, and
8Da–8Di, it can be found that all of these compounds showed
decreased inhibitory activities against DPP IV, and a few completely
lost inhibitory activity. To some extent, compounds 8Ch, 8Ck, and
8Dc exhibited a better ability to inhibit DPP IV than the other com-
The initial compound 8Aa displayed potential inhibition activities
toward DPP IV, with 98.7% inhibition at the concentration of
20 lg ⁄ mL. Introducing electron-donating and electron-withdrawing
groups to the para-position of the benzene ring of R1 did not
improve the inhibition activities. For instance, compounds 8Ab
A
B
C
D
Figure 3: Stereoview of the
binding poses of compounds 6(A),
7(B), 8Aa(C), and 8Aj(D) at the
dimmer interface site of dipeptidyl
peptidase IV (DPP IV). Hydrogen
atoms have been omitted for clar-
ity. All structure figures were pre-
pared using PYMOL (http://
pymol.sourceforge.net/).
850
Chem Biol Drug Des 2012; 80: 843–852

Design, Synthesis, Biological Evaluation, and Molecular Modeling
pounds. These findings indicated that both the fluorine-substituted
Acknowledgments
benzene and the R2 substitutions play important roles in the inhibi-
tory activities against DPP IV.
We gratefully acknowledge financial support from National Basic
Research Program of China (Grants 2009CB940903 and
2009CB918502), the National Natural Science Foundation of China
(Grants 20721003 and 81025017), National S&T Major Projects
(2012ZX09103-101-072), and Silver Project (260644).
Furthermore, compounds 8Aa and 8Aj were screened against a
panel of four members of the DPPs family (Table 2). Both com-
pounds 8Aa and 8Aj exhibited high selectivities against other
DPPs (FAP, DPP 7, DPP 8, and DPP 9), with inhibition rates <50%
against DPPs at 20 lg ⁄ mL.
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