Design, synthesis, biological evaluation, and docking studies of (S)-phenylalanine derivatives with a 2-cyanopyrrolidine moiety as potent dipeptidyl peptidase 4 inhibitors

Article abstract of DOI:10.1111/cbdd.12139

A novel series of (S)-phenylalanine derivatives with a 2-cyanopyrrolidine moiety were designed and synthesized through a rational drug design strategy. Biological evaluation revealed that most tested compounds were potent dipeptidyl peptidase 4 (DPP-4) inhibitors; among them, the cyclopropyl-substituted phenylalanine derivative 11h displayed the most potent DPP-4 inhibitory activity with an IC₅₀ value of 0.247 μm. In addition, molecular docking analysis of the representative compounds 11h, 11k, and 15a were performed, which not only revealed the impact of binding modes on DPP-4 inhibitory activity but also provided additional methodological values for design and optimization. A novel series of (S)-phenylalanine derivatives with a 2-cyanopyrrolidine moiety were designed and synthesized. It was found that the cyclopropyl-substituted phenylalanine derivative 11h displayed the most potent DPP-4 inhibitory activity with an IC₅₀ value of 0.247 μm. Further docking analysis of the representative compounds 11h, 11k, and 15a not only revealed the impact of binding modes on DPP-4 inhibitory activity, but also provided additional methodological values for design and optimization.

Full text of DOI:10.1111/cbdd.12139

Chem Biol Drug Des 2013; 82: 140–146
Research Article
Design, Synthesis, Biological Evaluation, and Docking
Studies of (S)-Phenylalanine Derivatives with a
2-Cyanopyrrolidine Moiety as Potent Dipeptidyl
Peptidase 4 Inhibitors
Yang Liu1, Yizhe Wu1, Haoshu Wu2, Li Tang1,
significantly reduces the risk of appearance of hypoglyce-
mia (3). In addition, GLP-1 stimulates insulin secretion,
Peng Wu1, Tao Liu1,* and Yongzhou Hu1,*
improves b-cells functions, and inhibits glucagon secre-
tion; all these biological effects help to prevent or reverse
1ZJU-ENS Joint Laboratory of Medicinal Chemistry,
College of Pharmaceutical Sciences, Zhejiang University,
Hangzhou 310058, China
progression of diabetes (4). While the short half-life of
GLP-1, which is attributed to the rapid degradation
through a serine protease dipeptidyl peptidase 4 (DPP-4),
limits its application in practical therapy (5). Thus, small-
2Institute of Pharmacology and Toxicology, College of
Pharmaceutical Sciences, Zhejiang University, Hangzhou
310058, China
molecule DPP-4 inhibitors offer
a
new therapeutic
*Corresponding authors: Tao Liu, lt601@zju.edu.cn;
Yongzhou Hu, huyz@zju.edu.cn
approach for the treatment of type 2 diabetes because
DPP-4 inhibition leads to the extension of the half-life of
GLP-1, enhances insulin secretion and improves the glu-
cose tolerance (6). Efforts in this area have already led to
the discovery of a number of potent DPP-4 inhibitors, such
as marketed drugs, sitagliptin (1), vildagliptin (2), saxagliptin
(3), alogliptin (4), and linagliptin (5) (Figure 1). Several oth-
ers promising DPP-4 inhibitors are currently under clinical
trials of different phases (7–11).
A novel series of (S)-phenylalanine derivatives with a
2-cyanopyrrolidine moiety were designed and synthe-
sized through a rational drug design strategy. Biological
evaluation revealed that most tested compounds were
potent dipeptidyl peptidase 4 (DPP-4) inhibitors; among
them, the cyclopropyl-substituted phenylalanine deriva-
tive 11h displayed the most potent DPP-4 inhibitory
activity with an IC50 value of 0.247 lM. In addition,
molecular docking analysis of the representative com-
pounds 11h, 11k, and 15a were performed, which not
only revealed the impact of binding modes on DPP-4
inhibitory activity but also provided additional methodo-
logical values for design and optimization.
Due to the important role of glycinyl-2-cyanopyrrolidine
moiety in contributing to potency, selectivity, and pharma-
cokinetic properties, it has been widely used as a key
pharmacophore for many reported DPP-4 inhibitors, such
as marketed drugs vildagliptin and saxagliptin (12).
Although the phenylalanine derivative 6 containing this
moiety showed only weak DPP-4 inhibition (Ki = 1.2 lM) in
previous studies (13), it bore advantageous facets includ-
ing small molecular weight, low logP value, and flexible
positions for chemical modifications (14). Thus, compound
6 was selected as the starting point for our work reported
herein. Docking study of compound 6 with DPP-4 was first
performed using the FlexiDock module of Sybyl 6.9 ver-
sion. It was discovered that the benzyl group of 6 was in
close proximity to Phe357 and Arg358 residues, two
important binding sites, in S2 pocket of DPP-4 (15). But
the benzyl group of 6 did not form interaction with Phe357
and Arg358 residues. So, Phe357 and Arg358 were
selected as potential target amino acid residues for form-
ing additional interaction. We carried out structure-based
drug design based on the docking simulated interaction
mode of lead 6 with DPP-4. A series of phenylalanine
derivatives with 1,2,3-triazole substituent were designed,
synthesized, and evaluated for their DPP-4 inhibitory
activity.
Key words: (S)-phenylalanine, 1,2,3-triazole, 2-cyanopyrroli-
dine, Dipeptidyl peptidase 4 inhibitors, type 2 diabetes
Received 19 November 2012, revised 5 February 2013 and
accepted for publication 20 March 2013
Type 2 diabetes mellitus, which is expected to affect 366
million people by 2030 according to an estimate made by
World Health Organization (WHO), has become a major
concern for human health worldwide.a Current available
treatment options including insulin and several oral antidia-
betic agents have been found with drawbacks, such as
hypoglycemia, edema, gastrointestinal disturbances, and
weight gain (1,2). Therefore, the development of novel an-
tidiabetic agents with improved safety and tolerability
profile is of significant importance. The incretin glucagon-
like peptide-1 (GLP-1)-based therapy appears to be a
promising strategy because GLP-1 regulates blood glu-
cose homeostasis in a dose-dependent manner, which
140
ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12139

(S)-Phenylalanine Derivatives as Potent DPP-4 Inhibitors
Figure 1: Approved DPP-4
inhibitors.
diluted using the assay buffer solution (Tris–HCl, pH 7.5)
and was incubated for 10 min at 37 °C with different con-
centrations of the test compound solutions in a 96-well
flat-bottomed microtiter plate. Subsequently, the reaction
was initiated by the addition of Gly-Pro-pNA substrate
solution. Absorbance at 405 nm of each reaction mixture
was measured using a microplate reader at the start and
end time of reaction. The initial rate of DPP-4 activity was
calculated over the first 15-min of the reaction. According
to these inhibitory rates in different concentrations, IC50
values were determined by using the GraphPad Prism
software with three independent determinations.
Methods and Materials
Chemistry
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 distilled for use, respectively.
Other commercially available chemicals and solvents are
reagent grade and were used without further purification.
€
Melting points were determined on a Buchi B-540 appara-
€
tus (Buchi Labortechnik, Flawil, Switzerland) and were
uncorrected. 1H NMR spectra were recorded on Bruker
500 MHz instruments (Bruker Bioscience, Billerica, MA,
USA) with CDCl3 or DMSO-d6 as solvent and tetrameth-
ylsilane (TMS) as internal standard. ESI Mass spectral data
were obtained by Esquire-LC-00075 spectrometer (Bruker
Bioscience). Flash column chromatography was performed
using silica gel (200-300 mesh). All yields are unoptimized
and only represent one experiment’s result.
Molecular docking
The molecular docking study was performed using the
FlexiDock module of Sybyl 6.9 version.b The three-dimen-
sional structures of all ligands were constructed manually
and then energy minimized under a Tripos force field. The
docked ligands were allowed to rotate on all single bonds,
€
and Gasteiger–Huckel charges were assigned to the ligand
atoms. The X-ray co-crystal structure of DPP-4 in complex
with its inhibitor has been available from the RCSB Protein
Data Bank (PDB code: 1ORW). The protein receptor was
preprocessed by removing the initial ligand, and then, the
free protein was modified by removing all solvents and add-
ing hydrogen atoms. Kollman all-atom charges were loaded
to the DPP-4 protein. Before the molecular docking pro-
cess, a binding pocket was defined to cover all of the
Detailed synthetic procedures and characterization data
for all new compounds 7–9, 10a–k, 11a–k, 12, 14a–c,
and 15a–c are provided in the Supporting Information
(Appendix S1) of this article.
DPP-4 inhibitory assay
Porcine kidney DPP-4 (0.8 U/cm3, Sigma) and Gly-Pro-
pNA (Sigma) were used as the enzyme source and sub-
strate, respectively. Sitagliptin, the first approved DPP-4
inhibitor, was selected as positive control. The assay prin-
ciple was based on the cleavage of substrate by DPP-4 to
release the chromogenic p-nitroaniline, which has a char-
acteristic absorption peak at 405 nm. The absorption
intensity change compared with solvent control was taken
as the DPP-4 percent inhibition rate (16). Each tested
compound was dissolved in dimethyl sulfoxide (DMSO)
and diluted into a series of concentrations. DPP-4 was
ꢀ
amino acid residues within 4.0 A radius sphere centered by
the docked compound in the initial compound and DPP-4
complex. All the single bonds of residues side chains inside
the defined DPP-4 binding pocket were regarded as rotat-
able or flexible bonds. The structure optimization was per-
formed for 100 000 generation using a genetic algorithm,
and the 20 best scoring ligand–protein complexes were
kept for further analyses. The lowest energy conformation
was selected as a final model to analyze the composition of
key amino acid residues of DPP-4 involved in the interac-
Chem Biol Drug Des 2013; 82: 140–146
141

Liu et al.
tion with its inhibitor. The pictures were prepared using Py-
mol (http://pymol.sourceforge.net/).
impact of the relative positions of phenyl ring and the
1,2,3-triazole on DPP-4 inhibitory activity (Figure 3).
Results and Discussion
Synthesis
The general synthetic route for compounds 11a–k is out-
lined in Scheme 1. (S)-N-Boc-4-iodophenylalanine deriva-
tive 7 was obtained in three steps from the starting
material (S)-phenylalanine through halogenation, Boc pro-
tection, and condensation reaction (14). Sonogashira cou-
Structure-based drug design
Firstly, docking study of compound 6 with DPP-4 was per-
formed using the FlexiDock module of Sybyl 6.9 version
(Figure 2). The docking results revealed that the glycinyl-2-
cyanopyrrolidine moiety functioned as a key pharmaco-
phore that could form crucial interaction with DPP-4,
which was consistent with literature reports (15). For
example, 2-cyanopyrrolidine ring was buried in a hydro-
phobic S1 pocket, the carbonyl group formed a hydrogen
bond with Asn710, and the amino group formed two
another hydrogen bonds with Glu205 and Glu206. It was
noteworthy to mention that the benzyl group at a-C posi-
tion was adjacent to Phe357 and Arg358 residues, two
important binding sites, in S2 pocket of DPP-4. But the
benzyl group of 6 did not form interaction with Phe357
and Arg358 residues. Based on these observations, it was
speculated that the potency of DPP-4 inhibitors could be
improved through the optimization of the substituents on
benzyl group. Considering the structural features of the S2
pocket, we intend to make further modifications at 4-posi-
tion of the benzyl ring. 1,2,3-Triazole generated via click
chemistry was given priority to the introduction of 4-posi-
tion of the benzyl ring, because it was usually used in drug
design with a good tolerance in vivo (17). More impor-
tantly, as a hydrogen bonding acceptor, 1,2,3-triazole may
form hydrogen bonds with Arg358 residue of this pocket,
which could improve the potency of DPP-4 inhibition.
Therefore, a series of phenylalanine derivatives containing
1,2,3-triazole substituent were designed and synthesized
in this study. Moreover, 1,2,3-triazole-based alanine deriv-
atives were also prepared and evaluated to investigate the
pling of
7 with trimethylsilylacetylene (TMSA) in the
presence of Pd(PPh3)2Cl2 and CuI gave trimethylsilyl-pro-
tected compound 8, whose following desilylation with
tetra-n-butylammonium fluoride (TBAF) in THF at room
temperature afforded (S)-N-Boc-4-ethynylphenylalanine
derivative 9 in good yield. Subsequent click reaction of 9
with different alkyl azides (18,19) in EtOH at room tem-
perature in the presence of CuSO4Á5H2O and sodium
ascorbate led to the formation of 1,2,3-triazole derivatives
10a–k in excellent yields. Ultimate removal of the Boc-
protecting group in HCl/Et2O solution furnished target
compounds 11a–k in the form of hydrochloride salt.
The synthesis of compounds 15a–c is outlined in
Scheme 2. The (S)-N-Boc-azidoalanine 12 was easily
accessible in two steps starting from the commercially
available (S)-N-Boc-serine following a reported procedure
(20,21). Click reaction of (S)-N-Boc-azidoalanine 12 with
phenylacetylene or substituted phenylacetylene in the
presence of catalytic amount of CuSO4Á5H2O and sodium
ascorbate in EtOH afforded the corresponding triazolyl
amino acid compounds 13a–c in good yields (22). The
triazolyl amino acid compounds 13a–c were then
condensed with (S)-pyrrolidine-2-carbonitrile hydrochloride
utilizing EDC and HOBt in CH2Cl2 to afford compounds
14a–c. Removal of the Boc-protecting group of com-
pounds 14a–c in HCl/Et2O solution provided target
compounds 15a–c.
DPP-4 inhibitory activity
All synthesized target compounds were evaluated for their
DPP-4 inhibitory activity in vitro, and sitagliptin (MK-0431)
was used as the reference drug. The results were summa-
rized in Table 1. Most of the tested compounds showed
potent DPP-4 inhibitory activity with IC50 values in the sub-
micromolar range. On the whole, the inhibitory activity of
phenylalanine derivatives was significantly better than that
of alanine derivatives. Among the phenylalanine derivatives,
the substituents at the N À 1 position of 1,2,3-triazole ring
had a major impact on activity. When methyl group was
introduced into the N À 1 position, compound 11a dis-
played potent DPP-4 inhibitory activity (IC50 = 0.265 lM).
Increasing the number of methylene groups was found to
be unfavorable for activity (ethyl-substituted 11b, IC50
=
0.339 lM; n-propyl-substituted 11c, IC50 0.374 lM;
n-butyl-substituted 11d, IC50 = 0.331 lM). Accordingly,
the branched alkyl groups were detrimental to activity
=
Figure 2: Docking study of compound 6 (cyan) with DPP-4.
Hydrogen bonds are shown as dashed red lines. For clarity, only
key amino acid residues are shown.
142
Chem Biol Drug Des 2013; 82: 140–146

(S)-Phenylalanine Derivatives as Potent DPP-4 Inhibitors
Figure 3: Docking-assisted design of novel DPP-4 inhibitors.
Scheme 1: Reagents and conditions: (a) TMSA, Pd(PPh3)2Cl2, CuI, Et3N, rt; (b) TBAF, THF, rt; (c) R1N3, CuSO4Á5H2O, sodium ascorbate,
EtOH, rt; (d) HCl/Et2O, CH2Cl2, rt.
Scheme 2: Reagents and conditions: (a) CuSO4Á5H2O, sodium ascorbate, phenylacetylene or substituted phenylacetylene, EtOH, rt; (b)
HOBt, EDC, DIPEA, (S)-pyrrolidine-2-carbonitrile hydrochloride, CH2Cl2, rt; (c) HCl/Et2O, CH2Cl2, rt.
(i-propyl-substituted 11e, IC50 = 0.527 lM versus n-propyl-
substituted 11c, IC50 = 0.374 lM; i-butyl-substituted 11f,
cyclic group, at the N À 1 position was beneficial to
activity.
IC50 = 0.578 lM versus n-butyl-substituted 11d, IC50
=
0.331 lM). In contrast, small cyclopropyl group showed
the most potent inhibitory activity in the phenylalanine
derivatives (11h, IC50 = 0.247 lM). With the further expansion
of ring, the inhibitory activity was decreased (cyclobutyl-
substituted 11i, IC50 = 0.342 lM; cyclopentyl-substituted
11j, IC50 = 0.332 lM). In particular, incorporation of a
cyclohexyl ring at the N À 1 position resulted in a dramatic
loss of activity (11k, IC50 = 0.863 lM). In addition, com-
pound 11g with a 2-hydroxylethyl group at the N À 1
position showed the moderate inhibitory activity
(IC50 = 0.478 lM), but it was lower than the activity of
compound 11h. These results suggested that the pres-
ence of a small substituent, either a linear alkyl group or a
Among the alanine derivatives, all compounds showed
weak DPP-4 inhibitory activity (15a, IC50 = 22.5 lM; 15b,
IC50 = 9.4 lM; 15c, IC50 = 19.0 lM). Whether the electron-
withdrawing fluoro- or electron-donating ethoxyl group
was introduced into the phenyl ring of compound 15a, the
activity showed no significant enhancement. These results
led us to conclude that the phenylalanine scaffold played a
vital role in DPP-4 inhibitory activity.
Molecular docking
Based on results of biological evaluation in vitro, three rep-
resentative compounds, high potent compound 11h,
Chem Biol Drug Des 2013; 82: 140–146
143

Liu et al.
Table 1: The DPP-4 inhibitory activity of compounds 11a–k and 15a–c
Compound
Structure
R1 (R2)
IC50 (lM)a
11a
11b
11c
11d
11e
11f
CH3
C2H5
n-C3H7
n-C4H9
i-C3H7
0.265
0.339
0.374
0.331
0.527
0.578
0.478
0.247
0.342
0.332
0.863
i-C4H9
11g
11h
11i
CH2CH2OH
c-C3H5
c-C4H7
c-C5H9
c-C6H11
11j
11k
15a
15b
H
3-F
4-OEt
22.5
9.4
19.0
0.040
15c
Sitagliptin
aIC50 values are an average of three independent determinations.
moderate potent compound 11k, and low potent com-
pound 15a, were selected for further docking simulations
to analyze their interaction modes with DPP-4 using the
FlexiDock module of Sybyl 6.9 version. Figure 4 repre-
sented the docking simulated interaction modes of these
three compounds binding to the active pocket of DPP-4.
At first, the high potent compound 11h was analyzed of
its binding mode with DPP-4 from docking calculation.
From the docking results (Figure 4A), 2-cyanopyrrolidine
ring of 11h fully occupied the hydrophobic S1 pocket,
which was similar to the same group of lead compound 6.
The carbonyl group of 11h formed a hydrogen bond with
the side chain of Asn710. The amino group of compound
11h could form hydrogen bond interactions with the oxy-
gen atoms in the carboxyl groups of both residues,
Glu205 and Glu206, respectively. The position-1 nitrogen
of triazole group formed a hydrogen bond with the side
chain of Arg358, which confirmed our design conception.
This additional hydrogen bond interaction made com-
pound 11h more potent DPP-4 inhibition than lead com-
pound 6. Docking simulation revealed that compound 11h
(À4042.3 kcal/mol) had a lower docking energy with DPP-
4 than the lead 6 (À2739.5 kcal/mol). As illustrated in Fig-
ure 4B, compound 11k had a similar interaction mode
with compound 11h binding to DPP-4. However, the
amino group of compound 11k only forms a hydrogen
bond with Glu205 due to a little change of its binding con-
formation, which led compound 11k to the decrease in
activity compared with 11h. In addition, the docking
energy of 11h was much lower than that of 11k
(À3709.1 kcal/mol). As for the low potent compound 15a
(Figure 4C), docking simulation indicated that it had a dif-
ferent binding mode from compound 11h. The key hydro-
gen bond was not formed between its amino group and
A
B
C
Figure 4: Docking studies of compounds 11h (yellow), 11k (magenta), and 15a (orange) with DPP-4. Hydrogen bonds are shown as
dashed red lines. For clarity, only key amino acid residues are shown. (A) the interaction of compound 11h with the key residues of DPP-
4; (B) the interaction of compound 11k with the key residues of DPP-4; (C) the interaction of compound 15a with the key residues of
DPP-4.
144
Chem Biol Drug Des 2013; 82: 140–146

(S)-Phenylalanine Derivatives as Potent DPP-4 Inhibitors
Glu205 or Glu206 of DPP-4, but only a hydrogen bond
was formed between the carbonyl group and the side
chain of Asn710. Such an unfavorable binding mode of
15a resulted in loss of its activity, and the docking energy
of 15a (À922.3 kcal/mol) was also significantly higher than
that of compound 11h. These docking results could
explain why the potency of compounds 11k and 15a was
lower than that of compound 11h. Combining with the
results from biological evaluation, it can be hypothesized
that both Glu205 and Glu206 play critical roles in the bind-
ing of phenylalanine derivatives to the DPP-4.
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Notes
aWorld Health Organization at http://www.who.int/diabe-
tes/facts/world_figures/en/.
bSybyl molecular modeling software packages. Tripos
Associates Inc., St. Louis, MO63144, 2002.
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Additional Supporting Information may be found in the
online version of this article:
a-amino acids via b-lactones.
Soc;107:7105–7109.
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Appendix S1. Experimental details and characterization
for all new compounds 7–9, 10a–k, 11a–k, 12, 14a–c,
and 15a–c.
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