Synthesis, in vitro evaluation, and computational simulations studies of 1,2,3-triazole analogues as DPP-4 inhibitors

Article abstract of DOI:10.1016/j.bmc.2020.115861

Novel 1,2,3-triazole analogues (S7 ~ S10) were synthesized and evaluated for their inhibitory activity against hDPP-4. All the 1,2,3-triazole analogues exhibited moderate in vitro hDPP-4 inhibitory activities (265 ~ 780 nM). These results are somewhat less potent compared to those of known 1,2,3-triazole analogues (S1 ~ S6, 14 ~ 254 nM). S2 and S3 manifested excellent potency against hDPP-4 with IC₅₀s of 28 and 14 nM, respectively. The role of the 1,2,3-triazole moiety in binding the molecule to the target was investigated using combined 10 1,2,3-triazole analogues (S1 ~ S10). Molecular dynamics (MD) simulations following the aforementioned docking phase were performed to elucidate potential binding modes of sitagliptin's 1,2,3-triazole analogues in hDPP-4, with the use of a cocrystal structure of hDPP-4 with sitagliptin (PDB ID: 1X70). Docking and MD simulations of the complexes of hDPP-4 with sitagliptin, S2 and S3 suggest that Glu205, Glu206, Tyr662, and Tyr666 would be the key amino acid residues for the binding of the molecules with the receptor. Especially, S2 and S3 showed additional strong π-π interaction between Phe357 and 1,2,3-triazole. Same strong π-π interaction is also observed between Phe357 and the 1,2,4-triazole ring of sitagliptin. Furthermore, additional interactions with Tyr547, Cys551, and especially Arg358 would enhance the binding affinity of the compounds for the pocket of the enzyme. In overall, in vitro hDPP-4 inhibitory activities of synthetic 1,2,3-triazole analogues were well matched with results of computational simulations studies.

Full text of DOI:10.1016/j.bmc.2020.115861

Journal Pre-proofs
Synthesis, In Vitro Evaluation, and Computational Simulations Studies of
1,2,3-Triazole Analogues as DPP-4 Inhibitors
Duy-Viet Vo, Kwon Ho Hong, Jongkook Lee, Haeil Park
PII:
S0968-0896(20)30691-X
https://doi.org/10.1016/j.bmc.2020.115861
BMC 115861
DOI:
Reference:
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Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
17 April 2020
11 October 2020
1 November 2020
Please cite this article as: D-V. Vo, K. Ho Hong, J. Lee, H. Park, Synthesis, In Vitro Evaluation, and
Computational Simulations Studies of 1,2,3-Triazole Analogues as DPP-4 Inhibitors, Bioorganic & Medicinal
Chemistry (2020), doi: https://doi.org/10.1016/j.bmc.2020.115861
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Synthesis, In Vitro Evaluation, and Computational Simulations Studies of 1,2,3-
Triazole Analogues as DPP-4 Inhibitors
Duy-Viet Vo1,¶, Kwon Ho Hong2,¶ Jongkook Lee1 and Haeil Park1*
1College of Pharmacy, Kangwon National University, Chuncheon 24341, Republic of Korea; 2Department of
Medicinal Chemistry, University of Minnesota, MN 55414, USA; *Corresponding author:
¶
haeilp@kangwon.ac.kr; These authors equally contributed to this work
<Abstract> Novel 1,2,3-triazole analogues (S7~S10) were synthesized and evaluated for their inhibitory activity
against hDPP-4. All the 1,2,3-triazole analogues exhibited moderate in vitro hDPP-4 inhibitory activities (265~780
nM). These results are somewhat less potent compared to those of known 1,2,3-triazole analogues (S1~S6,
14~254 nM). S2 and S3 manifested excellent potency against hDPP-4 with IC50s of 28 and 14 nM, respectively.
The role of the 1,2,3-triazole moiety in binding the molecule to the target was investigated using combined
10 1,2,3-triazole analogues (S1~S10). Molecular dynamics (MD) simulations following the aforementioned
docking phase were performed to elucidate potential binding modes of sitagliptin’s 1,2,3-triazole analogues
in hDPP-4, with the use of a cocrystal structure of hDPP-4 with sitagliptin (PDB ID: 1X70). Docking and MD
simulations of the complexes of hDPP-4 with sitagliptin, S2 and S3 suggest that Glu205, Glu206, Tyr662,
and Tyr666 would be the key amino acid residues for the binding of the molecules with the receptor.
Especially, S2 and S3 showed additional strong - interaction between Phe357 and 1,2,3-triazole. Same
strong - interaction is also observed between Phe357 and the 1,2,4-triazole ring of sitagliptin. Furthermore,
additional interactions with Tyr547, Cys551, and especially Arg358 would enhance the binding affinity of
the compounds for the pocket of the enzyme. . In overall, in vitro hDPP-4 inhibitory activities of synthetic
1,2,3-triazole analogues were well matched with results of computational simulations studies.
1. Introduction
Dipeptidyl peptidase-4 (DPP-4) inhibitors have emerged as a novel class of antidiabetic drugs which prevent
the proteolytic degradation of incretin hormones and stimulate the glucose-dependent insulin secretion
associated with a reduction in body weight and the risk of hyperglycemia.1 Inhibition of DPP-4 results in an
increase in the half-life of glucagon-like peptide-1 (GLP-1) and in a sustained physiological action of this
hormone, leading to an improvement in the glycemic control.2 Sitagliptin is the first approved DPP-4
inhibitor in the United States.
Intensive efforts have been made to investigate type 2 antidiabetic drugs with extended half-lives and
better pharmacological profiles.3,4 Previous structure-activity relationship (SAR) studies of sitagliptin revealed
that the modification of the 1,2,4-triazolopiperazine substructure is crucial to its bioactivity. Based on these
findings, diverse DPP-4 inhibitors comprising different heterocycle scaffolds were explored and proved to
improve bioactivities (Figure 1).5-7

The successful precedent SAR results led us to explore novel heterocycle scaffolds as 1,2,4-triazolopipeprazine
surrogates of sitagliptin. As part of this research we designed novel sitagliptin analogues S1~S6 with 1,2,3-
(
)
triazole heterocycle scaffold since the electron-withdrawing effect of nitrogen in 1,2,3-triazole may increase
the number and strength of interactions between the ligand and hDPP-4 receptor. Our previous SAR study
showed that the bulky alkyl group at C4 position of the 1,2,3-triazole ring exhibited better
inhibitory activity than the small alkyl group (tert-Bu > Me > H). Our previous result also showed that
compounds with different orientation of nitrogen atom in the pyridine (Py) ring at C4 position exhibited
different bioactivities (4-Py > 3-Py > 2-Py) as shown in Figure 1.8 In summary, our previous SAR study
suggested two important information as followings: the steric hindrance (size) of C4 substituent as well as
orientation of nitrogen atom of pyridine ring at C4 position seemed to play critical role for bioactivity.
F
F
NH2
O
F
N
F
NH2
ref.6
F
F
NR
N
F
F
N
N
ref.3
NH2
O
F
F
NH2
O
R1
N
F
N
1
S1
R : 2-Py (143 nM,
3-Py (28 nM,
)
N
N
S2
)
CF3
S3
4-Py (14 nM,
)
F
X
N
S4
)
H (254 nM,
N
Sitagliptin
(7.8 nM)
N
S5
)
Me (133 nM,
N
X=CH2; ref.4
X=O; ref.5
tert-Bu (121 nM, S6)
CF3
Figure 1 – Sitagliptin and its analogues under research.
In continuation of our SAR study on DPP-4 inhibitors, novel 1,2,3-triazole analogues (S7~S10) were designed,
synthesized and evaluated for their bioactivity. Structures of 1,2,3-analogues S7 and S8 were gained by switching
the N-alkyl backbone and the C-4 substituent of analogues S2 and S3. Regioisomeric 1,2,3-triazole analogues S7
and S8 were expected to have different interactions with hDPP-4 receptor than those of analogues S2 and S3.
1,2,3-Triazole analogues with 5- and 6-quinoline (Qu) rings instead of 3- and 4-pyridine rings at N-1 (S9/S10)
were designed to investigate the influence of steric hindrance and also orientation of nitrogen atom at N-1
substituents (quinoline ring vs. pyridine ring) to bioactivity. In addition, we performed computational
simulations of 10 known and novel 1,2,3-triazole analogues (S1~S10) with the crystal structure of human
DPP-4 in order to obtain insights into binding mode, binding strength and bioactivity. Herein, we report
the synthesis and in vitro biological evaluation (S7~S10), molecular docking and molecular dynamics (MD)
simulations of 1,2,3-triazole analogues (S1~S10).
F
R2
N
N
2
S7
R : 3-Py (
4-Py (
)
F
NH2
S8
)
S9
5-Qu (
)
N
S10
6-Qu (
)
F
Figure 2 – Structures of synthesized novel 1,2,3-triazole analogues of sitagliptin (S7~S10).
2. Results and discussion

2.1. Chemistry
1,2,3-Triazole analogues (S1~S10) for SAR study were prepared as follows: The analogues (S1~S6) were
synthesized
from
the
commercially
available
(R)-3-
(benzyloxy)carbonyl)amino-4-(2,4,5-
trifluorophenyl)butanoic acid in 5 steps as described in our previous report.8 The regioisomeric analogues
(S7~S10) were synthesized from (R)-3- (benzyloxy)carbonyl)amino-4-(2,4,5-trifluorophenyl)butanoic acid (I)
in 5 steps. Esterification of the starting material with cerium ammonium nitrate (CAN),9 followed by DIBAL
reduction provided the aldehyde intermediate (III).10 The aldehyde intermediate was converted into the
corresponding alkyne intermediate (IV) with Bestmann-Ohira reagent.10 Reaction of the alkyne key
intermediate with corresponding arylazides in “click reaction” conditions,11 followed by the Cbz
deprotection12 gave the corresponding 1,2,3-triazole analogues with propan-2-amine backbone (S7~S10)
as shown in Scheme 1.
F
F
F
F
F
R2
N
N
R2
N
N
F
Cbz
H
F
F
F
Cbz
F
Cbz
H
d)
b)
c)
e)
N
O
NH2
NHCbz
NH
N
O
OR
N
H
N
F
:
F
F
F
F
R2
:
Va
3-Py( ); 4-Py(
Vb
R2
:
S7 S8
3-Py( ); 4-Py( )
)
I
R = H
III
a)
IV
II
Vc Vd
S9 S10
)
: R = CH3
5-Qu( ); 6-Qu(
)
5-Qu( ); 6-Qu(
Scheme 1. Synthesis of target compounds S7~S10. Reagents and conditions: a) CAN/MeOH, rt, 5 h, 97%; b) DIBAL, -78 oC,
3h, 86%; c) Bestmann-Ohira reagent, K2CO3, MeOH, rt, 14 h, 94%; d) arylazides, Cu(OAc)2, AcCN, rt, 3 h, 68%~82%; e)
HBr/AcOH, 80 oC, 14 h, 65%~85%.
2.2. DPP-4 inhibition assay
Table 1. DPP-4 inhibitory activities of synthesized 1,2,3-triazole analogues (S1~S10)
F
F
R2
N
N
F
NH2
F
NH2
N
N
N
N
F
R1
F
S1~S6
S7~S10
Compound
R1
2-pyridyl
3-pyridyl
4-pyridyl
R2
-
-
-
DPP-4 IC50 (nM)
S1
S2
S3
1438
288
148
S4
S5
S6
S7
S8
H
-
-
-
2548
1338
1218
305
427
265
780
7.88
Methyl
tert-Butyl
-
-
-
-
-
3-pyridyl
4-pyridyl
5-quinolinyl
6-quinolinyl
-
S9
S10
Sitagliptin
We studied the structural features of the butan-2-amine backbone in compounds S1~S6. The compounds
bearing the N-pyridyl group exhibited better potency than compounds with alkyl groups. Also our data
showed that there was a relationship between the DPP-4 inhibitory activities and the orientation of the

nitrogen atom in the N-pyridyl group, which was likely to play an important role in the interaction with the
binding site (IC50 S1~S3, R1: 4-pyridyl > 3-pyridyl > 2-pyridyl). Moreover, analogues (S2 and S3) containing
the 3-pyridyl or the 4-pyridyl group showed excellent DPP-4 inhibitory activities (IC50 = 28 and 14 nM,
respectively). Among them, compound S3 displayed a strong IC50 value comparable to that of sitagliptin
(IC50 = 7.8 nM). Our results also disclosed that the size of the R1 alkyl group affected on DPP-4 inhibitory
activities (tert-Bu > Me > H).
As shown in Table 1, propan-2-amine analogues (S7 and S8) possessing N-pyridyl were identified as weak
DPP-4 inhibitors (IC50 > 300 nM) compared with butan-2-amine backbone analogues (S1~S3; IC50 < 150
nM). Replacement of N-pyridyl with 5-quinolinyl improved the DPP-4 inhibitory activities slightly (S9; IC50
= 265 nM), but introduction of the 6-quinolinyl group significantly decreased the biological activities (S10;
IC50 = 780 nM). To sum up for conducting the further SAR and computational simulations of the sitagliptin
analogues, DPP-4 inhibitory activities of propan-2-amine backbone analogues (S7~S10) shown in Table 1
exhibited much reduced bioactivities compared to those of butan-2-amine backbone analogues (S1~S6) in
the aforementioned literature.8
2.3. Molecular Docking Studies
2.3.1. Choosing the DPP-4 receptor
The crystal structure of the DPP-4 receptor (PDB ID: 1X70, http://www.rcsb.org) used in this study consists
of two symmetrical chains of the same protein, chains A and B. They were separately prepared for our
computational study. The chain A was selected as representative for two reasons: (1) the obtained re-docked
poses of the native ligand (HET code: 715) into the receptor deviated from the original X-ray pose less than
those received when the chain B was used as template, and (2) the corresponding docking scores were
better, as shown in Table 2.
Table 2. Information details used to choose a representative chain for the computational simulation: The protein 1X70
chain A chosen as the representative of DPP-4 due to the docking scores. The RMSD values obtained from docking
simulations with chains A and B, with the native ligand and a conformation shown in the crystal structure. (a), the
energy-minimized native ligand (b), and the energy-minimized re-constructed native ligand (prepared like 10 1,2,3-
triazole analogues shown in Experimental sections) (c)
Protei
n
Method
Resolution Amino
Co-
crystallize
Chain Docking score (kJ/mol)
RMSD (Å)
(a) (b)
(Å)
acids
(a)
(b)
(c)
(c)
number d ligand
1X70
X-ray
diffractio
n
2.1
728
Sitagliptin
A
B
-
-29.9534
-
0.6730 0.7215 0.5769
31.5092
29.8130
-
-23.8946
-
0.6739 1.1092 0.8178
28.0125
26.6775
2.3.2. Molecular docking of 10 sitagliptin analogues into the chain A
The docking scores of 10 sitagliptin analogues were distributed from -34.5657 to -22.7799 kJ/mol. Three

structures S4~S6 (of the butan-2-amine backbone, each containing an alkyl group) gave higher scores
(indicating worse docking solutions) than analogues bearing the same backbone but containing other N-
pyridyl substituents (S1~S3). This is supported by bioactivity results, revealing that S4~S6 were indeed less
potent than S1~S3 (Figure 3). The butan-2-amine backbone analogues (S1~S6) have lower scores than the
propan-2-amine analogues (S7~S10), which also agrees with experimental biological data (Figure 3).
S3
S2
S1
S6
S5
S4
S7
S8
S10
S9
-20
-25
-30
-35
-40
-22.7799
-23.4347
-26.5842
-27.2049
-27.3252
-28.5609
-29.3521
-33.2943
-33.5672
-34.5657
kJ/mol
Figure 3 - The docking scores of S1~S10.
Analogues (S1~S10) were examined for their in vitro inhibitory activity against the recombinant human
DPP-IV using sitagliptin (SGT) as the reference sample. In the 3D docking model, the propan-2-amine
backbone was not well-suited to the DPP-4 ligand-binding pocket as the docking poses of all the
compounds bearing this backbone (S7~S10) did not coincide with that of the reference sitagliptin structure
of the original X-ray pose (Figure 4). S7~S10 also did not form as much strong binding affinity as the H-
bonding interactions between ligand and ligand-binding pocket compare with the butan-2-amine backbone
analogues, as shown in Figure 4. This explains why the analogues containing the butan-2-amine backbone
(S1~S6) were deemed more active than those with the propan-2-amine structure (S7~10).
a
b
c
d

Figure 4 - The docking models of DPP-4 protein with sitagliptin (orange) and analogues S2 (a,b); S7 (c,d).
The analogue S3 had the lowest docking score and its docking pose coincided with that of sitagliptin, as
shown in Figure 5. Besides, compound S3 formed an H-bonding interaction between the pyridine moiety
and the residue Arg358 of DPP-4 and three H-bonding interactions (Glu205, Glu206, and Tyr662) as well as
one π-π interaction (Phe357), but S8 did not form any corresponding H-bonding interaction except three
H-bonding interactions (Glu205, Glu206, and Tyr662) with the 2-amine group. It was clear that the H-
bonding with Arg358 was necessary for a possible bioactivity. The aforementioned observations were
backed up by the fact that the analogue S3 (IC50 = 14 nM) exhibited a comparable inhibitory activity
compared to that of sitagliptin (IC50 = 7.8 nM).8
a
b
c
d
Figure 5 - The docking models of DPP-4 protein with sitagliptin (orange) and analogues S3 (a,b); S8 (c,d).
For butan-2-amine backbone compounds, the alkyl 1,2,3-triazole substituted analogues exerted a lower
inhibitory activity than N-(1-yl-1H-1,2,3-triazole-4-yl)pyridine substituted analogues, because the N-
pyridine group gave lower docking scores and the docking poses deviated from that of sitagliptin less
than those obtained from the alkyl group, as shown in the 3D protein-ligand interactions (Figure 4 and
Figure 6). The SAR data for compounds S1~S3 show that the orientation of the H-bonding site (nitrogen
atom) that coincided with that of sitagliptin also plays an important role in the inhibitory activity (IC50 of
S1~S3 with nitrogen position in R1: para>meta>ortho). For the alkyl group cases, S4~S6, when the alkyl

group is large and branched (from -H to -CH3 and -tert-butyl), the corresponding bioactivity increased, as
the van der Waals interactions increased. However, the observed improvement in bioactivity was small
because van der Waals interactions were normally weak.
The reduced bioactivity of the analogues S7~10 might be attributed to the unfavorable conformation of
the propan-2-amine moiety. The replacement of the N-pyridine group with a sterically hindered quinoline
ring decreased the inhibitory activity (S7, S8 > S9 » S10). The difference between the inhibitory activities
of S9 and S10 implies that the steric factor and the orientation of the H-bonding site (5-quinolinyl vs. 6-
quinolinyl) might be important for the resulting bioactivity of the compounds.
S10 gave one of the highest docking scores and had the lowest bioactivity among analogues. As shown in
the 3D model in Figure 6, S10 exposes most atoms (6-quinolinyl and 1,2,3-triazole ring) to the outside of
the pocket, its structure did not totally coincide with sitagliptin, and also the nitrogen atom of 6-quinolinyl
could not form an H bond with Arg 358. Therefore, the bioactivity of S10 was significantly reduced
compared to those of S7, S8 and S9.
a
b
c
d
Figure 6 - The docking models of DPP-4 protein with sitagliptin (orange) and analogues S4 (a,b); S10 (c,d).
2.4. Molecular dynamics simulations studies
Novel sitagliptin analogues with the 1,2,3-triazole heterocyclic scaffold were designed to investigate the
role of the triazole moiety in binding the molecule to the target and to search for a surrogate of the 1,2,4-
triazolopiperazine substructure of sitagliptin that might enhance the π-π interaction with Phe357 (Figure
7). The designed sitagliptin 1,2,3-triazole analogues were synthesized as shown in Scheme 1, and had their

inhibitory activity against the recombinant human DPP-4 evaluated by biological assays. We also performed
molecular dynamics (MD) simulations following the aforementioned docking phase to obtain insights into
potential binding modes of sitagliptin’s 1,2,3-triazole analogues in hDPP-4, with the use of a cocrystal
structure of hDPP-4 with sitagliptin (PDB ID: 1X70, Figure 7).
Figure 7 - Binding mode of sitagliptin in hDPP-4 (PDB ID: 1X70).
MD simulations of the complexes of hDPP-4 with sitagliptin, S1, S2, and S3 showed H-bonding
interactions with Glu205, Glu206, Tyr662, and Tyr666 as the common key binding interactions between
these compounds and the binding pocket. In addition to these interactions, sitagliptin consistently
interacted with Phe357, Tyr547, and Cys551 during the MD simulations. This agrees with its binding mode
in the hDPP-4 crystal structure (PDB ID: 1X70), except for a water-mediated H-bonding interaction with
Cys551: the interaction between Cys551 and sitagliptin is not observed in the X-ray pose (Figures 8 and
9).
S1
sitagliptin
S2
S3
Figure 8 - Representative pose views of major clusters from MD simulations of sitagliptin, S1, S2, and S3.

The interaction frequency of S1 with Tyr547 significantly increased after the system was equilibrated
while its interaction with His126 was observed in the early time points of the MD simulation but disappeared
after 2 ns. S2 formed π-π interactions with Phe357 consistently during the MD simulations, reinforcing
binding interactions with the binding pocket. S3 formed consistent additional interactions with Phe357 and
Arg358: the H-bond between the 4-pyridyl group of S3 and Arg358 and its π-π interactions with Phe357
further reinforced its binding affinity with the pocket. The results from the computational simulations are
consistent with those from the hDPP-4 enzyme assay (Figures 8 and 9).
sitagliptin
S1
S2
S3
Figure 9 - Interaction frequencies of sitagliptin and its analogues with the binding pocket in hDPP-4.

3. Conclusion
All synthesized 1,2,3-triazole analogues (S1~S10) exerted submicromolar inhibitory activities against hDPP-4.
Especially, S2 and S3 manifested excellent potency against hDPP-4 with IC50s of 28 and 14 nM, respectively.
For the alkyl group cases, S4~S6, when the alkyl group is large and branched (from -H to -CH3 and -tert-
Bu), the corresponding bioactivity increased, as the van der Waals interactions increased. However, the
observed improvement in bioactivity was small because van der Waals interactions were normally weak.
The reduced bioactivity of the analogues S7~10 might be attributed to the unfavorable conformation of
the propan-2-amine moiety. The replacement of the N-pyridine group with a sterically hindered N-
quinoline ring decreased the inhibitory activity (S7, S8 > S9 » S10). The difference between the inhibitory
activities of S9 and S10 implies that the steric factor and the orientation of the H-bonding site (N-5-
quinolinyl vs. N-6-quinolinyl) might be important for the resulting bioactivity of the compounds.
Docking and MD simulations of the complexes of hDPP-4 with sitagliptin, S2 and S3 suggest that Glu205,
Glu206, Tyr662, and Tyr666 would be the key amino acid residues for the binding of the molecules with
the receptor. Especially, S2 and S3 showed additional strong - interaction between Phe357 and the 1,2,3-
triazole. Same strong - interaction is also observed between Phe357 and the 1,2,4-triazole ring of
sitagliptin. Furthermore, additional interactions with Tyr547, Cys551, and especially Arg358 would enhance
the binding affinity of the compounds for the pocket of the enzyme. In conclusion, in vitro hDPP-4 inhibitory
activities of 1,2,3-triazole analogues were well matched with results of computational simulations studies.
4. Experimental section
4.1. Molecular Docking studies
4.1.1. Ligand Preparation
Ten sitagliptin analogues were prepared as ligands for docking simulations. SMILES chains of the ligands
from ChemBioDrawUltra 12.0 were converted to the corresponding 3D structures using ChemBio3DUltra
12.0 and saved in sd file format. The ligands were next subject to a preparation process using Sybyl-
X 2.0.13,14 Their energy was first minimized using the Conj Grad method and the standard Tripos force field.
The maximum number of iterations to perform was 10,000. Energy minimization was automatically stopped
once the energy difference between iterations was smaller than 0.0001 kcal.mol-1.. The Gasteiger-Huckel
charges were employed to calculate atomic formal charges. The output structures would then enter a short
molecular dynamics (MD) simulation phase. Each was heated at high temperature (700 K) during 1000 ps,
and was then cooled down to 200 K in another 1000 ps. The MD process was automatically repeated 10
times to give various necessary configurations of the ligands, after which energy minimization was carried

out once again to find the structure with the optimized energy. Finally, the ligands after preparation were
converted to sd file format using Sybyl-X 2.0.
4.1.2. DPP-4 Model Preparation
In this study, DPP-4 was imported from RCSB PDB (PDB ID: 1X70, http://www.rcsb.org) with the sitagliptin
inhibitor. 1X70 contains a dimer of two chains, and only the monomeric unit was used in the docking
studies. The DPP-4 model in complex with ligand was protonated; tethered, and minimized by the LigX tool
in MOE,15 and the ligand was subsequently removed.
4.1.3. Molecular Docking
Ten selected DPP-4 inhibitors were docked into the ligand-binding pocket of DPP-4 (PDB ID: 1X70) using
the FlexX package in LeadIT 2.1.2 to provide insights into molecular recognition via protein-ligand
interactions. In the present process, the triangle matching algorithm was chosen for the place base fragment,
while the maximum numbers of solutions per iteration and per fragmentation were defined as 1000 and
200, respectively. After docking, the best docking scores and protein-ligand interactions of the best docking
scores poses were employed to explain the bioactivities of 10 synthesized analogues. Compounds with
good docking scores were more likely to be the hits for DPP-4 inhibitory activity.16,17
4.2. Molecular Dynamics simulations studies
Docking poses of the compounds in hDPP-4 were used to prepare the systems for MD simulations using
Desmond: water molecules and salts (0.15 M MgCl2) were added (orthorhombic box with the volume of 10
Å x 10 Å x 10 Å). The MD simulations of the complexes of hDPP-4 with inhibitors in 0.15 MgCl2 solution
water were performed for 5 ns with 2 fs time steps. After that, 18 ns MD simulations of the complexes were
performed using Desmond 3.0 employing 128 cores in a high performance computing system. Default
option in Desmond/2011 for integration, ensemble, interaction, restraints, output, and Misc were used
except Thermostat and Barostat methods in Ensemble; Berendsen method was used as Thermostat and
Barostat methods with relaxation time of 1.0 ps and isotropic coupling style for the Barostat.18-21
4.3. DPP-4 inhibition assay
To measure the activity of DPP4, fluorogenic assay was employed using Gly-Pro-AMC, which is cleaved by
the enzyme to release fluorescent aminoethylcoumarin (AMC). Compounds (S7~S10) were incubated with
recombinant DPP4 or Caco-2 lysate and 50 μM Gly-Pro-AMC in a buffer containing 25 mM Tris/HCl, pH
8.0 (1 mg/ml bovine serum albumin was added only for recombinant human DPP4). The assay was
performed at 25 °C for 1 h in a total reaction volume of 200 μl. Liberated AMC was detected using an
excitation wavelength of 360 nm and an emission wavelength of 465 nm.
4.4. Chemistry
All chemicals, solvents and reagents were obtained from commercial suppliers and used without further
purification, unless specified. Reactions were monitored by thin-layer chromatography performed on glass
packed silica gel plates (60 F-254) with UV light. Flash column chromatography was performed with silica

gel (100–200 mesh). 1H-NMR (300 MHz & 400 MHz) and 13C-NMR (100 MHz) spectra were recorded on
Bruker DPX 400 spectrometers (MA, USA), fully decoupled and chemical shifts are reported in parts per
million (ppm) downfield relative to tetramethylsilane as an internal standard. Peak splitting patterns are
abbreviated as s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublet),
and m (multiplet). Mass spectra were recorded on API 3200 MS system of AB SCIEX. Melting points were
recorded on Fisher-Johns microscopic scale melting point apparatus (NJ, USA). Optical rotation data were
collected by Jasco DIP-1000 digital polarimeter (MD, USA).
4.4.1. Methyl (R)-3-(((benzyloxy)carbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoate (I)
The solution of (R)-3-(((benzyloxy)carbonyl)amino-4-(2,4,5-trifluorophenyl)butanoic acid (1.8 g, 4.9 mmol) in
MeOH (30 mL) was added CAN (2.7 g, 4.9 mmol) at room temperature. The reaction was stirred for 5 h at
room temperature. The solution was extracted by ethyl acetate, washed with 10% NaHSO3 and brine, dried
with anhydrous MgSO4 and evaporated to give the title compound as white solid (1.8 g, 97 %). 1H-NMR
(300 MHz, CDCl3): δ = 7.39-7.28 (m, 5H), 7.02 (m, 1H), 6.87 (m, 1H), 5.37 (d, J = 8.7, 1H), 5.04 (m, 1H), 4.20
(m, 1H), 3.69 (s, 3H), 2.58 (d, J = 7.0, 2H), 2.57 (m, 2H).
4.4.2. Benzyl (R)-(4-oxo-1-(2,4,5-trifluorophenyl)butan-2-yl)carbamate (II)
To a solution of methyl (R)-3-(((benzyloxy)carbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoate (0.76 g, 2
mmol) in 15 mL of anhydrous dichloromethane (DCM) was added 1M DIBAL (4mL, 4mmol) at -78 ℃ under
N2. The reaction was stirred for 3 h at -78 ℃. The solution was quenched with saturated NH4Cl, extracted
by DCM, washed with saturated NaHCO3, dried with anhydrous MgSO4 and evaporated. The residue was
purified by column chromatography to give the title compound (0.6 g, 86 %). 1H-NMR (300 MHz, CDCl3): δ
= 9.74 (s, 1H), 7.38-7.26 (m, 5H), 7.02 (m, 1H), 6.88 (m, 1H), 5.17 (d, J = 7.8, 1H), 5.04 (m, 2H), 4.28 (m, 1H),
2.88 (d, J = 6.4, 2H), 2.72 (d, J = 5.2, 2H).
4.4.3. Benzyl (R)-(1-(2,4,5-trifluorophenyl)pent-4-yn-2-yl)carbamate (III)
To a solution of benzyl (R)-(4-oxo-1-(2,4,5-trifluorophenyl)butan-2-yl)carbamate (0.5 g, 1.4 mmol) in
anhydrous MeOH (10 mL) was added Bestmann-Ohira reagent (0.33 g, 1.7 mmol) and K2CO3 (0.39g, 2.8
mmol) respectively at room temperature under N2. The reaction was stirred at room temperature for
overnight. The solution was extracted by ethyl acetate, washed with brine, dried with anhydrous MgSO4
and evaporated. The residue was purified by column chromatography (DCM : MeOH = 50:1) to the title
compound (0.46 g, 94 %). 1H-NMR (300 MHz, CDCl3): δ = 7.32-7.23 (m, 5H), 6.99 (m, 1H), 6.81 (m, 1H), 4.98
(m, 2H), 4.93 (m, 1H), 4.00 (m, 1H), 2.82 (m, 2H), 2.47-2.26 (m, 2H), 2.03 (t, J = 2.5, 1H).
4.4.4. Benzyl (R)-(1-R2-1H-1,2,3-triazol-4-yl)-3-(2,4,5-trifluorophenyl)propan-2-yl)carbamate (IV)
To a solution of (S)-(1-(2,4,5-trifluorophenyl)pent-4-yn-2-yl)carbamate (1 mmol) in CH3CN (10 mL) was
added proper azides (1.2 mmol) and Cu(OAc)2 (0.1 mmol) respectively at room temperature. The reaction
was stirred at room temperature for 3h. The solution was extracted by ethyl acetate, washed with brine,

dried with anhydrous MgSO4 and evaporated. The residue was purified by column chromatography to give
the following compounds as solid.
Benzyl (R)-(1-(1-(pyridin-3-yl)-1H-1,2,3-triazol-4-yl)-3-(2,4,5-trifluorophenyl)propan-2-yl)carbamate
(Va)
68 %; 1H-NMR (300 MHz, CDCl3): δ = 8.95 (s, 1H), 8.71 (d, J = 4.0, 1H), 8.08 (m, 1H), 7.84 (s, 1H), 7.50 (dd,
J = 8.3, 4.8, 1H), 7.34-7.29 (m, 5H), 7.10 (m, 1H), 6.89 (m, 1H), 5.27 (d, J = 9.0, 1H), 5.03 (m, 2H), 4.25 (m,
1H), 3.15-2.82 (m, 4H).
Benzyl (R)-(1-(1-(pyridin-4-yl)-1H-1,2,3-triazol-4-yl)-3-(2,4,5-trifluorophenyl)propan-2-yl)carbamate
(Vb)
75 %; 1H NMR (300 MHz, CDCl3): δ 8.87 (d, 2H, J = 4.9 Hz), 7.90 (s, 1H), 7.50 (d, 2H, J =5.9 Hz), 7.25- 7.08
(m, 5H), 7.05- 6.95 (q, 1H, J =7.7 Hz), 6.85-6.74 (q, 1H, J = 6.9 Hz), 5.25 (d, 1H, J = 8.1 Hz), 4.95 (s, 2H), 4.20-
4.10 (m, 1H), 3.10-2.60 (m, 3H).
Benzyl (R)-(1-(1-(quinolin-5-yl)-1H-1,2,3-triazol-4-yl)-3-(2,4,5-trifluorophenyl)propan-2-yl)carbamate
(Vc)
82 %; 1H-NMR (300 MHz, CDCl3): δ = 9.01 (dd, J = 4.2, 1.6, 1H), 8.30 (d, J = 8.5, 1H), 8.06 (d, J = 8.6, 1H),
7.82 (t, J = 7.5, 1H), 7.73 (s, 1H), 7.58 (d, J = 7.4, 1H), 7.45 (dd, J = 8.7, 4.2, 1H), 7.25 (m, 5H), 7.14 (m, 1H),
6.89 (m, 1H), 5.47 (d, J = 8.6, 1H), 5.03 (m, 2H), 4.28 (m, 1H), 3.21-2.87 (m, 4H).
Benzyl (R)-(1-(1-(quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(2,4,5-trifluorophenyl)propan-2-yl)carbamate
Vd)
80 %;1H-NMR (300 MHz, CDCl3): δ = 8.99 (dd, J = 4.2, 1.6, 1H), 8.24 (m, 2H), 8.14 (d, J = 2.2, 1H), 8.04 (dd,
J = 9.1, 2.3, 1H), 7.95 (s, 1H), 7.51 (dd, J = 8.3, 4.2, 1H), 7.27 (m, 5H), 7.13 (m, 1H), 6.89 (m, 1H), 5.46 (d, J =
8.7, 1H), 5.04 (m, 2H), 4.27 (m, 1H), 3.17-2.84 (m, 4H).
4.4.5. (R)-1-(1-R2-1H-1,2,3-triazol-4-yl)-3-(2,4,5-trifluorophenyl)propan-2-amine (S7~S10)
To a solution of 1,2,3-triazole intermediates (1 mmol) in AcOH (10 mL) was added HBr solution (3 mL). The
reaction was stirred at 80 ℃ for overnight. The solution was extracted by ethyl acetate, washed with
saturated NaHCO3, dried with anhydrous MgSO4 and evaporated. The residue was purified by column
chromatography to give 1,2,3-triazole final products as solid (65 % - 85 %).
(R)-1-(1-(Pyridin-3-yl)-1H-1,2,3-triazol-4-yl)-3-(2,4,5-trifluorophenyl)propan-2-amine (S7)
65 %; 1H-NMR (400 MHz, CDCl3): δ = 9.0 (d, J = 2.4, 1H), 8.70 (dd, J = 4.9, 1.5, 1H), 8.15 (m, 1H), 7.92 (s,
1H), 7.50 (dd, J = 7.9, 4.4, 1H), 7.13 (m, 1H), 6.93 (m, 1H), 3.48 (m, 1H), 3.02 (dd, J = 14.7, 4.4, 1H), 2.88 (dd,
J = 13.8, 5.3, 1H), 2.80 (dd, J = 14.7, 8.2, 1H), 2.68 (dd, J = 13.7, 8.0, 1H). 13C-NMR (100 MHz, CDCl3): δ =
156.1 (m, J = 2.4, 1C), 149.9, 148.9 (m, J = 2.2, 1C), 146.5 (m, J = 2.2, 1C), 146.3, 141.4, 133.7, 128.0, 124.3,

25
122.1 (m, 1C), 120.1, 119.1 (m, 1C), 105.5 (m, 1C), 51.4, 36.8, 33.4; Pale yellow solid: M.P. 106-107 °C; [α]D
-4.5° (c 0.1, CHCl3); ESIMS: m/z 334.1 [M+H]+.
(R)-1-(1-(Pyridin-4-yl)-1H-1,2,3-triazol-4-yl)-3-(2,4,5-trifluorophenyl)propan-2-amine (S8)
74 %; 1H NMR (300 MHz, CDCl3): δ 8.75 (dd, 2H, J = 3.1and 1.6 Hz), 8.01 (s, 1H), 7.76 (dd, 2H, J = 3.1 and
1.6 Hz), 7.15-7.05 (m,1H), 6.98-6.85 (m,1H), 3.52-3.45 (m,1H), 3.10-2.95 (m,1H), 2.95-2.78 (m, 2H), 2.62-2. 58
(m,1H). 1.5 (s, 2H); 13C NMR (100 MHz, CDCl3): δ 157.2, 155.6, 152.0, 151.5, 148.0, 146.8, 145.5, 143.0, 122.6,
119.9, 119.3, 113.9, 105.8, 51.6, 37.1, 33.7; Colorless solid: M.P. 82-83 °C; [α]D25 -5.4° (c 0.25, CHCl3); ESIMS:
m/z 356.2 [M+Na]+.
(R)-1-(1-(Quinolin-5-yl)-1H-1,2,3-triazol-4-yl)-3-(2,4,5-trifluorophenyl)propan-2-amine (S9)
80 %; 1H-NMR (400 MHz, CDCl3): δ = 9.01 (d, J = 4.1, 1H), 8.30 (d, J = 8.5, 1H), 8.13 (d, J = 8.6, 1H), 7.84
(s, 1H), 7.81 (d, J = 7.7, 1H), 7.65 (d, J = 7.4, 1H), 7.49 (dd, J = 8.6, 4.2 1H), 7.19 (m, 1H), 6.93 (m, 1H),
3.63-3.57 (m, 3H), 3.09 (dd, J = 14.7, 4.4, 1H), 2.94 (m, 2H), 2.79 (dd, J = 13.7, 7.6, 1H); 13C-NMR (100 MHz,
CDCl3): δ = 156.6, 151.8, 149.3, 148.8, 146.9, 145.2, 133.8, 132.1, 131.9, 128.9, 125.1, 124.2, 123.8, 123.0,
122.2, 119.6, 105.9, 51.8, 36.4, 33.0; Pale yellow solid: M.P. 105-106 °C; [α]D25 -3.4° (c 0.1, CHCl3); ESIMS:m/z
383.8 [M+H]+.
(R)-1-(1-(Quinolin-6-yl)-1H-1,2,3-triazol-4-yl)-3-(2,4,5-trifluorophenyl)propan-2-amine (S10)
85 %; 1H-NMR (400 MHz, CDCl3): δ = 8.98 (dd, J=4.2, 1.6 1H), 8.27 (d, J = 8.8, 2H), 8.21 (d, J = 2.3, 1H),
8.12 (dd, J = 9.1, 2.4 1H), 8.09 (s, 1H), 7.52 (dd, J = 8.2, 4.2, 1H), 7.18 (m 1H), 6.94 (m, 1H), 3.58 (m, 1H),
3.07 (dd, J = 14.8, 4.4, 1H), 2.98 (s, 2H), 2.91 (dd, J = 14.8, 7.8, 1H), 2.80 (dd, J = 13.6, 7.5, 1H); 13C-NMR
(100 MHz, CDCl3): δ = 156.6, 151.6, 149.4, 147.7, 146.1, 145.7, 137.0, 135.1, 131.8, 128.7, 122.9, 122.6, 122.0,
25
121.2, 119.7, 118.5, 106.0, 51.6, 36.0, 32.5; Yellow solid: M.P. 116-117 °C; [α ]D -1.9° (c 0.1, MeOH);
ESIMS:m/z 383.9 [M+H]+.
Acknowledgements
This work was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of
Education, Science and Technology (NRF-2016-R1D1A1B03934926: Development of Antidiabetic Agents
with DPP-4 Inhibitory Activity). The authors thank to Drug Development Research Institute and Central
Laboratory of Kangwon National University for the use of analytical instruments and bioassay facilities.
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