Design, synthesis and biological evaluation of glycolamide, glycinamide, and β-amino carbonyl 1,2,4-triazole derivatives as DPP-4 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2021.105049

Through modification of the skeleton of Sitagliptin and Vildagliptin, we successfully synthesized and built-up four series of 1,2,4-triazole derivatives, containing N,O-disubstituted glycolamide, N,N′-disubstituted glycinamide, β-amino ester, and β-amino

Full text of DOI:10.1016/j.bioorg.2021.105049

Bioorganic Chemistry 114 (2021) 105049
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of glycolamide, glycinamide,
and β-amino carbonyl 1,2,4-triazole derivatives as DPP-4 inhibitors
,
,
c
,
,
Mao-Tsu Fuha 1, Ching-Chun Tsengb 1, Sin-Min Lid, Shuo-En Tsaib c, Tsung-Jui Chuange,
Chih-Hao Luf, Ya-Chen Yangg, Henry J. Tsaig, Fung Fuh Wongb
,
*
a Division of Metabolism, Department of Internal Medicine, China Medical University, Taichung 40402, Taiwan
b School of Pharmacy, China Medical University, No. 91, Hsueh-Shih Rd., Taichung 40402, Taiwan
c Ph.D. Program for Biotech Pharmaceutical Industry, China Medical University, No. 91, Hsueh-Shih Rd., Taichung 40402, Taiwan
d Institute of New Drug Development, China Medical University, No. 91 Hsueh-Shih Rd., Taichung 40402, Taiwan
e Master Program for Pharmaceutical Manufacture, China Medical University, No. 91, Hsueh-Shih Rd., Taichung 40402, Taiwan
f Graduate Institute of Basic Medical Science, China Medical University, Taichung 40402, Taiwan
g Department of Food Nutrition and Health Biotechnology, Asia University, Taichung 41354, Taiwan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Through modification of the skeleton of Sitagliptin and Vildagliptin, we successfully synthesized and built-up
four series of 1,2,4-triazole derivatives, containing N,O-disubstituted glycolamide, N,N′-disubstituted glycina-
mide, β-amino ester, and β-amino amide as linkers, for the development of new dipeptidyl peptidase 4 (DPP-4)
inhibitors. The synthetic strategy for glycolamides or glycinamides involved convenient two-steps reaction:
functionalized transformation of 2-chloro-N-(2,4,5-triflurophenyl)acetamide 9 (hydroxylation or amination) and
esterification or amidation of 1,2,4-triazole-3-carboxylic acid. On the other hand, the one-pot synthesis pro-
cedure, including substitution and deprotection, was developed for the preparation of β-amino carbonyl 1,2,4-
triazoles from (1H-1,2,4-triazol-3-yl)methanol 12 or (1H-1,2,4-triazol-3-yl)methanamine 13 and Boc-(R)-3-
amino-4-(2,4,5-trifluoro-phenyl)-butyric acid 14. All of glycolamides, glycinamides, and β-amino carbonyl 1,2,4-
triazoles were also evaluated against DPP-4 inhibitory activity. Based on the SAR study of DPP-4 inhibitory
capacity, β-amino ester 5n and β-amino amide 1,2,4-triazoles 6d and 6p possessed the significant inhibition of
DPP-4 (IC50 < 51.0 nM), particularly for compound 6d (IC50 = 34.4 nM). The selectivity evaluation indicated
compound 5n and 6p had excellent selectivity over QPP, DPP-8, and DPP-9. In addition, the docking results
1,2,4-Triazole
DPP-4 inhibitors
Glycolamides
Glycinamide
β-amino carbonyl
revealed compounds 5n and 6p provided stronger π–π stacking interaction with residue Phe357 than 1,5-disub-
stituted 1,2,4-triazole 6d and Sitagliptin 1. In summary, compounds 5n and 6p could be promising lead com-
pounds for further development of DPP-4 inhibitor.
1. Introduction
inhibitors have been gained attention due to the well-tolerated and
minimal side effects.
Dipeptidyl peptidase IV (DPP-4) inhibitors are a new type of oral
hypoglycemic drugs that inhibit the enzyme DPP-4 for treating patients
with type 2 diabetes [1–4]. DPP-4 is an abundant enzyme existing in most
cell types. It can deactivate glucagon-like peptide 1 (GLP-1) by cleaving
peptide bonds [5]. The GLP-1 has multiple physiological functions,
including insulin stimulation, glucagon inhibition, and delayed gastric
emptying [6]. Inhibition of DPP-4 can elevate the concentration of active
GLP-1 to increase insulin secretion and reduction in HbAlc without
weight gain and have a low risk of hypoglycemia [7–11]. Hence, DPP-4
Sitagliptin 1 is the first marketed selective inhibitor of DPP-4 [12],
followed by Vildagliptin, Saxagliptin, Linagliptin, and Alogliptin [13,14].
The chemical structure of Sitagliptin 1 is divided into three parts: 2,4,5-
triflorophenyl fragment pharmacophore, enantiomerically pure β-amino
carbonyl linker, and the 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyri-
dine fragment (Fig. 1) [15]. Although numerous literatures on the syn-
thesis of Sitagliptin have appeared [16–18], the enantioselective
enamine reduction via efficient catalytic route was widely utilized to
generate the pure β-amino carbonyl linker in Sitagliptin scaffold [19].
* Corresponding author.
E-mail address: ffwong@mail.cmu.edu.tw (F.F. Wong).
1
The authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.105049
Received 23 February 2021; Received in revised form 11 May 2021; Accepted 26 May 2021
Available online 31 May 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

M.-T. Fuh et al.
Bioorganic Chemistry 114 (2021) 105049
However, this method is an expensive and non-green procedure due to
using the metal reagent-based catalysts. Herein, we revaluated that the
β-amino carbonyl linker necessitated a simplified modification.
presented in Table 1. Based on the inhibitory activities against DPP-4,
1,3-disubstituted 1,2,4-triazoles 7a–e and 1,3,5-trisubstituted 1,2,4-
triazoles 7l–q were found possessed better inhibition rates (DPP-4
Inh@1mM ≧35%). Therefore, compounds 7a–e and 7l–q were further
selected as promising targets for synthesis of N,O-disubstituted gly-
colamides 3, N,N-disubstituted glycinamides 4, and β-amino carbonyl
1,2,4-triazoles 5 and 6.
Sitagliptin 1 is structurally a non-substrate-like DPP-4 inhibitor,
which is non-peptide mimetic compound that occupies the hydrophobic
pocket of the enzyme binding site via the trifluorophenyl moiety. On the
other hand, Vildagliptin 2 is a substrate-like DPP-4 inhibitor with a
cyanopyrrolidine ring that mimic DPP-4 substrates to occupy the same
hydrophobic pocket of the enzyme binding site [20]. Despite the diverse
chemical structures of DPP-4 inhibitors, the β-amino carbonyl linker of
Sitagliptin 1 and glycinamide linker of Vildagliptin 2 have common
interactions with key residues that play an important role in the inhib-
itory activity through hydrogen bonds [20,21]. Based on the different
structures but similar binding sites results of Sitagliptin 1 and Vilda-
gliptin 2, we assumed that the linker structure and length of DPP-4 in-
hibitors might be a key factor in the binding mode. Moreover, functional
glycinamide linkers also play a crucial role in the investigation of bio-
logical processes in modern pharmacology, biochemistry, and medicinal
chemistry [22,23]. Thus, we designed four series of compounds 3–6
with different linkers, including glycolamide, glycinamide, β-amino
amide, and β-amino ester groups in a design of functional 1,2,4-triazole
modules in which 2,4,5-triflorophenyl fragment was maintained into the
architecture for modulating the heterocyclic moiety to structural vari-
ation (Fig. 2). Based on the considerable attracted academic interest,
these designed 1,2,4-triazole compounds with various linkers were
evaluated DPP-4 inhibitory activity compared with Sitagliptin 1 [24].
The selectivity of DPP-4 over QPP, DPP-8, and DPP-9 for each compound
were also determined by the IC50 values in a plaque assay to discovery
novel DPP-4 inhibitors with increasing safety and durability for the
treatment of T2DM.
2.2. Chemistry
2.2.1. Synthesis of N,O-disubstituted glycolamides 3 and N,N-disubstituted
glycinamides 4
Because the triazolopiperazine moiety of Sitagliptin 1 was recog-
nized as an important pharmacophore that contributed to its good
pharmacokinetic profile, potency, and selectivity [12,27], a new series
of heterocycle moieties were investigated. Preliminarily, we focused
on the evaluation of the following variations on 1,2,4-triazole struc-
ture. As outlined retrosynthetically in Scheme 1, both product targets
including 1,2,4-triazole moiety N,O-disubstituted glycolamides 3 and
N,N-disubstituted glycinamides 4 could be accessed from key in-
termediates N-(2,4,5-trifluorophenyl)-2-hydroxyacetamide 10 or N-
(2,4,5-trifluorophenyl)-2-aminoacetamide 11 treated with 1H-1,2,4-
triazole-3-carboxylic acids 8 [28], which were hydrolyzed from
methyl 1H-1,2,4-triazole-3-carboxylates 7 [26]. Compounds 10 and
11 were obtained from their
α-chloro N-arylacetamide precursor 9
through hydrolysis with cesium formate [29] or amination with
aqueous ammonium hydroxide [30]. α-Chloro N-arylacetamide 9 was
prepared from 2,4,5-trifluorobenzenamine reacted towards 2-chloroa-
cetyl chloride via our previous published procedure [31].
With an effective synthesis of intermediates 2-hydroxyacetamide 10
and 2-aminoacetamide 11, we examined the two-steps synthesies
involving amidation and hydrolysis or amination from 2,4,5-trifluoroben-
zenamine (Scheme 2). At the outset, the reaction of 2,4,5-trifluorobenzen-
amine and 2-chloroacetyl chloride proceeded successfully in refluxing
2. Result and discussion
2.1. Design of N,O-disubstituted glycolamides 3, N,N-disubstituted
glycinamides 4, and β-amino carbonyl 1,2,4-triazoles 5 and 6
toluene for 3 h to give α-chloro N-arylacetamide 9 in 92% high yield [31].
Subsequently, the hydrolysis of N-arylacetamide 9 which was performed
at reflux for 6 h on treatment with 3.0 equivalents of cesium formate
(HCO2Cs) [29] in EtOH solution to afford 2-hydroxyacetamide 10 effi-
ciently in 86% yield. We then evaluated the scope of the amination using
N-arylacetamide 9 at 60 ◦C for 3 h under the EtOH/aqueous ammonium
hydroxide condition [30]. The desired 2-aminoacetamide product 11 was
obtained in 88% yield (Scheme 2). Subsequently, a series of methyl 1H-
1,2,4-triazole-3-carboxylates 7 were prepared by our reported literature
procedures [26] and hydrolyzed in presence of DBU/MeOH at room
temperature for 6 h to afford the corresponding 1H-1,2,4-triazole-3-car-
boxylic acids 8 in > 90% high yields [28]. Crude compounds 8 were
extracted with CH2Cl2 and washed with 10% HCl(aq) without purification
technique for further investigations.
To investigate the binding structural mode of Sitagliptin 1 and Vil-
dagliptin 2 to DPP-4, the advanced docking program iGEMDOCK v2.1
was used to simulate the binding domain between inhibitors and DPP-4
enzyme [25]. The design approach was followed the rational Sitagliptin
1 and Vildagliptin 2 and carried out by structural optimization to pursue
high in vitro activity, high selectivity and low cytotoxicity. To earn these
benefits, we calculated the length of the linker and the binding mode of
the kinase domain of DPP-4 to generate the new designed structures of
N,O-disubstituted glycolamides 3, N,N-disubstituted glycinamides 4,
and β-amino carbonyl 1,2,4-triazoles 5 and 6 grafting 2,4,5-trifloro-
phenyl fragment (Fig. 2). The linker length is ranged between 4.6 Å
and 8.5 Å from the standard drugs Sitagliptin 1 and Vildagliptin 2.
Glycolamides 3 and glycinamides 4 were divided into two frag-
ments, N-(2,4,5-trifluorophenyl)-2-hydroxyacetamide or N-(2,4,5-
trifluorophenyl)-2-aminoacetamide and 1,2,4-triazole heterocyclic
moiety 7 (Scheme 1 and Fig. 2). At first, both 1,3-disubstituted 1,2,4-
triazoles 7a–g [26a] and 1,3,5-trisubstituted 1,2,4-triazoles 7h–q
[26b] were synthesized by following our previous published literature
procedure [26]. Their preliminary DPP-4 inhibition data were
To evaluate the activated forms of carboxylic acid in optimization study
[32,33], compound 8a (1.0 equiv) and 2-hydroxyacetamide 10 (1.1 equiv)
was reacted in the presence of activating reagents (2.0 equiv), such as acid
halides (Oxalyl chloride (COCl)2 and thionyl chloride (SOCl2)) [34], acti-
vated amides (1,1′-carbonyldiimidazole, CDI) [35], activeesters (car
bodiimides: N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochl
oride (EDC) [36], and dicyclohexylcarbodiimide (DCC)) [37], and re
Fig. 1. The structure of Sitagliptin 1.
2

M.-T. Fuh et al.
Bioorganic Chemistry 114 (2021) 105049
Fig. 2. The new designed structures of N,O-disubstituted glycolamides 3, N,N-disubstituted glycinamides 4, and β-amino carbonyl 1,2,4-triazoles 5 and 6 in
comparison with Sitagliptin 1 and Vildagliptin 2.
Scheme 1. The retrosynthesis route of N,O-disubstituted glycolamide 1,2,4-triazoles 3 and N,N-disubstituted glycinamide 1,2,4-triazoles 4.
active phosphate agents (benzotriazol-1-yl-oxytripyrrolidinophosphonium
hexafluorophosphate (PyBOP) [38], and N,N-bis[2-oxo-3-oxazolidinyl]
phosphorodiamide chloride (BOP-Cl)) [39] in anhydrous THF or CH2Cl2
solution under basic condition at 0 ◦C to room temperature for 6 h [33].
However, most of these reactions afforded N,O-disubstituted glycolamides
product 3a in moderate yields (<72%). Remarkably, significant and
successful reaction was allowed to stir with BOP-Cl activating reagent (2.0
equiv) and 2.0 equiv of NEt3, and the yield of product 3a was significantly
increased to 85% good yield. Since, N,N-bis[2-oxo-3-oxazolidinyl]phos-
phorodiamide chloride (BOP-Cl) could be served as the best convenient and
efficient activating carboxyl groups reagent that has been employed in the
further esterification and amidation transformation to prepare N,O-
3

M.-T. Fuh et al.
Bioorganic Chemistry 114 (2021) 105049
reactivity with the activated acid 8 than 2-hydroxyacetamide 10. All
structures of N,O-disubstituted glycolamides 3 and N,N-disubstituted
glycinamides 4 were fully characterized by spectroscopic methods, for
Table 1
The inhibitory activity results against DPP-4 of 1,3-disubstituted 1,2,4-triazoles
example, compound 4m presents one doublet peak at δ 4.39 ppm of
1
– –
α
-carbon methylene( CH2 ) in H NMR and characteristic absorption
7a–g and 1,3,5-trisubstituted 1,2,4-triazoles 7h–q.
13
–
–
CH2 , at δ 167.3 ppm for
at δ 43.9 ppm for methylene carbon
13
–
–
–
–
glycinamide carbon N
(
C
O) CH2, and at δ 161.9 ppm for amide
13
–
–
–
(
carbon N
C
O) in the 13C NMR spectrum. Glycinamide 4m was also
Compounds No.
Y
Ar
DPP-4% Inh@1mMa
further characterized by X-rays crystallographic analysis (as the single-
crystal X-ray diffraction study, ORTEP) and shown in Fig. 3.
7a
7b
7c
7d
7e
7f
H
Ph
33.9
37.0
34.6
37.8
40.1
32.3
10.1
19.2
1.01
4.13
24.4
43.7
85.1
94.5
100
H
p-Me-Ph
p-Cl-Ph
p-CF3-Ph
m-CF3-Ph
o-CF3-Ph
p-F-Ph
H
2.2.2. Synthesis of β-amino carbonyl 1,2,4-triazoles 5 and 6
H
The enantiomerically β-amino carbonyl linker intensively plays an
important role in examining the designed structure. Therefore, this
result drove us to synthesize β-amino carbonyl Sitagliptin analogues
containing 1,2,4-triazole moiety 5 and 6 (Scheme 3 and 4). Based on the
preliminary DPP-4 inhibition data of substituted 1,2,4-triazole-3-car-
boxylates in Table 1, compounds 7d (DPP-4 Inh@1mM 37.8%) and
7m–q with good inhibition rates (DPP-4 Inh@1mM ≧85%) were
selected as promising targets. Initially, we chose 1,2,4-triazole-3-carbox-
ylate 7d (1.0 equiv) as the model substrates to carry out the reduction
reaction with NaBH4 (1.5 equiv) in THF/MeOH at room temperature for
5 h. The desired 1H-1,2,4-triazol-3-ylmethanol product 12d was ob-
tained in 91% yield (Scheme 3) [40]. The subsequent chlorination was
performed using thionylchloride (SOCl2, 1.5 equiv) to yield the chloried
intermediate [41]. Without further purification, the intermediate 3-
chloromethyl-1,2,4-triazole was treated with NH4OH(aq) in EtOH solu-
tion and smoothly converted to the amination (1H-1,2,4-triazol-3-yl)
methanamine product 13d in 73% yield for two steps (Scheme 3) [42].
Finally, β-amino carbonyl targets 5d and 6d were individually syn-
thesized from Boc-(R)-3-amino-4-(2,4,5-trifluoro-phenyl)-butyric acid
14 with (1H-1,2,4-triazol-3-yl)methanol 12d or (1H-1,2,4-triazol-3-yl)
methanamine 13d by employing N,N-bis[2-oxo-3-oxazolidinyl]phos-
phorodiamide chloride (BOP-Cl, 2.0 equiv) as activating agent in pres-
ence of 2.0 equiv of NEt3 in CH2Cl2 solution to afford the Boc-protected
intermediates 15d and 16d (Scheme 4) [39]. In this key substitution
H
H
7g
7h
7i
H
Me
p-Cl-Ph
p-Br-Ph
p-Cl-Ph
p-Cl-Ph
p-Cl-Ph
p-Cl-Ph
m-CF3-Ph
p-Cl-Ph
Ph
Me
7j
Et
7k
7l
i-Pr
Furanyl
Cyclopentyl
Pyrrolyl
n-Bu
Ph
7m
7n
7o
7p
7q
100
Ph
p-Me-Ph
100
a
Concentration of each compound is 1.0 mmol Lꢀ 1 in DMSO.
disubstituted glycolamides 3 and N,N-disubstituted glycinamides 4.
Following the above activated carboxylic acid optimization study,
the 1,2,4-triazole-3-carboxylates 7a–e and 7l–q were preliminarily
converted to the corresponding 1,2,4-triazole-3-carboxylic acids 8a–e
and 8l–q. Without further purification, compounds 8 were directly
reacted with 2-hydroxyacetamide 10 or 2-aminoacetamide 11 and 2.0
equiv of BOP-Cl in presence of 2.0 equiv of NEt3 to give the corre-
sponding glycolamides 3a–e and 3m and glycinamides products 4a–e
and 4l–q in moderate to good yields (71–88% for 3 and 72–92% for 4,
Table 2). In general, 2-aminoacetamide 11 possessed better substitution
Scheme 2. Synthesis of N,O-disubstituted glycolamides 3 and N,N-disubstituted glycinamides 4. Reagents and conditions: (a) DBU, MeOH, r.t., 6 h, > 90%; (b) 2-
chloroacetyl chloride, toluene, reflux, 3 h, 92%; (c) For 10: cesium formate, EtOH, reflux, 6 h, 86%; (d) For 11: NH4OH, EtOH, 40 ◦C, 4 h, 88%; (e) BOP-Cl, NEt3,
CH2Cl2, 4 h, 71–92%.
4

M.-T. Fuh et al.
Bioorganic Chemistry 114 (2021) 105049
CH2Cl2 acidic solution to obtain β-amino ester product 5d and β-amino
amide product 6d in 76% and 79% yields for two steps, respectively
(Scheme 4 and Table 3) [43]. On the other hand, Boc-protected β-amino
carbonyl product 15d was tried to isolate for further identification of
structural skeleton (Scheme 4). Compound 15d presents two singlet
peaks at δ 1.33 ppm for the tert-Boc group and δ 8.62 ppm for the 1,2,4-
Table 2
The results of N,O-disubstituted glycolamide 1,2,4-triazoles 3a–q and N,N-
disubstituted glycinamide 1,2,4-triazoles
4a–q.
–
–
–
–
triazolic ring (N CH
N N) and one doublet peak δ 5.32 ppm of the
–
ester (O CH2 ) in 1H NMR. In 13C NMR spectrum, compound 15d
– –
possessed characteristic absorptions at δ 170.8 ppm for 13
C
O, at δ 59.2
–
–
Compounds No.
X
Y
Ar
Yields (%)a
13
–
–
ppm for O
CH2 , and at δ 28.2 and 79.5 ppm for tert-Boc group
13C(13CH3)3.
–
O
3a
4a
3b
4b
3c
4c
3d
4d
3e
4e
4l
O
H
Ph
85
87
87
92
88
89
83
84
71
72
83
73
74
73
82
82
84
NH
O
Employing the above reliable procedure for synthesis of 12d and
H
H
H
H
p-Me-Ph
p-Cl-Ph
13d, we converted compounds 7m–q to 1H-1,2,4-triazol-3-ylmetha-
nols 12m–q in 92–96% yields and 1H-1,2,4-triazol-3-ylmethanamines
13m–q in 76–84% yields (Scheme 3) [40–42]. Subsequently, com-
pound 14 was directly reacted with 1H-1,2,4-triazol-3-ylmethanols
12m–q or 1H-1,2,4-triazol-3-ylmethanamines 13m–q to carried out
the substitution reaction for generation of intermediates 15m–q and
16m–q [39]. Without further purification, the deprotection of Boc-
protected group of intermediates 15m–q and 16m–q was carried out
in TFA/CH2Cl2 acidic solution [43]. The β-amino ester products 5m–q
were provided from 74% to 84% yields and β-amino amide products
6m–q were obtained from 74% to 88% yields in two steps, respectively
(Table 3 and Scheme 4).
NH
O
NH
O
p-CF3-Ph
m-CF3-Ph
NH
O
NH
NH
O
Furanyl
p-Cl-Ph
p-Cl-Ph
3m
4m
4n
4o
4p
4q
Cyclopentyl
NH
NH
NH
NH
NH
Pyrrolyl
n-Bu
Ph
m-CF3-Ph
p-Cl-Ph
Ph
Ph
p-Me-Ph
a
The calculated isolation yield was based on the amount of starting materials
2-hydroxyacetamide 10 or 2-aminoacetamide 11.
2.3. Biological activity
2.3.1. Pharmacological evaluation in vitro DPP-4 inhibition studies and SAR
analysis of N,O-disubstituted glycolamides 3, N,N-disubstituted glycinamides
4, and β-amino carbonyl 1,2,4-triazoles 5 and 6
reaction, we found that the amino group of compound 13d was more
nucleophilic reactivity than the hydroxyl group of 12d. Without further
purification, the one-pot synthesis procedure was proceeded to prepare
β-amino carbonyl target compounds 5d and 6d. The deprotection of Boc-
protected group of intermediates 15d and 16d was carried out in TFA/
Screening of glycolamides 3a–e and glycinamides 4a–e with 1,3-
disubstituted 1,2,4-triazole moiety were evaluated in vitro against a
panel of standard DPP-4. The inhibitory activity was executed using the
Fig. 3. The ORTEP diagram of 1-(4-chlorophenyl)-5-cyclopentyl-N-(2-oxo-2((2,4,5-trifluorophenyl)amino)ethyl)-1H-1,2,4-triazole-3-carboxamide 4m (CCDC
No. 1973637).
Scheme 3. Synthesis of (1H-1,2,4-triazol-3-yl)methanols 12d, 12m–q and (1H-1,2,4-triazol-3-yl)methanamine 13d, 13m–q. Reagents and conditions: (a) NaBH4,
THF, MeOH, r.t., 4 h, 91–96%; (b) SOCl2, CH2Cl2, r.t., 2 h; (c) NH4OH, EtOH, 30 ◦C, 6 h, 73–84%.
5

M.-T. Fuh et al.
Bioorganic Chemistry 114 (2021) 105049
Scheme 4. Synthesis of β-amino carbonyl target compounds containing 1,2,4-triazole analogues 5d, 5m–q and 6d, 6m–q. Reagents and conditions: (a) BOP-Cl, NEt3,
CH2Cl2, r.t., 4 h; (b) CF3COOH, CH2Cl2, r.t., 3 h, 74–88%.
glycolamides 3 and glycinamides 4 were unfavorable linkers related to
Table 3
the β-amino carbonyl of Sitagliptin 1. To sum up, we observed that the
The synthesis results of β-amino carbonyl 1,2,4-triazoles 5d, 5m–q and 6d,
enantiomerically β-amino carbonyl linker seems to intensively play an
important role. Therefore, the results drove us to the discovery of
β-amino carbonyl 1,2,4-triazoles 5 and 6.
For further optimization, introducing β-amino carbonyl as a linker
became a primary goal. Glycolamide 3d and glycinamide 4d possessed
better inhibitory activity in Table 4. Thus, β-amino carbonyl 1,3-disub-
stituteds 1,2,4-triazoles 5d and 6d were selected to optimize linkage
property for evaluating DPP-4 inhibitory capacity. Fortunately, β-amino
amide 6d showed superior inhibitory activity (IC50 = 34.4 nM, Table 5).
On the other hand, no significant improvement was observed for
β-amino ester 5d (IC50 = 775 nM, Table 5). For further demonstration of
bioactivity potency, two series of compounds including β-amino esters
5m–q and β-amino amides 6m–q were synthesized in parallel to modify
the linker and 1,3,5-trisubstituted 1,2,4-triazoles (Table 5). Most of
compounds 5m–q and 6m–q showed the moderate inhibitory activity
and their IC50 values were ranging from 49.9 nM to 497 nM (Table 5).
However, these results could not provide us conclusive potential
inhibitory tendency order between β-amino esters 5m–q and β-amino
amides 6m–q (Table 5). To sum up, compounds 5n and 6p also
exhibited the significant potent anti-DPP-4 activity with IC50 value of
49.9 nM and 50.4 nM, respectively (Table 5).
6m–q.
Compounds No.
X
Y
Ar
Yields (%)a
5d
6d
5m
6m
5n
6n
5o
6o
5p
6p
5q
6q
O
H
p-CF3-Ph
76
79
79
83
74
74
78
81
83
88
84
86
NH
O
Cyclopentyl
Pyrrolyl
n-Bu
p-Cl-Ph
m-CF3-Ph
p-Cl-Ph
Ph
NH
O
NH
O
NH
O
Ph
NH
O
Ph
p-Me-Ph
NH
a
The calculated isolation yield was based on the amount of reactant Boc-(R)-
3-amino-4-(2,4,5-trifluoro-phenyl)-butyric acid 14.
2.3.2. Selectivity DPP-4 over DPP-7, DPP-8, and DPP-9 of β-amino ester
5n and β-amino amides 6d and 6p
disc diffusion method and the measurement of the zone of growth in-
hibition for a concentration 1.0 mmol/L (DMSO) for each glycolamides
3a–e and glycinamides products 4a–e. Sitagliptin 1 was used as com-
parison model for the inhibitory activity study (100%, Table 4). Note-
worthily, compounds 3d and 4d with p-CF3Ph substituents on N-1
position of triazole ring possessed better inhibitory activity against DPP-
4 (57.2% and 58.2%, Table 4). Moreover, the inhibitory activity ten-
dency of DPP 4 was observed that most of glycinamides 4a–e presented
better inhibitory activity than glycolamides 3a–e. Based on this result,
glycinamides 4l–q with 1,3,5-trisubstituted 1,2,4-triazole moiety
bearing n-butyl, cyclopentyl, furanyl, and pyrrolyl on C-5 position and p-
Cl-Ph, m-CF3-Ph, Phenyl, and p-Me-Ph on N-1 position of triazole ring
were significantly synthesized for further investigation. However, the
poor inhibitory data was obtained from glycinamides 4l–q (≤48.8%,
Table 4). The unsatisfactory inhibitory results led to the conclusion that
The DPP-4 activity and/or structural homologue (DASH) family,
including quiescent cell proline dipeptidase (QPP, also known as DPP-2
or DPP-7), DPP-8, DPP-9, and FAP [45]. As reported in previous study,
the DPP-4 inhibitors containing β-amino acid fragment in the structure
resulted in the QPP off-target activity [12,46]. In addition, selective
inhibition of DPP-8/DPP-9 was associated with toxicity, such as alope-
cia, thrombocytopenia, anemia, and animal mortality [47]. Therefore,
selectivity against QPP, DPP-8, DPP-9 was important for the safety of
DPP-4 inhibitors. Based on the potency of DPP-4 inhibition, we selected
β-amino ester 5n and β-amino amides 6d and 6p (IC50 < 51 nM) to
evaluate selectivity across enzymes QPP, DPP-8, and DPP-9. As shown in
Table 6, both β-amino carbonyl 1,3,5-trisubstituted 1,2,4-triazoles 5n
and 6p showed excellent selectivity over QPP, DPP-8, and DPP-9
(>1950-fold), however, the selectivity of 1,5-disubstituted 1,2,4-tri-
azole 6d was lower (135-, 31-, and 105-fold selectivity over QPP, DPP-8,
6

M.-T. Fuh et al.
Bioorganic Chemistry 114 (2021) 105049
Table 4
The DPP-4 inhibitory activity results of N,O-disubstituted glycolamide 1,2,4-triazoles 3, N,N-disubstituted glycinamide 1,2,4-triazoles 4, and β-amino carbonyls 5 and
6.
Compounds No.
X
Y
Ar
DPP-4% Inh@1mMa
3a
4a
O
H
43.4
37.6
NH
3b
4b
O
H
H
H
H
28.4
34.1
NH
3c
4c
O
39.4
47.2
NH
3d
4d
O
57.2
58.2
NH
3e
4e
O
37.0
42.5
NH
4l
NH
33.5
3m
4m
OH
NH
39.3
48.8
4n
NH
34.0
4o
4p
4q
NH
NH
NH
36.6
32.5
39.2
100
Sitagliptin 1
a
Concentration of each used compound is 1.0 mmol L¡1 (DMSO).
and DPP-9, respectively). This result indicated that an aryl group
appeared to be essential for the 5-position of 1,2,4-triazole. Importantly,
the selectivity over DPP8 of compounds 5n and 6d (>1984-fold) was
better than Sitagliptin (1, 787-fold), making compounds 5n and 6d
suitable for further study.
the RMSD was calculated by using LigRMSD [48]. Sitagliptin 1 docked
in the DPP-4 binding site almost at the same position (RMSD = 0.53 Å)
as the co-crystallized data (PDB ID 1X70), which validated the iGEM-
DOCK protocol. The DPP-4 enzyme was docked with compounds 5n, 6d,
and 6p in Sitagliptin 1 binding site. As depicted in Fig. 4, the docking
model revealed that compounds 5n, 6d, and 6p were stabilized at DPP-4
active site similarly to Sitagliptin 1 through three hydrogen bond in-
2.4. Docking study of β-amino carbonyl target 5n, 6d, and 6p into the
teractions with residues Tyr662, Glu205, and Glu206, and two
π–π
active site of DPP-4
stacking interaction with residues Tyr662 and Phe357. β-amino amide
6d with superior DPP-4 inhibition achieves lower docking scores of –
150.4 kcal/mol (Table S2), because 4-(trifluoromethyl)phenyl group of
compound 6d possesses additional van der Waals interaction with res-
idue Arg358 in comparison with Sitagliptin 1 (Fig. 4A). On the other
hand, it has been reported that neither DPP-8 nor DPP-9 has a residue
corresponding to Phe357 of DPP-4 [49]. Based on the ducking result, we
found that 1,3,5-trisubstituted 1,2,4-triazoles 5n (ꢀ 26.4 kcal/mol) and
In order to gain an insight of the possible binding interactions of
β-amino ester 5n and β-amino amides 6d and 6p in the active site of
DPP-4 enzyme, molecular modelling was performed. The X-ray structure
of DPP-4 cocrystallized with Sitagliptin 1 (PDB ID 1X70, resolution 2.10
Å) was used to perform the docking study through docking program
iGEMDOCK v2.1 [12,25]. Re-docking of the co-crystallized ligand
Sitagliptin 1 into DPP-4 active site via iGEMDOCK was performed, and
7

M.-T. Fuh et al.
Bioorganic Chemistry 114 (2021) 105049
3. Conclusion
Table 5
The DPP-4 inhibitory activity results of β-amino carbonyls
5
and
We have successfully synthesized and built-up four series of 1,2,4-tri-
azole derivatives 3–6 containing N,O-disubstituted glycolamide, N,N′-
disubstituted glycinamides, β-amino ester, and β-amino amide as linkers
through modifying Sitagliptin 1 and Vildagliptin 2. Different linkers
grafted the same 2,4,5-triflorophenyl fragment into the architecture,
and 1,2,4-triazole heterocyclic moiety was modulated for structural
variation. The synthetic strategy for glycolamides or glycinamides
6.
involved functionalized transformation of
α-chloro N-arylacetamides
Compounds
No.
X
Y
Ar
DPP-4 IC50
(nM)a
(hydroxylation or amination) and esterification or amidation of 1,2,4-
triazole-3-carboxylic acid by activating with BOP-Cl reagent. We also
developed one-pot synthesis procedure, including treating (1H-1,2,4-
triazol-3-yl)methanol or (1H-1,2,4-triazol-3-yl)methylamine with Boc-
(R)-3-amino-4-(2,4,5-trifluoro-phenyl)-butyric acid and deprotection of
tert-Butyloxycarbonyl (BOC) group, for the preparation of β-amino
carbonyl 1,2,4-triazoles. For the preliminary screening in vitro, glyco-
lamides 3 and glycinamides 4 showed poor dipeptidyl peptidase 4
inhibitory activity (≦58.2% DPP-4 Inh@1mM). Most of β-amino ester 5
and β-amino amide 1,2,4-triazoles 6 possessed the moderate inhibition
of DPP 4 (IC50 < 775 nM). Especially, β-amino carbonyl 5n, 6d, and 6p
had satisfactory DPP-4 inhibitory activity (50.4, 34.4, 49.9 nM,
respectively). Docking study also revealed compounds 5n, 6d and 6p
have favorable binding mode with DPP-4 enzyme. Selectivity evaluation
indicated β-amino carbonyl 5n and 6p hold excellent selectivity over
QPP, DPP-8, and DPP-9 and superior to reference Sitagliptin. Further-
5d
6d
O
H
775 (nM)
34.4 (nM)
NH
5m
6m
O
80.3 (nM)
131 (nM)
NH
5n
6n
O
49.9 (nM)
99.8 (nM)
NH
5o
6o
O
81.6 (nM)
119 (nM)
NH
5p
6p
O
91.3 (nM)
50.4 (nM)
NH
5q
6q
O
497 (nM)
138 (nM)
NH
more, compounds 5n and 6p provided stronger π–π stacking interaction
with residue Phe357 than 1,5-disubstituted 1,2,4-triazole 6d and Sita-
gliptin 1 based on the molecular docking results. In summary, β-amino
carbonyl 5n and 6p exhibited good activity in vitro and excellent
selectively and might be a promising lead for the treatment of diabetes
mellitus.
Sitagliptin 1
28 (18–38
nM)b
a
IC50 is the concentration (nM) needed to cause 50% inhibition human re-
combinant DPP-4 enzymatic activity. Values represent the mean of double
determinations.
b
The IC50 value of Sitagliptin was between 18 and 38 nM in reported litera-
4. Experimental section
ture [12,44].
4.1. Chemistry
6p (ꢀ 23.8 kcal/mol) with additional aromatic ring on the triazole ring
provided stronger π–π stacking interaction with residue Phe357 than
All chemicals were purchased with reagent grade. Both 1,3-disubsti-
tuted 1,2,4-triazoles 7a–g [26a] and 1,3,5-trisubstituted 1,2,4-triazoles
7h–q [26b] were prepared following our reported procedure [26]. All
reactions were carried out under argon or nitrogen atmosphere and
monitored by TLC. Flash column chromatography was carried out on
1,5-disubstituted 1,2,4-triazole 6d (ꢀ 11.8 kcal/mol) and Sitagliptin 1
(ꢀ 19.4 kcal/mol, Fig. 4B and Table S3), which might explain com-
pounds 5n and 6p improved selectivity inhibition of DPP-4 over DPP-8
and DPP-9.
Table 6
Selectivity of β-amino ester 5n, β-amino amides 6d and 6p, Sitagliptin 1 against QPP, DPP-8, and DPP-9.
Compounds No.
X
Y
Ar
IC50 (nM)a
DPP-4
QPP
>105
DPP-8
>105
DPP-9
>105
5n
O
49.9
6d
6p
NH
NH
34.4
50.4
28
4630
>105
>105
1060
>105
22030
3610
>105
>105
Sitagliptin 1b
a
b
IC50 is the concentration (nM) needed to cause 50% inhibition human recombinant DPP-4 enzymatic activity. Values represent the mean of double determinations.
Sitagliptin was used as the reference drug in previous literature [12].
8

M.-T. Fuh et al.
Bioorganic Chemistry 114 (2021) 105049
Fig. 4. Docked pose of the active site of DPP-4 enzyme with (A) Sitagliptin 1 (yellow) and β-amino amide 6d (magenta) (B) β-amino ester 5n (green), β-amino amides
6d (magenta) and 6p (orange). The red dashed lines indicate hydrogen-bonding interactions. (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
silica gel (230–400 mesh). Analytical thin-layer chromatography (TLC)
was performed on precoated plates (silica gel 60F-254) purchased from
Merck Inc. Commercially available reagents were used without further
purification unless otherwise noted. Dichloromethane, ethyl acetate,
and hexane were purchased from Mallinckrodt Chemical Co. Dimethyl
sulfone (DMSO2, purity = 100%) was purchased from Tokyo Chemical
Industry Co. Proton NMR spectra were obtained on a Bruker (400 MHz
or 500 MHz) spectrometer by use of CDCl3 or DMSO‑d6 as solvent.
Carbon-13 NMR spectra were obtained on a Bruker (100 MHz or 125
MHz) spectrometer by used of CDCl3 or DMSO‑d6 as solvent. The
following abbreviations are used for spin multiplicity: s, singlet; d,
doublet; t, triplet; q, quartet; p, quintet/pentet; sept, septet; dd, doublet
of doublets; dt, triplet of doublets; m, multiplet; J, coupling constant
(Hz). The purities of the compounds 3–6 were determined by using the
q1HNMR method (absolute q1HNMR with internal calibration) [50].
Most of purities were more than > 98%, for example, compound 6n was
98.80% (Figure S91). The purity of compound 6n was calculated by
following equation:
corresponding crude 1H-1,2,4-triazole-3-carboxylic acids 8a–e and 8m.
A solution of crude 1H-1,2,4-triazole-3-carboxylic acids 8a–e and
8m (1.0 equiv from 7a–e and 7m) and 2-hydroxy-N-(2,4,5-tri-
fluorophenyl)acetamide 10 (1.1 equiv) was treated with N,N-bis[2-oxo-
3-oxazolidinyl]phosphorodiamide chloride (BOP-Cl, 2.0 equiv) in pres-
ence of NEt3 (2.0 equiv) in CH2Cl2 solution (20 mL) [39]. The reaction
mixture was stirred at room temperature for 4 h. When the reaction was
completed, the reaction mixture was washed with water (15 mL) and
extracted with CH2Cl2 (15 mL × 3). The combined organic layers were
washed sodium bicarbonate (15 mL × 3), dried over MgSO4 to give
crude N,O-disubstituted glycolamides 3a–e and 3m. The crude products
3a–e and 3m were purified by column chromatography on silica gel to
give the pure corresponding N,O-disubstituted glycolamides 3a–e and
3m in 71–88% yields.
4.1.1.1. 2-Oxo-2-((2,4,5-trifluorophenyl)amino)ethyl 1-phenyl-1H-1,2,4-
◦
triazole-3- carboxylate (3a). Yield 85%, white solid, m.p. 184–186 C,
1H NMR (400 MHz, DMSO‑d6) δ 5.08 (s, 2H, OCH2), 7.50 (t, J = 7.38 Hz,
1H, ArH), 7.62 (t, J = 7.86 Hz, 2H, ArH), 7.66–7.71 (m, 1H, ArH), 7.93
(d, J = 8.00 Hz, 2H, ArH), 8.03 (dt, J = 15.95, 6.15 Hz, 1H, ArH), 9.50 (s,
1H, ArH), 10.34 (br, 1H, NH). 13C{1H} NMR (100 MHz, DMSO‑d6) δ
63.12, 106.01 (dd, J = 25.63, 22.73 Hz), 111.60 (d, J = 24.08 Hz),
120.01 (2 × C), 122.37, 128.79, 129.93 (2 × C), 136.30, 144.27, 145.17
(d, J = 238.67 Hz), 147.55, 149.92, 154.11, 158.83, 165.75. IR (KBr):
3460, 3164, 1750, 1692, 1427, 1202, 883, 750 cmꢀ 1. EIMS m/z: 376
(M+, 5), 231 (25), 173 (20), 172 (100), 145 (22), 104 (11), 77 (13).
HRMS (EI) m/z: [M]+ Calcd for C17H11F3N4O3: 376.0783; Found
376.0785.
nic.intt.MWt.mic
nt.intic.MWic.ms
p(%) =
.pic
where MW is the molecular weight, P is the purity of internal calibrant,
mic is the amount of internal calibrant, ms is the amount of sample, int is
integral, and n is the number of protons giving rise to a given NMR
signal. The subscript ic and t represents the internal calibrant and target
analyte, respectively. Infrared (IR) spectra were measured on a Bomem
Michelson Series FT-IR spectrometer. The wavenumbers reported are
referenced to the polystyrene absorption at 1601 cmꢀ 1. Flash column
chromatography purification of compounds was carried out by gradient
elution using hexanes in ethyl acetate (EA) unless otherwise stated.
High-resolution mass spectra were obtained from a JEOL JMS-HX110
mass spectrometer.
4.1.1.2. 2-Oxo-2-((2,4,5-trifluorophenyl)amino)ethyl
1-(p-tolyl)-1H-
1,2,4-triazole- 3-carboxylate (3b). Yield 87%, light yellow solid, m.p.
186–188 ◦C, 1H NMR (500 MHz, DMSO‑d6) δ 2.39 (s, 3H, CH3), 5.06 (s,
2H, OCH2), 7.42 (d, J = 8.40 Hz, 2H, ArH), 7.68 (td, J = 10.72, 7.45 Hz,
1H, ArH), 7.80 (d, J = 8.40 Hz, 2H, ArH), 8.02 (dt, J = 15.96, 6.30 Hz,
1H, ArH), 9.44 (s, 1H, ArH), 10.34 (br, 1H, NH). 13C{1H} NMR (125
MHz, DMSO‑d6) δ 20.58, 63.12, 106.04 (t, J = 23.76 Hz), 111.65 (d, J =
23.52 Hz), 119.92 (2 × C), 120.19, 130.30 (2 × C), 134.08, 138.51,
144.06, 144.37, 146.04, 147.79, 153.97, 158.87, 165.78. IR (KBr):
3449, 3160, 2929, 1736, 1685, 1427, 1202, 883, 815 cmꢀ 1. EIMS m/z:
390 (M+, 6), 244 (22), 187 (20), 186 (100), 159 (30), 118 (15), 91 (12).
HRMS (EI) m/z: [M]+ Calcd for C18H14F3N4O3: 390.0940; Found
390.0945.
4.1.1. Standard procedure for synthesis of N,O-disubstituted glycolamides
3a–e and 3m [26,28,39]
A series of methyl 1H-1,2,4-triazole-3-carboxylates 7a–e and 7m
were prepared as the starting martials by following our previous pub-
lished procedure [26]. 1H-1,2,4-triazole-3-carboxylates 7a–e and 7m
(1.0 equiv, 5.5 mmol) was dissolved in MeOH (15 mL) and stirred at
room temperature. 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 3.0
equiv) [28] was slowly added to the reaction mixture at room temper-
ature. Then the mixture was stirred for 6 h. When the reaction was
completed, the reaction mixture was added to water (15 mL) and
extracted with ethyl acetate (15 mL × 3). The combined organic layers
were washed sodium bicarbonate (15 mL × 3), dried over MgSO4,
filtered, and concentrated under reduced pressure to afford the
4.1.1.3. 2-Oxo-2-((2,4,5-trifluorophenyl)amino)ethyl 1-(4-chlorophenyl)-
1H-1,2,4- triazole-3-carboxylate (3c). Yield: 88%, light yellow solid; m.
p. 182–183 ◦C, 1H NMR (500 MHz, DMSO‑d6) δ 5.07 (s, 2H, OCH2),
9

M.-T. Fuh et al.
Bioorganic Chemistry 114 (2021) 105049
7.65–7.67 (m, 1H, ArH), 7.69 (d, J = 8.91 Hz, 2H, ArH), 7.97 (d, J =
8.74 Hz, 2H, ArH), 8.02 (dt, J = 16.03, 6.19 Hz, 1H, ArH), 9.52 (s, 1H,
ArH), 10.33 (br, 1H, NH). 13C{1H} NMR (125 MHz, DMSO‑d6) δ 63.14,
106.01 (dd, J = 25.91, 22.40 Hz), 111.59 (d, J = 23.85 Hz), 121.76 (2 ×
C), 122.34 (t, J = 11.36 Hz), 129.88 (2 × C), 130.05, 135.14, 144.47,
145.24 (dd, J = 240.59, 12.65 Hz), 147.75 (d, J = 8.48 Hz), 149.70 (d, J
= 9.61 Hz), 154.17, 158.72, 165.69. IR (KBr): 3456, 3160, 2919, 2851,
1746, 1682, 1427, 1199, 883, 828 cmꢀ 1. EIMS m/z: 410 (M+, 6), 264
(26), 208 (35), 207 (10), 206 (100), 179 (20), 147 (11), 138 (11). HRMS
(EI) m/z: [M]+ Calcd for C17H10ClF3N4O3: 410.0394; Found 410.0390.
mixture was stirred for 6 h. When the reaction was completed, the reac-
tion mixture was added to water (15 mL) and extracted with ethyl acetate
(15 mL × 3). The combined organic layers were washed sodium bicar-
bonate (15 mL × 3), dried over MgSO4, filtered, and concentrated under
reduced pressure to afford the corresponding crude 1H-1,2,4-triazole-3-
carboxylic acids 8a–e and 8–q.
A solution of crude 1H-1,2,4-triazole-3-carboxylic acids 8a–e and
8l–q (1.0 equiv, from 7a–e and 7l–q) and 2-amino-N-(2,4,5-tri-
fluorophenyl)acetamide 11 (1.1 equiv) was treated with N,N-bis[2-oxo-
3-oxazolidinyl]phosphorodiamide chloride (BOP-Cl, 2.0 equiv) in pres-
ence of NEt3 (2.0 equiv) in CH2Cl2 solution (20 mL) [39]. The reaction
mixture was stirred at room temperature for 4 h. When the reaction was
completed, the reaction mixture was washed with water (15 mL) and
extracted with CH2Cl2 (15 mL × 3). The combined organic layers were
washed sodium bicarbonate (15 mL × 3), dried over MgSO4 to give
crude N,N-disubstituted glycinamides 4a–e and 4l–q. The crude prod-
ucts 4a–e and 4l–q were purified by column chromatography on silica
gel to give the pure corresponding N,N-disubstituted glycinamides 4a–e
and 4l–q in 72–92% yields.
4.1.1.4. 2-Oxo-2-((2,4,5-trifluorophenyl)amino)ethyl 1-(4-(trifluoromethyl)
phenyl)- 1H- 1,2,4-triazole-3-carboxylate (3d). Yield 83%, yellow solid, m.
p. 112–113 ◦C, 1H NMR (400 MHz, DMSO‑d6) δ 5.09 (s, 2H, OCH2), 7.69
(td, J = 10.65, 7.51 Hz, 1H, ArH), 8.00–8.07 (m, 3H, ArH), 8.19 (d, J =
8.46 Hz, 2H, ArH), 9.65 (s, 1H, ArH), 10.36 (br, 1H, NH). 13C{1H} NMR
(100 MHz, DMSO‑d6) δ 63.23, 106.07 (dd, J = 25.58, 22.85 Hz), 111.59
(d, J = 24.22 Hz), 120.53(2 × C), 122.36, 123.82 (d, J = 272.31 Hz),
127.26, 127.30, 128.75 (q, J = 31.49 Hz), 139.20, 144.98, 145.28 (dd, J
= 241.78, 12.67 Hz), 147.55, 149.94 (d, J = 7.37 Hz), 154.44, 158.70,
165.71. IR (KBr): 3439, 3221, 2953, 1746, 1678, 1430, 1321, 1127, 1066,
849 cmꢀ 1. EIMS m/z: 444 (M+, 2), 271 (15), 256 (13), 241 (13), 240
(100), 213 (26), 172 (16), 159 (22), 147 (14), 145 (18). HRMS (EI) m/z:
[M]+ Calcd for C18H10F6N4O3:444.0657; Found 444.0649.
4.1.2.1. 2-Oxo-2-((2,4,5-trifluorophenyl)amino)ethyl-1-phenyl-1H-1,2,4-
◦
triazole-3- carboxylate (4a). Yield 87%, white solid, m.p. 245–246 C,
1
–
–
H NMR (500 MHz, DMSO‑d6) δ 4.17 (d, J = 3.34 Hz, 2H, NHCH2 ),
7.48 (t, J = 7.44 Hz, 1H, ArH), 7.61 (t, J = 7.97 Hz, 2H, ArH), 7.64–7.68
(m, 1H, ArH), 7.92 (d, J = 7.53 Hz, 2H, ArH), 8.02 (dt, J = 16.03, 6.25
Hz, 1H, ArH), 8.85 (t, J = 5.44 Hz, 1H, NH), 9.43 (s, 1H, ArH), 10.14 (br,
1H, NH). 13C{1H} NMR (125 MHz, DMSO‑d6) δ 42.60, 105.91 (dd, J =
26.21, 22.28 Hz), 111.58 (d, J = 22.53 Hz), 119.82 (2 × C), 122.88 (t, J
= 11.12 Hz), 128.47, 129.87 (2 × C), 136.49, 143.48, 144.19, 146.09,
147.68, 157.08, 158.83, 168.04. IR (KBr): 3401, 3290, 2922, 1671,
1542, 1505, 1433, 1202, 873 cmꢀ 1. EIMS m/z: 375 (M+, 2), 230 (14),
229 (58), 202 (16), 201 (34), 173 (15), 172 (100), 147 (36), 145 (21),
104 (12), 77 (16). HRMS (EI) m/z: [M]+ Calcd for C17H12F3N5O2:
375.0943; Found 375.0951.
4.1.1.5. 2-Oxo-2-((2,4,5-trifluorophenyl)amino)ethyl 1-(3-(trifluoromethyl)
phenyl)- 1H-1,2,4-triazole-3-carboxylate (3e). Yield: 71%, white solid, m.
p. 163–164 ◦C, 1H NMR (500 MHz, DMSO‑d6) δ 5.08 (s, 2H, OCH2), 7.67
(td, J = 10.69, 7.44 Hz, 1H, ArH), 7.85–7.90 (m, 2H, ArH), 8.03 (dt, J =
15.98, 6.19 Hz, 1H, ArH), 8.25–8.27 (m, 1H, ArH), 8.31 (s, 1H, ArH), 9.65
(s, 1H, ArH), 10.35 (br, 1H, NH). 13C{1H} NMR (125 MHz, DMSO‑d6) δ
63.22, 106.05 (dd, J = 25.86, 22.09 Hz), 111.65 (d, J = 22.87 Hz), 116.76,
116.79, 123.59 (d, J = 272.83 Hz), 124.04, 125.34, 130.55 (q, J = 32.81
Hz), 131.42, 136.87, 144.91, 145.28 (dd, J = 241.05, 12.57 Hz), 147.85,
149.72, 154.31, 158.72, 165.71. IR (KBr): 3470, 3160, 1746, 1692, 1508,
1427, 1202, 1131, 883 cmꢀ 1. EIMS m/z: 444 (M+, 4), 298 (18), 241 (14),
240 (100), 213 (14), 172 (10), 147 (11), 145 (14). HRMS (EI) m/z: [M]+
Calcd for C18H10F6N4O3: 444.0657; Found 444.0665.
4.1.2.2. N-(2-Oxo-2-((2,4,5-trifluorophenyl)amino)ethyl)-1-(p-tolyl)-1H-
1,2,4- triazole-3-carboxamide (4b). Yield 92%, light yellow solid, m.p.
251–252 ◦C, 1H NMR (500 MHz, DMSO‑d6) δ 2.38 (s, 3H, CH3), 4.17 (d,
J = 5.95 Hz, 2H, OCH2), 7.41 (d, J = 8.30 Hz, 2H, ArH), 7.64 (td, J =
15.98, 7.45 Hz, 1H, ArH), 7.80 (d, J = 8.45 Hz, 2H, ArH), 8.02 (dt, J =
16.01, 6.23 Hz, 1H, ArH), 8.19 (t, J = 5.90 Hz, 1H, NH), 9.37 (s, 1H,
ArH), 10.11 (br, 1H, NH). 13C{1H} NMR (125 MHz, DMSO‑d6,) δ 20.54,
42.58, 105.91 (t, J = 24.20 Hz), 111.56 (d, J = 24.48 Hz), 119.71 (2 ×
C), 122.82, 130.20 (2 × C), 134.22, 138.10, 143.23, 145.20 (dd, J =
237.34, 12.60 Hz), 147.73, 149.60, 156.92, 158.87, 168.05. IR (KBr):
3395, 3286, 3113, 2919, 2854, 1671, 1546, 1513, 1430, 1202, 866
cmꢀ 1. EIMS m/z: 389 (M+, 2), 243 (51), 187 (13), 186 (100), 159 (24),
118 (14), 91 (14). HRMS (EI) m/z: [M]+ Calcd for C18H14F3N5O2:
389.1110; Found 389.1103.
4.1.1.6. 2-Oxo-2-((2,4,5-trifluorophenyl)amino)ethyl 1-(4-chlorophenyl)-
5- cyclopentyl-1H-1,2,4-triazole-3-carboxylate (3m). Yield 73%, white
1
solid, m.p. 68–69 ◦C, H NMR (400 MHz, CDCl3) δ 1.62–1.68 (m, 2H,
CH2), 1.85–1.94 (m, 2H, CH2), 1.96–2.01 (m, 4H, CH2), 3.13 (quint, J =
8.28 Hz, 1H, CH), 5.00 (s, 2H, OCH2), 6.97 (td, J = 9.97, 6.99 Hz, 1H,
ArH), 7.40 (d, J = 8.71 Hz, 2H, ArH), 7.53 (d, J = 8.72 Hz, 2H, ArH),
8.31 (dt, J = 15.73, 5.92 Hz, 1H, ArH), 8.46 (br, 1H, NH).13C{1H} NMR
(100 MHz, CDCl3) δ 25.77 (2 × C), 33.00 (2 × C), 36.26, 63.38, 104.93
(t, J = 23.47 Hz), 111.56 (d, J = 25.00 Hz), 121.64, 126.73 (2 × C),
129.88 (2 × C), 135.10, 136.05, 142.95, 145.05, 148.77, 152.91,
158.12, 162.87, 164.69. IR (KBr): 3303, 3072, 2960, 2875, 1756, 1715,
1549, 1501, 1426, 1192, 1097, 876, 839 cmꢀ 1. EIMS m/z: 480 (M+ + 2,
5), 479 (M+ + 1, 4), 478 (M+, 16), 305 (16), 277 (18), 276 (37), 275
(40), 274 (100), 249 (13), 248 (30), 247 (39), 246 (70), 232 (14), 206
(17), 160 (22), 153 (16), 151 (44), 147 (46), 146 (16), 138 (13), 129
(22), 127 (75), 125 (33), 119 (19), 113 (11), 111 (26), 99 (22). HRMS
(EI) m/z: [M]+ Calcd for C22H18ClF3N4O3: 478.1020; Found 478.1016.
4.1.2.3. 1-(4-Chlorophenyl)-N-(2-oxo-2-((2,4,5-trifluorophenyl)amino)
ethyl)-1H- 1,2,4-triazole-3-carboxamide (4c). Yield 89%, white solid, m.
p. 245–246 ◦C, 1H NMR (500 MHz, DMSO‑d6) δ 4.17 (d, J = 5.99 Hz, 2H,
NHCH2), 7.48 (t, J = 7.44 Hz, 1H, ArH), 7.63–7.67 (m, 1H, ArH), 7.69
(dd, J = 6.82, 2.12 Hz, ArH), 7.96 (dd, J = 6.82, 2.12 Hz, ArH), 8.02 (dt,
J = 16.02, 6.23 Hz, 1H, ArH), 8.86 (t, J = 5.97 Hz, 1H, NH), 9.45 (s, 1H,
ArH), 10.11 (br, 1H, NH). 13C{1H} NMR (125 MHz, DMSO‑d6) δ 42.58,
105.91 (dd, J = 25.86, 22.23 Hz), 111.54 (d, J = 24.94 Hz), 121.53 (2 ×
C), 122.79, 128.47, 129.82 (2 × C), 135.32, 143.70, 145.19 (dd, J =
240.40, 15.64 Hz), 147.72, 149.57, 157.14, 158.71, 168.00. IR (KBr):
3334, 3228, 3116, 2919, 1712, 1678, 1525, 1495, 825 cmꢀ 1. EIMS m/z:
412 (M+ + 2, 2), 411 (M+ + 1, 3), (M+, 4), 238 (13), 237 (22), 236 (26),
235 (38), 208 (41), 207 (20), 206 (100), 180 (10), 179 (20), 151 (11),
149 (12), 148 (47), 147 (68), 138 (21), 119 (12), 111 (18). HRMS (EI)
m/z: [M]+ Calcd for C17H11ClF3N5O2: 409.0553; Found 409.0545.
4.1.2. Standard procedure for synthesis of N,N-disubstituted glycinamides
4a–e and 4l–q [26,28,39]
A series of methyl 1H-1,2,4-triazole-3-carboxylates 7a–e and 7l–q
were prepared as the starting martials by following our previous pub-
lished procedure [26]. 1H-1,2,4-Triazole-3-carboxylates 7a–e and 7l–q
(1.0 equiv, 5.5 mmol) was dissolved in MeOH (15 mL) and stirred at room
temperature. 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU, 3.0 equiv) [28]
was slowly added to the reaction mixture at room temperature. Then the
10

M.-T. Fuh et al.
Bioorganic Chemistry 114 (2021) 105049
4.1.2.4. N-(2-Oxo-2-((2,4,5-trifluorophenyl)amino)ethyl)-1-(4-(tri-
(12), 127 (57), 125 (62), 111 (19). HRMS (EI) m/z: [M]+ Calcd for
fluoromethyl) phenyl)-1H-1,2,4-triazole-3-carboxamide (4d). Yield 84%,
C22H19ClF3N5O2: 477.1171; Found 477.1179.
1
◦
light yellow solid, m.p. 177–178 C, H NMR (500 MHz, DMSO‑d6) δ
4.18 (d, J = 5.50 Hz, 2H, NHCH2), 7.65 (q, J = 9.61 Hz, 1H, ArH), 8.01
(d, J = 7.65 Hz, 2H, ArH), 8.03–8.04 (m, 1H, ArH), 8.17 (d, J = 7.90 Hz,
2H, ArH), 8.92 (t, J = 5.30 Hz, 1H, NH), 9.57 (s, 1H, ArH), 10.12 (br, 1H,
NH). 13C{1H} NMR (125 MHz, DMSO‑d6) δ 42.63, 105.94 (dd, J = 25.98,
22.30 Hz), 111.62 (d, J = 23.94 Hz), 120.30 (2 × C), 122.78, 124.94,
127.21, 127.23, 128.49 (q, J = 32.50 Hz), 139.38, 144.19, 145.18 (dt, J
= 243.33, 13.50 Hz), 147.78, 149.64, 157.42, 158.69, 168.01. IR (KBr):
3426, 3293, 3099, 2929, 1691, 1671, 1512, 1437, 1321, 1121, 1063,
842. cmꢀ 1. EIMS m/z: 443 (M+, 2), 297 (16), 269 (27), 241 (10), 240
(100), 213 (12), 172 (16), 147 (78), 145 (21). HRMS (EI) m/z: [M]+
Calcd for C18H11F6N5O2: 443.0817; Found 443.0812.
4.1.2.8. N-(2-Oxo-2-((2,4,5-trifluorophenyl)amino)ethyl)-5-(1H-pyrrol-
2-yl)-1- (3-(trifluoromethyl)phenyl)-1H-1,2,4-triazole-3-carboxamide
(4n). Yield 73%, light yellow solid, m.p. 181–182 ◦C, 1H NMR (500
MHz, CDCl3) δ 4.38 (d, J = 5.36 Hz, 2H, NHCH2), 5.83 (s, 1H, ArH), 6.01
(dd, J = 6.00, 2.55 Hz, 1H, ArH), 6.90–6.96 (m, 2H, ArH), 7.65–7.71 (m,
2H, ArH), 7.80–7.83 (m, 2H, ArH), 7.94 (t, J = 5.20 Hz, 1H, NH), 8.16
(dt, J = 16.03, 5.60 Hz, 1H, ArH), 8.60 (br, 1H, NH), 9.78 (br, 1H, NH).
13C{1H} NMR (125 MHz, CDCl3) δ 44.38, 104.95 (t, J = 23.49 Hz),
110.60, 110.82 (d, J = 23.88 Hz), 111.49, 117.77, 122.02, 122.31, 123.
63 (d, J = 3.43 Hz), 124.19, 127.07 (d, J = 3.81 Hz), 129.68, 130.36,
132.47 (q, J = 33.77 Hz), 137.88,145.28, 146.92, 148.80, 150.06,
155.47, 160.12, 166.93. IR (KBr): 3276, 3079, 2926, 1675, 1549, 1501,
1437, 1328, 1131 cmꢀ 1. EIMS m/z: 508 (M+, 10), 362 (34), 361 (24),
335 (40), 334 (28), 306 (18), 305 (100), 278 (32), 277 (25), 237 (42),
185 (13), 147 (15), 145 (22), 92 (13). HRMS (EI) m/z: [M]+ Calcd for
4.1.2.5. N-(2-Oxo-2-((2,4,5-trifluorophenyl)amino)ethyl)-1-(3-(tri-
fluoromethyl) phenyl)-1H-1,2,4-triazole-3-carboxamide (4e). Yield 72%,
white solid, m.p. 288–289 ◦C, 1H NMR (500 MHz, DMSO‑d6) δ 4.18 (d, J
= 6.05 Hz, 2H, NHCH2), 7.66 (td, J = 10.71, 7.50 Hz, 1H, ArH),
7.86–7.87 (m, 2H, ArH), 8.03 (dt, J = 15.98, 6.24 Hz, 1H, ArH),
8.25–8.28 (m, 1H, ArH), 8.32 (s, 1H, ArH), 8.92 (t, J = 6.03 Hz, 1H, NH),
9.59 (s, 1H, ArH), 10.11 (br, 1H, NH). 13C{1H} NMR (125 MHz,
DMSO‑d6) δ 42.57, 105.92 (dd, J = 25.85, 22.19 Hz), 111.54 (d, J =
23.37 Hz), 116.46, 116.49, 123.60 (d, J = 272.57 Hz), 123.67, 124.92,
130.50 (q, J = 32.79 Hz), 131.29, 137.02, 144.10, 145.21 (dt, J =
223.08, 14.88 Hz), 147.69, 149.64, 157.23, 158.65, 167.99. IR (KBr):
C22H14F6N6O2: 508.1082; Found 508.1088.
4.1.2.9. 5-Butyl-1-(4-chlorophenyl)-N-(2-oxo-2-((2,4,5-trifluorophenyl)
amino) ethyl)-1H-1,2,4-triazole-3-carboxamide (4o). Yield 82%, white
solid, m.p. 131–132 ◦C, 1H NMR (500 MHz, CDCl3) δ 0.85 (t, J = 7.35
Hz, 3H, CH3), 1.30 (sext, J = 7.52 Hz, 2H, CH2), 1.70 (quint, J = 7.64 Hz,
2H, CH2), 2.74 (t, J = 7.73 Hz, 2H, CH2), 4.35 (d, J = 5.80 Hz, 2H,
NHCH2), 6.90 (td, J = 9.82, 7.21 Hz, 1H, ArH), 7.35 (d, J = 8.60 Hz, 2H,
ArH), 7.47 (d, J = 8.60 Hz, 2H, ArH), 7.94 (t, J = 5.50 Hz, 1H, NH), 8.14
(dt, J = 15.86, 5.85 Hz), 8.77 (br, 1H, NH). 13C{1H} NMR (125 MHz,
CDCl3) δ 13.54, 22.17, 26.20, 29.51, 44.34, 104.85 (t, J = 23.61 Hz),
111.89 (d, J = 24.60 Hz), 122.18, 126.38 (2 × C), 129.75 (2 × C),
135.19, 135.67, 145.83 (dt, J = 249.22, 12.94 Hz), 146.14 (ddd, J =
243.81, 12.74, 2.93 Hz), 148.81, 155.36, 157.87, 160.26, 167.13. IR
(KBr): 3313, 2963, 2933, 2871, 1729, 1678, 1546, 1495, 1433, 1216,
1090, 1015, 835 cmꢀ 1. EIMS m/z: 465 (M+, 6), 321 (18), 319 (51), 294
(26), 293 (27), 292 (76), 291 (47), 264 (33), 263 (20), 262 (100), 237
(13), 236 (11), 234 (29), 194 (13), 193 (19), 192 (12). HRMS (EI) m/z:
[M]+ Calcd for C21H19ClF3N5O2: 465.1179; Found 465.1176.
3402, 3230, 3106, 1671, 1560, 1505, 1433, 1321, 1131, 801 cmꢀ 1
.
EIMS m/z: 443 (M+, 2), 391 (21), 297 (15), 269 (23), 257 (50), 241 (10),
240 (87), 217 (92), 213 (14), 172 (26), 165 (95), 159 (68), 147 (100),
146 (18), 145 (21), 139 (11), 119 (21). HRMS (EI) m/z: [M]+ Calcd for
C
18H11F6N5O2: 443.0817; Found 443.0812.
4.1.2.6. 1-(4-Chlorophenyl)-5-(furan-2-yl)-N-(2-oxo-2-((2,4,5-tri-
fluorophenyl) amino) ethyl)-1H-1,2,4-triazole-3-carboxamide (4l). Yield
83%, white solid, m.p. 188–189 ◦C, 1H NMR (500 MHz, CDCl3) δ 4.32 (d,
J = 5.91 Hz, 2H, NHCH2), 6.48 (d, J = 1.94 Hz, 1H, ArH), 6.77 (d, J =
2.97 Hz, 1H, ArH), 6.95 (td, J = 9.78, 6.90 Hz, 1H, ArH), 7.41 (d, J =
8.35 Hz, 1H, ArH), 7.46 (s, 1H, ArH), 7.49 (d, J = 8.62 Hz, 2H, ArH),
7.87 (br, 1H, NH), 8.22 (dt, J = 15.59, 5.85 Hz, 1H, ArH), 8.37 (br, 1H,
NH). 13C{1H} NMR (125 MHz, CDCl3) δ 44.74, 105.91 (t, J = 24.23 Hz),
110.61 (d, J = 24.39 Hz), 112.05, 114.55, 122.14, 127.25 (2 × C),
129.60 (2 × C), 135.82, 136.23, 141.28, 145.14, 145.33, 146.90,
147.34, 147.38, 155.88, 159.94, 166.74. IR (KBr): 3405, 3123, 2926,
1708, 1678, 1613, 1549, 1491,1436, 1230, 1090, 835 cmꢀ 1. EIMS m/z:
475 (M+, 5), 331 (16), 329 (45),304 (17), 303 (19), 302 (52), 301 (33),
274 (33), 273 (18), 272 (100),247 (17), 245 (50), 206 (12), 204 (35),
151 (22), 147 (24), 111 (23). HRMS (EI) m/z: [M]+ Calcd for
C21H13ClF3N5O3: 475.0659; Found 475.0653.
4.1.2.10. N-(2-Oxo-2-((2,4,5-trifluorophenyl)amino)ethyl)-1,5-diphenyl-
1H-1,2,4- triazole-3-carboxamide (4p). Yield 82%, white solid, m.p.
1
◦
210–212 C, H NMR (400 MHz, CDCl3) δ 4.34 (d, J = 6.04 Hz, 2H,
NHCH2), 6.94 (td, J = 9.99, 7.02 Hz, 1H, ArH), 7.32–7.38 (m, 4H, ArH),
7.41–7.46 (m, 4H, ArH), 7.47–7.50 (m, 2H, ArH), 7.96 (t, J = 5.92 Hz,
1H, NH), 8.21 (dt, J = 15.77, 5.98 Hz, 1H, ArH), 8.55 (br, 1H, NH). 13
C
{1H} NMR (100 MHz, CDCl3) δ 44.70, 104.89 (t, J = 23.29 Hz), 110.66
(d, J = 24.61 Hz), 122.18, 125.45 (2 × C), 126.72, 128.72 (2 × C),
128.96 (2 × C), 129.51 (2 × C), 129.64, 130.70, 137.55,145.82 (d, J =
249.18 Hz), 146.41, 148.93, 155.24, 155.50, 160.40, 166.98. IR (KBr):
3286, 3069, 1671, 1546, 1498, 1433, 1209, 692 cmꢀ 1. EIMS m/z: 451
(M+, 5), 305 (39), 279 (14), 278 (92), 277 (31), 249 (19), 248 (100), 222
(13), 221 (58), 180 (56), 147 (12), 117 (19), 77 (22). HRMS (EI) m/z:
[M]+ Calcd for C23H16F3N5O2: 451.1256; Found 451.1251.
4.1.2.7. 1-(4-Chlorophenyl)-5-cyclopentyl-N-(2-oxo-2-((2,4,5-tri-
fluorophenyl) amino)ethyl)-1H-1,2,4-triazole-3-carboxamide (4m). Yield
74%, yellow solid, m.p. 159–160 ◦C, 1H NMR (400 MHz, CDCl3) δ
1.55–1.57 (m, 2H, CH2), 1.77–1.92 (m, 6H, CH2), 3.05 (quint, J = 8.20
Hz, 1H, CH), 4.39 (d, J = 5.60 Hz, 2H, NHCH2), 6.84 (td, J = 9.96, 7.07
Hz, 1H, ArH), 7.31 (dd, J = 6.78, 1.90 Hz, 2H, ArH), 7.43 (dd, J = 6.72,
1.92 Hz, 2H, ArH), 8.06 (dt, J = 15.45, 6.12 Hz, 1H, ArH), 9.12 (br, 1H,
NH). 13C{1H} NMR (100 MHz, CDCl3) δ 25.65 (2 × C), 32.80 (2 × C),
36.03, 43.93, 104.77 (t, J = 23.28 Hz), 111.06 (d, J = 24.19 Hz), 121.15,
126.62 (2 × C), 129.60 (2 × C), 135.18, 135.54, 145.60 (d, J = 250.82
Hz), 145.86 (dt, J = 247.30, 14.53 Hz), 149.26 (d, J = 10.65 Hz),
155.28, 160.21, 161.89, 167.33. IR (KBr): 3402, 3293, 2960, 1682,
1542, 1495, 1433, 1209, 835 cmꢀ 1. EIMS m/z: 477 (M+, 5), 333 (12),
331 (36), 306 (26), 305 (24), 304 (82), 303 (32), 291 (11), 276 (24), 275
(15), 274 (74), 252 (30), 251 (12), 250 (100), 248 (17), 247 (18), 246
(37), 232 (28), 206 (13), 151 (27), 147 (16), 141 (22), 138 (10), 129
4.1.2.11. N-(2-Oxo-2-((2,4,5-trifluorophenyl)amino)ethyl)-5-phenyl-1-
(p-tolyl)-1H- 1,2,4- triazole-3-carboxamide (4q). Yield 84%, yellow
solid, m.p. 121–122 ◦C, 1H NMR (400 MHz, CDCl3) δ 2.34 (s, 3H, CH3),
4.44 (d, J = 5.64 Hz, 2H, NHCH2), 6.80 (td, J = 9.91, 7.15 Hz, 1H, ArH),
7.14 (s, 4H, ArH), 7.26 (t, J = 7.52 Hz, 2H, ArH), 7.35 (t, J = 7.38 Hz,
1H, ArH), 7.42 (d, J = 7.80 Hz, 2H, ArH), 8.02 (dt, J = 15.79, 5.95 Hz,
1H, ArH), 8.21 (t, J = 5.62 Hz, 1H, NH), 9.22 (br, 1H, NH). 13C{1H}
NMR (100 MHz, CDCl3) δ 21.07, 43.88, 104.69 (t, J = 23.40 Hz), 111.25
(d, J = 23.75 Hz), 122.13, 125.03 (2 × C), 126.68, 128.45 (2 × C),
128.75 (2 × C), 129.87 (2 × C), 130.36, 134.92, 139.64, 144.67, 146.70,
149.45, 154.85, 155.45, 160.18, 167.44. IR (KBr): 3286, 3058, 2922,
1739, 1695, 1549, 1512, 1430, 1209 cmꢀ 1. EIMS m/z: 465 (M+, 5), 447
11

M.-T. Fuh et al.
Bioorganic Chemistry 114 (2021) 105049
(13), 391 (12), 319 (32), 293 (19), 292 (91), 291 (18), 279 (12), 263
(25), 262 (100), 236 (19), 235 (84), 195 (15), 194 (87), 147 (16), 132
(23), 131 (40), 129 (13), 105 (54), 104 (29), 103 (12). HRMS (EI) m/z:
[M]+ Calcd for C24H18F3N5O2: 465.1413; Found 465.1406.
cmꢀ 1. ESIMS m/z: 460 (22), 459 (M + H+, 100), 317 (3), 258 (4), 244
(7), 173 (3). HRMS (ESI) m/z: [M+H]+ Calcd for C20H16F6N4O2:
459.1177; Found 459.1244.
4.1.3.3. (1-(4-Chlorophenyl)-5-cyclopentyl-1H-1,2,4-triazol-3-yl)methyl
(R)-3- amino-4-(2,4,5-trifluorophenyl)butanoate (5m). Yield 79%, deep
yellow liquid 1H NMR (500 MHz, CDCl3) δ 1.57 (t, J = 5.50 Hz, 2H, CP),
1.64 (br, 2H, NH2), 1.80–1.93 (m, 6H, CP), 2.37 (dd, J = 15.69, 8.58 Hz,
1H, CH2), 2.54 (dd, J = 15.69, 4.04 Hz, 1H, CH2), 2.62 (dd, J = 13.66,
7.67 Hz, 1H, CH2), 2.71 (dd, J = 13.73, 5.87 Hz, 1H, CH2), 3.04 (quint, J
= 8.19 Hz, 1H, CP), 3.45 (quint, J = 6.50 Hz, 1H, CH), 5.19 (d, J = 2.19
Hz, 2H, OCH2), 6.86 (dd, J = 9.42, 6.86 Hz, 1H, ArH), 7.02 (dd, J =
16.91, 8.99 Hz, 1H, ArH), 7.32 (d, J = 8.56 Hz, 2H, ArH), 7.44 (d, J =
8.55 Hz, 2H, ArH). 13C{1H} NMR (125 MHz, CDCl3) δ 25.68 (2 × C),
32.90 (2 × C), 36.15, 36.20, 41.77, 48.74, 59.48, 105.37 (dd, J = 28.74,
20.68 Hz), 118.96 (dd, J = 18.86, 5.99 Hz), 121.88 (dt, J = 18.14, 4.49
Hz), 126 63 (2 × C), 129.59 (2 × C), 135.07, 135.70, 146.53 (ddd, J =
244.68, 12.43, 3.30 Hz), 148.70 (dt, J = 250.01, 13.44 Hz), 156.09 (dd,
J = 244.24, 9.06 Hz), 158.53, 161.37, 171.41. IR (KBr): 3374, 3296,
3057, 2958, 2869, 1736, 1519, 1488, 1151, 1093, 836 cmꢀ 1. ESIMS m/
z: 492 ([M+H]+ + 2, 34),494 (26), 493 ([M+H]+, 100), 381 (3), 292
(4). HRMS (ESI) m/z: [M+H]+ Calcd for C24H25ClF3N4O2: 493.1613;
Found 493.1601.
4.1.3. Standard procedure for synthesis of β-amino esters 5d and 5m–q
[39,43]
The 1H-1,2,4-triazol-3-ylmethanols 12d, and 12m–q (1.0 equiv, 3.0
mmol) were treated with Boc-(R)-3-amino-4-(2,4,5-trifluoro-phenyl)-
butyric acid 14 (1.1 equiv) and N,N-bis[2-oxo-3-oxazolidinyl]phos-
phorodiamide chloride (BOP-Cl, 2.0 equiv) in presence of NEt3 (2.0
equiv) in CH2Cl2 solution (20 mL) [39]. The reaction mixture was stirred
at room temperature for 4 h. When the reaction was completed, the
reaction mixture was washed with water (15 mL) and extracted with
CH2Cl2 (15 mL × 3). The combined organic layers were washed sodium
bicarbonate (15 mL × 3), dried over MgSO4 to give crude Boc-protected
β-amino esters 15d and 15m–q. The reaction mixture of crude Boc-
protected β-amino esters 15d was purified by column chromatography
on silica gel to give the pure corresponding β-amino esters 15d in 84%
yield. The crude intermediates 15m–q were directly carried out the
deprotection without the further purification.
Boc-Protected β-amino esters 15d and 15m–q (1.0 equiv) were
added with trifluoroacetic acid (TFA, 1.5 equiv) in CH2Cl2 solution (15
mL) [43]. The reaction mixture was stirred at room temperature for 3 h.
When the reaction was completed, the reaction mixture was nertulized
with sodium bicarbonate (15 mL) and extracted with CH2Cl2 (3 × 30
mL). The combined organic layers were dried over MgSO4, filtered, and
concentrated under reduced pressure to afford the corresponding crude
β-amino esters 5d and 5m–q. The crude products 5d and 5m–q were
purified by column chromatography on silica gel to give the pure cor-
responding β-amino esters 5d and 5m–q in 74–84% yields.
4.1.3.4. (5-(1H-Pyrrol-2-yl)-1-(3-(trifluoromethyl)phenyl)-1H-1,2,4-tri-
azol-3-yl) methyl(R)-3-amino-4-(2,4,5-trifluorophenyl)butanoate (5n).
1
Yield 74%, yellow liquid, H NMR (500 MHz, CDCl3) δ 2.01 (br, 2H,
NH2), 2.41 (dd, J = 15.61, 8.80 Hz, 1H, CH2), 2.59 (dd, J = 15.58, 3.98
Hz, 1H, CH2), 2.66 (dd, J = 13.73, 7.73 Hz, 1H, CH2), 2.74 (dd, J =
13.76, 5.90 Hz, 1H, CH2), 3.51 (quint, J = 6.58 Hz, 1H, CH), 5.25 (d, J =
6.55 Hz, 2H, OCH2), 5.81 (d, J = 3.35 Hz, 1H, ArH), 6.07 (t, J = 2.83 Hz,
1H, ArH), 6.84–6.89 (m, 2H, ArH), 7.02 (dd, J = 17.05, 8.85 Hz, 1H,
ArH), 7.64 (t, J = 7.80 Hz, 1H, ArH), 7.70 (d, J = 8.10 Hz, 1H, ArH), 7.78
(d, J = 7.90 Hz, 1H, ArH), 7.80 (s, 1H, ArH), 10.20 (br, 1H, NH). 13C{1H}
NMR (125 MHz, CDCl3) δ 36.21, 41.85, 48.82, 59.07, 105.44 (dd, J =
28.74, 20.71 Hz), 110.23, 110.87, 118.39, 118.96 (dd, J = 18.86, 5.87
Hz), 121.65, 121.83 (t, J = 4.51 Hz), 123.20 (d, J = 272.65 Hz), 123.58
(d, J = 3.52 Hz), 126.52 (d, J = 3.37 Hz), 129.68, 130.20, 132.29 (q, J =
33.46 Hz), 138.33, 146.58 (ddd, J = 244.74, 12.39, 3.26 Hz), 148.78
(dt, J = 250.09, 13.40 Hz), 149.50, 156.12 (dd, J = 243.94, 9.22 Hz),
158.96, 171.50. IR (KBr): 3360, 3289, 3071, 2926, 2855, 1738, 1599,
1520, 1329, 1175, 1134, 739 cmꢀ 1. EIMS m/z: 523 (M+, 5), 379 (17),
378 (100), 310 (14), 309 (95), 307 (20), 292 (21), 291 (95), 199 (13),
170 (23), 151 (41), 145 (24). HRMS (EI) m/z: [M]+ Calcd for
4.1.3.1. (1-(4-(Trifluoromethyl)phenyl)-1H-1,2,4-triazol-3-yl)methyl (R)-
3-((tert-
butoxycarbonyl)amino)-4-(2,4,5-trifluorophenyl)butanoate
(15d). Yield 84%, light yellow solid, m.p. 89–91 ◦C, 1H NMR (500
MHz, CDCl3): δ 1.33 (s, 9H, CH3), 2.60–2.69 (m, 2H, CH2), 2.86 (br, 2H,
CH2), 4.16 (br, 1H, CH), 5.32 (d, J = 6.15 Hz, 2H, CH2), 6.84 (dd, J =
15.36, 8.79 Hz, 1H, ArH), 7.03 (dd, J = 15.62, 8.73 Hz, 1H, ArH), 7.75
(d, J = 7.92 Hz, 2H, ArH), 7.80 (d, J = 8.30 Hz, 2H,ArH), 8.62 (s, 1H,
ArH). 13C{1H} NMR (CDCl3, 125 MHz): δ 28.18 (3 × C), 32.90, 37.92,
47.69, 59.16, 79.52, 105.27 (dd, J = 28.60, 20.84 Hz), 119.07 (d, J =
18.93 Hz), 119.85 (2 × C), 121.32 (d, J = 18.72 Hz), 123.47 (d, J =
272.17 Hz), 127.12, 127.14, 130.37 (q, J = 33.06 Hz), 139.14, 141.87,
145.57 (dd, J = 244.16, 12.51 Hz), 148.83 (dt, J = 251.26, 13.54 Hz),
155.14, 156.20 (dd, J = 244.02, 8.84 Hz), 160.78, 170.78. IR (KBr):
C24H19F6N5O2: 523.1443; Found 523.1448.
3354, 2977, 2929, 1739, 1705, 1692, 1522, 1328, 1168, 842 cmꢀ 1
.
ESIMS m/z: 581 ([M+Na]+, 15), 560 (25), 559 ([M+H]+, 100), 395
(11), 377 (14). HRMS (ESI) m/z: [M+H]+ Calcd for C25H25F6N4O4:
559.1775; Found 559.1785.
4.1.3.5. (5-Butyl-1-(4-chlorophenyl)-1H-1,2,4-triazol-3-yl)methyl (R)-3-
amino-4- (2,4,5-trifluorophenyl)butanoate (5o). Yield: 78%, orange
liquid, 1H NMR (400 MHz, CDCl3) δ 0.79 (t, J = 7.36 Hz, 3H, CH3), 1.25
(sext, J = 7.44 Hz, 2H, CH2CH3), 1.63 (quint, J = 7.67 Hz, 2H,
CH2CH2CH3), 1.83 (br, 2H, NH2), 2.34 (dd, J = 15.75, 8.58 Hz, 1H,
CH2), 2.51 (dd, J = 15.79, 3.90 Hz, 1H, CH2), 2.59 (dd, J = 13.56, 7.64
Hz, 1H, CH2), 2.65–2.70 (m, 3H, CH2CH2CH2CH3, CH2), 3.42 (br, 1H,
CH), 5.15 (s, 2H, OCH2), 6.81 (td, J = 9.57, 6.69 Hz, 1H, ArH), 6.98 (dd,
J = 17.05, 8.89 Hz, 1H, ArH), 7.28 (d, J = 8.70 Hz, 2H, ArH), 7.40 (d, J
= 8.68 Hz, 2H, ArH). 13C{1H} NMR (100 MHz, CDCl3) δ 13.40, 22.10,
26.06, 29.53, 36.03, 41.52, 48.58, 59.23, 105.25 (dd, J = 28.62, 20.78
Hz), 118.85 (dd, J = 18.71, 5.51 Hz), 121.73 (dt, J = 18.01, 4.22 Hz),
126 11 (2 × C), 129.50 (2 × C), 134.86, 135.45, 146.39 (dd, J = 244.75,
12.70 Hz), 148.55 (dt, J = 249.64, 13.41 Hz), 155.91 (d, J = 244.68 Hz),
157.21, 158.47, 171.30. IR (KBr): 3378, 3289, 3057, 2960, 2933, 1738,
1519, 1489, 1152, 1093, 837 cmꢀ 1. ESIMS m/z: 483 ([M+H]+ + 2, 35),
482 (26), 481 ([M+H]+, 100), 289 (4), 280 (5). HRMS (ESI) m/z:
[M+H]+ Calcd for C23H25ClF3N4O2: 481.1613; Found 481.1601.
4.1.3.2. (1-(4-(Trifluoromethyl)phenyl)-1H-1,2,4-triazol-3-yl)methyl (R)-
3-amino-4- (2,4,5-trifluorophenyl)butanoate (5d). Yield: 76%, light yel-
low solid, m.p. 135–136 ◦C, 1H NMR (CDCl3, 500 MHz): δ 2.36 (dd, J =
15.78, 8.63 Hz, 1H, CH2), 2.51 (dd, J = 15.76, 4.10 Hz, 1H, CH2), 2.59
(dd, J = 13.71, 7.65 Hz, 1H, CH2), 2.67 (dd, J = 13.76, 6.00 Hz, 1H,
CH2), 3.42 (quint, J = 6.53 Hz, 1H, CH), 5.22 (d, J = 1.60 Hz, 2H, OCH2),
6.78 (td, J = 9.52, 6.80 Hz, 1H, ArH), 6.98 (dd, J = 16.93, 8.98 Hz, 1H,
ArH), 7.65 (d, J = 8.60 Hz, 2H, ArH), 7.72 (d, J = 8.50 Hz, 2H, ArH),
8.60 (s, 1H, ArH). 13C{1H} NMR (CDCl3, 125 MHz): δ 35.14, 41.34,
48.58, 58.84, 105.18 (dd, J = 28.77, 20.72 Hz), 118.79 (dd, J = 18.88,
6.00 Hz), 119.48 (2 × C), 121.60 (dt, J = 18.17, 4.55 Hz), 123.33 (d, J =
272.19 Hz), 126.82, 126.85, 129.87 (q, J = 33.17 Hz), 138.97, 141.2,
146.33 (ddd, J = 244.43, 12.33, 3.25 Hz), 148.51 (dt, J = 249.67, 13.35
Hz), 155.91 (dd, J = 244.11, 7.28 Hz), 160.65, 171.22. IR (KBr): 3221,
3116, 2922, 1729, 1617, 1549, 1522, 1335, 1179, 1138, 1049, 845
12

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Bioorganic Chemistry 114 (2021) 105049
4.1.3.6. (1,5-Diphenyl-1H-1,2,4-triazol-3-yl)methyl (R)-3-amino-4-(2,4,5-
trifluorophenyl)butanoate (5p). Yield: 83%, orange liquid, 1H NMR (500
MHz, CDCl3) δ 2.42 (dd, J = 15.76, 8.60 Hz, 1H, CH2), 2.58 (dd, J =
15.78, 4.08 Hz, 1H, CH2), 2.64 (dd, J = 13.76, 7.65 Hz, 1H, CH2), 2.73
(dd, J = 13.76, 5.90 Hz, 1H, CH2), 3.48 (quint, J = 6.55 Hz, 1H, CH), 5.30
(d, J = 4.35 Hz, 2H, OCH2), 6.85 (td, J = 9.60, 6.65 Hz, 1H, ArH), 7.02
(dd, J = 17.11, 8.85 Hz, 1H, ArH), 7.29–7.31 (m, 4H, ArH), 7.36–7.39 (m,
4H, ArH), 7.43–7.45 (m, 2H, ArH). 13C{1H} NMR (100 MHz, CDCl3) δ
36.20, 41.73, 48.71, 59.41, 105.36 (dd, J = 28.82, 20.59 Hz), 118.97 (dd,
J = 18.81, 6.12 Hz), 121.86 (d, J = 18.45 Hz), 125.25 (2 × C), 127.36,
128.51 (2 × C), 128.82 (2 × C), 129.00, 129.35 (2 × C), 130.16, 137.84,
146.52 (dd, J = 244.77, 12.59 Hz), 148.68 (dt, J = 249.87, 13.35 Hz),
154.84, 156.06 (dd, J = 243.48, 9.19 Hz), 158.92, 163.24, 171.46. IR
(KBr): 3370, 3299, 3064, 2929, 2858, 1738, 1519, 1424, 1152, 766, 694
cmꢀ 1. EsIMS m/z: 478 (27), 467 (M+, 100), 381 (2), 266 (6). HRMS (ESI)
m/z: [M+H]+ Calcd for C25H22F3N4O2: 467.1689; Found 467.1680.
CH2), 3.44 (d, J = 6.71 Hz, CH), 4.63 (d, J = 5.15 Hz, 2H, NHCH2), 6.89
(q, J = 8.47 Hz, 1H, ArH), 7.03 (q, J = 8.55 Hz, 1H, ArH), 7.72–7.77 (m,
4H, ArH), 8.54 (s, 1H, ArH). 13C{1H} NMR (CDCl3, 100 MHz): δ 37.19 (2
× C), 42.66, 49.09, 105.55 (dd, J = 28.73, 20.45 Hz), 118.99 (dd, J =
18.82, 5.92 Hz), 119.60 (2 × C), 121.57 (d, J = 17.93 Hz), 123.50 (d, J
= 273.01 Hz), 127.06, 127.10, 130.06 (d, J = 33.53 Hz), 139.21,
141.58, 146.54 (d, J = 243.56 Hz), 148.74 (dd, J = 249.08, 15.03 Hz),
156.06 (d, J = 245.32 Hz), 162.74, 171.46. IR (KBr): 3364, 3092, 2926,
1617, 1535, 1325, 1165, 1124, 841 cmꢀ 1. ESIMS m/z: 459 (23), 458
([M+H]+, 100), 285 (17), 243 (20). HRMS (EI) m/z: [M+H]+ Calcd for
C20H18F6N5O: 458.1410; Found 458.1414
4.1.4.2. (R)-3-Amino-N-((1-(4-chlorophenyl)-5-cyclopentyl-1H-1,2,4-tri-
azol-3-yl) methyl)-4-(2,4,5-trifluorophenyl)butanamide (6m). Yield 83%,
yellow liquid, 1H NMR (500 MHz, CDCl3) δ 1.59–1.61 (m, 2H, CH2),
1.85–1.90 (m, 4H, CH2), 1.94–1.96 (m, 2H, CH2), 2.29 (dd, J = 14.73,
8.78 Hz, 1H, CH2), 2.46 (dd, J = 14.66, 2.25 Hz, 1H, CH2), 2.68–2.71 (m,
1H, CH2), 2.73 (br, 2H, NH2), 2.79 (dd, J = 13.53, 5.63 Hz, 1H, CH2),
3.06 (quint, J = 7.95 Hz, 1H, CH), 3.49 (br, 1H, CH), 4.55 (t, J = 4.13 Hz,
2H, NCH2), 6.90 (dt, J = 9.43, 6.74 Hz, 1H, ArH), 7.07 (dd, J = 16.73,
8.98 Hz, 1H, ArH), 7.34 (d, J = 8.45 Hz, 2H, ArH), 7.47 (d, J = 8.45 Hz,
2H, ArH), 7.65 (br, 1H, NH2). 13C{1H} NMR (125 MHz, CDCl3) δ 25.54
(2 × C), 32.77 (2 × C), 36.01, 36.25, 37.25, 42.31, 48.98, 105.29 (dd, J
= 28.66, 20.70 Hz), 118.94 (dd, J = 18.86, 5.89 Hz), 121.56 (d, J =
18.37 Hz), 126 49 (2 × C), 129.46 (2 × C), 134.84, 135.62, 146.44 (dd,
J = 245, 12.10 Hz), 148.61 (dt, J = 250.07, 13.34 Hz), 155.97 (dd, J =
243.82, 7.98 Hz), 160.12, 161.03, 171.18. IR (KBr): 3434, 3349, 3289,
3054, 2957, 2872, 1659, 1518, 1424, 1210, 1093, 837 cmꢀ 1. EIMS m/z:
491 (M+, 1), 346 (10), 277 (21), 261 (18), 260 (21). HRMS (EI) m/z:
[M]+ Calcd for C24H25ClF3N5O: 491.1700; Found 491.1704.
4.1.3.7. (5-Phenyl-1-(p-tolyl)-1H-1,2,4-triazol-3-yl)methyl (R)-3-amino-
4-(2,4,5- trifluorophenyl)butanoate (5q). Yield: 84%, light yellow solid,
m.p. 71–73 ◦C, 1H NMR (500 MHz, CDCl3) δ 2.34 (s, 3H, CH3), 2.40 (dd,
J = 15.71, 8.60 Hz, 1H, CH2), 2.57 (dd, J = 15.68, 4.08 Hz, 1H, CH2),
2.62 (dd, J = 13.77, 7.70 Hz, 1H, CH2), 2.72 (dd, J = 13.76, 5.85 Hz, 1H,
CH2), 3.47 (quint, J = 6.47 Hz, 1H, CH), 5.28 (d, J = 4.90 Hz, 2H, OCH2),
6.83 (td, J = 14.32, 6.69 Hz, 1H, ArH), 7.01 (dd, J = 16.98, 8.98 Hz, 1H,
ArH), 7.16 (s, 4H, ArH), 7.27 (t, J = 7.50 Hz, 2H, ArH), 7.34 (t, J = 7.38
Hz, 1H, ArH), 7.43 (d, J = 7.30 Hz, 2H, ArH). 13C{1H} NMR (125 MHz,
CDCl3) δ 20.91, 36.03, 41.65, 48.61, 59.27, 105.17 (dd, J = 28.78,
20.72 Hz), 118.83 (dd, J = 18.82, 6.02 Hz), 121.81 (dt, J = 18.11, 4.55
Hz), 124.93 (2 × C), 127.34, 128.31 (2 × C), 128.63 (2 × C), 129.76 (2
× C), 129.90, 135.26, 139.00, 146.33 (dd, J = 245.24, 12.39 Hz),
148.49 (dt, J = 249.95, 13.62 Hz), 154.56, 155.92 (dd, J = 241.75, 8.93
Hz), 158.63, 171.29. IR (KBr): 3449, 3374, 3299, 3050, 2954, 2926,
2858, 1738, 1519, 1423, 1151, 823, 732, 696 cmꢀ 1. ESIMS m/z: 482,
(28), 481 ([M+H]+, 100), 468 (2), 280 (6). HRMS (ESI) m/z: [M+H]+
Calcd for C26H24F3N4O2: 481.1846; Found 481.1845.
4.1.4.3. (R)-N-((5-(1H-Pyrrol-2-yl)-1-(3-(trifluoromethyl)phenyl)-1H-
1,2,4-triazol- 3-yl)methyl)-3-amino-4-(2,4,5-trifluorophenyl)butanamide
(6n).. Yield 74%, light yellow liquid, 1H NMR (500 MHz, CDCl3) δ
2.30 (dd, J = 14.86, 9.25 Hz, 1H, CH2), 2.45 (dd, J = 14.86, 3.10 Hz, 1H,
CH2), 2.62–2.66 (m, 3H, CH2 and NH2), 2.73 (dd, J = 13.73, 5.83 Hz,
1H, CH2), 3.49 (d, J = 6.75 Hz, 1H, CH), 4.56 (qd, J = 15.91, 5.32 Hz,
2H, NCH2), 5.76 (d, J = 3.10 Hz, 1H, ArH), 6.03 (t, J = 3.10 Hz, 1H,
ArH), 6.81–6.86 (m, 2H, ArH), 6.97 (dd, J = 16.81, 8.90 Hz, 1H, ArH),
7.57–7.63 (m, 2H, ArH), 7.72–7.74 (m, 2H, ArH), 7.97 (t, J = 5.10 Hz,
1H, NH), 10.57 (br, 1H, NH). 13C{1H} NMR (125 MHz, CDCl3) δ 36.48,
37.11, 42.52, 49.13, 105.45 (dd, J = 28.70, 20.68 Hz), 110.08, 110.89,
118.24, 118.94 (dd, J = 18.81, 5.86 Hz), 121.48 (d, J = 18.07 Hz),
121.71, 123.19 (d, J = 272.62 Hz), 123.43 (d, J = 3.52 Hz), 126.31 (d, J
= 3.26 Hz), 129.57, 130.10, 132.14 (q, J = 33.41 Hz), 138.34, 146.57
(ddd, J = 244.91, 12.23, 3.43 Hz), 148.78 (dt, J = 250.27, 13.42 Hz),
149.32, 156.08 (dd, J = 242.58, 8.84 Hz), 160.63, 171.76. IR (KBr):
3358, 3285, 3075, 2926, 2855, 1738, 1649, 1597, 1519, 1329, 1176,
1133, 1070, 738 cmꢀ 1. EIMS m/z: 523 (M+, 3), 378 (14), 377 (80), 309
(17), 308 (100), 306 (27), 292 (20), 291 (57), 171 (10), 151 (18), 145
(11). HRMS (EI) m/z: [M]+ Calcd for C24H20F6N6O: 522.1603; Found
522.1612.
4.1.4. Standard procedure for synthesis of β-amino amides 6d and 6m–q
[39,43]
The 1H-1,2,4-triazol-3-ylmethanamines 13d and 13m–q (1.0 equiv,
3.0 mmol) were treated with Boc-(R)-3-amino-4-(2,4,5-trifluoro-phenyl)-
butyric acid 14 (1.1 equiv) and N,N-bis[2-oxo-3-oxazolidinyl]phosphor-
odiamide chloride (BOP-Cl, 2.0 equiv) in presence of NEt3 (2.0 equiv) in
CH2Cl2 solution (20 mL) [39]. The reaction mixture was stirred at room
temperature for 4 h. When the reaction was completed, the reaction
mixture was washed with water (15 mL) and extracted with CH2Cl2 (15
mL × 3). The combined organic layers were washed sodium bicarbonate
(15 mL × 3), dried over MgSO4 to give crude Boc-protected β-amino
amides 16d and 16m–q. Without further purification, the crude in-
termediates 16d and 16m–q were directly carried out the deprotection.
Boc-Protected β-amino amides 16d and 16m–q (1.0 equiv) were
added with trifluoroacetic acid (TFA, 1.5 equiv) in CH2Cl2 solution (15
mL) [43]. The reaction mixture was stirred at room temperature for 3 h.
When the reaction was completed, the reaction mixture was nertulized
with sodium bicarbonate (15 mL) and extracted with CH2Cl2 (3 × 30
mL). The combined organic layers were dried over MgSO4, filtered, and
concentrated under reduced pressure to afford the corresponding crude
β-amino amides 6d and 6m–q. The crude products 6d and 6m–q were
purified by column chromatography on silica gel to give the pure cor-
responding β-amino esters 6d and 6m–q in 74–88% yields.
4.1.4.4. (R)-3-Amino-N-((5-butyl-1-(4-chlorophenyl)-1H-1,2,4-triazol-3-
yl)methyl)- 4-(2,4,5-trifluorophenyl)butanamide (6o). Yield: 81%, yellow
liquid, 1H NMR (500 MHz, CDCl3) δ 0.82 (t, J = 7.33 Hz, 3H, CH3), 1.28
(sext, J = 7.60 Hz, 2H, CH2CH3), 1.62 (quint, J = 7.63 Hz, 2H,
CH2CH2CH3), 2.41 (br, 1H, CH2), 2.50–2.52 (m, 1H, CH2), 2.68 (t, J =
7.67 Hz, 2H, CH2), 2.86 (br, 2H, CH2), 3.58 (br, 1H, CH), 4.11 (br, 2H,
NH2), 4.52 (s, 2H, NCH2), 6.86 (td, J = 16.05, 9.32 Hz, 1H, ArH), 6.98
(dd, J = 15.14, 7.88 Hz, 1H, ArH), 7.30 (d, J = 8.41 Hz, 2H, ArH), 7.44
(d, J = 8.47 Hz, 2H, ArH) 7.60 (br, 1H, NH). 13C{1H} NMR (125 MHz,
CDCl3) δ 13.53, 22.22, 26.15, 29.78, 36.14, 37.35, 42.04, 49.18, 105.53
(dd, J = 28.50, 20.65 Hz), 119.13 (dd, J = 18.82, 5.74 Hz), 121.35 (d, J
= 20.24 Hz), 126 25 (2 × C), 129.67 (2 × C), 135.00, 135.67, 146.63 (d,
4.1.4.1. (R)-3-Amino-N-((1-(4-(trifluoromethyl)phenyl)-1H-1,2,4-triazol-
3-yl) methyl)-4-(2,4,5-trifluorophenyl)butanamide (6d). Yield 79%, light
yellow solid, m.p. 50–51 ◦C, 1H NMR (CDCl3, 400 MHz): δ 2.25 (dd, J =
15.09, 9.28 Hz, 1H, CH2), 2.46 (dd, J = 15.19, 1.90 Hz, 1H, CH2), 2.66
(dd, J = 13.41, 7.88 Hz, 1H, CH2), 2.76 (dd, J = 13.77, 5.35 Hz, 1H,
13

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Bioorganic Chemistry 114 (2021) 105049
J = 260.46 Hz), 148.88 (d, J = 250.23 Hz), 156.13 (d, J = 243.66 Hz),
157.12, 160.36, 171.15. IR (KBr): 3273, 3054, 2961, 2933, 2874, 1667,
1519, 1424, 1211, 1151, 1094, 838, 731 cmꢀ 1. EIMS m/z: 479 (M+, 1),
336 (14), 332 (42), 267 (30), 266 (16), 265 (100), 263 (16), 250 (11),
249 (18), 248 (26), 165 (15), 137 (13), 125 (10). HRMS (EI) m/z: [M]+
Calcd for C23H25F3N5O: 479.1700; Found 479.1705.
4.1.6. Standard procedure for synthesis of 2-hydroxy-N-(2,4,5-
trifluorophenyl)acetamide 10 [29]
2-Chloro-N-(2,4,5-trifluorophenyl)acetamide
9 (1.0 equiv, 4.0
mmol) and cesium formate (HCO2Cs, 3.0 equiv) in dry EtOH (10 mL)
was heated at reflux (ca. 77 ◦C) for 6 h. When the reaction was
completed, the solution was filtered to remove the excess amount of
HCO2Cs and the filtrate was concentrated under reduced pressure.
corresponding crude 2-hydroxyacetamide 10. The crude product 10 was
recrystallized in EtOH/hexane (3/1) solution to obtain the pure 2-
hydroxyacetamide 10 in 86% yield; white solid; m.p. 89–90 ◦C, 1H
NMR (CDCl3, 500 MHz): δ 2.93 (t, J = 5.15 Hz,1H, OH), 4.26 (d, J =
5.15 Hz, 2H, CH2NH2), 6.98 (td, J = 10.03, 7.00 Hz, 1H, ArH), 8.29 (dt,
J = 15.86, 5.95 Hz, 1H, ArH), 8.60 (br. 1H, NH). 13C{1H} NMR (125
MHz, CDCl3): δ 62.52, 104.95 (t, J = 23.53 Hz), 110.24 (d, J = 24.68
Hz), 121.79 (t, J = 10.82 Hz), 145.84 (ddd, J = 249.12, 14.60, 11.75
Hz), 146.34 (ddd, J = 243.43, 12.72, 3.63 Hz), 147.57 (ddd, J = 243.43,
8.98, 2.70 Hz), 169.53. IR (KBr): 3429, 3374, 3286, 1688, 1552, 1437,
1199, 876 cmꢀ 1. EIMS m/z: 205 (M+, 32), 147 (100), 146 (18), 119 (21),
71 (19). HRMS (EI) m/z: [M]+ Calcd for C8H6F3NO2: 205.0351; Found
205.0349.
4.1.4.5. (R)-3-Amino-N-((1,5-diphenyl-1H-1,2,4-triazol-3-yl)methyl)-4-
(2,4,5- trifluorophenyl)butanamide (6p). Yield 88%, orange liquid, 1H
NMR (500 MHz, CDCl3) δ 2.25 (dd, J = 14.98, 8.93 Hz, 1H, CH2), 2.45
(d, 2.58 J = 14.91 Hz, 1H, CH2), 2.65 (dd, J = 15.53, 7.93 Hz, 1H, CH2),
2.76 (dd, J = 13.68, 5.34 Hz, 1H, CH2), 3.45 (br, 1H, CH), 4.64 (d, J =
5.20 Hz, 2H, NCH2), 6.84–6.89 (m, 1H, ArH), 7.01 (dd, J = 17.08, 8.78
Hz, 1H, ArH), 7.28–7.31 (m, 4H, ArH), 7.35–7.38 (m, 4H, ArH),
7.41–7.42 (m, 2H, ArH), 7.46 (br, 1H, NH). 13C{1H} NMR (125 MHz,
CDCl3) δ 36.85, 37.42, 42.89, 49.07, 105.44 (dd, J = 28.77, 20.81 Hz),
119.03 (dd, J = 18.73, 6.30 Hz), 121.79 (d, J = 18.16 Hz), 125.26 (2 ×
C), 127.54, 128.57 (2 × C), 128.79 (2 × C), 128.93, 129.37 (2 × C),
130.15, 137.94, 146.63 (d, J = 241.14 Hz), 148.75 (d, J = 249.40 Hz),
154.63, 156.15 (dd, J = 244.04 Hz), 160.64, 171.35. IR (KBr): 3430,
3285, 3061, 2929, 1652, 1517, 1423, 1210, 1154, 765, 695 cmꢀ 1. EIMS
m/z: 465 (M+, 2), 321 (13), 321 (74), 252 (17), 251 (100), 249 (19), 235
(21), 234 (37), 131 (12), 103 (20). HRMS (EI) m/z: [M]+ Calcd for
4.1.7. Standard procedure for synthesis of 2-amino-N-(2,4,5-
trifluorophenyl)acetamide 11 [30]
2-Chloro-N-(2,4,5-trifluorophenyl)acetamide
9 (1.0 equiv, 4.0
C
25H22F3N5O: 465.1776; Found 465.1769.
mmol) was added with ammonium hydroxide solution (NH4OH, 15 mL)
◦
in ethanol solution (15 mL). The mixture was heated at 60 C under
4.1.4.6. (R)-3-Amino-N-((5-phenyl-1-(p-tolyl)-1H-1,2,4-triazol-3-yl)
methyl)-4- (2,4,5-trifluorophenyl)butanamide (6q). Yield 86%, yellow
liquid, 1H NMR (500 MHz, CDCl3) δ 2.35 (dd, J = 14.88, 8.93 Hz, 1H,
CH2), 2.47 (s, 3H, CH3), 2.54 (dd, J = 14.91, 3.30 Hz, 1H, CH2), 2.74 (dd,
J = 13.71, 7.90 Hz, 1H, CH2), 2.85 (dd, J = 13.68, 5.73 Hz, 1H, CH2),
3.53–3.59 (m, 1H, CH), 4.74 (d, J = 5.30 Hz, 2H, NCH2), 6.97 (td, J =
9.42, 6.84 Hz, 1H, ArH), 7.12 (dd, J = 16.51, 9.35 Hz, 1H, ArH), 7.27 (s,
4H, ArH), 7.38–7.42 (m, 2H, ArH), 7.47 (t, J = 7.08 Hz, 1H, ArH), 7.53
(d, J = 7.75 Hz, 2H, ArH), 7.69 (t, J = 4.83 Hz, 1H, NH). 13C{1H} NMR
(125 MHz, CDCl3) δ 21.02, 36.71, 37.30, 42.85, 49.00, 105.30 (dd, J =
28.69, 20.68 Hz), 118.93 (dd, J = 18.77, 6.01 Hz), 121.82 (dt, J = 18.20,
4.49 Hz), 124.99 (2 × C), 127.53, 128.43 (2 × C), 128.65 (2 × C),
129.84 (2 × C), 129.95, 135.38, 139.01, 146.44 (dd, J = 245.28, 12.82
Hz), 148.60 (dt, J = 250.12, 13.59 Hz), 154.41, 156.00 (dd, J = 244.24,
9.02 Hz), 160.41, 171.31. IR (KBr): 3424, 3286, 3050, 2926, 2865,
1651, 1518, 1423, 1210, 1151, 823, 698 cmꢀ 1. EIMS m/z: 479 (M+, 2),
335 (18), 334 (87), 266 (22), 265 (100), 263 (24), 249 (26), 248 (44),
145 (21), 117 (17), 105 (12). HRMS (EI) m/z: [M]+ Calcd for
sealed conditions for 3 h. When the reaction was completed, the reaction
mixture was concentrated and nertulized with 3% HCl(aq) and extracted
with CH2Cl2 (3 × 30 mL). The organic extracts were dried over MgSO4,
filtered, and concentrated under reduced pressure to afford the corre-
sponding crude 2-amino-N-(2,4,5-trifluorophenyl)acetamide 11. The
crude product 11 was recrystallized in EtOH/hexane (3/1) solution to
obtain the pure 2-aminoacetamide 11 in 88% yield; white solid; m.p.
69–70 ◦C, 1H NMR (CDCl3, 500 MHz): δ 3.44 (s, 2H, CH2NH2), 6.92 (q, J
= 9.07 Hz, 1H, ArH), 8.30 (dt, J = 16.38, 5.89 Hz, 1H, ArH), 9.73 (s, 2H,
NH2). 13C{1H} NMR (125 MHz, CDCl3): δ 44.98, 104.65 (t, J = 23.52
Hz), 109.53 (d, J = 24.65 Hz), 122.47 (t, J = 8.89 Hz), 145.22 (dt, J =
247.25, 12.96 Hz), 146.17 (dd, J = 242.46, 9.04 Hz), 147.42 (dd, J =
245.87, 8.97 Hz), 171.00. IR (KBr): 3503, 3364, 1678, 1569, 1501,
1426, 1206, 862 cmꢀ 1. EIMS m/z: 205 (11), 204 (M+, 46), 173 (26), 148
(16), 147 (100), 146 (18), 145 (15), 81 (11), 69 (16). HRMS (EI) m/z:
[M]+ Calcd for C8H7F3N2O: 204.0510; Found 287.1163.
4.1.8. Standard procedure for synthesis of (1H-1,2,4-triazol-3-yl)methanol
(12d, and 12m–q) [40]
C
26H24F3N5O: 479.1933; Found 479.1941.
The reliable procedure involved the treatment of 1H-1,2,4-triazole-3-
carboxylates 7d and 7m–q (1.0 equiv, 5.0 mmol) with NaBH4 (1.5 equiv,
7.5 mmol) in THF/MeOH (1/3, 12 mL) [40]. The reaction mixture was
stirred at room temperature for 5 h. When the reaction was completed,
the reaction mixture was concentrated under reduced pressure to afford
the crude residue. The residue was added water (15 mL) and extracted
with ethyl acetate (15 mL × 3). The combined organic layers were
washed sodium bicarbonate (15 mL × 3), dried over MgSO4 to give
crude (1H-1,2,4-triazol-3-yl)methanol product 12d and 12m–q. The
crude desired products 12d and 12m–q were recrystallized in n-hexane/
methanol (1/4) solution to obtain the pure (1H-1,2,4-triazol-3-yl)
methanol product 12d and 12m–q in 91–96% yields.
4.1.5. Standard procedure for synthesis of 2-chloro-N-(2,4,5-
trifluorophenyl)acetamide 9 [31,51]
2,4,5-Trifluorobenzenamine (1.0 equiv, 5.0 mmol) was dissolved in
toluene (20 mL) and stirred in an ice-bath. 2-Chloroacetyl chloride (1.1
equiv.) was slowly added to the reaction mixture under N2. Then the
mixture was heated and stirred at refliux (ca. 108 ◦C) within 3 h. When
the reaction was completed, the reaction mixture was added to water
(15 mL) and extracted with ethyl acetate (15 mL × 3). The combined
organic layers were washed sodium bicarbonate (15 mL × 3), dried over
MgSO4, filtered, and concentrated under reduced pressure to afford the
corresponding crude 2-chloro-N-(2,4,5-trifluorophenyl)acetamide 9.
The crude desired products 9 was recrystallized in EtOH/hexane (3/1)
solution to obtain the pure 2-chloro-N-(2,4,5-trifluorophenyl)acetamide
9 in 92% yield; 1H NMR (CDCl3, 500 MHz): δ 4.19 (s, CH2NH2), 6.99 (td,
J = 9.96, 7.00 Hz, 1H, ArH), 8.22 (dt, J = 15.81, 8.78 Hz, 1H, ArH), 8.44
(br. 1H, NH). 13C{1H} NMR (125 MHz, CDCl3): δ 42.69, 105.01 (t, J =
23.55 Hz), 110.30 (d, J = 24.81 Hz), 121.55 (t, J = 8.73 Hz), 146.07 (d,
J = 249.56 Hz), 146.27 (dt, J = 247.84, 11.89 Hz), 147.73 (ddd, J =
243.43, 9.11, 2.73 Hz), 163.88. Physical and spectral data were
consistent with those previously reported [51].
4.1.8.1. (1-(4-(Trifluoromethyl)phenyl)-1H-1,2,4-triazol-3-yl)methanol
1
◦
(12d). Yield 91%, white solid, m.p. 111–113 C, H NMR (400 MHz,
CDCl3) δ 2.84 (t, J = 5.97 Hz, 1H, OH), 4.85 (d, J = 5.55 Hz, 2H,
CH2OH), 7.76 (d, J = 8.79 Hz, 2H, ArH), 7.81 (d, J = 8.70 Hz, 2H, ArH),
8.59 (s, 1H, ArH). 13C{1H} NMR (100 MHz, CDCl3) δ 58.44, 119.70 (2 ×
C), 123.17 (d, J = 272.03 Hz), 127.13, 127.17, 130.17 (q, J = 33.02 Hz),
139.27, 141.59, 164.97. IR (KBr): 3210, 3114, 2922, 2851, 1335, 1176,
14

M.-T. Fuh et al.
Bioorganic Chemistry 114 (2021) 105049
1133, 1111, 1047, 848 cmꢀ 1. EIMS m/z: 243 (M+, 70), 242 (85), 224
(18), 214 (58), 187 (10), 172 (21), 159 (100), 145 (42), 140 (15), 139
(30), 109 (15). HRMS (EI) m/z: [M]+ Calcd for C10H8F3N3O: 243.0619;
Found 243.0625.
4.1.9. Standard procedure for synthesis of (1H-1,2,4-triazol-3-yl)
methanamine (13d, and 13m–q) [41,42]
The reliable procedure involved the treatment of 1H-1,2,4-triazol-3-
ylmethanol 12d, and 12m–q (1.0 equiv, 5.0 mmol) with thionyl chloride
(SOCl2, 1.5 equiv, 7.5 mmol) in CH2Cl2 solution (15 mL) [41]. The re-
action mixture was stirred at room temperature for 2 h under N2. When
the reaction was completed, the reaction mixture was concentrated
under reduced pressure, added with CH2Cl2 (15 mL), neutralized with
saturated sodium bicarbonate solution (15 mL × 3). The organic layer
was dried over MgSO4, filtered, then concentrated under reduced pres-
sure to afford crude residue. The residue was added with ammonium
hydroxide solution (NH4OH, 15 mL) in methanol solution (15 mL). The
mixture was heated at 60 ◦C under sealed conditions for 6.0 h [42].
When the amination reaction was completed, the reaction mixture was
concentrated and nertulized with 3% HCl(aq) and extracted with CH2Cl2
(3 × 30 mL). The organic extracts were dried over MgSO4, filtered, and
concentrated under reduced pressure to afford the corresponding crude
1H-1,2,4-triazol-3-ylmethanamines 13d and 13m–q. The crude prod-
ucts 13d and 13m–q were purified by column chromatography on silica
gel to give the pure corresponding β-amino esters 13d and 13m–q in
73–84% yields.
4.1.8.2. (1-(4-Chlorophenyl)-5-cyclopentyl-1H-1,2,4-triazol-3-yl)meth-
1
◦
anol (12m). Yield 94%, light yellow solid, m.p. 100–102 C, H NMR
(500 MHz, CDCl3) δ 1.59 (t, J = 5.35 Hz, 2H, CP), 1.83–1.95 (m, 6H, CP),
3.05 (quint, J = 8.18 Hz, 1H, CP), 3.41 (br, 1H, OH), 4.74 (s, 2H,
CH2OH), 7.34 (d, J = 8.47 Hz, 2H, ArH), 7.46 (d, J = 8.50 Hz, 2H, ArH).
13C{1H} NMR (125 MHz, CDCl3) δ 25.75 (2 × C), 32.96 (2 × C), 36.19,
58.29, 126.71 (2 × C), 129.64 (2 × C), 135.05, 135.82, 161.19, 162.83.
IR (KBr): 3264, 2955, 2869, 1519, 1486, 1092, 1059, 1011, 838 cmꢀ 1
.
EIMS m/z: 279 (M+ + 2, 5), 277 (M+, 14), 276 (14), 238 (24), 237 (10),
236 (77), 182 (18), 127 (32), 126 (13), 125 (100), 90 (22). HRMS (EI)
m/z: [M]+ Calcd for C14H16ClN3O: 277.0982; Found 277.0975.
4.1.8.3. (5-(1H-Pyrrol-2-yl)-1-(3-(trifluoromethyl)phenyl)-1H-1,2,4-tri-
azol-3-yl) methanol (12n). Yield 92%, white solid, m.p. 183–184 ◦C, 1H
NMR (500 MHz, CDCl3) δ 4.59 (br, 1H, OH), 4.83 (s, 1H, CH2OH), 5.81
(s, 1H, ArH), 6.09–6.11 (m, 1H, ArH), 6.92–6.93 (m, 1H, ArH),
7.65–7.71 (m, 2H, ArH), 7.80 (br, 2H, ArH), 10.01 (br, 2H, NH). 13C{1H}
NMR (125 MHz, CDCl3) δ 57.99, 110.36, 111.07, 118.49, 121.74,
123.25 (d, J = 272.61 Hz), 123.73, 126.59, 129.84, 130.28, 132.43 (d, J
= 33.19 Hz), 138.49, 149.38, 162.88. IR (KBr): 3317, 3128, 3114, 3054,
1499, 1327, 1161, 1112, 1070, 806, 753, 698 cmꢀ 1. EIMS m/z: 309 (19),
308 (M+, 93), 307 (40), 289 (53), 159 (100), 145 (14), 139 (11). HRMS
(EI) m/z: [M]+ Calcd for C14H11F3N4O: 308.0885; Found 308.8092.
4.1.9.1. (1-(4-(Trifluoromethyl)phenyl)-1H-1,2,4-triazol-3-yl)methan-
amine (13d). Yield 73%, light yellow solid, m.p. 75–77 ◦C 1H NMR (500
MHz, CDCl3) δ 1.98 (s, 2H, NH2), 3.98 (s, 2H, CH2NH2), 7.69 (d, J =
7.90 Hz, 2H, ArH), 7.75 (d, J = 7.85 Hz, 2H, ArH), 8.53 (s, 1H, ArH). 13
C
{1H} NMR (125 MHz, CDCl3) δ 39.76, 119.42 (2 × C), 123.42 (d, J =
272.15 Hz), 126.94, 126.97, 129.74 (q, J = 33.40 Hz), 139.37, 141.39,
166.82. IR (KBr): 3370, 3306, 3093, 2926, 2858, 1618, 1534, 1324,
1125, 983, 844 cmꢀ 1. EIMS m/z: 242 (M+, 54), 241 (100), 214 (77), 172
(12), 145 (14). HRMS (EI) m/z: [M]+ Calcd for C10H9F3N4: 242.0779;
Found 242.0770.
4.1.8.4. (5-Butyl-1-(4-chlorophenyl)-1H-1,2,4-triazol-3-yl)methanol
(12o). Yield 93%, orange solid, m.p. 48–50 ◦C, 1H NMR (500 MHz,
CDCl3) δ 0.85 (t, J = 7.35 Hz, 3H, CH3), 1.30 (sext, J = 7.44 Hz, 2H,
CH2CH3), 1.69 (q, J = 7.68 Hz, 2H, CH2CH2CH3), 2.72 (t, J = 7.78 Hz,
2H, CH2CH2CH2CH3), 3.97 (br, 1H, OH), 4.74 (s, 2H, CH2OH), 7.33 (dt,
J = 9.06, 2.60 Hz, 2H, ArH), 7.45 (dt, J = 9.45, 2.35 Hz, 2H, ArH). 13C
{1H} NMR (125 MHz, CDCl3) δ 13.56, 22.24, 26.15, 29.72, 57.99,
126.32 (2 × C), 129.66 (2 × C), 134.98, 135.70, 157.14, 162.98. IR
4.1.9.2. (1-(4-Chlorophenyl)-5-cyclopentyl-1H-1,2,4-triazol-3-yl)methan-
amine (13m). Yield 79%, orange liquid, 1H NMR (500 MHz, CDCl3) δ
1.37–1.42 (m, 2H, CP), 1.64–1.77 (m, 6H, CP), 2.09 (br, 2H, NH2), 2.89
(quint, J = 8.24 Hz, 1H, CP), 3.76 (br, 2H, CH2NH2), 7.18 (d, J = 8.79
Hz, 2H, ArH), 7.27 (d, J = 8.65 Hz, 2H, ArH). 13C{1H} NMR (125 MHz,
CDCl3) δ 25.31 (2 × C), 32.51 (2 × C), 35.76, 39.37, 126.22 (2 × C),
129.12 (2 × C), 134.28, 135.60, 160.42, 163.98; IR (KBr): 3372, 3302,
3050, 2957, 2870, 1513, 1487, 1092, 1012, 835 cmꢀ 1. EIMS m/z: 278
(M+ + 2, 26), 277 (30), 276 (M+, 92), 275 (57), 250 (15), 248 (53), 237
(27), 235 (100), 224 (12), 218 (20), 182 (14), 180 (45), 152 (22), 127
(20), 125 (62), 111 (13), 90 (15). HRMS (EI) m/z: [M]+ Calcd for
(KBr): 3292, 2958, 2931, 1520, 1487, 1092, 1055, 1012, 837 cmꢀ 1
.
EIMS m/z: 265 (M+, 5), 236 (18), 225 (32), 224 (12), 223 (100), 182
(13), 170 (35), 127 (22), 125 (54), 90 (19). HRMS (EI) m/z: [M]+ Calcd
for C13H16ClN3O: 265.0982; Found 265.0989.
4.1.8.5. (1,5-Diphenyl-1H-1,2,4-triazol-3-yl)methanol (12p). Yield 96%,
light yellow solid, m.p. 137–139 ◦C, 1H NMR (400 MHz, CDCl3) δ 3.70
(br, 1H, OH), 4.84 (s, 2H, CH2OH), 7.29–7.33 (m, 4H, ArH), 7.36–7.40
(m, 4H, ArH), 7.45–7.47 (m, 2H, ArH). 13C{1H} NMR (100 MHz, CDCl3)
δ 58.22, 125.33 (2 × C), 127.45, 128.59 (2 × C), 128.85 (2 × C), 128.96,
129.40 (2 × C), 130.17, 137.93, 154.65, 163.24. IR (KBr): 3307, 3061,
2922, 1595, 1511, 1449, 1050, 990, 774, 694 cmꢀ 1. EIMS m/z: 251 (M+,
46), 148 (36), 91 (100), 64 (11). HRMS (EI) m/z: [M]+ Calcd for
C15H13N3O: 251.1059; Found 251.1056.
C14H17ClN4: 276.1142; Found 2776.1147.
4.1.9.3. (5-(1H-Pyrrol-2-yl)-1-(3-(trifluoromethyl)phenyl)-1H-1,2,4-tri-
azol-3-yl) methanamine (13n). Yield 76%, light yellow liquid, 1H NMR
(500 MHz, CDCl3) δ 2.65 (br, 2H, NH2), 4.00 (br, 2H, CH2NH2), 5.80 (d,
J = 2.90 Hz, 1H, ArH), 6.05 (s, 1H, ArH), 6.87 (s, 1H, ArH), 7.60 (t, J =
7.85 Hz, 1H, ArH), 7.67 (d, J = 7.85 Hz, 1H, ArH), 7.75 (d, J = 7.70 Hz,
1H, ArH), 7.79 (br, 1H, ArH), 11.39 (s, 1H, NH). 13C{1H} NMR (125
MHz, CDCl3) δ 39.18, 19.84, 110.72, 118.62, 121.55, 123.18 (d, J =
272.61 Hz), 123.53, 126.18, 129.67, 130.05, 132.10 (q, J = 33.37 Hz),
138.55, 149.37, 164.59. IR (KBr): 3356, 3292, 2922, 2855, 1597, 1508,
1328, 1126, 1069, 748, 697 cmꢀ 1. EIMS m/z: 308 (19), 307 (M+, 100),
306 (53), 289 (26), 279 (20), 237 (12), 222 (18), 221 (13), 145 (12).
HRMS (EI) m/z: [M]+ Calcd for C14H12F3N5: 307.1045; Found 307.1039.
4.1.8.6. (5-Phenyl-1-(p-tolyl)-1H-1,2,4-triazol-3-yl)methanol
(12q).
Yield 96%, light yellow solid, m.p. 112–114 ◦C, 1H NMR (500 MHz,
CDCl3) δ 2.38 (s, 3H, CH3), 4.83 (s, 2H, CH2OH), 7.20 (s, 4H, ArH), 7.32
(t, J = 7.43 Hz, 2H, ArH), 7.38 (t, J = 7.35 Hz, 1H, ArH), 7.46–7.48 (m,
2H, ArH). 13C{1H} NMR (125 MHz, CDCl3) δ 21.18, 58.55, 125.18 (2 ×
C), 127.67, 128.56 (2 × C), 128.83 (2 × C), 129.99 (2 × C), 130.07,
135.55, 139.13, 154.57, 162.99. IR (KBr): 3209, 3039, 2922, 2858,
1519, 1453, 1050, 992, 825, 778, 696 cmꢀ 1. EIMS m/z: 266 (19), 265
(M+, 91), 162 (59), 106 (12), 105 (100), 104 (29). HRMS (EI) m/z: [M]+
Calcd for C16H15N3O: 265.1215; Found 265.1210.
4.1.9.4. (5-Butyl-1-(4-chlorophenyl)-1H-1,2,4-triazol-3-yl)methanamine
(13o). Yield: 81%, orange liquid, 1H NMR (500 MHz, CDCl3) δ 0.80 (t, J
= 7.35 Hz, 3H, CH3), 1.25 (sext, J = 7.40 Hz, 2H, CH2CH3), 1.63 (q, J =
7.67 Hz, 2H, CH2CH2CH3), 2.65 (t, J = 7.83 Hz, 2H, CH2CH2CH2CH3),
3.05 (br, 1H, NH2), 3.92 (br, 2H, CH2NH2), 7.29 (d, J = 8.70 Hz, 2H,
ArH), 7.39 (d, J = 8.65 Hz, 2H, ArH). 13C{1H} NMR (125 MHz, CDCl3) δ
15

M.-T. Fuh et al.
Bioorganic Chemistry 114 (2021) 105049
13.46, 22.16, 26.10, 29.68, 39.28, 126.14 (2 × C), 129.48 (2 × C),
substrate (AMC). The reaction rate was monitored by a fluorometer with
380-nm excitation and 460-nm emission filters, and determined by the
linear slope of the fluorescence accumulation. Inhibition constants
(IC50) were calculated from enzyme progress curves using standard
mathematical models.
134.61, 135.75, 156.82, 163.61. IR (KBr): 3406, 3296, 3046, 2959,
2930, 2630, 1504, 1488, 1092, 1012, 836 cmꢀ 1. EIMS m/z: 266 (M+
+
2, 19), 265 (26), 264 (57), 263 (53), 249 (19), 238 (19), 237 (15), 236
(59), 235 (21), 224 (33), 223 (25), 222 (100), 205 (25), 191 (32), 182
(15), 180 (40), 170 (63), 152 (28), 130 (21), 129 (18), 128 (64), 127
(62), 125 (94), 111 (25), 90 (31). HRMS (EI) m/z: [M]+ Calcd for
4.4. Molecular modeling [12,25]
C
13H17ClN4: 264.1142; Found 264.1134.
Computational studies were performed with Sitagliptin 1 and
β-amino carbonyl target 5n, 6d, and 6p. The lowest energy conformer
for chemical structure was saved in mol2 files by Chemdraw before use
in docking studies. The structure of DPP-4 enzyme, encoded PDB ID:
1X70 was downloaded from the protein data bank (PDB) before per-
forming docking studies [12]. Molecular docking studies were per-
formed using iGemdock 2.1 [25]. iGemdock docking calculations were
performed at the drug screening docking accuracy setting with param-
eters set for population size, generation, and number of solutions as 800,
80, and 10, respectively, and a Gemdock score function of hydrophobic
and electrostatic (1:1 preference). Residue interaction figure was pro-
duced by Pymol to demonstrate the binding pocket.
4.1.9.5. (1,5-Diphenyl-1H-1,2,4-triazol-3-yl)methanamine (13p).. Yield
82%, yellow liquid, 1H NMR (500 MHz, CDCl3) δ 1.94 (s, 2H, NH2), 3.99
(s, 2H, CH2NH2), 7.25–7.30 (m, 4H, ArH), 7.31–7.36 (m, 4H, ArH),
7.41–7.43 (m, 2H, ArH). 13C{1H} NMR (125 MHz, CDCl3) δ 39.62,
124.93 (2 × C), 127.53, 128.18(2 × C), 128.39, 128.44 (2 × C), 128.99
(2 × C), 129.61, 137.81, 154.06, 164.78. IR (KBr): 3371, 3303, 3064,
2923, 2851, 1596, 1508, 1448, 989, 770, 694 cmꢀ 1. EIMS m/z: 251 (19),
250 (M+, 100), 249 (69), 235 (35). 222 (53), 180 (30), 146 (31), 118
(19), 104 (14), 103 (14), 92 (12), 91 (62). HRMS (EI) m/z: [M]+ Calcd
for C15H14N4: 250.1218; Found 250.1223.
4.1.9.6. (5-Phenyl-1-(p-tolyl)-1H-1,2,4-triazol-3-yl)methanamine (13q).
1
Yield 84%, yellow liquid, H NMR (500 MHz, CDCl3) δ 2.09 (br, 2H,
Declaration of Competing Interest
NH2), 2.37 (s, 3H, CH3), 4.03 (s, 2H, CH2NH2), 7.18 (d, J = 8.43 Hz, 2H,
ArH), 7.21 (d, J = 8.52 Hz, 2H, ArH), 7.31 (t, J = 7.39 Hz, 2H, ArH), 7.36
(t, J = 7.33 Hz, 1H, ArH), 749 (dd, J = 8.28, 1.10 Hz, 2H, ArH). 13C{1H}
NMR (125 MHz, CDCl3) δ 20.82, 39.58, 124.80 (2 × C), 127.61, 128.17
(2 × C), 128.45 (2 × C), 129.57, 129.60 (2 × C), 135.36, 138.53, 154.00,
164.54. IR (KBr): 3367, 3292, 3068, 2923, 2853, 1517, 1451, 990, 821,
696 cmꢀ 1. EIMS m/z: 265 (24), 264 (M+, 100), 263 (56), 249 (31), 236
(49), 194 (24), 160 (29), 132 (27), 106(13), 105 (87), 104 (32). HRMS
(EI) m/z: [M]+ Calcd for C16H16N4: 264.1375; Found 264.1385.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgment
We are grateful to the China Medical University (CMU109-ASIA-08
and CMU109-MF-86) and the Ministry of Science and Technology of
Taiwan (MOST 109-2113-M-039-003) for financial support. In addition,
we are also grateful to Yung Shin Pharmaceutical Industrial Co., Ltd for
the generous support with Sitagliptin.
4.2. In vitro dipeptidyl peptidase 4 assay
4.2.1. Preparation of the DPP-4 enzyme
Appendix A. Supplementary material
Human recombinant dipeptidyl peptidase IV and its substrate, Gly-
Pro-7-amido-4-methylcoumarin (GP- AMC) and its natural inhibitor,
Ile-Pro-Ile tripeptide (diprotin A), were purchased from R&D Systems
(Minneapolis, MN). All other chemicals were purchased from Sigma-
Aldrich (St. Louis, MO).
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105049.
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