Synthesis and biological evaluation of 1-benzyl-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamides as antimitotic agents

Article abstract of DOI:10.1016/j.bioorg.2018.11.002

A library of 1-benzyl-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamides (7a–al) have been designed, synthesized and screened for their anti-proliferative activity against some selected human cancer cell lines namely DU-145, A-549, MCF-7 and HeLa. Most of them have shown promising cytotoxicity against lung cancer cell line (A549), amongst them 7f was found to be the most potent anti-proliferative congener. Furthermore, 7f exhibited comparable tubulin polymerization inhibition (IC₅₀ value 2.04 μM) to the standard E7010 (IC₅₀ value 2.15 μM). Moreover, flow cytometric analysis revealed that this compound induced apoptosis via cell cycle arrest at G₂/M phase in A549 cells. Induction of apoptosis was further observed by examining the mitochondrial membrane potential and was also confirmed by Hoechst staining as well as Annexin V-FITC assays. Furthermore, molecular docking studies indicated that compound 7f binds to the colchicine binding site of the β-tubulin. Thus, 7f exhibits anti-proliferative properties by inhibiting the tubulin polymerization through the binding at the colchicine active site and by induction of apoptosis.

Full text of DOI:10.1016/j.bioorg.2018.11.002

Accepted Manuscript
Synthesis and Biological Evaluation of 1-Benzyl-N-(2-(phenylamino)pyri-
din-3-yl)-1H-1,2,3-triazole-4-carboxamides as Antimitotic Agents
Budaganaboyina Prasad, V. Lakshma Nayak, P.S. Srikanth, Mirza Feroz Baig,
N.V. Subba Reddy, Korrapati Suresh Babu, Ahmed Kamal
PII:
S0045-2068(18)31002-2
DOI:
https://doi.org/10.1016/j.bioorg.2018.11.002
YBIOO 2610
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 September 2018
31 October 2018
1 November 2018
Please cite this article as: B. Prasad, V. Lakshma Nayak, P.S. Srikanth, M. Feroz Baig, N.V. Subba Reddy, K. Suresh
Babu, A. Kamal, Synthesis and Biological Evaluation of 1-Benzyl-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-
triazole-4-carboxamides as Antimitotic Agents, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.
2018.11.002
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Synthesis and Biological Evaluation of 1-Benzyl-N-(2-(phenylamino)pyridin-3-
yl)-1H-1,2,3-triazole-4-carboxamides as Antimitotic Agents
Budaganaboyina Prasad,a,b V. Lakshma Nayak,a P. S. Srikanth,a Mirza Feroz Baig,a N. V. Subba
Reddy,a Korrapati Suresh Babu,a,b Ahmed Kamal a,c*
aMedicinal Chemistry and Biotechnology, CSIR-Indian Institute of Chemical Technology,
Hyderabad 500007, India
bDepartment of Chemistry, Osmania University, Hyderabad 500007, Telangana, India
cSchool of Pharmaceutical Education and Research (SPER), Jamia Hamdard, 110 062, New
Delhi, India
Abstract
A library of 1-benzyl-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamides
(7a‒al) have been designed, synthesized and screened for their anti-proliferative activity against
some selected human cancer cell lines namely DU-145, A-549, MCF-7 and HeLa. Most of them
have shown promising cytotoxicity against lung cancer cell line (A549), amongst them 7f was
found to be the most potent anti-proliferative congener. Furthermore, 7f exhibited comparable
tubulin polymerization inhibition (IC50 value 2.04µM) to the standard E7010 (IC50 value
2.15µM). Moreover, flow cytometric analysis revealed that this compound induced apoptosis via
cell cycle arrest at G2/M phase in A549 cells. Induction of apoptosis was further observed by
examining the mitochondrial membrane potential and was also confirmed by Hoechst staining as
well as Annexin V-FITC assays. Furthermore, molecular docking studies indicated that
compound 7f binds to the colchicine binding site of the β-tubulin. Thus, 7f exhibits anti-
proliferative properties by inhibiting the tubulin polymerization through the binding at the
colchicine active site and by induction of apoptosis.
Keywords: 2-anilino-3-aminopyridines, triazole-4-carboxilic acids, apoptosis, cell cycle,
colchicine binding, molecular docking and tubulin polymerization.
Corresponding authors Tel: +91-40-27193157; fax: +91-40-27193189, e-mail:
ahmedkamal@iict.res.in (A. Kamal)

1. Introduction
Microtubules are cytoskeleton part of the cell residing always in dynamic assemblies of
α-tubulin and β-tubulin heterodimers [1‒3]. They play a pivotal role during eukaryotic cellular
processes such as mitosis, cellular transportation, motility and segregation of chromosomes [4,
5]. The self-assembles of heterodimers form mitotic spindles in dynamic equilibrium, further
division of mitotic spindle at the metaphase/anaphase leads to the formation of daughter cells [6].
At this stage, tubulin binding agents (TBAs) disturbs the dynamic equilibrium by stabilizing or
destabilizing tubulin polymerization, which leads to apoptotic cell death [7, 8].
During the last few decades, natural products have received much attention as tubulin
modulators. Among these, taxanes (paclitaxel, docetaxel) are tubulin polymerization stabilizers/
tubulin polymerization promoters, vinca alkaloids (vinfluine, vinorelbine, vincristine, dolastatins,
halichondrins, hemiasterlins and cryptophysin) and colchicines [colchicine (1), 2-methoxy
estradiol, combretastatin] are tubulin polymerization destabilizers and inhibitors [9‒11]. Most of
the naturally occurring molecules have certain limitations with respect to their synthesis,
solubility, toxicity and resistance [12]. Therefore their derived synthetic molecules have gathered
immense acknowledgments across the world with less complexity in their structure, improved
solubility with similar biological properties. Combretastatin A-4 (CA-4), 2a is the simplest
naturally occurring tubulin polymerization inhibitor, owing to its limitations, its derived soluble
phosphate prodrug CA-4P (2b, phosbretabulin or fosbretabulin) emerged as clinical candidate in
combination with bevacizumab [13‒15].
In recent years, antitumour agents are classified as tubulin-binding tumour vascular
disturbing agents (VDAs), they preferentially damage the blood vessels (tissues) of the tumour
rather than normal tissues thereby leading to apoptotic cell death [16,17]. The mechanism of
tumor vascular disturbing agents is still uncertain however it shows some common
characteristics of tubulin polymerization inhibition and binding to the colchicine site of the
tubulin [18, 19].

Figure 1: Structures of synthetic and naturally occurring anticancer agents.
E7010 (3, Fig. 1) is a synthetic pyridine-sulfonamide derivative, that exhibits cytotoxic
activity by inhibiting tubulin polymerization [20]. It causes cell cycle arrest by promoting
dynamic instability at interphase and binds at β-tubulin colchicine site which leads to apoptosis
via cell cycle arrest at G2/M phase [21]. Structure-activity relationship (SAR) studies for E7010
indicate that the pyridine ring system with 2-anilino and 3-sulfonamido substitution improves the
solubility and toxicity [22]. Moreover, recent studies have shown that at 3rd position of pyridine
with sulfonamide, carbonyl and other functional groups including either open or cyclic partners
linking with 4th positions of pyridine (5 and 6 membered heterocyclic ring) are crucial for their
cytotoxic activity [23]. Several modifications to the E7010 scaffold have been carried out by
omitting the sulfonamide moiety to minimize its toxicity profile. However, E7010 is the only
molecule that binds at the three binding zones [24, 25]. Another molecule, N-phenyl
nicotinamide derivative (4) induces apoptosis through cell cycle arrest in G2/M phase as that of
E7010 [26].
Disubstituted 1,2,3-triazole/1,2,4-triazole derivatives possess several biological properties
such as histone deacetylase, HSP90 C-terminal and kinase inhibition, inhibition of tubulin
polymerization as well as antibacterial activity [27‒33]. Most of the biologically active
derivatives were prepared by molecular hybridization through the incorporation of 1,2,3-
triazole/1,2,4-triazole moiety between 4 or 5 membered heterocyclic ring systems to improve
their potency. In addition, N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)nicotinamide (5) is one of

the substituted triazole-oxazole carboxamide derivative, arrest cells at G2/M phase and acts as
potent tubulin polymerization inhibitor [31]. Moreover, its related triazole-nicotinamide scaffold
was previously developed from this laboratory, one of the compound (6) in the series exhibited
promising cytotoxicity and potent tubulin polymerization inhibition, Figure 1 [34].
Based on these observations, we have made an attempt to introduce the triazole-linked
carboxamide instead of a sulfonamide group in E7010 scaffold with a view to enhance the
biological activity and thereby leading to the target compounds (7a‒al, Fig. 2).
Figure 2: The design strategy.
2. Results and discussion
2.1. Chemistry
1-Benzyl-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamides
(7a‒al)
were prepared by acid and amine coupling reaction. The two intermediates, 1-substituted benzyl-
1,2,3-triazole-4-carboxilic acids (15a‒f) and 2-substituted anilinoanilines (10a‒i) were prepered
from the conventional procedures for the synthesis of desired products. One of the intermediates,
1-substituted benzyl-1,2,3-triazole-4-carboxilic acids (15a‒f) were synthesized by employing
different substituted benzyl alcohols (11a‒f) as a starting material and then reaction with
phosphorus tribromide at 0 °C produces substituted benzyl bromides (12a‒f) [35]. Its
corresponding substituted benzyl azides (13a‒f) were obtained by the reaction with NaN3 in
anhydrous DMSO [36]. The aromatic substituted 1,2,3-triazole methyl ester derivatives (14a‒f)
were obtained by the click reaction with methylpropiolate and substituted benzyl azides (13a‒f)
in the presence of Na-ascorbate, CuSO4.5H2O as catalyst in H2O/t-BuOH (1:2) solvent for 24 h
[37]. Finally aromatic substituted 1,2,3-triazole carboxylic acid derivatives (15a−f) were
obtained through base hydrolysis with 5N NaOH from its corresponding substituted 1,2,3-
triazole methyl ester derivatives (14a‒f) in good to excellent yields (Scheme 1).

Scheme 1: Synthetic pathway for the preparation of 1-benzyl-N-(2-(phenylamino)pyridin-3-yl)-
1H-1,2,3-triazole-4-carboxamides.
Reagents and conditions: (a) Substituted anilines, ethylene glycol, 160 °C, 6 h, 70-74%; (b)
SnCl2∙2H2O, EtOH, 60 °C, 6 h, 58-66%; (c) PBr3, ether, 0 °C; (d) NaN3, DMSO, rt, 55-98%; (e)
Na Ascorbate (10 mol %), CuSO4∙5H2O (5 mol %), H2O/t-BuOH (1:2), rt, 55-98%; (f) 5N
NaOH, EtOH, 70°C, 55-98%; (g) EDCI, HOBt, DMF, rt, 73-86%.

Other intermediates 2-anilino-3-aminopyridine derivatives (10a‒i) were synthesized
using 2-chloro-3-nitropyridine (1) as starting material, upon reaction with substituted anilines in
ethylene glycol at 160 °C to produce substituted 2-anilino-3-nitropyridine derivatives (9a‒i)
[38]. Further, these 2-anilino-3-nitropyridine derivatives (9a‒i) were subjected to the reduction
with SnCl2.5H2O to furnish the corresponding substituted 2-anilino-3-aminopyridines (10a‒i) in
good yield. The structure of all these intermediates like anilinoanilines (10a‒i) and triazolic acids
1
(15a‒f) were confirmed from their respective H and 13C NMR spectral data that was
corroborated with the literature data and was found in accordance [39, 40]. The final amide
coupling reaction between substituted acids (15a‒f) and amines (10a‒i) was carried out using
EDCI/ HOBt and DMAP in DMF solvent at room temperature to obtain the desired substituted-
1-benzyl-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamide derivatives (7a‒al)
1
in good yield. Structure of all the target compounds was established on the basis of their H, 13C
and HRMS spectral data (Scheme 1).
2.2. Biology
2.2.1. Cytotoxicity
To access the cell viability, MTT assay was performed on all the synthesized compounds
against four human cancer cell lines viz DU-145 (prostate), A549 (lung), MCF-7 (breast) and
HeLa (cervical). E7010 was employed as a standard (positive control) and the results are
summarized in Table 1. It is observed that these conjugates (7a‒al) show promising cytotoxicity
with IC50 values ranging from 1.02 to 42.15 µM against all the cell lines. Notably, compound 7f
was found to be the most active amongst the series with IC50 value of 1.02 µM against A549
cells.
Table 1: Cytotoxicity of target compounds (7a‒al) against selected human cancer cell lines (IC50
values in µM).a
Compounds codes
DU-145b
1.479
1.455
1.738
14.78
2.443
A549c
1.318
1.368
1.148
12.25
2.028
MCF-7d
6.462
9.00
HeLae
1.409
9.278
8.760
17.05
4.325
7a
7b
7c
7d
7e
9.143
21.33
8.030

2.138
8.767
7.893
10.66
8.455
16.67
33.86
6.276
8.531
14.33
6.857
17.62
15.79
30.43
17.48
18.12
5.623
13.70
23.74
31.12
18.61
20.20
23.88
19.14
11.59
17.49
21.82
24.80
38.23
22.65
17.77
42.15
32.64
1.230
7f
7g
1.549
2.884
2.642
15.22
3.802
9.750
18.25
1.514
2.051
2.410
2.399
3.090
3.548
25.47
14.21
15.71
5.623
6.457
20.42
15.28
17.08
19.61
14.29
10.00
4.074
11.99
10.95
15.26
26.95
18.83
16.32
20.65
19.10
1.714
1.023
1.811
1.435
7.50
1.337
2.291
2.938
10.81
9.054
12.11
16.21
6.724
6.857
13.30
2.510
8.972
10.47
19.94
16.16
19.89
10.36
4.677
21.81
12.34
21.53
39.81
20.06
24.19
12.47
11.96
10.28
15.96
27.29
16.74
13.09
11.71
26.29
2.188
7h
7i
7j
1.560
5.115
14.86
1.122
1.318
1.950
1.862
2.109
2.754
18.64
9.143
13.48
7.923
6.273
21.94
10.41
12.93
9.893
9.036
10.62
3.920
8.654
5.495
14.52
23.61
14.47
10.93
19.12
13.35
1.622
7k
7l
7m
7n
7o
7p
7q
7r
7s
7t
7u
7v
7w
7x
7y
7z
7aa
7ab
7ac
7ad
7ae
7af
7ag
7ah
7ai
7aj
7ak
7al
E7010
a50% Inhibitory concentration after 48h of compounds treatment. Human prostate cancer.
b
cHuman lung cancer. dHuman breast cancer. eHuman cervical cancer.

The structure activity relationship (SAR) of these 1-benzyl-N-(2-(phenylamino)pyridin-3-
yl)-1H-1,2,3-triazole-4-carboxamide derivatives (7a‒7al) has been studied. The synthesized
compounds consist of A, B, C and D ring systems and for all the compounds ring B and C are
kept common and the SAR was studied by varying the substitution pattern on ring A as well as
ring D. Various analogues have been prepared by placing electron withdrawing and electron
donating groups such as 4‒F, 4‒OH, 4‒OMe, 2,4‒diOMe, 3,4‒diOMe, 3,5‒diOMe,
3,4,5‒triOMe on ring A. Similarly, 4‒F, 3‒OMe, 3‒OPh, 4‒OMe and 3,4,5‒triOMe groups
incorporated on ring D. In case of the congeners having donating substituents on both the rings
(A and D) cytotoxicity values were pronounced. However, the compounds having halogen group
(4‒F) on D ring are comparably less active (7d, 7l, 7s, 7x, 7ab and 7ah). Furthermore, the
conjugates 7a, 7f, 7h, 7m and 7o containing 3‒OPh group on D ring and donating groups on A
ring have shown significant cytotoxic activity with IC50 values ranging from 1.023‒14.33 against
all the selected cancer cell lines. Amongst them, compound 7f that contains 3,5‒diOMe group on
ring A and 3‒OPh group on ring D was found to be the most potent hybrid. Replacing the
substitution pattern on A ring from 3,5‒diOMe to 3,4‒diOMe and 4‒OMe without changing the
substitution pattern on D ring (3‒OPh) reduces the in cytotoxic activity (7m and 7a). Moreover,
when 3, 4 and 5 positions of ring A masked by methoxy group without changing the substitution
pattern of ring D (3‒OPh), drastic fall in the cytotoxic activity has observed (7o). Furthermore,
removal of all the substituents or placing hydroxy group or electron withdrawing halogen such as
F on ring A without changing the substitution pattern on ring D i.e., 3‒OPh progressively
decreases the cytotoxicity (7t, 7y, and 7ad). Finally, based on the above results it can be
summarized that cytotoxic activity order for the presence of donating and withdrawing groups on
A ring with same substitution on D ring is as follows; 3,5‒diOMe > 3,4‒diOMe > 4‒OMe ~
2,4‒diOMe > 3,4,5‒triOMe > 4‒H > 4‒OH > 4‒F.
Replacing the 3‒OPh group on D ring with substitutions like 3‒OMe, 4‒OMe,
3,4,5‒triOMe, 4‒F and without changing the substitution pattern on ring A (2,4‒diOMe)
exhibited 2‒8 fold reduction in cytotoxicity (7h > 7j > 7k > 7i > 7l). Similarly, reduction in
cytotoxicity can be compared equally between substitutions on A ring with that of D ring
(decreasing order of cytotoxicity 7a‒7e, 7h‒7l, 7o‒7s, 7t‒7x, 7y‒7ac, 7ad‒7ah and 7ai‒7al).
Based on these observations, the effect of changing the substituents on D ring in relation to
constant substituent on A ring, it is observed that the cytotoxicity decreasing in the following

order 3‒OPh> 3‒OMe > 4‒OMe > 3,4,5‒triOMe > 4‒H (Fig. 3). Most of the synthesized
compounds have shown relatively improved cytotoxicity against A-549 cell line. Interestingly,
compounds with donating groups on both the A and D ring systems exhibit better activity than
the reference standard E7010 (7a, 7b, 7c, 7f, 7h, 7m and 7n). Therefore, it can be concluded that
electron donating groups on both the rings are necessary for prominent cytotoxic activity. Hence
detailed studies were carried out on one of the potent representative compound 7f.
Figure 3: Structure–activity relationship for the 1-benzyl-N-(2-(phenylamino)pyridin-3-yl)-1H-
1,2,3-triazole-4-carboxamide derivatives (7a‒al)
2.2.2. Cell cycle analysis
Many anticancer compounds exert their growth inhibitory effect either by arresting the
cell cycle at a particular checkpoint of cell cycle or by induction of apoptosis or a combination of
both [41]. The screening results revealed that compound 7f showed significant anticancer
activity against human lung cancer cell line (A549). Further, we performed a cell cycle analysis
to confirm whether the cell growth inhibition of human lung cancer cells was due to cell cycle
arrest. In this study, A549 cells were treated with the compound 7f for 48 h at 1 and 2 µM
concentrations and it indicates that 7f has shown G2/M cell cycle arrest in comparison with the
control cells. Compound 7f displayed 60.79 % of cell accumulation in G2/M phase at 1 µM

concentration, whereas it exhibited 77.31 % of cell accumulation at 2 µM concentration (Fig. 4
and Table 2).
Figure 4: Flow cytometric analysis in A549 cells after treatment with compound 7f at 1 and 2
µM concentrations for 48 h. A: Control cells (A549), B: E7010 (2 µM), C: 7f (1 µM) and D: 7f
(2 µM).
Table 2: Effect of compound 7f on cell cycle phase distribution in A549 cells
Sample
Sub G1 %
0.81
G0/G1 %
55.60
S %
3.64
1.72
1.96
G2/M %
38.04
A: Control cells
B: E7010 (2 µM)
C: 7f (1 µM)
0.99
20.41
74.73
1.92
33.38
60.79

1.05
18.41
1.43
77.31
D: 7f (2 µM)
2.2.3. Effect of compounds on tubulin polymerization
In general G2/M cell cycle arrest is associated with inhibition of tubulin polymerization
and since compound 7f causes cell cycle arrest at G2/M cell cycle arrest; thus its effect on
microtubule inhibitory function was also investigated. Tubulin subunits are known to
heterodimerize and self-assemble to form microtubules in a time dependent manner. Thus, the
progression of tubulin polymerization [42‒45] was examined by monitoring the increase in
fluorescence emission at 420 nm (excitation wavelength is 360 nm) in 384 well plate for 1 h at
37 °C with and without the compound at 3 µM concentration in comparison with reference
compound E7010. Compound 7f significantly inhibited tubulin polymerization by 64.77 %,
which is comparable with E7010 (Fig. 5). This was followed by the evaluation of IC50 value for
compound 7f and the experimental results revealed that this compound shows 2.04 µM IC50
value suggesting that it inhibits the tubulin polymerization.
Figure 5: Effect of compound 7f on tubulin polymerization: tubulin polymerization was
monitored by the increase in fluorescence at 360 nm (excitation) and 420 nM (emission) for 1 h
at 37 °C. E7010 was used as the reference compound in this study. Values indicated are the mean
± SD of two different experiments.

2.2.4. Hoechst staining assay
Apoptosis, with its classic characteristics of chromatin condensation and fragmented
nuclei is one of the major pathways that lead to the process of cell death [46]. The apoptosis
inducing capability of this potent compound was studied by Hoechst staining method. In this
study, A549 cells were treated with 7f at 1 and 2 µM concentrations for 48 h. From these results,
it can be inferred that this compound induced apoptotic cell death in human lung cancer cells
A549 (Fig. 6).
Figure 6: Hoechst staining in A549 cell line; A: Control cells, B: 7f (1 µM) and 7f (2 µM).
2.2.5. Measurement of mitochondrial membrane potential (∆Ψm)
The maintenance of mitochondrial membrane potential (∆Ψm) is important for the
mitochondrial integrity and bioenergetic function [47]. Mitochondrial changes, including loss of
mitochondrial membrane potential ((∆Ψm) are key events that take place during drug induced
apoptosis. In order to study the apoptosis inducing effect of compound 7f, mitochondrial
membrane potential ((∆Ψm) changes were detected using the fluorescent probe JC 1 (5,5,6,6-
tetrachloro-1,1,3,3-tetraethylbenzimidazolocarbocyanine iodide-5 mg/mL). After 48 h of the
treatment by the compound, it was observed that the mitochondrial membrane potential (∆Ψm)
of A549 cells reduced significantly (Fig. 7).

Figure 7: Effect of compound 7f on mitochondrial membrane potential ((∆Ψm): Drops in
membrane potential (∆Ψm) was assessed by JC-1 staining of A549 cells treated with test
compound and samples were then subjected to flow-cytometry analysis on a FACS (Becton
Dickinson) in the FL1, FL2 channel to detect mitochondrial potential. A: Control cells, B: 7f (1
µM) and C: 7f (2 µM).
2.2.6. Annexin V-FITC assay
The apoptotic effect of 7f was further evaluated by Annexin V FITC/PI (AV/PI) dual
staining assay [48] to examine the occurrence of phosphatidylserine externalization and also to
understand whether it is due to physiological apoptosis or nonspecific necrosis. In this study,
A549 cells were treated with this compound for 48 h at 1 and 2 µM concentrations to examine
the apoptotic effect. It was observed that this compound showed significant apoptosis against
A549 cells as shown in Fig. 4. These results indicated that compound 7f has shown 19.26 and
34.86 % at 1 and 2 µM concentrations respectively (Fig. 8 and Table 3).

Figure 8: Annexin V-FITC staining. A: Control cells (A549), B: Nocodazole (2 µM) and C: 7f
(1 µM) and D: 7f (2 µM).
Table 3: Annexin V-FITC assay.
Sample
UL %
UR %
LL%
LR %
A: Control cells
0.04
0.01
99.84
0.12
C: 7f (1 µM)
D: 7f (2 µM)
4.12
0.01
5.01
76.62
65.14
14.25
23.84
11.02
2.3. Molecular docking studies
Molecular docking is a vital component in bioinformatics to exploit the structure of
protein–small molecule complexes in structural detail, which plays a pivotal role in drug
discovery and development. Docking is an algorithm, which predicts the preferred relative
orientation of one molecule (ligand) when bound in an active site of another molecule (protein)
to form a stable complex such that free energy of the overall system is minimized. Docking

involves two consecutive steps, initially sampling conformations of the ligand in the binding
pocket of the protein and followed by ranking these conformations via a scoring function.
Cytotoxic results of these conjugates (especially compounds like 7a, 7b, 7c, 7f, 7g, 7h,
7j, 7m and 7n on human lung cancer cell lines) encouraged us to perform their molecular
docking studies. Here, molecular docking tool was used to predict the 3D complex of the 1-
benzyl-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamides conjugates within the
constraints of E7010 binding pocket present in the tubulin (PDB ID: 3HKC) [49]. The co-
ordinates of the crystal structure were obtained from RCSB-Protein Data Bank and suitable
corrections to it were made by using Protein Preparation Wizard from Schrödinger package. In
regard to the ligands, molecules were constructed using ChemBio3D Ultra 12.0 and their
geometries were optimized using molecular mechanics. Finally, docking studies were performed
for the most active conjugate 7f and reference ligand E7010 by using Auto Dock 4.2 [50] and the
results were visualized through PyMOL [51].
Figure 9: Surface binding pose of compound 7f in E7010 binding pocket present in β-subunit of
tubulin, where the protein is shown as a surface representation.
Docking studies revealed that conjugate 7f fit well in the colchicine binding site present
in β-subunit at α-β interface of the tubulin (Fig. 9). In addition, free binding energies for
compound 7f and E7010 were found to be -13.00 and -9.15 respectively. In spite of having
structural similarities with co-crystal ligand (E7010), conjugate 7f exhibited unique binding
poses (Fig. 10A). 1-(3-Phenoxybenzyl)-1H-1,2,3-triazole-4-carboxylic acid and methoxybenzene
sulfonamide moieties present in conjugate 7f and E7010 respectively protrude towards the α-
subunit, N2-substituted phenylpyridine-2,3-diamine motifs present in both the ligands precisely
fit into the β-subunit, pyridine-2,3-diamine ring present in the conjugate 7f is deeply embedded

in to the β-subunit compared to same ring present in E7010. Methoxy oxygen present in
conjugate 7f shows hydrogen bonding with amide protons of Leu252 amino acid residue of β-
subunit (red dash lines in Fig. 10B) and bond length is recorded as 2.64 Å. Further, conjugate 7f
showed several hydrophobic interactions with the side chain of Gln136, Asn167, Phe169,
Tyr202, Val238, Cys241, Leu242, Leu248, Leu255, Asn258, Met259, Lys352, Thr376, Ile378
amino acid residues of β-subunit and Asn101, Val181 residues present in α-subunit. In addition,
the same conjugate showed interactions with peptide backbones of Gly237, Val238, Thr239,
Ala250, Asn251, Leu252, Leu254, Leu255, Thr314, Val315, Ala316, Ala317, Val318, Asn350,
Val351, Lys352, Thr353, Ala354, Thr376, Phe377 and Ile378 residues present in the β-subunit
(Fig. 10B).
Figure 10: A) Binding pose comparison of conjugate 7f with co-crystal ligand (E70), B) Binding
pose of conjugate 7f, including hydrogen bonding and hydrophobic interactions in colchicine
binding pocket of tubulin where protein is shown as a cartoon representation. Conjugate 7f and
E7010 were shown in stick and coloured by the atom type. Carbon: cyan, yellow; oxygen: red;
hydrogen: white; nitrogen: blue; sulphur.
3. Conclusion
A series of substituted-1-benzyl-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-
carboxamides (7a‒al) were designed, synthesized and evaluated for their cytotoxic activity
against DU-145, A-549, MCF-7 and HeLa human cancer cell lines. Most of the compounds have
shown moderate to promising cytotoxicity. Interestingly, compounds with donating groups have
shown profound cytotoxic activity against A549 cell line. Among these, compound 7f was found

to be the most active congener against human lung cancer cells with IC50 value of 1.023 µM.
Interestingly, the cell cycle analysis has shown that these derivatives arrested the cells in G2/M
phase and also inhibited microtubule assembly effectively in comparison to standard E7010.
Moreover, Hoechst staining, mitochondrial membrane potential (∆Ψm) and Annexin V-FITC
assays in human lung cancer cells confirmed the induction of apoptosis. In addition, the
molecular docking studies revealed that 7f interacted efficiently with the tubulin protein via
binding at the E7010 binding site of the tubulin. On the basis of all these studies, it can be
concluded that conjugate 7f showed promising cytotoxicity by inhibiting tubulin polymerization.
This compound (7f) could be further explored in detail and has a potential to be considered as a
lead from this type of a scaffold.
4. Experimental Protocols
4.1. Materials and methods
All Chemicals and reagents were purchased from the commercial suppliers Alfa Aesar,
Sigma Aldrich and used without further purification. The reaction progress was monitored by
Thin layer chromatography (TLC) was performed using pre-coated silica gel 60 F254 MERCK.
TLC plates were visualized and analyzed by exposure to UV light or iodine vapors and aqueous
solution of ninhydrin. Column chromatography was performed with Merck flash silica gel with
60–120 mesh size. Melting points were determined on an Electro thermal melting point
1
apparatus and are uncorrected. Nuclear magnetic resonance spectra for H NMR was obtained on
Avance 300, 400 and 500 MHz and analyzed using MestReNova software and the chemical
shifts are reported in ppm from tetramethylsilane( 0 ppm) or the solvent resonance as the internal
standard (CDCl3 7.26 ppm, DMSO-d6 2.49 ppm) and for 13C NMR the chemical shifts are
reported in ppm from the solvent resonance as the internal standard (CDCl3 77ppm, DMSO-d6
39.3ppm). HRMS was performed on a Varian ESI- QTOF instrument.
4.2. General synthetic procedure for the preparation of compounds (7a‒7al)
To a solution of 1-substituted benzyl-1,2,3-triazole-4-carboxilic acid (15a‒f, 1 mmol) in
dry DMF, EDCI (1.2 mmol) and HOBT (1.2 mmol) were added and the reaction mixture was
stirred for 20 min. To the reaction mixture, 2-substituted anilinoaniline (10a‒i, 1 mmol) was
added and further stirred at room temperature for 12 h. The contents of the reaction mixture were

poured into ice-cold water (50 mL), extracted with ethyl acetate (50 mL). The organic layer was
washed with 5% HCl (2x20 mL), 5% NaHCO3 (2x20 mL), and brine solution (20 mL). The
organic layer was separated, dried over anhydrous Na2SO4, filtered and the solvent was removed
to give a crude product which was purified by chromatography (ethyl acetate/hexanes) to give
the desired product (7a‒al).
4.2.1.
N-(2-((4-methoxyphenyl)amino)pyridin-3-yl)-1-(3-phenoxybenzyl)-1H-1,2,3-triazole-4-
carboxamide (7a)
1
Light brown solid, Yield 82%, M.P: 138−140 °C; H NMR (500 MHz, CDCl3) δ 8.72 (s, 1H),
8.14 (dd, J1 = 4.8 Hz, J2 = 1.6 Hz, 1H), 8.09 (s, 1H), 7.75 (dd, J1 = 7.7 Hz, J2 = 1.6 Hz, 1H), 7.40
– 7.33 (m, 3H), 7.29 – 7.27 (m, 2H), 7.16 (t, J = 7.4 Hz, 1H), 7.04 – 6.98 (m, 4H), 6.96 (d, J =
1.9 Hz, 1H), 6.87 – 6.85 (m, 2H), 6.83 (dd, J1 = 7.7 Hz, J2 = 4.9 Hz, 1H), 6.69 (s, 1H), 5.56 (s,
2H), 3.78 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 158.92, 158.36, 156.27, 155.49, 150.71,
145.85, 143.01, 135.37, 133.69, 133.04, 130.76, 130.00, 126.00, 124.05, 122.63, 122.54, 119.42,
119.31, 118.91, 118.25, 115.20, 114.29, 55.54, 54.35; MS (ESI): m/z 493 [M+H]+; HRMS (ESI)
Calcd for C29H26N5O3 [M+H]+ 493.1983; Found: 493.1999.
4.2.2.
1-(3-Methoxybenzyl)-N-(2-((4-methoxyphenyl)amino)pyridin-3-yl)-1H-1,2,3-triazole-4-
carboxamide (7b)
White solid, Yield 81%, M.P: 145−147 °C; 1H NMR (300 MHz, CDCl3) δ 8.73 (s, 1H), 8.13 (dd,
J1 = 4.9 Hz, J2 = 1.6 Hz, 1H), 8.06 (s, 1H), 7.74 (dd, J1 = 7.7 Hz, J2 = 1.5 Hz, 1H), 7.33 (t, J =
7.6 Hz, 1H), 7.30 – 7.26 (m, 2H), 6.96 – 6.87 (m, 2H), 6.87 – 6.82 (m, 3H), 6.82 – 6.78 (m, 1H),
13
6.72 (s, 1H), 5.55 (s, 2H), 3.81 (s, 3H), 3.78 (s, 3H); C NMR (125 MHz, CDCl3) δ 160.27,
158.96, 155.47, 150.72, 145.82, 142.96, 134.92, 133.74, 133.03, 130.51, 125.98, 122.51, 120.50,
119.35, 115.19, 114.51, 114.29, 114.17, 55.55, 55.38, 54.65; MS (ESI): m/z 431 [M+H]+; HRMS
(ESI) Calcd for C29H26N5O3 [M+H]+ 431.1826; Found: 431.1849.
4.2.3.
1-(4-Methoxybenzyl)-N-(2-((4-methoxyphenyl)amino)pyridin-3-yl)-1H-1,2,3-triazole-4-
carboxamide (7c)
White solid, Yield 82%, M.P: 170−172 °C; 1H NMR (500 MHz, CDCl3) δ 8.72 (s, 1H), 8.12 (dd,
J1 = 4.9 Hz, J2 = 1.6 Hz, 1H), 8.01 (s, 1H), 7.73 (dd, J1 = 7.7 Hz, J2 = 1.5 Hz, 1H), 7.27 (d, J =

1.4 Hz, 2H), 7.25 (d, J = 3.5 Hz, 2H), 6.95 – 6.90 (m, 2H), 6.87 – 6.83 (m, 2H), 6.81 (dd, J1 =
13
7.7 Hz, J2 = 4.9 Hz, 1H), 6.73 (s, 1H), 5.51 (s, 2H), 3.82 (s, 3H), 3.77 (s, 3H); C NMR (125
MHz, CDCl3) δ 160.31, 159.00, 155.49, 150.71, 145.77, 142.85, 133.73, 133.04, 130.01, 125.73,
125.45, 122.52, 119.38, 115.18, 114.76, 114.30, 55.56, 55.42, 54.30; MS (ESI): m/z 431
[M+H]+; HRMS (ESI) Calcd for C29H26N5O3 [M+H]+ 431.1826; Found: 431.1847.
4.2.4.
1-(4-Fluorobenzyl)-N-(2-((4-methoxyphenyl)amino)pyridin-3-yl)-1H-1,2,3-triazole-4-
carboxamide (7d)
White solid, Yield 83%, M.P: 159−161 °C; 1H NMR (500 MHz, CDCl3) δ 8.73 (s, 1H), 8.13 (dd,
J1 = 4.8 Hz, J2 = 1.3 Hz, 1H), 8.06 (s, 1H), 7.76 – 7.72 (m, 1H), 7.31 (dd, J1 = 8.5 Hz, J2 = 5.2
Hz, 2H), 7.27 (d, J = 4.1 Hz, 2H), 7.10 (dd, J1 = 14.2 Hz, J2 = 5.7 Hz, 2H), 6.85 (d, J = 8.9 Hz,
13
2H), 6.82 (dd, J1 = 7.7 Hz, J2 = 4.9 Hz, 1H), 6.71 (s, 1H), 5.56 (s, 2H), 3.78 (s, 3H); C NMR
(125 MHz, CDCl3) δ 164.10, 162.12, 158.88, 155.50, 150.68, 145.85, 143.06, 133.72, 133.04,
130.31 (d, JC-F = 8.4 Hz), 125.86, 122.49, 119.34, 116.44 (d, JC-F = 21.9 Hz), 115.23, 114.30,
55.55, 53.94; MS (ESI): m/z 419 [M+H]+; HRMS (ESI) Calcd for C29H26N5O3 [M+H]+
419.1626; Found: 419.1649.
4.2.5.
N-(2-((4-methoxyphenyl)amino)pyridin-3-yl)-1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-
triazole-4-carboxamide (7e)
White solid, Yield 81%, M.P: 172−174 °C; 1H NMR (500 MHz, CDCl3) δ 8.74 (s, 1H), 8.13 (dd,
J1 = 4.8 Hz, J2 = 1.5 Hz, 1H), 8.08 (s, 1H), 7.75 (dd, J1 = 7.7 Hz, J2 = 1.5 Hz, 1H), 7.28 – 7.26
(m, 2H), 6.87 – 6.83 (m, 2H), 6.82 (dd, J1 = 7.7 Hz, J2 = 4.9 Hz, 1H), 6.73 (s, 1H), 6.54 (s, 2H),
13
5.49 (s, 2H), 3.85 (d, J = 1.0 Hz, 9H), 3.77 (s, 3H); C NMR (125 MHz, CDCl3) δ 158.96,
155.46, 153.88, 150.66, 145.85, 142.92, 138.65, 133.68, 132.98, 128.87, 125.88, 122.48, 119.33,
115.23, 114.26, 105.61, 60.91, 56.26, 55.53, 55.02; MS (ESI): m/z 491 [M+H]+; HRMS (ESI)
Calcd for C29H26N5O3 [M+H]+ 491.2037; Found: 491.2055.
4.2.6. N-(2-((3,5-dimethoxyphenyl)amino)pyridin-3-yl)-1-(3-phenoxybenzyl)-1H-1,2,3-triazole-4-
carboxamide (7f)
1
Brown solid, Yield 78%, M.P: 121−123 °C; H NMR (300 MHz, CDCl3) δ 8.87 (s, 1H), 8.21 –
8.13 (m, 1H), 8.08 (s, 1H), 7.83 (d, J = 6.6 Hz, 1H), 7.35 (q, J = 7.4 Hz, 3H), 7.14 (t, J = 7.4 Hz,

1H), 7.07 – 6.93 (m, 6H), 6.89 (dd, J1 = 7.7 Hz, J2 = 4.9 Hz, 1H), 6.56 (d, J = 2.1 Hz, 2H), 6.11
13
(t, J = 2.0 Hz, 1H), 5.52 (s, 2H), 3.73 (s, 6H); C NMR (125 MHz, CDCl3) δ 161.14, 158.95,
158.36, 156.27, 149.35, 145.69, 142.96, 142.73, 135.36, 132.87, 130.76, 129.99, 126.04, 124.04,
122.63, 120.85, 119.42, 118.91, 118.25, 116.47, 97.86, 94.58, 55.31, 54.36; MS (ESI): m/z 523
[M+H]+; HRMS (ESI) Calcd for C29H26N5O3 [M+H]+ 523.2088; Found: 523.2113.
4.2.7. N-(2-((3,5-dimethoxyphenyl)amino)pyridin-3-yl)-1-(3-methoxybenzyl)-1H-1,2,3-triazole-4-
carboxamide (7g)
White solid, Yield 84%, M.P: 152−154 °C; 1H NMR (500 MHz, CDCl3) δ 8.83 (s, 1H), 8.19 (dd,
J1 = 4.8 Hz, J2 = 1.7 Hz, 1H), 8.07 (s, 1H), 7.83 (dd, J1 = 7.8 Hz, J2 = 1.6 Hz, 1H), 7.36 – 7.30
(m, 1H), 6.91 (qd, J = 8.8, 3.3 Hz, 4H), 6.85 – 6.81 (m, 1H), 6.56 (d, J = 2.2 Hz, 2H), 6.12 (t, J =
13
2.2 Hz, 1H), 5.54 (s, 2H), 3.81 (s, 3H), 3.75 (s, 6H); C NMR (75 MHz, CDCl3) δ 161.11,
160.24, 159.00, 149.37, 145.68, 142.88, 142.73, 134.90, 132.92, 130.51, 126.04, 120.82, 120.51,
116.44, 114.51, 114.14, 97.82, 94.53, 55.37, 55.31, 54.65; MS (ESI): m/z 461 [M+H]+; HRMS
(ESI) Calcd for C29H26N5O3 [M+H]+ 461.1932; Found: 461.1948.
4.2.8. N-(2-((2,4-dimethoxyphenyl)amino)pyridin-3-yl)-1-(3-phenoxybenzyl)-1H-1,2,3-triazole-4-
carboxamide (7h)
White solid, Yield 84%, M.P: 141−143 °C; 1H NMR (500 MHz, CDCl3) δ 8.72 (s, 1H), 8.13 (dd,
J1 = 4.9 Hz, J2 = 1.5 Hz, 1H), 8.10 (s, 1H), 7.86 (dd, J1 = 12.7 Hz, J2 = 4.9 Hz, 2H), 7.36 (q, J =
7.7 Hz, 3H), 7.15 (t, J = 7.4 Hz, 1H), 7.05 – 6.94 (m, 5H), 6.82 (dd, J1 = 7.5 Hz, J2 = 5.1 Hz,
2H), 6.53 – 6.46 (m, 2H), 5.55 (s, 2H), 3.80 (s, 3H), 3.79 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
158.91, 158.37, 156.30, 155.36, 150.33, 150.28, 145.48, 143.25, 135.46, 132.70, 130.76, 130.00,
125.95, 124.05, 123.65, 122.64, 120.53, 119.85, 119.43, 118.92, 118.26, 115.19, 103.94, 99.06,
55.84, 55.63, 54.37; MS (ESI): m/z 523 [M+H]+; HRMS (ESI) Calcd for C29H26N5O3 [M+H]+
523.2088; Found: 523.2114.
4.2.9.
N-(2-((2,4-dimethoxyphenyl)amino)pyridin-3-yl)-1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-
triazole-4-carboxamide (7i)
White solid, Yield 76%, M.P: 184−186°C; 1H NMR (300 MHz, CDCl3) δ 8.73 (s, 1H), 8.13 (dd,
J1 = 5.7 Hz, J2 = 2.4 Hz, 2H), 7.91 – 7.79 (m, 2H), 6.83 (dd, J1 = 7.8 Hz, J2 = 5.0 Hz, 2H), 6.55

(s, 2H), 6.52 – 6.46 (m, 2H), 5.51 (s, 2H), 3.85 (d, J = 1.5 Hz, 9H), 3.81 (s, 3H), 3.79 (s, 3H); 13C
NMR (75 MHz, CDCl3) δ 158.94, 155.39, 153.90, 150.35, 150.21, 145.48, 143.17, 132.58,
128.94, 125.98, 125.83, 123.60, 120.60, 119.89, 115.24, 105.62, 103.96, 99.06, 60.92, 56.27,
55.82, 55.61, 55.03; MS (ESI): m/z 521 [M+H]+; HRMS (ESI) Calcd for C29H26N5O3 [M+H]+
521.2143; Found: 521.2178.
4.2.10. N-(2-((2,4-dimethoxyphenyl)amino)pyridin-3-yl)-1-(3-methoxybenzyl)-1H-1,2,3-triazole-
4-carboxamide (7j)
White solid, Yield 81%, M.P: 129−131 °C; 1H NMR (500 MHz, CDCl3) δ 8.71 (s, 1H), 8.13 (dd,
J1 = 4.9 Hz, J2 = 1.7 Hz, 1H), 8.08 (s, 1H), 7.86 (dd, J1 = 11.3 Hz, J2 = 5.0 Hz, 2H), 7.35 – 7.30
(m, 1H), 6.93 (dd, J1 = 8.1 Hz, J2 = 2.2 Hz, 1H), 6.89 (d, J = 7.6 Hz, 1H), 6.85 – 6.80 (m, 3H),
13
6.51 – 6.47 (m, 2H), 5.55 (s, 2H), 3.81 (s, 6H), 3.79 (s, 3H); C NMR (75 MHz, CDCl3) δ
160.27, 158.93, 155.35, 150.33, 150.27, 145.44, 143.18, 134.98, 132.66, 130.49, 125.92, 123.68,
120.49, 119.88, 115.17, 114.51, 114.13, 103.95, 102.67, 99.06, 55.83, 55.61, 55.37, 54.65; MS
(ESI): m/z 461 [M+H]+; HRMS (ESI) Calcd for C29H26N5O3 [M+H]+ 461.1932; Found:
461.1952.
4.2.11. N-(2-((2,4-dimethoxyphenyl)amino)pyridin-3-yl)-1-(4-methoxybenzyl)-1H-1,2,3-triazole-
4-carboxamide (7k)
1
Light brown solid, Yield 80%, M.P: 168−170 °C; H NMR (500 MHz, CDCl3) δ 8.70 (s, 1H),
8.12 (dd, J1 = 4.9 Hz, J2 = 1.7 Hz, 1H), 8.04 (s, 1H), 7.89 – 7.82 (m, 2H), 7.30 – 7.26 (m, 2H),
6.95 – 6.90 (m, 2H), 6.81 (dd, J1 = 7.6 Hz, J2 = 5.0 Hz, 2H), 6.51 – 6.47 (m, 2H), 5.52 (s, 2H),
13
3.82 (s, 3H), 3.80 (s, 3H), 3.79 (s, 3H); C NMR (75 MHz, CDCl3) δ 160.26, 159.00, 155.31,
150.27, 145.43, 143.05, 132.70, 130.00, 125.69, 125.50, 123.62, 120.51, 119.84, 115.15, 114.72,
114.24, 103.88, 99.02, 55.81, 55.61, 55.41, 54.27; MS (ESI): m/z 461 [M+H]+; HRMS (ESI)
Calcd for C29H26N5O3 [M+H]+ 461.1932; Found: 461.1961.
4.2.12. N-(2-((2,4-dimethoxyphenyl)amino)pyridin-3-yl)-1-(4-fluorobenzyl)-1H-1,2,3-triazole-4-
carboxamide (7l)
White solid, Yield 83%, M.P: 192−194 °C; 1H NMR (500 MHz, CDCl3) δ 8.70 (s, 1H), 8.13 (dd,
J1 = 4.9 Hz, J2 = 1.5 Hz, 1H), 8.09 (s, 1H), 7.87 (dd, J1 = 7.7 Hz, J2 = 1.5 Hz, 1H), 7.84 (d, J =

8.4 Hz, 1H), 7.35 – 7.30 (m, 2H), 7.14 – 7.08 (m, 2H), 6.85 – 6.79 (m, 2H), 6.53 – 6.47 (m, 2H),
13
5.57 (s, 2H), 3.80 (s, 3H), 3.79 (s, 3H); C NMR (75 MHz, CDCl3) δ 158.86, 155.38, 150.33,
150.25, 145.49, 132.65, 131.27, 130.29 (d, JC-F = 8.4 Hz), 129.56, 125.79, 123.65, 120.54,
119.86, 116.57, 116.28, 115.21, 103.98, 99.08, 55.83, 55.61, 53.95; MS (ESI): m/z 449 [M+H]+;
HRMS (ESI) Calcd for C29H26N5O3 [M+H]+ 449.1732; Found: 449.1758.
4.2.13. N-(2-((3,4-dimethoxyphenyl)amino)pyridin-3-yl)-1-(3-phenoxybenzyl)-1H-1,2,3-triazole-
4-carboxamide (7m)
1
Light yellow solid, Yield 77%, M.P: 155−157 °C; H NMR (300 MHz, CDCl3) δ 8.79 (s, 1H),
8.13 (dd, J1 = 4.8 Hz, J2 = 1.5 Hz, 1H), 8.07 (s, 1H), 7.76 (dd, J1 = 7.7 Hz, J2 = 1.4 Hz, 1H), 7.40
– 7.30 (m, 3H), 7.14 (t, J = 7.4 Hz, 1H), 7.04 – 6.92 (m, 6H), 6.83 (ddd, J1 = 18.2 Hz, J2 = 12.0
Hz, J3 = 5.5 Hz, 4H), 5.52 (s, 2H), 3.83 (s, 6H); 13C NMR (125 MHz, CDCl3) δ 158.89, 158.36,
156.24, 150.54, 149.15, 145.86, 144.98, 142.97, 135.32, 134.17, 133.01, 130.75, 129.98, 125.96,
124.04, 122.61, 119.56, 119.40, 118.90, 118.23, 115.42, 112.89, 111.75, 105.96, 56.19, 55.84,
54.36; MS (ESI): m/z 523 [M+H]+; HRMS (ESI) Calcd for C29H26N5O3 [M+H]+ 523.2088;
Found: 523.2111.
4.2.14. N-(2-((3,4-dimethoxyphenyl)amino)pyridin-3-yl)-1-(3-methoxybenzyl)-1H-1,2,3-triazole-
4-carboxamide (7n)
1
Yellow solid, Yield 85%, M.P: 160−162 °C; H NMR (500 MHz, CDCl3) δ 8.77 (s, 1H), 8.14
(dd, J1 = 4.8 Hz, J2 = 1.5 Hz, 1H), 8.07 (s, 1H), 7.76 (dd, J1 = 7.7 Hz, J2 = 1.4 Hz, 1H), 7.33 (t, J
= 7.9 Hz, 1H), 6.98 (d, J = 2.3 Hz, 1H), 6.93 (dd, J1 = 8.3 Hz, J2 = 2.3 Hz, 1H), 6.89 (d, J = 7.6
Hz, 1H), 6.85 (ddd, J1 = 7.4 Hz, J2 = 6.7 Hz, J3 = 2.6 Hz, 3H), 6.81 – 6.76 (m, 2H), 5.55 (s, 2H),
13
3.84 (d, J = 2.5 Hz, 6H), 3.81 (s, 3H); C NMR (75 MHz, CDCl3) δ 160.25, 158.95, 150.57,
149.12, 145.85, 144.94, 142.90, 134.87, 134.17, 133.06, 130.52, 125.98, 120.50, 119.56, 115.40,
114.48, 114.19, 112.87, 111.71, 105.92, 56.20, 55.85, 55.38, 54.67; MS (ESI): m/z 461 [M+H]+;
HRMS (ESI) Calcd for C29H26N5O3 [M+H]+ 461.1932; Found: 461.1947.
4.2.15.
1-(3-Phenoxybenzyl)-N-(2-((3,4,5-trimethoxyphenyl)amino)pyridin-3-yl)-1H-1,2,3-
triazole-4-carboxamide (7o)

1
Brown solid, Yield 83%, M.P: 118−120 °C; H NMR (300 MHz, CDCl3) δ 8.79 (s, 1H), 8.18
(dd, J1 = 4.9 Hz, J2 = 1.6 Hz, 1H), 8.09 (s, 1H), 7.78 (dd, J1 = 7.7 Hz, J2 = 1.4 Hz, 1H), 7.36 (td,
J = 8.0, 4.2 Hz, 3H), 7.15 (t, J = 7.4 Hz, 1H), 7.05 – 6.94 (m, 5H), 6.89 (dd, J1 = 7.7 Hz, J2 = 4.8
13
Hz, 1H), 6.87 (s, 1H), 6.61 (s, 2H), 5.55 (s, 2H), 3.82 (s, 6H), 3.79 (s, 3H); C NMR (75 MHz,
CDCl3) δ 158.97, 158.35, 156.24, 153.35, 149.98, 145.82, 142.90, 136.84, 135.35, 133.19,
130.77, 130.00, 126.05, 124.06, 122.64, 120.17, 119.40, 118.91, 118.27, 115.96, 97.92, 60.94,
56.04, 54.38, 29.73; MS (ESI): m/z 553 [M+H]+; HRMS (ESI) Calcd for C29H26N5O3 [M+H]+
55.2194.; Found: 553.2224.
4.2.16. 1-(3,4,5-Trimethoxybenzyl)-N-(2-((3,4,5-trimethoxyphenyl)amino)pyridin-3-yl)-1H-1,2,3-
triazole-4-carboxamide (7p)
1
Brown solid, Yield 76%, M.P: 110−112 °C; H NMR (300 MHz, CDCl3) δ 8.81 (s, 1H), 8.20
(dd, J1 = 4.8 Hz, J2 = 1.5 Hz, 1H), 8.11 (s, 1H), 7.85 – 7.76 (m, 1H), 6.91 (dd, J1 = 7.7 Hz, J2 =
4.9 Hz, 1H), 6.87 (s, 1H), 6.61 (s, 2H), 6.56 (s, 2H), 5.52 (s, 2H), 3.87 (d, J = 1.9 Hz, 9H), 3.83
13
(s, 6H), 3.80 (s, 3H); C NMR (125 MHz, CDCl3) δ 158.98, 153.92, 153.38, 149.87, 145.80,
142.82, 138.78, 136.83, 133.53, 133.07, 128.77, 125.87, 120.30, 116.06, 105.71, 97.93, 60.91,
56.28, 56.05, 55.07, 29.70; MS (ESI): m/z 551 [M+H]+; HRMS (ESI) Calcd for C29H26N5O3
[M+H]+ 551.2249; Found: 551.2277.
4.2.17.
1-(4-Methoxybenzyl)-N-(2-((3,4,5-trimethoxyphenyl)amino)pyridin-3-yl)-1H-1,2,3-
triazole-4-carboxamide (7q)
1
Brown solid, Yield 80%, M.P: 130−132 °C; H NMR (300 MHz, CDCl3) δ 8.77 (s, 1H), 8.18
(dd, J1 = 4.8 Hz, J2 = 1.5 Hz, 1H), 8.02 (s, 1H), 7.76 (dd, J1 = 7.7 Hz, J2 = 1.3 Hz, 1H), 7.28 (d, J
= 7.4 Hz, 2H), 6.93 (d, J = 8.6 Hz, 2H), 6.88 (dd, J1 = 7.9 Hz, J2 = 5.0 Hz, 1H), 6.85 (s, 1H),
6.60 (s, 2H), 5.52 (s, 2H), 3.82 (s, 3H), 3.81 (s, 6H), 3.78 (s, 3H); 13C NMR (75 MHz, CDCl3) δ
160.27, 159.08, 153.30, 150.00, 145.77, 142.70, 136.85, 133.24, 130.02, 125.81, 125.38, 120.12,
116.09, 115.89, 114.71, 97.84, 60.93, 56.02, 55.41, 54.29; MS (ESI): m/z 491 [M+H]+; HRMS
(ESI) Calcd for C29H26N5O3 [M+H]+ 491.2037; Found: 491.2059.
4.2.18.
1-(3-Methoxybenzyl)-N-(2-((3,4,5-trimethoxyphenyl)amino)pyridin-3-yl)-1H-1,2,3-
triazole-4-carboxamide (7r)

White solid, Yield 82%, M.P: 136−138°C; 1H NMR (500 MHz, CDCl3) δ 8.77 (s, 1H), 8.18 (dd,
J1 = 4.8 Hz, J2 = 1.7 Hz, 1H), 8.07 (s, 1H), 7.77 (dd, J1 = 7.8 Hz, J2 = 1.6 Hz, 1H), 7.32 (dd, J1 =
15.0 Hz, J2 = 7.1 Hz, 1H), 6.93 (dd, J1 = 8.3 Hz, J2 = 2.1 Hz, 1H), 6.91 – 6.87 (m, 2H), 6.83 (s,
13
2H), 6.61 (s, 2H), 5.55 (s, 2H), 3.81 (s, 6H), 3.81 (s, 3H), 3.78 (s, 3H); C NMR (125 MHz,
CDCl3) δ 160.27, 159.00, 153.35, 149.98, 145.81, 142.85, 136.85, 134.87, 133.49, 133.15,
130.53, 126.01, 120.49, 120.17, 118.35, 115.94, 114.46, 114.23, 97.94, 60.91, 56.04, 55.36,
54.66; MS (ESI): m/z 491 [M+H]+; HRMS (ESI) Calcd for C29H26N5O3 [M+H]+ 491.2037;
Found: 491.2059.
4.2.19. 1-(4-Fluorobenzyl)-N-(2-((3,4,5-trimethoxyphenyl)amino)pyridin-3-yl)-1H-1,2,3-triazole-
4-carboxamide (7s)
White solid, Yield 86%, M.P: 169−171 °C; 1H NMR (500 MHz, CDCl3) δ 8.76 (s, 1H), 8.18 (dd,
J1 = 4.8 Hz, J2 = 1.7 Hz, 1H), 8.06 (s, 1H), 7.78 (dd, J1 = 7.8 Hz, J2 = 1.6 Hz, 1H), 7.32 (ddd, J1
= 6.6 Hz, J2 = 4.3 Hz, J3 = 1.7 Hz, 2H), 7.14 – 7.08 (m, 2H), 6.89 (dd, J1 = 7.8 Hz, J2 = 4.9 Hz,
13
1H), 6.81 (s, 1H), 6.59 (s, 2H), 5.56 (s, 2H), 3.81 (s, 6H), 3.78 (s, 3H); C NMR (100 MHz,
CDCl3) δ 163.14 (d, JC-F = 249.5 Hz), 158.91, 153.38, 149.91, 145.80, 142.96, 136.83, 133.55,
133.15, 130.33 (d, JC-F = 8.4 Hz), 129.40, 125.87, 120.25, 116.47 (d, JC-F = 21.8 Hz), 116.04,
97.97, 60.94, 56.07, 53.99.; MS (ESI): m/z 479 [M+H]+; HRMS (ESI) Calcd for C29H26N5O3
[M+H]+ 479.1838; Found: 479.1859.
4.2.20. 1-(3-Phenoxybenzyl)-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamide
(7t)
White solid, Yield 76%, M.P: 150−152 °C; 1H NMR (300 MHz, CDCl3) δ 8.79 (s, 1H), 8.18 (d,
J = 4.7 Hz, 1H), 8.08 (s, 1H), 7.81 (d, J = 7.6 Hz, 1H), 7.43 – 7.22 (m, 7H), 7.20 – 7.11 (m, 1H),
7.10 – 6.81 (m, 8H), 5.54 (s, 2H); 13C NMR (75 MHz, CDCl3) δ 158.92, 158.37, 156.26, 149.65,
145.71, 142.97, 140.78, 135.35, 132.92, 130.77, 130.00, 128.94, 126.03, 124.06, 122.64, 122.18,
121.78, 120.35, 119.62, 119.43, 118.93, 118.25, 116.14, 54.38; MS (ESI): m/z 463 [M+H]+;
HRMS (ESI) Calcd for C29H26N5O3 [M+H]+ 463.1877; Found: 463.1892.
4.2.21.
N-(2-(phenylamino)pyridin-3-yl)-1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazole-4-
carboxamide (7u)

1
White solid, Yield 75%, M.P: 142−144 °C; H NMR (500 MHz, CDCl3); δ 8.81 (s, 1H), 8.18
(dd, J1 = 4.7 Hz, J2 = 1.3 Hz, 1H), 8.09 (s, 1H), 7.82 (d, J = 7.7 Hz, 1H), 7.34 (d, J = 7.8 Hz,
2H), 7.28 (d, J = 7.5 Hz, 2H), 6.97 (t, J = 7.3 Hz, 1H), 6.92 (s, 1H), 6.89 (dd, J1 = 7.8 Hz, J2 =
13
4.9 Hz, 1H), 6.54 (s, 2H), 5.49 (s, 2H), 3.85 (s, 9H); C NMR (125 MHz, CDCl3); δ 158.96,
153.91, 149.59, 145.69, 142.91, 140.82, 138.74, 132.81, 128.93, 128.83, 125.91, 122.18, 120.44,
119.58, 116.18, 105.66, 60.91, 56.28, 55.04; MS (ESI): m/z 461 [M+H]+; HRMS (ESI) Calcd for
C29H26N5O3 [M+H]+ 461.1932; Found: 461.1948.
4.2.22. 1-(3-Methoxybenzyl)-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamide
(7v)
White solid, Yield 76%, M.P: 124−126 °C; 1H NMR (500 MHz, CDCl3) δ 8.79 (s, 1H), 8.18 (dd,
J1 = 4.8 Hz, J2 = 1.7 Hz, 1H), 8.06 (s, 1H), 7.80 (dd, J1 = 7.8 Hz, J2 = 1.6 Hz, 1H), 7.36 – 7.31
(m, 3H), 7.29 – 7.26 (m, 2H), 6.99 – 6.95 (m, 1H), 6.93 (dd, J1 = 8.6 Hz, J2 = 2.6 Hz, 2H), 6.88
(dd, J1 = 7.8 Hz, J2 = 4.8 Hz, 2H), 6.84 – 6.81 (m, 1H), 5.54 (s, 2H), 3.80 (s, 3H); 13C NMR (75
MHz, CDCl3) δ 158.99, 149.69, 145.67, 142.89, 140.80, 134.90, 132.97, 130.51, 128.92, 126.04,
122.14, 120.51, 120.33, 119.61, 116.09, 114.49, 114.16, 55.38, 54.65; MS (ESI): m/z 401
[M+H]+; HRMS (ESI) Calcd for C29H26N5O3 [M+H]+ 401.1721; Found: 401.1748.
4.2.23. 1-(4-Methoxybenzyl)-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamide
(7w)
1
White solid, Yield 79%, M.P: 145−147 °C; H NMR (500 MHz, CDCl3) δ 8.79 (s, 1H), 8.23 –
8.13 (m, 1H), 8.02 (s, 1H), 7.80 (dd, J1 = 7.8 Hz, J2 = 1.4 Hz, 1H), 7.34 (d, J = 7.7 Hz, 2H), 7.30
– 7.25 (m, 4H), 6.97 (t, J = 7.3 Hz, 1H), 6.93 (dd, J1 = 6.7 Hz, J2 = 1.9 Hz, 3H), 6.88 (dd, J1 =
13
7.8 Hz, J2 = 4.9 Hz, 1H), 5.51 (s, 2H), 3.82 (s, 3H); C NMR (75 MHz, CDCl3) δ 160.28,
159.04, 149.71, 145.66, 142.79, 140.81, 132.95, 130.03, 128.91, 125.78, 125.41, 122.12, 120.34,
119.60, 116.07, 114.73, 55.42, 54.29; MS (ESI): m/z 401 [M+H]+; HRMS (ESI) Calcd for
C29H26N5O3 [M+H]+ 401.1721; Found: 401.1739.
4.2.24. 1-(4-Fluorobenzyl)-N-(2-(phenylamino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamide
(7x)

White solid, Yield 77%, M.P: 165−167 °C; 1H NMR (500 MHz, CDCl3) δ 8.79 (s, 1H), 8.18 (dd,
J1 = 4.8 Hz, J2 = 1.6 Hz, 1H), 8.06 (s, 1H), 7.81 (dd, J1 = 7.8 Hz, J2 = 1.6 Hz, 1H), 7.32 (dd, J1 =
15.6 Hz, J2 = 6.8 Hz, 4H), 7.28 (d, J = 7.4 Hz, 2H), 7.13 – 7.07 (m, 2H), 6.97 (t, J = 7.3 Hz, 1H),
6.89 (dd, J1 = 7.7 Hz, J2 = 4.8 Hz, 2H), 5.55 (s, 2H); 13C NMR (125 MHz, CDCl3) δ 163.12 (d, J
= 249.2 Hz), 158.91, 149.66, 145.68, 143.02, 140.82, 132.94, 130.33 (d, J = 8.4 Hz), 129.46,
128.94, 125.91, 122.19, 120.39, 119.61, 116.45 (d, J = 21.9 Hz), 116.15, 53.96; MS (ESI): m/z
389 [M+H]+; HRMS (ESI) Calcd for C29H26N5O3 [M+H]+ 389.1521; Found: 389.1548.
4.2.25.
N-(2-((4-fluorophenyl)amino)pyridin-3-yl)-1-(3-phenoxybenzyl)-1H-1,2,3-triazole-4-
carboxamide (7y)
White solid, Yield 76%, M.P: 146−148 °C; 1H NMR (500 MHz, CDCl3) δ 8.77 (s, 1H), 8.16 (dd,
J1 = 4.9 Hz, J2 = 1.6 Hz, 1H), 8.09 (s, 1H), 7.75 (dd, J1 = 7.8 Hz, J2 = 1.6 Hz, 1H), 7.39 – 7.33
(m, 3H), 7.31 (ddd, J1 = 10.5 Hz, J2 = 5.3 Hz, J3 = 2.9 Hz, 2H), 7.15 (t, J = 7.4 Hz, 1H), 7.04 –
6.94 (m, 7H), 6.92 (s, 1H), 6.87 (dd, J1 = 7.7 Hz, J2 = 4.9 Hz, 1H), 5.56 (s, 2H).; 13C NMR (125
MHz, CDCl3) δ 158.92, 158.47 (d, JC-F = 240.7 Hz), 158.40, 156.25, 150.08, 145.82, 142.90,
136.69, 135.30, 133.11, 130.79, 130.01, 126.02, 124.07, 122.62, 121.78, 121.72, 119.76, 119.43,
118.95, 118.25, 115.86, 115.57, 115.39, 54.41; MS (ESI): m/z 481 [M+H]+; HRMS (ESI) Calcd
for C29H26N5O3 [M+H]+ 481.1783; Found: 481.1799.
4.2.26. N-(2-((4-fluorophenyl)amino)pyridin-3-yl)-1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazole-
4-carboxamide (7z)
White solid, Yield 75%, M.P: 190−192 °C; 1H NMR (300 MHz, CDCl3) δ 8.78 (s, 1H), 8.16 (dd,
J1 = 4.8 Hz, J2 = 1.5 Hz, 1H), 8.09 (s, 1H), 7.75 (dd, J1 = 7.7 Hz, J2 = 1.4 Hz, 1H), 7.35 – 7.27
(m, 2H), 6.97 (dd, J1 = 14.9 Hz, J2 = 6.1 Hz, 3H), 6.88 (dd, J1 = 7.7 Hz, J2 = 4.9 Hz, 1H), 6.55 (s,
13
2H), 5.50 (s, 2H), 3.86 (s, 9H); C NMR (125 MHz, CDCl3) δ 158.96, 158.46 (d, JC-F = 240.8
Hz), 153.94, 150.01, 145.79, 142.83, 138.81, 136.72, 133.02, 128.77, 125.88, 121.71 (d, JC-F
=
7.7 Hz), 119.86, 115.92, 115.48 (d, JC-F = 22.4 Hz), 105.72, 60.92, 56.30, 55.09; MS (ESI): m/z
479 [M+H]+; HRMS (ESI) Calcd for C29H26N5O3 [M+H]+ 479.1838; Found: 479.1853.
4.2.27.
N-(2-((4-fluorophenyl)amino)pyridin-3-yl)-1-(4-methoxybenzyl)-1H-1,2,3-triazole-4-
carboxamide (7aa)

White solid, Yield 75%, M.P: 162−164 °C; 1H NMR (300 MHz, CDCl3) δ 8.80 (s, 1H), 8.16 (dd,
J1 = 4.8 Hz, J2 = 1.6 Hz, 1H), 8.03 (s, 1H), 7.75 (dd, J1 = 7.7 Hz, J2 = 1.4 Hz, 1H), 7.36 – 7.30
(m, 2H), 7.28 (d, J = 2.9 Hz, 2H), 7.04 – 6.91 (m, 5H), 6.87 (dd, J1 = 7.7 Hz, J2 = 4.9 Hz, 1H),
5.53 (s, 2H), 3.84 (s, 3H); 13C NMR (100MHz, CDCl3) δ 160.32, 159.04, 158.42 (d, JC-F = 240.7
Hz), 150.10, 145.75, 142.72, 136.70, 133.16, 130.04, 125.79, 125.36, 121.73 (d, JC-F = 7.7 Hz),
119.76, 115.81, 115.46 (d, JC-F = 22.4 Hz), 114.76, 55.43, 54.34; MS (ESI): m/z 419 [M+H]+;
HRMS (ESI) Calcd for C29H26N5O3 [M+H]+ 419.1626; Found: 419.1641.
4.2.28.
1-(4-Fluorobenzyl)-N-(2-((4-fluorophenyl)amino)pyridin-3-yl)-1H-1,2,3-triazole-4-
carboxamide (7ab)
White solid, Yield 78%, M.P: 186−188 °C; 1H NMR (300 MHz, CDCl3) δ 8.76 (s, 1H), 8.15 (dd,
J1 = 4.9 Hz, J2 = 1.6 Hz, 1H), 8.06 (s, 1H), 7.74 (dd, J1 = 7.8 Hz, J2 = 1.5 Hz, 1H), 7.31 (ddd, J1
= 9.0 Hz, J2 = 5.4 Hz, J3 = 2.2 Hz, 4H), 7.16 – 7.06 (m, 2H), 7.03 – 6.93 (m, 2H), 6.92 (s, 1H),
13
6.87 (dd, J1 = 7.7 Hz, J2 = 4.9 Hz, 1H), 5.56 (s, 2H); C NMR (125 MHz, CDCl3) δ 164.14,
162.16, 159.43, 158.88, 157.51, 150.04, 145.82, 142.96, 136.70, 133.09, 130.32 (d, JC-F = 8.3
Hz), 129.40, 125.87, 121.72 (d, JC-F = 7.6 Hz), 119.80, 116.49 (d, JC-F = 21.9 Hz), 115.90,
115.48 (d, JC-F = 22.4 Hz), 54.01; MS (ESI): m/z 407.1426 [M+H]+; HRMS (ESI) Calcd for
C29H26N5O3 [M+H]+ 493.1983; Found: 407.1452.
4.2.29.
N-(2-((4-fluorophenyl)amino)pyridin-3-yl)-1-(3-methoxybenzyl)-1H-1,2,3-triazole-4-
carboxamide (7ac)
White solid, Yield 79%, M.P: 165−167 °C; 1H NMR (500 MHz, CDCl3) δ 8.77 (s, 1H), 8.15 (dd,
J1 = 4.9 Hz, J2 = 1.7 Hz, 1H), 8.06 (s, 1H), 7.74 (dd, J1 = 7.8 Hz, J2 = 1.6 Hz, 1H), 7.35 – 7.32
(m, 1H), 7.32 – 7.29 (m, 2H), 7.00 – 6.95 (m, 2H), 6.94 (s, 1H), 6.94 – 6.91 (m, 1H), 6.89 (d, J =
7.5 Hz, 1H), 6.86 (dd, J1 = 7.7 Hz, J2 = 4.9 Hz, 1H), 6.84 – 6.81 (m, 1H), 5.55 (s, 2H), 3.81 (s,
13
3H); C NMR (75 MHz, CDCl3) δ 160.27, 160.04, 158.97, 156.84, 150.07, 145.77, 142.84,
136.69, 134.83, 133.14, 130.55, 121.74 (d, JC-F = 7.7 Hz), 120.51, 119.74, 115.83, 115.47 (d, JC-F
= 22.4 Hz), 114.49, 114.21, 55.38, 54.70; MS (ESI): m/z 419 [M+H]+; HRMS (ESI) Calcd for
C29H26N5O3 [M+H]+ 419.1626; Found: 419.1650.
4.2.30. N-(2-((4-hydroxyphenyl)amino)pyridine-3-yl)-1-(3-phenoxybenzyl)-1H-1,2,3-triazole-4-
carboxamide (7ad)

1
Light brown solid, Yield 74%, M.P: 196−198 °C; H NMR (300 MHz, CDCl3 + DMSO-d6) δ
9.89 (s, 1H), 8.83 (s, 1H), 8.61 (s, 1H), 8.37 (d, J = 3.6 Hz, 1H), 8.09 (d, J = 7.4 Hz, 1H), 7.81 (s,
1H), 7.69 (dd, J1 = 9.2 Hz, J2 = 6.1 Hz, 4H), 7.55 (d, J = 8.6 Hz, 2H), 7.47 (t, J = 7.4 Hz, 1H),
7.43 – 7.22 (m, 6H), 7.09 (dd, J1 = 10.8 Hz, J2 = 5.7 Hz, 3H), 5.92 (s, 2H); 13C NMR (100 MHz,
CDCl3) δ 158.32, 156.52, 155.33, 151.48, 149.79, 143.52, 142.19, 135.68, 132.50, 131.77,
129.37, 128.84, 125.94, 122.65, 121.76, 121.15, 118.25, 117.88, 117.42, 117.25, 114.22, 112.85,
52.52; MS (ESI): m/z 479 [M+H]+; HRMS (ESI) Calcd for C29H26N5O3 [M+H]+ 479.1826;
Found: 479.1844.
4.2.31.
N-(2-((4-hydroxyphenyl)amino)pyridin-3-yl)-1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-
triazole-4-carboxamide (7ae)
1
Light brown solid, Yield 73%, M.P: 191−193 °C; H NMR (300 MHz, CDCl3 + DMSO-d6) δ
9.86 (s, 1H), 8.69 (s, 1H), 8.55 (s, 1H), 7.97 (d, J = 2.8 Hz, 1H), 7.73 (d, J = 7.7 Hz, 2H), 7.30 –
7.18 (m, 2H), 6.72 (dd, J1 = 9.5 Hz, J2 = 7.0 Hz, 5H), 5.56 (s, 2H), 3.84 (d, J = 2.5 Hz, 6H), 3.76
(d, J = 2.7 Hz, 3H); 13C NMR (100 MHz, CDCl3 + DMSO-d6) δ 158.81, 152.95, 152.08, 150.28,
144.35, 142.66, 137.59, 132.88, 132.17, 129.13, 125.99, 121.81, 118.69, 114.88, 113.56, 105.11,
60.06, 55.59, 53.96; MS (ESI): m/z 477 [M+H]+; HRMS (ESI) Calcd for C29H26N5O3 [M+H]+
477.1881; Found: 477.1898.
4.2.32. N-(2-((4-hydroxyphenyl)amino)pyridin-3-yl)-1-(3-methoxybenzyl)-1H-1,2,3-triazole-4-
carboxamide (7af)
1
Light brown solid, Yield 77%, M.P: 175−177 °C; H NMR (400 MHz, CDCl3) δ 9.56 (s, 1H),
8.56 (s, 1H), 8.29 (d, J = 4.8 Hz, 1H), 8.03 (d, J = 6.7 Hz, 1H), 7.77 (t, J = 8.2 Hz, 1H), 7.39 (s,
1H), 7.35 – 7.17 (m, 3H), 6.97 – 6.83 (m, 3H), 6.77 (d, J = 8.5 Hz, 3H), 5.58 (s, 2H), 3.80 (s,
13
3H); C NMR (100 MHz, CDCl3) δ 159.25, 158.66, 151.97, 150.13, 143.95, 142.55, 135.09,
132.79, 132.02, 129.48, 126.02, 125.89, 121.70, 119.63, 118.64, 114.73, 113.37, 113.28, 54.56,
53.41; MS (ESI): m/z 417 [M+H]+; HRMS (ESI) Calcd for C29H26N5O3 [M+H]+ 417.1670;
Found: 417.1683.
4.2.33. N-(2-((4-hydroxyphenyl)amino)pyridin-3-yl)-1-(4-methoxybenzyl)-1H-1,2,3-triazole-4-
carboxamide (7ag)

1
Light brown solid, Yield 79%, M.P: 170−172 °C; H NMR (300 MHz, CDCl3 + DMSO-d6) δ
9.87 (s, 1H), 8.74 (s, 1H), 8.55 (s, 1H), 7.95 (d, J = 3.4 Hz, 1H), 7.81 (s, 1H), 7.70 (d, J = 7.6 Hz,
1H), 7.35 (d, J = 8.6 Hz, 2H), 7.24 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.6 Hz, 2H), 6.76 – 6.64 (m,
13
3H), 5.58 (s, 2H), 3.78 (s, 3H); C NMR (100 MHz, CDCl3) δ 164.69, 164.26, 157.44, 155.70,
149.54, 148.03, 138.37, 137.66, 134.70, 131.27, 131.19, 127.13, 124.18, 120.22, 119.24, 118.86,
60.13, 58.56; MS (ESI): m/z 417 [M+H]+; HRMS (ESI) Calcd for C29H26N5O3 [M+H]+
417.1670; Found: 417.1683.
4.2.34.
1-(4-Fluorobenzyl)-N-(2-((4-hydroxyphenyl)amino)pyridin-3-yl)-1H-1,2,3-triazole-4-
carboxamide (7ah)
1
Light brown solid, Yield 80%, M.P: 204−206 °C; H NMR (300 MHz, CDCl3 + DMSO-d6) δ
9.88 (s, 1H), 8.65 (dd, J1 = 32.2 Hz, J2 = 7.1 Hz, 2H), 7.96 (d, J = 6.5 Hz, 1H), 7.80 – 7.66 (m,
2H), 7.49 – 7.36 (m, 2H), 7.24 (d, J = 8.7 Hz, 2H), 7.11 (t, J = 8.6 Hz, 2H), 6.72 (dd, J1 = 12.4
13
Hz, J2 = 6.8 Hz, 3H), 5.65 (s, 2H); C NMR (100 MHz, CDCl3) δ 168.72, 166.26, 164.18,
157.46, 155.70, 149.68, 148.16, 138.36, 137.63, 135.16 (d, JC-F = 8.2 Hz), 131.48 (d, JC-F = 11.3
Hz), 127.16, 124.12, 120.79 (d, JC-F = 21.5 Hz), 120.24, 118.89, 58.20; MS (ESI): m/z 405
[M+H]+; HRMS (ESI) Calcd for C29H26N5O3 [M+H]+ 405.1470; Found: 405.1488.
4.2.35.
1-Benzyl-N-(2-((4-fluorophenyl)amino)pyridin-3-yl)-1H-1,2,3-triazole-4-carboxamide
(7ai)
White solid, Yield 81%, M.P: 158−160 °C; 1H NMR (300 MHz, CDCl3) δ 8.81 (s, 1H), 8.18 (dd,
J1 = 4.8 Hz, J2 = 1.4 Hz, 1H), 8.07 (s, 1H), 7.86 – 7.72 (m, 1H), 7.38 – 7.27 (m, 5H), 7.26 – 7.19
(m, 1H), 7.10 (t, J = 8.5 Hz, 2H), 6.97 (t, J = 7.2 Hz, 1H), 6.92 (s, 1H), 6.89 (dd, J1 = 7.7 Hz, J2
= 4.9 Hz, 1H), 5.55 (s, 2H); 13C NMR (125MHz, CDCl3) δ 163.12 (d, JC-F = 249.3 Hz), 158.88,
149.65, 145.71, 143.02, 140.81, 132.89, 130.31 (d, JC-F = 8.3 Hz), 128.93, 125.88, 122.17,
120.84, 120.36, 119.58, 116.45 (d, JC-F = 21.9 Hz), 116.15, 53.95; MS (ESI): m/z 389 [M+H]+;
HRMS (ESI) Calcd for C29H26N5O3 [M+H]+ 389.1521; Found: 389.1517.
4.2.36.
1-Benzyl-N-(2-((3,4,5-trimethoxyphenyl)amino)pyridin-3-yl)-1H-1,2,3-triazole-4-
carboxamide (7aj)