Synthesis and biological evaluation of novel (E)-N'-benzylidene hydrazides as novel c-Met inhibitors through fragment based virtual screening

Article abstract of DOI:10.1080/14756366.2019.1702655

C-Met plays a crucial role in the development and progression of neoplastic disease. Type II c-Met inhibitors recognise the inactive DFG-out conformation of the kinase, result in better anti-tumour effects due to synergistic effect against the other kinas

Full text of DOI:10.1080/14756366.2019.1702655

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Synthesis and biological evaluation of novel (E)-N'-
benzylidene hydrazides as novel c-Met inhibitors
through fragment based virtual screening
Jing-wei Liang, Shi-long Li, Shan Wang, Wan-qiu Li & Fan-hao Meng
To cite this article: Jing-wei Liang, Shi-long Li, Shan Wang, Wan-qiu Li & Fan-hao Meng (2020)
Synthesis and biological evaluation of novel (E)-N'-benzylidene hydrazides as novel c-Met inhibitors
through fragment based virtual screening, Journal of Enzyme Inhibition and Medicinal Chemistry,
35:1, 468-477, DOI: 10.1080/14756366.2019.1702655
To link to this article: https://doi.org/10.1080/14756366.2019.1702655
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 468–477
https://doi.org/10.1080/14756366.2019.1702655
RESEARCH PAPER
Synthesis and biological evaluation of novel (E)-N’-benzylidene hydrazides as
novel c-Met inhibitors through fragment based virtual screening
ꢀ
ꢀ
Jing-wei Liang , Shi-long Li , Shan Wang, Wan-qiu Li and Fan-hao Meng
School of Pharmacy, China Medical University, Shen Yang, China
ABSTRACT
ARTICLE HISTORY
Received 13 September 2019
Revised 29 November 2019
Accepted 2 December 2019
C-Met plays a crucial role in the development and progression of neoplastic disease. Type II c-Met inhibi-
tors recognise the inactive DFG-out conformation of the kinase, result in better anti-tumour effects due to
synergistic effect against the other kinases. According to our previous works, an (E)-N’-benzylidene group
was selected as the initial fragment. Two series of (E)-N’-benzylidene hydrazides were designed by frag-
ment growth method. The inhibitory activities were in vitro investigated against c-Met and VEGFR-2.
Compound 10b exhibited the most potent inhibitory activity against the c-Met inhibitor (IC50 ¼ 0.37 nM).
Compound 11b exhibited multi-target c-Met kinase inhibitory activity as a potential type II c-Met inhibitor
(IC50 ¼ 3.41 nM against c-Met; 25.34 nM against VEGFR-2). The two compounds also demonstrate the feasi-
bility of fragment-based virtual screening method for drug discovery.
KEYWORDS
Virtual screening; c-Met
inhibitor; QSAR;
benzylidene hydrazides
1. Introduction
decreases tumour invasion and metastasis10,11. Studies on the
multi-target c-Met kinase inhibitors show that it is different from
the type I inhibitor which possesses high selectivity. The multi-
target c-Met kinase inhibitor binds to the inactive conformation of
Hepatocyte growth factor receptor (HGFR/c-Met) is a transmem-
brane heterodimer comprising two disulphide-linked chains,
including an outer a-chain (50 kDa) and a transmembrane b-chain
(140 kDa)1, encoded by c-Met gene2. Under normal physiological
conditions, it regulates important cellular processes, such as differ-
entiation, proliferation, cell cycle, motility, and apoptosis. The
intracellular portion of c-Met comprises a catalytic tyrosine kinase
domain, which contains multifunctional docking sites3. Many
downstream pathways such as PI3K, MAPK, and STAT3 will be acti-
vated, when the HGF binds to the extracellular domains of c-Met,
followed by autophosphorylation of tyrosine kinase residues in
the catalytic domain4. Abnormal activation in the c-Met pathway
induces excessive cell proliferation and is related to the develop-
ment and progression of the neoplastic disease5.
the kinase and belongs to the type II c-Met kinase inhibitor12,13
,
which binds to not only c-Met but also VEGFR, FGFR, ALK, EGFR,
MAT1R. All these studies suggest that simultaneous inhibition of
the two tyrosine kinase receptors results in better anti-tumour
effects14,15
.
The number of multi-target c-Met inhibitors that entering the
clinical stage is small and the skeleton of them is lacking in diver-
sity at present, it is easy to cause over-fitting when building vir-
tual screening models according to the ligand16. We established a
fragment-based multistage screening method for discovering
VEGFR-2 inhibitors in previous work17,18, obtained a novel mol-
ecule with a high antineoplastic effect. Not affected by the ligand
conditions, the fragment-based theory held that the active pock-
ets of drug targets were made up of multiple sub-pockets, and
the fragments are units that combine with these sub-pockets.
Finding these fragments and linking them together often led to
higher active compounds19,20,21. In this study, we intended to use
this method to establish a virtual screening model for discovering
multi-target c-Met inhibitors, increasing the structural diversity of
these inhibitors.
With the development of research on the mechanism in drug
resistance to cancer cells, the crossing talk between various mem-
brane receptors and c-Met is monitored in tumours and takes part
in the resistance to EGFR-tyrosine kinase inhibitors including gefiti-
nib and erlotinib6,7. Continuous angiogenesis is a key mechanism
of tumour growth. Therefore, anti-angiogenesis plays an important
role in cancer therapy. VEGF has a strong stimulating effect on
the proliferation of endothelial cells through increasing vascular
permeability and promoting the formation of tumour blood ves-
sels8. It specifically binds to the extracellular domain of VEGFR-2
and is involved in endothelial cell sprouting, migration, vascular
permeability, and tumour cell survival. However, VEGFR-2 inhibi-
tors can lead to c-Met-dependent invasion and metastasis of
tumour cells by increasing the degree of hypoxia when used
2. Results and discussions
2.1. Structural analysis and site map of the binding pocket
The crystal structures of c-Met and VEGFR-2 kinase are available in
alone9. While the combination of sunitinib and c-Met inhibitor PF- the Protein Data Bank (PDB), both of them are in their inactive
04217903 inhibits both VEGFR and c-Met pathways, significantly conformations (PDB IDs: 3LQ8 and 3VHE)22,23. The ATP-binding
CONTACT Fan-hao Meng
These authors contributed equally to this work.
fhmeng@cmu.edu.cn
School of Pharmacy, China Medical University, Shen Yang 110122, China
ꢀ
Supplemental data for this article can be accessed here.
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
469
inactive pocket of c-Met is generally divided into a back pocket 2.2. Fragment-based drug design
behind the gatekeeper (c-Met: Phe1223), and a front pocket
within the solvent-accessible area (Figure 1(a)). The back pocket is
the unique pocket of the inactive conformation of tyrosine kinase,
namely the DFG-out site. The individual pockets are constructed
by several amino acids, the surrounding amino acids of each
pocket impart distinct properties. The back pocket in c-Met is
characterised as a hydrophobic pocket due to the surrounding
hydrophobic amino acids: Phe1134, Leu1195, and Phe1200
(Figure 1(b)). Meanwhile, the front-pocket is located in a solvent-
accessible area and is surrounded by Ile1084, Lys1161 and
His1162 (Figure 1(b)). Analysis of protein-ligand interaction of c-
Met inhibitors (exemplified by GSK1363089 in c-Met) showed that
the common interaction patterns include: a conserved hydrogen
bond with the backbone of Asp1222, an arene–arene stacking
interaction with the side chain of Phe1223.
2.2.1. Fragment growth based on aromatic acyl hydrazine
For non-competitive inhibitors combining with the DFG-out site of
tyrosine kinases, an ASP and a Lys around gatekeeper are key
residues24,25. In our previous work, we found that the trans-
acylhydrazine can also form hydrogen bonds with Asp1110 and
Lys1222 of c-Met. Given that the hydrophobic residues were in
the back pocket, the aromatic acyl hydrazine was used as the initial
fragment (Figure 2(a)). Then the fragment growth method in MOE
was used to generate novel molecules. The newly generated small
molecules were then filtered by the screening model of VEGFR-2.
Finally, a novel skeleton that linked a phenyl group to the aromatic
acyl hydrazine with a triazole fragment was achieved. It was worth
noting that a series of small molecules with large substituents on
the phenyl group were abandoned by the second model, which
Figure 1. Structure of the kinase domain of c-Met bind to GSK1363089, (a) 3D-view image; (b) pose-view image.
Figure 2. (a) Fragment growing based virtual screening; (b) the hit compound docked into c-Met.

470
J.-W. LIANG ET AL.
indicated that large substituents were not beneficial for increasing anhydride. The important intermediates (4, 5) were obtained by
the inhibitory activity of small molecules against VEGFR-2.
the corresponding aryl alkyne (1, 3) and ethyl 2-azidoacetate (2).
Compounds 4 and 5 were subjected to hydrolysis to give inter-
mediates 6 and 7. The preparation of different (E)-benzylidenehy-
drazine (9a–9k) involved benzaldehyde derivatives reaction with
hydrazine hydrate. The intermediates (6, 7) were treated by EDCI
and HOBt. Then different (E)-benzylidenehydrazine (9a–9k) and
Et3N were added with stirring to give the compounds
10a–10k, 11a–11k.
In the previously designed route, we used intermediate 5
reacted with hydrazine hydrate and then form the target com-
pounds with benzaldehyde derivatives. We found an interesting
phenomenon during the previous synthetic route exploration.
There will be cis–trans isomers generated in this process. So we
improved the synthetic route to get the pure target compounds
(Scheme 1).
2.2.2. Docking
The hit compound was docked into the inactive conformation of
c-Met (Figure 2(b)). The halogenated aromatic ring was in the
hydrophobic region at the bottom of the pocket and the trans-
acylhydrazine fragment formed two hydrogen bonds with
Asp1110 and Lys1222, respectively. On the basis of the new skel-
eton, the halogen atom at the tail of the novel compound was
further changed, exploring the approach to increase the activity
of this small molecule. Owing to the large substituents on the
phenyl group seem to play an opposite role in the c-Met and
VEGFR-2 inhibitory activities of the small molecule, an acetylamino
group was taken into account as a compromise.
The difficulties in the synthesis of the target compounds
(10a–10k, 11a–11k) lie in the cis–trans isomerisation of the acyl
hydrazine fragment. To synthesise a pure single conformation is
2.3. Chemistry
The compounds (9a–9k, 10a–10k, 11a–11k) were achieved the focus of synthesis. It was found that acyl hydrazine fragment
through the route as illustrated in Scheme 1. N-(3-ethynylpheny- synthesised by click chemistry link to another fragment results in
l)acetamide (1) was prepared by 3-ethynylaniline and acetic the appearance of cis–trans isomers due to the direction of the
Scheme 1. Reagents and conditions: (i) CuSO4, Vitamin C, EtOH/H2O, 40 ꢁC, 5–10 h; (ii) NaOH, 6 h, HCl; (iii) hydrazine hydrate, rt, 5 h; (iv) EDCI, HOBt, Et3N,
35 ꢁC, 8–10 h.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
471
Table 1. Novel compounds and their activities against c-Met.
linkage. The mixture is difficult to separate according to the previ-
ous synthesis studies conducted by our group. The yields of the
cis and trans configurations were equal and were confirmed in
nuclear magnetics. This was also verified in the study of cis–trans
isomers by Jian Wu26, Mohammad Sayed Alam27. In response to
the above problems, we improved the synthetic route, the benzal-
dehyde derivatives were used to form the (E)-benzylidenehydra-
zine derivatives fragment with hydrating hydrazine. For another
fragment, phenylacetylene and 3-ethynylaniline were used as raw
materials and obtained by click chemistry and hydrolysis. The new
synthetic method solved the problem of generating cis–trans iso-
mers, simplified the synthetic method, reduced the synthetic steps
and has great reference value for the synthesis and purification of
cis–trans isomers.
IC50(nM)
c-Met
Compound
R1
R2
R3
R4
R5
a
11a
11c
11d
11e
11g
11h
11j
11k
10a
10c
10d
10e
10g
10h
10i
H
H
H
H
H
H
H
H
H
H
OH
NO2
OH
OH
H
H
H
H
OH
NO2
OH
OH
OCH3
H
H
H
H
H
Br
Cl
OH
H
H
H
H
H
H
–
5681.98
–
H
4276.61
–
11.93
3076.1
–
Br
Br
H
OCH3
H
H
H
OCH3
OCH3
H
H
H
H
Br
H
H
OCH3
OCH3
Cl
OH
H
H
H
2.4. QSAR study
NHCOCH3
NHCOCH3
NHCOCH3
NHCOCH3
NHCOCH3
NHCOCH3
NHCOCH3
NHCOCH3
NHCOCH3
1.70
The capacity of the novel compounds for inhibiting c-Met activity
was evaluated using a cell-free assay. The evaluation results are
summarised in Table 1.
H
H
H
Br
Br
H
11184.1
266.01
5857.3
8933.14
–
H
OCH3
OCH3
OCH3
1.73
2.4.1. PCA-based small molecule descriptor selection
To find out the molecules with the optimal structure, a 3D-QSAR
10j
10k
OCH3
OCH3
H
5.42
H
OCH3
91.52
0.6
study on the derivatives was carried out using the molecular GSK1363089
a–No inhibitory effect.
descriptors method in Molecular Operation Environment System
(MOE; Chemical Computing Group Inc., Montreal, QC, Canada).
Based on the molecular model, which showed a significant correl-
ation with the experimental IC50 activity data, further improve-
ment of this correlation was analysed by molecular descriptors.
The available molecular descriptors implemented in MOE were
tested. As a result, the molecular descriptors were used to analyse
Table 2. Descriptors and relative importance of descriptors.
Descriptors
Relative importance of descriptors
AM1Eele
PM3E
0.904317
0.500991
0.281605
0.258684
0.122461
0.071553
0.041214
0.032288
0.017582
0.017462
the structure-activity relationship of each series. To reduce the AM1E
PM3Eele
model noise and eliminate the interaction between descriptors,
the PCA-based small molecule descriptor method was adopted.
According to the significant correlation with IC50, 10 descriptors
were screened out from 379 descriptors by the PCA method
(RMSE ¼ 22124.5 R2 ¼ 0.746558). Target compounds correlation
coefficients are shown in Table 2. After calculation, the normalised
linear model displayed significant relation to descriptors. The fol-
lowing equation showed the generated QSAR model that included
eleven descriptors (AM1E, AM1Eele, MNDOE, PM3E, PM3Eele, pmi,
pmi2, pmi3, pmiX, pmiY) to predict IC50/SD values of c-Met inhibi-
tors.
pmiY
pmiX
pmi3
pmi2
MNDOE
pmi
energy of the molecule rotating around the centre of mass);
AM1Eele and PM3 represented electronic energy; pmi, pmi2,
Eele
pmi3, pmiX and pmiY showed principal moment of inertia (meas-
urement of inertia of a rigid body rotating around an axis). The
magnitude of the value indicated its correlation with IC50.
According to the QSAR model, the activity of the compounds was
mainly related to electronic energy (AM1Eele and PM3Eele).
PredIC50
QSAR :
ð
Þ
SD IC50
PM3Eele
SDðPM3Eele
AM1Eele
SDðAM1Eele
Þ
¼ 6:20846 ꢂ 16:95238ꢀ
þ 15:93245ꢀ
Þ
2.4.2. QSAR suggestion
MNDOE
pmiY
SDðpmiYÞ
pmiX
SDðpmiXÞ
pmi
SDðpmiÞ
pmi3
SDðpmi3Þ
þ 1:14920ꢀ
ꢂ 0:93352ꢀ
After analysis of the contribution of the substituents to the activ-
ities by MOE, a QSAR suggestion was given as a set of compounds
with more potential activities. The compounds in the QSAR sug-
gestion set combined the advantages of the synthesised com-
pounds and discarded their disadvantages. The results of the
QSAR study are shown in Figure 3. The activities of the com-
pounds in series 10 were better than that of series 11. For the
compounds in series 11, the substituents on the benzene ring
preferred for halogen atoms, such as bromine atom. This could be
explained by high total energy and electronic energy of com-
ð
Þ
SD MNDOE
PM3E
ꢂ 5:46574ꢀ
þ 4:91929ꢀ
ꢂ 0:21423ꢀ
þ 0:76667ꢀ
ꢂ 0:03510ꢀ
ꢂ 0:03081ꢀ
ð
Þ
SD PM3E
AM1E
SDðAM1EÞ
pmi2
SDðpmi2Þ
In the QSAR model above, AM1E, MNDOE, and PM3E were used
to describe total energy (molecular dynamic kinetic energy in pounds that bromine atom brought. While the introduction of
space, the energy of the electrons of a molecule, intramolecular hydroxy group reduced the activities of the compounds. As a
energy, vibrational energy between atoms in a molecule, the result of the principal moment of inertia, the activities of the

472
J.-W. LIANG ET AL.
Figure 3. Important structural requirements of (E)-N’-benzylidene hydrazides by means of the ligand based 3D-QSAR.
Table 3. The inhibitory activities of the five compounds against VEGFR-2 and
c-Met.
meta-substituted compounds were better than that of the ortho-
substituted and para-substituted compounds. However, the activ-
ity of the compound 11h was superior to 11g, prompting that it
could be dominated by the increased total energy rather than the
principal moment of inertia. Therefore, the QSAR model suggested
meta-chloro substitution (11b). For the compounds in series 10,
the contribution of ortho-substituents was uncertain. However, the
combination of hydroxyl and chlorine atoms could make the mol-
ecule have better physicochemical properties since the QSAR sug-
gestion gave 10f, 11f, and 11i. The compound 10i, 10j, and 10k
substituted by methoxy showed different inhibitory effects. It may
due to the different position and number of substituents brought
the change of compounds’ energy and moment of inertia.
The virtual screening model of VEGFR-2 was used to screen the
QSAR Suggestion, five molecules were screened out. Followed by
synthesising and detecting their kinase activities against c-Met
and VEGFR-2, respectively, 11b and 10f showed better inhibitory
activity than the others as shown in Table 3. It was consistent
with the QSAR predicted results. Unfortunately, compound 10b
did not exhibited expected activity against VEGFR-2, which may
be related to acetyl substitution. It was not accidental that mole-
cules with activity against c-Met can also inhibit VEGFR-2, the
multi-target inhibitors against c-Met and VEGFR-2 have a physio-
logical basis. Both of the proteins belong to the family of tyrosine
kinase, which catalyse the phosphorylation of tyrosine. The similar
functions make them possess similar active sites. While subtle dif-
ferences may make small molecules show different activities
against the two kinases. So we superimposed the two kinases in
three dimensions to explore the reason.
Name
R1
R2
R3
R4
R5
VEGFR-2 (nM)
25.34
c-Met (nM)
11b
10b
11f
10f
11i
H
H
H
OH
OH
OCH3
Cl
Cl
H
H
H
H
H
H
H
OCH3
H
H
Cl
Cl
H
3.41
0.37
–
1.19
–
a
NHCOCH3
–
H
NHCOCH3
H
9855.76
139.44
849.27
a–No inhibitory effect.
3. Experimental
3.1. Synthesis and characterisation of novel compounds
For all target compounds, 1H-NMR and 13C-NMR spectra were
recorded on a Varian NMR spectrometer operating at 600 MHz for
1H, and 150 MHz for 13C. All chemical shifts were measured in
DMSO-d6 as solvents. All chemicals were purchased from
Sinoreagent Chemical Reagent (Beijing, China) and were used as
received, unless stated otherwise. Analytical TLC is performed on
silica gel 60 F254 plates (Qingdao Haiyang Chemical Company,
Qingdao, Shandong, China) and visualised by UV. Flash column
chromatography is performed on gel 60 (40–63 mm) (Qingdao
Haiyang Chemical Company, Qingdao, Shandong, China). Melting
points were determined with an Electro thermal melting point
1
apparatus, are uncorrected. H-NMR and 13C-NMR of all the target
compounds are described in Supplementary material.
General procedure for the synthesis of the ethyl 2–(4-(3-acet-
amidophenyl)-1H-1, 2, 3-triazol-1-yl) acetate (4) and ethyl 2–(4-
phenyl-1H-1, 2, 3-triazol-1-yl) acetate (5):
A
mixture of N-(3-ethynylphenyl)acetamide (1) (5 g,
31.41 mmol) and ethanol (CH3CH2OH) (20 ml) was stirred at 40 ꢁC
for 15 min. The water solution of copper sulphate pentahydrate
(0.3 g) and ascorbic acid (0.3 g) was added and stirred for 5 more
minutes. Then ethyl 2-azidoacetate (2) (4.87 g, 37.69 mmol) was
added. The reaction was stirred at 40 ꢁC for 8 h and monitored by
TLC. After completion of the reaction, the solvent was removed in
vacuum. Adding water and ethyl acetate, the organic phase was
separated and dried over anhydrous sodium sulphate. The solvent
was removed in vacuum and the residue was purified by silica gel
column to give the pure compound (4), as a yellow solid.
Compound 5 was prepared with the same method using ethy-
nylbenzene (3), to give pure compound as a pale yellow oily liquid.
General procedure for the synthesis of the 2-(4-(3-acetamido-
phenyl)-1H-1, 2, 3-triazol-1-yl) acetic acid (6) and 2-(4-phenyl-1H-1,
2, 3-triazol-1-yl) acetic acid (7):
The structures of c-Met and VEGFR-2 were superposed in 3D
space as shown in Figure 4(a). The RMSD results of the superpos-
ition were 1.63 Å, indicating that the similarity of the two confor-
mations was very high. Moreover, the back pockets in the two
kinases, named the DFG out site, possessed higher similarity
(Figure 4(c), the residues marked with red circle). This also pro-
vides the basis for the discovery of dual target drugs. However,
when the active site was amplified, the difference between the
two pockets was detected. The opening of c-Met pocket was
wide, which can accommodate small molecules. So the inhibitor
of c-Met was longer. While the opening of VEGFR-2 pocket was
more narrow that it could not accommodate any group
(Figure 4(b), the ribbons marked with red arrows). So the inhibitor
of VEGFR-2 was not as long as that of c-Met. So it was suggested
that the opening of VEGFR-2 pocket could not accommodate the
acetylamino group, causing compound 10b to show promising
inhibitory activity against c-Met but no activity against VEGFR-2.
To a solution of ethyl 2-(4-(3-acetamidophenyl)-1H-1, 2, 3-tria-
zol-1-yl) acetate (4) (6 g, 20.81 mmol), sodium hydroxide (3.33 g,

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
473
Figure 4. (a) The superposing of c-Met and VEGFR-2 kinase; (b) the active sites of the two kinases; (c) the RMSD value of the superposing conformation.
83.24 mmol) in 30 ml water was added. The reaction solution was
Compounds 11a–11k were prepared with the same method
stirred at 40 ꢁC for 5 h. After completion of the reaction, dilute by using the corresponding intermediate 7.
hydrochloric acid was slowly added to adjust the solution pH to 2.
The obtained solid was filtered and dried to give the compound
2-(4-(3-acetamidophenyl)-1H-1, 2, 3-triazol-1-yl) acetic acid (6) as a
light yellow solid.
Compound 7 was prepared with the same method by using
corresponding raw materials 5.
General procedure for the synthesis of the (E)-benzylidenehy-
drazine derivatives (9a–9k):
3.1.1. (E)-2-(4-(3-acetamidophenyl)-1H-1, 2, 3-triazol-1-yl)-N’-(4-
chlorobenzylidene) (10a)
A white solid, yield: 61.24%. Mp:234.6–234.9 ꢁC 1H NMR (600 MHz,
DMSO-d6) d 11.96 (s, 1H, –N–H), 10.07 (s, 1H, –N–H), 8.50 (s, 1H,
–N–CH¼), 8.16 (s, 1H, Ar–H), 8.07 (s, 1H, –N ¼ CH–), 7.81 (d,
J ¼ 8.5 Hz, 2H, Ar–H), 7.58 (d, J ¼ 7.4 Hz, 1H, Ar–H), 7.53 (d,
A solution of 80% hydrazine hydrate (8.9 g, 177.85 mmol) was J ¼ 8.5 Hz, 2H, Ar–H), 7.49 (d, J ¼ 7.4 Hz, 1H, Ar–H), 7.38 (t,
J ¼ 7.9 Hz, 1H, Ar–H), 5.77 (s, 2H, CH2), 2.08 (s, 3H, CH3). 13 C NMR
added dropwise to 4-chlorobenzaldehyde (8a) (5.0 g, 35.57 mmol)
in 10 ml water. The reaction mixture was stirred at room tempera-
ture for 5 h and monitored by TLC. After completion of the reac-
tion, the reaction mixture was poured into 50 ml water and stirred
for 20 min. The reaction mixture was filtered off. Filter cake was
washed with water, and dried to give the compound 9a as a
white solid, yield: 81.27%.
(150 MHz, DMSO-d6) d 167.84, 166.92, 145.49, 142.65, 139.31,
134.00, 132.21, 130.58, 128.73, 128.32, 128.13, 122.59, 119.35,
117.79, 114.91, 50.12, 23.48.
3.1.2. (E)-2-(4-(3-acetamidophenyl)-1H-1, 2, 3-triazol-1-yl)-N’-(3-
chlorobenzylidene) (10b)
Compounds 9b to 9k were prepared with the same method
by using corresponding raw materials 8b to 8k.
A white solid, yield: 57.21%. Mp:255.8–256.3 ꢁC 1H NMR (600 MHz,
General procedure for preparation of target compounds DMSO-d6) d 12.01 (s, 1H, –N–H), 10.09 (s, 1H, –N–H), 8.51 (s, 1H,
(10a–10k, 11a–11k):
–N–CH¼), 8.17 (s, 1H, Ar–H), 8.07 (s, 1H, –N ¼ CH–), 7.94 (s, 1H,
Ar–H), 7.89 (s, 1H, Ar–H), 7.86 (d, J ¼ 7.6 Hz, 1H, Ar–H), 7.59 (d,
J ¼ 7.6 Hz, 1H, Ar–H), 7.56 (d, J ¼ 7.7 Hz, 1H, Ar–H), 7.37 (m, 2H,
Ar–H), 5.80 (s, 2H, CH2), 2.09 (s, 3H, CH3). 13 C NMR (150 MHz,
DMSO-d6) d 168.91, 168.13, 161.12, 146.56, 143.38, 140.38, 136.25,
134.19, 131.64, 131.34, 130.24, 129.79, 128.34, 127.40, 123.65,
120.43, 115.99, 51.26, 24.55.
A solution of 2-(4-(3-acetamidophenyl)-1H-1, 2, 3-triazol-1-yl)
acetic acid (6) (2.46 mmol) and 1-hydroxybenzotriazole (2.58 mmol)
in 20 ml DMF was stirred at ꢂ5 ꢁC for 20 min. Then 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide hydrochloride (2.58 mmol)
was added and stirred for 15 min. The reaction mixture was stirred
in 40 ꢁC oil bath and compounds 9a–9k (2.58 mmol), triethylami-
ne(7.38 mmol) were added. The reaction was stirred at 40 ꢁC for
8–10 h and monitored by TLC. After completion of the reaction,
slowly pour the liquid into 150 ml water. After adding ethyl acet-
ate in the mixture, the organic phase was separated and dried
over anhydrous sodium sulphate. The solvent was removed in vac-
3.1.3. (E)-2-(4-(3-acetamidophenyl)-1H-1, 2, 3-triazol-1-yl)-N’-(4-
hydroxybenzylidene) acetohydrazide (10c)
A white solid, yield: 52.11%. Mp:260.0–260.8 ꢁC 1H NMR (600 MHz,
uum. The residue was purified by silica gel column to give the tar- DMSO-d6) d 11.70 (s, 1H, –N–H), 10.07 (s, 1H, –O–H), 10.00 (s, 1H,
get compounds (10a–10k).
–N–H), 8.50 (s, 1H, –N–CH¼), 8.16 (s, 1H, Ar–H), 7.97 (s, 1H,

474
J.-W. LIANG ET AL.
–N ¼ CH–), 7.60 (d, J ¼ 8.7 Hz, 2H, Ar–H), 7.56 (d, J ¼ 8.7 Hz, 1H, –N–H), 8.47 (s, 1H, –N–CH¼), 8.30 (s, 1H, –N ¼ CH–), 8.14 (s, 1H,
Ar–H), 7.49 (d, J ¼ 7.6 Hz, 1H, Ar–H), 7.37 (t, J ¼ 7.9 Hz, 1H, Ar–H),
6.85 (d, J ¼ 8.7 Hz, 2H, Ar–H), 5.71 (s, 2H, CH2), 2.08 (s, 3H, CH3).
13C NMR (150 MHz, DMSO-d6) d 168.91, 167.45, 159.94, 148.69,
146.52, 145.29, 140.37, 131.68, 129.80, 129.52, 129.28, 125.31,
123.68, 120.42, 118.85, 116.18, 115.97, 51.14, 24.55.
Ar–H), 7.95 (s, 1H, Ar–H), 7.57 (d, J ¼ 4.9 Hz, 1H, Ar–H), 7.48 (d,
J ¼ 7.8 Hz, 1H, Ar–H), 7.43–7.34 (m, 3H, Ar–H), 5.77 (s, 2H, CH2),
2.07 (s, 3H, CH3). 13C NMR (150 MHz, DMSO-d6) d 168.89, 167.90,
162.78, 156.11, 146.49, 140.37, 140.10, 134.11, 131.68, 129.80,
128.32, 123.63, 122.92, 120.42, 118.87, 115.96, 111.39, 51.27, 24.55.
3.1.4. (E)-2-(4-(3-acetamidophenyl)-1H-1,2,3-triazol-1-yl)-N’-(2-hydro
3.1.9. (E)-2-(4-(3-acetamidophenyl)-1H-1,2,3-triazol-1-yl)-N’-(2,4-dime
xybenzylidene) acetohydrazide (10d)
1
A white solid, yield: 53.24%. Mp:253.8–254.2 ꢁC H NMR (600 MHz,
thoxybenzylidene) acetohydrazide (10i)
1
A yellow solid, yield: 59.36%. Mp:210.9–211.3 ꢁC H NMR (600 MHz,
DMSO-d6) d 11.81 (s, 1H, –N–H), 10.07 (s, 2H, –N–H, –O–H), 8.50 (s,
1H, –N–CH¼), 8.39 (s, 1H, –N ¼ CH–), 8.16 (s, 1H, Ar–H), 7.58 (d,
J ¼ 5.5 Hz, 1H, Ar–H), 7.49 (d, J ¼ 7.4 Hz, 1H, Ar–H), 7.38 (t,
J ¼ 7.9 Hz, 1H, Ar–H), 7.27 (t, J ¼ 7.0 Hz, 1H, Ar–H), 6.92 (m, 2H,
Ar–H), 6.88 (t, J ¼ 7.5 Hz, 1H, Ar–H), 5.72 (s, 2H, CH2), 2.08 (s, 3H,
CH3). 13C NMR (150 MHz, DMSO-d6) d 168.91, 167.57, 162.48,
DMSO-d6) d 10.05 (s, 1H, –N–H), 8.83 (s, 1H, –N–CH¼), 8.42 (s, 1H,
–N ¼ CH–), 8.16 (s, 1H, Ar–H), 7.91 (d, J ¼ 8.6 Hz, 1H, Ar–H), 7.55 (d,
J ¼ 8.0 Hz, 1H, Ar–H), 7.48 (d, J ¼ 7.7 Hz, 1H, Ar–H), 7.36 (t,
J ¼ 7.9 Hz, 1H, Ar–H), 6.66 (s, 1H, Ar–H), 6.64 (d, J ¼ 8.6 Hz, 1H,
Ar–H), 5.47 (s, 2H, CH2), 3.88 (s, 3H, CH3), 3.84 (s, 3H, CH3), 2.07 (s,
156.95, 148.23, 146.53, 142.15, 140.38, 131.89, 129.80, 126.69, 3H, CH3). 13C NMR (150 MHz, DMSO-d6) d 167.81, 163.92, 162.77,
123.67, 120.49, 119.89, 116.84, 116.63, 115.97, 51.18, 24.55.
159.54, 154.92, 145.34, 139.28, 130.64, 128.69, 127.10, 122.60,
119.32, 117.71, 114.85, 114.09, 105.94, 97.63, 55.26, 54.92,
50.05, 23.47.
3.1.5. (E)-2-(4-(3-acetamidophenyl)-1H-1,2,3-triazol-1-yl)-N’-(2-nitro
benzylidene)acetohydrazide (10e)
A
yellow solid, yield: 45.21%. Mp: 233.2–233.8 ꢁC 1H NMR
3.1.10. (E)-2-(4-(3-acetamidophenyl)-1H-1,2,3-triazol-1-yl)-N’-(3,4-
dimethoxybenzylidene) acetohydrazide (10j)
(600 MHz, DMSO-d6) d 12.17 (s, 1H, –N–H), 10.07 (s, 1H, –N–H),
8.51 (s, 1H, –N–CH¼), 8.47 (s, 1H, –N ¼ CH–), 8.17 (s, 1H, Ar–H),
7.82 (t, J ¼ 7.5 Hz, 1H, Ar–H), 7.69 (t, J ¼ 7.3 Hz, 1H, Ar–H), 7.59 (m,
2H, Ar–H), 7.50 (d, J ¼ 7.4 Hz, 1H, Ar–H), 7.40 – 7.35 (m, 2H, Ar–H),
5.77 (s, 2H, CH2), 2.08 (s, 3H, CH3). 13C NMR (150 MHz, DMSO-d6) d
167.85, 167.18, 147.49, 145.54, 139.60, 139.32, 133.04, 130.16,
128.74, 127.94, 127.51, 124.01, 122.59, 121.48, 119.37, 117.81,
114.92, 50.13, 23.49.
A white solid, yield: 54.32%. Mp:230.0–230.6 ꢁC 1H NMR (600 MHz,
DMSO-d6) d 11.80 (s, 1H, –N–H), 10.08 (s, 1H, –N–H), 8.52 (s, 1H,
–N–CH¼), 8.17 (s, 1H, Ar–H), 8.00 (s, 1H, –N ¼ CH–), 7.59 (d,
J ¼ 7.7 Hz, 1H, Ar–H), 7.50 (d, J ¼ 7.3 Hz, 1H, Ar–H), 7.41 (s, 1H,
Ar–H), 7.38 (t, J ¼ 7.9 Hz, 1H, Ar–H), 7.23 (d, J ¼ 6.7 Hz, 1H, Ar–H),
7.02 (d, J ¼ 8.2 Hz, 1H, Ar–H), 5.77 (s, 2H, CH2), 3.83 (s, 3H, CH3),
3.81 (s, 3H, CH3), 2.08 (s, 3H, CH3). 13C NMR (150 MHz, DMSO-d6) d
167.86, 166.61, 150.12, 148.46, 145.50, 143.99, 139.32, 130.62,
128.74, 125.98, 122.60, 121.01, 119.37, 117.79, 114.91, 110.82,
107.89, 54.95, 54.90, 50.20, 23.48.
3.1.6. (E)-2-(4-(3-acetamidophenyl)-1H-1,2,3-triazol-1-yl)-N’-(5-chloro-
2-hydroxybenzylidene) acetohydrazide (10f)
A white solid, yield: 58.29%. Mp:234.6–234.9 ꢁC H NMR (600 MHz,
1
DMSO-d6) d 11.88 (s, 1H, –N–H), 10.44 (s, 1H, –O–H), 10.06 (s, 1H,
–N–H), 8.48 (s, 1H, –N–CH¼), 8.32 (s, 1H, –N ¼ CH–), 8.15 (s, 1H,
Ar–H), 7.58 (d, J ¼ 7.5 Hz, 1H, Ar–H), 7.49 (d, J ¼ 7.3 Hz, 1H, Ar–H),
7.37 (t, J ¼ 7.8 Hz, 1H, Ar–H), 7.09 (s, 1H, Ar–H), 7.01 (d, J ¼ 8.8 Hz,
1H, Ar–H), 6.94 (d, J ¼ 8.8 Hz, 1H, Ar–H), 5.78 (s, 2H, CH2), 2.08 (s,
3H, CH3). 13C NMR (150 MHz, DMSO-d6) d 168.90, 167.90, 161.33,
140.37, 133.21, 131.27, 129.79, 129.09, 128.27, 127.15, 123.78,
123.63, 122.37, 120.46, 118.99, 117.87, 115.97, 51.27, 24.54.
3.1.11. (E)-2-(4-(3-acetamidophenyl)-1H-1,2,3-triazol-1-yl)-N’-(3,4,5-
trimethoxybenzylidene) acetohydrazide (10k)
1
A yellow solid, yield: 58.21%. Mp:210.2–212.9 ꢁC H NMR (600 MHz,
DMSO-d6) d 11.90 (s, 1H, –N–H), 10.05 (s, 1H, –N–H), 8.51 (s, 1H,
–N–CH¼), 8.16 (s, 1H, Ar–H), 7.99 (s, 1H, –N ¼ CH–), 7.58 (d,
J ¼ 8.0 Hz, 1H, Ar–H), 7.49 (d, J ¼ 7.7 Hz, 1H, Ar–H), 7.37 (t,
J ¼ 7.9 Hz, 1H, Ar–H), 7.08 (s, 2H, Ar–H), 5.78 (s, 2H, CH2), 3.85 (d,
9H, CH3), 2.08 (s, 3H, CH3). 13C NMR (150 MHz, DMSO-d6) d 168.89,
167.87, 162.76, 161.67, 153.66, 146.58, 144.82, 140.38, 139.71,
131.68, 129.77, 123.63, 120.42, 118.88, 116.03, 106.09, 104.84,
60.58, 56.46, 36.23, 31.22, 24.51.
3.1.7. (E)-2-(4-(3-acetamidophenyl)-1H-1,2,3-triazol-1-yl)-N’-(3,5-dib
romo-2-hydroxybenzylidene)acetohydrazide (10g)
A white solid, yield: 62.31%. Mp:237.0–237.5 ꢁC 1H NMR (600 MHz,
DMSO-d6) d 12.63 (s, 1H, –N–H), 12.23 (s, 1H, –O–H), 12.04 (s, 1H,
–N–H), 8.56 (s, 1H, –N–CH¼), 8.46 (s, 1H, –N ¼ CH–), 8.42 (s, 1H,
Ar–H), 8.31 (s, 1H, Ar–H), 7.94 (s, 1H, Ar–H), 7.58 (d, J ¼ 8.0 Hz, 1H,
Ar–H), 7.50 (d, J ¼ 7.2 Hz, 1H, Ar–H), 7.37 (s, 1H, Ar–H), 5.81 (s, 2H,
CH2), 2.08 (s, 3H, CH3). 13C NMR (150 MHz, DMSO-d6) d 168.89,
163.02, 154.00, 152.70, 147.84, 146.75, 140.38, 136.31, 132.67, 131.49,
129.79, 123.51, 121.35, 120.50, 116.07, 111.77, 111.01, 51.35, 24.54.
3.1.12. (E)-N’-(4-chlorobenzylidene)-2-(4-phenyl-1H-1,2,3-triazol-1-
yl)acetohydrazide (11a)
A
white solid, yield: 66.72%. Mp: 259.0–259.6 ꢁC. 1H NMR
(600 MHz, DMSO-d6) d 11.94 (s, 1H, –N–H), 8.56 (s, 1H, –N–CH¼),
8.07 (s, 1H, –N ¼ CH–), 7.87 (d, J ¼ 7.1 Hz, 2H, Ar–H), 7.80 (d,
J ¼ 8.5 Hz, 2H, Ar–H), 7.52 (d, J ¼ 8.5 Hz, 2H, Ar–H), 7.47 (t,
J ¼ 7.7 Hz, 2H, Ar–H), 7.35 (t, J ¼ 7.4 Hz, 1H, Ar–H), 5.76 (s, 2H, CH2).
13C NMR (150 MHz, DMSO-d6) d 167.94, 162.68, 147.15, 146.62,
143.75, 135.07, 133.29, 131.27, 129.41, 129.21, 128.29, 125.59,
3.1.8. (E)-2-(4-(3-acetamidophenyl)-1H-1,2,3-triazol-1-yl)-N’-(5-bromo-
2-hydroxybenzylidene) acetohydrazide (10h)
1
A white solid, yield: 57.64%. Mp:228.9–229.3 ꢁC H NMR (600 MHz,
DMSO-d6) d 11.85 (s, 1H, –N–H), 10.39 (s, 1H, –O–H), 10.04 (s, 1H, 123.64, 51.16.

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475
3.1.13. (E)-N’-(3-chlorobenzylidene)-2-(4-phenyl-1H-1,2,3-triazol-1- 8.29 (s, 1H, Ar–H), 7.47 (m, 3H, Ar–H), 7.35 (d, J ¼ 1.9 Hz, 2H, Ar–H),
5.81 (s, 2H, CH2). 13C NMR (150 MHz, DMSO-d6) d 167.85, 163.01,
147.88, 146.81, 141.75, 136.12, 132.68, 131.11, 129.42, 128.39,
yl)acetohydrazide (11b)
A
white solid, yield: 63.45%. Mp: 259.0–259.8 ꢁC. 1H NMR
125.65, 125.60, 123.49, 121.34, 51.38.
(600 MHz, DMSO-d6) d 11.99 (s, 1H, –N–H), 8.55 (s, 1H, –N–CH¼),
8.05 (s, 1H, –N ¼ CH–), 7.87 (m, 3H, Ar–H), 7.73–7.69 (t, 1H, Ar–H),
7.51–7.44 (m, 5H, Ar–H), 7.34 (t, J ¼ 7.4 Hz, 1H, Ar–H), 5.77 (s, 2H,
CH2). 13C NMR (150 MHz, DMSO-d6) d 168.25, 146.58, 143.25,
136.71, 134.19, 131.15, 130.14, 129.41, 128.27, 126.63, 126.44,
125.58, 123.63, 51.26.
3.1.19. (E)-N’-(5-bromo-2-hydroxybenzylidene)-2-(4-phenyl-1H-1,2,3-
triazol-1-yl)acetohydrazide (11h)
A
white solid, yield: 58.55%. Mp: 233.0–233.5 ꢁC. 1H NMR
(600 MHz, DMSO-d6) d 11.85 (s, 1H, –N–H), 10.39 (s, 1H, –O–H),
8.54 (s, 1H, –N–CH¼), 8.30 (s, 1H, –N ¼ CH–), 7.95 (s, 1H, Ar–H),
7.87 (m, 2H, Ar–H), 7.46 (m, 3H, Ar–H), 7.41 (d, J ¼ 8.7 Hz, 1H,
Ar–H), 7.35 (d, J ¼ 7.4 Hz, 1H, Ar–H), 5.77 (s, 2H, CH2). 13C NMR
(150 MHz, DMSO-d6) d 167.18, 155.49, 145.94, 139.60, 133.45,
130.68, 128.76, 127.62, 125.36, 124.96, 122.96, 122.31, 118.26,
110.76, 50.62.
3.1.14. (E)-N’-(4-hydroxybenzylidene)-2-(4-phenyl-1H-1,2,3-triazol-
1-yl)acetohydrazide (11c)
A
white solid, yield: 58.23%. Mp: 234.0–234.5 ꢁC. 1H NMR
(600 MHz, DMSO-d6) d 11.68 (s, 1H, –N–H), 9.94 (s, 1H, –O–H), 8.56
(s, 1H, –N–CH¼), 7.98 (s, 1H, –N ¼ CH–), 7.89 (d, 2H, Ar–H), 7.60 (d,
J ¼ 7.4 Hz, 2H, Ar–H), 7.49–7.34 (m, 3H, Ar–H), 6.85 (d, J ¼ 6.7 Hz,
2H, Ar–H), 5.71 (s, 2H, CH2). 13C NMR (150 MHz, DMSO-d6) d
167.41, 159.94, 146.60, 145.33, 131.30, 129.40, 129.29, 128.27,
125.59, 125.34, 123.67, 116.18, 51.13.
3.1.20. (E)-N’-(2,4-dimethoxybenzylidene)-2-(4-phenyl-1H-1,2,3-tria-
zol-1-yl)acetohydrazide (11i)
A
yellow solid, yield: 49.52%. Mp: 230.0–230.4 ꢁC. 1H NMR
(600 MHz, DMSO-d6) d 11.79 (s, 1H, –N–H), 8.64 (s, 1H, –N ¼ CH–),
8.57 (s, 1H, –N–CH¼), 7.99 (s, 1H, Ar–H), 7.87 (d, J ¼ 7.6 Hz, 2H,
Ar–H), 7.49 (d, J ¼ 1.3 Hz, 1H, Ar–H), 7.46 (d, J ¼ 7.7 Hz, 1H, Ar–H),
7.40–7.37 (m, 2H, Ar–H), 7.35 (t, J ¼ 7.4 Hz, 1H, Ar–H), 5.76 (s, 2H,
CH2), 3.82 (s, 6H, CH3). 13C NMR (150 MHz, DMSO-d6) d 163.22,
160.00, 157.87, 155.31, 148.89, 146.34, 133.50, 129.89, 127.55,
122.84, 122.15, 117.82, 114.60, 106.41, 99.36, 98.13, 56.55,
55.73, 55.37.
3.1.15. (E)-N’-(2-hydroxybenzylidene)-2-(4-phenyl-1H-1,2,3-triazol-
1-yl)acetohydrazide (11d)
A
white solid, yield: 63.45%. Mp: 239.0–239.6 ꢁC. 1H NMR
(600 MHz, DMSO-d6) d 11.78 (s, 1H, –N–H), 10.04 (s, 1H, –O–H),
8.55 (s, 1H, –N–CH¼), 8.38 (s, 1H, –N ¼ CH–), 7.90–7.85 (m, 3H,
Ar–H), 7.80 (d, J ¼ 6.3 Hz, 1H, Ar–H), 7.46 (m, 3H, Ar–H), 7.35 (d,
J ¼ 7.5 Hz, 1H, Ar–H), 7.29–7.24 (t, J ¼ 7.8 Hz,1H, Ar–H), 5.73 (s, 2H,
CH2). 13C NMR (150 MHz, DMSO-d6) d 167.53, 156.95, 146.59,
142.22, 131.88, 131.30, 129.41, 128.28, 126.74, 125.59, 123.66,
120.51, 119.89, 116.64, 51.15.
3.1.21. (E)-N’-(3,4-dimethoxybenzylidene)-2-(4-phenyl-1H-1,2,3-tria-
zol-1-yl)acetohydrazide (11j)
A
white solid, yield: 67.14%. Mp: 232.1–232.5 ꢁC. 1H NMR
(600 MHz, DMSO-d6) d 11.80 (s, 1H, –N–H), 8.65 (s, 1H, –N ¼ CH–),
8.58 (s, 1H, –N–CH¼), 8.00 (s, 1H, Ar–H), 7.88 (d, J ¼ 7.1 Hz, 2H,
Ar–H), 7.47 (m, 3H, Ar–H), 7.41 (d, J ¼ 1.7 Hz, 1H, Ar–H), 7.36 (d,
J ¼ 7.4 Hz, 1H, Ar–H), 5.77 (s, 2H, CH2), 3.83 (s, 6H, CH3). 13C NMR
(150 MHz, DMSO-d6) d 167.62, 151.23, 149.56, 146.62, 145.09,
131.30, 129.41, 128.28, 127.07, 125.59, 123.64, 122.06, 111.95,
109.08, 56.05, 51.24.
3.1.16.
yl)acetohydrazide (11e)
(E)-N’-(2-nitrobenzylidene)-2-(4-phenyl-1H-1,2,3-triazol-1-
A light yellow solid, yield: 53.14%. Mp: 225.2–225.6 ꢁC. 1H NMR
(600 MHz, DMSO-d6) d 12.15 (s, 1H, –N–H), 8.56 (s, 1H, –N–CH¼),
8.47 (s, 1H, –N ¼ CH–), 7.88 (d, J ¼ 6.5 Hz, 2H, Ar–H), 7.81 (t,
J ¼ 7.6 Hz, 1H, Ar–H), 7.69 (t, J ¼ 7.8 Hz, 1H, Ar–H), 7.49–7.45 (m,
3H, Ar–H), 7.35 (t, J ¼ 7.3 Hz, 2H, Ar–H), 5.77 (s, 2H, CH2). 13 C NMR
(150 MHz, DMSO-d6) d 168.20, 148.57, 146.88, 146.67, 140.65,
134.08, 131.22, 129.41, 129.02, 128.45, 125.65, 125.05, 123.64,
123.22, 53.06.
3.1.22. (E)-2-(4-phenyl-1H-1,2,3-triazol-1-yl)-N’-(3,4,5-trimethoxybenzy
lidene)acetohydrazide (11k)
A
white solid, yield: 65.31%. Mp: 233.5–233.8 ꢁC. 1H NMR
(600 MHz, DMSO-d6) d 11.91 (s, 1H, –N–H), 8.66 (s, 1H, –N ¼ CH–),
8.57 (s, 1H, –N–CH¼), 7.87 (d, J ¼ 7.2 Hz, 2H, Ar–H), 7.47 (m, 2H,
Ar–H), 7.35 (t, J ¼ 7.2 Hz, 1H, Ar–H), 7.22 (s, 2H, Ar–H), 5.77 (s, 2H,
CH2), 3.74 (s, 6H, CH3), 3.71 (s, 3H, CH3). 13C NMR (150 MHz,
DMSO-d6) d 167.86, 161.68, 153.65, 146.62, 144.83, 140.72, 139.71,
131.29, 129.74, 129.42, 128.29, 125.58, 123.64, 106.10, 104.86,
102.78, 100.00, 60.65, 56.43, 51.29.
3.1.17. (E)-N’-(5-chloro-2-hydroxybenzylidene)-2-(4-phenyl-1H-1,2,3-
triazol-1-yl)acetohydrazide (11f)
A
white solid, yield: 48.59%. Mp: 240.0–240.6 ꢁC. 1H NMR
(600 MHz, DMSO-d6) d 11.86 (s, 1H, –N–H), 10.37 (s, 1H, –O–H),
8.54 (s, 1H, –N–CH¼), 8.32 (s, 1H, –N ¼ CH–), 7.87 (d, J ¼ 7.4 Hz, 2H,
Ar–H), 7.83 (d, J ¼ 2.6 Hz, 1H, Ar–H), 7.46 (m, 3H, Ar–H), 7.35 (d,
J ¼ 7.5 Hz, 1H, Ar–H), 7.29 (d, J ¼ 8.8 Hz, 1H, Ar–H), 5.77 (s, 2H,
CH2). 13C NMR (150 MHz, DMSO-d6) d 167.89, 162.65, 155.69,
146.56, 140.20, 131.29, 129.42, 128.28, 125.57, 125.44, 123.80,
123.64, 122.37, 118.42, 51.27.
3.2. Cell-free detection of c-Met kinase activity
The compounds inhibitory activities of inhibitors against c-Met
were determined using ADP-Glo assay. The reaction buffer was
deployed by 50 mM HEPES, pH 7.5, 10 mM MgCl2, 0.1 mg/mL BSA,
2 mM DTT and 1% DMSO. The recombinant c-Met kinase was
3.1.18. (E)-N’-(3,5-dibromo-2-hydroxybenzylidene)-2-(4-phenyl-1H-
1,2,3-triazol-1-yl) acetohydrazide (11g)
A light yellow solid, yield: 55.47%. Mp: 231.0–231.5 ꢁC. 1H NMR diluted to 2.2 lg/mL using reaction buffer (50 mM HEPES, pH 7.5,
(600 MHz, DMSO-d6) d 12.58 (s, 1H, –O–H), 12.02 (s, 1H, –N–H), 10 mM MgCl2, 0.1 mg/mL BSA, 2 mM DTT, 1% DMSO), and ATP
8.62 (s, 1H, –N ¼ CH–), 8.52 (s, 1H, –N–CH¼), 8.41 (s, 1H, Ar–H), was diluted with reaction buffer (10 mM) to 250 lM, the test

476
J.-W. LIANG ET AL.
compounds and positive drug (GSK1363089) were formulated into synthesised and evaluated for their inhibitory activities against c-
four concentration gradient solutions (6 ꢃ 10ꢂ2 M, 6 ꢃ 10ꢂ4 M, Met. The QSAR results showed that halogens and acetyl of aro-
6 ꢃ 10 ꢂ6 M, 6 ꢃ 10ꢂ8 M). The reaction was started by sequentially matic rings could improve the activity of the compounds against
c-Met. However, the compounds with acetyl groups did not per-
form well in the activity test of VEGFR-2. Nevertheless, we have
obtained a highly active type I c-Met inhibitor 10b and a multi-
target c-Met kinase inhibitor 11b. They could be potential antitu-
mor agents that deserve further research.
adding 2 lL of a ATP solution, 1 lL of a drug solution, and 2 lL of
an enzyme solution in 96 wells. The assay was conducted for 1 h
at 37 ꢁC before addition of 5 lL ADP-Glo reagent and incubation
for 40 min at room temperature. About 10 lL kinase detection
reagent was added and incubated for 30 min at room temperature
before the luminescence value was measured using a chemilumin-
escence module of a full-wave length multi-function micro-
plate reader.
Disclosure statement
No potential conflict of interest was reported by the authors.
3.3. Fragment-based drug design
Funding
Fragment based drug design was performed using the Add Group
to Ligand module in Molecular Operating Environment package
(MOE; Chemical Computing Group Inc., Montreal, QC, Canada).
The aromatic acyl hydrazine was used as the initial fragment, the
hydrogen atom at the head of the acetylhydrazine group was
selected as the connection point. During the generation, a filter of
Lipinski’s rule of five was toggled ON. The novel small molecules
were generated based on the shape of the active pocket.
This work was supported by National Natural Science Foundation
of China [81274182 and 81573687].
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