Design, synthesis and biological evaluation of benzohydrazide derivatives containing dihydropyrazoles as potential EGFR kinase inhibitors

Article abstract of DOI:10.3390/molecules21081012

A series of novel benzohydrazide derivatives containing dihydropyrazoles have been synthesized as potential epidermal growth factor receptor (EGFR) kinase inhibitors and their biological activities as potential antiproliferative agents have been evaluated. Among these compounds, compound H20 exhibited the most potent antiproliferative activity against four cancer cell line variants (A549, MCF-7, HeLa, HepG2) with IC50 values of 0.46, 0.29, 0.15 and 0.21 μM respectively, which showed the most potent EGFR inhibition activities (IC50 = 0.08 μM for EGFR). Molecular modeling simulation studies were performed in order to predict the biological activity and activity relationship (SAR) of these benzohydrazide derivatives. These results suggested that compound H20 may be a promising anticancer agent.

Full text of DOI:10.3390/molecules21081012

molecules
Article
Design, Synthesis and Biological Evaluation of
Benzohydrazide Derivatives Containing
Dihydropyrazoles as Potential EGFR
Kinase Inhibitors
Hai-Chao Wang 1,2, Xiao-Qiang Yan 2, Tian-Long Yan 2, Hong-Xia Li 1, Zhong-Chang Wang 2,
*
and Hai-Liang Zhu 2,
*
1
College of Biology and Food Engineering, Suzhou University, Suzhou 234000, China;
wanghc0823@163.com (H.-C.W.); hxl96hx@163.com (H.-X.L.)
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China;
18120176083@163.com (X.-Q.Y.); ytlnju@163.com (T.-L.Y.)
2
*
Correspondence: wangzhongchang2006@163.com (Z.-C.W.); zhuhl@nju.edu.cn (H.-L.Z.);
Tel.: +86-177-1436-9196 (Z.-C.W.); Tel./Fax: +86-25-8968-2672 (H.-L.Z.)
Academic Editor: James W. Leahy
Received: 2 June 2016; Accepted: 29 July 2016; Published: 3 August 2016
Abstract: A series of novel benzohydrazide derivatives containing dihydropyrazoles have been
synthesized as potential epidermal growth factor receptor (EGFR) kinase inhibitors and their
biological activities as potential antiproliferative agents have been evaluated. Among these
compounds, compound H20 exhibited the most potent antiproliferative activity against four cancer
cell line variants (A549, MCF-7, HeLa, HepG2) with IC50 values of 0.46, 0.29, 0.15 and 0.21
µM
respectively, which showed the most potent EGFR inhibition activities (IC50 = 0.08 M for EGFR).
µ
Molecular modeling simulation studies were performed in order to predict the biological activity
and activity relationship (SAR) of these benzohydrazide derivatives. These results suggested that
compound H20 may be a promising anticancer agent.
Keywords: benzohydrazide derivatives; EGFR inhibitor; antiproliferative activity; cytotoxicity;
molecular docking
1. Introduction
Cancer has become one of the most serious diseases which represents a great threat to human
health around the world [
1]. Although there are currently a large number of anticancer drugs, more and
more people continue to die of cancer [
2
]. Epidermal growth factor receptor (EGFR) plays important
roles in human cancer. As a member of the HER family, EGFR is a tyrosine kinase receptor which plays
an essential role in normal cell growth and differentiation, and is involved in tumor proliferation and
survival [
ErbB-3 (HER3), and ErbB-4 (HER4) [
research and its over-expression or abnormal activation often causes cell malignant transformation [
The over-expression of EGFR has been observed in many solid tumors, such as colon [ ], ovarian [
breast and non-small cell lung cancer (NSCLC) [ ]. Therefore, EGFR inhibition has been developed as
3,
4
]. The HER family comprises four members: EGFR (HER1/ErbB-1), ErbB-2 (HER2/neu),
]. EGFR is one of the most important targets in current cancer
].
],
5
6
8
7
9
one of the most efficient strategies for cancer therapy.
Dihydropyrazole, a small bioactive molecule, is a prominent structural motif found in
numerous pharmaceutically active compounds. Some representatives had been demonstrated to
have important biological activities such as antiviral/antitumor [10], antibacterial [11], fungistatic [12],
antihyperglycemic activity [13], antimalarial [14] and antitubercular effects [15]. Biological evaluation
Molecules 2016, 21, 1012; doi:10.3390/molecules21081012
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1012
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indicated that some of the synthesized compounds were potent inhibitors of EGFR. Some novel
compounds containing the dihydropyrazole skeletons were reported as potent anticancer agents
against EGFR tyrosine kinase. It had been reported that anticancer compounds such as 3-alkoxy-1H-
pyrazolo[3,4-d]pyrimidines analogues (3A-1-PP, Figure 1) showed potent EGFR kinase inhibitory
activity, with IC50 values reaching single digit nanomolar values [16]. Compounds
(Figure 1) displayed the most potent EGFR inhibitory activity, with IC50 values of 0.07, 0.24, 0.06 and
0.26 M respectively, which were comparable to the positive control erlotinib (IC50 = 0.03 M) [17 20].
Compound containing the dihydropyrazole and naphthalene ring showed the highest inhibitory
EGFR activity (IC50 = 0.12 µM) [21].
a, b, c and d
µ
µ
–
e
Figure 1. Chemical structures of some reported compounds.
Some naphthalene derivatives have been reported as potent microtubule inhibitors, apoptosis
inducers, glutamamide analogues or P-glycoprotein inhibitors [22 23]. On the other hand, the
,
naphthalene ring can be recognized in various biologically active compounds with clinical applications.
Naftifine and terbinafine, allylamine antifungal agents, are widely used for the treatment of fungal
infections [24–26]. Propranolol is an anti-angina drug, duloxetine is used for the treatment of
depression, nafimidone and its oxime ester derivative can be further developed for the treatment of
epilepsy [27]. Therefore, preparation and extensive biological evaluations on naphthalene derivatives
have continuously attracted our attention [28]. Besides, benzohydrazides are reported to possess
a wide variety of biological activities like antiglycation [29], antioxidant [30], antileishmanial [22],
antibacterial [31], antifungal [23], antitumor [32] and anticonvulsant [24]. Benzothiazole Schiff bases
with diverse biological activities have also been reported [26].

Molecules 2016, 21, 1012
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The three combined substructures—the dihydropyrazole along with the naphthalene ring and
benzohydrazides—might exhibit synergistic anticancer effects. All of these facts encouraged us to
integrate these three moieties and screen new benzohydrazide derivatives containing dihydropyrazoles
as potential EGFR inhibitory agents. Herein we disclose the design and synthesis of some novel
benzohydrazide derivatives containing dihydropyrazoles as potential EGFR kinase inhibitors, and
their antitumor activity against the A549 (human lung cancer), MCF-7 (human breast cancer), HeLa
(human cervical cancer) and HepG2 (human hepatocellular cancer) cancer cell lines.
2. Results and Discussion
2.1. Chemistry
A series of novel benzohydrazide derivatives containing dihydropyrazole moieties were
synthesized by the routes outlined in Scheme 1. The structures of the compounds are listed in
Table 1. Methyl 4-hydrazinylbenzoate (
B) was synthesized from 4-hydrazinylbenzoic acid in methanol
at room temperature. The diverse substituted chalcones E1
–E16 were obtained by stirring substituted
acetophenones and naphthaldehyde in ethanol at room temperature for 6–8 h. The cyclization different
chalcones and methyl 4-hydrazinylbenzoate in refluxing ethanol gives compounds F1–F16.
Table 1. Structures of compounds H1–H32.
Compound
R1
R2
R3
R4
R5
R6
H1
H2
H3
H4
H5
H6
H7
H8
H9
H
CH3
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
CH3
H
H
H
H
H
H
F
H
H
CH3
H
H
H
OCH3
H
H
F
H
F
Cl
H
Cl
H
H
H
CH3
H
H
H
OCH3
H
H
F
H
F
Cl
H
Cl
H
H
H
H
CH3
CH2CH3
OCH3
OCH3
OCH2CH3
H
H
H
H
H
H
OCH3
H
H
H
H
F
H
H
H
H
H
H
H
H
H
H
OCH3
H
H
H
H
F
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
F
H
H
Cl
Cl
Br
H10
H11
H12
H13
H14
H15
H16
H17
H18
H19
H20
H21
H22
H23
H24
H25
H26
H27
H28
H29
H30
H31
H32
H
H
H
CH3
CH2CH3
OCH3
OCH3
OCH2CH3
H
H
F
H
H
Cl
Cl
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H

Molecules 2016, 21, 1012
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O
O
O
i
HN
H2N
HN
H2N
OH
A
B
iii
O
R1
R4
R4
R2
O
CHO
R2
R3
ii
R3
+
R1
C
D1-D16
E1-E16
O
O
O
NH-NH2
iv
N
N
N
N
R4
R3
R4
R3
R1
R1
R2
R2
G1-G16
F1-F16
R6
O
B
R5
NH
N
CH
N
N
v
R4
A
R3
R1
R2
H1-H32
Scheme 1. The synthetic routes to compounds H1
room temperature, 20–24 h; (ii) EtOH, KOH, room temperature, 6–8 h; (iii) EtOH, HAc, 80 ˝C, 20–24 h;
iv) MeOH, N2H4 ) RCHO, EtOH, HOAc, room temperature, 6–8 h.
H2O (80%), DMF, 70 ˝C, 20–24 h; (
–H32. Reagents and conditions: (i) MeOH, SOCl2,
(
¨
v
Compounds F1
intermediate G1 G16. The desired compounds H1
G1 G16 with substituted benzaldehydes in ethanol for 6–8 h. Purified compounds H1
finally obtained by chromatography. All synthesized compounds H1 H32 gave satisfactory analytical
–
F16 further reacted with excess hydrazine hydrate (80%) to give the requisite
H32 were generated by the reaction of compounds
H32 were
–
–
–
–
–
and spectroscopic data in full accordance with their depicted structures, and other data published in a
Chinese patent [33].
2.2. Bioassays
2.2.1. Antiproliferative Assay
The antiproliferative activities of the newly synthesized derivatives H1–H32 were evaluated
under identical conditions by the MTT assay against four cultured cell lines (A549, MCF-7, HeLa and
HepG2) with erlotinib as control. The IC50 values of the compounds against these human cancer cells

Molecules 2016, 21, 1012
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are summarized in Table 2. As shown, the results revealed that all of the target compounds exhibited
significant antiproliferative activities, ranging from 0.15 to 100 µM.
Table 2. In vitro antiproliferative activities (IC50, µM a) of all compounds against tumor cell lines.
A549 b
MCF-7 b
HeLa b
HepG2 b
Compounds
H1
H2
H3
14.52
1.41
3.02
20.96
1.52
2.89
22.47
0.60
2.45
28.76
0.67
3.54
H4
H5
0.43
5.49
0.69
7.72
0.45
8.71
0.51
6.16
H6
H7
H8
H9
8.32
10.17
95.32
15.87
70.36
80.12
60.79
55.64
51.28
42.46
35.24
31.85
18.39
1.63
13.16
>100
17.08
63.78
84.12
56.21
50.34
46.38
37.43
33.62
26.51
20.73
0.78
11.45
>100
19.75
76.48
92.50
60.86
52.30
45.37
40.20
38.93
34.19
26.67
0.56
78.32
10.71
50.65
58.59
45.16
41.48
35.25
30.14
26.57
21.78
13.30
1.22
2.24
0.46
4.54
7.12
75.66
9.92
48.55
56.39
44.11
39.42
33.25
28.64
24.77
19.71
0.20
H10
H11
H12
H13
H14
H15
H16
H17
H18
H19
H20
H21
H22
H23
H24
H25
H26
H27
H28
H29
H30
H31
H32
Erlotinib
2.45
0.29
6.33
9.69
1.19
0.15
7.93
3.26
0.21
5.06
12.31
94.10
15.28
61.87
85.15
54.22
47.67
42.95
35.34
31.82
25.55
0.17
10.54
89.47
18.56
71.10
80.07
62.94
48.06
40.99
38.20
36.33
32.91
0.10
93.23
13.79
65.53
77.49
58.98
56.14
50.88
40.40
34.44
30.25
0.12
a
Antiproliferation activity was measured using the MTT assay. Values are the average of six independent
experiments run in triplicate. Variation was generally 5%–10%; b Cancer cells kindly supplied by State Key
Laboratory of Pharmaceutical Biotechnology, Nanjing University.
Among them, compound H20, which had IC50 values of 0.46, 0.29, 0.15 and 0.21 µM against A549,
MCF-7, HeLa and HepG2, respectively, displayed the most potent antiproliferative activity, comparable
to that of the positive control drug erlotinib (with corresponding IC50 values of 0.20, 0.12, 0.17 and
0.10 µM). These results suggested that compound H20 is more potent than the other compounds overall.
Modification of substituents such as methyl, halogen, methoxyl, ethyl and ethoxy was performed
to explore the structure-activity relationships of these compounds. Compounds bearing one methyl
group at the para-position in the A ring showed stronger antiproliferative activity (the IC50 values of
H4 and H20: 0.45 and 0.21
µM against HeLa cells) than those with hydrogen (H1 and H17), fluorine
(H11 and H27), chlorine (H14 and H30), bromine (H16 and H32), ethyl (H5 and H21), ethoxy (H8 and
H24) and methoxyl (H6 and H22) substituents, in the order of Me > H > Br > Cl > F, Me > OMe and
Et > OEt. This means that compounds with electron-donating groups at the para-position of the A ring
had better antiproliferative activity than those with electron-withdrawing groups. A comparison of
the composition on the A ring was as follows: for compounds H2
–H4, H9–H11, H13–H14, H18–H20,
H25 H27 and H29 H30, the potency order of substituent groups on the A ring was found to be
–
–

Molecules 2016, 21, 1012
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para > ortho > meta. Compounds H9–H15 and H25
–H31 with halogen groups on the A ring exhibited
antiproliferative activities in the order of disubstituted > monosubstituted. Interestingly, compounds
H6 and H22 with one methoxyl group on the A ring showed stronger antiproliferative activities than
compounds H7 and H23 with three methoxyl groups, so we concluded that more electron-donating
groups could reduce the electron density of the A ring and make it difficult to form
amino acids containing aromatic groups.
π-π bonds with
In the case of constant A ring substituents, changes of substituents on the B ring can also
affect the activities of these compounds. Contrary to the A ring, derivatives H17 H32 with a
methoxyl at the para-position displayed better activities than at the meta-position (compounds H1 H16).
–
–
These indicated that the position of the substituent had an influence on the antitumoral activity.
2.2.2. Kinase Inhibitory Activity
All these newly synthesized compounds were evaluated for their inhibitory activities against
EGFR kinases using a solid-phase ELISA assay. The approved EGFR inhibitor drug erlotinib was used
as a positive control. The inhibition constants (IC50) of the compounds were summarized in Table 3.
Table 3. Inhibition activities (IC50, µM a) of all compounds against epidermal growth factor receptor (EGFR).
Compounds
IC50, µM a
Compounds
IC50, µM a
H1
H2
H3
H4
H5
H6
H7
H8
H9
8.47
0.65
2.12
0.23
4.31
5.96
26.94
7.20
16.20
19.95
14.10
12.81
11.86
10.33
9.87
H17
H18
H19
H20
H21
H22
H23
H24
H25
H26
H27
H28
H29
H30
H31
H32
7.89
0.48
1.25
0.08
3.56
5.07
24.58
6.65
15.33
18.55
13.78
12.34
11.24
10.02
9.66
H10
H11
H12
H13
H14
H15
H16
Erlotinib
9.01
0.03
8.64
a
EGFR inhibitory activity was measured by the solid-phase ELISA assay. Values are the average of
six independent experiments run in triplicate. Variation was generally 5%–10%.
The EGFR kinase inhibitory activity results of the tested compounds correlated with the structural
relationships (SAR) of their inhibitory effects on the cell proliferation assay. Among the tested
compounds, compound H20 showed potent anticancer activity with IC50 of 0.08
comparable to the positive control erlotinib (IC50 = 0.03 M). Other tested compounds displayed
moderate inhibitory activities, with IC50 values ranging from 0.35 M to 24.58 M. This suggests that
µM, which was
µ
µ
µ
the potent inhibitory effects of the synthetic compounds on the cell proliferation assay were basically
related to their kinase inhibitory activities.
2.2.3. Cytotoxicity Test
All the target compounds H1–H32 were evaluated for their toxicity against 293T human kidney
epithelial cells at the median cytotoxic concentration (CC50) value of the tested compounds determined
by the MTT assay. As displayed in Table 4, these compounds were tested at multiple doses to study
the viability of 293T cells. Judging from the median cytotoxic concentration (CC50) data, all of the

Molecules 2016, 21, 1012
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tested compounds demonstrated similar cytotoxic activities as erlotinib in vitro against human kidney
epithelial 293T cell.
Table 4. The median cytotoxic concentration (CC50, µM a) data of tested compounds
Compounds
CC50, µM a
Compounds
CC50, µM a
H1
H2
H3
H4
H5
H6
H7
H8
H9
98.56
87.45
92.32
98.83
92.50
81.75
92.93
79.69
83.43
95.79
86.52
80.24
93.35
90.14
88.23
81.87
89.32
H17
H18
H19
H20
H21
H22
H23
H24
H25
H26
H27
H28
H29
H30
H31
H32
94.17
90.64
92.58
89.86
82.61
84.67
85.76
81.29
85.60
94.30
89.42
82.16
94.37
88.54
84.77
83.63
H10
H11
H12
H13
H14
H15
H16
Erlotinib
a The cytotoxicity of each compound was expressed as the concentration of compound that reduced cell viability
to 50% (CC50).
2.2.4. Analysis of Apoptosis
To verify whether the inhibition of cell growth of HeLa cell lines was related to cell apoptosis,
we decided to use flow cytometry with an Annexin V-FITC/PI Apoptosis Detection Kit to induce the
HeLa cell apoptosis with compound H20. The uptake of Annexin V-FITC/PI markedly increased, and
the uptake of normal cells was significantly decreased in a time-dependent manner. According to
the annotated data, the percentage of apoptotic cells was significantly elevated directly and in a
dose-dependent manner. The results are shown in Figures 2 and 3. As can be seen, the percentages
of cell apoptosis were 9.01%, 28.7%, 34.9%, 47.7%, 58.12%, in response to 0, 2.0, 4.0, 6.0, 8.0 µM
concentrations of compound H20, respectively. This leads to the conclusion that the percentage of
apoptotic cells significantly increased after treatment with high doses of H20.
2.3. Molecular Docking
To gain better understanding of the potency of the 32 compounds and guide further SAR studies,
we examined the interaction of these compounds with EGFR (PDB code: 1M17) by molecular docking.
A simulation of binding between the compounds and the ATP binding sites in EGFR was performed.
All docking runs used the DS 4.5 software (Discovery Studio 4.5, Accelrys, Co. Ltd., Beijing, China).
The obtained results, presented in Figures 4 and 5, show the optimal binding mode of compound H20
interacting with the 1M17 protein. The amino acid residues of EGFR which had interactions with
compound H20 were labeled. In the proposed binding mode, compound H20 was nicely bound to the
ATP binding pocket of EGFR through hydrogen bond interactions and hydrophobic interactions.
In the binding model, compound H20 was well bound to the EGFR protein with Gln767, Thr766,
Arg752, Val702, Leu694, Leu820, Ala719, the seven amino acids located in the binding pocket of the
protein, playing an important role in the combination with compound H20. As we can see from
Figures 4 and 5, Gln767 formed conventional hydrogen bonds and Thr766 formed Van der Waals
bonds with the nitrogen atom of the Schiff base group, which enhanced the combination activity of
compound H20. Moreover, a carbon–hydrogen bond and
π-sulfur bonds were found between Arg828
and Cys751 with a benzene ring. Meanwhile the Leu694 formed three alkyl bonds with the methyl,

Molecules 2016, 21, 1012
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benzene ring and dihydropyrazole ring. Alkyl and
π
-alkyl bonds were displayed in presence of Leu820
and dihydropyrazole ring and benzene ring, respectively. Moreover, Ala719 also formed
π-alkyl bonds
with the benzene ring. Naphthalene was also bonded with Val702 by a -alkyl bond. In molecular
π
docking 3D modeling, compound H20 was nicely bound to the ATP binding site through the hydrogen
bond with the backbone of Gln767 (distance = 2.4 Å) which increased the binding affinity dramatically
in theory, as displayed in Figure 5.
Figure 2. Compound H20 induced apoptosis in Hela cells with the density of 0, 2.0, 4.0, 6.0, 8.0
). HeLa cells were treated with for 24 h. Values represent the mean S.D, n = 3, p < 0.05 versus
control. The percentage of cells in each part was indicated.
µM
(
a–
e
˘
Figure 3. The percentage of early apoptotic cells and late apoptotic cells located in the upper right
quadrant. Images were representative of three independent experiments. Values represent the
mean ˘ S.D, n = 3, p < 0.05 versus control.

Molecules 2016, 21, 1012
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Figure 4. Molecular docking 2D model of the interaction between compound H20 and EGFR enzyme:
for clarity, only interacting residues are displayed.
Figure 5. Molecular docking 3D modeling of compound H20 with the EGFR enzyme: for clarity, only
interacting residues are displayed.
In addition, the predicted binding interaction energy was used as the criterion for ranking; the
estimated interaction energies of other synthesized compounds ranged from
as displayed in Figure 6 with a histogram. The selected compound of H20 had a best estimated binding
´
64.95 to 52.99 kcal/mol,
´

Molecules 2016, 21, 1012
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free energy of
´
64.95 kcal/mol for EGFR. These molecular docking results, along with the biological
assay data, suggested that compound H20 is a potential inhibitor of EGFR.
Figure 6. The histogram about CDOCKER_INTERACTION_ENERGY (
(H1–H32) for EGFR.
´
kcal/mol) of compounds
3. Experimental Section
3.1. General Information
All chemicals and reagents used in current study were commercially available analytical or
chemically pure grade reagents, unless otherwise indicated. All the reactions were monitored by
thin-layer chromatography (TLC) on glass-backed silica gel sheets (silica GF 254) and visualized using
UV (254 or 365 nm). Column chromatography separations were performed on silica gel (200–300 mesh)
using EtOAc/petroleum ether as eluents. Melting points (uncorrected) were determined using an X-4
MP apparatus (Taike Corp, Beijing, China). All the 1H-NMR and 13C-NMR spectra were recorded
using a model DPX400 spectrometer (Bruker, Billerica, MA, USA) in DMSO-d6 and chemical shifts
were reported in
δ (ppm) units relative to the internal standard tetramethylsilane (TMS). Mass spectra
(MS) were recorded using a Mariner System 5304 mass spectrometer (Isoprime, Manchester, UK).
3.2. General Synthesis Procedure of Benzohydrazide Derivatives Containing Dihydropyrazole Moieties
3.2.1. Synthesis of Methyl 4-Hydrazinylbenzoate (B)
4-Hydrazinylbenzoic acid (3.0 g, 0.020 mol) was suspended in anhydrous methanol (30 mL) and
˝
thionyl chloride (5 mL) was added dropwise at 0 C over 30 min. Then the reaction mixture was stirred
at room temperature for 20–24 h, the precipitate that formed was filtered, washed with petroleum
ether and ethanol (5:1) three times to remove residual 4-hydrazinylbenzoic acid, and dried to give the
title 4-hydrazinylbenzoate.

Molecules 2016, 21, 1012
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3.2.2. General Synthetic Procedure for Diverse Substituted Chalcones E1–E16
The diverse substituted chalcones E1–E16 were synthesized by reacting the appropriate
substituted acetophenone (3.0 mmol) and one equivalent of naphthaldehyde (0.5 g, 3.0 mmol) using 40%
potassium hydroxide (2 mL) as catalyst in ethanol (20 mL). The reaction mixture was stirred at room
temperature for 6–8 h, the yellow precipitate that formed was filtered, washed with petroleum ether
and ethanol (5:1) three times to remove impurities, and dried to give the diverse substituted chalcones.
3.2.3. General Synthetic Procedure for Diverse Substituted Chalcones F1–F16
Added acetic acid (1 mL) was used as an catalytic agent. The cyclization reaction of different
substituted chalcones E1
–
E16 (3.0 mmol) and methyl 4-hydrazinylbenzoate (3.0 mmol) was performed
F16, as monitored by thin
in refluxing ethanol (20 mL) at 80 ˝C for 20–24 h to afford compounds F1
–
layer chromatography (TLC). The crude products were purified in good yields by recrystallization
from ethanol, ethyl acetate and acetone (1:1:0.05).
3.2.4. General Synthetic Procedure for Compounds G1–G16
Compounds F1
(80%, 5 mL) was added dropwise in methanol (20 mL). The solution was refluxed at 70 C for 20–24 h,
and then poured into water (50 mL) and extracted with ethyl acetate (3 30 mL). The organic layer
–F16 (3.0 mmol) were dissolved in DMF (10 mL) and excess hydrazine hydrate
˝
ˆ
was collected, and washed with brine. The organic layer was dried over anhydrous sodium sulfate
and concentrated in vacuo to obtain yields 60%–80% of crude products G1–G16.
3.2.5. General Synthetic Procedure for the Title Compounds H1–H32
Equimolar amounts of compounds G1–G16 (2.0 mmol) and 3- or 4-methoxybenzaldehyde
(2.0 mmol) were dissolved in ethanol (20 mL), then acetic acid (1 mL) was added as catalytic agent.
The reaction mixture solution continued to stirred at room temperature for 6–8 h. The resulting
solid was extracted with petroleum ether for column chromatography. Column chromatography was
performed using silica gel (200–300 mesh), eluting with ethyl acetate and petroleum ether (1:2, v/v),
1
to give the title products. All of the target compounds gave satisfactory analytical and H-NMR
(in Supplementary Materials) and MS spectroscopic data, in accordance with their depicted structures.
1
(E)-N -(3-Methoxybenzylidene)-4-(5-(naphthalen-2-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzo-hydrazide
˝
H1). Yellow powder. Yield: 75.01%. m.p. 238–240 C. 1H-NMR (DMSO-d6): 3.28–3.34 (dd, J1 = 5.44 Hz
,
(
J2 = 17.72 Hz, 1H); 3.79 (s, 3H); 4.04–4.11 (dd, J1 = 12.32 Hz, J2 = 17.56 Hz, 1H); 5.81–5.85
(dd, J1 = 5.40 Hz, J2 = 12.12 Hz, 1H); 6.97–6.99 (d, J = 7.96 Hz, 1H); 7.13–7.15 (d, J = 8.88 Hz, 2H); 7.23
(s, 1H); 7.32–7.36 (m, 1H); 7.38–7.53 (m, 6H); 7.73–7.75 (d, J = 8.80 Hz, 2H); 7.83–7.84 (m, 2H); 7.88–7.93
(m, 4H); 8.33 (s, 1H); 11.55 (s, 1H). 13C-NMR (DMSO-d6): 163.07, 161.08, 149.84, 139.88, 133.40, 132.34,
129.71, 129.66, 129.40, 129.21, 128.13, 128.08, 127.57, 126.97, 126.50, 125.07, 124.26, 123.28, 114.75, 112.49,
63.20, 60.40, 55.72, 43.22. MS m/z: [M + H]+ 525.6 (C34H28N4O2). Anal. Calcd. for C34H28N4O2:
C, 78.34; H, 5.45; N, 10.38. Found: C, 77.84; H, 5.38; N, 10.68.
1
(E)-N -(3-Methoxybenzylidene)-4-(5-(naphthalen-2-yl)-3-(o-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzo-hydrazide
H2).Yellow powder. Yield: 84.15%. m.p. 242–244 ˝C. 1H-NMR (DMSO-d6): 2.76 (s,3H); 3.34–3.39
(
(dd, J1 = 5.48 Hz, J2 = 11.92 Hz, 1H); 3.78 (s, 3H); 4.11–4.18 (dd, J1 = 12.12 Hz, J2 = 17.84 Hz, 1H);
5.74–5.79 (dd, J1 = 5.56 Hz, J2 = 12.36 Hz, 1H); 6.97–6.99 (d, J = 7.72 Hz, 1H); 7.09–7.11 (d, J = 8.80 Hz,
2H); 7.23–7.41 (m, 7H); 7.48–7.53 (m, 3H); 7.73–7.75 (d, J = 8.72 Hz, 2H); 7.87–7.92 (m, 4H); 8.32 (s, 1H);
11.54 (s, 1H). 13C-NMR (DMSO-d6): 163.12, 161.08, 148.84, 139.90, 133.46, 132.34, 129.71, 129.66, 129.50,
129.21, 128.13, 127.57, 126.97, 126.50, 125.07, 124.26, 123.28, 114.75, 112.49, 60.33, 55.73, 43.42. MS m/z:
[M + H]+ 539.6 (C35H30N4O2). Anal. Calcd. for C35H30N4O2: C, 78.04; H, 5.61; N, 10.40. Found: C,
77.92; H, 5.56; N, 10.34.

Molecules 2016, 21, 1012
12 of 21
1
(E)-N -(3-Methoxybenzylidene)-4-(5-(naphthalen-2-yl)-3-(m-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzo-hydrazide
H3). Yellow powder. Yield: 87.67%. m.p. 232–234 ˝C. 1H-NMR (DMSO-d6): 2.37 (s, 3H); 3.26–3.32
(
(dd, J1 = 5.12 Hz, J2 = 17.64 Hz, 1H); 3.79 (s, 3H); 4.01–4.09 (dd, J1 = 12.16 Hz, J2 = 17.64 Hz, 1H);
4.37–4.40 (t, 1H); 5.80–5.84 (dd, J1 = 5.36 Hz, J2 = 12.16 Hz, 1H); 6.97–6.99 (d, J = 8.08 Hz, 1H); 7.13–7.15
(
(
d, J = 8.68 Hz, 2H); 7.23–7.24 (d, J = 6.12 Hz, 2H); 7.33–7.39 (m, 3H); 7.49–7.51 (m, 2H); 7.60–7.62
d, J = 7.72 Hz, 1H); 7.68 (s, 1H); 7.73–7.76 (d, J = 8.60 Hz, 2H); 7.86–7.93 (m, 4H); 8.33 (s, 1H); 11.55
(s, 1H). 13C-NMR (DMSO-d6): 163.07, 161.07, 149.84, 139.88, 133.40, 132.34, 129.71, 129.66, 129.40,
129.21, 128.13, 128.08, 127.57, 126.97, 126.50, 125.07, 124.26, 123.28, 114.75, 112.49, 60.23, 55.72, 43.35.
MS m/z: [M + H]+ 539.6 (C35H30N4O2). Anal. Calcd. for C35H30N4O2: C, 78.04; H, 5.61; N, 10.40.
Found: C, 77.94; H, 5.54; N, 10.35.
1
(E)-N -(3-Methoxybenzylidene)-4-(5-(naphthalen-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzo-hydrazide
H4). Yellow powder. Yield: 87.18%. m.p. 233–235 ˝C. 1H-NMR (DMSO-d6): 2.35 (s, 3H); 3.25–3.30
(
(dd, J1 = 4.24 Hz, J2 = 17.60 Hz, 1H); 3.79 (s, 3H); 4.02–4.08 (dd, J1 = 12.52 Hz, J2 = 17.80 Hz, 1H);
5.78–5.82 (dd, J1 = 5.52 Hz, J2 = 12.24 Hz, 1H); 6.97–6.99 (d, J = 8.04 Hz, 1H); 7.11–7.13 (d, J = 8.80 Hz,
2H); 7.23–7.29 (m, 4H); 7.32–7.40 (m, 2H); 7.49–7.51 (m, 2H); 7.71–7.75 (m, J1 = 4.12 Hz, J2 = 4.88 Hz,
4H); 7.87–7.93 (m, 4H); 8.33 (s, 1H); 11.54 (s, 1H). 13C-NMR (DMSO-d6): 163.09, 161.05, 150.14, 138.88,
133.40, 129.71, 129.66, 129.40, 129.21, 128.13, 128.08, 127.57, 126.97, 126.50, 125.07, 124.26, 123.28, 114.75,
112.49, 60.54, 55.72, 43.35. ESI- MS m/z: [M + H]+ 539.6 (C35H30N4O2). Anal. Calcd. for C35H30N4O2:
C, 78.04; H, 5.61; N, 10.40. Found: C, 77.95; H, 5.56; N, 10.36.
(E)-4-(3-(4-Ethylphenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N1-(3-methoxybenzylidene)-
benzohydrazide (H5). Yellow powder. Yield: 76.02%. m.p. 241–243 ˝C. 1H-NMR (DMSO-d6): 3.25–3.30
(dd, J1 = 5.20 Hz, J2 = 17.64 Hz, 1H); 3.79–3.81 (d, J = 9.12 Hz, 6H); 4.00–4.08 (dd, J1 = 12.04 Hz,
J2 = 17.52 Hz, 1H); 5.76–5.80 (dd, J1 = 5.16 Hz, J2 = 12.08 Hz, 1H); 6.97–7.04 (m, 3H); 7.09–7.11
(d, J = 8.76 Hz, 2H); 7.23 (s, 2H); 7.30–7.40 (m, 3H); 7.49–7.51 (m, 2H); 7.71–7.78 (m, 4H); 7.87–7.93
(m, 4H); 8.32 (s, 1H); 11.53 (s, 1H). 13C-NMR (DMSO-d6): 163.07, 161.06, 160.56, 149.95, 149.85, 146.85,
146.77, 145.72, 140.01, 139.94, 133.40, 132.88, 129.62, 129.39, 128.92, 128.61, 128.23, 128.07, 127.59, 126.95,
126.63, 126.57, 125.02, 124.91, 124.27, 122.85, 114.75, 114.67, 112.26, 60.22, 55.72, 43.57, 43.44. MS m/z:
[M + H]+ 553.2 (C36H32N4O2). Anal. Calcd. for C36H32N4O2: C, 78.24; H, 5.84; N, 10.14. Found: C,
78.12; H, 5.79; N, 10.11.
(E)-N1-(3-Methoxybenzylidene)-4-(3-(4-methoxyphenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-
benzohydrazide (H6). Yellow powder. Yield: 63.64%. m.p. 234–236 ˝C. 1H-NMR (DMSO-d6): 2.62–2.67
(dd, J1 = 7.52 Hz, J2 = 15.08 Hz, 2H); 3.25–3.31 (dd, J1 = 5.12Hz, J2 = 17.56 Hz, 1H); 3.79–3.84 (t, 3H);
4.00–4.08 (dd, J1 = 12.08 Hz, J2 = 17.92 Hz, 1H); 5.77–5.82 (dd, J1 = 5.36 Hz, J2 = 12.16 Hz, 1H); 6.97–6.99
(d, J = 7.84 Hz, 1H); 7.10–7.14 (m, 2H); 7.23 (s, 2H); 7.29–7.40 (m, 4H); 7.49–7.51 (m, 2H); 7.73–7.75
(
d, J = 8.08 Hz, 4H); 7.87–7.92 (m, 5H); 8.33 (s, 1H); 11.54 (s, 1H). 13C-NMR (DMSO-d6): 163.07, 161.06,
149.96, 146.81, 146.72, 145.73, 139.95, 133.40, 132.88, 129.86, 129.64, 129.38, 128.92, 128.62, 128.24, 128.07,
127.58, 126.96, 126.64, 125.04, 124.26, 123.09, 114.75, 112.38, 63.08, 55.72, 43.57, 43.42. MS m/z: [M + H]+
555.6 (C35H30N4O3). Anal. Calcd. for C35H30N4O3: C, 75.79; H, 5.45; N, 10.10. Found: C, 75.67; H, 5.43;
N, 10.02.
1
(E)-N -(3-Methoxybenzylidene)-4-(5-(naphthalen-2-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzohydrazide (H7). Yellow powder. Yield: 77.42%. m.p. 230–232 ˝C. 1H-NMR (DMSO-d6): 3.35–3.41
(dd, J1 = 5.68 Hz, J2 = 17.56 Hz, 1H); 3.70 (s, 3H); 3.79 (s, 3H); 3.86 (s, 6H); 4.01–4.05 (dd, J1 = 6.28 Hz
,
J2 = 15.16 Hz, 1H); 5.82–5.86 (dd, J1 = 5.12 Hz, J2 = 16.40 Hz, 1H); 6.97–6.99 (d, J = 8.04 Hz, 1H);
7.10–7.11 (m, 4H); 7.23 (s, 2H); 7.33–7.40 (m, 2H); 7.49–7.51 (m, 2H); 7.72–7.74 (d, J = 8.68 Hz, 2H);
7.85–7.94 (m, 4H); 8.33 (s, 1H); 11.53 (s, 1H). 13C-NMR (DMSO-d6): 163.92, 163.63, 162.97, 162.32, 162.23,
161.09, 160.70, 147.83, 146.98, 146.16, 139.53, 136.01, 135.94, 135.87, 133.37, 132.92, 129.70, 129.64, 129.37,
128.95, 128.08, 127.53, 127.13, 126.67, 125.14, 124.24, 123.98, 114.75, 112.91, 109.54, 109.36, 104.85, 104.68,

Molecules 2016, 21, 1012
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104.51, 63.60, 55.72, 42.98. MS m/z: [M + H]+ 615.7 (C37H34N4O5). Anal. Calcd. for C37H34N4O5: C,
72.30; H, 5.58; N, 9.11. Found: C, 72.18; H, 5.56; N, 9.08.
(E)-4-(3-(4-Ethoxyphenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N1-(3-methoxybenzylidene)-
benzohydrazide (H8). Yellow powder. Yield: 82.31%. m.p. 233–235 ˝C. 1H-NMR (DMSO-d6): 1.32–1.36
(t, 3H); 3.23–3.29 (dd, J1 = 5.12 Hz, J2 = 17.56 Hz, 1H); 3.78 (s, 3H); 3.99–4.10 (m, 3H); 5.74–5.78
(
(
dd, J1 = 5.52 Hz, J2 = 12.08 Hz, 1H); 6.96–7.02 (dd, J1 = 1.76 Hz, J2 = 11.40 Hz, 3H); 7.09–7.11
d, J = 8.84 Hz, 2H); 7.23 (s, 2H); 7.32–7.40 (m, 2H); 7.47–7.52 (m, 2H); 7.73–7.77 (m, 4H); 7.86–7.92
(m, 4H); 8.33 (s, 1H); 11.53 (s, 1H). 13C-NMR (DMSO-d6): 163.01, 161.07, 159.98, 149.86, 146.85, 146.76,
140.01, 133.40, 132.87, 129.62, 129.38, 128.91, 128.43, 128.17, 128.07, 127.59, 126.95, 126.56, 125.02,
124.75, 124.27, 122.82, 115.08, 112.24, 63.69, 63.24, 55.72, 43.45. MS m/z: [M + H]+ 569.6 (C36H32N4O3).
Anal. Calcd. for C36H32N4O3: C, 76.04; H, 5.67; N, 9.85. Found: C, 75.90; H, 5.65; N, 9.81.
(E)-4-(3-(2-Fluorophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N1-(3-methoxybenzylidene)-
benzohydrazide (H9). Yellow powder. Yield: 83.1%. m.p. 235–237 ˝C. 1H-NMR (DMSO-d6): 3.31–3.36
(dd, J1 = 5.52 Hz, J2 = 17.64 Hz, 1H); 3.79 (s, 3H); 4.11–4.19 (dd, J1 = 12.52 Hz, J2 = 18.84 Hz, 1H);
5.80–5.85 (dd, J1 = 5.12 Hz, J2 = 12.16 Hz, 1H); 6.97–6.99 (d, J = 7.68 Hz, 1H); 7.14–7.16 (d, J = 8.48 Hz,
2H); 7.23 (s, 2H); 7.29–7.33 (m, 3H); 7.40–7.52 (m, 4H); 7.74–7.76 (d, J = 8.44 Hz, 2H); 7.88–8.02 (m, 5H);
8.33 (s, 1H); 11.55 (s, 1H). 13C-NMR (DMSO-d6): 163.97, 163.08, 161.91, 161.07, 149.52, 146.87, 146.76,
139.32, 133.39, 132.90, 129.67, 129.40, 128.98, 128.97, 128.94, 128.75, 128.24, 128.07, 127.57, 126.61, 125.08,
124.24, 123.30, 116.16, 114.75, 112.49, 63.42, 60.43, 55.72, 43.42. MS m/z: [M + H]+ 543.6 (C34H27FN4O2).
Anal. Calcd. for C34H27FN4O2: C, 75.26; H, 5.02; F, 3.50; N, 10.33. Found: C, 75.16; H, 5.01; F, 3.48;
N, 10.30.
(E)-4-(3-(3-Fluorophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N1-(3-methoxybenzylidene)-
˝
benzohydrazide (H10). Yellow powder. Yield: 74.96%. m.p. 241–243 C.1H-NMR (DMSO-d6): 3.30–3.36
(dd, J1 = 5.28 Hz, J2 = 17.72 Hz, 1H); 3.79 (s, 3H); 4.02–4.10 (dd, J1 = 12.96 Hz, J2 = 18.32 Hz, 1H);
5.84–5.89 (dd, J1 = 5.36 Hz, J2 = 12.28 Hz, 1H); 6.97–6.99 (d, J = 7.68 Hz, 1H); 7.15–7.17 (d, J = 8.76 Hz,
2H); 7.24–7.28 (m, 3H); 7.33–7.40 (m, 2H); 7.49–7.51 (m, 3H); 7.63–7.67 (m, 2H); 7.73–7.76 (d, J = 8.72 Hz,
2H); 7.88–7.93 (m, 4H); 8.33 (s, 1H); 11.56 (s, 1H). 13C-NMR (DMSO-d6): 163.87, 163.08, 162.31, 161.07,
149.32, 146.87, 146.66, 139.82, 133.39, 132.90, 129.66, 129.40, 128.99, 128.97, 128.94, 128.77, 128.71, 128.24,
128.07, 127.57, 126.61, 125.08, 124.24, 123.30, 116.16, 114.74, 112.49, 63.42, 60.23, 55.72, 43.42.MS m/z:
[M + H]+ 543.6 (C34H27FN4O2). Anal. Calcd. for C34H27FN4O2: C, 75.26; H, 5.02; N, 10.33. Found: C,
75.14; H, 5.00; N, 10.31.
(E)-4-(3-(4-Fluorophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N1-(3-methoxybenzylidene)-
˝
benzohydrazide (H11). Yellow powder. Yield: 80.56%. m.p. 235–237 C. 1H-NMR (DMSO-d6): 3.28–3.32
(dd, J1 = 5.16 Hz, J2 = 17.84 Hz, 1H); 3.79 (s, 3H); 4.03–4.10 (dd, J1 = 10.04 Hz, J2 = 17.64 Hz, 1H);
5.81–5.85 (dd, J1 = 6.40 Hz, J2 = 12.28 Hz, 1H); 6.97–6.99 (d, J = 7.68 Hz, 1H); 7.12–7.14 (d, J = 8.80 Hz
2H); 7.23 (s, 2H); 7.29–7.34 (m, 3H); 7.38–7.40 (d, J = 9.88 Hz, 1H); 7.50–7.53 (m, 2H); 7.72–7.75
d, J = 8.72 Hz, 2H); 7.86–7.93 (m, 6H); 8.32 (s, 1H); 11.54 (s, 1H). 13C-NMR (DMSO-d6): 163.87, 162.35,
,
(
161.07, 150.32, 146.87, 146.66, 139.82, 134.39, 132.90, 129.66, 129.40, 128.99, 128.97 128.94, 128.77, 128.71,
128.24, 128.07, 127.57, 126.61, 125.08, 124.24, 123.30, 116.16, 114.74, 112.49, 63.45, 60.33, 55.74, 43.45.
MS m/z: [M + H]+ 543.6 (C34H27FN4O2). Anal. Calcd. for C34H27FN4O2: C, 75.26; H, 5.02; N, 10.33.
Found: C, 75.15; H, 4.99; N, 10.29.
(E)-4-(3-(3,5-Difluorophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N1-(3-methoxybenzylidene)
˝
benzohydrazide (H12). Yellow powder. Yield: 77.14%. m.p. 225–227 C. 1H-NMR (DMSO-d6): 3.30–3.36
(dd, J1 = 5.44 Hz, J2 = 18.08 Hz, 1H); 3.79 (s, 3H); 4.00–4.07 (dd, J1 = 12.40 Hz, J2 = 17.80 Hz, 1H);
5.88–5.92 (dd, J1 = 5.60 Hz, J2 = 12.40 Hz, 1H); 6.97–6.99 (d, J = 8.04 Hz, 1H); 7.17–7.23 (m, 4H);
7.28–7.40 (m, 3H); 7.48–7.54 (m, 4H); 7.73–7.76 (d, J = 8.78 Hz, 2H); 7.88–7.94 (m, 4H); 8.33 (s, 1H); 11.57
(s, 1H). 13C-NMR (DMSO-d6): 163.08, 161.01, 153.43, 149.96, 146.85, 146.58, 133.93, 139.14, 133.41,
132.89, 129.66, 129.63, 129.37, 128.82, 128.22, 128.07, 127.86, 127.56, 126.98, 126.59, 124.92, 124.26, 123.18,

Molecules 2016, 21, 1012
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114.75, 112.44, 104.02, 63.15, 56.46, 55.72, 43.52. MS m/z: [M + H]+ 561.6 (C34H26F2N4O2). Anal. Calcd.
for C34H26F2N4O2: C, 72.85; H, 4.67; N, 9.99. Found: C, 72.74; H, 4.66; N, 9.96.
(E)-4-(3-(3-Chlorophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyraz˝ol-1-yl)-N1-(3-methoxybenzylidene)-
benzohydrazide (H13). Yellow powder. Yield: 87.17%. m.p. 231–233 C. 1H-NMR (DMSO-d6): 3.30–3.36
(dd, J1 = 5.44 Hz, J2 = 17.76 Hz, 1H); 3.79 (s, 3H); 4.01–4.08 (dd, J1 = 12.16 Hz, J2 = 15.84 Hz, 1H);
5.84–5.88 (dd, J1 = 5.52 Hz, J2 = 12.36 Hz, 1H); 6.97–6.99 (d, J = 8.08 Hz, 1H); 7.15–7.17 (d, J = 8.84 Hz
2H); 7.23 (s, 2H); 7.33–7.44 (m, 3H); 7.49–7.51 (m, 2H) 7.60–7.62 (d, J = 8.84 Hz, 1H); 7.74–7.76
d, J = 8.76 Hz, 2H); 7.80–7.82 (d, J = 7.88 Hz, 1H); 7.87–7.93 (m, 4H); 8.01 (s, 1H); 8.33 (s, 1H); 11.57
,
(
(s, 1H). 13C-NMR (DMSO-d6): 170.09, 163.07, 161.08, 149.72, 146.89, 146.43, 139.54, 134.12, 133.39,
132.90, 131.37, 129.67, 129.40, 129.25, 128.20, 128.08, 127.56, 126.98, 126.63, 125.11, 124.24, 123.51,
114.75, 112.60, 63.20, 60.32, 55.72, 43.42. MS m/z: [M + H]+ 560.0 (75%), [M + 2 + H]+ 562.0 (25%),
(C34H27ClN4O2). Anal. Calcd. for C34H27ClN4O2: C, 73.05; H, 4.87; N, 10.02;. Found: C, 72.90; H, 4.85;
N, 9.98.
(E)-4-(3-(4-Chlorophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N1-(3-methoxybenzylidene)-
˝
benzohydrazide (H14). Yellow powder. Yield: 72.41%. m.p. 236–238 C. 1H-NMR (DMSO-d6): 3.32–3.38
(dd, J1 = 5.40 Hz, J2 = 17.60 Hz, 1H); 3.79 (s, 3H); 4.02–4.09 (dd, J1 = 11.84 Hz, J2 = 15.40 Hz, 1H);
5.82–5.87 (dd, J1 = 6.96 Hz, J2 = 12.44 Hz, 1H); 6.97–6.99 (d, J = 7.68 Hz, 1H); 7.13–7.15 (d, J = 8.80 Hz,
2H); 7.23 (s, 2H); 7.36–7.40 (m, 2H); 7.49–7.54 (m, 4H); 7.73–7.75 (d, J = 9.08 Hz, 2H); 7.83–7.93 (m, 6H);
8.33 (s, 1H); 11.58 (s, 1H). 13C-NMR (DMSO-d6): 170.11, 163.08, 161.07, 148.79, 146.89, 146.47, 139.53,
134.11, 133.39, 132.90, 131.27, 129.67, 129.40, 129.25, 128.20, 128.08, 127.56, 126.98, 126.63, 125.11, 124.24,
123.51, 114.75, 112.60, 63.20, 60.42, 55.72, 43.42. MS m/z: [M + H]+ 560.0 (75%), [M + 2 + H]+ 562.0
(25%), (C34H27ClN4O2). Anal. Calcd. for C34H27ClN4O2: C, 73.05; H, 4.87; N, 10.02. Found: C, 72.91;
H, 4.86; N, 9.99.
(E)-4-(3-(3,4-Dichlorophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N1-(3-methoxy-benzylidene)
˝
benzohydrazide (H15). Yellow powder. Yield: 71.11%. m.p. 234–236 C. 1H-NMR (DMSO-d6): 3.32–3.37
(dd, J1 = 5.44 Hz, J2 = 16.48 Hz, 1H); 3.79 (s, 3H); 4.01–4.09 (dd, J1 = 12.36 Hz, J2 = 17.72 Hz, 1H);
5.86–5.90 (dd, J1 = 5.72 Hz, J2 = 12.32 Hz, 1H); 6.97–6.99 (d, J = 7.96 Hz, 1H); 7.16–7.18 (d, J = 8.80 Hz,
2H); 7.23 (s, 2H); 7.33–7.40 (m, 2H); 7.50–7.52 (m, 2H); 7.71–7.75 (m, 3H); 7.80–7.83 (m, 1H); 7.87–7.93
(m, 4H); 8.01–8.02 (m, 1H); 8.32 (s, 1H); 11.56 (s, 1H). 13C-NMR (DMSO-d6): 163.07, 161.08, 149.79,
146.78, 146.47, 139.52, 134.12, 133.39, 132.87, 131.27, 129.67, 129.40, 129.25, 128.20, 128.08, 127.56,
126.98, 126.63, 125.11, 124.24, 123.51, 114.75, 112.60, 60.52, 55.72, 43.42. MS m/z: [M + H]+ 594.5 (75%),
[M + 2 + H]+ 598.5 (25%), (C34H26Cl2N4O2). Anal. Calcd. for C34H26Cl2N4O2: C, 68.81; H, 4.42;
N, 9.44; O, 5.39. Found: C, 68.69; H, 4.41; N, 9.41.
(E)-4-(3-(4-Bromophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyraz˝ol-1-yl)-N1-(3-methoxybenzylidene)-
benzohydrazide (H16). Yellow powder. Yield: 77.83%. m.p. 245–247 C. 1H-NMR (DMSO-d6): 3.27–3.33
(dd, J1 = 5.40 Hz, J2 = 17.60 Hz, 1H); 3.79 (s, 3H); 4.02–4.09 (dd, J1 = 12.28 Hz, J2 = 17.68 Hz, 1H);
5.82–5.87 (dd, J1 = 5.64 Hz, J2 = 12.28 Hz, 1H); 6.97–6.99 (d, J = 7.80 Hz, 1H); 7.13–7.15 (d, J = 8.76 Hz,
2H); 7.23 (s, 2H); 7.32–7.40 (m, 2H); 7.49–7.51 (m, 2H); 7.65–7.67 (d, J = 8.52 Hz, 2H); 7.72–7.78 (m, 4H);
7.87–7.93 (m, 4H); 8.32 (s, 1H); 11.55 (s,1H). 13C-NMR (DMSO-d6): 163.07, 161.08, 148.56, 146.82, 146.47,
139.71, 133.38, 132.90, 131.60, 129.67, 129.40, 128.94, 128.43, 128.25, 127.55, 126.98, 126.63, 125.11, 124.23,
123.53, 122.83, 114.75, 112.61, 63.40, 55.73, 43.12. MS m/z: [M + H]+ 604.5 (50%), [M + 2 + H]+ 606.5
(50%), (C34H27BrN4O2). Anal. Calcd. for C34H27BrN4O2: C, 67.67; H, 4.51; N, 9.28. Found: C, 67.54;
H, 4.50; N, 9.25.
1
(E)-N -(4-Methoxybenzylidene)-4-(5-(naphthalen-2-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzo-hydrazide
(
(
H17). Orange powder. Yield: 75.36%. m.p. 234–236 ˝C. 1H-NMR (DMSO-d6): 3.27–3.33
dd, J1 = 4.20 Hz, J2 = 17.68 Hz, 1H); 3.79 (s, 3H); 4.04–4.11 (dd, J1 = 10.44 Hz, J2 = 17.56 Hz, 1H);
5.80–5.85 (dd, J1 = 5.56 Hz, J2 = 12.24 Hz, 1H); 6.98–7.00 (d, J = 8.44 Hz, 2H); 7.12–7.14 (d, J = 8.80 Hz
,
2H); 7.39–7.53 (m, 6H); 7.61–7.63 (d, J = 8.20 Hz, 2H); 7.72–7.74 (d, J = 8.68 Hz, 2H); 7.82–7.84

Molecules 2016, 21, 1012
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(
d, J = 7.04 Hz, 2H); 7.88–7.93 (m, 4H); 8.29 (s,1H); 11.41 (s, 1H). 13C-NMR (DMSO-d6): 163.03, 161.01,
149.84, 139.88, 133.40, 132.34, 129.71, 129.66, 129.40, 129.21, 128.13, 128.08, 127.57, 126.97, 126.50,
125.07, 124.26, 123.28, 114.75, 112.49, 63.20, 60.23, 55.73, 43.35. MS m/z: [M + H]+ 525.6 (C34H28N4O2).
Anal. Calcd. for C34H28N4O2: C, 77.84; H, 5.38; N, 10.68. Found: C, 76.69; H, 5.39; N, 10.42.
1
(E)-N -(4-Methoxybenzylidene)-4-(5-(naphthalen-2-yl)-3-(o-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzo-hydrazide
H18). Yellow powder. Yield: 70.12%. m.p. 240–242 ˝C. 1H-NMR (DMSO-d6): 2.76 (s, 3H); 3.33–3.39
(
(dd, J1 = 5.44 Hz, J2 = 17.28 Hz, 1H); 3.79 (s, 3H); 4.10–4.18 (dd, J1 = 12.08 Hz, J2 = 17.44 Hz, 1H);
5.74–5.80 (dd, J1 = 5.44 Hz, J2 = 12.16 Hz, 1H); 6.98–7.00 (d, J = 8.40 Hz, 2H); 7.08–7.10 (d, J = 8.76 Hz,
2H); 7.25–7.32 (m, 2H); 7.36–7.41 (m, 2H); 7.48–7.53 (m, 3H); 7.61–7.63 (d, J = 8.12 Hz, 2H); 7.72–7.74
(
d, J = 8.68 Hz, 2H); 7.87–7.93 (m, 4H); 8.29 (s, 1H); 11.39 (s, 1H). 13C-NMR (DMSO-d6): 163.08, 161.07,
149.88, 139.92, 133.45, 132.34, 129.73, 129.65, 129.41, 129.21, 128.13, 128.08, 127.57, 126.97, 126.50, 125.07,
124.26, 123.28, 114.75, 112.49, 63.22, 55.72, 43.42. MS m/z: [M + H]+ 539.6 (C35H30N4O2). Anal. Calcd.
for C35H30N4O2: C, 78.04; H, 5.61; N, 10.40. Found: C, 77.91; H, 5.60; N, 10.36.
1
(E)-N -(4-Methoxybenzylidene)-4-(5-(naphthalen-2-yl)-3-(m-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzo-hydrazide
H19). Yellow powder. Yield: 82.68%. m.p. 230–232 ˝C. 1H-NMR (DMSO-d6): 2.37 (s, 3H); 3.26–3.32
(
(dd, J1 = 5.24 Hz, J2 = 17.56 Hz, 1H); 3.79 (s, 3H); 4.01–4.09 (dd, J1 = 12.08 Hz, J2 = 15.72 Hz, 1H);
5.80–5.84 (dd, J1 = 5.48 Hz, J2 = 12.32 Hz, 1H); 6.98–7.00 (d, J = 8.56 Hz, 2H); 7.12–7.14 (d, J = 8.84 Hz,
2H); 7.23–7.25 (d, J = 7.64 Hz, 1H); 7.33–7.40 (m, 2H); 7.49–7.51 (t, 2H); 7.60–7.63 (dd, J1 = 3.44 Hz,
J2 = 4.32 Hz, 3H); 7.68 (s, 1H); 7.71–7.74 (d, J = 8.64 Hz, 2H); 7.86–7.93 (m, 4H); 8.29 (s, 1H); 11.40
(
s, 1H). 13C-NMR (DMSO-d6): 163.03, 161.01, 149.84, 139.88, 133.40, 132.34, 129.71, 129.66, 129.40,
129.21, 128.13, 128.08, 127.57, 126.97, 126.50, 125.07, 124.26, 123.28, 114.75, 112.49, 63.20, 55.73, 43.35.
MS m/z: [M + H]+ 539.6 (C35H30N4O2). Anal. Calcd. for C35H30N4O2: C, 78.04; H, 5.61; N, 10.40.
Found: C, 77.66; H, 6.71; N, 9.46.
1
(E)-N -(4-Methoxybenzylidene)-4-(5-(naphthalen-2-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzo-hydrazide
H20). Yellow powder. Yield: 73.91%. m.p. 241–243 ˝C. 1H-NMR (DMSO-d6): 2.35 (s, 3H); 3.23–3.29
(dd, J1 = 4.24 Hz, J2 = 17.64 Hz, 1H); 3.79 (s, 3H); 4.00–4.07 (dd, J1 = 12.20 Hz, J2 = 17.64 Hz, 1H);
5.76–5.81 (dd, J1 = 5.56 Hz, J2 = 12.16 Hz, 1H); 6.98–7.00 (d, J = 8.48 Hz, 2H); 7.10–7.13 (d, J = 8.72 Hz
2H); 7.26–7.28 (d, J = 8.04 Hz, 2H); 7.37–7.40 (d, J = 8.40 Hz, 1H); 7.49–7.51 (m, 2H); 7.61–7.63
d, J = 8.12 Hz, 2H); 7.71–7.73 (d, J = 8.04 Hz, 4H); 7.87–7.92 (m, 4H); 8.30 (s, 1H); 11.39 (s, 1H).
(
,
(
13C-NMR (DMSO-d6): 163.09, 161.05, 150.14, 138.58, 133.40, 129.71, 129.66, 129.40, 129.21, 128.13,
128.08, 127.57, 126.97, 126.50, 125.07, 124.26, 123.28, 114.75, 112.49, 60.54, 55.72, 43.35. MS m/z: [M + H]+
539.6 (C35H30N4O2). Anal. Calcd. for C35H30N4O2: C, 78.04; H, 5.61; N, 10.40. Found: C, 77.60; H, 5.43;
N, 10.36.
(E)-4-(3-(4-Ethylphenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N1-(4-methoxybenzylidene)-
˝
benzohydrazide (H21). Orange powder. Yield: 76.19%. m.p. 239–241 C. 1H-NMR (DMSO-d6): 3.24–3.29
(dd, J1 = 4.40 Hz, J2 = 17.36 Hz, 2H); 3.79–3.81 (d, J = 2.84 Hz, 6H); 3.99–4.07 (dd, J1 = 11.36 Hz
,
J2 = 17.80 Hz, 1H); 5.74–5.78 (dd, J1 = 5.20 Hz, J2 = 11.60 Hz, 1H); 6.98–7.03 (m, 4H); 7.09–7.11
(d, J = 8.52 Hz, 2H); 7.38–7.40 (d, J = 8.52 Hz, 1H); 7.49–7.51 (m, 2H); 7.61–7.63 (d, J = 7.88 Hz, 2H);
7.71–7.78 (m, 4H); 7.86–7.93 (m, 5H); 8.29 (s, 1H); 11.40 (s, 1H). 13C-NMR (DMSO-d6): 163.07, 161.06,
160.70, 149.95, 149.85, 146.85, 146.77, 145.72, 140.01, 139.94, 133.40, 132.88, 129.62, 129.39, 128.92, 128.61,
128.23, 128.07, 127.59, 126.95, 126.63, 126.57, 125.02, 124.91, 124.27, 122.85, 114.75, 114.67, 112.26, 63.06,
55.76, 43.57, 43.44. MS m/z: [M + H]+ 553.2 (C36H32N4O2). Anal. Calcd. for C36H32N4O2: C, 78.24; H,
5.84; N, 10.14. Found: C, 74.84; H, 5.53; N, 9.85.
(E)-N1-(4-Methoxybenzylidene)-4-(3-(4-methoxyphenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-
˝
benzohydrazide (H22). Yellow powder. Yield: 69.13%. m.p. 243–245 C. 1H-NMR (DMSO-d6): 2.62–2.67
(dd, J1 = 7.56 Hz, J2 = 15.16 Hz, 2H); 3.25–3.30 (dd, J1 = 5.40 Hz, J2 = 7.64 Hz, 1H); 3.79–3.83 (t, 3H);
4.01–4.08 (dd, J1 = 12.36 Hz, J2 = 17.72 Hz, 1H); 5.77–5.82 (dd, J1 = 5.48 Hz, J2 = 12.12 Hz, 1H); 6.98–7.00
(d, J = 8.52 Hz, 2H); 7.10–7.13 (d, J = 8.80 Hz,2H); 7.29–7.31 (d, J = 8.20 Hz, 2H); 7.38–7.40 (d, J = 8.48 Hz
,

Molecules 2016, 21, 1012
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1H); 7.49–7.51 (m, 2H); 7.61–7.63 (d, J = 8.28 Hz, 2H); 7.71–7.75 (m, 4H); 7.87–7.92 (m, 5H); 8.29 (s, 1H);
11.40 (s, 1H). 13C- NMR (DMSO-d6): 163.05, 161.06, 149.96, 146.81, 146.72, 145.73, 139.95, 133.40, 132.88,
129.86, 129.64, 129.38, 128.92, 128.62, 128.24, 128.07, 127.58, 126.96, 126.64, 125.04, 124.26, 123.09, 114.75,
112.38, 63.08, 55.77, 55.72, 43.57, 43.44. MS m/z: [M + H]+ 555.6 (C35H30N4O3). Anal. Calcd. for
C35H30N4O3: C, 75.79; H, 5.45; N, 10.10. Found: C, 74.90; H, 5.99; N, 9.31.
1
(E)-N -(4-Methoxybenzylidene)-4-(5-(naphthalen-2-yl)-3-(3,4,5-trimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)
˝
benzohydrazide (H23). Yellow powder. Yield: 62.58%. m.p. 237–239 C. 1H-NMR (DMSO-d6): 3.35–3.40
(dd, J1 = 5.64 Hz, J2 = 17.56 Hz, 1H); 3.70 (s,3H); 3.79 (s,3H); 3.86 (s, 6H); 4.01–4.08 (dd, J1 = 12.60 Hz
J2 = 17.56 Hz, 1H); 5.80–5.85 (dd, J1 = 5.20 Hz, J2 = 12.12 Hz, 1H); 6.98–7.00 (d, J = 8.60 Hz, 2H);
7.10–7.14 (m, 4H); 7.38–7.40 (d, J = 8.52 Hz, 1H); 7.49–7.51 (m, 2H); 7.61–7.63 (d, J = 8.20 Hz, 2H);
7.70–7.72 (d, J = 8.76 Hz, 2H); 7.85–7.94 (m, 4H); 8.29 (s, 1H); 11.39 (s, 1H). 13C-NMR (DMSO-d6):
163.95, 163.66, 162.97, 162.32, 162.23, 161.09, 160.70, 147.83, 146.98, 146.16, 139.53, 136.01, 135.94, 135.87,
133.37, 132.92, 129.70, 129.64, 129.37, 128.95, 128.08, 127.53, 127.00, 126.67, 125.14, 124.24, 123.98, 114.75,
112.91, 109.54, 109.36, 104.85, 104.68, 104.51, 63.65, 55.72, 42.98. MS m/z: [M + H]+ 615.7 (C37H34N4O5).
Anal. Calcd. for C37H34N4O5: C, 72.30; H, 5.58; N, 9.11. Found: C, 71.41; H, 6.06; N, 8.31.
,
(E)-4-(3-(4-Ethoxyphenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N1-(4-methoxybenzylidene)-
˝
benzohydrazide (H24). Yellow powder. Yield: 76.15%. m.p. 237–239 C. 1H-NMR (DMSO-d6): 1.32–1.36
(t, 3H); 3.23–3.29 (dd, J1 = 5.16 Hz, J2 = 17.60 Hz, 1H); 3.79 (s, 3H); 3.99–4.10 (m,3H); 5.74–5.79
(dd, J1 = 5.56 Hz, J2 = 12.12 Hz, 1H); 6.98–7.02 (m, 4H); 7.08–7.10 (d, J = 8.84 Hz, 2H); 7.37–7.40 (m, 1H);
7.49–7.51 (m, 2H); 7.61–7.63 (d, J = 8.24 Hz, 2H); 7.70–7.77 (m, 4H); 7.86–7.9 2(m, 4H); 8.29 (s ,1H); 11.39
(s, 1H). 13C- NMR (DMSO-d6): 163.07, 161.05, 159.98, 149.86, 146.85, 146.76, 140.01, 133.40, 132.87,
129.62, 129.38, 128.91, 128.23, 128.17, 128.07, 127.59, 126.95, 126.56, 125.02, 124.75, 124.27, 122.82, 115.08,
112.24, 63.69, 63.04, 55.72, 43.55. MS m/z: [M + H]+ 569.6 (C36H32N4O3). Anal. Calcd. for C36H32N4O3:
C, 76.04; H, 5.67; N, 9.85. Found: C, 75.04; H, 5.71; N, 9.58.
(E)-4-(3-(2-Fluorophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyraz˝ol-1-yl)-N1-(4-methoxybenzylidene)-
benzohydrazide (H25). Yellow powder. Yield: 85.32%. m.p. 244–246 C. 1H-NMR (DMSO-d6): 3.29–3.34
(dd, J1 = 5.52 Hz, J2 = 17.64 Hz, 1H); 3.79 (s, 3H); 4.11–4.18 (dd, J1 = 12.20 Hz, J2 = 17.68 Hz, 1H);
5.80–5.84 (dd, J1 = 5.60 Hz, J2 = 12.32 Hz, 1H); 6.99–7.01 (d, J = 8.16 Hz, 2H); 7.13–7.15 (d, J = 8.48 Hz,
2H); 7.28–7.33 (m, 2H); 7.40–7.52 (m, 5H); 7.61–7.63 (d, J = 7.92 Hz, 2H); 7.73–7.75 (d, J = 8.40 Hz,2H);
7.88–7.94 (m, 3H); 7.98–8.02 (m, 1H); 8.30 (s, 1H); 11.42 (s, 1H). 13C-NMR (DMSO-d6): 163.53, 162.31,
161.08, 149.22, 146.85, 146.76, 139.62, 133.42, 132.88, 129.66, 129.40, 128.99 128.97, 128.94, 128.77, 128.71,
128.24, 128.07, 127.57, 126.61, 125.08, 124.24, 123.30, 116.16, 114.74, 112.49, 63.23, 60.43, 55.73, 43.42.
MS m/z: [M + H]+ 543.6 (C34H27FN4O2). Anal. Calcd. for C34H27FN4O2: C, 75.26; H, 5.02; N, 10.33.
Found: C, 75.13; H, 5.01; N, 10.30.
(E)-4-(3-(3-Fluorophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N1-(4-methoxybenzylidene)-
benzohydrazide (H26). Yellow powder. Yield: 75.6%. m.p. 240–242 ˝C. 1H-NMR (DMSO-d6): 3.29–3.35
(dd, J1 = 5.56 Hz, J2 = 17.80 Hz, 1H); 3.79 (s, 3H); 4.02–4.09 (dd, J1 = 12.28 Hz, J2 = 17.72 Hz, 1H);
5.84–5.88 (dd, J1 = 5.64 Hz, J2 = 12.36 Hz, 1H); 6.99–7.01 (d, J = 8.48 Hz, 2H); 7.14–7.17 (d, J = 8.84 Hz,
2H); 7.24–7.28 (m, 1H); 7.38–7.40 (d, J = 8.52 Hz, 1H ); 7.48–7.54 (m, 3H); 7.61–7.67 (m, 4H); 7.72–7.74
(
d, J = 8.68 Hz, 2H); 7.88–7.93 (m, 4H); 8.29 (s, 1H); 11.42 (s, 1H). 13C-NMR (DMSO-d6): 163.08, 162.32,
161.09, 149.11, 146.82, 146.64, 139.82, 133.32, 132.90, 129.65, 129.40, 128.99, 128.97, 128.95, 128.76, 128.72,
128.24, 128.07, 127.57, 126.65, 125.02, 124.24, 123.30, 116.16, 114.72, 112.45, 63.36, 60.24, 55.72, 43.42.
MS m/z: [M + H]+ 543.6 (C34H27FN4O2). Anal. Calcd. for C34H27FN4O2: C, 75.26; H, 5.02; N, 10.33.
Found: C, 75.14; H, 5.00; N, 10.31.
(E)-4-(3-(4-Fluorophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyraz˝ol-1-yl)-N
'-(4-methoxybenzylidene)-
benzohydrazide (H27). Yellow powder. Yield: 62.50%. m.p. 236–238 C. 1H-NMR (400 MHz, DMSO-d6):
3.28–3.34 (dd, J1 = 5.52 Hz, J2 = 17.72 Hz, 1H); 3.79 (s, 3H); 4.02–4.09 (dd, J1 = 7.08 Hz, J2 = 17.80 Hz,1H);
5.80–5.84 (dd, J1 = 5.68 Hz, J2 = 12.28 Hz, 1H); 6.99–7.01 (d, J = 8.48 Hz, 2H); 7.11–7.13 (d, J = 8.80 Hz,

Molecules 2016, 21, 1012
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2H); 7.29–7.33 (t,2H); 7.38–7.40 (m, 1H); 7.49–7.51 (m, 2H); 7.61–7.63 (d, J = 8.28 Hz, 2H); 7.72–7.74
d, J = 8.68 Hz, 2H); 7.86–7.93 (m, 6H); 8.29 (s, 1H); 11.41 (s, 1H). 13C-NMR (100 MHz, DMSO-d6):
(
163.93, 163.06, 162.29, 161.07, 149.00, 146.87, 146.66, 139.82, 133.39, 132.90, 129.66, 129.40, 128.99, 128.97,
128.94, 128.94, 128.77, 128.71, 128.24, 128.07, 127.57, 126.61, 125.08, 124.24, 123.30, 116.16, 114.74, 112.49,
63.33, 60.23, 55.71, 43.42. MS m/z: [M + H]+ 543.6 (C34H27FN4O2). Anal. Calcd. for C34H27FN4O2: C,
75.26; H, 5.02; N, 10.33. Found: C, 73.87; H, 4.97; N, 10.06.
(E)-4-(3-(3,5-Difluorophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N1-(4-methoxy-benzylidene)
˝
benzohydrazide (H28). Yellow powder. Yield: 69.3%. m.p. 238–240 C. 1H-NMR (400 MHz, DMSO-d6):
3.30–3.36 (dd, J1 = 5.52 Hz, J2 = 17.88 Hz, 1H); 3.79 (s, 3H); 3.99–4.07 (dd, J1 = 12.36 Hz, J2 = 17.84 Hz
,
1H); 5.87–5.92 (dd, J1 = 5.64 Hz, J2 = 12.44 Hz, 1H); 6.99–7.01 (d, J = 8.40 Hz, 2H); 7.17–7.19
(
(
d, J = 8.80 Hz, 2H); 7.29–7.33 (t, 1H); 7.38–7.40 (d, J = 8.48 Hz, 1H); 7.48–7.54 (m, 4H); 7.61–7.63
d, J = 8.28 Hz, 2H); 7.72–7.74 (d, J = 8.68 Hz, 2H); 7.88–7.94 (m, 4H); 8.29 (s, 1H); 11.43 (s, 1H).
13C-NMR (100 MHz, DMSO-d6): 163.09, 161.07, 153.53, 149.96, 146.85, 146.58, 133.93, 139.14, 133.41,
132.89, 129.66, 129.63, 129.37, 128.92, 128.22, 128.07, 127.86, 127.56, 126.98, 126.59, 124.92, 124.26, 123.18,
114.75, 112.44, 104.02, 63.17, 56.46, 55.73, 43.51. MS m/z: [M + H]+ 561.6 (C34H26F2N4O2). Anal. Calcd.
for C34H26F2N4O2: C, 72.85; H, 4.67; N, 9.99;. Found: C, 70.13; H, 4.92; N, 9.59.
(E)-4-(3-(3-Chlorophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N1-(4-methoxybenzylidene)-
1
benzohydrazide (H29). Orange powder. Yield: 87.21%. m.p. 241–243 ˝C. H-NMR (400 MHz,
DMSO-d6): 3.29–3.34 (dd, J1 = 5.48 Hz, J2 = 17.64 Hz, 1H); 3.79 (s, 3H); 4.01–4.08 (dd, J1 = 12.16 Hz,
J2 = 17.48 Hz, 1H); 5.84–5.88 (dd, J1 = 5.52 Hz, J2 = 12.32 Hz, 1H); 6.98–7.00 (d, J = 8.48 Hz, 2H);
7.14–7.16 (d, J = 8.88 Hz, 2H); 7.38–7.44 (m, 2H); 7.49–7.51 (m, 2H); 7.60–7.62 (d, J = 7.12 Hz, 3H);
7.72–7.74 (d, J = 8.76 Hz, 2H); 7.80–7.82 (d, J = 7.80 Hz, 1H); 7.87–7.93 (m, 4H); 8.01 (s, 1H); 8.29 (s, 1H);
11.41 (s, 1H). 13C-NMR (100 MHz, DMSO-d6): 170.21, 163.07, 161.09, 149.72, 14690, 146.43, 139.52,
134.32, 133.32, 132.90, 131.37, 129.67, 129.40, 129.35, 128.28, 128.08, 127.56, 126.98, 126.66, 125.11, 124.24,
123.51, 114.75, 112.60, 63.20, 55.72, 43.42. MS m/z: [M + H]+ 560.0 (75%), [M + 2 + H]+ 562.0 (25%),
(C34H27ClN4O2). Anal. Calcd. for C34H27ClN4O2: C, 73.05; H, 4.87; N, 10.02. Found: C, 72.91; H, 4.86;
N, 9.99.
(E)-4-(3-(4-Chlorophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N1-(4-methoxybenzylidene)-
1
benzohydrazide (H30). Orange powder. Yield: 66.67%. m.p. 237–239 ˝C. H-NMR (400 MHz,
DMSO-d6): 3.27–3.32 (dd, J1 = 5.44 Hz, J2 = 17.72 Hz, 1H); 3.79 (s, 3H); 4.01–4.06 (dd, J1 = 5.92 Hz
,
J2 = 17.48 Hz, 1H); 5.81–5.86 (dd, J1 = 5.72 Hz, J2 = 12.32 Hz, 1H); 6.98–7.00 (d, J = 8.48 Hz, 2H);
7.13–7.15 (d, J = 8.80 Hz, 2H); 7.38–7.40 (m, 1H); 7.49–7.54 (m, J1 = 4.00 Hz, J2 = 8.64 Hz, 4H); 7.61–7.63
(d, J = 8.28 Hz, 2H); 7.72–7.74 (d, J = 8.68 Hz, 2H); 7.83–7.93 (m, 6H); 8.30 (s, 1H); 11.42 (s, 1H).
13C-NMR (100 MHz, DMSO-d6): 170.81, 163.01, 161.08, 148.79, 146.89, 146.47, 139.73, 134.11, 133.39,
132.90, 131.27, 129.67, 129.40, 129.25, 128.20, 128.08, 127.56, 126.98, 126.63, 125.11, 124.24, 123.51,
114.75, 112.60, 63.40, 60.22, 55.72, 43.19. MS m/z: [M + H]+ 560.0 (75%), [M + 2 + H]+ 562.0 (25%),
(C34H27ClN4O2). Anal. Calcd. for C34H27ClN4O2: C, 73.05; H, 4.87; N, 10.02. Found: C, 72.00; H, 4.95;
N, 9.71.
(E)-4-(3-(3,4-Dichlorophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyra˝zol-1-yl)-N
'-(4-methoxy-benzylidene)
benzohydrazide (H31). Orange powder. Yield: 73.45%. m.p. 243–245 C. 1H NMR (400 MHz, DMSO-d6):
3.32–3.37 (dd, J1 = 5.44 Hz, J2 = 17.92 Hz, 1H); 3.79 (s, 3H); 4.01–4.08 (dd, J1 = 12.60 Hz, J2 = 17.72
Hz, 1H); 5.85–5.90 (dd, J1 = 5.80 Hz, J2 = 12.36 Hz, 1H); 6.98–7.00 (d, J = 8.48 Hz, 2H); 7.15–7.17
(d, J = 8.80 Hz, 2H); 7.38–7.40 (d, J = 8.48 Hz, 1H); 7.49–7.51 (m, 2H); 7.61–7.63 (d, J = 8.20 Hz, 2H);
7.71–7.74 (m, 3H); 7.79–7.82 (m, 1H); 7.87–7.93 (m, 4H); 8.02 (s, 1H); 8.29 (s, 1H); 11.42 (s, 1H). 13C NMR
(100 MHz, DMSO-d6): 163.08, 161.07, 149.89, 146.78, 146.47, 139.52, 134.32, 133.39, 132.87, 131.27, 129.67,
129.40, 129.25, 128.20, 128.08, 127.56, 126.98, 126.33, 125.11, 124.24, 123.51, 114.75, 112.60, 60.52, 55.72,
43.42. MS m/z: [M + H]+ 594.5 (75%), [M + 2 + H]+ 598.5 (25%), (C34H26Cl2N4O2). Anal. Calcd. for
C34H26Cl2N4O2: C, 68.81; H, 4.42; N, 9.44. Found: C, 68.68; H, 4.40; N, 9.42.

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(E)-4-(3-(4-Bromophenyl)-5-(naphthalen-2-yl)-4,5-dihydro-1H-pyrazol-1-yl)-N1-(4-methoxybenzylidene)-
˝
benzohydrazide (H32). Yellow powder. Yield: 70.90%. m.p. 238–240 C. 1H-NMR (400 MHz, DMSO-d6):
3.26–3.32 (dd, J1 = 5.44 Hz, J2 = 17.80 Hz, 1H); 3.79 (s, 3H); 4.01–4.08 (dd, J1 = 11.96 Hz, J2 = 17.84 Hz
1H); 5.81–5.85 (dd, J1 = 5.60 Hz, J2 = 12.20 Hz, 1H); 6.98–7.00 (d, J = 8.00 Hz, 2H); 7.13–7.15
d, J = 8.48 Hz, 2H); 7.38–7.40 (d, J = 8.32 Hz, 1H); 7.49–7.51 (m, 2H); 7.61–7.67 (m, 4H); 7.72–7.77
,
(
(m, 4H); 7.87–7.93 (m, 4H); 8.29 (s,1H); 11.41 (s, 1H). 13C-NMR (100 MHz, DMSO-d6): 163.00, 161.08,
148.86, 146.89, 146.47, 139.71, 133.38, 132.90, 131.60, 129.67, 129.40, 128.94, 128.43, 128.25, 127.55, 126.98,
126.63, 125.11, 124.23, 123.53, 122.83, 114.75, 112.61, 63.40, 55.73, 43.12. MS m/z: [M + H]+ 604.5 (50%),
[M + 2 + H]+ 606.5 (50%), (C34H27BrN4O2). Anal. Calcd. for C34H27BrN4O2: C, 67.67; H, 4.51; N, 9.28.
Found: C, 66.49; H, 6.35; N, 7.86.
3.3. Biological Assays
3.3.1. Antiproliferation Activity
IC50 values of the test compounds against A549, MCF-7, Hela and HepG2 cells were obtained
from State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, and were determined
by a standard (MTT)-based colorimetric assay (Beyotime Inst. Biotech., Nanjing, China). Tumor cell
lines were grown to mid-log phase and then diluted to 5
were seeded in 96-well plates (Philas, Nanjing, China) at a density of 5
well. The outer wells containing 1 PBS solution of the plate are not used for the assay to avoid
the possibility of edge effects. The plate was incubated at 37 ˝C in a 5% CO2 incubator in DMEM
containing 10% fetal bovine serum (FBS, Gibco, Waltham, MA, USA) for 24 h. Then, we added a 100-
series concentration of drug-containing medium into the wells to maintain the final concentration
of drug as 100, 10, 1 and 0.1 mol/L. After 12 h, cell survival was determined by the addition of a
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl trtrazolium bromide) solution (10 L of 5 mg/mL
MTT in 1 PBS). We waited for 4 h proliferation, discarded the medium and quickly added 150
ˆ
103–1
ˆ
104 cells/mL making sure they
103–1 104 cells per
ˆ
ˆ
ˆ
µL
µ
µ
ˆ
µL
DMSO per well, shaking the plates well for 10 min to ensure complete dissolution. Optical absorbance
was measured at 570 nm on a microplate reader (Bio-Rad, Hercules, CA, USA). Survival ratios are
expressed in percentages with respect to untreated cells. The experiments were replicated at least
three times to verify the methodology reproducibility when using the above-mentioned conditions.
The results were summarized in Table 2.
3.3.2. Kinase Assay
EGFR tyrosine kinase activity was determined by an enzyme linked-immunosorbent assay (ELISA)
in 96-well plates precoated in a 10 mL reaction volume including 4 mL diluted test compounds, 4 mL
substrate and 2 mL ATP. After incubation for 1 h at 37 ˝C, the reactions were stopped with 10 mL 2%
(v/v) H3PO4. The plates were then aspirated and washed twice with 200 mL 0.9% (w/v) NaCl, and
incorporation of Pi was determined with an LX300 Epson Diagnostic microplate reader. The residual
kinase activities for each concentration of compound and the compound IC50 values were expressed in
percentages with respect to untreated cells. The experiments were replicated at least three times to
verify the methodology reproducibility when using the above-mentioned conditions. The results were
summarized in Table 3.
3.3.3. Cytotoxicity Assay
The cytotoxic activity in vitro was measured by the colorimetric MTT assay. Cells were incubated
in a 96-well plate at a density of 105 cells per well with various concentrations of compounds (160, 40,
10, 2.5, 0.25
(5 mg/mL) was added per well 4 h before the end of the incubation. After removing the supernatant,
200 L DMSO was added to dissolve the formazan crystals. The absorbance at 570 nm was read on an
µM) in distilled 10% DMSO (10 µL) for 24 h. For the cytotoxicity assay, 20 µL of MTT
µ
λ

Molecules 2016, 21, 1012
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LX300 Epson Diagnostic microplate reader, untreated cells were used as negative controls. The results
are summarized in Table 4.
3.3.4. Cell Apoptosis Assay by Flow Cytometry
For Annexin V/PI assays, HeLa cells were stained with Annexin VFITC and PI and then monitored
for apoptosis by flow cytometry. Briefly, 5
were treated with H20 (2.0, 4.0, 6.0, 8.0 M) for 24 h. Then, cells were collected and washed twice with
PBS and stained with 5 L Annexin V-FITC and 5 L PI (5 g/mL) in 1 binding buffer (10 mM HEPES,
ˆ
103 cells were seeded in 6-well plates for 24 h and then
µ
µ
µ
µ
ˆ
pH 7.4, 140 mM NaOH, 2.5 mM CaCl2) for 15 min at room temperature in the dark. Apoptotic cells were
quantified using a BD Accuri C6 Flow Cytometer (Becton, Dickinson and Company, Clifton, NJ, USA).
Statistical analysis was done using Flowjo 7.6.1 software. Both early apoptotic (AnnexinV-positive,
PI-negative) and late apoptotic (double positive of Annexin V and PI) cells were detected.
3.4. Docking Simulations
The crystal structures of the proteins complex were retrieved from the RCSB Protein Data Bank
(PDB code: 1M17). Then, three-dimensional structures of the aforementioned compounds were
constructed using Chem 3D Ultra 14.0 software (Cambridge Soft Corporation, Boston MA, USA), then
they were energetically minimized by using MMFF94 with 5000 iterations and minimum RMS gradient
of 0.10. Molecular docking of compound H20 into the three-dimensional EGFR complex structure
was carried out using the Discovery Studio (Discovery Studio 4.5, Accelrys, Co. Ltd., Beijing, China)
software as implemented through the graphical user interface DS-CDOCKER protocol. All bound
waters and ligands were eliminated from the protein and the polar hydrogen was added to the proteins.
Briefly, we define the EGFR complex as a receptor, then perform simulation of the compound into the
ATP binding site in EGFR by replacing ATP molecule with our compound H20.
4. Conclusions
A series of benzohydrazide derivatives H1–H32 containing dihydropyrazoles were synthesized
and evaluated in vitro for anticancer activities against the A549, MCF-7, HeLa and HepG2 cell lines,
EGFR inhibitory activities, as well as MTT assays. These compounds exhibited potent EGFR inhibitory
activities and antiproliferative activities against the above four cell lines. Among them, compound H20
showed the most potent EGFR inhibition activities (IC50 = 0.08
A549, MCF-7, HeLa and HepG2 (IC50 = 0.46, 0.29, 0.15 and 0.21
µ
M) and anticancer activities against
µM, respectively). Docking simulation
of the binding model of compound H20 with EGFR indicated that conventional hydrogen bonds and
Van der Waals interactions with the protein residues in the ATP binding site might play a crucial role
in its EGFR inhibition and antiproliferative activities. Therefore, compound H20 may be developed as
a potential antitumor agent.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
8/1012/s1.
Acknowledgments: The work was financed by a grant (No. J1103512) from National Natural Science Foundation
of China and the Projects (No. CXY1409 & CG1305) from the Science & Technology Bureau of Lianyungang City
of Jiangsu Province.
Author Contributions: Haichao Wang and Hailiang Zhu conceived, designed and performed the synthetic
experiment part; Hongxia Li analyzed the data; Xiaoqiang Yan conceived, designed and performed the
docking simulation study part; Tianlong Yan conceived, designed and performed the pharmacological test
part; Haichao Wang wrote the paper. Xiaoqiang Yan, Zhongchang Wang and Hailiang Zhu assisted with the
paper revision.
Conflicts of Interest: The authors declare no conflict of interest.

Molecules 2016, 21, 1012
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Sample Availability: Samples of the compounds H1–H32 are available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).