Discovery of novel double pyrazole Schiff base derivatives as anti-tobacco mosaic virus (TMV) agents

Article abstract of DOI:10.1016/j.cclet.2016.10.029

Many pyrazole derivatives were reported to exhibit highly activity towards tobacco mosaic virus (TMV). In this work, an optimized pyrazole Schiff base scaffold was designed and introduced to derive novel potential TMV inhibitors. Thirty-six compounds were synthesized, characterized by elemental analysis, mass spectra and nuclear magnetic resonance (NMR) spectroscopy and evaluated by biological experiments. The bioassay results showed that some of the synthesized compounds exhibited excellent anti-TMV activities. Especially, 5-chloro-3-methyl-1H-pyrazole contained compound 4j showed ningnanmycin comparable inhibitory activity and can be considered as potential anti-TMV candidate agent. With molecular docking, compound 4j insert into nucleotide sequence (GAAGUU) of OriRNA stably which revealed nucleotide could be a target of these compounds.

Full text of DOI:10.1016/j.cclet.2016.10.029

G Model
CCLET 3864 1–6
Chinese Chemical Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
1
2
Original article
3
4
Discovery of novel double pyrazole Schiff base derivatives as
anti-tobacco mosaic virus (TMV) agents
a
a
c,d
b,
b
5
6
Q1 Xian-Hai Lv , Zi-Li Ren , Dong-Dong Li , Ban-Feng Ruan *, Qing-Shan Li ,
a
a
a
a
a,
Ming-Jie Chu , Cheng-Ying Ai , Dao-Hong Liu , Kai Mo , Hai-Qun Cao *
a
7
8
9
1
School of Plant Protection, Anhui Agricultural University, Hefei 230036, China
b
School of Biological and Medical Engineering, Hefei University of Technology, Hefei 230009, China
c
School of Chemical Engineering, Nanjing Forestry University, Nanjing 210073, China
d
0
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Many pyrazole derivatives were reported to exhibit highly activity towards tobacco mosaic virus (TMV).
In this work, an optimized pyrazole Schiff base scaffold was designed and introduced to derive novel
potential TMV inhibitors. Thirty-six compounds were synthesized, characterized by elemental analysis,
mass spectra and nuclear magnetic resonance (NMR) spectroscopy and evaluated by biological
experiments. The bioassay results showed that some of the synthesized compounds exhibited excellent
anti-TMV activities. Especially, 5-chloro-3-methyl-1H-pyrazole contained compound 4j showed
ningnanmycin comparable inhibitory activity and can be considered as potential anti-TMV candidate
agent. With molecular docking, compound 4j insert into nucleotide sequence (GAAGUU) of OriRNA
stably which revealed nucleotide could be a target of these compounds.
Received 14 June 2016
Received in revised form 22 August 2016
Accepted 23 September 2016
Available online xxx
Keywords:
Pyrazole
Schiff base
Anti-TMV
ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.
Published by Elsevier B.V. All rights reserved.
Molecule docking
1
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2
1. Introduction
production of MP (movement protein), increases the size of the 29
VRC and promotes TMV replication and dissemination [10,11]. Be- 30
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15
16
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21
22
23
24
25
26
27
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Tobacco mosaic virus (TMV) is widely distributed and highly
destructive especially for tobacco, which can cause serious damage
and large economic losses [1]. For the prevention of tobacco mosaic
virus, a lot of work were reported [2,3], but did not achieve desired
effect. So the identification of new antiviral agent with novel
mechanisms is critically needed to prevent this viral infection.
Recently, different targets in plant viruses that could be new
weapons to discover and design new antiviral drugs have been
studied and validated. Most antiviral drugs exhibit their activities
work by interaction with viral proteins [4,5]. Previous reports
demonstrated that a large accumulation of TMV coat protein (CP)
inside chloroplasts may affected photosynthesis in virus-infected
plants by inhibiting photo system II activity [6,7]. Similarly, in the
replication process of TMV, one or more viral proteins direct the
assembly of virus replication complexes (VRCS), which are in
association with host-derived membranes [8,9]. CP enhances the
sides, the favourable interaction with the origin of TMV RNA 31
(OriRNA) which leads to interference of initiation of virus assembly 32
is a new anti-TMV strategy. According to Xi et al.’s report [12], a Q233
fine analogue can selectively bind with DNA and RNA bulged 34
structures against TMV. Inspired by the above notion, we hope to 35
design a series of new compounds based on the common pyrazole 36
scaffold which could be able to target TMV RNA to inhibit viral 37
assembly.
38
Pyrazole are widely used as core motifs for a large number of 39
compounds in various applications such as agrochemicals and 40
medicine, due to their broad range of biological activities 41
[13,14]. The attractiveness of pyrazole and its derivatives is their 42
versatility that allows for synthesis of a series of analogues with 43
different moieties, thus affecting the electronics and by extension 44
the properties of the resultant compounds [15]. The molecules of 45
many modern drugs in the pesticide area contain pyrazole rings. 46
Compound with a pyrazole moiety reported by Song et al. (Fig. 1A) Q347
has excellent anti-TMV activity [16]. In addition, Schiff base has 48
antibacterial [17], anti-oxidation [18] and anti-tumour activity. 49
Although many drugs containing pyrazole and Schiff bases have 50
been reported [19,20], pyrazole Schiff bases at 4-position are 51
*
Corresponding authors.
E-mail addresses: ruanbf@huft.edu.cn (B.-F. Ruan), haiquncao@163.com
(H.-Q. Cao).
http://dx.doi.org/10.1016/j.cclet.2016.10.029
1
001-8417/ß 2016 Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: X.-H. Lv, et al., Discovery of novel double pyrazole Schiff base derivatives as anti-tobacco mosaic virus
(TMV) agents, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.10.029

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Fig. 1. Design of the target compounds.
Scheme 1. General synthesis of compounds 4a–4t and 5a–5p. Reagents and conditions: (a) H
2 3
O, ethanol, 60 8C, TLC; (b) DMF, POCl , 80–85 8C, 5 h; (c) sodium acetate, ethanol,
r.t., TLC; (d) DMF, POCl , 80–85 8C, 5 h; (e) H O, NaOH, ethanol, 3 h, reflux; (f) ethanol, 2 h, reflux; (g) ethanol, 2 h, reflux.
3
2
Please cite this article in press as: X.-H. Lv, et al., Discovery of novel double pyrazole Schiff base derivatives as anti-tobacco mosaic virus
TMV) agents, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.10.029
(

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X.-H. Lv et al. / Chinese Chemical Letters xxx (2016) xxx–xxx
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Table 1
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60
61
62
63
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68
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71
72
73
scarcely evaluated for their antiviral activities. Base on above
information, we inferred that if Schiff bases are linked at 4-position
in the pyrazole ring, and the retaining pyrazole pharmacophore is
introduced into another pyrazole scaffold, it could lead to
significant increases in the potency and physicochemical proper-
ties [21] and the resulting compounds may have better bioactiv-
ities. Furthermore, the nitrile group can improve bioavailability,
enhance the selectivity and binding affinity to target proteins by
hydrogen bond interactions, covalent interactions, polar interac-
The structure of compounds.
Compound
R
1
R
3
Compound
R
2
R
3
4a
H
H
5a
5b
5c
5d
5e
5f
H
H
H
H
F
H
4b
4c
H
F
F
H
Cl
CH
Cl
CH
H
4
4
4
4
d
e
f
H
3
3
3
3
3
3
3
H
CF
H
3
F
F
F
g
F
F
5g
5h
5i
F
Cl
CH
H
tions, and
p
–
p
interactions [22]. In order to develop a new class of
4h
F
Cl
CH
F
4
4
4
4
i
F
Cl
Cl
Cl
Cl
CH3
agrochemicals, two pyrazole rings were connected by a Schiff base
and a nitrile group (Fig. 1B) on the basis of the reported molecular
structure by Song et al. [16]. In addition, introduction of different
substituents in the pyrazole ring can bring about significant
changes in bioactivity [23] and we found that substituted benzene
with pyrazole having excellent biological activity according to our
previous reports [24]. Therefore, we further introduced the
benzene into the pyrazole ring (Fig. 1C), attempting to improve
the activity of the target compound. All the target compounds were
synthesized and evaluated their antiviral property. In addition,
molecular docking study revealed that this kind of compounds
could interact well with TMV RNA.
j
F
CF
H
3
5j
F
k
l
Cl
Cl
Cl
Cl
Cl
CH
CH
CH
5k
5l
Cl
CH
H
F
4m
4n
Cl
CH
5m
5n
5o
5p
CH
CH
CH
3
3
3
F
4o
4p
4q
4r
CF
H
F
3
Cl
CH
3
3
3
Cl
4s
4t
CH3
CH
CH3
CF
3
3
Table 2
Crystal data of compound 5d.
74
75
2. Results and discussion
Compound
Formula
5d
2.1. Chemistry
27 20 6
C H N
ꢀ
1
MW (g mol
)
428.490
Triclinic
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
The synthetic route is shown in Scheme 1. Compounds 1a–1d
were prepared in accordance with the literature procedure,
starting from the reactions of substituted phenylhydrazines with
ethyl acetoacetate in anhydrous ethanol medium. Then the
reaction mixtures were added to a cold solution of DMF and
Crystal system
a
b
g
a (A)
b ( A˚ )
(8)
(8)
(8)
64.837(3)
82.289(3)
75.083(3)
8.0726(7)
˚
12.3161(13)
12.7650(12)
1109.5(2)
2
c ( A˚ )
3
POCl , to give the 5-chloro-1-aryl-3-methyl-1H-pyrazole-4-car-
V (A˚
3
)
boxylic acids 1a–1d. Compounds 2a–2d were prepared by the
reaction of the phenyl hydrazones and Vilsmeier–Haack reagent
Z
3
(DMF/POCl ) respectively. The compounds 3a–3e were obtained
via refluxing the mixture of hydrazine hydrochlorides and 2-(-
ethoxymethylene) malononitrile in ethanol medium for 3 h. The
target compounds were synthesized by mixing 5-amino-1-aryl-
1H-pyrazole-4-carbonitrile 3a–3e in ethanol medium at reflux
temperature with compounds 1a–1d and 2a–2d (Scheme 1). The
the optimum compound 4j exhibited nearly equal inhibitory effect 111
to Ningnanmycin at 500 mg/L and 4o inhibitory effect slightly 112
lower than Ningnanmycin.
113
In vivo, protection and inactivation effects of compound 4j are 114
equivalent to Ningnanmycin, while its curative effect is lower than 115
Ningnanmycin. For compound 4o, protection effect is higher than 116
Ningnanmycin, but inactivation and curative effects are both lower 117
1
structures of the target compounds were characterized by H NMR,
mass spectroscopy and element analysis. The structures of the
synthetic compounds were shown in Table 1.
9
3
2.2. Single crystal XRD studies of compound 5d
94
95
96
97
98
Compound 5d was successfully crystallized with solvent of
acetonitrile and its structure was determined by single-crystal X-
ray diffraction analysis. The crystal data and refinement parameter
the compound is listed in Table 2. The derivative 5d crystallizes in
triclinic space group P-1(2). The crystal structure is shown in Fig. 2.
9
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2.3. Bioactivity
100
101
102
103
104
105
106
107
108
109
110
The commercially available plant virucide Ningnanmycin was
used as the positive control. The antiviral bioassay against TMV is
assayed by the reported method and the antiviral results of all the
compounds are listed in Table 3. The results showed that most of
target compounds present excellent anti-TMV activities at 500 mg/
L.
In vitro, compounds 4a–4t exhibited excellent inhibitory
activities of 31.4–78.4% at 500 mg/L, and compounds 5a–5p
showed only 19–59.4% inhibitory activities. The commercial plant
virucide Ningnanmycin, perhaps the most successful registered
antiplant viral agent, exhibited 79.2% inhibitory effect at 500 mg/L;
Fig. 2. The molecular structure of 5d showing 50% probability displacement
ellipsoids.
Please cite this article in press as: X.-H. Lv, et al., Discovery of novel double pyrazole Schiff base derivatives as anti-tobacco mosaic virus
TMV) agents, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.10.029
(

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X.-H. Lv et al. / Chinese Chemical Letters xxx (2016) xxx–xxx
Table 3
Anti-TMV activity of compounds in vitro and in vivo at 500 mg/mL.
a
Compound
In vitro
In vivo inhibition rate (%)
a
inhibition rate (%)
Protection Inactivation Curative
effect
effect
effect
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
34.1 ꢁ 2.1
46.5 ꢁ 1.4
32.1 ꢁ 2.3
31.4 ꢁ 0.5
55.3 ꢁ 2.5
56.3 ꢁ 2.8
61.6 ꢁ 0.5
64.3 ꢁ 0.9
54.2 ꢁ 2.2
78.4 ꢁ 0.2
48.6 ꢁ 1.5
50.6 ꢁ 0.6
59.7 ꢁ 5.4
37.9 ꢁ 0.3
65.4 ꢁ 3.0
35.4 ꢁ 1.6
45.3 ꢁ 3.5
36.4 ꢁ 0.9
24.1 ꢁ 2.5
37.5 ꢁ 4.2
19.4 ꢁ 1.6
40.1 ꢁ 5.2
35.1 ꢁ 1.6
22.4 ꢁ 1.8
27.6 ꢁ 3.9
59.4 ꢁ 2.6
52.4 ꢁ 3.6
41.5 ꢁ 5.5
29.1 ꢁ 2.5
54.0 ꢁ 4.2
51.2 ꢁ 1.5
34.2 ꢁ 3.1
29.4 ꢁ 3.3
48.2 ꢁ 2.6
39.4 ꢁ 3.5
38.4 ꢁ 5.2
25.3 ꢁ 3.5
34.7 ꢁ 0.6
28.5 ꢁ 0.2
23.8 ꢁ 2.4
49.8 ꢁ 3.5
52.4 ꢁ 1.2
67.5 ꢁ 5.6
54.8 ꢁ 4.7
26.7 ꢁ 3.2
61.8 ꢁ 2.5
53.4 ꢁ 2.5
49.3 ꢁ 6.5
61.3 ꢁ 3.3
36.4 ꢁ 2.5
68.5 ꢁ 3.9
26.8 ꢁ 4.7
35.4 ꢁ 1.5
37.8 ꢁ 2.5
39.4 ꢁ 3.6
25.9 ꢁ 2.2
22.6 ꢁ 2.5
38.7 ꢁ 3.5
38.2 ꢁ 2.5
25.8 ꢁ 2.4
31.2 ꢁ 0.6
60.4 ꢁ 1.5
52.1 ꢁ 1.6
47.1 ꢁ 4.2
34.2 ꢁ 1.2
49.7 ꢁ 3.5
35.7 ꢁ 2.4
38.4 ꢁ 1.5
28.7 ꢁ 0.9
47.2 ꢁ 3.9
47.6 ꢁ 2.5
29.4 ꢁ 3.6
63.5 ꢁ 5.2
32.5 ꢁ 1.5
27.8 ꢁ 3.4
19.6 ꢁ 1.5
15.3 ꢁ 4.3
57.2 ꢁ 2.5
56.7 ꢁ 0.8
58.7 ꢁ 6.5
51.4 ꢁ 6.2
46.5 ꢁ 3.5
72.4 ꢁ 3.6
58.6 ꢁ 5.5
37.9 ꢁ 7.9
29.6 ꢁ 4.2
46.8 ꢁ 2.0
52.7 ꢁ 0.9
19.2 ꢁ 0.5
24.8 ꢁ 3.5
24.4 ꢁ 2.5
19.8 ꢁ 4.6
39.7 ꢁ 3.8
21.5 ꢁ 1.9
35.7 ꢁ 1.6
38.1 ꢁ 2.1
34.2 ꢁ 1.7
31.5 ꢁ 1.5
57.5 ꢁ 3.2
55.4 ꢁ 2.5
37.2 ꢁ 3.2
37.5 ꢁ 3.2
53.6 ꢁ 2.9
46.7 ꢁ 3.8
41.3 ꢁ 5.5
35.1 ꢁ 2.7
38.1 ꢁ 4.5
43.5 ꢁ 3.0
35.2 ꢁ 0.3
76.3 ꢁ 3.5
20.4 ꢁ 3.5
25.4 ꢁ 1.4
23.8 ꢁ 3.1
22.7 ꢁ 2.6
46.5 ꢁ 5.7
53.2 ꢁ 3.4
63.2 ꢁ 1.5
54.2 ꢁ 0.6
53.6 ꢁ 5.1
65.7 ꢁ 3.1
37.8 ꢁ 3.5
68.7 ꢁ 6.8
34.9 ꢁ 1.3
57.5 ꢁ 1.5
49.5 ꢁ 5.8
24.3 ꢁ 6.5
31.5 ꢁ 0.6
28.9 ꢁ 1.0
25.7 ꢁ 2.7
38.5 ꢁ 0.4
28.7 ꢁ 2.1
37.2 ꢁ 3.5
40.5 ꢁ 0.2
35.1 ꢁ 2.1
35.2 ꢁ 0.2
57.0 ꢁ 3.5
57.1 ꢁ 2.5
47.2 ꢁ 0.5
28.1 ꢁ 2.1
54.3 ꢁ 3.1
50.2 ꢁ 2.5
39.5 ꢁ 3.5
31.2 ꢁ 1.4
44.2 ꢁ 2.5
37.1 ꢁ 3.5
34.8 ꢁ 2.1
57.8 ꢁ 5.9
g
h
i
j
k
l
m
n
o
p
q
r
s
t
a
b
c
d
e
f
g
h
i
j
Fig. 3. (A) The tertiary structure of OriRNA (the orange one represented OriRNA
produced by RNAComposer; the green one represented OriRNA influenced by the
induced fit docking based on the orange one). The box described X-ray crystal
structure of coat protein in complexation with nucleotide GAA of OriRNA (PDB ID:
k
l
m
n
o
p
2TMV). (B) The interaction mode of compound 4j at the binding site of OriRNA.
2.4. Molecular docking
145
Ningnanmycin 79.2 ꢁ 2.5
a
Average of three replicates.
The assembly origin of TMV RNA was called OriRNA, which
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
0
located about 900 nucleotides from the 3 -end of the genome. As
shown in Fig. 3A, The OriRNA can form a putative hairpin loop
structure, the trinucleotide GAA of which is reported to bind
strongly to TMV CP for initiating viral assembly [26]. Therefore, this
could be selected as one important binding site of anti-TMV
compounds. In addition, the tertiary structure of OriRNA was built
on the RNA composer algorithm [27]. In order to explore that
binding mode between Pyrazole derivatives and TMV RNA, the most
potent compound 4j was docked into the binding site of OriRNA.
Induced fit docking displayed that compound 4j can fit the pocket
composed by the nucleotide sequence (GAAGUU) of OriRNA
(Fig. 3B). Obliviously, the conformations of the three key nucleotides
GAA that interacted with CP have greatly changed. The RMSD of the
postdocking conformations of GAA relative to the native structure
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
than Ningnanmycin. In short, the anti-TMV activity of 4j is better
than 4o. For target compounds 5a–5p, protection effects of
compound 5f is equivalent to 4j and Ningnanmycin, and its
curative effect is equivalent to Ningnanmycin, while its inactiva-
tion effect is lower than 4j, 4g and Ningnanmycin. In addition,
inhibitory activities of target compounds 5a–5p are lower than
target compounds 4a–4t whether in vivo or in vitro. The probable
reason is that substituted benzene reduced activity and chlorine
atom and methyl having excellent biological activity on pyrazole.
In general, when R
5a–5p, then, if R is an electron-withdrawing substituents, the
activity has a certain promotion compared to the electron-
donating substituent, and when the substituent is CF , the activity
of the compound is the best. The electron-withdrawing sub-
stituents activity order is: CF > F > Cl > CH . Likewise, when R is
the same, the activity would be optimized if R is substituted by an
electron withdrawing group. It has the same order of activity:
F > Cl > CH . As a whole, the optimal substituent for both R and R
could be concluded as follows: CF > F > Cl > CH . Therefore, we
1
is the same for target compounds 4a–4t and
2
3
˚
were 1.59, 3.10, and 3.30 A, respectively. This kind of result could
3
3
2
lead to the loss of RNA affinity to TMV CP in the assembly process.
1
3. Conclusion
163
3
1
2
3
3
In order to find a target of anti-TMV drugs in TMV CP, two series
of new 5-chloro-3-methyl-1-phenyl-pyrazole derivatives with
cyano substituent were designed and synthesized. Anti-TMV
activity biological evaluation showed that most of these compounds
performed excellent anti-TMV activity. Compound 4j showed the
most potent biological activity against TMV and nearly equal
inhibitory effect compared to the commercial agent Ningnanmycin.
Preliminary studies showed that compound 4j has the most
potent biological activity against TMV, and also showed that the
164
165
166
167
168
169
170
171
172
can say that the activities of the compounds increase along with
the electronic absorption ability of substituents.
Furthermore, their anti-TMV activities are similar to pyrazole
amide compounds which we previously reported [25]. This will
provide a favourable reference for our further work. For these
target compounds, compound 4j showed the most potent
biological activity against TMV and nearly equal inhibitory effect
compared to the commercial agent Ningnanmycin.
Please cite this article in press as: X.-H. Lv, et al., Discovery of novel double pyrazole Schiff base derivatives as anti-tobacco mosaic virus
TMV) agents, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.10.029
(

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5
173
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175
compound 4j is structurally against TMV by exhibiting a higher
affinity for TMV RNA, compound 4j is a potential anti-TMV
candidate.
[28]. This method is to measure the antiviral activity of compounds 232
in vitro and test the protective effect, the inactivation effect and the 233
curative effect in vivo against TMV.
234
Purification of tobacco mosaic virus was found by using the 235
Gooding’s method [28]. The absorbance values were estimated at 236
1
76
77
4. Experimental
260 nm by using an ultraviolet spectrophotometer.
237
2
38
1
4.1. Materials and measurements
A
260ꢂdilution ratio
Virus concentration ðmg=mLÞ ¼
0
:1%; 260 nm
178
179
180
181
182
183
184
185
All chemicals (reagent grade) used were commercially avail-
able. All the H NMR spectra were measured on an Agilent DD2
600 Hz spectrometer at 25 8C and referenced to Me4Si. Chemical
shifts were reported in parts per million (d). ESI-MS spectra were
recorded on a Mariner System 5304 Mass spectrometer. Elemental
analyses were performed on a CHN-O-Rapid instrument and were
within 0.4% of the theoretical values. Melting points were
measured on a XT4 MP apparatus (Taike Corp, Beijing, China).
E
1
cm
1
2
241
34 90
4
.3.1. Antiviral activity of target compounds in vitro
Fresh 5–6 growth stage of tobacco leaves (Nicotiana tabacum 242
var. Xanthi NC) were selected for the test. The tobacco was 243
inoculated by the juice-leaf rubbing method, and the concentration 244
mg/mL. The leaves were cut into halves 245
along the main vein, and a solution of the compounds with a 246
of TMV was 5.88 ꢂ 10ꢀ
2
concentration of 500
mg/mL was smeared on the halves, and then 247
1
86
4.2. Synthesis
cultured at 25 8C for 72 h. Each compound was tested three times. 248
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
Synthesis of 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbal-
dehyde 1a–1d: Para-substituted phenyl hydrazine (0.025 mol) was
dissolved in anhydrous ethanol, ethyl acetoacetate (0.025 mol) was
slowly added and stirred at 70–80 8C for 5 h, then the anhydrous
ethanol was removed under reduced pressure to form a solid, which
was dissolved in DMF (20 mL) and phosphorus oxychloride (16 mL)
of cold mixed solution and stirred at 80–85 8C for 5 h. The resulting
mixture was poured into ice-cold water, the resulting solid was
separated by filtration to give the light yellow solid.
Synthesis of 1-(4-substituented phenyl)-3-phenyl-1H-pyra-
zole-4-carbaldehyde 2a–2d: Para-substituted acetophenone
(20 mmol) interact with phenylhydrazine hydrochloride
(20 mmol) in anhydrous ethanol to form 1-phenyl-2-(1-pheny-
lethylidene) hydrazine, which was then dissolved in a cold mixed
4
.3.2. Protective effect of target compounds against TMV in vivo
The test compound solution was smeared on the left side, and 250
the solvent served as control on the right side of growing tobacco 251
Nicotine tabacum var. Xanthium NC). After 12 h, TMV at a 252
concentration of 6.0 g/mL was inoculated on the leaves which 253
249
(
m
were previously scattered with silicon carbide through the above 254
juice-leaf rubbing method. Then the leaves were again immersed 255
into water and rubbed softly along the nervation once or twice. The 256
local lesion numbers showing 3–4 days after inoculation were 257
counted. Each compound was tested in three replicates.
258
4
.3.3. Inactivate effect of target compounds against TMV in vivo
259
The virus was inhibited by mixing with the target compound 260
solution at the same volume for 30 min. Then the mixture was 261
inoculated on the left side of the host tobacco leaves (Nicotiana 262
tabacum var. Xanthi NC), and a mixture of solvent and the virus 263
was inoculated on the right side to serve as control. The local lesion 264
numbers showing 3–4 days after inoculation were counted. Each 265
3
solution of DMF (20 mL) and POCl (16 mL), stirred at 80–85 8C for
5 h. The resulting mixture was poured into ice-cold water, a
saturated solution of sodium hydroxide was added to neutralize
the mixture, and the solid precipitate was filtered, washed with
water, dried and recrystallized from ethanol.
compound was tested in three replicates.
266
Synthesis of 5-amino-1-aryl-1H-pyrazole-4-carbonitriles 3a–
3e:
A
stirred mixture of para-substituted phenylhydrazine
O (30 mL), then
4
.3.4. Curative effect of target compounds against TMV in vivo
267
hydrochloride (0.025 mol) was dissolved in H
2
Host plant tobacco leaves (Nicotiana tabacum var. Xanthi NC) of 268
the same age growing at the six-leaf stage were selected for the 269
test. TMV at a concentration of 6.0 g/mL was inoculated on the 270
whole leaves. The leaves were washed with water, and dried in a 271
greenhouse. The target compound solution was smeared on the left 272
side, and the solvent served as control on the right side. The local 273
lesion numbers showing 3–4 days after inoculation were counted. 274
the pH of the mixture was adjusted to pH 7–8 by the dropwise
addition of 10% NaOH solution to form the free para-substituted
phenyl hydrazines, which were then refluxed for 3 h with ethoxy
methylene malononitrile in an ethanol medium. After completion
of the reaction, the reaction mixture was allowed to cool at room
temperature, and the solid were filtered under vacuum. The crude
products obtained were recrystallized from anhydrous ethanol to
give the light yellow solid.
Synthesis of the target compounds 4a–4t: Equimolar portions of
the intermediate compounds 1 (1 mmol) and the intermediate
compounds 3 (1 mmol) were dissolved in approximately 8 mL of
ethanol. The reaction solution was allowed to stir at 80 8C for 2 h
until the reaction was complete. The reaction was monitored by TLC.
Mostly, a precipitate formed and was then collected by suction
filtration.
m
Each compound was tested in three replicates.
275
The inhibition rates of the compound in vitro and in vivo were 276
calculated according to the following formula (controls were not 277
treated with compound):
278
279
Inhibition rate ð%Þ ¼
2
3
average local lesion number of control
6
4
ꢀaverage local lesion number of drug treated7
5ꢂ100
(1)
average local lesion number of control
Synthesis of the target compounds 5a–5p: A mixture of
the intermediate compounds 2 (1 mmol) and 3 (1 mmol) in
ethanol (10 mL) was stirred at reflux for 2 h. After cooling to room
temperature, the precipitated solid was filtered, and then recrys-
tallized from ethanol to give the title compounds 5a–5p.
2801
282
4.4. X-ray crystallography
Compound 5d was successfully crystallized with solvent of 283
acetonitrile and its structure was determined by single-crystal X- 284
ray diffraction analysis. The structure was solved by direct 285
methods and refined on F by full-matrix least-squares methods 286
using ELXL-97 [29]. The unit cell dimensions and intensity data 287
2
29
4.3. Antiviral biological assay
2
2
2
30
The anti-TMV activities of the synthesized pure compounds
31 Q4 activity was tested by using the method reported by Thorson et al.
Please cite this article in press as: X.-H. Lv, et al., Discovery of novel double pyrazole Schiff base derivatives as anti-tobacco mosaic virus
TMV) agents, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.10.029
(

G Model
CCLET 3864 1–6
6
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were recorded at 293 K. The program SAINT/XPREP was used for
data reduction and APEX2/SAINT for cell refinement [30]. All non-
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4.5. Molecular docking
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Three dimensional structure of the TMV RNA was generated by
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Drawing Standard, Cambridge Soft Corporation, Cambridge, MA,
USA), then they were energetically minimized by using MMFF94
with 5000 iterations and minimum RMS gradient of 0.10. The
crystal structures of protein complex complexity were retrieved
from the RCSB Protein Data Bank (http://www.rcsb.org/pdb/home/
home.do) and prepared by Discovery Studio 3.1 with all bound
waters and ligands eliminated from the protein and the polar
hydrogen added to the protein. The molecular docking procedure
was performed by using CDOCKER protocol for receptor–ligand
interactions section of DS 3.1.
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3
3
3
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3
14 Q5
This work was supported by National Natural Science Founda-
tion of China (No. 21302002), Anhui Provincial Natural Science
Foundation (No. 1408085QB33) and Key Scientific and Technolog-
ical Project of Anhui Provincial Tobacoo Company (No.
20150551007).
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in
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derivatives, RSC Adv. 5 (2015) 55179–55185.
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Please cite this article in press as: X.-H. Lv, et al., Discovery of novel double pyrazole Schiff base derivatives as anti-tobacco mosaic virus
TMV) agents, Chin. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.cclet.2016.10.029
(