English

• 1.   Introduction

  Tobacco mosaic virus (TMV) is widely distributed and highly destructive especially for tobacco, which can cause serious damage and large economic losses . 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 production of MP (movement protein), increases the size of the VRC and promotes TMV replication and dissemination [10, 11]. Besides, the favourable interaction with the origin of TMV RNA (OriRNA) which leads to interference of initiation of virus assembly is a new anti-TMV strategy. According to Xi et al.'s report [12], a fine analogue can selectively bind with DNA and RNA bulged structures against TMV. Inspired by the above notion, we hope to design a series of new compounds based on the common pyrazole scaffold which could be able to target TMV RNA to inhibit viral assembly.

  Pyrazole are widely used as core motifs for a large number of compounds in various applications such as agrochemicals and medicine, due to their broad range of biological activities [13, 14]. The attractiveness of pyrazole and its derivatives is their versatility that allows for synthesis of a series of analogues with different moieties, thus affecting the electronics and by extension the properties of the resultant compounds [15]. The molecules of many modern drugs in the pesticide area contain pyrazole rings. Compound with a pyrazole moiety reported by Song et al. (Fig. 1A) has excellent anti-TMV activity [16]. In addition, Schiff base has antibacterial [17], anti-oxidation [18] and anti-tumour activity. Although many drugs containing pyrazole and Schiff bases have been reported [19, 20], pyrazole Schiff bases at 4-position are 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 properties [21] and the resulting compounds may have better bioactivities. Furthermore, the nitrile group can improve bioavailability, enhance the selectivity and binding affinity to target proteins by hydrogen bond interactions, covalent interactions, polar interactions, and π-π interactions [22]. In order to develop a new class of 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.

  图 1

  

  图 1  Design of the target compounds.

  Figure 1.  Design of the target compounds.

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  2.   Results and discussion

  2.1   Chemistry

  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 POCl₃, to give the 5-chloro-1-aryl-3-methyl-1H-pyrazole-4-carboxylic acids 1a-1d. Compounds 2a-2d were prepared by the reaction of the phenyl hydrazones and Vilsmeier-Haack reagent (DMF/POCl₃) 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 or 2a-2d (Scheme 1). The structures of the target compounds were characterized by ¹H NMR, mass spectroscopy and element analysis. The structures of the synthetic compounds were shown in Table 1.

  Scheme1

  

  Scheme1  General synthesis of compounds 4a-4t and 5a-5p. Reagents and conditions: (a) H₂O, ethanol, 60 ℃, TLC; (b) DMF, POCl₃, 80-85 ℃, 5 h; (c) sodium acetate, ethanol, r.t., TLC; (d) DMF, POCl₃, 80-85 ℃, 5 h; (e) H₂O, NaOH, ethanol, 3 h, reflux; (f) ethanol, 2 h, reflux; (g) ethanol, 2 h, reflux.

  Scheme1.  General synthesis of compounds 4a-4t and 5a-5p. Reagents and conditions: (a) H₂O, ethanol, 60 ℃, TLC; (b) DMF, POCl₃, 80-85 ℃, 5 h; (c) sodium acetate, ethanol, r.t., TLC; (d) DMF, POCl₃, 80-85 ℃, 5 h; (e) H₂O, NaOH, ethanol, 3 h, reflux; (f) ethanol, 2 h, reflux; (g) ethanol, 2 h, reflux.

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  表 1

  

  表 1  The structure of compounds.

  Table 1.  The structure of compounds.

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  2.2   Single crystal XRD studies of compound 5d

  Compound 5d was successfully crystallized with solvent of acetonitrile and its structure was determined by single-crystal Xray 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.

  表 2

  

  表 2  Crystal data of compound 5d.

  Table 2.  Crystal data of compound 5d.

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  图 2

  

  图 2  The molecular structure of 5d showing 50% probability displacement ellipsoids.

  Figure 2.  The molecular structure of 5d showing 50% probability displacement ellipsoids.

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  2.3   Bioactivity

  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.

  表 3

  

  表 3  Anti-TMV activity of compounds in vitro and in vivo at 500 mg/mL.

  Table 3.  Anti-TMV activity of compounds in vitro and in vivo at 500 mg/mL.

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  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; the optimum compound 4j exhibited nearly equal inhibitory effect to Ningnanmycin at 500 mg/L and 4o inhibitory effect slightly lower than Ningnanmycin.

  In vivo, protection and inactivation effects of compound 4j are equivalent to Ningnanmycin, while its curative effect is lower than Ningnanmycin. For compound 4o, protection effect is higher than Ningnanmycin, but inactivation and curative effects are both lower 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 inactivation 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 R₁ is the same for target compounds 4a-4t and 5a-5p, then, if R₂ is an electron-withdrawing substituents, the activity has a certain promotion compared to the electrondonating substituent, and when the substituent is CF₃, the activity of the compound is the best. The electron-withdrawing substituents activity order is: CF₃ > F > Cl > CH₃. Likewise, when R₂ is the same, the activity would be optimized if R₁ is substituted by an electron withdrawing group. It has the same order of activity: F > Cl > CH₃. As a whole, the optimal substituent for both R₁ and R₂ could be concluded as follows: CF₃ > F > Cl > CH₃. Therefore, we 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 [24]. 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.

  2.4   Molecular docking

  The assembly origin of TMV RNA was called OriRNA, which 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 [25]. 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 [26]. 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 were 1.59, 3.10, and 3.30 Å , respectively. This kind of result could lead to the loss of RNA affinity to TMV CP in the assembly process.

  图 3

  

  图 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: 2TMV). (B) The interaction mode of compound 4j at the binding site of OriRNA.

  Figure 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: 2TMV). (B) The interaction mode of compound 4j at the binding site of OriRNA.

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  3.   Conclusion

  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 compound 4j is structurally against TMV by exhibiting a higher affinity for TMV RNA, compound 4j is a potential anti-TMV candidate.

  4.   Experimental

  4.1   Materials and measurements

  All chemicals (reagent grade) used were commercially available. All the 1H NMR spectra were measured on an Agilent DD₂ 600 Hz spectrometer at 25 ℃ and referenced to Me4Si. Chemical shifts were reported in parts per million (δ). 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).

  4.2   Synthesis

  Synthesis of 5-chloro-3-methyl-1-phenyl-1H-pyrazole-4-carbaldehyde 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 ℃ 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 ℃ 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-pyrazole-4-carbaldehyde 2a-2d: Para-substituted acetophenone (20 mmol) interact with phenylhydrazine hydrochloride (20 mmol) in anhydrous ethanol to form 1-phenyl-2- (1-phenylethylidene) hydrazine, which was then dissolved in a cold mixed solution of DMF (20 mL) and POCl₃ (16 mL), stirred at 80-85 ℃ 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.

  Synthesis of 5-amino-1-aryl-1H-pyrazole-4-carbonitriles 3a- 3e: A stirred mixture of para-substituted phenylhydrazine hydrochloride (0.025 mol) was dissolved in H₂O (30 mL), then 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 ℃ for 2 h until the reaction was complete. The reaction was monitored by TLC.Mostly, aprecipitateformed andwas thencollectedbysuction filtration.

  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 recrystallized from ethanol to give the title compounds 5a-5p.

  4.3   Antiviral biological assay

  The anti-TMV activities of the synthesized pure compounds activity was tested by using the method reported by Thorson et al. [27]. This method is to measure the antiviral activity of compounds in vitro and test the protective effect, the inactivation effect and the curative effect in vivo against TMV.

  Purification of tobacco mosaic virus was found by using the Gooding's method [28]. The absorbance values were estimated at 260 nm by using an ultraviolet spectrophotometer.

  Virus concentration (mg=mL) =$\frac{{{A_{260}} \times {\text{dilution ratio}}}}{{E_{1{\text{cm}}}^{0.1{\text{ }}\% {\text{ ,260nm}}}}}$

  4.3.1   Antiviral activity of target compounds in vitro

  Fresh 5-6 growth stage of tobacco leaves (Nicotiana tabacum var. Xanthi NC) were selected for the test. The tobacco was inoculatedbythejuice-leafrubbingmethod, andtheconcentration of TMV was 5.88 × 10^-2 μg/mL. The leaves were cut into halves along the main vein, and a solution of the compounds with a concentration of 500 mg/mL was smeared on the halves, and then cultured at 25 ℃ for 72 h. Each compound was tested three times.

  4.3.2   Protective effect of target compounds against TMV in vivo

  The test compound solution was smeared on the left side, and the solvent served as control on the right side of growing tobacco (Nicotine tabacum var. Xanthium NC). After 12 h, TMV at a concentration of 6.0 μg/mL was inoculated on the leaves which were previously scattered with silicon carbide through the above juice-leaf rubbing method. Then the leaves were again immersed into water and rubbed softly along the nervation once or twice. The local lesion numbers showing 3-4 days after inoculation were counted. Each compound was tested in three replicates.

  4.3.3   Inactivate effect of target compounds against TMV in vivo

  The virus was inhibited by mixing with the target compound solution at the same volume for 30 min. Then the mixture was inoculated on the left side of the host tobacco leaves (Nicotiana tabacum var. Xanthi NC), and a mixture of solvent and the virus was inoculated on the right side to serve as control. The local lesion numbers showing 3-4 days after inoculation were counted. Each compound was tested in three replicates.

  4.3.4   Curative effect of target compounds against TMV in vivo

  Host plant tobacco leaves (Nicotiana tabacum var. Xanthi NC) of the same age growing at the six-leaf stage were selected for the test. TMV at a concentration of 6.0 μg/mL was inoculated on the whole leaves. The leaves were washed with water, and dried in a greenhouse.Thetarget compoundsolution wassmeared onthe left side, and the solvent served as control on the right side. The local lesion numbers showing 3-4 days after inoculation were counted. Each compound was tested in three replicates.

  The inhibition rates of the compound in vitro and in vivo were calculated according to the following formula (controls were not treated with compound):

  $\left[ \frac{\begin{align} & \rm{average local lesion number of control}- \\ & \rm{average local lesion number of drug treated} \\ \end{align}}{\rm{average local lesion number of control}} \right]\times 100\%$

  4.4   X-ray crystallography

  Compound 5d was successfully crystallized with solvent of acetonitrile and its structure was determined by single-crystal Xray diffraction analysis. The structure was solved by direct methods and refined on F² by full-matrix least-squares methods using ELXL-97 [29]. The unit cell dimensions and intensity data were recorded at 293 K. The program SAINT/XPREP was used for data reduction and APEX2/SAINT for cell refinement [30]. All nonhydrogen atoms were refined with anisotropic thermal parameters. All hydrogen atoms with the exception of those on nitrogen atoms were geometrically fixed and refined using a riding model. Molecular graphics employed were MERCURY and PLATON [31].

  4.5   Molecular docking

  Three dimensional structure of the TMV RNA was generated by the RNAComposer Server (http://rnacomposer.ibch.poznan.pl/Home). Molecular docking of compound 4j into the three dimensional structure of the TMV RNA structure was carried out by using the Discovery Studio (DS) (version 3.1, Neo Trident Corporation, Beijing, China) software. The three-dimensional structures of the aforementioned compounds were constructed by using Chem. 3D ultra 12.0 software (Chemical Structure 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.

  Acknowledgments

  This work was supported by National Natural Science Foundation of China (No. 21302002), Anhui Provincial Natural Science Foundation (No. 1408085QB33) and Key Scientific and Technological Project of Anhui Provincial Tobacoo Company (No. 20150551007).

  Appendix A. Supplementary data

  Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2016.10.029.

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  Figure 1  Design of the target compounds.

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  Scheme1  General synthesis of compounds 4a-4t and 5a-5p. Reagents and conditions: (a) H₂O, ethanol, 60 ℃, TLC; (b) DMF, POCl₃, 80-85 ℃, 5 h; (c) sodium acetate, ethanol, r.t., TLC; (d) DMF, POCl₃, 80-85 ℃, 5 h; (e) H₂O, NaOH, ethanol, 3 h, reflux; (f) ethanol, 2 h, reflux; (g) ethanol, 2 h, reflux.

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  Figure 2  The molecular structure of 5d showing 50% probability displacement ellipsoids.

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  Figure 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: 2TMV). (B) The interaction mode of compound 4j at the binding site of OriRNA.

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  Table 1.  The structure of compounds.

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  Table 2.  Crystal data of compound 5d.

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  Table 3.  Anti-TMV activity of compounds in vitro and in vivo at 500 mg/mL.

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•