Design, Synthesis and Anti-Tobacco Mosaic Virus (TMV) Activity of 5-Chloro-N-(4-cyano-1-aryl-1H-pyrazol-5-yl)-1-aryl-3-methyl-1H-pyrazole-4-carboxamide Derivatives

A series of novel pyrazole amide derivatives 3a–3p which take TMV PC protein as the target has been designed and synthesized by the reactions of 5-chloro-1-aryl-3-methyl-1H-pyrazole-4-carboxylic acids with 5-amino-1-aryl-1H-pyrazole-4-carbonitriles. All the compounds were characterized by 1H-NMR, mass spectroscopy and elemental analysis. Preliminary bioassays indicated that all the compounds acted against the tobacco mosaic virus (TMV) with different in vivo and in vitro modes at 500 μg/mL and were found to possess promising activity. Especially, compound 3p showed the most potent biological activity against tobacco mosaic virus (TMV) compared to ningnanmycin, and a molecular docking study was performed and the binding model revealed that the pyrazole amide moiety was tightly embedded in the binding sites of TMV PC (PDB code: 2OM3).


Introduction
Tobacco mosaic virus (TMV) is one of the most extensively studied among a number of plant viruses. Its infections are very widely distributed [1][2][3] and can cause serious damage and large economic losses. TMV is a growing problem in the contemporary pesticide field, as only a few antiviral agents are currently available in practice, and almost no treatment can control TMV effectively [4], so the identification of new antiviral agent with novel mechanisms of action is critically needed to overcome this viral infections.
Recently, different targets essential to plant viruses that could be new weapons to design and discover new antiviral drugs have been studied and validated. Most antiviral drugs exhibit their activities through interaction with viral proteins [5,6]. Coat protein-mediated resistance to TMV involves mainly two independent mechanisms: (i) interference by transgenic CP with disassembly of challenge virus; and (ii) interference of transgenic CP with formation of replication complexes, thus interfering with virus movement [7]. CP is an important part of the genomic structure of the tobacco mosaic virus (TMV), and its main function not only includes the protection of DNA from degradation, but it also has a close relationship with the long-distance movement of TMV and host symptoms [8]. Therefore, coat protein (PC) is usually used as a molecular target for drug action, and the study of TMV CP mainly focuses on CP-mediated resistance, interference the polymerization of CP by agents and so on. Many small molecule ligands. including pyrazole derivatives, that combine with molecular targets have been reported [9][10][11][12]. Inspired by the above notion, we hope to design a new compound which is able to target TMV PC to inhibit viral assembly through small molecule-coat protein interactions.
Pyrazole derivatives have great importance in the medicinal and pesticide fields [13][14][15], due to their broad range of biological activity [16][17][18]. The molecules of many modern drugs with antiphlogistic, antidiabetic, analgesic, and acaricidal activity in medicine [19] and herbicidal [20][21][22], antibacterial [23][24][25], acaricidal and insecticide [26,27] in the pesticide area contain pyrazole rings as a structural fragment. As shown in Figure 1, several pyrazole derivative products have been launched or announced, and they all contain several common structural features that are essential to their activity, including an amide bond, pyrazole and an aromatic ring attached to the carbonyl of the amide bond. Modification of the structural profile by the change of substituents at the 1-, 3-, or 4-positions in the pyrazole ring can bring about significant changes in bioactivity [28], and linking the pyrazole group with a structurally diverse side chain is an effective way to obtain new heterocyclic derivatives with high antiviral activities [29]. We assumed that if nitrile-containing pyrazole pharmacophores were introduced into the pyrazole scaffold, thus transforming the structure reported by Ouyang et al. [30], the resulting compounds may be interesting lead structures for antiviral agent development (Figure 2), because the nitrile can modulate the physicochemical and pharmacokinetic properties to improve bioavailability, enhance the selectivity and binding affinity to target proteins by hydrogen bond interactions, covalent interactions, polar interactions, and π-π interactions [31][32][33], and cyano substitution in the design of drugs has become one of the important research strategies in optimizing the structure of lead compounds [34].
The title structure Ouyang et al. reported structure We describe herein the synthesis and antiviral activity of a series of new pyrazole amide derivatives ( Figure 3). Molecular docking was performed and the binding model revealed that the title pyrazole amide was tightly embeded in the binding sites of TMV PC (PDB code: 2OM3) ( Figure 4). The anti-TMV activities of these compounds were subsequently evaluated, and a structure-activity relationship was established in terms of their anti-TMV activity.

Chemistry
The synthetic route is shown in Schemes 1-3. Compounds 1a-1d were prepared in accordance with the literature procedure [35] from the reactions of substituted phenylhydrazines with ethyl acetoacetate in anhydrous ethanol medium. Then the reaction mixtures was added to a cold solution of DMF and POCl3. Finally, the products were oxidized with KMnO4, to give the 5-chloro-1-aryl-3-methyl-1Hpyrazole-4-carboxylic acids 1 (Scheme 1).

Bioactivity
To judgme the antiviral potency of the synthesized compounds 3a-3p, the commercially available plant virucide ningnanmycin was used as the control. The antiviral bioassay against TMV was assayed by the method reported by Thorson et al. [37].
The in vivo and in vitro bioassay results of compounds 3a-3p against TMV are given in Table 1. The results indicated that the title compounds showed curative rates ranging from 22.6%-86.5%. Among them, compound 3p showed the most potent biological activity against TMV and it exhibited slightly higher activities compared to the commercial agent ningnanmycin.

Molecular Docking
Molecular docking of compound 4p into the three dimensional X-ray structure of the TMV PC protein-ligand complex crystal structure (PDB code: 2OM3) was carried out using the Discovery Studio (DS) (version 3.1, NeoTrident Corporation, Beijing, China) software [38] as implemented through the graphical user interface DS-CDOCKER protocol. The three-dimensional structures of the aforementioned compounds were constructed using Chem. 3D ultra 12.0 software (Chemical Structure Drawing Standard, CambridgeSoft 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 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.

3D-QSAR
In order to further explain of structure and activity relationships of these compounds, the in vitro antiviral activity data were used to build the 3D-QSAR models, and the 3D-QSAR study was performed by means of the DS 3.5 software. The correlation coefficient r 2 between observed and predicted activity of training set was found to be 0.899, which meant the dependability of linear fit. The molecules aligned with the iso-surfaces of the 3D-QSAR model coefficients on electrostatic potential grids (Figure 5a) and van der Waals grids (Figure 5b) are listed. As shown in Figure 5, compounds with electron-withdrawing atoms not only circumvented the red subregion but also got more close to the favorable green spaces. This figure showed that a halogen substituted group was a better choice than a methyl substituted group and F was a better substituent than Cl, so this QSAR study could predict that electron-withdrawing groupa substituted in the aromatic ring could have good interaction with the protein with nice molecule electrostatic and steric features. However, the chlorine atom is also a strong electron withdrawing-group but its ability is slight weaker than that of a F atom, therefore, the compounds with Cl atom substituted in the aromatic ring (e.g., 3f, 3h) have similar inhibition as 3p. The 3D QSAR models fitted the inhibitory activity well and thus provide us the directions for further modification.

General Information
Melting points (uncorrected) were determined on an XT4 MP apparatus (Taike Corp., Beijing, China). ESI mass spectra were obtained on a Mariner System 5304 mass spectrometer (Applied Biosystems, Foster City, CA, USA). 1 H-NMR spectra were recorded on a Bruker PX400 spectrometer (Bruker Corporation, Rheinstetten, Germany) at 25 °C with TMS and solvent signals assigned as internal references, 13 C-NMR spectra and 1 H-NMR spectra of compound 3e, 3n were recorded on an Agilent DD2 PX600 spectrometer (Agilent Technologies Corporation, Santa Clara, CA, USA). Chemical shifts were reported in ppm (δ). Elemental analyses were performed on a CHN-O-Rapid instrument (Leco, Tres Cantos, Madrid, Spain) and were within 0.4% of the theoretical values.

General Procedure for Synthesis of 5-Chloro-1-aryl-3-methyl-1H-pyrazole-4-carboxylic Acids 1a-1d
The intermediate 5-chloro-1-aryl-3-methyl-1H-pyrazole-4-carboxylic acids 1a-1d were synthesized as the following: para-substituted phenylhydrazines (0.025 mol) were reacted with ethyl acetoacetate (0.025 mol) in anhydrous ethanol to form a solid, which was then dissolved in a cold mixed solution of DMF (20 mL) and POCl3 (16 mL), and stirred at 50-60 °C. 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, and washed with water. Then above product was oxidized by KMnO4 solution, stirred at 70-80 °C. After cooling to room temperature the pH of the reaction mixture was adjusted to pH 7-8 by the dropwise addition of KOH solution, and the solution was filtered, HCl solution was added to the solution and solid 1a-1d eventually separated out. The crude product obtained was recrystallized from DMF to afford the pure product. All the reactions were monitored by TLC.

General Procedure for Synthesis of 5-Amino-1-aryl-1H-pyrazole-4-carbonitriles 2a-2d
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 ethoxymethylene malononitrile in an ethanol medium. After completion of the reaction, the reaction mixture was allowed to cool at room temperature, and the solid 2a-2d were filtered under vacuum. The crude products obtained were recrystallized from DMF to afford the pure products.

Antiviral Biological Assay
The anti-TMV activity of the synthesized pure compounds were tested by using the method reported by Thorson et al. [37]. This method is to measure the antiviral activity of compounds in vitro and to test the protective effect, the inactivation effect and the curative effect in vivo against TMV.

Antiviral Activity of Target Compounds in Vitro
Fresh 5-6 growth stage of tobacco leaves (Nicotianatabacum var. XanthiNC) were selected for the test. The tobacco was inoculated by the juice-leaf rubbing method, and the concentration of TMV was 5.88 × 10 −2 μg/mL. The leaves was cut into halves along the main vein, and a solution of the compounds with a concentration of 500 μg/mL was smeared on the halves, and then cultured at 25 °C for 72 h. Each compound was tested three times.

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 (Nicotianatabacum var. XanthiNC). 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.

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 (Nicotianatabacum var. XanthiNC), 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.

Curative Effect of Target Compounds against TMV in Vivo
Host plant tobacco leaves (Nicotianatabacum var. XanthiNC) 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. The target compound solution was smeared on the 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): Inhibition rate (%) = [(average local lesion number of control − average local lesion number of drug treated)/average local lesion number of control] × 100% (1)

Conclusions
In conclusion, a series of novel pyrazole amide compounds has been designed and synthesized, based on the skeleton of previously reported pyrazole derivatives. The title compounds 3a-3p were evaluated as potent antiviral agents against TMV and it was proved that compound 3p showed a significant activity.
Structure-activity relationships in these pyrazole amide derivatives were evaluated using the DS approach to proceed with structure optimization, The data demonstrated that compounds with para electron-withdrawing substituents (e.g., 3f, 3h, 3n, 3p) in the A-ring showed more potent activities than those with electron-donating substituents (e. g., 3a, 3d, 3i, 3k). In comparison the activity of compound with para substituents on the phenyl ring showed that an electron-withdrawing group gave slightly improved antiviral activity and the potency order is F > Cl. Among all the compounds, 3p with a para-F group showed more activity, while 3k showed the lowest activity.
For the binding model of compound 3p, an H-bond (2.49 Å and 109.02°) was established between its fluorine atom and Gin47, and it also formed a π-cation interaction (4.65 Å). The result of molecular docking suggests that introduction of a fluorine atom facilitates the adoption of a complementary shape of the designed ligands with TMV PC, and this possibly improves the anti-TMV inhibitory potency.