Synthesis, acaricidal activities and docking study of novel acrylonitrile derivatives

A series of novel acrylonitrile derivatives containing a flexible chain were designed using cyflumetofen as lead compound. They were synthesized via two steps. The structures were confirmed by 1 H NMR, 13 C NMR and HRMS. The title acrylonitrile derivatives exhibited good acaricidal activity against Tetranchus urticae . In particular, 3j possessed excellent activity in the green house, at the same level as the control Cyflumetofen at 4 days or 7 days

The mechanism (Figure 1) whereby cyflumetofen inhibits respiration of SDH was analysed by pesticidal physiological scientists. 21The final metabolite of cyflumetofen is AB-1 (Cyflumetofen de-esterification) and has better activity than cyflumetofen at different concentrations.So the cyflumetofen could be as a pro-pesticide in the acaricide discovery.In view of these facts, and also as a part of our work on the synthesis of bioactive lead compounds for drug discovery, [22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39] the acaricide cyenopyrafen was selected as a lead compound, and a series of cyenopyrafen analogues were designed and synthesized.A flexible chain was introduced onto the OH group.The acaricidal activity was tested and a docking study was also carried out.

Synthesis and Spectra
The synthetic route to the title acrylonitrile derivatives is shown in Scheme 1.In the key intermediate 2-(4tert-butylphenyl)-3-hydroxy-3-(2-trifluoromethylphenyl) acrylonitrile 1, the acrylonitrile (HO-C=C-CN) structure can exist either as the enol, or ketone tautomeric forms.From the 1 H NMR of 2-(4-tert-butylphenyl)-3hydroxy-3-(2-(rifluoromethylphenyl) acrylonitrile 1, we found two t-Bu signals at 1.30 ppm and 1.36 ppm respectively, with peak areas in the ratio of 1:1.The CHCN signal was found at 5.21 ppm.The aromatic signals integrated for 16, which indicated that the compound 1 contains enol and ketone tautomeric forms.From the 1 H NMR data, we can conclude that the ratio of the two tautomeric forms is 1:1.For the synthesis of final compounds, there is still a little Z-isomer, which is the same as in related references. 40,41The polarity of this Zisomer is small, so it can be easily removed by column chromatography.The residue is the cis product.If we used the potassium salt of intermediate 1 to prepare the final compounds, it still worked well.Scheme 1.The synthetic route of title compounds.
All the structures of compounds 2a-2k were determined using 1 H and 13 C NMR, and HRMS analysis.In the 1 H NMR spectra, doublets at δ 7.4 ppm and 7.7 ppm correspond to the t-Bu-ring protons, with a coupling constant of J 8.4 Hz.The signals around 7.6 ppm are a multiplet for the protons of the CF3-ring.In the 13 C NMR spectrum, the CF3 showed a quartet signal with a coupling constant of 274 Hz.With regard to the high resolution mass spectra, all the acrylonitrile derivatives showed M+H + signals with the standard error m/z± 0.0030.

Acaricidal activity
The acaricidal activity was determined and the results are listed in Table 1.Most of these compounds exhibited excellent activity against Tetranchus urticae, with compounds 2c, 2f and 2g having the best activity (100%).Compounds 2a and 2b also exhibited good activity (>90%).The compounds 2d and 2e had no activity.From these data, we find that when the alkyl group is t-Bu or 2-methyl-Bu, the activity is lower.Because of the high acarcidal activity, four compounds were tested for acarcidal activity in the green house.Table 2, shows that these compounds exhibited excellent acarcidal activity at 5 mg/L or 2.5 mg/L for 4 days, 7 days or 14 days.The activity is higher than that of control cyflumetofen.At 1.25 mg/L, only compound 2k exhibited low activity, which is lower than control.Even at 0.625 mg/L, compounds 2i and 2j still exhibited moderate activity, which is the same as the control.

Molecular docking
To further explore the interaction of the novel compounds with SDH at the molecular level, a docking study was carried out by using the Discovery studio 2.5.The reported crystal structure of SDH (PDB code: 1ZOY) was selected for docking simulation.As shown in Figure 2, intermediate 1 is well-matched to the active pocket of procine SDH.In intermediate 1 there are four hydrogen bonds between carboxyl group, cyano group, trifluoromethyl group, shown by the yellow dotted lines in Figure 2. Firstly, an intermolecular hydrogen bonding is observed between O of C=O and Thr58 with thae distance of 2.3 Å.Secondly, a hydrogen bond can be seen between F of CF3 group and Gly31 with a length of 2.5 Å.Another two intermolecular hydrogen bonds are shown between the nitrogen atom of the cyano group with hydrogen of hydroxyl of Ser230 and amino group of Gly216 with lengths of 2.2 Å and 2.2 Å respectively.

Experimental Section
General. 1 H NMR and 13 C NMR spectra were determined on a BRUKER Avance 500 MHz spectrometer using CDCl3 as the solvent.HR-ESI-MS were measured using an Agilent 1100 HPLC-JEOL AccuTOF instrument.All the reagents were analytical grade or synthesized in our lab.

Synthesis
To a solution of 2-(4-tert-butylphenyl)acetonitrile (1.73 g, 0.01 mol) and t-BuOK (1.232 g, 0.011 mol) in THF at 0 o C, 2-trifluoromethylbenzoyl chloride (2.08 g, 0.01 mol) was added dropwise.The mixture was stirred at room temperature.After 2 h, the solvent was evaporated and the residue was treated with H2O (100 mL).The solution was neutralized with HCl to afford a precipitated solid 1 which, without purification, was filtered off and dried (3.1 g, 90 %).Synthesis of compound 2a.To a solution of intermediate 1 (3.45 g, 0.01 mol) and Na2CO3 (1.59 g, 0.015 mol), NaI (0.15 g, 0.001 mol) in CH3CN (50 mL), the 1-chloroethyl ethyl carbonate (0.011 mol) was added.The mixture was refluxed for 5 h.After the reaction was completed, the solvent was removed and the residue was purified by column chromatography to give a colourless oil 2a (2.1 g, 45%-.The following compounds 2b-2k were prepared in a similar manner.

Acaricidal activity
All bioassays were performed on representative test organisms reared in the laboratory or greenhouse.The bioassay was repeated at 25 ± 1 o C according to statistical requirements.Lab test.The acaricidal activities of the title compounds 2a-2g were tested against Spider Mites (Tetranychus cinnabarinus).The leaves of horse bean plants were punched into 3cm-diameter discs, using leaf punch, and placed onto moist filter paper in a 7 cm-diameter styrol cup.Ten larvae spider mites (Tetranychus cinnabarinus)were put onto each leaf.A 5% emulsion of a compound was diluted with water containing a spreading agent to give a 1-10 ppm solution of the compound.The solution was sprayed over each cup in an amount of 2 mL/cup, using a rotary sprinkler.After 48 h, the mites in each cup were observed, and mortality of the mites was determined.Assessments were made on a dead/alive basis, and mortality rates were corrected using Abbott's formula.Evaluations are based on a percentage scale of 0-100 in which 0 = no activity and 100 = total kill.The results of acaricidal activity are listed in Table 1.Greenhouse test.The greenhouse test against Tetranychus cinnabarinus was carried out in horse bean plants.One bean seedlings with 2 to 3 broad leaves were transplanted into a plastic bowl with a diameter of 9 cm, and the seedlings were inoculated with Tetranychus cinnabarinus.Each pot of seedlings is a plot, and each concentration was repeated 3 times, randomly arranged, and clear water was used as a blank control.The number of spider mites on each plant was investigated, recorded and calculated to give a relative efficacy (%) AUTHOR(S) after 4 days, 7 days and 14 days.The spray method was used to spray broad bean seedlings with a spray volume of 2 mL per treatment.

Molecular Docking
The molecular docking simulations were carried to analyze interactions using Discovery Studio 2.5 software according to the reported method. 42,43From the mechanism of cyflumetofen, the cyflumetofen is metabolised in vivo to the acarcidal active intermediate 3-oxo-2-phenyl-3-(2-trifluoromethylphenyl)propanenitrile. 40ccoridngly, the structure of intermediate 2-(4-tert-butylphenyl)-3-oxo-3-(2trifluoromethylphenyl)propanenitrile 1 was selected as ligand and input into the Discovery Studio 2.5 software.The procine SDH structure (PDB code: 1ZOY) was selected for docking simulation and binding sites were generated from the protein complex.CHARMM and MMFF94 force fields were applied in compound and SDH.Then the docking process was carried out by using CDOCKER protocol.Finally, docking scores (-CDOCKER interaction energy) of the best-fitted interactions with the active site at the SDH structure were recorded.

Figure 1 .
Figure 1.The mode of action of cyflumetofen.

Figure 2 .
Figure 2. Plot depicting interaction of intermediate 1 with active site of SDH.

Table 1 .
The acaricidal activities of some acrylonitrile derivatives at 2.5 mg/L

Table 2 .
The control effect of further synthetic acrylonitrile derivatives at different concentrations