Synthesis and Fungicidal Activities of (Z/E)-3,7-Dimethyl-2,6-octadienamide and Its 6,7-Epoxy Analogues

In order to find new lead compounds with high fungicidal activity, (Z/E)-3,7-dimethyl-2,6-octadienoic acids were synthesized via selective two-step oxidation using the commercially available geraniol/nerol as raw materials. Twenty-eight different (Z/E)-3,7-dimethyl-2,6-octadienamide derivatives were prepared by reactions of (Z/E)-carboxylic acid with various aromatic and aliphatic amines, followed by oxidation of peroxyacetic acid to afford their 6,7-epoxy analogues. All of the compounds were characterized by HR-ESI-MS and 1H-NMR spectral data. The preliminary bioassays showed that some of these compounds exhibited good fungicidal activities against Rhizoctonia solani (R. solani) at a concentration of 50 µg/mL. For example, 5C, 5I and 6b had 94.0%, 93.4% and 91.5% inhibition rates against R. solani, respectively. Compound 5f displayed EC50 values of 4.3 and 9.7 µM against Fusahum graminearum and R. Solani, respectively.

In the 1 H-NMR of compounds 5a-5n, the olefin protons on C-2 exhibited a singlet with the chemical shifts δ 5.47-5.77, the olefin protons on C-6 displayed a multiplet at δ 4.90-5.20 due to the coupling with the adjacent methylene protons at C-5 and long range coupling with CH3 at C-7. The methyls on C-3 had a doublet at δ 2.23-2.05 with the coupling constant 1.2 Hz due to the long range coupling with the proton on C-2, and the amide protons had the broad singlet with the chemical shifts δ 5.27-7.57. While for the cis isomers 5A-5N, the amide protons had the similar chemical shifts as the trans isomers, but the chemical shift of the protons on C-2 and C-6 shifted to the downfield about δ 0.02-0.10, and the methyls on C-3 shifted to the upfield about δ 0.30-0.40.
In the 1 H-NMR of 6,7-epoxy compounds 6a-6j, the protons on C-2, the amide protons and the methyls on C-3 also showed the similar chemical shifts and coupling constants as that of compounds 5a-5n. However, the protons on C-6 had the chemical shifts δ 2.53-2.76 due to existence of 6,7-epoxy group and the peaks split into the double of doublet due to the coupling with the two protons at C-5 Scheme 1. Structures of (Z/E)-3,7-dimethyl-2,6-octadienoic acid and its 1,6-olide.
In the 1 H-NMR of compounds 5a-5n, the olefin protons on C-2 exhibited a singlet with the chemical shifts δ 5.47-5.77, the olefin protons on C-6 displayed a multiplet at δ 4.90-5.20 due to the coupling with the adjacent methylene protons at C-5 and long range coupling with CH3 at C-7. The methyls on C-3 had a doublet at δ 2.23-2.05 with the coupling constant 1.2 Hz due to the long range coupling with the proton on C-2, and the amide protons had the broad singlet with the chemical shifts δ 5.27-7.57. While for the cis isomers 5A-5N, the amide protons had the similar chemical shifts as the trans isomers, but the chemical shift of the protons on C-2 and C-6 shifted to the downfield about δ 0.02-0.10, and the methyls on C-3 shifted to the upfield about δ 0.30-0.40.
In the 1 H-NMR of 6,7-epoxy compounds 6a-6j, the protons on C-2, the amide protons and the methyls on C-3 also showed the similar chemical shifts and coupling constants as that of compounds 5a-5n. However, the protons on C-6 had the chemical shifts δ 2.53-2.76 due to existence of 6,7-epoxy group and the peaks split into the double of doublet due to the coupling with the two protons at C-5 Scheme 2. Synthetic route of (Z/E)-3,7-dimethyl-2,6-octadienamide and their 6,7-epoxy analogues.
In the 1 H-NMR of compounds 5a-5n, the olefin protons on C-2 exhibited a singlet with the chemical shifts δ 5.47-5.77, the olefin protons on C-6 displayed a multiplet at δ 4.90-5.20 due to the coupling with the adjacent methylene protons at C-5 and long range coupling with CH 3 at C-7. The methyls on C-3 had a doublet at δ 2.23-2.05 with the coupling constant 1.2 Hz due to the long range coupling with the proton on C-2, and the amide protons had the broad singlet with the chemical shifts δ 5.27-7.57. While for the cis isomers 5A-5N, the amide protons had the similar chemical shifts as the trans isomers, but the chemical shift of the protons on C-2 and C-6 shifted to the downfield about δ 0.02-0.10, and the methyls on C-3 shifted to the upfield about δ 0.30-0.40.
In the 1 H-NMR of 6,7-epoxy compounds 6a-6j, the protons on C-2, the amide protons and the methyls on C-3 also showed the similar chemical shifts and coupling constants as that of compounds 5a-5n. However, the protons on C-6 had the chemical shifts δ 2.53-2.76 due to existence of 6,7-epoxy group and the peaks split into the double of doublet due to the coupling with the two protons at C-5 with the coupling constants 5.5 and 7.0 Hz, the chemical shifts of the two methyls at C-7 shifted to upfield about δ 0.30-0.35. Similarly, for the cis isomers 6A-6J, the protons on C-2, the protons on C-6, the amide protons, the methyls on C-3, and the two methyls at C-7 had the similar chemical shifts and coupling constants. All of the new compounds were also characterized by HR-ESI-MS, and the [M + H] + peaks were detected; their exact mass numbers matched well with the calculated molecule weights.
In the Z/E-amides, comparison of the inhibition rates of 5l, 5m, 5n, 5L, 5M and 5N with 5a-5k and 5A-5K, we found that the aromatic amides showed much better fungicidal activities than the aliphatic amides against R. solani and A. solani. It seemed that the aromatic substituted group contributed a lot to the fungicidal activities. Thus, the different aromatic groups such as phenyl, substituted phenyl, benzyl and substituted benzyl groups were selected to optimize the structure. By comparing the inhibition rates of compounds 5b-5g, 5B-5G with compound 5a and 5A, we found that the ortho-substitution (Cl, F) was beneficial to improve the fungicidal activities such as 5c, 5d, 5C and 5D, while the activities at the para-substitution were kept or reduced such as 5b, 5e, 5f, 5B, 5e and 5F. However, one more N-methyl group did not significantly change the activity comparing 5a and 5A with 5g and 5G. So we concluded that the para-substitution was not helpful to improve the activity, especially the electron-withdraw substitution groups. Further, the amides with benzyl and substituted benzyl groups (compounds 5h-5k and 5H-5K) were synthesized and assayed. The results in Table 1 indicated that amides with (substituted) benzyl groups had similar or increased fungicidal activities against R. solani and A. solani comparing with compound 5a and 5A. Thus, the (substituted) benzyl groups have similar effects on the fungicidal activities as the (substituted) phenyl groups. The effect of the double bond configuration at C 2 and C 3 on the inhibition rates did not indicate significant differences by comparison the inhibition data of 5a-5n and 5A-5N.
From the data in Table 1, it was very clear that compounds 5 showed much better fungicidal activities against all tested phytopathgens than compounds 6. While compounds 6b and 6B were two exceptions, which showed the inhibition rates of 91.5% and 82.7% against R. solani, respectively, much higher than the 59.5% and 55.4% inhibition rates of 5b and 5B. The similar effects were observed for the aromatic and aliphatic amides, the substitution on the benzene ring of phenyl and benzyl groups, and the configuration of the double bond at C 2 and C 3 . The double bond at C 6 and C 7 or adjacent 6,7-dihydroxy played an essential role for a better fungicidal activity when comparison the inhibition rate data of compounds 5 and 6.
Based on the above results, the EC 50 values (EC 50 is the concentration of inhibition 50% fungus growth at tested condition.) were determined further for these compounds with more than 70% inhibition rates. The typical inhibition rates changing with the concentration could be seen in Figure 1. The data in Table 2 confirmed that most of compounds exhibited an inhibition against R. solani and A. solani with EC 50 values between 9.7 and 677.8 µM, and several compounds were active against F. graminearum, S. sclerotiorum and B. cinerea with EC 50 values between 4.3 and 92.9 µM. Among them, compound 5f had the best fungicidal activities with EC 50 values of 4.3 and 9.7 µM against F. graminearum and R. solani, respectively, and compounds 5g and 5I had the broad-spectrum of fungicidal activities against four phytopathgens with EC 50 values between 17.1 and 61.2 µM, most of the other compounds have EC 50 values between 13.4 and 97.9 µM against R. solani and A. solani except 6d, 6f, 5M and 6H. These results indicated that there would be the possible improvement of fungicidal activities against R. solani and A. solani if the chemical structures were further modified, especially on the structures of 5f, 5g and 5I. Optimizations on the aromatic amine moieties around compound 5 are in progress.

General Information
All reactions were performed with magnetic stirring. Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. Organic solutions were

General Information
All reactions were performed with magnetic stirring. Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. Organic solutions were concentrated under reduced pressure using a rotary evaporator or oil pump. Melting points were measured on a Yanagimoto apparatus (Yanagimoto MFG Co., Kyoto, Japan) and uncorrected. 1 H-NMR spectra were obtained on Bruker DPX 300 spectrometer (Bruker Biospin Co., Stuttgart, Germany) with CDCl 3 as a solvent and TMS as an internal standard. High-resolution mass spectral analysis was performed on a LTQ Orbitrap instrument (ThermoFisher scientific Inc., Waltham, MA, USA).

General Procedure for the Synthesis of Compounds 5
Take compound 5a as an example: according to the procedure in the literature [22], SOCl 2 (1.4 mL) was added to a solution of (E)-3,7-dimethyl-2,6-octadienoic acid (0.8 g, 5 mmol) in 40 mL DCM in a 100 mL flask at room temperature. The mixture was stirred and heated at 40˝C for 4 h. Then cool down to room temperature and remove the solvent under reduced pressure. The residue was dissolved in 10 mL DCM, the 10 mL DCM solution of aniline (0.92 mL, 10 mmol) was added and stirred for 10 h at room temperature. Quenched the reaction with water, extracted with DCM, and the organic layer was washed with brine, dried over anhydrous Na 2 SO 4 . Removed the solvent under reduced pressure and the residue was purified by column chromatography to afford compound 5a.

General Procedure for the Synthesis of Compounds 6
Take compound 6a as an example: according to the approach in the literature [25,32], compound 5a 0.24 g (1 mmol), CH 3 COOOH (2 mL), Na 2 CO 3 (0.7 g) and DCM (10 mL) were added to a 50 mL flask and stirred at room temperature for 2-4 h, quenched the reaction with water, and extracted with DCM. The organic layer was washed with brine and dried over anhydrous Na 2 SO 4 . The solvent was removed under reduce pressure, and the residue was purified by column chromatography to give the compounds 6a.

Bioassay of Fungicidal Activity
The preliminary fungicidal activities of compounds 5-6 against F. graminearum, R. solani, A. solani, S. sclerotiorum, and B. cinerea were evaluated using methods in the references [33][34][35][36][37] by the mycelium growth rate [38]. The culture was incubated at 25˘0.5˝C. Procedure for inhibition rate: The stock 2000 mg/L DMSO solution of tested compounds were prepared in advance. Then hot PDA culture medium was added into a plate, added sample solution or blank DMSO to the plate and mix with PDA culture medium, made the final concentration as desired. When plate was made, put a 5 mm diameter fungus cake into the center of plate, incubated them at 25˘0.5˝C for 24-48 h, checked the growth status and calculated the inhibition rate according to the reference. Three replicates were performed and the mean measurements were calculated from the three replicates for each compound.
The EC 50 values were determined from the inhibition rates of five different concentrations based on the statistics method of [39] for the compounds that had more than 70% inhibition rates. Procedure for EC 50 determination: the inhibition rates of compounds against different fungus at five concentrations were evaluated as before. Toxicity regression equations were obtained by statistics analysis and the EC 50 values (µM) were calculated from the regression equations with excel program. Carbendazim and Chlorothalonil were used as positive control in the mycelium growth rate test.

Conclusions
(Z/E)-3,7-dimethyl-2,6-octadienamide derivatives and their 6,7-epoxy analogues were synthesized in moderate to excellent yields in four steps with the commercially available nerol/geraniol as raw materials. All the compounds were characterized by HR-ESI-MS and 1 H-NMR spectral data. The preliminary bioassays showed that some of these compounds, such as 5C, 5I and 6b exhibit 94.0%, 93.4% and 91.5% inhibition rates against R. solani at the concentration of 50 µg/mL, respectively. The EC 50 values of compounds 5f and 5G were 9.7 and 13.4 µM against R. solani, respectively, while compound 5f had EC 50 value of 4.3 µM against F. graminearum. Further syntheses and structure optimization studies on the replacement of aromatic and aliphatic amines with nitrogen-containing heterocyclic amines are in progress in our laboratory.