Discovery of Novel Cinnamide Fungicidal Leads with Optical Hydroxyl Side Chain

In order to overcome the resistance of phytopathogens to commercial fungicides, a series of optical 2-methyl-2,3-diol-5-pentyl-based cinnamamide derivatives were rationally designed, synthesized, characterized, and evaluated for their in vitro and in vivo fungicidal activities. The bioassay results indicated that the EC50 (concentration for 50% of maximal effect) values of (R)-11f, (R)-11m, (S)-11m and (R)-11n were 0.16, 0.28, 0.41 and 0.47 µg/mL in the in vitro evaluation against Sclerotinia sclerotiorum, respectively, while compounds (R)- and (S)-11i, (R)- and (S)-11j exhibited excellent in vivo fungicidal activity against Pseudoperonspera cubensis with inhibition rates of 100% at 400 μg/mL. These findings supported the idea that optical 2-methyl-2,3-diol-5-pentyl-containing cinnamamides (R)- and (S)-11i, (R)- and (S)-11j with 2-chloro-4-trifluoromethyl aniline and 2-(4-chlorophenyl) aniline showed excellent in vivo fungicidal activity against S. sclerotiorum and P. cubensis and were promising fungicide candidates.


Introduction
Dimethomorph, flumorph and pyrimorph are widely used excellent amide fungicides in agriculture fields (Scheme 1). However, resistance of phytopathogens to them has developed due to their wide application and their similar structures [1][2][3][4][5][6][7]. The resistance mechanisms have been confirmed to relate with point mutation in cellulose synthase 3 (CesA3) [1,4]. In order to overcome this problem, some works addressing this issue have been published, and several compounds had good fungicidal activities against the tested phytopathogens [8][9][10], but all of these molecules retained two (hetero)aryl groups such as benzene, pyridine and isothiazole. For example, isothiamorph was found to exhibit excellent in vivo fungicidal activity against Pseudoperonspera cubensis with both fungicidal activity and systemic acquired resistance [10]. In practice, how to find the novel chemical structures to overcome resistances is difficult and a challenge for agricultural chemists, and the costs of developments are high. To address this issue, our initial strategy was to replace one of the aryl groups in the molecules of dimethomorph, flumorph and pyrimorph with non-aryl groups, and change the morpholine motif into the other amines. However, we faced the question of how to find a suitable functional group.
These structures were different from the dimethomorph, flumorph and pyrimorph, as they could not only improve the in vitro and in vivo fungicidal activities against phytopathogens, but also could overcome the resistance issue. The synthetic route is shown in Scheme 5, and the fungicidal activity evaluation is reported in this article.
These structures were different from the dimethomorph, flumorph and pyrimorph, as they could not only improve the in vitro and in vivo fungicidal activities against phytopathogens, but also could overcome the resistance issue. The synthetic route is shown in Scheme 5, and the fungicidal activity evaluation is reported in this article. Scheme 5. The synthetic route of optical 6,7-dihydroxy-3-aryl-7-methyloct-2-enamides 11a-11p.

Chemistry
As indicated in a previous report, lactone 2 was designed [26]. The lactone 2 and analogues were synthesized and evaluated for their fungicidal activities [22]. It was found that (R)-2 was the most active compound with EC50 values in the range of 0.2-13.5 µg/mL against the tested phytopathgens, better than its (S)-isomer and racemic mixture. The scanning electron microscope (SEM) and transmission electron microscope (TEM) observations indicated that compounds (S)-2 had a significant impact on the structure and function of the hyphal cell wall of S. sclerotiorum mycelium [22]. With comparison of those data with that of naturally occurring (R)-1, it was found that the C3-aryl significantly improved the fungicidal activities of this type of seven-membered lactone [22]. The amides 4, 5, and 6 having C3-CH3 were also synthesized and evaluated for their fungicidal activities. Some of them exhibited in vitro fungicidal activities against the tested phytopathogens, but were much weaker than those of (R)-1, pyrimorph and dimethomorph, while several compounds showed in vivo fungicidal activities against P. cubensis and Erysiphe graminis, but which were also weaker than that of the chiral acid (Z, R)-2 and (Z, S)-2 [24,25]. Therefore, we attempted to replace the C3-CH3 with similar aryl groups as in the previous report [22], and hope to improve the in vitro and in vivo fun-Scheme 5. The synthetic route of optical 6,7-dihydroxy-3-aryl-7-methyloct-2-enamides 11a-11p.

Chemistry
As indicated in a previous report, lactone 2 was designed [26]. The lactone 2 and analogues were synthesized and evaluated for their fungicidal activities [22]. It was found that (R)-2 was the most active compound with EC 50 values in the range of 0.2-13.5 µg/mL against the tested phytopathgens, better than its (S)-isomer and racemic mixture. The scanning electron microscope (SEM) and transmission electron microscope (TEM) observations indicated that compounds (S)-2 had a significant impact on the structure and function of the hyphal cell wall of S. sclerotiorum mycelium [22]. With comparison of those data with that of naturally occurring (R)-1, it was found that the C 3 -aryl significantly improved the fungicidal activities of this type of seven-membered lactone [22]. The amides 4, 5, and 6 having C 3 -CH 3 were also synthesized and evaluated for their fungicidal activities. Some of them exhibited in vitro fungicidal activities against the tested phytopathogens, but were much weaker than those of (R)-1, pyrimorph and dimethomorph, while several compounds showed in vivo fungicidal activities against P. cubensis and Erysiphe graminis, but which were also weaker than that of the chiral acid (Z, R)-2 and (Z, S)-2 [24,25]. Therefore, we attempted to replace the C 3 -CH 3 with similar aryl groups as in the previous report [22], and hope to improve the in vitro and in vivo fungicidal activities; thus, the amides 11a-11p were designed, synthesized and evaluated for their fungicidal activities in this article.

The In Vitro and In Vivo Fungicidal Activities
After completion of synthesis, the in vitro and in vivo fungicidal activities of compounds 11a-11p were evaluated, as shown in Tables 1-3. The data in Table 1 indicate that all of compounds ((R)-11a-11p and (S)-11a-11p) with dihydroxyl had weak fungicidal activities against A. solani, P. capsici, B. cinerea and R. solani, while some of them (eg. (R)-11f and (R)-11n) had excellent fungicidal activities against S. sclerotiorum. The EC 50 values of these compounds having strong fungicidal activities against S. sclerotiorum were determined and provided in Table 2. These data indicated that (R)-and (S)-11a, 11c, 11e and 11g almost lost their fungicidal activities against S. sclerotiorum after dihydroxylation, however (R)-and (S)-11b, (R)-and (S)-11f, and (R)-11h had good fungicidal activities with EC 50 values of 0.16-67.8 µg/mL against S. sclerotiorum after dihydroxylation. These compounds ((R)-and (S)-11m, 11n, 11o) with 2-chloro-4-trifluoromethylaniline and 2-(4chlorophenyl)aniline exhibited excellent fungicidal activities against S. sclerotiorum with EC 50 values of 0.28-11.4 µg/mL, which indicated that the dihydroxyl groups significantly improved their in vitro fungicidal activities. To our surprise, compounds (R)-and (S)-11e (R 1 + R 2 = morpholino) had very weak in vitro fungicidal activities against five phytopathagens, so we primarily deduced that the α-naphthyl group had a bigger hindrance than the 4-tert-butyl-phenyl, 4-phenyl-phenyl and β-naphthyl group, as they cannot enter the active site of the target. All the data in Table 2 showed that the R-configuration is much better than the S-configuration for in vitro fungicidal activities; the chiral amides have much better in vitro fungicidal activities than the seven-membered lactones such as 2, (R)and (S)-2 [22]. Among these compounds, the EC 50 values of (R)-11f, (R)-11m, (S)-11m and (R)-11n were 0.16, 0.28, 0.41 and 0.47 µg/mL against S. sclerotiorum, respectively. They exhibited the best in vitro fungicidal activities in comparison with the chiral lactone lead (R)-2, (S)-2 [22] and the chiral amides 6 [25].    In order to confirm their fungicidal activities, the in vivo fungicidal activities of compounds 11a-11p were assessed, and the results are provided in Table 3. For (R)-and (S)-11m, 11n, they only showed 50-60% efficacy against P. cubensis, weaker than that of positive control flumorph, pyrimorph, and the lead (R)-3 and (S)-3. To our surprise, com-pounds (R)-and (S)-11i and 11j, with weak in vitro fungicidal activities, exhibited excellent in vivo fungicidal activities with 100% efficacies at 400 µg/mL, better than that of the positive control flumorph. They still had 20-98% efficacies when concentration decreased to 100 µg/mL, much better than the chiral acid leads (R)-3 and (S)-3. Notably, (R)-and (S)-11j remained at 5% and 10% efficacies when concentration decreased to 6.25 µg/mL. These results showed that (R)-and (S)-11i, 11j, 11m and 11n were excellent lead compounds worthy of further optimization. This work is currently under way in our group.

General Information
All reactions were performed with magnetic stirring. Unless otherwise stated, all reagents were purchased from commercial suppliers (Energy Chemical, Shanghai, China) and used without further purification. Organic solutions were concentrated under reduced pressure using a rotary evaporator or oil pump. Flash column chromatography was performed using Qingdao Haiyang silica gel (200-300 mesh). Melting points were measured on a Yanagimoto apparatus (Yanagimoto MFG Co., Kyoto, Japan) and are uncorrected. 1 H and 13 C 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; chemical shifts were presented with δ. HR-ESI-MS spectra were analyzed on Bruker Apex II mass spectrometer (Bruker Co., Bremen, Germany). The solvents were analytical grade and newly distilled before usage. The e.e values were analyzed by an Agilent LC 1100 HPLC instrument equipped with a chiral Chiralpak AD column (250 mm × 4.

Synthesis of the Olefin Acids 7 and 8a-8d
The olefin acid 7 was prepared through 5-step reactions using 2-methylbut-3-en-2-ol as the starting material following the procedures. The olefin acids 8a-8d were prepared through the stereoselective Mizoroki-Heck arylation of 7 with 4-(tert-butyl)-iodobenzene, 4phenyl-iodobenzene, 1-iodonaphthalene and 2-iodonaphthalene according to the protocol in the previous reports, and their spectral data were identical with that reported in the literature [22,26].

Synthesis of the Amides 9a and 9b
Synthesis of the amides 9a and 9b: The olefin acid 7 (1.0 g, 6.5 mmol) and 100 mL CH 2 Cl 2 were added into a 250 mL single-necked flask in an ice-water bath, then we added 1 mL oxalyl dichloride and 3 drops of DMF in a stirred condition. After the bubble disappeared, we removed the ice-water bath, and reacted 1-2 h. The solvent was removed in vacuo to afford the acid chloride. The acid chloride CH 2 Cl 2 (10 mL) solution and pyridine (1 mL) were added dropwise into the 20 mL CH 2 Cl 2 solution of 2-chloro-4-(trifluoromethyl)aniline (2.00 g, 10.2 mmol) or 2-(4-chlorophenyl) aniline (2.07 g, 10.2 mmol) at the ambient temperature and stirred for 8-10 h. After the reaction was completed, 30 mL water was added into the mixture, poured into the separatory funnel, shaken and separated into the organic phase. Then, the water phase was extracted with CH 2 Cl 2 (3 × 30 mL), combined with the organic phase, and the organic phase was dried over anhydrous Na 2 SO 4 . The solvent was removed in vacuo, and the residue was recrystallized using petroleum ether to give white solid 9a or 9b.

Synthesis of the Amides 10a-10p
The general synthetic method (A): 50 mL CH 2 Cl 2 and olefin acid 8a (686 mg, 2.4 mmol) were added into a 250 mL single-necked flask, then EDCI (652 mg, 3.4 mmol) and HOBt (458 mg, 3.4 mmol) were added into the mixture and stirred. After the mixture was clear, morpholine (0.5 mL, 5.75 mmol) was added and reacted for 10 h. Then, 30 mL water was added into the mixture, poured into the separatory funnel, shaken and separated into the organic phase. Then the water phase was extracted with CH 2 Cl 2 (3 × 30 mL) and the organic phase was combined and dried over anhydrous Na 2 SO 4 . The solvent was removed in vacuo, and the residue was subjected to a flash silica gel chromatography and washed with petroleum ether/EtOAc (v:v = 3:1) to give a colorless liquid 10a. Compounds 10b-10h were prepared in a similar way.
The general synthetic method (B): To add 9a (400 mg, 1.20 mmol), Pd(OAc) 2 (14 mg, 0.06 mmol), P(o-MeC 6 H 4 ) 3 (42 mg, 0.14 mmol), 4-phenyl-iodobenzene (640 mg, 2.29 mmol), and 6 mL N(C 2 H 5 ) 3 into a 25 mL three-necked flask under N 2 atmosphere. The mixture was stirred and heated to 110 • C for 20 h, then cooled down to the room temperature; we adjusted pH to 2 using 1M HCl solution and added 30 mL water. The water phase was extracted with EtOAc (30 mL × 3), the organic phase was combined and dried over anhydrous Na 2 SO 4 . The solvent was removed under vacuum, and the residue was subjected to a flash silica gel chromatography and washed with petroleum ether/EtOAc (v:v = 100:1) to afford a white solid 10i. Compounds 10j-10p were prepared in a similar approach.

Synthesis of the Chiral Amides 11a-11p
The general synthetic method: A 50 mL round-bottomed flask equipped with a magnetic stirring bar was charged with AD-mix-β (1.4 g), water (7.5 mL) and tert-butyl alcohol (7.5 mL). The resulting mixture was stirred at room temperature to produce two clear phases. Methanesulfonamide (68 mg, 0.7 mmol) was added in one portion and the reaction mixture was stirred for 1.5 h. The reaction mixture was cooled to 0 • C. Compound 10a (356 mg, 1.0 mmol) was added at once, and the heterogeneous slurry was stirred vigorously at 0 • C for 40 h. The saturated Na 2 S 2 O 3 solution (15 mL) was added at 0 • C, and the mixture was allowed to reach room temperature and stirred for 30 min. EtOAc (50 mL) and water (20 mL) were added to the reaction mixture. The organic layer was separated and the aqueous layer was re-extracted with EtOAc (50 mL×3). The combined organic phase was dried over with anhydrous Na 2 SO 4 and the solvent was removed to give the crude product. This product was purified by flash chromatography on silica gel with petroleum ether/EtOAc (V:V = 3:1) as the eluent to give a colorless oil chiral diol amide (R)-11a 335 mg, yield 86%. In a similar way, the chiral diol amides (R)-11b-(S)-11p were prepared. (6R,2E)-3-(4-(tert-Butylphenyl)-6,7-dihydroxy-7-methyl-1-morpholinooct-2-en-

Fungicidal Activity of the Amides 11a-11p
The in vitro fungicidal activities of compounds 11a-11p against R. Solani, A. Solani, F. graminearum, S. Sclerotiorum, B. cinerea and P. capsici were evaluated using methods in the references [27,28] by the mycelium growth rate. Procedure for inhibition rate: The stock 2000 µg/mL DMSO solutions of tested compounds were prepared in advance. Then hot potato dextrose agar (PDA) culture medium (9.75 mL) was added into a plate, and we added sample solution (0.25 mL) or blank DMSO (0.25 mL) to the plate and mixed with PDA culture medium, to make the final concentration 50 µg/mL. When the plate was made, we 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 concentration. The EC 50 values were determined from the inhibition rates of six different concentrations (100, 25.0, 6.25, 1.56, 0.39, 0.10 µg/mL) based on the statistics method for the compounds which had more than 70% inhibition rates in the preliminary evaluation. Dimethomorph and pyrimorph were used as the positive control in the mycelium growth rate test.
The in vivo fungicidal activities of compounds 11a-11p against Pseudoperonospora cubensis, Erysiphe graminis, Puccinia sorghi and Colletotrichum gloeosporioides were evaluated using the potted plant method in a greenhouse [29,30]. Flumorph and pyrimorph were used as the positive control. The evaluation experiments were performed by State Key Laboratory of the Discovery and Development of Novel Pesticide, Shenyang Sinochem Agrochemicals R&D Co. Ltd., Shenyang, China.

Conclusions
In conclusion, novel cinnamide fungicidal leads with optical hydroxyl side chain (R)-11a-11p and (S)-11a-11p were designed and synthesized through amidation of the olefin acids 7 and 8a-8d or Mizoroki-Heck arylation of the amides 9a and 9b, and stereoselective synthesis of optical isomers of 3-aryl-7-methyl-6,7-dihydroxyoct-2-enamide with Sharpless asymmetric dihydroxylation as the key steps. Their structures were characterized by the 1 H, 13 C NMR and HR-ESI-MS spectra data, and the e.e values were analyzed by chiral HPLC. The EC 50 values of (R)-11f, (R)-11m, (S)-11m and (R)-11n were 0.16, 0.28, 0.41 and 0.47 µg/mL against S. sclerotiorum in the in vitro evaluation, respectively. The efficacies of (R)-and (S)-11i and 11j against P. cubensis in the in vivo evaluation were 100% at 400 µg/mL, which showed they were the most active compounds and could be used as the potential lead structures for the further modification.