Synthesis and Antifungal Activity of Novel 3-Caren-5-One Oxime Esters

A series of novel 3-caren-5-one oxime esters were designed and synthesized by multi-step reactions in an attempt to develop potent antifungal agents. Two E-Z stereoisomers of the intermediate 3-caren-5-one oxime were separated by column chromatography for the first time. The structures of all the intermediates and target compounds were confirmed by UV-Vis, FTIR, NMR, ESI-MS, and elemental analysis. The antifungal activity of the target compounds was preliminarily evaluated by the in vitro method against Fusarium oxysporum f. sp. cucumerinum, Physalospora piricola, Alternaria solani, Cercospora arachidicola, Gibberella zeae, Rhizoeotnia solani, Bipolaris maydis, and Colleterichum orbicalare at 50 µg/mL. The target compounds exhibited best antifungal activity against P. piricola, in which compounds (Z)-4r (R = β-pyridyl), (Z)-4q (R = α-thienyl), (E)-4f′ (R = p-F Ph), (Z)-4i (R = m-Me Ph), (Z)-4j (R = p-Me Ph), and (Z)-4p (R = α-furyl) had inhibition rates of 97.1%, 87.4%, 87.4%, 85.0%, 81.9%, and 77.7%, respectively, showing better antifungal activity than that of the commercial fungicide chlorothanil. Also, compound (Z)-4r (R = β-pyridyl) displayed remarkable antifungal activity against all the tested fungi, with inhibition rates of 76.7%, 82.7%, 97.1%, 66.3%, 74.7%, 93.9%, 76.7% and 93.3%, respectively, showing better or comparable antifungal activity than that of the commercial fungicide chlorothanil. Besides, the E-Z isomers of the target oxime esters were found to show obvious differences in antifungal activity. These results provide an encouraging framework that could lead to the development of potent novel antifungal agents.

3-Caren-5-one oxime (3, isomeric mixture) was prepared by oximation of compound 2 with NH 2 OH·HCl in a mixed solvent (EtOH:H 2 O = 5:1, v/v). Sodium acetate was added into the reaction system as an additive to neutralize HCl and form a buffer system. Two stereoisomers, (Z)-3-caren-5-one oxime ((Z)-3a) and (E)-3-caren-5-one oxime ((E)-3b), were isolated from compound 3 (3a:3b = 7:1, w/w, GC). In the 1 H-NMR spectrum of (Z)-3a, the olefinic proton (C 4 -H) in the 3-carene skeleton showed signals at about 5.88 and the signals of the C 6 -H appeared at 1.99 ppm. However, the corresponding signals of another isomer (E)-3b were shifted to 6.65 ppm and 1.50 ppm, respectively ( Figure 1). Due to the hydrogen bond interaction of the hydroxyl group oxygen with the olefinic proton (C 4 -H) or the C 6 -H, the signal of olefinic proton (C 4 -H) in the (E)-isomer and the signal of the C 6 -H in the (Z)-isomer shifted to downfield [38]. The structures of the two stereo-isomers were further confirmed by NOESY spectra. In the NOESY spectrum of (Z)-3a, no peak related to the coupling of hydroxyl hydrogen with C 4 -H was observed, while a peak corresponding to this space interaction in (E)-3b was found ( Figure 2).  The target oxime esters were characterized by UV-Vis, IR, 1 H-NMR, 13 C-NMR, ESI-MS, and elemental analysis, and all the related spectra can be found in the Supplementary Materials. It was found that the molar extinction coefficient of all target compounds ranged from 3.89 to 4.42 (log ε), implying that a conjugated system existed in these compounds. In the IR spectra, the weak absorption bands at about 3018 cm −1 were attributed to the stretching vibrations of the unsaturated C-H in the 3-carene moiety. The strong absorption bands at about 1738 cm −1 were assigned to the vibrations of C=O in the ester moiety. The moderate absorption peaks at about 1649 cm −1 were due to the vibrations of C=N. In the 1 H-NMR spectra, the protons of benzene ring showed signals at 7. 10-8.20 ppm. The olefinic protons of the 3-carene scaffold showed signals at about 6.10 ppm for the (Z)-isomers and the (E)-isomers showed signals at about 6.60 ppm. The protons bonded to the saturated carbons displayed signals in the range of 0.50-3.00 ppm. The 13 C-NMR spectra of the target compounds showed peaks for the olefinic carbons of the 3-carene moiety at 117.92-119.05 ppm (C4) and 147.49-149.74 ppm (C3) for the (Z)-isomers, however, the corresponding signals for (E)-isomers showed at about 113.20 (C4) ppm and 153.03 ppm (C3). The other saturated carbons displayed signals in the region of 13.59-55.94 ppm. The other signal peaks were correlated to the carbons of C=O, C=N, and the benzene ring. The molecular weights of the intermediates and target oxime esters were confirmed by the ESI-MS.

Antifungal Activity
The antifungal activities of the target compounds (Z)-4a-4w and (E)-4f′ were evaluated by in vitro method against fusarium wilt on cucumber (Fusarium oxysporum f. sp. cucumerinum), speckle on peanut (Cercospora arachidicola), apple ring rot (Physalospora piricola), tomato early blight (Alternaria solani), wheat scab (Gibberella zeae), rice sheath blight (Rhizoeotnia solani), corn southern leaf blight (Bipolaris maydis), and watermelon anthracnose (Colleterichum orbicalare) at 50 µg/mL. The results are listed in Table 1.  The target oxime esters were characterized by UV-Vis, IR, 1 H-NMR, 13 C-NMR, ESI-MS, and elemental analysis, and all the related spectra can be found in the Supplementary Materials. It was found that the molar extinction coefficient of all target compounds ranged from 3.89 to 4.42 (log ε), implying that a conjugated system existed in these compounds. In the IR spectra, the weak absorption bands at about 3018 cm −1 were attributed to the stretching vibrations of the unsaturated C-H in the 3-carene moiety. The strong absorption bands at about 1738 cm −1 were assigned to the vibrations of C=O in the ester moiety. The moderate absorption peaks at about 1649 cm −1 were due to the vibrations of C=N. In the 1 H-NMR spectra, the protons of benzene ring showed signals at 7.10-8.20 ppm. The olefinic protons of the 3-carene scaffold showed signals at about 6.10 ppm for the (Z)-isomers and the (E)-isomers showed signals at about 6.60 ppm. The protons bonded to the saturated carbons displayed signals in the range of 0.50-3.00 ppm. The 13 C-NMR spectra of the target compounds showed peaks for the olefinic carbons of the 3-carene moiety at 117.92-119.05 ppm (C4) and 147.49-149.74 ppm (C3) for the (Z)-isomers, however, the corresponding signals for (E)-isomers showed at about 113.20 (C4) ppm and 153.03 ppm (C3). The other saturated carbons displayed signals in the region of 13.59-55.94 ppm. The other signal peaks were correlated to the carbons of C=O, C=N, and the benzene ring. The molecular weights of the intermediates and target oxime esters were confirmed by the ESI-MS.

Antifungal Activity
The antifungal activities of the target compounds (Z)-4a-4w and (E)-4f′ were evaluated by in vitro method against fusarium wilt on cucumber (Fusarium oxysporum f. sp. cucumerinum), speckle on peanut (Cercospora arachidicola), apple ring rot (Physalospora piricola), tomato early blight (Alternaria solani), wheat scab (Gibberella zeae), rice sheath blight (Rhizoeotnia solani), corn southern leaf blight (Bipolaris maydis), and watermelon anthracnose (Colleterichum orbicalare) at 50 µg/mL. The results are listed in Table 1. The target oxime esters were characterized by UV-Vis, IR, 1 H-NMR, 13 C-NMR, ESI-MS, and elemental analysis, and all the related spectra can be found in the Supplementary Materials. It was found that the molar extinction coefficient of all target compounds ranged from 3.89 to 4.42 (log ε), implying that a conjugated system existed in these compounds. In the IR spectra, the weak absorption bands at about 3018 cm −1 were attributed to the stretching vibrations of the unsaturated C-H in the 3-carene moiety. The strong absorption bands at about 1738 cm −1 were assigned to the vibrations of C=O in the ester moiety. The moderate absorption peaks at about 1649 cm −1 were due to the vibrations of C=N. In the 1 H-NMR spectra, the protons of benzene ring showed signals at 7.10-8.20 ppm. The olefinic protons of the 3-carene scaffold showed signals at about 6.10 ppm for the (Z)-isomers and the (E)-isomers showed signals at about 6.60 ppm. The protons bonded to the saturated carbons displayed signals in the range of 0.50-3.00 ppm. The 13 C-NMR spectra of the target compounds showed peaks for the olefinic carbons of the 3-carene moiety at 117.92-119.05 ppm (C 4 ) and 147.49-149.74 ppm (C 3 ) for the (Z)-isomers, however, the corresponding signals for (E)-isomers showed at about 113.20 (C 4 ) ppm and 153.03 ppm (C 3 ). The other saturated carbons displayed signals in the region of 13.59-55.94 ppm. The other signal peaks were correlated to the carbons of C=O, C=N, and the benzene ring. The molecular weights of the intermediates and target oxime esters were confirmed by the ESI-MS.

General Information
The GC analysis was performed on an Agilent 6890 GC (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with a HP-1 (30 m, 0.530 mm, 0.88 µm) column. IR spectra were recorded (KBr pellet method) on a Nicolet iS50 FT-IR spectrometer (Thermo Scientific Co., Ltd., Madison, WI, USA). NMR spectra (including 1 H-NMR, 13 C-NMR, NOESY) were recorded in CDCl 3 solvent on a Bruker Avance III HD 600 MHz spectrometer (Bruker Co., Ltd., Zurich, Switzerland) and chemical shifts are expressed in ppm (δ) downfield relative to TMS as an internal standard. MS spectra were obtained by means of the electrospray ionization (ESI) method on TSQ Quantum Access MAX HPLC-MS instrument (Thermo Scientific Co., Ltd., Waltham, MA, USA). Elemental analyses were measured using a PE 2400 II elemental analyzer (Perkin-Elmer Instruments Co., Ltd., USA). The UV spectra were measured on a Shimadzu UV-1800 spectrophotometer (Shimadzu Corp., Kyoto, Japan). Melting points were determined on a MP420 automatic melting point apparatus (Hanon Instruments Co., Ltd., Jinan, China) and were not corrected. Ultrasonic irradiation was carried out on XO-SM50 ultrasonic microwave reaction system (Nanjing Xianou Instrument Manufacturing Co., Ltd., Nanjing, China). 3-Carene (GC purity 98%, isomeric mixture) was provided by Wuzhou Pine Chemicals Co., Ltd. (Wuzhou, Guangxi, China). The 7Å HY zeolite was purchased from Nankai University Catalyst Co., Ltd. (Tianjin, China). Other reagents were purchased from commercial suppliers and used as received.

Preparation of Catalyst
Cu(II)HY zeolite catalyst used for the synthesis of 3-caren-5-one was prepared by the incipient wetness method [39]. HY zeolite with pore diameter of 7 Å was dried at 120 • C 3 h under air atmosphere. Then, a solution of Cu(NO 3 ) 2 ·3H 2 O (5.7 g) in deionized water based on the saturated water absorption amount of the zeolite (10.0 g) was added dropwise to the dried zeolite, with constant stirring and grinding. The resulting mixture was aged for 24 h, and then dried in vacuum at 120 • C for 2 h. The powder sample was calcinated at 550 • C for 4 h under air atmosphere. (2) 3-Carene (10 mL, 63 mmol), Cu(II)HY zeolite catalyst (2.7 g) and CH 3 CN (20 mL) were mixed and then irradiated under ultrasonic wave for 40 min. When the mixture was heated to 45 • C, a mixed solution of 40 mL tert-butyl hydroperoxide (70% in water, wt %) (288 mmol) and 20 mL CH 3 CN was added to the reaction system by the way of constant flowing at a rate of 1 mL/min. Afterwards, the reaction mixture was stirred for 18 h at 45 • C. When the reaction was completed, the reaction mixture was cooled to room temperature and appropriate amount of Na 2 S 2 O 3 was added into it to destroy the unreacted tert-butyl hydroperoxide. The solvent CH 3 CN was removed by vacuum distillation, and the residue was then extracted three times with EtOAc. The combined organic phase was washed with saturated sodium chloride and deionized water, respectively, and dried over with anhydrous Na 2 SO 4 . After the EtOAc was removed in vacuum, the crude product was purified by silica gel column chromatography (EtOAc−petroleum ether = 1:30, v/v) to obtain pale yellow liquid compound 2. Yield 20%. UV-Vis (EtOH) λ max (log ε): 226.50 (4.4.16) (3) 3-Caren-5-one (1.5 g, 10 mmol), NH 2 OH·HCl (1.04 g, 15 mmol), and NaOAc (4.08 g, 30 mmol) were dissolved in a mixed solvent (EtOH:H 2 O = 5:1, v/v). The reaction mixture was stirred at 80 • C for 3 h. The reaction process was monitored by TLC. When 3-carene-5-one was fully reacted, the mixture was cooled to room temperature. After the solvent EtOH was removed by rotary evaporation, the residue was extracted by EtOAc. The separated organic phase was washed with saturated NaCl solution three times, and concentrated under reduced pressure to obtain the crude product, which was purified by silica gel column chromatography using a mixed solvent (EtOAc−petroleum ether = 1:60, v/v) as eluent.  ,9.15;N,8.48. Found: C,72.71;H,9.17;N,8.49.  ,9.15;N,8.48. Found: C,72.60;H,9.18;N,8.46.

General Procedure for Synthesis of 3-Caren-5-One Oxime Esters (Z)-4a-4w, (E)-4f , 4l , 4r
A solution of acyl chloride (1.2 mmol) in CH 2 Cl 2 (3 mL) was added slowly to a solution of (Z)or (E)-3-carene-5-one oxime (1 mmol) and triethylamine in CH 2 Cl 2 (5 mL) with ice-water cooling. The reaction process was monitored by TLC. Upon completion, 5 mL deionized water was added to the reaction mixture to destroy the unreacted acyl chloride. Then, the organic layer was separated, washed with deionized water three times, and concentrated in vacuum. The crude product was further purified by silica gel chromatography to afford the target compounds (Z)-4a-4w, and (E)-4f , 4l , 4r .

Antifungal Activity Test
Antifungal activity of the target compounds was perform in vitro method [40]. The tested compound was dissolved in acetone. Sorporl-144 (200 µg/mL) emulsifier was added to dilute the solution to 500 µg/mL. Then, 1 mL solution of the tested compound was poured into a culture plate, and then 9 mL PSA culture medium was added to obtain the flat containing 50 µg/mL tested compound. A bacterium tray of 5-mm diameter cut along the external edge of the mycelium was transferred to the flat containing the tested compound and put in equilateral triangular style in duplicate. Later, the culture plate was cultured at 24 ± 1 • C and the expanded diameter of the bacterium tray was measured after 48 h and compared with that treated with aseptic distilled water to calculate the relative inhibition percentage: Relative inhibitory rate (%) = (CK − PT)/CK × 100% where CK is the extended diameter of the circle of mycelium during the blank assay and PT is the extended diameter of the circle of mycelium during testing.