Semisynthesis and Pesticidal Activities of Novel Cholesterol Ester Derivatives Containing Cinnamic Acid-like Fragments

Due to the extensive use of agrochemicals resulting in the emergence of pesticide resistance and ecological environment problems, the research and development of new alternatives for crop protection is highly desirable. In order to discover potent natural product-based insecticide candidates, a series of new cholesterol ester derivatives containing cinnamic acid-like fragments at the C-7 position were synthesized. Some derivatives showed potent pesticidal activities. Against Mythimna separata Walker, compounds 2a, Id, Ig, and IIg showed 2.1–2.4-fold growth-inhibitory activity of the precursor cholesterol. Against Plutella xylostella Linnaeus, compounds Ig, IIf, and IIi exhibited 1.9–2.1-fold insecticidal activity of cholesterol. These results will pave the way for the future synthesis of cholesterol-based derivatives as agrochemicals.


Introduction
Cholesterol (1), a biological endogenous substance, has shown a variety of antitumor, antioxidant, anti-inflammatory, and insecticide activities [1][2][3][4][5][6][7][8][9][10]. Cinnamic acid (Figure 1), as a main component of Cinnamomum cassia Presl, has displayed a range of pharmaceutical and agricultural activities [11][12][13]. In addition, several cholesterol oxime esters containing cinnamic acid-like fragments (1 ) have shown promising insecticidal activities [14]. Nowadays, due to the extensive and irrational use of chemical insecticides resulting in pest resistance and ecological environment problems, the research and development of new pesticides for crop protection is highly desirable [15][16][17][18]. Because of the characteristics of natural secondary metabolites as an unparalleled source of bioactive products, currently, the study of natural product-based insecticides as an important alternative to classic agrochemicals has received much attention [19][20][21]. Therefore, in order to develop prospective cholesterol-based pesticide candidates, a series of new cholesterol derivatives I and II (Figure 1) were designed and synthesized by a combination of cholesterol and cinnamic acid-like fragments via the ester bond at the C-7 position of cholesterol. Meanwhile, two typically crop-threatening insect pests, Mythimna separata Walker (Lepidoptera: Noctuidae) and Plutella xylostella Linnaeus (Lepidoptera: Plutellidae), were used as the tested biotargets for assessment of the insecticidal activities of cholesterol derivatives I and II [22,23].

Materials and Methods
Multi-generation sensitive strains of Mythimna separata and Plutella xylostella (3rd instar larvae) were raised in our lab. Cholesterol was purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). The melting point (mp) was determined using the XT-415 digital melting point apparatus (Beijing Tech Instruments, Ltd., Beijing, China) and was uncorrected. 1 H NMR spectra were obtained using Avance III 500 MHz equipment (Bruker, Germany). High-resolution mass spectra (HRMS) were obtained with a Mi-crOTOF Q II instrument (Bruker, Germany). Infrared spectroscopy (IR) was obtained with a TENSOR-27 instrument (Bruker, Germany).

Synthesis of Compounds 2a and 2b
A mixture of cholesterol (1, 5 mmol), triethylamine (10 mmol), and substituted acyl chloride (7 mmol) in dry CH2Cl2 (20 mL) was stirred at room temperature for 14 or 17 h. Then, CH2Cl2 (30 mL) was added to the mixture, which was washed with brine. The organic phase was dried over anhydrous Na2SO4 and purified by silica gel column chromatography eluting with petroleum ether/dichloromethane (4/1, v/v) to obtain 2a and 2b.

Materials and Methods
Multi-generation sensitive strains of Mythimna separata and Plutella xylostella (3rd instar larvae) were raised in our lab. Cholesterol was purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). The melting point (mp) was determined using the XT-415 digital melting point apparatus (Beijing Tech Instruments, Ltd., Beijing, China) and was uncorrected. 1 H NMR spectra (Supplementary Materials) were obtained using Avance III 500 MHz equipment (Bruker, Germany). High-resolution mass spectra (HRMS) were obtained with a MicrOTOF Q II instrument (Bruker, Germany). Infrared spectroscopy (IR) was obtained with a TENSOR-27 instrument (Bruker, Germany).

Synthesis of Compounds 2a and 2b
A mixture of cholesterol (1, 5 mmol), triethylamine (10 mmol), and substituted acyl chloride (7 mmol) in dry CH 2 Cl 2 (20 mL) was stirred at room temperature for 14 or 17 h. Then, CH 2 Cl 2 (30 mL) was added to the mixture, which was washed with brine. The organic phase was dried over anhydrous Na 2 SO 4 and purified by silica gel column chromatography eluting with petroleum ether/dichloromethane (4/1, v/v) to obtain 2a and 2b.

Synthesis of Compounds 4a and 4b
Compound 3a or b (4 mmol) was dissolved in methanol/dichloromethane (1/1, v/v), and sodium borohydride (6 mmol) was added in batches under an ice bath. Then, it was raised naturally to room temperature. When the reaction was complete, methanol was removed and ethyl acetate (30 mL) was added to the mixture, which was washed with brine. The organic phase was dried over anhydrous Na 2 SO 4 , concentrated, and purified by silica gel column chromatography eluting with petroleum ether/dichloromethane (1/1, v/v) to obtain 4a and 4b.

General Procedure for Synthesis of Target Compounds I(a-i)-II(a-i)
Compound 4a or 4b (0.4 mmol) reacted with cinnamic acids (5a-i, 0.6 mmol) in dry CH 2 Cl 2 (5 mL) in the presence of EDCI (0.6 mmol) and DMAP (0.2 mmol) at room temperature for 10-34 h. Then, the mixture was diluted with CH 2 Cl 2 (20 mL). It was washed with brine, dried over anhydrous Na 2

Conclusions
In summary, to develop new natural product-based pesticide candidates, a series of new cholesterol ester derivatives containing cinnamic acid-like fragments at the C-7 position were prepared. Against M. separata, compounds 2a, Id, Ig, and IIg showed 2.1-2.4 times more potent growth-inhibitory activity than cholesterol. Against P. xylostella, compounds Ig, IIf, and IIi exhibited 1.9-2.1 times more promising insecticidal activity than cholesterol. The structure-activity relationships of the compounds suggest that the introduction of the fluorine atom or CF 3 on the para position of the phenyl of Ic and IIc was very important for the insecticidal activity against M. separata; and against P. xylostella, the introduction of the fluorine atom or CF 3 on the para position of the phenyl of Ic was necessary for insecticidal activity, whereas the introduction of the bromine atom or methoxy on the para position of the phenyl of IIc was vital for insecticidal activity. These results will provide a basis for guiding the synthesis of novel cholesterol derivatives as new pesticidal agents in the future.