Synthesis and Antifungal Activity of Chimonanthus praecox Derivatives

To search for efficient agricultural antifungal lead compounds, 39 Chimonanthus praecox derivatives were designed, synthesized, and evaluated for their antifungal activities. The structures of target compounds were fully characterized by 1H NMR, 13C NMR, and MS spectra. The preliminary bioassays revealed that some compounds exhibited excellent antifungal activities in vitro. For example, the minimum inhibitory concentration (MIC) of compound b15 against Phytophthora infestans was 1.95 µg mL−1, and the minimum inhibitory concentration (MIC) of compound b17 against Sclerotinia sclerotiorum was 1.95 µg mL−1. Therefore, compounds b15 and b17 were identified as the most promising candidates for further study.


Introduction
One billion tons of crops in the world are destroyed by diseases and insect pests every year according to statistics, resulting in a 20~30% reduction in crop production [1]. The traditional chemical pesticides play a great role in food protection, but they also lead to serious ecological and environmental problems. The development of new pesticides with active natural products as lead compounds can not only find analogues with better activity but also help the products meet the needs of environmental protection and national development requirements of carbon peak and carbon neutralization [2].
The research on the synthesis and activity of Chimonanthus praecox alkaloids focuses mainly on the research and development of medicine and less on the antifungal activity of agriculture. However, pesticide research and pharmaceutical research have been learning from each other, and they have many similarities. Pesticide researchers have found that Chimonanthus praecox has significant inhibitory activity against Watermelon fusariumwil, Fusarium oxysportium, Bipolaris maydis, Exserohilum turcium and Alternaria solanit [9][10][11][12][13]. Because of their broad spectrum of biological properties, a number of studies aimed at the synthesis and antimicrobial activity of Chimonanthus praecox alkaloids have been reported [14][15][16][17][18]. In the past few years, our research group has been committed to the synthesis of Chimonanthus praecox alkaloids and research on their activity against plant pathogens. The biological testing has shown that several of the synthesized compounds have exhibited diverse and promising bioactivities ( Figure 2) [19][20][21][22][23][24]; for instance, compound i performed better against Verticillium dahlia compared with chlorothalonil, with the minimum inhibitory concentration (MIC) value of 7.81 µg ml −1 , and compounds j and k revealed potent activity against acetylcholinesterase, with IC 50 values of 0.01 and 0.1 ng ml −1 , respectively [23]. These findings inspired us to further combine the structure of Chimonanthus praecox-based natural product with nicotine functional group so as to acquire potential agrochemical leads for plant disease control.

Chemistry
A series of Chimonanthus praecox analogues have been efficiently synthesized based on the methods developed in our previous work. The synthetic route to the Chimonanthus praecox analogues is shown in Scheme 1. Compound 1 was obtained using indole-3acetonitrile as the starting material. Compound 1 reacted with an excess of R 1 Br or R 2 Br at the N-position and C-3 position using tetrahydrofuran (THF) as the solvent and sodium hydride(NaH) as a base to obtain compound 2 or 4, respectively. Compound 3 or 5 was synthesized from 2 or 4 using lithium aluminum hydride (LiAlH 4 ) as the reducing agent. Then, the expected 39 compounds were obtained based on intermediate 3 or 5. The synthesized compounds were characterized by 1 H NMR, 13 C NMR, and MS. The spectra of all compounds are in the Supplementary Materials.

Antifungal Acitivity
The inhibitory effects of the Chimonanthus praecox analogues towards six plant pathogen fungi are outlined in Table 1. The MIC values were evaluated with Carbendazim and Amphotericin B as the positive controls to assay the activity of the prepared Chimonanthus praecox analogues against Sclerotinia sclerotiorum, Altenaria solani, Verticillium dahliae, Colletotrichum orbiculare, Cytospora juglandis, and Curvularia lunata. The preliminary bioassays showed that most of the synthesized compounds exhibited fungicidal activity. Compound b15 exhibited significant antifungal activity against A. solani, with a MIC value of 1.95 µg mL −1 . Compound b17 showed the strongest antifungal activity against S. sclerotiorum, with a MIC value of 1.95 µg mL −1 . Compounds b12, b13, and b17 exhibited significant antifungal activities against A. solani, with the same MIC value of 3.91 µg mL −1 . Compounds a5 and b10 revealed improved activity against F. oxysporum compared with Carbendazim and Amphotericin B, with the same MIC value of 15.16 µg mL −1 . Compounds b15, b16, b17, and b19 manifested much more activity against C. lunata than Carbendazim and Amphotericin B, all with the same MIC value of 15.16 µg mL −1 . The activity of compounds a6, b8, and b10 was more potent than Carbendazim and Amphotericin B against A. solani, all with the same MIC value of 15.63 µg mL −1 . The activity of compound b11 was more potent than Carbendazim and Amphotericin B against V. dahliae, with a MIC value of 15.63 µg mL −1 . Compounds a2, a3, a10, b16, and b17 manifested much more activity against V. dahliae than Carbendazim and Amphotericin B, all with the same MIC value of 15.63 µg mL −1 . Scheme 1. Synthesis of Chimonanthus praecox derivatives. Although it is difficult to extract clear structure-activity relationships from the biological data, some conclusions can still be drawn ( Figure 3). Firstly, the analysis of the relationship between structure and activity showed that compounds a6, a10, b4, b11, b15, b16, and b17 contained Cl atom, and the Cl atoms that were located at C-2, C-5, or C-6 positions showed excellent antifungal activities. Compound b16 contained two Cl atoms, and the antifungal activity was stronger than that of the target compound with one Cl. Secondly, when trifluoromethyl was introduced into the benzene ring of R 3 group, the antifungal effect was significantly improved, and when the aromatic ring of R 1 group was chloropyridine, the antifungal effect was significantly enhanced.

Instruments and Chemicals
All reagents and solvents were reagent grade or purified according to standard methods before use. Analytical thin-layer chromatography (TLC) was performed with silica gel plates using silica gel 60 GF 254 (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). The 1 H−NMR (400 MHz) and 13 C−NMR (100 MHz) were obtained on an AM−500 FT−NMR spectrometer (Bruker Corporation, Fällanden, Switzerland) with CDCl 3 , acetone-d 6 , or DMSO-d 6 as the solvent and TMS as the internal standard. MS were recorded under ESI conditions using a LCQ Fleet instrument (Thermo Fisher, Waltham, MA, USA).

Synthesis
The general synthetic methods for the compounds a1-a20 and b1-b19 are depicted in Scheme 1.

Synthesis of Compound 1
The 3-indole acetonitrile (2.0 g, 12.8 mmol) was dissolved in dimethyl sulfoxide (DMSO) (30 mL, 281.6 mmol). Then, 100 mL 37% HCl was added. The mixture was stirred at r.t. for 2 h. The reaction mixture was evaporated under reduced pressure to remove the solvent to obtain the white solid 1 without further purification (2.1 g, 93%).

Biological Activity
The antifungal activity of Chimonanthus praecox analogues was measured according to the previously reported method [17,18].

Conclusions
In summary, a total of 39 Chimonanthus praecox alkaloids (a1~a20 and b1~b19) were synthesized from indole-3-acetonitrile via alkylation reaction, reduction reaction, and acylation reaction. Their antifungal activities were studied, and the relationship between the antifungal activity of the target compounds and their structures was discussed. Compound b15 showed the best inhibitory effect on A. solani, and its minimum inhibitory concentration (MIC) value is 1.95 µg mL −1 ; compound b17 displayed the best effect on S. sclerotiorum, and its minimum inhibitory concentration (MIC) value is 1.95 µg mL −1 . These results will lay a foundation for subsequent research and development.
Author Contributions: S.Z. designed research; Y.W. and J.C. performed research; Y.Y. and Y.Z. performed statistical analysis; Y.Y. wrote the paper; R.Z. reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.