Synthesis and Structure–Activity Analysis of Novel Potential Antifungal Cyclotryptamine Alkaloid Derivatives

A total of 39 novel cyclotryptamine alkaloid derivatives were prepared from 2-(1H-indol-3-yl) acetonitrile. The prepared compounds were evaluated against six plant pathogen fungi. Bioassay results revealed that most of the compounds displayed higher in vitro antifungal activities than the positive control. Notably, compound b2 displayed the broadest and most effective activity among the tested cyclotryptamine alkaloid derivatives and might be a novel potential leading compound for further development as an antifungal agent.


Introduction
The abuse of chemical pesticides has had a serious impact on the environment and caused great harm to the survival of human beings and other organisms. Therefore, it is urgent to develop an efficient, low toxicity and environment-friendly pesticide [1,2].
Cyclotryptamine alkaloids (Figure 1), which can be isolated from plants, microorganisms and marine organisms, are a class of natural products with specific structural units of hexahydropyrrolo [2,3-b]indole [3]. The family is large and has a rich and important biological activity. One example is (-)-physistigmine, an alkaloid derived from the seeds of lentils grown in Africa, which now can be artificially synthesized and has an inhibitory effect on cholinesterase. It can shrink pupils and reduce intraocular pressure clinically. (-)-Folicanthine has very good biological activity. (-)-Win-64745 is a good neurokinin antagonist. (-)-Asperdimin has antiviral activity [4][5][6][7][8]. Cyclotryptamine alkaloids have unique structures and various medicinal values, which have attracted great interest among many synthetic chemists [8][9][10][11][12][13][14]. Research on the synthesis and activity of cyclotryptamine alkaloids mainly focuses more on the research and development of medicine, but less on the antifungal activity of agriculture. Pesticide research and pharmaceutical research have been learning from each other, and they have many similarities. Pesticide scientists have studied the agricultural antifungal activity of cyclotryptamine alkaloid compounds in recent years. The research shows that monomer cyclotryptamine alkaloids also have a strong inhibitory effect on many agricultural pathogenic fungi; (+)-D calycanthine has significant antibacterial activity against Watermelon Fusarium Wilt and other pathogens. Compound c2 showed strong activity against acetylcholinesterase; the IC 50 value was 0.01 ng mL −1 [15]. In addition, our group has reported the synthesis and biological profiling of a wide variety of halfcyclotryptamine alkaloid derivatives. It showed that changing substituents on heterocycles can affect biological activity [16][17][18].
Therefore, we put emphasis on the structural optimization of cyclotryptamine alkaloids. The aim is that compounds with better activity could be obtained. Herein, 39 cyclotryptamine alkaloid analogs were synthesized in good yield.

Synthesis of Cyclotryptamine Alkaloids
Cyclotryptamine alkaloids were synthesized as depicted in Scheme 1. The target compounds were prepared starting from the inexpensive and readily obtainable 2-(1H-indol-3yl) acetonitrile according to the route development in our group [19]. A total of 39 derivatives of cyclotryptamine alkaloids were prepared and characterized by 1 H-NMR, 13 C-NMR and ESI-MS. ( 1 H and 13 C NMR Spectra could be found in the Supplementary Materials). Scheme 1. Synthesis route to the title compounds a1-a20 and b1-b19.

Antifungal Acitivity
Antifungal activity tests of the target compounds are shown in Table 1. We use carbendazim and amphotericin B as positive controls, and MIC values were determined to evaluate the biological activities of cyclotryptamine alkaloids against Sclerotinia sclerotiorum, Altenaria solani, Verticillium dahliae, Fusarium oxysporum, Walnut pythium and Curvularia lunata. It is observed that most of the compounds generally exhibited more effective antifungal activity than the positive controls. Compounds b2, b4, b6, b10, b13 and b15 showed significant antifungal activity against Sclerotinia sclerotiorum, of which b2 and b6 were the most effective compared with carbendazim and amphotericin B, with the same MIC values of 1.90 µg/mL. Compounds b2, b4, b6, b10, b15 and b17 revealed improved activity against Altenaria solani compared with the positive controls carbendazim and amphotericin B, with the same MIC value of 1.90 µg mL −1 . Compounds b2, b10 and b17 manifested much more activity against Verticillium dahliae than carbendazim; b10 and b17 were the most effective, with a MIC value of 1.9 µg mL −1 . The activity of compounds b4, b5 and b15 was more potent than carbendazim and amphotericin B against Fusarium oxysporum, all with the same MIC value of 3.90 µg mL −1 . The activity of compound b13 is more potent than carbendazim and amphotericin B against Walnut pythium, with a MIC value of 7.80 µg mL −1 . Compounds a2, a9, a11, a12, b3, b4, b8, b9, b11, b13, b16 and b18 manifested much more activity against Curvularia lunata than carbendazim and amphotericin B, with MIC values between 7.8 and 31.3 µg mL −1 . Compound b4 in particular exhibited significant antifungal activity against Curvularia lunata, with a MIC value of 7.80 µg mL −1 .
Although it is difficult to extract clear structure-activity relationships from the biological data, some conclusions can still be drawn. Firstly, when the position of two, five or six in the substituent is a Cl atom, such as in compounds a13, b2, b4, b10 and b13, excellent antifungal effects are displayed. Secondly, when the position in the substituent is methyl group, such as in compounds a3, a5, a15, b3, b5 and b15, an improved antifungal effect was shown. Thirdly, from compounds a11, a12, b11 and b12, it can be seen that when the compound contains pyrazine, the increase of the number of N atoms in the compound has a promoting effect on the antibacterial activity. Finally, as far as the parent structure is concerned, when the compound contains 3-F benzyl bromide, it is less active than compounds containing methyl bromide.

Instruments and Chemicals
All solvents and substances used were commercially purchased and not further purified. The reactions were monitored by thin-layer chromatography (TLC) with silica gel plates using silica gel 60 GF 254 (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). 1 H−NMR (400 MHz) and 13 C−NMR (100 MHz) were measured by 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 was recorded under ESI conditions using LCQ Fleet instruments (Thermo Fisher, Waltham, MA, USA). The yields of the reactions were measured before optimization.

Synthesis
The general synthesis method of compounds a1-a20 and b1-b19 is shown in Scheme 1.

General Procedures for the Synthesis of Target Compound 1
Concentrated hydrochloric acid (37%, 100 mL) was added slowly to a stirred solution of 3-Indoleacetonitrile (3.12 g, 20 mmol) in dimethyl sulfoxide (25 mL) at 0 • C. The resulting mixture was allowed to warm to room temperature for 1 h. The solvents were removed to obtain crude compound 1 (3.20 g) with a yield of 93%.

General Procedures for the Synthesis of Target Compound 2
Under a N 2 atmosphere, NaH (1.8 g, 75 mmol) was added portionwise to a stirred solution of compound 1 (2.58 g, 15 mmol) in dry THF through an injector. 2-methylbenzyl bromide (6.45 g, 37.5 mmol) was added dropwise into the flask in an ice bath. The resulting solution was stirred for 11 h at room temperature. Then, it was quenched with ammonium chloride (3 mL) and stirred for 10 min. The reaction mixture was continuously extracted with acetic ether (3 × 50 mL), and the combined organic layer was washed with saturated salt water. The organic extracts were combined, washed with brine, dried over Na 2 SO 4 and concentrated. The residue was purified by flash column chromatography (V PE : V EA = 6:1) to obtain compound 2 (4.28 g, 75%).

General Procedures for the Synthesis of Target Compound 3
LiAlH 4 (60 mmol, 7.5 eq) was added to compound 2 (3.52 g, 10 mmol) in anhydrous THF (25 mL) at −78 • C by a low temperature reactor under a nitrogen and anhydrous atmosphere. After stirring at room temperature for 1 h, the reaction mixture was heated to 75 • C for 4 h. When TLC monitoring indicated that the starting material, compound 2, had disappeared, the mixture was cooled to room temperature and quenched by acetic ether and water dropwise in an ice bath. The resulting solution was filtered to remove the deposition, and the filtrate was extracted with acetic ether (3 × 50 mL). The combined organic layer was washed with saturate salt water and dried over Na 2 SO 4 . The residue was purified by flash column chromatography (V PE :V EA :V Et3N = 100:50:2) to obtain compound 3 (2.50 g, 68%).

General Procedures for the Synthesis of Target Compound 4
Under a N 2 atmosphere, NaH (1.8 g, 75 mmol) was added portionwise to a stirred solution of compound 1 (2.58 g, 15 mmol) in dry THF through an injector. 3-fluorobenzyl bromide (7.05 g, 37.5 mmol) was added dropwise into the flask in an ice bath. The resulting solution was stirred for 11 h at room temperature. Then, it was quenched with ammonium chloride (3 mL) and stirred for 10 min. The reaction mixture was continuously extracted with acetic ether (3 × 50 mL), and the combined organic layer was washed with saturated salt water. The organic extracts were combined, washed with brine, dried over Na 2 SO 4 and concentrated. The residue was purified by flash column chromatography (silica gel, 12.5% petroleum ether/acetic ether) to obtain compound 4 (4.13 g, 71%).

General Procedures for the Synthesis of Target Compound 5
LiAlH 4 (60 mmol, 7.5 eq) was added to compound 4 (3.88 g, 10 mmol) in anhydrous THF (25 mL) at −78 • C by a low temperature reactor under a nitrogen and anhydrous atmosphere. After stirring at room temperature for 1 h, the reaction mixture was heated to 75 • C for 4 h. When TLC monitoring indicated that the starting material, compound 4, had disappeared, the mixture was cooled to room temperature and quenched by acetic ether and water dropwise in an ice bath. The resulting solution was filtered to remove the deposition, and the filtrate was extracted with acetic ether (3 × 50 mL). The combined organic layer was washed with saturate salt water and dried over Na 2 SO 4 . The residue was purified by flash column chromatography (V PE :V EA :V Et3N = 100:50:2) to obtain compound 5 (2.74 g, 73%).
3.2.6. General Procedure for the Synthesis of a1-a20 and b1-b19 The corresponding desired reagent (2.5 eq) was dissolved in dry CH 2 Cl 2 (10 mL). Then, sulfoxide chloride (4 eq) was added to the solution, and the solution was refluxed at 55 • C for 2 h. After the solvent had been evaporated under reduced pressure, the reagent of sulfonyl chlorination was afforded.