Synthesis and antifungal activity of novel chiral indole analogues

Abstract Starting from L-tryptophan, 19 new N-substituted chiral indole analogs were synthesized. The prepared compounds were evaluated for biological activity against Sclerotinia sclerotiorum, Alternaria solani, Verticillium dahliae, Colletotrichum orbiculare, Cytospora juglandis and Curvularia lunata. The preliminary bioassays showed that most of the synthesized compounds exhibited fungicidal activity. Compound b13 in particular exhibited significant antifungal activity against Verticillium dahliae and Sclerotinia sclerotiorum, with the MIC value of 1.95 µg mL−1. Compound b13 also showed excellent activity against six plant pathogen fungi, and was identified as the most active on the biological assays, and will be studied further. Graphical Abstract


Introduction
The annual loss of crops caused by various diseases and insect pests is roughly equivalent to one third of the annual harvest of the world. Although the traditional chemical control can play an excellent control effect on grain protection, it has led to serious ecological and environmental problems. Plant derived pesticides developed from natural products have the advantages of low toxicity, easy degradation and not easy to produce resistance. It is an effective method to study and develop new pesticide varieties with natural products as lead compounds (Flight 2013, Wang et al. 2019. The research of chiral pesticides is of great significance for the scientific and rational use of chiral pesticides, the maximum mitigation, control or repair of the damage of pesticide residues to the ecosystem, and the development of new environmentally friendly and efficient pesticide varieties. In particular, nitrogen-containing chiral pesticides have outstanding biological activities (Liu et al. 2009;Davison and Sperry 2017;Zhang, Jia, et al. 2017;Singh and Singh 2018;Hilgeroth et al. 2019;Zhu, Zhao, et al. 2021).
In the past few years, our research group has been committed to the synthesis of indoles and the activity against plant pathogens. The biological testing showed that several of the synthesized compounds exhibited diverse and promising bioactivities ( Figure 1) Zheng et al. 2017;Zheng, Zhou et al. 2016;Zheng, Li, et al. 2016;Zheng, Gu, et al. 2018;Zheng, Yang, et al. 2018;Zheng et al. 2019;Zheng et al. 2021;, such as, compound a1 showed better against Verticillium dahlia compared with chlorothalonil, with a MIC value of 7.81 mg ml À1 ; Compounds a2 and a3 revealed potent activity against acetylcholinesterase, with MIC values of 0.01 and 0.1 ng ml À1 , respectively (Zheng et al. 2019). These findings inspired us to further modify the structure of indole-based natural product with nicotine functional group so as to acquire potential agrochemical leads for plant disease control.
As a continuation of the development of new natural-product-based antifungal agents, a series of N-substituted chiral indole analogs were designed and synthesized.

Synthesis
The synthetic route to the compounds is given in Scheme 1. L-tryptophan methyl ester 1 was obtained by esterification of L-tryptophan with methanol. Compound 2 was obtained by nucleophilic substitution reaction between L-tryptophan methyl ester and isopropyl chloroformate. Compound 2 forms ring closing product 3 under the action of phosphoric acid. Then, the expected 19 compounds were obtained by reacting with 19 different nicotinic acid analogues with ring closure intermediate 3. The synthesized compounds were characterized by 1 H NMR, 13 C NMR and MS.

Fungicidal acitivity
The inhibitory effects of the indole analogues towards six plant pathogen fungi are outlined in Tables 1. The MIC values were evaluated with carbendazim and amphotericin B as positive controls, to assay the activity of the prepared indole analogues against Sclerotinia sclerotiorum, Altenaria solani, Verticillium dahliae, Colletotrichum orbiculare, Cytospora juglandis and Curvularia lunata. The activity of compound b13 is more potent than Carbendazim and Amphotericin B against S. sclerotiorum, with a MIC value of 1.95 mg mL À1 . Compounds b4, b7, b11 and b13 showed higher inhibitory activity against A. solani than Carbendazim and Amphotericin B, with MIC values of 7.8, 3.9, 7.8 and 3.9 mg mL À1 respectively. Compounds b13 and b17 exhibited significant antifungal activity against V. dahliae, with the same MIC value of 1.95 mg mL À1 .Compounds b11, b13, b17 and b19 revealed improved activity against C. orbiculare compared with the positive control Carbendazim and Amphotericin B, with the same MIC value of 15.16 mg mL À1 . Compounds b7, b8, b12, b14 and b16 revealed improved activity against C. orbiculare compared with the positive control Carbendazim and Amphotericin B, with the same MIC value of 31.3 mg mL À1 . Compounds b7, b13 and b17 manifested much more activity against C. juglandis than Carbendazim and Amphotericin B, all with the same MIC value of 3.9 mg mL À1 .The activity of Compounds b11, b13, b17 and b19 is more potent than Carbendazim and Amphotericin B against C. orbiculare, all with the same MIC value of 15.63 mg mL À1 .Compounds b1, b4, b7, b13, b14, b15, b17 and b19 revealed improved activity against C. lunata compared with the positive control Carbendazim and Amphotericin B, with MIC values of 31.25, 31.3, 31.3, 15.63, 31.3, 31.3, 3.9 and 31.25 mg mL À1 , respectively.
Although it is difficult to extract clear structure-activity relationships from the biological data, some broad conclusions can still be drawn (Figure 2). First, when the position of 2 in the substituent is Cl atom, compound b13 has a good antifungal effect on S. sclerotiorum, A. solani and C. juglandis, and has the same significant antifungal activity against V. dahliae. Compound b7 showed good antifungal activity against A. solani, C. juglandis and C. orbiculare when the position of 2 in the substituent is F atom. When the position of 2 in the substituent is hydroxyl, compound b17 has a strong inhibitory effect on S. sclerotiorum and A. solani, which is equivalent to Amphotericin B in the control group. It has the same significant antifungal activity against V. dahliae as carbendazim in the positive control group, and is also superior to Scheme 1. Synthetic route to the title compounds b1-b19.
C. juglandis and C. lunata. Compound b19 showed better biological activity against C. lunata than the positive controls. Secondly, when the position of 1 and 2 in the substituent is Cl, F or OH group, it shows excellent antifungal activity. When the ortho position of N is replaced by electron rich amine group, the activity decreases. When the substituents contain benzene ring, naphthalene ring, 2-pyridine and 4-pyridine, they have poor antifungal effect on six plant pathogenic fungi, and even have no antifungal activity on some fungi. Thirdly, Most of compounds showed better antifungal activity against V. dahliae and C. juglandis than Amphotericin B.

Instruments
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 À NMR (400 MHz), and 13 C À NMR (100 MHz) were obtained on an AM À 500 FT À NMR spectrometer (Bruker Corporation, Switzerland) with CDCl 3 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). Reaction yields were not optimized.

Synthesis
The general synthetic methods for the compounds b1-b19 are depicted in Scheme 1.
Synthesis of the compound 1. Starting material L-tryptophan (2.04 g, 10 mmol) in 30 mL of methanol was added to thionyl chloride (0.87 mL, 12 mmol, 1.2 eq.) at 0 C. Then the mixture was stirred at room temperature for 1.5 h. When TLC monitoring showed that the starting material had disappeared, the reaction mixture was evaporated under reduced pressure to remove the solvent to obtain the white solid 1(2.05 g, 94% yield). The crude product 1 was used for the next step without further purification.
Synthesis of the compound 2. A solution of compound 1 (2.0 g, 9.2 mmol) in 15 mL of pyridine was added isopropyl chloroformate (1.35 g, 11 mmol, 1.2 eq.) dropwise at 0 C. When TLC monitoring showed that the compound 1 had disappeared, the reaction mixture was evaporated under reduced pressure to remove the solvent. Purification by flash chromatography (petroleum ether-ethyl acetate ¼ 1:1) on silica gel afforded compound 2 (2.11 g, 75% yield). Synthesis of the compound 3. A solution of compound 2 (4.56 g, 15 mmol) in THF (20 mL) was added 25 mL of H 3 PO 4 (85%). The mixture was stirred until TLC monitoring indicated the disappearance of the material 2 (6 h.).The reaction mixture was evaporated under reduced pressure to remove the solvent. Purification by flash chromatography (petroleum ether-ethyl acetate ¼ 2:1) on silica gel afforded compound 3 (3.89 g, 85% yield).

General procedure for the preparation of the compounds b 1-b 19
To a solution of the compound 3 (0.55 g, 2 mmol) in acetonitrile (20 mL) was added Et 3 N (0.31 mL, 3 mmol, 1.5 eq.) and the corresponding desired reagents (3 mmol, 1.5 eq.). The resulting mixture was refluxed for 2 ～ 6 h., then extracted three times with dichloromethane. The organic extracts were combined, washed with brine, dried over Na 2 SO 4 , and concentrated. Purification by flash chromatography on silica gel afforded the compounds b1-b19. The data of the other compounds and the NMR spectral details can be found in the Supplementary data.

Biological assay
The antimicrobial activity of calycanthaceous alkaloids analogues were measured according to the previously reported method (Zhang et al. 2009;2013).
The tested compounds dissolved in 5% dimethyl sulfoxide (DMSO), to a concentration of 1.02 mg/mL., 100 lL of the solutions were added to the first well and serially diluted from first well by taking 100 lL into second. This two-fold dilution was continued down the plate and 100 lL from the 8th column of the plated discarded. The 9th column of the plate was reserved for negative control wells (without inocula) and the 10th column, for the positive growth control wells (without antifungal agent). The antifungal concentrations were 256,128,64,32,16,8,4 and 2 lg/mL, respectively. The antifungal test plates were incubated aerobically at 37 C for 24 h, the antifungal test plates were incubated aerobically at 28 C for 48 h. The MICs were examined. All tests were performed in triplicate and repeated if the results differed.

Conclusions
In summary, 19 chiral indole analogs were synthesized, and evaluated for their antimicrobial activity against six plant pathogen fungi. The in vitro assessment of target compounds revealed that most of compounds showed certain biological activity, compounds b13 in particular showed excellent activity against six plant pathogen fungi, especially compound b13 exhibited significant antifungal activity against V. dahliae and S. sclerotiorum better than Carbendazim and Amphotericin B with the same MIC value of 1.95 mg mL À1 , and was identified as the most active on the biological assays, and will be studied further.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by the Zhoushan Public Welfare Science and Technology Project (2019C31066).