Design, Synthesis, and Antifungal Activity of Novel Aryl-1,2,3-Triazole-β-Carboline Hybrids

The copper catalytic azide and terminal alkyne cycloaddition reaction, namely “click chemistry”, gives a new and convenient way to create l,4-disubstitutd-l,2,3-triazoles. In this work, 2-pyrrolecarbaldiminato–Cu(II) complexes were established as efficient catalysts for the three-component 1,3-dipolar cycloaddition reaction of arylboronic acid and sodium azide (NaN3) with terminal alkynes in ethanol at room temperature to 50 °C, 1,4-disubstituted 1,2,3-triazoles were synthesized. Following the optimized protocol, two series of new aryl-1,2,3-triazole-β-carboline hybrids have been designed and synthesized, and the chemical structures were characterized by 1H NMR, 13C NMR, and high-resolution mass spectrometry (HRMS). All of the target compounds were evaluated in vitro for their antifungal activity against Rhizoctorzia solani, Fusarium oxysporum, Botrytis cinerea Pers., sunflower sclerotinia rot, and rape sclerotinia rot by mycelia growth inhibition assay at 50 μg/mL. The antifungal evaluation of the novel hybrids showed that, among the tested compounds, 5a, 5b, 5c, and 9b showed good antifungal activity against sunflower sclerotinia rot. Specifically, compound 9b also exhibited high broad-spectrum fungicidal against all the tested fungi with inhibition rates of 58.3%, 18.52%, 63.07%, 84.47%, and 81.23%. However, for F. oxysporum, all the target compounds showed no in vitro antifungal activities with an inhibition rate lower than 20%. These results provide an encouraging framework that could lead to the development of potent novel antifungal agents.


Introduction
Plant pathogenic microorganisms could infect crops and cause local or whole plant disease, which leads to significant economic losses [1]. In recent years, the potential impact of synthetic pesticides on the environment and human health has been of great concern, which highlights the need for environmentally-friendly pesticides to protect crops from insect infestation [2]. Therefore, plant-derived extracts and their bioactive natural compounds have been considered bio-rational alternatives [3]. Additionally, further modification and structural optimization of novel insecticides leading from the plant origin have recently been important methods for the research and development of new pesticides [4]. Harmine, harman, and harmol, belonging to the β-carboline alkaloid class, are

Chemistry
The synthesis of the desired key intermediate 1-methyl-9-(prop-2-yn-1-yl)-β-carboline (3) was performed in three steps starting from L-tryptophan, which was outlined in Scheme 1. The synthetic step involved the Pictet-Spengler condensation [6], and was followed by oxidation and decarboxylation to afford the intermediate 1-methyl-β-carboline (2). In the next step, the N 9 -alkylated of compound 2 was prepared by the action of sodium hydride (NaH) in anhydrous N,Ndimethylformamide (DMF) followed by addition of propargyl bromide to afford compound 3, which incorporates an alkynyl group required for click chemistry.

Chemistry
The synthesis of the desired key intermediate 1-methyl-9-(prop-2-yn-1-yl)-β-carboline (3) was performed in three steps starting from L-tryptophan, which was outlined in Scheme 1. The synthetic step involved the Pictet-Spengler condensation [6], and was followed by oxidation and decarboxylation to afford the intermediate 1-methyl-β-carboline (2). In the next step, the N 9 -alkylated of compound 2 was prepared by the action of sodium hydride (NaH) in anhydrous N,N-dimethylformamide (DMF) followed by addition of propargyl bromide to afford compound 3, which incorporates an alkynyl group required for click chemistry.
A number of synthetic methodologies [19,26,27] are available in the literature for the synthesis of 1,2,3-triazole. In our previous investigation [28,29], we have found that 2-pyrrolecarbaldiminato-Cu(II) complexes are efficient catalysts, which affords the 1-benzyl-1,2,3-triazoles in good yields. In order to improve the selectivity of the reaction, we have studied the reaction conditions by screening various catalysts. Initially, the cycloaddition reaction between phenylboronic acid, NaN 3 , and 1-methyl-9-propargyl-β-carboline (3) was selected as a model reaction to investigate the catalytic activity of four different 2-pyrrolecarbaldiminato-Cu(II) complexes, and the results are summarized in Table 1. It was found that the azidonation reaction of phenylboronic acid with NaN 3 proceeded smoothly within 8 h in the presence of the four Cu(II) complexes with 1 mol % loading. Subsequently, we added intermediate 3 to the reaction mixture, and the solution was heated at 50 • C for 2 h. The click cyclization reaction was completed to give the 1,4-disubstituted 1,2,3-triazoles in the yields of 69% to 84%, and Cu(II)-complex L 1 was found to be the best (Entries 1-4). The control experiment indicated that the reaction could not occur without the Cu(II)-complex (Entry 5). When the amount of the Cu(II)-complex, L 1 , was reduced from 1 mol % to 0.5 mol %, it resulted in a lower yield (Entry 6). Therefore, the optimal conditions for aryl-1,2,3-triazole-β-carboline hybrid synthesis involves the use of 1 mol % Cu(II)-complex L 1 as the catalyst. and ethanol as the solvent.

Chemistry
The synthesis of the desired key intermediate 1-methyl-9-(prop-2-yn-1-yl)-β-carboline (3) was performed in three steps starting from L-tryptophan, which was outlined in Scheme 1. The synthetic step involved the Pictet-Spengler condensation [6], and was followed by oxidation and decarboxylation to afford the intermediate 1-methyl-β-carboline (2). In the next step, the N 9 -alkylated of compound 2 was prepared by the action of sodium hydride (NaH) in anhydrous N,Ndimethylformamide (DMF) followed by addition of propargyl bromide to afford compound 3, which incorporates an alkynyl group required for click chemistry.  A number of synthetic methodologies [19,26,27] are available in the literature for the synthesis of 1,2,3-triazole. In our previous investigation [28,29], we have found that 2-pyrrolecarbaldiminato-Cu(II) complexes are efficient catalysts, which affords the 1-benzyl-1,2,3-triazoles in good yields. In order to improve the selectivity of the reaction, we have studied the reaction conditions by screening various catalysts. Initially, the cycloaddition reaction between phenylboronic acid, NaN3, and 1methyl-9-propargyl-β-carboline (3) was selected as a model reaction to investigate the catalytic activity of four different 2-pyrrolecarbaldiminato-Cu(II) complexes, and the results are summarized in Table 1. It was found that the azidonation reaction of phenylboronic acid with NaN3 proceeded smoothly within 8 h in the presence of the four Cu(II) complexes with 1 mol % loading. Subsequently, we added intermediate 3 to the reaction mixture, and the solution was heated at 50 °C for 2 h. The click cyclization reaction was completed to give the 1,4-disubstituted 1,2,3-triazoles in the yields of 69% to 84%, and Cu(II)-complex L1 was found to be the best (Entries 1-4). The control experiment indicated that the reaction could not occur without the Cu(II)-complex (Entry 5). When the amount of the Cu(II)-complex, L1, was reduced from 1 mol % to 0.5 mol %, it resulted in a lower yield (Entry 6). Therefore, the optimal conditions for aryl-1,2,3-triazole-β-carboline hybrid synthesis involves the use of 1 mol % Cu(II)-complex L1 as the catalyst. and ethanol as the solvent. Table 1. Cu(II)-complex-catalyzed one-pot synthesis of aryl-1,2,3-triazole-β-carboline hybrids from phenylboronic acid in ethanol: optimization of the catalytic conditions.  The generality of the optimized reaction condition was studied with a wide range of substrates, using various substituted phenylboronic acid bearing electron-withdrawing and electron-donating substituents, NaN3, and 1 mol % Cu(II)-complex L1 with 1-methyl-9-propargyl-β-carboline 3 to afford 9-(1,2,3-triazolyl)-β-carboline hybrids 5a-k, which are shown in Scheme 2. The synthetic routes of novel 7-(1,2,3-triazolyl)-β-carboline hybrids 9a-f are outlined in Scheme 3. The N 9 -alkylated harmine derivative 6 was prepared according to the synthetic protocol described by our group [30]. The preparation of compound 7 followed a common synthetic scheme and was characterized by demethylation of compound 6 using hydrobromic acid and acetic acid as the reaction solvent. Compound 8, bearing alkoxy in postion-7 of β-carboline core, was synthesized from compound 7 by the action of NaH in dry DMF followed by addition of propargyl bromide in 81% yield. Lastly, the synthesis of compounds 9a-f was carried out following the general procedure for the synthesis of compounds 5a-k. All structures of the final products were determined by 1 H NMR, 13  The generality of the optimized reaction condition was studied with a wide range of substrates, using various substituted phenylboronic acid bearing electron-withdrawing and electron-donating substituents, NaN 3 , and 1 mol % Cu(II)-complex L 1 with 1-methyl-9-propargyl-β-carboline 3 to afford 9-(1,2,3-triazolyl)-β-carboline hybrids 5a-k, which are shown in Scheme 2. The synthetic routes of novel 7-(1,2,3-triazolyl)-β-carboline hybrids 9a-f are outlined in Scheme 3. The N 9 -alkylated harmine derivative 6 was prepared according to the synthetic protocol described by our group [30]. The preparation of compound 7 followed a common synthetic scheme and was characterized by demethylation of compound 6 using hydrobromic acid and acetic acid as the reaction solvent. Compound 8, bearing alkoxy in postion-7 of β-carboline core, was synthesized from compound 7 by the action of NaH in dry DMF followed by addition of propargyl bromide in 81% yield. Lastly, the synthesis of compounds 9a-f was carried out following the general procedure for the synthesis of compounds 5a-k. All structures of the final products were determined by 1 H NMR, 13

Fungicidal Activities
From the synthetic route mentioned above, we obtained two series of novel aryl-1,2,3-triazoleβ-carboline hybrids 5a-k, 9a-f. These compounds were evaluated in a series of fungicidal tests in vitro against a range of phytopathogenic species including R. solani, Fusarium oxysporum, Botrytis cinerea Pers., sunflower sclerotinia rot, and rape sclerotinia rot. The activity results obtained as an inhibition rate are summarized in Table 2.

Fungicidal Activities
From the synthetic route mentioned above, we obtained two series of novel aryl-1,2,3-triazoleβ-carboline hybrids 5a-k, 9a-f. These compounds were evaluated in a series of fungicidal tests in vitro against a range of phytopathogenic species including R. solani, Fusarium oxysporum, Botrytis cinerea Pers., sunflower sclerotinia rot, and rape sclerotinia rot. The activity results obtained as an inhibition rate are summarized in Table 2.
Generally, at 50 μg/mL, the target compounds exhibited different levels of antifungal activity against these five tested fungi. Compared with that of the commercial fungicide carbendazim and

Fungicidal Activities
From the synthetic route mentioned above, we obtained two series of novel aryl-1,2,3-triazole-βcarboline hybrids 5a-k, 9a-f. These compounds were evaluated in a series of fungicidal tests in vitro against a range of phytopathogenic species including R. solani, Fusarium oxysporum, Botrytis cinerea Pers., sunflower sclerotinia rot, and rape sclerotinia rot. The activity results obtained as an inhibition rate are summarized in Table 2.
Generally, at 50 µg/mL, the target compounds exhibited different levels of antifungal activity against these five tested fungi. Compared with that of the commercial fungicide carbendazim and azoxystrobin, these compounds have exhibited a significant inhibitory effect against sunflower sclerotinia rot (SCR) in which compounds 5a (Ar = phenyl), 5b (Ar = 4-trifluoromethylphenyl), 5c (Ar = 3,4,5-trifluorophenyl), and 9b (Ar = 3,4,5-trifluorophenyl) had inhibitory rates of 85.04%, 86.93%, 85.98%, and 84.47%, respectively, which displayed comparable antifungal activity than that of the positive control, with an inhibition rate of 89.77% and 88.07%. In addition, compounds 5d-g, 5i-k, 9c-d, and 9f displayed moderate activity, with an inhibition rate ranging from 50% to 80%. For F. oxysporum, all the target compounds showed inactive in vitro antifungal activities with an inhibition rate lower than 20%. Similarly, for R. solani, the compounds showed weak antifungal activities with an inhibition rate ranging from 20% to 50%, except for 9b, which exhibited moderate activity with an inhibition rate of 58.30%. However, it was not as clear as the one drawn from the RSR data. Some of the compounds exhibited significant activities in vitro toward RSR in which the compound 9b had control efficacy rates of 81.23% and most of them showed weak to moderate activity. Of all aryl-1,2,3-triazole-β-carboline hybrids, compound 9b displayed as broad a fungicidal spectrum as azoxystrobin and carbendazim against these phytopathogens. The data in bold are used to emphasize that these compounds showed good activity. b significant inhibitory effect: inhibitory rate ≥ 80%, moderate: inhibition rate ranges from 50% to 80%, weak: inhibition rate ranges from 20% to 50%. c ClogP represent the calculated n-octanol/water partition coefficient (log Pow), and the values produced by Chemdraw software.

General Information
All the reactions were monitored by TLC on silica gel F254 plates (Qingdao Haiyang Inc., Qingdao, China) for detection of the spot. Column chromatography was performed with silica gel (200-300 mesh). NMR spectra were recorded at room temperature on a Bruker Avance III HD 400 instrument at 400 MHz for 1 H NMR and 100 MHz for 13 C NMR (Bruker Company, Bremen, Gemany). CDCl 3 , DMSO-d 6 , Methanol-d 4 or Pyridine-d 5 was used as the solvent and TMS as the internal standard. High-resolution mass spectrometry (HRMS) were measured on Bruker ultrafleXtreme MALDI-TOF/TOF-MS and HCCA (alpha-cyano-4-hydroxycinnamic acid) is used as matrix.

Biological Assays
The antifungal activity of the synthesized compounds was performed according to previously reported procedures [33]. The fungicidal activity of the target compounds against R. solani, F. oxysporum, B. cinerea Pers., sunflower sclerotinia rot and rape sclerotinia rot were evaluated using a mycelium growth rate test [17]. Carbendazim and azoxystrobin standard purchased from J&K Scientific Ltd. (Beijing, China), were used as a control, treating it in the same way. The relative inhibition ratio (%) was calculated using the following equation: The relative inhibition ratio (%) = Colony diameter of control − colony diameter of treated) colony diameter of control mycelial disk diameter × 100%.
Supplementary Materials: The following are available online, 1 H and 13 C NMR spectra for the target compounds are available online.