English

• 1.   Introduction

  Each year, plant diseases are responsible for billions of dollars in economic loss worldwide [1]. Therefore, agrochemicals play a critical role in the development of agriculture [2]. However, at present, traditional pesticides are less effective and will result in resistance in host plants and damages to the environment. So, the search for alternative pesticides which are effective and environmentally friendly is urgent [3]. Plant activator as an ecological pesticide has attracted more and more attention, because it has no direct bactericidal activity in vitro, but could activate the immune system[4-7] of the plant itself, such as systemic acquired resistance (SAR) [8-12], which is dependent on salicylic acid (SA), which could induce the expression of the pathogen-related proteins (PR proteins), and then provide defense against a broad spectrum of diseases including fungi, bacteria, viruses and insects without destroying the ecosystem [13-16]. So, it possesses better potential as a green and eco-friendly pesticide. Up to now, a variety of plant activators has been reported (Fig. 1), and some of which have come into the market, among them, benzothiadiazole (BTH) [17-19], 2, 6-dichlomisonicotinic acid (INA) [20], tiadinil (TDL) [21, 22], and isotianil activate SAR at the downstream of SA, conversely, probenazole (PBZ) [23] activates SAR at the upstream of SA. As the most successful compound, BTH has been exploited in agriculture for the control of downy and powdery mildew [17]. Based on the structure of BTH, our group has also reported two series of compounds, respectively were fluorine-containing derivatives of BTH and1, 2, 3-thiadiazole-6-carboxylate derivatives, all of them showed a potent activity [3, 4]. Besides, Fan group has also explored some plant activators based on the structure of BTH and TDL with excellent systemic acquired resistance [24-26]. Although the study of plant activators has attracted more and more attention, most of the plant activators are still developed based on the scaffold of 1, 2, 3-benzothiadiazole due to its potent bioactivity.

  图 1

  

  图 1  Structures of reported plant activators.

  Figure 1.  Structures of reported plant activators.

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  So, it'sinteresting to find some novel chemotypes which could act as plant activators. With the development of computer technology, computer aided drug design has attracted more and more attention, and it has been wildly used in discovering pesticides and predicting the toxicity of pesticide metabolites [27-29]. So, our motivation in this work is to find some new scaffolds as plant activators with the help of virtual screening. According to the chemical structures of salicylic acid (SA) and the reported plant activators, a novel benzotriazole scaffold was firstly identified as promising plant activator by scaffold hopping with the facility of virtual screening against the MayBridge database using SHAFTS [30, 31] (Scheme 1). SHAFTS is a free software designed to calculate 3D molecular similarity taking both molecular shape and molecular pharmacophore profiles into consideration, which has been successfully applied in the area of drug design [32, 33].

  Scheme 1

  

  图 Scheme 1  Virtual screening process.

  Scheme 1.  Virtual screening process.

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  As a result, three benzotriazole scaffolds were discovered by virtual screening (Fig. 2), among them, compound L1 was the most potent according to the activity results of the in-vivo bioassay (Table 1). In addition, the triazole family is also a group of pharmacophoric molecules with high bioactivity and broad spectrum of biological properties, including antifungal [34], anticancer [35], and antiviral [36] activities in the field of medicine, and acting as bactericide [37], herbicide [38], and insecticide [39] in the field of pesticide. To date, large numbers of triazole derivatives have been reported but still received considerable attention. So, the triazole family has the potential to be developed as plant activator, and L1 was selected as the lead compound for the subsequent optimization.

  图 2

  

  图 2  Structures of lead compounds.

  Figure 2.  Structures of lead compounds.

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  表 1

  

  表 1  In-vivo activities of the lead compounds.

  Table 1.  In-vivo activities of the lead compounds.

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  On the basis of these considerations, a series of novel benzotriazole derivatives were designed and synthesized (Scheme 2), and their activities as plant activators were evaluated both in vivo and in vitro against several plant diseases to confirm the prediction of the virtual screening results.

  Scheme 2

  

  图 Scheme 2  Design of target compounds.

  Scheme 2.  Design of target compounds.

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  2.   Results and discussion

  2.1   Discovery of novel plant activators through computational scaffold hopping

  3D molecular similarity based virtual screening against the MayBrige database by SHAFTS provided not only a ranked list of similarity score but also a corresponding molecular file containing the well-aligned chemical structures that facilitates further visual inspection. According to the screening results of SHAFTS in this study, we did a visual analysis of the top-ranked 500 molecules and purchased 190 compounds for bioactivity assay. Three compounds of benzotriazole (Fig. 2) were found with potent bioactivity. By comprehensively comparing the activity against four diseases (Table 1) as well as the synthesis route for each of the three hits, L1 was chosen as the ultimate lead compound.

  The superimposition of template BTH and compound L1 in Fig. 3(A) showed that although different in 2D topological structures, compound L1 and BTH share a similar molecular shape in three-dimension. Besides, according to Fig. 3(B), the two molecules are quite alike in terms of the pharmacophore features, including aromatic rings and hydrogen bond acceptors. However, there is one more hydrophobic centroid at 70-position of BTH. Therefore, the introduction of an additional hydrophobic group at 50-position of L1 could generate a hydrophobic centroid resembling that of BTH at 70-position. The substituted alkyl group at 10-position of L1 could also enhance the hydrophobic profiles of benzotriazole group in L1 to resemble that of benzothiadiazole. In summary, hydrophobic groups decorated at 10-position as well as 50-position of L1 would benefit the in vivo efficacy of the derivatives designed based on the scaffold of lead L1.

  2.2   Preparation of benzotriazole carboxylate derivatives

  The general method for the preparation of the target compounds is shown in Scheme 3. All compounds were synthesized with yields of 35%-83%. All compounds were separated and purified by recrystallization or silica gel chromatography, and their structures were determined by ¹H NMR, ¹³C NMR, and HRMS, and compounds containing fluorine atoms were also identified by ¹⁹F NMR.

  Scheme 3

  

  图 Scheme 3  The synthetic route of targeted compounds.

  Scheme 3.  The synthetic route of targeted compounds.

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  2.3   Bioactivity

  The in vivo inducing activities of the synthetic molecules were screened against seven diseases, and the results were shown in Table 2. Due to the positive control 50% procymidone (WP) showed 41.87% efficacy against B. cinerea, so, in order to compare the efficacy of the novel compounds, we defined herein the efficacy above 40% as promising active efficacy against the relevant disease.

  表 2

  

  表 2  In-vivo activities of the target compounds.

  Table 2.  In-vivo activities of the target compounds.

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  For series of benzotriazole derivatives, all compounds with carboxyl substitution at the 70-position (1a-c) exhibited weak SARinducing activities, only showed efficacy against one out seven tested diseases. However, comparing 1a with 2b, when the carboxyl was moved to the 50-position, it showed efficacy against two of the tested diseases, and the efficacy against M. melonis (MM) was 100%, better than the positive control 50% kresoxim-methyl (WG), which proved that for the benzotriazole scaffold, carboxyl was more helpful when it was substituted at the 50-position, which was same to the lead compound L1. Among all the derivatives whose carboxyl were substituted at 50-position, 3a achieved the highest hybrid score of 1.465 (Table 3), and showed excellent efficacy.

  表 3

  

  表 3  The similarity scores of derivatives of benzotriazole.

  Table 3.  The similarity scores of derivatives of benzotriazole.

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  As the triazole moiety of L1 is more hydrophilic than the thiadizole moiety of BTH, we expected to decrease the hydrophilicity by introducing alkyl groups to 10-position of benzotriazole. Among the derivatives whose 10-position were alkyl groups, 3a and 4a which were substituted with methyl and ethyl respectively, all showed efficacy against four out seven tested diseases, which was equal to the commercial plant activator BTH, all showed efficacy against a broad spectrum of diseases. The superimposition profiles in Fig. 3(A) showed that the ester groups of L1 and BTH were located at the same orientation, however, the ester group in L1 was less hydrophobic relative to the thioester group. Thus several more hydrophobic groups at 50-position such as ethyl and aromatic benzene ring were expected to improve the efficacy, and this has been proved by the excellent efficacy of 3e and 4a. An interesting phenomenon was observed that, the efficacy of 3d whose structure was similar to that of 3e, was evidently worse than 3e, the reason may be that the phynolic hydroxy group in 3d may somewhat decrease the hydrophobicity of the ester group. Comparing to 3a and 4a, when the 50-position was substituted with some fluorinecontaining polar groups (3c, 4b, 4c), the efficacy would reduce slightly although these groups are with strong hydrophobicity. Therefore, we concluded that the 50-position substitution was better than the 70-position substitution, and that the introduction of some small ester groups, such as carbomethoxy (3a) and carbethoxy (4a), increased the efficacy while fluorine-containing groups, such as 3c, 4b, 4c, decreased the activity, Furthermore, introduction of alkyl groups at 10-position was beneficial to their activities.

  图 3

  

  图 3  The synthetic route of targeted compounds.

  Figure 3.  The synthetic route of targeted compounds.

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  Next, 3a and 4a, with good in vivo efficacy were chosen for in vitro assays, which were conducted against four diseases including P. infestans, C. cassiicola, M. melonis and R. solani. The results can be seen in Table 4, comparing with the positive controls, all of the novel compounds (3a, 4a) had no direct fungicidal activity in vitro, and that accorded with the feature of the plant activator.

  表 4

  

  表 4  In-vitro anti-microbial activity of the target compounds.

  Table 4.  In-vitro anti-microbial activity of the target compounds.

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  In order to directly reflect the character of benzotriazole derivatives as plant activators, the expression of PR1, which is a symbolic defense gene in SAR was determined by quantitative real time PCR after treatment of compound 3a. The results were shown in Fig. 4. According to the results, compound 3a and BTH both could induce the overexpression of PR1. From the results above, we could conclude that, like BTH, benzotriazole derivatives also induce the disease resistance through SA-dependent SAR. This also demonstrated that the benzotriazole derivatives indeed acted as plant activators, and the detailed mechanism would be investigated in our following studies.

  图 4

  

  图 4  The expression of PR1after treatment of chemicals.

  Figure 4.  The expression of PR1after treatment of chemicals.

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  3.   Conclusion

  In summary, with the help of virtual screening, we have designed and synthesized a novel series of benzotriazole derivatives as plant activators, and their inducing activities were evaluated against seven diseases, and the prediction of the virtual screening results was confirmed by bioassay. In addition, the quantitative real time PCR results showed that benzotriazole derivatives could indeed induce the overexpression of defense gene PR1 in SA-dependent SAR, which was consistent with our expectation. Among them, compounds 3a and 4a were the most potent candidates, just like BTH, it showed efficacy against a broad spectrum of diseases, both showed efficacy against four out seven diseases, and had no direct activity in vitro. So, compounds 3a and 4a have the potential to be exploited as an excellent plant activator with a novel skeleton.

  4.   Experimental

  4.1   Materials

  Melting points were recorded on a Buchi B540 apparatus (Buchi Labortechnik AG, Flawil, Switzerland) and are uncorrected. The NMR spectra were recorded on a Brucker AM-400 (400 MHz) spectrometer with DMSO-d₆ or CDCl₃ as the solvent and TMS as internal standard. Chemical shifts are reported in δ (parts per million) values. The MS spectra were measured with a HRMS Micromass GCT CA 055 spectrometer. Triethylamine was dried over KOH and distilled. All other solvents and chemicals were of reagent grade and used without further purification.

  4.2   Virtual screening and scaffold hopping

  Four bioactive compounds (SA, MeSA, BTH and TDL) were selected as the query templates (Fig. 1). Because the bioactive conformations of these templates are not known presently, the energy minimized three-dimensional conformations of templates computed by LigPrep [32] module in Maestro 9.0 were applied as the query conformations. Then the conformations of all the compounds in the commercially available compound database MayBridge [33] were generated by in-house program Cyndi [40] which is an molecule conformation generation program based on the multi-objective evolution algorithm, with default parameters. Finally, SHAFTS was utilized to align and evaluate the similarity of shape and pharmacophore between the templates and those conformers of the compounds in MayBridge database to find hits with new scaffolds. According to the scores, the top-ranked 500 compounds were retained, and three molecules were selected as candidate leads after bioassay. The implementation of SHAFTS could be found at a freely accessed web-server named ChemMapper [41] (http://lilab.ecust.edu.cn/chemmapper/) with the MayBridge database integrated for automatic and efficient hits discovery based on the metric of 3D SHAFTS similarity.

  4.3   Synthesis of the benzotriazole derivatives [42]

  Compounds 1a-4c were prepared from s1 via five steps including esterification, reduction, diazotization, hydrolysis and reesterification (Scheme 3). Here we took the compound 3b as example to illustrate the general procedure of the synthesis of compounds 1a-4c, which are shown below:

  Synthesis of methyl 4-(methylamino)-3-nitrobenzoate (s2): 3.6 g (18 mmol) 4-methylamino-3-nitro-benzoic acid (s1) was dissolved in 35 mL methanol, 1 mL concentrated sulfuric acid was added. The mixture was heated and maintained at refluxing temperature of 70 ℃ for 6 h, then cooled and poured into 100 mL water, neutralized with saturated solution of sodium bicarbonate, extracted with 100 mL ethyl acetate, washed with 50 mL water and dried with anhydrous sodium sulfate overnight. The filtrate was concentrated by vacuum distillation to obtain yellow solid of s2 3 g, yield 77%. MS calcd. for: C₉H₁₀N₂O₄: 210.06, GC/MS: 210.

  Synthesis of methyl 3-amino-4-(methylamino) benzoate (s3): 1 g (4.7 mmol) s2 was dissolved in 25 mL ethyl acetate, 15 mg 10% Pd/C was added. The mixture was stirred at room temperature for 3 h under hydrogen atmosphere. The filtrate was concentrated by vacuum distillation to obtain pale green oil of s3 800 mg, yield 93%. MS calcd. for: C₉H₁₂N₂O₂: 180.09, GC/MS: 180.

  Synthesis of methyl 1-methyl-1H-benzo[d][1,2,3]triazole-5-carboxylate (s4): 800 mg (4.4 mmol) s3 was dissolved in 10 mL acetic acid, 336 mg (4.8 mmol) sodium nitrite was added slowly. The mixture was stirred for 18 h at room temperature, then poured into 30 mL water, neutralized with saturated solution of sodium bicarbonate, extracted with 50 mL ethyl acetate, washed with 20 mL water, dried with anhydrous sodium sulfate overnight. The filtrate was concentrated by vacuum distillation to obtain brown solid, recrystallized from ethyl acetate and hexane to afford pale brown solid of s4 300 mg, yield 35%. ¹H NMR(400 MHz, CDCl₃): δ 8.78 (s, 1H, ArH), 8.19 (d, 1H, J = 8.8 Hz, ArH), 7.55 (d, 1H, J = 8.8 Hz, ArH), 4.33 (s, 3H, OCH₃), 3.98 (s, 3H, NCH₃); ¹³C NMR (100 MHz, CDCl₃): δ 166.6, 145.6, 135.6, 128.2, 126.3, 122.9, 109.0, 52.4, 34.4. HRMS (EI) calcd. for C₉H₉N₃O₂ [M]⁺: 191.0695, found 191.0697.

  Synthesis of 1-methyl-1H-benzo[d][1,2,3]triazole-5-carboxylic acid (s5): 1 g (4.8 mmol) s4 was suspended in 20 mL water, 20 mL NaOH (2N) aqueous solution was added. The mixture was heated and maintained at 40 ℃ for 4 h, cooled, neutralized with HCl (2 mol/L) and extracted with 50 mL ethyl acetate. The filtrate was concentrated by vacuum distillation to obtain brown solid of s5 500 mg, yield 53%. MS calcd. for: C₈H₇N₃O₂: 177.05, GC/MS: 177.

  Synthesis of ethyl 1-methyl-1H-benzo[d][1,2,3]triazole-5-carboxylate (3b): 100 mg (0.56 mmol) s5 was suspended in 5 mL CH₂Cl₂, 0.5 mL (0.27 mmol) oxalyl chloride was added slowly. The mixture was stirred at room temperature for 1 h. Solvent was removed by vacuum distillation, the residue was dissolved in 5 mL CH₂Cl₂ and added dropwise to a mixture containing 0.6 mmol ethanol, 1.8 mmol triethylamine and 5 mL CH₂Cl₂. The mixture was stirred at room temperature for another 5 h. Then, the solvent was removed by vacuum distillation, the residue was recrystallized from ethyl acetate and hexane to give white solid 3b 65 mg. Yield 44%. mp 104-105 ℃. ¹H NMR(400 MHz, CDCl₃): δ 8.80 (s, 1H, ArH), 8.20 (d, 1H, J = 8.4 Hz, ArH), 7.55 (d, 1H, J = 8.8 Hz, ArH), 4.44 (q, 2H, J = 7.2 Hz, OCH₂), 4.34 (s, 3H, NCH₃), 1.44 (t, 3H, J = 7.2 Hz, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ 166.1, 145.7, 135.6, 128.2, 126.6, 122.8, 108.9, 61.4, 34.4, 14.3. HRMS (ESI) calcd. for C₁₀H₁₁N₃O₂ [M+H]⁺: 205.0851, found 205.0850.

  Synthesis of ethyl 1H-benzo[d][1,2,3]triazole-7-carboxylate (1a): Pale white solid. Yield 83%. mp 159-160 ℃. ¹H NMR(400 MHz, CDCl₃): δ 13.72-13.67 (m, 1H, NH), 8.35 (d, 1H, J = 8.4 Hz, ArH), 8.19 (d, 1H, J = 7.6 Hz, ArH), 7.50-7.45 (m, 1H, ArH), 4.62 (q, 2H, J = 7.2 Hz, OCH₂), 1.50 (t, 3H, J = 7.2 Hz, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ 165.6, 145.6, 132.3, 129.7, 125.2, 123.6, 114.2, 62.1, 14.3. HRMS (ESI) calcd. for C₉H₉N₃O₂ [M+H]⁺: 192.0773, found 192.0765.

  Synthesis of trifluoroethyl 1H-benzo[d][1,2,3]triazole-7-carboxylate (1b): White solid. Yield 76%. mp 251-253 ℃. ¹H NMR(400 MHz, CDCl₃): δ 13.15-13.12 (m, 1H, NH), 8.43 (d, 1H, J = 8.4 Hz, ArH), 8.27 (d, 1H, J = 7.2 Hz, ArH), 7.54 (dd, 1H, J₁ = 8.4 Hz, J₂ = 7.2 Hz, ArH), 4.87 (q, 2H, J = 8.0 Hz, OCH₂CF₃); ¹⁹F NMR (376 MHz, CDCl₃): δ -73.50. HRMS (ESI) calcd. for C₉H₉F₃N₃O₂ [M+H]⁺: 254.0412, found 254.0409.

  Synthesis of hexafluoroisopropyl 1H-benzo[d][1,2,3]triazole-7-carboxylate (1c): White solid. Yield 57%. mp 137-138 ℃. ¹H NMR (400 MHz, CDCl₃): δ 12.97-12.93 (m, 1H, NH), 8.49 (d, 1H, J = 8.0 Hz, ArH), 8.32 (d, 1H, J = 7.2 Hz, ArH), 7.58 (dd, 1H, J₁ = 8.0 Hz, J₂ = 7.2 Hz, ArH), 6.15-6.09 (m, 1H, OCH(CF₃)₂); ¹⁹F NMR (376 MHz, CDCl₃): δ -73.03. HRMS (ESI) calcd. for C₁₀H₅F₆N₃O₂ [M+H]⁺: 313.0286, found 313.0284.

  Synthesis of methyl 1H-benzo[d][1,2,3]triazole-5-carboxylate (2a): Pale white solid. Yield 72%. mp 169-170 ℃. ¹H NMR(400 MHz, CDCl₃): δ 8.59 (s, 1H, ArH), 8.09-8.03 (m, 2H, ArH), 3.94 (s, 3H, OCH₃). HRMS (ESI) calcd. for C₈H₇N₃O₂ [M+H]⁺: 177.0538, found 177.0540.

  Synthesis of ethyl 1H-benzo[d][1,2,3]triazole-5-carboxylate (2b): Pale brown solid. Yield 55%. mp 112-112 ℃. ¹H NMR(400 MHz, CDCl₃): δ 8.77 (s, 1H, ArH), 8.20 (d, 1H, J = 8.4 Hz, ArH), 7.92 (d, 1H, J = 8.8 Hz, ArH), 4.47 (q, 2H, J = 7.2 Hz, OCH₂), 1.46 (t, 3H, J = 7.2 Hz, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ 165.6, 145.6, 132.3, 129.7, 125.2, 123.6, 114.2, 62.1, 14.3. HRMS (ESI) calcd. for C₉H₉N₃O₂ [M+H]⁺: 191.0695, found 191.0697.

  Synthesis of methyl 1-methyl-1H-benzo[d][1,2,3]triazole-5-carboxylate (3a): Brown solid. Yield 35%. mp 177-178 ℃. ¹H NMR(400 MHz, CDCl₃): δ 8.78 (s, 1H, ArH), 8.19 (d, 1H, J = 8.8 Hz, ArH), 7.55 (d, 1H, J = 8.8 Hz, ArH), 4.33 (s, 3H, OCH₃), 3.98 (s, 3H, NCH₃); ¹³C NMR (100 MHz, CDCl₃): δ 166.6, 145.6, 135.6, 128.2, 126.3, 122.9, 109.0, 52.4, 34.4. HRMS (ESI) calcd. for C₉H₉N₃O₂ [M +H]⁺: 191.0695, found 191.0697.

  Synthesis of trifluoroethyl 1-methyl-1H-benzo[d][1,2,3]triazole-5-carboxylate (3c): White solid. Yield 48%. mp 98-99 ℃. ¹H NMR(400 MHz, CDCl₃): δ 8.85 (s, 1H, ArH), 8.20 (d, 1H, J = 8.4 Hz, ArH), 7.60 (d, 1H, J = 8.8 Hz, ArH), 4.77 (q, 2H, J = 8.4 Hz, OCH₂CF₃), 4.36 (s, 3H, NCH₃); ¹³C NMR (100 MHz, CDCl₃): δ 164.4, 145.6, 136.1, 128.2, 124.4, 123.7, 121.6, 109.4, 61.2, 60.8, 14.3. ¹⁹F NMR (376 MHz, CDCl₃): δ -73.62. HRMS (ESI) calcd. for C₁₀H₈F₃N₃O₂ [M+H]⁺: 259.0569, found 259.0573.

  Synthesis of 2-(2-hydroxylbenzoyloxy)-ethyl1-methyl-1Hbenzo[d-1, 2, 3] triazole -5-carboxylate (3d): Pale brown solid. Yield 38%. mp 135-137 ℃. ¹H NMR(400 MHz, CDCl₃): δ 10.63 (s, 1H, OH), 8.83 (s, 1H, ArH), 8.20 (d, 1H, J = 8.8 Hz, ArH), 7.88 (dd, 1H, J₁ = 1.6 Hz, J₂ = 8.0 Hz, ArH), 7.56 (d, 1H, J = 8.4 Hz, ArH), 7.47 (dd, 1H, J₁ = 8.4 Hz, J₂ = 8.8 Hz, ArH), 6.98 (d, 1H, J = 8.0 Hz, ArH), 6.89 (dd, 1H, J₁ = 8.0 Hz, J₂ = 7.2 Hz, ArH), 4.73 (s, 4H, CH₂CH₂), 4.34 (s, 3H, NCH₃); ¹³C NMR (100 MHz, CDCl₃): δ 169.9, 165.8, 161.7, 145.7, 136.0, 135.8, 130.0, 128.2, 125.7, 123.2, 119.3, 117.6, 112.0, 109.2, 63.0, 62.8, 34.4. HRMS (ESI) calcd. for C₁₇H₁₅N₃O₅ [M+H]⁺: 341.1012, found 341.1017.

  Synthesis of 2-(4-chlorobenzoyloxy)-ethyl-1-methyl-1H-benzo [d][1,2,3] triazole -5-carboxylate (3e): White solid. Yield 44%. mp 152.2-152.6 ℃. ¹H NMR(400 MHz, CDCl₃): δ 8.80 (s, 1H, ArH), 8.19 (d, 1H, J = 7.6 Hz, ArH), 7.99 (d, 2H, J = 8.4 Hz, ArH), 7.55 (d, 1H, J = 8.8 Hz, ArH), 7.40 (d, 2H, J = 8.8 Hz, ArH), 4.71 (s, 4H, CH₂CH₂), 4.33 (s, 3H, NCH₃); ¹³C NMR (100 MHz, CDCl₃): δ 165.8, 165.5, 145.6, 139.6, 135.8, 131.1, 128.8, 128.2, 125.8, 123.1, 109.2, 63.0, 62.9, 34.4. HRMS (ESI) calcd. for C₁₇H₁₄C_lN₃O₄ [M+H]⁺: 359.0673, found 359.0674.

  Synthesis of ethyl 1-ethyl-1H-benzo[d][1,2,3]triazole-5-carboxylate (4a): Pale white solid. Yield 83%. mp 58-60 ℃. ¹H NMR (400 MHz, CDCl₃): δ 165.8, 165.5, 145.6, 8.83 (s, 1H, ArH), 8.21 (d, 1H, J = 8.4 Hz, ArH), 7.58 (d, 1H, J = 8.8 Hz, ArH), 4.74 (q, 2H, J = 7.6 Hz, OCH₂), 4.46 (q, 2H, J = 7.2 Hz, NCH₂), 1.68 (t, 3H, J = 7.6 Hz, OCH₂CH₃), 1.46 (t, 3H, J = 7.2 Hz, NCH₂CH₃); ¹³C NMR (100 MHz, CDCl₃): δ 165.8, 165.5, 145.6, 166.1, 145.8, 134.8, 128.0, 126.5, 122.8, 109.0, 61.3, 43.4, 14.9, 14.3. HRMS (ESI) calcd. for C₁₁H₁₃N₃O₂ [M+H]⁺: 220.1086, found 220.1077.

  Synthesis of trifluoroethyl 1-ethyl-1H-benzo[d][1,2,3]triazole-5-carboxylate (4b): White solid. Yield 60%. mp 106-107 ℃. ¹H NMR (400 MHz, DMSO-d₆): δ 165.8, 165.5, 145.6, 8.88 (s, 1H, ArH), 8.21 (d, 1H, J = 8.8 Hz, ArH), 7.62(d, 1H, J = 8.8 Hz, ArH), 4.82-4.72 (m, 4H, OCH₂CF₃, NCH₂), 1.69 (t, 3H, J = 7.2 Hz, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ 164.5, 145.8, 135.3, 128.0, 124.3, 123.9, 109.4, 61.2, 60.8, 43.5, 14.9. ¹⁹F NMR (376 MHz, CDCl₃): δ -73.63. HRMS (ESI) calcd. for C₁₁H₁₀F₃N₃O₂ [M +H]⁺: 273.0728, found 273.0725.

  Synthesis of hexafluoroisopropyl 1-ethyl-1H-benzo[d][1,2,3] triazole-5-carboxylate (4c): White solid. Yield 58%. mp 63-65 ℃. 1H NMR(400 MHz, CDCl₃): δ 8.94 (s, ¹H, ArH), 8.23 (d, 1H, J = 8.8 Hz, ArH), 7.66 (d, 1H, J = 8.8 Hz, ArH), 6.11-6.04 (m, 1H, OCH(CF₃)₂), 4.80-4.74 (q, 2H, J = 7.6 Hz, NCH₂), 1.69 (t, 3H, J = 7.6 Hz, CH₃); ¹³C NMR (100 MHz, CDCl₃): δ 162.9, 145.7, 135.6, 128.2, 124.7, 122.6, 109.8, 67.5, 67.2, 66.8, 43.5, 14.9. ¹⁹F NMR (376 MHz, CDCl₃): δ -73.16. HRMS (ESI) calcd. for C₁₂H₉F₆N₃O₂ [M+H]⁺: 341.0599, found 341.0596.

  4.4   Biological assay screening

  The inducing activity of the target compounds was evaluated according to the following procedures which referred to the work we did before [3, 4].

  4.4.1   In-vivo screening

  During the in-vivo screening of lead compounds, Corynespora cassiicola, Cladosporium cucumerinum, Fusarium oxysporum, and Pseudoperonospron cubensis were used on cucumber. For the screening of the target compounds, Mycosphaerella melonis, Corynespora cassiicola, Pseudomonas syringae pv. lachrymans, Botrytis cinerea, and Fusarium oxysporum were used on cucumber, Phytophthora infestans was used on tomato, and Rhizoctonia solani was used on rice. Among them, M. melonis, C. cassiicola, P. syringae pv. lachrymans, P. infestans, and F. oxysporum were inoculated by leaf spray, and B. cinerea, R. solani were inoculated by irrigation. Additionally, commercialized 50% kresoxim-methyl (WG), 50% Dimethomorph (WP), 75% chlorothalonil (WP), 20% bismerthlazol (WP), 5% validamycin A (WP), 50% procymidone (WP), and 70% Mildothane (WP) were used as positive controls against M. melonis, P. infestans, C. cassiicola, P. syringae pv. lachrymans, R. solani, B. cinerea and F. oxysporum, respectively. BTH was used as a positive plant activator control. All test compounds and the positive control BTH were dissolved in acetone, and diluted with water to a concentration of 100 mg/L, solvent was used as blank control. During the test, the agents were sprayed on the plant leaves, the first application was conducted at proper life stage of plants, another two applications were conducted every seven days. Then, the inoculations were conducted three days after the third application. During the screening, the plants were cultured in green house maintaining the temperature between 24-26 ℃. Each test was repeated four times, and in every replication, five plants were used for the test. No other germicide or insecticide was used during the screening. After the disease conditions of controls became stable, the number of lesions on test plant leaves were recorded, and classified into six levels as below:

  Level 0: no lesion;

  Level 1: lesions occupied less than 5% of the entire leaf area;

  Level 2: lesions occupied 6%-10% of the entire leaf area;

  Level 5: lesions occupied 11%-25% of the entire leaf area;

  Level 7: lesions occupied 26%-50% of the entire leaf area;

  Level 9: lesions occupied more than 50% of the entire leaf area.

  The disease index and efficacy were calculated according to the Eqs. (1) and (2) respectively.

  Where I is the disease index, Y is the leaf number of each level, C is the level number of the corresponding lesion, N is the number of the total investigated leaves. I_c is the disease index of the control plants, and I_a is the disease index of the treated plants.

  4.4.2   In-vitro assay

  During the in-vitro assay, M. melonis, C. cassiicola, P. infestans and R. solani were used. All test compounds were dissolved in N, N-dimethylformamide (DMF) to prepare a stock solution with a concentration of 1000 mg/L. Then, 1 mL stock solution and 9 mL potato dextrose agar (PDA) culture media were mixed thoroughly in a petri dish to prepare a working solution of 100 mg/L. The PDA medium was thoroughly mixed by turning around the petri dish in the sterilized operation desk 5 times to scatter the compounds in PDA evenly. After solidification, the micro-organism was inoculated on the plate and incubated in the culture tank at 24-26 ℃, and solvent was used as blank control. After two days, the diameter of the bacterial colony was measured, and the inhibition efficacy was calculated according to the Eq. (3). Three replications were conducted for each test.

  Where D_c is the average diameter of the water control plants, and D_a is the average diameter of the treated plants.

  4.4.3   Quantitative real time PCR

  Total RNAs were extracted from 3-week old Arabidopsis thaliana after treatment of compound 3a, BTH and solvent using Trizol reagent (Ambion). The concentrations of all chemicals were 100 mg L^-1. The RNAs were quantitated using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific) and were used to synthesize cDNAs using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). Quantitative real-time PCR was performed with SYBR Green (Takara) using a CFX Real-Time System (Bio-Rad). ACTIN2 mRNA was detected as an internal control for data normalization. Experiments were repeated three times with similar results.

  Acknowledgments

  This work was financially supported by the National Basic Research Program of China (973 Program, No. 2010CB126100), the National High Technology Research and Development Program of China (863 Program, No. 2011AA10A207), the Shanghai Leading Academic Discipline Project (B507), and the Fundamental Research Funds for the Central Universities.

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• 

  Figure 1  Structures of reported plant activators.

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  Scheme 1  Virtual screening process.

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  Figure 2  Structures of lead compounds.

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  Scheme 2  Design of target compounds.

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  Scheme 3  The synthetic route of targeted compounds.

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  Figure 3  The synthetic route of targeted compounds.

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  Figure 4  The expression of PR1after treatment of chemicals.

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  Table 1.  In-vivo activities of the lead compounds.

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  Table 2.  In-vivo activities of the target compounds.

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  Table 3.  The similarity scores of derivatives of benzotriazole.

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  Table 4.  In-vitro anti-microbial activity of the target compounds.

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•