The discovery of new scaffold of plant activators: From salicylic acid to benzotriazole

Article abstract of DOI:10.1016/j.cclet.2017.02.004

Started from salicylic acid (SA) and related commercialized plant activators, based on molecular three-dimensional shape and pharmacophore similarity comparison (SHAFTS), a new lead compound benzotriazole was predicted and a series of benzotriazole derivatives were designed and synthesized. The bioassay showed that benzotriazole had high activity against a broad spectrum of diseases including fungi and oomycetes in vivo, but no activity in vitro. And the introduction of proper groups at the 1’-position and 5’-position was beneficial to the activity. So, they had the potential to be exploited as novel plant activators.

Full text of DOI:10.1016/j.cclet.2017.02.004

Accepted Manuscript
Title: The discovery of new scaffold of plant activators: From
salicylic acid to benzotriazole
Authors: Kang Chang, Jian-Qin Chen, Yan-Xia Shi, Mei-Jian
Sun, Peng-Fei Li, Zhen-Jiang Zhao, Wei-Ping Zhu, Hong-Lin
Li, Yu-Fang Xu, Bao-Ju Li, Xu-Hong Qian
PII:
S1001-8417(17)30048-7
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.cclet.2017.02.004
CCLET 3977
To appear in:
Chinese Chemical Letters
Received date:
Accepted date:
2-2-2017
2-2-2017
Please cite this article as: Kang Chang, Jian-Qin Chen, Yan-Xia Shi, Mei-Jian Sun,
Peng-Fei Li, Zhen-Jiang Zhao, Wei-Ping Zhu, Hong-Lin Li, Yu-Fang Xu, Bao-Ju Li,
Xu-Hong Qian, The discovery of new scaffold of plant activators: From salicylic acid to
benzotriazole, Chinese Chemical Letters http://dx.doi.org/10.1016/j.cclet.2017.02.004
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<DOCHEAD>Original article
<AT>The discovery of new scaffold of plant activators: from salicylic acid to benzotriazole
<AU>Kang Changa, Jian-Qin Chena, Yan-Xia Shic, Mei-Jian Sunb, Peng-Fei Lia, Zhen-Jiang Zhaob, Wei-
Ping Zhua, Hong-Lin Lib, Yu-Fang Xua* ##Email##yfxu@ecust.edu.cn##/Email##, Bao-Ju Lic*
##Email##libaoju@caas.cn##/Email##, Xu-Hong Qiana* ##Email##xhqian@ecust.edu.cn##/Email##
<AU>
<AFF>aState Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical Biology,
School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
<AFF>bState Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of New Drug Design,
School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China
<AFF>cInstitute of Vegetables and Flowers, Chinese Academy of Agricultural Science, Beijing 100081,
China
<AFF>
<PA>Corresponding authors.
<ABS-Head><ABS-HEAD>Graphical abstract
<ABS-P>► A series of novel plant activators possessing benzotriazole scaffold was developed with the help of SHAFTS.
<ABS-HEAD>ABSTRACT
<ABS-P>Started from salicylic acid (SA) and related commercialized plant activators, based on molecular
three-dimensional shape and pharmacophore similarity comparison (SHAFTS), a new lead compound
benzotriazole was predicted and a series of benzotriazole derivatives were designed and synthesized. The
bioassay showed that benzotriazole had high activity against a broad spectrum of diseases including fungi
and oomycetes in vivo, but no activity in vitro. And the introduction of proper groups at the 1'-position and
5'-position was beneficial to the activity. So, they had the potential to be exploited as novel plant
activators.
<KWD>Keywords: Plant activator Systemic acquired resistance Salicylic acid Benzotriazole Virtual screening
<H1>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.
So, it’s interesting 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].
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.
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.
<H1>2. Results and discussion
<H2>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 7’-position of BTH. Therefore, the introduction of an additional hydrophobic group at 5’-position of L1
could generate a hydrophobic centroid resembling that of BTH at 7’-position. The substituted alkyl group
at 1’-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 1’-position as well as 5’-position
of L1 would benefit the in vivo efficacy of the derivatives designed based on the scaffold of lead L1.
<H2>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.
<H2>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.
For series of benzotriazole derivatives, all compounds with carboxyl substitution at the 7’-position (1a-c) exhibited weak SAR-
inducing activities, only showed efficacy against one out seven tested diseases. However, comparing 1a with 2b, when the carboxyl
was moved to the 5’-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 5’- position, which was same to the lead compound L1. Among all the derivatives whose
carboxyl were substituted at 5’-position, 3a achieved the highest hybrid score of 1.465 (Table 3), and showed excellent efficacy.
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 1’-position of benzotriazole. Among the
derivatives whose 1’-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 5’-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 5’-position was substituted with some fluorine-containing
polar groups (3c, 4b, 4c), the efficacy would reduce slightly although these groups are with strong
hydrophobicity. Therefore, we concluded that the 5’-position substitution was better than the 7’-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 1’-position was beneficial to their activities.
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.
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.
<H1>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.
<H1>4. Experimental
<H2>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.
<H2>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.
<H2>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 oC 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 oC for 4 h, cooled, neutralized with HCl (2N) and extracted with 50 mL ethyl acetate. The filtrate was
concentrated by vacuum distillation to obtain brown solid of s5 500mg, 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 oC. 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): δ166.1, 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 oC. 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 oC. 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 oC. 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 oC. 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 oC. 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 oC. 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 oC. 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-1H-benzo[d]-[1,2,3] triazole -5-carboxylate (3d):
Pale brown solid. Yield 38%. mp 135-137 oC. 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 oC. 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
C17H14ClN3O4 [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 oC. 1H NMR(400 MHz，CDCl3): δ 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 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 oC. 1H NMR(400 MHz，DMSO-d6): δ 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 oC. 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.
<H2>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].
<H3>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 oC. 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 equations (1) and (2) respectively.

Ic  Ia
Ic
(YC)
I  
N 9
Efficacy 
100
(1)
(2)
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. Ic is the disease index of the control plants, and Ia is
the disease index of the treated plants.
<H3>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 oC, 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
equation (3). Three replications were conducted for each test.
Dc  Da
Dc
(3)
Efficacy 
Where Dc is the average diameter of the water control plants, and Da is the average diameter of the treated plants.
<H3>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.
Acknowledgment
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>Fig. 1. Structures of reported plant activators
<Figure>Fig. 2. Structures of lead compounds
<Figure>Fig. 3. (A) The superimposition of BTH and the lead L1. (B) Proposed key pharmacophore features for the benzotriazole analogs as potent plant
activators.
<Figure>Fig. 4. The expression of PR1after treatment of chemicals.
<Figure>Scheme. 1. Virtual screening process
<Figure>Scheme 2. Design of target compounds
<Figure>Scheme 3. The synthetic route of targeted compounds
<Table>Table 1. In-vivo activities of the lead compounds
Compd.a
Efficacy (%)b

C. cassiicola
C. cucumerinum
62.93
F. oxysporum
14.08
P. cubensis
3.92
L1
L2
L3
57.04
0
67.13
23.03
-12.50
1.71
29,86
13.61
ND
70% Mildothane
(WP)
60.55
70.98
26.76
28.47
75% Chlorothalonil
(WP)
64.06
62.28
34.82
80.36
37.35
59.61
84.51
32.24
-4.23
66.67
93.46
0
50%
(WP)
Dimethomorph
5% Validamycin A
(WP)
aThe concentration of compounds is 100 mg/L;
bND means not detected, efficacy is the average of all repeated tests.
<Table>Table 2. In-vivo activities of the target compounds
Compd.a
Efficacy (%)b
MM
CC
PL
PI
RS
BC
FO
1a
87.22
100.00
85.28
89.74
100.00
95.06
93.88
72.61
80.02
88.92
98.61
93.06
99.72
88.06
98.43
-1.64
-14.43
1.64
-9.32
23.58
8.73
39.61
11.02
1.37
-6.40
-2.86
0.10
15.05
36.16
-2.24
3.45
19.37
3.92
1b
1c
-2.13
58.97
-7.50
73.46
82.05
89.74
87.18
76.92
74.36
-7.50
0.56
2a
ND
NDc
8.33
13.35
-4.63
52.52
16.91
3.86
2b
79.02
18.28
55.12
18.77
7.75
37.91
16.34
18.80
37.31
7.43
30.32
47.92
19.44
9.72
16.71
11.24
2.35
3a
3b
3c
13.31
-5.44
4.56
3d
16.67
93.75
70.70
24.24
8.52
-5.64
44.21
-5.81
-6.40
-5.67
16.06
3e
18.24
54.75
14.75
58.14
-8.20
34.22
17.92
-13.92
5.19
4a
-1.58
4.63
4b
4c
-4.41
46.80
BTH
75.59
64.98
19.37
50% Kresoxim-methyl(WG)
75% Chlorothalonil(WP)
90.16

20% Bismerthlazol(WP)
50% Dimethomorph(WP)
5% Validamycin A(WP)
50% Procymidone(WP)
70% Mildothane(WP)
67.80
96.78
83.38
41.87
75.14
aBTH represents S-methyl benzo[1,2,3]thiadiazole-7-carbothioate and was synthesized in our lab; bMicro-organisms used: Mycosphaerella melonis (MM),
Corynespora cassiicola (CC), Pseudomonas syringae pv．Lachrymans (PL), Phytophthora infestans (PI), Rhizoctonia solani (RS), Botrytis cinerea (BC), and
Fusarium oxysporum (FO); efficacy is the average of all repeated tests.
cND means not detected.
<Table>Table 3. The similarity scores of derivatives of benzotriazole
Shape
Number
Compd.
Hybird score
Feature score
score
1
1.465
1.428
1.382
1.370
1.361
1.343
1.330
1.249
1.084
1.056
0.8827
0.5822
0.5434
0.5812
0.4885
0.5384
0.5449
0.458
3a (or L1)
2
3b
3c
2a
4a
4b
2b
4c
3e
3d
0.8850
0.8007
0.8812
0.8229
0.7977
0.8721
0.7435
0.6553
0.6481
3
4
5
6
7
8
0.5053
0.4290
0.4076
9
10
<Table>Table 4. In-vitro anti-microbial activity of the target compounds
Efficacy (%)b
compda
MM
-2
1
CC
15
12
20
PI
22
0
RS
9
3a
4a
2
BTH
4
12
48
50% Kresoxim-methyl
71
(WG)
75% Chlorothalonil
(WP)
21
50% Dimethomorph
(WP)
80
5% Validamycin A
(WP)
50
aBTH represents S-methyl benzo[1,2,3]thiadiazole-7-carbothioate and was synthesized in our lab; bMicro-organisms used: Mycosphaerella melonis (MM),
Corynespora cassiicola (CC), Phytophthora infestans (PI), Rhizoctonia solani (RS); efficacy is the average of all repeated tests.

TDENDOFDOCTD