5h-[1,2,4]triazinyl[5,6-b]indole derivatives containing a disulfide structure, and preparation method and application thereof

Abstract

This invention discloses a class of 5H-[1,2,4]triazinyl[5,6-b]indole-3-thione derivatives with disulfide structures, using 5H-[1,2,4]triazinyl[5,6-b]indole structures as lead compounds and alkyl or aryl thiols as linking groups. Different pharmacophores are then added to these compounds to synthesize a series of 5H-[1,2,4]triazinyl[5,6-b]indole derivatives. These compounds exhibit excellent inhibitory effects against plant pathogenic bacteria such as *Bacillus thuringiensis* (rice bacterial blight) and *Citrus canker* (citrus canker). Some compounds show excellent inhibitory activity against *Bacillus thuringiensis*, with EC50... 50 The concentration can reach 0.77–0.85 μg / mL; some compounds showed excellent inhibitory activity against *Citrus canker*, the causal agent of citrus canker, with EC50 levels of [missing information]. 50 The concentration is 2.62 μg / mL. The disulfide structure 5H-[1,2,4]triazine[5,6-b]indole derivative disclosed in this invention can be used to prepare pesticides against plant pathogenic bacteria and fungi, and has extremely high research value.

Description

Technical Field

[0001] This invention relates to the field of medicinal chemistry, specifically to 5H-[1,2,4]triazine[5,6-b]indole derivatives containing disulfide structures, their preparation methods, and applications. Background Technology

[0002] Bacterial plant diseases are among the most destructive plant diseases, affecting not only the healthy and stable development of agriculture but also posing a significant threat to food safety. Among them, bacterial diseases caused by *Xanthomonas oryzae* pv. *Oryzae* (Xoo), *Xanthomonas axonopodis* pv. *Citri* (Xac), and *Pseudomonas syringae* pv. *Actinidiae* (Psa) result in agricultural losses of hundreds of millions of dollars annually.

[0003] The application of chemical pesticides has long been a primary measure for controlling bacterial diseases in plants, offering advantages such as broad-spectrum bactericidal activity, rapid efficacy, and low cost. However, long-term use of chemical pesticides inevitably leads to pesticide residues and environmental pollution, posing significant challenges to human health and ecological balance. Furthermore, many commercially available pesticides have developed varying degrees of resistance. Therefore, there is an urgent need to find highly active, low-resistance compounds for combating plant pathogenic bacteria, and to develop green new pesticides with independent intellectual property rights, providing candidate drugs for the control of bacterial diseases in crops.

[0004] Currently, there are numerous studies on the bioactivity of 5H-[1,2,4]triazinyl[5,6-b]indole compounds and nitrogen-containing heterocyclic compounds. Therefore, how to continue using the 5H-[1,2,4]triazinyl[5,6-b]indole structure as a lead compound and perform skeletal transitions using disulfide groups to find highly effective bactericidal target compounds is an urgent problem to be solved. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a triazine compound containing a disulfide structure, its preparation method, and its application. Using 5H-[1,2,4]triazinyl[5,6-b]indole-3-thione as the parent structure, a series of triazine compounds containing a disulfide structure are synthesized using a skeletal transition method. Their biological activities are investigated, providing an important scientific basis for the research and development and creation of new pesticides.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention discloses a triazine compound containing a disulfide structure, with the general structural formula shown in formula (I) below:

[0008]

[0009] Wherein, R1 is selected from any substituted or unsubstituted alkyl, any substituted or unsubstituted cycloalkyl, and any substituted or unsubstituted aryl or benzyl; R2 is selected from methyl; R3 is selected from methyl or fluorine; R4, R5 and R6 are each independently selected from fluorine or chlorine.

[0010] Preferably, R1 is selected from C2-C8 alkyl, substituted or unsubstituted C6-C7 aryl, substituted or unsubstituted benzyl, wherein the substitution refers to one or more of halogen, methyl, methoxy, and trifluoromethyl; R2 is selected from methyl; R3 is selected from methyl or fluorine; and R4, R5 and R6 are each independently selected from fluorine or chlorine.

[0011] Preferably, R1 is selected from ethyl, propyl, allyl, n-butyl, n-hexyl, n-heptyl, octyl, decanyl, dodecyl, substituted or unsubstituted phenyl, substituted or unsubstituted benzyl, wherein the substitution refers to one or more of methyl, methoxy, halogen, and trifluoromethyl; R2 is selected from methyl; R3 is selected from methyl or fluorine; and R4, R5, and R6 are each independently selected from fluorine or chlorine.

[0012] This invention discloses a triazine compound containing a disulfide structure, specifically including the following compounds:

[0013]

[0014] The present invention discloses a method for preparing triazine compounds containing a disulfide structure, comprising the following steps:

[0015] (1) Indigo and potassium carbonate were dissolved in water by stirring. Thioaminourea was slowly added and the mixture was refluxed and stirred at 100°C. After the reaction was completed, the mixture was filtered. The pH of the liquid was adjusted with acetic acid. At the same time, solid precipitated out. The mixture was filtered again and washed with a large amount of water to obtain the compound.

[0016] (2) Dissolve the compound obtained in step (1) in dimethyl sulfoxide, then add it to a dichloromethane solution. After stirring until homogeneous, slowly add 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and stir the reaction under ice bath conditions. After the reaction is complete, filter the mixture to obtain the target compound. The chemical equation is as follows:

[0017]

[0018] A composition comprising a compound of formula (I) above, a stereoisomer thereof, a salt thereof, or a solvate thereof, or a prepared compound of formula (I) above, a stereoisomer thereof, a salt thereof, or a solvate thereof, and an agricultural adjuvant, fungicide, insecticide, or herbicide; the dosage form of the composition is selected from one or more of the following: emulsifiable concentrate (EC), powder (DP), wettable powder (WP), granules (GR), aqueous solution (AS), suspension concentrate (SC), ultra-low volume spray (ULV), soluble powder (SP), microcapsule (MC), fumigant (FU), emulsion (EW), or water-dispersible granules (WG).

[0019] The application of the compound represented by formula (I), or the prepared compound represented by formula (I), or the composition thereof, in the prevention and control of agricultural pests and diseases. The agricultural pests and diseases are bacterial and fungal plant diseases. These include plant pathogenic bacteria such as *Rhizoctonia solani* (rice bacterial leaf blight pathogen), *Xanthomonas oryzae* (rice leaf spot pathogen), *Xanthomonas tarda* (carpet pathogen), *Xanthomonas spp.* (rapeseed pathogen), *Pseudomonas syringae* (tomato leaf spot pathogen), *Pseudomonas syringae* (kiwifruit pathogen), *Ralstonia solanacearum* (cuckoo angular leaf spot pathogen), *Rhizoctonia solani* (soybean pathogen), or *Corynebacterium micranthum* (canker pathogen), and plant pathogenic fungi such as *Staphylococcus aureus*, *Fusarium oxysporum*, *Colletotrichum candida*, *Colletotrichum gloeosporioides*, *Colletotrichum gloeosporioides*, *Colletotrichum gloeosporioides*, and *Colletotrichum sorghum*.

[0020] Preferably, the bacterial disease is any one of rice bacterial blight, citrus canker, and kiwifruit canker. Preferably, the fungal disease is any one of *Botrytis cinerea*, *Fusarium graminearum*, and *Rhizoctonia solani*.

[0021] The present invention also provides a method for controlling agricultural pests and diseases using the triazine compound containing the disulfide structure and the composition thereof, comprising applying the triazine compound containing the disulfide structure thereof or its salt or its solvate, or the composition thereof, to the harmful substance or its living environment.

[0022] The term "alkyl" as used here refers to a straight-chain saturated or unsaturated hydrocarbon group having a specific number of carbon atoms. For example, "C2-8 alkyl" (or allyl) refers to C2, C3, C4, C5, C6, C7, and C8 alkyl groups.

[0023] The term "substituted" as used herein refers to the substitution of one or more hydrogen atoms on a specified atom or group by a chosen specified group, provided that the substitution does not exceed the general valence of the specified atom. Unless otherwise specified, substituents are named to the central structure. For example, it can be understood that when (cycloalkyl)alkyl is a possible substituent, the substituent's connection point to the central structure is within the alkyl moiety. Cyclic double bonds as used herein are double bonds formed between two adjacent ring atoms (e.g., C=C, C=N, or N=N). When substitution is mentioned, especially polysubstitution, it refers to the substitution of multiple substituents at various positions on a specified group, such as dichlorophenyl referring to 1,2-dichlorophenyl, 1,3-dichlorophenyl, and 1,4-dichlorophenyl.

[0024] Combinations of substituents and / or variables are permitted only when these combinations yield stable compounds or useful synthetic intermediates. A stable compound or stable structure implies that the compound is sufficiently stable to be isolated from the reaction mixture with useful purity, subsequently formulated to form an effective therapeutic agent. Preferably, the compound does not currently contain -F / -Cl halogens, N-CH3, allyl, aromatic, benzyl, or C3-C8 alkane chains.

[0025] The term "aryl" refers to a monocyclic aromatic hydrocarbon group having 6 or 7 carbon atoms in the ring moiety, such as phenyl and benzyl.

[0026] The term "halogen" or "halogen atom" refers to chlorine and fluorine.

[0027] Unless otherwise specified, the compounds of this invention are understood to include both their free state and their salts. The term "salt" means an acidic and / or basic salt formed from an inorganic and / or organic acid and base. Additionally, the term "salt" may include zwitterions (internal salts), such as when a compound of formula I contains a basic segment such as an amine or pyridine or imidazole ring, and an acidic segment such as a carboxylic acid. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, such as acceptable metal and amine salts, wherein the cation does not significantly contribute to toxicity or the biological activity of the salt. However, other salts may be useful, such as those prepared using separation or purification steps, and are therefore also included within the scope of this invention.

[0028] Compared with existing technologies, this invention has the following advantages: This invention uses a 5H-[1,2,4]triazinyl[5,6-b]indole-3-thione structure as a lead compound, and then uses alkyl or aryl thiols as linking groups, on which different pharmacophores are added. A series of 5H-[1,2,4]triazinyl[5,6-b]indole derivatives containing disulfide structures were synthesized. These compounds exhibit excellent inhibitory effects on plant pathogenic bacteria such as *Bacillus thuringiensis* (rice bacterial blight) and *Citrus canker* (citrus canker).

[0029] (1) In in vitro experiments, the target compounds showed good antibacterial activity against *Xanthomonas oryzae* pv. oryzae (Xoo) and *Xanthomonas citrus canker*. Compounds 1 and 33 showed excellent inhibitory activity against *Xanthomonas oryzae* pv. oryzae (Xoo) with EC50 values ​​of 100%. 50 The concentration was 0.77-0.85 μg / mL; Compound 1 showed excellent inhibitory activity against Xanthomonas axonopodis pv. citri (Xac) , with an EC50 concentration of 0.77-0.85 μg / mL. 50 It has a concentration of 2.62 μg / mL and can be used to prepare pesticides against pathogenic bacteria in plants.

[0030] (2) The target compounds exhibited good inhibitory activity against plant pathogenic bacteria such as *Bacillus thuringiensis*, the causal agent of rice bacterial blight. Most of the compounds containing an ethyl group showed good inhibitory activity against the EC50 of *Bacillus thuringiensis*. 50 All were below 10, especially compound 1 against the EC50 of rice bacterial blight pathogen. 50 The concentration of compound 16 was 0.77 μg / mL, and its EC50 concentration against *Citrus aurantium* was [missing value]. 50 The concentration was 3.43 μg / mL; Compound 6 showed an EC50 concentration against *Actinidia kiwifruit* causal agent. 50 The concentration was 2.86 μg / mL. The target compounds also exhibited certain inhibitory activity against plant pathogenic fungi. At a concentration of 50 μg / mL, all target compounds showed good bioactivity against *Rhizoctonia solani* (rice). Among them, compound 33 showed excellent inhibitory activity against *Rhizoctonia solani*, *Botrytis cinerea*, and *Fusarium graminearum* (wheat scab), with inhibition rates of 92.35%, 75.81%, and 61.33%, respectively. Therefore, these compounds can be used as novel pesticides against plant pathogenic bacteria and fungi, possessing extremely high research value and providing an important scientific basis for the research and development of new pesticides. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0032] Example 1

[0033] (1) Preparation of intermediate 5H-[1,2,4]triazine[5,6-b]indole-3-thione:

[0034]

[0035] In a 250 mL round-bottom flask, indigo (3.00 g, 20 mmol) and potassium carbonate (4.23 g, 30 mmol) were added, followed by water and stirring. Then, aminothiourea (1.86 g, 20 mmol) was added, and the mixture was refluxed at 100 °C with stirring. The reaction was monitored by TLC. After the reaction was complete, the solid and liquid phases were separated, filtered, and washed with water. The mixture was dried to give the intermediate 5H-[1,2,4]triazinyl[5,6-b]indole-3-thione, in yield 46.50%. Its NMR data are as follows: 1 H NMR (400MHz, DMSO-d6) δ13.48(s,1H,-NH),13.10(s,1H,-NH),7.32(dd,J=7.8,1.6Hz,1H,Ar-H),7.09(ddd ,J=8.6,7.1,1.6Hz,1H,Ar-H),6.68(dd,J=8.2,1.2Hz,1H,Ar-H),6.53(ddd,J=8.1,7.2,1.2Hz,1H,Ar-H); 13 C NMR(101MHz,DMSO-d6)δ173.41,153.33,148.17,147.74,131.03,130.80,115.89,115.29,115.22; HRMS(ESI)[M+H] + calcd for C9H7N4S:203.0386,found:203.0378.

[0036] (2) Preparation of intermediate 5-methyl-5H-[1,2,4]triazine[5,6-b]indole-3-thione:

[0037]

[0038] In a 250 mL round-bottom flask, 1-methylindigo (3.00 g, 20 mmol) and potassium carbonate (3.90 g, 30 mmol) were added, followed by water and stirring. Then, aminothiourea (1.71 g, 20 mmol) was added, and the mixture was refluxed at 100 °C with stirring. The reaction was monitored by TLC. After the reaction was complete, the solid and liquid phases were separated, filtered, and washed with water. The mixture was dried to give the intermediate 5-methyl-5H-[1,2,4]triazinyl[5,6-b]indole-3-thione, in yield 32.70%. Its NMR data are as follows: 1 H NMR (600MHz, DMSO-d6) δ8.12–7.95(m,1H,Ar-H),7.78–7.59(m,2H,Ar-H),7.48–7.33(m,1H,Ar-H),3.65(s,3H,-CH3). 13C NMR(151MHz,DMSO-d6)δ179.51,148.69,144.82,135.81,132.30,124.00,122.20,117.85,112.09,27.98.HRMS(ESI)[M+H] + calcd for C 10 H9N4S:217.0542,found:217.0535.

[0039] (3) Preparation of intermediate 6-methyl-5H-[1,2,4]triazinyl[5,6-b]indole-3-thione:

[0040]

[0041] In a 250 mL round-bottom flask, 7-methylindigo (3.00 g, 20 mmol) and potassium carbonate (3.90 g, 30 mmol) were added, followed by water and stirring. Then, aminothiourea (1.71 g, 20 mmol) was added, and the mixture was refluxed at 100 °C with stirring. The reaction was monitored by TLC. After the reaction was complete, the solid and liquid phases were separated, filtered, and washed with water. The mixture was dried to give the intermediate 6-methyl-5H-[1,2,4]triazinyl[5,6-b]indole-3-thione, in yield 33.50%. Its NMR data are as follows: 1 H NMR (600MHz, DMSO-d6) δ12.45(s,1H,-NH),7.34(d,J=114.6Hz,1H,Ar-H),7.05(d,J=44.8Hz,2H,Ar-H),6.49(s,1H,Ar-H),2.09(s,3H,-CH3); 13 C NMR(151MHz,DMSO-d6)δ173.65,153.45,148.84,145.57,131.74,128.86,123.59,122.54,119.70,115.28,18.45.HRMS(ESI)[M+H] + calcd forC 10 H9N4S:217.0542,found:217.0533.

[0042] (4) Preparation of intermediate 6-fluoro-5H-[1,2,4]triazine[5,6-b]indole-3-thione:

[0043]

[0044] In a 250 mL round-bottom flask, 7-fluoroindigo (3.00 g, 20 mmol) and potassium carbonate (3.80 g, 30 mmol) were added, followed by water and stirring. Then, aminothiourea (1.67 g, 20 mmol) was added, and the mixture was refluxed at 100 °C with stirring. The reaction was monitored by TLC. After the reaction was complete, the solid and liquid phases were separated, filtered, and washed with water. The mixture was dried to give the intermediate 6-fluoro-5H-[1,2,4]triazinyl[5,6-b]indole-3-thione, in yield 40.00%. Its NMR data are as follows: 1 H NMR (600MHz, DMSO-d6) δ7.82 (d, J=7.8Hz, 1H, Ar-H), 7.53 (dd, J=10.9, 8.2Hz, 1H, Ar-H), 7.32 (td, J=8.2, 4.6Hz, 1H, Ar-H). 13 C NMR(151MHz,DMSO-d6)δ179.93,150.16(d, 1 J C-F =228.5Hz),148.21,135.87,130.96(d, 2 J C-F =14.5Hz), 124.43(d, 3 J C-F =6.2Hz), 121.59(d, 3 J C-F =5.9Hz), 118.42(d, 4 J C-F =3.7Hz), 118.26(d, 2 J C-F =17.1Hz). 19 F NMR(565MHz,DMSO-d6)δ-131.09.HRMS(ESI)[M+H] + calcd for C9H6N4FS:221.0292,found:221.0284.

[0045] (5) Preparation of intermediate 7-fluoro-5H-[1,2,4]triazine[5,6-b]indole-indole-3-thione:

[0046]

[0047] In a 250 mL round-bottom flask, 6-fluoroindigo (3.00 g, 20 mmol) and potassium carbonate (3.80 g, 30 mmol) were added, followed by water and stirring. Then, aminothiourea (1.67 g, 20 mmol) was added, and the mixture was refluxed at 100 °C with stirring. The reaction was monitored by TLC. After the reaction was complete, the solid and liquid phases were separated, filtered, and washed with water. The mixture was dried to give the intermediate 7-fluoro-5H-[1,2,4]triazinyl[5,6-b]indole-3-thione, in yield 49.80%. Its NMR data are as follows: 1 H NMR (500MHz, Trifluoroacetic Acid-d) δ8.78–8.68(m,1H,Ar-H),7.87(dd,J=8.1,2.2Hz,1H,Ar-H),7.80–7.72(m,1H,Ar-H). 13 C NMR(126MHz,Trifluoroacetic Acid-d)δ168.88,166.07(d, 1 J C-F =236.8Hz), 147.58, 144.68 (d, 3 J C-F =13.1Hz), 140.29, 126.36 (d, 4 J C-F =3.2Hz), 115.24(d, 2 J C-F =24.9Hz), 113.00(d, 3 J C-F =11.2Hz), 102.80(d, 2 J C-F =28.7Hz). 19 FNMR(471MHz,Trifluoroacetic Acid-d)δ-77.53.HRMS(ESI)[M+H] + calcd for C9H6N4FS:221.0292,found:221.0284.

[0048] (6) Preparation of intermediate 8-fluoro-5H-[1,2,4]triazine[5,6-b]indole-3-thione:

[0049]

[0050] In a 250 mL round-bottom flask, 6-fluoroindigo (3.00 g, 20 mmol) and potassium carbonate (3.80 g, 30 mmol) were added, followed by water and stirring. Then, aminothiourea (1.67 g, 20 mmol) was added, and the mixture was refluxed at 100 °C with stirring. The reaction was monitored by TLC. After the reaction was complete, the solid and liquid phases were separated, filtered, and washed with water. The mixture was dried to give the intermediate 7-fluoro-5H-[1,2,4]triazinyl[5,6-b]indole-3-thione, in yield 55.50%. Its NMR data are as follows: 1 H NMR (600MHz, DMSO-d6) δ7.25 (dd, J=10.3, 2.9Hz, 1H, Ar-H), 6.99 (td, J=8.7, 3.3Hz, 1H, Ar-H), 6.68 (dd, J=9.0, 4.9Hz, 1H, Ar-H). 13 C NMR(151MHz,DMSO-d6)δ173.56,154.24,153.01(d, 1 J C-F =238.5Hz), 146.96, 144.55 (d, 4 J C-F =4.6Hz), 117.91(d, 2 J C-F =22.5Hz), 116.97(d, 3 J C-F =8.3Hz), 116.73(d, 2 J C-F =23.6Hz), 115.24(d, 3 J C-F =7.5Hz); 19 F NMR(565MHz,DMSO-d6)δ-130.32.HRMS(ESI)[M+H] + calcd for C9H6N4FS:221.0292,found:221.0283.

[0051] (7) Preparation of intermediate 9-chloro-5H-[1,2,4]triazine[5,6-b]indole-3-thione:

[0052]

[0053] In a 250 mL round-bottom flask, 4-chloroindigo (3.00 g, 20 mmol) and potassium carbonate (3.46 g, 30 mmol) were added, followed by water and stirring. Then, aminothiourea (1.52 g, 20 mmol) was added, and the mixture was refluxed at 100 °C with stirring. The reaction was monitored by TLC. After the reaction was complete, the solid and liquid phases were separated, filtered, and washed thoroughly with plenty of water. The solution was dried to give an orange-red intermediate, 9-chloro-5H-[1,2,4]triazinyl[5,6-b]indole-3-thione, in a yield of 25.90%. Its NMR data are as follows: 1 H NMR (600MHz, DMSO-d6) δ7.60 (t, J = 7.9 Hz, 1H, Ar-H), 7.39 (t, J = 8.0 Hz, 2H, Ar-H). 13 C NMR(151MHz,DMSO-d6)δ179.65,149.60,144.82,135.25,133.17,128.79,123.98,116.18,112.25.HRMS(ESI)[M+H] + calcd for C9H6ClS:236.9996,found:236.9985.

[0054] (8) Preparation of target compound 1:

[0055]

[0056] In a 50 mL round-bottom flask, intermediate (1) (100.00 mg, 1 mmol) was added first, followed by dichloromethane and stirring. Then, ethanethiol (47.00 mg, 1.5 mmol) was added, and the mixture was stirred at 0 °C for 5 min. Finally, DDQ (113.38 mg, 1 mmol) was slowly added, and the reaction was monitored by TLC. After the reaction was complete, the solid and liquid phases were separated, filtered, and washed with a large amount of dichloromethane. After drying, the pale yellow target compound 1 was obtained, with a yield of 87.90%.

[0057] (9) Preparation of target compound 2:

[0058]

[0059] In a 50 mL round-bottom flask, intermediate (1) (100.00 mg, 1 mmol) was added first, followed by dichloromethane and stirring. Then, propanethiol (57.64 mg, 1.5 mmol) was added, and the mixture was stirred at 0 °C for 5 min. Finally, DDQ (113.38 mg, 1 mmol) was slowly added, and the reaction was monitored by TLC. After the reaction was complete, the solid and liquid phases were separated, filtered, and washed with a large amount of dichloromethane. After drying, the pale yellow target compound 2 was obtained, with a yield of 90.14%.

[0060] (10) Preparation of target compound 3:

[0061]

[0062] In a 50 mL round-bottom flask, intermediate (1) (100.00 mg, 1 mmol) was added first, followed by dichloromethane and stirring. Then, propanethiol (109.98 mg, 1.5 mmol) was added, and the mixture was stirred at 0 °C for 5 min. Finally, DDQ (113.38 mg, 1 mmol) was slowly added, and the reaction was monitored by TLC. After the reaction was complete, the solid and liquid phases were separated, filtered, and washed with a large amount of dichloromethane. After drying, the pale yellow target compound 3 was obtained, with a yield of 92.67%.

[0063] (11) Preparation of target compound 31:

[0064]

[0065] In a 50 mL round-bottom flask, intermediate (2) (100.00 mg, 1 mmol) was added first, followed by dichloromethane and stirring. Then, ethanethiol (43.97 mg, 1.5 mmol) was added, and the mixture was stirred at 0 °C for 5 min. Finally, DDQ (106.03 mg, 1 mmol) was slowly added, and the reaction was monitored by TLC. After the reaction was complete, the solid and liquid phases were separated, filtered, and washed with a large amount of dichloromethane. After drying, the pale yellow target compound 31 was obtained, with a yield of 75.45%.

[0066] (12) Preparation of target compound 32:

[0067]

[0068] In a 50 mL round-bottom flask, intermediate (3) (100.00 mg, 1 mmol) was added first, followed by dichloromethane and stirring. Then, ethanethiol (43.97 mg, 1.5 mmol) was added, and the mixture was stirred at 0 °C for 5 min. Finally, DDQ (106.03 mg, 1 mmol) was slowly added, and the reaction was monitored by TLC. After the reaction was complete, the solid and liquid phases were separated, filtered, and washed with a large amount of dichloromethane. After drying, the pale yellow target compound 32 was obtained, with a yield of 89.32%.

[0069] (13) Preparation of target compound 33:

[0070]

[0071] In a 50 mL round-bottom flask, intermediate (4) (100.00 mg, 1 mmol) was added first, followed by dichloromethane and stirring. Then, ethanethiol (43.18 mg, 1.5 mmol) was added, and the mixture was stirred at 0 °C for 5 min. Finally, DDQ (104.12 mg, 1 mmol) was slowly added, and the reaction was monitored by TLC. After the reaction was complete, the solid and liquid phases were separated, filtered, and washed with a large amount of dichloromethane. After drying, the pale yellow target compound 33 was obtained, with a yield of 78.89%.

[0072] (14) Preparation of target compound 34:

[0073]

[0074] In a 50 mL round-bottom flask, intermediate (5) (100.00 mg, 1 mmol) was added first, followed by dichloromethane and stirring. Then, ethanethiol (43.18 mg, 1.5 mmol) was added, and the mixture was stirred at 0 °C for 5 min. Finally, DDQ (104.12 mg, 1 mmol) was slowly added, and the reaction was monitored by TLC. After the reaction was complete, the solid and liquid phases were separated, filtered, and washed with a large amount of dichloromethane. After drying, the pale yellow target compound 34 was obtained, with a yield of 88.65%.

[0075] (15) Preparation of target compound 35:

[0076]

[0077] In a 50 mL round-bottom flask, intermediate (6) (100.00 mg, 1 mmol) was added first, followed by dichloromethane and stirring. Then, ethanethiol (43.18 mg, 1.5 mmol) was added, and the mixture was stirred at 0 °C for 5 min. Finally, DDQ (104.12 mg, 1 mmol) was slowly added, and the reaction was monitored by TLC. After the reaction was complete, the solid and liquid phases were separated, filtered, and washed with a large amount of dichloromethane. After drying, the pale yellow target compound 35 was obtained, with a yield of 90.65%.

[0078] (16) Preparation of target compound 36:

[0079]

[0080] In a 50 mL round-bottom flask, intermediate (7) (100.00 mg, 1 mmol) was added first, followed by dichloromethane and stirring. Then, ethanethiol (40.18 mg, 1.5 mmol) was added, and the mixture was stirred at 0 °C for 5 min. Finally, DDQ (96.88 mg, 1 mmol) was slowly added, and the reaction was monitored by TLC. After the reaction was complete, the solid and liquid phases were separated, filtered, and washed with a large amount of dichloromethane. After drying, the pale yellow target compound 36 was obtained, with a yield of 56.78%.

[0081] The structures containing disulfides synthesized above, along with their 1H and 1C NMR spectra, are shown in Table 1, and their physicochemical properties are shown in Table 2.

[0082] Table 1: 1H NMR, 1C NMR, and high-resolution mass spectrometry data of the compounds

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098] Table 2: Physicochemical properties of the target compound

[0099]

[0100]

[0101] Example 2

[0102] EC 50Median effective concentration (MEC) is an important indicator for evaluating the sensitivity of plant pathogens to compounds, and it is also a crucial parameter for setting the compound concentration when studying the mechanism of action of target compounds. In concentration gradient experiments, five appropriate concentrations were set using the two-fold dilution method. Finally, the inhibition rate of the agent against the plant pathogen and the agent concentration were converted into logarithmic values, and the toxicity curve was obtained through regression analysis using SPSS software to calculate the EC50. 50 .

[0103] The effective medium concentration (EC) of the target compound against plant pathogens was determined using a turbidimetric method. 50 The experimental subjects were *Xoo*, *Xac*, and *Psa*, the pathogens of rice bacterial blight. DMSO was dissolved in the culture medium as a blank control. *Xoo* (the pathogen of rice bacterial blight was on M210 solid medium) was placed in NB medium and cultured in a constant temperature shaker at 28℃ and 180 rpm until the logarithmic growth phase. *Xac* (the pathogen of rice bacterial blight) was placed in NB medium. *Psa* (the pathogen of rice bacterial blight) was placed in NB medium and cultured in a constant temperature shaker at 28℃ and 180 rpm until the logarithmic growth phase. Prepare different concentrations (e.g., 100, 50, 25, 12.5, 6.25 μg / mL) of the drug (compound) in 5 mL of NB liquid culture medium containing the pathogen and add them to test tubes. Add 40 μL of NB liquid culture medium containing plant pathogen bacteria to each tube. Incubate in a constant temperature shaker at 28-30℃ and 180 rpm for 48 h for rice bacterial blight pathogens, 48 ​​h for citrus canker pathogens, and 36 h for rice bacterial leaf streak pathogens. Measure the OD595 value of each concentration of bacterial suspension using a microplate reader, and also separately measure the OD595 value of the corresponding concentration of sterile NB liquid culture medium containing the pathogen.

[0104] Corrected OD value = OD value of bacterial culture medium - OD value of sterile culture medium

[0105] Inhibition rate % = [(OD value of bacterial suspension in the corrected control medium - OD value of the corrected virus-containing medium) / OD value of bacterial suspension in the corrected control medium] × 100

[0106] The embodiments of this invention are provided to illustrate the technical solutions of this invention, but the content of the embodiments is not limited thereto. The experimental results of the target compound are shown in Table 3.

[0107] Table 3: EC50 of disulfide-substituted unsaturated nitrogen-containing heterocyclic compounds against plant pathogenic bacteria 50

[0108]

[0109]

[0110] As shown in Table 3, the target compounds exhibited good antibacterial activity against *Xanthomonas oryzae* var. *oryzae* and *Xanthomonas citrus canker* in in vitro experiments. Compounds 1 and 33 showed excellent inhibitory activity against *Xanthomonas oryzae* var. *oryzae* (Xoo), with EC50 values ​​of [missing data]. 50 The concentration was 0.77-0.85 μg / mL; Compound 1 showed excellent inhibitory activity against Xanthomonas axonopodis pv. citri (Xac) , the causal agent of citrus canker, with an EC50 of 0.77-0.85 μg / mL. 50 It is 2.62 μg / mL.

[0111] Example 3

[0112] Inhibitory activity of the compound against plant pathogenic fungi (50 μg / mL)

[0113] The mycelial growth rate method, also known as the toxic medium method, is one of the routine methods for determining the toxicity of fungicides. The main principle is to mix the test agent with a culture medium and measure the toxicity of the agent by the rate at which colonies grow on the toxic medium. In this example, *Botrytis cinerea*, *Rhizoctonia solani*, and *Fusarium graminearum* were used as test subjects, with DMSO as a blank control. The specific operation is as follows: 1) Weigh an appropriate amount of drug according to the test concentration, dissolve it with DMSO (the amount should not exceed 1% of the final toxic medium), then add 0.1% Tween 20 solution to make up to 10 mL, pour it into 90 mL of melted PDA medium, mix well and pour into 9 petri dishes for later use; 2) Sterilize the punch (with an inner diameter of 5 mm) by flame, and after it cools, punch holes in the hyphae near the edge of the pre-activated strain, and use an inoculation needle to place the hyphae on the center of the toxic medium. After the treatment is completed, place them in a culture at 25℃; 3) After the colony diameter of the control group grows to 5.5-6.6 cm, use the cross-cross method to determine the colony diameter of the control group and each drug treatment group; 4) Calculate the inhibition rate (%) using the following formula: Inhibition rate % = (CT) / (C-0.5) × 100; where C is the colony diameter of the control group, T is the colony diameter of the drug treatment group, and 0.5 is the diameter of the inoculated mycelium. Following the above method, the experimental results of the target compounds in Table 1 are shown in Table 4.

[0114] Table 4: Inhibitory activity of compounds against plant pathogenic fungi (50 μg / mL)

[0115]

[0116] In summary, as shown in Table 3, the target compounds exhibited good inhibitory activity against plant pathogenic bacteria such as *Bacillus thuringiensis*, the causal agent of rice bacterial blight, in in vitro experiments. Most of the compounds containing an ethyl group showed significant inhibitory activity against *Bacillus thuringiensis* at EC50. 50 All were below 10, especially compound 1 against the EC50 of rice bacterial blight pathogen. 50 The concentration of compound 16 was 0.77 μg / mL, and its EC50 concentration against *Citrus aurantium* was [missing value]. 50 The concentration was 3.43 μg / mL; Compound 6 showed an EC50 concentration against *Actinidia kiwifruit* causal agent. 50 The concentration was 2.86 μg / mL. As shown in Table 4, the target compounds also exhibited certain inhibitory activity against plant pathogenic fungi. At a concentration of 50 μg / mL, all target compounds showed good bioactivity against *Rhizoctonia solani*. Among them, compound 33 showed excellent inhibitory activity against *Rhizoctonia solani*, *Botrytis cinerea*, and *Fusarium graminearum*, with inhibition rates of 92.35%, 75.81%, and 61.33%, respectively. Therefore, these compounds can be used as novel pesticides against plant pathogenic bacteria and fungi, possessing extremely high research value.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention. All such modifications or substitutions should be covered by the scope of the claims of the present invention, and the protection scope of this application should be determined by the protection scope of the claims.

Claims

1. A class of 5H-[1,2,4]triazinyl[5,6-b]indole derivatives containing a disulfide structure, characterized in that... The compound has the structure shown in general formula (Ⅰ): Among them, compound 1: R1 is ethyl, and R2~R6 are all hydrogen; compound 2: R1 is propyl, and R2~R6 are all hydrogen; compound 3: R1 is allyl, and R2~R6 are all hydrogen; compound 12: R1 is 4-methoxyphenyl, and R2~R6 are all hydrogen; compound 13: R1 is 4-chlorophenyl, and R2~R6 are all hydrogen; compound 31: R1 is ethyl, R2 is methyl, and R3, R4, R5, and R6 are all hydrogen; compound 32: R1 is ethyl, R3 is methyl, and R2, R4, R5, and R6 are all hydrogen; compound 33: R1 is ethyl, R3 is F, and R2, R4, R5, and R6 are all hydrogen; compound 34: R1 is ethyl, R4 is fluorine, and R2, R3, R5, and R6 are all hydrogen; compound 36: R1 is ethyl, R6 is Cl, and R2, R3, R4, and R5 are all hydrogen.

2. The method for preparing the 5H-[1,2,4]triazine[5,6-b]indole derivative containing a disulfide structure as described in claim 1, characterized in that: The preparation method includes the following steps: Among them, compound 1: R1 is ethyl, and R2~R6 are all hydrogen; compound 2: R1 is propyl, and R2~R6 are all hydrogen; compound 3: R1 is allyl, and R2~R6 are all hydrogen; compound 12: R1 is 4-methoxyphenyl, and R2~R6 are all hydrogen; compound 13: R1 is 4-chlorophenyl, and R2~R6 are all hydrogen; compound 31: R1 is ethyl, R2 is methyl, and R3, R4, R5, and R6 are all hydrogen; compound 32: R1 is ethyl, R3 is methyl, and R2, R4, R5, and R6 are all hydrogen; compound 33: R1 is ethyl, R3 is F, and R2, R4, R5, and R6 are all hydrogen; compound 34: R1 is ethyl, R4 is fluorine, and R2, R3, R5, and R6 are all hydrogen; compound 36: R1 is ethyl, R6 is Cl, and R2, R3, R4, and R5 are all hydrogen.

3. A composition, characterized in that: It contains the compound according to any one of claims 1-2, as well as agricultural adjuvants or fungicides.

4. The use of the compound according to any one of claims 1-2, or the composition according to claim 3, in the preparation of a medicament for the prevention and control of plant bacterial diseases.

5. The application according to claim 4, characterized in that... The plant bacterial diseases mentioned are plant leaf blight and plant canker.

6. The application according to claim 4, characterized in that: The plant bacterial diseases mentioned are any one of rice bacterial blight, citrus canker, and kiwifruit canker.

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