Indigo derivatives, processes for their preparation and use in the preparation of plant antibacterial agents

Abstract

This invention discloses a class of indigo derivatives, their preparation methods, and their applications in preparing plant antibacterial drugs. The general structural formula of these derivatives is: where X represents any one of hydrogen, fluorine, chlorine, bromine, or methoxy; R1 represents any one of diethylamino, morpholino, pyrrolyl, or 4-methylpiperazinyl; R2 represents any one of methoxy or thiourea; and n takes the value 2 or 3. The indigo derivatives of this invention exhibit a certain inhibitory effect on the proliferation of *Culicoides zeolite* and *Pseudomonas aeruginosa*, and can be used to prepare plant antibacterial drugs.

Description

Technical Field

[0001] This invention belongs to the field of plant antibacterial drug technology, specifically relating to a class of indigo derivatives, as well as the preparation method and uses of the derivatives. Background Technology

[0002] Currently, plant pathogenic fungi have become the most significant pathogens affecting plants. Whether it's crops like corn or fruit trees like apples, all are susceptible to fungal invasion. Infected plants typically exhibit round or oval lesions that are white, yellowish-brown, dark brown, or black. Plant fungi are extremely destructive; once they invade plants, the planting area becomes a severely affected zone, causing significant yield reduction. Therefore, finding natural fungicides with low phytotoxicity and strong bactericidal effects is crucial for ensuring healthy plant growth.

[0003] Indigo, also known as 1-hydro-indole-2,3-dione, is an endogenous heterocyclic molecule found in human tissues and a natural active substance essential for the survival of marine organisms. It possesses various pharmacological activities, including antibacterial, antitumor, antiviral, and hypoglycemic effects. Current research on indigo includes substitution of the nitrogen atom at the 1-position, modification of the carbonyl carbons at the 2- and 3-positions, and modification of the benzene ring. Compounds modified at the 3-position carbonyl carbon exhibit better antibacterial activity, such as 3-arylmethylene indigo derivatives, indigo-3-hydrazone derivatives, indigo-3-imine metal complexes, and C3-indigo spirocyclic derivatives.

[0004] Since indigo itself is a natural, low-toxicity substance with good antibacterial activity, it can be used as a core and modified in a reasonable way to synthesize indigo derivatives with better antibacterial activity. Summary of the Invention

[0005] The purpose of this invention is to provide a class of indigo derivatives with antibacterial activity.

[0006] The structural formula of the indigo derivative provided by this invention is shown below:

[0007]

[0008] In the formula, X represents any one of hydrogen, fluorine, chlorine, bromine, and methoxy; R1 represents any one of diethylamino, pyrrolidinyl, and 4-methylpiperazinyl; R2 represents any one of methoxy and thiourea; and n takes the value of 2 or 3.

[0009] The aforementioned indigo derivatives are preferably any one of the following compounds 1 to 22:

[0010] Compound 1: (Z)-1-(3-(diethylamino)propyl)-3-(methoxyimino)indololin-2-one

[0011]

[0012] Compound 2: (Z)-5-chloro-1-(3-(diethylamino)propyl)-3-(methoxyimino)indoline-2-one

[0013]

[0014] Compound 3: (Z)-5-bromo-1-(3-(diethylamino)propyl)-3-(methoxyimino)indoline-2-one

[0015]

[0016] Compound 4: (Z)-5-bromo-1-(3-(morpholinyl)propyl)-3-(methoxyimino)indololin-2-one

[0017]

[0018] Compound 5: (Z)-1-(3-(pyrrolidinyl)propyl)-3-(methoxyimino)indoline-2-one

[0019]

[0020] Compound 6: (Z)-5-fluoro-1-(3-(pyrrolidinyl)propyl)-3-(methoxyimino)indoline-2-one

[0021]

[0022] Compound 7: (Z)-5-methoxy-1-(3-(pyrrolidinyl)propyl)-3-(methoxyimino)indoline-2-one

[0023]

[0024] Compound 8: (Z)-5-fluoro-1-(3-(4-methylpiperazinyl)propyl)-3-(methoxyimino)indoline-2-one

[0025]

[0026] Compound 9: (Z)-5-chloro-1-(3-(4-methylpiperazinyl)propyl)-3-(methoxyimino)indoline-2-one

[0027]

[0028] Compound 10: (Z)-5-bromo-1-(3-(4-methylpiperazinyl)propyl)-3-(methoxyimino)indoline-2-one

[0029]

[0030] Compound 11: (Z)-2-(5-chloro-1-(2-(diethylamino)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamate

[0031]

[0032] Compound 12: (Z)-2-(5-bromo-1-(2-(diethylamino)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamate

[0033]

[0034] Compound 13: (Z)-2-(5-fluoro-1-(2-(morpholinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamate

[0035]

[0036] Compound 14: (Z)-2-(5-chloro-1-(2-(morpholinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamate

[0037]

[0038] Compound 15: (Z)-2-(5-bromo-1-(2-(morpholinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamate

[0039]

[0040] Compound 16: (Z)-2-(5-chloro-1-(2-(pyrrolidinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbonyl

[0041]

[0042] Compound 17: (Z)-2-(5-bromo-1-(2-(pyrrolidinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbonyl

[0043]

[0044] Compound 18: (Z)-2-(1-(2-(4-methylpiperazinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamide

[0045]

[0046] Compound 19: (Z)-2-(5-fluoro-1-(2-(4-methylpiperazinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamide

[0047]

[0048] Compound 20: (Z)-2-(5-chloro-1-(2-(4-methylpiperazinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamate

[0049]

[0050] Compound 21: (Z)-2-(5-bromo-1-(2-(4-methylpiperazinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamide

[0051]

[0052] Compound 22: (Z)-2-(5-methoxy-1-(2-(4-methylpiperazinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamide

[0053]

[0054] When R2 represents a methoxy group in the above-mentioned indigo derivatives, the preparation method includes the following steps:

[0055] Step 1: React 5-substituted indigo (Formula I) with methoxyamine hydrochloride in methanol at room temperature under dipotassium hydrogen phosphate as a base to generate (Z)-5-substituted-3-(methoxyimino)indoline-2-one (Formula II).

[0056] Step 2: (Z)-5-substituted-3-(methoxyimino)indololin-2-one is reacted with 1-bromo-3-chloropropane or 1-bromo-2-chloroethane in N,N-dimethylformamide at room temperature under potassium carbonate as a base to produce (Z)-5-substituted-1-chloroalkyl-3-(methoxyimino)indololin-2-one as shown in Formula III.

[0057] Step 3: The (Z)-5-substituted-1-chloroalkyl-3-(methoxyimino)indoline-2-one and the N-containing compound shown in Formula IV are reacted by heating in N,N-dimethylformamide under potassium carbonate as a base to generate the target indigo derivative. The reaction equation is shown below:

[0058]

[0059] In the formula, H in R1H is hydrogen attached to the nitrogen atom in R1.

[0060] In step 1 above, the preferred molar ratio of 5-substituted indigo to methoxyamine hydrochloride to dipotassium hydrogen phosphate is 1:1.05-1.2:1-1.2.

[0061] In step 2 above, the preferred molar ratio of (Z)-5-substituted-3-(methoxyimino)indoline-2-one to 1-bromo-3-chloropropane or 1-bromo-2-chloroethane and potassium carbonate is 1:2 to 3:1.3 to 1.5.

[0062] In step 3 above, the preferred molar ratio of (Z)-5-substituted-1-chloroalkyl-3-(methoxyimino)indoline-2-one to the N-containing compound and potassium carbonate is 1:3 to 5:1.5 to 2.5, and the heating temperature is 60°C.

[0063] When R2 represents a thiourea group in the above-mentioned indigo derivatives, its preparation method includes the following steps:

[0064] Step 1: React the 5-substituted indigo shown in Formula I with the hydrochloride salt of the chloroalkyl N-containing compound shown in Formula V in a mixed solvent of chloroform and water or N,N-dimethylformamide under potassium carbonate as a base at room temperature or with heating to produce the 5-substituted-1-substituted indoline-2,3-dione shown in Formula VI.

[0065] Step 2: 5-substituted-1-substituted indoline-2,3-dione and aminothiourea are reacted with ethanol under heating in glacial acetic acid as a catalyst to generate the target indigo derivative. The reaction equation is shown below:

[0066]

[0067] In step 1 above, the preferred molar ratio of the 5-substituted indigo to the hydrochloride of the chloroalkyl N-containing compound and potassium carbonate is 1:1.1-2:2.2-4, and the heating temperature is 80°C.

[0068] In step 2 above, the preferred molar ratio of the 5-substituted-1-substituted indoline-2,3-dione to aminothiourea and glacial acetic acid is 1:1 to 1.3:0.08 to 0.12, and the heating temperature is 79 to 82°C.

[0069] When R2 represents a thiourea group in the above-mentioned indigo derivatives, they can also be prepared by the following method:

[0070] Step 1: React 5-substituted indigo as shown in Formula I with 1,2-dibromoethane or 1,3-dibromopropane in N,N-dimethylformamide at room temperature under potassium carbonate as a base to generate 5-substituted-1-(bromoalkyl)indoline-2,3-dione as shown in Formula VII.

[0071] Step 2: 5-substituted-1-(bromoalkyl)indoline-2,3-dione is reacted with the N-containing compound shown in Formula IV in N,N-dimethylformamide at room temperature under potassium carbonate as a base to produce 5-substituted-1-substituted indoline-2,3-dione shown in Formula VIII.

[0072] Step 3: 5-substituted-1-substituted indoline-2,3-dione and aminothiourea are reacted with ethanol under heating in glacial acetic acid as a catalyst to generate the target indigo derivative. The reaction equation is shown below:

[0073]

[0074] In step 1 above, the preferred molar ratio of 5-substituted indigo to 1,2-dibromoethane or 1,3-dibromopropane and potassium carbonate is 1:2 to 3:1.3 to 1.5.

[0075] In step 2 above, the preferred molar ratio of the 5-substituted-1-(bromoalkyl)indoline-2,3-dione to the N-containing compound and potassium carbonate is 1:3 to 5:1.5 to 2.5.

[0076] In step 3 above, the preferred molar ratio of the 5-substituted-1-substituted indoline-2,3-dione to aminothiourea and glacial acetic acid is 1:1 to 1.3:0.08 to 0.12, and the heating temperature is 79 to 82°C.

[0077] This invention also provides the use of indigo derivatives in the preparation of plant antibacterial drugs, wherein the pathogen is either *Curvularia micrantha* or *Phyllostachys edulis*.

[0078] The beneficial effects of this invention are as follows:

[0079] 1. In the indigo derivative of this invention, when R2 represents a methoxy group, it is synthesized by first oximeizing indigo at the 3-position and then attaching a chloroalkyl group at the 1-position; when R2 represents a thiourea group, one synthesis method is to first attach a hydrophilic group at the 1-position of indigo and then perform a condensation reaction with aminothiourea at the 3-position; another synthesis method is to first attach a bromoalkyl group at the 1-position of indigo and then use a simpler and easier potassium carbonate inclusion method to prepare the reaction intermediate 5-substituted-1-substituted indoline-2,3-dione, which does not require silica gel column chromatography separation, can obtain a relatively pure reaction intermediate, and has a high yield, and is expected to be used in industrial production.

[0080] 2. The indigo derivative of the present invention has a certain inhibitory effect on the proliferation of Curvularia micrantha and Pseudomonas macrantha, especially on Curvularia micrantha, and can be used to prepare plant antibacterial drugs. Detailed Implementation

[0081] The present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited to the following embodiments.

[0082] Example 1

[0083] Step 1: Add 0.736 g (5 mmol) of indigo and 10 mL of methanol to a reaction flask, then add 0.438 g (5.25 mmol) of methoxyamine hydrochloride and 1.14 g (5 mmol) of dipotassium hydrogen phosphate. Stir the mixture at room temperature for 2 h. After the reaction stops, remove the solvent by rotary evaporation under reduced pressure. Add water to the mixture to wash away impurities, yielding 0.871 g of a yellow solid compound (Z)-3-(methoxyimino)indoline-2-one, with a yield of 99%.

[0084]

[0085] Step 2: Add 0.705 g (4 mmol) of (Z)-3-(methoxyimino)indoline-2-one and 0.796 g (5.76 mmol) of potassium carbonate to a reaction flask, followed by 4 mL of DMF and 1.57 g (10 mmol) of 1-bromo-3-chloropropane. Stir the mixture at room temperature for 12 h. After the reaction is complete, add 10 mL of distilled water and extract with ethyl acetate (2 × 15 mL). Combine the organic phases and dry with anhydrous sodium sulfate. Remove the solvent by rotary evaporation under reduced pressure. Separate the concentrate by column chromatography (ethyl acetate / petroleum ether = 1 / 3, V / V) to obtain 0.844 g of a yellow solid compound (Z)-1-(3-chloropropyl)-3-(methoxyimino)indoline-2-one, with a yield of 83%.

[0086]

[0087] Step 3: Add 0.253 g (1 mmol) of (Z)-1-(3-chloropropane)-3-(methoxyimino)indoline-2-one, 0.293 g (4 mmol) of diethylamine, 0.276 g (2 mmol) of potassium carbonate, and 3 mL of DMF to the reaction flask. Stir the mixture at 60 °C for 12 h. After the reaction solution cools to room temperature, add 10 mL of water and extract with ethyl acetate (20 mL × 3). Combine the organic phases, dry with anhydrous sodium sulfate, filter, concentrate, and separate the residue by silica gel column chromatography (methanol / dichloromethane = 1 / 20, V / V) to obtain 0.122 g of yellow oily liquid compound 1, with a yield of approximately 42%.

[0088]

[0089] The structural characterization data of compound 1 are as follows: HRMS (C 16 H 23 N3O2)m / z[M+H]+ :290.1857 (calculated value 290.1863); 1 H NMR (600MHz, CDCl3) δ (ppm): 7.95 (d, J = 7.5Hz, 1H), 7.38 (t, J = 7.8Hz, 1H), 7.06 (t, J = 7.6Hz, 1H), 6.92 (d, J = 7.9Hz,1H),4.29(s,3H),3.80(t,J=7.2Hz,2H),2.69-2.51(m,6H),1.96-1.84(m,2H),1.05(t,J=7.1Hz,6H); 13 C NMR (151MHz, CDCl3) δ (ppm): 163.48 (s), 143.89 (s), 143.64 (s), 132.42 (s), 127.90 (s), 122.78 (s),115.76(s),108.77(s),64.68(s),50.21(s),46.69(s),38.22(s),25.20(s),11.39(s); IRν max (KBr)cm -1 :2948,2824,2715,1631,1595,1352,1155,774.

[0090] Example 2

[0091] In step 1 of this embodiment, the indigo in step 1 of Example 1 was replaced with an equimolar amount of 5-chloroindigo. The other steps were the same as in Example 1, yielding 0.150 g of yellow oily liquid compound 2 with a yield of 46%.

[0092]

[0093] The structural characterization data of compound 2 are as follows: HRMS(C 16 H 22 ClN3O2)m / z[M+H] + 324.1469 (calculated value 324.1473); 1 H NMR (600MHz, CDCl3) δ (ppm): 7.92 (d, J = 1.9Hz, 1H), 7.35 (dd, J = 8.4, 1.7Hz, 1H), 6.91 (d, J = 8.4Hz, 1H),3.80(t,J=7.1Hz,2H),2.63(dt,J=15.4,7.2Hz,6H),1.96-1.86(m,2H),1.07(t,J=7.2Hz,6H); 13C NMR (151MHz, CDCl3) δ (ppm): 163.09 (s), 142.73 (s), 142.16 (s), 132.06 (s), 128.12 (s), 127.81 (s),116.62(s),109.88(s),65.02(s),50.09(s),46.63(s),38.28(s),24.80(s),10.86(s); IRν max (KBr)cm -1 :2941,2831,2715,1631,1595,1389,1352,1147,767.

[0094] Example 3

[0095] In step 1 of this embodiment, the indigo in step 1 of Example 1 was replaced with an equimolar amount of 5-bromoindigo. The other steps were the same as in Example 1, yielding 0.134 g of yellow oily liquid compound 3 with a yield of 36%.

[0096]

[0097] The structural characterization data of compound 3 are as follows: HRMS(C 16 H 22 BrN3O2)m / z[M+H] + 368.0963 (calculated value 368.0968); 1 H NMR (600MHz, CDCl3) δ (ppm): 8.04 (s, 1H), 7.49 (d, J = 8.3Hz, 1H), 6.85 (d, J = 8.4Hz, 1H), 4.31 (s, 3 H),3.78(t,J=7.1Hz,2H),2.58(dt,J=16.1,7.2Hz,6H),1.96-1.80(m,2H),1.05(t,J=7.2Hz,6H); 13 C NMR (151MHz, CDCl3) δ (ppm): 162.87 (s), 142.65 (s), 142.57 (s), 134.86 (s), 130.44 (s), 116.98 (s),115.23(s),110.33(s),65.01(s),50.08(s),46.60(s),38.27(s),24.95(s),11.10(s); IRν max (KBr)cm -1 :2948,2831,2715,1631,1595,1352,1147,774.

[0098] Example 4

[0099] In step 3 of this embodiment, diethylamine in step 3 of Example 3 was replaced with equimolar morpholine. The residue was separated by silica gel column chromatography (methanol / dichloromethane = 1 / 80, V / V). The other steps were the same as in Example 3, yielding 0.305 g of yellow oily liquid compound 4, with a yield of 80%.

[0100]

[0101] The structural characterization data of compound 4 are as follows: HRMS(C 16 H 20 BrN3O3)m / z[M+H] + 382.0753 (calculated value 382.0761); 1 H NMR (600MHz, CDCl3) δ (ppm): 8.07 (d, J = 1.9Hz, 1H), 7.48 (dd, J = 8.4, 2.0Hz, 1H), 6.83 (d, J = 8.4Hz, 1H) ,4.31(s,3H),3.81(t,J=6.8Hz,2H),3.73-3.59(m,4H),2.38(t,J=6.7Hz,6H),1.84(m,J=6.7Hz,2H); 13 C NMR (151MHz, CDCl3) δ (ppm): 163.06 (s), 142.95 (s), 142.68 (s), 134.75 (s), 130.55 (s), 117.13 (s),115.19(s),110.19(s),66.89(s),65.06(s),55.71(s),53.61(s),38.20(s),24.09(s); IRν max (KBr)cm -1 :2948,2831,2721,1631,1595,1360,1147,774.

[0102] Example 5

[0103] In step 3 of this embodiment, diethylamine in step 3 of Example 1 was replaced with an equimolar amount of pyrrolidine. The other steps were the same as in Example 1, yielding 0.263 g of yellow oily liquid compound 5, with a yield of approximately 92%.

[0104]

[0105] The structural characterization data of compound 5 are as follows: HRMS (C 16 H 21 N3O2)m / z[M+H] + :288.1704 (calculated value 288.1707);1 H NMR (600MHz, CDCl3) δ (ppm): 7.93 (d, J = 7.4Hz, 1H), 7.36 (t, J = 8.1Hz, 1H), 7.04 (t, J = 8.1Hz, 1H), 6.92 (d, J = 7 .9Hz,1H),4.28(s,3H),3.82(t,J=6.8Hz,2H),2.54(dd,J=16.7,8.6Hz,6H),1.97-1.88(m,2H),1.77(s,4H); 13 C NMR (151MHz, CDCl3) δ (ppm): 163.54 (s), 143.95 (s), 143.63 (s), 132.42 (s), 127.86 (s), 122.75 (s),115.73(s),108.81(s),64.66(s),53.99(s),53.26(s),38.10(s),26.78(s),23.42(s); IRν max (KBr)cm -1 :2948,2832,2723,1633,1597,1357,1153,775.

[0106] Example 6

[0107] In step 1 of this embodiment, the indigo in step 1 of Example 5 was replaced with an equimolar amount of 5-fluoroindigo. The other steps were the same as in Example 5, yielding 0.297 g of yellow oily liquid compound 6 with a yield of 97%.

[0108]

[0109] The structural characterization data of compound 6 are as follows: HRMS(C 16 H 20 FN3O2)m / z[M+H + 306.1610 (calculated value 306.1612); 1 H NMR (600MHz, CDCl3) δ (ppm): 7.68 (dd, J=7.9, 2.6Hz, 1H), 7.08 (td, J=8.8, 2.7Hz, 1H), 6.88 (dd, J=8. 6,4.0Hz,1H),4.30(s,3H),3.81(t,J=6.9Hz,2H),2.53-2.49(m,6H),1.94-1.86(m,2H),1.77(m,4H); 13 C NMR (151MHz, CDCl3) δ (ppm): 163.35 (s), 159.50 (d, 1 J C-F=241Hz), 143.31(d, 4 J C-F =2.6Hz), 140.11(s), 118.67(d, 2 J C-F =24Hz), 116.25(d, 3 J C-F =9.5Hz), 115.44(d, 2 J C-F =26Hz), 109.46(d, 3 J C-F =7.9Hz),64.94(s),54.00(s),53.18(s),38.30(s),26.80(s),23.44(s); IRν max (KBr)cm -1 :2960,2828,2715,1600,1361,1149,778.

[0110] Example 7

[0111] In step 1 of this embodiment, indigo in step 1 of Example 5 was replaced with equimolar 5-methoxy-substituted indigo. The other steps were the same as in Example 5, yielding 0.304 g of orange-yellow oily liquid compound 7 with a yield of 96%.

[0112]

[0113] The structural characterization data of compound 7 are as follows: HRMS(C 17 H 23 N3O3)m / z[M+H] + 318.1809 (calculated value 318.1812); 1 H NMR (600MHz, CDCl3) δ (ppm): 7.56 (d, J = 2.6Hz, 1H), 6.91 (dd, J = 8.5, 2.6Hz, 1H), 6.83 (d, J = 8.5Hz, 1H) ,4.29(s,3H),3.80(s,3H),3.79(t,J=7.1Hz,2H),2.51(m,6H),1.94-1.85(m,2H),1.81-1.70(m,4H); 13C NMR(151MHz, CDCl3)δ(ppm):163.44(s),155.72(s),143.96(s),137.81(s),117.48(s),116.35(s),1 14.38(s),109.30(s),64.75(s),55.99(s),54.04(s),53.31(s),38.22(s),26.93(s),23.46(s); IRν max (KBr)cm -1 :2956,2832,2716,1597,1364,1153,775.

[0114] Example 8

[0115] In step 3 of this embodiment, the diethylamine in step 3 of Example 6 was replaced with an equimolar amount of 4-methylpiperazine. The other steps were the same as in Example 6, yielding 0.281 g of yellow oily liquid compound 8 with a yield of 84%.

[0116]

[0117] The structural characterization data of compound 8 are as follows: HRMS (C 17 H 23 FN4O2)m / z[M+H + 335.1879 (calculated value 335.1878); 1 H NMR (600MHz, CDCl3) δ (ppm): 7.68 (dd, J=7.2, 2.9Hz, 1H), 7.08 (td, J=8.8, 1.9Hz, 1H), 6.88 (dd, J=8.4, 2.3Hz, 1H ),4.31(s,3H),3.80(t,J=6.6Hz,2H),2.42(dd,J=25.1,18.4Hz,10H),2.31(s,3H),1.86(dd,J=13.2,6.5Hz,2H); 13 C NMR (151MHz, CDCl3) δ (ppm): 163.34 (s), 159.47 (d, 1 J C-F =241Hz), 143.31(d, 4 J C-F =2.3Hz), 140.12(s), 118.60(d, 2 J C-F =24Hz), 116.28(d, 3 J C-F =9.1Hz), 115.47(d, 2 JC-F =26Hz), 109.37(d, 3 J C-F =7.9Hz),64.94(s),55.11(s),54.89(s),52.74(s),45.75(s),38.19(s),24.51(s); IRν max (KBr)cm -1 :2956,2832,2723,1633,1597,1364,1153,775.

[0118] Example 9

[0119] In step 3 of this embodiment, the diethylamine in step 3 of Example 2 was replaced with an equimolar amount of 4-methylpiperazine. The other steps were the same as in Example 2, yielding 0.186 g of yellow oily liquid compound 9, with a yield of 53%.

[0120]

[0121] The structural characterization data of compound 9 are as follows: HRMS (C 17 H 23 ClN4O2)m / z[M+H] + :351.1582 (calculated value 351.1582); 1 H NMR (600MHz, CDCl3) δ (ppm): 7.94 (d, J=2.1Hz, 1H), 7.34 (dd, J=8.4, 2.2Hz, 1H), 6.89 (d, J= 8.4Hz,1H),4.31(s,3H),3.80(t,J=6.8Hz,2H),2.68-2.24(m,13H),1.84(p,J=6.8Hz,2H); 13 C NMR (151MHz, CDCl3) δ (ppm): 163.18 (s), 142.88 (s), 142.56 (s), 131.90 (s), 127.99 (s), 127.83 (s), 1 16.72(s),109.83(s),65.02(s),55.13(s),55.05(s),52.97(s),45.96(s),38.23(s),24.59(s); IRν max (KBr)cm -1 :2948,2824,2715,1631,1601,1360,1155,774.

[0122] Example 10

[0123] In step 3 of this embodiment, the diethylamine in step 3 of Example 3 was replaced with an equimolar amount of 4-methylpiperazine. The other steps were the same as in Example 3, yielding 0.343 g of yellow oily liquid compound 10, with a yield of 87%.

[0124]

[0125] The structural characterization data of compound 10 are as follows: HRMS (C 17 H 23 BrN4O2)m / z[M+H] + 395.1070 (calculated value 395.1077); 1 H NMR (600MHz, CDCl3) δ (ppm): 8.07 (d, J = 2.0Hz, 1H), 7.49 (dd, J = 8.4, 2.0Hz, 1H), 6.85 (d, J = 8.4Hz, 1H), 4.31(s,3H),3.79(t,J=6.8Hz,2H),2.39(dd,J=25.8,18.9Hz,10H),2.29(s,3H),1.84(p,J=6.7Hz,2H); 13 C NMR (151MHz, CDCl3) δ (ppm): 163.05 (s), 143.01 (s), 142.72 (s), 134.77 (s), 130.53 (s), 117.13 (s), 1 15.18(s),110.32(s),65.03(s),55.09(s),55.01(s),52.89(s),45.90(s),38.20(s),24.55(s); IRν max (KBr)cm -1 :2960,2832,2718,1608,1359,1145,768.

[0126] Example 11

[0127] Step 1: Add 0.726 g (4 mmol) of 5-chloroindigo, 10 mL of chloroform, and 2.21 g (16 mmol) of potassium carbonate to a reaction flask. Dissolve 1.38 g (8 mmol) of 2-(N,N-diethylamino)chloroethane hydrochloride in 9 mL of distilled water and add it to the reaction flask with stirring. Stir vigorously at room temperature for 10 h. After the reaction stops, extract with chloroform (3 × 15 mL), combine the organic phases, dry with anhydrous sodium sulfate, remove the solvent by rotary evaporation under reduced pressure, and separate the concentrate by column chromatography (methanol / dichloromethane = 1 / 80, V / V) to give 0.936 g of red oily liquid 5-chloro-1-(2-diethylaminoethyl)-indoline-2,3-dione, with a yield of 83%.

[0128]

[0129] Step 2: Add 0.280 g (1 mmol) of 5-chloro-1-(2-diethylaminoethyl)-indoline-2,3-dione, 0.091 g (1 mmol) of aminothiourea, 15 mL of ethanol, and 0.1 mmol of glacial acetic acid to a reaction flask, and stir under reflux at 79 °C for 4 h. After the reaction solution cools, remove the solvent by rotary evaporation under reduced pressure. Recrystallize the residue with 2 mL of ethanol, filter, and give 0.187 g of orange-yellow solid compound 11, with a yield of 53%.

[0130]

[0131] The structural characterization data of compound 11 are as follows: mp 143.1-143.6℃; HRMS (C 15 H 20 ClN5OS)m / z[M+H] + 354.1149 (calculated value 354.1150); 1 H NMR (600MHz, DMSO-d6) δ (ppm): 12.27 (s, 1H), 9.16 (s, 1H), 8.86 (s, 1H), 7.79 (d, J = 2.2Hz, 1H), 7.47 (dd, J = 8.4, 2.2Hz, 1H) ,7.23(d,J=8.5Hz,1H),3.80(t,J=6.4Hz,2H),2.62(t,J=5.6Hz,2H),2.46(dd,J=13.3,6.4Hz,4H),0.84(t,J=7.1Hz,6H); 13 C NMR(151MHz,DMSO-d6)δ(ppm):179.27(s),161.10(s),142.21(s),130.73(s),130.29(s),1 27.44(s),121.61(s),120.88(s),112.31(s),49.60(s),47.03(s),38.51(s),12.40(s); IRν max (KBr)cm -1 :2836,2717,1626,1600,1360,1151,772.

[0132] Example 12

[0133] In step 1 of this embodiment, 5-chloroindigo in step 1 of Example 11 was replaced with an equimolar amount of 5-bromoindigo. The other steps were the same as in Example 11, yielding 0.209 g of orange-yellow solid compound 12 with a yield of 53%.

[0134]

[0135] The structural characterization data of compound 12 are as follows: mp 145.2-145.6℃; HRMS (C 15 H 20 BrN5OS)m / z[M+H] + 398.0638 (calculated value 398.0645); 1 H NMR(600MHz,DMSO-d6)δ(ppm):12.24(s,1H),9.16(s,1H),8.87(s,1H),7.91(s,1 H),7.58(s,1H),7.16(s,1H),3.79(s,2H),2.61(s,2H),2.46(s,4H),0.83(s,6H); 13 CNMR(151MHz,DMSO-d6)δ(ppm):179.25(s),160.93(s),142.56(s),133.47(s),130.09(s),1 23.67(s),121.97(s),115.10(s),112.71(s),49.60(s),47.04(s),38.49(s),12.42(s); IRν max (KBr)cm -1 :2948,2825,2716,1597,1357,1153,768.

[0136] Example 13

[0137] Step 1: Add 0.660 g (4 mmol) of 5-fluoroindigo, 1.22 g (8.8 mmol) of potassium carbonate, and 5 mL of DMF to a reaction flask, and stir for 15 min at room temperature. Then add 0.819 g (4.4 mmol) of 4-(2-chloroethyl)morpholine hydrochloride to the solution and continue the reaction at 80 °C for 4 h. After the reaction is complete, add 10 mL of distilled water and extract with dichloromethane (3 × 15 mL). Combine the organic phases, dry with anhydrous sodium sulfate, remove the solvent by rotary evaporation under reduced pressure, and separate the concentrate by column chromatography (methanol / dichloromethane = 1 / 80, V / V) to obtain 0.430 g of red oily liquid 5-fluoro-1-(2-morpholinylethyl)-indoline-2,3-dione, with a yield of 39%.

[0138]

[0139] Step 2: Add 0.278 g (1 mmol) of 5-fluoro-1-(2-morpholinylethyl)-indoline-2,3-dione, 0.091 g (1 mmol) of aminothiourea, 15 mL of ethanol, and 0.1 mmol of glacial acetic acid to a reaction flask, and stir under reflux at 79 °C for 4 h. After the reaction solution cools, remove the solvent by rotary evaporation under reduced pressure. Recrystallize the residue with 2 mL of ethanol, filter, and give 0.186 g of orange-yellow solid compound 13, with a yield of 53%.

[0140]

[0141] The structural characterization data of compound 13 are as follows: mp 220.8-221.7℃; HRMS (C 15 H 18 FN5O2S)m / z[M+H + :352.1235 (calculated value 352.1238); 1 H NMR (600MHz, CDCl3) δ (ppm): 12.86 (s, 1H), 7.51 (s, 1H), 7.30 (dd, J=7.5, 2.5Hz, 1H), 7.10 (td, J=8.8, 2.6Hz, 1H), 6.87 ( dd,J=8.6,3.8Hz,1H),6.63(s,1H),3.87(t,J=6.6Hz,2H),3.70-3.64(m,4H),2.63(t,J=6.6Hz,2H),2.56-2.48(m,4H). 13 C NMR (151MHz, CDCl3) δ (ppm): 180.02 (s), 161.13 (s), 160.14 (d, 1 J C-F =243),139.26(d, 4 J C-F =1.8Hz), 131.37(s), 120.75(d, 3 J C-F =8.9Hz), 117.80(d, 2 J C-F =24.3Hz), 110.27(d, 3 J C-F =8.1Hz), 108.55(d, 2 J C-F =25.6Hz),66.90(s),55.38(s),53.70(s),37.38(s).IRν max (KBr)cm -1 :2825,2718,1636,1588,1385,1351,1162,764.

[0142] Example 14

[0143] In step 1 of this embodiment, 5-fluoroindigo in step 1 of Example 13 was replaced with an equimolar amount of 5-chloroindigo. The other steps were the same as in Example 13, yielding 0.271 g of orange-yellow solid compound 14 with a yield of 74%.

[0144]

[0145] The structural characterization data of compound 14 are as follows: mp 214.1-215.0℃; HRMS (C 15 H 18 ClN5O2S)m / z[M+H] + 368.0938 (calculated value 368.0942); 1 H NMR (600MHz, CDCl3) δ (ppm): 12.81 (s, 1H), 7.56 (d, J = 2.0Hz, 1H), 7.52 (s, 1H), 7.36 (dd, J = 8.4, 2.1Hz, 1H), 6.87 ( d,J=8.4Hz,1H),6.71(s,1H),3.87(t,J=6.5Hz,2H),3.69-3.63(m,4H),2.62(t,J=6.5Hz,2H),2.55-2.48(m,4H); 13 CNMR (151MHz, CDCl3) δ (ppm): 180.01 (s), 160.88 (s), 141.58 (s), 130.98 (s), 130.79 (s), 12 8.86(s),121.07(s),120.89(s),110.49(s),66.90(s),55.38(s),53.69(s),37.39(s); IRν max (KBr)cm -1 :2948,2818,2716,1633,1597,1357,1153,768.

[0146] Example 15

[0147] In step 1 of this embodiment, 5-fluoroindigo in step 1 of Example 13 was replaced with an equimolar amount of 5-bromoindigo. The other steps were the same as in Example 13, yielding 0.210 g of orange-yellow solid compound 15 with a yield of 51%.

[0148]

[0149] The structural characterization data of compound 15 are: mp 223.6-224.4℃; HRMS (C15 H 18 BrN5O2S)m / z[M+H] + :412.0428 (calculated value 412.0437); 1 H NMR (600MHz, DMSO-d6) δ (ppm): 12.23 (s, 1H), 9.15 (s, 1H), 8.86 (s, 1H), 7.93 (d, J = 1.5Hz, 1H), 7.59 (dd, J=8.4,1.5Hz,1H),7.19(d,J=8.4Hz,1H),3.86(t,J=6.3Hz,2H),3.50(s,4H),2.55(t,J=6.4Hz,2H),2.46 -2.38(m,4H); 13 CNMR(151MHz,DMSO-d6)δ(ppm):179.26(s),160.92(s),142.43(s),133.50(s),130.07(s),1 23.70(s),122.06(s),115.19(s),112.75(s),66.68(s),55.39(s),53.68(s),37.30(s); IRν max (KBr)cm -1 :2956,2832,2723,1597,1364,775.

[0150] Example 16

[0151] In step 1 of this embodiment, 4-(2-chloroethyl)morpholine hydrochloride in step 1 of Example 14 was replaced with equimolar 4-(2-chloroethyl)pyrrolidine hydrochloride. The other steps were the same as in Example 14, yielding 0.244 g of yellow solid compound 16 with a yield of 70%.

[0152]

[0153] The structural characterization data of compound 16 are as follows: mp 195.0-195.6℃; HRMS (C 15 H 18 ClN5OS)m / z[M+H] + 352.0992 (calculated value 352.0993); 1H NMR (600MHz, DMSO-d6) δ (ppm): 12.23 (s, 1H), 9.19 (s, 1H), 8.86 (s, 1H), 7.78 (s, 1H), 7.45 (d, J = 8.1Hz, 1H) ,7.20(d,J=8.3Hz,1H),3.85(t,J=5.6Hz,2H),2.67(t,J=5.7Hz,2H),2.52-2.47(m,4H),1.69-1.61(m,4H); 13 C NMR(151MHz,DMSO-d6)δ(ppm):179.24(s),160.89(s),141.89(s),130.74(s),130.10(s),1 27.56(s),121.65(s),120.92(s),112.08(s),54.10(s),52.88(s),39.16(s),23.65(s); IRν max (KBr)cm -1 :2956,2825,2716,1633,1597,1364,1153,775.

[0154] Example 17

[0155] In step 1 of this embodiment, 5-chloroindigo in step 1 of Example 16 was replaced with an equimolar amount of 5-bromoindigo. The other steps were the same as in Example 16, yielding 0.274 g of yellow solid compound 17, with a yield of 69%.

[0156]

[0157] The structural characterization data of compound 17 are: mp 211.7-212.2℃; HRMS (C 15 H 18 BrN5OS)m / z[M+H] + 396.0482 (calculated value 396.0488); 1 H NMR (600MHz, DMSO-d6) δ (ppm): 12.22 (s, 1H), 9.17 (s, 1H), 8.87 (s, 1H), 7.92 (s, 1H), 7.58 (d, J = 5.9Hz, 1H),7.16(d,J=6.6Hz,1H),3.92-3.76(m,2H),2.73-2.62(m,2H),2.52-2.43(m,4H),1.70-1.60(m,4H); 13C NMR(151MHz,DMSO-d6)δ(ppm):179.24(s),160.79(s),142.33(s),133.55(s),130.01(s),1 23.73(s),122.06(s),115.22(s),112.58(s),54.11(s),52.87(s),39.15(s),23.65(s); IRν max (KBr)cm -1 :2963,2831,2721,1623,1601,1360,1147,781.

[0158] Example 18

[0159] Step 1: Add 0.736 g (5 mmol) of indigo and 0.995 g (7.2 mmol) of potassium carbonate to a reaction flask, then add 5 mL of DMF and 2.35 g (12.5 mmol) of 1,2-dibromoethane. Stir the mixture at room temperature for 12 h. After the reaction is complete, add 10 mL of distilled water and extract with ethyl acetate (2 × 15 mL). Combine the organic phases and dry with anhydrous sodium sulfate. Remove the solvent by rotary evaporation under reduced pressure. Separate the concentrate by column chromatography (using dichloromethane as eluent) to obtain 0.864 g of orange-yellow solid 1-(2-bromoethyl)indoline-2,3-dione, with a yield of 68%.

[0160]

[0161] Step 2: Add 0.762 g (3 mmol) of 1-(2-bromoethyl)indoline-2,3-dione, 1.20 g (12 mmol) of N-methylpiperazine, and 0.829 g (6 mmol) of potassium carbonate to a reaction flask, then add 3 mL of DMF and stir at a constant speed at room temperature for 12 h. After the reaction is complete, filter to obtain a filter cake. Add 10 mL of distilled water to the filter cake and extract with ethyl acetate (2 × 15 mL). Combine the organic phases, dry with anhydrous sodium sulfate, and remove the solvent by rotary evaporation under reduced pressure to obtain 0.578 g of orange-yellow oily liquid 1-(2-(4-methylpiperazinyl)ethyl)indoline-2,3-dione, with a yield of 71%.

[0162]

[0163] Step 3: Add 0.273 g (1 mmol) of 1-(2-(4-methylpiperazinyl)ethyl)indoline-2,3-dione, 0.091 g (1 mmol) of aminothiourea, 15 mL of ethanol, and 0.1 mmol of glacial acetic acid to a reaction flask, and stir under reflux at 79 °C for 4 h. After the reaction solution cools, remove the solvent by rotary evaporation under reduced pressure. Recrystallize the residue with 2 mL of ethanol, filter, and give 0.196 g of yellow solid compound 18, with a yield of 57%.

[0164]

[0165] The structural characterization data of compound 18 are: mp 208.6-209.0℃; HRMS (C 16 H 22 N6OS)m / z[M+H] + :347.1641 (calculated value 347.1649); 1 H NMR (600MHz, DMSO-d6) δ (ppm): 12.41 (s, 1H), 9.07 (s, 1H), 8.72 (s, 1H), 7.70 (d, J = 7.4Hz, 1H), 7.42 (t, J = 7 .7Hz,1H),7.24-7.10(m,2H),3.85(t,J=6.4Hz,2H),2.56(t,J=6.4Hz,2H),2.51-2.16(m,8H),2.11(s,3H); 13 C NMR(151MHz,DMSO-d6)δ(ppm):179.20(s),161.28(s),143.38(s),131.62(s),131.56(s),123.26 (s),121.18(s),119.77(s),110.67(s),55.17(s),54.94(s),53.06(s),46.13(s),37.42(s); IRν max (KBr)cm -1 :2853,2816,2721,1635,1580,1385,1348,1167,780.

[0166] Example 19

[0167] In step 1 of this embodiment, the indigo in step 1 of Example 18 was replaced with an equimolar amount of 5-fluoroindigo. The other steps were the same as in Example 18, yielding 0.265 g of yellow solid compound 19 with a yield of 73%.

[0168]

[0169] The structural characterization data of compound 19 are as follows: mp 125.9-126.6℃; HRMS (C 16 H 21 FN6OS)m / z[M+H + 365.1548 (calculated value 365.1554); 1 H NMR (600MHz, DMSO-d6) δ (ppm): 12.27 (s, 1H), 9.17 (s, 1H), 8.80 (s, 1H), 7.55 (dd, J = 7.9, 2.5Hz, 1H), 7.27 (td, J = 9.1, 2.5Hz,1H),7.20(dd,J=8.6,4.0Hz,1H),3.84(t,J=6.4Hz,2H),2.54(t,J=6.4Hz,2H),2.53-2.15(m,8H),2.11(s,3H); 13 C NMR(151MHz,DMSO-d6)δ(ppm):179.28(s),161.27(s),159.81(d, 1 J C-F =238Hz), 139.64(s), 130.80(d, 4 J C-F =3.1Hz), 121.32(d, 3 J C-F =9.4Hz), 117.74(d, 2 J C-F =24Hz), 111.96(d, 3 J C-F =8.0Hz), 108.37(d, 2 J C-F =26Hz),55.18(s),54.92(s),53.06(s),46.12(s),37.59(s); IRν max (KBr)cm -1 :2946,2824,2721,1639,1594,1349,1149,776,627.

[0170] Example 20

[0171] In step 1 of this embodiment, the indigo in step 1 of Example 18 was replaced with an equimolar amount of 5-chloroindigo. The other steps were the same as in Example 18, yielding 0.290 g of yellow solid compound 20 with a yield of 77%.

[0172]

[0173] The structural characterization data of compound 20 are as follows: mp 200.3-200.8℃; HRMS (C 16 H 21 ClN6OS)m / z[M+H] + :381.1253 (calculated value 381.1259); 1 H NMR (600MHz, DMSO-d6) δ (ppm): 12.21 (s, 1H), 9.16 (s, 1H), 8.85 (s, 1H), 7.79 (d, J = 2.2Hz, 1H), 7.46 (dd, J = 8.4, 2.2Hz,1H),7.23(d,J=8.5Hz,1H),3.85(t,J=6.4Hz,2H),2.54(t,J=6.5Hz,2H),2.52-2.16(m,8H),2.12(s,3H); 13 C NMR(151MHz,DMSO-d6)δ(ppm):179.27(s),161.04(s),142.08(s),130.72(s),130.24(s),127.51 (s),121.68(s),120.89(s),112.33(s),55.17(s),54.92(s),53.04(s),46.10(s),37.63(s); IRν max (KBr)cm -1 :2963,2832,2709,1597,1364,1153,775.

[0174] Example 21

[0175] In step 1 of this embodiment, the indigo in step 1 of Example 18 was replaced with an equimolar amount of 5-bromoindigo. The other steps were the same as in Example 18, yielding 0.278 g of yellow solid compound 21 with a yield of 66%.

[0176]

[0177] The structural characterization data of compound 21 are as follows: mp 201.0-201.7℃; HRMS (C 16 H 21 BrN6OS)m / z[M+H] + :425.0750 (calculated value 425.0754); 1H NMR (600MHz, DMSO-d6) δ (ppm): 12.23 (s, 1H), 9.17 (s, 1H), 8.87 (s, 1H), 7.92 (d, J = 2.0Hz, 1H), 7.58 (dd, J = 8.4, 2.0Hz,1H),7.17(d,J=8.4Hz,1H),3.84(t,J=6.4Hz,2H),2.53(t,J=6.4Hz,2H),2.52-2.15(m,8H),2.11(s,3H); 13 C NMR(151MHz,DMSO-d6)δ(ppm):179.26(s),160.89(s),142.44(s),133.49(s),130.08(s),123.68 (s),122.04(s),115.17(s),112.75(s),55.18(s),54.92(s),53.06(s),46.12(s),37.60(s); IRν max (KBr)cm -1 :2958,2827,2714,1632,1581,1361,1151,777.

[0178] Example 22

[0179] In step 1 of this embodiment, indigo in step 1 of Example 18 was replaced with equimolar 5-methoxy-substituted indigo. The other steps were the same as in Example 18, yielding 0.197 g of orange-red solid compound 22 with a yield of 52%.

[0180]

[0181] The structural characterization data of compound 22 are as follows: mp 192.8-193.3℃; HRMS (C 17 H 24 N6O2S)m / z[M+H] + :377.1753 (calculated value 377.1754); 1 H NMR (600MHz, DMSO-d6) δ (ppm): 12.37 (s, 1H), 9.10 (s, 1H), 8.76 (s, 1H), 7.37 (d, J = 2.4Hz, 1H), 7.09 (d, J = 8.6Hz, 1H), 6 .98(dd,J=8.6,2.5Hz,1H),3.81(t,J=6.4Hz,2H),3.77(s,3H),2.54(d,J=6.4Hz,2H),2.52-2.14(m,8H),2.11(s,3H); 13C NMR(151MHz,DMSO-d6)δ(ppm):179.23(s),161.24(s),156.14(s),137.09(s),131.72(s),120.69(s), 117.37(s),111.49(s),106.73(s),56.10(s),55.19(s),54.97(s),53.07(s),46.15(s),37.45(s); IRν max (KBr)cm -1 :2959,2832,2719,1602,1361,1154,773.

[0182] Example 23

[0183] The application of the indigo derivative of the present invention in the preparation of antibacterial drugs is specifically tested as follows:

[0184] 1. Test strains

[0185] Corn curvularia lunata Walk and apple rot fungus (Valsa mali Miyabeet Yamada).

[0186] 2. Reagents and Materials

[0187] Acetone (AR), DMSO (AR), 75% alcohol, 95% alcohol, distilled water, glucose, agar powder, potato, gauze, absorbent cotton, plastic wrap.

[0188] 3. Experimental Procedure

[0189] (1) Activation and culture of the test bacteria

[0190] The prepared PDA medium (200g peeled potato cubes, added to 1000mL distilled water, boiled for 30min, filtered through 4 layers of gauze, the filtrate was then added to 20g glucose and 18g agar powder, heated until completely dissolved, and then diluted to 1000mL with distilled water, and then poured into 300mL Erlenmeyer flasks, 90mL in each Erlenmeyer flask) was autoclaved, plated, inoculated with the test bacteria, and then placed in an incubator at 26±1℃ for inverted culture.

[0191] (2) Preliminary determination of antibacterial activity

[0192] The antibacterial activity was preliminarily determined using the mycelial growth rate method. The prepared PDA medium (400g peeled and diced potatoes, added to 2000mL distilled water, boiled for 30min, filtered through four layers of gauze, the filtrate was then mixed with 40g glucose and 36g agar powder, heated until completely dissolved, and then diluted to 2000mL with distilled water, then poured into 300mL Erlenmeyer flasks, 90mL per flask) was autoclaved. 4.5mg of the required test compound was weighed, dissolved in 10% of the target volume of DMSO, and then diluted to 2mL with acetone. The prepared solution was added to each 90mL sterilized medium, mixed thoroughly, and used to prepare 50μg / mL toxic medium. A blank control was prepared using 2mL of acetone (containing 10% DMSO). The medium was diluted to 15mL per plate using stoppered test tubes, and the medium was poured into three plates for each treatment. The plates were then cooled to prepare the toxic medium. The cultured test bacteria plates were perforated along the edge using a 4mm inner diameter punch to create discs containing mycelia. Then, using aseptic technique, the mycelial discs were inoculated into the center of the corresponding virus-containing medium using an inoculation needle. One mycelial disc was placed on each plate, and the process was repeated three times. The inoculated plates were then placed upside down in an incubator at 26±1℃ and incubated. The size of the mycelial growth was observed at 72h and 96h, and the colony diameter (expressed in mm) was measured using the cross-sectional method. The mean value was used to represent the colony size. (Note:)

[0193] Colony diameter (mm) = Average measurement (mm) - 4mm

[0194] Antibacterial rate = [(average colony diameter of control group - average colony diameter of treatment group) / average colony diameter of control group] × 100%

[0195] 4. Experimental Results

[0196] Using carbendazim technical grade as a positive control, the experimental results are shown in Tables 1 and 2.

[0197] Table 1. Inhibition rate (%) of the tested compounds against two plant pathogens after 72 h.

[0198]

[0199]

[0200] Note: The average number of repeated experiments is three, and all results are expressed as ±SE.

[0201] Table 2. Inhibition rates (%) of the tested compounds against two plant pathogens after 96 h.

[0202]

[0203]

[0204] Note: The average number of repeated experiments is three, and all results are expressed as ±SE.

[0205] As shown in Tables 1 and 2, all tested compounds exhibited some inhibitory activity against both plant pathogens. Compounds 2, 9, 10, 18, 19, and 20 showed good inhibitory activity against *Cudrania tricuspidata*, with inhibition rates of 67.0% ± 0.3%–77.9% ± 0.7% at 72 h and 70.1% ± 0.4%–79.7% ± 0.2% at 96 h, all superior to the positive control, carbendazim. Compound 18 showed the most significant antibacterial activity, with inhibition rates of 77.9% ± 0.7% at 72 h and 79.7% ± 0.2% at 96 h. Tested compounds 3, 8, 13, and 16 showed some inhibitory activity against *Cudrania tricuspidata*, the causal agent of apple rot. Furthermore, comparing Tables 1 and 2 reveals that the inhibitory activity of all tested compounds against *Cudrania tricuspidata* decreased to some extent with prolonged administration time, while the inhibitory activity against *Cudrania tricuspidata* increased for most of the tested compounds. Antibacterial activity data show that the compounds of this invention have certain inhibitory effects on Curvularia micrantha and Bacillus rotundus, especially compounds 2, 9, 10, 18, 19, and 20, which show outstanding inhibitory effects on Curvularia micrantha and can be used to prepare antibacterial drugs for corn. Compounds 3, 8, 13, and 16 have good inhibitory effects on Bacillus rotundus and can be used to prepare antibacterial drugs for apples.

Claims

1. A class of indigo derivatives, characterized in that... The structural formula of the derivative is shown below: In the formula, X represents any one of hydrogen, fluorine, chlorine, bromine, and methoxy; R1 represents any one of diethylamino, morpholino, pyrrolidinyl, and 4-methylpiperazinyl; R2 represents methoxy; and n takes the value of 2 or 3. Alternatively, X can represent any one of hydrogen, fluorine, chlorine, bromine, or methoxy; R1 can represent any one of diethylamino, morpholino, pyrrolidinyl, or 4-methylpiperazinyl; R2 can represent thiourea; and n can be 3. Alternatively, X can represent any one of fluorine, chlorine, bromine, or methoxy; R1 can represent any one of diethylamino, morpholino, pyrrolidinyl, or 4-methylpiperazinyl; R2 can represent thiourea; and n can be 2. Alternatively, X represents hydrogen; R1 represents any one of morpholino, pyrrolidinyl, or 4-methylpiperazinyl; R2 represents thiourea; and n takes the value 2.

2. The indigo derivative according to claim 1, characterized in that... The derivative is any one of the following compounds 1 to 22: Compound 1: (Z)-1-(3-(diethylamino)propyl)-3-(methoxyimino)indololin-2-one Compound 2: (Z)-5-chloro-1-(3-(diethylamino)propyl)-3-(methoxyimino)indoline-2-one Compound 3: (Z)-5-bromo-1-(3-(diethylamino)propyl)-3-(methoxyimino)indoline-2-one Compound 4: (Z)-5-bromo-1-(3-(morpholinyl)propyl)-3-(methoxyimino)indololin-2-one Compound 5: (Z)-1-(3-(pyrrolidinyl)propyl)-3-(methoxyimino)indoline-2-one Compound 6: (Z)-5-fluoro-1-(3-(pyrrolidinyl)propyl)-3-(methoxyimino)indoline-2-one Compound 7: (Z)-5-methoxy-1-(3-(pyrrolidinyl)propyl)-3-(methoxyimino)indoline-2-one Compound 8: (Z)-5-fluoro-1-(3-(4-methylpiperazinyl)propyl)-3-(methoxyimino)indoline-2-one Compound 9: (Z)-5-chloro-1-(3-(4-methylpiperazinyl)propyl)-3-(methoxyimino)indoline-2-one Compound 10: (Z)-5-bromo-1-(3-(4-methylpiperazinyl)propyl)-3-(methoxyimino)indoline-2-one Compound 11: (Z)-2-(5-chloro-1-(2-(diethylamino)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamate Compound 12: (Z)-2-(5-bromo-1-(2-(diethylamino)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamate Compound 13: (Z)-2-(5-fluoro-1-(2-(morpholinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamate Compound 14: (Z)-2-(5-chloro-1-(2-(morpholinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamate Compound 15: (Z)-2-(5-bromo-1-(2-(morpholinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamate Compound 16: (Z)-2-(5-chloro-1-(2-(pyrrolidinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamate Compound 17: (Z)-2-(5-bromo-1-(2-(pyrrolidinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamate Compound 18: (Z)-2-(1-(2-(4-methylpiperazinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamide Compound 19: (Z)-2-(5-fluoro-1-(2-(4-methylpiperazinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamide Compound 20: (Z)-2-(5-chloro-1-(2-(4-methylpiperazinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamate Compound 21: (Z)-2-(5-bromo-1-(2-(4-methylpiperazinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamide Compound 22: (Z)-2-(5-methoxy-1-(2-(4-methylpiperazinyl)ethyl)-2-indoline-3-ylidene)hydrazine-1-thiocarbamide 。 3. A method for preparing the indigo derivative according to claim 1, wherein R2 represents a methoxy group, characterized in that: Step 1: 5-substituted indigo as shown in Formula I is reacted with methoxyamine hydrochloride in methanol at room temperature under the condition of dipotassium hydrogen phosphate as base to generate (Z)-5-substituted-3-(methoxyimino)indoline-2-one as shown in Formula II. Step 2: (Z)-5-substituted-3-(methoxyimino)indololin-2-one is reacted with 1-bromo-3-chloropropane or 1-bromo-2-chloroethane in N,N-dimethylformamide at room temperature under potassium carbonate as a base to produce (Z)-5-substituted-1-chloroalkyl-3-(methoxyimino)indololin-2-one as shown in Formula III; Step 3: (Z)-5-substituted-1-chloroalkyl-3-(methoxyimino)indololin-2-one and the N-containing compound shown in Formula IV are reacted by heating in N,N-dimethylformamide under potassium carbonate as a base to generate the target indigo derivative. In the formula, X represents any one of hydrogen, fluorine, chlorine, bromine, and methoxy; R1 represents any one of diethylamino, morpholino, pyrrolidinyl, and 4-methylpiperazinyl; H in R1H is hydrogen attached to the nitrogen atom in R1; and n takes the value of 2 or 3.

4. The method for preparing the indigo derivative according to claim 3, characterized in that: In step 1, the molar ratio of 5-substituted indigo to methoxyamine hydrochloride to dipotassium hydrogen phosphate is 1:1.05-1.2:1-1.2; in step 2, the molar ratio of (Z)-5-substituted-3-(methoxyimino)indololin-2-one to 1-bromo-3-chloropropane or 1-bromo-2-chloroethane and potassium carbonate is 1:2-3:1.3-1.5; in step 3, the molar ratio of (Z)-5-substituted-1-chloroalkyl-3-(methoxyimino)indololin-2-one to an N-containing compound and potassium carbonate is 1:3-5:1.5-2.5, and the reaction temperature is 60°C.

5. A method for preparing the indigo derivative of claim 1, wherein R2 represents a thiourea group, characterized in that: Step 1: React the 5-substituted indigo shown in Formula I with the hydrochloride salt of the chloroalkyl N-containing compound shown in Formula V in a mixed solvent of chloroform and water or N,N-dimethylformamide under potassium carbonate as a base at room temperature or with heating to produce the 5-substituted-1-substituted indoline-2,3-dione shown in Formula VI. Step 2: 5-substituted-1-substituted indoline-2,3-dione and aminothiourea are reacted with ethanol under the condition of glacial acetic acid as catalyst to generate the target indigo derivative. In the formula, X represents any one of hydrogen, fluorine, chlorine, bromine, and methoxy; R1 represents any one of diethylamino, morpholino, pyrrolidinyl, and 4-methylpiperazinyl; and n takes the value of 3. Alternatively, X can represent any one of fluorine, chlorine, bromine, or methoxy; R1 can represent any one of diethylamino, morpholino, pyrrolidinyl, or 4-methylpiperazinyl; and n can be 2. Alternatively, X represents hydrogen; R1 represents any one of morpholino, pyrrolidinyl, or 4-methylpiperazinyl; and n takes the value 2.

6. The method for preparing the indigo derivative according to claim 5, characterized in that: In step 1, the molar ratio of the 5-substituted indigo to the hydrochloride of the chloroalkyl N-containing compound and potassium carbonate is 1:1.1-2:2.2-4, and the reaction temperature is 80°C. In step 2, the molar ratio of the 5-substituted-1-substituted indoline-2,3-dione to aminothiourea and glacial acetic acid is 1:1-1.3:0.08-0.12, and the reaction temperature is 79-82°C.

7. A method for preparing the indigo derivative of claim 1, wherein R2 represents a thiourea group, characterized in that: Step 1: React 5-substituted indigo as shown in Formula I with 1,2-dibromoethane or 1,3-dibromopropane in N,N-dimethylformamide at room temperature under potassium carbonate as a base to generate 5-substituted-1-(bromoalkyl)indoline-2,3-dione as shown in Formula VII. Step 2: 5-substituted-1-(bromoalkyl)indoline-2,3-dione is reacted with the N-containing compound shown in Formula IV in N,N-dimethylformamide at room temperature under potassium carbonate as a base to generate 5-substituted-1-substituted indoline-2,3-dione shown in Formula VIII. Step 3: 5-substituted-1-substituted indoline-2,3-dione and aminothiourea are reacted with ethanol under the condition of glacial acetic acid as catalyst to generate the target indigo derivative. In the formula, X represents any one of hydrogen, fluorine, chlorine, bromine, and methoxy; R1 represents any one of diethylamino, morpholino, pyrrolidinyl, and 4-methylpiperazinyl; and n takes the value of 3. Alternatively, X can represent any one of fluorine, chlorine, bromine, or methoxy; R1 can represent any one of diethylamino, morpholino, pyrrolidinyl, or 4-methylpiperazinyl; and n can be 2. Alternatively, X represents hydrogen; R1 represents any one of morpholino, pyrrolidinyl, or 4-methylpiperazinyl; and n takes the value 2. In Formula IV, the H in R1H is hydrogen attached to the nitrogen atom in R1.

8. The method for preparing the indigo derivative according to claim 7, characterized in that: In step 1, the molar ratio of 5-substituted indigo to 1,2-dibromoethane or 1,3-dibromopropane and potassium carbonate is 1:2-3:1.3-1.5; in step 2, the molar ratio of 5-substituted-1-(bromoalkyl)indoline-2,3-dione to an N-containing compound and potassium carbonate is 1:3-5:1.5-2.5; in step 3, the molar ratio of 5-substituted-1-substituted indoline-2,3-dione to aminothiourea and glacial acetic acid is 1:1-1.3:0.08-0.12, and the heating temperature is 79-82°C.

9. The use of the indigo derivative according to claim 1 in the preparation of plant antibacterial drugs, characterized in that: The fungus mentioned is either *Curvularia micrantha* or *Pseudomonas macrantha*.

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