Discovery and Mechanism of Novel 7-Aliphatic Amine Tryptanthrin Derivatives against Phytopathogenic Bacteria

Rice bacterial leaf blight is a destructive bacterial disease caused by Xanthomonas oryzae pv. oryzae (Xoo) that seriously threatens crop yields and their associated economic benefits. In this study, a series of improved dissolubility 7-aliphatic amine tryptanthrin derivatives was designed and synthesized, and their potency in antibacterial applications was investigated. Notably, compound 6e exhibited excellent activity against Xoo, with an EC50 value of 2.55 μg/mL, compared with the positive control bismerthiazol (EC50 = 35.0 μg/mL) and thiodiazole copper (EC50 = 79.4 μg/mL). In vivo assays demonstrated that 6e exhibited a significant protective effect on rice leaves. After exposure, the morphology of the bacteria was partially atrophied by SEM. Furthermore, 6e increased the accumulation of intracellular reactive oxygen species, causing cell apoptosis and the formation of bacterial biofilms. All the results indicated that 6e could be a potential agrochemical bactericide for controlling phytopathogenic bacteria.


Introduction
Oryza sativa L. is a staple food crop, feeding more than half of the world's population. However, it is often attacked by various pathogens, such as bacterial leaf blight in rice, during production, which seriously affects rice yield and quality and even endangers global food security and economic development [1][2][3]. Xoo, Xanthomonas axonopodis pv. Citri (Xac), and Pseudomonas syringae pv. Actinidiae (Psa) are the three major diseases threatening the health of agricultural crops. Among them, bacterial leaf blight is one of the three most serious rice diseases caused by Xoo, which harms the leaves and leaf sheaths of rice owing to its high epidemic potential and the lack of effective bactericides. In severe epidemics outbreaks, crop losses may be as high as 75%, and millions of hectares of rice are severely infected annually. Existing commercial antibacterials agents such as bismerthiazol (BT) and thiodiazole copper (TC) are incapable of effectively controlling the spread of bacterial diseases [4][5][6][7].
Moreover, the long-term, excessive use of traditional agrochemicals has led to imbalances of soil nutrients and a decline in soil fertility and organic matter, so the problem of soil and water pollution is becoming increasingly prominent. A large number of toxic substances' residues also pose serious safety risks that threaten the safety of agricultural substances' residues also pose serious safety risks that threaten the safety of agricultural products and the environment. Therefore, driven by the importance of food security and increasing environmental protection, the creation of green pesticides with high efficiency, low toxicity, and low amounts of residue based on bio-derived pesticides is critical to solving traditional pesticide problems [8][9][10][11][12]. Natural compounds play an important role in drug discovery and are rich sources of lead compounds or pharmacophores used in new drug discovery. However, due to the limited resources of natural products, their low concentrations of active components, their poor specificity of action, and their inadequate pharmacokinetic properties, it is necessary to modify and optimize the structure of lead compounds via chemical synthesis to make them into more ideal drugs [13,14].
Tryptanthrin is a kind of indolequinazoline alkaloid that can be isolated from a variety of natural materials, including Isatis tinctoria, Strobilanthes cusia, Polygonum tinctorium, and Wrightia tinctoria. Tryptanthrin and its derivatives offer a wide range of biological activities, including antibacterial, anti-inflammatory, antileishmanial, antiviral, and antimalarial activity [15][16][17][18][19]. They also have excellent research and application prospects, as evidenced by their use in compounds such as Phaitanthrin A, Phaitanthrin B, and Phaitanthrin C ( Figure 1A) [20][21][22][23]. Therefore, our group introduced novel 7-aliphatic amine of flexible fragments to the tryptanthrin master ring skeleton, expecting to obtain candidate compounds with stronger antibacterial activity ( Figure 1B). Surprisingly, compound 6e showed significant improvement against the three plant bacteria compared to the positive control drugs BT and TC, especially against Xoo. A valuable finding was that compound 6e showed effective therapeutic and protective activity in vivo toward rice. The preliminary antibacterial mechanism of compound 6e was also investigated using scanning electron microscopy (SEM), analyzing the accumulation of reactive oxygen species (ROS), examining rates of apoptosis, and through a biofilm assay.

Chemistry
The titular compounds 6a-6z were prepared using a series of optimized traditional methods (as depicted in Scheme 1). In brief, the classic Sandmeyer reaction steps were adopted, wherein substituted anilines were employed as the starting materials. they reacted with chloral hydrate and hydroxylamine hydrochloride to yield the oxime group acetylaniline compound 2, and the cyclization reaction was carried out with concentrated sulfuric acid, which was hydrolyzed to yield intermediates 3a-3i. Meanwhile, various substituted isatoic anhydrides (4a-4h) were prepared via an oxidation reaction in dichloromethane solvent using m-chloroperbenzoic acid. Then, using acetonitrile as the solvent, using triethylamine as catalyst, the optimized Bergman method was used to perform reflux reaction and recrystallize methanol to obtain 7-chlorotryptanthrin derivatives containing various substituent groups, appearing yellow needle-shaped crystal compounds. Finally, through the traditional nucleophilic substitution reaction, aliphatic amines with a flexible structure were introduced into the tryptanthrin skeletons to obtain the target compounds 6a-6z, which offer superior antibacterial activity against plant pathogens.

Chemistry
The titular compounds 6a-6z were prepared using a series of optimized traditional methods (as depicted in Scheme 1). In brief, the classic Sandmeyer reaction steps were adopted, wherein substituted anilines were employed as the starting materials. they reacted with chloral hydrate and hydroxylamine hydrochloride to yield the oxime group acetylaniline compound 2, and the cyclization reaction was carried out with concentrated sulfuric acid, which was hydrolyzed to yield intermediates 3a-3i. Meanwhile, various substituted isatoic anhydrides (4a-4h) were prepared via an oxidation reaction in dichloromethane solvent using m-chloroperbenzoic acid. Then, using acetonitrile as the solvent, using triethylamine as catalyst, the optimized Bergman method was used to perform reflux reaction and recrystallize methanol to obtain 7-chlorotryptanthrin derivatives containing various substituent groups, appearing yellow needle-shaped crystal compounds. Finally, through the traditional nucleophilic substitution reaction, aliphatic amines with a flexible structure were introduced into the tryptanthrin skeletons to obtain the target compounds 6a-6z, which offer superior antibacterial activity against plant pathogens. Scheme 1. Synthetic route for the titular compounds 6a-6z. Scheme 1. Synthetic route for the titular compounds 6a-6z.

Antibacterial Activity of Target Compounds 6a-6z
The activity of 7-aliphatic amine tryptanthrin derivatives was evaluated in vitro against three types of pathogenic bacteria using the turbidimetric method. Commercialized BT and TC served as reference substances. The resulting EC 50 values are shown in Table 1; they suggest that most of the target compounds had prominent in vitro anti-Xoo, anti-Xac, and anti-Psa activity compared with the original skeleton of tryptanthrin and the control agents BT and TC. Notably, compound 6e exhibited the strongest antibacterial activity, with EC 50 values = 2.55 µg/mL and 4.01 µg/mL against Xoo and Xac, respectively. Compared to tryptanthrin, which presented EC 50 values = 117 µg/mL and 126 µg/mL, amounting to 45. 8 Analysis of antibacterial activity. Combined with EC 50 values in Table 1, the introduction of 7-aliphatic amine to tryptanthrin significantly increased antibacterial activity. For instance, when the 7-position substituent group was N1, N1-dimethylpropane-1,3-diamine, superior inhibitory activity toward Xoo was observed. The 2-substitution of tryptanthrin appears to be associated with higher antibacterial effects against Xoo and increased activity, presenting an (R 2 ) of (6e) F > (6i) OCH 3 > (6g) Br> (6b) H> (6j) CH 3 > (6k) NO 2 . As a cycloaliphatic amine substituent, piperazine is often used as a linker to link active substructures with promising biological activities, allowing for the significant improvement of the antibacterial activity of the corresponding compounds. However, the inhibitory activity of methylpiperazine substituents was weaker than that of piperazine. When the substitution group was morpholine, the antibacterial activity was weaker and almost non-existent.

In Vivo Bioassay Results of Compound 6e against Rice Bacterial Leaf Blight
Given the superior in vitro bioactivity of compound 6e against Xoo (EC 50 = 2.55 µg/mL), in vivo bioassays concerning compound 6e were further conducted, and the results are shown in Figure 2 and Table 2. Notably, compound 6e presented better control efficacy with respect to anti-Xoo activity (45.02% of protective activity and 43.75% of curative activity) toward rice bacterial blight under controllable greenhouse conditions at a dosage of 200 µg/mL; this activity is superior to that of commercialized BT (42.18% of protective activity and 47.32% of curative activity). After bacterial infection, the degree of leaf wilt in the blank control group and the 6e treatment group changed, as shown in Figure 2. Most of the leaves in the untreated group presented chlorosis and withered leaves, while only a small amount of bacterial contamination occurred in the tips of the leaves in the treated group. Compound 6e presented significant therapeutic and protective effects in the in vivo rice experiments. Therefore, it has desirable applicational prospects in the control of diseases caused by rice bacteria.

Effect of Compound 6e on the Morphology of Xoo Cells
In order to further verify the cells morphological changes induced by compound 6e on the Xoo bacteria, SEM imaging was performed (Figure 3). At a compound 6e concentration of 10 × EC 50 , it could be observed that part of the membrane of Xoo cells had assumed a folded, rod-shaped form and begun to shrink and collapse. When the concentration of compound 6e was increased to 20 × EC 50 , the cell morphology began to show severe shrinkage and deformation. However, compared to the untreated Xoo cells, there were complete cell membranes and complete rod shapes. Especially in the areas marked by red arrows, the collapse and contraction of cells were clearly evident. These results suggested that higher concentrations of compound 6e could affect the membrane integrity of Xoo cells and ultimately lead to bacterial apoptosis. The SEM results showed that compound 6e had a concentration-dependent effect on cell morphology. Since there are many factors that affect the alteration of cell morphology, we studied their interactions in terms of the elevation of ROS, the rate of apoptosis, and the influence on the formation of bacterial biofilms.

Compound 6e Induced ROS Accumulation
On the one hand, reactive oxygen species (ROS) play an important role in maintaining important biological functions of cells; On the other hand, an excessive accumulation of ROS can inhibit normal growth and even promote the apoptosis of cells. Therefore, to explore whether the significant anti-proliferative activity of compound 6e was related to ROS levels, Xoo cells were treated with 6e for 12 h ((0, 1 × EC 50 , 2 × EC 50 , 5 × EC 50 , and 10 × EC 50 ) and then loaded with 10 mM DCFH-DA for the examination of ROS using kit. Intracellular ROS can oxidize non-fluorescent DCFH to produce fluorescent DCF, and the fluorescence value of DCF can be detected to determine the levels of intracellular ROS. The results demonstrated that compound 6e significantly increased intracellular ROS accumulation in a dose-dependent manner, especially at a concentration of 10 × EC 50 , which the amount of ROS reached the maximum value of 157; Even at a lower concentration of 1 × EC 50 , ROS also increased significantly ( Figure 4). Therefore, it was concluded that the significant inhibitory effect of compound 6e on Xoo pathogens may be related to the accumulation of intracellular ROS.   In order to further verify the cells morphological changes induced by compound 6e on the Xoo bacteria, SEM imaging was performed (Figure 3). At a compound 6e concentration of 10 × EC50, it could be observed that part of the membrane of Xoo cells had assumed a folded, rod-shaped form and begun to shrink and collapse. When the concentration of compound 6e was increased to 20 × EC50, the cell morphology began to show severe shrinkage and deformation. However, compared to the untreated Xoo cells, there were complete cell membranes and complete rod shapes. Especially in the areas marked by red arrows, the collapse and contraction of cells were clearly evident. These results suggested that higher concentrations of compound 6e could affect the membrane integrity of Xoo cells and ultimately lead to bacterial apoptosis. The SEM results showed that compound 6e had a concentration-dependent effect on cell morphology. Since there are many factors that affect the alteration of cell morphology, we studied their interactions in terms of the elevation of ROS, the rate of apoptosis, and the influence on the formation of bacterial biofilms.

Compound 6e Induced ROS Accumulation
On the one hand, reactive oxygen species (ROS) play an important role in maintaining important biological functions of cells; On the other hand, an excessive accumulation of ROS can inhibit normal growth and even promote the apoptosis of cells. Therefore, to explore whether the significant anti-proliferative activity of compound 6e was related to ROS levels, Xoo cells were treated with 6e for 12 h ((0, 1 × EC50, 2 × EC50, 5 × EC50, and 10 × EC50) and then loaded with 10 mM DCFH-DA for the examination of ROS using kit. Intracellular ROS can oxidize non-fluorescent DCFH to produce fluorescent DCF, and the fluorescence value of DCF can be detected to determine the levels of intracellular ROS. The results demonstrated that compound 6e significantly increased intracellular ROS accumulation in a dose-dependent manner, especially at a concentration of 10 × EC50, which the amount of ROS reached the maximum value of 157; Even at a lower concentration of 1 × EC50, ROS also increased significantly ( Figure 4). Therefore, it was concluded that the significant inhibitory effect of compound 6e on Xoo pathogens may be related to the accumulation of intracellular ROS.

Compound 6e Induced Bacterial Cells' Apoptosis
The accumulation of intracellular reactive oxygen species may eventually lead to cell apoptosis. Therefore, to further verify that 6e induced apoptosis, Xoo cells were treated with either DMSO or various other concentrations of compound 6e. Then, the cells were harvested and stained with Annexin V-FITC and propidium iodide (PI), and the percentage of apoptotic cells was analyzed using flow cytometry. As shown in Figure 5, the apop-

Compound 6e Induced Bacterial Cells' Apoptosis
The accumulation of intracellular reactive oxygen species may eventually lead to cell apoptosis. Therefore, to further verify that 6e induced apoptosis, Xoo cells were treated with either DMSO or various other concentrations of compound 6e. Then, the cells were harvested and stained with Annexin V-FITC and propidium iodide (PI), and the percentage of apoptotic cells was analyzed using flow cytometry. As shown in Figure 5, the apoptotic rates of the drug-treated cells were positively correlated with the concentration changes. After treatment with different concentrations of 1 × EC 50 , 2 × EC 50 , 5 × EC 50 , and 10 × EC 50 , the total apoptotic rates were 18.76%, 22.53%, 32.87%, and 65.24%, respectively, whichcan be compared with that of 8.87% in the negative control group, thus showing that compound 6e effectively induced apoptosis in Xoo cells.

Compound 6e Inhibited the Formation of Bacterial Biofilms
Biofilms are mostly composed of microcolonies encased in an EPS matrix, proteins, and extracellular DNA, which adhere to each other in very fine ways, forming membranes that act as barriers and space-occupying forms of protection, preventing foreign germs from colonizing the body and invading through the portal [24]. Therefore, through a crystal violet staining experiment, the OD value was measured at the 570 nm wavelength, and the biofilm was quantitatively measured to study whether compound 6e could achieve an antibacterial effect by inhibiting the formation of Xoo bacterial biofilm; the corresponding results are shown in Figure 6. Obviously, the degree of biofilm formation was greater in the blank control sample, in which a bright blue biofilm was presented, and the maximum average OD value was 2.48. With the addition of compound 6e, the degree of biofilm formation gradually decreased, and the bright blue color gradually became lighter. When the concentration increased to 10 × EC50, the color was at its lightest, and the minimum average OD value was 0.507. These results suggest that compound 6e may act on Xoo bacteria in a dose-dependent manner by influencing the formation of bacterial biofilms.

Compound 6e Inhibited the Formation of Bacterial Biofilms
Biofilms are mostly composed of microcolonies encased in an EPS matrix, proteins, and extracellular DNA, which adhere to each other in very fine ways, forming membranes that act as barriers and space-occupying forms of protection, preventing foreign germs from colonizing the body and invading through the portal [24]. Therefore, through a crystal violet staining experiment, the OD value was measured at the 570 nm wavelength, and the biofilm was quantitatively measured to study whether compound 6e could achieve an antibacterial effect by inhibiting the formation of Xoo bacterial biofilm; the corresponding results are shown in Figure 6. Obviously, the degree of biofilm formation was greater in the blank control sample, in which a bright blue biofilm was presented, and the maximum average OD value was 2.48. With the addition of compound 6e, the degree of biofilm formation gradually decreased, and the bright blue color gradually became lighter. When the concentration increased to 10 × EC 50 , the color was at its lightest, and the minimum average OD value was 0.507. These results suggest that compound 6e may act on Xoo bacteria in a dose-dependent manner by influencing the formation of bacterial biofilms.
Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 10 of 14 Figure 6. Inhibition of biofilm formation incubated with compound 6e was analyzed using crystal violet assay. The symbols ** and *** stand for p < 0.01 and p < 0.001, respectively, compared with the controls.

Chemistry
The 1 H and 13 C NMR data were obtained using a Bruker Avance III 400 MHz NMR spectrometer (Bruker Optics, Billerica, MA, USA), for which CDCl3 was used as a solvent and the chemical shift (δ) was expressed in parts per million (ppm). High-resolution mass spectra (ESI-HRMS) were acquired using a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, St. Louis, MO, USA). The melting points (mp.) for all the titular compounds were detected using a (X-4D) digital micromelting point apparatus (Cewei Optoelectronic Technology Co., Ltd., Shanghai, China). The data on ROS and apoptosis were obtained using a BD FACSCalibur flow cytometer(Beckman Coulter, Inc. 250S. Kraemer Blvd. Brea, CA, USA). All the starting materials, reagents, and solvents were purchased from commercial sources without further purification. Detailed characterization data are presented in the Supporting Information (Figures S1-S78).

General Synthesis Procedure for Intermediates 3a-3i and 4a-4h
The syntheses of intermediates 3a-3i were conducted by referring to the literature [25][26][27]. Indoline-2,3-dione derivatives were synthesized using a lightly optimized procedure for provoking the Sandmeyer reaction, in which the intermediate compound 2 was obtained through a mixture of chloral hydrate, substituted anilines, hydroxylamine hydrochloride, hydrochloric acid, and sulfate-saturated aqueousa. Additionally, concentrated sulfuric acid was stirred in a heating system, with the temperature strictly maintained at 90 °C for 15 min, and then poured into ice water for the cyclic reaction for 30 min to obtain 3a-3i. Compounds 4a-4h were obtained via reflux reaction with 1.2 equivalents of m-chloroperoxybenzoic acid in dichloromethane solvent.

General Synthesis Procedure for Intermediates 5a-5h
Intermediates 5a-5h, with various substituent groups of 7-chlorotryptanthrin, were prepared according to a previously reported method, which simple optimization was carried out [28,29]. Figure 6. Inhibition of biofilm formation incubated with compound 6e was analyzed using crystal violet assay. The symbols ** and *** stand for p < 0.01 and p < 0.001, respectively, compared with the controls.

Chemistry
The 1 H and 13 C NMR data were obtained using a Bruker Avance III 400 MHz NMR spectrometer (Bruker Optics, Billerica, MA, USA), for which CDCl 3 was used as a solvent and the chemical shift (δ) was expressed in parts per million (ppm). High-resolution mass spectra (ESI-HRMS) were acquired using a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, St. Louis, MO, USA). The melting points (mp.) for all the titular compounds were detected using a (X-4D) digital micro-melting point apparatus (Cewei Optoelectronic Technology Co., Ltd., Shanghai, China). The data on ROS and apoptosis were obtained using a BD FACSCalibur flow cytometer(Beckman Coulter, Inc. 250S. Kraemer Blvd. Brea, CA, USA). All the starting materials, reagents, and solvents were purchased from commercial sources without further purification. Detailed characterization data are presented in the Supporting Information (Figures S1-S78).

General Synthesis Procedure for Intermediates 3a-3i and 4a-4h
The syntheses of intermediates 3a-3i were conducted by referring to the literature [25][26][27]. Indoline-2,3-dione derivatives were synthesized using a lightly optimized procedure for provoking the Sandmeyer reaction, in which the intermediate compound 2 was obtained through a mixture of chloral hydrate, substituted anilines, hydroxylamine hydrochloride, hydrochloric acid, and sulfate-saturated aqueousa. Additionally, concentrated sulfuric acid was stirred in a heating system, with the temperature strictly maintained at 90 • C for 15 min, and then poured into ice water for the cyclic reaction for 30 min to obtain 3a-3i. Compounds 4a-4h were obtained via reflux reaction with 1.2 equivalents of m-chloroperoxybenzoic acid in dichloromethane solvent.

General Synthesis Procedure for Intermediates 5a-5h
Intermediates 5a-5h, with various substituent groups of 7-chlorotryptanthrin, were prepared according to a previously reported method, which simple optimization was carried out [28,29].

General Procedures for the Synthesis of Target Compounds 6a-6z
Potassium carbonate (3.0 mmol, 0.414 g) was added in batches to a mixture of 7chlorotryptanthrin and a set of its derivatives, namely, 5a-5h (1.0 mmol, 0.282 g), in DMF (5.0 mL) and stirred at room temperature for 30 min. Then, the corresponding aliphatic amines (3.0 mmol) were added, and the resulting mixture was refluxed for 1-3 h. Finally, the precipitated solid was filtered and washed carefully with cold, distilled water and methanol to obtain the desired target compounds 6a-6z.

In Vitro Antibacterial Bioassay
In vitro antibacterial activities of title compounds 6a-6z against three pathogenic bacteria, namely, Xoo, Xac, and Psa, were determined using the classical turbidimetric method [30][31][32]. TC and BT were employed as representative commercial drugs serving as positive controls, and DMSO was used as the blank control. The inhibition rate (I) was calculated as follows: I (%) = (C − T)/C × 100. C is obtained by correcting the turbidity value of bacterial growth on the treated NB, and T is the drug-containing sample. The inhibitory activity of all target compounds against three strains of bacteria at different concentrations was further determined. The EC 50 values were obtained by measuring the optical density value at 595 nm, and SPSS 19.0 software was used for statistics.

In Vivo Assay against Rice Bacterial Blight
The protective and curative activities of compound 6e against rice bacterial leaf blight were determined using a previously reported method with some slight modifications [33]. "Fengyouxiangzhan" rice variety was grown until the tillering stage (28 • C and 90% RH) under greenhouse conditions to perform the antimicrobial experiment. After inoculation using a shear method, the therapeutic and protective effects of rice were tested using a spraying method. BT (90% white powder) and TC (20% suspending agent) were used as positive control agents. The rice plants cultured under controlled greenhouse conditions were subjected to in vivo experiments conducted on rice leaves at the tillering stage. The control efficiency was calculated using the following formula after 14 days of activity at a drug concentration of 200 µg/mL: I (%) = (C − T)/C × 100%. In the formula, C is the disease index for the negative control, and T is the disease index for the treatment group.

Morphological Investigations Using SEM
A bacterial morphology assay was carried out using previous research as a reference [34][35][36]. Briefly, 1.5 mL of Xoo cell suspension grown until the logarithmic stage was centrifuged, collected, washed with cooled PBS buffer solution 3 times, and then re-suspended with the same volume of PBS. Various concentrations of compound 6e (0, 10 × EC 50 , and 20 × EC 50 ) were added and incubated for 8 h at 28 • C/180 rpm. Then, the samples were washed with PBS 3 times once again. The Xoo cells were fixed overnight with 2.5% glutaraldehyde, dehydrated with gradient ethanol, replaced with tert-butanol solvent, and finally vacuum freeze-dried. Morphological changes of the Xoo bacteria were observed using scanning electron microscopy.

Detection of Reactive Oxygen Species
To assess the intracellular production of ROS, the Xoo pathogenic bacterial cells were treated with different concentrations of 6e (0, 1 × EC 50 , 2 × EC 50 , 5 × EC 50 , and 10 × EC 50 ) using a previously reported method with a slight modification [37]. After treatment for 12 h, cells were harvested and stained with 10 µM of DCFH-DA for 30 min at room temperature using a cell-based ROS assay kit. Next, cells were collected and measured using a BD FACSCalibur flow cytometer (Beckman Coulter, Inc. 250S. Kraemer Blvd., Brea, CA, USA).

Induction of Apoptosis in Pathogenic Bacterial Cells
To investigate whether the cell growth inhibition was associated with apoptosis, the plant pathogenic bacterial cells were treated with either DMSO or various concentrations of the target compound for 15 h using previous reported methods with slight modifications [38,39]. After treatment with different concentrations of 6e (0, 1 × EC 50 , 2 × EC 50 , and 5 × EC 50 ), the cells were washed twice with cold PBS, centrifuged, and collected. Then, the cells were stained with Annexin V-FITC and propidium iodide (PI) and analyzed using a flow cytometer to detect the apoptosis ratio. Quadrant panels were separated into four gates labeled Q1 (necrotic cells), Q2 (late apoptotic cells), Q3 (early apoptotic cells), and Q4 (viable cells). The control group consisted of untreated cells.

Bacterial Biofilm Assay
All of the strains of bacteria tested were provided by the Laboratory of Plant Disease Control at Guizhou University. NA media containing Xoo bacteria (OD 595 = 0.2) and different concentrations of compound 6e (0, 1 × EC 50 , 2 × EC 50 , 5 × EC 50 , and 10 × EC 50 ) were prepared in 5 mL glass test tubes and then added to 96-well polystyrene plates, as previously described [40,41]. After being cultured in a constant temperature incubator (28 • C) for 3 days, the culture medium was removed and cleaned twice with sterile, secondary distilled water. Next, after being dried at room temperature, crystal purple solution (0.1%) was added for a 10 min staining period. The staining agent was discarded and cleaned twice with sterile secondary distilled water again. Finally, the absorbance OD value was measured at 570 nm after the sample was dried again and fully dissolved with 95% ethanol.

Conclusions
In this study, a series of 7-aliphatic amine tryptanthrin derivatives, 6a-6z, was designed and synthesized by combining flexible small molecular fragments of aliphatic amines, which enhanced the solubility of the target compounds and broke the structures of planar aromatic molecules. The antibacterial bioassay results showed that most of the target compounds exhibited obvious antibacterial activity against three species of plant bacteria in vitro. Furthermore, compound 6e displayed the greatest antibacterial activity against Xoo in vitro and was effective in reducing rice bacterial leaf blight in vivo. The preliminary mechanism of compound 6e was investigated, and the results demonstrated that compound 6e could precipitate obvious changes in bacterial morphology, induce ROS accumulation, stimulate cell apoptosis, and inhibit the formation of biofilms. The current work provides valuable insights into tryptanthrin and its derivatives for use in the field of natural agricultural bactericides.