Identification of acylhydrazone derivatives with isopropanolamine moiety as potent anti‐virulence agents for combating bacterial infection

Zhi‐Jun Luo; Tai‐Hong Zhang; Jia‐Rui Rao; Pan Xu; Yan‐Yan Wen; San Tu; Qing‐Rong Bai; Xiang Zhou; Li‐Wei Liu; Zhi‐Bing Wu; Song Yang

doi:10.1002/ps.70963

. 2026 May 25;82(9):9185–9200. doi: 10.1002/ps.70963

Identification of acylhydrazone derivatives with isopropanolamine moiety as potent anti‐virulence agents for combating bacterial infection

Zhi‐Jun Luo ^1,^#, Tai‐Hong Zhang ^1,^#, Jia‐Rui Rao ¹, Pan Xu ¹, Yan‐Yan Wen ¹, San Tu ¹, Qing‐Rong Bai ¹, Xiang Zhou ^1,^✉, Li‐Wei Liu ¹, Zhi‐Bing Wu ¹, Song Yang ^1,^✉

PMCID: PMC13453249 PMID: 42185751

Abstract

BACKGROUND

Quorum sensing (QS)‐regulated virulence factors including biofilms are key drivers of phytopathogen pathogenicity and antimicrobial resistance. Unlike conventional bactericides that exert selective pressure leading to resistance evolution, anti‐virulence agents offer a promising strategy to control bacterial diseases without promoting resistance, having significant potential in the creation of new bactericides.

RESULTS

In this study, hydroxybenzaldehyde was used as the starting material to synthesize a series of 39 novel acylhydrazone derivatives containing flexible isopropanolamine moieties. Among these, compound C19 exhibited excellent in vitro activity against the rice bacterial blight pathogens Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas axonopodis pv. citri (Xac), with in vitro EC₅₀ (Half maximal effective concentration) values of 1.66 μg/mL and 1.19 μg/mL, respectively—significantly outperforming commercial bactericides bismerthiazol (BT EC₅₀ = 45.74 μg/mL for Xoo; 51.23 μg/mL for Xac) and thiodiazole‐copper (TC EC₅₀ = 108.10 μg/mL for Xoo; 81.54 μg/mL for Xac). At a concentration of 200 μg/mL, compound C19 showed protective and curative efficacies of 47.3% and 46.2%, respectively, both of which were superior to those of the commercial bactericides BT (44.3% and 44.5%) and TC (43.8% and 42.5%). Mechanistically, compound C19 effectively inhibited Xoo biofilm formation, bacterial motility and extracellular enzyme secretion, thereby reducing pathogen virulence and thus suppressing the bacterial infection process.

CONCLUSION

This study successfully designed and synthesized a series of novel isopropanolamine‐modified acylhydrazone derivatives as potent anti‐virulence agents. Compound C19, with its excellent antibacterial activity, favorable safety profile and unique anti‐virulence performance, represents a promising lead compound for the development of novel environmentally friendly bactericides to combat bacterial resistance. © 2026 Society of Chemical Industry.

Keywords: acylhydrazone derivatives, biofilm, virulence factors, antibacterial activity, drug resistance

Novel acylhydrazone compounds bearing an isopropanolamine substructure exhibit inhibitory effects on bacterial virulence factors. This study demonstrates excellent potential in combating bacterial infections, and laying an important foundation for the design of acylhydrazone‐based bactericides.

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1. INTRODUCTION

To date, bacterial plant diseases have posed a severe threat to crop growth and development, impairing harvest quality and reducing yields by 10–50% while inflicting substantial economic losses. ¹ , ² Notably, Gram‐negative bacteria—particularly species of the genus Xanthomonas—rank among the most intractable phytopathogens worldwide. ³ , ⁴ Representative examples include Xanthomonas oryzae pv. oryzae (Xoo), ⁵ , ⁶ the causal agent of rice bacterial leaf blight (BLB), and Xanthomonas axonopodis pv. citri (Xac), ⁷ , ⁸ the pathogen responsible for citrus canker; both exhibit strong pathogenicity toward their respective host plants. ⁹ Early studies have confirmed that Xanthomonas spp. can form biofilms on both abiotic and biotic surfaces, a trait that further enhances their environmental adaptability and pathogenicity. ¹⁰ , ¹¹ However, the long‐term, frequent and repetitive application of traditional bactericides has accelerated the evolutionary rate of phytopathogenic bacteria and the development of pesticide resistance, thereby diminishing the control efficacy of these agents. ¹² , ¹³ , ¹⁴ , ¹⁵ Furthermore, increasing pesticide dosages to ensure harvest yields inevitably exacerbates environmental contamination, which runs counter to the objectives of sustainable and green agricultural development. Therefore, there is an urgent need to develop novel bactericides characterized by unique modes of action, environmental benignity, and the ability to mitigate the risk of bacterial resistance.

Notably, biofilms formed by phytopathogens—especially those produced by Xanthomonas spp.—play a pivotal role in the infection process. ¹⁶ Taking Xoo as an example, the biofilms it synthesizes, in conjunction with other virulence factors [e.g., extracellular polysaccharides (EPS), extracellular enzymes, and type III secretion system (T3SS) effectors], protect the pathogen from adverse environmental conditions and facilitate its invasion of host plants. ¹⁷ , ¹⁸ Recent studies have further indicated that biofilm formation during bacterial infection represents an effective evolutionary strategy for evading antimicrobial agents. ¹⁹ , ²⁰ Bacteria within biofilms form well‐organized aggregates embedded in an EPS matrix, exhibiting 10–1000‐fold higher resistance to antimicrobials compared with their planktonic counterparts. ²¹ , ²² Intriguingly, a growing body of evidence has shown that anti‐quorum sensing (anti‐QS) inhibitors can disrupt bacterial infection processes while minimizing the development of drug resistance. This is because such inhibitors avoid the selective pressure imposed by bactericidal agents; instead of killing bacteria, they only attenuate the pathogens’ infectivity. ²³ Thus, targeting bacterial virulence—either by inhibiting biofilm formation or suppressing key virulence factors—has emerged as a novel strategy for developing high‐efficiency bactericides with unique mechanisms to control bacterial plant diseases.

Notably, acylhydrazones, as a distinct class of hydrazine derivatives, possess remarkable structural diversity and broad application prospects. ²⁴ , ²⁵ Their abundant reactive sites not only establish this molecular scaffold as a privileged structure in organic chemistry, but also render it a crucial scaffold for the synthesis of pharmaceutically valuable compounds. ²⁶ , ²⁷ Carbazochrome, a commercially available drug, acts as an efficient hemostatic agent; additionally, nifuroxazide—another antibiotic based on the nitroacylhydrazones scaffold—has been officially approved for the clinical treatment of colitis and diarrhea in adult patients. Intriguingly, Polovic et al. demonstrated that specific acylhydrazone derivatives (compounds 4 and 5) exerted prominent antibacterial effects by significantly attenuating various bacterial virulence factors involved in the bacterial infection process. ²⁸ Interestingly, He et al.'s group reported that compound 6, as a potential photosynthetic inhibitor, exhibited excellent algicidal activity and low toxicity, making it a promising candidate for developing effective and safe algicides. ²⁹ These compelling findings fully illustrate that acylhydrazones possess substantial pharmaceutical potential and promising anti‐biofilm activity, but their application in combating plant bacterial pathogens has rarely been reported. On the other hand, the isopropanolamine fragment has been extensively documented to exhibit potent antibacterial activity alongside remarkable anti‐QS properties. For instance, Feng et al., ³⁰ Zhang et al., ³¹ and Chu et al. ³² independently reported that compounds 7, 8, and 9 displayed significant in vivo anti‐Xoo efficacy, with their underlying mechanisms primarily associated with the inhibition of pathogenic factors such as biofilm formation. Consequently, strategic introduction of the isopropanolamine moiety into the acylhydrazone pharmacophore is hypothesized to hold great potential for discovering novel anti‐virulence agents.

Based on the aforementioned research findings, we rationally designed and synthesized a series of novel isopropanolamine‐modified hydroxybenzaldehyde derivatives, using p‐hydroxybenzaldehyde (PHBA) as the starting material and introducing both the isopropanolamine moiety and acylhydrazone groups into its molecular structure. Subsequently, we conducted a systematic and in‐depth investigation on the bioactivities of these newly synthesized derivatives, as well as the intrinsic molecular mechanisms governing their anti‐biofilm effects, aiming to provide valuable insights for the development of novel, high‐efficiency antibacterial agents.

2. MATERIALS AND METHODS

2.1. Instruments and chemicals

Nuclear magnetic resonance (NMR, ¹H NMR, ¹³C NMR, and ¹⁹F NMR) spectra of the target compounds were recorded on a Bruker Biospin‐AG‐400 spectrometer (Bruker Optics, Ettlingen, Germany), with DMSO‐d ₆ or CDCl₃ as the solvent and tetramethylsilane (TMS) as the internal standard. High‐resolution mass spectrometry (HRMS) data were obtained using an UltiMate 3000 instrument (Thermo Scientific, Thermo Scientific, Waltham, Massachusetts, USA). Scanning electron microscopy (SEM) images were captured with an FEI Nova NanoSEM 450 microscope (FEI, Hillsboro, Oregon, USA). A Cytation™ 5 multimode microplate reader (BioTek Instruments, Inc., USA) was used to measure the optical density at 595 nm (OD₅₉₅). Hypersensitivity reaction (HR) symptoms were imaged using the Bio‐Rad Chemi Doc MP gel imaging system (Bio‐Rad Laboratories, Hercules, CA, USA). Crystal violet, soluble starch, sodium carboxymethyl cellulose, and Congo red were purchased from Sangon Biotech (Shanghai, China) Co., Ltd. Bromopropylene oxide, p‐hydroxybenzaldehyde, and various hydrazides were supplied by Shanghai Bide Pharmaceutical Technology Co., Ltd (Shanghai, China). All other chemical reagents and solvents were acquired from commercial vendors.

2.2. Strain sources and preservation

The wild‐type Xoo strain ZJ173 was generously provided by Professor Ming‐Guo Zhou from Nanjing Agricultural University (Nanjing, China). Xac strains were preserved in our laboratory.

2.3. Antibacterial activity evaluation

The in vitro antimicrobial activity of all target compounds against two pathogenic strains (Xoo and Xac) was assessed using the classical turbidimetric method, as previously described in the literature. ³³ Commercial bactericides bismerthiazol (BT) and thiodiazole‐copper (TC) served as positive controls. In addition, the in vivo inhibitory effect of the compounds on rice leaf blight was examined following our previously reported protocol. ³⁴

2.4. Growth curve assay

Based on the in vitro bioassay results, the active compound C19 was selected to explore its influence on Xoo growth by conducting the growth curve assay. ³⁵ Briefly, Xoo cells were first cultured overnight in nutrient broth (NB) medium to reach the logarithmic growth phase (OD₅₉₅ = 0.6–0.8). The bacterial suspension was then diluted with sterile NB medium to an OD₅₉₅ value of 0.1, and immediately co‐incubated with different concentrations of compound C19 (0, EC₅₀, 2.5 EC₅₀, 5 EC₅₀) (EC₅₀, Half maximal effective concentration) in a shaker at 28 °C and 180 rpm. During the 30 h incubation period, the OD₅₉₅ values of all treatments were measured every 3 h using a microplate reader. Each treatment was set up with three biological replicates. Finally, growth curves were plotted, and the data were subjected to statistical analysis.

2.5. Inhibition of biofilm formation

The ability of compound C19 to inhibit Xoo biofilm formation was determined using the classical crystal violet staining method reported in the literature. ³⁶ Xoo cells from overnight cultures (OD₅₉₅ = 0.6–0.8) were adjusted to OD₅₉₅ = 0.1 with sterile NB medium; growth inhibition curves were used to verify that this concentration did not affect bacterial growth. Subsequently, 200 μL of the adjusted Xoo suspension was transferred to a 96‐well plate, followed by the addition of compound C19 at various concentrations [0, 1/8 minimal inhibitory concentration (MIC), 1/4 MIC, 1/2 MIC, MIC and 2 MIC]. Furthermore, the plate was incubated at 28 °C for 72 h. After incubation, the excess NB medium was carefully aspirated, and the wells were washed three times with sterile water before drying in a desiccator at 50 °C for 1 h. Then, 200 μL of 0.1% crystal violet staining solution was added to each well for 30 min, followed by three additional washes with sterile water to remove unbound dye. Finally, the stained biofilms were dissolved in 95% ethanol, and the OD₅₇₀ values were measured using a microplate reader.

2.6. Biofilm morphology analysis

Biofilm morphology was observed via SEM according to the method described previously. ³⁶ Briefly, 200 μL of Xoo suspension (OD₅₉₅ = 0.1) mixed with different concentrations of compound C19 (0, 1/4 MIC, 1/2 MIC, MIC and 2 MIC) was transferred to a six‐well polystyrene plate containing round glass coverslips and incubated at 28 °C for 5 days. The coverslips coated with biofilms were then removed and fixed overnight in 2.5% glutaraldehyde solution. After fixation, the samples were gently washed three times with phosphate‐buffered saline and sterile distilled water, followed by gradient dehydration with ethanol. Finally, the dehydrated samples were freeze‐dried, sputter‐coated with gold and observed under an SEM (FEI Nova NanoSEM 450).

2.7. Motility assay

Swimming motility assays were performed with appropriate modifications to the assay parameters, based on previously reported methods. ³⁷ , ³⁸ Briefly, 2 μL of Xoo suspension (OD₅₉₅ = 0.1) mixed with different concentrations of compound C19 (0, 1/8 MIC, 1/4 MIC, 1/2 MIC, MIC and 2 MIC) was inoculated into the center of a ix‐well polystyrene plate containing specially prepared NA medium (0.3% beef extract, 0.5% peptone, 0.1% yeast extract, 1% glucose, 0.1% agar powder, pH 7.1–7.2). After static incubation at 28 °C for 5 days, bacterial motility was observed and recorded. For bacterial aggregation assays, 2 μL of Xoo suspension (OD₅₉₅ = 1.0) mixed with the same concentrations of compound C19 was inoculated onto the same NA medium, and bacterial aggregation was evaluated after static incubation at 28 °C for 5 days.

2.8. Extracellular enzymatic assay

To investigate the effect of compound C19 on Xoo extracellular enzymes, the inhibitory activities against cellulase and amylase were preliminarily evaluated. Both enzyme activities were measured according to previously reported methods. ³⁰ , ³⁹

2.8.1. Extracellular cellulase

NA medium supplemented with 0.5% (w/v) sodium carboxymethyl cellulose (CMC) was sterilized under high temperature and pressure. After sterilization, the medium was mixed with different concentrations of compound C19 (0, 1/8 MIC, 1/4 MIC, 1/2 MIC, MIC and 2 MIC) and poured into Petri dishes to solidify. Subsequently, 2 μL of Xoo suspension (OD₅₉₅ = 0.5) was spotted onto the solidified medium, followed by incubation at 28 °C for 5 days. Post‐incubation, the plates were stained with 10 mL of 0.1% Congo red solution for 15 min, then decolorized twice with 1 mol/L NaCl solution (10 min per wash). Finally, the results were observed and photographed.

2.8.2. Amylase assay

NA medium containing 0.1% (w/v) soluble starch was sterilized under high temperature and pressure, then mixed with compound C19 to achieve final concentrations of 0, 1/8 MIC, 1/4 MIC, 1/2 MIC, MIC, and 2 MIC. After solidification, 2 μL of Xoo suspension (OD₅₉₅ = 0.5) was spotted onto the medium and incubated at 28 °C for 5 days. The plates were then stained with 10 mL of 1% I₂/KI solution for 10 min, followed by two rounds of decolorization with 70% ethanol (10 min each). All experimental results were observed, photographed and repeated in triplicate.

2.9. Pathogenicity assays and pathological sections of Xoo on rice

Pathogenicity assays were performed according to previously published protocols. ³⁰ , ⁴⁰ Briefly, Xoo suspension (OD₅₉₅ = 0.1) was added to conical flasks supplemented with compound C19 at final concentrations of 0, 1/4 MIC, 1/2 MIC, MIC and 2 MIC, then incubated at 28 °C for 12–14 h. The treated bacterial suspensions were subsequently inoculated onto leaves of 2‐month‐old rice plants using the leaf‐clipping method, and inoculated rice seedlings were maintained in a controlled greenhouse. Fourteen days post‐inoculation, tissue segments were excised from the lesion–healthy tissue boundary. Samples were processed via fixation, embedding, dewaxing, rehydration, staining, dehydration, clearing, and mounting prior to microscopic examination. Following toluidine blue O (TBO) staining, lignified cell walls stained blue–green, cellulose cell walls stained violet–blue, and bacterial cells stained deep purple.

2.10. Hypersensitive response assay

The hypersensitive response (HR) assay was conducted as described previously. ²⁹ Briefly, Xoo suspension (OD₅₉₅ = 0.1) was co‐incubated with compound C19 at final concentrations of 0, 1/4 MIC, 1/2 MIC, MIC, and 2 MIC in a shaking incubator at 28 °C for 2 h. Treated suspensions were then infiltrated into tobacco leaves using a needleless syringe until the infiltrated zone reached 1 cm in diameter. Inoculated tobacco plants were incubated in a plant growth chamber at 25 °C for 48 h, and HR symptoms were observed and imaged using a Bio‐Rad gel imaging system (Bio‐Rad Laboratories).

2.11. Statistical analysis

All data are expressed as mean ± standard deviation. Statistical analyses were performed using one‐way and two‐way analysis of variance in IBM SPSS Statistics 26.0 software (SPSS Inc., Chicago, IL, USA). Significance levels were set as *P < 0.05, **P < 0.01, and ***P < 0.001.

3. RESULTS AND DISCUSSION

3.1. Synthesis of intermediate and target compounds

To further excavate new anti‐virulence agents, A series acylhydrazone derivatives bearing flexible isopropanolamine moieties were synthesized by integrating a biologically active acylhydrazone scaffold with isopropanolamine‐based side chains. As shown in Fig. 1(b), a concise synthetic route for the target compound was designed. Specifically, the target compound was prepared via a straightforward three‐step synthetic route (Schemes 1 and 2), with intermediate 2 obtained through the alkylation reaction of p‐hydroxybenzaldehyde. This intermediate then underwent a ring‐opening reaction with various amine‐containing skeletons to yield intermediate 3. Finally, intermediate 3 reacted with a series of acylhydrazones via nucleophilic addition to afford the target compounds A1–A14, B1–B5 and C1–C20. All synthesized compounds were structurally characterized by NMR spectroscopy and HRMS, with the corresponding spectra provided in the Supplementary Information (Figs S1–S124).

(a) Molecules containing acylhydrazone or isopropanolamine groups. (b) The design concept for the title compounds.

Scheme 1 — General synthetic routes for A1–**A14** and B1–B5.

Scheme 2 — General synthetic routes for C1–**C20**.

3.2. Antimicrobial evaluation and structure–activity relationship analysis of target compounds

To investigate the antimicrobial potential of the designed compounds, and based on prior literature, ⁴¹ a series of target compounds A1–A14 and B1–B5 were initially synthesized using 3,5‐dimethoxyphenyl and 3,5‐dichlorophenyl groups as R ₁ substituents (Table 1). Their in vitro antibacterial activities were evaluated against Xoo and Xac using the turbidity method, with EC₅₀ values used to assess antibacterial potency. Commercial bactericides BT and TC were used as positive controls. Preliminary screening results are summarized in Supporting Information, Table S1.

Table 1.

In vitro antibacterial potency of compounds A1−A14 and B1–B5 toward plant pathogens Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas axonopodis pv. citri (Xac)

Compd	Xoo			Xac
Compd	Regression equation	EC₅₀ (μg/mL)	R	Regression equation	EC₅₀ (μg/mL)	R
A1		>50			>50
A2		>50			>50
A3		>50		y = 1.8653x + 1.9492	33.56 ± 0.35	1
A4		>50			>50
A5		>50			>50
A6		>50			>50
A7	y = 8.6534x − 3.5805	3.25 ± 0.1	0.98	y = 2.9449x + 1.4836	15.60 ± 0.80	1.0
A8	y = 4.5822x − 1.173	22.2 ± 0.8	0.93	y = 1.7392x + 2.4811	28.10 ± 1.54	0.95
A9	y = 3.911x +s 0.5595	13.7 ± 0.34	0.99	y = 0.8944x + 3.7733	23.53 ± 2.22	0.94
A10		>50			>50
A11		>50			>50
A12	y = 1.5766x + 1.5655	31.09 ± 0.12	0.91		>50
A13	y = 5.8172x − 3.7496	31.9 ± 0.53	1	y = 1.9636x + 1.8998	37.99 ± 0.65	0.99
A14		>50			>50
B1		>50			>50
B2	y = 5.6496x − 2.4816	20.12 ± 0.82	0.92		>50
B3	y = 7.9749x − 5.7834	22.49 ± 0.71	0.95		>50
B4		>50			>50
B5	y = 7.7243x + 0.2898	4.07 ± 0.08	1	y = 7.5093x − 2.6799	10.54 ± 0.15	0.96
BT	y = 7.5364x + 2.5835	45.74 ± 0.63	0.91	y = 10.135x − 15.3445	51.23 ± 0.93	0.90
TC	y = 4.3306x − 3.8076	108.10 ± 1.48	0.99	y = 4.5273x − 3.6534	81.54 ± 1.4	0.99

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Note: EC₅₀, Half maximal effective concentration; R, Pearson correlation coefficient.

For the A series compounds (A1–A14, Scheme 1), structure–activity relationship (SAR) analysis was conducted based on the results of in vitro anti‐Xoo assays (Table 1). Detailed analysis is as follows: (i) When R₂ is an aliphatic or heterocyclic amine group (e.g., A1, A3, and A13), antibacterial activity against Xoo is negligible. (ii) When R₂ is substituted with N‐methylbenzylamine (A7–A12), compounds bearing a para‐substituted electron‐donating group on the benzene ring (minimal steric hindrance) exhibit the strongest overall antibacterial activity against Xoo. A7 (N‐methyl‐4‐(p‐tolyl)methylamine, EC₅₀ = 3.25 μg/mL) > A9 (1‐(4‐fluorophenyl)‐N‐methylmethylamine, EC₅₀ = 13.7 μg/mL) > A8 (1‐(2‐methoxyphenyl)‐N‐methylmethylamine, EC₅₀ = 22.2 μg/mL) > A10 (1‐(2‐chlorophenyl)‐N‐methylmethylamine, EC₅₀ > 50 μg/mL). Data indicate that in vitro anti‐Xoo activity is optimal when R₂ is N‐methyl‐4‐(p‐tolyl)methylamine group (A7, EC₅₀ = 3.25 μg/mL). Similarly, evaluation of the B series compounds (B1–B5, Scheme 1) revealed that optimal anti‐Xoo activity was achieved when R₂ was the electron‐donating group N‐methyl‐4‐(p‐tolyl)methylamine (EC₅₀ = 4.07 μg/mL). When fatty amines or heterocyclic substituents were introduced at the R₂ position, compound activity was significantly reduced (EC₅₀ > 20 μg/mL). These results collectively indicate that the N‐methyl‐4‐(p‐tolyl)methylamine fragment at the R₂ position is a key pharmacophore for anti‐Xoo activity.

Based on the SAR trends observed in the A and B series, the N‐methyl‐4‐(p‐tolyl)methylamine moiety was fixed at the R₂ position, and a series of C1–C20 derivatives were synthesized to further explore the influence of R₁ substituents on antibacterial activity (Scheme 2). The in vitro anti‐Xoo activity results (Table 2) revealed distinct SAR patterns, which are summarized as follows: (i) When R₁ was a pyridine‐derived group, the position of the N atom exerts a significant impact on the activity, with the activity order being 2‐pyridin‐1‐yl (C3, EC₅₀ = 11.63 μg/mL) > 3‐pyridinyl (C4, EC₅₀ > 50 μg/mL) and 4‐pyridyl (C5, EC₅₀ > 50 μg/mL). (ii) When R₁ was a phenol‐derived group, the anti‐Xoo activity is correlated with the position of the electron‐donating hydroxyl group, and the activity order was determined as C11 (3‐hydroxyphenyl, EC₅₀ = 47.34 μg/mL) > C10 (2‐hydroxyphenyl, EC₅₀ > 50 μg/mL). (iii) When R₁ was a benzene ring substituted with electron‐withdrawing groups, compounds bearing a double electron‐withdrawing group on the benzene ring exhibited superior activity. For instance, C15 (2,4‐dichlorophenyl, EC₅₀ = 5.22 μg/mL), C16 (3‐bromo‐4‐fluorophenyl, EC₅₀ = 5.33 μg/mL) and C17 (2‐nitro‐4‐trifluoromethylphenyl, EC₅₀ = 21.39 μg/mL) showed higher activity than the single‐substituted compounds C12 (4‐fluorophenyl, EC₅₀ > 50 μg/mL), C13 (2‐nitrophenyl, EC₅₀ > 50 μg/mL) and C14 (3‐nitrophenyl substituent, EC₅₀ > 50 μg/mL). In addition, their activity was also higher than that of compounds with electron‐donating groups, such as C7 (3‐aminophenyl), C8 (4‐methoxyphenyl), and C11 (3‐hydroxyphenyl), all of which had an EC₅₀ > 50 μg/mL. (iv) When R₁ contained a diphenyl ether group, the 3‐phenoxyphenyl derivative (C19, EC₅₀ = 1.66 μg/mL) exhibited higher activity than the 2‐phenoxyphenyl (C18, EC₅₀ > 50 μg/mL) and 4‐phenoxyphenyl (C20, EC₅₀ > 50 μg/mL) analogs.

Table 2.

In vitro antibacterial potency of title compounds C1 − C20 toward plant pathogens Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas axonopodis pv. citri (Xac)

Compd	Xoo			Xac
Compd	Regression equation	EC₅₀ (μg/mL)	R	Regression equation	EC₅₀ (μg/mL)	R
C1		>50		y = 8.3298x − 8.0014	36.38 ± 0.66	0.9
C2		>50			>50
C3	y = 4.2075x + 0.5163	11.63 ± 1.67	1	y = 5.5645x − 1.1558	12.77 ± 0.12	0.96
C4		>50			>50
C5		>50			>50
C6	y = 6.8547x − 3.8764	19.72 ± 0.41	0.93	y = 1.9023x + 2.4836	21.03 ± 1.35	0.92
C7		>50			>50
C8		>50		y = 4.7362x − 0.7234	16.16 ± 0.9331	0.97
C9		>50		y = 7.516x − 5.0825	21.95 ± 1.059	0.91
C10		>50		y = 7.2614x − 2.4585	11.08 ± 2.64	0.9
C11	y = 7.4116x + 2.3348	47.34 ± 0.74	0.90	y = 6.8287x − 5.9283	39.84 ± 1.365	0.92
C12		>50			>50
C13		>50			>50
C14		>50		y = 5.2184x − 1.0413	14.38 ± 0.4479	0.92
C15	y = 7.1473x − 0.1266	5.22 ± 0.2419	0.91
C16	y = 7.2614x − 0.2755	5.33 ± 0.04	0.91	y = 7.6447x − 0.4753	5.26 ± 0.5637	0.98
C17	y = 7.9445x − 5.5679	21.39 ± 0.3851	0.98	y = 6.9271x + 0.1823	4.96 ± 0.088	0.94
C18		>50			>50
C19	y = 7.3121x + 3.3942	1.66 ± 0.009	0.95	y = 5.4207x + 4.6002	1.19 ± 0.034	0.95
C20		>50			>50
BT	y = 7.5364x + 2.5835	45.74 ± 0.63	0.91	y = 10.135x − 15.3445	51.23 ± 0.936	0.90
TC	y = 4.3306x − 3.8076	108.10 ± 1.48	0.99	y = 4.5273x − 3.6534	81.54 ± 1.414	0.99

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Note: EC₅₀, Half maximal effective concentration; R, Pearson correlation coefficient.

Furthermore, when the introduced hydrazone group possesses a macrocyclic electron‐donating group positioned at the meta‐position of the benzene ring, the compound exhibited significantly enhanced anti‐Xoo activity (C19 > A7). The above data indicated that the highest activity was observed when the R₂ group was N‐methyl‐4‐(p‐tolyl)methylamine; therefore, this group was fixed to investigate the effect of the hydrazide group on compound activity. Interestingly, the highest activity was achieved when the hydrazide group was a mesityl hydrazide (C19), demonstrating that the hydrazone parent structure and the isopropanolamine group exert a synergistic effect in enhancing antibacterial activity. The above‐mentioned outcomes were further illustrated as SAR study (Fig. 2). Finally, compound C19, featuring an N‐methyl‐4‐methylbenzylamine moiety linked to a meta‐phenoxy hydrazide, demonstrated significant inhibitory effects against the rice bacterial blight pathogen. Therefore, compound C19 was selected for subsequent mechanism studies.

Structure–activity relationship (SAR) study.

3.3. Effect of compound C19 on Xoo growth

Based on the in vitro antibacterial evaluation, the effect of compound C19 on the growth of Xoo was preferentially investigated to clarify its potential anti‐virulence mechanism. Bacterial growth curves were generated by monitoring OD₅₉₅ values at 3‐h intervals over a 30‐h incubation period. As shown in Fig. 3(a), compared with the blank control group, compound C19 at concentrations below 2.5 EC₅₀ had no significant effect on Xoo growth rate, whereas the 5 EC₅₀ concentration severely inhibited Xoo cell growth. These results indicate that C19 exerts a mild subinhibitory effect on Xoo within the concentration range of 0–2.5 EC₅₀, providing a basis for subsequent mechanistic studies. Furthermore, when investigating the effect of compounds on biofilm formation, the MIC was uniformly used to replace the EC₅₀ value (where MIC = 2 EC₅₀) for mechanism exploration.

(a) Growth curves of *Xanthomonas oryzae* pv. *oryzae* (*Xoo*) triggered by compounds **C19**. (b) Crystal violet assay of compounds **C19**. (c) Scanning electron microscopy images of *Xoo* biofilms after incubation with compounds **C19** at various dosages. Scale bars, 5 μm. Error bars indicate the mean ± standard deviation. **P < 0.01 and ****P < 0.0001 were used as the levels of significant differences.

3.4. Inhibition of biofilm formation

Some literature has demonstrated that biofilms protect bacteria against adverse environmental conditions and antimicrobial agents, facilitate bacterial colonization on both biotic and abiotic surfaces, and act as a key driver of the development of bacterial resistance. ⁴² Given that biofilms represent a critical virulence factor of Xoo, we investigated the regulatory effect of compound C19 on Xoo biofilm formation. A series of gradient concentrations of C19 were selected to analyze its impact on biofilm formation, with reference to the subinhibitory concentration range identified in the growth curve assay. As shown in Fig. 3(b), treatment with C19 at concentrations ranging from 1/8 MIC to MIC did not significantly affect bacterial growth compared with the untreated control, whereas biofilm formation was markedly suppressed in a concentration‐dependent manner. These results indicate that C19 effectively inhibits biofilm formation without impairing bacterial proliferation, thereby preventing the establishment of a protective biofilm matrix and increasing bacterial exposure to antimicrobial stress.

Furthermore, SEM was used as a complementary method to visually characterize bacterial morphology and biofilm structure. As shown in Fig. 3(c), the blank control group formed a dense biofilm that fully covered the Xoo cells. With increasing C19 concentrations, the biofilm structure was progressively disrupted, accompanied by an increase in exposed bacterial cells and obvious changes in bacterial morphology. These observations further confirm that C19 exerts a significant inhibitory effect on Xoo biofilm formation.

3.5. Swimming and swarming motility assays of Xoo

Swarming motility—a specialized form of bacterial surface translocation—enables rapid transition from a planktonic state to surface colonization, representing one of the fastest characterized bacterial surface migration mechanisms. This motility not only facilitates efficient colonization of nutrient‐rich niches and host tissues, but also drives the physiological and morphological differentiation of bacteria into colony‐forming cells. Intriguingly, biofilm formation exerts a regulatory effect that can indirectly modulate bacterial motility.

Accordingly, we further evaluated the impacts of C19 on the swimming and swarming motility of Xoo. As shown in Fig. 4(a), (c), the blank control group exhibited a maximum average swimming diameter of 22.46 mm. After treatment with C19 at concentrations of 1/8 MIC, ¼ MIC, ½ MIC, MIC, and 2 MIC, the average swimming diameters decreased to 12.06, 8.30, 7.50, 4.23, and 0.43 mm, respectively, corresponding to inhibition rates of 46.31%, 63.05%, 66.61%, 81.17% and 98.09% compared with the control. Similar concentration‐dependent inhibitory effects were observed in the swarming assay (Fig. 4(b), (d)). The blank control group showed a maximum average swarming diameter of 33.10 mm, while treatment with C19 at the same concentration gradient reduced the average diameters to 27.50, 22.25, 17.25, 9.25, and 0.625 mm, with inhibition rates of 16.92%, 32.78%, 47.89%, 72.05%, and 98.11%, respectively. Collectively, these results demonstrate that C19 inhibits Xoo swimming and swarming motility in a concentration‐dependent manner, which may contribute to its suppression of biofilm formation.

Effects of compound **C19** on the swimming and swarming motility of *Xanthomonas oryzae* pv. *oryzae* (*Xoo*). (a) Representative images of swimming motility. (b) Representative images of swarming motility. (c) Statistical analysis of swimming motility diameters. (d) Statistical analysis of swarming motility diameters. Error bars represent the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

3.6. Determination of the extracellular enzymes

Xoo secretes a variety of extracellular enzymes, including amylase and cellulase, which facilitate the degradation of plant cell walls and promote the invasion, colonization, and dissemination of pathogens within host tissues. ⁴³ To further elucidate the anti‐virulence mechanism of C19, its effects on the secretion of these key extracellular enzymes were assessed.

First, amylase activity was determined by culturing Xoo on semi‐solid medium containing 0.1% (w/v) soluble starch; amylase secreted by Xoo degrades starch, forming transparent zones that can be visualized by staining with 1% I₂/KI solution. Enzyme activity was quantified by measuring the diameter of these transparent zones, which showed a concentration‐dependent relationship with C19. As shown in Fig. 5(a), the control group exhibited an average amylase hydrolysis zone diameter of 18.83 mm. Treatment with C19 at 1/8 MIC, 1/4 MIC, 1/2 MIC, MIC, and 2 MIC reduced the average diameters to 13.1, 10.83, 7.50, 4.1, and 2.3 mm, with inhibition rates of 30.43%, 42.49%, 60.17%, 78.23%, and 87.79%, respectively.

Analysis of extracellular enzyme activity in *Xanthomonas oryzae* pv. *oryzae* (*Xoo*) treated with different concentrations of **C19**. (a) Extracellular amylase activity (cultured on swarm plates at 28 °C for 72 h). (b) Extracellular cellulase activity (cultured on swarm plates at 28 °C for 72 h). (c) Statistical analysis of amylase hydrolysis zone diameters, (d) Statistical analysis of cellulase hydrolysis zone diameters. Scale bars, 10 mm. Error bars represent the mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Furthermore, consistent results were obtained for cellulase activity (Fig. 5(b)). Cellulose can be stained with Congo red, and hydrolysis of cellulose by cellulase results in the formation of transparent zones. The control group showed an average cellulase hydrolysis zone diameter of 12.57 mm, whereas treatment with C19 at the same concentration gradient reduced the diameters to 7.5, 6.27, 4, 3.17, and 0.9 mm, corresponding to inhibition rates of 40.33%, 50.12%, 68.18%, 74.78%, and 92.8%. Statistical analyses of the enzyme activity data are presented in Fig. 5(c), (d). Overall, these results demonstrate that C19 suppresses the secretion of key extracellular enzymes by Xoo, which hinders biofilm formation and impairs bacterial invasion of host plants.

3.7. Pathogenicity analysis of Xoo on rice

Based on the aforementioned findings—that C19 potently inhibits multiple pathogenic factors of Xoo (including motility, biofilm formation, and extracellular enzyme secretion, which are crucial for plant tissue degradation and pathogen invasion)—we further evaluated C19's efficacy in suppressing Xoo pathogenicity in rice. As shown in Fig. 6(a), C19 treatment significantly reduced Xoo infection in rice plants. Compared with the average lesion length of 16.86 cm in the blank control group, the positive control group (TC) exhibited a reduced average lesion length of 12.08 cm, achieving an inhibition efficiency of 28.35%. By contrast, rice plants treated with C19 at concentrations of 1/4 MIC, 1/2 MIC, MIC, and 2 MIC exhibited significantly reduced average lesion lengths of 6.64, 4.0, 2.5, and 0.16 cm, respectively, corresponding to inhibition efficiencies of 60.62%, 76.28%, 85.17%, and 99.05%.

(a) Pathogenicity of *Xanthomonas oryzae* pv. *oryzae* (*Xoo* 14) days after treating rice plants with different concentrations of **C19** [0, 1/4 minimal inhibitory concentration (MIC), 1/2 MIC, MIC and 2 MIC]. (b) Histopathological sections of rice tissue stained with toluidine blue O. (c) Hypersensitivity reaction (HR) induced by *Xoo* on tobacco (*Nicotiana benthamiana*) leaves after treatment with different concentrations of **C19**.

Furthermore, a TBO staining assay was performed to visualize the colonization dynamics of Xoo within host rice tissues. As presented in Fig. 6(b), dense bacterial populations were clearly observed in the rice tissues of the blank control group. In stark contrast, bacterial abundance decreased progressively in groups treated with C19 at 1/4 MIC, 1/2 MIC, and MIC; notably, almost no viable bacteria were detectable in the 2 MIC treatment group. Collectively, these results confirm that C19 exerts a multifaceted inhibitory effect on Xoo virulence factors (e.g., biofilms and extracellular enzymes), thereby effectively impeding bacterial invasion and colonization in rice plants.

3.8. HR assay and phytotoxicity evaluation

The HR is a typical plant immune reaction to pathogen infection, characterized by localized programmed cell death that restricts pathogen spread. HR usually appears as rapid necrosis at the infection site, effectively preventing bacterial dissemination to surrounding healthy tissues. To further evaluate the effect of compound C19 on Xoo pathogenicity and its potential phytotoxicity, an HR assay was performed using tobacco leaves. Briefly, an Xoo suspension (OD₅₉₅ = 0.1) was co‐incubated with different concentrations of C19 and subsequently infiltrated into tobacco leaves. As shown in Fig. 6(c), the necrotic area on tobacco leaves gradually decreased with increasing concentrations of C19. In contrast to the pronounced HR observed in the untreated control, C19‐treated leaves exhibited significantly attenuated necrotic symptoms. These results indicate that C19 effectively suppresses virulence factors in Xoo, and thereby inhibits the Xoo‐elicited HR in plants by attenuating bacterial virulence rather than exerting direct phytotoxicity. Collectively, these observations substantiate its potential as a safe anti‐virulence candidate for plant disease management.

3.9. Toxicity assessment of C19

To comprehensively evaluate the safety and druggability of C19, we performed early pharmacokinetic assessment (ADMET), non‐target organism toxicity testing, and phytotoxicity evaluation. Specifically, ADMETlab 3.0 software was used to analyze the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties as well as the druggability of C19 (Fig. 7(b); Supporting Information, Table S2). The prediction results confirmed that C19 possessed low toxicity and favorable pharmacokinetic profiles, with a low risk of development failure attributed to toxicity. Furthermore, phytotoxicity is a critical indicator in pesticide development, so we initially evaluated the potential phytotoxicity of C19 to rice under greenhouse conditions. Rice plants were sprayed with 200 μg/mL C19 and cultured for 7 days, and no significant changes in the phenotypic characteristics of rice leaves were observed (Fig. 7(a)). This finding indicates that C19 is essentially non‐phytotoxic to rice, and when combined with its strong inhibitory activity against plant pathogens, it highlights the potential of C19 as a candidate agricultural bactericide.

(a) Phytotoxicity of **C19** (200 μg/mL) to rice leaves. (b) Physicochemical properties of compound **C19**. (c) Survival rate of earthworms after 14 days exposure to **C19** (0.1, 1 and 10 mg/kg).

To further assess environmental safety, acute toxicity tests were conducted on earthworms, a representative non‐target organism. As shown in Fig. 7(c), all earthworms maintained 100% survival after 14 days of exposure to 10 mg/kg C19, indicating an LC₅₀ (Half lethal concentration) > 10 mg/kg. According to standard toxicity classification, this confirms low environmental toxicity of C19 and minimal impact on non‐target organisms.

3.10. Compound C19 on controlling rice BLB

Based on the in vitro antimicrobial bioassay results, compound C19 was selected for evaluating its in vivo inhibitory activity against rice BLB under greenhouse conditions. Experimental data are summarized in Table 3 and Fig. 8, which collectively demonstrate that C19 exerted superior control efficacy against BLB. Specifically, at a concentration of 200 μg/mL, C19 achieved a curative efficacy of 46.2% and a protective efficacy of 47.3%, outperforming the commercial bactericides BT (curative efficacy: 44.5%; protective efficacy: 44.3%) and TC (curative efficacy: 42.5%; protective efficacy: 43.8%). These results confirm that C19 holds considerable application potential in BLB control, thereby qualifying it as a promising lead compound for the development of novel antibacterial agents.

Table 3.

Effect of C19 on the control of bacterial blight in rice at 200 μg/mL

Treatment	Curative activity (%)		Protective activity (%)
Treatment	Disease index	Control efficiency	Disease index	Control efficiency
C19	46.1	46.2b	44.3	47.3b
BT	47.6	44.5b	47.8	44.3b
TC	49.3	42.5b	48.2	43.8b
CK	85.8	/	85.8	/

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Note: BT, bismerthiazol; TC, thiodiazole‐copper; CK, blank control. Statistical analysis was performed by one‐way analysis of variance, with equal variances assumed (P > 0.05) and unequal variances not assumed (P < 0.05). Different lowercase letters indicate significant differences in bioactivity among treatment groups at P < 0.05.

*In vivo* control efficacy of compound **C19**, commercialized bismerthiazol (BT), and thiodiazole‐copper (TC) against rice bacterial leaf blight at 200 μg/mL.

3.11. The stability of compound C19

Hydrolysis and thermal decomposition are the main causes of pesticide instability, and hydrolysis largely determines the effective duration of pesticides after application ³⁴ , ⁴⁴ , ⁴⁵ , ⁴⁶ , ⁴⁷ , ⁴⁸ . To evaluate the stability of compound C19, hydrolysis assays and thermal stability tests were carried out in this study. The ultraviolet absorption spectra (Supporting Information, Fig. S125) showed negligible absorbance changes during the experiment, confirming the good hydrolytic stability of hydrazone C19. Furthermore, thermogravimetric analysis was further adopted to evaluate its thermal performance. As shown in Supporting Information, Fig. S126, no obvious weight loss was observed from room temperature to 300 °C, revealing excellent thermal stability of C19 in this range and enabling its long‐term storage at ambient temperature. Overall, compound C19 presents satisfactory stability.

4. CONCLUSION

In this study, a series of novel isopropanolamine‐modified acylhydrazone derivatives were designed and synthesized, and their antibacterial activities against Xoo and Xac were systematically evaluated. Among these compounds, C19 exhibited the most potent in vitro antibacterial activity, with EC₅₀ values of 1.66 μg/mL against Xoo and 1.19 μg/mL against Xac. Further mechanistic investigations were conducted to clarify its antibacterial mode of action (Fig. 9), which compound C19 exerted its antibacterial effects primarily by inhibiting multiple virulence‐related factors of Xoo, including biofilm formation, bacterial motility, flagellar‐associated behaviors, and extracellular enzyme secretion, thereby attenuating bacterial infectivity. Consistent with these findings ⁴⁹ , ⁵⁰ , ⁵¹ , in vivo assays further confirmed that C19 effectively suppressed Xoo infection in rice plants. At a concentration of 200 μg/mL, C19 achieved curative and protective efficacies of 46.2% and 47.3%, respectively, against rice BLB. In addition, toxicity evaluations indicated that C19 exhibited a favorable safety profile toward non‐target organisms under the tested conditions. Overall, this study demonstrates that acylhydrazone‐based small molecules can effectively interfere with bacterial infection by targeting biofilm formation and other key virulence factors. These results provide experimental evidence supporting the potential of such compounds for the development of environmentally friendly bactericides for the control of bacterial plant diseases. These findings underscore their value in addressing bacterial resistance and broad prospects for agricultural application, laying a solid theoretical and experimental foundation for the development of novel green bactericides.

Proposed mechanism for the suppression of bacterial infection by compound **C19**.

CONFLICT OF INTEREST

The authors claim that there is no conflict of interest.

Supporting information

Fig. S1. ¹H NMR spectra (CDCl₃, 400 MHz) of target Compound A1.

Fig. S2. ¹³ C NMR spectra (CDCl₃, 101 MHz) of target Compound A1.

Fig. S3. HRMS spectra of target compound A1.

Fig. S4. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A2.

Fig. S5. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A2.

Fig. S6. HRMS Spectra of target compound A2.

Fig. S7. ¹ H NMR spectra (CDCl₃, 400 MHz) of target Compound A3.

Fig. S8. ¹³ C NMR spectra (CDCl₃, 126 MHz) of target compound A3.

Fig. S9. HRMS spectra of target compound A3.

Fig. S10. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A4.

Fig. S11. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A4.

Fig. S12. HRMS spectra of target compound A4.

Fig. S13. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A5.

Fig. S14. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A5.

Fig. S15. HRMS spectra of target compound A5.

Fig. S16. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A6.

Fig. S17. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A6.

Fig. S18. HRMS spectra of target compound A6.

Fig. S19. ¹H NMR spectra (CDCl₃, 400 MHz) of target tompound A7.

Fig. S20. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A7.

Fig. S21. HRMS spectra of compound A7.

Fig. S22. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A8.

Fig. S23. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A8.

Fig. S24. HRMS spectra of compound A8.

Fig. S25. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A9.

Fig. S26. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A9.

Fig. S27. ¹⁹F NMR spectra (CDCl₃, 377 MHz) of target compound A9.

Fig. S28. HRMS spectra of target compound A9.

Fig. S29. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A10.

Fig. S30. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A10.

Fig. S31. HRMS spectra of target compound A10.

Fig. S32. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A11.

Fig. S33. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A11.

Fig. S34. ¹⁹F NMR spectra (CDCl₃, 377 MHz) of target compound A11.

Fig. S35. HRMS spectra of target compound A11.

Fig. S36. ¹H NMR spectra (CDCl₃, 400 MHz) of target Compound A12.

Fig. S37. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A12.

Fig. S38. HRMS spectra of target compound A12.

Fig. S39. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A13.

Fig. S40. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A13.

Fig. S41. HRMS spectra of target compound A13.

Fig. S42. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A14.

Fig. S43. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A14.

Fig. S44. ¹⁹F NMR spectra (CDCl₃, 471 MHz) of target compound A14.

Fig. S45. HRMS spectra of target compound A14.

Fig. S46. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound B1.

Fig. S47. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound B1.

Fig. S48. HRMS spectra of target compound B1.

Fig. S49. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound B2.

Fig. S50. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound B2.

Fig. S51. HRMS spectra of target compound B2.

Fig. S52. ¹H NMR spectra (DMSO‐d ₆, 400 MHz) of target compound B3.

Fig. S53. ¹³C NMR spectra (DMSO‐d ₆, 126 MHz) of target compound B3.

Fig. S54. HRMS spectra of target compound B3.

Fig. S55. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound B4.

Fig. S56. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound B4.

Fig. S57. ¹⁹F NMR spectra (CDCl₃, 471 MHz) of target compound B4.

Fig. S58. HRMS spectra of target compound B4.

Fig. S59. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound B5.

Fig. S60. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound B5.

Fig. S61. HRMS spectra of target compound B5.

Fig. S62. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C1.

Fig. S63. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C1.

Fig. S64. HRMS spectra of target compound C1.

Fig. S65. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C2.

Fig. S66. ¹³C NMR spectra (CDCl₃, 101 MHz) of target tompound C2.

Fig. S67. HRMS spectra of target compound C2.

Fig. S68. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C3.

Fig. S69. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C3.

Fig. S70. HRMS spectra of compound C3.

Fig. S71. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C4.

Fig. S72. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C4.

Fig. S73. HRMS spectra of target compound C4.

Fig. S74. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C5.

Fig. S75. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C5.

Fig. S76. HRMS spectra of compound C5.

Fig. S77. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C6.

Fig. S78. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C6.

Fig. S79. HRMS spectra of target compound C6.

Fig. S80. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C7.

Fig. S81. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C7.

Fig. S82. HRMS spectra of target compound C7.

Fig. S83. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C8.

Fig. S84. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C8.

Fig. S85. HRMS spectra of target compound C8.

Fig. S86. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C9.

Fig. S87. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C9.

Fig. S88. HRMS spectra of target compound C9.

Fig. S89. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C10.

Fig. S90. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C10.

Fig. S91. HRMS spectra of target compound C10.

Fig. S92. ¹H NMR spectra (DMSO‐d ₆, 400 MHz) of target compound C11.

Fig. S93. ¹³C NMR Spectra (DMSO‐d ₆, 101 MHz) of Target Compound C11.

Fig. S94. HRMS spectra of target compound C11.

Fig. S95. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C12.

Fig. S96. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C12.

Fig. S97. ¹⁹F NMR spectra (CDCl₃, 377 MHz) of target compound C12.

Fig. S98. HRMS spectra of target compound C12.

Fig. S99. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C13.

Fig. S100. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C13.

Fig. S101. HRMS spectra of target compound C13.

Fig. S102. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C14.

Fig. S103. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C14.

Fig. S104. HRMS spectra of target compound C14.

Fig. S105. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C15.

Fig. S106. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C15.

Fig. S107. HRMS spectra of target compound C15.

Fig. S108. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C16.

Fig. S109. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C16.

Fig. S110. ¹⁹F NMR spectra (CDCl₃, 377 MHz) of target compound C16.

Fig. S111. HRMS spectra of target compound C16.

Fig. S112. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C17.

Fig. S113. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C17.

Fig. S114. ¹⁹F NMR spectra (CDCl₃, 377 MHz) of target compound C17.

Fig. S115. HRMS spectra of target compound C17.

Fig. S116. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C18.

Fig. S117. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C18.

Fig. S118. HRMS spectra of target compound C18.

Fig. S119. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C19.

Fig. S120. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C19.

Fig. S121. HRMS spectra of target compound C19.

Fig. S122. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C20.

Fig. S123. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C20.

Fig. S124. HRMS spectra of target compound C20.

Table S1. Preliminary screening of compound A1‐A14, B1‐B5 and C1‐C20 for in vitro bioactivity against the plant pathogen Xoo, Xac,Psa.

Table S1. Preliminary screening of compound A1‐A14, B1‐B5 and C1‐C20 for in vitro bioactivity against the plant pathogen Xoo, Xac, Psa (continuation).

Table S2. Predicted ADMET and drug‐like characteristics of compound C19 (ADMETlab 3.0).

Fig. S125. UV–vis spectra of compound C19 during hydrolysis.

Fig. S126. TG diagram of C19 in N₂ atmosphere.

PS-82-9185-s001.docx^{(10.8MB, docx)}

ACKNOWLEDGEMENTS

This work was supported by National Key Research and Development Program of China (2022YFD1700300), National Natural Science Foundation of China (32372610, U23A20201, 32160661, 32202359), National Key Research and Development Program of China (2024YFE0214300), the Central Government Guides Local Science and Technology Development Fund Projects [Qiankehezhongyindi (2023) 001], [Qiankehezhongyindi (2024) 007], Scientific and Technological Innovation Platform Research Project of Guizhou Province (CXPTXM [2025] 012), Major Scientific and Technological Achievement Transformation Project of Guizhou Province (Qian Ke He Zhong Yin Di [2024]027), Qiankehe Platform DLSYS9 (2025) Major 001, Outstanding Young Scientific and Technological Talent Project of Guizhou Province (Qian Ke He Ping Tai Ren Cai YQK [2023]004), QiankexieKJLYRC‐[2026]088.

Contributor Information

Xiang Zhou, Email: jhzx.msm@gmail.com, Email: syang@gzu.edu.cn, Email: xiangzhou@gzu.edu.cn, Email: zhoux1534@163.com.

Song Yang, Email: syang@gzu.edu.cn.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1. ¹H NMR spectra (CDCl₃, 400 MHz) of target Compound A1.

Fig. S2. ¹³ C NMR spectra (CDCl₃, 101 MHz) of target Compound A1.

Fig. S3. HRMS spectra of target compound A1.

Fig. S4. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A2.

Fig. S5. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A2.

Fig. S6. HRMS Spectra of target compound A2.

Fig. S7. ¹ H NMR spectra (CDCl₃, 400 MHz) of target Compound A3.

Fig. S8. ¹³ C NMR spectra (CDCl₃, 126 MHz) of target compound A3.

Fig. S9. HRMS spectra of target compound A3.

Fig. S10. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A4.

Fig. S11. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A4.

Fig. S12. HRMS spectra of target compound A4.

Fig. S13. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A5.

Fig. S14. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A5.

Fig. S15. HRMS spectra of target compound A5.

Fig. S16. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A6.

Fig. S17. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A6.

Fig. S18. HRMS spectra of target compound A6.

Fig. S19. ¹H NMR spectra (CDCl₃, 400 MHz) of target tompound A7.

Fig. S20. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A7.

Fig. S21. HRMS spectra of compound A7.

Fig. S22. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A8.

Fig. S23. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A8.

Fig. S24. HRMS spectra of compound A8.

Fig. S25. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A9.

Fig. S26. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A9.

Fig. S27. ¹⁹F NMR spectra (CDCl₃, 377 MHz) of target compound A9.

Fig. S28. HRMS spectra of target compound A9.

Fig. S29. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A10.

Fig. S30. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A10.

Fig. S31. HRMS spectra of target compound A10.

Fig. S32. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A11.

Fig. S33. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A11.

Fig. S34. ¹⁹F NMR spectra (CDCl₃, 377 MHz) of target compound A11.

Fig. S35. HRMS spectra of target compound A11.

Fig. S36. ¹H NMR spectra (CDCl₃, 400 MHz) of target Compound A12.

Fig. S37. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A12.

Fig. S38. HRMS spectra of target compound A12.

Fig. S39. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A13.

Fig. S40. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A13.

Fig. S41. HRMS spectra of target compound A13.

Fig. S42. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound A14.

Fig. S43. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound A14.

Fig. S44. ¹⁹F NMR spectra (CDCl₃, 471 MHz) of target compound A14.

Fig. S45. HRMS spectra of target compound A14.

Fig. S46. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound B1.

Fig. S47. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound B1.

Fig. S48. HRMS spectra of target compound B1.

Fig. S49. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound B2.

Fig. S50. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound B2.

Fig. S51. HRMS spectra of target compound B2.

Fig. S52. ¹H NMR spectra (DMSO‐d ₆, 400 MHz) of target compound B3.

Fig. S53. ¹³C NMR spectra (DMSO‐d ₆, 126 MHz) of target compound B3.

Fig. S54. HRMS spectra of target compound B3.

Fig. S55. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound B4.

Fig. S56. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound B4.

Fig. S57. ¹⁹F NMR spectra (CDCl₃, 471 MHz) of target compound B4.

Fig. S58. HRMS spectra of target compound B4.

Fig. S59. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound B5.

Fig. S60. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound B5.

Fig. S61. HRMS spectra of target compound B5.

Fig. S62. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C1.

Fig. S63. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C1.

Fig. S64. HRMS spectra of target compound C1.

Fig. S65. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C2.

Fig. S66. ¹³C NMR spectra (CDCl₃, 101 MHz) of target tompound C2.

Fig. S67. HRMS spectra of target compound C2.

Fig. S68. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C3.

Fig. S69. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C3.

Fig. S70. HRMS spectra of compound C3.

Fig. S71. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C4.

Fig. S72. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C4.

Fig. S73. HRMS spectra of target compound C4.

Fig. S74. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C5.

Fig. S75. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C5.

Fig. S76. HRMS spectra of compound C5.

Fig. S77. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C6.

Fig. S78. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C6.

Fig. S79. HRMS spectra of target compound C6.

Fig. S80. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C7.

Fig. S81. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C7.

Fig. S82. HRMS spectra of target compound C7.

Fig. S83. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C8.

Fig. S84. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C8.

Fig. S85. HRMS spectra of target compound C8.

Fig. S86. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C9.

Fig. S87. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C9.

Fig. S88. HRMS spectra of target compound C9.

Fig. S89. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C10.

Fig. S90. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C10.

Fig. S91. HRMS spectra of target compound C10.

Fig. S92. ¹H NMR spectra (DMSO‐d ₆, 400 MHz) of target compound C11.

Fig. S93. ¹³C NMR Spectra (DMSO‐d ₆, 101 MHz) of Target Compound C11.

Fig. S94. HRMS spectra of target compound C11.

Fig. S95. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C12.

Fig. S96. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C12.

Fig. S97. ¹⁹F NMR spectra (CDCl₃, 377 MHz) of target compound C12.

Fig. S98. HRMS spectra of target compound C12.

Fig. S99. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C13.

Fig. S100. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C13.

Fig. S101. HRMS spectra of target compound C13.

Fig. S102. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C14.

Fig. S103. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C14.

Fig. S104. HRMS spectra of target compound C14.

Fig. S105. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C15.

Fig. S106. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C15.

Fig. S107. HRMS spectra of target compound C15.

Fig. S108. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C16.

Fig. S109. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C16.

Fig. S110. ¹⁹F NMR spectra (CDCl₃, 377 MHz) of target compound C16.

Fig. S111. HRMS spectra of target compound C16.

Fig. S112. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C17.

Fig. S113. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C17.

Fig. S114. ¹⁹F NMR spectra (CDCl₃, 377 MHz) of target compound C17.

Fig. S115. HRMS spectra of target compound C17.

Fig. S116. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C18.

Fig. S117. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C18.

Fig. S118. HRMS spectra of target compound C18.

Fig. S119. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C19.

Fig. S120. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C19.

Fig. S121. HRMS spectra of target compound C19.

Fig. S122. ¹H NMR spectra (CDCl₃, 400 MHz) of target compound C20.

Fig. S123. ¹³C NMR spectra (CDCl₃, 101 MHz) of target compound C20.

Fig. S124. HRMS spectra of target compound C20.

Table S1. Preliminary screening of compound A1‐A14, B1‐B5 and C1‐C20 for in vitro bioactivity against the plant pathogen Xoo, Xac,Psa.

Table S1. Preliminary screening of compound A1‐A14, B1‐B5 and C1‐C20 for in vitro bioactivity against the plant pathogen Xoo, Xac, Psa (continuation).

Table S2. Predicted ADMET and drug‐like characteristics of compound C19 (ADMETlab 3.0).

Fig. S125. UV–vis spectra of compound C19 during hydrolysis.

Fig. S126. TG diagram of C19 in N₂ atmosphere.

PS-82-9185-s001.docx^{(10.8MB, docx)}

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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PERMALINK

Identification of acylhydrazone derivatives with isopropanolamine moiety as potent anti‐virulence agents for combating bacterial infection

Zhi‐Jun Luo

Tai‐Hong Zhang

Jia‐Rui Rao

Pan Xu

Yan‐Yan Wen

San Tu

Qing‐Rong Bai

Xiang Zhou

Li‐Wei Liu

Zhi‐Bing Wu

Song Yang

Abstract

BACKGROUND

RESULTS

CONCLUSION

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1. Instruments and chemicals

2.2. Strain sources and preservation

2.3. Antibacterial activity evaluation

2.4. Growth curve assay

2.5. Inhibition of biofilm formation

2.6. Biofilm morphology analysis

2.7. Motility assay

2.8. Extracellular enzymatic assay

2.8.1. Extracellular cellulase

2.8.2. Amylase assay

2.9. Pathogenicity assays and pathological sections of Xoo on rice

2.10. Hypersensitive response assay

2.11. Statistical analysis

3. RESULTS AND DISCUSSION

3.1. Synthesis of intermediate and target compounds

Figure 1.

Scheme 1.

Scheme 2.

3.2. Antimicrobial evaluation and structure–activity relationship analysis of target compounds

Table 1.

Table 2.

Figure 2.

3.3. Effect of compound C19 on Xoo growth

Figure 3.

3.4. Inhibition of biofilm formation

3.5. Swimming and swarming motility assays of Xoo

Figure 4.

3.6. Determination of the extracellular enzymes

Figure 5.

3.7. Pathogenicity analysis of Xoo on rice

Figure 6.

3.8. HR assay and phytotoxicity evaluation

3.9. Toxicity assessment of C19

Figure 7.

3.10. Compound C19 on controlling rice BLB

Table 3.

Figure 8.

3.11. The stability of compound C19

4. CONCLUSION

Figure 9.

CONFLICT OF INTEREST

Supporting information

ACKNOWLEDGEMENTS

Contributor Information

DATA AVAILABILITY STATEMENT

REFERENCES

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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