Quaternary ammonium salt derivative containing thymol, preparation method of quaternary ammonium salt derivative, drug-loaded antibacterial liposome of quaternary ammonium salt derivative and application of drug-loaded antibacterial liposome

Abstract

The structural formula of the thymol-containing quaternary ammonium salt derivative is shown as (1), wherein R1 is 2-isopropyl-5-methylphenyl, ethyl or a benzene ring, R2 is 2-chloro-5-methylthiophene, 3-methyl-2-methoxycarbonyl furan, 4-methylnaphthalene and differently substituted benzyl, X-is Cl-and Br-, and n is equal to 3, 5, 7, 8 and 9. The preparation method of the drug-loaded antibacterial liposome comprises the step of jointly assembling hydrogenated soybean phosphatidylcholine, cholesterol hemisuccinic acid monoester, phosphatidyl ethanolamine and a target compound thymol-containing quaternary ammonium salt derivative into the drug-loaded antibacterial liposome. Part of the compounds have excellent antibacterial activity on Xanthomonas oryzae pv. Oryzae, Xanthomonas citri and Xanthomonas syringae pv. Kiwifruit. Meanwhile, the compound also has excellent biological activity on six fungi such as fusarium oxysporum and TMV viruses. A field test on Xoo shows that the protection effect of the drug-loaded antibacterial liposome is improved by 1.94 times, the treatment effect is improved by 1.56 times, and the drug-loaded antibacterial liposome shows remarkable pH-triggered release characteristics and colloidal stability. (1)

Description

Technical Field

[0001] This invention relates to the fields of chemistry, materials and pesticide science, specifically to a method for preparing a class of quaternary ammonium salt derivatives containing thymol, and their drug-loaded antibacterial liposomes and applications. Background Technology

[0002] The widespread application of liposomes in precision drug delivery, particularly in the medical field, highlights the enormous potential of this technology. Traditional pesticide formulations are already struggling to address challenges such as off-target deposition, resistance accumulation, and cascading ecotoxicity because they are inherently unstable in complex agricultural environments and are highly susceptible to photodegradation and microbial degradation. To date, among numerous delivery platform materials, lipids have attracted significant attention in the context of green agriculture due to their targeting ability, small particle size, strong biocompatibility, biodegradability, and low toxicity. However, it must be emphasized that for these early research findings to truly be applied in the field as agrochemicals, the key lies in redesigning lipid nanoparticle (LNP)-based delivery platforms. For example, thermosensitive liposomes can trigger pesticide release through phase transitions at 20 °C and 40 °C, increasing efficacy by 140% and enabling precise nighttime insecticidal action; PEGylation systems improve leaf penetration by 38.7% by enhancing the affinity of lipids for the cuticle; and PEI-conjugated formulations can activate RNAi, reducing pesticide usage by up to 75%. Although some experimental data support the application of LNPs in agriculture, significant challenges remain. Due to the pathological differences of plant bacterial diseases, its application in the prevention and control of plant bacterial diseases has not been explored to a large extent.

[0003] In recent years, the spread of plant diseases has threatened global food security because these diseases can severely reduce the yields of major crops such as rice, wheat, and corn. For example, rice bacterial blight (… Xanthomonas oryzae pv. oryzae , Xoo Rice (Clostridium difficile) is a highly destructive bacterial pathogen that primarily infects grasses, especially rice. It degrades and infects leaf tissues through extracellular enzymes and virulence proteins, creating a weakly acidic microenvironment (pH 5.5–6.5) on the leaf surface, thereby enhancing pathogenicity and potentially leading to annual yield losses of up to 50%. Worse still, the frequent and large-scale application of traditional fungicides such as tebuconazole and thiamethoxam has led to the rapid emergence of resistance and a significant decrease in efficacy. Concerningly, the various organic / inorganic adjuvants in these formulations often only address water solubility issues, potentially causing secondary pollution to already fragile ecosystems, ultimately increasing environmental toxicity and related hazards. Given these dilemmas in pesticide production and application, crop management is facing increasingly limited options. Simultaneously, developing novel fungicide candidates with innovative mechanisms of action and intelligent delivery systems has become a crucial solution for improving efficacy, reducing the risk of resistance, and providing biocompatible and environmentally friendly solutions.

[0004] Quaternary ammonium (QA) compounds, with their positively charged structure, can strongly disrupt negatively charged microbial membranes and have been widely used as effective sterilizing agents in medical and industrial devices, thus becoming promising antibacterial candidates. However, their potential phytotoxicity hinders further application in agriculture. Most lipid materials are positively charged or ionizable and can form nanoparticles with charged QA compounds through electrostatic interactions. This delivery strategy not only protects plants from QA phytotoxicity but also introduces stimulus-responsive lipids to enhance their targeted release and antibacterial properties. In addition to the QA cation, this antibacterial agent design incorporates the natural product thymol to enhance antibacterial activity and modulate liposome fluidity. By modifying the functional components of the liposomes, adjusting charge, particle size, and drug loading, pH responsiveness and functional targeting can be achieved in the weakly acidic microenvironment of Xoo, thereby improving the therapeutic effect on plant diseases.

[0005] Leveraging the properties of liposome nanomaterials, we designed a series of positively charged quaternary ammonium salt derivatives that bind to lipids through electrostatic and other interactions, achieving higher drug loading capacity compared to traditional compounding methods. This study focuses on the synthesis of quaternary ammonium salt derivatives, the regulation of targeting behavior through changes in lipid structure and composition, and the validation of the antibacterial efficacy of drug-loaded liposomes. During the study, techniques such as UV-Vis spectrophotometry, Zeta potential analysis, X-ray photoelectron spectroscopy (XPS), energy dispersive spectroscopy (EDS), and transmission electron microscopy (TEM) were used to characterize the assembly behavior and responsive release performance of the LNP system. Furthermore, this study explored the main challenges of using LNPs for the control of plant bacterial diseases, including system stability, drug loading capacity, pH-responsive release, leaf adhesion, and potential toxicity to plants and animals. Subsequent results showed that QA not only exhibited significant antibacterial activity against Xoo both in vitro and in vivo, but also effectively inhibited biofilm formation, further weakening pathogen virulence and reducing the risk of pesticide resistance. To enhance the advantages of this type of molecule, we introduced a pH-responsive drug delivery system based on LNPs to achieve more precise delivery of QA molecules. This system not only reduces the phytotoxicity of QA (a type of pesticide) but also exhibits certain growth-promoting effects. This targeted and environmentally friendly delivery system provides a new strategy for the control of plant bacterial diseases and has broad application prospects in pesticide development. Summary of the Invention

[0006] The technical problem this invention aims to solve is to provide a pesticide delivery system with strong environmental compatibility, responsive release, and high loading rate to improve the efficacy of pesticide delivery. Simultaneously, addressing the issue of resistance to bacteria, fungi, and viruses associated with traditional pesticides, this invention develops thymol derivatives containing quaternary ammonium salts and uses them in conjunction with a drug delivery system to verify the actual effectiveness of the liposome-based drug delivery system.

[0007] The technical solution of this invention is a liposome nanodelivery system developed for plant bacterial diseases. The specific formulation is as follows: hydrogenated soybean phosphatidylcholine (HSPC), cholesterol hemisuccinate monoester (CHEMS), and phosphatidylethanolamine (PE) are co-assembled into C2@LNP, constructing a multifunctional drug-loaded antibacterial liposome system (HSPC:CHEMS:PE:C2=6:2.7:2.7:1). The material exhibits the ability to release drugs under weakly acidic conditions, is environmentally friendly, reduces surface tension, and improves leaf adhesion.

[0008] The technical solution of the present invention: a class of quaternary ammonium salt derivatives containing thymol, the structural formula of which is shown in (1): (1) Where R1 is 2-isopropyl-5-methylphenyl, ethyl, or a benzene ring, R2 is 2-chloro-5-methylthiophene, 3-methyl-2-methoxycarbonylfuran, 4-methylnaphthalene, and benzyl groups with different substitutions, X - For Cl - and Br - n=3, 5, 7, 8, 9.

[0009] A quaternary ammonium salt derivative containing thymol, where R1 is 2-isopropyl-5-methylphenyl.

[0010] A quaternary ammonium salt derivative containing thymol, where R1 is 2-isopropyl-5-methylphenyl, R2 is benzyl, and X - For Cl - n=7.

[0011] The preparation method of quaternary ammonium salt derivatives containing thymol is shown in the following reaction formula: The specific synthetic routes for different compounds can be broken down into the following synthetic equations: Synthetic routes for quaternary ammonium salt derivatives with different benzyl and other substituent groups Synthetic routes for quaternary ammonium salt derivatives by replacing thymol with phenyl and ethyl compounds. Synthetic routes for quaternary ammonium salt derivatives with different chain lengths and substituted groups such as benzyl. This invention relates to drug-loaded antibacterial liposomes containing thymol quaternary ammonium salt derivatives, which are assembled by co-assembling hydrogenated soybean phosphatidylcholine, cholesterol hemisuccinate monoester, phosphatidylethanolamine, and the target compound containing thymol quaternary ammonium salt derivatives into drug-loaded antibacterial liposomes.

[0012] This invention relates to drug-loaded antibacterial liposomes containing a quaternary ammonium salt derivative of thymol. The mass ratio of hydrogenated soybean phosphatidylcholine, cholesterol hemisuccinate monoester, phosphatidylethanolamine, and the target compound containing the quaternary ammonium salt derivative of thymol is 6:2.7:2.7:1. The specific preparation process is as follows: the above four components are mixed and dissolved in 10 ml of ethanol, then rotary evaporated to dryness to form an oil film, and then 10 ml of deionized water is added. The mixture is then hydrated at 55°C for 60 minutes, and the liposomes are obtained.

[0013] This invention relates to the application of thymol-containing quaternary ammonium salt derivatives or their drug-loaded antibacterial liposomes in antibacterial agents.

[0014] Preferably, the antibacterial agent is resistant to rice bacterial blight.

[0015] The beneficial effects of this invention compared to existing technologies are as follows: This invention first introduces thymol into the quaternary ammonium salt structure to synthesize a series of derivatives (taking Xoo as an example), by disrupting... Xoo A novel antibacterial mechanism inducing apoptosis through inner and outer membrane permeability was developed to address the resistance problem of Xoo caused by long-term application of traditional pesticides. Simultaneously, an innovative liposome-based drug delivery system was developed, and through optimized formulation, liposomes loaded with C2 showed significantly better efficacy in controlling plant bacterial diseases than C2 alone. This system not only improved drug bioavailability but also significantly enhanced crop growth and health by reducing drug toxicity to plants and aquatic organisms. Optimizing the surface properties of the liposomes (such as contact angle and surface tension) improved the system's adhesion to plant surfaces, further enhancing efficacy. In practical applications, C2@LNP demonstrated excellent protective and curative effects, with significantly better control than traditional drug forms. Furthermore, it exhibited advantages such as being environmentally friendly, low in toxicity, and highly effective in controlling crop diseases. In addition, the system promoted plant growth, significantly increasing plant height and fresh weight, providing a more environmentally friendly and efficient solution for the control of plant bacterial diseases. Attached Figure Description

[0016] Figure 1 Schematic diagram of liposome co-assembly; Figure 2 Comparison of compound C2 before and after co-assembly; Figure 3 Release effect of C2@LNP at different pH levels; Figure 4 Different components at 5×EC 50 The following is a plate coating experiment; Figure 5 Toxicity test of each lipid component on rice growth at 100 μg / mL; Figure 6 Toxicity experiments of C2, LNP and C2@LNP on zebrafish at different concentrations; Figure 7 In vivo protective and therapeutic activities of C2, LNP and C2@LNP at 100 μg / mL. Detailed Implementation

[0017] Example 1 Synthesis of intermediates and target compounds Starting with 4-hydroxypyridine, intermediate 1 was obtained via electrophilic attack. Intermediate 1 was deprotected to give intermediate 2, which was then esterified to give intermediate 3. Finally, intermediate 3 was reacted with various benzyl compounds to give compounds A1-A17.

[0018] Preparation of intermediate 1 (intermediates 5–8 were prepared using the same method) Preparation of 9-(ethylene oxide-2-ylmethyl)-9H-carbazole 4-Hydroxypyridine (5.26 mmol) and KOH (6.31 mmol) were dissolved in acetonitrile (MeCN, 25 mL) and stirred at 80 °C for 30 min. Ethyl 6-bromohexanoate (5.26 mmol) was then slowly added dropwise, and the mixture was refluxed for 8 h. After the reaction was complete (monitored by TLC), the mixture was diluted with ethyl acetate (50 mL). The organic phase was washed sequentially with saturated NH4Cl aqueous solution (3 × 25 mL), dried over anhydrous Na2SO4, and concentrated under reduced pressure. Purification by column chromatography (CH2Cl2 / MeOH = 40:1) yielded intermediate 1 (using the general method for intermediates 5–8).

[0019] White, transparent liquid, yield 95.43%; 1 H NMR (400 MHz, CDCl3) δ 7.22 (d, J = 7.6 Hz,2H, N-(CH)2), 6.25 (d, J = 7.6 Hz, 2H, N-(CH)2-(CH)2), 3.99 (q, J = 7.2 Hz, 2H,CH3-CH2), 3.69 (t, J = 7.2 Hz, 2H, pyridine-O-CH2), 2.19 (t, J = 7.3 Hz, 2H, O=C-CH2), 1.67 (p, J= 7.4 Hz, 2H, pyridine-OC-CH2), 1.54 (p, J = 7.4 Hz, 2H, O=CC-CH2), 1.22 (p, J = 7.8 Hz, 2H, O=CCC-CH2), 1.12 (t, J = 7.1 Hz, 3H, -CH3). 13 C NMR(101 MHz, CDCl3) δ 178.81, 173.25, 139.97, 118.42, 60.32, 56.63, 33.75,30.48, 25.50, 24.09, 14.14. HR-MS (ESI): m / z calcd for C 13 H 19 NO3([M+H)) + )238.14377, found 238.14450. Preparation of intermediate 2 (intermediates 9–12 were prepared using the same method) Preparation of 6-(pyridine-4-oxy)hexanoic acid Intermediate 1 (2.11 mmol) and NaOH (3.16 mmol) were dissolved in H2O / MeCN (v / v = 1:1, 20 mL) and stirred at 60 °C for 5 h. After the reaction was confirmed by TLC, the solvent was removed by vacuum distillation. The residue was purified by column chromatography (CH2Cl2 / MeOH = 10:1) to give intermediate 2 (the general method for intermediates 9–12).

[0020] White solid, yield 45.94%, melting point 126-127 °C; 1 H NMR (400 MHz, DMSO- d 6) δ 7.68(d, J = 7.6 Hz, 2H, N-(CH)2), 6.06 (d, J = 7.6 Hz, 2H, N-(CH)2-(CH)2), 3.81 (t, J =7.1 Hz, 2H, pyridine-O-CH2), 2.12 (t, J = 7.3 Hz, 2H, O=C-CH2), 1.61 (p, J= 7.3Hz, 2H, pyridine-OC-CH2), 1.44 (p, J = 7.4 Hz, 2H, O=CC-CH2), 1.23 – 1.09 (m,2H, O=CCC-CH2). 13 C NMR (101 MHz, DMSO- d 6) δ 177.78, 175.13, 141.59, 117.69,55.67, 34.26, 30.61, 25.58, 24.52. HR-MS (ESI): m / z calcd for C 11 H 15 NO3([M+H)) + )210.11247, found 210.11341. Preparation of intermediate 3 (intermediates 4, 13–16 were prepared using the same method) Preparation of 2-isopropyl-5-methylphenyl 6-(pyridine-4-oxy)hexanoate Intermediate 2 (2.39 mmol) was dissolved in MeCN (20 mL), followed by the addition of EDCI (2.87 mmol), DMAP (2.87 mmol), and thymol (2.39 mmol). The mixture was stirred overnight at room temperature. After the reaction was complete (monitored by TLC), ethyl acetate (60 mL) was added. The organic phase was washed with saturated NH4Cl aqueous solution (30 mL × 3), dried over anhydrous Na2SO4, and concentrated under reduced pressure. The solution was purified by column chromatography (CH2Cl2 / MeOH = 60:1) to obtain intermediate 3 (using the general method for intermediates 4, 13–16).

[0021] White, transparent liquid, yield 82.94%; 1 H NMR (400 MHz, CDCl3) δ 7.32 (d, J = 7.6 Hz,2H, N-(CH)2), 7.19 (d, J = 7.9 Hz, 1H, CH3-CH), 7.01 (d, J = 8.0 Hz, 1H, CH3-C-CH), 6.77 (s, 1H, CH3-CH-C), 6.34 (d, J = 7.6 Hz, 2H, N-(CH)2-(CH)2), 3.77 (t, J=7.2 Hz, 2H, pyridine-O-CH2), 2.93 (p, J = 7.0 Hz, 1H, -CH-), 2.59 (t, J = 7.3 Hz,2H, O=C-CH2), 2.30 (s, 3H, -CH3), 1.79 (h, J = 7.6 Hz, 4H, pyridine-OC-CH2, O=CC-CH2), 1.42 (p, J = 7.8 Hz, 2H, pyridine-OCC-CH2), 1.17 (d, J = 7.0 Hz, 6H, -C(CH3)2). 13 C NMR (101 MHz, CDCl3) δ 178.99, 172.08, 154.56, 147.78, 140.14,127.21, 126.48, 125.77, 122.63, 119.93, 118.51, 116.15, 56.80, 33.82, 30.61,27.12, 25.68, 24.22, 23.03, 22.83, 20.82. HR-MS (ESI): m / z calcd for C 21 H 27 NO3([M+H)) + ) 342.20637, found 342.20613. Synthesis of target compounds A1-A17, B1-B2 and C1-C4 (taking the synthesis of compound A1 as an example) Intermediate 3 (879 μmol) and various substituted benzyl derivatives (3.51 mmol) were dissolved in MeCN and heated at 80 °C for 10 h. After the reaction was completed, the solvent was removed by vacuum distillation, and the product was purified by column chromatography using ethyl acetate as the eluent to obtain compounds A1–A17 (the general method for compounds B1, B2 and C1–C4).

[0022] Target compound A1 Brown liquid, yield 74.43%; 1 H NMR (400 MHz, CDCl3) δ 8.60 (d, J= 7.3 Hz, 2H,N-(CH)2), 7.52 (d, J = 7.3 Hz, 2H, N-(CH)2-(CH)2), 7.18 (d, J = 7.9 Hz, 1H, Cl-S-C-CH), 7.14 (d, J = 3.7 Hz, 1H, CH3-C-CH), 7.00 (d, J = 8.1 Hz, 1H, Cl-C-CH),6.85 (d, J = 3.9 Hz, 1H, CH3-C-CH-C-O), 6.78 (s, 1H, CH3-C-CH), 5.54 (s, 2H, N-CH2), 4.53 (t, J = 7.5 Hz, 2H, pyridine-O-CH2), 2.91 (p, J = 6.9 Hz, 1H, -CH-),2.66 (t, J = 7.2 Hz, 2H, O=C-CH2), 2.28 (s, 3H, -CH3), 2.11 (p, J = 7.5 Hz, 2H,pyridine-O-C-CH2), 1.84 (p, J = 7.3 Hz, 2H, O=C-C-CH2), 1.56 (p, J = 7.9 Hz, 2H, O=C-C-C-CH2), 1.15 (d, J = 6.9 Hz, 6H, -C(CH3)2). 13 C NMR (101 MHz, CDCl3) δ172.61, 169.70, 147.87, 145.92, 137.14, 136.73, 133.17, 133.04, 129.90,127.37, 126.61, 126.54, 122.88, 115.43, 68.37, 61.19, 53.64, 33.92, 31.31,27.20, 25.78, 24.22, 23.30, 21.05. HR-MS (ESI): m / z calcd for C 26 H 30 NO3S ([M-Cl - ] -) 472.17077, found 472.16867. Target compound A2 Brown liquid, yield 68.12%; 1 H NMR (400 MHz, CDCl3) δ 8.64 (d, J = 7.3 Hz, 2H,N-(CH)2), 7.54 (d, J = 7.4 Hz, 2H, N-(CH)2-(CH)2), 7.17 (m, 2H, Furan-OC-CH,(CH3)2CH-C-CH), 6.99 (d, J = 7.9 Hz, 1H, (CH3)2CH-CC-CH), 6.83 (d, J = 3.6 Hz,1H, (CH3)2CH-C-CH-CH), 6.78 (s, 1H, Furan-OC-CH-CH), 5.46 (s, 2H,-CH2-), 4.53(t, J = 7.5 Hz, 2H, pyridine-O-CH2), 3.88 (s, 3H, O-CH3), 2.90 (p, J = 6.8 Hz, 1H,(CH3)2CH-), 2.65 (t, J = 7.3 Hz, 2H, O=C-CH2), 2.27 (s, 3H, -CH3), 2.11 (p, J =7.7 Hz, 2H, pyridine-OC-CH2), 1.83 (p, J = 7.4 Hz, 2H, O=CC-CH2), 1.55 (p, J =7.9 Hz, 2H, O=CCC-CH2), 1.13 (d, J = 6.9 Hz, 6H, (CH3)2CH-). 1313C NMR (101 MHz, CDCl3) δ 172.54, 169.61, 158.93, 150.16, 147.88, 146.08, 145.75, 137.15, 136.70, 127.33, 126.58, 122.89, 118.90, 115.26, 115.07, 65.09, 61.17, 60.55, 53.67, 52.49, 33.94, 31.28, 27.17, 25.80, 24.26, 23.28, 21.23, 21.02, 14.32. HR-MS (ESI): m / z calcd for C 28 H 33 NO6([M - Cl - - ) 480.23806, found 480.23742. Target compound A3 Brown liquid, yield 54.67%; 1 1H NMR (400 MHz, CDCl3) δ 8.47 (d, J J = 7.1 Hz, 2H, N-(CH)2), 7.99 (s, 1H, N-CH-C-CH-CH), 7.89 (dd, J J = 9.0, 5.6 Hz, 2H, N-CH-C-CH-CH-CH-CH-CH-CH), 7.85 – 7.81 (m, 1H, N-CH-C-CH-CH-CH-CH-CH-CH-CH), 7.55 (dd, J J = 8.4, 1.8 Hz, 1Hz, N-CH-C-CH), 7.50 (dd, J J = 6.2, 3.2 Hz, 2H, N-(CH)2-(CH)2), 7.46 (d, J J = 7.5 Hz, 2H, N-CH-C-CH-CH-CH-CH-CH), 7.15 (d, J J = 7.8 Hz, 1H, (CH3)2CH-C-CH), 6.99 (d, J ​= 6.5 Hz, 1H, (CH3)2CH-C-CH-CH), 6.78 (s, 1H, (CH3)2CH-CC-CH), 5.55 (s, 2H, -CH2-), 4.40 (t, J = 7.5 Hz, 2H, pyridine-O-CH2), 2.90 (p, J =7.0 Hz, 1H, (CH3)2CH-), 2.62 (t, J = 7.3 Hz, 2H, O=C-CH2), 2.27 (s, 3H, -CH3),2.09 – 1.95 (m, 2H, pyridine-OC-CH2), 1.80 (p, J = 7.4 Hz, 2H, O=CC-CH2), 1.49(p, J = 7.7, 6.9 Hz, 2H, O=CCC-CH2), 1.14 (d, J = 6.9 Hz, 6H, (CH3)2CH-). 13 C NMR(101 MHz, CDCl3) δ 172.50, 170.24, 147.88, 145.69, 137.63, 137.14, 136.69,133.54, 133.11, 130.46, 129.23, 128.45, 128.24, 127.97, 127.32, 127.14,126.94, 126.57, 125.67, 122.89, 115.34, 73.77, 60.94, 60.57, 53.65, 33.91,31.25, 27.18, 25.75, 24.23, 23.29, 21.04, 14.33. HR-MS (ESI): m / z calcd forC 32 H 35 NO3([M-Br - ] - ) 482.26897, found 482.26676. Target compound A4 Brown liquid, yield 68.12%; 1H NMR (400 MHz, CDCl3) δ 8.59 – 8.56 (m, 2H, N-(CH)2), 7.51 – 7.47 (m, 4H, N-(CH)2-(CH)2, N-CH-C-(CH)2), 7.41 (t, J = 7.6 Hz,3H, N-CH-C-(CH)2-(CH)2-CH), 7.17 (d, J = 7.8 Hz, 1H, (CH3)2CH-C-CH), 7.00 (d, J =7.8 Hz, 1H, (CH3)2CH-C-CH-CH), 6.78 (s, 1H, (CH3)2CH-C-C-CH), 5.44 (s, 2H, -CH2-), 4.52 (t, J = 7.5 Hz, 2H, pyridine-O-CH2), 2.91 (p, J = 6.9 Hz, 1H, (CH3)2CH-), 2.65 (t, J = 7.1 Hz, 2H, O=C-CH2), 2.28 (s, 3H, -CH3), 2.09 (p, J = 7.6 Hz,2H, pyridine-O-C-CH2), 1.83 (p, J = 7.4 Hz, 2H, O=C-C-CH2), 1.55 (p, J = 8.4, 8.0Hz, 2H, O=C-C-C-CH2), 1.14 (d, J = 6.9 Hz, 6H, (CH3)2CH-). 13 C NMR NMR (126 MHz,CDCl3) δ 172.47, 170.41, 147.88, 145.82, 137.12, 136.70, 132.96, 129.59,129.24, 128.63, 127.32, 126.57, 122.85, 115.33, 73.71, 61.03, 53.59, 33.84,31.25, 27.19, 25.73, 24.15, 23.25, 21.00. HR-MS (ESI): m / z calcd for C 24 H 33 NO3([M-Cl - ] -) 432.25332, found 432.25265. Target compound A5 Brown liquid, yield 62.44%; 1 H NMR (400 MHz, CDCl3) δ 8.57 (s, 2H, N-(CH)2),7.49 (t, J = 7.3 Hz, 4H, N-CH-C-(CH)2-(CH)2), 7.43 (s, 1H, N-CH-C-(CH)2-(C)2-CH), 7.41 (s, 2H, N-(CH)2-(CH)2), 7.17 (d, J = 7.9 Hz, 1H, (CH3)2CH-C-CH), 7.00(d, J = 7.9 Hz, 1H, (CH3)2CH-CC-CH), 6.78 (d, J = 1.7 Hz, 1H, (CH3)2CH-C-CH-CH),5.45 (s, 2H, -CH2-), 4.53 (t, J = 7.5 Hz, 2H, pyridine-O-CH2), 2.93 – 2.88 (m,1H, -CH-), 2.65 (t, J = 7.1 Hz, 2H, O=C-CH2), 2.28 (s, 3H, -CH3), 1.83 (dt, J =15.1, 7.2 Hz, 3H, pyridine-OC-CH2, O=CC-CH), 1.59 – 1.51 (m, 3H, O=CC-CH,pyridine-OCC-CH2), 1.14 (d, J = 6.9 Hz, 6H, (CH3)2CH-). 13 C NMR (101 MHz, CDCl3)δ 172.40, 170.36, 147.80, 145.75, 137.05, 136.63, 132.89, 129.53, 129.18,128.56, 127.25, 126.50, 122.77, 115.28, 73.66, 60.98, 33.76, 31.17, 27.12,25.65, 24.07, 23.18, 20.93. HR-MS (ESI):m / z calcd for C 28 H 33 NO3([M-Br)) - )432.25332, found 432.25241. Target compound A6 Brown liquid, yield 68.53%; 1 H NMR (400 MHz, CDCl3) δ 8.61 (d, J = 7.4 Hz, 2H,N-(CH)2), 7.52 (d, J = 7.4 Hz, 2H, N-(CH)2-(CH)2), 7.46 (d, J = 8.5 Hz, 2H, N-CH-C-(CH)2, 7.40 (d, J = 8.5 Hz, 2H, (CH3)2CH-C-CH-CH)), 7.18 (d, J = 7.9 Hz, 1H, N-CH-CC-CH), 7.01 (d, J = 6.2 Hz, 1H, N-CH-CC-CH), 6.80 (s, 1H, (CH3)2CH-CC-CH)), 5.44 (s, 2H, -CH2-), 4.53 (t, J = 7.5 Hz, 2H, pyridine-O-CH2), 2.92 (p, J =6.9 Hz, 1H, -CH-), 2.66 (t, J = 7.2 Hz, 2H, O=C-CH2), 2.29 (s, 3H, -CH3), 2.11(p, J = 7.6 Hz, 2H, pyridine-OC-CH2), 1.84 (p, J = 7.3 Hz, 2H, O=CC-CH2), 1.56(p, J = 7.8 Hz, 2H, O=CCC-CH2), 1.16 (d, J = 6.9 Hz, 6H, (CH3)2CH-). 13 C NMR (101MHz, CDCl3) δ 171.26 (d, 1 J F= 231.8 Hz), 147.82, 145.83, 137.07, 136.63,135.38, 131.49, 130.07, 129.33, 127.26, 126.52, 122.81, 115.34, 72.81, 61.02,33.83, 31.18, 27.12, 25.69, 24.14, 23.21, 20.96. 19 F NMR (376 MHz, CDCl3) δ -117.87. HR-MS (ESI): m / z calcd for C 28 H 32 FNO3([M-Cl)) - ) 450.24389, found 450.24328. Target compound A7 Brown liquid, yield 70.32%; 1 H NMR (400 MHz, CDCl3) δ 8.59 (d, J = 7.3 Hz, 2H,N-(CH)2), 7.53 (d, J = 7.4 Hz, 2H, N-(CH)2-(CH)2), 7.47 – 7.36 (m, 1H, N-CH-C-CH-CH), 7.30 (d, J = 7.6 Hz, 1H, (CH3)2CH-CC-CH)), 7.19 (d, J = 8.7 Hz, 2H,(CH3)2CH-C-CH-CH)), 7.16 (s, 1H, N-CH-C-CH), 7.10 (t, J = 8.4 Hz, 2H, N-CH-C-(CH)2), 7.01 (d, J = 8.1 Hz, 1H, (CH3)2CH-CC-CH)), 6.78 (s, 1H, N-CH-C-CH-CH-CH-CF), 5.46 (s, 2H, -CH2-), 2.92 (dt, J = 14.0, 6.7 Hz, 1H, -CH-), 2.67 (t, J =7.2 Hz, 2H, pyridine-O-CH2), 2.29 (s, 3H, -CH3), 2.14 (p,J = 7.8 Hz, 2H, O=C-CH2), 1.86 (dt, J = 15.1, 7.3 Hz, 2H, pyridine-OC-CH2), 1.66 (s, 2H, O=CC-CH2), 1.58 (p, J = 7.9 Hz, 2H, O=CCC-CH2), 1.15 (d, J = 6.9 Hz, 6H, (CH3)2CH-). 13 C NMR (101 MHz, CDCl3) δ 172.54, 170.24, 163.02 (d, 1 J F = 247.6 Hz), 147.86,145.83, 137.12, 136.72, 135.28 (d, 3 J F = 7.5 Hz), 131.04 (d, 3 J F = 8.3 Hz),127.34, 126.59, 124.14, 122.84, 116.58 (d, 2 J F = 21.0 Hz), 115.45, 115.28 (d, 2 J F = 22.3 Hz), 72.82, 61.22, 33.86, 31.27, 27.21, 25.77, 24.16, 23.27, 21.01. 19 FNMR (376 MHz, CDCl3) δ -111.24. HR-MS (ESI): m / z calcd for C 28 H 32 FNO3([M-Cl)) - )450.24389, found 450.24388. Target compound A8 Brown liquid, yield 73.08%; 1 H NMR (400 MHz, CDCl3) δ 8.61 (d, J= 7.1 Hz, 2H,N-(CH)2), 7.53 (t, J = 7.5 Hz, 1H, (CH3)2CH-C-C-CH)), 7.47 (d, J = 7.2 Hz, 2H, N-(CH)2-(CH)2), 7.40 (q, J = 6.8, 5.9 Hz, 1H, F-C-CH-CH-CH), 7.20 (t, J = 7.6 Hz,1H, F-C-CH), 7.12 (dd, J = 20.6, 8.4 Hz, 2H, (CH3)2CH-C-CH-CH)), 6.97 (d, J = 7.7Hz, 1H, F-C-CH-CH-CH), 6.78 (s, 1H, F-C-CH), 5.43 (s, 2H, -CH2-), 4.50 (t, J =7.5 Hz, 2H, pyridine-O-CH2), 2.90 (p, J = 6.9 Hz, 1H, -CH-), 2.64 (t, J = 7.3 Hz,2H, O=C-CH2), 2.25 (s, 3H, -CH3), 2.09 (dd, J = 16.2, 8.7 Hz, 2H, pyridine-O-C-CH2), 1.83 (p, J = 7.5 Hz, 2H, O=C-C-CH2), 1.61 – 1.47 (m, 2H, O=C-C-C-CH2),1.13 (d, J = 6.9 Hz, 6H, (CH3)2C-). 13 C NMR (101 MHz, CDCl3) δ 172.54, 171.37,170.08, 162.34, 159.86, 147.93, 146.01, 137.19, 136.66, 131.96, 131.88,131.51, 127.31, 126.57, 124.99, 124.95, 122.94, 120.28, 116.11, 115.90,115.10, 67.62, 60.56, 34.01, 31.37, 27.16, 25.82, 24.34, 23.31, 21.03,14.34.13 C NMR (101 MHz, CDCl3) δ 172.54, 171.37, 170.08, 161.10 (d, 1 J F = 249.6Hz), 147.93, 146.01, 137.19, 136.66, 131.92 (d, 3 J F = 8.0 Hz), 131.51, 127.31,126.57, 124.97 (d, 4 J F = 3.4 Hz), 122.94, 120.28, 116.00 (d, 2 J F = 20.8 Hz),115.10, 67.62, 60.56, 34.01, 31.37, 27.16, 25.82, 24.34, 23.31, 21.03,14.34. 19 F NMR (376 MHz, CDCl3) δ -116.81. HR-MS (ESI): m / z calcd for C 28 H 32 FNO3([M-Cl)) - ) 450.24389, found 450.24262. Target compound A9 Brown liquid, yield 58.11%; 1 H NMR (400 MHz, CDCl3) δ 8.60 (d, J = 7.5 Hz, 2H,N-(CH)2), 7.71 – 7.59 (m, 4H, N-(CH)2-(CH)2, CF3-C-(CH)2), 7.53 (d, J = 7.4 Hz,2H, CF3-CC-(CH)2), 7.16 (d, J = 7.9 Hz, 1H, (CH3)2CH-C-CH)), 6.99 (d, J= 6.2 Hz,1H, (CH3)2CH-C-CH-CH)), 6.79 (s, 1H, (CH3)2CH-CC-CH)), 5.51 (s, 2H, -CH2-),4.50 (t, J = 7.5 Hz, 2H, pyridine-O-CH2), 2.91 (p, J = 7.0 Hz, 1H, -CH-), 2.65(t, J = 7.3 Hz, 2H, O=C-CH2), 2.26 (s, 3H, -CH3), 2.10 (p, J = 7.7 Hz, 2H,pyridine-OC-CH2), 1.83 (p, J = 7.4 Hz, 2H, O=CC-CH2), 1.61 – 1.47 (m, 2H, O=CCC-CH2), 1.14 (d, J = 6.9 Hz, 6H, (CH3)2C-). 13 C NMR (101 MHz, CDCl3) δ 172.56,170.04, 147.90, 145.99, 137.16, 137.07, 136.70, 131.32 (q, 2 J F = 32.6 Hz),128.77, 127.36, 126.60, 126.10 (q, 3 J F = 3.6 Hz), 123.94 (q, 1 J F = 272.4 Hz),122.90, 115.45, 72.62, 61.14, 33.96, 31.29, 27.18, 25.79, 24.27, 23.29,21.04. 19 F NMR (376 MHz, CDCl3) δ -62.61. HR-MS (ESI): m / z calcd for C 29 H 32 F3NO3([M-Br)) - ) 500.24071, found 500.23989. Target compound A10 Brown liquid, yield 66.72%; 1 H NMR (400 MHz, DMSO- d 6) δ 8.87 (d, J J = 7.4 Hz, 2H, N-(CH)2), 7.68 (d, J J = 7.4 Hz, 2H, N-(CH)2-(CH)2), 7.49 (d, J J = 2.7 Hz, 4H, Cl-C-(CH)2-(CH)2), 7.19 (d, J J = 7.9 Hz, 1H, (CH3)2CH-C-CH)), 7.00 (d, J J = 7.9 Hz, 1H,(CH3)2CH-C-CH-CH)), 6.75 (s, 1H, (CH3)2CH-C-C-CH)), 5.42 (s, 2H, -CH2-), 4.41(t, J J = 7.4 Hz, 2H, pyridine-O-CH2), 2.83 (p, J J = 7.0 Hz, 1H, -CH-), 2.59 (t, J J =7.3 Hz, 2H, O=C-CH2), 2.22 (s, 3H, -CH3), 1.91 – 1.85 (m, 2H, pyridine-O-C-CH2), 1.65 (p, J J = 7.4 Hz, 2H, O=C-C-CH2), 1.32 (p, J J = 7.7 Hz, 2H, O=C-C-C-CH2),1.06 (d, J J = 6.9 Hz, 6H, (CH3)2C-). 13 C NMR (126 MHz, DMSO- d 6) δ 172.41, 170.05,148.10, 146.63, 138.90, 137.19, 136.67, 131.83, 129.83, 129.20, 127.46,126.90, 123.25, 114.43, 72.51, 59.18, 33.64, 30.61, 27.00, 25.34, 24.29,23.44, 21.39, 20.87. HR-MS (ESI): m / z calcd for C28 H 32 ClNO3([M-Cl)) - ) 466.21435, found 466.21421. Target compound A11 Brown liquid, yield 79.52%; 1 H NMR (400 MHz, CDCl3) δ 8.57 (d, J = 7.2 Hz, 2H,N-(CH)2), 7.52 (d, J = 8.4 Hz, 2H, Br-C-(CH)2), 7.49 (d, J = 7.3 Hz, 2H, N-(CH)2-(CH)2), 7.38 (d, J = 8.3 Hz, 2H, Br-C-(C)2-(CH)2), 7.16 (d, J = 7.9 Hz, 1H, (CH3)2CH-C-CH)), 6.99 (d, J = 7.8 Hz, 1H, (CH3)2CH-CC-CH)), 6.78 (s, 1H, (CH3)2CH-C-CH-CH)), 5.39 (s, 2H, -CH2-), 4.48 (t, J = 7.5 Hz, 2H, pyridine-OC-CH2), 2.91(p, J = 7.0 Hz, 1H, -CH-), 2.64 (t, J = 7.3 Hz, 2H, O=C-CH2), 2.27 (s, 3H, -CH3), 2.08 (dd, J = 15.9, 6.8 Hz, 2H, pyridine-OCC-CH2), 1.82 (p, J = 7.4 Hz, 2H, O=CC-CH2), 1.60 – 1.46 (m, 2H, O=CCC-CH2), 1.14 (d, J = 6.8 Hz, 6H, (CH3)2C-). 13C NMR (101 MHz, CDCl3) δ 172.52, 170.09, 147.90, 145.89, 137.16, 136.69,132.33, 132.13, 130.42, 127.35, 126.60, 123.61, 122.91, 115.39, 72.85, 61.05,53.70, 33.97, 31.29, 27.19, 25.79, 24.29, 23.32, 21.07. HR-MS (ESI): m / z calcdfor C 28 H 32 BrNO3([M-Br] - ) 510.16383, found 510.16257. Target compound A12 Brown liquid, yield 63.07%; 1 H NMR (400 MHz, CDCl3) δ 8.55 (d, J = 7.1 Hz, 2H,N-(CH)2), 7.73 (d, J = 8.2 Hz, 2H, IC-(CH)2), 7.48 (d, J = 7.1 Hz, 2H, N-(CH)2-(CH)2), 7.27 – 7.23 (m, 2H, IC-(C)2-(CH)2), 7.17 (d, J = 8.0 Hz, 1H, (CH3)2CH-C-CH)), 6.99 (d, J = 7.8 Hz, 1H, (CH3)2CH-CC-CH)), 6.79 (s, 1H, (CH3)2CH-C-CH-CH)), 5.37 (s, 2H, -CH2-), 4.47 (t, J = 7.5 Hz, 2H, pyridine-O-CH2), 2.91 (p, J =7.0 Hz, 1H, -CH-), 2.65 (t, J = 7.3 Hz, 2H, O=C-CH2), 2.27 (s, 3H, -CH3), 2.07(dt, J = 16.0, 7.9 Hz, 2H, pyridine-OC-CH2), 1.82 (p,J = 7.5 Hz, 2H, O=CC-CH2),1.60 – 1.46 (m, 2H, O=CCC-CH2), 1.14 (d, J = 6.9 Hz, 6H, (CH3)2C-). 13 C NMR (101MHz, CDCl3) δ 172.55, 170.09, 147.90, 145.86, 138.28, 137.17, 136.70, 132.75,130.53, 127.37, 126.62, 122.93, 115.43, 95.66, 72.96, 61.09, 53.70, 34.00,31.31, 27.20, 25.82, 24.32, 23.36, 21.11. HR-MS (ESI): m / z calcd for C 28 H 32 INO3([M-Br - ] - ) 558.14996, found 558.14896. Target compound A13 Brown liquid, yield 53.41%; 1 H NMR (400 MHz, CDCl3) δ 8.60 (d, J = 7.2 Hz, 2H,N-(CH)2), 8.20 (d, J = 8.7 Hz, 2H, NO2-C-(CH)2), 7.70 (d, J = 8.5 Hz, 2H, NO2-C-(C)2-(CH)2), 7.56 (d, J = 7.2 Hz, 2H, N-(CH)2-(CH)2), 7.15 (d, J = 7.9 Hz, 1H,(CH3)2CH-C-CH)), 6.97 (d, J = 6.1 Hz, 1H, (CH3)2CH-C-CH)), 6.77 (s, 1H, (CH3)2CH-C-CH-CH)), 5.56 (s, 2H, -CH2-), 4.49 (t, J = 7.5 Hz, 2H, pyridine-O-CH2), 2.89(p, J= 7.0 Hz, 1H, -CH-), 2.64 (t, J = 7.4 Hz, 2H, O=C-CH2), 2.24 (s, 3H, -CH3), 2.13 – 2.02 (m, 2H, pyridine-OC-CH2), 1.80 (dd, J = 15.3, 7.5 Hz, 2H, O=CC-CH2), 1.53 (p, J = 8.1, 7.6 Hz, 2H, O=CCC-CH2), 1.12 (d, J = 6.9 Hz, 6H, (CH3)2C-). 13 C NMR (101 MHz, CDCl3) δ 172.62, 169.82, 147.90, 146.02, 140.48,137.17, 136.68, 129.12, 127.37, 126.63, 124.33, 122.92, 115.54, 72.03, 60.58,53.70, 34.03, 31.27, 27.19, 25.84, 24.35, 23.34, 21.10, 14.32. HR-MS (ESI): m / z calcd for C 28 H 32 N2O5([M-Br)) - ) 477.23839, found 477.23708. Target compound A14 Brown liquid, yield 62.15%; 1 H NMR (400 MHz, DMSO- d 6) δ 8.85 (d, J = 7.2 Hz, 2H,N-(CH)2), 7.67 (d, J = 7.2 Hz, 2H, N-(CH)2-(CH)2), 7.36 (d, J = 7.9 Hz, 2H,phenmethyl-C-(CH)2), 7.23 – 7.18 (m, 3H, phenmethyl-C-(CH)2-(CH)2, (CH3)2CH-C-CH)), 7.00 (d, J= 7.8 Hz, 1H, (CH3)2CH-CC-CH)), 6.75 (s, 1H, (CH3)2CH-C-CH-CH)), 5.37 (s, 2H, -CH2-), 4.40 (t, J = 7.3 Hz, 2H, pyridine-O-CH2), 2.83 (p, J =7.0 Hz, 1H, -CH-), 2.59 (t, J = 7.4 Hz, 2H, O=C-CH2), 2.29 (s, 3H, benzene-CH3), 2.22 (s, 3H, -CH3), 1.88 (p, J = 7.5 Hz, 2H, pyridine-OC-CH2), 1.65 (p, J = 7.5Hz, 2H, O=CC-CH2), 1.32 (dt, J = 15.2, 7.5 Hz, 2H, O=CCC-CH2), 1.06 (d, J = 6.9Hz, 6H, (CH3)2C-). 13 C NMR (126 MHz, DMSO- D 6) δ 172.41, 170.05, 148.10, 146.63,138.90, 137.19, 136.67, 131.83, 129.83, 129.20, 127.46, 126.90, 123.25,114.43, 72.51, 59.18, 33.64, 30.61, 27.00, 25.34, 24.29, 23.44, 21.39, 20.87.HR-MS (ESI): m / z calcd for C 29 H 35 NO3([M-Cl] - ) 446.26897, found 446.26887. Target compound A15 Brown liquid, yield 65.73%; 1 H NMR (400 MHz, CDCl3) δ 8.57 (d, J= 7.2 Hz, 2H,N-(CH)2), 7.45 (s, 2H, N-(CH)2-(CH)2), 7.40 (d, J = 7.9 Hz, 2H, phenethyl-C-(CH)2), 7.27 – 7.23 (m, 2H, phenethyl-C-(CH)2-(CH)2), 7.17 (d, J = 7.9 Hz, 1H,(CH3)2CH-C-CH)), 6.99 (d, J = 7.9 Hz, 1H, (CH3)2CH-C-C-CH)), 6.79 (s, 1H, (CH3)2CH-CH-CH)), 5.38 (s, 2H, -CH2-), 4.48 (t, J = 7.5 Hz, 2H, pyridine-O-CH2), 2.92(p, J = 7.0 Hz, 1H, -CH-), 2.65 (tt, J = 7.8, 4.2 Hz, 4H, benzene-CH2-CH3, O=C-CH2), 2.27 (s, 3H, -CH3), 2.08 (p, J = 8.9, 8.2 Hz, 2H, pyridine-O-C-CH2), 1.84(p, J = 7.5 Hz, 2H, O=C-C-CH2), 1.54 (p, J = 7.8 Hz, 2H, O=C-C-C-CH2), 1.22 (t, J =7.6 Hz, 3H, benzene-CH2-CH3), 1.15 (d, J = 6.9 Hz, 6H, (CH3)2C-). 13 C NMR (101MHz, CDCl3) δ 172.54, 170.34, 147.93, 145.85, 137.19, 136.68, 130.25, 128.94,128.74, 127.33, 126.59, 122.95, 73.67, 60.94, 34.01, 31.36, 28.81, 27.18,25.82, 24.34, 23.33, 21.07, 15.71. HR-MS (ESI): m / z calcd for C 30 H37 NO3([M-Cl - ] - )460.28462, found 460.28447. Target compound A16 Brown liquid, yield 77.14%; 1 H NMR (400 MHz, CDCl3) δ 8.54 (d, J = 7.1 Hz, 2H,N-(CH)2), 7.44 (dd, J = 10.7, 7.7 Hz, 4H, N-(CH)2-(CH)2, benzyloxy-C-(CH)2-(CH)2), 7.17 (d, J = 7.9 Hz, 1H, (CH3)2CH-C-CH)), 7.00 (d, J = 7.8 Hz, 1H, (CH3)2CH-CC-CH)), 6.93 (d, J = 8.7 Hz, 2H, benzyloxy-C-(CH)2), 6.79 (s, 1H, (CH3)2CH-C-CH-CH)), 5.35 (s, 2H, -CH2-), 4.47 (t, J = 7.5 Hz, 2H, pyridine-O-CH2),3.80 (s, 3H, O-CH3), 2.92 (p, J = 6.9 Hz, 1H, -CH-), 2.65 (t, J = 7.3 Hz, 2H, O=C-CH2), 2.28 (s, 3H, -CH3), 2.07 (t, J = 9.5 Hz, 2H, pyridine-OC-CH2), 1.83 (p, J = 7.6 Hz, 2H, O=CC-CH2), 1.54 (p, J = 7.3, 6.5 Hz, 2H, O=CCC-CH2), 1.15 (d, J = 6.8 Hz, 6H, (CH3)2C-). 13C NMR (101 MHz, CDCl3) δ 172.55, 170.36, 160.53,147.91, 145.73, 137.17, 136.69, 130.74, 127.33, 126.58, 124.97, 122.92,115.28, 114.58, 73.63, 60.94, 55.62, 33.97, 31.32, 27.19, 25.80, 24.30,23.31, 21.05. HR-MS (ESI): m / z calcd for C 29 H 35 NO4([M-Cl] - ) 462.26389, found462.26264. Target compound A17 Brown liquid, yield 46.81%; 1 H NMR (400 MHz, CDCl3) δ 8.59 (d, J = 7.2 Hz, 2H,N-(CH)2), 7.69 (d, J = 8.2 Hz, 2H, CN-C-(CH)2), 7.64 (d, J = 8.2 Hz, 2H, N-(CH)2-(CH)2), 7.55 (d, J = 6.9 Hz, 2H, CN-C-(CH)2-(CH)2), 7.16 (d, J = 7.9 Hz, 1H, (CH3)2CH-C-CH)), 6.99 (d, J = 6.4 Hz, 1H, (CH3)2CH-CC-CH)), 6.78 (s, 1H, (CH3)2CH-C-CH-CH)), 5.52 (s, 2H, -CH2-), 4.50 (t, J = 7.5 Hz, 2H, pyridine-O-CH2), 2.90 (p, J = 6.9 Hz, 1H, -CH-), 2.65 (t, J = 7.3 Hz, 2H, O=C-CH2), 2.26 (s, 3H, -CH3), 2.09 (p, J= 7.7 Hz, 2H, pyridine-OC-CH2), 1.82 (p, J = 7.5 Hz, 2H, O=CC-CH2),1.54 (p, J = 8.2 Hz, 2H, O=CCC-CH2), 1.13 (d, J = 7.0 Hz, 6H, (CH3)2C-). 13 C NMR(101 MHz, CDCl3) δ 172.59, 169.91, 147.88, 146.00, 138.38, 137.15, 136.71,132.99, 128.93, 127.39, 126.63, 122.90, 115.53, 72.37, 61.18, 60.57, 53.66,33.97, 31.27, 27.20, 25.80, 24.28, 23.33, 21.09. HR-MS (ESI): m / z calcd forC 29 H 32 N2O3([M-Br)) - ) 457.24857, found 457.24758. Intermediate 4 White, transparent liquid, yield 74.65%; 1 H NMR (400 MHz, CDCl3) δ 7.38 (t, J = 7.9 Hz,2H, N-(CH)2), 7.30 (d, J = 7.6 Hz, 2H, -OC-(C)2-(CH)2), 7.22 (d, J = 7.1 Hz, 1H,-OC-(C)2-(C)2-CH), 7.05 (d, J = 8.2 Hz, 2H, -OC-(CH)2), 6.37 (d, J = 7.7 Hz, 2H,N-(CH)2-(CH)2), 3.78 (t, J = 7.2 Hz, 2H, pyridine-O-CH2), 2.58 (t, J = 7.2 Hz, 2H,O=C-CH2), 1.79 (dp, J= 15.3, 7.4 Hz, 4H, pyridine-OC-CH2, O=CC-CH2), 1.43 (p, J = 7.8 Hz, 2H, pyridine-OCC-CH2). 13 C NMR (101 MHz, CDCl3) δ 178.89, 171.82,150.55, 139.87, 129.47, 125.90, 121.47, 118.64, 56.64, 33.86, 30.55, 25.58,24.12. HR-MS (ESI): m / z calcd for C 17 H 19 NO3([M+H)) + ) 286.14377, found 286.14252. Target compound B1 Light brown liquid, yield 53.54%; 1 H NMR (400 MHz, CDCl3) δ 8.52 (d, J = 7.4 Hz, 2H,N-(CH)2), 7.49 (d, J = 6.0 Hz, 2H, N-(CH)2-(CH)2), 7.47 – 7.38 (m, 5H, N-CH2-C-(CH)2-(CH)2, O=COC-CH-CH), 7.34 (t, J = 7.9 Hz, 2H, O=COC-CH-CH, N-CH2-C-(CH)2-(CH)2-CH), 7.19 (t, J = 7.4 Hz, 1H, O=COC-(CH)2-(CH)2-CH), 7.07 (d, J =7.7 Hz, 2H, O=COC-(CH)2), 5.40 (s, 2H, -CH2-), 4.44 (t, J = 7.6 Hz, 2H,pyridine-O-CH2), 2.63 (t, J = 7.3 Hz, 2H, O=C-CH2), 2.05 (p, J = 7.7 Hz, 2H,pyridine-OC-CH2), 1.81 (p, J= 7.4 Hz, 2H, O=CC-CH2), 1.59 – 1.46 (m, 2H, O=CCC-CH2). 13 C NMR (101 MHz, CDCl3) δ 172.22, 170.18, 150.67, 145.73, 133.06,129.60, 129.46, 129.16, 128.59, 125.97, 121.91, 115.19, 73.51, 60.86, 34.01,31.15, 25.66, 24.18. HR-MS (ESI): m / z calcd for C 24 H 25 NO3([M-Cl - ] - ) 376.19072, found 376.18984. Target compound B2 Brown liquid, yield 69.84%; 1 H NMR (400 MHz, CDCl3) δ 8.63 (d, J = 7.4 Hz, 2H,N-(CH)2), 7.56 (d, J = 7.4 Hz, 2H, N-(CH)2-(CH)2), 7.52 (d, J = 6.0 Hz, 2H, N-CH2-C-(CH)2), 7.49 – 7.37 (m, 3H, N-CH2-C-(CH)2-(CH)2-CH), 5.50 (s, 2H, -CH2-),4.54 (t, J = 7.5 Hz, 2H, O-CH2-), 4.11 (q, J = 7.1 Hz, 2H, pyridine-O-CH2), 2.35(t, J = 7.2 Hz, 2H, O=C-CH2), 2.08 (p, J = 7.6 Hz, 2H, pyridine-OC-CH2), 1.73(dd, J = 15.4, 7.7 Hz, 2H, O=CC-CH2), 1.48 (p, J = 7.8 Hz, 2H, O=CCC-CH2), 1.25(t, J= 7.1 Hz, 3H, -CH3). 13 C NMR (101 MHz, CDCl3) δ 173.48, 170.39, 145.77,132.93, 129.50, 129.16, 128.55, 115.32, 73.69, 61.03, 60.56, 33.78, 31.10,25.61, 24.03, 14.34. HR-MS (ESI): m / z calcd for C 20 H 25 NO3([M-Cl - ] - ) 328.19072, found 328.18977. Intermediate 5 White, transparent liquid, yield 94.15%; 1 H NMR (400 MHz, CDCl3) δ 7.16 (d, J = 7.5 Hz,2H, N-(CH)2), 6.08 (d, J = 7.6 Hz, 2H, N-(CH)2-(CH)2), 3.86 (q, J = 7.1 Hz, 2H,CH3-CH2), 3.65 (t, J = 7.3 Hz, 2H, pyridine-O-CH2), 2.10 (t, J = 7.1 Hz, 2H, O=C-CH2), 1.83 (p, J = 7.2 Hz, 2H, O=CC-CH2), 0.99 (t, J = 7.2 Hz, 3H, -CH3). 13 C NMR (126 MHz, CDCl3) δ 178.67, 172.08, 140.06, 118.42, 60.69, 55.57, 53.63,30.16, 25.89, 14.06. HR-MS (ESI): m / z calcd for C 11 H 15 NO3([M+H)) + ) 210.11247, found 210.11279. Intermediate 6 White, transparent liquid, yield 93.27%; 1 H NMR (400 MHz, CDCl3) δ 7.19 (d, J = 7.6 Hz,2H, N-(CH)2), 6.20 (d, J = 7.6 Hz, 2H, N-(CH)2-(CH)2), 3.96 (q, J = 7.2 Hz, 2H,CH3-CH2), 3.64 (t, J = 7.2 Hz, 2H, pyridine-O-CH2), 2.13 (t, J = 7.5 Hz, 2H, O=C-CH2), 1.62 (p, J = 6.9 Hz, 2H, pyridine-OC-CH2), 1.45 (p, J = 7.3 Hz, 2H, O=CC-CH2), 1.18 (s, 6H, pyridine-OCC-CH2, O=CC-CH2-CH2), 1.10 (t, J = 7.2 Hz, 3H,-CH3). 13 C NMR (126 MHz, CDCl3) δ 173.80, 172.36, 144.57, 115.64, 60.28, 59.60,34.16, 31.26, 28.76, 25.88, 24.71, 14.28. HR-MS (ESI): m / z calcd for C 15 H 23 NO3([M+H)) + ) 266.17507, found 266.17479. Intermediate 7 White, transparent liquid, yield 92.14%; 1 H NMR (400 MHz, CDCl3) δ 7.06 (d, J = 7.2 Hz,2H, N-(CH)2), 5.94 (d, J = 8.3 Hz, 2H, N-(CH)2-(CH)2), 3.71 (p, J = 7.9, 6.7 Hz,2H, CH3-CH2), 3.45 (q,J = 6.5 Hz, 2H, pyridine-O-CH2), 1.88 (q, J = 7.3 Hz, 2H, O=C-CH2), 1.38 (s, 2H, pyridine-OC-CH2), 1.21 (s, 2H, O=CC-CH2), 0.91 (s, 8H,pyridine-OCC-CH2-CH2-CH2-CH2), 0.85 (d, J = 4.0 Hz, 3H, -CH3). 13 C NMR (126 MHz, CDCl3) δ 178.45, 173.33, 140.01, 118.02, 59.80, 56.55, 53.55, 49.43, 33.88,30.57, 28.54, 25.77, 24.52, 13.98. HR-MS (ESI): m / z calcd for C 16 H 25 NO3([M+H)) + )280.19072, found 280.18978. Intermediate 8 White, transparent liquid, yield 91.83%; 1 H NMR (400 MHz, CDCl3) δ 7.24 (d, J = 7.1 Hz,2H, N-(CH)2), 6.30 (d, J = 7.1 Hz, 2H, N-(CH)2-(CH)2), 4.03 (q, J = 7.1 Hz, 2H,CH3-CH2), 3.70 (t, J = 7.2 Hz, 2H, pyridine-O-CH2), 2.19 (t, J = 7.5 Hz, 2H, O=C-CH2), 1.67 (p, J = 7.1 Hz, 2H, pyridine-OC-CH2), 1.51 (p, J = 7.2 Hz, 2H, O=CC-CH2), 1.20 (s, 8H, pyridine-OCC-CH2-CH2-CH2-CH2-CH2), 1.16 (t, J= 7.2 Hz, 3H,-CH3). 13 C NMR (126 MHz, CDCl3) δ 178.97, 173.95, 139.95, 118.61, 60.25, 57.12,34.32, 30.92, 29.01, 26.18, 24.88, 14.29. HR-MS (ESI): m / z calcd for C 17 H 27 NO3([M+H)) + ) 294.20637, found 294.20613. Intermediate 9 White solid, yield 43.04%; mp 122-123 °C; 1 H NMR (400 MHz, CD3OD) δ 8.55(d, J = 6.6 Hz, 2H, N-(CH)2), 7.24 (d, J = 6.5 Hz, 2H, N-(CH)2-(CH)2), 4.41 (t, J =7.4 Hz, 2H, pyridine-O-CH2), 2.40 (t, J = 6.9 Hz, 2H, O=C-CH2), 2.18 (p, J = 7.5Hz, 2H, O=CC-CH2). 13 C NMR NMR (126 MHz, CD3OD) δ 174.33, 145.44, 115.04, 65.96, 59.65, 30.04, 26.06. HR-MS (ESI): m / z calcd for C9H 11 NO3([M+H)) + )182.08117, found 182.08037. Intermediate 10 White solid, yield 45.31%; mp 129-130 °C; 1 H NMR (400 MHz, CD3OD) δ 8.30(d, J = 6.7 Hz, 2H, N-(CH)2), 6.94 (d,J = 6.8 Hz, 2H, N-(CH)2-(CH)2), 4.21 (t, J =7.4 Hz, 2H, pyridine-O-CH2), 2.25 (t, J = 7.4 Hz, 2H, O=C-CH2), 1.86 (p, J = 7.4Hz, 2H, pyridine-OC-CH2), 1.57 (p, J = 7.4 Hz, 2H, O=CC-CH2), 1.34 (h, J = 7.2,6.0 Hz, 6H, pyridine-OCC-CH2-CH2-CH2). 13 C NMR NMR (126 MHz, CD3OD) δ 176.34,144.22, 115.51, 58.41, 33.50, 30.63, 28.57, 28.41, 25.61, 24.53. HR-MS (ESI): m / z calcd for C 13 H 19 NO3([M+H)) + ) 238.14377, found 238.14265. Intermediate 11 White solid, yield 40.41%; mp 131-132 °C; 1 H NMR (400 MHz, CD3OD) δ 7.82(d, J = 7.1 Hz, 2H, N-(CH)2), 6.44 (d, J = 7.1 Hz, 2H, N-(CH)2-(CH)2), 3.96 (t, J =7.3 Hz, 2H, pyridine-O-CH2), 2.24 (t, J = 7.4 Hz, 2H, O=C-CH2), 1.77 (p, J = 7.3Hz, 2H, pyridine-OC-CH2), 1.56 (p, J = 7.1 Hz, 2H, O=CC-CH2), 1.30 (s, 8H, O=CCC-CH2-CH2-CH2-CH2). 13C NMR NMR (126 MHz, CD3OD) δ 179.21, 176.41, 141.97,117.03, 56.99, 33.67, 30.61, 28.88, 28.74, 28.66, 25.81, 24.71. HR-MS (ESI): m / z calcd for C 14 H 21 NO3([M+H)) + ) 252.15942, found 252.15918. Intermediate 12 White solid, yield 41.87%; mp 134-135 °C; 1 H NMR (400 MHz, CD3OD) δ 8.57(d, J = 7.4 Hz, 2H, N-(CH)2), 7.25 (d, J = 7.4 Hz, 2H, N-(CH)2-(CH)2), 4.35 (t, J =7.5 Hz, 2H, pyridine-O-CH2), 3.58 (q, J = 7.1 Hz, 2H, O=C-CH2), 2.25 (t, J = 7.4Hz, 2H, pyridine-OC-CH2), 1.90 (p, J = 7.4 Hz, 2H, O=CC-CH2), 1.56 (p, J = 7.1Hz, 2H, pyridine-OCC-CH2), 1.32 (d, J = 18.8 Hz, 8H, O=CCC-CH2-CH2-CH2). 13 CNMR NMR (126 MHz, CD3OD) δ 174.70, 171.15, 145.57, 114.48, 59.39, 57.00,53.54, 47.19, 33.42, 30.77, 28.87, 28.75, 25.76, 24.63, 17.07. HR-MS (ESI): m / z calcd for C 15 H 23 NO3([M+H))+ ) 266.17507, found 266.17456. Intermediate 13 White, transparent liquid, yield 91.17%; 1 H NMR (400 MHz, CDCl3) δ 7.39 (d, J = 7.7 Hz,2H, N-(CH)2), 7.20 (d, J = 7.9 Hz, 1H, (CH3)2CH-C-CH-CH), 7.02 (dd, J = 8.0, 1.8Hz, 1H, (CH3)2CH-C-CH), 6.79 (d, J = 1.9 Hz, 1H, (CH3)2CH-CC-CH), 6.36 (d, J =7.6 Hz, 2H, N-(CH)2-(CH)2), 3.91 (dd, J = 7.8, 6.6 Hz, 2H, pyridine-O-CH2), 2.92(p, J = 6.9 Hz, 1H, -CH-), 2.62 (t, J = 7.1 Hz, 2H, O=C-CH2), 2.29 (s, 3H, -CH3),2.16 (p, J = 7.1 Hz, 2H, O=CC-CH2), 1.18 (d, J = 7.0 Hz, 6H, -C(CH3)2). 13 C NMR(101 MHz, CDCl3) δ 178.65, 171.10, 147.50, 140.02, 136.72, 136.53, 127.26,126.43, 122.45, 118.53, 55.27, 53.60, 30.04, 26.99, 25.82, 22.94, 20.67. HR-MS (ESI): m / z calcd for C 19 H 23 NO3([M+H)) + ) 314.17507, found 314.17419. Intermediate 14 White, transparent liquid, yield 84.22%; 1 H NMR (400 MHz, CDCl3) δ 8.46 (s, 2H, N-(CH)2), 7.42 (s, 2H, N-(CH)2-(CH)2), 7.19 (d, J = 7.9 Hz, 1H, (CH3)2CH-C-CH),7.01 (d, J = 7.8 Hz, 1H, (CH3)2CH-CC-CH), 6.78 (s, 1H, (CH3)2CH-C-CH-CH), 4.39(s, 2H, pyridine-O-CH2), 2.94 (p, J = 6.9 Hz, 1H, -CH-), 2.57 (t, J = 7.4 Hz, 2H,O=C-CH2), 2.29 (s, 3H, -CH3), 1.91 (s, 2H, pyridine-OC-CH2), 1.73 (s, 2H, O=CC-CH2), 1.39 (s, 6H, pyridine-OCC-CH2-CH2-CH2), 1.17 (d, J = 7.1 Hz, 6H, -C(CH3)2). 13 C NMR (101 MHz, CDCl3) δ 172.48, 147.83, 144.42, 136.96, 136.45,127.05, 126.37, 122.67, 115.54, 59.32, 34.07, 31.15, 28.74, 28.60, 27.00,25.78, 24.67, 22.99, 20.77. HR-MS (ESI): m / z calcd for C 23 H 31 NO3([M+H)) + )370.23767, found 370.23742. Intermediate 15 White, transparent liquid, yield 76.53%; 1 H NMR (400 MHz, CDCl3) δ 8.42 (s, 2H, N-(CH)2), 7.45 (s, 2H, N-(CH)2-(CH)2), 7.09 (d, J= 7.8 Hz, 1H, (CH3)2CH-C-CH),6.91 (d, J = 7.8 Hz, 1H, (CH3)2CH-CC-CH), 6.67 (s, 1H, (CH3)2CH-C-CH-CH), 4.32(s, 2H, pyridine-O-CH2), 2.86 – 2.82 (m, 1H, -CH-), 2.46 (s, 2H, O=C-CH2),2.19 (s, 3H, -CH3), 1.81 (s, 2H, pyridine-OC-CH2), 1.63 (s, 2H, O=CC-CH2),1.24 (s, 8H, O=CCC-CH2-CH2-CH2-CH2), 1.06 (d, J = 6.8 Hz, 6H, -C(CH3)2). 13 C NMR(126 MHz, CDCl3) δ 172.66, 171.84, 147.91, 144.78, 137.06, 136.55, 127.13,126.46, 122.76, 115.50, 59.84, 53.72, 34.26, 31.37, 28.98, 28.87, 27.06,26.05, 24.89, 23.09, 20.88. HR-MS (ESI): m / z calcd for C 24 H 33 NO3([M+H)) + )384.25332, found 384.25299. Intermediate 16 White, transparent liquid, yield 54.42%; 1 H NMR (400 MHz, CDCl3) δ 7.29 (d, J = 7.6 Hz,2H, N-(CH)2), 7.19 (d, J = 7.9 Hz, 1H, (CH3)2CH-C-CH), 7.01 (d, J = 7.9 Hz, 1H,(CH3)2CH-C-CH-CH), 6.78 (s, 1H, (CH3)2CH-CC-CH), 6.38 (d, J= 7.6 Hz, 2H, N-(CH)2-(CH)2), 3.75 (t, J = 7.2 Hz, 2H, pyridine-O-CH2), 2.98 – 2.90 (m, 1H, -CH-), 2.57 (t, J = 7.5 Hz, 2H, O=C-CH2), 2.30 (s, 3H, -CH3), 1.76 (p, J = 7.2 Hz, 4H, pyridine-OC-CH2, O=CC-CH2), 1.46 – 1.37 (m, 2H, pyridine-OCC-CH2), 1.32 (s, 8H, O=CCC-CH2-CH2-CH2-CH2), 1.18 (d, J = 7.0 Hz, 6H, -C(CH3)2). 13 CNMR (101 MHz, CDCl3) δ 178.94, 172.53, 147.89, 139.84, 136.98, 136.51,127.06, 126.39, 122.71, 118.59, 57.03, 34.28, 30.85, 29.14, 29.05, 29.00,28.94, 27.05, 26.15, 24.90, 23.03, 20.82. HR-MS (ESI): m / z calcd for C 25 H 35 NO3([M+H)) + ) 398.26897, found 398.26950. Target compound C1 Brown liquid, yield 87.18%; 1 H NMR (400 MHz, CDCl3) δ 8.63 (d, J = 5.9 Hz, 2H,N-(CH)2), 7.49 (t, J = 5.7 Hz, 4H, N-(CH)2-(CH)2, N-CH-C-(CH)2), 7.41 (q, J = 7.4Hz, 3H, N-CH-C-(CH)2-(CH)2-CH), 7.17 (d, J = 7.9 Hz, 1H, (CH3)2CH-C-CH), 7.01(d,J = 7.9 Hz, 1H, (CH3)2CH-C-CH-CH), 6.84 (s, 1H, (CH3)2CH-CC-CH), 5.43 (s,2H, -CH2-), 4.62 (t, J = 7.8 Hz, 2H, pyridine-O-CH2), 2.92 (p, J = 6.9 Hz, 1H, -CH-), 2.85 (t, J = 6.9 Hz, 2H, O=C-CH2), 2.44 (t, J = 7.6 Hz, 2H, O=CC-CH2), 2.28(s, 3H, -CH3), 1.16 (d, J = 6.9 Hz, 6H, (CH3)2CH-). 13 C NMR (126 MHz, CDCl3) δ171.53, 170.50, 147.67, 145.98, 137.11, 136.85, 132.99, 129.57, 129.24,128.69, 127.63, 126.70, 122.82, 115.46, 73.73, 59.91, 30.62, 27.25, 26.85,23.39, 21.03. HR-MS (ESI): m / z calcd for C 26 H 29 NO3([M-Cl - ] - ) 404.22202, found404.22135. Target compound C2 Brown liquid, yield 84.37%; 1 H NMR (400 MHz, CDCl3) δ 8.55 (d, J = 6.9 Hz, 2H,N-(CH)2), 7.49 (d, J = 7.2 Hz, 4H, N-(CH)2-(CH)2, N-CH-C-(CH)2), 7.40 (p, J = 6.3Hz, 3H, N-CH-C-(CH)2-(CH)2-CH), 7.16 (d, J= 7.9 Hz, 1H, (CH3)2CH-C-CH-CH), 6.99(d, J = 7.9 Hz, 1H, (CH3)2CH-C-CH), 6.77 (s, 1H, (CH3)2CH-CC-CH), 5.45 (s, 2H,-CH2-), 4.46 (t, J = 7.6 Hz, 2H, pyridine-O-CH2), 2.92 (p, J = 6.9 Hz, 1H, -CH-), 2.57 (t, J = 7.5 Hz, 2H, O=C-CH2), 2.27 (s, 3H, -CH3), 2.01 (d, J = 8.7 Hz, 2H,pyridine-OC-CH2), 1.74 (p, J = 7.2 Hz, 2H, O=CC-CH2), 1.44 (s, 6H, O=CCC-CH2-CH2-CH2), 1.15 (d, J = 6.9 Hz, 6H, (CH3)2CH-). 13 C NMR (126 MHz, CDCl3) δ172.76, 171.34, 170.32, 147.99, 145.78, 137.16, 136.63, 133.04, 129.56,129.23, 128.66, 127.21, HR-MS (ESI): m / z calcd for C 30 H 37 NO3([M-Cl - ] - ) 460.28462, found 460.28483. Target compound C3 Brown liquid, yield 86.12%; 1 H NMR (400 MHz, CDCl3) δ 8.57 (d, J= 7.4 Hz, 2H,N-(CH)2), 7.51 (t, J = 6.6 Hz, 4H, N-(CH)2-(CH)2, N-CH-C-(CH)2), 7.49 – 7.38 (m,3H, N-CH-C-(CH)2-(CH)2-CH), 7.18 (d, J = 7.9 Hz, 1H, (CH3)2CH-C-CH-CH), 7.00 (d, J = 7.9 Hz, 1H, (CH3)2CH-C-CH), 6.79 (s, 1H, (CH3)2CH-C-C-CH), 5.48 (s, 2H, -CH2-), 4.50 (t, J = 7.5 Hz, 2H, pyridine-O-CH2), 2.95 (p, J = 6.9 Hz, 1H, -CH-),2.58 (t, J = 7.4 Hz, 2H, O=C-CH2), 2.30 (s, 3H, -CH3), 2.02 (d, J = 7.8 Hz, 2H,pyridine-O-C-CH2), 1.75 (p, J = 7.2 Hz, 2H, O=C-C-CH2), 1.41 (s, 8H, O=C-C-C-CH2-CH2-CH2-CH2), 1.17 (d, J = 6.9 Hz, 6H, (CH3)2CH-). 13 C NMR (101 MHz, CDCl3) δ172.77, 170.26, 147.98, 145.69, 137.11, 136.54, 133.00, 129.47, 129.16,128.58, 127.10, 126.45, 122.83, 115.27, 73.63, 61.36, 34.38, 31.39, 29.06,28.91, 27.10, 26.21, 24.96, 23.14, 20.93. HR-MS (ESI): m / z calcd for C 31 H 39 NO3([M-Cl - ] - ) 474.30027, found 474.29978. Target compound C4 Brown liquid, yield 79.81%; 1 H NMR (400 MHz, CDCl3) δ 8.55 (d, J = 7.4 Hz, 2H,N-(CH)2), 7.51 (d, J = 7.6 Hz, 4H, N-(CH)2-(CH)2, N-CH-C-(CH)2), 7.48 – 7.36 (m,3H, N-CH-C-(CH)2-(CH)2-CH), 7.18 (d, J = 7.9 Hz, 1H, (CH3)2CH-C-CH-CH), 7.00 (d, J = 8.0 Hz, 1H, (CH3)2CH-C-CH), 6.79 (s, 1H, (CH3)2CH-CC-CH), 5.47 (s, 2H, -CH2-), 4.47 (t, J = 7.5 Hz, 2H, pyridine-O-CH2), 2.95 (p, J = 6.9 Hz, 1H, -CH-), 2.57 (t, J = 7.5 Hz, 2H, O=C-CH2), 2.29 (s, 3H, -CH3), 2.01 (q, J = 6.8, 5.6 Hz,2H, pyridine-OC-CH2), 1.75 (p, J = 7.4 Hz, 2H, O=CC-CH2), 1.38 (d, J = 20.1 Hz,10H, O=CCC-CH2-CH2-CH2-CH2-CH2), 1.17 (d, J = 6.9 Hz, 6H, (CH3)2CH-). 13C NMR(101 MHz, CDCl3) δ 172.70, 170.31, 147.95, 145.67, 137.09, 136.55, 132.95,129.49, 129.16, 128.54, 127.10, 126.43, 122.80, 115.26, 76.75, 73.65, 61.39,53.49, 34.29, 31.33, 28.91, 28.74, 27.09, 26.12, 24.86, 23.12, 20.89. HR-MS(ESI): m / z calcd for C 32 H 41 NO3([M-Cl - ] - ) 488.31592, found 488.31542. Example 2 In vitro antibacterial activity assay: turbidimetric method and plate method Culture medium preparation: NA medium contains 20 g glucose, 6 g beef extract, 10 g peptone, 2 g yeast extract, and 30 g agar, dissolved in 2 L deionized water, and adjusted to pH 7.0 with 5 mol / L NaOH; NB medium has the same composition as NA medium but does not contain agar. Sterilize at 121 °C for 20 min. EC 50 Assay (turbidimetric method): Two concentration gradients were set up (8, 4, 2, 1, 0.5 μg / mL and 100, 50, 25, 12.5, 6.25 μg / mL). 1 mL of each concentration solution was mixed with 4 mL of sterile NB, and 40 μL of Xoo bacterial suspension (OD) was added. 595 ≈ 0.6 (logarithmic phase), incubated at 28 °C with shaking at 180 rpm for 24–48 h. Absorbance at 595 nm (OD) was measured using Cytation™ 5. 595 Using DMSO-containing culture medium as a negative control and a commercially available fungicide as a positive control, a linear regression (y = ax + b) was performed on the log concentration using probit analysis to derive the virulence equation and EC50. 50 Plate method: Mix 5 mL of each drug solution with 45 mL of NA medium melted and cooled to 50–55 °C, and pour into 3 sterile Petri dishes. After solidification, take 20 μL of the diluted bacterial solution (OD of the original bacterial solution). 595 = 0.6, diluted sequentially to 10⁻ 5Spread evenly, incubate at 28 °C for 4 days, and observe the colony inhibition with the naked eye.

[0023] Table 1. Effects of the target compound on the body at a concentration of 40 μg / mL Xoo Its antibacterial activity. a

[0024] a: Average of three repeated trials; BT: Dimethicone; TC: Copper thiadiazole.

[0025] b: Antimicrobial agents used to compare antimicrobial activity.

[0026] Table 2. Target compound on Xoo EC 50 value. a

[0027] a: Average of three repeated trials; BT: Dimethicone; TC: Copper thiadiazole.

[0028] b: Antimicrobial agents used to compare antimicrobial activity.

[0029] Table 3. Effects of the target compound on the body at a concentration of 40 μg / mL Xac Its antibacterial activity. a

[0030] a: Average of three repeated trials; BT: Dimethicone; TC: Copper thiadiazole.

[0031] b: Antimicrobial agents used to compare antimicrobial activity.

[0032] Table 4. Effects of the target compound on the body at a concentration of 40 μg / mL Psa Its antibacterial activity. a

[0033] a: Average of three repeated trials; BT: Dimethicone; TC: Copper thiadiazole.

[0034] b: Antimicrobial agents used to compare antimicrobial activity.

[0035] Biological tests showed that the synthesized compound could completely inhibit the pathogen at a concentration of 40 μg / mL. Xoo The normal growth of these compounds was observed, with specific data shown in Table 1. Further screening results revealed that these compounds had EC50... 50The values ​​ranged from 0.78 μg / mL to 13.80 μg / mL (see Tables 1 and 2), showing significant superiority over the commercial fungicide bismuth subtilis (BT, EC). 50 = 36.05 μg / mL) and thiamethoxam (TC, EC) 50 = 88.79 μg / mL). The structure-activity relationship (SAR) analysis yielded the following conclusions: 1) The benzyl substitution on the pyridinium nitrogen significantly enhanced the resistance... Xoo Activity, compared to thiapyr, furopyr or naphthylpyryl (A4(EC) 50 = 0.81 μg / mL) > A1 (EC 50 =2.15 μg / mL) > A2 (EC 50 = 13.80 μg / mL) > A3 (EC 50 = 2.82 μg / mL); 2) Substituents on the benzyl ring reduce antibacterial activity, A4 (EC 50 = 0.81 μg / mL) > A6 (EC 50 = 0.86 μg / mL) > A14 (EC 50 = 1.79 μg / mL); 3) The introduction of thymol is more beneficial to enhancing activity than ethyl or phenyl, A4 (EC 50 = 0.81 μg / mL) > B2 (EC) 50 = 2.53μg / mL)>B1 (EC) 50 = 57.08 μg / mL); 4) Regarding alkyl chain length, 8-carbon chain derivatives showed the best antibacterial efficacy, such as C2 (EC). 50 = 0.78 μg / mL) > C1 (EC 50 = 0.86 μg / mL) > C3 (EC 50 = 1.11 μg / mL).

[0036] Biological tests showed that the synthesized compound was effective against pathogens at a concentration of 40 μg / mL. Xac and Psa All showed good inhibitory activity, with some compounds even approaching or achieving complete inhibition. Further screening results showed that their activity was significantly superior to the commercial fungicides bismuth subtilis (BT) and thiamethoxam (TC), as detailed in Tables 3 and 4. The following conclusions were drawn from the structure-activity relationship (SAR): 1) For Xac Compounds with benzyl substitution on pyridinium nitrogen still showed significant advantages, with A4 exhibiting superior antibacterial activity compared to A1 and A2, indicating that benzyl substitution helps enhance antibacterial activity. Xac Function; 2) Substituents on the benzyl ring XacThe activity of these compounds was inhibited; for example, the inhibitory effect of A4 was significantly higher than that of A6 and A14, suggesting that electron-withdrawing groups are more conducive to maintaining activity; 3) Compounds incorporating thymol showed an inhibitory effect. Xac In the experiment, the performance of A4 was better than that of ethyl or phenyl substituents, while B1 showed the weakest activity, indicating that the aromaticity of the substituent has a significant impact on the activity; 4) In Psa Among them, the A-series and C-series compounds showed the most outstanding performance, with A12, A13, and C2 all showing near-complete inhibition. In contrast, the B-series and some numbered compounds (such as 1, 2, 5–12) exhibited lower activity, indicating that longer carbon chains and benzyl substitution enhance the resistance. Psa Significant activity; 5) In comprehensive comparison, C2 (8-carbon chain) and A4 (benzyl substitution) both showed stable and excellent activity in the two pathogen systems, suggesting that these two types of structures are key skeletons for further optimization.

[0037] Table 5. Effects of the target compound on the body at a concentration of 40 μg / mL Sclerotinia sclerotiorum Its antibacterial activity. a

[0038] a: the average of three repeated trials; CBZ: sulfone benzyl.

[0039] b: Antimicrobial agents used to compare antimicrobial activity.

[0040] Table 6. Effects of the target compound on the body at a concentration of 40 μg / mL Gibberella zeae Its antibacterial activity. a

[0041] a: the average of three repeated trials; CBZ: sulfone benzyl.

[0042] b: Antimicrobial agents used to compare antimicrobial activity.

[0043] Biological tests showed that the synthesized compounds exhibited good inhibitory activity against the fungi Sclerotinias clerotiorum and Gibberella zeae at a concentration of 40 μg / mL (see Tables 5 and 6). The A and C series were more prominent overall, and some samples were close to the level of the control agent CBZ. Based on the results, the structure-activity relationship (SAR) can be obtained as follows: 1) The introduction of a benzyl group onto pyridine significantly enhances antifungal efficacy. A1 and A2 both showed high inhibition rates in both fungal systems, significantly better than other non-benzyl substitutions; 2) Further introduction of substituents into the benzyl aromatic ring weakens the activity. A4, A6, and A14 all showed varying degrees of decrease compared to A1, indicating that keeping the benzyl group simple is beneficial to activity; 3) The introduction of thymol / small alkyl groups (B series) has limited contribution to fungi. B1 is the weakest, and B2 has only moderate inhibition against S. sclerotiorum and further decreased inhibition against G. zeae, suggesting that larger-volume substitutions containing phenolic hydroxyl groups are not conducive to antifungal activity; 4) The C series shows a significant alkyl chain length effect: against S. sclerotiorum, the inhibition rate of C1–C3 is generally above 72%, comparable to CBZ; against G. zeae, the inhibition rate of C1–C3… The activity is approximately 56%–60%, and further variations (such as C4) lead to a decrease in activity, indicating that a medium chain length is optimal. In summary, the N-benzylpyridinium skeleton (A1 / A2) and medium-chain quaternary ammonium salts (C1–C3) are the preferred lead structures for further optimization. Fine-tuning around the benzyl electronic effect and alkyl chain length can be used to obtain stronger and more stable antifungal activity.

[0044] Table 7. Antibacterial activity of the target compound against four fungi at a concentration of 40 μg / mL. a

[0045] a: Bipolaris drechsleri ( B. d. ); Fusarium oxysporum ( F. o .); Rhizoctonia solani ( R. s. ); Verticillium dahlia ( V. d. ). As can be seen from Table 7, we selected four typical plant pathogenic fungi ( Bipolaris drechsleri , Fusarium oxysporum , Rhizoctonia solani , Verticillium dahliae Using these compounds as test subjects, the antifungal activity of C1–C4 compounds was determined using the growth rate method. The results showed that this series of compounds generally exhibited good antifungal effects. Among them, C2 showed high inhibition rates against all four fungi.F. oxysporum Up to 64.51%, V. dahliae The inhibitory effect reached 60.22%, demonstrating a broad-spectrum and prominent inhibitory effect. C3 was second, maintaining an inhibitory level of over 50% on multiple pathogens. In contrast, C1... F. oxysporum It has some activity (55.51%), but... R. solani The activity was relatively weak, at only 37.74%. Overall, C4 activity was low, especially in... F. oxysporum The effective rate was only 39.52%. This indicates that C2 performed best among the four tested fungi, demonstrating that this structure possesses strong inhibitory capabilities against a variety of plant pathogenic fungi. Therefore, this type of compound holds promise as a candidate lead structure for the further development of broad-spectrum agricultural fungicides, particularly showing application potential in the control of important soil-borne diseases such as Fusarium and Verticillium.

[0046] Table 8. Inhibition rate of compounds against TMV

[0047] Table 8 shows that the compounds exhibited certain inhibitory activity against tobacco mosaic virus (TMV) at different concentrations. At 500 μg / mL, C1 showed the highest therapeutic activity (66.81%), generally superior to C2–C4; however, at 100 μg / mL, the inhibition rates of all compounds generally decreased, with C1 still maintaining a relative advantage (38.92%). Regarding protective activity, C1 and C2 were more effective than C3 and C4, with C1 still achieving 48.52% at high concentrations. Compared to the control drug ningnanmycin, although there was a certain absolute difference (ningnanmycin exceeded 80% under both conditions), some compounds, such as C1, approached the effective level of the reference drug in terms of therapeutic activity.

[0048] Example 3 1. Co-assembly and characterization of liposomes The liposome formulation was based on conventional lipids: hydrogenated soybean phosphatidylcholine (HSPC), cholesterol succinate monoester (CHEMS), and reported pH-responsive components: oleic acid (OA), lauric acid (LA), stearic acid (SA), azelaic acid (AA), decanoic acid (DA), and phosphatidylethanolamine (PE), with a fixed mass ratio of HSPC:CHEMS:responsive component:C2 = 6:2:2:1. 6 mg of HSPC, 2 mg of CHEMS, 2 mg of pH-responsive lipid, and 1 mg of the target compound C2 were dissolved in 10 mL of ethanol and rotary evaporated at 40 °C to form a thin film. 10 mL of ultrapure water was added, and the mixture was hydrated at 55 °C for 45 min to obtain C2@LNP. The blank LNP did not contain C2. Liposome hydration size (DLS), polydispersity index (PDI), and zeta potential were measured at 25 °C using a Nano Brook Omni analyzer, repeated three times. Screening criteria were PDI ≤ 0.3 and |Zeta potential| ≥ 30 mV. Encapsulation efficiency (EE) and drug loading (DL) were determined by dialysis-HPLC. Liposomes were dialyzed in 1000 Da dialysis bags for 12 h, freeze-dried, and reconstituted in methanol. HPLC quantitative calculation formulas were as follows: Encapsulation efficiency (%) = (actual encapsulated drug amount / drug dosage) × 100%; Drug loading (%) = (actual encapsulated drug amount / total liposome mass) × 100%. UV-Vis absorption spectra of C2-loaded liposomes were collected using a Shimadzu UV-2600i spectrophotometer. XPS involved uniformly dispersing freeze-dried liposome powder onto a silicon wafer, air-drying at room temperature, and then directly testing. The instrument used was a Thermo Scientific ESCALAB Xi+, a monochromatic AlKα X-ray source (1486.6 eV), with a pass energy of 20 eV and a step size of 0.1 eV. Charge correction was based on C 1s 284.8 eV. Full-spectrum data (0–1350 eV) and high-resolution spectra (P 2p, N 1s, O 1s, C 1s, Cl 2p) were acquired. Data were peaked and semi-quantitatively analyzed using Avantage software. For TEM observation of liposome morphology, liposomes were diluted 5-fold with ultrapure water, and 10 μL was dropped onto a 200-mesh carbon-supported copper mesh. The liposomes were air-dried at room temperature for 10 min, negatively stained with 2% phosphotungstic acid for 1 min, and then dried at room temperature. Observation was performed using a Hitachi HT7800 transmission electron microscope (120 kV). EDS analysis was performed in the same region using an Oxford X-MaxN 80T energy dispersive spectroscopy probe with an acquisition time of 60 s and an accelerating voltage of 20 kV to verify the elemental distribution and structural integrity.

[0049] Six pH-responsive liposome formulations were prepared using the above method, based on OA, LA, SA, AA, DA, and PE, and characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), zeta potential, encapsulation efficiency (EE), drug loading (DL), and UV-Vis spectroscopy. Among these, the PE-based liposomes exhibited the best overall performance and were selected for further optimization of the PE, CHEMS, and HSPC ratios. This resulted in an optimized formulation with HSPC:CHEMS:PE:C2 = 6:2.7:2.7:1, a hydration temperature of 55 °C, a hydration time of 1 h, a DLS size of 611.24 nm, a zeta potential of 65.28 mV, an EE of 82.41%, and a DL of 6.34%. Free C2 appeared as a clear yellow solution, while LNP formed a heterogeneous emulsion; their mixture resulted in a heterogeneous yellow dispersion. Notably, during the hydration-driven co-assembly process, the yellow color of C2 disappeared, the resulting solution became homogeneous, and exhibited a pronounced Tyndall effect. After co-assembly, the Cl 2p peak almost disappeared, and the N⁺:NH₂ ratio in LNP increased from 1:1 to 2:1 in C2@LNP. Furthermore, the C=O:CO:COOH ratio changed from 3:2:1 in LNP to 6:3:1 in C2@LNP, providing strong evidence for successful co-assembly. Elemental imaging (EDS) revealed a distinct bilayer structure in the C2@LNP liposomes; although the chloride ion content was low, chloride ions from C2 were still clearly visible within the bilayer. These experiments effectively determined the optimal liposome formulation and confirmed successful encapsulation.

[0050] 2. In vitro antibacterial activity of the compound and its liposomes The following methods were used: turbidimetric assay and plate count assay. The specific methods were the same as in Example 2. The test results are shown in Table 3 below: Table 3. Target compounds and their liposome pairs ​ EC 50 value. a

[0051] a: Average of three repeated trials; BT: Dimethicone; TC: Copper thiadiazole.

[0052] b: Antimicrobial agents used to compare antimicrobial activity.

[0053] In vitro antibacterial activity (turbidimetric method): To verify the simulation results, the activity of C2@LNP and its components against Xoo was determined by turbidimetric method (all C2 groups were converted to C2 mass to ensure comparability of active ingredients). The results (Table 3) show that the EC50 of C2@LNP... 50 It was 3.85 μg / mL, approximately equal to the free C2 (EC) concentration. 50 = 0.78 μg / mL) is 5 times higher. This difference may be related to the inhibitory effect of neutral medium on drug release from C2@LNP. It is worth noting that the EC of HSPC, CHEMS, and PE... 50 All concentrations were >200 μg / mL, offering almost no antibacterial contribution. Thymol EC 50 It exhibits moderate activity at 18.75 μg / mL.

[0054] 3. pH-responsive release experiment The pH release curve was obtained by dispersing prepared liposomes in phosphate-buffered saline (PBS) at different pH values ​​(7.4, 7.0, 6.0, 5.0) and placing them in dialysis bags with a molecular weight cutoff of 1 kDa. The dialysis bags were placed in PBS release media of the corresponding pH and shaken at 37 ℃ and 100 rpm. Fresh media were replaced daily, and the dialysate was collected, concentrated by rotary evaporation, reconstituted with methanol, and analyzed using an Agilent 1260 Infinity II HPLC system (C18 column; mobile phase: acetonitrile / water = 70:30, v / v; flow rate 1.0 mL / min; detection wavelength 254 nm). Monitoring was performed continuously for 7 days, and the cumulative release percentage was calculated using the following formula: Cumulative release rate (%) = (cumulative drug release / initial drug loading) × 100%. The HR experiment used five-leaf stage Nicotiana benthamiana as material, and six treatment zones were symmetrically divided along the midrib on the lower epidermis of the leaves. 50 μL of the mixed treatment solution was slowly injected using a 26 G syringe (injection diameter 5.0 ± 0.2 mm). The treatment solution consisted of Xoo bacterial suspension (OD200). 595 =0.6) was mixed with an equal volume of the test solution to prepare: both C2@LNP group and C2 group contained 5 times EC. 50 C2 (3.9 μg / mL) was used, with a final DMSO concentration of 0.5%. Six groups were established: (1) C2@LNP + Xoo; (2) C2 + Xoo; (3) Xoo + DMSO; (4) Xoo + H2O; (5) Xoo; (6) H2O. After inoculation, the tissues were incubated at 28 ℃ for 24 h. HR was assessed using a dual-mode method: the area of ​​necrotic patches was measured under natural light, and the autofluorescence intensity of the tissues was determined under 365 nm UV excitation.

[0055] We observed that C2@LNP remained relatively stable and milky white under neutral conditions. However, under weakly acidic conditions (pH=5.0 and 6.0), it turned a cloudy yellow after the release of C2. The release kinetics of C2@LNP under different pH conditions were investigated by adjusting the pH of the solution. C2@LNP showed high stability at pH 7 and 7.4, releasing only about 55% of C2 within 7 days. At pH 6 and 5, C2 release accelerated significantly, exceeding 50% on day 1 and over 70% on day 2. Notably, the release at pH 5 was consistently about 10% higher than at pH 6, but from day 2 onwards, the two values ​​converged, reaching approximately 95% within 7 days.

[0056] Growth curves were used at 1×EC 50 and 2×EC 50 At the concentration of 0.5 × EC, the growth rate of Xoo was significantly lower than that at 0.5 × EC. 50 Processing group, 5×EC 50 At the specified concentration, almost no growth was observed, indicating that C2@LNP can effectively release C2 and exert its antibacterial effect under liquid conditions. The agar plate method was performed at 5×EC... 50 Concentration tests showed that the antibacterial rates of LNP, HSPC, CHEMS, and PE were all below 10%, while C2@LNP achieved an antibacterial rate of 85.31%, slightly lower than C2 (87.32%). This indicates that the co-assembled C2 can be effectively released and exert an antibacterial effect on Xoo under solid conditions. Further hypersensitivity experiments showed that both C2@LNP and C2 could almost completely heal lesions under natural and ultraviolet light, further confirming their antibacterial effects under real-world conditions. Transmission electron microscopy (TEM) was used to observe the morphological changes of C2@LNP at different pH values. At pH 7.4 and 7, C2@LNP maintained a complete spherical structure, and the phospholipid bilayer was clearly visible; however, at pH 6, the bilayer began to break down; and at pH 5, the structure completely disintegrated. These experimental results further validated the pH-responsive release characteristics of C2@LNP and provided a preliminary assessment of its antibacterial activity after release.

[0057] 4. Leaf adhesion properties and in vivo anti-Xoo activity C2@LNP was prepared as described above, with free C2 dissolved in ultrapure water (final concentration 100 μg / mL). Mature rice leaves at the 60-day tillering stage were fixed on the sample stage, and the contact angle was measured using a JC-2000D contact angle meter. 5 μL of the solution was added to the midrib of the leaf using a microsyringe, and the static contact angle was calculated by fitting the Young-Laplace equation using the instrument software. Droplet rebound experiment: A 5 μL droplet was freely dropped from 15 cm above the leaf surface, and the impact process was recorded using a high-speed camera (Phantom VEO 410L, 1280×800, 1000 fps). Surface tension was measured using the pendant drop method at 25 ± 1 ℃, with deionized water (72.8 ± 0.5 mN / m) as the reference. All experiments were performed in triplicate.

[0058] Rice cultivation: Fengyouxiangzhan rice seeds were germinated in deionized water at 28 ℃ and 90% RH for 7 days. After transplanting, they continued to grow under the same conditions for 45–60 days, reaching a plant height of approximately 35 cm. Assay methods: The modified Schaad method was used to evaluate the protective and curative activities of C2, with commercial fungicides BT (20% wettable powder) and TC (20% suspension) as positive controls. Experimental design: Potted rice plants were divided into two groups. Treatment group: Leaves were dipped in Xoo bacterial suspension (OD = 0.6) for 15 seconds, followed by foliar spraying with C2 (100 μg / mL), BT, or TC. Protection group: Sprayed with C2, BT, or TC first, then inoculated using the same method. After treatment, the plants were cultured at 28 ℃ and 90% RH, and disease severity was assessed after 14 days. Disease severity grading: Classified into 5 grades based on the ratio of lesion area to leaf area: Grade 1: <5%; Grade 3: 6–10%; Grade 5: 11–20%; Grade 7: 21–50%; Grade 9: >50%. Disease index (DI) = [Σ(number of diseased leaves at each grade × grade value) / (total number of leaves × highest grade value)] × 100. Control efficacy (%) = [(control DI – treatment DI) / control DI] × 100.

[0059] This study elucidated the reasons for the enhanced antibacterial efficacy of C2@LNP under simulated field conditions through systematic analysis of interfacial behavior (contact angle, droplet rebound dynamics, and surface tension). The contact angle of C2 on rice leaves was 123.4°, only slightly lower than that of deionized water (124.4°); while the wettability of C2@LNP was significantly improved, with the contact angle decreasing to 67.7°. High-speed camera analysis revealed different droplet rebound behaviors. Significant splashing (rebound height > 5 mm) occurred after the impact of water and C2 droplets, while LNP and C2@LNP significantly suppressed rebound. Specifically, the maximum rebound height of C2@LNP was approximately half that of water 45 ms after impact, and it remained completely stationary on the leaf surface within 60 ms. Surface tension measurements quantified the interface optimization effect: the surface tension of C2@LNP was 51.35 mN / m, lower than that of H2O (73.34 mN / m), C2 (70.74 mN / m), and LNP (54.47 mN / m), representing a 42.8% reduction compared to H2O and a 6.1% reduction compared to LNP. Mechanistically, we hypothesize that the pyridine cation of C2 is amphiphilic, preferentially locating at the air-water interface and influencing the hydrogen bond network between water molecules through charge-dipole interactions, thereby reducing the interfacial energy. Simulated field spraying experiments confirmed that, thanks to its optimized wettability, C2@LNP formed uniformly distributed small droplets; while water and C2 droplets coalesced into larger droplets. C2@LNP reduced droplet size and achieved uniform coverage through stronger leaf adhesion, ultimately reducing the loss of active ingredients with runoff and minimizing pesticide waste.

[0060] C2 exhibits excellent in vitro antibacterial activity (ECG). 50= 0.78 μg / mL), therefore, the in vivo experimental concentration was significantly reduced to 100 μg / mL in this study. This optimized dosage combines the superior leaf retention capacity of C2@LNP with its pH-triggered release mechanism, forming an eco-friendly disease control strategy that can minimize ecological risks. C2@LNP showed significant performance improvements. In terms of protective activity, the efficacy of C2@LNP was 65.42%, which was 1.94 times, 2.49 times, and 3.89 times that of C2 (33.65%), conventional agents BT (26.17%), and TC (16.82%), respectively; in terms of curative activity, the efficacy of C2@LNP was 67.82%, which was 1.56 times, 2.05 times, and 3.84 times that of C2 (43.48%), BT (33.04%), and TC (17.39%), respectively. The experiment simulated standard agricultural operations using field conditions such as spray irrigation and natural ventilation. Although the in vitro activity of C2@LNP was slightly lower than that of C2 in both turbidimetric and agar plate methods, under simulated field conditions, the antibacterial activity of C2@LNP was significantly superior to that of C2.

[0061] 5. Toxicity test Pesticide development is time-consuming and labor-intensive, making early screening of low-toxicity candidates crucial. The ADMETlab 2.0 platform was used to evaluate the pharmacokinetic properties and toxicity risks of C2, focusing on phytotoxicity, absorption potential, human health hazards (hepatotoxicity, mutagenicity), and environmental impacts (bioaccumulation, soil persistence). This platform can predict target organ exposure levels and provide dosage recommendations for toxicity studies, enabling the rational selection of agricultural candidates. Rice toxicity tests were conducted using rice plants of uniform growth, divided into 9 groups: a control (0.1% Tween 80) and treatment groups (HSPC, CHEMS, PE, LNP, C2, C2@LNP, BT, TC, all at a concentration of 100 μg / mL). Foliar spraying began at the early tillering stage, with 10 mL applied every 3 days for a total of 3 applications. The plants were cultured at 28 ℃ and 90% RH, and leaf yellowing was observed 14 days after the last spray. Growth promotion experiment: During the early tillering stage, plants were randomly divided into 5 groups: control (0.1% Tween 80) and treatments (HSPC, CHEMS, PE, LNP, 100 μg / mL). The spraying method was the same as above. After 14 days, plant height, leaf length, fresh weight, and dry weight after drying at 80 ℃ to constant weight were measured. Data are expressed as relative values ​​to the control. Zebrafish toxicity experiment: 4 groups were set (control, C2, LNP, C2@LNP), with 20 fish in each group. Concentration settings: C2 and C2@LNP were 0.1, 1, and 10 mg / L based on C2 content; LNP was converted to 1.5, 15, and 150 mg / L based on a 7.3% drug loading. The experimental water was dechlorinated tap water, with a temperature of 21–25℃, dissolved oxygen ≥60% saturation, and pH 6.0–8.5. Observe for death and poisoning symptoms throughout the process, and record data at 24 h, 48 h, 72 h, and 96 h.

[0062] ADMET toxicity prediction of compound C2 showed significant safety advantages. Cardiotoxicity (hERG blocking risk: 0.727), hepatotoxicity (H-HT: 0.032; DILI: 0.184), and genotoxicity (Ames mutagenicity: 0.016) were all at low risk levels; acute toxicity (oral in rats: 0.061), carcinogenicity (0.176), eye irritation (0.029), and respiratory toxicity (0.198) also showed good performance. In terms of environmental safety, C2 had a low bioaccumulation factor (BCF 2.81) and moderate aquatic toxicity. To evaluate the effects of C2 quaternary ammonium salt on rice growth, C2 and C2@LNP, along with their individual components, were sprayed three times consecutively at a concentration of 100 μg / mL. The results showed that C2 had no significant effect on plant height, but leaf tip chlorosis of about 5 cm appeared (compared to 10 cm for BT and TC). After C2 was assembled into C2@LNP, the chlorosis almost disappeared, indicating that LNP effectively shielded the phytotoxicity of C2. Further evaluation of the safety of C2 for aquatic organisms was conducted using zebrafish as a model. Based on C2 content, C2 and C2@LNP were set at 10 mg / L, and LNP was converted to 150 mg / L based on a 7.3% loading, with an exposure time of 96 h. Only one zebrafish (n=20) died after 96 h in the C2 group. 50 >10 mg / L (96 h) confirmed that C2 has low toxicity to zebrafish; the toxicity further decreased after assembly, indicating that LNP shielded the intrinsic toxicity of C2 through an encapsulation mechanism. Finally, the effect of liposomes on rice growth was verified: HSPC, CHEMS, PE, and LNP were sprayed three times consecutively (10 mL each time), with a 3-day interval, and observed for 14 days. Data analysis showed that the HSPC treatment had the most significant growth-promoting effect: plant height was 59 cm (22.9% increase compared to CK 48 cm), leaf length was 35 cm (45.8% increase), fresh weight was 290 g, and dry weight was 78 g (both increased by 47.2%), far exceeding the CK and other treatment groups. The CHEMS and PE groups had plant heights of 51 cm and 49 cm, leaf lengths of 27 cm and 25 cm, respectively, with fresh and dry weight increases of less than 15%. The LNP group, composed of three components, was the second best: plant height 53 cm, leaf length 29 cm, fresh weight 221 g, and dry weight 59 g, which were 10.4%, 20.8%, 12.2%, and 11.3% higher than the CK, respectively. Although slightly lower than HSPC alone, it was still better than other single components and the CK. The results indicate that HSPC may indirectly promote rice growth and development by improving nutrient absorption efficiency and cell membrane fluidity.

[0063] 6. Antibacterial mechanism We evaluated a total of three bacteria ( ​ , ​ , ​ ) and two fungi ( ​ , ​ The antibacterial activity of compound C2 was evaluated; since compound C2 showed the best performance in all systems, it was selected. ​ To investigate its antibacterial mechanism as a unified target, we employed experiments related to quorum sensing and membrane permeability for verification. Crystal violet assay showed that C2 significantly inhibited... ​ Biofilm formation, OD 570 The value decreased from 1.5 to 0.2; the motility test showed that the diameter of the swimming ring was within 1×EC. 50 and 2×EC 50 The diameters of the treated cells decreased from 23.9 mm in the control to 15.2 mm and 8.9 mm, respectively, indicating impaired colonization and dispersal capabilities. C2 also significantly inhibited the activity of extracellular enzymes regulated by quorum sensing: the diameters of the amylase and cellulase hydrolytic zones decreased from 7.9 mm and 15.9 mm to 3.8 mm and 10.2 mm, respectively (1×EC). 50 The thickness was further reduced to 2.5 mm and 8.1 mm (2×EC). 50 Electrolyte leakage measurements showed that after 8 hours of treatment, at 5×EC 50 Under these conditions, the extracellular conductivity increased to 165.3 μS / cm, close to the 192.7 μS / cm of the boiled positive control, indicating significant membrane disruption. ONPG and NPN assays further confirmed that the inner and outer membrane permeability were approximately 2-fold and 14.7-fold higher than the control, respectively. AO / EB staining showed that the control group consisted entirely of viable cells; under 1×EC... 50 Surviving and apoptotic cells coexisted at 5×EC 50 Only dead cells were observed. Annexin V-FITC / PI flow cytometry confirmed a time-dependent increase in apoptosis, with the proportion of apoptotic cells increasing from 59.8% at 1 h to 80.0% at 8 h. Scanning electron microscopy revealed 5× EC50 cells. 50 The lower cell membrane showed significant contraction. From this, we can infer that compounds like C2 mainly achieve broad-spectrum antibacterial activity by disrupting the orderly arrangement of lipids in the cell membrane, significantly increasing the permeability of the inner and outer membranes, and inducing osmotic homeostasis imbalance. At low doses, they first inhibit quorum sensing-related adhesion, motility, and extracellular enzyme secretion, thereby weakening pathogenicity, while at higher doses they further cause irreversible membrane damage and trigger programmed cell death.

Claims

1. A class of quaternary ammonium salt derivatives containing thymol, characterized in that: Its structural formula is shown in (1): （1） Where R1 is 2-isopropyl-5-methylphenyl, ethyl, or a benzene ring, R2 is 2-chloro-5-methylthiophene, 3-methyl-2-methoxycarbonylfuran, 4-methylnaphthalene, and benzyl groups with different substitutions, X - For Cl - and Br - n=3, 5, 7, 8, 9.

2. The quaternary ammonium salt derivative containing thymol according to claim 1, characterized in that: R1 is 2-isopropyl-5-methylphenyl.

3. The quaternary ammonium salt derivative containing thymol according to claim 2, characterized in that: R2 is benzyl, X - For Cl - n=7.

4. The method for preparing a class of thymol-containing quaternary ammonium salt derivatives according to claims 1-3, wherein the synthetic reaction equation is as follows:

5. The drug-loaded antibacterial liposome containing a quaternary ammonium salt derivative of thymol as described in claim 1, characterized in that: Hydrogenated soybean phosphatidylcholine, cholesterol hemisuccinate monoester, phosphatidylethanolamine, and a quaternary ammonium salt derivative of the target compound containing thymol were co-assembled into drug-loaded antibacterial liposomes.

6. The drug-loaded antibacterial liposome containing a quaternary ammonium salt derivative of thymol as described in claim 5, characterized in that: The mass ratio of hydrogenated soybean phosphatidylcholine, cholesterol hemisuccinate monoester, phosphatidylethanolamine, and the target compound containing a thymol quaternary ammonium salt derivative was 6:2.7:2.7:

1. The specific preparation process was as follows: the above four components were mixed and dissolved in 10 ml of ethanol, then rotary evaporated to dryness to form an oil film, and then 10 ml of deionized water was added. The mixture was then hydrated at 55 °C for 60 minutes, and liposomes were obtained.

7. The application of a class of thymol-containing quaternary ammonium salt derivatives or their drug-loaded antibacterial liposomes as described in claim 1 or 5, characterized in that: Application in antibacterial agents.

8. The application of a class of thymol-containing quaternary ammonium salt derivatives or their drug-loaded antibacterial liposomes as described in claim 7, characterized in that: The antibacterial agents are effective against rice bacterial blight, citrus canker, kiwifruit canker, bipolar sclerotium, sclerotium, Fusarium oxysporum, Rhizoctonia solani, zearalenone, verticillium, and tobacco mosaic virus.

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