N-(3-fluorobenzyl)-3-methylbutyl-2-acrylamide compound as well as preparation method and application thereof

Abstract

The invention discloses an N-(3-fluorobenzyl)-3-methylbutyl-2-acrylamide compound as well as a preparation method and application thereof, and belongs to the technical field of pesticides. The preparation method comprises the following steps: by taking 3-methylbutyl-2-olefine acid as an initial raw material, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine, dissolving in dichloromethane, and stirring to obtain a mixed solution; and adding an amination reagent into the mixed solution to carry out condensation reaction, washing the reaction solution, drying, filtering and concentrating an organic phase, and purifying through column chromatography to obtain the N-(3-fluorobenzyl)-3-methylbutyl-2-enamide. The synthesis process is simple and efficient, mild in condition, good in yield and beneficial to green production; the compound shows a remarkable inhibition effect on 13 common and severely harmful plant pathogenic fungi, shows broad-spectrum and efficient antibacterial activity, and particularly has a remarkable effect on specific pathogenic bacteria.

Description

Technical Field

[0001] This invention belongs to the field of pesticide technology, specifically relating to an N-(3-fluorobenzyl)-3-methylbut-2-enamide compound, its preparation method, and its application. Background Technology

[0002] Microbial contamination leading to food spoilage, crop diseases, and clinical infections poses a serious threat to human health and the safety of industrial and agricultural production. The widespread use of traditional antibiotics and synthetic bactericides has resulted in increasing and spreading drug resistance in pathogenic microorganisms. This not only significantly weakens the efficacy of existing drugs but also leads to problems such as environmental residues and toxicity accumulation. Against this backdrop, the limitations of traditional antimicrobial agents are becoming increasingly apparent, failing to meet the urgent need for highly efficient, low-toxicity, and environmentally friendly antimicrobial materials. Therefore, the development of novel antimicrobial molecules with novel chemical structures, high antimicrobial activity, and ease of preparation has become a research hotspot in the interdisciplinary fields of chemistry, medicine, and materials science.

[0003] Amide compounds have attracted significant attention in medicinal chemistry and antibacterial agent development due to their stable chemical structures, diverse substituent modification spaces, and broad bioactivity. Acrylamides and their derivatives, containing olefinic bonds, are particularly noteworthy. The amide bond (-CONH-) and unsaturated double bonds in their structure endow the molecules with excellent reactivity and the potential to bind to biological targets. Numerous studies have demonstrated that these compounds possess a variety of pharmacological activities, including bactericidal, herbicidal, anti-inflammatory, and antitumor effects. In drug molecule design, introducing fluorine atoms is a key strategy for optimizing the performance of lead compounds, as it can effectively regulate the compound's lipophilicity, electron cloud density, and metabolic stability, thereby significantly enhancing bioactivity and targeting. Therefore, the rational design and synthesis of fluorinated amide derivatives has become an important direction in the development of novel antibacterial agents.

[0004] However, existing technologies still have shortcomings: First, some synthetic methods rely on expensive or highly toxic condensing agents and organic solvents, which are not conducive to green synthesis and large-scale production; second, most studies lack in-depth exploration of the systematic antibacterial spectrum of single compounds; third, the activity of reported fluorinated amide antibacterial agents (especially against plant pathogens) still has room for improvement, and the poor stability or solubility of some compounds restricts their practical application potential; fourth, the synthesis process often involves the separation and purification of intermediates, resulting in low overall reaction yields and room for improvement in process economy. Therefore, based on existing research, developing a simple, mild, and high-yield synthetic method, and systematically evaluating the inhibitory activity and mechanism of action of the obtained compounds against important plant pathogens, is of great significance for promoting the development of novel and highly effective antibacterial agents. Summary of the Invention

[0005] The first technical problem to be solved by this invention is to provide a method for preparing an N-(3-fluorobenzyl)-3-methylbut-2-enamide compound. This method uses (3-fluorophenyl)methylamine as an amination reagent and employs an EDCI / DMAP dehydration condensation system to efficiently construct amide bonds in dichloromethane solvent. The compound is then obtained after extraction, drying, concentration, and column chromatography purification. The second technical problem to be solved by this invention is to provide an N-(3-fluorobenzyl)-3-methylbut-2-enamide compound with excellent antibacterial activity. The third technical problem to be solved by this invention is to provide the application of this compound in the preparation of inhibitors for plant pathogenic fungi.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for preparing an N-(3-fluorobenzyl)-3-methylbut-2-eneamide compound comprises using 3-methylbut-2-enoic acid as a starting material, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine dissolved in dichloromethane, stirring to obtain a mixture; then adding an amination reagent to the mixture to carry out a condensation reaction, washing the reaction solution, drying, filtering, concentrating the organic phase, and purifying it by column chromatography to obtain N-(3-fluorobenzyl)-3-methylbut-2-eneamide.

[0008] Furthermore, the amination agent is (3-fluorophenyl)methylamine.

[0009] Furthermore, the condensation reaction time is 16 hours and the temperature is room temperature.

[0010] Furthermore, the molar ratio of 3-methylbut-2-enoic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 4~5:5~6:2~3.

[0011] Furthermore, the molar ratio of the 3-methylbut-2-enoic acid to the amination reagent is 4~5:5~6.

[0012] Furthermore, the N-(3-fluorobenzyl)-3-methylbut-2-enamide compound prepared by the method described above is an N-(3-fluorobenzyl)-3-methylbut-2-enamide compound.

[0013] Furthermore, the application of the N-(3-fluorobenzyl)-3-methylbut-2-enamide compound in the preparation of plant pathogenic fungal inhibitors.

[0014] Furthermore, the plant pathogenic fungi are selected from one or more of the following: *Anthracis bananaensis*, *Colletotrichum gloeosporioides*, *Fusarium cladosporium*, *Fusarium oxysporum*, *Fusarium equisetifolium*, *Rhizoctonia solani*, *Fusarium tumefaciens*, *Fusarium solani*, *Colletotrichum spp.*, *Colletotrichum spp.*, *Fusarium graminearum*, *Fusarium solani*, and *Fusarium heterosporum*.

[0015] Furthermore, the concentration of N-(3-fluorobenzyl)-3-methylbut-2-enamide compound used in the inhibitor is 0.01~0.02 g / mL.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] (1) The synthesis process of this invention is simple, efficient, and mild, with good yield, which is conducive to green production. This invention adopts a one-step dehydration condensation method, using 3-methylbut-2-enoic acid and (3-fluorophenyl)methylamine as raw materials, and reacts at room temperature in an EDCI / DMAP catalytic system and dichloromethane solvent to directly obtain the target product. This process route is concise, avoiding multi-step synthesis and separation and purification of complex intermediates, thus simplifying the operation process. The reaction is carried out at room temperature, without the need for heating or high pressure, resulting in low energy consumption and safe and convenient operation. Using this method, a mass yield of 61.8% can be obtained, and the product purity is as high as 99.64%. Compared with some existing technologies that require the use of more expensive or more toxic condensing agents (such as HATU), or require multi-step reactions with low overall yields (often below 50%), this method is significantly more efficient.

[0018] (2) The target compound obtained in this invention has a well-defined structure and a clear and reliable synthetic route. This invention has fully confirmed the structure of the target compound N-(3-fluorobenzyl)-3-methylbut-2-enamide using techniques such as ¹H NMR and ¹³C NMR, resulting in clear data and a well-defined structure. This provides a reliable material basis and quality assurance for subsequent activity studies, structure-activity relationship analysis, and potential application development.

[0019] (3) The target compounds of this invention exhibit broad-spectrum and highly efficient antibacterial activity, especially against specific pathogens. Systematic in vitro antibacterial experiments (mycelial growth rate method) demonstrated that the compounds of this invention showed significant inhibitory effects against 13 common and seriously harmful plant pathogenic fungi. This activity exhibits a clear concentration dependence; at a concentration of 20 mg / mL, the inhibitory effect on most tested strains was significantly enhanced. In particular, the compounds showed good inhibitory ability against *M. fructigena* and *F. proliferatum*, with inhibition rates as high as 79.00% and 54.80%, respectively, at a concentration of 20 mg / mL, showing great potential as novel lead compounds for the control of such diseases. Furthermore, they also showed moderate to strong inhibitory effects (inhibition rates between 30% and 48%) against several other important pathogens, such as *T. cucumeris* and *C. gloeosporioides*. This broad-spectrum and selective antibacterial property solves the problem that some existing fluorinated amide antibacterial agents have a narrow activity spectrum or unsatisfactory inhibitory effect on key plant pathogens. Attached Figure Description

[0020] Figure 1 The N-(3-fluorobenzyl)-3-methylbut-2-enamide compound prepared in Example 1 of this application 1 H NMR spectrum;

[0021] Figure 2 The N-(3-fluorobenzyl)-3-methylbut-2-enamide compound prepared in Example 1 of this application 13 C NMR spectrum;

[0022] Figure 3 The diagram shows the inhibitory effect of the N-(3-fluorobenzyl)-3-methylbut-2-enamide compound of this application on the mycelial growth of 13 plant pathogenic fungi.

[0023] In the figure, a is *F. banana*; b is *Fusarium* cladocerans; c is *Fusarium equisetifolium*; d is *Fusarium fusiforme*; e is *Colletotrichum spp.*; f is *Fusarium graminearum*; g is *Fusarium heterospora*; h is *Colletotrichum gloeosporioides*; i is *Fusarium oxysporum*; j is *Fusarium solani*; k is *Fusarium laminarum*; l is *Cladosporium sclerotiorum*; m is *Rhizoctonia solani*.

[0024] Figure (am) 1 shows a substance with a concentration of 10 mg / mL, and Figure (am) 2 shows a substance with a concentration of 20 mg / mL;

[0025] Figure a0 shows the control of *Anthracis bananaensis*; b0 shows the control of *Fusarium cladosporum*; c0 shows the control of *Fusarium equisetifolium*; d0 shows the control of *Fusarium tumefaciens*; e0 shows the control of *Colletotrichum spp.*; f0 shows the control of *Fusarium graminearum*; g0 shows the control of *Fusarium heterosporum*; h0 shows the control of *Colletotrichum gloeosporioides*; i0 shows the control of *Fusarium oxysporum*; j0 shows the control of *Fusarium solani*; k0 shows the control of *Fusarium lataniae*; l0 shows the control of *Cladosporium sclerotiorum*; m0 shows the control of *Rhizoctonia solani*. The control plates contained only dimethyl sulfoxide and no other compounds. Detailed Implementation

[0026] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are implemented based on the technical solutions of the present invention, and it should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0027] The following examples used 3-methylbut-2-enoic acid (Shaoyuan Technology Co., Ltd., 98% purity), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (Leyan, 98% purity), 4-dimethylaminopyridine (Adamas-beta, 99% purity), and (3-fluorophenyl)methylamine (Leyan, 97% purity). All of these reagents are commercially available.

[0028] Example 1

[0029] A method for preparing an N-(3-fluorobenzyl)-3-methylbut-2-enamide compound includes the following steps:

[0030] The reaction formula is:

[0031]

[0032] 500 mg of 3-methylbut-2-enoic acid, 1.12 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), and 305 mg of 4-dimethylaminopyridine (DMAP) were dissolved in 15 mL of dichloromethane and stirred at room temperature for 15 min. Then, 750 mg of (3-fluorophenyl)methylamine was added to the reaction system, and the mixture was stirred continuously at room temperature for 16 h. After the reaction was complete, the reaction solution was washed successively with 20 mL of water and 20 mL of saturated brine. The resulting organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. Finally, the crude product was purified by column chromatography (eluent: petroleum ether / ethyl acetate = 4:1, v / v) to obtain 640 mg of the colorless oily target compound, with a yield of 61.8% and a purity of 99.64%. N-(3-fluorobenzyl)-3-methylbut-2-enoamide 1 H NMR image as follows Figure 1 As shown, N-(3-fluorobenzyl)-3-methylbut-2-eneamide 13C NMR spectrum as shown Figure 2 As shown.

[0033] The spectral and mass spectrometry data are as follows:

[0034] 1 H NMR (400 MHz, DMSO-d6) δ 8.30 (t, J=5.6 Hz, 1H), 7.41–7.30 (m, 1H), 7.14– 6.99 (m, 3H), 5.73 (d, J=1.0 Hz, 1H), 4.31 (d, J=6.1 Hz, 2H), 2.10 (s,3H), 1.80 (s,3H). 13 C NMR (126 MHz, DMSO-d6) δ 166.51, 163.64, 149.83,143.41, 130.65, 123.61, 119.13, 114.32, 113.94, 41.70, 27.28, 19.77. MS-ESIm / z: 208.1 [M+H] + .

[0035] Application examples

[0036] 1. The 13 pathogenic fungi used in this experiment, namely *Colletotrichum musae*, *Colletotrichum gloeosporioides*, *Fusarium sporotrichioides*, *Fusarium oxysporum*, *Gibberella intricans*, *Thanatephorus cucumeris*, *Fusarium fujikuroi*, *Fusarium proliferatum*, *Colletotrichum lagenarium*, *Monilinia fructigena*, *Fusarium graminearum*, *Fusarium solani*, and *Fusarium heterosporum*, were all purchased from the Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains.

[0037] 2. Experimental Procedure

[0038] 2.1 Culture medium preparation

[0039] The preparation steps for potato dextrose agar (PDA) medium are as follows: Cut 200g of peeled potatoes into chunks, boil them in water until soft, and then filter the mixture through double-layered gauze. Add 20g of glucose and 16g of agar to the filtrate, and bring the volume to 1L with distilled water. Dispense the mixture into Erlenmeyer flasks. Finally, after autoclaving at 121℃ for 20min, pour the medium into plates in a clean bench.

[0040] 2.2 Strain activation

[0041] Take a small amount of mycelium from the preservation slant with an inoculation loop, inoculate it onto a PDA plate, and incubate at 28℃ for 5-7 days. It can be used after the mycelium has fully grown on the plate.

[0042] 2.3 Determination of antibacterial activity

[0043] The in vitro antibacterial effect of N-(3-fluorobenzyl)-3-methylbut-2-enamide was evaluated using a mycelial growth rate assay. Under aseptic conditions, N-(3-fluorobenzyl)-3-methylbut-2-enamide dissolved in DMSO was added to molten PDA to prepare drug-containing plates with final concentrations of 0.01 g / mL and 0.02 g / mL; the control group received only an equal amount of DMSO. Mycelial blocks of pathogens were obtained using a 6 mm diameter punch and inoculated into the center of the drug-containing plates, with the mycelial side facing down. Each treatment was repeated three times. After incubation at 28°C for 5–7 days, the colony diameter was measured using the cross-cross method. The inhibition rate was calculated using the following formula:

[0044] Inhibition rate / % = [(Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group] × 100%

[0045] 3. Results

[0046] The antibacterial activity of compound N-(3-fluorobenzyl)-3-methylbut-2-enamide was determined using the mycelial growth rate method. The results are shown in Table 1. The results indicated that both 10 mg / mL and 20 mg / mL of compound N-(3-fluorobenzyl)-3-methylbut-2-enamide effectively inhibited the mycelial growth of 13 tested pathogenic fungi. The specific growth patterns of these pathogens on the drug-containing plates are shown in Table 1. Figure 1 .

[0047] Table 1. Inhibition rate of compounds against 13 plant pathogenic fungi at different concentrations

[0048]

[0049] From Table 1 and Figure 3It was found that the compound N-(3-fluorobenzyl)-3-methylbut-2-enamide exhibited significant antifungal activity against 13 plant pathogenic fungi, showing a clear concentration-dependent and strain-specific effect. Overall, the antifungal effect at a concentration of 20 mg / mL was generally better than that at 10 mg / mL. Except for *C. musae*, the inhibition rate of the other 12 strains increased with increasing compound concentration, demonstrating a typical positive correlation between concentration and effect. Meanwhile, the sensitivity of different strains to concentration changes varied significantly. For example, *C. gloeosporioides* showed a 43.38 percentage point increase in inhibition rate when the compound concentration increased from 10 mg / mL to 20 mg / mL, while the increase for *G. intricans* was only 4.94 percentage points.

[0050] Based on the inhibition rate at a concentration of 20 mg / mL, the 13 tested strains can be divided into three categories: highly sensitive strains (inhibition rate ≥ 50%), including *M. fructigena* (79.00%) and *F. proliferatum* (54.80%), among which *M. fructigena* showed the strongest inhibitory effect at a concentration of 10 mg / mL, reaching an inhibition rate of 63.00%; moderately sensitive strains (30% ≤ inhibition rate < 50%), including *Rhizoctonia solani* and *Colletotrichum gloeosporioides*, with inhibition rates concentrated between 30% and 48%, exhibiting stable and moderate inhibitory effects; and low-sensitive strains (inhibition rate < 30%), including *F. fujikuroi* and *C. lagenarium*, which showed weak sensitivity to this compound. This difference suggests that the compound has a preferred inhibitory activity against some strains of the genera *Sclerotinia* and *Fusarium*, and has potential for further development in the prevention and control of plant diseases caused by these strains.

[0051] Some strains exhibited unique response patterns: *C. musae* was the only strain to show a 'concentration-negative correlation,' with an inhibition rate of 38.21% at 10 mg / mL significantly higher than that at 20 mg / mL (9.48%), which is related to the strain's metabolic detoxification ability or the stability changes of high-concentration compounds; *C. gloeosporioides*, on the other hand, showed a 'low concentration, low efficacy; high concentration, high efficacy' jump response, with an inhibition rate of only 2.34% at 10 mg / mL, which jumped to 45.72% at 20 mg / mL, suggesting that this strain has a concentration threshold effect on this compound, that is, a specific concentration must be reached to exert a significant antibacterial effect.

[0052] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A process for the preparation of a compound of N-(3-fluorobenzyl)-3-methylbut-2-enamide, characterized in that, Starting with 3-methylbut-2-enoic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in dichloromethane and stirred to obtain a mixture. Then, an amination reagent was added to the mixture to carry out a condensation reaction. The reaction solution was washed, and the organic phase was dried, filtered, concentrated, and purified by column chromatography to obtain N-(3-fluorobenzyl)-3-methylbut-2-enoamide.

2. Process for the preparation of N-(3-fluorobenzyl)-3-methylbut-2-enamide compounds according to claim 1, characterized in that: The amination reagent is (3-fluorophenyl)methylamine.

3. The method for preparing the N-(3-fluorobenzyl)-3-methylbut-2-enamide compound according to claim 1, characterized in that: The condensation reaction took 16 hours at room temperature.

4. The method for preparing the N-(3-fluorobenzyl)-3-methylbut-2-enamide compound according to claim 1, characterized in that: The molar ratio of 3-methylbut-2-enoic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 4-dimethylaminopyridine is 4~5:5~6:2~3.

5. The process for the preparation of N-(3-fluorobenzyl)-3-methylbut-2-enamide compound as claimed in claim 1, wherein: The molar ratio of 3-methylbut-2-enoic acid to the amination reagent is 4~5:5~6.

6. The N-(3-fluorobenzyl)-3-methylbut-2-enamide compound prepared by the method according to any one of claims 1 to 5.

7. The use of the N-(3-fluorobenzyl)-3-methylbut-2-enamide compound according to claim 6 in the preparation of plant pathogenic fungal inhibitors.

8. Use of the N-(3-fluorobenzyl)-3-methylbut-2-enamide compound according to claim 7 for the preparation of a plant pathogenic fungi inhibitor, characterized by: The plant pathogenic fungi are selected from one or more of the following: *Anthracis bananaensis*, *Colletotrichum gloeosporioides*, *Fusarium cladosporium*, *Fusarium oxysporum*, *Fusarium equisetifolium*, *Rhizoctonia solani*, *Fusarium tumefaciens*, *Fusarium solani*, *Colletotrichum spp.*, *Colletotrichum spp.*, *Fusarium graminearum*, *Fusarium solani*, and *Fusarium heterosporum*.

9. Use of the N-(3-fluorobenzyl)-3-methylbut-2-enamide compound according to claim 7 for the preparation of a plant pathogenic fungi inhibitor, characterized by: The concentration of N-(3-fluorobenzyl)-3-methylbut-2-enamide compounds used in inhibitors is 0.01~0.02 g / mL.

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