A propranolol derivative and its use in combating plant fungal diseases

Abstract

The application discloses a propranolol derivative with the following formula (I), and develops a series of derivatives with phosphatase inhibiting activity by substituting phenolic and amine parts at two ends of the isopropyl alcohol amine chain of propranolol and combining computer-aided drug design, and finally obtains a compound with better anti-plant fungal disease effect through anti-fungal activity screening, wherein the inhibiting rate of the compound to multiple fungi such as alternaria alternata and botrytis cinerea is significantly improved, and the compound is easy to realize chiral resolution, has the advantages of simple synthesis process and good industrial application prospect, etc.

Description

Technical Field

[0001] This invention relates to an isopropanolamine derivative and its application in combating plant fungal diseases, belonging to the field of crop disease control. Background Technology

[0002] Isopropanolamine is a widely found chemical structural unit in pharmaceutical and pesticide molecules that plays an important role in biological activity. Molecules with the isopropanolamine structure exhibit unique biological activities in anticancer, antibacterial, and antifungal applications. In isopropanolamine derivatives, there is a chiral carbon atom at the center of the chain. This results in isopropanolamine derivatives having two different chiral enantiomers, (R) and (S), exhibiting different optical activities and thus different biological activities in the biological environment.

[0003] Since the discovery of the bioactivity of isopropanolamine derivatives, structural modifications and derivatization of related compounds have been continuously carried out. Yang Song et al. from Guizhou University have expanded a series of isopropanolamine derivatives with anti-plant pathogenic bacteria and viruses; Duan Hongxia et al. from China Agricultural University have developed a batch of isopropanolamine derivatives with MoTPS1 inhibitory effects and the ability to inhibit the growth of rice blast fungus.

[0004] The isopropanolamine compound propranolol was first reported to have inhibitory activity against phosphatidylphosphatase, a protein that is widely present in plant pathogenic fungi and plays an important physiological role. This protein mainly acts on the fungal lipid metabolism pathway, catalyzing the conversion of phosphatidic acid into diglycerides and phosphatidylinositol. Its structure is highly conserved and it is one of the important targets of antifungal agents.

[0005]

[0006] The applicant modified the chemical structure of propranolol, especially by substituting the phenolic and amine portions at both ends of the isopropanolamine chain, and combined this with computer-aided drug design to develop a series of isopropanolamine derivatives with phosphatidylphosphatase inhibitory activity. Through antifungal activity screening, the applicant finally obtained novel compounds with better antifungal effects against plant fungal diseases. Summary of the Invention

[0007] The purpose of this invention is to provide a propranolol derivative with better resistance to plant fungal diseases.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] Propranolol derivatives with the following structure (Ⅰ):

[0010]

[0011] Wherein, R is a conventional substituent on the benzene ring, including alkyl, alkoxy, cyano, halogen, and trifluoromethyl.

[0012] Preferably, R is a para-substituted trifluoromethyl group.

[0013] More preferably, the propranolol derivative is a chiral isomer, especially when it is an R-chiral isomer, it has better inhibitory activity against Fusarium graminearum; when it is an S-chiral isomer, it has better inhibitory activity against Sclerotinia sclerotiorum and Sclerotinia sclerotiorum.

[0014] The present invention further provides the application of the aforementioned propranolol derivative in the resistance to plant fungal diseases.

[0015] Furthermore, the plant fungal diseases mentioned include Fusarium graminearum, Botrytis cinerea, Sclerotinia sclerotiorum, Monilinia fructicola, Bipolaris maydis (maize leaf spot fungus), Rhizoctonia solani, Pestalotiopsis theae, and Altermaria alternata.

[0016] The present invention also provides a drug for resisting plant fungal diseases, wherein the active ingredient of the drug is the propranolol derivative described above.

[0017] The beneficial effects of this invention are:

[0018] The compounds provided by this invention inhibit the activity of plant fungal diseases by binding to phosphatidylphosphatase. Compared with previously reported compounds, the derivatives provided by this invention have significantly improved the inhibition rate against a variety of fungi such as Alternaria alternata and Fusarium graminearum. The compounds are easy to chirally resolve and have the advantages of simple synthesis process and good industrial application prospects. Attached Figure Description

[0019] Figure 1 Schematic diagram of the molecular docking results of compounds (R)-C23 and (S)-C23 with phosphatidylphosphatase.

[0020] Figure 2 The control effect of compound C23 on brown rot disease in two types of peaches is shown in the figure. A is a nectarine; B is a golden autumn red honey peach.

[0021] Figure 3 : Effect of compound C23 on gray mold disease in tomatoes.

[0022] Figure 4The inhibition rate of compound C23 on brown rot of peach, brown rot of Jinqiuhongmi peach, and gray mold of tomato. Detailed Implementation

[0023] To better understand this invention, the technical solution of this invention will be fully and clearly described and explained below through specific embodiments. It should be noted that, unless otherwise specified, all reagents used in the experiments were obtained from commercial sources, and compounds without chiral isomers were racemic mixtures.

[0024] In our previous study, we substituted the amine portion of propranolol with p-fluorobenzylamine and the phenol portion with a monobenzyl ring. The results showed that most phenolic compounds with a monobenzyl ring could not improve the bioactivity of isopropanolamine derivatives. Only one compound with 2,4-diCl substitution showed stronger bioactivity than propranolol. This compound has been granted a Chinese invention patent, application number 202211153968.9.

[0025] Next, we added more steric hindrance and conjugated structures to the phenolic moiety and found that the naphthalene ring generally exhibited significantly higher activity than the benzene ring. Furthermore, the bioactivity of the compound could be further enhanced by modifying it with inactivating groups (Cl and Br). In exploring the influence of steric effects on the bioactivity of the compound, the cyclization strategy provided another important approach for successfully modifying the compound. Compared to 3,5-dimethylphenol, 5,6,7,8-tetrahydronaphthol showed significantly higher bioactivity, indicating that the bioactivity of the compound is strictly limited by its spatial structure.

[0026] In the modification of the amine moiety, we found that the introduction of the aromatic ring can significantly improve the antifungal activity of the compound. Benzylamine exhibits different biological activities at different positions on the benzene ring and with different substituents. The Hammett constant (σ) indicates that groups with absolute values ​​between σ and σ have better biological activity, while substituents with excessively large (CN, σ = 1.00) or excessively small (H, σ = 0.00) Hammett constants do not enhance the biological activity of the compound. Similarly, para-substitution exhibits better biological activity based on different positions, and this rule applies to both electron-donating (CH3) and electron-withdrawing (Br) groups.

[0027] Molecular docking results also showed that isopropanolamine derivatives generally exhibited high binding levels to phosphatidylphosphatase protein, with compound C23 showing particularly significant binding. The binding of the two different configurations of compounds (R)-C23 and (S)-C23 to phosphatidylphosphatase (MfPAP) from *Prunus cerevisiae* was significantly different. Both compounds bound to the phosphatidylphosphatase cavity via the trifluoromethyl group of the amine moiety, but due to their different configurations, the binding in the catalytic pockets P2 and P3 differed. Furthermore, the hydrogen bond interactions formed between the two isomers and phosphatidylphosphatase were also completely different. Figure 1 This also indicates that there is a difference in bioactivity between the two in terms of antifungal effects.

[0028] Molecular dynamics further confirmed this conclusion. In 50-nanosecond molecular dynamics simulations with MfPAP, the binding free energy of compound (R)-C23 was -10.39 kcal / mol, and that of (S)-C23 was -20.02 kcal / mol. Quantum chemical calculations showed that the surface electrostatic potential of compound C23 changed significantly compared to the original compound. The positive and negative charge centers of both molecules were concentrated near the core isopropanolamine chain, while the positive charge polarity of compound C23 was enhanced and the negative charge polarity was weakened. This allowed C23 to bind more strongly to the negative charge pocket of phosphatidylphosphatase.

[0029] In summary, through computer-aided drug design, we discovered compound C23, which exhibits a stronger binding interaction with phosphatidylphosphatase. Based on this, we synthesized C23 and verified its antifungal activity.

[0030] Example 1: Synthesis of compound 1-(5,6,7,8-tetrahydronaphth-1-yloxy)-3-({[4-(trifluoromethyl)phenyl]methyl}amino)prop-2-ol (C23)

[0031] S1: Synthesis of intermediate 2-[(5,6,7,8-tetrahydronaphthyl-1-yloxy)methyl]oxacyclopropane (A16):

[0032]

[0033] Add 1 equivalent of 5,6,7,8-tetrahydro-1-naphthol (5000 mg, 33.7 mmol) and 1.2 equivalents of anhydrous potassium carbonate (6.99 mg, 50.6 mmol) to a 150 mL three-necked flask. Under a nitrogen atmosphere, add 5 equivalents of epichlorohydrin (15.6 g, 13.2 mL, 169 mmol) and reflux at 110 °C for 24 h. Monitor the reaction by TLC until completion. Cool the reaction system to room temperature and wash with saturated sodium chloride aqueous solution and ethyl acetate (3 × 15 mL). Combine the organic phases and dry them with anhydrous sodium sulfate. Filter and remove the solvent by vacuum distillation to obtain the crude product. Purify the crude product using silica gel column chromatography with ethyl acetate:petroleum ether as eluent (1:5) to give the intermediate (colorless oily liquid, 5.03 mg, 24.6 mmol, yield 73.0%).

[0034] S2: Synthesis of target compound C23:

[0035]

[0036] Under a nitrogen atmosphere, 1 equivalent of 2-[(5,6,7,8-tetrahydronaphthyl-1-yloxy)methyl]oxecyclopropane intermediate (A16, 310 mg, 1.5 mmol) and 3 equivalents of [4-(trifluoromethyl)phenyl]methaneamine (788.2 mg, 0.64 mL, 4.5 mmol) were added to a stopcock-type reaction tube with a ground glass joint. The reaction was stirred at 50 °C for 24 h, and the reaction was monitored by TLC until complete. Recrystallization was performed using ethyl acetate:petroleum ether = 1:20 (v / v) to give the target compound C23 (white solid, 455 mg, 1.2 mmol, yield 82.1%). The proton, carbon, fluorine, and high-resolution mass spectrometry data of the compound are shown below:

[0037] 1 H NMR (600MHz, CDCl3) δ7.58 (d, J=7.8Hz, 2H, Phenyl-H ), 7.45 (d, J = 7.8 Hz, 2H, Phenyl-H ), 7.04(t, J = 7.8 Hz, 1H, Phenyl-H ), 6.71 (d, J = 7.8 Hz, 1H, Phenyl-H ), 6.62 (d, J = 7.8 Hz, 1H, Phenyl-H ), 4.14-4.08(m, 1H, CH2- CH (-CH2)-OH), 4.01-3.95(m,2H,O- CH 2-CH),3.90(q,J=13.8Hz,2H,NH- CH 2-Phenyl), 2.92-2.88 (m, 1H, CH- CH 2-NH), 2.86-2.81(m, 1H, CH- CH 2-NH), 2.77-2.71(m,2H, CH 2-Phenyl), 2.65-2.53(m,2H, CH 2-Phenyl), 1.75(s, 4H, 2× CH 2-CH2-Phenyl);

[0038] 13 C NMR (151MHz, CDCl3) δ156.29,144.24,138.89,129.52(q,J=32.2Hz),128.42,126.09,125.86,125.51( q, J=3.6Hz), 124.34 (q, J=271.9Hz), 107.94, 70.37, 68.83, 53.47, 51.56, 29.72, 23.31, 22.88, 22.83;

[0039] 19 F NMR (565MHz, CDCl3): δ-62.39;

[0040] HRMS(ESI):m / z Anal calcd.for C 21 H 25 F3NO2[M+H] + =380.1832,Found:380.1834.

[0041] Example 2: Synthesis of chiral compounds (R)-C23 and (S)-C23

[0042] S1: Synthesis of two different chiral intermediates (R)-A16 and (S)-A16:

[0043]

[0044] Under nitrogen protection, 3 equivalents of sodium hydroxide (600 mg, 15 mmol) and 20 mL of methanol were added to a 150 mL three-necked flask, heated to 35 °C, and stirred continuously until the solid was completely dissolved. 1 equivalent of 5,6,7,8-tetrahydronaphthyl-1-phenol (741 mg, 5 mmol) was dissolved in methanol and slowly added dropwise to the system. After the addition was complete, the mixture was stirred for 30 min, and then 3 equivalents of chiral epichlorohydrin (1387.8 mg, 1.17 mL, 15 mmol) was slowly added to the system. After the addition was complete, the reaction was maintained at 35 °C for 12 h. The reaction was monitored by TLC until completion. The reaction system was cooled to room temperature and washed with saturated sodium chloride solution and ethyl acetate (3 × 15 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by vacuum distillation to obtain the crude product. The intermediate (colorless oily liquid, 967 mg, 4.73 mmol, yield 94.6%) was purified by silica gel column chromatography using ethyl acetate:petroleum ether = 1:5 (v / v) as eluent. The enantiomeric excess (ee) value of the intermediate was monitored using a chiral column and high-performance liquid chromatography (HPLC). Using an Agilent HPLC system with a Daicel Chiralpak IC-3 (4.6 mm × 250 mm, 3 μm) column, at 25 °C and a flow rate of 0.7 mL / s, with hexane:isopropanol = 95:5 (v / v) as the mobile phase, the signal at 220 nm was monitored using a VWD detector. The retention times of the racemic intermediates were 12.20 min and 13.89 min, respectively. The retention time of (R)-A16 synthesized using chiral epichlorohydrin was 12.24 min, and the retention time of (S)-A16 was 14.29 min. All of them had ee values ​​>99%.

[0045] S2: Synthesis of two target compounds with different configurations, (R)-C23 and (S)-C23:

[0046]

[0047] Under a nitrogen atmosphere, one equivalent of chiral 2-[(5,6,7,8-tetrahydronaphthyl-1-yloxy)methyl]oxetine intermediate (A16, 310 mg, 1.5 mmol) and three equivalents of [4-(trifluoromethyl)phenyl]methaneamine (788.2 mg, 0.64 mL, 4.5 mmol) were added to a piston-shaped reaction tube with a ground glass joint. The reaction was stirred at 50 °C for 24 h, and the reaction was monitored by TLC until complete. Recrystallization was performed using ethyl acetate:petroleum ether = 1:20 (v / v) to obtain the target compound (R)-C23 (white solid, 512.3 mg, 1.35 mmol, yield 90.1%) or (S)-C23 (white solid, 482 mg, 1.27 mmol, yield 84.7%), depending on the chiral starting material used.

[0048] Based on the above reaction principle, this invention designs and synthesizes a series of compounds, some of which have the following structural formulas:

[0049]

[0050]

[0051]

[0052] Example 3: In vitro inhibitory activity of the synthesized isopropanolamine derivative C23 against eight plant pathogenic fungi.

[0053] 1. Culture of plant pathogenic fungi: *Fusarium graminearum* (wheat scab, PH-1), *Botrytis cinerea* (B05.10), *Sclerotinias clerotiorum* (rapeseed sclerotiorum, 1980UF-70), *Monilinia fructicola* (peach brown rot, 2YTF2-2), *Bipolaris maydis* (maize leaf spot, TM17), *Rhizoctonia solani* (rice sheath blight, WH-1), *Pestalotiopsis theae* (Pt1), and *Altermaria alternata* were all inoculated onto PDA medium and cultured in a constant temperature incubator at 25±0.1℃ for 3-6 days. Once the mycelium had grown well, they were ready for use.

[0054] 2. The plate method was used to test the inhibitory activity of different compounds against eight plant pathogenic fungi at certain concentrations. Commercially available fungicides Azoxystrobin (Azo.) and Boscalid (Bos.) were used as positive controls.

[0055] 3. Accurately weigh 20 mg (±0.1 mg) of the test compound into a 1.5 mL centrifuge tube. Add a certain amount of chromatographically pure DMSO to the centrifuge tube using a pipette, shake well to fully dissolve it into a 100 mmol / L stock solution, filter through a sterile filter, and transfer to a sterile centrifuge tube for later use. Add 6.9 g of potato dextrose agar (PDA) medium powder to a 250 mL Erlenmeyer flask, add 150 mL of ultrapure water, and shake well. Seal with a rubber band and a heat-resistant tissue culture sealing film, and autoclave at 121°C for 20 minutes. Heat the sterilized culture medium in a microwave oven on high for 3 minutes until completely melted into a liquid. After sterilizing under UV light for 30 minutes in a clean bench, accurately pipette 75 μL of the compound stock solution, inserting the pipette tip below the surface of the culture medium, and add the compound to the medium. Shake well on the same surface to prepare a 50 μmol / L drug-containing culture medium. Add the drug-containing culture medium evenly to a disposable petri dish, gently shake to mix until the bottom of the medium is covered. Label the cover plate with the time and compound number.

[0056] 4. After the drug-containing culture medium has cooled to room temperature and solidified, take the activated fungus and, using a 1.5 mm agar gel punch, cut several 1.5 mm diameter pieces of culture medium containing mycelia onto the medium in which it grows. Using a sterile scalpel, lift the mycelia-containing culture medium and inoculate it, hyphae side down, at the center of the solidified drug-containing culture medium. Invert the petri dishes in a constant temperature incubator and incubate at 22°C until the diameter of the blank control group reaches 80% of the culture medium.

[0057] 5. After cultivation, measure the diameter of the bacterial strains in the petri dishes using the cross-multiplication method and take the average value. For each compound and each bacterial strain in parallel test petri dishes, select the petri dishes with complete growth and the median diameter.

[0058] D = Colony diameter - Mushroom cake diameter

[0059] Inhibition rate = (D blank control - D drug treatment) / (D blank control - D solvent control) * 100%

[0060] 6. All experiments were conducted with three parallel control groups, and all experimental data were repeated three times.

[0061] Experimental results:

[0062]

[0063]

[0064] Experimental results showed that compounds C18, C22, and C23 generally exhibited higher antibacterial activity compared to other compounds, demonstrating that the modification strategy of increasing the steric hindrance of the isopropanolamine phenol portion was very successful and effective. Specifically, compound C23, at a concentration of 50 μmol / L, showed inhibition rates of 78.25%, 87.50%, 100%, 77.73%, 100%, 76.24%, 100%, and 100% against *Alternaria alternata*, *Scutellaria baicalensis*, *Botrytis cinerea*, *Fusarium graminearum*, *Sclerotinia sclerotiorum*, *Polytrichum sclerotiorum*, *Rhizoctonia solani*, and *Sclerotinia sclerotiorum*, respectively. At a concentration of 20 μmol / L, the inhibition rates against these eight plant pathogenic fungi were 75.69%, 86.76%, 92.00%, 67.33%, 100%, 65.39%, 89.71%, and 96.98%, respectively. These results indicate that isopropanolamine derivatives exhibit excellent broad-spectrum antifungal activity against plant pathogenic fungi.

[0065] Meanwhile, compared to previously synthesized and structurally similar compounds, C23 also exhibits superior bioactivity. At 50 μmol / L, C23 significantly enhances the inhibition of fungi such as Alternaria alternata and Gibberellin compared to previously reported compounds C10 and B15.

[0066] Example 4: Half-effective concentration test of chiral isopropanolamine derivatives (R)-C23 and (S)-C23 against four plant pathogenic fungi

[0067] The testing method was the same as in Example 3 above. The relevant compounds were prepared as stock solutions of different concentrations, and five different concentration gradients of drug-containing culture media were prepared under the condition that the DMSO content was consistent in all culture media. The inhibition rate of the compounds against fungi at different concentrations was observed and measured. A regression equation for the toxicity of the compounds against fungi was fitted, and the EC50 was calculated based on the independent regression equation. 50 value.

[0068] Experimental results:

[0069]

[0070] Compound (R)-C23 has EC50-like effects on Botrytis cinerea, Fusarium graminearum, Prunus sylvestris var. chinensis, and Sclerotinia sclerotiorum. 50 The values ​​were 6.119, 9.672, 4.708, and 8.598 μmol / L, respectively. The EC50 values ​​of compound (S)-C23 against the above four fungi were... 50The values ​​were 6.402, 14.32, 3.557, and 7.525 μmol / L, respectively. Among them, (R)-C23 showed 2.828 times the inhibitory effect on *Fusarium graminearum* and 30.38 times that of azoxystrobin; its inhibitory effect on *Prunus persica* was 17.62 times that of azoxystrobin and 2.444 times that of azoxystrobin. The inhibitory effect of (R)-C23 on *Fusarium graminearum* was significantly better than that of (S)-C23 (P<0.05), while (S)-C23 showed significantly better inhibitory effect on *Prunus persica* than (R)-C23 (P<0.05), and better inhibitory effect on *Sclerotinia sclerotiorum* than (R)-C23 (P>0.05). The results indicate that both chiral configurations of compound C23 possess excellent biological activity, and their inhibitory effects on different plant fungi vary.

[0071] Example 5: Evaluation of the inhibitory effect of isopropanolamine derivative C23 on brown rot disease of two peach fruits and gray mold disease of tomato.

[0072] 1. Culture of plant pathogenic fungi: Peach brown rot fungus cultured on PDA medium for 7 days and Botrytis cinerea cultured on V8 medium for 7 days were selected. For Peach brown rot fungus, holes were punched using a 5 cm diameter punch, and for Botrytis cinerea, spores were collected using PDB liquid medium.

[0073] 2. Select two types of peaches: Water Peaches and Golden Autumn Red Honey Peaches. Disinfect them by soaking them in 75% ethanol for 30 seconds, then rinse the fruit surface thoroughly with running water and let them air dry. Use large red tomatoes, rinse the fruit surface thoroughly with running water, and let them air dry.

[0074] 3. Make a 1cm deep, 2mm diameter hole in the peach fruit using a sterilized toothpick. Add 10μL of a compound solution with a concentration of 20, 50, or 100μmol / L into the hole. Apply the mycelial side of the peach brown rot fungus cake to the wound and seal with plastic wrap. Place in an artificial climate chamber and incubate at 25℃, 90% humidity, and 5000Lx light. After one day, remove the plastic wrap and continue incubation for 7 days under alternating light and dark conditions (12h + 12h).

[0075] 4. Using a sterilized scalpel, make a 1cm deep, 2cm long cross-shaped incision on the tomato fruit. Inject a suspension of the compound and Botrytis cinerea spores into the incision. The concentrations of the compound in the suspension are 20, 50, and 100 μmol / L. Place the treated tomato fruit in an artificial climate chamber and culture for 7 days at 25℃, 90% humidity, and 5000 Lx light, with alternating light and dark conditions for 12h + 12h.

[0076] Experimental results: Compound C23, at concentrations of 20, 50, and 100 μmol / L, showed inhibition rates of 35.83%, 71.87%, and 99.43% against *Prunus persica* infection in peaches, respectively; inhibition rates against *Prunus persica* infection in *Golden Autumn Red Honey* were 52.27%, 90.35%, and 99.57%, respectively; and inhibition rates against *Botrytis cinerea* infection in large red tomatoes were 18.02%, 50.95%, and 66.2%, respectively. Figure 4 The results showed that the compound had good inhibitory activity against plant pathogenic fungi in vivo, and was superior to commercially available fungicides of the same concentration.

[0077] The described embodiments are merely a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Modifications and improvements made by those skilled in the art based on the disclosure of the present invention to the embodiments of the present invention are also within the scope of protection claimed by the present invention.

Claims

1. The application of propranolol derivatives with the structure shown in formula (Ⅰ) in the resistance to plant fungal diseases, wherein the propranolol derivative is an R-type chiral isomer, and the plant fungal disease is Fusarium graminearum (… Fusarium gramineae ), formula (Ⅰ).

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