Compound with antifungal activity as well as preparation method and application thereof

Abstract

The invention relates to a compound with antifungal activity and a preparation method and application thereof, and discloses a compound targeting AAA ATP enzyme and a preparation method and application thereof, the compound targeting AAA ATP enzyme has a structure shown as a formula (I) or a formula (II), and tests show that three compounds AH-003, AH-006 and AH-008 have activity of inhibiting growth of plant pathogenic fungi. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of agricultural drug chemistry, and specifically relates to screening compounds with antibacterial activity through molecular docking and rational design. Background Art

[0002] The development and utilization of new green pesticides are conducive to increasing agricultural production and income and sustainable development, and the rational molecular design based on target proteins is a hot topic in the creation of new pesticides. Green fungicides play an important role in preventing and controlling various crop diseases, ensuring the yield and quality of agricultural products, and ensuring food security. The macrolide substance HSAF is a natural product isolated from Lysobacter enzymogenes, which has a wide range of inhibitory effects on fungi and oomycetes. The VpVeb1 protein encoded by the VpVEB1 gene of the pear rot pathogen has an AAA (ATPases associated with various cellular activities) domain and belongs to the AAA protein family. Our team has confirmed that HSAF can inhibit the growth of pear rot pathogen by inhibiting the activity of the AAA ATPase VpVeb1 (VpVeb1-ATPase). Based on the interaction between HSAF and VpVeb1-ATPase, new antifungal compounds are designed and synthesized, providing a new direction for the development of drugs for the prevention and control of plant fungal diseases. Summary of the invention

[0003] The object of the present invention is to design and synthesize novel antifungal compounds that bind to AAA ATPases.

[0004] First, the present invention discloses a compound having antifungal activity, which has a structure of the following formula (I) or formula (II):

[0005]

[0006] Wherein: R1 is selected from -NH2, -NHR a , preferably, R1 is -NHR a ; R a is selected from methyl, ethyl, propyl or butyl; R2 is selected from -NH2, -NHR b , R b is selected from methyl, ethyl, propyl or butyl; R3 is selected from methyl, ethyl, propyl or butyl.

[0007] In a preferred embodiment, the compound having antifungal activity is selected from any of the following structures:

[0008]

[0009] The present invention also provides the use of the compound of the present invention in the preparation of pesticides.

[0010] The present invention also provides the use of the compound of the invention in the preparation of fungicides. In a specific example, the use of the compound of the invention in the preparation of plant pathogenic fungi inhibitors.

[0011] The invention also discloses application of the compound in preventing and controlling plant fungal diseases.

[0012] In some specific examples, the plant fungal diseases described in the present invention include pear rot pathogens and pear anthracnose pathogens. Beneficial effects of the present invention:

[0013] The compounds of the present invention have an inhibitory effect on plant pathogenic fungi, and specifically have a good inhibitory effect on pear rot pathogens and pear anthracnose pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The binding mode of the representative molecule AH-003. A) The predicted binding mode of AH-003 and VpVeb1-ATPase; B) The superposition of the binding mode of AH-003 and VpVeb1-ATPase and the original crystal complex structure (PDB code: 6P12);

[0015] Figure 2 The inhibitory effects of AH-003, AH-006, and AH-008 on pear rot pathogens;

[0016] Figure 3 The inhibitory effects of AH-003, AH-006 and AH-008 on pear anthracnose. DETAILED DESCRIPTION

[0017] The following examples are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0018] Unless otherwise specified, the pear rot pathogen and pear anthracnose pathogen used in the following examples are strains preserved in this laboratory, which is open to the public.

[0019] Example 1 Synthesis of AH-003

[0020] 1. Synthesis route

[0021]

[0022] 2. Operation steps

[0023] Step 1: Synthesis of [(phenyloxy)(propylamino)methylidene]azanecarbonitrile

[0024]

[0025] Dissolve [bis(phenyloxy)methylidene]azanecarbonitrile (3 g, 12.6 mmol, 1 eq) in 15 mL tetrahydrofuran. Add TEA (3.82 g, 37.82 mmol, 3 eq) and propylamine hydrochloride (1.20 g, 12.6 mmol, 1 eq) to the reaction system and stir at room temperature for one hour. Monitor the reaction to completion and proceed to the next step without purification.

[0026] Step 2: Synthesis of 5-propylamino-2H-1,2,4-triazole-3-amine

[0027]

[0028] In the above reaction system, hydrazine hydrate (694 mg, 13.86 mmol, 1.1 eq) was added in an ice-water bath, and stirring was continued at 80°C for 1-2 hours. TLC monitoring showed that no raw material remained, and the reaction system was concentrated to dryness, and the residue was purified by silica gel (dichloromethane: methanol = 100: 0-10: 1) to obtain 1.5 g of solid, with a yield of 88%.

[0029] Step 3: Synthesis of [5-amino-3-propylamino-1,2,4-triazol-1-yl](2-fluoro-6-methoxyphenyl)methanone

[0030]

[0031] Under nitrogen protection, 5-propylamino-2H-1,2,4-triazole-3-amine (1g, 7.09mmol) was dissolved in 5mL dry dichloromethane, cooled to 0°C, triethylamine (2.14g, 21.2mmol, 3eq) was added, 2-fluoro-6-methoxybenzoyl chloride (1.33g, 7.09mmol) was slowly added dropwise, and stirring was continued at 0°C for 1 to 2 hours. TLC monitoring showed that there was no raw material remaining, 20mL of water was added to the reaction system to quench the reaction, ethyl acetate was added to extract the liquid, the organic phase was collected, the organic phase was washed again with 60mL of water, the organic phase was washed with 60mL of saturated sodium chloride solution, the organic phase was dried with anhydrous sodium sulfate, filtered, the filtrate was concentrated to dryness under reduced pressure, and the residue was purified by high pressure preparation to obtain 178mg of white solid with a purity of 99.6% and a yield of 8.5%.

[0032] LC-MS: m / z[M+H] + 294.3

[0033] 1H NMR (400MHz, DMSO-d6) δ7.62(s,2H),7.46(td,J=8.5,6.9Hz,1H),6.97(d,J=8.5Hz,1H),6.89(t,J=8.7Hz ,1H),6.28(t,J=5.8Hz,1H),3.79(s,3H),2.93-2.84(m,2H),1.40(h,J=7.3Hz,2H),0.79(t,J=7.4Hz,3H).

[0034] Example 2 Synthesis of AH-006

[0035] 1. Synthesis route:

[0036]

[0037] 2. Synthesis method:

[0038] Step 1: Synthesis of 2,2,2-trifluoro-N-{2-[(4-methylphenyl)dioxy-λ6-thio]-5-(2-methylpropan-2-yl)-4-phenylpyrazol-3-yl}acetamide

[0039] TFAA (0.68 g, 3.24 mmol) was added to a pyridine (10 mL) solution of 2-[(4-methylphenyl)dioxy-λ6-thio]-5-(2-methylprop-2-yl)-4-phenylpyrazole-3-amine (1 g, 2.70 mmol). The reaction mixture was stirred at room temperature for two hours. 50 mL of water was added to the reaction solution and extracted with ethyl acetate (100 mL×3). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by Flash silica gel column (petroleum ether / ethyl acetate: 0% to 60% elution) to obtain 0.9 g of white solid (yield 71.6%).

[0040] LC-MS: m / z[M+H] + 466.1

[0041] Step 2: Synthesis of 2,2,2-trifluoro-N-methyl-N-{2-[(4-methylphenyl)dioxy-λ6-thio]-5-(2-methylpropan-2-yl)-4-phenylpyrazol-3-yl}acetamide

[0042] Sodium hydride (122 mg, 3.05 mmol) was added to a DMF (10 mL) solution of 2,2,2-trifluoro-N-{2-[(4-methylphenyl)dioxy-λ6-sulfanyl]-5-(2-methylpropane-2-yl)-4-phenylpyrazol-3-yl}acetamide (0.9 g, 1.93 mmol), and the reaction mixture was stirred at room temperature for half an hour. Iodomethane (433 mg, 3.05 mmol) was added to the reaction solution, and the reaction mixture was stirred at room temperature for two hours. 50 mL of ice water was added to the reaction solution, and extracted with ethyl acetate (50 mL×3). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 0.9 g of brown solid (yield 96.8%).

[0043] LC-MS: m / z[M+H] + 480.1

[0044] Step 3: Synthesis of 5-(methylamino)-3-(2-methylpropan-2-yl)-4-phenyl-1H-pyrazole

[0045] Sodium hydroxide (0.91 g, 22.9 mmol, 5N) was added to a solution of 2,2,2-trifluoro-N-methyl-N-{2-[(4-methylphenyl)dioxy-λ6-thio]-5-(2-methylpropane-2-yl)-4-phenylpyrazol-3-yl}acetamide (0.9 g, 2.30 mmol) in methanol (10 mL), and the reaction mixture was stirred at room temperature overnight. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by reverse phase to obtain 150 mg of a yellow solid (yield 34.9%).

[0046] LC-MS: m / z[M+H] + 230.4

[0047] 1H NMR (400MHz, DMSO-d6) δ8.14(s,1H),7.36(td,J=7.3,6.3,1.3Hz,2H),7.30-7.24(m,1H),7.22-7.17(m,2H),2.59(s,3H),1.11(s,9H).

[0048] Example 3 Synthesis of AH-008

[0049] 1. Synthesis route:

[0050]

[0051] 2. Synthesis method:

[0052] Step 1: Synthesis of 2-[(4-methylphenyl)dioxy-λ6-thio]-5-(2-methylpropan-2-yl)pyrazol-3-amine

[0053] To a solution of 3-tert-butyl-pyrazole-5-amine (2.47 g, 17.74 mmol) in acetonitrile (10 mL) was added p-toluenesulfonyl chloride (3.5 g, 18.3 mmol) and sodium bicarbonate (1.9 g, 13.74 mmol). The reaction mixture was stirred at room temperature overnight. 50 mL of water was added to the reaction solution and extracted with ethyl acetate (100 mL × 3). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by Flash silica gel column (petroleum ether / ethyl acetate: 0% to 30% elution) to obtain 5.2 g of white solid (yield 96.1%).

[0054] LC-MS: m / z[M+H] + 294.1

[0055] Step 2: Synthesis of 4-bromo-2-[(4-methylphenyl)dioxy-λ6-thio]-5-(2-methylpropan-2-yl)pyrazol-3-amine

[0056] NBS (1.14 g, 8.20 mmol) was added to a DMSO (20 mL) solution of 2-[(4-methylphenyl)dioxy-λ6-thio]-5-(2-methylprop-2-yl)pyrazole-3-amine (2 g, 6.82 mmol), and the reaction mixture was stirred at room temperature for 2 hours. 100 mL of water was added to the reaction solution, and it was extracted with ethyl acetate (100 mL×3). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by Flash silica gel column (petroleum ether / ethyl acetate: 0% to 60% elution) to obtain 2.2 g of brown solid (yield 87.0%).

[0057] LC-MS: m / z[M+H] + 373.1

[0058] Step 3: Synthesis of 2-[(4-methylphenyl)dioxy-λ6-thio]-5-(2-methylprop-2-yl)-4-phenylpyrazol-3-amine

[0059] To a mixed solution of 4-bromo-2-[(4-methylphenyl)dioxyylidene-λ6-thio]-5-(2-methylpropan-2-yl)pyrazol-3-amine (1.4 g, 3.76 mmol) in dioxane (15 mL) and water (1.5 mL) were added (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzene (1.54 g, 7.53 mmol), sodium carbonate (1.2 g, 11.3 mmol), chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-1,1'-biphenyl)[2-(2'-amino-1,1'-biphenyl)]palladium(II) (300 mg, 0.38 mmol) and 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (180 mg, 0.38 mmol). The reaction mixture was stirred at 90°C overnight under a nitrogen atmosphere. After cooling to room temperature, it was filtered and the filtrate was concentrated under reduced pressure to obtain a crude product. 50 mL of water was added to the reaction solution and extracted with ethyl acetate (100 mL × 3). The combined organic phase was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by Flash silica gel column (petroleum ether / ethyl acetate: 10% to 70% elution) to obtain 0.9 g of brown solid (yield 64.7%).

[0060] LC-MS: m / z[M+H] + 370.1

[0061] Step 4: Synthesis of 5-(2-methylpropan-2-yl)-4-phenyl-2H-pyrazol-3-amine

[0062] Sodium hydroxide (0.33 g, 8.25 mmol, 5 N) was added to a solution of 2-[(4-methylphenyl)dioxy-λ6-thio]-5-(2-methylprop-2-yl)-4-phenylpyrazole-3-amine (0.3 g, 0.806 mmol) in methanol (5 mL), and the reaction mixture was stirred at room temperature overnight. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by reverse phase column to obtain 107 mg of a white solid (yield 61.8%).

[0063] LC-MS: m / z[M+H] + 216.2

[0064] 1H NMR (400MHz, DMSO-d6) δ8.13(s,1H),7.41-7.33(m,2H),7.32-7.24(m,1H),7.24-7.16(m,2H),1.11(s,9H).

[0065] Example 4 Antibacterial activity of AH-003AH-006AH-008

[0066] Add different concentrations (0, 100, 200, 400 μM) of compounds to the PDA plate, use a 5mm puncher to take the mycelium of pear rot pathogen and pear anthracnose pathogen, and inoculate them in the center of the PDA plate containing different concentrations of drugs, and culture at 26°C. When the diameter of the colony on the CK plate exceeds half the diameter of the culture dish, take out the culture dish, measure the diameter of each colony by the cross method and record the results. Figure 2 and Figure 3 As shown in the figure, according to the recorded results, the inhibition rate of different conditions on the bacteria was calculated, the inhibition rate was converted into a probability value, the drug concentration in the culture medium was expressed as a logarithmic value, the toxicity regression curve was drawn by the graphical method, and the EC 50 The results showed that the EC50 of AH-003AH-006AH-008 against pear rot pathogens were 144.5μM, 288.4μM, and 380.2μM, respectively, and the EC50 of AH-003AH-006AH-008 against pear anthracnose pathogens were 128.8μM, 407.4μM, and 489.8μM, respectively. This indicates that the compounds of the present invention have a good inhibitory effect on plant pathogenic fungi.

[0067] Based on SBDD, the binding mode of the representative molecule AH-003 is as follows Figure 1 As shown, A is the predicted binding mode of AH-003 and VpVeb1-ATPase; B is the superposition of the binding mode of AH-003 and VpVeb1-ATPase and the original crystal complex structure (PDB code: 6P12), indicating that the compound of the present invention is a compound targeting AAA ATPase.

Claims

1. A compound having antifungal activity, which has a structure of the following formula (I) or formula (II): in: R1 is selected from -NH2, -NHR a , preferably, R1 is -NHR a ; R a is selected from methyl, ethyl, propyl or butyl; R2 is selected from -NH2, -NHR b , R b R is selected from methyl, ethyl, propyl or butyl; R3 is selected from methyl, ethyl, propyl or butyl.

2. The compound having antifungal activity is selected from any of the following structures:

3. Use of the compound according to claim 1 or 2 in the preparation of pesticides.

4. Use of the compound according to claim 1 or 2 in the preparation of a fungicide.

5. Use of the compound according to claim 1 or 2 in the preparation of a plant pathogenic fungus inhibitor.

6. The use according to claim 5, characterized in that: The plant fungal diseases include pear rot pathogen and pear anthracnose pathogen.

7. Use of the compound according to claim 1 or 2 in preventing and controlling plant fungal diseases.

8. The use according to claim 7, characterized in that: The plant fungal diseases include pear rot pathogen and pear anthracnose pathogen.

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