Compounds having antifungal activity and methods of making and using the same

Abstract

The application discloses a compound with antifungal activity and a preparation method and application thereof, and discloses a compound targeting AAA ATPase and a preparation method and application thereof. The compound targeting AAA ATPase has the following formula (I) or formula (II) structure, and tests show that three compounds, namely, AH-003, AH-006 and AH-008, have the activity of inhibiting the growth of plant pathogenic fungi.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of agricultural chemicals, and particularly relates to screening of compounds with antibacterial activity by molecular docking and rational design. BACKGROUND

[0002] Development and utilization of green new pesticides are conducive to agricultural yield increase and sustainable development, and rational molecular design based on target proteins is a hot spot in new pesticide creation. Green bactericides play an important role in preventing various crop diseases, ensuring agricultural product yield and quality, and ensuring food security. Macrolactin HSAF is a natural product isolated from Lysobacter enzymogenes, and has a broad inhibitory effect on fungi and oomycetes. VpVeb1 protein encoded by VpVEB1 gene of Valsa mali has an AAA (ATPases associated with various cellular activities) domain, and belongs to the AAA protein family. It has been confirmed by the team that HSAF can inhibit the growth of V. mali by inhibiting the activity of 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 development of plant fungal disease control drugs. SUMMARY

[0003] The purpose of the present application is to design and synthesize novel antifungal compounds that bind to AAA ATPase.

[0004] Firstly, the present application discloses a compound with antifungal activity, which has the structure of formula (I) or formula (II) as follows:

[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; and R3 is selected from methyl, ethyl, propyl or butyl.

[0007] In a preferred example, the compound with antifungal activity is selected from any one of the following structures:

[0008]

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

[0010] This invention also provides the use of the compound described herein in the preparation of fungicides. In a specific example, it is used in the preparation of inhibitors of plant pathogenic fungi.

[0011] The present invention also discloses the application of the compound in the prevention and control of plant fungal diseases.

[0012] In some specific examples, the plant fungal diseases described in this invention include pear rot fungus and pear anthracnose fungus. The beneficial effects of this invention are:

[0013] The compounds described in this invention exhibit inhibitory effects against plant pathogenic fungi. Specifically, they show good inhibitory effects against *Pyrus pyriformis* and *Pyrus anthracnose*. Attached Figure Description

[0014] Figure 1 The binding mode of the representative molecule AH-003 is shown. A) The predicted binding mode of AH-003 with VpVeb1-ATPase; B) The superposition of the binding mode of AH-003 with 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 fungus were studied. Detailed Implementation

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

[0018] Unless otherwise specified, the pear rot pathogen and pear anthracnose pathogen used in the following examples are strains preserved in our laboratory, and our laboratory agrees to make them publicly available.

[0019] Example 1: Synthesis of AH-003

[0020] 1. Synthetic route

[0021]

[0022] 2. Operating Procedures

[0023] Step 1: Synthesis of [(phenyloxy)(propylamino)methylethylene]azine carbide nitrile

[0024]

[0025] [bis(phenyloxy)methylethylene]azine carbide nitrile (3 g, 12.6 mmol, 1 eq) was dissolved in 15 mL of tetrahydrofuran. TEA (3.82 g, 37.82 mmol, 3 eq) and propylamine hydrochloride (1.20 g, 12.6 mmol, 1 eq) were added to the reaction mixture, and the mixture was stirred at room temperature for one hour. The reaction was monitored for completion; no purification was required before proceeding to the next step.

[0026] Step 2: Synthesis of 5-propylamino-2H-1,2,4-triazol-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 the mixture was stirred at 80 °C for 1–2 hours. TLC monitoring showed no raw material remaining. The reaction system was concentrated to dryness, and the residue was purified with silica gel (dichloromethane:methanol = 100:0–10:1) to give 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) ketone

[0030]

[0031] Under nitrogen protection, 5-propylamino-2H-1,2,4-triazol-3-amine (1 g, 7.09 mmol) was dissolved in 5 mL of dry dichloromethane. The mixture was cooled to 0 °C, and triethylamine (2.14 g, 21.2 mmol, 3 eq) was added. 2-fluoro-6-methoxybenzoyl chloride (1.33 g, 7.09 mmol) was slowly added dropwise, and the mixture was stirred at 0 °C for 1–2 hours. TLC monitoring showed no reactants remaining. The reaction was quenched with 20 mL of water, and the organic phase was collected by extraction with ethyl acetate. The organic phase was washed again with 60 mL of water and then with 60 mL of saturated sodium chloride solution. The organic phase was dried with anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The residue was purified by high-pressure pretreatment to give 178 mg of a 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. Synthetic route:

[0036]

[0037] 2. Synthesis method:

[0038] Step 1: Synthesis of 2,2,2-trifluoro-N-{2-[(4-methylphenyl)dioxane-λ6-thio]-5-(2-methylpropyl-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)dioxane-λ6-thio]-5-(2-methylpropyl-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 mixture, and the mixture was extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by Flash silica gel column chromatography (petroleum ether / ethyl acetate: 0%–60% elution) to give 0.9 g of a 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)dioxane-λ6-thio]-5-(2-methylpropyl-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)dioxane-λ6-thio]-5-(2-methylpropyl-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 mixture, and the reaction mixture was stirred at room temperature for two hours. 50 mL of ice water was added to the reaction mixture, and the mixture was extracted with ethyl acetate (50 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 0.9 g of a brown solid (yield 96.8%).

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

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

[0045] Sodium hydroxide (0.91 g, 22.9 mmol, 5 N) was added to a methanol (10 mL) solution of 2,2,2-trifluoro-N-methyl-N-{2-[(4-methylphenyl)dioxane-λ6-thio]-5-(2-methylpropyl-2-yl)-4-phenylpyrazol-3-yl}acetamide (0.9 g, 2.30 mmol). The reaction mixture was stirred overnight at room temperature. The filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by reverse-phase chromatography to give 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. Synthetic route:

[0050]

[0051] 2. Synthesis method:

[0052] Step 1: Synthesis of 2-[(4-methylphenyl)dioxane-λ6-thio]-5-(2-methylpropyl-2-yl)pyrazole-3-amine

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

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

[0055] Step 2: Synthesis of 4-bromo-2-[(4-methylphenyl)dioxane-λ6-thio]-5-(2-methylpropyl-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)dioxane-λ6-thio]-5-(2-methylpropyl-2-yl)pyrazol-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 mixture, and the mixture was extracted with ethyl acetate (100 mL × 3). The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by Flash silica gel column chromatography (eluting with petroleum ether / ethyl acetate: 0%–60%) to give 2.2 g of a brown solid (yield 87.0%).

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

[0058] Step 3: Synthesis of 2-[(4-methylphenyl)dioxane-λ6-thio]-5-(2-methylpropyl-2-yl)-4-phenylpyrazole-3-amine

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

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

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

[0062] Sodium hydroxide (0.33 g, 8.25 mmol, 5 N) was added to a methanol (5 mL) solution of 2-[(4-methylphenyl)dioxane-λ6-thio]-5-(2-methylpropyl-2-yl)-4-phenylpyrazole-3-amine (0.3 g, 0.806 mmol), and the reaction mixture was stirred overnight at room temperature. The filtrate was concentrated under reduced pressure to give a crude product. The crude product was purified by reverse-phase column chromatography to give 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] Antibacterial activity of AH-003AH-006AH-008 in Example 4

[0066] Different concentrations (0, 100, 200, 400 μM) of the compound were added to PDA plates. Mycelial blocks of *Pyrus pyriformis* and *Pyrus anthracnose* were collected using a 5 mm punch and inoculated into the center of each PDA plate containing a different concentration of the drug. The plates were then incubated at 26°C. When the colony diameter on the control (CK) plate exceeded half the diameter of the culture dish, the dish was removed, and the diameter of each colony was measured using the cross-sectional method, and the results were recorded. The results are as follows: Figure 2 and Figure 3 As shown, based on the recorded results, the inhibition rate of the pathogen under different conditions was calculated, and the inhibition rate was converted into a probability value. The drug concentration in the culture medium was expressed as a logarithm. The virulence regression curve was plotted graphically, and the EC50 was calculated. 50 The results showed that the EC50 values ​​of AH-003AH-006AH-008 against *Pyrus pyrifolia* were 144.5 μM, 288.4 μM, and 380.2 μM, respectively, and the EC50 values ​​of AH-003AH-006AH-008 against *Pyrus anthracnose* were 128.8 μM, 407.4 μM, and 489.8 μM, respectively. This indicates that the compounds of the present invention have good inhibitory effects 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 represents the predicted binding mode of AH-003 to VpVeb1-ATPase; B represents the superposition of the binding mode of AH-003 to VpVeb1-ATPase and the original crystal complex structure (PDB code: 6P12). This indicates that the compound of the present invention is a compound targeting AAA ATPase.

Claims

1. Compounds with antifungal activity are selected from any of the following structures: 。 2. The use of the compound of claim 1 in the preparation of inhibitors for plant pathogenic fungi.

3. The application according to claim 2, characterized in that, The plant pathogenic fungi mentioned are *Pyrus pyriformis* and *Pyrus anthracnose*.

4. The use of the compound according to claim 1 in the prevention and control of plant fungal diseases.

5. The application according to claim 4, characterized in that, The plant fungal diseases mentioned are pear rot fungus and pear anthracnose fungus.

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