Application of natural product iheyamine A and derivative thereof in treatment of plant virus disease

Abstract

The invention relates to application of bisindole alkaloid iheyamine A and derivatives thereof in treatment of plant virus pathogens, and finds that iheyamine A and derivatives I-1-I-15 thereof show good anti-plant virus pathogen activity for the first time, and can well inhibit tobacco mosaic virus (TMV), cucumber fusarium wilt, peanut brown spot, apple ring rot, wheat sheath blight, tomato early blight and rice blast. , phytophthora capsici and sclerotium of rape.

Description

Technical Field

[0001] This invention relates to the application of the natural product iheyamine A and its derivatives in the treatment of plant viral and bacterial diseases, belonging to the field of agricultural protection technology. Background Technology

[0002] Iheyamine A (reaction formula 1) alkaloids are a class of natural products with a nitrogen-containing [3,2-b:4,5-b']bisindole skeleton structure, composed of two six-membered rings, two five-membered rings, and one seven-membered ring. In 1999, the Higa research group of Japanese scientists (Tetrahedron lett. 1999, 40, 303-306) first isolated the alkaloids Iheyamines A and B from a sea squirt collected from Okinawa.

[0003]

[0004] In 2016, Sperry's group (Org. Lett. 2016, 18, 5404-5407) synthesized iheyamine A for the first time in four steps via a cross-Mannich reaction using 5-methoxy-3-acetoxyindole as a substrate, achieving an overall yield of 55.8% (Reaction Formula 1). This method cleverly utilizes the electronic effects of substituents to achieve reaction selectivity, but it may not be suitable for preparing iheyamine A analogs with diverse substituents. In 2018, Abe and Yamada (Org. Lett. 2018, 20, 1469-1472) developed an intramolecular dehydration Mannich reaction based on halides to construct an asymmetric 2,2'-bisindole framework, successfully synthesizing iheyamine A with an overall yield of 31.4% (Reaction Formula 2). In 2020, Cheon's research group (J. Org. Chem. 2020, 85, 8149-8156) reported the total synthesis of iheyamine A using ethyl 2-aminocinnamate as a starting material via a cyanide-catalyzed imine-Stetter reaction, with an overall yield of 38.2% (Reaction Formula 3). In 2023, Liu Sheng's research group at Guizhou Medical University (J. Org. Chem. 2023, 88, 1256-1259) referenced an acid-catalyzed rearrangement method for synthesizing iheyamine A isomers, achieving the total synthesis of iheyamine A in four steps from tryptamine and 5-OMe-N-PMB-indigo, with an overall yield of 72.2% (Reaction Formula 4).

[0005]

[0006] Biindole compounds possess a wide range of biological activities. While the detailed biological characteristics of iheyamine compounds remain to be studied, other similar bisindole compounds, such as vinblastine and vincristine, have been found to be effective antitumor drugs. To date, there are no reports on the use of iheyamine alkaloids in the control of plant viral and fungal diseases. Summary of the Invention

[0007] This invention provides the application of iheyamine A derivatives in the prevention and control of plant viral and fungal diseases. The iheyamine A derivatives of this patent exhibit excellent antiviral and antifungal activity against plant viruses and pathogens.

[0008]

[0009] The iheyamine A and its derivatives of the present invention are I-1 to I-15.

[0010] Compared with existing technologies, this invention is the first to discover that iheyamine A and its derivatives I-1 to I-15 exhibit excellent anti-plant virus and pathogen activity, and can effectively inhibit eight plant pathogens, including tobacco mosaic virus (TMV), cucumber wilt, peanut brown spot, apple ring rot, wheat sheath blight, tomato early blight, rice blast, pepper phytosis, and rapeseed sclerotium. Detailed Implementation

[0011] In this invention, iheyamine A and its derivatives I-1 to I-13 were prepared according to the following reference: J.Org.Chem.2023, 88, 1256-1259. I-14 and I-15 were prepared according to the following reference: Molecules 2023, 28(7), 3032.

[0012] Example 1: Experimental data of Iheyamine A and its derivatives I-1 to I-15

[0013] I-1: Purple-red solid, melting point: 243-244℃. 1H NMR (400MHz, DMSO-d6) δ13.55 (s, 1H, NH), 8.91 (d, J=6.5Hz, 1H, ArH), 8.79 (d, J=6.5Hz, 1H, ArH), 8.58 (d, J=7.6Hz, 1H, ArH), 7.93 (d, J=8.0Hz, 2H, ArH), 7.80 (dd, J=13.9, 8.3Hz, 2H, ArH), 7.52 (t, J=7.5Hz, 1H, ArH), 7.39 (dd, J=8.7, 2.5Hz, 1H, ArH), 3.96 (s, 3H, OCH3). 13 C NMR (100MHz, DMSO-d6) δ156.2, 139.3, 130.6, 130.0, 127.9, 126.8, 123.1, 122.7, 122.2, 120.2, 117.0, 114.4, 103.1, 56.1.HRMS (ESI): calcd for C 19 H 14 N3O + [M+H] + 300.1131, found 300.1137.

[0014] I-2: Purple-red solid, melting point: 236-237℃. 1 H NMR (400MHz, DMSO-d6) δ9.11 (d, J=6.6Hz, 1H, ArH), 8.58 (d, J=6.5Hz, 1H, ArH), 8.36 (d, J=7.7Hz, 2H, ArH), 7 .76 (d, J=8.1Hz, 1H, ArH), 7.67 (t, J=7.5Hz, 1H, ArH), 7.53 (dd, J=15.8, 8.2Hz, 3H, ArH), 2.61 (s, 3H, ArCH3). 13 C NMR (100MHz, DMSO-d6) δ140.0, 132.1, 131.6, 130.7, 127.3, 126.8, 122.9, 122.7, 122.3, 121.5, 117.2, 115.5, 21.8.HRMS(ESI)calcd for C 19 H 14 N3 + [M+H] + 284.1182, found 284.1186.

[0015] I-3: Purple-red solid, melting point: 245-246℃. 11H NMR (400 MHz, DMSO-d6) δ 8.91 (dd, J = 6.3, 3.4 Hz, 1H, ArH), 8.77 (dd, J = 7.4, 4.0 Hz, 1H, ArH), 8.65 - 8.49 (m, 1H, ArH), 8.13 - 8.06 (m, 1H, ArH), 7.94 (d, J = 8.0 Hz, 1H, ArH), 7.89 - 7.83 (m, 1H, ArH), 7.78 (t, J = 7.5 Hz, 1H, ArH), 7.62 - 7.50 (m, 2H, ArH). 19 19F NMR (376 MHz, DMSO-d6) δ -119.6. 13 13C NMR (100 MHz, DMSO-d6) δ 160.0, 157.7, 140.0, 130.8, 130.1, 128.0 (d, J = 9.4 Hz), 126.8, 123.4, 122.9, 122.3, 118.0 (d, J = 25.8 Hz), 117.3, 114.4, 106.8 (d, J = 24.0 Hz). HRMS (ESI) calcd for C 18 H 11 FN3 + [M + H] + 288.0932, found 288.0928.

[0016] I-4: Purple-red solid, melting point: 241 - 242 °C. 1 1H NMR (400 MHz, DMSO-d6) δ 8.93 (d, J = 6.4 Hz, 1H, ArH), 8.79 (d, J = 6.4 Hz, 1H, ArH), 8.56 (d, J = ~Hz, 1H, ArH), 8.32 (s, 1H, ArH), 7.93 (d, J = 8.0 Hz, 1H, ArH), 7.85 (d, J = 8.5 Hz, 1H, ArH), 7.79 (t, J = 7.6 Hz, 1H, ArH), 7.72 (d, J = 8.5 Hz, 1H, ArH), 7.53 (t, J = 7.4 Hz, 1H, ArH). 13 13C NMR (100 MHz, DMSO-d6) δ 140.2, 130.5, 129.3, 127.9, 126.7, 126.2, 123.1, 122.6, 121.9, 120.4, 117.0, 116.7, 113.9. HRMS (ESI) calcd for C 18 H 11 ClN3 + [M + H] +304.0636, found 304.0641.

[0017] I-5: Purplish-red solid, melting point: 252-253℃. 1 H NMR (400MHz, DMSO-d6) δ8.95 (dd, J=17.5, 6.4Hz, 1H, ArH), 8.82 (dd, J=24.6, 6.4Hz, 1H, ArH), 8.63- 8.45 (m, 2H, ArH), 7.99-7.90 (m, 1H, ArH), 7.90-7.74 (m, 3H, ArH), 7.54 (dd, J=16.3, 8.9Hz, 1H, ArH). 13 C NMR (100MHz, DMSO-d6) δ131.9, 130.5, 128.5, 126.3, 123.5, 123.1, 122.6, 121.9, 114.5, 113.9.HRMS (ESI)calcd for C 18 H 11 BrN3 + [M+H] + 348.0131, found 348.0137.

[0018] I-6: Purple-red solid, melting point: 199-200℃. 1 H NMR (400MHz, DMSO-d6) δ8.95 (d, J=6.8Hz, 1H, ArH), 8.83 (d, J=6.8Hz, 1H, ArH), 8.60 (d, J=7.9Hz, 1H, ArH), 8.48 (dd, J=8.6, 5.7Hz, 1H, ArH), 7.9 2 (d, J=8.1Hz, 1H, ArH), 7.81-7.76 (m, 1H, ArH), 7.66 (dd, J=9.7, 2.1Hz, 1H, ArH), 7.57-7.51 (m, 1H, ArH), 7.37 (ddd, J=9.6, 8.6, 2.3Hz, 1H, ArH). 19 F NMR(376MHz, DMSO-d6)δ-109.0. 13 C NMR (100MHz, DMSO-d6) δ157.7 (d, J=9.7Hz), 141.6, 140.0, 133.6, 129.8 (d, J=7.0Hz), 129 .6, 129.5, 125.0, 123.6, 123.1, 122.5 121.3, 113.9 (d, J=3.7Hz), 113.6.HRMS(ESI)calcd for C 18 H 11FN3 + [M+H] + 288.0932, found 288.0935.

[0019] I-7: Purplish-red solid, melting point: 229-230℃. 1 H NMR (400MHz, DMSO-d6) δ8.94 (d, J=6.6Hz, 1H, ArH), 8.81 (d, J=6.6Hz, 1H, ArH), 8.57 (d, J=7.8Hz, 1H, ArH ), 8.42 (d, J=8.3Hz, 1H, ArH), 7.97-7.87 (m, 2H, ArH), 7.78 (t, J=7.5Hz, 1H, ArH), 7.57-7.46 (m, 2H, ArH). 13 C NMR (100MHz, DMSO-d6) δ140.3, 134.8, 130.9, 130.0, 126.4, 126.1, 123.4, 123.3, 123.1, 122.4, 116.5, 116.2.HRMS (ESI)calcd for C 18 H 11 ClN3 + [M+H] + 304.0636, found 304.0642.

[0020] I-8: Purple-red solid, melting point: 270-271℃. 1 H NMR (400MHz, DMSO-d6) δ8.96 (s, 1H, ArH), 8.86 (d, J=6.9Hz, 1H, ArH), 8.60 (s, 1H, ArH), 8.37 (s, 1H, Ar H), 8.05 (s, 1H, ArH), 7.93 (s, 1H, ArH), 7.80 (s, 1H, ArH), 7.64 (s, 1H, ArH), 7.55 (d, J=4.4Hz, 1H, ArH). 13 C NMR (100MHz, DMSO-d6) δ139.7, 130.2, 125.8, 125.0, 122.9, 122.8, 122.6, 122.4, 121.7, 118.5, 116.0.HRMS (ESI)calcd for C 18 H 11 BrN3 + [M+H] + 348.0131, found 348.0136.

[0021] I-9: Purple-red solid, melting point: 171-172℃.1 H NMR (400MHz, DMSO-d6) δ8.88 (dd, J=16.5, 7.2Hz, 2H, ArH), 8.61 (d, J=7.9Hz, 1H, ArH), 8.33 (d, J=7.2Hz, 1H , ArH), 7.88 (d, J=8.1Hz, 1H, ArH), 7.78-7.73 (m, 1H, ArH), 7.69-7.64 (m, 1H, ArH), 7.57-7.50 (m, 2H, ArH). 19 FNMR(376MHz, DMSO-d6)δ-129.5. 13 C NMR (100MHz, DMSO-d6) δ137.6, 131.6 (d, J=5.5Hz), 130.3, 130.0, 125.6, 123.6, 123.5 ( d, J=5.9Hz), 123.0, 122.0, 118.5, 116.6 (d, J=17.7Hz), 114.4, 114.1.HRMS(ESI)calcd for C 18 H 11 FN3 + [M+H] + 288.0932, found 288.0937.

[0022] I-10: Purple-red solid, melting point: 132-133℃. 1 H NMR (400MHz, DMSO-d6) δ8.83 (ddd, J=19.7, 13.0, 7.5Hz, 1H, ArH), 8.64-8.34 (m, 1H, ArH), 8.04- 7.64 (m, 2H, ArH), 7.58-7.27 (m, 3H, ArH), 7.22 (d, J=8.2Hz, 1H, ArH), 6.89 (d, J=8.4Hz, 1H, ArH). 13 C NMR (100MHz, DMSO-d6) δ142.0, 140.4, 137.4, 134.1, 130.3, 130.1, 129.9, 125.5, 124.0, 123.5, 123.0, 121.8, 114.4, 114.1.HRMS(ESI)calcd for C 18 H 11 ClN3 + [M+H] + 304.0636, found 304.0633.

[0023] I-11: Purple-red solid, melting point: 136-137℃. 1H NMR (400MHz, DMSO-d6) δ 8.93-8.46 (m, 3H, ArH), 8.07-7.73 (m, 2H, ArH), 7.57-7.34 (m, 2H, ArH), 7.23 (dd, J=8.3, 6.0Hz, 1H, ArH), 6.95-6.83 (m, 1H, ArH). 13 C NMR (100MHz, DMSO-d6) δ136.9, 133.6, 129.8, 129.5, 129.4, 125.6, 124.98, 123.50, 123.0, 122.5, 121.3, 113.91, 113.87, 113.6.HRMS(ESI)calcd forC 18 H 11 BrN3 + [M+H] + 348.0131, found 348.0138.

[0024] I-12: Purple-red solid, melting point: 183-184℃. 1 H NMR (400MHz, DMSO-d6) δ9.00 (d, J=6.5Hz, 1H, ArH), 8.84 (d, J=6.5Hz, 1H, ArH), 8.61 (d, J=7.8Hz, 1H, ArH), 8.47 (d, J=7 .7Hz, 1H, ArH), 7.97 (d, J=8.1Hz, 1H, ArH), 7.89 (d, J=8.1Hz, 1H, ArH), 7.84-7.74 (m, 2H, ArH), 7.58-7.50 (m, 2H, ArH). 13 C NMR (100MHz, DMSO-d6) δ146.3, 140.7, 131.1, 130.2, 127.3, 127.0, 123.2, 123.0, 122.6, 122.0, 117.6, 115.8.HRMS(ESI)calcd for C 18 H 12 N3 + [M+H] + 270.1026, found 270.1032.

[0025] I-13: Purple-red solid, melting point: 166-167℃. 1H NMR (400MHz, DMSO-d6) δ8.96 (d, J=6.3Hz, 1H, ArH), 8.85 (d, J=6.4Hz, 1H, ArH), 8.43 (d, J=7.8Hz, 1H, ArH), 8.18 (d, J=2.3Hz, 1H, ArH) , 7.87 (dd, J=11.8, 8.4Hz, 2H, ArH), 7.73 (t, J=7.5Hz, 1H, ArH), 7.51 (t, J=7.4Hz, 1H, ArH), 7.44-7.41 (m, 1H, ArH), 3.96 (s, 3H, OCH3). 13 C NMR (100MHz, DMSO-d6) δ156.2, 140.3, 129.8, 127.6, 127.1, 123.4, 122.8, 121.6, 120.7, 118.4, 115.1, 114.4, 104.6, 56.3.HRMS(ESI)calcd for C 19 H 14 N3O + [M+H] + 300.1131, found 300.1134.

[0026] I-14: Purple-red solid, melting point: 103-104℃. 1 H NMR (400MHz, DMSO-d6) δ8.65 (dd, J=79.5, 5.8Hz, 1H, ArH), 8.47-8.37 (m, 2H, ArH), 7.88 (dd, J=48.7, 8.3Hz, 1H, ArH), 7.74 (d, J=2.7Hz, 1H, A rH), 7.68-7.64 (m, 1H, ArH), 7.45 (td, J=7.2, 4.4Hz, 1H, ArH), 7.29 (ddd, J=48.4, 8.7, 2.6Hz, 2H, ArH), 4.72 (s, 3H, NCH3), 3.89 (s, 3H, OCH3). 13 C NMR (100MHz, DMSO-d6) δ156.3142.5, 140.7, 138.1, 136.1, 134.5, 131.1, 128.9 ,127.8,126.8,124.9,121.9,119.9,111.4,103.5,56.0,34.2.HRMS(ESI)calcd for C 20 H 16 N3O + [M+H] + 314.1288, found 314.1293.

[0027] I-15: Purple-red solid, melting point: 180-181℃. 1 H NMR (400MHz, DMSO-d6) δ8.88 (dd, J=65.7, 5.7Hz, 1H, ArH), 8.63 (s, 1H, ArH), 8.55 (t, J=8.6Hz, 1H, ArH), 7.88 (dd, J=22.3, 14.7Hz, 2H, ArH), 7.79- 7.63 (m, 2H, ArH), 7.51 (dd, J=14.1, 7.0Hz, 1H, ArH), 7.25 (dd, J=15.1, 7.4Hz, 3H, ArH), 7.19 (s, 3H, ArH), 6.98 (s, 2H, NCH2Ar), 3.92 (s, 3H, OCH3). 13 C NMR (100MHz, DMSO-d6) δ157.6, 156.6, 151.2, 150.4, 142.8, 142.0, 138.7, 135.2, 131.5, 129.1, 127.5 ,125.5,123.1,122.2,121.3,120.2,119.2,113.3,112.3,103.7,102.4,56.1,49.2.HRMS(ESI)calcd for C 26 H 20 N3O + [M+H] + 390.1601, found 390.1607.

[0028] Example 2: Determination of activity against tobacco mosaic virus, the determination procedure is as follows:

[0029] 1. Virus purification and concentration determination:

[0030] Virus purification and concentration determination were performed in accordance with the SOP (Standard Operating Procedure) for tobacco mosaic virus prepared by the Bioassay Laboratory of the Institute of Elementochemistry, Nankai University. The crude virus extract was centrifuged twice with polyethylene glycol, and the concentration was determined. It was then stored at 4°C for later use.

[0031] 2. Preparation of compound solutions:

[0032] After weighing, the original drug was dissolved in DMF to obtain 1×10 5 The stock solution was diluted to μg / mL with an aqueous solution containing 1‰ Tween 80 to the required concentration; the ribavirin preparation was diluted directly with water.

[0033] 3. In vivo protection:

[0034] Select uniformly growing *Nicotiana santalinus* plants at the 3-5 leaf stage and spray the entire plant with the pesticide. Each treatment was repeated three times, with a 1‰ Tween 80 aqueous solution as a control. 24 hours later, sprinkle emery (500 mesh) on the leaves. Using a brush dipped in the virus solution, gently rub the entire leaf surface along the veins twice, supporting the underside of the leaf with the palm of your hand. The virus concentration was 10 μg / mL. Rinse with running water after inoculation. Record the number of lesions after 3 days and calculate the control efficacy.

[0035] 4. In vivo therapeutic effects:

[0036] Select uniformly growing *Nicotiana sambac* plants at the 3-5 leaf stage. Inoculate the entire leaf with the virus using a paintbrush at a concentration of 10 μg / mL. Rinse with running water after inoculation. After the leaves have dried, spray the entire plant with the pesticide. Each treatment is replicated three times, with a 1‰ Tween 80 aqueous solution as a control. Record the number of lesions after 3 days and calculate the control efficacy.

[0037] 5. In vivo passivation effect:

[0038] Select uniformly growing 3-5 leaf stage *Nicotiana sambac* plants. Mix the pesticide with an equal volume of virus sap, inactivate for 30 minutes, and then inoculate by friction. The virus concentration is 20 μg / mL. Rinse immediately with running water after inoculation. Repeat 3 times. Include a 1‰ Tween 80 aqueous solution as a control. Count the number of lesions after 3 days and calculate the results.

[0039] Inhibition rate (%) = [(Number of control necrotic spots - Number of treated necrotic spots) / Number of control necrotic spots] × 100%

[0040] First, the in vivo inactivation activity against tobacco mosaic virus (TMV) of all compounds was tested at a treatment dose of 500 μg / mL. Compounds with a relative inhibition rate greater than 40% were further tested for in vivo therapeutic and protective activity at a treatment dose of 500 μg / mL, and for in vivo inactivation, therapeutic, and protective activity against TMV at a treatment dose of 100 μg / mL. The positive control was the commercially available antiviral agent ribavirin.

[0041] Table 1. Results of anti-tobacco mosaic virus (TMV) activity tests of Iheyamine A and its derivatives I-1 to I-15.

[0042]

[0043] As can be seen from the data in Table 1, iheyamine A and its derivatives I-1 to I-15 all exhibited good anti-TMV activity. Among them, at 500 μg / mL, the activity of 6 compounds was comparable to that of ningnanmycin, which has great development value.

[0044] Example 3: Antibacterial activity test, the determination procedure is as follows:

[0045] In vitro sterilization test, bacterial growth rate determination method (plate method):

[0046] A certain amount of the drug was dissolved in an appropriate amount of acetone, and then diluted to the required concentration with an aqueous solution containing 200 μg / mL emulsifier. 1 mL of the drug solution was then injected into each petri dish, followed by 9 mL of culture medium. After mixing, a 50 μg / mL drug-containing plate was prepared. A plate with 1 mL of sterile water added served as a blank control. Mycelial discs were cut along the outer edge of the hyphae using a 4 mm diameter punch and transferred to the drug-containing plate. Each treatment was repeated three times. The petri dishes were incubated in a constant temperature incubator at 24 ± 1℃. After 48 hours, the diameter of the mycelial discs in each treatment was observed, the average value was calculated, and the relative inhibition rate was calculated by comparing it with the blank control.

[0047]

[0048] Table 2 Results of in vitro bactericidal activity tests of Iheyamine A and its derivatives I-1 to I-15:

[0049]

[0050] Iheyamine A and its derivatives exhibited broad-spectrum inhibitory activity against eight tested bacteria at a test concentration of 50 μg / mL. Compound I-8 showed an 81% inhibition rate against apple ring rot. Some compounds showed superior inhibitory activity against certain strains compared to the commercially available chlorothalonil, demonstrating significant development potential.

Claims

1. Application of natural product iheyamine A and its derivatives I-1 to I-15 in the control of tobacco mosaic virus disease.

2. The application of iheyamine A and its derivatives I-1 to I-15 in the prevention and control of plant pathogenic diseases as described in claim 1, characterized in that... The plant pathogens mentioned are Fusarium wilt of cucumber, brown spot of peanut, ring spot of apple, sheath blight of wheat, early blight of tomato, rice blast of rice, Phytophthora of pepper, or sclerotinia sclerotiorum of rapeseed.

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