阅读说明：本技术 白叶藤碱d环衍生物在制备防治或抗植物病害的药物中的应用 (Application of cryptolepine D-ring derivative in preparation of medicines for preventing and treating or resisting plant diseases ) 是由 刘映前 陈永甲 刘华 彭静文 赵中敏 朱佳凯 周锐 燕银芳 王仁轩 尹晓丹 孙钰 于 2019-09-03 设计创作，主要内容包括：本发明公开了一种D环修饰的白叶藤碱衍生物CL-1～CL-9中任一化合物在防治农业植物病害中的应用。活性测试结果表明,本发明所述化合物对油菜菌核病菌,番茄灰霉病菌,小麦赤霉病菌,立枯丝核病菌四种植物病害表现出潜在的抑制活性,尤其是对番茄灰霉病菌具有优异的抑菌效果,可望作为一种新型的特异性杀菌剂来开发。(The invention discloses application of any compound of D-ring modified cryptolepine derivatives CL-1-CL-9 in prevention and treatment of agricultural plant diseases. The activity test result shows that the compound of the invention has potential inhibitory activity on four plant diseases of sclerotinia sclerotiorum, botrytis cinerea, fusarium graminearum and rhizoctonia solani, especially has excellent bacteriostatic effect on botrytis cinerea, and is expected to be developed as a novel specific bactericide.)

1. The invention relates to application of a D-ring modified cryptolepine derivative in a medicine for preventing and treating agricultural diseases, and belongs to new application of the cryptolepine derivative.

2. The D-ring modified cryptolepine derivatives CL-1 to CL-9 according to claim 1 have the following chemical structural features:

3. the use of any one of the compounds CL-1 to CL-9, which are D-ring modified cryptolepine derivatives according to claim 2, in the preparation of a medicament for the control or resistance of diseases caused by sclerotinia sclerotiorum.

4. The use of any one of the compounds CL-1 to CL-9 of the D-ring modified cryptolepine derivative according to claim 2 in the preparation of a medicament for controlling or combating a disease caused by Botrytis cinerea.

5. The use of any one of the compounds CL-1 to L-9, which are D-ring modified cryptolepine derivatives according to claim 2, in the preparation of a medicament for preventing or treating diseases caused by fusarium graminearum.

6. The use of any one of the compounds CL-1 to CL-9 of the D-ring modified cryptolepine derivative according to claim 2 in the preparation of a medicament for controlling or combating a disease caused by Rhizoctonia solani.

Technical Field

The invention belongs to the field of natural medicinal chemistry, discloses a new application of a D-ring modified cryptolepine derivative, and particularly relates to an application of the D-ring modified cryptolepine derivative in preventing and treating sclerotinia sclerotiorum, botrytis cinerea, fusarium graminearum and rhizoctonia solani.

Background

Plant diseases are common and seriously harmful in agricultural production, are one of natural disasters affecting agricultural production, bring huge economic loss to agricultural production every year, and statistically cause 13-20% of losses of global crop yield caused by plant diseases. As one of the major agricultural countries in the world, plant diseases have great threat to agricultural development and grain safety, and grain loss caused by the plant diseases exceeds 11% of yield every year. For example, the potato production area in China is affected by late blight, the yield is reduced by 10 to 40 percent every year, and the wheat is affected by gibberellic disease, and the yield is reduced by 10 to 30 percent every year. The economic loss can be reduced by about 300 million yuan per year by using the pesticide. Although the use of chemical pesticides can effectively improve the crop yield and ensure the agricultural benefit and the economic benefit, the problems of pesticide residue, increased drug resistance, frequent phytotoxicity, environmental pollution and the like caused by the use of chemical pesticides seriously threaten the human health and the ecological environment. Therefore, the method conforms to social development and develops a new green, economic, efficient and safe pesticide product, thereby promoting the development of modern pesticides and making a contribution to reducing the loss of plant diseases and ensuring the production safety of crops.

The natural product has the characteristics of structural diversity, biodegradability, environmental friendliness, target specificity, low toxicity and the like, meets the requirement of ideal pesticides, is expected to replace chemical pesticides, and becomes one of the research hotspots in the pesticide field. The grapevine is a natural alkaloid separated from western nontraditional medicinal plant red blood white vine (Cryptolepis sanguinolenta), and the known biological activity is mainly shown in aspects of antimalaria, hyperglycemia, hypertension, tumor and the like ((1) J.Med.chem,1998, 41(15): 2754-. Preliminary studies of subject groups (CN 109090123A; CN109717198A) found that the neocryptophylline derivatives exhibit excellent inhibitory activity on the control of Pyricularia oryzae, Sclerotinia sclerotiorum and Botrytis cinerea, and in the further activity-oriented optimization process of similar structures, we found that the neocryptophylline derivatives also exhibit potential inhibitory activity on various fungi, particularly exhibit significant inhibitory activity on Botrytis cinerea. Therefore, a series of D-ring modified cryptolepine derivatives are designed and synthesized by taking cryptolepine as a lead model and further through a total synthesis strategy, and the derivatives are found to show different degrees of inhibition effects on various pathogenic bacteria, especially have excellent bactericidal effects on botrytis cinerea and can be used for developing a novel bactericide.

Disclosure of Invention

The invention aims to provide a new application of natural botanical fungicide cryptolepine for agricultural production aiming at the defects in the prior art, namely the application of cryptolepine alkaloid as a biological pesticide in preventing and treating agricultural plant diseases such as sclerotinia sclerotiorum, rhizoctonia solani, botrytis cinerea, fusarium graminearum and the like.

In order to achieve the purpose, the invention provides the following technical method: a medicine for resisting sclerotinia sclerotiorum, rhizoctonia solani, botrytis cinerea and gibberella graminis contains a therapeutically effective amount of a compound represented by any one of CL-1-CL-9, namely a byttylline derivative represented by a chemical formula I.

The preparation method of the D-ring modified cryptolepine derivatives CL-1-CL-9 comprises the following steps:

the synthesis method of the cryptolepine derivative is shown in the embodiment, a pure product is obtained by separation of conventional methods such as silica gel column chromatography for many times, and the cryptolepine derivative CL-1-CL-9 of the claims is determined by spectrum technologies such as mass spectrum and nuclear magnetic resonance, and the structural formula of the cryptolepine derivative is shown in chemical formula 1. The results of indoor biological activity tests show that the cryptolepine derivative has potential inhibition effects on sclerotinia sclerotiorum, botrytis cinerea, rhizoctonia solani and fusarium graminearum, and especially has excellent inhibition effects on botrytis cinerea.

Detailed Description

The foregoing and other aspects of the present invention will become more apparent from the following detailed description, given by way of example only, for purposes of illustrating the invention. This is not to be construed as limiting the invention. The experimental procedures described in the following examples are conventional unless otherwise specified.

EXAMPLE 1 Synthesis of the target Compound CL-1

See literature methods for synthesis: org.Lett.2017,19(16):4275-4278, which comprises the following steps:

synthesis of intermediate 1: dissolving 5-fluoroindole (30mmol) in acetonitrile, adding 60% sodium hydride (42mmol) at 0 ℃, stirring at low temperature for 10 minutes, adding p-toluenesulfonyl chloride (33mmol), reacting at normal temperature for 4 hours, adding saturated ammonium chloride aqueous solution, stopping reaction, extracting with ethyl acetate, rinsing with brine, combining organic phases, drying with magnesium sulfate, concentrating under reduced pressure to obtain solid residue, and purifying by column chromatography with petroleum ether/ethyl acetate as eluent to obtain white solid.

Synthesis of intermediate 2: adding the intermediate 1(11mol) and water (110mol) into 110mL of acetone, adding NBS (12mmol) for reaction until the raw materials are reacted completely, monitoring by TLC, adding triethylamine (12mmol), stirring for 1 hour, and spin-drying the solvent to obtain a solid without purification.

Synthesis of intermediate 3: dissolving the intermediate 2(0.5mmol), N-methylaniline (0.55mmol) and triethylamine (1.0mmol) in ethyl acetate, heating and refluxing for 0.5-6 hours, after the reaction is finished, adding water, extracting with ethyl acetate, rinsing the organic layer with brine, combining the organic phases, drying with magnesium sulfate, concentrating under reduced pressure, dissolving in ethyl acetate (5mL), adding boron trifluoride diethyl etherate (2.5mmol), stirring at 50 ℃ for 3 hours, cooling to room temperature, adding a saturated sodium bicarbonate solution, extracting with ethyl acetate, rinsing the organic layer with brine, combining the organic phases, drying with magnesium sulfate, concentrating under reduced pressure to obtain a solid residue, using petroleum ether/ethyl acetate as an eluent, and purifying by column chromatography to obtain a solid.

Synthesis of intermediate 4: dropping phosphorus oxychloride (2mmol) into DMF (1mL) at-16 ℃ and stirring for 0.5 hour, adding the intermediate 3 into the reaction solution and stirring for 1 hour at normal temperature, then adding a saturated sodium bicarbonate solution into the reaction solution, extracting with ethyl acetate, rinsing an organic layer with the saturated sodium bicarbonate solution, combining organic phases, drying with magnesium sulfate, concentrating under reduced pressure to obtain a solid residue, and purifying by column chromatography with dichloromethane/methanol as an eluent to obtain a yellow solid product.

Synthesis of Sinomenine CL-1: dissolving dimethylamine hydrochloride (0.1mol) in DMF (1mL), adding the intermediate 4(0.05mol) into a reaction system, heating and refluxing for 1.5 hours, cooling after the reaction is finished, adding 5% sodium carbonate (5mL), stirring for 10 minutes at normal temperature, extracting for 3 times by ethyl acetate, rinsing by saturated sodium carbonate, combining organic phases, drying by magnesium sulfate, concentrating under reduced pressure to obtain a solid residue, and purifying by column chromatography by using dichloromethane/methanol as an eluent to obtain a purple solid product. Yield: 38 percent; a purple powdered solid;¹H NMR(400MHz,DMSO-d₆)δ:9.01(s,1H),8.53(d,J＝9.1Hz,1H),8.40(d,J＝ 8.3Hz,1H),8.29(d,J＝10.5,Hz,1H),7.93(t,J＝7.9Hz,1H),7.70(m,2H),7.48(t,1H),4.88(s, 3H)；m/z:C₁₆H₁₁FN₂：251.0[M+H]⁺。

example 2 Synthesis of target Compound CL-2

The experimental procedure was the same as in example 1 except that 5-chloroindole was used instead of 5-fluoroindole. Yield: 31 percent; a magenta powdered solid;¹H NMR (400MHz,DMSO-d₆)δ：9.05(s,1H),8.65–8.55(m,2H),8.43(d,J＝8.2Hz,1H),7.96(t,J＝8.7 Hz,1H),7.72(m,2H),7.54(dd,J＝9.1,2.1Hz,1H),4.92(s,3H)；MS-ESI m/z:C₁₆H₁₁ClN₂：267.0 [M+H]⁺。

example 3 Synthesis of the target Compound CL-3

The experimental procedure was as in example 1, substituting 5-bromoindole for 5-fluoroindole only. Yield: 32 percent; a magenta powdered solid;¹H NMR (400MHz,DMSO-d₆)δ:9.05(s,1H),8.70(s,1H),8.58(d,J＝9.0Hz,1H),8.44(d,J＝8.3Hz, 1H),8.02–7.87(m,1H),7.74(t,J＝7.5Hz,1H),7.69–7.60(m,2H),4.92(s,3H)；MS-ESI m/z: C₁₆H₁₁BrN₂：311.0[M+H]⁺。

example 4 Synthesis of target Compound CL-4

The experimental procedure was the same as in example 1, except that 6-fluoroindole was used instead of 5-fluoroindole. Yield: 35 percent; a violet-black powdery solid;¹H NMR (400MHz,DMSO-d₆)δ:8.89(s,1H),8.54–8.49(m,2H),8.36(d,J＝8.3,1H),7.93(t,J＝6.8Hz, 1H),7.71(t,J＝7.9Hz,1H),7.29(d,J＝11.01H),6.94(t,J＝9.1Hz,1H),4.84(s,3H)；MS-ESI m/z:C16H11FN2：251.0[M+H]+。

EXAMPLE 5 Synthesis of the target Compound CL-5

The experimental procedure was the same as in example 1, except that 6-chloroindole was used instead of 5-fluoroindole. Yield: 31 percent; a purple powdered solid;¹H NMR (400MHz,DMSO-d₆)δ：9.03(s,1H),8.65–8.51(m,2H),8.43(d,J＝8.2Hz,1H),8.06–7.90(m, 1H),7.71(dd,J＝13.9,8.2Hz,2H),7.52(dd,J＝9.1,2.2Hz,1H),4.92(s,3H)；MS-ESI m/z: C₁₆H₁₁ClN₂：267.0[M+H]⁺。

example 6 Synthesis of target Compound CL-6

The experimental procedure was as in example 1, substituting only 6-bromoindole for 5-fluoroindole. Yield: 33%; a purple powdered solid; (ii) a¹H NMR (400MHz,DMSO-d₆)δ：8.98(s,1H),8.53(dd,J＝10.8,9.0Hz,2H),8.44–8.35(m,1H),7.94 (m,1H),7.81–7.56(m,2H),7.02(dd,J＝8.9,2.0Hz,1H),4.91(s,3H)；MS-ESI m/z: C₁₆H₁₁BrN₂：311.0[M+H]⁺。

Example 7 Synthesis of target Compound CL-7

The experimental procedure was the same as in example 1, except that 5-methylindole was used instead of 5-fluoroindole. Yield: 34 percent; a violet-black powdery solid;¹H NMR(400MHz,DMSO-d₆)δ：8.90(s,1H),8.50(d,J＝9.0Hz,1H),8.38(d,J＝8.2Hz,1H), 8.25(s,1H),7.91(t,J＝7.9Hz,1H),7.69(t,J＝7.5Hz,1H),7.58(d,J＝8.7Hz,1H),7.40(d,J＝ 8.7Hz,1H),4.88(s,3H),2.48(s,3H)；MS-ESI m/z:C₁₇H₁₄N₂：247.1[M+H]⁺。

example 8 Synthesis of the target Compound CL-8

The experimental procedure was the same as in example 1, except that 6-methylindole was used instead of 5-fluoroindole. Yield: 32 percent; a violet-black powdery solid;¹H NMR(400MHz,DMSO-d₆)δ：8.94(s,1H),8.52(d,J＝8.5Hz,1H),8.35(s,1H),8.31(d,J＝ 8.4Hz,1H),7.67(d,J＝8.4Hz,1H),7.58(d,J＝1.3Hz,1H),7.56(t,J＝1.6Hz,1H),7.09(m, 1H),4.90(s,3H),2.69(s,3H)；MS-ESI m/z:C₁₇H₁₄N₂:247.0[M+H]⁺

example 9 Synthesis of target Compound CL-9

The experimental procedure was as in example 1, except that 5-methoxyindole was used instead of 5-fluoroindole. Yield: 30 percent; a violet-black powdery solid;¹H NMR(400MHz,DMSO-d₆)δ:8.98(s,1H),8.54(d,J＝9.0Hz,1H),8.41(d,J＝8.2Hz,1H), 7.95(t,J＝7.9Hz,1H),7.88(d,J＝2.5Hz,1H),7.73–7.64(m,2H),7.35(dd,J＝2.5Hz,1H), 4.94(s,3H),3.93(s,3H)；MS-ESI m/z:C₁₇H₁₄N₂O：263.1[M+H]⁺

EXAMPLE 10 test method and results of anti-phytopathogenic fungi Activity of Compound CL-1-CL-9

The antibacterial activity assay of the present invention was performed using a potato dextrose agar medium (PDA medium). The preparation method comprises the following steps: firstly, cleaning and peeling potatoes, weighing 200g of potatoes, cutting the potatoes into small pieces, adding water, boiling the potatoes thoroughly (boiling for 20-30 minutes, the potato pieces can be punctured by a glass rod), filtering the potatoes by eight layers of gauze, heating the potatoes, adding 15g of agar, continuously heating, stirring the mixture evenly, adding glucose after the agar is dissolved, stirring the mixture evenly, slightly cooling the mixture, then supplementing the water to 1000 ml, subpackaging the mixture in conical bottles, plugging and binding the conical bottles, and sterilizing the mixture for 2 hours at 115 ℃ for later use. Respectively dissolving the compounds L01-43 by DMSO, adding into a culture medium, uniformly mixing to make the concentration of the compounds in the culture medium respectively 50 μ g/mL, taking DMSO with equal concentration as a blank control, and taking the above-mentioned azoxystrobin as a positive control. And (3) pouring the plates, cooling, inoculating bacteria respectively, culturing in an incubator at 23 ℃, and determining the bacteriostasis rate of each compound by taking blank control hypha to overgrow the culture dish as a limit. All experiments were performed in triplicate or in triplicate. The calculation of the bacteriostasis rate is carried out according to the following calculation formula:

the test results of the anti-plant pathogenic bacteria activity of the compounds CL-1 to CL-9 are shown in Table 1

As shown in the results of the antibacterial activity determination in Table 1, the prepared byssurine derivatives CL-1-CL-9 show inhibitory activities of different degrees to four plant pathogenic bacteria at concentrations of 50 ppm and 25ppm, and the antibacterial activity of partial compounds is superior to that of azoxystrobin, especially superior to that of botrytis cinerea, and the compounds are expected to be developed into novel bactericides, so that the compounds can be used for preparing and controlling the plant pathogenic bacteria.