Naphthoquinone derivative as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a naphthoquinone derivative and a preparation method and application thereof. A series of novel naphthoquinone compounds are found from Fusarium sp.L-3 secondary metabolites for the first time, and the influence of treatment of the naphthoquinone derivatives on the growth of purslane roots, the influence of treatment of the naphthoquinone derivatives on the integrity of purslane root cell membranes, the weeding spectrum, the action mechanism and the toxicity of the naphthoquinone derivatives are studied. Results show that the naphthoquinone derivative can significantly inhibit growth of portulaca oleracea roots, destroy plasma membrane integrity and cause root cell death, plays a role mainly through a photosynthetic transmission chain plasma quinone competitive site, is safe and non-toxic, has the advantages of wide herbicide controlling spectrum, significant effect and unobvious toxicity, and meanwhile, has a simple structure, is easy for industrial production and has a broad application prospect. The bacillus subtilis has the potential of being developed into biological herbicides.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, and particularly relates to a naphthoquinone derivative, a preparation method and application thereof. BACKGROUND

[0002] Weeds and plant diseases have been two major threats to global agriculture. It is reported that the loss caused by weeds accounts for about 34% of the global crop yield loss, and the loss caused by plant pathogens accounts for 16%. Since human beings began to engage in agricultural production, controlling weeds has been an important task. With the widespread use of herbicides, the rapidly developing herbicide-resistant weeds in farmland pose a serious threat to the control of weeds in farmland, the ecological environment and food safety. Therefore, developing herbicides with more chemical structures and new action mechanisms is the fundamental way to control herbicide-resistant weeds.

[0003] Microbial herbicides are a class of pesticides synthesized by artificial modification with microorganisms or their metabolites as core components, which belong to an important branch of biological pesticides. The types thereof include living pathogenic microbial preparations (including fungi, bacteria, actinomycetes and viruses, etc.) and microbial metabolite herbicides (such as polypeptide, terpenoid phytotoxin), which have the characteristics of environmental friendliness, safety, etc.

[0004] Among them, fungi can produce a variety of bioactive secondary metabolites. Fungal secondary metabolites with herbicidal activity can cause diseases or interfere with the physiological processes of host plants, and are a more environmentally friendly alternative method for weed control. SUMMARY

[0005] Therefore, the present application aims to solve the technical problem and provide a naphthoquinone derivative, a preparation method and application thereof. The naphthoquinone derivative provided has application prospects as a biological source herbicide.

[0006] To achieve the above-mentioned purpose, the present application provides a naphthoquinone derivative having the structure shown in formula I, formula II or formula III, or a stereoisomer thereof:

[0007]

[0008] Formula I Formula II Formula III

[0009] In Formula I, R1-R3 are independently preferably hydrogen, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy; more preferably hydrogen, hydroxyl, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy; further preferably hydrogen, hydroxyl, substituted or unsubstituted C1-C3 alkyl, substituted or unsubstituted C1-C3 alkoxy; in some embodiments, the alkyl is selected from methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, or t-butyl. In some embodiments, the alkoxy is selected from methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, or t-butoxy. The substitution is preferably by one or more of hydroxyl, amino.

[0010] In some embodiments, R1-R3 are independently preferably any of hydrogen, hydroxyl, methyl, -CH2CH(OH)CH3.

[0011] In another aspect of the application, any adjacent pair of R1-R3 and the carbon atoms to which they are attached together form a five- to six-membered saturated or unsaturated heterocyclyl group. The carbon atoms refer to the carbon atoms on the phenyl ring to which R1-R3 are attached.

[0012] The heteroatoms in the heterocyclyl group include, but are not limited to, one or more of oxygen atoms, nitrogen atoms.

[0013] The number of heteroatoms in the heterocyclyl group can be 1 to 3, for example, 1, 2, or 3.

[0014] In some embodiments, the five- to six-membered saturated or unsaturated heterocyclyl group has any of the following structures:

[0015]

[0016] The above groups are fused to the parent ring through any two adjacent carbon atoms. The position of fusion can be at the position of R1, R2, or the position of R2, R3.

[0017] Any one or more of the hydrogen atoms in the five- to six-membered saturated or unsaturated heterocyclyl group can optionally be substituted by one or more of hydroxyl, alkyl, alkoxy, preferably by one or more of hydroxyl, C1-C4 alkyl, C1-C4 alkoxy, in some embodiments, any one or more of the hydrogen atoms in the five- to six-membered saturated or unsaturated heterocyclyl group can optionally be substituted by one or more of hydroxyl, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, t-butyl, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, or t-butoxy.

[0018] In Formula II, R4-R5 are preferably substituted or unsubstituted alkyl groups, substituted or unsubstituted alkoxy groups, or -CH2COCH3; more preferably, they are substituted or unsubstituted C1-C4 alkyl groups, substituted or unsubstituted C1-C4 alkoxy groups, or -CH2COCH3. In some embodiments, the alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl. In some embodiments, the alkoxy group is selected from methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, or tert-butoxy. The substitution is preferably one or more of hydroxyl and amino groups.

[0019] In some specific embodiments, R4-R5 are preferably any of the following groups: methyl, hydroxymethyl, -CH2COCH3.

[0020] In another embodiment of the invention, R4 and R5, along with the carbon atoms they are attached to, together form a five- to six-membered saturated or unsaturated heterocyclic group. The carbon atom refers to the carbon atom on the benzene ring substituted by R4 and R5.

[0021] The heteroatoms in the heterocyclic group include, but are not limited to, one or more of oxygen atoms and nitrogen atoms.

[0022] The number of heteroatoms in the heterocyclic group can be 1 to 3, for example, 1, 2 or 3.

[0023] In some specific embodiments, the five- to six-membered saturated or unsaturated heterocyclic group has any of the following structures:

[0024] .

[0025] Preferred options are:

[0026] .

[0027] The above groups are fused with the parent ring through any two adjacent carbon atoms.

[0028] Any one or more hydrogen atoms in the five- to six-membered saturated or unsaturated heterocyclic group may optionally be substituted with hydroxyl, alkyl, or alkoxy groups; preferably substituted with hydroxyl, C1-C4 alkyl, or C1-C4 alkoxy groups. In some specific embodiments, any one or more hydrogen atoms in the five- to six-membered saturated or unsaturated heterocyclic group may optionally be substituted with one or more of hydroxyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, or tert-butoxy groups.

[0029] In Formula II, R6 is preferably hydrogen, hydroxyl, or a substituted or unsubstituted alkyl group; more preferably hydrogen, or a substituted or unsubstituted C1-C4 alkyl group; and even more preferably hydrogen, or a substituted or unsubstituted methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl group. The substitution is preferably one or more of hydroxyl and amino groups.

[0030] In some specific embodiments of the present invention, R6 is hydrogen or hydroxyl.

[0031] In Formula III, R7 is preferably a substituted or unsubstituted saturated alkyl or unsaturated alkyl group; more preferably, it is a substituted or unsubstituted C1-C4 saturated alkyl or unsaturated alkyl group, wherein the unsaturated alkyl group may be alkenyl or ynyl. Further preferably, it is substituted or unsubstituted methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or tert-butyl. The substitution is preferably one or more of hydroxyl or amino groups.

[0032] In some specific embodiments of the present invention, R7 is 2-propenyl or 1-hydroxyethyl.

[0033] In some specific embodiments of the present invention, the naphthoquinone derivatives have any one of the following structures:

[0034] .

[0035] The preferred compounds are compounds 1, 2, 3, 7, 9, and 10 mentioned above.

[0036] This invention provides a method for preparing the above-mentioned naphthoquinone derivatives, comprising the following steps:

[0037] The Fusarium sp. L-3 strain was fermented, soaked in ethyl acetate, evaporated under reduced pressure, dehydrated and deoiled, and subjected to forward / reverse / gel column chromatography for semi-preparative processing.

[0038] The Fusarium sp. L-3 strain used in this invention has the Genbank number PQ456123 and is deposited at the Institute of Tropical Biotechnology, Chinese Academy of Tropical Agricultural Sciences.

[0039] In some specific embodiments, the preparation method of the naphthoquinone derivative includes the following steps:

[0040] S1) The fermentation product of Fusarium sp. L-3 was extracted with ethyl acetate and the oil was removed to obtain the total extract;

[0041] S2) The total extract was separated by normal-phase silica gel chromatography to obtain components Fr.1~Fr.5;

[0042] S3) Take component Fr.3 and separate to obtain components Fr.3.1~Fr.3.10;

[0043] Take component Fr.3.10 and separate it to obtain compound 5;

[0044] Take component Fr.3.9 and separate it to obtain compound 6;

[0045] Fr.3.7 was taken and compounds 3 and 10 were separated.

[0046] Take component Fr.3.6 and separate it to obtain compound 7;

[0047] Take component Fr.3.5 and separate components Fr.3.5.1-Fr.3.5.6, of which Fr.3.5.3 is compound 14; take component Fr.3.5.4 and separate compound 2 and compound 1.

[0048] Take component Fr.3.4 and separate it to obtain compound 11;

[0049] Take component Fr.3.3 and separate it to obtain 12;

[0050] Take component Fr.1 and filter to obtain compound 9;

[0051] Take component Fr.4 and separate it to obtain compound 4;

[0052] Take component Fr.2 and separate to obtain components Fr.2.1~Fr.2.9;

[0053] Fr.2.5 was taken and compounds 13 and 8 were obtained by separation.

[0054] The present invention does not impose specific limitations on the method for obtaining the Fusarium sp. L-3 fermentation product, and any fermentation method well known to those skilled in the art can be used. In some specific embodiments of the present invention, the Fusarium sp. L-3 fermentation product is obtained according to the following method:

[0055] A) The activated mycelium of Fusarium sp. L-3 was inoculated into a fungal culture medium for cultivation to obtain the seed culture of the strain;

[0056] B) Inoculate the seed culture of the above strains into the culture medium and let it stand for incubation.

[0057] The preferred culture temperature is 28~30℃, and the preferred culture time is 2~4 days.

[0058] The culture medium includes, but is not limited to, rice solid fermentation culture medium.

[0059] The preferred temperature for static incubation is 28-30℃, and the preferred time is 25-35 days.

[0060] The present invention does not impose any special limitation on the degreasing treatment in step S1), and can use any degreasing method well known to those skilled in the art. In some specific embodiments of the present invention, methanol and petroleum ether are used for degreasing, and the methanol phase is taken for subsequent separation and purification.

[0061] In step S2), the mobile phase used for normal-phase silica gel chromatography is preferably a petroleum ether and ethyl acetate system. The preferred ratios of petroleum ether and ethyl acetate are 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, and 0:1 (V:V), and fractional collection is performed to obtain components Fr.1 to Fr.5.

[0062] The separation in step S3) is preferably performed using Rp-C 18 The gel is graded.

[0063] The preferred method for separating component Fr.3.10 is semi-preparative HPLC purification. Elution is preferably performed using a methanol / water mixed solvent, with a preferred volume ratio of methanol to water of 80:20.

[0064] The preferred method for separating component Fr.3.9 is semi-preparative HPLC purification. Elution is preferably performed using a methanol / water mixed solvent, with a preferred volume ratio of methanol to water of 80:20.

[0065] The preferred method for separating component Fr.3.7 is semi-preparative HPLC purification. Elution is preferably performed using a methanol / water mixed solvent, with a preferred volume ratio of methanol to water of 60:40.

[0066] The preferred method for separating component Fr.3.6 is semi-preparative HPLC purification. Elution is preferably performed using a methanol / water mixed solvent, with a preferred volume ratio of methanol to water of 60:40.

[0067] The preferred method for separating the component Fr.3.5 is semi-preparative HPLC purification.

[0068] The preferred method for separating component Fr.3.5.4 is semi-preparative HPLC purification, and the preferred elution solvent is a mixture of acetonitrile and water, wherein the volume ratio of acetonitrile to water is preferably 1:1.

[0069] The preferred method for separating component Fr.3.4 is semi-preparative HPLC purification. Elution is preferably performed using a methanol / water mixed solvent, with a preferred volume ratio of methanol to water of 40:60.

[0070] The preferred method for separating component Fr.3.3 is semi-preparative HPLC purification. Elution is preferably performed using a methanol / water mixed solvent, with a preferred volume ratio of methanol to water of 35:65.

[0071] The preferred method for separating component Fr.4 is semi-preparative HPLC purification. Elution is preferably performed using a methanol / water mixed solvent, with a preferred volume ratio of methanol to water of 75:25.

[0072] The preferred method for separating the component Fr.2 is reversed-phase column chromatography.

[0073] The preferred method for separating component Fr.2.5 is HPLC purification. Elution with a methanol / water mixed solvent is preferred, with a preferred volume ratio of methanol to water of 70:30 or 65:35.

[0074] The structures of the compounds obtained above were determined by proton NMR, carbon NMR, ultraviolet and infrared spectroscopy.

[0075] The present invention also provides the application of the above-mentioned naphthoquinone derivatives or naphthoquinone derivatives prepared by the above preparation method in the preparation of herbicides.

[0076] Experimental results showed that the above compounds could inhibit the growth of purslane. Different substituents of naphthoquinone derivatives exhibited varying inhibitory effects. Compound 7, in particular, showed significant inhibitory activity, affecting the IC50 of radicle, hypocotyl, and fresh weight. 50 The concentrations were 3.26, 8.54, and 0.90 μg / mL, respectively.

[0077] The aforementioned compounds inhibit the growth of purslane roots, disrupt the integrity of root cell membranes, and cause root cell death or damage. They inhibit electron transport activity by competing with photosynthetic chain quinones and binding to the D1 protein in photosystem II.

[0078] To further understand this invention, this experiment measured the effects of compound 7 on the root length and fresh weight of purslane seedlings. Evans Blue, SEM, and TEM were used to detect the degree of plasma membrane damage and root cell death in purslane roots after treatment with compound 7. This was used to evaluate the activity of naphthoquinone derivatives in inhibiting the growth of purslane roots, thus providing a theoretical basis for the research of bio-derived herbicides.

[0079] This invention is the first to discover the mechanism of action of naphthoquinone derivatives in biological weed control: compound 7 can significantly reduce the efficiency of photosynthetic electron transport, and its site of action is on the thylakoid membrane, where it binds to the D1 protein of photosystem II to inhibit electron transport activity.

[0080] To further understand this invention, this experiment conducted transcriptional analysis on the roots of purslane after treatment with compound 7, and verified the results using QPCR, thereby evaluating the herbicidal mechanism of naphthoquinone derivatives.

[0081] This invention investigates the herbicidal mechanism, herbicidal activity, and herbicidal spectrum of naphthoquinone derivatives. It is the first time that naphthoquinone derivatives have been discovered to act as photosynthesis inhibitors and have the potential to be developed into biological herbicides, which can be used to prepare biological herbicides.

[0082] Furthermore, the herbicides are applicable to one or more of the following weeds, including but not limited to: purslane, foxtail grass, crabgrass, beggar-ticks, reverse-branch amaranth, and plantain.

[0083] Currently commercially available herbicides such as paraquat and bromobenzonitrile are highly toxic, classifying them as moderately toxic. Naphthoquinone derivatives, on the other hand, offer advantages such as low toxicity, rapid action, low dosage, and minimal environmental pollution, making them highly effective bio-based herbicides. Furthermore, their simple structure facilitates in-depth structural optimization, modification, and artificial synthesis, offering advantages such as simplicity and reduced costs in industrialization processes.

[0084] Based on this, the present invention also provides a herbicide comprising the above-mentioned naphthoquinone derivatives or naphthoquinone derivatives prepared by the above-mentioned preparation method and adjuvants.

[0085] The present invention does not limit the type of adjuvants. Those skilled in the art can select agriculturally acceptable adjuvants according to the formulation, including but not limited to one or more of surfactants, antifreeze agents, and viscosity modifiers.

[0086] The present invention also provides a weed control method, comprising the following steps: contacting the seeds, germs and / or leaves of weeds with the above-mentioned naphthoquinone derivatives or naphthoquinone derivatives prepared by the above-mentioned preparation method or the above-mentioned herbicides.

[0087] In some specific implementation plans, soil spraying can be used for weed control.

[0088] Compared with the prior art, the present invention provides a naphthoquinone derivative having the structure shown in Formula I, Formula II or Formula III, or a stereoisomer thereof.

[0089] This invention marks the first discovery of a series of novel naphthoquinone compounds from the secondary metabolites of Fusarium sp. L-3. The effects of these naphthoquinone derivatives on the growth of purslane roots, their influence on cell membrane integrity, their herbicidal spectrum, mechanism of action, and toxicity were investigated. Results show that these naphthoquinone derivatives significantly inhibit purslane root growth, disrupt plasma membrane integrity, and cause root cell death. They primarily exert their effects by competing with photosynthetic chain naphthoquinones for sites. They are safe and non-toxic, possessing a broad herbicidal spectrum, significant efficacy, and minimal toxicity. Furthermore, due to their simple structure and ease of industrial production, they have the potential to be developed into biological herbicides. Attached Figure Description

[0090] Figure 1 For 5-dehydroxy-7-demethylfusarium oleoresin 1 H NMR spectrum;

[0091] Figure 2 For 5-dehydroxy-7-demethylfusarium oleoresin 13 C NMR spectrum;

[0092] Figure 3 HMBC of 5-dehydroxy-7-demethylfusarium oleoresin and 1 H- 1 H COSY;

[0093] Figure 4 The HRESIMS spectrum of 5-dehydroxy-7-demethylfusarium oleoresin;

[0094] Figure 5 The UV spectrum of 5-dehydroxy-7-demethylfusarium ol;

[0095] Figure 6 The IR spectrum of 5-dehydroxy-7-demethylfusarium oleoresin;

[0096] Figure 7 The inhibitory effect of naphthoquinone derivatives on purslane seedlings;

[0097] Figure 8 Growth of other weeds treated with dehydrated fusarium oxychloride;

[0098] Figure 9 Evans Blue staining was used to detect the damage to the cell membrane of purslane root cells caused by dehydrated fusarium oxysporum treatment.

[0099] Figure 10 The effect of dehydrated fusarium oxychloride treatment on root tissue damage in purslane;

[0100] Figure 11 The effect of dehydrated fusarium oxychloride treatment on the transcriptome of purslane roots. Detailed Implementation

[0101] To further illustrate the present invention, a detailed description is provided below with reference to embodiments. However, it should be understood that these descriptions are merely for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims.

[0102] There are no particular restrictions on the source of any raw materials used in this invention; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0103] Example 1

[0104] Isolation and purification of naphthoquinone derivatives

[0105] Prepare seed culture of Fusarium sp. L-3 strain, inoculate it into rice solid fermentation medium, and incubate statically at 28 ℃ for 30 days. Then, extract all fermentation products three times with ethyl acetate to obtain ethyl acetate extract; after degreasing the ethyl acetate extract, obtain total extract.

[0106] The total extract of Fusarium sp. L-3 was separated by normal-phase silica gel column chromatography using a gradient elution system of petroleum ether and ethyl acetate at volume ratios of 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, and 0:1 (V:V). The fractions were collected, analyzed by thin-layer chromatography and high-performance liquid chromatography, and then combined to obtain five fractions, Fr.1 to Fr.5.

[0107] Fr.3 uses Rp-C 18 The gel was fractionated to obtain fractions Fr.3.1–Fr.3.10. Fr.3.10 was purified by semi-preparative HPLC, eluting with methanol / water (80:20, v / v) to give compound 5. Compound 6 was obtained by semi-preparative HPLC (80% methanol / water) from Fr.3.9. Compounds 3 and 10 were separated by semi-preparative HPLC (60% methanol / water) from Fr.3.7. Compound 7 was obtained by 60% methanol / water from Fr.3.6. Compound 14 and the major fraction Fr.3.5.4 were separated from Fr.3.5, the latter being used by semi-preparative HPLC (50% MeCN / water) to obtain compounds 2 and 1. Compound 11 was obtained by semi-preparative HPLC (40% methanol / water) from Fr.3.4. Compound 12 was obtained by semi-preparative HPLC (35% methanol / water) from Fr.3.3. Compound 9 was obtained by filtration through filter paper from Fr.1. Fr.4 was further prepared by semi-preparative HPLC using methanol / water (75:25, v / v) to obtain compound 4. Fr.2 was prepared by reverse column chromatography to obtain fractions Fr.2.1 to Fr.2.9, and Fr.2.5 was prepared by semi-preparative HPLC to obtain compounds 13 (70% methanol / water) and 8 (65% methanol / water).

[0108] Example 2

[0109] Structural identification of compound 5-dehydroxy-7-demethylfusarium alcohol

[0110] The structures of all isolated compounds, 5-dehydroxy-7-demethylfusarium alcohol, were identified using a combination of modern spectroscopic techniques, including nuclear magnetic resonance (NMR), infrared spectroscopy (IR), and mass spectrometry, along with chemical methods. Compound 5-dehydroxy-7-demethylfusarium alcohol was identified as a novel naphthoquinone compound.

[0111] 5-Dehydroxy-7-demethylfusarium oleoresinol, a yellow powder, has a molecular weight of 285.0695 [M + Na] as determined by HRESIMS analysis. + , combined 13 C NMR, 1 Analysis by 1H NMR and MS determined its molecular formula to be C 14 H 14 O5 has an unsaturation degree of 8. 1 The 1H NMR spectrum shows that one methoxy (δ) H 3.88), one methyl group (δ) H 1.08), one methylene (δ H 2.74 and 2.68), one phenolic hydroxyl group (δ H 12.19), one hydroxyl group (δ) H 4.66), three olefin protons (δ) H The presence of signals such as 7.43, 7.18 and 6.32. 13 C10 NMR and DEPT spectra revealed 14 carbon signals, including one methoxy group (δc 56.9) and one methyl group (δc 56.9). C 23.4), two carbonyl groups (δc 179.1 and 190.6), one methylene group (δc 44.9), one methine group (δc 66.5), and eight sp groups. 2 Carbon signals (δc 109.5, 161.2, 130.5, 120.5, 149.0, 124.8, 161.2, 112.0). These are measured via UV absorption at 234, 257, 316, and 488 nm and at 1685, 1635, and 1605 cm⁻¹. -1 The infrared absorption spectrum indicates that the main structure of this compound contains a 1,4-naphthone structure. Due to the COSY correlation points H-13 / C-11 / C-12, the presence of H-11 with C-5 and C-7, and H-5 with C-7, as well as the presence of hydroxyl protons (δ... H 4.66) and the correlation numbers with C-11 and C-13 indicate that this is a 2-hydroxypropyl group, and this spin-coupled system fragment is linked to the 1,4-naphthone structural portion via C-6. The hydroxyl proton (δ) HThe signal at 12.19) showed an HMBC correlation with C-9 and C-7, indicating that C-8 was hydroxylated. Based on the HMBC correlation point, the correlation between δ 3.88 (OCH3) and δ 161.2 (C-2) indicated that the methoxy group was attached to C-2, thus confirming the planar structure of the compound. The NMR chemical shift and optical rotation of the compound were very similar to those of 5-dehydroxysolaninol ([α]20 D +12.0, compound; [α]20 D 18.7, 5-dehydroxysolaninol), indicating that the compound has the same configuration as 5-dehydroxysolaninol, lacking a methyl group from C-7. The compound was ultimately named 5-dehydroxy-7-demethylsolaninol. Based on the above, the structure of the compound 5-dehydroxy-7-demethylsolaninol was identified, and a search revealed it to be a novel naphthoquinone compound.

[0112] Table 1. 5-Dehydroxy-7-Demethylfusarium oleoresin 13 C NMR (125 MHz) and 1 1H NMR (500 MHz) data, solvent DMSO-d6.

[0113]

[0114] 5-Dehydroxy-7-demethylfusarium oleoresin, yellow powder; [α]20 D+12.0 (c 1.0, MeOH); UV(MeOH) λmax (log ε) 244 (3.12), 287 (3.03), 417 (2.58), and 480 (1.49) nm; IR(KBr) νmax: 1657, 1655, and 1632 cm⁻¹ -1 ; 1 H and 13 C10 NMR data are shown in Table 1; HRESIMS m / z 285.0695 [M+Na]+ (theoretical value C10) 14 H 14 NaO5, 285.0841).

[0115] Example 3

[0116] The compound of this invention exhibits activity in inhibiting the root length of purslane.

[0117] Purslane seeds were sterilized with 4% sodium hypochlorite for 5 minutes, then rinsed three times with sterile water. MS medium with a final concentration of 50 μg / mL was prepared as the treatment group, medium without any added compounds was used as a blank control, and DMSO plates were added as a negative control. Seeds were cultured alternately day and night at a constant temperature of 28°C for 12 hours to observe seed growth. At the end of the culture, the length of roots and shoots, the fresh weight of each seed, and the seed germination inhibition rate were measured.

[0118] The test results are as follows Figure 7 As shown, Figure 7 The bar graph shows the inhibition rate of naphthoquinone derivatives on purslane seedlings. Figure 7 It can be seen that compounds 1, 2, 3, 7, 9, and 10 significantly inhibited the growth of purslane, mainly in three aspects: radicle, hypocotyl, and fresh weight. Other compounds had almost no inhibitory effect on purslane at 50 μg / mL.

[0119] For compounds with better inhibitory activity, different concentrations of the compounds were designed, and IC50 values ​​were obtained. 50 Glyphosate was used as a positive control.

[0120] Among them, the IC50 of positive glyphosate on shoot length, root length and fresh weight 50 The values ​​were 10.45, 17.47, and 10.06 μg / mL, respectively, indicating that dehydrated fusarin (compound 7) exhibited significant activity, with IC50 values ​​for shoot length, root length, and fresh weight. 50 The values ​​were 3.26, 8.54 and 0.90 μg / mL, respectively.

[0121] Table 1. IC50 of compounds on purslane roots, stems, and fresh weight 50 (μg / mL)

[0122]

[0123] Example 4

[0124] This invention provides dehydrated Javan Fusarium toxin that acts on foxtail grass, amaranth, plantain, crabgrass, and beggar-ticks.

[0125] Seed disinfection. Plants were treated with dehydrated fusarium oxychloride at final concentrations of 5, 10, and 50 μg / mL, with glyphosate serving as a positive control.

[0126] The test results are as follows Figure 8 As shown.

[0127] Fusarium oxychloride showed some inhibitory effect on foxtail grass, with the inhibitory effect increasing with increasing dosage, but lower than that of the positive control herb glyphosate. It had a very strong inhibitory effect on plantain growth, significantly better than glyphosate; treatment of seeds at a concentration of 10 μg / mL essentially suppressed weed growth. It showed similar inhibitory effects on amaranth and crabgrass. The compound inhibited the growth of beggar-ticks with increasing dosage; compared to the control group, the treated roots did not produce new shoots, and the shoots and roots became more slender.

[0128] Example 5

[0129] The compound dehydrated fusarium oxysin of this invention exhibits its inhibitory activity on the root length of purslane.

[0130] 5-1) Experimental method for inhibiting root length in Arabidopsis thaliana

[0131] 5-1-1) Analysis of root plasma membrane integrity by microscopic observation and Evans Blue staining

[0132] Purslane seeds were cultured on MS medium containing the compound at concentrations of 5, 10, and 50 μg / mL. After 7 days, the seedlings were carefully removed with tweezers, the agar on the surface was washed off, the moisture was absorbed with filter paper, and the seedlings were stained with 0.125% Evans blue dye for 15 minutes. The dye on the surface was then thoroughly washed off with water, and the seedlings were observed under a VHX-6000 ultra-deep field 3D microscope.

[0133] 5-1-2) SEM and TEM analysis of the degree of damage to root cells

[0134] Seed germination inhibition experiments were conducted using the same method as described above. Treated purslane seeds were fixed in glutaraldehyde and embedded in resin. Sections with a thickness of 60–100 nm were obtained using a Leica EM UC7 and digitally photographed using a HITACHI HT7800 80 kV transmission electron microscope.

[0135] 5-2) Analysis of the results of purslane root length inhibition

[0136] 5-2-1) Effects of dehydrated fusarium oxysporin on the permeable membrane of purslane root tip cells

[0137] Fusarium oxychloride affects the root length of the taproot of purslane and influences the apical meristem. With increasing dosage, the number of cortical cells from the dormant center to the first elongating cell decreases, affecting root cell separation and thus root elongation and growth. According to... Figure 9The control group grew well and was barely stained by Evans blue. Visualization showed the dose-response effect of fusarium oxysporum on purslane; as the concentration increased, the plants became increasingly blue, indicating damage to the root plasma membrane and increased permeability of the apical cell membrane, allowing the dye to enter and turn deep blue. This was especially evident after treatment with 50 μg / mL, where almost all cells were stained blue. The results indicate that with increasing concentrations of fusarium oxysporum, the viability of purslane gradually decreased, ultimately inhibiting its growth.

[0138] 5-2-2) Effects of dehydrated fusarium oxysporum on root cell damage in purslane

[0139] like Figure 10 As shown, at 300×, the traditional longitudinal ridges on the root surface of the control group were clearly visible, while the regular longitudinal ridges on the root surface treated with dehydrated javanica fusarium (10 μg / mL) were broken and disappeared, exhibiting irregularity. At 8000×, the root surface of the control group was found to be very smooth, while the root surface treated with dehydrated javanica fusarium (10 μg / mL) showed significant shrinkage and became uneven. These morphological changes indicate that dehydrated javanica fusarium damages the subcellular tissues of the roots. Furthermore, ultrastructural changes in the root tips of purslane were observed using transmission electron microscopy, further confirming the damage caused by dehydrated javanica fusarium. Under normal growth conditions, cells are clearly visible, structurally intact, and organelles are highly identifiable. In contrast, dehydrated javanica fusarium severely disrupted the ultrastructure of root tip cells, causing cytoplasmic shrinkage, nuclear dissolution, and almost no intact organelles visible in the field of view. These results indicate that dehydrated javanica fusarium inhibits weed growth by disrupting the physiological structure of purslane.

[0140] Example 6

[0141] The mechanism by which the compound dehydrated fusarium oxysine inhibits root length in purslane.

[0142] 6-1) Experimental method for inhibiting root length in Arabidopsis thaliana

[0143] 6-1-1) Transcriptome analysis

[0144] Purslane seeds were treated with a compound and incubated for three days. They were then rapidly freeze-dried in liquid nitrogen and stored at -80°C. Total RNA was extracted, and its concentration, purity, and integrity were assessed. Reverse transcription, adapter ligation, PCR amplification, library construction, library quality control, and library normalization were performed.

[0145] 6-1-2) Quantitative RT-PCR analysis

[0146] To validate the transcriptional level results from RNA-Seq data analysis, 10 genes were selected for qRT-PCR validation. The primers for qRT are listed in Table 2. RNA was extracted using the Trizol method. Reverse transcription of RNA was performed using HiScript III RT SuperMix to obtain cDNA. qRT-PCR experiments were conducted using the ChamQ Universal SYBR qPCR Master Mix, following the instructions for the Bio-Rad CFX Opus 96 real-time PCR system. The cycling program was: 95°C for 5 minutes, followed by 40 cycles of 95°C for 10 seconds and 60°C for 30 seconds. The actin gene from purslane was used as a reference gene. The relative expression levels of the sample genes were analyzed using a 22 −ΔΔCT Methodological calculations. All treatments were set up with three biological replicates.

[0147] Table 2 Primer Sequences

[0148]

[0149] 6-2) Mechanism analysis of purslane root length inhibition

[0150] PsbY is mainly distributed near the two α-helices of cytochrome B559, with its amino terminus located directly on the surface of the thylakoid. Anhydrojavanicin may compete with plastoquinone for D1 protein, blocking electron transfer from the QA site to the QB site and inhibiting photosynthesis.

[0151] The results are as follows Figure 11 As shown.

[0152] Example 7

[0153] Safety evaluation of the compound dehydrated fusarium in this invention.

[0154] The toxicity of dehydrated fusarium was evaluated in model crop wheat (5 g gai / ha), Kunming mice (100 mg / kg, 7 d), and at the cellular level (Beas-2B and GES-1, 100 μM).

[0155] Dehydrated Javan fusarium is non-toxic to wheat, mice, and normal human cells.

[0156] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A naphthoquinone derivative having the structure shown in Formula I, Formula II or Formula III, or a stereoisomer thereof: Formula I Formula II Formula III in, R1-R3 are independently selected from hydrogen, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy; the substitution is one or more substitutions by hydroxyl or amino groups; Alternatively, any adjacent groups in R1-R3 and the carbon atoms attached to them can together form a five- or six-membered saturated or unsaturated heterocyclic group; the heteroatom in the heterocyclic group is one or more of oxygen and nitrogen atoms; any one or more hydrogen atoms in the five- or six-membered saturated or unsaturated heterocyclic group may be optionally replaced by hydroxyl, alkyl, or alkoxy groups; R4-R5 are independently selected from substituted or unsubstituted alkyl groups, substituted or unsubstituted alkoxy groups, and -CH2COCH3; the substitution is one or more substitutions by hydroxyl groups and amino groups; Alternatively, R4 and R5 and the carbon atoms attached thereto can together form a five- or six-membered saturated or unsaturated heterocyclic group; the heteroatom in the heterocyclic group is one or more of oxygen and nitrogen atoms; any one or more hydrogen atoms in the five- or six-membered saturated or unsaturated heterocyclic group may optionally be replaced by hydroxyl, alkyl, or alkoxy groups; R6 is selected from hydrogen, hydroxyl, substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy; wherein the substitution is one or more of hydroxyl and amino groups; R7 is selected from substituted or unsubstituted saturated alkyl or unsaturated alkyl groups; the substitution is one or more substitutions with hydroxyl or amino groups.

2. The naphthoquinone derivative according to claim 1, characterized in that, R1-R3 are independently selected from hydrogen, hydroxyl, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy; the substitution is one or more of hydroxyl and amino groups; Alternatively, any adjacent groups in R1-R3 and the carbon atoms attached to them can jointly form a five- or six-membered saturated or unsaturated heterocyclic group; the heteroatom in the heterocyclic group is one or more of oxygen and nitrogen atoms; any one or more hydrogen atoms in the five- or six-membered saturated or unsaturated heterocyclic group may be optionally substituted with hydroxyl, C1-C4 alkyl, or C1-C4 alkoxy groups. R4-R5 are independently selected from substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy, and -CH2COCH3; the substitution is one or more of hydroxyl and amino groups; Alternatively, R4 and R5 and the carbon atoms attached thereto can together form a five- or six-membered saturated or unsaturated heterocyclic group; the heteroatom in the heterocyclic group is one or more of oxygen and nitrogen atoms; any one or more hydrogen atoms in the five- or six-membered saturated or unsaturated heterocyclic group may be optionally substituted with hydroxyl, C1-C4 alkyl, or C1-C4 alkoxy groups. R6 is selected from hydrogen, hydroxyl, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C4 alkoxy; the substitution is one or more of hydroxyl and amino groups; R7 is selected from substituted or unsubstituted C1-C4 saturated or unsaturated alkyl groups; the substitution is one or more substitutions with hydroxyl or amino groups.

3. The naphthoquinone derivative according to claim 1, characterized in that, The R1-R3 are independently selected from any of the following groups: hydrogen, hydroxyl, methyl, -CH2CH(OH)CH3; Alternatively, any adjacent groups in R1-R3 and their attached carbon atoms may together form a five- or six-membered saturated or unsaturated heterocyclic group, wherein the five- or six-membered saturated or unsaturated heterocyclic group has any of the following structures: ； The above groups are fused with the parent ring through any two adjacent carbon atoms; Any one or more hydrogen atoms in the five- to six-membered saturated or unsaturated heterocyclic groups may optionally be substituted by one or more of hydroxyl, alkyl, and alkoxy groups; R4-R5 are independently selected from any of the following groups: methyl, hydroxymethyl, -CH2COCH3; Alternatively, R4 and R5, along with their attached carbon atoms, may together form a five- or six-membered saturated or unsaturated heterocyclic group, wherein the five- or six-membered saturated or unsaturated heterocyclic group has any of the following structures: ； The above groups are fused with the parent ring through any two adjacent carbon atoms; Any one or more hydrogen atoms in the five- to six-membered saturated or unsaturated heterocyclic groups may optionally be substituted by one or more of hydroxyl, alkyl, and alkoxy groups; R6 is selected from hydrogen or hydroxyl; R7 is selected from 2-propenyl or 1-hydroxyethyl.

4. The naphthoquinone derivative according to claim 1, characterized in that, The naphthoquinone derivatives have any of the structures shown in compounds 1-14 below: 。 5. A method for preparing the naphthoquinone derivative according to any one of claims 1 to 4, comprising the following steps: The Fusarium sp. L-3 strain was fermented, soaked in ethyl acetate, evaporated under reduced pressure, dehydrated and deoiled, and subjected to forward / reverse / gel column chromatography for semi-preparative processing.

6. The preparation method according to claim 5, characterized in that, Includes the following steps: S1) The fermentation product of Fusarium sp. L-3 was extracted with ethyl acetate and the oil was removed to obtain the total extract; S2) The total extract was separated by normal-phase silica gel chromatography to obtain components Fr.1~Fr.5; S3) Take component Fr.3 and separate to obtain components Fr.3.1~Fr.3.10; Take component Fr.3.10 and separate it to obtain compound 5; Take component Fr.3.9 and separate it to obtain compound 6; Fr.3.7 was taken and compounds 3 and 10 were separated. Take component Fr.3.6 and separate it to obtain compound 7; Take component Fr.3.5 and separate components Fr.3.5.1-Fr.3.5.6, of which Fr.3.5.3 is compound 14; take component Fr.3.5.4 and separate compound 2 and compound 1. Take component Fr.3.4 and separate it to obtain compound 11; Take component Fr.3.3 and separate it to obtain 12; Take component Fr.1 and filter to obtain compound 9; Take component Fr.4 and separate it to obtain compound 4; Take component Fr.2 and separate to obtain components Fr.2.1~Fr.2.9; Fr.2.5 was taken and compounds 13 and 8 were obtained by separation.

7. The use of naphthoquinone derivatives according to any one of claims 1 to 4 or naphthoquinone derivatives prepared by the preparation method according to any one of claims 5 to 6 in the preparation of herbicides.

8. The application according to claim 7, characterized in that, The herbicide is applicable to one or more of the following weeds: purslane, foxtail grass, crabgrass, beggar-ticks, retroflexus, and plantain.

9. A herbicide comprising a naphthoquinone derivative as described in any one of claims 1 to 4 or a naphthoquinone derivative prepared by the preparation method described in any one of claims 5 to 6, and an adjuvant.

10. A weeding method, comprising the following steps: The naphthoquinone derivatives according to any one of claims 1 to 4, or the naphthoquinone derivatives prepared by the preparation method according to any one of claims 5 to 6, or the herbicide according to claim 9, come into contact with the seeds, embryos, and / or leaves of weeds.

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