Cytochalasin derivatives and processes for their preparation

Abstract

The present application belongs to the technical field of microorganism and its natural products, and particularly relates to a cytochalasin derivative and a preparation method thereof. The present application takes Chaetomium madiri as raw material, and then carries out separation and extraction, and then carries out chemical modification and transformation by using various chemicals, and finally carries out purification to prepare multiple cytochalasin derivatives. The method adopted by the present application has the characteristics of high selectivity and environmental friendliness, and the extraction method is simple and low in cost, and the purity of the prepared cytochalasin derivative can reach 99.8%. Through in vitro anti-inflammatory test, it is concluded that the cytochalasin derivative has good application prospect in preparing anti-inflammatory drugs. Through Arabidopsis root growth inhibition test, it is concluded that the cytochalasin derivative has good application prospect in preparing herbicides.

Description

Technical Field

[0001] This invention belongs to the field of application technology of microorganisms and their natural products, specifically relating to a cell relaxin derivative and its preparation method. Background Technology

[0002] When the body is stimulated by physical, chemical, or traumatic events, or infected by bacteria or viruses, it triggers the release of inflammatory mediators by relevant immune cells, thus inducing a defensive inflammatory response. Moderate inflammation is beneficial to the body's tissues, but excessive inflammation can damage normal cells and tissues, inducing various acute and chronic inflammatory diseases, such as cardiovascular disease, cancer, sepsis, inflammatory bowel disease, arthritis, and diabetes, seriously threatening human health.

[0003] Cytokines can be classified into pro-inflammatory and anti-inflammatory cytokines based on their roles in the inflammatory response, and their balance plays a crucial role in the inflammatory response and the immune system. Among the many factors involved in the inflammatory response, the expression of TNF-1, IL-6, and IL-1 is closely related to the incidence of endotoxin shock and cell death. MAPKs are an important signaling system in eukaryotic cells, transmitting extracellular signals into the cell and inducing cellular responses. Extracellular stimuli activate the phosphorylation of p38ERK 1 / 2 and JNK through the MAPKKK-MAPKK-MAPK three-level kinase cascade, thereby regulating the secretion of IL-6, IL-1β, and other cytokines by affecting the activity of transcription factors.

[0004] Currently, commonly used anti-inflammatory drugs in clinical practice fall into two categories: nonsteroidal anti-inflammatory drugs (NSAIDs) and steroidal anti-inflammatory drugs (SADs). However, long-term, high-dose use can lead to a series of adverse reactions, side effects, and tolerance issues. Therefore, there is an urgent need to find novel exogenous anti-inflammatory drugs.

[0005] Cytochalasin is a class of fungal polyamino acid hybrid secondary metabolites with significant biological functions, and it is also a highly substituted isoindolone derivative. Its typical structural feature is a tricyclic skeleton, with one macrocycle fused to the isoindolone skeleton. The diversity of cytochalasin species and structures is determined by the different types of amino acids fused to the polyketone skeleton, as well as the type of macrocycle, the number of carbon atoms, and the substituents on the macrocycle. Studies have found that this class of compounds possesses broad-spectrum antitumor, antibacterial, and antiviral biological activities.

[0006] In agricultural production, weeds have always been a major biological factor causing crop yield reduction, leading to losses of 30% to 90%. Currently, chemical weeding remains one of the primary methods for weed control. However, with the development of intensive agriculture and the increasing use of herbicides, herbicide resistance is becoming a growing problem.

[0007] Natural products, with their diverse structures and environmentally friendly characteristics, are considered the most promising sources of pesticide lead compounds. Fungi, in particular, produce abundant secondary metabolites, from which many natural compounds with herbicidal activity have been discovered. For example, Macroeidin A, produced by the pathogenic fungus *Phoma macrostoma*, is a pyrrolic acid compound that can be used as a herbicide.

[0008] Arabidopsis thaliana, as a model organism in genetics and plant development research, plays a role in agricultural science much like mice and fruit flies in human biological research. Arabidopsis's strict self-pollination ensures the uniformity and stability of germplasm resources. Using Arabidopsis for screening herbicide lead compounds not only greatly improves screening sensitivity and reduces missed screenings, but also overcomes the limitations of using weed seeds due to limited seed resources and significant differences in seed quality.

[0009] The combination of natural product chemistry and diversity-oriented synthesis involves using natural product extracts as starting substrates and chemically modifying them to obtain diverse molecular libraries. By using natural products as resources, and through semi-synthetic or structural modification of extracts, novel molecular libraries of natural product analogs can be constructed. This not only compensates for the limitations of natural products but also allows for the direct, one-step generation of a large number of products with modified functional groups, and even novel skeletons. This promotes the chemical diversity of natural products, provides more active molecules for drug development, facilitates the study of effective active sites and structure-activity relationships in drug molecules, increases the probability of druggability, and thus shortens the drug development process. This is of great significance for discovering drugs with application value. Summary of the Invention

[0010] The purpose of this invention is to provide a method for preparing a cytochalasin derivative.

[0011] The present invention further provides the application of the aforementioned cytochalasin derivative in the preparation of anti-inflammatory drugs.

[0012] The present invention further provides the application of the aforementioned cytochalasin derivative in the preparation of herbicides.

[0013] To achieve the above objectives, this invention uses *Trichoderma Madrid* as raw material, first separating and extracting it, then chemically modifying it using various chemical substances, and finally purifying it to prepare multiple cytochalasin derivatives.

[0014] Specifically, this invention prepared 11 compounds with a cytochalasin skeleton structure, which are cytochalasin derivatives. The inventors divided the obtained compounds into two categories: one category is cytochalasin derivatives containing amino or isoxazole structures, and their structural formulas are as follows:

[0015]

[0016] Another type is nitro-containing cytochalasin derivatives, whose structural formula is as follows:

[0017]

[0018] The cytochalasin derivative is prepared by the following steps:

[0019] (1) Activation of strain: Inoculate *Chaetoceros madrica* onto PDA agar plates and culture at 25–30°C for 5–7 days to obtain activated strain;

[0020] (2) Fermentation culture: The activated strain obtained in step (1) is inoculated into rice solid culture medium and cultured statically at 23-27℃ for 32-35 days to obtain fermentation product;

[0021] (3) Ethyl acetate extraction: The fermentation product obtained in step (2) is extracted with ethyl acetate. The extract is concentrated under reduced pressure and the ethyl acetate is recovered to obtain crude extract.

[0022] (4) Diversity-enhancing chemical modification of extracts: First, the crude extract was reacted with 1,1,1-trifluoroacetone, sodium bicarbonate and potassium persulfate (Oxone) to obtain the reaction residue; then the residue was reacted with lanthanum trifluoromethanesulfonate (III)(La(OTf)3) and 2,6-di-tert-butylpyridine to finally obtain the chemically modified diversity-enhancing bacterial extract.

[0023] (5) ODS reversed-phase silica gel column chromatography for crude separation: The diversity-enhancing bacterial extract obtained in step (4) was subjected to ODS reversed-phase silica gel column chromatography to obtain four components, which were numbered A, B, C, and D in descending order of polarity.

[0024] (6) Separation and purification of monomeric compounds: The final products were obtained by Sephadex LH-20 gel column chromatography and semi-preparative HPLC purification of components B, C and D in step (5).

[0025] Specifically, the strain used in this invention is a bacterium isolated from desert soil in Hotan City, Xinjiang. Its taxonomic name is Chaetomium madrasense CLC375, and it is deposited at the China Center for Type Culture Collection (CCTCC), located at Wuhan University, Wuhan, China; deposited on July 3, 2019, with accession number CCTCC M2019517.

[0026] Specifically, the rice solid culture medium in step (2) is prepared by the following steps: 60g of rice and 100mL of water are placed in a 500mL Erlenmeyer flask and then sterilized at a temperature of 121℃ and a pressure of 0.12MPa.

[0027] Specifically, the ethyl acetate extraction process in step (3) is as follows: 300 mL of ethyl acetate is added to the fermentation product obtained in step (2), stirred, and filtered after standing for 12 to 16 hours. The above extraction method is repeated three times, and the extracts from the three extractions are combined. The extracts are concentrated under reduced pressure to obtain crude extract.

[0028] Specifically, step (4) chemical modification is as follows: take the crude extract and dissolve it in a mixed solvent of acetonitrile and water. Under the condition of -5 to 5℃, add 1,1,1-trifluoroacetone, sodium bicarbonate and potassium persulfate (Oxone) in sequence. Keep at -5 to 5℃ and stir for 2 to 3 hours. Then add a certain amount of potassium persulfate (Oxone) and stir at -5 to 5℃ for another 2 to 3 hours. Pour the reaction mixture into water and extract with ethyl acetate. Combine the organic layers, wash, dry and evaporate to obtain the reaction residue.

[0029] The residue was dissolved in 1,2-dichloroethane to obtain a solution. Lanthanum trifluoromethanesulfonate (III) (La(OTf)3) and 2,6-di-tert-butylpyridine were added to the solution at room temperature. After reflux for 10–15 h, a certain amount of lanthanum trifluoromethanesulfonate (III) (La(OTf)3) was added, and the mixture was refluxed for another 20–24 h. The reaction mixture was then poured into 0.2–0.5 M hydrochloric acid solution and extracted with ethyl acetate. The organic layers were combined, washed, and dried to obtain the diversity-enhancing bacterial extract.

[0030] Specifically, the mass ratio of crude extract to potassium persulfate (Oxone) is (0.48–0.53):1.

[0031] Specifically, the molar ratio of 1,1,1-trifluoroacetone, sodium bicarbonate and potassium persulfate (Oxone) is (1-2):2:(3-4).

[0032] Specifically, the mass ratio of crude extract to lanthanum trifluoromethanesulfonate (III) is 5:3 to 5:2.

[0033] Further preferred, the specific reaction steps of chemical modification in step (4) are as follows: 20g of crude extract is dissolved in a mixed solvent of 1200mL acetonitrile and 500mL water. Under 0℃ conditions, 20mL of 1,1,1-trifluoroacetone, 28g of sodium bicarbonate and 40g of potassium persulfate (Oxone) are added in sequence. The mixture is stirred at 0℃ for 3h. Then, 40g of potassium persulfate (Oxone) is added and stirred at 0℃ for another 3h. The reaction mixture is poured into water and extracted three times with ethyl acetate (2000mL). The organic layers are combined, washed with water and salt water, dried with sodium sulfate, and evaporated to obtain 23g of reaction residue.

[0034] 20 g of the residue obtained from evaporation was dissolved in 320 mL of 1,2-dichloroethane. 10 g of lanthanum trifluoromethanesulfonate (III) (La(OTf)3) and 30 mL of 2,6-di-tert-butylpyridine were added to the solution at room temperature. After reflux for 15 h, 10 g of lanthanum trifluoromethanesulfonate (III) (La(OTf)3) was added, and the mixture was refluxed for another 24 h. The reaction mixture was then poured into 0.5 M hydrochloric acid and extracted three times with ethyl acetate (500 mL). The organic layers were combined, washed with saturated sodium bicarbonate solution and brine, and dried with sodium sulfate to obtain the diversity-enhancing bacterial extract.

[0035] Specifically, in step (5), when performing ODS reversed-phase silica column chromatography, water and methanol are used as the mobile phase for gradient elution, with volume ratios (v:v) of 100:0, 80:20, 40:60, 20:80, and 0:100.

[0036] Specifically, the separation and purification methods for compounds 1, 2, 6, and 7 in step (6) are as follows:

[0037] Fragment B was separated into five subfractions, B-1 to B-5, by Sephadex LH-20 gel column chromatography using methanol as the mobile phase. Among them, B-1, B-2, B-4, and B-5 were purified by repeated HPLC to obtain compound 1, compound 2, compound 6, and compound 7, respectively. The flow rate during repeated HPLC purification was 2 mL / min.

[0038] Specifically, the separation and purification methods for compounds 8, 9, 10, and 11 in step (6) are as follows:

[0039] Fragment C was subjected to Sephadex LH-20 gel column chromatography to obtain three subfractions, C-1 to C-3. Subfraction C-2 was purified by semi-preparative HPLC. The mobile phase was water and acetonitrile (v / v ratio 62.5:37.5), the flow rate was 2 mL / min, and the retention time was t. R = 22.9 min, compound 8 was obtained; retention time t R =35.1 min, yielding compound 9;

[0040] Subfraction C-3 was purified by semi-preparative HPLC using water and acetonitrile (60:40 v / v) as the mobile phase, at a flow rate of 2 mL / min, and a retention time of t. R =29.1 min, compound 10 was obtained, retention time t R =30.7 min, compound 11 was obtained.

[0041] Specifically, the separation and purification methods for compounds 3, 4, and 5 in step (6) are as follows:

[0042] Fraction D was subjected to Sephadex LH-20 gel column chromatography to obtain three subfractions D-1 to D3. D-2 was further purified by semi-preparative HPLC using water and acetonitrile (60:40 v / v) as the mobile phase at a flow rate of 2 mL / min and a retention time t0. R =27.4 min, compound 3 was obtained; retention time t R =29.7 min, yielding compound 4;

[0043] D-3 was purified by semi-preparative HPLC using water and acetonitrile (v / v ratio 53:47) as the mobile phase, at a flow rate of 2 mL / min, and a retention time t. R =19.4 min, compound 5 was obtained.

[0044] Specifically, in step (6), in the above semi-preparative HPLC, YMC, 10mm×250mm, particle size 5μm column is selected, detection wavelength is 254, 220, 280, and the mobile phase ratio of each component in the purification process is volume ratio.

[0045] Testing showed that the compounds prepared by the above methods all achieved a purity of 99.8%.

[0046] Based on a general inventive concept, the present invention also proposes the application of the aforementioned cytochalasin derivative in the preparation of anti-inflammatory drugs.

[0047] Based on a general inventive concept, the present invention also proposes the application of the aforementioned cytochalasin derivative in the preparation of herbicides.

[0048] Compared with the prior art, the beneficial effects of the present invention are:

[0049] This invention uses *Chaetoceros madrica* as raw material and chemically modifies it using various chemicals (1,1,1-trifluoroacetone, sodium bicarbonate, potassium persulfate (Oxone), lanthanum(III) trifluoromethanesulfonate (La(OTf)3), and 2,6-di-tert-butylpyridine) to obtain chemically modified, diversity-enhancing bacterial extracts. After purification, multiple cytochalasin derivatives containing amino or isoxazole structures, as well as multiple cytochalasin derivatives containing nitro groups, are obtained. The purity of the cytochalasin derivatives prepared by the method of this invention can reach 99.8%.

[0050] In vitro anti-inflammatory experiments showed that compounds 1 and 2 could effectively inhibit the excessive production of NO in LPS-induced mouse macrophages RAW264.7, thus exerting an anti-inflammatory effect. Compound 1 could reduce the overexpression of IL-6 and iNOS, and dose-dependently affect the phosphorylation of p38, ERK1 / 2, and JNK. It exerts its anti-inflammatory function through MAPK signaling and has good application prospects in the preparation of anti-inflammatory drugs.

[0051] The method employed in this invention is characterized by high selectivity and environmental friendliness, yielding a high-yield crude extract, and the extraction method is simple and low-cost. Through Arabidopsis root growth inhibition experiments, compound 5 was found to have certain inhibitory activity on Arabidopsis root growth and significant Arabidopsis seed toxicity, indicating that the nitro-containing cytochalasin derivative of this invention has good application prospects in the preparation of herbicides. Attached Figure Description

[0052] Figure 1 Compounds 1, 2, 6, 8, and 9 prepared in Example 1 1 H- 1 H COSY and HMBC NMR;

[0053] Figure 2 Comparison of experimental ECD and calculated ECD for compound 1 prepared in Example 1;

[0054] Figure 3 Compounds 3, 4, 5, 7, 10, and 11 prepared in Example 1 1 H- 1 H COSY and HMBC NMR;

[0055] Figure 4 The effects of compounds 1 and 2 in Example 2 on the viability of RAW264.7 cells;

[0056] Figure 5 The effects of compounds 1 and 2 in Example 2 on LPS-induced NO secretion in RAW264.7 cells;

[0057] Figure 6The effect of compound 1 in Example 2 on the release of IL-6 stimulated by LPS;

[0058] Figure 7 Compound 1 in Example 2 was used to induce LPS-induced iNOS protein expression in RAW264.7 cells.

[0059] Figure 8 The effect of compound 1 in Example 2 on MAPK signaling pathway proteins;

[0060] Figure 9 This is a graph showing the inhibitory activity of compound 5 in Example 3 on Arabidopsis root growth;

[0061] Figure 10 This is a graph showing the inhibitory activity of compound chaetoglobosins D on Arabidopsis root growth in Example 3.

[0062] Figure 11 This is a graph showing the inhibitory activity of compound chaetoglobosins E on Arabidopsis root growth in Example 3.

[0063] Figure 12 This is a graph showing the inhibitory activity of compound chaetoglobosins G on Arabidopsis root growth in Example 3. Detailed Implementation

[0064] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0065] The *Chaetomium madrasense* used in the following examples was isolated from desert soil in Hotan, Xinjiang. Its taxonomic name is *Chaetomium madrasense* CLC375, and it is deposited at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China; deposit date: July 3, 2019; accession number: CCTCC M2019517. For specific isolation methods, morphological characteristics, and physiological and biochemical identification of the *Chaetomium madrasense*, please refer to Chinese Patent No. 1 (CN201910745629.1) and will not be repeated here.

[0066] The reagents used in the following examples are: chromatographic grade methanol, chromatographic grade acetonitrile (Tianjin Kemeio Co., Ltd.), silica gel (200-300 mesh; Qingdao Ocean Chemical Plant, Qingdao, China), reversed-phase (ODS) silica gel (Yamaha, Tokyo), Sephadex LH-20 (Uppsala GE Healthcare Biosciences, Sweden), methanol, dichloromethane, ethyl acetate (chemically pure, Tianjin Deen Chemical Reagent Co., Ltd.), deuterated chloroform, deuterated methanol, deuterated DMSO (Saen Chemical Technology (Shanghai) Co., Ltd.).

[0067] Unless otherwise specified, all other instruments and equipment used are commercially available standard instruments and equipment; all other reagents and materials used are commercially available standard reagents and materials.

[0068] The components and preparation methods of the PDA plate culture medium and DMEM complete culture medium in the following examples are conventional methods in the art and are not the inventive point of this invention, so they will not be described in detail.

[0069] Example 1

[0070] Example 1 provides a method for preparing the aforementioned cytochalasin derivative, the specific steps of which are as follows:

[0071] (1) Activation of bacterial strain: Chaetomium madrasense was inoculated onto PDA agar plates and cultured at 25°C for 7 days to obtain activated bacterial strain;

[0072] (2) Fermentation culture: The activated strain obtained in step (1) is inoculated into rice solid culture medium and cultured at 25℃ for 35 days to obtain fermentation product; the rice solid culture medium in step (2) is prepared by the following steps: 60g rice and 100mL water are placed in a 500mL Erlenmeyer flask and then sterilized at a temperature of 121℃ and a pressure of 0.12MPa.

[0073] (3) Ethyl acetate extraction: The fermentation product obtained in step (2) was extracted with ethyl acetate. The extract was concentrated under reduced pressure and the ethyl acetate was recovered to obtain 77g of crude extract.

[0074] The ethyl acetate extraction process described in step (3) is as follows: 300 mL of ethyl acetate is added to the fermentation product obtained in step (2), stirred, and filtered after standing for 12 hours. The above extraction method is repeated three times, and the extracts from the three extractions are combined. The extracts are concentrated under reduced pressure to obtain crude extract paste.

[0075] (4) Diversity-enhancing chemical modification of extracts: First, the crude extract was reacted with 1,1,1-trifluoroacetone, sodium bicarbonate and potassium persulfate (Oxone) to obtain the reaction residue; then the residue was reacted with lanthanum trifluoromethanesulfonate (III)(La(OTf)3) and 2,6-di-tert-butylpyridine to finally obtain the chemically modified diversity-enhancing bacterial extract.

[0076] The specific reaction steps of step (4) chemical modification are as follows: Take 20g of crude extract and dissolve it in a mixed solvent of 1200mL acetonitrile and 500mL water. Under 0℃ conditions, add 20mL of 1,1,1-trifluoroacetone, 28g of sodium bicarbonate and 40g of potassium persulfate (Oxone) in sequence. Keep stirring at 0℃ for 3h. Then add 40g of potassium persulfate (Oxone) and stir at 0℃ for another 3h. Pour the reaction mixture into water and extract it three times with ethyl acetate (2000mL). Combine the organic layers, wash with water and salt water, dry with sodium sulfate and evaporate to obtain 23g of reaction residue.

[0077] 20 g of the residue obtained from evaporation was dissolved in 320 mL of 1,2-dichloroethane. 10 g of lanthanum trifluoromethanesulfonate (III) (La(OTf)3) and 30 mL of 2,6-di-tert-butylpyridine were added to the solution at room temperature. After reflux for 15 h, 10 g of lanthanum trifluoromethanesulfonate (III) (La(OTf)3) was added, and the mixture was refluxed for another 24 h. The reaction mixture was then poured into 0.5 M hydrochloric acid solution and extracted three times with ethyl acetate (500 mL). The organic layers were combined, washed with saturated sodium bicarbonate solution and brine, and dried with sodium sulfate to obtain the diversity-enhancing bacterial extract.

[0078] (5) ODS reversed-phase silica gel column chromatography for crude separation: The diversity-enhancing bacterial extract obtained in step (4) was subjected to ODS reversed-phase silica gel column chromatography with water and methanol as the mobile phase for gradient elution (volume ratio (v:v) of 100:0, 80:20, 40:60, 20:80, 0:100 respectively) to obtain four components, which were numbered A, B, C, and D in descending order of polarity.

[0079] (6) Separation and purification of monomeric compounds: The components B, C and D in step (5) were purified by Sephadex LH-20 gel column chromatography and semi-preparative HPLC to obtain compounds 1-11;

[0080] Specifically, the separation and purification method for compounds 1-11 in step (6) is as follows:

[0081] Fragment B was separated into five subfractions, B-1 to B-5, by Sephadex LH-20 gel column chromatography using methanol as the mobile phase. Among them, B-1, B-2, B-4, and B-5 were purified by repeated HPLC to obtain compound 1 (5 mg), compound 2 (4 mg), compound 6 (3 mg), and compound 7 (2 mg), respectively. The flow rate was 2 mL / min during the repeated HPLC purification.

[0082] Fragment C was subjected to Sephadex LH-20 gel column chromatography to obtain three subfractions, C-1 to C-3. Subfraction C-2 was purified by semi-preparative HPLC using water and acetonitrile (62.5:37.5) as the mobile phase and 2 mL / min as the flow rate, yielding compound 8 (3 mg, retention time t). R =22.9min), compound 9 (6mg, retention time t) R =35.1min);

[0083] Subfraction C-3 was purified by semi-preparative HPLC using water and acetonitrile (60:40) as the mobile phase at a flow rate of 2 mL / min, yielding compound 10 (3 mg, retention time t). R =29.1 min), compound 11 (6 mg, retention time t) R =30.7min).

[0084] Fraction D was subjected to Sephadex LH-20 gel column chromatography to obtain three subfractions D-1 to D3. D-2 was further purified by semi-preparative HPLC using water and acetonitrile (60:40) as the mobile phase at a flow rate of 2 mL / min, yielding compound 3 (2.8 mg, retention time t). R =27.4 min), compound 4 (1.9 mg, retention time t) R =29.7min);

[0085] D-3 was purified by semi-preparative HPLC using water and acetonitrile (53:47) as the mobile phase at a flow rate of 2 mL / min, yielding compound 5 (7 mg, retention time t). R =19.4min).

[0086] In the above semi-preparative HPLC, a YMC column with a diameter of 10 mm × 250 mm and a diameter of 5 μm was selected, and the detection wavelengths were 254, 220, and 280 nm. The mobile phase ratios of each component in the purification process were all volume ratios.

[0087] Testing showed that the compounds prepared by the above methods all achieved a purity of 99.8%.

[0088] The structures of compounds 1-11 were identified in this invention, as detailed below:

[0089] Compound 1: White amorphous powder, UV spectrum (MeOH)λ max (logε) 315 (0.64), 269 (0.84); ECD(MeOH)λmax(Δε)=310nm (+12.7), 263 (-26.5); 1H and 1C NMR spectra are shown in Tables 1 and 2; High-resolution mass spectra m / z 531.2482 [M+H] + (Calculated value C)31 H 34 N₂O₆, 531.2495 [M+H] + The experimental ECD and calculated ECD comparison chart for compound 1 is shown below. Figure 2 As shown.

[0090] Compound 2: White amorphous powder; UV (MeOH) λmax (logε) 368 (0.40), 261 (0.96), 228 (1.59) nm; ECD (MeOH) λmax (Δε) = 329 (+5.5), 296 (-0.35), 276 (+0.35), 249 (-5.6), 207 (+21.7) nm; 1H and 1C NMR spectra are shown in Tables 1 and 2; High-resolution mass spectrometry (HR-ESI-MS) m / z 533.2640 [M+H] + (calcd for C 31 H 37 N₂O₆, 533.2652[M+H] + ).

[0091] Compound 3: White amorphous powder. UV (MeOH) λmax (logε) 249 (0.88), 222 (1.32), 217 (1.48) nm; ECD (MeOH) λmax (Δε) = 331 (+3.06), 293 (-5.42), 257 (-4.06), 204 (+38.40) nm. ¹H and ¹³C NMR spectra are shown in Tables 1 and 2. HR-ESI-MS m / z 595.2297 [M+H] + (calcd for C 31 H 35 N2O 10 595.2286[M+H] + ).

[0092] Compound 4: White amorphous powder; UV (MeOH) λmax (logε) 223 (1.33), 219 (1.41), 205 (0.44) nm; ECD (MeOH) λmax (Δε) = 331 (+4.71), 296 (-4.39), 246 (-6.84), 203 (+42.20) nm; 1H and 1C NMR spectra are shown in Tables 1 and 2; HR-ESI-MS m / z 611.2246 [M+H] + (calcd for C 31 H 35 N2O 11 611.2135[M+H] + ).

[0093] Compound 5: White amorphous powder, UV (MeOH)λmax(logε) 332 (1.26), 299 (1.51), 248 (1.54) nm, 1H and 1C NMR spectra are shown in Tables 1 and 2, HR-ESI-MS m / z 585.2208 [M+Na] + (calcd forC 31 H 34 N₂O₈Na, 585.22O₇[M+Na] + ).

[0094] Compound 6: White amorphous powder, UV (MeOH)λmax(logε) 368 (0.39), 230 (1.91) nm, 1H and 1C NMR spectra are shown in Tables 3 and 4, HR-ESI-MS m / z 535.2808 [M+H] + (calcd for C 31 H 39 N₂O₆, 535.28O₃[M+H] + ).

[0095] Compound 7: White amorphous powder, UV (MeOH)λmax(logε) 366 (0.19), 243 (1.74), 230 (2.02) nm, 1H and 1C NMR spectra are shown in Tables 3 and 4, HR-ESI-MS m / z 565.2533 [M+H] + (calcd forC 31 H 37 N₂O₈, 565.2544 [M+H] + ).

[0096] Compound 8: White amorphous powder, UV (MeOH)λmax(logε) 229 (0.57), 228 (0.64), 205 (0.71) nm, 1H and 1C NMR spectra are shown in Tables 3 and 4, HR-ESI-MS m / z 535.2814 [M+H] + (calcd forC 31 H 37 N₂O₆, 535.28O₃[M+H] + ).

[0097] Compound 9: White amorphous powder; UV (MeOH)λmax(logε) 325 (1.80), 308 (1.91), 248 (2.13) nm; 1H and 1C NMR spectra are shown in Tables 3 and 4; HR-ESI-MS m / z 533.2635 [M+H] + (calcd forC 32 H37 N₂O₆, 533.2646 [M+H] + ).

[0098] Compound 10: White amorphous powder, UV (MeOH)λmax(logε) 368 (0.53), 230 (2.53), 219 (1.85) nm, 1H and 1C NMR spectra are shown in Tables 3 and 4, HR-ESI-MS m / z 587.2371 [M+Na] + (calcd forC 31 H 37 N₂O₆, 587.2364 [M+Na] + ).

[0099] Compound 11: White amorphous powder; UV (MeOH) λmax (logε) 235 (0.14), 217 (0.17), 208 (0.19) nm; ECD (MeOH) λmax (Δε) = 288 (-1.49), 261 (+0.41), 230 (-14.26), 203 (+23.54) nm; 1H and 1C NMR spectra are shown in Tables 3 and 4; HR-ESI-MS m / z 581.2495 [M+H] + (calcd for C 31 H 37 N₂O₉, 581.2494 [M+H] + ).

[0100] Table 1. 1H NMR data of compounds 1, 2, 6, 8, and 9. Note: a) deuterated reagent is CDCl3, b) deuterated reagent is DMSO- d6

[0101]

[0102]

[0103] Table 2. Carbon spectral data of compounds 1, 2, 6, 8, and 9

[0104] Note: a) Deuterated reagent is CDCl3, b) Deuterated reagent is DMSO- d6

[0105]

[0106]

[0107] Compound 3: White amorphous powder, UV spectrum (MeOH)λ max(logε) 249(0.88), 222(1.32), 217(1.48) nm; 1H and 1C NMR spectra are shown in Tables 3 and 4; High-resolution mass spectra m / z 595.2297 [M+H] + (Calculated value C) 31 H 34 N2O 10 595.2247[M+H] + ).

[0108] Compound 4: White powder, UV spectrum (MeOH)λ max (logε)223(1.33),219(1.41),205(0.44)nm; 1H and 1C NMR spectra are shown in Tables 3 and 4; High-resolution mass spectra m / z 611.2246 [M+H] + (Calculated value C) 31 H 34 N2O 11 611.2196[M+H] + ).

[0109] Compound 5: White powder, UV spectrum (MeOH)λ max (logε) 332(1.26), 299(1.51), 248(1.54) nm; 1H and 1C NMR spectra are shown in Tables 3 and 4; High-resolution mass spectra m / z 585.2208 [M+Na] + (Calculated value C) 31 H 34 N₂O₈, 585.22O₇[M+Na] + ).

[0110] Compound 7: White powder, UV spectrum (MeOH)λ max (logε) 366 (0.19), 243 (1.74), 230 (2.02) nm; 1H and 1C NMR spectra are shown in Tables 3 and 4; High-resolution mass spectra m / z 565.2533 [M+H] + (Calculated value C) 31 H 36 N₂O₈, 565.2505 [M+H] + ).

[0111] Compound 10: White powder, UV spectrum (MeOH)λ max (logε) 229(0.57), 228(0.64), 205(0.71) nm; 1H and 1C NMR spectra are shown in Tables 3 and 4; High-resolution mass spectra m / z 587.2371 [M+Na] + (Calculated value C) 31 H 36N₂O₈, 587.2364 [M+Na] + ).

[0112] Compound 11: White powder, UV spectrum (MeOH)λ max (logε) 235(0.14), 217(0.17), 208(0.19) nm; 1H and 1C NMR spectra are shown in Tables 3 and 4; High-resolution mass spectra m / z 581.2495 [M+H] + (Calculated value C) 31 H 36 N₂O₉, 581.2454 [M+H] + ).

[0113] Table 3. Proton NMR data of compounds 3, 4, 5, 7, 10, and 11

[0114] Note: a) Deuterated reagent is CDCl3, b) Deuterated reagent is DMSO- d6

[0115]

[0116]

[0117] Table 4. Carbon spectral data of compounds 3, 4, 5, 7, 10, and 11. Note: a) Deuterated reagent is CDCl3; b) Deuterated reagent is DMSO- d6

[0118]

[0119] Based on the above analysis, compounds 1-11 obtained have the skeletal structure of cytochalasin-like compounds and are cytochalasin derivatives. The inventors divided the obtained compounds into two categories: one category consists of cytochalasin derivatives containing amino or isoxazole structures, including compounds 1, 2, 6, 8, and 9, as shown below:

[0120]

[0121] Another class consists of nitro-containing cytochalasin derivatives, including compounds 3, 4, 5, 7, 10, and 11, as shown below:

[0122]

[0123] Examples 2 and 3 below also measured the properties of some of the compounds.

[0124] Example 2: Study on the anti-inflammatory effects of compounds 1 and 2

[0125] (I) Cell Culture and Passaging

[0126] (1) Culture of RAW 264.7 macrophages: RAW 264.7 cells were cultured in DMEM complete medium containing 10% fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin and placed in an incubator at 37℃ and 5% CO2. When the cell density reached 80%, the cells were passaged.

[0127] (2) Passaging of RAW 264.7 macrophages: DMEM complete medium and PBS were removed from the 4°C freezer and placed at room temperature. In a clean bench, the supernatant was discarded, the cells were washed with PBS, and 2 mL of fresh complete medium was added. The medium was carefully pipetted onto the cells on the flask wall. Finally, the well-mixed cell suspension was transferred to a culture flask containing fresh complete medium and incubated at 37°C with 5% CO2.

[0128] (II) Cell cryopreservation and thawing

[0129] (1) Cryopreservation of RAW 264.7 macrophages: Collect cell suspension in 15 mL centrifuge tubes, centrifuge at 1500 rpm for 5 min and discard the culture medium. Then add 1 mL of cell cryopreservation solution (95% DMEM + 5% DMSO), mix well by pipetting, transfer to cryopreservation tubes, seal with sealing film, and label with the name of the cryopreserved cells and the cryopreservation date. Finally, place the cryopreservation tubes in a programmed cooling box and freeze at -80℃ for 24 h, then transfer to liquid nitrogen for long-term storage.

[0130] (2) Recovery of RAW 264.7 macrophages: 8 mL of LDM MEM complete medium was added to a 15 mL centrifuge tube in a laminar flow hood beforehand, and a 37°C water bath was prepared. The frozen cells were removed from liquid nitrogen and immediately placed in a 37°C water bath, where they were agitated to rapidly thaw. In the laminar flow hood, the thawed cell suspension was transferred to a prepared centrifuge tube, centrifuged at 1500 rpm for 5 min, the medium was discarded, and 1 mL of fresh complete medium was added and mixed thoroughly. Finally, the mixture was transferred to a culture flask and incubated at 37°C with 5% CO2.

[0131] (III) MTT assay for cell viability

[0132] First, cell viability was assessed using the MTT assay to determine the non-toxic concentration ranges for compounds 1 and 2. The MTT assay procedure is as follows:

[0133] (1) Following the cell passage method, logarithmically growing RAW264.7 cells were pipetted into a cell suspension. After counting under an inverted microscope, the cell density was adjusted to 1×10⁻⁶ cells / cells. 5 100 μL of the sample was inoculated into each well of a 96-well plate and incubated in an incubator for 24 h.

[0134] (2) Compounds 1 and 2 were diluted at 200 μM to four different concentrations. The culture supernatant was carefully discarded. 100 μL of different concentrations of the drug was given to each well in the experimental group, and an equal amount of DMEM complete culture medium was given to the blank group. Six replicates were set up in each group and incubated in an incubator for 24 h.

[0135] (3) Add 10 μL MTT solution to each well under dark conditions, continue incubation in the incubator for 4 h, discard the culture supernatant, add 200 μL LDMSO solution to each well, react in the dark for 10 min, and measure the absorbance value at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0136] (iv) Determination of NO content using a NO reagent kit

[0137] (1) Following the cell passage method, RAW 264.7 cells in the logarithmic growth phase were collected and pipetted into a cell suspension. After counting under an inverted microscope, the cell density was adjusted to 2 × 10⁻⁶ cells / cells. 5 Inoculate 500 μL per well in a 24-well plate and incubate for 24 h.

[0138] (2) Carefully discard the culture supernatant and set up the following groups: ① Blank group: given 500 μL of DMEM complete medium; ② LPS (lipopolysaccharide) group: given LPS at a final concentration of 1 μg / mL; ③ Experimental group: five dose groups (low, medium, and high) were given compounds 1 and 2 at concentrations of 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM, respectively, and then given LPS at a final concentration of 1 μg / mL after 1 h. Incubate in an incubator for 24 h.

[0139] (3) Take the cell culture supernatant, centrifuge at 6000 rpm for 5 min to remove a small number of floating cells, collect the supernatant and test the NO content according to the kit instructions.

[0140] (V) ELISA method for measuring the secretion of pro-inflammatory cytokines

[0141] (1) Cell culture and drug treatment are the same as above.

[0142] (2) The content of IL-6 (interleukin-6) in the collected supernatant was determined according to the instructions of the corresponding ELISA kit (manufacturer: Beijing 4A Biotechnology Co., Ltd.).

[0143] ① Remove the kit from the refrigerator 30 minutes in advance and allow it to equilibrate at room temperature. Dilute the supernatant sample 50 times with sample diluent (except for the blank group).

[0144] ② Set up the following groups respectively: the blank control group is not added with any reagents; the standard group is added with 100 μL of different concentrations of standard; the sample group is added with 100 μL of each sample (the sample here is compound 1).

[0145] ③ Add 50 μL of biotinylated antibody working solution to each well. After mixing, seal the reaction wells with a sealing membrane and incubate at room temperature for 2 hours. Wash each well 4 times with washing buffer, and pat dry any remaining liquid in the wells on absorbent paper.

[0146] ④ Add 100 μL of enzyme conjugate working solution per well and incubate at room temperature for 30 min. Wash the plate 4 times and pat dry any remaining liquid in the wells on absorbent paper.

[0147] ⑤ Add 100 μL of chromogenic reagent to each well under light-protected conditions and incubate at room temperature for 20 min. Finally, add 100 μL of stop solution to each well, mix well, and immediately measure the absorbance at 450 nm using a microplate reader.

[0148] (vi) Western blot detection

[0149] The reagents used are shown in Table 5.

[0150] Table 5. Reagents for Western blot detection

[0151]

[0152] The specific steps are as follows:

[0153] (1) Following the cell passage method, RAW 264.7 cells in the logarithmic growth phase were collected and pipetted into a cell suspension. After counting under an inverted microscope, the cell density was adjusted to 5 × 10⁻⁶ cells / year. 5 Inoculate 2 mL per well in a 6-well plate and incubate for 24 h.

[0154] (2) Discard the culture supernatant and set up the following groups: ① Blank group: 2 mL of complete culture medium (the culture medium here is a complete culture medium prepared by adding double or triple antibodies and serum to DMEM complete culture medium); ② LPS group: LPS with a final concentration of 1 μg / mL; ③ Experimental group: 25 μM, 50 μM and 100 μM of compound 1 were added to the low, medium and high dose groups respectively. After 1 h, LPS with a final concentration of 1 μg / mL was added. Incubate in an incubator for 12 h, discard the culture supernatant, and wash gently twice with PBS.

[0155] (3) Extraction of total cellular protein:

[0156] ① Prepare total protein lysis buffer with a ratio of RIPA lysis buffer: PMSF (phenylmethylsulfonyl fluoride): phosphatase inhibitor = 100:1:1. Add 200 μL to each cell sample, resuspend, and lyse on ice for 30 min.

[0157] ② Centrifuge at 4℃ and 12000rpm for 10min, then transfer the supernatant to a new EP tube, which is the total protein.

[0158] ③ Add 1 / 4 volume of 5× loading buffer to the protein, mix well, and then denature the protein in a 100℃ water bath for 5 min. Store at -20℃.

[0159] (4) Determination of protein concentration using the BCA method:

[0160] ① Prepare BCA working solution with a ratio of BCA:copper reagent = 50:1.

[0161] ② Add 2 μL of protein sample to each well of a 96-well plate and dilute it 10 times with PBS diluent, i.e., 18 μL of PBA diluent needs to be added to each well in advance, and each sample is set up in 3 replicates.

[0162] ③ Add 200 μL BCA working solution to each well, mix thoroughly to avoid generating air bubbles, place in a 37℃ incubator for 30 min, and measure the absorbance at 562 nm using an ELISA reader.

[0163] ④ Substitute the measured absorbance values ​​into the standard curve to calculate the protein concentration. Calculate the loading volume for each protein, using a loading amount of 30 μg.

[0164] (5) SDS-PAGE

[0165] ① Glue Preparation: After cleaning and drying the glass plate, fix it on the glue mixer and prepare the separating glue as required. Slowly add the separating glue into the pores of the glass plate. When it reaches the appropriate height, add isopropanol solution to cover the separating glue and remove air bubbles. Place it in a 37℃ incubator for 30 minutes. After the separating glue solidifies, pour out the isopropanol, gently rinse with distilled water, and then blot dry with absorbent paper. Prepare the stacking glue as required, insert the comb, and place it in a 37℃ incubator for 25 minutes. The preparation methods for the separating glue and the stacking glue are shown in the table below.

[0166] Table 6. Proportioning System for Separating Gel and Stacking Gel

[0167]

[0168] ② Sample loading: Fix the prepared gel onto the electrophoresis tank, add electrophoresis buffer to the tank, and use a micropipette to pick up the protein sample (30 μg) and marker (2 μL).

[0169] ③ Electrophoresis: After adding the sample, first adjust the voltage to 80V and perform electrophoresis on the stacking gel for 30 minutes. When the bands reach the boundary between the stacking gel and the separating gel, adjust the voltage to 120V and perform electrophoresis on the separating gel for 60 minutes.

[0170] ④ Transfer: After electrophoresis, cut the gel within the required protein molecular weight range according to the marker, cut a PVDF membrane of the same size as the gel, soak it in methanol to activate it, place it in the following order: positive electrode - sponge pad - filter paper - PVDF membrane - gel - filter paper - sponge pad - negative electrode, clamp the plate, put it into the transfer tank and add transfer buffer, place it in a -4℃ refrigerator, and transfer at 72V for 60min.

[0171] (6) Immunoblot development

[0172] ① Blocking: After the transfer is completed, the PVDF membrane is taken out and soaked in TBST buffer containing 5% skim milk powder, and then placed on a shaker for 2 hours for blocking.

[0173] ② Primary antibody incubation: Dilute the PVDF membrane with iNOS (nitric oxide synthase) / COX-2 (cyclooxygenase) / β-actin at a ratio of 1:1000:TBST. Cut the membrane according to the corresponding protein molecular weight and immerse it in the corresponding primary antibody dilution solution. Incubate overnight at 4°C. Wash the PVDF membrane 5 times with PBST for 5 minutes each time.

[0174] ③ Secondary antibody incubation: Dilute the rabbit / mouse antibody to TBST at a ratio of 1:2000, immerse the PVDF membrane in the corresponding secondary antibody dilution solution, and incubate at room temperature for 2 hours. Wash the PVDF membrane 5 times with PBST, 5 minutes each time.

[0175] ④ Color development: Developing solution is added to the PVDF film and placed in a chemiluminescence imager for development. Finally, ImageJ is used to measure the grayscale value for quantitative analysis.

[0176] (VII) Experimental Results:

[0177] (1) Effects of compounds 1 and 2 on the viability of RAW264.7 cells

[0178] like Figure 4 As shown, when the concentration of compounds 1 and 2 reached 200 μM, cell viability showed a highly significant decrease (P < 0.001), indicating that both compounds 1 and 2 at 200 μM were cytotoxic. Within the concentration range of 12.5–100 μM, compounds 1 and 2 had no significant effect on cell viability. Therefore, 6.25, 25, 50, and 100 μM were selected as the study doses for subsequent experiments with compounds 1 and 2.

[0179] (2) Effects of compounds 1 and 2 on LPS-induced NO secretion in RAW264.7 cells

[0180] like Figure 5As shown, compared with the blank, LPS stimulation significantly increased the amount of NO secreted by RAW264.7 cells, while 100 and 50 μM of compound 1 and 100 μM significantly inhibited the increase in NO secretion after LPS induction in the experimental group.

[0181] (3) Effect of compound 1 on LPS-induced expression of iNOS protein secreted by RAW264.7 cells

[0182] like Figure 7 As shown, compared with the blank group, LPS stimulation significantly promoted the expression of iNOS protein in RAW264.7 cells, while the 100 and 50 μM compound experimental groups significantly inhibited the expression of iNOS protein after LPS stimulation.

[0183] (4) Effect of compound 1 on LPS-induced IL-6 release in RAW264.7 cells

[0184] like Figure 6 As shown, compared with the control group, LPS stimulation significantly promoted the release of IL-6 in RAW264.7 cells, while the 100 μM compound experimental group significantly inhibited the release of IL-6 in RAW264.7 cells after LPS stimulation.

[0185] (5) Effects of compound 1 on MAPK signaling pathway proteins

[0186] like Figure 8 As shown, compared with the blank, at a concentration of 50 μM, it can significantly inhibit the phosphorylation of p38 and P-ERK proteins (P<0.001); at a concentration of 100 μM, it can simultaneously and significantly inhibit the phosphorylation of p38, ERK and JNK proteins (P<0.001). Therefore, the anti-inflammatory effect of compound 2 may be related to MAPK signaling pathway proteins.

[0187] Example 3: Study on the inhibitory effect of compound 5 on Arabidopsis root growth

[0188] (I) Experimental Procedure:

[0189] 1. Preparation of 1 / 2 MS medium (1 / 2 MS refers to MS medium without agar and sucrose): 2.17g MS (using commonly used components in the field, and not the point of invention of this invention, so it will not be described in detail) , 20g sucrose, 3g plant agar or 7g ordinary agar, adjust the pH to 5.9 with hydrochloric acid, calcium hydroxide or sodium hydroxide, autoclave at 121℃ for 30min, and set aside.

[0190] 2. Arabidopsis seed culture: Disinfect the seed surface with 5% sodium hypochlorite for 5 minutes, then wash 5 times in 200-300 μL of sterile water. Using a 10 μL pipette, aspirate the seeds, ensuring they are arranged in a linear pattern within the pipette tip, and slowly place them into 9 cm of culture medium (1 / 2 MS medium), with 30-50 seeds per medium. Seal the medium with sealing film and place it in a greenhouse at 22℃, with 16 hours of light and 8 hours of darkness. Observe the growth of true leaves after one week.

[0191] 3. Preparation of blank culture medium: Add 100 μg / mL DMSO solution to 25 mL of 1 / 2 MS medium with 0.8% agar, spread it into a petri dish, and set aside. This is the blank control group.

[0192] Preparation of drug-containing culture medium: Set up multiple experimental groups, add compound (compound 5) to 25 mL of 1 / 2 MS medium with 0.8% agar, and make the final concentrations 0.1 μg / mL, 1 μg / mL, 10 μg / mL and 100 μg / mL respectively, and spread them into petri dishes for later use; compound 5 was prepared into multiple final concentrations respectively.

[0193] 4. Inoculation with Arabidopsis thaliana: Select suitable true leaves (4-5 true leaves) and place them in blank and drug-treated square plates, lay them flat in a row in a linear arrangement, 5 in a row, seal with sealing film, place in a greenhouse, temperature 23±1℃, 16 hours of light and 8 hours of darkness treatment, and observe the growth (changes in roots and leaves) after one week.

[0194] Meanwhile, compounds chaetoglobosins D, chaetoglobosins E, and chaetoglobosins G were selected as control experiments. Among them, compounds chaetoglobosins D, chaetoglobosins E, and chaetoglobosins G were prepared according to the method in Chinese Patent A Preparation Method and Application of Chaetoglobosins Compounds (202210877007.6).

[0195] (II) Experimental Results:

[0196] The results showed that at a concentration of 100 μg / mL, compounds 5, chaetoglobosins D, E, and G all exhibited certain inhibitory activity against Arabidopsis root growth. Among them, chaetoglobosins D showed the best activity, inhibiting Arabidopsis growth at a concentration of 10 μg / mL.

Claims

1. A cytochalasin derivative, characterized in that, The structural formula is as follows: , , .

2. The method for preparing the cytochalasin derivative according to claim 1, characterized in that, Includes the following steps: (1) Activation of strain: Inoculate *Chaetoceros madrica* onto PDA agar plates and incubate at 25-30°C for 5-7 days to obtain activated strain; (2) Fermentation culture: The activated strain obtained in step (1) is inoculated into rice solid culture medium and cultured statically at 23~27℃ for 32~35 days to obtain fermentation product; (3) Ethyl acetate extraction: The fermentation product obtained in step (2) is extracted with ethyl acetate. The extract is concentrated under reduced pressure and the ethyl acetate is recovered to obtain crude extract. (4) Diversity-enhancing chemical modification of extracts: First, the crude extract was reacted with 1,1,1-trifluoroacetone, sodium bicarbonate and potassium persulfate to obtain the reaction residue; then the residue was reacted with lanthanum(III) trifluoromethanesulfonate and 2,6-di-tert-butylpyridine to finally obtain the chemically modified diversity-enhancing bacterial extract. (5) ODS reversed-phase silica gel column chromatography for crude separation: The diversity-enhancing bacterial extract obtained in step (4) was subjected to ODS reversed-phase silica gel column chromatography to obtain four components, which were numbered A, B, C, and D in descending order of polarity. (6) Separation and purification of monomeric compounds: The final products were obtained by Sephadex LH-20 gel column chromatography and semi-preparative HPLC purification of components B, C and D in step (5).

3. The preparation method according to claim 2, characterized in that, Step (4) Chemical modification: Dissolve the crude extract in a mixed solvent of acetonitrile and water, add 1,1,1-trifluoroacetone, sodium bicarbonate and potassium persulfate in sequence at -5~5 ℃, stir at -5~5 ℃ for 2~3 h, then add a certain amount of potassium persulfate, stir at -5~5 ℃ for another 2~3 h, pour the reaction mixture into water, extract with ethyl acetate, combine the organic layers, wash, dry, evaporate, and obtain the reaction residue; The residue was dissolved in 1,2-dichloroethane to obtain a solution. Lanthanum(III) trifluoromethanesulfonate and 2,6-di-tert-butylpyridine were added to the solution at room temperature. After reflux for 10-15 h, a certain amount of lanthanum(III) trifluoromethanesulfonate was added, and the mixture was refluxed for another 20-24 h. The reaction mixture was then poured into a 0.2-0.5 M hydrochloric acid solution and extracted with ethyl acetate. The organic layers were combined, washed, and dried to obtain the diversity-enhancing bacterial extract.

4. The preparation method according to claim 2, characterized in that, In step (4), the mass ratio of crude extract to potassium persulfate is (0.48-0.53):1; the molar ratio of 1,1,1-trifluoroacetone, sodium bicarbonate and potassium persulfate is (1-2):2:(3-4); and the mass ratio of crude extract to lanthanum(III) trifluoromethanesulfonate is 5:3-5:

2.

5. The preparation method according to claim 2, characterized in that, In step (5), when performing ODS reversed-phase silica column chromatography, water and methanol are used as the mobile phase for gradient elution with volume ratios of 100:0, 80:20, 40:60, 20:80, and 0:

100.

6. The preparation method according to claim 2, characterized in that, The separation and purification methods for compounds 1 and 2 in step (6) are as follows: Fraction B was separated into five subfractions, B-1 to B-5, by Sephadex LH-20 gel column chromatography using methanol as the mobile phase. Subfractions B-1 and B-2 were further purified by repeated HPLC to obtain compounds 1 and 2, respectively. The structural formulas of compounds 1 and 2 are as follows: , .

7. The preparation method according to claim 2, characterized in that, The method for separating and purifying compound 5 in step (6) is as follows: Fraction D was subjected to Sephadex LH-20 gel column chromatography to obtain three subfractions D-1 to D-3; D-3 was purified by semi-preparative HPLC with water and acetonitrile as the mobile phase. The retention time was... t R =19.4 min, yielding compound 5; The structural formula of compound 5 is as follows: .

8. The use of the cytochalasin derivative according to claim 1 in the preparation of anti-inflammatory drugs, characterized in that, The cytochalasin derivatives are compounds 1 and 2.

9. The use of the cytochalasin derivative according to claim 1 in the preparation of herbicides, characterized in that, The cytochalasin derivative is compound 5.

Images