5 / 8 / 5 clostridiofungin diterpenoid compounds, biosynthetic gene cluster, and uses thereof

Abstract

The present application discloses 5 / 8 / 5 chalicoscladiolide diterpenoid compounds, biosynthesis gene cluster and application thereof, relates to the field of biological medicine technology.The 5 / 8 / 5 chalicoscladiolide biosynthesis gene cluster in Trichoderma harzianum is identified by a genome mining system Thm , and the structural diversification of chalicoscladiolide diterpenoid compounds is obtained by means of heterologous expression, and the obtained compounds are comprehensively evaluated for anti-thrombosis activity, and the results show that compounds 6 and 31 can obviously inhibit vascular thrombosis, wherein, compound 6 has a significant inhibitory effect on collagen-induced platelet aggregation, and compound 31 shows a better inhibitory capacity on thrombin-induced platelet activation. Compounds 6 and 31 exhibit strong anti-thrombosis effect in the models of arterial, venous and pulmonary arterial thrombosis, thereby providing strong technical support for the development of anti-thrombosis drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to 5 / 8 / 5 clostridium diterpenoids, biosynthetic gene clusters, and their applications. Background Technology

[0002] Platelet activation is essential for physiological hemostasis; however, its pathological dysregulation is a fundamental cause of thrombotic diseases—a leading cause of disease and death worldwide. The increasing prevalence of metabolic diseases (especially diabetes and obesity) significantly increases the risk of thrombosis in these populations, creating an urgent clinical need for safer treatment options. Current first-line oral antiplatelet regimens mainly combine cyclooxygenase-1 (COX-1) inhibitors (aspirin) with ADP receptor (P2Y12) antagonists (clopidogrel, prasugrel, or ticagrelor), but are consistently hampered by clinically significant bleeding complications. These limitations stem from the non-selective and irreversible inhibition of basic hemostatic pathways by existing drugs. This persistent "efficacy-bleeding" trade-off has become a core unresolved problem in cardiovascular medicine, driving the academic community to actively explore next-generation antiplatelet drugs that can selectively inhibit pathological thrombus formation without affecting physiological hemostasis.

[0003] Natural products have long been a vital source of innovative drug molecule skeletons targeting complex signaling pathways. Clostridium diterpenes (FCs) are a class of natural products derived from fungi, bacteria, and plants, possessing diverse structural skeletons, including fused carbon-ring systems such as 5 / 8 / 5, 5 / 8 / 6, 5 / 9 / 4, and 5 / 9 / 5, with the 5 / 8 / 5 fused carbon-ring skeleton being the most representative. Some compounds exhibit significant biological activity; for example, alterbrassinic acid A alleviates mitochondrial dysfunction by inhibiting ROS and stabilizing mitochondrial membrane potential, while platelet-coated alterbrassicene A nanoparticles delay aortic valve calcification by inhibiting p65 NF-κB phosphorylation. These findings indicate that FCs have significant potential and clinical prospects in the treatment of cardiovascular diseases. Given the crucial role of platelets in the pathogenesis of atherosclerosis and the unmet clinical need regarding the bleeding risk of current antithrombotic drugs, selectively modulating platelet function is a feasible strategy to decouple antithrombotic efficacy from bleeding risk. Therefore, it is essential to screen for FCs that possess both antithrombotic activity and hemostatic safety in order to achieve the goal of cardiovascular protection.

[0004] The limited abundance of natural products in nature makes them difficult to obtain in large quantities through isolation methods, severely hindering their development and utilization. Introducing biosynthetic gene clusters of secondary metabolites into suitable chassis hosts for heterologous expression can increase the yield of target compounds or promote the discovery of new compounds. This invention aims to identify the 5 / 8 / 5 clostridium diterpenoid biosynthetic gene cluster in *Trichoderma harzianum* using a genome mining system. Thm Furthermore, heterologous expression was used to obtain structurally diverse FCs compounds, in order to screen for FCs compounds with high antithrombotic activity. Summary of the Invention

[0005] The purpose of this invention is to provide 5 / 8 / 5 clostridium diterpenoid compounds, biosynthetic gene clusters, and their applications to address the problems existing in the prior art. The 5 / 8 / 5 clostridium diterpenoid compounds provided by this invention exhibit excellent antithrombotic activity, thus providing strong technical support for the development of antithrombotic drugs.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides the application of 5 / 8 / 5 clostridium diterpenoids in the preparation of antithrombotic drugs, wherein the 5 / 8 / 5 clostridium diterpenoids are compound 6 or compound 31; The structural formulas of compounds 6 and 31 are shown below. Figure 3 .

[0007] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0008] Furthermore, the dosage form of the drug is powder, tablet, granule, capsule, pill, or oral liquid.

[0009] This invention also provides a 5 / 8 / 5 clostridium diterpenoid compound with antithrombotic activity, the structural formula of which is: .

[0010] The present invention also provides an antithrombotic drug, wherein the active ingredient comprises compound 6 or compound 31; The structural formulas of compounds 6 and 31 are shown below. Figure 3 .

[0011] This invention also provides a biosynthetic gene cluster for 5 / 8 / 5 clostridium diterpenoids, comprising genes with nucleotide sequences as shown in SEQ ID NO. 1-6. thmA , thmB , thmC , thmD , thmE and thmF .

[0012] The present invention also provides a combination of co-expression vectors for expressing the above-mentioned biosynthetic gene clusters.

[0013] The present invention also provides a recombinant microbial strain for synthesizing 5 / 8 / 5 clostridium diterpenoids, comprising the above-described co-expression vector combination.

[0014] The present invention also provides the application of the above-mentioned biosynthetic gene clusters, co-expression vector combinations or recombinant microbial strains in the preparation of 5 / 8 / 5 clostridium diterpenoids, wherein the 5 / 8 / 5 clostridium diterpenoids are compound 6 or compound 31; The structural formulas of compounds 6 and 31 are shown below. Figure 3 .

[0015] The present invention also provides a method for preparing a 5 / 8 / 5 clostridium diterpenoid compound, wherein the 5 / 8 / 5 clostridium diterpenoid compound is compound 6 or compound 31; The structural formulas of compounds 6 and 31 are shown below. Figure 3 .

[0016] The preparation method includes the following steps: The above-mentioned recombinant microbial strains were fermented to obtain a fermentation broth; The fermentation broth was extracted and separated to obtain compound 6 or compound 31.

[0017] The present invention discloses the following technical effects: This invention identifies the 5 / 8 / 5 clostridium diterpenoid biosynthesis gene cluster in Trichoderma harzianum using a genome mining system. Thm By employing heterologous expression, structurally diverse FC-like compounds were obtained, and their antithrombotic activity was comprehensively evaluated. The results showed that compounds 6 and 31 significantly inhibited vascular thrombosis. Compound 6 exhibited a significant inhibitory effect on collagen-induced platelet aggregation, while compound 31 showed superior inhibitory ability against thrombin-induced platelet activation. Compounds 6 and 31 demonstrated potent antithrombotic effects in arterial, venous, and pulmonary thrombosis models, thus providing strong technical support for the development of antithrombotic drugs. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 for Thm and Mg Comparative diagram of gene clusters (A) and transformant AO- Thm Extraction ion chromatogram of culture extract (B); Figure 2 Crystal structure diagrams of compounds 4, 5, 6, 11, 16, 24, 25, 26 and 29; Figure 3 The chemical structural formula of compound 1-33; Figure 4 The results of detecting the effect of 5 / 8 / 5 clostridium diterpenoids on platelet intracellular-outward signal transduction are shown in the figure. A shows the aggregation of washed human platelets after pre-incubation with compound 1-33 (200 μM) or a negative control (DMSO), measured by optical transmission aggregation assay (n = 3). B shows the aggregation curves and statistical graphs of the aggregation status recorded by optical transmission aggregation assay after pre-treating washed human platelets with a concentration gradient of compound 6 or DMSO for 5 minutes, followed by stimulation with collagen (1 μg / mL) and thrombin (0.08 U / mL) (n = 4). C shows the aggregation curves and statistical graphs of the aggregation status recorded by optical transmission aggregation assay after pre-treating washed human platelets with a concentration gradient of compound 31 or DMSO for 5 minutes, followed by stimulation with collagen (1 μg / mL) and thrombin (0.08 U / mL) (n = 4). D shows the flow cytometry analysis of platelets after pre-treating with compound 6 (80 μM) (200 μM) or a negative control (DMSO) for 5 minutes. The results of platelet CD62P (P-selectin) expression levels after treatment with DMSO (n = 3-6); E is the result of evaluating the cytotoxic effects of compounds 6 (80 μM) and 31 (80 μM) or DMSO on platelets by lactate dehydrogenase (LDH) release (n = 6); Con represents the group that was stimulated after DMSO pretreatment; Blk represents the group that was not stimulated after DMSO pretreatment. Figure 5The results of detecting the effects of compounds 6 and 31 on platelet outward-inward signal transduction are shown in the following figures: A shows the results of quantifying platelet adhesion on collagen after treatment with compounds 6 (80 μM), 31 (80 μM), or DMSO using alkaline phosphatase assay (n = 5-10); B and C are representative microscopic images (scale bar: 10 μm; n = 3) and statistical graphs of platelet adhesion area after 5 minutes of pretreatment with compounds 6, 31, or carrier, respectively; D is a classification map of platelet morphology based on pseudopodia and platelet pseudopodia (n = 3); E, G, and H are time-series images (n = 3), bar graphs of contraction rate at each time point, and line graphs of contraction rate at each time point, respectively, for the thrombus contraction process of compound 6; H and J are time-series images (n = 3) for the thrombus contraction process of compound 31. 3) Quantitative bar chart and quantitative line chart of contraction rate at each time point; Con represents the group that received stimulation after DMSO pretreatment; Blk represents the group that received no stimulation after DMSO pretreatment; Figure 6 The diagram shows the analysis of compound 6's inhibition of the platelet GPVI signaling pathway; where AB represents the Western blot results and quantitative protein level analysis of PLCγ2 and Syk, respectively; CD represents the Western blot results and quantitative protein level analysis of phosphorylation levels of integrin β3, Src, and FAK, respectively; E is a heatmap of phosphorylation profiles in the GPVI and β3 integrin pathways (based on quantitative phosphorylated proteomics analysis of platelet samples from each group (n = 3), with color markings indicating fold changes relative to the unstimulated control group); F is a molecular docking diagram of compound 6 and Syk (UniProt P43405); G is a platelet cell thermal displacement analysis result showing that compound 6 can stabilize Syk protein within different temperature ranges. Figure 7 The diagram shows the molecular docking of compound 6 with Src (P12931) (A) and FAK (Q05397) (B); Figure 8The diagram shows the analysis of how compound 31 inhibits platelet activation by targeting cytoskeletal regulatory proteins. A and B are Western blot diagrams and quantitative protein level analysis diagrams of the phosphorylation levels of MYPT1, MLCK, Drebrin, and Dematin, respectively. C is a heatmap showing the phosphorylation profile of the RhoA / ROCK / MLCP pathway (based on quantitative analysis of phosphorylated proteomics in each treatment group; values ​​are expressed as normalized fold changes relative to the unstimulated control group). D is a molecular docking diagram of compound 31 with the RhoA (UniProt P61586) GTP binding pocket. E is a diagram showing the results of CETSA analysis of the thermostability of compound 31 on RhoA in platelets under a temperature gradient (n = 3). Figure 9 The following are analytical figures illustrating the effects of compounds 6 and 31 on thrombosis in mice: A shows the detection of vascular occlusion time in a FeCl3-induced mouse carotid artery injury model (n = 6); B shows the quantitative statistical graph of carotid artery occlusion time (n = 6); C shows representative MSB-stained microscopic images (scale bar: 100 μm) of carotid artery thrombus sections from the control group, the compound 6 treatment group, and the compound 31 treatment group; D shows representative MSB-stained images (scale bar: 100 μm) of inferior vena cava thrombus sections from a deep vein thrombosis model; EF shows statistical graphs of thrombus length and weight in the deep vein thrombosis model (n = 6); G shows statistical graphs of survival rate of mice in a bovine thrombin (2500 IU / kg)-induced pulmonary embolism model after pretreatment with DMSO, compound 6 (10 mg / kg), 31 (10 mg / kg), or aspirin (100 mg / kg) (mortality recorded within 15 minutes); H shows representative H&E-stained images of lung tissue sections. Figure 10 The graphs show the effects of compounds 6 and 31 on hemostatic function and platelet biochemical parameters. Among them, AB are statistical graphs of total bleeding time and rebleeding frequency, respectively; CF are statistical graphs of mean platelet volume (MPV), platelet distribution width (PDW), platelet hematocrit (PCT), and large platelet ratio (P-LCR) after 7 consecutive days of administration. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1 Engineered Aspergillus oryzae strain AO- Thm Construction 1. Protofungi Fungal isolation was performed on soil samples collected in August 2020 from Cangyuan Wa Autonomous County, Yunnan Province, China. Based on morphological and rDNA ITS sequence analysis, *Trichoderma harzianum* was isolated and identified. Trichoderma harzianum , GenBank accession No. OR743463).

[0026] 2. Construction of engineered strains Through bioinformatics analysis, *Trichoderma harzianum* isolated above was cloned... Thm Gene clusters (including genes with nucleotide sequences as shown in SEQ ID NO. 1-6) thmA , thmB , thmC , thmD , thmE and thmF Its core diterpene synthase gene. thmA With known Mg Gene cluster core synthase MgMs Having similarity ( Figure 1 (A)

[0027] These genes were constructed into four different Aspergillus oryzae expression plasmids (pAdeA2, pUARA2, pUNA2, and pUSA2) using Gibson assembly technology, forming a co-expression vector combination. thmA and GGPP The recombinant plasmid pAdeA2- was constructed onto pAdeA2. thmA-GGPP (in GGPP This is a gene used to synthesize precursors for 5 / 8 / 5 clostridium diterpenoids, the nucleotide sequence of which is shown in SEQ ID NO.7); thmB , thmC The recombinant plasmid pUARA2 was constructed onto pUARA2, resulting in pUARA2- thmBC ;Will thmD The recombinant plasmid pUNA2 was constructed onto pUNA2 to obtain pUNA2- thmD ;Will thmE and thmF The recombinant plasmid pUSA2 was constructed onto pUSA2, resulting in pUSA2- thmEF Subsequently, using the protoplast-polyethylene glycol (PEG) transformation method, these four recombinant Aspergillus oryzae expression plasmids were co-introduced into the tetratrophic Aspergillus oryzae host strain NSAR1, ultimately successfully obtaining a strain capable of heterologous expression of the entire Aspergillus oryzae strain. Thm Engineered Aspergillus oryzae strain AO- Thm .

[0028] Example 2: Preparation and structural identification of compounds 1-33 1. Preparation of compounds 1-33 (1) The obtained engineered Aspergillus oryzae strain AO- Thm Seed cultures were prepared by culturing on potato dextrose agar (PDA) at 25°C for 4 days. Then, the agar fragments containing the inoculum were inoculated into 100 Erlenmeyer flasks (1L), each containing 200g of rice and 200ml of distilled water (autoclaved). All flasks were incubated at 25°C for 35 days. The fermented rice substrate was extracted eight times at room temperature in a 95% ethanol aqueous solution. The total ethanol extract was obtained by evaporation under vacuum, extracted ten times with ethyl acetate:water = 1:1, and then distilled under reduced pressure to obtain 280g of total extract.

[0029] (2) The total ethyl acetate extract was subjected to silica gel column chromatography (100-200 mesh, 550 g) using petroleum ether:ethyl acetate:methanol (100:0:0, 70:1:0, 50:1:0, 30:1:0, 10:1:0, 8:1:0, 5:1:0, 3:1:0, 1:1:0, 50:50:1, 10:10:1, v / v / v). Similar fractions were analyzed by TLC to obtain 6 fractions (Fr. 1-6). The extract ion chromatograms (EIC) of the extracts are shown in [reference needed]. Figure 1 Compounds 1-33 were obtained by separation of B. Fr.1-Fr.6 using different normal-phase chromatography (petroleum ether / ethyl acetate, petroleum ether / dichloromethane or dichloromethane / methanol system), thin-layer chromatography (petroleum ether / ethyl acetate or dichloromethane / methanol system), reversed-phase chromatography (methanol / water system), gel chromatography (pure methanol or dichloromethane / methanol, 1 / 1, v / v) and high-performance liquid chromatography (methanol / water or acetonitrile / water system), and were named as shown in Table 1.

[0030] Table 1. Nomenclature of Compounds

[0031] 2. Compound structure identification A comprehensive analysis of high-resolution mass spectrometry, ultraviolet spectroscopy, infrared spectroscopy, optical rotation, nuclear magnetic resonance, circular dichroism spectroscopy, and X-ray single-crystal diffraction data of compounds 1-30 was performed to determine the structure of compounds 1-30 (structural formulas are shown in [link to structural formula]). Figure 3 ).

[0032] Compound 1: Colorless oil; - 142 (c 0.1, CH3OH); ECD (c 1, CH3CN) λmax(Δε) = 199 (+5.72), 222 (-9.35) nm; UV (CH3CN) λmax (log ε) = 193 (4.07),220 (3.73); IR (KBr) νmax 3418, 2924, 2855, 1671, 1381, 1037 and 885 cm -1 HRESIMS m / z 398.2306 [M+Na] + (calcd for C 22 H 33 NO4Na, 398.2307); 1 H NMR (600 MHz) and 13 The C NMR (150 MHz) data are shown in Table 2.

[0033] Compound 2: Colorless oil; +142 ( c 0.1, CH3OH), ECD ( c 1, CH3CN) λ max (Δ e )= 204 (+2.37), 224 (-2.63) nm; UV (CH3CN) l max (log ε) = 194 (4.15); IR (KBr) n max 3387, 2923, 2852, 1444, 1065, and 884 cm -1 HRESIMS m / z 400.2462 [M+Na] + (calcd. for C 22 H 35 NO4Na, 400.2464). 1 H NMR (400 MHz) and 13 The C NMR (100 MHz) data are shown in Table 2.

[0034] Compound 3: Colorless oil; -12 ( c 0.1, CH3OH); ECD ( c 0.5, CH3CN) l max (Δ e = 197 (+3.45); UV (CH3CN) l max (log e = 191 (3.90) nm; IR (KBr) n max 3445, 2961,2947, 2930, 1383, 1031, and 882 cm -1 HRESIMS m / z 343.2254 [M+Na] + (calcd. forC 20 H 32 O3Na, 343.2249); 1 H NMR (400 MHz) and 13 The C NMR (100 MHz) data are shown in Table 2.

[0035] Compound 4: Colorless crystal; mp 130.3-133.5℃; +167 ( c 0.1, CH3OH); ECD ( c 0.5 mg / mL, CH3CN) l max (Δ e ) = 217 (+4.62), 328 (+1.57); UV (CH3CN) l max (log e = 192 (3.95) nm; IR (KBr) n max 2952, 2927, 2871, 1749, 1454, 1379 and 864 cm -1 HRESIMS m / z 325.2138 [M+Na] + (calcd. for C 2. H 30 O2Na, 325.2143); 1 H NMR (400 MHz) and 13 The C10 NMR (100 MHz) data are shown in Table 2. Crystal data for compound 4 are as follows: Figure 2 As shown.

[0036] Compound 5: Colorless crystal; mp 130.3-133.5℃; -147 ( c 0.1, CH3OH); ECD ( c 0.5 mg / mL, CH3CN) l max (Δ e ) = 191 (+7.63); UV (CH3CN) l max (log e = 192(4.15) nm; IR (KBr) n max 3435, 2964, 2955, 2910, 2872,1095, 1024, and 885 cm -1 HRESIMS m / z 357.2399 [M+Na] +(calcd. for C 21 H 34 O3Na, 357.2406); 1 H NMR (400 MHz) and 13 C10 NMR (100 MHz) data are shown in Table 3; crystal data for compound 5 are as follows: Figure 2 As shown.

[0037] Compound 6: Colorless crystal; mp 110.1-113.5℃; +243 ( c 0.1, CH3OH); ECD ( c 0.5 mg / mL, CH3CN) l max (Δ e ) = 194 (+5.52); UV (CH3CN) l max (log e = 192(3.92) nm; IR (KBr) n max 3444, 2960, 2945, 2923,1119, 1074, and 902 cm -1 HRESIMS m / z 357.2406 [M+Na] + (calcd. for C 21 H 34 O3Na, 357.2406); 1 H NMR (400 MHz) and 13 C10 NMR (100 MHz) data are shown in Table 3; crystal data for compound 6 are as follows: Figure 2 As shown.

[0038] Compound 7: Colorless oil; -40 ( c 0.1, CH3OH); ECD ( c 0.5 mg / mL, CH3CN) l max (Δ e ) = 192 (+3.01); UV (CH3CN) l max (log e = 192 (4.00) nm; IR (KBr) n max3277,2959, 2940, 2921, 1057, 1025, and 890 cm -1 HRESIMS m / z 373.2354 [M+Na] + (calcd.for C 21 H 34 O4Na, 373.2355); 1 H NMR (400 MHz) and 13 The C NMR (100 MHz) data are shown in Table 3.

[0039] Compound 8: Colorless oil; -20 ( c 0.1, CH3OH); ECD ( c 1 mg / mL, CH3CN) l max (Δ e ) = 196 (+1.41); UV (CH3CN) l max (log e = 192 (3.91) nm; IR (KBr) n max 3446,2957, 2924, 2852, 1047, and 884 cm -1 HRESIMS m / z 373.2357 [M+Na] + (calcd. forC 21 H 34 O4Na, 373.2355); 1 H NMR (400 MHz) and 13 The C NMR (100 MHz) data are shown in Table 3.

[0040] Compound 9: Colorless oil; -twenty four ( c 0.1, CH3OH); ECD ( c 0.5 mg / mL, CH3CN) l max (Δ e ) = 191 (+1.50); ​​UV (CH3CN) l max (log e = 192 (4.01) nm; IR (KBr) n max 3489,2971, 2922, 2876, 1324, 1020, and 886 cm -1 HRESIMS m / z 373.2353 [M+Na] + (calcd.for C 21 H 34 O4Na, 373.2355); 1 H NMR (400 MHz) and 13 The C NMR (100 MHz) data are shown in Table 4.

[0041] Compound 10: Colorless oil; -112 ( c 0.1, CH3OH); ECD ( c 1 mg / mL, CH3CN) l max (Δ e ) = 198 (+2.54); UV (CH3CN) l max (log e = 193 (4.00) nm; IR (KBr) n max 3446,2955, 2926, 1096, 1063, and 891 cm -1 HRESIMS m / z 373.2355 [M+Na] + (calcd. forC 21 H 34 O4Na, 373.2355); 1 H NMR (400 MHz) and 13 The C NMR (100 MHz) data are shown in Table 4.

[0042] Compound 11: Colorless crystal; mp 128.5-132.4℃; -93 ( c 0.1, CH3OH); ECD ( c 0.5 mg / mL, CH3CN) l max (Δ e ) = 192 (+6.79); UV (CH3CN) lmax (log e = 193(4.00) nm; IR (KBr) n max 3474, 3416, 2953, 2919, 1113, 1040, and 885 cm -1 HRESIMS m / z 343.2242 [M+Na] + (calcd. for C 20 H 32 O3Na, 343.2249); 1 H NMR (400 MHz) and 13 The C10 NMR (100 MHz) data are shown in Table 4; the crystal data of compound 11 are as follows: Figure 2 As shown.

[0043] Compound 12: Colorless oil; -20 ( c 0.1, CH3OH); ECD ( c 1 mg / mL, CH3CN) l max (Δ e ) = 191 (+1.40); UV (CH3CN) l max (log e = 192 (3.82) nm; IR (KBr) n max 3417,2961, 2925, 2872, 1668, 1380, and 887 cm -1 HRESIMS m / z 359.2193 [M+Na] + (calcd.for C 20 H 32 O3Na, 359.2198); 1 H NMR (600 MHz) and 13 The C NMR (150 MHz) data are shown in Table 4.

[0044] Compound 13: Colorless oil; -twenty one ( c 0.1, CH3OH); ECD ( c 1 mg / mL, CH3CN) l max (Δ e ) = 194 (+0.84), 213 (+1.37); UV (CH3CN) l max (log e = 193 (3.67) nm; IR(KBr) n max 3422, 2922, 2862, 2872, 1658, and 1049 cm -1 HRESIMS m / z 357.2040 [M+Na] + (calcd. for C 20 H 30 O3Na, 357.2042); 1 H NMR (600 MHz) and 13 The C NMR (150 MHz) data are shown in Table 5.

[0045] Compound 14: Colorless oil; -170 ( c 0.1, CH3OH); ECD ( c 1 mg / mL, CH3CN) l max (Δ e ) = 213 (+1.55), 231 (-1.07); UV (CH3CN) l max (log e = 193 (4.11) nm; IR(KBr) n max 3366, 2955, 2927, 1758, 1206, and 1010 cm -1 HRESIMS m / z 355.1882 [M+Na] + (calcd. for C 20 H 28 O4Na, 355.1885); 1 H NMR (600 MHz) and 13 The C NMR (150 MHz) data are shown in Table 5.

[0046] Compound 15: Colorless oil; +125 ( c 0.1, CH3OH); ECD (c 1 mg / mL, CH3CN) l max (Δ e ) = 203 (+3.30), 309 (+0.35); UV (CH3CN) l max (log e ) = 192 (4.05) nm; IR(KBr) n max 3422, 2951, 2929, 2871, 1695, 1451, 1379, and 887 cm -1 HRESIMS m / z 313.2143 [M+Na] + (calcd. for C 19 H 30 O2Na, 313.2143); 1 H NMR (400 MHz) and 13 The C NMR (100 MHz) data are shown in Table 5.

[0047] Compound 16: Colorless crystal; mp 74.2-77.6℃; +72 ( c 0.1, CH3OH); ECD ( c 0.5 mg / mL, CH3CN) l max (Δ e ) = 210 (+1.23), 260 (+3.96); UV (CH3CN) l max (log e ) = 191 (4.08), 259 (3.94) nm; IR (KBr) n max 2924, 2870, 1708, 1376, 1179, and887 cm -1 HRESIMS m / z 309.2195 [M+Na] + (calcd. for C 20 H 30 ONa, 316.2194); 1 H NMR (400MHz) and 13C10 NMR (100 MHz) data are shown in Table 5; crystal data for compound 16 are shown in Table 5. Figure 2 As shown.

[0048] Compound 17: Colorless oil; +45 ( c 0.1, CH3OH); ECD ( c 0.5 mg / mL, CH3CN) l max (Δ e ) = 209 (+1.54), 262 (+3.87); UV (CH3CN) l max (log e ) = 191 (4.02), 258(3.86) nm; IR (KBr) n max 3298, 2947, 2931, 1705, 1639, 1186, 1098, and 890 cm -1 HRESIMS m / z 325.2142 [M+Na] + (calcd. for C 20 H 30 O2Na, 325.2143); 1 H NMR (400 MHz) and 13 The C NMR (100 MHz) data are shown in Table 6.

[0049] Compound 18: Colorless oil; +66 ( c 0.1, CH3OH); ECD ( c 0.25 mg / mL, CH3CN) l max (Δ e ) = 210 (+1.23), 260 (+3.96); UV (CH3CN) l max (log e ) = 191 (3.88), 259(3.72) nm; IR (KBr) n max 3434,3352, 2921, 2853, 1703, 1630, 1181, 1060, and 888cm -1 HRESIMS m / z325.2144 [M+Na] + (calcd. for C 20 H 30 O2Na, 325.2143); 1 H NMR (400MHz) and 13 The C NMR (100 MHz) data are shown in Table 6.

[0050] Compound 19: Colorless oil; +222 ( c 0.1, CH3OH); ECD ( c 0.25 mg / mL, CH3CN) l max (Δ e ) = 213 (+1.17), 260 (+6.20); UV (CH3CN) l max (log e ) = 192 (4.06),259 (3.95) nm; IR (KBr) n max 3274, 2952, 2925, 2868, 1707, 1632, 1179, 1064, and890 cm -1 HRESIMS m / z 325.2147 [M+Na] + (calcd. for C 20 H 30 O2Na, 325.2143); 1 H NMR (400 MHz) and 13 The C NMR (100 MHz) data are shown in Table 6.

[0051] Compound 20: Colorless oil; +35 ( c 0.1, CH3OH); ECD ( c 0.25 mg / mL, CH3CN) l max (Δ e ) = 215 (+1.48), 259 (+6.89); UV (CH3CN) l max (log e ) = 192 (4.12), 256(4.04) nm; IR (KBr) nmax 3466, 2956, 2927, 2870, 1692, 1627, 1184, and 894 cm -1 HRESIMS m / z 325.2146 [M+Na] + (calcd. for C 20 H 30 O2Na, 325.2143); 1 H NMR (400 MHz) and 13 The C NMR (100 MHz) data are shown in Table 6.

[0052] Compound 21: Colorless oil; +96 ( c 0.1, CH3OH); ECD ( c 0.5 mg / mL, CH3CN) l max (Δ e ) = 215 (+1.45), 260 (+5.69); UV (CH3CN) l max (log e ) = 192 (3.65), 260(3.91) nm; IR (KBr) n max 3435, 2927, 2873, 1705, 1634, 1376, 1181, and 938 cm -1 HRESIMS m / z 327.2303 [M+Na] + (calcd. for C 20 H 32 O2Na, 327.2300); 1 H NMR (400 MHz) and 13 The C NMR (100 MHz) data are shown in Table 7.

[0053] Compound 22: Colorless oil; -598 ( c 0.1, CH3OH); ECD ( c 0.25 mg / mL, CH3CN) l max (Δ e) = 191 (+6.73), 209 (-5.17), 260 (-10.01); UV (CH3CN) l max (log e ) =194 (3.95), 262 (3.88) nm; IR (KBr) n max 3433, 2956, 2926, 2864, 1704, 1616,1380, 1166 and 1000 cm -1 HRESIMS m / z 325.2139 [M+Na] + (calcd. for C 20 H 30 O2Na, 325.2143); 1 H NMR (400 MHz) and 13 The C NMR (100 MHz) data are shown in Table 7.

[0054] Compound 23: Colorless oil; +22 ( c 0.1, CH3OH); ECD ( c 0.25 mg / mL, CH3CN) l max (Δ e ) = 194 (-6.55), 242 (+5.04); UV (CH3CN) l max (log e ) = 192 (4.07), 244(4.01) nm; IR (KBr) n max 3411, 2954, 2925, 2871, 1675, 1613, 1457, and 886 cm -1 HRESIMS m / z 325.2148 [M+Na] + (calcd. for C 20 H 30 O2Na, 325.2143); 1 H NMR (600 MHz) and 13 The C NMR (150 MHz) data are shown in Table 7.

[0055] Compound 24: Colorless crystal; mp 81.2-82.3 ℃; -100 ( c 0.1, CH3OH); ECD ( c 0.5 mg / mL, CH3CN) l max (Δ e ) = 192 (+3.84); UV (CH3CN) l max (log e = 192(3.97); IR (KBr) n max 3389, 2922, 2852, 1446, 1380, 1104, and 885 cm -1 HRESIMS m / z 345.2406 [M+Na] + (calcd. for C 20 H 34 O3Na, 345.2406); 1 H NMR (400 MHz) and 13 The C10 NMR (100 MHz) data are shown in Table 7; the crystal data for compound 24 are as follows: Figure 2 As shown.

[0056] Compound 25: Colorless crystal; mp 116.8-119.9 ℃; -172 ( c 0.1,CH3OH); ECD ( c 0.5 mg / mL, CH3CN) l max (Δ e ) = 196 (+4.74); UV (CH3CN) l max (log e =192 (4.10); IR (KBr) n max 3431, 2958, 2927, 2859, 1641, 1380 and 888 cm -1 HRESIMS m / z 329.2450 [M+Na] + (calcd. for C 20 H34 O2Na, 329.2457); 1 H NMR (400 MHz) and 13 C10 NMR (100 MHz) data are shown in Table 8; crystal data for compound 24 are as follows: Figure 2 As shown.

[0057] Compound 26: Colorless crystal; mp 193.7-194.4 ℃; -91 ( c 0.1,CH3OH); ECD ( c 0.5 mg / mL, CH3CN) l max (Δ e ) = 197 (+5.24); UV (CH3CN) l max (log e =192 (3.86); IR (KBr) n max 3374, 2947, 2932, 2968, 1466, 1378, 1010, and 882 cm -1 HRESIMS m / z 329.2457 [M+Na] + (calcd. for C 20 H 34 O2Na, 329.2457); 1 H NMR (600 MHz) and 13 C10 NMR (150 MHz) data are shown in Table 8; crystal data for compound 26 are shown in Table 8. Figure 2 As shown.

[0058] Compound 27: Colorless oil; -84 ( c 0.1, CH3OH); ECD ( c 0.5 mg / mL, CH3CN) l max (Δ e ) = 195 (+3.58); UV (CH3CN) l max (log e = 192 (4.00); IR (KBr) n max3440,2949, 2919, 2661, 1457, 1381, 1173, and 884 cm -1 HRESIMS m / z 329.2458 [M+Na] + (calcd. for C 20 H 34 O2Na, 329.2457); 1 H NMR (600 MHz) and 13 The C NMR (150 MHz) data are shown in Table 8.

[0059] Compound 28: Colorless crystal; -28 ( c 0.1, CH3OH); ECD ( c 0.5 mg / mL, CH3CN) l max (Δ e ) = 194 (+2.62); UV (CH3CN) l max (log e = 192 (3.98); IR (KBr) n max 3410, 2956, 2924, 2855, 1468, 1377, 1072, and 885 cm -1 HRESIMS m / z 329.2454 [M+Na] + (calcd. for C 20 H 34 O2Na, 329.2457); 1 H NMR (400 MHz) and 13 The C NMR (100 MHz) data are shown in Table 8.

[0060] Compound 29: Colorless crystal; mp 113.3-115.5 ℃; -75 ( c 0.1,CH3OH); ECD ( c 0.5 mg / mL, CH3CN) l max (Δ e ) = 192 (+3.86); UV (CH3CN) l max(log e =191 (4.02); IR (KBr) n max 3355, 2956, 2885, 2870, 1376, 1055, 1302, and 896 cm -1 HRESIMS m / z 329.2455 [M+Na] + (calcd. for C 20 H 34 O2Na, 329.2457); 1 H NMR (400 MHz) and 13 C10 NMR (100 MHz) data are shown in Table 9; crystal data for compound 29 are as follows: Figure 2 As shown.

[0061] Compound 30: Colorless oil; -15 ( c 0.1, CH3OH); HRESIMS m / z 343.2244 [M+Na] + (calcd. for C 20 H 32 O3Na, 343.2249); 1 H NMR (600 MHz) and 13 The C NMR (150 MHz) data are shown in Table 9.

[0062] Table 2. Compounds 1-4 1 H and 13 C NMR data ( d in ppm, J (in Hz)

[0063] Note: a Recorded in DMSO- d 6. b Recorded in CD13 c Signals were overlapped. Table 3. Compounds 5-8 1 H and 13 C NMR data ( d in ppm, J (in Hz)

[0064] Note: a Recorded in CD13 b Recorded on CD3OD. c Signals were overlapped. Table 4. Compounds 9-12 1 H and 13 C NMR data ( d in ppm, J (in Hz)

[0065] Note: a Recorded in DMSO- d 6. b Recorded in CD13. c Signals were overlapped. Table 5. Compounds 13-16 1 H and 13 C NMR data ( d in ppm, J (in Hz)

[0066] Note: a Recorded in DMSO- d 6. b Recorded in CD13 c Signals were overlapped. Table 6. Compounds 17-20 1 H and 13 C NMR data ( d in ppm, J (in Hz)

[0067] Note: a Recorded in CD13 b Recorded on CD3OD. c Signals were overlapped. Table 7. Compounds 17-20 1 H and 13 C NMR data ( d in ppm, J (in Hz, deuterated chloroform)

[0068] Note: a Signals were overlapped. Table 8. Compounds 25-28 1 H and 13 C NMR data ( d in ppm, J (in Hz)

[0069] Note: a Recorded in DMSO- d 6. b Recorded in CD13. c Signals were overlapped. Table 9. Compounds 29 and 30 1 H and 13 C NMR data ( d in ppm, J (in Hz)

[0070] Note: a Recorded in DMSO- d 6. b Recorded on CD3OD. c Signals were overlapped. Example 3: Effects of compounds 1-33 on intra- and out-of-platelet signal transduction To evaluate the antithrombotic potential of compounds 1-33, this invention first examined their effect on thrombin-induced platelet aggregation. Washed human platelets were pre-incubated with compound 1-33 (200 μM) or a negative control (DMSO) for 5 minutes, followed by stimulation of platelet aggregation with thrombin (0.08 U / mL). Aggregation was measured by optical transmission aggregation assay (n=3), and the results are shown below. Figure 4 In the initial screening, compounds 5, 6, 31, and 33 significantly inhibited thrombin-induced platelet aggregation.

[0071] To evaluate functional selectivity, this invention further analyzed the effects of compounds 6 and 31 on various physiological platelet agonists, including ADP, collagen, and thrombin. Human platelets were washed and pretreated for 5 minutes with concentration gradients of compounds 6, 31, or DMSO, followed by stimulation with collagen (1 μg / mL) and thrombin (0.08 U / mL); a solvent control group was also included, pretreated with DMSO without any stimulation. Aggregation curves were recorded using optical transmission aggregation assay (n = 4). Flow cytometry was used to analyze the expression levels of CD62P (P-selectin) in platelets treated with compounds 6 (80 μM), 31 (80 μM), or DMSO. n = 3-6). The cytotoxic effects of compounds 6 (80 μM) and 31 (80 μM) or DMSO on platelets were assessed by assessing the release of lactate dehydrogenase (LDH). n = 6).

[0072] The test results showed that both compounds exhibited agonist-dependent inhibitory characteristics, with compound 6 showing a stronger inhibitory effect on collagen-induced aggregation. Figure 4 Compound B); while compound 31 is more effective against thrombin-induced aggregation ( Figure 4 (C). P-selectin translocates from α-granules to the platelet surface during platelet activation and is used as a sensitive marker of platelet activation. Flow cytometry analysis confirmed that compounds 6 and 31 significantly inhibited collagen-induced P-selectin exposure. Figure 4 (D). Furthermore, no significant increase in lactate dehydrogenase (LDH) was detected, indicating that this antiplatelet effect was not caused by cytotoxicity. Figure 4 (E).

[0073] Example 4: Effects of compounds 6 and 31 on platelet outward-inward signal transduction Platelet out-to-in signal transduction is a crucial component of platelet activation. This mechanism transmits integrin-mediated extracellular signals, thereby enhancing platelet adhesion, aggregation, and secretion. Furthermore, it plays a key role in regulating hemostasis and thrombosis. To test the effects of compounds 6 and 31 on platelet out-to-in signal transduction, this invention used an alkaline phosphatase assay to quantify platelet adhesion to collagen after treatment with compounds 6 (80 μM), 31 (80 μM), or DMSO; a solvent control group treated with DMSO without any stimulation was also included. The results showed that in the presence of compounds 6 and 31, collagen-mediated platelet adhesion (… Figure 5 (A) and spreading ( Figure 5 Both compounds significantly inhibited the activity of platelets (B). They also significantly reduced the average spreading area of ​​individual platelets. Figure 5(C), and inhibited the formation of filopodia and platypodia ( Figure 5 These results indicate that compounds 6 and 31 strongly inhibit key functional aspects of platelet out-in signal transduction. To further evaluate the effects of these compounds on mouse platelet out-in signal transduction, we performed a clot retraction experiment over time. Compared with the control group, platelets treated with compounds 6 and 31 showed a significant inhibitory effect (D). Figure 5 (China EJ).

[0074] Example 5: Compound 6 inhibits GPVI-mediated platelet signaling pathway Given the potent inhibitory effect of compound 6 on collagen-induced platelet activation, its mechanism of action was further explored by examining key downstream signaling events of the collagen receptor. Human platelets were pretreated with compound 6 (80 µM) and then stimulated with collagen for cell lysis. The phosphorylation levels of PLCγ2, Syk, integrin β3, Src, and FAK were detected by Western blotting. The results showed that compound 6 significantly inhibited the phosphorylation of Syk, PLCγ2, β3 integrin, Src, and FAK. Figure 6 The presence of AD in the compound suggests that these signaling molecules are involved in its inhibitory activity.

[0075] Phosphorylated proteomics analysis further revealed significant downregulation of key phosphorylation sites, including the PLCγ2-T4 site and Src-S51 site, which are crucial for PLCγ2 activity, as well as the autophosphorylation sites Y570, Y576, S29, and S910 of FAK. Figure 6 These results indicate that compound 6 weakens downstream pathways crucial for platelet activation and thrombosis by interfering with upstream signaling mediators (Syk, PLCγ2, β3 integrin, Src, and FAK).

[0076] Molecular docking analysis showed that compound 6 had strong binding affinity to Syk (-7.0 kcal / mol), Src (-7.7 kcal / mol), and FAK (-7.7 kcal / mol). Figure 6 China F and Figure 7 This supports its multi-target mechanism of action. Although Syk's binding energy is slightly lower, its inhibition still has key functional significance due to its central role in the collagen / GPVI pathway. The interaction with Syk was further validated by cellular thermal displacement analysis (CETSA): under thermal denaturation conditions of 65-70℃, compound 6 stabilized Syk protein and reduced its degradation compared to the DMSO control group. Figure 6(G). This specific stabilizing effect on Syk, combined with the balanced inhibition of multiple kinases such as Syk, PLCγ2, integrin β3, Src, and FAK, constitutes the molecular basis for compound 6's effective blocking of the early GPVI-mediated signaling pathway.

[0077] Example 6: Compound 31 inhibits platelet activation by targeting cytoskeletal regulatory proteins. Based on the potent antiplatelet effect of compound 31 against thrombin-induced aggregation, key signaling molecules involved in cytoskeleton remodeling were systematically analyzed to further investigate its mechanism of action. Human platelets were pretreated with compound 31 (80 µM) and washed, then lysed after collagen stimulation. The phosphorylation levels of MYPT1, MLCK, Drebrin, and Dematin were detected by Western blotting. The results showed that this compound specifically targets core proteins involved in the dynamic regulation of the cytoskeleton—a process fundamental to platelet activation. It significantly inhibited the phosphorylation of MYPT1 and MLCK, key regulators of myosin light chain phosphatase activity and actin contractility, respectively. Simultaneously, compound 31 downregulated the expression of actin-binding proteins Drebrin (F-actin stabilizing factor) and Dematin (involved in actin tufting). Figure 8 (A and B). Phosphorylated proteomics analysis identified the RhoA / ROCK / MLCP pathway as its core target: compound 31 weakens actomyosin contractility by inhibiting RhoA activation, reducing ROCK1-dependent MYPT1 phosphorylation, enhancing MLCP activity, and attenuating MLC phosphorylation. Furthermore, inhibition of Drebrin and Dematin expression disrupts actin cytoskeleton stability and filopodia formation. Figure 8 (C). Molecular docking analysis confirmed that compound 31 has a high affinity for the RhoA GTP binding pocket (P61586), with a binding energy of -7.0 kcal / mol. Figure 8 (D). The thermostabilizing effect of compound 31 on RhoA in platelets under a temperature gradient was detected by cellular thermal displacement analysis (CETSA). The results showed that, under thermal denaturation conditions of 65-70℃, compound 31 significantly stabilized RhoA protein (D) compared with the DMSO control group. Figure 8 (E).

[0078] Example 7 Effects of compounds 6 and 31 on thrombosis in mice This invention evaluated the antithrombotic efficacy of compounds 6 and 31 using three established mouse thrombosis models. After treatment with DMSO, compound 6 (10 mg / kg), compound 31 (10 mg / kg), or aspirin (100 mg / kg), respectively, in a carotid artery injury model, 1 mm × 1 mm filter paper was soaked in 10% ferric chloride solution and then attached to the blood vessel surface for 1 minute to induce thrombus formation. Hemodynamic changes were then monitored in real time using Doppler ultrasound until a sustained and significant reduction in blood flow was observed (maximum recording time 20 minutes). In a deep vein thrombosis model, mice were grouped and administered the same medication regimen. After isoflurane inhalation anesthesia, the inferior vena cava was exposed via laparotomy and ligated with a 0.2-0.3 mm diameter blunt-tipped wire to establish a standardized stenosis model. The thrombus was removed 48 hours post-surgery for weighing and histological analysis. In the thrombin-induced acute pulmonary embolism model, the mice were grouped and administered the same drugs as before. After 30 minutes, bovine thrombin (2500 IU / kg) was injected into the tail vein to induce thrombus formation. The behavioral changes and survival of the experimental animals were continuously observed. Mice that survived for more than 15 minutes were humanely euthanized, and lung tissue was taken for perfusion fixation, paraffin embedding, and tissue staining analysis.

[0079] Under Doppler flow monitoring, the vascular occlusion time in a FeCl3-induced mouse carotid artery injury model was measured after treatment with DMSO, compound 6 (10 mg / kg), 31 (10 mg / kg), or aspirin (100 mg / kg). The results showed that in the FeCl3-induced carotid artery injury model, both compounds (10 mg / kg) significantly prolonged vascular occlusion time and reduced thrombus formation. Notably, aspirin (positive control) required a ten-fold higher dose (100 mg / kg) to achieve a comparable antithrombotic effect. Figure 9 (A and B). Similar therapeutic effects were also observed in MSB staining results of mouse carotid artery thrombus sections ( Figure 9 (C). In an inferior vena cava stenosis model, treatment with compounds 6 and 31 (10 mg / kg) significantly reduced thrombus weight and length, with efficacy comparable to 100 mg / kg aspirin. Figure 9 In a thrombin-induced pulmonary embolism model, both compounds (10 mg / kg) significantly improved survival and reduced pulmonary thrombus burden compared to 100 mg / kg aspirin. Figure 9 (G). Histological analysis further confirmed that, compared with the thrombin-only group, mice treated with compounds 6 and 31 showed a significant reduction in pulmonary vascular thrombosis (G). Figure 9 (H). In summary, these results indicate that compounds 6 and 31 exhibit potent antithrombotic effects in arterial, venous, and pulmonary thrombosis models, and are superior to aspirin at significantly lower doses.

[0080] Example 8: Effects of compounds 6 and 31 on hemostatic function and platelet biochemical parameters The in vivo safety of compounds 6 and 31 was systematically evaluated to investigate their potential side effects. Administered 30 minutes preoperatively (compounds 6 and 31, 10 mg / kg or DMSO), followed by a tail transection hemorrhage test. Results showed that, compared with the control group, neither compound significantly prolonged bleeding time or increased the incidence of rebleeding. Figure 10 The presence of AB indicates that it has no significant effect on physiological hemostasis. To determine whether repeated administration affects platelet production or baseline hematological parameters, mice were injected daily with each compound for 7 days—covering the complete platelet turnover cycle. Peripheral blood analysis showed no significant changes in mean platelet volume (MPV), platelet distribution width (PDW), plateletcrit (PCT), and large platelet ratio (PLCR). Figure 10 The results (CF) indicate that neither compound 6 nor 31 impairs platelet production or alters the integrity of circulating platelets. These results suggest that compounds 6 and 31 have good safety profiles, minimal impact on hemostasis, and no adverse effects on platelet production or baseline hematological parameters even after prolonged treatment.

[0081] In summary, compound 6 significantly inhibited collagen-induced platelet aggregation, while compound 31 showed superior inhibitory activity against thrombin-induced platelet activation. The antiplatelet effects of these two natural small molecule compounds were further validated in various animal models of thrombosis. Mechanistic studies revealed the dual-pathway inhibitory effect of compound 6: (1) by inhibiting Syk tyrosine kinase autophosphorylation and PLCγ2 activation, it weakened GPVI / FcRγ-ITAM signaling; (2) by inhibiting β3 subunit phosphorylation and subsequent FAK / Src complex dissociation, it disrupted the bidirectional signaling of αIIbβ3 integrin. Compound 31 inhibited platelet activation by inhibiting the Rho GTPase / ROCK1 signaling axis, reducing MLC phosphorylation levels and Derbrin / Dematin expression, and affecting GSK3β activity, thereby disrupting cytoskeleton remodeling.

[0082] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

The application of 1.5 / 8 / 5 clostridium diterpenoids in the preparation of antithrombotic drugs, characterized in that, The 5 / 8 / 5 clostridium diterpenoid compound is compound 6; The structural formula of compound 6 is: .

2. The application according to claim 1, characterized in that, The drug also includes pharmaceutically acceptable excipients.

3. The application according to claim 2, characterized in that, The dosage form of the drug is powder, tablet, granule, capsule, pill or oral liquid.

4. A 5 / 8 / 5 clostridium diterpenoid compound with antithrombotic activity, characterized in that, Its structural formula is: 。 5. An antithrombotic drug, characterized in that, The active ingredient includes compound 6; The structural formula of compound 6 is: .

6. A biosynthetic gene cluster for a 5 / 8 / 5 clostridium diterpenoid compound, characterized in that, Genes including those with nucleotide sequences as shown in SEQ ID NO. 1-6 thmA , thmB , thmC , thmD , thmE and thmF .

7. A co-expression vector for expressing the biosynthetic gene cluster of claim 6, characterized in that, The co-expression vector comprises the biosynthetic gene cluster described in claim 6.

8. A recombinant microbial strain for synthesizing 5 / 8 / 5 clostridium diterpenoids, characterized in that, Includes the co-expression vector as described in claim 7.

9. The application of the biosynthetic gene cluster as described in claim 6, the co-expression vector as described in claim 7, or the recombinant microbial strain as described in claim 8 in the preparation of 5 / 8 / 5 clostridium diterpenoid compounds, characterized in that, The 5 / 8 / 5 clostridium diterpenoid compound is compound 6; The structural formula of compound 6 is: .

10. A method for preparing a 5 / 8 / 5 clostridium diterpenoid compound, characterized in that, The 5 / 8 / 5 clostridium diterpenoid compound is compound 6; The structural formula of compound 6 is: ; The preparation method includes the following steps: The recombinant microbial strain described in claim 8 is fermented to obtain a fermentation broth; The fermentation broth was extracted and separated to obtain compound 6.

Images