5 / 8 / 5 chitin diterpenoid compound, biosynthetic gene cluster and application of biosynthetic gene cluster

Abstract

The invention discloses a 5 / 8 / 5 chitosan clostridium diterpenoid compound, a biosynthetic gene cluster and application of the biosynthetic gene cluster, and relates to the technical field of biological medicines. A 5 / 8 / 5 chitin type diterpenoid biosynthetic gene cluster Thm in trichoderma harzianum is identified through a genome mining system, chitin diterpenoid compounds with diversified structures are obtained by means of heterologous expression, the obtained compounds are subjected to anti-thrombotic activity comprehensive evaluation, and the result shows that the compounds 6 and 31 can obviously inhibit vascular thrombosis, the compound 6 has a remarkable inhibition effect on collagen-induced platelet aggregation, and the compound 31 has a better inhibition capability on thrombin-induced platelet activation. The compounds 6 and 31 show powerful antithrombotic effects in artery, vein and pulmonary artery thrombosis models, so that powerful technical support is provided for development of antithrombotic drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to 5 / 8 / 5 chalcidicin diterpenoids, biosynthetic gene cluster and application thereof. BACKGROUND

[0002] Platelet activation is indispensable in physiological hemostasis, while its pathological disorder is the fundamental cause of thrombotic diseases, which are the main culprits of morbidity and mortality worldwide. The increasing prevalence of metabolic diseases (especially diabetes and obesity) significantly increases the risk of thrombosis in this population, thus giving rise to an urgent clinical need for safer treatment options. The current first-line oral antiplatelet regimen mainly combines the cyclooxygenase-1 (COX-1) inhibitor (aspirin) and the ADP receptor (P2Y12) antagonist (clopidogrel, prasugrel or ticagrelor), but is always plagued by clinically significant bleeding complications. These limitations are due to the non-selective and irreversible inhibition of the existing drugs on the basic hemostatic pathway. This persistent "efficacy-bleeding" trade-off paradox has become a core problem in the cardiovascular medicine field, which has prompted the academic community to actively explore the next generation of antiplatelet drugs that can selectively inhibit pathological thrombosis while not affecting physiological hemostatic function.

[0003] Natural products have always been an important source of developing innovative drug molecules targeting complex signaling pathways. Chalcidicin diterpenoids (FCs) are a class of natural products that can be derived from fungi, bacteria and plants, with diverse structural scaffolds, including 5 / 8 / 5, 5 / 8 / 6, 5 / 9 / 4 and 5 / 9 / 5 fused carbon ring systems, among which the 5 / 8 / 5 fused carbon ring scaffold is the most representative. Some compounds exhibit significant biological activity, such as alterbrassinic acid A, which reduces mitochondrial dysfunction by inhibiting ROS and stabilizing mitochondrial membrane potential, and platelet membrane-coated alterbrassicene A nanoparticles, which delay aortic valve calcification by inhibiting p65 NF-κB phosphorylation, which indicates that FCs have significant potential and clinical prospects in the treatment of cardiovascular diseases. Given the key role of platelets in the pathogenesis of atherosclerosis and the unmet clinical need for bleeding risk of current anti-thrombotic drugs, selective regulation of platelet function is a feasible strategy to decouple anti-thrombotic efficacy and bleeding risk. Therefore, it is necessary to screen FCs with anti-thrombotic activity and hemostatic safety to achieve the goal of cardiovascular protection.

[0004] Due to the limited content of natural products in nature, it is difficult to obtain a large amount by separation means, which seriously restricts the development and utilization process. The biosynthesis gene cluster of secondary metabolites is introduced into a suitable chassis host for heterologous expression, which can improve the yield of target compounds or promote the discovery of new compounds. The present application aims to identify the 5 / 8 / 5 chalcidicum diterpene biosynthesis gene cluster in Trichoderma harzianum through genome mining system Thm , and obtain structurally diversified FCs compounds by heterologous expression, so as to screen FCs compounds with higher antithrombotic activity. SUMMARY

[0005] The purpose of the present application is to provide 5 / 8 / 5 chalcidicum diterpenoid compounds, biosynthesis gene clusters and their applications, in order to solve the problems existing in the prior art. The 5 / 8 / 5 chalcidicum diterpenoid compounds provided by the present application have excellent antithrombotic effect, thereby providing strong technical support for the development of antithrombotic drugs.

[0006] To achieve the above purpose, the present application provides the following scheme: The present application provides the application of 5 / 8 / 5 chalcidicum diterpenoid compounds in the preparation of antithrombotic drugs, wherein the 5 / 8 / 5 chalcidicum diterpenoid compounds are compound 6 or compound 31; The structural formula of the compound 6 and the compound 31 is shown in Figure 3 .

[0007] Further, the medicine further comprises a pharmaceutically acceptable excipient.

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

[0009] The present application also provides a 5 / 8 / 5 chalcidicum diterpenoid compound with antithrombotic effect, and the structural formula is .

[0010] The present application also provides an antithrombotic drug, and the active ingredient comprises compound 6 or compound 31; The structural formula of the compound 6 and the compound 31 is shown in Figure 3 .

[0011] The present application also provides a 5 / 8 / 5 chalcidicum diterpenoid compound biosynthesis gene cluster, which comprises genes with nucleotide sequences as shown in SEQ ID NO. 1-6 thmA 、 thmB 、 thmC 、 thmD 、 thmE and thmF .

[0012] The application also provides a co-expression vector combination for expressing the biosynthetic gene cluster.

[0013] The application also provides a recombinant microbial strain for synthesizing 5 / 8 / 5 chalcidicum diterpenoid compounds, comprising the co-expression vector combination.

[0014] The application also provides application of the biosynthetic gene cluster, the co-expression vector combination or the recombinant microbial strain in preparation of 5 / 8 / 5 chalcidicum diterpenoid compounds, wherein the 5 / 8 / 5 chalcidicum diterpenoid compounds are compound 6 or compound 31. The structural formulae of the compound 6 and the compound 31 are shown in Figure 3 .

[0015] The application also provides a preparation method of 5 / 8 / 5 chalcidicum diterpenoid compounds, wherein the 5 / 8 / 5 chalcidicum diterpenoid compounds are compound 6 or compound 31. The structural formulae of the compound 6 and the compound 31 are shown in Figure 3 .

[0016] The preparation method comprises the following steps: Fermenting and culturing the recombinant microbial strain to obtain a fermentation liquor; Extracting and separating the fermentation liquor to obtain the compound 6 or the compound 31.

[0017] The application discloses the following technical effects: The application identifies 5 / 8 / 5 chalcidicum diterpenoid biosynthetic gene clusters in Trichoderma harzianum through a genome mining system Thm , and obtains FCs compounds with structural diversity by means of heterologous expression, and comprehensively evaluates the anti-thrombosis activity of the obtained compounds, and it is found that the compound 6 and the compound 31 can obviously inhibit vascular thrombosis, wherein the compound 6 has a significant inhibitory effect on collagen-induced platelet aggregation, and the compound 31 has a better inhibitory capacity on thrombin-induced platelet activation. The compound 6 and the compound 31 exhibit strong anti-thrombosis effects in arterial, venous and pulmonary arterial thrombosis models, thereby providing strong technical support for the development of anti-thrombosis drugs. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0019] Figure 1 ForThm with Mg Comparison of the gene cluster (A) and the transformed strain AO- Thm Extracted ion chromatogram of the culture extract (B); Figure 2 Crystal structure of compounds 4, 5, 6, 11, 16, 24, 25, 26 and 29; Figure 3 Chemical structures of compounds 1-33; Figure 4 Results of detecting the effect of 5 / 8 / 5 clostridolide diterpenoids on the inside-out signaling of platelets; A, the results of measuring the aggregation by light transmission aggregometry after preincubating washed human platelets with compounds 1-33 (200 μM) or negative control (DMSO) (n = 3); B, C) the aggregation curves and statistical plots recorded by light transmission aggregometry after pre-treating washed human platelets with a concentration gradient of compound 6 or DMSO for 5 min, followed by stimulation with collagen (1 μg / mL) and thrombin (0.08 U / mL) (n = 4); C, the aggregation curves and statistical plots recorded by light transmission aggregometry after pre-treating washed human platelets with a concentration gradient of compound 31 or DMSO for 5 min, followed by stimulation with collagen (1 μg / mL) and thrombin (0.08 U / mL) (n = 4); D, the results of flow cytometry analysis of the CD62P (P-selectin) expression level of platelets treated with compound 6 (80 μM), 31 (80 μM) or DMSO (n = 3-6); E, the results of evaluating the cytotoxic effect of compounds 6 (80 μM) and 31 (80 μM) or DMSO on platelets by the release of lactate dehydrogenase (LDH) (n = 6); Con, the group pre-treated with DMSO and then stimulated; Blk, the group pre-treated with DMSO and not stimulated; Figure 5Figure 6 is a graph showing the results of detecting the effect of compound 6 and 31 on platelet outward-to-inward signaling; wherein A is a graph showing the results of quantifying the adhesion of platelets on collagen after treatment with compound 6 (80 mM), 31 (80 mM) or DMSO using an alkaline phosphatase detection method (n = 5-10); B and C are, respectively, representative micrographs of platelet spreading on collagen surface (scale bar: 10 pm; n = 3) and a statistical graph of the area of adherent platelets after 5 minutes of pre-treatment with compound 6, 31 or vehicle; D is a graph showing the classification of platelet morphology based on the formation of pseudopodia and lamellipodia (n = 3); E-G are, respectively, time-lapse images (n = 3), a bar graph quantification of the contraction rate at each time point and a line graph quantification of the contraction rate at each time point for the thrombus contraction process of compound 6; H-J are, respectively, time-lapse images (n = 3), a bar graph quantification of the contraction rate at each time point and a line graph quantification of the contraction rate at each time point for the thrombus contraction process of compound 31; Con represents the group stimulated after pre-treatment with DMSO; Blk represents the group without any stimulation after pre-treatment with DMSO; Figure 6 Figure 7 is a graph showing the analysis of compound 6 inhibiting platelet GPVI signaling pathway; wherein A-B are, respectively, the results of Western blot detection and protein level quantification analysis of PLCy2 and Syk; C-D are, respectively, the results of Western blot detection and protein level quantification analysis of the phosphorylation level of integrin b3, Src and FAK; E is a heatmap of the phosphorylation profile of GPVI and b3 integrin pathway (based on quantitative phospho-proteomic analysis of platelet samples of each group (n = 3), the color scale represents the fold change relative to the unstimulated control group); F is a molecular docking diagram of compound 6 and Syk (UniProt P43405); G is a graph showing the results of platelet cell thermal shift assay that compound 6 can stabilize Syk protein in different temperature ranges; Figure 7 Figure 8 is a molecular docking diagram of compound 6 and Src (P12931) (A) and FAK (Q05397) (B); Figure 8Figure 6A-6H. Analysis of the inhibitory effect of compound 31 on platelet activation by targeting cytoskeleton regulatory proteins; A and B are the western blotting detection and protein level quantification analysis of the phosphorylation level of MYPT1, MLCK, Drebrin and Dematin, respectively; C is a heatmap showing the phosphorylation profile of RhoA / ROCK / MLCP pathway (quantitative analysis results based on the phosphoproteomics of each treatment group, the numerical value represents the normalized fold change relative to the unstimulated control group); D is a molecular docking diagram of compound 31 and the GTP binding pocket of RhoA (UniProt P61586); E is a result diagram of detecting the thermal stability of RhoA in platelets of compound 31 under temperature gradient by cell thermal shift assay (CETSA) (n = 3); Figure 9 Figure 7A-7H. Analysis of the effect of compounds 6 and 31 on thrombosis in mice; A is a detection diagram of the vessel occlusion time in the FeCl3-induced mouse carotid artery injury model (n = 6); B is a quantitative statistical diagram of the carotid artery occlusion time (n = 6); C is a representative microscopic image of MSB staining of carotid artery thrombus sections of mice in the control group, compound 6 and compound 31 treatment groups (scale bar: 100 μm); D is a representative MSB staining image of inferior vena cava thrombus sections in the deep vein thrombosis model (scale bar: 100 μm); E-F are the statistical diagrams of the length and weight of thrombus in the deep vein thrombosis model (n = 6); G is a statistical diagram of the survival rate of mice in the pulmonary embolism model induced by bovine thrombin (2500 IU / kg) after pretreatment with DMSO, compound 6 (10 mg / kg), 31 (10 mg / kg) or aspirin (100 mg / kg) (the mortality recording time is within 15 minutes); H is a representative H&E staining image of lung tissue sections; Figure 10 Figure 8A-8F. Analysis of the effect of compounds 6 and 31 on hemostatic function and platelet biochemical indicators; A-B are the statistical diagrams of total bleeding time and rebleeding frequency, respectively; C-F are the statistical diagrams of mean platelet volume (MPV), platelet distribution width (PDW), platelet hematocrit (PCT) and large platelet ratio (P-LCR) after 7 consecutive days of administration, respectively. DETAILED DESCRIPTION

[0020] The detailed description set forth below will be better understood in conjunction with the drawings as follows:

[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 knownMg Core synthase of a gene cluster MgMs having similarity (s) Figure 1 in A).

[0027] These genes were constructed into four different A. oryzae expression plasmids (pAdeA2, pUARA2, pUNA2 and pUSA2) respectively using Gibson assembly technology, forming co-expression vector combinations, in which thmA and GGPP were constructed into pAdeA2, obtaining recombinant plasmid pAdeA2- thmA-GGPP (wherein GGPP is a gene for synthesizing the precursor of 5 / 8 / 5 cembranoid compounds, the nucleotide sequence of which is shown as SEQ ID NO. 7); and thmB , thmC were constructed into pUARA2, obtaining recombinant plasmid pUARA2- thmBC ; and thmD were constructed into pUNA2, obtaining recombinant plasmid pUNA2- thmD ; and thmE and thmF were constructed into pUSA2, obtaining recombinant plasmid pUSA2- thmEF . Subsequently, these four recombinant A. oryzae expression plasmids were co-introduced into the quadruple auxotrophic A. oryzae host strain NSAR1 by protoplast-PEG transformation method, and finally the engineered A. oryzae strain AO- Thm capable of heterologously expressing the entire Thm gene cluster was successfully obtained.

[0028] Example 2 Preparation and structural identification of compounds 1-33 1. Preparation of compounds 1-33 (1) The obtained engineered A. oryzae strain AO- Thm was cultured on potato dextrose agar (PDA) at 25°C for 4 days to prepare seed culture. Then the agar fragments with the seed were inoculated into 100 conical flasks (1L) containing 200 grams of rice and 200 milliliters of distilled water (which had been sterilized using an autoclave) in a clean bench, and all the conical flasks were incubated at 25°C for 35 days. The fermented rice substrate was extracted in 95% ethanol aqueous solution at room temperature for 8 times, and the total extract was evaporated under vacuum to obtain 280g of total extract paste, which was extracted with ethyl acetate: water = 1:1 for 10 times, and then distilled under reduced pressure to obtain the total extract paste.

[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 ε )= 204 (+2.37), 224 (-2.63) nm; UV (CH3CN) λ max (log ε) = 194 (4.15); IR (KBr) ν 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) λ max (Δ ε = 197 (+3.45); UV (CH3CN) λ max (log ε = 191 (3.90) nm; IR (KBr) ν 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) λ max (Δ ε ) = 217 (+4.62), 328 (+1.57); UV (CH3CN) λ max (log ε = 192 (3.95) nm; IR (KBr) ν 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) λ max (Δ ε ) = 191 (+7.63); UV (CH3CN) λ max (log ε = 192(4.15) nm; IR (KBr) ν 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 C NMR (100 MHz) data are listed in Table 3; crystal data of compound 5 are shown in Figure 2

[0037] Compound 6: Colorless crystal; m. p. 110.1-113.5℃; +243 ( c 0.1, CH3OH); ECD ( c 0.5 mg / mL, CH3CN) λ max (Δ ε ) = 194 (+5.52); UV (CH3CN) λ max (log ε ) = 192(3.92) nm; IR (KBr) ν 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 C NMR (100 MHz) data are listed in Table 3; crystal data of compound 6 are shown in Figure 2

[0038] Compound 7: Colorless oil; -40 ( c 0.1, CH3OH); ECD ( c 0.5 mg / mL, CH3CN) λ max (Δ ε ) = 192 (+3.01); UV (CH3CN) λ max (log ε ) = 192 (4.00) nm; IR (KBr) ν max ​​3277,2959, 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) λ max (Δ ε ) = 196 (+1.41); UV (CH3CN) λ max (log ε = 192 (3.91) nm; IR (KBr) ν 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) λ max (Δ ε ) = 191 (+1.50); ​​UV (CH3CN) λ max (log ε = 192 (4.01) nm; IR (KBr)ν 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) λ max (Δ ε ) = 198 (+2.54); UV (CH3CN) λ max (log ε = 193 (4.00) nm; IR (KBr) ν 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) λ max (Δ ε ) = 192 (+6.79); UV (CH3CN) λmax (log ε = 193(4.00) nm; IR (KBr) ν 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) λ max (Δ ε ) = 191 (+1.40); UV (CH3CN) λ max (log ε = 192 (3.82) nm; IR (KBr) ν 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) λ max (Δε ) = 194 (+0.84), 213 (+1.37); UV (CH3CN) λ max (log ε = 193 (3.67) nm; IR(KBr) ν 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) λ max (Δ ε ) = 213 (+1.55), 231 (-1.07); UV (CH3CN) λ max (log ε = 193 (4.11) nm; IR(KBr) ν 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) λ max (Δ ε ) = 203 (+3.30), 309 (+0.35); UV (CH3CN) λ max (log ε ) = 192 (4.05) nm; IR(KBr) ν 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) λ max (Δ ε ) = 210 (+1.23), 260 (+3.96); UV (CH3CN) λ max (log ε ) = 191 (4.08), 259 (3.94) nm; IR (KBr) ν 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 13C NMR (100 MHz) data are listed in Table 5; crystal data for compound 16 are as shown in Figure 2

[0048] Compound 17: Colorless oil; +45 ( c 0.1, CH3OH); ECD ( c 0.5 mg / mL, CH3CN) λ max (Δ ε ) = 209 (+1.54), 262 (+3.87); UV (CH3CN) λ max (log ε ) = 191 (4.02), 258(3.86) nm; IR (KBr) ν max 3298, 2947, 2931, 1705, 1639, 1186, 1098, and 888 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 C NMR (100 MHz) data are listed in Table 6.

[0049] Compound 18: Colorless oil; +66 ( c 0.1, CH3OH); ECD ( c 0.25 mg / mL, CH3CN) λ max (Δ ε ) = 210 (+1.23), 260 (+3.96); UV (CH3CN) λ max (log ε ) = 191 (3.88), 259(3.72) nm; IR (KBr) ν max 3434,3352, 2921, 2853, 1703, 1630, 1181, 1060, and 888cm -1 ; HRESIMS m / z ​325.2144 [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) λ max (Δ ε ) = 213 (+1.17), 260 (+6.20); UV (CH3CN) λ max (log ε ) = 192 (4.06),259 (3.95) nm; IR (KBr) ν 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) λ max (Δ ε ) = 215 (+1.48), 259 (+6.89); UV (CH3CN) λ max (log ε ) = 192 (4.12), 256(4.04) nm; IR (KBr) νmax 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) λ max (Δ ε ) = 215 (+1.45), 260 (+5.69); UV (CH3CN) λ max (log ε ) = 192 (3.65), 260(3.91) nm; IR (KBr) ν 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) λ max (Δ ε) = 191 (+6.73), 209 (-5.17), 260 (-10.01); UV (CH3CN) λ max (log ε ) =194 (3.95), 262 (3.88) nm; IR (KBr) ν 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) λ max (Δ ε ) = 194 (-6.55), 242 (+5.04); UV (CH3CN) λ max (log ε ) = 192 (4.07), 244(4.01) nm; IR (KBr) ν 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) λ max (Δ ε ) = 192 (+3.84); UV (CH3CN) λ max (log ε = 192(3.97); IR (KBr) ν 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) λ max (Δ ε ) = 196 (+4.74); UV (CH3CN) λ max (log ε =192 (4.10); IR (KBr) ν 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 C NMR (100 MHz) data are listed in Table 8; crystal data of compound 24 are shown as Figure 2 indicated.

[0057] Compound 26: Colorless crystal; m. p. 193.7-194.4 ℃; -91 ( c 0.1, CH3OH); ECD ( c 0.5 mg / mL, CH3CN) λ max (Δ ε ) = 197 (+5.24); UV (CH3CN) λ max (log ε ) =192 (3.86); IR (KBr) ν 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 C NMR (150 MHz) data are listed in Table 8; crystal data of compound 26 are shown as Figure 2 indicated.

[0058] Compound 27: Colorless oil; -84 ( c 0.1, CH3OH); ECD ( c 0.5 mg / mL, CH3CN) λ max (Δ ε ) = 195 (+3.58); UV (CH3CN) λ max (log ε ) = 192 (4.00); IR (KBr) ν 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) λ max (Δ ε ) = 194 (+2.62); UV (CH3CN) λ max (log ε = 192 (3.98); IR (KBr) ν 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) λ max (Δ ε ) = 192 (+3.86); UV (CH3CN) λ max(log ε =191 (4.02); IR (KBr) ν 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 ( δ 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 ( δ in ppm, J (in Hz)

[0064] Note: a Recorded in CDCl3 b Recorded in CD3OD. c Signals were overlapped. Table 4.1H and13C NMR data of compounds 9-12 1 H and 13 C NMR data (ppm, deuterated chloroform) δ in ppm, J in Hz)

[0065] Note: a Recorded in DMSO- d 6. b Recorded in CDCl3. c Signals were overlapped. Table 5.1H and13C NMR data of compounds 13-16 1 H and 13 C NMR data (ppm, deuterated chloroform) δ in ppm, J in Hz)

[0066] Note: a Recorded in DMSO- d 6. b Recorded in CDCl3 c Signals were overlapped. Table 6.1H and13C NMR data of compounds 17-20 1 H and 13 C NMR data (ppm, deuterated chloroform) δ in ppm, J in Hz)

[0067] Note: a Recorded in CDCl3 b Recorded in CD3OD. c Signals were overlapped. Table 7.1H and13C NMR data of compounds 17-20 1 H and 13 C NMR data (ppm, deuterated chloroform) δ in ppm, J in Hz, deuterated chloroform)

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

[0069] Note: a Recorded in DMSO- d 6. b Recorded in CDCl3. c Signals were overlapped. Table 9. 1H NMR data of compounds 29 and 30 1 H and 13 C NMR data ( δ in ppm, J in Hz)

[0070] Note: a Recorded in DMSO- d 6. b Recorded in CD3OD. c Signals were overlapped. Example 3. Effect of compounds 1-33 on platelet inside-out signaling To evaluate the anti-thrombotic potential of compounds 1-33, the present application first examined their effects on thrombin-induced platelet aggregation. After pre-incubation of washed human platelets with compounds 1-33 (200 μΜ) or negative control (DMSO) for 5 min, the platelets were stimulated with thrombin (0.08 U / mL) to induce aggregation. The aggregation was measured by light transmission aggregometry (n = 3), and the results are shown in Figure 4 The primary screening results showed that compounds 5, 6, 31 and 33 significantly inhibited thrombin-induced platelet aggregation.

[0071] To assess functional selectivity, the present application further analyzed the effects of compounds 6 and 31 on various physiological platelet agonists, including ADP, collagen, and thrombin. Washed human platelets were pre-treated with a concentration gradient of compound 6, 31, or DMSO for 5 min, followed by stimulation with collagen (1 μg / mL) and thrombin (0.08 U / mL); a solvent control group pre-treated with DMSO without any stimulation was also set up. Aggregation curves were recorded using light transmission aggregometry (n = 4). Flow cytometry was used to analyze the CD62P (P-selectin) expression level of platelets treated with compound 6 (80 μM), 31 (80 μM), or DMSO (n = 3-6). The cytotoxic effects of compound 6 (80 μM) and 31 (80 μM) or DMSO on platelets were evaluated by lactate dehydrogenase (LDH) release (n = 6). n n

[0072] The results showed that both compounds exhibited agonist-dependent inhibitory profiles, with compound 6 showing stronger inhibition of collagen-induced aggregation (Fig. 2A) and compound 31 being more effective in inhibiting thrombin-induced aggregation (Fig. 2B). P-selectin translocates from the alpha granules to the platelet surface upon platelet activation and was used as a sensitive marker of platelet activation. Flow cytometry analysis confirmed that both compounds 6 and 31 significantly inhibited collagen-induced P-selectin exposure (Fig. 2C). Moreover, no significant increase in lactate dehydrogenase (LDH) release was detected, indicating that the anti-platelet effects were not caused by cytotoxicity (Fig. 2D). Figure 4 Figure 4 Figure 4 Figure 4

[0073] Example 4 Effects of compounds 6 and 31 on platelet outside-in signaling Platelet outside-in signaling is an important component of the platelet activation process. This mechanism transmits extracellular signals mediated by integrins, thereby enhancing platelet adhesion, aggregation, and secretion functions. In addition, it plays a key role in regulating hemostasis and thrombus formation. To test the effects of compounds 6 and 31 on platelet outside-in signaling, the present application quantified platelet adhesion to collagen in the presence of compound 6 (80 μM), 31 (80 μM), or DMSO using an alkaline phosphatase assay; a solvent control group treated with DMSO without any stimulation was also set up. The results showed that collagen-mediated platelet adhesion (Fig. 3A) and spreading (Fig. 3B) were significantly inhibited in the presence of compounds 6 and 31. Both compounds also significantly reduced the average spreading area of individual platelets (Fig. 3C). Figure 5 Figure 5 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 6Syk, PLCy2, integrin b3, Src and FAK, which together constitute the molecular basis of compound 6 to effectively block the early GPVI-mediated signaling pathway.

[0077] Example 6 Compound 31 inhibits platelet activation by targeting cytoskeleton regulatory proteins Based on the strong anti-platelet effect of compound 31 on thrombin-induced aggregation, the key signaling molecules of cytoskeleton remodeling were systematically analyzed to further study its mechanism of action. Human platelets were pretreated with compound 31 (80 µM) and lysed after collagen stimulation. The phosphorylation levels of MYPT1, MLCK, Drebrin and Dematin were detected by Western blotting. The results showed that the compound could specifically target the core protein of cytoskeleton dynamic regulation, which is the basis of platelet activation. It significantly inhibited the phosphorylation of MYPT1 and MLCK, which are key regulators of myosin light chain phosphatase activity and myosin contractility, respectively. At the same time, compound 31 also down-regulated the expression of actin-binding proteins Drebrin (F-actin stabilizing factor) and Dematin (involved in actin bundling) Figure 8 Central A and B). Phosphoproteomic analysis identified the RhoA / ROCK / MLCP pathway as its core target: compound 31 weakened myosin contractility by inhibiting RhoA activation, reducing ROCK1-dependent MYPT1 phosphorylation, enhancing MLCP activity and attenuating MLC phosphorylation. In addition, the inhibition of Drebrin and Dematin expression also disrupted the stability of the actin cytoskeleton and filopodia formation Figure 8 Central C). Molecular docking analysis confirmed that compound 31 has high affinity for the RhoA GTP-binding pocket (P61586) with a binding energy of -7.0 kcal / mol Figure 8 Central D). Compound 31 was detected for its thermal stability effect on RhoA in platelets under temperature gradient by cellular thermal shift assay (CETSA), and the results showed that compared with the DMSO control group under heat denaturation conditions at 65-70°C, compound 31 could significantly stabilize the RhoA protein Figure 8 Central E).

[0078] Example 7 Effect of compounds 6 and 31 on thrombus formation in mice The anti-thrombotic efficacy of compounds 6 and 31 was evaluated using three established mouse thrombosis models. In the FeCl3-induced carotid artery injury model, after treatment with DMSO, compound 6 (10 mg / kg), 31 (10 mg / kg), or aspirin (100 mg / kg), a filter paper (1 mm x 1 mm) was soaked in 10% FeCl3solution and attached to the vessel surface for 1 min to induce thrombosis. The hemodynamic changes were monitored in real time by ultrasound Doppler flowmetry until a persistent reduction in blood flow was observed (maximum recording time 20 min). In the deep vein thrombosis model, the mice were grouped and dosed as before. After isoflurane inhalation anesthesia, the inferior vena cava was exposed and ligated with a 0.2-0.3 mm diameter blunt steel wire to establish a standardized stenosis model. The thrombus was removed 48 h after surgery for weighing and histological analysis. In the thrombin-induced acute pulmonary embolism model, the mice were grouped and dosed as before. After 30 min, bovine thrombin (2500 IU / kg) was injected into the tail vein to induce thrombosis. The behavioral changes and survival of the experimental animals were continuously observed. Mice that survived for more than 15 min were humanely sacrificed, and the lung tissue was fixed by perfusion, paraffin-embedded, and stained for histological analysis.

[0079] Under Doppler flow monitoring, the vessel occlusion time in the FeCl3-induced carotid artery injury model after treatment with DMSO, compound 6 (10 mg / kg), 31 (10 mg / kg), or aspirin (100 mg / kg) was determined. The results showed that in the FeCl3-induced carotid artery injury model, both compounds (10 mg / kg) significantly prolonged the vessel occlusion time and reduced thrombosis. Notably, aspirin (positive control) required a ten-fold higher dose (100 mg / kg) to achieve a comparable anti-thrombotic effect. Figure 9 Similar efficacy was also observed in the MSB staining results of mouse carotid artery thrombus sections Figure 9 In the inferior vena cava stenosis model, compound 6 and 31 (10 mg / kg) significantly reduced the thrombus weight and length, with an efficacy comparable to that of 100 mg / kg aspirin Figure 9 In the thrombin-induced pulmonary embolism model, compared with 100 mg / kg aspirin, both compounds (10 mg / kg) significantly improved the survival rate and reduced the pulmonary thrombus load Figure 9 Histological analysis further confirmed that the pulmonary vascular thrombosis in the compound 6 and 31 treatment groups was significantly reduced compared with the simple thrombin group Figure 9 In summary, these results indicate that compounds 6 and 31 exhibit strong anti-thrombotic effects in arterial, venous, and pulmonary arterial 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 indicators The in vivo safety of compounds 6 and 31 was evaluated to investigate their potential side effects. The tail transection bleeding test was performed after pre-dosing (10 mg / kg of each compound 6, 31 or DMSO) 30 min before. The results showed that neither of the two compounds significantly prolonged the bleeding time or increased the incidence of rebleeding compared with the control group (Fig. 8A-B), indicating that they had no significant effect on physiological hemostatic function. To determine whether repeated dosing affected platelet production or basic hematological parameters, mice were injected with each compound daily for 7 days, covering the entire platelet renewal cycle. Peripheral blood analysis showed that there were no significant changes in mean platelet volume (MPV), platelet distribution width (PDW), platelet hematocrit (PCT), and large platelet ratio (PLCR) (Fig. 8C-F), indicating that neither of the two compounds impaired platelet production or changed the integrity of circulating platelets. These results indicated that compounds 6 and 31 had good safety, had minimal effect on hemostatic function, and had no adverse effects on platelet production and basic hematological parameters after prolonged treatment. Figure 10 Figure 10 In summary, compound 6 had a significant inhibitory effect on collagen-induced platelet aggregation, while compound 31 showed better inhibitory capacity on thrombin-induced platelet activation. The anti-platelet effects of these two natural small molecule compounds were further verified in various thrombosis animal models. Mechanism studies revealed the dual-pathway inhibitory effect of compound 6: (1) by inhibiting Syk tyrosine kinase autophosphorylation and PLCy2 activation, weakening GPVI / FcRy-ITAM signaling; (2) by inhibiting β3 subunit phosphorylation and subsequent FAK / Src complex dissociation, disrupting the bidirectional signaling of αIIbβ3 integrin. Compound 31, on the other hand, 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 cytoskeletal remodeling.

[0081] In summary, compound 6 had a significant inhibitory effect on collagen-induced platelet aggregation, while compound 31 showed better inhibitory capacity on thrombin-induced platelet activation. The anti-platelet effects of these two natural small molecule compounds were further verified in various thrombosis animal models. Mechanism studies revealed the dual-pathway inhibitory effect of compound 6: (1) by inhibiting Syk tyrosine kinase autophosphorylation and PLCy2 activation, weakening GPVI / FcRy-ITAM signaling; (2) by inhibiting β3 subunit phosphorylation and subsequent FAK / Src complex dissociation, disrupting the bidirectional signaling of αIIbβ3 integrin. Compound 31, on the other hand, 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 cytoskeletal remodeling.

[0082] The above-described embodiments are merely preferred modes of the present application and are not intended to limit the scope of the present application, which is defined by the appended claims. Various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art without departing from the design spirit of the present application shall fall within the scope of protection of the present application.​

Claims

1. Use of a 1.5 / 8 / 5 desulfovicosin diterpene compound for the preparation of an antithrombotic medicament, characterized in that, The 5 / 8 / 5 clostron diterpenoid compound is compound 6 or compound 31. The structural formula of the compound 6 is: ; The structural formula of the compound 31 is: .

2. Use according to claim 1, characterized in that, The medicine also includes a pharmaceutically acceptable excipient.

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

4. A 5 / 8 / 5 chalcidicum diterpene compound having an antithrombotic effect, characterized by, The structural formula is: .

5. An antithrombotic agent, characterized by comprising a compound of the formula (I) or a salt thereof. The active ingredient includes compound 6 or compound 31. The structural formula of the compound 6 is: ; The structural formula of the compound 31 is: .

6. A biosynthetic gene cluster of 5 / 8 / 5-cembranoid diterpenoids, characterized in that, A gene comprising a nucleotide sequence as set forth in SEQ ID NO. 1-6 thmA , thmB , thmC , thmD , thmE and thmF .

7. A co-expression vector combination for expressing the biosynthetic gene cluster of claim 6.

8. A recombinant microbial strain for synthesizing 5 / 8 / 5 chalcidicin diterpenoid compounds, characterized in that, The co-expression vector combination of claim 7 is included.

9. Use of the biosynthetic gene cluster of claim 6, the co-expression vector combination of claim 7 or the recombinant microbial strain of claim 8 for the preparation of 5 / 8 / 5 chalcidicin diterpenoid compounds, characterized in that, The 5 / 8 / 5 clostron diterpenoid compound is compound 6 or compound 31. The structural formula of the compound 6 is: ; The structural formula of the compound 31 is: .

10. A method for preparing 5 / 8 / 5 clostridinone diterpenoid compound, characterized in that, The 5 / 8 / 5 clostron diterpenoid compound is compound 6 or compound 31. The structural formula of the compound 6 is: ; The structural formula of the compound 31 is: ; The preparation method comprises the following steps: The recombinant microbial strain of claim 8 is subjected to fermentation culture to obtain a fermentation liquor; The fermentation liquor is subjected to extraction and separation treatment to obtain the compound 6 or the compound 31. The preparation method comprises the following steps: The recombinant microbial strain of claim 8 is subjected to fermentation culture to obtain a fermentation liquor; The fermentation liquor is subjected to extraction and separation treatment to obtain the compound 6 or the compound 31.

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