A guaiane sesquiterpene compound and application thereof

Abstract

The application discloses a kind of guaiane type sesquiterpene compounds and application thereof, including the sesquiterpene compound with five and seven ring coupling structure, most of which contain 4,10-dimethyl-7-isopropylvinyl group.The guaiane sesquiterpenes provided by the application have significant anti-immune activity, and have good inhibitory effect on the targeting cGAS-STING signal pathway.The application provides a new lead compound for researching and developing new anti-immune drugs.Meanwhile, the guaiane sesquiterpenes described in the application and pharmaceutical excipients can be made into tablets, capsules and injections, and can be applied in the field of medicine, and have good application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of guaiane sesquiterpene compound preparation, and particularly relates to a guaiane sesquiterpene compound and application thereof. BACKGROUND

[0002] Autoimmune diseases affect about 10% of the global population, and current drugs cannot completely cure the disease, so it is urgent to develop high-efficiency targeted drugs. The cGAS-STING signaling pathway is a new target for treating autoimmune diseases, and finding inhibitors of the pathway is a new direction for developing drugs for treating autoimmune diseases. It is found for the first time that guaiane sesquiterpene is an inhibitor of the cGAS-STING signaling pathway, which can interact with key molecules in the cGAS-STING signaling pathway to exert immunosuppressive activity. The cGAS-STING pathway signal pathway regulation mode is as shown in Figure 2 SUMMARY

[0003] The technical problem to be solved by the application is to provide a guaiane sesquiterpene compound and application thereof.

[0004] The purpose of the application is achieved by the following technical solutions.

[0005] A guaiane sesquiterpene compound and application thereof for inhibiting the cGAS-STING signaling pathway to resist autoimmune diseases.

[0006] Preferably, the guaiane sesquiterpene compound is a sesquiterpene compound with a five-membered and seven-membered ring coupled structure, most of which contains 4,10-dimethyl-7-isopropenyl,

[0007]

[0008] Preferably, the guaiane sesquiterpene compound has any one of the following structural formulae in the preparation as an application for resisting autoimmune diseases.

[0009]

[0010] Preferably, the drug for resisting autoimmune diseases is applied by inhibiting the cGAS-STING signaling pathway.

[0011] Preferably, the preparation method of the guaiane sesquiterpene compound comprises the following steps.

[0012] (1) The dry aboveground parts of artemisia vulgaris are cut into segments, solvent extraction is performed, and the extract is concentrated to obtain a crude extract;

[0013] ​(2) The crude extract is suspended in water, extracted with an organic solvent, and concentrated to obtain a crude extract infusion;

[0014] (3) The extract infusion is prepared into a compound by column chromatography and thin layer chromatography.

[0015] In the step (1), the solvent is 95% ethanol, and the extraction is carried out at room temperature in batches, repeated three times, each time for 7 days.

[0016] In the step (2), the organic solvent extraction is ethyl acetate and n-butanol in sequence.

[0017] In the step (3), the column chromatography includes macroporous resin, MCI, reverse phase column chromatography, dextran gel and normal phase silica gel, and the elution system is mainly methanol-water, petroleum ether-ethyl acetate, etc.

[0018] In the step (3), the thin layer chromatography uses normal phase silica gel chromatography technology for analysis, and the developing agent is dichloromethane-methanol, petroleum ether-acetone, petroleum ether-ethyl acetate, etc.

[0019] In the step (2), the ethyl acetate part (AaSEA) is preliminarily segmented by D101 macroporous adsorption resin, eluted with 30%, 50%, 80%, 95% ethanol-water gradient, and concentrated under reduced pressure. The 80% ethanol elution part (AaEA80) infusion is separated by normal phase silica gel, gradient eluted with 10%-100% (every 10% is a gradient) petroleum ether-ethyl acetate, and combined according to thin layer chromatography plate (TLC) sample analysis to obtain components AaEA80A-AaEA80C. A crystal is obtained, which is compound Aa-s-1.

[0020] The AaEA80A, AaEA80B, AaEA80C part in step (3) is separated by Sephadex LH-20 (95% methanol elution), and after sample analysis by thin layer chromatography plate (TLC), seven components A1, A2, B1, B2, B3, C1 and C2 and two monomer compounds Aa-s-2 and Aa-s-3 are obtained. The A1 part is separated and purified by semi-preparative HPLC (methanol / water gradient elution, 40%, 2 mL / min) to obtain compounds Aa-s-4 (tR=24 min), Aa-s-5 (tR=31 min) and Aa-s-6 (tR=43 min). The A2, B1, C1 part is separated and purified by semi-preparative HPLC (methanol / water gradient elution, 2 mL / min) to obtain compounds Aa-s-4 (tR=31 min), Aa-s-7 (tR=36 min), Aa-s-12 (tR=42 min), Aa-s-13 (tR=15 min), Aa-s-14 (tR=27 min), Aa-s-15 (tR=18 min) and Aa-s-16 (tR=24 min). The B2, C2 part is separated and purified by semi-preparative HPLC (acetonitrile / water gradient elution, 2 mL / min) to obtain compounds Aa-s-10 (tR=16 min), Aa-s-11 (tR=21 min), Aa-s-15 (tR=31 min) and Aa-s-17 (tR=25 min).

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] The guaiane sesquiterpenoid compound provided by the application has significant anti-immune activity and good inhibition effect on the cGAS-STING signal pathway, and provides a new lead compound for research and development of new anti-immune drugs. Meanwhile, the guaiane sesquiterpenoid compound and the pharmaceutical excipients can be made into tablets, capsules and injections, and can be applied in the field of medicine, and has good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The molecular structural formula of the compound Aa-s-1-Aa-s-17 is shown in the following table:

[0024] Figure 2 The cGAS / STING signal pathway regulation mode diagram is shown in the following table:

[0025] Figure 3 The action mode of Aa-s-1 and cGAS is shown in the following table:

[0026] Figure 4 The action mode of Aa-s-3 and cGAS is shown in the following table:

[0027] Figure 5 Mode of action of Aa-s-1 with STING;

[0028] Figure 6 Mode of action of Aa-s-3 with STING;

[0029] Figure 7 Modulation of key genes of cGAS / STING pathway by compound Aa-s-1 and Aa-s-3 (n=3);

[0030] Figure 8 cGAS, STING, TRF3, TBK1 gene expression levels, expression level display chart after compound intervention. DETAILED DESCRIPTION

[0031] The embodiments of the present application will be described in detail below with the accompanying drawings and examples, so that the implementation process of how to apply technical means to solve technical problems and achieve technical effects of the present application can be fully understood and implemented.

[0032] The present application provides a guaiane sesquiterpene compound and its application.

[0033] Compound identification of example 1

[0034] Compound Aa-s-1: colorless crystal, its spectral data are as follows: 1 H-NMR (CDCl3, 400 MHz) δ H : 3.86 (d, J = 1.2 Hz, H-2), 4.10 (d, J = 0.8 Hz, H-3), 2.81 (d, J = 10.8, H-5), 4.38 (dd, J = 9.6, 11 Hz, H-6), 3.58 (m, H-7), 2.32 (m, H-8α), 1.62 (m, H-8β), 1.91 (m, H-9), 6.19 (d, J = 3.6 Hz, H-13α), 5.46 (d, J = 3.4 Hz, H-13β), 1.23 (s, H-14), 1.57 (s, H-15).

[0035] 13 C NMR (101 MHz, CDCl3) δ C : 73.0 (C-1), 63.5 (C-2), 64.0 (C-3), 80.0 (C-4), 50.0 (C-5), 78.5 (C-6), 43.5 (C-7), 22.5 (C-8), 33.5 (C-9), 72.0 (C-10), 140.5 (C-11), 170.5 (C-12), 28.0 (C-14), 24.0 (C-15).

[0036] The above 1 H-NMR and 13 The C-NMR data are in accordance with the literature

[25] The compound was identified as 3α-chloro-4β,10α-dihydroxy-1β,2β-epoxy-5α,7αH-guai-11(13)-en-12,6α-olide, with the molecular formula of C 15 H 19 C105, with a molecular weight of 314.09, and its structural formula is as Figure 1 Compound Aa-s-1.

[0037] Compound Aa-s-3: colorless crystal, and its spectral data are as follows. 1 H-NMR (CDC13, 400 MHz) δ H : 3.54 (brs, H-2), 4.31 (s, H-3), 2.44 (d, J = 11.1, H-5), 4.43 (dd, J = 10.2, 10.8 Hz, H-6), 2.98 (m, H-7), 1.70 (m, H-8α), 2.22 (m, H-8β), 1.90 (m, H-9α), 1.85 (m, H-9β), 5.43 (d, J = 3.0 Hz, H-13α), 6.16 (d, J = 3.6 Hz, H-13β), 1.40 (s, H-14), 1.51 (s, H-15).

[0038] 13 C NMR (101 MHz, CDC13) δ C : 75.1 (C-1), 63.2 (C-2), 63.6 (C-3), 80.3 (C-4), 50.9 (C-5), 77.2 (C-6), 44.5 (C-7), 22.1 (C-8), 35.5 (C-9), 69.8 (C-10), 139.5 (C-11), 169.0 (C-12), 119.9 (C-13), 21.9 (C-14), 24.0 (C-15).

[0039] The above 1 H-NMR and 13 The C-NMR data are in accordance with the literature

[27] The compound was identified as (rel)-3β-chloro-4β,10β-dihydroxy-1α,2α-epoxy-5α,7αH-guai-11(13)-en-12,6α-olide, with the molecular formula of C 15 H 19 O5Cl, with a molecular weight of 332.125, and its structural formula is asFigure 1 Compound Aa-s-3.

[0040] Molecular docking of compounds Aa-s-1 and Aa-s-3 of Example 2

[0041] 1. Reagents

[0042] RNA extraction kit (Savant Technology Co., Ltd., approval number: SW203), cDNA synthesis kit (Jiangsu Kaygen Biotech Co., Ltd., approval number: KGA1311), PerfectStart Green qPCR SuperMix (Jiangsu Kaygen Biotech Co., Ltd., approval number: KFG2110), PAGE gel rapid preparation kit (Shanghai Yezheng Biomedicine Technology Co., Ltd., approval number: GP210), cGAS (primary antibody) (Wuhan Three-Ring Biotech Co., Ltd., approval number: 29958-1), STING (primary antibody) (Wuhan Three-Ring Biotech Co., Ltd., approval number: 80165-1-RR), GAPDH (primary antibody) (Wuhan Three-Ring Biotech Co., Ltd., approval number: 10494-1-AP), RIPA protein lysis buffer (Shanghai Biyun Tian Biotechnology Co., Ltd., approval number: P0013B), BCA protein concentration determination kit (Jiangsu Kaygen Biotech Co., Ltd., approval number: KGP902), Protein Marker (Thermo Fisher Scientific, approval number: 26616), rapid transfer buffer (Wuhan Saiver Biotechnology Co., Ltd., approval number: G2028), rapid electrophoresis buffer (Wuhan Saiver Biotechnology Co., Ltd., approval number: G2081), RIPA protein lysis buffer (Shanghai Biyun Tian Biotechnology Co., Ltd., approval number: P0013B), HRP-Goat Anti-Rabbit IgG (Wuhan Three-Ring Biotech Co., Ltd., approval number: SA00001-2).

[0043] 2. Instruments

[0044] High-speed centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.), PCR instrument (Dongsheng Technology Biomedical Innovation Co., Ltd.), fluorescent quantitative PCR instrument (BIO-RAD, USA), SDS-PAGE electrophoresis instrument (BIO-RAD, USA), chemiluminescence imaging analysis system (BIO-RAD, USA).

[0045] 3. Experimental methods

[0046] Molecular docking was used to further explore the interaction of the compound with STING and cGAS proteins, and to provide a direction for future structural optimization design.

[0047] 4. Specific experimental steps

[0048] The docking was completed using MOE software, and the top 5 docking modes were saved in the.mdb file. In the.mdb column File>Browse, open the docking mode browser, and observe the docking mode one by one in the three-dimensional rendering area of the MOE software and analyze the interaction between the ligand and the receptor. Finally, in the Pymol software environment, select the most suitable binding model, import the corresponding receptor protein and ligand molecule, and analyze the interaction between the molecule and the protein. The cells were collected by centrifugation, total RNA was extracted, and then total RNA with a concentration of 2 μg / μL was selected for reverse transcription and quantitative PCR detection, and finally the expression level of the key protein in the cGAS / STING signaling pathway was determined.

[0049] 5. Results

[0050] Compound Aa-s-1, in the process of interacting with cGAS, affects the amino acid residues around the protein when the compound is present, and interacts with the hydrophilic amino acids around it that can be affected by hydrogen bonding. The hydroxyl group connected to C-10 can form a hydrogen bond with the amino acid residue Ser434 on the chain. At the end of the molecular structure, it can form a hydrogen bond with the amino acid residue Asn482 on the chain. The docking results are shown in Figure 3 .

[0051] Compound Aa-s-3, the hydrophobic end of the molecular structure, is inserted into the approximately cylindrical deep hydrophobic pore of the cGAS protein, and acts on the same binding pocket. Further analysis of the binding mode of Aa-s-3 and Figures 3-4 The binding mode of the cGAS protein is shown. When the compound is present, it can change the amino acid residues around the protein, and interact with the amino acids that can change their hydrophilicity by hydrogen bonding. In addition, it can form hydrogen bonds with His437, Ser434 and Ser162, thereby causing changes in the protein structure. The docking results are shown in Figure 4 .

[0052] The deep hydrophobic pore of the STING protein of compound Aa-s-1, which is approximately cylindrical, is inserted by the hydrophobic end of the molecular structure. Further analysis of the binding mode of Aa-s-1 and the STING protein, when the compound is present, it can affect the amino acid residues around the protein, affect the hydrophilic amino acids around it by hydrogen bonding, and form hydrogen bonds with SerA162 and ThrA267, causing changes in the conformation of the protein. The docking results are shown in Figure 5 .

[0053] Compound Aa-s-3, when interacting with STING, these groups are surrounded by hydrophobic and hydrophilic amino acids, and are visualized in two dimensions as shown in Figures 3-5When the compound is present, it can change the amino acid residues around the protein and affect the hydrophilic amino acids around it by means of hydrogen bonds. The hydroxyl group connected to C2 of the compound can form a hydrogen bond with the amino acid residue SerB162 on the chain. At the end of the molecular structure, both alcohol hydroxyl groups can form hydrogen bonds with the amino acid residue SerA162 on the chain. The docking results are as shown in Figure 6 .

[0054] As shown in Figure 7 , compared with the model group, the expressions of cGAS and STING proteins were down-regulated after the intervention of the compounds, and the difference was significant (P<0.001). As shown in Figure 8 , compared with the model group, the expression levels of cGAS, STING, TRF3 and TBK1 genes were significantly down-regulated after the intervention of the compounds, which was consistent with the protein expression results, and there was a concentration-dependent effect. It is shown that the two compounds have inhibitory effect on the cGAS / STING signaling pathway, thereby exerting immunosuppressive activity.

[0055] 6. Compound structure-activity relationship analysis

[0056] For guaiane sesquiterpenes Aa-s-1-Aa-s-17, the activity of compound Aa-s-1-Aa-s-7 is better than that of Aa-s-12, Aa-s-13 and Aa-s-14, which indicates that the formation of a three-membered oxygen ring at C-1 and C-2 is a key functional group. Compounds Aa-s-1 and Aa-s-3 show stronger inhibitory effect than compound Aa-s-2, which indicates that the chlorine connected to C-3 is important. Compounds Aa-s-8 and Aa-s-9 both have an oxygen ring connected to C-3 and C-4, and the activity of compound Aa-s-9 is obviously stronger than that of Aa-s-8, which indicates that the chlorine connected to C-1 is a key functional group. Compounds Aa-s-15 and Aa-s-16 and Aa-s-17 all have strong inhibitory effect, but the activity of Aa-s-17 is stronger than that of compounds Aa-s-15 and Aa-s-16. It is shown that the i-val connected to C-8 is important.

[0057] The above only describes the preferred embodiments of the present application, and does not limit the present application in any form. Any simple modification, equivalent change and modification made on the basis of the technical essence of the present application to the above embodiments are within the scope of the technical solutions of the present application.

Claims

1. A method for preparing a guaiane sesquiterpene compound, characterized by: Comprising the following steps: (1) taking dry overground parts of Artemisia argyi, cutting into sections, mixing with 95% ethanol, extracting in batches at room temperature, repeating three times, each time for 7 days, combining the alcohol extract and reducing pressure to concentrate to obtain ethanol extract; (2) uniformly dispersing the ethanol extract in warm water, extracting with ethyl acetate for 3 times, reducing pressure to concentrate the ethyl acetate layer to obtain the ethyl acetate extract, uniformly dispersing the water layer extract with an equal volume of n-butanol, extracting for 3 times, reducing pressure to concentrate the n-butanol layer to obtain the n-butanol extract, and reducing pressure to concentrate the water layer to obtain the water phase extract; The ethyl acetate extract in step (2) is preliminarily separated by D101 macroporous adsorption resin, eluted with 30%, 50%, 80% and 95% ethanol-water gradient, reduced pressure concentrated, and the 80% ethanol elution part AaEA80 extract is separated by normal phase silica gel, gradient eluted with 10%-100% petroleum ether-ethyl acetate, each 10% as a gradient, combined according to thin layer chromatography plate sample analysis to obtain components AaEA80A-AaEA80C, and a crystal is obtained, which is compound Aa-s-1; The AaEA80 A part is further separated by Sephadex LH-20, 95% methanol elution, combined according to thin layer chromatography plate sample analysis to obtain two components A1 and A2, and a monomer compound Aa-s-2, the A1 part is separated and purified by semi-preparative HPLC, methanol / water gradient elution, 40%, 2mL / min, to obtain compounds Aa-s-4, tR=24min, Aa-s-5, tR=31min and Aa-s-6, tR=43min, and the A2 part is separated and purified by semi-preparative HPLC, methanol / water gradient elution, 2mL / min, to obtain compounds Aa-s-4, tR=31min, Aa-s-7, tR=36min and Aa-s-12, tR=42min; The AaEA80B part obtains three components B1, B2 and B3, and a monomer compound Aa-s-3, the B1 part obtains compounds Aa-s-13, tR=15min, and Aa-s-14, tR=27min by the above steps, the B2 part is separated and purified by semi-preparative HPLC, acetonitrile / water gradient elution, 2mL / min, to obtain compounds Aa-s-10, tR=16min, Aa-s-11, tR=21min and Aa-s-15, tR=31min; The AaEA80C part obtains two components C1 and C2, the C1 part obtains compounds Aa-s-15, tR=18min and Aa-s-16, tR=24min by step 4, and the B2 part obtains compound Aa-s-17, tR=25min by the above steps; The guaiane type sesquiterpenoids have any one of the following structural formulae,

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