A semi-oleanane diterpenoid alkaloid compound and a preparation method and application thereof

Abstract

The application provides a semi-labdanum type diterpene alkaloid compound and a preparation method and application thereof. The application provides three semi-labdanum type diterpene alkaloid compounds with new skeletons, wherein the semi-labdanum type diterpene alkaloid compounds are three new skeleton compounds Diaporlabanoids A-C with immunosuppressive activity which are separated from an endophytic Diaporthe phaseolorum H3-2 fungus in a mangrove plant Xanthophyllum coriaceum. The application also carries out experimental research on cytotoxicity and immunosuppressive activity of the separated compounds, and the results show that Diaporlabanoid A has strong immunosuppressive activity on ConA-induced mouse T lymphocytes and low cytotoxicity on normal spleen cells, and can be used for preparing immunosuppressive drugs targeting two cascade signal pathways of CaN / NFAT and PKC theta / NF-kappa B. The semi-labdanum type diterpene alkaloid compound provided by the application can be applied to preparation of immunosuppressive drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a halenaquinol type diterpene alkaloid compound and a preparation method and application thereof. BACKGROUND

[0002] Immunosuppressive drugs are mainly used in improving transplantation medicine, treating autoimmune diseases, immune-related diseases and the like. Research on immunosuppressive drugs helps to improve the survival rate and quality of life of patients.

[0003] Halenaquinol type diterpene alkaloids are mainly extracted from plants, and have wide biological activities and pharmacological effects, mainly including anticancer, anti-inflammatory, antibacterial, antiviral activities, and also have immunomodulatory effects, as well as antioxidant, antithrombotic, hypotensive, analgesic and other biological activities. However, different halenaquinol type diterpene alkaloids have great differences in activity level.

[0004] In summary, it is of great significance to study how to obtain halenaquinol type diterpene alkaloids with good immunosuppressive activity. SUMMARY

[0005] In view of this, the present application provides a halenaquinol type diterpene alkaloid compound and a preparation method and application thereof.

[0006] The halenaquinol type diterpene alkaloid compound provided by the present application is three new skeleton compounds Diaporlabanoids A-C with immunosuppressive activity isolated from the endophytic Diaporthe of the mangrove plant Xanthophyllum glauca for the first time. Diaporthe phaseolorum H3-2.

[0007] The technical scheme of the present application is as follows:

[0008] A halenaquinol type diterpene alkaloid compound, the structural formula of the halenaquinol type diterpene alkaloid compound is shown in formula 1 or formula 2 or formula 3:

[0009] .

[0010] Further, the halenaquinol type diterpene alkaloid compound of the present application is prepared from Diaporthe phaseolorum H3-2, Diaporthe phaseolorum H3-2 is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: M20231234.

[0011] Further, the preparation method of the halenaquinol type diterpene alkaloid compound of the present application comprises the following steps:

[0012] (1) First, the PDA culture medium is used to activate Diaporthe phaseolorumH3-2, when the colony morphology is stable and a single colony is formed, inoculate the single colony into PDA medium for expansion culture; inoculate the single colony into sterilized rice medium, and ferment at 27-29°C for 28-30 days; after fermentation, extract 2-4 times with ethyl acetate, and obtain the crude extract after concentration under reduced pressure;

[0013] (2) Mix the crude extract obtained in step (1) with 85-95% v / v methanol-water, and extract 2-4 times with an equal volume of petroleum ether; remove the petroleum ether layer, and concentrate under reduced pressure to obtain the extract;

[0014] (3) Mix the extract obtained in step (2) with 100-200 mesh normal silica gel powder by grinding, and the mass ratio of silica gel powder to extract is 1.4-1.6:1; perform gradient elution with CH2Cl2-MeOH as the eluent, the volume ratio is 100-0:0-100, elute the silica gel column, and after TLC point plate detection, combine similar components, and obtain 7 components Fr. 1-Fr. 7 in total;

[0015] The specific elution gradient of the gradient elution is as follows: the volume ratio of CH2Cl2-MeOH is 100:0, 100:2, 100:4, 100:6, 100:8, 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, and 0:100, a total of 12 gradients, and the elution volume of each gradient is one column volume; collect 12 samples by concentration under reduced pressure, and after TLC point plate detection, combine similar components, and obtain 7 components Fr. 1-Fr. 7 in total;

[0016] (4) Isocratically elute the component Fr. 2 obtained in step (3) by silica gel column chromatography, and the eluent is composed of petroleum ether-ethyl acetate with a volume ratio of 0.9-1.1:0.9-1.1; after TLC point plate detection, combine similar components to obtain 6 components Fr. 2.1-Fr. 2.6;

[0017] (5) Separate the component Fr. 2.1 obtained in step (4) by Sephadex LH-20 column using CH2Cl2-MeOH with a volume ratio of 0.9-1.1:0.9-1.1 as the eluent, concentrate under reduced pressure, combine similar components after TLC point plate detection, and obtain 5 components Fr. 2.1.1-Fr. 2.1.5;

[0018] (6) Isocratically elute the component Fr. 2.1.3 obtained in step (5) by silica gel column chromatography, and the eluent is PE-EtOAc with a volume ratio of 0.9-1.1:0.9-1.1; after TLC point plate detection, combine similar components, concentrate under reduced pressure, and dry to obtain Diaporlabanoid B;

[0019] (7) The component Fr. 2.3 obtained in step (4) is purified by a Sephadex LH-20 column with CH2Cl2-MeOH as eluent in a volume ratio of 0.9-1.1:0.9-1.1, concentrated under reduced pressure, and dried to obtain compound Diaporlabanoid A;

[0020] (8) The component Fr. 2.4 obtained in step (4) is separated by high performance liquid chromatography with MeOH-H2O as mobile phase in a linear gradient elution with a volume ratio of 70:30-100:0 to obtain Diaporlabanoid C.

[0021] Further, the rice culture medium is prepared from the following raw materials by weight: rice 95-105 parts, protein 0.5-0.7 parts, coarse sea salt 2-4 parts, and water 95-105 parts.

[0022] Further, the high performance liquid chromatography conditions are as follows: the chromatographic column is Waters, the specification of the chromatographic column is (5 μm, 10*150 mm), the filler of the chromatographic column is C18, the column temperature is 24-26 ℃, the flow rate is 1.5-2.5 mL / min, and the running time is 25-35 minutes, preferably the column temperature is 25 ℃, the flow rate is 2 mL / min, and the running time is 30 minutes.

[0023] The semi-oleanane diterpene alkaloid compounds are applied in the preparation of immunosuppressive drugs.

[0024] Further, the semi-oleanane diterpene alkaloid compounds are applied in the preparation of immunosuppressive drugs targeting the CaN / NFAT and PKCθ / NF-κB two cascade signal pathways.

[0025] Further, the semi-oleanane diterpene alkaloid compounds are compound Diaporlabanoid A.

[0026] Compared with the prior art, the present application has the following beneficial effects:

[0027] (1) The present application provides three new semi-oleanane diterpene alkaloid compounds with good immunosuppressive activity, which can be applied in the preparation of immunosuppressive drugs.

[0028] (2) The Diaporlabanoids A (compound B-5) provided by the present application not only has strong immunosuppressive activity, but also has low toxicity to normal spleen cells, and can be applied in the preparation of a new type of high-efficiency and low-toxicity immunosuppressive agent.

[0029] (3) The experimental study shows that Diaporlabanoid A has strong immunosuppressive activity on ConA-induced mouse T lymphocytes and low toxicity on normal spleen cells, and can be used for preparing immunosuppressive drugs targeting CaN / NFAT and PKCθ / NF-κB two cascade signal pathways. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 Chemical structures of Diaporlabanoids A-C (1-3);

[0031] Figure 2 COSY and HMBC correlations of compounds 1-3;

[0032] Figure 3 NOESY correlations of compounds 1-3;

[0033] Figure 4 ECD calculated relative configuration of compounds 1-3;

[0034] Figure 5 Single crystal of compound 1;

[0035] Figure 6 Toxic effects of compounds 1-3 on mouse spleen cells;

[0036] Figure 7 Inhibition of ConA-induced spleen cell proliferation by compounds 1-3;

[0037] Figure 8 Inhibition of CaN activity by B-5;

[0038] Figure 9 Effect of B-5 on one-way mixed lymphocyte reaction;

[0039] Figure 10 Effect of B-5 on ConA-induced spleen lymphocyte apoptosis;

[0040] Figure 11 Effect of B-5 on ConA-induced surface activation marker CD25 of mouse T lymphocytes;

[0041] Figure 12 Effect of B-5 on ConA-induced surface activation marker CD69 of mouse T lymphocytes;

[0042] Figure 13 Effect of B-5 on ConA-induced spleen lymphocyte cycle;

[0043] Figure 14 Effect of B-5 on CD4 +Effect on T cell ratio

[0044] Figure 15 Effect of B-5 on CaN protein expression in ConA-induced spleen lymphocytes 2+ Effect on concentration

[0045] Figure 16 Effect of B-5 on CaN protein expression in ConA-induced spleen lymphocytes

[0046] Figure 17 Effect of B-5 on NFAT protein expression in ConA-induced spleen lymphocytes

[0047] Figure 18 Effect of B-5 on NF-κB protein expression in ConA-induced spleen lymphocytes

[0048] Figure 19 Effect of B-5 on PKCθ protein expression in ConA-induced spleen lymphocytes

[0049] Figure 20 Effect of B-5 on IL-2, IL-4, IFN-γ, IL-6 in cell supernatant

[0050] Figure 21 Effect of B-5 on IL-2, IFN-γ, IL-4, IL-6 gene transcription in ConA-stimulated T lymphocytes

[0051] Figure 22 B-5 inhibits the expression level of NFAT protein in the nucleus

[0052] Figure 23 B-5 inhibits the expression level of NF-κB protein in the nucleus, DAPI is blue to represent the nucleus, NFAT is green, NF-κB is red, and the microscope magnification is 200x).

[0053] Note: Diaporlabanoids A-C correspond to compounds 1-3, respectively, and Diaporlabanoids B-5, 6-13 and HX- correspond to compounds 4-10 and 11, respectively. 22。 DETAILED DESCRIPTION

[0054] In order to better understand the technical content of the present application, the following specific examples are provided to further illustrate the present application.

[0055] The experimental methods used in the embodiments of the present application are all conventional methods unless otherwise specified.

[0056] The materials, reagents, etc. used in the embodiments of the present application can be obtained from commercial channels unless otherwise specified.

[0057] The Chinese names of some compounds used in the present application: PE petroleum ether; EtOAc ethyl acetate; CH2Cl2 dichloromethane; MeOH methanol.

[0058] 1. Research method

[0059] 1.1 Strain source

[0060] Endophytic fungi Diaporthe phaseolorum H3-2 is a fungus of the genus Colletotrichum obtained from fresh hypocotyl tissues of the mangrove plant Excoecaria aculeata collected in Hainan Province. Through observation of the morphological characteristics of the strain and molecular biology methods (ITS-rDNA sequence alignment), the strain was finally identified as Colletotrichum truncatum. Ceriops tagal Diaporthe H3-2 (GenBank No: MH707091.1) is preserved in the China Center for Type Culture Collection (CCTCC) (PDA medium, -20°C storage). The strain H3-2 is selected as the target strain, and the fungus is now preserved in the China Center for Type Culture Collection (CCTCC), located at Wuhan University, No. 29, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, with the preservation number CCTCC NO: M20231234 and the preservation date July 7, 2023. phaseolorum Diaporthe phaseolorum

[0061] 1.2. Fermentation extraction and separation process

[0062] (1) First, activate H3-2 in a petri dish with PDA medium. When the colony morphology is stable and a single colony is formed, inoculate a 1cm*1cm single colony in PDA medium for expansion culture. Inoculate a 2cm*2cm single colony into sterilized rice medium (100 1L conical flasks, each containing 100g of rice, 0.6g of peptone, 3g of coarse sea salt, 100mL of water, 121°C sterilization for 30min), and ferment at 28°C for 28 days. After fermentation, extract each flask with 400ml of ethyl acetate for 3 times, and concentrate under reduced pressure to obtain 35g of crude extract. Diaporthe phaseolorum (2) In order to remove the fatty acid impurities in the crude extract, mix the crude extract of step (1) with 300ml of 90%v / v methanol-water, and extract with 300ml of petroleum ether three times. Remove the petroleum ether layer and concentrate under reduced pressure to obtain 20g of extract, i.e. the extract.

[0063]

[0064] ​​​(3) The extract of step (2) was mixed with 100-200 mesh normal silica gel powder (mass of silica gel powder: mass of extract = 1.5:1) and gradient eluted with CH2Cl2-MeOH as eluent, and the silica gel column was eluted. The elution gradient was CH2Cl2-MeOH (100:0, 100:2, 100:4, 100:6, 100:8, 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 0:100, v / v), and the elution volume was 6 column volumes for each gradient. A total of 12 samples were collected by vacuum concentration. After thin layer chromatography (TLC) detection, similar components were combined, and a total of 7 components, Fr. 1-Fr. 7, were obtained.

[0065] (4) Component Fr. 2 was isocratically eluted with a silica gel column (petroleum ether: ethyl acetate 1:1, v / v), and similar components were combined after TLC detection, to obtain 6 components, Fr. 2.1-Fr. 2.6.

[0066] (5) Component Fr. 2.1 was separated by Sephadex LH-20 column using CH2Cl2-MeOH (1:1, v / v) as eluent, vacuum concentrated, and similar components were combined after TLC detection, to obtain 5 components, Fr. 2.1.1-Fr. 2.1.5.

[0067] (6) Component Fr. 2.1.3 was 10 mg of white powder, which was isocratically eluted with a silica gel column (PE-EtOAc (1:1, v / v)), and similar components were combined after TLC detection, vacuum concentrated, and dried to obtain Diaporlabanoid B (compound 2) (5 mg).

[0068] (7) Component Fr. 2.3 was 20 mg of white powder, and Fr. 2.3 was purified by Sephadex LH-20 column using CH2Cl2-MeOH (1:1, v / v) as eluent, vacuum concentrated, and dried to obtain compound Diaporlabanoid A (compound 1) (15 mg).

[0069] (8) Component Fr. 2.4 was 15 mg of white powder, which was separated by high performance liquid chromatography (MeOH-H2O, 70:30-100:0, v / v, linear gradient elution) to obtain Diaporlabanoid C (compound 3) (6 mg).

[0070] The conditions of the above high performance liquid chromatography: the chromatographic column is Waters, the specification of the chromatographic column is (5 μm, 10 x 150 mm), the filler of the chromatographic column is C18 column, the column temperature is 25 ℃, the flow rate is 2 mL / min, and the running time is 30 minutes.

[0071] 1.3 Compound structure analysis:

[0072] Diaporlabanoid A (1) is a colorless columnar crystal, [α] 20 D =+30 ( c 0.0001, MeOH), positive reaction to bismuth potassium iodine reagent, ultraviolet absorption UV (MeOH) λmax 204, 205, 206 nm. HR-ESI-MS [M+H] + m / z 404.2068 (calculated value 404.2073), indicating that the molecular formula of compound Diaporlabanoid A (1) is C 22 H 29 NO6, unsaturation is 9, containing nine double bond equivalent (DBE). Combined 1 H and 13 C NMR data (Table 1) and DEPT spectrum data, it is found that there are four sp 3 methyl, four sp 3 methylenes, seven sp 3 methylene (one of which contains an oxygen methylene signal at δ C 77.2), three sp 3 quaternary carbons (two of which contain oxygen quaternary carbon signals at δ C 68.4 and 80.0) and four sp 2 quaternary carbon signals (two ketone carbonyl functional groups signals at δ C 206.1 and 207.9, two carbonyl signals of imide functional group at δ C 175.1 and 178.1). The above NMR data accounts for four unsaturations (total unsaturation is 9), which can be inferred that Diaporlabanoid A (1) has a five-ring skeleton (accounting for the remaining 5 unsaturations). The planar structure of Diaporlabanoid A (1) is confirmed by two-dimensional nuclear magnetic resonance, in which 1 H- 1 H COSY spectrum ( Figure 2 ) confirms two proton carrying fragments drawn in bold, combined with HMBC spectrum (Figure 2 The correlation between the quaternary carbon atom and the oxygen atom was confirmed, thus determining the skeleton of Diaporlabanoid A (1). H3-21 / C-2, C-3, and C-4; H2-4, H-5, and H2-7 / C-6; H3-20 / C-7, C-8, and C-9; H-9 and H-10 / C-11; and H-13 / C-8, C-11, and C-18 showed HMBC correlation, as indicated by the arrows from H to C, confirming the connection segment from ring A to ring C in substructure I. The above NMR data are similar to the characteristic proton and / or carbon signals of labdane type diterpenes (reference), suggesting that the compound type is dinorlabdane diterpene. By amide proton ( δ H The presence of a unique succinimide molecule (ring E, part II) can be clearly inferred from the HMBC correlations of 11.49 (s, NH) with C-12, C-16, C-17, and C-18. The planar structure of Diaporlabanoid A (1) was determined by connecting the substructures of parts I and II via ring D through HMBC correlations of H2-14 / C-11, C-12, H-15, and H-16 / C-17 and the proton-containing fragment CH(13)-CH2(14)-CH(15)-CH3(19). Diaporlabanoid A (1) is a novel skeleton belonging to a unique class of dinorlabdane diterpene alkaloids with a 6 / 6 / 6 / 5 / 5 pentacyclic (A / B / C / D / E rings) core containing a rare succinimide unit. This was further confirmed by analyzing NOESY spectra (…). Figure 3 The analysis determined the relative configurations of Diaporlabanoid A (1), where H-1 / H3-20, H-5 / H3-20, H-5 / H3-21, H3-21 / H a -4, H-9 / H3-20, and H3-19 / H-16 showed NOE correlation, while H-9 / H-13 and H3-20 / H-13 showed no NOE correlation, indicating that H-1, H3-20, H-5, H3-21, and H... a -4, H-9, H3-19, and H-16 have the same β orientation. Furthermore, 3-OH / H aThe NOE correlation between -4, H3-22 / H-10 and H-13 / H-15 indicates that these protons are coplanar and have an α orientation. These results show that the pentacyclic rings are fused together in trans (A / B), cis (B / C), trans (C / D) and cis (D / E) configurations. Therefore, the relative configuration of Diaporlabanoid A (1) was determined. The calculated ECD spectrum of Diaporlabanoid A (1) is in good agreement with the experimental spectrum. Figure 4 The absolute configuration of Diaporlabanoid A(1) was determined by single-crystal X-ray crystallography using Cu Kα rays. This not only confirmed the above-mentioned planar structure, but also determined that the absolute configuration of Diaporlabanoid A(1) is 1. R , 3 S , 5 S , 8 S , 9 R , 10 S , 12 S , 13 S , 15 R , 16 S ( Figure 5 ).

[0073] Diaporlabanoid B (2) is a white powder, [α] 20 D +50 ( c 0.0001, MeOH), showed a positive reaction to potassium bismuth iodide reagent, with UV absorption of UV(MeOH) λmax at 204, 205, and 206 nm. Based on HR-ESI-MS [M+H] + m / z The calculated value is 388.2117 (calculated value 388.2124). The molecular formula of the compound is determined to be C. 22 H 29 NO5 indicates that Diaporlabanoid B (2) has one less hydroxyl group than Diaporlabanoid A (1). Its NMR spectral data are very similar to those of Diaporlabanoid A (1) (Table 1). However, the NMR spectrum of Diaporlabanoid B (2) lacks... δ H 4.42 hydroxyl signal of (3-OH) and δ C Oxygen-containing quaternary carbon signal at 68.4 (C-3). Diaporlabanoid B (2) at C-2 ( δ C33.2), C-3 ( δ C 25.8), C-4 ( δ C 34.7) shifted, H3-21 signal changed from singlet in Diaporlabanoid A (1) to doublet (J 21,3 = 6.5 Hz) in Diaporlabanoid B (2), which proved the absence of 3a-OH in Diaporlabanoid B (2). In the HMBC spectrum ( Figure 2 ) of Diaporlabanoid B (2), the correlations of H3-21 / C-2, C-3, and C-4; H-3 and H-4 / C-3, and in 1 H- 1 The CH(9)-CH(10)-CH(1)-CH2(2)-CH(3)-CH2(4)-CH(5) correlation observed in the H a -4 and H-3 / H b -4 NOESY correlations ( Figure 3 ) and the NMR similarity between Diaporlabanoid B (2) and Diaporlabanoid A (1) except for C-3 and C-5, which determined the relative configuration of the stereocenters in Diaporlabanoid B (2). This speculation was further confirmed by the very good agreement between the calculated ECD spectrum of Diaporlabanoid B (2) and the experimental one ( Figure 4 ), which verified the absolute configuration of Diaporlabanoid B (2). Therefore, the absolute configurations of nine steroidal centers were defined as 1 R , 3 R , 5 R ,8 S , 9 R , 10 S , 12 S , 13 S , 15 R , 16 S , which led to the complete structure of Diaporlabanoid B (2) as shown in Figure 1

[0074] Diaporlabanoid C (3) was a white powder, [a] 20 D +60 ( c ​0.0001, MeOH), showed a positive reaction to potassium bismuth iodide reagent, with UV absorption of UV(MeOH) λmax at 203, 204, and 205 nm. Based on HR-ESI-MS [M+H] + m / z 388.2118 (calculated value 388.2124) confirms that compound 3 has the same molecular formula as Diaporlabanoid B (2), C 22 H 29 NO5. Diaporlabanoid C (3) 1 H and 13 C NMR data (Table 1) and Diaporlabanoid B (2) 1 Hand 13 The C10 NMR data are very similar, except that the shifts between CH-1, CH2-2, CH-3, CH3-21, C-9, and CH-10 of Diaporlabanoid C(3) differ more than those of other parts of Diaporlabanoid B(2), suggesting that Diaporlabanoid C(3) may be a diastereomer of Diaporlabanoid B(2) on the six-membered A / B ring. One-dimensional and two-dimensional NMR spectra of Diaporlabanoid C(3) confirm this hypothesis, therefore the planar structure of Diaporlabanoid C(3) is the same as that of Diaporlabanoid B(2). In NOESY spectra (… Figure 3 H-5 / H-10, H-5 / H3-20, H-5 / H3-21, H-5 / H appear in ) b -4, H-3 / H a The correlation between -4, H3-22 / H-10, H-9 / H-10, and H-9 / H3-20 was established. Their configurations were verified by ECD calculations. Figure 4 ), which determined that Diaporlabanoid C(3) has 1 S , 3 R , 5 S , 8 S , 9 R , 10 R ,12 S , 13 S , 15 R , 16 S Configuration.

[0075] Diaporlabanoids A-C (1-3) are new skeleton compounds, which are different from the previously reported diptercane diterpenoids with a classic 6 / 6 / 6 fused tricyclic core skeleton, and contain a rare succinimide unit. The putative biosynthetic pathway of 1-3 can be traced back to manoyl oxide, which is obtained from geranylfarnesyl pyrophosphate (GFPP) through a series of enzymatic reactions.

[0076] Table 1. Compound 1-3 of 1 H and 13 C NMR data δ / ppm, J / Hz)

[0077]

[0078] 1. Preliminary screening of immunosuppressive activity of compounds 1-3 on mouse spleen cells

[0079] 1.1 Experimental materials

[0080] Animal material: 6-8 weeks old female BLAB / c mice raised under SPF conditions.

[0081] Experimental reagents: RPMI-1640 medium (Boster), fetal bovine serum (Sijiqing), CCK-8 reagent (Biosharp), calcineurin (CaN) kit (Shanghai Fusen), calcineurin (CaN) test box (Nanjing Jiancheng), cell cycle kit (Lianke Biology), Annexin V-FITC / PI apoptosis kit (Vazyme Cat. A211-01), PE Conjugate-Anti-Mouse CD8 (Elabscience), APC Conjugate-Anti-Mouse CD69 (Elabscience), PE Conjugate-Anti-Mouse CD25 (Elabscience), FITC Conjugate-Anti-Mouse CD4 (Elabscience), DMSO for cells (Boster), concanavalin A (ConA) (Sigma), erythrocyte lysis solution (Biosharp), mitomycin C (Shanghai Shifeng), compounds 1-3 (Diaporlabanoid A-C).

[0082] 1.2 Preparation of mouse spleen cell suspension

[0083] 1) Prepare the dissection box, 5 mL syringe needle, etc. in the super near workbench. Kill the mouse by cervical dislocation, soak it in alcohol, spray alcohol on it, put it in the workbench, cut a small opening from the middle of the left abdominal side of the mouse, expose the abdominal wall, lift the peritoneum, expose the spleen, take out the spleen with tweezers, and put it into a 15 mL centrifuge tube containing 5 mL of culture medium.

[0084] 2) Cut the spleen into several pieces on a 200-mesh stainless steel mesh, gently crush the spleen with a syringe needle core and gently rinse the mesh with culture medium to obtain a spleen cell suspension.

[0085] 3) After centrifugation at 1000 rpm for 5 min, discard the supernatant, lyse the red blood cells with red blood cell lysis solution for 5 min, centrifuge for 5 min, and add 4 mL of RPMI-1640 complete medium to prepare a cell suspension. Then count and prepare a cell suspension with a density of 1*10 7 6 / mL.

[0086] 1.3 Toxicity test

[0087] 1) Take a 96-well cell culture plate, inoculate 100 μL of lymphocyte suspension with a concentration of 1*10 7 6 / mL per well, and incubate overnight at 37°C in a 5% CO2 incubator to stabilize the cells.

[0088] 2) Add compound B-5 or positive control (CsA) diluted with complete medium to each well at different concentrations, so that the final concentrations are 1, 5, 10, 15, 20, 30 and 40 μM, respectively. The blank control group is added with complete medium containing 0.2% DMSO, and each concentration has 3 replicate wells. After incubation in the incubator for 72 h, 20 μL of CCK-8 reagent is added to each well. Incubate at 37°C for 4 h, and read OD 450 with a microplate reader. The cell survival rate is calculated by the formula:

[0089] Spleen cell survival rate = OD 实验组 / OD 空白对照组 x 100%.

[0090] Experimental results:

[0091] Table 2 Toxicity of compounds B-5, 6-13 and HX-22 to normal mouse spleen lymphocytes

[0092]

[0093] Experimental analysis: Figure 6 and Table 2 show that the IC 50 50 of compound B-5 to normal mouse spleen lymphocytes is 108.31 ± 2.54 μM; the IC50 = 13.13 ± 2.033 μM; the IC50 of compound HX-22 against normal mouse spleen lymphocytes was 25.62 ± 1.791 μM 50 = 13.13 ± 2.033 μM; the IC50 of compound HX-22 against normal mouse spleen lymphocytes was 25.62 ± 1.791 μM 50 = 11.34 ± 0.67 μM, indicating that the toxicity of compound B-5 to spleen cells was much lower than that of CsA in a certain concentration range.

[0094] 1.4. Effect of compound B-5 on the immunosuppressive activity of ConA-induced mouse spleen cells

[0095] 1) The method for preparing the spleen cell suspension was the same as that in 1.1. A 96-well cell culture plate was used, and the spleen cell suspension with a concentration of 5 x 10 6 was inoculated in each well at 100 μL, and the plate was cultured in a 37°C, 5% CO2 incubator.

[0096] 2) Grouping: blank control group: 200 μL of complete RPMI-1640 culture solution; negative control group: 100 μL of cell suspension and 100 μL of complete RPMI-1640 culture solution; ConA positive control group: 100 μL of cell suspension and 100 μL of complete RPMI-1640 culture solution containing ConA (final concentration of 5 μg / mL); drug control group: 100 μL of complete RPMI-1640 culture solution, 50 μL of ConA (final concentration of 5 μg / mL), and 50 μL of compound B-5 with different concentrations; drug group: 100 μL of cell suspension, 50 μL of ConA (final concentration of 5 μg / mL), and 50 μL of complete RPMI-1640 culture solution containing compound B-5 (final concentration of 15 μM, 30 μM, 45 μM, 60 μM, or 75 μM); three replicate wells were set in each well, and the plate was cultured in a 37°C, 5% CO2 incubator for 48 h. Then, 20 μL of CCK-8 reagent was added to each well of the 96-well plate, and the plate was further incubated in the incubator for 4 h in the dark. The absorbance value was measured at 450 nm using a microplate reader.

[0097]

[0098] Experimental results:

[0099] Table 3. Immunosuppressive activity of compounds B-5, 6-13, and HX-22

[0100]

[0101] Experimental analysis:

[0102] In the ConA-induced spleen cell proliferation experiment, the group with only ConA but without drugs was set as the cell survival rate of 100%, and the rest of the groups with drugs were treated with ConA at a final concentration of 5 μg / mL for proliferation induction, and the cell survival rate was calculated by the following formula: Figure 7 As shown in Table 2, compounds B-5, 6-13 and HX-22 had certain inhibitory effect on ConA-induced spleen lymphocyte proliferation, and the higher the concentration, the smaller the cell survival rate, showing a concentration-dependent manner. The results preliminarily indicated that compounds B-5, 6-13 and HX-22 all had certain immunosuppressive activity, and the immunosuppressive activity of compound B-5 was the best.

[0103] It was found from the cytotoxicity experiment that compared with CsA, compound B-5 had little toxicity to spleen lymphocytes, and the half-inhibitory concentration (IC 50 ) value was (108.31 ± 2.54) μM, while the skeleton analog compounds 6-13, HX-22 and the positive drug (CsA) had strong killing effect on mice, and the half-inhibitory concentration of toxicity was (IC 50 = 11.34 ± 0.67, 13.13 ± 2.033, 13.13 ± 2.033 μM), respectively. In the ConA-induced spleen cell proliferation experiment, compound B-5 inhibited the ConA-induced spleen lymphocyte proliferation, and the half-inhibitory concentration IC 50 = 47.28 ± 0.71 μM. (Note: p <0.05,** p <0.01 compared with the Control group). Therefore, among the three new skeleton compounds B-5, 6-13 and HX-22, compound B-5 was selected for further study on the immunosuppressive activity of ConA-induced spleen cell proliferation.

[0104] 2. Phenotype study on the immunosuppressive activity of compound 1 (B-5) on mouse spleen cells

[0105] 2.1. Effect of compound B-5 on CaN enzyme activity in vitro

[0106] The operation steps are as follows:

[0107] 1) The final concentration of compound B-5 was 1 μM, 2.5 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 50 μM, 70 μM, 90 μM, 120 μM; dissolved in DMSO, and the final DMSO content was 0.2%.

[0108] 2) CaN enzyme activity test

[0109] Table 4

[0110]

[0111] Table 5

[0112]

[0113] Calculation formula: Relative inhibition rate (%) = [1 - (ODdrug+enzyme- ODdrugcontrol) / ODenzyme] x 100%

[0114] Experimental analysis:

[0115] The in vitro enzyme activity experiment was carried out with pNPP as a substrate to determine the relative inhibition rate of monomeric compound B-5 on CaN, and the results were as follows: Figure 8 It can be seen that the higher the concentration of compound B-5, the higher the inhibition rate on CaN, which presents a concentration dependence, and the IC 50 was 59.18 ± 2.21 μM.

[0116] 2.2. Experiment of one-way mixed lymphocyte reaction

[0117] The operation steps were as follows:

[0118] 1) Cell treatment:

[0119] The mice were sacrificed by cervical dislocation, and the spleen cell suspension was prepared according to the method of 1.1 (the cell density was 1 x 10 7 6 / mL). Among them, the C57BL mouse spleen cells were treated with mitomycin C and set as reaction cells (the final concentration of mitomycin C was 50 μg / mL), incubated at 37°C for 45 min, and set as No. 1 reaction cells; the rest was not treated with mitomycin C and set as No. 2 reaction cells, and the BALB / c mouse spleen cells were set as stimulating cells.

[0120] 2) Co-incubation of drugs and cells

[0121] The two kinds of spleen cell suspensions were added into 96-well plates respectively, and were cultured with different concentrations of drugs. Among them, the blank negative control group: 200 μL of RPMI-1640 culture solution containing 10% serum; the reaction cell control group: 50 μL of reaction cell suspension, 50 μL of RPMI-1640 culture solution containing serum, 100 μL of RPMI-1640 solution; the stimulated cell control group: 50 μL of stimulated cell suspension, 50 μL of RPMI-1640 culture solution containing serum, 100 μL of RPMI-1640 solution; the reaction cell + stimulated cell group positive control: 50 μL of reaction cell suspension, 50 μL of stimulated cell suspension and 100 μL of RPMI-1640; the drug control group: 100 μL of RPMI-1640 culture solution containing serum, 100 μL of drug diluted with RPMI-1640; the drug group: 50 μL of reaction cell suspension, 50 μL of stimulated cell suspension and 100 μL of drug diluted with RPMI-1640. After being cultured in the incubator for 48 h, 20 μL of CCK-8 was added to each well, and the plate was incubated in the incubator for 4 h. The OD 450 .

[0122] Experimental analysis:

[0123] In the process of drug research, the one-way MLR experiment is an in vitro experiment that can be used to evaluate the immunomodulatory activity of compounds, and the most common one is the T lymphocyte-based one-way MLR experiment. Two groups of allogeneic T lymphocytes are mixed and cultured, the stimulation group is treated with mitomycin C to remove its proliferation ability, so as to allow the T lymphocytes in the reaction group to proliferate in response to foreign histocompatibility antigens, and the immunomodulatory activity of the compound is evaluated by adding the compound for treatment. In the one-way mixed lymphocyte reaction experiment, the negative control without drug is set as the cell survival rate of 100%, and the experimental results are as follows Figure 9 Compound B-5 has a certain inhibitory effect in the one-way mixed lymphocyte reaction experiment. With the continuous increase of the concentration of compound B-5, the cell survival rate is significantly reduced, and it shows a dose-dependent inhibition of the rejection reaction of the host to the graft. The half-inhibitory concentration of compound B-5 in the one-way mixed lymphocyte reaction is IC 50 46.71±2.03 μM (Note: * p <0.05, ** p <0.01 compared with the Control group).

[0124] 2.3. Effect of compound B-5 on spleen cell apoptosis

[0125] 1) The preparation method of the spleen cell suspension is the same as that in 1.1, and the final cell concentration is 1*10 7Cell suspension was prepared as described in 1.1. The final concentration of cell suspension was 1*10 o C, 5% CO2 incubator overnight.

[0126] 2) Other reagents were added as follows: negative control group: 1 mL RPMI-1640 complete culture solution. ConA positive control group: 1 mL RPMI-1640 complete culture solution containing ConA (final concentration 5 μg / mL). Drug group: 1 mL RPMI-1640 complete culture solution containing ConA (final concentration 5 μg / mL) and compound B-5 (final concentration 20 μM, 40 μM, 60 μM, respectively). 37 o C, 5% CO2 incubator for 48 h, then collect cells, stain, and detect by flow cytometry.

[0127] Experimental analysis:

[0128] As shown in Figure 10 Figure 2, after 48 hours of treatment with compound B-5, flow cytometry was used to detect apoptosis. As can be seen from the figure, with increasing concentration, there was no apoptosis trend in the cells, i.e. compound B-5 had no pro-apoptotic effect on ConA-induced spleen lymphocytes, which revealed that the inhibition of T lymphocyte proliferation by compound B-5 was not caused by inducing cell apoptosis. # p <0.05, ## p <0.01 compared with the Control group; * p <0.05, ** p <0.01 compared with the ConA group).

[0129] 2.4 Effect of compound B-5 on T lymphocyte surface activation markers CD25 and CD69

[0130] 1) Spleen cell suspension was prepared as described in 1.1. The final concentration of cell suspension was 1*10 7 Cell suspension was prepared as described in 1.1. The final concentration of cell suspension was 1*10 o C, 5% CO2 incubator overnight.

[0131] 2) Other reagent group plus reagent as follows: negative control group: plus 1 mL RPMI-1640 complete culture solution. ConA positive control group: 1 mL RPMI-1640 complete culture solution containing ConA (final concentration 5 μg / mL). Drug group: 1 mL RPMI-1640 complete culture solution containing ConA (final concentration 5 μg / mL) and compound B-5 (final concentration 20 μM, 40 μM, 60 μM respectively).37 o After 12 h culture in a 37 °C, 5% CO2 incubator, the cells were collected, and the samples were respectively stained with CD4\FITC, CD25\PE, CD69\APC, and detected by flow cytometry and analyzed.

[0132] Experimental analysis:

[0133] Results as shown in Figure 11 showed that the expression level of CD25 of the cells after ConA stimulation was significantly increased, and compound B-5 had a significant inhibitory effect on the expression of CD25 of the cells induced by ConA. Similarly, as shown in Figure 12 , the expression level of CD69 of the cells after ConA stimulation was significantly increased, and compound B-5 significantly inhibited the expression of CD69 of the cells induced by ConA. It is shown that compound B-5 inhibited the activation of CD4 + T cells induced by ConA. (Note: # p <0.05, ## p <0.01 compared with the Control group; * p <0.05, ** p <0.01 compared with the ConA group).

[0134] 2.5 Cell cycle detection experiment

[0135] The method for preparing the spleen cell suspension was the same as that in 1.1, and the cell density was 5×10 6The cells were treated with the compound B-5 (20, 40, 60 μM) and cultured for 24 h in the incubator. Then, the cells were collected in 1.5 mL EP tubes, centrifuged, and the supernatant was carefully aspirated. The cells were collected. 50 μL of the culture solution was reserved. The cells were washed with PBS and added to 1 mL of pre-cooled 70% ethanol, mixed gently, and fixed at -20°C for 24 h. The cells were centrifuged and the supernatant was aspirated. The cells were washed once more with PBS. 500 μL of the prepared PI staining solution was added to each sample, and the cells were mixed and incubated at 37°C for 30 min in the dark. The flow cytometry was used for detection.

[0136] Experimental results:

[0137] Results are shown in Figure 13 It is shown that most of the cells were in the G1 phase before the ConA treatment, and did not enter the cell division phase. After the ConA stimulation, the cells in the S and G2 phases were significantly increased, indicating that the cells entered the division phase. The process of the cells entering the S and G2 phases from the G1 phase was significantly inhibited after the treatment with the compound B-5. Therefore, the compound B-5 can inhibit the proliferation of the T lymphocytes induced by ConA by inhibiting the transformation of the T lymphocytes from the G1 phase to the S and G2 phases. # p <0.05, ## p <0.01 compared with the Control group; * p <0.05, ** p <0.01 compared with the ConA group).

[0138] 2.6. Effect of the compound B-5 on the CD4 + T cells in the ConA-induced spleen lymphocytes

[0139] 1) The preparation method of the spleen cell suspension was the same as that in 1.1, and the final concentration of the cell suspension was 1*10 7 cells / mL. 500 μL was added to each well of the 24-well plate and cultured overnight at 37°C in a 5% CO2 incubator. o

[0140] ​2) Other reagent group added reagent as follows: negative control group: added 1 mL RPMI-1640 complete culture solution. ConA positive control group: 1 mL RPMI-1640 complete culture solution containing ConA (final concentration 5 μg / mL). Drug group: 1 mL RPMI-1640 complete culture solution containing ConA (final concentration 5 μg / mL) and compound B-5 (final concentration 200 μM, 40 μM, 60 μM respectively).37 o C, 5% CO2 incubator for 24 h.

[0141] 3) Staining: two blank groups were respectively labeled with fluorescent antibodies CD4 / FITC, CD8 / PE; another group was not added any antibody; the rest of the samples needed to add the above two antibodies.4 o C After 30 min of staining in the dark, take out and wash with PBS for three times. Finally, add 500 μL PBS and detect by flow cytometry. Staining ratio: 100 μL PBS + 5 μL antibody.

[0142] Experimental analysis:

[0143] As Figure 14 shown, compared with the blank group, after the action of ConA, the ratio of CD4 + T cells increased obviously, while after the treatment of compound B-5, the proliferation of CD4 + T cells induced by ConA was inhibited obviously, and the proliferation of CD8 + T cells did not show obvious inhibition. It is indicated that compound B-5 inhibits the proliferation of T cells and is inclined to inhibit the proliferation of CD4 + T cell subpopulation. (Note: # p <0.05, ## p <0.01 compared with the Control group; * p <0.05, ** p <0.01 compared with the ConA group).

[0144] Three, mechanism of immune suppression activity of compound B-5 on mouse spleen cells

[0145] Animal material: 6-8 weeks old female BLAB / c mice raised under SPF conditions.

[0146] Experimental reagents: RPMI 1640 medium (Boster), fetal bovine serum (Sijiqing), cell-specific DMSO (Boster), (ConA) concanavalin A (Sigma), erythrocyte lysis buffer (Biosharp), Fluo-4 AM calcium ion fluorescent probe (Beyotime S1060), Tris-glycine-SDS electrophoresis buffer (Boster), Tris-glycine-SDS transfer buffer (Boster), SDS-PAGE gel preparation kit (Boster), ultrasensitive ECL chemiluminescence ready-to-use substrate (Boster), 10X TBST buffer (Solarbio), 0.45 μm PVDF membrane (Biosharp), NFAT (Affinity Biosciences), 4% paraformaldehyde (Biosharp), enhanced RIPA lysis buffer (Boster), ELISA kit for mouse interleukin-4 (IL-4), ELISA kit for mouse interferon-γ (IFN-γ), ELISA kit for mouse interleukin-2 (IL-2) (Shanghai Fusheng), Q711ChamQ Universal SYBR qPCR Master Mix, RC101 FastPure Cell / Tissue Total RNAIsolation Kit, R323-01 HiScript III® All in one RT SuperMix perfect for qPCR (Vazyme), 4% paraformaldehyde (Biosharp), Triton X-100 (Beyotime), Cy3-bound IgG (Boster), compound B-5, etc.

[0147] 3.1. Effect of B-5 on intracellular calcium ion concentration

[0148] 1) The method for preparing the spleen cell suspension is the same as in 1.1, and the final cell concentration is 1*102. 7 Cell suspension of 500 μL / ml was added to each well of a 24-well plate and incubated at 37°C. o Incubate overnight in a 5% CO2 incubator.

[0149] 2) Other reagents were added to the groups as follows: Negative control group: 1 mL of RPMI-1640 complete culture medium. ConA positive control group: 1 mL of RPMI-1640 complete culture medium containing ConA (final concentration 5 μg / mL). Drug group: 1 mL of RPMI-1640 complete culture medium containing ConA (final concentration 5 μg / mL) and compound B-5 (final concentration 60 μM). 37 oAfter culturing in a 5% CO2 incubator for 1 hour, cells were collected, washed three times with PBS, and then incubated at 37°C. o After staining with C Fluo-4 AM in the dark for 30 min, the cells were washed three times with PBS, incubated in the dark for 15 min, and the intracellular calcium ion fluorescence was detected under a laser confocal microscope.

[0150] Experimental results:

[0151] Experimental Analysis:

[0152] Ca 2+ As an important second messenger within T lymphocytes, it can act as a sensor to receive extracellular molecular stimuli, directly mediating the activation of the downstream CaN / NFAT signaling pathway, thereby inducing the transcription and secretion of related cytokines required for T lymphocyte activation and proliferation. To investigate whether compound B-5 affects the CaN / NFAT of T lymphocytes... 2+ Influx was observed, and cells treated with the drug were stained using the Fluo-4-AM probe. Changes in the mean fluorescence intensity of the cells were then observed using a laser confocal microscope. Figure 15 As shown, stimulation of mouse T cells with ConA significantly increased the intracellular calcium ion concentration. p <0.01); After treatment with compound B-5 (20, 40, 60 μM), there was no significant change in intracellular calcium ion concentration ( p >0.05). This indicates that compound B-5 does not affect ConA-induced calcium in T lymphocytes. 2+ internal flow. (Note: # p <0.05, ## p <0.01 compared with the Control group; * p <0.05, ** p <0.01 compared with ConA group; microscope magnification 20×).

[0153] 3.2. Western blot analysis of CaN protein, NFAT protein, and NF-κB protein expression.

[0154] 1) The method for preparing the spleen cell suspension is the same as in 1.1, and the final cell concentration is 1*102. 7 Cell suspension of 2 cells / mL was added to each well of a 6-well plate at 37°C. o Incubate overnight in a 5% CO2 incubator.

[0155] 2) Other reagent group plus reagent: negative control group: plus 2 mL RPMI-1640 complete culture solution; ConA positive control group: 2 mL RPMI-1640 complete culture solution containing ConA (final concentration 5 μg / mL); CsA group: 2 mL RPMI-1640 complete culture solution containing ConA (final concentration 5 μg / mL) and CsA (final concentration 5 μM); drug group: 2 mL RPMI-1640 complete culture solution containing ConA (final concentration 5 μg / mL) and compound B-5 (final concentration 20 μM, 40 μM, 60 μM, respectively).37 o C, 5% CO2 incubator for 24 h.

[0156] 3) Collect cells, centrifugal to remove supernatant. After pre-cooled PBS washing twice, transfer to EP tube.

[0157] 4) Protein extraction and concentration determination: use Doctor's strong lysis solution to extract protein, 150 μL per tube, carefully blow the cells, react on ice for 13 min, 14000 g, low-temperature centrifugal for 5 min, collect supernatant. Use BCA protein quantitative kit to determine protein concentration, then use 5x SDS-PAGE Loading Buffer and pre-cooled PBS to adjust protein concentration.100 o C, boil for 8 min, after cooling, put into -20 o C refrigerator. Protein loading amount is 30 μg, loading volume is 15 μL.

[0158] 5) Electrophoresis and membrane transfer: configure gel according to SDS-PAGE gel preparation kit, insert comb, carefully pull out comb after gel solidification. Connect electrophoresis device, add electrophoresis liquid, carefully add protein sample into slot hole. Connect power supply (parameters: U=80 V; I=120 mA; P=50 W; T=15 min), when protein electrophoresis reaches the upper edge of separation gel, change electrophoresis condition (parameters: U=120 V; I=150 mA; P=50 W; T=50 min). After protein sample electrophoresis to the bottom of gel plate, turn off electrophoresis, take out gel, according to its size, trim PVDF membrane, PVDF membrane is soaked in methanol for 1 min. According to sequence, place from negative electrode to positive electrode (sponge - filter paper - gel - PVDF membrane - filter paper - sponge). Put into membrane transfer liquid for membrane transfer (parameters: U=220 V; I=250 mA; P=50 W; T=90 min).

[0159] 6) Blocking: put PVDF membrane into incubation box containing 5% skimmed milk powder TBST solution, 4 o C, incubate on shaker overnight.

[0160] 7) Add antibody incubation: The next day, remove the PVDF membrane and wash it three times for 10 minutes each. Dilute the primary and secondary antibodies according to the manufacturer's instructions. Incubate the primary antibody at room temperature for 2 hours, then wash the PVDF membrane three times for 10 minutes each. Incubate the secondary antibody at room temperature for 1 hour, then wash the PVDF membrane three times.

[0161] 8) Development: Take 3 mL of ECL luminescent solution A and B, mix them in equal volumes, and place the PVDF membrane face down in the dark for 1 minute. Then place the PVDF membrane face up in the dark box and expose it for 10-15 seconds.

[0162] 9) Process data, Image J for gray scale analysis.

[0163] Experimental analysis:

[0164] From the bands Figure 16 , 17 , 18, 19, it can be seen that after ConA induction, the expression levels of p-PKCθ, CaN, NFAT, and NF-κB proteins in spleen cells increased significantly. After adding different concentrations of compound B-5, the expression levels of p-PKCθ, CaN, NFAT, and NF-κB proteins decreased, and showed a concentration-dependent manner. It is shown that compound B-5 can inhibit the expression of related proteins in the CaN / NFAT and PKCθ / NF-κB cascade signal pathways, to play an immunosuppressive role. (Note: # p <0.05, ## p <0.01 compared with the Control group; * p <0.05, ** p <0.01 compared with the ConA group).

[0165] 3.3. ELISA method for detecting cytokines in cell supernatant

[0166] 1) The preparation method of the spleen cell suspension is the same as 1.1, and the final cell concentration is 1*10 7 cells / mL of cell suspension. Add 500 μL to a 24-well plate and incubate at 37 o C in a 5% CO2 incubator overnight.

[0167] 2) Reagents were added to the groups as follows: Negative control group: 1 mL of RPMI-1640 complete culture medium; ConA positive control group: 1 mL of RPMI-1640 complete culture medium containing ConA (final concentration 5 μg / mL); CsA group: 1 mL of RPMI-1640 complete culture medium containing ConA (final concentration 5 μg / mL) and CsA (final concentration 5 μM); Drug group: 1 mL of RPMI-1640 complete culture medium containing ConA (final concentration 5 μg / mL) and compound B-5 (final concentrations of 20 μM, 40 μM, and 60 μM, respectively). 37 o Incubate at 5% CO2 for 24 hours.

[0168] 3) Collect in sterile EP tubes, centrifuge at 3000 rpm for 20 min, collect the supernatant, and dilute each cytokine standard according to the instructions.

[0169] 4) Sample addition: Set up blank wells: no sample or enzyme-labeled reagent added, 50 μL of sample diluent, all other steps unchanged; standard wells: 50 μL of diluted standard; sample wells: 40 μL of sample diluent + 10 μL of sample. Store in the dark at 37°C. o After incubating at C for 30 min, discard the liquid, shake dry, and wash five times with diluted washing buffer. Remove the blank wells, add 50 μL of enzyme-labeled reagent to the remaining wells, and incubate at 37°C in the dark. o Incubate at C for 30 min, discard the liquid, wash five times, then add 50 μL of colorimetric reagent A to each well, followed by 50 μL of colorimetric reagent B. Incubate at 37°C in the dark. o Incubate at C for 10 min, terminate the reaction with 50 μL of stop solution, zero the instrument with a blank well, and then detect the OD value at 450 nm.

[0170] Experimental Analysis:

[0171] After ConA induction, spleen cells were rapidly activated, and the levels of IL-2, IL-4, and IFN-γ were significantly increased. Different concentrations of compound B-5 and a positive control (CsA) were added to the cells for further treatment. Figure 20 The results showed that compound B-5 significantly reduced the levels of IL-2, IL-4, IFN-γ, and IL-6; the higher the concentration, the greater the inhibitory effect, exhibiting a concentration-dependent pattern. Compound B-5 inhibited the production of downstream cytokines IL-2, IL-4, IFN-γ, and IL-6 in the CaN / NFAT and PKCθ / NF-κB cascade signaling pathways, thus demonstrating good immunosuppressive activity. (Note:) # p <0.05, ## p<0.01 compared with the Control group; * p <0.05, ** p <0.01 compared with the ConA group).

[0172] 3.4. Real-time fluorescent quantitative PCR detection

[0173] 1) The method for preparing the spleen cell suspension was the same as that in 1.1, and the final cell concentration was 1*10 7 cells / mL of cell suspension. The cell suspension was added to a 6-well plate, 2 mL per well, and cultured overnight at 37 o C in a 5% CO2 incubator.

[0174] 2) Other reagents were added as follows: negative control group: 2 mL of RPMI-1640 complete culture solution; ConA positive control group: 2 mL of RPMI-1640 complete culture solution containing ConA (final concentration 5 μg / mL); CsA group: 2 mL of RPMI-1640 complete culture solution containing ConA (final concentration 5 μg / mL) and CsA (final concentration 5 μM); drug group: 2 mL of RPMI-1640 complete culture solution containing ConA (final concentration 5 μg / mL) and compound B-5 (final concentration 20 μM, 40 μM, and 60 μM, respectively). 37 o C in a 5% CO2 incubator for 24 h.

[0175] 3) The cells were collected, total RNA was extracted using the FastPure Cell / Tissue Total RNA Isolation Kit reagent kit, and then the concentration was determined, and the total RNA concentration was adjusted to 1 μg / μL. Then reverse transcription was performed using HiScript III®All in one RT SuperMix perfect for qPCR. The following mixture was prepared in an RNase-free centrifuge tube: 14 μL of RNase-free ddH2O, 4 μL of 5*All-in-one SuperMix, 1 μL of Enzyme Mix, and 1 μL of total RNA. The reverse transcription program was as follows: 42 o C, 60 min, 70 o C, 5 min.

[0176] 4) Real-time fluorescent quantitative PCR

[0177] IL-2 gene primers: 5'-CCTGAGCAGGATGGAGAATTACA-3' (forward) and 5'- CCGCAGAGGTCCAAGTTCA-3' (reverse);

[0178] IL-4 gene primers: 5'-GATAAGCTGCACCATGAATGAGT-3' (forward) and 5'- CCATTTGCATGATGCTCTTTAGG-3' (reverse);

[0179] IL-6 gene primers: 5'-CCCCAATTTCCAATGCTCTCC-3' (forward) and 5'- CGCACTAGGTTTGCCGAGTA-3' (reverse);

[0180] IFN-γ gene primers: 5'-CTCAAGTGGCATAGATGTGGAAG-3' (forward) and 5'- TGACCTCAAACTTGGCAATACTC-3' (reverse);

[0181] β-actin primers: 5'-CCTCTGACGTCCATCATCTA-3' (forward) and 5'- ATCTTCTGCTGCCGTCGCTT-3' (reverse).

[0182] Sterile enzyme-free qPCR tubes were configured as follows:

[0183] 2 x ChamQ Universal SYBR qPCR Master Mix plus 2 μL; 2.5 μM gene product plus 1.5 μL; reverse transcription product plus 2 μL; ddH2O plus 4 μL. Reaction procedure: pre-denaturation 95 o C, 10 min; cycle 40 times, 95 o C, 15 s - 60 o C, 1 min; melting curve 60 o C - 95 o C, each 15 s, 0.3 o C. Finally, data analysis was performed according to the C t values of the results.

[0184] Experimental analysis:

[0185] The effect of compound B-5 on the transcription of NFAT downstream cytokines IL-2, IFN-γ, IL-4, and IL-6 in cells was detected by real-time quantitative PCR. Figure 21The results show that compound B-5 can effectively reduce the transcription of the mRNA genes of IL-2, IFN-γ, IL-4, and IL-6 in cells, and ultimately reduce the secretion of IL-2, IFN-γ, IL-4, and IL-6, thereby achieving an immunosuppressive effect. # p <0.05, ## p <0.01 compared with the Control group; * p <0.05, ** p <0.01 compared with the ConA group.

[0186] 3.5. Immunofluorescence detection

[0187] 1) The method for preparing the spleen cell suspension was the same as that in 1.1, and the final cell concentration was 1*10 7 6 / mL of cell suspension. Add 2 mL to each well of a 6-well plate and incubate overnight at 37 o C in a 5% CO2 incubator.

[0188] 2) Add the following reagents to the other groups: negative control group: add 2 mL of RPMI-1640 complete culture medium; ConA positive control group: 2 mL of RPMI-1640 complete culture medium containing ConA (final concentration 5 μg / mL); drug group: 2 mL of RPMI-1640 complete culture medium containing ConA (final concentration 5 μg / mL) and compound B-5 (final concentration 60 μM). Incubate at 37 o C in a 5% CO2 incubator for 24 h.

[0189] 3) Collect the treated cells in an EP tube, centrifuge, discard the supernatant, and add 1 mL of 4% paraformaldehyde for fixation for 15 min. Centrifuge at 400 x g for 5 min. Wash with PBS three times, 10 min each time. Add 1 mL of 0.1% Triton X-100 at room temperature for 15 min to permeabilize the cell membrane. Centrifuge at 400 x g for 5 min. Wash with PBS three times, 10 min each time.

[0190] 4) Block with 5% BSA for 1 h to reduce non-specific binding signals.

[0191] 5) 400 x g centrifugation 5 min. After the supernatant is discarded, 500 μL of diluted NFAT / NF-κB primary antibody (1:100 dilution in PBS) is added, and the cells are stained at room temperature for 2 h in the dark. 400 x g centrifugation 5 min. PBS is used to wash the cells three times, each time for 10 min. 500 μL of FITC-conjugated or Cy3-conjugated IgG is added as a secondary antibody (1:500), and the cells are incubated at room temperature for 1 h in the dark. 400 x g centrifugation 5 min. PBS is used to wash the cells three times, each time for 10 min. 500 μL of DAPI nuclear staining solution is added, and the cells are stained at room temperature for 5 min in the dark. PBS is used to wash the cells three times, and the last time, about 100 μL of PBS remains, the cells are dropped onto a glass slide, and the cells are allowed to stand for 2-3 min, a cover glass is placed on the cells, and the cells are observed and photographed under a laser confocal microscope.

[0192] Experimental analysis:

[0193] In order to better study the expression level of NFAT and NF-κB in the spleen cells, the effect of compound B-5 on the expression of NFAT and NF-κB in the nucleus was detected by immunofluorescence. The results are shown in Figs. 6 and 7. Figure 22 and Figure 23 As shown in Figs. 6 and 7, in the control group, the NFAT and NF-κB proteins in the spleen cells mainly exist in the cytoplasm; after ConA stimulation, the cells proliferate rapidly, and a large amount of NFAT and NF-κB proteins enter the nucleus to express. However, after the spleen cells are treated with 60 μM of compound B-5, the NFAT and NF-κB proteins are significantly reduced, indicating that compound B-5 can inhibit the transfer of NFAT and NF-κB proteins in the cytoplasm to the nucleus. (Note: DAPI is blue, indicating the nucleus, NFAT is green, and NF-κB is red, and the microscope magnification is 200x).

[0194] Therefore, compound B-5 is a potential immunosuppressant that can inhibit the CaN / NFAT and PKCθ / NF-κB cascade signal pathways in cells. B-5 does not affect the viability of normal mouse spleen cells, effectively inhibits the activation, proliferation and differentiation ability of ConA-induced T lymphocytes in a concentration-dependent manner without promoting the apoptosis of ConA-induced T lymphocytes; by inhibiting CD4 + T cell activation and proliferation, inhibiting the CaN / NFAT and PKCθ / NF-κB cascade signal pathways, reducing the transcription and secretion of IL-2, IFN-γ, IL-4, IL-6 and other cytokines, thereby playing an immunosuppressive role. Compound B-5 can be applied to the preparation of a new type of highly effective and low-toxicity immunosuppressant.

[0195] The above merely describes preferred embodiments of the present application and is not used to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A semi-meridional flower diterpenoid alkaloid compound, characterized in that, The structural formula of the semi-heliotrope diterpene alkaloid compound is shown in formula 1 or formula 2 or formula 3: 。 2. The process for the preparation of the semi-esteranoid diterpene alkaloid according to claim 1, characterized in that, The compound is prepared from Diaporthe phaseolorum H3-2 was prepared, Diaporthe phaseolorum H3-2 was deposited at China Center for Type Culture Collection with the accession number CCTCC NO: M20231234.

3. The method for preparing the hemispherane-type diterpenoid alkaloid compound according to claim 2, characterized in that, The method comprises the following steps: (1) First, use PDA medium to activate Diaporthe phaseolorum H3-2, when the colony morphology is stable and a single colony is formed, inoculate a single colony in PDA medium for expansion culture; inoculate a single colony in sterilized rice medium and ferment at 27~29℃ for 28~30 days; after fermentation, extract 2~4 times with ethyl acetate, and obtain a crude extract after concentration under reduced pressure; (2) The crude extract obtained in step (1) is mixed with 85-95% v / v methanol-water, and an equal volume of petroleum ether is added for extraction 2-4 times; the petroleum ether layer is removed, and the extract is obtained by concentration under reduced pressure; (3) The extract obtained in step (2) is mixed with 100-200 mesh normal silica gel powder, and the mass ratio of the silica gel powder and the extract is 1.4-1.6:1; gradient elution is performed with CH2Cl2-MeOH as the eluent, the mass ratio of CH2Cl2-MeOH is 100-0:0-100, and the eluent is reduced and concentrated; similar components are combined, and 7 components Fr.1-Fr.7 are obtained; (4) The component Fr.2 obtained in step (3) is subjected to silica gel column chromatography isocratic elution, and the eluent is composed of petroleum ether-ethyl acetate with a volume ratio of 0.9-1.1:0.9-1.1; after detection by thin layer chromatography point plate, similar components are combined, and 6 components Fr.2.1-Fr.2.6 are obtained; (5) The component Fr.2.1 obtained in step (4) is separated by Sephadex LH-20 column with CH2Cl2-MeOH as the eluent, the volume ratio of CH2Cl2-MeOH is 0.9-1.1:0.9-1.1; after reduction and concentration, similar components are combined, and 5 components Fr.2.1.1-Fr.2.1.5 are obtained; (6) The component Fr.2.1.3 obtained in step (5) is subjected to silica gel column chromatography isocratic elution, and the eluent is PE-EtOAc with a volume ratio of 0.9-1.1:0.9-1.1; after detection by thin layer chromatography point plate, similar components are combined, and Diaporlabanoid B is obtained after reduction and concentration and drying; (7) The component Fr.2.3 obtained in step (4) is purified by Sephadex LH-20 column with CH2Cl2-MeOH as the eluent, the volume ratio of CH2Cl2-MeOH is 0.9-1.1:0.9-1.1; after reduction and concentration and drying, the compound Diaporlabanoid A is obtained, and the structural formula of the compound is shown in formula 1; (8) The component Fr.2.4 obtained in step (4) is separated by high performance liquid chromatography, and linear gradient elution is performed with MeOH-H2O as the mobile phase, the volume ratio of MeOH-H2O is 70:30-100:0, and Diaporlabanoid C is obtained, and the structural formula of the compound is shown in formula 3.

4. The method for preparing the hemispherane-type diterpenoid alkaloid compound according to claim 3, characterized in that, The rice culture medium is prepared from the following raw materials in parts by weight: rice 95-105 parts, protein 0.5-0.7 parts, coarse sea salt 2-4 parts, and water 95-105 parts.

5. The method for preparing the hemispherane-type diterpenoid alkaloid compound according to claim 3, characterized in that, In step (3), the specific elution gradient is performed according to CH2Cl2-MeOH in a volume ratio of 100:0, 100:2, 100:4, 100:6, 100:8, 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, and 0:

100.

6. The method for preparing the hemispherane-type diterpenoid alkaloid compound according to claim 3, characterized in that, In step (8), the high performance liquid chromatography conditions are as follows: the chromatographic column is Waters, the specification of the chromatographic column is 10 mm*150 mm, the particle size of the chromatographic column filler is 5 μm, the chromatographic column filler is a C18 column, the column temperature is 24-26 ℃, the flow rate is 1.5-2.5 mL / min, and the operation time is 25-35 min.

7. The semi-annual flower diterpene alkaloid compound of claim 1 is used for preparing an immunosuppressive drug.

8. Use according to claim 7, characterized in that, The semi-annual flower diterpene alkaloid compound is used for preparing an immunosuppressive drug targeting the CaN / NFAT and PKCθ / NF-κB two cascade signal pathways.

9. Use according to claim 8, characterized in that, The semi-annual flower diterpene alkaloid compound is compound Diaporla banoid A.

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