CN111202737B

CN111202737B - Application of tripterine amide derivative in preparation of medicine for treating autoimmune diseases

Info

Publication number: CN111202737B
Application number: CN202010202188.3A
Authority: CN
Inventors: 汪豪; 胡晓龙; 冯家豪; 何其伟
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2020-03-20
Filing date: 2020-03-20
Publication date: 2022-08-05
Anticipated expiration: 2040-03-20
Also published as: CN111202737A

Abstract

The invention discloses application of a tripterine amide derivative shown as a formula III or pharmaceutically acceptable salts thereof in preparing a medicament for treating autoimmune diseases, wherein R is selected from C1-C6 alkyl; the autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis. The invention also discloses a medicament for treating autoimmune diseases, which comprises an effective treatment dose of the tripterine amide derivative or pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable auxiliary materials. The tripterine amide derivative has high efficiency and low toxicity, can be used as a novel high-efficiency and low-toxicity immunosuppressant, and has the prospect of treating autoimmune diseases, especially rheumatoid arthritis.

Description

Application of tripterine amide derivative in preparation of medicine for treating autoimmune diseases

Technical Field

The invention belongs to the field of pharmaceutical chemistry and pharmacotherapeutics, and relates to an application of tripterine amide derivatives in preparing medicaments for treating autoimmune diseases,

background

Autoimmune Diseases (AD) is a disease caused by the damage of self-tissues due to the immune reaction of the body to self-antigens, is one of the emerging non-infectious diseases, seriously harms human health, becomes the third leading cause and death of human, and is second to heart disease and cancer. Common AD includes Rheumatoid arthritis (Rheumatoid arthritis), Systemic lupus erythematosus (Systemic lupus erythematosus), Multiple sclerosis (Multiple sclerosis), etc. Currently, immunosuppressive agents (immunosuppressive agents) are the main drugs in clinical treatment of AD. Most representative immunosuppressive drugs are developed from microbial secondary metabolites, such as cyclosporin a, rapamycin, tacrolimus, and the like. Although the above immunosuppressive agents have satisfactory therapeutic effects on organ transplantation and autoimmune diseases, their serious side effects such as renal or hepatic toxicity, infection, malignancy and other adverse reactions limit their clinical use. Therefore, the search and development of low-toxicity and high-efficiency immunosuppressive drugs are a major subject of the medical field at present.

Tripterygium wilfordii hook.f. is a plant of Tripterygium of Celastraceae, has effects of clearing heat and detoxicating, dispelling pathogenic wind, dredging collaterals, relieving rigidity of muscles and promoting blood circulation, and can be used for treating rheumatoid arthritis, nephritis, lupus erythematosus, and thrombocytopenic purpura. In China, a single herbal preparation, tripterygium wilfordii tablet, has been used clinically to treat autoimmune diseases such as systemic lupus erythematosus, dermatomyositis, rheumatoid arthritis, and the like for half a century. Tripterine (celastrol) is a friedelane triterpenoid, is one of main active ingredients in tripterygium wilfordii, and has various biological activities, including anti-inflammatory, anticancer, obesity treatment, antifungal and antiviral activities. In recent years, cellstrol has been proved to exert immunosuppressive action by inhibiting T cell proliferation and activation of Th17 cells, and is an immunosuppressant with a completely new structural type. But its toxicity and narrow safe therapeutic range limit its clinical application. Therefore, the method has important significance for reducing the toxic and side effects of the tripterygium wilfordii friendship compounds and improving the activity, namely 'attenuation and synergy'.

Disclosure of Invention

The inventor evaluates the immunosuppressive activity and cytotoxicity of the tripterine amide derivative and the IC of the tripterine amide derivative on T cells through a concanavalin A (ConA) induced spleen cell differentiation model ₅₀ The value is obviously stronger than tripterine (IC) ₅₀ 0.104 μ M) and methotrexate (IC) ₅₀ 0.126 μ M), and toxigenic tripterine (CC) ₅₀ Value of 0.131. mu.M) and methotrexate (CC) ₅₀ Value of 1.48 μ M), the stimulation index of the tripterine amide derivative is higher than that of tripterine and methotrexate. Research shows that the tripterine amide derivative can reduce the secretion of immune cell factors by promoting the apoptosis of T cells and regulating Lck and ZAP-70 mediated signal channels, influence the activation and the function of the T cells and further play the role of immunosuppression. The inventor further adopts a collagen-induced arthritis mouse model to carry out in-vivo anti-rheumatoid arthritis research, and the effect of the tripterine amide derivative is obviously superior to that of methotrexate. In conclusion, the tripterine amide derivative has high efficiency and low toxicity, can be used as a novel high-efficiency and low-toxicity immunosuppressant, and has the prospect of treating autoimmune diseases, especially rheumatoid arthritis.

The invention aims to provide application of the tripterine amide derivative shown in the formula III or pharmaceutically acceptable salt thereof in preparing a medicament for treating autoimmune diseases,

r is selected from C1-C6 straight chain or branched chain saturated or unsaturated alkyl.

Specifically, R is selected from methyl, ethyl, propyl, butyl, pentyl and hexyl; preferably, R is selected from methyl, ethyl.

The autoimmune diseases include rheumatoid arthritis, systemic lupus erythematosus and multiple sclerosis.

The invention also aims to provide a medicament for treating autoimmune diseases, which comprises an effective treatment dose of the tripterine amide derivative shown in the formula III or pharmaceutically acceptable salts thereof and one or more pharmaceutically acceptable auxiliary materials.

The medicine is prepared into an oral preparation or an injection preparation by adding pharmaceutically acceptable auxiliary materials and preparing the tripterine amide derivative or the pharmaceutically acceptable salt thereof.

Preferably, the oral preparation is any one of tablets, capsules, granules, suspensions, dripping pills, pills and oral liquid preparations; the injection preparation is injection or powder injection.

The tablet is a common oral tablet, a dispersible tablet, an orally disintegrating tablet or a sustained release tablet.

The invention has the beneficial effects that:

the invention discovers that the tripterine amide derivative can be used for treating autoimmune diseases, particularly rheumatoid arthritis for the first time, and pharmacodynamic tests prove that the tripterine amide derivative can effectively relieve collagen-induced rheumatoid arthritis, and has remarkable curative effect and small toxic and side effects.

Drawings

FIG. 1 shows the effect of compound I on T-cell factor secretion.

FIG. 2 shows the effect of compound I on ZAP-70 grade LCK protein expression.

FIG. 3 shows the effect of tripterine amide derivatives, tripterine and methotrexate on pathological morphology of mouse ankle joints; wherein, A is a control group, B is a model group, C is a compound I group, D is a compound II group, and E is methotrexate group.

Detailed Description

The following are the synthesis, in vitro immunosuppression and toxicity test research of the tripterine amide derivative, and the drug effect test research of the in vivo anti-collagen induced mouse rheumatoid arthritis of the tripterine amide derivative.

Test materials

Tripterine was purchased from Nanjing spring and autumn bioengineering Co., Ltd. PyBop, N, N-diisopropylethylamine and ethylamine hydrochloride were obtained from Ainshi chemical technology, Inc. Methylamine hydrochloride was purchased from shanghai meirui chemical technology ltd.

Tripterine was purchased from Nanjing spring and autumn bioengineering Co., Ltd. PyBop, N, N-diisopropylethylamine and ethylamine hydrochloride were purchased from Exhibit (Shanghai) chemical technology, Inc. Methylamine hydrochloride and ethylamine hydrochloride are available from Michelle chemical technology, Inc. of Shanghai.

Example 1

Tripterine 22mg (0.048mmol) was dissolved in 1mL of anhydrous DMF, and PyBop (50mg,0.096mmol), N-diisopropylethylamine (20. mu.L, 0.12mmol), methylamine hydrochloride (0.05mmol) were added in this order and reacted at room temperature for 24 hours. Adding deionized water 15ml, extracting with ethyl acetate for three times, combining organic phases, drying with anhydrous magnesium sulfate, filtering, and spin-drying to obtain a crude product. Column chromatography (dichloromethane/methanol) gave a red solid (compound i) in 61% yield.

¹ H NMR(600MHz,CDCl ₃ )δ _H 7.00(1H,d,J＝8.0Hz,H-6),6.99(1H,s),6.51(1H,s,H-1),6.32(1H,d,J＝8.0Hz,H-7),5.80(1H,s,NH),2.66(3H,s,NHCH3),2.19(3H,s,H-23),1.42(3H,s,H-25),1.24(3H,s,H-26),1.14(3H,s,H-30),1.11(3H,s,H-28),0.60(3H,s,H-27)； ¹³ C NMR(100MHz,CDCl ₃ )δ _C 178.5(C-2),178.3(C-29),170.4(C-8),164.8(C-10),146.0(C-3),134.1(C-6),127.4(C-5),119.5(C-1),118.0(C-7),117.1(C-4),45.1(C-14),44.4(C-18),43.0(C-9),40.3(C-20),39.4(C-13),38.2(C-25),36.4(C-16),35.0(C-22),33.6(C-11),33.5(C-28),31.6(C-19),31.3(C-17),30.8(C-21),30.1(C-30),29.4(C-12),28.7(C-15),26.5(NHCH3),21.7(C-26),18.1(C-27),10.3(C-23)；(+)-HR-ESI-MS m/z 464.3199[M+H]+(calcd for C ₃₀ H ₄₂ NO ₃ ,464.3159).

Example 2

Tripterine 22mg (0.048mmol) was dissolved in 1mL of anhydrous DMF, and PyBop (50mg,0.096mmol), N-diisopropylethylamine (20. mu.L, 0.12mmol), and ethylamine hydrochloride (0.05mmol) were added in this order and reacted at room temperature for 24 hours. Adding deionized water 15ml, extracting with ethyl acetate for three times, combining organic phases, drying with anhydrous magnesium sulfate, filtering, and spin-drying to obtain a crude product. Column chromatography (dichloromethane/methanol) gave a red solid (compound ii) in 54% yield.

¹ H NMR(400MHz,CDCl ₃ )δ _H 7.02(1H,d,J＝8.0Hz,H-6),7.01(1H,s),6.53(1H,s),6.34(1H,d,J＝8.0Hz,H-7),5.69(1H,s,NH),3.16(2H,m,H-1'),2.21(3H,s,H-23),1.43(3H,s,H-25),1.26(3H,s,H-26),1.15(3H,s,H-30),1.12(3H,s,H-28),0.65(3H,s,H-27)； ¹³ C NMR(100MHz,CDCl ₃ )δ _C 178.1(C-2),177.5(C-29),170.5(C-8),164.8(C-10),145.9(C-3),134.2(C-6),127.2(C-5),119.3(C-1),117.9(C-7),117.0(C-4),44.9(C-14),44.2(C-18),42.9(C-9),40.0(C-20),39.2(C-13),38.1(C-25),36.2(C-16),34.9(C-22),34.3(C-1'),33.7(C-11),33.4(C-28),31.5(C-19),30.9(C-17),30.7(C-21),30.0(C-30),29.3(C-12),28.5(C-15),21.6(C-26),18.2(C-27),14.5(C-2'),10.1(C-23)；(+)-HRESIMS m/z478.3342[M+H] ⁺ (calcd for C ₃₁ H ₄₄ NO ₃ ,478.3316).

Example 3

In vitro cell activity research of tripterine amide derivative

Test materials

CCK-8 cell survival assay kits were purchased from Dojindo Laboratories. Fetal bovine serum and RPMI 1640 medium were purchased from Invitrogen. Methotrexate is purchased from Shanghai Oriental drug science and technology laboratory, Inc. Erythrocyte lysates were purchased from Jiangsu Kai Biotech, Inc. Canavarin A (ConA) was purchased from Sigma. Male BALB/c mice (6-8 weeks old, 18-25g) were purchased from the university of Nanjing model animal center.

Test method

Balbc mice were sacrificed by cervical dislocation, soaked in 75% alcohol for 3 minutes, and spleens were separated. The spleen was ground and sieved through a 200 mesh sterile filter. After removal of cell debris and clumps, single cells were preparedAnd (3) suspending the cells. Erythrocytes were lysed in an erythrocyte lysate. Then, the cell pellet was washed 3 times with RPMI 1640 medium and resuspended in RPMI 1640 medium containing 10% fetal bovine serum. Subsequently, splenocytes were treated at 1 × 10 ⁵ The density of cells/well was plated into 96-well plates and stimulated for 48h with ConA (5. mu.g/mL) or blank medium in the presence of different concentrations (0.1-100. mu.M) of test compound or methotrexate. Detecting absorbance at 540nm according to the requirement of a CCK-8 detection kit, and calculating the cell survival rate to obtain IC ₅ 0 value and CC ₅₀ The value is obtained.

Results and discussion

The results are shown in Table 1, and compounds I and II have potent immunosuppressive activity, IC on T cells ₅₀ The values are 0.025 μ M and 0.031 μ M respectively, and the inhibition effect is obviously stronger than that of tripterine (IC) ₅₀ 0.104 μ M) and methotrexate (IC) ₅₀ 0.126 μ M). Toxicity of Compounds I and II (CC) ₅₀ The values were 2.43. mu.M and 1.78. mu.M, respectively), bitripterine (CC) ₅₀ Value of 0.131. mu.M) and methotrexate (CC) ₅₀ Value of 1.48 μ M) weak. In conclusion, the immunosuppressive activity of the compounds I and II is obviously stronger than that of the tripterine and the methotrexate, and the toxicity is obviously reduced compared with the tripterine and the methotrexate, so that the stimulation index is obviously increased, and the compounds have obvious difference. The tripterine amide derivative has high efficiency and low toxicity, and the treatment effect is superior to tripterine and the marketed drugs.

TABLE 1 immunosuppression and toxicity of test Compounds

Example 4

Study of Effect of Compound I on T-cell factor secretion

Test materials

Fetal bovine serum and RPMI 1640 medium were purchased from Invitrogen. Erythrocyte lysates were purchased from Jiangsu Kai Biotech, Inc. Canavarin A was purchased from Sigma. Male BALB/c mice (6-8 weeks old, 18-25g) were purchased from the university of Nanjing model animal center. Elisa test kits (IL-4, IL-2 and IFN-. gamma.) were purchased from Wuhan Huamei, Inc.

Test method

BALB/c mouse splenocytes were isolated (as in example 3), and the cells were cultured at 1X 10 ⁵ The density of cells/well was seeded into 96-well plates. Subsequently, different concentrations (0.01. mu.M, 0.1. mu.M, 1. mu.M) of Compound I and ConA (5. mu.g/mL) were added to the respective wells for stimulation for 48 h. Then, according to the requirements of the kit, collecting the supernatant, detecting, and obtaining the concentration of IL-4, IL-2 and IFN-gamma in the supernatant.

Results and discussion

After T cells are activated, T cells can secrete a large amount of 'helper cytokines' such as IL-4, IL-2, IFN-gamma and the like, and participate in T cell mediated immune response. As shown in FIG. 1, ConA can significantly promote T cell activation and secrete IL-4, IL-2 and IFN-gamma. While administration of lower doses of compound i significantly reduced the secretion of the above cytokines, suggesting that compound i has immunosuppressive effects in inhibiting T cell activation.

Example 5

Study on influence of compound I on ZAP-70 LCK protein expression

Test materials

Fetal bovine serum and RPMI 1640 medium were purchased from Invitrogen. Erythrocyte lysates were purchased from Jiangsu Kai Biotech, Inc. Canavarin A was purchased from Sigma. Male BALB/c mice (6-8 weeks old, 18-25g) were purchased from the university of Nanjing model animal center. The RIPA lysate and BCA protein quantification kit is purchased from Biyuntian biology company. ZAP-70, p-ZAP-70, LCK and p-LCK are available from Abcam. Beta-actin, goat anti-rabbit secondary antibody, goat anti-mouse secondary antibody were purchased from Santa Cruz.

Test method

On the basis of a ConA induced spleen cell differentiation model, a western-blot method is adopted to detect the expression of a related signal channel. The specific process is as follows: BALB/c mouse splenocytes were isolated (as in example 3), and the cells were cultured at 1X 10 ⁵ The density of cells/well was seeded into 96-well plates. Subsequently, different concentrations (0, 0.01. mu.M, 0.1. mu.M, 1. mu.M) of Compound I and ConA (5. mu.g/mL) were added to the respective wells for 48h stimulation without addition ofThe normal control group (control) was prepared by adding compound I and ConA. After the drug stimulation is finished, the culture medium is removed, PBS is washed for 2 times, RIPA cell lysate is added, cell protein is extracted, protein concentration is determined by a BCA protein quantitative kit, and SDS-PAGE gel electrophoresis is carried out on a protein sample diluted to a proper concentration. Then transferring to PVDF membrane, sealing in 5% skimmed milk powder sealing solution at room temperature for 1h, adding ZAP-70/p-ZAP-70 and LCK/p-LCK pathway related protein antibody, and standing at 4 deg.C overnight. Then, a secondary antibody (1:2000) was added thereto and incubated at room temperature for 2 hours, followed by development by an electrochemiluminescence detection method (ECL method). And (3) carrying out gray value analysis on the bands, correcting the protein expression level of the target gene according to the protein expression level of the reference gene beta-actin, and calculating the protein expression level of each group of the rest target genes.

Results and discussion

Zeta-chain-associated protein kinase (ZAP-70) is expressed only in T cells and natural killer cells and is required for activation of the T Cell Receptor (TCR). In the TCR/CD 3-mediated T cell activation signaling pathway, ZAP-70 aggregates to the TCR/CD3 complex and activates, initiating recruitment and phosphorylation of downstream adaptor or scaffold proteins, leading to T cell activation. As shown in FIG. 2, the compound I can significantly reduce p-ZAP-70 and p-LCK, and further proves that the compound I has immunosuppressive activity based on T cell inhibition.

Example 6

Research on in vivo anti-rheumatoid arthritis of tripterine amide derivative

Test materials

SPF grade male DBA/1J mice, 50, 6-7 weeks old, were purchased from Nanjing university model animal center.

Bovine type II collagen, complete Freund's adjuvant, incomplete Freund's adjuvant, and methotrexate were purchased from Shanghai Oriental drug science and technology laboratory, Inc.

Test method

50 DBA/1J mice 6-7 weeks old were adaptively fed for 1 week, and randomly divided into a control group, a model group, a compound I group (20mg/kg), a compound II group (20mg/kg), and methotrexate group (20mg/kg) using SPSS software, 10 mice per group, and 40 mice except for the 10 control groups were subjected to collagen-induced arthritis (CIA) modeling. Dissolving 10mg bovine type II collagen in 5mL of 0.01mmol/L acetic acid solution, shaking overnight at 4 ℃, taking 5mL complete Freund's adjuvant containing 2mg/mL, mixing in equal volume, sucking back and forth for 2000 times through a 20mL syringe connecting device at 4 ℃ for sufficient emulsification to prepare antigen emulsion, and storing the antigen emulsion in a refrigerator at 4 ℃ for later use. The first immunization is to inject 0.1mL of II type collagen emulsion into the root of the tail of the mouse subcutaneously, and the emulsion mixed by 0.1mLII type collagen and incomplete Freund adjuvant is taken to strengthen the immunization for 1 time on the 21 st day of model building; the normal group was injected with an equal volume of saline. Administration was started on day 1 after the second immunization, and the test compound group and the methotrexate group were each administered 0.2mL of suspension (formulated with physiological saline) and gavage (20mg/kg) 1 time per day, while the control group and the model group were each administered 0.2mL of physiological saline and gavage for 30 days. Then, the swelling thickness of the feet of the mice is observed and the pathological change of the ankle joints is detected.

Results and discussion

(1) Effect of swelling degree of feet in CIA mice

After 20 days of molding, mice in the model group, compound i group, compound II group and methotrexate group all exhibited different degrees of palm swelling compared to the control group. After 30 days after treatment, compared with the model group, the swelling thickness of the soles of the mice in the test compound group and the methotrexate group is obviously reduced, the effect of the test compound group is obviously better than that of the methotrexate group (table 2), and the difference has statistical significance.

TABLE 2 Effect of test Compounds I and II on swelling of the mouse feet

(2) Pathological morphological effects of ankle joints in CIA mice

30 days after treatment, the ankle joints of the mice are obtained by conventional materials, dehydrated, embedded, sliced and HE stained. As shown in FIG. 2, pathological sections of the ankle joints of the control mice showed normal and clear joint spaces, smooth cartilage surfaces, no obvious synovial hyperplasia and infiltration of inflammatory cells; the ankle joints of the model group mice show infiltration of a large number of inflammatory cells and proliferation of synovial membranes, and destruction of articular cartilage and bone; compared with the model group, the infiltration of ankle joint inflammatory cells, synovial hyperplasia and joint cartilage and bone damage of the 2 groups of test compounds and the methotrexate group are obviously reduced, and the effect of the test compounds is obviously better than that of the methotrexate group.

Claims

1. The application of the tripterine amide derivative shown in formula I or formula II or pharmaceutically acceptable salt thereof in preparing a medicament for treating autoimmune diseases, wherein the autoimmune diseases are rheumatoid arthritis;

formula I

And (5) formula II.

2. The use of claim 1, wherein the medicament comprises a tripterine amide derivative or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

3. The use according to claim 1, wherein the medicament is an oral formulation or an injectable formulation.

4. The use according to claim 3, characterized in that the oral formulation is a tablet, capsule, granule, suspension, pill, oral liquid; the injection preparation is injection or powder injection.

5. The use of claim 4, wherein the pill is a drop pill.