WO2021037188A1 - Pharmaceutical use of pyrimido[5,4-b]pyrrolizine compound - Google Patents

Abstract

Use of a pyrimido[5,4-b]pyrrolizine compound or a pharmaceutically acceptable salt, a solvate or a hydrate thereof in the preparation of drugs for treating autoimmune diseases comprising joint inflammatory diseases, rheumatoid arthritis or systemic lupus erythematosus. The compound has a therapeutic effect on rheumatoid arthritis, joint inflammatory diseases having pathological features of bone destruction and bone erosion, and systemic lupus erythematosus.

Description

Pharmaceutical use of pyrimido[5,4-b]pyrine compounds Technical field

The present invention relates to the pharmaceutical use of a pyrimido[5,4-b]pyrine compound, in particular to a pyrimido[5,4-b]pyrine compound used in the preparation for the treatment of diseases including joint inflammation, Use of drugs for rheumatoid arthritis and autoimmune diseases of systemic lupus erythematosus.

Background technique

Chinese patent application 201910334619.9 discloses the inhibitory activity of a class of pyrimido[5,4-b]pyrine compounds on the kinase BTK, and also discloses its in vivo anti-tumor activity on human B lymphoma cells. Non-Patent Literature (Discovery of 4,7-Diamino-5-(4-phenoxyphenyl)-6-methylene-pyrimido[5,4-b]pyrrolizines as Novel Bruton's Tyrosine Kinase Inhibitors, Yu Xue et al., J Med Chem. 2018 May 24; 61(10):4608-4627) also disclosed the anti-tumor inhibitory activity of pyrimido[5,4-b]pyrine compounds on BTK kinase. However, the prior art has not yet disclosed the therapeutic effects of such compounds on specific autoimmune diseases. Therefore, it is necessary to select a drug from this class of compounds that can be used in the treatment of specific autoimmune diseases.

Summary of the invention

The technical purpose of the present invention is to select a drug with strong efficacy for autoimmune diseases from the existing BTK inhibitors.

The present invention provides compound S17016 having the following structural formula or a pharmaceutically acceptable salt, solvate or hydrate thereof, which is prepared for the treatment of autoimmune diseases including joint inflammation, rheumatoid arthritis and systemic lupus erythematosus Uses in medicines:

In a specific embodiment, the pharmaceutically acceptable salt includes a conventional non-toxic salt formed by the reaction of the compound with an inorganic acid or an organic acid. For example, a conventional non-toxic salt can be prepared by reacting a compound with an inorganic acid or an organic acid. The inorganic acid includes hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, aminosulfonic acid, phosphoric acid, etc., and the organic acid includes lemon. Acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, maleic acid, malic acid, malonic acid, fumaric acid Acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2 -Acetoxybenzoic acid and isethion acid, etc.

Preferably, the joint inflammation disease is a joint inflammation disease characterized by bone destruction and bone erosion.

The drug may be a pharmaceutical composition containing a therapeutically effective amount of the compound S17016 or a pharmaceutically acceptable salt, solvate or hydrate thereof, and one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier includes, for example, an odorant, a fragrance and the like.

The pharmaceutical composition preferably contains 1 to 99% by weight, preferably 65% to 99%, of the compound S17016 or a pharmaceutically acceptable salt, solvate or hydrate thereof as an active ingredient, and the remainder is, for example, a Medicinal carrier.

The compound and pharmaceutical composition of the present invention can be prepared into a variety of dosage forms, such as tablets, capsules, powders, syrups, solutions, suspensions and aerosols, and solid tablets are particularly preferred. In addition, the compound and pharmaceutical composition of the present invention may be present in a suitable solid or liquid carrier or diluent and in a suitable sterilization device for injection or drip infusion.

Various dosage forms of the pharmaceutical composition can be prepared according to conventional preparation methods in the pharmaceutical field. The unit dose of the preparation formula contains 0.05-1000 mg, preferably 0.1 mg-500 mg of the compound S17016 or a pharmaceutically acceptable salt, solvate or hydrate thereof.

The compound and pharmaceutical composition of the present invention can be used clinically on mammals, including humans and animals, and can be administered through oral, nasal, skin, lung, or gastrointestinal administration routes. Most preferred is oral administration. The most preferred daily dose is 0.01-1000 mg/kg body weight, taken in one time, or 0.01-500 mg/kg body weight in divided doses. Regardless of the method of administration, the individual's optimal dosage should be determined based on the specific treatment. Usually, start with a small dose and gradually increase the dose until the most suitable dose is found.

The present invention provides a method for treating autoimmune diseases including joint inflammation diseases, rheumatoid arthritis and systemic lupus erythematosus, which comprises administering to a subject in need of such treatment a therapeutically effective amount of a compound selected from the group S17016 and its pharmacy One or more of the above acceptable salts, solvates or hydrates or the above-mentioned pharmaceutical compositions.

Beneficial effect

The present invention has been verified through experiments, and compared with the positive drug ibrutinib, the compound S17016 of the present invention shows a significantly superior treatment in the treatment of autoimmune diseases including joint inflammation, rheumatoid arthritis and systemic lupus erythematosus Therefore, it is expected to be developed as a potential drug for the treatment of these diseases.

Description of the drawings

Fig. 1 is a schematic diagram showing the experimental results of the inhibitory effect of the compound S17016 and the positive control compound according to the present invention on the secretion of mouse antigen-specific serum antibodies induced by chicken ovalbumin.

Figure 2 is a schematic diagram showing the experimental results of the inhibitory effect of the compound S17016 of the present invention and the positive control compound on the mouse antigen-specific immune response induced by chicken ovalbumin.

Figure 3 is a schematic diagram showing the experimental results of the therapeutic effect of the compound S17016 of the present invention and the positive control compound on bovine type II collagen-induced arthritis in mice.

Fig. 4 is a schematic diagram showing the experimental results of the inhibitory effect of compound S17016 and the positive control compound according to the present invention on arthritic infiltration, cartilage erosion, and synovial hyperplasia in mouse arthritis induced by bovine type II collagen.

Figure 5 is a schematic diagram showing the experimental results of the protective effect of the compound S17016 of the present invention and the positive control compound on bone destruction in mouse arthritis induced by bovine type II collagen.

Fig. 6 is a schematic diagram showing the experimental results of the protective effect of the compound S17016 of the present invention and the positive control compound on the osteolytic lesions in mouse arthritis induced by bovine type II collagen.

Figure 7 is a schematic diagram showing the experimental results of the inhibitory effect of the compound S17016 of the present invention and the positive control compound on the secretion of collagen-specific antibodies in mice induced by bovine collagen type II arthritis.

Fig. 8 is a schematic diagram showing the experimental results of the inhibitory effect of the compound S17016 and the positive control compound on RANKL/M-CSF-induced osteoclast differentiation according to the present invention.

Figure 9 is a schematic diagram showing the experimental results of the therapeutic effect of compound S17016 and the positive control compound according to the present invention on spontaneous systemic lupus erythematosus mouse nephritis.

Fig. 10 is a schematic diagram showing the experimental results showing the effect of compound S17016 and the positive control compound according to the present invention on improving the skin damage of spontaneous systemic lupus erythematosus mice.

Figure 11 is a schematic diagram showing the experimental results of the inhibitory effect of compound S17016 and the positive control compound according to the present invention on lymph node enlargement in spontaneous systemic lupus erythematosus mice.

detailed description

In order to enable persons with ordinary knowledge in the field to understand the characteristics and effects of the present invention, the following general descriptions and definitions are made on the terms and terms mentioned in the specification and the scope of the patent application. Unless otherwise specified, all technical and scientific terms used in the text have the usual meanings understood by those skilled in the art for the present invention. In case of conflict, the definition in this specification shall prevail.

The following specific embodiments are merely illustrative in nature, and are not intended to limit the present invention and its uses. In addition, this document is not limited by any theory described in the foregoing prior art or invention content or the following specific embodiments or examples.

Preparation examples

Synthesis of compound S17016

Step 1) Synthesis of compound 2

Add N-{(3S)-4-{5-{4-[N-(2-pyridyl)carbamoyl]phenyl}-4-amino-7H-pyrrolo[2, 3-d]pyrimidin-7-yl}buten-3-yl}tert-butyl carbamate (1,32.7g, 1eq), add 600mL DMF as a solvent. Slowly add N-bromosuccinimide (12.8 g, 1.1 eq) under stirring, and stir overnight at room temperature.

After the reaction is completed, water and ethyl acetate are added for extraction, the ester layers are combined, back-extracted with water once, and washed with saturated brine. Dry with anhydrous sodium sulfate. In the dry method, first use a mixture of CHCl ₃ :MeOH=50:1 as the eluent, and then change the mixture of CHCl ₃ :MeOH=30:1 as the eluent. The product N-{(3S)-4-{5-{4-[N-(2-pyridyl)carbamoyl]phenyl}-4-amino-6-bromo-7H-pyrrolo[2,3 -d] pyrimidin-7-yl}buten-3-yl}tert-butyl carbamate 2, the yield was 68%.

¹ H NMR(400MHz,CDCl ₃ )δ8.89(s,1H), 8.41(d,J=8.4Hz,1H), 8.30(d,J=4.0Hz,1H), 8.27(s,1H), 8.06 (d,J=8.5Hz,2H),7.83-7.75(m,1H),7.62(d,J=8.0Hz,2H),7.10(dd,J=6.9,5.3Hz,1H),5.95-5.84( m, 1H), 5.41–5.30(m, 2H), 5.24(d, J = 10.3Hz, 1H), 5.16(s, 2H), 4.69(s, 1H), 4.53–4.43(m, 1H), 4.37 --4.29(m,1H),1.24(s,9H).

Step 2) Synthesis of compound 3

Add N-{(3S)-4-{5-{4-[N-(2-pyridyl)carbamoyl]phenyl}-4-amino-6-bromo-7H-pyrrole to a 250mL round bottom flask And [2,3-d]pyrimidin-7-yl}buten-3-yl}tert-butyl carbamate (2,11.9g,1eq) and [1,1'-bis(diphenylphosphine) dicene Iron] Palladium dichloride (1.57 g, 0.11 eq), add 200 mL of tetrahydrofuran (THF) as a solvent, and replace with nitrogen, and replace it several times to ensure completeness. Under stirring, 4M sodium hydroxide solution (15.4 mL, 3 eq) was added. Reflux and stir for 15h at 85°C.

After the reaction was completed, water and ethyl acetate were added for extraction, and the ester layers were combined and washed with saturated brine. Dry with anhydrous sodium sulfate. Dry column (eluent: CHCl ₃ :MeOH=30:1) to obtain the product N-{(7S)-6-methylene-5-{4-[N-(2-pyridyl)carbamoyl ]Phenyl}-4-amino-7,8-dihydro-6H-pyrimido[5,4-b]pyrin-7-yl}carbamic acid tert-butyl ester 3, the yield was 85%.

¹ H NMR(400MHz,CDCl ₃ )δ8.77(s,1H), 8.41(d,J=8.3Hz,1H), 8.32(d,J=4.2Hz,1H), 8.28(s,1H), 8.06 (d,J=8.3Hz,2H),7.79(t,J=7.8Hz,1H),7.68(d,J=8.1Hz,2H),7.13-7.07(m,1H),5.52(s,1H) , 5.35 (s, 1H), 5.22 (s, 1H), 5.08 (s, 3H), 4.68 (dd, J = 11.4, 8.2 Hz, 1H), 3.99 (s, 1H), 1.48 (s, 9H).

Step 3) Synthesis of compound 4

Add N-{(7S)-6-methylene-5-{4-[N-(2-pyridyl)carbamoyl]phenyl}-4-amino-7,8- to a 50mL round bottom flask Tert-butyl dihydro-6H-pyrimido[5,4-b]pyrin-7-yl}carbamate (3,249mg, 1eq), 10mL DCM was added as a solvent. Trifluoroacetic acid (1 mL) was added dropwise with stirring. Stir at room temperature for 3h. After the reaction is completed, the reaction solution is directly spin-dried, and the trifluoroacetic acid is taken out with dichloromethane several times, and the crude product 4 is obtained after spin-drying.

Step 4) Synthesis of compound S17016

Add 4-[(7S)-6-methylene-4,7-diamino-7,8-dihydro-6H-pyrimido[5,4-b]pyrine-5- to a 50mL round bottom flask Yl]-N-(2-pyridyl)benzamide (4, 2.83g, 1eq), triethylamine (1eq), stir for five minutes and then add 2-butynoic acid (511mg, 1.1eq) and 2- (7-benzotriazole oxide)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) (2.31 g, 1.1 eq), 100 mL of DCM was added as a solvent. The temperature in an ice-water bath was cooled to 0°C, and triethylamine (1.54mL+0.77mL) was added dropwise. Gradually warm to room temperature, and stir at room temperature for 1.5 h. The reaction liquid was slightly yellow. Water and DCM were added for extraction, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and passed through the column by dry method _{. The product was eluted with CHCl 3} :MeOH=30:1, and the yield was 73%.

¹ H NMR(400MHz,CDCl3)δ8.71(s,1H), 8.41(d,J=8.0Hz,1H), 8.37-8.25(m,2H), 8.07(d,J=8.2Hz,2H), 7.80(t,J=7.1Hz,1H), 7.68(d,J=8.2Hz,2H), 7.15–7.07(m,1H), 6.23(d,J=8.0Hz,1H), 5.65(s,1H ),5.55(s,1H),5.24(s,1H),5.03(s,2H),4.70(dd,J=11.4,8.2Hz,1H),4.00(dd,J=11.7,5.1Hz,1H) ,1.97(s,3H).

Experimental example

Experimental Example 1: Evaluation of the immunosuppressive activity of compound S17016 through the non-specific toxicity and proliferation reaction of mouse lymphocytes

Toxicity test: After the mouse is sacrificed, its spleen is aseptically taken to prepare a single cell suspension, the red blood cell lysate is used to remove red blood cells and adjust the cell concentration.

Mouse spleen lymphocyte suspension 8×10 ⁵ /well was inoculated in a 96-well plate, and at the same time, different concentrations of compound S17016 were added, and the corresponding vehicle control (cell control) and culture medium background control (blank control) were also set up. Incubate in a 37°C, 5% CO ₂ incubator for 48 hours. MTT solution was added 4 hours before the end of the culture. At the end of the culture, the supernatant was aspirated and DMSO was added to each well to dissolve the purple crystals, and the OD value was measured at 570 nM of the microplate reader.

Immunosuppressive activity test:

Mouse spleen lymphocyte suspension 5×10 ⁵ /well was inoculated into 96-well plates, LPS (10 μg/ml), different concentrations of compounds were added, and corresponding negative control wells without LPS stimulation and positive control wells without drugs were set up. Incubate in a 37°C, 5% CO ₂ incubator for 48 hours. 8 hours before the end of the culture, ³ H-thymine nucleotides were added to each well. Continue to cultivate until the end of the experiment. ^{After adding the scintillation fluid, the amount of 3} H-TdR incorporated into the cell DNA was read on the Beta counter, and the cpm value was used to represent the cell proliferation.

Result evaluation:

The non-specific toxicity of compound S17016 on spleen cells was determined by dividing the OD value of the sample to be tested by the OD value of the cell control well, which was marked as cell survival rate (%). The proliferation reaction of lymphocytes takes the cpm value of the positive control sample minus the cpm value of the tested sample, and then divides it by the cpm value of the positive control sample, which is marked as the proliferation inhibition rate (%). The CC ₅₀ and IC ₅₀ values were fitted using GraphPad Prism 6 software.

The results are shown in Table 1.

Table 1. Spleen lymphocyte toxicity and inhibitory activity on B cells of prepared compound S17016

*Select index SI=CC ₅₀ /IC ₅₀

Table 2. The in vivo pharmacokinetic parameters of compound S17016 prepared

The above results indicate that the compound S17016 of the present invention has obvious inhibitory activity on LPS-induced B cell proliferation, and its activity is higher than that of the positive reference compounds Ibrutinib and ACP-196, and it has less toxicity to mouse spleen lymphocytes. The selection index is high and the safety is high. In addition, compound S17016 has better in vivo pharmacokinetic characteristics than the positive reference ibrutinib, and its oral bioavailability is 6 times that of ibrutinib.

Experimental Example 2: Evaluation of the inhibitory activity of compound S17016 on antigen-specific immune response in vivo through a mouse model induced by chicken ovalbumin

(1) Normal group, model group, positive reference ibrutinib group, positive reference APC-196 group and compound S17016 group. The main experimental materials and their sources are as follows:

1) Animals

SPF female C57BL/6 mice, weighing 18-20 g, were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences and raised in the SPF animal room of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

2) Main experimental drugs and reagents

Chicken ovalbumin (Sigma); Freund's complete adjuvant (Sigma); Freund's incomplete adjuvant (Sigma); Ibrutinib (Shanghai North Carolina Medical Technology Co., Ltd.); ACP-196 (Chengdu Ji'an) Special Pharmaceutical Technology Co., Ltd.); the rest of the reagents are all domestic analytical reagents.

3) Main instruments

Full-wavelength microplate reader (Molecular Device)

(2) Experimental method

1) Construction of antigen-specific immune response model

The mice were divided into 2 groups, a normal group and a model group. Fully emulsify chicken ovalbumin (OVA) and an equal volume of Freund’s complete adjuvant (CFA), and inject 200 μl/mouse subcutaneously into the model group of mice; on the 17th day of the experiment, OVA and equal volume of Freund’s incomplete adjuvant The IFA was fully emulsified, and the mice in the model group were injected subcutaneously with 200 μl/mouse for booster immunization.

2) The effect of drugs on antigen-specific immune response

a. The model mice are divided into 5 groups evenly. The daily doses of the model control group (double distilled water) and the 4 groups of administration group are: Ibrutinib group (25mg/kg), APC-196 group (25mg/kg) ), compound S17016 high-dose group (25mg/kg), compound S17016 low-dose group (12.5mg/kg). Start on the day of the first immunization, and gavage every day for a period of 24 days.

b. Detection of serum OVA-specific antibodies: at the end of the experiment, remove the eyeballs to collect blood, separate the serum, and detect the contents of OVA-specific antibodies IgG and each subtype in the serum of individual mice in each group (Figure 1) (Note: with the model group Comparison *=P<0.05, **=P<0.01, ***=P<0.001).

c. Spleen index: At the end of the experiment, the mouse spleen was weighed, and the spleen index was calculated by (spleen weight/body weight) × 10 (Figure 2). (Note: Compared with the model group *=P<0.05, **=P<0.01, ***=P<0.001)

(3) Experimental results

1) As shown in Figure 1, compound S17016 can significantly reduce the level of antigen-specific antibodies in the serum of model mice. After the administration of compound S17016 at a dose of 12.5 mg/kg, the degree of reduction of specific antibodies in the serum is comparable to that of 25 mg/kg. The reference substance was equivalent, and the compound S17016 at a dose of 25 mg/kg showed a better inhibitory effect.

2) As shown in Figure 2, compound S17016 can significantly inhibit the spleen enlargement of model mice induced by allogeneic antigen. The inhibitory effect of compound S17016 at doses of 25 mg/kg and 12.5 mg/kg is better than that of the positive reference at 25 mg/kg. .

The above results indicate that compound S17016 has a significant inhibitory effect on antigen-specific immune responses, and is better than the positive reference.

Experimental Example 3: Evaluation of the therapeutic effect of compound S17016 on rheumatoid arthritis through a mouse model of arthritis induced by bovine type II collagen

(1) Normal group, model group, positive reference ibrutinib group, positive reference APC-196 group and compound S17016 group. The main experimental materials and their sources are as follows:

1) Animals

SPF-grade male DBA/1 mice, weighing 18-20g, were purchased from Beijing Weitonglihua Animal Technology Co., Ltd. and raised in the SPF-grade animal room of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

2) Main experimental drugs and reagents

Bovine Type II Collagen (Tokyo Collagen Research Center, Japan); Freund's complete adjuvant (Sigma); Freund's incomplete adjuvant (Sigma); Ibrutinib (Shanghai North Carolina Medical Technology Co., Ltd.); ACP-196 (Chengdu Jiante Pharmaceutical Technology Co., Ltd.); the rest of the reagents are all domestic analytical reagents.

3) Main instruments

Full-wavelength microplate reader (Molecular Device); small animal micro-computer tomography (Micro-CT) (Siemens); fully automatic closed tissue dehydration machine (Wuhan Junjie Electronics Co., Ltd.); Paraffin embedding machine (Wuhan Junjie Electronics Co., Ltd.) Company); semi-automatic rotary microtome (Leica company); special water bath for pathological section (Leica company); upright optical microscope (Japan Nikon); imaging system (Japan Nikon).

(2) Experimental method

1) Construction of a mouse arthritis (CIA) model induced by bovine type Ⅱ collagen

The mice were divided into 2 groups, a normal group and a model group. Fully emulsify bovine type II collagen (CII) swollen with glacial acetic acid and an equal volume of Freund’s complete adjuvant (CFA), and subcutaneously inject 50μl/mouse into the tail base of the model group of mice. After 3 weeks, combine CII with an equal volume of Freund's incomplete adjuvant (IFA) was fully emulsified, and 50μl/mouse was injected subcutaneously into the tail base of the model group for booster immunity.

2) Therapeutic effect of drugs on mouse arthritis induced by bovine type Ⅱ collagen

a. The model mice were divided into 6 groups evenly. The daily doses of the model control group (double distilled water) and the 5 groups were: Ibrutinib group (25mg/kg), APC-196 group (25mg/kg) ), compound S17016 high-dose group (25 mg/kg), compound S17016 middle-dose group (12.5 mg/kg), compound S17016 low-dose group (6.25 mg/kg). Starting 10 days after the booster immunization, intragastric administration is given every day, and the administration period is 15 days.

b. Evaluation of the incidence of arthritis in mice: From the day of administration, the degree of joint disease in the limbs of the mice was scored once a day (Figure 3) (Note: each dose group of compound S17016 is compared with the model group *=P<0.05, ** =P<0.01, ***=P<0.001; comparison between ibrutinib group and model group #=P<0.05, ##=P<0.01, ###=P<0.001; ACP-196 group and model group Compare Δ=P<0.05, ΔΔ=P<0.01, ΔΔΔ=P<0.001).

c. Evaluation of joint pathological damage and bone erosion in mice: At the end of the experiment, a sample of the hind limbs of the mouse was taken, fixed by soaking in formalin, and then subjected to histopathological analysis (Figure 4) and microcomputer tomography analysis (Figure 5) .

d. Evaluation of the number of osteoclasts in mouse joint tissues: At the end of the experiment, a sample of the hind limbs of the mouse was taken and fixed with formalin. Tartrate-resistant acid phosphatase (TRAP) stained osteoclasts at the joints and counted positive staining cells ( Figure 6) (Note: Comparison of each treatment group with model group***=P<0.001).

e. Detection of serum CⅡ specific antibodies: at the end of the experiment, take the eyeballs and collect blood, separate the serum, and detect the levels of CⅡ specific antibody IgG and each subtype in the serum of each group of mice (Figure 7) (Note: each treatment group Compared with the model group *=P<0.05, **=P<0.01, ***=P<0.001).

(3) Experimental results

1) As shown in Figure 3, gavage of CIA model mice with compound S17016 can significantly alleviate the degree of joint disease in mice. Among them, compound S17016 at doses of 25 mg/kg and 12.5 mg/kg improved the joint comprehensive score better than The 25 mg/kg dose of ibrutinib and ACP-196, and the 6.25 mg/kg dose of compound S17016 had the same effect as the 25 mg/kg dose of the positive reference.

2) As shown in Figure 4 and Figure 5, by HE staining and Micro-CT tomographic reconstruction analysis, the experimental results show that the treatment of compound S17016 and the positive reference substance has an effect on joint damage, inflammatory infiltration, pannus formation, and slippage in CIA mice. Membrane hyperplasia, cartilage erosion and other pathological changes have a significant improvement effect. Among them, the 25mg/kg dose of compound S17016 can improve the pathological changes of joint bone erosion and bone loss more significantly than the positive reference.

3) As shown in Figure 6, the mouse joint tissues were stained with tartrate-resistant acid phosphatase to label osteoclasts. The experimental results showed that the treatment of compound S17016 and the positive reference substance significantly improved the joint osteolytic lesions of CIA mice. The 25mg/kg and 12.5mg/kg doses of compound S17016 had better effects on joint osteoclasts than the 25mg/kg dose of ibrutinib and ACP-196, while the 6.25mg/kg dose of compound S17016 had the same effect as 25mg The positive reference at the dose per kg is equivalent.

4) As shown in Figure 7, by detecting the content of specific antibodies and subtypes of anti-bovine type II collagen in the serum of mice, the experimental results show that the content of collagen specific antibodies in the model group is increased, the compound S17016 treatment group and the positive reference substance The content of total antibody IgG in the serum of mice in the treatment group was significantly reduced. Among them, compound S17016 treatment has an inhibitory effect on IgG2b type antibodies in mouse serum, while the positive reference does not have the inhibitory effect. Compound S17016 inhibits IgG1 subtype antibodies The effect is better than the positive reference.

The above results indicate that compound S17016 has a significant inhibitory effect on autoantibody levels, has a protective effect on joint pathological damage, and has an ideal therapeutic effect on rheumatoid arthritis. The therapeutic activity of compound S17016 on rheumatoid arthritis disease model in vivo is equivalent to 4 times that of the positive reference substance, and it can be developed as a therapeutic drug for rheumatoid arthritis.

Experimental Example 4: Evaluation of the inhibitory activity of compound S17016 on the differentiation of osteoclast precursors by inducing the differentiation of mouse macrophage cell lines into osteoclasts

Mouse macrophage cell line RAW264.7 cells 1500 cells/well were seeded in a 96-well plate, pre-incubated in a 37°C, 5% CO2 incubator for 12 hours. After 12 hours, the stimulants RANKL and M-CSF (50ng/ml) and different concentrations of compounds were added, and the corresponding stimulus control and negative control (no stimulant were added) were added, and cultured for 6 days. Tartrate-resistant acid phosphatase staining was performed at the end of the culture, and the number of positively stained cells was counted under a microscope.

Result evaluation:

Count the number of TRAP-positive stained cells containing ≥3 nuclei in the field of each culture well under an optical microscope to evaluate the inhibitory effect of compound S17016 on the differentiation of macrophages into osteoclasts (Figure 8) (Note: each treatment group and stimulation Comparison of cells in the control group *=P<0.05, **=P<0.01, ***=P<0.001).

The results shown in Figure 8 show that the compound S17016 prepared in Preparation Example 3 of the present invention has obvious inhibitory activity on RANKL/M-CSF-induced osteoclast differentiation of macrophages. At concentrations of 1000 nM, 100 nM and 10 nM, The inhibitory activity of compound S17016 was stronger than the positive reference.

The above results indicate that compound S17016 has significant inhibitory activity on osteoclast formation, and is better than the positive reference material, and can be developed as a therapeutic drug for joint diseases with bone destruction and bone erosion as pathological features.

Experimental Example 5: Evaluation of the therapeutic effect of compound S17016 on systemic lupus erythematosus through spontaneous lupus model MRL/lpr mice

(1) Normal group, model group, positive reference ibrutinib group, positive reference APC-196 group and compound S17016 group. The main experimental materials and their sources are as follows:

1) Animals

SPF female MRL/lpr mice, 6 weeks old, purchased from Shanghai Lingchang Biotechnology Co., Ltd., and raised in the SPF animal room of the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

2) Main experimental drugs and reagents

Protein quantitative detection dye (Bio-Rad); Ibrutinib (Shanghai North Card Pharmaceutical Technology Co., Ltd.); ACP-196 (Chengdu Jiante Pharmaceutical Technology Co., Ltd.); other reagents are domestic analytical reagents.

3) Main instruments

Full-wavelength microplate reader (Molecular Device).

(2) Experimental method

1) The model mice are equally divided into 5 groups with 10 mice in each group. The daily doses of the model control group (double distilled water) and the 4 groups were: Ibrutinib group (25mg/kg), compound S17016 high-dose group (25mg/kg), compound S17016 medium-dose group (12.5 mg/kg), compound S17016 low-dose group (6.25 mg/kg). The treatment started at 6 weeks of age, and the administration was given by intragastric administration every day, and the administration period was 15 weeks.

2) Evaluation of indicators of lupus nephritis in mice: The protein content of mouse urine was detected weekly (Figure 9) (Note: Comparison of each dose group of compound S17016 with the model group *=P<0.05, **=P<0.01, ***=P<0.001; comparison between ibrutinib group and model group #=P<0.05, ##=P<0.01, ###=P<0.001).

3) Evaluation of lupus skin lesions in mice: Starting from 12 weeks of age, the area of skin lesions on the body surface was scored (Figure 10). (Note: Comparison of each dose group of compound S17016 with model group *=P<0.05, **=P<0.01, ***=P<0.001; comparison between ibrutinib group and model group#=P<0.05,## =P<0.01, ###=P<0.001).

4) Evaluation of the degree of lymph node swelling in mice: The peripheral lymph node volume was scored from the 12 weeks of age in mice (Figure 11) (Note: Comparing each dose group of compound S17016 with the model group *=P<0.05, **=P <0.01, ***=P<0.001; comparison between ibrutinib group and model group #=P<0.05, ##=P<0.01, ###=P<0.001).

(3) Experimental results

1) As shown in Figure 9, each dose group of compound S17016 has a significant effect on reducing the level of urine protein, an important indicator of kidney injury in spontaneous lupus model MRL/lpr mice. As the disease progresses, the urine protein level of mice in the model group continues to increase. After 13 weeks of drug treatment, compound S17016 and the positive reference substance showed a significant and continuous inhibitory effect on the urine protein level of mice.

2) As shown in Figure 10, 14-week-old MRL/lpr mice have obvious hair loss and epidermal damage as the disease progresses, which is one of the typical pathological phenomena of systemic lupus erythematosus. By measuring the comprehensive score of the skin damage area of each group of mice, the results show that the treatment of compound S17016 and the positive reference substance can improve the skin damage of MRL/lpr mice, and the compound S17016 at the dose of 25mg/kg and 12.5mg/kg has the improvement effect. It is better than the 25 mg/kg dose of ibrutinib, and the 6.25 mg/kg dose of compound S17016 has the same effect as the 25 mg/kg dose of the positive reference. In particular, the compound S17016 has the function of restoring the normal phenotype of the skin of the mouse individuals who have suffered skin damage.

3) As shown in Figure 11, since 13 weeks of age, MRL/lpr mice have obvious lymph node enlargement as the disease progresses. The mice in each group were evaluated for the comprehensive score of peripheral lymph node diameter measurement. The results showed that the compound S17016 and the positive reference substance The treatment has a significant inhibitory effect on lymphadenopathy in MRL/lpr mice. The inhibitory effect of compound S17016 at doses of 25 mg/kg and 12.5 mg/kg is better than that of ibrutinib at 25 mg/kg, while at 6.25 mg/kg. The effect of compound S17016 is equivalent to that of the positive reference substance at a dose of 25 mg/kg.

The above results show that the compound S17016 has a significant improvement effect on lupus nephritis, and has a significantly better effect on the autoimmune skin damage typical of systemic lupus erythematosus than the positive reference substance, and has an improvement effect on the lymph caused by the abnormal proliferation of autoreactive lymphocytes. Organ enlargement has a better inhibitory effect than the positive reference, and can be developed as a therapeutic drug for systemic lupus erythematosus.

In summary, the compound S17016 of the present invention has shown a good therapeutic effect on rheumatoid arthritis, joint diseases characterized by bone destruction and bone erosion and systemic lupus erythematosus in animal models, and therefore has an ideal clinical application prospect .

Claims (10)

1. Use of a compound having the following structural formula or a pharmaceutically acceptable salt, solvate or hydrate thereof in the preparation of a medicament for the treatment of autoimmune diseases including joint inflammation diseases, rheumatoid arthritis and systemic lupus erythematosus:

   
2. The use according to claim 1, wherein the pharmaceutically acceptable salt includes a salt formed by reaction with an inorganic acid or an organic acid.
3. The use according to claim 2, wherein:

   The inorganic acid is one or more selected from hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, aminosulfonic acid and phosphoric acid, and

   The organic acid is selected from citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalene disulfonic acid, maleic acid, apple Acid, malonic acid, fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, One or more of p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid and isethionic acid.
4. The use according to claim 1, wherein the joint inflammation disease is a joint inflammation disease characterized by bone destruction and bone erosion.
5. The use according to claim 1, wherein the drug is a pharmaceutical composition containing a therapeutically effective amount of the compound or a pharmaceutically acceptable salt, solvate or hydrate thereof, and a Or a variety of pharmaceutically acceptable carriers.
6. The use according to claim 5, wherein the pharmaceutical composition contains 1 to 99% by weight of the compound or a pharmaceutically acceptable salt, solvate or hydrate thereof, and the remainder is pharmaceutically acceptable a.
7. The use according to claim 6, wherein the pharmaceutical composition contains 65% to 99% by weight of the compound or a pharmaceutically acceptable salt, solvate or hydrate thereof.
8. The use according to any one of claims 1-7, wherein the dosage form of the drug is a tablet, capsule, powder, syrup, solution, suspension or aerosol.
9. The use according to claim 8, wherein the unit dose of the dosage form of the drug contains 0.05-1000 mg of the compound or a pharmaceutically acceptable salt, solvate or hydrate thereof.
10. The use according to claim 9, wherein the unit dose of the dosage form of the drug contains 0.1 mg to 500 mg of the compound or a pharmaceutically acceptable salt, solvate or hydrate thereof.

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