Application of benzothiazinone vinylidene indene compound in preparation of medicine for inhibiting osteoclast differentiation

Abstract

The invention belongs to the field of medicines, and relates to application of a benzothiazinone vinylidene indene compound in preparation of an osteoclast differentiation inhibitor. Through tartrate-resistant acid phosphatase (TRAP) staining and reverse transcription real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) technologies, it is proved for the first time that the series of compounds can significantly inhibit RANKL-induced osteoclast differentiation and polykaryotic osteoclast formation, and have no cytotoxicity at the concentration of 5 [mu] M or below. Based on the discoveries, the benzothiazinone vinylidene indene compound provided by the invention can be used as a candidate drug molecule of a novel osteoclast differentiation inhibitor, and is used for preventing and / or treating osteoporosis, periprosthetic osteolysis, tumor metastasis bone destruction, rheumatoid arthritis and other bone metabolism diseases caused by overactivity of osteoclasts.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the application of a benzothiazinone vinylidene indene compound in the preparation of a drug for inhibiting osteoclast differentiation. Background Art

[0002] Osteoclasts (Oc) are the only known bone-resorbing cells in the skeletal system, and their differentiation and maturation processes are completed by precursor cells (such as bone marrow monocytes / macrophages) derived from hematopoietic stem cells under the synergistic action of a variety of regulatory factors. In this process, RANKL (receptor activator of nuclear factor-κB ligand) plays a central role. RANKL binds to the RANK receptor on the surface of osteoclast precursor cells, triggering the activation of signaling pathways such as NF-κB, AKT, and MAPK, further promoting the upregulation of transcription factors such as NFATc1 and c-Fos, thereby regulating the expression of osteoclast-specific genes, and ultimately achieving the differentiation and activation of osteoclasts. Under normal physiological conditions, osteoclasts maintain the homeostasis of bone by precisely regulating the dynamic balance between bone resorption and bone formation, thus playing an indispensable role in bone development and bone remodeling. However, when the number of osteoclasts increases abnormally or their functions get out of control, it may lead to various bone metabolic diseases such as osteoporosis, rheumatoid arthritis, periprosthetic osteolysis, and bone destruction caused by cancer metastasis. Therefore, inhibiting the differentiation and function of osteoclasts has become an important strategy for treating these diseases.

[0003] Currently, commonly used antiresorptive drugs in clinical practice include bisphosphonates, RANKL monoclonal antibodies (denosumab), calcitonin, and selective estrogen receptor modulators (raloxifene). These drugs help increase bone density, relieve bone pain symptoms, and reduce the risk of fractures by inhibiting osteoclast activity. However, each drug has significant differences in indications and safety, and is accompanied by certain toxic side effects. For example, although bisphosphonates are the first-line treatment option, long-term use may cause rare but serious complications such as osteonecrosis of the jaw or atypical femoral fractures; although denosumab can continuously inhibit bone resorption, bone turnover markers may rebound after drug withdrawal, and similar to bisphosphonates, there are potential bone-related risks. The long-term use of calcitonin preparations has been restricted due to potential carcinogenic risks, while raloxifene may slightly increase the probability of venous thromboembolic events. Given the above problems of existing drugs, the field of bone metabolism research is actively developing new osteoclast inhibitors. An ideal new drug should have clear efficacy, low toxicity, easy access and use, so as to break through the limitations of traditional treatments and meet the unmet clinical needs. Summary of the Invention

[0004] In view of this, an embodiment of the present application provides an application of a benzothiazinone vinylidene indene compound in the preparation of a drug for inhibiting osteoclast differentiation.

[0005] According to an embodiment of the present application, the benzothiazinone vinylidene indene compound has a structural formula as shown in formula (1) or (2):

[0006] Wherein, the R 1 is methyl or phenyl; R 2 is hydrogen, methyleneoxy, cyano or trifluoromethyl; R 3 is methyl, methoxy, halogen or forms a quinolinyl group with the skeleton.

[0007] More preferably, the benzothiazinone vinylidene indene compound can be any one of the following specific structural formulas 4d, 4e, 4f, 4i, 4p, 4q, 4u, and 4v: .

[0008] The above-mentioned application of the benzothiazinone vinylidene indene compound in the preparation of an osteoclast differentiation inhibitor.

[0009] The osteoclast differentiation inhibitor can be a drug for treating osteolytic diseases to inhibit the over-activation of osteoclasts in osteolytic diseases; the osteoclast differentiation inhibitor can be a drug for treating osteoporosis, periprosthetic osteolysis, bone destruction caused by cancer metastasis or rheumatoid arthritis.

[0010] The dosage form of the drug can be various forms such as tablets, pills, powders, capsules, injections, oral liquids, ointments, creams, etc., and drugs in various dosage forms can be prepared according to conventional methods in the pharmaceutical field. The administration method can be oral, injection or topical.

[0011] The drug includes an active ingredient of a benzothiazinone vinylidene indene compound and a pharmaceutically acceptable pharmaceutical excipient. The excipients include conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0012] The above-mentioned application of the benzothiazinone vinylidene indene compound in the preparation of a drug for inhibiting the expression of osteoclast differentiation marker genes, and the osteoclast differentiation marker genes are Cfos, Ctsk, Acp5, Oscar, Dcstamp, Mmp9 or Atp6v0d2 .

[0013] The present invention uses a mouse bone marrow macrophage osteoclast differentiation model induced by RANKL. Experiments prove that the benzothiazinone vinylidene indene compound can inhibit osteoclast differentiation marker genes (including Cfos, Ctsk, Acp5, Oscar,Dcstamp, Mmp9 and Atp6v0d2 The expression of ) can directly and effectively inhibit the formation of osteoclasts from bone marrow macrophages without obvious cytotoxicity, making it a candidate compound for developing novel osteoclast differentiation inhibitors to treat diseases caused by overactivation of osteoclasts. Therefore, the present invention has great clinical significance and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The drawings herein are incorporated into and constitute a part of the specification, used to show embodiments in line with the present application, and jointly explain the technical principles of the present application with the specification.

[0015] Figure 1 : The results of the cytotoxicity experiment of 5 μM benzothiazinone vinyl indene compounds (4d, 4e, 4f, 4i, 4p, 4q, 4u, 4v) on bone marrow mononuclear / macrophages (BMMs) detected by the CCK-8 method. Data are presented as mean ± standard deviation, n = 6 for each group.

[0016] Figure 2 : The staining result diagram for evaluating the inhibitory effect of 5 μM benzothiazinone vinyl indene compounds (4d, 4e, 4f, 4i, 4p, 4q, 4u, 4v) on osteoclast differentiation by tartrate-resistant acid phosphatase (TRAP) staining.

[0017] Figure 3 : Evaluation of the dose effect of benzothiazinone vinyl indene compound 4q on osteoclast differentiation by TRAP staining. A. The TRAP staining result diagram; B. The quantitative analysis result of the number of osteoclasts; C. The quantitative analysis result of the relative size of osteoclasts. Data are presented as mean ± standard deviation, n = 3 for each group. Compared with the RANKL treatment group without adding 4q, the statistical significance is marked as ****: p <0.0001.

[0018] Figure 4 : The analysis result for detecting the effect of benzothiazinone vinyl indene compound 4q on the expression of genes related to osteoclast differentiation by reverse transcription real-time fluorescence quantitative PCR (qPCR) method. Data are presented as mean ± standard deviation, n = 3 for each group. Compared with the RANKL treatment group without adding 4q, the statistical significance is marked as *: p <0.05, **: p <0.01, ***: p <0.001, ****: p <0.0001. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] The present invention will be further described below in conjunction with specific embodiments. The following examples are only illustrative of the present invention and do not limit the present invention in any way. The following examples provided can serve as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0020] The preparation methods of the benzothiazinone vinylidene indene compounds (4d, 4e, 4f, 4i, 4p, 4q, 4u, 4v) in the following examples can be referred to CN118955428A. Other experimental methods in the examples, unless otherwise specified, are all conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels. Unless otherwise specified, in the following examples, quantitative tests are all set with three repeated experiments, and the results are averaged. Figures 1 to 4 The experimental results of each example are respectively shown, and it can be seen the non-toxicity of the compounds and the effect of inhibiting osteoclast differentiation.

[0021] Example 1: Detection of the cytotoxicity of benzothiazinone vinylidene indene derivatives on bone marrow macrophages by CCK-8 method A. Experimental purpose To evaluate the cytotoxicity of 5 μM benzothiazinone vinylidene indene derivatives (4d, 4e, 4f, 4i, 4p, 4q, 4u, 4v) on mouse bone marrow macrophages.

[0022] B. Experimental method 1. Isolation and culture of bone marrow macrophages Take the femurs and tibias of 8-12-week-old mice, remove the muscle tissue and cartilage, and obtain the bone marrow by instantaneous centrifugation; suspend the bone marrow cells in α-MEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (referred to as complete medium), culture for 1 day and then centrifuge to collect the precipitate; culture with complete medium containing 15 ng / mL M-CSF (referred to as BMM medium) for 2 days to obtain adherent bone marrow macrophages (BMMs).

[0023] 2. Cell seeding and treatment Seed BMM cells in a 96-well plate at a density of 5000 cells / well, and after culturing in BMM medium for 1 day, divide them into 9 groups: a control group (adding dimethyl sulfoxide, i.e., DMSO solvent) and 8 experimental groups (adding 5 μM of 4d, 4e, 4f, 4i, 4p, 4q, 4u, 4v) respectively, with 6 replicates in each group; replace the BMM medium containing the corresponding compound or DMSO solvent every 24 hours, and detect at 24 h, 48 h, and 72 h respectively.

[0024] 3. CCK-8 detection Remove the culture medium, wash with DPBS, and add 100 μL of 10% CCK-8 reagent to each well. Incubate at 37 °C in the dark for 30 minutes; measure the absorbance (OD 450 value) at 450 nm using a microplate reader.

[0025] C. Experimental results Compared with the control group, the OD 450 values of each experimental group at 24 h, 48 h, and 72 h showed no difference or significant increase ( Figure 1 ), indicating that the 5 μM benzothiazinone vinyl indene derivatives (4d, 4e, 4f, 4i, 4p, 4q, 4u, 4v) have no cytotoxicity to bone marrow macrophages.

[0026] Example 2: Evaluation of the inhibitory effect of benzothiazinone vinyl indene derivatives on osteoclast differentiation by TRAP staining A. Experimental purpose Evaluate the effect of benzothiazinone vinyl indene derivatives (4d, 4e, 4f, 4i, 4p, 4q, 4u, 4v) on RANKL-induced osteoclast differentiation.

[0027] B. Experimental method 1. Cell treatment Seed bone marrow macrophages (BMMs) at a density of 1×10 5 cells / well in a 24-well culture plate, with 3 replicates per group. After culturing in BMM medium for 1 day, change to osteoclast induction medium containing 5 μM of the target compound or DMSO only (this medium is a complete medium containing 30 ng / mL M-CSF and 50 ng / mL RANKL). Change the culture medium every 2 days and continue culturing for 5 days.

[0028] 2. TRAP staining After culturing for 5 days, fix the cells with 4% paraformaldehyde, and then perform tartrate-resistant acid phosphatase (TRAP) staining using the TRAP staining kit from Suzhou Bizhong Biotechnology Co., Ltd. The specific operation steps are carried out strictly according to the kit instructions.

[0029] C. Experimental results Compared with the control group (DMSO treatment group), the number of osteoclasts in the treatment groups with 5 μM benzothiazinone vinyl indene derivatives (4d, 4e, 4f, 4i, 4p, 4q, 4u, and 4v) was significantly reduced ( Figure 2 ), indicating that this series of compounds can effectively inhibit osteoclast formation.

[0030] Example 3: Detection of the dose-effect of compound 4q on inhibiting osteoclast differentiation by TRAP staining A. Experimental purpose Analyze the inhibitory effect of compound 4q on osteoclast formation at different concentrations.

[0031] B. Experimental method The bone marrow macrophages were divided into 8 groups, namely NM group, 0 μM 4q treatment group, 0.3125 μM 4q treatment group, 0.625 μM 4q treatment group, 1.25 μM 4q treatment group, 2.5 μM 4q treatment group, and 5 μM 4q treatment group. They were seeded in 24-well culture plates at a density of 1×10 5 cells per well, and 3 replicate wells were set up for each group. After culturing in BMM medium for 1 day, the NM group was replaced with BMM medium containing DMSO, the 0 μM 4q group was replaced with osteoclast induction medium containing DMSO, and the other groups were replaced with osteoclast induction medium containing the corresponding concentration of compound 4q and DMSO. The volumes of the media in each group were equal, and the volume of DMSO was 1 / 1000 of the volume of the medium. The culture medium was changed every 2 days for 5 consecutive days. After the culture was completed, the cells were fixed with 4% paraformaldehyde, and then tartrate-resistant acid phosphatase (TRAP) staining was performed using the TRAP staining kit of Suzhou Bizhong Biotechnology Co., Ltd. The staining steps were carried out according to the kit instructions. After staining, the number of osteoclasts with TRAP positive and the number of nuclei ≥ 3 was counted.

[0032] C. Experimental results Compound 4q inhibited osteoclast formation in a dose-dependent manner: at a concentration of 0.3125 μM, the number and relative size of osteoclasts were significantly reduced; when the concentration ≥ 2.5 μM, compound 4q almost completely inhibited osteoclast formation (see Figure 3 ).

[0033] Example 4: Detection of the effect of compound 4q on the expression of osteoclast differentiation marker genes by qPCR method A. Experimental purpose Analyze the effect of compound 4q on the expression of osteoclast differentiation-related genes at different concentrations, so as to further determine the inhibitory effect of 4q on osteoclast formation.

[0034] B. Experimental method The bone marrow macrophages were divided into 8 groups (NM group and 0, 0.3125, 0.625, 1.25, 2.5, 5 μM 4q treatment groups), and seeded at 4×10 5Cells were seeded in 6-well plates at a density of [quantity] per well, with three replicates per group. After culturing in BMM medium for 1 day, the NM group was changed to BMM medium containing DMSO, the 0 μM 4q group was changed to osteoclast induction medium containing DMSO, and the other groups were changed to osteoclast induction medium containing the corresponding concentration of compound 4q and DMSO. The volume of the medium in each group was the same, and the volume of DMSO was 1 / 1000 of the volume of the medium. The culture medium was changed every 2 days for 5 days. After the culture, total RNA of the cells in each group was extracted using Trizol reagent, reverse transcribed into cDNA, and then real-time fluorescence quantitative PCR was performed to detect Cfos, Ctsk, Acp5, Oscar, Dcstamp, Mmp9, Atp6v0d2 mRNA expression levels.

[0035] C. Experimental results The mRNA levels of the above genes decreased significantly with the increase in the concentration of 4q ( Figure 4 ), confirming that 4q plays a role by inhibiting the expression of genes related to osteoclast differentiation.

Claims

1. Use of benzothiazinone vinyl indene compounds in the preparation of osteoclast differentiation inhibitors, wherein the structural formula of the benzothiazinone vinyl indene compounds is shown in Formula (1) or Formula (2): ， Said R 1 is methyl or phenyl; R 2 is hydrogen, cyano, trifluoromethyl or methylenedioxy; R 3 is methyl, methoxy, halogen or forms a quinolinyl group with the skeleton.

2. The application according to claim 1, characterized in that The benzothiazinone vinyl indene compounds are used in the preparation of drugs for preventing and / or treating osteolytic diseases.

3. The application according to claim 1, wherein The benzothiazinone vinyl indene compounds are used in the preparation of drugs for preventing and / or treating osteoporosis, periprosthetic osteolysis, bone destruction caused by cancer metastasis or rheumatoid arthritis.

4. The application according to claim 1, characterized in that, The benzothiazinone vinyl indene compounds are used in the preparation of drugs for inhibiting the expression of osteoclast differentiation marker genes.

5. The application according to any one of claims 1-4, characterized in that, The benzothiazinone vinyl indene compounds are any one of the following formulas: 。 6. The application according to any one of claims 1-4, characterized in that: The dosage forms of the inhibitor or drug are tablets, pills, powders, capsules, injections, oral liquids, ointments or creams, and the administration methods are oral, injection or topical.

7. The application according to any one of claims 1 to 4, characterized in that: The inhibitor or drug comprises an active ingredient of benzothiazinone vinyl indene compounds and a pharmaceutically acceptable excipient.

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