Application of benzothiazinone vinylidene indene compounds in the preparation of drugs for inhibiting osteoclast differentiation

Abstract

The present invention belongs to the field of medicine and relates to the use of benzothiazinone vinylidene indene compounds in the preparation of osteoclast differentiation inhibitors. The present invention uses tartrate-resistant acid phosphatase (TRAP) staining and reverse transcription real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) technology to confirm for the first time that this series of compounds can significantly inhibit RANKL-induced osteoclast differentiation and the formation of multinucleated osteoclasts, and are non-cytotoxic at concentrations of 5μM and below. Based on these findings, the benzothiazinone vinylidene indene compounds provided by the present invention can be used as candidate drug molecules for novel osteoclast differentiation inhibitors, for the prevention and / or treatment of bone metabolic diseases caused by excessive activation of osteoclasts, such as osteoporosis, periprosthetic bone dissolution, tumor metastasis bone destruction, and rheumatoid arthritis.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to the use 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. Their differentiation and maturation are initiated by hematopoietic stem cell-derived precursor cells (such as bone marrow monocytes and macrophages) under the coordinated action of multiple regulatory factors. RANKL (receptor activator of nuclear factor κB ligand) plays a central role in this process. By binding to the RANK receptor on the surface of osteoclast precursor cells, RANKL triggers activation of signaling pathways such as NF-κB, AKT, and MAPK. This further promotes the upregulation of transcription factors such as NFATc1 and c-Fos, thereby regulating the expression of osteoclast-specific genes and ultimately leading to osteoclast differentiation and activation. Under normal physiological conditions, osteoclasts maintain skeletal homeostasis by precisely regulating the dynamic balance between bone resorption and formation, thus playing an essential role in bone development and remodeling. However, abnormal osteoclast abundance or dysfunctional osteoclast function can lead to various bone metabolic diseases, including 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 such diseases.

[0003] Currently, commonly used antiresorptive drugs in clinical practice include bisphosphonates, the RANKL monoclonal antibody (denosumab), calcitonin, and the selective estrogen receptor modulator (raloxifene). These drugs inhibit osteoclast activity, helping to increase bone density, alleviate bone pain, and reduce fracture risk. However, each drug has significant differences in indications and safety profiles, and is associated with certain toxic side effects. For example, while bisphosphonates are a first-line treatment option, long-term use can lead to rare but serious complications, such as osteonecrosis of the jaw or atypical femoral fractures. Denosumab, while providing sustained inhibition of bone resorption, can cause rebound in bone turnover markers after discontinuation and, like bisphosphonates, carries potential skeletal risks. Long-term use of calcitonins is restricted due to potential carcinogenicity, while raloxifene may slightly increase the risk of venous thrombosis. Given these issues with existing drugs, the field of bone metabolism is actively developing novel osteoclast inhibitors. Ideal new drugs should have clear efficacy, low toxicity, and be easy to obtain and use, so as to break through the limitations of traditional treatments and meet unmet clinical needs. Summary of the Invention

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

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

[0006]

[0007] Among them, the R 1 is methyl or phenyl; R 2 is hydrogen, methyleneoxy, cyano or trifluoromethyl; R 3 It is methyl, methoxy, halogen or forms a quinolyl group with the skeleton.

[0008] 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:

[0009] .

[0010] Application of the above-mentioned benzothiazinone vinylidene indene compounds in the preparation of osteoclast differentiation inhibitors.

[0011] The osteoclast differentiation inhibitor may be a drug for treating osteolytic diseases to inhibit the excessive activation of osteoclasts in osteolytic diseases; the osteoclast differentiation inhibitor may be a drug for treating osteoporosis, periprosthetic bone dissolution, bone destruction caused by cancer metastasis, or rheumatoid arthritis.

[0012] The drug may be in the form of tablets, pills, powders, capsules, injections, oral solutions, ointments, creams, etc., and the various drug forms may be prepared according to conventional methods in the pharmaceutical field. The drug may be administered orally, by injection, or for external use.

[0013] The drug comprises an active ingredient, a benzothiazinone vinylidene indene compound, and medically acceptable pharmaceutical excipients, including conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, and the like in the pharmaceutical field.

[0014] The use of the above-mentioned benzothiazinone vinylidene indene compound in the preparation of a drug for inhibiting the expression of osteoclast differentiation marker genes, wherein the osteoclast differentiation marker gene is Cfos, Ctsk, Acp5, Oscar, Dcstamp, Mmp9 or Atp6v0d2 .

[0015] The present invention adopts the RANKL-induced mouse bone marrow macrophage osteoclast differentiation model, and experiments have shown that benzothiazinone vinylindene compounds can inhibit osteoclast differentiation marker genes (including Cfos, Ctsk, Acp5, Oscar, Dcstamp, Mmp9, and Atp6v0d2 ) expression, thereby directly and effectively inhibiting the formation of osteoclasts from bone marrow macrophages without significant cytotoxicity. This makes it a candidate compound for the development of novel osteoclast differentiation inhibitors to treat diseases caused by overactive osteoclasts. Therefore, this invention has significant clinical significance and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The drawings herein are incorporated into the specification and constitute a part of the specification, and are used to illustrate embodiments consistent with the present application and, together with the specification, explain the technical principles of the present application.

[0017] Figure 1 Cytotoxicity of 5 μM benzothiazinone vinylidene indene compounds (4d, 4e, 4f, 4i, 4p, 4q, 4u, and 4v) against bone marrow mononuclear / macrophage cells (BMMs) was assessed using the CCK-8 assay. Data are presented as mean ± SD; n = 6 per group.

[0018] Figure 2 : Tartrate-resistant acid phosphatase (TRAP) staining was used to evaluate the inhibitory effect of 5 μM benzothiazinone vinylidene indene compounds (4d, 4e, 4f, 4i, 4p, 4q, 4u, 4v) on osteoclast differentiation.

[0019] Figure 3 : Evaluation of the dose-effect of the benzothiazinone vinylidene indene compound 4q on osteoclast differentiation by TRAP staining. A. TRAP staining results; B. Quantitative analysis of osteoclast number; C. Quantitative analysis of osteoclast relative size. Data are presented as mean ± SD, n = 3 per group. Statistically significant differences compared to the RANKL-treated group without 4q are indicated with ****. p <0.0001.

[0020] Figure 4 Results of analysis of the effects of the benzothiazinone vinylidene indene compound 4q on the expression of genes associated with osteoclast differentiation, as assessed by reverse transcription real-time fluorescence quantitative PCR (qPCR). Data are presented as mean ± SD, n = 3 per group. Statistically significant differences compared to the RANKL-treated group without 4q are indicated with *. p <0.05, **: p <0.01, ***: p <0.001, ****: p <0.0001. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with specific embodiments. The following examples are merely illustrative of the present invention and are not intended to limit the present invention in any way. The examples provided below may serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0022] The preparation methods of the benzothiazinone vinylidene indene compounds (4d, 4e, 4f, 4i, 4p, 4q, 4u, and 4v) described in the following examples can be found in CN118955428A. All other experimental methods in the examples, unless otherwise specified, are conventional methods, performed according to techniques or conditions described in literature in the field or according to product specifications. The materials and reagents used in the following examples, unless otherwise specified, are commercially available. Unless otherwise noted, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged. Figures 1 to 4 The experimental results of each example are shown respectively, and it can be seen that the compound is non-toxic and has the effect of inhibiting osteoclast differentiation.

[0023] Example 1: Detection of cytotoxicity of benzothiazinone vinylidene indene derivatives on bone marrow macrophages by CCK-8 assay

[0024] A. Experimental Purpose

[0025] The cytotoxicity of 5 μM benzothiazinone vinylidene indene derivatives (4d, 4e, 4f, 4i, 4p, 4q, 4u, 4v) on mouse bone marrow macrophages was evaluated.

[0026] B. Experimental Methods

[0027] 1. Isolation and Culture of Bone Marrow Macrophages

[0028] Femurs and tibias were obtained from 8-12 week old mice, muscle tissue and cartilage were removed, and bone marrow was obtained by instant centrifugation. Bone marrow cells were suspended in α-MEM medium (complete medium) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. After 1 day of culture, the precipitate was collected by centrifugation. Adherent bone marrow macrophages (BMMs) were obtained by culturing in complete medium containing 15 ng / mL M-CSF (BMM medium) for 2 days.

[0029] 2. Cell Plating and Treatment

[0030] BMM cells were seeded in 96-well plates at a density of 5000 cells / well and cultured in BMM medium for 1 day. The cells were divided into 9 groups: a control group (added with dimethyl sulfoxide (DMSO) solvent) and 8 experimental groups (added with 5 μM 4d, 4e, 4f, 4i, 4p, 4q, 4u, and 4v, respectively), with 6 replicate wells in each group. The BMM medium containing the corresponding compound or DMSO solvent was replaced every 24 hours, and the cells were tested at 24 h, 48 h, and 72 h, respectively.

[0031] 3. CCK-8 Assay

[0032] After removing the culture medium and washing with DPBS, 100 μL of 10% CCK-8 reagent was added to each well and incubated at 37°C in the dark for 30 minutes. The absorbance at 450 nm (OD 450 value).

[0033] C. Experimental Results

[0034] Compared with the control group, the OD values of each experimental group at 24 h, 48 h, and 72 h were significantly higher than those of the control group. 450 There was no difference or a significant increase in the values ( Figure 1 ), indicating that 5 μM benzothiazinone vinylidene indene derivatives (4d, 4e, 4f, 4i, 4p, 4q, 4u, 4v) had no cytotoxicity to bone marrow macrophages.

[0035] Example 2: Evaluation of the inhibitory effect of benzothiazinone vinylidene indene derivatives on osteoclast differentiation by TRAP staining

[0036] A. Experimental Purpose

[0037] To evaluate the effects of benzothiazinone vinylidene indene derivatives (4d, 4e, 4f, 4i, 4p, 4q, 4u, 4v) on RANKL-induced osteoclast differentiation.

[0038] B. Experimental Methods

[0039] 1. Cell Treatment

[0040] Bone marrow macrophages (BMMs) were cultured at a rate of 1×10 5 Cells were seeded at a density of 100 μM per well in a 24-well culture plate, with triplicate wells set up for each group. After one day of culture in BMM medium, the cells were replaced with osteoclast induction medium (complete medium containing 30 ng / mL M-CSF and 50 ng / mL RANKL) containing either 5 μM of the target compound or DMSO alone. The medium was changed every two days for five days.

[0041] 2.TRAP Staining

[0042] After 5 days of culture, the cells were fixed with 4% paraformaldehyde and then stained for tartrate-resistant acid phosphatase (TRAP) using the TRAP staining kit from Suzhou Bizhong Biotechnology Co., Ltd. The specific operation steps were strictly followed according to the kit instructions.

[0043] C. Experimental Results

[0044] Compared with the control group (DMSO-treated group), the number of osteoclasts in the group treated with benzothiazinone vinylidene indene derivatives (4d, 4e, 4f, 4i, 4p, 4q, 4u, and 4v) at a concentration of 5 μM was significantly reduced ( Figure 2 ), indicating that this series of compounds can effectively inhibit the formation of osteoclasts.

[0045] Example 3: TRAP staining assay to detect the dose effect of compound 4q in inhibiting osteoclast differentiation

[0046] A. Experimental Purpose

[0047] The inhibitory effect of compound 4q on osteoclast formation at different concentrations was analyzed.

[0048] B. Experimental Methods

[0049] Bone marrow macrophages were divided into 8 groups, namely NM group, 0μM 4q treatment group, 0.3125μM 4q treatment group, 0.625μM 4q treatment group, 1.25μM 4q treatment group, 2.5μM 4q treatment group and 5μM 4q treatment group. 5 Cells were seeded at a density of 100 μM in 24-well culture plates, with triplicate wells set up for each group. After one day of culture in BMM medium, 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 medium volume in each group was equal, and the volume of DMSO was 1 / 1000 of the medium volume. The culture medium was replaced every 2 days for 5 days. After the incubation period, the cells were fixed with 4% paraformaldehyde and stained for tartrate-resistant acid phosphatase (TRAP) using the TRAP staining kit from Suzhou Bizhong Biotechnology Co., Ltd., according to the kit's instructions. After staining, the number of TRAP-positive osteoclasts with ≥3 nuclei was counted.

[0050] C. Experimental Results

[0051] 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 was ≥ 2.5 μM, compound 4q almost completely inhibited osteoclast formation (see Figure 3 ).

[0052] Example 4: qPCR detection of the effect of compound 4q on the expression of osteoclast differentiation marker genes

[0053] A. Experimental Purpose

[0054] The effects of compound 4q at different concentrations on the expression of osteoclast differentiation-related genes were analyzed to further determine the inhibitory effect of 4q on osteoclast formation.

[0055] B. Experimental Methods

[0056] Bone marrow macrophages were divided into 8 groups (NM group and 0, 0.3125, 0.625, 1.25, 2.5, and 5 μM 4q treatment groups), with 4 × 10 5 The cells were seeded in 6-well plates at a density of 100 cells / well, with three replicates per 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 volume of culture medium in each group was equal, and the volume of DMSO was 1 / 1000 of the volume of the culture medium. The culture medium was replaced every 2 days and cultured for 5 days. After the culture was completed, the total RNA of each group of cells was extracted using Trizol reagent, reverse transcribed into cDNA, and then subjected to real-time fluorescence quantitative PCR to detect Cfos, Ctsk, Acp5, Oscar, Dcstamp, Mmp9, Atp6v0d2 mRNA expression levels.

[0057] C. Experimental Results

[0058] The mRNA levels of the above genes decreased significantly with the increase of 4q concentration ( Figure 4 ), confirmed that 4q plays a role by inhibiting the expression of osteoclast differentiation-related genes.

Claims

1. Use of a benzothiazinone vinylidene indene compound in the preparation of a drug for treating osteolytic diseases, wherein the structural formula of the benzothiazinone vinylidene indene compound is shown in Formula (1) or Formula (2): ， The R 1 is methyl or phenyl; R 2 is hydrogen, cyano, trifluoromethyl or methylenedioxy; R 3 It is methyl, methoxy, halogen or forms a quinolyl group with the skeleton.

2. The use according to claim 1, characterized in that The benzothiazinone vinylidene indene compound is used for preparing medicine for treating osteoporosis, periprosthetic bone dissolution, bone destruction caused by cancer metastasis or rheumatoid arthritis.

3. The use according to any one of claims 1-2, characterized in that The benzothiazinone vinylidene indene compound is any one of the following formulas: 。 4. The use according to any one of claims 1-2, characterized in that The dosage form of the medicine is tablet, pill, powder, capsule, injection, oral solution, ointment or cream.

5. The use according to any one of claims 1-2, characterized in that The drug is administered orally, by injection or external use.

6. The use according to any one of claims 1-2, characterized in that The medicine comprises a benzothiazinone vinylidene indene compound as an active ingredient and medically acceptable pharmaceutical excipients.

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