Application of N-sulfinyl formylhydrazone derivative in preparation of medicine for inhibiting osteoclast generation and bone resorption

Abstract

The invention relates to an application of N-sulfinyl formylhydrazone derivatives in preparation of drugs for inhibiting osteoclast generation and bone resorption, the N-sulfinyl formylhydrazone derivatives HTM-13, HTM-17, HTM-18, HTM-25 and HTM-37 can directly and effectively inhibit bone marrow macrophages from differentiating to osteoclasts by inhibiting the expression of osteoclast differentiation marker genes, and can be used for preparing drugs for inhibiting osteoclast generation and bone resorption. The cell toxicity is not shown when the concentration is not higher than 5 mu M. The N-sulfinyl formylhydrazone derivative has the beneficial effects that the N-sulfinyl formylhydrazone derivative provided by the invention can be used as a candidate drug molecule of a novel osteoclast differentiation inhibitor, and is used for developing a novel anti-bone resorption drug and treating diseases caused by overexpression of osteoclasts. Therefore, the invention has important clinical significance and application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and in particular relates to the application of N-sulfinylformylhydrazone derivatives in the preparation of drugs that inhibit osteoclast formation and bone resorption. Background Technology

[0002] The maintenance of skeletal homeostasis depends on the dynamic balance between bone formation and bone resorption during bone remodeling. Osteoclasts, as the only multinucleated giant cells in the body with bone resorption function, play a crucial role in this process. Originating from hematopoietic stem cell lines, their differentiation, activation, and function are precisely regulated by a series of cytokines. Among them, macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL), expressed by osteoblasts / stromal cells, are two core regulatory factors. After RANKL binds to its receptor RANK on the osteoclast precursor cell membrane, it activates multiple downstream signaling pathways, including NF-κB, MAPK (such as ERK, p38, JNK), and PI3K / AKT, through the adaptor protein TRAF6, thereby inducing the expression of core transcription factors NFATc1 and c-Fos. These transcription factors then upregulate the transcription of a series of osteoclast differentiation and function-related genes (such as Ctsk, Acp5, Oscar, Dcstamp, Mmp9, and Atp6v0d2), ultimately driving the differentiation, fusion, and activation of osteoclast precursor cells into mature osteoclasts. Mature osteoclasts attach to the bone surface through their unique folded edges, forming closed bone resorption lacunae. They then complete bone resorption by secreting acids and various proteases (such as cathepsin K) to dissolve bone minerals and degrade collagen in the bone matrix.

[0003] In various pathological conditions, such as postmenopausal osteoporosis (PMOP), rheumatoid arthritis, bone metastasis of tumors, and periprosthetic osteolysis, the above-mentioned regulatory mechanisms are imbalanced, leading to abnormal activation of osteoclasts. Specifically, this manifests as a pathological increase in their number, significantly enhanced activity, and prolonged lifespan, causing the rate of bone resorption to far exceed that of bone formation, ultimately resulting in progressive bone loss, destruction of bone microstructure, and an increased risk of fracture.

[0004] Currently, the main anti-bone resorption drugs used in clinical practice for the above-mentioned diseases, especially PMOP, include bisphosphonates (such as alendronate sodium, risedronate sodium, and zoledronic acid), RANKL monoclonal antibodies (denosumab), estrogen therapy, and selective estrogen receptor modulators (such as raloxifene). While these drugs can effectively inhibit osteoclast activity and delay bone loss, significant limitations and safety issues have been revealed in long-term use. For example, bisphosphonates and denosumab carry the risk of osteonecrosis of the jaw and atypical femoral fractures; denosumab may cause rapid bone loss and a rebound risk of multiple vertebral fractures after discontinuation; and estrogen and related therapies may increase the risk of breast cancer and thrombotic events. Therefore, the development of novel osteoclast inhibitors that can efficiently and precisely inhibit pathological bone resorption while maintaining good safety is of urgent clinical need and significant scientific value. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide the application of N-sulfinylformylhydrazone derivatives in the preparation of drugs that inhibit osteoclast formation and bone resorption.

[0006] Firstly, an N-sulfinylformylhydrazone derivative is provided, with the following structural formula:

[0007] 1.

[0008] R1 is selected from 3-thienyl, 3-benzothienyl, 3-benzofuranyl, 4-chlorophenyl or 4-fluorophenyl; R2 is selected from 4-tolyl or 5-(dimethylamino)naphthyl.

[0009] Preferably, the N-sulfinylformylhydrazone derivative is any one of the following formulas:

[0010] .

[0011] In a second aspect, the use of the N-sulfinylformylhydrazone derivative as described in the first aspect in the preparation of a medicament for inhibiting the formation of mature osteoclasts and bone resorption is provided.

[0012] Preferably, the drug is used to prevent or treat osteolytic diseases, including osteoporosis, bone destruction due to tumor metastasis, periprosthetic osteolysis, and rheumatoid arthritis.

[0013] Preferably, the drug is used to inhibit osteoclast differentiation, including: inhibiting the expression of osteoclast differentiation marker genes.

[0014] Preferably, the osteoclast differentiation marker gene is selected from one or more of Nfatc1, Ctsk, Acp5, Oscar, Dcstamp, and Atp6v0d2.

[0015] Preferably, the dosage form of the drug is tablets, pills, powders, capsules, injections, oral liquids, ointments, or creams; the administration method of the drug is oral, injection, or external application.

[0016] Thirdly, a medicament as described in any of the second aspects is provided, the medicament comprising an active ingredient N-sulfinylformylhydrazone derivative and pharmaceutical excipients; the pharmaceutical excipients comprising one or more of diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, and lubricants.

[0017] The beneficial effects of this invention are as follows: Using a RANKL-induced mouse bone marrow macrophage osteoclast differentiation model, experiments confirmed that N-sulfinylformylhydrazone derivatives HTM-13, HTM-17, HTM-18, HTM-25, and HTM-37 can directly and effectively inhibit the differentiation of bone marrow macrophages into osteoclasts by suppressing the expression of osteoclast differentiation marker genes (including Nfatc1, Ctsk, Acp5, Oscar, Dcstamp, and Atp6v0d2), and no cytotoxicity was observed at concentrations not exceeding 5 μM. Based on these findings, the N-sulfinylformylhydrazone derivatives provided by this invention can serve as candidate drug molecules for novel osteoclast differentiation inhibitors, used in the development of novel anti-bone resorption drugs to treat diseases caused by excessive activation of osteoclasts. Therefore, this invention has significant clinical significance and application prospects. Attached Figure Description

[0018] Figure 1 Inhibitory effects of N-sulfinylformylhydrazone derivatives HTM-13, HTM-17, HTM-18, HTM-25 and HTM-37 on osteoclast formation at a concentration of 5 μM (assessed by TRAP staining).

[0019] Figure 2 A graph showing the quantitative statistical results of the number and size of mature osteoclasts (TRAP positive and ≥3 nuclei).

[0020] Figure 3 The graph shows the cytotoxicity of N-sulfinylformylhydrazone derivatives HTM-13, HTM-17, HTM-18, HTM-25 and HTM-37 on bone marrow-derived monocytes / macrophages (BMMs) at a concentration of 5 μM (based on the CCK-8 assay).

[0021] Figure 4 Dose-response plot of N-sulfinylformylhydrazone derivative HTM-17 inhibiting osteoclast formation (assessed by TRAP staining).

[0022] Figure 5 A graph showing the quantitative statistical results of the number and size of mature osteoclasts (TRAP positive and ≥3 nuclei).

[0023] Figure 6 The effect of N-sulfinylformylhydrazone derivative HTM-17 on the expression of osteoclast differentiation-related genes (detected by qPCR). Detailed Implementation

[0024] The present invention will be further described below with reference to embodiments. The descriptions of the embodiments below are merely for the purpose of helping to understand the present invention. It should be noted that those skilled in the art can make various modifications to the present invention without departing from its principles, and these modifications and improvements also fall within the scope of protection of the claims of the present invention. Other experimental methods in the embodiments, unless otherwise specified, are conventional methods, performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc., used in the following embodiments, unless otherwise specified, are commercially available. Unless otherwise specified, the quantitative experiments in the following embodiments are all performed in triplicate, and the results are averaged. Figures 1 to 6 The experimental results of each embodiment are presented, demonstrating the non-toxicity of the compound and its effect in inhibiting osteoclast differentiation.

[0025] Example 1: TRAP staining to evaluate the inhibitory effects of N-sulfinylformylhydrazone derivatives HTM-13, HTM-17, HTM-18, HTM-25, and HTM-37 on RANKL-induced osteoclast differentiation.

[0026] (1) Obtaining primary mouse bone marrow mononuclear / macrophages (BMMs)

[0027] A. Bone marrow cell isolation and culture: Six- to ten-week-old C57BL / 6 mice were euthanized by cervical dislocation. The femur and tibia were separated, and surrounding tissues were removed and cleaned. The bones were cut open at both ends and placed in a pre-made sterile nested centrifuge apparatus (a 0.6 mL centrifuge tube with a hole punched in the bottom, nested inside a 1.5 mL centrifuge tube). The cells were then briefly centrifuged at high speed (stopping at 10,000 rpm) to collect the bone marrow cells into the lower centrifuge tube. The bone marrow cells were resuspended in alpha-MEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S), seeded in 10 cm cell culture dishes, and cultured at 37°C and 5% CO2 for 24 hours.

[0028] B. M-CSF-induced adherence: The culture supernatant was collected, centrifuged, and the cells were resuspended in α-MEM complete medium (hereinafter referred to as BMM medium) containing 30 ng / mL macrophage colony-stimulating factor (M-CSF) and re-seeded. After stimulation with M-CSF, the suspended BMMs gradually adhered and grew.

[0029] C. BMMs Purification and Expansion: After culturing for 24 hours, the culture medium was discarded, and the culture dishes were gently washed twice with Dulbecco phosphate-buffered saline (DPBS) to thoroughly remove non-adherent cells. The remaining adherent cells were the desired primary bone marrow-derived macrophages (BMMs). The culture was then replaced with fresh BMM medium. When the cells reached approximately 90% confluence, they were digested with trypsin, neutralized with the aforementioned BMM medium, and resuspended to obtain a single-cell suspension. After determining the cell concentration using a cell counting chamber, the cells were reseeded into new culture plates at the required experimental density for subsequent experiments.

[0030] (2) Osteoclast induction differentiation and drug intervention methods

[0031] The counted BMMs were divided into 1×10 per hole. 5 Cells were seeded at a density of [number] cells per well in 24-well cell culture plates. Pre-cultured for 24 hours in alpha-MEM complete medium containing 15 ng / mL M-CSF to ensure full cell adhesion. Subsequently, experimental groups were established, including experimental and solvent control groups, with at least three replicates per group. The experimental group received osteoclast induction complete medium (composed of alpha-MEM complete medium containing 30 ng / mL M-CSF and 50 ng / mL RANKL) containing 5 μM of the test compound (HTM-13, HTM-17, HTM-18, HTM-25, or HTM-37 dissolved in DMSO). The solvent control group received an equal volume of osteoclast induction complete medium containing an equal volume of DMSO. During induction, the corresponding fresh medium was replaced every 48 hours. Culture was terminated after typical multinucleated osteoclasts formed in the solvent control group (usually on day 5-6 of induction). At the end of the treatment, discard the culture medium, wash each well once with DPBS, add 300 μL of 4% paraformaldehyde (PFA), and fix at room temperature for 10 minutes. After fixation, wash once with DPBS, and then perform tartrate-resistant acid phosphatase (TRAP) staining using the TRAP staining kit from Suzhou Bizhong Biotechnology Co., Ltd., following the kit instructions.

[0032] The results are as follows Figure 1As shown: A large number of typical multinucleated mature osteoclasts were observed in the solvent control group; however, almost no mature osteoclasts were observed in the 5 μM HTM-13, HTM-17, and HTM-37 treatment groups; and the number and size of osteoclasts were significantly reduced in the HTM-18 and HTM-25 treatment groups. Quantitative results further indicated that HTM-18 or HTM-25 reduced the number and size of mature osteoclasts (data are expressed as mean ± standard deviation, n=3). Compared with the solvent control group (DMSO group), both the 5 μM HTM-18 and HTM-25 groups showed significant inhibitory effects. p < 0.01, p < 0.001, p < 0.0001) Figure 2 The above results indicate that, at a concentration of 5 μM, all tested compounds HTM-13, HTM-17, HTM-18, HTM-25, and HTM-37 effectively inhibited osteoclast formation.

[0033] Example 2: CCK-8 assay for the cytotoxicity of 5 μM N-sulfinylformylhydrazone derivatives HTM-13, HTM-17, HTM-18, HTM-25 and HTM-37 on bone marrow macrophages.

[0034] The separated BMMs were divided into 5×10 pores per hole. 3 Cells were seeded at a density of 100 μL in 96-well plates and cultured for 24 h in complete medium containing 15 ng / mL M-CSF. After cell attachment, experimental groups and a solvent control group were established, with 6 replicates per group. The experimental group was cultured in BMM medium (alpha-MEM complete medium containing 30 ng / mL M-CSF) containing 5 μM of the test compound (HTM-13, HTM-17, HTM-18, HTM-25, or HTM-37 dissolved in DMSO); the solvent control group was cultured in an equal volume of BMM medium containing an equal volume of DMSO. The corresponding fresh medium was replaced every 48 h during the treatment. After 24 h and 72 h of treatment, the medium was discarded, the cells were washed twice with DPBS, and 100 μL of 10% CCK-8 reagent was quickly added to each well. The cells were incubated at 37°C in the dark for 1 h, and the absorbance (OD value) at 450 nm was measured using a microplate reader.

[0035] The results are as follows Figure 3As shown, at a concentration of 5 μM, HTM-13, HTM-17, HTM-18, HTM-25, and HTM-37 did not exhibit significant cytotoxicity against BMMs within 24 h and 72 h (data are expressed as mean ± standard deviation, n=6). This indicates that the effect of the compounds on osteoclast number at this concentration stems from their specific inhibition of osteoclast differentiation, rather than from cytotoxicity.

[0036] Example 3: TRAP staining to detect the dose-effect of N-sulfinylformylhydrazone derivative HTM-17 in inhibiting osteoclast formation

[0037] The separated BMMs were divided into 1×10⁻⁶ per pore. 5 Cells were seeded at a density of 100 μM / mL in 24-well plates and pre-cultured for 24 h in α-MEM complete medium containing 15 ng / mL M-CSF. After cell attachment, cells were divided into experimental and solvent control groups, with at least 3 replicates per group. The experimental group was treated with osteoclast induction medium containing 0.625, 1.25, 2.5, or 5 μM HTM-17 (α-MEM complete medium containing 30 ng / mL M-CSF and 50 ng / mL RANKL); the solvent control group was treated with an equal volume of induction medium containing an equal volume of DMSO. During induction, the corresponding fresh medium was replaced every 48 h. The experiment was terminated when typical multinucleated osteoclasts were formed in the solvent control group (usually day 5-6). The medium was discarded, the cells were washed once with DPBS, and 300 μL of 4% paraformaldehyde (PFA) was added to each well for fixation at room temperature for 10 min. After fixation, the cells were washed once with DPBS, and then tartrate-resistant acid phosphatase (TRAP) staining was performed according to the instructions of the TRAP staining kit from Suzhou Bizhong Biotechnology Co., Ltd. After staining, the number of mature osteoclasts with TRAP-positive cells and ≥3 nuclei in each group was counted.

[0038] The results are as follows Figure 4 As shown, HTM-17 can dose-dependently inhibit the formation of mature osteoclasts. Quantitative analysis further indicates that ( Figure 5 Within the concentration range of 0.625–5 μM, HTM-17 gradually reduced the number and size of mature osteoclasts with increasing concentration (data are expressed as mean ± standard deviation, n=3). Compared with the solvent control group (0 μM HTM-17), all concentration groups showed significant inhibitory effects. p < 0.05, p < 0.01, p < 0.001, p < 0.0001).

[0039] Example 4: qPCR detection of the effect of N-sulfinylformylhydrazone derivative HTM-17 on the expression of osteoclast differentiation and function-related genes.

[0040] The separated BMMs were divided into 4×10 pores per well. 5 Cells were seeded at a density of 1000 μM / mL in 6-well plates and pre-cultured for 24 h in alpha-MEM complete medium containing 15 ng / mL M-CSF. After cell attachment, cells were divided into three groups: a normal medium control group (NM group, containing 30 ng / mL M-CSF and 0.1% DMSO), a RANKL-induced solvent control group (0 μM HTM-17 group, containing 30 ng / mL M-CSF, 50 ng / mL RANKL, and 0.1% DMSO), and RANKL-induced groups containing 0.625, 1.25, 2.5, and 5 μM HTM-17 (all components were the same as the 0 μM group, only with the corresponding concentration of HTM-17 added). Each group had three replicates. The corresponding fresh medium was replaced every 48 h. Induction was terminated when a large number of mature osteoclasts appeared in the 0 μM HTM-17 group (approximately day 5). The medium was discarded, the cells were washed twice with DPBS, and total RNA was extracted using Trizol reagent. RNA was dissolved in 20 μL of DEPC water. After determining the concentration, 500 ng of RNA was used for reverse transcription to obtain 100 μL of cDNA. Real-time quantitative PCR was performed using a Bio-Rad CFX96 system. The reaction mixture contained 2 μL of cDNA, 0.5 μL of upstream and downstream primer mixture, 10 μL of SYBR Green qPCR Mix, and 7.5 μL of ddH2O. After the reaction, 2... －△△CT The relative mRNA expression levels of each target gene were calculated using this method.

[0041] The results are as follows Figure 6 As shown: Compared with the RANKL treatment group without HTM-17 (0 μM group), the mRNA expression levels of osteoclast marker genes Nfatc1, Ctsk, Atp6v0d2, Acp5, Oscar, and Dcstamp decreased in a dose-dependent manner with increasing HTM-17 concentration (data are expressed as mean ± standard deviation, n=3;). p < 0.05, p < 0.01, p < 0.001, (p < 0.0001). This result further demonstrates that HTM-17 can effectively inhibit osteoclast differentiation by downregulating the expression of Nfatc1, Ctsk, Atp6v0d2, Acp5, Oscar, and Dcstamp.

[0042] In summary, this invention provides a novel class of compounds with the general formula N-sulfinylformylhydrazone derivatives. Experiments show that compounds HTM-13, HTM-17, HTM-18, HTM-25, and HTM-37 can inhibit the formation of mature osteoclasts; and they do not exhibit cytotoxicity at concentrations not exceeding 5 μM. Based on these findings, the N-sulfinylformylhydrazone derivatives provided by this invention can serve as candidate drug molecules for novel osteoclast differentiation inhibitors, used in the development of novel anti-bone resorption drugs to treat diseases caused by excessive activation of osteoclasts.

Claims

1. An N-sulfinylformylhydrazone derivative, characterized in that, The structural formula is as follows: R1 is selected from 3-thienyl, 3-benzothienyl, 3-benzofuranyl, 4-chlorophenyl or 4-fluorophenyl; R2 is selected from 4-tolyl or 5-(dimethylamino)naphthyl.

2. The N-sulfinylformylhydrazone derivative according to claim 1, characterized in that, The N-sulfinylformylhydrazone derivative is any one of the following formulas: 。 3. The use of the N-sulfinylformylhydrazone derivative as described in claim 1 in the preparation of a medicament for inhibiting the formation of mature osteoclasts and bone resorption.

4. The application according to claim 3, characterized in that, The drug is used to prevent or treat osteolytic diseases, including osteoporosis, bone destruction due to tumor metastasis, periprosthetic osteolysis, and rheumatoid arthritis.

5. The application according to claim 4, characterized in that, The drug is used to inhibit osteoclast differentiation, including inhibiting the expression of osteoclast differentiation marker genes.

6. The application according to claim 5, characterized in that, The osteoclast differentiation marker gene is selected from one or more of Nfatc1, Ctsk, Acp5, Oscar, Dcstamp and Atp6v0d2.

7. The application according to any one of claims 3-6, characterized in that, The dosage form of the drug is tablets, pills, powders, capsules, injections, oral liquids, ointments, or creams; the administration method of the drug is oral, injection, or external application.

8. A medicament as described in claim 7, characterized in that, The drug comprises an active ingredient, N-sulfinylformylhydrazone derivative, and pharmaceutical excipients; the pharmaceutical excipients comprise one or more of the following: diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, and lubricants.

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