Application of trisubstituted pyrazole compounds in the preparation of drugs for inhibiting osteoclast differentiation

Abstract

The present invention discloses an application of a trisubstituted pyrazole compound in the preparation of a drug for inhibiting osteoclast differentiation, and belongs to the field of medicine. The present invention uses a variety of experimental methods such as tartrate-resistant acid phosphatase (TRAP) staining, reverse transcription real-time fluorescence quantitative PCR (qPCR), protein immunoblotting (Western blot), etc., and proves for the first time that the trisubstituted pyrazole compound of the following formula can significantly increase the expression of antioxidant proteins HO-1 and Catalase, thereby effectively inhibiting the differentiation and generation of osteoclasts. Therefore, the trisubstituted pyrazole compound provided by the present invention can be used as a candidate compound for the development of a new osteoclast differentiation inhibitor to treat diseases caused by excessive osteoclast activation.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to the application of a trisubstituted pyrazole compound in the preparation of a drug for inhibiting osteoclast differentiation. Background Art

[0002] Osteoclasts (Ocs) are the only known cells in the body responsible for bone resorption. They are differentiated and matured from osteoclast precursor cells (such as bone marrow monocytes / macrophages) derived from hematopoietic stem cells under the action of multiple factors, among which RANKL plays a particularly critical role. RANKL activates downstream signaling pathways such as NF-κB and MAPK by binding to the receptor RANK on osteoclast precursor cells, thereby upregulating transcription factors such as NFATc1 and c-Fos, thereby regulating the transcription of osteoclast-specific genes, and ultimately promoting osteoclast formation and activation. Osteoclasts play a vital role in normal bone development and reconstruction. However, abnormal increases in the number and / or activity of osteoclasts can lead to a variety of osteolytic diseases such as osteoporosis, rheumatoid arthritis, periprosthetic bone dissolution, and bone destruction caused by cancer metastasis. Inhibiting osteoclast differentiation and bone resorption is currently recognized as one of the main means of treating such diseases.

[0003] At present, the most widely used anti-bone resorption drugs in clinical practice include bisphosphonates, RANKL monoclonal antibody Denosumab, calcitonin, selective estrogen receptor modulator (raloxifene), etc. The use of bisphosphonates and Denosumab has the risk of mandibular osteonecrosis and atypical femoral fractures; once Denosumab is discontinued, there will be a rebound after withdrawal; long-term use of calcitonin has the potential to increase the risk of tumors; and raloxifene has a slight increase in the risk of venous thrombosis. It can be seen that the existing anti-bone resorption drugs have obvious shortcomings. Therefore, there is an urgent need for a safe and effective new drug that can treat osteolytic diseases such as osteoporosis by inhibiting osteoclast differentiation and function.

[0004] Intracellular reactive oxygen species (ROS) play a crucial role in osteoclast formation and bone resorption. Osteoclast precursor cells produce endogenous ROS after being stimulated by RANKL, and the application of ROS scavengers such as N-acetylcysteine ​​(NAC) can inhibit RANKL-mediated ROS generation, thereby inhibiting osteoclastogenesis, indicating that ROS is required for osteoclast differentiation. There are a variety of antioxidant enzymes in cells that antagonize ROS, such as heme oxygenase-1 (HO-1) and catalase, and enhancing the expression of the above antioxidant enzymes can clear ROS, thereby inhibiting osteoclast formation. Therefore, targeted inhibition of ROS can be used as a new strategy to inhibit osteoclast differentiation and thus treat osteolytic diseases such as osteoporosis. Summary of the invention

[0005] In view of this, an embodiment of the present application provides a use of a trisubstituted pyrazole compound in the preparation of a drug for inhibiting osteoclast differentiation.

[0006] According to the embodiment of the present application, the trisubstituted pyrazole compound has a structural formula as shown in formula (1):

[0007]

[0008] Among them, R 1 is phenyl or substituted phenyl, the substituent is selected from halogen such as fluorine, chlorine, and methoxy, R 2 It is phenyl or substituted phenyl, and the substituent is selected from halogen such as fluorine and chlorine, methyl, ethyl and phenyl.

[0009] More preferably, the trisubstituted pyrazole compound may be any one of the following specific structural formulas Y-1, Y-2, Y-3, Y-4 and Y-5:

[0010] .

[0011] Application of the above trisubstituted pyrazole compound in the preparation of osteoclast differentiation inhibitors.

[0012] By inhibiting osteoclast differentiation, the trisubstituted pyrazole compound can be used to prepare drugs for treating osteolytic diseases to inhibit the excessive activation of osteoclasts in osteolytic diseases; the trisubstituted pyrazole compound can also be used to prepare drugs for treating osteoporosis, tumor metastasis bone destruction or rheumatoid arthritis.

[0013] The drug may be in the form of tablets, pills, powders, capsules, injections, oral solutions, ointments, creams, etc., and the drugs in various dosage forms may be prepared according to conventional methods in the pharmaceutical field. The administration method may be oral, injection or external use.

[0014] The drug comprises an active ingredient trisubstituted pyrazole compound and medically acceptable pharmaceutical excipients, wherein the excipients include conventional diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc. in the pharmaceutical field.

[0015] The trisubstituted pyrazole compound is used in the preparation of a drug for inhibiting the expression of a key protein for osteoclast differentiation, wherein the key protein for osteoclast differentiation is c-Fos, Nfatc1, Src or Ctsk protein.

[0016] The trisubstituted pyrazole compound is used in the preparation of drugs for inhibiting ROS; and is used in the preparation of drugs for increasing the expression of antioxidant proteins such as HO-1 and Catalase.

[0017] The present invention adopts a RANKL-induced mouse bone marrow macrophage osteoclast differentiation model, and experiments prove that the trisubstituted pyrazole compound can directly and effectively inhibit the differentiation of bone marrow macrophages into osteoclasts by increasing the expression of antioxidant proteins such as HO-1 and Catalase and downregulating the expression of key osteoclast differentiation proteins such as c-Fos and Nfatc1, without obvious cytotoxicity, so that it can be used as a candidate compound for the development of a new osteoclast differentiation inhibitor to treat diseases caused by excessive osteoclast activation. Therefore, the present invention has great clinical significance and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments consistent with the present application and, together with the description, serve to explain the principles of the present application.

[0019] Figure 1 The inhibitory effects of 5 μM trisubstituted pyrazole compounds Y-1, Y-2, Y-3, Y-4, and Y-5 on RANKL-induced osteoclast differentiation were evaluated by tartrate-resistant acid phosphatase (TRAP) staining.

[0020] Figure 2 The experimental results of the CCK-8 method to detect the cytotoxicity of trisubstituted pyrazole compound Y-3 on bone marrow macrophages. The data are expressed as mean ± standard deviation; compared with the control group without Y-3, the OD 450 There were no significant differences in the values.

[0021] Figure 3 Tartrate-resistant acid phosphatase (TRAP) staining was used to evaluate the dose effect of trisubstituted pyrazole compound Y-3 in inhibiting osteoclast differentiation. A. TRAP staining results; B. Quantitative analysis results. Data are expressed as mean ± standard deviation; compared with the RANKL treatment group without Y-3, *: p <0.05, **: p <0.01; ***: p <0.001; ****: p <0.0001.

[0022] Figure 4 Reverse transcription real-time fluorescence quantitative PCR (qPCR) was used to detect the effect of trisubstituted pyrazole compound Y-3 on the expression of genes related to RANKL-induced osteoclast differentiation. Compared with the RANKL treatment group without Y-3, ****: p <0.0001.

[0023] Figure 5Western blot was used to detect the effect of trisubstituted pyrazole compound Y-3 on the expression of osteoclast differentiation-related proteins and antioxidant proteins during osteoclast differentiation. A. The effect of 5 μM Y-3 on the expression of osteoclast differentiation-related proteins such as Nfatc1, c-Fos, Ctsk and Src; B. The effect of 5 μM Y-3 on the expression of antioxidant proteins Catalase and HO-1. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with specific embodiments. The following examples are only intended to illustrate the present invention, but are not intended to limit the present invention in any way. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and are not intended to limit the present invention in any way.

[0025] The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. The quantitative tests in the following examples are repeated three times, and the results are averaged.

[0026] Example 1 Preparation and Identification of Trisubstituted Pyrazole Compounds

[0027]

[0028] Take a clean 25mL schlenk tube, add 1.0 mmol, 2.0 equivalents of sulfur ylide 1, 0.5 mmol, 1.0 equivalents of tetrafluoroborate diazonium salt 2 and 1.0 mmol, 1.5 mmol, 3.0 equivalents of NaOH and dissolve in 10 mL of acetonitrile, and stir at room temperature for 48 hours. After the TLC plate shows that the reaction is complete, the reaction solution is concentrated under reduced pressure, and the crude product is directly mixed with silica gel and passed through a column (ethyl acetate: petroleum ether = 12.5%) to obtain the target product 3, which can be specifically:

[0029]

[0030] (1-(p-Tolyl)-1 H -Pyrazole-3,4-diyl)bis(phenyl ketone)

[0031] White powder (98 mg), 80% yield; melting point: 99.3−101.0 °C; 1 H NMR (400 MHz, CDCl 3 ): δ8.30 (s, 1H), 8.14−7.99 (m, 2H), 7.89−7.76 (m, 2H), 7.67 (d, J = 8.4 Hz, 2H),7.60−7.48 (m, 2H), 7.48−7.35 (m, 4H), 7.31 (d, J = 8.0 Hz, 2H), 2.42 (s, 3H) 13 CNMR (100 MHz, CDCl 3 ): δ 189.2, 188.1, 151.2, 138.3, 138.2, 136.7, 136.7, 133.2,132.9, 130.2, 130.2, 130.1, 129.1, 128.5, 128.3, 125.0, 119.9, 21.0. HRMS(ESI): m / z calcd for [M+H] + : 367.1441, found: 367.1438.

[0032]

[0033] (1-(3-Fluorophenyl)-1 H -Pyrazole-3,4-diyl)bis(phenyl ketone)

[0034] White powder (79 mg), 64% yield; melting point: 151.0−151.9 °C; 1 H NMR (400 MHz, DMSO- d 6 ): δ 9.24 (s, 1H), 8.02 (d, J = 7.6 Hz, 2H), 7.97−7.85 (m, 4H), 7.73−7.66 (m,2H), 7.64−7.50 (m, 5H), 7.31 (t, J = 8.2 Hz, 1H). 13 C NMR (100 MHz, DMSO- d 6 ): δ 193.4, 193.1, 167.7 (d, J = 244.4 Hz), 156.6, 145.3 (d, J= 10.6 Hz), 142.5,141.3, 139.1, 138.5, 136.8 (d, J = 9.7 Hz), 135.0, 134.4, 134.0 (d, J = 3.5 Hz),129.2, 120.9, 119.9 (d, J = 21.0 Hz), 112.5 (d, J = 26.3 Hz), 112.3 (d, J = 3.5Hz). HRMS (ESI): m / z calcd for [M+H] + : 371.1190, found: 371.1192.

[0035]

[0036] (1-(4-Fluorophenyl)-1 H -Pyrazole-3,4-diyl)bis(phenyl ketone)

[0037] White powder (80 mg), 65% yield; melting point: 92.3−94.1 °C; 1 H NMR (400 MHz, CDCl 3 ): δ 8.29 (s, 1H), 8.06 (d, J = 7.2 Hz, 2H), 7.81 (m, 4H), 7.64−7.48 (m, 2H), 7.46−7.32 (m, 4H), 7.30−6.98 (m, 2H). 13 C NMR (100 MHz, CDCl 3 ): δ 189.0, 188.0, 162.1(d, J = 248.6 Hz), 151.6, 138.1, 136.6, 135.3 (d, J = 2.8 Hz), 133.4, 133.0,130.2, 129.1, 128.5, 128.4, 125.3, 121.9 (d, J = 8.5 Hz), 116.7 (d, J = 23.3 Hz). HRMS (ESI): m / z calcd for [M+H] +: 371.1190, found: 371.1196.

[0038]

[0039] (1-phenyl-1 H -pyrazole-3,4-diyl)bis((4-fluorophenyl)methanone)

[0040] White powder (84 mg), 65% yield; melting point: 134.5−136.5 °C; 1 H NMR (400 MHz, CDCl 3 ): δ 8.32 (s, 1H), 8.26−8.18 (m, 2H), 7.92−7.85 (m, 2H), 7.78 (d, J = 7.6 Hz, 2H),7.54 (t, J = 7.6 Hz, 2H), 7.46−7.41 (m, 1H), 7.14 (t, J = 8.6 Hz, 2H), 7.09 (t, J = 8.6 Hz, 2H). 13 C NMR (100 MHz, CDCl 3 ): δ 187.9, 186.1, 167.16 (d, J = 254.1 Hz),164.6 (d, J = 253.5 Hz), 151.1, 139.0, 134.5 (d, J = 2.9 Hz), 133.3 (d, J = 9.4Hz), 132.9 (d, J = 2.9 Hz), 131.9 (d, J = 9.4 Hz), 130.1, 130.0, 128.5, 125.3,120.1, 115.9 (d, J = 21.9 Hz), 115.7 (d, J = 21.7 Hz). HRMS (ESI): m / z calcd for[2M + Na] + : 799.1939, found: 799.1946.

[0041]

[0042] (1-[1,1'-diphenyl]-2-yl)-1 H -pyrazole-3,4-diethyl)-bis(phenyl ketone)

[0043] White powder (101 mg), 71% yield; melting point: 115.3−116.5 °C; 1 H NMR (400 MHz, CDCl 3 ): δ 7.90 (d, J = 4.4 Hz, 2H), 7.72 (s, 1H), 7.59−7.50 (m, 7H), 7.49−7.46 (m, 4H), 7.43−7.38 (m, 2H), 7.35−7.27 (m, 2H), 7.26−7.22 (m, 2H). 13 C NMR (100 MHz, CDCl 3 ): δ 188.4, 188.0, 151.1, 138.3, 137.9, 137.4, 137.3, 136.6, 135.0, 133.2,132.8, 131.3, 130.3, 129.6, 129.2, 129.0, 128.8, 128.7, 128.4, 128.3, 128.0,126.4, 123.9. HRMS (ESI): m / z calcd for [M+H] + : 429.1598, found: 429.1603.

[0044] Example 2 TRAP staining to evaluate the inhibitory effect of trisubstituted pyrazole compounds Y-1, Y-2, Y-3, Y-4 and Y-5 on RANKL-induced osteoclast differentiation

[0045] (1) Isolation, culture, digestion and plating of mouse bone marrow macrophages

[0046] The 6-8 week old mice were killed by cervical dislocation and the whole body of the mice was disinfected with alcohol. The femur and tibia of the hind limbs of the mice were removed, the meat on the bones was removed, and the cartilage on the knee joint was removed. Then, the bone marrow was obtained by rapid centrifugation in a centrifuge (stopped when the number of revolutions reached 10000 rpm). The bone marrow was evenly blown with alpha-MEM culture medium containing 10% fetal bovine serum and 1% penicillin / streptomycin double antibody (referred to as complete culture medium), and the bone marrow was spread in a 10 cm cell culture dish for 1 day. The supernatant was taken and centrifuged at 1000 rpm for 4 min, and the supernatant was discarded. The obtained precipitate was evenly blown and cultured with complete culture medium containing 15 ng / mL M-CSF for 2 days. The adherent cells were bone marrow macrophages. When the bone marrow macrophages grew to about 90%, they were washed twice with preheated Dulbecco's phosphate buffer solution (DPBS), 2 mL of trypsin was added to the culture dish, and it was kept in a 37°C incubator away from light for 2 min. After most of the cells have become round, tap the cell dish to loosen the attached cells. Immediately add 2 mL of complete culture medium to terminate the trypsin reaction, and repeatedly blow the attached cells with the tip of a gun to make all the cells fall off the bottom of the dish. Collect the cells in a centrifuge tube, centrifuge at 1000 rpm for 4 min, discard the supernatant, and blow the precipitate evenly with an appropriate amount of complete culture medium to obtain a bone marrow macrophage suspension. Pipette 10 μL and count under a 10x microscope using a cell counting plate. According to the 6-well plate 4×10 5 / well, 24-well plate 1×10 5 / well, 96-well plate with a cell density of 5000 / well;

[0047] (2) Osteoclast differentiation induction and TRAP staining experiments

[0048] The bone marrow macrophages were cultured at a rate of 1×10 5 Cells were plated in 24-well plates. They were cultured in complete medium containing 15 ng / mL M-CSF for 1 day. After the cells attached to the wall, the cells were replaced with osteoclast differentiation induction medium (complete medium containing 15 ng / mL M-CSF and 100 ng / mL RANKL) containing 5 μM of different trisubstituted pyrazole compounds (Y-1, Y-2, Y-3, Y-4 or Y-5) dissolved in dimethyl sulfoxide (DMSO) or an equal volume of dimethyl sulfoxide (DMSO) solvent. The medium was changed every 2 days. When a large number of osteoclasts appeared in the control group (i.e., the DMSO-only group) (about 5 days), the cells in the 24-well plate were washed twice with DPBS, fixed with 300 μL of 4% PFA at room temperature for 10 min, and then washed once with DPBS. After removing DPBS, the cells were stained with tartrate-resistant acid phosphatase (TRAP) to evaluate the formation of osteoclasts (i.e., cells with TRAP+ and more than 3 nuclei) in each group. The results are as follows Figure 1As shown, 5 μM trisubstituted pyrazole compounds Y-1, Y-2, Y-3, Y-4 and Y-5 had a significant inhibitory effect on the formation of osteoclasts.

[0049] Experimental Example 3 CCK-8 detection of the cytotoxicity of trisubstituted pyrazole compound Y-3 on bone marrow macrophages

[0050] Mouse bone marrow macrophages were isolated and cultured from mouse bone marrow according to the above method, and 5000 cells were plated into 96-well plates per well, and cultured for 1 day with complete medium containing 15 ng / mL M-CSF. The cells were divided into 7 groups, namely 0, 0.3125, 0.625, 1.25, 2.5, 5 or 10 μM Y-3 groups, with 6 replicates in each group; after the cells adhered to the wall, the culture medium was replaced with complete medium containing 15 ng / mL M-CSF and the above-mentioned different concentrations of Y-3 for cell culture; the complete medium was removed at 24 h, 48 h and 72 h of treatment, and the cells were washed 1~2 times with appropriate amount of DPBS, and 100 μL of 10% CCK-8 reagent was added to each well, and cultured in a 37°C incubator in the dark for 30 min; finally, the OD value at 450 nm was detected with an enzyme marker. The results are as follows Figure 2 As shown, compared with the group without Y-3 addition, the OD values ​​of the Y-3 treatment group had no significant differences at 24h, 48h, and 72h, indicating that Y-3 at concentrations of 10 μM and below had no cytotoxicity to bone marrow macrophages.

[0051] Experimental Example 4 TRAP staining to detect the dose effect of trisubstituted pyrazole compound Y-3 in inhibiting osteoclast formation

[0052] Mouse bone marrow macrophages were isolated and cultured from mouse bone marrow according to the above method, and 1×10 5 The cells were plated in 24-well plates and divided into 8 groups (including NM group and 0, 0.3125, 0.625, 1.25, 2.5, 5 or 10 μM Y-3 treatment groups, each with 3 replicates), and cultured for 1 day in complete medium containing 15 ng / mL M-CSF. After the cells adhered to the wall, the cells were replaced with fresh complete medium (NM group) or osteoclast differentiation induction medium containing the above-mentioned different concentrations of Y-3. The medium was changed every 2 days. When a large number of osteoclasts appeared in the control group (i.e., 0 μM Y-3 group) (about 5 days), the formation of osteoclasts in each group (i.e., TRAP+ and cells with more than 3 nuclei) was evaluated by tartrate-resistant acid phosphatase (TRAP) staining. TRAP staining results showed ( Figure 3Y-3 inhibited osteoclast formation in a dose-dependent manner. The number and size of osteoclasts were significantly reduced under 0.625 μM treatment, and the inhibition was more obvious under 1.25 and 2.5 μM treatments. The formation of osteoclasts was almost completely inhibited at concentrations of 5 μM and above.

[0053] Experimental Example 5 qPCR detection of the effect of trisubstituted pyrazole compound Y-3 on the expression of osteoclast differentiation-related genes

[0054] P2 BMM cells isolated from mouse bone marrow cells were cultured at 4 × 10 5 The cells were plated in 6-well plates and divided into 6 groups (including NM group and 0, 1.25, 2.5, 5 or 10 μM Y-3 treatment groups, 3 replicates in each group); the cells were cultured with complete medium containing 15 ng / mL M-CSF for 1 day, and after the cells attached to the wall, the cells were replaced with fresh complete medium (NM group) or osteoclast differentiation induction medium containing the above-mentioned different concentrations of Y-3. The medium was changed every other day until a large number of osteoclasts appeared in the control group (i.e., 0 μM Y-3 group) (about 5-6 days); the medium in the 6-well plate was discarded, and the plate was washed with DPBS twice. The total RNA was extracted using the Trizol kit. After the concentration was determined, 500 ng RNA was taken and reverse transcribed into cDNA in the reaction system; then real-time fluorescence quantitative PCR reaction was performed on the Bio-Rad CFX96 instrument, and the reaction system was 2 μL cDNA template, 0.5 μL upstream and downstream primer mixture, 10 μL SYBR Green qPCR Mix and 7.5 μL ddH 2 O; After the reaction is completed, use 2 –△△Ct The relative mRNA expression of each target gene was calculated by the method. Figure 4 As shown, osteoclast differentiation-related genes Nfatc1, Ctsk, Acp5, Oscar, Dcstamp, Mmp9 and Atp6v0d2 The mRNA expression level of decreased in a concentration-dependent manner with the increase of Y-3 concentration, further indicating that Y-3 inhibits osteoclast differentiation.

[0055] Experimental Example 6 Western blot detection of the effect of trisubstituted pyrazole compound Y-3 on the expression of osteoclast differentiation-related proteins and antioxidant proteins

[0056] According to 4×10 per well 5Bone marrow macrophages were plated in 6-well plates with a cell number of 100 cells / well; divided into control group and drug treatment group, with 3 replicates in each group; cultured with complete medium containing 15 ng / mL M-CSF for 1 day, the medium was replaced with osteoclast differentiation induction medium, and 5 μM Y-3 was added to the drug treatment group, and an equal amount of dimethyl sulfoxide solvent was added to the control group. The medium was changed every other day, and protein samples were collected on days 0, 1, 3, and 5. The specific steps of collecting samples are as follows: discard the medium in the 6-well plate, wash twice with DPBS, add 100 μL of RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors to each well, scrape the cells in the well plate, collect them in a centrifuge tube, lyse on ice for 10 min, centrifuge at 13000 rpm for 15 min, and collect the supernatant as the protein sample. After the concentration of the above protein samples was determined by BCA kit, equal amounts of protein samples were taken for electrophoresis on 10% SDS-PAGE gel. After the protein bands were separated, they were transferred to NC membranes. The NC membranes were then blocked with blocking solution (TBST containing 5% skim milk) at room temperature for 1 hour, and then anti-Ctsk, Src, c-Fos, Nfatc1, Catalase or HO-1 antibodies were added, respectively, and incubated at 4°C overnight; the next day, after washing 3 times with 1×TBST on a shaker, the corresponding secondary antibodies were added and incubated at room temperature for 1 hour, and then washed 3 times with 1×TBST and developed. The results are shown in Figure 2. Figure 5 As shown in the results, 5 μM Y-3 could significantly reverse the induction of RANKL on osteoclast differentiation-related proteins Ctsk, c-Fos, Src, and Nfatc1, as well as its inhibitory effect on antioxidant proteins Catalase and HO-1, indicating that Y-3 inhibits osteoclast differentiation by upregulating antioxidant proteins and downregulating key factors of osteoclast differentiation.

Claims

1. Use of a trisubstituted pyrazole compound in the preparation of an osteoclast differentiation inhibitor, wherein the trisubstituted pyrazole compound has a structural formula as shown in formula (1): ； The R1 is phenyl or substituted phenyl, the substituent is selected from halogen, and R2 is phenyl or substituted phenyl, the substituent is selected from halogen, methyl, ethyl and phenyl.

2. The use according to claim 1, characterized in that The trisubstituted pyrazole compound is used for preparing a drug for treating osteolytic diseases.

3. The use according to claim 1, characterized in that The trisubstituted pyrazole compound is used for preparing medicine for preventing or treating osteoporosis, tumor metastasis bone destruction or rheumatoid arthritis.

4. The use according to claim 1, characterized in that The trisubstituted pyrazole compound is used for preparing a drug for inhibiting the expression of a key protein for osteoclast differentiation.

5. The use according to claim 1, characterized in that The trisubstituted pyrazole compound is used for preparing a drug for enhancing the expression of antioxidant proteins to target and inhibit ROS.

6. The use according to any one of claims 1 to 5, characterized in that: The trisubstituted pyrazole compound is any one of the following formulas: 。 7. The use according to any one of claims 1 to 5, characterized in that: The inhibitor or the drug is in the form of tablets, pills, powders, capsules, injections, oral solutions, ointments or creams, and is administered orally, by injection or external use.

8. The use according to any one of claims 1 to 5, characterized in that: The inhibitor or the drug comprises an active ingredient, a tri-substituted pyrazole compound and a medically acceptable pharmaceutical excipient.

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