Application of a germarane-type sesquiterpene lactone compound

Abstract

The present invention relates to the application of a germarane-type sesquiterpene lactone compound. Biological activity experiments show that the compound has an osteoblast differentiation inhibitory effect, and the activity is better than that of the positive drug Pamidronate. The compound can be used to prevent and / or treat osteoporosis, osteolysis, periprosthetic bone dissolution, tumor bone metastasis, inflammatory bone loss, periodontitis inflammatory bone resorption and other diseases.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to pharmaceutical use of a germarane-type sesquiterpene lactone compound. Background Art

[0002] Normal bone metabolism is a metabolic balance between bone formation mediated by osteoblasts and bone resorption mediated by osteoclasts. Overactive osteoclasts can lead to an imbalance in bone metabolism, such as osteoporosis, osteolysis (Gorham-Stout syndrome), periprosthetic osteolysis, tumor bone metastasis, inflammatory bone loss, inflammatory bone resorption in periodontitis, etc., which are closely related to overactive osteoclasts.

[0003] For example, osteoporosis (OP) is a systemic metabolic bone disease characterized by decreased bone mass and destruction of bone microstructure, which is manifested by increased bone fragility and prone to fractures. Osteoporosis is mainly caused by the fact that osteoclast-mediated bone catabolism exceeds osteoblast-mediated bone anabolism. Therefore, inhibiting osteoclast-mediated bone resorption is an effective way to treat osteoporosis.

[0004] Osteolysis caused by wear particles around the prosthesis and the resulting aseptic loosening have become the most important long-term complications after hip and knee replacement surgery, affecting the service life of the prosthesis implant and the quality of life of the patient. It is the primary reason for revision after total joint replacement. There is currently no effective method to prevent and treat this disease. During the occurrence and development of the above complications, debris particles lead to macrophage activation and further macrophage aggregation, and then release osteolytic factors such as TNF-α, IL-1, IL-6, etc. The increase in related factors will increase RANK, and then increase the activation of RANK and RANKL, leading to abnormal recruitment and activation of osteoclasts and significant upregulation of bone resorption function, leading to osteolysis. Osteolysis around the prosthesis will cause micro-motion of the prosthesis, further leading to increased particle wear and further loosening of the prosthesis. Therefore, inhibiting osteoclasts is an effective way to treat periprosthetic osteolysis.

[0005] Tumor bone metastasis is a type of disease caused by the progression of malignant tumors, in which tumor cells metastasize to the skeletal system and grow locally in the bones, resulting in bone destruction and fractures, sudden fractures during normal limb activities, nerve compression, difficulty in limb movement, limb numbness, and paralysis in severe cases. In fact, bone is the third most common site of tumor metastasis, among which breast cancer, prostate cancer, lung cancer, multiple myeloma, etc. are the most common types of tumors with bone metastasis, and more than 40% of related advanced cancer patients are affected by this type of bone metastasis. Bone metastasis leads to bone metabolism disorders and causes bone-related events, such as spinal cord compression, pathological fractures, hypercalcemia, etc., which seriously reduce the quality of life of patients and increase their economic burden and mortality. When tumor cells metastasize to bones, they affect the metabolic balance between bone cells, eventually leading to excessive activation of osteoclasts and enhanced bone resorption. At the same time, osteoclast activation will release related growth factors, and some growth factors will induce the tumor to continue to grow in the bone. For example, prostate cancer is one of the most common malignant tumors in men. Since the early clinical symptoms of prostate cancer are atypical and prostate cancer is an osteophilic tumor, nearly 70% of patients have bone metastasis when they seek medical treatment. Bone metastasis leads to bone metabolism disorders and causes bone-related events, such as spinal cord compression, pathological fractures, and hypercalcemia, which seriously reduce the quality of life of patients and increase their economic burden and mortality. Prostate cancer bone metastasis mainly occurs through the hematogenous metastasis pathway, among which the spine, ribs, and femur are common metastatic sites of prostate cancer. Recent studies have shown that after prostate cancer cells enter the bone microenvironment through the hematogenous metastasis pathway, they pathologically increase the expression of nuclear factor-κB receptor activator ligand (RANKL). RANKL binds to nuclear factor-κB receptor activator (RANK) on the surface of osteoclasts to activate osteoclasts, degrade bone matrix, and cause osteolytic changes; while bone matrix dissolution releases growth factors such as transforming growth factor β (TGF-β), which promotes the growth of prostate cancer cells and forms a vicious cycle. Therefore, inhibiting osteoclast differentiation is an effective treatment to prevent tumor osteolytic lesions.

[0006] The main cause of inflammatory bone loss is that chronic persistent inflammation activates osteoclasts to increase bone absorption, leading to bone loss. Osteoporosis and osteolysis are the most common complications in patients with AIDS and tuberculosis. A large number of studies have shown that HIV or active tuberculosis can increase the level of nuclear factor κB receptor activator ligand (RANKL) and the RANKL / osteoprotegerin (OPG) ratio in serum, activate the function of osteoclasts, increase bone absorption, and reduce the body's bone mass. Clinically, osteoporosis is prone to brittle fractures, mainly in the hip, forearm, thoracic and lumbar spine, while bone tuberculosis often causes osteolytic destruction of bone joints and loss of joint function. Fractures or loss of joint function can cause disability in patients, and in severe cases even lead to death, bringing a huge burden to society and patients' families.

[0007] Existing osteoclast bone resorption inhibitors mainly include bisphosphonates, calcitonins, estrogens, selective estrogen receptor modulators, RANKL inhibitors, etc., which can prevent and treat osteoporosis to varying degrees, relieve pain, and reduce the incidence of fractures. However, these drugs also have problems such as uncertain efficacy, adverse side effects, and high prices. Such as tumors, infections, cardiovascular diseases, delayed fracture healing, hypocalcemia, jaw osteonecrosis, and injection side effects. Therefore, drug developers in the field of bone disease research are committed to finding new, effective, low-toxic and easy-to-obtain bone disease prevention and treatment drugs.

[0008] Natural products are secondary metabolites that have survived a long period of evolution and natural selection in nature. They have biodiversity, chemical diversity, and drug-like properties. Many clinical drugs such as artemisinin and paclitaxel are derived from natural products or their derivatives. Natural products have unique mechanisms of action; discovering and elucidating unique mechanisms of action can open up new directions for the development of new drugs, thereby developing more new drugs and generating huge economic value and social effects. The way natural products act on organisms is special and complex. They may act directly on a single or multiple targets in the form of original drugs, or act on specific targets in the form of metabolites after entering the body. Some natural products exert pharmacological effects by regulating endogenous substances after entering the body.

[0009] Germarane-type sesquiterpene lactones are a class of 10 Sesquiterpenes with macrocyclic skeletons have a five-membered lactone ring structure at positions 6, 7 or 7, 8. With more and more research on germarane-type sesquiterpenoid lactones, people have found that they have anti-tumor, anti-viral, anti-atherosclerotic and other pharmacological effects, and can be used to treat diseases such as liver cancer, ovarian cancer, breast cancer and bile duct cancer, but there are no reports on the inhibition of diseases related to excessive osteoclast activation by germarane-type sesquiterpenoid lactones. Summary of the invention

[0010] In order to solve the shortcomings and deficiencies of the prior art and to find osteoclast differentiation inhibitors with stronger pharmacological effects, the main purpose of the present invention is to provide a use of a germarane-type sesquiterpene lactone compound or its pharmaceutically acceptable salts, stereoisomers, and tautomers in the preparation of osteoblast differentiation inhibitors.

[0011] In order to achieve the above-mentioned invention object, the present invention adopts the following technical scheme:

[0012] A use of a germarane-type sesquiterpene lactone compound or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof in the preparation of an osteoblast differentiation inhibitor, characterized in that the germarane-type sesquiterpene lactone compound has a structural formula as shown in formula (I) or formula (II) or formula (III):

[0013] ;

[0014] Wherein, R1 and R2 are selected from H, CH3 or CH2CH3.

[0015] In one embodiment, in the structural formula of the germarane-type sesquiterpene lactone compound, R1 is selected from H or CH3, and R2 is selected from H, CH3 or CH2CH3.

[0016] In another embodiment, the germarane-type sesquiterpene lactone compound has a stereo configuration as shown in formula (IV), formula (V) or formula (VI):

[0017] ;

[0018] In another embodiment, the osteoclast differentiation inhibitor is used to prepare one or more drugs for preventing and / or treating osteoporosis, osteolysis, periprosthetic bone dissolution, tumor bone metastasis, inflammatory bone loss, and periodontitis inflammatory bone resorption diseases, including but not limited to the above-mentioned diseases.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects: the compound shown in the present invention is a class of compounds of the germarane-type sesquiterpene lactone class, and the present invention expands the pharmaceutical use of such compounds. The biological activity test experiment shows that the germarane-type sesquiterpene lactone compound of the present invention has significant activity in inhibiting osteoclast differentiation, IC 50 is 1.361*10 -8 M, reaching nM level, the activity is very prominent among the natural osteoclast active compounds, and is stronger than the existing bisphosphonates, such as zoledronic acid, the IC50 reported in the literature is about 0.1-2.5 μM. These natural germarane-type sesquiterpene lactone compounds can be used to prevent and / or treat osteoporosis, osteolysis, periprosthetic bone dissolution, tumor bone metastasis, inflammatory bone loss, periodontitis inflammatory bone resorption and other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram showing the effects of the compounds 1-8 provided by the present invention on osteoclast differentiation of osteoclast precursor cells, i.e., macrophage cell line RAW264.7 cells;

[0021] Figure 2 Schematic diagram of the multi-concentration gradient results of the effect of compound 1 on osteoclast differentiation induction of osteoclast precursor cells RAW264.7.

[0022] Figure 3 This is the IC50 curve of compound 1 in inhibiting osteoclast differentiation.

[0023] Figure 4 This is the effect of compound 1 on the expression levels of key genes of bone resorption function in osteoclast precursor cells RAW264.7.

[0024] Figure 5 This is the effect of compound 1 on the expression levels of key genes in osteoclast differentiation and bone resorption function of mouse primary macrophages BMMs.

[0025] Figure 6 Schematic diagram of the inhibitory effect of compound 1 on osteolysis. DETAILED DESCRIPTION

[0026] Elephantopus tomentosus L. is the dried whole herb of Elephantopus tomentosus L., a plant of the genus Elephantopus in the Asteraceae family, also known as ox tongue grass. The present invention uses a variety of chemical methods to extract a series of germarane-type sesquiterpene lactone compounds represented by formula (I), formula (II) or formula (III) from Elephantopus tomentosus L., uses a variety of spectroscopic methods to identify them, and proves the inhibitory effect of such compounds on osteoblast differentiation through cell experiments.

[0027] The technical solution of the present invention is further described below in conjunction with embodiments, but the present invention is not limited to these embodiments.

[0028] In the following examples, the microplate reader is the American BioTEK Synergy H1 Hybrid Multi-Mode Reader; the blot imaging analyzer is developed by Bio-Rad; the confocal microscope is Leica; the real-time fluorescence quantitative PCR system is RocheLight Cycler 480 Ⅱ,

[0029] Representative results from biological experiments were selected and presented in the context figures. Data were presented as mean ± SD as specified in the figure. All experiments were repeated at least three times. Data were analyzed using GraphPad Prism 8.0.2. The differences in mean values ​​between two groups and more than two groups were compared using t-test and analysis of variance, respectively. P < 0.05 was considered a significant difference.

[0030] Example 1 Structural Identification of Germarane-type Sesquiterpene Lactone Compounds

[0031] The structures of the obtained series of compounds were identified as germarane-type sesquiterpene lactone compounds. The structural information of the obtained compounds is as follows:

[0032] ;

[0033] Wherein, R1 and R2 are selected from H, CH3 or CH2CH3.

[0034] Some representative compound information is shown in Table 1 below:

[0035] Table 1: Information of some representative compounds

[0036]

[0037] The physicochemical data of compounds 1-8 are as follows:

[0038] Compound 1: white powder (methanol), vanillin-concentrated sulfuric acid turns purple when heated, HR-ESI-MS m / z :383.1465 [M+Na] + (calcd for C 20 H 24 O6Na, 383.1465); UV (MeOH) l max : 208 nm; IR (KBr) n max : 2958, 2924, 1770, 1716, 1655, 1431, 1276, 1157, 1090, 1056 cm -1 ; 1 H NMR (400 MHz, CDCl3) d 6.27 (1H, d, J = 2.6 Hz), 6.12 (1H, s,), 5.66 (2H, s), 5.75(1H, d, J = 2.6 Hz), 5.44 (1H, s), 5.20 (1H, s), 5.19 (1H, d, J = 4.0 Hz), 4.62(1H, dd, J = 5.9, 4.0 Hz), 3.68 (1H, d, J = 14.4 Hz), 3.17 (3H, s), 3.16 (1H, m), 1.94 (3H, s), 1.74 (3H, s), 1.69 (3H, s); 13 C NMR (100 MHz, CDCl3) d169.1,165.8, 139.2, 136.3, 135.2, 133.9, 129.2, 127.3, 127.2, 126.6, 114.2, 85.9,79.4, 76.7, 49.7, 39.1, 32.6, 32.6, 28.6, 18.4, 13.1.

[0039] Compound 2: white powder (methanol), vanillin-concentrated sulfuric acid turns purple when heated, HR-ESI-MS m / z :383.1454 [M+Na] + (calcd for C 20 H 24 O6Na, 383.1454); UV (MeOH) l max : 208 nm; IR (KBr) n max : 2955, 2927, 1771, 1717, 1655, 1433, 1280, 1157, 1091 1056 cm -1 ; 1 H NMR (400 MHz, CDCl3) d 6.92 (1H, qd, J = 6.1, 5.3, 1.6 Hz), 6.36 (1H, d, J = 2.6 Hz),6.20 (1H, s), 6.01 (1H, t, J = 1.5 Hz), 5.82 (1H, d, J = 2.2 Hz), 5.45 (1H, s), 5.25 (1H, td, J = 10.8, 3.9 Hz), 4.24 (1H, d, J = 3.8 Hz), 3.38 (1H, dq, J = 10.6,2.7 Hz), 2.75 (1H, dd, J = 12.1, 4.0 Hz), 2.48 (1H, t, J = 11.5 Hz), 2.01 (3H, d, J = 1.5 Hz), 1.84 (6H, d, J = 5.8 Hz), 1.79 (s, 3H); 13 C NMR (100 MHz, CDCl3) d .7, 19.8, 15.7, 20.2, 19.1, 14.7, 12.2.

[0040] Compound 3: white powder (methanol), purple color after heating with vanillin-concentrated sulfuric acid, HR-ESI-MS m / z: 397.1621 [M+Na] + (calcd for C 21 H 26 O6Na, 397.1622); UV (MeOH) λ max : 209 nm; IR (KBr)ν max : 2929, 1772, 1715, 1648, 1384, 1273, 1153, 1033 cm -1 ; 1 H NMR (400 MHz,CDCl3) d 6.91 (1H, dq, J = 7.0, 2.0 Hz), 6.28 (1H, d, J = 3.1 Hz), 5.76 (1H, d, J = 3.1 Hz), 5.60 (1H, s), 5.45 (1H, s), 5.22 (1H, d, J = 4.0 Hz), 5.12 (1H, m), 4.63 (1H, dd, J = 6.0, 4.0 Hz), 3.68 (1H, d, 14.3), 3.19 (3H, s), 3.12 (1H, m), 2.20 (1H, dd, J = 14.3, 4.3 Hz), 1.86 (3H, d, J = 7.0, 2.0 Hz), 1.75 (3H, s), 1.71 (3H, s), 1.82 (3H, s); 13 C NMR (100 MHz, CDCl3) d8.2, 168.2, 166.4, 139.3,138.8, 135.3, 134.2, 129.2, 128.6, 127.2, 127.2, 114.2, 85.9, 79.6, 76.5,49.5, 39.2, 32.9, 28.2, 14.8, 13.4, 12.3.

[0041] Compound 4: white powder (methanol), vanillin-concentrated sulfuric acid turns purple when heated, [α]25 D +47.9° (c1.0, MeOH), HR-ESI-MS m / z: 387.1415 [M+Na] + (calcd for C 19 H 24 O7Na, 387.1414); UV(MeOH) λ max : 206 nm; IR (KBr) ν max : 3438, 2970, 2917, 1752, 1716, 1292, 1158,1110, 1039 cm -1 ; 1 H NMR (400 MHz, CDCl3) d 6.21 (1H, s), 6.14 (1H, s), 5.80 (1H,s), 5.74 (1H, s), 5.40 (1H, s), 5.22 (1H, s), 5.13 (1H, d, J = 12.0 Hz), 4.32(1H, dd, J = 4.0, 6.0 Hz), 4.25 (1H, d, J = 4.0 Hz), 3.97 (1H, m), 2.54 (1H, d, J =15.0 Hz), 2.42 (1H, d, J = 15.0 Hz), 2.72 (2H, d, J = 15.0 Hz), 2.67 (1H, d, J =15.0 Hz), 2.01 (3H, s), 1.90 (1H, d, J = 15.0 Hz), 1.62 (3H, s); 13 C NMR (100MHz, CDCl3) d8.2, 50.0, 41.3, 35.4, 32.2, 18.7.

[0042] Compound 5: white powder (methanol), purple color after heating with vanillin-concentrated sulfuric acid, HR-ESI-MS m / z: 369.1308 [M+Na] + (calcd for C 19 H 22 O6Na, 369.1309); UV (MeOH) λ max : 205 nm; IR (KBr)ν max : 3447, 2928, 1760, 1700, 1641, 1384, 1274, 1162, 1050 cm -1 ; 1 H NMR (400MHz, CDCl3) d 6.36 (1H, d, J = 3.0 Hz), 6.19 (1H, s), 6.13(1H, s), 6.00 (1H, s), 5.81 (1H, d, J = 3.0 Hz), 5.66(1H, s), 5.43 (1H, s), 5.22 (1H, m), 4.22 (1H, d, J = 3.7 Hz), 3.37 (1H, m), 2.75 (1H, dd, J = 12.0, 3.7 Hz), 2.50 (1H, d, J = 12.0Hz), 2.00(3H, s), 1.94(3H, s), 1.77(3H, s); 13 C NMR (100 MHz, CDCl3) d 8.5, 195.2, 169.5, 165.8, 158.9, 137.1, 135.8, 133.5, 133.1, 129.5, 128.5, 126.8, 81.3, 73.9, 72.5, 49.7, 45.6, 20.0, 19.1, 18.5.

[0043] Compound 6: white powder (methanol), purple color after heating with vanillin-concentrated sulfuric acid, HR-ESI-MS m / z: 369.1410 [M+Na] + (calcd for C 19 H 22 O6Na, 369.1410); UV (MeOH) λ max : 205 nm; IR (KBr)ν max : 3447, 2929, 1762, 1703, 1640, 1384, 1277, 1169, 1055 cm -1 ; 1 H NMR (400MHz, CDCl3) d 6.27 (1H, d, J = 3.5 Hz), 6.19 (1H, d, J = 7.2 Hz), 5.77 (1H, d, J =2.8 Hz), 5.73 (1H, d, J = 6.8 Hz), 5.36 (1H, s), 5.24 – 5.14 (4H, m), 5.07 (1H,d, J = 9.7 Hz), 4.34 (1H, dd, J = 6.6, 3.5 Hz), 3.28 – 3.24 (1H, m), 3.21 (1H,dt, J = 6.1, 3.5 Hz), 2.79 – 2.52 (7H, m), 2.01 (3H, d, J = 7.5 Hz); 13 C NMR (100MHz, CDCl3) d .8, 18.6, 169.1, 165.7, 143.7, 140.3, 136.4, 135.6, 126.5, 126.5, 121.8,111.2, 107.9, 81.1, 79.1, 75.2, 50.9, 41.9, 39.4, 34.8, 18.6.

[0044] Compound 7: white needles (chloroform); [α]25D + 25.5 (c 1.0, MeOH); IR (KBr) ν max :1785, 1589, 1472, 1308, 1164, 1137, 1096, 1063, 922, 806 cm-1 ; UV (MeOH) λ max :216 nm; HR-ESI-MS m / z: 383.1444 [M+Na] + (calcd for C 20 H 24 O6Na, 383.1471); 1 H NMR (400 MHz, CDCl3) d 7.09 (1H, s), 6.21 (1H, m), 5.33 (1H, d, J = 4.8 Hz), 4.68(1H, dt, J = 11.3, 4.6 Hz), 5.05 (1H, m), 4.85 (1H, t, J = 9.9 Hz), 2.92 (1H, m), 2.76 (1H, d, J = 12.5 Hz), 2.70 (1H, dd, J = 12.5, 4.4 Hz), 2.43 (1H, dd, J = 12.5,6.6 Hz), 2.38 (1H, m), 2.32 (1H, dd, J = 14.6, 4.9 Hz), 2.03 (3H, m), 1.91 (3H,m), 1.74 (3H, d, J = 1.5 Hz), 1.36 (3H, d, J = 6.6 Hz); 13 C NMR (100 MHz, CDCl3) d 5.5, 177.9, 174.4, 167.3, 148.9, 141.6, 134.8, 131.6, 126.7, 125.5, 79.6, 78.7,72.3, 54.3, 40.5, 39.0, 30.8, 21.8, 20.7, 16.2, 15.5.

[0045] Compound 8: white powder (chloroform); IR (KBr) n max : 2930, 1776, 1713, 1665, 1446,1309, 1192, 1158, 981 cm -1 ; UV (MeOH) l max: 209 nm; HR-ESI-MS m / z : 383.1444 [M+Na] + (calcd for C 20 H 24 O6Na, 383.1471); 1 H NMR (400 MHz, CDCl3) d 7.12 (1H, s), 6.26 (1H, d, J = 3.2 Hz), 5.35 (1H, d, J = 4.6 Hz), 5.10 (1H, d, J = 10.4 Hz), 5.07 (1H,d, J = 3.2 Hz), 5.06 (1H, m), 4.45 (1H, m), 3.10 (1H, m), 2.65 (1H, dd, J = 12.6,3.8 Hz), 2.95 (1H, m), 2.92 (1H, d, J = 14.7 Hz), 2.36 (1H, dd, J = 14.7, 5.2Hz), 2.31 (1H, dd, J = 14.0, 7.0 Hz), 1.75 (3H, s), 1.66 (1H, m), 1.39 (1H,ddd, J = 14.4, 14.4, 7.3 Hz), 0.86 (3H, t, J = 7.4 Hz), 1.17 (3H, d, J = 7.1 Hz); 13 CNMR (100 MHz, CDCl3) d 8.0, 176.2, 174.5, 169.7, 149.4, 135.5, 134.0, 131.8, 125.6,124.0, 79.7, 79.0, 73.5, 49.8, 41.2, 40.3, 30.4, 26.3, 21.9, 17.2, 12.0.

[0046] Example 2 Effects of Compounds 1-8 on Differentiation and Maturation of Osteoclast Precursor Cells, Macrophage Cell Line RAW264.7

[0047] RAW264.7 cells in the logarithmic growth phase were taken and seeded in 96-well plates at a density of 3000 cells / well. They were cultured in DMEM medium containing 10% FBS (fetal bovine serum) and 1% antibiotics. After 6 hours of culture, the medium was replaced with α-MEM medium containing osteoclast induction factor M-CSF (30 ng / mL), RANKL (50 ng / mL) and different concentrations of compounds 1-8 (10-10 M~10-7 M). Three replicate wells were set up in each group and cultured for 4 days. New medium and drugs were replaced every 2 days. After a total of 4 days of culture, the medium containing the compound was removed, the medium was aspirated, and the cells were rinsed with cold PBS, fixed with 4% paraformaldehyde solution, and finally stained with TRAP (tartrate-resistant acid phosphatase). The nuclei of mature multinucleated osteoclasts induced can be stained dark by TRAP staining reagent, so the cells with more than 3 nuclei produced by differentiation were counted and the area was quantified under a microscope.

[0048] Test results such as Figure 1 The results showed that compared with the blank control group Ctrl(-), the number of osteoclasts in the control group (RANKL-induced group Ctrl(+)) increased significantly, and compound 1 -8 ~10 -6 At a dose of 10 M, compound 4 and compound 8 -7 ~10 -6 At a dose of 10 M, compound 2, compound 3, compound 5, compound 6, and compound 7 -6 At the M dose, the number of multinuclear osteoclasts generated ( Figure 1 A) showed different degrees of inhibitory activity, and compound 1 -8 ~10 -6 At a dose of 10 M, compound 4 -7 ~10 -6 At a dose of 10 M, compound 2, compound 3, compound 5, compound 6, and compound 8 -6 M doses showed different degrees of inhibition on the area of ​​multinuclear osteoclasts ( Figure 1 The above results prove that this class of sesquiterpene lactone compounds has a significant inhibitory effect on osteoclast differentiation.

[0049] Example 3 Experiment on the effect of compound 1 on differentiation and maturation of osteoclast precursor cells, namely macrophage cell line RAW264.7 cells

[0050] RAW264.7 cells in the logarithmic growth phase were seeded at a density of 3000 cells / well in a 96-well plate and cultured in DMEM medium containing 10% FBS (fetal bovine serum) and 1% antibiotics. After 6 h of culture, the medium was replaced with osteoclast induction factor M-CSF (30 ng / mL), RANKL (50 ng / mL) and different concentrations of BC-1 (10 -9 , 2*10 -9 , 4*10 -9 , 6*10 -9 , 8*10 -9 , 10 -8 , 2*10 -8 , 4*10 -8 , 6*10 -8 , 8*10 -8 , 10 -7 M) α-MEM complete medium, 5 replicates were set up in each group, and the culture was carried out for 3 days. The new medium and drug were replaced every 2 days. After a total of 3 days of administration, the medium containing the compound was removed, and then rinsed twice with cold PBS, fixed with 4% paraformaldehyde solution for 30 minutes, and finally stained with TRAP detection kit, i.e., tartrate-resistant acid phosphatase detection kit. The nuclei of the induced mature multinucleated osteoclasts can be stained wine red by the TRAP staining kit, and finally the cells with more than 3 nuclei produced by differentiation were counted and the area was quantified under a microscope.

[0051] Test results such as Figure 2-3 shown. Figure 2 A in the figure is a picture of RAW264.7 cells after osteoclast differentiation and TRAP staining taken under a low-power microscope. The cells with darker staining and larger volume are mature differentiated multinucleated osteoclasts. The blank control group (Ctrl(-)) was used as the negative control. Compared with the blank control group (Ctrl(-)), the addition of RANKL-induced control group Ctrl(+) induced a large number of multinucleated osteoclasts. Figure 2 B in the figure is the quantitative result of the number of multinuclear osteoclasts. Figure 2 C in the figure is the quantitative result of multinuclear osteoclast area. The results showed that after different compound 1 treatment groups, compound 1 increased from 6*10 -9 M began to show an inhibitory trend on the number and area of ​​osteoclasts, but no statistical difference was found (*P>0.05). -8 M, the inhibition rate of osteoclast number exceeded 90%, (P < 0.05), and compound 1 was -8M began to inhibit the osteoclast area by more than 80% (P < 0.001). By fitting the activity results, the IC50 of compound 1 for inhibiting osteoclast area was finally obtained to be 1.361*10 -8 M (see Figure 3 ), with activity reaching nM level, which is very rare in natural products and more active than related drugs such as bisphosphonates. For example, the IC50 of zoledronic acid in osteoclast differentiation inhibition reported in the literature is about 0.1-2.5 μM.

[0052] Example 4 Effect of Compound 1 on the mRNA Levels of Bone Resorption-Related Proteases after Differentiation of Osteoclast Precursor Cells RAW264.7 into Osteoclasts

[0053] RAW264.7 cells in the logarithmic growth phase were taken and the cells were cultured at 4*10 4 The cells were seeded at a density of 100 cells / well in a 12-well plate and cultured in DMEM medium containing 10% FBS (fetal bovine serum) and 1% double antibody. After 6 h of culture, the medium was replaced with osteoclast induction factor M-CSF (20 ng / mL), RANKL (10 ng / mL) and different concentrations of BC-1 (10 -8 M ~ 8*10 -8 M) α-MEM complete medium. New medium and drugs were replaced every 2 days. RNA was extracted after 3 days of administration, and q-PCR experiments were performed to detect the mRNA expression levels of corresponding indicators.

[0054] Test results such as Figure 4 As shown in the figure, Cathepsin K (CTSK) is one of the main enzymes that is specifically and highly expressed in osteoclasts to perform bone resorption. Its main function is to degrade type I and type II collagen, osteopontin, etc., which account for more than 90% of the bone matrix content in an acidic microenvironment. MMP9 is another secreted matrix metalloproteinase, which is related to the dissolution of bone matrix, recruitment and migration of osteoclasts during the bone resorption stage. Studies have shown that the body can induce secretory autophagy and enhance MMP9 secretion under stress. According to the q-PCR results in the figure below, it can be seen that compound 1 exhibits significant concentration-dependent inhibitory activity on CTSK and MMP9 ( P <0.001).

[0055] Example 5 Effect of Compound 1 on the expression level of key genes for osteoclast differentiation and bone resorption in primary mouse macrophages BMMs

[0056] Macrophages from bone marrow of 4-6 week-old C57BL / 6J mice were isolated and cultured at 10 5The cells were seeded at a density of 100 cells / well in a 6-well plate and cultured in α-MEM medium containing 20 ng / mL M-CSF, 10% FBS (fetal bovine serum) and 1% P / S dual antibody. After 24 h of culture, the medium was replaced with osteoclast induction factor M-CSF (20 ng / mL), RANKL (15 ng / mL) and 2*10 -8 M of compound 1 in α-MEM complete medium. New medium and drugs were replaced every 2 days. RNA was extracted after 5 days of administration, and q-PCR experiments were performed to detect the mRNA expression levels of corresponding indicators.

[0057] Test results such as Figure 5 The results showed that compound 1 could reduce the expression of TRAP, TRAF6, NFATc1, CTSK, MMP9, OSCAR, DC-stamp, c-SRC, and c-fos, indicating that compound 1 could reduce the expression of TRAP, TRAF6, NFATc1, CTSK, MMP9, OSCAR, DC-stamp, c-SRC, and c-fos in 2*10 -8 M concentration can inhibit osteoclast differentiation and key factors of bone resorption function.

[0058] Example 6 Experimental evaluation of the inhibitory effect of compound 1 on mouse skull osteolysis

[0059] Eight-week-old male C57BL / 6J mice weighing 25±2g were selected and housed in an SPF animal room with free access to food and water. After one week of adaptive feeding, the mice were randomly divided into 5 groups: sham group, osteolysis model group, osteolysis model group + low-dose compound 1 group (BC-1 LD low-dose group), osteolysis model group + medium-dose compound 1 group (BC-1MD medium-dose group), and osteolysis model group + high-dose compound 1 group (BC-1 HD high-dose group), with 8 mice in each group, for a total of 40 mice. After the mice were anesthetized with isoflurane, the hair on the top of the skull of the mouse was shaved with a shaver, and the skin of the top of the skull was disinfected with 75% ethanol. Then, a scalpel was used to make an incision of about 1.5 cm long along the midline of the skull. 30 mg Ti particles (30 μL) were evenly dropped on the surface of the skull, and the surgical incision was sutured to establish a mouse skull osteolysis model. In the sham group, an equal volume of sterile PBS was dropped on the surface of the skull. Drugs were administered by intraperitoneal injection starting from the second day after surgery. The sham group (Sham) and the osteolysis model group (Model) were injected with an equal amount of drug solvents, once a day, for 14 consecutive days. The experimental animals were killed 14 days later, and the skull specimens were collected and fixed with 4% paraformaldehyde for 48 hours. Ti particles on the skull surface were carefully removed, and a high-resolution Micro-CT instrument (Skyscan 1176; Bruker, Antwerp, Belgium) was used with a scanning resolution of 10.4μm, an X-ray voltage of 55kV, and a current of 145μA. The circular area centered on the median suture of the skull was used as the region of interest (ROI). Bone mineral density (BMD), bone volume fraction (BV / TV), bone surface area to bone volume ratio (BS / BV), and trabecular thickness (Tb. Th) were calculated using CTAn software; 3D reconstruction images were constructed using CTvox.

[0060] The results are as follows Figure 6As shown. The results showed that a large amount of bone loss was caused in the skull of mice in the osteolysis model group (Model). Compared with the sham operation group (Sham), BMD, BV / TV and Th. Tb were significantly reduced, BS / BV was significantly increased, and the difference was statistically significant (**p<0.01, ***p<0.001). After the administration of compound 1, bone loss was significantly improved. Compared with the osteolysis model group (Model), BMD, BV / TV and Th. Tb were significantly increased, BS / BV was significantly decreased, and the difference was statistically significant (#p<0.05, ##p<0.01, ###p<0.001). Further tissue sections were taken at the site of bone dissolution, and the results of HE staining showed that there was some inflammatory reaction on the skull surface of mice in the osteolysis model group (Model), with visible infiltration of inflammatory cells such as clustered macrophages and lymphocytes, uneven bone surface, and obvious bone dissolution; while the inflammatory reaction on the skull surface of the low-dose compound 1 (BC-1 LD low-dose) group and the medium-dose BC-1 (MD medium-dose) group was significantly improved, with significantly reduced infiltration of inflammatory cells such as clustered macrophages and lymphocytes, slightly flattened bone surface, and significantly weakened bone dissolution; no obvious inflammatory reaction was observed on the skull surface of the high-dose compound 1 (BC-1 HD high-dose) group, no infiltration of inflammatory cells such as clustered macrophages and lymphocytes was observed, the bone surface was flat, and bone dissolution was almost completely blocked.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Use of a germarane-type sesquiterpene lactone compound in the preparation of one or more drugs for preventing and / or treating osteoporosis, osteolysis, tumor bone metastasis, inflammatory bone loss, characterized in that The germarane-type sesquiterpene lactone compound is a compound 1 or a compound 3 or a structural formula shown in formula (III): ； Wherein, R1 and R2 are selected from H, CH3 or CH2CH3.

2. The use according to claim 1, characterized in that The formula (III) has a stereo configuration as shown in formula (VI): 。 3. The use according to claim 1 or 2, characterized in that The osteolysis is periprosthetic osteolysis, and the inflammatory bone loss is periodontitis-induced inflammatory bone resorption.

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