Application of phenylacetic acid compounds in the preparation of osteoclast differentiation inhibitors

Abstract

The present invention provides the use of phenylacetic acid compounds in the preparation of osteoclast differentiation inhibitors, and relates to the field of marine natural products. A phenylacetic acid compound is disclosed, and its structural formula is shown in formula (I), and it is named 4-butoxy-benzeneacetic acid. It has a significant inhibitory effect on LPS-induced NF-κB luciferase at a concentration of 10 μM, and can significantly inhibit the differentiation of RANKL-induced osteoclast precursor cells RAW264.7 cells and BMMs cells into osteoclasts at a concentration of 6 μM. It has a protective effect on bone destruction in mice with osteoporosis caused by ovariectomy by inhibiting osteoclasts in vivo. It can effectively replace existing bisphosphonates and denoximab, inhibit the bone resorption activity of osteoclasts, and is an ideal candidate compound for development into a new osteoclast differentiation inhibitor drug. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the field of marine natural products, and in particular to the application of phenylacetic acid compounds in the preparation of osteoclast differentiation inhibitors. Background Art

[0002] Osteoporosis is a common bone degenerative disease, which is mainly manifested by increased bone brittleness, decreased bone mass and destruction of bone microstructure, which seriously threatens the health of the aging population and postmenopausal women. Osteoclasts (OCs) are special cells formed by the fusion of monocyte / macrophage hematopoietic lineage precursor cells. They are the only cells in the human body that have bone resorption function. Defective osteoclast activity leads to osteosclerosis and bone marrow failure, and excessive activation can lead to osteolytic diseases such as osteoporosis, rheumatoid arthritis, and tumor bone metastasis. Inhibiting the formation and resorption function of OCs is one of the main strategies for the treatment of osteoporosis. Marine microorganisms are a new ideal resource for discovering new drug-derived molecules. There are few reports on the anti-osteoclast differentiation inhibitors of marine microbial natural products, so their anti-osteoclast differentiation potential needs to be further explored.

[0003] The formation of osteoclasts is a step-by-step process, initiated by the binding of the receptor activator of nuclear factor-κB receptor activator ligand (RANKL) to its receptor RANK on monocyte / macrophage precursors. Currently, the osteoclast differentiation-related inhibitors used in clinical practice are mainly denoximab and bisphosphonates, but both have certain complications and side effects. Therefore, it is urgent to find a safe, effective, quality-controlled, and economical drug that targets and inhibits osteoclast formation and bone resorption, effectively solving the huge demand for osteoclast differentiation inhibitors in clinical practice. Summary of the invention

[0004] In view of the above problems, the present invention provides a new application of a phenylacetic acid compound (named as 4-butoxy-benzeneacetic acid).

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The use of the phenylacetic acid compound of the present invention in the preparation of an osteoclast differentiation inhibitor, wherein the structural formula of the phenylacetic acid compound is shown in formula (I), and the phenylacetic acid compound is named 4-butoxy-benzeneacetic acid:

[0007]

[0008] In the present invention, it is further described that the osteoclast differentiation inhibitor is a drug for treating osteolytic diseases caused by excessive activation of osteoclasts.

[0009] In the present invention, it is further described that the osteoclast differentiation inhibitor is a drug for treating osteoporosis, rheumatoid arthritis, and bone destruction caused by tumor metastasis.

[0010] The present invention also provides the use of phenylacetic acid compounds in the preparation of NF-κB nuclear factor expression inhibitor drugs.

[0011] In the present invention, it is further described that the osteoclast differentiation inhibitor or NF-κB nuclear factor expression inhibitor drug comprises an effective amount of a phenylacetic acid compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or adjuvant.

[0012] In the present invention, it is further described that the osteoclast differentiation inhibitor or NF-κB nuclear factor expression inhibitor drug is an oral preparation, an injection formulation or an external dosage form.

[0013] The present invention also provides a method for preparing phenylacetic acid compounds. The phenylacetic acid compounds are prepared and separated from the fermentation culture of Acremonium sclerotigenum C2F21.

[0014] In the present invention, the method for preparing phenylacetic acid compounds is further described, and the specific steps are as follows:

[0015] a), preparing a fermentation culture of Acremonium sclerotigenum C2F21, soaking the fermentation culture with ethyl acetate, cutting the fermentation into small pieces, ultrasonically extracting for 15 minutes, filtering with a Buchner funnel, and distilling and concentrating the filtrate to obtain extract A; the filter residue was continuously extracted with ethyl acetate for 3 times, and extract B was obtained after distillation and concentration;

[0016] b) The crude extract of extract A and extract B was subjected to medium pressure normal phase liquid chromatography, using petroleum ether / dichloromethane as eluent, and gradient elution was performed from a volume ratio of 100:0 to 0:100. The fractions eluted with a volume ratio of petroleum ether / dichloromethane of 80:20 were collected and then subjected to medium pressure reverse phase C 18 Column chromatography was performed using methanol / water as the eluent, with gradient elution from a volume ratio of 10:90 to 100:0, and fractions eluted with a volume ratio of methanol / water of 58:42 were collected. The collected fractions were purified to obtain phenylacetic acid compounds.

[0017] In the present invention, it is further described that the step a) of preparing the fermentation culture of Acremonium sclerotigenum C2F21 is to inoculate the activated Acremonium sclerotigenum C2F21 into a seed culture medium, culture at 25°C, 180rpm, for 72h to obtain a seed solution, inoculate the seed solution into a fermentation culture medium at an inoculation rate of 5%, and statically culture at 25°C for 100 days to obtain a fermentation culture.

[0018] In the present invention, it is further described that the seed culture medium formula contains: 15g malt extract powder, 20g sodium bromide, and the balance is water, pH 7.5 per 1L of culture medium.

[0019] In the present invention, it is further described that the formula of the fermentation medium is that each 1L of the Erlenmeyer flask medium contains: 130g of rice, 3g of sodium bromide, 150mL of water, and pH 7.5.

[0020] The present invention also provides application of Acremonium sclerotigenum C2F21 in preparing phenylacetic acid compounds.

[0021] The present invention obtains through experiments that: the phenylacetic acid compound 4-butoxy-benzeneacetic acid has a significant inhibitory effect on LPS-induced NF-κB luciferase at a concentration of 10 μM (p<0.001), and its activity is equivalent to that of the positive drug BAY, and can be used as a lead compound for developing NF-κB nuclear factor expression inhibitors.

[0022] The present invention obtains through experiments that the phenylacetic acid compound 4-butoxy-benzeneacetic acid significantly inhibits the differentiation of RANKL-induced osteoclast precursor cells RAW264.7 (mouse mononuclear macrophages) and BMMs (Bone marrow macrophage cells) into osteoclasts in a dose-dependent manner at a concentration of 6 μM, and has no obvious cytotoxicity. Therefore, it is expected to be developed into a safe and effective new osteoclast differentiation inhibitor drug.

[0023] The present invention has obtained through experiments that the phenylacetic acid compound 4-butoxy-benzeneacetic acid has a significant protective effect on bone loss in the classic model of osteoclast activation leading to bone destruction - ovariectomized osteoporotic mice, reduces the area of ​​bone depression formed by osteoclasts on bone slices, inhibits the bone absorption activity of osteoclasts, and has the advantages of simple structure, convenient administration, and economical and effective. Therefore, it can effectively replace the existing bisphosphonate drugs and denoximab.

[0024] One object of the present invention is to provide the use of phenylacetic acid compound 4-butoxy-benzeneacetic acid or its pharmaceutical salt in the preparation of NF-κB nuclear factor expression inhibitor or osteoclast differentiation inhibitor drugs. The anti-osteoclast differentiation drug is a drug for treating the clinical indications of bisphosphonates and denoximab, and is used to prevent and treat osteolytic diseases such as osteoporosis, rheumatoid arthritis, and tumor metastasis bone destruction.

[0025] Another object of the present invention is to provide a drug for NF-κB nuclear factor expression inhibitor or osteoclast differentiation inhibitor, comprising an effective amount of a phenylacetic acid compound 4-butoxy-benzeneacetic acid or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier or adjuvant.

[0026] Due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0027] In the process of studying the secondary metabolites of the symbiotic fungus Acremonium sclerotigenum C2F21 of the antler cup coral (Pocillopora damicornis) from Weizhou Island, Guangxi, the present invention separated and obtained a phenylacetic acid compound 4-butoxy-benzeneacetic acid, which has a significant inhibitory effect on LPS-induced NF-κB luciferase, and significantly inhibits the differentiation of RANKL-induced osteoclast precursor cells RAW264.7 and BMMs cells into osteoclasts in a dose-dependent manner, without producing cytotoxicity, and has a significant improvement effect on the bone loss of the classic model of osteoclast activation leading to bone destruction-ovariectomized osteoporotic mice in vivo, reduces the bone depression area formed by osteoclasts on bone slices, and inhibits the bone absorption activity of osteoclasts. Therefore, it is an ideal candidate compound for development into a new NF-κB nuclear factor expression inhibitor or osteoclast differentiation inhibitor drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a comparison chart of the inhibitory activity of 4-butoxy-benzeneacetic acid (BBA) at a concentration of 10 μM on NF-κB luciferase induced by lipopolysaccharide (LPS) in RAW264.7 cells. BAY is a positive control. ### Indicates p<0.001 vs. control group; *** indicates p<0.001 vs. LPS group;

[0029] Figure 2The effect of phenylacetic acid compound 4-butoxy-benzeneacetic acid on the cell viability of RAW264.7 and BMMs cells;

[0030] Figure 3 The experimental results of the effect of phenylacetic acid compound 4-butoxy-benzeneacetic acid (BBA) on the differentiation of osteoclast precursor RAW264.7 cells into osteoclasts, where: RANKL is a ligand that activates the nuclear factor NF-κB receptor. Compared with the blank control group, ###P<0.001; compared with the RANKL group, *P<0.05;

[0031] Figure 4 The results are the experimental results of the effect of phenylacetic acid compound 4-butoxy-benzeneacetic acid (BBA) on the differentiation of mouse bone marrow macrophages (BMMs) into osteoclasts, where RANKL is the ligand for activating nuclear factor NF-κB receptor, and M-CSF is macrophage colony stimulating factor. Compared with the blank control group, ### P<0.001; compared with RANKL group, *P<0.05 **P<0.01;

[0032] Figure 5 The Micro-CT scan results of the effect of 4-butoxy-benzeneacetic acid (BBA) on the femur of ovariectomized osteoporotic mice, where: Sham is sham surgery, OVX is ovariectomy, 5 mg / kg (low dose) and 10 mg / kg (high dose) are the dosages of phenylacetic acid compounds, and Ez is the positive drug estradiol;

[0033] Figure 6 The results of the statistical analysis of bone parameters of the effect of 4-butoxy-benzeneacetic acid (BBA) on the femur of ovariectomized osteoporotic mice are shown in Table 1. Sham is a sham operation, OVX is ovariectomized, the high and low doses of 4-butoxy-benzeneacetic acid are 10 mg / kg and 5 mg / kg, respectively, and Ez is the positive drug estradiol; ConnD represents the connection density of trabeculae, BS / BV represents the area of ​​bone tissue per unit volume, and Tb / Th represents the thickness of trabeculae;

[0034] Figure 7The results of HE and TRAP staining of femoral sections of ovariectomized osteoporotic mice by 4-butoxy-benzeneacetic acid, a phenylacetic acid compound, where Sham is the sham operation group, OVX is the ovariectomized osteoporosis model group, OVX+5mg / kg and OVX+10mg / kg are the low-dose and high-dose groups of phenylacetic acid compounds, respectively, and Ez is the positive drug estradiol group;

[0035] Figure 8 The results are the quantitative analysis results of the effect of 4-butoxy-benzeneacetic acid (BBA) on the number of osteoclasts and the surface area of ​​osteoclasts per unit bone area in femoral slices of ovariectomized osteoporotic mice, where Sham is a sham operation, OVX is an ovariectomized osteoporosis model group, OVX+5mg / kg and OVX+10mg / kg are low-dose and high-dose groups of phenylacetic acid compounds, respectively, and Ez is a positive drug estradiol group. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0037] The present invention is an application of a phenylacetic acid compound 4-butoxy-benzeneacetic acid in the preparation of an anti-osteoclast differentiation inhibitor or NF-κB nuclear factor expression inhibitor drug. The osteoclast differentiation inhibitor or NF-κB nuclear factor expression inhibitor drug is an oral preparation, an injection dosage form or an external dosage form; it comprises an active ingredient phenylacetic acid compound 4-butoxy-benzeneacetic acid and a medically acceptable pharmaceutical excipient. The anti-osteoclast differentiation drug can treat, but is not limited to: postmenopausal osteoporosis, tumor metastasis bone destruction and other clinical indications for which known osteoclast inhibitor drugs such as bisphosphonates and denoximab are approved for treatment.

[0038] Generally speaking, as a drug, it is clinically applied after being prepared into a preparation. The effective active ingredient phenylacetic acid compound described in the present invention can be prepared according to methods known in the art as an effective active ingredient phenylacetic acid compound. Any dosage form suitable for human or animal use can be prepared by combining the effective active ingredient phenylacetic acid compound of the present invention with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants. The effective active ingredient phenylacetic acid compound of the present invention or the effective active ingredient phenylacetic acid compound containing it can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

[0039] The oral preparation is preferably a capsule. In order to prepare the dosing unit into a capsule, the active ingredient, the phenylacetic acid compound of the present invention, can be mixed with a diluent and a glidant, and the mixture can be directly placed in a hard capsule or a soft capsule. The active ingredient, the phenylacetic acid compound of the present invention, can also be first made into granules or pellets with a diluent, a binder, and a disintegrant, and then placed in a hard capsule or a soft capsule. The diluents, binders, wetting agents, disintegrants, and glidants used to prepare the tablets of the active ingredient phenylacetic acid compound of the present invention can also be used to prepare the capsules of the active ingredient phenylacetic acid compound of the present invention.

[0040] In order to prepare the effective active ingredient phenylacetic acid compounds of the present invention into injections, water, ethanol, isopropanol, propylene glycol or a mixture thereof can be used as a solvent and an appropriate amount of a solubilizer, a cosolvent, a pH adjuster, and an osmotic pressure regulator commonly used in the art can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH adjuster can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. If a lyophilized powder injection is prepared, mannitol, glucose, etc. can also be added as a support agent.

[0041] Example 1 Acremonium sclerotigenum C2F21

[0042] Acremonium sclerotigenum C2F21 was isolated from Pocillopora damicornis collected from Weizhou Island, Guangxi, China, and was deposited in Guangdong Microbiological Culture Collection Center (GDMCC) on May 20, 2019. The address is: 5th Floor, Building 59, Guangdong Institute of Microbiology, No. 100, Xianlie Middle Road, Guangzhou, Guangdong, Guangdong, Guangdong Institute of Microbiology, with the deposit number: GDMCC No.60670.

[0043] Example 2 Preparation and Isolation of Phenylacetic Acid Compound 4-butoxy-benzeneacetic Acid

[0044] 1. Cultivation

[0045] 1.1 Seed culture medium: Each liter of culture medium contains 15g malt extract powder, 20g sodium bromide, and the balance is water, pH 7.5. Mix the above components and contents evenly, then sterilize at 121℃ for 30 minutes for later use.

[0046] 1.2. Fermentation medium: Each 1L triangular flask medium contains: 130g rice, 3g sodium bromide, 150mL water, pH 7.5. Mix the above components and contents evenly, then sterilize at 121℃ for 30min for later use.

[0047] 2. Fermentation

[0048] 2.1. Seed culture: The activated Acremonium sclerotigenum C2F21 was inoculated into 1 L triangular culture bottles each containing 300 mL of seed culture medium, and cultured at 25° C., 180 rpm, for 72 h to obtain seed solution.

[0049] 2.2. Fermentation culture: The seed liquid was inoculated into 120 bottles of fermentation medium triangle flasks at an inoculum amount of 5% (volume percentage), and statically cultured at 25° C. for 100 days to obtain a fermentation culture.

[0050] 3. Extraction: Soak the fermentation culture with ethyl acetate, cut the fermentation culture into small pieces, extract by ultrasonication for 15 minutes, filter with a Buchner funnel, and concentrate the filtrate by distillation to obtain extract A; continue to extract the filter residue with ethyl acetate for 3 times, and obtain extract B after distillation and concentration. Combine extract A and extract B to obtain the total extract (100g).

[0051] 4. Separation and purification of phenylacetic acid compound 4-butoxy-benzeneacetic acid

[0052] The crude extract (100 g) of extract A and extract B was subjected to medium pressure normal phase column chromatography (MPLC) with petroleum ether / dichloromethane as the eluent, and gradient elution was performed from a volume ratio of 100:0 to 0:100. The fraction eluted with a volume ratio of petroleum ether / dichloromethane of 80:20 (2.5 g) was collected and then subjected to medium pressure reverse phase C 18Column chromatography was performed using methanol / water as the eluent, with gradient elution from a volume ratio of 10:90 to 100:0, and the fractions eluted with a methanol / water volume ratio of 58:42 were collected. The fractions were finally finely separated by semi-preparative high performance liquid chromatography, and purified using an elution system of methanol / water (volume ratio of 58:42, with 0.3% trifluoroacetic acid added, YMC-pack ODS-A chromatographic column, 10×250mm, 5μm, 2mL / min) to obtain a phenylacetic acid compound 4-butoxy-benzeneacetic acid (46mg).

[0053] Example 3 Structural Identification of Phenylacetic Acid Compound 4-butoxy-benzeneacetic Acid

[0054] Structural identification: 4-butoxy-benzeneacetic acid is a pale yellow oil. NMR data showed that it was a para-substituted phenylacetic acid compound, including a triplet methyl group (δ H / C 0.91 / 13.8), 4 methylene groups (δ H / C 3.54 / 40.7, 4.10 / 65.1, 1.60 / 30.7, 1.36 / 19.2), 4 aromatic methines (δ H / C 7.10 / 130.5, 6.73 / 115.7), 2 aromatic quaternary carbons (δ C 155.1,125.9), and 1 carboxyl group (δ C 173.1). Data attributed to: 1 HNMR (700MHz,CDCl3):δ H 3.54(2H,s,H2-2),7.10(2H,d,J=8.4Hz,H-4,8),6.73(2H,d,J=8.4Hz,H-5,7),4.10(2H,t ,J=7.0Hz,H2-1′),1.60(2H,m,H2-2′),1.36(2H,m,H2-3′),0.91(3H,t,J=7.0Hz,H3-4′). 13 C NMR (175MHz,CDCl3):δ C173.1(C,C-1),40.7(CH2,C-2),125.9(C,C-3),130.5(CH,C-4,8),115.7(CH,C-5,7),155.1(C,C-6),65.1(CH2,C-1′),30.7(CH2,C-2′),19.2(CH2,C-3′),13.8(CH3,C-4′). Based on the data reported in [Journal of Sun Yat-sen University (Natural Science Edition), 2009,48(3):136-138], the compound was identified as 4-butoxy-benzeneacetic acid.

[0055] Example 4 Determination of the inhibitory activity of 4-butoxy-benzeneacetic acid on LPS-induced NF-κB luciferase

[0056] The main reference for NF-κB luciferase inhibitory activity assay is (British Journal of Pharmacology, 2020, 177: 4242–4260).

[0057] RAW264.7 cells stably transfected with NF-κB luciferase reporter gene were seeded in 96-well plates (1×10 4 / well), add 200 μL of DMEM medium containing 10% fetal bovine serum, 100 IU / mL penicillin and streptomycin and 0.1 μg / mL G418 to each well, add phenylacetic acid compound 4-butoxy-benzeneacetic acid after the cells are stably attached to the wall, and set up 6 duplicate wells. After incubation for 4 hours, except for the negative control group, each compound group (3 wells) and the positive control group (NF-κB inhibitor, BAY11-7082, 5 μM) were added with LPS and RANKL, respectively, so that the final concentration of each well was 100 ng / mL. After 8 hours of stimulation, the supernatant was discarded, 25 μL of cell lysis solution was added to each well, and the cells were fully lysed by low-speed shaking for 10 minutes, and then 20 μL was transferred to the white plate, and 50 μL of fluorescein solution was added to each well, and the Luciferase value was detected by a multifunctional microplate reader.

[0058] Conclusion: The study found that compared with the LPS blank group, the phenylacetic acid compound 4-butoxy-benzeneacetic acid at 10μM had a significant inhibitory effect on LPS-induced NF-κB luciferase (p<0.001)( Figure 1 ), the activity was equivalent to that of the positive control.

[0059] Example 5 Cytotoxic effects of phenylacetic acid compound 4-butoxy-benzeneacetic acid on osteoclast precursor RAW264.7 cells and BMMs

[0060] Main references for cell viability assay (British Journal of Pharmacology, 2020, 177: 4242–4260)

[0061] (1) MTT assay to detect the effect of irinotecan on the survival of osteoclast precursor RAW264.7 cells

[0062] RAW264.7 cells (1×10 3 100 μL of 4-butoxy-benzeneacetic acid were added to each well of the 96-well plate, and DMEM medium (containing 10% fetal bovine serum, 100 IU / mL penicillin and 100 IU / mL streptomycin) was added to each well to 200 μL, and incubated overnight at 37°C and 5% CO2. After the cells were stably attached to the wall, different concentrations of irinotecan were added to make the final concentration of the phenylacetic acid compound 4-butoxy-benzeneacetic acid in the wells 1 μM, 3 μM, and 6 μM. Three replicate wells were set up in each group and incubated for 4 days. After the incubation was completed, the supernatant was discarded, and 100 μL of 0.5 mg / mL MTT was added to each well, and the cells were incubated for 4 hours at 37°C and 5% CO2. After the incubation was completed, the supernatant was discarded, 150 μL of DMSO solution was added to each well, and the cells were shaken on a micro-oscillator for 15 minutes. The optical density value (OD value) at a wavelength of 570 nm was measured using a TECANGENiosPro multifunctional microplate reader to calculate the cell survival rate of each group.

[0063] The results are as follows Figure 2 After adding 1μM, 3μM, and 6μM of phenylacetic acid compound 4-butoxy-benzeneacetic acid, there was no significant difference in the survival rate of osteoclast precursor RAW264.7 cells, indicating that in in vitro experiments, phenylacetic acid compound 4-butoxy-benzeneacetic acid has no cytotoxicity to RAW264.7 cells.

[0064] (2) CCK-8 method to detect the effect of 4-butoxy-benzeneacetic acid on the survival of mouse bone marrow macrophages (BMMs)

[0065] a. Preparation of mouse bone marrow macrophages (BMMs): Under sterile conditions, take the femur of 8-12 week old C57BL / 6 female mice, cut the joints at both ends of the femur, and repeatedly rinse the femur with phenol red-free α-MEM medium (containing 10% fetal bovine serum, 100 IU / mL penicillin and 100 IU / mL streptomycin) until the femoral cavity turns white. Place the flushed femoral bone marrow cavity cells in a 37°C, 5% CO2 cell culture incubator for 2 hours, aspirate the supernatant, lyse the red blood cells with lysis buffer, centrifuge, and resuspend to obtain BMMs.

[0066] b. CCK-8 method to detect cell survival:

[0067] Take the BMMs (1×10 5 100 μL / well) of cells were placed in a 96-well plate, 200 μL of phenol red-free α-MEM medium (containing 10% fetal bovine serum, 100 IU / mL penicillin and 100 IU / mL streptomycin) was added to each well, and macrophage colony stimulating factor (M-CSF, final concentration of 50 ng / mL) was added to each well, and then the 96-well plate was placed in a cell culture incubator at 37°C and 5% CO2 for incubation overnight. After the cells were stably attached to the wall, different concentrations of phenylacetic acid compound 4-butoxy-benzeneacetic acid were added respectively, so that the final concentration of phenylacetic acid compound 4-butoxy-benzeneacetic acid in the well was 1 μM, 3 μM, and 6 μM, and 3 replicate wells were set for each group, and incubation was carried out for 4 days. After incubation, discard the supernatant (100 μL), add 5 μL of CCK-8 reagent (Cell Counting Kit-8 cell counting reagent) to each well, shake well, and continue incubation at 37°C, 5% CO2 for 3 hours. Use TECANGENiosPro multi-function microplate reader to measure the optical density value (OD value) at a wavelength of 450 nm, and calculate the cell survival rate of each group.

[0068] The results are as follows Figure 2 As shown, after adding 1 μM, 3 μM, and 6 μM of the phenylacetic acid compound 4-butoxy-benzeneacetic acid, there was no significant difference in the survival rate of BMMs cells, indicating that the phenylacetic acid compound 4-butoxy-benzeneacetic acid had no cytotoxicity to BMMs cells in in vitro experiments.

[0069] Example 6 Effect of 4-butoxy-benzeneacetic acid, a phenylacetic acid compound, on RANKL-induced differentiation of osteoclast precursors RAW264.7 and BMMs cells into osteoclasts

[0070] The main reference for the determination of RANKL-induced osteoclast precursor BMMs cell differentiation inhibitory activity is (British Journal of Pharmacology, 2020, 177: 4242–4260).

[0071] (1) Effect of 4-butoxy-benzeneacetic acid on the differentiation of RAW264.7 cells into osteoclasts

[0072] RAW264.7 cells (1×10 3 / well) were inoculated in a 96-well plate and incubated overnight at 37°C and 5% CO2. After the cells were stably attached to the wall, different concentrations of phenylacetic acid compound 4-butoxy-benzeneacetic acid were added to make the final concentration of phenylacetic acid compound 4-butoxy-benzeneacetic acid in the wells 1μM, 3μM and 6μM, with 3 replicate wells in each group, and incubated for 4h. RANKL (final concentration of 100ng / mL) was added, and the solution was changed every two days for 4-5d. TRAP staining was performed on the incubated cells, and the cells were photographed and counted under an inverted microscope. TRAP-positive cells with more than 3 nuclei were osteoclasts.

[0073] The results are as follows Figure 3 As shown, the phenylacetic acid compound 4-butoxy-benzeneacetic acid can exert an inhibitory effect at an effective concentration of 3 μM, and when the effective concentration of 4-butoxy-benzeneacetic acid reaches 6 μM, it can significantly inhibit RANKL-induced osteoclastogenesis from RAW264.7 osteoclast precursor cells.

[0074] (2) Effect of 4-butoxy-benzeneacetic acid on differentiation of BMMs into osteoclasts

[0075] Take the BMMs (2×10 4100 μL / well) in a 96-well plate, add phenol red-free α-MEM medium to each well to 200 μL (containing 10% fetal bovine serum, 100 IU / mL penicillin and 100 IU / mL streptomycin), and add macrophage colony stimulating factor (M-CSF, final concentration of 50 ng / mL) to each well, and then place the 96-well plate in a cell culture incubator at 37°C and 5% CO2 for incubation overnight. After the cells are stably attached to the wall, different concentrations of phenylacetic acid compound 4-butoxy-benzeneacetic acid are added respectively, so that the final concentration of phenylacetic acid compound 4-butoxy-benzeneacetic acid in the well is 1 μM, 3 μM and 6 μM, and 3 replicate wells are set for each group, and incubated for 4 hours. After the incubation is completed, RANKL (final concentration of 100 ng / mL) is added and cultured for 3-4 days. The incubated cells were stained with TRAP, photographed and counted under an inverted microscope, and TRAP-positive cells with more than 5 nuclei were osteoclasts.

[0076] The results are as follows Figure 4 As shown, 4-butoxy-benzeneacetic acid, a phenylacetic acid compound, can significantly inhibit RANKL-induced osteoclastogenesis in BMMs. At an effective concentration of 3 μM, RANKL-induced osteoclastogenesis in BMMs osteoclast precursor cells can be significantly inhibited.

[0077] In summary, the phenylacetic acid compound 4-butoxy-benzeneacetic acid can significantly inhibit the formation and activation of osteoclasts in vitro without obvious cytotoxicity.

[0078] Example 7 Effects of 4-butoxy-benzeneacetic acid, a phenylacetic acid compound, on Micro-CT bone destruction, bone parameters, and expression of osteoclast-related indices in osteoclast-related ovariectomized osteoporotic mice

[0079] Thirty female 10-week-old C57BL / 6 mice were randomly divided into two groups: sham operation group (n=6) and ovariectomy group (n=24). The mice were anesthetized by intraperitoneal injection of 10% chloral hydrate solution at a dose of 80 μL / mouse. The ovaries were completely removed from the dorsal side and hemostasis was sutured. Only a small amount of fat tissue was removed in the sham operation group. After surgery, 20,000 U / kg of penicillin saline was injected intramuscularly for 3 consecutive days. After feeding for one week, the ovariectomized mice were randomly divided into 4 groups. (1) Sham operation group (sham group): administered with an equal amount of saline for injection; (2) Ovariectomized mouse group (OVX+Vehicle group or OVX group for short): administered with an equal amount of saline for injection; (3) Phenylacetic acid compound 4-butoxy-benzeneacetic acid 5 mg / kg group (OVX+BBA 5 mg / kg group): administered with 4-butoxy-benzeneacetic acid 5 mg / kg by gavage; (4) Phenylacetic acid compound 4-butoxy-benzeneacetic acid 10 mg / kg group (OVX+BBA 10 mg / kg group): administered with 4-butoxy-benzeneacetic acid 10 mg / kg by gavage; (5) Positive drug control group (OVX+estradiol 0.02 mg / kg group or Ez 0.02 mg / kg group for short): administered with estradiol 0.02 mg / kg by subcutaneous injection. The positive drug and drug groups were administered once a day. After 12 consecutive weeks of administration. Eyeballs were removed to collect blood and serum samples were prepared. After killing, the right hind limb of the mouse was taken and immersed in formalin. CT scanning, 3D modeling and related bone parameter analysis were performed using a ZKKS small animal Micro-CT scanner. The left hind limb of the mouse was fixed, embedded, and sliced ​​for HE staining and TRAP osteoclast staining.

[0080] By analyzing the imaging characteristics of mice through Micro-CT, we can see that the phenylacetic acid compound 4-butoxy-benzeneacetic acid can reverse the degree of osteoporotic bone destruction caused by ovariectomy and significantly increase the bone mass of the mouse femur ( Figure 5 ), the results of quantitative analysis of mouse femoral bone parameters also showed that 4-butoxy-benzeneacetic acid significantly increased the thickness of the femur connection ConnD and the thickness of the trabecular bone Th.Tb, and reduced the area of ​​bone tissue per unit volume BS / BV. In particular, high doses of 4-butoxy-benzeneacetic acid showed consistent effects with the positive control estradiol drug ( Figure 6 ).

[0081] HE staining results also further confirmed the protective effect of phenylacetic acid compound 4-butoxy-benzeneacetic acid on osteoporotic bone destruction caused by ovariectomy ( Figure 7 ), reducing the area of ​​bone depression formed by osteoclasts on the bone slices. In addition, the number of osteoclasts in the ovariectomy model group increased significantly ( Figure 7 ), while the number of osteoclasts in both the high-dose and low-dose groups of phenylacetic acid compounds was significantly reduced, especially in the high-dose group, where the number of osteoclasts was comparable to that in the positive control group ( Figure 7 and Figure 8 ). Therefore, both static and dynamic parameters confirmed that the phenylacetic acid compound 4-butoxy-benzeneacetic acid has a significant protective effect on bone destruction in ovariectomized osteoporotic mice by inhibiting osteoclasts in vivo.

[0082] Results and discussion: 4-butoxy-benzeneacetic acid, a phenylacetic acid compound, significantly inhibited LPS-induced NF-κB luciferase and significantly inhibited RANKL-induced differentiation of osteoclast precursors RAW264.7 cells and BMMs cells into osteoclasts at a concentration of 6 μM without obvious toxicity. It also had a protective effect on bone destruction in ovariectomized osteoporotic mice by inhibiting osteoclasts in vivo, and could effectively replace existing bisphosphonates and denoximab to inhibit the bone resorption activity of osteoclasts. It can be developed as a new osteoclast differentiation inhibitor or NF-κB nuclear factor expression inhibitor for the prevention and treatment of osteolytic diseases such as osteoporosis.

[0083] In summary, the present invention provides new candidate compounds for the development of novel osteoclast differentiation inhibitors or NF-κB nuclear factor expression inhibitors, which is of great significance to the development of new drugs with independent intellectual property rights in China.

[0084] The above description is a detailed description of the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the patent application of the present invention. All equivalent changes or modifications completed under the technical concept suggested by the present invention should fall within the scope of the patent covered by the present invention.

Claims

1. Use of a phenylacetic acid compound in the preparation of an osteoclast differentiation inhibitor, wherein the phenylacetic acid compound has a structural formula as shown in formula (I), and is named 4-butoxy-benzeneacetic acid: Formula (I); The osteoclast differentiation inhibitor is a drug for treating osteoporosis.

2. The use according to claim 1, characterized in that: The osteoclast differentiation inhibitor comprises an effective amount of a phenylacetic acid compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.

3. A method for preparing the phenylacetic acid compound described in claim 1, characterized in that: The phenylacetic acid compound is obtained from Acremonium sclerotiorum ( Acremonium sclerotigenum ) is prepared and isolated from the fermentation culture of C2F21; the sclerotium-producing Acremonium ( Acremonium sclerotigenum )C2F21 deposit number: GDMCCNO. 60670; The method for preparing phenylacetic acid compounds comprises the following specific steps: a) Preparation of Acremonium sclerotiorum ( Acremonium sclerotigenum ) C2F21 fermentation culture, soak the fermentation product with ethyl acetate, cut the fermentation culture into small pieces, extract with ultrasonic for 15 min, filter with Buchner funnel, distill and concentrate the filtrate to obtain extract A; continue to extract with ethyl acetate for 3 times, distill and concentrate to obtain extract B; The sclerotinia Acremonium ( Acremonium sclerotigenum ) The fermentation culture of C2F21 is activated Acremonium sclerotiorum ( Acremonium sclerotigenum ) C2F21 was inoculated into a seed culture medium, and dynamically cultured at 25°C, 180 rpm for 72 h to obtain a seed solution, and the seed solution was inoculated into a fermentation culture medium at a 5% inoculation rate, and statically cultured at 25°C for 100 days to obtain a fermentation culture; the seed culture medium formula is that per 1L of culture medium contains: 15 g of malt extract powder, 20 g of sodium bromide, and the balance is water, pH 7.5; the fermentation culture medium formula is that per 1L of flask culture medium contains: 130 g of rice, 3 g of sodium bromide, 150 mL of water, pH 7.5; b) The crude extract of extract A and extract B was subjected to medium pressure normal phase liquid chromatography, using petroleum ether / dichloromethane as the eluent, and gradient elution was performed from a volume ratio of (100:0) to (0:100). The fractions eluted with a volume ratio of petroleum ether / dichloromethane of 80:20 were collected and continued to be subjected to medium pressure reverse phase C 18 Column chromatography was performed using methanol / water as the eluent, with gradient elution from a volume ratio of (10:90) to (100:0), and fractions eluted with a volume ratio of methanol / water of 58:42 were collected. The collected fractions were purified to obtain phenylacetic acid compounds.

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