CN115813900A - Application of phenylacetic acid compound in preparation of osteoclast differentiation inhibitor - Google Patents

Abstract

The invention provides the application 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, whose structural formula is shown in formula (I), named 4-butoxy-benzoneacetic acid, which has a significant inhibitory effect on LPS-induced NF-κB luciferase at a concentration of 10 μM, and at a concentration of 6 μM It can significantly inhibit RANKL-induced differentiation of osteoclast precursor cells RAW264.7 cells and BMMs cells into osteoclasts, and inhibit osteoclasts in vivo to protect bone damage in mice with osteoporosis caused by ovariectomy, which can effectively It can replace the existing bisphosphonate drugs and denoximab, inhibit the bone resorption activity of osteoclasts, and is an ideal candidate compound for the development of new osteoclast differentiation inhibitor drugs.

.

Description

Application of Phenylacetic Acid Compounds in the Preparation of Osteoclast Differentiation Inhibitors

technical field

The invention relates to the field of marine natural products, in particular to the application of phenylacetic acid compounds in the preparation of osteoclast differentiation inhibitors.

Background technique

Osteoporosis is a common bone degenerative disease, mainly manifested as increased bone fragility, 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, and are the only cells capable of bone resorption in the human body. Deficiency in osteoclast activity leads to bone sclerosis and bone marrow failure. Excessive activation can lead to osteolytic diseases such as osteoporosis, rheumatoid arthritis, and tumor bone metastasis. Inhibiting the formation and resorption of OCs is the key to the treatment of osteoporosis. one of the main strategies. Marine microorganisms are a new ideal resource for discovering new drug-derived molecules. There are few reports on anti-osteoclast differentiation inhibitors of marine microbial natural products, so their anti-osteoclast differentiation potential needs to be further explored.

Osteoclast formation is a stepwise process, resulting from the binding of the receptor activator of nuclear factor-κB receptor activator of nuclear appaB ligand (RANKL) to its receptor RANK on monocyte/macrophage precursors And start. The currently clinically used osteoclast differentiation-related inhibitors are mainly denoximab and bisphosphonates, but both have certain complications and side effects. Therefore, there is an urgent need to find a safe, effective, quality-controllable, and economical drug that targets osteoclast formation and bone resorption, and effectively solves the huge clinical demand for osteoclast differentiation inhibitors.

Contents of the invention

The present invention aims at the above problems and provides a new application of phenylacetic acid compound (named 4-butoxy-benzoneacetic acid).

In order to achieve the above object, the technical scheme adopted in the present invention is:

The application of the phenylacetic acid compound of the present invention in the preparation of an osteoclast differentiation inhibitor, the structural formula of the phenylacetic acid compound is as shown in formula (I), which is named 4-butoxy-benzoneacetic acid:

In the present invention, it is further explained that the osteoclast differentiation inhibitor is a drug for treating osteolytic diseases caused by overactivation of osteoclasts.

In the present invention, it is further explained that the osteoclast differentiation inhibitor is a drug for treating osteoporosis, rheumatoid arthritis, tumor metastasis and bone destruction.

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

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

In the present invention, it is further explained that the osteoclast differentiation inhibitor or NF-κB nuclear factor expression inhibitor drug is in the form of oral preparation, injection or external preparation.

The invention also provides a method for preparing phenylacetic acid compounds, which are prepared and isolated from the fermentation culture of Acremonium sclerotigenum C2F21.

In the present invention, it is further illustrated that the described method for preparing phenylacetic acid compounds, the specific steps are as follows:

a), prepare the fermentation culture of Acremonium sclerotigenum (Acremonium sclerotigenum) C2F21, soak the fermentation culture with ethyl acetate, cut the fermented product into small pieces, ultrasonically extract for 15min, filter with Buchner funnel, and distill the filtrate Extraction A was obtained after concentration; the filter residue was extracted three times with ethyl acetate, and extract B was obtained after distillation and concentration;

b) The combined crude extracts of Extract A and Extract B were subjected to medium-pressure normal phase liquid chromatography, using petroleum ether/dichloromethane as eluent, and gradient washing was carried out from a volume ratio of 100:0 to 0:100. Collect the fractions eluted with petroleum ether/dichloromethane gradient at a volume ratio of 80:20, and continue to pass through medium-pressure reversed-phase C ₁₈ column chromatography, using methanol/water as the eluent, from a volume ratio of 10:90 to 100 :0 for gradient elution, collect the fractions eluted from the methanol/water volume ratio of 58:42 gradient, collect the fractions and obtain phenylacetic acid compounds after purification.

In the present invention, it is further illustrated that the preparation of the fermentation culture of Acremonium sclerotigenum C2F21 in the step a) is to insert activated Acremonium sclerotigenum C2F21 into the seed medium , 25° C., 180 rpm, cultivated for 72 hours to obtain a seed liquid, and inserted the seed liquid into a fermentation medium with an inoculation amount of 5%, and cultured it statically for 100 days at 25° C. to obtain a fermentation culture.

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

In the present invention, it is further explained that the formula of the fermentation medium is as follows: 130 g of rice, 3 g of sodium bromide, 150 mL of water, and pH 7.5 per 1 L of Erlenmeyer flask culture medium.

The present invention also provides the application of Acremonium sclerotigenum C2F21 in the preparation of phenylacetic acid compounds.

The present invention obtains through tests: phenylacetic acid compound 4-butoxy-benzoneacetic 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. Lead compounds of NF-κB nuclear factor expression inhibitors.

The present invention obtains through experiments: phenylacetic acid compound 4-butoxy-benzoneacetic acid dose-dependently significantly inhibits RANKL-induced osteoclast precursor cells RAW264.7 (mouse mononuclear macrophages) and BMMs (Bone Marrow macrophage cells (bone marrow macrophages) differentiate into osteoclasts without obvious cytotoxicity, so it is expected to be developed as a safe and effective new osteoclast differentiation inhibitor drug.

The present invention obtains through experiments: phenylacetic acid compound 4-butoxy-benzoneacetic acid has a significant protective effect on bone loss in ovariectomized osteoporosis mice, a classic model of bone destruction caused by osteoclast activation, and reduces osteoclasts in bone. The bone depression area formed on the tablet inhibits the bone resorption activity of osteoclasts, and has the advantages of simple structure, convenient taking, cost-effectiveness and the like. Therefore, it can effectively replace the existing bisphosphonates and denoximab.

One object of the present invention is to provide the application of phenylacetic acid compound 4-butoxy-benzoneacetic acid or its pharmaceutically acceptable salt in the preparation of NF-κB nuclear factor expression inhibitor or osteoclast differentiation inhibitor drug. The anti-osteoclast differentiation drugs are drugs for the treatment of clinical indications of bisphosphonates and denosimab, and are used to prevent and treat osteolytic diseases such as osteoporosis, rheumatoid arthritis, and bone destruction caused by tumor metastasis.

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

Owing to adopting above-mentioned technical scheme, the present invention has following beneficial effect:

The present invention isolates and obtains a phenylacetic acid compound 4- butoxy-benzoneacetic acid, which has a significant inhibitory effect on LPS-induced NF-κB luciferase, significantly inhibits RANKL-induced differentiation of osteoclast precursor RAW264.7 and BMMs cells into osteoclasts in a dose-dependent manner, without producing cells Toxicity, the classical model of osteoclast activation leading to bone destruction in vivo can significantly improve the bone loss of ovariectomized osteoporotic mice, reduce the area of bone depressions formed by osteoclasts on bone slices, and inhibit the bone resorption of osteoclasts active. Therefore, it is an ideal candidate compound to be developed as a novel NF-κB nuclear factor expression inhibitor or an osteoclast differentiation inhibitor drug.

Description of drawings

Figure 1 is a comparison chart of the inhibitory activity of phenylacetic acid compound 4-butoxy-benzoneacetic acid (BBA) at a concentration of 10 μM on NF-κB luciferase induced by lipopolysaccharide (LPS) in RAW264.7 cells, BAY is positive Control, ^### means p<0.001vs.control group; *** means p<0.001vs.LPS group;

Figure 2 is the effect of phenylacetic acid compound 4-butoxy-benzoneacetic acid on the cell viability of RAW264.7 and BMMs cells;

Figure 3 is the experimental results of the effect of phenylacetic acid compound 4-butoxy-benzoneacetic acid (BBA) on the differentiation of osteoclast precursor RAW264.7 cells into osteoclasts, in which: RANKL is the activation of nuclear factor NF-κB receptor Ligand, compared with blank control group, ###P<0.001; compared with RANKL group, *P<0.05;

Figure 4 is the experimental results of the effect of phenylacetic acid compound 4-butoxy-benzoneacetic acid (BBA) on the differentiation of mouse bone marrow macrophages (BMMs) into osteoclasts, wherein: RANKL is the activation of nuclear factor NF-κB receptor M-CSF is macrophage colony-stimulating factor, compared with blank control group, ^### P<0.001; compared with RANKL group, *P<0.05**P<0.01;

Fig. 5 is the Micro-CT scan result of phenylacetic acid compound 4-butoxy-benzoneacetic acid (abbreviated as BBA) on the femur of ovariectomized osteoporosis mice, wherein: Sham is sham operation, OVX is ovariectomy, 5mg/kg ( Low dose) and 10mg/kg (high dose) are the dosage of phenylacetic acid compounds, and Ez is the positive drug estradiol;

Figure 6 is the statistical analysis results of the bone parameters of the phenylacetic acid compound 4-butoxy-benzoneacetic acid (BBA) on the femur of ovariectomized osteoporosis mice, wherein: Sham is a sham operation, OVX is ovariectomy, and the phenylacetic acid compound The high and low dosages are 10mg/kg and 5mg/kg respectively, Ez is the positive drug estradiol; ConnD represents the connection density of trabecular bone, BS/BV represents the area of bone tissue per unit volume, and Tb/Th represents the bone trabecular thickness;

Figure 7 shows the HE and TRAP staining results of phenylacetic acid compound 4-butoxy-benzoneacetic acid on femur sections of ovariectomized osteoporosis mice, in which Sham is the sham operation group, OVX is the ovariectomized osteoporosis model group, OVX+5mg /kg and OVX+10mg/kg are the low and high dose groups of phenylacetic acid compounds respectively, and Ez is the positive drug estradiol group;

Figure 8 is the quantitative analysis results of the number of osteoclasts per unit bone area and the surface area of osteoclasts in the femur slices of ovariectomized osteoporotic mice with phenylacetic acid compound 4-butoxy-benzoneacetic acid (BBA for short), wherein: Sham is false Surgery, OVX is the ovariectomized osteoporosis model group, OVX+5mg/kg and OVX+10mg/kg are the low-dose and high-dose phenylacetic acid compound groups, respectively, and Ez is the positive drug estradiol group.

Detailed ways

In order to make the object, technical solution and technical effect of the present invention clearer, the present invention will be further described in detail below in conjunction with the embodiments and accompanying drawings. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

The invention relates to the application of phenylacetic acid compound 4-butoxy-benzoneacetic acid in the preparation of 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, injection or external dosage form; it includes the active ingredient phenylacetic acid compound 4-butoxy-benzoneacetic acid and medically acceptable medicinal Accessories. Anti-osteoclast differentiation drugs can be used to treat clinical conditions including but not limited to: postmenopausal osteoporosis, tumor metastases and bone destruction, where known osteoclast inhibitors such as bisphosphonates and denoximab are approved indications.

Generally speaking, as a drug, it is used clinically after being prepared into a preparation. The effective active ingredient phenylacetic acid compounds in the present invention can be prepared according to methods known in the art as the effective active ingredient phenylacetic acid compounds. Any dosage form suitable for human or animal can be prepared by combining the 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 the form of a unit dose, and the route of administration can be enteral or parenteral, such as oral administration, intravenous injection, intramuscular injection, subcutaneous injection , nasal cavity, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

Oral formulations are preferably capsules. In order to make the administration unit into a capsule, the active ingredient phenylacetic acid compound of the present invention can be mixed with a diluent and a glidant, and the mixture is directly placed in a hard capsule or a soft capsule. The active ingredient phenylacetic acid compound of the present invention can also be made into granules or pellets with diluent, binder, and disintegrating agent, and then placed in hard capsules or soft capsules. Various diluents, binders, wetting agents, disintegrants, and glidants used to prepare the active ingredient phenylacetic acid compound tablets of the present invention can also be used to prepare capsules of the active active ingredient phenylacetic acid compound tablets of the present invention .

In order to make the effective active ingredient phenylacetic acid compound of the present invention into an injection, water, ethanol, isopropanol, propylene glycol or their mixture can be used as a solvent and an appropriate amount of commonly used solubilizers, cosolvents, pH regulators, osmotic pressure Conditioner. The solubilizer or co-solvent can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be Sodium chloride, mannitol, glucose, phosphate, acetate, etc. For preparation of freeze-dried powder injection, mannitol, glucose, etc. can also be added as proppants.

Embodiment 1 Sclerotinia acremonium (Acremonium sclerotigenum) C2F21

Acremonium sclerotigenum C2F21 was isolated from Pocillopora damicornis collected from Weizhou Island, Guangxi, China, and was deposited in Guangdong Microbial Culture Collection Center (GDMCC) on May 20, 2019 , Address: 5th Floor, Building 59, Guangdong Institute of Microbiology, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Guangdong Institute of Microbiology, deposit number: GDMCC No.60670.

Preparation and separation of embodiment 2 phenylacetic acid compound 4-butoxy-benzoneacetic acid

1. Cultivate

1.1. Seed culture medium: Each liter of culture medium contains 15g of malt extract powder, 20g of sodium bromide, the balance is water, pH7.5. Mix evenly according to the above-mentioned components and contents, and then sterilize at 121°C for 30 minutes for later use.

1.2. Fermentation medium: Each 1L Erlenmeyer flask medium contains: rice 130g, sodium bromide 3g, water 150mL, pH7.5. Mix evenly according to the above-mentioned components and contents, and then sterilize at 121°C for 30 minutes for later use.

2. Fermentation

2.1. Seed culture: Inoculate activated Acremonium sclerotigenum C2F21 into a 1L triangular culture flask containing 300 mL of seed culture medium in each bottle, culture at 25° C. and 180 rpm for 72 hours to obtain a seed solution.

2.2. Fermentation culture: the seed solution was inserted into 120 flasks of fermentation medium Erlenmeyer flasks with an inoculum amount of 5% (volume percentage), and cultured statically at 25° C. for 100 days to obtain a fermentation culture.

3. Extraction: Soak the fermentation culture with ethyl acetate, cut the fermentation culture into small pieces, extract by ultrasonic crushing for 15 minutes, filter with a Buchner funnel, and obtain the extract A after the filtrate is concentrated by distillation; continue to extract the filter residue with ethyl acetate 3 times, extract B was obtained after distillation and concentration, and extract A and extract B were combined to obtain the total extract (100g).

4. Separation and purification of phenylacetic acid compound 4-butoxy-benzoneacetic acid

The combined crude extract (100g) of Extract A and Extract B was subjected to medium-pressure normal phase column chromatography liquid chromatography (MPLC), using petroleum ether/dichloromethane as eluent, from a volume ratio of 100:0 to Gradient elution at 0:100, collect fractions (2.5g) eluted with a volume ratio of petroleum ether/dichloromethane of 80:20, and continue to pass through medium-pressure reversed-phase C ₁₈ column chromatography, using methanol/water as eluent , carry out gradient elution from the volume ratio of 10:90 to 100:0, and collect the fractions eluted from the methanol/water gradient ratio of 58:42. After purification of methanol/water (volume ratio 58:42, adding 0.3% trifluoroacetic acid, YMC-pack ODS-A column, 10×250mm, 5μm, 2mL/min), the phenylacetic acid compound 4-butoxy-benzoneacetic acid (46 mg).

Example 3 Structural identification of phenylacetic acid compound 4-butoxy-benzoneacetic acid

Structural identification: phenylacetic acid compound 4-butoxy-benzoneacetic acid is light yellow oil. The NMR data showed that it was a para-substituted phenylacetic acid compound, including 1 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 methine groups (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, CDCl ₃ ): δ _H 3.54(2H,s,H ₂ -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,H ₂ -1′),1.60(2H,m,H ₂ -2′),1.36(2H,m, H ₂ -3′), 0.91 (3H,t, J=7.0Hz, H ₃ -4′). ¹³ C NMR (175MHz, CDCl ₃ ): _δC 173.1(C,C-1), 40.7(CH ₂ ,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(CH ₂ , C-1′), 30.7 (CH ₂ , C-2′), 19.2 (CH ₂ , C-3′), 13.8 (CH ₃ , 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-benzoneacetic acid.

Example 4 Determination of the inhibitory activity of phenylacetic acid compound 4-butoxy-benzoneacetic acid on LPS-induced NF-κB luciferase

Main references for the determination of NF-κB luciferase inhibitory activity (British Journal of Pharmacology, 2020, 177:4242–4260).

RAW264.7 cells stably transfected with NF-κB luciferase reporter gene were inoculated in 96-well plates (1× ¹⁰⁴ /well), and each well was added with 10% fetal bovine serum, 100IU/mL penicillin and streptomycin Add 200 μL of DMEM medium with 0.1 μg/mL G418 and 0.1 μg/mL G418. After the cells adhere to the wall and stabilize, add phenylacetic acid compound 4-butoxy-benzoneacetic acid, and set up 6 replicate wells. After continuing to incubate for 4 hours, except the negative control group, each compound group (3 wells) and 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 100ng /mL, after stimulating the two for 8 hours, discard the supernatant, add 25 μL of cell lysate to each well, shake at low speed for 10 minutes to fully lyse the cells, then transfer 20 μL to a white plate, add 50 μL of fluorescein solution to each well, and use a multifunctional Microplate reader detects Luciferase value.

Experimental conclusion: the study found that compared with the LPS blank group, the phenylacetic acid compound 4-butoxy-benzoneacetic acid had a significant inhibitory effect on LPS-induced NF-κB luciferase at 10 μM (p<0.001) (Figure 1), and the activity was compared with the positive control quite.

Example 5 Effect of phenylacetic acid compound 4-butoxy-benzoneacetic acid on osteoclast precursor RAW264.7 cells and BMMs cytotoxicity

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

(1) MTT method was used to detect the effect of iquamycin on the survival of osteoclast precursor RAW264.7 cells

RAW264.7 cells (1×10 cells/ ^well ) in good growth state were inoculated into 96-well plates, and DMEM medium (containing 10% fetal bovine serum, 100IU/mL penicillin and 100IU/mL streptomycin) was added to each well. ) to 200 μL, and incubated overnight at 37°C, 5% CO ₂ . After the cells were firmly attached to the wall, different concentrations of ikamycin were added to make the final concentration of the phenylacetic acid compound 4-butoxy-benzoneacetic acid in the wells to be 1 μM, 3 μM, and 6 μM. Three replicate wells were set up in each group and incubated for 4 sky. After the incubation was completed, the supernatant was discarded, 100 μL of 0.5 mg/mL MTT was added to each well, and the incubation was continued for 4 h at 37° C. and 5% CO ₂ . After the incubation is complete, discard the supernatant, add 150 μL of DMSO solution to each well, shake on a micro shaker for 15 minutes, measure the optical density value (OD value) at 570 nm wavelength with TECANGENiosPro multi-functional microplate reader, and calculate the cell survival rate of each group .

The result is shown in Figure 2. After adding 1 μM, 3 μM, and 6 μM of phenylacetic acid compound 4-butoxy-benzoneacetic acid, there was no significant difference in the survival rate of osteoclast precursor RAW264.7 cells, indicating that in vitro experiments, phenylacetic acid compound 4-butoxy- Benzeneacetic acid has no cytotoxicity to RAW264.7 cells.

(2) CCK-8 method to detect the effect of phenylacetic acid compound 4-butoxy-benzoneacetic acid on the survival of mouse bone marrow macrophages (BMMs)

a. Preparation of mouse bone marrow macrophages (BMMs): Under sterile conditions, take femurs of 8-12-week-old C57BL/6 female mice, cut off the joints at both ends of the femur, and culture them with phenol red-free α-MEM Base (containing 10% fetal bovine serum, 100IU/mL penicillin and 100IU/mL streptomycin) repeatedly washed the femur until the femoral cavity turned white. The washed femoral bone marrow coelomocytes were incubated in a 37°C, 5% CO ₂ cell incubator for 2 hours, the supernatant was aspirated, red blood cells were lysed with a lysate, centrifuged, and resuspended to obtain BMMs.

b. CCK-8 method to detect cell survival:

Take the BMMs prepared in step a (1× ¹⁰⁵ /well) in a 96-well plate, add phenol red-free α-MEM medium to 200 μL (containing 10% fetal bovine serum, 100IU/mL penicillin and 100IU/mL mL streptomycin), and macrophage colony-stimulating factor (M-CSF, final concentration 50ng/mL) was added to each well, and then the 96-well plate was placed in a cell culture incubator at 37°C and 5% CO ₂ for overnight incubation . After the cells adhered stably, different concentrations of phenylacetic acid compound 4-butoxy-benzoneacetic acid were added to make the final concentration of phenylacetic acid compound 4-butoxy-benzoneacetic acid in the wells 1 μM, 3 μM, and 6 μM. 3 duplicate wells, incubated for 4 days. After the incubation was completed, 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 to incubate at 37°C and 5% CO2 for 3 h. TECANGENiosPro multifunctional microplate reader measures the optical density value (OD value) at the wavelength of 450nm, and calculates the cell survival rate of each group.

The results are shown in Figure 2. After adding 1 μM, 3 μM, and 6 μM of phenylacetic acid compound 4-butoxy-benzoneacetic acid, there was no significant difference in the survival rate of BMMs cells, indicating that in vitro experiments phenylacetic acid compound 4-butoxy- Benzeneacetic acid has no cytotoxicity to BMMs cells.

Example 6 Effect of phenylacetic acid compound 4-butoxy-benzoneacetic acid on RANKL-induced differentiation of osteoclast precursor RAW264.7 and BMMs cells into osteoclasts

Main references for the determination of RANKL-induced inhibitory activity of osteoclast precursor BMMs cell differentiation (British Journal of Pharmacology, 2020, 177:4242–4260).

(1) Effect of phenylacetic acid compound 4-butoxy-benzoneacetic acid on the differentiation of RAW264.7 cells into osteoclasts

RAW264.7 cells (1×10 ³ cells/well) in good growth state were inoculated into 96-well plates, and incubated overnight at 37° C. and 5% CO ₂ environment. After the cells were firmly attached to the wall, different concentrations of phenylacetic acid compound 4-butoxy-benzoneacetic acid were added to make the final concentration of phenylacetic acid compound 4-butoxy-benzoneacetic acid in the wells 1 μM, 3 μM and 6 μM, and 3 wells were set up in each group. Repeat wells and incubate for 4h. Add RANKL (final concentration is 100ng/mL), change the solution every two days, and culture for 4-5d. The incubated cells were stained with TRAP, photographed and counted under an inverted microscope, and TRAP-positive cells with more than 3 nuclei were osteoclasts.

The results are shown in Figure 3. The phenylacetic acid compound 4-butoxy-benzoneacetic 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 RAW264.7 osteoclastosis Precursor cells generate osteoclasts.

(2) Effect of phenylacetic acid compound 4-butoxy-benzoneacetic acid on the differentiation of BMMs into osteoclasts

Take the BMMs prepared in step a above (2× ¹⁰⁴ /well) in a 96-well plate, add phenol red-free α-MEM medium to 200 μL (containing 10% fetal bovine serum, 100IU/mL penicillin and 100IU /mL streptomycin), and macrophage colony-stimulating factor (M-CSF, final concentration 50ng/mL) was added to each well, and then the 96-well plate was placed in a cell culture incubator at 37°C and 5% CO ₂ for incubation. overnight. After the cells adhered stably, different concentrations of phenylacetic acid compound 4-butoxy-benzoneacetic acid were added respectively, so that the final concentration of phenylacetic acid compound 4-butoxy-benzoneacetic acid in the wells was 1 μM, 3 μM and 6 μM, and each group set 3 Multiple wells were incubated for 4 h. After incubation, add RANKL (final concentration: 100ng/mL) and culture for 3-4d. 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.

The results are shown in Figure 4, the phenylacetic acid compound 4-butoxy-benzoneacetic acid can significantly inhibit RANKL-induced osteoclast formation in BMMs. At an effective concentration of 3 μM, RANKL can significantly inhibit the induction of osteoclasts from BMMs osteoclast precursor cells.

Based on the above results, the phenylacetic acid compound 4-butoxy-benzoneacetic acid can significantly inhibit the formation and activation of osteoclasts in vitro without obvious cytotoxicity.

Example 7 Effects of phenylacetic acid compound 4-butoxy-benzoneacetic acid on the expression of Micro-CT bone destruction, bone parameters, and osteoclast-related indicators in osteoclast-associated ovariectomized osteoporosis mice

Thirty female 10-week-old C57BL/6 mice were randomly divided into two groups: 6 mice in the sham operation group and 24 mice in the ovariectomy group. The rats were anesthetized by intraperitoneal injection of 10% chloral hydrate solution at a dose of 80 μL/cause, inserted from the dorsal side, and the bilateral ovaries were completely removed, and sutured to stop bleeding. In the sham group, only a small amount of fat tissue was removed. After the operation, 20,000 U/kg of penicillin saline was intramuscularly injected for 3 consecutive days. After feeding for one week, the ovariectomized mice were randomly divided into 4 groups. (1) Sham operation group (sham group): give the same amount of normal saline for injection; (2) ovariectomized mouse group (OVX+Vehicle group or OVX group for short): give the same amount of normal saline for injection; (3 ) phenylacetic acid compound 4-butoxy-benzoneacetic acid 5mg/kg group (OVX+BBA 5mg/kg group): intragastric administration of 4-butoxy-benzeneacetic acid 5mg/kg; (4) phenylacetic acid compound 4-butoxy-benzoneacetic acid acid 10mg/kg group (OVX+BBA 10mg/kg group): intragastric administration of 4-butoxy-benzoneacetic acid 10mg/kg; (5) positive drug control group (OVX+estradiol 0.02mg/kg group or Ez 0.02mg for short /kg group): subcutaneous injection of 0.02 mg/kg of estradiol. The positive drug and drug group were administered once a day. After 12 weeks of continuous administration. Eyeballs were removed to collect blood, and serum samples were prepared. After sacrifice, the right hind limbs of the mice were taken, soaked in formalin, and used for CT scanning with ZKKS small animal Micro-CT scanner, 3D modeling and analysis of relevant bone parameters. The left hind limb of the mouse was fixed, embedded and sectioned for HE staining and TRAP osteoclast staining.

By Micro-CT analysis of the imaging characteristics of mice, we can see that the phenylacetic acid compound 4-butoxy-benzoneacetic 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 the quantitative analysis of mouse femoral bone parameters also show that phenylacetic acid compound 4-butoxy-benzoneacetic acid can significantly increase the connection thickness ConnD and trabecular thickness Th.Tb of the mouse femur, and the bone tissue per unit volume The area size BS/BV was reduced, especially the high dose of phenylacetic acid compound 4-butoxy-benzoneacetic acid showed a consistent effect with the positive control estradiol drug (Figure 6).

The results of HE staining further confirmed the protective effect of phenylacetic acid compound 4-butoxy-benzoneacetic acid on osteoporotic bone damage caused by ovariectomy (Fig. 7), reducing the area of bone depressions formed by osteoclasts on bone slices. The number of osteoclasts in the resection model group was significantly increased (Figure 7), while the number of osteoclasts in both the high-dose group and the low-dose group of phenylacetic acid compounds was significantly reduced, especially in the high-dose group, the number of osteoclasts Comparable to the positive control group (Figure 7 and Figure 8). Therefore, both static parameters and dynamic parameters have confirmed that phenylacetic acid compound 4-butoxy-benzoneacetic acid has a significant protective effect on bone destruction in ovariectomy-induced osteoporosis mice by inhibiting osteoclasts in vivo.

Discussion of the results: phenylacetic acid compound 4-butoxy-benzoneacetic acid has a significant inhibitory effect on LPS-induced NF-κB luciferase, and can significantly inhibit RANKL-induced osteoclast precursor RAW264.7 cells and BMMs cells at a concentration of 6 μM Differentiate into osteoclasts without obvious toxic effects. In vivo, by inhibiting osteoclasts, it has a protective effect on bone destruction in mice with osteoporosis caused by ovariectomy, and can effectively replace the existing bisphosphonate drugs and denoximab, and inhibit the bone resorption activity of osteoclasts. It can be developed as a new type of osteoclast differentiation inhibitor or NF-κB nuclear factor expression inhibitor to prevent and treat osteolytic diseases such as osteoporosis.

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

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

Claims (9)

1. The application of phenylacetic acid compounds in preparing osteoclast differentiation inhibitors is characterized in that the structural formula of the phenylacetic acid compounds is shown as a formula (I), and the phenylacetic acid compounds are named as 4-butoxy-bezeneacetic acid:

2. use according to claim 1, characterized in that: the osteoclast differentiation inhibitor is a medicament for treating osteolytic diseases caused by over-activation of osteoclasts.

3. Use according to claim 1, characterized in that: the osteoclast differentiation inhibitor is a medicament for treating osteoporosis, rheumatoid arthritis and tumor metastasis bone destruction.

4. Use according to claim 1, characterized in that: the phenylacetic acid compound can also be applied to the preparation of NF-kB nuclear factor expression inhibitor drugs.

5. The use according to claim 1, wherein the osteoclast differentiation inhibitor or NF- κ B nuclear factor expression inhibitor medicament comprises an effective amount of a phenylacetic acid compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or adjuvant.

6. An Acremonium sclerotiorum (Acremonium sclerotium) C2F21 with a deposit number of: GDMCC NO.60670.

7. A process for preparing a phenylacetic acid compound according to claim 1, wherein the phenylacetic acid compound is isolated from a fermentation culture of Acremonium sclerotiorum (Acremonium sclerotiogenum) C2F 21.

8. The method according to claim 7, wherein the method for preparing phenylacetic acid compounds comprises the following specific steps:

a) Preparing a fermentation culture of Acremonium sclerotiorum (Acremonium sclerogenum) C2F21, soaking the fermentation product in ethyl acetate, cutting the fermentation culture into small pieces, ultrasonically extracting for 15min, filtering by using a Buchner funnel, and distilling and concentrating the filtrate to obtain an extract A; extracting the filter residue with ethyl acetate for 3 times, and distilling and concentrating to obtain extract B;

b) And carrying out medium-pressure normal phase liquid chromatography on the combined crude extract of the extract A and the extract B, and using petroleum ether/dichloromethane as an eluent to obtain a mixture of the crude extract and the extract B in a volume ratio (100: 0) To (0: 100 Gradient elution is carried out), the petroleum ether/dichloromethane volume ratio is collected from 80:20 fractions eluted in a gradient are passed on to medium-pressure reverse phase C ₁₈ Column chromatography, gradient elution was performed from volume ratios (10: 42, collecting the flow fraction eluted in a gradient manner, and purifying the flow fraction to obtain the phenylacetic acid compound.

9. The method according to claim 8, wherein the fermentation culture of the Acremonium sclerotiorum (Acremonium sclerotium) C2F21 prepared in the step a) is prepared by inoculating the activated Acremonium sclerotiorum (Acremonium sclerotium) C2F21 into a seed culture medium, dynamically culturing at 25 ℃ and 180rpm for 72 hours to prepare a seed solution, inoculating the seed solution into the fermentation culture medium at a inoculation amount of 5%, and statically culturing at 25 ℃ for 100 days to prepare the fermentation culture; the formula of the seed culture medium is that each 1L of culture medium contains: 15g of malt extract powder, 20g of sodium bromide and the balance of water, wherein the pH value is 7.5;

the formula of the fermentation medium is that every 1L of the triangular flask culture medium contains: 130g of rice, 3g of sodium bromide, 150mL of water and 7.5 of pH.

Images