CN115990155A - Application of Bromoazone Compounds in the Preparation of Osteoclast Differentiation Inhibitors - Google Patents

Abstract

The invention provides the application of bromoazone compounds in the preparation of osteoclast differentiation inhibitors, which belongs to the field of biomedicine. Disclosed is a bromoazone compound whose structural formula is shown in formula (I), named isochromophiloneG, which has a significant inhibitory effect on NF-κB luciferase induced by LPS, and can significantly inhibit it at a concentration of 20 μM RANKL-induced differentiation of BMMs cells into osteoclasts does not produce cytotoxicity at concentrations of 20 μM or below. Bromoazone compounds can be used as an osteoclast differentiation inhibitor drug to prevent and treat osteolytic diseases such as osteoporosis.

Description

Application of Bromoazone Compounds in the Preparation of Osteoclast Differentiation Inhibitors

technical field

The invention relates to the field of biomedicine, in particular to the application of bromoazone compounds in the preparation of osteoclast differentiation inhibitors.

Background technique

Osteoporosis is a common bone lytic disease in the aging population and postmenopausal women. Bone is a highly dynamic tissue that is constantly remodeling and balanced by osteoclastic bone resorption and osteoblastic bone formation processes. Osteoclasts (OCs) are composed of the receptor activator of nuclear factor-κB receptor activator of nuclearappa B ligand (RANKL) and its receptor RANK on monocyte/macrophage precursors It is the only cell with bone resorption function 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 important strategies.

The currently clinically used osteoclast differentiation-related inhibitors are mainly denoximab and bisphosphonates, but both have certain complications and side effects. Therefore, it is of great value to develop safe and effective new osteoclast differentiation inhibitors. Azotone compounds (azaphilones or azaphilonoids) are polyketide secondary metabolites of fungal origin. The chemical structure contains the basic skeleton of isochromane and its derivative isoquinoline. It usually has halogen substitution, and bromination is relatively rare. In recent years, it has been reported to have anti-tumor, anti-inflammatory, anti-microbial and enzyme inhibitory activities and as a pigment additive. There are few reports on the anti-osteoclast differentiation activity of azone compounds, so their anti-osteoclast differentiation potential needs to be further explored.

Contents of the invention

The first object of the present invention is to provide a bromoazone compound for the above problems.

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

Application of the brominated azone compound of the present invention in the preparation of an osteoclast differentiation inhibitor, the structural formula of the brominated azone compound is as shown in formula (I), which is named as isochromophilone G:

It is further illustrated that the osteoclast differentiation inhibitor is a drug for treating osteolytic diseases caused by overactivation of osteoclasts.

The present invention obtains through experiments: the bromoazone compound has significant inhibitory effect on LPS-induced NF-κB luciferase, and the half-inhibitory concentration ( _IC50 value) is 28 μ M, which can be used as an inhibitor of NF-κB nuclear factor expression lead compound.

The present invention obtains through experiments: brominated azone compounds can inhibit the differentiation of BMMs (Bonemarrow macrophage cells, bone marrow macrophage cells) cells into osteoclasts induced by RANKL, and have no obvious cytotoxicity to BMMs cells, so it is expected to be developed into Safe and effective new osteoclast differentiation inhibitor drugs.

The present invention also provides the application of the bromoazone compound or its pharmaceutically acceptable salt in the preparation of NF-κB nuclear factor expression inhibitor or osteoclast differentiation inhibitor drug. It is used for the prevention and treatment of osteolytic diseases such as osteoporosis, rheumatoid arthritis, tumor metastasis and bone destruction caused by excessive activation of osteoclasts.

The present invention also provides a NF-κB nuclear factor expression inhibitor or an osteoclast differentiation inhibitor drug, including an effective amount of bromoazone compound or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier or accessories.

To further illustrate, the osteoclast differentiation inhibitor is in the form of oral preparation, injection or external preparation.

The present invention also provides a Diaporthe sp. BGS4 used for preparing the above-mentioned bromoazone compounds, and its preservation number is: GDMCC No.60671.

The present invention also provides a method for preparing bromoazone compounds, and the bromoazone compounds are prepared and isolated from the fermentation culture of Diaporthe sp. BGS4.

In the present invention, it is further explained that the method for preparing bromoazone compounds, the specific steps are as follows:

a), preparing the fermentation culture of Diaporthe sp. BGS4, soaking the fermentation product with ethyl acetate, cutting the fermentation culture into small pieces, ultrasonically extracting for 15min, and obtaining the total extract after distillation and concentration;

b) The total extract was subjected to medium-pressure normal-phase liquid chromatography, using petroleum ether/dichloromethane as the eluent, and gradient elution was performed from the volume ratio (100:0) to (0:100), and the petroleum ether was collected /Dichloromethane volume ratio 70:30 gradient eluted fractions, continue to pass through medium-pressure reversed-phase C18 column chromatography, use methanol/water as eluent, from volume ratio (10:90) to (100:0) Gradient elution was carried out, and the fractions eluted with a methanol/water volume ratio of 30:70 were collected, and the collected fractions were purified by high-performance liquid chromatography to obtain bromoazone compounds.

In the present invention, it is further illustrated that the fermentation culture of preparing Diaporthe sp. BGS4 in the step a) is to insert activated Diaporthe sp. BGS4 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 30 days at 25° C. to obtain a fermentation culture.

In the present invention, it is further illustrated that the formula of the seed culture medium contains: 15 g of malt extract powder, 15 g of sodium bromide, the balance is water, and pH 7.5 in every 1 liter of culture medium.

In the present invention, it is further illustrated that the formula of the fermentation medium is that every 1L of Erlenmeyer flask culture medium contains: wheat 150g, sodium bromide 2.7g, soybean peptone 1.8g, water 180mL, pH 7.5.

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

In the present invention, during the research on secondary metabolites of Rhizophora stylosa (Rhizophora stylosa) symbiotic Diaporthesp. LPS-induced NF-κB luciferase has a significant inhibitory effect (IC ₅₀ value of 28 μM), which can inhibit RANKL-induced differentiation of BMMs cells into osteoclasts, so it is a novel inhibitor of NF-κB nuclear factor expression or osteoclasts Ideal candidate compounds for inhibitors of cellular differentiation drugs.

Description of drawings

Figure 1 is the key HMBC (single arrow), COZY (bold line) and NOESY (double arrow dotted line) correlation diagram of bromoazone compound isochromophilone G;

Fig. 2 is the measured and calculated ECD spectrum of bromoazone compound isochromophilone G;

Figure 3 is a comparison chart of the inhibitory activity of bromoazone compound isochromophilone G (20 μM) on NF-κB luciferase induced by lipopolysaccharide (LPS) in RAW264.7 cells, BAY 11-7028 is a positive control, ^{# ##} means p<0.001vs.control group; *** means p<0.001vs.LPS group;

Figure 4 is a schematic diagram of the TRAP staining results of the differentiation of the osteoclast precursor BMMs cells of the bromoazone compound isochromophilone G;

Figure 5 is the effect of the bromoazone compound isochromophilone G (icp-G for short) on the differentiation of osteoclast precursor BMMs cells, compared with the blank control group, ^### P<0.001; compared with the RANKL group, * P<0.05;

Fig. 6 is a diagram of the activity of bromoazone compounds on osteoclast precursor BMMs cells (72h).

Detailed ways

In order to make the object, technical solution and advantages 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.

Example 1 Between the shell fungus (Diaporthe sp.) BGS4

Diaporthe sp. BGS4 was isolated from Rhizophora stylosa collected in Sanya, Hainan, China, and was preserved in Guangdong Microbial Culture Collection Center (GDMCC) on May 20, 2019. Address: Guangdong Province 5th Floor, Building 59, Guangdong Institute of Microbiology, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Institute of Microbiology, deposit number: GDMCC No.60671.

Preparation and separation of embodiment 2 bromoazone compound isochromophilone G

1. Cultivate

1.1. Seed culture medium: Each liter of culture medium contains 15g of malt extract powder, 15g 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: wheat 150g, sodium bromide 2.7g, soybean peptone 1.8g, water 180mL, pH 7.5. 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 Diaporthe sp. BGS4 into 1L triangular culture flasks each containing 300 mL of seed medium, 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 60 flasks of fermentation medium Erlenmeyer flasks with 5% inoculum amount (volume percentage), and cultured statically for 30 days at 25°C to obtain a fermentation culture.

3. Extraction: Soak the fermentation product with ethyl acetate, cut the fermentation culture into small pieces, extract it ultrasonically for 15 minutes, and obtain the total extract (50 g) after distillation and concentration.

4. Separation and purification of bromoazone compound isochromophilone G

The total extract (50g) was subjected to medium pressure normal phase column chromatography liquid chromatography (MPLC), using petroleum ether/dichloromethane as eluent, and a gradient from volume ratio (100:0) to (0:100) Elution, collect sherwood oil/dichloromethane volume ratio and be that the flow fraction (2.5g) of 70:30 elutions, continue to cross medium pressure reverse phase C ₁₈ column chromatography, use methanol/water as eluent, from volume ratio ( 10:90)～(100:0) for gradient elution, collect the fractions eluted with methanol/water volume ratio 30:70 gradient, the fractions are finally separated and purified by semi-preparative high performance liquid phase, in the elution system After purification of methanol/water (volume ratio 79:21, adding 0.3% trifluoroacetic acid, YMC-pack ODS-A chromatographic column, 10×250mm, 5μm, 2mL/min), the bromoazone compound isochromophilone G was obtained (5 mg).

Example 3 Structural identification of bromoazone compound isochromophilone G

Structural identification: yellow oil. High-resolution mass spectrometry HR-ESIMS gave a pair of isotopic quasi-molecular ion peaks m/z 435.1177/437.1154 ([M+H] ⁺ ) with a peak height ratio of nearly 1:1 (M:M+2), suggesting that the compound contains a The bromine atom, combined with ¹³ C NMR and DEPT spectra, determined that its molecular formula is C ₂₂ H ₂₇ BrO ₄ , and its degree of unsaturation is 9. Careful analysis of its nuclear magnetic resonance data found that it is very similar to the data of epi-isochromophilone II [Journal of Natural Products, 2018, 81(4): 934–941.], a chlorinated azone compound that has been reported in this strain. The main difference is The chemical shift of C-5[δ _C 99.7 (bromoazone compound isochromophilone G), 109.4 (epi-isochromophilone II)] changes greatly, combined with the molecular formula, it is speculated that the bromoazone compound isochromophilone G in C- The bromine atom attached to the 5-position replaced the chlorine atom in epi-isochromophilone II, which was confirmed by the H-4/C-5 correlation and molecular formula in the HMBC spectrum (Figure 1). Based on the proton coupling constant (J=15.4Hz) and NOESY spectrum (Fig. 1) related signals, it was determined that the carbon-carbon double bonds at C-9 and C-11 were both in the E configuration. In addition, the correlation signal of H ₃ -18 (δ _H 1.10)/H ₂ -1′ (δ _H 3.24,2.75) in the NOESY spectrum indicated that Me-18 and methylene (C-1′) were located on the same side. Therefore, the relative configuration of isochromophilone G was determined to be rel-(7R,8S). Based on the consideration of biological sources, the 13-position chirality of the azotone compound is in the S configuration. Finally, by comparing the calculated and measured ECD spectra (Figure 2), it is confirmed that the absolute configuration of the brominated azone compound isochromophilone G is 7R , 8S, 13S.

Physicochemical data of isochromophilone G: yellow solid; [α]25D+154(c 0.09, MeOH); UV(MeOH)λ _max (logε)389(3.33),250(3.29),200( 3.38) nm; ECD (0.25mg/mL, MeOH) λ _max (Δε) 389 (+5.59), 340 (+0.54), 311 (+1.90), 255 (-1.49), 226 (+2.11) nm; IR (film)ν _max 3429,2927,1716,1662,1514,1367,1165cm ^-1 ; ¹ H NMR (700MHz, CDCl ₃ ): δ _H 6.88(1H,s,H-1),6.58(1H,s, H-4),3.35(1H,dd,J＝9.8,2.1Hz,H-8),6.07(1H,d,J＝15.4Hz,H-9),7.02(1H,d,J＝15.4Hz, H-10), 5.63 (1H, d, J=9.8Hz, H-12), 2.48 (1H, m, H-13), 1.41 (1H, m, H-14a), 1.29 (1H, m, H -14b),0.86(3H,t,J=7.0Hz,H ₃ -15),1.01(3H,d,J=7.0Hz,H ₃ -16),1.83(3H,s,H ₃ -17), 1.10(3H,s,H ₃ -18),3.24(1H,dd,J=16.8,2.1,Hz,H-1′a),2.75(1H,dd,J=16.8,9.8Hz,H-1′ b), 2.28 (3H, s, H ₃ -3′); ¹³ C NMR (175MHz, CDCl ₃ ): δ _C 143.7 (CH, C-1), 158.5 (qC, C-3), 107.7 (CH, C-4),145.9(qC,C-4a),99.7(qC,C-5),192.5(qC,C-6),74.1(qC,C-7),40.5(CH,C-8), 120.3 (qC, C-8a), 116.5 (CH, C-9), 142.2 (CH, C-10), 132.1 (qC, C-11), 147.9 (CH, C-12), 35.2 (CH, C -13),30.2(CH ₂ ,C-14),12.1(CH ₃ ,C-15),20.4(CH ₃ ,C-16),12.5(CH ₃ ,C-17),21.4(CH ₃ ,C -18), 40.0(CH ₂ ,C-1′), 207.1(qC,C-2′), 30.0(CH ₃ ,C-3′); HR-ESIMS m/z 435.1177[M+H] ⁺ ( calcd for C ₂₂ H ₂₈ BrO ₄ , 435.1171).

Example 4 Determination of the Inhibitory Activity of LPS-Induced NF-κB Luciferase by Bromoazone Compound Isochromophilone G

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 200 μL of DMEM medium. After the cells adhere to the wall, add isochromophilone G, a bromoazone compound, 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 bromoazone compound isochromophilone G had a significant inhibitory effect on LPS-induced NF-κB luciferase at 20 μM (p<0.001) (Figure 3), and the half-inhibitory concentration ( _IC50 value) was 28 μM.

Example 5 Effect of bromoazone compound isochromophilone G on RANKL-induced differentiation of osteoclast precursor BMMs cells

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

RAW264.7 cells in good growth state were inoculated in 96-well plates at a density of 1× ¹⁰³ /well, and cultured in DMEM containing 10% fetal bovine serum, 100IU/mL penicillin and 100IU/mL streptomycin in each well Then, the 96-well plate was placed in a cell culture incubator at 37°C and 5% CO ₂ for incubation. After the cells were stable overnight, the bromoazone compound isochromophilone G (5, 10 and 20 μM ), with 3 replicate wells in each group. After 4 hours, except for the negative control group, all other groups were added with RANKL to a final concentration of 100 ng/mL per well, and the solution was changed every two days. After 4-5 days of stimulation with RANKL, the supernatant was discarded and stained with TRAP. Take photos and count them under an inverted microscope, and TRAP-positive cells with more than 3 nuclei are osteoclasts.

Test results: The study found that compared with the RANKL group, the bromoazone compound isochromophilone G could inhibit the differentiation of BMMs cells into osteoclasts induced by RANKL (Figure 4 and Figure 5), and had no obvious cytotoxicity to BMMs cells (Figure 5). 6).

Discussion of the results: The bromoazone compound isochromophilone G has a significant inhibitory effect on LPS-induced NF-κB luciferase (IC ₅₀ value is 28 μM), and can significantly inhibit RANKL-induced BMMs cell differentiation into broken cells at a concentration of 20 μM. bone cells, and has no obvious toxic effect on BMMs cells. 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 the bromoazone compound in preparing the osteoclast differentiation inhibitor is characterized in that the bromoazone compound has a structural formula shown in a formula (I) and is named as isochromophilone G:

2. the use according to claim 1, wherein the inhibitor of osteoclast differentiation is a medicament for the treatment of osteolytic diseases caused by overactivation of osteoclasts.

3. The use according to claim, wherein: the osteoclast differentiation inhibitor is a medicament for treating osteoporosis, rheumatoid arthritis and tumor metastasis bone destruction.

4. The use of a bromoazone compound or a pharmaceutically acceptable salt thereof according to claim 1 for the preparation of an osteoclast differentiation inhibitor or NF- κb nuclear factor expression inhibitor drug, characterized in that: the osteoclast differentiation inhibitor is in the form of oral preparation, injection or external preparation.

5. The use according to claim 4, wherein the osteoclast differentiation inhibitor or NF- κb nuclear factor expression inhibitor medicament comprises an effective amount of a bromoazone compound or a pharmaceutically acceptable salt thereof, and a pharmaceutically or pharmaceutically acceptable carrier or adjuvant.

6. A backset fungus (Diaporthe sp.) BGS4 for use in the preparation of a bromoazone compound of claim 1, deposited under the accession number: GDMCC No.60671.

7. A method for preparing a bromoazone compound according to claim 1, wherein the bromoazone compound is prepared and isolated from a fermentation culture of chaetomium globosum (Diaporthe sp.) BGS 4.

8. The method according to claim 7, wherein the method for preparing the bromoazone compound comprises the following specific steps:

a) Preparing a fermentation culture of the chaetomium globosum (Diaporthe sp.) BGS4, soaking a fermentation product in ethyl acetate, cutting the fermentation culture into small pieces, performing ultrasonic extraction for 15min, and performing distillation and concentration to obtain a total extract;

b) The total extract was purified by medium pressure normal phase liquid chromatography using petroleum ether/dichloromethane as eluent from the volume ratio (100: 0) (0): 100 Gradient elution, collection of petroleum ether/dichloromethane volume ratio 70): 30 gradient eluted fraction, continuing to pass medium-pressure reversed phase C ₁₈ Column chromatography, using methanol/water as eluent, gradient elution from volume ratio (10:90) - (100:0), collecting methanol/water volume ratio 30:70, collecting the fractions eluted by gradient, and purifying the collected fractions by high performance liquid chromatography to obtain the bromoazone compound.

9. The method according to claim 8, wherein the fermentation culture for preparing the chaetomium globosum (Diaporthe sp.) BGS4 in the step a) is prepared by inoculating activated chaetomium globosum (Diaporthe sp.) BGS4 into a seed culture medium, dynamically culturing at 25 ℃ and 180rpm for 72 hours to obtain a seed solution, inoculating the seed solution into the fermentation culture medium at 5% of the inoculum size, and statically culturing at 25 ℃ for 30 days to obtain a fermentation culture; the seed culture medium formula comprises the following components in each 1L of culture medium: 15g of malt extract powder, 15g of sodium bromide and the balance of water, wherein the pH value is 7.5; the formula of the fermentation medium comprises the following components in each 1L of triangular flask medium: 150g of wheat, 2.7g of sodium bromide, 1.8g of soybean peptone, 180mL of water and pH7.5.

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