Application of brominated azodinidin compounds in the preparation of osteoclast differentiation inhibitors

Abstract

The present invention provides the application of brominated azodil compounds in the preparation of osteoclast differentiation inhibitors, belonging to the field of biomedicine. A brominated azodil compound is disclosed, and its structural formula is shown in formula (I), which is named isochromophiloneG, which has a significant inhibitory effect on LPS-induced NF-κB luciferase, and can significantly inhibit RANKL-induced BMMs cell differentiation into osteoclasts at a concentration of 20 μM, and does not produce cytotoxicity at a concentration of 20 μM or below. Brominated azodil compounds can be used as an osteoclast differentiation inhibitor drug for the prevention and treatment of osteolytic diseases such as osteoporosis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of brominated azodrin compounds in the preparation of osteoclast differentiation inhibitors. Background Art

[0002] Osteoporosis is a common osteolytic disease in the aging population and postmenopausal women. Bone is a highly dynamic tissue that is constantly remodeled and balanced through osteoclast bone resorption and osteoblast bone formation. Osteoclasts (OCs) are initiated by the binding of receptor activator of nuclear kappa B ligand (RANKL) to its receptor RANK on monocyte / macrophage precursors. Osteoclasts are the only cells in the human body with bone resorption function. Defective osteoclast activity leads to osteosclerosis and bone marrow failure, while overactivation 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 important strategies for the treatment of osteoporosis.

[0003] At present, the main osteoclast differentiation inhibitors used in clinical practice are 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. Azaphilones (azaphilonoids) are secondary metabolites of fungal polyketides. Their chemical structure contains the basic skeleton of isochromane and its derivative isoquinoline. They usually have halogen substitutions, and bromination is relatively rare. In recent years, they have been reported to have anti-tumor, anti-inflammatory, antimicrobial and enzyme inhibition activities and as pigment additives. There are few reports on the anti-osteoclast differentiation activity of azaphilones, so their anti-osteoclast differentiation potential needs to be further explored. Summary of the invention

[0004] The first object of the present invention is to provide a brominated azodine compound in view of the above problems.

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

[0006] The brominated azobenzoic acid ketone compound of the present invention is used in the preparation of an osteoclast differentiation inhibitor. The structural formula of the brominated azobenzoic acid ketone compound is shown in formula (I), and it is named isochromophilone G:

[0007]

[0008] It is further explained that the osteoclast differentiation inhibitor is a drug for treating osteolytic diseases caused by excessive activation of osteoclasts.

[0009] The present invention has obtained through experiments that brominated azodine compounds have a significant inhibitory effect on LPS-induced NF-κB luciferase, and the half inhibitory concentration (IC 50 The value) was 28 μM, which can be used as a lead compound for the development of NF-κB nuclear factor expression inhibitors.

[0010] The present invention obtains through experiments that brominated azodine compounds can inhibit RANKL-induced BMMs (Bonemarrow macrophage cells) cell differentiation into osteoclasts, and have no obvious cytotoxicity to BMMs cells, so they are expected to be developed into safe and effective new osteoclast differentiation inhibitor drugs.

[0011] The present invention also provides the use of brominated azodine compounds or pharmaceutical salts thereof in the preparation of NF-κB nuclear factor expression inhibitors or osteoclast differentiation inhibitors, which are used to prevent and treat osteolytic diseases such as osteoporosis, rheumatoid arthritis, and tumor metastasis bone destruction caused by excessive osteoclast activation.

[0012] The present invention also provides a NF-κB nuclear factor expression inhibitor or osteoclast differentiation inhibitor drug, comprising an effective amount of a brominated azodine compound or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier or auxiliary material.

[0013] It is further described that the osteoclast differentiation inhibitor is an oral preparation, an injection formulation or an external formulation.

[0014] The present invention also provides a Diaporthe sp. BGS4 for preparing the above-mentioned brominated azodrone compounds, and its deposit number is GDMCC No.60671.

[0015] The present invention also provides a method for preparing brominated azodrin compounds. The brominated azodrin compounds are prepared and separated from the fermentation culture of Diaporthe sp. BGS4.

[0016] In the present invention, it is further described that the method for preparing brominated azodine compounds comprises the following specific steps:

[0017] a), preparing a fermentation culture of Diaporthe sp. BGS4, soaking the fermentation product with ethyl acetate, cutting the fermentation culture into small pieces, ultrasonically extracting for 15 minutes, and obtaining a total extract after distillation and concentration;

[0018] b) The total extract is subjected to medium pressure normal phase liquid chromatography, using petroleum ether / dichloromethane as eluent, and gradient elution is performed from a volume ratio of (100:0) to (0:100), and the fractions eluted from the gradient elution of petroleum ether / dichloromethane volume ratio of 70:30 are collected, and then subjected to medium pressure reverse phase C18 column chromatography, using methanol / water as eluent, and gradient elution is performed from a volume ratio of (10:90) to (100:0), and the fractions eluted from the gradient elution of methanol / water volume ratio of 30:70 are collected, and the collected fractions are purified by high performance liquid chromatography to obtain brominated azodine compounds.

[0019] In the present invention, it is further described that the step a) of preparing the fermentation culture of Diaporthe sp. BGS4 is to inoculate the activated Diaporthe sp. BGS4 into a seed culture medium, culture at 25° C., 180 rpm, for 72 hours 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 30 days to obtain a fermentation culture.

[0020] In the present invention, it is further described that the seed culture medium formula contains: 15g malt extract powder, 15g sodium bromide, and the balance is water, pH 7.5 per 1L of culture medium.

[0021] In the present invention, it is further described that the formula of the fermentation medium is that each 1L of Erlenmeyer flask medium contains: 150g wheat, 2.7g sodium bromide, 1.8g soy peptone, 180mL water, pH 7.5.

[0022] Due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0023] In the process of studying the secondary metabolites of Diaporthesp. BGS4 from Rhizophora stylosa in Sanya, Hainan, a novel and rare brominated azodil compound was isolated and obtained, which has a significant inhibitory effect on LPS-induced NF-κB luciferase (IC 50 value is 28μM), which can inhibit RANKL-induced BMMs cell differentiation into osteoclasts, and is therefore an ideal candidate compound for the development of a new NF-κB nuclear factor expression inhibitor or osteoclast differentiation inhibitor drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the key HMBC (single arrow), COSY (bold line) and NOESY (double arrow dashed line) correlation diagram of the brominated azodophilic ketone compound isochromophilone G;

[0025] Figure 2It is the measured and calculated ECD spectra of isochromophilone G, a brominated azodophilic ketone compound;

[0026] Figure 3 This is a comparison of the inhibitory activity of isochromophilone G (20 μM) on NF-κB luciferase induced by lipopolysaccharide (LPS) in RAW264.7 cells. BAY 11-7028 is a positive control. ### Indicates p<0.001 vs. control group; *** indicates p<0.001 vs. LPS group;

[0027] Figure 4 This is a schematic diagram of the TRAP staining results of the osteoclast precursor BMMs cell differentiation using the brominated azodipine compound isochromophilone G;

[0028] Figure 5 This is the result of the effect of isochromophilone G (abbreviated as icp-G) on the differentiation of osteoclast precursor BMMs cells. Compared with the blank control group, ### P<0.001; compared with RANKL group, *P<0.05;

[0029] Figure 6 This is a graph showing the effect of brominated azodine compounds on the viability of osteoclast precursor BMMs cells (72h). DETAILED DESCRIPTION

[0030] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, 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.

[0031] Example 1 Diaporthe sp. BGS4

[0032] Diaporthe sp. BGS4 was isolated from Rhizophora stylosa collected from Sanya, Hainan, 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.60671.

[0033] Example 2 Preparation and Isolation of Brominated Azodipine Compound Isochromophilone G

[0034] 1. Cultivation

[0035] 1.1 Seed culture medium: Each liter of culture medium contains 15g malt extract powder, 15g 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.

[0036] 1.2. Fermentation medium: Each 1L Erlenmeyer flask medium contains: 150g wheat, 2.7g sodium bromide, 1.8g soy peptone, 180mL water, pH 7.5. Mix the above components and contents evenly, then sterilize at 121℃ for 30min for later use.

[0037] 2. Fermentation

[0038] 2.1. Seed culture: The activated Diaporthe sp. BGS4 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.

[0039] 2.2. Fermentation culture: The seed liquid was inoculated into 60 bottles of fermentation medium triangle flasks at an inoculum amount of 5% (volume percentage), and statically cultured at 25° C. for 30 days to obtain a fermentation culture.

[0040] 3. Extraction: Soak the fermentation product with ethyl acetate, cut the fermentation culture into small pieces, extract by ultrasonic for 15 minutes, and obtain the total extract (50 g) after distillation and concentration.

[0041] 4. Isolation and purification of isochromophilone G, a brominated azodipine compound

[0042] The total extract (50 g) 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 70:30 (2.5 g) was 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 the fractions eluted with a methanol / water volume ratio of 30:70 were collected. The fractions were further separated and purified using semi-preparative high performance liquid chromatography, and the brominated azodipine compound isochromophilone G (5 mg) was obtained after purification in an elution system of methanol / water (volume ratio 79:21, with 0.3% trifluoroacetic acid added, YMC-pack ODS-A column, 10×250 mm, 5 μm, 2 mL / min).

[0043] Example 3 Structural Identification of Brominated Azotoxin Compound Isochromophilone G

[0044] Structural identification: Yellow oil. High-resolution mass spectrometry HR-ESIMS gave a pair of isotopic quasi-molecular ion peaks with a peak height ratio of nearly 1:1 (M:M+2) m / z 435.1177 / 437.1154 ([M+H] + ), it is speculated that the compound contains a bromine atom, combined with 13 C NMR and DEPT spectra confirmed that its molecular formula is C 22 H 27 BrO4, with an unsaturation of 9. A careful analysis of its NMR data revealed that it was extremely similar to the data of the chloro-azocyanide compound epi-isochromophilone II reported in this strain [Journal of Natural Products, 2018, 81(4):934–941.], with the main difference being C-5[δ C 99.7 (bromo-nitrogen ketone compound isochromophilone G), 109.4 (epi-isochromophilone II)] chemical shift changes greatly. Combined with the molecular formula, it is speculated that the bromine atom at the C-5 position in the bromo-nitrogen ketone compound isochromophilone G replaces the chlorine atom in epi-isochromophilone II. This inference is confirmed by the HMBC spectrum ( Figure 1 ) was confirmed by the H-4 / C-5 correlation and molecular formula. Based on the proton coupling constant (J = 15.4 Hz) and NOESY spectrum ( Figure 1 ) related signals confirmed that the carbon-carbon double bonds at C-9 and C-11 were both in E-form. In addition, H3-18 (δ H 1.10) / H2-1′(δ H 3.24,2.75) related signals indicate that Me-18 and methylene (C-1′) are located on the same side. Therefore, the relative configuration of the brominated azodipine compound isochromophilone G is determined to be rel-(7R,8S). Based on the biogenic pathway, the chirality of the 13-position of the azodipine compound is all S-configuration. Finally, the calculated and measured ECD spectra ( Figure 2 ) The absolute configuration of isochromophilone G, a brominated azodipine compound, was determined to be 7R, 8S, 13S.

[0045] Physical and chemical data of isochromophilone G: yellow solid; [α]25D+154 (c 0.09, MeOH); UV (MeOH) λmax (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,CDCl3):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,H3-15),1.01(3H,d,J=7.0Hz,H3-16),1.83(3H,s,H3-17),1.10(3H,s,H3-18),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,H3-3′); 13 C NMR(175MHz,CDCl3):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(CH2,C-14),12.1(CH3,C-15),20.4(CH3,C-16),12.5(CH3,C-17),21.4(CH3,C-18),40.0(CH2,C-1′),207.1(qC,C-2′),30.0(CH3,C-3′);HR-ESIMS m / z 435.1177[M+H]+ (calcd for C 22 H 28 BrO4,435.1171).

[0046] Example 4 Determination of the inhibitory activity of isochromophilone G on LPS-induced NF-κB luciferase

[0047] The main reference for NF-κB luciferase inhibitory activity assay is (British Journal of Pharmacology, 2020, 177: 4242–4260).

[0048] 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 the brominated azodipine compound isochromophilone G 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.

[0049] Conclusion: The study found that compared with the LPS blank group, the brominated azodipine compound isochromophilone G at 20μM had a significant inhibitory effect on LPS-induced NF-κB luciferase (p<0.001)( Figure 3 ), half inhibitory concentration (IC 50 value) was 28 μM.

[0050] Example 5 Effect of isochromophilone G, a brominated azodone compound, on RANKL-induced differentiation of osteoclast precursor BMMs cells

[0051] 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).

[0052] RAW264.7 cells with good growth status were taken and 1×10 3 The cells were inoculated at a density of 100 μL / well in a 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. The 96-well plate was then placed in a cell culture incubator at 37°C and 5% CO2 for incubation. After the cells were attached to the wall and stabilized overnight, bromo-azobenzene compounds isochromophilone G (5, 10 and 20 μM) were added respectively, and 3 replicates were set for each group. After 4 hours, RANKL was added to all groups except the negative control group to a final concentration of 100 ng / mL per well. The solution was changed every two days. After RANKL stimulation for 4-5 days, the supernatant was discarded and TRAP staining was performed. Pictures were taken and counted under an inverted microscope. TRAP-positive cells with more than 3 nuclei were osteoclasts.

[0053] Experimental results: The study found that compared with the RANKL group, the brominated azodine compound isochromophilone G can inhibit RANKL-induced BMMs cell differentiation into osteoclasts ( Figure 4 and Figure 5 ) and had no obvious cytotoxicity to BMMs cells ( Figure 6 ).

[0054] Results and discussion: The brominated azobenzoic acid compound isochromophilone G has a significant inhibitory effect on LPS-induced NF-κB luciferase (IC 50 The value is 28μM), and at a concentration of 20μM, it can significantly inhibit the differentiation of BMMs cells into osteoclasts induced by RANKL, and has no obvious toxic effect on BMMs cells. 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.

[0055] 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.

[0056] 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 brominated azobenzoic acid compound in the preparation of an osteoclast differentiation inhibitor, wherein the brominated azobenzoic acid compound has a structural formula as shown in formula (I), and is named isochromophilone G: 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 brominated azodine compound or a pharmaceutically acceptable salt thereof, and medically or pharmaceutically acceptable auxiliary materials.

3. A method for preparing the brominated azodine compound described in claim 1, characterized in that: The brominated azotoxin compounds are obtained from Diaporthe sp.) BGS4 fermentation culture prepared and isolated; the interseat shell fungus ( Diaporthe sp.) BGS4, whose deposit number is: GDMCC No. 60671; The method for preparing the brominated azodine compounds comprises the following specific steps: a) Preparation of Metarhizium Diaporthe sp.) BGS4, soak the fermentation product with ethyl acetate, cut the fermentation culture into small pieces, extract by ultrasonic for 15 min, and obtain the total extract after distillation and concentration; The step a) of preparing the Diaporthe sp.) BGS4 fermentation culture is activated by Diaporthe sp.) BGS4 was inoculated into a seed culture medium, and the culture was carried out dynamically at 25°C and 180 rpm for 72 h to obtain a seed solution, and the seed solution was inoculated into a fermentation culture medium at a rate of 5%, and the culture was carried out statically at 25°C for 30 days to obtain a fermentation culture; the seed culture medium formula is that per 1L of the culture medium contains: 15 g of malt extract powder, 15 g of sodium bromide, and the balance is water, and the pH value is 7.5; the fermentation culture medium formula is that per 1L of the flask culture medium contains: 150 g of wheat, 2.7 g of sodium bromide, 1.8 g of soy peptone, 180 mL of water, and the pH value is 7.5; 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 a volume ratio of (100:0) to (0:100). The fractions eluted with a volume ratio of petroleum ether / dichloromethane of 70:30 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 from the gradient elution with a volume ratio of methanol / water of 30:70 were collected. The collected fractions were then purified by high performance liquid chromatography to obtain brominated azodine compounds.

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