Hydroxyindanone compound and application thereof in preparation of osteoclast differentiation inhibitor

Abstract

The invention provides a novel hydroxyl indanone compound and application thereof in preparation of an osteoclast differentiation inhibitor, and belongs to the field of marine natural products. The invention discloses a hydroxyl indanone compound which is named as (R)-3, 4-dihydroxy-2, 3-dihydro-1H-indan-1-one, the structural formula of the hydroxyl indanone compound is shown as a formula (I), the hydroxyl indanone compound has a remarkable inhibition effect on LPS (Lipopolysaccharide) induced NF-kappa B luciferase, can remarkably inhibit RANKL induced bone marrow macrophages from being differentiated into osteoclasts at the concentration of 20 mu M or below, and does not generate cytotoxicity at the concentration of 20 mu M or below. The hydroxyindanone compound can be used as an osteoclast differentiation inhibitor medicine and is used for preventing and treating osteolytic diseases such as osteoporosis. # imgabs0 #

Description

Technical Field

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

[0002] Osteoclasts (OCs) are the only cells in the body capable of bone resorption, initiated by the binding of receptor activator of nuclear kappa B ligand (RANKL) to its receptor, RANK, on ​​monocyte / macrophage precursors. The maintenance of bone homeostasis depends on a dynamic balance between osteoclasts (OCs) and osteoblasts. As the only cell in the body capable of bone resorption, aberrant activation or uncontrolled differentiation of osteoclasts can lead to osteolytic diseases such as osteoporosis and osteosclerosis, resulting in high disability rates and a heavy socioeconomic burden. Currently, mainstream osteoclast-targeted therapeutics include bisphosphonates and denoximab (a RANKL antibody), but their application has significant limitations: long-term use of bisphosphonates inhibits natural bone turnover and increases the risk of brittle fractures; denoximab requires subcutaneous administration, which is costly and can induce serious complications such as osteonecrosis of the jaw. Although existing therapies can inhibit osteoclast differentiation or bone resorption function, they cannot balance efficacy and safety. The development of new, low-toxic, highly effective and economically feasible anti-osteoclast differentiation drugs has become an urgent clinical need.

[0003] In recent years, indanone compounds, owing to their unique indene ring structure, have demonstrated a wide range of biological activities, including antitumor, antiviral, and neuroprotective effects. Hydroxyidanones, a key subclass of indanone derivatives, possess a molecular backbone that combines a rigid planar structure with modifiable sites, showing great potential in drug design. However, existing research has primarily focused on their applications in anti-Alzheimer's disease and antimicrobial applications, while their mechanisms of action in regulating osteoclast differentiation and their activity against osteolytic diseases remain largely unexplored. Of particular note, hydroxyyidanones may potentially intervene through multi-target effects, interfering with the RANKL / RANK / OPG signaling pathway, NF-κB activation, or osteoclast-specific gene expression, thereby circumventing the drug resistance and side effect risks of existing single-target drugs in the process of inhibiting osteoclast differentiation. Furthermore, their broad natural resource base and highly tunable structure offer unique advantages for the development of novel small-molecule inhibitors with low toxicity and high oral bioavailability.

[0004] It can be seen that there are few reports on the anti-osteoclast differentiation activity of hydroxyindanone compounds, so their anti-osteoclast differentiation potential needs to be further explored. Summary of the Invention

[0005] In order to solve the above problems, a hydroxyindane compound and its application in the preparation of osteoclast differentiation inhibitors are provided.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The hydroxyindenone compound of the present invention has a structural formula as shown in formula (I), and is named (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one:

[0008]

[0009] The present invention also provides a Diaporthe sp. BGS4 for preparing the hydroxyindanone compound, and its deposit number is GDMCC No. 60671.

[0010] The present invention also provides a method for preparing hydroxyindanone compounds. The hydroxyindanone compounds are prepared and separated from the fermentation culture of Diaporthe sp. BGS4.

[0011] In the present invention, the method for preparing hydroxyindanone compounds is further described, and the specific steps are as follows:

[0012] 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 concentrating by distillation to obtain a total extract;

[0013] b) The total extract was subjected to medium-pressure normal-phase liquid chromatography, using petroleum ether / dichloromethane as an eluent, and gradient elution was performed from a volume ratio of (100:0) to (0:100), and the fractions eluted with a petroleum ether / dichloromethane volume ratio of 20:80 were collected. The total extract was further subjected to medium-pressure reverse-phase C18 column chromatography, using methanol / water as an eluent, and gradient elution was performed from a volume ratio of (10:90) to (100:0), and the fractions eluted with a methanol / water volume ratio of 20:80 were collected. The collected fractions were then purified by high-performance liquid chromatography to obtain aromatic ketone compounds.

[0014] In the present invention, it is further described that the preparation of the fermentation culture of Diaporthe sp. BGS4 in step a) is to inoculate activated Diaporthe sp. BGS4 into a seed culture medium, culture at 25°C, 180 rpm, and obtain a seed liquid for 72 hours, inoculate the seed liquid into a fermentation medium at an inoculum rate of 5%, and statically culture at 25°C for 30 days to obtain a fermentation culture.

[0015] In the present invention, it is further described that the seed culture medium formula contains: 15g malt extract powder, 15g sea salt, and the balance is water per 1L of culture medium, with a pH of 7.5.

[0016] In the present invention, it is further described that the formula of the fermentation medium is as follows: 120g rice, 3g sea salt, 150mL water, pH 7.5 per 1L Erlenmeyer flask medium.

[0017] Another object of the present invention is to provide the osteoclast differentiation inhibitor as a drug for treating osteolytic diseases caused by excessive osteoclast activation.

[0018] The present invention obtains through experiments that: the hydroxyindanone compound has a significant inhibitory effect on LPS-induced NF-κB luciferase at 20 μM, and can be used as a lead compound for developing NF-κB nuclear factor expression inhibitors.

[0019] The present invention has obtained through experiments that hydroxyindanone compounds can inhibit RANKL-induced BMMs (Bonemarrow macrophage cells) differentiation into osteoclasts, and have no obvious cytotoxicity to BMMs cells. Therefore, they are expected to be developed into safe and effective new osteoclast differentiation inhibitor drugs.

[0020] The present invention also provides the use of hydroxyindanone compounds or pharmaceutically acceptable salts thereof in the preparation of NF-κB nuclear factor expression inhibitors or osteoclast differentiation inhibitors for preventing and treating osteolytic diseases such as osteoporosis, rheumatoid arthritis, and tumor metastasis bone destruction caused by excessive osteoclast activation.

[0021] The present invention also provides a NF-κB nuclear factor expression inhibitor or osteoclast differentiation inhibitor drug, comprising an effective amount of a hydroxyindone compound or a pharmaceutically acceptable salt thereof as an active ingredient, and a pharmaceutically acceptable carrier or excipient.

[0022] It is further stated that the osteoclast differentiation inhibitor is in the form of an oral preparation, an injection or an external use preparation.

[0023] Due to the adoption of the above technical solution, the present invention has the following beneficial effects:

[0024] The present invention relates to a novel hydroxyindone compound isolated during the study of secondary metabolites of the symbiotic fungus Diaporthe sp. BGS4 of Rhizophora stylosa. The compound has significant NF-κB pathway inhibitory activity and osteoclast differentiation inhibitory effect, and exhibits outstanding technical advantages and clinical application potential in the field of drug development. The compound is a new hydroxyindone structure isolated from nature for the first time. Its unique chemical skeleton is different from the existing reported NF-κB inhibitors and bone metabolism regulators. Experiments show that it can significantly inhibit LPS-induced NF-κB luciferase activity at a low concentration (20 μM), and has a significant inhibitory effect on RANKL-induced BMMs cell differentiation into osteoclasts, suggesting that it has an efficient dual mechanism of action. Compared with traditional synthetic inhibitors, this natural product molecule may have better biocompatibility and lower risk of toxic side effects. By effectively blocking RANKL-induced osteoclast differentiation, this compound can intervene in the pathological bone resorption process and provide a new treatment strategy for bone-destructive diseases such as osteoporosis, bone metastasis, and rheumatoid arthritis.

[0025] In summary, the present invention not only provides a natural active compound with novel structure and significant activity, but also opens up a new direction for the development of NF-κB pathway inhibitors and osteoclast differentiation inhibitors. It has outstanding technological progress and industrial application value in terms of drug innovation and wide application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The key HMBC (single arrow) of the hydroxyindenone compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one and 1 H- 1 H COSY (bold line) correlation diagram;

[0027] Figure 2 The measured and calculated ECD spectra of the hydroxyindenone compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one are shown;

[0028] Figure 3 This is a comparison of the inhibitory activity of the hydroxyindane compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one (20μM) on NF-κB luciferase induced by lipopolysaccharide (LPS) in RAW264.7 cells. BAY 11-7028 (BAY) is a positive control. ###p<0.001vs.control(CON)group; ***p<0.001,**p<0.05vs.LPSgroup;

[0029] Figure 4 This is a schematic diagram of the TRAP staining results of the hydroxyindane compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one osteoclast precursor BMMs cell differentiation;

[0030] Figure 5 This is the result of the effect of hydroxyindane compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one 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;

[0031] Figure 6 This is a graph showing the effect of hydroxyindone compounds on the viability of osteoclast precursor BMMs cells (72h). DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be 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 intended to limit the present invention.

[0033] Example 1 Diaporthe sp. BGS4

[0034] Diaporthe sp. BGS4 was isolated from Rhizophora stylosa collected from Sanya, Hainan, China, and deposited on May 20, 2019, at the Guangdong Microbial Culture Collection Center (GDMCC), Building 59, 5th Floor, Guangdong Institute of Microbiology, 100 Xianlie Middle Road, Guangzhou, Guangdong, China, with the deposit number: GDMCC No. 60671.

[0035] Example 2 Preparation and Isolation of Hydroxyindenone Compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one

[0036] 1. Cultivation

[0037] 1.1 Seed Culture Medium: Each liter of culture medium contains 15g of malt extract powder, 15g of sea salt, and the balance is water. pH 7.5. Mix the above ingredients and proportions uniformly, then sterilize at 121°C for 30 minutes.

[0038] 1.2 Fermentation medium: Each 1L Erlenmeyer flask contains the following: 120g rice, 3g sea salt, 150mL water, pH 7.5. Mix the above ingredients and proportions uniformly, then sterilize at 121°C for 30 minutes.

[0039] 2. Fermentation

[0040] 2.1. Seed culture: Activated Diaporthe sp. BGS4 was inoculated into 1 L triangular culture flasks containing 300 mL of seed culture medium each, and cultured at 25° C., 180 rpm, for 72 h to prepare seed solution.

[0041] 2.2. Fermentation culture: The seed liquid was inoculated into 48 bottles of fermentation medium triangular flasks at an inoculum amount of 5% (volume percentage) and cultured statically at 25° C. for 30 days to obtain a fermentation culture.

[0042] 3. Extraction: Soak the fermentation product with ethyl acetate, cut the fermentation culture into small pieces, extract with ultrasound for 15 minutes, and obtain the total extract (150 g) after distillation and concentration.

[0043] 4. Isolation and purification of hydroxyindenone compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one

[0044] The total extract (150 g) was subjected to medium pressure normal phase column chromatography (MPLC) with petroleum ether / dichloromethane as eluent, and gradient elution was performed from a volume ratio of (100:0) to (0:100). The fraction (2 g) eluted with a volume ratio of petroleum ether / dichloromethane of 20:80 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 20:80 were collected. The fractions were further separated and purified by semi-preparative high performance liquid chromatography, and the hydroxyindanone compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one (6 mg) was obtained after purification in an elution system of methanol / water (volume ratio 18:82, YMC-pack ODS-A column, 10×250 mm, 5 μm, 2 mL / min).

[0045] Example 3 Structural Identification of Hydroxyindenone Compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one

[0046] Structural identification: colorless crystals. High-resolution mass spectrometry HR-ESIMS gave a quasi-molecular ion peak m / z 163.0392 ([MH]-, calculated value 163.0395), combined with 13 C NMR spectrum confirmed its molecular formula to be C9H8O3 with a degree of unsaturation of 6. A careful analysis of its NMR data revealed that it is extremely similar to the NMR data of the known compound (S)-3,7-dihydroxy-1-indanone [Phytochemistry Letters, 2021, 44, 106–109.], with the main difference being the differences in the chemical shifts of C-4, C-5, C-6, and C-7 of the compound, which is speculated to be a positional isomer. The chemical shift of C-4 in the compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one shifts downfield (δ C 158.3), indicating that C-4 contains hydroxyl substitution, which is also confirmed by the H-3, H-6 / C-4 related signals in the HMBC spectrum ( Figure 1 Since the compound contains only one chiral center, ECD calculation was used to determine its absolute configuration. Figure 2 ) It was found that the calculated ECD spectrum of the R configuration was basically consistent with the measured ECD spectrum trend, so the absolute configuration of the compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one was identified as R.

[0047] Physical and chemical data of compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one: colorless crystals; (c0.1,MeOH); UV(MeOH)λ max (logε)310(3.02),253(3.42),217(3.82)nm; ECD(0.2mg / mL,MeOH)λ max (Δε)320(-6.83),253(+15.9),222(+22.0),208(-23.31)nm; 1 H NMR (500MHz,CDCl3):δ H 3.08(1H,dd,J=18.5,6.5Hz,H-2a),2.53(1H,dd,J=18.5,2.5Hz,H-2b),5.31(1H,dd,J=7.0,3 .0Hz,H-3),7.17(1H,d,J=7.5Hz,H-5),7.58(1H,t,J=7.5Hz,H-6),6.83(1H,d,J=7.5Hz,H-7); 13C NMR (125MHz,CDCl3):δ C 206.6(qC,C-1),47.7(CH2,C-2),68.9(CH,C-3),157.5(qC,C-3a),158.3(qC,C-4) ,117.9(CH,C-5),138.9(CH,C-6),116.4(CH,C-7),123.3(qC,C-7a); HR-ESIMSm / z 163.0392[MH] - (calcd for C9H7O4,163.0395).

[0048] Example 4 Determination of the inhibitory activity of hydroxyindenone compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one on LPS-induced NF-κB luciferase

[0049] The determination of NF-κB luciferase inhibitory activity mainly refers to the literature method previously reported by our team (Journal of Medicinal Chemistry, 2024, 67(4): 2602–2618.).

[0050] RAW264.7 cells stably transfected with NF-κB luciferase reporter gene were seeded in 96-well plates (1×10 4 Each well was incubated with 200 μL of DMEM medium supplemented with 10% fetal bovine serum, 100 IU / mL penicillin and streptomycin, and 0.1 μg / mL G418. After stable cell attachment, the hydroxyindane compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one was added, with six replicates set up. After a further 4 hours of incubation, LPS and RANKL were added to each compound group (three wells), except for the negative control group, and to a positive control group (NF-κB inhibitor, BAY11-7082, 5 μM), respectively, to a final concentration of 100 ng / mL per well. After 8 hours of stimulation with both, the supernatant was discarded, and 25 μL of cell lysis buffer was added to each well. The cells were shaken at low speed for 10 minutes to fully lyse the cells. Then, 20 μL of the sample was transferred to a white plate, and 50 μL of luciferin solution was added to each well. Luciferase activity was measured using a multi-function microplate reader.

[0051] Conclusion: The study found that compared with the LPS blank group, the hydroxyindane compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one at 20μM had a significant inhibitory effect on LPS-induced NF-κB luciferase (p<0.05) ( Figure 3 ).

[0052] Example 5 Effect of Hydroxyindanone Compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one on RANKL-Induced Osteoclast Precursor BMMs Cell Differentiation

[0053] The main reference for the determination of RANKL-induced osteoclast precursor BMMs cell differentiation inhibitory activity is (Journal of Medicinal Chemistry, 2024, 67(4): 2602–2618.).

[0054] RAW264.7 cells with good growth status were taken and 1×10 3 Cells were seeded at a density of 100 μL / well in a 96-well plate. DMEM medium supplemented with 10% fetal bovine serum, 100 IU / mL penicillin, and 100 IU / mL streptomycin was added to 200 μL per well. The 96-well plates were then incubated in a 37°C, 5% CO2 cell culture incubator. After the cells had stabilized and adhered overnight, the hydroxyindane compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one (5, 10, and 20 μM) was added, with triplicate wells per group. Four hours later, RANKL was added to a final concentration of 100 ng / mL per well in all groups except the negative control. The medium was changed every two days. After 4-5 days of RANKL stimulation, the supernatant was discarded and the cells were stained for TRAP. Cells were photographed and counted under an inverted microscope. TRAP-positive cells with more than three nuclei were identified as osteoclasts.

[0055] Experimental results: The study found that compared with the RANKL group, the hydroxyindane compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one could inhibit RANKL-induced BMMs cell differentiation into osteoclasts ( Figure 4 and Figure 5 ) and had no obvious cytotoxicity to BMMs cells ( Figure 6 ).

[0056] Results and Discussion: The hydroxyindanone compound (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one significantly inhibited LPS-induced NF-κB luciferase activity and, at a concentration of 20 μM, significantly inhibited RANKL-induced osteoclast differentiation of BMMs without significant cytotoxicity to BMMs. It could be developed as a novel inhibitor of osteoclast differentiation or NF-κB expression for the prevention and treatment of osteolytic diseases such as osteoporosis.

[0057] 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.

[0058] 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. A hydroxyindane compound, characterized in that: The structural formula of the hydroxyindenone compound is shown in formula (I), and it is named (R)-3,4-dihydroxy-2,3-dihydro-1H-inden-1-one:

2. A Diaporthe sp. BGS4 for preparing the hydroxyindanone compound according to claim 1, whose deposit number is GDMCC No. 60671.

3. A method for preparing the hydroxyindanone compound according to claim 1, characterized in that: The hydroxyindanone compound is prepared and separated from the fermentation culture of Diaporthe sp. BGS4.

4. The method according to claim 3, characterized in that The method for preparing hydroxyindanone compounds comprises the following specific steps: 1) 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 distilling and concentrating to obtain a total extract; 2) The total extract 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 20:80 were collected and continued to be eluted by 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 methanol / water volume ratio of 20:80 were collected. The collected fractions were then purified by high performance liquid chromatography to obtain hydroxyindanone compounds.

5. The method according to claim 4, characterized in that In step 1), the fermentation culture of Diaporthe sp. BGS4 is prepared by inoculating activated Diaporthe sp. BGS4 into a seed culture medium, dynamically culturing at 25° C., 180 rpm, for 72 hours to obtain a seed solution, inoculating the seed solution into a fermentation culture medium at a 5% inoculum rate, and statically culturing at 25° C. for 30 days to obtain a fermentation culture. The seed culture medium comprises: 15 g malt extract powder, 15 g sea salt, and the balance water per 1 L of culture medium, with a pH of 7.

5. The fermentation culture medium comprises: 120 g rice, 3 g sea salt, and 150 mL water per 1 L of culture medium in a triangular flask, with a pH of 7.

5.

6. Use of the hydroxyindanone compound according to claim 1 in the preparation of an osteoclast differentiation inhibitor.

7. The use according to claim 6, characterized in that The osteoclast differentiation inhibitor is a drug for treating osteolytic diseases caused by excessive activation of osteoclasts.

8. The use according to claim 6, characterized in that: The osteoclast differentiation inhibitor is a drug for treating osteoporosis, rheumatoid arthritis and bone destruction caused by tumor metastasis.

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

10. The use according to claim 9, characterized in that The osteoclast differentiation inhibitor or NF-κB nuclear factor expression inhibitor drug comprises an effective amount of a hydroxyindone compound or a pharmaceutically acceptable salt thereof, and a medically or pharmaceutically acceptable carrier or auxiliary material.

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