Deep sea naphthyridine pyran alkaloid compound and application thereof in preparation of medicine for preventing or treating bone metabolic diseases

Abstract

The invention belongs to the technical field of biological medicines, and discloses a deep-sea naphthyridine pyran alkaloid compound, a preparation method thereof and application of the deep-sea naphthyridine pyran alkaloid compound in preparation of medicines for preventing and / or treating bone metabolism diseases, particularly bone metabolism diseases such as bone fracture, osteoporosis, rickets, endocrine bone diseases, osteitis deformans and hereditary bone diseases. The new skeleton compound composed of 2, 7-naphthyridinone and pyranol is found for the first time, can promote mineralization of osteogenesis by targeting beta-catenin protein so as to achieve an obvious effect of promoting fracture repair, has obvious activity of promoting osteoblast mineralization and anti-bone metabolism activity, can be used for preparing an osteoblast activator, and can be used for preparing a bone repair material. And the compound has a good application prospect in the aspect of preparing medicines for preventing and / or treating bone metabolic diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to a class of deep-sea naphthidine pyran alkaloid compounds, their preparation methods, and their application in the preparation of drugs for the prevention and / or treatment of bone metabolic diseases, particularly bone metabolic diseases such as fractures, osteoporosis, rickets, endocrine bone diseases, osteomalacia, and hereditary bone diseases. Background Technology

[0002] The maintenance and repair of bone are regulated by two main types of cells: osteoblasts and osteoclasts. Osteoblasts are responsible for generating new bone tissue, while osteoclasts are responsible for degrading aging bone tissue. Under normal circumstances, there is a dynamic balance between osteoblasts and osteoclasts to maintain the health of the bone. When this balance is disrupted, leading to increased osteoclast activity and decreased osteoblast activity, a significant loss of bone tissue occurs, resulting in bone metabolic diseases. Bone metabolic diseases include osteoporosis, fractures, osteogenesis imperfecta, rickets, endocrine osteodystrophy, and osteitis deformans.

[0003] Currently, most drugs for treating bone metabolism focus on inhibiting the function and differentiation of osteoclasts, and cannot directly act on osteoblasts to enhance the rate of bone formation and thus increase bone mass accumulation. However, simply inhibiting bone resorption does not directly promote the formation of new bone in the body and may cause drug-related side effects. For example, bisphosphonates, a representative drug targeting osteoclasts in clinical practice, are associated with serious complications. Compared with anti-resorption drugs, anabolic drugs have greater anti-fracture efficacy and can produce greater increases in bone density. Unfortunately, there are no natural small molecule drugs targeting osteogenic activity in clinical practice, and to date, there are few research reports on natural active small molecules that promote osteoblast differentiation and mineralization.

[0004] The WNT signaling pathway is the most classic and crucial signaling pathway for developing drugs targeting bone anabolic metabolism. Activating the WNT signaling pathway can increase bone strength and regulate bone homeostasis. Therefore, the search for novel therapeutic drugs for bone metabolism, especially novel anti-bone metabolic diseases drugs that promote osteoblast activity by regulating the WNT signaling pathway, is urgently needed. Summary of the Invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide a class of naphthidine pyran alkaloids or their pharmaceutically acceptable salts.

[0006] The naphthidine pyran alkaloids of the present invention, or their pharmaceutically acceptable salts, can promote osteoblast mineralization by targeting β-catenin protein, exhibiting significant osteoblast mineralization-promoting activity, and can be used in the preparation and development of drugs for treating osteometabolic diseases.

[0007] Another object of the present invention is to provide the use of the above-mentioned naphthidine pyran alkaloid compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention or treatment of bone metabolic diseases.

[0008] The objective of this invention is achieved through the following solution:

[0009] In a first aspect, a class of naphthidine pyran alkaloids or pharmaceutically acceptable salts thereof, having the structure shown in Formula I:

[0010]

[0011] Wherein, R1 is H, halogen, hydroxyl, carboxyl, cyano, nitro, amino, carbonyl, ester, substituted or unsubstituted C1-C20 straight-chain or branched alkyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 alkylene, C1-C20 haloalkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C3-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C2-C20 alynylene, substituted or unsubstituted C1-C20 hydroxyl group, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted benzyloxy, substituted or unsubstituted C2-C20 heterocyclic group or heterocyclic aryl containing one or more of N, O and S.

[0012] R2 and R3 are, independently, H, carbonyl, substituted or unsubstituted C1-C20 straight-chain or branched alkyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 alkylene, C1-C20 haloalkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C3-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C2-C20 alynylene, substituted or unsubstituted C1-C20 alkyloxy, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted benzyloxy, substituted or unsubstituted C2-C20 heterocyclic or heterocyclic aryl containing one or more of N, O and S;

[0013] R4-R7 are, independently, H, halogen, hydroxyl, carboxyl, cyano, nitro, amino, carbonyl, ester, substituted or unsubstituted C1-C20 straight-chain or branched alkyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 alkylene, C1-C20 haloalkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C3-C20 alkenyl, or substituted or unsubstituted C2-C20 alkyne. The group includes: alkyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C1-C20 alkyloxy, substituted or unsubstituted C1-C20 N-alkyl-substituted amino, substituted or unsubstituted C1-C20 N,N-dialkyl-substituted amino, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted benzyloxy, substituted or unsubstituted C2-C20 heterocyclic group or heterocyclic aryl containing one or more of N, O and S.

[0014] Furthermore, the above substitution refers to the substitution of one or more hydrogen atoms by substituents, which are independently selected from one or more of hydrogen, halogen, hydroxyl, cyano, nitro, amino, C1-C20 straight-chain or branched alkyl, C3-C20 cycloalkyl, C1-C20 haloalkyl, C2-C20 ester, C2-C20 alkenyl, C2-C20 alkynyl, C1-C20 hydroxyloxy, aryl, C1-C20 N-alkyl-substituted amino, and C1-C20 N,N-dialkyl-substituted amino.

[0015] Furthermore, one or more hydrogen atoms in R1-R7 may be substituted with fluorine, chlorine, bromine, iodine, oxygen, alkenyl, alkynyl, aryl, hydroxyl, amino, carbonyl, carboxyl, ester, cyano, C1-C20 straight-chain or branched alkyl, C3-C20 cycloalkyl, C1-C20 alkyloxy or nitro groups.

[0016] Furthermore, the number of carbon atoms in the aforementioned aryl groups ranges from C6 to C20.

[0017] The naphthidine-pyran alkaloids or their pharmaceutically acceptable salts of the present invention, specifically 2,7-diazabenzylpyran skeleton compounds, represent a class of novel alkaloid secondary metabolites. Among the 10 isomers of naphthidine, 1,8-naphthidine is common, but 2,7-naphthidine is extremely rare in nature. This invention is the first to discover a novel skeleton compound composed of 2,7-naphthidine ketone and pyranol, which is of great significance for discovering and studying new targets for osteolytic metabolic diseases. The compounds of this invention can promote osteoblast mineralization by targeting β-catenin protein, exhibiting significant osteoblast-promoting activity.

[0018] A second aspect of the present invention provides a method for preparing the above-mentioned naphthidine pyran alkaloid compound or a pharmaceutically acceptable salt thereof, obtained by isolating it from Penicillium fermentation products.

[0019] The Penicillium allii-sativi described has the accession number MCCC 3A00580 and is deposited at the Marine Culture Collection of China (MCCC).

[0020] The Penicillium ferment can be obtained by fermenting Penicillium in a fermentation medium.

[0021] The fermentation can be carried out at room temperature.

[0022] The fermentation time can be 25-35 days.

[0023] The composition of the fermentation medium, in parts by mass and parts by volume (g, mL), may be: 80-120 parts by mass of oats, 3.0-3.6 parts by mass of sea salt, and 80-120 parts by volume of water.

[0024] The Penicillium is preferably first cultured on a PDA plate (e.g., cultured at room temperature for 3-4 days) to obtain mycelium, and then the mycelium is inoculated into PDB culture medium to obtain seed culture. Finally, the seed culture is inoculated into fermentation medium for culture.

[0025] Furthermore, the separation method includes the following steps:

[0026] The Penicillium fermentation product was extracted with ethyl acetate. The organic extract was passed through a normal-phase column chromatography column and eluted with petroleum ether, dichloromethane, and methanol, respectively. The dichloromethane layer was concentrated to obtain a crude extract. The crude extract was separated by normal-phase silica gel column chromatography with gradient elution using a petroleum ether-ethyl acetate system to obtain eight crude fractions: Fr.1 to Fr.8. The crude fraction Fr.6 was separated by dextran gel column chromatography (pure methanol) to obtain three crude fractions: Fr.6.1 to Fr.6.3. The crude fraction Fr.6.3 was purified by recrystallization to obtain the compound of this invention.

[0027] The preferred solvent for recrystallization is methanol-water (9:1).

[0028] The compound of this invention can be isolated from Penicillium, and has the advantages of being environmentally friendly, having a simple process, and producing a high-purity product.

[0029] A third aspect of the present invention provides the use of the above-mentioned naphthidine pyran alkaloid compounds or pharmaceutically acceptable salts thereof in the preparation of osteoblast activators.

[0030] Furthermore, the osteoblasts include bone marrow mesenchymal stem cells and mouse embryonic osteoblast precursor cells MC3T3-E1.

[0031] A fourth aspect of the invention provides the use of the above-mentioned naphthidine pyran alkaloid compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention and / or treatment of bone metabolic diseases.

[0032] Furthermore, the bone metabolic diseases include fractures, osteoporosis (including primary osteoporosis, such as postmenopausal osteoporosis, senile osteoporosis, and osteoporosis secondary to various endocrine diseases or disuse), rickets, endocrine bone diseases, osteodegenerative osteitis, hereditary bone diseases, etc.

[0033] Furthermore, the drugs, whether identical or different, comprise therapeutically effective amounts of naphthidine pyran alkaloids or pharmaceutically acceptable salts thereof.

[0034] Furthermore, the aforementioned drug can be prepared into various pharmaceutical dosage forms using conventional methods. These dosage forms include: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, powders, pills, elixirs, suspensions, tinctures, drops, and other oral dosage forms, as well as injections and other non-oral dosage forms, such as injections.

[0035] Furthermore, the drug may also contain one or more pharmaceutically acceptable carriers or excipients.

[0036] Furthermore, the carrier or excipient may include diluents, adhesives, surfactants, humectants, adsorbents, lubricants, fillers, disintegrants, preservatives, etc.

[0037] The present invention also provides a pharmaceutical composition for the prevention and / or treatment of bone metabolic diseases, comprising a naphthidine pyran alkaloid compound or a pharmaceutically acceptable salt thereof.

[0038] The present invention also provides the use of the above-mentioned naphthidine pyran alkaloid compounds or pharmaceutically acceptable salts thereof in the preparation of health products for the prevention and / or treatment of bone metabolic diseases.

[0039] The activity of the naphthidine pyran alkaloids or their pharmaceutically acceptable salts of the present invention was tested using methods such as CCK-8 assay, Alizarin Red S staining, quantitative real-time PCR (QPCR), RNA transcriptome sequencing (RNA-seq), immunofluorescence, molecular docking, and animal models of bone metabolism. These methods demonstrated that the compounds of the present invention can significantly promote fracture repair by targeting β-catenin protein to promote osteogenic mineralization, thereby exerting anti-bone metabolism activity. They have good application prospects in the preparation of drugs for the prevention or treatment of bone metabolic diseases. Attached Figure Description

[0040] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 This is a single-crystal diffraction structure diagram of compound formula I of the present invention.

[0042] Figure 2 The effects of the compounds of this invention on osteoblast mineralization and toxicity are shown. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 vs DMSO.

[0043] Figure 3 The results of RNA-seq analysis of the compounds of this invention are shown.

[0044] Figures 4-6 The effects of the compounds of this invention on proteins and genes closely related to osteogenic differentiation.

[0045] Figure 7 This describes the interaction between the compound of the present invention and the β-catenin protein.

[0046] Figure 8 The effect of the compounds of this invention on fracture repair.

[0047] Figure 9 This invention provides a quantitative method for measuring the amount of callus in fracture repair using the compounds of this invention. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, all materials involved in the following embodiments are commercially available. Unless otherwise specified, all methods described are conventional methods.

[0049] Example 1: Preparation and structural characterization of naphthidine pyran alkaloids:

[0050] (1) Penicillium allii-sativi (deposited at China Marine Microbial Culture Collection Center, accession number MCCC 3A00580) was cultured on PDA plates at 28℃ for 3-4 days; then fresh mycelium was inoculated into a culture medium containing 400mL PDB; after 24 hours, 10mL of seed culture was inoculated into 1L of fermentation medium and fermented at room temperature for 25-35 days to obtain Penicillium fermentation product; the composition of the fermentation medium, in parts by weight and parts by volume (g, mL), can be: 80-120 parts by weight of oats, 3.0-3.6 parts by weight of sea salt, and 80-120 parts by volume of water.

[0051] (2) The fermentation product obtained in step (1) was extracted three times with ethyl acetate, and the organic solvent was evaporated under reduced pressure to obtain organic extract (200g); these extracts were passed through a normal phase column chromatography column and eluted with petroleum ether, dichloromethane and methanol respectively; the dichloromethane layer was concentrated to obtain crude extract (63.0g).

[0052] (3) The crude extract obtained in step (2) was separated by normal phase silica gel column chromatography and eluted with a petroleum ether-ethyl acetate system to obtain 8 crude fractions (Fr.1~Fr.8);

[0053] (4) The crude fraction Fr.6 (7.0 g of which was obtained by elution at a ratio of 5:1) from step (3) was purified by dextran gel chromatography (pure methanol) and recrystallization (methanol-water, 9:1) to obtain compound I (22.7 mg).

[0054] (5) The planar structure of the compound of formula I obtained in the above steps was determined by 1D and 2D NMR spectra and high-resolution mass spectrometry, respectively. Then, its absolute configuration was determined by X-ray single-crystal diffraction, as detailed below:

[0055] Compound I is a yellow needle-like crystal. Its molecular formula was determined to be C636 based on the main ion peak in its high-resolution mass spectrometry. 11 H 10 N2O4. 1 H, 13 The C10 NMR data (Table 1) and DEPT and HMBC spectra show 11 carbon signals, including one oxymethylene (δ¹⁰) group. C 65.6 t), two oxygen-containing methines (δ C 62.9 d, 64.8 d), three olefin bonds (δ C 114.2d, 150.0d, 151.1d) and five aprotic carbons (δ CThe planar structure of the compound was determined using two-dimensional data (159.4 s, 120.0 s, 125.2 s, 130.2 s, 136.8 s), and finally, X-ray single-crystal diffraction was used. Figure 1 The absolute configuration of compound I was determined and named penicinaphthyridine A, PTA.

[0056] Table 1

[0057]

[0058] Note: 400M, dimethyl sulfoxide-d6.

[0059] Example 2: Cytotoxic activity of compound PTA against osteoblasts.

[0060] This embodiment sets up the following 3 groups:

[0061] Blank control group: equal volume of culture medium, containing no cells and without the addition of PTA;

[0062] Solvent control group: equal volume of culture medium, 0.1% dimethyl sulfoxide, containing cells, without the addition of PTA;

[0063] Experimental group: Equal volume of culture medium containing cells, 0.1% dimethyl sulfoxide + PTA;

[0064] The specific steps include: BMSCs cells in the logarithmic growth phase are inoculated at a rate of 2 × 10⁻⁶. 5 Cells were seeded at a density of 5000 cells / well for MC3T3-E1 cells in 96-well plates. After 24 h, control solvent or corresponding differentiation inducer and test compound (0, 1, 2.5, 5, 10, 20 μM) were added, with three replicates per sample. After 48 h of incubation, 10 μL of CCK-8 solution was added to each well, and the plates were incubated at 37 °C for 4 h. The OD value was measured at 450 nm using a microplate reader.

[0065] The results are as follows Figure 2 In A and 2D, the naphthidine pyran alkaloid compound PTA of the present invention still showed no cytotoxic activity against BMSCs and MC3T3-E1 at concentrations as high as 20 μM.

[0066] Example 3: Compound PTA promotes osteoblast mineralization

[0067] Mineralized nodules are a marker of osteoblast differentiation and maturation. Because these nodules are rich in calcium, they can be specifically stained bright red by Alizarin Red S. This embodiment uses Alizarin Red staining to detect the mineralization activity of PTA on osteoblasts under inducing agent conditions and to quantify the mineralization capacity of osteoblasts.

[0068] This embodiment sets up the following 5 groups:

[0069] Blank control group (NC): Equal volume of normal culture medium, without the addition of PTA;

[0070] Inducer control group (Diff): Equal volume of induction culture medium, without the addition of PTA;

[0071] Solvent control group (DMSO): equal volume of induction culture medium, 0.1% dimethyl sulfoxide, no PTA added;

[0072] Positive control group (PM): equal volume of induction culture medium, 0.1% dimethyl sulfoxide, and 1 μM Urmorphamine added;

[0073] Experimental group (PTA): equal volume of induction culture medium, 0.1% dimethyl sulfoxide + different concentrations of PTA (2.5, 5, 10, 20 μM);

[0074] The specific steps include: Three-week-old wild-type mice were euthanized by cervical dislocation, and the femur and tibia were removed and placed in sterile PBS. In a sterile laminar flow hood, muscles and fascia were removed, the metaphysis was removed, and the bone marrow was rinsed with α-MEM culture medium (containing 10% FBS + 0.1% penicillin / streptomycin) using a 5mL syringe, followed by thorough pipetting. The bone marrow was then transferred to a culture dish for culture. After 4–6 days, once the cells adhered to the dish, bone marrow mesenchymal stem cells (BMSCs) were obtained and passaged for propagation. The BMSCs were digested, counted, and diluted to 2 × 10⁻⁶ cells / mL. 5 Cell suspension was added at 100 μL / well in 96-well plates. After 24 hours, once cells had adhered, drug treatment could begin. Simultaneously, the normal culture medium was replaced with induction medium (containing 10% FBS + 0.1% penicillin-drug antibiotics + 50 μg / mL vitamin C + 5 mM sodium β-glycerophosphate). The medium was changed and drug treatment was performed every two days. On day 10, cells were collected and stained with Alizarin Red S. The culture medium was removed, and cells were fixed with 4% paraformaldehyde for 20 min, washed three times with 70% ethanol, and the ethanol was discarded until completely dry. Then, 50 μL of 2% Alizarin Red S solution was added to cover the cells, and they were cultured at room temperature for 3-5 min. Next, 200 μL of 50% ethanol was added, and the cells were washed three times, air-dried completely, and stored at room temperature. Microscopic images were then taken and recorded, and the size, number, and morphology of nodules were used to assess the cells' mineralization capacity. Simultaneously, the stained deep red complex was dissolved in 10% acetic acid and transferred to a new 96-well plate. The OD value was measured at 405 nm, and the mineralization capacity was quantified. Mineralization activity (%) = ([(OD value)] / ([(OD value)])) 405 Dosing port - OD 405 Blank hole) / (OD) 405 Reference Hole - OD 405 Blank hole)])×100%.

[0075] MC3T3-E1 cells were seeded into 96-well plates at a density of 5000 cells / well and cultured for 21 days, with medium changed every two days. The drug addition settings and staining procedures were the same as those for osteogenic mineralization of BMSCs described above.

[0076] The results are shown below. Figure 2 . Figure 2 A- Figure 2 C represents the experimental results for BMSCs. Figure 2 D- Figure 2 F represents the experimental results for MC3T3-E1. As can be seen from the figure, Figure 2 B-2C indicates that the naphthidine pyran alkaloid compound PTA of this invention can significantly promote osteogenic mineralization of BMSCs, and exhibits a concentration-dependent effect. Secondly, Figure 2 E-2F indicates that the naphthidine pyran alkaloid compound PTA of the present invention can still promote osteogenic mineralization of MC3T3-E1 in a concentration-dependent manner, which further confirms the osteogenic mineralization-promoting activity of the naphthidine pyran alkaloid compound of the present invention.

[0077] Example 4: PTA promotes osteogenic mineralization by activating the WNT signaling pathway

[0078] To gain a deeper understanding of the role of PTA in osteogenic processes, RNA sequencing (RNA-seq) transcriptomic analysis was performed in this embodiment. MC3T3-E1 cells were seeded at a density of 150,000 per well in 6-well plates and cultured overnight. Osteogenic induction medium with or without PTA (10 μM) was added, and the cells were cultured for another 8 days, with the medium changed every two days.

[0079] The specific steps include:

[0080] (1) RNA extraction and library construction

[0081] Total RNA was extracted using TRIzol reagent according to the manufacturer's instructions. RNA purity and quantification were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA), and RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Transcriptome libraries were constructed using the VAHTS Universal V6 RNA-seq Library Prep kit according to the manufacturer's instructions.

[0082] (2) RNA sequencing and differentially expressed gene analysis

[0083] Library sequencing was performed using the Llumina Novaseq 6000 sequencing platform, generating 150bp paired-end reads. Raw reads in FASTQ format were processed using FASTP software to remove low-quality reads, resulting in clean reads for subsequent data analysis. Reference genome alignment was performed using HISAT2 software, and gene expression levels (FPKM) were calculated. Read counts for each gene were obtained using HTSeq-count. PCA analysis and plotting were performed on genes using R (v 3.2.0) to assess sample biological replicates. Differentially expressed genes (DEGs) were analyzed using DESeq2 software, where genes meeting the thresholds of q-value < 0.05 and foldchange > 2 or foldchange < 0.5 were defined as DEGs. Hierarchical clustering analysis was performed on DEGs using R (v 3.2.0) to visualize gene expression patterns across different groups and samples. Subsequently, GO and KEGGPathway enrichment analyses were performed on differentially expressed genes based on the hypergeometric distribution algorithm to screen for significantly enriched functional entries. The graphs were plotted using the Microbioinformatics visualization platform (https: / / www.bioinformatics.com.cn / ). The results are shown below. Figure 3 .

[0084] As shown in the figure, PCA analysis revealed good intragroup consistency and differences between the control group and the PTA group. Figure 3 A). Statistical and volcano plot analyses showed that 93 genes were significantly upregulated by PTA, while 178 genes were significantly downregulated. Figure 3 B- Figure 3 C). According to heatmap analysis, there was extensive differential gene expression between the two groups. Figure 3 D). GO enrichment analysis of differentially expressed genes revealed transcriptional features positively regulated by bone marrow cell differentiation, skeletal development, skeletal system development, WNT signaling bodies, and extracellular matrix. Figure 3 E). Gene set enrichment analysis (GSEA) revealed transcriptional signatures positively regulating bone marrow cell differentiation in PTA. KEGG analysis showed that DEG was associated with functional annotation changes via the MAPK, WNT, and Hedgehog signaling pathways. Based on enrichment levels and corresponding p-values, the WNT signaling pathway was considered the most likely mechanism by which PTA induces osteogenic changes. Figure 3 F).

[0085] Example 5: PTA promotes the expression of β-catenin, a key protein in the WNT signaling pathway.

[0086] To further verify the mechanism, protein-protein interaction (PPI) analysis, Western blotting, and immunofluorescence assay were performed.

[0087] 5.1 Protein-protein interaction (PPI) analysis

[0088] The specific steps include: 250 genes from the Wnt / β-catenin signaling pathway were obtained from GeneCards (https: / / pathcards.genecards.org). The obtained genes were uploaded to String database version 12.0 (https: / / cn.string-db.org / ) to construct a PPI network. The resulting network was visualized using Cytoscape 3.7.0 and 3.9.1 software, and the network topology parameters (degree, spacing, and proximity) of the identified targets were determined using network analysis tools in Cytoscope.

[0089] result Figure 4 A indicates that, based on the node size and number of edges in the WNT signaling pathway, CTNNB1 (β-catenin protein) is indeed the core target in the WNT / β-catenin signaling pathway.

[0090] 5.2 Western blot for protein immunoblotting

[0091] The specific steps include: seeding BMSCs at a density of 500,000 per well into 6-well plates and culturing overnight. Adding osteogenic induction medium with or without PTA (10 μM) and culturing for another 8 days, changing the medium every two days. Washing cells twice with pre-chilled PBS buffer. Adding 200 μL of cell lysis buffer containing protease and phosphatase inhibitors to each well, incubating on ice for 10 min, scraping cells thoroughly from the wells with a cell scraper, and transferring to 1.5 mL centrifuge tubes. Performing the process on ice, vortexing for 5 seconds every 5 min for a total of 3 times, then centrifuging at 14000 rpm for 30 min at 4°C, collecting the supernatant, and determining the protein concentration using a BCA kit. Next, taking 50 μg of protein sample, adding 25% volume of 5x Loading buffer (containing 1% β-mercaptoethanol), heating in a 105°C metal bath for 7 min, centrifuging, and electrophoresis in Tris-glycine buffer (80V-120V). The electroporation buffer was pre-cooled at 4°C. The PVDF membrane (activated by soaking in methanol solution for 20 seconds) and filter paper were pre-immersed in the buffer. Following the order of filter paper-gel-PVDF membrane-filter paper, air bubbles were removed. The membranes were then placed in the electroporation tank with the positive electrode facing upwards. The pre-cooled buffer and ice packs were added, and electroporation was performed on ice (100V, 60min). The electroporated PVDF membrane was washed three times with ultrapure water, then blocked in 5% skim milk on a shaker at room temperature for 1 hour. It was washed once with TBST, then incubated overnight at 4°C with primary antibody. Finally, the corresponding secondary antibody solution (diluted with 5% skim milk) was added, and the membrane was shaken on a shaker at room temperature for 1 hour. The membrane was washed four times with TBST for 8 minutes each time. Solution A and solution B of the luminescent substrate ECL solution were mixed in a 1:1 ratio. A small amount of the mixture was added to cover the protein surface, incubated for 2 minutes, and immediately exposed in a dark room. The bands were recorded.

[0092] Western blot analysis showed that PTA promoted the expression of β-catenin and osteopontin (OPN) in BMSCs. Figure 4 B-4C).

[0093] 5.3 Immunofluorescence assay

[0094] The specific steps include: seeding BMSCs at a density of 300,000 per well into confocal 6-well plates (NEST, 801001-1, 20mm, TC) and culturing overnight. Adding osteogenic induction medium with or without PTA (10μM) and culturing for another 5 days, changing the medium every two days. Incubating cells in 4% PFA at room temperature for 10 min, then washing cells three times with ice-cold PBS. Incubating the sample with PBS (containing 0.25% Triton X-100) for 10 min, then washing cells three times with PBS, 5 min each time. Incubating cells for 30 min with PBST (PBS + 0.1% Tween 20) containing 1% BSA and 22.52 mg / mL glycine, blocking non-specific antibody binding. Incubating overnight at 4°C with primary antibody in a humidified chamber at room temperature. Washing cells three times with PBS, 5 min each time. Incubating cells with the corresponding secondary antibody in the dark for 1 hour. Washing cells three times with PBS in the dark, 5 min each time. Finally, the cells were incubated with 1 μg / mL DAPI (DNA staining) for 30 min, washed with PBS 3 times for 5 min each time, and photographed using a confocal fluorescence microscope after completion. The cells were then stored at 4°C in the dark.

[0095] Immunofluorescence assays showed that, compared with the control group, PTA-treated BMSCs cells exhibited significantly increased levels of OPN and β-catenin protein. Figure 5 ).

[0096] Example 6: PTA upregulates the mRNA expression levels of osteogenic differentiation markers Alpl, Runx2, Ibsp, Bglap, and Opn under differentiation-inducing conditions.

[0097] Key genes involved in the differentiation of bone marrow mesenchymal stem cells into osteoblasts include alkaline phosphatase (Alp1), Opn, and integrin-binding sialoprotein (Ibsp). These key genes play important roles in osteoblast differentiation and mineralization. Alkaline phosphatase activity is the most widely recognized marker of osteoblast activity and a typical protein product of osteoblast phenotype and differentiation. Bone sialoprotein appears after alkaline phosphatase expression, is located in the mineralized matrix, and can promote the nucleation of hydroxyapatite mineralization in vitro, as well as increase calcium binding and the formation of calcium nodules. Osteopontin is one of the most abundant non-collagenous proteins in the bone matrix produced by osteoblasts. In this embodiment, the osteogenic activity of the compound of the present invention was evaluated by detecting the expression level of PTA in the mRNA of these osteoblast-specific genes using qPCR.

[0098] This embodiment sets up the following two groups:

[0099] Control group: equal volume of induction culture medium, 0.1% dimethyl sulfoxide, no PTA added;

[0100] Experimental group: equal volume of induction culture medium, 0.1% dimethyl sulfoxide + PTA;

[0101] The specific steps include:

[0102] 1. Cell Culture: BMSCs were seeded at a density of 500,000 per well in 6-well plates and cultured overnight. Osteogenic induction medium with or without PTA (10 μM) was added and cultured for another 5 days, with the medium changed every two days.

[0103] 2. Total RNA Extraction: Wash cells once with pre-chilled PBS, then add 200 μL of Trizol to each well and lyse on ice for 10 min. Collect cells into 1.5 mL EP tubes, add 0.2 mL of chloroform, vortex vigorously for at least 15 seconds, and incubate at room temperature for 2–5 min. Then centrifuge at low temperature (12000 rpm, 4℃, 15 min). Take the supernatant (400 μL) and transfer it to a new enzyme-free EP tube. Add an equal volume of isopropanol to the aspirated supernatant, invert and mix well, and incubate on ice for 15 min. Centrifuge at low temperature (12000 rpm, 4℃, 15 min). Slowly aspirate the supernatant without touching the bottom of the EP tube, add 1 mL of pre-chilled 75% ethanol DEPC water. Gently shake by inverting and centrifuge at low temperature (12000 rpm, 4℃, 15 min). Repeat the above steps once. After centrifugation, remove the EP tube; a white RNA precipitate will be visible at the bottom. Aspirate the supernatant and air dry in a clean bench for about 5–10 minutes. Add 20 μL of pre-chilled DEPC water, incubate on ice to dissolve the RNA, and then mix thoroughly with a pipette.

[0104] 3. Determination of RNA concentration and purity: RNA concentration, A260 / A280 ratio and A260 / A230 ratio were detected using an ultra-micro UV-Vis spectrophotometer.

[0105] 4. Reverse transcription: DNA was synthesized using RNA as a template using the Novizan reverse transcription kit. In a 200 μL L Nase-free centrifuge tube, prepare 4 μL of 4X g DNAwiper mix, template RNA (Total RNA: 1 μg), and DEPC, bringing the total volume to 16 μL. Mix thoroughly by pipetting and centrifuge. Place the tube in a gradient RCR system and set the program to 42℃ for 2 min.

[0106] 5. Perform reverse transcription: Remove the centrifuge tube, add 4 μL of 5X HiScrippt q RT Super Mix reagent, mix well by pipetting, centrifuge, and place in a gradient RCR instrument. Set the program: 50℃, 5 min; 85℃, 5 s. After the program is complete, remove the centrifuge tube and store it in a -20℃ freezer for later use.

[0107] 6. Real-time quantitative PCR: Primer preparation: Prepare a 10 μL reaction mixture using the following amounts: 3.6 μL LEPC water, 0.2 μL each of primers (10 μM), 5 μL SYBR, and 1 μL cDNA. Add these to a 96-well plate and centrifuge (1600 rpm, 10 min). PCR reaction conditions: 94℃ for 5 min pre-denaturation; 94℃ for 30 s denaturation, 59℃ for 45 s annealing, 72℃ for 1 min extension, 39 cycles; 72℃ for 10 min extension. Calculate the fold change using the HPRT internal control after the reaction. The calculation formula is:

[0108]

[0109] Table 2. Primer sequences used in Q-PCR

[0110]

[0111] qPCR results ( Figure 6 The results showed that PTA can promote the expression of key osteogenic genes Alpl, Runx2, Ibsp, Bglap, and Opn in BMSCs, indicating that PTA can exert its anti-osteometabolic activity by upregulating the expression of these osteogenic-specific genes to promote osteogenic activity.

[0112] Example 7: PTA targets β-catenin protein

[0113] To assess the affinity of PTA for the active target β-catenin, the interaction between PTA and the β-catenin protein was analyzed using molecular docking.

[0114] The specific steps include: finding and downloading the target protein from the PBD library; preparing the ligand and generating a grid file using the OPLS3e force field in the receptor grid generation of the Schrodinger software; and then preparing the protein through restriction minimization using the OPLS3e force field. The receptor mesh generator creates the mesh location with a docking length of [value missing]. The center of the grid box coincides with the center of the ligand in the crystal file. Glid uses standard precision SP for semi-flexible docking, and the Epik module scores the docking scoring function. Molecular docking is performed using LigandDocking in the Schrodinger Glide module. The docking score is reported in kcal / mol; the more negative the value, the better the binding.

[0115] Molecular docking results showed that PTA could successfully enter the binding pocket of β-catenin. Figure 7 A), the docking score was -7.541 kcal / mol ( Figure 7 B), this strong binding activity is achieved through hydrogen bonding with valine residue at position 135 of the β-catenin protein. Figure 7 C- Figure 7 E).

[0116] Example 8: PTA can effectively promote fracture repair

[0117] This embodiment sets up the following two groups:

[0118] Control group: An equal volume of aqueous solution containing 0.1% dimethyl sulfoxide + PBS, without the addition of PTA;

[0119] Experimental group: Equal volume of PBS containing 0.1% dimethyl sulfoxide + 10 mg / kg PTA;

[0120] The specific steps included: Six-week-old WT mice were selected for fracture modeling. Mice were randomly divided into two groups of five. Mice were anesthetized with isoflurane inhalation. Hair was shaved from the left hind leg knee joint to the midline of the back, exposing the entire femur. The skin was disinfected three times with iodine using cotton swabs. A small incision was made in the middle of the left hind leg, and the skin was bluntly dissected with forceps to expose the quadriceps femoris muscle. The muscle was then bluntly dissected at the intermuscular space to expose the femur. A small power tool saw was used to cut the middle section of the femur, creating a linear fracture. A sterile 1mL needle was inserted parallel to the femur at the knee joint, connecting the fractured ends. The needle was removed with the small tool saw, leaving it fixed within the femur. 20μL of a matrix adhesive, with or without the compound, was applied directly to the fracture site. After the adhesive solidified, the muscles and skin were sutured together. The wound was disinfected twice more with iodine. After the surgery was completed and the mice stabilized, they were carefully returned to their cages, maintaining the same environment as described above. The drug was administered intraperitoneally every 2 days. Samples were collected 14 days later, including serum and femur fragments at the fracture sites. All fractured femurs were scanned using μ-CT.

[0121] μ-CT reconstructed images show that PTA can significantly promote fracture repair, as evidenced by an increase in callus volume. Figures 8-9The study results indicate that the deep-sea compound PTA shows strong potential for anti-bone metabolism activity.

[0122] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0123] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A class of naphthidine pyran alkaloids or their pharmaceutically acceptable salts, characterized in that... It has the structure shown in Equation I: Wherein, R1 is H, halogen, hydroxyl, carboxyl, cyano, nitro, amino, carbonyl, ester, substituted or unsubstituted C1-C20 straight-chain or branched alkyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 alkylene, C1-C20 haloalkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C3-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C2-C20 alynylene, substituted or unsubstituted C1-C20 hydroxyl group, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted benzyloxy, substituted or unsubstituted C2-C20 heterocyclic group or heterocyclic aryl containing one or more of N, O and S. R2 and R3 are, independently, H, carbonyl, substituted or unsubstituted C1-C20 straight-chain or branched alkyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 alkylene, C1-C20 haloalkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C3-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C2-C20 alynylene, substituted or unsubstituted C1-C20 alkyloxy, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted benzyloxy, substituted or unsubstituted C2-C20 heterocyclic or heterocyclic aryl containing one or more of N, O and S; R4-R7 are, independently, H, halogen, hydroxyl, carboxyl, cyano, nitro, amino, carbonyl, ester, substituted or unsubstituted C1-C20 straight-chain or branched alkyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C1-C20 alkylene, C1-C20 haloalkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C3-C20 alkenyl, or substituted or unsubstituted C2-C20 alkyne. The group includes: alkyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C1-C20 alkyloxy, substituted or unsubstituted C1-C20 N-alkyl-substituted amino, substituted or unsubstituted C1-C20 N,N-dialkyl-substituted amino, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted benzyloxy, substituted or unsubstituted C2-C20 heterocyclic group or heterocyclic aryl containing one or more of N, O and S.

2. The naphthidine pyran alkaloid compound or its pharmaceutically acceptable salt according to claim 1, characterized in that... The substitution refers to the substitution of one or more hydrogen atoms by substituents, which are independently selected from one or more of hydrogen, halogen, hydroxyl, cyano, nitro, amino, C1-C20 straight-chain or branched alkyl, C3-C20 cycloalkyl, C1-C20 haloalkyl, C2-C20 ester, C2-C20 alkenyl, C2-C20 alkynyl, C1-C20 hydroxyloxy, aryl, C1-C20 N-alkyl-substituted amino, and C1-C20 N,N-dialkyl-substituted amino.

3. The naphthidine pyran alkaloid compound or its pharmaceutically acceptable salt according to claim 1, characterized in that: One or more hydrogen atoms in R1-R7 are substituted with fluorine, chlorine, bromine, iodine, oxygen, alkenyl, alkynyl, aryl, hydroxyl, amino, carbonyl, carboxyl, ester, cyano, C1-C20 straight-chain or branched alkyl, C3-C20 cycloalkyl, C1-C20 alkyloxy, or nitro groups.

4. The naphthidine pyran alkaloid compound or its pharmaceutically acceptable salt according to any one of claims 1-3, characterized in that: The number of carbon atoms in the aryl group ranges from C6 to C20.

5. A method for preparing the naphthidine pyran alkaloid compound or its pharmaceutically acceptable salt according to any one of claims 1-4, characterized in that... It was isolated from Penicillium fermentation products.

6. The use of the naphthidine pyran alkaloid compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-4 in the preparation of osteoblast activators.

7. The use of the naphthidine pyran alkaloid compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-4 in the preparation of medicaments for the prevention and / or treatment of bone metabolic diseases.

8. The application according to claim 7, characterized in that: The bone metabolic diseases include at least one of the following: fracture, osteoporosis, rickets, endocrine bone disease, osteitis deformans, and hereditary bone disease.

9. A pharmaceutical composition for the prevention and / or treatment of bone metabolic diseases, characterized in that... It comprises a naphthidine pyran alkaloid compound as described in any one of claims 1-4, or a pharmaceutically acceptable salt thereof.

10. The use of the naphthidine pyran alkaloid compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-4 in the preparation of health products for the prevention and / or treatment of bone metabolic diseases.

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