Indole diterpene alkaloid compounds, and methods of making and using the same

Abstract

The application provides indole diterpene alkaloid compounds and a preparation method and application thereof, and relates to the field of marine drugs. Two novel indole diterpene alkaloid compounds are disclosed, which are named brefeldindole E and brefeldindole F, the structural formulae of which are shown as formula (I) and formula (II) respectively. Both of them have a significant inhibitory effect on LPS-induced NF-kappa B luciferase at a concentration of 10 micromoles, further inhibit RANKL-induced osteoclast precursor cell BMMs cell differentiation into osteoclasts, and have no obvious cytotoxicity. The compounds can effectively replace existing bisphosphonate drugs and denosumab, inhibit the bone resorption activity of osteoclasts, are ideal candidate compounds for developing new osteoclast differentiation inhibitor drugs, and are used for preventing and treating osteoclast-related osteolytic diseases.

Description

Technical Field

[0001] This invention relates to the field of marine pharmaceuticals, specifically to indole diterpenoid alkaloids, their preparation methods, and applications. Background Technology

[0002] Osteoporosis is a common degenerative bone disease, mainly characterized by increased bone fragility, decreased bone mass, and destruction of bone microstructure, seriously threatening the health of the aging population and postmenopausal women. Osteoclasts (OCs) are specialized cells formed by the fusion of monocyte / macrophage hematopoietic lineage precursor cells and 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 bone metastases from tumors. Inhibiting the formation and resorption function of OCs is one of the main strategies for treating osteoporosis. Osteoclast formation is a stepwise process initiated by the binding of the receptor activator of nuclear kappaB ligand (RANKL) to its receptor RANK on monocyte / macrophage precursors. Currently, the main osteoclast differentiation-related inhibitors used clinically are denoximab and bisphosphonates, but both have certain complications and side effects. Therefore, there is an urgent need to find a safe, effective, quality-controllable, and economical drug that targets and inhibits osteoclast formation and bone resorption, so as to effectively address the huge clinical demand for osteoclast differentiation inhibitors.

[0003] Marine natural products possess unique advantages such as novel structures and excellent activity, making them an important source of lead compounds for novel drugs. Currently, only one cyclic indole diterpenoid compound, peniditerpenoid A, has been found to have relevant effects among marine-derived osteoclast differentiation inhibitors (see Chinese Invention Patent Application 202310241947.0, "Marine Fungal-Derived Open-Ring Indole Diterpenoid A, its Preparation Method and Application"), and no other indole diterpenoid compounds have been found to have relevant effects. Marine microbial resources are abundant, making them an ideal resource for discovering novel anti-osteoporosis lead compounds. Therefore, researching and developing novel, highly effective, and low-toxicity osteoclast differentiation inhibitors from marine microbial resources is of great significance for the prevention and treatment of osteolytic diseases. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides new applications for indole diterpenoid alkaloid compounds (named brefeldindole E and brefeldindole F).

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A novel class of indole diterpenoid alkaloids, named brefeldindole E and brefeldindole F respectively; the structural formula of brefeldindole E is shown in formula (I), and the structural formula of brefeldindole F is shown in formula (II):

[0007]

[0008] Another object of the present invention is to protect a strain of *Penicillium brefeldianum* GXIMD 02511 used for preparing the above-mentioned indole diterpenoid alkaloids, which was deposited on September 27, 2022, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, Guangdong Institute of Microbiology, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No. 62843.

[0009] Another object of the present invention is to protect a method for preparing indole diterpenoid alkaloids, wherein the indole diterpenoid alkaloids are prepared and isolated from the fermentation culture of Penicillium brefeldianum GXIMD02511.

[0010] Furthermore, the method includes the following steps:

[0011] Preparation of S1 fermentation culture: Fermentation culture of strain Penicillium brefeldianum GXIMD 02511 was prepared;

[0012] S2 extraction: The fermentation culture was soaked in ethyl acetate, then chopped or pulverized and ultrasonically extracted. The residue and filtrate were obtained by filtration. The filtrate and residue were extracted with ethyl acetate separately and then concentrated to remove the ethyl acetate. The extracts obtained by extraction were combined.

[0013] S3 Separation and Purification: The extract was subjected to medium-pressure normal-phase liquid chromatography with petroleum ether / dichloromethane as the eluent, eluting in a gradient from 100:0 to 0:10 (v / v). The fraction eluted with petroleum ether:dichloromethane at a v / v ratio of 80:20 was collected and further purified by medium-pressure reversed-phase C20 chromatography. 18 Column chromatography was performed using methanol / water as the eluent, with gradient elution from a volume ratio of 10:90 to 100:0. The fractions eluted from the methanol:water gradient at a volume ratio of 58:42 were collected, and the fractions were then purified to obtain indole diterpenoid alkaloids.

[0014] Furthermore, in the preparation of the S1 fermentation culture, the fermentation culture is prepared by the following method: Activated strain *Penicillium brefeldianum* GXIMD 02511 is inoculated into a seed culture medium and dynamically cultured at 25°C and 180 rpm for 72 h to obtain a seed solution; the seed solution is inoculated into the fermentation culture medium at a 5% inoculation rate and statically cultured at 25°C for 30 days to obtain the fermentation culture; the seed culture medium formula is: 15g malt extract powder per 1L of medium, with the remainder being water, pH 7.5; the fermentation culture medium formula is: 200g rice, 2g sea salt, 200mL water per 1L Erlenmeyer flask, pH 7.5.

[0015] Another object of the present invention is to protect the use of the aforementioned indole diterpenoid alkaloid compounds in the preparation of osteoclast differentiation inhibitors.

[0016] Furthermore, the osteoclast differentiation inhibitor is a drug for treating osteolytic diseases caused by excessive activation of osteoclasts.

[0017] Furthermore, the osteoclast differentiation inhibitor is a drug for treating osteoporosis, rheumatoid arthritis, and bone destruction caused by tumor metastasis.

[0018] Another object of the present invention is to protect the use of the indole diterpenoid alkaloid compound in the preparation of NF-κB nuclear factor expression inhibitors.

[0019] Another object of the present invention is to provide a pharmaceutical composition, which can be an NF-κB nuclear factor expression inhibitor or an osteoclast differentiation inhibitor, said pharmaceutical composition comprising an effective amount of an active ingredient and a pharmaceutically acceptable carrier or excipient. The active pharmaceutical ingredient is an indole diterpenoid alkaloid compound and / or its pharmaceutically acceptable salt, specifically brefeldindole E and / or its pharmaceutically acceptable salt and / or brefeldindole F and / or its pharmaceutically acceptable salt.

[0020] This invention demonstrates through experiments that the indole diterpenoid alkaloids brefeldindole E and brefeldindole F inhibit LPS-induced NF-κB luciferase at a concentration of 10 μM (p < 0.01). These compounds can serve as lead compounds for the development of NF-κB nuclear factor expression inhibitors. Furthermore, they significantly inhibit RANKL-induced differentiation of osteoclast precursor cells (BMMs) into osteoclasts without significant cytotoxicity. Therefore, they hold promise for development into safe and effective novel osteoclast differentiation inhibitors.

[0021] By adopting the above technical solution, the present invention has the following beneficial effects:

[0022] In the course of studying the secondary metabolites of *Penicillium brefeldianum* GXIMD 02511 from the rhizosphere sediment of the Beilun River Estuary Mangrove Nature Reserve in Guangxi, this invention isolated two novel indole diterpenoid alkaloid compounds, brefeldindole E and brefeldindole F. Both compounds significantly inhibited LPS-induced NF-κB luciferase and, in a dose-dependent manner, significantly inhibited RANKL-induced differentiation of osteoclast precursor cells (BMMs) into osteoclasts, without cytotoxicity. Therefore, they are ideal candidate compounds for development into novel NF-κB nuclear factor expression inhibitors or osteoclast differentiation inhibitors. Attached Figure Description

[0023] Figure 1 This is a comparison of the inhibitory activities of the indole diterpenoid alkaloids brefeldindole E and brefeldindole F at a concentration of 10 μM on lipopolysaccharide (LPS)-induced NF-κB luciferase in RAW264.7 cells (mouse monocytes / macrophages). BAY is the positive control. ### p<0.001vs.control group; ***p<0.001vs.LPS group;

[0024] Figure 2 The effects of indole diterpenoid alkaloids brefeldindole E and brefeldindole F on cell viability of mouse bone marrow macrophages (BMMs);

[0025] Figure 3These are the experimental results of the effects of indole diterpenoid alkaloids brefeldindole E and brefeldindole F on the differentiation of osteoclast precursor cells (BMMs) into osteoclasts. Among them, RANKL is a ligand that activates the nuclear factor NF-κB receptor. Compared with the blank control group, ###P<0.001; compared with the RANKL group, *P<0.05, **P<0.01; Detailed Implementation

[0026] To make the objectives, technical solutions, and technical effects of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] Example 1

[0028] A novel class of indole diterpenoid alkaloids, named brefeldindole E and brefeldindole F respectively; the structural formula of brefeldindole E is shown in formula (I), and the structural formula of brefeldindole F is shown in formula (II):

[0029]

[0030] Example 2: Penicillium brefeldianum GXIMD 02511

[0031] The strain *Penicillium brefeldianum* GXIMD 02511, isolated from rhizosphere sediment collected from the Beilun River Estuary Mangrove Nature Reserve in Guangxi, China, was deposited on September 27, 2022, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, Guangdong Institute of Microbiology, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, with accession number GDMCC No. 62843.

[0032] The strain Penicillium brefeldianum GXIMD 02511 in this example can be used to prepare the indole diterpenoid alkaloids brefeldindole E and brefeldindole F in Example 1.

[0033] Example 3: Preparation and isolation of indole diterpenoid alkaloids brefeldindole E and brefeldindole F

[0034] 1. Culture medium

[0035] 1.1 Seed Culture Medium: Each 1L of culture medium contains 15g of malt extract powder, with the remainder being water, pH 7.5. Mix the above components and concentrations thoroughly, then sterilize at 121℃ for 30min before use.

[0036] 1.2 Fermentation medium: Each 1L Erlenmeyer flask contains: 200g rice, 2g sea salt, 200mL water, pH 7.5. Mix the above components and contents thoroughly, then sterilize at 121℃ for 30min.

[0037] 2. Fermentation

[0038] 2.1 Seed culture: The activated strain Penicillium brefeldianum GXIMD 02511 was inoculated into 1L Erlenmeyer flasks containing 300mL of seed culture medium and cultured at 25℃ and 180rpm for 72h to obtain seed solution.

[0039] 2.2 Fermentation culture: The seed culture was inoculated into 120 Erlenmeyer flasks containing fermentation culture medium at an inoculation rate of 5% (volume percentage). The culture was statically cultured at 25°C for 100 days to obtain the fermentation culture.

[0040] 3. Extraction: The fermentation culture was soaked in 2 times its volume of ethyl acetate for nearly 24 hours. Then, it was chopped or crushed and ultrasonically extracted for 20 minutes. The extract was filtered through 8 layers of gauze to obtain filtrate and residue. The filtrate was concentrated using a rotary evaporator to remove the organic solvent, and then extracted multiple times with ethyl acetate until the aqueous phase became lighter in color. The ethyl acetate was then concentrated to remove it. The residue was further extracted multiple times with ethyl acetate, and the ethyl acetate was then concentrated to remove it. Finally, the two portions of brownish-yellow extract, approximately 132g, were combined.

[0041] 4. Isolation and purification of indole diterpenoid alkaloids brefeldindole E and brefeldindole F

[0042] The extract (132g) was subjected to medium-pressure normal-phase liquid chromatography (MPLC) with petroleum ether / dichloromethane as the eluent, eluting at a gradient ratio from (100:0) to (0:100). The fraction eluted by the petroleum ether / dichloromethane gradient at a volume ratio of 80:20 was collected and further eluted by medium-pressure reversed-phase CLC. 18Column chromatography was performed using methanol / water as the eluent, with gradient elution from (10:90) to (100:0) by volume. The fraction eluted by the methanol / water gradient at a volume ratio of 58:42 was collected. This fraction was then finely separated by semi-preparative high-performance liquid chromatography (HPLC). After purification in a methanol / water elution system (volume ratio 58:42, YMC-pack ODS-A column, 10×250mm, 5μm, 2mL / min), indole diterpenoid alkaloids brefeldindole E (2mg) and brefeldindole F (2mg) were obtained.

[0043] The indole diterpenoid alkaloids brefeldindole E and brefeldindole F obtained from the above extraction and separation were subjected to the following structural identification and experiments:

[0044] Structural assessment:

[0045] The indole diterpenoid compound brefeldindole E is a pale yellow oil. NMR data show it to be characteristic of an indole diterpenoid containing three aromatic protons, namely H-21(δ...). H 6.79, d, J = 7.0 Hz), H-22 (δ) H 6.94,t,7.6Hz), H-23 (δ) H 7.16, d, J = 8.1 Hz); 5 methines, H-14 (δ H 6.17), H-11(δ) H 5.98), H-6 (δ) H 5.76), H-16 (δ) H 2.98), and H-9 (δ) H 4.30); 6 singlet methyl groups, H3-33 (δ H 1.34), H3-34(δ) H 1.34), H3-28(δ) H 1.31), H3-29(δ) H 1.33), H3-25 (δ) H 1.04), H3-26 (δ) H 1.22), and 6 methylene groups. Analysis was performed. 13 The 32 carbons in the C NMR data consist of 6 methyl groups (δ¹⁴). C 29.4, 29.2, 26.7, 25.3, 23.1, 15.2), 6 methylene groups (δ C 47.6, 34.6, 30.2, 29.9, 29.9, 28.9), 7 methines (δ C132.6, 122.2, 121.5, 119.5, 110.5, 79.3, 46.8), and 13 quaternary carbons (δ). C 201.1, 163.8, 149.1, 142.7, 142.4, 134.8, 125.3, 117.3, 95.3, 73.6, 71.6, 49.9, 43.9). The data belongs to: 1 H NMR (500MHz, CD3OD): δ H 2.44(2H,m,H-5,15),2.34(1H,dt,J=18.5,5.0Hz,H-5),2.26(1H,td,J=14.0,3.7Hz,H-6),2.17(1H,dt,J=14.0,3.7Hz,H-6),4.30(1H,br s,H-9),5.98(1H,br s,H-11),6.17(1H,dd,J=5.4,2.3Hz,H-14),2.03(1H,m,H-15),2.98(3H,m,H-16,17 ,30),2.65(1H,dd,J=12.4,9.5Hz,H-17),6.79(1H,d,J=7.0Hz,H-21),6.94(1H,t,J =7.6Hz,H-22),7.16(1H,d,J=8.1Hz,H-23),1.04(3H,s,H3-25),1.22(3H,s,H3-26) ,1.31(3H,s,H3-28),1.33(3H,s,H3-29),1.34(6H,s,H3-33,34),1.84(1H,m,H-31). 13 C NMR (125MHz, CD3OD): δ C149.1(C,C-2),49.0(C,C-3),43.9(C,C-4),28.9(CH,C-5),34.6(CH2,C-6),95.3(C,C-7),79.3(CH,C-9),201.1(C,C-10),122.2(CH, C-11),163.8(C,C-12),142.7(C,C-13),132.6(C,C-14),29.9(CH2,C-15),46.8(CH,C-16),30.2(CH2,C-17),117.3(C,C-18),125.3(C ,C-19),134.8(C,C-20),119.5(CH,C-21),121.5(CH,C-22),110.5(CH,C-23),142.4(C,C-24),15.2(CH3,C-25),23.1(CH3,C-26),73.6(C,C-27),25.3(CH3,C-28),26.7(CH3,C-29),29.9(CH2,C-30),47.6(CH2,C-31),71.6(C,C-32),29.4(CH3,C-33),29.2(CH3,C-34). This compound was identified as brefeldindole E. The structural formula of brefeldindole E is shown in formula (Ⅰ) below:

[0046]

[0047] The indole diterpenoid compound brefeldindole F is a white amorphous powder. NMR data show it to be characteristic of an indole diterpenoid alkaloid containing three aromatic protons, namely H-21(δ). H 6.74, dd, J=7.2, 0.9Hz), H-22 (δ H 6.88,dd,8.1,7.2Hz), H-23 (δ) H 7.13,dd,J=8.1,0.9Hz); 3 methines, H-11 (δ H 5.81), H-16 (δ) H 2.85), and H-9 (δ H 4.30); 6 singlet methyl groups, H3-33 (δ H 1.30), H3-34(δ) H 1.30), H3-28 (δ) H 1.14), H3-29(δ) H 1.42), H3-25(δ) H 1.39), H3-26 (δ) H1.23), and 6 methylene groups. Analysis was performed. 13 The 32 carbons in the C NMR data consist of 6 methyl groups (δ¹⁴). C 29.3, 29.2, 29.2, 23.8, 23.4, 16.6), 7 methylene groups (δ C 47.6, 33.9, 30.7, 29.9, 29.4, 27.7, 22.4), 6 methines (δ C 121.1, 119.3, 118.2, 110.5, 89.2, 50.3), and 13 quaternary carbons (δ). C 199.5, 172.4, 153.2, 142.0, 134.6, 125.7, 116.1, 106.2, 79.5, 77.7, 71.6, 52.5, 40.9). The data belongs to: 1 H NMR (700MHz, CD3OD): δ H 2.86(2H,m,H-5,6),2.63(1H,t,J=11.2Hz,H-5),1.95(1H,m,H-6),4.30(1H,d,J=1.3Hz,H-9),5.81(1H,br s,H-11),2.07(1H,m,H-14),1.78(1H,m,H-14),2.02(1H,m,H-15),1.94(1H,m,H-15),2.85(1H,m ,H-16),2.86(1H,m,H-17),2.55(1H,m,H-17),6.74(1H,dd,J=7.2,0.9Hz,H-21),6.88(1H,dd,J=8 .1,7.2Hz,H-22),7.13(1H,dd,J=8.1,0.9Hz,H-23),1.39(3H,s,H3-25),1.23(3H,s,H3-26),1.14 (3H,s,H3-28),1.42(3H,s,H3-29),2.89(1H,m,H-30),1.78(1H,m,H-31),1.30(6H,s,H3-33,34). 13 C NMR (175MHz, CD3OD): δ C153.2(C,C-2),52.5(C,C-3),40.9(C,C-4),27.7(CH2,C-5),29.4(CH2,C-6),106.2(C,C-7),89.2(CH,C-9),199.5(C,C-10),118.2(CH ,C-11),172.4(C,C-12),77.7(C,C-13),22.4(CH2,C-14),33.9(CH2,C-15),50.3(CH,C-16),30.7(CH2,C-17),116.1(C,C-18),125.7( C,C-19), 134.6(C,C-20), 119.3(CH,C-21), 121.1(CH,C-22), 110.5(CH,C-23), 142.0(C,C-24), 16.6(CH3,C-25), 23.4(CH3,C-26), 79.5(C,C-27), 23.8(CH3,C-28), 29.3(CH3,C-29), 29.9(CH2,C-30), 47.6(CH2,C-31), 71.6(C,C-32), 29.2(CH3,C-33), 29.2(CH3,C-34). This compound was identified as brefeldindole F. The structure of brefeldindole F is shown in formula (Ⅱ):

[0048]

[0049] Experiment 1: Determination of the inhibitory activity of the indole diterpenoid alkaloids brefeldindole E and brefeldindole F obtained in Example 3 against LPS-induced NF-κB luciferase.

[0050] The main reference for the determination of NF-κB luciferase inhibitory activity is (Marine Drugs, 2022, 20(3):178.).

[0051] RAW264.7 cells stably transfected with the NF-κB luciferase reporter gene were seeded into 96-well plates (1×10⁻⁶ cells per well). 4Cells were incubated in 200 μL of DMEM medium containing 10% fetal bovine serum, 100 IU / mL penicillin and streptomycin, and 0.1 μg / mL G418 per well. After the cells adhered and stabilized, indole diterpenoid alkaloids brefeldindole E and brefeldindole F were added, with 6 replicates. After incubation for another 4 h, except for the negative control group, LPS and RANKL were added to each compound group (3 wells) and the positive control group (NF-κB inhibitor, BAY11-7082, 5 μM), respectively, to a final concentration of 100 ng / mL per well. After stimulation for 8 h, the supernatant was discarded, and 25 μL of cell lysis buffer was added to each well. The cells were shaken slowly for 10 min to fully lyse the cells. Then, 20 μL of the lysate was transferred to a white plate, and 50 μL of luciferase solution was added to each well. The luciferase value was detected using a multi-mode microplate reader.

[0052] Experimental conclusion: The results are as follows Figure 1 As shown, the study found that, compared with the LPS blank group, the indole diterpenoid alkaloids brefeldindole E and brefeldindole F had a significant inhibitory effect on LPS-induced NF-κB luciferase at 10 μM (p<0.001).

[0053] Experiment 2: Effects of the indole diterpenoid alkaloids brefeldindole E and brefeldindole F obtained in Example 3 on the cytotoxicity of osteoclast precursor RAW264.7 cells and BMMs.

[0054] The main reference for cell viability assay is (Marine Drugs, 2022, 20(3):178.).

[0055] The effects of indole diterpenoid alkaloids brefeldindole E and brefeldindole F on the viability of mouse bone marrow macrophages (BMMs) were detected by the CCK-8 assay.

[0056] a. Preparation of mouse bone marrow macrophages (BMMs): Under aseptic conditions, femurs were taken from 8-12 week old female C57BL / 6 mice. The joints at both ends of the femur were cut off, and the femurs were repeatedly washed with phenol red-free α-MEM medium (containing 10% fetal bovine serum, 100 IU / mL penicillin, and 100 IU / mL streptomycin) until the femoral cavity turned white. The washed femoral bone marrow cells were incubated at 37°C in a 5% CO2 cell culture incubator for 2 hours. The supernatant was collected, and the red blood cells were lysed with lysis buffer, centrifuged, and resuspended to obtain BMMs.

[0057] b. CCK-8 assay for cell viability:

[0058] Take the BMMs (1×10) prepared in step a 5 Macrophages were cultured in 96-well plates (cells / well) and phenol red-free α-MEM medium (containing 10% fetal bovine serum, 100 IU / mL penicillin, and 100 IU / mL streptomycin) was added to each well to a final concentration of 200 μL. Macrophage colony-stimulating factor (M-CSF, final concentration 50 ng / mL) was also added to each well. The 96-well plates were then incubated overnight at 37°C and 5% CO2. After cell adhesion was stable, different concentrations of indole diterpenoid alkaloids brefeldindole E and brefeldindole F were added to bring the final concentrations of brefeldindole E and brefeldindole F in the wells to 10 μM and 15 μM, respectively. Each group was divided into three replicates, and the plates were incubated for 4 days. After incubation, discard the supernatant (100 μL), add 5 μL of CCK-8 reagent (Cell Counting Kit-8) to each well, shake well, and incubate for another 3 hours at 37°C and 5% CO2. Measure the optical density (OD value) at 450 nm using a TECANGENiosPro multi-functional microplate reader and calculate the cell viability of each group.

[0059] The results are as follows Figure 2 As shown, there was no significant difference in the survival rate of BMMs cells after the addition of 10 μM and 15 μM indole diterpenoid alkaloids brefeldindole E and brefeldindole F, indicating that in vitro, indole diterpenoid alkaloids brefeldindole E and brefeldindole F had no cytotoxicity to BMMs cells.

[0060] Experiment 3: Effects of indole diterpenoid alkaloids brefeldindole E and brefeldindole F obtained in Example 3 on RANKL-induced differentiation of osteoclast precursor cells (BMMs) into osteoclasts.

[0061] The main reference for the assay of RANKL-induced inhibitory activity of osteoclast precursor BMMs on differentiation is (Marine Drugs, 2022, 20(3):178.).

[0062] Take the BMMs (2×10) prepared in step a above. 4Macrophages were cultured in 96-well plates, with each well containing 200 μL of phenol red-free α-MEM medium (containing 10% fetal bovine serum, 100 IU / mL penicillin, and 100 IU / mL streptomycin) and macrophage colony-stimulating factor (M-CSF, final concentration 50 ng / mL). The plates were then incubated overnight at 37°C and 5% CO2. After cell adhesion was stable, different concentrations of indole diterpenoid alkaloids brefeldindole E and brefeldindole F were added to the wells, resulting in final concentrations of 5 μM, 10 μM, and 15 μM, respectively. Each group was divided into three replicates, and the plates were incubated for 4 h. After incubation, RANKL (final concentration 100 ng / mL) was added, and the plates were cultured for 3-4 days. After incubation, the cells were stained with TRAP, photographed and counted under an inverted microscope, and TRAP-positive cells with more than 5 nuclei were identified as osteoclasts.

[0063] The results are as follows Figure 3 As shown, the indole diterpenoid alkaloids brefeldindole E and brefeldindole F significantly inhibited RANKL-induced osteoclast formation in BMMs. Even at an effective concentration of 10 μM, they significantly inhibited RANKL-induced osteoclast formation from BMM osteoclast precursor cells.

[0064] The above experimental results indicate that the indole diterpenoid alkaloids brefeldindole E and brefeldindole F significantly inhibit LPS-induced NF-κB luciferase, and at a concentration of 10 μM, they significantly inhibit RANKL-induced differentiation of osteoclast precursor cells (BMMs) into osteoclasts. They significantly inhibit osteoclast generation and activation without significant toxicity, and can effectively replace existing bisphosphonates and denoximab to inhibit osteoclast bone resorption activity. They can be developed as novel osteoclast differentiation inhibitors or NF-κB nuclear factor expression inhibitors for the prevention and treatment of osteoporosis and other osteolytic diseases.

[0065] Based on the above experiments, it can be demonstrated that the two indole diterpenoid alkaloid compounds, brefeldindole E and brefeldindole F, of the present invention can be used in the preparation of drugs that inhibit osteoclast differentiation or NF-κB nuclear factor expression. The osteoclast differentiation inhibitors or NF-κB nuclear factor expression inhibitors are oral, injectable, or topical formulations; comprising the active ingredients, indole diterpenoid alkaloid compounds brefeldindole E and brefeldindole F, and medically acceptable pharmaceutical excipients. Anti-osteoclast differentiation drugs can treat, but are not limited to, postmenopausal osteoporosis, bone destruction due to tumor metastasis, and other known clinical indications for which osteoclast inhibitors such as bisphosphonates and denoximab are approved.

[0066] Generally, pharmaceuticals are clinically used only after being formulated into a finished product. The active ingredient of this invention, the indole diterpenoid alkaloid compound, can be prepared according to methods known in the art. It can be formulated into any dosage form suitable for human or animal use by combining the active ingredient of this invention with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants. The active ingredient of this invention, the indole diterpenoid alkaloid compound, or an active ingredient containing it, can be administered in unit doses via enteral or non-enteric routes, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0067] Oral formulations are preferably capsules. To formulate the drug delivery unit into capsules, the active ingredient, the phenylacetic acid compound of this invention, can be mixed with a diluent and a flow aid, and the mixture can be directly placed into hard or soft capsules. Alternatively, the active ingredient, the indole diterpene alkaloid compound of this invention, can be first formed into granules or microspheres with a diluent, binder, and disintegrant, and then placed into hard or soft capsules. The diluents, binders, wetting agents, disintegrants, and flow aids used to prepare tablets of the indole diterpene alkaloid compound of this invention can also be used to prepare capsules of the indole diterpene alkaloid compound of this invention.

[0068] To prepare the effective active ingredient of this invention, the indole diterpenoid alkaloid compound, into an injection, water, ethanol, isopropanol, propylene glycol, or mixtures thereof can be used as solvents, and appropriate amounts of commonly used solubilizers, co-solvents, pH adjusters, and osmotic pressure regulators can be added. Solubilizers or co-solvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; pH adjusters can be phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; osmotic pressure regulators can be sodium chloride, mannitol, glucose, phosphates, acetates, etc. If preparing a lyophilized powder injection, mannitol, glucose, etc., can also be added as a support agent.

[0069] In summary, this 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 for the development of new drugs with independent intellectual property rights in China.

[0070] The above description is a detailed explanation of 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 made under the technical concept presented in the present invention should fall within the patent scope covered by the present invention.

Claims

1. A class of indole diterpenoid alkaloids, characterized in that, The indole diterpenoid alkaloids are named brefeldindole E and brefeldindole F, respectively; the structural formula of brefeldindole E is shown in formula (I), and the structural formula of brefeldindole F is shown in formula (II).

2. A method for preparing the indole diterpenoid alkaloid compound of claim 1, characterized in that, The indole diterpenoid alkaloids were prepared and isolated from the fermentation culture of Penicillium brefeldianum GXIMD02511; the preservation number of Penicillium brefeldianum GXIMD02511 is: GDMCC NO.62843; The method specifically includes the following steps: Preparation of S1 fermentation culture: Fermentation culture of strain Penicillium brefeldianum GXIMD02511 was prepared; S2 extraction: The fermentation culture was soaked in ethyl acetate, then chopped or pulverized and ultrasonically extracted. The residue and filtrate were obtained by filtration. The filtrate and residue were extracted with ethyl acetate separately and then concentrated to remove the ethyl acetate. The extracts obtained by extraction were combined. S3 Separation and Purification: The extract was subjected to medium-pressure normal-phase liquid chromatography with petroleum ether / dichloromethane as the eluent, eluting in a gradient from 100:0 to 0:10 (v / v). The fraction eluted with petroleum ether:dichloromethane at a v / v ratio of 80:20 was collected and further purified by medium-pressure reversed-phase C20 chromatography. 18 Column chromatography was performed using methanol / water as the eluent, with gradient elution from a volume ratio of 10:90 to 100:

0. The fractions eluted by the methanol:water gradient at a volume ratio of 58:42 were collected, and the fractions were then purified to obtain the indole diterpenoid alkaloids. In the preparation of the S1 fermentation culture described above, the fermentation culture is prepared by the following method: An activated strain of *Penicillium brefeldianum* GXIMD 02511 is inoculated into a seed culture medium and dynamically cultured at 25°C and 180 rpm for 72 hours to obtain a seed solution; the seed solution is inoculated into the fermentation culture medium at a 5% inoculation rate and statically cultured at 25°C for 30 days to obtain the fermentation culture; the seed culture medium formula is: 15g of malt extract powder per 1L of medium, with the remainder being water, pH 7.5; the fermentation culture medium formula is: 200g of rice, 2g of sea salt, 200mL of water per 1L of Erlenmeyer flask medium, pH 7.

5.

3. The application of the indole diterpenoid alkaloid compound according to claim 1 in the preparation of osteoclast differentiation inhibitors.

4. The application according to claim 3, characterized in that: The osteoclast differentiation inhibitor is a drug for treating osteolytic diseases caused by excessive activation of osteoclasts.

5. The application according to claim 3, characterized in that: The osteoclast differentiation inhibitor is a drug used to treat osteoporosis, rheumatoid arthritis, and bone destruction caused by tumor metastasis.

6. The application of the indole diterpenoid alkaloid compound according to claim 1 in the preparation of NF-κB nuclear factor expression inhibitors.

7. A pharmaceutical composition, characterized in that, It includes an effective amount of the active pharmaceutical ingredient and a pharmaceutically acceptable carrier or excipient; the active pharmaceutical ingredient is the indole diterpenoid alkaloid compound as described in claim 1 and / or its pharmaceutical salt.

Images