Pyrrolidine-2, 4-diketone compound, preparation method thereof and application of pyrrolidine-2, 4-diketone compound in preparation of osteoclast differentiation inhibitor

Abstract

The invention provides a marine fungus-derived pyrrolidine-2, 4-diketone compound and application thereof in prevention and treatment of osteoclast differentiation and other related osteolytic diseases, and relates to the field of marine natural products. The pyrrolidine-2, 4-diketone compound is named montagnulan A, the structural formula of the pyrrolidine-2, 4-diketone compound is shown in a formula (I), the pyrrolidine-2, 4-diketone compound has a remarkable inhibition effect on LPS-induced NF-kappa B luciferase under a certain concentration, further inhibits RANKL-induced osteoclast precursor cell BMMs cells from being differentiated into osteoclasts, has no obvious cytotoxicity, can effectively replace existing diphosphate drugs and dioxyximab, and can be used for preparing osteoclasts. The compound can inhibit osteoclast differentiation, inhibit osteoclast bone resorption activity, is an ideal candidate compound developed into a novel osteoclast differentiation inhibitor drug, and is used for preventing and treating osteoclast-related osteolytic diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of marine natural products, and in particular to a novel pyrrolidine-2,4-dione compound derived from leucine and application thereof in the preparation of an osteoclast differentiation inhibitor. Background Art

[0002] Osteoclasts (OCs) are the only cells in the body that have the function of bone absorption. The delicate interaction between osteoclasts and osteoblasts can maintain bone homeostasis. However, abnormal osteoclast function can lead to a variety of osteolytic diseases, such as osteoporosis, osteosclerosis, and pycnogenol dysplasia, which are the main causes of fractures and disability worldwide, and impose a heavy burden on the economy and public health.

[0003] At present, the main drugs that can effectively treat various osteolytic diseases caused by abnormal osteoclasts are bisphosphonates and denoximab, but both have serious complications and side effects. For example, long-term use of bisphosphonates will inhibit the formation of new bone tissue and easily cause fractures; denoximab is not only expensive, but long-term use can cause complications such as mandibular necrotizing lesions.

[0004] At present, the inhibitors targeting osteoclast differentiation that can be used clinically are denoximab (RANKL (Receptor activator of nuclear kappa B ligand, nuclear factor κB receptor activator ligand) antibody) and bisphosphonates that can inhibit the bone resorption function of osteoclasts. Although these two types of drugs can inhibit the differentiation or function of osteoclasts, they both have serious complications and side effects that are difficult for patients to tolerate. For example, long-term use of bisphosphonates will inhibit the natural renewal of bone tissue, which can easily cause fractures; denoximab requires subcutaneous injection, is expensive, and long-term use of denoximab has the risk of causing serious complications such as mandibular necrosis. Therefore, there is an urgent need to develop a cost-effective drug with few toxic and side effects to treat osteolytic diseases caused by abnormal osteoclast differentiation.

[0005] Marine natural products have novel and diverse structures and important biological activities, and have good application value and prospects in the field of drug development and application. In recent years, a variety of marine natural products have been found to have the activity of inhibiting osteoclast differentiation, indicating that they are an important source for discovering therapeutic drugs for osteoclast-related osteolytic diseases. Summary of the invention

[0006] In view of the problems of high price and strong side effects of the prior art nuclear factor κB receptor activator ligand antibody denoximab and bisphosphonates that can inhibit the bone resorption function of osteoclasts, the present invention provides a novel leucine-derived pyrrolidine-2,4-dione compound named montagnulan A, and its use in the preparation of osteoclast differentiation inhibitors. The inventors of the present application found through extensive research that montagnulan A inhibits RANKL-induced osteoclast differentiation by inhibiting the NF-κB signaling pathway, and has potential medicinal value for preventing and treating osteolytic diseases such as osteoporosis.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A pyrrolidine-2,4-dione compound, named as compound montagnulan A, has a chemical structural formula as shown in formula (I):

[0009]

[0010] The second object of the present invention is to protect a source strain Montagnula sp. GXIMD 02514 for preparing the above-mentioned pyrrolidine-2,4-dione compounds, and its deposit number is: GDMCC No.65210.

[0011] The second object of the present invention is to protect a method for preparing the above-mentioned pyrrolidine-2,4-dione compounds, wherein the pyrrolidine-2,4-dione compounds are prepared and separated from the fermentation culture of the marine fungus Montagnula sp. GXIMD 02514.

[0012] Further, the method for preparing pyrrolidine-2,4-dione compounds comprises the following specific steps:

[0013] The strain Montagnula sp. GXIMD 02514 was inoculated from the stored slant medium into the MB plate. After the mycelium grew, it was inoculated into the MB seed medium (malt extract powder 1.5%, sea salt 2%, water 1000mL, pH 7.4) in a shaking table for 3 days (180rpm); then inoculated into a sterilized rice culture medium for fermentation, a total of 155 bottles (tissue culture bottles) were fermented, the rice culture medium formula: 60g rice, 2% sea salt, 0.1% methionine, 0.2% corn steep liquor, 70mL water, and fermented at room temperature for 60 days; after the fermentation was completed, the product was extracted with ethyl acetate, and the obtained extract was subjected to gradient elution by normal phase silica gel chromatography to obtain 9 fractions Frs.1-9; fraction Fr.7 was selected for reverse phase silica gel column chromatography separation, and gradient elution obtained 17 sub-fractions Fr.7-1-Fr.7-17; fraction Fr.7-8 was separated by semi-preparative HPLC to obtain sub-fraction Fr.7-8-6, and sub-fraction Fr.7-8-6 was further separated by normal phase silica gel column chromatography to obtain 23 fractions, wherein fraction Fr.7-8-6-15 was separated by semi-preparative HPLC to obtain compound montagnulan A.

[0014] Another object of the present invention is to provide the use of the pyrrolidine-2,4-dione compound in the preparation of osteoclast differentiation inhibitors.

[0015] It is further explained that the osteoclast differentiation inhibitor is a drug for treating osteolytic diseases such as osteoporosis, rheumatoid arthritis, and tumor metastasis bone destruction caused by excessive osteoclast activation.

[0016] It is further specified that the montagnulan A is selected from the monomer form of montagnulan A, or is a natural product extract containing montagnulan A.

[0017] It is further explained that the drug is prepared into a clinically acceptable drug preparation with montagnulan A as the main ingredient, and a pharmaceutically acceptable carrier or excipient, or a pharmaceutically acceptable excipient or auxiliary ingredient.

[0018] It is further described that the dosage form of the drug includes a dosage form for gastrointestinal administration and a dosage form for parenteral administration.

[0019] Furthermore, the pharmaceutically acceptable carrier or excipient is selected from solvents, diluents, dispersants, suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, adhesives, lubricants, stabilizers, hydrating agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, release agents, coating agents, flavoring agents, and antioxidants.

[0020] The drug can be prepared into any dosage form of powder, tablet, powder, capsule, pill, pellet, injection, emulsion, suspension or tincture, and the drugs in the above dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0021] The "pharmaceutically acceptable carrier or excipient" used in the drug of the present invention can be any conventional carrier in the field of pharmaceutical preparations, and the selection of a specific carrier will depend on the mode of administration or the type and state of disease used to treat a specific patient. The preparation of suitable drugs for a specific mode of administration is completely within the knowledge of those skilled in the art of medicine.

[0022] The term "drug" as used in this application has its general meaning. In addition, the "drug" of the present invention can also exist or provide in the form of health products, functional foods, foods, food additives, etc. The conventional techniques in the pharmaceutical field, particularly the field of preparations, can be used to obtain the active ingredients of the raw materials of the medicine of the present invention by the extraction, separation and purification means commonly used in the production of medicines, optionally mixed with one or more pharmaceutically acceptable carriers or excipients, and then formed into the required dosage form to prepare the medicine of the present invention. According to the medicine of the present invention, it is a pharmaceutical preparation that can be applicable to oral administration, parenteral administration or topical administration, external administration, and is particularly suitable for oral administration. The dosage form for oral administration may include, for example, tablets, pills, hard or soft capsules, solutions, suspensions, emulsions, syrups, powders, powders, fine particles, granules, pellets, elixirs, etc., and is not limited thereto. In addition to the active ingredients, these preparations may also contain diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and glycine), lubricants (e.g., silicon dioxide, talc, stearic acid or its magnesium salt, calcium salt, and polyethylene glycol). Tablets may also contain binders such as aluminum magnesium silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and polyvinylpyrrolidine. If necessary, they may also contain pharmaceutical additives such as disintegrants (e.g., starch, agar, alginic acid or its sodium salt), absorbents, colorants, flavoring agents, sweeteners, and the like. Tablets may be prepared according to commonly used mixing, granulation, or coating methods.

[0023] The present invention has the following beneficial effects:

[0024] The compound montagnulan A extracted from the fermentation product of the marine fungus Montagnula sp. GXIMD 02514 in the present invention can be used to prepare an osteoclast differentiation inhibitor. Experiments have shown that the compound inhibits NF-κB luciferase activity and then inhibits RANK-induced osteoclast differentiation, thereby curbing osteoclast differentiation and maturation, and has no obvious cytotoxicity. The compound montagnulan A of the present application is of great significance for the preparation of a method for preventing and / or treating osteoclast-related osteolytic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Chemical structure of montagnulan A in the present invention (left) and two-dimensional (HMBC, 1 H- 1 H COSY and NOESY) NMR correlation diagram (right)

[0026] Figure 2 H NMR spectrum (Chloroform-d) of montagnulan A in the examples of the present invention;

[0027] Figure 3 The carbon NMR spectrum (Chloroform-d) of montagnulan A in the examples of the present invention;

[0028] Figure 4 HSQC spectrum of montagnulan A in the embodiment of the present invention (Chloroform-d);

[0029] Figure 5 HMBC spectrum (Chloroform-d) of montagnulan A in the examples of the present invention;

[0030] Figure 6 In the embodiment of the present invention, montagnulan A 1 H- 1 H COSY spectrum (Chloroform-d);

[0031] Figure 7 NOESY spectrum (Chloroform-d) of montagnulan A in the examples of the present invention;

[0032] Figure 8 is the measured and calculated ECD spectra of montagnulan A in the embodiments of the present invention;

[0033] Fig. 9 is the number of TRAP-positive multinucleated cells quantified in the present invention; BAY 11-7028 is a positive control, ### Indicates p<0.001 vs. control group; * indicates p<0.05 vs. LPS group;

[0034] Fig.10 The cell viability of 27 at different concentrations in BMMs for 72 hours was determined by CCK-8 in the embodiment of the present invention;

[0035] Fig.11In the examples of the present invention, montagnulan A inhibits RANKL-induced osteoclast differentiation in BMMs in a dose-dependent manner. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0037] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

[0038] Example 1: Preparation of montagnulan A

[0039] Experiment 1: Isolation and identification of marine fungus Montagnula sp. GXIMD 02514:

[0040] In this embodiment, the marine fungus Montagnula sp. GXIMD 02514 was isolated from the antler coral of Weizhou Island, Beibu Gulf, Guangxi Zhuang Autonomous Region. The ITS sequence of the strain was obtained by PCR amplification and DNA sequencing, and was preliminarily identified as Montagnula sp. after comparison with the gene sequence in GenBank, and the strain was named Montagnula sp. GXIMD 02514. It was deposited in Guangdong Microbiological Culture Collection Center (GDMCC) on September 26, 2024, address: Guangdong Institute of Microbiology, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Guangdong Institute of Microbiology, 5th Floor, Building 59, Guangdong Provincial Institute of Microbiology, Guangdong Province, with a deposit number of GDMCC No. 65210.

[0041] Experiment 2: Isolation and identification of montagnulan A

[0042] The strain Montagnula sp.GXIMD 02514 was inoculated from the stored slant medium into the MB plate, and after the mycelium grew, it was inoculated into the MB seed medium (1.5% malt extract powder, 2% sea salt, 1000 mL water, pH 7.4) and cultured in a shaking table for 3 days (180 rpm). Then it was inoculated into the sterilized rice medium for fermentation, and a total of 155 bottles (tissue culture bottles) were fermented. The rice medium formula was: 60 g rice, 2% sea salt, 0.1% methionine, 0.2% corn steep liquor, 70 mL water, and fermented at room temperature for 60 days.

[0043] After the fermentation, the product was extracted with ethyl acetate (EtOAc), and the obtained extract was subjected to gradient elution by normal phase silica gel chromatography to obtain 9 fractions (Frs.1-9). Fraction Fr.7 was selected for reverse phase silica gel column chromatography, and 17 sub-fractions Fr.7-1-Fr.7-17 were obtained by gradient elution. Fraction Fr.7-8 was separated by semi-preparative HPLC to obtain sub-fraction Fr.7-8-6, and sub-fraction Fr.7-8-6 was further separated by normal phase silica gel column chromatography to obtain 23 fractions, among which Fr.7-8-6-15 was separated by semi-preparative HPLC to obtain compound montagnulan A.

[0044] Compound Montagnulan A: colorless oil; easily soluble in methanol and chloroform. According to the HR-ESIMS spectrum signal peak m / z316.1526[M+Na] + (calcd for C 16 H 23 NO4Na, 316.1525) to determine the molecular formula is C 16 H 23 NO4. 1 H NMR (500 MHz) and 13 C NMR (125 MHz) spectra combined with HSQC spectra showed a series of signals, including four methyl [δ H 1.12(3H,d,J=6.6Hz,H3-8),1.10(3H,dd,J=6.6Hz,H3-9),1.34(3H,d,J=6.3Hz,H3-15),0.84(3H,t,J=7.4Hz,H3-17); δ C 22.2(CH3,C-8),22.2(CH3,C-9),20.8(CH3,C-15),8.7(CH3,C-17)], 3 methylene groups [δ H 1.85(2H,q,J=7.4Hz,H2-16),δ C 26.0(CH2,C-16);δ H2.41–2.66(2H,m,H2-14),δ C 30.5(CH2,C-14);δ H 1.98(1H,m,H-11a),1.73(1H,m,H-11b)δ C 30.3(CH2,C-11)], 4 methine groups [δ H 2.4–2.66 (1H, m, H-7), δ C 27.1(CH,C-7);δ H 5.61(1H,d,J=9.8Hz,H-6), 119.5(CH,C-6); δ H 2.41–2.66 (1H, m, H-10), δ C 33.2(CH,C-10);δ H 4.45(1H,m,H-12),δ C 73.7 (CH, C-12)], and 5 quaternary carbons (δ C 132.0, C-5; 57.1, C-3; 3 carbonyl delta C 174.8, 200.0 and 171.7). The above NMR data show the structural characteristics of pyrrolidine-2,4-dione compounds, which are similar to the chemical structure of the reported compound lecanicilliumin A (Marine Drugs, 2022, 20, 255.), with the main difference being the differences in the groups connected at positions 3 and 5 on the pyrrolidine-2,4-dione skeleton. 1 H- 1 H COSY spectrum ( Figure 1 ) and the related signals of H-10, H-12, H2-14 / C-13, H-10 / C-2, C-4 and H3-17 / C-3 in the HMBC spectrum, combined with their chemical shifts, indicate that the C-3 position of compound montagnulan A is connected to an ethyl group and a six-membered lactone ring containing a methyl group substituted at the 12th position. 1 H- 1 The H3-8 / H-7 / H3-9 related signals and H-7, H-6 / C-5 related signals in the H COSY spectrum, combined with the chemical shift, indicate that an isobutyl group is connected to the 5-position via a carbon-carbon double bond. The one-dimensional and two-dimensional NMR spectra of compound montagnulan A are shown in Figure 1. Figure 2 to Figure 7 .

[0045] The relative configuration of compound montagnulan A is mainly determined by NOESY correlation. The correlation between H-6 / H3-18 and H-10 / H3-15 in the NOESY spectrum indicates that Me-8 and H-6 are located on the same side of the double bond, and H-10 and Me-15 are located on the same side of the six-membered lactone ring. Therefore, compound montagnulan A also has four isomers. By calculating the ECD spectra of the four isomers, the results show that the calculated ECD spectrum of the configuration (3S, 10R, 12R)-montagnulan A is consistent with the measured ECD spectrum ( Figure 8 ) are basically consistent, so the absolute configuration of montagnulan A is determined to be 3S, 10R, 12R.

[0046] Physicochemical data of compound montagnulan A: ECD (0.25 mg / mL, MeOH) max (Δε)200(-8.07),215(-3.09),226(-8.09),242(-6.33),298(+1.44)nm. 1 H NMRand 13 CNMR data, see Table 1; HR-ESIMS m / z 316.1527 [M+Na] + (calcd for C 16 H 23 NO4Na316.1525).

[0047] Compound montagnulan A 1 H(500MHz) and 13 C (125MHz) NMR data (Chloroform-d) are shown in Table 1:

[0048] Table 1

[0049]

[0050]

[0051] "s" means single peak; "d" means double peak; "t" means triple peak; "m" means multiple peak

[0052] Example 2: Effect of montagnulan A in inhibiting osteoclast differentiation

[0053] Experiment 1: Evaluation of the inhibitory activity of montagnulan A on LPS-induced NF-κB activation in RAW264.7 cells using luciferase reporter gene technology

[0054] RAW264.7 cells stably transfected with NF-κB luciferase reporter gene were cultured with compound montagnulan A (20μM) or NF-κB inhibitor (BAY11-7082, 5μM) for 4 hours, and then stimulated with LPS (100ng / mL) for 6 hours. The results showed that compound montagnulan A (20μM) could significantly inhibit NF-κB luciferase activity (P<0.05).

[0055] Experiment 2: Osteoclast precursor BMMs were used as experimental subjects to detect the effect of montagnulan A on cell differentiation into osteoclasts

[0056] Take BMMs cells (1×10 3 / well) were inoculated in a 96-well plate and incubated overnight at 37°C and 5% CO2. After the cells were stably attached to the wall, different concentrations of montagnulan A were added to make the final concentration of montagnulan A in the wells 1μM, 5μM and 10μM. Three replicate wells were set up in each group and incubated for 4h. RANKL (final concentration of 100ng / mL) was added, and the solution was changed every two days for 4-5 days. TRAP staining was performed on the incubated cells, and the cells were photographed and counted under an inverted microscope. TRAP-positive cells with more than 5 nuclei were osteoclasts. The results are shown in Fig. 9 and Fig.11 shown.

[0057] Experiment 3: CCK-8 method to detect cell survival

[0058] Take BMMs (1×10 5 Each well was added with 200 μL of α-MEM medium without phenol red (containing 10% fetal bovine serum, 100 IU / mL penicillin and 100 IU / mL streptomycin), and macrophage colony stimulating factor (M-CSF, final concentration of 50 ng / mL) was added to each well. Then the 96-well plate was placed in a cell culture incubator at 37°C and 5% CO2 for overnight incubation. After the cells were stably attached to the wall, different concentrations of montagnulan A were added to make the final concentration of montagnulan A in the wells 1 μM, 5 μM and 10 μM, and 3 replicate wells were set for each group and incubated for 4 days. After the incubation, the supernatant (100 μL) was discarded, 5 μL of CCK-8 reagent (Cell Counting Kit-8 cell counting reagent) was added to each well, shaken, and incubated for 3 h at 37°C and 5% CO2. The optical density value (OD value) at a wavelength of 450 nm was measured using a TECANGENiosPro multifunctional microplate reader to calculate the cell survival rate of each group. Fig.10 Shown

[0059] The results show that:

[0060] The compound montagnulan A showed an inhibitory effect on LPS-induced NF-κB activation in RAW 264.7 macrophages at 20 μM.

[0061] The compound montagnulan A inhibited RANKL-induced osteoclast differentiation in a dose-dependent manner and had no cytotoxicity.

[0062] In summary, the application of the compound montagnulan A in the preparation of osteoclast-related osteolytic diseases suggests that montagnulan A has potential therapeutic effects on osteoclast-related osteolytic diseases and has good clinical application prospects.

[0063] The above is a detailed introduction to the use of the compound montagnulan A provided by the present invention in the preparation of osteoclast differentiation inhibitors for the prevention and treatment of osteoclast-related osteolytic diseases. Specific examples are used in this application to illustrate the principles and implementation methods of the present invention. The description of the above examples is only used to help understand the method of the present invention and its central idea. It should be pointed out that for ordinary technicians in this field, without departing from the principles of the present invention, the present invention can also be improved and modified in a number of ways, and these improvements and modifications also fall within the protection of the claims of the present invention.

Claims

1. A pyrrolidine-2,4-dione compound, characterized in that: The pyrrolidine-2,4-dione compound is named montagnulan A, and the chemical structure of the compound montagnulan A is as shown in formula (I):

2. A source strain Montagnulasp.GXIMD 02514 for preparing the pyrrolidine-2,4-dione compound according to claim 1, whose deposit number is: GDMCC No.65210.

3. A method for preparing the pyrrolidine-2,4-dione compound according to claim 1, characterized in that: The pyrrolidine-2,4-dione compound is prepared and separated from the fermentation culture of the marine fungus Montagnula sp. GXIMD 02514.

4. The method according to claim 3, characterized in that The method for preparing pyrrolidine-2,4-dione compounds comprises the following specific steps: The strain Montagnula sp.GXIMD 02514 was inoculated from the preserved slant medium into the MB plate, and after the mycelium grew, it was inoculated into the MB seed medium for shaking culture for 3 days; then inoculated into the sterilized rice medium for fermentation, and a total of 155 bottles were fermented. The rice medium formula was: 60g rice, 2% sea salt, 0.1% methionine, 0.2% corn steep liquor, 70mL water, and fermented at room temperature for 60 days; After the fermentation is completed, the fermentation product is extracted with ethyl acetate, and the obtained extract is subjected to gradient elution by normal phase silica gel chromatography to obtain 9 fractions Frs.1~9; fraction Fr.7 is selected for separation by reverse phase silica gel column chromatography, and 17 sub-fractions Fr.7-1~Fr.7-17 are obtained by gradient elution; fraction Fr.7-8 is separated by semi-preparative HPLC to obtain sub-fraction Fr.7-8-6, and sub-fraction Fr.7-8-6 is further separated by normal phase silica gel column chromatography to obtain 23 fractions, among which fraction Fr.7-8-6-15 is separated by semi-preparative HPLC to obtain compound montagnulan A.

5. Use of the pyrrolidine-2,4-dione compound according to claim 1 in the preparation of an osteoclast differentiation inhibitor.

6. The use according to claim 5, characterized in that: The osteoclast differentiation inhibitor is a drug for treating osteolytic diseases such as osteoporosis, rheumatoid arthritis, and tumor metastasis bone destruction caused by excessive osteoclast activation.

7. The use according to claim 5, characterized in that: It is selected from the monomeric form of montagnulanA, or a natural product extract containing montagnulanA.

8. The use according to claim 5, characterized in that: The drug is prepared into a clinically acceptable drug preparation by taking montagnulan A as the main component and adding a pharmaceutically acceptable carrier or excipient, or adding pharmaceutically acceptable auxiliary materials or auxiliary components.

9. The use according to claim 8, characterized in that: The dosage form of the drug includes a dosage form for gastrointestinal administration and a dosage form for parenteral administration.

Images