Application of furanodtraenoic acid compounds and pharmaceutical compositions thereof

Abstract

The present invention discloses the application of a furotetraenoic acid compound having the structure of formula (1) and its pharmaceutical composition in the preparation of a GLS1 inhibitor drug. Through effective protein binding, it exerts an inhibitory effect on GLS1 protein, achieving significant anti-tumor activity in vitro and in vivo. The IC 50 values of its GLS1 inhibition and cytotoxic activity are both less than 10 μM. At the animal level, it can significantly inhibit the volume and weight of tumors, and has multiple effects in preventing and treating tumors. The active ingredient is isolated from natural microbial fermentation products, without residual harmful substances, safe and effective, and the applicable pharmaceutical dosage forms are flexible and diverse, and the dosage is easy to adjust, suitable for various drug-using populations and conditions, and has a wide range of application scenarios. #imgabs0#

Description

Technical Field

[0001] The present invention relates to applications of a furanodtraenoic acid compound and a pharmaceutical composition thereof, and in particular to applications of a furanodtraenoic acid compound and a pharmaceutical composition thereof in the preparation of a GLS1 inhibitor drug. Background Art

[0002] Metabolic reprogramming, as one of the hallmarks of tumors, is an adaptive mechanism of tumor cells that can promote the occurrence and development of tumor cells and compete with other cells for glucose and nutrients. There are two main pathways for tumor metabolic reprogramming: glucose glycolysis and glutamine-dependent replenishment of the tricarboxylic acid cycle. Among them, glutamine is the most abundant non-essential amino acid in human blood, an essential component in the process of tumor cell culture, and an important metabolic substrate for tumor cells and the second largest energy source after glucose. Tumor cells can only continue to grow and survive by taking up glutamine, showing the so-called "glutamine addiction". After glutamine enters the cell, it is mainly converted into glutamate by glutaminase (GLS), and glutamate is then metabolized to produce α-ketoglutarate, which can enter the tricarboxylic acid cycle to produce ATP to provide energy for tumor cell growth. In addition, glutamine metabolism can also be used as a precursor for the synthesis of biological macromolecules such as proteins, nucleotides, and lipids, which plays a vital role in the survival and growth of tumor cells. Mammalian GLS proteins are divided into two types: kidney type (GLS1) and liver type (GLS2), of which GLS1 is more widely expressed in tissues and serves as a clear target for tumor suppression.

[0003] Currently, new small molecule regulators targeting various nodes in the glutamine metabolic pathway are constantly being developed, such as DON, CB-839, BPTES, 968, etc. However, these small molecules are all artificially synthesized products with a single structural type, poor applicability, and many side effects. In addition, only CB-839 is in clinical research, which limits the development and utilization of this type of inhibitors. Summary of the invention

[0004] Purpose of the invention: In view of the problems of existing small molecule drugs such as limited structural types, insufficient efficacy, and strong side effects, the present invention aims to provide a furanodtraenoic acid compound with novel structure and excellent in vitro and in vivo anti-tumor activity and its pharmaceutical composition for use in the preparation of GLS1 inhibitor drugs.

[0005] Technical solution: As the first aspect of the present invention, furanodtraenoic acid compounds having the structure of formula (1) are used in the preparation of GLS1 inhibitor drugs.

[0006]

[0007] Compared with synthetic drug molecules, natural products have more skeleton types, less resistance, less toxicity, and stronger adaptability. They play an important role in drug development and are an important source of new chemical entity drugs. For example, artemisinin and paclitaxel are derived from natural products. Therefore, discovering GLS1-specific inhibitors with novel structural types and good drugability from natural products is conducive to the development of new anti-tumor metabolic drugs.

[0008] The present invention studies the metabolites of the aster plant endophytic fungus Pestalotiopsis neglecta S3, and obtains a furanoatetraenoic acid compound pestalotic acid G(1) by using a variety of separation and purification methods, including silica gel column chromatography, RP-18 and Sephadex LH-20 gel column chromatography. Subsequently, the inhibitory activity of the furanoatetraenoic acid compound pestalotic acid G(1) on glutaminase GLS1 is evaluated by an in vitro enzyme activity experiment, and the binding ability of the compound to glutaminase GLS1 is evaluated in SW620 cells by a cell thermal shift (CETSA) binding experiment. The above results show that the furanoatetraenoic acid compound is a new type of natural GLS1 inhibitor; subsequently, the in vitro and in vivo antitumor activity of the furanoatetraenoic acid compound pestalotic acid G(1) is evaluated by using colon cancer cell SW620 and nude mouse transplanted tumor models, and the results show that the furanoatetraenoic acid has in vitro and in vivo antitumor activity. The above results indicate that the furanodatetraenoic acid compound pestalotic acidG(1) has important applications in drugs for the treatment and prevention of glutaminase GLS1-related diseases and cancers.

[0009] Specifically, the above-mentioned GLS1 inhibitor drug is preferably a drug for treating and / or preventing tumors, and more preferably a drug for treating and / or preventing basal cell carcinoma, medulloblastoma, rhabdomyosarcoma, nevus-like basal cell carcinoma syndrome, small cell lung cancer, non-small cell lung cancer, metastatic prostate cancer, pancreatic cancer, chondrosarcoma, osteosarcoma, melanoma, glioma, breast cancer, ovarian cancer, esophageal cancer, gastric cancer, colon cancer, bile duct cancer, liver cancer, bladder cancer, hemangioma, chronic myeloid leukemia, acute lymphocytic leukemia, multiple myeloma, Hodgkin's lymphoma or non-Hodgkin's lymphoma.

[0010] In the application process of the above furandatetraenoic acid compounds, the daily dosage is preferably 0.01 to 15 mg / kg body weight, more preferably 0.1 to 10 mg / kg body weight. The administration may be one or more times.

[0011] The above-mentioned furanatetraenoic acid compound is prepared by the following steps:

[0012] (1) The endophytic fungus Pestalotiopsis neglecta S3 of aster plants was fermented in rice solid medium and extracted with ethyl acetate to obtain an extract;

[0013] (2) separating and purifying the extract obtained in step (1) to obtain the furanodtraenoic acid compound.

[0014] The specific method is to ferment the endophytic fungus Pestalotiopsis neglecta S3 (GenBank No. MT377727.1, CGMCC No. 3.20123) of aster plant at 28°C in rice solid culture medium for 35 days, then soak it in ethyl acetate overnight and fully extract it to obtain ethyl acetate extract; various separation materials and techniques such as silica gel, RP-18 and Sephadex LH-20 are used to separate and purify it to obtain furanodtraenoic acid compounds.

[0015] A more specific method is to activate the strain Pestalotiopsis neglecta S3 to grow in PDA culture medium, cut it into small pieces after the mycelium has grown all over the plate, and inoculate it into rice culture medium for fermentation under sterile conditions; the rice fermentation product of the strain is extracted with ethyl acetate three times, and the extract is concentrated under reduced pressure to obtain a total extract; the total extract is chromatographed on a silica gel column and gradient eluted with petroleum ether / acetone (1:0, 20:1, 2:1 and 1:1) to obtain 4 subfractions (Fr.1~Fr.4); Fr.2 is chromatographed on an RP-18 silica gel column and gradient eluted with methanol / water (10%~90%) to obtain 5 subfractions (Fr.2-1~Fr.2-5); Fr.2-3 is chromatographed on a Sephadex column and gradient eluted with methanol / water (10%~90%) to obtain 5 subfractions (Fr.2-1~Fr.2-5); LH-20 column chromatography with isocratic elution using chloroform / methanol (1:1) gave 6 subcomponents (Fr.2-3-1 to Fr.2-3-6). Fr.2-3-2 was subjected to semi-preparative HPLC to give the furanodtraenoic acid compound pestalotic acid G (1).

[0016] As a second aspect of the present invention, the pharmaceutical composition formed by the furanodtraenoic acid compound having the structure of formula (1) and a pharmaceutically acceptable carrier is used in the preparation of a GLS1 inhibitor drug.

[0017] The weight ratio of the furanatetraenoic acid compound is preferably 0.1% to 99.5%, more preferably 0.5% to 95%.

[0018] Specifically, the pharmaceutically acceptable carriers mentioned above refer to conventional drug carriers in the pharmaceutical field, such as: diluents, excipients such as water, fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin and polyvinyl pyrrolidone; wetting agents such as glycerol; disintegrants such as agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium stearate and magnesium stearate, and polyethylene glycol, etc. In addition, other adjuvants such as flavoring agents, sweeteners, etc. may be added to the composition.

[0019] The compounds of the present invention can be administered to patients in need of such treatment in the form of a composition by oral administration, nasal inhalation, rectal administration or parenteral administration. When used for oral administration, it can be prepared into conventional solid preparations such as tablets, powders, granules, capsules, etc., and into liquid preparations such as water or oil suspensions or other liquid preparations such as syrups, elixirs, etc.; when used for parenteral administration, it can be prepared into solutions for injection, water or oil suspensions, etc. Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the pharmaceutical field. For example, the active ingredient is mixed with one or more carriers and then prepared into the desired dosage form.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0021] 1. The furanoatetraenoic acid compound can effectively inhibit the activity of GLS1 protein, has significant in vitro and in vivo anti-tumor activity and protein binding effect, and its IC of GLS1 inhibition and cytotoxic activity is 50 The values ​​are all less than 10μM, which can significantly inhibit the volume and weight of tumors at the animal level, and can play multiple roles in preventing and treating tumors;

[0022] 2. The active ingredients used are separated from natural microbial fermentation products, have no harmful residues, are safe and effective, and the applicable pharmaceutical compositions have flexible and diverse dosage forms, and the dosage is easy to adjust. They are suitable for a variety of populations and conditions, and have a wide range of application scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The flowchart is a preparation process of the furanodtraenoic acid compound pestalotic acid G (1);

[0024] Figure 2 The inhibitory activity structure of pestalotic acid G(1), a furanoatetraenoic acid compound, on glutaminase GLS1;

[0025] Figure 3 The results are for the binding ability of furanodtraenoic acid compound pestalotic acid G(1) to glutaminase GLS1;

[0026] Figure 4 The results are for the in vitro antitumor activity of pestalotic acid G(1), a furanodtraenoic acid compound;

[0027] Figure 5 The results are for the in vivo antitumor activity of pestalotic acid G(1), a furanodtraenoic acid compound. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below in conjunction with embodiments.

[0029] Example 1: Preparation of furanodtraenoic acid compound pestalotic acid G (1)

[0030] Pestalotiopsis neglecta S3 (GenBank No. MT377727.1, CGMCC No. 3.20123) was grown in PDA medium at 28°C for 7 days. After the mycelium covered the plate, it was cut into small pieces and inoculated into 45 1L conical flasks containing rice medium (180g rice, 220mL tap water, autoclaved at 121°C for 30min) under sterile conditions. The pieces were then fermented at 28°C for 35 days.

[0031] The rice fermentation product of strain Pestalotiopsis neglecta S3 was extracted with ethyl acetate three times (15L×3 times), and the extract was concentrated under reduced pressure to obtain a total extract (69g). The total extract was chromatographed on a silica gel column and eluted with petroleum ether / acetone (1:0, 20:1, 2:1 and 1:1) gradient to obtain 4 fractions (Fr.1~Fr.4). Fr.2 (24g) was chromatographed on an RP-18 silica gel column and eluted with methanol / water (10%–90%) gradient to obtain 5 fractions (Fr.2-1~Fr.2-5). Fr.2-3 (15g) was chromatographed on a Sephadex LH-20 column and eluted isocratically with chloroform / methanol (1:1) to obtain 6 fractions (Fr.2-3-1~Fr.2-3-6). Fr.2-3-2 (7g) was purified by YMC-Pack ODS-A C 18 The furanodtraenoic acid compound pestalotic acid G(1) (943 mg) was obtained by semi-preparative HPLC separation using a 4% HPLC column (10×250mm, 5μm).

[0032] Example 2: Evaluation of the inhibitory activity of pestalotic acid G(1) against glutaminase GLS1 by enzyme inhibition assay

[0033] Experimental principle: GLS protein hydrolyzes its substrate glutamine into glutamate, which, under the action of glutamate dehydrogenase, transfers hydrogen atoms to NAD to generate NADH. The absorbance of NADH at 340nm is detected to reflect the activity of GLS protein.

[0034] Experimental method: Human GLS1 protein (0.1mM hGAC) and a certain concentration of compound were incubated in system 1 (50mMTris-Acetate pH=8.6, 0.2mM EDTA) at 25°C for 10min. Then, 200mM glutamine was added to start the first step of the reaction, and the reaction was carried out at 37°C for 60min. 0.6M hydrochloric acid was added to quench the reaction. Then system 2 (3.7units GDH, 160mMTris-Acetate pH=9.4, 400mM hydrazine, 5mM ADP, 2mM NAD + ) and incubated at 25°C for 30 min. Finally, the absorbance of the sample was measured at 340 nm.

[0035] The experimental results are shown in Figure 2 , NADH decreased significantly with the increase of concentration, pestalotic acid G(1) had inhibitory activity against GLS1, IC 50 It is 0.96μM.

[0036] Example 3: Evaluation of the binding ability of pestalotic acid G(1) to glutaminase GLS1 using protein thermal stability shift assay

[0037] Experimental principle: As the temperature rises, the protein will undergo denaturation, structural changes and even coagulation. The protein content in the cell can be detected by methods such as western blot. After the protein binds to the corresponding ligand drug small molecule, its thermal stability will be enhanced to a certain extent. Using the same heat treatment and measurement method, obvious thermal migration can be observed, and the degree of thermal migration can directly characterize the degree of binding between the protein and the small molecule.

[0038] Experimental method: SW620 cells in good condition were inoculated in 6-well plates, and after adherence, they were divided into groups and dosed with blank group, CB839 (10μM) and furanodtraenoic acid compound pestalotic acid G (1, 10μM) for 24h; cells in each well were collected, washed 2-3 times with PBS pre-cooled at 4℃, and the supernatant was discarded. An equal amount of PBS buffer containing protease inhibitors and phosphatase inhibitors was added to each group, and the mixture was thoroughly blown and mixed, and then divided into four equal parts; one aliquot from each group was heated at the specified temperature (40℃, 50℃, 60℃, 65℃) for 3min, and then restored to room temperature for 3min; it was subjected to liquid nitrogen quick freezing for 1min-room temperature thawing for 1min for 3-4 cycles, and finally the cell lysates were uniformly placed in a 4℃ centrifuge, centrifuged at 12000rpm for 30min, the supernatant was added to the loading buffer, boiled for 10min, and then subjected to protein immunoblotting analysis.

[0039] The results are as follows Figure 3 The results showed that the furanoderate acid compound pestalotic acid G(1) can bind well to the GLS1 protein and is a new type of natural inhibitor of glutaminase GLS1.

[0040] Example 4: Evaluation of the cytotoxic activity of pestalotic acid G (1) in colon cancer cells SW620 using SRB colorimetric assay

[0041] Experimental principle: Sulforhodamine B (SRB) is a pink anionic dye that is easily soluble in water. The basic amino acids that make up proteins in cells can specifically bind to this ion under acidic conditions and produce an absorption peak at a wavelength of 540nm. Its absorbance value is linearly positively correlated with the number of living cells and can be used to quantitatively detect the number of living cells.

[0042] Experimental method: SW620 cells were prepared into cell suspension in RPMI 1640 medium containing 10% fetal bovine serum, and 4000 cells were seeded in each well of a 96-well plate. After 24 hours, different concentrations of furanodtraenoic acid compound pestalotic acid G (1) were added to each well, so that the final concentrations were 20μM, 10μM, 5μM, 2μM, 1μM, 0.5μM and 0.25μM, respectively, and 3 replicate wells were set for each concentration. After the furanodtraenoic acid compound pestalotic acid G (1) was exposed to the medium for 24h or 48h, 25μL of 50% acetic acid solution was added to each well, fixed at room temperature for 1h, and then washed and dried. 100μL of 0.4% SRB staining solution was added to each well and stained at room temperature for 30min. Afterwards, the stain solution was discarded and the cells were washed three times with 1% acetic acid solution, dried, and 100 μL of 10 mM Tris was added to each well for dissolution. The absorbance was measured at 540 nm, and the inhibition rate and IC were calculated. 50 value.

[0043] The experimental results are shown in Figure 4 With the increase of exposure time and concentration, the survival rate of SW620 cells decreased significantly. The IC 50 It was 7.56±0.61μM (24h) and 3.60±0.20μM (48h).

[0044] Example 5: Evaluation of the in vivo antitumor activity of pestalotic acid G(1) using a SW620 nude mouse xenograft tumor model

[0045] SW620 cells were prepared with serum-free RPMI1640 medium at a concentration of 2×10 5 The cells / mL cell suspension was inoculated into the subcutaneous tissue of the left axilla of BABL / c nude mice. After 8 days of growth, a tumor-bearing mouse model was formed. The well-growing tumor-bearing mice were randomly divided into groups and intraperitoneally injected with pestalotic acid G(1) (20 mg / kg) and CB839 (20 mg / kg, positive control) once every two days. After 12 days of administration, the mice were killed, the tumors were removed and weighed, and statistical processing was performed.

[0046] The experimental results are shown in Figure 5 , where 5a is the line graph of tumor volume change, 5b is the tumor weight, Figure 5 c is a photo of the tumor after peeling. The results showed that after treatment with pestalotic acid G(1), the tumor volume and tumor weight of nude mice were reduced compared with the control group, indicating that pestalotic acid G(1) is a new type of strong natural anti-tumor drug.

[0047] The pestalotic acid G(1) of the present invention is a furanodtraenoic acid compound isolated from the endophytic fungus Pestalotiopsis neglecta S3 of Aster tataricus, and as a novel glutaminase GLS1 inhibitor, has good in vivo and in vitro anti-tumor activity. It is used in the preparation of targeted glutaminase GLS1 inhibitor drugs and anti-tumor drug preparations and prodrugs, and has the advantages of low preparation cost, low toxicity, good anti-tumor effect, etc.

[0048] Example 6: Preparation of tablets

[0049] Prescription: furanodtraenoic acid compound pestalotic acid G(1) 10mg, lactose 180mg, starch 55mg, magnesium stearate 5mg.

[0050] Preparation method: Mix the compound, lactose and starch, moisten them evenly with water, sieve the moistened mixture and dry it, sieve it again, add magnesium stearate, and then compress the mixture into tablets, each tablet weighing 250 mg and containing 10 mg of the compound.

[0051] Example 7: Preparation of ampoule

[0052] Prescription: furanodtraenoic acid compound pestalotic acid G(1) 2mg, sodium chloride 10mg.

[0053] Preparation method: Dissolve the compound and sodium chloride in an appropriate amount of water for injection, filter the resulting solution, and fill it into an ampoule under sterile conditions.

[0054] Example 8: Preparation of lyophilized agent

[0055] Prescription: pestalotic acid G(1) 10mg, sodium bicarbonate 2mg, mannitol 252mg.

[0056] Preparation method: Sodium bicarbonate and mannitol are dissolved in water for injection, activated carbon is added for adsorption for 30 minutes to remove pyrogens, the activated carbon is filtered to remove, the compound is added to the filtrate, ultrasonic treatment is performed to dissolve, the pH is adjusted to 5.0-7.0 with 1N hydrochloric acid, filtered through a microporous filter membrane, water for injection is added, packaged, freeze-dried, plugged, and capped to obtain the product.

[0057] Example 9: Preparation of capsules

[0058] Prescription: furanodtraenoic acid compound pestalotic acid G(1) 10mg, lactose 187mg, magnesium stearate 3mg.

[0059] Preparation method: Mix the compound with the cosolvent, sieve, mix evenly, and fill the resulting mixture into hard gelatin capsules. Each capsule weighs 200 mg and contains 10 mg of active ingredient.

Claims

1. Use of a furanodtraenoic acid compound having the structure of formula (1) in the preparation of a GLS1 inhibitor drug, wherein the GLS1 inhibitor drug is a drug for treating and / or preventing colon cancer. Formula (1).

2. The use according to claim 1, characterized in that: The daily dose of furanodtraenoic acid compounds is 0.01~15 mg / kg body weight.

3. The use according to claim 2, characterized in that: The daily dosage of furanodtraenoic acid compounds is 0.1-10 mg / kg body weight.

4. The use according to claim 1, characterized in that: The preparation method of the furan tetraenoic acid compound comprises the following steps: (1) Endophytic fungi in Aster plants Pestalotiopsis neglecta S3 was fermented with rice solid medium and extracted with ethyl acetate to obtain an extract; (2) Separating and purifying the extract obtained in step (1) to obtain the furanodtraenoic acid compound.

5. Use of a pharmaceutical composition comprising a furanodtraenoic acid compound of formula (1) and a pharmaceutically acceptable carrier in the preparation of a GLS1 inhibitor drug, wherein the GLS1 inhibitor drug is a drug for treating and / or preventing colon cancer. Formula (1).

6. The use according to claim 5, characterized in that: The weight ratio of the furanatetraenoic acid compound in the pharmaceutical composition is 0.1% to 99.5%.

7. The use according to claim 6, characterized in that: The weight ratio of the furanatetraenoic acid compound in the pharmaceutical composition is 0.5% to 95%.

8. The use according to claim 5, characterized in that: The pharmaceutically acceptable carrier is selected from one or more of starch, sucrose, alginate, gelatin, polyvinyl pyrrolidone, glycerol, agar, calcium carbonate, sodium bicarbonate, quaternary ammonium compounds, cetyl alcohol, kaolin, bentonite, talc, calcium stearate, magnesium stearate, polyethylene glycol, and flavoring agents.

9. The use according to claim 5, characterized in that: The pharmaceutical composition is administered orally, by inhalation, rectally or parenterally.

10. The use according to claim 5, characterized in that: The pharmaceutical composition is in the form of tablets, powders, granules, capsules, oral aqueous suspensions, oral oily suspensions, syrups, elixirs, injection solutions, injection aqueous suspensions, and injection oily suspensions.

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