Application of sterone compound in uniflower swisscentaury root in preparation of medicine for inhibiting protein tyrosine phosphatase

Abstract

The invention belongs to the technical field of medicines, and provides application of sterone compounds in uniflower swisscentaury roots in preparation of medicines for inhibiting protein tyrosine phosphatase. Experimental research finds that the sterone compounds shown in the formula I and the formula II have inhibitory activity on T-cell protein tyrosine phosphatase (TCPTP) and protein tyrosine phosphatase 1B (PTP1B), and can be used for preparing drugs for inhibiting TCPTP and / or PTP1B and drugs for preventing, delaying or treating type II diabetes mellitus and / or obesity; the sterone compounds with the structural formulas as shown in the formula II and the formula III have inhibitory activity on non-receptor protein tyrosine phosphatase 9 (MEG2), can be used for preparing a medicine for inhibiting MEG2, preparing a medicine for preventing, delaying or treating chronic granulocytic leukemia, and also can be used for preparing a medicine for preventing, delaying or treating type II diabetes mellitus and / or obesity. The new application lays a foundation for deep research of active ingredients of uniflower swisscentaury roots and development of new drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and relates to use of a sterol ketone compound from Qizhou radix elegans in preparing a drug for inhibiting protein tyrosine phosphatase. Background Art

[0002] Protein-tyrosine phosphatases (PTPs) are key enzymes in regulating cell signaling. They control processes throughout the cell cycle, including growth, differentiation, and metabolism, by regulating intracellular protein tyrosine phosphorylation, and participate in numerous physiological functions in the human body. Disturbances in PTPase activity can lead to a variety of diseases, such as diabetes and obesity (Kishihara et al., Cell, 1993, 74:143-156; Desai et al., Cell, 1996, 84:599-609). Currently, certain protein-tyrosine phosphatases (such as PTP1B, a therapeutic target for diabetes and obesity) have become recognized new drug targets and are a hot topic for research and development in drug development institutions worldwide.

[0003] Current research indicates that PTP1B (Protein tyrosine Phosphatase 1B) is most closely linked to the insulin signaling pathway (Brown-Shimer S., et al., Proc Nat Acad Sci, 1990, 87: 5148-5152). PTP1B is associated with the endoplasmic reticulum via its catalytically inactive C-terminal domain. It has enhanced IRβ dephosphorylation and IRS phosphorylation activities, thereby inhibiting the formation of the IRS-PI3K p85 subunit complex, as well as the phosphorylation of Akt and MAPK. Furthermore, it inhibits insulin-induced glycogen synthesis. Therefore, PTP1B may negatively regulate multiple sites in the insulin signaling cascade. Overexpression of PTP1B can increase insulin receptor dephosphorylation and downregulate post-receptor signaling; conversely, it can enhance insulin signaling (Elchebly M., et al., Science, 1999, 283: 1544-1548). Animal experiments have shown that PTP1B gene knockout mice have increased basal metabolic rate and total energy expenditure, reduced body weight, and enhanced insulin sensitivity (Klaman L.D., et al. Molecular and Cellular Biology, 2000, 20(15), 5479-5489). Elchebly et al. reported that PTP1B gene knockout mice are phenotypically healthy and have lower fasting blood insulin and blood glucose levels, and a significant decrease in blood glucose in insulin tolerance tests, which show increased insulin sensitivity. This sensitization effect is tissue-specific, increasing glucose uptake in skeletal muscle but having no significant effect on adipose tissue. Even more exciting is that the knockout mice do not become obese or gain weight when fed a high-fat diet, nor do they experience increased IR (Elchebly M., et al., Science 1999, 283: 1544-1548). Studies have shown that inhibiting PTP1B can effectively solve the two main problems of metabolic syndrome: impaired glucose tolerance and obesity. At the same time, increasing evidence shows that PTP1B plays an important role in regulating insulin sensitivity and energy metabolism, and is a new target for drug research in diabetes, obesity, etc. (Darry A.J., Future Med Chem, 2010, 2(10): 1563-1576).

[0004] Studies have found that knocking out PTP-MEG2 in the liver of diabetic mice can lead to insulin sensitization and normalization of hyperglycemia, and PTP-MEG2 inhibitors can enhance insulin action and improve insulin sensitivity and blood glucose homeostasis in diet-induced obese mice, indicating that inhibiting PTP-MEG2 activity is an effective strategy for treating type 2 diabetes.

[0005] Therefore, in recent years, the research on protein tyrosine phosphatase inhibitors has attracted widespread interest. Generally speaking, they can be roughly divided into the following four categories: metal vanadium compounds; irreversible inhibitors; natural product inhibitors; and structure-based inhibitors. Among these four types of PTP inhibitors, vanadium-containing compounds have many drawbacks in their clinical application as drugs: they are non-selective inhibitors and can cause toxicity due to excessive accumulation in the body. These drawbacks limit their clinical application. For example, Chinese patent application number CN202111194384.1 provides specific inhibitors targeting a single PTPase, which can improve enzyme inhibitory activity while reducing its effective concentration. However, long-term use may still cause hepatotoxicity and renal toxicity. Existing irreversible inhibitors and structure-based inhibitors also have certain toxic side effects. Natural product inhibitors, however, have attracted considerable attention as pharmaceuticals due to their structural diversity, biological activity, and low toxicity.

[0006] Rhaponticum uniflorum (L.) DC., a perennial herb in the Asteraceae family, is listed as a top-grade medicinal herb in the Shennong's Herbal Classic. It is primarily found in Anguo City, Hebei Province. Rhaponticum uniflorum has numerous therapeutic effects, including clearing heat and detoxifying, providing antioxidant benefits, regulating blood lipids, fighting tumors, providing anti-inflammatory effects, relieving pain, and promoting lactation.

[0007] At present, domestic and foreign scholars have conducted a series of studies on the chemical components of Qizhou radix scutellariae. The chemical components discovered mainly include phytoecdysones, triterpenes, and thiophenes. In addition, there are flavonoids and volatile oil components such as cyperasterone, cyperasterone, and tursterone. As ecdysone compounds, they have attracted the attention of scholars. CN1334093A discloses the medical uses of the phytosterone components of the traditional Chinese medicine radix scutellariae, which provides new uses for the phytosterone components of radix scutellariae and provides a new approach for the development of radix scutellariae. The phytosterone components of radix scutellariae can be used to prepare drugs for treating osteoporosis, can also be used to prepare drugs with bone resorption inhibition activity and chondrocyte DNA promotion activity, and can also be used to prepare drugs with intelligence-enhancing and antioxidant effects. CN1224023A discloses that the main component of the total sterones of radix scutellariae is β-ecdysone, which is mainly used to improve memory impairment caused by brain dysfunction. CN 116813683 A studied the active ingredients of the herb Lophatherum chinense. Ten sterol compounds were isolated from the root of Lophatherum chinense, two of which were discovered for the first time. Antitumor activity of the isolated sterol compounds was studied, and it was found that the compounds had inhibitory activity against multiple tumor cell lines.

[0008] Since the types and efficacy of sterones in Qizhou Lophatherum need to be further explored, the present invention has carried out relevant research. Summary of the Invention

[0009] The invention provides use of a sterone compound from Qizhou radix elegans in preparing a drug for inhibiting protein tyrosine phosphatase.

[0010] The technical solution of the present invention is achieved as follows:

[0011] The present invention provides a use of steroid ketone compounds having the structural formulas shown in Formula I and Formula II in the preparation of drugs for inhibiting protein tyrosine phosphatase, wherein the protein tyrosine phosphatase is T-cell protein tyrosine phosphatase and / or protein tyrosine phosphatase 1B;

[0012] Formula I;

[0013] Formula II.

[0014] The present invention also provides the use of steroid ketone compounds of the structural formulas shown in Formula I and Formula II in the preparation of drugs for preventing, delaying or treating type II diabetes and / or obesity.

[0015] The present invention also provides the use of steroid ketone compounds of the structural formulas shown in Formula II and Formula III in the preparation of drugs for inhibiting protein tyrosine phosphatase, wherein the protein tyrosine phosphatase is a non-receptor protein tyrosine phosphatase 9;

[0016] Formula II;

[0017] Formula III.

[0018] The present invention also provides the use of steroid ketone compounds with the structural formulas shown in Formula II and Formula III in the preparation of drugs for preventing, delaying or treating chronic myeloid leukemia.

[0019] The present invention also provides the use of steroid ketone compounds of the structural formulas shown in Formula II and Formula III in the preparation of drugs for preventing, delaying or treating type II diabetes and / or obesity.

[0020] As a further improvement of the present invention, the drug for the above-mentioned use further comprises at least one pharmaceutically acceptable carrier or excipient.

[0021] In the preparation of the medicament of the present invention, the sterone compound can be administered independently or in combination with a pharmaceutically acceptable carrier and excipient. "Pharmaceutically acceptable carriers or excipients" include: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, taste masking agents, coloring agents, anti-caking agents, humectants, chelating agents, plasticizers, tackifiers, antioxidants, preservatives, stabilizers, surfactants and buffers. Those skilled in the art will understand that certain pharmaceutically acceptable excipients can be used with more than one function and with alternative functions, depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation. There are many resources available to those skilled in the art that describe pharmaceutically acceptable excipients and that can be used to select suitable pharmaceutically acceptable excipients, such as books such as "Remington's Pharmaceutical Science," "Chinese Pharmaceutical Annals," and "Pharmaceutics."

[0022] As a further improvement of the present invention, the drug described in the above use is administered orally, by subcutaneous injection, intravenous injection or nasal administration.

[0023] As a further improvement of the present invention, the drug for the above use is prepared into one of the following dosage forms: tablets, capsules, granules, powders, suspensions, emulsions, powders, solutions, gels, syrups, pills, tinctures, wines, decoctions, lozenges, mixtures, suppositories, injections, inhalants or sprays.

[0024] The beneficial effects of the present invention are:

[0025] 1. The present invention provides a novel application for the study of sterol compounds isolated from the roots of Qizhou phragmites for inhibiting the activity of PTPs, including T-cell protein tyrosine phosphatase (TCPTP), protein tyrosine phosphatase 1B (PTP1B), and non-receptor protein tyrosine phosphatase 9 (MEG2). Experimental studies have found that the sterol compounds represented by Formula I and Formula II have inhibitory activity against T-cell protein tyrosine phosphatase (TCPTP) and protein tyrosine phosphatase 1B (PTP1B) and can be used to prepare drugs that inhibit TCPTP and / or PTP1B. The sterol compounds represented by Formula II and Formula III also have inhibitory activity against non-receptor protein tyrosine phosphatase 9 (MEG2) and can be used to prepare drugs that inhibit MEG2.

[0026] 2. T-cell protein tyrosine phosphatase (TCPTP), protein tyrosine phosphatase 1B (PTP1B), and non-receptor protein tyrosine phosphatase 9 (MEG2) mediate related diseases, including diabetes and neurological diseases. Among them, TCPTP and PTP1B are the main targets for the development of drugs for the treatment of type 2 diabetes. MEG2 affects the proliferation and survival of leukemia cells by dephosphorylating BCR-ABL (an oncogenic protein in chronic myeloid leukemia). Therefore, the sterone compounds represented by Formula I and Formula II can be used to prepare drugs for preventing, delaying, or treating type 2 diabetes and / or obesity; the sterone compounds represented by Formula II and Formula III can be used to prepare drugs for preventing, delaying, or treating chronic myeloid leukemia, as well as drugs for preventing, delaying, or treating type 2 diabetes and / or obesity. The new uses provided by this invention lay the foundation for in-depth research on the active ingredients of Qizhou Loulu and the development of new drugs. DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0028] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the technical field of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The experimental techniques or test methods involved in the embodiments of the present invention, unless otherwise specified, are conventional methods in the prior art, and their names and / or abbreviations are conventional names in this area, and are very clear and definite in the relevant application fields. Those skilled in the art can understand conventional process steps and apply corresponding equipment according to the names, and implement them according to conventional conditions or conditions recommended by the manufacturer. The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention have no special restrictions on source, are conventional products that can be purchased through regular commercial channels, and can also be prepared according to conventional methods well known to those skilled in the art.

[0029] Preparation Example 1 Compound Extraction (Same as Chinese Patent Application Publication No. CN 116813683 A)

[0030] The roots of Qizhou Phragmites australis are appropriately crushed and then extracted with 95% ethanol under reflux, the extract is filtered and concentrated under reduced pressure to an extract. The extract is dispersed in distilled water and extracted with petroleum ether, dichloromethane, and ethyl acetate, respectively. The ethyl acetate phase and the aqueous phase are concentrated under reduced pressure to obtain an ethyl acetate enrichment and an aqueous phase enrichment.

[0031] (1) Separation of aqueous phase enrichment:

[0032] S1 medium-pressure ODS column separation, chromatographic conditions: methanol-water (0:100→100:0) gradient elution for 24 h (the medium-pressure and high-pressure liquid chromatography involved in this preparation example, the gradient elution is a uniform gradient change within the set time), flow rate 80 mL / min, column volume of approximately 5 L, detection wavelength 254 nm, 20 components were obtained, respectively recorded as components 1-20.

[0033] S2 Preparative HPLC Separation:

[0034] The component 6 obtained in S1 was separated using the chromatographic conditions of 25% acetonitrile water isocratic elution for 35 min to obtain compound 1 (t R =15.7 min) and compound 2 (t R =28.3min);

[0035] The fraction 5 obtained in S1 was separated using the following chromatographic conditions: 15%-35% acetonitrile-water gradient elution for 40 min to obtain compound 10 (35.9 mg, t R =32.3min).

[0036] (2) Separation of ethyl acetate enrichment:

[0037] S1 was separated by a primary silica gel column at normal pressure: gradient elution with dichloromethane-methanol (100:0→0:100) to obtain 5 components, which were respectively recorded as components 1-5.

[0038] S2 secondary normal pressure silica gel column separation: fraction 2 obtained in S1 was loaded onto the column, and gradient eluted with dichloromethane-methanol (98:2→0:100). Six fractions were separated and combined, and recorded as fractions Fr1-6 respectively.

[0039] S3 medium pressure ODS column chromatography separation:

[0040] S3.1 Use component Fr3 obtained in S2 to load the column, and gradient elute with methanol-water (10%-100%) for 5 hours, a flow rate of 20 mL / min, and a column volume of 600 mL. Combine similar components to obtain a total of 10 components, which are recorded as components 1-10 respectively.

[0041] S3.2 Component Fr6 obtained in S2 was chromatographed on a medium-pressure ODS column with a gradient elution of methanol-water (0:100→100:0) for 6 h at a flow rate of 20 mL / min and a column volume of 1000 mL. Similar components were combined to obtain a total of 13 components, which were recorded as components 1-13.

[0042] S4 Preparative HPLC Separation:

[0043] The component 9 obtained in S3.1 was separated using the following chromatographic conditions: 20%-45% acetonitrile-water gradient elution for 40 min to obtain compound 3 (t R =15.7 min) and compound 4 (t R =30.2min).

[0044] The fraction 12 obtained in S3.2 was separated using the following chromatographic conditions: 18%-40% acetonitrile gradient elution for 35 min to obtain compound 5 (t R =12.7min), compound 6 (t R =15.8min) and compound 7 (t R =28.5min).

[0045] The component 13 obtained in S3.2 was separated using the following chromatographic conditions: 10%-55% acetonitrile-water gradient elution for 45 min, flow rate 20 mL / min, detection wavelength 254 nm) to give compound 8 (t R =21.4 min) and compound 9 (t R =39.5min).

[0046] According to the above method, the obtained compound 9 is makisterone C, namely formula II, with the structural formula

[0047] ;

[0048] Compound 6 is (24R, 25R)-amamasterono A, i.e., formula III, with the structural formula

[0049] ;

[0050] Compound Carthamosterone, i.e. Formula I, has the structural formula

[0051]

[0052] The extraction method of formula I is as follows:

[0053] The roots of Qizhou Phragmites australis were crushed and extracted with 95% ethanol under reflux. The extract was filtered and concentrated under reduced pressure. Distilled water was added to the concentrate, and the extract was extracted with petroleum ether, dichloromethane and ethyl acetate in sequence. The ethyl acetate phase was concentrated under reduced pressure to obtain an ethyl acetate extract.

[0054] The ethyl acetate extract was separated by normal pressure silica gel column chromatography: dichloromethane-methanol (100:0→0:100) gradient elution, and a fraction of about 200 mL was collected. Similar fractions were combined to obtain 16 components, which were respectively recorded as components Fr1-16.

[0055] Fractions Fr8-16 were combined and separated by medium-pressure ODS column chromatography. Chromatographic conditions: 25%-45% acetonitrile-water gradient elution for 40 min, flow rate 20 mL / min, to obtain Formula I (t R =20.5 min).

[0056] Example 1 Determination of inhibitory activity against T-cell protein tyrosine phosphatase (TCPTP), protein tyrosine phosphatase 1B (PTP1B) and non-receptor protein tyrosine phosphatase 9 (MEG2)

[0057] TCPTP, PTP1B, and MEG2 are all recombinant human protein tyrosine phosphatases that were expressed in Escherichia coli using genetic engineering methods and purified by affinity chromatography.

[0058] After the PTP enzymes react with the substrate, nitrophenyl dihydrogen phosphate (pNPP), the chromophore-containing pNPP is catalyzed by the phosphatase to form p-nitrophenol (pNP) and phosphate. Since pNP has an absorption peak at 405 nm, UV spectrophotometry can be used to measure the absorbance of pNP generated by the enzyme-catalyzed reaction to determine the degree of inhibition of the PTP enzyme activity by the compound. The specific procedure is as follows: 1 μL of compound is added to a 96-well plate, followed by 50 μL of buffer containing the enzyme (50 mM Tris, 2 mM DTT, and 2 mM EDTA at pH 6.0), followed by 50 μL of buffer containing pNPP (final concentration 2.5 mM). The mixture is incubated at 37°C for 30 minutes. The reaction is terminated by adding 2M NaOH, and the absorbance change at 405 nm is measured using a microplate reader.

[0059] Inhibition rate = (1-average absorbance of experimental group / average absorbance of blank group) × 100%

[0060] After testing, the results are shown in Table 1 below.

[0061]

[0062] The test results show that the compound of formula I and podocarpone C (formula II) have significant inhibitory activity against PTP1B and TCPTP. TCPTP and PTP1B are the main targets for developing drugs for the treatment of type 2 diabetes. It can be seen that the above two compounds have the potential to be developed into drugs for type 2 diabetes, and can be used to prepare drugs for preventing, delaying or treating diseases mediated by TCPTP and PTP1B, especially type 2 diabetes and obesity, or as lead compounds for such drugs.

[0063] Compounds of Formula I, podocarpone C (Formula II), and (24R,25R)-purpurasterone A (Formula III) exhibit significant inhibitory activity against MEG2. Compound (24R,25R)-purpurasterone A (Formula III) is particularly noteworthy for its high selectivity for MEG2, lacking inhibitory activity against TCPTP and PTP1B. This provides a foundation for the further development of MEG2 inhibitors. MEG2 dephosphorylates BCR-ABL (an oncogenic protein in chronic myeloid leukemia), affecting the proliferation and survival of leukemia cells. These compounds have the potential to be developed as leukemia therapeutics. MEG2 is also a target for diabetes treatment. Therefore, compound (24R,25R)-purpurasterone A (Formula III) can be used to prepare drugs for the prevention, delay, or treatment of type 2 diabetes and / or obesity.

[0064] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of the steroid ketone compounds of formula I and formula II in the preparation of drugs for inhibiting protein tyrosine phosphatase, characterized in that: The protein tyrosine phosphatase is T-cell protein tyrosine phosphatase and / or protein tyrosine phosphatase 1B; Formula I; Formula II.

2. Use of the sterol ketone compound according to claim 1 in the preparation of a medicament for preventing, delaying or treating type II diabetes and / or obesity.

3. Use of the sterol ketone compounds of formula II and formula III in the preparation of drugs for inhibiting protein tyrosine phosphatase, characterized in that: The protein tyrosine phosphatase is a non-receptor protein tyrosine phosphatase 9; Formula II; Formula III.

4. Use of the sterol ketone compound according to claim 3 in the preparation of a medicament for preventing, delaying or treating chronic myeloid leukemia.

5. Use of the sterol ketone compound of formula III as claimed in claim 3 in the preparation of a drug for preventing, delaying or treating type II diabetes and / or obesity.

6. The use according to any one of claims 1 to 5, characterized in that The medicament further comprises at least one pharmaceutically acceptable carrier or excipient.

7. The use according to any one of claims 1 to 5, characterized in that The drug is administered orally, subcutaneously, intravenously and / or nasally.

8. The use according to any one of claims 1 to 5, characterized in that The drug is prepared into one of the following dosage forms: tablets, capsules, granules, powders, suspensions, emulsions, powders, solutions, gels, syrups, pills, tinctures, wines, decoctions, lozenges, mixtures, suppositories, injections, inhalants or sprays.

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