Use of a benzofuran dimer compound in preparing a drug for treating diseases mediated by protein tyrosine phosphatase 1B

Abstract

This invention discloses the use of a benzofuran dimer compound in the preparation of a drug for treating diseases mediated by protein tyrosine phosphatase 1B, wherein the benzofuran dimer compound is derived from Vaseline (…). Eupatorium chinense The extract was isolated from the roots of *Eupatorium fortunei*. Four benzofuran dimer compounds were obtained by separating the extract using macroporous adsorption resin, semi-preparative liquid chromatography, and chiral HPLC. The protein tyrosine phosphatase 1B (PTP1B) inhibitory activity of these benzofuran dimer compounds was then tested. The results showed that three of the four benzofuran dimer compounds exhibited good inhibitory activity and could be used to formulate drugs for the prevention, delay, or treatment of PTP1B-mediated diseases, particularly type II diabetes and obesity, or as lead compounds for such drugs.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the preparation of benzofuran dimer compounds and their use in the preparation of drugs for treating diseases mediated by protein tyrosine phosphatase 1B. Background Technology

[0002] Protein-tyrosine phosphatases (PTPs) are a family of receptor-like cytoplasmic signal transduction enzymes that regulate metabolic processes in various cells and maintain homeostasis by dephosphorylating their substrate tyrosine residues (Kimet al., Int. J. Mol. Sci. 2018, 19: 2708). Protein-tyrosine phosphatase 1B (PTP1B) is a typical non-transmembrane tyrosine phosphatase and a negative transducer of insulin signaling. Insulin resistance, especially in adipose tissue and muscle, is closely related to the development of type 2 diabetes mellitus (T2DM) (Minoura et al., Eur. J. Pharmacol . 2005, 519: 182-190; Kim et al., J. Clin. Invest . 2000, 105: 1791-1797).

[0003] With globalization, modernization, and changes in human lifestyles, the incidence of diabetes and obesity is increasing year by year (Xiao et al., Curr. Med. Chem (2015, 22: 23-38). According to the International Diabetes Federation (IDF), approximately 451 million people worldwide are affected by diabetes, and this number is estimated to reach 693 million by 2045 (Choet et al., 2015, 22: 23-38). Diabetes Res. Clin. Pract (2018, 138: 271-281). Disruption of insulin production and / or function can lead to diabetes. PTP1B is an important member of protein tyrosine phosphatases (PTPs) and has a negative regulatory effect on the insulin metabolic pathway. Overexpression of PTP1B may lead to decreased phosphorylation of the insulin receptor (Kenner et al., 2018, 138: 271-281). Mol. Cell. Biochem (1998, 182: 101-108), and studies have shown that mutations in the PTP1B gene can lead to diabetes (Meshkani et al., 1998, 182: 101-108). Clin. Chem (Johnson et al. 2007, 53: 1585-1592). Therefore, PTP1B is considered an important drug target for the treatment of diabetes and obesity-related diseases (Johnson et al., 2007, 53: 1585-1592). Nat. Rev. Drug. Discov(2002, 1: 696-709). Developing specific inhibitors of PTP1B shows promising potential for the treatment of type 2 diabetes and obesity.

[0004] Furthermore, PTP1B overexpression has been detected in studies of breast cancer, ovarian cancer, and prostate cancer, and it can be detected at all stages of tumor development (Kenneth et al., Mol. Cell. Biol .1998, 18, 2965-2975; Weiner et al., J. Natl. Cancer Inst (1996, 86, 372-378), indicating that PTP1B is closely related to many important tumorigenesis signaling pathways. Therefore, PTP1B inhibitors can be used to treat or prevent cancer or to slow its progression during cancer development.

[0005] Natural products (especially those derived from plants) have historically been an important source for discovering innovative drugs with novel structures and mechanisms of action (Newman et al., J.Nat.Prod (2020, 83: 770-803). Plant active ingredients are characterized by diverse skeletons, novel structures, multiple targets, and low toxicity; they are often the result of natural selection and evolution, enabling them to effectively bind to biomolecules and exhibit good activity. Therefore, screening and discovering novel and highly effective PTP1B inhibitors from plant-derived active ingredients has significant research value. Summary of the Invention

[0006] The first objective of this invention is to provide the use of a benzofuran dimer compound in the preparation of a medicament for the prevention or treatment of diseases mediated by protein tyrosine phosphatase PTP1B, and the second objective is to provide a method for its preparation.

[0007] A benzofuran dimer compound having a structural formula comprising any one or more combinations of the following:

[0008] , ,

[0009] .

[0010] The technical solution of the present invention is the application of the benzofuran dimer compound in the preparation of drugs for preventing, delaying or treating diseases mediated by protein tyrosine phosphatase PTP1B.

[0011] Another technical solution of the present invention is the use of the benzofuran dimer compound in the preparation of a drug for the prevention, delay or treatment of diabetes, obesity or its complications.

[0012] The method for preparing the benzofuran dimer compound includes the following steps:

[0013] A. Solvent extraction: The roots of Zephyranthes cusia were dried and pulverized, and then extracted with 50-70% ethanol at room temperature and concentrated to obtain extract a.

[0014] B. Ethyl acetate extraction: Extract a is suspended in water and then extracted sequentially with petroleum ether and ethyl acetate. The resulting ethyl acetate extract is then concentrated to obtain ethyl acetate extract b.

[0015] C. Macroporous adsorption column chromatography separation: Ethyl acetate extract b is dissolved in water and loaded onto a macroporous adsorption resin chromatography column. First, it is eluted with water, and then the column is eluted sequentially with 20%-95% ethanol. The 50%-70% ethanol eluent is collected, concentrated, and dried to obtain extract c.

[0016] D. HPLC separation and purification: The extract was separated by preparative HPLC to obtain racemic mixture A and racemic mixture B;

[0017] E. Racemic mixture A was separated by chiral HPLC to obtain compounds I and II; racemic mixture B was separated by chiral HPLC to obtain compound III.

[0018] In a preferred embodiment, the organic solvent in step A is 70% ethanol.

[0019] In step C, the ethanol gradient elution concentrations are 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 95%.

[0020] The chiral HPLC separation conditions for racemic mixture A were as follows: using an AD-H chiral column and performing liquid phase separation through an 85-90% methanol-water solution.

[0021] The chiral HPLC separation conditions for racemic mixture B were as follows: using an AD-H chiral column and performing liquid phase separation through a 75-80% methanol aqueous solution.

[0022] The racemic mixture A was separated by chiral HPLC using an 88% methanol aqueous solution for liquid phase separation.

[0023] The racemic mixture B was separated by chiral HPLC using an 80% methanol aqueous solution for liquid phase separation.

[0024] Compared with the prior art, this application has at least one of the following beneficial effects:

[0025] 1. For the first time, three benzofuran dimer compounds extracted and isolated from the roots of Zephyranthes cusia have been discovered to be PTP1B inhibitors, which have broad application prospects for development into drugs that can prevent or treat PTP1B-mediated diseases such as diabetes and obesity.

[0026] 2. The compound of this application has significant PTP1B inhibitory activity, and there are no reports in the existing literature on the PTP1B inhibitory activity of the compound and its application in related diseases. Detailed Implementation

[0027] The following embodiments are used to further explain and illustrate the present invention. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0028] Example 1: Preparation of benzofuran dimer compounds

[0029] The preparation method includes the following steps:

[0030] Step A: The roots of Zephyranthes cusia (8.0 kg) were dried and crushed. They were then extracted three times with 70% ethanol at room temperature. The extract was filtered through a Buchner funnel and concentrated by a rotary evaporator to obtain a brown extract a (830 g).

[0031] Step B: Extract a is suspended in water and then extracted four times with petroleum ether and ethyl acetate respectively. The ethyl acetate extract is concentrated to obtain ethyl acetate extract b (96 g).

[0032] Step C: Dissolve extract b in water, load it onto a macroporous adsorption resin chromatography column, elute with water first, then elute the column with a gradient of 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 95% ethanol, collect the 50%-70% ethanol eluent, concentrate and dry to obtain extract c (30 g).

[0033] Step D: The extract c is separated by preparative HPLC to obtain racemate A and racemate B. The racemic mixture A is then separated into enantiomer I (t...) by chiral HPLC. R =18.6 min) (22 mg, accounting for 0.0028‰ of the medicinal material content) and II (t R =19.2 min)(20 mg, accounting for 0.0025‰ of the medicinal material content), the chiral HPLC analysis conditions were: column: AD-H chiral column, mobile phase: 88% CH3OH / water, flow rate: 1 mL / min. The racemic mixture B was separated into enantiomer III (t) by chiral HPLC.R =22.4 min)(32 mg, accounting for 0.004‰ of the medicinal material content) and IV (t R =23.2 min)(30 mg, accounting for 0.00375‰ of the medicinal material content). The chiral HPLC analysis conditions were: column: AD-H chiral column, mobile phase: 80% CH3OH / water, flow rate: 1 mL / min.

[0034] Example 2: Determination of inhibitory activity of protein tyrosine phosphatase 1B

[0035] 1. Experimental Materials

[0036] 1.1 Experimental instruments: Electronic balance (AL204-IC, Shanghai Mettler Toledo Instruments Co., Ltd.); InfiniteM200 Pro microplate reader (TECAN Group, Switzerland).

[0037] 1.2 Experimental drugs: recombinant human PTP1B protein, sodium alum (Na3VO4), dithiothreitol (DTT), p-nitrophenyl phosphate (p-NPP), ethylenediaminetetraacetic acid (EDTA), sodium hydroxide, citric acid, and sodium chloride.

[0038] 2. Experimental Methods:

[0039] 2.1 The determination method of PTP1B was based on the literature (Xu J, Cao JQ, Yue JY, et al. Newtriterpenoids from acorns of Quercus liaotungensis and their inhibitory activity against α-glucosidase, α-amylase and protein-tyrosine phosphatase1B). J. Funct. Foods , 2018, 41:232-239; Hajini Sha, Abdurehman Nurula, Li Gairu, et al. Chemical constituents of quince seeds and their PTP1B inhibitory activity. Acta Pharmaceutica Sinica , 2019, 54(3): 510-513.)

[0040] 2.2 10 μL of compounds (compounds I, II, III, and IV isolated above) were added to 170 μL of reaction buffer solution (composed of 50 mM citric acid (pH 7.4), 50 mM NaCl, 2 mM dithiolitol (DTT), and 1.1 mM EDTA). 20 μL of recombinant PTP1B solution (1 mg / mL, 1 μL) was added and mixed into each well. The reaction mixture was preheated to 37°C using a block heater for 15 min. 10 μL of the reaction substrate p-nitrophenyl phosphate (pNPP) (33 mM) was added, and the reaction was carried out at 37°C for 15 min. NaOH solution (10 μL, 0.1 M) was added to stop the reaction. The absorbance was recorded at 405 nm.

[0041] 2.3 For step 2.2, set up the enzyme activity group (enzyme + reaction buffer solution + substrate), enzyme blank group (reaction buffer solution + reaction substrate), sample group (sample + reaction buffer solution + enzyme + reaction substrate), and sample blank group (sample + reaction buffer solution + reaction substrate), with sodium orthovanadate aqueous solution as positive control.

[0042] 2.4 The inhibition rate is calculated according to the following formula: Inhibition rate = 1 - (Sample A - Blank Sample A) / (Enzyme A activity - Blank Sample A)

[0043] 3. Experimental Results: The PTP1B activity of compounds I-IV was tested, and the results are shown in Table 1.

[0044] Table 1. Data on the inhibitory activity of the tested compounds against PTP1B.

[0045]

[0046] The test results showed that all three benzofuran dimer compounds exhibited significant inhibitory activity, with compound III showing the highest IC50 value. 50 Up to 3.0 μM, IC50 of compound I 50 The concentration can reach 5.1 μM, but the enantiomer of compound I, compound II, has no inhibitory activity against PTP1B. The compounds described in this invention can be used to prepare drugs for treating diabetes, obesity, and their complications, or as lead compounds for such drugs.

[0047] Although the present invention has been described in detail above through general description, specific embodiments, and activity experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The use of a benzofuran dimer compound in the preparation of drugs for the prevention, delay, or treatment of diseases mediated by protein tyrosine phosphatase PTP1B, characterized in that, Benzofuran dimer compounds include any one or more combinations of the following structural formulas: 、 。 2. The use of benzofuran dimer compounds in the preparation of medicaments for the prevention, delay, or treatment of diabetes, obesity, or its complications, characterized in that, Benzofuran dimer compounds include any one or more combinations of the following structural formulas: 、 。

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