Preparation of acetylene components in gynura divaricata and application of the acetylene components in treating diabetes

Abstract

The application discloses preparation of a chemical component of Adenocline excelsa and application of the chemical component in resisting diabetes, wherein the chemical component is separated from an extraction part of Adenocline excelsa. Eupatorium chinense The root extract of Adenocline excelsa is separated by using thin layer silica gel chromatography, Sephadex LH-20 gel chromatography and high performance liquid chromatography, and two acetylene compounds are obtained. The two acetylene compounds obtained by separation are tested for inhibitory activity of diabetes related target points α -glucosidase and protein tyrosine phosphatase 1B (PTP1B), and the experimental results show that the two acetylene compounds have good inhibitory activity on the two enzymes, and part of the data is stronger than that of a positive drug. Molecular docking experiments also show that the two compounds have good binding energy with the two target proteins α -glucosidase and protein tyrosine phosphatase 1B, so the two acetylene compounds can be used to prepare medicines for preventing, delaying or treating diseases mediated by α -glucosidase or PTP1B, in particular type II diabetes and obesity, or as a lead compound of the medicines.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the preparation, isolation, and application of alkyne compounds from the roots of *Zephyranthes bidentata* in the preparation of therapeutic agents. α - Use in drugs for diseases mediated by glucosidase or protein tyrosine phosphatase 1B. Background Technology

[0002] With globalization, modernization, and changes in human lifestyles, the incidence of diabetes and obesity is increasing year by year. 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. Diabetes Res. Clin. Pract . 2018, 138: 271-281).

[0003] α -glucosidase ( α β-glucosidase, present in the small intestinal mucosa, is one of the main target enzymes controlling postprandial blood glucose. It converts dietary carbohydrates into glucose and reversibly competes with sugar molecules for enzyme binding sites on the brush border epithelial cells of the small intestine, thereby delaying monosaccharide formation and achieving blood glucose balance. Currently... α - Glucosidase inhibitors are one of the first-line drugs for the clinical treatment of type 2 diabetes.

[0004] The protein-tyrosine phosphatases (PTPs) family is a class of receptor-like cytoplasmic signal transduction enzymes that dephosphorylate their substrate tyrosine residues (Kim et al., Int. J. Mol. Sci 2018, 19: 2708), thereby regulating the metabolic processes of various cells and maintaining homeostasis. 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 occurrence of type 2 diabetes mellitus (T2DM). Eur. J. Pharmacol . 2005, 519: 182-190; Kim et al., J. Clin. Invest . 2000, 105: 1791-1797). Furthermore, PTP1B overexpression has been detected in studies of breast cancer, ovarian cancer, and prostate cancer, and at all stages of tumor development. Mol. Cell. Biol 1998, 18, 2965-2975; J. Natl. Cancer Institute (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. Simultaneously inhibiting both α-glucosidase and PTP1B targets can lower postprandial blood glucose and enhance insulin sensitivity, reduce pancreatic β-cell load, and jointly maintain stable blood glucose levels. Furthermore, it can reduce body weight in obese diabetic patients by increasing leptin levels.

[0005] Natural products (especially plant-derived natural products) have historically been an important source for discovering innovative drugs with novel structures and mechanisms of action. 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 efficient α-glucosidase and PTP1B inhibitors from plant-derived active ingredients is of significant research value. Summary of the Invention

[0006] The first objective of this invention is to provide two alkyne compounds from the roots of *Zephyranthes bidentata* and their preparation methods. The second objective is to provide their applications in the preparation of preventative or therapeutic compounds. α - Use in drugs that mediate diseases mediated by glucosidase or protein tyrosine phosphatase PTP1B.

[0007] Two types with inhibitory α - Compounds with glucosidase or PTP1B activity, having a structural formula including any one or more combinations of the following:

[0008]

[0009] The technical solution of this invention is to prepare two alkyne compounds from the roots of *Eupatorium fortunei* for prevention, delay, or treatment. α - Application in drugs that mediate diseases mediated by glucosidase or protein tyrosine phosphatase PTP1B.

[0010] The method for preparing the aforementioned acetylenoid compound includes the following steps:

[0011] A. Solvent extraction: After being pulverized, the extract was successively extracted with methanol, petroleum ether, and ethyl acetate, and then concentrated to obtain extract a;

[0012] B1. Crude fractionation of extract a by chromatography column: Take extract a, elute with dichloromethane-methanol at a volume concentration of 90:10, and collect the eluted fraction.

[0013] C1. The distillate was eluted with Sephadex LH-20, and the product was purified and separated by semi-preparative HPLC after methanol to obtain compound 1.

[0014] B2. Crude fractionation of extract a by chromatography column: Take extract a, elute with dichloromethane-methanol at a volume concentration of 40:60, and collect the eluted fraction.

[0015] C2. Compound 2 was obtained by semi-preparative HPLC purification and separation of the distillate product after passing it through methanol.

[0016] The volume ratio of the Sephadex LH-20 elution solvent, dichloromethane, to methanol is 2:1 to 1:2.

[0017] Compared with the prior art, this application has at least one of the following beneficial effects:

[0018] 1. The two compounds in this application have significant α -Glucosidase or PTP1B inhibitory activity, no publicly reported information regarding the compound having... α - Inhibitory activity of glucosidase or PTP1B and its application in related diseases.

[0019] 2. Two inhibitory agents isolated from the ethyl acetate extract of *Eupatorium fortunei* roots were discovered for the first time. α Compounds with glucosidase or PTP1B activity. Potential for development into compounds with preventative or therapeutic effects. α Drugs for diseases mediated by glucosidase or PTP1B, such as diabetes and obesity, have broad application prospects. Attached Figure Description

[0020] Figure 1 The diagram shows the interaction analysis between compounds 1 and 2 and α-glucosidase protein, where A: compound 1; B: compound 2.

[0021] Figure 2 The diagram shows the interaction analysis of compounds 1 and 2 with PTP1B protein, where A: compound 1; B: compound 2. Detailed Implementation

[0022] 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.

[0023] Example 1: Isolation and purification of two alkyne compounds from the roots of Zephyranthes bidentata

[0024] The preparation method includes the following steps:

[0025] Step A, Solvent extraction: After drying and crushing the roots of Zephyranthes cusia, extract them three times overnight with 70-100% methanol-water at room temperature. After concentration by rotary evaporator, extract with petroleum ether to remove fat, and then repeat the extraction three times with ethyl acetate. Concentrate the extract to obtain extract a.

[0026] Step B, coarse fractionation of the chromatography column: Take extract a, add 16 g of silica gel and mix. Take another 500 g of normal phase silica gel (200-300 mesh), soak it in dichloromethane for 1 hour, and then pack it into a column using a wet method. The volume of the chromatography column is 750 mL. After dry loading, use a gradient elution of dichloromethane-methanol = 100:0 → 0:100 (volume ratio). The amount of eluent used for each gradient is 2 column volumes. Combine the same fractions, and finally elute to obtain 8 fractions (Fr.1~Fr.8).

[0027] The dichloromethane-methanol elution ratios corresponding to the Fr.1~Fr.8 gradient fractions are 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, and 0:100, respectively.

[0028] Step C, HPLC separation and purification: Fragment Fr.2 was eluted with Sephadex LH-20 (elution solvent: dichloromethane: methanol = 1:1, volume ratio) to obtain 5 fragments (Frr.1~Frr.5, which were combined to obtain a mixture). The mixture was dissolved in methanol and repeatedly purified and separated by semi-preparative HPLC to obtain compound 1; Fragment Fr.7 was dissolved in methanol and repeatedly purified by semi-preparative HPLC to obtain compound 2.

[0029] In the embodiments of this application, it was found that step B, 100:0, did not collect the product of this case.

[0030] In the embodiments of this application, it was found that the product of this case was not collected in step B, 80:20.

[0031] In the embodiments of this application, it was found that the product of this case was not collected in step B, 70:30.

[0032] In the embodiments of this application, it was found that the product of this case was not collected in step B, 50:50.

[0033] In the embodiments of this application, it was found that the product of this case was not collected in step B, 0:100.

[0034] The nuclear magnetic resonance (NMR) and mass spectrometry (MS) data for compounds 1 and 2 are as follows:

[0035] Compound 1: Yellow powder, molecular formula: C 14 H 14 O3; EI-MS: m / z 230.1 [M] + ; 1 H-NMR (400 MHz, CDCl3- d 6) δ H 7.55 (1H, d, J = 1.8 Hz, H-6), 7.42 (1H, d, J = 1.8 Hz, H-2), 6.40 (1H,s, OH), 5.25 (1H, s, H-13a), 5.12 (1H, s, H-13b), 4.02 (3H, s, H-14), 2.47 (3H,s, H-8), 2.06 (3H, s, H-12); 13 C-NMR (100 MHz, CDCl3- d 6) δ C Compound 1 has the following chromatogram values: 195.2 (C-7), 150.8 (C-4), 145.2 (C-3), 128.3 (C-1), 126.0 (C-6), 125.4 (C-11), 121.6 (C-13), 109.1 (C-2,5), 95.8 (C-10), 81.6 (C-9), 56.2 (C-14), 25.8 (C-8), 22.2 (C-12). 1 H-NMR spectrum and 13 C-NMR spectroscopy and literature ( Journal of Natural Products The results were consistent with those in 2001, 64(08): 1048-1051, therefore the compound was identified as 1-[4-hydroxy-3-methoxy-5-(3-methylbut-3-en-1-ynyl) phenyl]ethanone.

[0036] Compound 2: Pale yellow oily substance, molecular formula: C 10 H 10 O3; EI-MS: m / z 178.1 [M] + ; 1 H-NMR (400MHz, DMSO- d 6) δ H 5.87 (1H, dd, J=17.4, 11.35 Hz, H-9), 5.69 (1H, dd, J =17.4, 2.1Hz, H-10), 5.68 (1H, m, H-3), 5.58 (1H, m, H-2), 5.57 (1H, dd, J =11.3, 2.1 Hz, H-10), 4.54 (1H, brs, H-1), 4.28 (1H, brd, J =4.8 Hz, H-4), 3.50 (1H, t, J =2.1 Hz, H-5); 13 C-NMR (100 MHz, DMSO- d 6) δ C : 129.43(C-2), 128.43(C-10), 126(C-3), 110.72(C-9), 87.67(C-7), 83(C-8), 63.38(C-1), 62.11(C-5), 56.11(C-4), 56.06(C-6).

[0037] Compound 2 1 H-NMR spectrum and 13 C-NMR spectroscopy and literature ( Phytochemistry The results were consistent with those in 2011, 72(9): 923-928, therefore the compound was identified as Speciosin L.

[0038] Example 2: Two compounds α -Assay on glucosidase and PTP1B inhibitory activity

[0039] Experimental methods

[0040] α - Glucosidase activity assay

[0041] The method for α-glucosidase assay was modified based on the method of Tao et al. (Biomedical chromatography: BMC.2013, 27(2): 148-155). The p-nitrophenyl-β-galactopyranoside method was used, in which pNPG, as a substrate, was decomposed into pNPG and glucose under the catalysis of α-glucosidase. pNPG showed a yellow color and strong UV absorption at 405 nm. Acarbose was used as a positive control. The amount of product after sample addition was measured to determine the inhibition rate of the sample. 80 μL of sample solution (PBS buffer at pH 7.2) and 20 μL of... were added to the wells of a 96-well plate. α- Glucosidase (2 U / mL, PBS buffer, pH 7.2), mixed thoroughly. After incubation at 37 °C for 15 min, add 20 μL of 5 mmol / L pNPG to start the reaction. After incubation for 15 min, add 80 μL of 1 mol / L Na2CO3 to terminate the reaction. Add 80 μL of 1% PBS to the blank control group instead of the sample solution. Quantify the release of pNPG by absorbance (A) at 405 nm. Set up enzyme activity group (20 μL enzyme + 80 μL buffer + 20 μL substrate), enzyme blank group (100 μL buffer + 20 μL substrate), sample group (80 μL sample + 20 μL enzyme + 20 μL substrate), and sample blank group (80 μL sample + 20 μL reaction buffer + 20 μL substrate), with acarbose solution as a positive control. Inhibition rate is calculated using the formula: Inhibition rate = 1 - (A sample - A blank sample) / (A enzyme activity - A enzyme blank).

[0042] PTP1B enzyme activity assay

[0043] The determination method for PTP1B is as described in the literature ( Funct. Foods , 2018, 41:232-239; Acta Pharmaceutica Sinica, 2019, 54(3): 510-513.). 10 μL of compounds (compounds I, II, III, and IV isolated above) were added to 170 μL of reaction buffer (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 to each well. The reaction mixture was preheated using a block heater at 37°C 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. For the above steps, the enzyme activity group (20 μL enzyme + 170 μL buffer solution + 10 μL substrate), enzyme blank group (190 μL buffer solution + 10 μL reaction substrate), sample group (170 μL sample + 20 μL enzyme + 10 μL reaction substrate), and sample blank group (170 μL sample + 20 μL buffer solution + 10 μL reaction substrate) were set up, with sodium orthovanadate aqueous solution and oleanolic acid solution as positive controls.

[0044] The inhibition rate is calculated using the following formula: Inhibition rate = 1 - (Sample A - Blank Sample A) / (Enzyme A activity - Blank Sample A)

[0045] Experimental results:

[0046] To the compound α - The results of the glucosidase and PTP1B inhibitory activity assays are shown in Tables 1-2:

[0047] Table 1. Inhibition of two compounds α - Glucosidase activity results

[0048]

[0049] Test results show that both of the above-mentioned alkyne compounds from the roots of *Zephyranthes cusia* have good efficacy. α -Glucosidase inhibitory activity, and compound 1 α - Its glucosidase inhibitory activity is superior to that of the positive control drug acarbose.

[0050] Table 2. Results of the two compounds inhibiting PTP1B activity

[0051]

[0052] The test results showed that both of the alkyne compounds from the roots of Zephyranthes cusia exhibited good PTP1B enzyme inhibitory activity, and compound 2 showed better inhibitory activity than the positive control agents sodium orthovanadate and oleanolic acid.

[0053] Example 3: Docking two alkyne compounds with two protein target molecules

[0054] Experimental methods

[0055] Compounds 1 and 2, used in this docking experiment, were constructed using ChemDraw, then imported into Chem3D software for optimization and energy minimization using the MM2 module, and saved as SDF files as ligand molecules for molecular docking. They were subsequently imported into Pymol and Autodock software for further optimization and exported as PDBQT files. α - The protein structures of glucosidase (PDB ID: 5ZCE) and PTP1B (PDB ID: 5QG3) are derived from the RCSB database. https: / / www.rcsb.org / The protein structure was processed using Pymol and Autodock platforms, including water molecule removal, ligand removal, and hydrogenation. Energy minimization and geometric optimization were performed on the protein, and the results were exported as a PDBQT file. Molecular docking was handled and optimized using the Grid module in Autodock software. The PDBQT file from Example 2 was imported into the software, allowing the container to fully encapsulate the protein before molecular docking. Furthermore, the protein-small molecule complexes were visualized and analyzed using Pymol.

[0056] docking results

[0057] Molecular docking results

[0058] The molecular docking results obtained using the above methods are shown in Table 3. Additionally, the protein-small molecule complexes were visualized and analyzed using Pymol 2.1, as shown below. Figure 1 and Figure 2 .

[0059] Table 3. Docking results of compounds 1 and 2 with the target protein

[0060]

[0061] Compound-protein interaction analysis

[0062] This experiment will combine compounds 1 and 2 with... α Molecular docking was performed between the compound and the target protein PTP1B. The docking results showed that the compound and the target protein had a good binding affinity and a high degree of match. Figure 1 and Figure 2 The binding energies were all less than -6 kcal / mol. The complexes formed by the docking compounds and proteins were visualized using Pymol 2.1 software to obtain the binding patterns. Based on these patterns, it was clearly observed that the amino acid residues binding to the protein pocket formed multiple hydrogen bonds or hydrophobic interactions, indicating strong binding ability and playing an important role in anchoring small molecules within the protein pocket. Figure 1 ).

[0063] Compound 1 forms multiple hydrogen bonds or hydrophobic interactions with multiple amino acid residues at the active site of PTP1B protein, exhibiting strong binding ability and playing an important role in anchoring small molecules in the protein pocket; Compound 2 also forms multiple hydrogen bonds or hydrophobic interactions with multiple amino acid residues at the active site of PTP1B protein, exhibiting strong binding ability and playing an important role in anchoring small molecules in the protein pocket. Figure 2 ).

[0064] 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. Alkyne compounds in the preparation of inhibitory α Its use in drugs with β-glucosidase and / or PTP1B activity, characterized in that, The structural formula of alkyne compounds is selected from any one of the following: 。 2. The application of alkyne compounds in the preparation of drugs for treating type II diabetes, characterized in that, The structural formula of alkyne compounds is selected from any one of the following: 。

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