Isoastragaloside derivative as well as preparation method and application thereof

Abstract

The invention relates to the field of chemical medicines, in particular to an isoastragaloside derivative as well as a preparation method and application thereof. The invention discloses an isoastragaloside water-soluble derivative, namely, isoastragaloside acid with a structure as shown in a formula 1 or medicinal salt, ester and amide of the isoastragaloside acid. According to the invention, C6 '-CH2OH in glucosyl groups in isoastragaloside molecules is selectively oxidized into C6'-COOH to prepare the isoastragaloside water-soluble derivative isoastragaloside acid, so that the problems of poor water solubility and low bioavailability of isoastragaloside are solved, and the isoastragaloside water-soluble derivative isoastragaloside acid has a wide application prospect in the aspect of preparing antibacterial and anti-inflammatory drugs.

Description

Technical Field

[0001] The present invention relates to the field of chemical medicine, and in particular to an isoastragaloside IV derivative and a preparation method and application thereof. Background Art

[0002] Astragalus is the first tonic, belonging to the genus Astragalus of the Leguminosae family. In recent years, with the development of modern separation and identification technology, a large number of triterpenoid saponins, flavonoid compounds and polysaccharide components have been isolated from Astragalus. Later biological and biochemical experiments showed that the medicinal effects of Astragalus are mainly manifested through astragalosides. Recent studies have shown that astragalosides have shown extremely promising medicinal prospects in terms of immune regulation, hypoglycemic, improving insulin resistance, anti-tumor, cardiovascular system regulation, antiviral and antioxidant activities, and among the many astragalosides, astragaloside I and isoastragaloside I have the best activity.

[0003] Isosafragin, also known as 6-O-β-D-glucosyl-cycloastragenol, is formed by removing a molecule of xylose from astragaloside IV. The content of isoastragaloside IV in Astragalus is very low, but in the process of immunological research, it was found that isoastragaloside IV is superior to astragaloside IV in promoting the secretion of interleukin-2 (IL-2), IL-1β and IL-8 (Yesilada E, Bedir E, Calis I, et al. Effects of triterpene saponins from Astragalus species on in vitro cytokine release [J]. J Ethnopharmacology, 2005, 96 (1-2): 71-77.). The study also shows that isoastragaloside IV can improve the phagocytic function of macrophages, promote the proliferation of lymphocytes, promote T cell activation, and play an important role in improving specific immunity.

[0004] However, the similar structure of isoastragaloside Ⅳ and astragaloside Ⅳ also leads to the extremely low solubility of isoastragaloside Ⅳ in the body, and its poor bioavailability after oral administration, which greatly limits its clinical application. Therefore, there is an urgent need for a substitute that can both ensure its therapeutic effect and improve its solubility. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a derivative of isoastragaloside IV and a preparation method and application thereof, as follows:

[0006] The first aspect of the present invention provides a compound having a structure of Formula 1 (named as astragaloside) or a pharmaceutically acceptable salt, ester and amide thereof, the structural formula of which is:

[0007]

[0008] Furthermore, the salt is formed by a compound having a structure of Formula 1 and an alkali metal, an alkaline earth metal, or an inorganic ammonia under conventional salt-forming conditions; the ester is formed by a compound having a structure of Formula 1 and an alcohol under conventional esterification conditions; and the amide is formed by a compound having a structure of Formula 1 and an inorganic ammonia or an organic amine under conventional amide-forming conditions.

[0009] Furthermore, the metal cation of the salt is selected from at least one of lithium, sodium, potassium, calcium, magnesium, zinc, and iron ions; the alcohol is at least one of methanol, ethanol, propanol, isopropanol, propylene glycol, butanol, tert-butanol, or glycerol; and the organic amine is selected from at least one of methylamine, ethylamine, piperazine, imidazole, pyridine, pyrrole, and piperidine.

[0010] The second aspect of the present invention provides the use of the compound or its pharmaceutically acceptable salts, esters and amides in promoting the secretion of interleukins.

[0011] Furthermore, the present invention provides the use of the compound or its pharmaceutically acceptable salts, esters and amides in the preparation of antibacterial and anti-inflammatory drugs.

[0012] In a third aspect, the present invention provides a method for preparing the compound or its pharmaceutically acceptable salts, esters and amides, which realizes the directional oxidation of specific alcoholic hydroxyl groups in the isoastragaloside molecule to carboxyl groups to obtain isoastragaloside. The method has simple steps, mild reaction conditions and is suitable for large-scale industrial production.

[0013] Specifically, the method comprises the following steps:

[0014] Take a certain amount of isoastragaloside IV, add it to 1 to 20 times the mass of the first solvent, add 0.1 to 10 times the mass of the halogenated base, 0.1 to 10 times the mass of the piperidine nitrogen oxide compound, and 0.1 to 10 times the mass of the hypohalous acid or its salt in sequence, react at -10 to 50° C. for 30 to 300 minutes, filter, adjust the pH of the obtained filtrate to acidic with acid, concentrate after the solid precipitates, separate the solid and the liquid, dissolve the solid with a second solvent, transfer, and concentrate to dryness to obtain a compound having the structure of formula 1.

[0015] Furthermore, the first solvent is selected from at least one of DMSO aqueous solution, DMF aqueous solution, acetonitrile aqueous solution or pyridine aqueous solution with a concentration of 50% to 90%; the second solvent is selected from at least one of DMSO aqueous solution, DMF aqueous solution, acetonitrile aqueous solution or pyridine aqueous solution with a concentration of 50% to 90%. The alkali halide is selected from NaCl, KCl, MgCl2, CaCl2, NaBr, KBr, MgBr2 or CaBr2. The piperidine nitrogen oxide compound is selected from at least one of 2,2,6,6-tetramethylpiperidine-1-oxyl free radical, 4-oxy-TEMPO, nitrogen oxide free radical piperidinol, TEMPO methyl methacrylate, 4-(2-bromoacetamide)-TEMPO, 4-amino-2,2,6,6-tetramethylpiperidinyloxy free radical, 4-phosphonooxy-2,2,6,6-tetramethyl-1-piperidone, 4-cyano-2,2,6,6-tetramethylpiperidinyl-1-oxyl free radical, 4-carboxyl-2,2,6,6-tetramethylazacyclohexane-1-oxyl free radical, 4-(2-chloroacetylamino)-2,2,6,6-tetramethylpiperidinyl-1-oxyl free radical, 4-(2-iodoacetylamino)-2,2,6,6-tetramethylpiperidinyl-1-oxyl free radical) or 4-methoxy-2,2,6,6-tetramethylpiperidinyl-1-oxyl free radical.

[0016] The hypohalous acid is selected from at least one of hypochlorous acid and hypobromous acid; the hypohalite is selected from at least one of sodium hypochlorite, potassium hypochlorite, calcium hypochlorite, magnesium hypochlorite, sodium hypobromite, potassium hypobromite, calcium hypobromite or magnesium hypobromite.

[0017] In a fourth aspect, the present invention provides a pharmaceutical composition for antibacterial and anti-inflammatory purposes, which is a preparation prepared by adding an effective dose of the compound having the structure of Formula 1 or its pharmaceutically acceptable salts, esters and amides as active ingredients and pharmaceutically acceptable excipients. The preparation is selected from at least one of an oral preparation or an injectable preparation.

[0018] The beneficial effects of the present invention include but are not limited to:

[0019] 1. The compound having the structure of Formula 1 (named as astragaloside IV) provided by the present invention or its pharmaceutically acceptable salts, esters and amides have good water solubility compared with the traditional astragaloside IV, and greatly improve the drugability of astragaloside IV;

[0020] 2. The compound having the structure of Formula 1 (named as astragaloside) or its pharmaceutically acceptable salts, esters and amides provided by the present invention is better than traditional astragaloside in promoting interleukin secretion and antibacterial and anti-inflammatory effects, providing a new option for immune promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0022] Figure 1 It is a schematic diagram of HMBC related signals of some key structural units of the compound having the structure of Formula 1. DETAILED DESCRIPTION

[0023] The present invention is described in detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present invention are purchased through commercial channels.

[0024] Example 1

[0025] (1) Preparation of compounds:

[0026] Compound preparation: Take 100 mg of isoastragaloside IV (Ruan Ming, Zhang Liyang, Yu Bin, et al. Conversion of astragaloside IV during bidirectional solid fermentation of Astragalus [J]. Chinese Medicinal Materials, 2010, 33 (3): 339-343.), add it to 500 mL of 10% acetonitrile aqueous solution, add 25 mg of potassium bromide, 5 mg of 4-amino-2,2,6,6-tetramethylpiperidinyloxy free radical, and 1 mL of sodium hypochlorite in sequence, react at 0°C for 120 min, filter, adjust the filtrate to pH 3 with concentrated hydrochloric acid, concentrate to 20 mL after solid precipitation, separate the solid and liquid, dissolve the solid in 10% methanol and transfer, and concentrate to dryness to obtain 83.68 mg of white powder, which is the compound having the structure of formula 1 (HPLC purity 96.5%).

[0027] (2) Structural identification

[0028] White powder, after TLC development, sprayed with 5% concentrated sulfuric acid / ethanol to show purple red; IR (KBr): Vmax: 3388.7, 2968.6, 872.4, 1454.1, HR-ESI-MS quasi-ion peak m / z 689.4682 [M+Na] + The molecular formula is inferred to be C 36 H 58 O 11 .

[0029] 1 H NMR showed one anomeric hydrogen signal of the glycosyl group: δ H 5.05, 13 C NMR also showed a corresponding carbon signal: δ C107.4, based on which it is inferred that the compound contains one sugar fragment. Negative ion ESI-MS showed m / z 675[MH]-, and MSn fragment peaks were m / z 608[(MH)-177] (one glucuronic acid group was lost); after acid hydrolysis of the compound, TLC showed a color spot at the same position of glucuronic acid; these results indicate that the sugar chain is composed of glucuronic acid.

[0030] 1 H-NMR spectrum shows that H There are 7 methyl signal peaks at 1.96, 1.53, 1.35, 1.29, 1.26, 1.24 and 0.96. 13 C NMR data comparison confirmed that the parent nucleus of the compound had not changed and was still cycloartane-type. Combined with the above analysis of the sugar group, it was shown that the aglycone was isoastragalosapogenin.

[0031] Compared with isoastragaloside IV, the 13 C NMR shows a carboxyl signal peak δ C 172.7, no glucosyl 6" signal peak of isoastragaloside IV; 13 C NMR showed that the C-6 of the aglycone underwent glycosylation shifts, which were δ C 88.2 and δ C 79.0, thus confirming that the sugar group of the compound should be connected to the C-6 position of the aglycone; the long-range correlation between glucuronic acid H-1 and aglycone C-6 in the HMBC spectrum confirms this speculation. The key long-range correlation of the HMBC spectrum is shown in Figure 1 .

[0032] Comprehensive ESI-MSn, HR-ESI-MS, 1 H NMR, 13 C NMR, DEPT, 1 H- 1 The structure of the compound was identified as formula 1 by H COSY, HMBC, HMQC and other spectral analyses and compared with isoastragaloside IV, and was named isoastragaloside.

[0033] Example 2

[0034] Compound preparation: Take 100 mg of isoastragaloside IV (Ruan Ming, Zhang Liyang, Yu Bin, et al. Conversion of astragaloside IV during bidirectional solid fermentation of Astragalus [J]. Chinese Medicinal Materials, 2010, 33 (3): 339-343.), add it to 50 mL of 10% acetonitrile aqueous solution, add 25 mg of potassium bromide, 10 mg of 4-amino-2,2,6,6-tetramethylpiperidinyloxy free radical, and 0.25 mL of sodium hypochlorite in sequence, react at 20°C for 120 min, filter, adjust the filtrate to pH 2.5 with concentrated hydrochloric acid, and after solid precipitation, concentrate to 10 mL, separate the solid and liquid, dissolve the solid in 10% ethanol and transfer, and concentrate to dryness to obtain 85.86 mg of white powder, which is the compound having the structure of formula 1 (HPLC purity 97.2%).

[0035] Example 3

[0036] Compound preparation: Take 100 mg of isoastragaloside IV (Ruan Ming, Zhang Liyang, Yu Bin, et al. Conversion of astragaloside IV during bidirectional solid fermentation of Astragalus [J]. Chinese Medicinal Materials, 2010, 33 (3): 339-343.), add it to 20 mL of 10% piperidine aqueous solution, add 20 mg of potassium bromide, 8 mg of 4-amino-2,2,6,6-tetramethylpiperidinyloxy free radical, and 0.5 mL of sodium hypochlorite in sequence, react at 0°C for 120 min, filter, adjust the filtrate to pH 3 with concentrated hydrochloric acid, concentrate to 10 mL after solid precipitation, separate the solid and liquid, dissolve the solid in 10% ethanol and transfer, and concentrate to dryness to obtain 71.38 mg of white powder, which is the compound with the structure of formula 1 (HPLC purity 92.4%).

[0037] Example 4

[0038] Effects of the compound of formula 1 on the immune function of macrophages

[0039] (1) Preparation, grouping and administration of macrophages Five mice were given 2 mL of serum-free RPMI 1640 culture medium ip. The mice were killed by cervical dislocation 15 minutes later and soaked in 75% alcohol for 15 minutes. The intraperitoneal fluid was collected and the cells were collected by centrifugation at 1000 rpm for 5 minutes. The cell concentration was adjusted to 1×10 with RPMI 1640 culture medium containing 10% inactivated fetal bovine serum. 6 / L. Take 1 mL of cell suspension to each well of a 24-well culture plate, place it in a CO2 incubator and culture it for 2 hours, pour off the supernatant, wash off the non-adherent cells with phenol red-free RPMI 1640 culture medium, and the culture wells contain macrophages. The experiment was divided into 7 groups, namely, a blank control group (PBS solution), a positive control group (isastragaloside group with a final concentration of 5, 10, and 50 μmol / L), and an isastragaloside group (compound of formula 1 structure) with a final concentration of 5, 10, and 50 μmol / L. Each group was added with 1 mL of phenol red-free RPMI 1640 10% FBS culture medium containing a series of concentrations of the test drug.

[0040] (2) Neutral red test to detect macrophage phagocytic function Take the cells cultured for 24 hours as above, set up 6 replicate wells in each group, add neutral red saline to each well to make the final mass concentration 1g / L, and continue to culture for 20 minutes. Wash the cells 3 times with PBS, add 1mL of cell lysis solution (acetic acid-anhydrous ethanol 50:50) to each well, and leave at room temperature for 2 hours. After the cells are dissolved, take 0.2mL of supernatant and measure the absorbance (A) at 540nm on the microplate reader. This value represents the strength of macrophage phagocytic function. (3) Determination of IL-1, IL-2, and IL-6 production by macrophages Take the culture supernatant of the cells cultured for 24 hours as above, set up 6 replicate wells in each group, and measure the indicators according to the instructions of the ELISA kits for IL-1, IL-2, and IL-6.

[0041] (4) Data were expressed as x±s and analyzed using SPSS16.0 statistical software. One-way analysis of variance was used for comparison between groups. P<0.05 was considered statistically significant. The results are shown in Table 1.

[0042] Table 1

[0043]

[0044]

[0045] As shown in Table 1, compared with the blank control group, the low, medium and high dose groups of the compound of formula 1 also showed significant increases in neutral red A value and IL-6 level (P < 0.05, 0.01), the medium and high dose groups showed significant increases in IL-2 level (P < 0.05, 0.01), and the high dose group showed significant increases in IL-1 level (P < 0.05). Compared with the positive control isoastragaloside IV, the effect is slightly better, which proves that the compound of formula 1 of the present invention does not reduce the medicinal properties of the original isoastragaloside IV, but increases the water solubility and has great medicinal value.

[0046] The above description is only an embodiment of the present invention. The protection scope of the present invention is not limited by these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent substitution, improvement, etc. made within the technical ideas and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A compound having a structure of Formula 1 or a pharmaceutically acceptable salt, ester and amide thereof, wherein the structural formula is:

2. The compound according to claim 1 or its pharmaceutically acceptable salt, ester and amide, characterized in that: The salt is formed by reacting a compound having a structure of Formula 1 with an alkali metal, an alkaline earth metal, or an inorganic ammonia under conventional salt-forming conditions; the ester is formed by reacting a compound having a structure of Formula 1 with an alcohol under conventional esterification conditions; and the amide is formed by reacting a compound having a structure of Formula 1 with an inorganic ammonia or an organic amine under conventional amide-forming conditions.

3. The compound according to claim 2 or its pharmaceutically acceptable salt, ester and amide, characterized in that: The metal cation of the salt is selected from at least one of lithium, sodium, potassium, calcium, magnesium, zinc, and iron ions; the alcohol is at least one of methanol, ethanol, propanol, isopropanol, propylene glycol, butanol, tert-butanol, or glycerol; and the organic amine is selected from at least one of methylamine, ethylamine, piperazine, imidazole, pyridine, pyrrole, and piperidine.

4. Use of the compound according to any one of claims 1 to 3 or its pharmaceutically acceptable salts, esters and amides in promoting interleukin secretion.

5. Use of the compound according to any one of claims 1 to 3 or its pharmaceutically acceptable salts, esters and amides in the preparation of antibacterial and anti-inflammatory drugs.

6. A method for preparing the compound according to any one of claims 1 to 3 or its pharmaceutically acceptable salts, esters and amides, characterized in that: The following steps are involved: Take a certain amount of isoastragaloside IV, add it to 0.1 to 10 times the mass of the first solvent, add 0.01 to 5 times the mass of the halogenated base, 0.01 to 5 times the mass of the piperidine nitrogen oxide compound, and 0.01 to 5 times the mass of the hypohalous acid or its salt in sequence, react at -10 to 50° C. for 30 to 300 minutes, filter, adjust the pH of the obtained filtrate to acidic with acid, concentrate after the solid precipitates, separate the solid and the liquid, dissolve the solid with a second solvent, transfer, and concentrate to dryness to obtain a compound having the structure of formula 1.

7. The method according to claim 6, characterized in that The first solvent is selected from at least one of acetonitrile aqueous solution or pyridine aqueous solution with a concentration of 0% to 50%; the second solvent is selected from at least one of alcohol aqueous solution, acetonitrile aqueous solution or pyridine aqueous solution with a concentration of 0% to 50%.

8. The method according to claim 6, characterized in that The alkali halide is selected from NaCl, KCl, MgCl2, CaCl2, NaBr, KBr, MgBr2 or CaBr2.

9. The method according to claim 6, characterized in that The piperidine nitrogen oxide compound is selected from 2,2,6,6-tetramethylpiperidin-1-oxyl free radical, 4-oxy-TEMPO, nitrogen oxide free radical piperidinol, TEMPO methyl methacrylate, 4-(2-bromoacetamide)-TEMPO, 4-amino-2,2,6,6-tetramethylpiperidinyloxy free radical, 4-phosphonooxy -2,2,6,6-tetramethyl-1-piperidone, 4-cyano-2,2,6,6-tetramethylpiperidin-1-oxyl free radical, 4-carboxy-2,2,6,6-tetramethylazacyclohexane-1-oxyl free radical, 4-(2-chloroacetylamino)-2,2,6,6-tetramethylpiperidin-1-oxyl free radical, 4-(2-iodoacetylamino)-2,2,6,6-tetramethylpiperidin-1-oxyl free radical) or at least one of 4-methoxy-2,2,6,6-tetramethylpiperidin-1-oxyl free radical.

10. The method according to claim 6, characterized in that The hypohalous acid is selected from at least one of hypochlorous acid and hypobromous acid; the hypohalite is selected from at least one of sodium hypochlorite, potassium hypochlorite, calcium hypochlorite, magnesium hypochlorite, sodium hypobromite, potassium hypobromite, calcium hypobromite or magnesium hypobromite.

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