Meglutol derivatives, and preparation method, pharmaceutical composition and application thereof

Abstract

The invention belongs to the technical field of medicines, particularly discloses a Meglutol derivative and a preparation method, a pharmaceutical composition and application thereof, and particularly relates to a compound shown in a formula (I) or pharmaceutically acceptable salt thereof and a preparation method thereof, and a composition containing the compound, the compound has the effect of remarkably reversing polarization activity of M2 macrophages, and has the advantages that the effect of remarkably reversing polarization of the M2 macrophages is achieved; therefore, the compound can be used for preparing drugs for resisting tumors, fibrosis diseases, viruses, bacteria, infection and other related diseases. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a class of Meglutol derivatives and application thereof in reversing M2 macrophage polarization. Background Art

[0002] When performing specific functions, macrophages will polarize under different physiological conditions and tissue microenvironments. According to the different biological functions they perform, they can be roughly divided into M1 and M2 types. They play an important role in many diseases, including tumors, infections (bacteria and viruses) and various fibrotic diseases. The main function of M1 is to activate immune response to respond to the invasion of foreign antigens and to eliminate abnormal cells in the body. M2 mainly mediates immune tolerance and damage repair.

[0003] During tumor occurrence and progression, tumor-associated macrophages (TAMs) are abundantly infiltrated in the tumor microenvironment, which has an important impact on tumor metastasis, angiogenesis, and immune escape. At the same time, the high density of TAMs in tumors of most patients is closely related to their poor prognosis. Therefore, TAMs targeted therapy is a hot strategy in anti-tumor treatment. In the tumor microenvironment, macrophages are mainly M2 type according to their phenotype. The M2 phenotype can promote tumor cell growth, while the M1 phenotype can inhibit tumor growth and stimulate anti-tumor immune response. Therefore, it is an important direction for the development of tumor immunotherapy to find drugs that inhibit M2 macrophages in the tumor microenvironment, induce them to repolarize from M2 to M1, and then enhance the inhibitory effect of macrophages on tumor cells. In addition, in non-tumor diseases, the infiltration of M2 macrophages in diseased organs is also an important cause of fibrosis caused by many organ damage, such as end-stage fibrosis of the lungs, liver, or kidneys. Therefore, reversing the increase of M2 macrophages is also one of the important means to improve fibrotic diseases. Furthermore, reversing M2 and increasing M1 macrophages is also an important means to enhance immunity and fight against infectious diseases such as bacteria, viruses, and mycoplasma.

[0004] Sophora tonkinensis is taken from the dried roots and rhizomes of the leguminous plant Sophora tonkinensis Gapnea. The Kaibao Materia Medica records that Sophora tonkinensis has the effects of "detoxifying various drugs, relieving pain, eliminating sores and swelling, treating acute jaundice, fever and cough, and killing small insects." The Bencao Qiuzhen calls Sophora tonkinensis "the first medicine for purging the heart and protecting the lungs, and reducing the reverse fire of the yin meridians, and relieving sore throat." Therefore, it is generally believed that Sophora tonkinensis has the effects of clearing heat and detoxifying, reducing swelling and relieving sore throat. It is a traditional Chinese medicine used to treat sore throat, swollen gums, hepatitis, cancer and other diseases. According to literature reports, more than 200 natural products have been found from Sophora tonkinensis, the main components of which are alkaloids and flavonoid compounds, which have anti-tumor, anti-inflammatory, liver protection and antiviral biological activities.

[0005] This patent isolated a novel class of Meglutol derivatives from Sophora subprostrata. Pharmacological experiments have demonstrated that they can inhibit the polarization of M2 macrophages and promote their polarization towards M1 macrophages, that is, they can induce macrophages in the tumor microenvironment to repolarize from M2 to M1, thereby enhancing the phagocytic ability of macrophages against tumor cells. Currently, there are no research reports on the antitumor effect of Meglutol derivatives reversing M2 macrophage polarization. Summary of the Invention

[0006] The technical problem solved by this invention is to provide a new class of Meglutol derivatives, their preparation methods, and pharmaceutical compositions, which can reverse the polarization of M2 macrophages, promote macrophage phagocytosis, and thus play roles in anti-tumor cell growth, anti-fibrotic diseases, anti-virus, anti-bacteria, anti-infection, etc., as well as their applications in the preparation of drugs for treating the above diseases.

[0007] A series of Meglutol derivatives were found in Sophora subprostrata. These compounds all contain the Meglutol structure, namely 3-methyl-3-hydroxyglutaric acid, with one side carboxylic acid esterified with glucose in flavonoid glycosides, lignan glycosides, etc., and the other side being carboxylic acid or ester.

[0008] The first aspect of the technical solution of this invention is to provide a compound of formula (Ⅰ) or its pharmaceutically acceptable salt,

[0009]

[0010] wherein R1 independently selects from flavonoids and their glycosides, isoflavonoids and their glycosides, lignans and their glycosides; R2 independently selects from H or C 1-3 alkyl; independently selects from or

[0011] Alternatively, preferably, wherein R1 has a fragment of formula II,

[0012]

[0013] The parent nucleus I can be independently linked to the A1 or A2 position of formula II; wherein A1 and A2 independently select from H, OH, C1-C3 alkoxy groups or monosaccharides, where the alkoxy group is preferably a straight-chain or branched-chain alkoxy group, further preferably OCH3 or OC2H5, and the monosaccharide is preferably glucose, xylose or apiose.

[0014] Alternatively, preferably, wherein R1 has a fragment of formula III,

[0015]

[0016] The mother nucleus I can be independently linked to the B1, B2, B3 or B4 position of formula III; B1, B2, B3 and B4 are independently selected from H, OH, alkoxy groups having 1 to 3 carbon atoms or monosaccharides, wherein the alkoxy group is preferably a linear or branched alkoxy group, more preferably OCH3 or OC2H5, and the monosaccharide is preferably glucose, xylose or apiose.

[0017] Alternatively, preferably, wherein R1 has a fragment of formula IV,

[0018]

[0019] The mother nucleus I can be independently linked to the C1, C2, C3, C4 or C5 position of formula IV; C1, C2, C3, C4 and C5 are independently selected from H, OH, alkoxy groups having 1 to 3 carbon atoms or monosaccharides, wherein the alkoxy group is preferably a linear or branched alkoxy group, more preferably OCH3 or OC2H5, and the monosaccharide is preferably glucose, xylose or apiose; independently selected from or

[0020] Furthermore, the compounds are selected from:

[0021]

[0022] The pharmaceutically acceptable salts of the above compounds are selected from the salts formed by the compounds and organic bases, inorganic bases. The organic bases include meglumine, tris(hydroxymethyl)aminomethane and N-methyl-glucosamine, methylamine, ethylamine, diethylamine, triethylamine, propylamine, butylamine, octylamine, hexamethylenediamine, ethylenediamine, propylenediamine, butylenediamine, benzylamine, phenethylamine, o-phthalylenediamine, p-phthalylenediamine, and the inorganic bases include sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide, copper hydroxide, iron hydroxide, ammonium hydroxide, sodium bicarbonate, potassium bicarbonate, sodium carbonate, potassium carbonate.

[0023] The second aspect of the technical solution of the present invention is to provide a preparation method of the compound described in the first aspect or its pharmaceutically acceptable salt, and the preparation method is as follows: The Sophora subprostrata root medicinal material is crushed and extracted with a solvent. After the extract is concentrated, the above compound is obtained through organic solvent extraction and column chromatography separation.

[0024] The extraction solvent includes water, ethanol and ethanol aqueous solutions with any concentration.

[0025] The extraction solvent described above is an organic solvent immiscible with water or a mixed solvent in any proportion. The organic solvents include, but are not limited to, n-butanol, ethyl acetate, chloroform, dichloromethane, ether, petroleum ether, cyclohexane, and n-hexane. The mixed solvents include, but are not limited to, a mixture of two or more of the above organic solvents in any proportion and a mixture of two or more of the above solvents and solvents such as methanol, ethanol, and acetone in any proportion.

[0026] The column chromatography described above includes normal pressure column chromatography, medium pressure column chromatography, and high performance liquid column chromatography. The fillers used include normal phase fillers, reverse phase fillers, gel fillers, and ion exchange resins. The normal phase fillers include fillers such as silica gel, diol, cyano, and amino; the reverse phase fillers include macroporous resins, microporous resins, C18, C8, phenyl, etc.; the gel fillers include HW type gels and Sephadex type gels.

[0027] The third aspect of the technical solution of the present invention provides a pharmaceutical composition, which includes one or more compounds in an effective dose described in the first aspect of the present invention or their pharmaceutically acceptable salts and pharmaceutically acceptable carriers or excipients. The dosage forms of the pharmaceutical composition include tablets, capsules, pills, granules, powders, dripping pills, oral liquids, or suspensions. The pharmaceutical composition can be made into ordinary preparations, or can also be sustained release preparations, controlled release preparations, targeted preparations, and various microparticle drug delivery systems.

[0028] In order to prepare the compounds of the present invention into tablets, various excipients well-known in the art can be widely used, including diluents, binders, wetting agents, disintegrants, lubricants, and glidants. The diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agents can be water, ethanol, isopropanol, etc.; the binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia mucilage, gelatin mucilage, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; the disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium dodecyl sulfate, etc.; the lubricants and glidants can be talc powder, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc. The tablets can be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer tablets and multilayer tablets. In order to prepare the dosage unit into capsules, the active ingredient, the compound of the present invention, can be mixed with a diluent and a glidant, and the mixture can be directly placed into hard capsules or soft capsules. The varieties of each diluent, binder, wetting agent, disintegrant, and glidant used for preparing the tablets of the compounds of the present invention can also be used for preparing the capsules of the compounds of the present invention. In addition, if necessary, colorants, preservatives, fragrances, flavoring agents, or other additives can also be added to the pharmaceutical preparations. For the purpose of medication and enhancing the therapeutic effect, the drugs or pharmaceutical compositions of the present invention can be administered by any well-known administration methods.

[0029] The fourth aspect of the technical solution of the present invention is to provide the use of the compound described in the first aspect of the present invention or its pharmaceutically acceptable salt in the preparation of drugs for treating anti-tumor, anti-fibrotic diseases, anti-viral, anti-bacterial, anti-infective and other diseases. The described diseases that can be treated include tumors, organ fibrosis, various infections (including viruses, bacteria, and mycoplasmas), etc.

[0030] Beneficial technical effects:

[0031] A novel class of Meglutol derivatives discovered from Sophora subprostrata in the present invention has a significant effect of inhibiting the polarization of M2 macrophages, and can promote the phagocytosis of tumor cells by macrophages, thereby exerting an anti-tumor therapeutic effect. The effect of reversing M2 macrophage polarization is also related to the effects of various organ fibrosis and anti-infection. Therefore, this class of components has the application prospect of being further developed into anti-tumor, anti-fibrotic disease, anti-viral, anti-bacterial, and anti-infective drugs. Detailed implementation manners

[0032] The following examples and pharmacological activity experiments are used to further illustrate the present invention, but this does not mean any limitation to the present invention.

[0033] Preparation of Meglutol derivatives from Sophora subprostrata

[0034] The Sophora subprostrata medicinal materials (50 kg) were crushed and refluxed with distilled water for extraction three times, 2 h each time. The extract was concentrated and subjected to macroporous resin column chromatography, and eluted successively with ethanol (0 - 95%). The eluate of 30% ethanol was concentrated and then passed through an MCI column, and eluted successively with ethanol (0 - 75%). The fractions of 10% and 20% ethanol were subjected to silica gel (60 - 100 mesh) column chromatography and eluted with a gradient of chloroform - methanol (99:1 - 1:1) to obtain Fr.1 - 41 and Fr.G - 1 - 32. Fr.18 - 24 and Fr.G - 11 - 18 were combined and eluted through a Diol Flash column (CHCl3 - MeOH, 20:1 - 2:1) to obtain M7 - D1 - 26.

[0035] Selected M7 - D4 - 6 was purified by preparative high - performance liquid chromatography. The liquid chromatography conditions were: (1) Chromatographic column: PFP chromatographic column (250×10 mm, 5 μm); (2) Mobile phase: water / methanol solution; (3) Flow rate: 2.5 mL / min; (5) Detection wavelength: 205 nm, and the above - mentioned compound 1 (61% methanol, t R = 20.5 min), 2 (60% methanol, t R = 16.0 min), 3 (45% methanol, t R = 25.0 min) were obtained.

[0036] The structures of the compounds were determined by spectroscopic methods such as UV, IR, NMR, MS and CD.

[0037] The spectral information and NMR signal assignments of the above - mentioned compounds are as follows:

[0038] Compound 1, Arjungenin B 6””'-O-(3”-hydroxy - 3”-methylglutaryl methyl ester)-7”'-O-β - D - glucopyranoside

[0039]

[0040] Pale yellow powder; (c 0.07, MeOH); ECD (MeOH) λ max (Δε) 200 (-3.09), 239 (-1.02), 310 (-0.97) nm; UV (MeOH) λ max (logε) 205 (4.53), 229 (4.25), 255 (3.96), 310 (4.23), 327 (4.27) nm; IRνmax 3364, 2924, 2853, 1731, 1626, 1607, 1446, 1380, 1255, 1178, 1073 cm -1 ; 1 H NMR and 13 C NMR are shown in Table 1-1; (-)-HRESIMS m / z 573.1618 [M-H] - (calcd for C 28 H 29 O 13 - , 573.1614).

[0041] Compound 2 7''',3'''-dihydroxy-8''',4''-dimethoxyisoflavone 6'''-O-(3''-hydroxy-3''-methylglutaryl methyl ester)-3''-O-β-D-glucopyranoside

[0042]

[0043] Pale yellow powder; (c 0.10, MeOH); ECD (MeOH) λ max (Δε) 224 (-1.86), 298 (-0.19) nm; UV (MeOH) λ max (logε) 203 (4.14), 253 (3.92) nm; IR ν max 3362, 2921, 2850, 1731, 1596, 1515, 1454, 1269, 1076 cm -1 ; 1 H NMR and 13 C NMR are shown in Table 1-1; (-)-HRESIMS m / z 633.1829 [M-H] - (calcd for C 30 H 33 O 15 - , 633.1825).

[0044] Compound 3 Syringaresinol 6'''-O-(3''-hydroxy-3''-methylglutaryl)-4''',4''-di-O-β-D-glucopyranoside

[0045]

[0046] White powder; UV (MeOH) λ max (logε) 208 (5.89), 271 (4.32) nm; IR ν max3426, 2938, 1728, 1595, 1505, 1462, 1419, 1230, 1126, 1071 cm -1 ; 1 1H NMR and 13 13C NMR are shown in Table 1-2; (-)-HRESIMS m / z 885.3042 [M-H] - (calcd for C 40 45 53 18 22 - , 885.3034).

[0047] Table 1-1 1H NMR and 1 13C NMR (126 MHz) data of Compounds 1 and 2 (CD3OD) 13 Determined at 500 MHz and

[0048]

[0049] At a 500 MHz, b 400 MHz; c,d Signals may be interchangeable; e Overlapped with the solvent and determined in HSQC or HMBC.

[0050] Table 1-2 1H NMR (500 Hz) and 1 13C NMR (101 MHz) data of Compound 3 (CD3OD) 13 Determined at 500 MHz and

[0051]

[0052] e-k,m,n,p-s Signals may be interchangeable; c Overlapped with the water peak

[0053] Pharmacological experiments

[0054] Effect Example 1 Inhibitory effect on M2 macrophages

[0055] Mouse macrophages Ana-1 were cultured in 1640 medium with 10% FBS and seeded in 6-well plates. The test samples were pre-incubated with the cells at a concentration of 10 μM for 2 h, and Ana-1 cells were induced to polarize into M2 type with 20 ng / mL of IL-4 and co-incubated for 24 h. The cells were collected and centrifuged, and the cells were resuspended in 100 μL. Each group of cells was first blocked with CD16 / 32 to block non-specific binding sites, then APC-CD206 antibody was added, and incubated for 30 min under light protection conditions. After washing with PBS, the proportion of M2 macrophages was measured by flow cytometry. The results (Table 2-1) showed that Compounds 1 and 2 had the effect of reducing the proportion of M2 macrophages at a concentration of 10.0 μM.

[0056] Table 2-1 Inhibitory effect of compounds (10.0 μM) on M2 macrophages.

[0057]

[0058] **P < 0.01, compared with the model group

[0059] Effect Example 2 Dose-dependence of the inhibitory effect on M2 macrophage polarization

[0060] To study the dose-dependence of the active compounds, the activity of Compound 2 at different concentrations (1 μM, 2.5 μM, 10 μM) was further measured. Mouse macrophages Ana-1 were cultured in 1640 medium with 10% FBS and seeded in 6-well plates. The test samples were pre-incubated with the cells at concentrations of 1, 2.5, and 10 μM for 2 h, and Ana-1 cells were induced to polarize into M2 type with 20 ng / mL of IL-4 and co-incubated for 24 h. The cells were collected and centrifuged, and the cells were resuspended in 100 μL. Each group of cells was first blocked with CD16 / 32 to block non-specific binding sites, then APC-CD206 antibody was added, and incubated for 30 min under light protection conditions. After washing with PBS, the proportion of M2 macrophages was measured by flow cytometry. The results are shown in Table 2-2.

[0061] Table 2-2 Inhibitory effect of compounds at different concentrations on M2 macrophages

[0062]

[0063] *P < 0.05, **P < 0.01, compared with the model group

[0064] Effect Example 3 Phagocytosis of tumor cells

[0065] Bone marrow cells were isolated from the hindlimb femurs of mice and induced into primary macrophages using CSF1. The macrophages were co-cultured with tumor cells labeled with CFSE dye. After treatment at 37 °C for 2 h, the macrophages were stained with APC-conjugated F4 / 80 and analyzed by flow cytometry. A total of 10,000 cells were analyzed in each sample. Unstained controls and single-stained cells were prepared for gating. The phagocytosis was calculated as the percentage of F4 / 80+CFSE+ cells among F4 / 80+ cells.

[0066] Table 2-3 Phagocytosis of tumor cells by macrophages

[0067]

[0068] *P<0.05, **P<0.01, compared with the control group

[0069] As can be seen from Effect Examples 1 and 2, the Meglutol derivatives (1, 2) obtained from Sophora flavescens Ait. showed varying degrees of inhibition on M2 macrophages. Among them, compound 2 had the strongest activity and had a significant reversal effect at 1.0 μM.

[0070] As can be seen from Effect Example 3: Compound 2 had a promoting effect on the phagocytosis of macrophages at different concentrations and had significant activity at a concentration of 2.0 μM.

[0071] The above indicates that such compounds can significantly reverse M2 macrophage polarization, thereby promoting macrophage phagocytosis, and have the prospect of being developed into drugs for treating related diseases such as anti-tumor, anti-fibrotic diseases, anti-viral, anti-bacterial, and anti-infective diseases.

Claims

1. A class of Meglutol derivatives represented by formula (I) or a pharmaceutically acceptable salt thereof, characterized in that, The parent nucleus of this class of compounds has the structure of general formula I, wherein R1 is independently selected from flavonoids and their glycosides, isoflavonoids and their glycosides, lignans and their glycosides; R2 is independently selected from H or C 1-3 alkyl; is independently selected from 2. The Meglutol derivative (I) or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, wherein when R1 has a fragment of formula II, the parent nucleus I can be independently linked to the A1 or A2 position of formula II; wherein A1 and A2 are independently selected from H, OH, alkoxy groups with 1 to 3 carbon atoms or monosaccharides; wherein when R1 has a fragment of formula III, the parent nucleus I can be independently linked to the B1, B2, B3 or B4 position of formula III; B1, B2, B3 and B4 are independently selected from H, OH, alkoxy groups with 1 to 3 carbon atoms or monosaccharides; wherein when R1 has a fragment of formula IV, The mother nucleus I can be independently connected to the C1, C2, C3, C4, C5 or C6 position of formula IV; C1, C2, C3, C4, C5 and C6 are independently selected from H, OH, alkoxy groups having 1 to 3 carbon atoms or monosaccharides; independently selected from 3. The Meglutol derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1-2, characterized in that, the alkoxy groups in A1 and A2 are selected from OCH3 or OC2H5, and the monosaccharides are selected from glucose, xylose or apiose; the alkoxy groups in B1, B2, B3 and B4 are selected from OCH3 or OC2H5, and the monosaccharides are selected from glucose, xylose or apiose; the alkoxy groups in C1, C2, C3, C4, C5 and C6 are selected from OCH3 or OC2H5, and the monosaccharides are selected from glucose, xylose or apiose.

4. The Meglutol derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1-3, characterized in that, The described compounds are selected from:

5. The Meglutol derivative or a pharmaceutically acceptable salt thereof according to any one of claims 1-4, characterized in that, The pharmaceutically acceptable salts are selected from the salts formed by the reaction of the compounds with inorganic bases and organic bases.

6. A method for preparing the Meglutol derivative according to any one of claims 1-5, characterized in that, The preparation method is as follows: After the Sophora subprostrata root medicinal material is extracted with water and / or ethanol, the extract is separated and purified by extraction and column chromatography to obtain the described compound.

7. A pharmaceutical composition, characterized in that, The described pharmaceutical composition comprises the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient.

8. The pharmaceutical composition according to claim 7, characterized in that, The dosage forms of the described pharmaceutical composition include tablets, capsules, pills, granules, powders, dripping pills, oral liquids or suspensions.

9. Use of the pharmaceutical compound according to any one of claims 1-5 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition according to any one of claims 7-8 in the preparation of a drug for treating diseases related to M2 macrophage polarization.

10. According to the use of claim 9, the diseases related to M2 macrophage polarization include tumor diseases, fibrotic diseases, and viral, bacterial and other microbial infectious diseases.

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