WO2024051666A1 - 二苄基丁内酯糖苷类化合物、其制备方法和应用 - Google Patents

二苄基丁内酯糖苷类化合物、其制备方法和应用 Download PDF

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WO2024051666A1
WO2024051666A1 PCT/CN2023/116894 CN2023116894W WO2024051666A1 WO 2024051666 A1 WO2024051666 A1 WO 2024051666A1 CN 2023116894 W CN2023116894 W CN 2023116894W WO 2024051666 A1 WO2024051666 A1 WO 2024051666A1
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compound
monosaccharide
derivatives
dibenzylbutyrolactone
preparation
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French (fr)
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许叶春
唐炜
陈国峰
冯春兰
刘佳缘
杨涛
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中国科学院上海药物研究所
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    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
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    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings

Definitions

  • the present application relates to the field of medicinal chemistry, specifically to a dibenzylbutyrolactone glycoside compound represented by formula (I), its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, which Preparation method, pharmaceutical composition containing the same, and use thereof in the preparation of phosphodiesterase 4 inhibitors and in the preparation of diseases related to abnormal phosphodiesterase 4 activity levels or expression levels uses in medicines.
  • a dibenzylbutyrolactone glycoside compound represented by formula (I) its stereoisomers, tautomers or pharmaceutically acceptable salts thereof
  • Cyclic nucleotide phosphodiesterases can specifically catalyze the hydrolysis of the intracellular second messenger cyclic adenosine monophosphate (cAMP) or cyclic guanosine monophosphate (cGMP) and regulate the concentration of cAMP or cGMP in the cell. , thereby regulating a series of physiological and pathological processes mediated by second messenger molecules, such as cell cycle control, cell differentiation, inflammation, heart function, smooth muscle relaxation and contraction, visual signal transmission, learning and memory, etc. PDEs are important regulatory proteins in the cyclic nucleotide signaling pathway.
  • PDEs are an important family of drug targets, and PDEs inhibitors have been widely used in the research and treatment of pathological mechanisms of various diseases (Maurice DH, et al. Nat. Rev. Drug Discov. 2014, 13: 290-314; Menniti FS ,et al.Nat.Rev.Drug Discov.2006,5(8),660-670).
  • PDE4 specifically hydrolyzes the intracellular second messenger molecule cAMP. It is a member of the 11 subfamilies of the PDE superfamily, including four subtypes of PDE4A/B/C/D. It is widely distributed in the body and is mainly expressed in a variety of immune systems. Related cells such as neutrophils, eosinophils, and monocytes. Therefore, PDE4 has become an important target for the research of major anti-inflammatory drugs in immune and inflammation-related diseases.
  • PDE4 is involved in a wide variety of diseases, among which diseases related to the role of PDE4 in the inflammatory process include chronic obstructive pulmonary disease, asthma, psoriasis, allergic rhinitis, idiopathic pulmonary fibrosis, and rheumatoid arthritis.
  • Diseases involving the nervous system include Alzheimer's disease, Parkinson's disease, depression and schizophrenia (Menniti FS, et al. Nat. Rev. Drug Discov. 2006, 5: 660-670; Burgin A B, et al. al. Nat. Biotechnol. 2010, 28: 63-70; Garcia OstaA, et al. ACS Chem. Neurosci. 2012, 3: 832-844). Since PDE4 is involved in a variety of important diseases and some PDE4 inhibitors have been used for clinical treatment, the design and discovery of new inhibitors of PDE4 is a hot topic in the field of new drug research and development.
  • Psoriasis is a common chronic relapsing inflammatory skin disease with typical clinical manifestations of well-defined erythema, rash, plaques, and scales. The pathogenesis of the disease is complex and the cause is unknown.
  • Tumor necrosis factor ⁇ (TNF- ⁇ ) is highly expressed in psoriasis, and TNF- ⁇ blocking therapy has significant clinical effects.
  • Targeted inhibition of PDE4 hydrolysis activity can reduce the expression of pro-inflammatory factors such as TNF- ⁇ by increasing intracellular cAMP concentration, thereby alleviating the symptoms of psoriasis.
  • the treatment of psoriasis is mainly based on local treatment.
  • Arctiin and its aglycone Arctigenin are derived from burdock. They are dibenzylbutyrolactone compounds and have a variety of pharmacological activities, including immunomodulation, anti-diabetes, anti-tumor, neuroprotection, etc. effect. Its structural formula is as follows:
  • arctiin and arctigenin can inhibit the release of TNF- ⁇ , IL-1 ⁇ , IL-6 and other inflammatory factors by inhibiting the release of NO and PGE2, inhibiting COX2 activity, inhibiting the generation of reactive oxygen species, etc. Anti-inflammatory effect (Gao, et al. Acta Pharmacol. Sin. 2018, 39: 787-801).
  • studies have shown that arctigenin can inhibit PDE4D, increase cAMP levels in the body, and exert significant anti-inflammatory and anti-psoriasis effects (Li, et al. J. Adv. Res. 2021, 33: 241-251 ), but it also has problems such as poor solubility and rapid metabolism, which hinders its potential as a drug. Further structural modification of arctigenin is expected to obtain candidate compounds with better anti-inflammatory activity and better druggability.
  • the purpose of this application is to provide a dibenzylbutyrolactone glycoside compound represented by formula (I), its stereoisomer, tautomer or its pharmaceutically acceptable salt, and its preparation method, Pharmaceutical compositions containing the same, and their use in the preparation of phosphodiesterase 4 inhibitors and in the preparation of medicaments for the prevention, treatment or adjuvant treatment of diseases associated with abnormal phosphodiesterase 4 activity levels or expression levels the use of.
  • the dibenzylbutyrolactone glycoside compound represented by formula (I) of the present application has strong inhibitory activity against PDE4D, and its anti-inflammatory effect is also significantly better than the natural product source of the parent compound arctigenin - burdock.
  • Arctiin has broad application prospects in the clinical treatment of diseases related to abnormal phosphodiesterase 4 activity levels or expression levels.
  • this application provides a dibenzylbutyrolactone glycoside compound represented by formula (I), its stereoisomer, tautomer or its pharmaceutically acceptable salt:
  • R is selected from the group consisting of: monosaccharides, monosaccharide derivatives, disaccharides consisting of monosaccharide units connected by glycosidic bonds, oligosaccharides consisting of three to five monosaccharide units connected by glycosidic bonds.
  • Sugar wherein the monosaccharide does not comprise D- ⁇ -glucose, but the monosaccharide derivative comprises a D- ⁇ -glucose derivative, and wherein the R is bonded to a di-glycosidic bond through an ⁇ or ⁇ -type O-glycosidic bond.
  • the main body of benzyl butyrolactone compounds is connected.
  • the monosaccharide is selected from: furanose and pyranose;
  • the monosaccharide is D-form or L-form.
  • the monosaccharide derivative is selected from the group consisting of: oxidized sugars, halo-substituted sugars, unsaturated sugars, deoxy sugars, aminodeoxy sugars, anhydrosaccharides, sulfated sugars, phosphorylated sugars , heterosaccharides, glycosyl alkylated derivatives and glycosyl esterified derivatives;
  • the oxidized sugar is selected from monosaccharide derivatives obtained by independently oxidizing any 1, 2, 3, 4 or 5 hydroxyl groups on a monosaccharide or a monosaccharide derivative to an aldehyde or acid;
  • the halosugar is selected from monosaccharides or monosaccharide derivatives in which any 1, 2, 3, 4 or 5 hydroxyl groups are independently substituted by halogens, and the halogens are optionally selected. from fluorine, chlorine, bromine or iodine;
  • the unsaturated sugar is selected from monosaccharides or monosaccharide derivatives obtained by changing any two adjacent hydroxyl groups on a monosaccharide derivative into unsaturated double bonds;
  • the deoxysugar or aminodeoxysugar is selected from monosaccharides or monosaccharide derivatives in which any 1, 2, 3, 4 or 5 hydroxyl groups are independently replaced with methyl groups. ;
  • the anhydrous sugar is selected from monosaccharides or monosaccharide derivatives in which any 1, 2, 3, 4 or 5 hydroxyl groups on the monosaccharide derivatives are independently replaced with hydrogen atoms;
  • the sulfated sugar or phosphorylated sugar is selected from monosaccharides or monosaccharide derivatives in which any 1, 2, 3, 4 or 5 hydroxyl groups are independently modified with sulfate groups or phosphate groups. thing;
  • the halo-chain sugar is selected from monosaccharides or monosaccharide derivatives obtained by independently having a carbon substituent on any 1, 2, 3, 4 or 5 non-terminal carbon atoms.
  • the carbon substituent can be replaced by a hydrogen atom or a hydroxyl group;
  • the glycosyl alkylated derivative is selected from monosaccharides or monosaccharide derivatives in which any 1, 2, 3, 4 or 5 hydroxyl groups are independently modified with linear or branched alkyl groups.
  • the alkyl group is C 1 -C 10 alkyl or alkylene, C 1 -C 8 haloalkyl, C 3 -C 10 cycloalkyl, 3-8 membered heterocycloalkyl, C 6 -C 10 aryl, 3-8 membered aromatic heterocyclyl, 3-8 membered heterocycloalkyl C 1 -C 8 alkylene, 3-8 membered cycloalkyl C 1 -C 8 alkylene or C 6 -C 10 aryl C 1 -C 8 alkylene;
  • the glycosyl esterified derivative is selected from a monosaccharide or a monosaccharide derivative in which any 1, 2, 3, 4 or 5 hydroxyl groups independently form an ester bond with a linear or branched acyl group.
  • the acyl groups are each independently C 1 to C 10 alkyl acyl, C 1 -C 8 haloalkyl acyl, C 3 -C 10 cycloalkyl acyl, 3-8 membered heterocycloalkyl acyl, C 6 -C 10 aryl acyl, 3-8 membered aromatic heterocyclyl acyl, 3-8 membered heterocycloalkyl C 1 -C 8 alkylene acyl, 3-8 membered cycloalkyl C 1 -C 8 sub Alkyloyl or C 6 -C 10 aryl C 1 -C 8 alkylene acyl is preferably acetyl, propionyl, isobutyryl or benzoyl.
  • the monosaccharide derivative is selected from the group consisting of halosugar, unsaturated sugar, glycosyl alkylated derivatives and glycosyl esterified derivatives;
  • the halosugar is selected from monosaccharides or monosaccharide derivatives in which any 1, 2, 3, 4 or 5 hydroxyl groups are independently substituted by fluorine, chlorine, bromine or iodine atoms;
  • the unsaturated sugar is selected from monosaccharides or monosaccharide derivatives obtained by changing any two adjacent hydroxyl groups on a monosaccharide derivative into unsaturated double bonds;
  • the glycosyl alkylated derivative is selected from the group consisting of monosaccharides or monosaccharide derivatives in which any 1, 2, 3, 4 or 5 hydroxyl groups are independently replaced by methyl, ethyl, cyclopropyl, benzyl, etc. Or monosaccharide derivatives substituted by p-methoxybenzyl, or any two hydroxyl groups are formed into a cyclic acetal or ketal through methylene, ethylene, isopropylidene, or benzylidene;
  • the glycosyl esterification derivative is selected from the group consisting of monosaccharides or monosaccharide derivatives in which any 1, 2, 3, 4 or 5 hydroxyl groups are independently combined with acetyl, propionyl, isobutyryl or benzoyl.
  • said R in formula (I) is selected from the following glycosyl or glycosyl derivatives:
  • dibenzylbutyrolactone glycoside compound represented by formula (I) is selected from the group consisting of the following compounds:
  • the compound is selected from the following:
  • the compound is selected from the following:
  • the compound is:
  • the present application provides a method for preparing the dibenzylbutyrolactone glycoside compound, its stereoisomer, tautomer or its pharmaceutically acceptable salt as described in the first aspect, so The preparation method includes the following steps:
  • the catalyst is selected from: trimethylsilyl triflate, trifluoromethanesulfonic acid, boron trifluoride ether, ferric chloride, preferably trifluoromethanesulfonate. Boron fluoride ether;
  • step (1) the substitution reaction is carried out in a solvent, and the solvent used is selected from: Dichloromethane, 1,2-dichloroethane, 1,4-dioxane, preferably dichloromethane;
  • step (1) the substitution reaction is carried out at a temperature of -60°C to 0°C, preferably -50°C to 0°C;
  • the molar ratio of arctigenin to acetyl-protected sugar is 0.05-3, preferably 0.1-2; the molar ratio of catalyst to arctigenin is 0.008 ⁇ 1.2, preferably 0.01-1.
  • the solvent used is selected from: methylene chloride, acetone, ethyl acetate, methanol, ethanol, preferably methanol;
  • the reaction temperature is 20 to 30°C, preferably room temperature.
  • the acetyl-protected sugar has the following structural formula:
  • the preparation method follows the following process route:
  • Ra is selected from: H, -CH 2 OAc, -CH 3 ;
  • Rh When Ra is H, Rh is also H; when Ra is -CH 2 OAc, Rh is -CH 2 OH; when Ra is -CH 3 , Rh is -CH 3 .
  • the present application provides a pharmaceutical composition, which includes the dibenzylbutyrolactone glycoside compound described in the first aspect, its stereoisomer, tautomer or its pharmaceutically acceptable Acceptable salt, as well as pharmaceutically acceptable Excipients accepted.
  • the specific gravity of the dibenzylbutyrolactone glycoside compound, its stereoisomer, tautomer or its pharmaceutically acceptable salt and the pharmaceutically acceptable excipient is from 0.001 to Within the range of 100, preferably within the range of 0.001 to 10.
  • the present application provides the use of dibenzylbutyrolactone glycoside compounds, their stereoisomers, tautomers or their pharmaceutically acceptable salts as described in the first aspect above in the preparation of phosphodiesters.
  • the application provides dibenzylbutyrolactone glycoside compounds as described in the first aspect, their stereoisomers, tautomers or pharmaceutically acceptable salts thereof, or the third aspect as described above.
  • PDE4 phosphodiesterase 4
  • the PDE4 is PDE4A, 4B, 4C or 4D, preferably PDE4D;
  • the disease is an immune and/or inflammatory disease associated with abnormal activity levels and/or expression levels of PDE4A, 4B, 4C or 4D (especially PDE4D);
  • the disease is selected from the group consisting of: psoriasis, psoriatic arthritis, atopic dermatitis, Behcet's disease, seborrheic dermatitis, atopic dermatitis, chronic obstructive pulmonary disease, asthma, Allergic rhinitis, ankylosing spondylitis, systemic lupus erythematosus, rheumatoid arthritis, rheumatoid arthritis, inflammatory bowel disease, malignant glioma, pulmonary fibrosis, amyotrophic lateral sclerosis, multiple sclerosis Alzheimer's disease, Huntington's disease, Parkinson's disease, ADHD, depression and schizophrenia.
  • Arctiin is a natural product source glycoside that is superior to the parent compound arctiin.
  • dibenzylbutyrolactone compounds as shown in formula (I) realizes dibenzylbutyrolactone glycosides by improving the catalyst dosage and reaction temperature and based on the principle of thermodynamic control.
  • the size of the compound Batch stereoselective preparation has very important practical significance and good application prospects for the clinical medicinal use of such compounds.
  • Figure 1 is the half-inhibitory concentration curve of compound AG09 described in Example 8 on the PDE4D catalytic domain
  • Figure 2 is the half-inhibitory concentration curve of compound AG09 described in Example 9 on the secretion of inflammatory factor TNF- ⁇ in human PBMC cells;
  • Figure 3 shows the appearance changes of skin lesions in psoriasis mice treated with AG09 ointment as described in Example 11; from left to right are the normal control group, model control group, AG09 ointment 2% treatment group, and AG09 ointment. 5% treatment group;
  • Figure 4 shows the pathological tissue photo changes of skin lesions in psoriasis mice treated with AG09 ointment as described in Example 11; from left to right are the normal control group, the model control group, the AG09 ointment 2% treatment group, and the AG09 Ointment 5% treatment group;
  • Figure 5 shows the changes in skin thickness of psoriasis mice treated with AG09 ointment as described in Example 11; from left to right are the normal control group, the model control group, the AG09 ointment 2% treatment group, and the AG09 ointment 5%. therapy group.
  • the compounds used can be obtained from commercial sources or can be prepared by using commercially available raw materials and reagents as follows: Synthesized by conventional methods.
  • dibenzylbutyrolactone glycoside compound AG02 of the present application is prepared according to the above reaction formula:
  • dibenzylbutyrolactone glycoside compound AG03 of the present application is prepared according to the above reaction formula:
  • dibenzylbutyrolactone glycoside compounds AG04 and AG05 of the present application are prepared according to the above reaction formula:
  • dibenzylbutyrolactone glycoside compounds AG06 and AG07 of the present application are prepared according to the above reaction formula:
  • compound 5 was obtained -1, which is a white solid, and the yield is 370 mg, and the yield is 32.7%;
  • Compound 6-1 is obtained, which is a colorless solid, and the yield is 470 mg, and the yield is 41.6%.
  • dibenzylbutyrolactone glycoside compounds AG08 and AG09 of the present application are prepared according to the above reaction formula:
  • compound 7 was obtained -1, which is a white solid, and the yield is 435 mg, and the yield is 33.5%;
  • Compound 8-1 is obtained, which is a colorless solid, and the yield is 585 mg, and the yield is 45.8%.
  • dibenzylbutyrolactone glycoside compound AG10 of the present application is prepared according to the above reaction formula:
  • dibenzylbutyrolactone glycoside compound AG31 of the present application is prepared according to the above reaction formula:
  • Arctigenin 2.5g, 6.71mmol
  • 1,2,3,4-tetra-O-acetyl- ⁇ -D-xylopyranose (1.42g, 4.48mmol) were mixed with oven-dried
  • the molecular sieve was dissolved in anhydrous DCM (50 mL), and 1 mol/L boron trifluoride ether solution (0.448 mL, 0.448 mmol) was slowly added dropwise at -20°C under N2 protection, and the reaction was maintained at -20°C for 12 hours.
  • dibenzylbutyrolactone glycoside compound AG32 of the present application is prepared according to the above reaction formula:
  • dibenzylbutyrolactone glycoside compound AG33 of the present application is prepared according to the above reaction formula:
  • dibenzylbutyrolactone glycoside compounds AG14 and AG15 of the present application are prepared according to the above reaction formula:
  • dibenzylbutyrolactone glycoside compounds AG16, AG17, AG24 and AG25 of the present application are prepared according to the above reaction formula:
  • dibenzylbutyrolactone glycoside compounds AG18 and AG19 of the present application are prepared according to the above reaction formula:
  • dibenzylbutyrolactone glycoside compounds AG20 and AG21 of the present application are prepared according to the above reaction formula:
  • dibenzylbutyrolactone glycoside compounds AG22 and AG23 of the present application are prepared according to the above reaction formula:
  • the synthesis of intermediates 14-1 to 14-6 refers to the synthesis method of intermediates 13-1 to 13-6, using ⁇ -methyl-D-mannoside as the starting material.
  • dibenzylbutyrolactone glycoside compound AG26 of the present application is prepared according to the above reaction formula:
  • dibenzylbutyrolactone glycoside compounds AG27 and AG28 of the present application are prepared according to the above reaction formula:
  • dibenzylbutyrolactone glycoside compounds AG29 and AG30 of the present application are prepared according to the above reaction formula:
  • the rate is 54.8%.
  • Example 18 Stereoselective preparation of the compound of the present application is achieved through the preparation method described in the present application.
  • Step 2 Referring to the preparation method of AG02, 1.55g of white solid was prepared from the intermediate compound obtained in step 1, with a yield of 68.6%. The product was confirmed by NMR and MS to be a single compound 8 (AG09) with ⁇ -glycosidic bond.
  • the cDNA sequence encoding the human PDE4D catalytic domain (GenBank: NM_001197221.1; coding region: T86-S413) was cloned into the expression vector pET15b and transformed into the Escherichia coli BL21 (DE3) expression strain at 16°C. Low temperature induces the expression of target protein. After collecting the bacterial cells at room temperature at 5000 rpm, resuspend and crush under high pressure, centrifuge at 12000 rpm for 45 min and take the supernatant.
  • Ni-NTA affinity chromatography column Pass the Ni-NTA affinity chromatography column, wash the column with a buffer of 50mM NaH 2 PO 4 (pH 7.5), 200mM NaCl and 50mM Imidazole, and then elute with a buffer containing 200mM imidazole to obtain the target protein. Further, high-purity target protein PDE4D catalytic domain (purity >95%) was gradually obtained through anion affinity chromatography and size exclusion chromatography for subsequent experiments.
  • SPA Scintillation Proximity Assay
  • the half inhibitory concentration curve of compound AG09 on PDE4D is shown in Figure 1. It can be seen from Figure 1 that the half inhibitory concentration (IC 50 value) of compound AG09 on PDE4D can reach hundreds of nanomoles. Compared with the positive compound Arctiin (AG01 ) increased by as much as 21 times.
  • the compounds AG15, AG17, AG26, AG27, AG28, and AG29 of the present application are measured at 5 ⁇ M and 0.5 ⁇ M respectively.
  • AG31, AG32, AG33 and positive control compound Arctiin (AG01) inhibition rate of PDE4 is as follows:
  • Inhibition rate (Absorbance value of the control group - Absorbance value of the test group) / (Absorbance value of the control group - Absorbance value of the blank group) ⁇ 100%
  • Example 20 Effect of the compound of the present application on TNF- ⁇ secretion in PBMC
  • PDE4 is widely expressed in immune- and inflammation-related cells, such as neutrophils, eosinophils, and monocytes, making PDE4 an important target for the study of major anti-inflammatory drugs in immune- and inflammation-related diseases.
  • Lipopolysaccharide (LPS) is a component of the cell wall of Gram-negative bacteria. It can significantly stimulate the expression of the inflammatory factor TNF- ⁇ through MAPK and other signaling pathways, thereby evaluating the cellular activity of some PDE4 inhibitors and simulating inflammation in vitro. effect.
  • the inhibitory effect of partial PDE4 inhibitors on TNF- ⁇ expression in human PBMC cells mainly refers to the work of George W. Muller et al.
  • test compounds including compounds AG04, AG06, AG08, AG09, AG10 and positive control compound Arctiin (AG01) of the present application
  • test compounds including compounds AG04, AG06, AG08, AG09, AG10 and positive control compound Arctiin (AG01) of the present application
  • TNF- ⁇ uses dual wavelengths to measure the absorbance value to eliminate measurement interference during single-wavelength detection.
  • the OD450nm and OD570nm photometric values are measured using a microplate reader (OD450nm is the detection wavelength; OD570nm is the reference wavelength).
  • the calculation formula for the inhibition rate of compounds on TNF- ⁇ secretion by human PBMC cells is as follows:
  • TNF- ⁇ inhibition rate % (absorbance value of the control group - absorbance value of the test group) / (absorbance value of the control group - absorbance value of the blank group) ⁇ 100%.
  • the EC 50 values of the compounds AG04, AG06, AG08, AG09, and AG10 of the present application for inhibiting the secretion of TNF- ⁇ by PBMC cells are in the range of 3.51-24.04 ⁇ M, while the EC 50 value of the positive compound AG01 is as high as 97.58 ⁇ M, indicating that it inhibits the secretion of TNF- ⁇ by PBMC cells.
  • the inhibitory effect of TNF- ⁇ secreted by cells is significantly weaker than that of a series of compounds of this application.
  • compound AG09 has the best activity, and its half-inhibitory concentration curve for the secretion of the inflammatory factor TNF- ⁇ in human PBMC cells is shown in Figure 2.
  • Figure 2 shows that the half-inhibitory concentration (EC 50) of compound AG09 for TNF- ⁇ secretion value) reaches 3.51 ⁇ M, which is nearly 1/28 of the positive control compound AG01, indicating that its inhibitory activity on the secretion of TNF- ⁇ by PBMC cells is about 28 times that of the positive control compound Arctiin (AG01), that is, it inhibits the secretion of TNF- ⁇ The activity is significantly better than that of Arctiin(AG01).
  • Example 21 Effect of the compound of the present application on TNF- ⁇ secretion in RAW264.7
  • Tumor necrosis factor as an important inflammatory mediator in the development of inflammation, autoimmune diseases and other diseases, is mainly produced by activated monocytes/macrophages. It can mediate the occurrence of a variety of inflammatory reactions and accelerate the progression of the disease. .
  • the mouse mononuclear/macrophage leukemia cell line RAW 264.7 is one of the commonly used inflammatory cell models. After LPS-induced activation, it can release TNF- ⁇ and other inflammatory mediators.
  • RAW 264.7 cells (1 ⁇ 10 5 cells/well) were seeded in a 96-well plate, and after incubation for 24 hours, 1 ⁇ g/mL LPS was added for induction. After the RAW 264.7 cells are polarized, add different concentrations of the compounds to be tested (including a series of compounds of this application and the positive control compound Arctiin (AG01)), and incubate for 18 hours at 37°C in a 5% CO 2 incubator. No stimulant is provided. The total volume of the background control and stimulation control wells is 200 ⁇ L at 300 g/min. After centrifugation for 10 min, the supernatant is collected, and the ELISA method is used to detect the secretion level of TNF- ⁇ in the culture supernatant.
  • the compounds to be tested including a series of compounds of this application and the positive control compound Arctiin (AG01)
  • Example 22 Therapeutic effect of the compound of the present application on psoriasis-like animal models
  • Modeling drugs Imiquimod ointment, produced by Sichuan Mingxin Lidi Pharmaceutical Co., Ltd., national drug approval number H20030128, product batch number 15060139.
  • mice BALB/c mice, female, weighing 18-22g, provided by Shanghai Slack Experimental Animal Co., Ltd.
  • Psoriasis-like model modeling method After the back hair of the mice is removed, 62.5 mg imiquimod ointment is applied to the back skin every day at about 7:30 every day for 8 consecutive days. At this time, Psoriasis-like skin lesions were at a more severe level, as shown in the model control group in Figure 3 .
  • the experiment is divided into four groups:
  • mice i.e., normal healthy mice
  • Model control group i.e., psoriasis-like mouse model without any treatment
  • AG09 ointment 5% treatment group i.e., psoriasis-like mouse model treated with 5% AG09 ointment
  • AG09 ointment 2% treatment group i.e., psoriasis-like mouse model treated with 2% AG09 ointment, 10 animals in each group;
  • AG09 ointment 5% and 2% treatment groups were treated as follows: 62.5 mg of 5% AG09 ointment and 2% AG09 ointment were applied to the back skin of the psoriasis-like mouse model daily, and the application time was approximately 18:30 every day, for 8 consecutive days.
  • mice from each group were taken for H&E staining; specifically, skin samples were collected, fixed in 10% formalin tissue fixative, embedded in paraffin, cut into 3 ⁇ m sections and sliced. H&E skin tissue staining was performed, and skin epidermal thickening and inflammatory cell infiltration were observed under an Olympus IX73 microscope; the results are shown in Figure 4;
  • the dibenzylbutyrolactone glycoside compound represented by formula (I) provided by this application has strong inhibitory activity against type 4 phosphodiesterase (PDE4) and has strong anti-inflammatory activity and can be used for Prevention, treatment or auxiliary treatment of diseases related to abnormal activity levels and/or expression levels of PDE4, especially immune and inflammatory diseases related to them; in addition, the preparation method of dibenzylbutyrolactone glycoside compounds provided in this application Its stereoselective preparation can be achieved.
  • PDE4 type 4 phosphodiesterase

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Abstract

公开了一种如式(I)所示的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐，其制备方法，包含其的药物组合物，以及其在制备预防、治疗或辅助治疗与磷酸二酯酶4(PDE4)活性水平或表达水平异常相关的疾病的药物中的用途。公开的二苄基丁内酯糖苷类化合物对PDE4具有很强的抑制活性，并具有很强的抗炎活性，可以用于预防、治疗或辅助治疗与PDE4的活性和/或表达水平异常相关的疾病；此外，公开的的二苄基丁内酯糖苷类化合物的制备方法可以实现其立体选择性制备。

Description

二苄基丁内酯糖苷类化合物、其制备方法和应用
交叉引用
本申请要求于2022年9月5日提交的、申请号为202211078981.2、发明名称为“二苄基丁内酯糖苷类化合物、其制备方法和应用”的中国专利申请的优先权，其全部内容通过引用并入本文。
技术领域
本申请涉及药物化学领域，具体涉及一种如式(I)所示的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐，其制备方法，包含其的药物组合物，以及其在制备磷酸二酯酶4抑制剂中的用途以及在制备用于预防、治疗或辅助治疗与磷酸二酯酶4活性水平或表达水平异常相关的疾病的药物中的用途。
背景技术
环核苷酸磷酸二酯酶(Cyclic nucleotide phosphodiesterases,PDEs)可以特异性催化水解细胞内第二信使环磷酸腺苷(cAMP)或环磷酸鸟苷(cGMP)，调节cAMP或cGMP在细胞内的浓度，从而调控第二信使分子所介导的一系列生理病理过程，如细胞周期控制、细胞分化、炎症、心脏功能、平滑肌舒张与收缩、视觉信号传导、学习与记忆等。PDEs是环核苷酸信号传导通路中的重要调控蛋白，抑制其水解活性，可以提高细胞内cAMP或cGMP浓度，抑制多种炎症介质活性、抑制细胞粘附因子的上调和表达、诱导细胞凋亡以及诱导儿茶酚胺类物质和内源性激素的释放等。因此，PDEs是重要的药物作用靶标家族，PDEs抑制剂已经广泛应用于多种疾病病理机制研究及治疗(Maurice DH,et al.Nat.Rev.Drug Discov.2014,13:290-314；Menniti FS,et al.Nat.Rev.Drug Discov.2006,5(8),660-670)。
PDE4特异性水解细胞内第二信使分子cAMP，是PDE超家族中11个亚家族中的一员，包含PDE4A/B/C/D四种亚型，在体内分布广泛，主要表达于多种免疫相关细胞如中性粒细胞、嗜酸性粒细胞和单核细胞等。因此，PDE4成为免疫及炎症相关疾病中主要的抗炎药物研究的重要靶点。PDE4涉及的疾病种类繁多，其中与PDE4在炎症进程中的作用相关的有慢性阻塞性肺疾病、哮喘、银屑病、过敏性鼻炎、特发性肺纤维化和风湿性关节炎等疾病。涉及神经系统的疾病包括阿尔兹海默症、帕金森氏症、抑郁症和精神分裂症等(Menniti FS,et al.Nat.Rev.Drug Discov.2006,5:660-670；BurginA B,et al.Nat.Biotechnol.2010,28:63-70；Garcia OstaA,et al.ACS Chem.Neurosci.2012,3:832-844)。 由于PDE4涉及多种重要疾病且已有部分PDE4抑制剂用于临床治疗，设计和发现PDE4的新型抑制剂是新药研发领域的一大热点。
银屑病是一种常见的慢性复发性炎症性皮肤病，典型临床表现为边界清楚的红斑、皮疹、斑块、鳞屑，该病发病机制复杂，病因未明。肿瘤坏死因子α(TNF-α)在银屑病中高度表达，针对TNF-α的阻断治疗在临床上效果显著。靶向抑制PDE4水解活性可以通过增加细胞内cAMP浓度下调TNF-α等促炎因子的表达，缓解银屑病症状。目前银屑病治疗多以局部治疗为主，类固醇类激素如丁酸氢化可的松、糠酸莫米松等在治疗银屑病中应用广泛，但长期使用副作用大，影响其广泛应用；他克莫司、卡波三醇等由于其昂贵的价格也限制了其在临床上的应用。虽然目前为止用于治疗银屑病的PDE4抑制剂逐步上市，如Crisaborole和Apremilast，但仍在巨大缺口，迫切需要研发新的副作用更少、经济成本更低以及疗效更好药物。
牛蒡子苷(Arctiin)及其苷元牛蒡子苷元(Arctigenin)来源于牛蒡，属于二苄基丁内酯类化合物，具有多种药理活性，包括免疫调节、抗糖尿病、抗肿瘤、神经保护等作用。其结构式如下所示：
许多研究表明，牛蒡子苷和牛蒡子苷元可以通过抑制NO和PGE2释放、抑制COX2活性、抑制活性氧生成等多种途径抑制TNF-α、IL-1β、IL-6等炎症因子释放，发挥抗炎效果(Gao,et al.Acta Pharmacol.Sin.2018,39:787-801)。近年来研究表明，牛蒡子苷元可以通过抑制PDE4D，升高体内cAMP水平，发挥显著的抗炎与抗银屑病效果(Li,et al.J.Adv.Res.2021,33:241-251)，但其也存在溶解性差、代谢迅速等问题，阻碍了其作为药物开发的潜力，对牛蒡子苷元进行进一步结构改造有望获得具有更好抗炎活性、更好成药性的候选化合物。
在药物化学领域，糖类药物的研究越来越受到重视。糖类化合物结构的复杂与多样性赋予了母体药物多样化的理化性质与药理活性。天然产物的糖基化修饰可以通过增加溶解性、调节血浆半衰期、提高结合特异性等方式改变母体药物的药理活性(蔡孟深,李中军.糖化学[M].化学工业出版社,2007，315-324)。然而，牛蒡子苷元天然来源的 糖苷——牛蒡子苷对PDE4D的抑制活性偏弱，细胞水平的抗炎活性微弱。
目前，尚未有对此类二苄基丁内酯类化合物进行的多种糖基化修饰报道，不同糖基化修饰对该类化合物的药理活性的影响仍有待探究。
此外，区域及立体选择性地构筑糖苷键在糖类化合物合成领域一直是很大的挑战(Shivatare,et al.J.Org.Chem.2020,85,15780-15800)。目前也尚未报道以二苄基丁内酯类化合物和糖基供体为原料选择性构筑糖苷键的方法，由此带来立体异构体拆分困难的问题，极大限制了此类糖苷的大规模制备。
发明内容
本申请的目的在于提供一种如式(I)所示的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐，其制备方法，包含其的药物组合物，以及其在制备磷酸二酯酶4抑制剂中的用途以及在制备用于预防、治疗或辅助治疗与磷酸二酯酶4活性水平或表达水平异常相关的疾病的药物中的用途。本申请的如式(I)所示的二苄基丁内酯糖苷类化合物对于PDE4D具有很强的抑制活性，其抗炎效果也显著优于母体化合物牛蒡子苷元的天然产物来源——牛蒡子苷(Arctiin)，在与磷酸二酯酶4活性水平或表达水平异常相关的疾病的临床治疗中具有广阔的应用前景。
本申请的上述目的通过以下技术方案来实现：
第一方面，本申请提供了一种如式(I)所示的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐：
所述式(I)中，R选自由以下组成的组：单糖、单糖衍生物、由单糖单元通过糖苷键连接的双糖、由三至五个单糖单元通过糖苷键连接的寡糖；其中，所述单糖不包含D-β-葡萄糖，但所述单糖衍生物包含D-β-葡萄糖衍生物，并且，其中，所述R通过α或β型O-糖苷键与二苄基丁内酯类化合物主体相连。
在优选的实施方案中，所述单糖选自：呋喃糖和吡喃糖；
优选地，所述单糖为D型或L型。
在优选的实施方案中，所述单糖衍生物选自由以下组成的组：氧化糖、卤代糖、不饱和糖、去氧糖、氨基去氧糖、脱水糖、硫酸化糖、磷酸化糖、歧链糖、糖基烷基化衍生物和糖基酯化衍生物；
进一步优选地，所述氧化糖选自单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地被氧化为醛或酸所得的单糖衍生物；
进一步优选地，所述卤代糖选自单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地被卤素取代所得的单糖衍生物，所述卤素任意地选自氟、氯、溴或碘；
进一步优选地，所述不饱和糖选自单糖或单糖衍生物上任意相邻两个羟基变为不饱和双键所得的单糖衍生物；
进一步优选地，所述去氧糖或氨基去氧糖选自单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地被替换为甲基所得的单糖衍生物；
进一步优选地，所述脱水糖选自单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地被替换为氢原子所得的单糖衍生物；
进一步优选地，所述硫酸化糖或磷酸化糖选自单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地被硫酸基或磷酸基修饰所得的单糖衍生物；
进一步优选地，所述歧链糖选自单糖或单糖衍生物上任意1，2，3，4或5个非端基碳原子上各自独立地有一个碳取代基所得的单糖衍生物，所述碳取代基可以置换为一个氢原子或一个羟基；
进一步优选地，所述糖基烷基化衍生物选自单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地被直链或支链的烷基修饰所得的单糖衍生物，所述烷基为C1-C10烷基或亚烷基、C1-C8卤代烷基、C3-C10环烷基、3-8元杂环烷基、C6-C10芳基、3-8元芳杂环基、3-8元杂环烷基C1-C8亚烷基、3-8元环烷基C1-C8亚烷基或C6-C10芳基C1-C8亚烷基；
进一步优选地，所述糖基酯化衍生物选自由单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地与直链或支链的酰基组成酯键所得的单糖衍生物，所述酰基各自独立地为C1～C10烷基酰基、C1-C8卤代烷基酰基、C3-C10环烷基酰基、3-8元杂环烷基酰基、C6-C10芳基酰基、3-8元芳杂环基酰基、3-8元杂环烷基C1-C8亚烷基酰基、3-8元环烷基C1-C8亚烷基酰基或C6-C10芳基C1-C8亚烷基酰基，优选为乙酰基、丙酰基、异丁酰基、苯甲酰基。
在进一步优选的实施方案中，所述单糖衍生物选自由以下组成的组：卤代糖、不饱和糖、糖基烷基化衍生物和糖基酯化衍生物；
优选地，所述卤代糖选自单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地被氟、氯、溴、碘原子取代；
优选地，所述不饱和糖选自单糖或单糖衍生物上任意相邻两个羟基变为不饱和双键所得的单糖衍生物；
优选地，所述糖基烷基化衍生物选自单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地被甲基、乙基、环丙基、苄基或对甲氧基苄基所取代，或任意两个羟基通过亚甲基、亚乙基、异亚丙基、苯亚甲基组成环状缩醛或缩酮所得的单糖衍生物；
优选地，所述糖基酯化衍生物选自由单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地与乙酰基、丙酰基、异丁酰基或苯甲酰基组成酯键所得的单糖衍生物。
在进一步优选的实施方案中，式(I)中的所述R选自如下糖基或糖基衍生物：
柔红霉糖、阿洛糖、阿卓糖、古洛糖、半乳糖、氨基半乳糖、氨基葡萄糖、甘露糖、艾杜糖、塔洛糖、来苏糖、阿拉伯糖、核糖、木糖、岩藻糖、鼠李糖、异鼠李糖、橄榄霉糖、毛地黄毒糖、加拿大麻糖、奎诺糖、艾杜糖、6-脱氧艾杜糖、N-乙酰葡萄糖胺、N-乙酰半乳糖胺、果糖，或前述任意一项的衍生物，或D-β-葡萄糖衍生物。
在优选的具体实施方案中，如式(I)所示的二苄基丁内酯糖苷类化合物选自由如下化合物组成的组：





进一步优选地，所述化合物选自以下：



更进一步优选地，所述化合物选自以下：


更进一步优选地，所述化合物为：

第二方面，本申请提供了如上述第一方面所述的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐的制备方法，所述制备方法包括以下步骤：
(1)牛蒡子苷元与乙酰基保护的糖，在氮气保护下，在催化剂作用下，经取代反应，形成乙酰基保护的牛蒡子糖苷化合物；
(2)在碳酸钾或甲醇钠存在下，使步骤(1)所得乙酰基保护的牛蒡子糖苷化合物脱去乙酰基保护，得到所述二苄基丁内酯糖苷类化合物。
在优选的实施方案中，步骤(1)中，所述催化剂选自：三氟甲磺酸三甲基硅酯、三氟甲磺酸、三氟化硼乙醚、三氯化铁，优选为三氟化硼乙醚；
在优选的实施方案中，步骤(1)中，所述取代反应在溶剂中进行，所用溶剂选自： 二氯甲烷、1,2-二氯乙烷、1,4-二氧六环，优选为二氯甲烷；
在优选的实施方案中，步骤(1)中，所述取代反应在-60℃～0℃，优选-50℃～0℃的温度下进行；
在优选的实施方案中，步骤(1)中，所述牛蒡子苷元与乙酰基保护的糖的摩尔比为0.05～3，优选为0.1～2；催化剂与牛蒡子苷元的摩尔比为0.008～1.2，优选为0.01～1。
在优选的实施方案中，步骤(2)中，所用溶剂选自：二氯甲烷、丙酮、乙酸乙酯、甲醇、乙醇，优选为甲醇；
在优选的实施方案中，步骤(2)中，反应温度为20～30℃，优选为室温。
在一些优选的具体实施方案中，步骤(1)中，所述乙酰基保护的糖具有如下结构式：
所述制备方法按如下工艺路线：
其中，Ra选自：H、-CH2OAc、-CH3；
当Ra为H时，Rh也为H；当Ra为-CH2OAc时，Rh为-CH2OH；当Ra为-CH3时，Rh为-CH3。
第三方面，本申请提供了一种药物组合物，其包括如上述第一方面所述的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐，以及药学上可 接受的辅料。
在优选的实施方案中，所述二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐与药学上可接受的辅料的比重在0.001至100的范围内，优选在0.001至10范围内。
第四方面，本申请提供了如上述第一方面所述的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐在制备磷酸二酯酶4抑制剂、优选磷酸二酯酶4D抑制剂中的用途。
第五方面，本申请提供了如上述第一方面所述的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐或者如上述第三方面所述的药物组合物在制备用于预防、治疗或辅助治疗与磷酸二酯酶4(PDE4)活性水平和/或表达水平异常相关的疾病的药物中的用途。
优选地，所述PDE4为PDE4A、4B、4C或4D，优选PDE4D；
优选地，所述疾病为与PDE4A、4B、4C或4D(特别是与PDE4D)活性水平和/或表达水平异常相关的免疫和/或炎症性疾病；
进一步优选地，所述疾病选自由以下组成的组：银屑病、银屑病关节炎、特应性皮炎、白塞氏病、脂溢性皮炎、过敏性皮炎、慢性阻塞性肺病、哮喘、过敏性鼻炎、强直性脊柱炎、系统性红斑狼疮、风湿性关节炎、类风湿性关节炎、炎症性肠病、恶性胶质瘤、肺纤维化、肌萎缩性侧索硬化症、多发性硬化症、阿尔兹海默症、亨廷顿舞蹈症、帕金森氏症、多动症、抑郁症和精神分裂症。
有益效果
本申请的发明人经过长期而深入的研究，制备得到了一类能够抑制磷酸二酯酶(PDE4)的式I化合物；本申请的如式(I)所示的特定糖基修饰的二苄基丁内酯类化合物对四型磷酸二酯酶(PDE4)、特别是PDE4D具有很强的抑制活性，并具有很强的抗炎活性，可以用于预防、治疗或辅助治疗与磷酸二酯酶4的活性和/或表达水平异常相关的疾病，例如银屑病、银屑病关节炎、特应性皮炎、白塞氏病、脂溢性皮炎、过敏性皮炎、慢性阻塞性肺病、哮喘、过敏性鼻炎、强直性脊柱炎、系统性红斑狼疮、风湿性关节炎、类风湿性关节炎、炎症性肠病、恶性胶质瘤、肺纤维化、肌萎缩性侧索硬化症、多发性硬化症、阿尔兹海默症、亨廷顿舞蹈症、帕金森氏症、多动症、抑郁症和精神分裂症等疾病，尤其是与PDE4相关的免疫和炎症性疾病如银屑病和关节炎等，其效果显著优于母体化合物牛蒡子苷元的天然产物来源糖苷——牛蒡子苷(Arctiin)。
此外，本申请所提供的如式(I)所示二苄基丁内酯类化合物的制备方法，通过改进催化剂用量和反应温度并基于热力学控制原理等，实现了二苄基丁内酯糖苷类化合物的大 批量立体选择性制备，这对于此类化合物的临床药用具有非常重要的现实意义与良好的应用前景。
附图说明
一个或多个实施例通过与之对应的附图中的图片进行示例性说明，这些示例性说明并不构成对实施例的限定。在这里，专用的词“示例性”意为“用作例子、实施例或说明性”。在这里，作为“示例性”所说明的任何实施例不必解释为优于或好于其它实施例。
图1为实施例8中记载的化合物AG09对PDE4D催化结构域的半抑制浓度曲线；
图2为实施例9中记载的化合物AG09对人PBMC细胞中炎症因子TNF-α分泌的半抑制浓度曲线；
图3为实施例11中记载的AG09软膏剂治疗银屑病小鼠的皮肤损伤的外观变化；从左至右分别为正常对照组，模型对照组，AG09软膏剂2％治疗组、AG09软膏剂5％治疗组；
图4为实施例11中记载的AG09软膏剂治疗银屑病小鼠的皮肤损伤的病理组织照片变化；从左至右分别为正常对照组，模型对照组，AG09软膏剂2％治疗组、AG09软膏剂5％治疗组；
图5为实施例11中记载的AG09软膏剂治疗银屑病小鼠的皮肤厚度变化；从左至右分别为正常对照组，模型对照组，AG09软膏剂2％治疗组、AG09软膏剂5％治疗组。
具体实施方式
为使本申请的目的、技术方案和优点更加清楚，下面将对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本申请保护的范围。
另外，为了更好的说明本申请，在下文的具体实施方式中给出了众多的具体细节。本领域技术人员应当理解，没有某些具体细节，本申请同样可以实施。在一些实施例中，对于本领域技术人员熟知的原料、方法等未作详细描述，以便于凸显本申请的主旨。
除非另有其它明确表示，否则在整个说明书和权利要求书中，术语“包括”或其变换如“包含”或“包括有”等等将被理解为包括所陈述的组成部分，而并未排除其它组成部分。
此外，需要说明的是，本申请所有数值指定均可理解为前面有术语“约”。
以下实施例中，未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件；除非另外说明，否则百分比和份数按重量计算。
以下实施例中，所用化合物可以从市售获得或可通过以下使用市售的原料和试剂的 常规方法合成。
以下实施例中，1H NMR谱与13C NMR采用Bruker-500MHz型核磁共振仪测定，MS谱在Finnigan LTQ线性离子阱质谱仪上测定，反应用TLC跟踪，HPLC制备和纯度测定(乙腈、水为流动相，添加1‰三氟乙酸)在大连依利特P3140A/P型仪器上进行，所有反应未作特别说明均在空气氛围下进行。
此外，实施例中使用了以下缩略语：
PE        石油醚
EA        乙酸乙酯
TEA       三乙胺
DCM       二氯甲烷
TMSOTf    三氟甲磺酸三甲基硅酯。
实施例1
本实施例中，按照上述反应式，制备本申请的二苄基丁内酯糖苷类化合物AG02：
中间体2R，3R，4S，5R，6S-2-乙酰氧基甲基-6-(3R，4R)-4-(3，4-二甲氧基苄基)-2- 氧代四氢呋喃-3-基)甲基-2-甲氧基苯氧基四氢-2H-吡喃-3，4，5-三基三乙酸酯(化合物 1-1)的合成：
将牛蒡子苷元(100mg,0.268mmol)和2,3,4,6-四-O-乙酰基-β-D-吡喃半乳糖酰基-2,2,2-三氯代亚氨乙酸酯(132mg,0.268mmol)加入溶于无水二氯甲烷(3mL)中,N2保护，0℃下缓慢滴加TMSOTf(6mg,0.027mmol)，缓慢升至室温后反应12h。反应完成后，旋干有机相，快速柱层析分离(PE:EA＝1:1)得无色油状物，即为中间体化合物1-1，其产量为130mg，收率为69.1％。
化合物3R，4R-4-(3，4-二甲氧基苄基)-3-(3-甲氧基-4-(2S，3R，4S，5S，6R)-3，4， 5-三羟基-6-羟甲基四氢-2H-吡喃-2-基)苄基)二氢呋喃-2(3H)-酮(AG02)的制备：
将化合物1-1(100mg,0.142mmol)溶于甲醇(2mL)中，加入无水碳酸钾(49mg,0.355mmol)，室温反应3h。反应完成后，旋干有机相，柱层析分离(DCM:MeOH＝10:1)，得白色固体，即为化合物AG02，其产量为55mg，收率为72.4％。1H NMR(500MHz,Chloroform-d)δ6.97(d,J＝8.1Hz,1H),6.75(dd,J＝8.3,2.8Hz,1H),6.65–6.58(m,2H), 6.55(dd,J＝8.1,2.1Hz,1H),6.48(d,J＝2.1Hz,1H),4.72(d,J＝7.6Hz,1H,1-H),4.18–4.07(m,2H),4.01–3.93(m,1H),3.89–3.81(m,6H),3.79(s,3H),3.74–3.71(m,1H),3.69(s,3H),3.61–3.54(m,1H),2.88–2.84(m,2H),2.68–2.62(m,1H),2.59–2.51(m,2H),2.51–2.44(m,1H).13C NMR(126MHz,CDCl3)δ178.16,149.03,148.53,147.37,144.52,132.91,129.85,121.40,120.22,117.33,112.64,111.46,110.96,102.26(1-C),74.17,72.68,70.74,70.57,68.32,61.00,55.44,55.42,55.37,45.94,40.63,37.58,33.98.HRMS:m/z for C27H34NaO11[M+Na]+calculated:557.1993,found:557.1987.
实施例2
本实施例中，按照上述反应式，制备本申请的二苄基丁内酯糖苷类化合物AG03：
中间体2R，3R，4S，5S，6R-2-乙酰氧基甲基-6-(4-(3R，4R)-4-(3，4-二甲氧基苄基)-2- 氧代四氢呋喃-3-基)甲基)-2-甲氧基苯氧基-2H-吡喃-3，4，5-三基三乙酸酯(化合物2-1) 的合成：
将牛蒡子苷元(200mg,0.537mmol)和1,2,3,4,6-O-五乙酰基-α-甘露糖(105mg,0.269mmol)溶于无水1,2-二氯乙烷(5mL)中,N2保护，0℃下缓慢滴加三氟甲磺酸(3mg,0.02mmol)，缓慢升至室温后反应12h。反应完成后，旋干有机相，快速柱层析分离(PE:EA＝1:1)得无色油状物，即为中间体化合物2-1，其产量为135mg，收率为71.8％。
化合物3R，4R-4-(3，4-二甲氧基苄基)-3-(3-甲氧基-4-(2R，3S，4S，5S，6R)-3，4， 5-三羟基-6-羟甲基四氢-2H-吡喃-2-基)氧基)苄基)二氢呋喃-2(3H)-酮(AG03)的制备：
参照AG02的制备方法，得白色固体，即为化合物AG03，其产量为45mg，收率为74.3％。1H NMR(500MHz,Chloroform-d)δ6.96(d,J＝8.0Hz,1H),6.75(d,J＝8.1Hz,1H),6.67–6.62(m,1H),6.59(d,J＝8.1Hz,1H),6.55(dd,J＝8.1,1.9Hz,1H),6.49(d,J＝1.9Hz,1H),5.46(s,1H),4.24(s,1H,1-H),4.15–4.10(m,2H),4.00–3.93(m,2H),3.87–3.82(m,4H),3.81–3.76(m,4H),3.74–3.66(m,4H),2.88(d,J＝5.7Hz,2H),2.64(dd,J＝13.6,6.1Hz,1H),2.56(dt,J＝8.2,5.4Hz,2H),2.47(q,J＝7.5Hz,1H).13C NMR(126MHz,CDCl3)δ178.63,150.29,149.04,147.89,144.16,133.25,130.38,121.67,120.68,118.12,113.43,111.97,111.46,99.78(1-C),73.14,71.44,71.22,70.90,66.12,60.88,55.92,55.91,55.86,46.47,41.11,38.09,34.46.HRMS:m/z for C27H34NaO11[M+Na]+calculated: 557.1993,found:557.1990.
实施例3
本实施例中，按照上述反应式，制备本申请的二苄基丁内酯糖苷类化合物AG04和AG05：
中间体2R，3R，4S，5R-2-乙酰氧基甲基-5-(4-(3R，4R)-4-(3，4-二甲氧基苄基)-2- 氧代四氢呋喃-3-基)甲基-2-甲氧基苯氧基四氢呋喃-3，4-二基二乙酸酯(化合物3-1)和中 间体2R，3R，4S，5S-2-乙酰氧基甲基-5-(4-(3S，4S)-4-(3，4-二甲氧基苄基)-2-氧代四 氢呋喃-3-基甲基)-2-甲氧基苯氧基四氢呋喃-3，4-二基二乙酸酯(化合物4-1)的制备：
将牛蒡子苷元(1.0g,2.69mmol)和四乙酰阿拉伯呋喃糖(570mg,1.79mmol)溶于无水DCM(20mL)中，N2保护，0℃下缓慢滴加1mol/L三氟化硼乙醚溶液(3.58mL,3.58mmol),升至室温后反应12h。反应完成后，旋干有机相，快速柱层析分离(PE:EA＝1:1)，分别得到两种立体异构体化合物：化合物3-1和化合物4-1。化合物3-1为白色固体，产量为350mg，收率为30.1％；化合物4-1为白色固体，产量为430mg，收率为38.1％。
化合物3R，4R-3-(4-(2R，3S，4S，5R)-3，4-二羟基-5-羟甲基四氢呋喃-2-基)氧基-3- 甲氧基苄基)-4-(3，4-二甲氧基苄基)二氢呋喃-2(3H)-酮(AG04)的制备：
参照AG02的制备方法，得白色固体，即为化合物AG04，其产量为57mg，收率为71.6％。1H NMR(500MHz,Chloroform-d)δ6.98(d,J＝8.0Hz,1H),6.75(d,J＝8.1Hz,1H),6.71–6.66(m,2H),6.54(dd,J＝8.1,2.0Hz,1H),6.50(d,J＝2.2Hz,1H),5.72(s,1H，1-H),4.32(s,1H),4.29–4.24(m,1H),4.16(dd,J＝9.1,7.1Hz,1H),4.12(s,1H),3.93–3.83(m,6H),3.82(s,3H),3.78(s,3H),2.98–2.91(m,2H),2.65(dd,J＝13.3,6.1Hz,1H),2.62–2.55(m,2H),2.54–2.48(m,1H).13C NMR(126MHz,CDCl3)δ178.07,150.54,148.56,147.43,142.06,132.90,129.83,121.25,120.11,117.51,112.40,111.45,110.91,106.75(1-C),87.92,78.43,77.71,70.78,61.51,55.42,55.41,55.16,46.03,40.58,37.67,34.09.HRMS:m/z for C26H32NaO10[M+Na]+calculated:527.1888,found:557.1885.
化合物3R，4R-3-(4-(2S，3S，4S，5R)-3，4-二羟基-5-羟甲基四氢呋喃-2-基)氧基-3- 甲氧基苄基)-4-(3，4-二甲氧基苄基)二氢呋喃-2(3H)-酮(AG05)的制备：
参照AG02的制备方法，得白色固体，即为化合物AG05，其产量为66mg，收率为63.5％。1H NMR(500MHz,Chloroform-d)δ7.05(d,J＝8.1Hz,1H),6.76(dd,J＝8.2,2.3Hz,1H),6.71(d,J＝2.0Hz,1H),6.63(dd,J＝8.2,1.9Hz,1H),6.56(dd,J＝8.1,2.0Hz,1H),6.49(t,J＝2.5Hz,1H),4.75(d,J＝6.5Hz,1H,1-H),4.15(dd,J＝9.1,7.4Hz,1H),4.10–4.01(m,1H),4.00–3.95(m,2H),3.91–3.84(m,4H),3.82(s,3H),3.81–3.74(m,4H),3.60(dd,J＝12.4,1.8Hz,1H),2.93(d,J＝5.8Hz,2H),2.65(dd,J＝13.8,6.5Hz,1H),2.61–2.53(m,2H),2.52–2.43(m,1H).13C NMR(126MHz,CDCl3)δ178.07,149.81,148.58,147.44,143.92,133.51,129.83,121.31,120.13,118.74,112.57,111.40,110.88,102.19(1-C),72.15,70.76,70.10,66.75,64.77,55.43,55.42,55.41,46.03,40.52,37.70,34.03.HRMS:m/z for C26H32NaO10[M+Na]+calculated:527.1888,found:557.1876.
实施例4
本实施例中，按照上述反应式，制备本申请的二苄基丁内酯糖苷类化合物AG06和AG07：
中间体2-乙酰氧基甲基-5-(3R，4R)-4-(3，4-二甲氧基苄基)-2-氧代四氢呋喃-3-基甲 基)-2-甲氧基苯氧基-3-二氢呋喃-3-二乙酸酯(化合物5-1)和中间体2-乙酰氧基甲基-5-(3S， 4S)-4-(3，4-二甲氧基苄基)-2-氧代四氢呋喃-3-基甲基)-2-甲氧基苯氧基-3，4-二氢呋喃-3， 4-二基二乙酸酯(化合物6-1)的制备：
参照化合物3-1和化合物4-1的制备方法，以牛蒡子苷元和3，4，5-三乙酰氧基氧杂环戊环-2-基乙酸甲酯为起始原料，得化合物5-1，其为白色固体，产量为370mg，收率为32.7％；得化合物6-1，其为无色固体，产量为470mg，收率为41.6％。
化合物3R，4R-3-(4-(2R，3S，4R，5S)-3，4-二羟基-5-羟甲基四氢呋喃-2-基)氧基-3- 甲氧基苄基)-4-(3，4-二甲氧基苄基)二氢呋喃-2(3H)-酮(AG06)的制备：
参照AG02的制备方法，得无色固体，即为化合物AG06，其产量为72mg，收率为77.3％。1H NMR(500MHz,Chloroform-d)δ6.94(d,J＝8.1Hz,1H),6.77–6.73(m,1H),6.69(d,J＝2.0Hz,1H),6.64–6.60(m,1H),6.57–6.53(m,1H),6.50–6.46(m,1H),5.53(s,1H,1-H),4.68(t,J＝5.5Hz,1H),4.39(d,J＝4.9Hz,1H),4.21–4.13(m,2H),3.90(dd,J＝9.1,7.6Hz,1H),3.87–3.83(m,4H),3.82(s,3H),3.79(s,3H),3.71(dd,J＝12.4,3.0Hz,1H),2.96–2.90(m,2H),2.67–2.61(m,1H),2.59–2.53(m,2H),2.52–2.45(m,1H).13C NMR(126MHz,CDCl3)δ178.12,150.11,148.52,147.40,144.12,133.05,129.84,121.49,120.15,118.25,112.78,111.37,110.86,107.88(1-C),84.86,75.50,70.84,69.74,61.39,55.42,55.37,55.22,46.04,40.52,37.78,34.04.HRMS:m/z for C26H32NaO10[M+Na]+calculated:527.1888,found:557.1879.
化合物3R，4R-3-(4-(2S，3S，4R，5S)-3，4-二羟基-5-羟甲基四氢呋喃-2-基)氧基-3- 甲氧基苄基)-4-(3，4-二甲氧基苄基)二氢呋喃-2(3H)-酮(AG07)的制备：
参照AG02的制备方法，得无色固体，即为化合物AG07，其产量为68mg，收率为68.9％。1H NMR(500MHz,Chloroform-d)δ6.82(d,J＝1.9Hz,1H),6.74(d,J＝8.1Hz,1H),6.64(d,J＝2.0Hz,1H),6.63–6.55(m,2H),6.51(dd,J＝8.8,2.1Hz,1H),5.34(t,J＝4.5Hz,1H，1-H),4.49(t,J＝4.0Hz,1H),4.38(dd,J＝7.9,4.6Hz,1H),4.15(dt,J＝9.0,6.5Hz,1H),4.06(dt,J＝7.8,3.7Hz,1H),3.97(dd,J＝11.9,3.1Hz,1H),3.90(dt,J＝12.8,4.6Hz,1H),3.85(d,J＝5.2Hz,7H),3.82(d,J＝2.3Hz,3H),2.97–2.90(m,2H),2.70(dd,J＝13.4,6.3Hz,1H),2.57(dq,J＝8.5,5.1Hz,2H),2.50(q,J＝7.4Hz,2H).13C NMR(126MHz,CDCl3)δ178.26,148.53,147.50,145.87,140.46,130.01,128.77,122.13,120.38,119.86,112.02,111.02,110.65,81.93,78.72,72.67,72.06,70.88,62.05,55.73,55.61,55.46,46.19,40.52,37.71,34.18.HRMS:m/z for C26H32NaO10[M+Na]+calculated:527.1888,found:557.1885.
实施例5
本实施例中，按照上述反应式，制备本申请的二苄基丁内酯糖苷类化合物AG08和AG09：
中间体(2R,3R,4S,5R)-2-(4-(((3R,4R)-4-(3,4-甲氧基苄基)-2-氧代四氢呋喃-3- 基)甲基)-2-甲氧基苯氧基)四氢-2H-吡喃-3,4,5-三乙酸三酯(化合物7-1)和化合物 (2S,3R,4S,5R)-2-(4-(((3R,4R)-4-(3,4-甲氧基苄基)-2-氧代四氢呋喃-3-基)甲基)-2- 甲氧基苯氧基)四氢-2H-吡喃-3,4,5-三乙酸三酯(化合物8-1)的制备：
参照化合物3-1和化合物4-1的制备方法，以牛蒡子苷元和1,2,3,4-四-O-乙酰-β-D-吡喃木糖为起始原料，得化合物7-1，其为白色固体，产量为435mg，收率为33.5％；得化合物8-1，其为无色固体，产量为585mg，收率为45.8％。
化合物3R，4R-4-(3，4-二甲氧基苄基)-3-(3-甲氧基-4-(2R，3R，4S，5R)-3，4，5- 三羟基四氢-2H-吡喃-2-基)氧基苄基)二氢呋喃-2(3H)-酮(AG08)的制备：
参照AG02的制备方法，得无色固体，即为化合物AG08，其产量为230mg，收率为73.9％。1H NMR(500MHz,Chloroform-d)δ7.04(d,J＝8.0Hz,1H),6.76(dd,J＝8.3,2.1Hz,1H),6.68(d,J＝1.9Hz,1H),6.63(dd,J＝8.2,1.9Hz,1H),6.56(dd,J＝8.1,2.0Hz,1H),6.47(d,J＝2.0Hz,1H),5.24(d,J＝3.7Hz,1H,1-H),4.16(dd,J＝9.1,7.4Hz,1H),3.94–3.87(m,1H),3.85(s,3H),3.82–3.79(m,6H),3.79–3.77(m,4H),3.60(dd,J＝9.3,3.7Hz,1H),2.93(dd,J＝6.1,3.0Hz,2H),2.65–2.62(m,1H),2.59–2.55(m,2H),2.49–2.45(m,1H).13C NMR(126MHz,CDCl3)δ178.07,149.96,148.57,147.41,144.48,133.47,129.85,121.41,120.19,118.84,112.39,111.37,110.87,100.16(1-C),74.67,71.92,70.79,69.19,62.07,55.44,55.40,55.37,46.02,40.50,37.73,34.07.HRMS:m/z for C26H32NaO10[M+Na]+calculated:527.1888,found:557.1875.
化合物3R，4R-4-(3，4-二甲氧基苄基)-3-(3-甲氧基-4-(2S，3R，4S，5R)-3，4，5- 三羟基四氢-2H-吡喃-2-基)氧基)苄基二氢呋喃-2(3H)-酮(AG09)的制备：
参照AG02的制备方法，得无色固体，即为化合物AG09，其产量为320mg，收率为70.7％。1H NMR(400MHz,Chloroform-d)δ7.03(d,J＝8.1Hz,1H),6.79–6.69(m,2H),6.64(dd,J＝8.1,2.0Hz,1H),6.55(dd,J＝8.1,2.0Hz,1H),6.49(d,J＝2.0Hz,1H),4.91(d,J＝5.3Hz,1H,1-H),4.17(dd,J＝9.1,7.3Hz,1H),4.10(dd,J＝11.9,4.3Hz,1H),3.93–3.84(m,5H),3.83–3.78(m,6H),3.74–3.65(m,2H),3.40(dd,J＝12.0,7.9Hz,1H),2.99–2.90(m,2H),2.65(dd,J＝13.7,6.7Hz,1H),2.61–2.54(m,2H),2.50–2.42(m,1H).13C NMR(101MHz,CDCl3)δ178.56,150.22,149.08,147.94,144.26,133.93,130.31,121.80,120.63,118.83,113.05,111.92,111.43,102.54(1-C),74.53,72.11,71.28,69.41,64.57,55.93,55.90,55.89,46.50,41.03,38.20,34.54.HRMS:m/z for C26H32NaO10[M+Na]+calculated:527.1888,found:557.1888.
实施例6
本实施例中，按照上述反应式，制备本申请的二苄基丁内酯糖苷类化合物AG10：
中间体2S，3S，4R，5R，6S-2-(3R，4R)-4-(3，4-二甲氧基苄基)-2-氧代四氢呋喃-3- 基)甲基-2-甲氧基苯氧基-6-甲基四氢-2H-吡喃-3，4，5-三基三乙酸酯(化合物9-1)的制备：
参照化合物3-1和化合物4-1的制备方法，以牛蒡子苷元和1,2,3,4-四-O-乙酰基-α-L-岩藻吡喃糖苷为起始原料，得白色固体，即为化合物9-1，其产量为365mg，收率为67.7％。
化合物3R，4R-(3，4-二甲氧基苄基)-3-(3-甲氧基-4-(2S，3S，4R，5S，6S)-3，4， 5-三羟基-6-甲基四氢-2H-吡喃-2-氧基)苄基)二氢呋喃-2(3H)-酮(AG10)的制备：
参照AG02的制备方法，得白色固体，即为化合物AG10，其收率为73.6％。1H NMR(500MHz,Chloroform-d)δ7.07(d,J＝8.0Hz,1H),6.76(d,J＝8.0Hz,1H),6.72(d,J＝1.9Hz,1H),6.67–6.60(m,1H),6.57(dd,J＝8.1,2.0Hz,1H),6.50(d,J＝2.0Hz,1H),5.25(d,J＝3.7Hz,1H,1-H),4.27(q,J＝6.7Hz,1H),4.18–4.12(m,1H),4.02(dd,J＝9.6,3.1Hz,1H),3.94–3.88(m,3H),3.86(s,3H),3.82(s,3H),3.80(s,3H),2.99–2.90(m,2H),2.67–2.64(m,1H),2.61–2.55(m,2H),2.48(q,J＝7.7Hz,1H),1.34(d,J＝6.6Hz,3H).13C NMR(126MHz,CDCl3)δ178.05,150.11,148.60,147.47,144.94,133.41,129.83,121.46,120.13,119.06,112.33,111.39,110.93,100.83(1-C),71.00(2C),70.74,68.90,66.74,55.45, 55.39,55.28,46.03,40.50,37.66,34.00,15.74.ESI-MS:[M+Na]+:541.4.
实施例7
本实施例中，按照上述反应式，制备本申请的二苄基丁内酯糖苷类化合AG31：
化合物(2S，3R，4S，5R)-2-(4-(((3R，4R)-4-(3,4-二甲氧基苄基)-2-氧 代四氢呋喃-3-基)甲基)-2-甲氧基苯氧基)四氢-2H-吡喃-3,4,5-三基三乙酸酯(AG31) 的制备
将牛蒡子苷元(2.5g，6.71mmol)和1,2,3,4-四-O-乙酰-β-D-吡喃木糖(1.42g，4.48mmol)和烘干的分子筛溶于无水DCM(50mL)中，N2保护下-20℃缓慢滴加1mol/L三氟化硼乙醚溶液(0.448mL，0.448mmol)，保持-20℃低温反应12h。反应完成后旋干有机相，快速柱层析分离(PE:EA＝1:1)，得白色固体1.90g，即为化合物AG31，收率为67.4％。1H NMR(500MHz,Chloroform-d)δ6.99(d,J＝8.1Hz,1H),6.75(d,J＝8.1Hz,1H),6.69(d,J＝2.0Hz,1H),6.63(dd,J＝8.2,2.0Hz,1H),6.54(dd,J＝8.2,2.0Hz,1H),6.49(d,J＝2.0Hz,1H),5.25–5.17(m,2H),5.07(d,J＝5.7Hz,1H),5.04–4.98(m,1H),4.23(dd,J＝12.1,4.7Hz,1H),4.17(dd,J＝9.1,7.2Hz,1H),3.91(dd,J＝9.1,7.4Hz,1H),3.86(s,3H),3.82(s,3H),3.76(s,3H),3.48–3.45(m,1H),2.99–2.88(m,2H),2.68–2.57(m,2H),2.56–2.44(m,2H),2.11(s,3H),2.09(s,3H),2.08(s,3H).13C NMR(126MHz,CDCl3)δ179.00,170.24,170.05,169.64,150.75,149.01,147.87,144.59,133.96,130.29,121.57,120.59,119.48,113.49,111.88,111.41,100.00,71.44,70.63,70.21,68.65,61.87,55.91,55.91,55.87,46.53,41.05,38.16,34.55,20.79,20.75(2C).HRMS:m/z for C32H38NaO13[M+Na]+calculated:653.2205,found:653.2208.
实施例8
本实施例中，按照上述反应式，制备本申请的二苄基丁内酯糖苷类化合AG32：
化合物(2S，3R，4S，5R)-2-(4-(((3R，4R)-4-(3,4-二甲氧基苄基)-2-氧 代四氢呋喃-3-基)甲基)-2-甲氧基苯氧基)四氢-2H-吡喃-3,4,5-三基三(2-甲基丙酸酯) (AG32)的制备
将化合物AG09(60mg，0.119mmol)，吡啶(86.6μL，1.07mmol)加入无水二氯甲烷(2mL)中，N2保护，0℃下逐滴加入异丁酰氯(68.5μL，0.654mmol)，滴加完毕后缓慢升至室温反应12h。反应完成后用EA萃取，饱和食盐水洗涤。有机相旋干后快速柱层析分离(PE:EA＝1.5:1)，得白色固体51mg，即为化合物AG32，收率60.0％。1H NMR(600MHz,Chloroform-d)δ6.99(d,J＝8.1Hz,1H),6.75(d,J＝8.1Hz,1H),6.69(d,J＝2.0Hz,1H),6.63(dd,J＝8.1,2.0Hz,1H),6.53(dd,J＝8.1,2.0Hz,1H),6.49(d,J＝2.0Hz,1H),5.29(t,J＝8.4Hz,1H),5.23(dd,J＝8.7,6.5Hz,1H),5.08–5.01(m,2H),4.20(dd,J＝11.9,5.0Hz,1H),4.14(dd,J＝9.1,7.2Hz,1H),3.88(dd,J＝9.1,7.6Hz,1H),3.85(s,3H),3.82(s,3H),3.75(s,3H),3.43(dd,J＝11.9,8.4Hz,1H),2.99–2.89(m,2H),2.65(dd,J＝13.3,5.8Hz,1H),2.60–2.47(m,6H),1.17–1.11(m,18H).13C NMR(151MHz,CDCl3)δ178.62,175.95,175.93,175.37,150.70,149.05,147.91,144.67,133.89,130.31,121.49,120.56,119.44,113.49,111.89,111.40,100.37,71.26,70.78,70.27,68.53,62.30,55.93,55.88,55.79,46.48,41.08,38.14,34.52,33.92(2C),33.88,18.90,18.84,18.82(2C),18.73(2C).HRMS:m/z for C38H50NaO13[M+Na]+calculated:737.3144,found:737.3141.
实施例9
本实施例中，按照上述反应式，制备本申请的二苄基丁内酯糖苷类化合AG33：
化合物(3R，4R)-3-(4-(((2R，4aR，6S，7R，8R，8aS)-7,8-二羟基-2-苯基 六氢吡喃[3,2-][1,3]二氧杂环己烷-6-基)氧基)-3-甲氧基苄基)-4-(3,4-二甲氧基苄基) 二氢呋喃-2(3H)-酮(AG33)的制备
将牛蒡子苷(400mg，0.187mmol)溶于乙腈(5mL)中，加入苯甲酸二甲缩醛(225μL，0.374mmol)和(±)-10-樟脑磺酸(2mg，0.002mmol)，室温反应12h。反应完成后旋干乙腈，快速柱层析分离(PE:EA＝1:2)，得白色固体350mg，即为化合物AG33，收率75.1％。1H NMR(500MHz,Chloroform-d)δ7.52–7.46(m,2H),7.37(dd,J＝5.2,2.0Hz,3H),7.04(d,J＝8.1Hz,1H),6.76(d,J＝8.1Hz,1H),6.71(d,J＝2.0Hz,1H),6.65(dd,J＝8.1,2.0Hz,1H),6.56(dd,J＝8.1,2.0Hz,1H),6.49(s,1H),5.56(s,1H),4.79(d,J＝7.8Hz,1H),4.37(dd,J＝10.5,4.9Hz,1H),4.20(dd,J＝9.2,7.4Hz,1H),3.96–3.89(m,2H),3.88–3.74(m,11H),3.64(t,J＝9.3Hz,1H),3.53(td,J＝9.7,5.0Hz,1H),3.00–2.88(m,2H),2.70–2.57(m,3H),2.55–2.45(m,1H).13C NMR(126MHz,CDCl3)δ178.86,150.45,149.02,147.90,144.69,136.79,134.69,129.84,129.03,128.37,126.27,121.91,120.64,120.37,113.13,111.89,111.40,103.93,102.00,80.19,74.41,73.12,71.43,68.58,66.75,55.93(2C),55.88,46.55,41.01,38.21,34.59.
实施例10
本实施例中，按照上述反应式，制备本申请的二苄基丁内酯糖苷类化合AG14和AG15：
中间体四-O-乙酰基-L-吡喃鼠李糖(10-1)的制备
将L-鼠李糖(2g，11mmol)加入吡啶(10mL)中，0℃下缓慢滴加乙酸酐(5.2mL，54.9mmol)，升至室温后反应12h。反应完全后浓缩反应液，有机相用EA萃取，1N盐酸和饱和食盐水洗涤。合并有机相后旋干，柱层析分离，得无色透明油状物3.1g，即为中间体10-1，收率76.6％。
化合物(2S，3R，4R，5S，6S)-2-(4-(((3R，4R)-4-(3,4-二甲氧基苄基) -2-氧代四氢呋喃-3-基)甲基)-2-甲氧基苯氧基)-6-甲基四氢-2H-吡喃-3,4,5-三基三乙酸 酯(AG15)的制备
将牛蒡子苷元(500mg，1.34mmol)和中间体10-1(669mg，2.01mmol)和烘干的分子筛溶于无水DCM(10mL)中，N2保护下缓慢滴加1mol/L的三氟化硼乙醚溶液(111μL，1.01mmol)，室温反应12h。反应完成后旋干有机相，快速柱层析分离(PE:EA＝1:1)，得白色固体285g，即为化合物AG15，收率为32.9％。1H NMR(500MHz,Chloroform-d)δ6.98(d,J＝8.1Hz,1H),6.76(d,J＝8.1Hz,1H),6.71(d,J＝2.0Hz,1H),6.62(dd,J＝8.1,2.0Hz,1H),6.54(dd,J＝8.1,2.1Hz,1H),6.50(d,J＝2.0Hz,1H),5.58–5.49(m,2H),5.34(d,J＝1.8Hz,1H),5.15(t,J＝9.8Hz,1H),4.22–4.11(m,2H),3.93–3.87(m,1H),3.86(s,3H),3.83(s,3H),3.79(s,3H),3.00–2.89(m,2H),2.68–2.49(m,4H),2.18(s,3H),2.07(s,3H),2.03(s,3H),1.20(d,J＝6.3Hz,3H).13C NMR(126MHz,CDCl3)δ178.79,170.15(2C),170.11,150.83,149.02,147.88,144.06,133.78,130.31,121.53,120.55,118.89,113.53,111.86,111.38,97.58,71.32,71.07,69.79,68.99,67.36,55.92,55.88(2C),46.52,41.04,38.15,34.52,20.95,20.84,20.76,17.37.
化合物(3R，4R)-4-(3,4-二甲氧基苄基)-3-(3-甲氧基-4-(((2S，3R，5R， 6S)-3,4,5-三羟基-6-甲基四氢-2H-吡喃-2-基)氧基)苄基)二氢呋喃-2(3H)-酮(AG14) 的制备
将化合物AG15(100mg，0.077mmol)溶于甲醇(2mL)中，加入无水碳酸钾(27mg，0.193mmol)，室温反应2.5h。反应完成后，旋干有机相，柱层析分离(DCM:MeOH＝10:1)，得白色固体28mg，即为化合物AG14，收率为69.6％。1H NMR(500MHz,Chloroform-d)δ7.01(d,J＝8.1Hz,1H),6.75(d,J＝8.1Hz,1H),6.71(d,J＝1.9Hz,1H),6.62(dd,J＝8.1,2.0Hz,1H),6.55(dd,J＝8.1,1.9Hz,1H),6.49(d,J＝2.0Hz,1H),5.42(s,1H),4.23(s,1H),4.15(dd,J＝9.0,7.1Hz,1H),4.09–3.99(m,1H),3.94–3.86(m,2H),3.86–3.80(m,6H),3.77(s,3H),3.56(t,J＝9.3Hz,1H),2.93(qd,J＝14.1,5.9Hz,2H),2.67–2.47(m,4H),1.28(d,J＝6.2Hz,3H).13C NMR(126MHz,CDCl3)δ178.67,150.40,149.01,147.85,144.26,133.05,130.37,121.63,120.60,118.10,113.35,111.82,111.35,99.20,73.33,71.60,71.30,70.78,68.82,55.92(2C),55.86,46.52,41.07,38.19,34.55,17.45.HRMS:m/z for C27H34NaO10[M+Na]+calculated:541.2044,found:541.2046.
实施例11
本实施例中，按照上述反应式，制备本申请的二苄基丁内酯糖苷类化合AG16、AG17、AG24和AG25：
中间体四-O-乙酰基-D-吡喃来苏糖(11-1)的制备
参照中间体10-1的制备方法，以D-来苏糖为起始原料，透明油状物872mg，收率72.3％。1H NMR(500MHz,Chloroform-d)δ6.00(d,J＝3.4Hz,1H),5.37(dd,J＝9.0,3.5Hz,1H),5.25(t,J＝3.4Hz,1H),5.20(td,J＝8.9,4.9Hz,1H),4.01(dd,J＝11.6,5.0Hz,1H),3.71(dd,J＝11.5,8.8Hz,1H),2.16(s,3H),2.14(s,3H),2.08(s,3H),2.06(s,3H).
化合物(2R，3S，4S，5R)-2-(4-(((3R，4R)-4-(3，4-二甲氧基苄基)-2- 氧代四氢呋喃-3-基)甲基)-2-甲氧基苯氧基)四氢-2H-吡喃-3，4，5-三基三乙酸酯(AG17) 和化合物(2S，3S，4S，5R)-2-(4-(((3R，4R)-4-(3，4-二甲氧基苄基)-2-氧 代四氢呋喃-3-基)甲基)-2-甲氧基苯氧基)四氢-2H-吡喃-3，4，5-三基三乙酸酯(AG25) 的制备
参照化合物AG15的制备方法，以中间体11-1为起始原料，得α型糖苷与β型糖苷的混合物，经柱层析分离(PE:EA＝1.5:1)得118mg白色固体(AG17)与24mg白色固体(AG25)，收率分别为53.8％和10.8％。AG17的谱图：1H NMR(500MHz,Chloroform-d)δ6.97(d,J＝8.1Hz,1H),6.76(d,J＝8.1Hz,1H),6.69(d,J＝2.0Hz,1H),6.61(dd,J＝8.1,2.0Hz,1H),6.54(dd,J＝8.1,2.0Hz,1H),6.50(d,J＝2.1Hz,1H),5.57(dd,J＝9.8,3.4Hz,1H),5.52(dd,J＝3.5,2.3Hz,1H),5.37(d,J＝2.4Hz,1H),5.28(td,J＝ 9.8,5.8Hz,1H),4.16(dd,J＝9.2,7.3Hz,1H),3.97–3.87(m,3H),3.86(s,3H),3.83(s,3H),3.79(s,3H),2.98–2.87(m,2H),2.67–2.45(m,4H),2.17(s,3H),2.08(s,3H),2.06(s,3H).13C NMR(126MHz,CDCl3)δ179.04,170.35,170.12(2C),150.81,149.00,147.87,143.92,133.87,130.29,121.54,120.59,119.07,113.57,111.87,111.39,97.67,71.44,69.45,68.49,66.78,60.50,55.91,55.89,55.88,46.57,41.01,38.18,34.51,20.87,20.84,20.80.AG25的谱图：1H NMR(500MHz,Chloroform-d)δ6.98(d,J＝8.1Hz,1H),6.78(d,J＝8.2Hz,1H),6.74(d,J＝2.0Hz,1H),6.66(dd,J＝8.1,2.0Hz,1H),6.58(dd,J＝8.1,2.0Hz,1H),6.52(d,J＝2.1Hz,1H),5.46(t,J＝3.3Hz,1H),5.43(d,J＝3.1Hz,1H),5.30(dd,J＝5.9,3.5Hz,1H),5.07(ddd,J＝6.0,4.4,3.0Hz,1H),4.34(dd,J＝12.6,3.0Hz,1H),4.16(dd,J＝9.2,7.2Hz,1H),3.93–3.90(m,1H),3.88(s,3H),3.85(s,3H),3.80(s,3H),3.57(dd,J＝12.6,4.4Hz,1H),2.96(dd,J＝6.0,1.8Hz,2H),2.71–2.55(m,4H),2.20–2.15(m,6H),2.14(s,3H).13C NMR(126MHz,CDCl3)δ178.81,170.33,170.17,169.94,150.86,149.04,147.89,144.59,133.69,130.34,121.70,120.58,119.41,113.85,111.87,111.39,96.30,71.34,68.37,67.71,67.04,59.97,55.98,55.92,55.86,46.53,41.04,38.16,34.50,20.95,20.84,20.81.
化合物(3R，4R)-4-(3,4-二甲氧基苄基)-3-(3-甲氧基-4-(((2R，3S，4S， 5R)-3,4,5-三羟基四氢-2H-吡喃-2-基)氧基)苄基)二氢呋喃-2(3H)-酮(AG16)的 制备
参照化合物AG14的合成方法，以化合物AG17为起始原料，得白色固体28mg，即为化合物AG16，收率58.3％。1H NMR(500MHz,Chloroform-d)δ7.02(d,J＝8.1Hz,1H),6.76(d,J＝8.1Hz,1H),6.70(d,J＝2.0Hz,1H),6.63(dd,J＝8.1,1.9Hz,1H),6.56(dd,J＝8.1,2.0Hz,1H),6.48(d,J＝1.9Hz,1H),5.24(d,J＝4.0Hz,1H),4.18–4.13(m,2H),4.12–4.08(m,1H),3.95(tt,J＝7.5,3.6Hz,1H),3.91–3.86(m,2H),3.85(s,3H),3.82(s,3H),3.80–3.74(m,4H),2.93(d,J＝5.9Hz,2H),2.65(dd,J＝13.6,6.5Hz,1H),2.62–2.53(m,2H),2.52–2.44(m,1H).13C NMR(126MHz,CDCl3)δ178.65,150.44,149.03,147.88,144.48,133.62,130.35,121.74,120.63,118.88,113.22,111.83,111.31,100.54,71.39,71.31,69.90,68.06,64.19,55.92,55.89,55.87,46.53,41.04,38.21,34.52.HRMS:m/z for C26H32NaO10[M+Na]+calculated:527.1888,found:527.1890.
化合物(3R，4R)-4-(3,4-二甲氧基苄基)-3-(3-甲氧基-4-(((2S，3S，4S， 5R)-3,4,5-三羟基四氢-2H-吡喃-2-基)氧基)苄基)二氢呋喃-2(3H)-酮(AG24)的 制备
参照化合物AG14的合成方法，以化合物AG25为起始原料，得白色固体8mg，即为化合物AG24，收率53.3％。1H NMR(500MHz,Chloroform-d)δ7.00(d,J＝7.9Hz,1H),6.81–6.70(m,2H),6.63(d,J＝7.9Hz,1H),6.56(d,J＝8.0Hz,1H),6.49(s,1H),5.43(s, 1H),4.22–4.08(m,3H),4.05–3.95(m,2H),3.93–3.88(m,1H),3.86(s,3H),3.84–3.78(m,6H),3.54(d,J＝12.8Hz,1H),2.94(d,J＝5.4Hz,2H),2.72–2.41(m,4H).13C NMR(126MHz,CDCl3)δ178.55,150.11,149.07,147.94,143.82,133.60,130.30,121.71,120.61,117.36,112.80,111.85,111.36,100.10,71.66,71.29,69.88,65.60,60.61,55.94(2C),55.89,46.55,41.04,38.22,34.53.
实施例12
本实施例中，按照上述反应式，制备本申请的二苄基丁内酯糖苷类化合AG18和AG19：
中间体四-O-乙酰基-D-吡喃来苏糖(12-1)的制备
参照中间体10-1的制备方法，以D-阿拉伯糖为起始原料，得透明油状物3.9g，即为中间体12-1，收率76.5％。
化合物(2R，3S，4R，5R)-2-(4-(((3S，4S)-4-(3,4-二甲氧基苄基)-2-氧 代四氢呋喃-3-基)甲基)-2-甲氧基苯氧基)四氢-2H-吡喃-3,4,5-三基乙酸酯(AG19) 的制备
参照化合物AG15的制备方法，以中间体12-1为起始原料，得白色固体99mg，即为化合物AG19，收率16.5％。1H NMR(500MHz,Chloroform-d)δ6.94(d,J＝8.1Hz,1H),6.79(d,J＝8.1Hz,1H),6.73(d,J＝2.0Hz,1H),6.65(dd,J＝8.1,2.0Hz,1H),6.58(dd,J＝8.1,2.1Hz,1H),6.52(d,J＝2.0Hz,1H),5.71(d,J＝3.5Hz,1H),5.61(dd,J＝10.9,3.5Hz,1H),5.47(dd,J＝3.6,1.9Hz,1H),5.29(dd,J＝10.9,3.6Hz,1H),4.32(dd,J＝13.1,1.4Hz,1H),4.17(dd,J＝9.1,7.3Hz,1H),3.93–3.87(m,4H),3.85(s,3H),3.79(s,3H),3.76(d,J＝1.3Hz,1H),2.96(d,J＝6.0Hz,2H),2.69–2.52(m,4H),2.19(s,3H),2.15(s,3H),2.07(s,3H).13C NMR(126MHz,CDCl3)δ178.74,170.67,170.47,170.16,151.08,149.05,147.91,144.18,134.06,130.30,121.66,120.58,119.98,113.58,111.84,111.39,96.94,71.31,69.07, 68.32,67.07,61.27,55.92,55.86,55.78,46.50,41.02,38.16,34.48,20.97,20.85,20.78.
化合物(3R，4R)-4-(3,4-二甲氧基苄基)-3-(3-甲氧基-4-(((2R，3S，4R， 5R)-3,4,5-三羟基四氢-2H-吡喃-2-基)氧基)苄基)二氢呋喃-2(3H)-酮(AG18)的 制备
参照化合物AG14的合成方法，以AG15为起始原料，得19mg白色固体，即为化合物AG18，收率79.2％。1H NMR(500MHz,Chloroform-d)δ7.07(d,J＝8.1Hz,1H),6.76(d,J＝8.1Hz,1H),6.72(d,J＝2.1Hz,1H),6.63(dd,J＝8.1,2.0Hz,1H),6.57(dd,J＝8.1,2.1Hz,1H),6.50(d,J＝2.0Hz,1H),5.29(d,J＝3.7Hz,1H),4.19–4.08(m,3H),4.04(dd,J＝9.6,3.4Hz,1H),3.94–3.87(m,3H),3.86(s,3H),3.83(s,3H),3.81(s,3H),2.94(dt,J＝7.1,4.1Hz,2H),2.66(dd,J＝13.7,6.5Hz,1H),2.61–2.55(m,2H),2.51–2.43(m,1H).13C NMR(126MHz,CDCl3)δ178.56,150.60,149.06,147.93,145.27,134.04,130.29,121.95,120.60,119.65,112.81,111.82,111.36,101.53,71.26,70.66,69.72,68.76,63.27,55.92,55.86,55.77,46.52,40.98,38.17,34.48.HRMS:m/z for C26H32NaO10[M+Na]+calculated:527.1888,found:527.1891.
实施例13
本实施例中，按照上述反应式，制备本申请的二苄基丁内酯糖苷类化合AG20和AG21：
中间体(2S，3S，4S，5R，6S)-2-(碘甲基)-6-甲氧基四氢-2H-吡喃-3,4,5-三醇(13-1) 的制备
将甲基α-D-吡喃葡萄糖苷(3g，15.45mmol)，三苯基膦(6.08g，23.17mmol)和咪唑(2.10g，30.90mmol)溶于乙腈(30ml)中，分批加入碘颗(4.9g，19.31mmol)，加入完毕后升温至70℃反应10h。反应完成后加入三乙胺(3.1mL)淬灭反应，浓缩除 去乙腈。向反应混合物中加入水(6mL)于45℃搅拌45min，继续加入水(24mL)搅拌1h，有白色固体析出。抽滤，收集滤液，并将白色固体用水充分洗涤以收集固体中的残留糖液，合并滤液即为中间体13-1的水溶液。不经纯化直接将13-1的水溶液用于下一步反应。
中间体(2S，3R，4S，5S，6R)-2-甲氧基-6-甲基四氢-2H-吡喃-3,4,5-三醇(13-2) 的制备
将中间体13-1的水溶液使用N2保护，后加入次亚磷酸钙(1.14g，10.82mmol)与VA-044(500mg，1.55mmol)，升温至70℃反应1.5h。反应完成后冷却至室温，加入氢氧化钙(494mg，8.5mmol)搅拌30min使钙盐沉淀。滤出不溶性沉淀后，旋干滤液，得粗产物13-2，直接用于下一步反应。
中间体(2S，3R，4S，5R，6R)-2-甲氧基-6-甲基四氢-2H-吡喃-3,4,5-三乙酸三酯 (13-3)的制备
向中间体13-2中加入乙腈(15mL)，0℃下加入乙酸酐(8mL，84mmol)，三乙胺(11.7mL，84mmol)和二甲氨基吡啶(68mg，0.56mmol)，缓慢升至室温后反应12h。反应完成后浓缩反应液，乙酸乙酯萃取，先后用饱和碳酸氢钠、饱和亚硫酸钠和饱和食盐水洗涤。有机相旋干后柱层析分离(PE:EA＝4:1)，得无色透明油状物2.2g，即为中间体13-3，收率46.9％。1H NMR(500MHz,Chloroform-d)δ5.47–5.41(m,1H),4.91–4.84(m,2H),4.80(t,J＝9.6Hz,1H),3.92–3.86(m,1H),3.40(s,3H),2.08(s,3H),2.04(s,3H),2.01(d,J＝1.0Hz,3H),1.20(d,J＝6.3Hz,3H).
中间体1,2,3,4-四-O-乙酰基-6-脱氧-α/β-D-吡喃葡萄糖(13-4)的制备
0℃下，将13-3(1.5g，4.93mmol)溶于冰醋酸(935μL)中，加入等体积乙酸酐(935μL，9.86mmol)，逐滴加入浓硫酸(40μL，0.074mmol)，升至室温反应3h。反应完成后，冷却至0℃，用饱和碳酸氢钠调pH至近中性，EA萃取，有机相用饱和食盐水洗涤，旋干有机相，柱层析(PE:EA＝5:1)得白色固体1g，即为化合物13-4，为α和β构型混合物，收率61.1％。α异构型谱图：1H NMR(500MHz,Chloroform-d)δ6.21(d,J＝3.7Hz,1H),5.41–5.35(m,1H),5.01(dd,J＝10.3,3.7Hz,1H),4.82–4.78(m,1H),3.95(dq,J＝10.0,6.2Hz,1H),2.00(s,3H),1.97(s,3H),1.95(s,3H),1.15(d,J＝6.2Hz,3H).
中间体(3R，4S，5R，6R)-2-羟基-6-甲基四氢-2H-吡喃-3,4,5-三乙酸三酯(13-5) 的制备
将中间体13-4(550mg，1.65mmol)溶于四氢呋喃(5mL)后，加入乙酸肼(167mg，1.83mmol)，升温至50℃反应1.5h。反应完成后直接旋干，柱层析分离(PE:EA＝1:1)，得白色固体190mg，即为中间体13-5，收率39.6％。1H NMR(500MHz, Chloroform-d)δ5.51(dd,J＝10.2,9.5Hz,1H),5.42(t,J＝3.5Hz,1H),4.91(dd,J＝10.1,3.7,1H),4.86–4.82(m,1H),4.17–4.11(m,1H),2.93(dd,J＝3.5,1.2Hz,1H),2.10(s,3H),2.07(s,3H),2.04(s,3H),1.21(d,J＝6.3Hz,3H).
中间体(2R，3R，4S，5R)-2-甲基-6-(((三氯甲基)氨基)氧基)四氢-2H-吡 喃-3,4,5-三乙酸酯(13-6)的制备
将中间体13-5(190mg，0.66mmol)溶于无水二氯甲烷中，N2保护并降温至0℃，加入DBU(31.2μL，0.21mmol)，反应5min后逐滴加入三氯乙腈(197μL，1.96mmol)，缓慢升至室温反应12h。反应完成后直接旋干，柱层析分离(PE:EA＝20:1)得260mg黄色固体，即为中间体13-6，收率92.9％。1H NMR(500MHz,Chloroform-d)δ8.64(s,1H),6.49(d,J＝3.7Hz,1H),5.53(t,J＝9.9Hz,1H),5.10(dd,J＝10.2,3.8Hz,1H),4.93–4.86(m,1H),4.16–4.08(m,1H),2.06(s,3H),2.03(s,3H),2.01(s,3H),1.23(d,J＝6.2Hz,3H).
化合物(2R，3R，4S，5R，6R)-2-(4-(((3R，4R)-4-(3,4-二甲氧基苄基) -2-氧代四氢呋喃-3-基)甲基)-2-甲氧基苯氧基)-6-甲基四氢-2H-吡喃-3,4,5-三基三乙酸 酯(AG21)的制备
将牛蒡子苷元(110mg，0.295mmol)，中间体13-6(128mg，0.295mmol)和分子筛加入无水二氯甲烷(5mL)中，N2保护，0℃下缓慢加入三氟甲磺酸三甲基硅酯(8μL，0.004mmol)，缓慢升至室温反应12h。反应完成后快速柱层析(PE:EA＝1:1)得白色固体23mg，即为化合物AG21，收率12.1％。1H NMR(500MHz,Chloroform-d)δ6.91(d,J＝8.0Hz,1H),6.77(d,J＝8.2Hz,1H),6.71(d,J＝2.0Hz,1H),6.62(dd,J＝8.1,2.0Hz,1H),6.55(dd,J＝8.1,2.0Hz,1H),6.51(d,J＝2.1Hz,1H),5.68(dd,J＝10.3,9.4Hz,1H),5.59(d,J＝3.7Hz,1H),4.95(dd,J＝10.3,3.8Hz,1H),4.87(t,J＝9.7Hz,1H),4.30(dt,J＝10.0,6.3Hz,1H),4.15(dd,J＝9.2,7.4Hz,1H),3.91–3.85(m,4H),3.84(s,3H),3.76(s,3H),2.99–2.90(m,2H),2.67(dd,J＝13.6,6.2Hz,1H),2.63–2.53(m,2H),2.48(dt,J＝15.8,7.8Hz,1H),2.11(s,3H),2.08(s,3H),2.05(s,3H),1.18(d,J＝6.3Hz,3H).13C NMR(126MHz,CDCl3)δ178.93,170.50,170.39,170.10,151.14,149.05,147.92,144.20,134.20,130.31,121.69,120.58,120.43,113.73,111.88,111.42,96.11,73.78,71.36,71.24,69.93,66.09,55.93,55.90,55.82,46.56,40.96,38.13,34.40,20.79,20.76,20.76,17.12.
化合物(3R，4R)-4-(3,4-二甲氧基苄基)-3-(3-甲氧基-4-(((2R，3R，4S， 5S，6R)-3,4,5-三羟基-6-甲基四氢-2H-吡喃-2-基)氧基)苄基)二氢呋喃-2(3H)-酮 (AG20)的制备
参照化合物AG14的制备方法，以AG21为起始原料，得白色固体24mg，即为化合物AG20，收率80.6％。1H NMR(500MHz,Chloroform-d)δ6.99(d,J＝8.1Hz,1H),6.70(d,J＝8.1Hz,1H),6.63(d,J＝2.0Hz,1H),6.58(dd,J＝8.1,2.0Hz,1H),6.50(dd,J＝8.2, 2.0Hz,1H),6.42(d,J＝2.0Hz,1H),5.13(d,J＝3.8Hz,1H),4.10(dd,J＝9.2,7.4Hz,1H),3.95(dq,J＝9.6,6.3Hz,1H),3.87–3.81(m,2H),3.79(s,3H),3.76(s,3H),3.73(s,3H),3.53(dd,J＝9.5,3.8Hz,1H),3.23(t,J＝9.3Hz,1H),2.93–2.83(m,2H),2.62–2.56(m,1H),2.55–2.47(m,2H),2.45–2.38(m,1H),1.27(d,J＝6.3Hz,3H).13C NMR(126MHz,CDCl3)δ178.56,150.63,149.05,147.91,145.27,134.06,130.31,121.87,120.61,119.74,112.80,111.81,111.32,100.93,75.17,74.88,72.73,71.28,68.55,55.92,55.87,55.78,46.53,41.01,38.22,34.58,17.53.HRMS:m/z for C27H34NaO10[M+Na]+calculated:541.2044,found:541.2051.
实施例14
本实施例中，按照上述反应式，制备本申请的二苄基丁内酯糖苷类化合AG22和AG23：
中间体14-1至14-6的合成参照中间体13-1至13-6的合成方法，以α-甲基-D-甘露糖苷为起始原料。
化合物(2R，3S，4S，5R，6R)-2-(4-(((3R，4R)-4-(3,4-二甲氧基苄基) -2-氧代四氢呋喃-3-基)甲基)-2-甲氧基苯氧基)-6-甲基四氢-2H-吡喃-3,4,5-三基三乙酸 酯(AG23)的制备
参照化合物AG21的制备方法，以中间体14-1为起始原料，得白色固体150mg，即为化合物AG23，收率76.7％。1H NMR(500MHz,Chloroform-d)δ6.97(d,J＝8.1Hz,1H),6.75(d,J＝8.1Hz,1H),6.69(d,J＝2.0Hz,1H),6.63(dd,J＝8.1,2.0Hz,1H),6.53(dd,J＝8.1,2.1Hz,1H),6.50(d,J＝2.0Hz,1H),5.57–5.50(m,2H),5.35(d,J＝1.8Hz,1H),5.14(t,J＝9.9Hz,1H),4.21–4.10(m,2H),3.91–3.85(m,4H),3.83(s,3H),3.79(s,3H),3.00–2.88(m,2H),2.67–2.45(m,4H),2.18(s,3H),2.07(s,3H),2.02(s,3H),1.19(d,J＝6.3Hz,3H).
化合物(3R，4R)-4-(3,4-二甲氧基苄基)-3-(3-甲氧基-4-(((2R，3S，4S， 5S，6R)-3,4,5-三羟基-6-甲基四氢-2H-吡喃-2-基)氧基)苄基)二氢呋喃-2(3H)-酮 (AG22)的制备
参照化合物AG14的制备方法，以化合物AG23为起始原料，得白色固体60mg，即为化合物AG22，收率74.6％。1H NMR(500MHz,Chloroform-d)δ7.00(d,J＝8.1Hz,1H),6.75(d,J＝8.1Hz,1H),6.69(d,J＝2.0Hz,1H),6.63(dd,J＝8.1,2.0Hz,1H),6.55(dd,J＝8.2,2.0Hz,1H),6.49(d,J＝2.0Hz,1H),5.42(d,J＝1.6Hz,1H),4.23(dd,J＝3.5,1.7Hz,1H),4.18–4.10(m,1H),4.10–4.01(m,1H),3.89(ddd,J＝14.3,9.3,6.8Hz,2H),3.84(s,3H),3.82(s,3H),3.76(s,3H),3.58(q,J＝9.9,9.4Hz,1H),2.99–2.86(m,2H),2.67–2.45(m,4H),1.27(d,J＝6.3Hz,3H).13C NMR(126MHz,CDCl3)δ178.68,150.46,149.01,147.86,144.25,133.12,130.37,121.64,120.61,118.23,113.32,111.84,111.36,99.34,73.22,71.58,71.29,70.80,68.90,55.91(2C),55.88,46.52,41.11,38.17,34.55,17.46.
实施例15
本实施例中，按照上述反应式，制备本申请的二苄基丁内酯糖苷类化合AG26：
化合物(3R，4R)-4-(3,4-二甲氧基苄基)-3-(3-甲氧基-4-(((2S，3R，4S， 5R)-3,4,5-三甲氧基四氢-2H-吡喃-2-基)氧基)苄基)二氢呋喃-2(3H)-酮(AG26) 的制备
将化合物AG09(60mg，0.119mmol)与2,6-二叔丁基吡啶(252μL，1.14mmol)溶于1,2-二氯乙烷(2mL)中，升温至70℃反应2h。反应完成后直接旋干，柱层析分离(PE:EA＝2:1)得白色固体16mg，即为化合物AG26，收率24.6％。1H NMR(500MHz,Chloroform-d)δ6.98(d,J＝8.1Hz,1H),6.75(d,J＝8.1Hz,1H),6.71(d,J＝2.0Hz,1H),6.63(dd,J＝8.2,2.0Hz,1H),6.54(dd,J＝8.1,2.0Hz,1H),6.49(d,J＝2.0Hz,1H),4.84(d,J＝7.2Hz,1H),4.14(dd,J＝9.2,7.1Hz,1H),4.05(dd,J＝11.7,5.0Hz,1H),3.90–3.86(m,1H),3.86(s,3H),3.82(s,3H),3.80(s,3H),3.68(s,3H),3.64(s,3H),3.48(s,3H),3.38–3.30(m,2H),3.27–3.20(m,2H),2.99–2.90(m,2H),2.66(dd,J＝13.0,5.5Hz,1H),2.62–2.52(m,2H),2.51–2.46(m,1H).13C NMR(126MHz,CDCl3)δ178.69,150.34,149.03,147.87, 145.46,132.95,130.37,121.53,120.56,117.73,113.31,111.86,111.36,102.94,84.66,82.92,79.15,71.26,63.20,60.62,60.46,58.66,55.98,55.91,55.86,46.51,41.00,38.16,34.49.HRMS:m/z for C29H28NaO10[M+Na]+calculated:569.2357,found:569.2365.
实施例16
本实施例中，按照上述反应式，制备本申请的二苄基丁内酯糖苷类化合AG27和AG28：
化合物(2S，3R，4S，5R，6R)-2-(4-((((3R，4R)-4-(3,4-二甲氧基苄基) -2-氧代四氢呋喃-3-基)甲基)-2-甲氧基苯氧基)-6-甲基四氢-2H-吡喃-3,4,5-三基三乙酸 酯(AG28)的制备：
将牛蒡子苷元(100mg，0.268mmol)，中间体13-6(123mg，0.282mmol)和分子筛加入无水二氯甲烷(5mL)中，N2保护，0℃下缓慢加入三氟化硼乙醚(7μL，0.054mmol)，缓慢升至室温反应12h。反应完成后旋干，快速柱层析分离(PE:EA＝1:1)，得白色固体86mg，即为化合物AG28，收率49.7％。1H NMR(500MHz,Chloroform-d)δ7.01(d,J＝8.0Hz,1H),6.75(d,J＝8.1Hz,1H),6.67(d,J＝2.0Hz,1H),6.64(dd,J＝8.1,2.0Hz,1H),6.53(dd,J＝8.1,2.0Hz,1H),6.48(d,J＝2.0Hz,1H),5.26–5.22(m,2H),4.96–4.88(m,2H),4.17(dd,J＝9.1,7.2Hz,1H),3.90(dd,J＝9.1,7.3Hz,1H),3.85(s,3H),3.82(s,3H),3.75(s,3H),3.63(dq,J＝9.8,6.2Hz,1H),2.99–2.87(m,2H),2.65–2.48(m,4H),2.07(s,3H),2.05(s,3H),2.03(s,3H),1.27(d,J＝6.2Hz,3H).13C NMR(126MHz,CDCl3)δ178.80,170.51,169.78,169.59,150.77,149.02,147.88,145.09,134.12,130.27,121.52,120.56,119.88,113.55,111.88,111.40,100.62,73.27,72.69,71.65,71.38,70.22,55.98,55.93,55.88,46.50,41.08,38.18,34.61,20.70(3C),17.44.HRMS:m/z for C33H40NaO13[M+Na]+calculated:667.2361,found:667.2366.
化合物(3R，4R)-4-(3,4-二甲氧基苄基)-3-(3-甲氧基-4-(((2S，3R，4S， 5S，6R)-3,4,5-三羟基-6-甲基四氢-2H-吡喃-2-基)氧基)苄基)二氢呋喃-2(3H)-酮 (AG27)的制备：
参照化合物AG14的制备方法，以AG28为起始原料，得白色固体28mg，即为化合物AG27，收率68.2％。1H NMR(500MHz,Chloroform-d)δ7.04(d,J＝8.0Hz,1H),6.75 (d,J＝8.1Hz,1H),6.69(d,J＝1.9Hz,1H),6.64(dd,J＝8.1,1.9Hz,1H),6.54(dd,J＝8.0,1.9Hz,1H),6.48(d,J＝2.0Hz,1H),4.66(d,J＝7.5Hz,1H),4.22–4.13(m,1H),3.93–3.87(m,1H),3.85(s,3H),3.81(s,3H),3.80(s,3H),3.59(dt,J＝28.3,9.0Hz,2H),3.51–3.41(m,1H),3.31(t,J＝8.8Hz,1H),3.00–2.88(m,2H),2.69–2.63(m,1H),2.61–2.53(m,2H),2.52–2.47(m,1H),1.38(d,J＝6.0Hz,3H).13C NMR(126MHz,CDCl3)δ178.58,150.26,149.02,147.89,145.06,134.04,130.27,121.90,120.61,119.58,113.01,111.86,111.37,103.13,76.31,74.93,73.81,72.19,71.30,55.93(2C),55.89,46.51,40.98,38.20,34.55,17.70.HRMS:m/z for C27H34NaO10[M+Na]+calculated:541.2044,found:541.2054.
实施例17
本实施例中，按照上述反应式，制备本申请的二苄基丁内酯糖苷类化合AG29和AG30：
中间体6-溴-6-脱氧-1,2:3,5-二-O-异亚丙基-α-D-呋喃葡萄糖(15-1)的制备
0℃下，将NBS(6.8g，38.42mmol)溶于DMF(140mL)，加入三苯基膦(9.8g，37.46mmol)反应10min，后加入二丙酮-D-葡萄糖(5g，19.21mmol)反应3h，并升温至105℃继续反应2h。反应完成后，浓缩反应液，用EA萃取，有机相用5％碳酸氢钠、饱和食盐水洗涤。旋干有机相，柱层析分离(PE:EA＝10:1)得无水油状物3.5g，即为中间体15-1，收率56.4％。1H NMR(500MHz,Chloroform-d)δ6.00(d,J＝3.7Hz,1H),4.59(d,J＝3.6Hz,1H),4.33(dd,J＝7.2,3.9Hz,1H),4.24(d,J＝3.9Hz,1H),3.74(td,J＝7.3,3.3Hz,1H),3.62(dd,J＝10.9,3.2Hz,1H),3.44(dd,J＝10.9,7.4Hz,1H),1.50(s,3H),1.39(s,3H),1.37(s,3H),1.33(s,3H).
中间体(3aR，3bS，7aS，8aR)-2,2,5,5-四甲基-7-亚甲基四氢-7H-[1,3]二氧杂环[4'， 5':4,5]呋喃[3,2-d][1,3]二恶英(15-2)的制备
将中间体15-1(3.52g，10.89mmol)溶于甲苯(22mL)中，加入DBU(3.25mL，21.78mmol)，升温至80℃反应12h。反应完成后，滤出白色沉淀，将滤液旋干，柱层析分离(PE:EA＝10:1)得无色油状物2.3g，即为中间体15-2，收率87.16％。1H NMR(500MHz,Chloroform-d)δ6.01(t,J＝4.0Hz,1H),4.79(d,J＝0.9Hz,1H),4.71(d,J＝0.9Hz,1H),4.61–4.56(m,1H),4.42–4.35(m,2H),1.55(s,3H),1.50(s,3H),1.42(s,3H),1.35(s,3H).
中间体脱氧-1,2:3,5-二-O-异亚丙基-L-呋喃艾杜糖(15-3)的制备
将中间体15-2(2g，8.26mmol)溶于乙醇(30mL)中，加入10％钯碳(200mg)，氢气氛围下反应18h。反应完成后，滤出固体，滤液旋干后柱层析分离(PE:EA＝20:1)，得无色透明油状物888mg，即为中间体15-3，收率44.03％。
中间体2,3,4-四-O-乙酰基-6-脱氧-L-吡喃艾杜糖(15-4)的制备
将中间体15-3(888mg，3.64mmol)和离子交换树脂(320mg)加入水(10mL)中，回流反应18h。反应完成后，冷却至室温，抽滤，将滤液分别用乙醇与甲苯共沸，得黄色油状物。将黄色油状物溶于吡啶(8mL)中，0℃下缓慢滴加乙酸酐，滴加完毕后缓慢升至室温反应18h。反应完成后，反应液用EA萃取，饱和食盐水洗涤，旋干有机相后柱层析分离(PE:EA＝3:1)，得无色油状物662mg，即为中间体15-4，收率54.8％。
化合物(2S，3R，4S，5R，6S)-2-(4-((((3R，4R)-4-(3,4-二甲氧基苄基) -2-氧代四氢呋喃-3-基)甲基)-2-甲氧基苯氧基)-6-甲基四氢-2H-吡喃-3,4,5-三基三乙酸 酯(AG30)的制备
参照化合物AG31的制备方法，以中间体15-4为起始原料，得白色固体30mg，即为化合物AG30，收率15.8％。1H NMR(500MHz,Chloroform-d)δ7.01(d,J＝8.1Hz,1H),6.78(d,J＝8.1Hz,1H),6.74(d,J＝2.0Hz,1H),6.65(dd,J＝8.2,2.0Hz,1H),6.58(dd,J＝8.1,2.0Hz,1H),6.53(d,J＝2.0Hz,1H),5.45(dd,J＝2.2,0.9Hz,1H),5.17(ddd,J＝4.3,2.2,0.7Hz,1H),5.11–5.06(m,1H),4.91–4.86(m,1H),4.58(qd,J＝6.5,2.2Hz,1H),4.17–4.12(m,1H),3.92–3.89(m,1H),3.88(s,3H),3.85(s,3H),3.80(s,3H),2.97(d,J＝5.9Hz,2H),2.68–2.51(m,4H),2.18(s,3H),2.17(s,3H),2.15(s,3H),1.19(d,J＝6.6Hz,3H).13C NMR(126MHz,CDCl3)δ178.86,170.22,169.58,169.37,150.51,149.04,147.89,144.49,132.94,130.38,121.61,120.58,117.64,113.44,111.87,111.40,97.23,71.32,69.43,67.80,66.96,63.40,55.93,55.87(2C),46.56,41.05,38.14,34.48,20.89,20.80,20.76,15.60.HRMS:m/z for C33H40NaO13[M+Na]+calculated:667.2361,found:667.2368.
化合物(3R，4R)-4-(3,4-二甲氧基苄基)-3-(3-甲氧基-4-(((2S，3R，4S， 5S，6S)-3,4,5-三羟基-6-甲基四氢-2H-吡喃-2-基)氧基)苄基)二氢呋喃-2(3H)-酮 (AG29)的制备
参照化合物AG14的制备方法，以AG30为起始原料，得白色固体22mg，即为化合物AG29，收率72.3％。1H NMR(500MHz,Chloroform-d)δ7.00(d,J＝8.1Hz,1H),6.79–6.72(m,2H),6.64(dd,J＝8.2,2.0Hz,1H),6.56(dd,J＝8.2,2.0Hz,1H),6.50(d,J＝2.0Hz,1H),5.59(s,1H),4.26(qd,J＝6.6,1.6Hz,1H),4.14(dd,J＝9.1,7.2Hz,1H),4.05(d,J＝4.2Hz,1H),3.95(p,J＝1.5Hz,1H),3.89(dd,J＝9.1,7.7Hz,1H),3.86(s,3H),3.83(s,3H),3.80(s,3H),3.69–3.65(m,1H),3.00–2.90(m,2H),2.67–2.44(m,4H),1.18(d,J＝6.7Hz,3H).13C NMR(126MHz,CDCl3)δ178.62,149.85,149.05,147.91,142.71,132.85,130.35,121.49,120.59,116.03,112.78,111.84,111.36,99.19,71.78,71.28,69.44,67.87,63.38,55.93,55.86,55.80,46.53,41.14,38.19,34.60,15.92.HRMS:m/z for C27H34NaO10[M+Na]+calculated:541.2044,found:541.2053.
实施例18、通过本申请所述制备方法，实现了本申请化合物的立体选择性制备
牛蒡子苷β-D-木糖衍生物(即，化合物AG09)的立体选择性制备：
步骤一：将牛蒡子苷元(2.5g,6.71mmol)和1,2,3,4-四-O-乙酰-β-D-吡喃木糖(1.42g,4.48mmol)溶于无水DCM(50mL)中，N2保护，-20℃下缓慢滴加1mol/L三氟化硼乙醚溶液(0.448mL,0.448mmol),保持-20℃低温反应12h。反应完成后，旋干有机相，快速柱层析分离(PE:EA＝1:1)，得上图所示中间体化合物，其为白色固体，产量为1.90g，收率为67.4％。1H NMR(500MHz,Chloroform-d)δ7.02(d,J＝8.1Hz,1H),6.78(d,J＝8.1Hz,1H),6.72(d,J＝2.0Hz,1H),6.66(dd,J＝8.2,2.0Hz,1H),6.56(dd,J＝8.1,2.0Hz,1H),6.51(d,J＝2.1Hz,1H),5.28–5.20(m,2H),5.09(d,J＝5.7Hz,1H,1-H),5.03(td,J＝7.5,4.7Hz,1H),4.25(dd,J＝12.1,4.7Hz,1H),4.18(dd,J＝9.1,7.2Hz,1H),3.91(dd,J＝ 9.1,7.6Hz,1H),3.88(s,3H),3.85(s,3H),3.79(s,3H),3.48(dd,J＝12.1,7.8Hz,1H),3.02–2.91(m,2H),2.67(dd,J＝13.4,6.0Hz,1H),2.64–2.54(m,2H),2.53–2.46(m,1H),2.13(s,3H),2.11(s,3H),2.10(s,3H).
步骤二：参照AG02的制备方法，由步骤一所得中间体化合物制得白色固体1.55g，收率68.6％。产物经NMR与MS确证为单一的、具有β-糖苷键的化合物8(AG09)。
实施例19、本申请化合物对PDE4的抑制活性
本实施例中，将编码人源PDE4D催化结构域的cDNA序列(GenBank:NM_001197221.1；coding region:T86-S413)克隆到表达载体pET15b中，转化至Escherichia coli BL21(DE3)表达菌株，16℃低温诱导目的蛋白表达。5000转/分钟室温收集菌体后，重悬经高压破碎，以12000转/每分钟离心45min后取上清。通过Ni-NTA亲和层析柱，经50mM NaH2PO4(pH 7.5)、200mM NaCl和50mM Imidazole的缓冲液洗柱后，以含200mM咪唑的缓冲液洗脱得到目的蛋白后。进一步由阴离子亲和层析和分子排阻层析逐步获得高纯度目的蛋白PDE4D催化结构域(纯度>95％)，用于后续实验。
1.抑制PDE4D的IC50测定
采用临近闪烁实验(Scintillation Proximity Assay,SPA)方法测定本申请的一系列化合物(AG03、AG04、AG06、AG08、AG09、AG10)和阳性对照化合物Arctiin(AG01)对PDE4D催化结构域的酶活抑制效果(即，IC50值)。在实验中，使用分析缓冲液(50mM Tris pH 7.5、8.3mM MgCl2和1.7mM EGTA)制备浓度为0.2-0.4nM的蛋白质溶液。在总体积为125μL/孔的96孔板中进行反应。将待测化合物溶解在DMSO(最终浓度为1％(v/v))中，设置一系列浓度梯度，并将其以10μL的体积添加到板中，然后添加80μL蛋白质溶液和10μL[3H]-cAMP(0.5μCi/mL)。在30℃下孵育10分钟后，通过添加25μL磷酸二酯酶SPA珠(RPNQ0150，Perkineller Inc.)终止反应，适当振摇，静置20min后，使用微孔板闪烁计数器读数。并将抑制活性达50％时抑制剂的浓度定为IC50值。实验设至少2个复孔。
实验结果如表1所示。
表1本申请化合物对PDE4D的抑制活性
表1结果表明，本申请化合物对PDE4D具有强烈的抑制作用，显著优于阳性化合物Arctiin(AG01)。
特别是，化合物AG09对PDE4D的半抑制浓度曲线如图1所示，由图1可知，化合物AG09对PDE4D的半抑制浓度(IC50值)可达百纳摩尔，相较于阳性化合物Arctiin(AG01)提高了21倍之多。
2.在特定浓度下对于PDE4D的抑制率测定
采用与上述测定IC50值基本相同的方法(除了将待测化合物终浓度分别调整为5μM和0.5μM)，分别测定5μM和0.5μM下，本申请化合物AG15、AG17、AG26、AG27、AG28、AG29、AG31、AG32、AG33和阳性对照化合物Arctiin(AG01)对PDE4的抑制率。对PDE4D的抑制率的计算公式如下：
抑制率＝(对照组吸光度值-测试组吸光度值)/(对照组吸光度值-空白组吸光度值)×100％
实验结果如表2所示。
表2特定浓度下本申请化合物对PDE4D的抑制率
表2结果表明，本申请化合物对PDE4D具有极高的抑制率，特别是在5μM下，其抑制率均在90％以上，最高可达99.93％，接近100％；此外，无论在5μM还是在0.5μM下，本申请化合物对PDE4D的抑制率均显著优于阳性化合物Arctiin(AG01)。
结论：
以上结果表明，本申请化合物对PDE4D具有极高的抑制活性；由于PDE4D与PDE4A、PDE4B、PDE4C的催化结构域具有非常高的序列同一性，因此，可由上述结果获知，本申请化合物对PDE4A、PDE4B、PDE4C也均具有较高的抑制活性。
实施例20、本申请化合物对PBMC中TNF-α分泌的影响
PDE4广泛表达于免疫和炎症相关细胞，如中性粒细胞、嗜酸性粒细胞和单核细胞，使PDE4成为免疫及炎症相关疾病中主要的抗炎药物研究的重要靶点。PDE4通过调节 cAMP信号通路水平，改变炎症介质的表达，如TNF-α、干扰素-γ、IL-6和IL-12。脂多糖(Lipopolysaccharide,LPS)是革兰氏阴性菌胞壁的成份，可通过MAPK等多种信号通路显著激发致炎因子TNF-α的表达，从而评价PDE4部分抑制剂的细胞活性，模拟体外炎症效应。PDE4部分抑制剂对人PBMC细胞TNF-α表达的抑制效应主要参照George W.Muller等人的工作。
将冻存的人PBMC细胞解冻后，离心收集细胞沉淀，以6*106个/mL重新悬浮在含有RPMI1640和10％FBS新鲜培养基的96孔板中，并在37℃，5％CO2恒温箱中孵育3-4h。将梯度浓度的待测化合物(包括本申请的化合物AG04、AG06、AG08、AG09、AG10和阳性对照化合物Arctiin(AG01))加入人PBMC中，确保每孔DMSO的终浓度为0.1％。于37℃，5％CO2恒温箱中孵育1-1.5h。加入终浓度为1μg/mL的LPS，于37℃，5％CO2中孵育24h刺激细胞产生细胞因子。设置无刺激剂本底对照及刺激对照孔，总体积为200μl，DMSO终浓度为0.1％。收集上清液，用hTNF-αELISA试剂盒测定化合物对人PBMC细胞分泌TNF-α的抑制活性。使用GraphPad Prism 8.0版(GraphPad Inc.)对测定数据进行分析。化合物对人PBMC细胞分泌TNF-α的抑制活性以其对TNF-α分泌的半抑制浓度(即EC50值)表示。
此外，采用与上述测定EC50值基本相同的方法(除了将待测化合物终浓度分别调整为50μM和10μM)，测试了上述本申请化合物在50μM、10μM下对人PBMC细胞中炎症因子TNF-α分泌的抑制率。TNF-α采用双波长测定吸光值，以排除单波长检测时的测定干扰，经酶标仪分别检测OD450nm和OD570nm光度值(OD450nm为检测波长；OD570nm为参考波长)。得到的原始数据消除非特异性吸收(OD450nm-OD570nm)并根据标准曲线拟合公式转换成相应浓度后，再计算抑制率。化合物对人PBMC细胞分泌TNF-α的抑制率的计算公式如下：
TNF-α抑制率％＝(对照组吸光度值-测试组吸光度值)/(对照组吸光度值-空白组吸光度值)×100％。
各待测化合物对人PBMC细胞中炎症因子TNF-α分泌的抑制实验结果如表3所示。
表3化合物对PBMC细胞中炎症因子TNF-α分泌的抑制实验结果
表3结果表明，本申请的一系列化合物AG04、AG06、AG08、AG09、AG10均表现出显著的抑制人PBMC细胞分泌TNF-α的效应，特别是，在50μM浓度下，它们可以 明显抑制人PBMC中TNF-α的分泌，其对人PBMC分泌TNF-α的抑制率远远高于阳性对照化合物AG01。
并且，本申请化合物AG04、AG06、AG08、AG09、AG10抑制PBMC细胞分泌TNF-α的EC50值在3.51-24.04μM范围内，而阳性化合物AG01的EC50值则高达97.58μM，表明其对PBMC细胞分泌TNF-α的抑制效应显著弱于本申请的一系列化合物。
特别是，化合物AG09活性最优，其对人PBMC细胞中炎症因子TNF-α分泌的半抑制浓度曲线如图2所示，图2显示，化合物AG09对TNF-α分泌的半抑制浓度(EC50值)达3.51μM，为阳性对照化合物AG01的将近1/28，表明其对PBMC细胞分泌TNF-α的抑制活性为阳性对照化合物Arctiin(AG01)的约28倍，即，其抑制TNF-α分泌的活性明显优于Arctiin(AG01)。
此外，采用与上述相同的方法，还测试了本申请化合物AG09、AG26、AG27、AG28、AG29、AG31、AG32、AG33在5μM、1μM下对人PBMC细胞中炎症因子TNF-α分泌的抑制率，结果如以下表4所示。
表4化合物对PBMC细胞中炎症因子TNF-α分泌的抑制率
表4结果表明，本申请化合物AG09、AG26、AG27、AG28、AG29、AG31、AG32、AG33在5μM下也表现出对人PBMC细胞中炎症因子TNF-α分泌的极高的抑制率，特别是AG26、AG28、AG29、AG31、AG32、AG33；此外，部分本申请化合物(例如AG26、AG28、AG31、AG32、AG33)在1μM下也可以明显抑制人PBMC中TNF-α的分泌。
上述结果进一步证明，本申请化合物可以显著抑制人PBMC细胞中炎症因子TNF-α的分泌。
实施例21、本申请化合物对RAW264.7中TNF-α分泌的影响
肿瘤坏死因子(TNF-α)作为炎症、自身免疫病等疾病发展过程中重要的炎症介质，主要由活化的单核/巨噬细胞产生，可介导多种炎症反应的发生，加速恶化疾病进程。小鼠单核/巨噬细胞白血病细胞株RAW 264.7是常用的炎症细胞模型之一，在LPS诱导激活后，可释放TNF-α等多种炎症介质的释放。
将RAW 264.7细胞(1×105个/孔)接种于96孔板，孵育24h后，加入1μg/mL LPS诱导 RAW 264.7细胞极化后，加入不同浓度的待测化合物(包括本申请的一系列化合物和阳性对照化合物Arctiin(AG01)，于37℃，5％CO2培养箱中孵育18h。另设无刺激剂本底对照及刺激对照孔，总体积为200μL。300g/分钟，离心10min后收取上清，使用ELISA法检测培养上清中TNF-α的分泌水平。
实验结果如表5所示。
表5化合物对RAW 264.7细胞中炎症因子TNF-α分泌的抑制实验结果
表5结果表明，本申请化合物在100μM浓度下，可以明显抑制对RAW 264.7细胞中TNF-α的分泌，特别是化合物AG08、AG09，其活性优于Arctiin(AG01)将近2倍，提示：本申请化合物具有明显优于Arctiin(AG01)的抗炎效果。
实施例22、本申请化合物对银屑病样动物模型的治疗作用
1.实验材料：包含实施例5制得的AG09作为活性成分的软膏剂，所述软膏剂的制备方法如下：
(1)将液体石蜡15份，凡士林50份，单硬脂酸甘油酯50份，单硬脂酸100份，混合加热至70℃，得到油相A；
(2)将甘油100份，三乙醇胺2份，十二烷基硫酸钠2份，尼泊金乙酯1份溶于450份纯化水中，加热至70℃，加入40.5份AG09(即5％AG09)，迅速搅拌溶解，得水相B；
(3)将油相A缓慢加入水相B中，不断搅拌，制成AG09软膏剂，静置，冷却。
2.造模药物：咪喹莫特软膏，由四川明欣利迪药业有限责任公司生产，国药准字H20030128，产品批号15060139。
3.实验动物：BALB/c小鼠，雌性，体重18-22g，由上海斯莱克实验动物有限公司提供。
4.银屑病样模型造模方法：小鼠剔除背部毛发后，对其背部皮肤每日涂抹62.5mg咪喹莫特软膏，涂抹时间约每日7:30，连续进行8天，此时，银屑病样皮损处于较为严重的水平，如图3的模型对照组所示。
5.实验方法：
实验共分四组：
(1)正常对照组(即，正常健康小鼠)；
(2)模型对照组(即，未经任何治疗的银屑病样小鼠模型)；
(3)AG09软膏剂5％治疗组(即，经5％AG09软膏剂治疗的银屑病样小鼠模型)；
(4)AG09软膏剂2％治疗组(即，经2％AG09软膏剂治疗的银屑病样小鼠模型)，每组10只动物；
其中：AG09软膏剂5％和2％治疗组的处理方式如下：对银屑病样小鼠模型的背部皮肤每日分别涂抹5％AG09软膏剂和2％AG09软膏剂62.5mg，涂抹时间约为每日18:30，连续进行8天。
6.评价方法：
通过以下方法，评价AG09软膏制剂对银屑病样动物模型的治疗作用：
1)实验终点，对各组小鼠拍照，记录皮肤出现红斑、增厚、鳞屑现象；其结果如图3所示；
2)实验终点，取各组小鼠皮肤组织，以进行H&E染色；具体地，收集皮肤样品，将其固定于10％福尔马林组织固定液中，包埋于石蜡，切成3μm切片并进行H&E皮肤组织染色，在Olympus IX73显微镜观察皮肤表皮增厚及炎症细胞浸润等；其结果如图4所示；
3)实验终点，取各组小鼠皮肤组织，以制作病理切片(病理切片由扫描仪扫描组织进行切片)，并使用Image-Pro Plus软件(Silver Springs,MD,USA)对每个皮肤切片的表皮厚度进行定量；具体地，收集皮肤样品，将其固定于10％福尔马林组织固定液中，包埋于石蜡，切成3μm切片并进行H&E皮肤组织染色，使用Image-Pro Plus软件(Silver Springs,MD,USA)对每个皮肤切片的表皮厚度进行定量；其结果如图5所示。
图3-5结果表明：AG09软膏制剂(2％，5％)均能够有效缓解咪喹莫特诱导的小鼠银屑病样皮损，表现在：
I)如图3所示，模型对照组小鼠皮肤出现红斑、增厚、鳞屑现象，与模型对照组相比，外用AG09软膏制剂(2％，5％)对于咪喹莫特诱导银屑病小鼠皮肤的红斑、增厚及鳞屑具有明显的改善作用。
II)如图4所示，模型对照组小鼠皮肤角质形成细胞过度增殖、炎症细胞浸润明显，外用AG09软膏制剂(2％，5％)对表皮过度增殖和炎症细胞浸润有明显缓解。
III)如图5所示，与正常对照组相比，咪喹莫特诱导的银屑病样模型小鼠(即，模型对照组)表现出明显的表皮增厚，而用AG09软膏制剂(2％，5％)处理后，明显改善了这种表皮增厚，并且表现出剂量依赖效应。
上述结果均提示：本申请化合物AG09对于银屑病样小鼠模型具有明显的治疗效应，并且，随着活性化合物含量的增高，其治疗效力也明显增高。
最后应说明的是：以上实施例仅用以说明本申请的技术方案，而非对其限制；尽管参照前述实施例对本申请进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本申请各实施例技术方案的精神和范围。
工业实用性
本申请提供的如式(I)所示的二苄基丁内酯糖苷类化合物对四型磷酸二酯酶(PDE4)具有很强的抑制活性，并具有很强的抗炎活性，可以用于预防、治疗或辅助治疗与PDE4的活性水平和/或表达水平异常相关的疾病，特别是与其相关的免疫和炎症性疾病；此外，本申请提供的二苄基丁内酯糖苷类化合物的制备方法可以实现其立体选择性制备。

Claims (15)

  1. 如式(I)所示的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐：
    所述式(I)中，R选自由以下组成的组：单糖、单糖衍生物、由单糖单元通过糖苷键连接的双糖、由三至五个单糖单元通过糖苷键连接的寡糖；其中，所述单糖不包含D-β-葡萄糖，但所述单糖衍生物包含D-β-葡萄糖衍生物，并且，其中，所述R通过α或β型O-糖苷键与二苄基丁内酯类化合物主体相连。
  2. 根据权利要求1所述的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐，其特征在于，所述单糖选自：呋喃糖和吡喃糖；
    优选地，所述单糖为D型或L型。
  3. 根据权利要求1所述的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐，其特征在于，所述单糖衍生物选自由以下组成的组：氧化糖、卤代糖、不饱和糖、去氧糖、氨基去氧糖、脱水糖、硫酸化糖、磷酸化糖、歧链糖、糖基烷基化衍生物和糖基酯化衍生物；
    优选地，所述氧化糖选自单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地被氧化为醛或酸所得的单糖衍生物；
    优选地，所述卤代糖选自单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地被卤素取代所得的单糖衍生物，所述卤素任意地选自氟、氯、溴或碘；
    优选地，所述不饱和糖选自单糖或单糖衍生物上任意相邻两个羟基变为不饱和双键所得的单糖衍生物；
    优选地，所述去氧糖或氨基去氧糖选自单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地被替换为甲基所得的单糖衍生物；
    优选地，所述脱水糖选自单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地被替换为氢原子所得的单糖衍生物；
    优选地，所述硫酸化糖或磷酸化糖选自单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地被硫酸基或磷酸基修饰所得的单糖衍生物；
    优选地，所述歧链糖选自单糖或单糖衍生物上任意1，2，3，4或5个非端基碳原子上各自独立地有一个碳取代基所得的单糖衍生物，所述碳取代基可以置换为一个氢原子或一个羟基；
    优选地，所述糖基烷基化衍生物选自单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地被直链或支链的烷基修饰所得的单糖衍生物，所述烷基为C1-C10烷基或亚烷基、C1-C8卤代烷基、C3-C10环烷基、3-8元杂环烷基、C6-C10芳基、3-8元芳杂环基、3-8元杂环烷基C1-C8亚烷基、3-8元环烷基C1-C8亚烷基或C6-C10芳基C1-C8亚烷基；
    优选地，所述糖基酯化衍生物选自由单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地与直链或支链的酰基组成酯键所得的单糖衍生物，所述酰基各自独立地为C1～C10烷基酰基、C1-C8卤代烷基酰基、C3-C10环烷基酰基、3-8元杂环烷基酰基、C6-C10芳基酰基、3-8元芳杂环基酰基、3-8元杂环烷基C1-C8亚烷基酰基、3-8元环烷基C1-C8亚烷基酰基或C6-C10芳基C1-C8亚烷基酰基，优选为乙酰基、丙酰基、异丁酰基、苯甲酰基。
  4. 根据权利要求3所述的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐，其特征在于，所述单糖衍生物选自由以下组成的组：卤代糖、不饱和糖、糖基烷基化衍生物和糖基酯化衍生物；
    优选地，所述卤代糖选自单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地被氟、氯、溴、碘原子取代；
    优选地，所述不饱和糖选自单糖或单糖衍生物上任意相邻两个羟基变为不饱和双键所得的单糖衍生物；
    优选地，所述糖基烷基化衍生物选自单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地被甲基、乙基、环丙基、苄基或对甲氧基苄基所取代，或任意两个羟基通过亚甲基、亚乙基、异亚丙基、苯亚甲基组成环状缩醛或缩酮所得的单糖衍生物；
    优选地，所述糖基酯化衍生物选自由单糖或单糖衍生物上任意1，2，3，4或5个羟基各自独立地与乙酰基、丙酰基、异丁酰基或苯甲酰基组成酯键所得的单糖衍生物。
  5. 根据权利要求1所述的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐，其特征在于，所述R选自如下糖基或糖基衍生物：
    柔红霉糖、阿洛糖、阿卓糖、古洛糖、半乳糖、氨基半乳糖、氨基葡萄糖、甘露糖、 艾杜糖、塔洛糖、来苏糖、阿拉伯糖、核糖、木糖、岩藻糖、鼠李糖、异鼠李糖、橄榄霉糖、毛地黄毒糖、加拿大麻糖、奎诺糖、艾杜糖、6-脱氧艾杜糖、N-乙酰葡萄糖胺、N-乙酰半乳糖胺、果糖，或前述任意一项的衍生物，或D-β-葡萄糖衍生物。
  6. 根据权利要求1所述的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐，其为选自以下的化合物：





    优选地，所述化合物选自以下：



    进一步优选地，所述化合物选自以下：

    更进一步优选地，所述化合物为：


  7. 一种如权利要求1-6任一项所述的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐的制备方法，其特征在于，所述制备方法包括以下步骤：
    (1)牛蒡子苷元与乙酰基保护的糖，在氮气保护下，在催化剂作用下，经取代反应，形成乙酰基保护的牛蒡子糖苷化合物；
    (2)在碳酸钾或甲醇钠存在下，使步骤(1)所得乙酰基保护的牛蒡子糖苷化合物脱去乙酰基保护，得到所述二苄基丁内酯糖苷类化合物。
  8. 根据权利要求7所述的制备方法，其特征在于，步骤(1)中，所述催化剂选自：三氟甲磺酸三甲基硅酯、三氟甲磺酸、三氟化硼乙醚、三氯化铁，优选为三氟化硼乙醚；
    和/或，步骤(1)中，所述取代反应在溶剂中进行，所用溶剂选自：二氯甲烷、1,2-二氯乙烷、1,4-二氧六环，优选为二氯甲烷；
    和/或，步骤(1)中，所述取代反应在-60℃～0℃，优选-50℃～0℃的温度下进行；
    和/或，步骤(1)中，所述牛蒡子苷元与乙酰基保护的糖的摩尔比为0.05～3，优选为0.1～2；催化剂与牛蒡子苷元的摩尔比为0.008～1.2，优选为0.01～1。
  9. 根据权利要求7或8所述的制备方法，其特征在于，步骤(2)中，所用溶剂选自：二氯甲烷、丙酮、乙酸乙酯、甲醇、乙醇，优选为甲醇；
    和/或，步骤(2)中，反应温度为20～30℃，优选为室温。
  10. 根据权利要求7-9任一项所述的制备方法，其特征在于，步骤(1)中，所述乙酰基保护的糖具有如下结构式：
    所述制备方法按如下工艺路线：
    其中，Ra选自：H、-CH2OAc、-CH3；
    当Ra为H时，Rh也为H；当Ra为-CH2OAc时，Rh为-CH2OH；当Ra为-CH3时，Rh为-CH3。
  11. 一种药物组合物，其包括如权利要求1-6任一项所述的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐，以及药学上可接受的辅料。
  12. 根据权利要求11所述的药物组合物，其特征在于，所述二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐与药学上可接受的辅料的比重在0.001至100的范围内，优选在0.001至10范围内。
  13. 如权利要求1-6任一项所述的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐在制备磷酸二酯酶4抑制剂、优选磷酸二酯酶4D抑制剂中的用途。
  14. 如权利要求1-6任一项所述的二苄基丁内酯糖苷类化合物、其立体异构体、互变异构体或其药学上可接受的盐或者如权利要求11-12任一项所述的药物组合物在制备用于预防、治疗或辅助治疗与磷酸二酯酶4活性水平和/或表达水平异常相关的疾病的药物中的用途。
  15. 根据权利要求14所述的用途，其特征在于，所述磷酸二酯酶4为磷酸二酯酶4A、4B、4C或4D；
    优选地，所述疾病为与磷酸二酯酶4活性水平和/或表达水平异常相关的免疫和/或炎症性疾病；
    优选地，所述疾病为与磷酸二酯酶4A、4B、4C或4D活性水平和/或表达水平异常相关的免疫和/或炎症性疾病；
    进一步优选地，所述疾病选自由以下组成的组：银屑病、银屑病关节炎、特应性皮炎、白塞氏病、脂溢性皮炎、过敏性皮炎、慢性阻塞性肺病、哮喘、过敏性鼻炎、强直性脊柱炎、系统性红斑狼疮、风湿性关节炎、类风湿性关节炎、炎症性肠病、恶性胶质瘤、肺纤维化、肌萎缩性侧索硬化症、多发性硬化症、阿尔兹海默症、亨廷顿舞蹈症、帕金森氏症、多动症、抑郁症和精神分裂症。
PCT/CN2023/116894 2022-09-05 2023-09-05 二苄基丁内酯糖苷类化合物、其制备方法和应用 WO2024051666A1 (zh)

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