CN113980071B - Salidroside derivatives and their applications - Google Patents

Abstract

The invention provides a salidroside derivative and application thereof, wherein the derivative has the following structure:according to the invention, a plurality of series of glycoside analogues are extended on the basis of the Salidroside (Salidroside) structure, so that the medicinal activity of the Salidroside is effectively improved.

Description

Salidroside derivatives and their applications

Technical Field

The present invention belongs to the field of pharmaceutical chemistry, and in particular, relates to a salidroside derivative and application thereof.

Background Art

Peripheral Artery Disease refers to the blockage of arteries due to atherosclerosis and other diseases, which limits the blood supply to tissues outside the heart. Limb Ischemia is the most common peripheral artery disease. It is caused by blood vessel blockage, thrombosis, high blood sugar and other reasons, resulting in insufficient blood supply to the distal (lower limbs). Since oxygen is transported to various tissues and organs by red blood cells in the blood, insufficient blood supply results in hypoxia and malnutrition of lower limb tissues. In severe cases, it can lead to tissue gangrene, tissue loss and even death of the individual.

Currently, there is no effective treatment or control method for lower limb ischemic disease. In severe cases, amputation is required, which causes great pain and loss to patients. The treatment method currently considered to have a good effect is to promote angiogenesis and improve distal blood supply, thereby preventing tissue necrosis and improving lower limb function.

There are currently first-generation and second-generation therapeutic drugs for lower limb ischemic diseases. Among them, the first-generation therapeutic drugs use a single angiogenesis factor (vascular endothelial growth factor (VEGF) etc.), but the clinical trial results are not ideal, the new blood vessels are immature, leaky, and lack functionality (see: Therapeutic angiogenesis for critical limbischemia. Nature Reviews Cardiology, 2013, 10 (7): 387-96). The reason for the unsatisfactory treatment results is believed to be that vascular remodeling is a complex process involving multiple factors. The second-generation therapeutic drugs use a combination of multiple angiogenesis factors (fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGF); VEGF and angiopoietin-1 (ANG1)). Although they have certain effects, there are many types of angiogenesis factors, and they play different roles in different stages of vascular remodeling. Therefore, there are difficulties in selecting the type of angiogenic factors, the ratio when combining, when to administer the drugs, etc. In addition, current treatments for lower limb ischemic diseases also face the problem that exogenous angiogenic factors are localized and therefore insufficient to induce sufficient new mature blood vessels in a wide area of ischemia and hypoxia.

Diabetic foot is one of the most common and serious complications of diabetes. Due to poor blood sugar control, peripheral vascular disease in the lower limbs occurs, which causes insufficient blood supply to the lower limbs and hypoxia of the lower limb tissue cells. In addition, the ability of tissue repair and wound healing is significantly reduced under high sugar conditions. In severe cases, tissue gangrene, tissue loss, and even death may occur. Clinically, severe patients often require amputation. The ideal treatment for diabetic foot is to improve blood supply. Currently, the methods for treating vascular lesions include the use of stents, bypass surgery, and balloon dilatation. In addition, recently, due to its advantages such as non-invasiveness, promoting vascular remodeling is considered to be the best treatment approach.

In the preliminary study of the present invention, a drug for treating lower limb ischemic disease has been found, which contains salidroside as an active ingredient. In addition, in the preliminary study of the present invention, it has been found that in the diabetic foot model of mice, salidroside specifically promotes the expression and secretion of angiogenesis factors under high sugar and hypoxic conditions. The relevant research results have been granted Chinese authorized patents CN105687216B and CN105535001B. However, how to further improve salidroside in the treatment of diabetes and lower limb ischemic disease is still a technical problem to be solved in this field.

The hypoxia stress protein hypoxia inducible factor-1α (HIF-1α) is a key factor for cells and organisms to cope with hypoxia/hypoxia. HIF-1a is hydroxylated by the prolyl hydroxylase domain family (PHD family; including PHD1, PHD2 and PHD3) under normoxic conditions using oxygen as a substrate, and the hydroxylated HIF-1a will be degraded through the ubiquitination/protease degradation pathway, causing it to be at a low expression level under normoxic conditions. Under hypoxia/hypoxia conditions, the hydroxylation induced by the PHD family decreases, resulting in the stabilization of HIF-1a protein. The accumulation of HIF-1a protein promotes the increase in the expression of its downstream factors, which include angiogenesis factors such as VEGF and PDGF-BB, as well as other hypoxia stress-related factors such as EPO (erythropoietin) and HO-1 (see: Hypoxia-inducible factors in physiology and medicine. Cell, 2012, 148: 399–408). Therefore, the increase of HIF1-a protein is conducive to promoting angiogenesis and thus treating or improving lower limb ischemic diseases. In addition, the increase of red blood cells induced by EPO is conducive to increasing oxygen carrying capacity, thereby alleviating diseases and physical discomfort caused by hypoxia such as altitude sickness together with angiogenesis (see: Regulation of erythropoiesis by hypoxia-inducible factors. Blood Reviews, 2013, 27(1): 41-53).

Summary of the invention

The purpose of the present invention is to improve the activity of salidroside in treating diabetic foot and lower limb ischemic diseases, and the present invention is achieved by extending a series of glycoside analogs based on the structure of salidroside. In order to achieve the purpose of the present invention, the present invention intends to adopt the following technical solutions:

One aspect of the present invention relates to a salidroside derivative, wherein the derivative has the following structure:

Wherein R ₁ ＝H, OH, OMe, F;

R ₂ ＝H, OH;

R ₃ , R ₄ , R ₅ , and R ₆ are all hydroxyl groups, or any one of these four substituents is H or -OMe;

A＝-(CH ₂ )n-, n＝2 or 3;

-O-(CH ₂ )n-, n=2 or 3;

-O-(CH ₂ CH(CH ₃ ) ₂ )-, -O-(CH ₂ CH ₂ CH(CH ₃ ) ₂ )-, -O-(CH ₂ C(CH ₂ ) ₃ )-,

-C ₆ H ₄ -CH ₂ -, -OC ₆ H ₄ -, -OC ₆ H ₄ -CH ₂ -;

In a preferred embodiment of the present invention, A is -OC ₆ H ₄ -, -OC ₆ H ₄ -CH ₂ -, and R ₁ and R ₂ are located at the ortho position, meta position and/or para position of A on the benzene ring.

In a preferred embodiment of the present invention, R ₁ is -OMe, R ₂ is H, and R ₁ is located at the meta position and/or para position relative to X on the benzene ring.

In a preferred embodiment of the present invention, the derivative is selected from any one of C-1 to C-33.

In a preferred embodiment of the present invention, the derivative is selected from C-30 or C-31.

Another aspect of the present invention also relates to the use of the above derivatives in the preparation of HIF-1α inducers.

Another aspect of the present invention also relates to the use of the above derivatives in the preparation of drugs for treating ischemic diseases and/or relieving altitude sickness.

In a preferred embodiment of the present invention, the derivative is used to promote the expression level of HIF-1α.

In a preferred embodiment of the present invention, the derivative is used to promote the expression level of HIF-1α protein.

In a preferred embodiment of the present invention, the derivative is used to promote the expression level of HIF-1α by inhibiting the expression level of PHD3.

In a preferred embodiment of the present invention, the derivative is used to promote the expression level of HIF-1α protein by inhibiting the expression level of PHD3.

In a preferred embodiment of the present invention, the medicament is used for angiogenesis.

In a preferred embodiment of the present invention, the medicament is used for blood perfusion restoration.

In a preferred embodiment of the present invention, the drug is used to promote skeletal muscle cell activity under hyperglycemic and non-hyperglycemic conditions.

In a preferred embodiment of the present invention, the drug is used to enhance the secretion and migration potential of angiogenic factors in skeletal muscle cells under hyperglycemic and non-hyperglycemic conditions.

In a preferred embodiment of the present invention, the drug is used to enhance the angiogenesis ability of skeletal muscle cells under hyperglycemic and non-hyperglycemic conditions.

In a preferred embodiment of the present invention, the drug is used to enhance the proliferation and migration ability of vascular endothelial cells and smooth muscle cells under hyperglycemic and non-hyperglycemic conditions.

In a preferred embodiment of the present invention, the drug is an intramuscular injection preparation.

Another aspect of the present invention also relates to the use of the above derivatives in preparing medicines for lower limb ischemic diseases.

Effects of the Invention

The present invention effectively improves the pharmaceutical activity of salidroside by modifying the structure of salidroside.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: (A) Schematic diagram of structural modification of each part of salidroside. (B) First round of screening using 5×HRE-Luc reporter gene activity detection. (C) Second round of screening of compounds with greater activity than salidroside in the first round of 5×HRE-Luc reporter gene screening to investigate their activity against the PDG-B promoter-driven firefly luciferase reporter gene (PDGF-B-Luc). (D) Western blotting to detect the protein expression levels of angiogenic factors in C2C12 cells treated with SA, C-30 or C-31 at the same concentration (300 μM). Luciferase reporter gene activity was calculated as the ratio of the chemiluminescence value of firefly luciferase to that of Renilla luciferase. The relative luciferase reporter gene activity was obtained by normalizing the luciferase reporter gene activity of cells treated with each compound to that of cells in the control group. β-Actin was used as the loading reference for western blotting. All experiments were performed under hyperglycemia and data are presented as mean ± SD (n = 3). SA relative to blank control, ##P<0.01; analogs relative to SA, *P<0.05; **P<0.01; NS: no significant difference; SA: Salidroside.

Figure 2: C-30 and C-31 can promote skeletal muscle cell viability under hyperglycemia. (A) Total number of C2C12 cells treated with different concentrations of C-30 and C-31 at different time points under hyperglycemia (n=3). (B and C) EdU incorporation assay to detect the proliferation of C2C12 cells treated with different concentrations of C-30 under hyperglycemia. (B) Representative images, scale bar: 200 μm. (C) Relative cell proliferation rate was obtained by comparing the ratio of EdU-positive cells to Hoechst-positive cells with that of the control group (n=6). (D and E) EdU incorporation assay to detect the proliferation of C2C12 cells treated with different concentrations of C-31 under hyperglycemia. (D) Representative images, scale bar: 200 μm. (E) Relative cell proliferation rate was obtained by comparing the ratio of EdU-positive cells to Hoechst-positive cells with that of the control group (n=6). (F and G) Apoptosis rate of C2C12 cells treated with C-30 and C-31 (final concentration: 15 μM and 30 μM) under hyperglycemic conditions, Annexin-V/PI double staining, and flow cytometry analysis results. (F) shows representative images and (G) quantitative results (n = 3). Data are expressed as mean ± SD. Con: control group cells; **P < 0.01; *P < 0.05; NS: no significant difference.

Figure 3: C-30 increases the total number of skeletal muscle cells under hyperglycemia. Total number of C2C12 cells after treatment with different concentrations of C-30 and salidroside under hyperglycemia. Data are presented as mean ± SD (n = 3). H-Glu: blank group cells cultured under hyperglycemia. **P < 0.01; NS: no significant difference.

Figure 4: C-30 increases skeletal muscle cell proliferation and inhibits apoptosis under normoglycemic conditions. (A and B) EdU-incorporation assay to detect the proliferation capacity of C2C12 cells treated with 30 μM C-30 under normoglycemic conditions. Representative images (scale: 200 μm: A), statistical results of the ratio of EdU-positive cells to Hoechst-positive cells (n=6; B). (C and D) Annexin-V/PI-staining, flow cytometric analysis of the apoptosis rate of C2C12 cells treated with 30 μM C-30 under normoglycemic conditions. Representative images (C) and statistical results (n=3, D). Data are expressed as mean ± SD. N-Glu: C2C12 cells cultured under normoglycemic conditions. **P<0.01.

Figure 5: Transcriptome analysis of skeletal muscle cells treated with C-30 under high glucose. (A) RNA sequencing analysis results of C2C12 treated with 30μM C-30 for 12 hours under high glucose conditions, heat map represents the results of differential gene set clustering analysis. (B) Gene ontology (GO) enrichment analysis of up-regulated genes. (C) KEGG pathway enrichment analysis of up-regulated genes. (D) Fold change of key genes for angiogenesis. (E and F) Validation of 30μM C-30 on the expression of key angiogenic factors in C2C12 cells. (E) mRNA expression levels were analyzed by qRT-PCR, and (F) protein levels were determined by western blotting. β-Actin was used for qRT-PCR normalization and as an internal reference control for western blotting. Data are expressed as mean ± SD (n = 3). **P < 0.01.

Figure 6: C-30 enhances the secretion and migration potential of angiogenic factors in skeletal muscle cells under hyperglycemia. (A) Western blotting to detect the protein expression levels of angiogenic factors in C2C12 cells treated with 30 μM C-30 or salidroside. (B and C) ELISA was used to detect the secretion of VEGF-A (B) and PDGF-BB (C) in the culture medium of C2C12 cells treated with 30 μM C-30 or salidroside under high glucose conditions. (D and E) Phalloidin staining to analyze the F-actin polymerization in C2C12 cells treated with 30 μM C-30 or salidroside under hyperglycemia; (D) Representative images (scale bar: 100 μm or 50 μm); (E) Fractal dimension quantitative analysis (n=6). (F and G) Transwell chamber assay to detect the migration of C2C12 cells treated with 30 μM C-30 or salidroside. (F) Representative images, scale bar: 100 μm; (G) Statistical analysis of the number of cells that migrated to the lower chamber of the transwell (n=6). Data are expressed as mean ± SD (n=3). N-Glu: cells cultured with blank solvent under normoglycemic conditions; H-Glu, H-Glu+SA, H-Glu+C-30: cells cultured with blank solvent, 30 μM salidroside, or 30 μM C-30 under hyperglycemic conditions. **P<0.01; *P<0.05.

Figure 7: Knockdown efficiency of shRNA targeting HIF-1α. (A) mRNA expression level of HIF-1α in C2C12 cells transfected with shHIF-1α expression plasmid. (B) Protein expression level after shHIF-1α expression plasmid transfected C2C12 cells. Representative images (left) and quantitative statistical results (n=3; right). β-Actin was used as an internal reference for qRT-PCR and a control for protein immunoblotting experiments. Data are presented as mean ± SD (n=3). **P<0.01.

Figure 8: HIF-1α is essential for C-30 to enhance the angiogenesis of skeletal muscle cells under hyperglycemia. (A) Western blotting to detect the expression levels of HIF-1α protein and angiogenic factors in C2C12 cells with HIF-1α knockdown treated with 30μM C-30. The left side is a representative image, and the right side is the quantitative result (n=3). (B and C) EdU-incorporation method to detect the proliferation ability of C2C12 cells with HIF-1α knockdown treated with 30μM C-30. B is a representative image (scale: 200μm), and (C) is the quantitative analysis result (n=6). (D and E) Annexin-V/PI double staining followed by flow cytometry analysis of the apoptosis rate of C2C12 cells with HIF-1α knockdown treated with 30μM C-30. D is a representative image, and E is the quantitative analysis result (n=3). (F and G) Transwell assay to detect the migration ability of C2C12 cells after HIF-1α knockdown treated with 30μM C-30. (F) is a representative image (scale bar: 100μm), and (G) shows the statistical results of the number of cells that migrated to the lower chamber of the transwell (n=6). Data are expressed as mean ± SD (n=3). Con: C2C12 cells transfected with shCon; C-30: C2C12 cells treated with 30μM C-30; shHIF-1α: C2C12 cells transfected with anti-HIF-1α shRNA expression vector; **P<0.01; *P<0.05.

Figure 9: C-30 inhibits the expression level of PHD3. (A) qRT-PCR was used to analyze the mRNA expression levels of PHD1, PHD2 and PHD3 in C2C12 cells treated with 30 μM salidroside or C-30. (B) After skeletal muscle cells were treated with 30 μM salidroside and C-30, the protein expression levels of HIF-1α, PHD1, PHD2 and PHD3 were detected by western blotting. β-Actin was used as the internal control for qRT-PCR normalization and protein loading. Data are expressed as mean + SD difference, (n = 3) *P < 0.05; **P < 0.01; NS: no significant difference.

Figure 10: Preparation of skeletal muscle cell conditioned medium. (A) Schematic diagram of preparation of C2C12 cell conditioned medium (CM-N) treated with blank solvent under normal blood glucose culture conditions. (B) Schematic diagram of preparation of C2C12 cell conditioned medium (CM-H), (CM-H/SA) or (CM-H/C-30) treated with blank solvent, 30 μM SA or 30 μM C-30 under high blood glucose culture conditions.

Figure 11: C-30 treatment of skeletal muscle cells secretes and enhances the proliferation and migration of endothelial cells and smooth muscle cells under hyperglycemia. (A-D) The proliferation capacity of HUVECs (A and B) and MOVAS cells (C and D) cultured with CM-H/C-30 under hyperglycemia was detected by EdU-incorporation method. (A and C) are representative images (scale bar: 200 μm), (B and D) are the relative values of the ratio of EdU-positive cells to Hoechst-positive cells compared with the control group (n=6). (E-H) Phalloidin staining was used to analyze F-actin polymerization in HUVECs (E and F) and MOVAS cells (G and H) cultured with CM-H/C-30 under hyperglycemia. (E and G) are representative pictures (scale bar: 100 μm, inset 50 μm); (F and H) are quantitative analysis of F-actin fractal dimension (n=6). (I-L) Transwell chamber assay was used to detect the migration of HUVECs (I and J) and MOVAS cells (K and L) cultured in CM-H/C-30 under hyperglycemia. (I and K) are representative images (marker: 100m); (J and L) are statistical analysis of the number of cells that migrated to the lower chamber of the transwell chamber (n=6). Data are expressed as mean ± SD, (n=3). CM-N: conditioned medium obtained from C2C12 cells treated with blank solvent under normoglycemic conditions; CM-H, CM-H/SA, CM-H/C30: conditioned medium obtained from C2C12 cells treated with blank solvent, salidroside, and C-30, respectively, under hyperglycemic conditions. **P<0.01; *P<0.05; NS: no significant difference.

Figure 12: HIF-1α is essential for the role of C-30 in promoting the proliferation and migration of endothelial cells and smooth muscle cells under hyperglycemia through secretion from skeletal muscle cells. (A-D) The proliferation potential of HUVEC (A and B) and MOVAS cells (C and D) cultured with conditioned medium from C2C12 cells with HIF-1α silenced under hyperglycemia was determined by EdU incorporation. Representative images (scale bar: 200 μm; A and C) and the ratio of EdU-positive cells to Hoechst-positive cells (n=6; B and D). (E-H) The migration potential of HUVEC (E and F) and MOVAS cells (G and H) cultured with conditioned medium from C2C12 cells with HIF-1α silenced under hyperglycemia was determined by transwell migration assay. Representative images (scale bar: 100 μm; E and G) and the number of cells that migrated to the subchamber of the transwell chamber (n=6; F and H). CM-H/shCon and CM-H/shCon+C-30: conditioned medium obtained from C2C12 cells treated with blank solution and C-30, respectively; CM-H/shHIF-1α and CM-H/shHIF-1α+C-30: conditioned medium obtained from HIF-1α-silenced C2C12 cells treated with vehicle and C-30, respectively; data are expressed as mean ± SD. *P < 0.05; **P < 0.01; NS: not significant.

Figure 13: VEGF-A and PDGF-BB are essential for the interaction between skeletal muscle cells and angiogenic cells induced by C-30 under hyperglycemia culture conditions. (A-D) The proliferation potential of HUVECs (A and B) and MOVAS cells (C and D) cultured in CM-H/C-30 after treatment with VEGF receptor inhibitor Ki8751 or PDGF-BB receptor inhibitor CP868596 was detected by EdU-incorporation assay. (A and C) are representative images (scale bar: 200 μm), and (B and D) represent the relative value of the ratio of EdU-positive cells to Hoechst-positive cells compared with the control group (n=6). (E-H) The migration ability of HUVECs (E and F) and MOVAS cells (G and H) cultured in CM-H/C-30 after treatment with VEGF receptor inhibitor Ki8751 or PDGF-BB receptor inhibitor CP868596 was detected by transwell chamber migration assay. (E and G) are representative images (scale bar: 100 μm), (F and H) are statistical analysis of the number of cells that migrated to the lower chamber of the transwell (n = 6). Data are expressed as mean ± SD (n = 3). CM-N: conditioned medium obtained by treating C2C12 cells with blank solution under normoglycemic conditions; CM-H and CM-H/C30: conditioned medium obtained by treating C2C12 cells with blank solvent or C-30 under hyperglycemic conditions; **P < 0.01.

Figure 14: Intramuscular injection of C-30 promotes blood perfusion recovery in diabetic HLI mice. (A) Schematic diagram of HLI induced by left femoral artery resection. (B and C) Blood flow recovery after intramuscular injection of indicated concentrations of C-30 or salidroside into ischemic hindlimbs of diabetic HLI mice at indicated time points. (B) Representative images obtained by laser Doppler imaging system, (C) Comparative analysis of blood perfusion between ischemic hindlimbs (left) and non-ischemic hindlimbs (right). (D) Morphological evaluation of ischemic hindlimbs of diabetic HLI mice after intramuscular injection of indicated concentrations of C-30 or salidroside at indicated time points (0 = no difference from control group; 1 = slight color change; 2 = moderate color change; 3 = severe discoloration, necrosis, subcutaneous tissue loss; 4 = lower limb amputation; n = 8 per group). (E and F) Immunofluorescence of vascular endothelial cells (PECAM-1 positive) and smooth muscle cells (α-SMA positive) in the gastrocnemius muscle of ischemic hindlimbs of diabetic HLI mice. (E) is a representative image (scale bar: 100 μm), and (F) is the result of quantitative analysis (n=6). Data are expressed as mean ± SD (n=3). (G) Western blotting was used to detect the protein expression levels of HIF-1α and angiogenic factors 21 days after intramuscular injection of the indicated concentrations of C-30 or salidroside into the ischemic hindlimbs of diabetic HLI mice. Con: control group; SA: mice intramuscularly injected with salidroside; C-30: mice intramuscularly injected with C-30. **P<0.01; *P<0.05; NS: no significant difference.

Figure 15: Effects of C-30 on various tissues of diabetic HLI mice. (A) TUNEL analysis of apoptosis rate of gastrocnemius muscle in ischemic hindlimb of diabetic HLI mice injected with corresponding concentrations of C-30 and salidroside. Representative images (scale bar: 100 μm, left), ratio of TUNEL-positive cells to DAPI-positive cells (n=6; right). (B) Immunohistochemistry of heart, liver, spleen, lung, and kidney of diabetic HLI mice in the 30 mg/kg C-30 administration group. Scale bar: 100 μm. Data are expressed as mean ± standard deviation. **P<0.01.

Figure 16: Schematic diagram of the pharmacological activity of salidroside analog C-30 in promoting angiogenesis and blood perfusion recovery in diabetic HLI mice by enhancing skeletal muscle cell viability, migration and secretion.

Figure 17: C-30 promotes blood perfusion recovery in non-diabetic HLI model mice. (A) Blood perfusion of ischemic hindlimbs of non-diabetic HLI mice after intramuscular injection of 30 mg/kg C-30 at designated time points, representative images measured by laser Doppler imaging system. (B) Blood perfusion ratio of ischemic (left) hindlimbs and non-ischemic (right) hindlimbs of non-diabetic HLI mice after intramuscular injection of 30 mg/kg C-30 at designated time points (n=4). Con: mice injected with blank solvent. **P<0.01.

DETAILED DESCRIPTION

In order to further understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be purchased through commercial channels.

The present invention also includes isotope-labeled compounds, which are equivalent to those described in formula C-1 to C-33, but one or more atoms are replaced by atoms having an atomic mass or mass number different from the atomic mass or mass number common in nature. Examples of isotopes that can be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, and oxygen, such as ² H, ³ H, ¹³ C, ¹¹ C, ¹⁴ C, ¹⁸ O, and ¹⁷ O, respectively.

The drug for treating diabetes and/or the drug for treating lower limb ischemic disease in the present invention may also contain one or more excipients. The excipients are not limited, such as solvents, isotonic agents, pH adjusters, antioxidants, preservatives and other excipients commonly used in the art.

Examples of the solvent include distilled water for injection, physiological saline, vegetable oil, and alcohols such as propylene glycol, polyethylene glycol, ethanol, and glycerol.

Examples of the isotonic agent include isotonic agents commonly used in the art, such as sorbitol, sodium chloride, and glucose.

Examples of the pH adjuster include hydrochloric acid, citric acid, sodium hydroxide, potassium hydroxide, sodium bicarbonate, and disodium hydrogen phosphate.

Examples of the antioxidant include sodium sulfite, sodium bisulfite, and ascorbic acid.

Examples of the preservative include preservatives commonly used in the art, such as parabens, sorbic acid and its salts.

Synthesis Example:

1) The main synthetic routes of C-1 to C-6, C-17 to C-22, C-26 to C-33:

Synthesis route 1 Synthesis of β-O-glycoside compounds C-1 to C-6, C-17 to C-22, C-26 to C-33. Reagents and conditions: (a) Boron trifluoride etherate, dichloromethane, -10°C to room temperature, overnight; (b) Sodium methoxide, methanol, hydrochloric acid, 2-step yield 30-65%; (c) 10% palladium carbon, H ₂ , methanol, yield 90-95%.

Synthesis route 1 describes the synthesis of β-O-glycoside compounds C-1 to C-6, C-17 to C-22, C-26 to C-33. According to the literature method (Das, SK; et al, Carbohydr. Res. 2007, 342, 2309-2315.), the corresponding alcohol or phenol intermediates (34a-t) are efficiently coupled with β-D-glucose pentaacetate in a dry dichloromethane solution under the catalysis of Lewis acid boron trifluoride ether to form O-glycoside compounds. Although the coupling product inevitably contains two anomeric isomers, α and β, they do not need to be separated immediately. After removing all acetyl groups in an excess of sodium methoxide/methanol solution, the reaction solution was acidified with hydrochloric acid to pH = 1-2, and then stirred at room temperature. Most of the α-isomer glycosidic bonds were hydrolyzed again, and the relatively stable β-isomer was purified by silica gel column chromatography (if necessary, further protected by Pd/C catalytic hydrogenation debenzylation) to obtain the target compound. In the H NMR spectrum, the coupling constant of the hydrogen atoms at positions 1 and 2 of the sugar ring of the target product was J _1,2 = 7-8 Hz, confirming that all products were β-configuration.

2) The main synthetic route of β-C-glycoside compounds C-15, C-16, C-23 to C-25:

Synthesis route 2 Synthesis of β-C-glycoside compounds C-15, C-16, C-23 to C-25. Reagents and conditions: (a) magnesium powder or n-butyl lithium, ether or tetrahydrofuran, -78°C; (b) boron trifluoride etherate, triethylsilane, acetonitrile, -30°C; (c) 10% palladium carbon, hydrogen, methanol, 6-16 hours. Total yield 35-60%.

Synthesis route 2 describes the synthesis of β-C-glycoside compounds C-15, C-16, C-23 to C-25. According to the method described in the literature (Ho, T.C., et al, Org. Lett. 2016, 18, 4488-4490), the starting material bromoaryl or alkane 37a-e is first prepared with magnesium powder or n-butyl lithium to form a Grignard reagent, and then reacted with an excess of sugar lactone intermediate 36 to form hemiketal intermediate 38a-e. The hemiketal intermediate is then reduced at low temperature by a boron trifluoride etherate/triethylsilane system to obtain intermediate 39a-e, and finally the benzyl group is removed by hydrogenation under 10% palladium carbon catalysis to obtain the target compound.

3) The main synthetic route of β-O-glycoside compounds C-7, C-8, C-11 and C-12:

Synthesis route 3 Synthesis of β-O-glycoside compounds C-7, C-8, C-11 and C-12. Reagents and conditions: (a) benzaldehyde, p-toluenesulfonic acid, N,N-dimethylformamide, triethyl orthoformate, 78.6%; (b) 1.2 equivalents, benzyl bromide, 1.5 equivalents sodium hydroxide, 80°C, 6 hours, silica gel column chromatography to separate regional isomers, yields: 41d 18%, 41u 22%, 41 39%; (c) i. sodium hydride, N,N-dimethylformamide, 0°C, 30 minutes, iodomethane, room temperature, overnight; ii. 10% palladium on carbon, hydrogen, ethyl acetate/methanol (4:1), 2-step yields 48-76%; (d) i. thiocarbonyldiimidazole, dimethylaminopyridine, diisopropylethylamine, ii. tributyltin hydrogen, azobisisobutyronitrile, toluene, 80°C, 4 hours, iii. 10% palladium on carbon, hydrogen, ethyl acetate/methanol (4:1), 3-step yields 39-65%.

Synthesis route 3 shows the synthesis route of β-O-glycoside compounds C-7, C-8, C-11 and C-12. First, benzaldehyde selectively reacts with the two hydroxyl groups at positions 4 and 6 on the sugar ring of compound C-1 to obtain 4,6-O-phenylmethylene-β-pyranoglucoside intermediate 40. The remaining two hydroxyl groups at positions 2 and 3 of the sugar ring are then non-selectively protected by benzyl bromide and separated by column chromatography to obtain two regioisomers: 41d protected by benzyl at position 3 and 41u protected by benzyl at position 2. The unprotected hydroxyl groups in 41d and 41u are methylated, and then hydrogenated to remove the benzyl protection to obtain the target compounds C-7 and C-8; at the same time, 41d and 41u are subjected to Barton-McCombie deoxygenation reaction to reduce the unprotected hydroxyl groups to methylene, and then catalytic hydrogenation is used to remove the benzyl protection to finally obtain the target compounds C-11 and C-12.

4) The main synthetic route of β-O-glycoside compounds C-9, C-10, C-13 and C-14:

Synthesis route 4. Synthesis of β-O-glycoside compounds C-9, C-10, C-13 and C-14. Reagents and conditions: (a) benzyl bromide, sodium hydroxide, 76%; (b) triethylsilane, I ₂ , 0°C, 83%; (c) trimethylsilyl trifluoromethanesulfonate, borane tetrahydrofuran, dichloromethane, Molecular sieves, room temperature, 80%; (d) i. Sodium hydride, N,N-dimethylformamide, 0°C, 30 minutes, iodomethane, room temperature, overnight; ii. 10% palladium on carbon, hydrogen, ethyl acetate/methanol (4:1), 2-step yield 48-76%. (e) i. Thiocarbonyl diimidazole, dimethylaminopyridine, diisopropylethylamine, ii. Tributyltin hydride, azobisisobutyronitrile, toluene, 80°C, 4 hours, iii. 10% palladium on carbon, hydrogen, ethyl acetate/methanol (4:1), 3-step yield 65%. (f) i. p-Toluenesulfonyl chloride, triethylamine, dichloromethane, pyridine, dimethylaminopyridine, 0°C→room temperature, overnight; ii. Lithium aluminum hydride, tetrahydrofuran, 40°C, 2 hours; iii. 10% palladium on carbon, ethyl acetate/methanol, (4:1), 3-step yield 47.8%.

Synthesis route 4 shows the synthesis route of β-O-glycoside compounds 9-10, 13-14. First, the two remaining hydroxyl groups of intermediate 40 are completely protected with excess benzyl bromide to obtain intermediate 42. Then, the benzyl acetal ring structure of 42 is regioselectively opened under different reduction reaction systems. Among them, after opening with triethylsilane/iodine, 4-OH intermediate 43 is obtained, while reduction with trimethylsilyl trifluoromethanesulfonic acid/borane tetrahydrofuran system releases 6-OH to obtain intermediate 44. The exposed hydroxyl groups in 43 and 44 are methylated with iodomethane, and then hydrogenated to remove the benzyl protection to obtain the desired compounds C-9 and C-10 respectively; in addition, intermediate 43 is subjected to Barton-McCombie deoxygenation reduction reaction to reduce the exposed hydroxyl group to methylene, and then hydrogenated to remove the benzyl protection to obtain compound C-13. The 6-OH of 44 was esterified with p-toluenesulfonyl chloride, and then the _p -toluenesulfonate was reduced with LiAlH4. After hydrogenation to remove the benzyl protection, the target compound C-14 was obtained in a high yield.

5) Detailed synthesis method of the main synthesis route of compounds C-1 to C-33

Unless otherwise specified, all commercially purchased chemicals were used without further purification. The progress of each chemical reaction was monitored by thin layer chromatography using purchased Merck Kiesel gel 60F254 silica gel plates (Merck, Darmstadt, Germany), which could be observed under ultraviolet light at 254 nm or heated after soaking in phosphomolybdic acid/ethanol solution to develop color. Column chromatography purification used silica gel (200-300 mesh) columns. Agilent 400MR nuclear magnetic resonance instrument (Agilent Technologies, Santa Clara, CA) was used to collect ¹ H NMR spectra, and Agilent 400MR DD2 (100 MHz) proton decoupling was used to obtain ¹³ C NMR spectra, chemical shifts were expressed in parts per million (ppm), coupling constants (J) were expressed in Hertz (Hz), and signals were described as singlets (s), doublets (d), triplets (t) and multiplets (m). High-resolution mass spectra (HRMS) were obtained by a Bruker SolariX 7.0T spectrometer (Bruker, Karlsruhe, Germany). The melting points of the compounds were detected by a WRS-2A digital melting point instrument (Shanghai Youyi, Shanghai, China) without correction. The optical rotation was determined by an automatic polarimeter (Autopol I, Rudolph, Hackettstown, NJ). The purity was determined by high-performance liquid chromatography (HPLC) (Agilent Technologies, Santa Clara, CA), and the purity of all final compounds was not less than 95%. Purity analysis method 1: Agilent 1260 Infinity II, Inertsil ODS-3 4.6×250 mm, 5 μm. Water plus 0.1% formic acid (mobile phase A), acetonitrile (mobile phase B), gradient mobile phase B: 0min 20.0%, 5min 20.0%, 15min 70%, 15.1min 20.0%, 20min, 20%. The flow rate was 1 mL/min. The detection wavelength was 210 nm. Analysis method 2: Agilent 1260 Infinity, ZORBAX SB-C18, 4.6×150 mm, 5 μm, water plus 0.1% formic acid (mobile phase A), acetonitrile plus 0.1% formic acid (mobile phase B), gradient mobile phase B: 0 min 10.0%, 10 min 90.0%, 15 min 90%, 15.1 min 20.0%, 20 min 20%. The flow rate was 1 mL/min. The detection wavelength was 210 nm.

Procedure A: Synthesis of β-D-O-glycoside compounds C-1 to C-5, C-17 to C-22, C-26 to C-33

Under nitrogen protection, anhydrous dichloromethane and Molecular sieves, then add alcohol or phenol intermediate 34a-t (1.0 equivalent), α-D-glucose pentaacetate (2.0 equivalents), cool to 5°C, boron trifluoride etherate (2.2 equivalents) is added dropwise to the solution, then stirred at 5°C for 1 hour, warmed to room temperature and continued to stir for 16 hours. Filter off the molecular sieves, wash the filtrate with 10% sodium bicarbonate, dry over anhydrous sodium sulfate and concentrate to dryness. The residue is dissolved in methanol, sodium methoxide solution (10.0 equivalents) is added, stirred at room temperature for 4-6 hours, concentrated to remove the organic solvent, and the residue is acidified to pH = 1-2 with 3N hydrochloric acid aqueous solution, and stirred for 30 minutes. The product is extracted with ethyl acetate containing 5% methanol, the combined organic phases are washed with saturated sodium chloride brine, dried over anhydrous sodium sulfate and concentrated. The concentrated residue is purified by silica gel column chromatography to obtain the desired compound.

Procedure B: Debenzylation method

The benzyl-protected compound (100 mg) was dissolved in 5 mL of a mixture of ethyl acetate and methanol (4:1, v/v), and 30 mg of 10% palladium on carbon was added. The mixture was stirred continuously for more than 4 hours in a balloon hydrogen environment until the debenzylation reaction was completed. The product was purified by silica gel column chromatography (ethyl acetate/methanol, 10:0-1, v/v) to obtain a debenzylated product.

Procedure C: Synthesis of β-D-C-glycoside compounds C-15, C-16, C-23 to C-25

In the synthesis of C-15 and C-16, the alkyl halide intermediate 37a or 37b (2.0 equivalents) was first stirred with magnesium powder (4.0 equivalents) in dry tetrahydrofuran at 50°C for 2 hours to prepare a Grignard reagent, then cooled to room temperature, and a tetrahydrofuran solution of intermediate 36 (1.0 equivalents) was added dropwise. After stirring at room temperature for 1 hour, the reaction was quenched with water, and the product was extracted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, and concentrated to dryness.

In the synthesis of C-23, C-24 and C-25, the halogenated aromatic intermediate 37c, 37b or 37e (1.0 equivalent) was dissolved in dry tetrahydrofuran, cooled to -78°C, and n-BuLi (1.2 equivalent) was added dropwise and stirred at -78°C for 1 hour. Then, a tetrahydrofuran solution of intermediate 36 (1.0 equivalent) was added and stirred at -78°C for 2 hours. A small amount of methanol was added to quench the reaction, and the product was extracted with ethyl acetate. The combined organic phase was washed with brine, dried over anhydrous sodium sulfate and concentrated to dryness.

The residue obtained after the above concentration was dissolved in a mixture of acetonitrile and dichloromethane (1:1, v/v), cooled to -30°C, triethylsilane (2.0 equivalents) was added to the solution, followed by boron trifluoride etherate (2.0 equivalents), and stirred at -30°C for 2 hours. The reaction was quenched with water, and the product was extracted with ethyl acetate. The combined organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated. The remaining product was debenzylated and purified according to step B to obtain the target compound.

Procedure D: Synthesis of Intermediates 40-44

Synthetic intermediate 40

Compound C-1 (2.6 g, 6.67 mmol), benzaldehyde (918 mg, 8.67 mmol), p-toluenesulfonic acid (316 mg, 1.67 mmol) and triethyl orthoformate (1.23 g, 8.3 mmol) were dissolved in 20 mL of N, N-dimethylformamide and stirred at room temperature overnight. Ethyl acetate was added, and the organic phase was washed with water and brine successively, dried over anhydrous sodium sulfate and concentrated to dryness. The residue was dissolved by heating with 20 mL of ethyl acetate, and 150 mL of n-hexane was added and stirred at room temperature for 1 hour. The obtained white solid was collected by filtration and dried under vacuum to obtain intermediate 40. (2.5 g, 78.6%) ¹ H NMR (400 MHz, cdcl ₃ )δ7.48(dd,J＝6.5,2.9Hz,2H),7.41(t,J＝7.3Hz,3H),7.39–7.34(m,4H),7.34–7.28(m,1H),7.14(d,J＝8.5Hz,2H),6.92(d,J＝8.5Hz,2H),5.52(s,1H),5 .04(s,2H),4.37(d,J＝7.7Hz,1H),4.33( dd,J＝10.5,4.9Hz,1H),4.12(dd,J＝15.1,7.9Hz,1H),3.79(dd,J＝9.6,4.9Hz,1H),3.77–3.68(m,2H),3.54(t,J＝9.3Hz,1H),3.50–3.46(m,1H),3.46–3.3 9(m,1H),2.90(dd,J＝10.3,5.1Hz,2H). ¹³ C NMR (101MHz, cdcl ₃ ) δ157.49,137.03,136.91,130.39,129.83,129.26,128.55,128.32,127.91,127.43,126.25,114.92,103.29,101.91,80.5 3,74.57,73.10,71.28,70.03,68.64,66.42,35.22.

Synthesis of intermediate 41

Intermediate 40 (4.5 g, 9.4 mmol), sodium hydroxide (564 mg, 14.1 mmol), benzyl bromide (1.93 g, 11.2 mmol) were completely dissolved in a mixed solution (acetonitrile/N,N-dimethylformamide/H ₂ O, 10:1:0.1, v/v/v), heated to 80°C and stirred for 6 hours. The reaction solution was diluted with water and the product was extracted with ethyl acetate. The combined organic phases were washed with water and brine, dried over anhydrous sodium sulfate and concentrated. The residue was purified by column chromatography (n-hexane/ethyl acetate/dichloromethane, 10:1:2, v/v/v) and the regional isomers were separated (TLC: n-hexane/ethyl acetate, 5:1; Rf: 0.2 and 0.3) to give 41u (1.2 g, 22%, Rf: 0.3), 41d (963 mg, 18%, Rf: 0.2) and their mixture 41 (2.2 g, 39%).

Synthesis of intermediate 42

Intermediate 40 (2.4 g, 5 mmol), 60% sodium hydride (700 mg, 17.5 mmol), benzyl bromide (3.0 g, 17.5 mmol), and 20 mg of tetrabutylammonium bromide were dissolved in 30 mL of N,N-dimethylformamide and stirred at room temperature overnight. Water was added to quench the reaction, and the product was extracted with ethyl acetate. After the organic phase was dried and concentrated, n-hexane was added to the residue and stirred for crystallization to obtain intermediate 42 (2.5 g, 76%). ¹ H NMR (400 MHz, cdcl ₃ )δ7.48(d,J＝6.3Hz,2H),7.35(dt,J＝15.0,6.6Hz,10H),7.26(dt,J＝13.7,6.9Hz,8H),7.14(d,J＝8.2Hz,2H),6.86(d,J＝8.2Hz,2H),5.56(s,1H),4.96(s ,2H),4.84(dd,J＝43.3,11.4Hz,2H),4.66(t ,J＝15.3Hz,2H),4.51(d,J＝7.6Hz,1H),4.34(dd,J＝10.4,4.8Hz,1H),4.14(dd,J＝15.6,6.7Hz,1H),3.72(ddd,J＝20.8,19.4,9.8Hz,4H),3.47–3.42(m,1H) ,3.42–3.36(m,1H),2.90(t,J=6.9Hz,2H). ¹³ CNMR (101MHz, cdcl ₃ ) δ157.44,138.51,138.39,137.32,137.11,130.77,129.86,128.92,128.54,128.26,128.22,128.04,128.00,127.88,127. 60,127.58,127.42,125.99,114.82,104.07,101.12,82.16,81.46,80.81,75.21,75.08,71.23,69.97,68.80,66.02,35.36.

Synthesis of intermediate 43

Intermediate 42 (1.0 g, 1.5 mmol) was dissolved in 8.0 mL of dry acetonitrile, cooled to 0°C, triethylsilane (0.48 mL 3.0 mmol) was added, and elemental iodine (500 mg, 2 mmol) was added in portions to keep the reaction solution deep red. The reaction progress was monitored by TLC until the starting material was completely converted. The reaction solution was diluted with water and the product was extracted with ethyl acetate. The combined organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated. The residue was purified by silica gel column chromatography (n-hexane/ethyl acetate/dichloromethane, 10:2:1) to obtain intermediate 43 (832 mg, 83%). ¹ H NMR (400 MHz, cdcl ₃ )δ7.44–7.25(m,21H),7.17(d,J＝8.5Hz,2H),6.89(d,J＝8.5Hz,2H),4.99(s,2H),4.94(d,J＝11.4Hz,1H),4.75(t,J＝11.8Hz,2H),4.64–4.57(m,3H),4. 45(d,J＝7.1Hz,1H),4.19(dt,J＝9.2,6.6Hz,1H),3.79(dd,J＝10.4,3.6Hz,1H),3.77–3.70(m,2H),3.64–3.58(m,1H),3.49–3.41(m,3H),2.94(t,J＝7. 0Hz,2H). ¹³ C NMR (101MHz, cdcl ₃ ) δ157.40,138.63,138.47,137.95,137.15,131.00,129.87,128.55,128.53,128.42,128.30,128.11,127.96,127.89,127.82,1 27.71,127.61,127.43,114.81,103.68,84.01,81.75,75.25,74.64,74.09,73.67,71.53,70.91,70.29,69.98,35.39.

Synthesis of intermediate 44

Intermediate 42 (1.5 g, 2.27 mmol) was dissolved in 30 mL of dichloromethane and added Molecular sieves, then add borane tetrahydrofuran (11.5 mL, 11.3 mmol) and trimethylsilyl trifluoromethanesulfonate 100 μl, stir and react at room temperature for 2.5 hours, add 10 mL methanol and 15 mL triethylamine to quench the reaction, wash the organic phase with 10% sodium bicarbonate and brine, dry over anhydrous sodium sulfate and evaporate the solvent under vacuum. The residue is purified by silica gel column chromatography (n-hexane/ethyl acetate, 4:1, v/v) to obtain intermediate 44 (1.2 g, 80%).

¹ H NMR (400MHz, cdcl ₃ ) δ7.43–7.33 (m, 4H), 7.33–7.24 (m, 14H), 7.24–7.20 (m, 2H), 7.14 (d, J = 8.5Hz, 2H), 6.86(d,J＝8.5Hz,2H),4.95(d,J＝8.9Hz,2H),4.88(dd,J＝23.6,11.0Hz,2H),4.76(dd,J＝21.8,11.0Hz,2H ),4.62(t,J＝11.1Hz,2H) ,4.44(d,J＝7.8Hz,1H),4.13(dt,J＝9.2,6.6Hz,1H),3.85(ddd,J＝11.7,5.6,2.6Hz,1H),3.77–3.68(m,2H ),3.60(dt,J＝18.6,9.0Hz,2H),3.44–3.37(m,1H),3.34(ddd,J＝9.3,4.2,2.8Hz,1H),2.90(t,J＝6.9Hz, 2H). ¹³ C NMR (101MHz, cdcl ₃ ) δ157.45,138.53,138.42,137.98,137.12,130.81,129.85,128.55,128.49,128.37,128.31,128.07,127.92,127.89,127. 86,127.62,127.43,114.83,103.66,84.46,82.33 ,77.54,75.67,75.08,75.04,74.80,71.08,69.98,62.05,35.41.

Procedure E: Synthesis of methylated analogs C-7 to C-10

41d, 41u, 43 or 44 (1.0 equivalent) was dissolved in N,N-dimethylformamide in a sealed tube, and 60% sodium hydride (3.0 equivalent) was added in portions. After stirring at room temperature for 20 minutes, iodomethane (6.0 equivalent) was added and the reaction was sealed at room temperature until the raw material was completely converted. The reaction was terminated with water, and the product was extracted with ethyl acetate and purified by debenzylation according to process B to obtain the target compound.

Procedure F: Synthesis of deoxy compounds C-11, C-12 and C-13

Intermediate 41d, 41u or 43 (1.0 equivalent), 1,1'-thiocarbonyldiimidazole (2.0 equivalent), dimethylaminopyridine (0.2 equivalent), diisopropylethylamine (2.0 equivalent) were stirred in N,N-dimethylformamide at room temperature for 20 hours. After adding water to quench the reaction, the product was extracted with ethyl acetate. The organic layer was washed with brine, dried over anhydrous sodium sulfate and concentrated. After adding n-hexane, a solid of thiocarbonyl imidazole ester was precipitated. After vacuum drying, the solid was refluxed at 80°C in toluene with tributyltin hydride (1.1 equivalent) and azobisisobutyronitrile (0.25 equivalent) for 4 hours. Toluene was evaporated under reduced pressure, n-hexane was added and stirred vigorously, and the precipitated solid was collected by filtration, and the target compound was obtained by debenzylation and purification in step B.

7. Specific examples:

(2R,3R,4S,5S,6R)-2-(4-(Benzyloxy)phenethoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (C-1) White solid, melting point 95.8-97.5℃, (c 0.16, methanol). HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₂₁ H ₂₆ O ₇ Na, 413.1576, found 413.1574. HPLC analysis method 1, main peak retention time = 12.39 min, 95.33%.

(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-(3-hydroxyphenethoxy)tetrahydro-2H-pyran-3,4,5-triol (C-2)

C-2 (92 mg, 32.0% for 3 steps) was obtained from intermediate 34b (228 mg, 1.0 mmol) according to Procedure A and B. White solid, melting point 71.5-73.4 °C, (c 0.28, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.07 (t, J = 7.8 Hz, 1H), 6.71 (d, J = 7.4 Hz, 2H), 6.65–6.58 (m, 1H), 4.30 (d, J = 7.8 Hz, 1H), 4.07 (dd, J = 17.0, 7.6 Hz, 1H), 3.87 (d, J = 12. 2Hz,1H),3.77–3.71(m,1H),3.71–3.64(m,1H),3.36(t,J＝8.6Hz,1H),3.28(d,J＝7.6Hz,2H),3.19(t,J＝8.4Hz,1H),2.86(t,J＝6.9Hz,2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 156.95, 140.06, 128.90, 119.81, 115.40, 112.71, 102.93, 76.64, 76.50, 73.66, 70.25, 70.16, 61.28, 35.74. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₄ H ₂₀ O ₇ Na, 323.1107, found 323.1105. HPLC analysis method 2, main peak retention time = 4.73 min, 97.80%.

(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-(2-hydroxyphenethoxy)tetrahydro-2H-pyran-3,4,5-triol (C-3)

C-3 (110 mg, 33.3% for 3 steps) was obtained from intermediate 34C (250 mg, 1.1 mmol) according to Procedure A and B. Viscous foam, (c 0.3, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.12 (d, J = 7.1Hz, 1H), 7.01 (t, J = 7.5Hz, 1H), 6.74 (t, J = 6.9Hz, 2H), 4.32 (d, J = 7.8Hz, 1H), 4.05 (dd, J = 16.4, 8.1Hz, 1H), 3.86 (d, J = 1 1.2Hz,1H),3.77(dd,J＝15.9,8.4Hz,1H),3.67(dd,J＝11.9,5.0Hz,1H),3.39– 3.34(m,1H),3.29(dd,J＝15.4,7.0Hz,2H),3.19(t,J＝8.3Hz,1H),2.93(t,J＝7. 4Hz,2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 155.11, 130.42, 127.06, 124.63, 119.19, 114.60, 103.00, 76.61, 76.48, 73.67, 70.13, 69.35, 61.26, 30.47. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₄ H ₂₀ O ₇ Na, 323.1107, found 323.1105. HPLC analysis method 2, main peak retention time = 4.95 min, 98.45%.

(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-phenylethoxytetrahydro-2H-pyran-3,4,5-triol (C-4)

C-4 (165 mg, 35.6% for 2 steps) was obtained from intermediate 34d (200 mg, 1.63 mmol) according to Procedure A. White solid, melting point 113.8-115.9 °C, (c 0.28, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.25 (d, J = 4.3 Hz, 4H), 7.16 (dt, J = 8.4, 4.1 Hz, 1H), 4.30 (d, J = 7.8 Hz, 1H), 4.09 (dd, J = 16.8, 7.7 Hz, 1H), 3.86 (d, J = 12.1 Hz, 1H), 3. 75(dd,J＝16.5,7.9Hz,1H), 3.66(dd,J＝11.8,4.9Hz,1H), 3.37–3.27(m,3H), 3.18(t,J＝8.4Hz,1H), 2.93(t,J＝7.1Hz,2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 138.61, 128.58, 127.90, 125.77, 102.94, 76.65, 76.53, 73.66, 70.28, 70.19, 61.31, 35.80. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₄ H ₂₀ O ₆ Na, 307.1158, found 307.1155. HPLC analysis method 1, main peak retention time = 3.12 min, 99.67%.

(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-(4-methoxyphenethoxy)tetrahydro-2H-pyran-3,4,5-triol (C-5)

C-5 (296 mg, 46.3% for 2 steps) was obtained from intermediate 34e (310 mg, 2.04 mmol) according to Procedure A. White solid, mp 98.1-100.5 °C, (c 0.3, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.17 (s, 1H), 7.15 (s, 1H), 6.83 (s, 1H), 6.81 (s, 1H), 4.29 (d, J = 7.8Hz, 1H), 4.05 (dd, J = 17.0, 7.6Hz, 1H), 3.86 (d, J = 10.5Hz, 1H) ,3.74(s,3H),3.73–3.63(m,2H),3.40–3.22(m,3H),3.19(t,J=8.4Hz,1H),2.91–2.81(m,2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 158.19, 130.53, 129.53, 113.34, 102.92, 76.64, 76.50, 73.66, 70.54, 70.18, 61.31, 54.22, 34.90. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₅ H ₂₂ O ₇ Na, 337.1263, found 337.1261. HPLC analysis method 1, main peak retention time = 3.11 min, 97.52%.

(2R,3R,4S,5S,6R)-2-(4-Fluorophenethoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (C-6)

C-6, 124 mg (38.6% for 2 steps) was obtained from intermediate 34f (150 mg, 1.07 mmol) according to Procedure A. White solid, melting point 114.1-116.5 °C, (c 0.27, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.27(dd,J＝8.3,5.6Hz,2H),6.98(t,J＝8.8Hz,2H),4.29(d,J＝7.8Hz,1H),4.08(dt,J＝9.4,7.3Hz,1H),3.91–3.82(m,1H),3.74(d t,J＝9.5,7.3Hz,1H),3.70–3.62(m,1H),3.35(t,J＝8.8Hz,1H),3.31–3.25(m,2H),3.18(t,J＝8.4Hz,1H),2.92(t,J＝7.1Hz,2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 162.71, 160.30, 134.66, 130.30, 130.23, 114.52, 114.31, 102.94, 76.66, 76.53, 73.65, 70.19, 70.15, 61.32, 34.90. HRMS (ESI) m/z: [M+Na] ⁺ calculated for C ₁₄ H ₁₉ FO ₆ Na, 325.1063, found 325.1061. HPLC analysis method 1, main peak retention time = 3.32 min, 98.87%.

(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-(4-hydroxyphenethoxy)-5-methoxytetrahydro-2H-pyran-3,4-diol (C-7)

C-7 (90 mg, 53.8% for 2 steps) was obtained from intermediate 41d (300 mg, 0.53 mmol) according to Procedure E. White solid, melting point 97.2-99.8°C, (c 0.25, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.06 (d, J = 8.3 Hz, 2H), 6.69 (d, J = 8.3 Hz, 2H), 4.31 (d, J = 7.8 Hz, 1H), 4.09 (dt, J = 9.3, 6.8 Hz, 1H), 3.85 (dd, J = 11.9, 1.8 Hz, 1H), 3.7 3–3.62(m,2H),3.45(s,3H),3.37–3.24(m,3H),3.24–3.16(m,1H),2.81(t,J＝6.6Hz,2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 155.34, 129.49, 114.61, 103.09, 83.42, 76.31, 76.03, 70.45, 70.14, 61.25, 59.51, 34.97. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₅ H ₂₂ O ₇ Na, 337.1263, found 337.1262. HPLC analysis method 1, main peak retention time = 3.46 min, 97.91%.

(2R,3R,4S,5R,6R)-2-(Hydroxymethyl)-6-(4-hydroxyphenethoxy)-4-methoxytetrahydro-2H-pyran-3,5-diol (C-8)

From intermediate 41u (300 mg, 0.53 mmol), C-8 (81 mg, 48.8% for 2 steps) was obtained according to Procedure E. It was viscous and foamy. (c 0.15, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.06(d,J＝8.3Hz,2H),6.69(d,J＝8.4Hz,2H),4.29(d,J＝7.8Hz,1H),4.02(dd,J＝16.6,8.0Hz,1H),3.85(dd,J＝11.8,1.8Hz,1H),3.7 3–3.64(m,2H),3.63(s,3H),3.35(t,J＝9.3Hz,1H),3.28–3.20(m,2H),3.06(d,J＝9.0Hz,1H),2.83(t,J＝8.2Hz,2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 155.35, 129.50, 129.29, 114.67, 102.88, 86.44, 76.37, 73.49, 70.68, 69.71, 61.18, 59.62, 34.92. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₅ H ₂₂ O ₇ Na, 337.1263, found 337.1262. HPLC analysis method 1, main peak retention time = 3.50 min, 98.68%.

(2R,3R,4R,5S,6R)-6-(Hydroxymethyl)-2-(4-hydroxyphenethoxy)-5-methoxytetrahydro-2H-pyran-3,4-diol (C-9)

C-9 (121 mg, 76.9% for 2 steps) was obtained from intermediate 43 (330 mg 0.5 mmol) according to Procedure E. White solid, melting point 177.0-179.5°C, (c 0.29, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.05(d,J＝8.3Hz,2H),6.69(d,J＝8.4Hz,2H),4.26(d,J＝7.8Hz,1H),4.01(dd,J＝16.6,8.1Hz,1H),3.81(dd,J＝11.9,1.7Hz,1H),3.7 0(dd,J＝15.1,6.0Hz,2H),3.55(s,3H),3.45(t,J＝9.1Hz,1H),3.26–3.14(m,2H),3.09(t,J＝9.3Hz,1H),2.82(t,J＝7.4Hz,2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 155.34, 129.50, 129.27, 114.69, 102.83, 79.43, 76.67, 75.57, 73.77, 70.67, 60.80, 59.42, 34.93. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₅ H ₂₂ O ₇ Na, 337.1263, found 337.1262. HPLC analysis method 1, main peak retention time = 3.35 min, 98.60%.

(2R,3R,4S,5S,6R)-2-(4-Hydroxyphenethoxy)-6-(methoxymethyl)tetrahydro-2H-pyran-3,4,5-triol (C-10)

C-10 (118 mg, 75.2% for 2 steps) was obtained from intermediate 44 (330 mg 0.5 mmol) according to Procedure E. Viscous foam, (c 0.42, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.05(d,J＝8.3Hz,2H),6.69(d,J＝8.4Hz,2H),4.27(d,J＝7.8Hz,1H),3.98(dd,J＝17.0,7.6Hz,1H),3.74–3.61(m,2H),3.56(dd,J＝ 10.8,5.5Hz,1H),3.38(s,3H),3.34(dd,J＝8.0,6.4Hz,2H),3.31–3.25(m,1H),3.18(t,J＝8.3Hz,1H),2.83(t,J＝8.7Hz,2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 155.34, 129.51, 129.26, 114.70, 102.95, 76.58, 75.27, 73.59, 71.63, 70.78, 70.16, 58.18, 34.94. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₅ H ₂₂ O ₇ Na, 337.1263, found 337.1262. HPLC analysis method 1, main peak retention time = 3.52 min, 95.41%.

(2R,3S,4R,6R)-2-(Hydroxymethyl)-6-(4-hydroxyphenethoxy)tetrahydro-2H-pyran-3,4-diol (C-11)

From intermediate 41d (350 mg, 0.51 mmol) according to Procedure F, C-11 (89 mg, 51.0% for 3 steps) was obtained. (c 0.3, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.02 (d, J = 8.3Hz, 2H), 6.67 (d, J = 8.3Hz, 2H), 4.50 (d, J = 8.4Hz, 1H), 4.02 (dd, J = 12.0, 4.7Hz, 1H), 3.84 (t, J = 8.8Hz, 1H), 3.69–3.58 ( m,2H),3.51(dt,J＝11.6,4.9Hz,1H),3.20–3.10(m,2H),2.75(t,J＝7.2Hz,2H),2.07(dd,J＝12.4,3.7Hz,1H),1.45(dd,J＝21.9,12.0Hz,1H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 155.35, 129.47, 129.43, 114.62, 99.77, 76.60, 71.65, 71.09, 70.11, 61.49, 38.95, 34.99. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₄ H ₂₀ O ₆ Na, 307.1158, found 307.1156. HPLC analysis method 1, main peak retention time = 3.49 min, >98.0%.

(2R,3S,5R,6R)-2-(Hydroxymethyl)-6-(4-hydroxyphenethoxy)tetrahydro-2H-pyran-3,5-diol (C-12)

Intermediate 41u (400 mg, 0.59 mmol) was used according to Procedure F to obtain C-12 (81 mg, 65.3% for 3 steps). White solid, melting point 134.8-136.2°C, (c 0.5, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.06(d,J＝8.4Hz,2H),6.69(d,J＝8.4Hz,2H),4.23(d,J＝7.6Hz,1H),4.03(dd,J＝16.6,8.1Hz,1H),3.84(dd,J＝11.8,2.3Hz,1H),3.6 6(ddd,J＝15.7,13.8,7.0Hz ,2H),3.54–3.44(m,1H),3.37(ddd,J＝12.1,7.5,5.0Hz,1H),3.26–3.20(m,1H),2.82(dd,J＝10.8,4.2Hz,2H),2.28(dt,J＝12.0,4.8Hz,1H),1.47(q,J＝ 11.7Hz,1H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 155.33, 129.51, 129.34, 114.69, 104.98, 80.27, 70.46, 67.91, 64.73, 61.40, 39.14, 34.96. HRMS (ESI) m/z: [M+Na] ⁺ calculated for C ₁₄ H ₂₀ O ₆ Na, 307.1158, found 307.1156 HPLC analysis method 2, main peak retention time = 3.75 min, 97.39%.

(2R,3R,4S,6S)-6-(Hydroxymethyl)-2-(4-hydroxyphenethoxy)tetrahydro-2H-pyran-3,4-diol (C-13)

C-13 (56 mg, 39.4% for 3 steps) was obtained from intermediate 43 (330 mg, 0.5 mmol) according to Procedure F. White solid, melting point 155.3-157.7 °C, (c 0.35, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.06(d,J＝8.4Hz,2H),6.69(d,J＝8.4Hz,2H),4.23(d,J＝7.7Hz,1H),4.01(dd,J＝16.3,8.3Hz,1H),3.68(dd,J＝16.2,8.4Hz,1H),3.6 3–3.47(m,4H),3.12–3.04(m,1H),2.90–2.74(m,2H),1.91(dd,J＝11.8,5.0Hz,1H),1.36(dd,J＝23.7,11.5Hz,1H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 155.33, 129.49, 129.30, 114.68, 103.26, 75.47, 72.46, 70.75, 70.65, 64.10, 34.99, 34.96. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₄ H ₂₀ O ₆ Na, 307.1158, found 307.1156. HPLC analysis method 2, main peak retention time = 4.69 min, 96.91%.

(2R,3R,4S,5S,6R)-2-(4-Hydroxyphenethoxy)-6-methyltetrahydro-2H-pyran-3,4,5-triol (C-14)

Intermediate 44 (330 mg, 0.5 mmol), pyridine (2.0 mL), dimethylaminopyridine (30 mg), dichloromethane (2.0 mL), cooled to 0 ° C, added p-toluenesulfonyl chloride (285 mg, 3.0 mmol) and stirred at room temperature overnight. Ethyl acetate was added to the reaction solution, washed with 2N hydrochloric acid, 10% sodium bicarbonate and brine, dried over anhydrous sodium sulfate and concentrated to dryness. The residue was dissolved in dry tetrahydrofuran (10.0 mL), and lithium aluminum hydride (76 mg, 2.0 mmol) was added in portions and reacted at 40 ° C for 2 hours. Ethyl acetate (20 mL) and 20% sodium hydroxide (0.8 mL) were added to quench the reaction, the solid was filtered off, and the residue was concentrated. Debenzylation according to Procedure B to obtain C-14 (68 mg, 47.8% for 3 steps). Viscous foam, (c 0.15, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.05(d,J＝8.3Hz,2H),6.69(d,J＝8.3Hz,2H),4.26(d,J＝7.8Hz,1H),3.95(dd,J＝16.2,8.4Hz,1H),3.67(dd,J＝16.0,8.7Hz,1H),3.3 0–3.25(m,2H),3.17(t,J＝8.5Hz,1H),2.99(t,J＝9.1Hz,1H),2.86–2.77(m,2H),1.27(d,J＝6.1Hz,3H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 155.36, 129.46, 129.21, 114.68, 102.83, 76.31, 75.57, 73.90, 71.82, 70.73, 34.97, 16.63. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₄ H ₂₀ O ₆ Na, 307.1158, found 307.1156. HPLC analysis method 2, main peak retention time = 4.29 min, 97.72%.

(2R,3S,4R,5R,6S)-2-(Hydroxymethyl)-6-(4-hydroxyphenethoxy)tetrahydro-2H-pyran-3,4,5-triol (C-15)

C-15 (97 mg, 35.2% for 3 steps) was obtained from intermediate 37a (580 mg, 2.0 mmol) and 36 (520 mg, 0.96 mmol) according to Procedure C. White solid, melting point 139.3-141.5 °C, (c0.20, methanol). ¹ HNMR(400MHz,CD ₃ OD)δ7.02(d,J＝8.4Hz,2H),6.68(d,J＝8.4Hz,2H),3.88(dd,J＝11.9,2.0Hz,1H),3.66(dd,J＝11.9,5.8Hz,1H),3.32–3.24(m,2H),3.18(dd,J＝5.6,1.9Hz,1H),3.12–3.00(m,2H),2.77(ddd,J＝13.8,9.6,4.5Hz,1H),2.62(dt,J＝13.6,8.4Hz,1H),2.08(dt,J＝15.9,8.4Hz,1H),1.72–1.55(m,1H)。 ¹³ C NMR (101 MHz, CD ₃ OD) δ 154.84, 132.99, 129.05, 114.60, 80.19, 78.44, 78.23, 74.14, 70.68, 61.78, 33.69, 30.08. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₄ H ₂₀ O ₆ Na, 307.1158, found 307.1156. HPLC analysis method 2, main peak retention time = 5.71 min, 96.61%.

(2R,3S,4R,5R,6S)-2-(Hydroxymethyl)-6-(3-(4-hydroxyphenyl)propyl)tetrahydro-2H-pyran-3,4,5-triol (C-16)

C-16 (150 mg, 34.0% for 3 steps) was obtained from intermediate 37b (900 mg, 2.96 mmol) and 36 (800 mg, 1.48 mmol) according to Procedure C. White solid, melting point 136.2-138.5 °C, (c 0.32, methanol). ¹ H NMR (400 MHz, CD ₃ OD)δ6.99(d,J＝8.4Hz,2H),6.67(d,J＝8.4Hz,2H),3.83(dd,J＝11.8,2.0Hz,1H),3.63(dd,J＝11.8,5.6Hz,1H),3.32(dd,J＝12.2,6.0Hz,1H),3.29–3.24(m,1H ),3.20–3.15(m,1H),3.12(d,J＝7.9Hz,1H),3.04(t,J＝8.9Hz,1H),2.52(t,J＝6.9Hz,2H),1.91–1.79(m,2H),1.70–1.54(m,1H),1.42(dd,J＝18.9,9.1Hz ,1H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 154.77, 133.34, 128.90, 114.55, 80.14, 79.37, 78.45, 74.10, 70.59, 61.69, 34.72, 31.16, 27.36. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₅ H ₂₂ O ₆ Na, 321.1314, found 321.1320. HPLC analysis method 2, main peak retention time = 6.01 min, 95.89%.

(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-(3-(4-hydroxyphenyl)propoxy)tetrahydro-2H-pyran-3,4,5-triol (C-17)

C-17 (200 mg, 43.3% for 3 steps) was obtained from intermediate 34g (600 mg, 1.47 mmol) according to Procedure A and B. White solid, melting point 65.2-67.3°C, (c 0.34, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.02(d,J＝8.3Hz,2H),6.69(d,J＝8.3Hz,2H),4.25(d,J＝7.8Hz,1H),3.96–3.82(m,2H),3.67(dd,J＝11.8,5.3Hz,1H),3.57–3.47( m,1H),3.40–3.29(m,2H),3.29–3.24(m,1H),3.20(t,J＝8.4Hz,1H),2.61(t,J＝7.5Hz,2H),1.93–1.79(m,2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 154.89, 132.71, 128.99, 114.64, 102.98, 76.65, 76.42, 73.71, 70.20, 68.67, 61.31, 31.49, 30.81. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₅ H ₂₂ O ₇ Na, 337.1263, found 337.1261. HPLC analysis method 1, main peak retention time = 3.45 min, 99.73%.

(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-(2-(4-hydroxyphenyl)-2-methylpropoxy)tetrahydro-2H-pyran-3,4,5-triol (C-18)

C-18 (160 mg, 57.0% for 3 steps) was obtained from intermediate 34h (220 mg, 0.86 mmol) according to Procedure A and B. White solid, melting point 135.0-136.9 ° C, (c 0.42, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.22(d,J＝8.6Hz,2H),6.70(d,J＝8.6Hz,2H),4.20(d,J＝7.8Hz,1H),3.94(d,J＝9.4Hz,1H),3.84(dd,J＝11.8,1.5Hz,1H),3.65(dd,J ＝11.9,5.5Hz,1H),3.44(d,J＝9.5Hz,1H),3.31(dd,J＝18.8,9.6Hz,2H),3.26–3.20(m,1H),3.19–3.14(m,1H),1.30(s,6H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 154.87, 138.22, 126.77, 114.33, 103.53, 79.03, 76.63, 76.42, 73.73, 70.17, 61.30, 37.93, 25.25, 25.22. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₆ H ₂₄ O ₇ Na, 351.1420, found 351.1418. HPLC analysis method 1, main peak retention time = 3.62 min, 98.76%.

(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-((1-(4-hydroxyphenyl)cyclobutyl)methoxy)tetrahydro-2H-pyran-3,4,5-triol (C-19)

C-19 (320 mg, 53.8% for 3 steps) was obtained from intermediate 34i (640 mg, 2.38 mmol) according to Procedure A and B. Viscous foam, (c 0.23, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.04(d,J＝8.5Hz,2H),6.70(d,J＝8.5Hz,2H),4.20(d,J＝7.7Hz,1H),4.02(d,J＝9.6Hz,1H),3.84(d,J＝11.9Hz,1H),3.65(dd,J＝11.9 ,5.5Hz,1H),3.53(d,J＝9.6 Hz,1H),3.32–3.26(m,2H),3.18(dt,J＝17.1,8.4Hz,2H),2.37(td,J＝8.7,4.8Hz,2H),2.30–2.17(m,2H),2.06(dq,J＝16.9,8.6Hz,1H),1.81(qd,J＝9. 4,4.7Hz,1H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 154.78, 138.95, 126.84, 114.22, 103.44, 77.22, 76.67, 76.43, 73.76, 70.11, 61.24, 45.85, 29.67, 29.52, 15.13. HRMS (ESI) m/z: [M+Na] ⁺ calculated for C ₁₇ H ₂₄ O ₇ Na, 363.1420, found 363.1418. HPLC analysis method 2, main peak retention time = 4.91 min, 98.78%.

(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-(2-methyl-2-phenylpropoxy)tetrahydro-2H-pyran-3,4,5-triol (C-20)

C-20 (452 mg, 48.3% for 2 steps) was obtained from intermediate 34j (450 mg, 3.0 mmol) according to Procedure A. Viscous foam, (c 0.24, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.43 (d, J = 7.5Hz, 2H), 7.27 (t, J = 7.7Hz, 2H), 7.15 (t, J = 7.3Hz, 1H), 4.22 (d, J = 7.8Hz, 1H), 4.02 (d, J = 9.5Hz, 1H), 3.85 (dd, J = 11.9, 1 .9Hz,1H),3.66(dd,J＝11.9,5.4Hz,1H),3.51(d,J＝9.5Hz,1H),3.34–3.30(m ,1H),3.26(d,J＝8.4Hz,1H),3.24–3.19(m,1H),3.16(t,J＝8.3Hz,1H),1.37(s ,6H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 147.32, 127.63, 125.77, 125.42, 103.53, 78.68, 76.66, 76.46, 73.71, 70.19, 61.32, 38.60, 25.15, 25.09. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₆ H ₂₄ O ₆ Na, 335.1471, found 335.1468. HPLC analysis method 1, main peak retention time = 6.88 min, 94.86%.

(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-(2-(4-methoxyphenyl)-2-methylpropoxy)tetrahydro-2H-pyran-3,4,5-triol (C-21)

C-21 (320 mg, 35.8% for 2 steps) was obtained from intermediate 34k (470 mg, 2.61 mmol) according to Procedure A. White solid, melting point 68.3-70.2 °C, (c 0.13, methanol). ¹ H NMR (400MHz, cdcl ₃ ) δ7.28(d,J＝8.7Hz,2H),6.83(d,J＝8.7Hz,2H),4.17(d,J＝7.6Hz,1H),3.97–3.81(m,2H),3.75(s,4H),3.49(d,J＝7.3Hz,1H),3.46 –3.37(m,2H),3.30(t,J＝8.0Hz,1H),3.16(d,J＝9.3Hz,1H),1.31(d,J＝9.2Hz,6H). ¹³ C NMR (101 MHz, cdCl ₃ ) δ 157.68, 139.06, 127.07, 113.52, 103.59, 79.86, 76.12, 75.41, 73.37, 69.37, 61.39, 55.19, 38.46, 26.09, 25.89. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₇ H ₂₆ O ₇ Na, 365.1576, found 365.1574. HPLC analysis method 1, main peak retention time = 7.09 min, 97.39%.

(2R,3S,4S,5R,6R)-2-(Hydroxymethyl)-6-(3-(4-methoxyphenyl)-3-methylbutyloxy)tetrahydro-2H-pyran-3,4,5-triol (C-22)

C-22 (322 mg, 29.3% for 2 steps) was obtained from intermediate 341 (600 mg, 3.09 mmol) according to Procedure A. White solid, melting point 102.5-104.7 °C, (c 0.30, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.27(d,J＝8.8Hz,2H),6.83(d,J＝8.8Hz,2H),4.11(d,J＝7.8Hz,1H),3.81(d,J＝11.9Hz,1H),3.75(s,3H),3.74–3.68(m,1H),3.63 (dd,J＝11.9,5.5Hz,1H),3.35–3.23(m,3H),3.21–3.14(m,1H),3.11(t,J＝8.3Hz,1H),2.07–1.91(m,2H),1.30(s,6H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 157.57, 140.52, 126.31, 113.05, 102.99, 76.62, 76.38, 73.64, 70.11, 66.96, 61.20, 54.19, 43.08, 35.42, 28.74, 28.45. HRMS (ESI) m/z: [M+Na] ⁺ calcd for _{C 18} H ₂₈ O ₇ Na, 379.1733, found 379.1731. HPLC analysis method 1, main peak retention time = 9.22 min, 99.26%.

(2R,3S,4R,5R,6S)-2-(Hydroxymethyl)-6-(2-(4-methoxybenzyl)phenyl)tetrahydro-2H-pyran-3,4,5-triol (C-23)

C-23 (211 mg, 45.1% for 3 steps) was obtained from intermediate 37C (370 mg, 1.3 mmol) according to Procedure C. Viscous foam, (c 0.10, methanol). ¹ H NMR (400MHz, cdcl ₃ ) δ7.35 (s, 1H), 7.09 (d, J = 2.6Hz, 2H), 6.92 (d, J = 8.4Hz, 3H), 6.71 (d, J = 8.1Hz, 2H), 4.37 (d, J = 8.7Hz, 1H), 3.98 (d, J = 15.8Hz, 1H), 3 .84(d,J＝15.9Hz,1H), 3.64(s,3H), 3.54(d,J＝23.8Hz,5H), 3.05(d,J＝7.5Hz,1H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 158.14, 137.32, 136.91, 132.57, 129.82, 129.22, 128.52, 126.94, 125.55, 113.52, 81.19, 76.62, 75.33, 71.13, 67.33, 61.73, 54.25, 37.53. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₂₀ H ₂₄ O ₆ Na, 383.1471, found 383.1469. HPLC analysis method 1, main peak retention time = 9.09 min, 97.96%.

(2R,3S,4R,5R,6S)-2-(Methoxy)-6-(3-(4-methoxybenzyl)phenyl)tetrahydro-2H-pyran-3,4,5-triol (C-24)

C-24 (288 mg, 62.5% for 3 steps) was obtained from intermediate 37d (355 mg, 1.28 mmol) according to Procedure C. Viscous foam, (c 0.28, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.27(s,1H),7.25–7.19(m,2H),7.09(d,J＝8.5Hz,3H),6.80(d,J＝8.6Hz,2H),4.09(d,J＝9.4Hz,1H),3.89(s,2H),3.85(s,1H), 3.73(s,3H),3.68(dd,J＝12.0,5.2Hz,1H),3.45(dd,J＝15.7,6.9Hz,1H),3.38(t,J＝8.2Hz,2H),3.34(s,1H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 158.05, 141.52, 139.47, 133.27, 129.41, 128.14, 128.05, 127.72, 125.24, 113.38, 82.33, 80.79, 78.38, 74.96, 70.53, 61.75, 54.22, 40.51. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₂₀ H ₂₄ O ₆ Na, 383.1471, found 383.1469. HPLC analysis method 2, main peak retention time = 6.19 min, 96.48%.

(2R,3S,4R,5R,6S)-2-(Hydroxymethyl)-6-(4-(4-methoxybenzyl)phenyl)tetrahydro-2H-pyran-3,4,5-triol (C-25)

C-25 (151 mg, 32.7% for 3 steps) was obtained from intermediate 37e (355 mg, 1.28 mmol) according to Procedure C. White solid, melting point 95.3-97.8 °C, (c 0.32, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.32(d,J＝8.0Hz,2H),7.15(d,J＝7.9Hz,2H),7.07(d,J＝8.5Hz,2H),6.79(d,J＝8.5Hz,2H),4.09(d,J＝9.4Hz,1H),3.86(s,2H),3. 86-3.82(m,1H),3.72(s,3H),3.68(dd,J＝11.9,5.1Hz,1H),3.46(dd,J＝16.5,7.9Hz,1H),3.40-3.32(m,3H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 158.04, 141.61, 137.00, 133.35, 129.35, 128.09, 127.69, 113.39, 82.06, 80.73, 78.35, 74.92, 70.53, 61.76, 54.24, 40.29. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₂₀ H ₂₄ O ₆ Na, 383.1573, found 383.1469. HPLC analysis method 1, main peak retention time = 10.23 min, 98.93%.

(2R,3S,4S,5R,6S)-2-(Hydroxymethyl)-6-((4'-methoxy-[1,1'-biphenyl]-2-yl)oxy)tetrahydro-2H-pyran-3,4,5-triol (C-26)

C-26 (450 mg, 49.7% for 2 steps) was obtained from intermediate 34m (500 mg, 2.5 mmol) according to Procedure A. White solid, melting point 55.2-58.1 °C, (c 0.32, DMSO). ¹ H NMR (400MHz, CD ₃ OD) δ7.52(d,J＝8.7Hz,2H),7.25(dd,J＝9.0,6.2Hz,3H),7.10–7.00(m,1H),6.93(d,J＝8.7Hz,2H),5.02(d,J＝7.3Hz,1H),3.86(dd,J＝11. 9,1.3Hz,1H),3.79(s,3H),3.68(dd,J=12.0,5.3Hz,1H),3.46–3.35(m,4H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 158.74, 154.05, 130.96, 130.66, 130.47, 130.25, 127.76, 122.04, 114.95, 112.96, 100.33, 76.93, 76.68, 73.50, 69.83, 61.08, 54.27. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₉ H ₂₂ O ₇ Na, 385.1263, found 385.1261. HPLC analysis method 1, main peak retention time = 10.59 min, 95.04%.

(2R,3S,4S,5R,6S)-2-(Hydroxymethyl)-6-((4'-methoxy-[1,1'-biphenyl]-3-yl)oxy)tetrahydro-2H-pyran-3,4,5-triol (C-27)

C-27 (700 mg, 51.5% for 2 steps) was obtained from intermediate 34n (750 mg, 3.75 mmol) according to Procedure A. White solid, melting point 199.1-201.5 °C, (c 0.25, DMSO). ¹ H NMR(400MHz,dmso)δ7.62(d,J＝8.7Hz,2H),7.34(t,J＝7.9Hz,1H),7.30–7.23(m,2H),6.99(dd,J＝15.4,5.3Hz,3H),4.95(d,J＝7.2Hz,1H),3.79(s,3H),3 .73(dd,J＝11.6,3.5Hz,1H),3.48(dt,J＝11.7,5.9Hz,1H),3.38–3.30(m,1H),3.33–3.25(m,2H),3.18(dt,J＝13.6,7.0Hz,1H). ¹³ C NMR (101 MHz, dmso) δ 159.41, 158.38, 141.61, 132.65, 130.22, 128.24, 120.11, 115.07, 114.74, 114.46, 100.89, 77.55, 77.10, 73.74, 70.30, 61.21, 55.60. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₉ H ₂₂ O ₇ Na, 385.1263, found 385.1262. HPLC analysis method 1, main peak retention time = 10.28 min, 96.25%.

(2R,3S,4S,5R,6S)-2-(Hydroxymethyl)-6-((4'-methoxy-[1,1'-biphenyl]-4-yl)oxy)tetrahydro-2H-pyran-3,4,5-triol (C-28)

C-28 (500 mg, 46.8% for 2 steps) was obtained from intermediate 34O (600 mg, 3.0 mmol) according to Procedure A. White solid, melting point 227.5-229.2 °C, (c 0.15, DMSO). ¹ H NMR (400MHz, DMSO) δ7.54 (dd, J＝8.5, 6.6Hz, 4H), 7.10 (d, J＝8.6Hz, 2H), 7.00 (d, J＝8.7Hz, 2H), 4.89 (d, J＝7.2Hz, 1H), 3.78 (s, 3H), 3.72 (dd, J＝10.5, 5.2Hz, 1 H),3.49(dt,J＝11.7,5.9Hz,1H),3.33-35(m,1H),3.32–3.23(m,2H),3.19(dt,J＝14.2,7.1Hz,1H). ¹³ C NMR (101 MHz, dmso) δ 158.89, 156.98, 133.97, 132.62, 127.76, 127.52, 117.10, 114.73, 100.96, 77.51, 77.05, 73.70, 70.15, 61.16, 55.57. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₉ H ₂₂ O ₇ Na, 385.1263, found 385.1258. HPLC analysis method 1, main peak retention time = 10.38 min, 97.69%.

(2R,3S,4S,5R,6S)-2-(Hydroxymethyl)-6-(2-(4-methoxybenzyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triol (C-29)

C-29 (200 mg, 6.9% for 2 steps) was obtained from intermediate 34p (200 mg, 0.93 mmol) according to Procedure A. White solid, melting point 112.5-114.7 °C, (c 0.30, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.19–7.09(m,4H),7.03(d,J＝7.4Hz,1H),6.94–6.87(m,1H),6.78(d,J＝8.5Hz,2H),4.91(d,J＝7.3Hz,1H),4.03(d,J＝15.0Hz,1H ),3.95–3.85(m,2H),3.72(s,3H),3.71–3.65(m,1H),3.50(dd,J=16.9,9.1Hz,2H),3.40–3.35(m,2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 157.87, 155.28, 133.16, 131.18, 129.83, 129.61, 126.92, 121.88, 114.82, 113.24, 101.24, 76.80, 76.67, 73.62, 69.93, 61.11, 54.22, 34.31. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₂₀ H ₂₄ O ₇ Na, 399.1420, found 399.1418. HPLC analysis method 2, main peak retention time = 7.66 min, 97.51%.

(2R,3S,4S,5R,6S)-2-(Hydroxymethyl)-6-(3-(4-methoxybenzyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triol (C-30)

C-30 (121 mg, 57.1% for 2 steps) was obtained from intermediate 34q (120 mg, 0.56 mmol) according to Procedure A. White solid, melting point 132.1-133.5 °C, (c 0.25, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.15(t,J＝7.7Hz,1H),7.08(d,J＝8.4Hz,2H),6.89(d,J＝9.0Hz,2H),6.81(t,J＝7.7Hz,3H),4.84(d,J＝7.2Hz,1H),3.84(s,2H),3. 73 (s, 3H), 3.67 (dd, J = 12.0, 4.5Hz, 1H), 3.43 (dd, J = 9.8, 7.0Hz, 2H), 3.36 (t, J = 9.8Hz, 2H), 3.28 (d, J = 9.4Hz, 1H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 158.09, 157.75, 143.41, 133.02, 129.48, 128.86, 122.36, 116.70, 113.66, 113.41, 100.72, 76.59, 76.55, 73.45, 69.84, 60.96, 54.23, 40.37. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₂₀ H ₂₄ O ₇ Na, 399.1420, found 399.1419. HPLC analysis method 1, main peak retention time = 11.29 min, 97.53%.

(2R,3S,4S,5R,6S)-2-(Hydroxymethyl)-6-(4-(4-methoxybenzyl)phenoxy)tetrahydro-2H-pyran-3,4,5-triol (C-31)

Intermediate 34r (428 mg, 2.0 mmol) was used according to Procedure A to obtain C-31 (450 mg, 59.8% for 2 steps), a white solid, melting point 126.2-128.3 ° C, (c 0.34, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.05(t,J＝8.2Hz,4H),6.99(d,J＝8.6Hz,2H),6.79(d,J＝8.6Hz,2H),4.84(d,J＝7.2Hz,1H),3.89–3.84(m,1H),3.80(s,2H),3.72 (s,3H),3.67(dd,J＝12.1,4.9Hz,1H),3.43(dd,J＝8.1,4.0Hz,2H),3.41–3.35(m,2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 157.99, 155.95, 135.74, 133.58, 129.29, 129.26, 116.34, 113.38, 101.01, 76.63, 76.54, 73.50, 69.94, 61.07, 54.23, 39.67. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₂₀ H ₂₄ O ₇ Na, 399.1420, found 399.1419. HPLC analysis method 1, main peak retention time = 11.42 min, 97.87%.

(2S,3R,4S,5S,6R)-2-(4-(4-hydroxybenzyl)phenoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (C-32)

C-32 (150 mg, 60.0% for 2 steps) was obtained from intermediate 34s (200 mg, 0.69 mmol) according to Procedure A. White solid, melting point 215.1-217.2 °C, (c 0.20, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.06 (d, J = 8.5 Hz, 2H), 6.98 (d, J = 8.6 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H), 6.67 (d, J = 8.4 Hz, 2H), 4.85 (d, J = 7.4 Hz, 1H), 3.87 (d, J = 11.6 Hz, 1H), 3.78 (s, 2H), 3.67 (dd, J = 12.0, 4.8Hz, 1H), 3.44 (dd, J = 10.7, 7.9Hz, 2H), 3.37 (d, J = 7.0Hz, 2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 155.90, 155.12, 135.94, 132.41, 129.30, 129.22, 116.28, 114.69, 100.99, 76.63, 76.53, 73.49, 69.93, 61.06, 39.70. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₉ H ₂₂ O ₇ Na, 385.1263, found 385.1262. HPLC analysis method 2, main peak retention time = 5.16 min, 97.54%.

(2S,3R,4S,5S,6R)-2-(4-(3,4-dihydroxybenzyl)phenoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (C-33)

C-33 (100 mg, 42.0% for 2 steps) was obtained from intermediate 34t (250 mg, 0.63 mmol) according to Procedure A. White solid, melting point 215.1-217.2 °C, (c 0.25, methanol). ¹ H NMR (400MHz, CD ₃ OD) δ7.05(d,J＝8.5Hz,2H),6.97(d,J＝8.5Hz,2H),6.64(d,J＝8.0Hz,1H),6.55(d,J＝1.5Hz,1H),6.46(dd,J＝8.0,1.4Hz,1H),4.83(d,J＝ 7.3Hz,1H),3.85(d,J＝11.6Hz,1H),3.71(s,2H),3.66(dd,J＝12.0,4.7Hz,1H),3.46-3.40(m,2H),3.39-3.35(m,2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 155.88, 144.73, 142.97, 135.92, 133.21, 129.26, 119.65, 116.25, 115.55, 114.81, 101.01, 76.61, 76.53, 73.50, 69.94, 61.06, 39.93. HRMS (ESI) m/z: [M+Na] ⁺ calcd for C ₁₉ H ₂₂ O ₈ Na, 401.1212, found 401.1210. HPLC analysis method 2, main peak retention time = 4.60 min, 96.65%.

Pharmacological activity, cell and animal experiments

1 Preliminary physicochemical properties and absorption, distribution, metabolism and excretion (ADME) studies of the compounds

This part of the study was conducted at Shenyang Pharmaceutical University. The experimental animals involved were male Sprague-Dawley rats (7 weeks, weighing 200±20g), purchased from the Experimental Animal Center of Shenyang Pharmaceutical University, and approved by the Animal Experiment Ethics Committee of Shenyang Pharmaceutical University. Human plasma was obtained from the Shenyang Military Region General Hospital and stored in the Shenyang Military Region General Hospital Biological Specimen Bank. The collection of human plasma was approved by the Research Ethics Committee of Shenyang Military Region General Hospital.

①LgP _O/W

2 mL of drug aqueous solution of a certain concentration (C ₀ ) and 2 mL of octanol were added to a 5 mL glass tube. The mixture was shaken at room temperature for 3 days, centrifuged at 2500 rpm for 10 min, and the organic phase was discarded. The drug concentration in the liquid phase (C _w ) was determined by HPLC (L-2320, Hitachi, Japan) and L-2420 UV detector (Hitachi). The chromatographic column was Diamonsil·C18 (200 mm×4.6 mm, 5 μm) (Dima Technology, Beijing, China). Detection conditions: wavelength 275 nm; column temperature: 35.0°C; flow rate: 0.7 mL/min; injection volume: 20 μL; mobile phase ratio (methanol/water (v/v)): 91/18 salidroside, 70/30 C-29, C-30 and C-31, 50/50 C-32 and C-33. The apparent oil-water distribution coefficient ( _PO/W ) is calculated according to the formula: PO _/W = ( _C0 - _CW )/ _CW

②Solubility

Excessive drug was added to water, sonicated for 5 min, shaken at room temperature for 3 days, and centrifuged at 10,000 rpm for 5 min. The supernatant was diluted with water, and then 20 μL of the dilution was injected into HPLC (L-2320, Hitachi, Japan) to record the peak area and calculate the apparent solubility of the drug using the calibration curve.

③pKa

The absorbance of each drug at different pH values was determined by spectrophotometry, and then the pKa value was obtained through the Ig[In-]/[HIn]~pH linear curve.

④ Plasma stability

Add 100 μL of rat plasma to a 1.5 mL EP tube, and add 10 μL of sample solution to make the concentrations of SA and C-30 6.25, 12.5, and 25 mg/L, respectively (repeat 3 times). After mixing for 30 seconds, let stand at room temperature for 12 hours, and add 300 mL of methanol to quench the reaction. Vortex for 2 minutes, centrifuge at 10,000 rpm for 5 minutes, and determine the drug concentration in the supernatant by HPLC.

⑤ Plasma binding rate

The plasma protein binding rate was determined by equilibrium dialysis. 1 mL of human plasma was added to a dialysis bag that had been soaked in PBS buffer (pH 7.4) for several hours. The dialysis bag was soaked in 10 mL of the drug solution and placed at 4°C until the drug diffusion reached equilibrium. The dialysate with a positive protein test was discarded. HPLC (L-2320, Hitachi, Japan) was used to determine the drug concentration before and after dialysis. The plasma protein binding rate was calculated according to the following formula:

PPB=(D _t -D _f )/D _t ×100%=(1-D _f /D _t )×100%

PPB: plasma protein binding rate; D _t : pre-dialysis drug concentration; D _f : post-dialysis free drug concentration.

⑥Muscle stability

Reagents and instruments were precooled at 4°C. After the rats were killed, the gastrocnemius muscles were taken. Muscle homogenate was prepared by grinding 100 mg of muscle in 400 mL of normal saline. 50 μL of the homogenate was added to 250 μL of the drug solution, incubated at 37°C for 30 min, 900 mL of methanol was added to stop the reaction, and centrifuged at 10,000 rpm for 5 min. The drug concentration in the supernatant was determined by HPLC (L-2320, Hitachi, Japan).

⑦ Liver microsome stability

Rats were fasted overnight and then sacrificed. Liver homogenate was prepared by mixing 100 mg of liver with 400 mL of sucrose solution using a tissue homogenizer in an ice-water bath. The homogenate was centrifuged at 20,000 × g, 4°C for 20 min. The supernatant was centrifuged at 100,000 × g, 4°C for 60 min. The precipitate was resuspended in Tris-HCl buffer (pH 7.4, containing 10 Mm MgCl ₂ and 10 mM KCl). 10 μL of microsomes were added to different concentrations of drug solution and pre-incubated at 37°C for 10 min. The reaction was started by adding 1 mM NADPH. After incubation for 30 min, 3 volumes of methanol were added to stop the reaction. The mixture was centrifuged at 10,000 rpm for 5 min. The drug concentration in the supernatant was determined by HPLC (L-2320, Hitachi, Japan).

⑧Caco-2 cell permeability assay

Caco-2 cells at passage number 35 to 45 were seeded in Transwell chambers (1.12 cm2 surface, 0.4 μm pore size, 12 mm diameter; Corning Costar, Corning, NY) in 12-well plates and grown at a density of 1-1.5×105 cells/cm2 for 21 days. The TEER of the monolayer cells was measured using Millicell ERS-2 (Millipore, Billerica, MA), and the resistance TEER value should exceed 400Ω·cm2. The apparent permeability coefficient of phenol red, P _app (AB), and the efflux rate (ER) of digoxin (a P-gp substrate) were used as reference controls for the successful establishment of the Caco-2 monolayer model.

All compounds were dissolved in 100% HBSS to provide 50 μg/mL and used as drug solutions. Prior to the permeation experiment, the Caco-2 cell monolayer was gently rinsed twice with preheated (37°C) HBSS and incubated at 37°C for 30 minutes. For experiments of transport from the apical (AP) side to the basolateral (BL) side, 0.5 mL of drug solution was added to the AP side and 1.5 mL of HBSS was added to the BL side. After incubation at 37°C for 120 minutes, samples (0.4 mL) were collected from the BL side. For permeation experiments from BL to AP, 1.5 mL of sample was added to the BL side and 0.5 mL of HBSS was added to the AP side. 0.4 mL of sample was collected from the AP side at the above time intervals. The drug concentration in the sample was determined by HPLC-UV analysis. All incubations were performed in triplicate.

2 Cell culture

① Cell culture

Mouse myoblasts C2C12, vascular endothelial cells human umbilical vascular endothelial cells (HUVEC), smooth muscle cells MOVAS and human colon epithelial cell line Caco-2 cells were purchased from American Type Culture Collection (ATCC). All cells were cultured in Dulbecco's modified Eagle medium (DMEM) (Gibco, Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Biological Industries, Beit Hameke, Israel) in a high humidity incubator (37°C, 5% CO2). Before use, C2C12 cells were cultured in DMEM containing 1% fetal bovine serum for 5 days to differentiate into mature skeletal muscle cells (Watanabe Y, et al., Proc. Natl. Acad. Sci. U.S.A. 2016, 113, 6011-6016).

②Gene knockdown experiment

In the HIF-1α knockout experiment, C2C12 cells were transfected with a blank vector (shCon) or a shRNA expression vector targeting HIF-1α (shHIF-1α) according to the instructions of Lipofectamine 2000 (Invitrogen, Grand Island, NY). After 6 hours, the medium was changed and incubated for another 24 hours, and then the non-transfected cells were eliminated with puromycin (2.5 μg/mL, 36 hours).

③Hypoxia experiment under hyperglycemia or normoglycemia conditions

The cells were cultured in DMEM medium containing 25 μM (high glucose concentration) or 5.5 μM (normal glucose concentration) glucose for 24 h, and then the corresponding DMEM without FBS was added. After 12 h of treatment with or without drug addition, the cells were placed in a hypoxic box (Anaeropouch Box, 0.1% O2, Mitsubishi Gas Chemical, Tokyo, Japan), and then the hypoxic box was placed in an incubator and cultured until the set experimental time.

3. Plasmid construction

① Construction of dual luciferase reporter gene plasmid

1) 5×HRE-Luc reporter gene plasmid

The luciferase reporter gene plasmid is a luciferase reporter vector plasmid driven by five tandem hypoxia response elements (5×HRE).

2) PDGF-B-Luc reporter gene plasmid

a. Genomic DNA was extracted from mouse C2C12 cells using the TIANamp Genomic DNA Kit (Tiangen Biotechnology, Beijing, China) as a template, and the -673 to +268 region of mouse PDGF-B (NC_000081.7) was used Max DNA polymerase (Takara Bio, Dalian, China) was used for amplification (upstream primer: 5'-CGC GATATC GCATCTTGGTGGCAGTCCTT-3', downstream primer: 5'-CTG AAGCTT GCTCGGGTCAGTCTGTCTAT-3'), and the amplified products were separated by gel electrophoresis and recovered to obtain the purified target DNA fragment.

b. The amplified DNA fragment and the PGL4.13 reporter gene vector plasmid were double-digested with EcoRI and HindIII, and the double-digested vector plasmid was subjected to gel electrophoresis to obtain the desired vector fragment through a gel recovery kit.

c. The target DNA fragment after double enzyme digestion was linked with the purified vector fragment by enzyme, transformed into Escherichia coli, plated, and cultured at 37°C overnight.

d. Pick a certain number of single colonies and perform colony PCR.

e. Separate the products obtained by colony PCR by agarose gel electrophoresis, and culture the colonies that can amplify the target band in a small amount at 37°C overnight.

f. After the plasmid is extracted with a small amount extraction kit, it is double-digested with EcoRI and HindIII, and then the result is identified by agarose gel electrophoresis. The plasmid that can electrophores out the target fragment after double digestion is sequenced.

g. Compare the sequencing results with the target fragment sequence. If the sequences are consistent, it means that the plasmid construction has been successful.

②Construction of shHIF-1α knockdown plasmid

For knockdown of HIF-1α, shRNA expression vectors targeting mouse HIF-1α (NM_001313919) were constructed according to the method described in the literature (Miyagishi M, et al., Oligonucleotides, 2003, 13(5): 325-33). For the blank vector (shCon), a vector containing 7 thymines downstream of the U6 promoter was used. 4 Dual luciferase reporter gene activity screening experiment

① 1×10 ⁴ C2C12 cells were seeded into each well of a 24-well plate and cultured under high glucose conditions for 24 hours.

② According to the instructions of Lipofectamine 2000 (Invitrogen, Grand Island, NY), the 5xHRE-luc or PDGF-B-Luc reporter gene vector and the Renilla luciferase expression plasmid (pRL-SV40, Promega, Fitchburg, WI) as an internal reference were mixed and co-transfected into C2C12 cells.

③ After 6 hours of plasmid transfection, replace the serum-free high-glucose medium, add the specified concentration of compound or blank solvent, continue to incubate stably for 12 hours, and then place it in a hypoxia box for further incubation for 24 hours.

④ Take out the 24-well plate from the hypoxia box, add 100 μL of reporter gene cell lysis solution to each well, and shake for 15-20 minutes to fully lyse the cells.

⑤ Collect the lysate, centrifuge at 10,000 rpm for 5 minutes, take the supernatant for the next step of measurement, or place it at -80℃ for further measurement.

⑥ Under light-proof conditions: Take 20 μL of the lysate supernatant from each well and add it to a 96-well white detection plate. Simultaneously add 30 μL of LARII solution to each well, measure the first chemiluminescence value, and record the data.

⑦ Add 30 μL Stop&Glo detection solution to the above detection wells, measure the second chemiluminescence value data, and record the data.

⑧ Compare the first measured value with the second measured value to calculate the relative luminescence value.

5RNA extraction and real-time fluorescence quantitative PCR (qPCR)

① RNA extraction and reverse transcription

1) C2C12 cells treated with the compound or its blank solvent for 12 hours under high glucose conditions in a 6-well plate were cultured under hypoxic conditions for 12 hours.

2) Remove the cells, quickly wash twice with PBS, add 1.0 mL of Trizol lysis buffer to each well, gently blow the cells to ensure that all cells are completely detached, and collect the lysis buffer into a 1.5 mL sterile EP tube.

3) Add 500 μL of chloroform to the lysate, vortex vigorously for 30 seconds, and place in an ice bath for 5 minutes.

4) Set the tube to centrifuge at 10,000 rpm for 15 min at 4°C, then transfer the supernatant to a new 1.5 mL sterile EP tube, add 500 μL of isopropanol, mix by inverting three times, let stand in an ice bath for 10 min, and then centrifuge at 10,000 rpm for 15 min at 4°C.

5) Pour off the supernatant, carefully wash the white solid at the bottom of the EP tube with 1 mL of 75% ethanol, and then centrifuge at 10,000 rpm for 5 minutes at 4°C.

6) Pour off the upper layer of ethanol, place the EP tube on an ice box, carefully remove or air dry the residual ethanol on the tube wall, and use an appropriate amount of DEPC water to completely dissolve the precipitate at the bottom of the EP tube to obtain the sample RNA solution.

7) The concentration of each RNA sample was determined using Nanodrop 2000 (Thermo, Waltham, MA), and 1 μg of RNA was taken for reverse transcription according to the instructions of the PrimeScript Reagent Kit with gDNA Eraser (Takara Bio, Dalian, China). The excess RNA was stored at -80°C. The cDNA solution obtained by reverse transcription was diluted 10 times with DEPC water and used as the template solution for subsequent real-time fluorescence quantification, and stored at -20°C for later use.

② Real-time fluorescence quantitative PCR (qPCR)

1) Prepare the PCR reaction solution for each well according to Table 1:

Table 1. qPCR reaction system

2) Perform qPCR reaction in a Bio-Rad fluorescent quantitative PCR instrument by setting the following temperature and time control process according to Table 2. In this embodiment, the internal reference gene used in the qPCR experiment is β-Actin.

Table 2. Temperature and time control process for qPCR

6. Protein Extraction and Western Blot Analysis

① C2C12 cells treated with the compound or its blank solvent for 12 hours under normal or high glucose conditions were cultured under hypoxic conditions for 24 hours.

② Wash the cells and add RIPA lysis buffer containing a mixture of protease inhibitors (PMSF and phosphatase inhibitors (complete cocktail). Scrape the cells and transfer all the cells and lysis buffer into a 1.5 mL EP tube and place it in an ice bath for 10 minutes.

③ Centrifuge at 10,000 rpm and 4°C for 15 min and transfer the supernatant to a new 1.5 mL EP tube.

④The protein concentration of the samples was detected using the BCA protein quantification kit (Biyuntian, Shanghai, China).

⑤ Take equal amounts of protein and separate them on SDS-PAGE gel electrophoresis. After the separation, transfer the protein bands to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA) with a pore size of 0.45 μm or 0.22 μm (for proteins ≤10 kDa), place the entire membrane with the protein side facing up, add 5% skim milk powder/PBS solution, and block at 4°C overnight.

⑥ Pour away the milk, add the primary antibody diluent prepared according to the ratio, shake slowly at room temperature for 1.5 hours, and wash the membrane 3 times with TBST, each time for 5 to 10 minutes.

⑦Add secondary antibody diluent at room temperature, shake slowly for 1.5 hours, and wash 3 times with TBST, each time for 5-10 minutes.

⑧Use SuperSignal West Femto Maximum Sensitivity Substrate developer (Thermo, Waltham, MA) for protein development and photography.

7. Cell Viability Assay

① The cells were seeded in 96-well plates (4x10 ³ cells/well), cultured under high blood glucose conditions for 24 hours, and treated with the specified concentration of compounds or blanks. After 12 hours, the wells were placed in a hypoxic box and incubated in an incubator for 12 hours, 24 hours, or 36 hours. The standard curve was prepared using the CCK-8 kit (Dojindo, Kumamoto, Japan) according to the instructions.

② Detect the absorbance of each well in each plate at the specified time, and use the absorbance to calculate the number of cells in each well according to the standard curve.

8EdU cell proliferation assay (5-ethyl-2'-deoxyuridine incorporation labeling)

According to the Cell-Light ^TM EdU The assay was performed according to the instruction manual of 488 In Vitro Imaging Kit (Ruibo Biotech, Guangzhou, China). The specific experimental steps are as follows:

① Sample preparation: C2C12 cells were treated with the specified compounds or their blank solvents in a 24-well plate under normal or high glucose conditions for 12 hours, and then incubated under hypoxia for 24 hours.

② Dilute the EdU reagent with culture medium at 1:1,000 and add it to each well of cells to a final concentration of 25 μM.

③ Place in the incubator for 1.5 hours, remove the culture medium, and wash the cells twice with PBS, each time for 5 minutes.

④ Add 200 μL of 4% paraformaldehyde solution to each well and fix for 10 minutes. Wash the cells twice with PBS, each time for 5 minutes.

⑤ Add 200 μL of permeabilization agent (0.3% TritonX-100) to each well and let stand for 10 minutes. Wash the cells twice with PBS, each time for 5 minutes.

⑥ Add 200 μL of Azide 488 reaction solution prepared according to the instructions, react at room temperature in the dark for 30 minutes, and wash the cells with PBS three times, 5 minutes each time.

⑦Add 200 μL of Hoechst 3342 dilution to stain the cell nucleus and let stand at room temperature for 10 minutes. Wash the cells with PBS

3 times, 5 minutes each time.

⑧ Add 200 μL of anti-fluorescence quencher to each well.

⑨ Images were collected using a fluorescence microscope DMI6000B (Leica, Heidelberg, Germany), and the software Image J was used for counting and quantification.

9 Cell migration assay

① C2C12 cells were cultured under normal or high blood sugar conditions, treated with the specified compounds or blank solvent for 12 hours, and then cultured under hypoxia for 12 hours.

② After hypoxia, the cells were inoculated in the upper chamber of the transwell (Corning Costar, Corning, NY) (8×10 ³ cells/chamber), and the corresponding culture medium was added to the lower chamber. The cells were then placed in a hypoxic state for 24 hours to allow the cells to migrate.

③ Use a cotton swab to gently wipe off the cells that have not migrated in the upper chamber. After the cells that have migrated to the lower chamber (outside the transwell) are stained with crystal violet for 1 hour, the crystal violet staining solution is aspirated, and the inside and outside of the transwell are washed once each with PBS (Note: Multiple washing is not recommended, as the stained cells will also be washed away), and excess water on the chamber is aspirated with a cotton swab or filter paper.

④ The cells that migrated to the lower chamber were photographed using Olympus IX71 (Olympus, Tokyo, Japan), and the number of cells in each photo was recorded for statistics.

In experiments using conditioned medium (CM), HUVECs or MOVAS cells were seeded in the upper chamber, and the corresponding conditioned medium was also added to the lower chamber of the transwell, which was then placed in a hypoxic environment for 24 hours.

10 ELISA

The contents of VEGF-A and PDGF-BB in the conditioned medium were determined using a mouse VEGF-A ELISA kit (Xinbosheng Biotechnology, Shenzhen, China) and a mouse PDGF-BB ELISA kit (Sizhengbai Biotechnology, Beijing, China), respectively.

11F-actin phalloidin staining

① Cells were seeded in 15 mm glass-bottomed cell culture dishes at 8×10 ³ /well, treated with designated compounds or blank solvents under designated culture medium conditions for 12 hours, and then placed in hypoxia for another 12 hours.

② Remove the culture medium, wash twice with PBST, add 500 μL 4% paraformaldehyde to each culture dish to fix the cells for 10 minutes, and wash twice with PBST.

③Permeabilize with 0.1% Triton X-100 for 10 min and wash twice with PBST.

④ Block with 1% bovine serum albumin for 1 hour.

⑤ Pour off the blocking solution and add 200 μL of phalloidin dilution solution (5 μL phalloidin + 195 μL 1% bovine serum albumin) to each culture dish according to the instructions of phalloidin (Invitrogen, Grand Island, NY). Stain for 30 minutes at room temperature in the dark.

⑥ Remove the staining solution, wash twice with PBST, and add 200 μL of anti-fluorescence quenching solution.

⑦ A confocal microscope (Microsystems-TCS SP5, Leica, Wetzlar, Germany) was used to obtain cell F-actin images, and the fractal dimension was quantified using Image J software.

In experiments using conditioned medium, HUVECs or MOVAS cells were seeded in the specified conditioned medium, incubated under hypoxic conditions for 12 h, and then F-actin staining was performed according to the above procedure.

12 Apoptosis rate determination

C2C12 cells were treated with the indicated compounds or blank solution under normal or high blood sugar for 12 h and then incubated under hypoxia for 24 h. Cells were stained with Annexin V/PI (Yisheng Biotechnology, Shanghai, China), and the apoptosis rate was determined by flow cytometry.

13 In vivo experiments on lower limb ischemia model

①Establishment of diabetic mouse model

Male C57 BL/6 mice (8 weeks) were purchased from the Third Military Medical University, Chongqing, China. All animal experiments were performed at the Third Military Medical University and approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University and in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Ministry of Health. Mice were anesthetized by intraperitoneal injection of ketamine/xylazine (5 mg and 80 mg per kg body weight, respectively).

The diabetic mouse model was established as described in the literature (Surwit R S, et al., Diabetes, 1991, 40 (1): 82-7). Briefly, mice were randomly divided into groups, fed a high-fat diet (20% protein, 20% sugar, 60% fat) for 3 weeks, and then injected with freshly prepared streptozotocin (45 mg/kg, Sigma Aldrich, St. Louis, MO) for 5 consecutive days. After waiting for 1 week after the last injection, the mice were fasted overnight, and blood was taken from the tail. The blood glucose concentration of the mice was measured using an Accu-Chek Integra blood glucose meter (Roche Diagnostics, Shanghai, China). If the blood glucose level was ≥16.7 mmol/L, diabetes was considered to be successfully induced.

②Establishment of diabetic or non-diabetic HLI model mice, administration cycle and injury assessment

To establish the HLI model, diabetic or non-diabetic mice were anesthetized as described above, and then the hind limb area was depilated and the proximal end of the left femoral artery was completely removed (Scholz D, et al., J Mol Cell Cardiol, 2002, 34(7):775-87; Stabile E, et al., Circulation, 2003, 108(2):205-10; Wu S, et al., Mol Ther, 2008, 16(7):1227-34). The right femoral artery was not operated on and served as a control. Starting from the day after arterectomy, the designated compounds or blank solution were injected into the ischemic lower limb muscles every 3 days. Mice were randomly assigned after surgery, and the researchers were unaware of the group status during the evaluation process. Ischemic injury was assessed on a scale of 0-4, as described in the literature, 0 = no difference from the non-ischemic hindlimb; 1 = mild discoloration; 2 = moderate discoloration; 3 = severe discoloration, subcutaneous tissue loss or necrosis; 4 = amputation, up to 21 days after arteriotomy (Stabile E, et al., Circulation, 2003, 108(2):205-10.).

③ Laser Doppler blood perfusion imaging

Mice were anesthetized as described above. Blood perfusion was measured in the ischemic (left) and non-ischemic (right) hindlimbs before and after surgery and at designated times (days 0, 3, 7, 14, and 21) using a laser Doppler perfusion imager (Moor Instruments Ltd, Axminster, Devon, UK). The ratio of the blood perfusion measured in the left ischemic hindlimb to the blood perfusion measured in the right non-ischemic hindlimb was the restored blood perfusion rate.

14 Hematoxylin-eosin (H&E) and immunofluorescence staining For H&E staining, tissues were fixed with 4% paraformaldehyde and then embedded in paraffin. They were then sectioned at 10 μm thickness in a cryostat. Sections were dewaxed and rehydrated with xylene. Hematoxylin and eosin (Biyuntian, Shanghai, China) were used for staining as directed.

For immunofluorescence staining, transverse sections of gastrocnemius muscle tissue of mouse lower limbs with a thickness of 10 μm were taken and incubated with PECAM-1 antibody for 1 hour. Then, monoclonal antibody α-SMA (Sigma-Aldrich, St. Louis, MO) conjugated with Cy3 dye and goat anti-rat IgG (Beyond Sky, Shanghai, China) were incubated with Alexa Fluor 488 (Invitrogen, Grand Island, NY). Images were obtained by Microsystems-TPS SP8 (Leica, Heidelberg, Germany).

15TUNEL assay

Tissue slides were permeabilized in 0.1% Triton X-100 and TUNEL-positive cells were detected according to the manufacturer's instructions of the Fluorescein in situ Cell Death Detection Kit (Roche Applied Science, Mannheim, Germany). Images were captured using a Pannoramic Midi (3DHistech, Budapest, Hungary).

16 Statistical analysis

The experimental results are expressed as mean ± SD. The experimental results are shown in Tables 3-6. SPSS Statistics v.23.0 was used for statistical analysis. One-Way ANOVA was used for statistical analysis in cell experiments. The non-parametric Mann-Whitney test was used for animal experiments. The differences between the two groups were analyzed by Student's t test for statistical significance. *P<0.05 was considered significant.

Table 3. Results of activity test of derivatives

Table 4. Caco-2 cell permeability test results of salidroside and its derivatives

The apparent permeability (P _app ) was calculated using the following formula: dC/dt×V is the transfer rate on the acceptor side (μg/s), where d is the detection concentration after sample permeation, dt is the sampling time, and V is the sampling volume; A is the membrane surface area (1.12 cm ² ), and C ₀ is the initial drug concentration in the donor compartment (μg/mL);

The outflow rate (ER), the ratio of P _app (basal to apical) to P _app (apical to basal), was calculated using the following formula:

All data represent mean ± standard error (three independent experiments)

Table 5. Preliminary ADME studies of salidroside and C-30

Table 6. Blood glucose in diabetic hind limb ischemic disease mice

The above describes the preferred embodiments of the present invention, but it is not intended to limit the present invention. Those skilled in the art may make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims (5)

1. The application of salidroside derivative selected from C-30 and/or C-31 in preparing medicine for treating lower limb ischemic diseases and/or relieving altitude stress,

2. the use according to claim 1, the medicament being for angiogenesis.

3. The use according to claim 1, the medicament being for use in blood perfusion recovery.

4. The use according to claim 1, wherein the medicament is an intramuscular injection formulation.

5. The use according to claim 1, wherein the salidroside derivative is used as a HIF-1 a inducer.