Preparation of glycoside compound and application of glycoside compound in acute kidney injury

Abstract

The invention belongs to the technical field of medicines, and discloses preparation of a glycoside compound and application of the glycoside compound in acute kidney injury, and the structural formula of the glycoside compound K30 is shown in the specification. The K30 compound is obtained by improving the chemical structure of the glycoside compound and the corresponding preparation method, and the K30 compound can be particularly used for treating acute kidney injury.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and more specifically, relates to the preparation of a glycoside compound and its application in acute kidney injury. This glycoside compound can treat acute kidney injury and provides a lead compound for the development of drugs for the treatment of acute kidney injury. Background Technology

[0002] Kidney disease is a public health problem affecting millions of people worldwide, including acute kidney injury and chronic kidney disease (Reference: Bao YW, Yuan Y, Chen JH, Lin WQ. Kidney disease models: tools to identify mechanisms and potential therapeutic targets[J]. Zool Res). Acute kidney injury is a clinical syndrome with high mortality caused by one or more factors. It is a common critical illness characterized by a rapid decline in kidney function and a decrease in glomerular filtration rate, specifically manifested as a significant increase in serum urea nitrogen and creatinine levels, and an increase in urinary albumin levels (Reference: Gonsalez SR, Cortês AL, Silva RCD, Lowe J, Prieto MC, Silva Lara LD. Acute kidney injury overview: From basic findings to new prevention and therapy strategies[J]. Pharmacol Ther). Currently, no specific drugs for the treatment of acute kidney injury have been successfully developed, and the focus of clinical intervention remains on prevention and early diagnosis. Early intervention can reduce the degree of renal function damage in patients. For patients diagnosed with acute kidney injury, the main clinical intervention is renal replacement therapy (including hemodialysis, peritoneal dialysis, or continuous renal replacement therapy). Although renal replacement therapy has made significant progress in recent years, the mortality rate of patients with acute kidney injury remains high, and the treatment cost remains high (Reference: Moore PK, Hsu RK, Liu KD. Management of Acute Kidney Injury: Core Curriculum 2018[J]. Am J Kidney Dis). Therefore, developing an effective drug for the treatment of acute kidney injury is of great significance in the clinical treatment of acute kidney injury.

[0003] The inventors previously reported the application of a glycoside compound in the preparation of a drug for treating kidney disease (see Chinese patent application CN117442633A). Using the natural product Kinsenoside (KD) with the structure shown below, kidney disease was treated. Furthermore, in treating folic acid-induced acute kidney injury, the optimal daily dose of KD corresponding to the best efficacy was 20 mg / kg (i.e., 20 mg of KD applied per kg of organism per day). Given that KD does not show a significant advantage in kidney disease treatment compared to the positive control drug telmisartan, further research is needed to obtain new compounds with better therapeutic effects on kidney disease.

[0004] Summary of the Invention

[0005] In view of the above-mentioned defects or improvement needs of the prior art, the purpose of this invention is to provide a method for preparing glycoside compounds and their application in acute kidney injury. By improving the chemical structure of glycoside compounds and the corresponding preparation method, K30 compound is obtained, which can be used to treat acute kidney injury and has significant advantages over KD.

[0006] To achieve the above objectives, according to one aspect of the present invention, a glycoside compound K30 is provided, characterized in that its structural formula is as follows:

[0007]

[0008] According to another aspect of the present invention, the present invention provides a method for synthesizing the above-mentioned glycoside compound K30, characterized in that the synthetic route is as follows:

[0009]

[0010] Specifically, the following steps are included:

[0011] (a) Compound 1 was dissolved in pyridine, and TMSCI was added dropwise to react and generate compound 2;

[0012] (b) Compound 2 was dissolved in DCM and anhydrous ethanol was added, followed by the dropwise addition of NH4OAC to react and generate compound 3;

[0013] (c) Compound 3 was dissolved in ultra-dry pyridine to obtain a pyridine solution of compound 3; at the same time, 3,3-dimethylacryloyl chloride was dissolved in ultra-dry DCM to obtain a DCM solution of 3,3-dimethylacryloyl chloride; at 0°C, the DCM solution of 3,3-dimethylacryloyl chloride was slowly added dropwise to the pyridine solution of compound 3, and then the reaction was carried out at room temperature to generate compound 4;

[0014] (d) Compound 4 was dissolved in DCM, and BF3·Et2O was added dropwise to produce compound K30.

[0015] According to another aspect of the present invention, the present invention provides the use of the above-mentioned glycoside compound K30 in the preparation of a drug for treating acute kidney injury.

[0016] As a further preferred embodiment of the present invention, the acute kidney injury is specifically cisplatin-induced acute kidney injury.

[0017] As a further preferred embodiment of the present invention, the acute kidney injury is specifically folic acid-induced acute kidney injury.

[0018] As a further preferred embodiment of the present invention, the glycoside compound K30 exerts its therapeutic effect on acute kidney injury by improving mitochondrial function.

[0019] As a further preferred embodiment of the present invention, the glycoside compound K30 exerts its therapeutic effect on acute kidney injury by inhibiting MST1.

[0020] As a further preferred embodiment of the present invention, the dose of the glycoside compound K30 applied per kg of organism per day is 200 mg.

[0021] Compared with the prior art, the present invention obtains a new glycoside compound K30 (structural formula shown below) by modifying the structure of KD, and evaluates its anti-acute kidney injury effect and mechanism for the first time, which can be used to treat acute kidney injury.

[0022]

[0023] The glycoside compound K30 can be used to treat cisplatin- and folic acid-induced acute kidney injury. It can exert its therapeutic effect on acute kidney injury by improving mitochondrial function and by inhibiting MST1.

[0024] As illustrated in the examples below, by evaluating the activity of the glycoside compound K30 in treating cisplatin- and folic acid-induced acute kidney injury, it was found that compound K30 can effectively inhibit the development of acute kidney injury in mice. In the cisplatin- and folic acid-induced mouse acute kidney injury model, it has activities such as improving renal function, inhibiting mitochondrial damage, inhibiting oxidative stress, and reducing the secretion of inflammatory factors, thereby effectively inhibiting inflammatory infiltration and oxidative stress damage in renal tissue, reducing renal tissue damage, and delaying the development of acute kidney injury.

[0025] In summary, this invention demonstrates that the K30 compound can effectively inhibit mitochondrial damage, inflammatory infiltration of renal tissue, and oxidative stress damage, thereby reducing renal tissue damage. It can serve as a lead compound for the development of drugs to treat acute kidney injury. Attached Figure Description

[0026] Figure 1 The protective effect of glycoside compound K30 against cisplatin-induced acute kidney injury was demonstrated, wherein:

[0027] Figure 1 In the figure, A corresponds to the serum creatinine and blood urea nitrogen levels in mice. In the control group (Con, physiological saline solution), cisplatin model group (CDDP, cisplatin 20 mg / kg), low-dose K30 group (CDDP-K30L, cisplatin: 20 mg / kg + K30: 50 mg / kg), medium-dose K30 group (CDDP-K30M, cisplatin: 20 mg / kg + K30: 100 mg / kg), high-dose K30 group (CDDP-K30H, cisplatin: 20 mg / kg + K30: 200 mg / kg), and positive control KD group (CDDP-KD, cisplatin: 20 mg / kg + KD: 200 mg / kg), K30 significantly reduced serum creatinine and blood urea nitrogen levels in mice, with a significantly better effect than the positive control KD.

[0028] Figure 1 In this context, B corresponds to the expression of NGAL and KIM-1 mRNA, markers of kidney injury, in mouse kidney tissue. K30 can downregulate the expression levels of NGAL and KIM-1 mRNA, markers of kidney injury, in mouse kidney tissue.

[0029] Figure 1 The "C" in the figure corresponds to the H&E staining results of mouse kidney tissue. K30 can significantly reduce renal tubular damage, inflammatory cell infiltration, and kidney tissue damage, and its effect is significantly better than that of the positive control drug KD.

[0030] Figure 1 The "D" in the figure corresponds to the PAS staining results of mouse kidney tissue. K30 can significantly reduce mesangial proliferation, reduce droplet degeneration, and reduce focal cell hypertrophy and tubular diameter increase in renal tubules.

[0031] Figure 1 The E in the figure corresponds to the TUNEL staining results of mouse kidney tissue. K30 can significantly inhibit apoptosis in kidney tissue.

[0032] Figure 1 The F in the figure corresponds to the detection results of apoptosis-related proteins in mouse kidney tissue. K30 can significantly reduce the expression of CDDP-induced apoptosis-related proteins PARP, Bax, and Cleaved Caspase 3, while upregulating the expression of apoptosis-inhibiting proteins Bcl-2 and Bcl-xl, and its effect is better than that of the positive control drug KD.

[0033] Figure 2 The study demonstrated that the glycoside compound K30 inhibits the cisplatin-induced inflammatory response, wherein:

[0034] Figure 2 The "A" in the figure corresponds to the detection results of inflammatory factor levels in mouse serum. The levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), chemokine ligand 2 (CCL2), tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGF-β) in mouse serum were measured. The results showed that K30 significantly downregulated the levels of inflammatory factors TGF-β, TNF-α, CCL2, IL-6, and IL-1β in mouse serum, with a significantly better effect than the positive control drug KD.

[0035] Figure 2 The "B" in the figure corresponds to the detection results of the mRNA expression levels of inflammatory factors in mouse kidney tissue. The expression of inflammatory factors TGF-β, TNF-α, CCL2, IL-6, and IL-1β mRNA in mouse kidney tissue was detected. The results showed that K30 significantly reduced the expression of TGF-β, TNF-α, CCL2, IL-6, and IL-1β mRNA in mouse kidney tissue, with an effect comparable to that of positive control drugs.

[0036] Figure 2 The "C" in the figure corresponds to the detection results of macrophage infiltration in mouse kidney tissue. Immunohistochemical detection of the levels of macrophage surface markers CD68 and F4 / 80 in kidney tissue showed that K30 could significantly reduce macrophage infiltration in mouse kidney tissue, with an effect comparable to that of positive control drugs.

[0037] Figure 2 The "D" in the figure corresponds to the detection results of expression of inflammatory signal-related proteins in mouse kidney tissue. Western blot analysis of IL-1β, TNF-α, COX2, C-JUN, p-ERK, p-JNK, p-P38, and p-P65 protein expression in mouse kidney tissue showed that K30 significantly inhibited the expression of these proteins in mouse kidney tissue, with a better effect than the positive control drug KD.

[0038] Figure 3 The study showed that the glycoside compound K30 inhibits cisplatin-induced renal oxidative stress, wherein:

[0039] Figure 3 The "A" in the table corresponds to the detection results of oxidative factors in mouse kidney tissue. The kit detects the levels of glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) in mouse kidney tissue homogenates. K30 significantly reduces the level of MDA, a lipid peroxidation product, in mouse kidney tissue, upregulates the levels of antioxidant enzymes GSH and SOD in the kidney, and inhibits renal oxidative stress, with effects comparable to positive control drugs.

[0040] Figure 3The value of B in the table corresponds to the detection results of 4-HNE, ROS, and CAT in mouse kidney tissue. Tissue section staining was used to observe the levels of 4-HNE, ROS, and CAT in mouse kidney tissue. K30 significantly reduced the levels of 4-HNE and ROS in kidney tissue and increased the level of CAT, showing a better effect than the positive control drug KD.

[0041] Figure 3 The C in the figure corresponds to the detection results of oxidative stress-related proteins in mouse kidney tissue. Western blot was used to detect the expression of NRF2, KEAP1, HO-1, NQO1, NOX4 and CAT proteins in mouse kidney tissue. The results showed that K30 could reverse the changes in protein expression induced by cisplatin.

[0042] Figure 3 The "D" in the diagram corresponds to the mitochondrial structure of mouse kidneys. Transmission electron microscopy revealed that mitochondrial morphology in mouse kidney tissue significantly inhibited cisplatin-induced mitochondrial structural damage.

[0043] Figure 3 The "E" in the figure corresponds to the results of detection of mitochondrial function-related proteins in mouse kidneys. Western blot analysis of mitochondrial function-related proteins COX-IV, ATP5B, SDHA, VDAC1 / 2, and PGC-1α showed that K30 could significantly reverse the changes in protein expression levels induced by cisplatin and inhibit mitochondrial functional damage.

[0044] Figure 4 The glycoside compound K30 is shown to target MST1, where:

[0045] Figure 4 The A in the sample corresponds to a possible target of K30 by mass spectrometry. After co-incubating the cells with the drug and control solvent (DMSO), the cells were treated at 54°C for 3 min, and mass spectrometry was performed. The results showed that K30 significantly increased the thermostability of DNM1, UFD1, MSI2, Cul4a, SWOAHA, MST1, TOM1, PPP1R2, and H2AC21 proteins, suggesting that K30 may have a direct interaction with these proteins.

[0046] Figure 4 The B in the equation corresponds to the molecular docking evaluation of the binding between K30 and MST1. The molecular docking calculation shows that the binding energy between K30 and MST1 is -7.9 kcal / mol. Hydrogen bonds may exist between K30 and the Ser109, Asp112, Gly153, Glu38, and Asp167 sites of MST1.

[0047] Figure 4 The C in the formula corresponds to the DARTS experiment, which verifies that K30 enhances the stability of the MST1 enzyme.

[0048] Figure 4 The D in the figure corresponds to the CETSA experiment, which verifies that K30 enhances the thermal stability of MST1.

[0049] Figure 4 The E value corresponds to the SPR experiment, which determined that the KD value between K30 and MST1 is 5.84 × 10⁻⁶. -5 M.

[0050] Figure 5 The study showed that the glycoside compound K30 targets MST1 to regulate mitochondrial homeostasis and mitophagy, wherein:

[0051] Figure 5 The effect of K30 on Drp1 expression and distribution was observed using immunofluorescence staining, corresponding to the A value in the diagram. K30 significantly inhibited Drp1 protein expression and reduced Drp1 mitochondrial translocation.

[0052] Figure 5 The B in the diagram corresponds to the quantitative map of Drp1 in mitochondria.

[0053] Figure 5 The "C" in the figure corresponds to the results of mitochondrial dynamics and mitophagy-related protein detection in mouse kidney tissue. Western blot analysis of the effects of K30 on mitochondrial dynamics and mitophagy-related protein expression showed that K30 significantly inhibited the expression of mitochondrial fission regulatory proteins p-JNK, p-Drp1, Drp1, MFF, Fis1 and mitophagy regulatory proteins LC3, Parkin, PINK, while promoting the expression of mitochondrial fusion proteins OPA1, MFN1, MFN2, and inhibiting excessive mitochondrial fission and autophagy.

[0054] Figure 5 The "D" in the figure corresponds to the results of mRNA expression detection in mouse kidney tissue related to mitochondrial dynamics. qPCR results showed that K30 significantly inhibited the mRNA expression of mitochondrial fission regulatory proteins JNK, Drp1, MFF, and Fis1, while restoring the mRNA expression of mitochondrial fusion proteins OPA1, MFN1, and MFN2, thus inhibiting excessive mitochondrial fission.

[0055] Figure 6 The study demonstrated that the glycoside compound K30, targeting MST1, improves folic acid-induced acute kidney injury, wherein:

[0056] Figure 6In the figure, A corresponds to the results of serum creatinine and blood urea nitrogen (BUN) levels in mice. Among the control group (Con, NaHCO3 solution), folic acid model group (FA-Model, folic acid: 250 mg / kg), low-dose K30 group (FA-K30L, folic acid: 250 mg / kg + K30: 50 mg / kg), medium-dose K30 group (FA-K30M, folic acid: 250 mg / kg + K30: 100 mg / kg), high-dose K30 group (FA-K30H, folic acid: 250 mg / kg + K30: 200 mg / kg), and positive control KD group (FA-KD, folic acid: 250 mg / kg + KD: 20 mg / kg), K30 significantly reduced serum creatinine and BUN levels in mice and improved renal function, with effects significantly superior to the positive control KD.

[0057] Figure 6 In this context, B corresponds to the expression of NGAL and KIM-1 mRNA, markers of kidney injury, in mouse kidney tissue. K30 can downregulate the expression levels of NGAL and KIM-1 mRNA, markers of kidney injury, in mouse kidney tissue.

[0058] Figure 6 The "C" in the table corresponds to the detection results of oxidative factors in mouse kidney tissue. The kit detects the contents of GSH, SOD, and MDA in mouse kidney tissue homogenate. K30 can significantly reduce the level of lipid peroxidation product MDA in mouse kidney tissue, upregulate the levels of antioxidant enzymes GSH and SOD in the kidney, and reduce oxidative stress in kidney tissue, with effects comparable to the positive control drug KD.

[0059] Figure 6 The "D" in the graph corresponds to the quantitative assessment of kidney injury scores in mice. K30 significantly inhibits renal tubular injury and reduces kidney injury scores, with a significantly better effect than KD.

[0060] Figure 6 The "E" in the figure corresponds to the H&E, PAS, and Masson staining results of mouse kidney tissue. K30 can significantly reduce renal tubular damage, inflammatory cell infiltration, mesangial proliferation, droplet degeneration, and collagen fiber deposition in mice, and inhibit renal tissue lesions and fibrosis.

[0061] Figure 6 The F in the figure corresponds to the detection results of mitochondrial function-related proteins in mouse kidney tissue. In a folic acid-induced acute kidney injury model, K30 can significantly inhibit MST1 protein expression, while upregulating the expression of COX-IV, NDUFV2, ATP5B, SDHA, VDAC1 / 2, and PGC-1α proteins, thus improving mitochondrial function.

[0062] Figure 6The G in the figure corresponds to the expression results of mitochondrial dynamics-related proteins in mouse kidney tissue. In a folic acid-induced acute kidney injury model, K30 significantly inhibited the expression of Drp1, p-Drp1, MFF, and Fis1, while upregulating the expression of MFN1, MFN2, and OPA1, thus inhibiting excessive mitochondrial fission.

[0063] Figure 7 It is the K30 compound obtained in Example 1. 1 H NMR spectrum.

[0064] Figure 8 It is the K30 compound obtained in Example 1. 13 C NMR spectrum.

[0065] Figure 9 This is a diagram illustrating the mechanism by which K30 compounds exert their anti-acute kidney injury effects. Detailed Implementation

[0066] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0067] Example 1

[0068] The synthesis and structural identification of the glycoside compound K30 are shown below.

[0069]

[0070] Includes the following steps:

[0071] (a) Compound 1 (i.e., KD; 5.28 g) was dissolved in pyridine (50 mg: 2 mL; i.e., 2 mL of pyridine for every 50 mg of compound 1), and TMSCl (5.02 eq) was added dropwise. TLC was used for detection. The reaction was completed after 36 hours. The target product was extracted. 27.7 g of product, i.e., compound 2, was obtained, with a yield of 70%.

[0072] (b) Compound 2 (7.7 g) was dissolved in a mixture of DCM and ethanol (volume ratio 1:1, compound 2 concentration: 0.2 M), and then NH4OAC (2 eq) was added dropwise. The reaction was monitored by TLC. The reaction was allowed to proceed for 24 h. The reaction was complete. 36.1 g of product, compound 3, was obtained, with a yield of 91%.

[0073] (c) Compound 3 (2.0 g) was dissolved in ultradry pyridine (2.5 eq) in a reaction flask. 3,3-Dimethylacrylyl chloride (i.e., (CH3)2C=CHCOCl; 1 eq) was dissolved in ultradry DCM (0.1 M). The acrylyl chloride DCM solution was slowly added dropwise to the reaction flask at 0 °C, and then the reaction was allowed to proceed to room temperature. TLC analysis was performed. The final product was 42.2 g, namely compound 4, with a yield of 94%.

[0074] (d) Compound 4 (2.2 g) was dissolved in DCM (0.1 M), and BF3·Et2O (4 eq) was added dropwise. The reaction was detected by TLC. The reaction was stopped after 30 min. Purification was performed by column chromatography using dichloromethane / ethanol (v / v 15:1) as the eluent, yielding 1.3 g of a white solid K3O (96% yield).

[0075] The H and C spectra of product K30 are as follows: 1H NMR(400MHz,D2O)δ5.81(q,J=1.3Hz,1H),4.61–4.52(m,3H),4.44(dd,J=12.3 ,2.4Hz,1H),4.34(dd,J=12.3,5.5Hz,1H),3.69(ddd,J=8.0,5.4,2.2Hz,1H), 3.56–3.42(m,2H),3.28(dd,J=9.0,7.9Hz,1H),2.99(dd,J=18.5,6.3Hz,1H), 2.71(dd,J=18.4,1.7Hz,1H),2.14(d,J=1.3Hz,3H),1.95(d,J=1.4Hz,3H).13C NMR(101MHz,D2O)δ179.6,168.5,161.0,114.2,101.5(C-1),75.6,75.3,75.1,73.6,72.8,69.6,62.2,35.2,26.7,19.9.HRESIMSm / z calcd.for C15H22O9Na369.1162[M+Na]+,found 369.1155.such as Figure 7 , Figure 8 As shown.

[0076] The product K30 was found to have a purity >99.0% by HPLC.

[0077] Example 2

[0078] The glycoside compound K30 obtained in Example 1 was used to treat cisplatin-induced acute kidney injury. Sixty SPF-grade male 8-week-old C57BL / 6 mice were housed in the SPF-grade Laboratory Animal Center of Tongji Medical College, Huazhong University of Science and Technology, and were provided with standard diet and water. The room temperature was controlled at 23±2℃. The experimental protocol involving mice was approved by the Animal Experiment Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. After 7 days of acclimatization, mice were randomly divided into a control group (Con, saline), a model group (CDDP, cisplatin: 20 mg / kg), a low-dose K30 group (CDDP-K30L, cisplatin: 20 mg / kg + K30: 50 mg / kg), a medium-dose K30 group (CDDP-K30M, cisplatin: 20 mg / kg + K30: 100 mg / kg), a high-dose K30 group (CDDP-K30H, cisplatin: 20 mg / kg + K30: 200 mg / kg), and a positive control group (CDDP-KD, cisplatin: 20 mg / kg + KD: 200 mg / kg), with 10 mice in each group. Each group was given the corresponding dose of the test drug once a day for 3 days. 30 minutes after the first administration, an acute kidney injury model was established by intraperitoneal injection of 20 mg / kg cisplatin solution. The mice were harvested after 3 days of administration. The results of renal function tests in mice showed that K30 could significantly reduce serum creatinine and blood urea nitrogen levels in mice and improve renal dysfunction in mice (see...). Figure 1 (A) The results of kidney injury marker detection showed that the expression of KIM-1 and NGAL mRNA in mouse tissue samples was significantly downregulated after K30 treatment (see A). Figure 1 (B in the text). Kidney H&E staining results showed that K30 reduced renal tubular damage and inflammatory cell infiltration in renal tissue in mice, and improved renal pathological changes (see [reference]). Figure 1 (C) Kidney PAS staining showed that K30 significantly reduced mesangial proliferation, decreased droplet degeneration, reduced focal tubular cell hypertrophy, and decreased tubular diameter thickening (see C). Figure 1 (D in the text). Kidney TUNEL staining results showed that K30 significantly reduced kidney cell apoptosis (see D). Figure 1 The results of renal tissue apoptosis-related protein expression level detection showed that K30 could significantly reverse the changes in apoptosis-related protein expression levels induced by cisplatin (see E). Figure 1 (F in the middle).

[0079] The detection of inflammation-related markers in mice showed that K30 significantly reduced the levels of inflammatory factors IL-1β, IL-6, MCP-1, TNF-α, and TGF-β in mouse serum (see...). Figure 2 (A) The expression levels of IL-1β, IL-6, MCP-1, TNF-α, and TGF-β mRNA in mouse kidney tissue samples were detected. The results showed that K30 could downregulate the expression of inflammatory cytokine mRNA in mouse kidney tissue (see A). Figure 2 (B) Macrophage infiltration in mouse kidney tissue was detected. The results showed that K30 significantly reduced the expression of macrophage markers CD68 and F4 / 80 in kidney tissue and reduced macrophage infiltration (see B). Figure 2 (C in the text). The expression of proteins related to inflammatory cytokine signaling pathways was detected, and the results showed that K30 significantly inhibited cisplatin-induced activation of inflammatory signaling pathways (see [link to article]). Figure 2 (D in the middle).

[0080] The results of oxidative stress-related marker detection showed that K30 significantly upregulated the levels of antioxidant enzymes GSH and SOD in mouse kidney tissue homogenate, while downregulating the level of lipid peroxidation product MDA in kidney tissue (see...). Figure 3 (A) Tissue section staining was used to detect 4-HNA, ROS, and CAT levels. The results showed that K30 significantly downregulated 4-HNA and ROS levels in kidney tissue and upregulated CAT levels in kidney tissue (see A). Figure 3 (B in the text) The expression changes of oxidative stress-related signaling pathways in mouse kidney tissue were detected, and the results showed that K30 could significantly reverse the changes in oxidative stress-related protein expression induced by cisplatin (see [link to article B]). Figure 3 (C in the text). Transmission electron microscopy was used to observe changes in mouse mitochondrial structure, and the results showed that K30 could significantly inhibit cisplatin-induced mitochondrial structural damage (see C). Figure 3 (D in the text). The expression of mitochondrial function-related proteins in mouse kidney tissue was detected, and the results showed that K30 significantly upregulated the expression levels of mitochondrial respiratory chain and function-related proteins (see D). Figure 3 (E in the text)

[0081] The target of compound K30 was investigated and identified. HK2 cells were treated with K30 at 37℃ for 1 h, cell samples were collected, and the cells were then treated in a PCR instrument at 54℃ for 3 min. After three freeze-thaw cycles in liquid nitrogen, the protein solution was collected by centrifugation. Mass spectrometry was used to detect differentially expressed proteins between the K30-treated group and the blank control group. The results showed that K30 significantly upregulated the protein levels of DNM1, UFD1, MSI2, Cul4a, SWOAHA, MST1, TOM1, PPP1R2, and H2AC21, suggesting that K30 may interact with these proteins (see...). Figure 4 (A) Molecular docking calculations were performed to determine the binding affinity between K30 and MST1. The results showed that the lowest binding energy between K30 and MST1 was -7.9 kcal / mol, and possible binding sites for K30 and MST1 were predicted (see A). Figure 4 (B in the text). DARTS and CETSA confirmed that K30 can increase the stability and thermostability of MST1 enzyme (see B). Figure 4 C and Figure 4 In the D), the SPR experimental results showed that the KD value of K30 combined with MSTI was 5.84 × 10⁻⁶.-5 M, the results show that K30 and MST1 have a direct binding.

[0082] Immunofluorescence staining was used to observe the expression and distribution of Drp1 in kidney tissue. The results showed that K30 significantly inhibited Drp1 protein expression and also inhibited Drp1 translocation to mitochondria (see...). Figure 5 A and Figure 5 (B in the text) The expression of downstream proteins involved in the regulation of mitochondrial dynamics by MST1 was examined. The results showed that K30 significantly downregulated the expression of p-JNK, Drp1, p-Drp1, MFF, and Fis1, while upregulating the expression of OPA1, MFN1, and MFN2, thus inhibiting excessive mitochondrial fission (see [link to article B]). Figure 5 C and Figure 5 (D in the text). The effect of K30 on mitophagy was examined, and the results showed that K30 significantly inhibited the expression of LC3, Parkin, and PINK, thus suppressing excessive mitophagy (see D). Figure 5 (C in the middle).

[0083] Example 3

[0084] The glycoside compound K30 obtained in Example 1 was used to treat folic acid-induced acute kidney injury. Sixty SPF-grade male 8-week-old C57BL / 6 mice were housed in the SPF-grade Laboratory Animal Center of Tongji Medical College, Huazhong University of Science and Technology, and were provided with standard diet and water. The room temperature was controlled at 23±2℃. The experimental protocol involving mice was approved by the Animal Experiment Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. After 7 days of acclimatization, mice were randomly divided into four groups of 10 mice each: a control group (Con, NaHCO3 solution), a model group (FA-Model, folic acid: 250 mg / kg), a low-dose K30 group (FA-K30L, folic acid: 250 mg / kg + K30: 50 mg / kg), a medium-dose K30 group (FA-K30M, folic acid: 250 mg / kg + K30: 100 mg / kg), a high-dose K30 group (FA-K30H, folic acid: 250 mg / kg + K30: 200 mg / kg), and a positive control group (FA-KD, folic acid: 250 mg / kg + KD: 20 mg / kg). Each group was given the corresponding dose of the test drug once a day for 3 days. 30 minutes after the first administration, 250 mg / kg folic acid solution was injected intraperitoneally to establish an acute kidney injury model. Samples were collected after 14 days of administration. The results showed that K30 significantly reduced serum creatinine and blood urea nitrogen levels and improved renal function disorders in mice (see [link to study]). Figure 6 (A) The expression of kidney injury markers in mice was detected, and the results showed that K30 significantly reduced the expression of KIM-1 and NGAL mRNA in mouse kidney tissue (see A). Figure 6(See section B). The expression levels of oxidative stress-related markers GSH, SOD, and MDA in mouse kidney tissue were detected. The results showed that K30 significantly upregulated GSH and SOD expression while inhibiting MDA levels, thus reducing renal oxidative stress (see section B). Figure 6 (C in the text). Kidney H&E staining results showed that K30 reduced renal tubular damage and inflammatory cell infiltration in mouse kidney tissue; kidney PAS staining showed that K30 significantly reduced mesangial proliferation, droplet degeneration, focal tubular cell hypertrophy, and tubular diameter thickening (see...). Figure 6 D and Figure 6 E in the text). Masson staining results for the kidneys showed that K30 significantly inhibited renal collagen fiber deposition and reduced renal fibrosis (see [link to article]). Figure 6 The expression levels of mitochondrial function-related proteins in kidney tissue were detected. The results showed that K30 significantly upregulated the expression of COX-IV, NDUFV2, ATP5B, SDHA, VDAC1 / 2, and PGC-1α proteins, and improved mitochondrial function (see E). Figure 6 (F in the text). The expression of mitochondrial dynamics-related proteins was detected. The results showed that K30 significantly inhibited the expression of Drp1, p-DRP1, MFF, and Fis1 proteins, while upregulating the expression of MFN1, MFN2, and OPA1, thus inhibiting excessive mitochondrial fission (see F in the text). Figure 6 (G in the middle).

[0085] It can be seen that, as Figure 9 As shown, the mechanism by which K30 compounds exert their anti-acute kidney injury effects includes: K30 targets and inhibits MST1, inhibits JNK-mediated Drp1 phosphorylation and mitochondrial translocation, blocks excessive mitochondrial fission, thereby reducing mtROS production, maintaining the integrity of the mitochondrial respiratory chain and mtDNA, maintaining mitochondrial structural and functional homeostasis, thereby reducing oxidative stress, inflammatory response and apoptosis, and alleviating kidney injury.

[0086] Through the above embodiments and Figure 1 - Figure 6 It is evident that the glycoside compound K30 obtained in this invention has a good therapeutic effect on cisplatin- and folic acid-induced acute kidney injury. It can effectively improve cisplatin- and folic acid-induced renal dysfunction, reduce renal tubular damage, renal inflammatory cell infiltration, oxidative stress, and mitochondrial damage. The mechanism is that this compound inhibits excessive mitochondrial fission by targeting MST1, reducing mitochondrial structural and functional damage, and subsequently inhibiting oxidative stress and inflammatory response caused by mitochondrial damage. It can serve as a lead compound for the development of drugs to treat acute kidney injury.

[0087] Furthermore, as can be seen from the above examples, regardless of whether it is acute kidney injury induced by cisplatin or folic acid, the overall efficacy is optimal when the dose of K30 applied per kg of organism per day is 200 mg.

[0088] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A glycoside compound K30, characterized in that, Its structural formula is shown below:

2. The method for synthesizing the glycoside compound K30 as described in claim 1, characterized in that, The synthesis route is as follows: Specifically, the following steps are included: (a) Compound 1 was dissolved in pyridine, and TMSCI was added dropwise to react and generate compound 2; (b) Compound 2 was dissolved in DCM and anhydrous ethanol was added, followed by the dropwise addition of NH4OAC to react and generate compound 3; (c) Compound 3 was dissolved in ultra-dry pyridine to obtain a pyridine solution of compound 3; at the same time, 3,3-dimethylacryloyl chloride was dissolved in ultra-dry DCM to obtain a DCM solution of 3,3-dimethylacryloyl chloride; at 0°C, the DCM solution of 3,3-dimethylacryloyl chloride was slowly added dropwise to the pyridine solution of compound 3, and then the reaction was carried out at room temperature to generate compound 4; (d) Compound 4 was dissolved in DCM, and BF3·Et2O was added dropwise to produce compound K30.

3. The use of the glycoside compound K30 as described in claim 1 in the preparation of a drug for treating acute kidney injury.

4. The application as described in claim 3, characterized in that, The acute kidney injury specifically refers to cisplatin-induced acute kidney injury.

5. The application as described in claim 3, characterized in that, The acute kidney injury mentioned above specifically refers to folic acid-induced acute kidney injury.

6. The application as described in claim 3, characterized in that, The glycoside compound K30 exerts its therapeutic effect on acute kidney injury by improving mitochondrial function.

7. The application as described in claim 3, characterized in that, The glycoside compound K30 exerts its therapeutic effect on acute kidney injury by inhibiting MST1.

8. The application as described in claim 3, characterized in that, The daily dose of the glycoside compound K30 applied per kg of organism is 200 mg.

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