A method for preparing a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes and its application.

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a method for preparing a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes and its application. The preparation method includes the following steps: dissolving atorvastatin in dichloromethane, adding a condensing agent and an organic base, stirring the reaction, adding a dichloromethane solution of 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine, continuing the reaction, adding dilute hydrochloric acid solution to terminate the reaction, and extracting the mixture with dichloromethane and concentrating under reduced pressure to obtain a crude product; adding N,N-dimethylformamide, a polypeptide, a condensing agent, and an organic base, stirring the reaction, concentrating under reduced pressure, and then separating the crude product using high-performance liquid chromatography to obtain the reactive oxygen species-responsive atorvastatin prodrug FWW-TK-Ato targeting cardiomyocytes. The product prepared by this invention can be effectively used in the preparation of drugs for treating myocardial injury in sepsis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes and its application. Background Technology

[0002] Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection, and is one of the leading causes of death in intensive care unit patients. The cardiovascular system is the most frequently affected system in sepsis; approximately 40%-50% of sepsis patients develop septic cardiomyopathy, characterized by left ventricular dilation, reduced ejection fraction, and myocardial depression unresponsive to fluid resuscitation. The pathological mechanisms of myocardial injury in sepsis are extremely complex, and current research suggests that they mainly involve direct myocardial depression caused by cytokine storms, energy metabolism disorders caused by mitochondrial dysfunction, oxidative stress damage, and microcirculatory disturbances. Despite a deepening understanding of the pathophysiological processes of sepsis, there is still a lack of specific therapeutic drugs in clinical practice that can effectively intervene in the progression of myocardial injury.

[0003] Statins are 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors, widely used clinically for lipid-lowering therapy. In recent years, the pleiotropic effects of statins have attracted considerable attention. Studies have shown that statins, represented by atorvastatin, have significant anti-inflammatory effects. They can inhibit the mevalonate pathway, blocking the isoprenelation of GTPases, thereby inhibiting the activation of nuclear transcription factor-κB, reducing the expression of pro-inflammatory factors such as tumor necrosis factor-α and interleukin-6, and lowering C-reactive protein levels. Furthermore, atorvastatin can improve endothelial function by activating endothelial nitric oxide synthase and alleviate oxidative stress damage by inhibiting NADPH oxidase activity, thus demonstrating potential cardioprotective effects in a sepsis-induced myocardial injury model. However, at conventional doses, atorvastatin lacks tissue selectivity in its distribution within the body, making it difficult to accumulate locally in the myocardium to achieve optimal therapeutic concentrations. This, to some extent, limits its efficacy in treating sepsis-induced myocardial injury. Therefore, improving the selective distribution of atorvastatin in myocardial tissue and enhancing its local efficacy in lesions has become an important research direction.

[0004] Prodrug strategies provide an effective means to improve drug targeting and efficacy. By linking drugs to specific targeting ligands, drugs can be guided to selectively distribute to target organs or cells. Studies have shown that the cardiac targeting peptide FWWYSSLPR has a good affinity for cardiomyocytes and can specifically bind to corresponding receptors on the surface of cardiomyocyte membranes. It can serve as a navigation head for cardiac targeted delivery and is expected to improve the selective accumulation of drugs in myocardial tissue.

[0005] On the other hand, during sepsis, the myocardial microenvironment exhibits distinct pathological characteristics, with a significant increase in reactive oxygen species (ROS) levels. Excessive ROS accumulation is a key factor leading to mitochondrial damage and apoptosis, and is also an important marker of myocardial injury in sepsis. Utilizing the characteristics of the pathological microenvironment as triggers to design intelligent responsive prodrugs can achieve precise drug release at the lesion site, thereby improving efficacy while reducing impact on normal tissues. Thioacetate bonds, a type of chemical linkage sensitive to ROS, are relatively stable under normal physiological conditions but can specifically break in highly reactive oxygen environments, and have been widely used to construct ROS-responsive drug delivery systems.

[0006] Against this backdrop, if a novel compound can be constructed that actively targets myocardial tissue and intelligently releases atorvastatin within the pathological microenvironment of septic myocardial injury by combining myocardial targeting peptides with a reactive oxygen species (ROS) release mechanism, it is expected to significantly enhance the local efficacy of atorvastatin in the myocardium. Therefore, this invention aims to provide a method for preparing a ROS-responsive atorvastatin prodrug targeting cardiomyocytes and its application. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing a reactive oxygen species (ROS)-responsive atorvastatin prodrug targeting cardiomyocytes and its application. A novel compound, FWW-TK-Ato, is synthesized by linking the cardiomyocyte-targeting peptide FWWYSSLPR to atorvastatin via an ROS-sensitive thiophanate bond. This compound can specifically recognize and bind to cardiomyocytes and responsively release atorvastatin in the high ROS environment at the site of septic myocardial injury, thereby achieving precise drug delivery and on-demand release, significantly improving the efficacy of atorvastatin against septic myocardial injury.

[0008] This invention provides a method for preparing a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes, comprising the following steps: (1) Atorvastatin was dissolved in dichloromethane, and then the condensing agent N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea and organic base were added. After stirring the reaction, a dichloromethane solution of 2,2'-(propane-2,2-diylbis(sulfadiyl))diethylamine was added to continue the reaction. Then, dilute hydrochloric acid solution was added to terminate the reaction. The mixture was extracted with dichloromethane and concentrated under reduced pressure to obtain the crude product. The crude product NH2-TK-Ato was then separated by rapid column chromatography. (2) N,N-dimethylformamide, polypeptide FWWYSSLPR, condensing agent N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate and organic base were added to the crude product NH2-TK-Ato. After stirring and reacting, the product was concentrated under reduced pressure. The crude product was then prepared and separated by high performance liquid chromatography to obtain the reactive oxygen species-responsive atorvastatin prodrug FWW-TK-Ato that targets cardiomyocytes.

[0009] Furthermore, the organic base is N,N-diisopropylethylamine.

[0010] Further, in step (1), the ratio of the amount of atorvastatin, dichloromethane, condensing agent, organic base and dichloromethane solution of 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine is 1 mol: (10-20) mL: (1-2) mol: (1-2) mol: (1-1.5) mol.

[0011] Furthermore, the concentration of the dichloromethane solution of 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine is 10-100 mg / mL.

[0012] Further, in step (1), the concentration of the dilute hydrochloric acid solution is 0.05-0.5M, and the amount added is [missing information].

[0013] Furthermore, in step (1), the temperature is controlled at -5℃ to 5℃ when the condensing agent is added to the system.

[0014] Further, in step (2), the ratio of crude NH2-TK-Ato, N,N-dimethylformamide, polypeptide FWWYSSLPR, condensing agent and organic base is 1 mol: (10-20) mL: (1-1.5) mol: (1-1.5) mol: (1-1.5) mol.

[0015] Furthermore, in step (2), the temperature is controlled at -5℃ to 5℃ when the polypeptide FWWYSSLPR, condensing agent and organic base are added to the system.

[0016] On the other hand, the present invention also provides a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes, which is prepared using the aforementioned preparation method.

[0017] On the other hand, the present invention also provides the application of the reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes prepared by the aforementioned preparation method in the preparation of a drug for treating myocardial injury in sepsis.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention introduces the myocardial targeting peptide FWWYSSLPR into the atorvastatin prodrug molecule. Utilizing the peptide's specific affinity for the surface of cardiomyocytes, the drug can actively navigate and accumulate in myocardial tissue. Compared to the indiscriminate distribution of traditional atorvastatin throughout the body, this technique significantly improves drug accumulation in damaged myocardium and reduces drug distribution in non-target organs, thereby reducing potential systemic side effects.

[0019] This invention ingeniously constructs a reactive oxygen species (ROS)-sensitive thiophene bond between atorvastatin and the targeting peptide. Under normal physiological conditions, this prodrug remains stable; however, in the pathological microenvironment of septic myocardial injury, due to abnormally elevated ROS levels, the thiophene bond undergoes specific cleavage, thereby triggering the precise release of atorvastatin at the lesion site. This responsive release mechanism ensures a positive correlation between drug release and disease severity, achieving intelligent and precise treatment.

[0020] The prodrug prepared in this invention (FWW-TK-Ato) showed significantly better performance than the same dose of conventional atorvastatin in reducing serum myocardial injury markers such as cTnI and CK-MB levels, restoring ATP levels, and inhibiting the release of inflammatory factors such as TNF-α and IL-6. HE staining of cardiac tissue also confirmed that this regimen more effectively reduced myocardial fiber disorder and inflammatory cell infiltration, restoring the myocardial tissue structure to near normal. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below.

[0022] Figure 1 Synthetic route diagram for FWW-TK-Ato, a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes; Figure 2 The 1H NMR spectrum (500 MHz, DMSO) of FWW-TK-Ato, a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes. Figure 3 The HPLC chromatogram of FWW-TK-Ato, a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes, after co-incubation with hydrogen peroxide. Figure 4 Figure showing the uptake of FWW-TK-Ato, a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes, in H9c2 cardiomyocytes; Figure 5 Figure showing the effect of FWW-TK-Ato, a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes, on LPS-induced intracellular ROS levels in H9c2 cells. Scale bar = 100 μm. Figure 6 Figure showing the effect of FWW-TK-Ato, a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes, on LPS-induced intracellular ATP levels in H9c2 cells; Figure 7 Figure showing the effect of FWW-TK-Ato, a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes, on LPS-induced release of tumor necrosis factor-α, an inflammatory cytokine, from H9c2 cells. Figure 8 Figure showing the effect of FWW-TK-Ato, a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes, on LPS-induced release of the inflammatory cytokine interleukin-6 from H9c2 cells; Figure 9 A graph showing serum cardiac troponin I levels; Figure 10 This is a graph showing serum creatine kinase isoenzyme levels. Figure 11 Image of HE staining of cardiac tissue. Detailed Implementation

[0023] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] It should also be noted that the dichloromethane, N,N-dimethylformamide, atorvastatin, condensing agent, organic base and 2,2'-(propane-2,2-dimethylbis(sulfadiyl))diethylamine used in this invention were all purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0025] Example 1 The present invention provides a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes, the preparation method of which includes the following steps: Atorvastatin (Ato, 0.5 mmol, 280 mg) was placed in a round-bottom flask and dissolved in 20 mL of dichloromethane. The condensing agent N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea (HATU, 0.7 mmol, 268 mg) and the organic base N,N-diisopropylethylamine (DIPEA, 0.7 mmol, 92 mg) were added to the reaction system at 0 °C. After stirring at 650 rpm and 25 °C for 1 hour, 20 mL of a dichloromethane solution of 2,2'-(propane-2,2-diylbis(sulfadiyl))diethylamine (NH2-TK-NH2, 1.2 mmol, 233 mg) was slowly added dropwise at a rate of 1 mL / min, and the reaction was continued at 25 °C for 8 hours. The reaction was then terminated by adding 20 mL of 0.1 M dilute hydrochloric acid solution. The mixture was extracted with dichloromethane and concentrated under reduced pressure at 0.1 atm to obtain the crude product. The crude product NH2-TK-Ato was then separated by rapid column chromatography (ethyl acetate / petroleum ether volume ratio of 20 / 80, v / v) with a mass of 560 mg, which was directly used in the next step of the reaction.

[0026] 16 mL of N,N-dimethylformamide was added to the above solid, followed by the addition of peptide FWWYSSLPR (0.2 mmol, 252 mg), condensing agent HATU (0.6 mmol, 228 mg), and organic base DIPEA (0.6 mmol, 78 mg) at 0 °C. The mixture was heated to 25 °C and stirred at 650 rpm for 12 hours. After concentration under reduced pressure at 0.1 atm, the crude product was prepared and separated by high performance liquid chromatography (ethyl acetate / petroleum ether volume ratio of 20 / 80, v / v) to obtain 145 mg of pale yellow solid. The overall yield of the two steps was 37%.

[0027] 1H NMR (500 MHz, DMSO-d6) δ 10.77 (s, 2H), 10.35 (s, 1H), 9.06 (s,1H), 8.30 (s, 1H), 7.97 (s, 1H), 7.91 (s, 1H), 7.81 (d, J = 4.7 Hz, 4H), 7.71(d, J = 14.5 Hz, 3H), 7.65 – 7.59 (m, 2H), 7.58 – 7.49 (m, 5H), 7.32 – 7.18(m, 13H), 7.17 (dd, J = 4.0, 1.4 Hz, 2H), 7.10 – 6.99 (m, 9H), 6.78 (s, 1H),6.62 (dd, J = 6.1, 1.6 Hz, 3H), 6.44 (s, 1H), 5.44 (s, 1H), 4.90 (t, J = 5.4Hz, 2H), 4.81 (d, J = 4.9 Hz, 1H), 4.73 (d, J = 4.9 Hz, 1H), 4.56 (s, 2H),4.45 (d, J = 16.7 Hz, 3H), 4.37 (s, 1H), 4.31 (s, 1H), 4.13 (d, J = 12.2 Hz,1H), 4.07 – 4.01 (m, 2H), 3.75 (d, J = 4.9 Hz, 1H), 3.70 – 3.63 (m, 2H), 3.63– 3.56 (m, 2H), 3.51 (d, J = 5.4 Hz, 5H), 3.41 – 3.28 (m, 4H), 3.19 (d, J =12.4 Hz, 1H), 3.14 – 2.96 (m, 9H), 2.96 – 2.89 (m, 1H), 2.86 – 2.79 (m, 2H),2.75 (d, J = 12.4 Hz, 1H), 2.65 (d, J = 12.4 Hz, 1H), 2.38 – 2.28 (m, 2H),2.25 (d, J = 12.4 Hz, 1H), 2.15 (d, J = 12.4 Hz, 1H), 2.02 (s, 1H), 1.96 (s,1H), 1.82 (d, J = 7.5 Hz, 2H), 1.75 (d, J = 12.5 Hz, 1H), 1.68 (d, J = 1.0Hz, 2H), 1.61 – 1.54 (m, 4H), 1.52 (d, J = 12.1 Hz, 1H), 1.33 (s, 3H), 1.28(s, 3H), 1.19 (s, 3H), 1.14 (s, 3H), 0.88 (s, 3H), 0.83 (s, 3H). (ESI):[M+2H]2+ m / z calcd 979.9719, found 979.9732. .

[0028] High performance liquid chromatography (HPLC) Figure 3 The figure shows that after co-incubation of compound FWW-TK-Ato with hydrogen peroxide, the two main characteristic peaks are those of the drug atorvastatin (Rt = 10.27 min) and compound FWW-TK-Ato (Rt = 13.58 min). This result indicates that compound FWW-TK-Ato can cleave the TK bond of thioacetate under the action of reactive oxygen species (hydrogen peroxide) and responsively release the drug atorvastatin.

[0029] High-performance liquid chromatography (HPLC) was used to detect the uptake of FWW-TK-Ato by H9c2 cardiomyocytes. H9c2 cells were seeded in 6-well plates and cultured until 80% confluence. Atorvastatin (Ato) and the compound FWW-TK-Ato (both at a final concentration of 10 μM) were added, and the cells were incubated at 37°C and 5% CO2 for 6 hours. After incubation, the culture medium was discarded, and the cells were washed three times with pre-cooled PBS to remove adsorbed drug from the surface. The cells were collected and lysed. After protein precipitation, the supernatant of the cell lysate was analyzed by HPLC. The relative drug concentration in the cells was calculated based on a standard curve, and normalization was performed using a control group (Ato group).

[0030] To investigate the cardiomyocyte targeting properties of FWW-TK-Ato, HPLC was used to detect the drug uptake in H9c2 cells. The results are as follows: Figure 4 As shown, compared with the Ato group, the relative drug distribution in H9c2 cells was significantly increased in the FWW-TK-Ato group. Quantitative analysis revealed that the relative intracellular drug content in the Ato group was 1.00 ± 0.03 (normalized value), while the relative content in the FWW-TK-Ato group reached 5.36 ± 0.44, approximately 5.4 times that of the Ato group. These results indicate that the introduction of the cardiac targeting peptide FWWYSSLPR significantly enhanced the accumulation of atorvastatin in cardiomyocytes, demonstrating that FWW-TK-Ato has good cardiomyocyte targeting properties.

[0031] The intracellular reactive oxygen species (ROS) level in H9c2 cells was detected using the DCFH-DA fluorescent probe, and the antioxidant effect of FWW-TK-Ato was evaluated. H9c2 cells were seeded in 24-well plates and cultured until 80% confluence. Cells were then divided into control, model, Ato, and FWW-TK-Ato groups. Except for the control group, all other groups were stimulated with lipopolysaccharide (LPS, 1 μg / mL). The Ato and FWW-TK-Ato groups were respectively incubated with 10 μM Ato or FWW-TK-Ato. All cells were incubated at 37℃ in a 5% CO2 incubator for 24 h. After incubation, the culture medium was discarded, and serum-free culture medium containing 10 μM DCFH-DA probe was added to each well. The cells were incubated at 37℃ in the dark for 20 min. The probe solution was discarded, and the cells were washed three times with serum-free culture medium. Cells were then observed and images were acquired under a fluorescence microscope, with the intensity of green fluorescence reflecting the intracellular ROS level.

[0032] Changes in intracellular ROS levels in H9c2 cells of each group were observed using fluorescence microscopy. Results showed that cells in the control group exhibited weak background green fluorescence; however, cells in the model group showed significantly enhanced green fluorescence intensity after LPS stimulation, indicating successful induction of intracellular oxidative stress. Compared to the model group, the fluorescence intensity of cells treated with atorvastatin was reduced, while the reduction was more significant in the FWW-TK-Ato treatment group, and cell morphology was significantly improved compared to the model group. These results indicate that FWW-TK-Ato can effectively alleviate LPS-induced ROS levels in cardiomyocytes, and its anti-oxidative stress effect is superior to that of the same concentration of atorvastatin.

[0033] The ATP level in H9c2 cells was measured using an ATP assay kit to evaluate the protective effect of FWW-TK-Ato on mitochondrial function. H9c2 cells were seeded in 96-well plates and cultured until 80% confluence. Cells were then divided into control, model, Ato, and FWW-TK-Ato groups. Except for the control group, all other groups were stimulated with lipopolysaccharide (LPS, 1 μg / mL). The Ato and FWW-TK-Ato groups were simultaneously incubated with atorvastatin (10 μM) or FWW-TK-Ato (10 μM), respectively. All cells were incubated at 37°C and 5% CO2 for 24 h. After incubation, the culture medium was discarded, and 100 μL of ATP lysis buffer was added to each well. Cells were lysed on ice. The lysis buffer was centrifuged at 12000 g for 5 min at 4°C. The supernatant was collected and analyzed according to the ATP assay kit instructions. The chemiluminescence value of each well was detected using a chemiluminescence method, and the relative intracellular ATP content was calculated based on a standard curve.

[0034] To investigate the effect of FWW-TK-Ato on energy metabolism in septic cardiomyocytes, chemiluminescence immunoassay was used to detect intracellular ATP levels in H9c2 cells of each group. The results are as follows: Figure 6As shown, the control group had higher intracellular ATP levels; the model group showed a significant decrease in intracellular ATP levels after LPS stimulation, indicating impaired mitochondrial function. Compared with the model group, the ATP levels in the Ato treatment group rebounded, while the rebound was more significant in the FWW-TK-Ato treatment group. Quantitative analysis showed that the relative ATP content in the model group was 0.011 ± 0.002, in the Ato group it was 0.024 ± 0.003, and in the FWW-TK-Ato group it was 0.037 ± 0.003, approximately 3.4 times that of the model group and significantly higher than that of the Ato group. These results indicate that FWW-TK-Ato can effectively improve LPS-induced cardiomyocyte energy metabolism disorders, and its protective effect on mitochondrial function is superior to that of atorvastatin at the same concentration.

[0035] The anti-inflammatory effect of FWW-TK-Ato was evaluated by detecting the levels of inflammatory factors in cell culture supernatant using ELISA. H9c2 cells were seeded in 24-well plates and cultured until 80% confluence. Cells were then divided into control, model, Ato, and FWW-TK-Ato groups. Except for the control group, all other groups were stimulated with lipopolysaccharide (LPS, 1 μg / mL). The Ato and FWW-TK-Ato groups were simultaneously incubated with atorvastatin (10 μM) or FWW-TK-Ato, respectively. All cells were incubated at 37°C and 5% CO2 for 24 h. After incubation, the cell culture supernatant was collected from each well, and the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured according to the ELISA kit instructions. Each sample was tested in triplicate, and the absolute concentrations of each inflammatory factor were calculated using a standard curve.

[0036] To investigate the inhibitory effect of FWW-TK-Ato on the inflammatory response of septic cardiomyocytes, the levels of TNF-α and IL-6 in the culture supernatant of H9c2 cells in each group were detected by ELISA. The results are as follows: Figure 7 and 8As shown, the control group had lower levels of TNF-α and IL-6; after LPS stimulation, the levels of TNF-α and IL-6 in the model group significantly increased, indicating successful induction of the inflammatory response. Compared with the model group, the Ato treatment group showed a decrease in TNF-α and IL-6 levels, while the decrease was more significant in the FWW-TK-Ato treatment group. Quantitative analysis revealed that the TNF-α level was 90.9 ± 9.2 pg / mL in the model group, 67.5 ± 10.0 pg / mL in the Ato group, and further decreased to 22.5 ± 4.9 pg / mL in the FWW-TK-Ato group, approaching the control group level; the IL-6 level was 202.0 ± 11.9 pg / mL in the model group, 144.2 ± 18.5 pg / mL in the Ato group, and decreased to 91.2 ± 20.6 pg / mL in the FWW-TK-Ato group. The results showed that FWW-TK-Ato could effectively inhibit the release of LPS-induced inflammatory factors from cardiomyocytes, and its anti-inflammatory effect was significantly better than that of atorvastatin at the same concentration.

[0037] The in vivo pharmacodynamic effects of FWW-TK-Ato were evaluated using a lipopolysaccharide-induced sepsis mouse model. Male Balb / c mice were acclimatized for one week and then randomly divided into four groups (n=6 per group): sham-operated group, model group, Ato treatment group, and FWW-TK-Ato treatment group. Except for the sham-operated group, all other groups established a sepsis myocardial injury model by intraperitoneal injection of lipopolysaccharide (LPS, 6 mg / kg). After modeling, the Ato treatment group and the FWW-TK-Ato treatment group received atorvastatin or FWW-TK-Ato (10 mg / kg) via tail vein injection, while the sham-operated group and the model group received an equal volume of physiological saline. Twenty-four hours after administration, orbital blood was collected from the mice, serum was separated, and the levels of cardiac troponin I and creatine kinase isoenzymes in the serum were detected using an automated biochemical chemiluminescence immunoassay analyzer. Simultaneously, mouse heart tissue was collected for HE staining to observe histopathological changes in the myocardial tissue.

[0038] To investigate the protective effect of FWW-TK-Ato against septic myocardial injury, the serum levels of myocardial injury markers in each group of mice were measured. The results are as follows: Figure 9 and 10As shown, the sham-operated group had lower levels of cTnI and CK-MB; after LPS stimulation, the levels of cTnI and CK-MB in the model group significantly increased, indicating that the sepsis-induced myocardial injury model was successfully established. Compared with the model group, the levels of cTnI and CK-MB decreased in the Ato treatment group, while the decrease was more significant in the FWW-TK-Ato treatment group. Quantitative analysis showed that the cTnI level was 146.2 ± 11.0 ng / L in the model group, 105.4 ± 18.6 ng / L in the Ato group, and further decreased to 44.8 ± 9.1 ng / L in the FWW-TK-Ato group; the CK-MB level was 11.5 ± 1.5 ng / mL in the model group, 6.9 ± 0.7 ng / mL in the Ato group, and decreased to 3.7 ± 1.2 ng / mL in the FWW-TK-Ato group. HE staining results of cardiac tissue ( Figure 11 The results showed that the model group exhibited disordered myocardial fiber arrangement and significant inflammatory cell infiltration; the Ato treatment group showed some relief of these pathological changes; and the FWW-TK-Ato treatment group showed near-normal myocardial tissue structure and significantly reduced inflammatory cell infiltration. These results indicate that FWW-TK-Ato effectively reduces myocardial damage in septic mice, and its protective effect is significantly superior to that of the same dose of atorvastatin.

[0039] Finally, it should be noted that the above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention; those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention; and all technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes, characterized in that the steps include... include: (1) Atorvastatin was dissolved in dichloromethane, and then the condensing agent N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate urea and organic base were added. After stirring the reaction, a dichloromethane solution of 2,2'-(propane-2,2-diylbis(sulfadiyl))diethylamine was added to continue the reaction. Then, dilute hydrochloric acid solution was added to terminate the reaction. The mixture was extracted with dichloromethane and concentrated under reduced pressure to obtain the crude product. The crude product NH2-TK-Ato was then separated by rapid column chromatography. (2) N,N-dimethylformamide, polypeptide FWWYSSLPR, condensing agent N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate and organic base were added to the crude product NH2-TK-Ato. After stirring and reacting, the product was concentrated under reduced pressure. The crude product was then prepared and separated by high performance liquid chromatography to obtain the reactive oxygen species-responsive atorvastatin prodrug FWW-TK-Ato that targets cardiomyocytes.

2. The method for preparing a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes according to claim 1, characterized in that, The organic base is N,N-diisopropylethylamine.

3. The method for preparing a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes according to claim 1, characterized in that, In step (1), the ratio of the amount of atorvastatin, dichloromethane, condensing agent, organic base and dichloromethane solution of 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine is 1 mol: (10-20) mL: (1-2) mol: (1-2) mol: (1-1.5) mol.

4. The method for preparing a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes according to claim 2, characterized in that, The concentration of the dichloromethane solution of 2,2'-(propane-2,2-dimethylbis(sulfonamide dimethyl))diethylamine is 10-100 mg / mL.

5. The method for preparing a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes according to claim 1, characterized in that, In step (1), the concentration of the dilute hydrochloric acid solution is 0.05-0.5 M, and the amount added is as follows.

6. The method for preparing a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes according to claim 1, characterized in that, In step (1), the temperature is controlled at -5℃ to 5℃ when the condensing agent is added to the system.

7. The method for preparing a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes according to claim 1, characterized in that, In step (2), the ratio of crude NH2-TK-Ato, N,N-dimethylformamide, polypeptide FWWYSSLPR, condensing agent and organic base is 1 mol: (10-20) mL: (1-1.5) mol: (1-1.5) mol: (1-1.5) mol.

8. The method for preparing a reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes according to claim 1, characterized in that, In step (2), the temperature is controlled at -5℃ to 5℃ when the polypeptide FWWYSSLPR, condensing agent and organic base are added to the system.

9. A reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes, characterized in that, It is prepared using the preparation method according to any one of claims 1-8.

10. The use of the reactive oxygen species-responsive atorvastatin prodrug targeting cardiomyocytes prepared by any one of the preparation methods of claims 1-9 in the preparation of a drug for treating myocardial injury in sepsis.

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