1-Deoxynojirimycin Derivatives and Uses Thereof

Abstract

The present invention discloses 1-deoxynojirimycin derivatives and their uses. Through screening, several 1-deoxynojirimycin derivatives were prepared. Compared with the lead compound 1-deoxynojirimycin, these derivatives better bind to the relevant amino acid site of the target protein OPA1 and are stabilized in the binding pocket of the dimer interface. They promote the formation of OPA1 dimers and restore mitochondrial ultrastructure, thereby significantly maintaining mitochondrial function and effectively improving cellular physiological status. The 1-deoxynojirimycin derivatives of the present invention are useful for preparing drugs for treating diseases associated with an imbalance in OPA1 dimer formation, and are promising therapeutic agents for, for example, mitochondrial cardiomyopathy and other mitochondrial diseases.

Description

[Technical Field]

[0001] The present invention relates to the technical field of biomedicine, and in particular to 1-deoxynojirimycin derivatives and uses thereof. [Background technology]

[0002] Mitochondria are important organelles that act as cellular energy factories, supplying ATP to the body, as well as regulating the production of reactive oxygen species, cellular apoptosis, and Ca 2+ Mitochondrial dysfunction is also important for signal transduction. Various clinical diseases caused by mitochondrial dysfunction are collectively called mitochondrial diseases.

[0003] Mitochondrial cardiomyopathy (MIM510000) is a mitochondrial disease affecting the heart and skeletal muscle due to abnormalities in mitochondrial structure or function. Patients often suffer from severe heart failure. Common clinical symptoms include exertional dyspnea, tachycardia, generalized muscle weakness with severe generalized edema, and cardiac and hepatic hypertrophy. The heart, constantly contracting and relaxing, is the organ that consumes the most oxygen and energy in the body. Cardiomyocytes require rapid and efficient oxidation to generate the ATP needed to maintain normal pumping function. Myocardial energy metabolism is primarily carried out by mitochondria, which synthesize ATP through oxidative phosphorylation. Therefore, mitochondria are crucial for maintaining homeostasis of energy metabolism in cardiomyocytes and meeting cardiac energy needs. Mitochondrial dysfunction leads to abnormal energy metabolism and oxidative stress, which can lead to the development and progression of cardiovascular diseases such as dilated and hypertrophic cardiomyopathy and promote the progression of heart failure. We have previously successfully established mitochondrial hypertrophic cardiomyopathy (HCM)-specific induced pluripotent stem cells and their directed differentiation into cardiomyocytes (iPSC-CMs) (Li S, et al. Mitochondrial dysfunctions contribute to hypertrophic cardiomyopathy in patient iPSC-derived cardiomyocytes with MT-RNR2 mutation. Stem Cell Reports. 2018; 10: 808-821.). These cardiomyocytes have a pathological phenotype similar to that of cardiomyocytes from HCM patients, exhibiting clear mitochondrial dysfunction.

[0004] Maintaining mitochondrial homeostasis is crucial for regulating cardiac energy metabolism, so targeting mitochondria to improve mitochondrial function is crucial for the prevention and treatment of cardiovascular disease. Currently, the usual therapeutic agents for cardiomyopathy are β-blockers, Ca 2+Although channel blockers, antiarrhythmics, calcium-sensitizing agents, and metabolic and contractile modulators are currently used, most of these drugs are inhibitors and are specialized for specific types of diseases. In particular, their applicability to mitochondrial cardiomyopathy is limited, resulting in insufficient therapeutic efficacy. Mitochondria are one of the important targets for the treatment of cardiomyopathy. However, current mitochondrial therapeutics mostly focus on symptomatic treatment through single pathways, such as regulating intracellular mitochondrial biogenesis, NAD+ levels, and mitochondrial reactive oxygen species levels, or inducing mitochondrial metabolic reprogramming, and are unable to fundamentally improve mitochondrial function. Therefore, developing innovative drugs that directly target mitochondria, reconstruct mitochondrial ultrastructure, and restore mitochondrial function is highly significant and offers new hope for the treatment of mitochondrial cardiomyopathy and other mitochondrial diseases. [ka] Formula I

[0005] 1-Deoxynojirimycin (DNJ) is a polyhydroxy alkaloid (see Formula I) that primarily acts as an α-glucosidase inhibitor, regulating glucose metabolism and also possessing functions such as hypoglycemic, hypolipidemic, antitumor, and antiviral effects. Previous research has used a two-stage screening strategy using transmitochondrial cells (cybrids) and iPSC-cardiomyocytes specific to patients with mitochondrial cardiomyopathy. We have found that the compound DNJ targets the mitochondrial protein OPA1 and promotes the formation of OPA1 dimers, thereby improving mitochondrial ultrastructure and function in patients with mitochondrial cardiomyopathy (patent pending, substantive review). However, DNJ extraction and application have several drawbacks: (1) the low content and extraction rate of DNJ in natural products result in high DNJ production costs; (2) chemical synthesis is complex, time-consuming, and results in low yields and environmental pollution; and (3) DNJ has low bioavailability and lipophilicity, making it difficult for it to maintain effective and sustained activity in the body. Therefore, developing new DNJ derivatives to improve the lipophilicity of DNJ, increase its bioavailability, enhance tissue affinity, improve targeting, etc., is an effective strategy to promote clinical application.

[0006] Conventional DNJ derivatives are primarily classified into three categories based on the modification site: N-substituted, C-substituted, and O-substituted derivatives. Among these, the most common and classic are N-substituted derivatives such as miglitol and miglustat. Both of these derivatives are used as potent α-glucosidase inhibitors and are widely used in the treatment of type 2 diabetes. Miglustat, in particular, received marketing approval in Europe in 2003. It crosses the blood-brain barrier and inhibits glycolipid synthesis, providing a novel solution for the treatment of various lysosomal storage diseases associated with the central nervous system. Although DNJ derivatives with better performance are continuously being developed, their previously known pharmacological activities, such as antidiabetic, antitumor, and antiviral, are still limited.

[0007] Optic atrophy protein-1 (OPA1) is a motor protein primarily found in the inner mitochondrial membrane. OPA1 multimers are essential for maintaining mitochondrial morphology. OPA1 dysfunction can cause mitochondrial crest failure and mitochondrial fission, potentially leading to a range of diseases, including optic atrophy, progressive external ophthalmoplegia and ataxia, progressive myoclonic epilepsy, spastic paraplegia and intestinal dysmotility, and retinal degeneration. Currently, there are few drugs targeting OPA1, and most of them are inhibitors. MYLS22 has been reported to suppress tumor growth by inhibiting OPA1 expression.

[0008] Currently, there are no novel DNJ derivatives that target OPA1, improve mitochondrial ultrastructure and biological function, and act as agonists to treat mitochondrial diseases such as mitochondrial cardiomyopathy, hearing loss, optic atrophy, progressive external ophthalmoplegia and ataxia, progressive myoclonic epilepsy, spastic paraplegia and intestinal motility disorders, and retinal degeneration. This patent brings innovation to DNJ derivatives and their uses. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention addresses the shortcomings of the prior art by providing 1-deoxynojirimycin derivatives that have superior target protein OPA1 binding activity compared to the lead compound 1-deoxynojirimycin, and uses thereof. [Means for solving the problem]

[0010] The 1-deoxynojirimycin derivative is one of the following: (1) DNJ-1 having the structure represented by Formula II [ka] Formula II (2) DNJ-5a, the structure of which is represented by Formula VII [ka] Formula VII (3) DNJ-5c, the structure of which is represented by Formula VIII [ka] Formula VIII (4) The structure is represented by general formula II [ka] Formula II wherein R is selected from —H, —NO, —X, where X is F, Cl, Br, or I.

[0011] Preferably, R is selected from -X, where X is F.

[0012] When R is selected from -H, the structure is DNJ-3a, as shown in Formula III. [ka] Formula III When R is selected from -F, the structure is DNJ-3b, as shown in Formula IV. [ka] Formula IV

[0013] When R is selected from -NO2, the structure is DNJ-3c, as shown in Formula V. [ka] Formula V

[0014] The present invention also provides use of the 1-deoxynojirimycin derivative or a pharmaceutically acceptable salt thereof in the preparation of an OPA1 agonist, wherein the 1-deoxynojirimycin derivative or a pharmaceutically acceptable salt thereof is capable of promoting the polymerization of OPA1 monomers into multimers.

[0015] The present invention also provides a 1-deoxynojirimycin derivative or a pharmaceutically acceptable salt thereof for use in the preparation of a drug for treating a disease associated with an imbalance in OPA1 oligomerization.Preferably, the disease associated with an imbalance in OPA1 oligomerization is hypertrophic cardiomyopathy, hearing loss, optic atrophy, progressive external ophthalmoplegia and ataxia, progressive myoclonic epilepsy, spastic paraplegia and intestinal motility disorder, or retinal degeneration.More preferably, the disease associated with an imbalance in OPA1 oligomerization is caused by mitochondrial dysfunction.

[0016] The present invention also provides a drug for treating a disease associated with an imbalance in OPA1 oligomerization, wherein the active ingredient is the 1-deoxynojirimycin derivative or a pharmaceutically acceptable salt thereof. Preferably, the disease associated with an imbalance in OPA1 oligomerization is hypertrophic cardiomyopathy, hearing loss, optic atrophy, progressive external ophthalmoplegia and ataxia, progressive myoclonic epilepsy, spastic paraplegia and intestinal motility disorder, or retinal degeneration. More preferably, the disease associated with an imbalance in OPA1 oligomerization is caused by mitochondrial dysfunction.

[0017] The present invention also provides a method for preparing the 1-deoxynojirimycin derivative. When the 1-deoxynojirimycin derivative is the above (1), compounds a and b are subjected to a condensation reaction in a solvent to obtain the corresponding 1-deoxynojirimycin derivative, wherein compound a is 1-deoxynojirimycin and compound b is [ka] and When the 1-deoxynojirimycin derivative is the above (2), first, the nitrogen position of the piperidylidene of 1-deoxynojirimycin is protected, and then the condensation reaction of the hydroxymethyl position is carried out. For this purpose, first, 1-deoxynojirimycin is reacted with benzyl chloroformate to produce intermediate product 1, [ka] Next, intermediate product 1 is reacted with PivCl to obtain intermediate product 2, [ka] Finally, intermediate product 2 is reduced to obtain the 1-deoxynojirimycin derivative. During reduction, intermediate product 2 is first dissolved in MeOH, then Pd / C is added, and the reaction is carried out under H2 protection. When the 1-deoxynojirimycin derivative is (3), 1-deoxynojirimycin is first reacted with 2-bromoethylbenzene to generate an intermediate product, [ka] Next, the intermediate product is reacted with PivCl to obtain the 1-deoxynojirimycin derivative.

[0018] When the 1-deoxynojirimycin derivative is the above (4), the compounds a and b4 are subjected to a condensation reaction in a solvent to obtain the corresponding 1-deoxynojirimycin derivative, wherein the compound a is 1-deoxynojirimycin and the compound b4 is [ka] is.

[0019] The present invention discloses 1-deoxynojirimycin derivatives and their uses. Through screening, several 1-deoxynojirimycin derivatives were prepared. Compared with the lead compound 1-deoxynojirimycin, these derivatives better bind to the relevant amino acid site of the target protein OPA1 and are stabilized in the binding pocket of the dimer interface. They promote the formation of OPA1 dimers and restore mitochondrial ultrastructure, thereby significantly maintaining mitochondrial function and effectively improving cellular physiological status. The 1-deoxynojirimycin derivatives of the present invention are useful for preparing drugs for treating diseases associated with an imbalance in OPA1 dimer formation, and are promising therapeutic agents for, for example, mitochondrial cardiomyopathy and other mitochondrial diseases. [Brief explanation of the drawings]

[0020] [Figure 1] This is the reaction scheme for structural formula II. [Figure 2] FIG. 1 is a mass spectrum analysis diagram of DNJ-1. [Figure 3] This is the reaction scheme for structural formula III. [Figure 4] NMR resonance spectrum of DNJ-3a. [Figure 5] This is the reaction scheme for structural formula IV. [Figure 6] NMR resonance spectrum of DNJ-3b. [Figure 7] This is the reaction formula for structural formula V. [Figure 8] NMR resonance spectrum of DNJ-3c. [Figure 9] This is the reaction scheme for structural formula VI. [Figure 10] NMR resonance spectrum of DNJ-4d. [Figure 11] This is the reaction scheme for structural formula VII. [Figure 12] NMR resonance spectrum of DNJ-5a. [Figure 13] This is the reaction scheme for structural formula VIII. [Figure 14] FIG. 1 is a mass spectrum analysis diagram of DNJ-5c. [Figure 15] FIG. 1 shows the results of immunofluorescence detection of cardiomyocytes differentiated from wild-type and mitochondrial HCM-specific iPSCs. [Figure 16] FIG. 1 shows the results of preliminary screening of various DNJ derivatives on the mitochondrial membrane potential of mitochondrial HCM-specific Cybrids. [Figure 17]Figure 1 shows the results of multiple clone rescreening of various DNJ derivatives on the mitochondrial membrane potential of mitochondrial HCM-specific Cybrids. A: Effect of DNJ derivatives on improving mitochondrial membrane potential. B: Effect of derivatives DNJ-1 and DNJ-5a on the mitochondrial membrane potential of control group (Con) and HCM Cybrids. n=9, ***P<0.001. [Figure 18] Figure 1 shows the effects of several derivatives on the mitochondrial activity of mitochondrial HCM-specific iPSC-CMs compared with the lead compound, DNJ. A: Cell viability after culturing in galactose medium for various days; B: Cell viability on day 3 after drug addition. n=3. ###P<0.001 compared with the Con-CM group; *P<0.05, **P<0.01, ***P<0.001 compared with the HCM-CM group. [Figure 19] Figure 1 shows the results of detecting the median effective concentrations of derivatives DNJ-1 and DNJ-5a compared to DNJ in HCM iPSC-CMs. A: represents the median effective concentration of DNJ, B: represents the median effective concentration of DNJ-1, and C: represents the median effective concentration of DNJ-5a. [Figure 20] This figure shows the results of detecting the affinity of the purified OPA1-EGFP protein with the derivatives DNJ-1 and DNJ-5a compared to DNJ. A: Coomassie Brilliant Blue identification results of the purified protein; B: affinity curve of DNJ; C: affinity curve of DNJ-1; D: affinity curve of DNJ-5a. [Figure 21] The restorative effects of derivatives DNJ-1 and DNJ-5a on the action potential of iPSC-CMs are shown. A: Statistics of the abnormal electrophysiological state of cardiomyocytes. B: Representative electrophysiological diagrams of cardiomyocytes treated with DNJ-5a. [Figure 22]Echocardiographic results of mice with Ang II-induced myocardial hypertrophy treated with DNJ-5a derivative were compared with those of DNJ. A: Representative image of mouse echocardiographic results. B-E: Statistical results of relevant parameters of mouse echocardiography. Sham, n = 8; Sham + DNJ-5a, n = 7; Ang II, n = 8; Ang II + DNJ, n = 9; Ang II + DNJ-5a, n = 9. *P < 0.05, **P < 0.01, ***P < 0.001. [Figure 23] Figure 1 shows the size and weight of mouse hearts. A: Representative images of the largest cross-sections of the hearts stained with HE staining from each group of mice. B: Statistical results of mouse heart weight (mg) / body weight (g). Sham, n = 8; Sham + DNJ-5a, n = 7; Ang II, n = 8; Ang II + DNJ, n = 9; Ang II + DNJ-5a, n = 9. *P < 0.05, **P < 0.01, ***P < 0.001. [Figure 24] Representative transmission electron microscopy images of mitochondria in mouse heart tissue. DETAILED DESCRIPTION OF THE INVENTION

[0021] The main considerations for structural optimization of DNJ are as follows: introducing aryl and aryl alkyl groups to improve the lipid solubility, bioavailability, and activity of the compound, and introducing amino acids to increase tissue affinity and improve targeting.

[0022] Depending on the structure of natural products that can enhance DNJ and mitochondrial membrane potential, the following strategies are mainly used to modify the structure of DNJ:

[0023] (1) Using the principles of splicing, we select structural fragments of salidroside and thioctic acid, compounds that increase mitochondrial membrane potential, splice them with DNJ, and examine their effect on the activity of the target compound.

[0024] (2) Introducing aryl or arylalkyl groups onto the nitrogen atom to increase the lipid solubility, bioavailability, and activity of the compound.

[0025] (3) Introducing amino acids into the nitrogen atom to increase tissue affinity and improve targeting.

[0026] (4) In order to improve the affinity of the compound for the mitochondrial membrane and improve its targeting to mitochondria, a quaternary ammonium ion is introduced.

[0027] In the actual synthesis, a total of 10 compounds in 4 series were obtained, and the DNJ structurally optimized compounds obtained by the actual synthesis had a purity of 90% or more after purification. Example 1

[0028] (1) Compound name: DNJ-1; Molecular formula C 14 H 25 NO5S2, structural name: 5-((R)-1,2-dithiolan-3-yl)-1-((2R,3R,4R,5S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperidin-1-yl)pentan-1-one; the structure is shown in Formula II. [ka] Formula II The reaction scheme is shown in Figure 1. Compounds a (193.97 mg, 1.19 mmol) and b (245 mg, 1.19 mmol) were placed in a round-bottom flask, and DCM (10 mL) was added. The temperature was lowered to 0°C, and HOBT (0.177 g, 1.31 mmol) and EDCl (0.251 g, 1.31 mmol) were added. The mixture was then allowed to react at room temperature for 10 hours. After the reaction was completed, the solvent was evaporated and the product was separated by column chromatography to obtain the product. FIG. 2 is a mass spectrum analysis diagram of DNJ-1.

[0029] (2) Compound name: DNJ-3a; molecular formula C 14 H 21 NO4, structural name: (2R,3R,4R,5S)-2-(hydroxymethyl)-1-phenethylpiperidine-3,4,5-triol; the structure is shown in Formula III. [ka] Formula III The reaction scheme is shown in FIG. Compound a (163 mg, 1 mmol), compound b (220.79 mg, 1.2 mmol), and potassium carbonate (414 mg, 3 mmol) were weighed into a sealed tube, and DMF (5 mL) was added as a solvent. The mixture was heated at 80 °C for 4 h, and the completion of the reaction was monitored by TLC. The solvent was evaporated and the mixture was subjected to column chromatography to obtain approximately 80 mg of the product. FIG. 4 is the NMR resonance spectrum of DNJ-3a.

[0030] (3) Compound name: DNJ-3b; Molecular formula C 14 H 20 FNO4, structural name: (2R,3R,4R,5S)-1-(4-fluorophenethyl)-2-(hydroxymethyl)piperidine-3,4,5-triol; the structure is shown in Formula IV. [ka] Formula IV The reaction scheme is shown in FIG. Compound a (163 mg, 1 mmol), compound b (242.38 mg, 1.2 mmol), and potassium carbonate (414 mg, 3 mmol) were weighed into a sealed tube and reacted in DMF at 80°C for 4 h. The reaction completion was monitored by thin-layer chromatography (TLC) plate. The solvent was evaporated by rotary evaporation, and column chromatography was performed to obtain approximately 70 mg of the product. FIG. 6 is the NMR resonance spectrum of DNJ-3b.

[0031] (4) Compound name: DNJ-3c; molecular formula C 14 H 20 N2O6, structural name: (2R,3R,4R,5S)-2-(hydroxymethyl)-1-(4-nitrophenethyl)piperidine-3,4,5-triol; the structure is shown in Formula V. [ka] Formula V The reaction scheme is shown in FIG. Compound a (163 mg, 1 mmol), compound b (274.8 mg, 1.2 mmol), and potassium carbonate (414 mg, 3 mmol) were weighed into a sealed tube and reacted in DMF at 80°C for 4 h. The reaction completion was monitored by thin-layer chromatography (TLC) plate. The solvent was evaporated by rotary evaporation, and column chromatography was performed to obtain approximately 120 mg of the product. FIG. 8 is the NMR resonance spectrum of DNJ-3c.

[0032] (5) Compound name: DNJ-4d; Molecular formula C 15 H 18N2O5, structural name: 4-(2-((2R,3R,4R,5S)-3,4,5-trihydroxy-2-(hydroxymethyl)piperidin-1-yl)acetyl)benzonitrile; the structure is shown in Formula VI. [ka] Equation VI The reaction scheme is shown in FIG. Compound a (163 mg, 1 mmol), compound b (267.6 mg, 1.2 mmol), and potassium carbonate (414 mg, 3 mmol) were weighed into a sealed tube and reacted in DMF at 80°C for 4 h. The reaction completion was monitored by thin-layer chromatography (TLC) plate. The solvent was evaporated by rotary evaporation, and column chromatography was performed to obtain approximately 90 mg of the product. FIG. 10 is the NMR resonance spectrum of DNJ-4d.

[0033] (6) Compound name: DNJ-5a; Molecular formula C 11 H 21 NO5, structural name: ((2R,3R,4R,5S)-3,4,5-trihydroxypiperidin-2-yl)methyl pivalate; the structure is shown in Formula VII. [ka] Formula VII The reaction scheme is shown in FIG. Compound a (115 mg, 0.7 mmol) was weighed into a mixture of dioxane / water (1:1, 12 mL), followed by the addition of sodium chloride (1.75 equivalents) and benzyl chloroformate (1.54 equivalents) and stirring at room temperature for 18 hours. The dioxane was then removed by rotary evaporation, extracted with dichloromethane, and the organic layer was retained. Product b was obtained by column chromatography. Compound b (149 mg, 0.5 mmol) was dissolved in pyridine (3 mL), and PivCl (2 equivalents) was added slowly at 0 °C. After 1 hour, another PivCl (2 equivalents) was added, and after 3 hours, the reaction mixture was diluted with ethyl acetate and methanol. The solvent was removed under reduced pressure, and compound c was obtained by column chromatography. Compound c (150 mg, 0.39 mmol) was dissolved in MeOH (5 mL), and then 10% Pd / C (30 mg) was added. The mixture was stirred at room temperature and protected with H overnight. Compound d was obtained by column chromatography. FIG. 12 is the NMR resonance spectrum of DNJ-5a.

[0034] (7) Compound name: DNJ-5c; Molecular formula C 19 H 29 NO5, structural name: ((2R,3R,4R,5S)-3,4,5-trihydroxy-1-phenethylpiperidin-2-yl)methyl pivalate; the structure is shown in Formula VIII. [ka] Formula VIII The reaction scheme is shown in FIG. Compound a (163 mg, 1 mmol), 2-bromoethylbenzene (220.79 mg, 1.2 mmol), and potassium carbonate (414 mg, 3 mmol) were weighed into a sealed tube, and the mixture was heated in DMF as a solvent at 80°C for 4 h to react. The completion of the reaction was monitored by TLC thin-layer chromatography (TLC) plate. The solvent was evaporated by rotary evaporation, and column chromatography was performed to obtain approximately 80 mg of the intermediate product (compound b). Compound b (133.5 mg, 0.5 mmol) was dissolved in pyridine (3 mL) and PivCl (2 equivalents) was added slowly at 0 °C. After 1 h, PivCl (2 equivalents) was added again, and after 3 h, the reaction mixture was diluted with ethyl acetate and methanol. The solvent was removed under reduced pressure, and compound c was obtained by column chromatography. FIG. 14 is a mass spectrum analysis diagram of DNJ-5c. Example 2

[0035] Cytoplasts of immortalized lymphocytes isolated from normal individuals and patients with MT-RNR2 mutations were fused with mitochondrial DNA-deficient ρ0 cells to construct mitochondria-specific mitochondrial cells (Cybrids) with the same nuclear background. (For the specific method, see our previous paper: Li D, Sun Y, et al. Mitochondrial dysfunction caused by the m.2336T>C mutation with hypertrophic cardiomyopathy in cybrid cell lines. Mitochondrion. 2019 May;46:313-320.) This cell line was previously constructed and preserved in our laboratory. Urinary cells from a patient with hypertrophic cardiomyopathy carrying the MT-RNR2 mutation were induced to become iPSCs and differentiated into iPSC-CMs (for specific methods, see a paper previously published by our laboratory: Li S, Pan H, Tan C, et al. Mitochondrial dysfunctions contribute to hypertrophic cardiomyopathy in patient iPSC-derived cardiomyocytes with MT-RNR2 mutation. Stem Cell Reports. 2018; 10: 808-821.). Briefly, 3-4 days before differentiation induction, iPSCs were digested into single cells with Accutase (Stem Cell) and resuspended in mTeSR1 (Stem Cell) culture medium. 10 5Cells were evenly seeded onto a 6-well plate with a Matrigel (BD) bottom. On day 0, when cell density reached approximately 95%, the medium was replaced with RPMI / B27-insulin (Gibco, Catalog No. A1895601) + 12 μM CHIR99021 (Selleck, Catalog No. CT99021) and cultured for 24 hours. On day 1, the intracellular CHIR99021 was removed, and the medium was replaced with RPMI / B27-insulin medium. On days 2-3, the cells were treated with 5 μmol / L IWP2 (Tocris, Catalog No. 3533) dissolved in RPMI / B27-insulin for 2 days. On days 3-4, the intracellular IWP2 was removed, and the culture was continued in RPMI / B27-insulin medium for 2 days. The medium was then replaced with RPMI / B27 (Gibco), and spontaneously beating cardiomyocytes were observed between days 7-12.

[0036] Cells were seeded onto 24-well slides. After washing three times with PBS, the slides were fixed with 4% paraformaldehyde for 15 minutes at room temperature. Next, the slides were permeabilized with 0.2% Triton-X100 for 15 minutes at room temperature. Next, 3% BSA was added for 1 hour at room temperature for blocking. Anti-α-ACTIN (Sigma, 1:200) and TNNT2 (Abcam, 1:200) were added and incubated overnight at 4°C. Fluorescent secondary antibodies (goat anti-rabbit IgG H&L (Alexa Fluor® 488) (Abcam) and goat anti-mouse IgG H&L (Alexa Fluor® 594) (Abcam)) were added and incubated for 1 hour at room temperature in the dark. 0.5 mL of 1 μg / mL DAPI was added, incubated for 5 minutes at room temperature in the dark, and mounted with 50% glycerol. After washing three times with PBS at each step, the results were finally observed under a confocal microscope. As shown in Figure 15 , the immunofluorescence results showed that all induced cardiomyocytes could normally express the cardiac marker proteins anti-α-ACTIN and TNNT2, indicating that the above steps could successfully differentiate HCM-iPSCs with MT-RNR2 mutations into cardiomyocytes.

[0037] Mitochondrial drug screening using patient-specific transmitochondrial cells (Cybrids) combined with cells differentiated from iPSCs has been a fruitful attempt. Transmitochondrial cells eliminate the influence of nuclear genes and intuitively reflect the impact of mitochondrial gene mutations on mitochondrial function. Meanwhile, iPSC-CMs, a more powerful model, provide more physiological results for more accurate drug evaluation. This two-stage screening method combining transmitochondrial cells and iPSC-CMs creates the conditions for conducting mechanistic studies on mitochondrial rescue and cardiomyocyte function recovery, and may facilitate progress in research on mitochondrial-targeted drugs for mitochondrial diseases. Example 3

[0038] Transmitochondrial cells carrying the MT-RNR2 mutation were treated with various DNJ derivatives and the mitochondrial membrane potential was measured. This experiment was performed using the JC-10 Mitochondrial Membrane Potential Assay Kit (Abcam, ab112134).

[0039] JC-10 has the ability to selectively enter mitochondria, and its color reversibly changes from green to red with increasing membrane potential. In normal cells, JC-10 is concentrated in the mitochondrial matrix and forms red fluorescent aggregates. However, in apoptotic or necrotic cells, JC-10 diffuses out of the mitochondria and becomes a monomeric form, resulting in green fluorescent staining of the cells. Fluorescence values ​​corresponding to Ex / Em = 490nm / 525nm and 540nm / 590nm were detected using a microplate reader, which can reflect the level of mitochondrial membrane potential. A lower ratio indicates more severe mitochondrial damage.

[0040] In a 96-well black clear-bottom plate, 0.8 x 10 transmyochondrial cells were cultured. 4Cells were seeded at 100x per well. Three replicate wells were set up per group. 16 hours after seeding, the medium was replaced with fresh complete medium (DMEM + 10% FBS). 30 μM of each DNJ derivative was added to the HCM-Cybrid experimental group, and an equal volume of DMSO was added to the HCM-Cybrid control group. The cells were incubated at 37°C and 5% CO2 for 24 hours. After 24 hours, 100x JC-10 was diluted with Assay Buffer A. 25 μl was added to each well of the plate and incubated at 37°C and 5% CO2 for 30 minutes. Before loading, 25 μL of JC-10 Buffer B was added to each well. Fluorescence values ​​corresponding to Ex / Em = 490 nm / 525 nm and 540 nm / 590 nm were detected using a microplate reader. PL590 / PL525 were compared after subtracting the corresponding blank control from each well.

[0041] The results are shown in Figure 16. Here, HCM + DMSO indicates the control group with DMSO added, HCM + DNJ indicates the group with the lead compound DNJ added, HCM + 3a indicates the DNJ derivative added was DNJ-3a, and the other groups indicate the addition of different DNJ derivatives. The results showed that derivatives DNJ-4a, 4b, 4c, and 4d were not very effective in increasing the mitochondrial membrane potential of HCM-Cybrids with the MT-RNR2 mutation, whereas DNJ-3a, 3b, 3c, 1, 5a, and 5c significantly increased the mitochondrial membrane potential and could be included in the second round of screening. Example 4

[0042] The results of multiple clone rescreening of various DNJ derivatives on the mitochondrial membrane potential of MT-RNR2 mutant HCM-Cybrids were measured.

[0043] The mitochondrial membrane potential detection method was as described in Example 3. The difference here is that, to increase the reliability of the results, detection was performed using three HCM-Cybrid cell clones. As shown in Figure 17A, DNJ-1 and DNJ-5a significantly increased mitochondrial membrane potential, while DNJ-3a, DNJ-3b, and DNJ-3c also increased the potential to a certain extent, although the effect was not significant. Next, as shown in Figure 17B, we focused on the effects of DNJ-1 and DNJ-5a and the addition of FCCP in different groups to improve the membrane potential in normal Cybrid cells.

[0044] The procedure for Figure 17B was as follows. 24 hours after adding 30 μM DNJ-1 and DNJ-5a drugs to Con-Cybrid and HCM-Cybrid cells, 50 μL of 10 μM FCCP diluted in medium was added to triplicate wells of each group. FCCP may act as an uncoupler and inhibit the formation of mitochondrial membrane potential. 50 μL of medium was added to triplicate wells of each clone in each group and incubated at 37°C and 5% CO2 for 30 minutes. 100x Compound A (JC-10) was diluted with Compound B (JC-10 Buffer A), and 25 μL was added to each well of the well plate. The incubation was at 37°C and 5% CO2 for 30 minutes. Before loading, 25 μL of JC-10 Buffer B was added to each well. Fluorescence values ​​corresponding to Ex / Em = 490 nm / 525 nm and 540 nm / 590 nm were detected using a microplate reader. PL590 / PL525 were compared after subtracting the corresponding blank control group from each well.

[0045] The results showed that the derivatives DNJ-3a, DNJ-3b, and DNJ-3c increased the mitochondrial membrane potential of HCM-Cybrid to some extent, but DNJ-1 and DNJ-5a had the most significant increasing effect, and these two derivatives had no effect on the mitochondrial membrane potential of Con-Cybrid. Example 5

[0046] HCM-iPSC-CMs carrying the MT-RNR2 mutation were treated with various DNJ derivatives and DNJ, and the effects of the derivatives on cell viability were detected by galactose-induced cell death experiments.

[0047] 2 x 10 cells in a 24-well plate 4 Cells were seeded at 1000 kJ / well and cultured in L-15 medium (Gibco) with B27 cell culture supplements in a 37°C, 5% CO2 incubator. After 16 hours, the medium of the HCM-CM group was replaced with L-15 medium supplemented with drugs (final concentration 30 μmol / L). Con-CM and HCM-CM groups were also treated with L-15 medium supplemented with an equal volume of DMSO as controls, with three replicate wells per group. Cells from each well were collected every 24 hours and counted three times consecutively using a hemocytometer. Glycogen in L-15 medium is primarily galactose, and in this culture environment, cells primarily source energy through mitochondria.

[0048] The results are shown in Figure 18. DNJ-1 and DNJ-5a showed the most potent effects, significantly improving cell viability of HCM-iPSC-CMs carrying the MT-RNR2 mutation in galactose medium by improving mitochondrial function. They also performed better than the lead compound. DNJ-3a, DNJ-3b, and DNJ-3c also showed some improvement. Example 6

[0049] HCM-iPSC-CMs carrying the MT-RNR2 mutation were treated with various concentrations of the derivatives DNJ, DNJ-1, and DNJ-5a, and their half-effective concentrations (EC50) were obtained by measuring the mitochondrial membrane potential.

[0050] The drug was diluted 10-fold, and the effect of treatment with DNJ-1 at various concentrations (0.0003, 0.003, 0.03, 0.3, 3, and 30 μM) on mitochondrial membrane potential was observed. The mitochondrial membrane potential was measured according to the method described in Example 4 above.

[0051] The results are shown in Figure 19. The median effective concentrations of DNJ, DNJ-1, and DNJ-5a were 69.6 nM, 21.2 nM, and 38.02 nM, respectively, indicating that 50% of the maximum effect could be achieved at these concentrations. This indicates that, compared with the lead compound DNJ, DNJ-1 and DNJ-5a have a faster onset of action and higher pharmacological activity. Example 7

[0052] Using purified eukaryotic OPA1-EGFP protein, we detected the interaction between target proteins and small molecule compounds by microscale thermophoresis (MST) and obtained two affinity parameters, namely, KD values.

[0053] The full-length sequence of OPA1 (NM_015560.2) was obtained from HEK293T cDNA by PCR and then homologously recombined into the pcDNA3.1-Flag / His-EGFP empty vector to construct the pcDNA3.1-OPA1-Flag / His-EGFP eukaryotic expression vector.

[0054] Flag / His-OPA1-EGFP protein was overexpressed in HEK293T cells. 48 hours after transfection, cells were harvested and lysed. The protein supernatant was transferred to a new 15 mL centrifuge tube, and Flag beads were added. The mixture was incubated on a rotating platform at 4 °C for 6–8 hours. The supernatant was then discarded using a magnetic stand, lysis buffer was added, transferred to a new 1.5 mL EP tube, and incubated on a rotating platform at 4 °C for 3 minutes. This step was then aspirated and discarded. This step was repeated two more times. PBS and 3x Flag peptide were added, and the mixture was incubated on a rotating platform at 4 °C overnight. After the incubation, the liquid was transferred to a new EP tube using a magnetic stand. This liquid represents the purified target protein. The purified protein was identified and quantified by electrophoresis and Coomassie Brilliant Blue staining (Figure 20A).

[0055] Derivative DNJ-1 (stock solution 30 mM / L) was diluted 150-fold with binding buffer to form the initial concentration in tube 1. Sixteen PCR tubes were prepared, and the above DNJ-1 was serially diluted. 10 μL of binding buffer was added to tubes 2–16. 20 μL of diluted DNJ-1 was aspirated into PCR tube 1 and mixed uniformly by pipetting. 10 μL was then aspirated from tube 1 and added to tube 2. After mixing the mixture uniformly, 10 μL was added to tube 3, and so on. 10 μL of 40 nM purified EGFP-OPA1 was added to each tube, mixed, and analyzed using a Monolith NT.115 instrument. The Kd values ​​were then calculated.

[0056] Microscale thermophoresis (MST) is a classical method used to detect interactions between proteins and small molecules. MST detects the affinity between a ligand and a target protein by observing the conformational change of the ligand after binding to the target protein, i.e., the ligand proximity effect, based on the thermophoretic effect. As shown in Figure 20, the Kd values ​​of DNJ-1 and DNJ-5a were significantly reduced compared to the lead compound, indicating that DNJ-1 and DNJ-5a have superior targeting properties. Example 8

[0057] After induction, TrypLE was added to the cardiomyocytes and digested at 37°C for 3 minutes, followed by the addition of complete RPMI medium. The cells were centrifuged at low speed, resuspended in RPMI / B27, and seeded onto Matrigel-coated 8mm slides at a 1:10 ratio. Cultured at 37°C in a 5% CO2 environment. 16 hours after seeding, the medium was replaced with fresh RPMI / B27 medium, and 30 μM small molecule drugs were added to the experimental group, while an equal volume of DMSO was added to the control group. After 48 hours of culture, action potentials of the cardiomyocytes were recorded. The cell slides were removed, placed in a thermostatic patch clamp bath at 37°C, and continuously perfused with extracellular solution. The cell membranes of the cardiomyocytes were ruptured with negative pressure, and action potentials of spontaneously beating cells were recorded. Data were collected and analyzed using Patch Master (HEKA), Fit Master (HEKA), Igor Pro (Wavemetrics), and Origin 6.1 (Microcal) software. As shown in Figure 21, DNJ-5a can significantly improve the proportion of cells with electrophysiological abnormalities in HCM-iPSC-CMs compared with DNJ-1. Example 9

[0058] A model was established by subcutaneously implanting an Alzet Osmotic Pump2004 slow-release pump delivering angiotensin II (AngII) / saline in C57BL / 6J wild-type mice. Continuous feeding modeling and drug administration over a 4-week period yielded relevant statistical results.

[0059] Model construction: The mouse was weighed, anesthetized, and the surgical site was prepared and disinfected. A 1 cm incision was made on the front leg above the scapula using surgical scissors. The incision was perpendicular to the tail. The skin was carefully incised to avoid damaging the underlying tissue, and a subcutaneous tunnel was created under the skin using hemostat. The pump with the regulator tip was fully inserted into the pocket (facing the end of the mouse's tail), and the incision was sutured.

[0060] Administration method: intraperitoneal injection, drug dose 10 mg / kg / d, dissolved in phosphate buffered saline (PBS), administered twice daily, morning and evening. The placebo group received an equal volume of phosphate buffered saline (PBS).

[0061] Cardiac ultrasound examination of mice was performed 4 weeks after modeling and drug administration. Briefly, (1) Skin preparation: Prior to ultrasound examination, the mouse's chest was depilated from the xiphoid process to the left axilla using a hair removal cream. (2) Anesthesia: The mouse was anesthetized with isoflurane inhalation. Once the mouse was lightly anesthetized, the isoflurane flow rate was rapidly reduced. The mouse was then placed on a 37°C thermostatic bench. The mouse's limbs were fixed with tape until the mouse's heart rate was controlled at approximately 480-540 beats per minute. At this time, the mouse's anesthesia weakened, its heart rate recovered, and it was able to cooperate with the ultrasound procedure before ultrasound examination began.

[0062] In this experiment, a Vinno 6LAB small animal high-resolution ultrasound detector was used to perform two-dimensional (2D) parasternal short-axis M-mode detection. Under image guidance, the ultrasound probe was rotated and adjusted until a clear short-axis view of the left ventricle was obtained, and images were collected. Each index was examined over 3 to 6 cardiac cycles, and the average value of the measured cardiac cycles was calculated.

[0063] The statistical results of several key parameters in echocardiography are shown in Figure 22. Compared with the control group, key indicators such as ejection fraction (EF) and fractional shortening (FS) significantly decreased in the modeling group, and left ventricular diastolic wall thickness (LVPWd) and left ventricular systolic wall thickness (LVPWs) significantly improved. In the treatment group, relevant pathological phenotypes significantly improved after intervention, and DNJ-5a was superior to the lead drug, DNJ, indicating that DNJ-5a has a superior in vivo effect than the lead drug, DNJ. Example 10

[0064] After 4 weeks of continuous feeding, modeling, and drug administration, the mice were subjected to weight and other indicators before collection. They were then deeply anesthetized with isoflurane and sacrificed after complete relief of pain. After sacrifice, the hearts were removed and heart weight and other indicators were measured.

[0065] For histological analysis, mouse hearts were harvested, cut along the maximal cross-section, and fixed in 4% paraformaldehyde. Subsequently, they were paraffin-embedded, sectioned, and stained with hematoxylin-eosin (HE). The steps were as follows: 1) xylene (I) for 9 minutes; 2) xylene (II) for 9 minutes; 3) xylene (III) for 9 minutes; 4) absolute ethanol I for 5 minutes; absolute ethanol II for 5 minutes; 5) 85% ethanol for 5 minutes; 6) 85% ethanol for 5 minutes; 7) washed 2–3 times with distilled water for 4–5 minutes each; 8) stained with hematoxylin for 5 minutes; 9) washed with running water for 1–3 seconds to remove hematoxylin; 10) differentiated with 1% HCl ethanol for 3 seconds; 11) washed with double-distilled water for 10–30 seconds; 12) washed with PBS for 1–2 seconds; and 13) stained with 0.5% eosin for 2 minutes. 14) The slices were washed with distilled water for 2 seconds. 15) They were differentiated in 85% ethanol for 4 seconds. 16) 95% alcohol (I) for 2 minutes; 17) 95% alcohol (II) for 3 minutes; 18) absolute ethanol for 11 minutes; 19) carbolic acid xylene for approximately 8-9 minutes; 20) xylene (I) for 4 minutes; 21) xylene (II) for 4 minutes; 22) xylene (III) for 4.5 minutes; 23) Finally, they were mounted using neutral gum for long-term storage. The slices were scanned with a scanner (3DHISTECH) and acquired and measured using slideviewer software.

[0066] The results are shown in Figure 23. The heart weight / body weight ratio of the modeling group was significantly higher than that of the control group. After drug intervention, this ratio was significantly reduced. Compared with the lead drug DNJ, DNJ-5a is more effective. Example 11

[0067] After sacrificing the mice, a small portion of the left ventricular tissue was harvested and the myocardium was cut into 1 mm pieces. 3The tissue was cut into cubes. The tissue was collected along the long and short axes of the heart, in accordance with the orientation of mitochondria in the myocardial tissue, and samples for transmission electron microscopy were prepared.

[0068] The steps were as follows: 1) Fixation: The collected myocardial tissue specimens were placed in 2.5% glutaraldehyde in PBS buffer and fixed for 2 hours at room temperature, followed by overnight fixation at 4°C. 2) Rinsing was performed three times with approximately 1 ml of 0.1 M PBS for 10 minutes each. 3) Fixation was performed for 1 hour in approximately 50-100 μl (covering the sample) of 1% osmic acid. 4) Rinsing was performed three times with water for 10 minutes each. 5) Fixation / staining was performed for 30 minutes in approximately 100 μl of 2% uranyl acetate solution. 6) Dehydration: Dehydration was performed twice, with 50%, 70%, and 90% ethanol for 15 minutes each, 100% ethanol for 20 minutes, and 100% acetone for 20 minutes. 7) Infiltration: The specimens were immersed in a 1:1 mixture of embedding medium and pure acetone at room temperature for 2 hours, or overnight in a 3:1 mixture of embedding medium and pure acetone. 8) Embedding: Replace with pure embedding medium, place at 30-37 degrees, and embed in the correct direction.

[0069] After polymerization, ultrathin sectioning and staining were performed at the Cryo-Electron Microscopy Center of Zhejiang University, and then a Talos 120KV cryo-transmission electron microscope was used to image the tissue mitochondria.

[0070] As shown in Figure 24, compared with the control group, cardiac mitochondria in the modeling group mice were significantly damaged, with swollen and vacuolated mitochondrial cristae and disorganized myofilaments. Meanwhile, the treated mice showed a significant improvement in mitochondrial ultrastructure. Compared with the lead drug DNJ, DNJ-5a more significantly improved the ultrastructure of mitochondrial cristae and reduced the rate of vacuolation.

Claims

1. 1. A 1-deoxynojirimycin derivative, characterized in that it is one of the following: (1) DNJ-1, whose structure is represented by Formula II 【Chemistry 1】 Formula II (2) DNJ-5a, whose structure is shown in VII 【Chemistry 2】 Formula VII (3) DNJ-5c, whose structure is represented by Formula VIII 【Transformation 3】 Formula VIII (4) The structure is represented by general formula II 【Chemistry 4】 Formula I-I (wherein R is -H, -NO ２ , —X, where X is F, Cl, Br, or I.

2. 2. The 1-deoxynojirimycin derivative according to claim 1, wherein R is selected from -X, and X is F.

3. Use of the 1-deoxynojirimycin derivative or a pharmaceutically acceptable salt thereof according to claim 1 or 2 in the preparation of an agonist of OPA1.

4. 3. Use of the 1-deoxynojirimycin derivative or a pharmaceutically acceptable salt thereof according to claim 1 or 2 in the preparation of a drug for treating a disease associated with an imbalance in OPA1 multimer formation.

5. The use according to claim 4, wherein the diseases associated with an imbalance in OPA1 multimer formation are hypertrophic cardiomyopathy, hearing loss, optic nerve atrophy, progressive external ophthalmoplegia and ataxia, progressive myoclonic epilepsy, spastic paraplegia and intestinal motility disorder, and retinal degeneration.

6. The use according to claim 5, wherein the disease associated with an imbalance in OPA1 multimer formation is caused by mitochondrial dysfunction.

7. A drug for treating a disease associated with an imbalance in OPA1 multimer formation, characterized in that the active ingredient is the 1-deoxynojirimycin derivative or a pharmaceutically acceptable salt thereof according to claim 1 or 2.

8. The drug described in claim 7, characterized in that the diseases associated with an imbalance in OPA1 multimer formation are hypertrophic cardiomyopathy, hearing loss, optic nerve atrophy, progressive external ophthalmoplegia and ataxia, progressive myoclonic epilepsy, spastic paraplegia and intestinal motility disorder, and retinal degeneration.

9. The drug of claim 8, wherein the hypertrophic cardiomyopathy, hearing loss, optic atrophy, progressive external ophthalmoplegia and ataxia, progressive myoclonic epilepsy, spastic paraplegia and intestinal motility disorder, and retinal degeneration are caused by mitochondrial dysfunction.

10. (i) When the 1-deoxynojirimycin derivative is (1), compounds a and b1 are subjected to a condensation reaction in a solvent to obtain the corresponding 1-deoxynojirimycin derivative, wherein compound a is 1-deoxynojirimycin and compound b1 is 【Transformation 5】 and (ii) When the 1-deoxynojirimycin derivative is (2), first, 1-deoxynojirimycin is reacted with benzyl chloroformate to produce intermediate product 1; 【Transformation 6】 Next, intermediate product 1 is reacted with PivCl to obtain intermediate product 2, 【Transformation 7】 Finally, intermediate product 2 is reduced to give the 1-deoxynojirimycin derivative. (iii) When the 1-deoxynojirimycin derivative is (3), first, 1-deoxynojirimycin is reacted with 2-bromoethylbenzene to produce an intermediate product; 【Transformation 8】 Next, the intermediate product is reacted with PivCl to obtain the 1-deoxynojirimycin derivative, (iv) When the 1-deoxynojirimycin derivative is (4), compounds a and b4 are subjected to a condensation reaction in a solvent to obtain the corresponding 1-deoxynojirimycin derivative, in which compound a is 1-deoxynojirimycin and compound b4 is 【Chemistry 9】 2. The method for preparing a 1-deoxynojirimycin derivative according to claim 1, wherein

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