Mitochondria-targeting isoketal / isolevuglandin scavenger

Abstract

Compounds and methods for treating, preventing, and ameliorating at least one of vascular oxidative stress, vascular function, or hypertension, comprising administering to a subject a compound that targets mitochondrial CypD to inhibit vascular oxidative stress, improve vascular function, and / or reduce hypertension.

Description

Background Art

[0001] Without government support.

[0002] Background of the Invention The present invention relates to scavengers that target mitochondria of highly reactive lipid dicarbonyls derived from arachidonic acid and other polyunsaturated fatty acids, isolevuglandin (isoLG, also known as isoketal or gamma ketoaldehyde), pharmaceutical compositions containing such compounds, and methods of treating conditions including inflammation, oxidative stress, and / or mitochondrial dysfunction.

[0003] One aspect of the present invention is novel compounds that target mitochondria. Without being bound by mechanisms or theories, these compounds are not typical antioxidants, but remove products of inflammation and protect endothelial-dependent relaxation.

Technical Field

[0004] Highly reactive lipid dicarbonyls such as isoLG cause cellular dysfunction, cytotoxicity, and immunogenicity, and promote inflammation and tissue damage in cardiovascular disease, hypertension, cancer, and neurodegeneration.

[0005] Cardiovascular disease and cancer are the leading causes of death in Western societies. In 2002, more than 450,000 Americans under 85 years old died of cancer and of heart disease. The present invention meets the long-standing need for the treatment of both cardiovascular disease and cancer, based on the contribution of oxidative stress in both pathological conditions.

[0006] In the United States, approximately 50 million people suffer from overt hypertension, and up to 60% of the population has prehypertension. Hypertension is a major healthcare concern because it significantly increases the risk of death from stroke, ischemic heart disease, and other vascular diseases. An important mediator is the hormone angiotensin II, which increases thirst, promotes salt retention by the kidneys, causes blood vessel constriction, and promotes the release of catecholamines from nerves and the adrenal glands. Angiotensin II also directly promotes the development of inflammation and atherosclerosis. Hypertension is associated with oxidative stress in blood vessels and the accumulation of reactive lipid dicarbonyls such as isolG. Removal of reactive lipid dicarbonyls by salicylamine (2-HOBA) reduces hypertension, prevents inflammation, and protects endothelial-dependent relaxation in animal experiments.

Summary of the Invention

[0007] Summary of the Invention One embodiment of the present invention is a compound that is a scavenger targeting mitochondrial lipid dicarbonyls. In one embodiment, the following formula

Chemical formula

[0008] In another embodiment, the scavenger targeting mitochondria has the following formula

Chemical formula

[0009] In another embodiment, the scavenger targeting mitochondria has the following formula ​​[ka] (In the formula, R is C1~C) 12 (It is a substituted or unsubstituted alkyl group.) These are compounds, their stereoisomers, and pharmaceutical salts.

[0010] In another embodiment, a scavenger that targets mitochondria is given by the following formula [ka] (In the formula, R is C1~C) 12 (It is a substituted or unsubstituted alkyl group.) These are compounds, their stereoisomers, and pharmaceutical salts.

[0011] In another embodiment, a scavenger that targets mitochondria is given by the following formula [ka] (In the formula, X is a bond, -O-, or -CH2-, R is C1~C 12 It is a substituted or unsubstituted alkyl group. R1 is C1~C 12 (It is a substituted or unsubstituted alkyl or acetoxymethyl molecule.) These are compounds, their stereoisomers, and pharmaceutical salts.

[0012] In another embodiment, a scavenger that targets mitochondria is given by the following formula [ka] (In the formula, each R is independent and C1~C 12 Selected from substituted or unsubstituted alkyl groups, Each R1 is independent and C1~C 12 (Selected from substituted or unsubstituted alkyl or acetoxymethyl) These are compounds, their stereoisomers, and pharmaceutical salts.

[0013] In another embodiment, the scavenger targeting mitochondria has the following formula

Chemical formula

Chemical formula

[0014] In yet another embodiment, there is a compound of the following formula

Chemical formula

[0015] In another embodiment of the present invention, there is provided a method for treating, preventing, and alleviating hypertension in a subject, which includes administering an effective amount of the scavenger targeting mitochondria of the present invention or a pharmaceutically acceptable salt thereof.

[0016] In another embodiment of the present invention, there is provided a method for treating, preventing, and alleviating vascular oxidative stress in a subject, which includes administering an effective amount of the scavenger targeting mitochondria of the present invention or a pharmaceutically acceptable salt thereof.

[0017] Another embodiment of the present invention is a method for treating, preventing, and alleviating at least one of vascular oxidative stress, improving vascular function, and / or reducing hypertension, which includes administering a compound targeting mitochondrial CypD to a subject to inhibit vascular oxidative stress, improve vascular function, and / or reduce hypertension.

[0018] Another embodiment of the present invention is for use in treating, preventing, and alleviating hypertension in a subject, and has the following formula

Chemical formula

[0019] Another embodiment of the present invention relates to a formula used to treat, prevent, and relieve a target hypertension, the following formula [ka] (In the formula, each R is independently C1~C 12 Selected from substituted or unsubstituted alkyl groups, Each R1 is independently C1~C 12 (Selected from substituted or unsubstituted alkyl or acetoxymethyl) These are compounds, their stereoisomers, and pharmaceutical salts.

[0020] In another embodiment, a mitochondrial-targeting scavenger used to treat, prevent, and relieve a target hypertension is given by the following formula: [ka] (In the formula, R is C1~C) 12 It is a substituted or unsubstituted alkyl group. R2 is selected from -P-Ph3, or [ka] (is) These are compounds, their stereoisomers, and pharmaceutical salts.

[0021] Another embodiment of the present invention relates to a formula used to treat, prevent, and relieve a target hypertension, the following formula [ka] These are compounds, their stereoisomers, and pharmaceutical salts. [Brief explanation of the drawing]

[0022] [Figure 1]Figure 1 is a schematic diagram illustrating hyperacetylation of CypD, vascular oxidative stress, and hypertension. The inventors have discovered that excessive acetylation of CypD promotes vascular oxidative stress and contributes to hypertension, and that means of reducing CypD acetylation and CypD inhibition improve vascular function and alleviate hypertension. [Figure 2] Figures 2A–2D are a series of graphs showing aortic vasodilation induced by angiotensin II-induced hypertension (A), vascular mitochondrial O2 (B), and acetylcholine (C) or NO donor SNPs (D). Blood pressure was measured by telemetry. After 14 days of infusion with saline or Ang II (0.7 mg / kg / day), mice were sacrificed and the aorta isolated to test mitochondrial O2 and vasodilation. * P < 0.01 vs. Sham, ** P < 0.01 vs. WT + Ang II, *** P < 0.01 vs. WT (n = 8). [Figure 3] Figures 3A–3D are a series of graphs illustrating examples of CypD targeting in hypertension. Blood pressure in C57Bl / 6J mice infused with saline (sham), Ang II (0.7 mg / kg / day), or treated with the CypD blocker sangliferin A (SFA) after the onset of Ang II-induced hypertension (Ang II + SFA). Blood pressure was measured using the tail cuff method (A)3. Fourteen days after saline or Ang II infusion, mice were sacrificed and the aorta isolated to test mitochondrial O2·_ using MitoSOX and HPLC, or to test vasodilation. Results are mean ± SEM (n = 6–8). * P < 0.01 vs. sham, ** P < 0.01 vs. Ang II, *** P < 0.01 vs. Ang II + SFA (n = 8). [Figure 4]Figures 4A–4D are a series of graphs showing mitochondrial O2 (A) and vasodilation (B, C, D) of blood vessels treated ex vivo with a combination of Ang II (10 nM), IL17A (10 ng / ml), and TNFα (1 ng / ml) (ATI) for 24 hours. Aorta was isolated from C57Bl / 6J (WT), CypD- / -, TgSOD2, or mCAT mice. Mitochondrial O2 was measured by MitoSOX and HPLC.3 Results are mean ± SEM (n = 8). * P < 0.01 vs WT, ** P < 0.05 vs WT + ATI. [Figure 5] Figure 5 shows Western blots of Sirt3 expression and mitochondrial protein (mitoAc-K) acetylation in patients with essential hypertension compared to a normal blood pressure control group. Results are mean ± SEM (n = 6). * P < 0.01 vs. normal blood pressure, ** P < 0.001 vs. normal blood pressure. [Figure 6] Figure 6 shows mitochondrial hyperacetylation and CypD acetylation in hypertension. Western blots of mitochondria isolated from the aorta dissected from AngII-injected C57Bl / 6J and CypD- / - mice. CypD acetylation was measured by CypD immunoprecipitation and Western blotting using anti-acetyllysine antibody. Figure 6 also shows representative blots obtained from three experiments. [Figure 7] Figure 7 shows that depletion of CypD or GCN5L1 acetylase hinders the simulation of mitochondrial O2·_, while depletion of Sirt3 leads to overproduction of O2·_. HAECs were treated with AngII (10 ng / ml) and TNFα (1 ng / ml) for 24 hours, and mitochondrial O2·_ was measured by MitoSOX and HPLC. This figure also shows a typical CypD Western blot analysis. Results are mean ± SEM (n = 6). * P < 0.01 vs. Sham, ** P < 0.05 vs. NS, *** P < 0.05 vs. NS, § P < 0.05 vs. Sham. [Figure 8]Figure 8 shows mitochondrial swelling (A) and respiratory impairment (B) induced by isoLG or isoLG-PE. Untreated mouse kidney mitochondria containing glutamate and malate were incubated (5 minutes) with ethanol, isoLG (1 μM), or isoLG-PE (1 μM) as the vehicle, followed by the addition of ADP (50 μM) and measurement of oxygen consumption. * P < 0.001 vs. control, ** P < 0.03 vs. isoLG. [Figure 9] Figure 9 shows the suppression of mitochondrial oxidative stress by mito2HOBA. HAECs were treated with mito2HOBA (50 nM), 2HOBA, or isoLG inactive 4HOBA and incubated with Ang II (100 nM) and TNFα (10 nM) for 24 hours. MitoSOX-mediated mitochondrial O2·_ was then measured using HPLC (A) and cardiolipin oxidation by LC / MS (B). * P < 0.001 vs. control, ** P < 0.01 vs. Ang II / TNFα. [Figure 10] Figure 10 shows the effects of mito2HOBA on Ang II-induced hypertension, isoLG adducts, and CypD acetylation in aortic mitochondria. (A) Blood pressure of C57Bl / 6J mice infused with physiological saline (sham) or Ang II (0.7 mg / kg / ml). Mito2HOBA was supplemented with drinking water (0.1 g / L). (B) Western blot of aortic mitochondria for isoLG adducts (D11 antibody), CypD, GCN5L1, Sirt3, and CypD acetylation (CypD ip and anti-acetyl-K WB). Results are mean ± SEM. * P < 0.01 vs. sham, ** P < 0.01 vs. Ang II (n = 8). [Figure 11]Figures 11A-11B show LS / MS / MS analysis of mitochondrial isoLG-Lys-lactam protein adducts. (A) Representative LC / MS / MS chromatogram. (B) isoLG-Lys-lactam levels in mitochondria isolated from the kidneys of mice ingested with water (sham), mito2HOBA (0.1 g / L), and injected with Ang II (0.7 mg / kg / day). Results are mean ± SEM (n = 3). * P < 0.05 vs. Ang II. [Figure 12] Figure 12 shows that mito2HOBA reduces mPTP pores and prevents mitochondrial dysfunction. C57Bl / 6J mice were infused with Ang II (0.7 mg / kg / ml) and mito2HOBA (0.1 g / L) in drinking water. Fourteen days after Ang II infusion, the animals were sacrificed and kidneys were isolated for mitochondrial testing. Addition of CaCl2 to mitochondria exceeding their Ca2+ holding capacity causes mPTP pores and mitochondrial swelling. Mitochondria isolated from Ang II-infused mice showed a significant decrease in Ca2+ capacity due to increased mPTP pores, and cyclosporine A (CsA), a CypD inhibitor, restored (rescue) their Ca2+ holding capacity (A). The respiratory control ratio (state 3 / state 4) was measured in isolated kidney mitochondria containing glutamate and malate (B). Control levels are 100%. (B) Renal ATP was measured in newly isolated tissues by a luciferase-based luminescence assay. Results are mean ± SEM. * P < 0.01 vs. Sham, ** P < 0.01 vs. Ang II (n = 3-8). [Figure 13]Figure 13 shows the effects of mito2HOBA on aortic O2·₂ (A) and endothelial NO (B) in mice injected with Ang II. (A) Aortic O2·₂ was measured by DHE probe and HPLC. (B) Endothelial NO was analyzed by ESR and Fe(DETC)2. C57Bl / 6J mice were injected with Ang II (0.7 mg / kg / ml) and provided with mito2HOBA in drinking water (0.1 g / L). Results are mean ± SEM. * P < 0.01 vs. sham, ** P < 0.01 vs. Ang II (n = 6). [Figure 14] Figure 14 is a schematic diagram illustrating the finding that isolevgrandin activates CypD, which is a contributing factor to mitochondrial dysfunction, vascular oxidative stress, and hypertension, and that the removal of mitochondrial isoLG reduces endothelial dysfunction and alleviates hypertension. [Figure 15] Figure 15 shows the reaction of isoLG with protein lysine and phosphatidylethanolamine (PE), and the removal of isoLG by 2-hydroxybenzylamine (2HOBA) or its mitochondrial-targeting analog, mito2HOBA. [Figure 16] Figure 16 shows the effect of mito2HOBA on angiotensin II-induced hypertension and the accumulation of isoLG mitochondrial protein adducts. (A) Blood pressure of C57Bl / 6J wild-type mice infused with either saline (sham) or Ang II (0.7 mg / kg / ml). Mito2HOBA was supplemented with drinking water (0.1 g / L). (B) Mitochondrial isoLG was measured by Western blotting of cardiac mitochondria using D11 antibody, as previously described. Results are mean ± SEM. * P < 0.01 vs. sham, ** P < 0.01 vs. Ang II (n = 8). [Figure 17]Figure 17 shows mitochondrial oxidative stress in hypertension. (A) Systolic blood pressure of C57Bl / 6J wild-type (WT) and mCAT mice infused with physiological saline (sham) or Ang II (0.3 mg / kg / day). (B) Measurement of cardiolipin oxidation by LC-MS36. Fourteen days after infusion of physiological saline or Ang II, mice were sacrificed and hearts isolated for measurement of cardiolipin oxidation. * P < 0.01 vs. sham, ** P < 0.01 vs. Ang II (n = 6). [Figure 18] Figure 18 shows a Western blot analysis of isoLG protein adducts from mitochondria isolated from the aorta dissected from C57Bl / 6J mice injected with AngII and treated with mito2HOBA (A). CypD-isoLG modifications were measured by CypD immunoprecipitation and Western blotting using an anti-isoLG D11 antibody (B). This figure shows representative blots obtained from three experiments. [Figure 19] Figure 19 shows that mito2HOBA reduces mitochondrial dysfunction. (A) Respiratory control ratio (state 3 / state 4) was measured in isolated kidney mitochondria containing glutamate and malate. Control level is 100%. (B) Kidney ATP levels were measured in freshly isolated tissue by a luciferase-based luminescence assay. C57Bl / 6J mice were infused with AngII (0.7 mg / kg / ml) and supplied with mito2HOBA in drinking water (0.1 g / L). Fourteen days after Ang II infusion, the animals were sacrificed and kidneys were isolated for mitochondrial and ATP testing. Results are mean ± SEM. * P < 0.01 vs. Siamese, ** P < 0.01 vs. Ang II (n = 3-8). [Modes for carrying out the invention]

[0023] Before disclosing and describing the compounds, compositions, articles, systems, apparatus and / or methods of the present invention, it should be understood that these synthesis methods or agents are not limited to any particular ones unless otherwise indicated, as they are naturally subject to change. It should also be understood that the terminology used herein is intended to describe only specific aspects and is not intended to be restrictive. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but only illustrative methods and materials are described here.

[0024] All publications referenced herein are incorporated herein by reference to disclose and illustrate methods and / or materials relating to the matters relating to which such publications are referenced. Publications referenced herein are provided only with respect to disclosures prior to the filing date of this application. This herein shall not be construed as an admission that the present invention is not entitled to precede such publications for the purposes of prior invention. Furthermore, the publication dates of publications provided herein may differ from the actual publication dates and should be verified independently.

[0025] As used herein and in the claims thereof, the singular form includes plural references unless explicitly stated otherwise. For example, a reference to “functional group,” “alkyl,” or “residue” includes combinations of two or more such functional groups, alkyls, or residues.

[0026] A range may be expressed herein as a range from a particular value preceded by "approximately" and / or to another particular value preceded by "approximately". When such a range is expressed, the further perspective includes that particular value and / or that other particular value. Similarly, when a value is expressed as an approximation using the antecedent "approximately", that particular value is understood to constitute the further perspective. Furthermore, each endpoint of a range is understood to be significant with respect to the other endpoints, and independently of the other endpoints. It is also understood that there are several values ​​disclosed herein, and each value is disclosed herein not only as the value itself but also as that particular value preceded by "approximately". For example, if the value "10" is disclosed, "approximately 10" is also disclosed. Each unit between two particular units is also understood to be disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13 and 14 are also disclosed.

[0027] As used herein, the terms "optionally have" or "optionally have" mean that the event or situation described thereafter may or may not occur, and that such description includes both examples of cases in which the event or situation occurs and examples in which it does not occur.

[0028] As used herein, the term “subject” means the target of administration. The subjects of the methods disclosed herein may be vertebrates (e.g., mammals), fish, birds, reptiles, or amphibians. Thus, the subjects of the methods disclosed herein may be humans, non-human primates, horses, pigs, rabbits, dogs, sheep, goats, cattle, cattle, guinea pigs, or rodents. This term does not imply any particular age or sex. Thus, adult and neonatal subjects, as well as fetuses / fetuses, are intended to be included, regardless of whether they are male or female. “Patient” means a subject suffering from a disease or disorder. The term “patient” includes human and veterinary subjects.

[0029] As used herein, the term “treatment” means the medical management of a patient with the intention of curing, relieving, stabilizing or preventing a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically directed toward improvement of a disease, pathological condition, or disorder, and causal treatment, i.e., treatment directed toward the elimination of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes reactive treatment, i.e., treatment planned for symptom relief rather than cure of a disease, pathological condition, or disorder; preventive treatment, i.e., treatment directed toward minimizing the associated disease, pathological condition, or disorder or partially or completely preventing their onset; and supportive treatment, i.e., treatment used to complement other specific treatments directed toward improvement of the associated disease, pathological condition, or disorder.

[0030] As used herein, the terms “prevent” or “prevention” mean preventing, avoiding, removing, taking preventative measures against, stopping or hindering the occurrence of something, particularly by prior action. Where “reduce,” “inhibit,” or “prevent” are used herein, the use of the other two terms is also understood to be expressly disclosed unless otherwise specifically indicated. As can be understood herein, there is overlap between the definitions of treatment and prevention.

[0031] As used herein, the term “diagnosed” means that a person has undergone a medical examination by a person skilled in the art (e.g., a physician) and has been found to have a pathological condition that can be diagnosed or treated by the compounds, compositions, or methods disclosed herein. As used herein, the phrase “identified as needing treatment for a disorder” means the selection of a person based on the need for treatment for the disorder. For example, a person may be identified as needing treatment for a disorder (e.g., an inflammation-related disorder) based on an early diagnosis by a person skilled in the art, and subsequently receive treatment for the disorder. In one respect, the identification may be performed by a person other than the person making the diagnosis. In a further respect, the treatment may be performed by a person who subsequently performs the treatment.

[0032] As used herein, the terms “administering” and “administering” mean any method by which a pharmaceutical preparation is provided to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral administration, transdermal administration, inhalation administration, nasal administration, topical administration, vaginal administration, ocular administration, intraotoral administration, intracerebral administration, rectal administration and parenteral administration (including injectable methods such as intravenous, intra-arterial, intramuscular, and subcutaneous administration). Administration may be continuous or intermittent. In various aspects, preparations may be administered therapeutically; that is, to treat an existing disease or pathological condition. In further various aspects, preparations may be administered prophylactically; that is, to prevent a disease or pathological condition.

[0033] As used herein, the term “effective dose” means a quantity sufficient to achieve a desired outcome or exert a certain effect against an undesirable condition. For example, “therapeutic dose” means a quantity sufficient to achieve a desired therapeutic outcome or exert a certain effect against an undesirable symptom, but generally insufficient to cause adverse side effects. The specific therapeutic dose level for any particular patient depends on various factors (including factors well known in the medical field, such as the disorder to be treated and its severity; the specific composition used; the patient’s age, weight, general health, sex, and diet; the number of doses; the route of administration; the rate of excretion of the specific compound used; the duration of treatment; and drugs intentionally used in combination with or incidentally administered simultaneously with the specific compound used). For example, it is well known to those skilled in the art to start with a dose of a compound lower than the level required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. If desired, an effective daily dose may be divided into multiple doses for administration purposes. As a result, a single-dose composition may contain a fraction of its amount that constitutes such an amount or daily dose. Dosage may be adjusted by individual physicians in the event of any contraindications. Dosage may vary and may be administered once or twice daily for one or several days. Guidance can be found in the literature on appropriate dosing for a given class of drugs. In various further respects, preparations may be administered in a “preventive effective dose”; that is, a dose effective in preventing disease or pathological condition.

[0034] As used herein, the term “pharmaceutically acceptable carrier” means a sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion immediately before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Adequate fluidity can be maintained, for example, by the use of a coating agent (e.g., lecithin), and in the case of dispersions, by maintaining the required particle size and using a surfactant. These compositions may also contain adjuvant agents, such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of microbial action can be ensured by including various antibacterial and antimicrobial agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may be desirable to include isotonic agents, such as sugars or sodium chloride. Extended absorption of injectable dosage forms can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin. Injectable depot dosage forms are manufactured by forming a microcapsule matrix of the drug in biodegradable polymers, such as polylactide-polyglycolide, poly(orthoester), and poly(anhydrous). The drug release rate can be controlled depending on the drug-to-polymer ratio and the properties of the specific polymer used. Depot-type injectable formulations can also be manufactured by encapsulating the drug in liposomes or microemulsions that are compatible with body tissues. These injectable formulations can be sterilized, for example, by filtration through a bacterial retention filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed immediately before use in sterile water or other sterile injectable media. Suitable inert carriers include sugars, such as lactose. Preferably, at least 95% by weight of the active ingredient particles have an effective particle size in the range of 0.01 to 10 micrometers.

[0035] As used herein, the terms “scavenger” or “scavenging” refer to a chemical substance that can be administered to remove or inactivate impurities or unwanted reaction products. For example, isoketals specifically and irreversibly add to lysyl residues of proteins. The isoketal scavengers of the present invention react with isoketals before they can add to lysyl residues. Thus, the compounds of the present invention prevent isoketals from adding to proteins by “scavenging” them.

[0036] As used herein, the term “substituted” includes all permitted substituents of an organic compound. In one broad view, permitted substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and non-aromatic substituents of an organic compound. Exemplary substituents include, for example, the following. Permitted substituents may be one or more identical or different for a given organic compound. For purposes of this disclosure, a heteroatom (e.g., nitrogen) may have hydrogen substituents and / or any permitted substituents of the organic compounds described herein (satisfying the valency of the heteroatom). This disclosure is not intended to be limited in any way by the permitted substituents of an organic compound. Furthermore, the terms “substituted” or “substituted with” imply that the substitution conforms to the permitted valencies of the substituted atom and substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation (e.g., rearrangement, cyclization, removal, etc.).

[0037] The term “alkyl” as used herein refers to a branched or unbranched saturated hydrocarbon group having 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, etc. Alkyl groups may be cyclic or acyclic. Alkyl groups may be branched or unbranched. Alkyl groups may also be substituted or unsubstituted. For example, an alkyl group may be substituted with one or more of the groups described herein (including, but not limited to, optionally substituted alkyl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfo-oxo, or thiol). A “lower alkyl” group is an alkyl group having 1 to 6 (e.g., 1 to 4) carbon atoms.

[0038] Throughout this specification, “alkyl” is generally used to refer to both unsubstituted and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by specifying the specific substituents of the alkyl group. For example, the term “alkyl halide” specifically refers to an alkyl group substituted with one or more halides (e.g., fluorine, chlorine, bromine, or iodine). The term “alkoxyalkyl” specifically refers to an alkyl group substituted with one or more alkoxy groups as listed below. The term “alkylamino” specifically refers to an alkyl group substituted with one or more amino groups as listed below. And so on. If “alkyl” is used in one example and a specific term, such as “alkyl alcohol,” is used in another example, it does not mean that the term “alkyl” also refers to the specific term, such as “alkyl alcohol.” And so on.

[0039] Embodiments of the present invention include methods for treating, preventing, and relieving a target hypertension.

[0040] Other embodiments of the present invention include methods for treating, preventing, and alleviating oxidative stress in target blood vessels.

[0041] Other embodiments of the present invention include methods for targeting mitochondrial CypD to suppress oxidative stress in blood vessels, improve vascular function, and / or alleviate hypertension.

[0042] This invention is the first to define CypD as a therapeutic target for hypertension. For the past 30 years, there have been no novel therapeutic methods for this disease. The antihypertensive agent of this invention targets CypD and can be added to currently available therapeutic devices to improve the treatment of hypertension.

[0043] Free radical oxidation of arachidonic acid generates highly reactive isolevgrandins (isoLGs) that cause mitochondrial dysfunction by opening mitochondrial permeability transition pores (mPTPs). Inhibition of cyclophyllin D, an mPTP regulatory subunit, reduces isoLG-induced mitochondrial dysfunction (Free Radic Biol Med 2010; 49(4):567-79). The inventors have shown that depletion of cyclophyllin D leads to mitochondrial O2 ·_ We discovered that it reduces mitochondrial isoLG, improves vasodilation, and reduces hypertension (Hypertension 2016; 67(6):1218-27). The inventors tested their hypotheses that hypertension is associated with the accumulation of mitochondrial isoLG and that mitochondrial-targeting isoLG scavengers reduce vascular oxidative stress and alleviate hypertension. The inventors developed a novel mitochondrial-targeting isoLG scavenger compound containing 4-(aminomethyl)-3-hydroxyphenoxy)butyl)triphenylphosphonium (mito2HOBA) by conjugating the lipophilic cation triphenylmethylphosphonium to 2-hydroxybenzylamine (2HOBA). Mito2HOBA is a water-soluble compound and was well tolerated by cultured human aortic endothelial cells (HAECs) and mice administered with Mito2HOBA in drinking water. mito2HOBA (50 nM) reduces mitochondrial O2·_ It was found that 2HOBA (50 nM) inhibited the production of mitochondrial isoLG (MitoSOX / HPLC) and prevented cardiolipin oxidation (LS-MS) in HEAC incubated with TNFα and angiotensin II, but had no effect. The functional role of mitochondrial isoLG was tested in vivo using an angiotensin II model of hypertension. C57Bl / 6J mice were infused with angiotensin II (0.7 mg / kg / day) or saline (sham) and administered mito2HOBA (0.1 g / L) in drinking water. It was also found that mito2HOBA significantly reduced angiotensin II-induced hypertension. Hypertension was associated with increased mitochondrial isoLG formation, as measured by Western blot analysis of cardiac mitochondria using D11 antibody, and mito2HOBA reduced the accumulation of isoLG adducts in cardiac mitochondria of angiotensin II-infused mice. A decrease in NO is characteristic of endothelial dysfunction in hypertension due to oxidative stress of blood vessels.

[0044] The inventors of this invention have discovered fluorescent O2 ·_ Aortic O2 using probe DHE (dihydroethidium) and HPLC ·_ Vascular oxidative stress was investigated by measuring vascular O2 in mice injected with angiotensin II, and by analyzing endothelial NO using electron spin resonance and specific NO spin trap Fe(DETC)2. ·_ It was discovered that this reduces endothelial NO and maintains endothelial NO levels.

[0045] 2-aminomethylphenols, such as 2-hydroxybenzylamine (2-HOBA, salicylate), show exceptional responsiveness to diseases that cause dicarbonylation. [ka] Mitochondria are oxidative sites and can be damaged by the products of oxidative stress, which is responsible for many diseases, including multiple sclerosis. 2-HOBA is expected to remove these reactive molecules and provide protection, but it needs to be modified to access mitochondria. Certain cationic attachments have been successfully used in the past to achieve this.

[0046] Embodiments of the present invention include a series of mitochondria-targeting compounds. One embodiment of the present invention is a compound with the following formula [ka] (wherein X is a bond, -O-, or -CH2-, R is C1~C 12 (It is a substituted or unsubstituted alkyl group.) These are compounds, their stereoisomers, and pharmaceutical salts.

[0047] Another embodiment of the present invention is given by the following formula [ka] (In the formula, R is C1~C) 12 (It is a substituted or unsubstituted alkyl group.) These are compounds, their stereoisomers, and pharmaceutical salts.

[0048] Another embodiment of the present invention is given by the following formula [ka] (In the formula, R is C1~C) 12 (It is a substituted or unsubstituted alkyl group.) These are compounds, their stereoisomers, and pharmaceutical salts.

[0049] Another embodiment of the present invention is given by the following formula [ka] (In the formula, R is C1~C) 12 (It is a substituted or unsubstituted alkyl group.) These are compounds, their stereoisomers, and pharmaceutical salts.

[0050] Another embodiment of the present invention is given by the following formula [ka] (wherein X is a bond, -O-, or -CH2-, R is C1~C 12 It is a substituted or unsubstituted alkyl group. R1 is C1~C 12 (It is a substituted or unsubstituted alkyl or acetoxymethyl molecule.) These are compounds, their stereoisomers, and pharmaceutical salts.

[0051] Another embodiment of the present invention is given by the following formula [ka] (In the formula, each R is independently C1~C 12 Selected from substituted or unsubstituted alkyl groups, Each R1 is independently C1~C 12 (Selected from substituted or unsubstituted alkyl or acetoxymethyl) These are compounds, their stereoisomers, and pharmaceutical salts.

[0052] Another embodiment of the present invention involves using a lipophilic ester (preferably acetoxymethyl) to transport the “prodrug” across the membrane and hydrolyzing it by intracellular esterase-releasing ionic acids that are captured within the cellular compartment. [ka]

[0053] Another aspect of the present invention is an additional compound that increases the ionicity of the resulting 2-HOBA during hydrolysis. [ka]

[0054] Another embodiment of the present invention is given by the following formula [ka] These are compounds, their stereoisomers, and pharmaceutical salts.

[0055] An embodiment of the present invention is given by the following formula [ka] This includes compounds, their stereoisomers, and pharmaceutical salts.

[0056] I. Hypertension Hypertension is a major health problem in Western societies and a risk factor for stroke, myocardial infarction, and heart failure. Despite the use of multiple medications, one-third of patients have poorly controlled blood pressure, likely due to a mechanism contributing to hypertension that is unaffected by current treatments. In recent years, studies have shown that mitochondria are dysfunctional in hypertension, and that mitochondrial superoxide (O2) plays a role in this disease. ·_ The inventors defined a new role for mitochondrial O2 by angiotensin II and cytokines. ·_ The invention demonstrated the crucial role of mitochondrial cyclophyllin D (CypD) in the stimulation of mitochondrial membrane permeability transition pores (mPTPs). The inventors found that treatment with CypD inhibitors after the onset of hypertension lowered blood pressure, while genetic CypD depletion alleviated hypertension. One aspect of the invention is to define the role of vascular CypD and the potential of CypD-targeted therapies in vascular dysfunction and hypertension. The inventors' studies in animal and human subjects with essential hypertension showed CypD activation by K166 acetylation due to an imbalance between GCN5L1 acetyltransferase and reduced Sirt3 deacetylase activity. Sirt3 levels are reduced in hypertension, and residual Sirt3 is blocked by the highly reactive mitochondrial lipid dicarbonyl isolevgrandin (isoLG), and isolG scavenging prevents hyperacetylation of CypD and alleviates hypertension (Figure 1).

[0057] This invention defines CypD as a novel target for the treatment of hypertension. For the past 30 years, there have been no mechanistically novel therapies for this disease. A new class of antihypertensive drugs targeting CypD could be added to the currently available therapeutic equipment to improve the treatment of hypertension. Without being bound by mechanisms or theories, the inventors have found that specific CypD depletion in endothelium and smooth muscle reduces oxidative stress on blood vessels, protects vasodilation, and alleviates hypertension. Acetylation of CypD-K166 contributes to vascular dysfunction and hypertension. CypD inhibition and blockade of CypD hyperacetylation after the onset of hypertension improve vascular function.

[0058] Clinical data show that one-third of the adult population suffers from hypertension, and an estimated 1.4 billion people worldwide have hypertension, thus this invention meets a long-standing need. This disease is a major risk factor for stroke, myocardial infarction, and heart failure. Despite treatment with multiple drugs, one-third of hypertensive patients remain hypertensive, possibly due to mechanisms unaffected by current treatments. However, for the past 30 years, there have been no new mechanistic treatments for this disease. Therefore, a new class of antihypertensive drugs can be added to the currently available therapeutic equipment to improve the treatment of hypertension.

[0059] Hypertension is a multifactorial disorder. However, in almost all experimental models of hypertension, reactive oxygen species (ROS: O2) are involved. ·_The production of ROS (and H2O2) is increased in multiple organs. In the brain, ROS promote neuronal firing and increase sympathetic nerve outflow. In the kidneys, ROS act in multiple sites to promote sodium absorption and volume retention. In the vascular system, ROS promote vasoconstriction and reformation, increasing systemic vascular resistance. Our group has identified several causes of ROS that contribute to hypertension, including NADPH oxidase, unbound nitric oxide synthase, and mitochondria, and defined their interactions. Overproduction of ROS leads to oxidative stress, which promotes target organ damage in hypertension. Antioxidant therapies are not currently available, and common antioxidants such as ascorbate and vitamin E are ineffective in preventing cardiovascular disease and hypertension, but therapies that target mitochondria in particular are a promising strategy for reducing target organ damage.

[0060] Mitochondrial dysfunction is involved in the pathogenesis of hypertension and cardiovascular disease. However, despite the central role of mitochondria in human health and disease, there are no approved drugs that directly target mitochondria. Mitochondrial dysfunction is characterized by impaired ATP production and increased oxidative stress leading to cellular dysfunction and apoptosis. Mitochondrial membrane permeability transition pores (mPTPs) play a crucial role in mitochondrial dysfunction and terminal organ damage in hypertension. We have discovered that depletion or inhibition of cyclophyllin D (CypD), a regulatory subunit of mPTP pores, improves vascular function and alleviates hypertension. Previous studies have shown that CypD is involved in cell death, and we have shown that CypD is important in vascular oxidative stress and endothelial dysfunction. Our data suggest a novel role for CypD acetylation and reactive isoleviglandins (isoLGs) in mPTP pores and vascular dysfunction.

[0061] The inventors have found that inhibition of CypD in mitochondria isolated from endothelial cells leads to superoxide (O2) ·_Having previously reported preventing the overproduction of ), in this study we propose that gene deletion of vascular CypD or specific inhibition of CypD reduces vascular oxidative stress, improves endothelial function, and alleviates hypertension.

[0062] The inventors of this invention have developed a CypD knockout mouse (CypD - / - CypD deficiency in ) is associated with mitochondrial O2 in angiotensin II (AngII) injected mice. ·_ It prevented the overproduction of (Figure 2A), reduced hypertension (Figure 2B), and improved endothelium-dependent and endothelium-independent vasodilation compared to wild-type C57Bl / 6J mice (Figures 2C, D).

[0063] One aspect of the present invention is to target CypD after the onset of hypertension. The inventors implanted an osmotic pump containing Ang II (0.7 mg / kg / day) into wild-type mice and initiated treatment with sangliferin A after the onset of Ang II-induced hypertension (Figure 3A). In fact, when hypertensive mice were treated with the CypD inhibitor sangliferin A (ip 10 mg / kg / day), blood pressure decreased (Figure 3A), and mitochondrial O2 ·_ Production is normalized (Figure 3B), and vasodilation is improved (Figures 3C, D).

[0064] The inventors have discovered that angiotensin II and cytokines work together to induce CypD-dependent vascular dysfunction. IL17A and TNFα are required for AngII-induced hypertension. These cytokines are generally associated with human hypertension and contribute to the pathogenesis of this disease. This invention relates that Ang II, IL17A, and TNFα interact in mitochondrial O2 in endothelial cells. ·_ This indicates that it coordinately induces CypD-dependent vascular oxidative stress. The functional role of mitochondrial O2 ·_ Scavenger SOD2 (Tg SOD2The study was conducted using aortic sections isolated from mice overexpressing ) or mitochondrial-targeting H2O2 scavenger catalase (mCAT). Our data suggest that aortic vascular treatment with Ang II, IL17A, and TNFα (ATI) is effective in treating CypD - / - We have shown that this leads to severe impairment of endothelium-dependent vasodilation, which is prevented in the aorta isolated from mice. Interestingly, overexpression of SOD2 or expression of mitochondrial-targeting catalase significantly reduced the impairment of vasodilation, similar to the protection induced by CypD deletion (Figure 4).

[0065] These data indicate that the prooxidative environment of Ang II and cytokines causes severe vascular oxidative stress, reducing endothelial NO and impairing vasodilation. This is because Tg SOD2 and CypD depletion or mitochondrial O2 in the blood vessels of mCAT mice ·_ This is prevented by the removal of H2O2. Furthermore, our data confirm the important role of CypD in regulating vascular oxidative stress and in stimulating mitochondrial oxidative stress and vascular dysfunction in hypertension. However, the specific pathway of CypD activation is not clear.

[0066] The increase in hypertension with age is associated with decreased Sirt3 expression. The inventors discovered that Sirt3 inactivation contributes to mitochondrial hyperacetylation in human hypertension. The inventors analyzed Sirt3 expression and acetylated mitochondrial protein in human subjects with essential hypertension. Western blotting of peripheral blood mononuclear cells showed a 1.4-fold decrease in Sirt3 protein levels and a 2.6-fold increase in mitochondrial acetylation in hypertensive subjects (Figure 5).

[0067] The inventors recently reported mitochondrial hyperacetylation in human hypertension and mouse models of hypertension, measured by mass spectrometry and Western blotting. Mitochondrial hyperacetylation in hypertension is accompanied by CypD acetylation, which represents gain of function and promotes mPTP pore opening. To test this, the inventors measured total acetylation and specific CypD acetylation of mitochondrial proteins in aortic mitochondria isolated from normotensive and hypertensive mice. - / - Mice were injected with AngII (0.7 mg / kg / day) or physiological saline (vehicle) for 14 days, and mitochondria were isolated from the aorta for Western blotting. Western blotting analysis showed a potent increase in total lysine acetylation (Ac-K ab) in mitochondrial lysates isolated from hypertensive mice injected with AngII, compared to Siamese wild-type mice. After CypD immunoprecipitation, Western blotting showed no change in CypD levels (WT CypD) in normotensive and hypertensive mice. However, CypD acetylation was significantly increased in aortic mitochondria isolated from hypertensive mice. - / - The mice were protected from hypertension and endothelial dysfunction (Figures 2 and 4), and Western blot showed CypD - / - No CypD or CypD acetylation was observed in the mouse samples, confirming the specificity of the Western blot.

[0068] The role of CypD acetylation in endothelial dysfunction is unclear. However, acetylation of lysine 166 acquires the function of promoting mPTP pore opening, and mitochondrial Sirt3 deacetylates CypD-K166. CypD inhibitors such as cyclosporine A bind near K166 and prevent CypD-mediated mPTP pore opening. In muscle cells, mutations of K166 to arginine (CypD-K166R) mimic deacetylation and weaken mPTP pore opening, while mutations of K166 to glutamine (K166Q) mimic acetylation, increasing mPTP pore opening and exacerbating ischemia-reperfusion injury. GCN5L1-mediated acetylation de-regulates Sirt3-mediated deacetylation. We have shown that in endothelial cells, depletion of GCN5L1 is associated with mitochondrial O2 · While decreasing, Sirt3 deacetylase depletion leads to mitochondrial O2 · This study shows that it enhances the production of O2. Human aortic endothelial cells (HAECs) were transfected with non-silencing siRNA (NS), GCN5L1 siRNA, Sirt3 siRNA, or CypD siRNA. Three days after transfection, the cells were stimulated with Ang II and TNFα, and O2 · MitoSOX O2 · The specific product is Mito-2OH-E + It was measured by HPLC analysis. Depletion of GCN5L1 is similar to depletion of CypD, as is O2 · While neutralizing the overproduction of Sirt3, Sirt3 depletion reduces the O2 levels in basal and stimulated mitochondria. · It was found that both increased (Figure 7). These data support the role of CypD acetylation in endothelial oxidative stress.

[0069] The inventors also learned that isolevgrandin (isoLG), a highly reactive lipid dicarbonyl derived from arachidonic acid, mechanistically links pathogenic reactive oxygen species with disease progression, and discovered that acute isoLG exposure to mitochondria induces CypD-dependent mPTP pore opening and inhibits mitochondrial respiration (Figure 8). Reactive isoLG generates protein-lysine adducts and cytotoxic isoLG-phosphatidylethanolamine adducts (isoLG-PE), which can independently contribute to mitochondrial dysfunction. Indeed, acute treatment of isolated mitochondria with isoLG-PE suppressed respiration by 41%, and similar doses of isoLG reduced respiration by 74%, supporting the potential roles of both isoLG-PE and isoLG protein-lysine adducts in mitochondrial dysfunction.

[0070] The inventors developed mito2HOBA, an isoLG scavenger that targets mitochondria, by conjugating the lipophilic cation triphenylphosphonium to 2HOBA. The membrane potential of mitochondria in living cells is negative internally (-150mV). Because this membrane potential is much higher than that of other organelles in the cell, the lipophilic cation triphenylphosphonium accumulates in the mitochondrial matrix more than 500 times more selectively.

[0071] The inventors of this invention have found that Mito2HOBA is a mitochondrial O2 · We discovered that reducing production inhibits cardiolipin oxidation, demonstrating that mitochondrial oxidative stress produces isoLG, and that removing isoLG improves mitochondrial function. Mitochondrial O2 in cultured human aortic endothelial cells (HAEC) · Cardiolipin oxidation (a specific marker of mitochondrial dysfunction and oxidative stress) was incubated with Ang II and TNFα. These drugs were selected in combination because both have shown contributions to endothelial dysfunction in hypertension. Ang II and TNFα contribute to mitochondrial O2 ·Mitochondrial oxidative stress was induced, as measured by a twofold increase in isoLG and a 2.5-fold increase in cardiolipin oxidation. Treatment with the mitochondrial-targeted isoLG scavenger mito2HOBA (50 nM) reduced mitochondrial oxidative stress (Figure 9A), but the concentrations of non-targeted 2HOBA, which required similar protection, were much higher. Mito2HOBA was more effective than 2HOBA in preventing cardiolipin oxidation (Figure 9B). These data support a previously unidentified role of isoLG in mitochondrial dysfunction and demonstrate the feasibility of using very low doses of mito2HOBA for therapeutic purposes.

[0072] The inventors demonstrate that Mito2HOBA alleviates hypertension, reduces mitochondrial isoLG, prevents hyperacetylation of CypD, and that treatment with the mitochondrial-targeting isoLG scavenger mito2HOBA reduces vascular oxidative stress, protects endothelial function, and alleviates hypertension. Mice injected with Sham or AngII were supplemented with drinking water (0.1 g / L) supplemented with mito2HOBA or plain water. Mito2HOBA significantly reduced Ang II-induced hypertension (Figure 10A). Fourteen days after Ang II injection, mice were sacrificed and the aorta was isolated for Western blotting. Hypertension was associated with a potent increase in mitochondrial isoLG adducts as measured by D11 antibody, and mito2HOBA prevented the accumulation of isoLG adducts. CypD expression remained unchanged. However, acetyltransferase GCN5L1 was increased and deacetylase Sirt3 was decreased in hypertensive mice. This leads to an imbalance between the mitochondrial acetylation and deacetylation pathways, resulting in hyperacetylation of mitochondrial proteins, as measured by hyperacetylation of Ac-K and CypD. Mito2HOBA corrects the imbalance between GCN5L1 and Sirt3, reducing mitochondrial Ac-K and preventing CypD hyperacetylation, in which mitochondrial isoLG is involved in CypD acetylation (Figure 10B).

[0073] Mito2HOBA prevents the accumulation of isoLG-lysyl-lactam protein adducts in mitochondria by Ang II. As described above, the inventors measured isoLG-lysyl-lactam adducts by liquid chromatography-tandem mass spectrometry (LC / MS) after proteolytic digestion of extracted proteins. Hypertension was found to be associated with a four-fold increase in mitochondrial isoLG-lysyl-lactam protein adducts. Supplementation with the mitochondrial-targeting isoLG scavenger mito2HOBA inactivated the formation of isoLG-lysyl-lactam adducts in mitochondria (Figure 11).

[0074] Hypertension impairs mitochondrial function, and mito2HOBA alleviates mitochondrial dysfunction. The accumulation of mitochondrial isoLG in hypertension promotes CypD acetylation and mPTP pore opening, impairing mitochondrial respiration and reducing ATP. Removal of mitochondrial isoLG by mito2HOBA prevents these harmful effects. The inventors analyzed kidney tissue isolated from control mice (sham), mice ingesting mito2HOBA, mice injected with Ang II, and Ang II-injected mice supplemented with mito2HOBA (mito2HOBA and Ang II). Indeed, Ang II injection was found to reduce Ca 2+ It reduced retention capacity, increased mPTP pores, impaired mitochondrial respiration, and decreased renal ATP. However, supplementation with mito2HOBA reduced CypD acetylation, weakened mPTP pores, protected mitochondrial respiration, and maintained normal ATP levels (Figures 10, 12). These data demonstrate the pathophysiological role of mitochondrial isoLG in mitochondrial dysfunction and hypertension.

[0075] Therefore, embodiments of the present invention are Mito2HOBA compounds that reduce oxidative stress in blood vessels and improve endothelial function. The inventors believe that blood vessel O2 · We were the first to show that overproduction contributes to endothelial dysfunction in hypertension. Among other effects, O2· This inactivates endothelial nitric oxide (NO), promoting vasoconstriction and vascular regeneration, ultimately increasing systemic vascular resistance. Therefore, the reduced bioavailability of NO is characteristic of endothelial oxidative stress in hypertension, due to NO oxidation, decreased NO production, and uncoupling of endothelial NO synthase. As mentioned above, fluorescent O2 · Aortic O2 using probe DHE and HPLC · Endothelial NO was measured. Endothelial NO was quantified by electron spin resonance (ESR) and specific NO spin trap Fe(DETC)2. As shown in Figure 13, we found that mito2HOBA was used in vascular O2 of Ang II-injected mice. · We discovered that it reduces NO and maintains its bioavailability. These data indicate a previously unrecognized role of mitochondrial isoLG in vascular oxidative stress and endothelial dysfunction.

[0076] II. Oxidative Stress in Blood Vessels The inventors discovered that mitochondria are dysfunctional in hypertension and defined a new role of mitochondrial oxidative stress in this disease. Mitochondria are involved in superoxide radical (O2) ·_ It is a major source of ) and is rich in unsaturated fatty acids. Free radical oxidation of arachidonic acid produces highly reactive isolegulandins (isoLG). This is what we discovered, that opening of mitochondrial membrane permeable transition pores (mPTPs) causes mitochondrial dysfunction, and inhibition of the mPTP regulatory subunit cyclophyllin D (CypD) reduces isoLG-induced mitochondrial dysfunction. Recently, we found that inhibition of mPTP opening by CypD depletion or inhibition of CypD leads to mitochondrial O2 ·_They discovered that it reduces vasodilation and alleviates hypertension. The inventors also developed a novel isoLG scavenger compound mito2HOBA that targets mitochondria. This novel compound reduces mitochondrial isoLG protein adducts, suppresses the oxidation of cardiolipin, a specific marker of mitochondrial oxidative stress, and reduces vascular O2 ·_ This reduces mitochondrial isoLG, normalizes endothelial nitric oxide, and reduces hypertension. These data are consistent with feedforward stimulation of mitochondrial oxidative stress and demonstrate the therapeutic benefits of mitochondrial isoLG targeting in the treatment of cardiovascular disease. IsoLG causes mitochondrial dysfunction via CypD, leading to organelle damage. Measures to reduce mitochondrial isoLG reduce CypD activation and improve vascular function. This novel concept may lead to a paradigm shift in defining mitochondrial isoLG as a new target in the treatment of cardiovascular disease (Figure 14).

[0077] Reactive oxygen species (ROS: O2) · The production of ROS (and H2O2) increases in hypertension in multiple organs, including key centers of the brain, vascular system, and kidneys. The inventors have identified several sources of ROS contributing to hypertension, including NADPH oxidase, unbound nitric oxide synthase, and mitochondria, and have defined their interactions. On the other hand, antioxidant therapy is not currently available, and common antioxidants such as ascorbate and vitamin E are ineffective in preventing cardiovascular disease and hypertension because they do not easily reach critical ROS production sites such as mitochondria. Furthermore, the inventors have discovered a novel isoLG-dependent mechanism that causes mitochondrial dysfunction and terminal organ damage in hypertension. The compounds of the present invention target mitochondrial isoLG to reduce mitochondrial oxidative stress, improve vascular function, and reduce hypertension.

[0078] To demonstrate an example of removing isoLG from mitochondria to improve mitochondrial function, the mitochondria-targeted isoLG scavenger mito2HOBA compound was developed (see, for example, Figure 15). The membrane potential of mitochondria in living cells is negative internally (-150mV). Because this membrane potential is much higher than that of other organelles in the cell, lipophilic cations such as triphenylphosphonium (TPP) selectively accumulate in mitochondria. Therefore, molecules bound to TPP target mitochondria. For example, mitoTEMPO is enriched more than 500 times within the mitochondrial matrix.

[0079] The above examples are water-soluble compounds that can be supplied to cells in culture medium and to animals in drinking water. In our preliminary in vitro and in vivo experiments, mito2HOBA, contained in drinking water, was well tolerated by cultured human aortic endothelial cells (HAECs) when administered at doses of 0.1–0.3 g / L at concentrations up to 200 nM. Mass spectrometry of kidney and cardiac mitochondria isolated from mice administered drinking water containing mito2HOBA (0.1 g / L) for 5 days confirmed significant accumulation of mito2HOBA at the μM level in the mitochondrial fraction (80%). Similarly, incubation of isolated mitochondria with mito2HOBA (0.1 μM) resulted in 400–600-fold increased accumulation of mito2HOBA in the mitochondrial pellet (Figure 15, insert).

[0080] The inventors implanted osmotic minipumps containing Ang II (0.7 mg / kg / day) or physiological saline (sham) into C57Bl / 6J mice that were administered mito2HOBA (0.1 g / L) in drinking water or ordinary water. Supplementation with mito2HOBA was found to significantly reduce Ang II-induced hypertension (Figure 16A). Fourteen days after Ang II infusion, the mice were sacrificed, and their hearts were isolated for mitochondrial testing, and vascular O2 levels were measured. ·_The aorta was isolated for analysis of endothelial nitric oxide. As expected, hypertension was associated with increased mitochondrial isoLG formation, as measured by Western blotting analysis of cardiac mitochondria using D11 antibody. Furthermore, mito2HOBA reduced the accumulation of isoLG adducts in cardiac mitochondria of Ang II-injected mice (Figure 16B).

[0081] To demonstrate that hypertension is associated with mitochondrial oxidative stress and that mitochondrial H2O2 removal reduces mitochondrial dysfunction and alleviates hypertension, we tested C57Bl / 6J wild-type (WT) and transgenic mice expressing catalase (mCAT), a mitochondrial H2O2 scavenger. Infusion of a low dose of AngII (0.3 mg / kg / day) elevated blood pressure in wild-type mice (136 mm Hg) but not in mCAT mice (115 mm Hg) (Figure 17B). Fourteen days after Ang II infusion, the mice were sacrificed, and the hearts were isolated as described above to measure markers of mitochondrial oxidative stress and cardiolipin oxidation. As expected, hypertension was associated with increased cardiolipin oxidation in wild-type mice. Interestingly, cardiolipin oxidation was completely abolished in mCAT mice injected with Ang II (Figure 17B).

[0082] These data support previously unrecognized roles of mitochondrial oxidative stress and mitochondrial isoLG in endothelial dysfunction and hypertension. As shown above, the inventors of CypD - / - CypD deficiency in mice reduces Ang II-induced hypertension and mitochondrial O2 compared to wild-type C57Bl / 6J mice. ·_ This has been shown to prevent the overproduction of and improve endothelium-dependent and endothelium-independent vasodilation (Figure 9).

[0083] To define the potential role of CypD-isoLG interactions in vascular cell mitochondria, we isolated mitochondrial fractions from the aorta dissected from mice injected with Ang II and measured isoLG-protein adduct formation using an anti-isoLGD11 antibody. 32 Ang II-induced hypertension was associated with a potent increase in mitochondrial isoLG in the aorta, and mito2HOBA supplementation attenuated mitochondrial isoLG accumulation (Figure 18A). Potential CypD-isoLG adduct formation was measured by CypD immunoprecipitation and Western blotting with anti-isoLGD11 antibody. The data showed that Ang II injection increased both mitochondrial protein isoLG adducts and CypD-isoLG, while mito2HOBA decreased them (Figure 18B).

[0084] Hypertension impairs mitochondrial function, and mito2HOBA mitigates mitochondrial dysfunction. Based on the above results, the inventors hypothesized that the accumulation of mitochondrial isoLG in hypertension leads to impaired mitochondrial respiration and decreased ATP production, and that removal of mitochondrial isoLG by mito2HOBA improves mitochondrial respiration and maintains ATP synthesis. To demonstrate this, the inventors analyzed kidney tissue isolated from control mice (sham), mice ingesting mito2HOBA (mito2HOBA), mice injected with Ang II (Ang II), and Ang II injected mice supplemented with mito2HOBA (mito2HOBA and Ang II). In the kidneys of Ang II injected mice with hypertension, mitochondrial respiration was impaired and renal ATP levels were significantly reduced. Interestingly, supplementation of Ang II injected mice with mito2HOBA protected mitochondrial respiration and maintained normal ATP production (Figure 19). These data demonstrate the pathophysiological role of mitochondrial isoLG in mitochondrial dysfunction associated with hypertension.

[0085] Since the present invention is described in this manner, it is obvious that the invention can be modified in many ways. Such modifications, which are obvious to those skilled in the art, should be considered to be included in this disclosure.

[0086] Unless otherwise indicated, all numbers used herein to represent quantities of components, properties (e.g., reaction conditions), etc., should be understood in all cases to be modified by the term “approximately.” Therefore, unless otherwise indicated, the numerical parameters set forth herein and in the claims are approximations that may vary depending on the desired properties to be determined by the present invention.

[0087] Although the numerical ranges and parameters describing the broad scope of this invention are approximations, the numerical values ​​shown in the Experimental Section or the Examples Section have been described as accurately as possible. However, any numerical value inherently contains some degree of error, which is necessarily due to the standard deviation found in each test measurement.

Claims

1. The following formula 【Chemistry 1】 (In the equation, X is a bond and R is C) 1 (It is a substituted or unsubstituted alkyl group.) Compounds of the same, their stereoisomers, and pharmaceutically acceptable salts.

2. The compound of the following formula, its stereoisomers, and its pharmaceutically acceptable salts. 【Chemistry 2】

3. The following formula 【Transformation 3】 (In the formula, R is C 6 to C 12 R is a substituted or unsubstituted alkyl group. 2 is -P-Ph 3 (is) Compounds of the same, their stereoisomers, and pharmaceutically acceptable salts.

4. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof for treating, preventing, and relieving a target hypertension.

5. A pharmaceutical composition comprising a compound according to any one of claims 1 to 3 for treating, preventing, and alleviating oxidative stress in target blood vessels.

6. A compound according to any one of claims 1 to 3, for use in the treatment, prevention, and remission of the target hypertension.

7. A compound according to any one of claims 1 to 3, for use in the treatment, prevention, and remission of oxidative stress in target blood vessels.

8. The following formula 【Chemistry 4】 (In the formula, X is a bond, -O-, or -CH) 2 - and R is C 1 A substituted or unsubstituted alkyl group, or C 6 ~C 12 (It is a substituted or unsubstituted alkyl group.) A pharmaceutical composition comprising the compound, its stereoisomers, and pharmaceutically active salts, for the treatment, prevention, and remission of a target hypertension.

9. For use in the treatment, prevention, and remission of the target hypertension, The following formula 【Transformation 5】 (wherein X is a bond, -O-, or -CH 2 -, R is C 1 substituted or unsubstituted alkyl, or C 6 ~C 12 substituted or unsubstituted alkyl) Compounds of the same, their stereoisomers, and pharmaceutically acceptable salts.

10. A pharmaceutical composition comprising the compound described in any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

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