Agent for improving mitochondrial dysfunction

Abstract

The present invention addresses the problem of providing a mitochondrial dysfunction improving agent comprising a vitamin K derivative having high delivering efficiency to mitochondria. A mitochondrial dysfunction improving agent, an amyotrophic lateral sclerosis improving agent, a neurodegenerative disease improving agent, an Alzheimer's disease improving agent, and a Parkinson's disease improving agent comprising at least one selected from active vitamin K carboxylates represented by the general formula (1) (wherein R1 and R2 are each a hydrogen atom or a substituent selected from glycine, N-acylglycines, N-alkylglycines, N,N-dialkylglycines, N,N,N-trialkylglycines, acyl, dicarboxylic acid hemiesters, and salts thereof, at least one of R1 and R2 is glycine, N-acylglycine, N-alkylglycine, N,N-dialkylglycine, N,N,N-trialkylglycine, acyl, dicarboxylic acid hemiester, or a salt thereof, R3 represents a group represented by the general formula (2) or the general formula (3), and n represents an integer of 1-7) and salts thereof, and a mitochondrial dysfunction improving agent, an amyotrophic lateral sclerosis improving agent, a neurodegenerative disease improving agent, an Alzheimer's disease improving agent, and a Parkinson's disease improving agent comprising at least one selected from active vitamin K carboxylates represented by the general formula (1) (wherein R1 and R2 are a carboxylic acid residue selected from the group consisting of R4OOCCH2CH2CO- and R4OOCCH2CH2CH2CO-, R3 represents the general formula (2) or (3), and R4 is a C1-C3 alkyl group) and salts thereof.

Description

Related Application

[0001] This application claims the priority of Japanese Patent Application No. 2020-046864 filed on March 17, 2020, which is incorporated herein by reference.

Technical Field

[0002] The present invention relates to an agent for improving mitochondrial dysfunction, particularly an agent for improving mitochondrial dysfunction containing a vitamin K hydroquinone derivative as an active ingredient.

Background Art

[0003] Mitochondrial dysfunction is considered to be the cause of various diseases such as neurodegenerative diseases and type 2 diabetes. For example, Alzheimer's disease (AD) is the most common neurodegenerative disease, and is a devastating disease characterized pathologically by strong aggregation of amyloid-β peptide (Aβ) and hyperphosphorylated tau (p-tau). The development of an effective and safe treatment method that can suppress the progression targeting AD lesions is required. However, there is no effective treatment method yet. In clinical trials of AD, the focus is on the clearance of Aβ and the suppression of p-tau.

[0004] In sporadic and familial human AD brain samples, AD-induced pluripotent stem cell (iPSC)-derived neurons, and AD transgenic animal models, AD presents with impaired mitochondrial quality control and reduced mitochondrial function in neurons. The accumulation of these dysfunctional mitochondria contributes to the increase in the pathological factors Aβ and p-tau in AD. Moreover, the increase in Aβ and p-tau conversely exacerbates mitochondrial dysfunction (Non-Patent Documents 1 to 5). Energy deficiency and Aβ aggregation due to mitochondrial dysfunction lead to synaptotoxicity and memory loss (Non-Patent Documents 3, 6), and the increase in Aβ and p-tau induces axonal transport defects in mitochondria, resulting in synaptic starvation, ATP depletion, and ultimately neurodegeneration (Non-Patent Document 7). It has been revealed that by pharmaceutically or genetically restoring or enhancing dysfunctional mitochondria (mitochondrial function and mitochondrial quality control), Aβ toxicity, which is an AD lesion, can be reduced and tau hyperphosphorylation can be invalidated (Non-Patent Document 1).

[0005] UBIAD1 is an enzyme that generates menahydroquinone-4 (MKH) from vitamin K3 hydroquinone and geranylgeranyl pyrophosphate (Chemical Formula 1) (Non-Patent Documents 8, 9). In AD patients, the expression level of UBIAD1 in the brain is decreased and the MKH level is also decreased (Non-Patent Document 10). It has been clarified that UBIAD1 increases the membrane potential of depolarized mitochondria by a membrane potential depolarizing agent, suggesting that it restores mitochondrial dysfunction and enhances energy production (Non-Patent Document 11).

[0006]

Chemical Formula

[0007] Moreover, Parkinson's disease (PD) is a neurodegenerative disease of dopaminergic neurons. In PD as well, mitochondrial dysfunction and breakdown of mitochondrial quality control lead to neuronal cell death (Non-Patent Document 12). The proteins PINK1 / Parkin are essential for mitophagy involved in mitochondrial quality control in PD, and PINK1 / Parkin is also involved in mitophagy in AD. It has been reported that UBIAD1 also rescues mitochondrial quality control in a PD model (Non-Patent Document 13). Mitochondrial dysfunction in upper motor neurons has also been revealed in patients with amyotrophic lateral sclerosis (ALS) (Non-Patent Documents 14 and 15).

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

[0010] Based on the above background, the present inventor hypothesized that by delivering active menadione-4 (MKH) or active phylloquinone (PKH) to mitochondria, it would be possible to restore and enhance mitochondrial function and mitochondrial quality control, and as a result, reduce or reverse AD lesions (increase in Aβ and p-tau). Then, the present inventor aimed to develop a low-molecular compound capable of realizing the hypothesis as a drug discovery target, search for candidate compounds from prodrugs of MKH and PKH, develop an agent for improving mitochondrial dysfunction, and ultimately develop preventive and therapeutic drugs for AD, therapeutic drugs for PD, and therapeutic drugs for ALS.

[0011] Currently, Alzheimer's disease (AD) drugs include cholinesterase inhibitors and NMDA receptor antagonists, but they only manage AD symptoms and there is no effective treatment. There is a need to develop an effective and safe treatment that can target AD lesions and modify the progression. In AD clinical trials, the focus is on suppressing the increase in Aβ and p-tau. The present invention has been made in view of the above prior art, and the problem to be solved is to provide a vitamin K derivative that can effectively perform mitochondrial dysfunction and mitochondrial quality control.

Means for Solving the Problem

[0012] As described above, the present inventors have found that a vitamin K hydroquinone derivative having a specific structure is excellent in mitochondrial delivery, and have completed the present invention.

[0013] That is, the mitochondrial dysfunction improver (vitamin K hydroquinone derivative) according to the present invention is represented by the following general formula (1). General formula (1)

[0014]

Chemical formula

[0015]

Chemical formula

[0016]

Chemical formula

[0017] [Chemical formula] (In general formula (4), R1 and R2 are carboxylic acid residues selected from the group consisting of R4OOCCH2CH2CO- and R4OOCCH2CH2CH2CO-. R3 is The following has the meaning of general formula (2) or (3). R4 is a hydrogen atom, or a C1-C3 alkyl group.) [Chemical formula] However, n means an integer from 1 to 7. [Chemical formula]

[0018] The present invention relates to an agent for improving mitochondrial function, which contains at least one of a carboxylic acid ester of vitamin K hydroquinone, a salt thereof, or a vitamin K hydroquinone dicarboxylic acid double ester represented by the general formula (1) and general formula (4) as an active ingredient, and efficiently delivers active vitamin K to mitochondria, and is effective for the prevention and treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, and type 2 diabetes.

[0019] The compound represented by the general formula (1) can be contained in a formulation alone, or can be formulated in a formulation as a salt thereof. The compound represented by the general formula (4) can also be contained in a formulation alone. In the present invention, examples of the carboxylic acid residues R1 and R2 having a nitrogen substituent are as follows. Those in which a hydrogen atom or one or two alkyl groups or acyl groups are bonded to a nitrogen atom. Examples of the alkyl group include linear or branched alkyl groups having 1 to 6 carbon atoms, such as methyl group, ethyl group, n-propyl group, n-butyl group, n-pentyl group, n-hexyl group, isopropyl group, isobutyl group, 1-methylpropyl group, tert-butyl group, 1-ethylpropyl group, and isoamyl group. Among them, the methyl group and the ethyl group are preferred. Similarly, the hydrocarbon chain in the case of having an acyl group can be defined in the same way.

[0020] Preferably, the amino group and the carbonyl group are bonded by a linear, branched or cyclic alkylene group having 1 to 7 carbon atoms. Examples of the branched alkylene group include those derived from alkyl groups such as isopropyl, isobutyl, tert-butyl, and 1-ethylpropyl. Examples of the cyclic alkylene group include the following. Those containing a cyclopentane ring, a cyclohexane ring, or a methylcyclohexane ring in the structure. Among them, the methylene group or the ethylene group is particularly preferred as the alkylene group.

[0021] Preferred hydrogen halide acid salts include hydrochloride and hydrobromide. In the present invention, hydrogen halide acid salts often crystallize or solidify, which has the advantage of facilitating handling in formulation. Examples of other salts include the following. Examples of alkyl sulfonates include methanesulfonate, and examples of sugar acid salts include gluconate, glucoheptanoate, lactobionate, etc.

[0022] In the present invention, the dicarboxylic acid residues R1 and R2 are selected from the residues of dicarboxylic acids and their alkali metal salts or meglumine salts. The carbonyl groups of the dicarboxylic acid residues are bonded by a linear alkylene group having 2 to 4 carbon atoms. As the alkylene group, the ethylene group or the propylene group is particularly preferred. As the alkali metal salts, sodium salts and potassium salts are preferred.

[0023] In the present invention, in the dicarboxylic acid double ester, the carbonyl groups of the dicarboxylic acid residues are bonded by a linear alkylene group having 2 to 4 carbon atoms. As the alkylene group, an ethylene group or a propylene group is particularly preferable. The alcohol residue of the double ester is preferably an alkyl group having 1 to 3 carbon atoms, and particularly preferably an ethyl group.

[0024] In the present invention, various methods for producing the compounds represented by the general formula (1) and the general formula (4) can be considered. A typical method is as follows.

[0025]

Chemical formula

[0026] The vitamin Ks represented by the general formula (5) are reduced with a reducing agent to obtain vitamin K hydroquinone represented by the general formula (6). Then, the vitamin K hydroquinone is subjected to an esterification reaction with a carboxylic acid having a nitrogen substituent, or a reactive acid derivative thereof, or a hydrohalide salt thereof, or an acid anhydride, by a conventional method, to obtain the target substance (1) of the present invention. The reducing agent used here is one that reduces the naphthoquinone skeleton of vitamin Ks to a naphthohydroquinone skeleton, and examples thereof include sodium borohydride, sodium hydrosulfite, tri-n-butylphosphine, zinc chloride, stannous chloride, and zinc powder.

[0027] The esterification reaction of vitamin K hydroquinone follows conventional methods. However, when esterifying amino acids having a primary or secondary amino group or a hydroxyl or thiol group in the side chain, appropriate protecting groups such as tert-butoxycarbonyl group (hereinafter abbreviated as t-BOC group), benzyloxycarbonyl group (hereinafter abbreviated as Z group), 9-fluorenylmethoxycarbonyl group (hereinafter abbreviated as FMOC group), etc. are used for protection. For N,N-dialkyl amino acids, hydrohalic acid salts are used, and the reaction is preferably carried out in the presence of active esterification reagents such as dicyclohexylcarbodiimide (hereinafter abbreviated as DCC), N,N-disuccinimidyl oxalate (hereinafter abbreviated as DSO), etc. Anhydrous pyridine is preferably used as the solvent in this case. Also, in the method using a reactive acid derivative, the method using an acid halide, especially acid chloride, gives preferable results. An anhydrous benzene-anhydrous pyridine mixture is preferably used as the solvent in this case. Hydrohalic acid salts, alkyl sulfonates, and sugar acid salts are produced by reacting free vitamin K hydroquinone nitrogen-containing carboxylic acid esters with hydrohalic acids, alkyl sulfonic acids, and lactone forms of acidic sugars according to conventional methods. Also, after producing N-acyl amino acid esters, hydrohalic acid salts can be produced by deprotecting with hydrohalic acid according to conventional methods.

[0028] The dicarboxylic acid double ester represented by the general formula (4) is obtained by reacting a vitamin K compound represented by the general formula (5) with zinc powder and an acid anhydride under heating in acetic acid acidity to obtain a vitamin K hydroquinone carboxylic acid hemi-ester, and then esterifying with an alcohol under an acidic catalyst to obtain the target substance (4). Also, the vitamin K hydroquinone carboxylic acid hemi-ester obtained in the above general formula (1) is esterified with an alcohol under an acidic catalyst to obtain the target substance (4).

[0029] The vitamin K hydroquinone derivative of the present invention has a high degree of freedom in dosage forms, and oral administration, transdermal administration, nasal administration, injection administration, etc. can be adopted. As additives, an isotonic agent, a buffer, a pH adjuster, a solubilizer, a thickening agent (dispersant), a mucoadhesive, a stabilizer (antioxidant), a preservative (antiseptic), an absorption enhancer, etc. can be appropriately blended, and formulated by a well-known method. can be done. Further, by adding a pH adjuster, a thickening agent, a dispersant, etc. and suspending the drug, stable eye drops and nasal drops can also be obtained.

[0030] The isotonic agent is not particularly limited as long as it is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable. For example, as ionic isotonic agents, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc. can be mentioned. Examples of nonionic isotonic agents include glycerin, propylene glycol, sorbitol, mannitol, etc.

[0031] The buffer is not particularly limited as long as it is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable. For example, phosphoric acid, phosphate, citric acid, acetic acid or ε-aminocaproic acid can be mentioned.

[0032] The pH adjuster is not particularly limited as long as it is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable. For example, hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, potassium hydroxide, boric acid, borax, sodium carbonate, sodium hydrogen carbonate, etc. can be mentioned. The pH of the eye drops may be within the range acceptable for ophthalmic preparations, but is 4.0 to 9.0, and more preferably 5.5 to 8.5.

[0033] The solubilizer is not particularly limited as long as it is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable. For example, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, polyoxyethylene sorbitan fatty acid ester, vitamin E TPGS, polyoxyethylene fatty acid ester, polyoxyethylene polyoxypropylene glycol, sucrose fatty acid ester, etc. can be mentioned.

[0034] As the thickener, dispersant, and mucoadhesive agent, there is no particular limitation as long as it is pharmaceutically, pharmacologically (pharmaceutically), or physiologically acceptable. For example, cellulose polymers such as hydroxypropylmethylcellulose or hydroxypropylcellulose; polyvinyl alcohol; or polyvinylpyrrolidone can be mentioned.

[0035] As the stabilizer, there is no particular limitation as long as it is pharmaceutically, pharmacologically (pharmaceutically), or physiologically acceptable. For example, edetic acid, sodium edetate, disodium edetate, tetrasodium edetate, sodium citrate, etc. can be mentioned, and sodium edetate may be a hydrate.

[0036] As the antioxidant, there is no particular limitation as long as it is pharmaceutically, pharmacologically (pharmaceutically), or physiologically acceptable. For example, ascorbic acid, vitamin E, dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate, sodium sulfite, etc. can be mentioned.

[0037] As the preservative (antiseptic), there is no particular limitation as long as it is pharmaceutically, pharmacologically (pharmaceutically), or physiologically acceptable. For example, benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl paraben, propyl paraben, chlorobutanol, etc. can be mentioned, and these preservatives can also be used in combination.

Advantages of the Invention

[0038] As described above, the mitochondrial function disorder improver according to the present invention enables the delivery of active vitamin K to mitochondria and can improve various diseases caused by mitochondrial function disorders.

Brief Description of the Drawings

[0039]

Figure 1

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 4

Figure 5

Figure 6A

Figure 6B

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Mode for Carrying Out the Invention

[0040] Hereinafter, preferred embodiments of the present invention will be described in detail. In sporadic and familial human AD brain samples, AD-induced pluripotent stem cell (iPSC)-derived neurons, and AD transgenic animal models, it has been revealed that in AD, there are impairments in mitochondrial quality control of neurons and a decrease in mitochondrial function, and the accumulation of these dysfunctional mitochondria contributes to the increase in Aβ and p-tau, which are AD pathological factors (Non-Patent Documents 1 to 5). It has been clarified that by restoring or enhancing dysfunctional mitochondria (mitochondrial function and mitochondrial quality control) by pharmacological or genetic methods, Aβ toxicity can be reduced and tau hyperphosphorylation can be invalidated (Non-Patent Document 1). UBIAD1 is an enzyme that biosynthesizes menahydroquinone-4 (MKH) (Chemical Formula 1). In AD patients, the expression of UBIAD1 in the brain is decreased and the MKH level is also decreased. UBIAD1 has been shown to increase the membrane potential of polarized mitochondria, that is, to improve mitochondrial dysfunction (Non-Patent Document 13).

[0041] Therefore, the present inventor hypothesized that efficient delivery of MKH to the brain mitochondria of AD patients could restore and enhance mitochondrial function and mitochondrial quality control, and reduce or reverse AD lesions (Aβ and p-tau). Based on this hypothesis, the inventor aimed to develop small-molecule compounds that could enable MKH and PKH delivery and reduce or reverse AD lesions (increased Aβ and p-tau) as a drug discovery target. MKH is a type of Vitamin K (VK), vitamin K 2(20) (menaquinone-4, MK-4), which is a two-electron reductant. In the endoplasmic reticulum, as an active form of VK, it functions as a cofactor for the enzyme (GGCX) that carboxylates Glu residues to Gla residues in the post-translational modification of vitamin K-dependent proteins (VKDPs), which is one of the functions of vitamin K. Since MKH is extremely prone to oxidation, quinone-type MK-4 is used clinically. However, low photostability, high phototoxicity, poor water solubility, and the requirement for a reduction activation process have been issues in drug delivery of MKH by MK-4. The present inventor has made it possible to develop an MKH delivery agent with high photostability and no phototoxicity that does not require a reduction activation process through prodrug formation of MKH.

[0042] First, as a drug discovery target, an MKH prodrug was selected to verify its function and evaluate its potential as a preventive and therapeutic agent for AD in vitro by hypothesizing that MKH delivery could restore and enhance mitochondrial function and reduce Aβ protein toxicity in this study. Moreover, Parkinson's disease (PD) is a neurodegenerative disease of dopaminergic neurons. In PD, mitochondrial dysfunction and breakdown of mitochondrial quality control also lead to neuronal cell death. Furthermore, it has been reported that UBIAD1 rescues mitochondrial quality control in a PD model. Therefore, using an MKH prodrug as a drug discovery target for small-molecule compounds, it was verified whether mitochondrial dysfunction could be restored, and its potential as a therapeutic agent for PD was evaluated in vitro.

[0043] 1) Possibility as a preventive and therapeutic agent for Alzheimer's disease (AD) In a neuronal cell damage model treated with Aβ peptide by treating a single neuron-astrocyte co-culture specimen with Aβ peptide, the effect of MKH derivative on improving neurodegeneration was evaluated. Since the mitochondrial affinity of the derivative and the re-convertibility to MKH are predicted to affect the MKH delivery by the MKH derivative, a cationic derivative (MKH-DMG) expected to have high affinity, an anionic derivative (MKH-SUC) with high intracellular re-convertibility, and an oxidized form of MKH (MK-4) used clinically were evaluated (Chemical Formula 7).

[0044] [Chemical Formula] Quinone-type vitamin K and active (hydroquinone-type) vitamin K derivative

[0045] Improvement effect of MKH derivative on neuropathy caused by β-amyloid After culturing astrocytes isolated from the cerebral cortex of 0-1-day-old ICR mice for 2 weeks, the areas were divided into dots on a slide glass and cultured. After further culturing for 1 week, neurons derived from the hippocampus of 0-1-day-old ICR mice were seeded and co-cultured. From 1 day after neuron seeding, Aβ 25-35 (1 μM) and the test compound (0.3 μM) were exposed for 3 days, and the morphological changes of neurons were observed. The nerve dendrites (MAP2 antibody) and axons (Tau antibody) were stained and analyzed using the immunostaining method. The dendrites and axons were evaluated for changes in the branching and elongation of the dendrites and axons by the Sholl analysis method. The staining results of neurons are shown in Figure 1. The branching and elongation of nerve axons and the branching and elongation of nerve dendrites were both affected by Aβ 25-35It was significantly suppressed and neuropathy was observed. MKH derivatives (DMG (MKH-DMG) and SUC (MKH-SUC)) significantly improved both axonal and dendritic neuropathies (Figs. 2, 3), revealing an improvement effect on neuropathy. No improvement effect was observed for MK4 (MK-4) against any neuropathy. Since MKH derivatives have an improvement effect on β-amyloid neurodegeneration at a low concentration of 0.3 μM, high safety is expected.

[0046] 2) Improvement effect of MKH derivatives on mitochondrial dysfunction In nervous system cells, the effects of MKH derivatives and PKH derivatives on mitochondrial dysfunction induced by complex I inhibitor rotenone (10 mM), complex II inhibitor (3-nitro propionic acid (3-NP)), complex III inhibitor (antimycin A), and depolarizing agent (carbonyl cyanide-m-chlorophenyl hydrazone, CCCP) were evaluated. Cell death (survival measurement by cell titer blue), ATP production inhibition (ATP measurement by cell titer glo), and membrane potential decrease (JC-1 staining, depolarization (Green), hyperpolarization (Red)) were evaluated. Cell death induced by treatment with the complex I inhibitor rotenone was suppressed by administration of MKH derivatives (Fig. 4), ATP production inhibition was suppressed (Fig. 5), and the decrease in mitochondrial membrane potential was restored (Fig. 6). Cell death induced by rotenone treatment was suppressed by administration of PKH derivatives (Fig. 7), ATP production inhibition was suppressed (Fig. 8), and the decrease in mitochondrial membrane potential was restored (Fig. 9).

[0047] In addition, each derivative restored the decrease in membrane potential (Fig. 10). Furthermore, the decrease in survival rate, ATP production, and membrane potential induced by the complex III inhibitor (antimycin A) was restored (Fig. 11). In addition, the decrease in survival rate induced by the uncoupling agent (CCCP) was restored (Fig. 12). Also, each derivative increased the expression of PGC-1α (peroxisome proliferator-activated receptor-c coactivator-1α), which activates mitochondrial biogenesis. As a result of the above, it was revealed that the MKH derivative and the PKH derivative can recover mitochondrial dysfunction (mitochondria with decreased membrane potential).

[0048] 3) Possibility as a therapeutic agent for PD In sporadic PD, Complex I activity is decreased, and the rotenone complex I inhibition model causes morphological and functional damage in dopaminergic neurons in addition to the characteristic symptoms of PD such as bradykinesia, rigidity, and tremors. In addition, mitochondria damaged by Complex I inhibition (mitochondria with decreased membrane potential) are also used as an evaluation of hereditary PD because they induce mitochondrial quality control. Therefore, it was revealed from the obtained results that the MKH derivative and the PKH derivative are effective in the rotenone PD model, and the possibility as a therapeutic agent for PD was revealed.

[0049] From the above evaluation results, · It was revealed using primary neurons that MKH derivatives of MKH-DMG and MKH-SUC can improve neurotoxicity caused by Aβ at a low concentration of 0.3 μM. · It was revealed that MKH-DMG and MKH-SUC can improve mitochondrial function (respiratory chain) disorders. These results support the hypothesis that efficient delivery of MKH to the brain mitochondria of AD patients enables the recovery and enhancement of mitochondrial dysfunction and mitochondrial quality control, and the reduction and recovery of AD lesions (increase in Aβ and p-tau), and it was shown that the MKH derivative has a high possibility as a drug discovery target based on the hypothesis. In addition, the effect of the MKH derivative in the rotenone PD model supports the possibility as a therapeutic agent for PD by the recovery and enhancement of mitochondrial quality control. In addition, it supports the possibility as a therapeutic agent for amyotrophic lateral sclerosis by the recovery of mitochondrial function.

[0050] Therefore, the present inventors propose MKH derivatives as drug discovery targets for preventive and therapeutic agents for AD and therapeutic agents for PD, which act on the recovery and enhancement of mitochondrial function disorders and quality control. The present inventors hypothesized that by efficiently delivering MKH to the brain mitochondria of AD patients, it is possible to reduce AD lesions (increase in Aβ and p-tau) by recovering and enhancing mitochondrial function disorders and mitochondrial quality control, and developed a low-molecular compound that enables MKH delivery and reduces AD lesions (increase in Aβ and p-tau) as a drug discovery target. So far, an original MKH prodrug has been evaluated and its high potential has been clarified. Originality: Although the reduction of AD lesions (increase in Aβ and p-tau) by recovering and enhancing mitochondrial function disorders and mitochondrial quality control has been shown in AD model cells and AD model mice, there is no method of delivering MKH by an original MKH prodrug, and this method is the first in the world. High safety: The improvement of Aβ-induced neurotoxicity by an original MKH prodrug is effective at a drug concentration of 0.3 μM, so it can exert an effect at a low concentration. The MKH prodrug is a low-molecular compound, and it is expected to follow the same fate in the body as vitamin K2 and disappear after functioning as MKH. Therefore, it is considered to be a highly safe preventive and therapeutic agent with extremely low concern about harmful side effects.

Examples

[0051] The present invention will be further specifically described with the following examples, but the present invention is not limited thereto. Examples 1 to 34 The vitamin K hydroquinone derivatives shown in Tables 1 to 5 were produced by the methods shown in the following production methods A to I. Also, the mass spectra (ionization methods; FD method and FAB method) of the obtained substances, 1 1H-NMR spectra are shown in Tables 6 to 8.

[0052] Production method A Dissolve 0.1 mol of amino acid in 100 ml of distilled water - dioxane (1:1, v / v), add 30 ml of triethylamine, gradually add di-tert-butyl dicarbonate, and stir at room temperature for 30 minutes. Distill off dioxane under reduced pressure, add 50 ml of aqueous sodium hydrogen carbonate solution (0.5 M), and wash with 100 ml of ethyl acetate. Wash the ethyl acetate layer with 50 ml of sodium hydrogen carbonate solution, combine the aqueous layers, add aqueous citric acid solution (0.5 M) under ice-cooling to make it acidic (pH 3), saturate with sodium chloride, and then extract with ethyl acetate (100 ml × 3 times). After dehydrating the extract with anhydrous sodium sulfate, distill off the solvent under reduced pressure. Add isopropyl ether to the oily residue or crystallize by cooling to obtain N-t-BOC-amino acid. Dissolve 6.75 mmol of vitamin K in 40 ml of isopropyl ether, dissolve 47 mmol of sodium borohydride in 15 ml of methanol and add it, and stir at room temperature until the yellow color of the solution becomes colorless. Add 60 ml of isopropyl ether and 100 ml of distilled water to the reaction solution, separate the isopropyl ether layer, add another 100 ml of isopropyl ether to the aqueous layer to extract the soluble fraction, combine the isopropyl ether layers, dehydrate with anhydrous sodium sulfate, and concentrate under reduced pressure. Add n-hexane to the residue to precipitate a white solid to obtain vitamin K hydroquinone.

[0053] Add 13.55 mmol of vitamin K hydroquinone, 13.55 mmol of N-t-BOC-amino acid, and 13.55 mmol of DCC to 50 ml of anhydrous pyridine and stir at room temperature for 20 hours. Distill off the solvent under reduced pressure, add ethyl acetate to the residue to extract the soluble fraction (100 ml × 2 times), concentrate the extract under reduced pressure, and separate and purify the residue by silica gel column chromatography (elution solvent; n-hexane - isopropyl ether) to obtain vitamin K hydroquinone-1,4-bis-N-t-BOC-amino acid. Dissolve vitamin K hydroquinone-1,4-bis-N-t-BOC-amino acid in a small amount of acetone, add hydrochloric acid - dioxane (2.5 - 4.0 N) in an amount corresponding to about 20 times the molar amount of hydrochloric acid of the ester amount, stir for 1 hour, and then distill off the solvent under reduced pressure. Recrystallize the residue from an acetone - methanol system to obtain the hydrochloride of vitamin K hydroquinone-1,4-bis-amino acid ester.

[0054] Production method B Dissolve 6.75 mmol of vitamin K in 40 ml of isopropyl ether, dissolve 47 mmol of sodium borohydride in 15 ml of methanol and add it. Stir at room temperature until the yellow color of the solution becomes colorless. Add 60 ml of isopropyl ether and 100 ml of distilled water to the reaction solution, separate the isopropyl ether layer, add 100 ml of isopropyl ether to the aqueous layer to extract the soluble fraction, combine the isopropyl ether layers, dehydrate with anhydrous sodium sulfate, and concentrate under reduced pressure. Add n-hexane to the residue to precipitate a white precipitate to obtain vitamin K hydroquinone. Add vitamin K hydroquinone, 13.55 mmol of hydrochloric acid N,N-dialkylamino acid or 13.55 mmol of hydrochloric acid N,N,N-trialkylamino acid, and 13.55 mmol of DCC to 50 ml of anhydrous pyridine and stir at room temperature for 20 hours. Distill off the solvent under reduced pressure, suspend the residue in distilled water, add sodium hydrogen carbonate to adjust the pH of the solution to 7 - 8, and then extract with ethyl acetate (100 ml × 3 times). Dehydrate the extract with anhydrous sodium sulfate, distill off the solvent under reduced pressure, and separate and purify the residue by silica gel column chromatography (elution solvent; isopropyl ether - ethyl acetate) to obtain vitamin K hydroquinone-1,4-bis-N,N-dialkylamino acid ester or vitamin K hydroquinone-1,4-bis-N,N,N-trialkylamino acid ester.

[0055] Production method C Dissolve 6.75 mmol of vitamin K in 40 ml of isopropyl ether, dissolve 50 mmol of sodium hydrosulfite in 50 ml of distilled water and add it. Stir at room temperature until the isopropyl ether turns brown and then colorless. Separate the isopropyl ether layer, add 100 ml of isopropyl ether to the aqueous layer to extract the soluble fraction, combine the isopropyl ether layers, dehydrate with anhydrous sodium sulfate, and concentrate under reduced pressure. Add n-hexane to the residue to precipitate a white solid to obtain vitamin K hydroquinone. Add 6.75 mmol of N,N-dialkylamino acid hydrochloride and 6.75 mmol of DCC to vitamin K hydroquinone and stir in 50 ml of anhydrous pyridine for 20 hours. Distill off the solvent under reduced pressure, suspend the residue in distilled water, add sodium bicarbonate to adjust the pH of the solution to 7 - 8, and extract with ethyl acetate (100 ml × 3 times). Dehydrate the extract with anhydrous sodium sulfate, distill off the solvent under reduced pressure, and separate and purify the residue by silica gel column chromatography (eluent; isopropyl ether - ethyl acetate, 3:2) to obtain vitamin K hydroquinone-1-N,N-dialkylamino acid ester and vitamin K hydroquinone-4-N,N-dialkylamino acid ester.

[0056] Production method D Dissolve 6.75 mmol of vitamin K in 40 ml of isopropyl ether, dissolve 47 mmol of sodium borohydride in 15 ml of methanol and add it. Stir at room temperature until the yellow color of the solution becomes colorless. Add 60 ml of isopropyl ether and 100 ml of distilled water to the reaction solution, separate the isopropyl ether layer, add 100 ml of isopropyl ether to the aqueous layer to extract the soluble fraction, combine the isopropyl ether layers, dehydrate with anhydrous sodium sulfate and concentrate under reduced pressure. Add n-hexane to the residue to precipitate a white precipitate to obtain vitamin K hydroquinone. Dissolve vitamin K hydroquinone in 30 ml of anhydrous benzene-anhydrous pyridine (1:1, v / v), add pyridine carboxylic acid chloride hydrochloride and stir at room temperature for 3 hours. Remove the insoluble matter by filtration and concentrate the filtrate under reduced pressure. Suspend the residue in 100 ml of distilled water, add sodium hydrogen carbonate (pH 7-8), extract the soluble fraction with ethyl acetate (100 ml × 3 times), concentrate the extract under reduced pressure, and separate and purify the residue by silica gel column chromatography (elution solvent; isopropyl ether-ethyl acetate, 9:1) to obtain vitamin K hydroquinone-1,4-bis-pyridine carboxylic acid ester.

[0057] Production method E Dissolve 2 mmol of vitamin K hydroquinone-1,4-bis-N,N-dialkylamino acid ester or vitamin K hydroquinone-1,4-bis-pyridine carboxylic acid in 20 ml of acetone, add hydrochloric acid-dioxane (2.5 - 4.0 N) in an amount corresponding to 10 times the molar amount of hydrochloric acid of the ester, distill off the solvent under reduced pressure, and recrystallize the residue from acetone-methanol to obtain the hydrochloride of vitamin K hydroquinone-1,4-bis-N,N-dialkylamino acid or vitamin K hydroquinone-1,4-bis-pyridine carboxylic acid.

[0058] Production method F Dissolve 2 mmol of vitamin K hydroquinone-1,4-bis-N,N-dialkylamino acid or vitamin K hydroquinone-1,4-bis-pyridinecarboxylic acid in 20 ml of dichloromethane, add 2 mmol of alkylsulfonic acid and stir. Filter the precipitated crystals to obtain the alkylsulfonate of vitamin K hydroquinone-1,4-bis-N,N-dialkylamino acid ester or vitamin K hydroquinone-1,4-bis-pyridinecarboxylic acid ester.

[0059] Production Method G Dissolve 4.55 mmol of vitamin K in 40 ml of isopropyl ether, dissolve 31.5 mmol of sodium borohydride in 15 ml of methanol and add it. Stir at room temperature until the yellow color of the solution becomes colorless. Add 60 ml of isopropyl ether and 100 ml of purified water to the reaction solution, separate the isopropyl ether layer, add another 100 ml of isopropyl ether to the aqueous layer to extract the soluble fraction, combine the isopropyl ether layers, dehydrate with anhydrous sodium sulfate, and then distill off the solvent under reduced pressure. Add 8.97 mmol of dimethylaminopyridine and 18.0 mmol of dicarboxylic anhydride to the residue, dissolve it in 100 ml of isopropyl ether-dioxane (6:4, v / v), stir at room temperature for 3 hours, then react for 2 hours while heating to 50 - 60 °C, and further react for 10 hours while cooling to room temperature. Add 100 ml of purified water to the reaction solution, separate the isopropyl ether layer, dehydrate with anhydrous sodium sulfate, and distill off the solvent under reduced pressure. Suspend the residue in isopropyl ether, add 100 ml of ethyl acetate and 100 ml of purified water to the precipitate obtained by centrifugation to extract the ethyl acetate-soluble fraction, dehydrate with anhydrous sodium sulfate, and distill off the solvent under reduced pressure. Suspend the residue in isopropyl ether and recrystallize the insoluble matter with ethyl acetate to obtain vitamin K hydroquinone-1,4-bis-dicarboxylic acid hemiester.

[0060] Production Method H 6.75 mmol of vitamin K, 18.4 mmol of zinc, 33.0 mmol of dicarboxylic anhydride, 13.8 mmol of sodium acetate anhydride, and 161.5 mmol of acetic acid are placed in a 100 ml eggplant flask, fitted with a Dimroth condenser, and heated at 85 °C for 3 hours with thorough stirring. After cooling to room temperature, 200 ml of ethyl acetate and 100 ml of purified water are added to the resulting white solid, and the ethyl acetate-soluble fraction is extracted. After dehydration with anhydrous sodium sulfate, the solvent is distilled off under reduced pressure. The residue is recrystallized from ethyl acetate to obtain vitamin K hydroquinone-1,4-bis-dicarboxylic acid hemi-ester.

[0061] Production Method I 2 mmol of vitamin K hydroquinone-1,4-bis-dicarboxylic acid hemi-ester is added with 2-fold mol of 0.1N aqueous sodium hydroxide solution or 2-fold mol of meglumine aqueous solution, dissolved, and freeze-dried. Recrystallization is carried out with methanol-acetonitrile to obtain vitamin K hydroquinone-1,4-bis-dicarboxylic acid hemi-ester-bis-sodium salt or vitamin K hydroquinone-1,4-bis-dicarboxylic acid hemi-ester-bis-meglumine salt.

[0062] Production Method J Vitamin K hydroquinone-1,4-bis-dicarboxylic acid hemi-ester is dissolved in a primary or secondary alcohol and stirred under hydrochloric acid acidity. The solvent is distilled off under reduced pressure to obtain vitamin K hydroquinone-1,4-bis-dicarboxylic acid alcohol ester.

[0063] [Table 1]

[0064] [Table 2]

[0065] [Table 3]

[0066]

Table 4

[0067]

Table 5

[0068]

Table 6

[0069]

Table 7

[0070]

Table 8

Claims

1. A substituent selected from the following general formula (1) 【Chemical 1】 (wherein R １ and R ２ are each a hydrogen atom, or 【Chem.】 means, R １ and R ２ at least one of which is 【Chem.】 is a substituent selected from, R ３ is represented by the following general formula (2) [Chemical 2] or a group represented by the following general formula (3) [Chemical Formula 3] represents a group represented by). An agent for improving mitochondrial dysfunction comprising at least one of carboxylic acid esters of active vitamin K represented by the formula or a salt thereof.

2. The agent for improving mitochondrial dysfunction according to Claim 1, which improves mitochondrial dysfunction that is at least one of ATP production inhibition, mitochondrial membrane potential decrease, and cell death.

3. The following general formula (4) 【Chemical Formula 4】 (In general formula (4), R １ and R ２ are carboxylic acid residues selected from the group consisting of R ４ OOCC H ２ C H ２ CO- and R ４ OOCC H ２ C H ２ C H ２ CO-. R ３ has the meaning of the following general formula (2) or (3). R ４ is a hydrogen atom or a C1-C3 alkyl group.) Carboxylic acid esters of active vitamin K represented thereby or salts thereof. 【Chemical Formula 5】 However, n means an integer of 1 to 7. [Chemical Formula 6]

4. A neurodegeneration improving agent containing the compound represented by the general formula (1) according to Claim 1 or a salt thereof.

5. An amyotrophic lateral sclerosis improving agent containing the compound represented by the general formula (1) according to Claim 1 or a salt thereof.

6. An Alzheimer's disease improving agent containing the compound represented by the general formula (1) according to Claim 1 or a salt thereof.

7. A Parkinson's disease improving agent containing the compound represented by the general formula (1) according to Claim 1 or a salt thereof.

8. A neurodegeneration improving agent containing the compound represented by the general formula (4) according to Claim 3 or a salt thereof.

9. An amyotrophic lateral sclerosis improving agent containing the compound represented by the general formula (4) according to Claim 3 or a salt thereof.

10. An Alzheimer's disease improving agent containing the compound represented by the general formula (4) according to Claim 3 or a salt thereof.

11. A Parkinson's disease improving agent containing the compound represented by the general formula (4) according to Claim 3 or a salt thereof.

12. An agent for improving mitochondrial dysfunction containing the compound represented by the general formula (4) according to Claim 3 or a salt thereof.

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