Mitochondrial dysfunction improvers

Abstract

The object of the present invention is to provide a mitochondrial dysfunction improving agent comprising a vitamin K derivative with high delivery to mitochondria. A mitochondrial dysfunction improving agent comprising at least one of the carboxylic acid esters or salts thereof of active vitamin K represented by the following general formula (1) (wherein R1 and R2 are hydrogen atoms or substituents selected from glycine, N-acylglycine, N-alkylglycine, N,N-dialkylglycine, N,N,N-trialkylglycine, acyl groups, dicarboxylic acid half esters and salts thereof, at least one of R1 and R2 is glycine, N-acylglycine, N-alkylglycine, N,N-dialkylglycine, N,N,N-trialkylglycine, acyl groups, dicarboxylic acid half esters and salts thereof, R3 is a group represented by the following general formula (2) or the following general formula (3), and n is an integer from 1 to 7). An agent for improving mitochondrial dysfunction, an agent for improving amyotrophic lateral sclerosis, an agent for improving neurodegeneration, an agent for improving Alzheimer's disease and an agent for improving Parkinson's disease, and an ...

Description

[0001] Related Applications

[0002] This application claims the benefit of Japanese Patent Application No. 2020-046864 filed on March 17, 2020, which is hereby incorporated by reference herein. Technical Field

[0003] The present invention relates to a mitochondrial dysfunction improving agent, and in particular to a mitochondrial dysfunction improving agent containing a vitamin K hydroquinone derivative as an effective ingredient. Background Art

[0004] Mitochondrial dysfunction is thought to be a cause of various diseases, including neurodegenerative diseases and type 2 diabetes.

[0005] For example, Alzheimer's disease (AD) is the most common neurodegenerative disease and is a devastating disease characterized by strong aggregation of amyloid-β peptide (Aβ) and hyperphosphorylated tau (p-tau) as pathological features. It is necessary to develop effective and safe treatments for AD lesions that can inhibit the progression of the disease. However, there is no effective treatment to date. In clinical trials for AD, attention is paid to the clearance of Aβ and the inhibition of p-tau.

[0006] In sporadic and familial human AD brain samples, AD induced pluripotent stem cell (iPSC) derived neurons and AD transgenic animal models, AD is manifested as impaired mitochondrial quality control and reduced mitochondrial function in neurons. The accumulation of these dysfunctional mitochondria contributes to the increase of Aβ and p-tau, the pathological factors of AD, and the increase of Aβ and p-tau in turn exacerbates mitochondrial dysfunction (non-patent literature 1~5). Energy deficiency and Aβ aggregation caused by mitochondrial dysfunction lead to synaptic toxicity and memory loss (non-patent literature 3, 6), and the increase of Aβ and p-tau induces axonal transport defects of mitochondria, leading to synaptic starvation, ATP depletion, and ultimately neurodegeneration (non-patent literature 7). It is known that by restoring or enhancing dysfunctional mitochondria (mitochondrial function, mitochondrial quality control) with pharmaceutical or genetic methods, the toxicity of Aβ, which is an AD lesion, can be reduced, and tau hyperphosphorylation can be invalidated (non-patent literature 1).

[0007] UBIAD1 is an enzyme that generates menahydroquinone-4 (MKH) from vitamin K3 hydroquinone and geranylgeranyl pyrophosphate (chemical formula 1) (non-patent literature 8, 9). The expression of UBIAD1 in the brain of AD patients is reduced, and the level of MKH is also reduced (non-patent literature 10). It is known that UBIAD1 increases the membrane potential of mitochondria depolarized by membrane potential depolarizers, suggesting that it can restore mitochondrial dysfunction and enhance energy production (non-patent literature 11).

[0008] [Chemical formula 1]

[0009]

[0010] UBIAD1-based biosynthesis of MKH.

[0011] In addition, Parkinson's disease (PD) is a neurodegenerative disease of dopaminergic neurons. In PD, mitochondrial dysfunction or failure of mitochondrial quality control leads to neuronal cell death (Non-patent Document 12). Mitochondrial autophagy involved in mitochondrial quality control in PD requires PINK1 / Parkin protein, and PINK1 / Parkin is also included in mitochondrial autophagy in AD. It is reported that UBIAD1 can also rescue mitochondrial quality control in PD models (Non-patent Document 13). Mitochondrial dysfunction of upper motor neurons has also been found in patients with amyotrophic lateral sclerosis (ALS) (Non-patent Documents 14, 15).

[0012] Prior art literature

[0013] Patent Literature

[0014] Patent Document 1: Japanese Patent No. 3088137,

[0015] Patent document 2: Japanese Patent No. 4040082.

[0016] Non-patent literature

[0017] Non-patent literature 1: Fang, EF, et al., Mitophagy inhibits amyloid-β and taupathology and reverses cognitive deficits in models of Alzheimer's disease. Nature Neuroscience, 22, 401-412 (2019).

[0018] Non-patent literature 2: Scheibye-Knudsen, M., et al., Protecting the mitochondrial powerhouse. Trends Cell Biol. 25, 158-170 (2015).

[0019] Non-patent document 3: Kerr, JS et al. Mitophagy and Alzheimer's disease: cellular and molecular mechanisms. Trends Neurosci. 40, 151-166 (2017).

[0020] Non-patent literature 4: Selfridge, JE, et al., Role of mitochondrial homeostasis and dynamics in Alzheimer's disease. Neurobiol. Dis. 51, 3-12 (2013).

[0021] Non-patent document 5: Beck, JS, et al., Evidence for mitochondrial UPR gene activation in familial and sporadic Alzheimer's disease. Curr. Alzheimer Res. 13, 610-614 (2016).

[0022] Non-patent document 6: Mairet-Coello, G. et al. The CAMKK2-AMPK kinase pathway mediates the synaptotoxic effects of Aβ oligomers through Tauphosphorylation. Neuron 78, 94-108 (2013).

[0023] Non-patent literature 7: Vossel, KA et al. Tau reduction prevents Aβ-induced defects in axonal transport. Science 330, 198 (2010).

[0024] Non-patent literature 8: Nakagawa K, et al., Identification of UBIAD1 as a novel human menaquinone-4 biosynthetic enzyme. Nature. 468 (7320): 117-121 (2010).

[0025] Non-patent document 9: Hirota Y, et al., Menadione (vitamin K3) is a catabolicproduct of oral phylloquinone (vitamin K1) in the intestine and a circulating precursor of tissue menaquinone-4 (vitamin K2) in rats. J Biol Chem. 288(46): 33071-33080 (2013).

[0026] Non-patent literature 10: Hirota Yoshihisa, Elucidation of the mechanism of vitamin K synthesis in brain neurodegenerative diseases, Uehara Memorial Life Science Foundation Research Reports, 30 (2016) 109.

[0027] Non-patent literature 11: Fredericks WJ, et al., The TERE1 Protein InteractsWith Mitochondrial TBL2: Regulation of Trans-Membrane Potential, ROS / RNS andSXR Target Genes. J. Cell. Biochem., 114, 2170-2187 (2013).

[0028] Non-patent literature 12: Von Stockum, S., et al., Mitochondrial dynamics andmitophagy in Parkinson's disease: A fly point of view. Neurobiol Dis 90, 58-67 (2016).

[0029] Non-patent document 13: Vos M., et al., Vitamin K2 Is a Mitochondrial ElectronCarrier That Rescues Pink1 Deficiency. SCIENCE, 336, 1306-1310 (2012).

[0030] Non-patent literature 14: Gautam, M., et al., Mitoautophagy: A Unique Self-Destructive Path Mitochondria of Upper Motor Neurons With TDP-43 PathologyTake, Very Early in ALS. Front. Cell. Neurosci., 13, 489 (2019).

[0031] Non-patent literature 15: Gautam, M., et al., Mitochondria, ER, and nuclearmembrane defects reveal early mechanisms for upper motor neuron vulnerability with respect to TDP-43 pathology. Acta Neuropathol. 137, 47-69 (2019). Summary of the invention

[0032] Problems to be solved by the invention

[0033] Based on the above background, the present inventors established the following hypothesis: by delivering active menatetraenehydroquinone-4 (MKH) or active phyllohydroquinone (PKH) to mitochondria, mitochondrial dysfunction and the restoration of mitochondrial quality control can be enhanced, resulting in the alleviation or recovery of AD pathology (increase of Aβ and p-tau).

[0034] Then, the inventors set low-molecular compounds that can realize the hypothesis as new drug development targets, planned to explore candidate compounds from prodrugs of MKH and PKH, develop agents to improve mitochondrial dysfunction, and further develop preventive and therapeutic drugs for AD, therapeutic drugs for PD, and therapeutic drugs for ALS.

[0035] Currently, there are cholinesterase inhibitors and NMDA receptor antagonists in Alzheimer's disease (AD) drugs, but they only control AD ​​symptoms and there is no effective treatment. It is necessary to develop effective and safe treatments for AD lesions that can change the progression of the disease. In clinical trials of AD, attention is paid to inhibiting the increase of Aβ and p-tau.

[0036] The present invention has been made in view of the above-mentioned prior art, and an object of the present invention is to provide a vitamin K derivative that can effectively control mitochondrial dysfunction and mitochondrial quality.

[0037] Means of solving problems

[0038] As described above, the present inventors have found that a vitamin K hydroquinone derivative having a specific structure is excellent in the ability to be delivered to mitochondria, thereby completing the present invention.

[0039] That is, the mitochondrial dysfunction-improving agent (vitamin K hydroquinone derivative) of the present invention is represented by the following general formula (1).

[0040] Active vitamin K carboxylic acid esters or salts thereof represented by the general formula (1):

[0041] [Chemical formula 2]

[0042]

[0043] In the formula, R1 and R2 are respectively a hydrogen atom or a substituent selected from glycine, N-acylglycine, N-alkylglycine, N,N-dialkylglycine, N,N,N-trialkylglycine, an acyl group, a dicarboxylic acid half ester and a salt thereof; at least one of R1 and R2 is glycine, N-acylglycine, N-alkylglycine, N,N-dialkylglycine, N,N,N-trialkylglycine, an acyl group, a dicarboxylic acid half ester and a salt thereof; R3 is a group represented by the following general formula (2) or the following general formula (3); and n is an integer of 1 to 7.

[0044] [Chemical formula 3]

[0045]

[0046] [Chemical formula 4]

[0047] .

[0048] In addition, the vitamin K hydroquinone dicarboxylic acid diester of the present invention is represented by the following general formula (4):

[0049] [Chemical formula 5]

[0050]

[0051] In the general formula (4), R1 and R2 are carboxylic acid residues selected from R4OOCCH2CH2CO- and R4OOCCH2CH2CH2CO-, R3 has the meaning of the above general formula (2) or (3), and R4 is a C1~C3 alkyl group.

[0052] In the present invention, the mitochondrial dysfunction improving agent containing at least one of the carboxylic acid ester or salt thereof of vitamin K hydroquinone or the vitamin K hydroquinone dicarboxylic acid diester represented by the above-mentioned general formula (1) and general formula (4) as an effective ingredient can effectively deliver 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, type 2 diabetes, etc.

[0053] The compound represented by the above general formula (1) may be contained in the preparation alone or in the form of a salt thereof. The compound represented by the above general formula (4) may also be contained in the preparation alone. In the present invention, the carboxylic acid residues R1 and R2 having a nitrogen substituent may include the following groups.

[0054] A group in which a hydrogen atom or one or two alkyl groups or acyl groups are bonded to a nitrogen atom.

[0055] The alkyl group is a straight chain or branched alkyl group having 1 to 6 carbon atoms, and the following groups can be exemplified. Methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, 1-methylpropyl, tert-butyl, 1-ethylpropyl, isopentyl. Methyl and ethyl are preferred as the alkyl group. In addition, the hydrocarbon chain having an acyl group can also be defined in the same manner.

[0056] The amino group and the carbonyl group are preferably bonded via a linear, branched or cyclic alkylene group having 1 to 7 carbon atoms. Examples of the branched alkylene group include the following: Alkylene groups derived from alkyl groups such as isopropyl, isobutyl, tert-butyl and 1-ethylpropyl.

[0057] As the cyclic alkylene group, the following groups can be exemplified.

[0058] A group containing a cyclopentane ring, a cyclohexane ring, a methylcyclohexane ring, etc. in the structure. As the above-mentioned alkylene group, a methylene group or an ethylene group is particularly preferred.

[0059] As the hydrohalide salt, hydrochloride, hydrobromide, etc. are preferred. In the present invention, the hydrohalide salt is mostly crystallized or solidified, so it has the advantage of being easy to handle during preparation. Other salts can be exemplified by the following salts. As alkyl sulfonate, there are methanesulfonate, etc., as sugar acid salt, there are gluconate, glucoheptonate, lactobionate, etc.

[0060] In the present invention, the dicarboxylic acid residues R1 and R2 are selected from the residues of dicarboxylic acid and its alkali metal salt or meglumine salt. The carbonyl groups of the dicarboxylic acid residues are bonded with a straight-chain alkylene group having 2 to 4 carbon atoms. The alkylene group is particularly preferably an ethylene group or a propylene group. The alkali metal salt is preferably a sodium salt or a potassium salt.

[0061] In the present invention, the carbonyl groups of the dicarboxylic acid residues in the dicarboxylic acid diester are bonded by a straight-chain alkylene group having 2 to 4 carbon atoms. The alkylene group is particularly preferably an ethylene group or a propylene group. The alcohol residue of the diester is preferably an alkyl group having 1 to 3 carbon atoms, and particularly preferably an ethyl group.

[0062] In the present invention, various methods for producing the compounds represented by the general formula (1) and the general formula (4) are contemplated, and a representative method is as follows.

[0063] [Chemical formula 6]

[0064] .

[0065] The vitamin K represented by the general formula (5) is reduced with a reducing agent to produce vitamin K hydroquinone represented by the general formula (6), and the vitamin K hydroquinone is subjected to an esterification reaction with a carboxylic acid having a nitrogen substituent or its reactive acid derivative or their hydrohalide salt, or an acid anhydride by a conventional method, thereby obtaining the target substance (1) of the present invention. The reducing agent used here is a substance that reduces the naphthoquinone skeleton of the vitamin K to a naphthohydroquinone skeleton, and examples thereof include sodium borohydride, sodium dithionite, tri-n-butylphosphine, zinc chloride, stannous chloride, zinc powder, and the like.

[0066] The esterification reaction of vitamin K hydroquinone is carried out according to conventional methods, but when the primary or secondary amino group or amino acid having a hydroxyl group or a thiol group on the side chain is esterified, it is used after being protected with an appropriate protecting group such as tert-butyloxycarbonyl (hereinafter abbreviated as t-BOC group), benzyloxycarbonyl (hereinafter abbreviated as Z group), 9-fluorenylmethoxycarbonyl (hereinafter abbreviated as FMOC group), etc., and N,N-dialkylamino acid is used with a hydrohalide salt, and reacted in the presence of an active esterification reagent such as dicyclohexylcarbodiimide (hereinafter abbreviated as DCC), N,N-disuccinimide oxalate (hereinafter abbreviated as DSO), and preferred results can be obtained. At this time, anhydrous pyridine is preferably used as a solvent. In addition, in the method using a reactive acid derivative, a method using an acyl halide, especially an acyl chloride, can obtain preferred results. At this time, an anhydrous benzene-anhydrous pyridine mixture is preferably used as a solvent. Hydrohalide salts, alkyl sulfonates, and sugar salts are prepared by reacting free vitamin K hydroquinone nitrogen-containing carboxylic acid esters with hydrohalic acid, alkyl sulfonic acid, and lactones of acidic sugars by conventional methods. In addition, after preparing N-acylamino acid esters, deprotection groups are carried out with hydrohalic acid by conventional methods to prepare hydrohalide salts.

[0067] Regarding the dicarboxylic acid diester represented by the general formula (4), vitamin K represented by the general formula (5) is reacted with zinc powder and anhydride under heating in the presence of acetic acid to prepare vitamin K hydroquinone carboxylic acid half ester, which is then esterified with alcohol under an acidic catalyst to obtain the target substance (4). Alternatively, the vitamin K hydroquinone carboxylic acid half ester obtained by the general formula (1) is esterified with alcohol under an acidic catalyst to obtain the target substance (4).

[0068] The dosage form of the vitamin K hydroquinone derivative of the present invention has a high degree of freedom, and can be administered orally, transdermally, nasally, or by injection. As additives, isotonic agents, buffers, pH adjusters, solubilizers, thickeners (dispersants), mucoadhesives, stabilizers (antioxidants), preservatives (preservatives), absorption promoters, etc. can be appropriately combined, and thus, preparations can be made by well-known methods. In addition, by adding pH adjusters, thickeners, dispersants, etc. to suspend the drug, a stable eye drop or nasal drop liquid preparation can also be obtained.

[0069] As an isotonic agent, there is no particular limitation as long as it is a substance that is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable. For example, as an ionic isotonic agent, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc. can be listed. As a non-ionic isotonic agent, glycerin, propylene glycol, sorbitol, mannitol, etc. can be listed.

[0070] The buffer is not particularly limited as long as it is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable, and examples thereof include phosphoric acid, phosphates, citric acid, acetic acid, and ε-aminocaproic acid.

[0071] The pH adjuster is not particularly limited as long as it is a substance that is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable, and examples thereof include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, potassium hydroxide, boric acid, borax, sodium carbonate, sodium bicarbonate, etc. The pH of the eye drops may be within the range permitted for ophthalmic preparations, and may be 4.0 to 9.0, more preferably 5.5 to 8.5.

[0072] The solubilizing agent is not particularly limited as long as it is a substance that is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable, and examples thereof include 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, and the like.

[0073] There are no particular limitations on the thickener, dispersant, and mucoadhesive as long as they are pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable, and examples thereof include cellulose-based polymers such as hydroxypropylmethylcellulose and hydroxypropylcellulose, polyoxyethylene alcohol, and polyvinyl pyrrolidone.

[0074] There are no particular restrictions on the stabilizer as long as it is a substance that is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable. Examples include ethylenediaminetetraacetic acid (edetic acid), monosodium ethylenediaminetetraacetate, disodium ethylenediaminetetraacetate, tetrasodium ethylenediaminetetraacetate, sodium citrate, etc., and sodium ethylenediaminetetraacetate may be a hydrate.

[0075] The antioxidant is not particularly limited as long as it is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable, and examples thereof include ascorbic acid, vitamin E, butylated hydroxytoluene, butylated hydroxyanisole, sodium erythorbate, propyl gallate, sodium sulfite and the like.

[0076] There are no particular restrictions on the preservative (antiseptic) as long as it is a substance that is pharmaceutically, pharmacologically (pharmaceutically) or physiologically acceptable. Examples thereof include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methylparaben, propylparaben, chlorobutanol, etc. These preservatives may also be used in combination.

[0077] Effects of the Invention

[0078] As described above, the mitochondrial dysfunction-improving agent of the present invention can deliver active vitamin K to mitochondria, and can improve various diseases caused by mitochondrial dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] [ Figure 1 ] Illustration showing the improving effect of MKH derivatives on neurological disorders caused by β-amyloid protein.

[0080] [ Figure 2A ] Illustration showing the improving effect of MKH derivatives on neurological disorders caused by β-amyloid protein.

[0081] [ Figure 2B ] Illustration showing the improving effect of MKH derivatives on neurological disorders caused by β-amyloid protein.

[0082] [ Figure 3A ] Illustration showing the improving effect of MKH derivatives on neurological disorders caused by β-amyloid protein.

[0083] [ Figure 3B ] Illustration showing the improving effect of MKH derivatives on neurological disorders caused by β-amyloid protein.

[0084] [ Figure 4 ] Graph illustrating the improvement effect of MKH derivatives on cell death. Compared with control (**=p<0.01), compared with Rot (##=p<0.01)

[0085] [ Figure 5 ] Graph illustrating the improvement effect of MKH derivatives on ATP production inhibition. Compared with control (**=p<0.01), compared with Rot (##=p<0.01)

[0086] [ Fig. 6A ] Illustration of the state of membrane potential recovery caused by MKH derivatives.

[0087] [ Figure 6B ] Illustration of the recovery of membrane potential caused by MKH derivatives. Compared with control (**=p<0.01), compared with Rot (##=p<0.01)

[0088] [ Figure 7 ] Illustration of the cell death improvement effect caused by PKH derivatives. Compared with control (**=p<0.01), compared with Rot (##=p<0.01)

[0089] [ Figure 8 ] Illustration of the improvement effect of PKH derivatives on ATP production inhibition. Compared with control (**=p<0.01), compared with Rot (##=p<0.01)

[0090] [ Fig. 9 ] Illustration of the membrane potential recovery effect caused by PKH derivatives. Compared with control (**=p<0.01), compared with Rot (##=p<0.01)

[0091] [ Fig.10 ] Illustration of the membrane potential recovery effect of MKH derivatives against Complex II inhibitors. Compared with control (**=p<0.01), compared with 3-NP (##=p<0.01)

[0092] [ Fig.11 ] Illustration of the membrane potential recovery effect of MKH derivatives against Complex III inhibitors. Compared with control (**=p<0.01), compared with antimycin A (##=p<0.01)

[0093] [ Fig.12 ] Graph illustrating the effect of MKH derivatives on cell death induced by an uncoupler (carbonyl cyanide m-chlorophenylhydrazone, CCCP). Comparison with control (**=p<0.01), comparison with CCCP (##=p<0.01). DETAILED DESCRIPTION

[0094] Hereinafter, preferred embodiments of the present invention will be described in detail.

[0095] It is known that in sporadic and familial human AD brain samples, AD induced pluripotent stem cell (iPSC) derived neurons and AD transgenic animal models, AD is manifested as impaired mitochondrial quality control and reduced mitochondrial function in neurons. It is known that the accumulation of these dysfunctional mitochondria contributes to the increase of Aβ and p-tau as AD pathological factors (non-patent literature 1~5). It is known that by restoring or enhancing dysfunctional mitochondria (mitochondrial function, mitochondrial quality control) with pharmaceutical or genetic methods, Aβ toxicity can be reduced and tau hyperphosphorylation can be invalidated (non-patent literature 1). UBIAD1 is an enzyme (chemical formula 1) that biosynthesizes tetraene menadione-4 (MKH). UBIAD1 expression in the brain of AD patients is reduced, and MKH levels are also reduced. UBIAD1 increases the membrane potential of depolarized mitochondria, that is, it shows improvement of mitochondrial dysfunction (non-patent literature 13).

[0096] Therefore, the inventors established the hypothesis that by effectively delivering MKH to the brain nerve mitochondria of AD patients, mitochondrial dysfunction and mitochondrial quality control can be restored and enhanced, and AD pathology (Aβ and p-tau) can be alleviated or restored. The development of low-molecular compounds that can deliver MKH and PKH to alleviate or restore AD pathology (increase in Aβ and p-tau) is the goal of new drug development.

[0097] MKH is a vitamin K (VK) 2(20) The two-electron reduced form of (tetraene menaquinone-4, MK-4) acts as the active form of VK in the endoplasmic reticulum, and plays a role as a cofactor of the enzyme (GGCX) that carboxylates Glu residues to Gla residues in the post-translational modification of vitamin K-dependent proteins (VKDP), one of the effects of vitamin K. Since MKH is easily oxidized, quinone-type MK-4 is used clinically, but low photostability, high phototoxicity, poor water solubility, and the need for a reduction activation process are problems in the drug delivery of MKH based on MK-4. So far, the inventors have made it possible to realize a MKH delivery agent that does not require a reduction activation process, has high photostability, and is non-phototoxic through the prodrugization of MKH.

[0098] First, based on the MKH delivery hypothesized in this study, low-molecular-weight compounds that can lead to enhanced mitochondrial function recovery and reduced Aβ protein toxicity are targeted for new drug development. MKH prodrugs are obtained and their functions are verified, and their potential as preventive and therapeutic drugs for AD is evaluated in vitro.

[0099] In addition, Parkinson's disease (PD) is a neurodegenerative disease of dopaminergic neurons, in which mitochondrial dysfunction or failure of mitochondrial quality control leads to neuronal cell death. In addition, UBIAD1 has been reported to rescue mitochondrial quality control in PD models.

[0100] Therefore, we used MKH prodrug as a new drug development target for low-molecular-weight compounds to verify whether mitochondrial dysfunction can be restored and evaluated its potential as a therapeutic drug for PD in vitro.

[0101] 1) Possibility as a preventive and therapeutic drug for Alzheimer's disease (AD)

[0102] The neurodegeneration-improving effect of MKH derivatives was evaluated in a neuronal disorder model caused by Aβ peptide in which a single neuron-astrocyte co-culture specimen was treated with Aβ peptide. Since MKH delivery based on MKH derivatives is expected to be affected by the derivative's mitochondrial affinity and reconversion to MKH, a cationic derivative (MKH-DMG) with high expected affinity and an anionic derivative (MKH-SUC) with high intracellular reconversion, as well as a clinically used MKH oxidase (MK-4) were evaluated (Chemical Formula 7).

[0103] [Chemical formula 7]

[0104]

[0105] Quinone vitamin K and active (hydroquinone) vitamin K derivatives.

[0106] Improvement effect of MKH derivatives on neurological disorders caused by β-amyloid protein

[0107] Astrocytes isolated from the cerebral cortex of 0-1 day old ICR mice were cultured for 2 weeks and then cultured in dotted areas on a glass slide. After another week of culture, neurons from the hippocampus of 0-1 day old ICR mice were inoculated for co-culture. Aβ was injected into the neurons one day after the neurons were inoculated. 25-35 (1μM) and the test compound (0.3μM) were exposed for 3 days, and the morphological changes of neurons were observed. Dendrites (MAP2 antibody) and axons (Tau antibody) of the nerves were stained and analyzed using immunostaining. For dendrites and axons, the changes in branching and elongation of dendrites and axons were evaluated by Sholl analysis.

[0108] The neuronal staining results are shown in Figure 1 The branching and elongation of axons and dendrites are affected by Aβ 25-35Significant inhibition was observed for neurological disorders. MKH derivatives (DMG (MKH-DMG) and SUC (MKH-SUC)) significantly improved neurological disorders in axons and dendrites (Figures 2 and 3), showing an improvement effect on neurological disorders. MK4 (MK-4) did not have an improvement effect on any neurological disorder. MKH derivatives have an improvement effect on β-amyloid neurodegeneration at a low concentration of 0.3μM, so high safety can be expected.

[0109] 2) Improvement effect of MKH derivatives on mitochondrial dysfunction

[0110] In neurons, the effects of MKH derivatives and PKH derivatives on mitochondrial dysfunction caused by complex I inhibitor rotenone (10 mM), complex II inhibitor (3-nitropropionic acid (3-NP), complex III inhibitor (antimycin A), and depolarizing agent (carbonyl cyanide m-chlorophenylhydrazone, CCCP) were evaluated. The effects were evaluated by cell death (survival assay using cell titer blu), inhibition of ATP production (ATP assay using cell titer glo), and decrease in membrane potential (JC-1 staining, depolarization (green), hyperpolarization (red)).

[0111] Cell death induced by treatment with the complex I inhibitor rotenone was inhibited by administration of MKH derivatives ( Figure 4 ), ATP production inhibition ( Figure 5 ) and the decrease in mitochondrial membrane potential (Figure 6) were restored. In the administration of PKH derivatives, cell death caused by rotenone treatment was inhibited ( Figure 7 ), ATP production inhibition ( Figure 8 ) and decreased mitochondrial membrane potential ( Fig. 9 ) is restored.

[0112] In addition, each derivative decreased the membrane potential ( Fig.10 ) were restored. In addition, the decreased survival rate, decreased ATP production, and decreased membrane potential caused by the complex III inhibitor (antimycin A) were restored ( Fig.11 ). In addition, the decrease in survival rate caused by the uncoupler (CCCP) was restored ( Fig.12 In addition, each derivative increased the expression of PGC-1α (peroxisome proliferator-activated receptor c coactivator 1α) which activates mitochondrial biogenesis.

[0113] The above results indicate that MKH derivatives and PKH derivatives can restore mitochondrial dysfunction (mitochondria with reduced membrane potential).

[0114] 3) Possibility of using it as a therapeutic drug for PD

[0115] Complex I activity is reduced in sporadic PD, and the rotenone complex I inhibition model causes morphological and functional damage to dopaminergic neurons in addition to characteristic symptoms of PD such as bradykinesia, rigidity, and tremor. In addition, mitochondria damaged by complex I inhibition (mitochondria with reduced membrane potential) induce mitochondrial quality control, and are therefore also used to evaluate hereditary PD. Therefore, based on the results obtained, it can be seen that MKH derivatives and PKH derivatives are effective in the rotenone PD model, revealing their potential as PD therapeutic drugs.

[0116] According to the above evaluation results,

[0117] Using primary neural cells, it was revealed that MKH derivatives of MKH-DMG and MKH-SUC could ameliorate neurotoxicity caused by Aβ at a low concentration of 0.3 μM.

[0118] · Revealed that MKH-DMG and MKH-SUC can improve mitochondrial function (respiratory chain) disorders.

[0119] These results support the hypothesis that by effectively delivering MKH to the brain neural mitochondria of AD patients, mitochondrial dysfunction or mitochondrial quality control can be restored or enhanced, and AD pathology (increase in Aβ and p-tau) can be alleviated or restored, showing that MKH derivatives have a high potential as targets for hypothesis-based new drug development.

[0120] In addition, the effects of MKH derivatives in the rotenone PD model support their potential as PD therapeutics based on restoring and enhancing mitochondrial quality control.

[0121] In addition, it supports its potential as a therapeutic agent for ALS based on restoration of mitochondrial function.

[0122] Therefore, the present inventors propose to use MKH derivatives as new drug development targets for AD prevention and treatment drugs and PD treatment drugs with the mechanism of action of restoring or enhancing mitochondrial dysfunction or quality control.

[0123] The present inventors hypothesized that by effectively delivering MKH to brain nerve mitochondria of AD patients, mitochondrial dysfunction or mitochondrial quality control can be restored or enhanced, thereby reducing AD pathology (increase of Aβ and p-tau), and the development of low-molecular compounds that can deliver MKH and reduce AD ​​pathology (increase of Aβ and p-tau) is a new drug development target. So far, a unique MKH prodrug has been evaluated and revealed to have high potential.

[0124] Originality: Reduction of AD pathology (increase of Aβ and p-tau) caused by restoration or enhancement of mitochondrial dysfunction or mitochondrial quality control has been shown in AD model cells or AD model mice, but there is no method of delivering MKH using a unique MKH prodrug. This method is the first of its kind in the world.

[0125] High safety: The unique MKH prodrug improves neurotoxicity caused by Aβ, which is effective at a drug concentration of 0.3μM, so it can be effective at low concentrations. MKH prodrug is a low molecular weight compound, and after acting as MKH, it is expected to disappear in the body in the same way as vitamin K2. Therefore, it can be considered a highly safe preventive and therapeutic drug with extremely low risk of harmful side effects. Example

[0126] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

[0127] Embodiments 1 to 34

[0128] The vitamin K hydroquinone derivatives shown in Tables 1 to 5 were prepared by the methods shown in the following preparation methods A to I. In addition, the mass spectra (ionization method: FD method and FAB method) and 1 The H-NMR spectra are shown in Tables 6 to 8.

[0129] Manufacturing Method A

[0130] Dissolve 0.1 mol of amino acid in 100 ml of distilled water-dioxane (1:1, v / v), add 30 ml of triethylamine, slowly add di-tert-butyl dicarbonate, and stir at room temperature for 30 minutes. Dioxane is distilled off under reduced pressure, 50 ml of sodium bicarbonate aqueous solution (0.5 M) is added, and washed with 100 ml of ethyl acetate. Wash the ethyl acetate layer with 50 ml of sodium bicarbonate solution, combine the water layers, add citric acid aqueous solution (0.5 M) under ice cooling to acidify (pH 3), saturate with sodium chloride, extract with ethyl acetate (100 ml × 3 times), dehydrate the extract with anhydrous sodium sulfate, distill off the solvent under reduced pressure, add isopropyl ether or cool to crystallize the oily residue to obtain Nt-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, and stir at room temperature until the yellow color of the solution turns colorless. 60 ml of isopropyl ether and 100 ml of distilled water were added to the reaction solution, the isopropyl ether layer was separated, and 100 ml of isopropyl ether was added to the water layer to extract the soluble components, and the isopropyl ether layers were combined, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure. N-hexane was added to the residue to precipitate a white precipitate, thereby obtaining vitamin K hydroquinone.

[0131] Vitamin K hydroquinone, 13.55 mmol of Nt-BOC-amino acid, and 13.55 mmol of DCC were added to 50 ml of anhydrous pyridine and stirred at room temperature for 20 hours. The solvent was distilled off under reduced pressure, and ethyl acetate was added to the residue to extract the soluble component (100 ml × 2 times). The extract was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (eluting solvent: n-hexane-isopropyl ether) to obtain vitamin K hydroquinone-1,4-bis-Nt-BOC-amino acid. Vitamin K hydroquinone-1,4-bis-Nt-BOC-amino acid was dissolved in a small amount of acetone, and hydrochloric acid-dioxane (2.5~4.0N) was added in an amount corresponding to about 20 times the molar amount of hydrochloric acid to the ester amount. After stirring for 1 hour, the solvent was distilled off under reduced pressure. The residue was recrystallized in an acetone-methanol system to obtain the hydrochloride of vitamin K hydroquinone-1,4-bis-amino acid ester.

[0132] Manufacturing method B

[0133] 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, and stir at room temperature until the yellow color of the solution turns 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 water layer to extract the soluble components, 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, thereby obtaining vitamin K hydroquinone. Add vitamin K hydroquinone, 13.55 mmol of N,N-dialkylamino acid hydrochloride or 13.55 mmol of N,N,N-trialkylamino acid hydrochloride, and 13.55 mmol of DCC to 50 ml of anhydrous pyridine and stir at room temperature for 20 hours. The solvent is distilled off under reduced pressure, the residue is suspended in distilled water, sodium bicarbonate is added to adjust the pH of the solution to 7-8, and then extracted with ethyl acetate (100 ml x 3 times), the extract is dehydrated with anhydrous sodium sulfate, the solvent is distilled off under reduced pressure, and the residue is separated and purified by silica gel column chromatography (eluting solvent: isopropyl ether-ethyl acetate) to obtain vitamin K hydroquinone-1,4-bis-N,N-dialkyl amino acid ester or vitamin K hydroquinone-1,4-bis-N,N,N-trialkyl amino acid ester.

[0134] Manufacturing method C

[0135] Dissolve 6.75mmol of vitamin K in 40ml of isopropyl ether, dissolve 50mmol of sodium dithionite in 50ml of distilled water and add. The isopropyl ether is brown, and then stir at room temperature until it becomes colorless. Separate the isopropyl ether layer, add 100ml of isopropyl ether to the water layer to extract the soluble components, 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 6.75mmol of N,N-dialkylamino acid hydrochloride and 6.75mmol of DCC to vitamin K hydroquinone, and stir in 50ml of anhydrous pyridine for 20 hours. The solvent was distilled off under reduced pressure, and the residue was suspended in distilled water. Sodium bicarbonate was added to adjust the pH of the solution to 7-8, and then extracted with ethyl acetate (100 ml x 3 times). The extract was dehydrated with anhydrous sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was separated and purified by silica gel column chromatography (eluting solvent: isopropyl ether-ethyl acetate, 3:2) to obtain vitamin K hydroquinone-1-N,N-dialkyl amino acid ester and vitamin K hydroquinone-4-N,N-dialkyl amino acid ester.

[0136] Manufacturing method D

[0137] 6.75mmol of vitamin K was dissolved in 40ml of isopropyl ether, 47mmol of sodium borohydride was dissolved in 15ml of methanol and added, and stirred at room temperature until the yellow color of the solution turned colorless. 60ml of isopropyl ether and 100ml of distilled water were added to the reaction solution, the isopropyl ether layer was separated, and 100ml of isopropyl ether was added to the water layer to extract the soluble components, the isopropyl ether layers were combined, and the soluble components were concentrated under reduced pressure after dehydration with anhydrous sodium sulfate. N-hexane was added to the residue to precipitate a white precipitate, thereby obtaining vitamin K hydroquinone. Vitamin K hydroquinone was dissolved in 30ml of anhydrous benzene-anhydrous pyridine (1:1, v / v), pyridine hydrochloride was added, and stirred at room temperature for 3 hours. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure. The residue was suspended in 100 ml of distilled water, sodium bicarbonate (pH 7-8) was added, and the soluble components were extracted in ethyl acetate (100 ml x 3 times). The extract was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (eluting solvent: isopropyl ether-ethyl acetate, 9:1) to obtain vitamin K hydroquinone-1,4-bis-pyridinecarboxylate.

[0138] Manufacturing method E

[0139] 2 mmol of vitamin K hydroquinone-1,4-bis-N,N-dialkyl amino acid ester or vitamin K hydroquinone-1,4-bis-pyridine carboxylic acid is dissolved in 20 ml of acetone, and hydrochloric acid-dioxane (2.5~4.0N) is added, and the amount of hydrochloric acid is equivalent to 10 times the molar amount of the ester. The solvent is distilled off under reduced pressure, and the residue is recrystallized from acetone-methanol to obtain the hydrochloride of vitamin K hydroquinone-1,4-bis-N,N-dialkyl amino acid or vitamin K hydroquinone-1,4-bis-pyridine carboxylic acid.

[0140] Manufacturing method F

[0141] 2 mmol of vitamin K hydroquinone-1,4-bis-N,N-dialkyl amino acid or vitamin K hydroquinone-1,4-bis-pyridine carboxylic acid is dissolved in 20 ml of dichloromethane, and 2 mmol of alkyl sulfonic acid is added and stirred. The precipitated crystals are filtered to obtain alkyl sulfonate of vitamin K hydroquinone-1,4-bis-N,N-dialkyl amino acid ester or vitamin K hydroquinone-1,4-bis-pyridine carboxylic acid ester.

[0142] Manufacturing method G

[0143] Dissolve 4.55mmol of vitamin K in 40ml of isopropyl ether, dissolve 31.5mmol of sodium borohydride in 15ml of methanol and add, and stir at room temperature until the yellow color of the solution turns colorless. Add 60ml of isopropyl ether and 100ml of purified water to the reaction solution, separate the isopropyl ether layer, add 100ml of isopropyl ether to the water layer to extract the soluble component, combine the isopropyl ether layers, dehydrate with anhydrous sodium sulfate, and distill off the solvent under reduced pressure. Add 8.97mmol of dimethylaminopyridine and 18.0mmol of dicarboxylic anhydride to the residue, dissolve in 100ml of isopropyl ether-dioxane (6:4, v / v), stir at room temperature for 3 hours, react for 2 hours while heating to 50-60℃, and react for 10 hours while cooling at room temperature. Add 100ml 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. The residue was suspended in isopropyl ether, 100 ml of ethyl acetate and 100 ml of purified water were added to the precipitate obtained by centrifugation to extract the ethyl acetate soluble component, and after dehydration with anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. The residue was suspended in isopropyl ether, and the insoluble matter was recrystallized with ethyl acetate to obtain vitamin K hydroquinone-1,4-bis-dicarboxylic acid half ester.

[0144] Manufacturing method

[0145] 6.75 mmol of vitamin K, 18.4 mmol of zinc, 33.0 mmol of dicarboxylic anhydride, 13.8 mmol of anhydrous sodium acetate, and 161.5 mmol of acetic acid were added to a 100 ml eggplant flask, and a serpentine condenser was installed. The mixture was heated at 85°C for 3 hours while being fully stirred. 200 ml of ethyl acetate and 100 ml of purified water were added to the white solid generated by cooling at room temperature to extract the ethyl acetate soluble component, and after dehydration with anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. The residue was recrystallized with ethyl acetate to obtain vitamin K hydroquinone-1,4-bis-dicarboxylic acid half ester.

[0146] Manufacturing Method I

[0147] To 2 mmol of vitamin K hydroquinone-1,4-bis-dicarboxylic acid half ester, add 2 times mol of 0.1N sodium hydroxide aqueous solution or 2 times mol of meglumine aqueous solution to dissolve it, and freeze-dry it. Recrystallize it with methanol-acetonitrile to obtain vitamin K hydroquinone-1,4-bis-dicarboxylic acid half ester-bis-sodium salt or vitamin K hydroquinone-1,4-bis-dicarboxylic acid half ester-bis-meglumine salt.

[0148] Manufacturing method J

[0149] Vitamin K hydroquinone-1,4-bis-dicarboxylic acid half ester is dissolved in a primary alcohol or a secondary alcohol, and stirred under the acidification of hydrochloric acid. The solvent is distilled off under reduced pressure to obtain vitamin K hydroquinone-1,4-bis-dicarboxylic acid alcohol ester.

[0150] [Table 1]

[0151]

[0152] [Table 2]

[0153]

[0154] [Table 3]

[0155]

[0156] [Table 4]

[0157]

[0158] [Table 5]

[0159]

[0160] [Table 6]

[0161]

[0162] [Table 7]

[0163]

[0164] [Table 8]

[0165]

Claims

1. Use of at least one of the carboxylic acid esters of active vitamin K represented by the following general formula (1) or its salts for the manufacture of an agent for improving Alzheimer's disease or Parkinson's disease: [Chemical formula 1] In the formula, R1 and R2 are hydrogen atoms or selected from glycine, N-acylglycine, N-alkylglycine, N,N-dialkylglycine, N,N,N-trialkylglycine, Nt-BOC-NHCH2CH2CO-, Nt-BOC-N(CH3)CH2CO-, Nt-BOC-NH(CH2)5CO-, CH3NHCH2CO-, (CH3)3NCH2CO-, The substituents of dicarboxylic acid half esters and their salts, at least one of R1 and R2 is selected from glycine, N-acylglycine, N-alkylglycine, N,N-dialkylglycine, N,N,N-trialkylglycine, Nt-BOC-NHCH2CH2CO-, Nt-BOC-N(CH3)CH2CO-, Nt-BOC-NH(CH2)5CO-, CH3NHCH2CO-, (CH3)3NCH2CO-, Any of a dicarboxylic acid half ester and a salt thereof, wherein Nt-BOC represents an N-tert-butoxycarbonyl group, and in the dicarboxylic acid half ester, the carbonyl groups of the dicarboxylic acid residues are bonded via a straight-chain alkylene group having 2 to 4 carbon atoms, R3 represents a group represented by the following general formula (2) or the following general formula (3), and n represents an integer of 1 to 7. [Chemical formula 2] 2. Use of the carboxylic acid esters of active vitamin K represented by the following general formula (4) or their salts for the manufacture of an agent for improving Alzheimer's disease or Parkinson's disease: In the general formula (4), R1 and R2 are carboxylic acid residues selected from R4OOCCH2CH2CO- and R4OOCCH2CH2CH2CO-, R3 has the meaning of the general formula (2) or (3) defined in claim 1, and R4 is a C1-C3 alkyl group.

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