MITOCHONDRIAL DYSFUNCTION IMPROVING AGENT

Abstract

The object of the present invention is to provide a mitochondrial dysfunction improving agent comprising a vitamin K derivative having a high deliverability to mitochondria.

A mitochondrial dysfunction improving agent, neurodegeneration improving agent, amyotrophic lateral sclerosis improving agent, Alzheimer's disease improving agent, and Parkinson's disease improving agent comprising at least one of a carboxylic acid ester of an active vitamin K represented by a general formula (1) or a salt thereof, and

##STR00001##

(wherein, R.sub.1 and R.sub.2 are a hydrogen atom, respectively, or a substituent selected from glycine, N-acyl glycine, N-alkyl glycine, N,N-dialkyl glycine, N,N,N-trialkyl glycine, acyl, dicarboxylic acid hemiester, and salts thereof; and at least either of R.sub.1 and R.sub.2 is glycine, N-acyl glycine, N-alkyl glycine, N,N-dialkyl glycine, N,N,N-trialkyl glycine, acyl, dicarboxylic acid hemiester, and salts thereof. R.sub.3 is a group represented by a general formula (2), or a general formula (3). n is an integer of 1 to 7)

##STR00002##

a mitochondrial dysfunction improving agent, neurodegeneration improving agent, amyotrophic lateral sclerosis improving agent, Alzheimer's disease improving agent, and Parkinson's disease improving agent comprising a carboxylic acid ester of an active vitamin K or a salt thereof (in the general formula (1), R.sub.1 and R.sub.2 is a carboxylic acid residue selected from a group consisting of R.sub.4OOCCH.sub.2CH.sub.2CO— and R.sub.4OOCCH.sub.2CH.sub.2CH.sub.2CO—. R.sub.3 represents the above general formula (2) or (3). R.sub.4 is a C1-C3 alkyl group.).

Claims

1. A mitochondrial dysfunction improving agent comprising at least one of a carboxylic acid ester of an active vitamin K represented by a general formula (1) or a salt thereof. ##STR00062## (wherein, R.sub.1 and R.sub.2 are a hydrogen atom, respectively, or a substituent selected from glycine, N-acyl glycine, N-alkyl glycine, N,N-dialkyl glycine, N,N,N-trialkyl glycine, acyl, dicarboxylic acid hemiester, and salts thereof; and at least either of R.sub.1 and R.sub.2 is glycine, N-acyl glycine, N-alkyl glycine, N,N-dialkyl glycine, N,N,N-trialkyl glycine, acyl, dicarboxylic acid hemiester, and salts thereof. R.sub.3 is a group represented by a general formula (2), or a general formula (3). n is an integer of 1 to 7) ##STR00063##

2. A carboxylic acid ester of an active vitamin K represented by a general formula (4) or a salt thereof. ##STR00064## (in the general formula (4), R.sub.1 and R.sub.2 is a carboxylic acid residue selected from a group consisting of R.sub.4OOCCH.sub.2CH.sub.2CO— and R.sub.4OOCCH.sub.2CH.sub.2CH.sub.2CO—. R.sub.3 represents the above general formula (2) or (3). R.sub.4 is a C1-C3 alkyl group.)

3. A mitochondrial dysfunction improving agent according to claim 1, wherein said mitochondrial dysfunction is neurodegeneration.

4. A mitochondrial dysfunction improving agent according to claim 1, wherein said mitochondrial dysfunction is amyotrophic lateral sclerosis.

5. A mitochondrial dysfunction improving agent according to claim 1, wherein said mitochondrial dysfunction is Alzheimer's disease.

6. A mitochondrial dysfunction improving agent according to claim 1, wherein said mitochondrial dysfunction is Parkinson's disease.

7. A carboxylic acid ester of an active vitamin K according to claim 2, wherein said carboxylic acid ester is a neurodegeneration improving agent.

8. A carboxylic acid ester of an active vitamin K according to claim 2, wherein said carboxylic acid ester is an amyotrophic lateral sclerosis improving agent.

9. A carboxylic acid ester of an active vitamin K according to claim 2, wherein said carboxylic acid ester is an Alzheimer's disease improving agent.

10. A carboxylic acid ester of an active vitamin K according to claim 2, wherein said carboxylic acid ester is a Parkinson's disease improving agent.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0056] FIG. 1 illustrates the improvement effect by an MKH derivative to a neurological disorder caused by β-amyloid.

[0057] FIG. 2A illustrates the improvement effect by an MKH derivative to a neurological disorder caused by β-amyloid.

[0058] FIG. 2B illustrates the improvement effect by an MKH derivative to a neurological disorder caused by β-amyloid.

[0059] FIG. 3A illustrates the improvement effect by an MKH derivative to a neurological disorder caused by β-amyloid.

[0060] FIG. 3B illustrates the improvement effect by an MKH derivative to a neurological disorder caused by β-amyloid.

[0061] FIG. 4 illustrates the improvement effect of cell death by an MKH derivative. vs control (**=p<0.01), vs Rot (##=p<0.01)

[0062] FIG. 5 illustrates the improvement effect of suppression of ATP production by an MKH derivative. vs control (**=p<0.01), vs Rot (##=p<0.01)

[0063] FIG. 6A illustrates the recovery state of the membrane potential by an MKH derivative.

[0064] FIG. 6B illustrates the recovery state of the membrane potential by an MKH derivative. vs control (**=p<0.01), vs Rot (##=p<0.01)

[0065] FIG. 7 illustrates the improvement effect of cell death by a PKH derivative. vs control (**=p<0.01), vs Rot (##=p<0.01)

[0066] FIG. 8 illustrates the improvement effect of suppression of ATP production by a PKH derivative. vs control (**=p<0.01), vs Rot (##=p<0.01)

[0067] FIG. 9 illustrates the recovery effect of the membrane potential by a PKH derivative. vs control (**=p<0.01), vs Rot (##=p<0.01)

[0068] FIG. 10 illustrates the recovery effect of the membrane potential to Complex II inhibitor by an MKH derivative. vs control (**=p<0.01), vs 3-NP (##=p<0.01)

[0069] FIG. 11 illustrates the recovery effect of the membrane potential to Complex III inhibitor by an MKH derivative. vs control (**=p<0.01), vs antimycin A (##=p<0.01)

[0070] FIG. 12 illustrates the effect of an MKH derivative to cell death caused by an uncoupler (carbonylcyanide-m-chlorophenylhydrazone, CCCP). vs control (**=p<0.01), vs CCCP (##=p<0.01)

DESCRIPTION OF EMBODIMENTS

[0071] In the following, preferred embodiments of the present invention are described in detail.

[0072] In sporadic and familial human AD brain samples, AD-induced pluripotent stem cell (iPSC) derived neurons, and AD transgenic animal models, mitochondrial quality control disorder and mitochondrial function decline of neurons are exhibited in AD, and it was revealed that accumulation of these dysfunctional mitochondria contributes to an increase of Aβ and p-tau which are the factors of the AD lesion (Non-Patent Literatures 1-5). It was revealed that Aβ toxicity can be decreased and tau hyperphosphorylation can be invalidated by restoring or strengthening dysfunctional mitochondria (mitochondrial functions or mitochondrial quality control) with a pharmaceutical or genetic method (Non-Patent Literature 1). It is shown that UBIAD1 is an enzyme that biosynthesizes menahydroquinone-4 (MKH) (Chem. 1), UBIAD1 expression of the brain is decreased in AD patients, MKH level is also decreased, and UBIAD1 increases the membrane potential of polarized mitochondria, i.e., improvement in mitochondrial dysfunction (Non-Patent Literature 13).

[0073] Therefore, the inventors hypothesized that mitochondrial dysfunction or mitochondrial quality control can be restored and strengthened, and the AD lesion (Aβ and p-tau) can be reduced or restored by the effective delivery of MKH to mitochondria of cranial nerves of AD patients, and aimed to develop a low-molecular compound capable of MKH, PKH delivery and reducing or restoring the AD lesion (increase of Aβ and p-tau) as a drug target.

[0074] MKH is a two-electron reductant of vitamin K.sub.2(20) (menaquinone-4, MK-4) which is a type of vitamin K(VK). In an endoplasmic reticulum, MKH functions as a cofactor of an enzyme (GGCX) that carboxylates a Glu residue to a Gla residue in post-translational modification of a vitamin K-dependent protein (VKDP) which is one action of vitamin K as an activator of VK. Since MKH is extremely easily oxidized, a quinone-type MK-4 is used in clinical study; however, low photostability, high phototoxicity, low water solubility, and necessity of a reductive activation process were problems upon drug delivery of MKH by MK-4. The inventors succeeded to produce an MKH delivery agent that does not require a reductive activation process and has a high photostability and no phototoxicity by making MKH a prodrug.

[0075] First, a low-molecular compound capable of restoring and strengthening mitochondrial function and decreasing Aβ protein toxicity by the MKH delivery hypothesized in this study was aimed as a drug target, and an MKH prodrug was focused. Its functions were investigated, and possibility as AD prevention and a therapeutic agent was evaluated in vitro.

[0076] Moreover, Parkinson's disease (PD) is a neurodegenerative disease of dopaminergic neurons, and disruption of mitochondrial dysfunction and mitochondrial quality control lead to neuronal cell death in PD. Furthermore, it is reported that UBIAD1 rescues mitochondrial quality control in PD models.

[0077] Therefore, an MKH prodrug is used as a drug target of a low-molecular compound to investigate whether mitochondrial dysfunction can be restored and evaluate possibility as a therapeutic agent of PD in vitro.

(1) Possibility as Prevention and Therapeutic Agent for Alzheimer's Disease

[0078] In a neuronopathy model due to Aβ peptide of which a single neuron-astrocyte coculture sample is treated with Aβ peptide, an improvement effect in neurodegeneration of a MKH derivative was evaluated. Since mitochondrial affinity of the derivative and reconvertibility to MKH are expected affect the MKH delivery by an MKH derivative, a cationic derivative (MKH-DMG) presumed to have a high affinity, an anionic derivative (MKH-SUC) having a high intracellular reconversionability, and an oxidant of MKH (MK-4) used in clinical study were evaluated (Chem. 7).

TABLE-US-00001 [Chem. 7] [00009] Menaquinone-4 (MK-4) [00010] Phylloquinone (PK) [00011] Compounds R.sub.1 R.sub.2 MKH H [00012] MKH-DMG —OCCH.sub.2N(CH.sub.3).sub.2•HCl [00013] MKH-SUC —OCCH.sub.2CH.sub.2COOH [00014] PKH H [00015] PKH-DMG —OCCH.sub.2N(CH.sub.3).sub.2•HCl [00016]

Quinone-Type Vitamin K and Active (Hydroquinone-Type) Vitamin K Derivatives

Improvement Effect of MKH Derivative to Neuropathy Caused by β-Amyloid

[0079] After culturing (2 weeks) astrocytes isolated from the cerebral cortex of 0- to 1-day-old ICR mouse, regions were divided in dots on a slide glass and cultured. After culturing for one more week, hippocampus-derived neurons of 0- to 1-day-old ICR mouse were seeded and cocultured. From day 1 of seeding neuron, Aβ.sub.25-35 (1 μM) and a test compound (0.3 μM) were exposed for three days, and morphological change of neurons was observed. Dendrites (MAP2 antibody) and axons (Tau antibody) of nerves were stained by immunostaining and analyzed. Dendrites and axons were evaluated for changes in branches and elongation of dendrites and axons by Sholl analysis method.

[0080] Results of staining of neurons are shown in FIG. 1. Both of branches and elongation of nerve axons and branches and elongation of nerve dendrites were significantly suppressed by Aβ.sub.25-35, and neuropathy was observed. In MKH derivatives (DMG(MKH-DMG) and SUC(MKH-SUC)), both neuropathies of nerve axons and nerve dendrites were significantly improved (FIGS. 2 and 3), and the improvement effect of neuropathy was revealed. No improvement effect was observed for MK4 (MK-4) to both neuropathies. Since MKH derivatives have the improvement effect to β-amyloid at a low concentration of 0.3 μM, a high safety can be expected.

2) Improvement Effect of Mitochondrial Dysfunction by MKH Derivative

[0081] Effects of an MKH derivative and a PKH derivative to mitochondrial dysfunctions caused by Complex I inhibitor rotenone (10 Complex II inhibitor (3-nitro propionic acid (3-NP)), Complex III inhibitor (antimycin A), and a depolarizing agent (carbonylcyanide-m-chlorophenylhydrazone, CCCP) in neural cells were evaluated. The effects were evaluated with cell death (viability measurement with cell titer blue), inhibition of ATP production (ATP measurement with cell titer glo), and membrane potential reduction (JC-1 staining: depolarization (green), hyperpolarization (red)).

[0082] By MKH derivative administration, cell death caused by Complex I inhibitor rotenone treatment was suppressed (FIG. 4), and inhibition of ATP production (FIG. 5) and mitochondrial membrane potential reduction were restored (FIG. 6). In PKH derivative administration, cell death caused by rotenone treatment was suppressed (FIG. 7), and inhibition of ATP production (FIG. 8) and mitochondrial membrane potential reduction were restored (FIG. 9).

[0083] Moreover, each derivative restored membrane potential reduction (FIG. 10). Furthermore, each derivative restored reduction in viability, ATP production and membrane potential caused by Complex III inhibitor (antimycin A) (FIG. 11). In addition, each derivative restored reduction in viability caused by a depolarization agent (CCCP) (FIG. 12). Moreover, each derivative increased expression of PGC-1α (peroxisome proliferator-activated receptor-c coactivator-1α) that activates mitochondrial biogenesis.

[0084] From the above results, it was revealed that the MKH derivative and PKH derivative can restore mitochondrial dysfunction (membrane potential reduced mitochondria).

3) Possibility as PD Therapeutic Agent

[0085] Complex I activity is decreased in sporadic PD. In rotenone Complex I inhibitor models, morphological and functional damages are caused in dopaminergic neurons in addition to characteristic symptoms of PD such as bradykinesia, rigidity, and tremor. Moreover, since mitochondria damaged by Complex I inhibition (membrane potential reduced mitochondria) induces mitochondrial quality control, it is used for evaluation of hereditary PD. Therefore, the acquired results revealed that the MKH derivative and PKH derivative are effective in rotenone PD models, and possibility as a PD therapeutic agent was revealed.

[0086] From the above evaluation results: [0087] it was revealed that, by using primary neurons, MKH derivatives of MKH-DMG and MKH-SUC can improve neurotoxicity caused by Aβ at a low concentration of 0.3 μM; and [0088] it was revealed that MKH-DMG and MKH-SUC can improve mitochondrial function (respiratory chain) damage.

[0089] These results support the hypothesis that mitochondrial dysfunction or mitochondrial quality control can be restored or strengthened and the AD lesion (increase of Aβ and p-tau) can be reduced or restored by the effective delivery of MKH to cranial nerve mitochondria of AD patients, and it was shown that the MKH derivative have a high possibility of a drug target based on the hypothesis.

[0090] Moreover, the effect of the MKH derivative in rotenone PD models supports a possibility as a PD therapeutic agent by restoration and strengthening of mitochondrial quality control.

[0091] Moreover, it supports a possibility as a therapeutic agent for amyotrophic lateral sclerosis by mitochondrial function restoration.

[0092] Therefore, the inventors propose an MKH derivative as a drug target of a preventative/therapeutic agent for AD and a therapeutic agent for PD having restoration and strengthening of mitochondrial dysfunction or quality control as a mechanism of action.

[0093] The inventors hypothesize that the AD lesion (increase of Aβ and p-tau) can be decreased by enabling restoration and strengthening of mitochondrial dysfunction or mitochondrial quality control by the effective delivery of MKH to mitochondria of cranial nerves of AD patients, and aim to develop a low molecular compound that enables the MKH delivery and a reduction of the AD lesion (increase of Aβ and p-tau) as a drug target. So far, the inventors evaluated an original MKH prodrug, and succeeded to reveal a high possibility.

[0094] Originality: A reduction of the AD lesion (increase of Aβ and p-tau) by restoration and strengthening of mitochondrial dysfunction or mitochondrial quality control has been shown in AD model cells or AD model mice; however, there is no method using the MKH delivery by an original MHK prodrug, and this method is the first method in the world.

[0095] High safety: Since improvement of neurotoxicity caused by Aβ using an original MHK prodrug is effective at a drug concentration of 0.3 μM, the effect can be exhibited at a low concentration. An MKH prodrug is a low molecular compound, and it is expected that after functioning as an MKH, it follows the same fate as vitamin K and disappears. Therefore, an MKH prodrug is considered to be a highly safe prevention/therapeutic agent with extremely low risk for harmful side effects.

EXAMPLES

[0096] The present invention is described in further details with reference to Examples in the following; however, the present invention is not limited thereto.

Examples 1 to 34

[0097] Vitamin K hydroquinone derivatives shown in Tables 1 to 5 are produced by the methods A to I in the following. Moreover, mass spectra (ionizing method; FD method and FAB method) and .sup.1H-NMR spectra of the obtained substances are shown in Tables 6 to 8.

Production Method A

[0098] Amino acid (0.1 mol) is dissolved in 100 ml of distilled water-dioxane (1:1, v/v), and 30 ml of triethylamine is added thereto. Then, di-tert-butyl decarbonate is gradually added thereto, and the mixture is stirred for 30 minutes at room temperature. Dioxane is removed under reduced pressure. 50 ml of sodium hydrogen carbonate aqueous solution (0.5 M) is added thereto, and washed with 100 ml of ethyl acetate. The ethyl acetate layer is washed with 50 ml of sodium hydrogen carbonate solution. The aqueous layer is combined and adjusted to be acidic (pH 3) with a citric acid aqueous solution (0.5 M) under cooling with ice, saturated with sodium chloride, and then extracted with ethyl acetate (100 ml×3 times). The extract is dehydrated with anhydrous sodium sulfate, and the solvent is removed under reduced pressure. A N-t-BOC-amino acid is obtained by adding isopropyl ether to the oily residue or by the crystallization thereof under cooling. Vitamin K (6.75 mmol) is dissolved in 40 ml of isopropyl ether, and sodium borohydride (47 mmol) is dissolved in 15 ml of methanol and added thereto. The mixture is stirred at room temperature until the color of the solution changes from yellow to colorless. 60 ml of isopropyl ether and 100 ml of distilled water are added to the reaction solution, and the isopropyl ether layer is separated. 100 ml of isopropyl ether is further added to the aqueous phase to extract a soluble fraction. The isopropyl layer is combined, and the mixture is dehydrated with anhydrous sodium sulfate and then is concentrated under reduced pressure. n-Hexane is added to the residue and a white precipitate is deposited to obtain vitamin K hydroquinone.

[0099] Vitamin K hydroquinone, N-t-BOC-amino acid (13.55 mmol) and DCC (13.55 mmol) are added to 50 ml of anhydrous pyridine, and the mixture is stirred for 20 hours at room temperature. The solvent is removed under reduced pressure. Ethyl acetate is added to the residue to extract a soluble fraction (100 ml×2 times). The extract is concentrated under reduced pressure, and the residue is separated and purified by silica gel column chromatography (elution solvent: n-hexane-isopropyl ether) to obtain vitamin K hydroquinone-1,4-bis-N-t-BOC-amino acid. Vitamin hydroquinone-1,4-bis-N-t-BOC-amino acid is dissolved in a small amount of acetone. Hydrochloric acid-dioxane (2.5˜4.0 N) is added thereto so that the amount of hydrochloric acid is about 20 times of the ester in moles. After stirring for 1 hour, the solvent is removed under reduced pressure. The residue is recrystallized with acetone-methanol system to obtain a hydrochloride of vitamin K hydroquinone-1,4-bis-amino acid aster.

Production Method B

[0100] Vitamin K (6.75 mmol) is dissolved in 40 ml of isopropyl ether, and sodium borohydride (47 mmol) is dissolved in 15 ml of methanol and added thereto. The solution is stirred at room temperature until the color of the solution changes from yellow to colorless. 60 ml of isopropyl ether and 100 ml of distilled water are added to the reaction solution, and the isopropyl ether layer is separated. 100 ml isopropyl ether is further added to the aqueous phase to extract a soluble fraction, and the isopropyl layer is combined. After dehydrating with anhydrous sodium sulfate, the extract is concentrated under reduced pressure. n-Hexane is added to the residue and a white precipitated is deposited to obtain vitamin K hydroquinone. Vitamin K hydroquinone, N,N-dialkylamino acid hydrochloride (13.55 mmol) or N,N,N-trialkylamino acid hydrochloride (13.55 mmol), and DCC (13.55 mmol) are added to 50 ml of anhydrous pyridine, and the mixture is stirred for 20 hours at room temperature. The solvent is removed under reduced pressure, and the residue is suspended in distilled water, adjusted to pH 7 to 8 with sodium hydrogen carbonate, and then extracted with ethyl acetate (100 ml×3 times). After dehydrating the extract with anhydrous sodium sulfate, the solvent is removed under reduced pressure. The residue is separated and purified 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 aster.

Production Method C

[0101] Vitamin K (6.75 mmol) is dissolved in 40 ml of isopropyl ether, and hydrosulfite sodium (50 mmol) is dissolved in 50 ml of distilled water and added thereto. The mixture is stirred at room temperature until isopropyl ether exhibits brown color and then further changes to colorless. The isopropyl layer is separated, and 100 ml of isopropyl ether is further added to the aqueous phase to extract a soluble fraction. The isopropyl layer is combined, and after dehydrating with anhydrous sodium sulfate, the extract is concentrated under reduced pressure. n-Hexane is added to the residue and a white precipitate is deposited to obtain vitamin K hydroquinone. N,N-dialkylamino acid hydrochloride (6.75 mmol) and DCC (6.75 mmol) are added to vitamin K hydroquinone, and the mixture is stirred in 50 ml of anhydrous pyridine for 20 hours. The solvent is removed under reduced pressure, and the residue is suspended in distilled water, adjusted to pH 7 to 8 with sodium hydrogen carbonate, and then extracted with ethyl acetate (100 ml×3 times). After the extract is dehydrated with anhydrous sodium sulfate, the solvent is removed. The residue is separated and purified by silica gel column chromatography (elution solvent: 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.

Production Method D

[0102] Vitamin K (6.75 mmol) is dissolved in 40 ml of isopropyl ether, and sodium borohydride (47 mmol) is dissolved in 15 ml of methanol and added thereto. The solution is stirred until the color of the solution changes from yellow to colorless at room temperature. 60 ml of isopropyl ether and 100 ml of distilled water is added to the reaction solution, and the isopropyl ether layer is separated. 100 ml of isopropyl ether is further added to the aqueous layer to extract a soluble fraction, and the isopropyl ether layer is combined. After dehydrating with anhydrous sodium sulfate, the extract is concentrated under reduced pressure. n-Hexane is added to the residue and a white precipitate is deposited to obtain vitamin K hydroquinone. Vitamin K hydroquinone is dissolved in 30 ml of anhydrous benzene-anhydrous pyridine (1:1, v/v), pyridinecarbonyl chloride hydrochloride is added thereto, and stirred for 3 hours at room temperature. Insoluble matters are removed by filtration, and the filtrate is concentrated under reduced pressure. The residue is suspended in 100 ml of distilled water, and sodium hydrogen carbonate is added thereto (pH 7-8) to extract a soluble fraction to ethyl acetate (100 ml×3 times). The extract is concentrated under reduce pressure. The residue is separated and purified by silica gel column chromatography (elution solvent: isopropyl ether-ethyl acetate, 9:1) to obtain vitamin K hydroquinone-1,4-bis-pyridinecarboxylic acid ester.

Production Method E

[0103] Vitamin K hydroquinone-1,4-bis-N,N-dialkylamino acid ester or vitamin K hydroquinone-1,4-bis-pyridinedicarboxylic acid (2 mmol) is dissolved in 20 ml of acetone, and hydrochloric acid-dioxane (2.5˜4.0 N) is added thereto so that the amount of hydrochloric acid is 10 times of the ester in moles. The solvent is removed under reduced pressure. The residue is recrystallized with acetone-methanol to obtain a hydrochloride of vitamin K hydroquinone-1,4-bis-N,N-dialkyl amino acid or vitamin hydroquinone-1,4-bis-pyridinecarboxylic acid.

Production Method F

[0104] Vitamin K hydroquinone-1,4-bis-N,N-dialkyl amino acid or vitamin K hydroquinone-1,4-bis-pyridinedicarboxylic acid (2 mmol) is dissolved in 20 ml of dichloromethane. Alkyl sulfonic acid (2 mmol) is added thereto and stirred. Depositing crystals are filtered to obtain an alkylsulfonate salt of vitamin K hydroquinone-1,4-bis-N,N-dialkyl amino acid ester or vitamin hydroquinone-1,4-bis-pyridinedicarboxylic acid ester.

Production Method G

[0105] Vitamin K (4.55 mmol) is dissolved in 40 ml of isopropyl ether, and sodium borohydride (31.5 mmol) is dissolved in 15 ml of methanol and added thereto. The solution is stirred until the color of the solution changes from yellow to colorless at room temperature. 60 ml of isopropyl ether and 100 ml of purified water are added to the reaction solution, and the isopropyl ether layer is separated. 100 ml of isopropyl ether is further added to the aqueous phase to extract a soluble fraction, and the isopropyl layer is combined. After dehydrating with anhydrous sodium sulfate, the solvent is removed under reduced pressure. Dimethylaminopyridine (8.97 mmol) and dicarboxylic anhydride (18.0 mmol) are added to the residue, dissolved in 100 ml of isopropyl ether-dioxane (6:4, v/v), and stirred for 3 hours at room temperature. Then, the solution is heated to 50˜60° C. to react for 2 hours, and allowed to cool at room temperature to react for 10 hours. 100 ml of purified water is added to the reaction solution, and the isopropyl ether layer is separated. After dehydrating with anhydrous sodium sulfate, the solvent is removed under reduced pressure. The residue is suspended in isopropyl ether, and centrifuged to obtain a precipitate. 100 ml of ethyl acetate and 100 ml of purified water are added to the precipitate to extract an ethyl acetate soluble fraction. After dehydrating with anhydrous sodium sulfate, the solvent is removed under reduced pressure. The residue is suspended in isopropyl ether and insoluble matters are recrystallized with ethyl acetate to obtain a vitamin K hydroquinone-1,4-bis-dicarboxylic acid hemiester.

Production Method H

[0106] Vitamin K (6.75 mmol), zinc (18.4 mmol), anhydrous dicarboxylic acid (33.0 mmol), anhydrous sodium acetate (13.8 mmol), and acetic acid (161.5 mmol) are put into a 100 ml eggplant flask, Dimroth condenser is mounted thereto, and the mixture is heated at 85° C. for 3 hours while being stirred well. Then, the mixture is cooled at room temperature to obtain a white solid substance. 200 ml of ethyl acetate and 100 ml of purified water are added thereto to extract an ethyl acetate soluble fraction. After dehydrating with anhydrous sodium sulfate, the solvent is removed under reduced pressure. The residue is recrystallized with ethyl acetate to obtain a vitamin K hydroquinone-1,4-bis-dicarboxylic acid hemiester.

Production Method I

[0107] Vitamin K hydroquinone-1,4-bis-dicarboxylic acid hemiester (2 mmol) is added to 0.1 N sodium hydroxide aqueous solution (2 times in moles) or meglumine aqueous solution (2 times in moles), and freeze dried. It is recrystallized with methanol-acetonitrile to obtain vitamin K hydroquinone-1,4-bis-dicarboxylic acid hemiester-bis-sodium salt or vitamin K hydroquinone-1,4-bis-dicarboxylic acid hemiester-bis-meglumine salt.

Production Method J

[0108] Vitamin K hydroquinone-1,4-bis-dicarboxylic acid hemiester is dissolved in primary or secondary alcohol, and stirred under hydrochloric acid acidity. The solvent is removed under reduced pressure to obtain vitamin K hydroquinone-1,4-bis-dicarboxylic acid alcohol ester.

TABLE-US-00002 TABLE 1 Production Example Compound Name R.sub.1 R.sub.2 R.sub.3 n method 1 Vitamin K  hydroquinone- 1,4-bis-N-t-butoxycarbonyl glycinate N-t-BOC-NHCH.sub.2CO— N-t-BOC-NHCH.sub.2CO— [00017] 4 A 2 Vitamin K  hydroquinone- 1,4-bis-N-t-butoxycarbonyl- β-alaninate N-t-BOC-NHCH.sub.2CH.sub.2CO— N-t-BOC-NHCH.sub.2CH.sub.2CO— [00018] 4 A 3 Vitamin K  hydroquinone- 1,4-bis-N-t-butoxycarbonyl phenyl alaninate [00019] [00020] [00021] 4 A 4 Vitamin K  hydroquinone- 1,4-bis-N-t-butoxycarbonyl sarcosinate N-t-BOC-N(CH.sub.3)CH.sub.2CO— N-t-BOC-N(CH.sub.3)CH.sub.2CO— [00022] 4 A 5 Vitamin K  hydroquinone- 1,4-bis-N-t-butoxycarbonyl tranexamate [00023] [00024] [00025] 4 A 6 Vitamin K  hydroquinone- 1,4-bis-N-t-butoxycarbonyl- -aminocaproate N-t-BOC-NH(CH.sub.2).sub.5CO— N-t-BOC-NH(CH.sub.2).sub.5CO— [00026] 4 A indicates data missing or illegible when filed

TABLE-US-00003 TABLE 2 Ex- Production ample Compound Name R.sub.1 R.sub.2 R.sub.3 n method  7 Vitamin K  hydroquinone- 1,4-bis-N,N-dimethyl glycinate (CH.sub.3).sub.2NCH.sub.2CO— (CH.sub.3).sub.2NCH.sub.2CO— [00027] 4 B, C  8 Vitamin K  hydroquinone- 1-N,N-dimethyl glycinate (CH.sub.3).sub.2NCH.sub.2CO— H [00028] 4 B, C  9 Vitamin K  hydroquinone- 4-N,N-dimethyl glycinate H (CH.sub.3).sub.2NCH.sub.2CO— [00029] 4 B, C 10 Vitamin K  hydroquinone- 1,4-bis-N,N-dimethyl glycinate hydrochloride HCl•(CH.sub.3).sub.2NCH.sub.2CO— HCl•(CH.sub.3).sub.2NCH.sub.2CO— [00030] 4 E 11 Vitamin K  hydroquinone- 1-N,N-dimethyl glycinate hydrochloride HCl•(CH.sub.3).sub.2NCH.sub.2CO— H [00031] 4 E 12 Vitamin K  hydroquinone- 4-N,N-dimethyl glycinate hydrochloride H HCl•(CH.sub.3).sub.2NCH.sub.2CO— [00032] 4 E indicates data missing or illegible when filed

TABLE-US-00004 TABLE 3 Ex- Production ample Compound Name R.sub.1 R.sub.2 R.sub.3 n method 13 Vitamin K  hydroquinone- 1,4-bis-N,N-dimethyl glycinate methanesulfonate CH.sub.3SO.sub.3H•(CH.sub.3).sub.2NCH.sub.2CO— CH.sub.3SO.sub.3H•(CH.sub.3).sub.2NCH.sub.2CO— [00033] 4 B 14 Vitamin K  hydroquinone- 1,4-bis-glycinate hydrochloride HCl•NH.sub.2CH.sub.2CO— HCl•NH.sub.2CH.sub.2CO— [00034] 4 A 15 Vitamin K  hydroquinone- 1,4-bis-sarcosinate hydrochloride HCl•CH.sub.3NHCH.sub.2CO— HCl•CH.sub.3NHCH.sub.2CO— [00035] 4 A 16 Vitamin K  hydroquinone- 1,4-bis-  chloride Cl.sup.−•(CH.sub.3).sub.3N.sup.+CH.sub.2CO— Cl.sup.−•(CH.sub.3).sub.3N.sup.+CH.sub.2CO— [00036] 4 B 17 Vitamin K  hydroquinone- 1,4-bis-tranexamate hydrochloride [00037] [00038] [00039] 4 A 18 Vitamin K  hydroquinone- 1,4-bis- -aminocaproate hydrochloride HCl•NH.sub.2(CH.sub.2).sub.5CO— HCl•NH.sub.2(CH.sub.2).sub.5CO— [00040] 4 A indicates data missing or illegible when filed

TABLE-US-00005 TABLE 4 Ex- Production ample Compound Name R.sub.1 R.sub.2 R.sub.3 n method 19 Vitamin K  hydroquinone- 1,4-bis-nicotinate [00041] [00042] [00043] 4 D 20 Vitamin K  hydroquinone- 1,4-bis-nicotinate methanesulfonate [00044] [00045] [00046] 4 F 21 Vitamin K  hydroquinone- 1,4-bis-N,N-dimethyl glycinate (CH.sub.3).sub.2NCH.sub.2CO— (CH.sub.3).sub.2NCH.sub.2CO— [00047] — B, C 22 Vitamin K  hydroquinone- 1-N,N-dimethyl glycinate (CH.sub.3).sub.2NCH.sub.2CO— H [00048] — B, C 23 Vitamin K  hydroquinone- 4-N,N-dimethyl glycinate H (CH.sub.3).sub.2NCH.sub.2CO— [00049] — B, C 24 Vitamin K  hydroquinone- 1,4-bis-N,N-dimethyl glycinate hydrochloride HCl•(CH.sub.3).sub.2NCH.sub.2CO— HCl•(CH.sub.3).sub.2NCH.sub.2CO— [00050] — E 25 Vitamin K  hydroquinone- 1-N,N-dimethyl glycinate hydrochloride HCl•(CH.sub.3).sub.2NCH.sub.2CO— H [00051] E bis-  aminocaproate hydrochloride [00052] indicates data missing or illegible when filed

TABLE-US-00006 TABLE 5 Ex- Production ample Compound Name R.sub.1 R.sub.2 R.sub.3 n method 26 Vitamin K  hydroquinone-4- N,N-dimethyl glycinate hydrochloride H HCl•(CH.sub.3).sub.2NCH.sub.2CO— [00053] E 27 Vitamin K  hydroquinone- 1,4-bis-succinate HOOCCH.sub.2CH.sub.2CO— HOOCCH.sub.2CH.sub.2CO— [00054] 4 G, H 28 Vitamin K  hydroquinone- 1,4-bis-succinate meglamine salt HOH.sub.2C•(CH(OH)).sub.4CH.sub.2NHCH.sub.3• HOOCCH.sub.2CH.sub.2CO— HOH.sub.2C• (CH(OH)).sub.4CH.sub.2NHCH.sub.3• HOOCCH.sub.2CH.sub.2CO— [00055] 4 G, I 29 Vitamin K  hydroquinone- 1,4-bis-glutamate HOOCCH.sub.2CH.sub.2CH.sub.2CO— HOOCCH.sub.2CH.sub.2CH.sub.2CO— [00056] 4 G, H 30 Vitamin K  hydroquinone- 1,4-bis-succinate HOOCCH.sub.2CH.sub.2CO— HOOCCH.sub.2CH.sub.2CO— [00057] G, H 31 Vitamin K  hydroquinone- 1,4-bis-succinate meglamine salt HOH.sub.2C•(CH(OH)).sub.4CH.sub.2NHCH.sub.3• HOOCCH.sub.2CH.sub.2CO— HOH.sub.2C• (CH(OH)).sub.4CH.sub.2NHCH.sub.3• HOOCCH.sub.2CH.sub.2CO— [00058] G, I 32 Vitamin K  hydroquinone- 1,4-bis-glutamate HOOCCH.sub.2CH.sub.2CH.sub.2CO— HOOCCH.sub.2CH.sub.2CH.sub.2CO— [00059] G, H 33 Vitamin K  hydroquinone- 1,4-bis-succinate ethyl ester CH.sub.3CH.sub.2OOCCH.sub.2CH.sub.2CO— CH.sub.3CH.sub.2OOCCH.sub.2CH.sub.2CO— [00060] 4 G, H, J 34 Vitamin K  hydroquinone- 1,4-bis-succinate ethyl ester CH.sub.3CH.sub.2OOCCH.sub.2CH.sub.2CO— CH.sub.3CH.sub.2OOCCH.sub.2CH.sub.2CO— [00061] G, H, J indicates data missing or illegible when filed

TABLE-US-00007 TABLE 6 Mass spectrometry H-NMR Example (m/z) (δ ppm, internal standard TMS) 1 760 (FD-MS) (in CDC ) (M ) 7.70(2H, m), 7.47(2H, m), 5.15-5.01(6H, m), 4.36(4H, s), 3.40(2H, d), 2.23(3H, s), 2.05-1.93(12H, m), 1.77(3H, s), 1.66-1.57(12H, m), 1.48(18H, s) 2 788 (FD-MS) (in CDC ) (M ) 7.65(2H, m), 7.46(2H, m), 5.08(6H, m), 3.57(4H, ), 3.39(2H, d), 3.01,(4H, m), 2.23(3H, s), 2.05-1.95(12H, m), 1.77(3H, s), 1.67-1.57 (12H, m), 1.47(18H, s) 3 940 (FD-MS) (in CDC ) (M ) 7.39-7.29(14H, m), 5.09-4.99(8H, m), 3.42(2H, d), 3.26(4H, m), 2.12(3H, s), 2.06-1.93(12H, m), 1.73(3H, s), 1.67-1.57(12H, m), 1.44(9H, s), 1.42(9H, s) 4 788 (FD-MS) (M ) 5 924 (FD-MS) (in CDC ) (M ) 7.63(2H, m), 7.43(2H, m), 5.10-5.01(4H, m), 4.62(2H, s), 3.36(2H, s), 3.04(4H, s), 2.70(2H, m), 2.34(4H, m), 2.19(3H, s), 2.05-1.93(16H, m), 1.75-1.46(39H, m), 1.11(4H, m) 6 872 (FD-MS) (in CDC ) (M ) 7.64(2H, m), 7.44(2H, m), 5.07(4H, m), 4.51(2H, s), 3.39(2H, d), 3.16(4H, m), 2.75(4H, m), 2.21(3H, s), 2.07-1.85(16H, m), 1.76(3H, s), 1.67-1.45(38H, m) 7 616 (FD-MS) (in CDC ) (M ) 7.74(2H, m), 7.50(2H, m), 5.05(4H, m), 3.72(4H, s), 3.42(2H, d), 2.47(6H, s), 2.45(6H, s), 2.23(3H, s), 2.09 1.90(12H, m), 1.77(3H, s), 1.64-1.52(12H, m), 8 531 (FD-MS) (M ) 9 531 (FD-MS) (M+) 10 616 (FD-MS) (in CDC ) (M -2HC ) 7.90(2H, m), 7.61(2H, m), 5.05(4H, m), 4.90(4H, s), 3.52(2H, d), 3.13(6H, s), 3.12(6H, s), 2.32(3H, s), 2.10 1.89(12H, m), 1.83(3H, s)1.63 1.51(12H, m) indicates data missing or illegible when filed

TABLE-US-00008 TABLE 7 Mass spectrometry H-NMR Example (m/z) (δ ppm, internal standard TMS) 11 531 (FD-MS) (in CD OD) (M HC ) 8.20(1H, m), 7.71(1H, m), 7.46(2H, m)5.07(4H, m), 4.78(2H, s), 3.61(2H, d), 3.10(6H, s), 2.25(3H, s), 2.12-1.89(12H, m), 1.83(3H, s), 1.63-1.52(12H, m) 12 531 (FD-MS) (in CD OD) (M HC ) 8.22(1H, m), 7.68(1H, m), 7.45(2H, m), .04(4H, m), 4.76(2H, s), 3.42(2H, d), 3.09(6H, s), 2.34(3H, s), 2.10-1.89(12H, m), 1.81(3H ), 1.64-1.52(12H, m 13 616 (FD-MS) (M CH SO H) 14 560 (FD-MS) (in CD OD) (M 2HC ) 7.88(2H, m), 7.57(2H, m), .06(4H, m), 4.48(4H, m), 3.51(2H, d) 32(3H, s), 2.11-1.92(12H, m), 1.83(3H, s), 1.68-1.48(12H, m) 15 588 (FD-MS) (in CD OD) (M 2HC ) 7.90(2H, m), 7.60(2H, m), 5.05(4H, m), 4.64(4H, s), 3. 1(2H, d), 2.90(3H, s), 2.89(3H, s), 2.32(3H, s), 2.10-1.92(12H, m), 1.83(3H, s), 1.63-1.50(12H, m) 16 646(FD MS) (in CD OD) (M 2C ) 7.90(2H, m), 7.61(2H, m), 5.05(4H, m)4.90(4H, s), 3.52(2H, d), 3.13(9H, s), 3.12(9H, s), 2.32(3H, s), 2.10-1.92(12H, m), 1.83(3H, s), 1.63-1.51(12H, m) 17 724 (FD MS) (M 2HC ) 18 672 (FD MS) (in CD OD) (M 2HC ) 7.73(2H, m), 7.51(2H, m) .07(4H, m), 3.43(2H, d), 2.97(4H, m), 2.87(4H, m), 2.24(3H, s), 2.10-1.85(12H, m), 1.81-1.48(27H, m) 19 656 (FD-MS) (in CDC ) (M ) 9.56(2H, d), 8.94(2H, s), 8.58(2H, m), 7.74(2H, m), 7.54(2H, m), 7.46(2H, m), 5.13(1H ), 5.07(3H, m), 3.50(2H, s), 2.34(3H, s), 2.05-1.93(12H, m), 1.66-1.55(15H, m) 20 656 (FD MS) (in CD OD) (M CH SO H) 9.74(2H, d), 9.37(2H, m), 9.20(2H, s), 8.32(2H, m), 7.89(2H, m), 7.56(2H, m), 5.11(1H ), 5.04(3H, m), 3.60(2H, s), 2.71(6H, s), 2.39(3H, s), 2.05-1.91(12H, m), 1.66-1.52(15H, m) indicates data missing or illegible when filed

TABLE-US-00009 TABLE 8 Mass spectrometry H-NMR Example (m/ ) (δ ppm, internal standard TMS) 21 622 (F MS) (in CD OD) (M ) 7.74 7.50(2H ) 01( 2.45 2.44 2.22 1.94( 0.8 22 537 (F MS) (in CD OD) (M ) 8 (2H .70(2H 2.46 1.9 ( 1.80(3H 0.88 ) 23 537 (F MS) (in CD OD) (M ) 8.18 7.40(2H 5.0 3.69(2H 2.44( 1 ) 24 622 (F MS) (in CD OD) (M ) 7. (2H 2.32(3H 1 0.8 m) 25 5 7 (F MS) (in CD OD) (M ) 8.19 4.79(2H 3H 1 ) 26 5 7 (F MS) (in CD OD) (M ) 4.78 1 m) 27 669 (FA MS) (in CD OD) (M 7 s) 28 6 6 (F MS) (  CD OD) (M ) m) 1.70 s) 29 697 ) (in CD (M ) 7 2.6 ) 30 75 (F MS) (in ) (M 7 ) 31  (F MS) (in CD ) (M ) 7 1.68(3H ) 32 70  (FA MS) (in D (M 7 1.90 ) 33 725 (F MS) (in CD D) (M 7 3.32 2.16 1.20 m) 34 72  (FA MS) (in CD ) (M 7.6 (2H 2.16(3H 1.68(3H 1.44(1H 1 0.79( 2H m) indicates data missing or illegible when filed