WO2019065674A1

WO2019065674A1 - Osteoarthritis improving agent

Info

Publication number: WO2019065674A1
Application number: PCT/JP2018/035563
Authority: WO
Inventors: 横山　富久; 康宏西田
Original assignee: アスタファーマシューティカルズ株式会社
Priority date: 2017-09-27
Filing date: 2018-09-26
Publication date: 2019-04-04
Also published as: JP2021008404A

Abstract

To provide a novel osteoarthritis improving agent. An osteoarthritis improving agent which comprises a trans-astaxanthin derivative represented by formula (I), a geometric isomer thereof, a mixture of geometric isomers thereof, an optical isomer thereof or a salt thereof [in formula (I), m1, m2, n1 and n2 are the same or different and represent an integer of 1-6].

Description

Osteoarthritis improvement agent

The present invention relates to an agent for improving osteoarthritis comprising an astaxanthin derivative.

Astaxanthin is widely known to have potentially strong anti-inflammatory effects. For example, in the patent family of Patent Documents 1-5, omega-3 fatty acid-containing lipids, neutral fats, antioxidants such as α-tocopherol, pigments such as astaxanthin, extraction of krills containing metals such as zinc and / or marine organisms A high concentration capsule of the substance oil is orally administered to a group of patients with osteoarthritis, and in many of them there is a remarkable alleviation effect of pain and a remarkable improvement effect of the flexibility of large joints (lower spine, knees, shoulders) Have been reported (see Example 2 of each document).

WO 2002/102394 pamphlet Patent No. 5135568 gazette JP, 2010-090140, A Patent No. 5228185 gazette JP, 2013-079278, A

However, natural products such as Patent Documents 1 to 5 have problems such as the inclusion of impurities derived from the natural products and poor solubility, and it is possible for anyone with age, such as osteoarthritis, However, for possible symptoms, further improvement effects are required.
The main object of the present invention is to provide an osteoarthritis improving agent having a further improving effect on osteoarthritis.

The present inventors diligently studied to find new therapeutic agents as a remedy for osteoarthritis, and as a result, the trans-astaxanthin derivative represented by the formula (I), its geometric isomer, a mixture of these geometric isomers, The inventors have found that their optical isomers or their salts have excellent therapeutic effects on osteoarthritis and completed the present invention.

That is, the present invention provides the following inventions [1] to [4].

[1] An osteoarthritis improving agent containing a trans-astaxanthin derivative represented by the formula (I), a geometric isomer thereof, a mixture of these geometric isomers, an optical isomer thereof or a salt thereof.

(Wherein, m ₁ , m ₂ , n ₁ and n ₂ are the same or different and each represents an integer of 1 to 6).

[2] Use of the trans-astaxanthin derivative represented by the above formula (I), its geometric isomer, a mixture of these geometric isomers, their optical isomers or a salt thereof for producing an osteoarthritis improving agent .
[3] The trans-astaxanthin derivative represented by the above-mentioned formula (I), its geometric isomer, a mixture of these geometric isomers, their optical isomers or a salt thereof, which is used for the improvement of osteoarthritis.
[4] A deformability characterized by administering an effective amount of the trans-astaxanthin derivative represented by the above formula (I), its geometric isomer, a mixture of these geometric isomers, their optical isomers or their salts Methods of treating arthropathy.

The trans-astaxanthin derivative represented by the formula (I) of the present invention, its geometric isomer, a mixture of these geometric isomers, their optical isomers or a salt thereof are various animals such as humans, dogs, cats and horses in general. Of the astaxanthin derivative of the formula (I), its geometric isomer, a mixture of these geometric isomers, an optical isomer thereof or a salt thereof These pharmaceutical compositions are excellent as osteoarthritis improving agents.

It is a figure which shows the relationship of the weight load ratio with the administration days of a control group and a compound X administration group in an Example. It is a figure which shows the relationship of the administration day in a control group and a compound X administration group, and a pain threshold in an Example.

The osteoarthritis improving agent of the present invention comprises, as an active ingredient, the trans-astaxanthin derivative represented by the above formula (I), its geometric isomer, a mixture of these geometric isomers, their optical isomers or their salts contains. "Deroarthritis" is a joint disease mainly involving chronic arthritis that develops in joints such as wrists, elbows, shoulders, necks, hips, hips, knees, ankles, and toes, etc. It is a disease that causes destruction of cartilage and proliferative changes of bone and cartilage due to progressive degeneration. The improvement of osteoarthritis related to the present invention can be expected to have a remarkable effect especially in osteoarthritis of the knee. "Amelioration" of osteoarthritis is meant to include not only the improvement of the symptoms of osteoarthritis itself but also the alleviation or analgesia of pain caused by osteoarthritis.

(Wherein, m ₁ , m ₂ , n ₁ and n ₂ are the same or different and each represents an integer of 1 to 6)

Among the compounds of formula (I), preference is given to the case where m ₁ and m ₂ each represent an integer of ₁ and n ₁ and n ₂ each represent an integer of 3.

The compound according to formula (I), its geometric isomer, a mixture of these geometric isomers and optical isomers thereof have a common salt-forming reaction with a basic substance or a basic compound desired from having a carboxyl group in the molecule A pharmaceutically acceptable salt can be formed by Such salts include, for example, alkali metal salts such as sodium salts, potassium salts and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts, amino acids such as lysine salts, ornithine salts and arginine salts Salt can be mentioned.

In the chemical structural formula of the formula (I), the double bond part in the medium chain carbon chain part in the astaxanthin basic skeleton can take on the structure of trans and cis geometric isomers in terms of chemical structure. With regard to the active ingredient according to the present invention, not only the trans form of formula (I) but also cis forms represented by the following formula (Ia) and formula (Ib) are effective in the osteoarthritis improving agent according to the present invention It can be mentioned as an ingredient. The osteoarthritis improving agent of the present invention also includes, as an active ingredient, a mixture of trans form of formula (I) and cis form which is its geometric isomer in various mixing ratios.

(Wherein, m ₁ , m ₂ , n ₁ and n ₂ have the same meaning as described above)

In addition, the compound of the formula (I), its geometric isomer and a mixture of these geometric isomers may include the optical isomer (IA) represented by the following, and its enantiomer and a mixture thereof, All diastereomers are also included as active ingredients of the osteoarthritis improving agent according to the present invention.

Among the compounds of the formula (I), their geometric isomers, mixtures of these geometric isomers and their optical isomers, the compounds of the trans form of the above-mentioned formula (IA) are preferred.
Further, among the trans compounds of the above formula (IA), compounds in which m ₁ and m ₂ each represent an integer of ₁ and n ₁ and n ₂ each represent an integer of 3 are preferable.

As described above, an optically active trans-astaxanthin derivative represented by the formula (IA) or a salt thereof is more preferable, and an optically active cis-astaxanthin derivative corresponding to the optically active trans-astaxanthin derivative represented by the formula (IA) More preferred is a highly pure optically active trans-astaxanthin derivative substantially free of a salt or a salt thereof. Here, containing the active ingredient of the osteoarthritis improving agent according to the present invention in "high purity" means that the purity in the active ingredient is at least 95% or more, preferably 98% or more.

Compounds of the formula (I) according to the present invention, geometric isomers thereof, optical isomers thereof and salts thereof can be produced by appropriately using the production method described in WO 2015/178404 and the same method as the known methods. It can manufacture by combining. Among these production methods, the geometric isomer of the compound of the formula (I) and the production method of the optical isomer thereof will be described below by using the production method of the optical isomer of the above formula (IA) as a representative.

(1A) Deprotection reaction

(Wherein, m ₁ , m ₂ , n ₁ and n ₂ have the same meanings as described above, and R means a protecting group)

The target optically active trans-astaxanthin derivative of the formula (IA) can be produced by removing the protecting group of the compound of the formula (II) which is a starting compound.

The elimination reaction may be a conventional elimination reaction of a protecting group, and specifically, an elimination reaction with an acid can be mentioned.
As a protecting group, a tertiary butyl group, a trimethylsilyl group, a tetrahydropyranyl group etc. can be mentioned, A tertiary butyl group, a trimethylsilyl group etc. can be mentioned as a suitable thing.

In the case of the elimination reaction with acid, the compound of formula (IA) can be produced by reacting the compound of formula (II) with an acid in an inert solvent.
The solvent used is not particularly limited as long as it is inert to the reaction, and examples thereof include aliphatic hydrocarbons such as hexane, heptane, ligroin and petroleum ether; aromatics such as benzene, toluene and xylene Hydrocarbons; Halogenated hydrocarbons such as chloroform, methylene chloride, 1,2-dichloroethane and carbon tetrachloride; Nitriles such as acetonitrile and propionitrile; ethyl formate, isopropyl formate, isobutyl formate, ethyl acetate, Organic acid esters such as isobutyl acetate and butyl acetate; ethers such as diethyl ether, diisopropyl ether, tetrahydrofuran, dioxane, dimethoxyethane, diethylene glycol dimethyl ether; dimethylformamide, dimethylacetamide, hexamethyl phosphate triamide Amides such as: alcohols such as methanol, ethanol, propanol and isopropanol; organic acids such as trifluoroacetic acid, formic acid, acetic acid, propionic acid; water; or mixed solvents of these solvents, Preferably, halogenated hydrocarbons, nitriles, ethers, alcohols, organic acids, amides, water, or a mixed solvent of these solvents, more preferably halogenated hydrocarbons, nitriles Alcohol, organic acids, ethers, water or a mixed solvent of these solvents, and most preferably halogenated hydrocarbons, acetonitrile, water, methanol, ethanol, isopropanol, formic acid, dioxane, tetrahydrofuran or water Mixed solvent of organic solvents with these organic solvents (protecting group is C1-C6 alkyl group If) can be mentioned.
The acid which can be used is not particularly limited as long as it is used as an acid in a usual reaction, and, for example, inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, perchloric acid and phosphoric acid; acetic acid, formic acid Organic acids such as boric acid, methanesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, trifluoroacetic acid, trifluoromethanesulfonic acid; zinc chloride, tin tetrachloride, boron trichloride, boron trifluoride, boron tribromide Or a acidic ion exchange resin, preferably an inorganic or organic acid, most preferably hydrochloric acid, acetic acid, formic acid and trifluoroacetic acid.
The reaction temperature varies depending on the raw material compound to be reacted, the acid used, the solvent and the like, but is usually −20 ° C. to 150 ° C., preferably 0 ° C. to 100 ° C. The reaction time varies depending on the raw material compound, the solvent, the reaction temperature and the like, but is usually 30 minutes to 10 days, and preferably 30 minutes to 5 days. The amount of the solvent used may be 10 to 50 times, preferably 30 times the volume of the weight of the compound of formula (II). The amount of the acid used is usually 5 to 50 times by mole, preferably 10 to 30 times by mole, as long as it is an inorganic acid relative to the compound of the formula (II) which is the raw material, an organic acid In this case, the molar amount is usually 100 to 1000 times, preferably 200 to 600 times.

The product obtained by the above deprotection reaction can contain geometric isomers such as the above 9-cis form and 13-cis form, so that separation and purification means such as column chromatography, reprecipitation and crystallization can be obtained. By combining as appropriate according to the purpose, the same geometric isomer can be separated and removed, and the objective optically active trans-astaxanthin derivative of the formula (IA) can be isolated and manufactured with high purity.
Further, the separated cis-form can be isolated and obtained by appropriately combining the purification and separation methods as described above.

(1B) Method of converting cis form to trans form

Representative cis-forms used in this production method are the compounds of the formulas (IAa) and (IAb) as described above, which may be used as sole raw material compounds, as a mixture of cis-forms, or in excess of cis-forms. The desired optically active trans-astaxanthin derivative of the formula (IA) can be produced by dissolving it in an inert solvent as a mixture with the trans form and reacting it with a conversion reagent such as iodine. .

The solvent to be used is not particularly limited as long as it is inert to the reaction, and examples thereof include tetrahydrofuran, ethyl acetate, acetonitrile, acetone, water and the like.
Preferred examples of the conversion reagent include iodine.
The reaction temperature varies depending on the raw material compound to be reacted, the conversion reagent to be used, the solvent and the like, but is usually −20 ° C. to 150 ° C., preferably 10 ° C. to 100 ° C. The reaction time varies depending on the raw material compound, the solvent, the reaction temperature and the like, but is usually 30 minutes to 10 days, and preferably 30 minutes to 5 days. The amount of the solvent used may be 10 to 50 times the used weight of the compound of formula (IAa) or (IAb), and preferably 30 times the volume. The amount of conversion reagent used may be usually 0.01 times or more by mole, preferably 0.1 times or more by mole, of the compound of the formula (IAa) or formula (IAb) as a raw material.

Among products obtained by the above conversion reaction, methods such as column chromatography, reprecipitation, crystallization and the like can be mentioned as methods for separating geometric isomers such as 9-cis and 13-cis. By combining these methods as appropriate depending on the purpose, the same geometric isomer can be separated, and the objective optically active trans-astaxanthin derivative of the formula (IA) can be isolated and produced in high purity.
In addition, the separated cis form can also be isolated and manufactured as each cis form by using the above separation means in combination as appropriate.

Next, a typical production method of the above starting compound (II) will be described below.

(2A) A method for directly binding the entire side chain moiety to 3S, 3'S-astaxanthin

(Wherein, m ₁ and n ₁ have the same meaning as described above, and R means a protecting group (eg, tertiary butyl group).)

A compound of formula (II) is obtained by dissolving 3S, 3'S-astaxanthin in an inert solvent and then reacting the compound of formula (III) corresponding to the side chain moiety in the compound of formula (I) in the presence of a condensing reagent. Can be manufactured.

Examples of the solvent include organic solvents such as methylene chloride, chloroform and carbon tetrachloride.
As the condensation reagent, those used in ordinary condensation reactions can be used, and as a specific example, water-soluble carbodiimide hydrochloride (eg, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) And N, N-diisopropylcarbodiimide, carbonyldiimidazole, dicyclohexylcarbodiimide and the like. The amount of the condensation reagent used is usually at least 2 times the molar amount, preferably 2.5 times to 20 times the molar amount of the raw material 3S, 3'S-astaxanthin.
The compound of the formula (III) corresponding to the side chain moiety may be used usually in a 2-fold molar amount or more, preferably 2.5-fold molar to 20-fold molar amount with respect to 3S, 3'S-astaxanthin.
The reaction temperature varies depending on the raw material compound to be reacted, the condensation reagent to be used, the solvent and the like, but is usually −20 ° C. to 150 ° C., preferably −10 ° C. to 100 ° C. The reaction time varies depending on the raw material compound, the solvent, the reaction temperature and the like, but is usually 30 minutes to 10 days, and preferably 30 minutes to 5 days. The amount of the solvent used is usually 10 to 50 times the used weight of 3S, 3'S-astaxanthin, preferably 30 times the volume.

The compound of the formula (II) obtained can be usually purified and isolated by appropriately combining purification means such as column chromatography, reprecipitation, recrystallization and the like.
In addition, the whole side chain part can be manufactured by the following method.

(2A-1)

The desired compound of formula (III) can be produced by sequentially reacting the compound of formula (IV) with the compound of carbonyldiimidazole (V) and the compound of formula (VII).
Specifically, the compound of formula (IV) is an intermediate compound of formula (VI) by reacting carbonyldiimidazole (V) in the presence or absence of a reagent such as a base in an inert solvent. Compounds can be obtained. Furthermore, the desired compound of formula (III) can be produced by reacting the compound of formula (VII) with trimethylsilyl chloride in the presence of a reagent such as a base and then reacting with the compound of formula (VI) .

In the step of obtaining the compound of the formula (VI), organic solvents such as chloroform and methylene chloride can be exemplified as the solvent, and the amount of these organic solvents used is usually 5 times the used weight of the compound of the formula (IV) A volume of 30 to 30 volumes, preferably 15 volumes, may be used.
As the basic reagent, those used for ordinary condensation reactions can be used, and specific examples can include triethylamine, N, N-diisopropylethylamine, pyridine, N, N-dimethylaminopyridine and the like. .
The reaction temperature varies depending on the raw material compound to be reacted, the reagent used, the solvent and the like, but is usually −20 ° C. to 150 ° C., preferably 0 ° C. to 30 ° C. The reaction time varies depending on the raw material compound, the solvent, the reaction temperature and the like, but is usually 15 minutes to 10 days, and preferably 30 minutes to 2 days can be mentioned.
In the step of obtaining the desired compound of formula (III), organic solvents such as chloroform, methylene chloride, pyridine and the like can be mentioned as solvents for reacting trimethylsilyl chloride and the compound of formula (VII). The amount used is usually 5 times to 50 times the volume, preferably 20 times the volume of the used weight of the compound of the formula (VII).
As the base, those used for ordinary condensation reactions can be used, and specific examples can include triethylamine, N, N-diisopropylethylamine, pyridine, N, N-dimethylaminopyridine and the like. The amount of the base or reagent used is usually at least 2 moles, preferably 2.5 to 5.0 moles, per mole of the compound of the formula (VI) as the raw material.
The reaction temperature varies depending on the raw material compound to be reacted, the reagent used, the solvent and the like, but is usually −20 ° C. to 100 ° C., and preferably 0 ° C. to 30 ° C. The reaction time varies depending on the raw material compound, the solvent, the reaction temperature and the like, but is usually 15 minutes to 5 days, and preferably 30 minutes to 2 days can be mentioned. Next, the compound of formula (VI) is added and reacted at a reaction temperature which varies depending on the starting compound to be reacted, the reagent to be used, the solvent and the like, but is usually -20 ° C to 150 ° C, preferably 10 ° C. To 60 ° C. The reaction time varies depending on the raw material compound, the solvent, the reaction temperature and the like, but is usually 30 minutes to 10 days, and preferably 30 minutes to 4 days.

(2B) Method of sequentially bonding parts of side chain portion to 3S, 3'S-astaxanthin

(Wherein, m ₁ , m ₂ , n ₁ and n ₂ have the same meaning as described above, and R means a protecting group (eg, tertiary butyl group or trimethylsilyl group).)

In this production method, the side chain part (VIII) obtained by reacting the compound represented by the general formula (VII) with carbonyldiimidazole (V) is bound to 3S, 3'S-astaxanthin, This can then be achieved by coupling part (XI) of the side chain part to the obtained product (IX).

In the step using carbonyldiimidazole (V), various reaction conditions shown in the production method of the above (2A-1) may be used similarly.
As the solvent, organic solvents such as chloroform and methylene chloride can be mentioned, and the amount of these organic solvents used is usually 2-fold to 30-fold volume with respect to the weight used of the compound of formula (VII). You can use 7 times capacity for
The reaction temperature varies depending on the raw material compound to be reacted, the reagent to be used, the solvent and the like, but is usually −20 ° C. to 150 ° C., preferably −10 ° C. to 100 ° C. The reaction time varies depending on the raw material compound, the solvent, the reaction temperature and the like, but is usually 30 minutes to 10 days, and preferably 30 minutes to 5 days. Examples of bases include triethylamine, N, N-diisopropylethylamine, pyridine, N, N-dimethylaminopyridine and the like.
The compound of the formula (IX) can be produced by reacting the resulting part of the side chain part (VIII) with 3S, 3'S-astaxanthin in the same manner as in the reaction of 2A above. .

The step of obtaining the target general formula (II) can be achieved by reacting the compound having the general formula (IX) obtained above with the general formula (XI). This reaction is carried out according to the method for producing the above general formula (VIII).
The reaction temperature varies depending on the raw material compound to be reacted, the reagent to be used, the solvent and the like, but is usually −20 ° C. to 100 ° C., preferably 0 ° C. to 40 ° C. The reaction time varies depending on the raw material compound, the solvent, the reaction temperature and the like, but is usually 30 minutes to 10 days, and preferably 30 minutes to 30 hours.
The method for producing a compound having the general formula (XI) can be achieved according to the method for synthesizing t-butyl ester of generally known amino acid when (1) R is a t-butyl group, (2) When R is a trimethylsilyl group, it can be achieved by reacting a compound having the general formula (X) with trimethylsilyl chloride in the presence of a base in an inert solvent (to form a compound of the general formula (III) It can be achieved according to the method). The reaction of (2) can be achieved according to generally known methods for silylation of hydroxyl and carboxyl groups. When R in the general formula (XI) is a trimethylsilyl group, the trimethylsilyl group can be easily eliminated by using water or weakly acidic water for post-treatment of the reaction to generate the general formula (II). .

The desired compound of the formula (II) can be produced by using the obtained product in combination with purification methods such as ordinary column chromatography, reprecipitation, recrystallization and the like as appropriate.

The compounds of the formula (I), their geometric isomers, mixtures of these geometric isomers, their optical isomers, as described in the examples below, improve the symptoms of osteoarthritis, in particular alleviating pain or excellent pain It has an analgesic effect.
Here, as described above, osteoarthritis develops in various joints such as the wrist, elbow, shoulder, neck, waist, hips, hips, knees, ankles, fingers and toes, and is effective for any osteoarthritis in the present invention. However, it is more effective for osteoarthritis of the knee, osteoarthritis of the hip, and osteoarthritis, especially for osteoarthritis of the knee and osteoarthritis, especially osteoarthritis of the knee. In addition, symptoms of osteoarthritis include pain, swelling, deformation of joints, muscle weakness and the like, and in the present invention, it is effective for the alleviation of all the symptoms, and is particularly excellent in the pain improvement effect.

The compounds of the formula (I) according to the present invention, their geometric isomers, mixtures of these geometric isomers, optical isomers thereof or salts thereof are generally used in medical fields and foods, such as injections, oral agents, in vivo placement, etc. Any method used in the field can be administered without particular limitation. The administration time may be before eating, after eating, between meals, before going to bed, or a combination of these. The number of daily doses is also not particularly limited.
The injection can be produced by a usual formulation technique, using an appropriate combination of a pharmaceutically acceptable osmotic pressure regulator, a stabilizer, a solubilizer, a pH regulator and the like.
The oral preparation may be administered in any form such as tablets, capsules, granules, powders and the like, and is appropriately mixed with pharmaceutically acceptable excipients, disintegrants, binders and other pharmaceutical additives, It can be produced by using conventional formulation techniques.
The compound of the formula (I) according to the present invention, its geometric isomer, a mixture of these geometric isomers, their optical isomers or a salt thereof may be administered as an injection, as in existing pharmaceuticals, It may be administered as an agent, may be administered as an external preparation such as a patch, an ointment and the like, and may be administered in a topical administration form.

The compound of the formula (I) according to the present invention, its geometric isomer, a mixture of these geometric isomers, their optical isomers or their salts may be mixed with other drugs or pharmaceutical additives and co-administered.
The drug used in combination is not particularly limited, and drugs that can be used generally as pharmaceuticals, such as hyaluronic acid, digestive aids, intestinal stabilizers, antiulcer agents, antibiotics, hormones, enzymes, steroids, endocrine drugs, cardiovascular agents, anti drugs Rheumatic agents, anti-inflammatory agents, analgesics, antipyretics, anti-allergic agents, antitumor agents, and further, peptides such as interferons, interleukins, tumor necrosis factors and the like may be used.
The pharmaceutical additive is also not particularly limited, and any one that can be used as a pharmaceutical can be used.

When a compound of the formula (I) according to the present invention, a geometric isomer thereof, a mixture of these geometric isomers, an optical isomer thereof or a salt thereof is administered as an injection, it is usually per day for adults And 5.0 mg to 50 mg may be administered into the joint cavity, and may be increased or decreased appropriately according to the symptoms.
The compounds of the formula (I) according to the present invention, their geometric isomers, mixtures of these geometric isomers, their optical isomers or salts thereof are particularly problematic in terms of safety within the above-mentioned dose range. Absent.

Hereinafter, examples of the present invention will be described.
However, the scope of the present invention is not limited to the following examples.
In this example, an analgesic test was conducted using a rat model in which osteoarthritis (OA) was induced using monoiodoacetic acid sodium salt (MIA).

(1) Model Preparation Crl: CD (SD) rats were prepared, and Japanese Pharmacopoeia isoflurane (manufactured by Mylan Pharmaceutical Co., Ltd.) was prepared as an inhalation anesthetic. Osteoarthritis (OA) was administered 50 μL (2 mg / site) of monoiodoacetic acid sodium salt (MIA, manufactured by Sigma-Aldrich) into the right knee joint cavity of a rat under 2% inhalation anesthesia with inhalation anesthetic. Induced.

(2) Administration solution preparation and administration As a test substance (compound X), a lysine salt of the optically active trans-astaxanthin derivative of the above formula (IA) (m ₁ and m ₂ are each an integer of ₁ , n ₁ and n ₂ are each An integer of 3, purity 97.6%) was used. Compound X was prepared as described in the following preparation example. The necessary amount of compound X was weighed, and physiological saline was added to make up to a predetermined amount, and the solutions adjusted to 0.1, 1 and 10 mg / mL were used as administration solutions. The final concentration of each dosing solution at the time of dosing will be 0.01, 0.1 and 1 mg / knee. Saline alone was administered to the control group (control group).
100 μL of each administration solution was injected into the knee joint cavity as an injection from 7 days after MIA administration twice a week for 4 weeks.

(3) Production Example of Compound X In the chemical structural formula in the following description, the geometric isomer in the medium chain carbon chain portion of the astaxanthin skeleton is conveniently represented by a chemical structure of all trans form.

(3.1) Synthesis of t-butyl 4- (imidazol-1-ylcarbonylamino) butyrate (in this chemical name, t means tertiary, hereinafter the same shall apply):

Methylene chloride (79.6 kg) is added to carbonyldiimidazole (9.95 kg) and stirred to give a solution of t-butyl 4-aminobutyric acid hydrochloride (8.0 kg) in methylene chloride (53.1 kg) At 5 ° C., the reaction mixture was stirred for 30 minutes at the same temperature. The mixture was warmed to 15-25 ° C. and stirred at the same temperature for 1 hour. To the reaction mixture, water (40 kg) was added and stirred, and the organic layer was separated. The resulting solution was washed with 5% brine (42.1 kg), dried over anhydrous magnesium sulfate and concentrated under reduced pressure to give the crude title product (10.2 kg of concentrated residue).
NMR spectrum (δ ppm, CDCl ₃ ): 8.22 (1 H, s), 7.92 (1 H, br), 7. 28 (1 H, d, J = 0.8 Hz) 7.05 (1 H, d, J = 0.8 Hz), 3.45 (2 H, dt, J = 6.0, 6.0 Hz), 2.40 (2 H, t, J = 6.4), 1.92 (2 H, tt, J = 6.4, 6.4 Hz), 1.44 (9 H, s).
Mass spectrum (+ ESI, m / z): 254.00 (M + H) ^<+> .

(3.2) Synthesis of t-butyl 4- (3-carboxymethylureido) butyrate:

Methylene chloride (185.7 kg), glycine (7.4 kg), triethylamine (8 kg), t-butyl 4- (imidazol-1-ylcarbonylamino) butyrate (the compound of the above (3.1)) (10.4 kg). 3 kg) was added and stirred, chlorotrimethylsilane (8.9 kg) was added at -5 to 5 ° C, and the reaction mixture was stirred at 15 to 30 ° C for 60 hours. The reaction mixture was concentrated under reduced pressure, a mixture of ethyl acetate (208 kg), hydrochloric acid (5.66 kg) and 20% brine (106 kg) was added and stirred, and the organic layer was separated. The resulting organic layer solution was washed with a mixture of hydrochloric acid (5.66 kg) and 20% brine (106 kg). The aqueous layer was mixed with ethyl acetate (57.4 kg), extracted with ethyl acetate, and the organic layer and the extract were combined. The resulting solution was washed with 20% brine (100 kg), dried over anhydrous magnesium sulfate and concentrated under reduced pressure. Ethyl acetate (14.4 kg) was added and stirred to a homogeneous solution at 45-55 ° C. The solution was cooled to 20-30 ° C., n-heptane (108.7 kg) was added dropwise, and after confirming the precipitation of crystals, the solution was stirred for 1 hour. The precipitated crystals were collected by filtration to give the title compound (7.98 kg, purity 99.2%) as white crystals.
The purity of the product is determined by high performance liquid chromatography (column: YMC-Triart C18 ExRS manufactured by YMC Co. and mobile phase: acetonitrile / pH 8 phosphate buffer = 3/7, flow rate: 1 mL / min, detection wavelength : 210 nm).
NMR spectrum (δ ppm, CDCl ₃ ): 6.16 (1 H, t, J = 5.6 Hz), 6.01 (1 H, t, J = 5.6 Hz), 3.67 (2 H, d, J = 6) .0 Hz), 2.97 (2 H, dt, J = 6.4, 6.4 Hz), 2.17 (2 H, t, J = 7.2 Hz), 1.56 (2 H, tt, J = 7. 2, 7.2 Hz), 1.39 (9 H, s).
Mass spectrum (+ ESI, m / z): 260.92 (M + H) ^<+> .

(3.3) 4- (3- {4- [18- (4 (S)-[3- (3-t-butoxycarbonylpropyl) ureidoacetoxy] -2,6,6-trimethyl-3-oxocyclohexene S-1-enyl) -3,7,12,16-tetramethyloctadeca-1 (E), 3 (E), 5 (E), 7 (E), 9 (E), 11 (E), 13 (E), 15 (E), 17 (E) -nonanil] -3,5,5-trimethyl-2-oxocyclohex-3-enyl-1 (S) -oxycarbonylmethyl} ureido) butyric acid t- Synthesis of butyl:

3 (S), 3 '(S) -astaxanthin (1.8 kg), t-butyl 4- (3-carboxymethylureido) butyrate (compound of the above (3.2)) (2.75 kg), N, N-Dimethyl-4-aminopyridine (2.95 kg) and methylene chloride (71.6 kg) were added and stirred to give a solution. To the solution was added 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (4.63 kg) at -5 to 5 ° C, and stirred at the same temperature for 4 hours. Water (3.6 kg) was added to the reaction mixture and stirred, and ethyl acetate (48.4 kg) was further added, stirred and concentrated under reduced pressure. Ethyl acetate (48.4 kg) and water (45 kg) were added to the concentrated residue, and the mixture was stirred, and the organic layer was separated. The obtained solution was treated three times with hydrochloric acid (0.3 M, 46.4 kg), 10% saline (45.9 kg), aqueous sodium hydrogen carbonate solution (about 7%, 48.2 kg), 20% saline (45 kg) The extract was successively washed with water, dried over anhydrous magnesium sulfate, and concentrated to dryness under reduced pressure to obtain the title compound (concentrated residue, 3.26 kg, purity 98.1%).
The purity of the product is determined by high performance liquid chromatography (column: YMC-TriartC18 ExRS manufactured by YMC Co., Ltd. and mobile phase: acetonitrile containing 0.025% trifluoroacetic acid / 0.025% trifluoroacetic acid water = 30 to 98 / It was determined using 70-2, flow rate: 1 mL / min, detection wavelength: 474 nm).
NMR spectrum (δ ppm, CDCl ₃ ): 6.18-6.72 (14 H, m), 5.56 (2 H, dd, J = 6.4, 13.2 Hz), 5.04 (2 H, t, J = 4.8 Hz), 4.81 (2 H, t, 5.7 Hz), 4. 25 (2 H, dd, J = 18.1, 6.6 Hz), 4.03 (2 H, dd, J = 18. 3, 4.6 Hz), 3.19-3.26 (4 H, m), 2. 29 (4 H, t, J = 7.3 Hz), 2.02-2.13 (4 H, m), 99 (12H, s), 1.90 (3H, s), 1.76 to 1.83 (4H, m), 1.44 (18H, s), 1.34 (6H, s), 1.23 (6H, s).
Mass spectrum (+ ESI, m / z): 1081.88 (M + H) ^<+> , 1103.67 (M + Na) ^<+> .

(3.4) 4- (3- {4 (S)-[18- (4- [3- (3-carboxypropyl) ureidoacetoxy] -2,6,6-trimethyl-3-oxocyclohexa-1 -Enyl) -3,7,12,16-tetramethyloctadeca-1 (E), 3 (E), 5 (E), 7 (E), 9 (E), 11 (E), 13 (E) ), 15 (E), 17 (E) -nonaniel] -3,5,5-trimethyl-2-oxocyclohex-3-enyl-1 (S) -oxycarbonylmethyl} ureido) butyric acid:

4- (3- {4- [18- (4 (S)-[3- (3-t-butoxycarbonylpropyl) ureidoacetoxy] -2,6,6-trimethyl-3-oxocyclohex-1-enyl ) -3,7,12,16-Tetramethyloctadeca-1 (E), 3 (E), 5 (E), 7 (E), 9 (E), 11 (E), 13 (E), 15 (E), 17 (E) -nonanyl] -3,5,5-trimethyl-2-oxocyclohex-3-enyl-1 (S) -oxycarbonylmethyl} ureido t-butyl butyrate (above (3) Formic acid (272 mL) was added to the compound of 3) (18.12 g) and stirred at 25 to 35 ° C. for 1 hour. The reaction mixture was added to water (1087 mL) and stirred, ethyl acetate (1087 mL) was added and stirred, and the organic layer was separated. The organic layer was washed twice with water (543 mL), twice with 10% brine (543.4 g), dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The concentrated residue was dissolved in tetrahydrofuran (81.1 mL) and water (8.11 mL), acetonitrile (486.8 mL) was added dropwise to the solution, and precipitation of a solid was confirmed, followed by stirring for 1 hour. The precipitated solid was collected by filtration and dried to give the title compound (4.48 g, purity 90.7%) as a dark purple to dark red solid.
The purity of the product is determined by high performance liquid chromatography (column: YMC-TriartC18 ExRS manufactured by YMC Co., Ltd. and mobile phase: acetonitrile containing 0.025% trifluoroacetic acid / 0.025% trifluoroacetic acid water = 30 to 98 / It was determined using 70-2, flow rate: 1 mL / min, detection wavelength: 474 nm).

(3.5) 4- (3- {4 (S)-[18- (4- [3- (3-carboxypropyl) ureidoacetoxy] -2,6,6-trimethyl-3-oxocyclohexa-1 -Enyl) -3,7,12,16-tetramethyloctadeca-1 (E), 3 (E), 5 (E), 7 (E), 9 (E), 11 (E), 13 (E) ), 15 (E), 17 (E) -nonaniel] -3,5,5-trimethyl-2-oxocyclohex-3-enyl-1 (S) -oxycarbonylmethyl} ureido) butyric acid:

4- (3- {4 (S)-[18- (4- [3- (3-carboxypropyl) ureidoacetoxy] -2,6,6-trimethyl-3-oxocyclohex-1-enyl) -3 , 7, 12, 16-tetramethyloctadeca-1 (E), 3 (E), 5 (E), 7 (E), 9 (E), 11 (E), 13 (E), 15 (E ), 17 (E) -nonaniel] -3,5,5-trimethyl-2-oxocyclohex-3-enyl-1 (S) -oxycarbonylmethyl} ureido) butyric acid (crude form of (3.4) above) Tetrahydrofuran (18.4 mL) and water (2.0 mL) were added to substance (4.0 g), and the mixture was stirred and dissolved. Acetonitrile (60 mL) was added dropwise to the solution, and after confirming precipitation of a solid, the mixture was stirred for 1 hour. The precipitated solid was collected by filtration and dried to give a dark purple to dark red solid (3.31 g, purity 96.9%). To the obtained solid (3.26 g), tetrahydrofuran (15.0 mL) and water (1.6 mL) were added and stirred to dissolve. Acetonitrile (49 mL) was added dropwise to the solution, and after confirming precipitation of a solid, the mixture was stirred for 1 hour. The precipitated solid was collected by filtration and dried to give a dark purple to dark red solid (2.93 g, purity 98.9%). To the obtained solid (2.38 g), tetrahydrofuran (10.9 mL) and water (1.2 mL) were added and stirred to dissolve. Acetonitrile (36 mL) was added dropwise to the solution, and after confirming precipitation of a solid, the mixture was stirred for 1 hour. The precipitated solid was collected by filtration and dried to give the title compound (2.18 g, purity 99.3%) as a dark purple to dark red solid.
The purity of the product is determined by high performance liquid chromatography (column: YMC-TriartC18 ExRS manufactured by YMC Co., Ltd. and mobile phase: acetonitrile containing 0.025% trifluoroacetic acid / 0.025% trifluoroacetic acid water = 30 to 98 / It was determined using 70-2, flow rate: 1 mL / min, detection wavelength: 474 nm).

(3.6) 4- (3- {4 (S)-[18- (4- [3- (3-carboxypropyl) ureidoacetoxy] -2,6,6-trimethyl-3-oxocyclohexa-1 -Enyl) -3,7,12,16-tetramethyloctadeca-1 (E), 3 (E), 5 (E), 7 (E), 9 (E), 11 (E), 13 (E) ), 15 (E), 17 (E) -nonanil] -3,5,5-trimethyl-2-oxocyclohex-3-enyl-1 (S) -oxycarbonylmethyl} ureido) butyric acid dilysine salt; Synthesis of X:
4- (3- {4 (S)-[18- (4- [3- (3-carboxypropyl) ureidoacetoxy] -2,6,6-trimethyl-3-oxocyclohex-1-enyl) -3 , 7, 12, 16-tetramethyloctadeca-1 (E), 3 (E), 5 (E), 7 (E), 9 (E), 11 (E), 13 (E), 15 (E ), 17 (E) -nonanil] -3,5,5-trimethyl-2-oxocyclohex-3-enyl-1 (S) -oxycarbonylmethyl} ureido) butyric acid (compound of the above (3.5)) To (0.50 g,), ethanol (10 mL) and water (0.5 mL) were added and stirred. To the suspension was added a solution of L-lysine monohydrate (0.174 g,) in water (2 mL) at room temperature. Water (7.5 mL) was added to the reaction mixture and stirred to dissolve. Ethanol (32 mL) was added dropwise to the reaction mixture at room temperature, and after confirming precipitation of a solid, the mixture was stirred for 1 hour. The precipitated solid was collected by filtration and dried to give the title compound (0.47 g, purity 98.6%, optical purity 99.0% de) as a dark purple to dark red solid.
The purity of the product was determined using high performance liquid chromatography as described above. The optical purity is high performance liquid chromatography (column: YMC, YMC CHIRAL ART Amylase SA (5 μm, 4.6 mm ID x 250 mm), column temperature: 25 ° C and mobile phase: THF / water / TFA (40:60) : 0.1), flow rate: 1 mL / min, detection wavelength: 474 nm, column retention time: 15.4 minutes (S, S), 17.6 minutes (meso), 20.6 minutes (R, R)) Used to determine.

(4) Pain measurement 1
(4.1) Weight measurement of left and right hind limbs Weight of left and right hind paws of test animals, once a week (administered twice weekly for the test substance: 3 days after the first administration for the second administration), 7 days After 14 days, 21 days, 28 days, it measured over 4 weeks. Incapacitance Tester (manufactured by Linton Instrument) was used as a measurement device. In the weight measurement, the animals were placed in a dedicated folder, and the left and right hind limbs were placed on weight meters equipped at two places, respectively, to perform measurement. And the ratio (weight load ratio) of the weight of the left foot and the weight of the right foot was calculated to evaluate the degree of pain.

(4.2) Weight measurement results of left and right hind limbs The results are shown in Table 1 and FIG.
The weight-bearing ratio was 0.60, 0.61, 0.58 and 0.61 before administration, 7 days, 14 days, 21 days and 28 days after administration of the control group. The weight-loading ratio of Compound X administration group before, 7, 14, 21, 21 and 28 days after administration was 0.60, 0.62, 0.60, 0.60 at a compound X concentration of 0.01 mg / knee. And the compound X concentration is 0.60, 0.63, 0.61, 0.63, and 0.64 at 0.1 mg / knee, and the compound X concentration is 0.60 at 1 mg / knee. , 0.61, 0.63, 0.65 and 0.66. Compound X concentrations of 0.1 mg / knee and 1 mg / knee Compound X administration groups showed significantly higher values compared with the control group after 14, 21, and 28 days.
In Table 1, each calculated value of weight-loading ratio indicates the average value ± standard error of 10 rats. In Table 1 and FIG. 1, "*" is P value <0.05 (P value between control group and compound X administration group is less than 5%), "**" is P value It means that it is <0.01 (P value between the control group and the compound X administration group is less than 1%), respectively.

(5) Pain measurement 2
(5.1) Measurement of pain threshold (mechanical stimulation) The pain threshold (g) of the right footpad was measured once per animal on the same day as the weight measurement day of the left and right hind limbs. A Dynamic Plantar Aesthesiometer (Cat. No. 37400, manufactured by Ugo Basile) was used as a measurement device. The mechanical stimulation conditions were set to 30.0 g maximum pressure and 40 seconds to reach the maximum pressure.
Statistical analysis performed Student's t-test for every measurement time point. In both cases, a risk ratio of less than 5% (P <0.05) was determined to be significant on both sides. All results are shown as mean ± standard error. As analysis software, EXSUS Version 7.7.1 (CAC Corporation, Inc.) using SAS 9.1.3 (SAS Institute Japan Co., Ltd.) was used.

(5.2) Measurement result of pain threshold (mechanical stimulation) The results are shown in Table 2 and FIG.
The pain thresholds of the control group were 11.3, 10.4, 10.2, 10.0 and 9.9 g before, 7, 14, 21 and 28 days after administration. The pain threshold values before administration, 7, 14, 21, 21 and 28 days of the compound X administration group are 11.3, 10.9, 11.0, 10.6 and 0.01 at a compound X concentration of 0.01 mg / knee. It is 10.5 g, the compound X concentration is 11.3, 11.0, 11.4, 11.8 and 12.1 in the 0.1 mg / knee group, and the compound X concentration is 11.4 in the 1 mg / knee. , 10.5, 11.1, 13.3 and 13.4. The compound X concentration showed significantly high values compared with the control group after 21 days and 28 days in the compound X administration groups at 0.1 mg / knee and 1 mg / knee.
In addition, in Table 2, each measured value of a pain threshold value has shown the average value +/- standard error of ten rats. In Table 2 and FIG. 2, "*" is P value <0.05 (P value between control group and compound X administration group is less than 5%), "**" is P value It means that it is <0.01 (P value between the control group and the compound X administration group is less than 1%), respectively.

(6) Summary It is as follows when the result of a present Example is put together.
Using the rat MIA-induced OA model, the effects of Compound X on weight-bearing ratio and pain threshold were examined. It was determined that a model animal was appropriately produced because all of the individuals showed reductions in weight-bearing ratio and pain threshold. Compound X showed a significant suppressive effect from 3 weeks after the start of administration (21 days after) to 4 weeks (after 28 days) against the reduction of the weight-loading ratio and the pain threshold observed in the above-mentioned pathological model.

Claims

An osteoarthritis improving agent containing a trans-astaxanthin derivative represented by the formula (I), a geometric isomer thereof, a mixture of these geometric isomers, an optical isomer thereof or a salt thereof.

(Wherein, m 1 , m 2 , n 1 and n 2 are the same or different and each represents an integer of 1 to 6).
The osteoarthritis improving agent according to claim 1, wherein m 1 and m 2 are each an integer of 1 and n 1 and n 2 are each an integer of 3 in formula (I).
The osteoarthritis improving agent according to claim 1 or 2, wherein the salt is a lysine salt.
The osteoarthritis improving agent according to any one of claims 1 to 3, wherein the trans-astaxanthin derivative represented by the formula (I) is an optically active trans-astaxanthin derivative represented by the formula (IA).

(Wherein, m 1 , m 2 , n 1 and n 2 are the same or different and mean an integer of 1 to 6).
Use of a trans-astaxanthin derivative represented by the formula (I), a geometric isomer thereof, a mixture of these geometric isomers, an optical isomer thereof or a salt thereof for producing an osteoarthritis improving agent.

(Wherein, m 1 , m 2 , n 1 and n 2 are the same or different and each represents an integer of 1 to 6).
The use according to claim 5, wherein in formula (I), m 1 and m 2 are each an integer of 1 and n 1 and n 2 are each an integer of 3.
The use according to claim 5 or 6, wherein the salt is a lysine salt.
The use according to any one of claims 5 to 7, wherein the trans-astaxanthin derivative represented by the formula (I) is an optically active trans-astaxanthin derivative represented by the formula (IA).

(Wherein, m 1 , m 2 , n 1 and n 2 are the same or different and mean an integer of 1 to 6).
A trans-astaxanthin derivative represented by the formula (I), its geometric isomer, a mixture of these geometric isomers, an optical isomer thereof or a salt thereof used for the improvement of osteoarthritis.

(Wherein, m 1 , m 2 , n 1 and n 2 are the same or different and each represents an integer of 1 to 6).
The compound according to claim 9, wherein m 1 and m 2 each is an integer of 1 and n 1 and n 2 each is an integer of 3, their geometric isomers, mixtures of these geometric isomers, Their optical isomers or their salts.
11. The compound according to claim 9, wherein the salt is a lysine salt, a geometric isomer thereof, a mixture of these geometric isomers or a salt of an optical isomer thereof.
The trans-astaxanthin derivative represented by the formula (I) is an optically active trans-astaxanthin derivative represented by the formula (IA), a geometric isomer thereof, a mixture of these geometric isomers or a salt thereof The compound or its salt of any one statement.

(Wherein, m 1 , m 2 , n 1 and n 2 are the same or different and mean an integer of 1 to 6).
Improvement of osteoarthritis characterized by administering an effective amount of the trans-astaxanthin derivative represented by the formula (I), its geometric isomer, a mixture of these geometric isomers, their optical isomers or their salts Method.

(Wherein, m 1 , m 2 , n 1 and n 2 are the same or different and each represents an integer of 1 to 6).
The method according to claim 13, wherein in the formula (I), m 1 and m 2 are each an integer of 1, and n 1 and n 2 are each an integer of 3.
The method according to claim 13 or 14, wherein the salt is a lysine salt.
The method according to any one of claims 13 to 15, wherein the trans-astaxanthin derivative represented by the formula (I) is an optically active trans-astaxanthin derivative represented by the formula (IA).

(Wherein, m 1 , m 2 , n 1 and n 2 are the same or different and mean an integer of 1 to 6).