JP2012516285A - Pharmaceutical composition - Google Patents

Abstract

〔式（１）または式（２）において、Ｒ１からＲ８は独立に水素または低級アルキル基、−ＣＨ２ＣＨ２ＯＣＨ３、−ＣＨ２ＣＯＲ９（Ｒ９＝ＣＨ３、Ｃ２Ｈ５、ＮＨ２）、−ＣＨ２−ＯＣＯＣ２Ｈ５を；Ｘは−Ｎ（Ｒ１０）−（Ｒ１０＝ＣＨ３、Ｃ２Ｈ５、Ｐｈ、−ＣＨ２Ｐｈ、−ＣＨ（Ｐｈ）２、−ＣＯＣＨ３、−ＣＯＯＣＨ３、−ＳＯ２ＣＨ３）、−ＣＨ２−、−ＣＨ（ＣＨ３）−、−ＣＨ（Ｃ２Ｈ５）−、−Ｏ−、−Ｓ−を；示す。但しＰｈはフェニル基である〕A new ultraviolet absorber, ultraviolet protective agent and / or melanin production inhibitor is provided. 2-substituted-6-alkyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] of the formula (1)
Pyrimidine compounds, or 2-substituted-7-alkyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine compounds of formula (2) or pharmaceutically acceptable salts thereof UV-absorbing agent, UV-protecting agent and / or melanin production inhibitor containing as an active ingredient.

[In Formula (1) or Formula (2), R1 to R8 are independently hydrogen or a lower alkyl group, —CH2CH2OCH3, —CH2COR9 (R9═CH3, C2H5, NH2), —CH2—OCOC2H5; X is —N ( R10)-(R10 = CH3, C2H5, Ph, -CH2Ph, -CH (Ph) 2, -COCH3, -COOCH3, -SO2CH3), -CH2-, -CH (CH3)-, -CH (C2H5)-, -O- and -S- are shown. Where Ph is a phenyl group]

Description

The present invention is a novel ultraviolet absorber, novel dermatological agent, cosmetic agent, specifically, protects the skin from UV damage, UV protective agent, UV protective agent, or skin stains, freckles, inflammation, etc. It is related with the inhibitor or regulator of the production system of melanin which is, or the inhibitor or regulator of the function / activity of skin cells such as melanocytes and keratinocytes.

Pigment abnormalities such as spots on the skin, freckle formation, pigmentation after sunburn, sunburn, etc., differ in degree depending on the constitution, but exposure of pigmented cells (melanocytes) present in skin tissue to sunlight, etc. It is an expression of the result of melanin synthesis by the body defense activity against. Blots, freckles, and pigmentation after sunburn are both easy and difficult to do. For those who are easy to do, it is a big problem, and it is necessary to incorporate effective UV protection methods against UV exposure. Yes.

Due to the destruction of the ozone layer in the stratosphere above by chlorofluorocarbons, etc., there is a concern that the number of cancer patients will increase due to genetic effects, carcinogenic effects, etc. due to exposure to stronger ultraviolet rays. Human skin is sensitive to ultraviolet rays with a wavelength of 280 to 320 nm, and especially ultraviolet rays in the region of 305 to 310 nm cause skin cancer. It has been argued that the destruction of the ozone layer that absorbs ultraviolet rays in this wavelength range increases the amount of ultraviolet light that falls on the ground, leading to an increase in skin cancer as described above. Developing drugs to treat has also become an important issue.

In modern times, skin aging such as wrinkle formation due to UV exposure, prevention and treatment of melanoma, skin cancer and cataracts, etc. For the purpose of improvement, there is a growing need for more effective and desirable UV protection agents and UV protection cosmetics. When UV absorbers, UV blockers, and UV scattering agents are collectively referred to as UV protective agents, substances such as titanium dioxides, zinc oxides, cinnamic acid derivatives, and benzophenone derivatives have been conventionally used. In addition, ascorbic acids, hydrogen peroxide, hydroquinones, arbutin, ferulic acid, kojic acid, glutathione, whitening crude drug for the purpose of avoiding, preventing or improving skin cosmetic problems such as pigment abnormalities and pigmentation Skin external preparations, so-called whitening agents, containing the above-mentioned extracts and the like have been produced as cosmetics and used daily.

The present inventor has a scientific interest in moles, which are benign tumors of melanocytes, and `` links between resistance to allergic diseases / sensitivity and number of moles '', that is, `` mole (formation ability) is an allergy such as hay fever. It has been reported that it is a phenotypic characteristic (predisposition) of disease resistance, etc., and prevention and treatment of disease onset and progression through activation of skin conditions such as melanin synthesis and metabolic system activation of melanocytes A method has been proposed (Non-Patent Documents 1 and 2 and Patent Document 1).

As an extension of the study, the present inventor has come up with the idea of investigating the biological activity of a group of synthetic pyrimidine compounds that have been studied by the present inventors as a UV protective agent and as a whitening agent. As shown in Patent Documents 2 to 7 and Non-Patent Documents 3 to 5, these synthetic pyrimidine compounds are originally applied to peripheral nerve disorders and central nerve disorders, which have nerve cell regeneration and repair effects. It is a therapeutic agent for neurological diseases, has an effect of improving and recovering learning memory, is a therapeutic agent that can be applied to brain diseases such as dementia, and is expected to be developed as a therapeutic agent for wound healing A compound.

JP 2004-203855 A Japanese Patent No. 2628707 (Japanese Patent Laid-Open No. 1-139572, WO87 / 04928) US Pat. No. 4,959,368 Japanese Patent No. 2825092 (JP-A-1-40483) Japanese Examined Patent Publication No. 7-51566 (Japanese Patent Laid-Open No. 1-40469) JP-A-9-295946 US Pat. No. 5,976,523

Akira Awaya et.al: Microbiol Immunol. 2003; 47 (1): 101-3. Akira Ashiya: Allergy and Immunity Vol.12, No.8, 1214-1217,2005. Awaya, A. et.al: Biol. Pharm. Bull. 16 (3), 248-253, 1993. Koyama Y, Awaya A. et.al: Biol. Pharm. Bull. 20 (7), 823-825, 1997. Akira Ashiya: Tissue culture engineering 28 (3), 106-111, 2002.

It is an object of the present invention to provide a useful ultraviolet absorber, ultraviolet protective agent and / or melanin production inhibitor having a new chemical structural formula that has not existed before.

〔式（１）または式（２）において、Ｒ１からＲ８は独立に水素または低級アルキル基、−ＣＨ２ＣＨ２ＯＣＨ３、−ＣＨ２ＣＯＲ９（Ｒ９＝ＣＨ３、Ｃ２Ｈ５、ＮＨ２）、−ＣＨ２−ＯＣＯＣ２Ｈ５を；Ｘは−Ｎ（Ｒ１０）−（Ｒ１０＝ＣＨ３、Ｃ２Ｈ５、Ｐｈ、−ＣＨ２Ｐｈ、−ＣＨ（Ｐｈ）２、−ＣＯＣＨ３、−ＣＯＯＣＨ３、−ＳＯ２ＣＨ３）、−ＣＨ２−、−ＣＨ（ＣＨ３）−、−ＣＨ（Ｃ２Ｈ５）−、−Ｏ−、−Ｓ−を；示す。但しＰｈはフェニル基である〕 For this purpose, the inventors of the present invention have newly disclosed the UV-protecting activity and melanin of the pyrimidine compounds of the formula (1) or the formula (2) or the pharmaceutically acceptable salts thereof previously found by the inventors. By screening production inhibitory activity and finding that the compound of the present invention has excellent biological activity as expected, the intended object was achieved and the present invention was completed. That is, the present invention provides an ultraviolet absorber, an ultraviolet protective agent and / or a melanin production inhibitor containing the pyrimidine compound of formula (1) or formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient. Is.

[In Formula (1) or Formula (2), R1 to R8 are independently hydrogen or a lower alkyl group, —CH2CH2OCH3, —CH2COR9 (R9═CH3, C2H5, NH2), —CH2—OCOC2H5; X is —N ( R10)-(R10 = CH3, C2H5, Ph, -CH2Ph, -CH (Ph) 2, -COCH3, -COOCH3, -SO2CH3), -CH2-, -CH (CH3)-, -CH (C2H5)-, -O- and -S- are shown. Where Ph is a phenyl group]

 The present invention provides an ultraviolet absorber, an ultraviolet protective agent and / or a melanin production inhibitor comprising the pyrimidine compound of the above formula (1) or formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient. .

Synthetic pyrimidine compounds that can be provided as these agents according to the present invention include 2-substituted-6-alkyl-5-oxo-5,6-dihydro compounds described in Patent Documents 2 to 7, Non-Patent Documents 3 to 5, and the like. 7H) pyrrolo [3,4-d] pyrimidine compounds and 2-substituted-7-alkyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine compounds or salts thereof. . More specifically, it is a compound represented by the above formula (1) or formula (2). [In Formula (1) or Formula (2), R1 to R8 are independently hydrogen or a lower alkyl group, —CH2CH2OCH3, —CH2COR9 (R9═CH3, C2H5, NH2), —CH2—OCOC2H5; X is —N ( R10)-(R10 = CH3, C2H5, Ph, -CH2Ph, -CH (Ph) 2, -COCH3, -COOCH3, -SO2CH3), -CH2-, -CH (CH3)-, -CH (C2H5)-, -O- and -S- are shown. Where Ph is a phenyl group]

(In Formula (1) or Formula (2), R1 to R8 are independently hydrogen or a lower alkyl group, CH3OCH2CH2-, -CH2CONH2, -COCH3, -COC2H5, -CH2OCOC2H5, X is> NH,> N-CH3, > N-C2H5,> N-ph,> N-CH2-ph,> N-CH-ph2,> N-COCH3,> N-COOC2H5,> N-SO2CH3,> CH2,> CHCH3,> CHC2H5, -O -, -S-, where ph represents a phenyl group.) The lower alkyl group is preferably a lower alkyl group having 1 to 7 carbon atoms.

Representative compounds of formula (1) are listed below. 2-piperazino-6-methyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine, 2- (4-methylpiperazino) -6-methyl-5-oxo-5,6- Dihydro (7H) pyrrolo [3,4-d] pyrimidine, 2- (4-ethylpiperazino) -6-methyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine, 2- Piperidino-6-methyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine, 2- (4-methylpiperidino) -6-methyl-5-oxo-5,6-dihydro ( 7H) pyrrolo [3,4-d] pyrimidine, 2- (4-ethylpiperidino) -6-methyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine, 2-morpholino- 6-Methyl-5-o So-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine, 2-thiomorpholino-6-methyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine 2-piperazino-6-ethyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine,

2-piperazino-6-isopropyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine, 2-piperazino-6-n-butyl-5-oxo-5,6-dihydro ( 7H) pyrrolo [3,4-d] pyrimidine, 2-piperazino-6-sec-butyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine, 2-piperazino-6 t-butyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine, 2-piperazino-4,6-dimethyl-5-oxo-5,6-dihydro (7H) pyrrolo [ 3,4-d] pyrimidine, 2-piperazino-6,7-dimethyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine, 2-piperazino-6,7,7- Trimethyl-5-oxo-5, -Dihydro (7H) pyrrolo [3,4-d] pyrimidine, 2-piperidino-4,6-dimethyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine, 2-piperidino -6,7,7-trimethyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine, 2-piperazino-7-methyl-6-ethyl-5-oxo-5,6 -Dihydro (7H) pyrrolo [3,4-d] pyrimidine, 2-piperazino-4-methyl-6-ethyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine.

Representative compounds of formula (2) are listed below. 2-piperazino-7-methyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine, 2- (4-methylpiperazino) -7-methyl-6-oxo-5,6- Dihydro (7H) pyrrolo [2,3-d] pyrimidine, 2- (4-ethylpiperazino) -7-methyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine, 2- (4-N-acetylpiperazino) -7-methyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine, 2-piperidino-7-methyl-6-oxo-5 , 6-Dihydro (7H) pyrrolo [2,3-d] pyrimidine, 2- (4-methylpiperidino) -7-methyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine , 2- (4-Ethylpiperidino) 7-methyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine, 2-morpholino-7-methyl-6-oxo-5,6-dihydro (7H) pyrrolo [2, 3-d] pyrimidine, 2-thiomorpholino-7-methyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine, 2-piperidino-7-ethyl-6-oxo-5 , 6-dihydro (7H) pyrrolo [2,3-d] pyrimidine, 2-piperidino-7-n-propyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine, 2 -Piperidino-7-isopropyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine,

2-piperidino-7-n-butyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine, 2-piperidino-7-t-butyl-6-oxo-5,6- Dihydro (7H) pyrrolo [2,3-d] pyrimidine, 2-piperidino-5-methyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine, 2-piperazino-5 Methyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine, 2-piperazino-4,7-dimethyl-6-oxo-5,6-dihydro (7H) pyrrolo [2, 3-d] pyrimidine, 2-piperidino-5,7-dimethyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine, 2-piperidino-5,5,7-trimethyl- 6-oxo-5,6-dihydro ( H) pyrrolo [2,3-d] pyrimidine, 2-piperazino-5,7-dimethyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine, 2-piperazino-5 5,7-trimethyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine, 2-piperidino-4methyl-7-ethyl-6-oxo-5,6-dihydro (7H ) Pyrrolo [2,3-d] pyrimidine, 2-piperidino-5-methyl-7-ethyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine.

The pyrimidine compounds of the present invention have been studied in various in vitro and in vivo experimental systems as described below, and have been shown to effectively act in the process of UV damage and have UV protection and / or melanin production-inhibiting activities. It was. That is, the protective effect against UVB of 280-320 nm is obtained by adding 0-500 mM of the present compound before or after UVB irradiation to human epidermal keratinocyte HaCaT cell culture medium and exposing UVB (50, 100 mJ / cm2). The cell viability after 12 and 24 hours after UVB exposure was evaluated by measuring the amount of LDH leakage outside the cell and detecting DNA fragmentation (TUNEL staining, agarose gel electrophoresis). In addition, the production amount of TNF-α, which is an inflammatory cytokine, was measured by ELISA. With the compound of the present invention, the cell viability decrease and the induction of apoptosis by UVB exposure were suppressed in a concentration-dependent manner, and UVB-induced TNF-α production was also significantly suppressed. Further, when the effect of the compound of the present invention on the proliferation of HaCaT cells was examined, it was found that the compound of the present invention generally increases cell proliferation.

On the other hand, screening for the melanin production inhibitory effect was performed in an experimental system in which mouse B16 melanoma cells were cultured for 72 hours in the presence of various concentrations of the compound of the present invention, mainly at 100 μM. Intracellular melanin production was evaluated by solubilizing melanin under alkaline conditions and measuring its absorbance. In addition, the influence of the compound of the present invention on the expression level of intracellular Mitf and tyrosinase involved in melanin production was examined by the Westernblot method. The compound of the present invention significantly suppressed the amount of melanin produced in B16 cells, and the compound of the present invention further reduced the expression levels of intracellular Mitf and tyrosinase. The cytotoxicity of the compound of the present invention against mouse B16 melanoma cells was examined by the MTT method. The compound of the present invention was generally low in toxicity and was a highly safe compound.

The back hair of mice, rats, guinea pigs, etc. was shaved and irradiated with ultraviolet rays, and the effects of the compound of the present invention on skin reactions such as erythema and edema (redness, eczema, rash) were observed with the naked eye, and the ultraviolet protective effect was examined. However, the effect in vivo was noticeable.

Thus, the pyrimidine compounds of the formulas (1) and (2) of the present invention have a UVB protective action and / or a melanin production inhibitory action through suppression of intracellular Mitf and tyrosinase expression. It was shown that it is useful as an ultraviolet protective agent and / or a melanin production inhibitor.

The pyrimidine compounds of formula (1) and formula (2) or pharmaceutically acceptable salts thereof are usually used in the form of pharmaceutical compositions and cosmetic preparations, and are percutaneous, subcutaneous, intramuscular, oral, intravenous, It is dosed by various routes such as intranasal, transbronchial, transpulmonary and rectal routes.

The pharmaceutical composition containing the UV protective agent and / or the melanin production inhibitor of the present invention, the cosmetic for freckles and freckles, and the anti-pigmentation cosmetic after sunburn and sunburn are represented by the formula (1) or the formula (2). In addition to the compounds of the present invention, pharmaceutically acceptable fillers, binders, lubricants, and moisturizers that are generally used in pharmaceutical compositions or cosmetics as long as the effects of the present invention are not impaired as necessary. It can be mixed with excipients, additives, diluents or carriers such as agents and disintegrants. Examples of these components include surfactants, antioxidants, thickeners, preservatives, moisturizers, surfactants, antiseptics, antioxidants, bactericides, fragrances, dyes, powder components, fats and oils, waxes, Hydrocarbon oils, higher fatty acids, higher alcohols, synthetic ester oils, silicones and the like can be blended.

The present invention includes pharmaceutical formulations containing a pharmaceutically acceptable carrier and a compound of formula (1) and formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient. Examples of the pharmaceutically acceptable salts of the compounds of formula (1) and formula (2) of the present invention include hydrochloride, hydrobromide, sulfate, bisulfate, phosphate, and acidic phosphoric acid. Salt, acetate, maleate, fumarate, succinate, lactate, tartrate, benzoate, citrate, gluconate, saccharide, methanesulfonate, p-toluenesulfonate , Salts formed from acids that form non-toxic acid addition salts with pharmaceutically acceptable anions such as naphthalene sulfonate, or hydrates thereof, and quaternary ammonium (or amine) salts, or their Hydrates.

Compositions of the present invention include, for example, ointments, sterile injections, emulsions, emulsions, suspensions, aerosols, molded pops, tablets, capsules, powders, granules, troches, cashiers, elixirs, syrups, soft and hard gelatin capsules , Alzet® pump capsules, pellets, suppositories and sterile packaging powders.
For oral administration, the compounds of formula (1) and formula (2) are mixed with a carrier or diluent and prepared in the form of tablets, capsules and the like. For parenteral administration, the active ingredient is dissolved in 10% aqueous glucose solution, isotonic saline, sterile water, or a similar liquid and placed in a vial or ampoule to be administered intravenously by infusion or injection, or by intramuscular injection. Sealed. Advantageously, solubilizers, local anesthetics, preservatives and buffering agents can also be included in the medium. To increase the stability, the composition can be lyophilized after injection into a vial or ampoule. Other preparations for parenteral administration include preparations administered transdermally such as ointments and poultices. In this case, a molded patch or tape is advantageous. In addition, there are pellets and alzet (registered trademark) pump capsules that are directly embedded in damaged regions, tissues, bones, and the like. Examples of the resin used for pellet production include poly (2-hydroxyethyl methacrylate) and ethylene vinyl acetate copolymer. The composition can also be prepared so that the active ingredient is released rapidly, sustained or delayed after dosing to the patient.

Examples of pharmaceutically acceptable carriers (or diluents) are lactose, glucose, sucrose, sorbitol, mannitol, corn starch, crystalline cellulose, gum arabic, calcium phosphate, alginate, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone Tragacanth gum, gelatin, syrup, methylcellulose, carboxymethylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, inert polymers, water or mineral oil.

The compositions generally contain from 0.1 to 2000 mg, preferably from 0.5 to 1000 mg of active ingredient per unit dosage form. The compounds of formula (1) and formula (2) are effective over a wide dosage range. For example, the daily dosage is usually in the range of 0.03 mg / kg to 100 mg / kg. The amount of compound actually administered will be determined by the physician depending on the compound being administered and on the age, weight, response, severity of symptoms, route of administration, etc. of the individual patient. Accordingly, the above dosage ranges do not limit the scope of the invention. The preferred number of doses per day is usually 1-6 times, preferably 1-4 times. Further, the compound of the present invention is expected as a novel ultraviolet protective agent having a whitening effect, and is well known in the art as a skin external preparation or cosmetic preparation in the form of cream, lotion, ointment, pack, powder or skin lotion, emulsion, etc. Prepared by the preparation method. The preferred number of applications per day may be in accordance with the pharmaceutical composition.

The compounds of formulas (1) and (2) are effective UV protection agents and / or melanin production inhibitors per se, but may be administered in combination with one or more other synergists if necessary. It can also be applied. Examples of such additional drugs include UV protection agents and melanin production inhibitors as described above.
Since the present inventors have already filed patent applications and published papers for the preparation examples of the compounds of formula (1) and formula (2) used in the therapeutic agent of the present invention, we would like to refer to them (Patent Documents 2 to 2). 7 and Non-Patent Documents 3 and 4).
Hereinafter, the present invention will be described in more detail with reference to examples and experimental examples, but the present invention is not limited thereto.

Example 1
A tablet containing 10 mg of the active ingredient is produced as follows.
Per tablet
Active ingredient 10mg
Corn starch 55mg
Crystalline cellulose 35mg
Polyvinylpyrrolidone (as 10% aqueous solution) 5mg
Carboxymethylcellulose calcium 10mg
Magnesium stearate 4mg
Talc 1mg
───────────────────────────────
120mg total
The active ingredient, starch and crystalline cellulose are passed through an 80 mesh screen and mixed thoroughly. The resulting powder is mixed with a polyvinylpyrrolidone solution and granulated, and then passed through an 18-mesh sieve. The granules thus produced are dried at 50-60 ° C. and sized again with an 18 mesh screen. Carboxymethylcellulose calcium and magnesium stearate and talc, previously screened through an 80 mesh screen, are added to the granules, mixed, and then tableted into tablets weighing 120 mg each.

Example 2
A tablet containing 200 mg of the active ingredient is produced as follows.
Per tablet
Active ingredient 200mg
Corn starch 50mg
Crystalline cellulose 42mg
Light anhydrous silicic acid 7mg
Magnesium stearate 1mg
───────────────────────────────
300mg total
The ingredients are passed through an 80 mesh screen and mixed thoroughly. The obtained powder is compression-molded to produce a tablet having a weight of 300 mg.

Example 3
Capsules containing 100 mg of active ingredient are produced as follows.
Per capsule
Active ingredient 100mg
Corn starch 40mg
Lactose 5mg
Magnesium stearate 5mg
───────────────────────────────
150mg total
Mix the above ingredients and pass through 80 mesh sieve and mix thoroughly. 150 mg of the obtained powder is filled into a capsule.

Example 4
A vial-in-solution injection containing 5 mg of the active ingredient is produced as follows.
Per vial
Active ingredient 5mg
Mannitol 50mg
At the time of use, dissolve and use 1 ml of distilled water for injection.

Example 5
An ampoule-containing injection containing 50 mg of the active ingredient is produced as follows.
Per ampoule
Active ingredient 50mg
Sodium chloride 18mg
Distilled water for injection ───────────────────────────────
2ml total

Example 6
An adhesive patch preparation containing 17.5 mg of the active ingredient is produced as follows. 10 parts of ammonium polyacrylate are dissolved in 60 parts of water. On the other hand, 2 parts of glycerin diglycidyl ether is dissolved in 10 parts of water while heating. On the other hand, 10 parts of polyethylene glycol (grade 400), 10 parts of water, and 1 part of active ingredient are stirred and dissolved. Then, while stirring the aqueous solution of ammonium polyacrylate, the aqueous solution of drug-containing gel containing an aqueous solution of glycerin diglycidyl ether and an active ingredient of polyethylene glycol was added and mixed into a flexible plastic film with an active ingredient per square centimeter. It was applied to 0.5 mg, and the surface was covered with release paper and cut into 35 square centimeters to prepare a preparation.

Example 7
An adhesive patch preparation containing 10 mg of the active ingredient is produced as follows. A mixed aqueous sol solution of 100 parts of sodium polyacrylate, 100 parts of glycerin, 150 parts of water, 0.2 part of triepoxypropyl isocyanurate, 100 parts of ethanol, 25 parts of isopropyl myristate, 25 parts of propylene glycol and 15 parts of active ingredient Prepared. Next, this sol solution was applied to a nonwoven fabric surface of a composite film composed of a rayon nonwoven fabric and a polyethylene film in a thickness of 100 μm to form a drug-containing pressure-sensitive adhesive layer. The content of release auxiliary substances (isopropyl myristate and propylene glycol) contained in this layer was about 20% by weight. Thereafter, it was cross-linked at 25 ° C. for 24 hours, a release film was bonded to the pressure-sensitive adhesive interface, and this was further cut into 35 square centimeters to obtain a preparation.

Example 8
Coarse sandy crystals of poly (2-hydroxyethyl methacrylate) (Polysciences, Inc.) were placed in a mortar and finely pulverized aseptically in a clean bench using a pestle. 10 mg of this powder was taken in a sterile petri dish, 10 μl of 99% ethyl alcohol and 10 μl of an aqueous solution containing 0.5 mg of the active ingredient were added, stirred well to form a paste-like lump, and then dried.

Example 9
10% by weight of glyceryl monostearate, 10% by weight of liquid paraffin, 2% by weight of POE (30) cetyl ether, 5% by weight of cetyl alcohol, 4% by weight of petrolatum and γ-tocopherol used as a base component for the preparation of oil-in-water cream 1% by weight of the active ingredient was added and the ingredients were mixed and heated to 80 ° C. On the other hand, 56% by weight of purified water and 10% by weight of 1,3-butylene glycol were mixed and heated to 80 ° C. Both mixtures were mixed and stirred to emulsify, then cooled to 35 ° C. to produce an oil-in-water cream.

Example 10
Using the same various components as in Example 9, a transdermal preparation was produced by applying the Solid in Oil technique in which a hydrophilic compound was dissolved in an oily base in a solid state and encapsulated in an oil phase.

Example 11
Effects of compounds of the present invention on HaCaT cells exposed to ultraviolet light
HaB cells were irradiated on HaCaT cells cultured in 40 mm dish in DMEM medium. Various concentrations of the compound of the present invention were added before and after UVB irradiation of 50 to 100 mJ / cm 2, and after incubation for 24 hours, the LDH (lactate dehydrogenase) content of the supernatant was measured. As a control, the LDH content of the supernatant after culturing of cells not irradiated with UVB and not added with the compound of the present invention was used. Based on the LDH content, the cell viability% was determined, and the effects of the compound of the present invention are shown below. The significant difference with respect to the cell viability of the UVB irradiation only group is shown in parentheses. The cell survival rate of the untreated group was 91.39 ± 0.32%. The cell survival rate of the UVB50mJ / cm2 irradiation only group was 59.91 ± 2.50%, and the cell survival rate of the UVB100mJ / cm2 irradiation only group was 52.19 ± 2.07%.

When 2-piperazino-6-methyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine maleate is added, 50 mJ / cm 2 before UVB irradiation is added; They were 66.34 ± 1.72% (p <0.05), 76.55 ± 2.76% (p <0.005), and 83.31 ± 0.54% (p <0.005), respectively. In the case of addition after 50 mJ / cm 2 UVB irradiation, the values were 64.35 ± 3.29%, 66.61 ± 3.44%, and 70.81 ± 2.67% (p <0.01), respectively. When added to 100 mJ / cm2 before UVB irradiation, they were 57.59 ± 1.35% (p <0.05), 61.62 ± 4.71% (p <0.005), and 71.89 ± 1.09% (p <0.005), respectively. . In the case of addition after 100 mJ / cm 2 UVB irradiation, they were 49.93 ± 4.99%, 59.43 ± 1.97% (p <0.01), and 55.07 ± 3.08%, respectively. Thus, it was clarified that cell death by UVB is significantly suppressed by the compound of the present invention in a concentration-dependent manner.

Example 12
Effects of the compounds of the present invention on cell chromatin aggregation and DNA fragmentation by UVB irradiation of HaCaT cells
HaCaT cell culture was subjected to 500 μM 2-piperazino-6-methyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine maleate prior to 50-100 mJ / cm 2 UVB irradiation. The cell viability after 12 hours counted about 250 cells, but the UVB-irradiated group dropped to 70% compared to the untreated group, while the almost untreated group It recovered to the extent (p <0.005). In addition, the addition of the compound of the present invention inhibits the aggregation of chromatin after 12 hours due to UVB irradiation by staining cell nuclei with Hoechst33342, which was shown by fluorescence microscope observation of apoptotic cell-specific nuclear morphological changes. . On the other hand, it was shown by fluorescence microscope observation by Tunel staining that UV irradiation DNA fragmentation of HaCaT cells was suppressed by the addition of the compound of the present invention.
Further, UV-irradiated DNA fragmentation can be achieved by subjecting the extracted DNA fragment after cell lysis to agarose gel electrophoresis and detecting the fluorescence of DNA by Ethidium bromide staining using a UV transilluminator. It was clarified that the addition was suppressed.

Example 13
Effect of the compound of the present invention on TNF-α production by UVB irradiation to HaCaT cells
When TNF-α content of the culture supernatant of HaCaT cells was measured using a TNF-α-specific ELISA measurement system, the amount of TNF-α after UVB irradiation at 50 mJ / cm2 was 3.62 ± 0.37 pg / ml The amount of TNF-α after 24 hours of UVB irradiation at 100 mJ / cm 2 was 4.98 ± 0.31 pg / ml, and the untreated group was 2.67 ± 0.47 pg / ml. When 2-piperazino-6-methyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine maleate was added before 50 mJ / cm 2 irradiation, 50, 250, 500 μM, TNF-α amount is 2.02 ± 0.88pg / ml, 1.31 ± 0.57pg / ml
ml (p <0.01) 0.61 ± 0.57 pg / ml (p <0.005). The values in parentheses are p-values for a significant difference test with the 50 mJ / cm 2 UVB irradiation group. When 2-piperazino-6-methyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine maleate was added before 100 mJ / cm 2 irradiation, 50, 250, 500 μM, The amounts of TNF-α were 4.21 ± 0.27 pg / ml (p <0.05), 3.00 ± 0.80 pg / ml (p <0.05), and 2.67 ± 0.47 pg / ml (p <0.005). The values in parentheses are p-values for a significant difference test with the 100 mJ / cm 2 UVB irradiation group. It was revealed that UVB-induced TNF-α production was also significantly suppressed by the addition of the compound of the present invention.

Example 14
50 μM, 250 μM and 500 μM 2-piperazino-6-methyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine maleate was added to the HaCaT cell culture and cultured for 6 hours. After adding 1 μCi / dish of 3H-thymidine and examining the effect of the compound of the present invention on cell proliferation 18 hours later, it was found that cell proliferation was increased in a concentration-dependent manner.

Example 15
Effects of the compounds of the present invention on melanin production in B16 melanoma cells
B16 melanoma cells are spread in a 6-well plate at 1 x 105 cells / well in D-MEM medium, cultured for 24 hours, replaced with fresh medium, and at this time, the compound of the present invention dissolved in PBS is added to make a total volume of 2 ml. (1.8 ml of medium + 200 μl of the present compound / PBS). Subsequently, after culturing for 72 hours, the cells were washed, EDTA 0.025% was added at 600 μl / well, and the cells were peeled off and collected in a 1.5 ml tube. The tube was then centrifuged at 700 g for 5 minutes. Then, leaving about 50 μl containing cells, the supernatant was discarded, and 100 μl / tube of 1N NaOH was added. This was kept at 80 ° C. for 1 hour. Then, it was transferred to a well plate at 100 μl / well, and the absorbance was measured at a wavelength of 405 nm. When 100 μM 2-piperazino-6-methyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine maleate is added, the amount of melanin produced after protein amount correction is Compared to the control, it was 62.7% (P <0.005), and the melanin production inhibitory action of the compound of the present invention was remarkable.

Example 16
Effects of the compounds of the present invention on the expression levels of Mitf and tyrosinase in B16 melanoma cells
After lysing B16 cells cultured at about 80% confluence in a 60 mm dish, the effect of addition of the compound of the present invention on the expression level of intracellular Mitf and tyrosinase after 72 hours was examined by Western blot. , 50 μM, 100 μM 2-piperazino-6-methyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine maleate in a concentration-dependent manner, Mitf and tyrosinase The result which decreased the expression level of was obtained.

Example 17
The cytotoxicity of the compounds of the present invention against B16 melanoma cells was measured by MTT assay. When 2-piperazino-6-methyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine maleate is added and incubated for 72 hours, in the concentration range of 5-100 μM The cell viability was almost 90%, indicating that the substance is weakly toxic.

Example 18
Using 5 to 8 week-old ICR male mice, the hair on the back of the mice was shaved with a clipper, and then 20 μl of 100 μM, 250 μM to 500 μM 2-piperazino-6-methyl- 5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine maleate, 2- (4-methylpiperazino) -6-methyl-5-oxo-5,6-dihydro (7H) pyrrolo [3,4-d] pyrimidine maleate, 2-piperazino-7-methyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine maleate, 2-piperidino-7 -Methyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine maleate, 2- (4-ethylpiperidino) -7-methyl-6-oxo-5,6-dihydro ( 7H) Pirolo [2, -d] was applied compound pyrimidine maleate salt present invention. The coated part was dried with cold air using a dryer, and then irradiated with UVB ultraviolet rays having an irradiation intensity of 50 mJ / cm2. When the compound of the present invention was observed every day for 2 weeks after application, abnormal skin reactions such as redness, eczema and rash were not detected as compared with the non-application area. In addition, the body weight gain followed the same increasing trend as in the control group mice, and no abnormal findings were observed.

Example 19
UV absorption spectrum: 50 μM of each aqueous solution of the compound of the present invention was placed in a quartz cell (optical path length: 1 cm), and UV absorption was measured with an ultraviolet spectrophotometer. 2-piperazino-6-methyl-5-oxo-5 , 6-Dihydro (7H) pyrrolo [3,4-d] pyrimidine maleate has a maximum at 272.6 nm and is 2-piperazino-7-methyl-6-oxo-5,6-dihydro (7H) pyrrolo [2,3-d] pyrimidine maleate was shown to have a maximum at 301.0 nm.

Example 20
Glyceryl trioctanoate 5%, squalane 5%, glycerin 5%, liquid lanolin 3%, stearyl alcohol 2%, trimethylolpropane trioctanoate 2%, glyceryl monooleate 2%, polyoxyethylene monostearate (20 mol) sorbitan 1%, 1% triethanolamine, 0.2% carboxyvinyl polymer, 0.1% p-aminobenzoic acid, 0.1% vitamin C were mixed with 1% active ingredient in a weight ratio.
Further, a milky lotion was prepared by adding a small amount of perfume, a small amount of sodium bisulfate, a small amount of preservative, and the remaining distilled water.

Experimental example 1
The compounds of the present invention were orally administered to male ddy-type 5-week-old mice and male Wistar-type 8-week-old rats, and toxicity was determined after 24 hours. Each compound had an LD50 value of> 1000 mg / kg or less in mice. It was 550 to 1000 mg / kg, and it was also> 1700 mg / kg to 550 to 1000 mg / kg in rats, and it was generally considered to be a highly safe drug with low toxicity. The individual values are described in the specification of the patent application already filed or registered by the present inventors.

As described above, the compounds found by the present invention are excellent in use as ultraviolet absorbers, ultraviolet protective agents and / or melanin production inhibitors, and are highly safe pharmaceutical compositions, stains and freckles cosmetics, sunscreens, and the like. It is useful as an anti-pigmentation cosmetic after sunburn, and as a beautifying cosmetic. It is considered to be effective in preventing and suppressing the onset of malignant tumors such as melanoma and skin cancer, skin inflammation, skin wrinkle formation, and cataracts, which are considered to be UV damage.

Claims (1)

UV absorbers, UV protection agents and cosmetics comprising pyrimidine compounds for UV protection of formula (1) or formula (2).

[In Formula (1) or Formula (2), R1 to R8 are independently hydrogen or a lower alkyl group, —CH2CH2OCH3, —CH2COR9 (R9═CH3, C2H5, NH2), —CH2—OCOC2H5; X is —N ( R10)-(R10 = CH3, C2H5, Ph, -CH2Ph, -CH (Ph) 2, -COCH3, -COOCH3, -SO2CH3), -CH2-, -CH (CH3)-, -CH (C2H5)-, -O- and -S- are shown. Where Ph is a phenyl group]