WO2010087527A1 - Absorbeurs ultraviolets et compositions pharmaceutiques et produits cosmétiques contenant des composés pyridines pour protection contre les uv - Google Patents

Absorbeurs ultraviolets et compositions pharmaceutiques et produits cosmétiques contenant des composés pyridines pour protection contre les uv Download PDF

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WO2010087527A1
WO2010087527A1 PCT/JP2010/051725 JP2010051725W WO2010087527A1 WO 2010087527 A1 WO2010087527 A1 WO 2010087527A1 JP 2010051725 W JP2010051725 W JP 2010051725W WO 2010087527 A1 WO2010087527 A1 WO 2010087527A1
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pyrro
dihydro
oxo
pyrimidine
compounds
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PCT/JP2010/051725
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English (en)
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Akira Awaya
Shuji Kojima
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Akira Awaya
Shuji Kojima
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Priority to JP2011531691A priority Critical patent/JP2012516285A/ja
Publication of WO2010087527A1 publication Critical patent/WO2010087527A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/494—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom
    • A61K8/4953—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with more than one nitrogen as the only hetero atom containing pyrimidine ring derivatives, e.g. minoxidil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/16—Emollients or protectives, e.g. against radiation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/12—Ophthalmic agents for cataracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations

Definitions

  • the prsent invention relates to new ultraviolet absorbers, new agents for dermatological field and for cosmetics, for details agents for ultraviolet(UV) light protection and agents for protection of UV-induced disorders, to protect skin from disorders caused by UV rays, or inhibitors or modulators of melanogenesis from which pigmented spots, freckles and inflammations and the like are originated, or inhibitors or modulators of function and activity of epidermal cells such as melanocytes and keratinocytes.etc.
  • UV-protective cosmetics are growing more and more for the purpose of from the field of prevention and treatment of skin aging such as wrinkle formation and the like, and developing of melanomas and skin cancer, and cataractogenesis and the like caused by UV exposure, and for the purpose of the field of improvement of cosmetic troubles in head and neck, and no clothes-wearing portion of body and exposed area of skin in the body.
  • UV absorber, UV blocker and UV-scattering agent are as a whole collectively called as UV potector, substances such as titanium dioxide and the like, zinc oxide and the like, cinnamic acid derivatives and the like, benzophenone derivative and the like are used so far.
  • whitening agents For the purpose of avoiding and preventing, or improving cosmetic troubles such as abnormality in the formation of pigment in skin and pigmentation, topical products, so called whitening agents have been produced as cosmetics and routinely used, containing combination of ascorbic acid and the like, hydrogen peroxide, hydroquinone and the like, arbutin, ferulic acid, kojic acid, glutathione and the like and whitening extracts of herb and the like.
  • the inventor has a scientific interest in melanocyte nevi which are benign tumor cells of melanocytes, and he has released report on the link between allergy resistance or allergy sensitivity, and more or less nevi, so that nevi (forming activity) is a phenotype (cause) of resistance to allergic diseases including pollinosis, and other reports.
  • the inventor has proposed how to protect and treat developing and progress of diseases through activating the skin condition such as activation of melanin-synthesizing metabolisms and so (non-patent literature 1 ⁇ non-patent literature 2, and patent literature 1 ).
  • Patent literature 2 Japanese Patent2,628,707(Tokkaihei 1 — 139,572. WO87/04928)
  • Patent literature 4 Japanese Patent2,825,092(Tokkaihei 1 —40,483)
  • Patent literature 5 Tokkouhei 7 — 51,566(Tokkaihei1 -40,469)
  • the present invention relates to providing useful UV absorbers, UV protectors and/or melanogenesis inhibitors and whitening agents that have nonconventionally new chemical structures.
  • the present inventor began to exercise efforts for the purpose of screening newly UV absorbing action, UV protective activity and melanogenesis inhibiting activity of formula (1) or (2) of pyrimidine compounds or pharmaceutically acceptable salts thereof, and reached finding their superior biological activity, as expectedly.
  • the present invention has been completed based on such findings.
  • the present inventor provides UV absorbers, UV protectors and melanogenesis inhibitors containing formula (1) or (2) of pyrimidine compounds or pharmaceutically acceptable salts thereof, as active ingredients.
  • the present inventor provides UV absorbers, UV protectors and/or melanogenesis inhibitors containing above described formula (1 ) or (2) of pyrimidine compounds or pharmaceutically acceptable salts thereof, as active ingredients.
  • Synthetic pyrimidine compounds which can be provided as UV absorbers, UV protectors and/or melanogenesis inhibitors by the present invention include 2-substituted-6-alkyl-5-oxo-5,6-dihydro-(7H)pyrro[3,4-d]pyrimidine compounds, and 2-substituted-7-alkyl-6-oxo-5,6-dihydro-(7H)pyrro[2,3-d]pyrimidine compounds and salts thereof, which are shown by formula (1 ) or (2) .
  • R 1 to R 8 independently represent a hydrogen atom, a lower alkyl (especially C 1 -C 7alkyl) group, CH 3 OCH 2 CH 2 -, -CH 2 CONH 2 , -COCH 3 , -COC 2 H 5 or -CH 2 OCOC 2 H 5 , and X represents >NH, >N-CH 3 , >N-C 2 H 5 , >N-ph,>N-CH 2 -ph, >N-CH-ph 2 , >N-COCH 3 , >N-COOC 2 H 5 , >N ⁇ SO 2 CH 3 , >CH 2 , >CHCH 3 , >CHC 2 H 5 , -O- or -S- in which ph stands for a phenyl group.
  • Typical illustrative compounds of formula (1 ) include: 2-Piperazino-6-methyl-5-oxo-5,6-dihydro(7H)pyrro[3,4-d]pyrimidine, 2-(4-Methylpiperazino-6-methyl-5-oxo-5,6-dihydro(7H)pyrro[3,4 -d]pyrimidine, 2-(4-Ethylpiperazino-6-methyl-5-oxo-5,6-dihydro(7H)pyrro[3,4-d]pyrimidine, 2-Piperidino-6-methyl-5-oxo-5,6-dihydro(7H)pyrro[3,4-d]pyrimidine, 2-(4-Methylpiperidino)-6-methyl-5-oxo-5,6-dihydro(7H)pyrro[3,4-d]pyrimidine, 2-(4-Ethylpiperidino)-6-methyl-5-oxo-5,
  • Typical illustrative compounds of formula (2) include:
  • the present invented pyrimidine compounds were examined in various in vitro and in vivo experimental systems described below. It was clarified that those compounds had L ) V protective and/or melanogenesis inhibiting activity, through acting effectively on the process of UV disorders. That is, the protective effects of the present invented compounds on 280-320nm of UVB-irradiation were evaluated in the human epidermal keratinocyte
  • HaCaT cultures with the addition of 0-50OmM of those compounds before or after 50 or 100 mJ/cm2 of UVB irradiation.
  • Cell viability was observed 12 and 24 hrs after UVB exposure by means of assay of extracellular LDH leakage, and detection of DNA fragmentation (Tunel staining and electrophoresis on agarose-gel).
  • inflammatory cytokine, TNF- Qf production was measured by ELISA. The results were obtained that the decrease of cell viability and induction of apoptosis due to UVB exposure were inhibited dose-dependently by the present invented compounds, and TNF- Qf production induced by UVB was inhibited significantly.
  • UV protective effects were examined by the means of observation with the naked eye of effects on skin reactions such as erythemas, edemas(flare, eczema.rash) and the like. In vivo effects were also distinctly found in the tests.
  • formula (1 ) or (2) of the present invented compounds have UV protective effects and/or melanogenesis inhibiting effects through suppression of expression of intracellular MITF and tyrosinase, and so- ape * useful as UV protectors and/or melanogenesis inhibitors.
  • the compounds of formula (1) or formula (2) are used normally in the form of pharmaceutical compositions and cosmetic preparations, and administered through various routes, for example, skin permeation, subcutaneous, intramuscular, oral, intravenous, intranasal, transbronchial, pulmonary and intrarectal routes and the like.
  • compositions or cosmetics for pigmented spot and freckle, and skin-care products for prevention of skin pigmentation after suntan and sunburn containing present invented UV protectors and/or melanogenesis inhibitors may contain, in addition to the present invented compounds of formula (1) or formula (2) as exemplified above, as necessary, one or more of vehicles, additives, diluents or carriers such as fillers, binders, lubricants, wetting agents, disintegrants, emulsifying agents, suspending agents, preservatives, sweetening agents and flavoring agents etc., which are used generally, as pharmaceutically-acceptable in pharmaceutical compositions or cosmetics.
  • compositions for examples, surfactant, antioxidants, thickeners, antiseptics, moisturizers, disinfectants, fragrances, pigments, powder constituents, oils and fats, wax, hydrocarbon oils, higher fatty acids, higher alcohols, synthesized ester oils, silicones etc. can be blended.
  • the present invention also includes a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically-acceptable carrier and a compound of formula (1) or formula (2) or its pharmaceutically-acceptable salt as an active ingredient.
  • the pharmaceutically-acceptable salt of the compound of formula (1) or formula (2) include salts formed from acids capable of forming pharmaceutically-acceptable non-toxic acid-addition salts containing anions, such as the hydrochloride, hydrobromide, sulfate, bisulfite, phosphate, acidic phosphate, acetate, maleate, fumarate, succinate, lactate, tartrate, benzoate, citrate, gluconate, glucanate, methanesulfonate, p-toluenesulfonate and naphthalenesulfonate, and their hydrates, as well as the quaternary ammonium (or amine) salt and its hydrate.
  • composition of this invention may be formulated into ointments, sterilized injection solution, emulsions, solutions, suspensions, aerosols/tablets, capsules, powders, granules, troches, cachet wafer capsules, elixirs, syrups, molded cataplasmas, soft and hard gelatin capsules, "alzet " (trademark) pump capsules, pellets, suppositories, and aseptic packed powders.
  • the compound of formula (1 ) or formula (2) is mixed with a carrier or diluent and formed into tablets, capsules or the like.
  • the active ingredient is dissolved in a 10% aqueous solution of glucose, isotonic salt water, sterilized water or a like liquid, and hermetically filled in vials or ampoules for intravenous instillation or injection or intramuscular injection.
  • a dissolution aid, a local anesthetic agent, a preservative and a buffer may also be included into the medium.
  • preparations for parenteral administration include those administered percutaneously, such as ointments and cataplasms. In this case a molded cataplasm or a tape is advantageous.
  • Further examples include pellets for direct implantation in specific dyscratic parts, tissues or bones and " alzet " (trade name) pump capsules.
  • Illustrative resins usable for the preparation of such pellets include poly(2-hydroxyethylmethacrylate) ad ethylene-vinyl acetate copolymer.
  • the composition of this invention may be formulated such that after administration to a patient, the active ingredient is released rapidly, continuously or slowly.
  • Examples of the pharmaceutically-acceptable carrier include lactose, glucose, sucrose, sorbitol, mannitol, corn starch, crystalline cellulose, gum arabic, calcium phosphate, arginates, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, tragacanth gum, gelatin, syrup, methyl cellulose, carboxymethyl cellulose, methylhydroxybenzoic acid esters, propylhydroxybenzoic acid esters, talc, magnesium stearate, inert polymers, water, and mineral oils.
  • the composition of this invention contains generally 0.1 to 2,000 mg, preferably 0.5 to 1 ,000 mg of the active ingredient per unit dosage form.
  • the compound of formula (1) or formula (2) is effective over a wide dosage range.
  • the amount of the compound administered per day usually falls within a range of 0.03 mg/kg to 100 mg/kg.
  • the amount of the compound to be actually administered is determined by a physician depending, for example, upon the type of the compound administered, and the age, body weight, reaction, condition, etc. of the patient and the administration route.
  • the above dosage range therefore, does not limit the scope of the invention.
  • the suitable number of administrations is usually 1 to 6, preferably 1 to 4, daily.
  • Topical products for skin uses and cosmetic preparations are prepared in the form of creams, lotions, ointments, packs, powders or beauty washs, emulsions, by the widely known methods for preparation in such technical field. The number of times of application of those products per a day may conform to that of pharmaceutical compositions.
  • the compounds of formula (1) or formula (2) by themselves effective UV protectors and/or melanogenesis inhibitors. If required, they may be administered in combination with one or more other equally effective drugs. Examples of such additional drugs include such various UV protectors and/or melanogenesis inhibitors.
  • the examples of the production of compounds of formula (1) or formula (2) used in the present therapeutic agents and cosmetics have been already published in patent publications and papers. The present inventor refer to them (patent literatures 2 ⁇ 7, and non-patent literatures 3 and 4). The present invention will hereinafter be described in further detail by the following Examples and Experiments. It is however to be borne in mind that the present invention is not limited to them.
  • Tablets each containing 10 mg of an active ingredient were prepared by the following procedures.
  • Per tablet Active ingredient 10 mg+Cornstarch 55 mg +Crystalline cellulose 35 mg +Polyvinyl pyrrolidione (as 10% aq. soln.) 5 mg+ Carboxymethylcellulose calcium 10 mg +Magnesium stearate 4 mg +TaIc 1 mg: Total 120 mg.
  • the active ingredient, corn starch and crystalline cellulose were passed through an 80-mesh sieve for thorough mixing.
  • the mixed powder was granulated together with the polyvinyl pyrrolidone solution, and passed through an 18-mesh sieve.
  • Tablets each containing 200 mg of an active ingredient were produced by the following procedures. Per tablet: Active ingredient 200 mg ⁇ Cornstarch 50 mg +Crystalline cellulose 42 mg ⁇ Light silicic anhydride 7 mg +Magnesium stearatel mg: Total 300 mg. The above components were passed through an 80-mesh sieve for thorough hmixing. The resulting mixed powder was compression-molded to produce tablets each having a weight of 300 mg. [0031] EXAMPLE 3
  • Capsules each containing 100 mg of an active ingredient were produced by the following procedures.
  • Per capsule Active ingredient 100 mg +Corn starch 40 mg ⁇ Lactose 5 mg + Magnesium stearate 5 mg: Total 150 mg.
  • EXAMPLE 4 An injectable preparation in vials each containing 5 mg of an active ingredient were produced by the following procedures for dissolution just before use. Per vial : Active ingredient 5 mg ⁇ Mannitol 50 mg. Just prior to use, the preparation was dissolved in 1 ml of distilled water for injection, and administered.
  • An adhesive patch containing 17.5 mg of an active ingredient was produced by the following procedures. Ten parts of polyCammonium acrylate) were dissolved in 60 parts of water. Two parts of glycerin diglycidyl ether were dissolved under heat in 10 parts of water.
  • the resulting solution for a medicament-containing hydrogel was coated on flexible plastic film so that the rate of the active ingredient was 0.5 mg per cm2.
  • the surface was covered with releasing paper and cut into a size of 35 cm2 to form an adhesive patch.
  • An adhesive patch containing 10 mg of an active ingredient was produced by the following procedures.
  • An aqueous sol was prepared from 100 parts of polyCsodium acrylate), 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 the active ingredient.
  • the sol was then coated to a thickness of 100 ⁇ m on a non-woven fabric surface of a composite film composed of rayon non-woven fabric and a polyethylene film to form an adhesive layer containing the medicament.
  • the amount of the release aids were then coated to a thickness of 100 ⁇ m on a non-woven fabric surface of a composite film composed of rayon non-woven fabric and a polyethylene film to form an adhesive layer containing the medicament. The amount of the release aids
  • the adhesive layer was then crosslinked at 25degrees C. for 24 hours, and are leasing film was bonded to the adhesive layer surface.
  • the entire film was then cut into pieces each having an area of 35 cm2.
  • glyceryl monostearate 10% by weight, liquid paraffin10% by weight, POE(30)cetyl ether 2% by weight, cetyl alcohol 5% by weight, vaseline 4% by weight and 7-tocophero 1% by weight were used and were blended with active constituents 1 % by weight, and heated to 80degrees C.
  • purified water 56% by weight and 1 ,3-butylene glycol 10% by weight were mixed, and heated to ⁇ Odegrees C. Both mixtures were mixed each other, and stirred, and emulsified, and after that cooled to 35degrees C, and then oil in water cream was produced.
  • Transdermal formulation was using each component similar to that in EXAMPLE9 manufactured by application of Solid in Oil technology that makes hydrophilic compounds dissolve in solid state in grease base, and encapsulate them in oil phase.
  • Human epidermal keratinocyte HaCaT cells were cultured in Dulbecco' s modified Eagle' s medium (DMEM) containing 10% heat-inactivated FBS, 100units/l penicillin G and 100 units/I streptomycin with 5 % CO2 at 37degrees C.
  • DMEM Dulbecco' s modified Eagle' s medium
  • FBS heat-inactivated bovine serum
  • FBS heat-inactivated penicillin G
  • 100 units/I streptomycin 100 units/I streptomycin with 5 % CO2 at 37degrees C.
  • HaCaT cells were seeded on a 35 mm dish.
  • a UVB lamp(HP-30LM; Atto Co., Japan) with 280-320 nm emission peaking at 312 nm was used.
  • HaCaT cells were grown in the dish to confluence and treated with different concentrations of the present invented compounds in DMEM culture medium
  • HaCaT cells were determined by means of LDH assay. After incubation, culture supernatant was collected and centrifuged at 3,000 rpm for 3 min. The supernatant were subjected to the assay. LDH activity was determined colorimetrically with a Cytotoxicity Detection Kit.
  • HaCaT cells were treated with 2-Piperazino-6-methyl-5-oxo-5,6- dihydro(7H)pyrro[3,4-d]pyrimidine maleate before or after UVB-irradiation (50 and 100 mJ/cm2) at concentrations of 50, 250, and 500 mM.
  • UVB-irradiation 50 and 100 mJ/cm2
  • the viability of the cells was evaluated.
  • cell viability % were 66.34 ⁇ 1.72%(p ⁇ 0.05),76.55 ⁇ 2.76%(p ⁇ 0.005), 83.31 ⁇ 0.54%(p ⁇ 0.005), at 50, 250, 500 jl M, respectively.
  • cell viability % were 64.35 ⁇ 3.29%, 66.61 ⁇ 3.44%,70.81 ⁇ 2.67%(p ⁇ 0.01), respectively.
  • cell viability % were 57.59 ⁇ 1.35%(p ⁇ 0.05)
  • HaCaT cells were cultured on a glass coverslip, treated with 2-Piperazino-6-methyl-5-oxo-5,6- dihydro(7H)pyrro-[3,4-d]pyrimidine maleate and UVB-irradiated and incubated for 12 h.
  • UVB-irradiation was also shown by fluorescence microscope observation using Tunel staining in the addition of this compound. Furthermore, after cell lysis DNA extracts were electrophoresed on agarose-gel, and DNA fragmentation was also detected by fluorescence microscope observation using Ethidium bromide staining. The facts were also observed that pre-treatmentwith this compound could protect HaCaT cells from UVB-induced cell damage.
  • UVB induces production of inflammatory cytokines, such as TNF-Q? and IL- 10, in cells.
  • 2-Piperazino-6-methyl-5-oxo-5,6- dihydro(7H)pyrro[3,4-d]pyrimidine maleateon the UVB-induced elevation of TNF-Qf production in HaCaT cells was examined by EUSA.
  • the wells were washed 3 times, then 100 ml aliquots of standardCrecombinant mouse TNF- Qf ) and sample were added to each well, and the plates were incubated for 2 h at room temperature. The plate was washed 3 times with wash buffer, then biotin-labeled TNF- Qf mAb was added, and incubation was continued for 1 h at room temperature. The solutions in the wells werere moved and the wells were washed 5 times with wash buffer. Next, 100 ml of avidin-HRP was added to each well, and the plate was incubated for 30 min at room temperature. The color reaction was quenched with stop solution (50ml, 2.5 M H2SO4).
  • the absorbance at 450 nm was measured with a spectro fluorophotometer and sample concentration was determined from the absorbance with reference to a standard curve.
  • the determination range was 1.0-62.5 pg/ml, and samples were appropriately diluted to obtain a concentration within this range.
  • the amount of TNF- Of in HaCaT cells at 24 hours after UVB-irradiation at 50mJ/cm2 was 3.62 ⁇ 0.37pg/ml, and that at 100mJ/cm2 was 4.98 ⁇ 0.31 pg/ml, and that of non-treated cells was 2.67 ⁇ 0.47pg/ml.
  • the amount of TNF- Qf in 50mJ/cm2 of UVB-irradiated HaCaT cells treated by this compound before irradiation was 2.02 ⁇ 0.88pg/ml, 1.31 ⁇ 0.57pg/ml(p ⁇ 0.01),0.61 ⁇ 0.57pg/ml(p ⁇ 0.005),at 50, 250, 500// M, respectively.
  • the amount of TNF- Qf in 100mJ/cm2 of UVB-irradiated HaCaT cells treated by this compound before irradiation was 4.21 ⁇ 0.27pg/ml(p ⁇ 0.05), 3.00 ⁇ 0.80pg/ml(p ⁇ 0.05), 2.67 ⁇ 0.47pg/ml(p ⁇ 0.005), at 50, 250, 500 ⁇ M, respectively.
  • UVB-irradiation at 100 mJ/cm2 significantly elevated TNF- ⁇ ? production in HaCaT cells. This elevation was dose-dependently inhibited by pre-treatment with this compound.
  • pre-treatment of this compound suppressed the UVB-induced elevation of TNF- Qf production.
  • B16 melanoma cells were seeded in a 6-well plate at a density of 2.5 x 104cells per well and allowed to attach for 24 h. Then the cells were incubated in fresh medium containing 100 mM 2-Piperazino-6-methyl-5-oxo-5,6-dihydro(7H)pyrro[3,4-d]pyrimidine maleate for 72 h. After incubation, the cells were washed with PBS and detached by means of a short incubation with EDTA. After centrifugation, the cell pellets were photographed and solubilized in 100 ml of 1 M NaOH at 80 degrees C for 1 h.
  • the melanin contents were estimated from the absorbance at 405 nm, and values were corrected for the amount of proteins.
  • a dark pigment was observed in B16 cells not treated with this compound, while the accumulation was reduced to about 62.7 % (p ⁇ 0.005) of the control in the cells treated with this compound, indicating that this compound attenuated distinctly melanogenesis.
  • a cell-free system was used to investigate whether the inhibitory effect of this compound on melanogenesis involves direct inhibitory effects of this compound on tyrosinase activity.
  • MITF plays a crucial role in melanogenesis as a regulator of tyrosinase
  • expression of MITF and tyrosinase was analyzed by Western blotting.
  • B16 melanoma cells were treated with 100 mM of 2-Piperazino-6-methyl-5-oxo-5,6-dihydro(7H)pyrro[3,4-d]pyrimidine maleate for 72 h.
  • the cells were washed with cold PBS twice and lysed in cold lysis buffer (10 mM HEPES-NaOH pH 7.4, 1 % Triton X-100, 5 mM 2Na-EDTA, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium o-vanadate) containing protease inhibitor cocktail. Proteins (30 mg per lane) were separated by means of 10 % SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were blocked with 1% BSA in TBS-T buffer.
  • MITF, tyrosinase, ERK and pERK were detected with anti-MITF antibody (dilution 1:1 ,000, anti-tyrosinase antibody (dilution 1 :1 ,000), anti-rat ERK1/2 antibody, and anti-phosphorylated-ERK1 /2 antibody, respectively.
  • the nitrocellulose membrane was further incubated with anti-rabbit IgG HRP-conjugated antibody and anti-mouse IgG-HRP. Bound antibodies were detected using ECL Western blotting detection reagents according to the manufacturer' s instructions. Equal loading was confirmed using anti-j ⁇ -actin antibody to normalize the amount of total protein.
  • This compound suppressed expression of both MITF and tyrosinase in a dose-dependent manner. These data indicate that this compound inhibits melanogenesis via suppression of MITF and tyrosinase expression.
  • Activation of ERK by this compound was also examined in B16 melanoma cells, because ERK activation results in MITF phosphorylation, leading to ubiquitination and degradation of MITF. Sustained ERK activation was observed from 30 min after treatment with this compound(100 mM).
  • B16 melanoma cells were seeded in a 48-well plate at a density of 5 x 103 per well and allowed to attach for 24 h. Then the cells were incubated in fresh medium containing various concentrations of this compound for 72 h. After incubation, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added to each well at a final concentration of 0.05 %.
  • the compounds which are useful in the practice of the present invention were orally administered to 5-weeks old male ddy mice and 8-weeks old male Wistar rats. Their toxicity was determined 24 hours later. For the mice, those compounds were found to have an LD50 value larger than1 ,000mg/kg, or in a range of from 550 mg/kg to 1 ,000 mg/kg. For the rats, on the other hand, they were found to have an LD50 value larger than 1 ,700mg/kg, or in a range of from 550 mg/kg to 1 ,000 mg/kg. Accordingly, the compounds useful in the practice of the present invention can be regarded as medicaments having low toxicity and high safety in general. For the specific values of their LD50 values, reference may be made to the specifications of certain patent applications which were filed in the name of the present inventors and have already been laid open to the public or even registered.
  • the present invented compounds superior in use for UV protectors and/or melanogenesis inhibitors, and high-security pharmaceutical compositions, or cosmetics for pigmented spot and freckle or skin-care products for prevention of skin pigmentation after suntan and sunburn, or beauty products to make the skin beautiful and skin-whitening products.
  • These compounds can be thought to have effects also on prevention and inhibition of onsets of disorders that are thought to be caused by exposure to ultraviolet rays, ie. malignant tumors such as melanomas, skin cancer and the like, cutaneous inflammation, dermal wrinkle formation and cataract and the like.

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  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)
  • Cosmetics (AREA)

Abstract

La présente invention porte sur de nouveaux absorbeurs ultraviolets et sur de nouvelles compositions pharmaceutiques et de nouveaux produits cosmétiques contenant des composés pyrimidines destinés à une protection contre les ultraviolets et une inhibition de la mélano-genèse. La présente invention porte également sur des procédés de fabrication de ces agents. Ces composés pyrimidines sont des composés 2-substitués-6-alkyl-5-oxo-5,6-dihydro(7H)pyrro[3,4-d]pyrimidine, et des composés 2-substitués-7-alkyl-6-oxo- 5,6-dihydro(7H)pyrro[2,3-d]pyrimidine et des sels de ceux-ci, représentés par la formule (1) ou la formule (2) ci-dessous, dans lesquelles R1 à R3 représentent indépendamment un atome d'hydrogène, un groupe alkyle inférieur (notamment un alkyle en C1-C7), CH3OCH2CH2-, -CH2COR9(R9=CH3, C2H5. NH2), - CH2OCOC2H5; et X représente -N (R10) - (R10=CH3, C2H5, Ph,-CH2Ph,-CH (Ph)2,-COCH3, -COOCH3,-SO2CH3),-CH2-,-CH (CH3) -,-CH (C2H5) -,-O-,-S-, où ph représente un groupe phényle.
PCT/JP2010/051725 2009-01-29 2010-01-28 Absorbeurs ultraviolets et compositions pharmaceutiques et produits cosmétiques contenant des composés pyridines pour protection contre les uv WO2010087527A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01139572A (ja) * 1987-08-26 1989-06-01 Mitsui Pharmaceut Inc ピリミジン類及びその薬学的に許容される塩類
JP2005515232A (ja) * 2002-01-14 2005-05-26 ザ・ボード・オブ・トラスティーズ・オブ・ザ・ユニバーシティ・オブ・イリノイ 幹細胞の移動および増殖を促進するための修飾されたピリミジン化合物の使用
WO2006133876A1 (fr) * 2005-06-17 2006-12-21 Dsm Ip Assets B.V. Utilisation de derives pyrimidine dans des buts cosmetiques
WO2010033576A1 (fr) * 2008-09-16 2010-03-25 University Of Central Florida Research Foundation, Inc. Compositions pour traiter et retarder le début de chute de cheveux

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH01139572A (ja) * 1987-08-26 1989-06-01 Mitsui Pharmaceut Inc ピリミジン類及びその薬学的に許容される塩類
JP2005515232A (ja) * 2002-01-14 2005-05-26 ザ・ボード・オブ・トラスティーズ・オブ・ザ・ユニバーシティ・オブ・イリノイ 幹細胞の移動および増殖を促進するための修飾されたピリミジン化合物の使用
WO2006133876A1 (fr) * 2005-06-17 2006-12-21 Dsm Ip Assets B.V. Utilisation de derives pyrimidine dans des buts cosmetiques
WO2010033576A1 (fr) * 2008-09-16 2010-03-25 University Of Central Florida Research Foundation, Inc. Compositions pour traiter et retarder le début de chute de cheveux

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