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WO2005033109A1 - Compose triazinoindolamine - Google Patents

Compose triazinoindolamine Download PDF

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Publication number
WO2005033109A1
WO2005033109A1 PCT/JP2004/014695 JP2004014695W WO2005033109A1 WO 2005033109 A1 WO2005033109 A1 WO 2005033109A1 JP 2004014695 W JP2004014695 W JP 2004014695W WO 2005033109 A1 WO2005033109 A1 WO 2005033109A1
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WO
WIPO (PCT)
Prior art keywords
group
formula
compound
collagen
meo
Prior art date
Application number
PCT/JP2004/014695
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English (en)
Japanese (ja)
Inventor
Akiko Kakimizu
Chizuko Takahashi
Original Assignee
Sumitomo Chemical Company, Limited
Higashi, Kiyoshi
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sumitomo Chemical Company, Limited, Higashi, Kiyoshi filed Critical Sumitomo Chemical Company, Limited
Publication of WO2005033109A1 publication Critical patent/WO2005033109A1/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
    • A61K8/4966—Triazines or their condensed derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • 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
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • 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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations

Definitions

  • the present invention relates to a triazinoindoleamine compound and a compound containing the compound as an active ingredient for inhibiting collagen bleeding and Z or reduction! ; Essences etc. Background art
  • Estrogen which improves diseases such as osteoporosis, It has been reported that it synthesizes collagen and suppresses the production of matrix meta-oral protease (MMP) -1 (hereinafter sometimes referred to as MMP-1) (for example, J. Ce). l l. B i ⁇ chem., 86, 251, (202), Endocrine, 15, 291, (2001)).
  • MMP-1 matrix meta-oral protease
  • TGF-] 3 a type of cytokin, is a well-known factor that promotes the expression of extracellular matrices such as collagen. It is also referred to as MMPs below.
  • MMPs extracellular matrices such as collagen.
  • TGF-) 3 enhances the production of type I, III, and V collagen in human lung line cells, and the production of fibronectin in human fetal line cells, while MMP-1 and MMP-2 And suppresses the production of MMP-9 (see, for example, Lab. Invest., 63, 171, (1990), J. Invest. De Rma to l., 94, 365,).
  • a low ⁇ ? Compound having the effect of promoting collagen synthesis and / or suppressing collagen decrease by promoting collagen synthesis and inhibiting collagen content may be useful as a non-type collagen synthesis ⁇ agent.
  • containing the compound as an active ingredient as it can be expected to improve diseases or tissue conditions associated with a decrease in the amount of collagen in tissues such as wrinkles, abnormal wound healing, periodontal disease, osteoporosis, rheumatoid arthritis, etc. Is expected to lead to the development of drugs, quasi-drugs, cosmetics, and foods. Disclosure of the invention
  • X represents a hydrogen atom, an alkyl group, an alkoxy group, a halogen atom, a haloalkyl group, a haloalkoxy group, a nitro group or an amino group
  • ⁇ represents a hydrogen atom, an alkyl group or an aralkyl group
  • ⁇ represents an alkylene chain having 1 or 2 carbon atoms which may be substituted
  • R represents an aryl group which may be substituted or a heteroaryl group which may be substituted.
  • a composition for promoting and / or suppressing the decrease of collagen accumulation hereinafter sometimes referred to as the composition of the present invention
  • a method for promoting the accumulation and / or reduction of the amount of collagen in tissues characterized by administering the composition of the present invention (hereinafter, also referred to as the method of the present invention); and the like.
  • the compound (I) of the present invention includes an aminy conjugate represented by the following formula ( ⁇ ) and a triazinoindolethione compound represented by the formula (III), a methylsulfinyltriazinoindole compound represented by the formula (IV): It can be formed by condensing with a methylsulfonyltriazinoindole compound represented by the formula (V).
  • the above condensation method can be carried out, for example, according to the method described in J. Heterocyclic Chem., (1988) 25, 475, and Arch. Pharma. (1987), 320, 1196. it can.
  • the amount of the reagent used in the reaction is determined based on the triazinoindolethione compound represented by the formula (III),
  • the amine compound represented by the formula (II) is usually 1 to 100 mol per 1 mol of the methylsulfiertriazinoindole compound represented by the formula (IV) or the methylsulfonyltriazinoindole compound represented by the formula (V) It is a ratio of the degree.
  • the reaction time is usually in the range of 0 to 25 ⁇ ⁇ ⁇ , and the reaction time is usually in the range of 0;
  • the compound (I) of the present invention can be prepared by, for example, performing the following post-treatment operation.
  • the above-mentioned compound (I) of the present invention can be further purified by recrystallization, chromatography and the like.
  • the triazinoindolethione compound represented by the above formula (III) can be prepared, for example, by the method described in J. Medicina 1 Chem., (1972), 15 (3), 277, etc. It can be obtained accordingly. Further, the methylsulfinyltriazinoindole compound represented by the above formula (IV) and the methylsulfonyltriazinoindole compound represented by the formula (V) can be prepared, for example, by using a triamine represented by the above formula (III) using methyl iodide or the like. The compound can be obtained by methylating the dinoindolethione compound and then reacting it with an oxidizing agent such as 3-chloroperbenzoic acid.
  • an oxidizing agent such as 3-chloroperbenzoic acid.
  • the aryl group represented by R includes, for example, a phenyl group and an aryl group having 6 to 10 carbon atoms of naphthylline.
  • up to 10 heteroaryl groups up to 10 heteroaryl groups.
  • Examples of the substituent which may be substituted on the aryl group and the heteroaryl group include an alkyl group, an alkoxy group, a halogen atom, a haloalkyl group, a haloalkoxy group, an amino group, a cyano group, a nitro group, a hydroxyl group, Alkoxyl group, alkoxycarbonyl group, aminocarbonyl group, and alkylaminocarbonyl group, preferably an alkoxy group, a halogen atom, a haloalkyl group, a haloalkoxy group, an amino group, a cyano group, and a 7_acid group, and more preferably.
  • alkyl group examples include methyl, ethyl, ⁇ -propyl, isopropyl, ⁇ -butylyl, sec-butylyl, tert-butyl, n-pentyl, and n-hexylyl having 1 to 6 carbon atoms.
  • alkoxy group examples include those composed of the above-mentioned alkyl group and oxygen atom.
  • halogen atom examples include a fluorine atom, a chlorine atom, a bromine atom, and an iodine atom.
  • Examples of the octaalkyl group include those composed of the above alkyl group and a halogen atom, and examples thereof include a fluoromethyl group, a difluoromethyl group, a trifluoromethyl group, and a trichloromethyl group.
  • Examples of the haloalkoxy group include those composed of the above alkoxy group and a halogen atom, and include, for example, a chloromethoxy group, a fluoromethoxy group, a trifluoromethoxy group, and a ethoxy binding force.
  • alkoxycarbonyl group examples include those composed of the above-mentioned alkoxy group and carbonyl group. Examples thereof include a methoxycarbonyl group, an ethoxycarbonyl group, an n-propyloxycarbonyl group, an isopropyloxycarbonyl group, an n-butyloxycarbonyl group, sec-butyloxycarbonyl group, tert-butyloxycarbonyl group, n-pentyloxycarbonyl group, n-hexyloxycarbonyl group.
  • alkylaminocarbonyl group examples include those composed of the alkyl group and the aminocarbonyl group. Examples thereof include a methylaminocarbonyl group, an ethylaminocarbonyl group, an n-propylaminocarbonyl group, and isopropylamino. Examples include a carbonyl group, a dimethylaminocarbonyl group, and acetylaminocarbonyl.
  • the optionally substituted aryl group or the optionally substituted heteroaryl group represented by R is a phenyl group or a pyridyl group, which is unsubstituted or substituted with an alkoxy group, an octogen atom or an amino group. Is preferably used.
  • alkyl group, alkoxy group, halogen atom, haloalkyl group and haloalkoxy group represented by X are the same as those described above as the substituent which may be substituted on the aryl group and the heteroaryl group, respectively.
  • substituent which may be substituted on the aryl group and the heteroaryl group, respectively.
  • a hydrogen atom, an alkyl group, an alkoxy group or a hydrogen atom is preferable, and more preferably a substituent in which these substituents are bonded to the 8-position of the triazinoindole ring. .
  • Examples of the alkyl group represented by Y include the same linear or branched alkyl group having 1 to 6 carbon atoms as described above.
  • Examples of the aralkyl group include those composed of the above-mentioned alkyl group, phenyl group, and carboxylic acid, and include, for example, a benzyl group and a phenethyl group.
  • Y is preferably a hydrogen atom.
  • Examples of the alkylene group having 1 or 2 carbon atoms represented by Z include a methylene group and an ethylene group.
  • Examples of the substituent which may be substituted on the alkylene group include a methyl group, Examples include an engineered group, an n-propyl group, and an alkyl group having 1 to 3 carbon atoms in isopropyl.
  • Embodiments of the compound (I) of the present invention include, for example, the following compounds.
  • R is a phenyl group which may be substituted, and a triazinoindoleamine compound
  • a triazinindoleamine compound wherein R is a cyclophenyl group
  • a triazinindoleamine compound wherein R is an aminophenyl group
  • R is an unsubstituted phenyl group
  • a triazinoindoleamine compound in which X is an S halogen atom in the formula (I), a triazinindoleamine compound in which X is an alkoxy group; in the formula (I), X is an alkyl group Triazinyl amine compounds;
  • ⁇ ⁇ is an alkyl group.
  • ⁇ is an ethylene chain which may be substituted by an alkyl group having 1 to 3 carbon atoms.
  • a triadinoin'-lamine compound which is a methylene group which may be substituted with an alkyl group having 1 to 3 carbon atoms;
  • R is an unsubstituted phenyl group
  • X and ⁇ are hydrogen atoms.
  • R is a phenyl group which may be substituted, X is an alkoxy group, and ⁇ is a hydrogen atom; a triazinindoleamine compound;
  • R is a phenyl group which may be substituted, X-force, a logen atom, and a triazinoindoleamine compound which is a calcium atom;
  • R is an alkoxy-substituted phenyl group
  • X force V a logen atom
  • ⁇ is a hydrogen atom
  • R is a pyridyl group
  • X and ⁇ are hydrogen atoms.
  • R is a pyridyl group, X is a halogen atom, and Y is a hydrogen atom;
  • a triazinoindoleamine compound; and these compounds are pharmaceutically acceptable salts, for example, « Salts with inorganic acids such as salts and ⁇ -salts, and salts with organic acids such as acetates, acetates, triacetate salts, and salts of organic acids such as sodium chloride, etc. Conversion to such salts Can be done by conventional means.
  • Ph represents a phenyl group.
  • the composition of the present invention comprises, for example, one or more of the compound (I) of the present invention or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient, and / or pharmaceutical additive. Or a food additive or a cosmetic additive.
  • the compound (I) of the present invention or a pharmaceutically acceptable salt thereof, or the thread of the present invention stimulates the transcription of type I collagen and / or suppresses the transcription of MMP-1. Have the ability. This force increases the expression level of type I collagen ⁇ ? And / or decreases the expression level of type 1 collagen, thereby reducing the effect of collagen ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ 1 and / or suppression of the decrease in fiber. It is important to improve the disease or tissue condition associated with a decrease in the amount of collagen in the tissue by leading, and it can be used as a drug, quasi-drug, cosmetics, food, etc. for this purpose .
  • the amount of collagen in the fiber is decreased due to various causes.
  • the pharmaceutically acceptable carrier, excipient, and / or pharmaceutical additive, food additive or cosmetic additive to be used can be selected according to the specific use of the composition.
  • the form of the perishables can be, for example, various solids, liquids, etc., depending on the specific application.
  • composition of the present invention or the compound (I) of the present invention or a pharmaceutically acceptable salt thereof when used as a pharmaceutical, specific forms include, for example, powders, fine granules, granules, Oral preparations such as syrups, capsules, pills, emulsions, extracts, etc., transdermal absorption preparations (external preparations for skin) such as injections, external preparations, ointments, and parenteral preparations for topical use Etc. can be given.
  • Oral preparations include, for example, gelatin, sodium alginate, supplement, corn starch, sucrose, ⁇ , glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soy lecithin, sucrose, fatty acid ester, talc, magnesium stearate.
  • Excipients, binders, disintegrants, surfactants, lubricants, fluid ⁇ agents, diluents, preservatives, coloring agents, fragrances, stable Agent It can be produced according to a usual method using pharmaceutical additives such as a wetting agent, a preservative, and an antioxidant.
  • the dosage varies depending on the age of the mammal to be administered, tt3 ⁇ 4i ⁇ , body weight, degree of disease, the type of the extinct material of the present invention or the compound (I) of the present invention, the administration form, and the like.
  • about 1 mg to about 2 g, preferably about 5 mg to about 1 g, of the active ingredient may be administered per day.
  • the above-mentioned daily dose can be administered once or in several divided doses.
  • injections include menstruation 77, water-soluble ⁇ such as sterile water in Ringer's solution, water-insoluble ⁇ such as vegetable oils and fatty acid esters ⁇ , isotonic agents such as glucose and sodium chloride, and melting aids.
  • a pharmaceutical additive such as a stabilizing agent, a preservative, a preservative, an emulsifier, or an emulsifier according to an ordinary method.
  • Liquid preparations for external use, transdermal absorption agents such as gel-shaped ointments, suppositories for jffl administration, and the like can be prepared according to ordinary methods.
  • a topical agent can be produced, for example, by incorporating the compound (I) of the present invention into a pellet of a sustained-release polymer such as an ethylene vinyl acetic acid polymer.
  • the pellet may be surgically implanted into the tissue to be treated.
  • the dosage varies depending on the age of the baby to be administered, the body weight, the disease, the type of the thread substance of the present invention or the present invention (I), the administration form, and the like. In this case, it is sufficient to administer about 0.1 mg to about 500 mg as an effective ingredient in a human adult. In addition, the above-mentioned daily dose can be administered once or in several divided doses.
  • composition of the present invention or the compound (I) of the present invention is used as cosmetics
  • specific forms include, for example, creams, lotions and the like.
  • the lotion can be prepared according to an ordinary method using, for example, a cosmetic additive such as a suspending agent, an emulsifier, and a preservative.
  • the dose varies depending on the age, sex, body weight, degree of disease, the type of the composition of the present invention or the present invention, and the dosage form of the mammal to be administered.
  • the component may be administered in an amount of about 0.01 mg to about 5 Omg.
  • the above-mentioned daily dose can be administered once or in several divided doses.
  • composition of the present invention or the compound (I) of the present invention or a pharmaceutically acceptable salt thereof is used as a food
  • specific forms include, for example, powders, powders, beverages, Or mixed liquid with syrup, for example, seasonings, Japanese sweets, ⁇ ⁇ ⁇ ⁇ , frozen desserts, beverages, spreads, pastes, pickles, canned pins, processed meat, fish, processed fish, milk, egg processing Goods, processed vegetables, fruit processing May be used as general foods and drinks, foods, etc. It may also be used as a food additive to feed and feed for domestic animals such as poultry, poultry, bees, silkworms, and fish.
  • the dosage is the age of the mammal to be administered, the body weight, the degree of the disease, the type of the composition of the present invention or the compound (I) of the present invention or a pharmaceutically acceptable salt thereof, the type of the dosage form, the dosage form, etc. Usually, it is sufficient that about 0.1 mg to about 500 mg of the active ingredient is administered to a human adult.
  • the above-mentioned daily dose can be administered once or in several divided doses.
  • the obtained genomic DNA solution (equivalent to 1 g of genomic DNA *) was combined with an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 1 and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 2 (each 10 pmo 1 / PL ⁇ ) each, distilled water 291, TaKaRa LA Taq (Takara Shuzo, catalog number RR002A), buffer 51, Mg2 + solution 51, dNTP mix 5 n1, and TaKaRa LA Taq (Takara Shuzo Co., Ltd., Log No. RR002A) 0.5 1 was mixed.
  • the obtained mixture was kept at 94 for 5 minutes, and then at 94, for 1 minute, then at 60, for 1 minute, and further at 72 T: for 1 minute, and this was repeated 30 times.
  • the mixture was subjected to 1% agarose gel electrophoresis to recover about 3.5 kb of DNA.
  • the recovered DNA was treated with phenol / chloroform and then precipitated with ethanol to recover DNA.
  • the recovered DNA was dissolved in ⁇ Sfo, and Nhel 2.5t1 and Hindlll 2.51 were added on the night of the dissolution, followed by incubation at 37 for 3 hours. Next, the dissolution was subjected to 1% agarose gel electrophoresis to recover about 3.5 kb of DNA.
  • Luc vector DNA DNA Ligation kit kit Ver2 enzyme ⁇ was added thereto.
  • 5 Hda of Mi bacterium TO YOBO, Catalog No. DNA-903 was added, and the mixture was left on ice for 30 minutes and then incubated for 42 and 45 seconds.
  • Plasmid DNA was prepared from the obtained culture using AUTOMAT IC DNA I SOLATION SYSTEM PI-50 (KURABO). The nucleotide sequence of the prepared plasmid DNA was analyzed using a DNA sequencer.
  • the plasmid contained one of 3,500 to 57 (one of the transcription start sites +1) of one of the transcription regulatory regions of human type I collagen ⁇ 2 chain. It was confirmed that there was a sequence in which a nucleotide sequence encoding firefly luciferase was connected downstream of the nucleotide sequence.
  • the vesicles were cultured at 37 in a 5% C02 atmosphere. Six hours later, the culture supernatant was removed from the dish, the cells were washed twice with PBS, and 1 ml of PBS containing 0.25% trypsin was added to detach the cells. After adding D-MEM (+) to the cells and mixing well, dispense the lactose solution into a 12-well plate and incubate at 37 with 5% CO 2 atmosphere overnight. did. The next day, wash each well twice with D-MEM (—), and then use Du 1 becco's—MEM medium containing 0.1% FBS (hereinafter referred to as D-MEM (0.1%) The medium was changed to 1 ml.
  • D-MEM Du 1 becco's—MEM medium containing 0.1% FBS
  • DMSO dimethyl sulfoxide
  • D-MEM 10% dimethyl sulfoxide
  • 10 l 10 l
  • 101 of 10% D MSO was added.
  • TGF- / 3 PeproTec r
  • * H * 5 ng / ml The cells were further cultured at 37 in a 5% C2 atmosphere for 40 hours. After washing the cultured cells twice with PBS, a cell lysing agent (Toyo Ink, catalog No.
  • the recovered supernatant or cell lysing agent 51 was previously dispensed into a 96-well plate with j3-ga1 substrate ⁇ ⁇ M (5.8 mM o-nitropheny l-be ta-D-ga l ac t opyr anos ide, 1 mM MgC 12, 45 mM 2—mercaptoethanol) 5 After incubation for 2 hours at 37 in addition to 01, the absorbance at 420 nm in each well was measured using a microplate reader. Based on the obtained values, the transcription activity was calculated according to the following equation.
  • Transgenicity [amount of luminescence (supernatant added area)-amount of luminescence (cell lysate added area)] / [420 nmW. (Supplemented area)-420 credential (cell lysate added area) )]
  • the transcription key ability of the compound of the present invention (I) for the type I collagen relative to the control was defined in the following three stages, and the results are shown in Table 29.
  • the control expression level was 100 and the sex control expression level was 100, the type I collagen gene expression level (relative value on a correction basis) was
  • the repo overnight activity of the type I collagen gene of the compound (I) of the present invention is as shown in Table 29, and the ability to promote the transcription of the type I collagen gene was confirmed.
  • TGF- / 3 As a positive control, TGF- / 3 It was added so that the final concentration was 5 ng / ml. After culturing for 2 days in an atmosphere of 37, 5 C02, the cells were washed with PBS (-), and total RNA (about 301) was extracted using RNeasy Minikit (QIAGEN, catalog number 74106). . To the extracted total RNA 5 ⁇ 1 (5 Ong), 20 20 oligo dT 1 u1 and RNase-free distilled water 41 were added, and the mixture was incubated at 65 for 5 minutes and immediately cooled with ice.
  • MMP-1 expression level (correction value) measured MMP-1 gene expression M / GAPDH level
  • the expression of the type I collagen and the expression level of the MMP-11 gene of the compound (I) of the present invention are as shown in Table 30, and the transcription of the type I collagen gene was reduced by 1 and / or the MMP-1 gene was expressed. The ability to suppress transcription was recognized.
  • Test Example 3 (measurement of the effect of the present compound (I) on the spread or suppression of collagen) using the protein amount of non-type I collagen as an index
  • PVDF membrane 250 mA, 90 minutes. After incubating the protein-transferred PVDF membrane for 30 minutes with shaking in a 5% skim milk solution, 0.1 l% Tween After washing with a PBS solution containing en20 (hereinafter referred to as PBS-T) and adding a 1000-fold diluted rabbit herb anti-type I collagen antibody (Polyscience, cat. no. 23706), add room temperature for 1 hour. Incubated.
  • PBS-T PBS solution containing en20
  • the PVDF membrane was washed with PBS-T, and then incubated with a 2M glycine-HC1 solution (pH 2.8) at room temperature for 1 hour to remove the antibody.
  • a 2M glycine-HC1 solution pH 2.8
  • 1000-fold diluted Egret anti-bovine serum albumin antibody-HRP Cape 1, Cat.No. 55285 was added and incubated at room temperature for 1 hour.
  • the luminescence of the approximately 60 kDa portion corresponding to the band of bovine serum albumin was quantified using Image Gauge in the same manner as described above.
  • the amount of non-degraded type I collagen protein was calculated according to the following equation.
  • the amount of the non-type I collagen protein of the compound (I) of the present invention relative to the control was defined in the following three stages. The results are shown in Table 31.
  • the protein content of the non-degraded type I collagen of the compound (I) of the present invention is as shown in Table 31, and the ability of collagen was confirmed. '
  • Test Example 4 Measurement of the ability of the compound (I) of the present invention to activate the intracellular signal e3 ⁇ 4 pathway using the expression level of the repo overnight gene as an index
  • An oligonucleotide consisting of the base sequence represented by SEQ ID NO: 9 and an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 10 were mixed at a ratio of 20 i 1 each. After keeping the mixture warm for 5 minutes, it was cooled to room temperature (hereinafter referred to as DNA containing the Smad3 binding sequence (SBE)).
  • SBE Smad3 binding sequence
  • a vector obtained by adding a TATA sequence to pGL3 (promega, catalog number E1751) having a nucleotide sequence encoding luciferase was digested with NheI and Xhol, followed by agarose gel electrolysis. It was subjected to electrophoresis, and about 5 kb of DNA was recovered. The DNA was recovered again by subjecting the recovered DNA to ethanol.
  • 5Hda (TOYOBO, Cat. No. DNA-903) was added to the mixture, and the mixture was left on ice for 30 minutes, and then incubated at 42 and 45 seconds.
  • the obtained bacteria were 50 igZml ampicillin sodium. (Nacalai Tesque, Catalog No. 027-39) and inoculated on an LB plate. The single colony that appeared was cultured in 2 ml of LB medium containing 50 jtig / ml ampicillin at 37 for 12 hours.
  • Plasmid DNA was prepared from the obtained culture using AUTOMAT IC DNA I SOLATION SYST EM PI-50 (KURABO). The nucleotide sequence of the prepared plasmid DNA was analyzed using a DNA sequencer.
  • the plasmid (hereinafter, referred to as SBE-TATA-Luc) is composed of a base comprising a TATA sequence and a base sequence coding for luciferase downstream of the Smad3 binding sequence. You were confirmed to have the sequence.
  • Blast cells Normal human fetal skin «Blast cells 5x105 cells are seeded on a 60 mm dish, and are incubated in D-MEM (+) at 37 ° in a 5% CO 2 atmosphere! Nourished. Then, the medium was replaced with 2 ml of D-MEM (-).
  • SBE-TATA-Luc and pCMV-i3-ga1 were added to D-MEM (-) IOOI, and the resulting mixture was left at room temperature for 5 minutes ( ⁇ 1).
  • Lipofectine8xl was added to D-MEM (-) ⁇ , and the resulting mixture was left at room temperature for 45 minutes (Part 2).
  • Nada 1 and Job 2 were mixed, and the mixture was allowed to stand at room temperature for 10 minutes.
  • the mixed solution was added to the normal human fetal skin fibroblasts. Culture was performed under an atmosphere. Six hours later, the culture supernatant was removed from the dish, the cells were washed twice with D-MEM (-), and 4 ml of D-MEM (0.1%) was added, followed by culturing for 1 hour.
  • DMSO 251 Conte As the roll, 41 pieces of DMSO and 41 pieces of D-MEM (0.1%) were added.
  • the cells were cultured at 37 in a 5% CO 2 atmosphere for an additional 40 hours, washed twice with PBS, and added with a cell lysing agent (150 ⁇ l) to detach the cells. After collecting the obtained cell suspension, the cell suspension was centrifuged (15,000 rpm, 4 for 5 minutes) to collect the supernatant.
  • the recovered supernatant or cell lysing agent 501 was previously dispensed into a 96-well plate.
  • ga 1 substrate ⁇ S (5.8 mM on it ropheny l-beta d-galac topyranos After incubation for 2 hours at 37 in addition to Ide, 1 mM MgC12, 45 mM 2-mercaptoethanol) 501, the degree of 420 nm in each well was measured using a microplate reader. Based on the obtained values, the transmissivity was calculated according to the following equation.
  • Inversion ratio [Luminescence (supernatant added)-Luminescence (cell lysing agent added)] / [420 nm (supernatant added)-420 nm absorbance (cell lysing agent added)]
  • Table 32 shows the control as 100% based on the calculated transcription activity.
  • Test Example 5 Measurement of the ability of compound (I) of the present invention to activate the intracellular signal transmission pathway using the amount of Smad3 in the nucleus as an index
  • Controls include 0.1% DM SO 0 —: ⁇ 1 £ 1 ⁇ (0.
  • TGF-) 3Nada 5ngZml was added as a positive control.
  • the plate was then washed three times with cold PBS (-), and 0.4 ml of cold methanol was added.
  • Table 33 shows the percentage of cells in which the fluorescence was observed only in the nucleus of 100 cells.
  • the compound (I) of the present invention or a pharmaceutically acceptable salt thereof which has the effect of promoting collagen accumulation and suppressing Z or decrease in collagen, is useful in fibers such as wrinkles, abnormal wound healing, periodontal disease, osteoporosis, and rheumatoid arthritis. It can be expected to improve the 3 ⁇ 4 or ⁇ condition accompanied by a decrease in the amount of collagen, and this may lead to the development of drugs, quasi-drugs, cosmetics, foods, etc. containing this as an active ingredient. In addition, among the compound (I) of the present invention or a pharmaceutically acceptable salt thereof, one having an effect of suppressing collagen decrease can be expected to suppress the migration. It can lead to development. Sequence Listing Free Text Sequence No. 1
  • Oligonucleotide primer designed to amplify collagen promoter DN DN SEQ ID NO: 2
  • Oligonucleotide primer designed to amplify collagen promoter DNA SEQ ID NO: 3
  • Oligonucleotide primer designed to detect type I collagen DNA SEQ ID NO: 4
  • Oligonucleotide primer designed to detect type I collagen DNA SEQ ID NO: 5
  • Oligonucleotide probe designed to detect type I collagen DNA SEQ ID NO: 6
  • MMP oligonucleotide primer designed to detect 1 DNA SEQ ID NO: 7
  • Oligonucleotide primer designed to detect MMP-1 DNA SEQ ID NO: 8
  • Oligonucleotide designed to generate reporter plasmid containing Smad 3 DNA binding sequence SEQ ID NO: 10
  • Oligonucleotides designed to generate reporter plasmids containing the Smad3 DNA binding sequence are designed to generate reporter plasmids containing the Smad3 DNA binding sequence

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Abstract

L'invention concerne un composé triazinoindolamine correspondant à la formule (I), ainsi qu'une composition destinée à accélérer l'accumulation de collagène et/ou empêcher la diminution de collagène, laquelle se caractérise en ce qu'elle contient soit ledit composé, soit un sel pharmaceutiquement acceptable de celui-ci.
PCT/JP2004/014695 2003-10-03 2004-09-29 Compose triazinoindolamine WO2005033109A1 (fr)

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JP2003-345472 2003-10-03

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013041859A1 (fr) * 2011-09-19 2013-03-28 Isis Innovation Limited Agonistes du récepteur des cannabinoïdes 2

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1023720A (en) * 1963-09-17 1966-03-23 Allen & Hanburys Ltd as-triazino (5,6-b)indoles
JPS62132882A (ja) * 1985-12-04 1987-06-16 Sumitomo Chem Co Ltd トリアジノインド−ル誘導体、その製造法およびそれを有効成分とする除草剤
JPH11246333A (ja) * 1997-12-19 1999-09-14 L'oreal Sa 収斂用化粧料におけるケイ皮酸またはその誘導体の用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1023720A (en) * 1963-09-17 1966-03-23 Allen & Hanburys Ltd as-triazino (5,6-b)indoles
JPS62132882A (ja) * 1985-12-04 1987-06-16 Sumitomo Chem Co Ltd トリアジノインド−ル誘導体、その製造法およびそれを有効成分とする除草剤
JPH11246333A (ja) * 1997-12-19 1999-09-14 L'oreal Sa 収斂用化粧料におけるケイ皮酸またはその誘導体の用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GLADYCH J.M.Z. ET AL.: "Antiviral agents. 5H-as-triazino[5,6-b]indoles", JOURNAL OF MEDICINAL CHEMISTRY, vol. 15, no. 3, 1972, pages 277 - 281, XP002984250 *
MBAGWU G.O. ET AL.: "Indole derivatives. 1. Synthesis of tetracyclic mesoionic pyramido[3,2:2',3']-as-triazino [5',6'-b]indole-2,4-diones", JOURNAL OF HETEROCYCLIC CHEMISTRY, vol. 25, no. 2, 1988, pages 475 - 478, XP002984251 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013041859A1 (fr) * 2011-09-19 2013-03-28 Isis Innovation Limited Agonistes du récepteur des cannabinoïdes 2

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