WO2003080592A1

WO2003080592A1 - 2-pyrone compounds and use thereof

Info

Publication number: WO2003080592A1
Application number: PCT/JP2003/003392
Authority: WO
Inventors: Junya Takahashi; Kiyoshi Higashi
Original assignee: Sumitomo Chemical Company, Limited
Priority date: 2002-03-27
Filing date: 2003-03-20
Publication date: 2003-10-02
Also published as: AU2003220931A1

Abstract

It is intended to provide 2-pyrone compounds represented by the following general formula (I): (I) wherein X represents halogeno, C1-4 alkyl optionally substituted by C1-4 alkoxy, etc.; and R represents C1-4 alkyl, etc.; compositions inhibiting I type collagen gene transcription which contain the above 2-pyrone compound as the active ingredient together with an inert carrier; a treatment method of improving tissue fibrosis characterized by administering an effective dose of the above 2-pyrone compound to a mammalian subject with a need of such treatment, etc.

Description

2-Pyrone compounds and their uses

Technical field

The present invention relates to 2-pyrone compounds and uses thereof. Background art

Diseases such as cirrhosis, interstitial lung disease, chronic renal failure (or diseases that lead to chronic renal failure), hyperplasia scars after inflammation, postoperative scars-burn scars, scleroderma, arteriosclerosis, hypertension, etc. Under abnormal conditions, excessive accumulation of extracellular matrix, such as collagen, causes tissue fibrosis and hardening, resulting in reduced organ and tissue function and scar formation. Such excessive accumulation of extracellular matrix is induced by enhanced collagen production based on the breakdown of the balance between biosynthesis and degradation of collagen and the like. In fact, in fibrotic tissues, it has been observed that the expression level of collagen genes, especially the type I collagen gene, is increased [J. Invest. De rmatol., 94, 365, Natl. Acad. Sci. USA, 88, 6642, (1991)]. It has also been observed that the amount of TGF-0, one of the cytokines, is elevated in fibrotic tissues [J. Inves t. Dermatol., 94, 365, ( Sci. USA, 88, 6642, (1 991)]. TGF- has been shown to increase the expression level of type I collagen gene, enhance collagen production, and thus contribute to tissue fibrosis [Lab. Inves t., 63, 171, (1990), J. Invest. Derma tol., 94, 365, (1990)].

On the other hand, in various fibrosis model animals, administration of interferon τ / reduces the amount of type I collagen gene expression in tissues and reduces the amount of collagen to improve tissue fibrosis. [Ex p. Lung Re s., 21, 79 1-808, (1 995), Kidney Int., 47, 62-69, (1 995), J. He atol., 28, 471-479, (1998), J. He atol., 26, 894-903, (1997)]. Therefore, there is a strong need for the development of a drug that improves tissue fibrosis by reducing the expression level of the type I collagen gene in tissues and reducing the amount of collagen accumulation (ie, an inhibitor of collagen accumulation and a therapeutic agent for fibrosis). ing. Disclosure of the invention

Under such circumstances, the present inventors have conducted intensive studies, and as a result, have found that a 2-pyrone compound represented by the following formula (I) has the ability to suppress the transcription of a type I collagen gene, leading to the present invention. Was.

That is, the present invention

1. Formula (I)

[In the formula, X is a hydrogen atom, a halogen atom, a halogen atom or a C1-C4 alkyl group, a nitro group, a CI-C4 alkoxy group which may be substituted by a C1-C4 alkoxy group, or WR represents ^one group (W represents an oxygen atom or a sulfur atom, R ¹ represents a C 1 -C 4 alkyl group substituted with a halogen atom), and R represents a C 1 -C 4 alkyl group, C 3- Represents a C 4 alkenyl group or a C 3 -C 4 alkynyl group. ]

2-pyrone compound [hereinafter may be referred to as compound (I) of the present invention. ];

2. The 2-pyrone compound according to the above 1, wherein R is a C1-C4 alkyl group or a C3-C4 alkenyl group;

3. The 2-pyrone compound according to the above 1, wherein R is a methyl group, an ethyl group, or an aryl group; 4. C 1—C wherein X may be substituted with a hydrogen atom, a halogen atom, or a halogen atom. 4 2-pyrone compound according to the above 1, which is an alkyl group or a dinitro group;

5. The 2-pyrone compound according to the above 1, wherein X is a hydrogen atom, a halogen atom, a C 1 -C 4 alkyl group, a ditoluene group or a trifluoromethyl group;

6. The 2-pyrone compound according to the above 1, wherein X is a hydrogen atom, a chlorine atom, a methyl group, a nitro group or a trifluoromethyl group; 7. The 2-pyrone compound according to the above 4, wherein R is a methyl group;

8. The 2-pyrone compound according to the above item 4, wherein X is a chlorine atom, a methyl group or a trifluoromethyl group, and R is an ethyl group or an aryl group;

9. The 2-pyrone compound according to the above ^{1, wherein} X is — WR ¹ group (W represents an oxygen atom or a sulfur atom, and R ¹ represents a C 1 -C 4 alkyl group substituted with a halogen atom). ;

10. 2-pyrone compound as described in the preceding item 9, wherein R ¹ is a C 1 -C 4 alkyl group substituted with one or more fluorine atoms;

11. The 2-pyrone compound according to the above item 9, wherein R ¹ is a difluoromethyl group, a trifluoromethyl group or a 1,1,2,2-tetrafluoroethyl group;

12. The 2-pyrone compound according to the above item 9, wherein W is an oxygen atom;

13. The 2-pyrrone compound according to the above item 9, wherein W is a sulfur atom and R ¹ is a trifluoromethyl group;

14. the 2-pyrone compound according to the above item 9, wherein R is a methyl group;

15. The 2-pyrone compound according to the above 1, wherein X is a C 1 -C 4 alkoxy group;

16. The 2-pyrone compound according to the above 15, wherein X is a methoxy group or an ethoxy group;

17. The 2-pyrone compound as described in 15 above, wherein R is a methyl group, an ethyl group, or an aryl group.

Wherein, R ^a represents a Echiru group or Ariru group. ]

A 2-pyrone compound represented by:

19. The 2-pyrone compound according to the above 1, wherein X is a C 1 -C 4 alkyl group substituted with a C 1 -C 4 alkoxy group;

20. The 2-pyrone compound according to the above 19, wherein X is a methoxymethyl group;

21. Formula (III)

A 4-hydroxy-2-pyrone compound represented by the formula:

2 2. Formula (IV)

[In the formula, R ² represents an ethoxy group, a methoxymethyl group, or a 1,2,2-tetraphenylolethoxy group. ]

A 4-hydroxy-2-pyrone compound represented by the formula:

2 3. Equation (V)

A 4-hydroxy-2-pyrone compound represented by the formula:

2 4. Equation (VI)

A 4-hydroxy-2-pyrone compound represented by the formula:

25. Use of the 2-pyrone compound described in 1 above for suppressing transcription of a type I collagen gene;

26. A type I collagen gene transcription inhibitory composition comprising the 2-pyrone compound according to item 1 as an active ingredient and an inert carrier (hereinafter referred to as the transcription inhibitory composition of the present invention) is there. ) ;

27. The transcription of the type I collagen gene according to the above item 26 for improving the fibrosis of tissues by reducing the expression level of the type I collagen gene and leading to a decrease in the amount of collagen accumulation. Use of an inhibitory composition;

28. A composition for improving tissue fibrosis characterized by containing the 2-pyrone compound described in 1 above as an active ingredient and an inert carrier (hereinafter referred to as the fibrosis improving composition of the present invention) There is.);

29. A method for improving tissue fibrosis, which comprises administering to a mammal patient in need of such treatment an effective amount of the 2-pyrone compound described in 1 above. How to

30. Use of the 2-pyrone compound described in 1 above for suppressing the action of TGF—3;

31. A composition for inhibiting the action of TGF-] 3, characterized by containing the 2-pyrone compound described in 1 above as an active ingredient and an inert carrier (hereinafter referred to as the TGF-inhibiting composition of the present invention) There is.);

32. Use of the composition for suppressing the action of TGF- according to the preceding item 31, for obtaining a hair-growing effect by inhibiting the promotion of the transition of the hair to the catagen phase by TGF- / 3 and leading to prolongation of the hair growth period. ;

33. A hair-growing composition characterized by containing the 2-pyrone compound described in 1 above as an active ingredient and an inert carrier (hereinafter sometimes referred to as the hair-growing composition of the present invention); A hair-growing method comprising administering an effective amount of a 2-pyrone compound according to item 1 to a mammalian patient in need of hair-growing treatment;

Is provided. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

In the present invention,

Examples of the halogen atom in X include a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.

In X, a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom) or a CI—C4 alkyl group which may be substituted by a CI—C4 alkoxy group is, for example, a methyl group, a trifluoromethyl And a methoxymethyl group. Examples of the C1-C4 ^ -oxy group in X include a methoxy group and an ethoxy group.

Halogen atom in R ¹ (a fluorine atom, a chlorine atom, a bromine atom, an iodine atom) Examples of the C 1-C 4 alkyl group substituted by, for example, one or more C 1 one C 4 alkyl group substituted with a fluorine atom Difluoromethyl group, trifluoromethyl group, 1,1,2,2-tetrafluoroethyl group and the like.

- WR ¹ group (. W represents an oxygen atom or a sulfur atom, R ¹ is representative of a C 1 one C 4 alkyl group substituted by a halogen atom), for example, substituted with one or more fluorine atoms C 1-C4 alkoxy groups such as difluoromethoxy, trifluoromethoxy, trifluoromethylthio, 1,1,2,2-tetrafluoroethoxy, etc.

Examples of the C 1 -C 4 alkyl group, C 3 -C 4 alkyl group or C 3 -C 4 alkyl group in R include a methyl group, an ethyl group, and an aryl group. Embodiments of the compound (I) of the present invention include the following embodiments _c

As typical examples of the compound (I) of the present invention,

Formula (VII)

[In the formula, R ³ represents a chlorine atom, a trifluorenomethyl group, a difluoromethoxy group or a trifluoromethoxy group, and R represents a C 1 -C 4 alkyl group, a C 3 -C 4 alkenyl group or a C 3 _C 4 represents an alcohol group. ]

A 2-pyrone compound represented by the formula: Formula (VIII)

[In the formula, R represents a C1-C4 alkyl group, a C3-C4 alkenyl group or a C3-C4 alkenyl group. ]

A 2-pyrone compound represented by the formula:

Equation (IX)

[In the formula, R represents a C 1 -C 4 alkyl group, a C 3 -C 4 alkyl group or a C 3 -C 4 alkyl group. ]

A 2-pyrone compound represented by the formula:

Expression (X)

[In the formula, R represents a C 1 -C 4 alkyl group, a C 3 -C 4 alkenyl group or a C 3 -C 4 alkenyl group. ]

A 2-pyrone compound represented by the formula:

Formula (XI)

[In the formula, R represents a C 1 -C 4 alkyl group, a C 3 -C 4 alkenyl group or a C 3 -C 4 alkyl group. ]

A 2-pyrone compound represented by the formula: Formula (XII)

[Wherein, R ⁵ is a halogen atom, a C 1 -C 4 alkyl group optionally substituted with a halogen atom or a C 1 -C 4 alkoxy group, or a C 1 -C 4 alkoxy group substituted with a halogen atom. R represents a C 1 -C 4 alkyl group, a C 3 -C 4 alkenyl group or a C 3-

Represents a C 4 alkynyl group. ].

A 2-pyrone compound represented by

Formula (II)

Wherein, R ^a represents a Echiru group or Ariru group. ]

And the like.

Here, examples of the C1-C4 alkyl group, C3-C4 alkenyl group or C3-C4 alkynyl group in R include, for example, a methyl group, an ethyl group, and an aryl group.

Further, in R ⁵ above,

Examples of the halogen atom include a chlorine atom and the like.

Examples of the C 1 -C 4 alkyl group which may be substituted with a halogen atom or a C 1 -C 4 alkoxy group include a methyl group, a trifluoromethyl group, and a methoxymethyl group.

Examples of the C 1 -C 4 alkoxy group substituted by a halogen atom include a difluoromethoxy group, a trifluoromethoxy group, and a 1,1,2,2-tetrafluoroethoxy group. The compound (I) of the present invention is represented by the formula (XIII), wherein X represents the same meaning as described above. (Hereinafter, may be referred to as the present intermediate (II)) by alkylation, alkenylanilide or alkynylani.

Examples of the method of alkylation, alkenyliyi or alkyliyirou include a compound represented by the formula (XIII) and a compound represented by the formula (XIV)

RY (XIV)

[Wherein, R represents the same meaning as described above, and Y represents a leaving group. ]

Or an alkenylating agent or an alkynylating agent [hereinafter may be referred to as compound (XIV). ] In the presence of a base. The reaction of the compound represented by the formula (XIII) with the compound (XIV) in the presence of a base is usually performed in a solvent. Examples of the solvent used include acid amides such as N, N-dimethylformamide and N, N-dimethylacetamide; sulfoxides such as dimethylsulfoxide; and phosphoric amide compounds such as hexamethylphosphoramide. And ketones such as aceton and methyl ethyl ketone.

Examples of the base used in the reaction include alkali metal hydrides such as sodium hydride and potassium hydride, carbonates of alkali metal such as sodium carbonate and carbonated carbonate, and silver oxide. .

Examples of the alkylating agent, alkenylating agent or alkynyl agent used in the reaction include alkyl sulfonic acid esters such as methyl methanesulfonate and aryl sulfonic acid esters such as ethyl p-toluenesulfonate. And sulphates such as dimethyl sulfate and halides such as butyl bromide, aryl bromide and propargyl bromide. The amount of the reagent used in the reaction is usually 1 mole to 2 moles per 1 mole of the compound represented by the formula (XIII), and the compound (XIV) is usually 1 mole to 2 moles. Ratio.

The reaction temperature is usually in the range of 0 ° C to 100 ° C, and the reaction time is usually 1 hour to 200 ° C. Within the time range.

After completion of the reaction, the compound (I) of the present invention can be isolated by performing post-treatment operations such as extraction of the reaction mixture with an organic solvent and drying and concentration of the organic layer. The isolated compound (I) of the present invention can be further purified by chromatography, recrystallization and the like. Of this intermediate (XIII),

Formula (III)

A 4-hydroxy-2-pyrone compound represented by

Formula (IV)

A 4-hydroxy-2-pyrone compound represented by the formula:

Equation (V)

A 4-hydroxy-2-pyrone compound represented by the formula:

Equation (VI)

The 4-hydroxy-2-pyrone compound represented by, for example, reacts dehydroacetic acid with a benzaldehyde compound (Indian J. Chem (1974), 12, 9 56).

In Table 1, _c- intermediate (XIII) which exemplifies the intermediate (XIII) represented by the compound numbers (i) to (xxiv)

Table 2 Compound No. (1) ~: _c illustrate the present invention compound (I) represented by (14) Table 2 Compound (I) of the present invention

Table 2

Table 3 illustrates the compounds (I) of the present invention represented by the compound numbers (15) to (28). _C Table 3 Compounds of the present invention (I)

(I)

The compound (I) of the present invention has an ability to suppress the transcription of a type I collagen gene. This ability is important for improving tissue fibrosis by reducing the expression of the type I collagen gene and leading to a decrease in collagen accumulation. Therefore, the compound (I) of the present invention is a composition (medicine, cosmetics, food additive, etc.) for improving tissue fibrosis by reducing the expression level of type I collagen gene and leading to a reduction in collagen accumulation. Etc.) can be used as active ingredients.

Diseases to which the transcription-suppressing composition of the present invention or the fibrosis-improving composition of the present invention can be applied include tissues that fibrillate and harden due to excessive accumulation of collagen, and as a result, the function of organs / tissues is reduced. Diseases that cause formation (ie, fibrosis) can be mentioned. For example, cirrhosis, interstitial lung disease, chronic renal failure (or a disease that leads to chronic renal failure), hyperplasia scars after inflammation, postoperative scars ゃ burn scars, scleroderma, arteriosclerosis, hypertension, etc. Illnesses and abnormalities 3392

14

The transcription-suppressing composition of the present invention and the fibrosis-improving composition of the present invention contain the present compound (I) and an inert carrier. The compound (I) of the present invention contained in these compositions is usually 0.01% to 99.99% by weight, and the inert carrier is usually 99.99% to 0.01% by weight. %. The inert carrier is a pharmaceutically acceptable carrier or excipient, and the transcription-suppressing composition and the fibrosis-improving composition of the present invention further include a pharmaceutical additive, a cosmetic additive, a food additive, and the like. May contain. In addition, the compound (I) of the present invention inhibits the ability of TGF- to promote the transcription of type I collagen gene, as shown in Example 54 described later. That is, the compound (I) of the present invention is a TGF- / S antagonist having the ability to suppress the action of T.GF-. Therefore, the compound (I) of the present invention can also be used as an active ingredient of a composition for inhibiting the action of TGF-3. TGF- / 3 is known to have the ability to promote the transition from the anagen phase to the catagen phase in the hair growth cycle [J. Invest. De Rma tol., 111, 948-954 ( 1998), FASEB J., 16, 1967-1969 (2002)]. Furthermore, it has been reported that anti-TGF-5 antibody and TGF-0 inhibitor F etuin and the like act antagonistically to the hair growth inhibitory effect of TGF-, and exhibit a hair growth promoting effect. [J. Invest. Dermatol., 1 18, 993-997 (2002), and Japanese Patent Laid-Open Publication No. 2000-342296]. Therefore, the composition for inhibiting the action of TGF-) 3, which contains the compound (I) of the present invention as an active ingredient, inhibits the promotion of the transition of the hair to the regression phase by the TGF-3 and leads to prolongation of the hair growth phase. Thus, it can be used as a composition (pharmaceuticals, cosmetics, food additives, etc.) for obtaining a hair-growth effect.

Such a TGF-suppressing composition of the present invention and a hair-growing composition of the present invention contain the present compound (I) and an inert carrier. The present compound (I) contained in these compositions is usually 0.01% to 99.99% by weight, and the inert carrier is usually 99.99% to 0.01%. % By weight. The inert carrier is a pharmaceutically acceptable carrier and an excipient, and the TGF-β inhibitory composition and the hair restoration composition of the present invention further include a pharmaceutical additive and cosmetics. It may contain additives, food additives, etc. Pharmaceutically acceptable carriers, excipients, pharmaceutical additives, food additives, cosmetic additives, etc. used in the composition can be appropriately selected according to the specific use of the composition. . In addition, the form of the composition may be, for example, various solids, liquids, and the like, depending on the specific application.

For example, when the compound (I) of the present invention is used as an active ingredient of a pharmaceutical, specific forms include, for example, powders, fine granules, granules, tablets, syrups, capsules, suspending agents, Examples include oral preparations such as emulsion preparations, extracts and pills, injection preparations, transdermal absorption preparations such as external preparations and ointments, and parenteral preparations such as suppositories and topical preparations.

Oral IJ includes, for example, gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polypyrrolepyrrolidone, crystalline cellulose, soy lecithin, sucrose, fatty acid esters, Carriers such as luk, magnesium stearate, polyethylene glycol, magnesium silicate, and caustic anhydride, excipients, binders, disintegrants, surfactants, lubricants, fluidity promoters, diluents, preservatives, coloring It can be manufactured according to a usual method using pharmaceutical additives such as agents, fragrances, stabilizers, humectants, preservatives, and antioxidants.

The dosage varies depending on the age, sex, body weight, degree of disease, type of the composition of the present invention, dosage form, etc. of the mammal to be administered. About 1 mg to about 2 g of the ingredient, preferably about 5 mg to about 1 g of the active ingredient may be administered. In addition, the above-mentioned daily dose can be administered once or in several divided doses.

Of the parenteral preparations, injections include water-soluble solvents such as physiological saline and sterilized Ringer's solution, water-insoluble solvents such as vegetable oils and fatty acid esters, isotonic agents such as pudose and sodium chloride, and dissolution aids. It can be produced according to a usual method using pharmaceutical additives such as agents, stabilizers, preservatives, suspending agents and emulsifiers. Liquid preparations for external use, transdermal absorption agents such as gel ointments, suppositories for rectal administration, and the like can also be produced according to ordinary methods. In order to administer such a parenteral preparation, injection (subcutaneous, intravenous, etc.), transdermal administration, or rectal administration may be used. local The 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 ethylene-butyl acetate polymer. The pellet may be surgically implanted into the tissue to be treated.

The dosage varies depending on the age, sex, body weight, degree of disease, the type of the composition of the present invention, the dosage form, etc. of the mammal to be administered. You may administer from 0.1 mg to about 50 mg. In addition, the above-mentioned daily dose can be administered once or in several divided doses.

When the compound (I) of the present invention is used by adding it to cosmetics, specific forms of the cosmetics to which the compound is added include, for example, liquid, milky, cream, lotion, ointment, gel, Aerosol, mousse, etc. can be mentioned. Lotions can be manufactured according to ordinary methods using, for example, cosmetic additives such as suspending agents, emulsifiers, and preservatives.

The dose varies depending on the age, sex, body weight, degree of disease, the type of the composition of the present invention, the dosage form, etc., of the mammal to be administered, but is usually about 0.01 mg as an active ingredient in a human adult. About 5 O mg may be administered. The above-mentioned daily dose can be administered once or in several divided doses.

When the compound (I) of the present invention is used as a food additive, specific forms of the food to which the additive is added include, for example, powders, tablets, drinks, ingestible gels and syrups. Mixed liquids, for example, seasonings, Japanese sweets, western confectionery, ice confectionery, beverages, spreads, pastes, pickles, canned bottles, processed meat, fish and fishery products, processed milk and eggs, processed vegetables, and processed fruit products And general foods and drinks such as processed cereals, etc.

. It can also be added to feed and feed for domestic animals, poultry, bees, silkworms, fish, and other domestic animals.

The dosage varies depending on the age, sex, weight, degree of disease, the type of the composition of the present invention, the dosage form, etc. of the mammal to be administered. About 500 mg may be administered. The above-mentioned daily dose can be administered once or in several divided doses. Example

Hereinafter, the present invention will be described more specifically with reference to examples. Example 1 (Production of Intermediate (XIII) (Part 1))

15.0 g of dehydroacetic acid, 9.47 g of benzaldehyde and 2 ml of piperidine were dissolved in 150 ml of chlorophonolem, and heated under reflux for 2 hours 30 minutes while removing water with a Soxhlet extractor filled with molecular sieves . Further, 15.0 g of dehydroacetic acid and 2 ml of piperidine were added, and the mixture was heated under reflux for 2 hours and 30 minutes while removing water. After cooling to room temperature, the reaction solution was washed sequentially with 10% hydrochloric acid and saturated saline, dried over anhydrous magnesium sulfate, and concentrated. The residue was subjected to silica gel column chromatography to obtain crystals. The crystals were recrystallized from a mixture of chloroform and hexane and then ethanol to give 4-hydroxy-3- [3-phenyl-1-oxo-2-prober] -6-methyl-2H- 8.15 g of pale yellow crystals of pyran-2-one [compound number (i)] were obtained.

Melting point: 130-133 ° C Example 2 (Production of this intermediate (XIII) (Part 2))

In the same manner as in Example 1 except that 32.32 g of 2-chlorobenzaldehyde was used in place of benzaldehyde, 4-hydroxy-3- [3- (2-chlorophenyl) -1- Oxo-2-propinyl] _6-methyl-2H-pyran-2-one (Compound No. (ii)) 7.77 g was obtained.

Melting point: 131-134 ° C Example 3 (Preparation of Intermediate (XIII) (Part 3))

In the same manner as in Example 1 except that 32.14 g of 3-cyclopentene benzaldehyde was used in place of benzaldehyde, 4-hydroxy-3- [3- (3-cyclobutene) — There was obtained 11.15 g of [1-oxo-2-propynyl] -1-methyl-2-H-pyran-12-one (compound number (iii)).

Melting point: 150.5-: 153 ° C 3392

18

Example 4 (Production of Intermediate (XIII) (Part 4))

In the same manner as in Example 1 except that 32.14 g of 4-cyclobenzaldehyde was used instead of benzaldehyde, 4-hydroxy-3- [3- (4-chlorophenyl) —1-— Oxo-2-probe] -6-methinole_2H-pyran-2-one (Compound No. (iv)) 9.20 g was obtained.

Melting point: 159-16.5 ° C Example 5 (Production of the present intermediate (XIII) (Part 5))

4-Hydroxy-3- [3- (2-methylphenyl) -1_oxo-2propane was prepared in the same manner as in Example 1 except that 25.00 g of 2-methylbenzaldehyde was used instead of benzaldehyde. Nyl] -6-methyl-2H-pyran-2-one (Compound No. (V)) 7.49 g was obtained.

Melting point: 143 ~ 144 ° C

1 H-NMR (300 MHz, CDC 1 3) δ (p pm): 2. 29 (s, 3 H), 2. 50 (s, 3H), 5. 96 (s, 1 H), 7. 20 ~ 7.35 (m, 3H), 7.79 (dd, 1H, J = l. 1, 8.2Hz), 8.23 (d, 1H, J = 15.6Hz), 8.30 (d, 1H) , J = 15.6 Hz), 12.32 (s, 1 H) Example 6 (Production of Intermediate (XIII) (Part 6))

In the same manner as in Example 1 except that 25.65 g of 3-methylbenzaldehyde was used instead of benzaldehyde, 4-hydroxy-3- [3- (3-methylphenyl) -1-oxo-2-pro- 5.27 g of 6-methinole-2H-pyran-2-one (compound number (vi)) was obtained.

Melting point: 135.5-: 136. C Example 7 (Production of this intermediate (XIII) (Part 7))

Instead of benzaldehyde, 4.08 g of 4-methoxybenzanolaldehyde was used Otherwise, in the same manner as in Example 1, 4-hydroxy-3- [3- (4-methoxyphenyl) -1-oxo-2-proberyl] -6-methyl-2H-pyran-2 0.77 g of -one (compound number (vii)) was obtained.

Melting point: 205 ~ 208.5 ° C Example 8 (Production of this intermediate (XIII) (No. 8))

4-Hydroxy-3- [3- (3-ethoxyphenyl) 1-1- was carried out in the same manner as in Example 1 except that 3.00 g of 3-ethoxybenzaldehyde was used instead of benzaldehyde. 2.07 g of pale yellow crystals of oxo-2-propynyl] _ 6-methyl-2H-pyran-2-one (Compound No. (viii)) were obtained.

Melting point: 136-137 ° C

'H-NMR (300MHz, CDC 1 3) δ (ρ pm): 1. 44 (t, 3Η, J = 7. 1Hz), 2. 28 (s, 3H), 4. 08 (q, 2 H, J = 7.1 Hz), 5.96 (s, 1H), 6.97 (dt, 1H, J = 1.7, 7.8Hz), 7.19 (d, 1H, J = 2.0Hz), 7. 26 to 7.36 (m, 2 H), 7.92 (d, 1 H, J = 15.7 Hz), .8.29 (d, 1 H, J = 15.7 Hz), 12.35 (s, 1H) Example 9 (Production of Intermediate (XIII) (Part 9))

In the same manner as in Example 1 except that 3-trifluoromethylbenzaldehyde 33.7 Og was used in place of benzaldehyde, 4-hydroxy-3- [3- (2-trifluoride port) was used. 3.14 g of methylphenyl) -1-oxo-2-propenyl] -6-methyl-2H-pyran-2-one (compound number (ix)) was obtained.

Melting point: 1 10-110.5 ° C Example 10 (Production of this intermediate (XIII) (part 10))

In the same manner as in Example 1 above, except that 3-trifluoromethylbenzaldehyde 33.4 Og was used instead of benzaldehyde, 4-hydroxy-3- [3- (3- 14.26 g of (methyl fluoride in trifluoromethyl) -1-oxo-2_propenyl) -6-methyl-2H-pyran-2-one (Compound No. (X)) was obtained.

Melting point: 133-13.5 ° C Example 11 (Production of this intermediate (XIII) (Part 11))

In the same manner as in Example 1 except that 4-monotrifluoromethylbenzaldehyde 17.4 Og was used in place of benzaldehyde, 4-hydroxy-3- [3- (4-trifluoromethyl methylphenate) was used. )-1-oxo-2-propenyl) -6-methyl-2H-pyran-2-one (Compound No. (xi)) 8.87 g was obtained.

Melting point: 149-: 150 ° C Example 12 (Production of this intermediate (XIII) (Part 1 2))

4-Hydroxy-3_ [3_ (2-nitrophenyl) -1-oxo- was obtained in the same manner as in Example 1 except that 29.15 g of 2-nitrobenzaldehyde was used instead of benzaldehyde. 2-Propenyl] -6-methyl-2H-pyran-2-one (Compound No. (xii)) 15.77 g was obtained.

Melting point: 158.5-: 159 ° C Example 13 (Production of this intermediate (XIII) (Part 13))

4-Hydroxy-3_ [3- (3-nitrophenyl)-1- was prepared in the same manner as in Example 1 except that 10.13 g of 3-nitrobenzaldehyde was used instead of benzaldehyde. Oxo-2-propinyl] -6-methyl-2H-pyran-2-one (Compound No. (xiii)) 7.67 g was obtained.

Melting point: 183 to 183.5 ° C Example 14 (Production of the intermediate (XIII) (Part 14))

In the same manner as in Example 1 except that 42.0 g of benzaldehyde was used instead of benzaldehyde, 4-hydroxy-3- [3- (4-ditrophenyl) was used. ) 1-oxo-2-propinyl] -6-methyl-2H-pyran-2-one (Compound No. (xiv)) 17.38 g was obtained. '

Melting point: 243.5 to 244 ° C Example 15 (Production of this intermediate (XIII) (part 15))

In the same manner as in Example 1 except that 7.87 g of 3-methoxymethylbenzylaldehyde was used instead of benzaldehyde, 4-hydroxy-3- [3- (3-methoxymethylphenyl) 1-oxo-2 —Propenyl) 6-methyl-2H-pyran-2-one (II-Drug Compound No. (XV)) 2.06 g was obtained.

Melting point: 18.5-: L 19 ° C

^{: H-NMR (300MHz, CDC} 1 3) δ (ρ m): 2. 29 (s, 3 Η), 3. 42 (s, 3Η), 4. 49 (s, 2 Η), 5. 96 ( s, 1Η), 7.35 to 7.45 (m, 2Η), 7.55 to 7.65 (m, 2Η), 7.97 (d, 1Η, J = 15.8Ηζ), 8 32 (d, 1Η, J = 15.7Ηζ), 12.35 (s, 1Η) Example 16 (Preparation of this intermediate (XIII) (16))

4-Hydroxy-3- [3- (2-difluoromethoxyphenyl) —1 was prepared in the same manner as in Example 1 except that 5.00 g of 2-difluoromethoxybenzaldehyde was used instead of benzaldehyde. -Oxo-2-propenyl] -6-methyl-2H-pyran-2-one (Compound No. (xvi)) (2.11 g) was obtained as pale yellow crystals.

Melting point: 178-179 ° C

^{1 H-NMR (30 OMH z} , CDC 1 3) δ (p pm): 2. 29 (s, 3 H), 5. 97 (s, 1H), 6. 59 (t, 1 H, J = 73 1Hz), 7.20 (d, 1H, J = 8.5Hz), 7.28 (t, 1H, J = 7.2Hz), 7.43 (dt, 1H, J = 1.8, 8.OH z), 7.87 (dd, 1 H, J = 1.4, 7.8 Hz), 8.24 (d, 1 H, J = 16.0 Hz), 8.35 (d, 1H , J = 16.1 Hz), 12.40 (s, 1 H) Example 17 (Production of Intermediate (XIII) (Part 17))

4-Hydroxy-3- [3- (3-difluoromethoxyphenyl) — 1-oxo —2-Propenyl) -6_methyl-2H-pyran-2-one (compound number (xvii)) 2.52 g was obtained.

Melting point: 122-123 ° C

^{1 H-NMR (30 OMH z} , CDC 1 3) δ (ρ pm): 2. 30 (s, 3 Η), 5. 98 (s, 1Η), 6. 55 (t, 1 Η, J = 73 5Ηζ), 7.19 (dd, 1 1, J = 1.8, 8.1 Hz), 7.38 to 7.48 (m, 2H), 7.55 (d, 1H, J = 7 7Hz), 7.89 (d, 1H, J = 15.8Hz), 8.30 (d, 1H, J = 15.7Hz), 12.56 (s, 1H) Of body (XIII) (18))

4-Hydroxy-3- [3- (4-difluoromethoxyphenyl) —1 was used in the same manner as in Example 1 except that 6.13 g of 4-difluoromethoxybenzaldehyde was used instead of benzaldehyde. _Oxo-2-propininole] -6-methyl-2H-pyran-2-one (compound number (xviii)) 4.33 g was obtained.

Melting point: 167 ~: 168 ° C

'H-NMR (30 ΟΜΗζ, CDC 1 3) δ (p pm): 2. 29 (s, 3 H), 5. 97 (s, 1H), 6. 57 (t, 1 H, J = 73. 3 Hz), 7.16 (d, 2H, J = 8.6 Hz), 7.70 (d, 2H, J = 8.6 Hz), 7.92 (d, 1H, J = 15.8 Hz), 8 26 (d, 1 H, J = 15.8 Hz), 12.43 (s, 1 H) Example 19 (Production of this intermediate (XIII) (part 19))

In the same manner as in Example 1 above, except that 5.0 g of 2-trifluoromethoxybenzaldehyde was used instead of benzaldehyde, 4-hydroxy-3- [3- (2-trichlorobenzene) was used. Fluorometoxyphenyl) 1-oxo_2_propininole] —6-methinole 2 H-pyran-12-one (compound number (xix)) (2.0 g) was obtained.

Melting point: 11.8-11.8 ° C

^{X H-NMR (30 0MH z} , CDC 1 3) δ (p pm): 2. 30 (s, 3H), 5. 9 8 (s, 1 H), 7. 3 0 (dd, 1 H, J = 1.3, 6.6Hz), 7.36 (t, 1H, J = 7.6Hz), 7.46 (dt, 1H, J = 2.0, 7.9Hz), 7 .90 (dd, 1H, J = l.4, 7.8Hz), 8.19 (d, 1H, J = 15.8Hz), 8.35 (d, 1H, J = 15.8Hz), 12.58 (s, 1H

) Example 20 (Production of the present intermediate (XIII) (No. 20))

4-hydroxy-3- [3- (3-trifluoromethoxyphenyl) was performed in the same manner as in Example 1 except that 5.0-Og of 3-trifluoromethylbenzaldehyde was used instead of benza / redide. ) -1 -oxo-2-propidinyl] -6-methyl-2H-pyran-2-one (Compound No. (XX)) 1.81 g was obtained.

Melting point: 108 ~ 108.5 ° C

¹ H-NMR (30 OMH z, CDC ") δ (p pm): 2.30 (s, 3 H), 5.98 (s, 1 H), 7.28 (d, 1 H , J = 7.6 Hz), 7.45 (t, 1 H, J = 7.9 Hz), 7.48 (s, 1 H), 7.63 (d, 1 H, J = 7.7 Hz) ), 7.89 (d, 1 H, J = 15.8 Hz), 8.31 (d, 1 H, J = 15.7 Hz), 12.61 (s, 1 H Example 21 (Preparation of Intermediate (XIII) (Part 21))

In the same manner as in Example 1 except that 6.77 g of 4-trifluoromethoxybenzaldehyde was used instead of benzaldehyde, 4-hydroxy-3- [3- (4-trifluoromethoxyphenyl) — 0.84 g of 1-oxo-2-propyl] -6-methyl-2H-pyran-12-one (compound number (xxi)) was obtained.

Melting point: 115-118 ° C ^{X H-NMR (300MHz, CDC} 1 3) δ (ppm): 2. 29 (s, 3 H), 5. 98 (s, 1H), 7. 26 (d, 2H, J = 8. 4Hz), 7.71 (d, 2H, J = 8.8Hz), 7.91 (d, 1H, J = 15.7Hz), 8.28 (d, 1H, J = 15.7Hz), 12 53 (s, 1H) Example 22 (Production of Intermediate (XIII) (Part 22))

4-hydroxy-3- [3_ (2-trifluoromethylthiophene) was prepared in the same manner as in Example 1 except that 5.OO g of 2-trifluoromethylthiobenzaldehyde was used instead of benzaldehyde. 1.76 g of (enyl) -1-oxo-2-propenyl) -6-methyl-2H-pyran-2-one (compound number (xxii)) was obtained.

Melting point: 128 ~ 128.5 ° C

_{iH- NMR (300MHz, CDC 1 3} ) δ (p pm): 2. 30 (s, 3H), 5. 99 (s, 1H), 7. 47 (dt, 1 H, J = 1. 3, 6 1 Hz), 7.58 (t, 1H, J = 7.1 Hz), 7.79 (d, 1 H, J = 7.7 Hz), 8.00 (dd, 1H, J = 1.2 , 7.9Hz), 8.31 (d, 1H, J = 15.6Hz), 8.60 (d, 1H, J = 15.7Hz), 12.59 (s, 1H) Example 23 (Production of this intermediate (XIII) (Part 23))

In the same manner as in Example 1 except that 25.0 g of 3- (1,1,2,2-tetrafluoroethoxy) benzaldehyde was used instead of benzaldehyde, 4-hydroxy-3 -{3_ [3- (1,1,2,2-tetrafluoroethoxy) phenyl] -1-oxo-2-propyl} -6-methyl-2H-pyran-2-one (compound Number (xxiii)) 10.6 was obtained.

Melting point: 118-119 ° C

1 H-NMR (300 MHz, CDC ") δ (ρ pm): 2.30 (s, 3 H), 5.94 (tt, 1 H, J = 2.8, 53.1 Hz), 5.98 (s , 1H), 7.28 (dd, 1H, J = 1., 9.2Hz), 7.44 (t, 1H, J = 7.9Hz), 7.49 (s, 1H), 7. 61 (d, 1 H, J = 7.8 Hz), 7.89 ( d, 1 H, J = 15.7 Hz), 8.31 (d, 1 H, J = 15.7 Hz), 12.58 (s, 1 H) Example 24 (this intermediate ( XIII) (Part 24))

In the same manner as in Example 1 except that 5.00 g of 4- (1,1,2,2-tetrafluoroethoxy) benzaldehyde was used instead of benzaldehyde, 4-hydroxy-3 -{3— [4 -— (1,1,2,2-tetrafluoroethoxy) phenyl]-1-oxo—2-produr}-6-methyl-2H-pyran-2-one (Compound No. (xxiv)) 1.79 g was obtained.

Melting point: 1 15.5-: 1 16 ° C

^{X H-NMR (30 0MHz,} CDC 1 3) δ (ρ pm): 2. 29 (s, 3 H),

5.92 (tt, 1H, J = 2.8, 52.9Hz), 5.97 (s, 1H), 7.

27 (d, 2H, J = 8.6Hz), 7.7 1 (d, 2H, J = 8.6Hz), 7.

92 (d, 1 H, J = 15.8 Hz), 8.29 (d, 1 H, J = 15.7 Hz), 12.48 (s, 1 H) Production of Compound (I) (Part 1)

Hexamethylphosphoramide 32. 74 g of sodium hydride (60% oil, 1 "raw) was suspended in 2 ml of 1-ml, and 4-hydroxy 3- [3-phen-nore 1- was suspended at about 0 ° C. Oxo-2-propioninole] —6-methyl-2H-pyran-2-one (4.77 g) was added, and the mixture was heated to room temperature and stirred for 37 minutes. After that, the mixture was stirred for 2 days at room temperature, and then the reaction mixture was poured into ice water and extracted with ethyl acetate.The organic layer was washed with saturated saline and dried over anhydrous magnesium sulfate. The residue was subjected to silica gel column chromatography and subjected to 4-methoxy-3- [3-phenyl-1-oxo-2-propylenyl] -6-methyl-2H-biran_2_one [ 1.96 g of a pale yellow oil of Compound No. (1) was obtained.

¹ H-NMR (300 MHz, CDC ") δ (p pm): 2.34 (s, 3H), 3.92 (s, 3 H), 6.13 (s, 1 H), 7 1 2 (d, 1 H, J = 16.1 Hz), 7.35 to 7.45 (m, 3H), 7.52—7.62 (m, 2H), 7. 61 (d, 1 H, J = 15.8 Hz) Example 26 (Production of Compound (I) of the Present Invention (Part 2))

4-Hydroxy _ 3— [3-Feninole 1-year-old oxo 2 —Propenyle] -1 6-Methinole 2H—Pyran—In place of 2-one, 4-hydroxy—3- [ 3- (2-chloro-2-phenyl) -1-oxo-2-propyl] -6-methinole-2H-pyran-2-one Same as Example 25 above except for using 7.00 g To give 4-methoxy-3- [3- (2-chloromethyl)-1-oxo-2-propenyl] -6-methyl-2H-pyran-2-one [compound number (2 )] 1.06 g was obtained.

Melting point: 161 ~: 162 ° C

'H-NMR (300MHz, CDC ") δ (ppm): 2.35 (s, 3H), 3.95 (s, 3H), 6.13 (s, 1H), 7.12 (d, 1H, J = 16.0 Hz), 7.20-7.40 (m, 2H), 7.40 to 7.45 (m, 1H), 7.66 to 7.75 (m, 1H ), 8.02 (d, 1 H, J = 16. OH z) Example 27 (Production of Compound (I) of the Present Invention (Part 3))

4-Hydroxy-3— [3-Feninole_1-oxo—2-propenile] —6 _Methyl-2H-pyran-2-one is replaced with 4-hydroxy-3— [3- (3-chloro Methyl phenyl) -1-oxo-2-propenyl] _6-methynole-2H-pyran-2-one In the same manner as in Example 25 described above, except that 8.00 g of 4-methoxy-3- [ 1.26 g of 3- (3-chlorophenyl) -1-1-oxo-2-propenyl] -6-methyl-2H-pyran-2-one [Compound No. (3)] was obtained.

Melting point: 122-123 ° C

^! H-NMR (300MHz, CDC ") δ (p pm): 2.35 (s, 3H), 3.95 (s, 3H), 6.13 (s, 1H), 7.15 (d, 1 H, J = 16.2 Hz), 7.26-7.40 (m, 2H), 7.40 to 7.50 (m, 1H), 7.56 to 7.58 (m, 1H ), 7.55 (d, 1H, J = 15.9 Hz) 0303392

27 Example 28 (Production of Compound (I) of the Present Invention (Part 4))

13.40 g of 4-hydroxy-3- [3- (3-chlorophenyl) -1-oxo-2-propenyl] -6-methyl-2H-pyran-2-one in 13 Oml of dimethylformamide 2.03 g of sodium hydride (60% oil) was suspended at about 0 ° C. The mixture was heated to room temperature and stirred for 30 minutes. Next, 8.37 g of aryl bromide was added, and the mixture was stirred at 55 ° C for 2 hours. Thereafter, the reaction mixture was poured into ice water and extracted with ethyl acetate. The organic layer was washed with saturated saline, dried over anhydrous magnesium sulfate, and concentrated. The residue was subjected to silica gel column chromatography, and the obtained solid was washed with t-butyl methyl ether to give 4-arinoleoxy-3- [3- (3-chlorophenyl) -1-oxo-2--2-. Propenyl] -6-methyl-2H-pyran-2-one [Compound No. (4)] 1.60 g was obtained.

Melting point: 81. 5-82. C

¹ H-NMR (400 MHz, CDC ") δ (ρ ρ m): 2.33 (s, 3Η), 4.69 (dt, 2Η, J = 1.5, 5.1 Hz), 5.34 ( dd, 1 H, J = 1.0, 10.8 Hz), 5.42 (dd, 1 H, J = 1.0, 17.4 Hz), 5.88 to 6.00 (m, 1H), 6 07 (s, 1 H), 7.14 (d, 1 H, J = 16.2 Hz), 7.31 (q, 1 H, J = 7.4 Hz), 7.34 (dt, 1 H , J = 2.0, 6.6 Hz), 7.43 (brd, 1 H, J = 7.1 Hz), 7.54 (d, 1H, J = 15.9 Hz), 7.54 (brs, 1H) Example 29 (Production of Compound (I) of the Present Invention (Part 5))

4-Hydroxy 3 -— [3-Hue-no-le-one-one-one-year-old 2-—Pro-Peninole] —6-Methino-le 2H-Pyran—In place of 2-one, 4-hydroxy _3-[3-(4 1-oxo-l-2-propidinyl] -6-methyl-2H-pyran-2-one In the same manner as in Example 25 except that 7.00 g was used, 4 2.06 g of -methoxy-3- [3- (4-chlorophenyl) -1-oxo-2-propenyl] -6-methyl-2H-pyran-2-one [compound number (5)] was obtained.

Melting point: 164-165 ° C

^{1 H-NMR (300MHz, CDC} 1 3) δ (ρ pm): 2. 35 (s, 3 H), 3.94 (s, 3H), 6.13 (s, 1H), 7.12 (d, 1H, J = 15.6 Hz), 7.34 (d, 2H, J = 8.5Hz) , 7.50 (d, 2H, J = 8.5 Hz), 7.56 (d, 1H, J = 16.0 Hz) Example 30 (Production of Compound (I) of the Present Invention (Part 6))

4-Hydroxy-3— [3-Feni / le-1-oxo-2-2-probe-nore] —6-methinole 2H-pyran-2-one Instead of 4-hydroxy-3 -— [3- (2 -Methylphenyl) -1-oxo-2-propenyl] -6_methyl-2H-pyran-2-one In the same manner as in Example 25 above, except that 5.49 g of 4-methoxy-3 -[3- (2-Methylphenyl) -1-oxo-2-propenyl] -6-methyl-2H-pyran-2-one [Compound No. (6)] 0.46 g was obtained.

Melting point: 132 ~ 132.5 ° C

¹ H-NMR (30 OMHz, CDC ") δ (p pm): 2.35 (s, 3H), 2.44 (s, 3H), 3.95 (s, 3H), 6.13 (s, 1H), 7.07 (d, 1H, J = 15.7Hz), 7.14 to 7.30 (m, 3H), 7.58 to 7.66 (m, 1H), 7 94 (d, 1 H, J = 15.8 Hz) Example 31 (Production of Compound (I) of the Present Invention (Part 7))

4-Hydroxy _ 3— [3-Feninole 1-oxo _ 2 _propininole] -1 6-methinole 2 Instead of 2H-pyran-2-one, 4-hydroxy _3-[3- (3 -Methylphenyl) -1-oxo-2-propenyl] -6-methyl-2H-pyran-2-one In the same manner as in Example 25 except that 3.87 g of 4-methoxy-3 was used. — [3- (3-Methylphenyl) -1-oxo-2-propenyl] _6-methyl-2 H-pyran-2-one [Compound No. (7)] 1.27 g was obtained.

Melting point: 113.5-15-1 ° C

'H-NMR (300MHz, CDC ") δ (pm): 2.34 (s, 3H), 2.36 (s, 3H), 3.93 (s, 3H), 6.12 (s, 1H), 7.11 (d, 1H, J = 15.9Hz), 7.15 to 7.30 (m, 2H), 7.35 to 7. 40 (m, 2H), 7.58 (d, 1H, J = 16.OHz) Example 32 (Production of the present invention compound (I) (No. 8))

4-Hydroxy-3_ [3- (3-methylphenyl) -l-oxo-2-propyl] _ 6-methyl-2H-pyran-2-one in dimethylformamide 10 Om 1 1. 19 g and 11.44 g of anhydrous potassium carbonate were suspended, and 10.37 g of acrylyl bromide was added thereto, followed by stirring at 50 ° C for 7 hours and 30 minutes. Then, after that, the reaction mixture was poured into ice water and extracted with ethyl acetate. The organic layer was washed with saturated saline, dried over anhydrous magnesium sulfate, and then concentrated. The residue was subjected to silica gel column chromatography, and the obtained solid was washed with t-butyl methyl ether to give 4-aryloxy-3- [3- (3-methylphenyl) -1-oxo-2-propenyl. ] -6-Methyl-2H-pyran-2-one [Compound No. (8)] 2.78 g was obtained.

Melting point: 70-70.5 ° C

'H-NMR (400MHz, CDC 1 3) δ (p pm): 2. 3 2 (s, 3H), 2. 3 6 (s, 3H), 4. 6 7 (dt, 2 H, J = 1 4, 5.2Hz), 5.32 (dd, 1H, J = l. 0, 10.8Hz), 5.40 (dd, 1H, J = 1.0, 17.4Hz) ), 5.70—5.98 (m, 1 H), 6.06 (s, 1H), 7.09 (d, 1H, J = l 6.1 Hz), 7.1 8 (d, 1H, J = 7.6 Hz), 7.25 (t, 1H, J = 7.8Hz), 7.33 to 7.42 (2H), 7.57 (d, 1 H, J = 16.2 Hz) Example 33 (Production of Compound (I) of the Present Invention (No. 9))

4-Hydroxy 3_ [3 -pheninole 1-oxo 2 -propenile] -1- 6 -methinole 2 H-pyran 2_one Instead of 4-hydroxy-3- [3--(4-methoxyphene) El) -l-oxo-2-propynyl] -6-methyl-2H-pyran-2-one In the same manner as in Example 25 except that 10.00 g of 4-methoxy- There were obtained 4.40 g of 3- [3- (4-methoxyphenyl) _1-oxo-2-propenyl] -6-methyl-2H-pyran-2-one (compound number (9)). Melting point: 1 18 ~: 1 18.5 ° C

Ή-NMR (300 MHz, CDC ") δ (p pm): 2.34 (s, 3H), 3.84 (s, 3H), 3.92 (s, 3H), 6.11 ( s, 1 H), 6.89 (dd, 2H, J = 1.9, 8.8 Hz), 6.98 (d, 1 H, J = 15.7 Hz), 7.5 1 ( dd, 2H, J = 2.0, 8.7 Hz), 7.55 (d, 1H, J = 16 .OHz) Example 34 (Production of Compound (I) of the Present Invention (10) )

4-Hydroxy—3— [3-Feninole 1—oxo—2—Propernole] —6—Methinole 2H—Pyran—instead of 2—one, 4-hydroxy-3-—3— (3 —Ethoxyphenyl) — 1-year-old xo-2-propenyl) -6-methyl-2H-pyran-2-one 1.14 g, and 0.70 g of getyl sulfate instead of dimethyl sulfate Ethoxy-3- [3- (3-ethoxyphenyl) -1-oxo-2-propenyl] -6-methyl-2H-pyran- 2 (Compound No. (10)) 0.18 g was obtained.

1 H-NMR (40 OMH z, CDC ") δ (p pm): 1.40 (t, 3Η, J = 7.6Η), 1.42 (t, 3H, J = 7.1Η) ζ), 2.33 (s, 3Η), 4.05 (q, 2H, J = 7.1 Hz), 4.19 (q, 2Η, J = 7.1 Hz) , 6.08 (s, 1H), 6.92 (dd, 1H,] = 2.7, 8.3Hz), 7.07 (d, 1H, J = 15.9 Hz ), 7.06 to 7.29 (m, 3H), 7.55 (d, 1H, J = 15.9 Hz) Example 35 (Production of compound (I) of the present invention (the 1 1))

4-Hydroxy-3— [3_pheninole 1-oxo-2_propioninole] -16-methinole 2H-pyran-2-one Instead of 4-hydroxy-3— [3— (3— Ethoxyphenyl) -1-oxo-2-propenyl] -6-methyl-2H-pyran-2-one 3.00 g, and allylic bromide 1.45 g instead of dimethyl sulfate Other than that, 4-arinoleoxy-3- [3- (3-ethoxyphenyl) -1-oxo-2-propynyl] -6-methyl-2H-pyran was prepared in the same manner as in Example 25. -2 (Compound No. (1 1)) 0.23 g was obtained. Melting point: 124 ~: 125 ° C

¹ H-NMR (300 MHz, CDC ") δ (p pm): 1.42 (t, 3 H, J = 6.9 Hz), 2.32 (s, 3 H), 4.05 (q, 2 H , J = 7.4 Hz), 4.67 to 4.68 (m, 2H), 5.33 (d, lH, J = 10.6 Hz), 5.41 (d, 1H, J = 17.1 Hz) , 5.87 to 5.98 (m, 1H), 6.07 (s, 1H), 6.92 (dd, 1H, J = 2.0, 7.9Hz), 7.08 to 7.33 ( m, 3H), 7.09 (d, 1 H, J = 15.8 Hz), 7.55 (d, 1 H, J = 15.9 Hz) Example 36 (Production of compound (I) of the present invention (part 12 ))

4-Hydroxy-3— [3-pheninole 1-oxo-2—probe-norre] —6-methinole 2H—Pyran-2-one Instead of 4-hydroxy-3_ [3— (2-trifluoro L-methylphen-)-1-oxo-2-propyl] -6-methyl-2H-pyran-2-one The procedure of Example 25 was repeated except that 2.50 g was used. -Methoxy-3- [3- (2-trifluoromethylphenyl)-1-oxo-2-propenyl] -6-methyl-2H-pyran-2-one [Compound No. (12)] 0.42 g Obtained.

Melting point: 149 ~ 149.5 ° C

'H-NMR (300MHz, CDC 1 3) δ (ρ pm): 2. 35 (s, 3 H), 3. 94 (s, 3H), 6. 13 (s, 1H), 7. 11 (d , 1 H, J = 15.7 Hz), 7.46 (t, 1 H, J = 7.5 Hz), 7.56 (t, 1 H, J = 7.4 Hz), 7.69 (d, 1 H, J = 7.8 Hz), 7.80 (d, 1 H, J = 7.7 Hz), 7.94 (dq, 1H, J = 2.1, 15.7 Hz) 37 (Production of Compound (I) of the Present Invention (Part 13))

4-Hydroxy— 3— [3-Feninole 1—oxo—2-propeninole] — 6—Methylenol 2H—Instead of pyran-2-one, 4-hydroxy-3- [3— (3-Trifluoromethylpheninole) 1-oxo-2-propinyl] -6-methinole-2H-pyran-2-one Same as Example 25 except that 5.00 was used. To give 4-methoxy-3_ [3- (3-trifluoromethy 0.41 g of 1-oxo-2-propininole] -6-methinole-2H-pyran-2-one [compound number (13)] was obtained.

Melting point: 120-122 ° C

^{X H-NMR (300MH z,} CDC 1 3) δ (ρ pm): 2. 3 6 (s, 3H), 3. 9 6 (s, 3H), 6. 14 (s, 1 H), 7. 2 2 (d, 1 H, J = 15.8 Hz), 7.50 (t, 1 H, J = 7.8 Hz), 7.62 (d, 1 H, J = 8.5 Hz), 7.63 (d, 1H, J = 15.8Hz), 7.74 (d, 1H, J = 7.7Hz), 7.80 (s, 1H) 38 (Production of Compound (I) of the Present Invention (Part 14))

4-Hydroxy—3 -— [3- (3-Methinolephenyno) 1-loxo-2-—propenol] -1-6-methyl-2-H-pyran-2-one Instead of 4-hydroxy- 3- [3- (3-trifluoromethylphenyl) -1-oxo-2-propyl] -6-methyl-2H-pyran-2-one The above example except that 5.0 Og was used. In the same manner as for 32, 4-aryloxy_3_ [3- (3-trifluoromethylphenyl) -1-oxo-2-propyl] -6-methyl-2H-pyran-2-one [compound No. (14)] 1.02 g was obtained.

Melting point: 102 ~: 103 ° C

^{1 H-NMR (400MH z,} CDC 1 3) δ (p pm): 2. 34 (s, 3H), 4. 70 (dt, 2H, J = 1. 5, 5. 1 H z), 5. 3 5 (dd, 1 H, J = 1.2, 10.5 Hz), 5.43 (dd, 1 H, J = 1.0, 17.3 Hz), 5.

9 0—6.00 (m, 1H), 6.09 (s, 1H), 7.21 (d, 1H, J = 16.1 Hz), 7.50 (t, 1 H, J = 7.8 Hz, 7.6 2 (d, 1 H, J = 8.8 Hz), 7.62 (d, 1 H, J = 1 5.9 Hz), 7.7 3 (d, 1 H, J = 7.8 Hz), 7.79 (s, 1 H) Example 39 (Production of Compound (I) of the Present Invention (Part 15))

4-Hydroxy—3— [3-Feninole 1-oxo—2-propenile] -1-6-methinole—2H—Pyran-2-one Instead of 4-hydroxy 3 -— [3 -— (4-trif Norelomethyl feninole ) — 1-oxo-2-propioninole] — 6-methinole 2 H-pyran-1-one In the same manner as in Example 25, except that 5.57 g, 4-methoxy-3- [3- (4-Trifluoromethylphenyl) _1-oxo-2-propenyl] -6-methyl-2H-pyran-2-one [Compound No. (15)] 1.22 g was obtained.

Melting point: 136 ~: 138 ° C

NMR (30 OMH z, CDC ") δ (p pm): 2.36 (s, 3H), 3.96 (s, 3H), 6.14 (s, 1H), 7.24 (d, 1 H, J = 16.0 Hz), 7.58 to 7.72 (m, 5 H) Example 40 (Production of Compound (I) of the Present Invention (Part 16))

4-Hydroxy-3-[3-pheninole-1-oxo-2--2-propane / re] -6-methinole 2 Instead of H-pyran-2-one, 4-hydroxy-3- [3- (2-nitrophenyl) — 1-oxo-2-propidinyl] -6-methyl-2H-pyran-2-one 10. 4-g-methoxy-3 -— [3-— (2-Ditropheuel) — 1-oxo-2-propenyl) -6-methyl-2H-pyran_2_one [Compound No. (16)] 0.97 g was obtained.

Melting point: 157-158 ° C

^{1 H-NMR (300MHz, CDC} 1 3) δ (p pm): 2. 35 (s, 3 H), 3. 98 (s, 3H), 6. 15 (s, 1H), 7. 03 (d , 1H, J = 16.0 Hz), 7.53 (dt, 1H, J = 1.3, 8.4Hz), 7.65 (dt, 1H, J = 0.9, 7.2Hz) , 7.74 (dd, 1 H, J = 1.1, 7.8 Hz), 7.98 (d, 1 H, J = 15.8 Hz), 8.03 (dd, 1 H, J = 1.2 , 9. OH z) Example 41 (Production of Compound (I) of the Present Invention (Part 17))

4-Hydroxy-3- (3-pheninole 1-oxo-2-2-propynole) -1-6-methinole-2H-pyran-2-one is replaced with 4-hydroxy-3— [3 -— (3— Nitrophenyl) —1-oxo —2-Propaninole] — 6-Methinole-2H-pyran-2-one 4.13 g In the same manner as in Example 25, 4-methoxy-3- [3- (3-nitrophenyl) -1-1-oxo-2-propenyl] -6-methyl_2H-pyran-2-one [ Compound number (17)] 1.12 g was obtained.

Melting point: 184-18.4 ° C

^{1 H-NMR (300MHz, CDC} 1 3) δ (p pm): 2. 37 (s, 3 H), 3. 98 (s, 3H), 6. 15 (s, 1H), 7. 30 (d , 1 H, J = -15.8 Hz), 7.56 (t, 1 H, J = 8.0 Hz), 7.65 (d, 1 H, J = 15.9 Hz), 7.88 (d, 1 H,] = 7.7 Hz), 8.22 (dd, 1 H, J = 1.8, 8.8 Hz), 8.40 (t, 1 H, J = 1.9 Hz) 42 (Production of compound (I) of the present invention (18))

4-Hydroxy-3— [3-Fe2 / re-1-oxo-2-propaninole] -6-methinole 2 Instead of H-pyran-2-one, 4-hydroxy-3— [3— (4 —Nitrophenyl) -1-1-oxo-2-propenyl] _6-methyl-2H-pyran-2-one 10. Except for using Og, 4-methoxy-3- 2.94 g of [3- (4-ditrophenyl) -1-oxo-2-propanidinyl] -6-methyl-2H-pyran-2-one [compound number (18)] was obtained.

Melting point: 222-22.5 ° C

^{1 H-NMR (300MHz, CDC} 1 3) δ (p pm): 2. 37 (s, 3 H), 3. 98 (s, 3H), 6. 15 (s, 1H), 7. 33 (d , 1 H, J = 16.0

Hz), 7.65 (d, 1H, J = 15.8Hz), 7.71 (d, 2H, J = 8.7Hz), 8.23 (d, 2H, J = 8.7Hz) 43 (Production of Compound (I) of the Present Invention (Part 19))

4-Hydroxy-3_ [3-pheninolei 1-oxo—2-propidinyl] -1-6-methylinole 2H-Pyran-2-one is replaced by 4-hydroxy-3- [3- (3-methoxymethyl Phenyl) -l-oxo-2-propenyl) -6-methinole-2H-pyran-l 2-one In the same manner as in Example 25 except that 4.34 g of 4-methoxy was used. -3- [3- (3-Methoxymethylphen Nyl) -1-oxo-2-propyl] -6-methyl-2H-pyran-2-one [1.95 g] of a pale yellow oil of [Compound No. (19)] was obtained.

^{1 H-NMR (3 00MH z} , CDC 1 3) δ (p pm): 2. 3 5 (s, 3 H), 3. 40 (s, 3H), 3. 9 3 (s, 3H), 4 46 (s, 2 H), 6.12 (s, 1 H), 7.14 (d, 1 H, J = 15.7 Hz), 7.32 to 7.38 (m, 2H), 7.45 to 7.52 (m, 1H), 7.54 (s, 1H), 7.61 (d, 1H, J = 16.1 Hz) Example 44 (Production of Compound (I) of the Present Invention (No. 20))

4-Hydroxy 3 _ [3 -Fenizole 1 -oxo 1 2 -propeninole] -6 -methinole-2 H-pyran-2-one Instead of 4-hydroxy-3- [3- ( 2-Difluoromethoxyphenyl) 1-oxo-2-probene] -6-methinole 2H-pyran-2-one Same as Example 25 except that 2.00 g was used. To give 4-methoxy-3- [3- (2-difluoromethoxyphenyl) -1_oxo-2-propenyl] -6-methyl-2H-pyran-2-one [Compound No. (20) 0.65 g of a pale yellow crystal was obtained.

Melting point: 12.6-126.5 ° C

^J H-NMR (30 ΟΜΗ, CDC ") δ (pm): 2.35 (s, 3H), 3.94 (s, 3H), 6.13 (s, 1H), 6 .5 3 (t, 1H, J = 73.6Hz), 7.13 to 7.26 (m, 3H), 7.38 (dt, 1H, J = 1.8, 7 9 Hz), 7.70 (dd, 1 H, J = 1.5, 7.8 Hz), 7.87 (d, 1 H, J = 16.1 Hz) Example 45 (compound of the present invention ( Production of I) (Part 2 1))

4-hydroxy-3— [3-phenyl-1-oxo-2-2-propeninole] -6-methinoleh 2H-pyran-2-one Instead of 4-hydroxy-3- [3- (3 —Difluoromethoxyphenyl) 1-1-oxo-2-propenyl] -6-methyl-2H-pyran-2-one Same as Example 25 except that 2.00 g was used. , 4-Methoxy-3- [3- (3-difluoromethoxyphenyl)-1-oxo-2--2-propyl}-6-methyl-2H-pyran-2-one [compound No. (21)] 0.20 g was obtained.

Melting point: 1 14.5-: 1 15 ° C

^{1 H-NMR (30 0MH z} , CDC 1 3) δ (pm): 2. 3 6 (s, 3 H), 3. 9 5 (s, 3H), 6. 1 3 (s, 1 H), 6.5 2 (t, 1 H, J = 7 3.8 Hz), 7.13 to 7.47 (m, 4 H), 7.15 (d, 1 H, J = 1 6. 2 Hz), 7.57 (d, 1 H, J = 15.9 Hz) Example 46 (Production of Compound (I) of the Present Invention (Part 2 2))

4-Hydroxy-3— [3-Fenii / lei 1-year-old oxo-2—propininole] -1-6-methinole 2H-pyran-2-one Instead of 4-hydroxy-3 -— [3- ( 4-Difluoromethoxy) phenol--1-oxo-2-propioninole-6_methinole-2H-pyran-2-one In the same manner as in Example 25 except that 2.55 was used. 4-methoxy-3- [3- (4-difluoromethox) pheninole 1_oxo-2-propidinyl] -6-methyl-2H-pyran-2-one [Compound No. (22)] 1.1 g were obtained.

Melting point: 105-: 105.5 ° C

¹ H-NMR (300 MHz, CDC ") δ (p pm): 2.35 (s, 3H), 3.94 (s, 3H), 6.13 (s, 1H), 6. 5 5 (t, 1H, J = 73.3Hz), 7.10 (d, 1H, J = 16.1Hz), 7.11 (d, 2H, J = 8 4Hz), 7.57 (d, 2H, J = 9.OHz), 7.58 (d, 1H, J = 15.2Hz) Example 47 (Compound (I) of the present invention) Manufacturing (part 2 3))

4-Hydroxy-3-[3-Feninole-1-oxo-2- 2-propeninole] -6-methinole 2 Instead of H-pyran-2-one, 4-hydroxy-3-[3- (2 —Triphnorelomethoxy) Hue-Lu 1-oxo-2-prob] -1-6-methyl-2H-pyran-2-one In the same manner as in Example 25 except that 1.85 g was used. , 4-Methoxy-3- [3- (2-trifluoromethoxy) phenyl-1-oxo-2-propenyl] -6-methyl-2H-pyran-2-one [Compound number (2 3) 0.52 g was obtained. Melting point: 123 ~ 123.5 ° C

^{1 H-NMR (30 ΟΜΗζ,} CDC 1 3) δ (ρ pm): 2. 35 (s, 3Η), 3. 94 (s, 3H), 6. 13 (s, 1H), 7. 19 (d , 1H, J = 16.2 Hz), 7.26 to 7.35 (m, 2H), 7.41 (dt, 1H, J = 1.7, 7.9 Hz), 7.74 (dd, 1H, J = 1.5, 7.7Hz), 7.84 (d, 1H, J = 16.1Hz) Example 48 (Production of Compound (I) of the Present Invention (Part 24))

4—Hydroxy—3— [3-Feni / re ———————————— / ————————————————————————————————————— instead of 4-hydroxy-3 — [3 -— (3-Triphnorelomethoxy) pheninole—1oxo—2—propininole] —6—methyl / le—2H—pyran—2-one Except for using 1.41 g, In the same manner as in Example 25 above, 4-methoxy-3- [3- (3-trifluoromethoxy) phenyl-1-oxo-2-propenyl] -6-methyl-2H-pyran-2-one [ Compound number (24)] 0.51 g was obtained.

Melting point: 98 ~ 98.5 ° C

^{1 H-NMR (300MHz, CDC} 1 3) δ (ρ pm): 2. 36 (s, 3 Η), 3. 95 (s, 3Η), 6. 13 (s, 1 Η), 7. 17 ( d, 1Η, J = 16.1 Hz), 7.22 (dd, 1Η, J = 1.6, 9.ΟΗζ), 7.36 to 7.46 (m, 2Η), 7.50 ( d, 1Η, J = 7.9 Hz), 7.58 (d, 1Η, J = l

Example 49 (Production of Compound (I) of the Present Invention (Part 25))

4-Hydroxy-3— [3-Feninole 1-year-old oxo—2-Propaninole] —6-Methinole-2H-pyran-2-one Instead of 4-hydroxy-3--[3--(4 -Trifluoromethoxy) feninole-1-oxo-2-propioninole-1-6-methinolee 2H-pyran-2-one In the same manner as in Example 25 except that 1.6 g was used. , 4-Methoxy-3- [3- (4-triphneolelomethoxy) pheninole-1-oxo-2-2-propeninole] -6-methinole-2H-pyran-2-one 25)] 1.39 g were obtained. 38 pale yellow oil

¹ H-NMR (30 OMHz, CDC ") δ (ppm): 2.36 (s, 3 H),

3.95 (s, 3H), 6.13 (s, 1H), 7.14 (d, 1H, J = 16.2 Hz), 7.22 (d, 2H, J = 8.5 Hz), 7.60 (d, 2H, J = 8.7 Hz), 7.60 (d, 1H, J = 15.8 Hz) Example 50 (Production of Compound (I) (Part 26))

4-Hydroxy-3- [3-Fenii / lei 1-sulfone 2-propidinole] -1-6-methinole-2ϋ-pyran-2-one Instead of 4-hydroxy-3- [3- ( Same as Example 25 above, except that 0.96 g of 2-triphnoreolomethylthio) ferro 1_oxo-2-propyl] -6-methyl-2Η-pyran-2-one was used. To give 4-methoxy-3- [3- (2-trifluoromethylthio) phenyl-1-oxo-2-propyl] -6-methyl-2H-pyran-2-one

[Compound No. (26)] 0.16 g was obtained.

Melting point: 11 1 ~: I 12 ° C

^{1 H-NMR (30 OMHz,} CDC 1 3) δ (ρ pm): 2. 36 (s, 3H), 3. 94 (s, 3H), 6. 14 (s, 1H), 7. 14 (d , 1H, J = 16.1 Hz), 7.42 (dt, 1H, J = 1.3, 7.7Hz), 7.53 (t, 1H, J = 7.6Hz), 7.75 (d, 1 H, J = 7.6 Hz), 7.84 (dd, 1 H, J = 1.1, 7.8 Hz), 8.23 (d, 1 H, J = 15.9 Hz) 51 (Production of Compound (I) of the Present Invention (Part 27))

4-Hydroxy-3— [3-Feninole—1oxo—2—Propaninole] —6—Methinole—2H-pyran—Instead of 2-one, 4-hydroxy—3— {3 -[3- (1,1,2,2-Tetrafunorolexy) pheninole] — 1-oxo—2—propininole) — 6-methinole 1 2H—pyran—2—one 1.38 g Except for using, in the same manner as in Example 25 described above, 4-methoxy-3- {3- [3- (1,1,2,2-tetrafuronoloethoxy) pheninole] -1_oxo- 2-Propylene} -1-6-methylino-2H-pyran-2-one [Compound No. (27)] 0.15 g was obtained. Melting point: 112 ~ 113C- ¹ H-NMR (300MHz, CDC ") δ (pm): 2.36 (s, 3H), 3.95 (s, 3H), 5.91 (tt, 1H, J = 2.8, 52) , 8Hz), 6.13 (s, 1H), 7.16 (d, 1H, J = 16.OHz), 7.20 to 7.53 (m, 4H), 7.58 (d, 1H) , J = 15.9 Hz) Example 52 (Production of Compound (I) of the Present Invention (Part 28))

4-Hydroxy 3— [3-Fenora 1—Kizo 1 2 _Propaninole] — 6—Methinole _ 2 Ti-Pyran 1—Instead of 2-one, 4-hydroxy-3- {3— [4— (1,1,2,2-tetrafluoroethoxy) phenyl] -1-oxo-2-propenyl} -6_methyl-2H-pyran-2-one The above procedure was followed except that 1.30 g was used. In the same manner as in Example 25, 4-methoxy-3- {3- [4- (1,1,2,2-tetrafluoroethoxy) phenyloxo-2-propyl} —6-methyl-2-H —Pyran-2-one [Compound No. (28)] 0.18 g was obtained. Pale yellow oil

¹ H-NMR (30 OMHz, CDC ") δ (p pm): 2.35 (s, 3 H), 3.95 (s, 3H), 5.91 (tt, 1 H, J = 2.8 , 52.7 Hz), 6.13 (s, 1H), 7.13 (d, 1H, J-15.9 Hz), 7.22 (d, 2H, J = 8.5 Hz), 7.58 ( d, 2H, J = 8.7 Hz), 7.60 (d, 1 H, J = 14.2 Hz) Example 53 (having a reporter gene linked to the transcription regulatory region of type I collagen gene) Preparation of plasmid)

1 × 10 ⁸ normal human fetal skin fibroblasts (C 10 ntech, Cat. No. CC-1509) were cultured at 37 ° C. in a 5% C ₂ atmosphere. After washing the cultured cells twice with PBS, 3 ml of PBS was added, and the cells were detached from the vessel wall using a cell scraper (Nalgen, force tag number 179693). The detached cells were collected by centrifugation (1,500 rpm, 4 ° C., 15 minutes), suspended in 20 ml of PBS, and centrifuged again. 11 μm of Solutio n2 of DNA Extraction on Kit (Stratagene, catalog number 200600) was added to the obtained precipitate. 1, 4.8 μl of each pronase was added, and the mixture was shaken at 60 ° C. for 1 hour, and the obtained mixture was left on ice for 10 minutes. Next, 4 ml of the Soulution 3 of the above kit was added to the mixed solution, mixed, and then left on ice for 5 minutes. After centrifugation (3,000 rpm, 4 ° C, 15 minutes), the supernatant was recovered. To the collected supernatant, 21 RNases per 1 ml of the supernatant was added, and left at 37 ° C for 15 minutes. To this mixed solution, two volumes of ethanol was added and mixed, and the white filamentous substance (genome DNA) that appeared was recovered. The collected genomic DNA was washed with 70% ethanol and air-dried. The air-dried genomic DNA was prematurely melted into 500 O1 of 1 OmM Tris-HC1, 1 mM EDTA (pH 8.0) (hereinafter referred to as TE).

The obtained genomic DNA solution (equivalent to genomic DNA), an oligonucleotide having the nucleotide sequence of SEQ ID NO: 1 and an oligonucleotide having the nucleotide sequence of SEQ ID NO: 2 (10 pmo 1 / μ 1) 1 μl each, distilled water 29 1, Τa Ka Ra LA Taq (Takara Shuzo, catalog number RRO 02A) buffer 5 μ1, Mg ²⁺ solution 51, dNTP miture 5 u 1 and 0.51 was mixed with TaKaRaLATaq (Takara Shuzo, catalog number RR0O2A). The obtained mixture was kept at 94 ° C for 5 minutes, and then 30 cycles of 94 ° C, 1 minute, then 60 ° C, 1 minute, and 72 ° C, 1 minute were taken as one cycle. . The mixture was subjected to 2% agarose gel electrophoresis to recover about 0.5 kb of DNA. The recovered DNA was treated with phenol / chloroform and then precipitated with ethanol to recover the DNA. The recovered DNA was dissolved in ultrapure water, Nhel 2.51 and Hindill 2.51 were added to the solution, and the mixture was kept at 37 ° C for 3 hours. Next, the lysate was subjected to 2% agarose gel electrophoresis to recover about 0.5 kb of DNA. The collected DNA was precipitated with ethanol to recover the DNA again (hereinafter, referred to as collagen mouth motor DNA).

On the other hand, after digesting vector pGL3 having a nucleotide sequence encoding firefly luciferase (Pupa Mega, Catalog No. E1751) with NheI and HindIII, it was subjected to agarose gel electrophoresis in the same manner as described above. kb of DNA was recovered. The collected DNA was precipitated with ethanol to recover the DNA again. To the recovered DNA Add distilled water 441, Buffer 51 attached to Alkaline P hosphatase (Takara Shuzo, Kataguchi No. 2 12 OA) and 1 μl of Alkaline P hosphatase (Takara Shuzo, catalog No. 2 12 OA) This mixture was kept at 65 ° C. for 30 minutes. Next, the mixed solution was treated twice with phenol'-cloth form and then precipitated with ethanol to recover DNA (hereinafter, referred to as Luc vector DNA). Next, after mixing about 20 ng of the above collagen promoter DNA and about 20 ng of Luc vector, the same amount of a DNA Ligationkit Ver 2 enzyme solution was added thereto, and the mixture was incubated at 16 ° C for 24 hours. Add Escherichia coli 5Hdα (ΤΟΥΟΒΟ, Cat. No. DNA-903) to the mixture, leave it on ice for 30 minutes, and incubate at 42 ° C for 45 seconds. The seeds were seeded on an LB plate containing g / m 1 ampicillin sodium (Nacalai, Cat. No. 02 7-39) and left at 37 ° C for 24 hours. The single colony that appeared was cultured at 37 ° C. for 12 hours in 2 ml of an LB medium containing 50 μg / m 1 ampicillin. Plasmid DNA was prepared from the resulting 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. As a result, the plasmid (hereinafter, referred to as COL-Luc) contains 342 to 1557 bases (the transcription start point is +1) of the transcription regulatory region of the human type I collagen α2 chain gene. It was confirmed that the DNA sequence had a nucleotide sequence connected to a nucleotide sequence encoding firefly luciferase downstream of the sequence. Example 54 (Measurement of transcriptional control ability of type I collagen gene of test compound using expression level of reporter gene as index)

Seeded normal human fetal skin, fibroblasts 1 X 1 0 ⁶ cells 1 0 Omm dish, inactivated fetal calf serum (hereinafter referred to as FBS. G ibco, Kata port grayed No. 2 1 1 40 - 0 7 9 ) Medium containing 10% (VZV)) (Nissui Pharmaceutical, Kataguchi No. 059 19) medium (hereinafter, the medium is referred to as D-MEM (+)). The cells were cultured at 37 ° C. in a 5 ° / ₀ CO ₂ atmosphere. Next, the medium was replaced with Dulbecco's-MEM medium without FBS (hereinafter, the medium is referred to as D-MEM (-)). Was.

12 g of COL-Luc was added to 300 μl of D-MEM (−), and the resulting mixture was allowed to stand at room temperature for 45 minutes (solution 1). Also, 20 μl of Lipofectine (Gibco, Cat. No. 18292-001) was added to 300 μl of D-MEM (-), and the resulting mixture was left at room temperature for 45 minutes. (Solution 2). Next, the solution 1 and the solution 2 were mixed, left at room temperature for 10 minutes, and 5.4 ml of D-MEM (-) was added to the mixed solution and mixed. After the mixture was added to the normal human fetal skin fibroblasts, the cells were cultured overnight at 37 ° C. in a 5% C ₂ atmosphere. The next day, the culture supernatant was removed from the dish, the cells were washed twice with PBS, and the cells were detached by adding PBS lm1 containing 0.25% trypsin. After adding D-MEM (+) to the cells and mixing well, the cell suspension was dispensed in 1 ml portions into a 12-well plate, and this was added at 37 ° C and 5% CO _2. The cells were cultured under an atmosphere for 1 hour.

The compound (I) of the present invention represented by the compound number (1) to (8) or (10) to (28) is added to the cells cultured in this manner so that the dimethyl sulfoxide is adjusted to 1 mM. (Hereinafter, referred to as DMSO) were added to each solution (final concentration: ΙμΜ). In the case of the compound (I) of the present invention represented by the compound number (9), a solution prepared by dissolving the compound in DMS O to 10 mM was added (final concentration: 10 M ). As a control, DMSO was added to the rice cake.

After 1 hour, TGF-j3 the (P epro T ech) 5 xg / ml aqueous solution or distilled water was 1 1 added and further cultured for 24 hours under 3 7 ° C, 5% CO z atmosphere. After washing the cultured cells twice with PBS, add a cell lysing agent (Toyo Ink, Cat. No. PD10) 150 / il and add a cell scraper (Na1gen, Cat. No. The cells were detached from the vessel wall using 1 79 6 9 3). After recovering the obtained cell suspension, the cell suspension was centrifuged (15,000 rpm, 4 ° C, 5 minutes) to recover the supernatant. After transferring each collected supernatant to a 96-well plate, use a Lucase solution (2 OmM Trinice (pH 7.0)) with MICR OLUMAT LB96P (EG & G BERTHOLD). 8), 2. 6 7 mM Mg S0 4, 0. ImM EDTA, 3 3. 3mM DTT, 2 70 C oenz yme A , 530 μm, 470 M Luciferin) 50 ul was automatically dispensed to the plate, and the luminescence in each cell was measured (De 1 ay: 1.6 seconds, Me as Interva 1: 5 seconds) ).

On the other hand, the recovered supernatant 5a1 or cell lysis reagent 51 is pre-dispensed into a 96-well plate and diluted 5-fold with a Protein Assay solution (Bio-Rad, Cat. No. 500-0006) 200 After shaking and mixing in addition to 1, the absorbance at 595 nm in each well was measured using a microplate reader (Bio-Rad, Benemark). Based on the obtained value, the transcription activity was calculated according to the following equation.

Transcription activity = [Light emission (supernatant added) One light emitted (cell lysing agent added)] / [595 nm absorbance (supernatant added)-595 nm absorbance (cell lysate added)]

Next, based on the calculated transcription activity, the inhibitory effect of the test compound on the transcription promoting ability of the type I collagen gene possessed by TGF- ^ S was calculated as the degree of inhibition according to the following formula.

P and degree of harm = transcriptional activity (DMSO and TGF-added test group)-transcription activity (compound and TGF-] 3 added test group) / [transcription activity (DMSO and TGF_ added test group) Transcription activity ( DM SO and TGF—] 3 free test)] xl OO

The degree of inhibition of the compound (I) of the present invention represented by the compound numbers (1) to (28) was 70 or more. It was confirmed that these compounds have the ability of TGF- to inhibit the type I collagen gene transcription promoting ability and suppress the type I collagen gene transcription. Example 55 (Improvement of diabetic nephropathy by administration of compound (I) of the present invention)

As a diabetic nephropathy model animal, 10-week-old 9-week-old male C57BL / KeJ Jcl-db / db mice [Nippon Challus Riva Co., Ltd.] were used. In addition, as control animals, 10 9-week-old male C57BL / KeJ Jcl_db / m mice [Japan Charl. Riva Co., Ltd.] were used. The present compound (I) represented by the compound number (7) [hereinafter, referred to as the present compound (7). Or a compound (T) of the present invention represented by compound number (27) [hereinafter, referred to as compound (7) of the present invention. ] And basic feed (CRF-1, Oriental Yeast Industry) After mixing uniformly, a feed containing the compound of the present invention was prepared. The body weight and food intake per mouse were measured every two weeks of breeding, and the amount of the test substance mixed with the basal feed was determined based on the measured values. The thus-prepared feed containing the compound of the present invention was fed freely to the above diabetic nephropathy model animal for 8 weeks (compound administration group). As a P-easy control administration group, the above diabetic nephropathy model animals were bred while freely feeding on a basal feed, and halofuginone, a TGF-iS inhibitor, was administered intraperitoneally every other day. As a non-administration group, one group of the above-mentioned diabetic nephropathy model animals was bred with free access to a basal feed. In addition, as a control group, one group of the above-described control animals was bred with free access to a basal feed.

After breeding for 8 weeks, urine was collected from each group of mice for one day, and urine volume was measured. The urinary albumin concentration of the collected urine was measured using A1bwe11M (EXOCELL), and the amount of urinary albumin excreted in one day was calculated. As statistical processing of the obtained results, the F-test of the variance ratio was performed between the two groups of the non-administration group and each of the other groups. If there was no significant difference in variance, Student's t test (one-sided) When there was a significant difference in variance, the Aspin-Welch test (one-sided) was performed. Table 4 shows the results. It was confirmed that the compound (7o) of the present invention and the compound (27) of the present invention have an ability to improve diabetic nephropathy. Table 4

Group Animal Administered substance Dose Urinary assay

A / Ref "Min amount

Non-administration group Nephropathy mote, 0 60.91

Animals

Positive control nephropathy mote, halofuki, non-p 0.01

Compound (7) nephropathy Mote "Le present compound 10 m _g / k _g body weight /曰38. 39 p <0. 01 administered group animals (7)

Compound (27) Nephropathy mote, compound of the present invention 10 _mg / kg body weight / day 44.12 p 0.05 0.05 administration group Animals (27)

Control group Control animal 0 37.21 p 0.05 Industrial applicability

According to the present invention, a composition for improving tissue fibrosis by reducing the expression level of type I collagen gene in tissue and reducing the amount of collagen accumulated (ie, a collagen accumulation inhibitor or a therapeutic agent for fibrosis) The development of 'offer is possible. In addition, it is possible to develop and provide a composition (namely, a hair restorer) for obtaining a hair-growth effect by inhibiting the promotion of the transition of the hair to the catagen phase by TGF- and leading to an extension of the hair growth period. Sequence listing free text

SEQ ID NO: 1

Oligonucleotide primers designed to amplify collagen promoter DNA

SEQ ID NO: 2

Oligonucleotide primers designed to amplify collagen promoter DNA

Claims

The scope of the claims

[In the formula, X is a hydrogen atom, a halogen atom, a halogen atom or a C 1 -C 4 alkyl group which may be substituted by a C 1 -C 4 alkoxy group, a nitro group, a CI-C 4 alkoxy group, or a WR. ^One group (W represents an oxygen atom or a sulfur atom, R ¹ represents a C 1 -C 4 alkyl group substituted with a halogen atom), and R represents a C 1 -C 4 alkyl group, C 3 -C 4 represents an alkenyl group or a C3-C4 alkynyl group. ]

A 2-pyrone compound represented by the formula: 2. The 2-pyrone compound according to claim 1, wherein R is a C1-C4 alkyl group or a C3-C4 alkenyl group.

3. The 2-pyrone compound according to claim 1, wherein R is a methyl group, an ethyl group or an aryl group. 4. The 2-pyrone compound according to claim 1, wherein X is a hydrogen atom, a halogen atom, a C1-C4 alkyl group optionally substituted with a halogen atom, or a ditro group.

5. The 2-pyrone compound according to claim 1, wherein X is a hydrogen atom, a halogen atom, a C1-C4 alkyl group, a di-to-mouth group or a trifluoro-to-methyl group.

6. The 2-pyrone compound according to claim 1, wherein X is a hydrogen atom, a chlorine atom, a methyl group, a nitro group, or a trifluoromethyl group.

7. The 2-pyrone compound according to claim 4, wherein R is a methyl group.

8. The 2-pyrone compound according to claim 4, wherein X is a chlorine atom, a methyl group or a trifluoromethyl group, and R is an ethyl group or an aryl group.

9. The 2-pyrone compound according to claim 1, wherein X is —WR ¹ group (W represents an oxygen atom or a sulfur atom, and R ¹ represents a C 1 -C 4 alkyl group substituted with a halogen atom). .

10. The 2-pyrone compound according to claim 9, wherein R ¹ is a C 1 -C 4 alkyl group substituted with one or more fluorine atoms. 11. The 2-pyrone compound according to claim 9, wherein R ¹ is a difluoromethyl group, a trifluoromethyl group or a 1,1,2,2-tetrafluoroethyl group.

12. The 2-pyrone compound according to claim 9, wherein W is an oxygen atom. 13. The 2-pyrone compound according to claim 9, wherein W is a sulfur atom, and R ¹ is a trifluoromethyl group.

14. The 2-pyrone compound according to claim 9, wherein R is a methyl group. 15. The 2-pyrone compound according to claim 1, wherein X is a C1-C4 alkoxy group.

16. The 2-pyrone compound according to claim 15, wherein X is a methoxy group or an ethoxy group.

17. The 2-pyrone compound according to claim 15, wherein R is a methyl group, an ethyl group, or an aryl group.

18. Equation (II)

Wherein, R ^a represents a Echiru group or Ariru group. ]

A 2-pyrone compound represented by the formula: 19. The 2-pyrone compound according to claim 1, wherein X is a C1-C4 alkyl group substituted with a C1-C4 alkoxy group.

20. The 2-pyrone compound according to claim 19, wherein X is a methoxymethyl group.

A 4-hydroxy-2-pyrone compound represented by the formula:

22. Equation (IV)

[In the formula, R ² represents an ethoxy group, a methoxymethyl group, or a 12,2-tetrafluoroethoxy group. ]

A 4-hydroxy-2-pyrone compound represented by the formula: 23. Equation (V)

A 4-hydroxy_2-pyrone compound represented by the formula:

2 4. Formula (VI)

A 4-hydroxy-2-pyrone compound represented by the formula:

25. Use of the 2-pyrone compound according to claim 1, for suppressing transcription of a type I collagen gene.

26. A composition for suppressing transcription of a type I collagen gene, comprising the 2-pyrone compound according to claim 1 as an active ingredient and an inert carrier.

27. The composition for inhibiting transcription of a type I collagen gene according to claim 26, which is used for improving tissue fibrosis by reducing the expression level of the type I collagen gene to reduce the amount of collagen accumulation. Use of.

28. A composition for improving tissue fibrosis, comprising the 2-pyrone compound according to claim 1 as an active ingredient and an inert carrier.

29. A treatment method for improving tissue fibrosis, which comprises administering to a mammal patient in need of such treatment an effective amount of the 2_pyrone compound according to claim 1. How to sign.

30. Use of the 2-pyrone compound of claim 1 for inhibiting the action of TGF-j3.

31. A composition for suppressing the action of TGF-J3, comprising the 2-pyrone compound according to claim 1 as an active ingredient and an inert carrier.

32. The effect-inhibiting composition of TGF- | 3 according to claim 31, for obtaining a hair-growing effect by inhibiting the promotion of the transition of the hair to the regression phase by TGF-] 3 and leading to prolongation of the hair growth phase. Use of things.

33. A hair-growing composition comprising the 2-pyrone compound according to claim 1 as an active ingredient and an inert carrier.

34. A hair-growing method comprising administering an effective amount of the 2-pin compound according to claim 1 to a mammalian patient in need of hair-growing treatment.