JP2002275063A - Hyperplasia inhibitor for capillary endothelium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a hyperplasia inhibitor for capillary endothelium, especially a medicine useful for treatment and prevention of restenosis detected after coronary angioplasty. SOLUTION: Disclosed is a hyperplasia inhibitor for capillary endothelium featured by containing as an active component a compound represented by formula (1) [wherein, X is oxygen atom or a general formula (2) (wherein, (n) is an integer from 0 to 2); R1 is hydrogen atom or an acyl group; R2 is hydrogen atom, a lower alkyl group or a lower alkenyl group; R3 is a lower alkyl group; R4 , R5 and R6 are each allowable to be same or different and each hydrogen atom or an alkyl group which may have a substituent; and R5 and R6 may also jointly form a cycloalkyl group].

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an agent for inhibiting intimal hyperplasia, and more particularly to an agent for inhibiting intimal hyperplasia containing a 2,6-di-t-butylphenol derivative as an active ingredient.

[0002]

2. Description of the Related Art It is known that coronary atherosclerosis is a major cause of ischemic heart diseases such as angina pectoris and myocardial infarction. Minimization of the vascular lumen caused by atherosclerotic intimal hyperplasia results in nutrient and oxygen deficiency in myocardial tissue and induces the above pathology. Percutaneous coronary angioplasty (Percuta) recently developed as a treatment for ischemic heart disease such as angina pectoris and myocardial infarction
neous Transluminal Corona
ryAngioplastic, hereinafter abbreviated as PTCA)
The technique involves inflating a balloon at a stenotic part of a coronary artery to physically dilate a blood vessel. However, as a problem from the early stage of development of this treatment method, it is known that even if a stenotic part of a coronary artery is dilated, restenosis of the part occurs at a frequency of about 40% 3 to 6 months after the operation ( Circulation 77,
361-371, 1988. ).

Until now, attempts have been made to prevent stenosis due to atherosclerotic intimal hyperplasia and restenosis after PTCA by using an anticoagulant, an antiplatelet agent, a drug having an inhibitory effect on medial smooth muscle proliferation and the like. . For example, various drugs have been searched (Japanese Patent Application Laid-Open Nos.
-83923, JP-A-3-118383, JP-A-4
-99775, JP-A-4-154720, JP-A-6
JP-A-135829, JP-A-6-206842, JP-A-7-25768, JP-A-7-149641, JP-A-7-223958 and the like. However, at present, there is no drug that has a clinically sufficient inhibitory effect on stenosis of blood vessels due to atherosclerotic intimal thickening and restenosis due to intimal thickening of blood vessel walls after PTCA ( Japanese clinical, 52 special edition, 869-872, 1994).

[0004]

Means for Solving the Problems The inventors of the present invention have made intensive studies to solve the above-mentioned problems, and as a result, the general formula (1)

[0005]

Embedded image (Where X is an oxygen atom or a general formula (2)

[0006]

Embedded image

Wherein n is an integer of 0 to 2, R ₁ is a hydrogen atom or an acyl group, and R ₂ is
Represents a hydrogen atom or a lower alkyl group or a lower alkenyl group, R ₃ represents a lower alkyl group, and R ₄ , R ₅ , R
₆ may be the same or different, a hydrogen atom, an alkyl group optionally having a substituent, an alkenyl group optionally having a substituent, an alkynyl group optionally having a substituent, And R ₅ and R ₆ are taken together to form a cycloalkyl group or one or more optional methylenes on the ring of the cycloalkyl group, A heterocyclic ring substituted by a nitrogen atom substituted by a sulfur atom or an alkyl group may be formed. Has been found to have a strong growth inhibitory effect on vascular smooth muscle-derived cells, and a strong inhibitory effect on intimal hyperplasia in a balloon-injured intimal hypertrophy model. Was. It has been disclosed that the compound represented by the general formula (1) is effective for treating and preventing ischemic organ disorders such as arteriosclerosis, myocardial infarction, and stroke (Japanese Patent Application Laid-Open No. 6-206842). , WO94-0893
0, WO95-27710).

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the definition of the compound represented by the general formula (1), the acyl group includes an acetyl group, a formyl group, a propionyl group, a benzoyl group, a benzyloxycarbonyl group, an aminoacetyl group, an N-methyl group. Examples include an aminoacetyl group and an N, N-dimethylaminoacetyl group. The lower alkyl group refers to a linear or branched alkyl group having 1 to 6 carbon atoms, such as a methyl group, an ethyl group, an n-propyl group, an i-propyl group, and an n-alkyl group.
Butyl, s-butyl, t-butyl and the like. The lower alkenyl group is a straight or branched alkenyl group having 2 to 6 carbon atoms, and examples thereof include a vinyl group, an allyl group, a butenyl group, and a pentenyl group.

The alkyl group represented by R ₄ , R ₅ and R ₆ is
A linear or branched alkyl group having 1 to 20 carbon atoms, such as a methyl group, an ethyl group, an n-propyl group,
Examples include i-propyl, n-butyl, s-butyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl groups. The alkenyl group refers to a linear or branched alkenyl group having 2 to 20 carbon atoms, and examples thereof include a vinyl group, an allyl group, a butenyl group, a pentenyl group, a geranyl group, and a farnesyl group. The alkynyl group refers to a linear or branched alkynyl group having 2 to 20 carbon atoms.
Examples include an ethynyl group, a propynyl group, and a butynyl group. The aryl group means a monovalent substituent obtained by removing one hydrogen atom from an aromatic hydrocarbon, and examples thereof include a phenyl group, a tolyl group, a xylyl group, a biphenyl group, a naphthyl group, an anthryl group, a phenanthryl group, and the like. is there. Substituents on an alkyl group which may have a substituent, an alkenyl group which may have a substituent, an alkynyl group which may have a substituent, and an aryl group which may have a substituent Examples thereof include a halogen atom, a lower alkyl group, a hydroxyl group, an amino group, an alkoxy group, an aryloxy group, a nitro group, and a trifluoromethyl group. The cycloalkyl group refers to a cycloalkyl group having 3 to 8 carbon atoms, and examples thereof include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, and a cyclooctyl group. Examples of the heterocyclic ring in which one or more arbitrary methylene on the ring of the cycloalkyl group is substituted by an oxygen atom, a sulfur atom, or a nitrogen atom substituted by an alkyl group include, for example, a tetrahydropyranyl group.

When X is an oxygen atom as the compound represented by the general formula (1), the following substituents are preferred. That is, R ₁ is preferably a hydrogen atom, an acetyl group, a benzyloxycarbonyl group, an aminoacetyl group, an N-methylaminoacetyl group, an N, N-dimethylaminoacetyl group. An N-dimethylaminoacetyl group is preferred. R ₂ is preferably a hydrogen atom, a methyl group, or an n-propyl group, and particularly preferably a hydrogen atom. R ₅ represents a hydrogen atom, a methyl group, an n-butyl group, an n-pentyl group, an n-hexyl group,
An i-amyl group is preferred, and an n-pentyl group is particularly preferred. R ₆ is preferably a hydrogen atom, a methyl group, an n-butyl group, an n-pentyl group, an n-hexyl group, an i-amyl group, and particularly preferably an n-pentyl group. R ₅ and R
_The ring formed by ₆ together is preferably a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group or a tetrahydropyranyl group, particularly preferably a cyclohexyl group, a cycloheptyl group or a cyclooctyl group. X represents a compound represented by the general formula (2) as a compound represented by the general formula (1).

[0011]

Embedded image

When n is an integer of 0 to 2, the following substituents are preferred. That is, R ₁ is preferably a hydrogen atom, an acetyl group, a benzyloxycarbonyl group, an aminoacetyl group, an N-methylaminoacetyl group, an N, N-dimethylaminoacetyl group, and particularly preferably a hydrogen atom, an acetyl group, an N, N An N-dimethylaminoacetyl group is preferred. R ₂ is preferably a hydrogen atom, a methyl group, or an n-propyl group, and particularly preferably a hydrogen atom. R ₅ is preferably a hydrogen atom, a methyl group, an n-butyl group, an n-pentyl group, an n-hexyl group, or an i-amyl group, and particularly preferably an n-pentyl group. R ₆ is preferably a hydrogen atom, a methyl group, an n-butyl group, an n-pentyl group, an n-hexyl group, an i-amyl group, and particularly preferably an n-pentyl group. As the ring formed by R ₅ and R ₆ together, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, and a tetrahydropyranyl group are preferable, and a cyclohexyl group, a cycloheptyl group, and a cyclooctyl group are particularly preferable. preferable.
n is preferably 0 or 1, and particularly preferably 0.

The compounds suitable for the purpose of the present invention include:
2,6-di-tert-butyl-3-methyl-4-propyloxyphenol, 4-allyloxy-2,6-di-t-
Butyl-3-methylphenol, 1,3-bis (3,5
-Di-tert-butyl-4-hydroxy-2-methylphenoxy) propane and the like.

The compound represented by the general formula (1) used in the present invention can be synthesized, for example, according to the methods described in JP-A-6-206842 and WO95-27710.

The agent for inhibiting intimal hyperplasia of the present invention can be used as various pharmaceutical compositions comprising a compound represented by the general formula (1) as an active ingredient and a physiologically harmless solid or liquid pharmaceutical carrier. Can be used. This pharmaceutical composition is prepared and used in various formulations depending on the administration method.
Formulation forms include tablets, granules, pills, capsules,
Examples include solutions, syrups, suspensions, emulsions or injections. As the pharmaceutical carrier, commonly used excipients, binders, disintegrants, lubricants, coating agents, dissolution aids, emulsifiers, suspending agents, stabilizers or solvents can be used.

The compound represented by the general formula (1) of the present invention and the above-mentioned pharmaceutical composition can be used for oral administration, parenteral administration such as intravenous injection, sustained-release administration by sustained-release preparation, or catheter for local administration. It can be used by local administration such as.

Further, according to the present invention, the actual dose of the compound represented by the general formula (1) required to suppress restenosis after percutaneous coronary angioplasty is determined by the patient's age and symptoms. The effective daily dose generally accepted is, for example, 1 to 1000 mg, preferably 100 to 300 mg, for an adult, depending on the seriousness, administration route and the like. It is preferable to administer such a dose to a patient in need of treatment once to three times a day.

[0018]

EXAMPLES The present invention will be further described below with reference to examples, but the present invention is not limited to these examples. Preparation of Compounds The following compounds 1 to 46 were synthesized by the method described in JP-A-6-206842. Compound 1 : 4,6-di-tert-butyl-5-hydroxy-
2,3-dihydrobenzofuran compound 2 : 4,6-di-tert-butyl-5-hydroxy-
2-Methyl-2,3-dihydrobenzofuran compound 3 : 5-acetoxy-4,6-di-tert-butyl
2,2-dimethyl-2,3-dihydrobenzofuran compound 4 : 4,6-di-tert-butyl-5-hydroxy-
2,2-dimethyl-2,3-dihydrobenzofuran compound 5 : 5-acetoxy-4,6-di-tert-butyl-
2,2-diethyl-2,3-dihydrobenzofuran

Compound 6 : 4,6-di-tert-butyl-2
2-Diethyl-5-hydroxy-2,3-dihydrobenzofuran compound 7 : 4,6-di-tert-butyl-2,2-di-n-
Propyl-5-hydroxy-2,3-dihydrobenzofuran compound 8 : 4,6-di-tert-butyl-2,2-di-n-
Butyl-5-hydroxy-2,3-dihydrobenzofuran compound 9 : 5-acetoxy-4,6-di-tert-butyl
2- (1-octenyl) benzofuran compound 10 : 5-acetoxy-4,6-di-tert-butyl-2-octylbenzofuran

Compound 11 : 4,6-di-tert-butyl-5
-Hydroxy-2-octylbenzofuran compound 12 : 4,6-di-t-butyl-5-hydroxy-2-octyl-2,3-dihydrobenzofuran compound 13 : 2,4,6-tri-t-butyl-5 -Hydroxy-2,3-dihydrobenzofuran compound 14 : 4,6-di-tert-butyl-2,2-di-i
-Propyl-5-hydroxy-2,3-dihydrobenzofuran compound 15 : 4,6-di-tert-butyl-2,2-diphenyl-5-hydroxy-2,3-dihydrobenzofuran

Compound 16 : 4,6-di-tert-butyl-
2,2-dibenzyl-5-hydroxy-2,3-dihydrobenzofuran compound 17 : 4,6-di-tert-butyl-2-chloromethyl-5-hydroxy-2,3-dihydrobenzofuran compound 18 : 4,6 -Di-tert-butyl-5-hydroxy-2,3-dihydrobenzofuran-2-spiro-1'-
Cyclopentane compound 19 : 4,6-di-tert-butyl-5-hydroxy-2,3-dihydrobenzofuran-2-spiro-1'-
Cyclohexane compound 20 : 4,6-di-tert-butyl-5-hydroxy-2,3-dihydrobenzofuran-2-spiro-1'-
Cycloheptane

Compound 21 : 4,6-di-t-butyl-5
-Hydroxy-2,3-dihydrobenzofuran-2-spiro-1'-cyclooctane compound 22 : 4,6-di-tert-butyl-5-hydroxy-2,3-dihydrobenzofuran-2-spiro-4'-
Tetrahydropyran compound 23 : 4-acetoxy-3,5-di-tert-butyl-1- (2-methyl-2-propenyloxy) -2-propylbenzene compound 24 : 5-acetoxy-4,6-di-t -Butyl-2,2-dimethyl-7-propyl-2,3-dihydrobenzofuran compound 25 : 5-hydroxy-4,6-di-tert-butyl-2,2-dimethyl-7-propyl-2,3- Dihydrobenzofuran

Compound 26 : 5-acetoxy-4,6-di-tert-butylbenzofuran Compound 27 : 4,6-di-tert-butyl-5-hydroxybenzofuran Compound 28 : 4,6-di-tert-butyl 5-Hydroxy-2-methylbenzofuran compound 29 : 2,4,6-tri-t-butyl-5-hydroxybenzofuran compound 30 : 1-acetoxy-2,6-di-t-butyl-3-methyl-4-methyl Propyloxybenzene

Compound 31 : 2,6-di-tert-butyl-3
-Methyl-4-propyloxyphenol compound 32 : 1-acetoxy-4-allyloxy-2,
6-Di-tert-butyl-3-methylbenzene compound 33 : 4-allyloxy-2,6-di-tert-butyl-3-methylphenol compound 34 : 1,3-bis (4-acetoxy-3,5-)
Di-tert-butyl-2-methylphenoxy) propane compound 35 : 1,3-bis (3,5-di-tert-butyl)
4-Hydroxy-2-methylphenoxy) propane

Compound 36 : 4,6-di-tert-butyl-
2,2-di-n-pentyl-5-hydroxy-2,3-
Dihydrobenzofuran compound 37 : 4,6-di-tert-butyl-2,2-di-n
-Octyl-5-hydroxy-2,3-dihydrobenzofuran compound 38 : 4,6-di-tert-butyl-2,2-di-n
-Heptyl-5-hydroxy-2,3-dihydrobenzofuran compound 39 : 4,6-di-tert-butyl-2,2-di-n
-Hexyl-5-hydroxy-2,3-dihydrobenzofuran compound 40 : 5-acetoxy-2,2-di-i-amyl-4,6-di-tert-butyl-2,3-dihydrobenzofuran

Compound 41 : 2,2-di-i-amyl-
4,6-Di-tert-butyl-5-hydroxy-2,3-dihydrobenzofuran compound 42 : 5-acetoxy-4,6-di-tert-butyl-2-methyl-2- (4,8,12- Trimethyltrideca-3 (E), 7 (E), 11-trienyl) -2,3
-Dihydrobenzofuran compound 43 : 4,6-di-tert-butyl-5-hydroxy-2-methyl-2- (4,8,12-trimethyltrideca-3 (E), 7 (E), 11-trienyl ) -2,3
-Dihydrobenzofuran compound 44 : 4,6-di-t-butyl-5-hydroxy-2-methyl-2- (4 ', 8', 12'-trimethyltridecyl) -2,3-dihydrobenzofuran compound 45 : 5-Acetoxy-4,6-di-tert-butyl-2- (5-hydroxy-4-methyl-3 (E) -pentenyl) -2-methyl-2,3-dihydrobenzofuran compound 46 : 4,6- Di-tert-butyl-5-hydroxy-2- (5-hydroxy-4-methyl-3- (E) -pentenyl) -2-methyl-2,3-dihydrobenzofuran

Compound 47 : 5-acetoxy-4,6-di-tert-butyl-2-hydroxymethyl-2-methyl-
2,3-Dihydrobenzofuran 10.0 g of 4-acetoxy-3,5-di-tert-butyl-2- (2-methyl-2-propenyl) phenol synthesized according to JP-A-6-206842 is dissolved in 200 ml of chloroform. , Metachloroperbenzoic acid 11.0
g was added and the mixture was heated and refluxed for one day. After cooling, a saturated aqueous solution of sodium thiosulfate was added to the reaction solution, and the mixture was extracted with chloroform. The organic layer was washed with brine, dried over anhydrous magnesium sulfate and concentrated. The concentrate was purified by silica gel column chromatography (n-hexane containing 25% ethyl acetate) to give 5-acetoxy-4,6-di-tert-butyl-2-hydroxymethyl-2-methyl-2,3-. 7.3 g (70% yield) of dihydrobenzofuran (a mixture of rotamers) was obtained as a colorless oil.

¹ H NMR (270 MHz, CDC
l ₃ ) δ ppm: 1.30 (s, 9H), 1.37
(S, 9H), 1.38 (s, 1.5H), 1.45
(S, 1.5H), 2.30 (s, 3H), 3.06
(D, 0.5H, J = 15.5 Hz), 3.16 (d,
0.5H, J = 15.5 Hz), 3.38 (d, 0.5
H, J = 15.5 Hz), 3.52 (d, 0.5H, J
= 15.5Hz), 3.58-3.72 (m, 2H),
6.75 (s, 0.5H), 6.76 (s, 0.5H) Mass: 334 (M ⁺ )

Compound 48 : 4,6-di-tert-butyl-5
-Hydroxy-2-hydroxymethyl-2-methyl-
2,3-dihydrobenzofuran 114 mg of lithium aluminum hydride under nitrogen atmosphere
Is suspended in 3 ml of tetrahydrofuran.
Acetoxy-4,6-di-tert-butyl-2-hydroxymethyl-2-methyl-2,3-dihydrobenzofuran 5
A solution of 00 mg in 7 ml of tetrahydrofuran was added dropwise.
After heating under reflux for 2 hours, the temperature was returned to room temperature, ethyl acetate was added dropwise, 10% hydrochloric acid was added, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and concentrated, and the concentrate was purified by silica gel chromatography (20% ethyl acetate-containing n-hexane) to give 4,6-di-t-butyl-5.
-Hydroxy-2-hydroxymethyl-2-methyl-
2,3-dihydrobenzofuran as a white solid 320
mg (73% yield).

Melting point: 126-128 ° C. ¹ H NMR (270 MHz, CDCl ₃ ) δ ppm:
1.38 (s, 3H), 1.40 (s, 9H), 1.4
9 (s, 9H), 2.04 (bs, 1H), 3.14
(D, 1H, J = 15.5 Hz), 3.45 (d, 1
H, J = 15.5 Hz), 3.59 (d, 2H, J =
1.65 Hz), 4.74 (s, 1H), 6.65
(S, 1H) IR (cm- ¹ ): 3648, 3448, 2960 Mass: 292 (M ⁺ )

The following compounds 49 to 67 were converted to WO95-27
No. 710 was synthesized. Compound 49 : 5-acetoxy-4,6-di-tert-butyl-2,2-di-n-pentyl-2,3-dihydrobenzothiophene Compound 50 : 4,6-di-tert-butyl-5-hydroxy -2,2-di-n-pentyl-2,3-dihydrobenzothiophene compound 51 : 4,6-di-tert-butyl-5-hydroxy-2-methyl-2,3-dihydrobenzothiophene compound 52 : 4 5,6-Di-tert-butyl-5-hydroxy-2,2-dimethyl-2,3-dihydrobenzothiophene compound 53 : 5-acetoxy-4,6-di-tert-butylbenzo [b] thiophene compound 54 : 4 , 6-Di-tert-butyl-5-hydroxybenzo [b] thiophene compound 55 : 5-acetoxy-4,6-di-tert-butylbenzo [b] thiophene-1,1-dioxide

Compound 56 : 5-acetoxy-4,6-di-tert-butyl-2,3-dihydrobenzothiophene
1,1-dioxide compound 57 : 4,6-di-tert-butyl-5-hydroxy-2,3-dihydrobenzothiophene compound 58 : 4,6-di-tert-butyl-5-hydroxy-2,3- Dihydrobenzothiophene-2-spiro-
1'-cyclohexane compound 59 : 5-acetoxy-4,6-di-tert-butyl-2-iodomethyl-2-methyl-2,3-dihydrobenzothiophene compound 60 : 5-acetoxy-4,6-di-t -Butyl-2- (N, N-dimethylaminomethyl) -2-methyl-2,3-dihydrobenzothiophene

Compound 61 : 4,6-di-t-butyl-5
-Hydroxy-2- (N, N-dimethylaminomethyl)
-2-Methyl-2,3-dihydrobenzothiophene compound 62 : 5-acetoxy-2-acetoxymethyl-
4,6-Di-tert-butyl-2-methyl-2,3-dihydrobenzothiophene compound 63 : 4,6-Di-tert-butyl-5-hydroxy-2-hydroxymethyl-2-methyl-2,3 -Dihydrobenzothiophene compound 64 : 4,6-di-tert-butyl-5-hydroxy-2-methyl-2- (4,8-dimethylnon-3
(E), 7-dienyl) -2,3-dihydrobenzothiophene compound 65 : 4,6-di-tert-butyl-5-hydroxy-2-methyl-2- (4,8-dimethylnonyl) -2
3-dihydrobenzothiophene compound 66 : 4,6-di-t-butyl-5-hydroxy-2-methyl-2- (4,8,12-trimethyltrideca-3 (E), 7 (E), 11 -Trienyl) -2,3
-Dihydrobenzothiophene compound 67 : 4,6-di-t-butyl-5-hydroxy-2-methyl-2- (4,8,12-trimethyltridecyl) -2,3-dihydrobenzothiophene

[0034]

[Test Example 1] Inhibition of proliferation of vascular smooth muscle cells (in v
In vitro) A7r5 cells derived from rat aorta (ATCC-C) were used as an inhibitory effect on the growth of vascular smooth muscle cells in vitro.
RL-1444) was examined for the inhibitory effect of Compound 36 on serum cell proliferation. DME containing 10% FBS
5 × 10 ³ cells in a 96-well plate using M medium
The cells were cultured by seeding at a cell number of /0.2 ml / well. On day 3 of the culture, the medium was replaced with a DMEM medium containing 2% FBS, and on day 4 of the culture, the medium was replaced with a DMEM medium containing 0.2% FBS. After culturing for 48 hours in a DMEM medium containing 0.2% FBS, the medium was removed by suction, and the DMEM medium (0.
1 ml) and compounds were added, and the serum was stimulated to grow (2
% FBS). The test compound was dissolved and suspended in ethanol, added at 0.5 μl / well, and the ethanol concentration in each well was set to 0.5%. BrdU for 16 to 38 hours and 38 to 64 hours after growth stimulation
(10 μM) and BrdU incorporation into DNA was measured as an indicator of cell proliferation. The amount of BrdU incorporation was measured using an EIA kit using an anti-BrdU antibody (5-Bromo-2'-deoxy-uri).
Dine Labeling and Detecti
on Kit III: BohringerMannh
eim Biochemical). Table 1 shows the results.

[0035]

[Table 1]

As shown in Table 1, Compound 36 inhibited serum-stimulated vascular smooth muscle cell proliferation in a concentration-dependent manner.

[0037]

[Test Example 2] Inhibition of proliferation of vascular smooth muscle cells (in v
In vitro) A7r5 cells derived from rat aorta (ATCC-C) were used as an inhibitory effect on the growth of vascular smooth muscle cells in vitro.
RL-1444) was examined for the inhibitory effect of Compound 36 on cell proliferation by serum or PDGF. Furthermore, a growth inhibitory effect on vascular smooth muscle cells has been reported (J. Biol. Chem, 266: 61).
88-6194, 1991. ) Comparative study with the action of α-tocopherol. 5 × 10 ³ cells / 0.2 m in a 96-well plate using 10% FBS-containing DMEM medium
The cells were cultured by seeding with 1 / well of cells.
On day 3 of the culture, the medium was replaced with a DMEM medium containing 2% FBS, and on day 4 of the culture, the medium was replaced with a DMEM medium containing 0.2% FBS. After culturing in a DMEM medium containing 0.2% FBS for 48 hours, the medium was removed by suction, and the DMEM medium (0.2 ml) was removed.
And a compound, and serum or PDGF (PDGF
-BB; human: Becton Dickinso
n Labware) (FBS 2% or PDGF 4 ng / ml). The test compound was dissolved and suspended in ethanol, and 1 μl / well was added.
The ethanol concentration in the well was set to 0.5%. Eight hours after growth stimulation, Hyd was adjusted to a final concentration of 1.5 mM.
Roxyurea was added to achieve a tighter synchronization of the cell cycle. After further culturing for 14 hours, Hydroxy
urea was removed and washed, and a DMEM medium, a compound and a growth stimulating substance were added again, and further [ ³ H] -thymid
ine (1 μCi / well: 20 μCi / mmol:
NEN Research) for 5 hours [ ³ H]
-Thymidine uptake was measured as an index of cell proliferation. Table 2 shows the results.

[0038]

[Table 2]

As shown in Table 2, Compound 36 suppressed serum and PDGF-stimulated proliferation of vascular smooth muscle cells. Also, the effect was stronger than α-tocopherol.

[0040]

[Test Example 3] Inhibition of proliferation of vascular smooth muscle cells (in v
In vitro) A7r5 cells derived from rat aorta (ATCC-C) were used as an inhibitory effect on the growth of vascular smooth muscle cells in vitro.
RL-1444) against compounds 1, 4, 20, 22, 27, 31, 36, 50, 5 against cell proliferation by serum
The inhibitory effects of 2,58 were compared and studied. 5 × 10 ³ ce was added to a 96-well plate using DMEM medium containing 10% FBS.
The cells were cultured by seeding at a cell number of lls / 0.2 ml / well. On day 3 of culture, DMEM containing 2% FBS
Replace the medium with DME containing 0.2% FBS on day 4 of culture.
The medium was replaced with M medium. After culturing for 24 hours in a DMEM medium containing 0.2% FBS, a test compound was added. The test compound was dissolved and suspended in ethanol, added at 1 μl / well, and the ethanol concentration in each well was set to 0.5%.
After further culturing for 24 hours, the medium was removed by suction, and DM
EM medium (0.2 ml) and the test compound again were added,
Growth stimulation with serum (FBS 2%) was performed. 48 hours after the growth stimulation, the acid phospha in the well was added.
Tase activity was measured using a kit (Abacus Cell Pr)
introduction Kit, Clontech
Lab. Inc. CA, USA) as an index of cell proliferation. Table 3 shows the results.

[0041]

[Table 3]

As shown in Table 3, all the test compounds inhibited the proliferation of vascular smooth muscle cells induced by serum.

[0043]

Test Example 4 Action on In Vivo Intimal Hyperplasia Model (1) As an action on the in vivo intimal hyperplasia model, the inhibitory effect of compound 36 on the rabbit balloon injury model was examined. Cholesterol diet loaded rabbit (J
W: CSK male, 11 weeks old) was used with 8 birds per group to prepare a rabbit balloon injury model. High cholesterol diet (cholesterol content 1%) 2 weeks before balloon treatment
Cholesterol loading was carried out by freely ingesting. Under anesthesia, a balloon catheter (3 Frenc)
h; Baxter) to perform balloon treatment by peeling the aortic intima five times. The compound was administered orally after suspending the compound in 1% CMC (200 mg / k).
g). The control group was orally administered only a 1% CMC solution. The compound was administered once a day from one week before the balloon treatment, and once a day after the balloon treatment, oral administration was continued until the day before the anatomical evaluation.

For measurement of intimal thickening, an aortic sample taken 4 weeks after balloon treatment was used for Elastica van.
It was Giesson-stained and measured as intimal hyperplasia width and hypertrophy intimal area by image analysis under a light microscope. For the stained aortic section, the average value of the intimal thickening width and the thickened intimal area was calculated for each individual. The measurement results were evaluated except for those who had dropped off during surgery and those whose serum cholesterol level was 3500 mg / dl or more. The number of individuals used for evaluation was 6 or 5 in each group. Table 4 shows the results.

[0045]

[Table 4]

As shown in Table 4, Compound 36 showed a significant inhibitory effect on intimal hyperplasia in any of the parameters of intimal thickness and intimal area in the rabbit balloon-injured intimal hyperplasia model.

[0047]

[Test Example 5] Effect on in vivo intimal hypertrophy model (2) As action on in vivo intimal hypertrophy model, inhibition of intimal hyperplasia of compound 36 on balloon injury model using rabbits of different age from Test Example 4 The effect was examined. Furthermore, an inhibitory effect on intimal hyperplasia in a balloon injury model has been reported (Proc. Natl. Aca).
d. Sci. USA, 89: 11312-11316,
1992. ) Comparison with the action of probucol. Cholesterol diet-loaded rabbit (JW: CSK male, 15 weeks old)
Was used in a group of 7 birds to prepare a rabbit balloon injury model. Cholesterol loading was performed by having a high cholesterol diet (cholesterol content 1%) freely taken two weeks before balloon treatment. Under anesthesia, balloon treatment was performed by peeling the aortic intima five times using a balloon catheter (4 French; Baxter). The compound was administered orally (200 mg / kg) by suspending the compound in a 1% CMC solution. The control group was orally administered only a 1% CMC solution. The compound was administered once a day from one week before the balloon treatment, and once a day after the balloon treatment, oral administration was continued until the day before the anatomical evaluation. In order to evaluate the degree of balloon injury, a non-balloon-treated group (three birds) was provided.

For measurement of intimal thickening, an aortic sample collected 4 weeks after balloon treatment was used for Elastica van.
It was Giesson-stained and measured as intimal hyperplasia width and hypertrophy intimal area by image analysis under a light microscope. For the stained aortic section, the average value of the intimal thickening width and the thickened intimal area was calculated for each individual. The measurement results were evaluated except for those who had dropped off during surgery and those whose serum cholesterol level was 3500 mg / dl or more. The number of individuals used for evaluation was 5 or 4 in each group. Table 5 shows the results.

[0049]

[Table 5]

As shown in Table 5, in the rabbit balloon-injured intimal hyperplasia model, Compound 36 showed a significant inhibitory effect on intimal hyperplasia in both parameters of intimal thickness and intimal area. Also, the effect was stronger than probucol.

[0051]

Industrial Applicability The agent for inhibiting intimal hyperplasia containing a 2,6-di-t-butylphenol derivative as an active ingredient according to the present invention can be used in serum and PDG in cultured cells derived from vascular smooth muscle.
F has a strong growth inhibitory effect on cell proliferation caused by F and a strong inhibitory effect on intimal thickening in a balloon-injured intimal thickening model, which is useful for treatment and prevention of restenosis due to intimal thickening of the vascular wall after PTCA It is.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) A61P 9/10 103 A61P 9/10 103 // C07D 307/80 C07D 307/80 333/54 333/54 493/10 493 / 10 C (72) Inventor Yoshiaki Kato 1-135, Komamon, Gotemba-shi, Shizuoka Chugai Pharmaceutical Co., Ltd. F-term (reference) 4C037 PA07 4C071 AA04 AA07 BB01 BB05 CC12 EE05 FF17 HH09 LL01 4C086 AA01 AA02 BA06 BB03 CA01 MA01 NA14 ZA36 ZA40 4C206 AA01 AA02 CA17 CA19 MA01 MA04 NA14 ZA36 ZA40

Claims (20)

[Claims] 1. A compound of the general formula (1) [In the formula, X is an oxygen atom or a general formula (2). Wherein n represents an integer of 0 to 2, R ₁ represents a hydrogen atom or an acyl group, R ₂ represents a hydrogen atom or a lower alkyl group or a lower alkenyl group, and R ₃ represents Represents a lower alkyl group, wherein R ₄ , R ₅ , and R ₆ may be the same or different and are a hydrogen atom, an alkyl group optionally having a substituent, or an alkenyl group optionally having a substituent; Represents an alkynyl group which may have a substituent, an aryl group which may have a substituent, and R ₅ and R ₆ together form a cycloalkyl group or a ring of a cycloalkyl group. One or more of the above optional methylenes may form a heterocycle substituted with an oxygen atom, a sulfur atom or a nitrogen atom substituted with an alkyl group. ] An intimal hyperplasia inhibitor comprising the compound of formula (I) as an active ingredient. 2. The agent for inhibiting intimal hyperplasia according to claim 1, wherein in the general formula (1), X is an oxygen atom. 3. In the general formula (1), X is an oxygen atom, and R ₄ , R ₅ and R ₆ may be the same or different, and may have 1 to 1 carbon atoms which may have a substituent. 20 alkyl groups, alkenyl groups having 2 to 20 carbon atoms which may have a substituent, alkynyl groups having 2 to 20 carbon atoms which may have a substituent, and optionally having a substituent An aryl group, or R ₅ and R ₆ are taken together and a cycloalkyl group or one or more optional methylene on the ring of the cycloalkyl group is substituted by an oxygen atom, a sulfur atom or an alkyl group; The agent for inhibiting intimal hyperplasia according to claim 1, wherein the agent forms a heterocycle substituted with a nitrogen atom. 4. In the general formula (1), X is an oxygen atom, R ₁ is a hydrogen atom, R ₂ is a hydrogen atom, and R ₄ , R ₅ and R ₆ are the same or different. An alkyl group having 1 to 20 carbon atoms which may have a substituent,
An alkenyl group having 2 to 20 carbon atoms which may have a substituent, an alkynyl group having 2 to 20 carbon atoms which may have a substituent, or an aryl group which may have a substituent Or R ₅ and R ₆ are taken together to replace a cycloalkyl group or one or more optional methylenes on the ring of the cycloalkyl group with a nitrogen atom substituted with an oxygen atom, a sulfur atom or an alkyl group. The agent for inhibiting intimal hyperplasia according to claim 1, which forms a heterocycle. 5. In the general formula (1), X is an oxygen atom, R ₁ is a hydrogen atom, R ₂ is a hydrogen atom, and R ₄ , R ₅ and R ₆ are the same or different. An alkyl group having 1 to 20 carbon atoms which may have a substituent,
An optionally substituted alkenyl group having 2 to 20 carbon atoms, or R ₅ and R _{6 taken} together as a cycloalkyl group,
The agent for suppressing intimal hyperplasia according to claim 1. 6. In the general formula (1), X is the general formula (2). (Wherein n represents an integer of 0 to 2).
The agent for suppressing intimal hyperplasia of the blood vessel. 7. The agent of claim 1, wherein X is a sulfur atom in the general formula (1). 8. In the general formula (1), X is a sulfur atom, and R ₄ , R ₅ and R ₆ may be the same or different, and may have a hydrogen atom or a substituent. An alkyl group, an optionally substituted alkenyl group, or R ₅ and R _{6 taken} together are a cycloalkyl group;
The agent for suppressing intimal hyperplasia according to claim 1. 9. In the general formula (1), X is a sulfur atom, R ₁ is a hydrogen atom, R ₂ is a hydrogen atom, and R ₄ , R ₅ and R ₆ are the same. _{May be} different, may be an alkyl group which may have a substituent, an alkenyl group which may have a substituent, or R ₅ and R ₆ together form a cycloalkyl group is there,
The agent for suppressing intimal hyperplasia according to claim 1. 10. The agent for suppressing intimal hyperplasia according to any one of claims 1 to 9, wherein the intimal hypertrophy is coronary restenosis observed after percutaneous coronary angioplasty. 11. A compound of the general formula (1) [In the formula, X is an oxygen atom or a general formula (2). Wherein n represents an integer of 0 to 2, R ₁ represents a hydrogen atom or an acyl group, R ₂ represents a hydrogen atom or a lower alkyl group or a lower alkenyl group, and R ₃ represents Represents a lower alkyl group, wherein R ₄ , R ₅ , and R ₆ may be the same or different and are a hydrogen atom, an alkyl group optionally having a substituent, or an alkenyl group optionally having a substituent; Represents an alkynyl group which may have a substituent or an aryl group which may have a substituent, and R ₅ and R ₆ together form a cycloalkyl group or a ring of a cycloalkyl group; One or more of the above optional methylenes may form a heterocycle substituted with an oxygen atom, a sulfur atom or a nitrogen atom substituted with an alkyl group. ] An agent for inhibiting restenosis after percutaneous coronary angioplasty (PTCA), which comprises the compound of formula (I) as an active ingredient. 12. The percutaneous coronary angioplasty (PTC) according to claim 11, wherein in the general formula (1), X is an oxygen atom.
A) Post-restenosis inhibitor. 13. In the general formula (1), X is an oxygen atom, and R ₄ , R ₅ and R ₆ may be the same or different and have 1 to 1 carbon atoms which may have a substituent. 20 alkyl groups,
An alkenyl group having 2 to 20 carbon atoms which may have a substituent, an alkynyl group having 2 to 20 carbon atoms which may have a substituent, or an aryl group which may have a substituent Or R ₅ and R ₆ are taken together to replace a cycloalkyl group or one or more optional methylenes on the ring of the cycloalkyl group with a nitrogen atom substituted with an oxygen atom, a sulfur atom or an alkyl group. 12. A modified heterocycle is formed.
The post-percutaneous coronary angioplasty (PTCA) restenosis inhibitor according to the above. 14. In the general formula (1), X is an oxygen atom, R ₁ is a hydrogen atom, R ₂ is a hydrogen atom, and R ₄ , R ₅ and R ₆ are the same or different. An alkyl group having 1 to 20 carbon atoms which may have a substituent,
An alkenyl group having 2 to 20 carbon atoms which may have a substituent, an alkynyl group having 2 to 20 carbon atoms which may have a substituent, or an aryl group which may have a substituent Or R ₅ and R ₆ are taken together to replace a cycloalkyl group or one or more optional methylenes on the ring of the cycloalkyl group with a nitrogen atom substituted with an oxygen atom, a sulfur atom or an alkyl group. 12. A modified heterocycle is formed.
The post-percutaneous coronary angioplasty (PTCA) restenosis inhibitor according to the above. 15. In the general formula (1), X is an oxygen atom, R ₁ is a hydrogen atom, R ₂ is a hydrogen atom, and R ₄ , R ₅ and R ₆ are the same or different. An alkyl group having 1 to 20 carbon atoms which may have a substituent,
An optionally substituted alkenyl group having 2 to 20 carbon atoms, or R ₅ and R _{6 taken} together as a cycloalkyl group,
The post-percutaneous coronary angioplasty (PTCA) restenosis inhibitor according to claim 11. 16. In the general formula (1), X is the general formula (2) (Wherein n represents an integer of 0 to 2).
The agent for suppressing restenosis after percutaneous coronary angioplasty (PTCA) according to claim 1. 17. The percutaneous coronary angioplasty (PT) according to claim 11, wherein in the general formula (1), X is a sulfur atom.
CA) Post-restenosis inhibitor. 18. In the general formula (1), X is a sulfur atom, and R ₄ , R ₅ and R ₆ may be the same or different, and may have a hydrogen atom or a substituent. An alkyl group, an optionally substituted alkenyl group, or R ₅ and R _{6 taken} together are a cycloalkyl group;
The post-percutaneous coronary angioplasty (PTCA) restenosis inhibitor according to claim 11. 19. In the general formula (1), X is a sulfur atom, R ₁ is a hydrogen atom, R ₂ is a hydrogen atom, and R ₄ , R ₅ and R ₆ are the same. _{May be} different, may be an alkyl group which may have a substituent, an alkenyl group which may have a substituent, or R ₅ and R ₆ together form a cycloalkyl group is there,
The post-percutaneous coronary angioplasty (PTCA) restenosis inhibitor according to claim 11. 20. The percutaneous coronary angioplasty (PTCA) according to any one of claims 11 to 19, wherein the intimal hyperplasia is coronary restenosis observed after percutaneous coronary angioplasty.
Post-restenosis inhibitor.