JPH07112930A - Vascular smooth muscle cell growth inhibitor - Google Patents

Abstract

PURPOSE:To obtain a cell proliferation inhibitor for vascular smooth muscle useful for preventing and treating arteriosclerosis, diabetic angiopathy, etc. CONSTITUTION:This cell proliferation inhlbitor (or vascular smooth muscle comprises a substance having farnesyltransferase inhibitory activity, preferably UCF1 derivative of formula I (R is an alkanoyl or alkenoyl; X<1> is group of formula V; X<2> is OH or X<1> and X<2> are linked to form O) as an active ingredient. A compound of formula II wherein X<1> and X<2> are bonded to show O in the compound of formula I is obtained by reacting 2-amino-4-methoxyphenol with 1-2 equivalent of an acyl halide, acyl anhydride or a mixed acid containing the objective acyl group in the presence of a proper base in a solvent to give a compound of formula III, oxidizing the compound III in the presence of an oxidizing agent in a solvent and oxidizing the prepared compound of formula IV in the presence of a hypochlorite in a solvent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a vascular smooth muscle cell proliferation inhibitor useful for the prevention or treatment of arteriosclerosis, diabetic angiopathy and the like.

[0002]

BACKGROUND OF THE INVENTION After injury of either the vascular endothelium or the vascular wall, smooth muscle cells in the vascular media of the vascular wall lead to a growing state, ie proliferation and extracellular matrix production.
In addition to DNA synthesis and mitosis, a portion of smooth muscle cells migrate to the intima of the vessel wall and proliferate there. A substance that suppresses this proliferation can be expected to be useful in the prevention or treatment of arteriosclerosis, diabetic vascular disorder, etc. (Pro
c. Natl. Acad. Sci. USA, 90, 8474-8478, 1993).

Examples of vascular smooth muscle cell migration or growth factors include platelet-derived growth factor (PDGF) and chemokines (Chemoki).
ne) and many other factors are known. In transducing growth signals from these factors, low molecular weight GTs such as p21 ras
It has been shown that P-binding proteins play an important role. Further, recently, it has been shown that post-translational processing of the C-terminal and translocation to the cell membrane are necessary for active expression of low-molecular weight G proteins such as p21 ras. C
Cyst, the first step in the end of processing
It was revealed that the reaction of S-farnesylation of the eine residue is essential for the functional expression of p21 ras, and since this reaction is catalyzed by farnesyl transferase, farnesyl transferase has attracted attention as a target for the search for new drugs. ing.

On the other hand, as inhibitors for arteriosclerosis or diabetic inner layer injury, calcium antagonists, angiotensin converting enzyme (ACE) inhibitors, heparin, antiplatelets, HMG-CoA reductase inhibitors, etc. Although known to be effective in models, clinical efficacy in humans is still inadequate and new drugs are needed. UCF1-C (Manumycin) and its related substances are known as substances having farnesyl transferase inhibitory activity (Proc. Natl. Acad. Sci. USA, 90, 2281-2285, 1993).
Year; JP-A-4-221377).

[0005]

The object of the present invention is to proliferate smooth muscle cells in the intima of arteries and / or the media of blood vessels,
Prevention of smooth muscle cell migration from the tunica media to the intimal artery and formation of extracellular matrix, and neoarterial intimal formation, angiogenesis after vascular injury, especially due to physical, chemical or surgical causes To provide a vascular smooth muscle cell proliferation inhibitor useful for the treatment and prevention of postoperative arterial restenosis or vascular wall thickening in arteries and veins, and useful for the prevention or treatment of arteriosclerosis, diabetic vascular disorder, etc. is there.

[0006]

The present invention relates to a vascular smooth muscle cell growth inhibitor containing a substance having farnesyl transferase inhibitory activity as an active ingredient. Examples of the substance having farnesyl transferase inhibitory activity include those represented by the formula (I)

[0007]

[Chemical 3]

(Wherein R represents alkanoyl or alkenoyl, and X ¹ is

[0009]

[Chemical 4]

And X ² represents hydroxy or X ¹
And X ² together represent O) UCF1 derivative [hereinafter, the compound represented by the formula (I) is referred to as compound I].
The same applies to compounds having other formula numbers]. In the definition of each group of the formula (I), alkanoyl includes linear or branched C2 to C25, for example, acetyl, propionyl, hexanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, 3,7,11-trimethyl. Lauroyl, 3,7,11,15-tetramethylpalmitoyl and the like can be mentioned. Examples of alkenoyl include linear or branched C3 to C25, for example, propenoyl, 2-hexenoyl, palmitoreoyl, linoleoyl, linolenoyl, 2,4-Dimethyl-2-octenoyl, 4,6-Dimethyl-2,4
-Decadienoyl, 2,4,6-trimethyl-2,4-decadienoyl, farnesoyl, geranylgeranoyl and the like can be mentioned.

More preferably, R in the formula (I) is 2,4-
Examples include UCF1 derivatives which are dimethyl-2-octenoyl, 4,6-dimethyl-2,4-decadienoyl, 2,4,6-trimethyl-2,4-decadienoyl, farnesoyl or geranylgeranoyl. Next, a method for producing compound I will be described. In compound I, compound II in which X ¹ and X ² together represent O is acylated 2-amino-4-methoxyphenol (step 1), oxidized to quinone (step 2) and then epoxidized. It can be produced according to each reaction step of (Step 3).

[0012]

[Chemical 5]

(Wherein R represents the same meaning as described above) Step 1 Compound III comprises 2-amino-4-methoxyphenol and 1-
2 equivalents of an acyl halide, an acyl hydride, or a mixed acid anhydride having a desired acyl group, and the like are treated with dimethylformamide in the presence of a suitable base such as pyridine, N, N-dimethylaniline, or N, N-diethylaniline. , Dimethylsulfoxide, chloroform, dichloromethane, toluene or the like or by reacting with a suitable base as a solvent. The reaction temperature is 0 to 50 ° C,
The temperature is preferably 20 to 30 ° C., and the reaction time varies depending on the reaction conditions, but the reaction is completed at the time when the raw materials disappear by thin layer chromatography, and is usually 1 to 48 hours.

Step 2 Compound IV is prepared by converting Compound III into tetrahydrofuran, diethyl ether, in the presence of 1 to 2 equivalents of an oxidizing agent such as lead tetraacetate, cerium (IV) ammonium nitrate, and bis (trifluoroacetyl) iodobenzene. It can be obtained by oxidation in a solvent such as dioxane, acetonitrile, chloroform, acetic acid, water or a mixed solvent thereof. The reaction temperature is 0 to 50 ° C., preferably 20 to 30 ° C., and the reaction time varies depending on the reaction conditions, but it ends when the raw material disappears in thin layer chromatography, and usually 10 to 120 minutes. Is.

Step 3 Compound II is obtained by oxidizing Compound IV in the presence of 1 to 2 equivalents of hypochlorite such as sodium hypochlorite and calcium hypochlorite in a solvent such as tetrahydrofuran and dioxane. It can be obtained by The reaction temperature is -10
The temperature is preferably -20 ° C, and the reaction time varies depending on the reaction conditions, but the reaction is terminated when an appropriate amount of the desired product is formed by thin layer chromatography, and is usually 10-60 minutes.

In compound I, R is 2,4-dimethyl-2-octenoyl, 4,6-dimethyl-2,4-decadienoyl or
The compound which is 2,4,6-trimethyl-2,4-decadienoyl can be obtained by the method disclosed in the above publication. Isolation and purification of the product in the above-mentioned production method are carried out by a method usually used in organic synthesis, for example, filtration, extraction, washing,
Drying, concentration, crystallization, various chromatographies and the like can be appropriately combined and performed.

The compound I or compound II may exist in the form of an adduct with water or various solvents,
These adducts are also used as the vascular smooth muscle cell proliferation inhibitor of the present invention. Specific examples are shown in Table 1.

[0018]

[Table 1]

[0019]

[Table 2]

Next, the pharmacological activity will be described with reference to test examples. Test Example 1: Antiproliferative effect on cultured arterial smooth muscle cells derived from streptozotocin diabetic rats. Chemical substances not noted were purchased from Sigma. [Method] The test compound was dissolved in 100 mM dimethylsulfoxide (DMSO) at a concentration of 10 mM, stored at -20 ° C, and dissolved before use.

Cultured arterial smooth muscle cells (VSMC) were isolated from streptozotocin (90 mg / kg) -induced diabetic Wistar male rats using the explant method. Graft (1
x1 mm), the endothelium and adventitia were taken out from a 7-week-old rat. Place the tissue graft on a 100 mm dish containing 10 ml of Delbeco's basal medium (DMEM; GIBCO) containing 20% fetal bovine serum (GIBCO) in 5% carbon dioxide, 95% air. After culturing for 2 weeks, the cells released by trypsin treatment were taken out and diluted 3-fold with the same medium. All experiments were performed with cells within passage 5.

[0022] (1) cytostatic effect: cytostatic effect of the test compounds, [5'- ³ H] thymidine ^([3 H] Thymidine; 18
5-740 GBq / mmol; manufactured by Amersham) was evaluated. Cultured arterial smooth muscle cells were plated on 96-well microplates.
x 103 / well Day 1, 0, 10, 15, 20, 25, 3
The cells were treated with a fresh culture medium containing 0, 40 µM of the test compound. [ ³ H] Thymidine (1 μCi / well) was added after 20 hours of culturing, and after further culturing for 4 hours, a semi-automatic cell harvester (Lab
Cells were collected on a glass filter using Mosh, Labo Sci., Tokyo, Japan). The radioactivity of each sample is
It measured using the liquid scintillation counter.

(2) Inhibition of isoprenylated protein formation: Isoprenylation of proteins was measured in cultured arterial smooth muscle cells by the modified Crowell method. Cells are 5 x 105 / plate
It was cultured for 3 days. Then 10 μM simvastatin (S
imvastatin; J. Med. Chem., 29, 849-852, 1986).
It was treated for 4 hours and then RS- [2- ¹⁴ C] mevalonolactone (RS-
[2- ¹⁴ C] Mevalonolactone; 1.48-2.22 GBq / mmol; New En
gland Nuclear) 10 μCi / ml, 10 μM Simvastatin
The cells were cultured for 3 hours in a fresh culture medium containing 0, 2, 20 µM of the test compound. The cells were treated with trypsin, washed twice with phosphate buffer-physiological saline, and then 10m at 4 ° C for 10 minutes.
M dipotassium hydrogen phosphate, 154mM sodium chloride, 12m
M sodium deoxycholate, 1 mM sodium fluoride, 0.1% sodium dodecyl sulfate (SDS), 31 mM sodium azide, 1% (v / v) Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 0.15 u / ml aprotinin
It was dissolved in a buffer containing (aprotinin) and 10 μg / ml leupeptin. After separating the cell extract for 10 minutes at 12,000 rpm,
Transfer the supernatant to a new tube and lower the protein contents to Lowry.
It was measured by the method. Furthermore, mix with an electrophoretic buffer (Bio-Rad), heat at 100 ° C for 5 minutes, and perform Laemmli method.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a 12% acrylamide gel. Coomassie Brilliant Blue (Coomass
ie Brilliant Blue; made by Bio-Rad) and amplified (Amplify; sensitizer for fluorography; Amersh
(manufactured by Am Co.), dried at 60 ° C under vacuum, and dried at -80 ° C for 30
Exposed to Kodak X-Omat AR film for a day. [Results] (1) Uptake of [ ³ H] Thymidine was suppressed to 71% at 20 μM UCF1-C and 47% at 30 μM UCF1-C compared to the control (FIG. 1).

(2) At 20 μM UCF1-C, formation of an isoprenylated protein (corresponding to a farnesylated protein) having a molecular weight of 21-26 kDa was obviously suppressed (FIG. 2).

Test Example 2: FTase inhibitory activity For FTase, DEAE-Sep was used as an extract obtained by crushing bovine brain.
Hacel (Pharmacia) column chromatography, and the active fraction was concentrated by ultrafiltration and dialyzed [20 mM Tr
What was is-HCl pH8.0, 50 mM NaCl, 20 mM ZnCl ₂ , 1 mM dithiothreitol (DTT), 0.2 mM phenylmethylsulfonylfluoride (PMSF)] was used as a crude enzyme solution.
For the activity measurement, the enzyme obtained by the above method was used, and FTase [ ³
H] SPA enzyme assay kit (Amersham) was used. Regarding the enzyme inhibitory activity, the inhibition of farnesylation of lamin B to the C-terminal peptide by the test sample was measured using the above reaction system. By comparing the enzyme inhibitory activities of the untreated group and the group treated with a known concentration of the sample, the concentration of the sample that inhibits farnesylation by 50% (IC ₅₀ ) was calculated.

The results are shown in Table 2.

[0027]

[Table 3]

According to Table 2, Compound I showed a clear enzyme inhibitory activity against FTase. The inhibition by UCF1-C was antagonistic with farnesyl pyrophosphate. The acute toxicity (LD ₅₀ ; 50% lethal dose) of UCF1-C was 20 mg / kg when administered intravenously to rats.

The compound of the present invention is orally or parenterally administered as it is or as various pharmaceutical compositions.
Examples of the dosage form of such a pharmaceutical composition include tablets, pills, powders, granules, capsules, suppositories, injections, and drip infusions. For the formulation of the above dosage form, generally known methods are applied, for example, various excipients, lubricants, binders, disintegrants, lubricants, suspending agents, isotonic agents, emulsifiers, It may contain an absorption promoter and the like.

Examples of the carrier used in the pharmaceutical composition include water, distilled water for injection, physiological saline, glucose, fructose, sucrose, mannitol, lactose, starch, corn starch, cellulose, methyl cellulose, carboxymethyl cellulose, Examples include hydroxypropyl cellulose, alginic acid, talc, sodium citrate, calcium carbonate, calcium hydrogen phosphate, magnesium stearate, urea, silicone resin, sorbitan fatty acid ester, glycerin fatty acid ester, etc., which are appropriately selected depending on the type of formulation. To be selected.

The dose and frequency of administration of the compound I used for the above-mentioned purpose will vary depending on the desired therapeutic effect, administration method, treatment period, age, body weight, etc., but will be oral or parenteral (eg injection, infusion, Oral administration by suppository, skin patch, etc.)
/ kg. The novel use of the substance having farnesyl transferase inhibitory activity according to the present invention can be clearly distinguished from the use as a known anticancer agent and antibacterial agent in the application range of the use.

The embodiments of the present invention will be described below with reference to Examples and Reference Examples.

[0033]

【Example】

Example 1 Tablet UCF1-C 30 g Lactose 40 g Corn starch 18 g Carboxymethyl cellulose calcium 10 g A 10% hydroxypropyl cellulose solution is added to the above mixture and kneaded. The kneading liquid is granulated by an extrusion granulator equipped with a 1.0 mm basket, and magnesium stearate is added to the granules for tableting. Then, according to the conventional method, 8m containing UCF1-C 30mg in one preparation (170mg)
Use tablets of m diameter.

Example 2 Injectable compound 1,1 g of the compound is dissolved in 20 ml of ethanol, and the mixture is sterilized by pressure filtration with a Millipore filter (pore size: 0.22 μl). An injection containing 1,15 mg of compound per vial is prepared by dispensing 5 ml of the resulting solution into a vial.

Example 3 Soft Capsules Compounds 2 and 3 g are dissolved in 100 g of soybean oil. Then, by injecting the resulting solution into a capsule by a conventional method, 1
Soft capsules containing 2,10 mg of compound per capsule are prepared.

Example 4 Tablet Compound 7 30 g Lactose 40 g Corn starch 18 g Carboxymethyl cellulose calcium 10 g A 10% hydroxypropyl cellulose solution is added to the above mixture and kneaded. The kneading liquid is granulated by an extrusion granulator equipped with a 1.0 mm basket, and magnesium stearate is added to the granules for tableting. Then, in the usual manner, one preparation (170 mg) contains 7,30 mg of compound
Use tablets with a diameter of 8 mm.

Reference Example 1 4-Methoxy-2-nitrophenol 4-methoxyphenol (124 mg) was dissolved in dichloromethane, silica gel (1 g) was added thereto, and the mixture was stirred, and concentrated nitric acid (0.077 ml) was added dropwise at room temperature. After confirming the completion of the reaction by thin layer chromatography, the mixture was filtered through Celite. The solvent was evaporated, the residue was purified by silica gel chromatography (dichloromethane), and 4-methoxy-2-nitrophenol 98.5 mg (58%) was obtained.

Rf 0.65 (1/9 ethyl acetate / toluene) ¹ HNMR (CDCl ₃ ) δ; 3.83 (s, 3H), 7.08 (d, 1H, J = 9.3
Hz), 7.24 (dd, 1H, J = 2.9, 9.3 Hz), 7.51 (d, 1H, J =
2.9 Hz), 10.33 (s, 1H). FAB-MS (M / Z); 168 (M-1) ^-

Reference Example 2 2-Amino-4-methoxyphenol 4-methoxy-2-nitrophenol 12 obtained in Reference Example 1
Dissolve 0 mg in 2.4 ml of ethyl acetate and add 12 mg of platinum oxide to this.
Was added, and the mixture was stirred under a hydrogen stream at room temperature for 1 hour. The reaction solution was filtered through Celite, and a hydrogen chloride ethyl acetate solution was added to the filtrate. The precipitate was filtered and dried to give 2-amino-4-methoxyphenol 111 mg (89%).

Rf 0.47 (1/9 chloroform / methanol) ¹ HNMR (CDCl ₃ ) δ; 3.75 (s, 3H), 6.90 (m, 3H).

Reference Example 3 4,5-Epoxy-1-farnesoylamino-3,6-dioxocyclohexene Farnesanoic acid 61.0 mg was dissolved in toluene, to which 0.04 ml of oxalyl chloride was added dropwise, and the mixture was allowed to stand at room temperature for 3 hours. It was stirred.
The solvent was distilled off to obtain farnesanoic acid chloride as a crude product.

2-Amino-4-methoxyphenol (31.6 mg) obtained in Reference Example 2 was dissolved in pyridine (2 ml), the farnesic acid chloride obtained above was added dropwise thereto, and the mixture was stirred at room temperature for 4 hours. Dilute the reaction solution with ethyl acetate and add 2M.
Hydrochloric acid was added. The organic layer was washed with water and then saturated aqueous sodium hydrogen carbonate solution, and dried over sodium sulfate. The solvent was distilled off, and the residue was subjected to silica gel chromatography (5/95
Purification with (ethyl acetate / toluene) yielded 46.0 mg (57%) of 2-farnesoylamino-4-methoxyphenol.

Rf 0.38 (1/9 ethyl acetate / toluene) ¹ HNMR (CDCl ₃ ) δ; 1.60 (s, 3H), 1.62 (s, 3H), 1.6
8 (d, 3H, J = 0.76 Hz), 1.99-2.23 (m, 8H), 2.24 (d,
3H, J = 0.84 Hz), 3.74 (s, 3H), 5.11 (m, 2H), 5.78
(br s, 1H), 6.59 (d, 1H, J = 2.9 Hz), 6.69 (dd, 1H,
J = 2.9, 8.9 Hz), 6.94 (d, 1H, J = 8.9 Hz), 7.33 (br s,
1H). FAB-MS (M / Z); 356 (M + 1) ⁺

2-farnesoylamino-4 obtained above
-Methoxyphenol 46.0 mg in tetrahydrofuran 2
ml of water and 2 ml of water and dissolve it in lead tetraacetate 69
mg was added, and the mixture was stirred at room temperature for 30 minutes. Dilute with ether,
After adding a saturated aqueous sodium hydrogen carbonate solution, the organic layer was washed with saturated saline and dried over sodium sulfate. The solvent was distilled off, and the residue was subjected to silica gel chromatography (5/95
Purification with (ethyl acetate / toluene) gave 2-farnesoylamino-1,4-quinone (15.7 mg, 36%).

Rf 0.56 (1/9 ethyl acetate / toluene) ¹ HNMR (CDCl ₃ ) δ; 1.60 (s, 3H), 1.62 (d, 3H, J = 1.
1 Hz), 1.68 (d, 3H, J = 1.1 Hz), 1.98-2.21 (m, 8H),
2.22 (d, 3H, J = 1.2 Hz), 5.06-5.11 (m, 2H), 5.75 (d,
1H, J = 1.1 Hz), 6.72 (dd, 1H, J = 2.3, 10.1 Hz), 6.7
6 (d, 1H, J = 10.1 Hz), 7.64 (d, 1H, J = 2.3 Hz), 7.92
(br s, 1H). FAB-MS (M / Z); 344 (M + 3) ⁺

2-farnesoylamino-obtained above
Dissolve 1,4-quinone (15.0 mg) in dioxane (0.05 ml),
To this, 0.045 ml of a sodium hypochlorite solution was added dropwise under water cooling, and the mixture was stirred at room temperature for 30 minutes. Phosphate buffer in the reaction solution
(PH 7) was added, the mixture was extracted with ethyl acetate, then the organic layer was washed with saturated brine and dried over sodium sulfate. The solvent was distilled off, and the residue was chromatographed on silica gel (1/7 ethyl acetate / n-hexane to 4/3 dichloromethane / toluene).
Purified with 4,5-epoxy-1-farnesoylamino-3,6
-1.7 mg (11%) of dioxocyclohexene was obtained.

Rf 0.29 (2/8 ethyl acetate / n-hexane) ¹ HNMR (CDCl ₃ ) δ; 1.59 (s, 3H), 1.61 (d, 3H, J = 0.
9 Hz), 1.67 (d, 3H, J = 1.0 Hz), 1.96-2.11 (m, 4H),
2.21 (s, 3H), 2.18-2.24 (m, 4H), 3.81 (dd, 1H, J = 2.
2, 3.7 Hz), 3.90 (d, 1H, J = 3.7 Hz), 5.08 (m, 2H),
5.70 (d, 1H, J = 1.0 Hz), 7.59 (d, 1H, J = 2.2 Hz),
7.73 (br s, 1H). FAB-MS (M / Z); 358 (M + 1) ⁺

[0048]

INDUSTRIAL APPLICABILITY The present invention provides a vascular smooth muscle cell proliferation inhibitor useful for preventing or treating arteriosclerosis, diabetic angiopathy and the like.

[Brief description of drawings]

FIG. 1 shows the effect of UCF1-C on Thymidine uptake into streptozotocin-treated rat arterial smooth muscle cells (using cells at passage 5).

FIG. 2 is a diagram showing the results of SDS gel analysis of p21 ras farnesylation.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Kengo Inoue 4-12-13 Kajino-cho, Koganei-shi, Tokyo (72) Inventor Akira Mihara 1079-22 Kiso-cho, Machida-shi, Tokyo (72) Inventor Nakano Hirofumi Tokyo Miyakomachida City Nakamachi 3-9-13

Claims (2)

[Claims] 1. A vascular smooth muscle cell proliferation inhibitor comprising a substance having farnesyl transferase inhibitory activity as an active ingredient. 2. A substance having farnesyl transferase inhibitory activity is represented by the formula (I): (In the formula, R represents alkanoyl or alkenoyl,
X ¹ is The stands, X ² is or represents hydroxy, X ¹ and vascular smooth muscle cell proliferation inhibitor according to claim 1 which is UCF1 derivative represented by X ² represents O together).