JPH02306971A - Phenetyl alcohol derivative and immune suppressing agent containing phenetyl alcohol derivative as active ingredient - Google Patents

Abstract

NEW MATERIAL:3,4,5-Trihydroxyphenetyl alcohol expressed by the formula. USE:A medicine. Active ingredient for immune suppressing agent. The alcohol derivative expressed by the formula has mitogen activity suppressing action and is formulated into a preparation such as tablet or granule by adding carrier or assistant thereto and the preparation is administered. The preparation is orally administered in dose of 50mg-5g (as active ingredient amount)/adult/day divided several times. PREPARATION:3,4,5-Trimethoxyphenyl acetic acid is heated in hydrobromic acid under reflux to methylate the above-mentioned compound or 3,4,5- trimethoxyphenyl acetic acid is directly protected with a protecting group such as benzyl bromide and then carboxyl group thereof is reduced and deprotected to provide the compound expressed by the formula.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to black-forming cells (hereinafter referred to as PFC).
The present invention relates to an immunosuppressant that has a suppressive action and a mitogen activity suppressing action, and is useful for immunosuppression during III organ transplantation, and for treatment by autoimmune doctors, etc.

[Conventional technologies and issues] Immunosuppressants are used clinically for the survival of transplanted organs, as well as for systemic lupus erythematosus, collagen diseases such as rheumatoid arthritis, glomerulonephritis, polymyopathy, and autoimmune hemolysis. It is used to treat so-called autoimmune diseases such as sexual anemia.

The immunosuppressants currently used clinically include:
There are steroids such as hydrocortiscin, purine derivatives such as azathioprine, and alkylating agents such as cyclosulfamide, but side effects have been reported such as bone marrow function suppression such as decreased white blood cell and platelet counts, and gastrointestinal disorders.

Recently, cyclosporin A IJ, an antibiotic extracted from fungi, has been attracting attention due to its extremely high survival rate in transplanted organs, and attempts have been made to use it for various autoimmune diseases. However, there have been reports of serious renal toxicity such as acute tubular necrosis and renal assimilation.

Therefore, it has been desired to develop an immunosuppressant that has no side effects or that can be used in combination with existing drugs to reduce side effects.

[Means for Solving the Problems] The present inventors sought a compound that has an immunosuppressive effect that is useful for immunosuppression during organ transplantation and for the treatment of autoimmune diseases, etc.
As a result of searching for active compounds mainly using mitogen activity suppressing effect as an indicator, the present invention was completed by discovering that phenethyl alcohol derivatives have mitogen activity suppressing effect.

That is, the present invention is as shown below.

(+) Formula I below ] A new phenethyl alcohol derivative represented by

(2) The following formula ■ (In the formula, R1 represents a hydroxymethyl group or a carboxyl group, R7 and R3 represent a hydrogen atom, a hydroxyl group, a methoxyl group, or together represent a methylenedioxy group, and R4 represents a methylenedioxy group. An immunosuppressant containing phenethyl alcohol derivatives represented by (representing a hydrogen atom or a hydroxyl group) as six active ingredients.

Hereinafter, the compounds of formula 1 and formula H will be collectively referred to as compounds of formula.

Among the compounds of the formula, the compound represented by the formula I can be obtained, for example, by the following method.

After demethylating 3.4.5-trimethoxyphenylacetic acid or directly protecting 3.4.5-trihydroquinphenylacetic acid with a protecting group such as benzyl bromide, the carboxyl group is reduced and deprotected. can be obtained.

In addition, among the compounds of the formula, R2 is hydroxymethyl, R
Compounds other than those in which 2, R1 and R3 are represented by a hydroxyl group are all known substances as shown below, and examples thereof are shown in Table 1.

However, all R are hydrogen atoms.

In order to obtain the above-mentioned compounds in Table 1, the following methods may be mentioned, for example.

p-hydroxyphenethyl alcohol, p-methquinphenethyl alcohol, 3-methoxy-4-hydroxyphenethyl alcohol, 3.4-dimethoxyphenethyl alcohol, and 3,4-dihydroxyphenylacetic acid are generally commercially available compounds (Nguma). It is.

3.4-Dihydroxyphenethyl alcohol is obtained by demethylation of 3-methquine-4-hydroxyphenethyl alcohol and also by acid hydrolysis of phenethyl alcohol glycosides such as actiocyto.

3-Hydroxy-4-methoxyphenethyl alcohol is obtained by partial demethylation of 3,4-dihydroxyphenethyl alcohol.

3.4-methylenezoxy-phenethyl alcohol is
It is derived by methylene dioxylation of 3.4-dihydroxyphenethyl alcohol or methylene dioxylation of 3.4-dihydroxyphenylacetic acid and reduction of the carboxyl group.

A specific example of the production of the compound of the formula is as follows.

Specific example I 3-methquin-4-hydroxyphenethyl alcohol 2
．． 09 (manufactured by Sigma) was dissolved in 100 d of acetonitrile, 6.09 g of sodium iodide and 6.09 g of aluminum chloride were added, and the mixture was stirred at room temperature for 6 hours. While cooling the reaction solution, water was added to decompose excess aluminum chloride, the solvent was distilled off under reduced pressure, and the mixture was partitioned between ethyl acetate and an aqueous sodium thiosulfate solution. After concentration, the ethyl acetate transfer portion was subjected to phosphoric gel column chromatography (manufactured by Merck & Co., Ltd.) and eluted with chloroform-methanol (20:I) to obtain a colorless oil. This f@color 71
The physical and chemical properties of the 11-like substance are described in the literature [A, 5akurai
et al., Bull, chem.

Soc, Jpn, 56.1573 (1983)].

Specific Example 2 3.4-dimethoxyphenethyl alcohol 209 (manufactured by Sigma) was dissolved in 100 d of dichloromethane, 6.09 d of aluminum chloride was added, and the mixture was stirred at room temperature for 4 hours. While cooling the reaction solution, water was added to decompose excess aluminum chloride, and then partitioned with chloroform. After concentrating the chloroform transition part, high performance liquid chromatography (
T S K gel OD S-120T, 2.1
i, d, Ta. The physical and chemical properties of this colorless needle crystal are described in the literature [H, 5asaki, et al.

Chem, Pharm, Bull, 26.2111 (
1984)] of 3-hydroxy-4-methoxy-phenethyl alcohol.

Specific Example 3 3.4-dihydroxyphenylacetic acid 2.09 (manufactured by Sigma) was dissolved in methanol 100-, diazomethane was added, the carboxyl group was rapidly methylated, and the mixture was concentrated to dryness under reduced pressure. This was dissolved in acetone+007, 6.09 g of potassium carbonate and 6.09 g of chloromethyl bromide were added, and after heating under reflux for 2 hours, the reaction solution was filtered. After concentrating the filtrate, 3.09 g of lithium aluminum hydride was added in anhydrous tetrahydrofuran, and after reduction for 2 hours, the mixture was extracted with ethyl acetate. After concentrating this ethyl acetate extract, it was subjected to silica gel column chromatography and eluted with chloroform to obtain 850 mW of colorless oil. This colorless oil is based on the following physical and chemical properties and smooth data.
．．・Identified as 1-methylenedioxyphenethyl alcohol.

Properties: Colorless oil ET-MS m/z (%): 166 (M') Proton nuclear magnetic resonance spectrum (δ ppm in acetone-do): 2.7
2 (2H, t, J=7.0Hz).

3.67 (2H, t, J = 7.0 Hz).

5.92 (2H, s).

6.68 (I H, dd, J = 8.1 /2.2
Hz).

6.71 (IH, d, J = 2.2 Hz).

6.74 (IH, d, J = 8.1 Hz) Next, it will be explained with experimental examples that the compound of the formula has mitogenic activity and antibody production suppressing effect, and is suitable for use as an immunosuppressant S. .

Experimental Example 1 (Reagent Preparation) A compound of the formula was dissolved in physiological saline, and after sterilization using a 0.45Ia membrane filter, it was used in an experiment.

Phytohemagglutinin (D
irco 1. manufactured by obo, hereinafter referred to as PHA. ) was used.

For measuring mitogenic activity, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (manufactured by Sigma, referred to as MTT) was used.
was adjusted to 5R9/d with a phosphate buffer solution of pH 7.2.

All culture solutions used were RPMII640 (manufactured by Hinaga Co., Ltd.).

(Cell Preparation) The viscera of a BALI13/c mouse (6-8 weeks old, female) was removed aseptically, and the cells were loosened on a 200 m2 stainless wire mesh in a culture medium, and the cells were isolated by pipetting. A suspension was prepared. After centrifugation, red blood cells were dissolved on the surface using 0.83% ammonium chloride solution, and then washed three times with culture medium. Cells were treated with 1O% fetal bovine serum (manufactured by Gibco, heated at 56°C for 30 minutes) and 5XIO-'
lIPMII640 with M mercaptoethanol added
After preparing the cells to be IxlO'/- using a culture solution, they were subjected to the following experiment.

2 X l O'/well of phlegm cells prepared as above were treated with T cell mitogen (phytohemagglutinin) or B cell mitogen (lipopolysaccharide).
and cultured at 37°C under 15% carbon dioxide for 48 hours.

The prepared test solution of the compound of the formula was added at the same time as the start of the culture. Before the end of the culture, add 20 copies of the MTT solution, and then add 60 copies of the MTT solution.
After incubation for hours, 10% 5DS-0.01N hydrochloric acid + 00I
The reaction was stopped by adding Ji.

After dissolving MTT formazan produced in proportion to activation, colorimetric determination (OD 540-690) was performed and Tw
The effect of compounds of formula on J- or B-cell proliferation was determined.

Table 2 shows the inhibition rate of the compound of the formula on T cell proliferation at a concentration of 1001/d, and the inhibition rate of the compound of the formula on B cells at a concentration of 100
The inhibition rate at skin/d1 degree is shown in Table 3.

Table 2 Table 3 Experimental Example 2 Vulgar cells (I) prepared according to the cell preparation method of Experimental Example 1
X 10 ll/rttfl/24 well) in the presence of 3XIO'' sheep red blood cells (hereinafter referred to as 5RBC),
After 4 days &'i@a by Mitschel and Dutton's method, anti-5RBC antibody-producing cells were measured by Cunningham and Sotsenberg's method, and the inhibition rate of anti-5RBC-PFC induction at 100#/ll1a degree was determined by the compound of the formula was measured.

The results are shown in Table 4.

Table 4 (As is clear from the above results on induction of anti-5RBC-PFC, the compound of the formula shows an inhibitory effect on cell proliferation and induction of antibody-producing cells,
Its usefulness as an immunosuppressant was confirmed.

Further, an acute toxicity test of the compound of the formula was conducted using ddY male mice, and there were no deaths after oral administration of 197 kW, confirming safety.

Therefore, the compounds of the formula, especially 3,4-dihydroquinphenylacetic acid and 3,4-dihydroxyphenethyl alcohol, are considered to be a new type of immunosuppressive agent because of their remarkable cell growth-inhibiting effect and suppressive effect on the induction of antibody-producing cells. expected to be.

Next, the dosage and formulation of compounds of formula will be described.

The compounds of the formula can be administered to animals and humans neat or with conventional pharmaceutical carriers.

The dosage form is not particularly limited and may be selected and used as required, including tablets, capsules, granules, fine granules,
Examples include oral preparations such as powders, parenteral preparations such as injections, and suppositories.

In order to exert the desired effect as an oral agent, although it varies depending on the age, weight, and severity of the disease of the patient, it is usual for an adult to administer 50m9 to 57m of the compound of the present invention divided into several times a day. It seems appropriate to take it.

Oral agents include, for example, starch, lactose, sucrose, mannitol,
It is manufactured using conventional methods using carboxymethyl cellulose, corn starch, inorganic salts, etc.

In addition to the excipients mentioned above, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, coloring agents, fragrances, and the like can be used in this type of preparation as appropriate. Specific examples of each are shown below.

[Binder] Starch, dextrin, gum arabic powder, gelatin,
Hydroquine propyl starch, methyl cellulose, sodium carboxyl methyl cellulose, hydroxypropyl cellulose, crystalline cellulose, ethyl cellulose, polyvinylpyrrolidone, macrogol.

[Disintegration process] Starch, hydroxypropyl starch, sodium carboxyl methylcellulose, calcium carboxyl methylcellulose, carboxymethyl cellulose, low substituted hydroxypropyl cellulose.

[Surfactant] Sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 80° [Lubricant] Talc, waxes, hydrogenated vegetable oil, N=JvJ fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate , polyethylene glycol.

[Fluidity promoter] Light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate.

In addition, the compound of formula 4 can be administered as a suspension, emulsion, syrup, or elixir, and these various drug layers may contain flavoring agents and coloring agents. .

In order to exert the desired effect as a parenteral agent, it is usually necessary to administer the compound of formula 0.1119 to I9 per day as an adult, although this will vary depending on the child's age, body weight, and degree of disease. Intravenous injection, intravenous drip, subcutaneous injection, and intramuscular injection are considered appropriate.

This parenteral preparation is manufactured according to a conventional method, and generally uses distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol, etc. as a diluent. be able to. Furthermore, a bactericide, a preservative, and a stabilizer may be added as necessary. In addition, from the viewpoint of stability, this parenteral preparation may be filled into a vial or the like, then frozen, water removed by ordinary freeze-drying techniques, and a liquid preparation prepared from the freeze-dried product immediately before use. Furthermore, isotonizing agents, stabilizers, preservatives, soothing agents, etc. may be added as appropriate.

Other parenteral preparations include liquid preparations for external use, liniments such as ointments, suppositories for rectal administration, and the like, which are manufactured according to conventional methods.

Next, the present invention will be explained in more detail with reference to Examples.
The present invention is not limited to this in any way.

Example 1 500 mg of 3.4.5-trimethoxyphenylacetic acid was heated under reflux in 45% hydrobromic acid 107 for 20 hours to perform demethylation. The obtained reaction solution was concentrated to dryness under reduced pressure and dissolved in dimethylformamide (DMF) I Od. Benzyl bromide 2- and 1 g of potassium carbonate were added to this solution.
was added and heated at 80°C for 10 hours. The reaction solution was filtered with Jβ
Thereafter, the filtrate was concentrated to dryness under reduced pressure to obtain a tetrabenzyl compound. This was dissolved in tetrahydrofuran 107 and cooled with water.
Lithium aluminum hydride 300 J11? -Tetrahydrofuran IOI/11 suspension and stirred for 1 hour. The reaction solution contains 0.3 d of water and 0.3 d of IN potassium hydroxide.
- was added to remove excess lithium aluminum hydride, and then dehydrated with 1 g of potassium carbonate. The precipitate was filtered off, concentrated to dryness, crystallized from aqueous ethanol, and purified to obtain a reduced product. This reduced product was dissolved in methanol 5d, added to palladium carbon-methanol suspension 57, and stirred for 2 hours at room temperature under a hydrogen stream to perform catalytic reduction. The reaction solution was filtered and the filtrate was evaporated to dryness to obtain 250 my of colorless oil. The physicochemical properties of this compound are as follows, and from these data, the structure of the compound of formula I, ie, 3.4.5-trihydroxyphenethyl alcohol, was determined.

Properties: Colorless oil FD-MS m/z: I 71 [M+H]' Proton nuclear magnetic resonance spectrum (δ ppm in CDJD): 2.60 (2H, t, J = 7.2 Hz).

3.66 (21-(, t, J=7.2Hz).

6.21 (2H, s) Example 2 ■Corn starch 449 ■Crystalline cellulose 40g ■Carboxymethylcellulose carunium 5g ■Light silicic anhydride 0.59■Magnesium stearate 0.59■Example 11? Power compound-1
0-L Total 100y According to the above recipe, ① to ① were mixed uniformly and compressed using a tablet machine to obtain a tablet of 200 xy.

This tablet-tablet contains the compound 2019 obtained in Example 1, and an adult should take 10 to 25 tablets in several doses per day.

Example 3 ■Crystalline cellulose 84.51? ■ Magnesium stearate 0.59 ■ Calcium carboxymethyl cellulose 59 ■ - As shown in Example 1 1 - Total
1009 According to the recipe in L, ■, ■, and a part of ■ are uniformly mixed, compressed and molded, then crushed, and the remaining amounts of ■ and ■ are added and mixed, and compressed and molded using a tablet machine. Two tablets of 200 parts were obtained.

These tablets contain 20=2 of the compound obtained in Example 1, and are taken by adults at 10 to 25 tablets in several doses per day.

Example 4 ■ Crystalline cellulose 49.59 ■ lO% hydroxypropyl cellulose ethanol solution 359 ■ Calcium carboxymethyl cellulose 59 ■ Magnesium stearate 0.59 ■ Compound obtained in Example 2 109 Total 1009 ■, ■ and ■ according to the above recipe After uniformly mixing, neutering by a conventional method, granulating with an extrusion granulator, drying and crushing, mix ■ and ■, compression mold with a key press, and make a portion of 200 R9. Got the tablets.

This tablet-tablet contains Compound 20j9 obtained in Example 2, and is taken in doses of 10 to 25 tablets in several doses per day for adults.

Example 5 ■Corn starch 34.590 Magnesium stearate 502 ■Calcium carboxymethyl cellulose 59 ■Light anhydrous silicic acid 0.59 ■Compound obtained in Example 3 109 Total 1009 Mix ■~■ uniformly according to the above recipe and compress After compression molding with a molding machine, it was crushed with a crusher and sieved to obtain granules.

This granule 19 contains compound 100 R obtained in specific example 3.
Adults should take 2 to 59 doses in several doses per day.

Example 6 ■ Crystalline cellulose 55 g ■ 10% hydroxypropyl cellulose ethanol solution 359 ■ Compound obtained in Example 1 IO total 1009 According to the above recipe, ■ to ■ were uniformly mixed and suspended. After going into an extrusion granulator and granulating, the mixture was dried and sieved to obtain granules.

This granule 19 contained 100 m of the compound obtained in Example I.
It contains 2 to 59 grams per day for adults, divided into several doses.

Example 7 ■Corn starch 89.59■Light silicic anhydride 0.59■Compound obtained in Specific Example I
+oy total 1009 Mix ω~■ uniformly according to the above recipe, 20019
was filled into a No. 2 capsule.

This capsule I capsule contains the compound 20!9 obtained in Specific Example I, and an adult should take IO to 25 capsules a day in several doses.

Example 8 ■ Soybean oil 59 ■ Distilled water for injection 89.5Li ■ Soybean phosphorus f
f 2.5g ■ Glycerin
29 ■ Compound obtained in Example 2 -17 Total amount 100g Dissolve ■ in ■ and ■ according to the above recipe, and add ■
The solutions of (1) and (2) were added and emulsified to obtain an injection.

Claims (2)

[Claims] (1) The following formula I ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (2) Formula II below ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (However, in the formula, R_1 represents a hydroxymethyl group or carboxyl group, R_2 and R_3 are hydrogen atoms,
It represents a hydroxyl group, a methoxyl group, or a methylenedioxy group together, and R_4 represents a hydrogen atom or a hydroxyl group. ) An immunosuppressant whose active ingredient is a phenethyl alcohol derivative.