JPH07330770A - Purine derivative and treating agent containing the derivative as active component - Google Patents

Abstract

PURPOSE:To obtain a new purine derivative suitable for continuous or long-term oral administration, exhibiting the action to inhibit the adhesion of leukocyte such as mature granulocyte to a tissue cell and effective especially for the treatment of inflammatory diseases such as rheumatism, autoimmune diseases and graft versus host diseases. CONSTITUTION:This purine derivative(salt) is expressed by formula I (R1 and R2 are each H or a 1-4C alkyl), e.g. 6-amino-9-benzyl-8bromopurine. The derivative can be produced e.g. by reacting 9-benzyl-6-chloropurine of formula II with an amine of the formula R1-NH-R2 in a solvent such as ethanol, dissolving the obtained 6-(alkyl)amino-9-benzyl-purine in sodium acetate and dropping bromine water to the solution.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel purine derivative and a therapeutic agent containing the same as an active ingredient. Particularly, the purine derivatives of the present invention are useful as leukocyte adhesion inhibitors or therapeutic agents for inflammatory diseases.

[0002]

BACKGROUND OF THE INVENTION Autoimmune diseases such as rheumatism, nephritis, and systemic lupus erythematosus (SLE), graft-versus-host disease (GVHD) known as rejection during organ transplantation, asthma and dermatitis. Allergic
Diseases, chronic inflammation such as osteoarthritis, and further injury during blood reperfusion after myocardial infarction, all of these diseases are caused by leukocytes in affected tissues, especially neutrophils. It is a sexually transmitted disease. In various inflammation sites, P
It is known that neutrophils in blood migrate to an inflammation site by stimulation of neutrophil chemotactic factors such as AF and LTB ₄ . Adhesion of neutrophils to target cells then occurs at the site of inflammation. In the process of adhesion of neutrophils to target cells, adhesion molecules are bonded by binding of adhesion molecules such as LFA-1 on neutrophil cells with ligands such as ICAM-1 on endothelial cells that are target cells. It has been reported to happen (Dust
in et al., J. Immunology, 137, 245-254 (1986) or Rothlein et al., J. Immunology, 137, 1270-1274.
(1986) etc.). Inflammation is caused by active oxygen released by the adhered neutrophils, inflammatory substances such as protease,
It is believed that the cells at the site of inflammation are damaged and occur.

It has been reported that the activity of neutrophil adhesion is enhanced when the above adhesion molecules are changed by stimulating substances such as PAF and IL-8 (Takuya Tamaya et al., Experimental Medicine, 9, 318 (1991)). Furthermore, the adhesive activity of neutrophils is also enhanced by stimulation with histamine, thrombin and the like.

It is already known that inflammation can be suppressed by inhibiting the adhesion of neutrophils to tissue cells, which is the first step of inflammation. For example, antibodies against the adhesion molecules LFA-1 and ICAM-1 have been researched and developed as therapeutic agents for autoimmune diseases such as rheumatism, and it has been reported that they effectively suppress inflammation (J.
Immunology, 147, 4167-4171 (1991)). However, the above-mentioned antibody against LFA-1, or ICAM-1
It has been reported that the antibody against E. coli requires a high dose of about 5 mg / kg as a therapeutic agent. These antibodies are
Since it is a polypeptide, it has a short half-life due to metabolism, and it is not easy to orally administer the dose required for maintaining drug efficacy. In addition, especially for therapeutic agents for rheumatism, orally administrable low molecular weight compounds, for example, cyclooxygenase inhibitors such as indomethacin are effective and widely used, but this indomethacin is not only in the affected area but also in the digestive tract. It also inhibits cyclooxygenase, and when it is continuously taken, it has a drawback that it frequently causes ulcers in the digestive tract.

In the above-mentioned inflammatory diseases such as autoimmune diseases, graft-versus-host disease (GVHD) known as rejection at the time of organ transplantation, allergic diseases, and chronic inflammation such as osteoarthritis, etc., a therapeutic agent Since the administration of the drug is continuous or long-term, an orally administrable drug containing a low molecular weight compound as an active ingredient is desired, and further, it plays a central role in the early stage of inflammation. There is a need for new drugs that produce a medicinal effect mainly by inhibiting the adhesion of neutrophils to tissue cells. That is, a novel therapeutic agent for inflammatory diseases, which is suitable for continuous or long-term oral administration and whose main pharmacological effect is inhibition of adhesion of neutrophils to tissue cells, is desired.

[0006]

DISCLOSURE OF THE INVENTION The present invention is to solve the above-mentioned problems, and an object of the present invention is to be suitable for continuous or long-term oral administration, and to obtain leukocytes such as neutrophils as a main pharmacological effect. It is an object of the present invention to provide a novel compound having an action of inhibiting adhesion to tissue cells, particularly a low molecular weight compound. Further, it is to provide a therapeutic agent for inflammatory diseases, which comprises a novel compound having an action of inhibiting the adhesion of leukocytes to the constituent cells, particularly a low molecular weight compound, as an active ingredient.

[0007]

Against this background, the present inventors have completed the present invention as a result of intensive research to create a novel low molecular weight compound having an adhesion-inhibiting effect. The compound of the present invention has the following general formula (1):

[Chemical 2] (In the formula, R ₁ and R ₂ each independently represent a group selected from the group consisting of a hydrogen atom, a linear alkyl group having 1 to 4 carbon atoms, and a branched alkyl group having 3 or 4 carbon atoms. And a purine derivative which is a pharmaceutically acceptable salt thereof.

Examples of the straight-chain alkyl having 1 to 4 carbon atoms in the present invention include methyl group, ethyl group, propyl group and butyl group. Further, a branched alkyl group having 3 or 4 carbon atoms includes an isopropyl group, a secondary butyl group,
A tertiary butyl group can be mentioned. As a preferred compound in the present invention, a purine derivative represented by the above general formula (1) in which either R ₁ or R ₂ is a hydrogen atom and the other is a hydrogen atom or a methyl group can be mentioned. A particularly preferred compound is 6-amino-9-
Benzyl-8-bromopurine, 6-methylamino-9-
Benzyl-8-bromopurine can be exemplified.

In the purine derivative of the present invention, preferable examples of the pharmaceutically acceptable salt thereof include salts with pharmacologically acceptable acids such as hydrochloride, sulfate, acetate and bromide. Includes hydrogen salts, phosphates, succinates, maleates, fumarates, citrates, gluconates, methanesulfonates, p-toluenesulfonates, and the like, and pharmacologically acceptable cations. Examples of salts include sodium salt, potassium salt, calcium salt and the like. The above-mentioned pharmaceutically acceptable salt can be prepared by a method such as recrystallization by mixing the derivative with a corresponding acid or a base containing a corresponding cation.

The present invention is also a leukocyte adhesion inhibitor containing the above purine derivative as an active ingredient, and further a therapeutic agent for inflammatory diseases containing the above purine derivative as an active ingredient, particularly rheumatism and nephritis. , An autoimmune disease or a graft-versus-host disease inflammatory disease. The therapeutic agent of the present invention can be a parenteral preparation such as an intravenous injection, an intramuscular injection, a suppository, and a patch, but it is most purposeful to prepare an oral administration preparation, and tablets and capsules. It is preferable to use a dosage form such as a powder. Then, it can be prepared into a preparation by a conventional means together with an excipient such as lactose or glucose commonly used for orally administered preparations, and a lubricant such as magnesium stearate or talc. Furthermore, it may be administered in other forms suitable for oral administration, such as a liquid preparation. The effective dose of the above-mentioned purine derivative and the number of administrations are appropriately determined depending on the body weight, age, and symptoms of the patient, but usually 1 to 1 times per day.
It is desirable to administer 100 mg / kg in one to several divided doses.

The compound represented by the above general formula (1) is
It can be prepared by the following synthetic method. First, 6
Known synthesis method of-(alkylamino) -9-benzylpurines (J. Heterocyclic Chem., 23, 1189-1193 (19
86) or J. Med. Chem., 31, 616-612 (1988) and the references described in the above document)), and once used as an intermediate material, 9-benzyl-6 represented by the formula (2). −
Chloropurine is synthesized, the intermediate raw material is reacted with the amine represented by the general formula (3), and the 6-amino substitution product obtained by substituting the chloro atom at the 6-position with an amino group, the purine represented by the general formula (4). A derivative, 6- (alkylamino) -9-benzyl-purine or 6-amino-9-benzyl-purine, is synthesized. Then, the purine derivative represented by the general formula (4) is treated with bromine water to synthesize a purine derivative represented by the general formula (1) in which a bromine atom is substituted at the 8-position.

[0012]

[Chemical 3]

[0013]

[Chemical 4] In the formula, R ₁ and R ₂ each represent the same group as in the above general formula (1). )

[0014]

[Chemical 5] (In the formula, R ₁ and R ₂ are each represented by the general formula (1) above.
Shows the same group as. )

Next, the main points of the synthesis process will be briefly described. Step 1 [Preparation of 5-amino-6-benzylamino-4-chloropyrimidine] One amount of 5-amino-4,6-dichloropyrimidine, an equimolar amount of benzylamine, and an equimolar amount of a trialkylamine such as triethylamine. A slight excess of the amount is dissolved in 1-butanol, and the resulting solution is heated to reflux. After a lapse of a predetermined time, the solution is allowed to cool to precipitate crystals of the reaction product 5-amino-6-benzylamino-4-chloropyrimidine. The crystals are separated by filtration, washed appropriately, dried and collected.

Step 2 [Preparation of 9-benzyl-6-chloropurine] One amount of 5-amino-6-benzylamino-4-chloropyrimidine,
A large excess of ethyl orthoformate (HC (OEt) ₃ ) and a catalytic amount of ethanesulfonic acid (EtSO ₃ H) are mixed, and the resulting solution is stirred at room temperature for a given time. Then, the reaction mixture is concentrated under reduced pressure, the remaining reaction product is dissolved in ethyl acetate, and the mixture is washed successively with 5% aqueous sodium hydrogen carbonate solution, water and brine.
An ethyl acetate solution in which this reaction product is dissolved is distilled under reduced pressure to obtain a reaction product 9-benzyl-6-
After washing chloropurine appropriately, it is dried and collected.

Step 3 [Preparation of 6- (alkylamino) -9-benzylpurine] One amount of 9-benzyl-6-chloropurine represented by the formula (2) and an excess amount of the amine represented by the general formula (3). Etano
The resulting solution is stirred at room temperature for a predetermined time.
Then, the solvent ethanol is distilled off under reduced pressure, water is added to the residue, and then the reaction product is subjected to solvent extraction with ethyl acetate or the like. The reaction product is isolated by a general-purpose means such as distillation under reduced pressure, and if necessary, washed and dried to recover 6- (alkylamino) -9-benzylpurine represented by the general formula (4) as the reaction product. To do.

In the general formula (4), R ₁ and R
6-Amino-9-benzylpurine in which ₂ is both hydrogen atoms is prepared by heating the reaction solution in an autoclave at 120 ° C. using ammonia as the amine represented by the general formula (3). Atoms can be replaced with amino groups.

Step 4 [Preparation of 6- (alkylamino) -9-benzyl-8-bromopurine] One amount of 6- (alkylamino) -9-benzylpurine represented by the general formula (4) and an excess amount of acetic acid. An excess amount of bromine water is added dropwise to an acetic acid solution that dissolves sodium under ice cooling. Next, this liquid is heated and stirred. After a lapse of a predetermined time, the solution is allowed to cool, the solvent acetic acid is distilled off under reduced pressure, then the reaction product is dissolved in ethyl acetate or the like, and the filtered filtrate is recovered. The filtrate is washed successively with saturated aqueous sodium hydrogen carbonate solution and water, the solvent ethyl acetate is distilled off under reduced pressure, and dried to give the desired compound represented by the general formula (1) 6
The-(alkylamino) -9-benzyl-8-bromopurine is recovered.

It should be noted that 6-in the above general formula (4)
(Alkylamino) -9-benzylpurine and purine derivatives having various substitutions on the benzene ring of the benzyl group are similar to the compounds of the present invention and are described in the above-mentioned reference J. Med. Chem., 31, 606-612 (1988). These purine derivatives having no substitution of the bromine atom at the 8-position have been shown to have anticonvulsant action, particularly anticonvulsant action against convulsions (seizures) caused by electric shock. There are no reports of effects on sexually transmitted diseases.

The purine derivative of the present invention is at the 8-position from the 6- (alkylamino) -9-benzylpurine represented by the general formula (4) and the purine derivative having various substitutions on the benzene ring of the benzyl group. Has a clear structural difference in that it has a substitution of a bromine atom, and as shown in the examples below, it has an inhibitory effect on the adhesion of neutrophils to tissue cells as a main pharmacological effect, and thus has a wide range of inflammatory properties. It is a useful drug for treating diseases. In particular, it is a therapeutic agent suitable for use in the treatment of inflammatory disease such as rheumatism, nephritis, autoimmune disease, or graft-versus-host disease, and further suitable for continuous or long-term oral administration. .

Next, the present invention will be specifically described with reference to examples.

Example 1 [6-Amino-9-benzyl-8-bromopurine] 6-amino-9-benzyl-8-bromo in which, in the general formula (1), R ₁ and R ₂ are both hydrogen atoms. Purines were synthesized according to the steps described below.

[Step 1.1] 9-benzyl-6-chloro
Preparation of lopurine 9-benzyl-6- chloropurine as an intermediate raw material was synthesized by the following procedure. 5-amino-4,6-dichloropyrimidine 60.0 g (0.36 mol), benzylamine 39.1 g
(0.36 mol), and triethylamine 38.0 g (0.38 m
ol) is dissolved in 700 ml of 1-butanol and the resulting solution is heated to reflux overnight (about 18 hours). Then, the solution is allowed to cool and the reaction product 5-amino-6-benzylamino-
Crystals of 4-chloropyrimidine were precipitated. The crystals were collected by filtration and dried to collect 5-amino-6-benzylamino-4-chloropyrimidine crystals. The weight of the recovered 5-amino-6-benzylamino-4-chloropyrimidine after drying was 77.8 g, and the starting material 5-amino-4,6-
The yield based on dichloropyrimidine was 92%.

5-Amino-6-benzylamino-4-chloropyrimidine obtained by the above method 77.8 g (0.33 m
ol), ethyl orthoformate (HC (OEt) ₃ ) 664 ml (7.4 mo
l) and ethanesulfonic acid (EtSO ₃ H) 602 mg (0.6 mm
ol) and mix the resulting solution at room temperature overnight (about 18 hours).
It was stirred. Then, the mixture was concentrated under reduced pressure, the remaining reaction product was dissolved in ethyl acetate, and the mixture was washed with 5% aqueous sodium hydrogen carbonate solution, water and brine in this order. The ethyl acetate solution was distilled under reduced pressure to obtain a reaction product 9-benzyl-6-chloropurine as a residue. After further drying, 9-
67.8 g of benzyl-6-chloropurine could be recovered, and the yield was 84% based on the starting material 5-amino-6-benzylamino-4-chloropyrimidine.

[Step 1.2] 6-amino-9-benzyl
Preparation of lu-8-bromopurine Using 9-benzyl-6-chloropurine as an intermediate raw material, it was synthesized by the following procedure.

17.0 g (70 mmol) of 9-benzyl-6-chloropurine as an intermediate raw material was previously mixed with 123 g of ammonia (
7.2 mol) was blown in and added to 1100 ml of dissolved ethanol, and the mixture was heated and stirred in an autoclave at 120 ° C. for 48 hours. Then, it cooled to room temperature and the crystal | crystallization of the reaction product was deposited. The precipitated crystals were collected by filtration, recrystallized with a solvent methanol and purified. After purification, 16.6 g was recovered as the dry weight of 6-amino-9-benzylpurine, and the yield was 9 based on 9-benzyl-6-chloropurine as an intermediate raw material.
It was 9%.

6-amino-9-benzylpurine 10.0 g
(42 mmol) and 67.4 g (0.80 mol) of sodium acetate were dissolved in 385 ml of acetic acid. Brominated water 90.
1 g (Br ₂ : 0.56 mol) was added dropwise and mixed. Next, this liquid was heated and stirred at 70 ° C. for 1.5 hours. Then, the solution was allowed to cool, the solvent acetic acid was distilled off under reduced pressure, ethyl acetate was added to the residue to dissolve the reaction product, and the filtered filtrate was recovered.
The filtrate was washed successively with saturated aqueous sodium hydrogen carbonate solution and water. The solvent ethyl acetate was distilled off under reduced pressure, and the residue was further dried to recover the target 6-amino-9-benzyl-8-bromopurine. The amount of 6-amino-9-benzyl-8-bromopurine recovered was 6.7 g, and the yield based on the starting material 6-amino-9-benzylpurine was 50%.

6-amino-prepared according to the above steps
9-benzyl-8-bromopurine exhibited the following physical properties. NMR spectrum ¹ H-NMR δ ppm (DMSO-d ₆ ): 5.28 (2H, s: -CH _2- ),
7.23 (5H, s: -C ₆ H ₅ ), 7.36 (2H, s: -NH ₂ ), 8.11 (1
H, s)

[0029]

Example 2 [9-Benzyl-8-bromo-6-methylaminopurine] In the above general formula (1), one of R ₁ and R ₂ is a hydrogen atom and the other is a methyl group 9- Benzyl-
8-Bromo-6-methylaminopurine was synthesized according to the steps described below.

17.0 g (70 m) of 9-benzyl-6-chloropurine as an intermediate raw material prepared according to the above [Step 1.1]
mol) and 40% methylamine aqueous solution 36.2 ml (CH ₃ NH ₂ :
0.47 mol) was dissolved in 150 ml of ethanol, and the resulting solution was stirred overnight (about 18 hours) at room temperature. From this solution, the solvent ethanol was distilled off under reduced pressure, and then water was added,
The reaction product was subjected to solvent extraction using ethyl acetate as a solvent. The solvent ethyl acetate was distilled off under reduced pressure from the extract, and the reaction product 9-benzyl-6-methylaminopurine obtained as a residue was washed with hexane. After drying, 13.7 g of 9-benzyl-6-methylaminopurine was recovered, and 9-
The yield based on benzyl-6-chloropurine was 82%.

9-benzyl-6-methylaminopurine
7.8 g (32.5 mmol) and sodium acetate 12.7 g (0.15
mol) was dissolved in 120 ml of acetic acid. This solution under ice cooling,
Bromine water 17.2 g (Br ₂ : 0.10 mol) was added dropwise to the mixture. Next, this solution was heated and stirred at 50 ° C. overnight (about 18 hours). Then, the solution was allowed to cool, the solvent acetic acid was distilled off under reduced pressure,
Next, ethyl acetate was added to the residue to dissolve the reaction product, and the filtered filtrate was recovered. The filtrate was washed successively with saturated aqueous sodium hydrogen carbonate solution and water. The solvent ethyl acetate was distilled off under reduced pressure to collect 9-benzyl-8-bromo-6-methylaminopurine as a reaction product. Further, silica gel column chromatography (eluent chloroform:
Purification was carried out with methanol = 100 volumes: 1 volume). After purification, the amount of 9-benzyl-8-bromo-6-methylaminopurine of interest was 6.1 g, and the yield was 59% based on 9-benzyl-6-methylaminopurine as a raw material.
Met.

The 9-benzyl-8-bromo-6-methylaminopurine prepared according to the above steps had the following physical properties. NMR spectrum 1H-NMR δ ppm (DMSO-d ₆ ): 2.95 (3H, d: -CH ₃ ), 5.
29 (2H, s: -CH _2- ), 7.21 (5H, s: -C ₆ H ₅ ), 7.80 (1H,
s: -NH-), 8.19 (1H, s)

[0033]

Example 3 [9-Benzyl-8-bromo-6-dimethylaminopurine] In the above general formula (1), 9-benzyl-8-bromo-6-dimethyl in which both R ₁ and R ₂ are methyl groups. Aminopurine was synthesized according to the steps described below.

22.5 g (92 m) of the intermediate raw material 9-benzyl-6-chloropurine prepared according to the above [Step 1.1]
mol) and 50% dimethylamine aqueous solution 58.2 ml ((CH ₃ )
₂ NH: 0.65 mol) was dissolved in 300 ml of ethanol, and the resulting solution was stirred overnight (about 18 hours) at room temperature. From this solution, the solvent ethanol was distilled off under reduced pressure, water was added, and then the reaction product was subjected to solvent extraction using ethyl acetate as a solvent. The solvent ethyl acetate was distilled off from the extract under reduced pressure, and the reaction product 9-benzyl-6-dimethylaminopurine obtained as a residue was washed with hexane. After drying, 20.3 g of 9-benzyl-6-dimethylaminopurine was recovered, and the yield based on the intermediate raw material 9-benzyl-6-chloropurine was
It was 87%.

9-benzyl-6-dimethylaminopurine
14.8 g (58 mmol) and sodium acetate 23.1 g (0.28 m
ol) was dissolved in 380 ml of acetic acid. 23.0 g (Br2: 0.14 mol) of bromine water was added dropwise to this solution under ice cooling. next,
This solution was heated and stirred at 50 ° C. for 1 hour. Then, the solution was allowed to cool, the solvent acetic acid was distilled off under reduced pressure, ethyl acetate was added to the residue to dissolve the reaction product, and the filtered filtrate was recovered. The filtrate was washed successively with saturated aqueous sodium hydrogen carbonate solution and water. The solvent ethyl acetate was distilled off under reduced pressure to collect 9-benzyl-8-bromo-6-dimethylaminopurine as a reaction product. Furthermore, silica gel column chromatography (eluent chloroform: methanol = 50)
Volume: 1 volume) was used for purification. After purification, the desired 9-
The recovered amount of benzyl-8-bromo-6-dimethylaminopurine was 5.1 g, and the yield based on the raw material 9-benzyl-6-dimethylaminopurine was 26%.

The 9-benzyl-8-bromo-6-dimethylaminopurine prepared according to the above steps had the following physical properties. NMR spectrum 1H-NMR δ ppm (DMSO- d 6): 3.47 (6H, s: -N (CH 3) 2)
, 5.33 (2H, s: -CH _2- ), 7.28 (5H, s: -C ₆ H ₅ ), 8.2
8 (1H, s)

[0037]

Example 4 (Neutrophil adhesion inhibitory action) In order to verify that the purine derivative of the present invention inhibits adhesion between neutrophils and endothelial cells,
The effect of preventing neutrophil adhesion to vascular endothelial cells was evaluated by the following method.

In this evaluation method, rat peritoneal neutrophils were used as neutrophils, and human umbilical vein vascular endothelial cells were used as vascular endothelial cells. In addition, this evaluation method is based on the method described in Biopharmaceutical Science Laboratory, Volume 12, "Inflammation and Allerky-2", Chapter 7, Neutrophil Hirokawa Shoten. The rat neutrophils were separated and purified, and cultured in advance in 199 medium. Next, it was labeled with ⁵¹ Cr, and the neutrophil suspension was 0.1%.
(W / v) BSA-199 medium with a given density (3 x 1
0 ⁶ pieces / 500 μl). Human umbilical vein vascular endothelial cells were separated and collected from the umbilical cord vein of human umbilical cord obtained from obstetrics and gynecology. The obtained endothelial cell pellet was cultured in 20% FBS-199 medium, and further 1
Subculture was performed in 0% FBS-199 medium.

The above-mentioned vascular endothelial cells (1.5 × 10 ⁵ cells /
Wells) were plated on 24-well dishes and cultured until they reached confluence. Confluent endothelial cells were treated with 0.1% (w / v) BSA-199 medium 500
Washed twice with μl. Then, 500 μl of 0.1% (w / v) BSA-199 medium containing the test compound prepared in advance and 1 U / ml of thrombin, an inducer of neutrophil adhesion,
And immediately add 500 μl of neutrophil suspension (3 × 10
⁶ ) were added and well dispersed. The test solution was added with 5% CO
₂ incubation - data - the use, and stationary culture for 20 min at 37 ° C.. Immediately after the completion of the culture, unadhered neutrophils were suspended and removed by suction with a pipette. After that, 400 μl of 0.25% trypsin-0.01% EDTA solution was added to add 37
Let stand for 10 minutes at ℃, the remaining adhered neutrophils,
Separated from endothelial cells. The test solution was ice-cooled to obtain 0.1% c
100 μl of 199 medium containing alf serum was added to stop the trypsin reaction. Then 0.1N NaOH
400 μl of the solution was added, and the separated neutrophils were transferred to a radioactivity measuring tube, and the total amount of radioactivity from neutrophils labeled with ⁵¹ Cr was measured. The index of adhesion was calculated from the ratio of this measured value to the total radioactivity measured from 500 μl of the initial neutrophil suspension. The inhibition rate was calculated from the difference between the adhesion index measured when the test compound was not dissolved in the reaction solution and the adhesion index measured when a predetermined amount of the test compound was dissolved. did. The inhibition rate was defined as 0% when the difference in the index of adhesion was zero, and the inhibition rate was defined as 100% when the index of adhesion was equal to the case where the inducer thrombin was not added (blank group).

Various final concentrations of the test compound were selected, the correlation between the final concentration and the inhibition rate was investigated, and the effect of preventing neutrophil adhesion to vascular endothelial cells was evaluated. Table 1
The results of selecting the final concentration of 10 μM are illustrated in FIG.

[0041]

Table 1 Final concentration of test compound (μM) Suppression rate (%) Compound of Example 1 10 65 Compound of Example 2 10 65

From the above results, it can be seen that the purine derivative has an action of suppressing the neutrophil-endothelial cell adhesion process.

[0043]

Example 5 (Preventive Effect on Adjuvant Arthritis) In order to verify that the purine derivative of the present invention has an effect as a therapeutic agent for inflammatory diseases, it is used as a model of rheumatoid arthritis, which is a typical inflammatory disease. The preventive effect against adjuvant arthritis was evaluated by the following method.

The number of model rats for adjuvant arthritis was 5
Using week-old male SD rats, 0.1% as an adjuvant.
A liquid paraffin suspension of 6% Mycobacterium butyricum (0.1% / animal) was subcutaneously injected into the foot pad (one hind leg).

The purine derivative of the test compound was suspended in 0.5% CMC-Na aqueous solution at a daily dose of 100 mg / kg and orally administered daily for 15 days from the day of induction. Further, as a positive control group, indomethacin, which is a non-steroidal anti-inflammatory drug, was dosed at 1 mg / kg per day, suspended in 0.5% CMC-Na aqueous solution, and orally administered in the same manner. As a reference group, only 0.5% CMC-Na aqueous solution was orally administered in the same manner. For the onset of arthritis, the foot volume of each foot was measured by a foot volume measuring device, and the foot volume measured on the day of induction was used as a reference to calculate the increase in foot volume due to arthritis. The increase in paw volume on day 18 after induction was divided by the paw volume on the day of induction, and the value expressed in% was taken as the edema rate. Further, the difference between the edema rate of the test compound and the positive control group with respect to the edema rate of the reference group was divided by the edema rate of the reference group, and the value expressed in% was defined as the inhibition rate. Table 2 shows the results of the hind limbs injected with the adjuvant, and Table 3 shows the results of the hind limbs not injected with the adjuvant. Each group had 6 animals, and the average value was calculated.

[0046]

[Table 2] [Results of hind limbs injected with adjuvant] Test compound Edema rate (%) Inhibition rate (%) Reference group (no administration) 179.2 ± 18.1-Compound of Example 1 79.0 ± 8.9 ** 56 Compound 92.0 ± 14.7 ** 49 Compound of Example 3 136.9 ± 21.5 24 Indomethacin 74.1 ± 9.5 ** 59 Note) **: P <0.01 There is a significant difference from the reference group

[0047]

[Table 3] [Results of hind limbs without injection of adjuvant] Test compound Edema rate (%) Inhibition rate (%) Reference group (no administration) 66.0 ± 15.2-Compound of Example 16.2 ± 0.8 ** 75 Example Compound 2 of 13.9 ± 2.1 ** 79 Compound of Example 3 56.3 ± 12.8 15 Indomethacin 15.6 ± 4.2 * 76 Note) **: P <0.01 Significant difference from the reference group *: P <0.05 Reference group Significant difference from

From the above results, at the dose tested,
It can be seen that the purine derivative of the present invention has the same protective effect against adjuvant arthritis as indomethacin. It is also found that the purine derivative of the present invention can be used as a therapeutic agent to be administered orally.

Adjuvant arthritis has been reported to be caused by T cells that are highly reactive with bacterial components of M. tuberculosis (mainly heat shock proteins derived from M. tuberculosis) (Science 219, 56 (1983)). reference). Similarly, it has been reported that T cells in synovial fluid of affected areas of rheumatoid arthritis also show high reactivity with heat shock proteins derived from Mycobacterium tuberculosis (L.
ancet 2, 478 (1988)). Inflammation is caused by the aggregation of these T cells by the antigens that the tissue cells of the joint present on their surface. Taking this into consideration, from the results of Example 5 and the results of this example described above, the purine derivative of the present invention inhibits the process in which leukocytes such as T cells and neutrophils adhere to tissue cells in the affected area. It is judged that the effects of suppressing and preventing the onset and exacerbation of inflammation will occur. That is, a nonsteroidal anti-inflammatory drug such as indomethacin suppresses the action of cyclooxygenase expressed by cells, as a result of mainly leukocytes such as T cells and neutrophils adhering to tissue cells in the affected area. Cyclic Peroxide (PGH ₂ ) Generated from Arachidonic Acid
The mechanism of action of suppressing the production of Escherichia coli is different from that of the purine derivative of the present invention. In addition, the disadvantages of non-steroidal anti-inflammatory drugs such as indomethacin that inhibit cyclooxygenase in the digestive tract in a non-tissue specific manner and cause ulcer in the digestive tract at high frequency are The purine derivative of the present invention inhibits the process of accumulation of leukocytes such as T cells and neutrophils and the process of adhesion to tissue cells by tissue-specific and disease-specific antigens, so that the above-mentioned drawbacks can be overcome. Is. This characteristic becomes remarkable in continuous or long-term oral administration.

[0050]

INDUSTRIAL APPLICABILITY The purine derivative of the present invention has an effect of effectively inhibiting the process in which white blood cells such as T cells and neutrophils adhere to tissue cells in the affected area, and therefore inflammation caused by this process of cell adhesion is caused. Diseases such as autoimmune diseases, graft-versus-host disease (GVH) known as rejection during organ transplantation
D), allergic diseases, and therapeutic agents having a therapeutic effect on chronic inflammation such as osteoarthritis of the knee. Also,
Since it is a low molecular weight compound and is easily absorbed in the digestive tract, it is a therapeutic agent suitable for oral administration. Furthermore, when it is used for the treatment of chronic inflammatory diseases or autoimmune diseases for which the administration is continuous or long-term, its mechanism of action has specificity to the affected tissue, so that conventional non-steroids such as indomethacin are used. Non-tissue-specific inhibition of gastrointestinal cyclooxygenase, which is often seen with sexual anti-inflammatory agents, and has the advantage of being a therapeutic agent that overcomes the drawbacks such as causing gastric tract ulcers at high frequency. Occurs.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display area A61K 31/52 ACV

Claims (5)

[Claims] 1. The following general formula (1): (In the formula, R ₁ and R ₂ each independently represent a group selected from the group consisting of a hydrogen atom, a linear alkyl group having 1 to 4 carbon atoms, and a branched alkyl group having 3 or 4 carbon atoms. ) A purine derivative which is a compound represented by the formula or a pharmaceutically acceptable salt thereof. _2. The purine derivative according to claim 1, wherein one of R ₁ and R ₂ is a hydrogen atom, and the other is a hydrogen atom or a methyl group. 3. A leukocyte adhesion inhibitor containing the purine derivative according to claim 1 or 2 as an active ingredient. 4. A therapeutic agent for an inflammatory disease, which comprises the purine derivative according to claim 1 or 2 as an active ingredient. 5. The above-mentioned inflammatory disease is rheumatism, nephritis,
The therapeutic agent according to claim 4, which is an inflammatory disease of either autoimmune disease or graft-versus-host disease.