JPH0345074B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to the general formula (): P-(CH ₂ ) _o -O-R ¹ () (wherein P is the general formula:

[Formula] (wherein R represents a hydrogen atom) hydroxypurine residue, n is an integer from 1 to 6, and R ¹ is a general formula:

Residues represented by [formula] (wherein R ² is a group bonded to the α-carbon of the α-amino acid, and R ³ is a hydrogen atom), general formula:

[Formula] (In the formula, m is an integer from 2 to 6, R ² is the same as above,
R ⁴ is OH) or general formula:

Residue or general formula represented by [Formula] (in which R ² and R ⁴ are the same as above): (However, in this case, in the general formula (), n is a residue represented by 2)) A method for producing the compound and an immunization agent containing at least one compound represented by the general formula () as an active ingredient. Concerning regulators. The "immunomodulating effect" of the present invention refers to an effect of interacting with cells having immune capacity and activating the immune capacity of the cells. Recently, in the field of immunopharmacology, a series of extracted polypeptides with thymic hormonal action have been investigated, and they can be used as antigen reinforcement agents in clinical medicine, such as the treatment of several infectious diseases, especially viral infections, or malignant tumors. used in the field. For example, a drug containing 12 different polypeptides extracted from bovine thymus is known. The drug is aimed at increasing the immunomodulatory effect on the attack of viruses, bacteria and even tumor cells. The amino acid sequence of the extracted polypeptide has not yet been elucidated, and it is not clear what specific action the polypeptide has in regulating immune action. As other immunopharmacology studies, adenosine derivatives or clinical consequences for many viruses.
We have succeeded in synthesizing a derivative of an effective component for the human immune system that has an immunostimulatory effect on DNA or viral RNA. However, even in these cases, activation of the immune system appears to be nonspecific and lacks selectivity for one or more lymphocyte functions (e.g., stimulating or suppressing the immune response). ). Similarly, activation of non-specific lymphocyte functions by the above-mentioned drugs is also uncertain. Therefore, the main objective of the present invention is to provide a compound that has a specific immunomodulatory effect that can activate only a cell fraction that has a specific function. R ² in the compound represented by the above general formula ()
An α-amino acid in which the carbon to which is bonded is the α-carbon can be used even if it is in the optically active d-form or racemic form, but the optically active l-form is preferable. Examples of α-amino acids in which the carbon to which R ² is bonded in the compound represented by the above general formula () are α-carbons include glycine, alanine, valine, leucine,
Examples include isoleucine, serine, threonine, aspartic acid, glutamic acid, arginine, lysine, cysteine, cystine, methionine, phenylalanine, tyrosine, tryptophan, proline, and histidine. The hydroxypyrimidine ring of the purine derivative of the present invention may be an enol type as represented by the general formula (), a keto type, or a mixture of an enol type and a keto type. The sequence and properties of the amino acids attached to the purine ring qualitatively change the hormone-like message,
Thereby, the compound () acts chemically on cells. Therefore, by using a compound represented by the general formula (), only a cell fraction having a specific function can be activated. Furthermore, compared to known analogous compounds, the compounds of the present invention can have more limited clinical use against the pathology of immune deficiencies, immune inactivations or autoimmune diseases, and malignant tumors themselves. As mentioned above, the present invention relates to a method for producing a compound represented by the general formula (), and can be summarized as follows. That is, the general formula (): P-(CH ₂ ) _o -OH () (wherein P is the general formula:

A 6-hydroxypurine residue represented by the following formula (wherein R is a hydrogen atom, n is an integer from 1 to 6) and the general formula (): (In the formula, R ² is the same as above, R ⁵ is an amino protecting group commonly used in peptide synthesis, such as t-
The carboxyl group is activated using a known condensing agent such as N,N'-carbonyldiimidazole, dicyclohexylcarbodiimide, etc. The general formula (): General formula (a) consisting of preparing an intermediate represented by (wherein P, n, R ² and R ⁵ are the same as above) and then removing the protection of the amino group: (In the formula, P, n, R ² and R ³ are the same as above) A method for producing a compound represented by the general formula () by reacting a compound represented by the general formula () with an activated dicarboxylic acid derivative, (): P-( _CH2 ) _o -O-CO-( _CH2 ) _n -COOH
An intermediate represented by () (wherein P, n, and m are the same as above) is prepared, and then the carboxyl group of this intermediate is activated by a known method, and if necessary, the carboxyl group can be replaced with, for example, a benzyl group. General formula (b) consisting of protecting with a protecting group such as and reacting with an amino acid: (In the formula, P, n, m, R ² and R ⁴ are the same as above)
Method for producing a compound represented by the formula () and the compound represented by the general formula () and phosgene (COCl ₂ )
and then α-amino acid or general formula (′): (In the formula, R ² is the same as above, and R ⁶ is a protecting group for the carboxyl group, such as a benzyl group.) By reacting with an α-amino acid whose carboxyl group is protected, the general formula (): General formula (c) consisting of preparing an intermediate represented by (wherein P, n, R ² and R ⁶ are the same as above) and further removing the protecting group: (wherein P, n, R ² and R ⁴ are the same as above). In addition, a compound represented by the general formula () having a side chain different from that shown above is produced according to a production method similar to that described above. Furthermore, the compounds of the present invention or corresponding intermediates can also be obtained by using a similar compound in which a chlorine atom is attached to the pyrimidine ring instead of a hydroxyl group as a starting material and converting the chlorine atom into a hydroxyl group by a known nucleophilic substitution reaction. The body is manufactured. The starting material, a compound represented by the general formula (): P-(CH ₂ ) _o -OH () (where n and P are the same as above), was prepared by Schaeffer HS et al.
al.) (J.Med.Chem., 21, (1968)). Next, the reaction formula is shown. Next, general formula ( ¹ ): P ¹ − (CH ₂ ) _o −OH ( ¹ ) (in the formula, P ¹ is

[Formula] n is an integer of 2 to 6) and general formula ( ¹ ) obtained by reacting them with succinic anhydride in pyridine: P ¹ -(CH ₂ ) _o -O-CO-( CH ₂ ) ₂ −COOH
Table 1 shows the melting point and elemental analysis position of the hemisuccinate ester represented by ( ¹ ) (wherein P ¹ and n are the same as above). Reference Compound ( ¹ ) mp: 262℃, Kondo et al. (Makromol.Chem.298−301 (1969))

【table】

【table】

[General formula ( ^V1-2 ): Represented by ^P1- ( _CH2 ) ₂ -O-CO-( _CH2 ) _2- COOH( ^V1-2 ) (in the formula, ^P1 is the same as above) Production of 9-(2-hydroxyethyl)-6-hydroxypurine hemisuccinate]

(A) 20ml of 1.24g (12.3m mole) of succinic anhydride
0.74 g of 9-(2-hydroxyethyl)-6-hydroxypurine was dissolved in warm pyridine.
(4.1 mmole) and a small amount of dimethylaminopyrimidine (hereinafter referred to as DMAP) were added using a spatula, and the mixture was refluxed for 22 hours while stirring. The pyridine was then distilled off, and the residue was washed with alcohol to remove excess succinic acid. After further filtration, the residue was dried to give the title compound (mp: 210-215°C). Furthermore, the compound is subjected to thin layer chromatography (hereinafter referred to as TLC).
(80% ethanol - 20% triethylamine) and the structure was determined by IR spectroscopy. (B) 0.20 g (1 mmole) of 9-(2-hydroxyethyl)-6-chloropurine and 1 g of succinic anhydride were reacted in the same manner as in Production Example (A), and after distilling off the pyridine, the residue was It was dissolved in 10 ml of formic acid and refluxed for 30 minutes. After cooling, add 20ml of methanol,
205 mg of the title compound was obtained by suctioning off the precipitate. Example 2 [General formula: ^P1- ( _CH2 ) ₂ -O-CO-( _CH2 ) _2-
Production of 6-hydroxy-9-(2-(2-chlorocarbonylethyl)-carbonyloxyethyl)-purine hydrochloride represented by COCl·HCl (in the formula, P ¹ is the same as above)] General formula (V ¹ 100 mg of hemisuccinate represented by -2) was dissolved in 5 ml of thionyl chloride at room temperature and stirred at room temperature for 24 hours. Excess thionyl chloride was then distilled off under reduced pressure, and the resulting white solid was dissolved in anhydrous ethyl ether and washed several times with ethyl ether by suction filtration to yield the title compound as white microcrystals. (Collapse temperature: over 200°C) The structure of the compound was determined by converting the compound into a methyl ester (mp: 183-185°C) by NMR and mass spectrometry. Example 3 [General formula (a-1): (In the formula, P ¹ is the same as above) Production of 6-hydroxy-9-(2-(2-amino-3-(4-hydroxyphenyl)propionyloxy)))-ethylpurine] (A) O-Benzyl-N-Boc-Tyrosine 371mg
(1 mmole) was dissolved in anhydrous dimethyl sulfoxide (hereinafter referred to as DMSO), 368 mg (2 mmole) of carbonyldiimidazole was added, and the mixture was heated at 70°C for 5 hours. The reaction mixture was then diluted with 5 ml of DMSO containing 20 mg of 4-dimethylaminopyridine and preheated to 70°C.
280 mg (1 mmole) of (2-hydroxyethyl)-6-hydroxypurine was added to the solution. The resulting reaction mixture was heated at 70-80° C. for about 2 hours, then cooled and 50 ml of water was added. Extract the oil that separates twice with 25 ml of ethyl acetate,
The extracted material was dried and ethyl acetate was removed by distillation under reduced pressure. The oily residue was dissolved in 20 ml of ethanol without further purification, then 5 ml of cyclohexane and 120 mg of palladium/charcol were added and refluxed for 2 hours with stirring. The precipitate deposited during refluxing was dissolved in acetic acid, and after removing the charcoal, 4 to 5 drops of trifluoroacetic acid were added and the mixture was refluxed for 30 minutes.
The acetic acid was then distilled off in vacuo, and the residue was washed with methanol to give the title compound as colorless crystals. (Yield: 87%, mp: 235-240°C) Next, the characteristic values of the title compound are shown. NMR spectrum (in DMSO, internal standard:
TMS) 3.0δ 2H (doublet) CH ₂ −CH 4.0δ 1H (triplet) CH −CH ₂ 4.10δ 4H (singlet) CH ₂ −CH ₂ 6.6~7.0δ 4H (AA′BB′)C ₆ H ₄ 7.9 and 8.0δ 2H (singlets) purine hydrogen atom 11.0δ 4H (broad band) variable hydrogen atom (B) O-benzyl-N-Boc-tyrosine 371 mg
(1 mmole) dissolved in anhydrous acetonitrile, 198 mg of 9-(2-hydroxyethyl)-6-chloropurine, 210 mg of dicyclohexylcarbodiimide and 25 mg of 4-pyrrolidinopyridine.
was added and heated at 60°C for 12 hours. After cooling the resulting reaction mixture, dicyclohexylurea was separated and the solvent in the liquid was distilled off under reduced pressure. The residue is purified without further methanol:
It was dissolved in acetic acid (50:50) mixture, and 100 mg of palladium/charcoal and 500 mg of ammonium formate were added thereto. After stirring at room temperature for 30 minutes,
The palladium/charcoal was separated and the solvent was distilled off under reduced pressure. The residue was treated with 50% trifluoroacetic acid containing 10% anisole for 1 hour, then 15 ml of methanol was added and left at 5°C for 24 hours. The title compound was obtained by filtering the resulting precipitate. Example 4 [General formula (Ib-1): ^P1- ( _CH2 ) ₂ -O-CO-( _CH2 ) _2- CO-NH
-CH ₂ -COOH (Ib-1) (wherein P ¹ is the same as above) of 6-hydroxy-9-(2-(2-(carboxymethylaminocarbonyl)ethyl)carbonyloxyethyl) purine Production] (A) 2.8 g (10 mmole) of the hemisuccinate of 9-(2-hydroxyethyl)-6-hydroxypurine obtained in Example 1 was converted into hydrochloride of chloride by the method shown in Example 2. did. The obtained amorphous powder was suspended in 20 ml of anhydrous methylene chloride,
4.95 g (30 mmole) of glycine benzyl ester dissolved in 30 ml of anhydrous methylene chloride was added.
The resulting reaction mixture was stirred at room temperature for 3 hours and then filtered. The liquid was washed with 30 ml of saturated sodium bicarbonate solution and 30 ml of saturated brine, dried over sodium sulfate, and then evaporated under reduced pressure.
The residue was purified by silica gel column chromatography using acetic acid:methanol (1:
1). Furthermore, by crystallizing from ethanol, it becomes a white powder crystal 6
-Hydroxy-9-(2-(2-(benzyloxy)carbonylmethylaminocarbonyl)ethyl)carbonyloxy)ethylpurine 2.3g
(yield: 52%, mp: 158-160°C). Next, the characteristic values of the obtained compound are shown. Elemental analysis value: C ₂₀ H ₂₁ N ₅ O ₆ Calculated value (%): C56.20 H4.91 N16.39 Actual value (%): C56.31 H4.73 N15.98 NMR spectrum ((CDCl ₃ + DMSO− d ⁶ ) middle,
Internal standard: TMS) 2.65δ 4H (singlet) CO−CH ₂ −CH ₂ −CO 3.95δ 2H (singlet) NH− CH ₂ −CO 4.40δ 4H (broad singlet) N−CH ₂ −CH ₂
-O 5.23δ 2H (singlet) CH ₂ -C ₆ H ₅ 7.26δ 5H (singlet) C ₆ H ₅ 7.95 and 8.10δ 2H (singlets) Purine hydrogen atoms 10δ 2H (broad band) NH and OH (B) Above implementation 220 mg of the compound obtained in Example 4 (A)
(0.5m mole) was dissolved in methanol, and while stirring, 86mg (1.6m mole) of ammonium formate was dissolved in methanol.
and 100 mg of palladium/charcoal were added. The resulting reaction mixture was further stirred at 40° C. for 1 hour, then 40 mg of ammonium formate was added, and stirring was continued for another hour. After confirming completion of the debenzylation reaction by TLC (ethyl acetate:methanol (1:1)), the reaction mixture was filtered through celite and the liquid was evaporated. The obtained crude product was treated with ethanol,
The precipitate obtained by filtration was crystallized from a mixture of methanol and water (90:10) to obtain 97 mg of the title compound (Ib-1) as white crystals (yield: 55
%, mp: 186-190℃). Next, the characteristic values of the obtained compound are shown. Elemental analysis value: C ₁₃ H ₁₅ N ₅ O ₆ Calculated value (%): C46.29 H4.45 N20.75 Actual value (%): C46.39 H4.25 N20.97 NMR spectrum ((CD ₃ OD + TFA) Medium, internal standard: TMS) 2.63δ 4H (singlet) CO−CH ₂ −CH ₂ −CO 3.98δ 2H (singlet) NH− CH ₂ −CO 4.65δ 4H (multiplet) N−CH ₂ −CH ₂ −O 8.2 and 9.25δ 2H (singlets) purine hydrogen atom Example 5 [General formula (Ib-2): (In the formula, P ¹ is the same as above) Production of 6-hydroxy-9-(2-(2-(1-carboxy)ethylaminocarbonylethyl)carbonyloxyethylpurine) (A) The general formula ( 9-( ²
-Hydroxyethyl)-6-hydroxypurine hemisuccinate (2.8 g (10 mmole)) was converted into a chloride hydrochloride by the method shown in Example 2, and 5.2 g of dl-alanine benzyl ester was prepared.
(30 m mole) dissolved in methylene chloride 30
Added to ml. The resulting reaction mixture was stirred at room temperature for 6 hours, treated in the same manner as in Example 4 (A), and crystallized from water to give white crystals of 6-hydroxy-9-(2-(2- 2.6 g of (1-benzyloxycarbonylethylaminocarbonyl)-ethyl)carbonyloxy)ethylpurine (yield: 57%, mp: 140-142°C) was obtained. Next, the characteristic values of the obtained compound are shown. Elemental analysis value: C ₂₁ H ₂₃ N ₅ O ₆ Calculated value (%): C57.14 H5.21 N15.87 Actual value (%): C57.01 H5.01 N15.69 NMR spectrum ((CDCl ₃ + DMSO) Medium, internal standard: TMS) 1.45δ 3H (doublet) CH ₃ −CH 2.65δ 4H (singlet) CO−OH ₂ −CH ₂ −CO 4.45δ 1H (quartet) CH −CH ₃ 4.58δ 4H (broad singlet) N −CH ₂ −CH ₂
-O 5.23δ 2H (singlet) CH ₂ −C ₆ H ₅ 7.25δ 5H (singlet) C ₆ H ₅ 7.95 and 8.08δ 2H (singlets) Purine hydrogen atom 10-11δ 2H (broad signal) NH and
OH (B) 226 mg of the compound obtained in (A) of Example 5 above
(0.5 mmole) was dissolved in 20 ml of methanol and debenzylated in the same manner as in Example 4 (B). It was then recrystallized from ethanol to give 103 mg of the title compound as a white crystalline solid (yield: 57%, mp:
172-178℃). Next, the characteristic values of the obtained compound are shown. Elemental analysis value: C ₁₄ H ₁₇ N ₅ O ₆ Calculated value (%): C47.86 H4.88 N21.30 Actual value (%): C48.18 H4.70 N21.60 NMR spectrum ((CD ₃ OD + TFA) Medium, internal standard: TMS) 1.45δ 3H (doublet) CH ₃ −CH 2.60δ 4H (singlet) CO−CH ₂ −CH ₂ −CO 4.40δ 1H (quartet) CH −CH ₃ 4.65δ 4H (broad singlet) N −CH ₂ −CH ₂
-O 8.10δ 2H (singlets) purine hydrogen atom Example 6 [General formula (Ib-3): (where P ¹ is the same as above) N〓−(3
-Production of -(2-(6-hydroxypurinyl-9)-ethoxy-carbonyl)-propionyl)lysine] (A) N〓-carbobenzyloxy-l-lysine-
3.70 g (10 m mole) of benzyl ester and 0.25 ml of triethylamine were dissolved in 50 mL of anhydrous methylene chloride.
The hydrochloride salt obtained from 2.8 g (10 mmole) of the hydroxyethyl derivative represented by the general formula (V ¹ -2) dissolved in 100 ml of chloride was added to the suspension in methylene chloride. The resulting reaction mixture was stirred at room temperature for 4 hours and then filtered. The liquid was distilled off under reduced pressure, and the residue was treated with ethanol to form a white precipitate, and further mixed with ethanol:water (90:
10) to obtain 3.28 g of N-carbobenzyloxy-N-(3-(2-(6-hydroxypurinyl-9)ethoxy-carbonyl)propionyl)lysine benzyl ester (yield: 52%) , mp: 134-136℃). Next, the characteristic values of the obtained compound are shown. Elemental analysis value: C ₃₂ H ₃₆ N ₆ O ₈ Calculated value (%): C60.75 H5.69 N13.29 Actual value (%): C60.35 H5.73 N13.20 NMR spectrum ((CDCl ₃ + DMSO) Medium, internal standard: TMS) 1 to 1.6δ 6H (multiplet) CH ₂ −CH ₂ −
CH ₂ 2.6δ 4H (singlet) CO−CH ₂ −CH ₂ −CO 3.1δ 2H (triplet) CH ₂ −NH 4.4δ 5H (broad singlet) N−CH ₂ −CH ₂
−O+CH−COOBz 5.03 and 5.13δ 4H (double singlet) CH ₂ −
C ₆ H ₅ 7.3δ 10H (singlet) C ₆ H ₅ 7.95δ 2H (singlet) Purine hydrogen atom 9-11δ 3H (broad sisnal) NH,OH (B) Compound obtained in (A) of Example 6 above was debenzylated in the same manner as in Example 4 (B) to obtain 403 mg of the title compound as a crude product. This was further crystallized from water to obtain 345 mg of the title compound (yield: 76%, mp unknown) as a white, hygroscopic powder. Next, the characteristic values of the obtained compound are shown. In NMR spectrum (DMSO + D ₂ O), internal standard: TMS) 1.8δ 6H (multiplet) CH ₂ −CH ₂ −CH ₂ 2.6δ 4H (multiplet) CO−CH ₂ −CH ₂ −
CO 3.2δ 2H (triplet) CH ₂ −NM ₂ 3.6δ 1H (triplet) CH −CH ₂ 4.45δ 4H (singlet) O−CH ₂ −CH ₂ −N 8.05δ 2H (singlet) Purine hydrogen atom example 7 [ General formula (Ib-4): (wherein P ¹ is the same as above)
-(2-(6-hydroxypurinyl-9)-ethoxycarbonyl)-propionyl)-N'-(4-carboxy-4-aminobutyl)guanidine] Represented by the above general formula (V ¹ -2) compound 300
N〓-z-l was added to a suspension of the hydrochloride obtained from mg in 5 ml of anhydrous dimethylformamide.
- 330 mg (1.07 mmole) of arginine and 0.2 ml of triethylamine dissolved in 4 ml of dimethylformamide were added under stirring. After stirring the resulting reaction mixture for 3 hours, the solvent was removed, and the resulting crude product was further purified by gel filtration. The eluate was first freeze-dried, then dissolved in 30 ml of methanol, and 100 mg of ammonium formate was added to this.
and 40 mg of palladium/charcol.
Stirred at ~45°C for 3 hours. The resulting reaction mixture was filtered through Celite and the solvent was saturated with HCl.
The resulting precipitate is then filtered, washed with isopropanol and dried under vacuum in the presence of phosphorus pentoxide to yield the white, amorphous and hygroscopic title compound.
195mg (collapse temperature: over 200℃) was obtained. Next, the characteristic values of the obtained compound are shown. NMR spectrum (in (DMSO + D ₂ O), internal standard: TMS) 1.78δ 4H (multiplet) −CH ₂ −CH ₂ − 2.55δ 4H (singlet) CO−CH ₂ −CH ₂ −CO 3.35δ 2H (triplet) CH ₂ -NH- 3.55δ 1H (triplet) CH -CH ₂ 4.40δ 4H (singlet) N-CH ₂ -CH ₂ -O 8.03 and 8.06δ 2H (singlet) Purine hydrogen atom Example 8 [General formula (Ib-5 ): (where P ¹ is the same as above) N〓−(3
Production of -(2-(6-hydroxypurinyl-9)ethoxy-carbonyl)-propionyl)-tyrosine 181 mg (1 mmole) of l-tyrosine was dissolved in deionized water.
In addition to 400 mg of sodium hydrogen carbonate dissolved in 10 ml, 340 mg of the hydrochloride salt obtained in Example 2 was added. However, the above reaction was carried out at 10°C or lower. Once the reaction is complete (takes approximately 30 minutes), 5%
The pH was adjusted to 7 with hydrochloric acid and freeze-dried. Then, dissolve the freeze-dried material in warm anhydrous methanol,
After cooling, the mixture was centrifuged and the solvent was distilled off from the resulting supernatant under vacuum to obtain fine white crystals. The characteristic values of this product matched those of the compound represented by general formula (Ib-5). Next, its characteristic values are shown. NMR spectrum (in DMSO, internal standard:
TMS) 2.5δ 4H (singlet) CO−CH ₂ −CH ₂ −CO 3.05δ 2H (doublet) CH ₂ −CH 3.98δ 1H (multiplet) CH −CH ₂ 4.45δ H (singlet) N−CH ₂ −CH ₂ -O 6.6-7.0δ 4H (AA′, BB′) −C ₆ H ₄ − 7.9 and 8.0δ 2H (singlets) Purine hydrogen atom 9δ 1H (doublet) NH 11.5δ 2H (broad band) OH Example 9 [General Formula (Ic-1): (wherein P ¹ is the same as above) 6-hydroxy-9-(6-(2-(carboxyethyl)
Production of aminocarbamoyloxy)hexyl)purine] (A) 2.2 g (9 mmole) of 9-(6-hydroxyhexyl)-hypoxanthine represented by formula ( ^1-6 ) was suspended in 50 ml of anhydrous toluene.
5 ml of 20% phosgene solution (in toluene) were added under stirring. The resulting reaction mixture was stirred at room temperature for 3 hours and then placed under a nitrogen atmosphere to remove excess phosgene.
5.2 g (30 mmole) was added to a solution in 30 ml of methylene chloride. After stirring for 1 hour, the resulting precipitate was filtered off, and the liquid was distilled off under reduced pressure. The residue was dissolved in ethanol and crystallized from water to give a white crystalline powder. (Yield: 7%, mp: 167-168°C) Next, the characteristic values of the obtained compound are shown. Elemental analysis value: C ₂₂ H ₂₇ N ₅ O ₅ Calculated value (%): C59.86 H6.12 N15.87 Actual value (%): C59.60 H6.02 N15.63 NMR spectrum (in DMSO, internal standard :
TMS) 1.35δ 8H (singlet) C-(CH ₂ ) ₄ −C 1.48δ 3H (doublet) CH ₃ −CH 4.00δ 4H (2triplets) CH ₂ −O and CH ₂
-N 4.43δ 1H (quartet) CH -CH ₃ 5.23δ 2H (singlet) CH ₂ -C ₆ H ₅ 7.30δ 5H (singlet) C ₆ H ₅ 7.80 and 8.30δ 2H (singlets) Purine hydrogen atoms 9-11δ 2H (broad signal) OH, NH (B) 440mg (1m mole) of the compound obtained in (A) above
was debenzylated in the same manner as in Example 4 (B) to give 318 mg of the title compound as a white powder.
(yield: 90%, mp: >220°C). Next, the characteristic values of the obtained compound are shown. Elemental analysis value: C ₁₅ H ₂₁ N ₅ O ₅ Calculated value (%): C51.28 H5.98 N19.94 Actual value (%): C51.00 H6.02 N20.31 NMR spectrum ((DMSO + D ₂ O) Medium, internal standard: TMS) 1.33δ 8H (broad singlet) C- (CH ₂ ) ₄ -
C 1.45δ 3H (doublet) CH ₃ −CH 3.94δ 2H (triplet) CH ₂ −N) 4.12δ 2H (triplet) CH ₂ −O 4.40δ 1H (quartet) CH −CH ₃ 7.95 and 8.12δ 2H (singlets) Purine hydrogen atom Example 10 [General formula (Ic-2): (wherein P ¹ is the same as above) 6-hydroxy-9-(5-(2-(carboxyethyl)
Production of amino-carbamoyloxy)pentyl)purine] (A) 6 by the same method as in Example 9 (A) using 9-(5-hydroxypentyl)-hypoxanthine represented by formula ( ^1-5 ). -Hydroxy-
9-(5-(2-(benzyloxycarbonyl-
Ethylamino-carbamoyl)oxy)pentyl)purine (mp, 184°C) was obtained. Next, the characteristic values of the obtained compound are shown. Elemental analysis value: C ₂₁ H ₂₅ N ₅ O ₅ Calculated value (%): C57.66 H5.72 N16.01 Actual value (%): C57.31 H5.62 N16.20 NMR spectrum (in DMSO, internal standard :
TMS) 1.36δ 6H (broad singlet) C-( _CH2 ) _3-
C 1.45δ 3H (doublet) CH ₃ −CH 4.03δ 4H (2triplets) CH ₂ −O and
CH ₂ N 4.43δ 1H (quartet) CH −CH ₃ 5.23δ 2H (singlet) CH ₂ −C ₆ H ₅ 7.3δ 5H (singlet) C ₆ H ₅ 7.80 and 8.28δ 2H (singlet) Purine hydrogen atom 9-11δ 2H (broad signal) OH, NH (B) Using the compound obtained in (A) above, perform the procedure of Example 9.
The title compound (collapse temperature: 200°C) was obtained by the same method as (B). Next, the characteristic values of the obtained compound are shown. Elemental analysis value: C ₁₄ H ₁₉ N ₅ O ₅ Calculated value (%): C49.85 H5.63 N20.87 Actual value (%): C49.80 H5.82 N20.45 NMR spectrum (DMSO, D ₂ O Medium, internal standard: TMS) 1.35δ 6H (broad singlet) C- (CH ₂ ) ₃ -
C 1.45δ 3H (doublet) CH ₃ −CH 3.95δ 2H (triplet) CH ₂ −N 4.12δ 2H (triplet) CH ₂ −O 4.40δ 1H (quartet) CH−CH ₃ 7.95 and 8.10δ 2H (singlets) Purine Hydrogen atom Example 11 [General formula (Ic-3): (wherein P ¹ is the same as above) N ² −(5
Production of -(hypoxanthine-9-yl)pentyloxycarbonyl)-L-arginine] Suspend 9-(5-hydroxypentyl)hypoxanthine (40 mmol) represented by formula ( ^1-5 ) in 200 ml of anhydrous toluene. 25 ml of 20% phosgene solution (in toluene) was added to the mixture under stirring. After stirring the resulting reaction mixture at room temperature for 24 hours,
The solvent was distilled off under reduced pressure. The obtained crude chlorocarbonate/hydrochloric acid was suspended in 100 ml of toluene, and a solution of 13.6 g of L-arginine dissolved in 70 ml of hot water was added to this suspension. The reaction mixture was stirred at room temperature for 12 hours. The aqueous layer was separated and its pH value was adjusted to 7 with _NaHCO3 . Water was distilled off under reduced pressure, and the residue was purified by silica gel chromatography (crude product/silica gel = 1:40) using methanol as an eluent (yield: 65%). Next, the characteristic values of the obtained compound are shown. [α] = 4.1° (C = 2%, in water) MS analysis (FD mode): 423 (MH ⁺ ) It has a specific immunomodulatory effect that is superior to that of drugs. This is a graph showing the general tendency of the effect of Compound (Ib-4) and the comparative compound methisopurinol on the rejuvenation of lymphocytes when the lymphocytes are rejuvenated by PHA (hereinafter referred to as PHA). The concentration (γ/ml) of the added compound (Ib-4) and the comparative compound methysopurinol is shown,
The vertical axis shows ^3H -labeled thymidine (hereinafter referred to as ^3H -thymidine) added to the medium by imaginal lymphocytes.
The amount taken in is expressed in cpm (count per minute). Note that PHA was used at a concentration of 0.01 ml/ml. The rejuvenation response of lymphocytes induced by PHA is lower than the normal range in lymphocytes of patients with Hodgkin's disease. In vitro effects of compound (Ib-4) and methisopurinol on lymphocyte blastogenesis induced by PHA
The effects on these compounds were investigated by culturing lymphocytes that had been transformed with PHA in the presence of various concentrations of these compounds. The concentrations used were: methysopurinol: 100, 200, 400 γ/ml; compound (Ib-4): 0.1, 0.5, 1, 5 γ/ml. As is clear from Figure 1, compound (Ib-4) is
At a concentration of 0.1 γ/ml, methisopurinol already enhances the lymphocyte blastogenesis response induced by PHA, whereas even at 200 to 400 γ/ml, compound (Ib-4)
It shows only a lower enhancing effect than . More importantly, the concentration of 0.1 to 1 γ/ml at which compound (Ib-4) exhibits an enhancing effect is a physiological concentration and can be applied in vivo. On the other hand, the concentration at which methisopurinol exhibits the maximum effect is too high for use in living organisms. Therefore, the results of this experiment demonstrate that one of the compounds of the present invention
It has been shown that one compound (Ib-4) can be used for more effective treatment than methisopurinol. FIG. 2 is a graph showing the effect of compound (Ib-4) on the oxidative metabolic activity of the membrane of imaginalized polymorphonuclear lymphocytes. That is, polymorphonuclear leukocytes (2.5 cells x 10 ⁴ /ml) were incubated in a phosphate buffer solution (hereinafter referred to as PBS).
After adding 50 μl of PHA, ferricytochrome C (1.5
mg/ml) for 15 minutes at 37°C. The amount of reduced cytochrome C in the cell-free supernatant was then determined by absorbance at 550 nm and O ₂ ^- according to the standard formula.
The amount released was calculated. The horizontal axis of the graph is the compound (Ib−
The concentration (γ/ml) of 4), the vertical axis shows the absorbance at 550 nm. From the results of statistically processing the data obtained in the above manner using a T test, compound (Ib-4) was found to be
It was revealed that a concentration of 1 γ/ml had a statistically sufficient effect of promoting superoxide production.
At higher concentrations, the amount of O ₂ ^- produced decreases slowly, probably due to a complex mechanism of metabolic control.
Increased production of superoxide anions indicates more effective phagocytosis. In addition, compound (b-4) is 0.001 to 40 μg/ml
It also has the effect of inhibiting the uptake of 3H-thymidine in splenocytes of BALB/C mice aged 8 to 10 weeks, which have been rejuvenated with PHA or lipopolysaccharides (hereinafter referred to as LPS). Compound (Ib-4) in
The inhibition of splenocyte proliferation in vitro is dose-dependent, and this effect is probably due to suppressor cell mechanisms. Next, the splenocytes of the BALB/C mice were transferred to PHA.
Table 2 shows the results of investigating the effect of the compound (Ib-4) on the uptake of ³ H-thymidine in 10 ⁶ cells/ml culture medium of cells that had been rejuvenated with LPS.

【table】

[Table] Furthermore, the effect of compound (Ib-4) on suppressor cells was investigated by mixed lymphocyte reaction using lymphocytes obtained from healthy volunteers. That is, the lymphocytes were incubated with compound (Ib-4) at various concentrations for 48 hours, washed, and then treated with autologous lymphocytes that had been transformed with concanavalin A (hereinafter referred to as Con A) for 3 days or treated with mitomycin. The cells were incubated with xenogeneic lymphocytes for 5 days. Compound used (Ib-4)
The concentration was 0.1-1.5 μg/ml, and the growth inhibition effect was approximately 60% compared to the control.
The effects of the compounds of the present invention on the ability of T-lymphocytes to form active E-rosettes have also been reported by Pasino et al. (Boll. Isc.
The investigation was conducted according to Sieroter. Mil. 60, 4, 297-301 (1981). Briefly, lymphocytes were incubated for 24 hours at 4°C, then isolated and incubated in the presence or absence of various concentrations of test compound before analysis.
Incubated for 90 minutes. The results were expressed as the percentage (%) of cells that formed rosettes. Compound (Ib−
2) was 35-50% at 1-3 μg/ml, and the percentage of active rosettes decreased at higher concentrations. Furthermore, the compounds of the present invention can be used in vivo (C57Be/6
When administered to mice at 0.1 to 5 mg/Kg, it has the effect of increasing anti-sheep red blood cell antibodies and anti-TNF (trinitrophenyl)-LPS antibodies. According to the trypan blue exclusion test, it was found that the compound of the present invention has no cytotoxicity (survival rate of 98% or more) in cultured human mononuclear peripheral blood cells even at extremely high concentrations of 60 to 120 μg/ml. The regulation of ³ H-thymidine uptake by murine and human mononuclear cells by a compound (Ic-3) was investigated. It has been difficult to understand whether and how to increase ³ H-thymidine uptake by human peripheral blood cells and/or murine splenocytes. This is probably due to adherent cells in each assay.
This may be due to the fact that the variable absolute number of is likely to significantly affect the test. However, when compound (Ic-3) is added to the culture medium, compound (Ic-3) increases the
In 18%, administration of suboptimal doses of PHA enhanced lymphocyte responses. The PHA responsiveness of each was low. In elderly diabetics, all blood donors have low initial
40-45 of them showed PHA reactivity.
% showed enhancement of PHA reaction. Similar results were obtained in the group of patients who received antitumor treatment for 7-10 days before the experiment. Next, we investigated the enhancement of chemiluminescence (CL) and chemotaxis by human polymorphonuclear leukocytes (PMNs) and alveolar macrophages. 83% of the tested blood donors responded, demonstrating that compound (Ic-3) modulates CL by human PMNs. Similar results were obtained in two experiments using purified alveolar macrophages obtained from bronchial aspirates. Compound (Ic-3) contains Ca ²⁺ and
When tested in the absence of Mg2 ⁺ , it was inactive, similar to Tuftsin. Additionally, high serum concentrations (10-15%) affected tuftsin, substance P and compound (Ic-3) activities. In fact, the increase in CL in PMNs caused by both compound (Ic-3) and tuftsin was significantly inhibited by fetal calf serum (FCS) or human AB serum at concentrations of 10% or higher. PMN chemotaxis was increased when compound (Ic-3) was added to the lower Boyden chamber and inhibited when mixed with cells in the upper chamber. Elderly patients with post-cytotoxic disease (post-
In vitro responses to mitogens and antigens in cytotoxic patients were evaluated in a total of 46 individuals. Antigen-induced proliferation is caused by compound (Ic-3)
PMN-induced chemiluminescence was shown to be significantly increased, while not affected by PMN-induced chemiluminescence.

[Brief explanation of drawings]

Figure 1 shows that the compound (Ib-
A graph showing the general trend of the effect of 4) and the comparative compound methysopurinol on the larval development of lymphocytes. Figure 2 shows the effect of a compound (Ib-4) on the oxidative metabolic activity of the membrane of larva-formed lymphocytes. This is a graph showing the effect of

Claims (1)

[Claims] 1 General formula (): P-(CH ₂ ) _o -0-R ¹ () (wherein P is the general formula: [Formula] (wherein, R is a hydrogen atom) The represented hydroxypurine group, n is an integer from 1 to 6, and R ¹ is a general formula: [Formula] (wherein, R ² is a group bonded to the α-carbon of the α-amino acid, and R ³ is a hydrogen atom. A residue represented ^by the ^general formula:
Residue or general formula represented by [Formula] (in which R ² and R ⁴ are the same as above): (However, in this case, in the general formula (), n is 2). 2. The compound according to claim 1, wherein ^R2 is a hydrogen atom, a methyl group, a P-hydroxybenzyl group, or an aminobutyl group. 3 α where the carbon to which R ² is bonded is α-carbon
- The compound according to claim 1, wherein the amino acid is an optically active l-form or d-form, or a racemate. 4 α where the carbon to which R ² is bonded is α-carbon
- The compound according to claim 1, wherein the amino acid is a natural amino acid. 5. The compound according to claim 1, wherein n is 2, R is a hydrogen atom, and R ¹ is [Formula]. 6. The compound according to claim 1, wherein n is 2, R is a hydrogen atom, and ^R1 is -CO-( _CH2 ) ₂ -CO-NH- _CH2- COOH. 7 The above n is 2, R is a hydrogen atom, and R ¹ is The compound according to claim 1, which is 8 The above n is 2, R is a hydrogen atom, and R ¹ is The compound according to claim 1, which is 9 The above n is 2, R is a hydrogen atom, R ¹ is The compound according to claim 1, which is 10. The compound according to claim 1, wherein n is 6, R is a hydrogen atom, and R ¹ is [Formula]. 11. The compound according to claim 1, wherein n is 5, R is a hydrogen atom, and R ¹ is [Formula]. 12 General formula (): P-(CH ₂ ) _o -OH () (wherein P is a hydroxypurine residue represented by the general formula: [formula] (wherein R is a hydrogen atom), n is 6-hydroxypurine represented by (an integer from 1 to 6) and the general formula (): (In the formula, R ² is a group bonded to the α-carbon of the α-amino acid, and R ⁵ is a protecting group for amino groups commonly used in peptide synthesis.) By reacting with an amino acid using a condensing agent, the general formula (): (In the formula, P, n, R ² and R ⁵ are the same as above) is prepared, and then the protecting group of the amino group is removed by a known method. General formula (): P-( CH ₂ ) _o -O-R ¹ () (wherein, P and n are the same as above, R ¹ is the general formula: [Formula] (wherein, R ² is the same as above, R ³ is a hydrogen atom ) A method for producing a compound represented by ). 13 General formula (): P-(CH ₂ ) _o -OH () (wherein P is a hydroxypurine residue represented by the general formula: [formula] (wherein R is a hydrogen atom), n is 6-hydroxypurine (an integer from 1 to 6) is reacted with an activated dicarboxylic acid derivative to obtain the general formula (): (wherein P and n are the same as above, m is an integer from 2 to 6) is prepared, and then this intermediate and α-amino acid or general formula (): [Formula] () (In the formula, R ² is a group bonded to the α-carbon of the α-amino acid, R ⁹
is a protecting group for a carboxyl group), and then, if the carboxyl group is protected, the protecting group for the carboxyl group is removed by a known method. General formula consisting of (): P-(CH ₂ ) _o -O-R ¹ () (wherein, P and n are the same as above, R ¹ is the general formula: [formula] (wherein, m, R ² is the same as above, R ⁴ is OH)
A method for producing a compound represented by (which is a residue represented by) 14 General formula (): P-(CH ₂ ) _o -OH () (wherein, P is a hydroxypurine residue represented by the general formula: [formula] (wherein, R represents a hydrogen atom), and n is 6-hydroxypurine (an integer from 1 to 6) is reacted with phosgene, and then the α-amino acid or the general formula (′): [Formula] (′) (where R ² is an α-amino acid) is reacted with phosgene. a group bonded to the α-carbon;
General formula () consisting of reacting with an α-amino acid whose carboxyl group is protected, represented by (R ⁶ is a protecting group for a carboxyl group), and then removing the protecting group from the carboxyl group by a known method: P- (CH ₂ ) _o -O-R ¹ () (In the formula, P and n are the same as above, R ¹ is the general formula: [Formula] (In the formula, R ² is the same as above, R ⁴ is OH ) A method for producing a compound represented by ). 15 As an active ingredient, a hydroxy compound represented by the general formula (): P-(CH ₂ ) _o -O-R ¹ () (wherein, P is the general formula: [formula] (wherein, R represents a hydrogen atom) a purine group, n is an integer from 1 to 6, and R ¹ is a general formula: [Formula] (wherein R ² is a group bonded to the α-carbon of the α-amino acid, and R ³ is a hydrogen atom); The residue represented by the general formula: [Formula] (where m is an integer of 2 to 6, R ² is the same as above, and R ⁴ represents OH), the general formula: [Formula] ( (wherein R ² and R ⁴ are the same as above) or a general formula: (However, in this case, in the general formula (), n is a residue represented by 2.) An immunomodulator containing at least one compound represented by the following.