JPH04352785A - New methotrexate derivative - Google Patents

Abstract

PURPOSE:To provide the subject new compound having low toxicity and useful as an antirheumatic agent. CONSTITUTION:The compound of formula I (R1 is 1-4C alkyl; R2 is H or together with R1 forms 2-3C methylene; R3 and R4 are H or 1-4C alkyl), e.g. N-[1-[(2,4-diamino-6-pteridinyl-)methyl]-indoline-5-carbonyl]-N-phthal oyl-ornithine methyl ester. The compound of formula I can be produced by condensing a compound of formula II with a compound of formula III, deprotecting the product, condensing the resultant compound of formula IV with a compound of formula V, deprotecting the product and reacting with a compound of formula VI.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to novel methotrexate derivatives, and more particularly to novel methotrexate derivatives useful as antirheumatic agents.

0002

[Prior Art/Problems to be Solved by the Invention] Methotrexate has long been used as a treatment for leukemia, but in 1951, Gubner et al.
Since its effectiveness was reported for A) and psoriasis, it has been used in Europe and America as a treatment for RA. Relatively recently, detailed studies on usage and dosage have been carried out, and it has become clear that low-dose methotrexate therapy has relatively few side effects and exhibits excellent efficacy. However, side effects such as liver damage and lung fibrosis caused by methotrexate administration cannot be ignored, so there is a desire for a drug with even fewer side effects and superior efficacy.

[003] So far, as a derivative in which the amino acid moiety of methotrexate is an Nδ amide form of ornithine, for example, the following formula

[C3]

[004] (JMed.chem.1988, 31, 1
332) etc. are known. The present inventors have discovered a compound with a stronger antirheumatic effect by alkylating N 2 or forming a ring with a benzene ring in this type of methotrexate derivative, and have accomplished the present invention.

005]

[Means for Solving the Problems] The present invention is based on the following general formula (
I)

[C4]

(In the formula, R1 represents a lower alkyl group having 1 to 4 carbon atoms; R2 represents a hydrogen atom or a methylene group having 2 or 3 carbon atoms bonded to R1; R3 and R4 represent a hydrogen atom or a methylene group having 2 or 3 carbon atoms; represents a lower alkyl group of numbers 1 to 4)

[
[007] and the following general formula (II)

[C5]

The present invention provides a methotrexate derivative represented by the formula (wherein R1, R2 and R3 have the same meanings as above).

The compounds of the present invention are all novel compounds that have not been described in any literature, and can be synthesized, for example, as follows. 1) When the COOR4 group in general formula (I) is at the meta or para position i) When R2 in general formula (I) represents a hydrogen atom

010]

[Chemical formula 6] ii) When R2 in general formula (I) combines with R1 to represent a methylene group having 2 or 3 carbon atoms.

011]

[C7]

2) When the COOR4 group in general formula (I) is at the ortho position

[Image Omitted] (In the formula, R1, R2, R3 and R4 have the same meanings as above, n represents an integer of 2 or 3, and X represents a halogen atom such as bromo or chloro)

In the case of 1)-i), the reaction to obtain the compound of general formula (3) from the compound of general formula (1) and the compound of general formula (2) is to react the compound of general formula (1) with chloroform, This is carried out by dissolving the compound in an aprotic solvent such as dichloromethane, tetrahydrofuran or dioxane, adding the compound of general formula (2), water and, for example, potassium carbonate, triethylamine, sodium hydrogen carbonate, pyridine, etc., and stirring at room temperature. This amidation may be performed by a mixed acid anhydride method, an active ester method, or an activated amide method.

The reaction for obtaining the compound of general formula (4) from the compound of general formula (3) is to dissolve the compound of general formula (3) in ethanol, methanol, tetrahydrofuran, dioxane, etc., and in the presence of palladium-carbon, This is carried out by stirring at room temperature under a hydrogen atmosphere.

The reaction to obtain the compound of general formula (6) from the compound of general formula (4) and the compound of general formula (5) is carried out by the reaction of general formula (4).
), add thionyl chloride and dimethylformamide and stir at room temperature, then add the compound of general formula (5), potassium carbonate or triethylamine, and water in a solvent such as dichloromethane, and stir at room temperature. Amidation may be carried out by the anhydride method, active ester or active amide method.

[016] In the reaction to obtain the compound of general formula (7) from the compound of general formula (6), hydrogen bromide-acetic acid in which phenol or anisole has been dissolved in advance is added to the compound of general formula (6), and the mixture is stirred at room temperature. Do by doing.

The reaction to obtain the compound of general formula (9) from the compound of general formula (7) and the compound of general formula (8) is carried out at 25 to 100°C in an aprotic polar solvent such as dimethylacetamide and dimethylformamide. Preferably 50-6
After stirring at 5° C., the mixture is stirred in water containing, for example, triethylamine, potassium carbonate, or sodium hydrogen carbonate.

In particular, when R3 is a hydrogen atom, a sodium hydroxide solution is further added in a solvent such as ethanol, and the mixture is stirred at room temperature to obtain the desired product.

In the case of 1)-ii), general formula (10)
The reaction to obtain the compound of general formula (11) from the compound of formula (10) and the compound of general formula (4) is carried out by adding thionyl chloride and dimethylformamide to the compound of general formula (10), stirring at room temperature, and then adding the compound to the compound of general formula (10) in a solvent such as dichloromethane. The amidation is carried out by stirring at room temperature in the presence of the compound of general formula (4) and triethylamine or potassium carbonate, but the amidation may also be carried out by a mixed acid anhydride method, an active ester method, or an active amide method.

[020] From the compound of general formula (11), general formula (12)
The reaction for obtaining the compound of formula (11) is carried out by adding hydrogen bromide-acetic acid in which phenol or anisole has been dissolved in advance to the compound of general formula (11), and stirring the mixture at room temperature.

The reaction to obtain the compound of general formula (13) from the compound of general formula (12) and the compound of general formula (8) is carried out at 25 to 100°C in an aprotic polar solvent such as dimethylacetamide or dimethylformamide. Preferably 50-6
After stirring at 5° C., the mixture is stirred at room temperature in water containing triethylamine, potassium carbonate or sodium hydrogen carbonate. In particular, when R3 is a hydrogen atom, the desired product is obtained by further stirring at room temperature in a solvent such as ethanol in the presence of an aqueous sodium hydroxide solution.

In the case of 2), the reaction to obtain the compound of general formula (15) from the compound of general formula (1) and the compound of formula (14) is to react the compound of general formula (1) with chloroform, dichloromethane, tetrahydrofuran, This is carried out by dissolving the compound in an aprotic solvent such as dioxane, adding the compound of formula (14), water and, for example, potassium carbonate, triethylamine, sodium hydrogen carbonate, pyridine, etc., and stirring at room temperature. The reaction for obtaining the compound of general formula (16) from the compound of general formula (15) is carried out by stirring at -30°C in a solvent such as methanol, adding thionyl chloride, and then refluxing.

[023] From the compound of general formula (16), general formula (17)
The reaction for obtaining the compound of formula (16) is carried out by dissolving the compound of general formula (16) in ethanol, methanol, tetrahydrofuran, dioxane, etc., and stirring the solution at room temperature under a hydrogen atmosphere in the presence of palladium-carbon.

[024] From the compound of general formula (18), general formula (19)
The reaction to obtain the compound of formula (18) is carried out by adding thionyl chloride and dimethylformamide to the compound of general formula (18) and stirring the mixture at room temperature.

The reaction to obtain the compound of general formula (20) from the compound of general formula (17) and the compound of general formula (19) is carried out by dissolving the compound of general formula (19) in dichloromethane, etc., and adding the compound of general formula (19) to this solution. Amidation is carried out by adding the compound of (17), potassium carbonate or triethylamine, and water and stirring at room temperature, but amidation may also be carried out by a mixed acid anhydride method, an active ester method, or an active amide method.

[026] From the compound of general formula (20), general formula (21)
The reaction for obtaining the compound of formula (20) is carried out by adding hydrogen bromide-acetic acid in which phenol or anisole has been dissolved in advance to the compound of general formula (20), and stirring the mixture at room temperature.

The reaction to obtain the compound of general formula (22) from the compound of general formula (8) and the compound of general formula (21) is preferably carried out at 25 to 100°C in an aprotic polar solvent such as dimethylacetamide and dimethylformamide. is 50~
After stirring at 65° C., the mixture is stirred in water containing, for example, triethylamine, potassium carbonate, or sodium hydrogen carbonate.

[028] From the compound of general formula (22), general formula (23)
The reaction to obtain the compound is carried out in a solvent such as ethanol by adding a sodium hydroxide solution and stirring at room temperature.

029]

[Function] The compound represented by the general formula (I) obtained according to the present invention has antirheumatic action. It is also less toxic than methotrexate. This effect will be investigated in the following experimental examples: "Lymphocyte proliferation inhibitory effect using human peripheral blood" and "Toxicity comparison between methotrexate (MTX) and the compound of the present invention by continuous intraperitoneal administration using rats." It was confirmed by this.

Experimental Example 1 "Lymphocyte proliferation inhibitory effect using human peripheral blood" (Method) Lymphocytes were separated from human peripheral blood using Ficoll-Paque R, and appropriately diluted drugs and the lymphocytes 105 cells were cultured in a 96-well ciltue plate for 2 days with PHA or 3 days with PWM. 5 hours before the end of culture, 3H-UdR (1 μc
i/well) and the incorporation of 3H-UdR into lymphocytes was measured using a scintillation counter.

[031] (Here, PHA is Phytohaemagg)
lutinin, PWM is pokeweed mit
ogen, UdR indicates deoxyuridine. ) The drugs used are as follows.

[Chemical formula 9]

(Results and Discussion) Figure 1 shows the percentage of 3H-UdR uptake taken as 100% by PHA-stimulated lymphocytes to which no drug was added. As is clear from FIG. 1, it was confirmed that the compound of the present invention has a superior lymphocyte proliferation inhibitory effect (anti-rheumatic effect) than the control compound.

Experimental Example 2 "Toxicity comparison between methotrexate (MTX) and the compound of the present invention by continuous intraperitoneal administration using rats"

034
(Method) MTX and the compound of the present invention were intraperitoneally administered to 8-week-old SD male rats once a day, 5 days a week for 5 consecutive weeks. The dosage is two doses of 0.25 and 0.5 mg/kg for both MTX and the compound of the present invention. A vehicle (phosphate buffer, pH 7.4) was similarly administered to the control group. The number of animals per group was 5.

[035] The inspection items are as follows. 1) Life and death and symptoms: Observed daily. 2) Weight change: Measured once a week. 3) Blood test: white blood cell count (WB) in peripheral blood during the week of final administration.
C), red blood cell count (RBC) was measured. 4) Liver weight and liver triglyceride (TG) content: After the final administration, liver weight and liver TG were determined using autopsy.
The content was measured. The drugs used are as follows.

036]

[Chemical formula 10]

(Results) 1) Life, death, and symptoms Among the MTX administration groups, there were no deaths in the 0.25 mg/kg group throughout the administration period, and no abnormal symptoms were observed. However, in the 0.5 mg/kg group, deterioration of nutritional status, anemia, decreased fecal volume, swelling around the mouth, etc. were observed from the 4th week, and 1 out of 5 patients died.

On the other hand, in the group administered with the compound of the present invention, 0.25
There were no deaths in either the mg/kg group or the 0.5 mg/kg group, and no abnormal symptoms were observed.

2) Change in body weight during rest Figure 2 shows the change in body weight. The compound of the present invention was less effective in suppressing weight gain than MTX.

3) Blood test Figure 3 shows the blood test results. The decrease in WBC and RBC was clearly weaker with the compound of the present invention than with MTX.

4) Liver weight and TG content in the liver Liver weight and TG content in the liver are shown in Figure 4. Both the increase in liver weight and the increase in TG content in the liver were weaker with the compound of the present invention than with MTX.

(Discussion) Using rats, toxicity due to continuous intraperitoneal administration was compared between MTX and the compound of the present invention. As a result, the compound of the present invention was considered to be clearly less toxic than MTX in terms of mortality rate, symptoms, weight change, blood test, liver weight, and liver TG content.

043]

【Example】

Reference example 1. N-phthaloyl-N-carbobenzoxy-ornithine
Synthesis of methyl ester Phthalic anhydride (2.0 g) was added to a solution of N-carbobenzoxy-L-ornithine (2.0 g) in dichloromethane (70 ml).
Then, water (70 ml) and potassium carbonate (1.12 g) were added, and the mixture was stirred at room temperature for 15 hours. The reaction solution was concentrated to 60 ml under reduced pressure and adjusted to pH=3 with 1N hydrochloric acid.
The precipitate prepared was collected by filtration and dried under vacuum. The obtained white solid was dissolved in low moisture methanol (80 ml),
The solution was cooled to -30°C and stirred for 10 minutes. Next, thionyl chloride (2 ml) was slowly added dropwise at the same temperature. The reaction solution was slowly returned to room temperature and refluxed for an additional 2 hours. The solvent was distilled off under reduced pressure, and the resulting residue was subjected to silica gel chromatography to obtain the desired product (2
．． 16g) was obtained.

1H-NMR (CDCl3, δ); 1.6-
2.0 (4H, m), 3.69 (5H, m), 4.20
(1H, m), 5.06 (2H, s), 5.70 (1H
, m), 7.29 (5H, m), 7.66 (2Hm),
7.81 (2H, m)

Reference example 2. Synthesis of N-phthaloyl-ornithine methyl ester A methanol solution (1
00ml), 10% palladium-carbon (500mg)
After adding, the mixture was stirred at room temperature under a hydrogen atmosphere for 20 hours. Palladium-carbon was filtered off using Celite, and the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography to obtain the desired product (223 mg) using chloroform:methanol=100:3 as an elution solvent.

1H-NMR (CDCl3, δ); 1.7-
2.1 (4H, m), 3.75 (2H, m), 3.83
(3H, s), 7.76 (2H, m), 7.84 (2H
, m)

Reference example 3. Synthesis of N-(1-carbobenzoxyindoline-5-carbonyl)phthaloyl-ornithine methyl ester Add 1-carbobenzoxyindoline-5-carboxylic acid (297 mg) to thionyl chloride (2.5 ml).
) was added to form a suspension, and a catalytic amount of dimethylformamide was added to this suspension, followed by stirring at room temperature for 2 hours. Next, the reaction solution was concentrated to dryness under reduced pressure. The obtained solid was dissolved in dichloromethane (7 ml), and the compound of Reference Example 2 (250 mg) and potassium carbonate (640 ml) were added to this solution.
g), water (7 ml) was added, and the mixture was vigorously stirred at room temperature for 12 hours. Next, the reaction solution was poured into water and extracted with chloroform, and the chloroform layer was further washed with 1N hydrochloric acid solution and dried over sodium sulfate. The solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography using chloroform:methanol=1 as the elution solvent.
The desired product (330 mg) was obtained using 00:3.

1H-NMR (CDCl3, δ); 1,6-
2.1 (4H, m), 3.12 (2H, t, J = 8.8
Hz), 3.72 (2H, m), 3.76 (3H, s)
,4.08(2H,t,J=8.8Hz),4.84(
1H, m), 5.27 (2H, S), 6.80 (1H,
d, J=7.8Hz), 7.1-7.5 (6H, m),
7.5-7.9 (6H, m)

Reference example 4. Synthesis of N-(indoline-5-carbonyl)-N-phthaloyl-ornithine methyl ester phenol (3
The compound of Reference Example 3 (330 mg) was added to a solution of 00 mg) in 30% hydrogen bromide-acetic acid (8 ml), and the mixture was stirred at room temperature for 4 hours. Next, a large amount of ether was added to the reaction solution, and a reddish-brown oily substance precipitated. Most of the ether layer was removed and the oil was suspended in chloroform, the suspension was washed with saturated sodium bicarbonate solution and extracted with chloroform. The chloroform layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain the desired product (147 mg).

1H-NMR (CDCl3, δ); 1.6-
2.1 (4H, m), 3.04 (2H, t, J=8.3
Hz), 3.62 (2H, t, J=8.3Hz), 3.
72 (2H, m), 3.75 (3H, s), 4.86 (
1H, m), 6.5-6.7 (2H, m), 7.52 (
2H, m), 7.68 (2H, m), 7.82 (2H,
m)

Example 1. N-[1-[(2,4=diamino-6-pteridinyl)
Synthesis of methyl]-indoline-5-carbonyl]-N-phthaloyl-ornithine methyl ester The compound of Reference Example 4 (146 mg) and 6-bromomethyl-2,4-diaminopteridine hydrobromide isopropanol adduct (115 mg) were Dimethylacetamide (1.0ml)
and stirred at 50 to 55°C for 4 hours. After cooling, water (4 ml) containing triethylamine (30 mg) of the reaction solution was added.
) was added, stirred, and then extracted with chloroform. After drying the chloroform layer over sodium sulfate, the solvent was distilled off under reduced pressure, and the resulting residue was subjected to silica gel chromatography, using chloroform:methanol=10 as the eluent.
: The desired product (140 mg) was obtained using a mixed solvent of 1:1.

1H-NMR (CDCl3, δ); 1.7-
2.2 (4H, m), 3.06 (2H, t, J=8.3
Hz), 3.56 (2H, t, J=8.3Hz), 3.
72 (2H, m), 3.76 (3H, s), 4.53 (
2H, s), 4.88 (1H, m), 6.45-6.6
2 (2H, m), 7.56 (2H, m), 7.71 (2
H, m), 7.84 (2H, m), 8.82 (1H, s
)

Example 2. N-[1-[(2,4-diamino-6-pteridinyl)
Synthesis of methyl]-indoline-5-carbonyl]-N-hemiphthaloyl-ornithine The compound of Example 1 (140 mg) was suspended in 2N sodium hydroxide solution (10 ml) and stirred at 30°C for 12 hours. The reaction solution was dried under reduced pressure, and the resulting yellow solid was dissolved in water (5 ml) and adjusted to pH=3 with 1N hydrochloric acid.
．． 7 and left in the refrigerator for 2 hours. The deposited precipitate was collected by filtration. The obtained yellow solid was subjected to silica gel chromatography, and the desired product (20 mg) was obtained using a mixed solvent of chloroform:methanol:28%-ammonia water = 5:4:1 as an elution solvent.

1H-NMR (DMSO-d6, δ); 1.
5-2.1 (4H, m), 3.14 (2H, t, J
= 8.3Hz), 3.58 (2H, t, J = 8.3Hz
), 4.38 (1H, m), 4.54 (2H, s), 6
．． 71 (1H, m), 7.3-7.6 (4H, m), 7
．． 6-7.8 (3H, m), 8.08 (1H, m), 8
．． 71 (1H, s) mp; 195-199℃ (de
c. )

Reference example 5. Synthesis of N-(4-methoxycarbonylbenzoyl)-N-carbobenzoxy-ornithine methyl esterN
Terephthalic acid monomethyl ester chloride (3.0 g) was added to a solution of -carbobenzoxy-L-ornithine (2.0 g) in dichloromethane (60 ml), and then water (
60 ml) and potassium carbonate (4.8 g) were added and stirred at room temperature for 15 hours. The reaction solution was concentrated to 50 ml under reduced pressure, adjusted to pH=3 with 1N hydrochloric acid, and the precipitate deposited was collected by filtration and dried in vacuum. The resulting white solid was dissolved in low water methanol (100 ml), and the solution was cooled to -30°C and stirred for 10 minutes. Next, at the same temperature, thionyl chloride (3
ml) was slowly added dropwise. The reaction solution was slowly returned to room temperature and refluxed for an additional 2 hours. The solvent was distilled off under reduced pressure, and the resulting residue was subjected to silica gel chromatography using chloroform:methanol=1 as the elution solvent.
The desired product (710 mg) was obtained using 00:1.

1H-NMR (CDCl3, δ); 1.6-
2.1 (4H, m), 3.51 (2H, m), 3.74
(3H, s), 3.94 (3H, s), 4.43 (1H
, m), 5.11 (2H, s), 5.53 (1H, m)
, 6.63 (1H, bs), 7.34 (5H, s), 7
．． 83 (2H, d, J = 8.8Hz), 8.08 (2H
, d, J=8.8Hz)

Reference example 6. Synthesis of N-(4-methoxycarbonylbenzoyl)-ornithine methyl ester A methanol solution (1
00ml), 10% palladium-carbon (100mg)
After adding, the mixture was stirred at room temperature under a hydrogen atmosphere for 20 hours. Palladium-carbon was filtered off using Celite, and the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography to obtain the desired product (410 mg) using chloroform:methanol=100:3 as an elution solvent.

1H-NMR (CDCl3, δ); 1.6-
2.1 (4H, m), 3.49 (3H, m), 3.73
(3H, s), 3.94 (3H, s), 7.20 (1H
, m), 7.84 (2H, d, J=8.3Hz), 8.
09 (2H, d, J=8.3Hz)

Reference example 7
．． Synthesis of N-(1-carbobenzoxyindoline-5-carbonyl)-N-(4-methoxycarbonylbenzoyl)-ornithine methyl ester 1-carbobenzoxyindoline-5-carboxylic acid (260 mg) was mixed with thionyl chloride (2 .5ml
) was added to form a suspension, and a catalytic amount of dimethylformamide was added to this suspension, followed by stirring at room temperature for 2 hours. Next, the reaction solution was concentrated to dryness under reduced pressure. The obtained solid was dissolved in dichloromethane (6 ml), and the compound of Reference Example 6 (245 mg) and potassium carbonate (812 ml) were added to this solution.
g), water (6 ml) was added, and the mixture was vigorously stirred at room temperature for 12 hours. Next, the reaction solution was poured into water and extracted with chloroform, and the chloroform layer was further washed with 1N hydrochloric acid solution and dried over sodium sulfate. The solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography, and the elution solvent was chloroform:methanol 10
The target product (310 mg) was obtained using 0:3.

1H-NMR (CDCl3, δ); 1.7-
2.2 (4H, m), 3.16 (2H, t, J = 8.3
Hz), 3.61 (1H, m), 3.79 (3H, s)
, 3.94 (3H, s), 4.11 (2H, t, J=8
．． 3Hz), 4.83 (1H, m), 5.29 (2H,
s), 6.89 (1H, d, J=7.3Hz), 7.0
2 (1H, m), 7.40 (5H, m), 7.66 (2
H, d, J = 7.3Hz), 7.90 (2H, d, J =
8.8Hz), 8.08 (2H, d, J=8.8Hz)

Reference example 8. Synthesis of N-(indoline-5-carbonyl)-N-(4-methoxycarbonylbenzoyl)-ornithine methyl ester Phenol (1.0 g) was dissolved in 30% hydrogen bromide-acetic acid (10
Add the compound of Reference Example 7 (400 mg) to the solution
The mixture was stirred at room temperature for an hour. Next, a large amount of ether was added to the reaction solution, and a reddish-brown oily substance precipitated. Most of the ether layer was removed and the oil was suspended in chloroform.
This suspension was washed with saturated sodium hydrogen carbonate solution, and the target product was extracted with chloroform. The chloroform layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained white solid was recrystallized from n-hexane-chloroform-methanol to obtain the desired product (145 mg).

1H-NMR (CDCl3, δ); 1.6-
2.1 (4H, m), 3.05 (2H, t, J = 8.3
Hz), 3.48 (2H, m), 3.62 (2H, t,
J=8.3Hz), 3.76(3H,s), 3.94(
3H, s), 4.75 (1H, m), 6.57 (1H,
d, J=7.8Hz), 7.19 (1H, d, J=7.
8Hz), 7.55 (2H, m), 7.89 (2H, d
, J=8.3Hz), 8.07(2H, d, J=8.3
Hz)

Example 3. N-[1-[(2,4-diamino-6-pteridinyl)
Synthesis of methyl]indoline-5-carbonyl]-N-(4-methoxycarbonylbenzoyl)-ornithine methyl ester Compound (140 mg) of Reference Example 8 and 6-bromomethyl
2,4-diaminopteridine hydrobromide (105 mg
) in dimethylacetamide (2.0 ml),
Stirred at 0-55°C for 4 hours. After cooling, water (4 ml) containing triethylamine (32 mg) as a reaction solution was added and stirred, and then the target product was extracted with chloroform. After drying the chloroform layer over sodium sulfate, the solvent was distilled off under reduced pressure, and the resulting residue was subjected to silica gel chromatography, using chloroform:methanol=10:1 as the elution solvent.
The desired product (180 mg) was obtained using a mixed solvent of .

1H-NMR (CDCl3, δ); 1.6-
2.1 (4H, m), 3.08 (2H, t, J = 8.8
Hz), 3.4-3.7 (4H, m), 3.79 (3H
, s), 3.94 (3H, s), 4.54 (2H, s)
, 4.83 (1H, m), 6.52 (1H, d, J=8
．． 3Hz), 6.79 (1H, d, J=6.8Hz),
7.17 (1H, m), 7.59 (2H, m), 7.9
2 (2H, d, J = 8.3Hz), 8.09 (2H, d
, J=8.3Hz), 8.81(1H,s)

065]
Example 4. N-[1-[(2,4-diamino-6-pteridinyl)
Synthesis of Example 3 (150 mg) was added to ethanol (24 m
1) and further diluted with 1N sodium hydroxide solution (0
．． 8 ml) was added thereto, and the mixture was stirred at 35°C for 4.5 hours. After stirring at 25° C. for 20 hours, water (1 ml) was added to the reaction solution, and the reaction solution was dried under reduced pressure. At this time, the external temperature was kept not to exceed 30°C. The obtained yellow solid was dissolved in water (5 ml), adjusted to pH=3.7 with 1N hydrochloric acid, and left in a refrigerator for 2 hours. The precipitate was collected by filtration to obtain the desired product (109 mg).

1H-NMR (CDCl3, δ); 1.5-
2.0 (4H, m), 3.00 (2H, t, J=8.8
Hz), 3.30 (2H, m), 3.58 (2H, t,
J = 8.8Hz), 4.36 (1H, m), 4.54 (
2H, s), 6.68 (1H, d, J=8.8Hz),
7.62 (2H, m), 7.91 (2H, d, J=8.
3Hz), 7.99 (2H, d, J=8.3Hz), 8
．． 10 (1H, d, J = 7.8Hz), 8.61 (1H
, m), 8.71 (1H, s) mp; 215-220°C
(dec.)

067]

[Brief explanation of the drawing]

Figure 1 shows 3H-Ud at each concentration of the test drug.
Represents the amount (ratio) of R incorporated. Figure 2 shows changes in body weight of rats when MTX and the compound of the present invention were administered, Figure 3 shows changes in white blood cell count (WBC) and red blood cell count (RBC), and Figure 4 shows liver weight and liver TG. Represents a change in content.

Claims (2)

[Claims] Claim 1: The following general formula (I) [Formula 1] (wherein, R1 represents a lower alkyl group having 1 to 4 carbon atoms; R2 is a hydrogen atom or a methylene group having 2 or 3 carbon atoms bonded to R1) R3 and R4 represent a hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms. 2. A methotrexate derivative represented by the following general formula (II): (wherein R1, R2 and R3 have the same meanings as above).