JP3207285B2 - New methotrexate derivatives - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel methotrexate derivative, and more particularly, to a novel methotrexate derivative useful as an antirheumatic agent.

[0002]

2. Description of the Related Art Methotrexate has been used as a therapeutic agent for leukemia for a long time, but in 1951, Gubner et al. Reported its efficacy in rheumatoid arthritis (RA) and psoriasis. Since then, it has been used in Europe and the United States as a therapeutic agent for RA. More recently, detailed studies of dosage and administration have revealed that low-dose methotrexate therapy has relatively few side effects and exhibits excellent efficacy. However, since side effects such as liver damage and pulmonary fibrosis caused by taking methotrexate cannot be ignored, there is a demand for a drug with less side effects and excellent efficacy.

[0003] The present inventors have conducted intensive studies in search of a compound having a better antirheumatic effect in a methotrexate derivative, and have accomplished the present invention.

[0004]

The present invention provides a compound represented by the following general formula (I):

Embedded image In the formula, R ¹ represents a lower alkyl group having 1 to 4 carbon atoms; R ² represents a lower alkyl group having 1 to 4 carbon atoms or a trifluoromethyl group; R ³ represents a hydrogen atom or a 1 carbon atom. R ⁴ represents a hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms; n represents an integer of 1 to 4; R ⁵ represents a general formula COOR ⁶ (wherein R ⁶ represents a hydrogen atom or a lower alkyl group having a carbon number of 1 to 4) a group represented by the group or SO ₃ H expressed by; methotrexate derivative represented by (wherein, R ¹ Is a methyl group, R ² is a trifluoromethyl group, R ³ is a hydrogen atom, R
⁴ is intended to provide a hydrogen atom, n is 2, R ⁵ is COOR ⁶ except Compound (R ⁶ is a hydrogen atom) is).

The compounds of the present invention are novel compounds which have not been published in any literature, and are synthesized, for example, as follows. (Method A)

Embedded image

(Method B)

Embedded image (Wherein, R ¹ , R ² , R ³ , R ⁴ , R ⁵ and n have the same meaning as described above, R ′ represents a lower alkyl group having 1 to 4 carbon atoms, and A ¹ and A ² are protected X represents a halogen atom.)

In the method A, the reaction for obtaining the compound of the general formula (2) from the compound of the general formula (1) is carried out, for example, in the presence of sodium hydrogen carbonate, calcium carbonate, potassium carbonate and the like in the presence of water-methanol, water-ethanol, water - a mixed solvent such as propanol, carried out by reacting the halogen represented by a compound of formula X ₂ in the general formula (1). General formula X
Examples of ₂ include fluorine, chlorine, bromine, iodine and the like. When R ² or R ³ in the compound of the general formula (1) is trifluoromethyl, the halogen to be reacted is preferably iodine.

In the reaction for obtaining the compound of the general formula (3) from the compound of the general formula (2), the compound of the general formula (2) is reacted with acetic anhydride in a solvent such as toluene, benzene, xylene, dioxane or pyridine. It is done by doing. The steps from the compound of the general formula (1) to the compound of the general formula (2) and the process from the compound of the general formula (2) to the compound of the general formula (3) are reversed. Is also good.

In the reaction for obtaining the compound of the general formula (4) from the compound of the general formula (3), the compound of the general formula (3) and copper cyanide are, for example, N-methylpyrrolidone, dimethylformamide, dimethylacetamide, dimethylpropylene The reaction is performed in a solvent such as urea and hexamethylphosphoric triamide.

The reaction for obtaining the compound of the general formula (5) from the compound of the general formula (4) is carried out by reacting the compound of the general formula (4) with 12N-
The reaction is performed by reacting hydrochloric acid, a 50% aqueous solution of sodium hydroxide, or the like.

The reaction for obtaining the compound of the general formula (7) from the compound of the general formula (5) and the compound of the general formula (6) is carried out in an alcohol such as methanol or ethanol in the presence of hydrochloric acid or sulfuric acid. Do. Alternatively, the reaction may be carried out in the presence of thionyl chloride in alcohol. As the alkyl group represented by R 'in the formula, a methyl group, an ethyl group and the like are preferable.

The reaction for obtaining the compound of the general formula (9) from the compound of the general formula (7) and the compound of the general formula (8) is carried out by dissolving the compound of the general formula (7) in a solvent such as pyridine or sodium hydride. The reaction is carried out by suspending in tetrahydrofuran, dimethylformamide, dimethoxyethane, dioxane, toluene or the like in the coexistence, adding the compound of the general formula (8), and stirring. In the formula, the protecting group represented by A ¹ includes
For example, a p-toluenesulfonyl group, a benzyloxycarbonyl group, an acetyl group and the like can be mentioned.

In the reaction for obtaining a compound of the general formula (11) from a compound of the general formula (9) and a compound of the general formula (10), the compound of the general formula (9) is converted into, for example, anhydrous dimethylformamide,
It is dissolved in tetrahydrofuran, dimethoxyethane, dioxane or the like, and added with, for example, sodium hydride under ice-cooling.
After stirring at 0 ° C to 120 ° C, preferably at room temperature, the compound of the general formula (11) is added, and the mixture is stirred at 10 ° C to 120 ° C, preferably at room temperature.

From the compound of the general formula (11), the compound of the general formula (1)
In the reaction for obtaining the compound of 2), the compound of the general formula (11) is reacted in a solvent such as methanol, ethanol or tetrahydrofuran in the presence of potassium hydroxide, an aqueous sodium hydroxide solution or the like at 10 ° C to 100 ° C, preferably at room temperature. The reaction is carried out.

From the compound of the general formula (12), the compound of the general formula (1)
In the reaction for obtaining the compound of 3), the compound of the general formula (12) is suspended in an acid halogenating agent such as thionyl chloride, oxalyl chloride and the like, and is suspended in the presence of a catalytic amount of dimethylformamide or the like.
It is performed by stirring at room temperature.

The compound of the general formula (13) and the compound of the general formula (14)
In the reaction for obtaining the compound of the general formula (15) from the compound of the formula, a solution of the compound of the general formula (13) in a solvent such as dichloromethane is added to an aqueous solution of the compound of the general formula (14) under ice cooling or water cooling. In addition, the reaction is carried out by stirring at room temperature in the presence of an inorganic base such as potassium carbonate, sodium hydroxide and sodium hydrogen carbonate.

From the compound of the general formula (15), the compound of the general formula (1)
The reaction for obtaining the compound of the formula 6) is performed by dissolving anisole or phenol in a solution of hydrogen bromide-acetic acid according to the general formula (15).
And stirring at 10 ° C. to 60 ° C., preferably at room temperature. When A ¹ is a carbobenzoxy group, the compound of the general formula (15)
The reaction for obtaining the compound of 6) may be carried out by dissolving the compound of the general formula (15) in a solvent such as methanol, ethanol or acetic acid, adding palladium-carbon, and then stirring at room temperature under a hydrogen atmosphere. Good.

The compound of the general formula (16) and the compound of the general formula (17)
In the reaction for obtaining the compound of the general formula (I) from the compound of the formula (I), the compound of the general formula (16) and the compound of the general formula (17) are dissolved in a solvent such as dimethylacetamide, dimethylformamide or the like.
C. to 100.degree. C., preferably 50.degree. Especially when R ⁴ is a hydrogen atom, further methanol or ethanol, in a solvent such as tetrahydrofuran 1N-
An aqueous solution of sodium hydroxide is added, and the mixture is stirred at 0 ° C to 60 ° C, preferably at 35 ° C to obtain the desired product.

In the method B, the reaction of obtaining the compound of the general formula (19) from the compound of the general formula (5) and the compound of the general formula (18) comprises the compound of the general formula (5) and the compound of the general formula (18) The reaction is carried out at 0 ° C to 100 ° C, preferably at room temperature. Wherein the protecting group represented by A ^2, trifluoroacetyl group, and a acetyl group.

The compound of the general formula (19) and the compound of the general formula (20)
Is obtained by adding potassium hydroxide to an acetone solution of the compound of the general formula (19) and the compound of the general formula (20), and adding 0 to 55 ° C., preferably 45 ° C. Perform by stirring at ~ 53 ° C.

From the compound of the general formula (21), the compound of the general formula (2)
The reaction for obtaining the compound of 2) is carried out by reacting the compound of the general formula (21) with potassium hydroxide or sodium hydroxide in water. Here, the compound of the above general formula (11) may be used instead of the compound of the general formula (21).

The compound of the general formula (22) and the compound of the general formula (17)
In the reaction for obtaining the compound of the general formula (23) from the compound of the general formula (23), the compound of the general formula (22) and the compound of the general formula (17) are dissolved in a solvent such as dimethylacetamide or dimethylformamide.
It is carried out by stirring at 0 ° C to 100 ° C, preferably at 40 ° C to 55 ° C.

The compound of the general formula (23) and the compound of the general formula (14)
The compound of general formula (I) is obtained by reacting the compound of general formula (23) with dimethylformamide, dimethylacetamide, N-methyl in the presence of cyanide diethylphosphate, dicyclohexylcarbodiimide and 1-hydroxybenzotriazole. After stirring in a solvent such as pyrrolidinone to prepare an active ester and a mixed acid anhydride, respectively, the compound of the general formula (14) is added, and the mixture is added at 0 ° C to 200 ° C.
It is preferably performed by stirring at 10 ° C to 80 ° C.
Alternatively, triethylamine is added to the compound of the general formula (23), then isobutyl chloroformate is added, the mixture is stirred in a solvent such as dimethylformamide, dimethylacetamide, N-methylpyrrolidinone, and the compound of the general formula (14) is added. C. to 50.degree. C., preferably -10.degree. In particular, when R ⁴ is a hydrogen atom, a 1N aqueous sodium hydroxide solution is further added in a solvent such as methanol or ethanol, and the mixture is stirred at 0 ° C. to 60 ° C., preferably at room temperature, to obtain the desired product.

[0024]

The compound represented by the general formula (I) obtained by the present invention has an antirheumatic effect. This effect was confirmed by examining the following experimental example "the effect of suppressing lymphocyte proliferation using human peripheral blood".

[0025]

【Example】

Lymphocytes were separated using a "growth inhibitory effects of human peripheral blood-derived lymphocyte" (Method) Ficoll-Paque ^R from human peripheral blood, moderately diluted drug and its lymphocyte 10 ⁵ pieces of PHA (0.3 [mu] g / Ml) for 2 days in a 96-well culture plate. Five hours before the end of the culture, ³ H-UdR (1 μCi /
well) was added, and the uptake of ³ H-UdR into lymphocytes was measured using a scintillation counter. (Where PHA is phytohaemamagglutinin, U
dR indicates deoxyurdine. ) The following drugs were used.

Embedded image

(Results and Discussion) FIG.
The ratio is shown with the ³ H-UdR uptake of stimulated lymphocytes as 100%. As is clear from FIG. 1, it was confirmed that the compound of the present invention has a superior lymphocyte proliferation inhibitory action (anti-rheumatic action) than the control compound.

[0027]

【Example】

[Reference Example 1] N-carbobenzoxy-4-amino-3-methylbenzoate
Acid synthesis

4-amino-3-methylbenzoic acid (5.0
g) was suspended in water (80 ml), and the suspension was cooled in ice with an ether solution (20 ml) of carbobenzoxychloride (3.7 g) and sodium hydrogen carbonate (3.6 g).
Were added alternately. After stirring at room temperature for 2.5 hours,
Further, an ether solution (95 ml) of carbobenzoxychloride (3.7 g) and sodium hydrogen carbonate (7.2 g)
Were alternately added under ice-cooling, and the mixture was stirred at room temperature for 1.5 hours. After the reaction solution was acidified with 4N-hydrochloric acid, the precipitated insolubles were collected by filtration. After vacuum drying the obtained solid, the target substance (2.
8 g) were obtained. ¹ H-NMR (CDCl ₃ , δ value): 2.38 (3H,
s), 5.20 (2H, s), 7.41 (5H, m),
7.97 (3H, m)

[0029]

Reference Example 2 N-carbobenzoxy-N-methyl-4-amino-3-
Synthesis of methyl benzoate methyl ester

After sodium hydride (1.4 g) was suspended in dimethylformamide (10 ml), a dimethylformamide solution (10 ml) of the compound of Reference Example 1 (2.8 g) was added to this suspension at 0 ° C. And slowly added. After the reaction solution was stirred at room temperature for 1 hour, methyl iodide was further added slowly, and the mixture was stirred at room temperature for 2 hours. Then, the reaction solution was poured into a saturated aqueous solution of sodium hydrogen carbonate, and extracted with toluene. After drying over sodium sulfate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography, and chloroform was used as an elution solvent to obtain the desired product (1.8 g). ¹ H-NMR (CDCl ₃ , δ value): 2.19 (3H,
s), 3.19 (3H, s), 3.87 (3H, s),
5.09 (2H, s) 7.21 (6H, m), 7.85
(2H, m)

[0031]

Reference Example 3 N-carbobenzoxy-N-methyl-4-amino-3-
Synthesis of methylbenzoic acid Ethanol (15 m) of the compound of Reference Example 2 (1.8 g)
l) A 2N aqueous solution of sodium hydroxide (15 ml) was added to the solution.
Was added and refluxed for 2 hours. Then, the reaction solution was concentrated under reduced pressure to 10 ml. After acidifying with 4N hydrochloric acid, the mixture was extracted with chloroform and dried over sodium sulfate. The solvent was distilled off under reduced pressure to obtain the desired product (1.5 g). ¹ H-NMR (CDCl ₃ , δ value): 2.02 (3H,
s), 3.04 (3H, s), 4.69 (2H, s),
7.08 (6H, m) 7.77 (2H, m)

[0032]

Reference Example 4 N- (N′-carbobenzoxy-N′-methyl-4-a
Mino-3-methylbenzoyl) -L-glutamic acid die
Synthesis of tyl ester The compound of Reference Example 3 (820 mg) was converted to thionyl chloride (5 m
After adding to l), a catalytic amount of dimethylformamide was further added, and the mixture was stirred at room temperature for 2 hours. Next, the reaction solution was concentrated to dryness under reduced pressure. The solid obtained was diluted with dichloromethane (2
0 ml), and glutamic acid diethyl ester hydrochloride (700 mg) and potassium carbonate (1.4 mg) were added to the solution.
g) was added, and water (20 ml) was further added, followed by vigorous stirring at room temperature for 12 hours. Next, the reaction solution was poured into water, extracted with chloroform, washed with 1N hydrochloric acid, and dried over sodium sulfate. The solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography, and the desired product (700 mg) was obtained using a mixed solvent of chloroform: methanol = 100: 3 as an elution solvent.

¹ H-NMR (CDCl ₃ , δ value):
1-1.5 (6H, m), 2.0-2.7 (4H,
m), 2.18 (3H, s), 3.19 (3H, s),
3.9-4.4 (4H, m), 4.80 (1H, m),
5.10 (2H, s), 7.0-7.5 (6H, m),
7.63 (2H, m)

[0034]

Reference Example 5 N- [N'-methyl- (4-amino-3-methylbenzo)
Il)] Synthesis of diethyl L-glutamic acid The compound of Reference Example 4 (700 mg) was added to a 30% hydrogen bromide-acetic acid (7 ml) solution containing anisole (700 mg), and the mixture was stirred at room temperature for 4 hours. Next, ether (200 ml) was added to the reaction solution, and a red-brown oily substance precipitated. After removing most of the ether layer, the oily substance was suspended in chloroform. This suspension was washed with a saturated sodium hydrogen carbonate solution, and the organic layer was separated. After the organic layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure to obtain the desired product (360).
mg).

¹ H-NMR (CDCl ₃ , δ value):
0-1.5 (6H, m), 2.11 (3H, s), 2.
90 (3H, s), 2.1-2.7 (4H, m), 3.
9-4.4 (4H, m), 4.80 (1H, m), 6.
54 (1H, d, J = 8 Hz), 6.80 (1H, d,
J = 7 Hz), 7.61 (2H, m)

[0036]

Example 1 N- {4- [N '-(2,4-diamino-6-pteridi)
Nyl) methyl-N'-methylamino] -3-methylben
Synthesis of zoyl} -L-glutamic acid diethyl ester The compound of Reference Example 5 (360 mg) and 6-bromomethyl-
2,4-Diaminopteridine hydrobromide isopropanol adduct (407 mg) was added to dimethylacetamide (6
ml) and stirred at 55-60 ° C for 4 hours. After cooling, water (30 ml) containing triethylamine (248 mg) was added to the reaction solution, stirred, and then extracted with chloroform. After the chloroform layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel chromatography, using a mixed solvent of chloroform: methanol = 10: 1 as an elution solvent (280 mg).
I got

¹ H-NMR (CDCl ₃ , δ value):
2-1.4 (6H, m), 2.0-2.6 (4H,
m), 2.41 (3H, s), 2.73 (3H, s),
4.0-4.3 (4H, m), 4.32 (2H, s),
4.76 (1H, m), 7.06 (1H, d, J = 8.
3 Hz), 7.41 (1H, d, J = 7.8 Hz),
7.65 (2H, m), 8.79 (1H, s)

[0038]

Example 2 N- {4- [N '-(2,4-diamino-6-pteridi)
Nyl) methyl-N'-methylamino] -3-methylben
Synthesis of zoyl} -L-glutamic acid (compound 1) Ethanol (20m2) of the compound of Example 1 (280mg)
1) Add 1N-sodium hydroxide aqueous solution (1.6 m
l) was added and the mixture was stirred at 35 ° C for 4 hours. Then at 25 ° C 2
After stirring for 0 hour, water (2 ml) was added to the reaction solution, and the reaction solution was dried under reduced pressure. At this time, the external temperature was kept below 30 ° C. The obtained yellow solid was washed with water (10 ml).
And adjusted to pH = 3.7 with 1N-hydrochloric acid and left in a refrigerator for 2 hours. The deposited precipitate was collected by filtration to obtain the desired product (180 mg).

¹ H-NMR (DMSO-d6, δ value):
1.9-2.2 (2H, m), 2.35 (2H, t, J
= 8.0 Hz), 2.42 (3H, s), 2.73 (3
H, s), 4.34 (2H, s), 4.41 (1H,
m), 7.10 (1H, d, J = 8.3 Hz), 7.7
2 (2H, m), 8.38 (1H, d, J = 7.8H
z), 8.65 (1H, s)

[0040]

Reference Example 6 N-carbobenzoxy-N-ethyl-4-amino-3-
Synthesis of methyl-benzoic acid ethyl ester Under a nitrogen atmosphere, sodium hydride (164 mg) was suspended in dimethylformamide (2 ml) and brought to 0 ° C. To this suspension was slowly added a solution of N-benzyloxycarbonyl-4-amino-3-methylbenzoic acid (650 mg) in dimethylformamide (2 ml), and then slowly added ethyl iodide (730 μl) at 0 ° C. added. After stirring at room temperature for 2.5 hours, the reaction solution was poured into a saturated sodium hydrogen carbonate solution, and extracted with toluene. The toluene layer was dried over magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain the desired product (680 mg).

¹ H-NMR (CDCl ₃ , δ value):
91-1.63 (6H, m), 2.16 (3H, s),
3.35-3.90 (2H, m), 4.10-4.55
(2H, m), 5.07 (2H, bs), 6.95-
7.35 (6H, m), 7.68-7.95 (2H,
m)

[0042]

Reference Example 7 N-benzyloxycarbonyl-N-ethyl-4-amido
Synthesis of no-3-methylbenzoic acid Ethanol (5m2) of the compound of Reference Example 6 (680mg)
l) To the solution, 2N-sodium hydroxide aqueous solution (5 ml)
Was added and refluxed for 2 hours. Then, the reaction solution was cooled to room temperature and dried under reduced pressure. The obtained residue was dissolved in a small amount of water, washed with ether, and then concentrated to pH 3 using concentrated hydrochloric acid.
And Extract the precipitated precipitate using chloroform,
Dried over sodium sulfate. The solvent was distilled off under reduced pressure to obtain the desired product (540 mg).

¹ H-NMR (CDCl ₃ , δ value):
16 (3H, t, J = 5.4 Hz), 1.20 (3H,
s), 3.38-3.94 (2H, m), 5.12 (2
H, bs), 6.93-7.44 (6H, m), 7.7.
2-8.08 (2H, m)

[0044]

Reference Example 8 N- [N'-ethyl-3-methyl- (4-aminobenzo
Yl)]-L-glutamic acid diethyl ester The compound of Reference Example 7 (500 mg) was treated with thionyl chloride (3 m
After 1), a catalytic amount of dimethylformamide was further added, and the mixture was stirred at room temperature for 2 hours. Next, the reaction solution was concentrated to dryness under reduced pressure. The obtained solid was dissolved in dichloromethane (13 ml), and glutamic acid diethyl ester (382 mg) and potassium carbonate (448 m) were added to the solution.
g) was added, and water (13 ml) was further added, followed by vigorous stirring at room temperature for 12 hours. The reaction solution was poured into a saturated aqueous solution of sodium hydrogen carbonate, extracted with chloroform, washed with 1N hydrochloric acid, and dried over sodium sulfate. The solvent was distilled off under reduced pressure to obtain a colorless oil (830 mg). Then
30% hydrogen bromide- was added to the obtained residue (830 mg).
A mixed solution of acetic acid (10 ml) and anisole (830 mg) was added, and the mixture was stirred at room temperature for 3 hours. After the reaction, a large amount of ether was added to the reaction solution, and a dark brown oily substance precipitated. After removing most of the ether layer, the oily substance was suspended in chloroform, and this suspension was washed with a saturated aqueous solution of sodium hydrogen carbonate. The organic layer was separated, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure.
g) was obtained.

¹ H-NMR (CDCl ₃ , δ value):
03-1.48 (9H, m), 1.83-2.62 (7
H, m), 2.96-3.43 (2H, m), 3.83.
-4.40 (4H, m), 4.48-4.98 (1H,
m), 6.33-6.92 (2H, m), 7.34-
7.73 (2H, m)

[0046]

Example 3 N- {4- [N '-(2,4-diamino-6-pteridi)
Nyl) methyl-N'-ethylamino] -3-methylben
Synthesis of zoyl} -L-glutamic acid diethyl ester The compound of Reference Example 8 (360 mg) and 6-bromomethyl-
A dimethylacetamide suspension (10 ml) of 2,4-diaminopteridine hydrobromide / isopropanol adduct (342 mg) was stirred at 60 ° C. for 4 hours, and then further treated with 6-bromomethyl-2,4-diaminopteridine bromide. A hydrochloride / isopropanol adduct (342 mg) was added, and the mixture was stirred at 70 ° C. for 1.5 hours. Then, the reaction solution was poured into a saturated aqueous solution of sodium hydrogen carbonate and extracted with chloroform. After the chloroform layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel column chromatography, using a mixed solvent of chloroform: methanol = 10: 1 as an elution solvent to obtain the desired compound ( 1
67 mg).

¹ H-NMR (CDCl ₃ , δ value):
06 (3H, t, J = 8.0 Hz), 1.15-1.4
2 (6H, m), 1.76 (3H, s), 2.37-
2.54 (4H, m), 3.08 (2H, q, J = 8.
0 Hz), 4.10 (2H, q, J = 8.0 Hz),
4.24 (2H, q, J = 8.0 Hz), 4.38 (2
H, s), 4.70-4.88 (1H, m), 5.32.
(2H, bs), 6.94 (1H, d, J = 7.4H
z), 7.05 (1H, d, J = 8.8 Hz), 7.5
6 (1H, d, J = 5.2 Hz), 7.67 (1H, b
s), 8.80 (1H, s)

[0048]

Example 4 N- {4- [N '-(2,4-diamino-6-pteridi)
Nyl) methyl-N'-ethylamino] -3-methylben
Synthesis of Zoyl} -L-glutamic acid (Compound 2) Ethanol (11 m) of the compound of Example 3 (150 mg)
l) 1N-sodium hydroxide aqueous solution (0.84m
l) was added and the mixture was stirred at 35 ° C for 4 hours. 20 at 25 ° C
After stirring for an hour, water (1 ml) was added to the reaction solution, and the reaction solution was dried under reduced pressure. At this time, the external temperature did not 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 deposited precipitate was collected by filtration and the desired product (1
21 mg).

¹ H-NMR (CDCl ₃ , δ value):
04 (3H, t, J = 6.8 Hz), 1.85-2.2
8 (2H, m), 2.30-2.48 (5H, m),
3.04 (2H, q, J = 6.8 Hz), 4.39 (2
H, s), 6.57 (2H, bs), 7.10 (1H,
d, J = 8.3 Hz), 7.61 (1H, d, J = 8.
3Hz), 7.72 (1H, s), 8.34 (1H,
d, J = 7.8 Hz), 8.57 (IH, s)

[0050]

Reference Example 9 N- (N'-carbobenzoxy-N'-methyl-4-a
Mino-3-methylbenzoyl) -L-2-aminoazibi
Synthesis of dimethyl acid ester The compound of Reference Example 3 (8.4 g) was converted to thionyl chloride (29 m
After adding to l), a catalytic amount of dimethylformamide was further added, and the mixture was stirred at room temperature for 2 hours. The reaction was then concentrated to dryness under reduced pressure. The obtained solid was dissolved in dichloromethane (150 ml), and L-2-aminoadipic acid dimethyl ester hydrochloride (6.6 g) and potassium carbonate (10 g) were added to the solution, and water (150 ml) was further added. Stirred vigorously at room temperature for 12 hours. The reaction solution was poured into water, extracted with chloroform, washed with 1N hydrochloric acid, and dried over sodium sulfate. The solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography, and chloroform: methanol = 1 as an elution solvent.
Using 00: 3, the desired product (9.7 g) was obtained.

¹ H-NMR (CDCl ₃ , δ value):
45-2.55 (9H, m), 3.15 (3H, s),
3.60 (3H, s), 3.72 (3H, s), 4.4
5-4.95 (1H, m), 5.05 (2H, bs),
6.80-7.35 (7H, m), 7.45-7.95
(2H, m)

[0052]

Reference Example 10 N- [N'-methyl- (4-amino-3-methylbenzo)
Yl)]-L-2-aminoadipate dimethyl ester
The compound of Reference Example 9 (9.5 g) was added to a 30% hydrogen bromide-acetic acid (100 ml) solution containing anisole (9.5 g), 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 red-brown oily substance precipitated. After removing most of the ether layer, the oily substance was suspended in chloroform, and the suspension was washed with a saturated aqueous solution of sodium hydrogen carbonate and extracted with chloroform. The chloroform layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure.
0 g). ¹ H-NMR (CDCl ₃ , δ value): 1.43-2.0
0 (4H, m), 2.03 (3H, s), 2.34 (2
H, t, J = 6 Hz), 2.90 (3H, s), 3.6
5 (3H, s), 3.75 (3H, s), 4.53-
5.03 (1H, m), 6.33-6.80 (2H,
m), 7.38-7.68 (2H, m)

[0053]

Example 5 N- {4- [N '-(2,4-diamino-6-pteridi)
Nyl) methyl-N'-methylamino] -3-methyldibenzo
Nzoyl-L-2-aminoadipate dimethyl ester
The compound of Reference Example 10 (3.83 g) and 6-bromomethyl-2,4-diaminopteridine hydrobromide isopropanol adduct (4.06 g) were suspended in dimethylacetamide (75 ml) and the suspension was heated at 55-60 ° C. Stir for 4 hours.
After cooling, the reaction solution was poured into a saturated aqueous solution of sodium hydrogen carbonate and extracted with chloroform. After the chloroform layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel chromatography, using a mixed solvent of chloroform: methanol = 10: 1 as an elution solvent (3. 41 g) were obtained.

¹ H-NMR (CDCl ₃ , δ value):
55-1.95 (4H, m), 2.26-2.46 (5
H, m), 2.74 (3H, s), 3.67 (3H,
s), 3.79 (3H, s), 4.33 (2H, s),
4.70-4.85 (1H, m), 5.34 (2H, b
s), 6.78 (1H, d, J = 6.8 Hz), 7.0
5 (1H, d, J = 8.4 Hz), 7.61 (1H,
d, J = 7.8 Hz), 7.68 (1H, s), 8.8
5 (1H, s)

[0055]

Example 6 N- {4- [N '-(2,4-diamino-
6-pteridinyl) methyl-N'-methylamino] -3
-Methyldibenzoyl-L-2-aminoadipic acid
Synthesis of Compound 3) Ethanol (160 m) of the compound of Example 5 (3.0 g)
l) 1N aqueous solution of sodium hydroxide (18 ml)
Was added and stirred at 35 ° C. for 4 hours. After further stirring at 25 ° C. for 20 hours, water (20 ml) was added to the reaction solution, and the reaction solution was dried under reduced pressure. At this time, the external temperature was kept below 30 ° C. The obtained yellow solid was dissolved in water (50 ml), adjusted to pH = 3.7 with 1N-hydrochloric acid, and left in a refrigerator for 2 hours. The deposited precipitate was collected by filtration to obtain the desired product (2.3 g).

¹ H-NMR (DMSO-d6, δ value):
1.40 = 1.88 (4H, m), 2.24 (2H,
t, J = 7.8 Hz), 2.41 (3H, s), 2.7
2 (3H, s), 4.23-4.43 (4H, m),
6.64 (2H, bs), 7.11 (1H, d, J =
8.8 Hz), 7.66 (1H, d, J = 8.3H)
z), 7.74 (1H, s), 8.38 (1H, d, J
= 8.2Hz), 8.64 (1H, s)

[0057]

Reference Example 11 4-2,4- [N '-(diamino-6-pteridinyl)
Methyl] -N'-methylamino-3-methylbenzoic acid
Under a synthetic nitrogen atmosphere, 6-bromomethyl-2,4-diaminopteridine hydrobromide isopropanol adduct (3.8
g) and 4-methylamino-3-methylbenzoic acid (4.
8 g) was suspended in dimethylacetamide (50 ml), stirred at 40 ° C. for 4 days, and then stirred at 55 ° C. for 2 hours. The reaction was poured into water (500 ml), the pH was adjusted to 5 with a 1N aqueous solution of sodium hydroxide, and the resulting precipitate was collected by filtration and washed with water and acetone to obtain the desired product (3.3).
g) was obtained. ¹ H-NMR (DMSO-d6, δ value): 2.40 (3
H, s), 2.74 (3H, s), 4.36 (2H,
s), 7.10 (1H, d, J = 8.3 Hz), 7.6
-7.8 (2H, m), 8.64 (1H, s)

[0058]

Example 7 N- {4- [N '-(2,4-diamino-6-pteridi)
Nyl) methyl-N'-methylamino] -3-methyldibenzo
Synthesis of ammonium salt of nzoyl-L-homocysteic acid Under a nitrogen atmosphere, a suspension of the compound of Reference Example 11 (2.4 g) in dimethylacetamide (250 ml) was added at 0 ° C. with triethylamine (2.7 ml) and isobutyl chloroformate (0 ml). .85 ml) and stirred for 5 minutes, and then isobutyl chloroformate (0.12 ml) was further added and stirred for 5 minutes. To this solution was added L-homocysteic acid methyl ester hydrochloride (1.5 g), and the mixture was stirred for 30 minutes. Then, triethylamine (1.35 ml) and isobutyl chloroformate (0.3 g) were added.
42 ml), methyl L-homocysteate hydrochloride (0.
75 g) was added in the same manner and stirred for 30 minutes. After gradually raising the temperature to room temperature and stirring for 45 minutes, the solvent was distilled off under reduced pressure so that the bath temperature did not exceed 40 ° C., and a 0.1N aqueous sodium hydroxide solution (400 ml) was added to the residue, and the mixture was stirred at room temperature for 2 minutes. After stirring for an hour, a 1N aqueous sodium hydroxide solution (8 m
l) was added and stirred for 20 minutes. The reaction solution was freeze-dried, the obtained residue was subjected to silica gel column chromatography, and eluted with chloroform: methanol: aqueous ammonia = 5: 4: 1 as an elution solvent, and the obtained crude product was further subjected to DEAE cellulose. The mixture was applied to a column, washed with water, and eluted with a 3% aqueous solution of ammonium bicarbonate to obtain the desired product (1.1 g).

¹ H-NMR (D ₂ O, δ value): 2.15
-2.25 (1H, m), 2.3-2.4 (1H,
m), 2.4 (3H, s), 2.8 (3H, s), 2.
9-3.1 (2H, m), 4.4-4.5 (1H,
m), 4.5 (2H, s) 7.1 (1H, d, J = 8.
5 Hz), 7.6 (1H, d, J = 8.5 Hz), 7.
7 (1H, m), 8.6 (1H, s)

[0060]

Reference Example 12 Np-toluenesulfonyl-4-amino-3-ethyl
Synthesis of benzoic acid methyl ester 3-ethyl-4-aminobenzoic acid methyl ester (1.
To a solution of 86 g) in pyridine (30 ml) was added p-toluenesulfonyl chloride (3 g), and the mixture was stirred at room temperature for 3 hours. After the reaction, the solvent was distilled off under reduced pressure, chloroform and 1N-hydrochloric acid were added to the residue, and the mixture was stirred, and then the organic layer was separated. After the organic layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure, and the resulting residue was subjected to silica gel chromatography, using a mixed solvent of hexane: ethyl acetate 4: 1 as an elution solvent (3 g). I got

¹ H-NMR (CDCl ₃ , δ value): 1.
10 (3H, t), 2.40 (3H, s), 2.1-
2.6 (2H, m), 3.90 (3H, s), 6.6
7.9 (7H, m)

[0062]

Reference Example 13 Np-toluenesulfonyl-N-methyl-4-amino
Synthesis of methyl 3-ethylbenzoate In a solution of the compound of Reference Example 12 (3 g) in anhydrous dimethylformamide (50 ml) was added sodium hydride (1.
3g) was added and stirred at room temperature for 10 minutes. Then, methyl iodide (3.8 g) was added to the reaction solution, and the mixture was returned to room temperature and stirred for 2 hours. After the reaction, water was added to the reaction solution, and extracted with ethyl acetate. After the ethyl acetate layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel column chromatography to obtain the desired product (1.56 g) using chloroform as an elution solvent. Was.

¹ H-NMR (CDCl ₃ , δ value):
25 (3H, t), 2.45 (3H, s), 2.7-
3.1 (2H, m) 3.15 (3H, s), 3.90
(3H, s), 6.6-8.1 (7H, m)

[0064]

Reference Example 14 Np-toluenesulfonyl-N-methyl-4-amino
Synthesis of -3-ethylbenzoic acid The compound (1.56 g) of Reference Example 13 in methanol (60
ml) solution, 1N aqueous sodium hydroxide solution (60 m
l) was added, and the mixture was stirred under reflux for 2 hours. The solvent was concentrated under reduced pressure while maintaining the temperature of the water bath at 30 ° C. or lower. 1N-hydrochloric acid was added to the reaction solution to adjust the pH to 2.5, and the deposited precipitate was collected by filtration to obtain the desired product (1.33 g).

¹ H-NMR (CDCl ₃ , δ value):
25 (3H, t), 2.45 (3H, s), 2.7-
3.1 (2H, m), 3.15 (3H, s), 6.6
8.2 (7H, m)

[0066]

[Reference Example 15] N- (N'-lawtoluenesulfonyl-N'-methyl-
4-amino-3-ethylbenzoyl) -L-glutamine
Synthesis of diethyl acid ester The compound of Reference Example 14 (1.33 g) was converted to thionyl chloride (5
ml), a catalytic amount of dimethylformamide was further added, and the mixture was stirred at room temperature for 2 hours. Next, the reaction solution was concentrated to dryness under reduced pressure. The obtained solid was dissolved in dichloromethane (35 ml), and glutamic acid diethyl ester hydrochloride (900 mg) and potassium carbonate (1.4 g) were added to the solution. Water (15 ml) was further added, followed by vigorous stirring at room temperature overnight. did. After the reaction solution was poured into water and extracted with chloroform, the chloroform layer was washed with a saturated aqueous solution of sodium hydrogen carbonate, 1N-hydrochloric acid, and dried over magnesium sulfate. The solvent was distilled off under reduced pressure, and the target compound (1.
5 g) were obtained.

¹ H-NMR (CDCl ₃ , δ value): 1.
0-1.4 (9H, m), 2.0-3.0 (6H,
m), 2.45 (3H, s), 3, 10 (3H, s),
3.9-4.4 (4H, m), 4.5-5.0 (1H,
m), 6.5-7.9 (7H, m)

[0068]

[Reference Example 16] N-(N'-methyl-4-amino-3-ethyl-benzoyl) -L- synthesis of glutamic acid diethyl ester

To a solution of anisole (1.5 g) in 30% hydrogen bromide-acetic acid (15 ml) was added the compound of Reference Example 15 (1.5 g).
g) was added and the mixture was stirred at room temperature for 4.5 hours. Next, ether (300 ml) was added to the reaction solution, and a red-brown oily substance precipitated. After removing most of the ether layer, the oily substance was suspended in chloroform. The suspension was washed with a saturated aqueous solution of sodium hydrogen carbonate, and the organic layer was separated. The organic layer was dried over magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain the desired product (1.0 g).

¹ H-NMR (CDCl ₃ , δ value):
0-1.4 (9H, m), 2.0-3.0 (6H,
m), 2.85 (3H, s), 3.7-4.3 (4H,
m), 4.5-4.9 (1H, m), 6.4-7.7
(3H, m)

[0071]

Example 8 N- {4- [N '-(2,4-diamino-6-pteridi)
Nyl) methyl-N'-methylamino] -3-ethylben
Synthesis of Zoyl｝ -L-glutamic acid diethyl ester

The compound of Reference Example 16 (1.0 g) and 6-bromomethyl-2,4-diaminopteridine hydrobromide / isopropanol adduct (1.1 g) were suspended in dimethylacetamide (10 ml). Stirred at C for 6 hours. After cooling, the reaction solution was poured into a saturated aqueous solution of sodium hydrogen carbonate and extracted with chloroform. After the organic layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography, and the desired product (671 mg) was obtained using a mixed solvent of chloroform: methanol = 10: 1 as an elution solvent.

¹ H-NMR (CD ₃ OD, δ value):
0-1.4 (9H, m), 2.0-3.0 (6H,
m), 2.75 (3H, s), 3.9-4.7 (7H,
m), 7.1-7.8 (3H, m), 8.70 (1H,
s)

[0074]

Example 9 N- {4- [N '-(2,4-diamino-6-pteridi)
Nyl) methyl-N'-methylamino] -3-ethylben
Synthesis of Zoyl} -L-glutamic acid (Compound 4)

To a solution of the compound of Example 8 (640 mg) in ethanol (22 ml) was added a 1N aqueous solution of sodium hydroxide (1.78 ml), and the mixture was stirred at 35 ° C. for 4.5 hours. After further stirring at 25 ° C. for 20 hours, water (2 ml) was added to the reaction mixture, and then the reaction mixture was cooled with 1N-water under ice-cooling.
The pH was adjusted to 3.7 with hydrochloric acid, and the mixture was left overnight in a cool place. The deposited precipitate was collected by filtration to obtain the desired product (340 mg).

¹ H-NMR (DMSO-d6, δ value):
1.26 (3H, m), 1.9-2.5 (4H, m),
2.69 (3H, s), 2.80 (2H, m), 4.2
9 (2H, s), 4.39 (1H, m), 7.22 (1
H, m), 7.71 (2H, m), 8.64 (1H,
s)

[0077]

[Reference Example 17] N-carbobenzoxy-4-amino-3,5-dimethyl
Synthesis of ethyl benzoate

Under a nitrogen atmosphere, sodium hydride (0.82
g) in tetrahydrofuran (THF) suspension
Amino-3,5-dimethylbenzoic acid ethyl ester (2.0 g) was added, and the mixture was stirred at room temperature for 30 minutes. Then, carbobenzoxychloride (4.4 ml) was added to the suspension, and the mixture was stirred overnight. After a small amount of water was added to the reaction solution, it was poured into ice water and extracted with ethyl acetate. After washing with water and drying (magnesium sulfate), the solvent was distilled off under reduced pressure. The precipitated crystals were added with ethanol, collected by filtration, and dried in vacuo to obtain the desired product (3.2 g).

¹ H-NMR (CDCl ₃ , δ value): 1.
39 (3H, t, J = 7 Hz), 2.05 (6H,
s), 4.35 (2H, q, J = 7 Hz), 5.15
(2H, s), 7.23 (5H, m), 7.75 (2
H, s)

[0080]

Reference Example 18 N-carbobenzoxy-N-methyl-4-amino-3,
Synthesis of 5-dimethylbenzoic acid ethyl ester

Under a nitrogen atmosphere, sodium hydride (1.2
g) was suspended in dimethylformamide (50 ml) and stirred. The compound of Reference Example 17 (3.2 g) was added to the suspension at room temperature, and the mixture was stirred for 30 minutes and then methyl iodide (1.8).
ml), and the mixture was further stirred at room temperature overnight. Next, a few drops of water were added to the reaction solution under ice cooling, and the reaction solution was poured into ice water.
Extracted with ethyl acetate and dried over magnesium sulfate. The solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel column chromatography, and using hexane: ethyl acetate = 9: 1 as an eluting solvent, the desired product (1.47 g).
I got

¹ H-NMR (CDCl ₃ , δ value):
38 (3H, t, J = 7 Hz), 2.21 (6H,
s), 3.17 (3H, s), 4.42 (2H, q, J
= 7 Hz), 5.13 (2H, s), 7.0-7.6,
(5H, m), 7.80 (2H, s)

[0083]

Reference Example 19 N-carbobenzoxy-N-methyl-4-amino-3,
Synthesis of 5-dimethylbenzoic acid

TH of the compound of Reference Example 18 (1.47 g)
A 1N aqueous solution of sodium hydroxide (12 ml) was added to the F (30 ml) solution, the mixture was heated at 60 ° C. for 6 hours, and further stirred at room temperature overnight. Then, the pH of the reaction solution was adjusted to about 5 with 3N-hydrochloric acid, and the mixture was concentrated under reduced pressure. After adjusting the pH to about 2, THF
And dried over magnesium sulfate. The solvent was distilled off under reduced pressure to obtain the desired product (1.34 g).

¹ H-NMR (CDCl ₃ , δ value):
18 (6H, s), 3.14 (3H, s), 5.04
(2H, s), 6.9-7.5 (5H, m), 7.76
(2H, s)

[0086]

Reference Example 20 N- (N'-carbobenzoxy-N'-methyl-4-a
Mino-3,5-dimethylbenzoyl) -L-glutamine
Of diethyl methacrylate

To the compound of Reference Example 19 (1.34 g) was added thionyl chloride (10 ml), and a catalytic amount of dimethylformamide was further added, followed by stirring at room temperature for 3 hours. Next, the reaction solution was concentrated to dryness under reduced pressure. The obtained solid was dissolved in dichloromethane (25 ml), and an aqueous solution (25 ml) of glutamic acid diethyl ester hydrochloride (1.0 g) and potassium carbonate (1.2 g) were added to the solution. After adjusting the pH of the solution to about 10,
Stir vigorously overnight at room temperature. The dichloromethane layer was separated, washed with 1N-hydrochloric acid, a saturated aqueous solution of sodium hydrogen carbonate and saturated saline in this order, and dried over sodium sulfate. The solvent was distilled off under reduced pressure to obtain the desired product (2.14 g).

¹ H-NMR (CDCl ₃ , δ value): 1.
23 (3H, t, J = 7 Hz), 1.31 (3H, t,
J = 7 Hz), 2.1-2.7 (4H, m), 2.17
(6H, s), 3.13 (3H, s), 4.13 (2
H, q, J = 7 Hz), 4.25 (2H, q, J = 7H)
z), 4.73 (1H, m), 5.07 (2H, s),
6.94 (1H, d, J = 8 Hz), 7.1-7.5
(5H, m), 7.51 (2H, s)

[0089]

Reference Example 21 N- [N'-methyl- (4-amino-3,5-dimethylbenzoyl)]-L- glutamic acid diethyl ester
Synthesis

To a solution of the compound of Reference Example 20 (2.1 g) in anisole (2.4 ml) was added a 30% hydrogen bromide-acetic acid (24 ml) solution, and the mixture was stirred at room temperature for 3 hours. Next, about 200 ml of ether was added to the reaction solution, and a red-brown oily substance precipitated. After removing most of the ether layer, the oily substance was suspended in chloroform. This suspension was washed with a saturated aqueous solution of sodium hydrogen carbonate, and the organic layer was separated. After the organic layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure to obtain the desired product (1.2 g).

¹ H-NMR (CDCl ₃ , δ value): 1.
21 (3H, t, J = 7 Hz), 1.29 (3H, t,
J = 7 Hz), 2.0-2.8 (4H, m), 2.29
(6H, s), 2.87 (3H, s), 3.11 (1
H, s), 4.11 (2H, q, J = 7 Hz), 4.2
3 (2H, q, J = 7 Hz), 4.83 (1H, m),
6.83 (1H, d, J = 8 Hz), 7.43 (2H,
s)

[0092]

Example 10 N- {4- [N '-(2,4-diamino-6-pteridi)
Nyl) methyl-N'-methylamino] -3,5-dimethyl
Of rubenzoyl} -L-glutamic acid diethyl ester
Synthesis

The compound of Reference Example 21 (1.2 g) and 6-bromomethyl-2,4-diaminopteridine hydrobromide isopropanol adduct (1.0 g) were suspended in dimethylacetamide (20 ml). Stir for 12 hours. After cooling, water was added to the reaction solution, the mixture was stirred, and then extracted with chloroform. After the chloroform layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel chromatography, using a mixed solvent of chloroform methanol = 99: 1 to 95: 5 as an elution solvent. (0.64 g).

¹ H-NMR (CDCl ₃ , + CD ₃ O
D, δ value): 1.25 (3H, t, J = 7 Hz);
32 (3H, t, J = 7 Hz), 2.0-2.7 (4
H, m), 2.42 (6H, s), 2.82 (3H,
s), 4.18 (2H, q, J = 7 Hz), 4.28
(2H, q, J = 7 Hz), 4.42 (2H, s),
4.78 (1H, m), 7.53 (2H, s), 8.8
9 (1H, s)

[0095]

Example 11 N- {4- [N '-(2,4-diamino-6-pteridi)
Nyl) methyl-N'-methylamino] -3,5-dimethyl
Synthesis of Rubenzoyl} -L-glutamic acid (Compound 5)

The compound of Example 10 (0.59 g) was dissolved in a mixed solvent of ethanol (40 ml) and THF (20 ml), and further dissolved in a 1N aqueous sodium hydroxide solution (3.5.
ml) and the mixture was stirred at room temperature overnight. The solvent was distilled off under reduced pressure, and the obtained yellow solid was dissolved in water (20 ml), adjusted to pH = 3.7 with 1N hydrochloric acid under ice cooling, and the deposited precipitate was collected by filtration. After washing in order of water and acetone, vacuum drying was performed to obtain the desired product (0.47 g).

¹ H-NMR (DMSO-d ₆ , δ value):
1.8-2.2 (2H, m), 2.3-2.5 (2H,
m), 2.34 (6H, s), 2.73 (3H, s),
4.3-4.5 (1H, m), 4.36 (2H, s),
6.66 (2H, s), 7.55 (2H, s), 8.4
0 (1H, d, J = 6 Hz), 8.74 (1H, s)

[0098]

Reference Example 22 of 4-methylamino-3-trifluoromethylbenzoic acid
Synthetic 4-amino-3-trifluoromethylbenzoic acid (3.6
g) was suspended in trifluoroacetic anhydride (90 ml) and stirred overnight. The solvent was distilled off under reduced pressure, and the obtained residue was dissolved in acetone (30 ml). To this solution was added methyl iodide (9.9 g), heated to 45 ° C. and slowly added powdered potassium hydroxide (6.8 g) with vigorous stirring. After continuing stirring at this temperature for another hour, the solvent was distilled off under reduced pressure. The obtained residue was dissolved in water (50 ml) and heated under reflux for 2 hours. Under ice cooling, the pH was adjusted to 3.5 using 1N-hydrochloric acid. The white precipitate was collected by filtration, washed with a small amount of water, and dried under vacuum to obtain the desired product (2.2 g). ¹ H-NMR (CDCl ₃ : CD ₃ OD = 1: 1, δ
Value): 2.95 (3H, s), 6.74 (1H, d, J
= 9.0 Hz), 7.96-8.12 (2H, m)

[0099]

[Reference Example 23] 4-Methylamino-3-trifluoromethylbenzoic acid
Synthesis of Cyl Ester A methanol solution of the compound of Reference Example 22 (2.2 g) (2
5 ml) was blown with hydrogen chloride gas under ice-cooling, and stirring was continued at room temperature for 4 hours. The solvent was distilled off under reduced pressure, and the obtained residue was dissolved in ethyl acetate. The solution was washed with a saturated aqueous solution of sodium hydrogen carbonate, dried over anhydrous magnesium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography, and the desired product (1.3 g) was obtained using ethyl acetate: normal hexane = 1: 3 as an elution solvent. ¹ H-NMR (CDCl ₃ , δ value): 2.91 (3H,
s), 3.83 (3H, s), 4.63 (1H, b
s), 6.65 (1H, d, J = 9.0 Hz), 7.9
2-8.99 (2H, m)

[0100]

Reference Example 24 4- [N- (2,4-diamino-6-pteridinyl) meth
Tyl-N-methyl] amino-3-trifluoromethyl ammonium
Synthesis of benzoic acid To a solution of the compound of Reference Example 23 (1.3 g) in hexamethylphosphoric triamide (20 ml) was added sodium hydride (0.26 g) under ice-cooling and nitrogen atmosphere.
For a minute. 6-Bromomethyl-2,4
-Diaminopteridine hydrobromide 1/3 isopropanol adduct (2.8 g) was added, and the mixture was stirred for 30 minutes, further stirred at 70 ° C for 6 hours and at room temperature for 2 days. The reaction solution was poured into diluted hydrochloric acid, neutralized with a saturated aqueous solution of sodium hydrogen carbonate,
The resulting brown precipitate was collected by filtration. After vacuum drying, the mixture was suspended in a mixed solvent of chloroform: methanol = 1: 1, and heated under reflux for 1 hour. The insoluble material was removed by filtration, and the filtrate was concentrated to dryness under reduced pressure. The obtained residue was dissolved in methanol (50 ml), a 1N aqueous solution of sodium hydroxide (20 ml) was added, and the mixture was stirred at 60 ° C for 1 hour. After methanol was distilled off under reduced pressure, insolubles were collected by filtration. Under ice cooling, the pH was adjusted to 4 using 1N-hydrochloric acid. The orange precipitate was collected by filtration, dried in vacuo, and then subjected to silica gel chromatography. Separation was performed using ethyl acetate as an elution solvent, and then using chloroform: methanol: 28% aqueous ammonia = 5: 4: 1. The target fraction was concentrated to dryness under reduced pressure, and the obtained residue was dissolved in water. After adjusting the pH to 4 using 1N-hydrochloric acid under ice cooling, the yellow precipitate was collected by filtration and dried in vacuo to obtain the desired product (102 mg).

¹ H-NMR (DMSO-d ₆ = CDCl
₃ = 7: 1, δ value): 2.79 (3H, s), 4.42
(2H, s), 7.61 (1H, d, J = 8.3H
z), 8.11-8.17 (2H, m), 8.69 (1
H, s)

[0102]

Example 12 N- {4- [N ′-(2,4-diamino-6-pteridi)
Nyl) methyl-N'-methyl] amino-3-trifluoro
Combination of romethylbenzoyl} -L-2-aminoadipic acid
Formation (L-2- {N- [4- [N '- (2,4- diamino -
6-pteridinyl) methyl-N'-methyl] amino-3
-Trifluoromethylbenzoyl] {synthesis of aminoadipic acid)

To a solution of the compound of Reference Example 24 (88 mg) in hexamethylphosphoric triamide (2 ml) was added:
Under ice cooling and nitrogen atmosphere, hydroxybenzotriazole (36 mg), dicyclohexylcarbodiimide (55
mg) and stirred for 30 minutes. Then, L-2-aminoadipic acid dimethyl ester hydrochloride (63 mg), N-
After adding methyl morpholine (34 mg), the mixture was gradually returned to room temperature and stirred for 2 days. The reaction solution was subjected to silica gel chromatography, and purified using chloroform as an elution solvent, and then using chloroform: methanol = 19: 1. The desired fraction was concentrated to dryness under reduced pressure, and methanol (2m
l). Under ice cooling and a nitrogen atmosphere, a 1N aqueous solution of sodium hydroxide (0.4 ml) was added, and the mixture was stirred for 5 hours.
The solvent was distilled off under reduced pressure, and the obtained residue was dissolved in water.
After filtering off the insoluble matter, pH = 3.5 using 1N-hydrochloric acid.
Was adjusted to The yellow precipitate was collected by filtration, washed with a small amount of water, and dried under vacuum. This crude product was subjected to preparative thin-layer chromatography (silica gel), and chloroform: methanol: 28% aqueous ammonia = 5:
Separated using 4: 1. The eluate was concentrated to dryness under reduced pressure and then dissolved in an aqueous sodium hydrogen carbonate solution. After filtering off the insoluble matter, the pH was adjusted to 3.5 using 1N-hydrochloric acid. The pale yellow precipitate was collected by filtration, dried in vacuo and the desired product (18
mg).

¹ H-NMR (DMSO-d ₆ = CDCl
₃ = 7: 1, δ value): 1.53-1, 93 (4H,
m), 2.25 (2H, t, J = 7.3 Hz), 2.7
5 (3H, s), 4.34-4.45 (3H, m),
6.91 (2H, bs), 7.63-7.67 (2H,
m), 7.95 (1H, bs), 8.16 (1H, d,
J = 8.3 Hz), 8.22 (1H, s), 8.72
(1H, s), 8.79 (1H, d, J = 7.8 Hz)

[Brief description of the drawings]

FIG. 1 shows ³ H-Ud at each concentration of the test drug.
Indicates the amount (ratio) of incorporation of R.

──────────────────────────────────────────────────続 き Continued on the front page (72) Keiichiro Tsuji 1-135, Komamon, Gotemba, Shizuoka Prefecture Inside Chugai Pharmaceutical Co., Ltd. (56) References ANTIMICROBIAL AGE NTS AND CHEMOTHERA PY, Vol. 35, No. 7 (July, 1991) p. 1348-1355 (58) Fields investigated (Int. Cl. ⁷ , DB name) C07D 475/08 CA (STN) CAOLD (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] 1. A compound of the general formula (I) In the formula, R ¹ represents a lower alkyl group having 1 to 4 carbon atoms; R ² represents a lower alkyl group having 1 to 4 carbon atoms or a trifluoromethyl group; R ³ represents a hydrogen atom or a 1 carbon atom. R ⁴ represents a hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms; n represents an integer of 1 to 4; R ⁵ represents a general formula COOR ⁶ (wherein R ⁶ is a hydrogen atom or having 1 to 4 lower alkyl represents a group atoms), a group represented by or SO ₃ H a group represented by a compound represented by (wherein, R ¹ is A methyl group, R ² is a trifluoromethyl group, R ³ is a hydrogen atom, R ⁴ is a hydrogen atom, n is 2, and R ⁵ is COOR ⁶ (excluding compounds in which R ⁶ is a hydrogen atom).