JP3281099B2 - Anti-rheumatic drug - 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]

BACKGROUND OF THE INVENTION 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] So far, as a methotrexate derivative to N ¹⁰ is alkyl groups other than methyl groups are introduced, for example, the following formula

Embedded image (J. Med. Chem., 22 , 862 (197)
9)) and expressions

Embedded image (J. Med. Chem., 25 , 877 (198)
2)), etc., but did not show satisfactory activity.

[0004] The present inventors have conducted intensive studies in search of a compound having better anti-rheumatic activity in this type of methotrexate derivative, and have accomplished the present invention.

[0005]

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

Embedded image Wherein R ₁ is CH ₂ , CH ₂ CH ₂ , CH ₂ O, CH ₂
Shows a member selected from the group consisting of S and CH _{2 SO;} R 2 represents a hydrogen atom or a lower alkyl group or a benzyl group having a carbon number of 1 to 4; n is an integer of from 1 to 4; R ₃ Is a general formula COOR ₄ (where R ₄ represents a hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms) or a general formula NHCOR ₅ (where R ₅ represents a phenyl group which may be substituted) or a general formula Formula CONR ₆ R
₇ (where R ₆ represents a hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms; R ₇ represents a lower alkyl group having 1 to 4 carbon atoms or a phenyl group or a carboxyalkyl group which may be substituted or a lower alkyl group) A sulfonyl group) or a group represented by PO ₃ H ₂ or SO ₃ H;
A methotrexate derivative represented by the formula:

[0006] 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 (Method C) When R ₃ in the general formula (I) is

Embedded image (R ′: hydrogen atom or lower alkyl group having 1 to 4 carbon atoms)

Embedded image

Embedded image (Wherein, R ₁ , R ₂ , R ₃ and n have the same meanings as described above, R ′ represents a hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms, and A ₁ and A ₂ represent a protecting group. , X represents a halogen atom.)

In the method A, the compound of the general formula (2) is obtained by suspending the compound of the general formula (1) in an acid halogenating agent such as thionyl chloride or oxalyl chloride. The reaction is carried out by stirring at room temperature in the presence of a catalytic amount of dimethylformamide or the like. Wherein the protecting group represented by A _1, carbobenzoxy group, a tosyl group, and a acetyl group.

The reaction for obtaining the compound of the general formula (4) from the compound of the general formula (2) and the compound of the general formula (3) is carried out by dissolving the compound of the general formula (2) in a solvent such as dichloromethane or the like. The reaction is carried out by adding an aqueous solution of the compound of the general formula (3) under cooling or water cooling, and stirring at room temperature in the coexistence of an inorganic base such as potassium carbonate, sodium hydroxide and sodium hydrogen carbonate.

In the reaction for obtaining the compound of the general formula (5) from the compound of the general formula (4), the compound of the general formula (4) is added to a solution of anisole and phenol dissolved in a hydrogen bromide-acetic acid solution. C. to 60.degree. C., preferably by stirring at room temperature. The reaction for obtaining the compound of the general formula (5) from the compound of the general formula (4) is carried out by dissolving the compound of the general formula (4) in a solvent such as methanol, ethanol or acetic acid, adding palladium-carbon, It may be performed by stirring at room temperature under a hydrogen atmosphere.

In the reaction for obtaining the compound of the general formula (7) from the compound of the general formula (6) and the compound of the general formula (5), the compound of the general formula (6) and the compound of the general formula (5) are converted to dimethylacetamide, 0 ° C to 1 in a solvent such as dimethylformamide
The stirring is carried out at 00 ° C, preferably at 50 ° C to 60 ° C. In particular, when R ₂ is a hydrogen atom, a 1N aqueous solution of sodium hydroxide is further added to a solvent such as methanol or ethanol, and the mixture is stirred at 0 ° C. to 60 ° C., preferably 35 ° C. to obtain the desired product. In the formula, examples of the halogen atom represented by X include a bromine atom and a chlorine atom.

In the method B, the reaction of obtaining the compound of the general formula (9) from the compound of the general formula (6) and the compound of the general formula (8) is carried out by the compound of the general formula (6) and the compound of the general formula (8) In a solvent such as dimethylacetamide or dimethylformamide at 0 ° C to 100 ° C, preferably at 55 ° C.

In the reaction for obtaining the compound of the general formula (7) from the compound of the general formula (9) and the compound of the general formula (3), the compound of the general formula (9) is reacted with cyanide diethylphosphate, dicyclohexylcarbodiimide and 1- After stirring in a solvent such as dimethylformamide, dimethylacetamide, or N-methylpyrrolidinone in the presence of hydroxybenzotriazole or the like, the compound of the general formula (3) is added, and the mixture is stirred at 0 ° C to 200 ° C, preferably at 10 ° C to 80 ° C. It is done by doing. In particular, when R ₂ is a hydrogen atom, a 1N aqueous solution of sodium hydroxide 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.

In the method C, the reaction of obtaining the compound of the general formula (12) from the compound of the general formula (10) and the compound of the formula (11) is performed by converting the compound of the general formula (10) to chloroform, dichloromethane, tetrahydrofuran, dioxane or the like. , A compound of the formula (11), water and, for example, potassium carbonate, triethylamine, sodium hydrogen carbonate, pyridine and the like are added, and the mixture is stirred at room temperature. Wherein the protecting group represented by A _2, carbobenzoxy group, a tosyl group, and a acetyl group.

From the compound of the general formula (12), the compound of the general formula (1)
The reaction for obtaining the compound of 3) is carried out in a solvent such as methanol,
The reaction is carried out by stirring at 60 ° C to -20 ° C, preferably at -30 ° C, adding thionyl chloride, and then refluxing.

From the compound of the general formula (13), the compound of the general formula (1)
The reaction for obtaining the compound 4) is carried out by dissolving the compound of the general formula (13) in ethanol, methanol, tetrahydrofuran, dioxane or the like, and stirring the mixture at room temperature under a hydrogen atmosphere in the presence of palladium-carbon.

In the reaction for obtaining the compound of the general formula (15) from the compound of the general formula (14) and the compound of the general formula (2), the compound of the general formula (2) is dissolved in dichloromethane or the like, and The compound (14), potassium carbonate or triethylamine and water are added and the mixture is stirred at room temperature, but may be amidated by a mixed acid anhydride method, an active ester or an active amide method.

From the compound of the general formula (15), the compound of the general formula (1)
The reaction for obtaining the compound 6) is carried out by adding hydrogen bromide-acetic acid in which phenol or anisole is dissolved in advance to the compound of the general formula (15) and stirring the mixture at room temperature.

The reaction for obtaining the compound of the general formula (17) from the compound of the general formula (6) and the compound of the general formula (16) is carried out at 25 ° C. to 100 ° C. in an aprotic polar solvent such as dimethylacetamide and dimethylformamide. Preferably 5
After stirring at 0 ° C. to 65 ° C., for example, triethylamine,
It is performed by stirring in water containing potassium carbonate or sodium hydrogen carbonate.

From the compound of the general formula (17), the compound of the general formula (I)
The reaction for obtaining the compound of the above is carried out by adding an aqueous solution of sodium hydroxide in a solvent such as ethanol and stirring at room temperature.

[0020]

The compound represented by the general formula (I) obtained by the present invention has an antirheumatic effect. It is also less toxic than methotrexate. This effect is examined in the following experimental examples: “Inhibition of lymphocyte proliferation using human peripheral blood” and “Comparison of toxicity between methotrexate (MTX) and compound of the present invention by continuous intraperitoneal administration using rats”. This was confirmed by:

[0021] Experimental Example 1 "antiproliferative activity of human peripheral blood-derived lymphocyte" (Method) Lymphocytes were separated using a Ficoll-Paque ^R from human peripheral blood, 10 ⁵ appropriate dilution drug and its lymphocytes Was cultured with PHA (0.3 μg / ml) for 2 days in a 96-well culture plate. Five hours before the end of the culture, ³ H-UdR (1 μCi / w
ell) was added, and the incorporation of ³ H-UdR into lymphocytes was measured with a scintillation counter.

(Where PHA is phytohaemag
glutinin, UdR is deoxyuridine
Is shown. The drugs used are as follows.

Embedded image

(Results and Discussion) FIGS. 1 to ³ show the uptake of ³ H-UdR by PHA-stimulated lymphocytes without drug.
The ratio was set to 00%. As is clear from FIGS. 1 to 3, it was confirmed that the compound of the present invention has a superior lymphocyte proliferation inhibitory action (anti-rheumatic action) than the control compound.

Experimental Example 2 "Comparison of toxicity between methotrexate (MTX) and compound of the present invention by continuous intraperitoneal administration in rats"

(Method) An 8-week-old male SD rat was treated with M
TX or the compound of the present invention was intraperitoneally administered once a day, 5 days a week for 5 consecutive weeks. The dose is two doses of 0.25, 0.5 mg / kg for both MTX and the compound of the present invention. The control group contained a solvent (phosphate buffer, pH7.
4) was similarly administered. The number of animals per group is five.

The inspection items are as follows. 1) Life and death and symptoms: Observed daily. 2) Changes in body weight: Measured once a week. 3) Blood test: White blood cell count in peripheral blood (WB
C), red blood cell count (RBC) was measured. 4) Liver weight and liver triglyceride (TG) content: After final administration, dissection was performed, and liver weight and liver TG
The content was measured. The following drugs were used.

[0027]

Embedded image

(Results) 1) Life and Death and Symptoms Among the MTX administration groups, there were no deaths and no abnormal symptoms were observed in the 0.25 mg / kg group throughout the administration period. However, in the 0.5 mg / kg group, deterioration of nutritional status, anemia, decrease in fecal volume, swelling around the mouth, etc. were observed from the fourth week, and one of five patients died.

On the other hand, in the compound administration group of the present invention, 0.2
No deaths occurred in any of the 5 mg / kg group and the 0.5 mg / kg group, and no abnormal symptoms were observed.

2) Weight Transition FIG. 4 shows the weight transition. The compound of the present invention showed less inhibition of weight gain than MTX.

3) Blood test FIG. 5 shows the results of the blood test. The reduction of WBC and RBC was clearly weaker for the compounds of the invention compared to MTX.

4) Liver Weight and Liver TG Content FIG. 6 shows liver weight and liver TG content. 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) The toxicity of MTX was compared with the compound of the present invention by continuous intraperitoneal administration using rats. As a result, the compound of the present invention was considered to be clearly less toxic than MTX in all respects of mortality, symptoms, changes in body weight, blood tests, liver weight and liver TG content.

[0034]

【Example】

Reference Example 1 Synthesis of 1-carbobenzoxy-5-carboxyindoline
Success

After 5-carboxyindoline (2.0 g) and sodium hydroxide (0.6 g) were added to a mixture of water (20 ml) and diethyl ether (20 ml), the mixture was cooled with ice-water and carbobenzoxycyclolide was added. (2.59 g) and sodium hydroxide (2.1 g) were added, water (10 ml) was alternately added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was acidified with 2N-hydrochloric acid, and the precipitated crystals were filtered. After washing with ether, it was air-dried to obtain the desired product (2.8 g).

¹ H-NMR (DMSO-d ₆ , δ):
3.10 (2H, t, J = 8 Hz), 4.03 (2H,
t, J = 8 Hz), 5.23 (2H, s), 7.2-
8.0 (8H, m) mp; 194-196 ° C

[0037]

Reference Example 2 N- (1-carbobenzoxyindoline-5-carboni
G) Synthesis of diethyl-L-glutamate

REFERENCE EXAMPLE 1 Was suspended in thionyl chloride (10 ml), a catalytic amount of dimethylformamide was added, and the mixture was stirred at room temperature for 30 minutes. Subsequently, excess thionyl chloride was distilled off under reduced pressure, and the residue was triturated with N-hexane. The obtained crystals were filtered, dissolved in dichloromethane (20 ml), and the dichloromethane solution was suspended under ice-water cooling in dichloromethane (50 ml) containing glutamic acid diethyl ester hydrochloride (3.0 g) and triethylamine (2.8 g). It was added dropwise to the suspension. After stirring at room temperature for 2.5 hours, the solvent was distilled off under reduced pressure, and ethyl acetate (200 ml) and diluted hydrochloric acid were added to the residue under ice-water cooling.
(200 ml) of the mixture was added. After stirring for 5 minutes,
The organic layer was separated. The organic layer was washed with a 5% aqueous sodium hydrogen carbonate solution and dried over magnesium sulfate. Then, ethyl acetate was distilled off under reduced pressure, and the obtained residue was subjected to silica gel column chromatography, using a mixed solvent of chloroform: methanol = 30: 1 as an elution solvent, to obtain the desired product (3.1 g). .

¹ H-NMR (CDCl ₃ , δ): 1.1
9 (3H, t, J = 7 Hz), 1.25 (3H, t, J)
= 7 Hz), 2.1-2.6 (4H, m), 3.06
(2H, t, J = 8 Hz), 3.8-4.3 (6H,
m), 4.75 (1H, m), 5.20 (2H, s),
6.79 (1H, d, J = 7 Hz), 7.2-7.7
(8H, m) mp; 120-121 ° C

[0040]

Reference Example 3 N- (Indoline-5-carbonyl) -L-glutamine
Of diethyl methacrylate

Reference Example 2 Was dissolved in tetrahydrofuran (80 ml), 10% palladium-carbon (0.4 g) was added, and the mixture was stirred under a hydrogen atmosphere at room temperature for 5 hours. The target product (1.2 g) was obtained by filtering off palladium-carbon using celite.

¹ H-NMR (CDCl ₃ , δ): 1.2
1 (3H, t, J = 7 Hz), 1.29 (3H, t, J
= 7 Hz), 2.0-2.6 (4H, m), 3.00
(2H, t, J = 8 Hz), 3.53 (2H, t, J =
8 Hz), 4.07 (2H, q, J = 7 Hz), 4.1
9 (2H, q, J = 7 Hz), 4.75 (1H, m),
6.47 (1H, d, J = 9 Hz), 6.68 (1H, d, J = 9 Hz)
d, J = 7 Hz), 7.45 (1H, d, J = 9H)
z), 7.49 (1H, s) mp; 96-97 ° C.

[0043]

Example 1 N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] indoline- 5-carbonyl] -L-glutami
Synthesis of diethyl phosphate

Reference Example 3 (214 mg) and 6-
Bromomethyl-2,4-diaminopteridine hydrobromide / isopropanol adduct (250 mg) was suspended in dimethylacetamide (3 ml) and stirred at 50 to 55 ° C for 4 hours. After cooling, triethylamine (12
Water (15 ml) containing 4 mg) was added and stirred, and then extracted with chloroform (350 ml) in four portions. After the organic 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 eluting solvent to obtain the desired compound. (200 mg) was obtained.

¹ H-NMR (DMSO-d ₆ , δ):
1.22 (3H, t, J = 7 Hz), 1, 30 (3H,
t, J = 7 Hz), 2.0-2.5 (4H, m), 3.
07 (2H, t, J = 8 Hz), 3.57 (2H, t,
J = 8 Hz), 4.10 (2H, q, J = 7 Hz),
4.23 (2H, q, J = 7 Hz), 4.79 (1H,
m), 5.24 (2H, s), 6.51 (1H, d, J
= 9 Hz), 6.76 (1H, d, J = 7 Hz), 7.
57 (1H, s), 7.59 (1H, d, J, = 9H
z), 8.82 (1H, s) mp;

[0046]

Example 2 N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] indoline-5-carbonyl] -L-glutami
Synthesis of acid (Compound 1)

Embodiment 1 (170 mg) was dissolved in ethanol (33 ml), a 1N aqueous sodium hydroxide solution (0.84 ml) was added at 35 ° C., and the mixture was heated at the same temperature to 4.5.
Stirred for hours. After further stirring at 25 ° C. for 20 hours,
Water (2 ml) was added to the reaction solution. The reaction solution was dried under reduced pressure. The obtained residue was dissolved in water (15 ml), and the reaction solution was pH-3.7 with 1N hydrochloric acid under ice-water cooling.
And left overnight in a cool place. The deposited precipitate was collected by filtration to obtain the desired product (130 mg).

¹ H = NMR (DMSO-d ₆ , δ):
1.94 (2H, m), 2.32 (2H, m), 2.9
8 (2H, t, J = 8 Hz), 3.56 (2H, t, J
= 8 Hz), 4.29 (1H, m), 4.53 (2H,
s), 6.71 (1H, d, J = 9 Hz), 7.57
(1H, s), 7.59 (1H, d, J = 9 Hz),
8.72 (1H, s) mp; 201-204 ° C (dec.)

[0049]

[Reference Example 4]N- (1-carbobenzoxyindolin-5-carboni
L) Synthesis of dimethyl-L-α-aminoadipate

After 1-carbobenzoxyindoline-5-carboxylic acid (3.1 mg) was suspended in thionyl chloride (10 ml), a catalytic amount of dimethylformamide was added, and the mixture was stirred at room temperature for 2 hours. Subsequently, excess thionyl chloride was distilled off under reduced pressure, and the residue was triturated with n-hexane. After filtering the obtained crystals, dichloromethane (30 ml) was used.
And the dichloromethane solution was cooled with ice-water,
It was added dropwise to an aqueous solution (30 ml) containing α-aminoadipic acid dimethyl hydrochloride (2.7 g). Further, potassium carbonate (5.6 g) was added to the reaction solution. After stirring overnight at room temperature, the reaction mixture was poured into a saturated aqueous solution of sodium hydrogen carbonate, and the organic matter was extracted with chloroform. The chloroform layer was washed with 1N-hydrochloric acid, dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography, and the desired product (3.1 mg) was obtained using a mixed solvent of chloroform: methanol = 100: 1 as an elution solvent.

¹ H-NMR (CDCl ₃ , δ): 1.6
−2.1 (4H, m), 2.36 (2H, t, J = 6.
8Hz), 3.13 (2H, m), 3.66 (3H,
s), 3.77 (3H, s), 4.09 (2H, m),
4.78 (1H, m), 5.27 (2H, bs), 6.
80 (1H, d, J = 7.8 Hz), 7.2-7.5
(6H, m), 7.63 (2H, m)

[0052]

Reference Example 5 N- (Indoline-5-carbonyl) -L-α-amino
Synthesis of dimethyl adipate

Reference Example 4 In a solution of anisole (1.5 g) in 30% hydrogen bromide-acetic acid (15 ml). Compound (1.5
g) was added and stirred at room temperature for 4 hours. Then, a large amount of ether was added to the reaction solution, and a red-brown oily substance precipitated. Most of the ether layer was removed, and the oily substance was suspended in chloroform. This suspension was washed with a saturated aqueous solution of sodium hydrogen carbonate, and the chloroform layer was separated. The chloroform layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure to obtain the desired product (960 mg).

¹ H-NMR (CDCl ₃ , δ): 1.6
−2.1 (4H, m), 2.36 (2H, t, J = 6.
8Hz), 3.07 (2H, m), 3.66 (2H,
m), 3.66 (3H, s), 3.77 (3H, s),
4.78 (1H, m), 6.62 (2H, m), 7.5
4 (2H, m)

[0055]

Example 3 N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] -indoline-5-carbonyl] -L-α-a
Synthesis of dimethyl minoadipate

Reference Example 5 (960 mg) and 6-
Bromomethyl-2,4-diaminopteridine hydrobromide / isopropanol adduct (1140 mg) was suspended in dimethylacetamide (20 ml) and stirred at 50 to 60 ° C for 6 hours. After cooling, the mixture was poured into a saturated aqueous solution of sodium hydrogen carbonate, and the desired product was extracted with chloroform three times. After the organic layer was dried over sodium 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 = 100: 10 as an elution solvent to obtain the desired compound ( 520m
g) was obtained.

¹ H-NMR (CDCl ₃ , δ): 1.6
−2.1 (4H, m), 2.38 (2H, t, J = 6.
8Hz), 3.07 (2H, m), 3.57 (2H,
m), 3.67 (3H, s), 3.78 (3H, s),
4.53 (2H, s), 4.74 (1H, m), 6.5
2 (1H, d, J = 8.3 Hz), 7.01 (1H, d, J = 8.3 Hz)
d, J = 7.8 Hz), 7.57 (2H, m), 8.7
7 (1H, s)

[0058]

Example 4 N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] -indoline-5-carbonyl] -L-α-a
Synthesis of minoadipic acid

Embodiment 3 FIG. Was dissolved in ethanol (22 ml), a 1N aqueous sodium hydroxide solution (3.1 ml) was added at 35 ° C., and the mixture was stirred at the same temperature for 4 hours. After stirring was further continued at 25 ° C. for 20 hours, water (3 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 resulting yellow solid was dissolved in water (10 ml) and 1
The pH was adjusted to 3.7 with N-hydrochloric acid and left in a refrigerator for 2 hours. The deposited precipitate was collected by filtration, and the desired product (320 m
g) was obtained.

¹ H-NMR (DMSO-d ₆ , δ):
1.4, -1.9 (4H, m), 2.23 (2H, t,
J = 6.8 Hz), 3.01 (2H, m), 3.58
(2H, m), 4.32 (1H, m), 4.55 (2
H, s), 6.69 (1H, d, J = 8.3 Hz),
7.63 (2H, m), 8.10 (1H, d, J = 8.
3Hz), 8.72 (1H, s)

[0061]

Reference Example 6 Nα- (1-carbobenzoxyindoline-5-carbo
Nyl) -Nδ-benzoyl-L-ornithine methyl
Steal Synthesis

Thionyl chloride (1.5 ml) was added to 1-carbobenzoxyindoline-5-carboxylic acid (180 mg).
Was added to obtain a suspension, and a catalytic amount of dimethylformamide was added to the 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 (4 ml), and Nδ-
Benzoyl-L-ornithine methyl ester (150m
g) and calcium carbonate (750 mg) were added, and water (4 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, and the chloroform layer was further washed with a 1N hydrochloric acid solution and dried over sodium sulfate. The solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel chromatography, using chloroform: methanol = 100: 3 as the elution solvent to obtain the desired compound.
(140 mg).

¹ H-NMR (CDCl ₃ , δ): 1.6
-2.2 (4H, m), 3.15 (2H, t, J = 8.
8Hz), 3.56 (2H, m), 3.78 (3H,
s), 4.10 (2H, t, J = 8.8 Hz), 4.8
2 (1H, m), 5.27 (2H, s), 6.70 (1
H, m), 6.89 (1H, d, J = 8.8 Hz),
7.20 (8H, m), 7.68 (2H, m), 7.8
0 (2H, m)

[0064]

Reference Example 7 Nα- (indoline-5-carbonyl) -Nδ-benzo
Synthesis of yl-L-ornithine methyl ester

Reference Example 6: A solution of phenol (150 mg) in 30% hydrogen bromide-acetic acid (2 ml). Of the compound (140
mg) and 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 to obtain the desired product (50 mg).

¹ H-NMR (CDCl ₃ , δ): 1.6
−2.1 (4H, m), 3.02 (2H, t, J = 8.
8Hz), 3.56 (2H, m), 3.62 (2H,
t, J = 8.8 Hz), 3.75 (3H, s), 4.7
9 (1H, m), 6.55 (1H, d, J = 7.8H
z), 6.86 (2H, m), 6.99 (1H, m),
7.1-7.6 (5H, m), 7.82 (2H, m)

[0067]

Embodiment 5Nα- [1-[(2,4-diamino-6-pteridini)
Ru) methyl] -indoline-5-carbonyl] -Nδ-
Synthesis of benzoyl-L-ornithine methyl ester

Reference Example 7 (50 mg) and 6-bromomethyl-2,4-diaminopteridine hydrobromide isopropanol adduct (44 mg) were suspended in dimethylacetamide (1 ml), and the mixture was stirred at 50 to 55 ° C for 4 hours. After cooling, triethylamine (22 mg) was added to the reaction mixture.
Was added and stirred, and then extracted with chloroform. After drying the chloroform layer with sodium sulfate,
The solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel chromatography, and chloroform:
Using a mixed solvent of methanol = 10: 1, the desired product (34 m
g) was obtained.

¹ H-NMR (CDCl ₃ , δ): 1.6
-2.2 (4H, m), 3.07 (2H, t, J = 8.
8Hz), 3.50 (2H, m), 3.56 (2H,
t, J = 8.8 Hz), 3.79 (3H, s), 4.5
3 (2H, s), 4.76 (1H, s), 6.52 (1
H, d, J = 8.3 Hz), 7.19 (1H, d, J =
7.6 Hz), 7.45 (3H, m), 7.62 (2
H, m), 7.80 (2H, m), 8.76 (1H,
s)

[0070]

Embodiment 6Nα- [1-[(2,4-diamino-6-pteridini)
Ru) methyl] -indoline-5-carbonyl] -Nδ-
Synthesis of benzoyl-L-ornithine (compound 2)

Embodiment 5 FIG. (34 mg) was dissolved in ethanol (5 ml), a 1N aqueous sodium hydroxide solution (0.1 ml) was added, and the mixture was stirred at 35 ° C. for 4.5 hours. After stirring at 25 ° C. for 20 hours, water (1
ml), 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), and pH was adjusted to pH = 1 with 1N hydrochloric acid.
It was adjusted to 3.7 and left in the refrigerator for 2 hours. The deposited precipitate was collected by filtration to obtain the desired product (26 mg).

¹ H-NMR (DMSO-d ₆ , δ):
1.64 (2H, m), 1.83 (2H, m), 2.9
8 (2H, t, J = 8.3 Hz), 3.57 (2H,
t, J = 8.3 Hz), 4.36 (1H, m), 4.5
3 (2H, s), 6.64 (2H, m), 7.45 (3
H, m), 7.59 (2H, m), 7.82 (2H,
m), 8.12 (1H, d, J = 8.6 Hz), 8.4
3 (1H, m), 8.69 (1H, s) mp; 179-183 ° C (dec.)

[0073]

Reference Example 8 Nδ-phthaloyl-Nα-carbobenzoxy-ornichi
Synthesis of methyl ester

To a solution of Nα-carbobenzoxy-L-ornithine (2.0 g) in dichloromethane (70 ml) was added phthalic anhydride (2.45 g), followed by water (70 ml).
l) and potassium carbonate (1.12 g) were added and stirred at room temperature for 15 hours. The reaction solution was concentrated under reduced pressure to 60 ml and
The pH was adjusted to 3 with N-hydrochloric acid, and the deposited precipitate was collected by filtration and dried under vacuum. The resulting white solid was washed with low-moisture methanol (8
0 ml), and the solution is cooled to -30 ° C.
Stirred for 0 minutes. Then, at the same temperature, thionyl chloride (2 ml)
Was slowly added dropwise. The reaction solution was slowly returned to room temperature, and further refluxed for 2 hours. The solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel chromatography, and chlororum: methanol = 100:
Using 1 to give the desired product (2.16 g).

¹ H-NMR (CDCl ₃ , δ): 1.6
-2.0 (4H, m), 3.69 (5H, m), 4.2
0 (1H, m), 5.06 (2H, s), 5.70 (1
H, m), 7.29 (5H, m), 7.66 (2H,
m), 7.81 (2H, m)

[0076]

Reference Example 9 Synthesis of Nδ-phthaloyl-ornithine methyl ester

Reference Example 8 10% palladium-carbon (500 mg) was added to a methanol solution (100 ml) of the compound of formula (2.16 g), and the mixture was added at room temperature under a hydrogen atmosphere at room temperature.
Stirred for hours. Palladium-carbon was removed by filtration using Celite, and the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography, and chloroform: methanol = 100: 3 was used as the elution solvent to obtain the desired product (22
3 mg).

¹ H-NMR (CDCl ₃ , δ): 1.7
−2.1 (4H, m), 3.75 (2H, m), 3.8
3 (3H, s), 7.76 (2H, m), 7.84 (2
H, m)

[0079]

Reference Example 10 Nα- (1-carbobenzoxyindoline-5-carbo
Nyl) -Nδ-phthaloyl-ornithine methyl ester
Synthesis of

Thionyl chloride (2.5 ml) was added to 1-carbobenzoxyindoline-5-carboxylic acid (297 mg) to form a suspension. A catalytic amount of dimethylformamide was further added to the suspension, and the mixture was cooled to room temperature. And stirred for 2 hours. Next, the reaction solution was concentrated to dryness under reduced pressure. The obtained solid was dissolved in dichloromethane (7 ml), and this solution was added to Reference Example 9. (250 mg) and potassium carbonate (640 mg) were added, and water (7 ml) was further added, followed by vigorous stirring at room temperature for 12 hours. Next, the reaction solution was poured into water and extracted with chloroform.
-Washed with 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 (330 mg) was obtained using chloroform: methanol = 100: 3 as the elution solvent.

¹ H-NMR (CDCl ₃ , δ): 1.6
−2.1 (4H, m), 3.12 (2H, t, J = 8.
8Hz), 3.72 (2H, m), 3.76 (3H,
s), 4.08 (2H, t, J = 8.8 Hz), 4.8
4 (1H, m), 5.27 (2H, s), 6.80 (1
H, d, J = 7.8 Hz), 7.1-7.5 (6H,
m), 7.5-7.9 (6H, m)

[0082]

Reference Example 11 Nα- (indoline-5-carbonyl) -Nδ-phthalo
Synthesis of yl-ornithine methyl ester

Reference Example 10: A solution of phenol (300 mg) in 30% hydrogen bromide-acetic acid (8 ml). Compound (33)
0 mg) and 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 to obtain the desired product (147 mg).

¹ H-NMR (CDCl ₃ , δ): 1.6
−2.1 (4H, m), 3.04 (2H, t, J = 8.
3Hz), 3.62 (2H, t, J = 8.3Hz),
3.72 (2H, m), 3.75 (3H, s), 4.8
6 (1H, m), 6.5-6.7 (2H, m), 7.5
2 (2H, m), 7.68 (2H, m), 7.82 (2
H, m)

[0085]

Embodiment 7Nα- [1-[(2,4-diamino-6-pteridini)
Ru) methyl] -indoline-5-carbonyl] -Nδ-
Synthesis of phthaloyl-ornithine methyl ester

Reference Example 11 Compound (146 mg) and 6
-Bromomethyl-2,4-diaminopteridine hydrobromide isopropanol adduct (115 mg) was suspended in dimethylacetamide (1.0 ml) 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, 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 (140 mg). I got

¹ H-NMR (CDCl ₃ , δ): 1.7
-2.2 (4H, m), 3.06 (2H, t, J = 8.
3Hz), 3.56 (2H, t, J = 8.3Hz),
3.72 (2H, m), 3.76 (3H, s), 4.5
3 (2H, s), 4.88 (1H, m), 6.45-
6.62 (2H, m), 7.56 (2H, m), 7.7
1 (2H, m), 7.84 (2H, m), 8.82 (1
H, s)

[0088]

Embodiment 8Nα- [1-[(2,4-diamino-6-pteridini)
Ru) methyl] -indoline-5-carbonyl] -Nδ-
Synthesis of Hemiphthaloyl-Ornithine (Compound 3)

Embodiment 7 FIG. Compound (140 mg) in 2N
-Suspended in aqueous sodium hydroxide solution (10 ml), 3
Stirred at 0 ° C. for 12 hours. The reaction solution was dried under reduced pressure, and the obtained yellow solid was dissolved in water (5 ml).
-Adjusted to pH = 3.7 with hydrochloric acid 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% -aqueous ammonia = 5: 4: 1 as an elution solvent.

¹ H-NMR (DMSO-d ₆ , δ):
1.5-2.1 (4H, m), 3.14 (2H, t, J
= 8.3 Hz), 3.58 (2H, t, = 8.3H)
z), 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 ° C (dec.)

[0091]

Reference Example 12 Nδ- (3-methoxycarbonylbenzoyl) -Nα-
Synthesis of carbobenzoxy-ornithine methyl ester

To a solution of Nα-carbobenzoxy-L-ornithine (2.4 g) in dichloromethane (40 ml) was added isophthalic acid monomethyl ester chloride (2.1 g), followed by water (40 ml) and potassium carbonate (2.4).
g) was added and the mixture was stirred at room temperature for 15 hours. The reaction solution was concentrated under reduced pressure to 30 ml, adjusted to pH = 3 with 1N-hydrochloric acid, and the deposited precipitate was collected by filtration and dried in vacuo. The obtained white solid was dissolved in low-moisture methanol (100 ml), and the solution was cooled to -30 ° C and stirred for 10 minutes. Next, thionyl chloride (3 ml) was slowly added dropwise at the same temperature. The reaction solution was slowly returned to room temperature, and further refluxed for 2 hours. The solvent was distilled off under reduced pressure, the obtained residue was subjected to silica gel chromatography, and chloroform: methanol = 100: 1 was used as the elution solvent to obtain the desired product (600 m
g) was obtained.

¹ H-NMR (CDCl ₃ , δ): 1.6
-2.0 (4H, m), 3.46 (2H, m), 3.7
0 (3H, s), 3.89 (3H, s), 4.34 (1
H, m), 5.08 (2H, s), 5.88 (1H,
d, J = 7.8 Hz), 7.31 (5H, s), 7.4
5 (1H, m), 8.05 (2H, m), 8.41 (1
H, s)

[0094]

Reference Example 13 Nδ- (3-methoxycarbonylbenzoyl) -orni
Synthesis of tin methyl ester

Reference Example 12 10% palladium-carbon (100 mg) was added to a methanol solution (100 ml) of the compound (600 mg) of the above (2), and then added at room temperature under a hydrogen atmosphere.
Stirred for 0 hours. Palladium-carbon was removed by filtration using Celite, and the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography, and chloroform: methanol = 100: 3 was used as the elution solvent to give the desired product (3
60 mg).

¹ H-NMR (CDCl ₃ , δ): 1.6
-2.0 (4H, m), 3.50 (3H, m), 3.7
2 (3H, s), 3.92 (3H, s), 7.49 (1
H, t, J = 7.8 Hz), 7.63 (1H, m),
8.0-8.2 (2H, m), 8.43 (1H, s)

[0097]

Reference Example 14 Nα- (1-carbobenzoxyindoline-5-carbo
Nyl) -Nδ- (3-methoxycarbonylbenzoyl)
-Synthesis of ornithine methyl ester

Thionyl chloride (5 ml) was added to 1-carbobenzoxyindoline-5-carboxylic acid (350 mg) to form a suspension, and a catalytic amount of dimethylformamide was added to the suspension. Stirred for hours. Next, the reaction solution was concentrated to dryness under reduced pressure. The obtained solid was dissolved in dichloromethane (7 ml). (360 mg) and potassium carbonate (650 mg) were added, and water (7 ml) was further added, followed by vigorous stirring at room temperature for 12 hours. Next, the reaction solution was poured into water and extracted with chloroform.
Washed with 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 (390 mg) was obtained using chloroform: methanol = 100: 3 as the elution solvent.

¹ H-NMR (CDCl ₃ , δ): 1.6
−2.1 (4H, m), 3.08 (2H, t, J = 8.
3Hz), 3.52 (2H, m), 3.75 (3H,
s), 3.88 (3H, s), 4.06 (2H, t, J
= 8.3 Hz), 4.78 (1H, m), 5.26 (2
H, bs), 7.16 (1H, d, J = 7.3 Hz),
7.2-7.6 (7H, m), 7.65 (2H, m),
7.9-8.1 (2H, m), 8.43 (1H, s)

[0100]

Reference Example 15 Nα- (indoline-5-carbonyl) -Nδ- (3-
Methoxycarbonylbenzoyl) -ornithine methyl
Steal Synthesis

Reference Example 14: A solution of anisole (0.5 g) in 30% hydrogen bromide acetic acid (6 ml). Compound (390m
g) was added and 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. The obtained white solid was treated with n-hexane-chloroform-
Recrystallization from methanol gave the desired product (192 mg).

¹ H-NMR (CDCl ₃ , δ): 1.7
−2.1 (4H, m), 3.05 (2H, t, J = 8.
3Hz), 3.53 (2H, m), 3.67 (2H,
t, J = 8.3 Hz), 3.75 (3H, s), 3.8
9 (3H, s), 4.75 (1H, m), 6.73 (1
H, d, J = 7.8 Hz), 7.16 (1H, d, J =
7.8 Hz), 7.47 (1H, m), 7.55 (2
H, m), 8.09 (2H, m), 8.45 (1H,
s)

[0103]

Example 9 Nα- [1 - [(2,4- diamino-6-pteridinyl) methyl] - indol down-5-carbonyl] -Enuderuta-
(3-methoxycarbonylbenzoyl) -ornithinemeth
Synthesis of tyl ester

Reference Example 15 Compound (192 mg) and 6
-Bromomethyl-2,4-diaminopteridine hydrobromide (142 mg) was added to dimethylacetamide (2.5 m
l) and stirred at 50-55 ° C for 4 hours. After cooling, water (5 ml) containing triethylamine (43 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 to obtain the desired compound (110 m
g) was obtained.

¹ H-NMR (CDCl ₃ + CD ₃ OD,
δ): 1.7-2.1 (4H, m), 3.06 (2H,
t, J = 8.3 Hz), 3.55 (4H, m), 3.7
8 (3H, s), 3.93 (3H, s), 4.52 (2
H, s), 4.79 (1H, m), 6.51 (1H,
d, J = 7.8 Hz), 7.05 (1H, d, J = 7.
8 Hz), 7.4-7.7 (3H, m), 8.0-8.
2 (2H, m), 8.45 (1H, m), 8.77 (1
H, s)

[0106]

Embodiment 10Nα- [1-[(2,4-diamino-6-pteridini)
Ru) methyl] -indoline-5-carbonyl] -Nδ-
Synthesis of isophthaloyl-ornithine

Embodiment 9 FIG. (110 mg) was dissolved in ethanol (14 ml), 1N-aqueous sodium hydroxide solution (0.48 ml) was added, and the mixture was added at 35 ° C. and 4.5.
Stirred for 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 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 allowed to stand in a refrigerator for 2 hours.
The deposited precipitate was collected by filtration to obtain the desired product (78 mg).

¹ H-NMR (DMSO-d ₆ , δ):
1.5-2.0 (4H, m), 3.00 (2H, t, J
= 8.8 Hz), 3.32 (2H, m), 3.59 (2
H, t, J = 8.8 Hz), 4.40 (1H, m),
4.56 (2H, s), 6.69 (1H, d, J = 8.
3Hz), 7.56 (1H, m), 7.63 (2H,
m), 8.0-8.2 (2H, m), 8.44 (1H,
s), 8.65 (1H, m), 8.73 (1H, s)

[0109]

Reference Example 16 Nα- (4-methoxycarbonylbenzoyl) -Nα-
Synthesis of carbobenzoxy-ornithine methyl ester

To a solution of Nα-carbobenzoxy-L-ornithine (2.0 g) in dichloromethane (60 ml) was added terephthalic acid monomethyl ester chloride (3.0 g), and then water (60 ml) and potassium carbonate (4.8).
g) was added and the mixture was stirred at room temperature for 15 hours. The reaction solution was concentrated under reduced pressure to 50 ml, adjusted to pH = 3 with 1N-hydrochloric acid, and the deposited precipitate was collected by filtration and dried in vacuo. The obtained white solid was dissolved in low-moisture methanol (100 ml), and the solution was cooled to −30 ° C. and stirred for 10 minutes. Next, thionyl chloride (3 ml) was slowly added dropwise at the same temperature. The reaction solution was slowly returned to room temperature, and further refluxed for 2 hours. The solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel chromatography, and chloroform: methanol = 100: 1 was used as the elution solvent to obtain the desired product (710 m
g) was obtained.

¹ H-NMR (CDCl ₃ , δ): 1.6
−2.1 (4H, m), 3.51 (2H, m), 3.7
4 (3H, s), 3.94 (3H, s), 4.43 (1
H, m), 5.11 (2H, s), 5.53 (1H,
m), 6.63 (1H, bs), 7.34 (5H,
s), 7.83 (2H, d, J = 8.8 Hz), 8.0
8 (2H, d, J = 8.8 Hz)

[0112]

Reference Example 17 Nδ- (4-methoxycarbonylbenzoyl) -orni
Synthesis of tin methyl ester

Reference Example 16 10% palladium on carbon (100 mg) was added to a methanol solution (100 ml) of the compound (710 mg), and then the mixture was added at room temperature in a hydrogen atmosphere at room temperature.
Stirred for hours. Palladium monocarbon was removed by filtration using Celite, and the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography, and chloroform: methanol = 100: 3 was used as the elution solvent to obtain the desired product (41).
0 mg).

¹ H-NMR (CDCl ₃ , δ): 1.6
−2.1 (4H, m), 3.49 (3H, m), 3.7
3 (3H, s), 3.94 (3H, s), 7.20 (1
H, m), 7.84 (2H, d, J = 8.3 Hz),
8.09 (2H, d, J = 8.3 Hz)

[0115]

Reference Example 18 Nα- (1-carbobenzoxyindoline-5-carbo
Nyl) -Nδ- (4-methoxycarbonylbenzoyl)
-Synthesis of ornithine methyl ester

Thionyl chloride (2.5 ml) was added to 1-carbobenzoxyindoline-5-carboxylic acid (260 mg) to form a suspension. A catalytic amount of dimethylformamide was further added to the suspension, and the mixture was brought to room temperature. And stirred for 2 hours. Next, the reaction solution was concentrated to dryness under reduced pressure. The obtained solid was dissolved in dichloromethane (6 ml). (245 mg) and potassium carbonate (812 mg) were added, and water (6 ml) was further added, followed by vigorous stirring at room temperature for 12 hours. Next, the reaction solution was poured into water and extracted with chloroform.
Washed with N-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 (310 mg) was obtained using chloroform: methanol 100: 3 as the elution solvent.

¹ H-NMR (CDCl ₃ , δ): 1.7
-2.2 (4H, m), 3.16 (2H, t, J = 8.
3Hz), 3.61 (1H, m), 3.79 (3H,
s), 3.94 (3H, s), 4.11 (2H, t, J
= 8.3 Hz), 4.83 (1H, m), 5.29 (2
H, s), 6.89 (1H, d, J = 7.3 Hz),
7.02 (1H, m), 7.40 (5H, m), 7.6
6 (2H, d, J = 7.3 Hz), 7.90 (2H,
d, J = 8.8 Hz), 8.08 (2H, d, J = 8.
8Hz)

[0118]

Reference Example 19 Nα- (indoline-5-carbonyl) -Nδ- (4-
Methoxycarbonylbenzoyl) -ornithine methyl
Steal Synthesis

Reference Example 18 A solution of phenol (1.0 g) in 30% hydrogen bromide-acetic acid (10 ml) was used. Compound (40)
0 mg) and 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. This suspension was washed with a saturated aqueous solution of sodium hydrogen carbonate, and the desired 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 m
g) was obtained.

¹ H = NMR (CDCl ₃ , δ): 1.6
−2.1 (4H, m), 3.05 (2H, t, J = 8.
3Hz), 3.48 (2H, m), 3.62 (2H,
t, J = 8.3 Hz), 3.76 (3H, s), 3.9
4 (3H, s), 4.75 (1H, m), 6.57 (1
H, d, J = 7.8 Hz), 7.19 (1H, d, J =
7.8 Hz), 7.55 (2H, m), 7.89 (2
H, d, J = 8.3 Hz), 8.07 (2H, d, J =
8.3Hz)

[0121]

Embodiment 11Nα- [1-[(2,4-diamino-6-pteridini)
Ru) methyl] indoline-5-carbonyl] -Nδ-
(4-methoxycarbonylbenzoyl) -ornithine
Synthesis of tyl ester

Reference Example 19 Compound (140 mg) and 6
-Bromomethyl-2,4-diaminopteridine hydrobromide (105 mg) was added to dimethylacetamide (2.0 m
l) and stirred at 50-55 ° C for 4 hours. After cooling, water (4 ml) containing triethylamine (32 mg) was added to the reaction solution, and the mixture was stirred, and then the desired product was extracted with chloroform. After drying the chloroform layer with sodium sulfate,
The solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel chromatography, and chloroform:
Using a mixed solvent of methanol = 10: 1, the target compound (180
mg).

¹ H-NMR (CDCl ₃ , δ): 1.6
−2.1 (4H, m), 3.08 (2H, t, 8.8H
z), 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.
3 Hz), 6.79 (1H, d, J = 6.8 Hz),
7.17 (1H, m), 7.59 (2H, m), 7.9
2 (2H, d, J = 8.3 Hz), 8.09 (2H,
d, J = 8.3 Hz), 8, 81 (1H, s)

[0124]

Embodiment 12Nα- [1-[(2,4-diamino-6-pteridini)
L) methyl] indoline-5-carbonyl] -Nδ-te
Synthesis of phthaloyl-ornithine

Embodiment 11 FIG. (150 mg) was dissolved in ethanol (24 ml), a 1N aqueous sodium hydroxide solution (0.8 ml) was added, and the mixture was added at 35 ° C. and 4.5.
Stirred for 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 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 allowed to stand in a refrigerator for 2 hours.
The deposited precipitate was collected by filtration to obtain the desired product (109 mg).

¹ H-NMR (CDCl ₃ , δ): 1.5
−2.0 (4H, m), 3.00 (2H, t, J = 8.
8Hz), 3.30 (2H, m), 3.58 (2H,
t, J = 8.8 Hz), 4.36 (1H, m), 4.5
4 (2H, s), 6.68 (1H, d, J = 8.8H)
z), 7.62 (2H, m), 7.91 (2H, d, J
= 8.3 Hz), 7.99 (2H, d, J = 8.3H)
z), 8.10 (1H, d, J = 7.8 Hz), 8.6
1 (1H, m), 8.71 (1H, s) mp; 215
-220 ° C (dec.)

[0127]

Reference Example 20 Nt-butoxycarbonyl-γ-anilide-L-glu
Synthesis of α-benzyl tamate

A solution of Nt-butoxycarbonyl-L-glutamic acid α-benzyl ester (2.2 g) and triethylamine (0.92 ml) in tetrahydrofuran (8
butylcarbonate (0.8 ml) in tetrahydrofuran (2 m
l) was added and stirred for 30 minutes. Then, aniline (0.5
ml) and stirred for 1 hour. The temperature was gradually returned to room temperature, and the mixture was further stirred for 20 hours. The solvent was distilled off under reduced pressure, and the obtained residue was dissolved in chloroform. The chloroform layer was washed with a saturated aqueous solution of sodium hydrogen carbonate, dried over sodium sulfate, and then concentrated under reduced pressure. The obtained residue was subjected to silica gel chromatography, and the desired product (1.7 g) was obtained using chloroform: methanol = 99: 1 as an elution solvent.

¹ H = NMR (CDCl ₃ , δ): 1.4
4 (9H, s), 1.79-1.97 (1H, m),
2.27-1.40 (2H, m), 4.32-4.44
(1H, m), 5.16 (2H, s), 5.22-5.
38 (1H, m), 7.08 (1H, t, J = 7.3H
z), 7.26-7.33 (7H, m), 7.55 (2
H, d, J = 7.8 Hz), 8.42 (1H, s)

[0130]

[Reference Example 21] γ-anilide-L-glutamic acid α-benzyl ester
Synthesis of

Reference Example 20 (1.7 g) was dissolved in trifluoroacetic acid (8 ml) under ice cooling and stirred for 30 minutes. The solvent was distilled off under reduced pressure, and the residue was dissolved in chloroform. The chloroform layer was washed with a saturated aqueous solution of sodium bicarbonate and then dried over sodium sulfate. The solvent was distilled off under reduced pressure to obtain the desired product (1.3 g).

¹ H-NMR (CDCl ₃ , δ): 1.8
6-2.00 (1H, m), 2.17-2.38 (1
H, m), 2.42-2.53 (2H, m), 3.53
-3.60 (1H, m), 5.15 (2H, s), 7.
07 (1H, t, J = 7.3 Hz), 7.29-7.3
5 (7H, m), 7.48 (2H, d, J = 7.8H
z), 8.27 (1H, bs)

[0133]

Reference Example 22 N- (1-carbobenzoxyindoline-5-carboni
L) -γ-anilide-L-glutamic acid α-benzyl ester
Steal Synthesis

Thionyl chloride (2 ml) was added to 1-carbobenzoxyindoline-5-carboxylic acid (419 mg), 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 is dissolved in dichloromethane (2 ml).
And dissolved in this solution. Of the compound (400
mg) and triethylamine (0.21 ml) in methylene chloride (2 ml) were added under ice-cooling and a nitrogen atmosphere, and the mixture was stirred overnight. The reaction solution was washed sequentially with 1N hydrochloric acid, a saturated aqueous solution of sodium hydrogen carbonate and water, dried over sodium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography, and the desired product (317 m
g) was obtained.

¹ H-NMR (CDCl ₃ , δ): 2.1
0-2.20 (1H, m), 2.29-2.51 (3
H, m), 3.05 (2H, t, J = 8.8 Hz),
4.06 (2H, t, J = 8.8 Hz), 4.76 −
4.88 (1H, m), 5.18 (2H, s), 5.2
8 (2H, bs), 7.05 (1H, t, J = 7.3H)
z), 7.18 (1H, d, J = 7.8 Hz), 7.2
3-7.30 (2H, m), 7.33 (5H, s),
7.39 (5H, s), 7.43-7.66 (4H,
m), 7.80-7.84 (1H, m), 8.57 (1
H, bs)

[0136]

Reference Example 23 N- (1-carbobenzoxyindoline-5-carboni
B) Synthesis of -γ-anilide-L-glutamic acid

Reference Example 22 Compound (580 mg) in a mixed solvent of chloroform: methanol = 1: 2 (30 m
l) and dissolved in a 1N aqueous sodium hydroxide solution (0.9
8 ml) and stirred at room temperature overnight. Water bath temperature 3
The solvent was distilled off under reduced pressure while maintaining the temperature at 0 ° C or lower. The obtained residue was dissolved in water, made acidic with 1N-hydrochloric acid, and extracted with chloroform. After the chloroform layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure to obtain the desired product (413 mg).

¹ H = NMR (CDCl ₃ , δ): 2.1
7-2.29 (2H, m), 2.52-2.56 (2
H, m), 2.79 (2H, t, J = 7.8 Hz),
3.81 (2H, t, J = 7.8 Hz), 4.51-
4.61 (1H, m), 5.17 (2H, s), 6.9
4 (1H, t, J = 7.3 Hz), 7.13 (2H,
t, J = 7.8 Hz), 7.34 (5H, s), 7.4
3-7.65 (5H, m), 8.03 (1H, bs),
9.02 (1H, s)

[0139]

Reference Example 24 N- (1-carbobenzoxyindoline-5-carbonyl) -γ-anilide -α-methyles L-glutamate
Tell synthesis

Reference Example 23 Was dissolved in dry methanol (20 ml), trimethylsilyldiazomethane (2 ml) was added, and the mixture was stirred for 10 hours. Further, trimethylsilyldiazomethane (3 ml) was added,
Stirred for 20 hours. After evaporating the solvent under reduced pressure, the obtained residue was subjected to silica gel chromatography, and the desired product (252 mg) was obtained using chloroform as an elution solvent, and then using chloroform: methanol = 199: 1.

¹ H-NMR (CDCl ₃ , δ): 2.0
6-2.21 (1H, m), 2.31-2.56 (3
H, m), 3.08 (2H, t, J = 8.8 Hz),
3.77 (3H, s), 4.08 (2H, t, J = 8.
8 Hz), 4.77-4.87 (1H, m), 5.28
(2H, s), 7.07 (1H, t, J = 7.3H
z), 7.15 (1H, d, J = 7.8 Hz), 7.2
8 (2H, t, J = 7.8 Hz), 7.37-7.43
(5H, m), 7.58 (2H, d, J = 7.8H
z), 7.62-7.68 (2H, m), 7.86 (1
H, bs), 8.67 (1H, bs)

[0142]

Reference Example 25 N- (Indoline-5-carbonyl) -γ-anilide-
Synthesis of L-glutamic acid α-methyl ester

Reference Example 24. 10% palladium-carbon (50 mg) was added to a methanol solution (10 ml) of the compound (250 mg), and the mixture was stirred at room temperature under a hydrogen atmosphere for 15 hours. The palladium-carbon was removed by filtration using Celite, and the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography, and the desired product (172 mg) was obtained using chloroform: methanol = 99: 1, and then chloroform: methanol = 19: 1 as an elution solvent.

¹ H = NMR (CDCl ₃ , δ): 1.9
9-2.13 (1H, m), 2.36-2.55 (3
H, m), 3.03 (2H, t, J = 8.8 Hz),
3.64 (2H, t, J = 8.8 Hz), 3.77 (3
H, s), 4.08 (1H, bs), 4.79-4.8.
8 (1H, m), 6.55 (1H, d, J = 8.3H
z), 6.92 (1H, d, J = 7.3 Hz), 7.0
8 (1H, t, J = 7.3 Hz), 7.30 (2H,
t, J = 7.8 Hz), 7.53-7.65 (4H,
m), 9.01 (1H, bs)

[0145]

Example 13 N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] -indoline-5-carbonyl] -γ-anili
Synthesis of do-L-glutamic acid α-methyl ester

Reference Example 25 Compound (169 mg) and 6
-Bromomethyl-2,4-diaminopteridine hydrobromide isopropanol adduct (223 mg) was suspended in dimethylacetamide (5 ml) and stirred at room temperature for 24 hours. The reaction solution was subjected to silica gel chromatography, and ethyl acetate and then chloroform were used as elution solvents.
The desired product (82 mg) was obtained using methanol = 19: 1.

¹ H-NMR (CDCl ₃ , δ): 2.0
1-2.09 (1H, m), 2.39-2.48 (3
H, m), 3.04 (2H, t, J = 8.8 Hz),
3.58 (2H, t, J = 8.8 Hz), 3.78 (3
H, s), 4.53 (2H, s), 4.79-4.89.
(1H, m), 6.48 (1H, d, J = 7.8H
z), 6.87 (1H, d, J = 7.8 Hz), 7.0
8 (1H, t, J = 7.8 Hz), 7.26-7.32
(2H, m), 7.59-7.64 (4H, m), 8.
81 (1H, s)

[0148]

Example 14 N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] -indoline -5-carbonyl] -γ-anili
Synthesis of do-L-glutamic acid

Embodiment 13 FIG. Is dissolved in a mixed solvent of ethanol: water = 2: 1 (15 ml),
A 1N aqueous solution of sodium hydroxide (0.15 ml) was added, and the mixture was stirred at room temperature for 60 hours. The solvent was distilled off under reduced pressure while maintaining the temperature of the water bath at 30 ° C. or lower. The obtained residue was dissolved in a saturated aqueous solution of sodium hydrogen carbonate, and the pH was adjusted to 3.7 using 1N hydrochloric acid. The precipitated orange precipitate was collected by filtration to obtain the desired product (61 mg).

¹ H = NMR (DMSO-d ₆ , δ):
1.99-2.23 (2H, m), 2.42-2.51
(2H, m), 2.98 (2H, t, J = 7.8H
z), 3.59 (2H, t, J = 8.3 Hz), 4.3
2-4.43 (1H, m), 4.56 (2H, s),
6.70 (1H, d, J = 7.8 Hz), 6.90 (2
H, bs), 7.00 (1H, t, J = 7.8 Hz),
7.26 (2H, t, J = 7.8 Hz), 7.55-
7.66 (4H, m), 7.72-7.85 (1H,
m), 7.89-7.97 (1H, m), 8.20 (1
H, d, J = 7.3 Hz), 8.75 (1H, s),
9.93 (1H, s)

[0151]

Reference Example 26 Nt-butoxycarbonyl-γ- (2-methoxycal
Bonylanilide) -L-glutamic acid α-benzyles
Tell synthesis

A solution of Nt-butoxycarbonyl-L-glutamic acid α-benzyl ester (496 mg) in tetrahydrofuran (5 ml) was ice-cooled under a nitrogen atmosphere.
N, N'-carbonyldiimidazole (262 mg) was added and the mixture was stirred for 1 hour. Subsequently, a solution of methyl o-aminobenzoate (0.19 ml) in tetrahydrofuran (2 m
After l) was added, the mixture was stirred for 5 hours. Gradually return to room temperature,
The mixture was further stirred for 48 hours. The solvent was distilled off under reduced pressure, and the obtained residue was subjected to silica gel chromatography, and using chloroform as an eluting solvent, the desired product (273 mg) was obtained.
I got

¹ H-NMR (CDCl ₃ , δ): 1.4
1 (9H, s), 2.04-2.18 (1H, m),
2.20-2.32 (1H, m), 2.48-2.57
(2H, m), 3.92 (3H, s), 4.34-4.
47 (1H, m), 5.18 (2H, s), 5.19-
5.27 (1H, m), 7.08 (1H, t, J = 7.
8Hz), 7.34 (5H, s), 7.53 (1H,
t, J = 7.3 Hz), 8.02 (1H, d, J = 7.
8 Hz), 8.68 (1H, d, J = 7.8 Hz), 1
1.06 (1H, s)

[0154]

Reference Example 27 γ- (2-methoxycarbonylanilide) -L-gluta
Synthesis of α-benzyl ester of formic acid

Reference Example 26 Compound (632 mg) was dissolved in trifluoroacetic acid (4.5 ml) under ice-cooling and stirred for 90 minutes. The solvent was distilled off under reduced pressure, and the residue was dissolved in chloroform. The chloroform layer was washed with a saturated aqueous solution of sodium hydrogen carbonate, dried over sodium sulfate, and then the solvent was distilled off under reduced pressure. The resulting residue was subjected to silica gel chromatography, and chloroform (chloroform: methanol = 97: 3) as an elution solvent was used to obtain the desired product (486 mg).

¹ H = NMR (CDCl ₃ , δ): 1.9
1-2.02 (1H, m), 2.20-2.30 (1
H, m), 2.55-2.64 (2H, m), 3.56
-3.62 (1H, m), 3.92 (3H, s), 5.
17 (2H, s), 7.07 (1H, t, J = 8.3H)
z), 7.35 (5H, s), 7.53 (1H, t, J
= 8.8 Hz), 8.02 (1H, d, J = 8.3H)
z), 8.69 (1H, d, J = 8.8 Hz), 11.
08 (1H, bs)

[0157]

Reference Example 28 Synthesis of 1-[(2,4-diamino-6-pteridinyl) methyl] -indoline-5- carboxylic acid

1-Carbobenzoxyindoline-5-carboxylic acid (87 mg) and 6-bromomethyl-2,4-diaminopteridine hydrobromide 1/2 isopropanol adduct (136 mg) were added to dimethylacetamide (2 m
1) and stirred at 55 ° C. for 4 hours. Water (20 ml) was added to the reaction solution, and the mixture was left overnight in a refrigerator. The precipitated solid was collected by filtration, dissolved in a small amount of a 1N aqueous solution of sodium hydroxide, and adjusted to pH = 6.5 with 1N-hydrochloric acid.
The precipitated brown precipitate was collected by filtration to obtain the desired product (56 mg).

¹ H-NMR (DMSO.d ₆ , δ):
3.01 (2H, t, J = 8.8 Hz), 3.64 (1
H, t, J = 8.8 Hz), 4.60 (2H, s),
6.68 (1H, d, J = 8.3 Hz), 7.16 (2
H, bs), 7.58 (1H, s), 7.65 (1H,
7. d, J = 8.3 Hz), 8.09 (1H, bs),
25 (1H, bs), 8.77 (1H, s)

[0160]

Example 15 N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] -indoline-5-carbonyl] -γ- (2-
Methoxycarbonylanilide) -L-glutamic acid α-
Synthesis of benzyl ester

Reference Example 28 Compound (134 mg) and 1
-Dicyclohexylcarbodiimide (123 mg) was added to a suspension of hydroxybenzotriazole (108 mg) in dimethylformamide (5 ml) under ice-cooling and a nitrogen atmosphere.
Dimethylformamide solution (1.5 ml)
Minutes. Next, Reference Example 27. Was added, and the mixture was gradually warmed to room temperature and stirred for 24 hours. The reaction solution was subjected to silica gel chromatography, and the desired product (29 mg) was obtained using ethyl acetate and then chloroform: methanol = 19: 1 as an elution solvent.

¹ H-NMR (CDCl ₃ , δ): 2.3
2-2.46 (2H, m), 2.46-2.70 (2
H, m), 2.96 (2H, t, J = 8.8 Hz),
3.49 (2H, t, J = 8.3 Hz), 3.89 (3
H, s), 4.46 (2H, s), 4.83-4.90.
(1H, m), 5.20 (2H, s), 6.40 (1
H, d, J = 8.3 Hz), 7.05 (1H, t, J =
8.3 Hz), 7.14 (1H, d, J = 7.8H)
z), 7.34 (5H, s), 7.41-7.59 (3
H, m), 7.96 (1H, d, J = 8.3 Hz),
8.64 (1H, d, J = 8.3 Hz), 8.77 (1
H, s)

[0163]

Example 16 N- [1 = [(2,4-diamino-6-pteridinyl)
Methyl] -indoline-5-carbonyl] -γ- (2-
Synthesis of (carboxyanilide) -L-glutamic acid

Embodiment 15 FIG. (28 mg) was suspended in methanol (2.5 ml), a 1N aqueous sodium hydroxide solution (0.41 ml) was added, and the mixture was stirred at 10 ° C. for 5 hours. The solvent was distilled off under reduced pressure while maintaining the temperature of the water bath at 30 ° C. or lower. The obtained residue was dissolved in a saturated aqueous solution of sodium hydrogen carbonate, and the pH was adjusted to 3.7 using 1N hydrochloric acid. The orange precipitate that precipitated was collected by filtration, and the desired product (21
mg).

¹ H-NMR (DMSO-d ₆ , δ):
1.95-2.25 (2H, m), 2.39-2.58
(2H, m), 2.98 (2H, t, J = 8.3H
z), 3.59 (2H, t, J = 8.3 Hz), 4.3
8-4.50 (1H, m), 4.57 (2H, s),
6.69 (1H, d, J = 8.3 Hz), 7.12 (1
H, t, J = 7.8 Hz), 7.24-7.35 (2
H, m), 7.51-7.64 (3H, m), 7.96.
(1H, d, J = 7.8 Hz), 8.04-8.39
(4H, m), 8.47 (1H, d, J = 7.8H
z), 8.78 (1H, s), 11.25 (2H, s)

[0166]

[Reference Example 29] N-t-butoxycarbonyl-L-glutamic acid α-meth
Synthesis of tyl γ-benzyl diester

After sodium hydrogen carbonate (2.5 g) was suspended in a solution of Nt-butoxycarbonyl-L-glutamic acid γ-benzyl ester (5.0 g) in dimethylformamide (75 ml), methyl iodide (10 g) was added. .5
A solution of 2 g) in dimethylformamide (75 ml) was added, and the mixture was stirred at room temperature for 24 hours. After the reaction solution was concentrated under reduced pressure, water (70 ml) was added, and the mixture was extracted with a 1: 1 solution of ethyl acetate: n-hexane. After the organic layer was washed with water 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 (5.2 g) was obtained using ethyl acetate: n-hexane = 1: 2 as an elution solvent.

¹ H = NMR (CDCl ₃ , δ): 1.4
3 (9H, s), 1.92-2.01 (1H, m),
2.11-2.37 (1H, m), 2.42-2.51
(2H, m), 3.73 (3H, s), 4.23-4.
40 (1H, m), 5.12 (2H, s), 7.35
(5H, s)

[0169]

Reference Example 30 Nt-butoxycarbonyl-L-glutamic acid α-meth
Synthesis of tyl ester

Reference Example 29 10% palladium-carbon (1.1 g) was added to a methanol solution (30 ml) of the compound (5.2 g), and the mixture was stirred under a hydrogen atmosphere at room temperature for 15 hours. The palladium-carbon was removed by filtration using Celite, and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in a saturated aqueous solution of sodium hydrogen carbonate and washed with chloroform. After separating the aqueous layer, pH = 4 using 5% citric acid.
And extracted with chloroform. After drying the chloroform layer with sodium sulfate, the solvent was distilled off under reduced pressure.
The desired product (3.9 g) was obtained.

¹ H-NMR (CDCl ₃ , δ): 1.4
4 (9H, s), 1.89-2.04 (1H, m),
2.09-2.27 (1H, m), 3.75 (3H,
s), 4.34 (1H, m), 5.17-5.21 (1
H, m), 9.38 (1H, bs)

[0172]

Reference Example 31 Nt-butoxycarbonyl-γ- (3-ethoxycal
Bonylanilide) -L-glutamic acid α-methyl ester
Synthesis

Reference Example 30 (1.6 g) and 1-
To a dichloromethane suspension (10 ml) of hydroxybenzotriazole (0.8 g) was added a dichloromethane solution (5 ml) of dicyclohexylcarbodiimide (1.5 g) under ice-cooling and a nitrogen atmosphere, followed by stirring for 30 minutes. Then
After adding ethyl m-aminobenzoate (1.5 g), the mixture was gradually returned to room temperature and stirred for 20 hours. The solvent was distilled off under reduced pressure, ethyl acetate was added to the obtained residue, and white insolubles were removed by filtration. The filtrate was concentrated under reduced pressure, and the obtained residue was subjected to silica gel chromatography to obtain the desired product (2.5 g) using chloroform as an elution solvent and then chloroform: methanol = 99: 1.

¹ H = NMR (CDCl ₃ , δ): 1.3
8 (3H, t, J = 7.1 Hz), 1.47 (9H,
s), 1.82 to 2.04 (1H, m), 2.20-
2.39 (1H, m), 2.45-2.51 (2H,
m), 3.74 (3H, s), 4.32-4.42 (1
H, m), 4.37 (2H, q, J = 7.1 Hz),
5.37 (1H, bd, J = 7.3 Hz), 7.40
(1H, t, J = 7.8 Hz), 7.78 (1H, d,
J = 7.8 Hz), 7.96 (1H, d, J = 9.3H)
z), 8.13 (1H, s), 8.83 (1H, bs)

[0175]

Reference Example 32 γ- (3-ethoxycarbonylanilide) -L-gluta
Synthesis of α-methyl ester of formic acid

Reference Example 31 (2.5 g) was dissolved in trifluoroacetic acid (15 ml) under ice cooling and stirred for 1 hour. The solvent was distilled off under reduced pressure, and the residue was dissolved in chloroform. The chloroform layer was washed with a saturated aqueous solution of sodium hydrogen carbonate, dried over sodium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography, and the desired product (1.6 g) was obtained using chloroform: methanol 19: 1 as an elution solvent.

¹ H-NMR (CDCl ₃ , δ): 1.3
9 (3H, t, J = 7.1 Hz), 1.74 (2H,
s), 1.82-1.99 (1H, m), 2.15
2.32 (1H, m), 2.52-2.62 (2H,
m), 3.54-3.60 (1H, m), 3.74 (3
H, s), 4.37 (2H, q, J = 7.1 Hz),
7.39 (1H, t, J = 7.8 Hz), 7.77 (1
H, d, J = 7.3 Hz), 7.94 (1H, d, J =
7.8 Hz), 7.99 (1H, s), 8.63 (1
H, bs)

[0178]

Reference Example 33 N- (1-carbobenzoxyindoline-5-carboni
) -Γ- (3-ethoxycarbonylanilide) -L-
Synthesis of glutamic acid α-methyl ester

Thionyl chloride (4 ml) was added to 1-carbobenzoxyindoline-5-carboxylic acid (612 mg), 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 is diluted with dichloromethane (4 ml)
In Reference Example 32. Compound (52)
9 mg) and a dichloromethane solution (4 ml) of triethylamine (0.36 ml) were added under ice cooling under a nitrogen atmosphere, and the mixture was stirred overnight. The reaction solution was washed sequentially with 2N-hydrochloric acid, a saturated aqueous solution of sodium hydrogen carbonate and water, dried over sodium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography, and the desired product (870 mg) was obtained using chloroform as the elution solvent and then using chloroform: methanol = 99: 1.

¹ H = NMR (CDCl ₃ , δ): 1.3
7 (3H, t, J = 7.1 Hz), 2.08-2.22
(1H, m), 2.36-2.57 (3H, m), 3.
06 (2H, t, J = 8.8 Hz), 3.77 (3H,
s), 4.07 (2H, t, J = 8.8 Hz), 4.3
0-4.40 (2H, q, J = 7.1 Hz), 4.78
-4.87 (1H, m), 5.28 (2H, bs),
7.13 (1H, d, J = 7.8 Hz), 7.30 −
7.43 (6H, m), 7.60-7.67 (2H,
m), 7.74 (1H, d, J = 7.8 Hz), 7.8
9 (1H, d, J = 7.8 Hz), 8.12 (1H, b
s), 8.96 (1H, bs)

[0181]

Reference Example 34 N- (Indoline-5-carbonyl) -γ- (3-ethoate
Xycarbonylanilide) -L-glutamic acid α-methyl
Synthesis of ester

Reference Example 33 10% palladium-carbon (0.17 g) was added to a methanol solution (11 ml) of the compound of formula (842 mg), and the mixture was added under hydrogen atmosphere at room temperature.
Stirred for hours. The palladium-carbon was removed by filtration using Celite, and the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography, and the desired product (448 mg) was obtained using chloroform as the elution solvent, and then using chloroform: methanol = 99: 1.

¹ H-NMR (CDCl ₃ , δ): 1.3
6 (3H, t, J = 7.1 Hz), 2.05-2.25
(1H, m), 2.25-2.48 (1H, m), 2.
48-2.59 (2H, m), 2.93 (2H, t, J
= 8.8 Hz), 3.57 (2H, J = 8.8 Hz),
3.71 (3H, s), 4.18 (1H, bs), 4.
34 (2H, q, J = 7.1 Hz), 4.73-4.8
4 (1H, m), 6.46 (1H, d, J = 8.8H)
z), 7.16 (1H, d, J = 7.8 Hz), 7.3
2 (1H, t, J = 7.8 Hz), 7.50-7.53
(2H, m), 7.73 (1H, d, J = 7.8H
z), 7.87 (1H, d, J = 7.3 Hz), 8.1
9 (1H, s), 9.39 (1H, bs)

[0184]

Example 17 N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] -indoline-5-carbonyl] -γ- (3-
Ethoxycarbonylanilide) -L-glutamic acid α-
Synthesis of methyl ester

Reference Example 34 Compound (448 mg) and 6
-Bromomethyl-2,4-diaminopteridine hydrobromide isopropanol adduct (587 mg) was suspended in dimethylacetamide (5 ml) and stirred at room temperature for 36 hours. The reaction solution was subjected to silica gel chromatography, and ethyl acetate and then chloroform were used as elution solvents.
The desired product (653 mg) was obtained using methanol = 9: 1.

¹ H-NMR (DMSO-d ₆ , δ):
1.34 (3H, t, J = 6.8 Hz), 1.96−
2.32 (2H, m), 2.32-2.59 (2H,
m), 3.00 (2H, t, J = 7.3 Hz), 3.5
6-3.66 (5H, m), 4.32 (2H, q, J =
6.5Hz), 4.40-4.56 (3H, m), 6.
66-6.71 (3H, bs), 7.39 (1H, t,
J = 7.8 Hz), 7.63-7.67 (3H, m),
7.84 (1H, d, J = 7.3 Hz), 8.22 (1
H, s), 8.32 (1H, d, J = 7.3 Hz),
8.73 (1H, s), 10.11 (1H, s)

[0187]

Example 18 N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] -indoline-5-carbonyl] -γ- (3-
Synthesis of (carboxyanilide) -L-glutamic acid

Embodiment 17 FIG. Was dissolved in methanol (10 ml), a 1N aqueous solution of sodium hydroxide (1.06 ml) was added, and the mixture was stirred at room temperature for 12 hours. Water (1 ml) was added, and the mixture was further stirred for 2 hours. The solvent was distilled off under reduced pressure while maintaining the temperature of the water bath at 30 ° C. or lower. The obtained residue was dissolved in a saturated aqueous solution of sodium hydrogen carbonate, and the pH was adjusted to 3.7 using 1N hydrochloric acid.
The deposited orange precipitate was collected by filtration to obtain the desired product (157 mg).

¹ H-NMR (DMSO-d ₆ , δ):
1.97-2.25 (2H, m), 2.42-2.58
(2H, m), 2.99 (2H, t, J = 8.8H
z), 3.59 (2H, t, J = 8.8 Hz), 4.3
3-4.45 (1H, m), 4.59 (2H, s),
6.71 (1H, d, J = 8.3 Hz), 6.93 (2
H, bs), 7.39 (1H, t, J = 7.8 Hz),
7.57-7.82 (5H, m), 7.88-8.04
(1H, m), 8.06-8.12 (1H, m), 8.
15-8.26 (2H, m), 8.75 (1H, s),
10.12 (1H, s)

[0190]

Reference Example 35 N-carbobenzoxy-γ-N ′, N′-dimethylamido
Synthesis of α-benzyl ester of do-L-glutamic acid

A solution of N-carbobenzoxy-L-glutamic acid α-benzyl ester (508 mg) and triethylamine (0.19 ml) in tetrahydrofuran (2 m
Ethyl chlorocarbonate (0.14 ml) was added to 1) under a nitrogen atmosphere at −20 ° C., followed by stirring for 30 minutes. Then, dimethyl hydrochloride (112 mg) and triethylamine (0.1
9 ml) of a dichloromethane solution (3 ml)
Stir for 1 hour. The temperature was gradually returned to room temperature, and the mixture was further stirred for 24 hours. The solvent was distilled off under reduced pressure, and the obtained residue was dissolved in chloroform. The chloroform layer was washed with a saturated sodium hydrogen carbonate solution and 1N-hydrochloric acid, dried over sodium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography, and chloroform: methanol = 49: 1 was used as the elution solvent to afford the desired product (4
53 mg).

¹ H-NMR (CDCl ₃ , δ): 1.9
7-2.37 (4H, m), 2.86 (3H, s),
2.90 (3H, s), 4.39-4.41 (1H,
m), 5.10 (2H, s), 5.17 (2H, s),
5.86-5.89 (1H, m), 7.34 (10H,
s)

[0193]

Reference Example 36 N-carbobenzoxy-γ-N ′, N′-dimethylamido
Synthesis of do-L-glutamic acid

Reference Example 35 To a methanol solution (5 ml) of the compound (450 mg) was added a 1N aqueous sodium hydroxide solution (1.15 ml), and the mixture was stirred at room temperature overnight. The solvent was distilled off under reduced pressure while maintaining the temperature of the water bath at 30 ° C. or lower. The obtained residue was dissolved in water, acidified with 1N-hydrochloric acid, and extracted with chloroform. After the chloroform layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure to obtain the desired product (341 mg).

¹ H-NMR (CDCl ₃ , δ): 1.9
3-2.06 (1H, m), 2.21-2.33 (1
H, m), 2.42-2.56 (1H, m), 2.76
-2.84 (1H, m), 2.98 (3H, s), 3.
00 (3H, s), 4.19-4.27 (1H, m),
5.09 (2H, s), 6.04-6.06 (1H,
m), 7.37 (10H, s)

[0196]

Reference Example 37 N-carbobenzoxy-γ-N ′, N′-dimethylami
Synthesis of do-L-glutamic acid α-methyl ester

Reference Example 36. Was dissolved in dry methanol (4 ml), trimethylsilyldiazomethane (5 ml) was added, and the mixture was stirred for 2 hours. The solvent was distilled off under reduced pressure to obtain the desired product (338 mg).

¹ H-NMR (CDCl ₃ , δ): 1.9
6-2.43 (4H, m), 2.92 (3H, s),
2.95 (3H, s), 3.74 (3H, s), 4.2
8-4.40 (1H, m), 5.10 (2H, s),
5.80 (1H, m), 7.35 (5H, s)

[0199]

Reference Example 38 γ-N ′, N′-dimethylamido-L-glutamic acid α
-Synthesis of methyl ester

Reference Example 37 After adding 10% palladium-carbon (70 mg) to a methanol solution (10 ml) of the compound (580 mg), the mixture was stirred at room temperature for 15 hours under a hydrogen atmosphere. The palladium-carbon was removed by filtration using Celite, and the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography, and chloroform: methanol = 19: 1 was used as the elution solvent to give the desired product (160 m
g) was obtained.

¹ H-NMR (CDCl ₃ , δ): 1.7
4-1.92 (1H, m), 2.06-2.23 (1
H, m), 2.47 (2H, t, J = 7.3 Hz),
2.95 (3H, s), 3.02 (3H, s), 3.4
9-3.56 (1H, m), 3.73 (3H, s)

[0202]

Reference Example 39 N- (1-carbobenzoxyindoline-5-carboni
) -Γ-N ', N'-dimethylamide-L-glutami
Of α-Methyl Ester

Thionyl chloride (2 ml) was added to 1-carbobenzoxyindoline-5-carboxylic acid (295 mg), 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 (2 ml). Of the compound (160
mg) and a dichloromethane solution (2.5 ml) of triethylamine (0.18 ml) were added under ice cooling under a nitrogen atmosphere, and the mixture was stirred overnight. The reaction solution was washed sequentially with 1N hydrochloric acid, a saturated aqueous solution of sodium hydrogen carbonate and water, dried over sodium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography, and chloroform was used as an elution solvent, and then chloroform: methanol = 99: 1.
Was used to obtain the desired product (359 mg).

¹ H-NMR (CDCl ₃ , δ): 2.2
2-2.32 (2H, m), 2.43-2.55 (2
H, m), 2.94 (3H, s), 2.98 (3H,
s), 3.15 (2H, t, J = 8, 8 Hz), 3.7
6 (3H, s), 4.10 (2H, t, J = 8.8H)
z), 4.58-4.68 (1H, m), 5, 28 (2
H, s), 7.36-7.41 (6H, m), 7.71.
(1H, s), 7.90 (1H, d, J = 5.9 Hz)

[0205]

Reference Example 40 N- (indoline-5-carbonyl) -γ-N ′, N ′
-Dimethylamide-L-glutamic acid α-methyl ester
Synthesis

Reference Example 39 10% palladium-carbon (7 ml) was added to a methanol solution (5 ml) of the compound (359 mg).
(0 mg), followed by stirring at room temperature for 15 hours under a hydrogen atmosphere. The palladium-carbon was removed by filtration using Celite, and the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel chromatography, and chloroform:
The desired product (131 mg) was obtained using methanol = 99: 1.

¹ H-NMR (CDCl ₃ , δ): 2.1
9-2.30 (2H, m), 2.44-2.53 (2
H, m), 2.93 (3H, s), 2.97 (3H,
s), 3.04 (2H, t, J = 8.3 Hz), 3.6.
2 (2H, t, J = 8.3 Hz), 3.75 (3H,
s), 4.14 (1H, bs), 4.60-4.70.
(1H, m), 6.56 (1H, d, J = 7.8H
z), 7.51-7.62 (3H, m)

[0208]

Example 19 N- [1-[(2,4-diamino-6-pteridinyl)]
Methyl] -indoline-5-carbonyl] -γ-N ′,
N'-dimethylamido-L-glutamic acid α-methyl ester
Steal Synthesis

Reference Example 40 Compound (131 mg) and 6
-Bromomethyl-2,4-diaminopteridine hydrobromide isopropanol adduct (233 mg) was suspended in dimethylacetamide (4 ml) and stirred at room temperature for 24 hours. The reaction solution was subjected to silica gel chromatography, and ethyl acetate and then chloroform were used as elution solvents.
The desired product (95 mg) was obtained using methanol = 9: 1.

¹ H-NMR (CDCl ₃ + DMSO-d
₆ , δ): 1.98-2.17 (2H, m), 2.42.
(2H, t, J = 6.8 Hz), 2.84 (3H,
s), 2.94 (3H, s), 3.00 (2H, t, J
= 8.3 Hz), 3.59 (2H, t, J = 8.3H)
z), 3.64 (3H, s), 4.30-4.42 (1
H, m), 4.55 (2H, s), 6.68 (1H,
d, J = 7.3 Hz), 7.60-7.64 (2H,
m), 8.34 (1H, d, J = 7.3 Hz), 8.7
1 (1H, s)

[0211]

Example 20 N- [1-[(2,4-diamino-6-pteridinyl)]
Methyl] -indoline-5-carbonyl] -γ-N ′,
Synthesis of N'-dimethylamido-L-glutamic acid

Embodiment 19 FIG. (70 mg) was dissolved in methanol (5 ml), a 1N aqueous sodium hydroxide solution (0.15 ml) was added, and the mixture was stirred at room temperature for 12 hours. Water (2 ml) was added, and the mixture was further stirred for 5 hours. The solvent was distilled off under reduced pressure while maintaining the temperature of the water bath at 30 ° C. or lower. The obtained residue was dissolved in a saturated aqueous solution of sodium hydrogen carbonate, and the pH was adjusted to 3.7 using 1N hydrochloric acid. The deposited orange precipitate was collected by filtration to obtain the desired product (16 mg).

¹ H-NMR (DMSO-d ₆ , δ):
1.96-2.08 (2H, m), 2.41 (2H,
t, J = 7.1 Hz), 2.82 (3H, s), 2.9
2 (3H, s), 3.01 (2H, t, J = 7.8H
z), 3.62 (2H, t, J = 7.8 Hz), 4.2
7-4.37 (1H, m), 4.62 (2H, s),
6.72 (1H, d, J = 8.8 Hz), 7.61-
7.64 (2H, m), 8.24 (1H, d, J = 6.
8Hz), 8.87 (1H, s), 12.46 (1H,
bs)

[0214]

[Reference Example 41]1-[(2,4-diamino-6-pteridinyl) methyl
L] 1,2,3,4-tetrahydro-6-quinolineka
Synthesis of rubonic acid

6-bromomethyl-2,4-diaminopteridine hydrobromide / isopropanol adduct (178
mg) and 1,2,3,4-tetrahydro-6-quinolinecarboxylic acid (55 mg) in dimethylacetamide (3 mg).
ml) and stirred at 60-65 ° C overnight. After cooling, water (10 ml) was added to the reaction solution, the reaction solution was adjusted to pH = 3.5 with 1N-hydrochloric acid under ice-water cooling, and left overnight in a cool place. The deposited precipitate was collected by filtration to obtain the desired product (70 mg).

¹ H-NMR (DMSO-d ₆ , δ):
2.08 (2H, t, J = 8 Hz), 2.88 (2H, t, J = 8 Hz)
t, J = 8 Hz), 3.68 (2H, t, J = 8H)
z), 4.72 (2H, s), 6.62 (1H, d, J
= 9 Hz), 7.48 (2H, m), 8.51 (1H,
s)

[0219]

Example 21 N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] 1,2,3,4-tetrahydro-6-quinoline carbonyl] -L- glutamate synthesis of diethyl

Triethylamine (60 mg) and diethylphosphorocyanidate (98 mg) were suspended in anhydrous dimethylformamide (6 ml). (60 mg) was added and stirred. After dissolution, the reaction solution was stirred at 80 ° C. for 3 minutes, then returned to room temperature and stirred for 10 minutes. Next, triethylamine (20 mg) and glutamic acid diethyl ester hydrochloride (40 mg) were added,
Stirred at 80 ° C. for 2 hours. After the reaction, the solvent was distilled off under reduced pressure, chloroform was added to the residue, and the chloroform layer was washed with saturated aqueous sodium hydrogen carbonate. 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 (50 mg). I got

¹ H-NMR (DMSO-d ₆ , δ):
1.1-1.4 (6H, m), 1.8-2.4 (4H,
m), 2.40 (2H, t, J = 8 Hz), 2.82
(2H, t, J = 8 Hz), 3.51 (2H, t, J =
8 Hz), 4.0-4.3 (4H, m), 4.64 (1
H, m), 4.75 (2H, s), 6.65 (1H,
d, J = 9 Hz), 7.47 (2H, m), 8.65
(1H, s)

[0220]

Example 22 N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] 1,2,3,4-tetrahydro-6-quinoli
Carbonyl] -L-glutamic acid

Embodiment 21 FIG. (50 mg) was dissolved in ethanol (10 ml), a 1N aqueous solution of sodium hydroxide (0.24 ml) was added at 35 ° C., and the mixture was added at the same temperature.
Stir for 5 hours. Furthermore, after stirring was continued at 25 ° C. for 20 hours, water (1 ml) was added to the reaction solution. Under ice-water cooling, the reaction solution was adjusted to pH = 3.7 with 1N-hydrochloric acid, and left overnight in a cool place. The deposited precipitate was collected by filtration, and the target product (27 m
g) was obtained.

¹ H-NMR (DMSO-d ₆ , δ):
1.8-2.2 (4H, m), 2.31 (2H, t, J
= 8 Hz), 2.75 (2H, t, J = 8 Hz), 3.
60 (2H, t, J = 8 Hz), 4.36 (1H,
m), 4.70 (2H, s), 6.62 (1H, d, J
= 9 Hz), 7.51 (2H, m), 8.62 (1H,
s) mp; 204-208 ° C (dec.)

[0223]

[Reference Example 42]4-[(2,4-diamino-6-pteridinyl) methyl
Ru] -3,4-dihydro-2H-1,4-benzoxa
Synthesis of methyl gin-7-carboxylate

6-bromomethyl-2,4-diaminopteridine hydrobromide / isopropanol adduct (410
mg) and methyl 3,4-dihydro-2H-1,4-benzoxazine-7-carboxylate (200 mg) were suspended in dimethylacetamide (10 ml).
And stirred at 90 ° C. for 19 hours. After cooling, dimethylacetamide was concentrated under reduced pressure, and chloroform and an aqueous solution of sodium hydrogen carbonate were added. After the precipitate was removed by filtration, the organic layer was washed with water, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography, and eluted with a mixed solvent of chloroform: methanol = 97: 3 to obtain the desired product (100 mg).

¹ H-NMR (CDCl ₃ : CD ₃ OD =
9: 1, δ): 3.63 (2H, brt), 3.84
(3H, s), 4.32 (2H, brt), 4.72
(2H, s), 6.68 (1H, d, J = 9.0H)
z), 7.47 (1H, s), 7.50 (1H, d, J
= 9.0 Hz), 8.71 (1H, s)

[0226]

[Reference Example 43]4-[(2,4-diamino-6-pteridinyl) methyl
Ru] -3,4-dihydro-2H-1,4-benzoxa
Synthesis of gin-7-carboxylic acid

Reference Example 42 Compound (60 mg) in 1N
-Suspended in a mixed solvent of aqueous sodium hydroxide solution (20 ml) and methanol (20 ml), and heated under reflux for 2.5 hours. After cooling, the solvent was distilled off, and water was added.
Adjusted to H = 5 (suspended). After being left overnight in a cool place, it was collected by filtration and dried to obtain the desired product (60.8 mg).

¹ H-NMR (DMSO-d ₆ , δ):
3.63 (2H, m), 4.22 (2H, m), 4.7
1 (2H, s), 7.21 (1H, s), 8.29 (1
H, s)

[0229]

Example 23 N- [4-[(2,4-diamino-6-pteridinyl)
Methyl] -3,4-dihydro-2H-1,4-benzoo
Xazin-7-carbonyl] -L-glutamic acid diethyl
Synthesis

Diphenyl phosphorocyanidate (64 μm)
l), triethylamine (60 μl) and a solution of dry dimethylformamide (5 ml). (50 mg) was added, and the mixture was stirred at 80 ° C. for 5 minutes under a nitrogen atmosphere. Cool to room temperature and triethylamine (20 μl)
And diethyl glutamate (34 mg) were added.
C. and stirred for 2.5 hours. After cooling, the mixture was extracted with chloroform and washed with an aqueous sodium hydrogen carbonate solution and brine. After drying over magnesium sulfate, the solvent was distilled off. The residue was subjected to silica gel column chromatography, and eluted with a mixed solvent of chloroform: methanol = 19: 1 to obtain the desired product (10 mg).

¹ H-NMR (CDCl ₃ : CD ₃ OD =
9: 1, δ): 1.23 (3H, t, J = 6.8H)
z), 1.29 (3H, t, J = 6.8 Hz), 3.6
0 (2H, m), 4.11 (2H, q, J = 6.8H
z), 4.22 (2H, q, J = 6.8 Hz), 4.3
2 (2H, m), 4.71 (2H, s), 6.69 (1
H, d, J = 10.0 Hz), 7.29 (1H, d, J)
= 10.0 Hz), 7.36 (1H, s), 8.70
(1H, s)

[0232]

Example 24 N- [4-[(2,4-diamino-6-pteridinyl)
Methyl] -3,4-dihydro-2H-1,4-benzoo
Synthesis of Xazin-7-carbonyl] -L-glutamic acid

Embodiment 23 FIG. (9 mg) was dissolved in ethanol (3 ml), a 1N aqueous sodium hydroxide solution was added, and the mixture was stirred at room temperature for 4 days. The solvent was distilled off, the residue was subjected to silica gel column chromatography, eluted with a mixed solvent of chloroform: methanol: aqueous ammonia = 5: 4: 1, and the fraction containing the target compound was collected.
The solvent was distilled off. Saturated aqueous sodium bicarbonate solution (20
0 μl), dissolve, add 1N-hydrochloric acid dropwise and adjust the pH.
Was adjusted to about 4 (suspended) and left in the cold overnight. The precipitate was collected by filtration and dried to give the desired product (2.8 mg).

¹ H-NMR (DMSO-d ₆ , δ):
1.86-1.95 (1H, m), 1.95-2.03
(1H, m), 2.24-2.38 (2H, m), 3.
67 (2H, t, J = 3.8 Hz), 4.25 (2H,
t, J = 3.8 Hz), 4.30 (1H, m), 4.7
0 (2H, s), 6.83 (1H, d, J = 8.6H)
z), 7.26 (1H, s), 7.30 (1H, d, J
= 8.6 Hz), 8.09 (1H, m), 8.70 (1
H, s) IR (KBr) νmax 3464, 1642 and
1512cm ^-1

[0235]

Example 25 N- [1-[(2,4-diamino-6-pteridinyl)]
Methyl] indoline-5-carbonyl] -DL-2-a
Synthesis of Mino-4-phosphonobutyric acid methyl ester

Triethylamine (272 mg) and diethyl cyanophosphonate (440 mg) were suspended in anhydrous dimethylformamide (30 ml). Next, 1-[(2,4-diamino-6-pteridinyl) methyl] indoline-5-carboxylic acid (303 mg) was added to the solution, and the mixture was stirred at room temperature overnight (solution A). Meanwhile, DL-2-amino-4-phosphonobutyric acid was dissolved in anhydrous methanol (5 ml) at 0 ° C., and thionyl chloride (1 ml) was dissolved at the same temperature.
Was slowly added. After returning the mixture to room temperature and stirring overnight, the solvent was distilled off under reduced pressure. The obtained residue was dissolved in anhydrous dimethylformamide (solution B). Next, the solution A and the solution B were combined. Further, triethylamine (505 mg) was added to this mixed solution, and the mixture was stirred at room temperature for 3 days. Then, the solvent was distilled off under reduced pressure to obtain a residue (700 mg). From 100 mg of the obtained residue, fractionation was performed by high performance liquid chromatography (column: YMC-A323), and water: acetonitrile: trifluoroacetic acid = 87.5: 12.5: 1 was used as a developing solvent. Thus, the desired product (2 mg) was obtained.

¹ H-NMR (D ₂ O, δ): 1.7-
2.3 (4H, m), 3.06 (2H, t, J = 8H
z), 3.58 (2H, t, J = 8 Hz), 3.84
(3H, s), 4.36 (1H, m), 4.70 (2
H, s), 6.63 (1H, d, J = 8.6 Hz),
7.60 (2H, m), 8.78 (1H, s)

[0238]

Reference Example 44 4-Amino-3-hydroxy-benzoic acid medium
Synthesis of tyl ester

4-Amino-3-hydroxy-benzoic acid (5.36 g) was suspended in methanol (40 ml), and hydrogen chloride gas was passed through the suspension for 10 minutes.
Stirred at room temperature for 6 hours. The deposited precipitate was collected by filtration, washed with ether three times, and then dried under vacuum to obtain the desired product (4.15).
g) was obtained.

¹ H-NMR (CDCl ₃ , δ): 3.8
3 (3H, s), 6.68 (1H, d, J = 8 Hz),
7.41 (2H, m)

[0241]

Reference Example 45 4-methoxycarbonyl-2-hydroxy-chloroacetate
Synthesis of tilanilide

The compound of Reference Example 44 (1.0 g) and triethylamine (4.0 ml) were added to dichloromethane (30 m
Dissolved in 1) and brought to 0 ° C. A dichloromethane solution (5 ml) of chloroacetyl chloride (1.0 ml) was added dropwise to the solution over 8 minutes under a nitrogen stream, and the mixture was stirred at the same temperature for 1 hour. Next, a saturated ammonium chloride aqueous solution was added to the reaction solution, and the resulting precipitate was collected by filtration. The desired product (0.83 g) was obtained by recrystallization from chloroform-acetone.

¹ H-NMR (CDCl ₃ , δ): 3.9
0 (3H, s), 4.25 (2H, s), 7.52 (2
H, m), 8.37 (1H, d, J = 8 Hz)

[0244]

Reference Example 46 3-oxo-3,4-dihydro-2H-1,4-benzo
Oxazine-7-carboxylic acid methyl S
Tell synthesis

The compound of Reference Example 45 (6.0 g) was suspended in ethanol (300 ml), potassium acetate (5.0 g) was added to the suspension, and the mixture was refluxed for 90 minutes under a nitrogen stream. After cooling the reaction solution, the precipitate was collected by filtration and dried under vacuum to obtain the desired product (5.2 g).

¹ H-NMR (DMSO-d ₆ , δ):
3.80 (3H, s), 4.63 (2H, s), 6.9
5 (1H, m), 7.43 (1H, m), 7.56 (1
H, m)

[0247]

Reference Example 47 3,4-Dihydro-2H-1,4-benzoxazine-
Synthesis of 7-carboxylic acid methyl ester

Under a nitrogen stream, tetrahydrofuran / borane complex (1 [M] solution, 10 ml) and the compound of Reference Example 46 (440 mg) were added at 0 ° C. in tetrahydrofuran (30 ml).
In addition to 1), the mixture was stirred at room temperature for 15 minutes, and then refluxed for 4 hours. The reaction solution was cooled to room temperature, and 6N-hydrochloric acid (2.7
ml) was added. After the reaction solution was concentrated under reduced pressure, it was poured into water and made alkaline with a 2N-sodium hydroxide aqueous solution. Next, the target substance was extracted with ethyl acetate, and the obtained organic layer was washed with brine and dried over magnesium sulfate. After evaporating the solvent under reduced pressure, the obtained residue was subjected to silica gel column chromatography. The mixture was developed and eluted with a mixed solution of hexane: ethyl acetate = 3: 2 to obtain the desired product (310 mg).

¹ H-NMR (CDCl ₃ , δ): 3.4
6 (2H, m), 3.84 (3H, s), 4.22 (2
H, t, J = 4.4 Hz), 4.30 (1H, m),
6.53 (1H, δ, J = 9.8 Hz), 7.45 (1
H, s), 7.47 (1H, d, J = 9.8 Hz)

[0250]

Reference Example 48 N-carbobenzoxy-3,4 dihydro-2H-1,4
-Benzoxazine-7-carboxylic acid
Synthesis of tyl ester

The compound of Reference Example 47 (1.4 g) was dissolved in tetrahydrofuran (50 ml), and sodium hydroxide (700 mg) was slowly added to the solution, followed by stirring at room temperature for 30 minutes. Then, carbobenzoxychloride (3 ml) was added to the reaction solution and stirred overnight. After a few drops of water were added to the reaction solution, the mixture was poured into cold water and the desired product was extracted with ethyl acetate. After the obtained organic layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure. The residue was recrystallized from hexane-ethyl acetate to obtain the desired product (1.79 g).

¹ H-NMR (CDCl ₃ , δ): 3.8
5 (3H, s), 3.91 (2H, m), 4.25 (2
H, m), 5.34 (2H, s), 7.35 (5H,
m), 7.53 (2H, m), 7.99 (1H, m)

[0253]

Reference Example 49 N-carbobenzoxy-3,4-dihydro-2H-1,
4-benzoxazine-7-carboxylic acid
Synthesis of

The compound of Reference Example 48 (1.79 g) was suspended in ethanol (50 ml), 1N-aqueous sodium hydroxide solution (8.2 ml) was added to the suspension, and the mixture was stirred overnight. After evaporating the solvent under reduced pressure, the obtained residue was dissolved in water (20 ml). Next, 1N-hydrochloric acid was slowly added to adjust the pH to 2, and the deposited precipitate was collected by filtration and dried under vacuum to obtain the desired product (1.39 g).

¹ H = NMR (CDCl ₃ , δ): 3.8
2 (2H, m), 4.14 (2H, m), 5.13 (2
H, s), 7.2-7.7 (7H, m), 7.95 (1
H, m)

[0256]

Reference Example 50 N- (3,4-dihydro-2H-1,4-benzoxa
(Zin-7-carbonyl) -L-α-amino-adipic
Synthesis of acid dimethyl ester

After suspending the compound of Reference Example 49 (800 mg) in thionyl chloride (5 ml), a catalytic amount of dimethylformamide was added, and the mixture was stirred at room temperature for 2 hours. Subsequently, excess thionyl chloride was distilled off under reduced pressure, and the residue was triturated with hexane. The obtained crystals were collected by filtration, dissolved in dichloromethane (20 ml), and the dichloromethane solution was cooled with ice-water and cooled to L-α-amino-adipic acid dimethyl ester hydrochloride (1.0 g) and triethylamine (1 g). 2.0 g) (20 ml)
Was added dropwise to a dichloromethane solution. After stirring at room temperature overnight and distilling off the solvent under reduced pressure, the residue was cooled with ice-water.
A mixture of ethyl acetate and dilute hydrochloric acid was added. After stirring for 5 minutes, the organic layer was separated, and then the organic layer was washed with a saturated aqueous sodium hydrogen carbonate solution. After drying over magnesium sulfate, the solvent was distilled off under reduced pressure. The obtained residue was dissolved in ethanol (80 ml), 10% palladium-carbon (400 mg) was added, and the mixture was stirred overnight at room temperature under a hydrogen atmosphere. Palladium-carbon was removed by filtration using Celite, and the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography, and the desired product (230 mg) was obtained using a mixed solvent of chloroform: methanol = 100: 1 as an elution solvent.

¹ H-NMR (CDCl ₃ , δ): 1.6
−2.1 (4H, m), 2.38 (2H, t, J = 6.
8Hz), 3.46 (2H, m), 3.68 (3H,
s), 3.77 (3H, s), 4.24 (2H, m),
4.72 (1H, m), 6.59 (1H, d, J = 8.
3Hz), 7.33 (2H, m)

[0259]

Example 26 N- [1-[((2,4-diamino) -6-pteridini)
M)]-3,4-dihydro-2H-1,4-ben
Zoxazine-7-carbonyl] -L-α-amino-a
Synthesis of dipic acid dimethyl ester

The compound of Reference Example 50 (200 mg) and 6-
Bromomethyl-2,4-diaminopteridine hydrobromide / isopropanol adduct (226 mg) was suspended in dimethylacetamide (3 ml) and stirred at 60 ° C. for 6 hours. After cooling, the reaction solution was poured into a saturated aqueous solution of sodium hydrogen carbonate and extracted three times 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,
The desired product (260 mg) was obtained using a mixed solvent of chloroform: methanol = 10: 1 as an elution solvent.

¹ H-NMR (CD ₃ OD, δ): 1.6
−2.0 (4H, m), 2.36 (2H, t, J = 6.
8Hz), 3.58 (2H, m), 3.66 (3H,
s), 3.76 (3H, s), 4.39 (2H, m),
4.67 (2H, bs), 4.73 (1H, m), 6.
66 (1H, d, J = 8.3 Hz), 6.99 (1H,
d, J = 7.3 Hz), 7.29 (2H, m), 8.7
0 (1H, s)

[0262]

Example 27 N- [1-[((2,4-diamino) -6-pteridini)
M)]-3,4-dihydro-2H-1,4-ben
Zoxazine-7-carbonyl] -L-α-amino-a
Synthesis of dipic acid

The compound of Example 26 (260 mg) was dissolved in ethanol (12 ml), 1N aqueous sodium hydroxide solution (0.45 ml) was added at 35 ° C.
Stirred for hours. After stirring at 25 ° C. for 20 hours, water (0.5 ml) was added to the reaction solution, and ethanol was distilled off under reduced pressure. The obtained residue was dissolved in water (6 ml), adjusted to pH = 3.7 with 1N hydrochloric acid under ice-water cooling, and allowed to stand overnight in a cool place. The deposited precipitate was collected by filtration and the desired product (1
76 mg).

¹ H-NMR (DMSO-d ₆ , δ):
1.5-2.0 (4H, m), 2.14 (2H, t, J
= 6.8 Hz), 3.68 (2H, m), 4.28 (3
H, m), 4.71 (2H, bs), 6.80 (1H,
d, J = 8.3 Hz), 7.31 (2H, m), 8.1
3 (1H, d, J = 7.3 Hz), 8.71 (1H,
s)

[0265]

[Reference Example 51] Nt-butoxycarbonyl-L-glutamic acid α-meth
Synthesis of tyl-γ-benzyl ester

After suspending sodium hydrogen carbonate (2.5 g) in a dimethylformamide solution (75 ml) of Nt-butoxycarbonyl-L-glutamic acid γ-benzyl ester (5.0 g), methyl iodide ( 10.5
g) in dimethylformamide (75 ml) was added and the mixture was stirred at room temperature for 24 hours. After concentrating the reaction solution under reduced pressure,
Pour into water (70 ml) and add ethyl acetate: n-hexane =
Extracted with a 1: 1 mixed solution. After the organic layer was washed with water and dried over sodium sulfate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography, and ethyl acetate: n-hexane = 1: 2 as an elution solvent.
Was used to obtain the desired product (5.2 g).

¹ H-NMR (CDCl ₃ , δ): 1.4
3 (9H, s), 1.92-2.01 (1H, m),
2.11-2.37 (1H, m), 2.42-2.51
(2H, m), 3.73 (3H, s), 4.23-4.
40 (1H, m), 5.12 (2H, s), 7.35
(5H, s)

[0268]

[Reference Example 52] N-t-butoxycarbonyl-L-glutamic acid α-meth
Synthesis of tyl ester

To a methanol solution (30 ml) of the compound of Reference Example 51 (5.2 g) was added 10% palladium-carbon (1.
After 1 g) was added, the mixture was stirred at room temperature under a hydrogen atmosphere for 15 hours. The palladium-carbon was removed by filtration using Celite, and the solvent was distilled off under reduced pressure. The obtained residue was dissolved in a saturated aqueous solution of sodium hydrogen carbonate and washed with chloroform. After separating the aqueous layer, the pH was adjusted to 4 using 5% citric acid and extracted with chloroform. After the chloroform layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure to obtain the desired product (3.9 g).

¹ H-NMR (CDCl ₃ , δ): 1.4
4 (9H, s), 1.89-2.04 (1H, m),
2.09-2.27 (1H, m), 2.33-2.59
(2H, m), 3.75 (3H, s), 4.24-4.
44 (1H, m), 5.17-5.21 (1H, m),
9.38 (1H, bs)

[0271]

Reference Example 53 Synthesis of methyl 4-amino-n-butyrate hydrochloride

Hydrogen chloride gas was passed through a methanol solution (20 ml) of 4-amino-n-butyric acid (1.0 g) for 10 minutes, followed by stirring at room temperature for 5 hours. The solvent was distilled off under reduced pressure to obtain the desired product (1.5 g).

¹ H-NMR (DMSO-d ₆ , δ):
1.83-2.24 (2H, m), 2.32-2.60
(2H, m), 2.76-3.26 (2H, m), 3.
61 (3H, s), 8.26 (2H, bs)

[0274]

Reference Example 54 Nt-butoxycarbonyl-γ- (3-methoxycal
Bonylpropylamide) -α-methyl L-glutamate
Ester synthesis

To a solution of the compound of Reference Example 52 (518 mg) and triethylamine (0.33 ml) in tetrahydrofuran (5 ml) was added a solution of isobutyl chlorocarbonate (0.31 ml) in tetrahydrofuran (1 ml) at -20 ° C. under a nitrogen atmosphere. Stir for 30 minutes. Then, Reference Example 5
Compound 3 (366 mg) in triethylamine (0.3
3 ml) of a tetrahydrofuran suspension (5 ml) was added, and the mixture was stirred for 1 hour. Gradually return to room temperature and add another 24
Stirred for hours. The solvent was distilled off under reduced pressure, and the obtained residue was dissolved in ethyl acetate. The ethyl acetate layer was sequentially washed with a 5% aqueous citric acid solution, a saturated aqueous sodium hydrogen carbonate solution and water, dried over sodium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography, and the desired product (575 mg) was obtained using chloroform: methanol = 99: 1 as an elution solvent.

¹ H-NMR (CDCl ₃ , δ): 1.4
4 (9H, s), 1.78-1.99 (3H, m),
2.16-2.19 (1H, m), 2.22-2.31
(2H, m), 2.39 (2H, t, J = 7.1H
z), 2.70 (2H, q, J = 6.5 Hz), 3.6
8 (3H, s), 3.74 (3H, s), 4.14-
4.31 (1H, m), 5.35 (1H, bs),
43 (1H, bs)

[0277]

Reference Example 55 γ- (3-methoxycarbonylpropylamide) -L-
Glutamic acid α-methyl ester trifluoroacetate
The compound of Synthesis Reference Example 54 (726 mg) was dissolved in trifluoroacetic acid (2 ml) under ice cooling and stirred for 1 hour. The solvent was distilled off under reduced pressure to obtain the desired product (754 mg).

¹ H-NMR (CDCl ₃ : CD ₃ OD =
20: 1, δ): 0.92 (2H, m), 2.04-
2.42 (4H, m), 2.50 (2H, t, J = 6.
3 Hz), 3.17-3.24 (2H, m), 3.67
(3H, s), 3.81 (3H, s), 4.09-4.
15 (1H, m), 7.44 (1H, t.J = 6.0H)
z)

[0279]

[Reference Example 56]N- (1-Carbonbenzoxyindoline-5-carbo
Nil) -γ- (3-methoxycarbonylpropylamido)
C) Synthesis of α-methyl ester of -L-glutamic acid

Thionyl chloride (5 ml) was added to 1-carbobenzoxyindoline-5-carboxylic acid (599 mg), 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 methylene chloride (7 ml), and the compound of Reference Example 55 (754 m
g) and an aqueous solution of sodium hydrogen carbonate (534 mg) (3
ml) at room temperature and stirred for 15 hours. PH of reaction solution
= 8, and the insoluble material was removed by filtration using Celite. Then, the methylene chloride layer was separated. The methylene chloride layer was sequentially washed with 1N hydrochloric acid and water, dried over sodium sulfate, and then the solvent was distilled off under reduced pressure.
The obtained residue was subjected to silica gel column chromatography, and chloroform: methanol = 9 as an elution solvent.
The desired product (474 mg) was obtained using 9: 1.

¹ H-NMR (CDCl ₃ , δ): 1.8
1 (2H, m, J = 7.0 Hz), 2.09-2.27
(4H, m), 2.34 (2H, t, J = 7.3H
z), 3.15 (2H, t, J = 9.0 Hz), 3.2
7 (2H, t, J = 5.4 Hz), 3.65 (3H,
s), 3.77 (3H, s), 4.10 (2H, t, J
= 8.8 Hz), 4.65-4.75 (1H, m) 5.
28 (2H, s), 6.38-6.45 (1H ,,
m), 7.36-7.46 (6H, m), 7.66-
7.68 (2H, m)

[0282]

Reference Example 57 N- (Indoline-5-carbonyl) -γ- (3-meth
Xycarbonylpropylamide) -L-glutamic acid α
-Synthesis of methyl ester

A 10% palladium-carbon (9%) solution was added to a methanol solution (10 ml) of the compound of Reference Example 56 (470 mg).
After adding 0 mg), the mixture was stirred at room temperature under a hydrogen atmosphere for 15 hours. After filtering off the palladium-carbon using celite, the solvent was distilled off under reduced pressure to obtain the desired product (350 mg).

¹ H = NMR (CDCl ₃ , δ): 1.6
6-2.48 (8H, m), 2.81-3.30 (4
H, m), 3.50 (2H, t, J = 6.0 Hz),
3.64 (3H, s), 3.73 (3H, s), 4.4
9-4.81 (1H, m), 6.51 (2H, d, J =
9,0 Hz), 6.62-6.84 (1H, m), 7.
21-7.33 (1H, m), 7.42-7.57 (2
H, m)

[0285]

Example 28 N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] -indoline-5-carbonyl] -γ- (3-
Methoxycarbonylpropylamide) -L-glutamine
Synthesis of acid α-methyl ester

The compound of Reference Example 57 (350 mg) and 6-
Bromomethyl-2,4-diaminopteridine hydrobromide isopropanol adduct (414 mg) was suspended in dimethylacetamide (7 ml) and stirred at room temperature for 24 hours. Triethylamine (0.29 ml) was added to the reaction mixture, and the mixture was stirred for 10 minutes. The mixture was subjected to silica gel column chromatography, and ethyl acetate and then chloroform: methanol = 9: 1 were used as elution solvents to obtain the desired compound (263).
mg).

¹ H-NMR (DMSO-d ₆ : CDCl
₃ = 7: 3, δ): 1.67 (2H, m, J = 6.9H)
z), 1.91-2.14 (2H, m), 2.19-
2.33 (4H, m), 2.97-3.13 (4H,
m), (3.56-3.59 (5H, m), 3.65)
(3H, s), 4.34-4.44 (1H, m), 4.
55 (2H, s), 6.67 (1H, d, J = 8.3H
z), 7.32 (4H, bs), 7.62-7.66.
(2H, m), 7.84 (1H, t, J = 8.3H
z), 8.33 (1H, d, J = 7.3 Hz), 8.7
1 (1H, s)

[0288]

Working Example 29 N- [1-[(2,4-diamino-6-pteridinyl)]
Methyl] -indoline-5-carbonyl] -γ- (3-
Synthesis of (carboxypropylamide) -L-glutamic acid

To a solution of the compound of Example 28 (250 ml) in methanol (5 ml) was added a 1N aqueous solution of sodium hydroxide (0.95 ml), and the mixture was stirred at room temperature for 20 hours.
The solvent was distilled off under reduced pressure while maintaining the temperature of the water bath at 30 ° C. or lower. The resulting residue was subjected to silica gel column chromatography to obtain a brown solid using chloroform: methanol: 28% aqueous ammonia = 5: 4: 1 as an elution solvent. The obtained solid was dissolved in water, the insolubles were removed by filtration, and the pH was adjusted to 3.7 using 1N hydrochloric acid.
The resulting precipitate was collected by filtration to give the desired product (144 mg).

¹ H-NMR (DMSO-d ₆ : CDCl
₃ = 9: 1, δ): 1.61 (2H, m), 1.89−
2.08 (2H, m), 2.13-2.30 (4H,
m), 2.95-3.10 (4H, m), (3.58
(2H, t, J = 8.0 Hz), 4.25-4.38
(1H, m), 4.55 (2H, s), 6.70 (1
H, d, J = 8.3 Hz), 7.61-7.65 (2
H, m), 7.85 (1H, t, J = 5.8 Hz),
8.21 (1H, d, J = 7.3 Hz), 8.73 (1
H, s)

[0291]

Working Example 30 N- [1-[(2,4-diamino-6-pteridinyl)]
Methyl] -indoline-5-carbonyl] -homocyste
Synthesis of ammonium formate

In a nitrogen atmosphere, triethylamine (870 μl) and chlorotrimethylsilane (630 μl) were added to a suspension of homocysteic acid hydrobromide (330 ml) in benzene (6 ml), and the mixture was stirred at room temperature for 3 days. The precipitate was separated by filtration, and the filtrate was concentrated to obtain silylated homocysteic acid (450 mg). Under a nitrogen atmosphere, diethyl cyanophosphonate (213 μl) and triethylamine (172 μl) were converted to dimethylformamide (18 ml).
And 1-[(2,4-diamino) -6-pteridinyl) methyl] indoline-5-carboxylic acid (1
70 mg) was added little by little, and the mixture was stirred at the same temperature for 3 hours.
To this solution was added silylated homocysteic acid (450 m
g) in dimethylformamide (2 ml) was added, and the mixture was stirred at room temperature for 2 days. Water (1 ml) was added to the reaction mixture, and the solvent was distilled off under reduced pressure. A 3% aqueous solution of ammonium bicarbonate was added to the residue, the insolubles were filtered off, the residue was applied to a DEAE-cellulose column, washed with water, and eluted with a 3% aqueous solution of ammonium bicarbonate to obtain the desired product (18 mg). Was.

¹ H-NMR (D ₂ O, δ): 2.0-
2.4 (2H, m), 3.15 (4H, m), 3.49
(2H, m), 4.34 (1H, m), 4.49 (2
H, s), 6.66 (1H, m), 7.55 (2H,
m) 8.69 (1H, s)

[0294]

Reference Example 58 3,4-Dihydro-2H-1,4-benzothiazinecar
Synthesis of methyl borate

Under a nitrogen atmosphere, 2-aminobenzothiazole-6-carboxylic acid (15 g) and potassium hydroxide (22 g)
g) and a mixture of water (22 g) were refluxed for 3 hours. The reaction solution was cooled to room temperature, water (20 ml), 1,2-dibromoethane (40 ml) and hexadecyltributylphosphonium bromide (3 g) were added, and the mixture was refluxed for 6 hours. The reaction solution was cooled to room temperature, water and chloroform were added, insolubles were removed by decantation, the aqueous layer was adjusted to pH = 3 with hydrochloric acid, and the chloroform layer was separated. The obtained chloroform layer was washed with saturated saline and then dried over anhydrous sodium sulfate. The residue obtained by evaporating the solvent under reduced pressure was dissolved in methanol, and hydrogen chloride gas was passed through the solution for 10 minutes, followed by stirring at room temperature overnight. Water and ethyl acetate were added to the residue obtained by evaporating the solvent under reduced pressure, the aqueous layer was adjusted to pH = 5 with a 1N aqueous sodium hydroxide solution, and extracted with ethyl acetate. The obtained organic layer was washed with a saturated saline solution and dried over anhydrous sodium sulfate. After evaporating the solvent under reduced pressure, the residue was subjected to silica gel column chromatography and the desired product (721 mg) was obtained using ethyl acetate: hexane = 1: 4 as the elution solvent.

¹ H-NMR (CDCl ₃ , δ): 2.9
-3.1 (2H, m), 3.6-3.9 (2H, m),
3,83 (3H, s), 4,0-5.0 (1H, b
r), 6.39 (1H, d, J = 8.4 Hz), 7.4
5-7.75 (2H, m),

[0297]

Reference Example 59 N-carbobenzoxy-3,4-dihydro-2H-1,
4-benzothiazine-7-carboxylic acid
Synthesis

The compound of Reference Example 58 (313 mg) was dissolved in tetrahydrofuran (10 ml), and sodium hydride (180 mg) was slowly added to the solution, followed by stirring at room temperature for 20 minutes. Then, carbobenzoxycyclolide (1.3 ml) was added to the reaction solution and stirred overnight. After adding water to the reaction solution, extraction was performed using ethyl acetate. After the obtained organic layer was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography, and ethyl acetate: hexane = 1: 10 was used as the elution solvent to obtain N-carbobenzoxy-3,
4-Dihydro-2H-1,4-benzothiazine-7-carboxylic acid methyl ester is suspended in ethanol (15 ml), 1N aqueous sodium hydroxide solution (1.9 ml) is added to the suspension, and the mixture is stirred overnight. did.
After evaporating the solvent under reduced pressure, the obtained residue was washed with water (20 m
l). Then slowly add 1N hydrochloric acid,
The mixture was adjusted to pH = 3 and extracted with chloroform. The organic layer is washed with saturated saline, dried over anhydrous sodium sulfate, and the solvent is distilled off under reduced pressure. The residue obtained is subjected to silica gel column chromatography, using chloroform: methanol = 95: 5 as the eluting solvent. (208 mg).

¹ H-NMR (CDCl ₃ : CD ₃ OD =
9: 1, δ): 3.0-3.3 (2H, m), 3.8-
4.1 (2H, m), 5.25 (2H, s), 7.3
7.9 (8H, m)

[0300]

Reference Example 60 N- (3,4-dihydro-2H-1,4-benzothiazi
-7-carbonyl) -L-glutamic acid diethyl ester
Synthesis of ter The compound of Reference Example 59 (208 mg), L-glutamic acid diethyl ester hydrochloride (151 mg), 1-hydroxybenzotriazole (90 mg), N-methylmorpholine (70 μl) in dimethylformamide solution (5 m
1) Dicyclohexylcarbodiimide (145) under ice-cooling
mg), and the mixture was stirred at the same temperature for 1 hour and then at room temperature overnight. Ethyl acetate was added to the reaction solution, the precipitate was filtered off, and the filtrate was washed with an aqueous solution of sodium hydrogen carbonate and saturated saline,
Dry over anhydrous sodium sulfate. The residue obtained by evaporating the solvent under reduced pressure was subjected to silica gel column chromatography, and N- [N'-carbobenzoxy-3,4 obtained using ethyl acetate: hexane = 1: 2 as an elution solvent.
-Dihydro-2H-1,4-benzothiazine-7-carbonyl] -L-glutamic acid diethyl ester was dissolved in ethanol (20 ml), 10% palladium-carbon (700 mg) was added, and the mixture was stirred at room temperature under a hydrogen atmosphere. Stirred overnight. Palladium-carbon was removed by filtration using Celite, and the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography, and the desired product (83 mg) was obtained using a mixed solvent of ethyl acetate: hexane = 2: 3 as an elution solvent.

¹ H-NMR (CDCl ₃ : CD ₃ OD =
9: 1, δ): 1.25 (6H, m), 2.0-2.7.
(4H, m), 2.9-3.1 (2H, m), 3.6-
3.8 (2H, m), 3.9-4.4 (5H, m),
6.45 (1H, d, J = 8.0 Hz), 7.2-7.
6 (2H, m)

[0302]

Example 31 N- [1-[((2,4-diamino) -6-pteridini)
M)]-3,4-dihydro-2H-1,4-ben
Zothiazin-7-carbonyl] -L-glutamic acid die
Synthesis of tyl ester

The compound of Reference Example 60 (83 mg) and 6-bromomethyl-2,4-diaminopteridine hydrobromide isopropanol adduct (85 mg) were suspended in dimethylacetamide (3 ml) and stirred at 60 ° C. for 3 hours. After that, the mixture was stirred at 100 ° C. for 30 minutes. After cooling, the reaction solution was poured into a saturated aqueous solution of sodium hydrogen carbonate and extracted three times with chloroform. After the organic layer was dried over anhydrous sodium sulfate, the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography, and using a mixed solvent of chloroform: methanol = 93: 7 as an elution solvent, the target compound (2
1 mg).

¹ H = NMR (CDCl ₃ : CD ₃ OD =
9: 1, δ): 1.26 (6H, m), 2.0-2.3.
(2H, m), 2.46 (2H, m), 3.12 (2
H, m), 3.91 (2H, m), 4.1-4.3 (4
H, m), 4.62 (1H, m), 4.78 (2H,
s), 6.69 (1H, d, J = 8.9 Hz), 7.4
0 (1H, m), 7.60 (1H, d, J = 2.2H
z), 8.66 (1H, s)

[0305]

EXAMPLE 32 N- [1 - [((2,4- diamino) -6-pteridinyl) methyl] -3,4-dihydro-2H-1,4-benzothiazine-7-carbonyl] -L- glutamic Acid combination
Success

The compound of Example 31 (20 mg) was dissolved in ethanol (2 ml), a 1N aqueous sodium hydroxide solution (170 μl) was added at 35 ° C., and the mixture was stirred at the same temperature for 4 hours. After continuing stirring at 25 ° C. for 20 hours, water (0.5 ml) was added to the reaction solution, and ethanol was distilled off under reduced pressure. The obtained residue was dissolved in water (6 ml) and iced.
The pH was adjusted to 3.7 with 1N hydrochloric acid while cooling with water, and the mixture was allowed to stand overnight in a cool place. The deposited precipitate was collected by filtration to obtain the desired product (18 mg).

¹ H-NMR (DMSO-d ₆ , δ):
1.8-2.2 (2H, m), 2.30 (2H, m),
3.18 (2H, m), 3.95 (2H, m), 4.3
7 (1H, m), 4.76 (2H, s), 6.79 (1
H, d, J = 8.8 Hz), 7.42 (1H, m),
7.59 (1H, d, J = 2.0 Hz), 8.22 (1
H, d, J = 7.3 Hz), 8.67 (1H, s)

[0308]

Working Example 33 N- [1-[(2,4-diamino-6-pteridinyl)]
Methyl] -1-oxo-3,4-dihydro-2H-1,
4-benzothiazine-7-carbonyl] -L-glutamic
Synthesis of acid

The compound of Example 32 (10 mg) was added to water (1
ml), and dissolved by adding a 1N aqueous sodium hydroxide solution. After cooling on ice, 0.5 M sodium metaperiodate (50 μl) was added, and the mixture was stirred at the same temperature for 5 hours. The pH was adjusted to 3.5 with 1N hydrochloric acid, and the resulting precipitate was collected by filtration to obtain the desired product (4 mg).

¹ H-NMR (DMSO-d ₆ , δ):
1.8-2.2 (2H, m), 2.33 (2H, m),
2.9-3.3 (2H, m), 3.8-4.0 (1H,
m), 4.2-4.5 (2H, m), 4.83 (1H,
d, J = 17.1 Hz), 5.19 (1H, d, J = 1)
7.1 Hz), 7.07 (1H, d, J = 9.3H)
z), 7.7-7.9 (1H, m), 8.1-8.2
(1H, m), 8.42 (1H, m), 8.72 (1
H, s) IR (KBr) νmax 2800-3600,164
4,1608,1552,1504 and 1008
cm ^-1 FAB MS 515 (M + 1) ⁺

[0311]

Reference Example 61 N ² -t-butoxycarbonyl-N ⁵ -methanesulfonate
Synthesis of Luglutamine Benzyl Ester

N, N'-carbonyldiimidazole (1
3.6 g) and [N- (t-butoxycarbonyl) glutamic acid] -α-benzyl ester (25 g) were dissolved in tetrahydrofuran (268 ml), and the mixture was stirred under ice cooling for 1 hour. Then the solution was treated with methanesulfonamide (2
0.5g) and 1,8-diazabicyclo [5,4,0]-
A solution of 7-undecene (32.9 g) in tetrahydrofuran (132 ml) was added dropwise under ice cooling. After the addition, the mixture was returned to room temperature and stirred for 4 days. 1N hydrochloric acid 500m
After adding l, the mixture was extracted with chloroform. After the organic layer was dried over anhydrous magnesium sulfate, the solvent was distilled off under reduced pressure to obtain the desired product (30 g).

¹ H-NMR (CDCl ₃ , δ): 1.4
2 (9H, s), 1.8-2.8 (4H, m), 3.2
5 (3H, s), 4.32 (1H, m), 5.14 (2
H, s), 7.40 (5H, s)

[0314]

[Reference Example 62] N 5 ^- · methanesulfonyl glutamine benzyl ester
Synthesis of trifluoroacetate

The compound of Reference Example 61 (4.65 g) was dissolved in trifluoroacetic acid (60 ml) and stirred at room temperature for 1 hour. Then, the reaction solution was concentrated at 30 ° C., and triturated with ether to obtain the desired product (4.5 g).

¹ H-NMR (CDCl ₃ , δ): 1.8
-2.8 (4H, m), 3.20 (3H, s), 4.5
0 (1H, m), 5.28 (2H, s), 7.42 (5
H, s)

[0317]

Embodiment 34 ^{N 2 - [1 - [(} 2,4- diamino-6-Puterijini
) Methyl] indolin-5-carbonyl] -N ⁵ - main
Synthesis of tansulfonylglutamine benzyl ester

In a nitrogen atmosphere, diethyl cyanophosphonate (213 μl) and triethylamine (172 μl) were dissolved in dimethylformamide (18 ml), and
[(2,4-Diamino-6-pteridinyl) methyl] indoline-5-carboxylic acid (160 mg) was added little by little, and the mixture was stirred at the same temperature for 3 hours. Reference Example 62.
(450 mg) and triethylamine (180 μ
A dimethylformamide solution (3 ml) containing 1) was added, and the mixture was stirred at room temperature for 3 days, water (1 ml) was added, and the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography, and chloroform:
The target product (71 mg) was obtained using a mixed solvent of methanol: aqueous ammonia = 15: 5: 1.

¹ H-NMR (DMSO-d ₆ , δ):
1.9-2.1 (2H, m), 2.3-2.5 (2H,
m), 2.98 (2H, t, J = 8.6 Hz), 3.2
8 (3H, s), 3.55 (2H, t, J = 8.6H)
z), 4.21 (1H, m), 4.53 (2H, s),
5.04 (2H, s), 6.71 (1H, d, J = 8.
6 Hz), 7, 34 (5H, s), 7.55 (2H,
m), 8.72 (1H, s)

[0320]

Example 35 ^{N 2 - [1 - [(} 2,4- diamino-6-pteridinyl) methyl] indolin-5-carbonyl] -N ⁵ - main
Synthesis of tansulfonylglutamine

The compound of Example 34 (25 mg) was suspended in ethanol (5 ml), 1N-aqueous sodium hydroxide solution (200 μl) was added, and the mixture was stirred at room temperature overnight. Then, water (0.5 ml) was added to the reaction solution, and ethanol was distilled off under reduced pressure. The obtained residue was dissolved in water (6 ml),
The pH was adjusted to 3.7 with 1N-hydrochloric acid under ice-water cooling, and the mixture was allowed to stand overnight in a cool place. The deposited precipitate was collected by filtration and the target product (43 m
g) was obtained.

¹ H-NMR (DMSO-d ₆ , δ):
1.9-2.1 (2H, m), 2.2-2.4 (2H,
m), 3.00 (2H, t, J = 8.6 Hz), 3.1
9 (3H, s), 3.60 (2H, t, J = 8.6H)
z), 4.35 (1H, m), 4.56 (2H, s),
6.70 (1H, d, J = 8.6 Hz), 7, 63 (2
H, m), 8.75 (1H, s)

[0323]

Reference Example 63 4-Nitro-3-hydroxy-benzoic acid medium
Synthesis of tyl ester

A suspension of 4-nitro-3-hydroxy-benzoic acid (2.0 g) in methanol (10 ml) was cooled to -78 ° C using dry ice, and thionyl chloride was slowly added at the same temperature. . After the reaction solution was gradually returned to room temperature, it was refluxed for about 1 hour. After cooling, the solvent was concentrated under reduced pressure, and a large amount of water was added to the concentrated liquid. The precipitated solid was collected by filtration, washed with water, and then dried in vacuum to obtain about 2.
0 g was obtained.

¹ H-NMR (δ value, CDCl ₃ ): 3.
98 (3H, s), 7.5-7.7 (1H, m), 7.
85 (1H, m), 8.20 (1H, d, J = 10H
z)

[0326]

Reference Example 64 4-Nitro-3- (2-chloroethoxy) -benzoic
Synthesis of quasid methyl ester

A solution of 4-nitro-3-hydroxy-benzoic acid methyl ester (43 g) in DMF (4
Bromochloroethane (91 ml) was added to
The temperature was raised to 100 ° C. with stirring, and potassium carbonate (61 g) was slowly added at the same temperature. After stirring at 100 ° C. for 2 hours, the reaction solution was poured into water (2 l) and extracted with toluene. After the toluene layer was washed with water and dried over sodium sulfate, the solvent was distilled off under reduced pressure to obtain 56 g of the desired product.

¹ H-NMR (CDCl ₃ , δ): 3.5
-4.0 (5H, m), 4.42 (2H, m), 7.6
-8.0 (3H, m)

[0329]

Reference Example 65 4-Amino-3- (2-chloroethoxy) -benzoic
Synthesis of quasid methyl ester

4-Nitro-3- (2-chloroethoxy)
A solution of benzoic acid methyl ester (56 g) in acetic acid (1.3 l) was brought to 5-10 ° C., and Zn (143
g) was added slowly. After completion of the heat generation, the mixture was stirred at room temperature for 2 hours, and then subjected to natural filtration. The filtrate was concentrated under reduced pressure, dissolved in chloroform (300 ml), and washed with a saturated aqueous sodium hydrogen carbonate solution (500 ml × 2).

Next, the chloroform layer was dried using sodium sulfate, and the solvent was distilled off under reduced pressure.
g) was obtained.

¹ H-NMR (CDCl ₃ , δ): 3.6
-4.0 (5H, m), 4.27 (2H, t, J = 6H
z), 6.68 (1H, d, J = 8 Hz), 7.4-
7.7 (2H, m)

[0333]

Reference Example 66 4-Tosylamino-3- (2-chloroethoxy) -ben
Synthesis of Zoic Acid Methyl Ester

4-Amino-3- (2-chloroethoxy)
A pyridine solution (500 ml) of -benzoic acid methyl ester (48 g) was added with p-toluenesulfonyl chloride (60 g), and the mixture was stirred overnight. Next, a saturated aqueous solution of ammonium chloride (2.5 l) was added to the reaction solution, and the precipitated white solid was collected by filtration. The obtained solid was suspended in chloroform (500 ml), and 1N hydrochloric acid (500 ml) was added.
ml) and water (500 ml). After the chloroform layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure. The residue was recrystallized from chloroform-n-hexane to obtain the desired product (45 g).

¹ H-NMR (CDCl ₃ , δ): 2.3
6 (3H, s), 3.76 (2H, t, J = 5.4H
z), 3.87 (3H, s), 4.21 (3H, t, J
= 5.4 Hz), 7.2-7.4 (4H, m), 7.6
-7.8 (4H, m)

[0336]

Reference Example 67 N-Tosyl-3,4-dihydro-2H-1,4-benzo
Synthesis of oxazine-7-carboxylic acid

4-Tosylamino-3- (2-chloroethoxy) -benzoic acid methyl ester (45
To a DMF solution (900 ml) of g), sodium hydride (11.3 g, 60%) was gradually added under ice-cooling, followed by stirring at the same temperature for 30 minutes. Next, a small amount of water was added to the reaction solution,
The reaction solution was poured into a large amount of water (3 l) and extracted with toluene (800 ml × 2). The toluene layer was dried using sodium sulfate, and the solvent was distilled off under reduced pressure. N-hexane was added to the obtained residue to remove nujol,
The residue was suspended in ethanol (500 ml), a 1N aqueous sodium hydroxide solution (500 ml) was added, and the mixture was stirred overnight.

Next, the solvent was distilled off under reduced pressure. The obtained white solid was dissolved in water (300 ml) and toluene (30 ml) was dissolved.
0 ml) to wash the aqueous layer. Further, the water layer is 4N-
The pH was adjusted to 2 using hydrochloric acid. The precipitated crystals were collected by filtration. It was dried under vacuum to obtain the desired product (36 g).

¹ H-NMR (CDCl ₃ , δ): 2.3
8 (3H, s), 3.83 (2H, m), 3.92 (2
H, m), 7.24 (2H, d, J = 8.3 Hz),
7.5-7.7 (3H, m), 7.93 (1H, d, J
= 8.8Hz)

[0340]

Reference Example 68 N- (N′-tosyl-3,4-dihydro-2H-1,4
-Benzoxazine-7-carbonyl) -L-α-amido
Synthesis of no-apicic acid dimethyl ester

N-tosyl 3,4-dihydro-2-1.4
Synthesis of -benzoxazine-7-carboxylic acid (2.98 g) in thionyl chloride suspension (15 ml)
Was added with a catalytic amount of DMF, and the mixture was stirred at room temperature for 2 hours. The solvent was distilled off under reduced pressure, and the obtained residue was dissolved in dichloromethane (40 ml) and neutralized with potassium carbonate while stirring with an aqueous solution (40 ml) of dimethyl homoglutamate hydrochloride (2.8 g). Next, 5.0 g of potassium carbonate was added, and the mixture was stirred vigorously at room temperature.

The organic layer was separated and washed with 1N HCl. After the organic layer was dried over sodium sulfate, the solvent was distilled off under reduced pressure. The obtained colorless oil was washed with CHCl ₃ : MeO
The mixture was subjected to silica gel column chromatography using a mixed solution of H = 100: 1 to obtain the desired product (4.3 g).

¹ H-NMR (CDCl ₃ , δ): 1.6
−2.1 (4H, m), 2.35 (2H, m), 2.3
9 (3H, s), 3.66 (3H, s), 3.72 (2
H, m), 3.77 (3H, s) 3.87 (2H,
m), 4.74 (1H, m), 6.73 (1H, d, J
= 7.3 Hz), 7.2-7.4 (4H, m), 7.5
1 (2H, d, J = 8.3 Hz), 7.93 (1H,
d, J = 8.8 Hz)

[0344]

Reference Example 69 N- (3,4-dihydro-2H-1,4-benzoxa)
(Zin-7-carbonyl) -L-α-amino-adipic
Synthesis of acid dimethyl ester

30% of anisole (4.3 g) in HBr
N- (N'-tosyl-3,4) in acetic acid solution (50 ml)
-Dihydro-2H-1,4-benzoxazine-7-carbonyl) -L-α-amino-adipacid dimethyl ester (4.3 g) was added, and the mixture was stirred at room temperature for 4 hours. Then, ether (400 ml) was poured, and the deposited precipitate was collected by Decantation. The obtained precipitate was suspended in a mixed solution of chloroform (100 ml) and a saturated aqueous solution of sodium hydrogen carbonate (100 ml), and the mixture was vigorously stirred. The organic layer was separated and dried over sodium sulfate. Next, the solution was distilled off under reduced pressure to obtain the desired product (1.4).
5 g) obtained.

¹ H-NMR (CDCl ₃ -CD ₃ OD,
δ): 1.6-2.1 (4H, m), 2.38 (2H,
t, J = 6.8 Hz), 3.46 (2H, m), 3.6
8 (3H, s), 3.77 (3H, s), 4.24 (2
H, m), 4.72 (1H, m), 6.59 (1H,
d, J = 8.3 Hz), 7.33 (2H, m)

[0347]

Working Example 36 N- [1-[((2,4-diamino) -6-pteridini)
M)]-3,4-dihydro-2H-1,4-ben
Zoxazine-7-carbonyl] -L-α-amino-a
Synthesis of dipic acid dimethyl ester

N- (3,4-dihydro-2H-1,4-
Benzoxazine-7-carbonyl) -L-α-amino-adipacid dimethyl ester (1.45 g)
And a suspension of 2,4-diaminopteridine-6-methylbromide hydrobromide / isopropanol (1.47 g) in dimethylacetamide (23 ml) were added at 60 ° C. for 6 hours.
Stirred for hours. Next, the mixture was poured into a saturated aqueous solution of sodium hydrogen carbonate (300 ml) and extracted with chloroform.
After the chloroform layer was dried over sodium sulfate, the solvent was distilled off. Using the obtained residue as a developing solvent, CHCl
₃ : The residue was subjected to silica gel column chromatography using MeOH = 10: 1 to obtain the desired compound (1.48 g).

¹ H-NMR (CDCl ₃ -CD ₃ OD,
δ): 1.6-2.0 (4H, m), 2.36 (2H,
t, J = 6.8 Hz), 3.58 (2H, m), 3.6
6 (3H, s), 3.76 (3H, s), 4.39 (2
H, m) 4.67 (2H, bs), 4.73 (1H,
m), 6.66 (1H, d, J = 8.3 Hz), 6.9
9 (1H, d, J = 7.3 Hz), 7.29 (2H,
m), 8.70 (1H, s)

[0350]

Working Example 37 N- [1-[((2,4-diamino) -6-pteridini]
M)]-3,4-dihydro-2H-1,4-ben
Zoxazine-7-carbonyl] -L-α-amino-a
Synthesis of dipic acid

After dissolving the compound of Example 36 (1.48 g) in ethanol (80 ml), 1N aqueous sodium hydroxide solution (8.5 ml) was added, and the mixture was stirred at 40 ° C. for 4 hours and further at room temperature. Stirred for 20 hours. Next, the reaction solution was concentrated to 5 ml under reduced pressure, redissolved in water (50 ml), and the insoluble material was filtered off.
H was set to 3.8 to 4.2, and the deposited precipitate was collected by filtration and dried in vacuo to obtain the desired product (1.26 g).

¹ H-NMR (DMSO-d ₆ , δ):
1.5-2.0 (4H, m), 2.14 (2H, t, J
= 6.8 Hz), 3.68 (2H, m), 4.28 (3
H, m), 4.71 (2H, bs), 6.80 (1H,
d, J = 8.3 Hz), 7.31 (2H, m), 8.1
3 (1H, d, J = 7.3 Hz), 8.71 (1H,
s)

[0353]

[Brief description of the drawings]

1-3, ³ at each concentration of the test drug H-
Indicates the amount (ratio) of incorporation of UdR. FIG. 4 shows changes in the weight of rats upon administration of MTX and the compound of the present invention, FIG. 5 shows changes in white blood cell count (WBC) and red blood cells (RBC), and FIG. 6 shows liver weight and TG content in liver. Represents the change.

──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Keiichiro Tsuji 1-135 Komamon, Gotemba, Shizuoka Prefecture Inside Chugai Pharmaceutical Co., Ltd. (72) Inventor Hiroshi Suzuki 3-41-8 Takada, Toshima-ku, Tokyo Chugai Pharmaceutical Co., Ltd. In-house (72) Inventor Yasuhisa Takeda 1-1135 Komamon, Gotemba-shi, Shizuoka Prefecture Inside Chugai Pharmaceutical Co., Ltd. Aki (56) References JP-A-60-115584 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 31/519 C07D 475/08 REGISTRY (STN) CA (STN) CAOLD (STN)

Claims (1)

(57) [Claims] 1. A compound of the general formula (I) Wherein R ₁ is CH ₂ , CH ₂ CH ₂ , CH ₂ O, CH ₂
Shows a member selected from the group consisting of S and CH _{2 SO;} R 2 represents a hydrogen atom or a lower alkyl group or a benzyl group having a carbon number of 1 to 4; n is an integer of from 1 to 4; R ₃ Is a general formula COOR ₄ (where R ₄ represents a hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms) or a general formula NHCOR ₅ (where R ₅ represents a phenyl group which may be substituted) or a general formula Formula CONR ₆ R
₇ (where R ₆ represents a hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms; R ₇ represents a lower alkyl group having 1 to 4 carbon atoms or a phenyl group or a carboxyalkyl group which may be substituted or a lower alkyl group) A sulfonyl group) or a group represented by PO ₃ H ₂ or SO ₃ H;
An antirheumatic agent comprising a compound represented by the formula (1) as an active ingredient.