JP3387943B2 - Anticancer drug - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] BACKGROUND OF THE INVENTION The present invention relates to a novel methotrexe
Derivatives, and more specifically, novel methods useful as anticancer agents.
It relates to a totrexate derivative. [0002] 2. Description of the Related Art Methotre
Kisate has been used as a treatment for leukemia since ancient times.
However, in 1951, Gubner et al.
(RA) and its efficacy in psoriasis
It has been used in Europe and the United States as a remedy. Relatively recently
Detailed study of dosage and administration was conducted,
Rexate therapy has relatively few side effects and is excellent
It has proven to be effective. But me
Liver damage and lung fibrosis caused by taking totrexate
Since side effects cannot be ignored, there are fewer side effects.
There is a demand for a drug with excellent efficacy. [0003] Until now, N¹⁰Other than a methyl group
As a methotrexate derivative with a kill group introduced
Is, for example, Embedded image (J. Med. Chem.,22, 862 (197)
9)) and expressions Embedded image (J. Med. Chem.,25, 877 (198
2)) etc. are known, but those showing satisfactory activity
Did not. [0004] We have discovered that this type of methotrexate
In search of compounds with superior anticancer action in derivatives
After intensive research, the present invention has been accomplished. [0005] According to the present invention, there is provided a compound represented by the following general formula:
(I) Embedded image Where R₁Is CH₂, CH₂CH₂, CH₂O, CH₂
S, CH₂SO, CH₂SO₂And CH₂Composed of NH
R represents a member selected from the group:₂Is a hydrogen atom or charcoal
Represents a lower alkyl group having a prime number of 1 to 4;
R represents an integer;₃Is the general formula COOR₄(Where R₄
Represents a hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms.
) Or the general formula NHCOR₅(Where R₅Is replaced
Or a phenyl group which may be represented by the general formula CONR
₆R₇(Where R₆Is a hydrogen atom or a group having 1 to 4 carbon atoms
R represents a lower alkyl group;₇Represents a lower alkyl group having 1 to 4 carbon atoms.
Represents an alkyl group or an optionally substituted phenyl group.
Or PO₃H₂, SO₃A group represented by H;
To provide a methotrexate derivative represented by
You. [0006] The compounds of the present invention are all new
It is a reference compound and is synthesized, for example, as follows. (Method A) Embedded image(Method B) Embedded image(Method C) R in the general formula (I)₃Is the general formula Embedded image (R ′: hydrogen atom or lower alkyl having 1 to 4 carbon atoms)
Base) Embedded image (Where R₁, R₂, R₃And n have the same meaning as above
R ′ is a hydrogen atom or a lower alkyl having 1 to 4 carbon atoms.
A kill group;₁And A₂Represents a protecting group, and X represents
Indicates a logen atom. ) In the method A, the compound of the general formula (1)
The reaction for obtaining the compound of the general formula (2) from the compound of the general formula (1)
The compound is converted to an acid halo such as thionyl chloride, oxalyl chloride, etc.
Suspended in a gentizing agent, and a catalytic amount of dimethylformamide
It is performed by stirring at room temperature in the coexistence. Where A₁so
The protecting groups shown include carbobenzoxy group, tosyl
Group, acetyl group and the like. The compound of the general formula (2) and the compound of the general formula (3)
The reaction for obtaining the compound of the general formula (4) from the compound is represented by the general formula
Compound (2) dissolved in a solvent such as dichloromethane
The water of the compound of the general formula (3) is cooled under ice-cooling or water-cooling.
In addition to the solution, potassium carbonate, sodium hydroxide, carbonated water
Stir at room temperature in the presence of an inorganic base such as sodium hydrogen
And so on. The compound of the general formula (4) is converted from the compound of the general formula (5)
The reaction to obtain the compound is bromination of anisole and phenol.
Compound of general formula (4) in a solution dissolved in hydrogen-acetic acid solution
And stirring at 10 ° C to 60 ° C, preferably at room temperature
Performed by Further, from the compound of the general formula (4),
The reaction for obtaining the compound of the formula (5) is carried out by reacting the compound of the general formula (4)
Dissolve in a solvent such as methanol, ethanol, acetic acid, etc.
After adding radium-carbon, stir at room temperature under hydrogen atmosphere
May be performed. The compound of the general formula (6) and the compound of the general formula (5)
The reaction for obtaining the compound of the general formula (7) from the compound is performed by the general formula
The compound of (6) and the compound of general formula (5) are dimethylacetate
0 ° C to 1 in a solvent such as toamide and dimethylformamide
The stirring is carried out at 00 ° C, preferably at 50 ° C to 60 ° C. In particular
R₂Is a hydrogen atom, methanol or ethanol
To a solvent such as ethanol.
The desired product is stirred at 0 ° C to 60 ° C, preferably at 35 ° C.
obtain. In the formula, the halogen atom represented by X is bromine
Atom, chlorine atom and the like. In the method B, the compound of the general formula (6)
A compound of the general formula (9) is obtained from a compound of the general formula (8)
The reaction is carried out by reacting the compound of the general formula (6) with the compound of the general formula (8)
With dimethylacetamide or dimethylformamide
Stir at 0 ° C to 100 ° C, preferably 55 ° C in a medium
Performed by The compound of the general formula (9) and the compound of the general formula (3)
The reaction for obtaining the compound of the general formula (7) from the compound is performed by the general formula
The compound of (9) is used as
Hexylcarbodiimide and 1-hydroxybenzotria
Dimethylformamide, dimethyla
Stir in a solvent such as Cetamide, N-methylpyrrolidinone, etc.
After that, the compound of the general formula (3) is added, and 0 ° C to 200 ° C is added.
It is preferably performed by stirring at 10 ° C to 80 ° C. Special
To R₂Is a hydrogen atom, methanol or ethanol
Add a 1N sodium hydroxide solution in a solvent such as
And stirring at 0 ° C to 60 ° C, preferably at room temperature to obtain the desired product
You. In the method C, the compound of the general formula (10)
And a compound of the general formula (12) from the compound of the formula (11)
The reaction is performed by converting the compound of the general formula (10) into chloroform and
Non-chloromethane, tetrahydrofuran, dioxane, etc.
Dissolve in a protic solvent and add the compound of formula (11) and water
For example, potassium carbonate, triethylamine, sodium hydrogen carbonate
And pyridine, etc., and stirred at room temperature.
U. Where A₂As the protecting group represented by, carboben
Examples include a zoxy group, a tosyl group, and an 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 mixture is stirred at 60 ° C to -20 ° C, preferably at -30 ° C.
It is carried out by refluxing after adding onyl. From the compound of the general formula (13), the compound of the general formula (1)
The reaction for obtaining the compound of 4) is carried out by reacting the compound of general formula (13)
Ethanol, methanol, tetrahydrofuran, diox
Dissolve in sun, etc., and in a hydrogen atmosphere in the presence of palladium-carbon
It is performed by stirring at room temperature under air. The compound of the general formula (14) and the compound of the general formula (2)
The reaction for obtaining the compound of the general formula (15) from the compound is carried out by the general
The compound of the formula (2) is dissolved in dichloromethane or the like.
Dissolve the compound of general formula (14) with potassium carbonate or
Add ethylamine and water and stir at room temperature
Perform the mixed anhydride method, active ester or active
The amidation may be carried out by the amide method. From the compound of the general formula (15), the compound of the general formula (1)
The reaction for obtaining the compound of 6) is carried out by reacting the compound of general formula (15)
Dissolve phenol or anisole in advance
Hydrogen bromide-acetic acid was added and stirred at room temperature.
U. The compound of the general formula (6) and the compound of the general formula (16)
The reaction for obtaining the compound of the general formula (17) from the compound is a
Such as tilacetamide and dimethylformamide
25 ° C. to 100 ° C., preferably 5 ° C. in an aprotic polar solvent
After stirring at 0 ° C. to 65 ° C., for example, triethylamine,
In water containing potassium carbonate or sodium hydrogen carbonate
This is performed by stirring. From the compound of the general formula (17), the compound of the general formula (I)
The reaction to obtain the compound of
Add sodium solution and stir at room temperature. [0020] The compound represented by the general formula (I) obtained by the present invention.
The compounds have an anticancer effect. This effect is demonstrated in the following experiment
Example: Confirmed by conducting a mouse cancer cell growth inhibition experiment
did. "Mouse cancer cell growth inhibition experiment (cancer
for)" (Method) RPMI 1640 medium (fetal bovine serum 5
%, 2-mercaptoethanol in 10^-6To be M
Mouse phosphorus suspended in
Oplasma P388 cells or mouse colon
26 carcinoma cells were transferred to a flat bottom microplate (Co
5 × 10 in each well of # 25870)
³Drug solution prepared in the same medium
Was added so that each well was 0.2 ml. This
This was placed in a carbon dioxide cultivation apparatus (carbon dioxide 5%, air 95%, wet
After culturing for 3 days in the MTT solution (3-%
(4,5-dimethylthiazol-2-yl) -2,5-
Diphenyltetrazolium bromide in phosphate buffered physiology
5 mg / ml with a common saline solution (pH 7.4, 10 mM).
Was added to each well in an amount of 10 μl.
After further culturing for 4 hours, the culture supernatant was
The resulting dye (formazan) is removed by D
MSO (dimethyl sulfoxide; 150 μl / well
) And absorb with a microplate spectrophotometer.
The degree (540 nm) was measured. The drugs used are as follows. Embedded image (Results and Discussion) Using P388 cells in FIG.
Example of the case where colon 26 is used in FIG. 2
showed that. Data are for wells with no drug added
Cell proliferation, that is, the absorbance is set to 100%, and the drug is added.
To which extent growth was suppressed
Is shown in%. As is apparent from FIG. 1 and FIG.
Ming's compound has better inhibitory effect on cancer cell growth than control drug
(Carcinostatic action). [0026] 【Example】 [Reference Example 1]Synthesis of 1-carbobenzoxy-5-carboxyindoline
Success 5-carboxyindoline (2.0 g)
Sodium hydroxide (0.6g) was added to water (20ml)
After adding to a mixture of chill ether (20 ml), ice-water
Under cooling, carbobenzoxycyclolide (2.59 g) and water
Sodium oxide (2.1 g) was added and water (10 ml) was exchanged.
They were added to each other and stirred at room temperature for 2 hours. Reaction solution is 2N-salt
The mixture was acidified with an acid, and the precipitated crystals were filtered. Wash with ether
After the purification, the product was air-dried to obtain the desired product (2.8 g). [0028]¹H-NMR (DMSO-d₆, 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 <0> C [0029] [Reference Example 2]N- (1-carbobenzoxyindoline-5-carboni
G) Synthesis of diethyl-L-glutamate Reference Example 1 Compound (2.5 g)
After suspension in onil (10 ml), a catalytic amount of dimethylform
The amide was added and stirred at room temperature for 30 minutes. Then under reduced pressure
The excess thionyl chloride is distilled off, and the residue is washed with N-hexane.
Triturated. After filtering the obtained crystals,
Dissolve in dichloromethane (20 ml) and add this dichloromethane
Glutamic acid diethyl ester hydrochloride under ice-water cooling
Contains salt (3.0 g) and triethylamine (2.8 g)
It was added dropwise to a dichloromethane (50 ml) suspension. At room temperature
After stirring for 2.5 hours, the solvent was distilled off under reduced pressure.
Under ice-water cooling, ethyl acetate (200 ml) and dilute hydrochloric acid (2
00 ml). After stirring for 5 minutes, organic
The layers were separated. Wash the organic layer with 5% aqueous sodium carbonate solution
After cleaning, it was dried with magnesium sulfate. Then ethyl acetate
Is distilled off under reduced pressure, and the obtained residue is
Chromatography and chloroform
Using a mixed solvent of methanol: methanol = 30: 1,
(3.1 g) was obtained. [0031]¹H-NMR (CDCl₃, 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 [0032] [Reference Example 3]N- (indoline-5-carbonyl) -L-glutamine
Of diethyl methacrylate Reference Example 2 Compound (1.8 g)
Dissolve in hydrofuran (80 ml) and add 10% palladium
-After adding carbon (0.4 g), at room temperature under hydrogen atmosphere
Stir for 5 hours. Palladium-carbon using celite
The desired product (1.2 g) was obtained by filtration. [0034]¹H-NMR (CDCl₃, 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), 3.59 (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 [0035] Embodiment 1N- [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 hydrobromic acid
Salt / isopropanol adduct (250 mg) in dimethyl
Suspend in acetamide (3 ml), 4 hours at 50-55 ° C
While stirring. After cooling, triethylamine (12
4 mg) in water (15 ml) and stirred.
Extraction was performed four times with loroform (350 ml). Yes
After drying the organic layer with magnesium sulfate, the solvent was distilled off under reduced pressure.
The residue obtained is separated by silica gel column chromatography.
And eluted with chloroform: methanol
= 10: 1 using a mixed solvent to give the desired product (200 mg)
Obtained. [0037]¹H-NMR (DMSO-d₆, 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; 168-170 ° C [0038] Embodiment 2N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] indolinecarbonyl] -L-glutamic acid
Synthesis of (Compound 1) Embodiment 1 Compound (170 mg)
Dissolved in ethanol (33 ml) at 35 ° C with 1N sodium hydroxide
An aqueous solution of lithium (0.84 ml) was added, and the mixture was heated at the same temperature for 4.5 hours.
Stirred for hours. After further stirring at 25 ° C. for 20 hours,
Water (2 ml) was added to the reaction solution. Reaction solution under ice-water cooling
Was adjusted to pH = 3.7 with 1N-hydrochloric acid and left overnight in a cool place
did. The deposited precipitate was collected by filtration and the desired product (130 mg)
I got [0040]¹H-NMR (DMSO-d₆, 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.) [0041] [Reference Example 4]N- (1-carbobenzoxyindolin-5-carboni
L) Synthesis of dimethyl-L-α-aminoadipate 1-carbobenzoxyindoline-5-ca
Rubonic acid (3.1 mg) in thionyl chloride (10 ml)
After suspension, add a catalytic amount of dimethylformamide and
Stir for 2 hours. The excess thionyl chloride is then removed under reduced pressure.
The residue was triturated with n-hexane. Profit
After filtering the obtained crystals, dichloromethane (30 ml) was used.
And dissolved in dichloromethane under ice-water cooling.
Including α-aminoadipate dimethylhydrochloride group (2.7 g)
The mixture was added dropwise with an aqueous solution (30 ml). Furthermore, this reaction
Potassium carbonate (5.8 g) was added to the solution. At room temperature,
After stirring overnight, convert the reaction mixture to saturated sodium bicarbonate solution.
The organic matter was extracted with chloroform. Chloroform
The layer was washed with 1N hydrochloric acid, dried over sodium sulfate and reduced.
The solvent was distilled off under pressure. The residue obtained is silica gel
Chromatography, and use chloroform as the elution solvent.
Using a mixed solvent of lum: methanol = 100: 1
(3.1 mg) was obtained. [0043]¹H-NMR (CDCl₃, 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) [0044] [Reference Example 5]N- (indoline-5-carbonyl) -L-α-amino
Synthesis of dimethyl adipate Anisole (1.5 g) 30% hydrogen bromide
-Reference Example 4 in acetic acid (15 ml) solution. Compound (1.5
g) was added and stirred at room temperature for 4 hours. Then, add
A reddish-brown oil precipitated upon addition of an amount of ether.
Was. Remove the oily substance from chloroform by removing most of the ether layer.
Suspended in saturated sodium bicarbonate solution
And the chloroform layer was separated. Chloroform layer
Was dried over sodium sulfate, and the solvent was distilled off under reduced pressure.
Target (960 mg) was obtained. [0046]¹H-NMR (CDCl₃, 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 (2H, s),
4.78 (1H, m), 6.62 (2H, m), 7.5
4 (2H, m) [0047] Embodiment 3N- [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 hydrobromic acid
Salt and isopropanol adduct (1140 mg)
Suspend in luacetamide (20 ml) and at 50-60 ° C
Stir for 6 hours. After cooling, saturated sodium bicarbonate solution
And extract the target substance in three portions with chloroform.
Was. After drying the organic layer over sodium sulfate, the solvent was removed under reduced pressure.
The residue obtained is distilled off and the resulting residue is purified by silica gel column chromatography.
And then eluted with chloroform: methanol
(100m) using a mixed solvent of 100: 10
g) was obtained. [0049]¹H-NMR (CDCl₃, 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) [0050] Embodiment 4N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] -indoline-5-carbonyl] -L-α-a
Synthesis of minoadipic acid Embodiment 3 FIG. Compound (400 mg)
Dissolved in ethanol (22 ml) and 1N-hydroxide at 35 ° C.
An aqueous solution of thorium (3.1 ml) is added, and the same temperature is applied for 4 hours.
Stirred. After further stirring at 25 ° C. for 20 hours,
Water (3 ml) was added to the reaction solution, and the reaction solution was separated under reduced pressure.
To dryness. At this time, make sure that the outside temperature does not exceed 30 ° C.
Was. The resulting yellow solid was dissolved in water (10 ml) and 1
Adjust the pH to 3.7 with N-hydrochloric acid and place in refrigerator for 2 hours
I left it. The deposited precipitate was collected by filtration, and the desired product (320 m
g) was obtained. [0052]¹H-NMR (CDCl₃, Δ): 1.4.
-1.9 (4H, m), 2.23 (2H, t, J = 6.
8Hz), 3.01 (2H, m), 3.58 (2H,
m), 4.32 (1H, m), 4.55 (2H, s),
6.69 (1H, d, J = 8.3 Hz), 7.63 (2
H, m), 8.10 (1H, d, J = 8.3 Hz),
8.72 (1H, s) [0053] [Reference Example 6]Nα- (1-carbobenzoxyindoline-5-carbo
Nyl) -Nδ-benzoyl-L-ornithine methyl
Steal Synthesis 1-carbobenzoxyindoline-5-ca
Thionyl chloride (1.5 ml) in rubonic acid (180 mg)
To make a suspension, and add a catalytic amount of
Tilformamide was added and the mixture was stirred at room temperature for 2 hours. Next
The reaction mixture was concentrated to dryness under reduced pressure. The resulting solid is
Dissolve in chloromethane (4 ml) and add Nδ-
Benzoyl-L-ornithine methyl ester (150m
g), calcium carbonate (750 mg) and water
(4 ml) was added and stirred vigorously at room temperature for 12 hours. Next
The reaction mixture was poured into water and extracted with chloroform.
The roloform layer is washed with a 1N hydrochloric acid solution and washed with sodium sulfate.
And dried. The solvent was distilled off under reduced pressure, and the obtained residue was
Silica gel chromatography, elution solvent
Using chloroform: methanol = 100: 3
(140 mg). [0055]¹H-NMR (CDCl₃, 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) [0056] [Reference Example 7] Nα- (indoline-5-carbonyl) -Nδ-benzo
Il-L-ornithineSynthesis of methyl ester Phenol (150 mg) in 30% bromide water
Reference Example 6 in a hydrogen-acetic acid (2 ml) solution. Of the compound (140
mg) and stirred at room temperature for 4 hours. Next, add
A reddish-brown oil precipitated upon addition of an amount of ether.
Was. Remove the oily substance from chloroform by removing most of the ether layer.
Suspended in saturated sodium bicarbonate solution
And extracted with chloroform. Chloroform layer
Dry over sodium sulfate and evaporate the solvent under reduced pressure
(50 mg). [0058]¹H-NMR (CDCl₃, 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) [0059] 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-butane
Lomomethyl-2,4-diaminopteridine hydrobromide
Isopropanol adduct (44 mg)
Suspend in amide (1 ml) and stir at 50-55 ° C for 4 hours
did. After cooling, triethylamine (22 mg) was added to the reaction mixture.
Water (3 ml) containing water and stirring, and then chloroform.
Extracted. After drying the chloroform layer with sodium sulfate,
The solvent was distilled off under reduced pressure, and the resulting residue was
After the chromatography, chloroform:
Using a mixed solvent of methanol = 10: 1, the desired product (34 m
g) was obtained. [0061]¹H-NMR (CDCl₃, 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), 4.6 (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) [0062] Embodiment 6Nα- [1-[(2,4-diamino-6-pteridini)
Ru) methyl] -indoline-5-carbonyl] -Nδ-
Synthesis of benzoyl-L-ornithine Embodiment 5 FIG. Compound (34 mg) in ethanol
(5 ml), and further 1N sodium hydroxide
Solution (0.1 ml) and stirred at 35 ° C. for 4.5 hours
did. After stirring at 25 ° C. for 20 hours, water (1 m
l) was added, and the reaction solution was dried under reduced pressure. This and
The outside temperature did not exceed 30 ° C. The yellow solid obtained
The form was dissolved in water (5 ml) and pH = 3 with 1N hydrochloric acid.
7 and left in the refrigerator for 2 hours. Deposited sediment
The residue was collected by filtration to obtain the desired product (26 mg). [0064]¹H-NMR (DMSO-d₆, 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.) [0065] [Reference Example 8]N-phthaloyl-N-carbobenzoxy-ornithine
Synthesis of methyl ester N-carbobenzoxy-L-ornithine
(2.0 g) in dichloromethane (70 ml) solution
Phthalic acid (2.45 g) was added, followed by water (70 m
l), potassium carbonate (1.12 g) was added and the mixture was kept at room temperature for 15 hours.
And stirred. 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 vacuumed.
Dried. 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. Return the reaction solution slowly to room temperature.
The mixture was refluxed for another 2 hours. Solvent is distilled off under reduced pressure
The residue obtained was subjected to silica gel chromatography.
And elution solvent: chloroform: methanol = 10
The desired product (2.16 g) was obtained using 0: 1. [0067]¹H-NMR (CDCl₃, 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) [0068] [Reference Example 9]Synthesis of N-phthaloyl-ornithine methyl ester Reference Example 8 Of the compound of formula (2.16 g)
10% palladium-carbon in ethanol solution (100 ml)
(500 mg) at room temperature in a hydrogen atmosphere at room temperature.
Stirred for hours. Filter palladium-carbon using Celite
The solvent was distilled off under reduced pressure. The resulting residue is
It is subjected to Kagel chromatography, and
The desired product (22) was obtained using chloroform: methanol = 100: 3.
3 mg). [0070]¹H-NMR (CDCl₃, 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) [0071] [Reference Example 10] N- (1-carbobenzoxyindoline-5-carboni
L) phthaloyl-olSynthesis of Nitin Methyl Ester 1-carbobenzoxyindoline-5-ca
Thionyl chloride in ruboxylic acid (297 mg)
(2.5 ml) to give a suspension, and this suspension
And add a catalytic amount of dimethylformamide to it at room temperature for 2 hours.
While stirring. Next, the reaction solution was concentrated to dryness under reduced pressure. Get
The solid thus obtained was dissolved in dichloromethane (7 ml).
Reference Example 9. Compound (250 mg), potassium carbonate
(640 mg) and water (7 ml).
Stirred vigorously at room temperature for 12 hours. Next, the reaction solution is
Extract with chloroform and further add 1N chloroform layer
-Washed with hydrochloric acid solution and dried over sodium sulfate. solvent
Was distilled off under reduced pressure. The residue obtained is purified by silica gel chromatography.
After the chromatography, chloroform:
The desired product (330 mg) was prepared using methanol = 100: 3.
Obtained. [0073]¹H-NMR (CDCl₃, 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) [0074] [Reference Example 11]N- (indoline-5-carbonyl) -N-phthaloyl
-Synthesis of ornithine methyl ester Phenol (300 mg) in 30% bromide water
Reference Example 10 in a hydrogen-acetic acid (8 ml) solution. Compound (33)
0 mg) and stirred at room temperature for 4 hours. Next, the reaction solution
When a large amount of ether was added, a reddish-brown oily substance precipitated.
I did it. Remove the oily substance from chloropho
Suspended in saturated sodium bicarbonate.
Washed with the solution and extracted with chloroform. Chloroform
Dry the layer with sodium sulfate and evaporate the solvent under reduced pressure
Then, the desired product (147 mg) was obtained. [0076]¹H-NMR (CDCl₃, 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) [0077] Embodiment 7N- [1- [2,4-diamino-6-pteridinyl) me
Tyl] -indoline-5-carbonyl] -N-phthaloy
Synthesis of ru-ornithine methyl ester Reference Example 11 Compound (146 mg) and 6
-Bromomethyl-2,4-diaminopteridine hydrogen bromide
Isopropanolate adduct (115 mg) in dimethyl
Suspended in acetamide (1.0 ml) at 50-55 ° C
Stir for 4 hours. After cooling, triethylamine
(30 mg) in water (4 ml), stir and then cool.
Extracted with loroform. Chloroform layer with sodium sulfate
After drying with a solvent, the solvent was distilled off under reduced pressure.
The gel is subjected to Ricagel chromatography and used as an elution solvent.
Using a mixed solvent of loroform: methanol = 10: 1
Was obtained (140 mg). [0079]¹H-NMR (CDCl₃, 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) [0080] Embodiment 8N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] -indoline-5-carbonyl] -N-hemif
Synthesis of Talloylu Ornithine (Compound 2) Embodiment 7 FIG. Compound (140 mg) in 2N
Suspended in sodium hydroxide solution (10 ml), 30
Stirred at C for 12 hours. Dry the reaction mixture under reduced pressure
The yellow solid obtained was dissolved in water (5 ml) and 1N
-Adjust to pH = 3.7 with hydrochloric acid and release for 2 hours in refrigerator
Placed. The deposited precipitate was collected by filtration. The obtained yellow solid
The product is subjected to silica gel chromatography,
And chloroform: methanol: 28% -ammonia water
= 5: 4: 1 using a mixed solvent to give the desired product (20 mg)
Was. [0082]¹H-NMR (DMSO-d₆, Δ):
1.5-2.1 (4H, m), 3.14 (2H, t, J
= 8.3 Hz), 3.58 (2H, t, J = 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.) [0083] [Reference Example 12]N- (3-methoxycarbonylbenzoyl) -N-cal
Synthesis of bobenzoxy-ornithine methyl ester N-carbobenzoxy-L-ornithine
(2.4 g) in dichloromethane (40 ml)
Phthalic acid monomethyl ester chloride (2.1 g)
Then water (40 ml), potassium carbonate (2.4
g) was added and the mixture was stirred at room temperature for 15 hours. Reduce the pressure of the reaction solution
To 30 ml and adjust to pH = 3 with 1N-hydrochloric acid.
The resulting precipitate was collected by filtration and dried under vacuum. White solid obtained
Is dissolved in low-moisture methanol (100 ml), and this solution is dissolved.
Was cooled to −30 ° C. and stirred for 10 minutes. Next, at the same temperature
Then, thionyl chloride (3 ml) was slowly added dropwise. reaction
The solution was slowly brought to room temperature and refluxed for another 2 hours.
Was. The solvent is distilled off under reduced pressure, and the obtained residue is silica gel.
Chromatography and chloroform as elution solvent
Using methanol: methanol = 100: 1 (600 m
g) was obtained. [0085]¹H-NMR (CDCl₃, Δ): 1.
6-2.0 (4H, m), 3.46 (2H, m), 3.
70 (3H, s), 3.89 (3H, s), 4.34
(1H, m), 5.08 (2H, s), 5.88 (1
H, d, J = 7.8 Hz), 7.31 (5H, s),
7.45 (1H, m), 8.05 (2H, m), 8.4
1 (1H, s) [0086] [Reference Example 13]N- (3-methoxycarbonylbenzoyl) -ornichi
Synthesis of methyl ester Reference Example 12 Of the compound (600 mg)
10% palladium-charcoal in a ethanol solution (100 ml)
After adding hydrogen (100 mg), the mixture was added at room temperature under a hydrogen atmosphere at room temperature.
Stirred for 0 hours. Palladium-carbon using celite
After filtration, the solvent was distilled off under reduced pressure. The resulting residue is
The gel is subjected to Ricagel chromatography and used as an elution solvent.
Using roroform: methanol = 100: 3, the desired product (3
60 mg). [0088]¹H-NMR (CDCl₃, 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) [0089] [Reference Example 14]N- (1-carbobenzoxyindoline-5-carboni
) -N- (3-methoxycarbonylbenzoyl) -o
Synthesis of lunitine methyl ester 1-carbobenzoxyindoline-5-ca
Thionyl chloride in ruboxylic acid (350mg)
(5 ml) to make a suspension, and then touch this suspension.
A medium amount of dimethylformamide was added and stirred at room temperature for 2 hours.
Stirred. Next, the reaction solution was concentrated to dryness under reduced pressure. Got
The solid was dissolved in dichloromethane (7 ml) and the solution dissolved.
Reference Example 13. Compound (360 mg), potassium carbonate
(650 mg) and water (7 ml)
Stirred vigorously at warm for 12 hours. Next, pour the reaction solution into water
The mixture was extracted with chloroform.
Washed with hydrochloric acid solution and dried over sodium sulfate. Solvent
The solvent was distilled off under reduced pressure. The residue obtained is chromatographed on silica gel.
After performing chromatography, chloroform:
The desired product (390 mg) was obtained using tanol = 100: 3.
Was. [0091]¹H-NMR (CDCl₃, 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) [0092] [Reference Example 15]N- (indoline-5-carbonyl) -N- (3-meth
(Xycarbonylbenzoyl) -ornithine methyl ester
Synthesis 30% hydrogen bromide of anisole (0.5 g)
Reference Example 14 in acetic acid (6 ml) solution. Compound (390m
g) was added and stirred at room temperature for 4 hours. Next, add
A reddish-brown oil precipitated upon addition of an amount of ether.
Was. Remove the oily substance from chloroform by removing most of the ether layer.
Suspended in saturated sodium bicarbonate solution
And extracted with chloroform. Chloroform layer
After drying over sodium sulfate, the solvent was distilled off under reduced pressure.
The obtained white solid was subjected to n-hexane-chloroform-meta
Recrystallization from knol gave the desired product (192 mg). [0094]¹H-NMR (CDCl₃, 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) [0095] Embodiment 9N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] -indoline-5-carbonyl] -N- (3-
Methoxycarbonylbenzoyl) -ornithine methyl
Steal Synthesis Reference Example 15 Compound (192 mg) and 6
-Bromomethyl-2,4-diaminopteridine hydrogen bromide
Acid salt (142 mg) was added to dimethylacetamide (2.5 m
l) and stirred at 50-55 ° C for 4 hours. cooling
Then, water containing triethylamine (43 mg) of the reaction solution
(5 ml), stirred and then extracted with chloroform.
Was. After drying the chloroform layer with sodium sulfate,
The solvent was distilled off under reduced pressure, and the resulting residue was purified by silica gel chromatography.
After performing chromatography, chloroform:
The target compound (110 m
g) was obtained. [0097]¹H-NMR (CDCl₃, Δ): 1.7.
−2.1 (4H, m), 3.06 (2H, t, J = 8.
3Hz), 3.55 (4H, m), 3.78 (3H,
s), 3.93 (3H, s), 4.52 (2H, 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 (1H, s) [0098] Embodiment 10N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] -indoline-5-carbonyl] -N-isofu
Synthesis of Taroyl-Ornithine Embodiment 9 FIG. Compound (110 mg)
Dissolved in ethanol (14 ml), and further added with 1N sodium hydroxide.
Lithium solution (0.48 ml) was added and the mixture was heated at 35 ° C for 4.5 hours
While stirring. After stirring at 25 ° C. for 20 hours, water was added to the reaction solution.
(1 ml) was added, and the reaction solution was dried under reduced pressure.
At this time, the external temperature did not exceed 30 ° C. Got
Dissolve the yellow solid in water (5 ml) and add 1N hydrochloric acid to pH
= 3.7 and left in the refrigerator for 2 hours. Precipitation
The resulting precipitate was collected by filtration to obtain the desired product (78 mg). [0100]¹H-NMR (DMSO-d₆, 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) [0101] [Reference Example 16]N- (4-methoxycarbonylbenzoyl) -N-cal
Synthesis of bobenzoxy-ornithine methyl ester N-carbobenzoxy-L-ornithine
(2.0 g) in dichloromethane (60 ml) solution
Phthalic acid monomethyl ester chloride (3.0 g)
Then water (60 ml), potassium carbonate (4.8)
g) was added and the mixture was stirred at room temperature for 15 hours. Reduce the pressure of the reaction solution
And concentrated to 50 ml, adjusted to pH = 3 with 1N hydrochloric acid and precipitated.
The resulting precipitate was collected by filtration and dried in vacuo. The obtained white solid
Dissolve in low-moisture methanol (100 ml) and add this solution
It cooled to -30 degreeC and stirred for 10 minutes. Next, at the same temperature
Thionyl chloride (3 ml) was slowly added dropwise. Reaction
The solution was slowly returned to room temperature and refluxed for another 2 hours.
Was. The solvent is distilled off under reduced pressure, and the obtained residue is silica gel.
Chromatography and chloroform as elution solvent
Using methanol: methanol = 100: 1 (710 m
g) was obtained. [0103]¹H-NMR (CDCl₃, 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) [0104] [Reference Example 17]N- (4-methoxycarbonylbenzoyl) -ornichi
Synthesis of methyl ester Reference Example 16 Of the compound (710 mg)
10% palladium on carbon in ethanol solution (100 ml)
(100 mg), and then added at room temperature under hydrogen atmosphere.
Stirred for hours. Filtration of palladium-carbon using Celite
The solvent was distilled off under reduced pressure. The resulting residue is
It is subjected to Kagel chromatography, and
The desired product (41) was obtained using chloroform: methanol = 100: 3.
0 mg). [0106]¹H-NMR (CDCl₃, 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) [0107] [Reference Example 18]N- (1-carbobenzoxyindoline-5-carboni
Ru) -N- (4-methoxycarbonylbenzoyl) -o
Synthesis of lunitine methyl ester 1-carbobenzoxyindoline-5-ca
Thionyl chloride in ruboxylic acid (260mg)
(2.5 ml) to give a suspension, and this suspension
And add a catalytic amount of dimethylformamide to it at room temperature for 2 hours.
While stirring. Next, the reaction solution was concentrated to dryness under reduced pressure. Get
The solid thus obtained was dissolved in dichloromethane (6 ml).
Reference Example 17. Compound (245 mg), potassium carbonate
(812 mg) and water (6 ml).
The mixture was stirred vigorously at room temperature for 12 hours. Next, add the reaction solution to water
Open and extract with chloroform.
Washed with N-hydrochloric acid solution and dried over sodium sulfate. Dissolution
The medium was distilled off under reduced pressure. The resulting residue is
Chromatography and chloroform
The target substance (310 mg) using methanol: methanol 100: 3
I got [0109]¹H-NMR (CDCl₃, 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) [0110] [Reference Example 19]N- (indoline-5-carbonyl) -N- (4-meth
(Xycarbonylbenzoyl) -ornithine methyl ester
Synthesis 30% hydrogen bromide of phenol (1.0 g)
-Reference Example 18 in acetic acid (10 ml) solution. Compound (40)
0 mg) and stirred at room temperature for 4 hours. Next, the reaction solution
When a large amount of ether was added, a reddish-brown oily substance precipitated.
I did it. Remove the oily substance from chloropho
Suspended in saturated sodium bicarbonate.
After washing with the solution, the desired product was extracted with chloroform. Black
The roform layer is dried over sodium sulfate, and the solvent is evaporated under reduced pressure.
Was distilled off. The obtained white solid was washed with n-hexane-chloroform.
Recrystallized from form-methanol to give the desired product (145 mg)
I got [0112]¹H-NMR (CDCl₃, 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) [0113] Embodiment 11N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] indoline-5-carbonyl] -N- (4-meth
Toxylcarbonylbenzoyl) -ornithine methyles
Tell synthesis Reference Example 19 Compound (140 mg) and 6
-Bromomethyl-2,4-diaminopteridine hydrogen bromide
Acid salt (105 mg) was added to dimethylacetamide (2.0 m
l) and stirred at 50-55 ° C for 4 hours. cooling
Then, water containing triethylamine (32 mg) of the reaction solution
(4 ml), and the mixture is stirred.
Extracted. After drying the chloroform layer with sodium sulfate,
The solvent was distilled off under reduced pressure, and the resulting residue was
After the chromatography, chloroform:
Using a mixed solvent of methanol = 10: 1, the target compound (180
mg). [0115]¹H-NMR (CDCl₃, 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) [0116] Embodiment 12 N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] indoline-5-carbonyl] -N-terephthalate
Synthesis of Loyl-Ornithine Embodiment 11 FIG. Compound (150 mg)
Dissolve in ethanol (24 ml) and add 1N
Add thorium solution (0.8ml) and at 35 ° C for 4.5 hours
While stirring. After stirring at 25 ° C. for 20 hours, water was added to the reaction solution.
(1 ml) was added, and the reaction solution was dried under reduced pressure.
At this time, the external temperature did not exceed 30 ° C. Got
Dissolve the yellow solid in water (5 ml) and add 1N hydrochloric acid to pH
= 3.7 and left in the refrigerator for 2 hours. Precipitation
The resulting precipitate was collected by filtration to obtain the desired product (109 mg). [0118]¹H-NMR (CDCl₃, 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.) [0119] [Reference Example 20]Nt-butoxycarbonyl-γ-anilide-L-glu
Synthesis of α-benzyl tamate Nt-butoxycarbonyl-L-gluta
Α-Benzyl ester (2.2 g) and triethyl
Amine (0.92 ml) in tetrahydrofuran (8
ml) at -20 ° C under nitrogen atmosphere.
Chill (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. Gradually return to room temperature
And stirred for a further 20 hours. The solvent was distilled off under reduced pressure to obtain
The obtained residue was dissolved in chloroform. Chloroform layer
Was washed with a saturated aqueous solution of sodium bicarbonate,
And dried under reduced pressure. The resulting residue
Silica gel chromatography, elution solvent
Using chloroform: methanol = 99: 1
(1.7 g) was obtained. [0121]¹H-NMR (CDCl₃, 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) [0122] [Reference Example 21]γ-anilide-L-glutamic acid α-benzyl ester
Synthesis of Reference Example 20 The compound (1.7 g) was cooled with ice
Dissolve in trifluoroacetic acid (8 ml) below and stir for 30 minutes
Was. The solvent is distilled off under reduced pressure, and the residue is dissolved in chloroform.
did. Chloroform layer is saturated aqueous sodium hydrogen carbonate
And then dried over sodium sulfate. Dissolve under reduced pressure
The solvent was distilled off to obtain the desired product (1.3 g). [0124]¹H-NMR (CDCl₃, 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) [0125] [Reference Example 22]N- (1-carbobenzoxyindoline-5-carboni
L) -γ-anilide-L-glutamic acid α-benzyl ester
Steal Synthesis 1-carbobenzoxyindoline-5-ca
Thionyl chloride (2 ml) was added to rubonic acid (419 mg).
The mixture was stirred at room temperature for 2 hours. Next, the reaction solution is concentrated under reduced pressure.
To dryness. The obtained solid is dissolved in dichloromethane (2 ml).
And dissolved in this solution. Of the compound (400
mg) and triethylamine (0.21 ml)
A water solution (2 ml) is added under ice-cooling and nitrogen atmosphere, and stirred overnight.
Stirred. The reaction solution is 1N hydrochloric acid, saturated sodium hydrogen carbonate
Washed sequentially with an aqueous solution and water, and dried over sodium sulfate
Thereafter, the solvent was distilled off under reduced pressure. The residue obtained is silica gel
Chromatography, and elution solvent
Using methane and then chloroform, the desired product (317 m
g) was obtained. [0127]¹H-NMR (CDCl₃, 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) [0128] [Reference Example 23]N- (1-carbobenzoxyindoline-5-carboni
B) Synthesis of -γ-anilide-L-glutamic acid Reference Example 22 Compound (580 mg)
A mixed solvent of roloform: 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. Obtained
Was dissolved in water and acidified with 1N hydrochloric acid.
Extracted with chloroform. Sulfuric acid in chloroform layer
After drying with sodium, the solvent was distilled off under reduced pressure to obtain the target compound.
(413 mg) was obtained. [0130]¹H-NMR (CDCl₃, 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) [0131] [Reference Example 24]N- (1-carbobenzoxyindoline-5-carboni
-)-Γ-anilide-L-glutamic acid α-methyles
Tell synthesis Reference Example 23 Compound (470 mg) was dried.
Dissolve in dry methanol (20 ml) and add trimethylsilyl
Diazomethane (2 ml) was added and the mixture was stirred for 10 hours. Sa
And trimethylsilyldiazomethane (3 ml).
Stirred for 20 hours. After evaporating the solvent under reduced pressure, the obtained
The residue was subjected to silica gel chromatography and eluted.
Chloroform as a medium, then chloroform: methanol
To obtain the desired product (252 mg). [0133]¹H-NMR (CDCl₃, 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) [0134] [Reference Example 25]N- (indoline-5-carbonyl) -γ-anilide-
Synthesis of L-glutamic acid α-methyl ester Reference Example 24. Of the compound (250 mg)
10% palladium on carbon in ethanol solution (10 ml)
(50 mg) at room temperature under a hydrogen atmosphere at 15:00
While stirring. Palladium-carbon is removed by filtration using Celite
Then, the solvent was distilled off under reduced pressure. The residue obtained is silica gel
Chlorophore as the eluting solvent.
Lum: methanol = 99: 1, then chloroform: me
The desired product (172 mg) was obtained using 19: 1 of ethanol.
Was. [0136]¹H-NMR (CDCl₃, 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) [0137] Embodiment 13N- [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 hydrogen bromide
Acid salt (223 mg) in dimethylacetamide (5 ml)
And stirred at room temperature for 24 hours. The reaction solution is silica
Gel chromatography was performed, and acetic acid was used as the elution solvent.
Chill, then use chloroform: methanol = 19: 1
Thus, the desired product (82 mg) was obtained. [0139]¹H-NMR (CDCl₃, 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) [0140] Embodiment 14N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] -indoline-5-carbonyl] -γ-anili
Synthesis of do-L-glutamic acid Embodiment 13 FIG. Compound (82 mg)
Dissolved in a mixed solvent (15 ml) of knol: water = 2: 1,
A 1N aqueous solution of sodium hydroxide (0.15 ml) was added.
Then, the mixture was stirred at room temperature for 60 hours. Water bath temperature below 30 ℃
The solvent was distilled off under reduced pressure while maintaining the pressure. The resulting residue
Dissolve in saturated aqueous sodium bicarbonate and add 1N hydrochloric acid
And adjusted to pH = 3.7. The deposited orange precipitate
Filtration yielded the desired product (61 mg). [0142]¹H-NMR (DMSO-d₆, 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) [0143] [Reference Example 26]Nt-butoxycarbonyl-γ- (2-methoxycal
Bonylanilide) -L-glutamic acid α-benzyles
Tell synthesis Nt-butoxycarbonyl-L-gluta
Tetrahydric acid α-benzyl ester (496 mg)
In a drofuran solution (5 ml), ice-cooled, under a nitrogen atmosphere,
N, N'-carbonyldiimidazole (262 mg)
The mixture was stirred for 1 hour. Next, 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 to obtain
The residue was subjected to silica gel chromatography and eluted.
Using chloroform as a solvent, the desired product (273 mg)
I got [0145]¹H-NMR (CDCl₃, 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) [0146] [Reference Example 27]γ- (2-methoxycarbonylanilide) -L-gluta
Synthesis of α-benzyl ester of formic acid Reference Example 26 Compound (632 mg) on ice
Dissolve in trifluoroacetic acid (4.5 ml) under cooling for 90 minutes
Stirred. The solvent was distilled off under reduced pressure.
Was dissolved. Chloroform layer is saturated sodium bicarbonate
After washing with an aqueous solution and drying over sodium sulfate,
The solvent was distilled off with. The obtained residue is subjected to silica gel chromatography.
Chromatography, chloroform as elution solvent, then
Using chloroform: methanol = 97: 3
(486 mg). [0148]¹H-NMR (CDCl₃, 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) [0149] [Reference Example 28]1-[(2,4-diamino-6-pteridinyl) methyl
[Indone-5-carboxylic acid] 1-carbobenzoxyindoline-5-ca
Rubonic acid (87 mg) and 6-bromomethyl-2,4-di
Aminopteridine hydrobromide 1/2 isopropanol
The adduct (136 mg) was converted to dimethylacetamide (2 m
1) and stirred at 55 ° C. for 4 hours. Water in the reaction solution
(20 ml) and left in the refrigerator overnight. Deposited
The solid is collected by filtration and added to a small amount of 1N aqueous sodium hydroxide solution.
It was dissolved and adjusted to pH = 6.5 using 1N-hydrochloric acid.
The precipitated brown precipitate was collected by filtration to obtain the desired product (56 mg).
Was. [0151]¹H-NMR (DMSO-d₆, 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) [0152] Embodiment 15N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] -indoline-5-carbonyl] -γ- (2-
Methoxycarbonylanilide) -L-glutamic acid α-
Synthesis of methyl ester Reference Example 28 Compound (134 mg) and 1
Of hydroxybenzotriazole (108 mg)
Ice-cooled, nitrogen atmosphere in chilled formamide suspension (5 ml)
Infrared, dicyclohexylcarbodiimide (123 mg)
Dimethylformamide solution (1.5 ml)
Minutes. Next, Reference Example 27. The compound dimethyl
After adding formamide solution (1.5 ml), slowly
The mixture was returned to warm temperature and stirred for 24 hours. The reaction solution was chromatographed on silica gel
Ethyl acetate as the elution solvent, then
Using chloroform: methanol = 19: 1
(29 mg) was obtained. [0154]¹H-NMR (CDCl₃, 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) [0155] Embodiment 16N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] -indoline-5-carbonyl] -γ- (2-
Synthesis of (carboxyanilide) -L-glutamic acid Embodiment 15 FIG. Compound (28 mg)
And 1N sodium hydroxide
Aqueous solution (0.41 ml) and stirred at 10 ° C for 5 hours.
Stirred. While maintaining the temperature of the water bath at 30 ° C or less, under reduced pressure
The solvent was distilled off. The resulting residue is washed with saturated sodium bicarbonate
And pH was adjusted to 3.7 using 1N hydrochloric acid.
It was adjusted. The orange precipitate that precipitated was collected by filtration, and the desired product (21
mg). [0157]¹H-NMR (DMSO-d₆, 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 (4
H, m), 8.47 (1H, d, J = 7.8 Hz),
8.78 (1H, s), 11.25 (2H, s) [0158] [Reference Example 29]Nt-butoxycarbonyl-L-glutamic acid α-meth
Synthesis of tyl γ-benzyl diester Nt-butoxycarbonyl-L-gluta
Dimethyl phosphoric acid γ-benzyl ester (5.0 g)
Lumamide (75 ml) solution in sodium bicarbonate
(2.5 g) was suspended in methyl iodide (10.5 g).
2 g) in dimethylformamide (75 ml)
Stirred at room temperature for 24 hours. The reaction was concentrated under reduced pressure
Thereafter, water (70 ml) was added, and ethyl acetate: n-hexane was added.
= 1: 1 solution. The organic layer is washed with water,
After drying with thorium, the solvent was distilled off under reduced pressure. Get
The residue was subjected to silica gel chromatography and eluted.
Using ethyl acetate: n-hexane = 1: 2 as a solvent
The desired product (5.2 g) was obtained. [0160]¹H-NMR (CDCl₃, Δ): 1.4.
3 (9H, s), 1.92-2.01 (1H, m),
2.11-2.37 (1H, m), 2.51-2.42
(2H, m), 3.73 (3H, s), 4.23-4.
40 (1H, m), 5.12 (2H, s), 7.35
(5H, s) [0161] [Reference Example 30]Nt-butoxycarbonyl-L-glutamic acid α-meth
Synthesis of tyl ester Reference Example 29 Of the compound (5.2 g) of
10% palladium-carbon in ethanol solution (30 ml)
(1.1 g) and then at room temperature under hydrogen atmosphere for 15:00
While stirring. Palladium-carbon is removed by filtration using Celite
Then, the solvent was distilled off under reduced pressure. The obtained residue is saturated with carbonic acid.
Dissolve in aqueous sodium hydrogen, wash with chloroform
Was. After separating the aqueous layer, pH = 4 using 5% citric acid.
And extracted with chloroform. Chloroform layer
After drying over sodium sulfate, the solvent was distilled off under reduced pressure,
The desired product (3.9 g) was obtained. [0163]¹H-NMR (CDCl₃, 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) [0164] [Reference Example 31]Nt-butoxycarbonyl-γ- (3-ethoxycal
Bonylanilide) -L-glutamic acid α-methyl ester
Synthesis Reference Example 30 (1.6 g) and 1-
Dichloro of hydroxybenzotriazole (0.8g)
To a methane suspension (10 ml) was added ice-cooled and nitrogen atmosphere.
Dichloro of cyclohexylcarbodiimide (1.5 g)
A methane solution (5 ml) was added and stirred for 30 minutes. Then
After addition of ethyl m-aminobenzoate (1.5 g),
The mixture was returned to room temperature and stirred for 20 hours. Evaporate the solvent under reduced pressure
Then, ethyl acetate was added to the obtained residue, and white insolubles were removed by filtration.
did. The filtrate is concentrated under reduced pressure, and the resulting residue is
Chlorophore as the eluting solvent.
Lum, then use chloroform: methanol = 99: 1
Thus, the desired product (2.5 g) was obtained. [0166]¹H-NMR (CDCl₃, 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) [0167] [Reference Example 32]γ- (3-ethoxycarbonylanilide) -L-gluta
Synthesis of α-methyl ester of formic acid Reference Example 31 Ice-cooled compound (2.5 g)
Dissolve in trifluoroacetic acid (15 ml) under stirring for 1 hour
did. The solvent was distilled off under reduced pressure, and the residue was dissolved in chloroform.
I understand. Chloroform layer is saturated aqueous sodium bicarbonate
After washing with liquid and drying over sodium sulfate, dissolve under reduced pressure.
The medium was distilled off. The obtained residue is subjected to silica gel chromatography.
And then eluted with chloroform: methanol
To obtain the desired product (1.6 g). [0169]¹H-NMR (CDCl₃, 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) [0170] [Reference Example 33]N- (1-carbobenzoxyindoline-5-carboni
) -Γ- (3-ethoxycarbonylanilide) -L-
Synthesis of glutamic acid α-methyl ester 1-carbobenzoxyindoline-5-ca
Thionyl chloride (4 ml) was added to rubonic acid (612 mg).
The mixture was stirred at room temperature for 2 hours. Next, the reaction solution is concentrated under reduced pressure.
To dryness. The solid obtained is diluted with dichloromethane (4 ml)
In Reference Example 32. Compound (52)
9mg) and triethylamine (0.36ml)
Methane solution (4 ml) was added under ice-cooling and nitrogen atmosphere.
Stirred overnight. The reaction solution was diluted with 2N hydrochloric acid and saturated sodium bicarbonate.
Wash sequentially with aqueous solution of water and water, dry over sodium sulfate
After that, the solvent was distilled off under reduced pressure. The obtained residue is silica
Gel chromatography.
Form, then chloroform: methanol = 99: 1
To give the desired product (870 mg). [0172]¹H-NMR (CDCl₃, Δ): 1.3.
7 (3H, t, J = 7.1 Hz), 2.22-2.08
(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) [0173] [Reference Example 34]N- (indoline-5-carbonyl) -γ- (3-etho
Xycarbonylanilide) -L-glutamic acid α-methyl
Synthesis of ester Reference Example 33 Of the compound (842 mg)
10% palladium on carbon in ethanol solution (11 ml)
(0.17 g) was added at room temperature under a hydrogen atmosphere at room temperature.
Stirred for hours. Filter palladium-carbon using Celite
The solvent was distilled off under reduced pressure. The obtained residue is silica
Gel chromatography.
Form, then chloroform: methanol = 99: 1
This gave the desired product (448 mg). [0175]¹H-NMR (CDCl₃, Δ): 1.3.
6 (3H, t, J = 7.1 Hz), 2.05-2.25
(1H, m), 2.25-2.48 (1H, m), 2.
25-2.48 (2H, m), 2.93 (2H, t, J
= 8.8 Hz), 3.57 (2H, t, J = 8.8H)
z), 3.71 (3H, s), 4.18 (1H, b
s), 4.34 (2H, t, q, J = 7.1 Hz),
4.73-4.84 (1H, m), 6.46 (1H,
d, J = 8.8 Hz), 7.16 (1H, d, J = 7.
8 Hz), 7.32 (1H, t, J = 7.8 Hz),
7.50-7.53 (2H, m), 7.73 (1H,
d, J = 7.8 Hz), 7.87 (1H, d, J = 7.
3 Hz), 8.19 (1H, s), 9.39 (1H, b
s) [0176] Embodiment 17N- [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 hydrogen bromide
Isopropanolate adduct (587 mg) in dimethyl
Suspend in acetamide (5 ml) and stir at room temperature for 36 hours.
Stirred. The reaction solution was subjected to silica gel chromatography.
And then eluted with ethyl acetate and then chloroform:
The desired product (653 mg) was obtained using methanol = 9: 1.
Was. [0178]¹H-NMR (DMSO-d₆, 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, s), 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) [0179] Embodiment 18N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] -indoline-5-carbonyl] -γ- (3-
Synthesis of (carboxyanilide) -L-glutamic acid Embodiment 17 FIG. Compound (303 mg)
Dissolve in ethanol (10 ml) and add 1N sodium hydroxide
Aqueous solution (1.06 ml) and stirred at room temperature for 12 hours
did. Water (1 ml) was added, and the mixture was further stirred for 2 hours. water
The solvent is distilled off under reduced pressure while keeping the temperature of the bath at 30 ° C or less
did. The residue obtained is taken up in a saturated aqueous sodium hydrogen carbonate solution.
It was dissolved and adjusted to pH = 3.7 using 1N-hydrochloric acid.
The precipitated orange precipitate was collected by filtration, and the desired product (157 mg) was obtained.
Obtained. [0181]¹H-NMR (DMSO-d₆, 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) [0182] [Reference Example 35]N-carbobenzoxy-γ-N ′, N′-dimethylami
Synthesis of α-benzyl ester of do-L-glutamic acid N-carbobenzoxy-L-glutamic acid
α-benzyl ester (508 mg) and triethylamido
(0.19 ml) in tetrahydrofuran (2 m
l) Ethyl chlorocarbonate at -20 ° C under nitrogen atmosphere
(0.14 ml) was added and stirred for 30 minutes. Next,
Tyl hydrochloride (112 mg) and triethylamine (0.1
9 ml) of a dichloromethane solution (3 ml)
Stir for 1 hour. Gradually return to room temperature and stir for an additional 24 hours.
Stirred. The solvent was distilled off under reduced pressure, and the resulting residue was
Dissolved in form. Saturated NaHCO 3 in chloroform layer
Washed with 1N-hydrochloric acid and dried over sodium sulfate
After drying, the solvent was distilled off under reduced pressure. The resulting residue is
The gel is subjected to Ricagel chromatography and used as an elution solvent.
Using roroform: methanol = 49: 1, the desired product (4
53 mg). [0184]¹H-NMR (CDCl₃, 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) [0185] [Reference Example 36]N-carbobenzoxy-γ-N ′, N′-dimethylami
Synthesis of do-L-glutamic acid Reference Example 35 Of the compound (450 mg)
1N-sodium hydroxide aqueous solution in a ethanol solution (5 ml)
The solution (1.15 ml) was added and stirred at room temperature overnight. water
The solvent is distilled off under reduced pressure while maintaining the temperature of the bath at 30 ° C or less.
did. The residue obtained is dissolved in water and acidified with 1N hydrochloric acid.
After that, extraction was performed using chloroform. Chlorophor
After drying the solvent layer with sodium sulfate, the solvent was distilled off under reduced pressure.
This gave the desired product (341 mg). [0187]¹H-NMR (CDCl₃, 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) [0188] [Reference Example 37]N-carbobenzoxy-γ-N ′, N′-dimethylami
Synthesis of do-L-glutamic acid α-methyl ester Reference Example 36. Compound (353 mg) was dried.
Dissolve in dry methanol (4 ml) and add
Azomethane (5 ml) was added and stirred for 2 hours. Under reduced pressure
The solvent was distilled off to obtain the desired product (338 mg). [0190]¹H-NMR (CDCl₃, 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) [0191] [Reference Example 38]γ-N ′, N′-dimethylamide-L-glutamic acid α
-Synthesis of methyl ester Reference Example 37 Of compound (580 mg)
10% palladium on carbon in ethanol solution (10 ml)
(70 mg) and then at room temperature under a hydrogen atmosphere at 15:00
While stirring. Palladium-carbon is removed by filtration using Celite
Then, the solvent was distilled off under reduced pressure. The residue obtained is silica gel
Chlorophore as the eluting solvent.
Lum: methanol = 19: 1 using the desired product (160 m
g) was obtained. [0193]¹H-NMR (CDCl₃, 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) [0194] [Reference Example 39]N- (1-carbobenzoxyindoline-5-carboni
) -Γ-N ', N'-dimethylamide-L-glutami
Of α-Methyl Ester 1-carbobenzoxyindoline-5-ca
Thionyl chloride (2 ml) was added to rubonic acid (295 mg).
The mixture was stirred at room temperature for 2 hours. Next, the reaction solution is concentrated and dried under reduced pressure.
Hardened. The solid obtained is taken up in dichloromethane (2 ml)
After dissolving in this solution, Reference Example 38. Of the compound (160
mg) and triethylamine (0.18 ml)
Add methane solution (2.5 ml) under ice-cooling and nitrogen atmosphere
Stirred overnight. The reaction solution was 1N-hydrochloric acid, saturated sodium bicarbonate
Wash sequentially with aqueous solution of lithium and water and dry over sodium sulfate
After that, the solvent was distilled off under reduced pressure. The resulting residue is
It is subjected to Kagel chromatography, and
Chloroform, then chloroform: methanol = 99: 1
Was used to obtain the desired product (359 mg). [0196]¹H-NMR (CDCl₃, 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) [0197] [Reference Example 40]N- (indoline-5-carbonyl) -γ-N ', N'
-Dimethylamide-L-glutamic acid α-methyl ester
Synthesis Reference Example 39 Of the compound (359 mg)
10% palladium-carbon (7 ml) was added to a ethanol solution (5 ml).
0 mg), and then stirred at room temperature for 15 hours under a hydrogen atmosphere.
did. The palladium-carbon was removed by filtration using Celite to reduce
The solvent was distilled off under pressure. The residue obtained is purified by silica gel chromatography.
After the chromatography, chloroform:
The desired product (131 mg) was prepared using methanol = 99: 1.
Obtained. [0199]¹H-NMR (CDCl₃, 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) [0200] Embodiment 19N- [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 hydrogen bromide
Isopropanolate adduct (233 mg) in dimethyl
Suspend in acetamide (4 ml) and stir at room temperature for 24 hours.
Stirred. The reaction solution was subjected to silica gel chromatography.
And then eluted with ethyl acetate and then chloroform:
The desired product (95 mg) was obtained using methanol = 9: 1.
Was. [0202]¹H-NMR (CDCl₃, Δ): 1.9.
8-2.17 (2H, m), 2, 42 (2H, t, J =
6.8 Hz), 2.84 (3H, s), 2.94 (3
H, s), 3.00 (2H, t, J = 8.3 Hz),
3.59 (2H, t, J = 8.3 Hz), 3.64 (3
H, s), 4.30-4.42 (1H, m), 4.55
(2H, s), 6.68 (1H, d, J = 7.3H
z), 7.60-7.64 (2H, m), 8.34 (1
H, d, J = 7.3 Hz), 8.71 (1H, s) [0203] Embodiment 20 N- [1-[(2,4-diamino-6-buteridinyl)
Methyl] -indoline-5-carbonyl] -γ-N ',
Synthesis of N'-dimethylamido-L-glutamic acid Embodiment 19 FIG. Compound (70 mg)
Dissolved in ethanol (5 ml) and 1N aqueous sodium hydroxide
Add the solution (0.15 ml) and stir at room temperature for 12 hours
Was. Water (2 ml) was added, and the mixture was further stirred for 5 hours. Bathing
The solvent was distilled off under reduced pressure while maintaining the temperature at 30 ° C or lower.
Was. The obtained residue is dissolved in a saturated aqueous sodium hydrogen carbonate solution.
Then, the pH was adjusted to 3.7 using 1N-hydrochloric acid. Occasionally
The resulting orange precipitate was collected by filtration to obtain the desired product (16 mg).
Was. [0205]¹H-NMR (DMSO-d₆, 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) [0206] [Reference Example 41]N-1-[(2,4-diamino-6-pteridinyl) me
Tyl] 1,2,3,4-tetrahydro-6-quinoline
Synthesis of carboxylic acid 6-bromomethyl-2,4-diaminopte
Lysine hydrobromide / isopropanol adduct (178
mg) and 1,2,3,4-tetrahydro-6-quinoli
Carboxylic acid (55 mg) was added to dimethylacetamide (3
ml) and stirred at 60-65 ° C overnight. After cooling
Water (10 ml) was added to the reaction mixture, and the reaction mixture was cooled with ice-water.
Is adjusted to pH = 3.5 with 1N-hydrochloric acid and left overnight in a cool place
did. The deposited precipitate was collected by filtration, and the desired product (70 mg) was obtained.
Obtained. [0208]¹H-NMR (DMSO-d₆, 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) [0209] Embodiment 21N-1-[(2,4-diamino-6-pteridinyl) me
Tyl] 1,2,3,4-tetrahydro-6-quinoline
Synthesis of carbonyl] -diethyl L-glutamate Triethylamine (60 mg) and diethyl
Phosphorocyanidate (98mg) in anhydrous dimethylform
Muamide (6 ml). of
The compound (60 mg) was added and stirred. After dissolution, reaction
Stir the solution at 80 ° C for 3 minutes, then return to room temperature for 10 minutes
Stirred. Then triethylamine (20mg) and glue
Tamamic acid diethyl ester hydrochloride (40 mg) was added,
Stirred at 80 ° C. for 2 hours. After the reaction, the solvent is distilled off under reduced pressure.
Chloroform was added to the residue, and the chloroform layer was saturated.
Washed with aqueous sodium bicarbonate. Chloroform layer with magnesium sulfate
After drying with, the solvent was distilled off under reduced pressure.
Gel column chromatography, elution solvent
Using a mixed solvent of chloroform: methanol = 10: 1
The desired product (50 mg) was obtained. [0211]¹H-NMR (DMSO-d₆, 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 = 3 Hz), 7.47 (2H, m), 8.65
(2H, s) [0212] Embodiment 22 N-1-[(2,4-diamino-6-pteridinyl) me
Chill] 1,2,3,4-Tetrahydro-6-quinoline
Synthesis of carbonyl] -L-glutamic acid Embodiment 21 FIG. Compound (50 mg)
L in ethanol (10 ml), 1N
An aqueous solution of thorium (0.24 ml) was added, and at the same temperature 4.
Stir for 5 hours. Further, stirring was continued at 25 ° C. for 20 hours.
Thereafter, water (1 ml) was added to the reaction solution. Ice-water cooling, anti
The reaction solution was adjusted to pH = 3.7 with 1N hydrochloric acid and left overnight in a cool place.
I left it. The deposited precipitate was collected by filtration, and the target product (27 m
g) was obtained. [0214]¹H = NMR (DMSO-d₆, 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.) [0215] [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-diaminopte
Lysine hydrobromide / isopropanol adduct (410
mg) and 3,4-dihydro-2H-1,4-benzooxy
Methyl sagin-7-carboxylate (200 mg)
Suspended in lucetamide (10 ml), bath temperature 65 ° C 4
And stirred at 90 ° C. for 19 hours. After cooling,
Cetamide is concentrated under reduced pressure, and chloroform and hydrogencarbonate are added.
An aqueous thorium solution was added. After filtering off the precipitate, the organic layer
After washing with water and drying over magnesium sulfate, the solvent is distilled off under reduced pressure.
did. The residue was subjected to silica gel column chromatography.
And a mixed solvent of chloroform: methanol = 97: 3.
Elution gave the desired product (100 mg). [0219]¹H-NMR (CDCl₃: CD₃OD =
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) [0218] [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
-Sodium hydroxide aqueous solution (20 ml) and methanol
(20 ml) in a mixed solvent and heated under reflux for 2.5 hours
did. After cooling, the solvent was distilled off, and water was added.
Adjusted to H = 5 (suspended). After leaving in a cool place overnight, filter
It was collected and dried to give the desired product (60.8 mg). [0220]¹H-NMR (DMSO-d₆, Δ):
3.63 (2H, m), 4.22 (2H, m), 4.7
1 (2H, s), 7.21 (1H, s), 8.29 (1
H, s) [0221] Embodiment 23N- [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), dry dimethyl
Reference Example 43 in a solution of Lumamide (5 ml). Compound of
(50 mg) and stirred at 80 ° C. for 5 minutes under a nitrogen atmosphere.
did. Cool to room temperature and triethylamine (20 μl)
And diethyl glutamate (34 mg) were added.
C. and stirred for 2.5 hours. After cooling, chloroform
Extracted with water and washed with aqueous sodium hydrogen carbonate and brine
did. After drying over magnesium sulfate, the solvent was distilled off. Remaining
The residue is subjected to silica gel column chromatography,
Elution with a mixed solvent of roform: methanol = 19: 1
Thus, the desired product (10 mg) was obtained. [0223]¹H-NMR (CDCl₃: CD₃OD =
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, z), 8.70
(1H, s) [0224] Embodiment 24N- [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. Compound (9 mg) in ethanol
(3 ml), and 1N aqueous solution of sodium hydroxide
The solution was added and stirred at room temperature for 4 days. The solvent is distilled off.
The column was subjected to Ricagel column chromatography.
Solvent: methanol: aqueous ammonia = 5: 4: 1 mixed solvent
Eluted, and collect the fraction containing the target substance,
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. Analysis
The product was collected by filtration and dried to obtain the desired product (2.8 mg). [0226]¹H-NMR (DMSO-d₆, 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       [0227]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows “P388 cell growth inhibitory action”, and FIG.
growth inhibition effect of olon26 ". The absorbance at each concentration of the test drug is shown as a percentage when the absorbance when no drug is added is 100%.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C07D 475/08 REGISTRY (STN) CA (STN)

Claims (1)

(57) [Claims] General formula (I) Wherein R ₁ is CH ₂ , CH ₂ CH ₂ , CH ₂ O, CH ₂
R represents a member selected from the group consisting of S, CH ₂ SO, CH ₂ SO ₂ and CH ₂ NH; R ₂ represents a hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms; Represents an integer; 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 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 an optionally substituted phenyl group) or PO A group represented by ₃ H ₂ or SO ₃ H;
An anticancer agent comprising a compound represented by the formula (1) as an active ingredient: