JP2000109482A - Monoglutamic acid modification of methotrexate derivative - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound with a glutamate group on the terminal of the homoglutamic acid moiety of a methotrexate derivative, useful as a therapeutic agent for rheumatism. SOLUTION: This new compound is shown by the formula (A is CH2, CH2O or the like; R1 to R3 are each H or a 1-4C alkyl), pref. 5-[4-(2,4- diaminopteridin-6-yl)methyl-3,4-dihydro-2H-1,4-benzothiazine-7-carbony lamino]-5- carboxypentanoyl-2-aminoglutaric acid. The compound of the formula is obtained, for example, for the above-illustrated compound, by alkylation between 5-(3,4-dihydro-2H-1,4-benzothiazine-7-carbonylamino)-5-ethoxycarbonylp entanoyl-2- aminoglutaric acid diethyl ester and 2,4-diaminopteridine-6-methyl bromide hydrobromide in dimethylacetamide followed by alkaline hydrolysis of the ester group.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an oxidized monoglutamine derivative of a methotrexate derivative. More specifically, the present invention relates to a monoglutamine oxidized form of a methotrexate derivative in which the amino acid portion of the methotrexate derivative is homoglutamic acid and has a benzene ring portion which is cyclized by bonding to a substituent at the nitrogen atom at the 10-position.

[0002]

2. Description of the Related Art Methotrexate has long been used as a therapeutic agent for leukemia.
Report the efficacy of aminopterin in rheumatoid arthritis (RA) and psoriasis (American Journal)
al of the Medical Science
s, 221, 176-182 (1951))
It has attracted attention as a therapeutic agent for RA and has been mainly used in Europe and the United States. More recently, detailed studies of dosage and administration have revealed that low-dose intermittent 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] Among many methotrexate derivatives,
Examples of methotrexate derivatives having homoglutamic acid in the amino acid portion include those described in J. Am. Med. Chem. , 40
(1), 105-111 (1997).
-[(2,4-diamino-6-pteridinyl) methyl]
-Indoline-5-carbonyl] -L-α-homoglutamic acid, N- [1-[(2,4-diamino-6-pteridinyl) methyl] -3,4-dihydro-2H-1,4-
Benzoxazine-7-carbonyl] -L-α-homoglutamic acid and N- [1-[(2,4-diamino-6)
-Pteridinyl) methyl] -3,4-dihydro-2H-
1,4-benzothiazine-7-carbonyl] -L-α-
Homoglutamic acid and the like are known. It is strongly suggested that these methotrexate derivatives having homoglutamic acid have an antirheumatic effect, and do not cause polyglutamination via monoglutamate, which is considered to be one of the main causes of side effects caused by methotrexate. ing. Among these, N- [1-
[(2,4-diamino-6-pteridinyl) methyl]-
3,4-dihydro-2H-1,4-benzothiazine-7
-Carbonyl] -L-α-homoglutamic acid is expected to be put to practical use as a therapeutic agent for rheumatism having a high synovial cell growth inhibitory effect.

Thus, N- [1-[(2,4-diamino-6-pteridinyl) methyl] -3,4-dihydro-2H-1,4-benzothiazine-7-carbonyl]-
While L-α-homoglutamic acid has been developed as a useful drug, its metabolism has been studied, but such N- [1-[(2,4-diamino-6-
Pteridinyl) methyl] -3,4-dihydro-2H-
1,4-benzothiazine-7-carbonyl] -L-α-
To clarify that among the metabolites of homoglutamic acid, there was no compound that caused polyglutamation via monoglutamation, as a standard,
N- [1-[(2,4-diamino-6-pteridinyl)
Methyl] -3,4-dihydro-2H-1,4-benzothiazine-7-carbonyl] -L-α-homoglutamic acid monoglutamate or polyglutamate is required. However, N- [1-[(2,4-diamino-
6-pteridinyl) methyl] -3,4-dihydro-2H
-1,4-benzothiazine-7-carbonyl] -L-α
-Synthesis of monoglutamate or polyglutamate of homoglutamic acid has not been reported so far.

[0005]

An object of the present invention is to provide a novel methotrexate derivative having a glutamate group at the terminal of the homoglutamic acid moiety of the methotrexate derivative.

[0006]

Means for Solving the Problems To solve the above-mentioned problems, the present inventor uses glutamyl homoglutamic acid obtained by binding a glutamic acid to a homoglutamic acid derivative as a starting material, and comprises an aminobenzoic acid moiety, pteridine By linking the moieties sequentially and finally hydrolyzing as needed, a new methotrexate derivative having a glutamate group at the terminal of the amino acid portion of the methotrexate derivative was successfully synthesized. Further, the compound is a DHFR (dihydrofolate redu).
ctase) It has been found that it has an inhibitory action and is useful as a medicament, particularly a therapeutic agent for rheumatism, and has completed the present invention. According to the present invention, general formula (I):

[0007]

Embedded image

Wherein A is —CH₂-Or -CH₂
O- or -CH₂Represents S-; R₁ , R₂, R₃Is the same
May be one or different, and may be a hydrogen atom or a carbon number 1 to
4 represents a lower alkyl group. ), Methotrek
An oxidized monoglutamine of a sate derivative is provided.

In the definition of the monoglutamine oxidized form of the methotrexate derivative represented by the general formula (I), R
₁ , R ₂ , and R _{3 each} represent a hydrogen atom and a carbon atom having 1 to 1;
4 lower alkyl groups, among which a hydrogen atom is preferable. Examples of the lower alkyl group having 1 to 4 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an i-propyl group, and an n-
Examples thereof include a butyl group, an i-butyl group, an s-butyl group, and a t-butyl group. Among them, an ethyl group and an i-propyl group are preferable, and an ethyl group is particularly preferable. R ₁ , R ₂ , and R
₃ may be the same or different from each other, but are preferably the same. Further, it is particularly preferred that R ₁ , R ₂ and R ₃ are all hydrogen atoms. As A,-
Examples include CH ₂ —, —CH ₂ O—, and —CH ₂ S—, with —CH ₂ S— being particularly preferred. The methotrexate derivative represented by the general formula (I) of the present invention includes 5
-[(4- (2,4-diaminopteridin-6-yl)
Methyl-3,4-dihydro-2H-1,4-benzothiazine-7-carbonyl) amino] -5-carboxylpentanoyl-2-aminoglutaric acid is preferred. Also,
The methotrexate derivative represented by the general formula (I) of the present invention has one or more asymmetric carbon atoms, and each of the asymmetric carbon atoms has an R configuration (D-amino acid residue), S Any of the configurations (L-amino acid residues) are included in the present invention, and preferred are those in which the configuration of the carbon atom corresponding to the α-carbon in the amino acid residues is S configuration (L-amino acid residues). It is.

The monoglutamine oxidized form of the methotrexate derivative represented by the general formula (I) of the present invention is a novel compound, for example, 1-benzyloxycarbonyl-indoline-5-carboxy described in JP-A-5-132485. Acid or 1-benzyloxycarbonyl-3,4
-Dihydro-2H-1,4-benzoxazine-7-carboxylic acid; Med. Chem. , 40
(1), 1-tosyl-3,4-dihydro-2H-1,4-benzothiazine-7-carboxylic acid described in 105-111 (1997) as one of the starting materials, and is exemplified by the following reaction formula. Based on the above method, it can be created by using a desired reagent as needed.

[0011]

Embedded image

(Wherein, R ₄ represents an amino-protecting group such as a t-butoxycarbonyl group, a benzyloxycarbonyl group, or an allyloxycarbonyl group. R ₅ represents a methyl group, an ethyl group, an n-propyl group) Group, i-propyl group,
an n-butyl group, an i-butyl group, an s-butyl group, a linear or branched lower alkyl group having 1 to 4 carbon atoms which is a t-butyl group, or an aralkyl group such as a benzyl group;
Represents an ester protecting group. The reaction for obtaining compound 2 from compound 1 is carried out by reacting compound 1 with a coupling reagent and then adding glutamic acid diethyl ester hydrochloride. The coupling reagent used is 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride, dicyclohexylcarbodiimide, diimidazolecarbodiimide, benzotriazol-1-yloxy-tris (dimethylamino) phosphonium hexafluorophosphate ,
Bromo-tris (pyrrolidino) phosphonium hexafluorophosphate, benzotriazol-1-yl-oxy-tris (pyrrolidino) phosphonium hexafluorophosphate, chloro-N, N, N ',
N'-bis (tetramethylene) formamidinium hexafluorophosphate and the like, preferably,
1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride is used. At this time, in order to promote the reactivity between compound 1 and the coupling reagent, 1
-Hydroxybenzotriazole, 3-hydroxy-
An accelerator such as 1,2,3-benzotriazin-4 (3H) -one can also be used. As the solvent, dimethylformamide, dimethylacetamide, dimethylsulfoxide, dichloromethane, chloroform, tetrahydrofuran, ethyl ether or a mixture thereof is used, but dimethylformamide is preferably used. The reaction temperature is between -50 ° C and 50 ° C, preferably,
Perform between -20 ° C and 0 ° C.

The reaction from compound 2 to compound 3 is carried out by deprotecting compound 2 with an acid, and then reacting hydrogen chloride gas in a linear or branched lower alcohol having 1 to 4 carbon atoms (preferably ethanol). To form a hydrochloride at the same time as the esterification. The acid used at this time is trifluoromethanesulfonic acid, methanesulfonic acid,
Examples thereof include trifluoroacetic acid, acetic acid, hydrochloric acid, hydrobromic acid, sulfuric acid, and nitric acid. Preferably, trifluoroacetic acid is used. The reaction temperature is between −78 ° C. and 50 ° C.,
Preferably, it is performed at a temperature between -10 ° C and 30 ° C. In the esterification and hydrochloride reaction, the deprotected compound of compound 2 is dissolved in a linear or branched lower alcohol (preferably ethanol) having 1 to 4 carbon atoms, and hydrogen chloride gas is bubbled therein. By doing. Reaction temperature is -30
C. to 50.degree. C., preferably 15 to 30.degree.
Do between. The compound 3 thus obtained has high purity and can be used for the next reaction without purification. In the reaction from compound 2 to compound 3, methanol, n-propanol, i
-Propanol, n-butanol, i-butanol, s
By using -butanol or t-butanol, R ₁ , R ₂ and R ₃ in the general formula (I) can be a methyl group, an n-propyl group, an i-propyl group, an n-butyl group, an i-butyl group. , A s-butyl group, or a t-butyl group. In addition, by using a mixture of these instead of ethanol alone, it is possible to lead to compounds having different R ₁ , R ₂ and R ₃ in the general formula (I). The reaction for obtaining the compound 4 from the compound 3 is performed by converting N- (4′-tolylsulfonyl) -3,4-dihydro-2H-1,4-benzothiazine-7-carboxylic acid into thionyl chloride or oxalyl chloride (preferably thionyl chloride). And then performing a coupling reaction with glutamic acid having a protected carboxyl group in the presence of a base. The solvent when preparing the acid chloride is toluene, benzene, dichloromethane, tetrachloromethane, or no solvent, preferably
Toluene or no solvent. To accelerate the reaction rate, a catalytic amount of dimethylformamide may be added. The reaction is carried out at a temperature between 0 ° C and 100 ° C.
Perform between 0 ° C and 25 ° C. As a solvent used in the coupling reaction, toluene, benzene, dichloromethane, chloroform, tetrahydrofuran, ethyl ether, or a mixture thereof with water is used, and preferably, a mixture of dichloromethane and water is used. Reaction is 0 ° C-10
It is carried out between 0 ° C, preferably between 10 ° C and 30 ° C. The base to be used includes pyridine, triethylamine, potassium carbonate, sodium hydrogen carbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide and the like, and preferably potassium carbonate is used. In addition, compound 3 to compound 4
In the reaction to obtain N- (4′-tolylsulfonyl) -3,4-dihydro-2H-1,4-benzothiazine-7-carboxylic acid, instead of N-benzyloxycarbonyl-3,4-dihydro-2H -1,4-benzothiazine-7-carboxylic acid, Nt-butyloxycarbonyl-3,4-dihydro-2H-1,4-benzothiazine-7-carboxylic acid, and N-pivaloyl-3,4-dihydro- 2H-1,4-benzothiazine-7-carboxylic acid or the like can be used. Further, instead of N- (4′-tolylsulfonyl) -3,4-dihydro-2H-1,4-benzothiazine-7-carboxylic acid, 1-benzyloxycarbonyl-indoline-5-carboxylic acid or 1-benzyloxycarbonyl-indoline-5-carboxylic acid is used. By using benzyloxycarbonyl-3,4-dihydro-2H-1,4-benzoxazine-7-carboxylic acid, A in the general formula (I) becomes -C
A compound that is H ₂ — or —CH ₂ O— can also be obtained.

The reaction from compound 4 to compound 5 is carried out by deprotecting the tosyl group of compound 4 with an acid. The acid used at this time includes a hydrobromic acid acetic acid solution, hydrochloric acid, sulfuric acid, methanesulfonic acid, trifluoromethanesulfonic acid and the like, and preferably a hydrobromic acid acetic acid solution is used.
The reaction is carried out at a temperature between 0 ° C and 100 ° C, preferably 1 ° C.
Perform between 5 ° C and 30 ° C. At this time, a cation scavenger such as cresol, anisole, or phenol may be added to capture the generated cation species.

In the reaction from compound 5 to compound 6, the alkylation reaction of compound 5 with 2,4-diaminopteridine-6-methylbromide is followed by alkaline hydrolysis of the ester group and further to the isoelectric point. Performed by In the alkylation reaction, dimethylformamide is used as a solvent,
There are dimethylacetamide, dimethylsulfoxide and the like, and preferably, dimethylacetamide is used.
The reaction temperature is between 0 ° C and 100 ° C, preferably between 20 ° C and 60 ° C. In the hydrolysis reaction, the base used is sodium hydroxide, potassium hydroxide,
Lithium hydroxide, potassium carbonate, sodium bicarbonate,
There are barium hydroxide, triethylamine, pyridine and the like, and preferably, sodium hydroxide and lithium hydroxide are used. The reaction temperature is between 0 ° C and 50 ° C, preferably between 10 ° C and 30 ° C. The reaction solvent to be used includes methanol, ethanol, tetrahydrofuran, ethyl ether, dimethoxyethane, dioxane, dimethylformamide, dimethylacetamide, dimethylsulfoxide, or a mixture thereof. Preferably, methanol or ethanol is used. As a method for leading to the isoelectric point, 0 ° C to 30 ° C, preferably 15 ° C to
At a reaction temperature of 20 ° C, the basic reaction solution after hydrolysis is
A method of adding an acid such as hydrochloric acid or sulfuric acid, preferably hydrochloric acid, may be mentioned. Further, a method of adding an acid such as hydrochloric acid or sulfuric acid to a basic reaction solution after hydrolysis to make the reaction solution acidic and then adding a base such as sodium hydroxide or potassium hydroxide can also be mentioned. In addition, in the reaction from compound 5 to compound 6, by omitting alkali hydrolysis of the ester group, R ₁ , R ₂ and R in general formula (I) can be omitted.
Compounds in which all ₃ are ethyl groups can be obtained.
The compounds of the present invention can also be synthesized by applying the specific production methods described in the examples.

The compound of the present invention is DHFR (dihydro)
(folate reduce) inhibitory action,
It is useful as a medicament, for example, a therapeutic agent for rheumatism. The pharmaceutical composition containing the compound of the present invention as an active ingredient can be orally administered (orally or inhaled) or parenterally administered (for example, intravenous administration,
Subcutaneous administration, transdermal administration, etc.), and at the time of administration, it can be prepared into a formulation suitable for each administration method.

The pharmaceutical composition containing the compound of the present invention as an active ingredient can be prepared by a conventional preparation technique.
Depending on its use, it can be used as solid and liquid preparations such as capsules, granules, creams, powders, syrups, tablets, injections, and ointments.

In the preparation of such preparations, for example, stabilizers, lubricants, buffers, bases, flavoring agents, and the like, which are nontoxic additive components usually used in the preparation of this type of drug,
Binders, antioxidants, coating agents, coloring agents, tonicity agents, excipients, dispersants, disintegrants, preservatives, solubilizing agents, flow agents and the like can be used.

As the stabilizer, sulfites (sodium bisulfite, sodium sulfite, etc.), edetates (sodium edetate, tetrasodium edetate, etc.), hydrogenated oil, sesame oil, chondroitin sulfate, dibutylhydroxytoluene, etc. No. Lubricants include dried aluminum hydroxide gel, glycerin, silicic acid and its salts (light anhydrous silicic acid, magnesium silicate, etc.), stearic acid and its salts (aluminum stearate, calcium stearate, magnesium stearate, etc.), Examples include talc, polyethylene glycols, phosphoric acid and the like. Examples of the buffer include acetic acid, tartaric acid, sodium carbonate, boric acid, phosphoric acid and salts thereof (trisodium phosphate, sodium hydrogen phosphate, dipotassium phosphate and the like).

Examples of the base include glycerin, vegetable oils (eg, olive oil, sesame oil, wheat germ oil), stearyl alcohol, cetanol, lard, white petrolatum, paraffin, bentonite, isopropyl lanolin fatty acid, and petrolatum. As a flavoring agent, L-aspartic acid and salts thereof (sodium L-aspartate, magnesium L-aspartate, etc.), sodium saccharin,
Sugars (lactose, sucrose, glucose, D-mannitol, etc.),
d1-menthol, 1-menthols and the like.
Examples of the binder include agar, stearyl alcohol, gelatin, cellulose and derivatives thereof (ethylcellulose, carboxymethylethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, etc.), starch and derivatives thereof (pregelatinized starch, oxidized starch, dextrin, etc.), Sugars (lactose, sucrose, microcrystalline cellulose, glucose, etc.), tragacanth, polyvinyl alcohol and the like.

As antioxidants, ascorbic acid, L-
Ascorbic acid stearate, sulfites (sodium sulfite, sodium bisulfite, sodium pyrosulfite, etc.), sodium edetate, erythorbic acid,
Examples include cysteine hydrochloride, dibutylhydroxytoluene, sodium thiomalate, concentrated mixed tocopherol, butylhydroxyanisole, propyl gallate and the like. Examples of the coating agent include shellac, cellulose derivatives (cellulose acetate, hydroxypropylcellulose, cellulose acetate phthalate, hydroxypropylmethylcellulose, etc.), polyvinylpyrrolidones, polyethylene glycol, polyethylene glycols, methacrylic acid copolymers, liquid paraffin, etc. No. Examples of the coloring agent include indicocarmine, caramel, riboflavin and the like. As the tonicity agent, potassium chloride, sodium chloride, glycerin, sodium bromide, D-sorbitol, nicotinamide, glucose,
Boric acid and the like. Excipients include silicates (synthetic aluminum silicate, magnesium silicate aluminate, calcium silicate, magnesium silicate, etc.), tartaric acid, potassium hydrogen tartrate, magnesium hydroxide, cellulose and derivatives thereof (hydroxypropylcellulose, Hydroxypropyl methylcellulose, methylcellulose, etc.), starch and derivatives thereof (sodium carboxymethyl starch, β-cyclodextrin, dextrin, hydroxypropyl starch, etc.), sugars (lactose, sucrose, glucose, D-mannitol, etc.), glyceryl monostearate, And sorbitan monostearate.

Examples of dispersants include gum arabic, propylene glycol alginate, stearic acid and salts thereof (zinc stearate, magnesium stearate, etc.), sorbitan sesquioleate, D-sorbitol, tragacanth, methylcellulose, aluminum monostearate, etc. Is mentioned. As disintegrants, agar, gelatin, cellulose and derivatives thereof (crystalline cellulose, cellulose acetate phthalate, hydroxypropylcellulose, hydroxypropylmethylcellulose, etc.),
Carbonates (calcium carbonate, sodium hydrogen carbonate, magnesium carbonate, etc.), starch and its derivatives (sodium carboxymethyl starch, hydroxypropyl starch, etc.), tragacanth and the like. As a preservative,
Alcohols (chlorobutanol, phenethyl alcohol, propylene glycol, benzyl alcohol, etc.),
Benzalkonium chloride, benzethonium chloride, dried sodium sulfite, dried sodium sulfate, cresol, chlorocresol, dibutylhydroxytoluene, potassium sorbate, sodium dehydroacetate, paraoxybenzoic acid esters (isobutyl paraoxybenzoate, ethyl paraoxybenzoate, paraoxyl Methyl benzoate, etc.), phenol, formalin, phosphoric acid and the like.

Examples of the solubilizer include sodium benzoate, ethylenediamine, citric acid, sodium citrate, glycerin, sodium acetate, sodium salicylate, sorbitan sesquioleate, nicotinamide,
Glucose, benzyl alcohol, polyvinylpyrrolidones and the like can be mentioned. Examples of the fluidizing agent include hydrous silicon dioxide, stearic acid and salts thereof (such as calcium stearate and magnesium stearate), talc, and anhydrous ethanol. In addition, a pharmaceutical component can be added in addition to these additional components.

The content of the compound of the present invention in such a preparation is as follows:
Although it depends on the dosage form, it is generally 0.00001 to 1
Desirably, it is contained at a concentration of 00% by weight. The pharmaceutical composition of the compound of the present invention can be widely varied depending on the kind of warm-blooded animals including human beings, severity of symptoms, diagnosis by a doctor, and the like. 0.001 to 50 per day
mg / kg, 0.000 / day for parenteral administration
01 to 10 mg / kg. In addition, the above dose is 1 to
It can be administered once or several times per 7 days in a lump or in divided doses, and can be appropriately changed depending on the severity of the symptoms, the judgment of the doctor, and the like.

[0025]

The present invention will be described more specifically with reference to the following examples. Further, in order to show the usefulness of the compound of the present invention,
Test examples show the results of DHFR inhibition tests for representative compounds of the present invention.

Reference Example 1: 5- (Nt-butoxycarbonylamino) -5-t-
Butoxycarbonylpentanoyl-2-aminoglutar
Acid diethyl ester Nt-butoxycarbonyl homoglutamic acid α-t
-Butyl ester (1.36 g, 4.28 mmol),
Glutamic acid diethyl ester hydrochloride (1.2 g, 4.
91 mmol) and 3-hydroxy-1,2,3-
Benzotriazin-4 (3H) -one (0.8 g, 4.
81 mmol) in dimethylformamide (50 ml) and triethylamine (1.3 ml,
After cooling to -17 ° C, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (0.92 g, 4.8 mmol) was added, and the mixture was stirred at the same temperature for 3 hours. Thereafter, the temperature was raised to 20 ° C to 25 ° C, and the mixture was further stirred for 3 hours. Ice water (400m
l) was added, and the organic matter was extracted with ethyl acetate. The obtained organic layer was sequentially washed with a 10% aqueous citric acid solution, water, 5% sodium hydrogen carbonate and water, dried over anhydrous sodium sulfate, and then the solvent was distilled off under reduced pressure. The obtained residue was subjected to silica gel column chromatography, and purified using hexane-ethyl acetate (9: 1) as a developing solvent.
The target product (1.62 g) was obtained as a colorless oil.

¹ H-NMR (CDCl ₃ , δ): 1.1
8 (6H, m), 1.37 (18H, m), 1.56
(4H, m), 1.82 (1H, m), 1.95 (1
H, m), 2.10 (2H, m), 2.38 (2H,
m), 3.76 (1H, m), 4.07 (4H, m),
4.23 (1H, m), 7.06 (1H, d, J = 8.
9 Hz), 8.19 (1H, d, J = 7.6 Hz).

Reference Example 2: 5- (N- (4'-tolylsulfonyl) -3,4-di
Hydro-2H-1,4-benzothiazine-7-carboni
Ruamino) -5-ethoxycarbonylpentanoyl-2
-Aminoglutaric acid diethyl ester The compound obtained in Reference Example 1 (1.61 g, 3.2 mmol)
While cooling with ice-methanol, trifluoroacetic acid (17.2 ml, 224 mmol) was added, and the mixture was stirred under the same conditions for 6 minutes. Then, the reaction solution was returned to 20 ° C to 25 ° C and stirred for 2 hours. The solvent was removed under reduced pressure, and the obtained residue was dissolved in ethanol (30 ml), and hydrogen chloride gas (16.8 g, 460 mmol) was bubbled therein under ice cooling. After the temperature was returned to 20 ° C to 25 ° C, the mixture was stirred for 12 hours. The solvent was removed under reduced pressure to obtain a colorless solid residue. This residue was dissolved in dichloromethane (20 ml) (solution A). Next, N- (4'-tolylsulfonyl) -3,4-dihydro-2H-1,4-benzothiazine-7-carboxylic acid (1.7 g, 4.87 mmol)
Was suspended in thionyl chloride (10 ml) and stirred at 20 ° C. to 25 ° C. for 3 hours in the presence of 0.1 equivalent of dimethylformamide. The solvent was removed under reduced pressure, and the obtained residue was dissolved in dichloromethane (20 ml) (solution B). Next, water (14 ml), solution B, and potassium carbonate (1.1 g, 7.92 mmol) were sequentially added to the solution A.
At 25 ° C., the reaction was stirred for 12 hours. The reaction solution was poured into a saturated aqueous solution of sodium hydrogen carbonate, and organic substances were extracted with chloroform. The organic layer was washed with 1N hydrochloric acid and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and the obtained residue was subjected to silica gel column chromatography,
Purification was performed using chloroform as a developing solvent to obtain the target compound as a colorless oil (1.80 g).

Reference Example 3: 5- (3,4-dihydro-2H-1,4-benzothiazi
-7-carbonylamino-5-ethoxycarbonylpe
The compound obtained in Reference Example 2 of nantanoyl-2-aminoglutaric acid diethyl ester (1.78 g, 2.52 mmol)
l) and anisole (1.73 g, 15.2 mmol) in 30% hydrogen bromide acetic acid solution (21 ml, 77.9 mmol).
l) and stirred at 20 ° C to 25 ° C for 4.5 hours. Next, dry ether (360 ml) was added to the reaction solution under ice cooling, and a precipitate was obtained by decantation. The precipitate is suspended in chloroform, the suspension is washed with a saturated aqueous solution of sodium hydrogen carbonate, dried over anhydrous sodium sulfate, and then the solvent is removed under reduced pressure to give the target compound as a colorless oil (1.24 g). ) Got.

Example 1 5- [4- (2,4-diaminopteridin-6-yl)
Methyl-3,4-dihydro-2H-1,4-benzothia
Zin-7-carbonylamino] -5-carboxyl pen
Tanoyl-2-aminoglutaric acid The compound obtained in Reference Example 3 (1.24 g, 2.25 mmol)
l) and 2,4-diaminopteridine-6-methylbromide hydrobromide (1.0 g, 2.65 mmol) were suspended in dimethylacetamide (30 ml), and the suspension was added at 20 ° C to 25 ° C.
The mixture was stirred for 15 hours at a temperature of 60 ° C. under light shielding, further heated to 60 ° C., and stirred for 8 hours. The reaction solution was poured into a saturated aqueous solution of hydrogencarbonate, and organic substances were extracted with a mixed solvent of chloroform: methanol = 10: 1. The obtained organic layer was dried over anhydrous sodium sulfate, and 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 = 10: 1 as a developing solvent, a main reaction product as a yellow solid (Rf value = 0.4)
I got The obtained solid was dissolved in ethanol, and water (5 ml) and sodium hydroxide (0.17 g,
4.08 mmol), and the mixture was stirred at 20 ° C to 25 ° C for 12 hours. The reaction solvent was distilled off under reduced pressure, and water (20 ml) was added to the obtained residue.
Was adjusted to 3.7 with 1N HCl, and the precipitate was collected by filtration to give the desired product as a tan solid (0.64 g).

¹ H-NMR (CDCl ₃ , δ): 1.4
-1.9 (5H, m), 1.93 (1H, m), 2.1
7 (2H, m), 2.29 (2H, m), 3.21 (2
H, s), 3.98 (2H, s), 4.20 (1H,
m), 4.30 (1H, m), 4.81 (2H, s),
6.83 (1H, m), 7.48 (1H, m), 7.6
2 (1H, m), 8.09 (1H, s), 8.22 (1
H, s), 8.70 (1H, s).

Test Example: A representative compound of the present invention was tested for inhibition of DHFR. 10 μM unlabeled dihydrofolate (Sigma), the method of Sheldon et al. (Analytical Biochemist)
ry, 103, 152-156 (1980)), folic acid labeled with tritium (manufactured by Amersham).
0.1 μM tritium-labeled dihydrofolate, 55 mM NADPH, 4% BSA (Int.
ergen), 10 mM 2-mercaptoethanol (Nacalai Tesque), 60 mM sodium
citrate (pH = 7.2), DHFR (Sig
ma (89 μunit / ml) and various concentrations of the test drug were added to make a total of 110 μl. 37 ℃
For 30 minutes, then 500 μl of 54 mM folic acid (Sigma), 100 μl of 1 M HCl, 100 μl
The reaction was stopped by sequentially adding μl of a 600 mM zinc sulfate solution, followed by centrifugation (25,000 × g, 6 minutes, 4 minutes).
° C). By centrifugation, the substrate and folic acid in the reaction solution precipitate, whereas tetrahydrofolate, a reaction product, remains in the centrifugation supernatant. The count in the centrifuged supernatant obtained here was measured with a liquid scintillation counter,
The obtained result was defined as DHFR activity. The results are shown in Table 1.

[0033]

[Table 1]

As is clear from Table 1, the compounds of the present invention have an inhibitory effect on DHFR.

[0035]

The compound of the present invention has an inhibitory effect on DHFR and is useful as a drug, for example, a therapeutic agent for diseases based on abnormal cell proliferation such as rheumatism. Further, the compound of the present invention is also useful as a sample for confirming that a methotrexate derivative having a homoglutamic acid at a terminal does not undergo glutamine oxidation in a living body.

Claims (6)

[Claims] 1. A compound of the general formula (I):(Wherein A is -CH₂-Or -CH₂O- or-
CH₂Represents S-; R₁ , R₂, And R₃Are the same but different
A hydrogen atom or a lower carbon atom having 1 to 4 carbon atoms
Shows an alkyl group. Methotrexate induction indicated by)
body. 2. The methotrexate derivative according to claim 1, wherein R ₁ , R ₂ , and R ₃ are all hydrogen atoms. 3. The methotrexate derivative according to claim 1, wherein A is —CH ₂ S—. 4. 5-[(4- (2,4-diaminopteridin-6-yl) methyl-3,4-dihydro-2H-
1,4-benzothiazine-7-carbonyl) amino 1
The methotrexate derivative according to any one of claims 1 to 3, which is 5-carboxylpentanoyl-2-aminoglutaric acid. 5. A pharmaceutical composition comprising the methotrexate derivative according to claim 1 as an active ingredient. 6. A therapeutic agent for rheumatism, comprising the methotrexate derivative according to claim 1 as an active ingredient.