JP2001335486A - Pterin derivative-containing apoptosis inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To newly find out that a pterin derivative which is not conventionally known to have apoptosis inhibitory actions at all has the above actions by carrying out intensive search for a compound having the apoptosis inhibitory actions and to thereby provide a new apoptosis inhibitor. SOLUTION: This pterin derivative is represented by formula (I) [wherein, R1 and R2 are each independently a 1-20C alkyl group which may occasionally be substituted with one or more groups selected from the group consisting of hydroxy group, amino group, phospate group and sulfo group, an acyl group represented by the formula; R3-CO (R3 is a 1-4C alkyl group), an amino acid residue represented by formula (a) or H; R4 is H or a linear or a branched 1-4C alkyl group or a linear or a branched 1-4C alkyl group substituted with one group selected from the group consisting of hydroxy group, carboxy group, carbamoyl group, mercapto group, methylthio group, amino group, guanidino group and indolyl group or the like as a substituent group; X is formyl group or hydroxymethyl group; A is a group represented by formula (Ia); and n is 0 or an integer of >=1].

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an apoptosis inhibitor containing a pterin derivative. The apoptosis inhibitor of the present invention is useful for the prevention or treatment of diseases that can be prevented or treated by inhibiting apoptosis, in particular, diseases caused by promoting apoptosis.

[0002]

2. Description of the Related Art Apoptosis refers to a series of processes leading to cell death caused by cells themselves under physiological conditions, and the abnormality is caused by various diseases such as fulminant hepatitis, AIDS, adult T-cell leukemia, Or, it is known to be closely related to the onset of influenza.

[0003] Examples of compounds having an apoptosis-suppressing effect include, for example, azolidinedione derivatives (Japanese Unexamined Patent Publication No.
263726), licoricidinol (Japanese Unexamined Patent Application Publication No.
JP-A-255650), triazepine derivatives or diazepine derivatives (JP-A-11-228576), isocarbacycline derivatives (JP-A-11-22).
2436), lycorine (JP-A-11-79992)
Publication), or berberine, palmatin, capillary lysine, 7-methylcapillarycin, 6,7-dimethylesculetin, alcapillin, capirin, capylene, or capillarin (the above-mentioned Japanese Patent Application Laid-Open No. 9-30983).
It has been known.

[0004]

DISCLOSURE OF THE INVENTION The present inventors diligently searched for a compound having an apoptosis-suppressing action, and found that a pterin derivative, which had hitherto never been known to have an apoptosis-suppressing action, has the above-mentioned action. Newly found. The present invention is based on such findings. Therefore, an object of the present invention is to provide a novel apoptosis inhibitor.

[0005]

The object of the present invention is to provide, according to the present invention, a compound of formula (I):

Embedded image [Wherein, R ¹ and R ² are each independently a carbon which may be substituted with one or more groups selected from the group consisting of a hydroxyl group, an amino group, a phosphate group, and a sulfonic acid group. An alkyl group of the formulas 1 to 20, or a formula R ³ —CO—
(R ³ is an alkyl group having 1 to 4 carbon atoms), an acyl group represented by the formula (a):

Embedded image An amino acid residue represented by or a hydrogen atom;
⁴ is a hydrogen atom or a linear or branched C 1
Or a group consisting of a hydroxyl group, a carboxyl group, a carbamoyl group, a mercapto group, a methylthio group, an amino group, a guanidino group, an imidazolyl group, a phenyl group, a hydroxyphenyl group, and an indolyl group. X is a formyl group or a hydroxymethyl group, which is a linear or branched alkyl group having 1 to 4 carbon atoms, which is substituted with one group selected from the group consisting of:
A is of the formula (Ia):

Embedded image N is 0 or an integer of 1 or more, provided that when X is a hydroxymethyl group, n is 0, and when n is an integer of 1 or more. In the formula, R ¹ and R ² are each a hydrogen atom, and X is a formyl group].
Alternatively, the problem can be solved by an apoptosis inhibitor characterized by containing a cyclic body or a salt thereof as an active ingredient.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The apoptosis inhibitor of the present invention comprises:
It contains a pterin derivative represented by the above formula (I) as an active ingredient. The alkyl group having 1 to 20 carbon atoms represented by R ¹ and R ² is a linear or branched alkyl group having 1 to 20 carbon atoms (preferably an alkyl group having 1 to 10 carbon atoms, more preferably Alkyl group having 1 to 4 carbon atoms). The alkyl group having 1 to 4 carbon atoms represented by R ³ is a linear or branched alkyl group having 1 to 4 carbon atoms. Examples of such an alkyl group include a methyl group, an ethyl group, an n-propyl group, an i-propyl group, an n-butyl group, an i-butyl group, a sec-butyl group, and a tert-butyl group. Groups or ethyl groups. In the present specification, "alkyl group which may be substituted"
The term means an unsubstituted alkyl group or an alkyl group substituted with one or more same or different substituents.

The amino acid residue represented by the formula (a) (ie, the group excluding the hydroxyl group of the α-carboxyl group of the amino acid) includes both L-form and D-form. The amino acid residue represented by the formula (a) is preferably alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, glycine, serine,
It can be derived from threonine, cysteine, tyrosine, asparagine, glutamine, lysine, histidine, arginine, aspartic acid, or glutamic acid.

In the compound represented by the formula (I),
R ¹ and R ² are each independently the same or different, and may be optionally substituted with one or more groups selected from the group consisting of a hydroxyl group, an amino group, a phosphate group, and a sulfonic acid group. alkyl group having 1 to 20 carbon atoms, wherein R ³ -
It may be an acyl group represented by CO- (R ³ is the above-mentioned alkyl group having 1 to 4 carbon atoms), an amino acid residue represented by the formula (a), or a hydrogen atom. In the formula (I), n is 0 or an integer of 1 or more, preferably 0 or an integer of 1 to 100, more preferably 0 or 1 to 1.
It is an integer of 10.

In the pterin derivative represented by the formula (I), R ¹ and R ² are each independently a group selected from the group consisting of a hydroxyl group, an amino group, a phosphate group and a sulfonic acid group. An alkyl group having 1 to 20 carbon atoms which may be substituted by one or more, a compound represented by the formula R ³ —CO— (R ³
Is an alkyl group having 1 to 4 carbon atoms), an amino acid residue represented by the formula (a), or a hydrogen atom, and A is represented by the formula (Ia) Group,
6-Formyl-pterin or a derivative thereof in which n is 0 or an integer of 1 or more and X is a formyl group is preferred. Further, as the pterin derivative represented by the formula (I),
At the 6-position of the 4-oxy-pteridine ring,-(CH = N-
A) Preferably, _n- X or -X is bonded.

Further, as the pterin derivative represented by the above formula (I), R ¹ and R ² are hydrogen atoms, and n
Is 0, that is, the compound represented by the formula (I),
Formula (II):

Embedded image 6-formyl-pterin is more preferable.

Since 6-formyl-pterin has an amino group and a formyl group, it can naturally form a Schiff base between a plurality of molecules in an aqueous solution to be an oligomer or polymer of 6-formyl-pterin. The oligomer or polymer includes not only a linear oligomer or polymer but also a cyclic oligomer or polymer. The reaction from the monomer to the oligomer or polymer is a reversible reaction, and under alkaline conditions, the 6-formyl-pterin oligomer or polymer readily decomposes to the monomer 6-formyl-pterin. Therefore, it is considered that a similar reversible reaction occurs in the living body or on the skin. The 6-formyl-pterin oligomer or polymer is
A compound represented by the above formula (I) wherein R ¹ and R ² are simultaneously a hydrogen atom, and n is an integer of 1 or more (preferably 1 to 100), and the active ingredient in the apoptosis inhibitor of the present invention. Can be used as

A derivative which can be easily converted in vivo into the pterin derivative represented by the above formula (I), ie, a prodrug, can also be used as the active ingredient of the present invention. Methods commonly used for the selection and manufacture of suitable prodrugs are described, for example, in Design of
Prodrugs, ed. H. Bundgaard, E
lsevier, 1985.

6-Formyl-pterin can be used as an active ingredient in the form of a free compound, and its salts, especially pharmaceutically acceptable salts thereof, such as inorganic acid salts (eg, hydrochloric acid) Salts or sulfates) or organic acid salts (eg, camphorsulfonate, tartrate,
Malate or oxalate). Further, it can be used in the form of the above-mentioned 6-formyl-pterin oligomer or polymer. 6
Formyl-pterin oligomers or polymers are known compounds and the free compounds can be converted into their salts or the salts into free compounds or other salts by methods known per se.

The pterin derivative represented by the above formula (I) can also be used as an active ingredient in the form of a free compound and a salt thereof, particularly a pharmaceutically acceptable salt thereof, for example, an inorganic salt. They can also be used in the form of acid addition salts with acids or organic acids, or salts with inorganic or organic bases. Examples of the inorganic acid salt include, for example, hydrochloride and sulfate, and examples of the organic acid salt include:
For example, camphorsulfonate, tartrate, malate, oxalate and the like can be mentioned. As the inorganic base suitable for forming the salt of the pterin derivative represented by the formula (I), for example, a hydroxide, carbonate, or bicarbonate such as ammonia, sodium, lithium, calcium, magnesium, or aluminum And the like. Examples of the salt with an organic base include a mono-, di-, or tri-alkylamine salt such as methylamine, dimethylamine, or triethylamine, a mono-, di-, or tri-hydroxyalkylamine salt, and a guanidine salt. , N-methylglucosamine salt,
Or an amino acid salt. The pterin derivative represented by the formula (I) is a known compound or can be prepared by a method known per se. In addition, the free compound can be converted into its salt, or the salt can be converted into the free compound or another salt, by a method known per se.

For example, a pterin derivative of the formula (I) wherein R ¹ and R ² are both hydrogen atoms, n is 0 and X is a hydroxymethyl group (ie, 6-hydroxymethyl-pterin) Is used as a starting compound, and after protecting the hydroxyl group with a suitable protecting group, the amino group at the 2-position of the 4-oxy-pteridine ring is optionally substituted with a hydroxyl group, an amino group, a phosphate group, and a sulfonic acid group. 1 carbon atom which may be substituted with 1 or more selected groups
To 20 alkyl groups, an acyl group represented by the formula R ³ —CO— (R ³ is the alkyl group having 1 to 4 carbon atoms), and an amino acid residue represented by the formula (a). A 6-hydroxymethyl-pterin derivative can be prepared by introducing a group 1 or 2 selected from the group and then removing the hydroxyl-protecting group. Further, by oxidizing the hydroxymethyl group in the 6-hydroxymethyl-pterin derivative obtained by the above method,
Formyl-pterin derivatives can be prepared.

A pterin derivative represented by the above formula (I),
Or a cyclic body thereof, or a pharmaceutically acceptable salt thereof, has an apoptosis-suppressing action or can induce a compound having an apoptosis-suppressing action. It can be used as an active ingredient of a pharmaceutical composition for preventing or treating a treatable disease (particularly, a disease caused by promotion of apoptosis) (hereinafter sometimes simply referred to as “pharmaceutical composition according to the present invention”). it can.

For example, in the pterin derivative represented by the above formula (I), n is 0 and either R ¹ or R ² is not a hydrogen atom, and the pterin derivative represented by the formula (I), or 6-Formyl-pterin [ie, a compound of formula (I) in which n is 0, R ¹ and R ² are simultaneously hydrogen atoms, and X is a formyl group] is itself apoptotic. Has an inhibitory action.

Further, of the pterin derivatives represented by the formula (I), the pterin derivative represented by the formula (I) in which n is an integer of 1 or more, particularly, n is an integer of 1 to 100, was administered. Since it is gradually decomposed in the body of the animal to release 6-formyl-pterin, a compound having an apoptosis inhibitory action can be induced.

Since the pharmaceutical composition of the present invention contains the pterin derivative represented by the above formula (I), its cyclic form, or a pharmaceutically acceptable salt thereof, it can inhibit apoptosis. Is possible. Therefore, the pharmaceutical composition according to the present invention provides a disease which can be prevented or treated by inhibiting apoptosis (particularly a disease caused by promotion of apoptosis) such as fulminant hepatitis, AIDS, adult T-cell leukemia, or influenza. It can be treated or prevented effectively.

The pharmaceutical composition according to the present invention may comprise a pterin derivative or a pharmaceutically acceptable salt thereof, alone or, preferably, a conventional pharmaceutically or veterinarily acceptable carrier. In addition, it can be administered orally or parenterally to animals, preferably mammals (particularly humans).

The dosage form is not particularly limited. For example, oral preparations such as powders, fine granules, granules, tablets, capsules, suspensions, emulsions, syrups, extracts and pills Or injection, topical solution, ointment, suppository, topical cream, jelly, gel, paste,
Or parenteral preparations such as eye drops can be mentioned.

These oral preparations include, for example, gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannitol, carboxymethylcellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soy lecithin, sucrose, fatty acid ester, talc Excipients, such as magnesium stearate, polyethylene glycol, magnesium silicate, silicic anhydride, or synthetic aluminum silicate, binders, disintegrants, surfactants, lubricants, fluidity promoters, diluents, and preservatives Agents, coloring agents,
It can be produced according to a conventional method using a flavor, a flavoring agent, a stabilizer, a humectant, a preservative, an antioxidant and the like.
For example, capsules filled with a mixture of 6-formyl-pterin and lactose are used.

Examples of the method of parenteral administration include topical administration by cream or ointment, injection (subcutaneous, intravenous, etc.), and rectal administration. Of these, injections are most preferably used.

For example, in preparing an injection, in addition to the pterin derivative as an active ingredient, for example, a water-soluble solvent such as physiological saline or Ringer's solution, a water-insoluble solvent such as vegetable oil or fatty acid ester, glucose or sodium chloride For example, an isotonic agent, a solubilizer, a stabilizer, a preservative, a suspending agent, an emulsifier, or the like can be used.

The pharmaceutical composition of the present invention may be administered using a sustained-release preparation technique using a sustained-release polymer or the like. For example, the pharmaceutical compositions of the present invention can be incorporated into a pellet of ethylene vinyl acetate polymer and the pellet can be surgically implanted into the tissue to be treated.

The pharmaceutical composition of the present invention comprises, but is not limited to, a pterin derivative or a pharmaceutically acceptable salt thereof in an amount of 0.01 to 99% by weight, preferably 0.1 to 99% by weight. It can be contained in an amount of up to 80% by weight.

When the pharmaceutical composition of the present invention is used by utilizing its apoptosis-suppressing action, the dosage varies depending on the type of disease, age, sex, body weight, symptom degree of the patient, or the administration method. The trader can determine it appropriately. Furthermore, the form is not limited to pharmaceuticals, and it can be provided in various forms, for example, in the form of food or drink as a functional food, a health food, or a feed.

When 6-formyl-pterin was subjected to an acute toxicity test using rats, 6 mg / kg body weight of 6-formyl-pterin was intravenously injected to 10-formyl-pterin.
Administered to rats and observed for 3 months, no deaths were observed.

[0029]

The action of the apoptosis inhibitor of the present invention is not limited to this, but the present inventors believe that it is based on the following action. That is, in apoptosis, DNA fragmentation is observed prior to final cell death. As a group of proteases involved in the DNA fragmentation, at least 10 types of caspases (caspase 1 to caspase 10) are known (B
iochem. J. , 326, 1-16, 1997),
Caspase 3 is a CAD (caspase activated DN) which is a final execution factor of DNA fragmentation.
ase) activator. Formula (I)
When the pterin derivative represented by is administered, DNA fragmentation is suppressed, and as a result, apoptosis is suppressed as shown in the Examples below. In addition, as shown in the examples described later, the administration of the pterin derivative also suppresses the activation of caspase-3. Therefore, it is currently unknown whether the pterin derivative represented by the formula (I) directly suppresses the activation of caspase 3 or the activation of a group of caspases further upstream of caspase 3. However, it is considered that by suppressing the activation of caspase 3, DNA fragmentation is suppressed, and as a result, apoptosis can be suppressed.

[0030]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention.

Example 1 In this example, one of the apoptosis-inducing factors
6-formyl-pterin (6F) in human cultured cells in which apoptosis was induced using anti-Fas antibody
The effect of P) was examined. Fas stimulation (eg, anti-Fas
Induction of apoptosis by the addition of antibodies)
It is considered a pathological model for AIDS, adult T-cell leukemia, or influenza. Cell line derived from human T-cell leukemia (Jurkat cell; 2 × 10 ⁶ cells / mL)
The anti-Fas antibody CH-11 (MBL) 200 ng /
mL and various concentrations (final concentration = 10-500 μmol)
/ L) and 6-formyl-pterin, and cultured for 3 hours. In the culture, an RPMI medium was used as a medium.

After completion of the culture, the cells are collected, a part thereof is subjected to nuclear staining with propium iodide (PI), and the degree of DNA fragmentation (the degree of apoptosis) is measured by flow cytometry. did. In addition, an antibody against activated caspase 3 (anti-activated caspase 3 antibody; Pharminge)
(Company n), and the degree of activation of caspase 3 was measured by flow cytometry. The degree of DNA fragmentation (unit =%) and caspase-3
The degree of activation (unit =%) was measured as a percentage of the number of relevant cells (ie, “cells in which DNA fragmentation has occurred” or “cells in which caspase 3 is activated”) relative to the total number of cells. . The caspase 3 is Fas
It is believed to be the ultimate executive of DNA fragmentation caused by various factors, including stimulation.

In Table 1, the results obtained when 6-formyl-pterin (6FP) was added at various concentrations (Experiment Nos. 3 to 8 in Table 1) are shown as the control results [ie, the anti-Fas antibody CH- 11 and 6-formyl-pterin-free (Experiment No. 1) results and anti-Fa
Results when only s antibody CH-11 was added (6-formyl-pterin was not added) (Experiment No. 2)]. In Table 1, the unit of “6FP addition concentration” is “μm
ol / L ", and each unit of" DNA fragmentation "and" caspase 3 activation "is"% ". Further, in the “anti-Fas antibody” column in Table 1, the symbol “−” indicates that the anti-Fas antibody
s antibody was not added, and the symbol “+” indicates that anti-F
It means that as antibody was added. FIG. 1 shows the results of graphing the data shown in Table 1. In FIG. 1, the black bar on the left side of each black bar consisting of two
The degree of “A fragmentation” is shown, and the black bar on the right is “Caspase 3”.
Indicates the degree of "activation". The numbers 1 to 8 on the horizontal axis in FIG. 1 correspond to the experiment numbers 1 to 8 in Table 1.

As is clear from Table 1 and FIG.
Upon stimulation (ie, addition of an anti-Fas antibody), the DNA
Although fragmentation and caspase 3 activation occurred (see Experiment No. 2), administration of 6-formyl-pterin suppressed DNA fragmentation and caspase 3 activation (see Experiment Nos. 3 to 8). ). In addition, these inhibitory effects of 6-formyl-pterin are 100 μmol / L.
Is the maximum (see Experiment No. 6), and the effect is attenuated when the addition amount is larger than this. The present results indicate that 6-formyl-pterin directly inhibited Fas-stimulated activation of caspase-3, or
It is considered that the suppression of the activation of the caspase group further upstream of caspase 3 suppressed the DNA fragmentation. In any case, it was revealed that 6-formyl-pterin suppressed the activation of caspase 3, thereby suppressing DNA fragmentation, and further suppressed apoptosis.

<< Table 1 >> Experiment No. 1 2 3 4 5 6 7 8 Concentration of anti-Fas antibody − ++++++++++ 6FP addition 0 0 10 25 50 100 250 500 DNA fragmentation 5.8 28.4 14.4 12.4 7.1 4.7 7.5 7.3 Caspase 3 activation 1.5 1.5 11.9 8.2 5.1 4.4 5.9 11.8

[0035]

The apoptosis inhibitor according to the present invention can suppress apoptosis. Therefore, the pharmaceutical composition according to the present invention containing the apoptosis inhibitor can effectively prevent or treat a disease that can be prevented or treated by inhibiting apoptosis (particularly, a disease caused by promotion of apoptosis). it can.

[Brief description of the drawings]

FIG. 1 is a graph showing the degree of DNA fragmentation and caspase 3 activation when 6-formyl-pterin was added at various concentrations.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 35/02 A61P 35/02 43/00 105 43/00 105

Claims (5)

[Claims] 1. A compound of formula (I): [Wherein, R ¹ and R ² are each independently a carbon which may be substituted with one or more groups selected from the group consisting of a hydroxyl group, an amino group, a phosphate group, and a sulfonic acid group. An alkyl group of Formulas 1 to 20, and a compound represented by the formula R ³ —CO— (R ³
Is an alkyl group having 1 to 4 carbon atoms), and an acyl group represented by the formula (a): An amino acid residue represented by or a hydrogen atom;
⁴ is a hydrogen atom or a linear or branched C 1
Or a group consisting of a hydroxyl group, a carboxyl group, a carbamoyl group, a mercapto group, a methylthio group, an amino group, a guanidino group, an imidazolyl group, a phenyl group, a hydroxyphenyl group, and an indolyl group. X is a formyl group or a hydroxymethyl group, which is a linear or branched alkyl group having 1 to 4 carbon atoms, which is substituted with one group selected from the group consisting of:
A is a compound of the formula (Ia): N is 0 or an integer of 1 or more, provided that when X is a hydroxymethyl group, n is 0, and when n is an integer of 1 or more. In the formula, R ¹ and R ² are each a hydrogen atom, and X is a formyl group].
Or an apoptosis inhibitor comprising a cyclic body thereof or a salt thereof as an active ingredient. 2. The compound of formula (II): The apoptosis inhibitor according to claim 1, which comprises 6-formyl-pterin or a salt thereof represented by the following formula: 3. A pterin derivative represented by the formula (I) according to claim 1, or a cyclic body thereof, or a pharmaceutically acceptable salt thereof, as an active ingredient. A pharmaceutical composition for preventing or treating a disease that can be prevented or treated by inhibiting apoptosis. 4. The pharmaceutical composition according to claim 3, wherein the disease that can be prevented or treated by suppressing apoptosis is fulminant hepatitis, AIDS, adult T-cell leukemia, or influenza. 5. An inhibitor of DNA fragmentation in apoptosis, comprising the pterin derivative represented by the formula (I) according to claim 1 or a cyclic body thereof or a salt thereof as an active ingredient. .