JP2003212894A - Telomerase inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new peptide having telomerase-inhibiting activities. <P>SOLUTION: A peptide containing an α-amino acid residue substituted with an intercalator such as a group having an acridine skeleton, a group having an anthraquinone skeleton and a group having an anthracene skeleton can strongly bond to four-stranded structure formed by a guanidine sequence of a telomea DNA to stabilize the four-stranded structure, and can be a strong telomerase inhibitor. As a result, the peptide can be used as an effective therapeutic agent for exuberant disease found in an exuberant cell having exasperated telomerase activity, e.g. cancer, psoriasis and Marfan's syndrome, and as a reagent for research. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a peptide having an telomerase inhibitory activity containing an α-amino acid residue substituted with an intercalator group, a derivative thereof having an N-terminal or C-terminal thereof substituted, or a salt thereof, and effective use thereof. The present invention relates to a telomerase inhibitor contained as a component.

[0002]

2. Description of the Related Art Telomere exists at both ends of chromosomes of eukaryotic cells and has a special structure composed of DNA and protein. Human telomeres have a unique TTAGGG nucleotide sequence repeated 2,000 to 3,000 times. It has a structure and is required for stabilization of the chromosome ends. In fact, chromosomes lacking telomeres are known to undergo fusion and translocation. In somatic cells, the DNA polymerase complex
In vitro or in vivo because the 5'end cannot be replicated
In, the telomere length shortens with each cell division, and it is said to be an end-replication problem. In this way, whenever cell division is repeated and DNA is replicated,
Since the telomere DNA becomes shorter, the telomere is also called a "life-saving clock".

Usually, in somatic cells and the like, shortening of telomeric DNA occurs every time cell division is repeated, but germ cells and immortalized cells retain the length of telomeric DNA. this is,
Telomerase, an enzyme that elongates telomeric DNA
This is because telomere DNA is synthesized by merase). The presence of this enzyme was first reported in 1984 by Greider et al. In Tetrahymena. This telomerase is an RNA-protein complex consisting of a template RNA having a sequence complementary to the telomere repeat sequence and two types of proteins.
It is a kind of reverse transcriptase. Human telomerase RNA was isolated and its template region contains 11 nucleotides (5'-CUAACCCUAA
C), which is known to be complementary to the human telomere sequence (TTAGGG) n. Normally, telomeres have a protruding 3'end as a single strand, and telomerase recognizes and binds to the terminal structure of the telomere DNA, and uses an endogenous template RNA to cultivate the 3'end of the telomere single-stranded DNA. The telomere repeat sequence is added to. Recently, it was revealed that this telomerase activity, which is not detected in normal human somatic cells, is highly activated (85 to 95%) in immortalized cultured cells and cells derived from cancer tissues, and immortalization of cells , It became clear that telomerase plays an important role in canceration or aging.

[0004]

From the above facts,
It is thought that substances that inhibit or suppress the activity of telomerase can be effective therapeutic agents for hyperproliferative diseases found in hyperproliferative cells with enhanced telomerase activity, such as cancer, psoriasis, and Marfan syndrome. Various telomerase inhibitors have been proposed so far. For example,
Based on antisense DNA or peptide nucleic acid (Nort
on et al., Nature Biotechnology 14: 615-619), pyridine compounds (JP-A-11-49676, JP-A-11-49678).
JP, JP 11-49679, JP 11-49777, etc.), catechins derived from green tea (Naasani et al., 249: 391-39
Many telomerase inhibitors have been proposed, such as 6, 1998).

On the other hand, since telomeric DNA has a repetitive sequence rich in guanine (G), four G sequences are gathered in the overhanging region on the 3'-terminal side (G-tetrad) to form a four-stranded structure ( Qu
Adruplex) has been revealed (Stu Bo
rman, October 5, 1998, C & EN, 42-46). Telomerase cannot bind to this four-stranded structure and does not show its activity.
Therefore, if this four-chain structure can be stabilized, the activity of telomerase can be inhibited, and it is considered that it effectively acts on cancer cells. Telomere like this
Potassium ions (Balagurumoorthy, P. et.al., 1994, J. Biol. C
hem., 269, 21858), anthracene derivative (Sun, D., et a
l., 1997. J. Med. Chem., 40, 2113; Perry, PJ, et al.,
J. Med. Chem., 41, 3253), perylene derivative (Fedoroff, O.
Y., et al., 1998. Biochemistry 37, 12367) and the like have been proposed. However, even these telomerase inhibitors cannot be said to be able to sufficiently inhibit the activity of telomerase, and the present situation is that a drug targeting telomerase has not been developed yet. Therefore, an object of the present invention is to provide a new substance capable of sufficiently inhibiting the activity of telomerase. Another object of the present invention is to provide a telomerase inhibitor containing this new substance as an active ingredient.

[0006]

[Means for Solving the Problems] The present inventor
As a result of intensive research aimed at obtaining a substance that sufficiently stabilizes the four-chain structure formed by gathering four G sequences existing in the 3'terminal overhanging region of A, as a result, a group having an acridine skeleton, anthraquinone Substitution with an intercalator group such as a group having a skeleton or a group having an anthracene skeleton, that is, an intercalator group that can be inserted in parallel with a planar structure formed by four guanidine bases in the four-stranded structure of telomeric DNA It was found that a peptide containing a selected α-amino acid residue can bind strongly to the four-stranded structure of telomeric DNA and stabilize the four-stranded structure, and thus can be a powerful telomerase inhibitor. Completed Therefore, the present invention is a peptide containing an intercalator group-substituted α-amino acid residue, which is represented by the following formula I, its N-terminal or C-terminal substituted derivative or a salt thereof.

[Chemical 3] (In the formula, X represents an intercalator group, Y represents a divalent group forming an α-amino acid, Z represents a monovalent group forming an α-amino acid, and n represents 0, 1, 2, Furthermore, the present invention is a telomerase inhibitor containing the above peptide, its N-terminal or C-terminal substituted derivative or a salt thereof as an active ingredient.

[0007]

DETAILED DESCRIPTION OF THE INVENTION In Formula I, X represents an intercalator group. Telomeres as intercalator groups
In the four-stranded structure formed by gathering four guanine sequences existing in the overhang region on the 3'end side of DNA, the guanidine base
Any group may be used as long as it can be inserted in parallel with the planar structure formed by four groups. Examples of the intercalator group include a group having an acridine skeleton which may have a substituent, a group having an anthraquinone skeleton which may have a substituent, and an anthracene skeleton which may have a substituent. Having a group, a group having a naphthalene imide skeleton that may have a substituent, a group having a naphthalene diimide skeleton that may have a substituent,
Examples thereof include a group having a pyrene skeleton which may have a substituent. Examples of the substituent here include a halogen atom such as chlorine, fluorine and bromine; a carbon atom which may be substituted with a halogen atom such as methyl, ethyl, propyl, butyl, pentyl, hexyl and trifluoromethyl. To 6 alkyl groups; methoxy, ethoxy,
1 carbon atom such as butoxy, pentoxy, hexoxy
To 6 alkoxy groups; formyl, acetyl, butanoyl, pentanoyl, hexanoyl, and other acyl groups having 1 to 7 carbon atoms; acetyloxy, butanoyloxy,
Pentanoyloxy, hexanoyloxy and other acyloxy groups having 2 to 7 carbon atoms; Aminomethyl, aminoethyl, aminopropyl, methylaminomethyl, dimethylaminomethyl, dimethylaminopropyl, dimethylaminobutyl and other carbon atoms having 1 to 7 carbon atoms Examples thereof include an aminoalkyl group having 1 to 6 carbon atoms, which may be substituted with one or two 6 alkyl groups. These substituents are
It may be substituted at any position of the acridine skeleton, anthraquinone skeleton, anthracene skeleton, etc., and two or more plural substituents may be substituted on these skeletons. The acridine skeleton, anthraquinone skeleton, anthracene skeleton and the like may have a functional group for bonding to Y in the peptide of formula I. Examples of such a functional group include methylene and ethylene. Examples thereof include an alkylene group having 1 to 6 carbon atoms, a carbonyl group, an alkylenecarbonyl group having 1 to 6 carbon atoms, a sulfonyl group and an amide group.

A group having an acridine skeleton that may have a substituent, a group having an anthraquinone skeleton that may have a substituent, a group having an anthracene skeleton that may have a substituent, Specific examples of a group having a naphthalene imide skeleton that may have a substituent, a group having a naphthalene diimide skeleton that may have a substituent, and a group having a pyrene skeleton that may have a substituent Examples include the following: A group having an acridine skeleton which may have a substituent

[Chemical 4] Having an anthraquinone skeleton which may have a substituent
Basis

[Chemical 5] A group having an anthracene skeleton which may have a substituent

[Chemical 6] Has a naphthalene imide skeleton that may have a substituent
The basis

[Chemical 7] Having a naphthalenediimide skeleton that may have a substituent
Group

[Chemical 8] A group having a pyrene skeleton which may have a substituent

[Chemical 9]

In the peptide of formula I, Y represents a divalent group constituting an α-amino acid, and Z represents an α-amino acid.
Represents a valence group. That, Y is -CH from α- amino acids (NH ₂₎ COOH
Represents a divalent group constituting the remaining α-amino acid except Z, and Z represents a monovalent group. Specifically, Y is a group represented by -AW-, wherein A represents a linear or branched alkylene group having 1 to 6 carbon atoms such as methylene, ethylene, propylene, and methylmethylene. W is a single bond, NH, C
Represents O, CONH, NHCO, O, S or HNC (NH) NH. Z is -AW-
It is a group represented by H. These Y and Z are α-amino acids such as alanine, lysine, norlysine, aspartic acid, glutamine, serine and cysteine.
Represents a divalent group or a monovalent group constituting the remaining α-amino acid excluding —CH (NH ₂ ) COOH. In the peptide of formula I, n represents 0, 1, 2, 3, 4 or 5.

The peptide of formula I may be its derivative substituted at its N-terminus or C-terminus or a salt thereof. Examples of N-terminal substituted derivatives include acyl groups having 1 to 7 carbon atoms such as formyl, acetyl, butanoyl, pentanoyl and hexanoyl; methoxycarbonyl, butoxycarbonyl, t-butoxycarbonyl, pentyloxycarbonyl, hexyloxy. Examples thereof include derivatives in which an alkoxycarbonyl group having 2 to 7 carbon atoms such as carbonyl is substituted with an N-terminal amino group. As the C-terminal substituted derivative, an alkyl group having 1 to 6 carbon atoms which may be substituted with a halogen atom such as methyl, ethyl, propyl, butyl, pentyl, hexyl and trifluoromethyl; aminomethyl, amino Aminoalkyl groups having 1 to 6 carbon atoms such as ethyl, aminopropyl, methylaminomethyl, dimethylaminomethyl, dimethylaminopropyl, dimethylaminobutyl; derivatives in which an amino group or the like is substituted with a C-terminal carbonyl group. To be Examples of the salt of the peptide of formula I or the salt of the derivative in which the N-terminal or C-terminal of the peptide is substituted include organic acids such as acetic acid, trifluoroacetic acid, citric acid and malic acid; inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid. Examples thereof include acid addition salts with acids and the like. Also included are sodium salts, potassium salts and the like of the peptide of formula I with the carboxyl group.

Particularly preferred examples of the peptide of formula I of the present invention include the peptides represented by the following formula II.

[Chemical 10]

The peptide of formula I of the present invention comprises a protected derivative of an amino group or carbonyl group of an α-amino acid substituted with an intercalator group of the following formula III and a derivative of protected amino group or carbonyl group of an α-amino acid of formula IV below. Then, it can be synthesized by performing a well-known peptide synthesis reaction.

[Chemical 11] Peptide synthesis can be performed by sequentially linking both protected derivatives by peptide bonds, or can be performed by a well-known solid phase method or liquid phase method, or can be performed by a peptide synthesizer. The intercalator group-substituted α-amino acid of the above formula III and the amino group or carbonyl group protected derivative of the above α of the formula IV include:
The thing protected by the above-mentioned protecting group is mentioned. The above formula
The intercalator group-substituted α-amino acid of III has the following formula V
It can be obtained by reacting an α-amino acid represented by the following formula with an intercalator derivative represented by the following formula VI (D in the formula VI represents a reactive group such as a halogen atom).

[Chemical 12] Examples of the α-amino acid of formula IV or V include alanine, lysine, norlysine, aspartic acid, glutamine, serine, cysteine and the like described above. Thus the formula
A peptide of I can be obtained. The N-terminal or C-terminal substituted derivative of the peptide of formula I or a salt thereof may be obtained at the stage of synthesis of the peptide of formula I, or obtained from the peptide of formula I by a method known per se. You can also

The peptide of formula I of the present invention, its N-terminal or C-terminal substituted derivative or salt thereof is:
It can be a strong telomerase inhibitor because it binds to a four-stranded structure formed by gathering four guanine sequences existing in the overhanging region on the 3'end side of telomeric DNA and sufficiently stabilizes it. Therefore, it can be used as an effective therapeutic agent for hyperproliferative diseases found in hyperproliferative cells with enhanced telomerase activity, such as cancer, psoriasis, and Marfan syndrome. The telomerase inhibitor of the present invention can be applied to mammals including human. Such a telomerase inhibitor can be mixed with excipients such as lactose and starch which are usually used for formulation, lubricants such as magnesium stearate and talc, and other additives to prepare tablets. In addition to tablets, pills, powders, liquids and the like can be used, and injections, external preparations and the like can also be used. These formulations can be prepared by a method known per se. A peptide of formula I according to the invention, wherein N
The dose of the derivative in which the C-terminal or the C-terminal is substituted, or a salt thereof may vary depending on the sex, age, symptoms, body weight, etc. of the subject to be administered, but is usually 0.00
The dose can be administered in the range of 1 to 100 mg / kg in a single dose or in divided doses. The telomerase inhibitor of the present invention, in addition to mammals including humans, has a linear chromosome, nematodes that require telomerase for its maintenance, yeast, fungi,
It can be applied as a growth inhibitor for protozoa and the like. In addition, the telomerase inhibitor of the present invention can also be used as a research reagent.

[0014]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Synthesis of tetrakis- acridinyl peptide A solid phase peptide (TAP) containing a lysine residue in which an acridine skeleton in which a methoxy group and a chlorine atom are substituted as an intercalator group shown in FIG. It was synthesized by a synthetic method. 1. Synthesis of Fmoc-Lys (Acr) -OH

[Chemical 13] a) Method 30 g of phenol was placed in a 50 ml beaker and dissolved by heating to 80 ° C. To this, 4.54 g (13.5 mmol) of 6-chloro-2-methoxy-9-phenoxyacridine was added and completely dissolved. Then, at 55-65 ° C., 4.42 g (12.0 mmol) of Fmoc-Lys-OH was added, and the mixture was stirred for 2 hours. It was then allowed to cool for a while and 300 ml of ether was poured with stirring. The obtained precipitate was suction filtered and dried under reduced pressure. b) Result Property: Yellow solid Yield: 7.57 g (Yield: 92%) As a result of identification by ¹ H-NMR and MALDI TOF MASS (mass spectrometry), a peak corresponding to a molecular weight of 610.11 of the target compound was 610.
Detected in 80.

2.Ac-Lys (Acr)-(Lys (Boc)) ₂ -Lys (Acr)-(Ly
s (Boc)) ₂ -Lys (Acr)-(Lys (Boc)) ₂ -Lys (Acr) -Resin (TAP extension reaction) a) Method Reaction Put 200 mg of Fmoc-NH SAL resin in the vessel and cartridge. Fmoc-Lys (Acr) -OH 0.33g on 1, 4, 7, 10
The cartridge, and put Fmoc-Lys (Bo
c) -OH was added. Then 431A from PE Applied Biosystems
The synthesis was performed using a peptide synthesizer. N in Fmoc method
The ends were acetylated. b) Resulting properties: Yellow solid Yield: 0.5003g

3.Ac-Lys (Acr)-(Lys (Boc)) ₂ -Lys (Acr)-(Ly
s (Boc)) ₂ -Lys (Acr)-(Lys (Boc)) ₂ -Lys (Acr) -Cleavage of the resin from the resin and deprotection of the side chain a) Method of 2) above in a 50 ml eggplant-shaped flask. 0.50 g of the resin synthesized by the experimental operation was added, m-cresol 0.10 ml, thioanisole 0.60 ml, and trifluoroacetic acid 4.3 ml were added thereto, and the mixture was stirred at room temperature for 1 hour. Then, TFA was distilled off under reduced pressure, and 20 ml of ether was added in an ice bath. After ultrasonic irradiation, the solution was left for a while and the supernatant was removed. Then, 20 ml of ethyl acetate was added, the mixture was irradiated with ultrasonic waves, and then left for a while. It was suction filtered and washed with ether. Then, it was dried under reduced pressure. b) Resulting properties: Yellow solid Yield: 0.3181g

4. Purity check and purification by HPLC The purity of TAP was checked under the following conditions. Detection wavelength: 210nm Column: Inertsil ODS Sample injection solvent: Ultrapure water Flow rate: 1 ml / min Eluent A: 0.1% TFA aqueous solution Eluent B: 70% acetonitrile / 0.1% TFA solution Gradient conditions

[Table 1] Identification was performed by TOF MASS. As a result of TOF MASS, a peak corresponding to a molecular weight of 2307.51 of the target compound was detected at 2309.03.

Example 2 Tm measurement of TAP by CD The CD (Circular dichroism: circular dichroism) spectrum shows the structure of the compound in solution, such as A-type, B-type, Z-type in the case of DNA, For proteins, information on the structures such as α-helix and β-sheet can be obtained. Therefore, the structure of the DNA in the solution varies greatly depending on whether it is a single-stranded structure, a double-stranded structure, or a four-stranded structure.
The structure of A in solution is known. a) Method Tm measurement by CD was performed under the following measurement conditions. Measurement solution: 10 mM MES, 1 mM EDTA (pH6.25), 50 mM KCl [telomere DNA] = 2.2 μM, DNA: dye = 1: 1 As the telomere DNA, the oligonucleotide having the following human telomere DNA sequence was used. 5′-CATGGTGGTTTGGGTTAGGGTTAGGGTTAGGGTTACCAC-3 ′ CD measurement was performed with the TAP of the present invention as a target compound.
The known compounds shown in 1 above were used. To the above measurement solution, telomere DNA was added to 2.2 μM, and TAP or each compound was also added at 2.2 μM to compare how the Tm curve (stability of the four-stranded structure) changes. .

B) The Tm curve obtained as a result is shown in FIG. FIG. 3 shows a situation in which the four-stranded structure formed by the telomeric DNA sequence is dissociated into a single-stranded structure in the presence of TAP or each known compound. In Fig. 3, the stability of the four-stranded structure is shown by the [θ] value of the CD spectrum. In Fig. 3, the temperature at which Normalized [θ] becomes exactly 0.5 is Tm (melting temperature). The higher the Tm value, the more stable the four-stranded structure. As is clear from FIG. 3, the highest Tm (melting temperature) is exhibited when the TAP of the present invention is used, and the quadruplex structure formed by the telomeric DNA sequence is highly stabilized by the TAP of the present invention. I know that As shown in FIG. 1, the intercalator group in the TAP molecule of the present invention is inserted into the planar structure of the four-stranded structure formed by the telomeric DNA sequence, and the four-stranded structure is highly stabilized. Conceivable.

[0021]

EFFECT OF THE INVENTION Peptides containing an α-amino acid residue substituted with an intercalator group such as a group having an acridine skeleton, a group having an anthraquinone skeleton and a group having an anthracene skeleton provided by the present invention are telomere DNAs.
Strongly binds to the four-stranded structure formed by the guanidine sequence of
It can stabilize the double-stranded structure and can be a potent telomerase inhibitor. Therefore, it can be used as an effective therapeutic agent for hyperproliferative diseases found in hyperproliferative cells with enhanced telomerase activity, such as cancer, psoriasis, and Marfan syndrome. It can also be used as a reagent for research.

[Brief description of drawings]

FIG. 1 is a chemical structural formula of TAP (tetrakis-acridinyl peptide), which is a typical peptide obtained in the present invention, and TAP has a strong 4-chain structure formed by the guanidine sequence of telomeric DNA. Shows the mechanism that binds to and stabilizes the four-stranded structure.

FIG. 2 shows a known compound having a telomerase inhibitory action, which was used as a target compound of the peptide of the present invention.

FIG. 3 shows the situation in which the four-stranded structure of telomeric DNA is changed to a single-stranded structure in the presence of TAP, which is a typical peptide obtained in the present invention, and a compound having a known telomerase inhibitory action. It is a Tm curve shown.

Continued front page    F-term (reference) 4C084 AA02 AA07 BA32 CA59 DC32                       MA35 MA52 MA55 MA66 NA14                       ZA891 ZB261 ZC201                 4H045 AA10 AA30 BA15 BA51 DA55                       EA22 EA28 FA33 GA40

Claims (6)

[Claims] 1. A peptide containing an α-amino acid residue substituted with an intercalator group, which is represented by the following formula I, its N-terminal or C-terminal substituted derivative or a salt thereof. [Chemical 1] (In the formula, X represents an intercalator group, Y represents a divalent group forming an α-amino acid, Z represents a monovalent group forming an α-amino acid, and n represents 0, 1, 2, Represents 3, 4, or 5) 2. The intercalator group has a group having an acridine skeleton which may have a substituent, a group having an anthraquinone skeleton which may have a substituent, and an anthracene which may have a substituent. Group having a skeleton, group having a naphthaleneimide skeleton that may have a substituent, a group having a naphthalene diimide skeleton that may have a substituent, or a pyrene skeleton that may have a substituent 2. The peptide according to claim 1, which is a group, a derivative thereof having an N-terminal or C-terminal thereof substituted, or a salt thereof. 3. Y is -AW- (A is a linear or branched alkylene group having 1 to 6 carbon atoms, W is a single bond, NH, C
O, CONH, NHCO, O, S or HNC (NH) NH), the peptide of claim 1 or 2, its N-terminal or C-terminal substituted derivative or a salt thereof. 4. The peptide according to any one of claims 1 to 3, wherein Z is -AWH (A and W are the same as defined in claim 3), a derivative thereof substituted at its N-terminal or C-terminal, or Those salts. 5. A peptide according to any one of claims 1 to 4 represented by the following formula II, a derivative thereof having an N-terminal or C-terminal thereof substituted, or a salt thereof. [Chemical 2] 6. A telomerase inhibitor containing the peptide according to any one of claims 1 to 5, a derivative thereof having an N-terminal or C-terminal thereof substituted, or a salt thereof as an active ingredient.