JP4629993B2 - Pyrazolone derivatives and telomerase inhibitors - Google Patents

Description

  The present invention relates to a pyrazolone derivative having telomerase inhibitory activity and antitumor activity.

  Development of new drugs targeting reverse transcriptase and telomerase, which are responsible for elongation of chromosome end sites (telomeres), has been actively conducted mainly for application to anticancer drugs and regenerative medicine. Especially for anticancer drugs, (1) telomerase activity is found only in germ cells and stem cells and not in somatic cells. (2) Many human cancer tissues have telomerase activity, while immortalized cancer. Inhibiting telomerase activity of cells causes shortening of telomeres, aging, cell death, and the like, and therefore, it is expected to have few side effects on normal cells while exhibiting effects on a wide variety of cancers.

  Various attempts have been made to inhibit telomerase enzyme activity. Drugs that suppress or inhibit the activity of telomerase enzymes that have been studied so far can be broadly divided into (1) antisense to telomerase, (2) ribozymes, (3) G-quadruplex stabilizers, and (4) other inhibitors. There are four agents. These telomerase inhibitors are briefly described below.

(1) Antisense against telomerase This is a method for suppressing the activity of telomerase enzyme by introducing single-stranded DNA or RNA having a sequence complementary to hTR in the telomerase enzyme complex. In many cases, RNA is expressed endogenously, and in the case of DNA, synthetic oligo DNA is used. In the case of synthetic oligo DNA, chemical modification is often introduced for the purpose of preventing enzymatic degradation. In humans, antisense to the first 185 bases in hTR is effective. Among synthetic oligo DNAs, peptide nucleic acids whose backbone consists of glycine have the advantage of being extremely resistant to degradation by proteases and nucleases.

(2) Ribozyme
An RNA enzyme (ribozyme) having an activity of degrading (cleaving) RNA at the trans position is used as a telomerase inhibitor. The point that the template sequence of hTR is the target of ribozyme is the application of antisense technology. A hammerhead ribozyme is used.

(3) G-quadruplex structure stabilizer
The G-quadruplex structure has been proposed as a structure that characterizes the telomere. In the human telomere sequence, d (AG3 (T2AG3) 3) is folded within the same molecule, and four identical sequences are paralleled to form a rope-like structure. Agents that stabilize the G-quadruplex structure inhibit telomerase activity because this structure must be dissociated when the telomeres extend. There are several types of G-quadruplex structures, some with different affinities depending on the drug.

(4) Others Inhibitors against telomerase enzyme proteins (catalytic inhibitors), those that interfere with the binding sites of subunits constituting the enzyme complex and inhibit complex formation, and competitively inhibit dNTP substrate binding to the enzyme Inactivated nucleotide (nucleoside analog), dominant-negative type hTERT or its expression vector is introduced.

  Of these, the following compounds are mainly known as telomerase inhibitors based on stabilization of the G-quadruplex structure.

Examples of perylene derivatives include perylene diimide and 3,4,9,10-perylene tetracarboxylic acid diimide.

Quinolines and quinoline related substances such as 2,4,6-triamino-1,3,5-triazine, 12459 (a kind of), 10H-indolo [3,2-b] quinoline, fluoroquinophenoxazine and anthraquinone It is done.

Examples of the porphyrin-related substance include TMPyP.

Inhibitors targeting the typical G-quadruplex structure of telomestatin (WO00 / 24747 pamphlet).

  Thus, various telomerase inhibitors have been developed that target different steps in the addition of telomeric repeats by telomerase enzymes. Since a more effective cancer treatment can be expected by combining these, development of a drug having a new inhibition mode or a novel structure is important.

International Publication No. 00/24747 Pamphlet

  An object of the present invention is to provide a novel telomerase inhibitor or antitumor agent.

  The present inventors have studied pyrazolone compounds that have not yet been reported as telomerase enzyme inhibitors. As a result, they have found that certain pyrazolone derivatives have telomerase enzyme inhibitory activity and have completed the present invention.

［式中、Ｘ_１〜Ｘ_５は、互いに独立して、水素原子又はハロゲン原子である。］
で表される化合物又はその薬学的に許容される塩を含むテロメラーゼ阻害剤。 That is, the present invention includes the following inventions.
(1) The following formula (1):

[Wherein, X _{1 to} X ₅ are each independently a hydrogen atom or a halogen atom. ]
Or a pharmaceutically acceptable salt thereof, a telomerase inhibitor.

(2) The telomerase inhibitor according to the above (1), wherein X ₃ and X ₅ are halogen atoms.

(3) The telomerase inhibitor according to the above (2), wherein X ₃ and X ₅ are chlorine.

［式中、Ｘ_１〜Ｘ_５は、互いに独立して、水素原子又はハロゲン原子である。］
で表される化合物又はその薬学的に許容される塩を含む抗腫瘍剤。 (4) The following formula (1):

[Wherein, X _{1 to} X ₅ are each independently a hydrogen atom or a halogen atom. ]
Or an antitumor agent comprising a pharmaceutically acceptable salt thereof.

(5) The antitumor agent according to (4), wherein X ₃ and X ₅ are halogen atoms.

(6) The antitumor agent according to (5), wherein X ₃ and X ₅ are chlorine.

［式中、Ｘ_１〜Ｘ_５は、互いに独立して、水素原子又はハロゲン原子である。但し、Ｘ_１〜Ｘ_５のうち少なくとも１つはハロゲン原子である。］
で表される化合物又はその薬学的に許容される塩。 (7) Formula (1) below:

[Wherein, X _{1 to} X ₅ are each independently a hydrogen atom or a halogen atom. However, at least one of X _{1 to} X ₅ is a halogen atom. ]
Or a pharmaceutically acceptable salt thereof.

(8) The compound according to (7) above, wherein X ₃ and X ₅ are halogen atoms.

(9) The compound according to (8), wherein X ₃ and X ₅ are chlorine.

［式中、Ｒは水素原子又は炭素数１〜６のアルキル基である。］
の化合物と、下記式(ii)：

［式中、Ｘ_１〜Ｘ_５は、互いに独立して、水素原子又はハロゲン原子である。］
の化合物とを反応させて、下記式(iii)：

［式中、Ｘ_１〜Ｘ_５は上記定義のとおりである。］
の化合物を得て、次いで化合物(iii)を塩素化剤で塩素化して下記式（１）：

［式中、Ｘ_１〜Ｘ_５は上記定義のとおりである。］
の化合物を得ることを含む式（１）の化合物の製造方法。 (10)
Formula (i) below

[Wherein, R represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. ]
And a compound of the following formula (ii):

[Wherein, X _{1 to} X ₅ are each independently a hydrogen atom or a halogen atom. ]
And a compound of the following formula (iii):

[Wherein, X _{1 to} X ₅ are as defined above. ]
Then, the compound (iii) is chlorinated with a chlorinating agent and the following formula (1):

[Wherein, X _{1 to} X ₅ are as defined above. ]
A process for producing a compound of formula (1) comprising obtaining a compound of formula (1):

  The pyrazolone derivative of formula (1) used in the present invention has a remarkable telomerase inhibitory activity, and the present invention provides a new class of telomerase inhibitors and antitumor agents.

  The present invention is described in detail below.

［式中、Ｘ_１〜Ｘ_５は、互いに独立して、水素原子又はハロゲン原子である。］
で表される化合物又はその薬学的に許容される塩を含有する。 The telomerase inhibitor and antitumor agent of the present invention have the following formula (1) as an active ingredient:

[Wherein, X _{1 to} X ₅ are each independently a hydrogen atom or a halogen atom. ]
Or a pharmaceutically acceptable salt thereof.

In the compound of the formula (1), X _{1 to} X ₅ are each independently a hydrogen atom or a halogen atom, and examples of the halogen atom include fluorine, chlorine, bromine and iodine. Preferably, at least one of X _{1 to} X ₅ is a halogen atom, more preferably X ₃ and X ₅ are both halogen atoms, most preferably X ₁ and X ₅ or both X ₃ and X ₅ are both It is a chlorine atom.

In addition, although the compound of the formula (1) in which all of X _{1 to} X ₅ are hydrogen atoms is known, the compound of the formula (1) in which at least one of X _{1 to} X ₅ is a halogen atom Is a novel pyrazolone derivative found by the authors.

  Examples of the pharmaceutically acceptable salt of the compound of formula (1) include inorganic acid salts such as hydrochloride, sulfate, and phosphate, acetate, maleate, fumarate, tartrate, and citric acid. Examples include acid addition salts such as organic acid salts such as salts and lactates.

  Next, the manufacturing method of the pyrazolone derivative represented by General formula (1) is demonstrated.

  As a method for synthesizing a pyrazolone skeleton having a phenyl group, a method by reaction of diketene and phenylhydrazine as shown below is known.

  In this method, when the phenyl group is unsubstituted, the desired phenylpyrazolone compound 2 can be obtained in good yield. However, when halogenated phenylhydrazine is used as a raw material as described below, the desired halogenated phenylpyrazolone compound 4 cannot be obtained, and a compound 5 in which two molecules of halogenated phenylhydrazine are mainly introduced into diketene is formed. I found out that

［式中、Ｒは水素原子又は炭素数１〜６のアルキル基である。］
の化合物と、下記式(ii)：

［式中、Ｘ_１〜Ｘ_５は、互いに独立して、水素原子又はハロゲン原子である。］
の化合物とを反応させて、下記式(iii)：

［式中、Ｘ_１〜Ｘ_５は上記定義のとおりである。］
の化合物を得て、次いで化合物(iii)を塩素化剤で塩素化して下記式（１）：

［式中、Ｘ_１〜Ｘ_５は上記定義のとおりである。］
の化合物を得ることを含む式（１）の化合物の製造方法である。 Therefore, new synthetic routes for producing the compounds of the present invention must be sought. As a result of intensive studies on a novel method for producing a phenylpyrazolone compound, the present inventors have obtained (halogenated) phenyl in a high yield by reacting β-ketoester (or β-keto acid) with (halogenated) phenylhydrazine. It has been found that pyrazolone compounds can be synthesized. That is, the production method of the present invention has the following formula (i):

[Wherein, R represents a hydrogen atom or an alkyl group having 1 to 6 carbon atoms. ]
And a compound of the following formula (ii):

[Wherein, X _{1 to} X ₅ are each independently a hydrogen atom or a halogen atom. ]
And a compound of the following formula (iii):

[Wherein, X _{1 to} X ₅ are as defined above. ]
Then, the compound (iii) is chlorinated with a chlorinating agent and the following formula (1):

[Wherein, X _{1 to} X ₅ are as defined above. ]
A method for producing a compound of formula (1) comprising obtaining a compound of formula (1):

In the above production method, R is a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, preferably a methyl group, an ethyl group or a propyl group. X _{1 to} X ₅ are each independently a hydrogen atom or a halogen atom, but preferably at least one of X _{1 to} X ₅ is a halogen atom, and more preferably X _{1 to} X ₅ . At least one of them is a chlorine atom.

  The reaction between the compound of formula (i) and the compound of formula (ii) is carried out in a solvent such as acetic acid, propionic acid, acetonitrile, toluene and the like at 70 ° C. to reflux temperature. The reaction time may be appropriately set according to the reaction temperature and the like, but is usually 2 to 20 hours. After completion of the reaction, the produced compound (iii) may be purified by usual purification means (for example, filtration, extraction with an organic solvent, column chromatography, recrystallization, etc.), but without isolation and purification from the reaction solution. It may be used for the next reaction.

  Next, compound (iii) can be chlorinated with a chlorinating agent to obtain compound (1).

やN-クロロスクシンイミド、塩素等が挙げられる。塩素化反応は溶媒として、例えば、酢酸、１，４−ジオキサン、ジエチルエーテル、テトラヒドロフラン等を用いて、０〜１００℃で１０〜６０分行なう。反応終了後、粗生成物を通常の精製手段（例えば、濾過、有機溶媒による抽出、カラムクロマトグラフィー、再結晶等）、好ましくは酢酸からの再結晶により化合物（１）を得る。 Examples of the chlorinating agent include 1,3-dichloro-4,4-dimethylhydantoin:

And N-chlorosuccinimide, chlorine and the like. The chlorination reaction is carried out at 0 to 100 ° C. for 10 to 60 minutes using, for example, acetic acid, 1,4-dioxane, diethyl ether, tetrahydrofuran or the like as a solvent. After completion of the reaction, the crude product is obtained by the usual purification means (for example, filtration, extraction with an organic solvent, column chromatography, recrystallization, etc.), preferably by recrystallization from acetic acid to obtain the compound (1).

  The pyrazolone derivative used in the present invention strongly stabilizes the G-quadruplex structure of the telomere part, thereby inhibiting telomerase activity. The telomerase inhibitor of the present invention is also useful as an antitumor agent having a broad spectrum by inhibiting the activity of telomerase. Telomerase is present in a wide range of malignant tumors (eg, skin, breast, lung, stomach, pancreas, ovary, cervix, uterus, kidney, bladder, colon, prostate, central nervous system (CNS), retinal and blood tumor cells Telomerase has been found in more than 85% of all malignant tumors including the system.) Since it is rarely present in normal somatic cells, it acts only on tumor cells.

  As the pharmaceutical dosage form when using the telomerase inhibitor or antitumor agent of the present invention, various pharmaceutical dosage forms can be adopted depending on the purpose, for example, tablets, capsules, powders, granules, fine granules , Liquids, pills, emulsions, suspensions and other oral preparations, injections, suppositories, ointments, plasters, patches, aerosols, eye drops, etc. Can be produced by conventional methods known to those skilled in the art.

  When an oral solid preparation is prepared, an excipient, if necessary, a binder, a disintegrant, a lubricant, a colorant, a corrigent, a flavoring agent and the like are added to the active ingredient, and then the tablet is prepared by a conventional method. , Capsules, powders, granules, fine granules and the like can be produced. Examples of excipients include lactose, sucrose, starch, talc, magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate, gum arabic, and the like, and binders include polyvinyl alcohol, polyvinyl ether, ethyl cellulose, arabic. Gum, shellac, sucrose, etc. are dry starch, sodium alginate, agar powder, sodium bicarbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, lactose etc. as disintegrants, magnesium stearate, talc as lubricants However, sucrose, orange peel, citric acid, tartaric acid and the like can be used as a flavoring agent. In addition, generally known colorants, flavoring agents, and the like can be used. The tablets should be ordinary tablets coated by a well-known method as necessary, for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, other double tablets, multilayer tablets Can do.

  When preparing an oral liquid preparation, an oral solution, a syrup, an elixir and the like can be produced by a conventional method by adding a corrigent, a buffer, a stabilizer, a corrigent and the like to the active ingredient. In this case, the flavoring agent may be those listed above. Sodium citrate or the like may be used as the buffer, and tragacanth, gum arabic, gelatin or the like may be used as the stabilizer.

  When preparing injections, add diluents, pH adjusters, buffers, stabilizers, tonicity agents, local anesthetics, etc. to the active ingredients, and use intravenous, intramuscular, subcutaneous in the usual way. Intradermal and intraperitoneal injections can be produced. As the diluent, for example, water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters and the like can be used. Sodium citrate, sodium acetate, sodium phosphate and the like can be used as the pH adjusting agent and buffer, and sodium pyrosulfite, ethylenediaminetetraacetic acid, thioglycolic acid, thiolactic acid and the like can be used as the stabilizer. As the isotonic agent, sodium chloride, glucose and the like can be used, and as the local anesthetic, procaine hydrochloride, lidocaine hydrochloride and the like can be used.

  When preparing a suppository, a suppository can be produced by a conventional method after adding a base to the active ingredient and, if necessary, a surfactant or the like. As the base, for example, an oily base such as macrogol, lanolin, cacao oil, fatty acid triglyceride, witepsol (manufactured by Dynamite Nobels) can be used.

  When preparing an ointment, bases, stabilizers, wetting agents, preservatives and the like that are usually used as active ingredients are blended as necessary, and mixed and formulated by a conventional method. As the base, liquid paraffin, white petrolatum, honey beeswax, octyldodecyl alcohol, paraffin and the like can be used, and as the preservative, methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate and the like can be used.

  When a patch is produced, the active ingredient and the ointment, cream, gel, paste or the like may be applied to a normal support by a conventional method. As the support, woven fabric, nonwoven fabric, soft vinyl chloride, polyethylene, polyurethane, or a foam sheet made of cotton, suf, or chemical fiber can be used.

  Furthermore, each of the above preparations may contain a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetening agent, and other pharmaceuticals as necessary.

  The amount of the active ingredient (that is, the pyrazolone derivative of the formula (1)) to be contained in the preparation of the present invention is not particularly limited and is appropriately selected depending on the administration form. It is good to set it to -70weight%.

  The administration method of the preparation of the present invention thus obtained is not particularly limited, and is appropriately determined according to various preparation forms, patient age, sex, other conditions, disease degree, and the like. For example, a pharmaceutical preparation in the form of an injection can be administered by intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal administration and the like. If necessary, it can be mixed with a normal replacement fluid such as glucose or amino acid and administered intravenously. The antitumor agent of the present invention in the form of solids such as tablets, pills, granules, capsules, or liquids for oral administration can be administered orally or enterally. Suppositories can be administered rectally.

  The amount of the active ingredient to be blended in each of the above dosage unit forms can be appropriately set according to the symptoms of the patient to which the dosage form is applied or the dosage form thereof, but generally about 1 to 1000 mg per oral dosage unit form. It is desirable that the dosage is about 0.1 to 500 mg for injections and about 5 to 1000 mg for suppositories.

  The daily dose of the drug having the above dosage form is appropriately selected according to the patient's symptoms, body weight, age, sex, other conditions, etc. The dose may be 1000 mg / kg, preferably about 1 to 100 mg / kg, which can be administered once a day or divided into about 2 to 4 times a day.

  The tumor that can be treated by administering the preparation of the present invention is not particularly limited, and examples thereof include head and neck cancer, esophageal cancer, stomach cancer, colon cancer, rectal cancer, liver cancer, gallbladder / Solids such as bile duct cancer, pancreatic cancer, kidney cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, testicular tumor, bone / soft tissue sarcoma, cervical cancer, skin cancer, brain tumor, etc. A malignant tumor, a malignant lymphoma, leukemia, etc. are mentioned, Preferably it is a solid malignant tumor.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

Example 1 Production of 4,4-dichloro-1- (2,4-dichlorophenyl) -3-methyl-5-pyrazolone

  22.9 g (176 mmol) of ethyl acetoacetate and 31.1 g (176 mmol) of 2,4-dichlorophenylhydrazine were dissolved in acetic acid and reacted at 110 to 111 ° C. for 16 hours. After completion of the reaction, the reaction solution was cooled to room temperature, washed with water and saturated brine, dried over sodium sulfate and evaporated to remove 1- (2,4-dichlorophenyl) -3-methyl-5-pyrazolone. (30.25 g, 71%) was obtained. Next, 15.0 g (61.7 mmol) of 1- (2,4-dichlorophenyl) -3-methyl-5-pyrazolone and 12.2 g (61.7 mmol) of 1,3-dichloro-4,4-dimethylhydantoin as a chlorinating agent Was dissolved in acetic acid and reacted at 15-20 ° C. for 30 minutes. After completion of the reaction, the reaction solution was washed with water and saturated brine, dried over sodium sulfate, and then the solvent was distilled off to obtain a crude product. Since the crude product was colored yellowish brown, it was recrystallized from acetic acid. The precipitated crystals were collected by filtration to give the title compound as a white solid (14.53 g, 76%, melting point 91.6 ° C.).

(Example 2)
Synthesis of 4,4-dichloro-1- (2,6-dichlorophenyl) -3-methyl-5-pyrazolone (# 17341)

  The title compound was synthesized in the same manner as in Example 1 except that 2,6-dichlorophenylhydrazine was used as the phenylhydrazine compound.

Kinetic analysis of telomerase inhibition by # 17341 (TRAP _EZE assay)
The telomerase enzyme activity was measured using Intergen's TRAP _EZE telomerase detection kit. This kit is based on the TRAP method. That is, a telomere repeat sequence is added to the template DNA sequence by telomerase enzyme activity, and then the extended telomere sequence is amplified by a PCR reaction. The TRAP _EZE kit contains an internal standard in the PCR reaction, which enables high-precision quantification over a wide concentration range, and is therefore widely used for detecting telomerase enzyme activity.

First, telomerase enzyme used in the assay was extracted from several types of cultured cancer cells. Since the Namalwa cell line, a cancer cell line derived from T cells, had already been confirmed to exhibit high telomerase activity, a cell extract was obtained from this cell line. The cell extract was obtained as follows. Cells in logarithmic growth phase (3 × 10 ⁷ cells) were washed twice with PBS, then 50 μL of lysis buffer (8.5 mM Tris-HCl (pH 7.5), 0.85 mM MgCl ₂ , 0.85 mM EGTA (pH 7.5), 4.25 mM) (β-ME, 0.425% CHAPS, 8.5% glycerol, 1 mM PMSF, 10 μg / mL leupepsin, 25 μg / mL pepstatin, 1 mM DTT, 10 mM NaF, 100 μMNa ₃ VO ₄ , 50 μg / mL Bowman-Birk) did. After standing at ice temperature for 30 minutes, the supernatant was collected by centrifugation at 55000 rpm, 4 ° C. for 30 minutes to obtain a cell extract. The obtained cell extract was measured for protein concentration by Bradford method, dispensed, and stored at -80 ° C.

Examination of assay system In order to conduct kinetic analysis, the reaction rate of an appropriate telomerase enzyme was determined. For this reason, the following checks were performed on the assay system. Three types of enzyme solutions were prepared (total amounts of 0.04, 0.1 and 0.25 μg per 50 μL), and a telomerase extension reaction was performed at 30 ° C. Samples that had passed 0, 5, 10, 15, 20, 30, 40, and 60 minutes after the start of the reaction were each PCR amplified. Within the examined range, it was shown that the final product amount with the TRAP _EZE kit increased linearly with time at any protein concentration. The protein concentration dependence of the reaction rate showed linearity within the studied range, that is, the final product amount was proportional to the reaction rate under the examined conditions. Based on this result, the assay was carried out using a protein solution having a concentration of 0.1 μg per 50 μL and measuring the product 30 minutes after the start of the reaction.

The assay assay was performed at a quarter of the manually specified scale (12.5 μL / reaction). The cell extract was diluted with distilled water so that the total protein concentration during the reaction was 0.025 μg / 12.5 μL. 10 × TRAP reaction Buffer 1.25 μl, 50 × dNTP Mix 0.25 μL, TS primer 0.25 μL, TRAP Primer Mix 0.25 μL, Taq polymerase 0.1 μL, and distilled water 7.9 μL were mixed at ice temperature (TRAP reaction solution). However, the TS primer was diluted with distilled water to an appropriate concentration in addition to the indicated concentration (1 × TS). 2.5 μL of an inhibitor (50 times the concentration at the time of reaction) and 22.5 μL of the cell extract were mixed, and 2.5 μL of this was mixed with the above TRAP reaction solution. After 30 ° C / 30 minutes treatment with a thermal cycler, 34 cycles of 94 ° C / 30 seconds-59 ° C / 30 seconds were performed. 1.25 μL of electrophoresis buffer was added to 12.5 μL of the sample after PCR.

  5 μL of electrophoresis buffer was added to 50 μL of the sample after PCR, and electrophoresis was performed on a 10% acrylamide gel (15 mA). SYBER Green 5 μL was diluted in 100 mL of 0.5 × TBE buffer, the gel was soaked, and shaken for 30 minutes. After rinsing with distilled water for 10 minutes, the fluorescence intensity was measured by FLA-2000. The results were analyzed using statistical software KYplot.

  For reaction kinetic analysis, prepare reaction solutions with stepwise concentrations of the substrate to be analyzed (TS primer, dATP, dGTP, or TTP), and use a thermal cycler for 30 ° C / 30 minutes. I did it. The telomerase enzyme was inactivated by treatment at 95 ° C. for 2 minutes, and then the target substrate was added to each sample so that the final concentrations were the same (final concentrations were 375 nM with TS primer and 75 μM with dNTP). The amount of liquid added was 1.5 μL (final liquid volume was 14 μL), and TRAP reaction buffer and a non-target substrate were also added so that these final concentrations were not affected. The subsequent PCR amplification reaction was performed as usual.

Mode of inhibition of telomerase by # 17341
i) In the case of TS primer substrate The influence of the TS primer concentration on the reaction rate was measured under the conditions of # 17341 of 0, 0.2, 0.5 and 1 μM. After the extension reaction, correction was performed so that the TS primer concentration during the PCR reaction was uniform. When the relationship between the TS primer concentration and the reaction rate in the presence of # 17341 was plotted, a hyperbolic TS primer concentration-response rate response curve was obtained (FIG. 1). The maximum reaction rate (Vmax) was obtained in the range where the TS primer concentration was higher than approximately 50 nM, and Vmax decreased obviously as the concentration of # 17341 increased. This suggested a non-antagonistic type of inhibition. Based on this data, a Lineweaver-Burk reciprocal plot was made (Fig. 2). Based on this result, if there was no # 17341 inhibitor, the Michaelis-Menten constant of the TS primer (Km respectively) And αKm), and when TS primer was not present, the binding constants of # 17341 (Ki and αKi, respectively) were determined for each of the cases, and it was found that αKm was approximately twice Km. That is, # 17341 was shown to inhibit the binding of TS primer to telomerase enzyme. On the other hand, αKi was slightly higher than Ki, indicating that # 17341 is more likely to bind to the enzyme alone than the enzyme-DNA complex.

ii) In case of dNTP substrate
Similar analysis was performed for dNTPs, which are substrates for telomerase enzymes as well as TS primers. Since the sequence added to the telomeres by the telomerase enzyme is (TTAGGG) n, dATP, dGTP and TTP are substrates for the enzyme. For these three substrates, the concentration dependence of the telomerase enzyme reaction rate was analyzed under the conditions of # 17341 of 0, 0.1 and 1.0 μM. As with the TS primer, a hyperbolic dNTPs concentration-response rate response curve was obtained (FIG. 3). For all the substrates, the maximum reaction rate (Vmax) was reached at about 5 μM, and Vmax decreased as the concentration of # 17341 increased. When line-weaver plotting was performed based on this data (FIG. 4), the Michaelis-Menten constants of dATP, dGTP, and TTP were 340 nM, 980 nM, and 530 nM, respectively. On the other hand, the plot moved in parallel as the # 17341 concentration increased. This is a characteristic seen in the non-antagonistic mode of inhibition. That is, # 17341 does not inhibit the binding of dNTPs to the enzyme, but binds to the enzyme-substrate complex and inhibits the reaction.

Model of telomerase inhibition The telomere elongation reaction by telomerase enzyme can be considered as a two-substrate-1 product reaction by TS primer and dNTP. The telomerase enzyme binds to the telomere end (corresponding to the TS primer), and further adds a telomere repeat sequence using dATP, dGTP and TTP as substrates. Based on the kinetic analysis described above, # 17341 showed a mixed antagonistic / non-antagonistic inhibition mode for the reaction using TS primer as a substrate. That is, for this substrate, # 17341 acts on the enzyme alone to inhibit the binding to the telomere sequence, and also inhibits the activity of the enzyme bound to the telomere sequence. On the other hand, # 17341 showed a non-antagonistic inhibition mode for the reaction using dNTP as a substrate. That is, # 17341 did not inhibit the binding of dNTP to the telomerase enzyme, but showed inhibition against the dNTP-bound telomerase enzyme. The binding constant of TS primer is about 5 nM, whereas that of dNTP is about 300-1000 nM, indicating that binding to TS primer is much more likely. In addition, analysis of TS primer showed inhibition against the enzyme alone and higher affinity to the enzyme alone than the enzyme-DNA complex, indicating that # 17341 has an inhibitory effect on telomerase enzyme protein. Conceivable.

(Example 3) Effect on cultured cancer cells
Cell culture method The cells used in the experiment were cultured as follows.
T cell-derived cancer cell lines Namalwa, MOLT-4, B lymphocyte-derived cancer cell line REH cell line, and fibroblast TIG-3 cells as cells without telomerase enzyme activity It was. Namalwa, MOLT-4 and REH cell lines (all floating cells) are RPMI1640 medium / 10% FBS in 10cm plastic culture dish (uncoated), TIG-3 cell line (adherent cells) is MEM medium / 10% FBS Using a 10 cm plastic culture dish (with coating), the cells were cultured in a constant temperature incubator at 37 ° C./5% CO ₂ . Namalwa, MOLT-4 and REH cells were passaged once every 4 days, and TIG-3 cells were passaged once every 7 days.

  When examining the inhibitory effect of # 17341 on cell proliferation, 6 cm plastic culture dishes were used for all cells. # 17341 to be added was prepared at 50 mM in methanol and stored at -20 ° C. Immediately before use, a 2 mM solution was prepared with 50% methanol and added to RPMI1640 medium (floating cancer cells) or MEM medium (TIG-3 cells) to give a predetermined concentration. The methanol concentration in the medium was all 0.5%. Four types of floating cancer cell groups were passaged once every 4 days, and TIG-3 cells were passaged once every 7 days in a freshly prepared medium containing # 17341. At that time, the number of cells was counted and microscopic photography was performed.

Cell growth inhibitory activity
Subculture for about 5 weeks in the presence of T17-derived cancer cell lines Namalwa and MOLT-4 and B lymphocyte-derived cancer cell line # 17341, cell growth and morphology The effect was examined regardless of # 17341 was added at 1 μM, 5 μM, 7.5 μM, 10 μM, 15 μM, and 20 μM. In addition, TIG-3 cells showing no telomerase enzyme activity were used as controls.

  The cell growth curve obtained for the cancer cells is shown in FIG. Inhibition of cell growth could not be confirmed with addition of 1 μM and 5 μM, but there was a correlation between the concentration of # 17341 and the cell growth rate in the range of 7.5 μM to 15 μM. At 7.5 μM, the growth rate was lower than at 5 μM, and at 15 μM, growth was almost stopped in REH and MOLT-4 cells. On the other hand, the time when the growth inhibitory effect appeared was almost the same for each cell line regardless of the concentration (8 (PD doubling (PD) = 5) and 8 (PD) for the Namalwa, REH, and MOLT-4 cell lines, respectively) = 4) and day 12 (PD = 10)). However, since the number of cells was confirmed at the time of subculture every 4-5 days, no shorter time lag was detected. Therefore, it is possible that a difference of several days occurred in the appearance of the inhibitory effect due to the concentration of # 17341. In addition, the difference in time until the inhibitory effect appears is considered to reflect the difference in telomere length among cell lines, but the sensitivity to # 17341 may vary depending on the cell line.

  The growth curve of TIG-3 fibroblasts that do not show telomerase enzyme activity is shown in FIG. When cultured in the presence of 20 μM # 17341, an inhibitory effect on cell proliferation was observed. However, when added at a concentration of 10 μM or less, a growth curve that was the same as when no additive was added was obtained.

  Based on FIG. 5 and FIG. 6, the concentration dependency of # 17341 on the growth inhibition of each cell was quantitatively evaluated. As a method for this, cell number doubling (PD) was determined for about 20 days in the latter half of the culture, and the PD per day was calculated (Table 1).

Based on Table 1, the relative PD at each concentration was calculated based on the PD per day in the control medium without # 17341. The result is shown in FIG. There was no change in the relative PD until the concentration of # 17341 was 7.5 μM, but the relative PD decreased in a concentration-dependent manner when it reached 10 μM or more. The concentration dependence curve of relative PD is different for each cell, but the slope of the curve is almost the same for the three cell lines. This suggests that the mechanism of growth inhibition is the same in all three cells (ie, inhibition of the telomerase enzyme). On the other hand, PD of TIG-3 cells is about 80% of control in the presence of 20 μM # 17341. Although not completely uninhibited, the degree of inhibition is much lower compared to the three cancer cell lines. That is, # 17341
(1) At a concentration of around 15 μM, it exhibits a growth inhibitory effect specific to cancer cells,
(2) This concentration dependence varies greatly depending on the cell type.
It was shown that.

Changes in cell morphology
When microscopic images of Namalwa and REH cell lines after 5 weeks of culture were observed, cells cultured in medium supplemented with # 17341 at 20 μM clearly had a smaller cell volume than cells in the target area. This suggested that the cells had undergone apoptosis.

  A microscopic image of TIG-3 cells was also observed in the same manner. As a result, even when # 17341 was added at 20 μM, a cell morphology that was the same as that of the control was observed. Some TIG-3 cells had shorter microprojections and fewer numbers than normal cells. Cells that did not form any microprojections or cells that did not adhere to the culture substrate and remained floating were also observed. That is, it is considered that # 17341 inhibited the adhesion of TIG-3 cells. Such adhesion inhibition was also suggested in the culture of floating cancer cells. The REH strain is relatively easy to form cell aggregates, but the addition of 20 μM suppressed the formation of aggregates. Adhesive cells such as the TIG-3 cell line are known to inhibit proliferation when adhesion is inhibited. When # 17341 was added at a concentration of 20 μM, growth inhibition of the TIG-3 strain was observed, which is considered to be due to inhibition of TIG-3 cell adhesion by # 17341. In the TIG-3 cell culture experiment, # 17341 was administered immediately after cell passage. Some inhibitors are known to inhibit the process by which suspended cells adhere, but not the growth of already adhered cells. In view of this, it can be expected that when administered to a living body, there is no inhibitory effect on normal cells already adhered.

  The novel pyrazolone derivative of the present invention has excellent telomerase inhibitory activity and is useful as an antitumor agent and the like.

It is a figure which shows the relationship between the reaction rate in the presence of # 17341, and TS primer concentration. The reaction rate depends on the amount of product obtained by the reaction for 30 minutes at 0.1 μg total protein / 50 μL. The concentrations of # 17341 are 0 μM (◯), 0.2 μM (●), 0.5 μM (□), and 1.0 μM (■), respectively. Bars are standard deviations (n = 2-3). It is a Lineweaver-Burk reciprocal plot derived from the result of FIG. The concentrations of # 17341 are 0 μM (◯), 0.2 μM (●), 0.5 μM (□), and 1.0 μM (■). Bars are standard deviations (n = 2-3). It is a figure which shows the relationship between the reaction rate in presence of # 17341, and dATP, dGTP, and TTP density | concentration. The reaction rate depends on the amount of product obtained after reaction for 30 minutes at 0.1 μg total protein / 50 μL. The concentrations of # 17341 are 0 μM (◯), 0.1 μM (●), and 1.0 μM (□). Bars are standard deviations (n = 4-5). 4 is a Lineweaver-Burk reciprocal plot derived from the results of FIG. The concentrations of # 17341 are 0 μM (◯), 0.1 μM (●), and 1.0 μM (□). Bars are standard deviations (n = 4-5). It is a figure which shows the growth curve of a cancer cell line in presence of # 17341. The cell number doubling of Namalwa, REH and MOLT-4 cells in media supplemented with various concentrations of # 17341 is plotted against the number of culture days. The concentrations of # 17341 are 0 μM (◯), 1 μM (□), 5 μM (■), 7.5 μM (Δ), 10 μM (▲), and 15 μM (▽). The plot is the average of the values determined by two independent culture experiments. Bars indicate standard deviation. It is a figure which shows the growth curve of TIG-3 cell line in presence of # 17341. The concentrations of # 17341 are 0 μM (◯), 1 μM (●), 5 μM (□), and 20 μM (■). It is a figure which shows # 17341 density | concentration dependence of the cell number doubling index in various cancer cell lines and TIG-3 cell lines. It is a microscope observation figure of the cancer cell line group after culture | cultivation for 5 weeks. From the top are Namalwa cells, REH cells, and MOLT-4 cells. In the left and right columns, # 17341 was added to the medium at 0 μM and 20 μM, respectively. The bar is 50 μm.

Claims (3)

Following formula (1):

[Wherein, X _{1 to} X ₅ are each independently a hydrogen atom or a halogen atom. ]
Or a pharmaceutically acceptable salt thereof, a telomerase inhibitor. The telomerase inhibitor according to claim 1, wherein X ₃ and X ₅ are halogen atoms. The telomerase inhibitor according to claim 2, wherein X ₃ and X ₅ are chlorine.

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