JP2002104973A - Protein kinase inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein kinase inhibitor useful as a carcinostatic agent. SOLUTION: This protein kinase inhibitor contains a nucleotide alkyl derivative represented by the formula (I) (wherein, R1 denotes a straight-chain or a branched chain alkyl group). The inhibitor is useful as the carcinostatic agent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a new use of a nucleotide alkyl derivative. More specifically, it relates to a protein kinase inhibitor containing a nucleotide alkyl derivative and the inhibitor for use as an anticancer agent.

[0002]

2. Description of the Related Art Protein kinases (protein kinases) are enzymes that phosphorylate the serine, threonine, or tyrosine hydroxyl groups of proteins, and are an important group of enzymes responsible for signal transmission such as cell growth and differentiation. . So far, more than 200 genes encoding protein kinases have been cloned. However, protein kinases are present in almost every place in cells, and are regulated in various aspects such as cell growth, differentiation and functional expression. And is thought to be deeply involved in control.

[0003] In recent years, with the progress of cancer molecular biology research, genes involved in canceration of cells have been clarified, and it has been found that many oncogenes encode protein kinases. In squamous cell carcinoma, ErbB-1 (EGF-
R) has been found to amplify receptor tyrosine kinases such as ErbB-2 in breast, gastric and ovarian cancers, suggesting a relationship with canceration. In the case of breast cancer, patients with tyrosine kinase amplification clearly have a poorer prognosis than patients without amplification, and these receptor-type tyrosine kinases are considered to be important targets in cancer treatment (Toi, M. et al., Eur. J. Ca
ncer, 26, 722, 1990, Yoneda, T. et al., Can
cer. Res., 51, 4430, 1991). In addition, human C
95% or more of ML (cell-mediated lymphocyte injury reaction), A
In about 20% or more cases of LL (acute lymphocytic leukemia), as a result of reciprocal translocation between chromosome 9 and 22, the abl gene and bcr on one chromosome (Ph1 chromosome)
It is known that gene fusion occurs. This chimeric gene product is an activated tyrosine kinase.

[0004] It is known that non-receptor tyrosine kinases are also activated in various cancers. For example, Bcr-abl of chronic myeloid leukemia, Jak-2 of acute lymphocytic leukemia, and c-cell of colon cancer and the like. -Src and c-Y
es tyrosine kinase and the like are considered to be targets, and inhibitors having selectivity for them have been reported (Okabe, M. et al., Leukemia Lymph., Vol. 12, p. 41,
1993, Clark, JW, Int. J. Cancer, Vol. 65, 186.
Page, 1996, Meydan, N. et al., Nature, 379, 64.
5 pages, 1996).

[0005] Regardless of whether a cancer gene is activated or a cancer control gene is inactivated, an excessive or abnormal signal flowing into the nucleus is closely related to canceration. Therefore, drugs that interfere with signaling pathways that are overexpressed in cancer cells are useful as novel anticancer agents.
Protein kinase is one of such molecular targets, and a substance obtained by searching for a compound having protein kinase inhibitory activity is useful as an anticancer agent. Cancer is a disease with the highest mortality rate in Japan. In recent years, advances in local therapies such as surgery and radiotherapy have been contributing to the improvement of the survival rate of cancer patients, but cancers are often found when they have spread throughout the body due to metastasis. It is urgently necessary to respond to cancer that has become a systemic disease. In this regard, expectations for the development of chemotherapy centered on anticancer drugs are increasing. Also,
Advances in molecular biological approaches to cancer cells have provided important guidance in elucidating the mechanism of action of anticancer drugs, which has not been elucidated until now.

[0006] In view of the above, screening of anticancer drugs having protein kinase inhibitory activity was carried out, and as a result, Herbimycin A (Uehara,
Y. et al., Jpn. J. Cancer res., 76, 672, 198
5 years), arbstatin (Umezawa, H. et al., J. Anti
biot., Vol. 39, p. 170, 1986), genistein (Ogaw)
ara, H. et al., J. Antibiot., 39, 606, 1986
Promising drug candidate substances such as

On the other hand, it is well known that a fluorouracil compound having fluorine in a molecule has an anticancer activity. Uridine 5′-alkyl phosphates inhibit sexual aggregation between α and α haploid (haploid) cells of yeast without affecting cell growth (FEMS Microbiol. Lett., No. 147). Volume, 17 pages, 199
7 years), having antifungal activity (Japanese Patent Laid-Open No. 10-21877)
No. 8 gazette). Furthermore, the present inventors have found that nucleoside 5′-alkyl phosphates have a calcium antagonism and are useful as platelet aggregation inhibitors (JP-A-2000-247891).
No.). However, no protein kinase inhibitory activity has been reported for a nucleoside 5'-alkyl phosphate derivative containing no fluorine, and there is no report that the derivative is useful as an anticancer agent.

[0008]

An object of the present invention is to provide a novel protein kinase inhibitor useful as an anticancer agent.

[0009]

Means for Solving the Problems In view of the above prior art, the present inventors have conducted intensive studies on various nucleoside derivatives, and as a result, have found that nucleoside 5'-alkyl phosphate derivatives, in particular, uridine 5'-alkyl phosphate. The fact that the derivative (UMPC) has a remarkable protein kinase inhibitory activity was found, and the present invention was completed based on this finding. That is, the present invention
(1) Formula (I):

[0010]

Embedded image (In the formula, R represents a linear or branched alkyl group.)
A protein kinase inhibitor comprising a nucleotide alkyl derivative represented by the formula: (2) the protein kinase inhibitor according to the above (1), wherein R is an alkyl group having no more than 30 carbon atoms, and (3) R having 4 to 4 carbon atoms. A protein kinase inhibitor according to the above (2), which is an alkyl group of 24;
(4) The above (3), wherein R is an alkyl group having 16 carbon atoms.
(5) The protein kinase inhibitor according to (1), wherein the nucleotide alkyl derivative is used in a free form or any pharmaceutically acceptable form capable of giving a free form in vivo.
The protein kinase inhibitor according to any one of (1) to (4), (6) the above (1) for use as an anticancer agent;
To (5) a protein kinase inhibitor according to any one of (1) to (5).

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION In the above formula (I), the alkyl group represented by R may be linear or branched. The carbon number does not usually exceed 30 and is preferably 4 to 24, and more preferably 16 to 20. Specific examples of such an alkyl group include butyl, pentyl, hexyl, heptyl, octyl, decyl, undecyl, dodecyl, tetradecyl, hexadecyl, heptadecyl, octadecyl, linear alkyl such as eicosyl, or geranyl, farnesyl and the like. Examples include branched alkyl. Currently considered the most preferred is when R represents a 16 carbon n-hexadecyl group, ie uridine 5′-hexadecyl phosphate (UMPC16).

[0012] The nucleotide alkyl derivative as an active ingredient is not only in free form, but also as long as it is pharmaceutically acceptable.
It may be used in any form capable of giving a free form in a living body, such as a hydrate, a salt, an ester and the like. Thus, in the following description, the compounds of formula (I) are meant to encompass not only the free form, but also any such pharmaceutically acceptable forms.

The nucleotide 5'-alkyl derivative which is a compound of the above formula (I) is generally prepared from the corresponding nucleotide 5'-monophosphate (a compound corresponding to R = H in the formula (I)) in a conventional manner ( FEBS Lett., Chapter
94, 339-341, 1978, Life Sci., 43, 437-44
4, 1988, FEBS Lett., 352, 353-355, 1994
Year). For example, in the case of a uridine 5′-alkyl derivative, it is produced by dissolving uridine 5′-monophosphate and various linear or branched alkyl alcohols in t-butyl alcohol and reacting in the presence of dicyclohexylcarbodiimide. can do. The reaction temperature varies depending on the type of the solvent, but is usually 60 to 100 ° C, preferably 70 to 100 ° C.
90 ° C. and the reaction time depends on the reaction temperature,
It is usually 2 to 20 hours, preferably 6 to 8 hours.

As used herein, "cancer" is used in its broadest sense and includes tumors, neoplasias, carcinomas, sarcomas, leukemias, lymphomas, and the like. As used herein, the term "anticancer" means prevention of cancerous lesions, delay of progression of cancerous lesions, suppression of generation of cancerous lesions, reduction of cancerous lesions, or removal of cancerous lesions. . As used herein, “protein kinase” refers to a group of enzymes that phosphorylate other proteins, and includes tyrosine kinase, calmodulin kinase III, A kinase, C kinase,
MAP kinase, serine / threonine kinase and the like.

[0015] It is considered that cancer cells are characterized by proliferation more rapidly than normal cells. Anticancer drugs are chemotherapy that uses drugs that take advantage of this characteristic and are toxic to proliferating cells. The protein kinase inhibitor of the present invention can be used as an anticancer agent that suppresses the growth of cancer cells or induces apoptosis, and can be used for malignant tumors (cancer) such as benign tumors, sarcomas, leukemias, lymphomas, and carcinomas. For treatment, they are usually administered systemically or locally, generally in oral or parenteral form. When administering the compound of formula (I) of the present invention, a solid composition for oral administration,
Liquid and other compositions, parenteral injection, infusion, buccal, rectal, or transdermal administration, i.e., administration in a convenient manner to apply the drug can do.

[0016] Solid compositions for oral administration include tablets,
Pills, capsules, powders, granules and the like are included, and capsules include soft capsules and hard capsules.
Liquid compositions include, for example, syrups, suspensions, and emulsions. The solid composition is a mixture of one or more active substances with at least one inert diluent. The composition may contain general additives other than the inert diluent, for example, a lubricant, a disintegrant, a stabilizer, and a solubilizing agent, according to a conventional method.

Tablets can be manufactured by a conventional method of preparing a solid formulation, by mixing the compound as it is or by adding excipients, binders, disintegrants or other suitable carriers and mixing them evenly. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose. In addition, a coloring agent, a flavoring agent, and the like can be added as necessary. Further, the skin may be applied with a gastric or enteric film such as sucrose, gelatin, hydroxypropylcellulose, or hydroxypropylmethylcellulose phthalate. Or two or more layers.

Compositions in capsule form can be prepared using conventional encapsulation procedures. For example, active ingredient-containing pellets can be prepared by using a standard carrier and then filled into hard gelatin capsules. Alternatively,
A dispersion or suspension can be prepared using any suitable pharmaceutical carrier, for example, aqueous gums, celluloses, silicates or oils, and then the dispersion or suspension can be filled into soft gelatin capsules.

Liquid compositions for oral administration include pharmaceutically acceptable suspensions, emulsions, syrups, elixirs and the like, and may contain a commonly used inert diluent. Such compositions may contain, in addition to inert diluents, stabilizers, buffers, flavoring agents, preservatives, dispersion stabilizers, or other suitable additives as needed.

Injections for parenteral administration according to the present invention include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Aqueous or non-aqueous solutions, suspensions are prepared by mixing one or more active substances with at least one inert diluent. Examples of the aqueous diluent include water for injection, physiological saline, Ringer's solution, glucose solution, or a mixture of physiological saline and glucose solution. Non-aqueous diluents include, for example, olive oil, sesame oil, soybean oil, camellia oil, rapeseed oil, corn oil, peanut oil, vegetable oils such as cottonseed oil, ethanol, propylene glycol, and organic solvents such as polyethylene glycols. No. Such a composition further includes additives such as antioxidants, chelating agents, buffers, stabilizers such as water-soluble organic solvents, soothing agents, preservatives, isotonic agents, emulsifiers, and solubilizing agents. May be included.

As antioxidants, sodium pyrosulfite, ascorbic acid, etc., and as chelating agents, EDT
Examples of buffers such as A, thioglycolic acid, and thiolactic acid include citrate, acetate, and phosphate. Many emulsifiers such as gum arabic, tragacanth, gelatin, and polysorbate 80 (registered trademark) are used in the preparation of suspensions and emulsions. These injections for parenteral administration are prepared by the heating method (dry heat method, high pressure steam method,
It is sterilized by a known sterilization means such as a flowing steam method, a boiling method, an intercalation method, a filtration method, and an irradiation method (radiation method, ultraviolet method, high-frequency method). They also produce sterile solid compositions (for example, by freeze-drying method) and are dissolved in sterile distilled water for injection or other solvents before use. You can also.

The dose of the compound of the formula (I) of the present invention varies depending on age, body weight, symptom, therapeutic effect, administration method, treatment time and the like.
It is orally administered once to several times a day in the range of 100 μg to 400 mg, or parenterally once to several times a day in the range of 10 μg to 100 mg per adult patient per time. . Also, 400mg per day
Up to doses may be administered by continuous intravenous infusion. Thus, the total daily dose by oral administration is 100
The daily dose by parenteral administration is in the range 10 μg to 400 mg, with the range from μg to 2000 mg. Suitably, the compounds will be administered for a period of continuous therapy, for example for a week or more. Of course, since the dosage varies depending on various conditions, a dosage smaller than the above-mentioned dosage may be sufficient, or may be required beyond the range. In the present invention, when the compound of the formula (I) is administered for the purpose of inhibiting protein kinase, no apparent toxicity is observed. Hereinafter, the present invention will be described in detail with reference to Production Examples, Formulation Examples, and Test Examples in the Examples, but these are merely examples, and the present invention is not limited thereto.

[0023]

EXAMPLES Production Example: uridine 5'-hexadecyl phosphate _{(R = C 16 H 33:} UMPC16) Preparation uridine 5'-monophosphate (600 [mu] mol) of and hexadecyl alcohol (6 mmol) 20 mL
Was dissolved in t-butyl alcohol, and 3 mmol of dicyclohexylcarbodiimide was added, followed by heating at 80 ° C. for 20 hours. After completion of the reaction, the formed precipitate was filtered off, and t-
Butyl alcohol was removed under reduced pressure. The residue was washed several times with a mixture of hexane and acetone (1: 1), dried and then mixed with a mixture of chloroform and methanol (95: 1).
Dissolved in 5). This was loaded on a silica gel column prepared with the same solution, and the column was sufficiently washed with the same solution to allow dicyclocarbodiimide to flow out. Thereafter, uridine 5′-hexadecyl phosphate was eluted by sequentially increasing the concentration of methanol. The solvent was removed from the eluate under reduced pressure to obtain a white precipitate. The yield per uridine 5'-monophosphate was 32.4.
%Met.

Formulation Example 1: Intravenous Injection Compound of formula (I) 1 to 40 mg Buffer pH up to about 7 Solvent up to 100 mL In Formulation Example 1 above, specific examples of the buffer include citrate, phosphate and Sodium hydroxide / hydrochloric acid and water as a specific example of the solvent can be mentioned.

Formulation Example 2: Tablets 1 to 40 mg of the compound of formula (I) Diluent / filler 50 to 250 mg Binder 5 to 25 mg Disintegrant 5 to 50 mg Lubricant 1 to 5 mg Specific examples of the filler include microcrystalline cellulose, lactose and starch, specific examples of the binder include polyvinylpyrrolidone and hydroxypropylmethylcellulose, specific examples of the disintegrant include sodium starch glycolate and crospovidone, and a lubricant. Specific examples include magnesium stearate and sodium stearyl fumarate.

Formulation Example 3: Oral suspension 1-40 mg of compound of formula (I) Precipitant 0.1-10 mg Diluent 20-60 mg Preservative 0.01-1.0 mg Buffer pH about 5-8 Up to cosolvent 0 to 40 mg Fragrance 0.01 to 1.0 mg Colorant 0.001 to 0.1 mg In Formulation Example 3 above, specific examples of the suspending agent include xanthin gum and microcrystalline cellulose, and specific examples of the diluent. As a sorbitol solution, typically water, a specific example of a preservative is sodium benzoate, a specific example of a buffer is citrate, and a specific example of a cosolvent is alcohol, propylene glycol, polyethylene glycol. And cyclodextrin.

Test Example 1: Measurement of protein kinase inhibitory activity of UMPC16 The protein kinase inhibitory activity of the nucleotide alkyl derivative of the present invention was determined by the Ministry of Education, Culture, Sports, Science and Technology's Cancer Specified Comprehensive Cancer / Cancer Drug Screening Committee, 1999 edition. "Molecular Target Screening of Anticancer Drugs-Overview and Usage-"
Based on the method described in “Assay for Protein Kinase Inhibitory Activity”, a method for detecting a plurality of protein kinase activities on the same gel using a cell lysate of cancer cells NIH3T3 / v-src (Fukazawa, H. et al. ., Anal. Biochem.,
212, 106, 1993, Pei-Ming Li et al., Antica
ncer Res., Vol. 13, pp. 1957-1964, 1993). In the above experimental system, NIH3T3 cell vs.
The rc transformant is crushed in a hypotonic buffer, then centrifuged and enucleated to obtain an enzyme solution. The sample dissolved in DMSO, cAMP, phorbol ester, [γ ³² P] AT
Add P and react at 25 ° C. for 20 minutes. After stopping the reaction,
The phosphorylated protein is analyzed by SDS-PAGE and autoradiography. By this method, A kinase, C kinase, tyrosine kinase of the src family, and calmodulin-dependent protein kinase I
At least four protein kinase activities of II can be detected simultaneously. The activity is evaluated by adding a sample to a final concentration of 0.1 mg / mL and determining the presence or absence of phosphorylation and the inhibition pattern. If inhibitory activity is observed, make a three-step dilution series. Specifically, the procedure was performed as follows. NIH3T3 cells transformed with v-src were treated with 1 mM Hepes buffer (pH
7.4), antipain is MgCl ₂ and protease inhibitors 5 mM, leupeptin, pepstatin A
Was cooled on ice for 10 minutes in a hypotonic buffer containing 25 μg / mL each. The swollen cells were mixed by stirring at room temperature for 2 minutes and suspended. Next, Hep is adjusted to 20 mM.
After adding es buffer (200 mM, pH 7.4)
Centrifugation at 500 g for 5 minutes to precipitate nuclei.
The supernatant was collected and a final concentration of 20 mM Hepes buffer (pH 7.4), 10 mM MgCl ₂ , 0.1 mM
Na ₃ VO ₄ , 10 mM β-glycerophosphate,
It was prepared to be 1 mM NaF and 2.5 mg / mL protein. Enucleated fraction 20 μL
, 5 μL of various kinase activators and UMPC16 in DMSO were added. 5 μL of [γ- ³² P]
ATP (75 μM, 10 μCi) was added to start the kinase reaction and incubated at 25 ° C. for 15 minutes.
The final concentration of activator is phorbol ester (PMA)
1 μM, CaCl ₂ 10 μM, DMSO 10% (v
/ V) and cAMP was 20 μM. Also, UMPC1
The final concentration of 6 is 0.1, 1, 10, 100 μg / mL
And The reaction was terminated by adding 10 μL of a 4 × concentration of SDS-PAGE sample buffer or heating in a boiling water bath for 5 minutes. The heat-treated mixture was cooled at room temperature, and centrifuged at 15,000 g for 30 minutes to precipitate denatured protein.
Was mixed. 9% SDS- phosphorylated protein
Separated using PAGE (6 cm long) and visualized by autoradiography. Table 1 shows the obtained results.

[0028]

[Table 1] PKA: A kinase, PKC: C kinase, PTK: Tyrosine kinase, CAMK: Calmodulin-dependent protein kinase I
II ++:> 70% inhibition, +: 40-70% inhibition,-: <4
0% inhibition (no inhibition) As shown in Table 1, UMPC16 inhibited various protein kinase activities in a concentration-dependent manner. Therefore,
The nucleotide 5′-alkyl derivative represented by the formula (I) of the present invention, including UMPC16, is useful as a protein kinase inhibitor, and is considered to be a useful substance as an anticancer agent.

[0029]

According to the present invention, a novel protein kinase inhibitor can be obtained by using a nucleotide alkyl derivative represented by the formula (I) as an active ingredient, which is useful as an anticancer agent.

Claims (6)

[Claims] 1. A compound of formula (I): (In the formula, R represents a linear or branched alkyl group.)
A protein kinase inhibitor comprising a nucleotide alkyl derivative represented by the formula: 2. The protein kinase inhibitor according to claim 1, wherein R is an alkyl group having no more than 30 carbon atoms. 3. The protein kinase inhibitor according to claim 2, wherein R is an alkyl group having 4 to 24 carbon atoms. 4. The protein kinase inhibitor according to claim 3, wherein R is an alkyl group having 16 carbon atoms. 5. The protein kinase inhibitor according to claim 1, wherein the nucleotide alkyl derivative is used in a free form or in any pharmaceutically acceptable form capable of giving a free form in vivo. . 6. The method according to claim 1, which is used as an anticancer drug.
The protein kinase inhibitor according to any one of claims 1 to 5, wherein