JP2006028104A - Histone deacetylase inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a histone deacetylase (HDAC) inhibitor or the like containing a mercaptoacetophenone derivative or a pharmaceutically acceptable salt thereof as an active ingredient, more concretely, the HDAC inhibitor useful for prophylaxis and/or therapy or the like of cancer, tumor, neurodegeneration disease or the like. <P>SOLUTION: The HDAC inhibitor contains the mercaptoacetophenone derivative represented by formula (I) [wherein, R<SP>1</SP>is a hydrogen atom, a substituted or nonsubstituted lower acyl, CONHCH(R<SP>3</SP>)CO<SB>2</SB>R<SP>4</SP>(wherein, R<SP>3</SP>is a hydrogen atom or a substituted or nonsubstituted lower alkyl; and R<SP>4</SP>is a hydrogen atom or a lower alkyl) or the like; R<SP>2</SP>is a hydrogen atom or a substituted or nonsubstituted lower alkyl; and Ar is a substituted or nonsubstituted aryl] or the like, or the pharmaceutically acceptable salt thereof as an active ingredient. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a histone deacetylase inhibitor.

  The nuclear DNA of higher organisms exists as a structure called a nucleosome, and the control of the higher order structure plays an important role in the expression of genetic information on the DNA. Nuclear DNA can exist in cells by taking a highly condensed structure, but it is a protein called histone that forms the basic structure of nucleosomes together with the nuclear DNA, and DNA consists of four types of core histones. It forms a compact structure (nucleosome structure) wrapped around an octamer structure consisting of (H2A, H2B, H3, H4). There are many basic amino acids in the N-terminal region of histone, and this part maintains the bond between DNA and histone by electrically interacting with the negatively charged phosphate backbone of DNA. [“Nature. Rev. Mol. Cell Biol.”, 2001, Vol. 2, pp.422-432].

  Moreover, it has been clarified that the change to a condensed structure of DNA via histone is affected by various modifications in the histone N-terminal region. In particular, reversible acetylation and deacetylation that occurs at lysine residues in the histone N-terminal region is known to play an important role in controlling histone-DNA or histone-non-histone protein interactions [ “Current Opinion in Genetics & Development”, 2001, Vol. 11, pp.155-161, “Current Opinion in Genetics & Development” Current Opinion in Genetics & Development) ”, 2001, Vol. 11, p.162-166]. Such reversible acetylation and deacetylation in the histone N-terminal region is performed by enzymes classified into two families called histone acetyltransferase (HAT) and histone deacetylase (HDAC). It is known that histone acetylation and transcriptional activation are closely related because the transcription factor complex has HAT activity and HDAC is recruited to the vicinity of the target gene promoter by transcriptional repressor. .

It is also known that deregulation of HAT and HDAC activity is involved in multiple human cancers. As a typical example, the t (15; 17) translocation of chromosomes frequently seen in acute promyelocytic leukemia (APL) expresses a chimeric protein (PML-RARα) in which a retinoic acid receptor is fused to a PML transcription factor It is known to do. Recruitment of HDAC by this chimeric protein is thought to suppress normal differentiation of hematopoietic cells by suppressing transcriptional activation by retinoic acid, leading to cell carcinoma [Nature Genet .) ”, 1998, Vol. 18, p. 126-135] Therefore, inhibiting HDAC function is expected to release transcriptional repression of cancerous cells and induce differentiation [“ Journal Of the National Cancer Institute (J. Natl. Cancer Inst.), 1998, Vol. 90, pp. 1621-1625]
Inhibiting HDAC function also causes various cellular responses through transcriptional activation of genes due to increased histone acetylation, and not only induces normal differentiation of the above leukemia cells but also solid cancer-derived cells. In some cases, it has been reported by a plurality of HDAC inhibitors that it has an antitumor activity accompanied with apoptosis induction by stopping the cell cycle.

  Furthermore, analysis of the relationship between HDAC and transcriptional regulation has progressed, and it is known that HDAC is widely involved in human diseases, not limited to cancer. For example, for neurodegenerative diseases such as Huntington's disease involving polyglutamine repeats, it is suggested that HDAC inhibitors may become new therapeutic agents by inhibiting transcriptional disorders caused by polyglutamine repeats. [Nature], 2001, 413, p.739-743, “Proc. Natl. Acad. Sci.”, 2003, 100 In addition, HDAC inhibitors have been reported to have neurodegeneration-inhibiting effects due to oxidative stress against neurodegenerative diseases caused by oxidative stress ["Proceeding National Academy. Of Science (Proc. Natl. Acad. Sci.) ", 2003, 100, p.4281-4286], HDAC inhibitors are attracting attention as effective drugs for diseases caused by neurodegeneration. On the other hand, valproic acid used as an antiepileptic drug was found to have HDAC inhibitory activity from a completely different approach, and the regulation of gene expression via HDAC inhibition may be involved in the antiepileptic effect of valproic acid [“The Journal of Biological Chemistry (J. Biol. Chem.)”, 2001, Vol. 276, p. 36734-36741, “The Embo Journal (EMBO. J.) ”, 2001, Vol. 20, p.6969-6978].

  Thus, HDAC inhibitors are not limited to application as anticancer agents through cell growth / differentiation regulation, but may be applied to, for example, neurodegenerative diseases through functional regulation of various tissues. Promising.

Conventionally, as a compound having an HDAC inhibitory action, many structures such as those derived from natural products and synthetic compounds have been reported (see Non-Patent Document 1, Non-Patent Document 2, etc.).
“J. Natl. Cancer Inst.”, 2000, Vol. 92, pp. 1210-1216 (Nature Rev. Cancer), 2001, Vol. 1, p.194-202)

  An object of the present invention is to provide an HDAC inhibitor containing a mercaptoacetophenone derivative or a pharmaceutically acceptable salt thereof as an active ingredient. More specifically, it is an object of the present invention to provide an HDAC inhibitor useful for the prevention and / or treatment of cancer, tumors, neurodegenerative diseases and the like.

The present invention relates to the following (1) to (7).
(1) Formula (I)

[Wherein, R ¹ represents a hydrogen atom, substituted or unsubstituted lower acyl, CONHCH (R ³ ) CO ₂ R ⁴ (wherein R ³ represents a hydrogen atom or substituted or unsubstituted lower alkyl, R ⁴ represents Represents a hydrogen atom or lower alkyl), formula (T)

(Wherein R ⁵ represents a hydrogen atom or a substituted or unsubstituted lower alkyl)
Or formula (U)

(Wherein R ⁶ represents lower alkyl, R ⁷ represents a substituted or unsubstituted aryl or a substituted or unsubstituted aromatic heterocyclic group),
R ² represents a hydrogen atom or a substituted or unsubstituted lower alkyl,
Ar represents substituted or unsubstituted aryl]. A histone deacetylase (HDAC) inhibitor containing a mercaptoacetophenone derivative represented by the formula: or a pharmaceutically acceptable salt thereof as an active ingredient.
(2) R ² is HDAC inhibitors described above (1), wherein the hydrogen atom.
(3) The HDAC inhibitor according to the above (1) or (2), wherein R ¹ is CONHCH (R ³ ) CO ₂ R ⁴ and R ³ is lower alkyl substituted with a phenyl group.
(4) The HDAC inhibitor according to any one of (1) to (3) above, wherein R ¹ is CONHCH (R ³ ) CO ₂ R ⁴ and R ⁴ is a hydrogen atom.
(5) R ¹ is the formula (T)

The HDAC inhibitor according to (1) or (2), wherein R ⁵ is a hydrogen atom or unsubstituted lower alkyl.
(6) R ¹ is the formula (U)

And the HDAC inhibitor according to the above (1) or (2), wherein R ⁷ is a substituted or unsubstituted aromatic heterocyclic group.
(7) The HDAC inhibitor according to the above (1) or (2), wherein R ¹ is a hydrogen atom or unsubstituted lower acyl.

  According to the present invention, there is provided an HDAC inhibitor containing a mercaptoacetophenone derivative or a pharmaceutically acceptable salt thereof as an active ingredient, which is useful for the prevention and / or treatment of cancer, tumors, neurodegenerative diseases and the like. .

Hereinafter, the compound represented by formula (I) is referred to as compound (I). The same applies to the compounds of other formula numbers.
In the definition of each group of compound (I),
(i) As the lower alkyl, for example, linear or branched alkyl having 1 to 6 carbon atoms, specifically methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, And isopentyl, neopentyl, tert-pentyl, hexyl and the like.
(ii) Examples of aryl include phenyl and naphthyl.
(iii) As the aromatic heterocyclic group, for example, a 5-membered or 6-membered monocyclic aromatic heterocyclic group containing at least one atom selected from a nitrogen atom, an oxygen atom and a sulfur atom, Examples include condensed bicyclic or tricyclic condensed rings containing at least one atom selected from a nitrogen atom, an oxygen atom, and a sulfur atom. Specific examples include furyl, thienyl, Benzothienyl, pyrrolyl, pyridyl, pyrazinyl, imidazolyl, pyrazolyl, triazolyl, thiazolyl, isothiazolyl, thiadiazolyl, oxazolyl, oxadiazolyl, pyrimidinyl, indolyl, isoindolyl, benzothiazolyl, benzoimidazolyl, benzotriazolyl, quinolyl, isoquinolyl, quinazolyl, quinazolyl Indolyl is preferable.
Examples of (iv) lower acyl include linear or branched acyl having 1 to 6 carbon atoms, specifically, formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl and the like.
(v) Substituents in substituted lower alkyl and substituted lower acyl are, for example, halogen, hydroxy, carboxy, cycloalkyl, aryl, substituted aryl, which are the same or different and have 1 to 3 substituents. And the same as the substituent in the substituted aryl.
The aryl shown here is synonymous with the aryl. In addition, examples of the halogen shown here include atoms such as fluorine, chlorine, bromine, and iodine. Examples of the cycloalkyl include cycloalkyl having 3 to 8 carbon atoms. Examples include propyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.

(vi) Examples of the substituent in the substituted aryl and the substituted aromatic heterocyclic group include, for example, halogen, hydroxy, nitro, cyano, carboxy, formyl, lower alkyl, lower alkoxy, substituted, Unsubstituted aryl (substituents in the substituted aryl include, for example, halogen, hydroxy, lower alkyl, lower alkoxy, cyano, nitro, carboxy, etc., which are the same or different and have 1 to 3 substitutions) It is done.
The lower alkyl part of lower alkyl and lower alkoxy shown here has the same meaning as the lower alkyl, and aryl has the same meaning as the aryl. Halogen has the same meaning as the above halogen.

  Pharmaceutically acceptable salts of compound (I) include, for example, pharmaceutically acceptable acid addition salts, metal salts, ammonium salts, organic amine addition salts, amino acid addition salts and the like. Examples of the pharmaceutically acceptable acid addition salt of compound (I) include inorganic acid salts such as hydrochloride, sulfate, and phosphate, acetate, maleate, fumarate, tartrate, and citrate. And organic acid salts such as lactate and succinate, and pharmaceutically acceptable metal salts include, for example, alkali metal salts such as sodium salt and potassium salt, alkaline earth such as magnesium salt and calcium salt Examples of the pharmaceutically acceptable ammonium salt include salts of ammonium, tetramethylammonium and the like, and examples of the pharmaceutically acceptable organic amine addition salt include metal salts, aluminum salts, zinc salts and the like. Examples include addition salts such as morpholine and piperidine, and pharmaceutically acceptable amino acid addition salts include, for example, lysine, glycine, phenylalanine, aspartic acid, glutamic acid. Addition salts such as phosphate and the like.

  Next, a method for producing compound (I) will be described. In the production method shown below, when the defined group changes under the conditions of the implementation method or is inappropriate for carrying out the method, a method for introducing and removing a protective group commonly used in organic synthetic chemistry [for example, , TW Greene, “Protective Groups in Organic Synthesis”, John Wiley & Sons Inc., 1981, etc. ] Can be used to obtain the target compound. Further, the order of reaction steps such as introduction of substituents can be changed as necessary.

Compound (I) can be produced according to the following reaction steps.
Production method 1
Among the compounds (I), the compound (Ia) in which R ¹ is a hydrogen atom is “New Experimental Chemistry Course”, Maruzen Co., Ltd., 1978, Vol. 14, p. 1699, “Synthesis-Stuttgart” ) ”, 1995, p. 373,“ Tetrahedron Lett. ”, 1999, Vol. 40, p. 603, and the like.

(Wherein R ² and Ar are as defined above, and X represents a fluorine atom, a chlorine atom, a bromine atom or an iodine atom)
Production method 2
Among the compounds (I), the compound (Ib) in which R ¹ is substituted or unsubstituted lower acyl is obtained from the compound (Ia) from “New Experimental Chemistry Course”, Maruzen Co., Ltd., 1978, Vol. 14, p. It can be synthesized according to the method described in .1823 etc.

(Wherein R ² and Ar are as defined above, and R ⁸ represents a substituted or unsubstituted lower acyl)
Production method 3
Among the compounds (I), the compound (Ic) in which R ¹ is CONHCH (R ³ ) CO ₂ R ⁴ is derived from the compound (Ia) from “New Experimental Chemistry Course”, Maruzen Co., Ltd., 1978, Vol. , p.1658 and the like.

(Wherein R ² , R ³ , R ⁴ and Ar are as defined above)
Production method 4
Among the compounds (I), R ¹ is represented by the formula (T)

(Wherein R ⁵ is as defined above)
The compound (Id) can be synthesized from the compound (II) according to the method described in “New Experimental Chemistry Course”, Maruzen Co., Ltd., 1978, Vol. 14, p.

(Wherein R ² , R ⁵ , Ar and X are as defined above)
Compound (III) can be synthesized according to the method described in “Collect Czech Chem. Commun.”, 1982, Vol. 47, p.
Manufacturing method 5
Among the compounds (I), the compound (Ie) in which R ¹ is C (═NR ⁶ ) R ⁷ is obtained from the compound (II) from “Synthesis”, 1987, p.817, “Journal of -It can be synthesized according to the method described in Chemical Society, Perkin Trans. 1 "(J. Chem. Soc., Perkin Trans. 1", 1972, p. 2332, etc.).

(Wherein R ² , R ⁶ , R ⁷ , Ar and X are as defined above)
Compound (IV) can be synthesized according to the method described in “New Experimental Chemistry Course”, Maruzen Co., Ltd., 1978, Vol. 14, p.

Conversion of the functional group contained in R ¹ , R ² , or Ar in the compound (I) may be carried out by other known methods other than the above steps [for example, “Complex Organic Organic Trans” by RC Larock. Comprehensive Organic Transformations ", 1989, etc.] or a method according to them.

  By appropriately combining the above methods, a compound (I) having a desired functional group at a desired position can be obtained.

  Intermediates and target compounds in the above production methods are purified methods commonly used in organic synthetic chemistry, such as filtration, extraction, washing, drying, concentration, recrystallization, high performance liquid chromatography, thin layer chromatography, silica gel chromatography, etc. It can be purified and isolated by subjecting it to various types of chromatography. In addition, the intermediate can be subjected to the next reaction without any particular purification.

  Some compounds (I) may have stereoisomers such as positional isomers, geometric isomers, optical isomers, tautomers, etc., but the HDAC inhibitor of the present invention includes these. All possible isomers and mixtures thereof can be used, including.

  In the case of obtaining a salt of compound (I), it may be purified as it is when compound (I) is obtained in the form of a salt. It may be dissolved or suspended in a solvent and isolated or purified by adding an acid or a base.

  Compound (I) or a pharmaceutically acceptable salt thereof may exist in the form of an adduct with water or various solvents, and these adducts can also be used for the HDAC inhibitor of the present invention. it can.

  Specific examples of compound (I) used in the HDAC inhibitor of the present invention are shown in Table 1. However, the compounds used in the present invention are not limited to these.

The mass spectrometric data of the compounds described in Table 1 were measured with an electrospray ionization mass spectrometer (ESIMS): Waters ZQ, and the instrument data are described below.
Compound 1 MS (ESI) m / z: 293 (M + H) ⁺
Compound 2 MS (ESI) m / z: 263 (M + H) ⁺
Compound 3 MS (ESI) m / z: 289 (M + H) ⁺
- Compound 4 MS (ESI) m / z : 344 (M + H) +
Compound 5 MS (ESI) m / z: 247 (M + H) ⁺
Compound 6 MS (ESI) m / z: 337 (M + H) ⁺
Compound 7 MS (ESI) m / z: 153 (M + H) ⁺

Next, the pharmacological activity of a typical compound (I) will be described in test examples.
Test Example 1 HDAC inhibitory activity
HDAC inhibitory activity was evaluated by the method described below. The HDAC enzyme reaction was performed by purchasing the HDAC Fluorescent Activity Assay / Drug Discovery Kit * -AK-500 (Biomol, catalog number AK-500). . The reaction was performed in the wells of a 96-well half well plate (Coaster, catalog number 3694). For the test drug sample, 10 mmol / L sample dissolved in dimethyl sulfoxide (DMSO) is diluted in order, and the drug is prepared so that the final concentration is 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30 μmol / L. did. DMSO in which the drug was not dissolved was used for the control and blank. 15 μL of HeLa cell extract diluted 30-fold was added to 10 μL of the diluted test drug sample. Further, 25 μL of a substrate solution adjusted to 500 μmol / L was added and mixed, and then reacted at 37 ° C. for 30 minutes. After the reaction, the HDAC enzyme reaction was stopped by adding 50 μL of a 20-fold diluted developer solution and reacting at 37 ° C. for 10 minutes. After stopping the reaction, fluorescence (460 nm) emitted with excitation light (390 nm) was measured using a multi-label counter (wallac 1420 ARVOsx) (Amersham Pharmacia Biotech). The concentration (IC50) of 50% inhibition of HDAC in each sample was determined from the measured values. The results obtained in Test Example 1 are shown in Table 2.

Test Example 2 Intracellular HDAC inhibitory activity and expression-inducing action of cyclin-dependent kinase inhibitor molecule (p21 molecule)
HDAC regulates histone acetylation levels through deacetylation of intracellular histone proteins, and it is known that acetylation of histones is enhanced in cells treated with HDAC inhibitors [“ The Journal of Biological Chemistry (J. Biol. Chem.), 1990, Vol. 265, p. 17174-17179]. In addition, the expression of multiple genes is enhanced with histone acetylation, and the expression of p21WAF1 / CIP1 (hereinafter abbreviated as p21), which is a cyclin-dependent kinase inhibitor involved in cell cycle regulation. It has been reported that induction plays an important role in the cytostatic activity of HDAC inhibitors [“J. Biol. Chem.”, 1999, No. 274, p. 34940-34947, “Procedure National Academy of Science (Proc., Natl., Acad., Sci.)”, 2000, 97, p. 10014-10019]. The intracellular HDAC inhibitory activity and p21 expression inducing action of a series of compounds including Compound 1 were verified by the methods shown below.

Human non-small cell lung cancer A549 cells (ATCC number: CCL-185) were used. RPMI164O medium containing 10% FBS was used for culturing A549 cells. 1 × 10 ⁶ A549 cells were inoculated in a 10 cm dish (Iwaki Glass Co., Catalog No. 3020-100) by 8 mL each, and cultured at 37 ° C. for 22 hours in a 5% carbon dioxide incubator. The test drug sample was prepared by sequentially diluting 10 mmol / L samples dissolved in DMSO and adjusting the drugs to final concentrations of 3, 10, and 30 μmol / L. DMSO in which no drug was dissolved was used as a control, and trichostatin A (abbreviated as TSA) 5 μmol / L was used as a positive drug. 20 hours after drug addition, cells were collected and lysed (Lysis solution) [50 mmol / L Hepes buffer solution (HEPES), 150 mmol / L NaCl, 1 mmol / L disodium ethylenediaminetetraacetate (EDTA), 2.5 mmol / L ethylene Glycol bis (2-aminoethyl ether) tetraacetate (EGTA), 0.1% polyoxyethylene sorbitan monolaurate (Tween 20), 10% glycerol, 0.1 mmol / L phenylmethylsulfonyl fluoride (PMSF), Suspended in 10 mmol / L beta-glycerophosphate, 1 mmol / L NaF, 0.1 mmol / L Na ₃ VO ₄ , 10 mg / mL aprotinin, 10 mmol / L leupeptin, pH 7.5] After stirring at 1 ° C. for 1 hour, the supernatant was collected by centrifugation at 12000 g for 10 minutes. The protein concentration of each collected cell extract was measured with a DC Protein Assay Kit (Biorad, catalog number 500-0116), and 20 μg of each cell extract was used for the following operations.

  The amount of acetylated histone and p21 protein increased by inhibition of HDAC contained in each cell extract was verified by Western blotting according to a conventional method. After 20 μg of cell extract was fractionated using a polyacrylamide gradient gel plate for electrophoresis 15-25% (Daiichi Kagaku, catalog number 301520), a polyvinylidene fluoride (PVDF) membrane (Millipore, The protein was transferred to catalog number IPVH304F0). Anti-acetylated histone H3 antibody (UBI, catalog number 06-599), anti-acetylated histone H3, H4, and p21 protein levels in the protein transcribed onto the PVDF membrane. After reacting with acetylated histone H4 antibody (UBI, catalog number 06-598) and anti-p21 antibody (Pharmingen, catalog number 65961A)), anti-reacts with both acetylated histone antibodies The reaction was performed with a rabbit antibody [Dako Cytomation (DAKO), catalog number P0217] or an anti-mouse antibody (Amersham Pharmacia, catalog number NA931V) that reacts with the p21 antibody. After the antibody reaction, the PVDF membrane is soaked in Super Signal West Pico Chemiluminescent Substrate solution (Pierce, catalog number 34077), and the fluorescence signal depending on the amount of antibody is applied to the X-ray film. Detected. Based on the detected signal intensity, it was determined whether a series of compounds including Compound 1 exhibited intracellular HDAC inhibitory activity and p21 expression inducing action.

  FIG. 1 shows the intracellular HDAC inhibitory activity and p21 expression inducing action of Compound 1 at 10 μmol / L.

  In FIG. 1, no signal is detected with the anti-acetylated histone antibody in the control, but when TSA 5 μmol / L, which is a positive drug of a deacetylation inhibitor, is added, a signal with the anti-acetylated histone antibody is detected. At this time, when Compound 1 is added at a concentration of 3, 10, or 30 μmol / L, a signal from the anti-acetylated histone antibody is detected from 10 μmol / L in a concentration-dependent manner. From this, it was considered that the test compound 1 exhibits histone deacetylation inhibitory activity also in the cells. In the control, no signal was detected with the anti-p21 antibody, but when TSA 5 μmol / L, which is a positive drug of a deacetylation inhibitor, was added, a signal with the anti-p21 antibody was detected. At this time, when test compound 1 is added at a concentration of 3, 10, or 30 μmol / L, a signal due to anti-p21 antibody is detected from 10 μmol / L in a concentration-dependent manner. From this, it was considered that the test compound 1 exhibits p21 expression inducing action.

Test Example 3 Effect on transcriptional repression by leukemia causative gene AML1 / ETO fusion gene The t (8; 21) translocation, which is the most frequently observed translocation in human acute leukemia, forms AML1 / ETO fusion protein and causes leukemia It is known to be involved. At that time, it is assumed that ETO induces HDAC and suppresses transcription of the target gene activated by AML1 as a cause of tumorigenicity, and HDAC inhibitor that specifically acts on AML1 / ETO is Expected as a new leukemia treatment. In order to verify the action of inhibiting AML1 / ETO-induced HDAC activity in cells, the compounds were evaluated by a reporter assay using the cultured cells shown below.

Monkey kidney-derived cell line COS-7 cells were seeded at 4 × 10 ⁴ cells per well in MULTIWELL [Falcon, catalog number 35-3043)]. Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS) was used as the medium, and the cells were cultured at 37 ° C. for 24 hours in a 5% carbon dioxide incubator. AML1, PEBP2β and AML1 / cloned in pME18S cloning vectors [“Mole. Cell. Biol., 1996, Vol. 16, p. 3967-3979] as effectors in these cells. Tww-tk-Luc ["Molecular and Cellular Biology (" Molecular and Cellular Biology "), a luciferase reporter that uses the ETO gene as a promoter and a T cell receptor beta chain enhancer (TCRβenhancer) having PEBP2 site, which is the DNA binding site of AML1 Mol. Cell. Biol.) ”1996, Vol. 16, p. 3967-3979] was introduced using Super Fect Transfection Reagent [QIAGEN, Catalog No. 301305], respectively. Two hours after introduction, the cells were washed twice with phosphate buffered saline (PBS), and then the compound dissolved in DMSO was added to DMEM containing 10% FCS to a final concentration of 10 μmol / L. The cells were cultured in a 5% carbon dioxide incubator for 24 hours, and as a control, DMEM containing 10% FCS with the same concentration (0.1%) of DMSO without drug was used. (Catalog No. PGL 1500) and Lumat LB 9507 (Berthold Japan (BERTHOLD)), and the results are shown in Fig. 2 (average values and standard deviations of two independent tests are shown. AML1, The luciferase activity by Tww-tk-Luc induced by PEBP2β is defined as 100%, and the activity value at the time of AML1 / ETO introduction and in the presence of the drug is displayed as a relative value).

  According to FIG. 2, Tww-tk-Luc activity induced by AML1 and PEBP2β is suppressed to about 30% by co-expression of AML1 / ETO. For example, the suppression is suppressed in the presence of Compound 3. It was shown to drop to about 50%. The release of this suppression was equivalent to or better than that of the known HDAC inhibitor TSA (Figure 2 shows the mean of two independent tests and its standard deviation. Tww- induced by AML1, PEBP2β. The luciferase activity by tk-Luc was taken as 100%, and the activity value when AML1 / ETO was introduced and in the presence of the drug was expressed as a relative value).

  As described above, from the analysis described in Test Examples 1 to 3, compound (I) has an HDAC inhibitory activity, a growth inhibitory action on cells (p21 inducing action), and a function of AML1 / ETO, which is one of the causative molecules of leukemia It was shown to have an inhibitory effect. From these analysis results, it was shown that the compound found in the present invention becomes a new therapeutic agent having HDAC inhibitory activity.

  Compound (I) or a pharmaceutically acceptable salt thereof can be administered alone as it is, but usually various pharmaceutical preparations, preferably antitumor agents, more preferably lung cancer, kidney or metastasis thereof. It is desirable to provide it as a therapeutic agent for cancer or leukemia. These pharmaceutical preparations are used for animals or humans.

  The pharmaceutical preparation according to the present invention may contain Compound (I) or a pharmaceutically acceptable salt thereof as an active ingredient alone or as a mixture with any other active ingredient for treatment. In addition, these pharmaceutical preparations are produced by any method well known in the technical field of pharmaceutics by mixing the active ingredient together with one or more pharmaceutically acceptable carriers.

  It is desirable to use the administration route that is most effective in the treatment, and can be oral or parenteral, such as intravenously.

  Examples of the dosage form include tablets, powders, granules, syrups, injections and the like.

  For example, tablets suitable for oral administration can be produced using excipients such as lactose, disintegrants such as corn starch, lubricants such as magnesium stearate, binders such as hydroxypropylcellulose, and the like.

  In the case of an injection suitable for parenteral administration, for example, it can be produced using a salt solution, a glucose solution or a mixed solution of a salt solution and a glucose solution.

  Compound (I) or a pharmaceutically acceptable salt thereof is usually administered systemically or locally in an oral or parenteral form when used for the above-mentioned purposes. The dose and frequency of administration vary depending on the administration form, patient age, body weight, nature or severity of symptoms to be treated, etc., but usually 0.1 to 1000 mg / kg per adult, preferably 0.5 It is administered orally or parenterally once to several times a day in the range of ˜500 mg / kg, or continuously administered intravenously in the range of 1 to 24 hours per day. However, these doses and the number of doses vary depending on the various conditions described above.

  Below, the aspect of this invention is demonstrated with an Example and a reference example. However, the scope of the present invention is not limited to these examples.

Reference Example 1: Synthesis step 1 of 1- (2′-fluorobiphenyl-4-yl) -2-mercaptoethanone (Compound 5)
To a suspension of aluminum chloride (12.7 g, 95.2 mmol) in dichloroethane (100 mL), acetyl chloride (4.40 mL, 61.8 mmol) and 2-fluorobiphenyl (8.20 g, 47.6 mmol) were sequentially added, and the mixture was stirred for 1 hour. . The reaction solution was poured onto ice and concentrated hydrochloric acid was added until the insoluble material was dissolved. Chloroform was added to the mixture, and the organic layer was extracted, washed with saturated brine, and dried over anhydrous magnesium sulfate. After removing the solvent, the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 30: 1) to give 1- (2'-fluorobiphenyl-4-yl) -ethanone (9.18 g, 90%) as colorless crystals. It was.
APCMS m / z: [M + H] ⁺ 215.

Process 2
The compound obtained in Step 1 (9.00 g, 42.0 mmol) was dissolved in ethyl acetate (100 mL), cupric bromide (18.7 g, 84.0 mmol) was added, and the mixture was heated to reflux for 6 hours. After cooling to room temperature, insolubles were removed by celite filtration. Water was added to the filtrate, and the organic layer was extracted, washed with saturated brine, and dried over anhydrous magnesium sulfate. After the solvent was distilled off, recrystallization from hexane-ethyl acetate gave 2-bromo-1- (2′-fluorobiphenyl-4-yl) -ethanone (9.11 g, 74%) as pale yellow crystals.
APCMS m / z: [M + H] ⁺ 293.

Process 3
The compound obtained in Step 2 (1.50 g, 5.12 mmol) was dissolved in DMF (30 mL), 70% aqueous sodium hydrogen sulfide solution (0.430 g, 5.37 mmol) was added under ice cooling, and the mixture was stirred at room temperature for 12 hours. Water and chloroform were added to the reaction solution, and the organic layer was extracted, washed with saturated brine, and dried over anhydrous magnesium sulfate. After evaporation of the solvent, recrystallization from hexane gave the title compound (0.825 g, 66%) as colorless crystals.
¹ H NMR (CDCl ₃ , d): 1.50 (s, 1H), 3.82 (s, 2H), 7.05-7.59 (m, 6H), 7.90 (d, J = 7.8 Hz, 2H).
APCMS m / z: [M + H] ⁺ 247.

Reference Example 2: Synthesis of S-2- (2′-fluorobiphenyl-4-yl) -2-oxoethyl thioacetate (Compound 3) The compound (0.500 g, 1.71 mmol) obtained in Step 2 of Reference Example 1 was It melt | dissolved in THF (10 mL), Acetic acid thiol (0.134 mL, 1.88 mmol) and triethylamine (0.262 mL, 1.88 mmol) were added, and it stirred at room temperature for 1 hour. Saturated aqueous sodium hydrogen carbonate and chloroform were added to the reaction mixture, and the organic layer was extracted, washed with saturated brine, and dried over anhydrous magnesium sulfate. After the solvent was distilled off, the residue was purified by silica gel column chromatography (hexane: ethyl acetate = 30: 1) to obtain the title compound (0.430 g, 87%) as a colorless oil.
¹ H NMR (CDCl ₃ , d): 2.35 (s, 3H), 3.98 (s, 2H), 7.10-7.56 (m, 6H), 7.95 (d, J = 7.8 Hz, 2H).
APCMS m / z: [M + H] ⁺ 289.

Reference Example 3: Synthesis step 1 of phenacyl mercaptan (compound 7)
Tetrahedron Letters Vol. 40, No. 4, pages 603 to 606 (1999) were used to dissolve commercially available phenacyl bromide (5.00 g, 25.1 mmol) in dimethylformamide (50 mL). , Potassium thioacetate (purity about 90%, 3.19 g, 25 mmol) was added, and the mixture was stirred at room temperature for 1 hour. After confirming the completion of the reaction by thin layer chromatography, workup was performed according to the method described in the literature to obtain a crude product of thioacetic acid S-phenacyl ester (5.03 g, quantitative yield).

Process 2
The total amount of the product obtained in Step 1 (5.03 g) was reacted according to the method described in Journal of Organic Chemistry Vol. 51, No. 9, pages 1556 to 1562 (1986) to give the title compound (550 mg, 12% yield) was obtained.

Tablet (Compound 1)
A tablet having the following composition is prepared by a conventional method. 40 g of Compound 1 is mixed with 286.8 g of lactose and 60 g of corn starch, and 120 g of a 10% aqueous solution of hydroxypropylcellulose is added thereto. This mixture is kneaded by a conventional method, granulated and dried, and then sized to obtain granules for tableting. 1.2 g of magnesium stearate is added and mixed, and then tableted with a tableting machine (Clean Press Collect 12 manufactured by Kikusui Seisakusho) with a 8 mm diameter punch and contains 20 mg of active ingredient per tablet. )
Formulation Compound 1 20 mg
Lactose 143.4 mg
Corn starch 30 mg
Hydroxypropylcellulose 6 mg
Magnesium stearate 0.6 mg
200 mg

Injection (Compound 7)
An injection having the following composition is prepared by a conventional method. 1 g of compound 7 and 9 g of sodium chloride are dissolved in 1000 mL with distilled water for injection by a conventional method. The resulting solution is aseptically filtered using a 0.2 μm disposable membrane filter and then aseptically filled in 2 mL glass vials to give an injection (containing 2 mg of active ingredient per vial).
Formulation Compound 7 2 mg
Sodium chloride 18 mg
Distilled water for injection
2.00mL

It is a figure showing the intracellular HDAC inhibitory activity of compound 1 and TSA, and the expression effect | action of p21 molecule | numerator. The item represents the type of molecule (acetylated histones H3 and H4, p21 antibody), and the numerical value represents the test concentration. It is the figure which showed the effect | action with respect to the transcriptional repression by the leukemia causative gene AML1 / ETO fusion gene of compound 3 and TSA. The vertical axis of the graph represents the activity value at the time of AML1 / ETO introduction and in the presence of the drug, and the luciferase activity by Tww-tk-Luc induced by AML1 and PEBP2β was defined as 100%.

Explanation of symbols

+: Data obtained under conditions in which each corresponding gene (AML1, PEBP2b, AML1 / ETO) was introduced into the cell.
-: The data under the condition where each corresponding gene (AML1, PEBP2b, AML1 / ETO) is not introduced into the cell is shown. Moreover, the term “medicine” indicates data under a condition where no chemical treatment is performed.

Claims (7)

Formula (I)

[Wherein, R ¹ represents a hydrogen atom, substituted or unsubstituted lower acyl, CONHCH (R ³ ) CO ₂ R ⁴ (wherein R ³ represents a hydrogen atom or substituted or unsubstituted lower alkyl, R ⁴ represents Represents a hydrogen atom or lower alkyl), formula (T)

(Wherein, R ⁵ represents a hydrogen atom or a substituted or unsubstituted lower alkyl)
Or formula (U)

(Wherein R ⁶ represents lower alkyl, R ⁷ represents a substituted or unsubstituted aryl or a substituted or unsubstituted aromatic heterocyclic group),
R ² represents a hydrogen atom or a substituted or unsubstituted lower alkyl,
Ar represents substituted or unsubstituted aryl]. A histone deacetylase (HDAC) inhibitor containing a mercaptoacetophenone derivative represented by the formula: or a pharmaceutically acceptable salt thereof as an active ingredient. ^2. The HDAC inhibitor according to claim 1, wherein R ² is a hydrogen atom. ^3. The HDAC inhibitor according to claim ¹ , wherein R ¹ is CONHCH (R ³ ) CO ₂ R ⁴ , and R ³ is lower alkyl substituted with a phenyl group. The HDAC inhibitor according to any one of claims 1 to 3, wherein R ¹ is CONHCH (R ³ ) CO ₂ R ⁴ and R ⁴ is a hydrogen atom. R ¹ is the formula (T)

The HDAC inhibitor according to claim 1 or 2, wherein R ⁵ is a hydrogen atom or unsubstituted lower alkyl. R ¹ is the formula (U)

The HDAC inhibitor according to claim 1 or 2, wherein R ⁷ is a substituted or unsubstituted aromatic heterocyclic group. 3. The HDAC inhibitor according to claim ¹ , wherein R ¹ is a hydrogen atom or unsubstituted lower acyl.

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