JP2016017040A - Hydroxamic acid derivatives and hdac8 inhibitors - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide compounds that are highly active and can highly selectively inhibit HDAC8 functionality, and HDAC8 inhibitors.SOLUTION: Each hydroxamic acid derivative comprise a compound of the general formula (1) in the figure, where φ represents a benzene ring that may have substituents, or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof. These hydroxamic acid derivatives (1) can selectively inhibit HDAC8 enzyme activity.SELECTED DRAWING: None

Description

  The present invention relates to a hydroxamic acid derivative capable of selectively inhibiting the function of HDAC8 and an HDAC8 inhibitor using the same.

  Histones are proteins that fold DNA in eukaryotes to form a chromatin structure, and are chemically modified by the action of various enzymes, which are thought to change the chromatin structure and control gene expression. . In recent years, various knowledge about such epigenetic gene regulation has been discovered.

  Recently discovered Histone Deacetylase (hereinafter referred to as “HDAC”) catalyzes the reaction of deacetylating lysine residues of acetylated histones and controls the expression of many genes. When the ε-amino group of a histone lysine residue is acetylated, the histone positive charge is neutralized and the nucleosome structure relaxes. That is, a transcriptional regulatory factor is easily accessible to the promoter region, and as a result, transcription is activated. Conversely, when histone deacetylation is enhanced, the nucleosome structure is condensed and transcription is suppressed.

  Eleven isoforms from HDAC1 to HDAC11 are known for HDAC. According to recent research, inhibition of blood system cancer cells and induction of differentiation of neuroblastoma are possible by inhibiting HDAC8. (Non-Patent Document 1, Non-Patent Document 2).

  However, there are still many unclear points regarding the biological significance, such as how HDAC8 affects the living body. For this reason, if a substance that inhibits the catalytic action of HDAC8 (ie, an HDAC8 inhibitor) is found, it can be used as a bioprobe to investigate the function of HDAC8 or as a new type of anticancer agent. I can expect.

Examples of the compounds that inhibit HDAC8 reported so far include SAHA (Non-patent Document 3), PCI-34051 (Non-patent Document 1), and the like.
The present inventors have also exhaustively searched for compounds having HDAC8 inhibitory activity using the Huisgen reaction, and have found compounds that selectively inhibit HDAC8 (Patent Document 1).

The compounds reported so far with HDAC8 inhibitory activity are listed below. Among these, the compound 7 (abbreviation “NCC-149”) reported in Patent Document 1 has excellent inhibitory activity and selectivity for HDAC8.

WO2011 / 089995

Leukemia 2008, 22, 1026-1034 Clin Cancer Res 2009, 15, 91-99 Proc. Natl. Acad. Sci. USA 95, 3003-3007, 1998

  The present invention has been made in view of the above-described conventional circumstances, and a problem to be solved by providing a compound and an HDAC8 inhibitor that are highly active and can selectively inhibit the function of HDAC8. It is said.

  The present inventors analyzed the interaction between HDAC8 and NCC149 using computational chemistry software Glide-Macromodel. As a result, it was suggested that the π-electron of the triazole ring present in NCC149 interacts with the benzylic hydrogen of Phe152 of HDAC8, which is deeply related to inhibitory activity and selectivity. (See FIG. 1). Therefore, various compounds having other cyclic structures having a π-electron conjugated system were synthesized in place of the triazole ring moiety in NCC149, and the inhibitory properties against HDAC8 were investigated. As a result, a new compound that selectively exhibits HDAC8 inhibitory activity was found and the present invention was completed.

That is, the hydroxamic acid derivative of the present invention is characterized by comprising the following general formula (1) or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof.

  The hydroxamic acid derivative (1) of the present invention can selectively inhibit the enzyme activity of HDAC8. For this reason, it can be suitably used as a biological tool for examining the function of HDAC8. It also has cancer cell growth inhibitory action and is expected to be used as an anticancer agent. The prodrug refers to a compound that is hydrolyzed in vivo to regenerate the hydroxamic acid derivative (1).

  φ may be an unsubstituted benzene ring. The inventors have confirmed that when φ is an unsubstituted benzene ring, it is surely highly active and highly selective as an HDAC8 inhibitor. In particular, a compound in which the triazole ring is bonded to carbon in the opposite direction to NCC149 and the other structural parts are the same as NCC149 was more active and more selective than NCC149 as an HDAC8 inhibitor.

It is a figure which shows the interaction between the pi electrons of NCC149 and HDAC analyzed using the computational chemistry software Glide-Macromodel. It is a photograph which shows the result of the electrophoresis in Western blot analysis. It is a figure which shows the principle of an MTS assay.

<Molecular design>
As described above, the triazole ring of NCC149 is considered to interact with the benzylic hydrogen of Phe152 of HDAC8 (see FIG. 1). Therefore, Compound 8 in which the triazole ring of NCC149 is converted into a 6-membered benzene ring, Compounds 9, 10, and 12 in which the 5-membered ring is the same as the triazole ring and is converted to a heterocycle having a different electron density, and the triazole ring are NCC149 Compound 11 was synthesized in which it was bonded to carbon in the opposite direction and the other structural parts were the same as NCC149 (see the structural formula below). The synthesis method is described in detail below.

Measuring method Melting points were measured using a willow moto micro melting point measuring apparatus or a Buehi 545 melting point measuring apparatus. ^{For 1} H-NMR and ¹³ C NMR, JEOL JNM-LA500 or JEOL JNM-A500 was used. Chemical shift (δ) was measured using TMS as an internal standard. Elemental analysis was performed using a Yanaco CHN CORDER NT-5 analyzer. For ElMS analysis, a JEOL JMS-SX102A mass spectrometer was used. FTIR was measured using Shimadzu FTIR84005. Reagents and solvents purchased from Aldrich, Tokyo Chemical Industry, Wako Pure Chemical Industries, and Kanto Chemical were used as they were without purification. Flash column chromatography was performed using Merk silica gel 60 (particle size: 0.046-0.063 mm).

Example 1
In Example 1, compound 8 was prepared by the following synthetic route. Details will be described below.

・ Synthesis of Compound 15
DMF (7 mL) of 3-iodobenzyl alcohol 13 (2.72 g, 11.6 mmol) and 3- (methoxycarbonyl) phenylboronic acid (14) (2.009, 11.1 mmol) and Pd (OAc) ₂ (56.2 ng, 0.250 mmol) ) 1M Na ₂ CO ₃ (34 mL) aqueous solution was added to the solution and heated at 55 ° C. for 1.5 hours. The reaction solution was diluted with chloroform and filtered. The chloroform layer was separated, washed with water and brine, and dried over anhydrous sodium sulfate. Further, the residue was concentrated in vacuo and purified by silica gel flash column (eluent: 0-80% ethyl acetate / n-hexane) to obtain 3.85 g of compound 15 as a light brown oil.
^l H NMR (500 MHz, CDCl ₃ ): δ = 8.27 (t, J = 1.7 Hz, 1H), 8.00 (dt, J = 1.5, 7.5 Hz, 1H), 7.78 (dt, J = 1.6, 7.8 Hz, 1H), 7.63 (5, 1H) .7.57-7.48 (m, 2H) .7.43 (t, J = 7.7 Hz, 1H), 7.37 (d, J = 7.0 Hz, 1H), 4.76 (d, J = 6.0 Hz, 2H) .3.94 ppm (s, 3H)
ElMS: m / z 242 [M ⁺ ].

- In a nitrogen gas stream under 0 ℃ compound _{16, PBr 3 (0.6 mL,} 6.38 mmol) is added dropwise alcohol 15 (3.85 g, 15.9mmol) in 10ml of anhydrous dioxane. The reaction solution is stirred at 0 ° C. for 1.5 hours and then poured into a mixture of ice and saturated sodium bicarbonate solution. The reaction solution was extracted with ethyl ether, washed with water and brine, and then dried over anhydrous sodium sulfate. Concentration in vacuo gave 4.01 g (83% yield) of compound 16 as a yellow oil.
^l H NMR (500 MHz, CDCl3): δ = 8.26 (s, 1H), 8.02 (d,) = 7.5 Hz, 1H), 7.76 (d, J = 8.0 Hz, 1H), 7.63 (5,1H), 7.56-7.47 (111,2 H), 7.44-7.38 (m, 2H), 4.55 (s, 2H), 3.94 ppm (5,3H); ElMS: m / z 304, 306 [M ⁺ ].

-Synthesis of Compound 17 An aqueous solution (15 mL) of sodium thiophenol (2.07 g, 15.7 mmol) and triethylamine (5 mL) were added to a solution of Compound 16 (3.70 g, 12.1 mmol) in acetone (35 mL). The mixture was stirred at room temperature for 19.5 hours. After the solvent was distilled off, the residue was poured into water and extracted with chloroform. The organic layer was washed with water and brine and then dried over anhydrous sodium sulfate. Then, it was concentrated in vacuo and purified by a silica gel flash column (eluent: 0-30% ethyl acetate / n-hexane) to obtain 3.85 g of compound 172.02 g (yield 50%) as a colorless oil.
^l H NMR (500 MHz, CDCl ₃ : δ = 8.20 (t, J = 1.7 Hz, 1H), 7.99 (dt, J = 1.4, 7.7 Hz, 1H), 7.68 (dt, J = 1.2, 7.2 Hz, 1H ), 7.47-7.44 trn, 3H), 7.35-7.18 (rn, 7H), 4.14 (s, 2H), 3.92 ppm (s, 3H); ElMS: m / z 334 [M ⁺ ].

Synthesis of compound 18Methyl ester 17 (2.02 g, 6.04 mmol) was dissolved in a mixed solvent of methanol (20 mL), THF (10 mL) and CHCl ₃ (10 mL), and an aqueous sodium hydroxide solution (2N.12 mL, 24.0 mmol) was poured. The reaction solution was stirred at room temperature for 5 hours. After removing the solvent, the residue was poured into 2N aqueous sodium hydroxide solution and extracted with chloroform. The organic layer was washed with water and brine, and then dried over anhydrous sodium sulfate. It was then concentrated in vacuo and purified on a silica gel flash column (elution was 0-40% ethyl acetate / n-hexane followed by 0-40% methanol / CHCl ₃ ) 957 mg of compound 18 as a white solid (yield 50%).
^I H NMR (500 MHz, CDCl ₃ ): δ = 8.26 (5,1 H), 8.07 (d, J = 7.5 Hz, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.53-7.21 (m , 10H), 4.18 ppm (s, 2H); ElMS: m / z 320 [M ⁺ ] ..

- Compound 18 Compound 19 (0.957 g, 2.99 mmol) , EDCl · HCl (1.70 g, 8.85 mmol) and _{HOBt · H 2 O (1.39 g} , 9.08 mmol) DMF (25 mL) of the solution to NH ₂ OTHP ( 1.00 g, 8.55 mmol). The mixture is stirred at room temperature for 30 hours, then poured into a saturated aqueous sodium bicarbonate solution and extracted with ethyl ether. The organic layer was washed with water and brine and then dried over anhydrous sodium sulfate. Then, it was concentrated in vacuo and purified by a silica gel flash column (eluent: 10-50% ethyl acetate / n-hexane) to obtain 1.06 g (yield 84%) of Compound 19 as a pale yellow amorphous solid.
^l H NMR (500 MHz, CDCl ₃ ): δ = 8.90 (s, 1H), 7.88 (s, 1H), 7.70 (d, J = 7.5 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H) , 7.497.45 (m, 3H), 7.37-7.27 (m, 6H), 7.21 (m, 1H), 5.11 (s, 1H), 4.16 (s, 2H), 4.02 (t, J = 10.2 Hz, 1 H), 3.68-3.66 (m, 1 H), 1.91-1.86 (m, 3H) 1.87-1.59 ppm (m, 3H); ElMS: m / z 419 [M ⁺ ].

- Compound 19 (264 mg, 0.630 mmol) of the compound 8 methanol (30 mL) was added _{TsOH · H 2 0 (12.0 mg} , 63μmol) and stirred 5.5 hours at room temperature was added the mixture. The solid obtained by removing the solvent was suspended in ethyl acetate and filtered to obtain 85.1 mg (yield 40%) of Compound 8 as a white solid. Further, this was recrystallized in THF to obtain 51 mg of colorless crystals of Compound 8.
mp: 167-168 ° C; ^l H NMR (500 MHz, [D ₆ ] DMSO): δ = 11.33 (s, 1H), 9.12 (s, 1 H), 7.98 (s, 1H), 7.76-7.73 (m , 2 H), 7.69 (s, 1H), 7.59-7.53 (m, 2 H), 7.44-7.37 (m, 4H), 7.31 (t, J = 7.7 Hz, 2H), 7.19 (t, J = 7.2 Hz, 1H), 4.33 ppm (s, 2H); ¹³ C NMR (125 MHz, [D ₆ ] DMSO): δ = 164.1, 140.0, 139.5, 138.4, 136.0, 133.5, 129.3, 129.2, 129.1, 129.0, 128.5 , 128.3,127.4, 126.1, 126.0, 125.6, 125.1, 36.7 ppm; ElMS: m / z 335 [M ⁺ ]; elemental analysis calculated as C ₂₀ H ₁₇ NO ₂ S ・ 0.5H ₂ O: C 69.74, H 5.27, N 4.07, found: C69.72, H 5.12, N 4.17.

(Example 2)
In Example 2, compound 9 was prepared by the following synthetic route. Details will be described below.

・ Synthesis of compound 21
Concentrated sulfuric acid (4 mL) was added to a solution of 3-acetylbenzoic acid 20 (2.02 g, 12.3 mmol) in methanol (60 mL), and the mixture was refluxed with stirring for 26.5 hours. After removing the solvent, the residue was poured into water and extracted with ethyl ether. The organic layer was washed with water and brine, and then dried over anhydrous sodium sulfate. The filtrate was concentrated in vacuo to obtain 2.12 g (yield 97%) of compound 21 as a white solid.
¹ H NMR (500 MHz, CDCl ₃ ): δ = 8.60 (s, 1H), 8.24 (t, J = 7.5 Hz, 1H), 8.17 (d, J = 7.5 Hz, 1H), 7.57 (t, J = 7.7 Hz, 1 H), 3.96 (s, 3 H), 2.66 ppm (s, 3H); ElMS: m / z 178 [M ⁺ ].

- Compound 21 (1.11 g, 6.24 mmol) of compound 22 as a 0 ℃ chloroform (5 mL) solution _{of, Br 2 (0.3 mL, 5.82} mmol) is added 25% HBr-AcOH in a catalytic amount. The reaction solution is stirred at room temperature for 2 hours, poured into a saturated aqueous sodium hydrogen carbonate solution and extracted with chloroform. The organic layer was washed with water and brine and then dried over anhydrous sodium sulfate. Then, it was filtered and concentrated in vacuo to obtain 1.48 g of compound 22 as a white solid.
ElMS: m / z 256, 258 [M ⁺ ].

Synthesis of Compound 24 A solution of phenylthioacetic acid 23 (3.01 g, 17.9 mmol) and oxalyl chloride (4 mL, 46.6 mmol) in dry THF (20 mL) is brought to 0 ° C., and a catalytic amount of DMF is added. After the mixture was stirred at 0 ° C. for 0.5 h, the solvent was evaporated under reduced pressure to give the corresponding acid chloride as a yellow solid. The acid chloride in THF (40 ml) is brought to 0 ° C., 25% aqueous ammonia (20 mL) is added, and after 1.5 hours, the reaction solution is poured into saturated aqueous sodium bicarbonate and extracted with ethyl acetate. The organic layer was washed with water and brine and then dried over anhydrous sodium sulfate. Then, it was filtered and concentrated in vacuo to obtain 2.46 g (yield 83%) of amide 24 as a white solid.
^l H NMR (500 MHz, CDCl ₃ ): δ = 7.34-7.30 (m, 3H), 7.25-7.21 (m, 2H), 6.69 (broad s, 1H), 5.45 (broad s, 1H), 3.63 ppm ( s, 2H); ElMS: m / z 167 [M ⁺ ].

-Synthesis of Compound 25 Lawson's reagent ((2.84 g, 7.02 mmol) was added to a dry toluene (100 mL) solution of amide 24 (2.46 g, 14.7 mmol) under a nitrogen stream, and the mixture was stirred at 80 ° C for 3.5 hours. After the solvent was distilled off, the residue was purified by a silica gel flash column (eluent: 15-50% ethyl acetate / n-hexane) to obtain 554 mg (yield 21%) of compound 25 as a crude white solid. : m / z 183 [M ⁺ ]

Synthesis of compound 26 Ethanol (10 mL) and chloroform (10 mL) mixed with compound 22 (1.52 g, 8.31 mmol) and 3 angstrom activated molecular sieve in ethanol (10 mL) solution of thioamide 25 (555 mg, 2.16 mmol) Into the solution under a stream of nitrogen. The mixture is stirred at 70 ° C. for 3 hours and filtered, and then the solvent is distilled off. The residue is poured into a saturated aqueous sodium bicarbonate solution and extracted with ethyl acetate. The organic layer was washed with water and brine and then dried over anhydrous sodium sulfate. The residue was purified by a silica gel flash column (eluent: 0-30% ethyl acetate / n-hexane) to obtain 636 mg (yield 78%) of Compound 26 as a white solid.
'H NMR (500MHz, CDCl ₃ ): δ = 8.45 (s, 2H), 8.11 (d, J = 7.5 Hz, 1H), 7.63-7.61 (m, 2H), 7.48 (d, J = 7.5 Hz, 2 H), 7.34 (t, J = 7.0 Hz, 2 H), 7.29-7.28 (m, 1H), 6.92 (s, 1H), 4.9 "(s, 2H), 3.97 ppm (s, 3H); ElMS: m / z 341 [M ⁺ ].

Synthesis of Compound 9 Compound 29 was synthesized from Compound 26 as a starting material by the same method as the synthesis method of Compound 8.
mp: 56-60 ° C; ^l H NMR (500 MHz, CD ₃ OD): δ = 8.25 (s, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.711 (s, 1H), 7.69 (d , J = 7.5 Hz, 1H), 7.50 (t, J = 7.7 Hz, lH), 7.41 (d, J = 8.0Hz, 2H), 7.28 (t, J = 7.5Hz, 2H), 7.21 (t, J = 8.0 Hz, 1H), 4.53 ppm (s, 2 H);
¹³ C NMR (125 MHz, CD ₃ OD): δ = 171.1, 168.0, 1 ~ .5.2, 136.1, 135.9, 134.2, 131.3, 130.4, 130.2, 130.1,128.2, 127.6, l: ~ 6.0, 116.3, 36.5 ppm ;
ElMS: m / z 343 [M ⁺ ];
Elemental analysis _{C 7 H 14 N 2 O 2} S 2 · H 2 O Calculated C56.65, H4.47, N7.77, Found C56.65, H4.42, N7.76.

(Example 3)
In Example 3, compound 10 was prepared by the following synthetic route. Details will be described below.

Synthesis of compound 28
A mixture of NH ₂ OH · HO (3.70 g, 53.3 mmol) and KOH (3.00 g, 53.5 mmol) is stirred in methanol (10 mL) for 30 minutes at room temperature. A solution of phenylthioacetonitrile 27 (2.00 g, 13.4 mmol) in methanol (14 mL) is added, and the mixture is further stirred at room temperature for 20 hours. After the mixture was filtered, the solvent was distilled off under reduced pressure, and the residue was purified by a silica gel flash column (eluent: 30-50% ethyl acetate / n-hexane). 43%).
'H NMR (500MHz, [D ₆ DMSO]: d = 9.18 (5, 1H), 7.39 (d, J = 8.5 Hz, 2H), 7.2g (t, J = 7.7 Hz, 2H), 7.17 (t, J = 6.7 Hz, 1H), 5.50 (s, 2H), 3.56 ppm (s, 2H); ElMS: m / z 182 [M ⁺ ].

-Synthesis of Compound 29 A solution of carbonyldiimidazole (CDI) (0.871 g, 5.37 mmol) in DMF (3 mL) is added to a solution of monomethyl isophthalate (0.937 g, 5.26 mmol) in DMF (4 mL). The mixture is stirred at room temperature for 30 minutes, then a solution of compound 28 (0.979 g, 5.37 mmol) in DMF (7 mL) is added, and the mixture is stirred overnight at room temperature. Heat the mixture in a microwave oven (140 ° C, 450 W for 2 minutes). The mixture is poured into water and extracted with ethyl ether. The organic layer was washed with water and brine and then dried over anhydrous sodium sulfate. Then, it was filtered, concentrated under reduced pressure, and purified by silica gel flash column (eluent was 5-50% ethyl acetate / n-hexane) to obtain 557 mg (yield 33%) of compound 29 as a yellow solid. .
¹ H NMR (500 MHz, CDCl ₃ ): δ = 8.79 (s, 1H), 8.30 (d, J '= 7.5Hz, 1H), 8.26 (d, J = 7.0Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.41.5 (d, J = 7.5 Hz, 2H), 7.31 (t, J = 7.7 Hz, 2H), 7.267.24 (m, 1H), 4.22 (s, 2H), 3.97 ppm (s, 3H); ElMS: m / z 326 [M ⁺ ].

Synthesis of Compound 10 Compound 10 was obtained by the same method as the synthesis method of Compound 8 using Compound 29 as a starting material (yield 17%)
mp: 157-158 ° C: ¹ HNMR (500 MHz, CDCl ₃ ): δ = 8.48 (t, J = 1.5 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H), 8.01 (dt, J = 1.5, 8.0 Hz, 1H), 7.69 (t, J = 8.0 Hz. 1H), 7.44 (d, J = 7.0 Hz, 2H), 7.30 (t, 1 = 7.5 Hz, 2H), 7.23 (t, J = 7.5 Hz, 1H), 4.26 ppm (s, 2 H);
¹³ C NMR (125 MHz, CD ₃ OD): δ = 176.6, 170.3, 166.6, 135.8, 135.0, 132.5, 131.9, 131.8, 130.9, 130.2, 128.3, 127.7, 125.7, 29.8 ppm; ElMS: m / z 327 [ M ⁺ ];
Elemental analysis Calculated as C ₁₆ H ₁₃ N ₃ O ₃ S: C 58.70, H 4.00, N 12.84, measured C58.31, H 4.14, N 12.53.

Example 4
In Example 4, Compound 11 was prepared according to the following synthetic route. Details will be described below.

Synthesis of compound 31
An aqueous solution (30 mL) of NaNO ₂ (3.69 g, 53.4 mmol) is added at 0 ° C. to a solution of ethyl 3-aminobenzoate 30 (2.02 g, 12.2 mmol) in TFA (17 mL). Then, an aqueous solution of sodium azide (4.05 g, 62.3 mmol) is added and stirred at room temperature for 2.5 hours. The reaction solution is poured into 2N hydrochloric acid and extracted with ethyl ether. The organic layer was washed with water and brine and then dried over anhydrous sodium sulfate. Then, it was filtered, concentrated under reduced pressure, and purified by silica gel flash column (eluent was 2-20% ethyl acetate / n-hexane) to obtain 2.20 g (yield 94%) of Compound 31 as a yellow oil. .
¹ H NMR (500 MHz, CDCl ₃ ): δ = 7.82 (dt, J = 1.0, 8.5 Hz. 1H), 7.70 (t, 1 = 1.7 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H) , 7.42 (t, J = 8.0 Hz, 1H), 7.19 (ddd, J = 0.9, 2.4, 6.4 Hz, 1H), 4.39 (q, J = 7.3 Hz, 2 H), 1.40 ppm (t, 1 = 7.0 Hz, 3H); FTIR (neat): ν = 2102 cm ^-1

Synthesis of Compound 32 An aqueous solution (10 mL) of copper sulfate (83.7 mg, 523 μmol) and sodium ascorbate (0.585 g, 2.95 mmol) was added to phenylpropargyl sulfide (0.775 g, 5.23 mmol) and azide 31 (0.980 g, 5.12). mmol) in methanol (10 mL). The reaction mixture was stirred at room temperature for 21 hours, the mixture was filtered through Celite, the solvent was evaporated, and the residue was purified by silica gel flash column (eluent: 5-40% ethyl acetate / n-hexane). As a solid, 1.17 g (yield 67%) of compound 32 was obtained.
¹ H NMR (500 MHz, CDCl ₃ ): δ = 8.27 (t, 1 = 1.7 Hz, 1H), 8.10 (dt, 1 = 1.2, 8.0 Hz, 1H), 7.95 (ddd, 1 = 1.0, 2.2, 6.2 Hz, 1H), 7.84 (s, 1H), 7.60 (t, J = 8.0 Hz, 1H), 7.38 (d, J = 7.5 Hz, 2 H), 7.30 (t. 1 = 7.7 Hz, 2 H), 7.22 (t, J = 7.5 Hz, 1H), 4.43 (q, J = 7.3 Hz, 2H), 4.33 (s, 2H), 1.43 ppm (t, 1 = 7.2 Hz, 3H); ElMS: m / z 339 [M ⁺ ].

Synthesis of Compound 11 Compound 11 was obtained by the same method as the synthesis method of Compound 8 using Compound 32 as a raw material (yield 48%).
mp: 179-180 ° C: ¹ H NMR (500 MHz, [D6] DMSO): δ = 11.40 (s, 1H), 9.22 (s, 1H), 8.74 (5, 1H), 8.22 (s, 1H) , 8.01 (d, 1 = 8.0 Hz.1H), 7.84 (d, J = 7.5Hz, lH), 7.67 (d, J = 8.0Hz, 1H), 7.41 (d, J = 8.0 Hz, 2H), 7.33 (t, 1 = 7.7 Hz, 2 H), 7.20 (t, 1 = 7.2 Hz, 1H), 4.39 ppm (s, 2H);
¹³ CNMR (125 MHz, [D6] DMSO): δ = 162.8, 144.9, 136.4,135.5, 134.3, 130.1, 129.0, 128.2, 126.8, 126.0, 122.4, 121.5, 118.3, 27.1 ppm; ElMS: m / z 326 [ M ⁺ ]; Elemental analysis Calculated as C ₁₆ H ₁₄ N ₄ O ₂ S: C58.88, H 4.32, N 17.17, Found: C 58.69, H 4.54, N 17.00.

(Example 5)
In Example 5, compound 12 was prepared by the following synthetic route. Details will be described below.

Synthesis of Compound 34 Compound 34 was synthesized by the same method as the synthesis method of Compound 15 using 3- (methoxycarbonyl) phenylboronic acid 14 and 2-iodo-3-methylthiophene 33 as raw materials.
¹ H NMR (500 MHz, CDCl ₃ ): δ = 8.22 (s, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.72 (d, 1 = 7.5 Hz, 1H), 7.42 (t, 1 = 7.7 Hz, 1H), 7.18 (d, J = 3.5 Hz, 1H), 6.756.74 (m, 1H), 3.94 (s, 3H), 2.52 ppm (s, 3H); ElMS: m / z 232 [M ⁺ ]).

・ Synthesis of Compound 35 NBS (0.554 g, 3.11 mmol) and AIBN (19.2 mg, 0.116 mmol) were added to a chloroform solution (20 mL) of Compound 34 (0.601 g, 2.61 mmol), and this mixture was refluxed with 1 Stir for hours. And it diluted with carbon tetrachloride and filtered. The filtrate was evaporated, poured into water and extracted with ethyl acetate. The organic layer was collected, washed with water and brine, and dried over anhydrous sodium sulfate. Then, it was filtered and concentrated in vacuo to obtain 811 mg of compound 35 as a brown oil.
¹ H NMR (500 MHz, CDCL3): δ = 8.24 (s, 1H), 7.96 (dt, J = 1.4, 7.7 Hz, 1H), 7.74 (ddd, .f = 1.2, 2.0, 6.1 Hz, 1H), 7.21 (d, 1 = 4.0 Hz, 1H), 7.10 (d, 1 = 3.5 Hz, 1;), 4.75 (s, 2H), 3.95 ppm (s, 3H); ElMS: m / z 310,312 [M ⁺ ] .

-Synthesis | combination of compound 12 Compound 12 was synthesize | combined by the method similar to the method of synthesize | combining compound 8 using compound 35 as a raw material (36% of yield).
mp154-155 ° C: ¹ H NMR (500 MHz, [D ₆ ] DMSO): δ = 11.32 (s, 1H), 7.92 (s, 1H), 7.72 (d, J = 7.5 Hz, 1H), 7.65 ( d, 1 = 8.5 Hz, 1H), 7.47 (t, J = 7.5 Hz, l H), 7.40-7.36 (rn, 3H), 7.32 o. J = 7.7Hz, 2H), 7.21 (t, 1 = 7.2 Hz, 1H), 7. (11 (d, J = 3.5 Hz, 1H), 4.51 ppm (s, 2H); ¹³ C NMR (125 MHz, CDCl ₃ ) δ = 163.6, 141.5, 141.5, 135.2, 133.7, 133.4, 129.2,128.9, 128.6, 1-7.9, 127.5, 126.2, 125.8, 123.7, 123.2, 31.6 ppm;
ElMS: m / z 341 [M ⁺ ]; elemental analysis C ₁₈ H ₁₅ NO ₂ S ₂ 0.5H ₂ 0: Calculated as C 61.19, H
4.60, N 4.00, found: C 61.77, H 4.76, N 4.10.

-Evaluation-
<Evaluation by fluorescent enzyme assay>
About the compound 8-12 of Examples 1-5 synthesize | combined as mentioned above, the inhibitory effect with respect to various HDACs (HDACS, HDAC1, HDAC2, HDAC3, HDAC4, HDAC6, and HDAC8) was investigated by the fluorescent enzyme assay, and each HDAC isozyme IC ₅₀ was sought. As Comparative Example 1, the same measurement was performed for Compound 7 (NCC149). Furthermore, as Comparative Example 2, the same measurement was performed for vorinostat (trade name: ZOLINZA, former name: SAHA), which was approved and approved for cutaneous T cell lymphoma in FAD.
In the fluorescent enzyme assay, an acetylated lysine analog labeled with a fluorescent substance was used as a substrate for HDAC. When HDAC deacetylates the fluorescently labeled substrate, lysine endopeptidase (LEP) recognizes the deacetylated lysine and hydrolyzes the amide bond behind lysine, releasing the fluorescent substance AMC. . The enzyme activity is evaluated by measuring the fluorescence intensity of the released AMC and calculating the ratio of control to the fluorescence intensity.
HDAC activity was measured using HDACs / HDAC8 deacetylase fluorescence analysis kit (CY-1150 / CY-1158, Cyclex Compary), (HDAC1 / HDAC6 fluorescence activity drug discoverly kit (AK-511 I AK-516, BIOMOL Research). Laboratories), Fluorescent SIRT1 Activity Assay / Drug Discovery (AK-555, BIOMOL Laboratories)), or Fluorogenic HDAC Class2α Assay Klt (BPS Bioscience), Total HDACs (CY-1150, Cyde), HDAC1 (SE456, BIOMOL Research) Laboratories), HDAC2 (SE-500, BIOMOL Research Laboratories), HDAC3 / NCOR1 complwx (SE-515, BIOMOL Research Laboratories), HDAC4 (BPS Bioscience), HDAC6 (SE508, BIOMOL Laboratories) and HDAC8 (CY-1158, Cydex Company Limited) and measured according to the manufacturer's instructions. In the fluorescence measurement, the excitation wavelength was 160 nm, and the detection wavelength was 460 nm. The concentration at 50% inhibitory activity (IC ₅₀ ) was determined from a graph of log vs. inhibitory activity at each concentration. The acetylation activity of Cohesin, a-tubulin and H3K9 was measured according to a conventional method. Each measurement was performed at least twice. The results are shown in Table 1.

As shown in Table 1, Compound 7 (NCC149) of Comparative Example 2 showed superior inhibitory activity and superior inhibitory selectivity compared to vorinostat of Comparative Example 1, which is widely used as an HDAC8 inhibitor (in HDAC8) whereas a IC ₅₀ = 0.070μM, HDACs in IC ₅₀ = 54μM, HDAC1 in IC ₅₀ = 38μM, HDAC2 the IC _50> 100 [mu] M, in HDAC3 68μM, the HDAC4 IC ₅₀ = 44μM, the HDAC6 IC ₅₀ = 2.4 μM).
Further, Compound 8 of Example 1 in which the triazole ring in Compound 7 (NCC149) of Comparative Example 2 is replaced with a benzene ring is 1/20 of HDAC8 inhibitory activity of Comparative Example 1, but selectively inhibits HDAC8 activity. I found out that
On the other hand, compounds 9 to 12 of Examples 2 to 5 showed excellent HDAC8 inhibitory activity. From these results, the 5-membered ring possessed by the compounds 9 to 12 of Examples 2 to 5 works favorably for binding to the Cap site of HDAC8, which contributes to excellent inhibitory activity and excellent selectivity. It was suggested that
In particular, compound 11 in Example 4 was found to exhibit greater HDAC8 inhibitory activity than NCC149 (7) (compound 11 had an IC ₅₀ = 0.053 μM, whereas compound 7 had an IC ₅₀ = 0.070).
Furthermore, compounds 9-12 of Examples 2-5 were found to selectively inhibit HDAC8 activity. In particular, the compound 11 of Example 4 was more selective for HDAC8 inhibitory activity than NCC149 (7) (11: HDAC1 ICsolHDAC8 ICso 1900; HDAC4 IC50 HDAC8 IC50 1900; HDAC6 ICsolHDAC8 IC50 = 42. 7: HDAC1 ICsolHDAC8 ICso = 540; HDAC4 ICso / HDAC8 ICso = 630; HDAC6 ICsolHDAC8 IC50 = 34). These results suggested that small geometric differences in heteroaryl rings with 5-membered rings affect not only HDAC8 inhibitory activity but also HDAC8 selectivity.
From the above results, it was found that the compound 11 of Example 4 was more potent and selective than the compound 7 (NCC149) of Comparative Example 1 as an HDAC8 inhibitor.

<Evaluation of intracellular HDAC8 inhibition>
In order to examine the selectivity of HDAC8 inhibition in cells, the compounds 9 to 11 of Examples 2 to 4 and the compound 7 of Comparative Example 1 were evaluated by Western blot analysis. HDAC8 is a cohesin deacetylase, and its inhibitory effect on HDAC8 was evaluated by measuring the amount of cohesin acetylated in Healer cells.
As shown in FIG. 2, the compounds 9 to 11 of Examples 2 to 4 and the compound 7 of Comparative Example 1 all induced an increase in acetylated cohesin and showed a strong HDAC8 inhibitory action in cells. While trichostatin A, a representative pan-HDAC inhibitor, has been reported to significantly increase acetylated H3K9 in Healer cells, compounds 9-11 of Examples 2-4 and compound 7 of Comparative Example 1 are H3K9 (ie, It did not promote acetylation of nuclear HDACs (eg, major substrates of HDAC1 and HDAC2). For acetylated α-tubulln and HDAC6, Compound 7 of Comparative Example 1 and Compounds 10 and 11 of Examples 3 and 4 promoted α-tubulln induction at a concentration of 10 μM or more. Α-tubulln was not induced. From these results, it was found that Compound 9 of Example 2 selectively prevents HDAC8 over other HDACs contained in cells.

<Proliferation inhibition test against T cell tumor cell line>
PCI-34051, known as an HDAC8 selective inhibitor, has been reported to show cell growth inhibitory activity against T cell tumors. Therefore, a growth inhibition test (MTS assay) on T cell tumor cell lines was performed for compounds 9 to 11 of Examples 2 to 4 and compound 7 of Comparative Example 1.

(MTS assay procedure)
([3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt]) is dehydrated in intracellular mitochondria Oxidation enzyme (NADH) and electron carrier PMS (phenazine methosulfate) coexist to cause an oxidation-reduction reaction, which is reduced to formazan (see FIG. 3). Since NADH is an enzyme related to the respiratory chain, the amount of Formazan resulting from this reaction corresponds to the number of viable cells. The number of cells was quantified by colorimetric determination of this amount of Formazan, and GI ₅₀ was calculated.

(Result)
The results of the MTS assay are shown in Table 2. Compound 9 and Compound 10 of Example 2 and Example 3 showed stronger cell growth inhibitory action against Jurkat, HH, MT4, and HeLa cells than Compound 7 (NCC149) of Comparative Example 1. Moreover, the compound 9 of Example 2 showed the cell growth inhibitory effect equivalent to the compound 7 (NCC149) of the comparative example 1 also in K562 and HUT78 cell. On the other hand, the isozyme selectivity to HDAC8 was improved, and the compound 10 of Example 3 had a cell growth inhibitory action lower than that of Compound 7 (NCC149) of Comparative Example 1. Moreover, the compound 9 and the compound 10 of Example 2 and Example 3 showed stronger cell growth inhibitory action than PCI-34051 which is known as the most excellent HDAC8 selective inhibitor to date.

<Evaluation with DATT, Daudi and A549 by MTT assay>
DATT (human medulloblastoma cell line), Daudi (malignant lymphoma cell line), and A549 (human alveolar basal epithelial adenocarcinoma cell line) were also subjected to the MTT assay by the same method as the MTS assay described above. The assay was performed at least three times for each compound.

(Result)
The results of the MTT assay are shown in Table 3. Compound 9 of Example 3 showed stronger cell growth inhibitory action than Compound 7 (NCC149) of Comparative Example 1 on all DAOY, Daudi and A549 cells. Moreover, the compound 10 of Example 4 showed the stronger cell growth inhibitory effect about Daudi and A549 than the compound 7 (NCC149) of the comparative example 1. Furthermore, the compound 11 of Example 5 showed the cell growth inhibitory effect comparable to the compound 7 (NCC149) of the comparative example 1 with respect to all the cells of DAOY, Daudi, and A549.

  It can be suitably used as a biological tool for examining the function of HDAC8.

Claims (3)

A hydroxamic acid derivative comprising the following general formula (1) or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof:
  The hydroxamic acid derivative according to claim 1, wherein φ is an unsubstituted benzene ring.   An HDAC8 inhibitor comprising the hydroxamic acid derivative according to claim 1 or 2 as an active ingredient.