KR20090033634A - Hsp90 inhibitors containing 3h-quinazolin-4-one derivatives and anti-cancer drugs using the hsp90 inhibitors - Google Patents

Abstract

An Hsp90(heat shock protein 90) inhibitor containing 3h-quinazolin-4-yl derivatives is provided to show pharmaceutically improved properties and effects for anti-cancer drugs. An Hsp90(heat shock protein 90) inhibitor comprises a compound represented by the formula 1 or pharmaceutically allowable salt thereof. In the formula 1, X1 is H and Y1 is one of 2-(4-chlorophenyl) ethynyl, 2-amino-1-(3-chloro-4-methyl phenyl)-4,5-dihydro-4-oxo-1H-pyrrol-3-yl, [2-[(2-furanylmethyl)amino]-2-oxoethyl]thio, [2-[(4-acetylphenyl)amino]-2-oxoethyl]thio or [[4-(methoxycarbonyl)phenyl]methyl]thio; X1 is methyl and Y1 is 2-(5-methoxy-1H-indol-3-yl)ethenyl; or X1 is 2-hydroxyethyl and Y1 is (dimethylamino)phenyl]ethenyl.

Description

Hsp90 inhibitors containing 3H-quinazolin-4-one derivatives and anticancer agents using the same Hs90 inhibitors containing 3H-quinazolin-4-one derivatives and anti-cancer drugs using the Hsp90 inhibitors

The present invention relates to an Hsp90 inhibitor and an anticancer agent using the same, and more particularly, to an Hsp90 inhibitor containing a derivative of 3H-quinazolin-4-one and an anticancer agent using the same.

Hsp90 is one of the most common molecular chaperones in eukaryotic cells and plays an important role in the folding, stabilization, activation and formation of protein complexes in metastable proteins called client proteins. Hsp90 client proteins include tumor-causing proteins such as Her2 / ErbB2, Akt, Raf-1, Hif-1α, hormone receptors, survivin, p53 mutations and hTERT.

Inhibition of Hsp90 induces the degradation of the tumor-inducing client proteins via proteasomes, which is why Hsp90 is emerging as a new target for anticancer drug development.

An example of a patent for Hsp90 inhibitors is described in Korean Patent Publication No. 10-2007-0045290 (published May 02, 2007). Related to imidazolone compounds and their salts are pharmaceutical preparations comprising benzoimidazolone compounds, novel benzoimidazolone compounds and novel methods for preparing benzoimidazolone compounds.

On the other hand, morphological changes of Hsp90 by ATP binding and hydrolysis in the N-terminal ATPase domain are essential for the chaperone function of Hsp90. Most of the known Hsp90 inhibitors inhibit Hsp90 ATPase activity by binding to N-terminal ATP binding sites. Hsp90 inhibitors of this kind include geldanamycin (GA), radicicol, and 17-allylamino-17-demethoxygeldanamycin derived from GA (17-allylamino-17-demethoxygeldanamycin; 17-). AAG), 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-dimethylaminoethylamino-17-demethoxygeldanamycin; 17-DMAG), purine skeletal derivatives PU3 and PU24FCl, pyrazoles, and Shepherdin, a peptidomimetic inhibitor of the bivin-Hsp90 complex.

Novobiocin and cisplain, on the other hand, inhibit Hsp90 by binding to a second ATP binding site, which is thought to be present at the Hsp90 C-terminus. Hsp90 present in tumor cells has higher ATPase activity than normal cells and has high binding capacity with Hsp90 inhibitors, thereby enabling Hsp90 inhibitors to specifically act on tumor cells. Antitumor activity of Hsp90 inhibitors is different in vitro (in vitro) and the cells (in vivo) has been proven in a model system, 17-AAG has shown promising results as a therapeutic agent in clinical trials for various cancers.

However, because 17-AAG has problems such as formulation, potential toxicity, and low water solubility, there is a need for continuous development of Hsp90 inhibitors with improved pharmaceutical properties and efficacy.

Accordingly, an object of the present invention is to provide a novel compound as an inhibitor for Hsp90.

The present invention to achieve the above object,

[Formula 1]

In Chemical Formula 1,

X ₁ is H, Y ₁ is 2- (4-chlorophenyl) ethynyl, 2-amino-1- (3-chloro-4-methylphenyl) -4,5- Dihydro-4-oxo-1H-pyrrol-3-yl (2-amino-1- (3-chloro-4-methylphenyl) -4,5-dihydro-4-oxo-1H-pyrrol-3-yl) , [2-[(2-furanylmethyl) amino] -2-oxoethyl] thio ([2-[(2-furanylmethyl) amino] -2-oxoethyl] thio), [2-[(4-acetylphenyl ) Amino] -2-oxoethyl] thio ([2-[(4-acetylphenyl) amino] -2-oxoethyl] thio) and [[4- (methoxycarbonyl) phenyl] methyl] thio ([[4- (methoxycarbonyl) phenyl] methyl] thio);

X ₁ is methyl and Y ₁ is 2- (5-methoxy-1H-indol-3-yl) ethynyl (2- (5-methoxy-1H-indol-3-yl) ethenyl);

X ₁ is 2-hydroxyethyl and Y ₁ is 2- [4- (dimethylamino) phenyl] ethynyl (2- [4- (dimethylamino) phenyl] ethenyl);

X ₁ is 4-methoxycyclohexa-2,6-diinoxy ethyl and Y ₁ is 4-methoxyphenyl;

X ₁ is 1-naphthalenyl and Y ₁ is 2- (2-hydroxyphenyl) ethenyl;

X ₁ is phenyl, Y ₁ is 2- (2-hydroxyphenyl) ethynyl, 2- (2-hydroxy-3-methoxyphenyl) ethynyl (2- (2-hydroxy-3-methoxyphenyl) ethenyl), 2- (4-hydroxy-3-methoxyphenyl) ethynyl (2- (4-hydroxy-3-methoxyphenyl) ethenyl), [2- ( 4-morpholinyl) -2-oxoethyl] thio ([2- (4-morpholinyl) -2-oxoethyl] thio) and [(4-nitrophenyl) methyl] thio ([(4-nitrophenyl) methyl] thio );

X ₁ is 4-pyridinylmethyl and Y ₁ is 2- (4-nitrophenyl) ethenyl;

X ₁ is 2-pyridinylmethyl, Y ₁ is 2- (4-fluorophenyl) ethenyl) and (isoindole-1,3-di One of (isoindol-1,3-dionyl) ethyl)

It provides a Hsp90 inhibitor characterized in that it contains the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient,

[Formula 2]

In Chemical Formula 2,

X ₂ is methoxy, Y ₂ is 2- (2-methyl-1H-indol-3-yl) ethynyl (2- (2-methyl-1H-indol-3-yl) ethenyl) and ( One of 2-pyridinylthio) methyl;

X ₂ is hydroxy, Y ₂ is 2- (2-furanyl) ethenyl and 2- (4-hydroxy-3-methoxyphenyl) ethynyl One of (2- (4-hydroxy-3-methoxyphenyl) ethenyl);

X ₂ is chloro, Y ₂ is 2- (3-pyridinyl) ethynyl and (2-pyridinylthio) methyl ),

It provides a Hsp90 inhibitor characterized in that it comprises a compound of Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient,

[Formula 3]

In Chemical Formula 3,

X ₃ is methoxy, Y ₃ is 2- [2- (dimethylamino) phenyl] ethynyl (2- [2- (dimethylamino) phenyl] ethenyl), 2- (4-hydroxyphenyl) To ethynyl (2- (4-hydroxyphenyl) ethenyl), 2- (4-nitrophenyl) ethynyl (2- (4-nitrophenyl) ethenyl), and 2- (4-hydroxy-3-methoxyphenyl) Tinyl (2- (4-hydroxy-3-methoxyphenyl) ethenyl);

X ₃ is hydroxy, Y ₃ is 2- [4- (dimethylamino) phenyl] ethynyl (2- [4- (dimethylamino) phenyl] ethenyl) and 2-phenylethenyl );

X ₃ is methyl and Y ₃ is [(1,2-dihydro-2-oxo-3H-indol-3-ylidin) methyl ([(1,2-dihydro-2-oxo-3H -indol-3-ylidene) methyl),

It provides a Hsp90 inhibitor characterized in that it comprises a compound of Formula 3 or a pharmaceutically acceptable salt thereof as an active ingredient,

[Formula 4]

In Chemical Formula 4,

X ₄ is methoxy, Y ₄ is 2- (2-hydroxy-3-methoxyphenyl) ethynyl, 2- (4-hydroxy Hydroxyphenyl) ethynyl (2- (4-hydroxyphenyl) ethenyl), (1H-benzimidazol-2-ylmethyl) thio ((1H-benzimidazol-2-ylmethyl) thio), [2- (4-morpholy Yl) -2-oxoethyl] thio ([2- (4-morpholinyl) -2-oxoethyl] thio), [2-[[4- (dimethylamino) phenyl] amino] -2-oxoethyl] thio ( [2-[[4- (dimethylamino) phenyl] amino] -2-oxoethyl] thio) and [(1,2-dihydro-2-oxo-3H-indol-3-ylidin) methyl ([(1 , 2-dihydro-2-oxo-3H-indol-3-ylidene) methyl);

X ₄ is ethoxy, Y ₄ is [(3-fluorophenyl) methyl] thio ([(3-fluorophenyl) methyl] thio) and (2-amino-2-oxoethyl) thio ((2 -amino-2-oxoethyl) thio);

X ₄ is nitro and Y ₄ is 2- (4-fluorophenyl) ethynyl;

X ₄ is methyl and Y ₄ is [2- (1,3-benzodioxol-5-ylamino) -2-oxoethyl] thio ([2- (1,3-benzodioxol-5-ylamino ) -2-oxoethyl] thio), (2-amino-2-oxoethyl) thio ((2-amino-2-oxoethyl) thio) and [2-oxo-2- (1-piperidinyl) ethyl] thio ([2-oxo-2- (1-piperidinyl) ethyl] thio);

X ₄ is chloro, Y ₄ is [2-[(4-methylphenyl) amino] -2-oxoethyl] thio ([2-[(4-methylphenyl) amino] -2-oxoethyl] thio), [2- (2,3-dihydro-1H-indol-1-yl) -2-oxoethyl] thio ([2- (2,3-dihydro-1H-indol-1-yl) -2-oxoethyl] thio), [1-[(6-indazolinyl) carbamoyl] methyl] thio ([1-[(6-indazolinyl) carbamoyl] methyl] thio) and [2- (1,3-benzodioxol- 5-yl-2-oxoethyl] thio ([2- (1,3-benzodioxol-5-yl) -2-oxoethyl] thio);

X ₄ is fluoro and Y ₄ is (isoindol-1,3-dionyl) ethyl ((isoindol-1,3-dionyl) ethyl);

X ₄ is carboxyl and Y ₄ is 2- (2-hydroxyphenyl) ethynyl

It provides an Hsp90 inhibitor characterized by containing the compound of Formula 4 or a pharmaceutically acceptable salt thereof as an active ingredient.

In another aspect, the present invention provides an anticancer agent characterized in that it contains any one of the compounds described above Hsp90 inhibitor as an active ingredient.

Hereinafter, the present invention will be described in more detail.

In the present invention, virtual screening through docking simulation was performed on 85,000 chemicals to selectively screen 300 chemicals. Of the 300, 285 chemicals were screened for Hsp90 inhibitors by investigating ATPase activity using yeast Hsp90.

Hsp90 ATPase activity was measured in 96-well plates by using a malachite green reagent to detect the release of inorganic phosphoric acid by color change. As a result, in 48 compounds of the present invention, which are 3H-quinazolin4-one derivatives, Hsp90 activity was 65% or less (meaning 35% or more inhibitory effect) at an addition concentration of 50 μM (some compounds were 40 μM). appear.

On the other hand, in Example 21 compound, compound 31, compound 48 to the intracellular ( in in vivo ) inhibition efficacy was tested. The effect of Her2 degradation on MCF-7 breast cancer cell line was investigated. Hsp90 inhibitory activity was investigated by observing the induction of Hsp70 with the degradation of Her2 protein, one of the Hsp90 client proteins. Each chemical was treated for 24 hours at a concentration of 30 μM in MCF-7 cells, and expression levels of Her2 and Hsp70 were examined by Western blotting. Compared with the control group (geldanamycin), it was confirmed that the Her2 protein level was reduced by more than 50% and the expression of Hsp70 was induced in the compound 21, 31 and 48 compound treated groups.

In addition, growth inhibition of cells was measured, and compounds 21, 31, and 48 exhibited GI ₅₀ values between 25.4 and 35.6 μM concentrations, which effectively inhibited the growth of MCF-7 cells. GI ₅₀ of Tanamycin It is only 2.6 ~ 3.7 times higher than (9.6 μM).

On the other hand, the present invention provides a medicament for treating cancer (in a broader sense, prophylactic), ie an anticancer agent, comprising one of the 48 compounds of the present invention.

An anticancer agent of the present invention is a mixture containing, as an active ingredient, a significant amount of a compound of the present invention or a pharmaceutically acceptable salt thereof, and containing or mixing one or more inorganic or organic solid or liquid pharmaceutically acceptable carriers. to be.

Pharmaceutical compositions according to the invention may be administered to mammals (particularly humans) orally or parenterally (eg intramuscular or intravenous injections). The dosage of active ingredient depends on the species, weight, age and individual condition of the mammal.

When administering a compound of the present invention or a pharmaceutically acceptable salt thereof to a mammal, eg, a human weighing approximately 70 kg, the daily dosage per person is preferably from about 3 mg to about 10 g, more preferably Preferably from about 10 mg to about 1.5 g, most preferably from about 100 mg to about 1000 mg. The frequency of administration is preferably administered in a day divided by the same amount 2-3 times. Generally, half of the adult dose is applied to children.

The anticancer agent of the present invention comprises from about 1% to about 95%, preferably from about 20% to about 90% of the active ingredient (compound of the present invention). Pharmaceutical compositions according to the invention may be in unit dosage forms such as ampoules, vials, suppositories, sugars, tablets or capsules.

The anticancer agent of the present invention may be prepared through conventional dissolution, lyophilization, mixing, granulation, or glycosylation processes in a known manner, and commercially available ones may be purchased and used.

Anticancer agents of the present invention may include excipients (eg, preservatives, stabilizers, wetting agents, emulsifiers, solubilizers, salts or buffers for controlling osmotic pressure). It may also comprise a viscosity increasing substance (eg sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatin).

Injection form anticancer agents of the present invention may be prepared in conventional manner under sterile conditions, and conventional methods may be used when the composition is introduced into an ampoule or vial and the container is sealed.

Tablets as an anticancer agent for oral administration of the present invention may be prepared by combining the active ingredient with a solid carrier, granulating the resulting mixture if necessary, and optionally processing the mixture after adding an appropriate excipient. Suitable carriers include starch paste with corn, wheat, rice or potato starch and the like, gelatin, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, carboxymethyl starch, crosslinked polyvinylpyrroli Money, agar, alginic acid or sodium alginate. Suitable excipients include flow conditioners and lubricants (eg, silicic acid, talc, stearic acid or salts thereof such as magnesium stearate or calcium stearate or polyethylene glycol).

As the anticancer agent for oral administration of the present invention, the capsule may be a dry-filled capsule made of gelatin and a soft sealed one made of gelatin and a plasticizer (for example, glycerol or sorbitol). Dry-filled capsules may be added together with a filler (eg lactose), a binder (eg starch), a glidant (eg talc or magnesium stearate) or a stabilizer to the active ingredient in granular form. In soft capsules, the active ingredient is preferably dissolved or suspended in suitable oily excipients (eg, fatty oils, paraffin oils or liquid polyethylene glycols), and stabilizers or antimicrobial agents may be added.

As described above, it was confirmed that the 3H-quinazolin-4-one derivative of the present invention has an inhibitory activity against the Hsp90 protein, and thus the present invention can be used as an anticancer agent, which is very useful in the pharmaceutical industry.

The present invention will be described in more detail with reference to the following examples. However, the scope of the present invention is not limited only to the following examples, and includes modifications of equivalent technical ideas.

On the other hand, in Examples 1 to 4 below, the ATPase activity inhibitory activity of the compounds selected in the present invention was measured, but the method was as follows.

Saccharomyces cerevisiae ) HSC82 ORF encoding the Hsp90 protein was amplified by PCR and cloned into the Nde I and Xho I positions of the pET23b plasmid. E. coli BL21 star (DE3) pLysS was transformed with this plasmid, followed by six his-tags at the C-terminus using Ni-NTA affinity chromatography and Mono Q 5/50 GL. tag) and yeast Hsp90 protein was purified by FPLC. Hsp90 ATPase activity was measured in 96-well plates by using a malachite green reagent to detect the release of inorganic phosphoric acid by color change.

Yeast Hsp90 (0.6 μM, final concentration) was added to the reaction buffer [1 mM ATP, 100 mM Tris-HCl (pH 7.4), 20 mM KCl, 6 mM MgCl ₂ , 0.4% DMSO] and dissolved in 0.4% DMSO When the compound of (indicated by Sample Nos. 1 to 48 in Examples 1 to 4 below) was added at a concentration of 40 μM (Compound No. 21, Compound 31, and Compound 48 were treated with 50 μM), the changed activity was investigated. After incubating the reaction mixture at 37 ° C. for 3 hours, the reaction was stopped by the addition of malachite green (bioassay system co.) Reagent. After incubation for 10 minutes at 37 ℃, the absorbance was measured at 620nm using a microplate reader (thermo labsystem co.). By comparing this with the absorbance of the untreated group, the inhibition degree of Hsp90 was expressed as 'Activity (%)' in Examples 1 to 4 below (all values indicated in Examples 1 to 5 below were recognized as significant data). Values)

On the other hand, geldanamycin, a positive control known as an ATPase inhibitory compound, exhibited 50% of its activity upon 2.3 μM treatment.

Example  1: of the compound derivative represented by the following formula (1) ATPase  Active measurement

[Formula 1]

Sample X ₁ Y ₁ activation(%) One   H 2- (4-chlorophenyl) ethynyl 50.16 2 2-amino-1- (3-chloro-4-methylphenyl) -4,5-dihydro-4-oxo-1H-pyrrol-3-yl 58.31 3 [2-[(2-furanylmethyl) amino] -2-oxoethyl] thio 55.80 4 [2-[(4-acetylphenyl) amino] -2-oxoethyl] thio 59.56 5 [[4- (methoxycarbonyl) phenyl] methyl] thio 57.68 6 methyl 2- (5-methoxy-1H-indol-3-yl) ethynyl 52.04 7  2-hydroxyethyl 2- [4- (dimethylamino) phenyl] ethynyl 58.93 8 4-methoxycyclohexa-2,6-diinoxyl ethyl 4-methoxyphenyl 49.22 9 1-naphthalenyl 2- (2-hydroxyphenyl) ethynyl 37.00 10   Phenyl 2- (2-hydroxyphenyl) ethynyl 56.11 11 2- (2-hydroxy-3-methoxyphenyl) ethynyl 55.80 12 2- (4-hydroxy-3-methoxyphenyl) ethynyl 56.74 13 [2- (4-morpholinyl) -2-oxoethyl] thio 54.86 14 [(4-nitrophenyl) methyl] thio 59.87 15 4-pyridinylmethyl 2- (4-nitrophenyl) ethynyl 50.16 16 2-pyridinylmethyl 2- (4-fluorophenyl) ethynyl 54.86 17 (Isoindole-1,3-dionyl) ethyl 15.6

Example  2: of the compound derivative represented by the following formula (2) ATPase  Active measurement

[Formula 2]

Sample X ₂ Y ₂ activation(%) 18 Methoxy  2- (2-methyl-1H-indol-3-yl) ethynyl 53.29 19 (2-pyridinylthio) methyl 47.65 20 Hydroxy 2- (2-furanyl) ethynyl 60.50 21  2- (4-hydroxy-3-methoxyphenyl) ethynyl 25.10 22 Chloro 2- (3-pyridinyl) ethynyl 59.25 23  (2-pyridinylthio) methyl 55.49

Note) Sample 21 is 50μM treatment group

Example  3: of a compound derivative represented by the following formula (3) ATPase  Active measurement

[Formula 3]

Sample X ₃ Y ₃ activation(%) 24  Methoxy 2- [2- (dimethylamino) phenyl] ethynyl 55.17 25 2- (4-hydroxyphenyl) ethynyl 55.49 26 2- (4-nitrophenyl) ethynyl 55.49 27 2- (4-hydroxy-3-methoxyphenyl) ethynyl 59.87 28 Hydroxy 2- [4- (dimethylamino) phenyl] ethynyl 58.31 29  2-phenylethynyl 60.50 30 methyl  [(1,2-Dihydro-2-oxo-3H-indol-3-ylidin) methyl 59.87

Example  4: of the compound derivative represented by the following formula (4) ATPase  Active measurement

[Formula 4]

Sample X ₄ Y ₄ activation(%) 31   Methoxy 2- (2-hydroxy-3-methoxyphenyl) ethynyl 21.30 32 2- (4-hydroxyphenyl) ethynyl 53.92 33 (1H-benzimidazol-2-ylmethyl) thio 50.78 34 [2- (4-morpholinyl) -2-oxoethyl] thio 53.61 35 [2-[[4- (dimethylamino) phenyl] amino] -2-oxoethyl] thio 51.10 36 [(1,2-Dihydro-2-oxo-3H-indol-3-ylidin) methyl 60.50 37 Ethoxy [(3-fluorophenyl) methyl] thio 22.30 38 (2-amino-2-oxoethyl) thio 47.02 39 Nitro 2- (4-fluorophenyl) ethynyl 46.71 40  methyl [2- (1,3-benzodioxol-5-ylamino) -2-oxoethyl] thio 53.92 41 (2-amino-2-oxoethyl) thio 58.31 42  [2-oxo-2- (1-piperidinyl) ethyl] thio 55.17 43  Chloro [2-[(4-methylphenyl) amino] -2-oxoethyl] thio 55.80 44 [2- (2,3-dihydro-1H-indol-1-yl) -2-oxoethyl] thio 57.05 45 [1-[(6-indazolinyl) carbamoyl] methyl] thio 59.87 46 [2- (1,3-benzodioxol-5-yl-2-oxoethyl] thio 52.66 47 Fluoro (Isoindole-1,3-dionyl) ethyl 52.66 48 Carboxyl  2- (2-hydroxyphenyl) ethynyl 30.30

Note) Samples 31 and 48 have 50μM treatment group

Example  5: above Example  Of compounds 21, 31 and 48 of the compounds described in 1 to 4; Intracellular ( in vivo Efficacy test

Compounds 21, 31 and 48 were examined for her2 degradation in MCF-7 breast cancer cell lines. The inhibition of Hsp90 was examined by observing the induction of Hsp70 with the degradation of the Her2 protein, one of the Hsp90 client proteins. Since the heat shock transcription factor (Hsf1) is inhibited by Hsp90, Hsf1 target genes including Hsp70 are induced by Hsp90 inhibitors including geldanamycin. .

Each chemical (No. 21, No. 31, No. 48) was treated with MCF-7 cells at a concentration of 30 μM for 24 hours, and expression levels of Her2 and Hsp70 were examined by Western blotting. First, MCF-7 cells were seeded in 6-well plates at a concentration of ⁵ × 10 ⁵ cells / well and cells were attached to the bottom for one day. The next day, cells dissolved in 0.4% DMSO were treated with cells, and the same amount of 0.4% DMSO was treated with cells as a control. After incubation for 24 hours, cells were treated with TNES buffer [Tris-HCl (50 mM; pH 7.4), NP40 (1%), EDTA (2 mM), NaCl (100 mM), phenylmethylsulfonyl fluoride (1 mM), protease inhibitor cocktail solution. (1%, sigma)], cell extracts of Her2, Hsp70, tubulin by using antibodies against Hsp70 (Stressgen Bioreagents), Her2 (Santa Cruz Biotechnology) and tubulin (Santa Cruz Biotechnology). Expression levels were analyzed.

Compared to the positive control (geldanamycin), Her2 protein levels were reduced by more than 50% and expression of Hsp70 was induced (FIG. 1: 1a compound 21, 1b compound 31 and 1c 48 in FIG. 1). Times compound). These three compounds all showed similar effects of the degradation of Her2.

On the other hand, in order to measure the growth inhibition of the cell was used a method using sulforhodamine B (SRB). MCF-7 cells grown in 96-well plates were treated with varying concentrations of geldanamycin (positive control), compound 21, compound 31 and compound 48 for 4 days. The cells were fixed with 10% trichloroacetic acid and stained with 0.4% SRB (Sigma-Aldrich) dissolved in 1% acetic acid for 30 minutes. The dye bound to the protein was dissolved in Tris base (10 mM; pH 10.5), and the absorbance was measured at 550 nm through a microplate reader. GI ₅₀ was calculated as the concentration at which cell growth was inhibited by 50% compared to the untreated negative control (Table 5).

compound Geldanamycin Compound 21 Compound 31 Compound 48 GI ₅₀ (μM) ^a 9.6 25.4 27.5 35.6 ^{a by} n = 2

As shown in Table 5, Compound 21, Compound 31, and Compound 48 showed GI ₅₀ values between 25.4 and 35.6 μM concentrations, effectively inhibiting the growth of MCF-7 cells. This is the GI _{50 of} geldanamycin. It is only 2.6 ~ 3.7 times higher than (9.6 μM).

Formulation example  1: tablet

Tablets of the following composition were prepared in a conventional manner.

mg Compound 48 as an active ingredient 100 Powdered lactose 95 White corn starch 35 Polyvinylpyrrolidone 8 Na carboxymethyl starch 10 Magnesium stearate 2 Tablet total weight 250

Formulation example  2: capsule

Capsules of the following composition were prepared in a conventional manner.

mg Compound 48 as an active ingredient 50 Crystalline lactose 60 Microcrystalline cellulose 34 talc 5 Magnesium stearate One Capsule Filling Gross Weight 150

Formulation example  3: injection

Injection solutions of the following compositions were prepared in a general manner.

The compound of Example 48 as an active ingredient 1.0 mg 1N HCl 30.0 μl Acetic acid 0.5 mg NaCl 8.0 mg phenol 10.0 mg 1N NaOH Appropriate amount up to pH 5 H ₂ O Appropriate amount up to 1 mL

1 shows Her2 degradation and Hsp70 inducing effects of Hsp90 inhibitors. In Fig. 1, geldanamycin (GA; positive control) at the indicated concentrations, compound 21 (1a), compound 31 (1b), compound 48 (1c) and 0.4% untreated negative control in 0.4% DMSO After DMSO (C) was treated with MCF-7 cells for 24 hours, the expression levels of Her2 and Hsp70 were analyzed by Western blotting. Tubulin was used as a control for quantitative analysis.

Claims (5)

An Hsp90 inhibitor characterized by containing a compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient:

In Chemical Formula 1, X ₁ is H, Y ₁ is 2- (4-chlorophenyl) ethynyl, 2-amino-1- (3-chloro-4-methylphenyl) -4,5-dihydro-4-oxo-1H-py Rol-3-yl, [2-[(2-furanylmethyl) amino] -2-oxoethyl] thio, [2-[(4-acetylphenyl) amino] -2-oxoethyl] thio and [[4 One of-(methoxycarbonyl) phenyl] methyl] thio; X ₁ is methyl and Y ₁ is 2- (5-methoxy-1H-indol-3-yl) ethynyl; X ₁ is 2-hydroxyethyl and Y ₁ is 2- [4- (dimethylamino) phenyl] ethynyl; X ₁ is 4-methoxycyclohexa-2,6-diinoxy ethyl and Y ₁ is 4-methoxyphenyl; X ₁ is 1-naphtharenyl, Y ₁ is 2- (2-hydroxyphenyl) ethynyl; X ₁ is phenyl, Y ₁ is 2- (2-hydroxyphenyl) ethynyl, 2- (2-hydroxy-3-methoxyphenyl) ethynyl, 2- (4-hydroxy-3-methoxy Phenyl) ethynyl, [2- (4-morpholinyl) -2-oxoethyl] thio and [(4-nitrophenyl) methyl] thio; X ₁ is 4-pyridinylmethyl and Y ₁ is 2- (4-nitrophenyl) ethynyl; X ₁ is 2-pyridinylmethyl and Y ₁ is _one of 2- (4-fluorophenyl) ethynyl and (isoindole-1,3-dionyl) ethyl. An Hsp90 inhibitor characterized by containing the compound of formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient:

In Chemical Formula 2, X ₂ is methoxy and Y ₂ is one of 2- (2-methyl-1H-indol-3-yl) ethynyl and (2-pyridinylthio) methyl; X ₂ is hydroxy and Y ₂ is one of 2- (2-furanyl) ethynyl and 2- (4-hydroxy-3-methoxyphenyl) ethynyl; X ₂ is chloro and Y ₂ is one of 2- (3-pyridinyl) ethynyl and (2-pyridinylthio) methyl. An Hsp90 inhibitor characterized by containing the compound of formula 3 or a pharmaceutically acceptable salt thereof as an active ingredient:

In Chemical Formula 3, X ₃ is methoxy, Y ₃ is 2- [2- (dimethylamino) phenyl] ethynyl, 2- (4-hydroxyphenyl) ethynyl, 2- (4-nitrophenyl) ethynyl and 2- One of (4-hydroxy-3-methoxyphenyl) ethynyl; X ₃ is hydroxy and Y ₃ is one of 2- [4- (dimethylamino) phenyl] ethynyl and 2-phenylethynyl; X ₃ is methyl and Y ₃ is [(1,2-dihydro-2-oxo-3H-indol-3-ylidin) methyl. An Hsp90 inhibitor characterized by containing the compound of formula (4) or a pharmaceutically acceptable salt thereof as an active ingredient:

In Chemical Formula 4, X ₄ is methoxy, Y ₄ is 2- (2-hydroxy-3-methoxyphenyl) ethynyl, 2- (4-hydroxyphenyl) ethynyl, (1H-benzimidazol-2-ylmethyl ) Thio, [2- (4-morpholinyl) -2-oxoethyl] thio, [2-[[4- (dimethylamino) phenyl] amino] -2-oxoethyl] thio and [(1,2 -Dihydro-2-oxo-3H-indol-3-ylidin) methyl; X ₄ is ethoxy and Y ₄ is one of [(3-fluorophenyl) methyl] thio and (2-amino-2-oxoethyl) thio; X ₄ is nitro and Y ₄ is 2- (4-fluorophenyl) ethynyl; X ₄ is methyl, Y ₄ is [2- (1,3-benzodioxol-5-ylamino) -2-oxoethyl] thio, (2-amino-2-oxoethyl) thio and [2-oxo -2- (1-piperidinyl) ethyl] thio; X ₄ is chloro, Y ₄ is [2-[(4-methylphenyl) amino] -2-oxoethyl] thio, [2- (2,3-dihydro-1H-indol-1-yl) -2- Oxoethyl] thio, [1-[(6-indazolinyl) carbamoyl] methyl] thio and [2- (1,3-benzodioxol-5-yl-2-oxoethyl] thio; X ₄ is fluoro and Y ₄ is (isoindole-1,3-dionyl) ethyl; X ₄ is carboxyl and Y ₄ is 2- (2-hydroxyphenyl) ethynyl. An anticancer agent comprising the Hsp90 inhibitor according to any one of claims 1 to 4 as an active ingredient.

Images