KR101456174B1 - The glycosylated non-quinone geldanamycin derivatives having increased solubility or pharmaceutically acceptable salts thereof, preparation method thereof and pharmaceutical composition for Heat shock protein(Hsp90) ATPase inhibitor containing same - Google Patents

Abstract

The present invention relates to a non-quinone geldanamycin derivative having increased solubility, a pharmaceutically acceptable salt thereof, a process for preparing the same, and a pharmaceutical composition for inhibiting thermal shock protein aptase activity. The present non-quinone geldanamycin glycoconjugate derivative according to the present invention can be used as a non-quinone geldanamycin derivative since glucose is introduced into a non-quinone geldanamycin derivative to increase the solubility and the bioavailability, Anticancer agents, antibiotics, antifungal agents, antiviral agents, immunosuppressants, therapeutic agents for degenerative neurological diseases, or anti-inflammatory agents.

Description

FIELD OF THE INVENTION The present invention relates to a non-quinone geldanamycin derivative having increased solubility, a pharmaceutically acceptable salt thereof, a process for preparing the same, and a pharmaceutical composition for inhibiting ATPase activity of a heat shock protein (Hsp90) (Hsp90) ATPase inhibitor containing same or a pharmaceutically acceptable salt thereof,

The present invention relates to a non-quinone geldanamycin derivative having increased solubility or a pharmaceutically acceptable salt thereof, a process for preparing the same, and a method for inhibiting the heat shock protein 90 (Hsp90) ATPase activity ≪ / RTI >

Geldanamycin is an ansamycin-based antibiotic of the macrocyclic lactam structure, as shown in the following formula: 3-aminocarbamate, such as herbimycin, reblastatin, mecbecin, (3-amino-5-hydroxybenzoic acid, AHBA) as a precursor.

[Chemical Formula]

The above compounds were identified as antibiotics, antifungal agents, antiviral agents and anticancer agents between 1970 and 2000.

Such biosynthesis of geldanamycin can be achieved by forming a polyketide basic skeleton by continuous addition of skeleton-extending precursors such as acetate, propionate and glycolate, starting from AHBA as a starting unit, And the geldanamycin was confirmed to bind to the ATP binding site of Heat shock protein 90 (Hsp90) having chaperone protein activity by Neckers et al. In 1994 .

Therefore, it has been proposed that geldanamycin does not exhibit anticancer activity by inhibiting the enzyme activity of tyrosine kinase having an oncogenic protein function, but may be a protein of a variety of Hsp90 including a tyrosine kinase ) Inhibits the function of Hsp90, which is important for the structural stability of the target proteins, and thus the stability of the proteins is inhibited.

Because of the physiological importance of Hsp90, Hsp90 activity inhibitors such as 17-allyl amino geldanamycin (17-AAG) and 17-DMAG, which are chemically synthesized derivatives of geldanamycin, have been developed as anticancer drugs. However, There is a problem that does not dissolve well.

Furthermore, due to the chemical characteristics of geldanamycin with benzoquinone rings, high hepatotoxicity is hampering the development of new drugs for these compounds. Carbon 19 in the benzoquinone ring of geldanamycin has been identified through the spontaneous chemical reaction in vivo and the possibility of covalent bonding with various unspecified proteins through the binding force of glutathione 4. Due to this highly reactive chemical structure, Is known to occur. In order to alleviate or eliminate it, studies have been conducted to develop a non-quinone geldanamycin derivative through a biosynthetic gene modification method. As this product, non-quinone geldanamycin derivatives have been reported in Korean Patent No. 10-860502. The non-quinone geldanamycin derivative is expected to lose the binding power of glutathione and to weaken hepatotoxicity, but still has a problem of being insoluble in water.

Accordingly, the inventors of the present invention have conducted studies to solve the above problems. As a result, the non-quinone geldanamycin derivative is improved in solubility of the non-quinone geldanamycin derivative by introducing glucose into the non-quinone geldanamycin derivative. The present invention has been completed.

1. Korean Registered Patent No. 10-860502

1. DeBoer C. et al. J. Antibiotic., 23 (9) 442-447, 1970; Omura, S. et al., J. Antibiotic. 32, 255-261, 1979; Muroi, M. et. al., J. Antibiotic. 33, 205-212, 1980; Neckers L. et al., Invest. New Drugs 17, 361-373,1999; Piper P.W., Curr. Opin. Investing Drugs 2 (11) 1606-1610, 2001 2. Whitesell L. et. al. Proc. Natl. Acad. Sci. USA, 91, 8324-8328, 1994; Prodromou C. et. al. Cell, 90, 65-75, 1997 3. Walter S. and Buchner J., Angew. Chem. Int. Ed. 41, 1098-1113, 2002; Piper P. W., Current Opinion in Investigational Drugs 2, 1606-1610, 2001 4. Cysyk, R. L. et al. Chem. Res. Toxicol. 19, 376-381, 2006; Maroney, A. C. et al. Biochemistry 45, 5678-5685, 2006. 5. Kim, W. et al. ChemBioChem. 8, 1491-1494, 2007; Kim, W. et al. ChemBioChem. 10, 1243-1251, 2009; Wu, C.-Z. et al. J. Antibiotic. 64, 461-463, 2011.

It is an object of the present invention to provide a non-quinone geldanamycin derivative or a pharmaceutically acceptable salt thereof.

Another object of the present invention is to provide a process for preparing the non-quinone geldanamycin derivative.

It is still another object of the present invention to provide a pharmaceutical composition for inhibiting the ATPase activity of a heat shock protein comprising a non-quinone geldanamycin derivative and a pharmaceutically acceptable salt thereof as an active ingredient.

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating cancer diseases, which comprises a non-quinone geldanamycin derivative and a pharmaceutically acceptable salt thereof as an active ingredient.

In order to achieve the above object, the present invention provides a non-quinone geldanamycin glycoside derivative represented by the following general formulas 1 to 5 or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

In addition,

Quinone geldanamycin having an increased solubility, comprising the steps of: preparing a non-quinone geldanamycin derivative by carrying out a glycosylation reaction using an enzyme of the present invention and a non-quinone geldanamycin derivative as a substrate, To provide a method for preparing the derivative.

Furthermore, the present invention provides a pharmaceutical composition for inhibiting the ATPase activity of a heat shock protein comprising a non-quinone geldanamycin derivative and a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention or treatment of cancer diseases, which comprises a non-quinone geldanamycin derivative and a pharmaceutically acceptable salt thereof as an active ingredient.

The present non-quinone geldanamycin glycoconjugate derivative according to the present invention can increase the solubility and bioavailability by introducing glucose into the non-quinone geldanamycin derivative, It can be effectively used as an anticancer agent, an antibiotic, an antifungal agent, an antiviral agent, an immunosuppressant agent, a therapeutic agent for degenerative nerve disease or an anti-inflammatory agent.

1 is a diagram showing LC / MS analysis data of the compound of Example 1 of the present invention.
2 is a diagram showing LC / MS analysis data of the compound of Example 2 of the present invention.
3 is a diagram showing LC / MS analysis data of the compound of Example 3 of the present invention.
4 is a diagram showing LC / MS analysis data of the compound of Example 4 of the present invention.
5 shows LC / MS analysis data of the compound of Example 5 of the present invention.
FIG. 6 is a graph showing the inhibitory effect of a compound according to an embodiment of the present invention on the expression of heat shock proteins. FIG.
7 is a graph showing HPLC analysis data for comparing the solubility of a compound according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a non-quinone geldanamycin glycoside derivative represented by the following general formulas 1 to 5, or a pharmaceutically acceptable salt thereof.

The present invention also relates to the non-quinone geldanamycin glycoconjugate derivatives represented by the above general formulas (1) to (5), as well as their pharmaceutically acceptable salts, possible solvates, hydrates, racemates or stereoisomers .

The non-quinone geldanamycin derivative represented by the general formulas (1) to (5) of the present invention can be used in the form of a pharmaceutically acceptable salt. Examples of the salt include pharmaceutically acceptable acid addition salts formed by free acid Salts are useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Sulfonate, methanesulfonate, propanesulfonate, naphthalene-1-sulphonate, naphthalene-1-sulphonate, , Naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be produced by a conventional method, for example, by dissolving the derivative of the non-quinone cinzincanamic acid derivative represented by Chemical Formulas 1 to 5 in an excess amount of an acid aqueous solution, For example, methanol, ethanol, acetone or acetonitrile.

The same amount of the non-quinone geldanamycin derivative represented by Chemical Formulas 1 to 5 and an acid aqueous solution or alcohol may be heated, followed by evaporating the mixture, followed by drying or precipitation of the precipitated salt by suction filtration.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the salt of the undissolved compound, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt.

The non-quinone geldanamycin derivative has an increased solubility (see Experimental Example 4) and an increased activity of the thermal shock proteinaceptase inhibitor (see Experimental Example 1).

In addition,

Quinone geldanamycin derivative, which comprises the step of preparing a non-quinone geldanamycin derivative by carrying out a glycosylation reaction using an enzyme of the present invention and a non-quinone geldanamycin derivative as a substrate, And a manufacturing method thereof.

The method for preparing a non-quinone geldanamycin glycoconjugate derivative according to the present invention may further include separating the glycosyltransferase from the Escherichia coli, wherein the glycosyltransferase can be isolated from Escherichia coli.

Specifically, the UDP-tagged enzyme derived from Bacillus lentorformis is cloned into an Escherichia coli expression vector, and 6 amino acid histines at the N-terminus are added to the Escherichia coli expression vector. And the UDP-tagged enzyme can be separated and used by using nickel-NTA affinity column chromatography.

The non-quinone geldanamycin glycosylation derivative can be prepared by subjecting glycosaminoglycans to a glycosylation reaction using a glycosidase and a non-quinone geldanamycin derivative as a substrate. Specifically, the sugar chain reaction can be carried out at 25-35 ° C for 11-13 hours after the addition of the UDP-tagged enzyme and UDP-glucose, and can be performed in an environment of pH 6.0-9.0 .

At this time, the non-quinone geldanamycin derivatives which can be used are one kind selected from the group consisting of the compounds represented by the following formulas (A), (B) and (C).

(A)

[Chemical Formula B]

≪ RTI ID = 0.0 &

Furthermore, the present invention provides a pharmaceutical composition for inhibiting the ATPase activity of a heat shock protein comprising a non-quinone geldanamycin derivative and a pharmaceutically acceptable salt thereof as an active ingredient.

(ATPase) inhibitory activity (IC ₅₀ ) of the heat shock protein according to the present invention is 0.75 μM in the case of the compound represented by the formula (1) of Example 1, 4 Was 2.14 [mu] M, which is superior to the conventional non-quinone geldanamycin. In particular, it was confirmed that the glycated compound exhibited similar ATPase inhibitory activity as that of the non-quinone geldanamycin derivative used as the original substrate (see Experimental Example 1).

Accordingly, the pharmaceutical composition can be used as an antibiotic, an antifungal agent, an antiviral agent, an immunosuppressant, a therapeutic agent for degenerative neurological disease or an anti-inflammatory agent by inhibiting the ATPase activity of heat shock protein.

The present invention also provides a pharmaceutical composition for the prevention or treatment of cancer diseases, which comprises a non-quinone geldanamycin derivative and a pharmaceutically acceptable salt thereof as an active ingredient.

The non-quinone geldanamycin glycoconjugate derivative according to the present invention is used as a control in Korean breast cancer cell lines, Korean Patent Registration No. 10-860502; It was confirmed that the IC ₅₀ value was lower than that of the compound represented by the chemical formula A of WK-88-2. However, since the IC ₅₀ value is excellent, it can be effectively used for an anticancer drug (see Experimental Example 2).

The cancer diseases may include breast cancer, liver cancer, stomach cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, anorectal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, Carcinoma, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma or central nervous system tumor.

Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

However, the following examples and experimental examples are illustrative of the present invention, but the present invention is not limited thereto.

< Example  1 to 3> non-quinone Zeldanamycin The party  Preparation of derivative 1

The party  Isolation of enzymes

The His-tagged enzyme in Escherichia coli expressing UDP-glycosyltransferase (BL-C), a peritonezyme derived from Bacillus licheniformis, was analyzed by nickel-NTA affinity column chromatography . Over-production of the protein was achieved by incubating E. coli BL21 (DE3) with an overexpressing plasmid in a 5 ml LB (Luria-Bertani) liquid medium containing kanamycin and chloramphenicol at a concentration of 50 μg / ml for about 17 hours Lt; / RTI &gt; It was diluted 1/60 with 30 ml of fresh LB medium and cultured for 2 to 3 hours to obtain an OD (optical density) value of about 0.5 to 0.6 at a UV spectrum of 600 nm. At this time, the cells were placed on ice for 10 minutes, and isopropyl β-D-thioglucopyranoside (IPTG) was added to a final concentration of 1 mM to induce overexpression, followed by further incubation at 20 ° C. for 17 hours . Escherichia coli thus obtained was obtained by centrifugation (3500 rpm, 30 minutes) and then E. coli was added to 3 ml of a buffer (100 mM sodium phosphate solution (pH 7.4), 20 mM KCl, 6 mM MgCl ₂ , 10 mM imidazole) Lt; / RTI &gt; Escherichia coli cells were pulverized using an ultrasonic pulverizer, followed by centrifugation (15,000 rpm, 30 minutes), and the pulverized liquid was subjected to SDS-PAGE to confirm protein production. The above crushed supernatant was mixed with 0.5 ml of Ni-NTA resin in an empty column, gently shaken at 4 ° C for 60 minutes, and then washed with 3 ml of wash buffer (100 mM sodium phosphate solution; 4), 20 mM KCl, 6 mM MgCl 2, 20 mM imidazole) the process three times, and finally eluting buffer (elution buffer; 100 mM Sodium phosphate solution (pH7.4), 20 mM KCl, 6 mM MgCl 2, 250 mM imidazole) was treated five times with 0.5 ml each. After confirming each fraction by SDS-PAGE, the enzyme was isolated.

Glycation  reaction

Among the compounds disclosed in Korean Patent No. 10-860502, which is a substrate of the sugar-transferred enzyme separated in the step 1, a compound having a benzene ring represented by the following formula A (non-quinone geldanamycin derivative: WK- 88-2).

This reaction was carried out by adding 1 mg of enzyme, 2 mM of UDP-glucose, 1 μM of MgCl 2 and a non-quinone geldanamycin derivative represented by the following formula A to 1 ml of 25 μM Tris-HCl (pH 8.0) Was added and the reaction was carried out at 30 ° C for 12 hours. After the reaction is completed, the reaction solution is first extracted with ethyl acetate (EtOAc), and the water layer is dried in a freeze drier for 24 hours and then dissolved in a small amount of methanol. These extracts and methanol concentrate were separated by preparative HPLC (Waters Delta Prep 3000 system, Waters, USA; USA) with increasing MeCN stepwise in 10% increments every 10 min starting from MeCN: H ₂ O (0.05% TFA) = 10: [YMC-Jsphere ODS-H80 250 X 10 mm id, 3 ml / min]). As a result, 20 mg of the compound represented by the formula (1), 6.7 mg of the compound represented by the formula (2) and 6.2 mg of the compound represented by the formula (3) were obtained.

(A)

Analysis

In order to identify the compound isolated in step 2, the following experiment was conducted.

The specific rotation ([?] _D ²⁵ ) was measured using a JASCO DIP-370 polarimeter (JASCO, Inc.) using an Electrothermal 9100 instrument (Electrothermal, UK) Japan), and UV was measured using a Shimadzu UV-1601 spectrophotometer (Shimadzu, Japan). ESI-MS was obtained from Finnigan LCQ Advantage Max Mass Spectrophotometer (Thermo, USA). HRESI-MS data were obtained from Korea Basic Science Research Institute and LC / MS analysis data were obtained from Shimadzu LCMS-IT -TOF mass spectrophotometer (Shimadzu Corp., Japan). The results are shown in Table 1 below.

At this time, whether the mass peak of the predicted compound to which glucose was added was observed in the LC / MS analysis was measured using the Xalibur software (version 1.3 SP2, Thermo Electron) program to measure the background ground peak produced by the Metabolite ID 2.0 software (Thermo Electron Co., USA). As a result, it was confirmed that the molecular weight of the compound represented by formulas (1) to (3) according to the present invention was higher than that of the compound represented by formula (A) before saccharification. The results are shown in the following Table 1 and Figs.

division rescue Analytical data


Example 1
(Formula 1)

White powder;
_{^{[α] 18 D: +49.98 (}} c, 0.10, MeOH);
UV (MeOH); ? max (log?): 206 (4.18), 256 (3.54);
HR-ESIMS (m / z): 703.3411 [M + Na] ⁺ , measured value 703.3412 (C ₃₄ H ₅₂ N ₂ O ₁₂ )



Example 2
(2)

White powder;
_{^{[α] 18 D: -6.46 (}} c, 0.10, MeOH);
UV (MeOH)? Max (log?): 256 (3.39), 204 (4.09);
HR-ESIMS (m / z): 865.3945 [M + Na] &lt; ⁺ &gt;, measured value; _{865.3941 (C 40 H62N 2 O 17} )


Example 3
(Formula 3)

White powder;
_{^{[α] 18 D: -4.62 (}} c, 0.10, MeOH);
UV (MeOH) [lambda] max (log [epsilon]): 259 (3.37), 204 (4.09);
HR-ESIMS (m / z): 703.3411 [M + Na] &lt; ⁺ &gt;, measured value; _{703.3412 (C 34 H 52 N 2} O 12)

< Example  4> Non-quinone Zeldanamycin The party  Preparation of derivative-2

Among the compounds disclosed in Korean Patent No. 10-860502 in Examples 1 to 3, a compound having a benzene ring (non-quinone geldanamycin derivative: WK-88-2) represented by the following formula A was used Quinone geldanamycin derivative derivative represented by the following formula (4) was obtained by carrying out the same procedure as the above except that the compound (WK-88-1) represented by the formula (B) was used instead of the compound represented by the formula The results are shown in Table 2 and FIG.

[Chemical Formula B]

The rescue Analytical data



Formula 4

White powder;
_{^{[α] 18 D: -3.24 (}} c, 0.10, MeOH);
UV (MeOH) [lambda] max (log [epsilon]): 205 (3.99), 251 (3.33);
HR-ESIMS (m / z): 687.3469 [M + Na] &lt; ⁺ &gt;, measured value; _{687.3463 (C 34 H 52 N 2} O 11)

< Example  5> Non-quinone Zeldanamycin The party  Preparation of derivative-3

Among the compounds disclosed in Korean Patent No. 10-860502 in Examples 1 to 3, a compound having a benzene ring (non-quinone geldanamycin derivative: WK-88-2) represented by the following formula A was used Quinone geldanamycin derivative derivative represented by the following formula (5) was obtained by carrying out the same process except that the compound represented by the following formula (C) (17-dimethoxy-levastatin) was used instead of the compound . The results are shown in Table 3 and FIG.

&Lt; RTI ID = 0.0 &

The rescue Analytical data



Formula 5

White powder;
_{^{[α] 18 D: +24.5 (}} c, 0.10, MeOH);
UV (MeOH) [lambda] max (log [epsilon]): 206 (4.68);
HR-ESIMS (m / z) 703.3410 [M + Na] &lt; ⁺ &gt;, measured value; _{703.3412 (C 34 H 52 N 2} O 12)

< Experimental Example  1> Thermal shock  Protein Atopy ( ATPase ) Inhibition activity measurement

The following experiment was conducted to measure the ATPase inhibitory activity of the derivative of non-quinone geldanamycin glycoconjugate according to the present invention.

Step 1: Thermal shock  Separation of protein purification

First, the heat shock protein (Hsp90) of yeast was separated and purified as follows. In order to clone the gene coding for a Hsp90 protein by introducing a Nde I restriction site 5'-CAT ATG GCT AGT GAA ACT TTT GAA TTT CAA G-3 ' introducing a 5 (forward primer) and a Sal I restriction site " -GCT GAC ATC TAC CTC TTC CAT TTC GGT GTC AG-3 '(reverse primer) and cloned into the pET-28a (+) vector. The prepared vector was transformed into Escherichia coli Rosetta (DE3) and expressed.

Overproduction of the protein was carried out in a 5 ml LB (Luria-Bertani) liquid medium containing kanamycin and chloramphenicol at a concentration of 50 μg / ml for about 17 hours Lt; / RTI &gt; It was diluted 1/60 with 30 ml of fresh LB medium and cultured for 2 to 3 hours to obtain an OD (optical density) value of about 0.5 to 0.6 at a UV spectrum of 600 nm. At this time, the cells were placed on ice for 10 minutes, and isopropyl β-D-thioglucopyranoside (IPTG) was added to a final concentration of 1 mM to induce overexpression, followed by further incubation at 20 ° C. for 17 hours . Escherichia coli thus obtained was obtained by centrifugation (3500 rpm, 30 minutes) and then E. coli was suspended in 3 ml of a buffer (100 mM Tris-HCl, 20 mM KCl, 6 mM MgCl ₂ , imidazole 10 mM, pH 7.4) Respectively. Escherichia coli cells were pulverized using an ultrasonic pulverizer, followed by centrifugation (15,000 rpm, 30 minutes), and the pulverized liquid was subjected to SDS-PAGE to confirm protein production. The crushed supernatant was mixed with 0.5 ml of Ni-NTA resin in an empty column, gently shaken at 4 ° C for 60 minutes, and then washed with 3 ml of wash buffer (100 mM Tris-HCl, 20 mM _{KCl, 6 mM MgCl 2, imidazole} 20 mM, pH 7.4) the process three times, and finally eluting buffer (elution buffer, 100 mM Tris- HCl, 20 mM KCl, 6 mM MgCl 2, imidazole 250 mM, pH 7.4) Were each treated five times with 0.5 ml each. After confirming each fraction by SDS-PAGE, the Hsp90 protein was isolated.

Step 2: Thermal shock  Protein Atopy ( ATPase ) Inhibition activity measurement

The heat-shock protein (Hsp90) of the yeast isolated and purified as described above was measured for its ATPase activity by a known method (B. Panaretou, C. Prodromou, SM Roe, R. O'Brien, JE Ladbury, PW Piper, , EMBO J.1998,17 (16), 4829-4836 .; Rowlands, MG, Newbatt, YM, Prodromou, C., Pearl, LH, Workman, P. and Aherne, W. Anal. Biochem 2004. 327,176- 183). The protein was dialyzed against a reaction buffer (100 mM Tris-HCl, 20 mM KCl, 6 mM MgCl ₂ , pH 7.4) to a final concentration of 0.3 mg / ml, and the malachite green reaction solution was prepared by a known method (5.72%, w / v, in 6 M HCl), and water in the ratio 2 (2%), green (0.0812%, w / v), polyvinyl alcohol (2.32%, w / v; dissolves with difficulty and requires heating), ammonium molybdate : 1: 1: 2).

ATPase inhibitory activity of the compounds of formulas (1) to (4) according to the present invention and geldanamycin, non-quinone geldanamycin A (Korea Patent No. 10-860502; WK- 0.11, 0.01, 0.001, 0.0001, 0.00001, and 0 μM, respectively, and 1 μl of each of the compounds was prepared. Was added to 10 μl of heat shock protein (1 μM) and 4 μl of reaction buffer (100 mM Tris-HCl, 20 mM KCl, 6 mM MgCl ₂ , pH 7.4) and the reaction was performed at 37 ° C. for one hour, 10 μl of 2.5 mM ATP was added thereto, followed by shaking for 1 minute. The reaction solution was allowed to react at 37 ° C for 3 hours, and then 80 μl of malachite reaction solution was added. 10 μl of 34% sodium citrate was added Allow to stand for 15 minutes at room temperature and stop the reaction. Next, as described in EMBO J.1998, 17 (16), 4829-4836; Anal. Biochem. 2004. 327, 176-183. The absorbance of this reaction solution was measured at a wavelength of 620 nm using a UV spectrum analyzer, and the ATPase inhibition IC ₅₀ value was calculated. The results are shown in Table 4 below.

compound ATPase inhibitory activity
(IC ₅₀ , μM ± SD) Formula 1 0.75 + 0.17 Formula 4 2.14 ± 0.67 Formula A (WK-88-2) 1.58 ± 0.65 Formula (B) (WK-88-1) 0.79 + - 0.12 Zeldanamycin 3.19 ± 0.65

As shown in Table 4, the ATPase inhibitory activity (IC ₅₀ ) of the non-quinone geldanamycin glycoconjugate derivative according to the present invention was 0.75 μM in the case of the compound represented by the formula (1) and 2.14 μM in the case of the compound represented by the formula mu] M, exhibited superior ATPase inhibitory activity than the conventional geldanamycin. In particular, the non-quinone geldanamycin glycoconjugate derivative compound according to the present invention was found to exhibit similar ATPase inhibitory activity compared to the compound used as the original substrate.

Therefore, the compound according to the present invention can be effectively used not only as an anticancer agent but also as an antibiotic, an antifungal agent, an antiviral agent, an immunosuppressant, a therapeutic agent for degenerative neurological diseases, an antiinflammatory agent and the like because it has excellent effect of inhibiting ATPase activity of heat shock protein.

< Experimental Example  2> Evaluation of anticancer activity

The following experiments were carried out to determine the cytotoxicity of the non-quinone geldanamycin glycoconjugate derivative represented by formula (1) according to the present invention to cancer cells.

Cancer cell lines used human breast cancer (SK-Br3). The SK-Br3 cell line is a cell line that overexpresses ErbB2 (kinase, cancer-related phosphorylation enzyme), a substrate protein of Hsp90 protein, and is widely used as a cell line for evaluating anticancer activity against existing geldanamycin derivatives. The cancer cell lines were divided into 10 ⁴ cancer cells per 96 well plate in a 96-well plate and cultured at 37 ° C in 5% CO ₂ for 24 hours. Then, 100, 10, 1, (Korean Registered Patent No. 10-860502; WK-88-2) as the control group and the compound represented by the formula (1) obtained in Example 1 at concentrations of 0.1, 0.01, 0.001, 0.0001, 0.00001, Respectively. The cells were incubated for 72 hours under the same conditions. Then, MTT reagent was added and reacted at 37 ° C for 4 hours to form formazan in the cells. In order to measure the absorbance of the formazan glass, a lysis buffer was added, and the mixture was allowed to stand at 37 ° C for one day. The absorbance was measured at 570 nm / 650 nm using a UV spectrum analyzer and IC ₅₀ was calculated. The results are shown in Table 5 below.

compound Breast cancer (SK-Br3)
(IC ₅₀ ; uM) Formula 1 19.0 [mu] M A
(WK-88-2) 9.43 [mu] M

As shown in Table 5, the non-quinone geldanamycin glycoconjugate derivative according to the present invention was used as a control group in breast cancer cell lines in Korean Patent No. 10-860502; It was confirmed that the IC ₅₀ value was lower than that of the compound represented by the chemical formula A of WK-88-2, but it can be usefully used for an anticancer drug because it has an excellent IC ₅₀ value.

< Experimental Example  3> Her2 , Akt  And c- Shelf  Evaluation of Protein Expression Inhibition

In order to determine the inhibitory effect of Her2 (kinase, cancer-associated phosphorylase), Akt and c-Raf on the substrate protein of the heat shock protein (Hsp90) of the non-quinone geldanamycin glycoprotein derivative represented by the compound 1 according to the present invention The following experiment was conducted.

Herb-overexpressed breast cancer cell line SK-Br3 was cultured in 5% CO ₂ at 37 ° C for 24 hours. As a control, the compound (WK-88-2 ) And 1 mu M of the derivative compound of the non-quinone geldanamycin glycoside derivative represented by Example 1 were dissolved in DMSO and treated for each concentration. The cultured cell lines were harvested and resuspended in lysis buffer (50 mM Tris Buffer pH 7.6, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% (v / v) # P8340)), the cells were ultrasonically pulverized to obtain pulverized material, and the pulverized material was quantitated by BCA method. Approximately 50 μg of the protein was loaded on SDS-PAGE and immunoblotted against Her2, Akt, c-Raf and β-actin as a control. The results are shown in Fig.

As shown in FIG. 6, the non-quinone geldanamycin glycoconjugate derivative represented by formula (1) according to the present invention binds to a heat shock protein (Hsp90) to inhibit the activity of Hsp90, Akt and c-Raf, respectively.

Therefore, the non-quinone geldanamycin glycosylated derivatives according to the present invention are excellent in the effect of inhibiting the expression of Her2, Akt, c-Raf, which is a substrate protein of a heat shock protein (Hsp90), and thus can be used as anticancer agents, antibiotics, Antiviral agents, immunosuppressive agents, therapeutic agents for degenerative neurological diseases, anti-inflammatory agents and the like.

< Experimental Example  4> Non-quinone Zeldanamycin The party  Determination of the solubility of derivatives

Conventionally, non-quinone type geldanamycin has excellent thermal shock protein inhibitory activity and low hepatic toxicity, but has a problem in evaluation of anticancer activity due to low solubility. In order to measure the solubility of the compound represented by formula (1) according to the present invention, the following experiment was conducted.

The solubility of the compound is determined by a method of HPLC analysis of the degree of the compound which extracts non-quinone geldanamycin and glycosylated non-quinone geldanamycin present in the buffer solution with ethyl acetate (EtOAc) solvent and moves to the water layer and the solvent layer Respectively.

As a result, the water solubility of the non-quinone geldanamycin glycoconjugate derivative according to the present invention was improved. Particularly, as shown in FIG. 7, a part of the glycation reaction solution reacted in the buffer solution was analyzed by HPLC Quinone geldanamycin derivative derivative peak according to the present invention shows a conversion of 48.8% from the non-quinone geldanamycin represented by the formula (A) used as a substrate before saccharification. However, the reaction solution was extracted with ethyl acetate (EtOAc) As a result of the extraction with a solvent and HPLC, peaks of the glycated compounds were found to be extremely small as shown in Fig. 7B, while peaks of the glycated compounds mostly present in the water layer after extraction were confirmed. This indicates that the non-quinone geldanamycin used as the substrate is not soluble in water, while the glycosylated compounds are well soluble in the water layer.

Therefore, the non-quinone geldanamycin derivative derivatives according to the present invention are not very superior in activity from the non-quinone geldanamycin used as a substrate, but they are very excellent in solubility, Because of the fact, it is possible to easily produce an injectable roll with high solubility of glucose-glycosylated compounds, and when administered in vivo, the glycosidase (the enzyme) present in the blood again causes the originally active structure of non-quinone zeldanas Can be advantageously used as prodrugs by taking advantage of the fact that they are converted into a mycine compound.

Claims (12)

A non-quinone geldanamycin derivative represented by the following general formulas (1) to (5): or a pharmaceutically acceptable salt thereof.
[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

delete Quinone geldanamycin derivative represented by the following formula (A), (B) or (C) as a substrate to prepare a non-quinone geldanamycin derivative Wherein the solubility of the non-quinone geldanamycin derivative is increased.
(A)

[Chemical Formula B]

&Lt; RTI ID = 0.0 &

4. The method according to claim 3, wherein the non-quinone geldanamycin derivative further comprises a step of separating the enzyme.
4. The method according to claim 3, wherein the glycoprotein is a UDP-glycosyltransferase derived from bacillus ricortosum.
[Claim 5] The method according to claim 3, wherein the sugar chain reaction is carried out at 25-35 &lt; [deg.] &Gt; C for 11-13 hours after addition of the UDP-tagged enzyme and UDP-glucose.
4. The method according to claim 3, wherein the glycation reaction is performed in an environment of pH 6.0-9.0.
delete An anticancer agent, an antibiotic, an antifungal agent, an antiviral agent, an immunosuppressant, a therapeutic agent for degenerative neurological disease or an anti-inflammatory agent containing a pharmaceutically acceptable salt of geldanamycin glycoside derivative represented by the following formula 1 or 4 as an active ingredient .
[Chemical Formula 1]

[Chemical Formula 4]

delete delete 10. The method of claim 9, wherein the cancer is selected from the group consisting of breast cancer, liver cancer, stomach cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, Wherein the cancer is a cervical cancer, a vaginal cancer, a vulvar carcinoma, a Hodgkin's disease, a prostate cancer, a bladder cancer, a renal cancer, a ureter cancer, a renal cell carcinoma, a renal pelvic carcinoma or a central nervous system tumor.

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