KR101456173B1 - Novel geldanamycin derivatives, pharmaceutically acceptable salt thereof, preparation method thereof and agent for the prevention and treatment of tumor containing the same as an active ingredient - Google Patents

Abstract

(상기 화학식 1에서, R은 본 명세서에서 정의된 바와 같다)The present invention relates to a novel geldanamycin derivative represented by the following general formula (1), a pharmaceutically acceptable salt thereof, a process for producing the same, and a prophylactic and therapeutic agent for cancer containing the same as an active ingredient. The geldanamycin derivative Inhibits the ATPase activity and inhibits the activity of Hsp90, a chaperon protein that plays an important role in the growth and metastasis of cancer cells. By inhibiting the expression of Her2 and c-raf proteins, it inhibits not only cancer treatment agents but also antibiotics, Antiviral agents, immunosuppressants, therapeutic agents for degenerative neurological diseases, anti-inflammatory agents and the like.
[Chemical Formula 1]

(In the above formula 1, R is as defined herein)

Description

TECHNICAL FIELD The present invention relates to a novel geldanamycin derivative, a pharmaceutically acceptable salt thereof, a process for producing the same, and a cancer preventing and treating agent containing the same as an active ingredient. tumor containing the same as an active ingredient}

The present invention relates to a geldanamycin derivative, a pharmaceutically acceptable salt thereof, a process for producing the same, and a prophylactic and therapeutic agent for cancer containing the same as an active ingredient.

Geldanamycin has been used as an initial precursor with 3-amino-5-hydroxybenzoic acid (AHBA) along with herbimycin, macbecin and reblastatin. The compounds have a polyketide skeleton structure biosynthesized. They were identified as antibiotics, antifungal agents, antiviral agents and anticancer agents between 1970 and 2000.

This geldanamycin was confirmed by Neckers et al. In 1994 to bind to the ATP binding site of heat shock protein 90 (Hsp90) having chaperone activity. Because of this discovery, the existing geldanamycin does not exhibit anticancer activity by inhibiting the enzyme activity of tyrosine kinase having an oncogenic protein function, but it is also known that the binding protein of various kinds of Hsp90 including tyrosine kinase inhibits the function of Hsp90, which is important for the structural stability of the client protein, and thus it is confirmed that the stability of these binding proteins is inhibited.

Because of the physiological importance of this Hsp90, the chemical synthetic derivatives of geldanamycin, 17-allyamino-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-dimethoxy geldanamycin (17- (dimethylaminoethylamino) -17-demethoxygeldanamycin, 17-DMAG) have been developed as anticancer drugs.

U.S. Patent Application No. 10 / 212,962 discloses benzoquinone ansamycin-like compounds useful for the treatment of cancer or other diseases caused by undesired hyperproliferation of cells and methods of making the same.

Korean Patent Application No. 2003-7008551 discloses a novel geldanamycin derivative and a preparation method thereof, and Korean Patent Application No. 2004-7004202 discloses a novel geldanamycin derivative using chemical synthesis of 17-allylaminogelnamamycin and other ansamycin And a manufacturing method thereof.

Korean Patent No. 860502 discloses a novel geldanamycin derivative from recombinant Streptomyces hygroscopicus mutants and a method for producing the same.

Accordingly, the inventors of the present invention focused on the fact that geldanamycin or a derivative thereof can be used as a therapeutic agent for various diseases related to cancer. Thus, while studying the recombinant Streptomyces hygroscopicus mutant, And found that the anticancer activity of a geldanamycin derivative is excellent, and the present invention has been completed.

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

Another object of the present invention is to provide a process for producing the geldanamycin derivative.

Yet another object of the present invention is to provide an anticancer agent containing the geldanamycin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide an antibiotic containing the geldanamycin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

It is still another object of the present invention to provide an immunosuppressant comprising the geldanamycin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a therapeutic agent for a degenerative neurological disease containing the geldanamycin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

Still another object of the present invention is to provide an anti-inflammatory agent containing the geldanamycin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide an antiviral agent containing the geldanamycin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to achieve the above object, the present invention provides a geldanamycin derivative represented by the following general formula (I) or a pharmaceutically acceptable salt thereof:

[Chemical Formula 1]

(here,

R is -OH or -OCONH _2).

The present invention also relates to a method for producing a recombinant Streptomyces hygroscopicus mutant, comprising the steps of: (1) preparing a culture solution by culturing a recombinant Streptomyces hygroscopicus mutant;

Extracting the culture medium of step 1 with ethyl acetate to prepare an extract (step 2);

Fractionating the extract of step 2 with ethyl acetate to obtain a fraction (step 3);

The fraction of step 3 is subjected to silica gel chromatography to prepare an active fraction (step 4); And

And isolating the compound of Formula 1 from the active fraction of Step 4 (Step 5).

Further, the present invention provides a pharmaceutical composition for preventing and treating cancer diseases, which comprises the geldanamycin derivative represented by the above formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides an antibiotic comprising the geldanamycin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

Further, the present invention provides a pharmaceutical composition for the prevention or treatment of immunosuppressive diseases containing the geldanamycin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating degenerative neurological diseases containing the geldanamycin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

Further, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease containing the geldanamycin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating viral diseases containing the geldanamycin derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

The geldanamycin derivatives represented by formula (1) according to the present invention are excellent in anticancer activity which inhibits ATPase activity and inhibits the activity of Hsp90, a chaperon protein that plays an important role in the growth and metastasis of cancer cells. Her2 and c-raf And thus can be effectively used as an antibiotic, an antifungal agent, an antiviral agent, an immunosuppressant, a therapeutic agent for degenerative neurological disease, an anti-inflammatory agent and the like, as well as a preventive and therapeutic agent for various cancer diseases.

1 is a fragmentary view according to one embodiment of the present invention.
2 is a diagram showing the result of SDS-PAGE in which a heat shock protein (Hsp90) of yeast is isolated and purified.
FIG. 3 is a graph showing the inhibitory effect of a compound according to an embodiment of the present invention on the expression of Her2 and c-raf, which are substrate proteins of a heat shock protein.

Hereinafter, the present invention will be described in detail.

The present invention provides a geldanamycin derivative represented by the following formula (I) or a pharmaceutically acceptable salt thereof.

(In the formula 1,

R is -OH or -OCONH _2. )

The present invention includes all of the novel geldanamycin derivatives represented by the above formula (1) or pharmaceutically acceptable salts thereof as well as possible solvates, hydrates or prodrugs thereof which can be prepared therefrom.

The derivatives of formula (I) of the present invention can be used in the form of pharmaceutically acceptable salts, and as salts, acid addition salts formed by pharmaceutically acceptable free acids 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 Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, 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 compound of formula (1) in an excess amount of an acid aqueous solution, and using this salt in a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile Followed by precipitation. By heating the same amount of the compound of formula (I) and an acid or alcohol in water, and then evaporating and drying the mixture, or by suction filtration of the precipitated salt.

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 an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

The present invention also provides a process for preparing a novel geldanamycin derivative of formula (1). Here, the geldanamycin derivative may be isolated from the recombinant Streptomyces hygroscopicus mutant extract, but is not limited thereto.

As an example, the geldanamycin derivative may be prepared by, for example,

Culturing the recombinant Streptomyces hygroscopicus mutant to prepare a culture (step 1);

Extracting the culture medium of step 1 with ethyl acetate to prepare an extract (step 2);

Fractionating the extract of step 2 with ethyl acetate to obtain a fraction (step 3);

The fraction of step 3 is subjected to silica gel chromatography to prepare an active fraction (step 4); And

Separating the compound of formula (1) from the active fraction of step (4) (step 5).

At this time, the fractionation process from step 3 to step 5 is shown in FIG.

Hereinafter, the production method of the present invention will be described step by step.

In step 1 according to the present invention,

To obtain a culture solution of a recombinant Streptomyces hygroscopicus mutant. Herein, the mutant is a "recombinant Streptomyces hygroscopicus mutant" which produces a compound similar to geldanamycin, and a variant known in Korean Patent Registration No. 860502 is used. Specifically, the gene cluster of the strain biosynthesizing geldanamycin contains three polyketide synthase genes ( gelA- C ), an amide synthase gene ( gelD ), a putative hydroxylase gene ( gel1 , 7 & 16 ) and carbamoyltransferase ( gel8 ). Among them, the gene function of the hydroxylase gene (gel7) disappeared together with the dehydratase domain of the polyketide synthetic gene (gelA) .

In step 2 according to the present invention,

And extracting the culture solution obtained in the step 1 with ethyl acetate to prepare an extract. Specifically, it can be produced by performing the following steps.

a) extracting the culture medium with an extraction solvent;

b) cooling and then filtering the extract of step a); And

c) concentrating the filtered extract of step b) under reduced pressure and drying.

In step 2, the extraction solvent in step a) may be water, ethyl acetate, alcohol or a mixture thereof. As the alcohol, C ₁ to C ₄ lower alcohol is preferably used, and ethyl acetate is most preferably used, but not limited thereto. Examples of the extraction method include, but are not limited to, filtration, hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction. The extraction solvent may be added 1 to 10 times, preferably 1 to 3 times, the weight of the culture liquid. The extraction temperature is preferably 20 to 30 占 폚, but is not limited thereto. The extraction time is preferably from 24 to 72 hours, but is not limited thereto.

In the step 2, it is preferable to use a vacuum decompression concentrator or a vacuum rotary evaporator for the decompression concentration in the step c), but it is not limited thereto. The drying is preferably performed under reduced pressure, vacuum drying, boiling, spray drying or freeze drying, but not always limited thereto.

In step 3 according to the present invention,

The step of extracting the culture broth obtained in the step 2 with ethyl acetate to obtain fractions. Specifically, water and ethyl acetate are added to the ethyl acetate extract, and the organic layer is separated into an aqueous layer and an organic layer using a separatory funnel. The organic layer fraction is concentrated under reduced pressure using a vacuum distillation to obtain an ethyl acetate fraction.

In step 4 according to the present invention,

The ethyl acetate fraction obtained in the step 3 is subjected to silica gel chromatography to separate active fractions. Specifically, the ethyl acetate fraction obtained in the above step 3 was separated into active fractions (A to F) by silica gel chromatography using a mixed solvent of CH ₂ Cl ₂ to MeOH = 100% to 100% as a mobile phase, Of these, fraction C was used as the active fraction in the next step.

In step 5 according to the present invention,

And isolating the compound of formula (I) from the active fraction obtained in step (4). Specifically, the active fraction C1 (Compound 1a) and C2 (Compound 1b) were obtained by subjecting the active compound of Formula 1 to active fraction C obtained in Step 4 using MeCN-H ₂ O as an eluting solvent have. The compounds 1a and 1b thus isolated can be identified by using an analyzer such as a mass spectrometer, NMR or IR.

Further, the present invention provides a pharmaceutical composition for the prevention and treatment of cancer diseases, which comprises the novel geldanamycin derivative represented by the above formula (1) as an active ingredient.

The cancer disease may be 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, rectum cancer, esophageal cancer, small bowel cancer, anal cancer, colon cancer, fallopian tube carcinoma, endometrial carcinoma, , Vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, renal cancer, ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor,

The present invention also provides an antibiotic comprising the novel geldanamycin derivative represented by the above formula (1) as an active ingredient. At this time, the antibiotic includes an antibacterial agent and an antifungal agent.

Further, the present invention provides a pharmaceutical composition for the prevention and treatment of immunosuppressive diseases containing the novel geldanamycin derivative represented by the above-mentioned formula (1) as an active ingredient. The immunosuppressive disease may be an autoimmune disease including organ transplantation and rheumatoid arthritis or an immune related disease associated with allergy such as atopic or allergic rhinitis.

The present invention also provides a pharmaceutical composition for preventing and treating degenerative neurological diseases containing the novel geldanamycin derivative represented by Formula 1 as an active ingredient. At this time, the degenerative neurological disease may be Alzheimer's disease, Down syndrome disease and the like.

Furthermore, the present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory diseases containing the novel geldanamycin derivative represented by the above formula (1) as an active ingredient. The inflammatory disease may be asthma, periodontitis, stomatitis, peritonitis, gastritis, enteritis, arthritis, nephritis, hepatitis and the like.

The present invention also provides a pharmaceutical composition for the prevention and treatment of viral diseases containing the novel geldanamycin derivative represented by the above formula (1) as an active ingredient. Herein, the viral disease may be a cockscomb virus disease caused by coxsack virus or a lynoviral disease caused by a lynovirus. The Coxsackie viral disease may be myocarditis, pericarditis, meningitis, otitis media, epidemic pleural effusion, etc., and the renoviral disease may be general cold.

The inventors of the present invention measured the effect of inhibiting the ATPase activity of the geldanamycin derivative according to an embodiment of the present invention and found that the inhibitory activity against ATPase activity was superior to that of the conventional compound DHQ3 as well as geldanamycin (see Table 3) ).

In addition, the inventors of the present invention measured the anticancer activity of geldanamycin derivatives according to one embodiment of the present invention and found that the anticancer activity was about 2.6 times higher than that of the conventional compound DHQ3 (see Table 4).

Further, the present inventors measured the inhibitory effect of the geldanamycin derivative on the expression of the cancerous protein according to an embodiment of the present invention, and found the effect of inhibiting the expression of c-raf protein including Her2, a cancer-associated phosphorylase enzyme 3).

Accordingly, the geldanamycin derivatives represented by formula (1) according to the present invention are excellent in anticancer activity which inhibits ATPase activity and inhibits the activity of Hsp90, a chaperon protein that plays an important role in cancer cell growth and metastasis, and Her2 and c -raf protein, it can be used not only as a preventive and therapeutic agent for various cancer diseases, 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.

When the composition of the present invention is used as a medicament, the pharmaceutical composition containing the novel geldanamycin derivative represented by the above formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient can be administered orally, The composition may be formulated and administered in a parenteral administration form, but is not limited thereto.

The pharmaceutical composition containing the novel geldanamycin derivative of the formula (1) or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient can be administered orally or parenterally, including oral or mucosal administration, intravenous administration, And can be provided in the form of a general pharmaceutical preparation. When the composition of the present invention is formulated, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, and surfactants that are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain one or more excipients such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stylactone are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for oral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-drying agents, suppositories, and the like. Non-aqueous solvents and suspensions may include esters such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and ethyl oleate, which can be administered by injection. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.

The composition containing the novel geldanamycin derivative of formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient according to the present invention is effective for inhibiting ATPase activity, Hsp90 activity, Her2 and c-raf protein expression And the like. For example, the composition of the present invention can be administered to a human in a single dose or several times, and the dose is 0.1 to 1000 mg, preferably 0.1 to 500 mg. However, it can be increased or decreased depending on route of administration, severity of disease, sex, weight, age and the like. Therefore, the dose is not intended to limit the scope of the present invention in any way.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited thereto.

< Example  1 to 2 > Zeldanamycin  Preparation of derivatives

AC15  Culture of strain

As the strain, AC15, which is a mutant strain known in the literature (Korean Patent Registration No. 860502), was used. Specifically, the gene cluster of the strain biosynthesizing geldanamycin contains three polyketide synthase genes ( gelA- C ), an amide synthase gene ( gelD ), a putative hydroxylase gene ( gel1 , 7 & 16 ) and carbamoyltransferase ( gel8 ). Among them, the gene function of the hydroxylase gene (gel7) disappeared together with the dehydratase domain of the polyketide synthetic gene (gelA) Lt; / RTI &gt; strain (AC15). In this strain (AC15), a compound having a skeleton such as geldanamycin, but a compound DHQ3 containing a hydroxyl group at the carbon 15 position and DHQ5 and DHQ6 in a tricyclic form (Hong et al. Tetrahedron letters 51, 351-353, 2010) were reported to be biosynthesized.

The transformed strain (AC15) was firstly cultured in a medium supplemented with yeast extract-malt extract to 300 ml of liquid glycerol for 3 days at 28 ° C using spores. The primary culture was inoculated in a 1 liter flask with 300 ml of the same medium as above to a final concentration of 5%, and then cultured at 28 ° C for 7 days at 150 rpm. Thirty flasks were cultured at the same time to obtain a culture solution of about 10 L of the strain.

Extraction of the compound of formula (1)

To prepare the compound of formula (1) according to the present invention, the following procedure was carried out.

10 liters of the culture prepared above was extracted twice with EtOAc in the same amount, and the extract was filtered to remove insolubles, which was then concentrated and then fractionated with EtOAc and water to obtain 2.7 g of an organic phase extract. Specifically, the mutant strains (AC15) cultured as described above were extracted with EtOAc and subjected to silica gel chromatography using CH ₂ Cl ₂ -MeOH as a mobile phase to obtain fractions (A to F). The fraction C obtained by performing silica gel chromatography using CH ₂ Cl ₂ : MeOH = 85: 15 as a mobile phase was eluted with 10% aqueous solution starting from MeCN: H ₂ O (0.05% TFA) = 10: (Waters Delta Prep 3000 system, Waters, USA; [YMC-J'sphere ODS-H80 250 X 10 mm id, 3 ml / min]) was added stepwise with increasing MeCN. Fraction C1 was obtained 30 minutes after the preparative HPLC and fraction C2 was obtained at 35 minutes. At this time, 31.8 mg of the compound of Formula 1a ( Example 1) and 17.6 mg of the compound of Formula 1b ( Example 2) were obtained from the fraction C1 obtained by performing the preparative HPLC.

&Lt; Analysis > Identification of compounds of formulas Ia and Ib

In order to identify the compound thus prepared, the melting point was measured without correction using Electrothermal 9100 equipment (Electrothermal, UK), and specific rotation ([?] _D ²⁵ ) was measured using JASCO DIP -370 Polarimeter (JASCO, Japan), and UV was measured using a Shimadzu UV-1601 spectrophotometer (Shimadzu, Japan). ESI-MS was obtained by Finnigan LCQ Advantage Max Mass Spectrophotometer (Thermo, USA). HRESI-MS was purchased from Shimadzu LCMS-IT-TOF mass spectrophotometer (Shimadzu Corp., Japan). The above analysis results are shown below.

Example 1 (Formula 1a): white powder; specific rotation [?] ²⁷ _D +11.1 (c, 0.10, MeOH); UV (MeOH)? Max (log?) 204 (4.55), 260 (3.77), 289 nm (3.69); ESI-MS m / z 555.8 [M + Na] &lt; ⁺ &gt;, 531.8 [MH]; HRESI-MS (m / z 555.2673 [M + Na] +, calculated 555.2677 for C 28 H 40 N 2 NaO 8).

Example 2 (Formula 1b): white powder; _{^{specific rotation [α] 27 D -24.8}} (c, 0.10, MeOH); UV (MeOH)? Max (log?) 203 (4.43), 262 (3.70), 295 nm (3.62); ESI-MS m / z 598.8 [M + Na] &lt; ⁺ &gt;, 574.8 [MH]; HRESI-MS ( m / z 598.2733 [M + Na] ⁺ , calculated 598.2735 for C ₂₉ H ₄₁ N ₃ NaO ₉ ).

In addition, NMR analyzes for Formulas Ia and Ib were measured with a Varian UNITY 400 spectrophotometer using DMSO-d ₆ . ^The results of ¹ H NMR analysis are shown in Table 1, and the results of ¹³ C NMR analysis are shown in Table 2 below.

¹ H NMR ^a zeldanamycin ^b Comparative Example 1 ^c Example 1 ^c Example 2

One 2 3 6.94 brd (9.9) 5.58 brd (7.2) 5.89 dd (4.8, 7.2) 6.01 t (6.0) 4 6.57 brt (10.2, 10.8) 2.04 m; 2.72 overlap 2.12 m; 2.23 m 2.15m 5 5.88 brt (9.0, 10.2) 1.11 m 1.42 m; 1.62 m 1.33 m; 1.45 m 6 4.32 d (9.0) 3.22 t (8.4) 2.96 t (8.4) 3.22 m 7 5.19 h 4.87 overlap 4.80 d (4.8) 4.79 d (8.8) 8 9 5.82 d (7.8) 5.16 d (9.6) 5.47 d (9.2) 5.31 d (10.4) 10 2.79m 2.34 m 2.57 m 2.82m 11 3.53 d (6.6) 3.64 dd (2.8, 9.2) 3.88 d (4.8) 4.98 brs 12 3.39 d (7.2) 3.07 dt (4.0, 11.2) 13 1.74 m 0.75 brt (10.8);
1.77 dt (3.6, 10.8) 5.15 d (10.4) 5.01 d (10.4) 14 1.68 brs 2.57 m 2.53 m 15 2.47 m 4.47 d (4.0) 4.39 brs 4.34 brs 16 17 6.37 s 6.27 s 6.26s 18 19 7.29 h 6.49 h 7.49 brs 7.48 s 20 21 6.73 brs 6.48 s 22 2.03 h 1.86 s 1.78 s 1.75s 23 1.80 s 1.37 s 1.50 s 1.59s 24 0.98 overlap 1.02 d (6.8) 0.77 d (6.4) 0.88 d (7.2) 25 0.98 overlap 0.65 d (7.2) 1.10 d (6.8) 1.11 d (6.8) 6-OCH ₃ 3.30s 3.42 s 3.38 s 3.37 s 12-OCH ₃ 3.36 h 3.35 s 2.68 s 2.77 s 17-OCH ₃ 4.13 h 7-OCONH ₂ 11-OCONH ₂ 11-OH 4.76 d (5.6) 15-OH 5.10 d (2.8) 5.07 d (3.2) 18-OH 9.13 s 9.19 s NH 8.72 s 9.33 h 9.22 s ^a CDCl _3; ^b CD ₃ OD; ^c DMSO-d ₆

¹³ C NMR ^a zeldanamycin ^b Comparative Example 1 ^c Example 1 ^c Example 2

One 169.19 s 176.01 s 168.32 s 168.45 s 2 134.83 s 131.83 s 132.43 s 132.05 s 3 127.26 d 135.56 d 136.73 d 136.90 d 4 126.28 d 23.88 t 23.75 t 23.92 t 5 136.46 d 31.19 t 30.64 t 30.80 t 6 81.28 d 80.75 d 79.89 d 80.60 d 7 81.72 d 83.97 d 81.14 d 81.40 d 8 133.29 s 131.68 s 130.02 s 131.93 h 9 133.15 d 134.28 d 136.10 d 133.47 d 10 32.33 d 36.22 d 32.66 d 31.60 d 11 72.70 d 74.12 d 71.83 d 73.70 d 12 81.02 d 80.30 d 154.98 s 150.26 s 13 34.69 t 31.61 t 111.86 d 112.64 d 14 27.93 d 36.22 d 37.35 d 37.39 d 15 32.74 t 79.25 d 76.75 d 76.75 d 16 127.61 s 144.85 s 146.80 s 146.86 s 17 157.00 h 113.41 d 108.07 d 108.42 d 18 184.13 s 158.52 s 156.05 s 156.93 s 19 111.74 d 109.90 d 103.76 d 103.48 d 20 138.08 s 141.45 s 139.42 s 139.50 s 21 184.97 s 116.28 d 110.25 d 109.35 d 22 12.51 q 14.23 q 12.62 q 12.48 q 23 12.86 q 12.14 q 11.47 q 11.64 q 24 12.40 q 18.43 q 12.17 q 12.80 q 25 22.93 q 12.25 q 18.71 q 19.07 q 6-OCH ₃ 57.23 q 59.89 q 59.21 q 58.74 q 12-OCH ₃ 56.72 q 57.12 q 56.57 q 56.66 q 17-OCH ₃ 61.66 q 7-OCONH ₂ 156.03 s 159.14 s 156.86 s 155.99 s 11-OCONH ₂ 156.95 s NH ^a CDCl3; ^b CD3OD; ^c DMSO-d ₆

From the above analysis results, it was confirmed that the active fraction C1 was the compound of formula (1a) (Example 1) and the active fraction C2 was the compound of formula (Ib) (Example 2). In addition, it was confirmed that the compound DHQ3 found in the prior art (Korean Patent No. 860502) and the compound 1a of Example 1 differ in the presence or absence of double bonds at positions 12 and 13 in terms of structure.

< Comparative Example  1> Compound DHQ3 Manufacturing

In the extraction step of the compound of Example 1, Compound DHQ3 was obtained from fraction B obtained by performing silica gel chromatography using CH ₂ Cl ₂ : MeOH = 85: 15 as a mobile phase. At this time, the compound DHQ3 was confirmed in the same manner as in the assay method used in Example 1, except that NMR analysis was performed using CD ₃ OD. The results are shown in Table 1 and Table 2 together with the results of NMR analysis of geldanamycin, Example 1 and Example 2.

< Experimental Example  1> ATPase  Measurement of inhibitory activity

In order to measure the ATPase inhibitory activity of the compounds according to the present invention, the following experiment was conducted.

One. Thermal shock  Separation of protein purification

First, the heat shock protein (Hsp90) of yeast was separated and purified as follows.

5'-CAT ATG GCT AGT GAA ACT TTT GAA TTT CAA G-3 '(forward primer) introduced with NdeI restriction enzyme site to clone gene encoding Hsp90 protein and 5'-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, 10 minutes), and E. coli was suspended in 3 ml of a buffer (100 mM Tris-HCl, 20 mM KCl, 6 mM MgCl ₂ , imidazole 10 mM, 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, three handles 6mM MgCl _2, imidazole20mM, pH7.4), and finally the elution buffer (elution buffer, 100mM Tris-HCl , 20mM KCl, 6mM MgCl 2, imidazole250mM, pH7.4) was treated five times by 0.5 ㎖ After each fraction was confirmed by SDS-PAGE, it was confirmed that the Hsp90 protein was isolated. The results are shown in Fig.

2 is a diagram showing the result of SDS-PAGE in which a heat shock protein (Hsp90) of yeast is isolated and purified.

2. 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 , and molybdate (5.72%, w / v, in 6 M HCl), and water in the ratio (0.0812%, w / v), polyvinyl alcohol (2.32%, w / v; dissolves with difficulty and requires heating) 2: 1: 1: 2).

ATPase inhibitory activity was determined by adding 1 μl of each compound (geldanamycin, DHQ3, 1a and 1b) at concentrations of 10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001 and 0 μM, 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) were prepared and reacted at 37 ° C. for one hour. After adding 10 μl of 2.5 mM ATP and shaking for 1 minute, the reaction solution was reacted at 37 ° C for 3 hours, then 80 μl of malachite reaction solution was added, and 10 μl of 34% sodium citrate was added thereto, And the reaction is stopped. 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 3 below.

compound

ATPase inhibition IC ₅₀ value (μM)
Zeldanamycin 3.19 Comparative Example 1 1.84 Example 1 1.75 Example 2 5.87

As shown in Table 3, the geldanamycin derivative according to the present invention showed an excellent ATPase inhibitory activity (IC ₅₀ ) of 1.75 μM in Example 1 and 5.87 μM in Example 2. In particular, the compound of Example 1 of the present invention exhibited a higher inhibitory activity than the conventionally known geldanamycin derivative compound DHQ3 (Comparative Example 1) than the ATPase inhibitory activity (1.84 [mu] M).

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

In order to measure the cytotoxicity of the compound of formula (1) according to the present invention to cancer cells, the following experiment was conducted.

Cancer cell lines used human ovarian cancer (A2780), breast cancer (SK-Br3 and BTB474). 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, 0.1, 0.01, 0.001, 0.0001, 0.00001, and 0 μM, respectively, and the compounds obtained in Comparative Example 1, Example 1 and Example 2 were added and treated 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 4 below.

Cancer cell survival IC ₅₀ value compound Ovarian cancer (A2780) Breast cancer (SK-Br3) Breast cancer (BTB474)
Comparative Example 1
24 μM
40 μM
16 μM
Example 1
13 μM
15 μM
8 μM
Example 2
17 μM
173 [mu] M
63 μM

As shown in Table 4, the compound 1a according to the present invention (Example 1) showed a maximum 2.6-fold increase in anticancer activity compared to the compound DHQ3 (Comparative Example 1) found in the prior art (Korean Patent No. 860502) Respectively.

Therefore, the compound according to the present invention has remarkably improved anticancer activity, and thus can be effectively used as an anticancer agent.

< Experimental Example  3> Her2  And c- shelf  Evaluation of Substrate Protein Expression Inhibition

In order to measure the inhibitory effect of the compounds 1a and 1b according to the present invention on the expression of Her2 (kinase, cancer-associated phosphorylase) and c-raf, which are substrate proteins of heat shock protein (Hsp90), 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. Compound DHQ3 (Korean Patent No. 860502) and Compound 1a of Example 1 were dissolved in DMSO (250, 150, 50, 1 μM). 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 onto SDS-PAGE and subjected to immunoblotting for β-actin as Her2, c-raf and a control group. The results are shown in Fig.

FIG. 2 is a graph showing the inhibitory effect of a compound according to an embodiment of the present invention on the expression of Her2 and c-raf, which are substrate proteins of a heat shock protein.

As shown in Fig. 2, Compound 1a of Example 1 binds to a heat shock protein (Hsp90) to inhibit the activity of Hsp90, confirming that the effect of inhibiting the expression of Her2 and c-raf, which are substrate proteins of Hsp90 I could.

Therefore, the compound according to the present invention is excellent in the effect of inhibiting the expression of Her2 and c-raf, which are substrate proteins of heat shock protein (Hsp90), and thus can be used not only as anticancer agent but also as antibiotic, antifungal, antiviral, immunosuppressive, Anti-inflammatory agents and the like.

Examples of formulations using the composition of the present invention are illustrated below.

&Lt; Formulation Example 1 > Preparation of pharmaceutical preparation

<1-1> Preparation of powder

2 g &lt; RTI ID = 0.0 &gt;

Lactose 1 g

After mixing the above components, the mixture was packed in an airtight container to prepare a powder.

<1-2> Preparation of tablets

100 mg of the compound of formula (1)

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

&Lt; 1-3 > Preparation of capsules

100 mg of the compound of formula (1)

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

&Lt; 1-4 >

1 g of the compound of formula (1)

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

<1-5> Preparation of granules

150 mg of the compound of formula (1)

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

<1-6> Preparation of Injection Solution

10 [mu] g / ml of the compound of formula (1)

Until dilute hydrochloric acid BP pH 3.5

Sodium chloride for injection Up to 1 ml

The compound of formula 1 was dissolved in a suitable volume of sodium chloride BP for injection. The pH of the resulting solution was adjusted to pH 3.5 with dilute hydrochloric acid BP, and the volume was adjusted using sodium chloride BP for injection and mixed thoroughly. The solution was filled in a 5 ml type I ampoule made of transparent glass, sealed in an upper lattice of air by dissolving the glass, sterilized by autoclaving at 120 DEG C for 15 minutes or longer, and an injection solution was prepared.

Claims (16)

A geldanamycin derivative represented by the following formula (1a) or a pharmaceutically acceptable salt thereof:
[Formula 1a]

Histidine in the functional amino acid of the dihydrotartrate of the module 1 involved in the formula of the geldanamycin carbon 15 of Streptomyces hygroscopicus subsp. Duamyceticus is replaced by glutamine, and the gel7 gene A step of culturing a recombinant Streptomyces hygroscopicus mutant transformed with a vector containing a mutated inactivated construct (step 1);
Extracting the culture medium of step 1 with ethyl acetate to prepare an extract (step 2);
Fractionating the extract of step 2 with ethyl acetate to obtain a fraction (step 3);
The fraction of step 3 is subjected to silica gel chromatography to prepare an active fraction (step 4); And
And separating the geldamycin derivative of claim 1 from the active fraction of step 4 (step 5).
A pharmaceutical composition for preventing and treating cancer diseases, which comprises the geldanamycin derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
The method according to claim 3, wherein the cancer is selected from the group consisting of breast cancer, liver cancer, stomach cancer, colon cancer, pancreatic cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, esophageal cancer, small intestine cancer, , A cancer selected from the group consisting of cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, prostate cancer, bladder cancer, kidney cancer, ureter cancer, kidney cell carcinoma, renal pelvic carcinoma and central nervous system tumor Or a pharmaceutically acceptable salt thereof.
An antibiotic comprising the geldanamycin derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
The antibiotic according to claim 5, wherein the antibiotic comprises an antimicrobial agent and an antifungal agent.
An immunosuppressant comprising the geldanamycin derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
8. The immunosuppressant according to claim 7, wherein said immunosuppressive agent is used for the treatment of autoimmune diseases or allergies.
A pharmaceutical composition for preventing or treating degenerative neurological diseases containing the geldanamycin derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
10. The pharmaceutical composition according to claim 9, wherein the degenerative neurological disease is Alzheimer's disease.
A pharmaceutical composition for preventing or treating an inflammatory disease containing the geldanamycin derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
12. The pharmaceutical composition according to claim 11, wherein the inflammatory disease is asthma, periodontitis, stomatitis, peritonitis, gastritis, enteritis, arthritis, nephritis or hepatitis.
A pharmaceutical composition for preventing or treating a viral disease comprising the geldanamycin derivative of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
14. The pharmaceutical composition for preventing or treating viral diseases according to claim 13, wherein the viral disease is a cockscomb virus disease caused by coxsack virus or a lignovirus disease caused by a rhinovirus.
15. The pharmaceutical composition for preventing or treating viral diseases according to claim 14, wherein the Coxsackie viral disease is myocarditis, pericarditis, meningitis, hydrocephalus or an infective pleural effusion.
15. The pharmaceutical composition for preventing or treating viral diseases according to claim 14, wherein the renoviral disease is general cold.

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