KR100225807B1 - 3-(5-(hexa-2,4-dienylidene)-2-oxo-5,6-dihydro-2h-pyran-3-yl)-propionic acid, process for the preparation thereof and composition for type iv collagenase inhibitor containing same - Google Patents

Abstract

The present invention is a novel compound that inhibits the activity of type IV collagenase (separated from the culture medium of Westerdykella multispora strain isolated from soil, type IV collagenase, a method for preparing the same and the same The present invention relates to a composition for inhibiting type IV collagenase activity, wherein the novel compound 3- (5- (hexa-2,4-dienili) separated and purified from the culture solution of Westerdella multispora according to the present invention. Den) -2-oxo-5,6-dihydro-2H-pyran-3-yl) -propionic acid has an excellent effect of inhibiting the activity of type IV collagenase, so it can be used as a preventive and therapeutic agent for cancer metastasis and tumors. Can be.

Description

3- (5- (hexa-2,4-dienylidene) -2-oxo-5,6-dihydro-2H-pyran-3-yl) -propionic acid, preparation method thereof and type IV collagen comprising the same Composition for inhibiting naze activity {3- (5- (HEXA-2,4-DIENYLIDENE) -2-OXO-5,6-DIHYDRO-2H-PYRAN-3-YL) -PROPIONIC ACID, PROCESS FOR THE PREPARATION THEREOF AND COMPOSITION FOR TYPE IV COLLAGENASE INHIBITOR CONTAINING SAME}

The present invention is a novel compound that inhibits the activity of type IV collagenase purified from the culture medium of Westerdykella multispora strain isolated from the soil, a preparation method thereof and IV comprising the same The present invention relates to a composition for inhibiting type IV collagenase activity.

The main cause of death in cancer patients is not due to early tumors, but to tumor cell infiltration and metastasis. An important part of cancer metastasis is the degradation of proteins in the extracellura matrix and basement membrane, the support structures through which tumor cells cannot pass normally ( Cell, 64 , 327-336 (1991)). . Type IV collagenase is one of the matrix metalloproteinases and the major structural component of the basement membrane, the first barrier of tumor cell metastasis, is an enzyme that breaks down type IV collagen. It is an important enzyme ( TIBTECH., 10, 200-207 (1992)). Therefore, the development of inhibitors specific for type IV collagenase may be used as a useful drug for diseases such as rheumatoid arthritis, periodontitis, etc., the degradation of collagen connective tissue is an important cause.

Type IV collagenase inhibitors include protein-derived inhibitors (TIMPs) secreted by tumor cells ( J. Biol. Chem., 266, 13070-13075 (1991)). There is a limit to development. As synthetic inhibitors, batimastat ( Cancer Res., 53, 2087-2091 (1993)) and peptide derivatives (GM 6001) ( Cancer Res., 54, 4715-4718 (1994)) have been shown to prevent cancer metastasis. It has been reported to be suppressed. Inhibitors produced by microorganisms include matlystatin ( J. Antibiotics, 45, 1723-1732 (1992)), which is isolated from strains of the genus Actinomadura, which inhibits fibrosarcoma infiltration. As reported, BE16627B ( Agents Actions, 389, 182-186 (1993)) has been isolated from Streptomyces strains.

The inventors of the present invention, while continuing to research the development of a substance showing a strong inhibitory activity against type IV collagenase, incubated the Westerdykella multispora strain isolated from the soil, and from the obtained culture By discovering that the isolated new compound inhibits the type IV collagenase activity, the present invention is isolated from the Westerdella multispora to isolate the new compound that can inhibit the type IV collagenase activity and to identify its chemical structure. To complete.

An object of the present invention is to provide a novel compound and method for preparing the same, which inhibit the activity of type IV collagenase isolated from the culture medium of Westerdykella multispora strain.

Another object of the present invention to provide a composition for inhibiting type IV collagenase activity containing the novel compound.

FIG. 1 shows the infrared spectrum of novel gelatinta A and isomer B compounds in potassium bromide (KBr) tablets.

FIG. 2 shows the proton nuclear magnetic resonance spectra of gelastatin A and isomer B thereof in deuterium methanol (CD ₃ OD) solution.

FIG. 3 shows the carbon nuclear magnetic resonance spectra of gelastatin A and its isomer B in deuterium methanol (CD ₃ OD) solution.

In order to achieve the above object, in the present invention, 3- (5- (hexa-2,4-dienylidene) -2-oxo-5,6-dihydro-2H-pyran-3-yl) -propionic acid and It provides Westerdykella multispora F-50733 strain to produce.

In the present invention, Westerdykella multispora ( Westdykella multispora ) strain, comprising the steps of culturing, extracting the culture or mycelium with an organic solvent, and concentrating and purifying the obtained extract, Provided are methods for preparing the compounds.

In order to achieve the above another object, the present invention provides a composition for inhibiting type IV collagenase activity comprising the compound effective amount and a pharmaceutically acceptable carrier.

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

3- (5- (hexa-2,4-dienylidene) -2-oxo-5,6 having type IV collagenase inhibitory activity isolated from the culture medium of the Westerdella multispora strain according to the present invention -Dihydro-2H-pyran-3-yl) -propionic acid (3- (5- (hexa-2,4-dienylidene) -2-oxo-5,6-dihydro-2H-pyran-3-yl) -propionic acid) compound is 3- (5E- (hexa-2E, 4E-dienylidene) -2-oxo-5,6-dihydro-2H- of formula (1) wherein the 5 position of the pyranone ring is E-form. 3- (5Z- (hexa-2E, 4E-dienylidene) -2-oxo-5 of Formula 2 wherein pyran-3-yl) -propionic acid and the optical isomer thereof have the 5-position of the pyranone ring in the Z-form; , 6-dihydro-2H-pyran-3-yl) -propionic acid, wherein the compound of Formula 1 is named gelastat A, and its isomer is represented by gelatin.

Westerdykella mulltispora F-50733 strain of the present invention has been deposited with the Korean National Institute of Science and Technology Biotechnology Research Institute Gene Bank as July 22, 1996 as Accession No. KCTC 0265BP. Like the other fungi, the Westerdella multispora F-50733 strain is prone to change in appearance, even though the mutant strain (naturally occurring or induced), fusion or genetic recombinant of F-50733 may contain All productive Westerticella multispora strains are included within the scope of the present invention.

The method for producing the compound gelastat A and isomer B thereof from the Westerdykella multispora strain will be described in detail. That is, nutrient sources (for example, glucose, lactose, etc. are used as carbon sources, tryptone, peptone, etc. are used as nitrogen sources, and magnesium sulfate and other inorganic salts are added as needed. Incubate the Westerdella multispora F-50733 strain (KCTC 0265BP) under appropriate conditions, for example, at a temperature in the range of 23 to 28 ° C. At this time, the culture method is preferably a culture method under aerobic conditions, in particular liquid immersion culture method. When the culture was completed, centrifugation was performed to purify the culture supernatant using solvent extraction, adsorption chromatography, gel filtration chromatography, and high pressure liquid chromatography to obtain compound gelastatin A and isomer B having collagenase inhibitory activity. can do.

The production of gelastatin A and its isomer B during culturing or purification can be confirmed using the IV inhibition of collagenase as follows.

That is, the activity detection enzyme having a molecular weight of 72,000 Daltons (Da) through a process such as ammonium sulfate precipitation, gel filtration chromatography, gelatin affinity chromatography, lectin affinity chromatography, heparin affinity high pressure liquid chromatography from serum-free cultures of human glioma cells. Phosphorus type IV collagenase was isolated ( J. Biochem. Mol. Biol., 28, 33-39 (1995)), and the type IV collagenase activity screening substrate was 7-methoxycumarin-4-acetyl-proline Samples were placed on a microplate using leucine-glycine-leucine- (3- [2,4-dinitrophenyl] -2,3-diaminopropyl) -alanine-arginine-amide (Sigma) Put the type IV collagenase enzyme and measure the amount of fluorescence with a spectrofluorimeter, and then enter the substrate on the microplate and react and calculate the amount of the type IV collagenase inhibition (%) by measuring the amount of fluorescence again. , IC ₅₀ The measures.

Since the novel compounds of the present invention can inhibit the activity of type IV collagenase, it can be used as a prophylactic and therapeutic agent for cancer metastasis and tumors.

That is, the unit, such as tablets, capsules, powders, granules, suspensions, emulsions or parenteral formulations, which are purified and separated from the culture solution of the Westerdykella multispora strain as described above by conventional methods. It can be formulated as a dosage form or multiple dose formulation and used as a prophylactic and therapeutic agent for cancer metastasis and tumors.

The composition of the present invention comprising the novel compound of the present invention as an active ingredient and a pharmaceutically acceptable carrier may be parenterally or orally administered as desired, and 5 to 20 mg of gelatin at 1 kg of body weight per day. It can be administered in an amount of. Dosage levels for a particular patient may vary depending on the particular compound to be used, weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, drug mixture and severity of the disease.

Hereinafter, the present invention will be described in more detail based on the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Experimental Example 1 Cultivation of Wester Della Multispora Strains

The pH of the medium containing glucose 1%, tryptone 0.5%, yeast extract 0.3%, and malt extract 0.3% was adjusted to 6.5, which was dispensed in a 500 ml Erlenmeyer flask by 100 ml and sterilized at 121 ° C. for 15 minutes. Thereafter, seed cultures were inoculated by inoculating 1-2 platinum of Westerdykella multispora F-50733 strain (KCTC 0265BP) and shaken at 25 ° C for 2 days. Subsequently, 3 L of the medium having the same composition as the seed culture medium was sterilized for 40 minutes at 121 ° C., and then placed in a 5 L incubator and inoculated with 100 mL of the obtained seed culture solution, followed by incubation at 25 ° C. for 5 days under aeration for 180 rpm. .

Example 2 Separation Purification and Structural Analysis of Gelastatin A and B

The culture solution obtained in Example 1 was centrifuged at 6000 rpm to obtain a culture supernatant. The culture supernatant was adsorbed onto DIION-20 and the inhibitory portion was collected with 80% methanol to remove the solvent under reduced pressure, and then dissolved in a small amount of water and extracted with ethyl acetate. Subsequently, the ethyl acetate extract was concentrated and then adsorbed onto silica gel and developed with chloroform / methanol (10/1) to collect and concentrate the portions having inhibitory activity, and then dissolved in a small amount of methanol to obtain 100% of Sephadex LH-20. The mixture was developed with methanol, gel chromatography was performed, and the active fractions were collected and concentrated. The concentrated activity fraction was subjected to high pressure liquid chromatography (column: YMC-pack ODS-AM, eluent: 0.05% trichloroacetic acid containing 35% acetonitrile, flow rate: 1.5 ml / min, detection: 254 nm). In 20 minutes, the new compounds, gelastatin A and B, were purely separated and the solvent was removed with a reduced pressure dryer to obtain a yellow oil well.

The physicochemical properties of the gelastatin A and B thus obtained are shown in Table 1 below.

Color and geometry Yellow oil well Molecular formula C ₁₄ H ₁₆ O ₄ High Resolution Mass Spectrometry (M + H) ⁺ Calculation 249, 1127 Found 249, 1099 UV absorption spectrum λ _max nm (ε) 263 (9500), 338 (18900) Infrared Absorption Spectrum (cm ^-1 , KBr) 2490, 1708, 1600, 14071209, 1106, 1037, 991

As shown in Table 1, the new compounds gelastatin A and B were yellow oil wells, but dissolved in organic solvents such as methanol and ethyl acetate, but not in water. As a result of high resolution mass spectrometry, it was found that the molecular weight is 248, and the structural formula is C ₁₄ H ₁₆ O ₄ . The maximum UV absorption wavelength in methanol was 263 and 338 nm, and the absorption spectrum of the α, β-unsaturated carbonyl group was observed at 1708 cm ⁻¹ . Carbon and proton nuclear magnetic resonance spectrum analysis results are shown in Table 2 below. From this, it can be seen that the active material is an isomer mixture in which two optical isomers, gelatin, A and B are mixed 2: 1, and proton-proton cozy ( ¹ H- ¹ H COZY) and HMBC experiments confirmed that the propionic acid and hexadienylene moieties were linked to α, β-unsaturated δ-lactones. Finally, each form of gelastatin A and B has carbon nuclear magnetic resonance spectra and NOESY results, so that the gelastatin A is in the form of E in the pyranone ring, and the gelatine B is in the Z form in the 5 position of the pyranone ring. It was confirmed that it is an isomer.

C number Gelastin A Gelastin B ¹³ C ¹ H ¹³ C ¹ H 2 167.2 167.0 3 126.9 127.7 4 137.0 7.54 143.4 7.00 5 129.6 128.0 6 72.2 4.89 68.0 5.20 One' 27.9 2.65 27.6 2.60 2' 34.0 2.54 34.0 2.51 3 ' 176.6 176.5 One 133.0 6.27 134.1 6.32 2 125.1 6.59 125.4 6.59 3 139.7 6.42 140.7 6.45 4 132.5 6.22 133.1 6.23 5 134.5 5.91 135.0 5.91 6 18.6 1.81 18.7 1.81

Example 3 Measurement of Type IV Collagenase Inhibitory Activity

Into a 96-hole microplate, add 87 μl of 50 mM Tris-HCl buffer solution (100 mM sodium chloride, 0.01% Breeze-35, 5 mM calcium chloride, pH 7.5) and 2 μl of sample dissolved in dimethyl sulfoxide (DMSO). 10 μl of type IV collagenase (final concentration 1nM), activated at 37 ° C. for 15 minutes with 1 mM aminophenyl mercuricacetate, was equipped with a microplate reader. spectrofluorimeter) was used to measure the amount of fluorescence at excitation 328 nm and emission 398 nm, and the fluorescent substrate 7-methoxycumarin-4-acetyl-proline-leucine-glycine-leucine- (3 -[2,4-dinitrophenyl-2,3-diaminopropyl) -alanine-arginine-amide (Sigma) 1μl (final concentration 10μM) was added and reacted for 30 minutes at 37 ℃ and then the amount of fluorescence again Measured. Enzyme inhibition rate (%) was calculated as in Equation 1 below, IC ₅₀ value was determined by the concentration of the inhibitor whose enzyme activity is 50% inhibition rate.

Here, A represents the amount of fluorescence before the reaction of the sample containing the inhibitor, B represents the amount of fluorescence after the reaction of the sample containing the inhibitor, C represents the amount of fluorescence before the reaction of the control without the inhibitor, And D represents the amount of fluorescence after the reaction of the control without the inhibitor.

As a result of measuring the concentration (IC ₅₀ ) that the mixture of the separated gelatinta inhibits the type IV collagenase activity by 50%, the IC ₅₀ value was 0.16 µg / ml, which was 0.63 µM in terms of molarity.

As described above, the novel compound 3- (5- (hexa-2,4-dienylidene) -2-oxo of the present invention purified from the culture medium of the Westerdykella multispora strain isolated from the soil Since -5,6-dihydro-2H-pyran-3-yl) -propionic acid is excellent in inhibiting the activity of type IV collagenase, it can be used as a prophylactic and therapeutic agent for cancer metastasis and tumors.

Claims (5)

3- (5- (hexa-2,4-dienylidene) -2-oxo-5,6-dihydro-2H-pyran-3-yl) -propionic acid. The compound of claim 1 , comprising culturing the Westerdykella multispora strain, extracting the culture or mycelium with an organic solvent, and concentrating and purifying the obtained extract. How to prepare. The method of claim 2, The Westerdella multispora strain is Westerdykella multispora F-50733 strain (KCTC 0265BP). A composition for inhibiting type IV collagenase activity comprising an effective amount of the compound of claim 1 and a pharmaceutically acceptable carrier. The method of claim 4, wherein The composition is an agent for preventing and treating cancer metastasis and tumors.

Images