KR0137687B1 - Novel fatty acid lactone derivatives and extracting method thereof - Google Patents

Abstract

The present invention relates to a novel fatty acid lactone derivative having anticancer effect and a method for extracting the same, wherein the fatty acid lactone derivative according to the present invention comprises the following structural formulas (I) and (II) Elution from organic animals, particularly Hydraroid Solanderia Secunda, as an organic solvent and separation and extraction by chromatographic methods, inhibiting the action of the Parnesyl Protein Transferase, It has been found to act as an anticancer agent by preventing internalization.

Description

Novel C22 fatty acid lactone derivatives and extraction methods thereof.

1 is a structural formula of a fatty acid lactone derivative (soladerlactone C) according to the present invention,

2 is a structural formula of another fatty acid lactone derivative (soladerlactone G) according to the present invention,

3 is a hydrogen NMR spectrum of solanderactone C,

b is the carbon NMR spectrum of solanderactone C,

c is the hydrogen COSY NMR spectrum of solanderactone C,

4 is a hydrogen NMR spectrum of solanderactone G,

b is the carbon NMR spectrum of solanderactone G,

c is the hydrogen COZY NMR spectrum of solanderactone G.

The present invention relates to a novel fatty acid lactone derivative having an anticancer effect and a method for extracting the same, and in particular, a novel substance first discovered by the present invention without being known, as a result of extracting and separating a natural product from a tonic animal and testing the extract. As a C ₂₂ fatty acid lactone with a novel cyclopropane (Cyclopropane) and tonic, which has been found to be anti-cancer by inhibiting the action of Farnesyl protein transferase (Farnesyl protein transferase) It relates to an extraction method for extracting from animals.

Recently, research on marine natural products, or metabolites of marine organisms, has been rapidly growing, and many new substances have been extracted by the growth of this field, which is related to organic chemistry, biology, ecology, medicine and pharmaceuticals. It also contributed greatly to the development of such fields. The present invention is also the same as the research on marine natural products, it is made through a steady research process of collecting and separating a variety of marine organisms from the sea, extracting and separating the natural products therefrom and experimenting with the structure and properties of the extract .

Structurally closest to the novel materials found in the present invention are several fatty acid lactone derivatives found and extracted from marine organisms (Higgs Mulheirn, 1981; Niwa et al. 1989; Nagle Gerwick, 1990; Ojika et al, 1990), all of which are composed of 20 carbon skeletons, each of which has six, nine, and thirteen hexagonal lactone rings, originating from Arachidonic acid, which forms a phospholilipid, prostaglandin. Prostaglandin, leucotriene and other prostanoids (Prostanoids) are known to be a change in the process of biosynthesis. In addition, these materials are poly Paget force _{A 2 (Phospolipase A 2),} 5- Lee epoxy agent and the (5-Lipoxygenase) such Araki donik Acid Cascade anti-inflammatory activity and hence for the various enzymes involved in (arachidonic acid cascade) It is known to have an effect.

Meanwhile, the Ras gene, which is related to the biological activity of the novel substance disclosed in the present invention, was originally discovered as a cancer expression gene of Harvey (HaSV) and Kirsten (Ki) rat sarcoma virus. It is found in more than 30% of all human cancer cells, including 50%, and it is found that Ras of this cancer cell exists in a modified form unlike that of normal cells. Therefore, if it could prevent the modification of Ras, it was expected to be able to treat more than 30% of human cancers.

In addition, many studies have shown that Ras is similar in function to G-proteins and must be present in the plasma membrane in combination with GTP (guanine phosphate) to be activated. However, since Ras itself does not have a special lipophilic group that can interact with the plasma membrane, Ras must have a lipophilic group in order to interact with the plasma membrane. In other words, the C-terminus of Ras is CA1A2X (C is cystine, A is an aliphatic amino acid, X is an amino acid of all kinds), and the action of parnesyl protein transferase (Farnesyl Protein Transferase) results in parsyl diphosphate ( The fasyl group of Farnesyl Pyrophosphate) is attached to the C-terminal cystine, and then the three C-terminal amino acids are hydrolyzed by hydrolase, and the C-terminal carboxy group is esterified by an esterification reaction. Should be changed to

The modified Ras binds strongly to the plasma membrane and continuously transmits signals to the nucleus in the cell. As a result, the cells grow abnormally and eventually develop into cancer cells.

Therefore, if there is a substance that inhibits the pasylation of Ras, it can prevent the transformation of normal Ras into cancer cells, so it may be developed as a new anticancer agent.

Accordingly, the present invention was made in response to the rapid growth of the marine natural products as described above and the possibility of developing a new anticancer agent through the development of inhibitors of fascil protein transferases. The aim is to provide novel fatty acid lactone derivatives that are totally different.

In addition, the present invention not only provides an extraction method for extracting and separating the fatty acid lactone derivatives disclosed by the present invention from marine life, but also reveals that the fatty acid lactone derivatives according to the present invention have a function of inhibiting the production of cancer cells. Another purpose is to provide a new anticancer agent using fatty acid lactone derivatives as an active ingredient.

The novel fatty acid lactone derivatives according to the present invention are highly useful among various natural products extracted from Hydraroid Solanderia Secunda and whose structure has been determined, and two of the following (I) and (II) There is a structure of form.

The inventors of the present invention extracted the compound from Hydra (Hydroid) Solanderia Secunda (Sorlanderia Secunda), and because it is a kind of fatty acid lactone derivative having a fatty acid lactone ring, the compound of the structure as described in (I) is solanderactone C As such, the compound having the same structure as (II) was named solanderactone G.

Solanderactone C and G having the above structure is C ₂₂ H ₃₆ O ₄ and C ₂₂ H ₃₄ O ₄ , respectively, the molecular weight of cyclopropane (Cyclopropane) and fatty acid lactone ring C ₂₂ fatty acid (Fatty Acid Lactones ), It has a new carbon skeleton different from the known fatty acid lactones. That is, as shown in Figs. 1 and 2 together with the carbon number (Numbering) the structural formula, the carbon skeleton consists of 22 compared to the conventional 20, the lactone ring 6, 9 and Unlike the 13-corner, it is uniquely octagonal. Solanderactone C and G all have five asymmetric points, including carbon 7, 8, 10, 11, 14, and have hydroxyl groups on carbon 11 and 14, and solanderactone C on carbon 12, 16, 19 , Solanderactone G has a double bond at carbon 4, 12, 16, 19 position.

In the present invention, the solanderactone C and G is not a substance derived from arachidonic acid in view of 22 carbon skeletons, but DHA (cis-4, 7, 10, 13, 16, 19-docosahexaenoic acid) It is believed to be a substance derived from. Nevertheless, the present solanderlactones C and G have the same functional groups in positions similar to the known C ₂₀ fatty acid lactones, and thus have a constant physiological activity, which will be described in detail later.

In addition, the physicochemical properties of solanderactone C and G are viscous liquids, generally stable at room temperature, and soluble in almost all organic solvents.

The extraction method of the novel fatty acid lactone derivatives as described above comprises the steps of: collecting tonic animals from the ocean to prepare an extraction material; Putting the extract raw material into a solvent to form a solution, and filtering and distilling the solution to separate the crude extract; The crude extract is further separated and purified by chromatography to separate the fatty acid lactone derivatives.

The tonic animals include coral, jellyfish, hydra, and the like, preferably hydra, and its scientific name is Solanderia Secunda.

The extraction method as described above will be further clarified by the following examples.

EXAMPLES Extraction Method of Fatty Acid Lactone Derivatives

From the sea, 5 kg of Hydraroid Solanderia Secunda, dried in the shade, are chopped to prepare an extract. Dichloromethane 21 was added to the extract and left for 48 hours, after which the solution was filtered. The solvent of this filtered solution was removed by vaporization under reduced pressure to separate 10.7 g of crude extract.

Next, the crude extract was separated into 16 fractions by vacuum silica flash chromatography, wherein the eluent was diluted 5% by ethyl acetate from 100% hexane. A mixture of hexane and ethyl acetate was used in order up to 100% ethyl acetate. Hydrogen nuclear magnetic resonance spectroscopy of each of these fractions confirmed the presence of an interesting metabolite in fractions 12-16 (50-70% ethyl acetate / hexane, 100% ethyl acetate). It was.

Fraction 14 isolated from the (170㎎), fraction 15 (140㎎) and the fraction 16 (130㎎) C ₁₈ reversed phase semi-preparative HPLC column _{(C 18 reversed phase semi-preprative} HPLC column) (YMC semi-prep ODS Chromatography on a 10 μm, 250 × 10 mm (length × inner diameter) and elution rate of 2.5 ml / min yielded 4.1 mg of solanderactone C as a liquid substance, wherein the eluting fluid was water and acetonitrile (1 A mixed solvent of: 1) was used, a refractive index detector was used as a detector, and the burn time was 18.5 minutes.

In addition, the fraction isolated from the 12 (100㎎), fraction 13 (70㎎) and the fraction 14 (170㎎) C ₁₈ reversed phase semi-preparative HPLC column _{(C 18 reversed phase semi-preprative} HPLC column) (YMC semi- Chromatography was carried out on a prep ODS, 10 μm, 250 × 10 mm (length × inner diameter) and elution rate 2.0 ml / min to obtain 16 mg of solaneractone G, a liquid substance, with an eluting fluid of 42% water / Acetonitrile mixed solution was used, and the shelf time was 19.8 minutes.

On the other hand, the material extracted in the above embodiments (solran having lactone C, solran having lactone G) structure NMR (Nuclear Magnetic Resonance) was determined by the spectrum and high resolution mass spectrometry data, the nuclear magnetic resonance spectrum ¹ of The assignment of the H-nmr signal and the ¹³ C-nmr signal was performed through COSY, TOCSY, HMQC, and HMBC experiments. The designations of the structures of these compounds are shown in Table 1 below, and the hydrogen NMR spectrum, carbon NMR spectrum, and hydrogen COSY NMR spectrum for solanderactone C are as shown in FIGS. 3 a, b and c. Hydrogen NMR spectra, carbon NMR spectra, and hydrogen COSY NMR spectra for delactone G are as shown in Figures 4 a, b and c. The structure determined in this way is the same as the above-mentioned structural formula (I) and (II).

Table 1 ¹ H and ¹³ C NMR Designations of Solanderlactone C and G

* The ¹ H and ¹³ C NMR spectra in Table 1 were measured at 500 MHz and 125 MHz in chloroform-d solvent, respectively. This is the case of chemical warfare 1H was based on tetramethylsilane peak (0 ppm), was prepared in accordance with the residual solvent peak (77ppm) in chloroform -d is the case of ¹³ C.

In addition, the solanderactone C and G was found to exhibit a constant physiological activity by the present invention, that is, while the conventional C ₂₀ fatty acid lactone derivatives have a constant anti-inflammatory effect, the C ₂₂ fatty acid lactone of the present invention is an enzyme This difference is seen as a result of the difference in carbon number and lactone ring structure.

That is, the physiological activity of solanderactone C and G revealed by the present invention is to inhibit the action of farnesyl protein transferase (Farnesyl Protein Transferase) to function to prevent the endosylation of the Ras gene. This was found in the following passil protein transferase inhibitory activity screening experiment.

Experimental Example Screening of Penalcil Protein Transferase Inhibitory Activity of Solanderlactone

As an enzyme source of PAS, the brains of rats (Male Sprague-Dowley 100-150 g) were separated, washed with physiological saline, and homogenized. Partial purification was used.

In addition, the activity of the enzyme was measured by the phosphate as a substrate ^{(3 H-Farnesyl Pyrophosphate, FPP} ) in ³ H- Ipanema chamber pie using a scintillation proximity assay Ke (Scintillation Proximity Assay, SPA) method.

In other words, FPP as a substrate was used to modify a part of the method such as Brow (Brow), 10 μl sample solution, 10 μl assay buffer solution (50 mM Tris-HCL pH 7.5, 25 mM MgCl ₂ , 2 mM KCL, 5 mM DTT, 5 mM Na ₂ HPO ₄ , 0.01% Triton X-100), 20 μl diluted ³ HFPP, 20 μl biotin-lamin B peptide, 40 μl farnesyl protein transferase, followed by reaction at room temperature for 30 minutes. 150 μl of SPA beads and a bead / stop reagent solution were added, and the degree of pansylation of the biotin-lamin B peptide was measured in units of CPM (Count Per Minute) using a liquid scintillation counter. Inhibitory activity of the endogenous protein transferase was calculated as follows.

In the above formula, the blank sample is the case where there is no enzyme and inhibitor samples, and the control sample is the CPM was measured after the reaction under optimum conditions in the absence of the inhibitor sample.

As a result of measuring the inhibitory activity of panesyl protein transferase, Lc60 was 80 µm for solanderactone C and 120 µm for solanderactone G.

Therefore, solanderactone C and G can prevent the transformation of normal Ras into cancer cells by inhibiting the parsylation of the Ras gene, it is a new anticancer agent as an active ingredient.

As described in detail above, the solanderlactones C and G of the present invention are cyclopropane and C ₂₂ fatty acid lactones having fatty acid lactone rings, which are completely different from the known fatty acid lactones. Fatty acid lactone derivative material. In addition, this substance can be eluted with organic solvents from tonic animals, especially Hydraroid Solanderia Secunda, and extracted by chromatographic methods. It has been shown to have anticancer activity by inhibiting the action of Protein Transferase) and preventing the parsylation of Ras gene.

Claims (8)

C ₂₂ fatty acid lactones having the following structural formula (I): C ₂₂ fatty acid lactones having the following structural formula (II). Collecting tonic animals from the ocean to prepare an extract; Putting the extract raw material into a solvent to form a solution, and filtering and distilling the solution to separate the crude extract; Method for extracting a C ₂₂ Fatty Acid Lactones derivative comprising the step of further separating and purifying the crude extract by chromatography. 4. The method of extracting C ₂₂ fatty acid lactone derivatives according to claim 3, wherein the tonic animal Hydra is Solanderia Secunda. The method of claim 3, wherein the solvent is dichloromethane extraction method of C ₂₂ Fatty Acid Lactones derivatives. According to claim 3, wherein the separation and purification of the crude extract by chromatography, using vacuum silica flash chromatography (Vacuum Silica Flash Chromatography), the eluate is 100% hexane (Ethylacetate) from ethyl acetate (Ethylacetate) 5 increments% using hexane and a mixed solution of ethyl acetate through to 100% ethyl acetate, and then separating the crude extract by several fractions, the fraction C ₁₈ reverse-phase semi-preparative HPLC column (C ₁₈ reversed phase A method for extracting a C ₂₂ fatty acid lactone derivative, characterized in that the separation is performed by chromatography on a semi-preprative HPLC column again. The method according to claim 6, wherein the fraction is 16, and fraction 14, fraction 15 and fraction 16 of the fraction are chromatographed using a mixed solvent of water and acetonitrile (1: 1) as an eluting fluid. C ₂₂ Extraction of Fatty Acid Lactones Derivatives. 7. The C ₂₂ fatty acid lactone according to claim 6, wherein the fraction is 16, and fraction 12, fraction 13 and fraction 14 of the fraction are chromatographed using a 42% water / acetonitrile mixed solution as an eluting fluid. Extraction method of (Fatty Acid Lactones) derivative.