KR20060104555A - Anti-cancer composition comprising cyclopentadione derivatives - Google Patents

Abstract

The present invention provides an anticancer composition comprising a cyclopentadione derivative of the formula (1) and a birch tree [ Linera erythrocarpa Makino (Lauraceae) to a method for separating and purifying cyclopentadione compounds.

The composition of the present invention can be usefully used for the prevention and treatment of cancer by effectively inhibiting the growth of human cancer cell lines and the activity of panesyl transferase which is an essential enzyme for Ras oncogene activation.

Formula 1

Wherein R ₁ and R ₂ are hydrogen or methoxy and R ₃ is hydroxy or methoxy.

Trees, cyclopentadione, las oncogenes, cancer prevention and treatment

Description

Anti-cancer composition comprising cyclopentadione derivatives

The present invention is an anticancer composition and a birch tree comprising a cyclopentadione derivative of Formula 1 [Linera erythrocarpa Makino] (Lauraceae) to a method for separating and purifying cyclopentadione compounds.

Although the incidence of cancer increases with the development of civilization, the treatment of cancer patients is dependent on chemotherapy by surgical surgery, radiation therapy, and the administration of about 40 kinds of cytotoxic drugs. Most of these treatments are limited to early cancer patients or certain cancers, and cancer deaths continue to increase.

In order to express the Ras cancer gene, which is related to about 30% of human cancer, the Ras protein must be bound to the plasma membrane, and for this, a Farnesyl group must be coupled to the C-terminus of the Ras protein. If this process can be inhibited, it is expected that the expression of the gene can be suppressed, and new anticancer drug development efforts are underway.

Ras protein is a protein that plays the most important role in the signaling process of growth regulators involved in cell growth. Its molecular weight is 21kDa and consists of 188-189 amino acids, which can bind to guanine nucleotides (GDP and GTP). Can be. Ras proteins are expressed by ras proto-oncogenes (progenitors) and ras genes were first discovered as oncogenic genes (carcinogens) in various human tumors. It is a gene and is known to be associated with human malignancies. This gene is classified into harvey- ras (H- ras ), kirsten- ras (K- ras ), and N- ras . More than 30% of most cancers found in humans have been shown to have ras genes with abnormally altered biological activities of Ras proteins (H-Ras, K-Ras, N-Ras), especially pancreatic cancers. 90%), colorectal cancer (50%) and lung cancer (50%) were found to have a high frequency of mutated ras genes (M. Barbacid, Ann. Rev. Biochem . 56, 779, 1987). ).

Ras proteins are intracellular signaling regulators that regulate cell proliferation and differentiation. Like many G proteins, Ras proteins act as signal transmitters when in GTP-coupled form (switch "on"), where GTP hydrolyzes native GTP. It is inactivated (switch "off") when it is hydrolyzed to GDP by GAP (GTPase Activating Protein), a protein that aids in activity or GTP hydrolysis (G. Bollag, Ann. Rev. Cell Biol . 7, 601, 1991 ).

Structural and biochemical studies of Ras protein revealed that in addition to GTP binding, the Ras protein is located in the cell membrane during the activation phase of Ras protein. In order to function as a signaling medium, the Ras protein must be placed on the plasma membrane after binding to GTP. When the Ras protein is not located on the membrane and is in the cytoplasm, it does not show its activity. That is, in order for the lath to exhibit biological activity, the C-terminus must be modified and then attached to the plasma membrane surface of the plasma membrane. To do this, Ras must be biosynthesized as a precursor of inactivation in the cytoplasm and then bound to the lipids produced during steroid biosynthesis, a series of reactions called "post-translational modification."

The binding of these signaling proteins to the plasma membrane is found in most G proteins, including the Ras protein, and this lipidation reaction occurs at the cysteine residues at the C-terminus. These proteins have a CAAX motif containing one cysteine, two aliphatic residues, and one carboxyl-terminal "X" residue at the C-terminus. have. Amino acids at the "X" position are limited to Ser, Ala, Met, Glu, and the like, and are important residues for the farnesyl transferase to recognize protein substrates. The composition of these residues determines the enzymes that interact with the lipid to which they are bound. There are two major types of lipidation reactions, Farnesylation and Geranylgeranylation. In the case of Ras protein, farnesylation occurs, and most lipid-binding proteins in cells Nylgeranyl group (geranylgeranyl group) is bonded. That is, geranyl geranylation occurs about 5-10 times more than the panesylation reaction (PJ Casey, Proc. Natl. Acad. Sci. USA 86, 8323, 1989). The isoprenyl group that participates in the isoprenylation is the biosynthesis process of isoprenoids that biosynthesize cholesterol, starting from 2-acetyl-CoA. (JL Goldstein and MS Brown, Nature, 343, 425, 1990).

To summarize the post-translational modifications necessary for plasma membrane binding of Ras proteins, the first step is to introduce a panesyl group into the cysteine at position 4 at the carboxy terminus, and secondly, the three carboxy-side amino acids of the cysteine Proteolytic cleavage, and finally a series of methylation reactions to the carboxy-terminated cysteine. Through this process, the C-terminus of Ras protein is transformed into a lipophilic property and is strongly bound to the plasma membrane. This C-terminal lipid acts like a frontal supporter in intracellular signaling. Three post-translational modifications involve three enzymes, namely, farnesyl transfer, peptide hydrolysis, and methylation enzymes. It is expected to be effectively controlled, suggesting the possibility of developing new anticancer drugs. The reason is that even if the Ras protein, which has been activated for a long time due to modification, does not bind to the plasma membrane, it cannot deliver signals necessary for cell growth and differentiation to Raf protein, the next-generation signaling protein.

Among these enzymes, peptide hydrolase has not yet been isolated and purified, and the exact mechanism of reaction has not been revealed, and researches for developing inhibitors of panesyl transfer and methylation enzymes have been actively conducted. However, research on the development of anticancer drugs through the search for inhibitors of panesyl transferase is the most active since the panesylation reaction is the first step in the modification of Ras.

In 1990, for the first time, it was discovered that the purely isolated farnesyl transferase from rats was competitively inhibited by compounds having a structure similar to that of Ras' C-terminal peptide, and inhibitors that mimic those structures began to be synthesized. (Y. Reiss, Cell, 62, 81, 1990). Among these peptidomimetic compounds, L-731,734 and L-731,735 (NE Kohl et al , Science, 260, 1934, 1993) developed by the Merck Co. laboratory, benzodiazepine from Genentech, etc. Has been shown to show good activity in overexpressed cell lines (GL James et al , Science, 260, 1937, 1993). Among them, L-744,822, developed by the Merck Co. laboratory, has been reported to strongly inhibit tumors caused by implantation of the Ras oncogene into nude mice (NE Kohl et al , Nature). Med. 1, 792, 1995). Synthetic inhibitors other than these include hydroxyfarnesyl phosphonic acid derivatives and SCH 44,342, a tricyclic compound published by Seringflower, USA (WR Bishop, et al , J. Biol). Chem. 270, 30661, 1995).

The exploration of many samples by enzymatic methods has led to the discovery of various natural-derived compounds that inhibit farnesyl transferase. The first natural-derived inhibitors are limonene, perillic and dihydroperillic acid isolated from plants, and microbial inhibitors are streptonigrin isolated from actinomycetes in 1992. 10'-desmethoxy streptonigrin, a derivative of C. oleracea, is the first inhibitor. Two new dicarboxylic acid derivatives, ketomellic acid A and ketomellic acid B, It was isolated from the fermentation broth of Ketomella acutiseta. Zaragozic acid A and B have been reported to be potent inhibitors of squalene synthase and have been found to be competitive inhibitors of farnesyl transferase. In addition to these, pepticinnamins and the like have been reported as inhibitors of farnesyl transferase (V. Manne, et al , Drug Devep. Res. 34, 121, 1995, S. Omura et al , Drug Future, 19, 751, 1994, JK Buolamwini, Curr. Opinion Chem. Biol. 3, 500, 1999).

Under this background, the inventors have isolated purified and purified methyllinderone, methyllucidone, lucidone, and linderone from the leaves and berries of the birch tree. By revealing the activity of panesyl transferase, an essential enzyme for oncogenic gene activation, and growth inhibitory activity against cancer cell lines, we confirmed that the cyclopentadione derivatives could be usefully used for preventing and treating cancer. The invention was completed.

An object of the present invention is to provide an anticancer composition comprising a cyclopentadione derivative of formula (1).

It is another object of the present invention to provide a method for purifying cyclopentadione derivatives of the formula (1) from a birch tree.

In one embodiment, the present invention relates to an anticancer composition comprising a cyclopentadione derivative having the structure of Formula (I):

Wherein R ₁ and R ₂ are OCH ₃ or H, and R ₃ is OCH ₃ or OH.

The anticancer effect of the compound of Formula 1 is a novel use that has not been identified before the present invention. The anticancer effect was confirmed. Activity measurements using panesyl transferase and panesylpyrophosphate isolated from rats showed IC ₅₀ values of 33 to 125 uM, and WST-1 assay for human cancer cell lines such as colon cancer, breast cancer and lung cancer. It showed a growth inhibitory effect (GI ₅₀ ) of 50% at 15 μg / ml.

As used herein, the term “anticancer” refers to both prevention and treatment of cancer. The composition of the present invention is preferably for the prevention of cancer due to overexpression of Las protein, such as colon cancer, stomach cancer, prostate cancer, breast cancer, kidney cancer, liver cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer, pancreatic cancer, or the like It can be used for treatment, but not limited to these.

The compound of formula 1 included in the composition of the present invention may also be used in the form of its salts, which salts include various organic or inorganic salts which are pharmacologically and physiologically acceptable. Preferred compounds of formula 1 are methyllinderone of formula 2, methyllucidone of formula 3, lucidone of formula 4, and linderone of formula 5:

The composition of the present invention may further comprise a pharmacologically or physiologically acceptable carrier, excipient, diluent in addition to the compound of formula (1).

The composition of the present invention may be formulated in various forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, oral formulations such as aerosols, sterile injectable solutions, and the like according to conventional methods for each purpose of use. It may be used orally or may be administered through various routes including intravenous, intraperitoneal, subcutaneous, rectal, topical administration and the like. Examples of suitable carriers, excipients and diluents that may be included in such compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, mineral oil and the like. The composition may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, and the like. Mix and formulate. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. Bases for injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives.

The amount of the composition of the present invention may vary depending on the age, sex, and weight of the patient, but in general, an amount of 1 to 100 mg, preferably 5 to 20 mg per kg body weight, is administered daily or every other day or 1 day. Administration may be divided into three to three times. However, it may increase or decrease depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any way.

Cyclopentadione derivatives of Formula 1 can be isolated from natural materials or synthesized using methods well known in the art. As a natural material capable of separating the compound of Formula 1, birch can be preferably used.

Lindera erythrocarpa is a Lauraceae plant that is distributed in 45 genera and 1,500 species worldwide, and 6 genera and 12 species are known to grow in Korea (Sun, BY and Chung, YH (1988) Monographic study of the Lauraceae in Korea.Kor. J. Plant Tax. 18 , 133-151). It is also known as boyan tree and white tree. It is 10m high and 30cm in diameter, and it is a mausoleum which grows light yellow flowers from April to May and forms a round red fruit with a diameter of 8mm in September. It is a deciduous tree with a height of 5m that grows in warm parts of Japan, China, southern part of Korea.

The dried fruit of the birch has an unusual aroma and bitter taste, so it is used in Japan as a painkiller for gastrointestinal drugs and neuralgia. In addition, it has been reported that the extracts of the leaves and fruits of the birch tree have antifungal activity, but it has not been disclosed that the terpene compounds isolated from the birch tree have anticancer effects.

In another aspect, the present invention comprises the steps of (1) alcohol extraction of the birch tree; (2) silica gel chromatography of the extract of step (1); (3) thin layer chromatography of the eluate of step (2) and (4) high performance liquid chromatography of the fractions of step (3), including methyllinderone, methylsilidone, lucidon or linderon. It relates to a method for purifying.

Alcohol extraction in this method can use the leaves, berries or both of the trees. The alcohol used for alcohol extraction is preferably methanol. Preferably, the chromatography of step (2) is carried out using a gradient of methylene chloride and methanol, and the chromatography of step (4) is carried out using a gradient of methanol and water.

In a specific embodiment, the leaves and fruits of the birch tree are extracted with methanol, and the extracts are loaded onto a silica gel chromatography column to elute with an gradient of methylene chloride and methanol, preferably from 99: 1 to 95: 5, to obtain active fractions. After separation, the separated active mixture is again adsorbed onto a series of chromatography, i.e., a C18 chromatography column, eluted with methanol and water, subjected to thin layer chromatography and loaded onto a high performance liquid chromatography column to obtain a gradient of methanol and water, preferably Preferably eluted with a gradient of 50:50 to 70:30 to separate purely. As described above, the purified compound was determined by UV absorbance measurement, IR absorbance measurement, high resolution mass spectrometry, and NMR analysis to determine that the compound was methylderon, mexilusidone, lucidon or linderon with anticancer activity.

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

Example 1 Extraction and Separation Purification of Cyclopentadione Compounds from Trees

Trees (collected to grow native to South Korea) finely pulverized leaves or berries, methanol was added to stand at room temperature for 48 hours, then stirred to separate the liquid and solid parts using a filter paper. The liquid phases were combined, concentrated under reduced pressure, and dissolved by adding methanol. The organic solvent layers containing the active material were combined and concentrated under reduced pressure. The concentrated extract was dissolved in methylene chloride, added to silica gel (Merck, Art No. 9385) to adsorb the active substance, and then activated by silica gel column chromatography, changing the ratio of methylene chloride and methanol from 99: 1 to 95: 5. Fractions were separated. The obtained fractions were adsorbed on a C18 column and then eluted with methanol and water to obtain a partially purified active material. After thin layer chromatography of this active material, the active fractions were finally eluted by high performance liquid chromatography (HPLC) (HPLC column: ODS (250 x 21.2 mm) from Phenomenex), methanol: water (gradient elution from 50:50 to 70:30). The following compound was isolated purely.

Finally, 1.68 kg of the leaves were extracted with methanol to obtain 75.32 g of the extract, from which the pure compounds methyllinderone and methyllucidone were obtained in 0.43 g and 1.45 g, respectively. 163 g of berries were extracted with methanol to obtain 8.91 g of extract, and 0.21 g, 0.91 g, 0.06 g and 0.04, respectively, methyllinderone, methyllucidone, lucidone, and linderone, respectively. g was separated

Example 2 Structural Analysis of Cyclopentadione Compounds

UV absorbance analysis, IR (infrared) absorbance analysis and high resolution mass spectrometry analysis were performed to determine the molecular weight and molecular formula of the pure purified compound. Specifically, UV absorbance analysis was measured by Shimadzu UV-265 spectrophotometer, and IR absorbance was measured by Bio-Rad's DigiLab Divzone FTS-80 spectrophotometer (Bio-Rad Digilab Division FTS- 80 spectrophotometer) and molecular weight and molecular formula were determined by measuring high resolution MS using VG70-SEQ mass spectrometry (MS). In addition, ¹ H, ¹³ C-NMR spectra were obtained using a nuclear magnetic resonance apparatus (Varian 300 MHz, 500 MHz NMR), and the structures were determined by comprehensive analysis of these spectra.

Methyllinderone Appearance Pale yellow Molecular formula C ₁₇ H ₁₆ O ₅ Molecular Weight 300.31 Melting Point (℃) 84-85 Solubility Availability Alcohols, DMSO Insoluble Hexane, H ₂ O

Methyllucidone Appearance Pale yellow Molecular formula C ₁₆ H ₁₄ O ₄ Molecular Weight 270.09 Melting Point (℃) 126-128 Solubility Availability Alcohols, DMSO Insoluble Hexane, H ₂ O

Lucidone Appearance Pale yellow Molecular formula C ₁₅ H ₁₂ O ₄ Molecular Weight 256.07 Melting Point (℃) 166.5-168.5 Solubility Availability Alcohols, DMSO Insoluble Hexane, H ₂ O

Linderone Appearance Pale yellow Molecular formula C ₁₆ H ₁₄ O ₅ Molecular Weight 286.08 Melting Point (℃) 92-94 Solubility Availability Alcohols, DMSO Insoluble Hexane, H ₂ O

Example 3 Measurement of Panesyl Transferase Inhibitory Activity of Cyclopentadione Compound

As an enzyme source of farnesyl transferase, a brain of a rat (Sprague Dawley male, 100-150 g in weight) was extracted, washed with physiological saline, homogenized, centrifuged, and the Q Sepharose high-speed column (Q). Partially purified using Sepharose Fast Flow column).

Activity of the enzyme was measured by ³ H- Ipanema onion which phosphate ^{(3 H-Farnesyl pyrophosphate, FPP} ) for grooving (Blow) method with some modifications to SPA (Scintillation Proximity Assay), such as a method of using as a substrate.

10 μl sample solution of the compound purified in Example 1, analytical buffer (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) 10 μl, diluted ³ H-FPP 20 μl, biotin-lamin B peptide 20 μl, and 40 μl of farnesyl protein transferase, followed by reaction at room temperature for 30 minutes and 150 μl of SPA beads. After the addition of the reaction solution (bead / stop reagent solution) was measured using the liquid scintillation counter (scintillation counter) the degree of pansylation of biotin-Lamine B peptide in units of CPM (count per minute), according to the following equation 1 Inhibitory activity of farnesyl protein transferase was calculated.

CPM (T): CPM with inhibitor sample and enzyme

CPM (C1): CPM when no inhibitor sample was added and enzyme was added

CPM (B): CPM without inhibitor sample and enzyme

As a result, IC ₅₀ (50% activity inhibition concentration) values are shown in Table 5 below.

Compounds IC ₅₀ (mM) Methyllinderone 43.3 Methyllucidone 40.7 Lucidone 125 Linderone 33.3

Example 4 Inhibition of Growth of Cancer Cell Lines

Inhibition of growth of human cancer cell lines of the compound purified in Example 1 was measured using WST-1. Human cancer cell lines were cultured at 37 ° C., 5% carbon dioxide in a medium containing 10% FBS, and cells were detached using 0.05% trypsin-EDTA. 4,000 (A549, MDA-MB-231), 5,000 (HEK293, NCI-H23) or 6,000 (HCA-7) calculated by hematocytometer in each well of a 96 well plate , HCT116, SW620, DU145) cells were added respectively. Incubated in a medium containing 10% FBS in a 37 ℃ incubator containing 5% carbon dioxide, and after 24 hours each control compound (0.1% DMSO) and each compound separated and purified in Example 1 by concentration (compound dissolved in DMSO Dilution with medium). After treatment for 48 hours, 10 μl of WST-1 (Roche) was added to each well and incubated for 2 hours, and then absorbance was measured at 450 nm using an ELISA reader (ELISA Reader, Bio-Rad).

As a result, a 50% growth inhibitory effect was observed at a concentration of 0.2 to 15 μg / ml, and each compound was sensitive to a specific cancer cell line. Among them, methyl Lucisidone showed the most effective inhibition of human colon cancer cells HCT116 and HCA-7 with GI ₅₀ of 0.26 μg / ml and 0.39 μg / ml, respectively. On the other hand, for other colon cancer cells SW620, lung cancer cells NCI-H23 and A549, prostate cancer cell DU145, breast cancer cell MDA-MB-231, and kidney cancer cell HEK293, GI ₅₀ was 2, 1, 15, 1.8, 3.5 and 4.8 ㎍ / Measured in ml. Methylderon has a GI ₅₀ of 0.45, 0.52, 3.5, 1.1, 3.2, and 3.8 for colorectal cancer cells HCT116, HCA-7, SW620, lung cancer cells NCI-H23 and A549, breast cancer cells MDA-MB-231, and kidney cancer cells HEK293. And 3 μg / ml.

As described above, the cyclopentadione derivative of Formula 1 is useful for treating and preventing cancer by inhibiting the growth of human cancer cell lines and the activity of panesyl transferase, an enzyme essential for Ras oncogene activation.

Claims (5)

An anticancer composition comprising the compound of formula (1). Formula 1

Wherein R 1 and R 2 are hydrogen or methoxy and R 3 is hydroxy or methoxy. 2. The anticancer composition according to claim 1, wherein the compound of formula 1 is a methyllinderone of formula 2, a methylsilidone of formula 3, a lucidone of formula 4, or a linderon of formula 5. 3. Formula 2

Formula 3

Formula 4

Formula 5

The anticancer composition according to claim 1, wherein the cancer is colorectal cancer, gastric cancer, prostate cancer, breast cancer, kidney cancer, liver cancer, brain tumor, lung cancer, uterine cancer, colon cancer, bladder cancer or pancreatic cancer. (1) alcohol extracting the birch tree; (2) silica gel chromatography of the extract of step (1); (3) thin layer chromatography of the eluate of step (2) and (4) high performance liquid chromatography of the fractions of step (3), including methyllinderone, methylsilidone, lucidon or linderon. How to refine. The method of claim 4, wherein the chromatography of step (2) is carried out using a gradient of methylene chloride and methanol, and the chromatography of step (4) is carried out using a gradient of methanol and water.