KR100282559B1 - Phospholipase inhibitory anticancer active compound - Google Patents

Abstract

The present invention relates to an anticancer substance, which is an inhibitor of phospholipase C? 1, produced by extracting an active ingredient from a colony belonging to the family Acanthopanax sp. And using an active screening method of phospholipase C? 1, And a novel anticancer substance showing high safety for colon cancer.

Description

Phospholipase inhibitory anticancer active compound

The present invention relates to a method for producing a novel anticancer substance by extracting an anticancer active ingredient from a horse chestnut belonging to the family Acanthopanax sp. And using an active screening method for phospholipase C? 1.

The general formula (I) of the phospholipase Cγ1 inhibiting active ingredient isolated by the method of fractionation using a bioassay from horse chestnut is as follows.

(Formula I)

In the general formula

R ₁ and R ₂ are each hydrogen or methyl (except when R ₁ and R ₂ are both hydrogen)

X and Y each represent hydrogen, a lower alkyl group having 1 to 4 carbon atoms, an alkoxy group, an amino group, a halogen or a hydroxy group.

(Provided that when R ₁ is methyl and R ₂ is hydrogen, X hydrogen and Y hydroxy groups are excluded;

When R ₁ is hydrogen and R ₂ is methyl, X hydrogen and Y hydroxy groups are excluded.)

From the past, hooked branches called Uncaria Hooks have been used medicinally. In spring and autumn, young branches with hooks were picked and dried in the sun or steamed and dried. Several major alkaloids reported to date have been reported as hypotensive substances, but no studies have been conducted on anticancer active ingredients.

In the present invention, in order to create new substances different from conventional anticancer drugs, anticancer substances were extracted from natural substances, which can be referred to as new substance reports, and a new search method capable of detecting anticancer activity was conducted. The phospholipase C? 1 active search method was introduced into natural products, especially herbal medicines.

Recent research results have shown that phospholipase C, particularly phospholipase Cγ1, is involved in cell proliferation and cancer production. Phospholipase C has been shown to be capable of transforming normal cells into cancer cells, and it has also been shown that the continuous stimulation of phospholipase C induces cancer cell production of the cells, and the overexpression of phospholipase C Lt; / RTI >

Aminochromene, ET-18-OCH ₃ , suramin, U-73122, and 3-F-PtdIns have been reported to have anticancer activity among the phospholipase C inhibitors thus far identified ET-10-OCH ₃ is in the clinical trial stage. In particular, myroridine K, streptothricin B, and edein, which are inhibitors of phospholipase Cγ1, exhibited selective cytotoxicity in breast cancer, colon cancer, malignant melanoma and the like (Hill et al., 1994), the PLCγ1 inhibitor cis-hinokiresinol isolated from Annemarrhena asphodeloides is more potent cytotoxic than human cancer cells in lung cancer, rectal cancer and breast cancer, (Lee et al., 1996b). In addition, three anacardic acids (three cardanols, three species of bilobols, etc.), PLCγ1 inhibitors isolated from the bank species (Ginkgo biloba sarcotesta) (Lee et al., 1998). Inhibition of growth was observed in overexpressed human cancer cells, especially in normal colon cells than in colon cancer cells (Lee et al., 1998).

There is a limitation that the cancer treatment agent currently used shows severe toxicity to normal cells. In the present invention, the inhibitory activity of phosphatidylinositol specific phospholipase Cγ1 (PLCγ1, PLCγ1), which is characteristic of phosphatidylinositol, has been studied in order to overcome these limitations. Phospholipase Cγ1 is an important enzyme for intracellular signal transduction and is essential for cell proliferation and differentiation. Recently, many research results suggest that inhibitors of phospholipase Cγ1 may be an effective tool for the development of anticancer drugs. Therefore, it was found that the activity of crude chloroform fraction was strong in searching for the phospholipase Cγ1 inhibitory substance of natural products.

Accordingly, the present invention aims to isolate the phospholipase C? 1 inhibitory activity and evaluate the phospholipase C? 1 inhibitory activity and anticancer activity to provide an anticancer substance having low toxicity to normal cells.

The present invention relates to an anticancer substance, which is an inhibitor of phospholipase C? 1, produced by extracting an active ingredient from a colony belonging to the family Acanthopanax sp. And using an active screening method of phospholipase C? 1, And developed a new anticancer substance showing high safety for colon cancer.

Accordingly, an object of the present invention is to provide an antitumor active phospholipase Cγ1 inhibiting anticancer active compound represented by the following general formula (I).

(Formula I)

In the general formula

R ₁ and R ₂ are each hydrogen or methyl (except when R ₁ and R ₂ are both hydrogen)

X and Y each represent hydrogen, a lower alkyl group having 1 to 4 carbon atoms, an alkoxy group, an amino group, a halogen or a hydroxy group.

(Provided that when R ₁ is methyl and R ₂ is hydrogen, X hydrogen and Y hydroxy groups are excluded;

When R ₁ is hydrogen and R ₂ is methyl, X hydrogen and Y hydroxy groups are excluded.)

A further object of the present invention is to provide a method for fractionating the fractions of Daisakusa by extracting with chloroform-methanol using a fractionation method using bioassay and fractionating the chloroform with silica gel column chromatography, Sephadex LH-20 and Semiprep HPLC And a method for separating and purifying the phospholipase Cγ1 inhibiting active ingredient represented by the above general formula (I).

The present invention relates to the synthesis of 3 beta-hydroxy-27-i-perylaryloxy-olea-12-en-28-oic acid (1 ) (3β-hydroxy-27- E- ferulyloxy-olea-12-ene-28-oic acid);

3-beta-hydroxy-27-i-peryllyloxy-ur-12-ene-

(3β-hydroxy-27- E- ferulyloxy-urs-12-ene-28-oic acid);

3-beta-hydroxy-27-jet-peryllyloxy-olea-12-ene-

(3β-hydroxy-27- Z- ferulyloxy-olea-12-ene-28-oic acid);

3-beta-hydroxy-27-jet-p-coumaroyloxy-ur-12-ene-

(3β-hydroxy-27- Z- coumaroyloxy-urs-12-ene-28-oic acid);

3-beta-hydroxy-27-iso-p-coumaroyloxy-olea-12-ene-

(3β-hydroxy-27- E- p-coumaroryloxy-olea-12-ene-28-oic acid);

3-beta-hydroxy-27-iso-p-coumaroyloxy-ur-12-ene-

(3β-hydroxy-27- E- coumaroyloxy-urs-12-ene-28-oic acid);

3-beta-hydroxy-27-jet-p-coumaroyloxy-olea-12-ene-

(3β-hydroxy-27- Z- p-coumaroryloxy-olea-12-ene-28-oic acid);

3-beta-hydroxy-27-jet-p-coumaroyloxy-ur-12-ene-

(3β-hydroxy-27- Z- p-coumaroryloxy-urs-12-ene-28-oic acid)

.

In the present invention, all of the compounds isolated by the present invention include p-coumaric and ferulic acid in the nucleus of 27-oxidized pentacyclic triterpene, These five compounds (1) to (5) are the first new substances separated in the natural world as uncaric acid A (1), unsaturated carboxylic acid C , uncaric acid C (2), uncaric acid B (3), uncaric acid D (4), and uncaric acid E (5) , The above three compounds (6) to (8) are the first substances isolated from the madder, and ¹³ C-NMR data of the compound (6) are first shown.

The compounds isolated in the present invention were evaluated for their phospholipase C? 1 inhibitory activity by in vitro experiments. As a result, IC ₅₀ showed a significant concentration-dependent activity _ranging from 5.89 to 28.88 μg / ml, The activity was stronger or similar to that of the inhibitor. Among them, Compound (6) showed the highest activity. In general, the ursane skeleton showed stronger activity than the trans and oleanane skeletons, and p-coumaryloxy group free of 6'-OCH ₃ rather than peryllyloxy group showed high activity . The phospholipase Cγ1 inhibitory activity for this class of compounds is reported for the first time.

In addition, the phospholipase Cγ is known to be activated by a growth factor such as PDGF (platelet-derived growth factor). In order to examine the activity of the present invention, the present invention provides the production of total IPs for stimulation of PDGF in NH3T3γ1 cells PI-turnover), the activity of IC ₅₀ was 15.01 ~ 48.62 ㎍ / ㎖, similar to the enzyme level.

In order to investigate the structural activity of the compounds isolated in the present invention, compound derivatives were synthesized and showed activity in the state of ester linkage of triterpene nucleus and phenylpropanoic acid, and 3-OH, 7 '-OH, 28-COOH, 2', and 3 'double bonds. It was also found that the activity of the compound esterified to 27-OH was superior to that of 3-OH or 28-OH.

In order to investigate the structural activity, the structure of the most active compound (6) was changed to evaluate the phospholipase Cγ activity. The activities of 27-hydroxy ursolic acid and p-coumaric acid produced by hydrolysis of the compound (6) were remarkably decreased by an IC ₅₀ of 250 μg / ml or more Respectively. This suggests that the triterpenoid nucleus and phenylpropanoic acid exhibit activity in the ester-bonded state. In addition, acetylation products produced when acetylation was not observed at a concentration of 250 μg / ml, suggesting that 3-OH and 7'-OH play an important role in the activity. On the other hand, the methylated methyl ester compound and the compound produced by reducing the double bond at the 2 'position exhibited weak activity as compared with the compound (6) at 82.34% and 54.24% at an IC ₅₀ of 100 μg / ml, respectively. This suggests that the double bond at the 28-COOH, 2 'position plays an important role in the activity. 3-OH, and 28-OH, respectively, exhibited weaker activity than the compound (6). From this, it can be seen that the compound which is esterified to 27-OH is more active than p-coumaric acid is bound to 3-OH or 28-OH.

Phospholipase γ1 is essential for the proliferation and progression of human cancer, and overexpression by this enzyme induces transformation of normal cells. The growth inhibitory activity against various compounds (A549, HCT15, MCF-7, HT-1197, p388) in the log phase was evaluated using MTT and SRB for the separated compounds (1) to (8) Respectively. As a result, the IC ₅₀ showed a strong activity at 0.55-6.52 / / ㎖ in the solid cancer cells (A549, HCT15, MCF-7, HT-1197) overexpressing phospholipase Cγ1 and normal cells and cancer cells (CCD-18Co) was 10 times more toxic than the corresponding rectal cancer cells.

To determine the mechanism of cytotoxicity of these compounds, cytotoxicity against confluent A549 and HCT-15 in a stationary phase was measured and compared with the results at log phase. Unlike the log phase, it showed almost no toxicity at a concentration of 50 μg / ml or less. Therefore, it was estimated that cancer cell growth inhibitory activity was caused not by cell killing but by growth inhibition. In order to confirm this fact, the HCT-15 cells were exposed to the SRB assay and a colony forming assay after two hours of exposure, respectively. As a result, the inhibitory activity against cancer cells was 50 μg / ml The inhibition of growth was observed at the concentrations below.

Caffeic acid, and so on. The DPPH test was carried out with antioxidant activity. As a result, the antioxidant activity was exhibited at a concentration of 100-150 mM.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples do not limit the scope of the present invention.

(Example 1) Extraction and fractionation of the compounds (1) to (8)

Twenty kilograms of the dried jowl were ultrasonically extracted with chloroform-methanol (2: 1) three times in 24 L portions, filtered, and concentrated under reduced pressure to obtain a total extract (800 g). This was suspended in distilled water, extracted three times with chloroform, and concentrated under reduced pressure to obtain a chloroform fraction (600 g). The remaining suspension was regarded as an aqueous layer (200 g). The inhibitory activity of phospholipase Cγ1 was measured at 100 μg / ml for each fraction.

600 g of the chloroform fraction having a good activity was subjected to silica gel column chromatography using chloroform and a solvent of chloroform-methanol (30: 1, 15: 1, 8: 1, 5: 1, 2: Silica gel column chromatography was used to divide the fractions into 6 fractions and the active fractions were determined by TLC using the method of measuring activity after preparative TLC for the 4th fraction having the highest activity. Six fractions were obtained by performing silica gel column chromatography with a chloroform-acetone (30: 1) solvent to remove alkaloids present in large amounts together with the active ingredient in the fourth fraction (120 g). Separately, fraction 10 (12 g) was subjected to vacuum column chromatography using chloroform-methanol-acetic acid (50: 1: 0.1) as a solvent in order to remove ursolic acid, ≪ / RTI >

Three fractions of the chloroform fraction of the crude fraction were subjected to column chromatography to obtain fraction 14 (3.2 g), which was further purified by vacuum column chromatography using a solvent of 22.5: 1: 1 as a solvent of nucleic acid-ethyl acetate-isopropyl alcohol To obtain five small fractions. The small fractions were purified using Sephadex LH-20 (nucleic acid-methylene chloride-isopropyl alcohol = 3: 4: 1) and semipreparative HPLC to yield compound 1 (52.3 mg) And Compound 3 (63.2 mg) and Compound 4 (115.4 mg) were obtained from the third small fraction.

Further, 15 fractions obtained in three chromatographic steps were further subjected to vacuum column chromatography (nucleic acid-ethyl acetate-isopropyl alcohol = 20: 1: 1) to obtain six small fractions. The small fractions were purified using Semiprep HPLC with Sephadex LH-20 (nucleic acid-methylene chloride-isopropyl alcohol = 3: 4: 1) to obtain Compound 5 (35.5 mg) and Compound 6 (70.0 mg ), And Compound 7 (43.0 mg) and Compound 8 (135.1 mg) were obtained from the fifth small fraction.

(Example 2) Synthesis of Derivative of Compound (6)

First, compound 6 was dissolved in pyrimidine and reacted with acetic anhydride to obtain an acetylated compound at the 3-OH, 7'-OH site. Compound 6 was methylated with CH ₂ N ₂ dissolved in the ester To obtain a methyl ester compound methylated in 28-COOH. Compounds were also obtained by alkaline hydrolysis of Compound 6 under KOH. The compounds were compared with a standard sample and spectra, and co-TLC was performed to determine 27-hydroxy ursolic acid and p (P-coumaric acid), and the 2 ', 3' double bond was reduced with hydrogen gas under Pd / C to obtain a derivative of compound 6.

Compound 9 was synthesized by using p-coumaric acid etherified compound at position 3 as a starting material. Protection and selective deprotection were carried out to protect the alumatic OH of p-coumaric acid. To obtain a TBS-modified compound in both OH and COOH by using TSDMSCL (tertiary butyl dimethyl silyl chloride) and imidazole and to selectively deprotect only TBS bound to COOH, a TBS-modified compound was obtained by using potassium carbonate solution , Esterified with DCC and DMAP to synthesize 3-OH-esterified compound, and TBS was deprotected with 1M-TBAF to synthesize compound 9.

Compound 10 was subjected to a selective esterification reaction only at 28 times after reducing the COOH of urushosenic acid in order to obtain the esterified compound at position 28. Ursolic acid was esterified with p-cumaric acid, DCC and DMAP by reduction of 28-COOH with LiAlH ₄ , synthesized by esterification with 28-OH, and TBS was removed by 1M-TBAF Compound 10 was synthesized.

(Example 3) Determination of structure of compounds

The compounds obtained from the above examples were reacted with anisaldehyde H₂SO₄(anisaldehyde H₂SO₄), FeCl₃, And the sequence of the compounds can be determined according to the Libermann-Burchard reaction. According to this method, anisaldehyde H₂SO₄Purple, FeCl₃, And it was predicted that it was a triterpene and phenolic compounds positively in the Libermann-Burchard reaction. The detailed structures of the respective compounds are the melting point, FT-IR, UV, H^One-NMR,¹³C-NMR, DEPT, H^One-H^OneCOZY, .H^One-¹³C COSY, HMBC, and 1D-NOE.

(Example 4) Measurement of phospholipase C? 1 activity in vitro

Rhee et al. (1991) were used. 5 μl of the enzyme solution containing the phospholipase Cγ1 in a buffer containing 50 mM HEPES / NaOH (pH 7.0), 3 mM CaCl ₂ and 1 mM EGTA, 10 μl of the sample solution, and 10 μl of [ ³ H-inositol] PI 50 μl of the substrate solution and 50 μl of the buffer solution at pH 7.0 were added and reacted at 37 ° C for 10 minutes. Then, 1 ml of the reaction termination solution A and 0.3 ml of the reaction termination solution B were added to stop the reaction. Centrifuged and divided into two layers. The radioactivity of [ ³ H-inositol] PI produced in the aqueous layer was measured by a liquid scintillation counter. As a background control, DMSO alone was used as a control, and amentoflavone was reported as a positive control (Lee et al., 1996) .

(Example 5) Measurement of the total amount of inositol phosphate (IPt) in NIH 3T3 gamma 1 cells

NIH 3T3γ1 cells were labeled for 24 hours in inositol-free DMEM media containing ¹ μCi / ml of myo- [2- ³ H] inositol. Cells were washed with PBSFH, incubated in the reaction medium for 15 min, and then added to the sample. After 20 minutes of sample addition, PDGF was added and incubated for 30 minutes. IPt was extracted with 5% HClO ₄ in an ice bath for 30 min and the acid solution was subjected to a Biorad AG 1-X8 anion exchange column. IPt was eluted by varying the concentration of the ammonium formate solution during the column chromatography. [ ³ H-inositol] The radioactivity of IPt was measured with a liquid scintillation counter. The measurement was carried out in the same manner as in Example 4.

(Example 6) Measurement of cancer cell growth inhibitory activity

NCI method (Monks et al., 1991). Cells in the log phase were added to a 96 well plate at a concentration of 5 to 10 x 10 ⁴ cells / ml determined in the preliminary experiment, and the cells were pre-cultured at 37 ° C for 24 hours. 100 μl of the medium containing the sample was added and reacted for 48 hours. In order to measure the cytotoxicity of the cells in the stationary phase, 190 μl of the cells were placed in a 96-well plate, pre-cultured for 72 to 96 hours, and reacted with 10 μl of the sample for 48 hours . Cell viability was measured using SRB (Skehan et al., 1990, Rubinstein et al., 1990) and MTT (Alley et al., 1998, Vistica et al., 1991) The index was expressed as IC ₅₀ and adriamycin was used as a positive control.

(Example 7) Measurement of antioxidative activity (DPPH assay)

Samples were dissolved in DMSO to a concentration of 10 mg / ml and serially diluted 5 times to give 6 concentrations. 5 μl of each sample was placed in duplicate in a 96-well plate, and 100% DMSO was used as a control. DPPH soln. (3.16 mM, EtOH) was diluted 10-fold with absolute EtOH immediately before use, and 95 [mu] l was added. The plate was covered with parafilm immediately after the addition, so that EtOH did not evaporate during incubation. After shaking for 1 min, incubation was carried out in a 37 ° C incubator for 1 hr and absorbance was measured with an ELISA reader at 515 nm. As% control was determined for IC _50.

The effect of the present invention is to produce an anticancer substance, which is an inhibitor of phospholipase C? 1, in a colostrum by extracting an active ingredient from a colony belonging to the family Acanthopanax sp. Using an active search method of phospholipase C? 1. Inhibiting activity, and has developed a new anticancer substance that shows high safety in colon cancer.

Claims (3)

The phospholipase C? 1 inhibitory anticancer active compound represented by the following general formula (I) (Formula I) In the general formula R ₁ and R ₂ are each hydrogen or methyl (except when R ₁ and R ₂ are both hydrogen) X and Y each represent hydrogen, a lower alkyl group having 1 to 4 carbon atoms, an alkoxy group, an amino group, a halogen or a hydroxy group. (Provided that when R ₁ is methyl and R ₂ is hydrogen, X hydrogen and Y hydroxy groups are excluded; When R ₁ is hydrogen and R ₂ is methyl, X hydrogen and Y hydroxy groups are excluded.) 3. The compound of claim 1, wherein the compound is 3beta-hydroxy-27-i-ferulyloxy-olea-12-en-28-oic acid (1); 3 beta -hydroxy-27-i-peryllyloxy-ur-12-en-28-oic acid (2); 3beta-hydroxy-27-jet-peryllyloxy-olea-12-ene-28-oic acid (3); 3beta-hydroxy-27-jet-p-coumaroyloxy-wur-12-en-28-oic acid (4); 3beta-hydroxy-27-iso-p-coumaroyloxy-olea-12-en-28-oic acid (5); 3 beta -hydroxy-27-iso-p-coumaroyloxy-ur-12-en-28-oic acid (6); 3beta-hydroxy-27-jet-p-coumaroyloxy-olea-12-en-28-oic acid (7); 3-beta-hydroxy-27-jet-p-coumaroyloxy-truss-12-en-28-oic acid (8) Using a fractionation method using a bioassay, Daesung belonging to the Acanthaceae family was extracted with chloroform-methanol, and the chloroform fraction was purified by silica gel column chromatography, Sephadex LH-20 and Semiprep HPLC to the general formula (I) A method for separating and purifying the displayed phospholipase C? 1 inhibiting active ingredient