KR19980077620A - Anticancer composition containing a diaryl heptanoid derivative and its salt as a main component - Google Patents

Abstract

The present invention provides the substance 1,7-bis- (4-hydroxyphenyl) -5- (β-D-glucopyranosyloxy) -3-heptanone and its derivatives represented by the following general formula (1) showing anticancer activity isolated from natural resources And it relates to an anticancer agent composition containing a pharmaceutically usable salt as an active ingredient. The material of the present invention isolated from natural resources is expected to have excellent anticancer activity, high safety, and greatly reduce side effects compared to conventional drugs.

In the formulas, R ₁ and R ₂ each represent hydrogen, a lower alkyl group, a lower alkanoyl group, and a monosaccharide having 5 to 6 carbon atoms.

Description

Anticancer composition containing a diaryl heptanoid derivative and its salt as a main component

The present invention relates to diaryl heptanoid compounds isolated from natural resources. More specifically, the present invention provides 1,7-bis (4-substitutedphenyl) -5- (beta-D-substituted or unsubstituted compound represented by the following general structural formula (1) showing anticancer activity isolated from alder It relates to an anticancer agent composition containing (3--3-heptanone) and a pharmaceutically usable salt as a main component.

In the formula, R ₁ , R ₂ represent hydrogen, lower alkyl group, acetyl group or monosaccharide containing 5-6 carbons.

1,7-bis- (4-hydroxyphenyl) -5- (O-beta-D-glucopyranosyl) -3-peptanone (hereinafter referred to as SJ-2704) in the compound of the general formula (1). Is a type of diaryl heptanoid, reported in 1973 as the first material isolated from Betula platyphylla by Teresa et al. [Mokuzai Gakkaishi 19 (1), 47 (1973)] However, there is no mention in this document that SJ-2704 has anticancer effects.

The present inventors have searched for efficacy against natural products for a long time to find a substance having anticancer activity from natural resources in Korea. Alder bark has been used for antipyretic, hemostasis, astringent action from whales, intestinal infections, diarrhea, traumatic bleeding, bloody stools, etc. (Kim Jae-gil: Metabolism of Color Natural Medicine, 1984)

The present inventors were able to observe the anticancer activity of the extract extracted from the leaves and bark of alder as a result of cytotoxicity test on cancer cell lines derived from humans, using a series of systematic solvent fractions and various column chromatography. The active substance (SJ-2704) was followed up. The compound was found to have the same or better efficacy as compared with the existing anticancer drugs, and the acute toxicity test of the animal showed a very high safety in oral and intraperitoneal administration. . Alders (genus Alnus) include A. mayri, A. hirsuta, Alder (A, hirstua var, sibirica), Dumble duck (A. fruticosa var, mandshurica) , A. maxim-owiczii, A. vermicularis, A. firma, A. pendula, A. japonica, etc. (Betulaceae), most of which grow in mountain or alpine areas. (Lee Chang-bok: Korea Plant Book, 1989)

It is an object of the present invention to provide an anticancer composition comprising the above general formula (1), its derivatives and salts thereof as main components.

The present invention is described in detail as follows.

The alder leaves were dried and pulverized, extracted with water and an alcohol mixed solvent, and the extract was concentrated under reduced pressure, and then systematic solvent fractionation was performed. Each of the fractionated solvents was removed to prepare a concentrated extract, which was used primarily as a sample for cytotoxic effect screening. As a result, ethyl acetate extract showed significant effect, and the extract was followed to isolate the compound of general structural formula (1) which is anticancer active ingredient by methods such as silica gel column chromatography and gel filtration by bioactive directed fractionation. The compound of the general formula (1) is a substance exhibiting an anticancer effect equal to or higher than that of a drug widely used as an anticancer agent. The solubility problem, which is one of the disadvantages of drugs, can be overcome, so that it is not only a great help in formulating but also a side effect is greatly reduced compared to a synthetic compound as a substance separated from nature.

The compound of the general formula (1) is formulated in the form of conventional formulations such as oral and injectables, which are commonly used pharmaceutically, in combination with conventional pharmaceutically used excipients, adjuvants, stabilizers, pH adjusting agents, and antioxidants. May be administered.

The compound of the general formula (1) may be used in accordance with the condition, age, sex, etc. of the patient, but can generally be administered in an amount of 10 to 1,000 mg once or several times a day. In addition, this component may be administered alone or in combination with other effective drugs in the form of commonly used alkali metal salts or conventional inorganic and organic acid salts.

The present invention is explained in more detail by the following examples and experimental examples.

Example 1

The dried alder leaf powder (10 kg) was reflux-extracted three times for three hours with 15 L of 70% ethanol, and the extract was concentrated under reduced pressure to obtain a concentrated extract. 1 liter of distilled water was added to the extract and suspended in an 80 ° C water bath, followed by systematic solvent fractionation at room temperature to prepare hexane (25 g), ethyl acetate (60 g), butanol (80 g) and water (360 g) extracts. It was used as a sample of drug search. In addition, SJ-2704 was isolated by silica gel column chromatography using an ethyl acetate fraction in which significant activity was observed in a mixed solvent of chloroform / methanol / water.

Example 2

The dried alder leaves (5 kg) were crushed and extracted three times at room temperature with 7 liters of methanol for 24 hours. The extracts were combined and concentrated. The extract was shaken with 90% methanol and hexane and the methanol was concentrated to chloroform / methanol / water. SJ-2704 was isolated by silica gel column chromatography using and gel filtration using methanol as a solvent.

Example 3

Alder bark (5 kg) was cut and extracted with methanol (7 L) three times at room temperature for 24 hours, and concentrated and fractionated with hexane / methanol. Methanol was further concentrated to give ethyl acetate fractions using ethyl acetate and water, which was concentrated, silica gel column chromatography using ethyl acetate / methanol / water mixed solvent and filtration with ethanol and water gave SJ-2704.

Instrumental Analysis Data of SJ-2704

mp: 165 ° C. (decomp.)

: 3400-3350 (OH), 2950-1850 (CH), 1700 (C = O),

1610, 1590, 1510 (Ph)

224 (4.15), 279 (3.54), 282 (3.8)

¹ H-NMR (CD ₃ OD, TMS, δ ppm)

1.74 (m, 1H, 6-H), 1.83 (m, 1H, 6-H), 2.59 (m, 3H), 2.74 to 2.81 (m, 5H, 1,2,4,7-H), 4.16 ( m, 1H, 5-H), 6.67 (dd, 4H, J = 8 Hz, Ar-H), 6.99 (d, 2H, J = 8.5 Hz, Ar-H), 7.01 (d, 2H, J = 8.5 Hz , Ar-H), 3.15 (m, 1H, 2'-H), 3.25 (m, 1H), 3.69 (dd, 1H, J = 12, 5 Hz, 6'-H), 3.86 (dd, 1H, J = 12, 2 Hz, 6'-H), 4.28 (d, 1H, J = 7.5 Hz, 1'-H)

¹³ C-NMR (Pyridine-d ₅ , TMS, δ ppm)

29.2 (1), 45.9 (2), 209.4 (3), 48.2 (4), 75.3 (5), 38.2 (6), 31.0 (7), 132.1 (1 '),

133.2 (1), 129.9 (2 '), 129.9 (2), 116.3 (3'), 116.3 (3), 156.9 (4 '), 157.1 (4),

116.2 (5 '), 116.2 (5), 130.1 (6'), 130.1 (6), 103.6 (1 '), 75.3 (2'), 78.5 (3 '), 71.7 (4'),

78.3 (5 '), 62.8 (6')

MS (m / z): 476 (M ⁺ ), 296 (M ⁺ -180)

Example 4

To 5 mg of 2% H ₂ SO _{4 in} 100mg of SJ-2704, reflux reaction was carried out for 2 hours, and the reaction solution was extracted with chloroform, and the extract was concentrated under reduced pressure. Graphitic and gel filtration was performed to yield 1,7-bis (4-hydroxyphenyl) -5-hydroxy-3-heptanone (1,7-bis (4-hydroxyphenyl) -5, a hydrolyzate of SJ-2704. 21 mg of -hydroxy-3-heptanone was obtained.

mp: 122 ° C. (decomp.)

: 3400 (OH), 1690 (C = O), 1600, 1500, 1210, 800

¹ H-NMR (CD ₃ OD, TMS, δ ppm)

1.66 (m, 2H, 6-H), 2.49-2.75 (m, 8H, 1-H, 2-H, 4-H, 7-H), 4.00 (m, 1H, 5-H),

6.68 (d, 2H, J = 7.6 Hz, 3'-H, 5'-H), 6.69 (d, 2H, J = 7.4 Hz, 3-H, 5-H), 6.98 (d, 4H, J = 8 Hz, 2'-, 6'-, 2-, 6-H)

¹³ C-NMR (CD ₃ OD, TMS, δ ppm)

29.9, 32.0, 40.6, 46.5, 51.4, 68.4, 212.0, 116.6, 116.3, 130.3, 130.4, 133.4, 134.2, 156.5, 156.7

MS (m / z): 314 (M ⁺ ), 296, 107 (100)

Example 5

20 mg of SJ-2704 is dissolved in 1 ml of anhydrous pyridine, 1 ml of acetic anhydride is added thereto, and the mixture is left to stand at room temperature for 24 hours. 50 ml of ice water was added to the reaction mixture, followed by extraction three times with 50 ml of chloroform. The extracts were combined and concentrated under reduced pressure under boiling point. Then, silica gel column chromatography was performed with chloroform / ethyl acetate to give 30 mg of SJ-2704 hexaacetate.

mp: 70 ° C (decomp.)

¹ H-NMR (CD ₃ OD, TMS, δ ppm)

2.28 (two s, 6H, aromatic-OAc × 2), 1.98, 2.01 and 2.03 (four s, 12H,

alcoholic-OAc × 4), 1.75-1.86 (m, 2H), 2.3-2.87 (m, 8H), 3.7 (m, 1H),

4.58 (d, 1H, J = 8 Hz, 1'-H), 4.6-5.2 (m, 7H), 6.96-7.19 (m, 8H, aromatic-H)

MS (m / z): 728 (M ⁺ ), 686, 331, 107

Experimental Example 1

Cytotoxicity Experiments: SRB Method.

Each cell line was prepared as a single cell suspension, and then the cell lines viable under a microscope in a hematocytometer chamber were counted. Water was added and preincubated for 24 hours at 37 ° C., 5% CO ₂ -95% air incubator. Add the test substance to be tested together with the control group, incubate for 48 hours in 37 ℃, 5% CO ₂ -95% air incubator, remove the medium of each well by aspiration, add cold TCA, and leave at 4 ℃ for 1 hour. Wash in running water five times and dry in air. 0.4% SRB was added to each dried well and allowed to stand at room temperature for 30 minutes, then washed 5 times with 1% HAc to remove excess SRB and dried at room temperature. Each well was dried for 5 minutes in a plate shaker and the optical density (OD) was measured at a wavelength of 540nm with an ELISA reader. The IC ₅₀ value (concentration required to inhibit 50% growth rate in μg / ml) in each cell line was determined as the concentration of test substance that resulted in a 50% reduction in control OD. All three were obtained by three or more replicates, and the average was calculated by the following formula.

(1-mean O.D. in test well / mean O.D. in control well) × 100 (%)

From this, the IC ₅₀ value of each test substance was determined using Quantal probit ananysis of the phamacological calculation program.

Table 1 Cytotxicity against human tumor cell lines (IC ₅₀ ; ㎍ / ml)

Human tumor cell lines: A549 (lung carcinoma), AGS (gastric adenocarcinoma), HCT15 (colon adenocarcinoma), SKOV3 (ovary adenocarcinoma), HEP3B (hepatocellular carcinoma), VERO (green monkey kidney normal cell)

From the results of Table 1, the overall cytotoxic effect of the compound SJ-2704 is recognized, and the toxicity to green cells (green monkey kidney normal cell) is expected to be more safe from weak compared to conventional drugs.

Experimental Example 2

Acute Toxicity Experiment

Five-week-old mice (ICR, males 25 ± 3.0 g) were freely fed solid feed and water at room temperature 23 ± 1 ° C. and 55 ± 1% humidity. Six animals per group were used for oral and parenteral drug administration (abdominal and intravenous). Observation was made for 14 days, and the appearance and death were recorded. The mortality was examined by necropsy. LD ₅₀ values were determined by Litchfield J. T. and Wilcoxon (Litchfield JT-Wilcoxon) method and the results are shown in Table 2.

TABLE 2 Acute toxicity (LD ₅₀ mg / kg)

Drugs in Japan (ethical drugs)

From Table 2, the LD ₅₀ value of Compound SJ-2704 was more than 2000 mg / kg in oral administration and more than 1000 mg / kg in intraperitoneal injection.

Formulation Example 1.

Preparation of tablets

SJ-2404 100mg

Corn Starch 50mg

Lactose 50mg

Sodium Starch Glycolate 40mg

H. P. C5 mg

Magnesium Stearate 5mg

The above ingredients are prepared into tablets by tableting into 250 mg tablets according to a conventional tablet production method.

Formulation Example 2.

Manufacturing method of capsule

SJ-2704 100mg

Lactose 80mg

Dicalcium Phosphate 100mg

H. P. C.5 mg

Magnesium Stearate 15mg

The above ingredients are prepared by filling into 300 mg capsules according to a conventional capsule preparation method.

Formulation Example 3.

Preparation of injectables (based on 2 ml)

SJ-2704 100mg

Sodium Chloride18mg

Benzyl Alcohol 45mg

Distilled water equivalent for injection

The above ingredients are filled into ampoules according to a conventional injection method to prepare an injection.

Formulation Example 4.

Preparation of fluid (250 ml basis)

SJ-2704 300mg

Sodium Chloride2250mg

Sodium Bisulfate250mg

Citric Acid 50mg

Sodium Citrate 50mg

Propylene Glycol12500mg

Distilled water equivalent for injection

The above components are filled into a 250 ml capacity container according to a conventional method of preparing a fluid to prepare a fluid.

Claims (1)

Formula (1) or a pharmaceutically acceptable salt containing the compound of the general formula (1) as an active ingredient in a conventional pharmaceutically acceptable excipient and adjuvant and formulated in a conventional pharmaceutical formulation in a conventional pharmaceutically acceptable manner Anticancer agent composition. In the formulas, R ₁ and R ₂ each represent hydrogen, a lower alkyl group, a lower alkanoyl group, and a monosaccharide having 5 to 6 carbon atoms.