KR20030059644A - (3R,6R)-4-methyl-6-(1-methylethyl)-3-phenylmethyl-1,4-perhydrooxazine-2,5-dione from the Fruiting Bodies of Isaria japonica and apoptosis-inducing composition containing the same - Google Patents

Abstract

PURPOSE: Provided is a novel compound, (3R,6R)-4-methyl-6-(1-methylethyl)-phenylethyl-1,4-perhydroxazine-2,5-dione(MMPP O), separated from fruit body extract of Isaria japonica Yasuda and inducing apoptosis. The compound is represented by the formula 1 and is used as an anti-cancer agent. CONSTITUTION: A composition containing the compound, (3R,6R)-4-methyl-6-(1-methylethyl)-phenylethyl-1,4-perhydroxazine-2,5-dione(MMPP O) characteristically leads to apoptosis in the cancer cell of leukemia, breast cancer, liver cancer, stomach cancer, ovary cancer, urinary bladder cancer, lung cancer and epithelioma cancer.

Description

(3R, 6R) -4-methyl-6- (1-methylethyl) -3-phenylmethyl-1,4-perhydrooxazine-2,5-dione and isolated apoptosis inducer {(3R, 6R) -4-methyl-6- (1-methylethyl) -3-phenylmethyl-1,4-perhydrooxazine-2,5-dione from the Fruiting Bodies of Isaria japonica and apoptosis-inducing composition containing the same }

The present invention relates to a novel compound isolated from the extract of the fruiting body of Isaria japonica Y _ASUDA , and more particularly, as a component isolated from the _{eyeflower inflorescence} (3R, 6R) -4-methyl-6- (1- It relates to an apoptosis inducer composition containing methylethyl) -3-phenylmethyl-1,4-perhydrooxazine-2,5-dione (hereinafter referred to as 'MMPPD').

Although cancer treatment has opened the way for considerable advances in medical technology, biotechnology and genetic engineering, cancer is still recognized as a disease that cannot be cured yet.

Many methods such as chemotherapy, radiation therapy, and incision have been developed as cancer treatment methods, but radiation therapy and incisions are mainly effective only when the initial diagnosis of cancer is performed. However, if progression to terminal cancer is seen, cancer should be treated with chemotherapy, but the cure rate is quite low.

Therefore, the clinical field requires a new anticancer agent that is more effective than conventional anticancer agents, but in order to develop such an anticancer agent, development of a new target of the anticancer agent is required.

Recently, various genes and proteins involved in apoptosis have been studied as targets of such anticancer agents. The initiation of this study began with the discovery that anticancer agents used for chemotherapy over the past few years can damage genes in cancer cells, leading to apoptosis, or programmed cell death.

Although the mechanisms by which tumor cells can be killed through apoptosis are controversial, finding a substance that can induce apoptosis has been recognized as a method for identifying new anticancer drugs.

Apoptosis is a form of cell death that appears in all cells, and when apoptosis occurs, distinct morphological and biochemical characteristics are manifested. This apoptosis apoptosis is usually caused by the action of several genes in the end-of-life normal cells, as important as the production of new cells from undifferentiated stem cells. Cells that undergo apoptosis through apoptosis do not have any effect on adjacent cells by inflammatory reactions or the like, and only the cells are destroyed by carrying out the planned killing. The physiological process of apoptosis is distinguished from necrotic cell death, which causes an inflammatory response and also destroys surrounding cells after severe cell damage, swelling and lysis.

Apoptosis is a planned apoptosis that effectively kills virus-infected cells and cells that have been transformed into tumors by natural mutations when the cells that have to be present briefly during life's development disappear. .

Therefore, the development of a substance capable of inducing apoptosis may be characterized by effectively killing only tumor cells without inducing death of normal cells through inflammatory reactions.

Therefore, the present inventors have conducted a lot of research to find a new substance that can induce apoptosis, 4-acetyl-12, 13-epoxy-9- as a newly separated component from the methanol extract of the fruiting body of Snow Cordyceps in the previous study A patent application dated Aug. 20, 2001 concerning an anticancer agent composition containing AETD as an active ingredient, discovering that trichothecin-3,15-diol (AETD) can induce apoptosis of cancer cells, especially leukemia cells. 2001-50001).

The inventors of the present invention conducted further studies on the medicinal components of the Snow Clover, as (3R, 6R) -4-methyl-6- (1-methylethyl) -3-phenyl as a novel compound isolated from the Snow Clover. It was found that methyl-1,4-perhydrooxazine-2,5-dione (hereinafter referred to as 'MMPPD') has very good apoptosis inducing activity, so that a composition containing it can be used as an apoptosis inducing agent composition. The present invention has been completed.

Accordingly, it is an object of the present invention to provide a novel compound isolated from snow whim fungus.

In addition, another object of the present invention is to provide an apoptosis inducer composition containing a compound isolated from a snow whim fungus as an active ingredient.

1 is a diagram showing by nuclear staining that apoptosis is induced in leukemia cell line HL-60 cells by treatment with a compound of the present invention (in FIG. 1, the asterisk body is typically seen in cells showing apoptosis). (apoptotic bodies).

FIG. 2 shows DNA fragmentation test showing that apoptosis is induced in leukemia cell line HL-60 cells by treatment with a compound of the present invention (in FIG. 2, Marker is a φx174 RF DNA / Hae III size marker. The size of 1,353 bp, 1,078 bp, 872 bp, 603 bp from above).

Figure 3 shows that the treatment of the compound of the present invention increases the transfer of the leukemia cell line HL-60 cells to the cell cycle of the sub-G1 phase, a typical feature of apoptosis.

In order to achieve the above object, the present invention is a novel compound isolated from Isaria japonica Y _ASUDA , (3R, 6R) -4-methyl-6- (1-methylethyl) ) -3-phenylmethyl-1,4-perhydrooxazine-2,5-dione (hereinafter referred to as 'MMPPD').

[Formula 1]

The effective amount of MMPPD as an active ingredient in the apoptosis inducer composition according to the present invention may be appropriately selected depending on the method of administration, the type of preparation, the age of the patient, the weight of the patient, the sensitivity of the patient, and the condition of the disease. Generally, however, MMPPD may be formulated to be administered at a concentration of 0.001-100 mg / kg body weight. Of course, the dose of the active ingredient may be an amount outside the above range.

Formulations for administering MMPPD within the above range may have a conventional form, for example, may be used in the form of pills, capsules or drinks, injections, pharmaceuticals and the like. They may be administered to the site to which apoptosis is induced through oral or various parenteral routes of administration, and are suitable for the dosage form and are well known to those skilled in the art, and various excipients and carriers can be easily selected by those skilled in the art. Or a diluent or the like.

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

Cordyceps sinensis, especially the fungus belonging to Isaria, is an anamorphic fungus of the genus Cordicef that infects insect larvae and then breeds in the corpse of the host. This form of fungal fruiting has traditionally been used to treat many diseases. Although many bioactive substances, such as cordycepin and adenosine derivatives, have been found in fungi of the genus Cordicep, not many substances have been reported in fungi of Isaaria except for powerful immunosuppressive agents (myriosin).

The present inventors conducted a study to find other components capable of inducing apoptosis in addition to 4-acetyl-12,13-epoxy-9-tricortesin-3,15-diol (AETD), which were revealed in previous studies, As a novel compound isolated from Cordyceps sinensis, it was confirmed that MMPPD had superior apoptosis inducing activity than existing substances without causing side effects. Accordingly, the present invention provides an apoptosis inducer composition containing MMPPD as an active ingredient based on these findings.

According to the present invention, the novelly disclosed MMPPD is characterized by the characteristics of apoptosis commonly known in the art in cancer cells, such as (1) morphological changes in typical apoptotic cells, including chromatin and fission; (2) fragmentation of cancer cell DNA by activation of DNases; And (3) activation of metastasis to sub-G1 groups in the cell cycle. Therefore, the composition according to the present invention can be usefully used for the purpose of inducing apoptosis in the treatment of various diseases.

The composition according to the present invention containing MMPPD as an active ingredient is a variety of cancers such as apoptosis, preferably leukemia, breast cancer, liver cancer, cervical cancer, stomach cancer, ovarian cancer, bladder cancer, lung cancer, epithelial cancer, and more preferably. Preferably it can induce apoptosis of cells that contribute to leukemia, and thus can be usefully administered in the treatment of cancer, in particular leukemia.

That is, cancer is most often caused by an imbalance between apoptosis-associated cell death and cell proliferation by intracellular signaling mechanisms. In general, in cancer tissues, signaling mechanisms involved in cell proliferation are activated, whereas intracellular cells involved in apoptosis are activated. It is well known in the art that signaling is inhibited. Thus, apoptosis inducers can treat cancer by activating apoptosis in cancer cells and inducing planned apoptosis in cancer cells.

In this respect, the apoptosis inducer composition according to the present invention can be usefully used for the treatment of cancer, preferably leukemia, by activating apoptosis in cancer cells, preferably leukemia cells. Is not limited thereto, and may also be usefully used for the treatment of diseases caused by oversuppression of apoptosis, which is commonly known in the art.

In addition, the apoptosis inducer composition according to the present invention may be administered alone when applied to cancer treatment, but may be used in combination with other cancer treatment or cancer treatment composition, drugs, and various other physical and medical treatment methods. By doing so, the effect can be maximized.

Hereinafter, the present invention will be described in more detail with reference to Examples and drawings, but the present invention is not limited thereto. Although the following examples show only the results of application to HL-60 cells in leukemia cells with respect to the apoptosis inducing effect of the compounds according to the present application, the scope of the compounds of the present invention is applied only to the HL-60 cells or leukemia cells. It will be apparent to those skilled in the art that the cancer disease can be treated by inducing apoptosis in various cancer cells, but not applicable.

Experimental examples of the following Examples are the results obtained by performing each experiment at least three times, using a statistical analysis using Student's t-test. The limit of significance was set based on the p value <0.05.

Reference Example 1 Material

The reagents and samples used in the examples below were obtained as follows.

RPMI-1640 and fetal bovine serum (FBS) were purchased from Gibco / BRL (Grand Island.NY.U.S.A). Dimethy sulfoxide (DMSO), 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide, trypan blue, DAPI and propidium ioda Propidium iodide (PI) was purchased from Sigma Chemical, St. Louis, Mo., USA. All other solvents were of analytical grade and were purchased from Merck (Darmstadt, Germany).

Reference Example 2 Cell Culture

The HL-60 cell line (CCL-240) was distributed from the Depositary of the American Type Culture Collection (ATCC), Rockville. MD. USA (ATCC), and unless otherwise stated below, the HL-60 cell line is 10% utea serum. In RPMI-1640 culture medium to which (FBS) was added, the culture was maintained at 37 ° C. in a humid atmosphere with 95% air and 5% carbon dioxide (CO ₂ ). Cells were incubated with fresh culture medium every 2-3 days to maintain exponential growth.

Reference Example 3 Collection and Cultivation of Snow Cordyceps Sinensis

The species of Cordyceps sinensis used in the present invention was used as a species isolated from Moaksan, Jeollabuk-do, Korea, and was identified as Snowflower Cordyceps by Professor Woo Deok-Hyun of Korea.

Identified Snow Cordyceps sinensis (I. japonica, voucher No. CHO-6133) was inoculated in potato glucose medium and cultured at 25 ° C. for about 10 days until the colonies had a diameter of 5 cm. The fungal colonies formed were gray.

The obtained bacterial colonies were stored at a temperature of about 4 ° C., used as stock cultures, and used for the following test cultures. Then, 1 ml flask was filled with 500 ml of culture medium, sterilized at 121 ° C. for 20 minutes, and then cooled to 15 ° C., and four 5 mm agar plugs obtained from the storage culture medium were placed. Second cultures were performed by inoculation. The flask culture was incubated at 140 ° C. for 3 days at 140 rpm and then for 4 days at 160 rpm. At this time, the culture medium was used consisting of 200g / ℓ of potatoes and 30g / ℓ of sugar. All cultures were performed in the dark.

Then, a third fermentation was carried out in 100 g silkworm pupa. The non-divided medium was sterilized at 121 ° C. for 40 minutes, and the sterilized non-divided medium was cooled to 15 ° C., and then inoculated with 5 ml of spore solution. Then, it was incubated for 8 days in the dark at 25 ℃. After incubation, the culture conditions were changed to 60% humidity and further incubated in the dark for 10 days, followed by further incubation for 30 days under the culture conditions maintained at 20 ° C., 80% humidity and 2000 lux brightness, and then the fruiting body of Snow Cordyceps Got it.

Example 1 Extraction and Separation

1,000 g of air dried fruiting body were extracted with 5 L of methanol for 48 hours. Then, the methanol extract was concentrated and suspended with water, and extracted sequentially with n-hexane, ethyl acetate and butanol to obtain 2.9 g of EtOAc soluble fraction. The EtOAc soluble fraction was then subjected to C ₁₈ flash column chromatography, followed by dilution with 0%, 20%, 40%, 50%, 60%, 70%, 90% and 100%. Elution was carried out with a methanol concentration gradient of (v / v) (8 fractions, 200 ml for each fraction). Then, fraction (305 mg) eluted in 90% methanol solution was applied to silica gel (Merck Kieselgel 60; 0.063-0.2 mm particles) column chromatography (silica gel column chromatography; 3 cm diameter, 20 cm height), Eluted with a CH ₂ Cl ₂ -MeOH concentration gradient (eluted with 0-50% methanol solution diluted with CH ₂ Cl ₂ ) (50 mL for each fraction; 15 fractions were obtained). Based on TLC analysis (silica gel, CH ₂ Cl ₂ / MeOH = 9: 1), fractions eluted with 4% and 5% methanol solutions diluted with CH ₂ Cl ₂ (fractions eluted between 300-400 mL; 164.2 Mg) and sub-mass HPLC (Alltech HS Hyperprep 100 BDS C ₁₈ (1.0 cm in diameter, 25 cm in height) using a gradient of 60-100% CH ₃ CN solution diluted with H ₂ O Compound 1 (42.5 mg, t _R = 36.8 min) was isolated purely by applying a particle size of -m; 2 ml / min; UV detection at 210 nm) for 60 minutes.

In addition, the molecular formula of Compound 1 was determined based on NMR and FABMS data analysis, and as a result, Compound 1 was (3R, 6R) -4-methyl-6- (1-methylethyl) -3-phenylmethyl-1,4 It was confirmed that it was perhydrooxazine-2,5-dione (MMPPD). The overall planar structure of Compound 1 was confirmed by analysis of two-dimensional NMR data (COSY and HMBC).

On the other hand, the results of the spectrum analysis of Compound 1 newly discovered in the present invention are as follows.

MMPPD (Compound 1):

[α] ²³ _D : 48.8 ^o (c 0.43, CHCl ₃ );

UV (MeOH): _max (log) = 209 (5056) nm;

IR (film): _max = 2964, 1745, 1660, 1483, 1413, 1371, 1263, 1180, 1109, 1018 cm ⁻¹ ;

¹ H and ¹³ C NMR: see Table 1 below;

C / H no. MMPPD ¹³ C ^a δ ¹ H _b δ (multiplicity, J in Hz) HMBC NOESY 2 170.2 - - - 3 57.6 5.47 (br d) 2 10 4-CH ₃ 32.6 3.00 (s) 3, 5 - 5 169.5 - - 6 75.7 4.92 (d, 8.7) 2, 7, 8 7 7 29.9 2.02 (m) 5, 6, 8 6 8 17.8 0.42 (d, 6.9) 6, 7, 9 9, 12, 13 9 18.5 0.80 (d, 6.9) 6, 7, 8 8 10 35.0 3.36 (dd, 14.5, 5.1) 2.98 (dd, 14.5, 11.9) 2, 3 3 11 136.9 - - - 12,16 129.1 7.23-7.26 (m) 10 8 13,15 128.8 7.23-7.26 (m) 10 8 14 127.0 7.17 (m) - - ^a Recorded at 500 MHz ^b Recorded at 125 MHz ^c Assignments may be interchanged

LRESIMS: m / z = 262 [M + H] ⁺

Example 2 Comparison of Spectrum Results with MMPPD and Conventional Compounds

Compound 1 has the appearance of white powder. Based on the results of carbon nuclear magnetic resonance and mass spectrometry, it was confirmed that the molecular formula of Compound 1 was C ₁₅ H ₁₉ NO ₃ .

On the other hand, the proton and carbon NMR analysis results of the compound 1 shown in Table 1 was almost the same as the results for the conventional latritin represented by the following formula (4) (11).

[Formula 4]

Although suspicion has been raised that the compounds of the present invention will have the structure of ratitatin (11), the structure of Compound 1 was once again confirmed through primary and secondary NMR data analysis.

In addition, by identifying the structure of Compound 2 produced (72% yield) under weak base reaction conditions (1N NaOH in MeOH, rt, 80 min), the structure of Compound 1 could be confirmed once again. The structure of the compound 2 is NMR and ESIMS material ^- have been identified through the ([MH] at m / z 278). The number of compound 2 was according to the manner used for compound 1. The spectral results and the structural formula of Compound 2 are as follows.

(3R, 6R) -N-methyl-[(1-hydroxy-2-methyl) propyl] phenylalanate:

[α] ²³ _D : + 25 ^o (c 0.12, CHCl ₃ );

UV (MeOH): _max (log) = 206 (5296) nm;

IR (film): _max = 3474, 2966, 1747, 1664, 1496, 1377, 1253, 1182, 1136, 1043 cm ^-1 ;

¹ H and ¹³ C NMR: see Table 2 below;

C / Hno. (3R, 6R) -N-methyl-[(1-hydroxy-2-methyl) propyl] phenylalanate ¹³ C ^a δ ¹ H ^b δ (multiplicity, J in Hz) 2 165.9 3 61.9 4.40 (dd, 5.1, 5.1) 4-CH3 32.9 2.99 (s) 5 164.5 - 6 83.8 4.36 (d, 7.4) 7 32.4 1.18 (m) 8 17.5 0.59 (d, 6.5) 9 18.9 0.85 (d, 6.9) 10 38.1 3.34 (dd, 14.2, 5.1) 11 134.8 - 12, 16 129.8 7.26-7.34 (m) c 13, 15 129.1 7.15-7.16 (m) c 7.26-7.34 (m) c 14 127.8 7.15-7.16 (m) c

LRESIMS: m / z = 278 [M-H] ^- .

[Formula 2]

The tertiary structure of Compound 1 has not been identified and was confirmed by analyzing the NOESY data (see Table 1) in the present invention. The NOESY link peak between the NMR signal (7.23-7.26) corresponding to the hydrogen of the aromatic structure and the NMR signal (0.42) of the hydrogen corresponding to the methyl group means that the two groups are close together in space. Thus, it can be seen that the benzyl group and the methyl group are located on the same side of the 1,4-oxazinane ring. It can be seen that the tertiary structure is different from basiatin having the formula (3) in which the relative orientations of the carbon 3 and 6 positions of the compound 1 are reported.

[Formula 3]

Through comparative analysis of the proton NMR data of compound 1 and the known proton NMR data (11), signals corresponding to H-6 (2.98 vs. 4.92) and H ₃ -9 (0.74 vs. 0.42) were remarkable. It can be seen that there is a difference.

In addition, the difference in the observed NMR signal was compared by comparing the molecular model of the x-ray structure of vaciatin (9) and MMPPD of the present invention. Based on the x-ray structure of the bastiatin, it can be seen that H-6 is located in the shielding region of the benzyl group, which is relatively high in up-field chemical shift) in the region. On the other hand, in the case of MMPPD, it can be seen that the methyl group is located in the masking region of the benzyl group rather than H-6, which indicates that the methyl group has resonance in the relatively higher chemical shift region. Therefore, it can be seen that the difference in NMR data of compound 1 and vaciatin is due to different relative orientations of the benzyl groups with respect to H-3 and methyl groups.

On the other hand, in order to determine the absolute orientation of C-3 and C-6 of the MMPPD of the present invention, the optical activity of the hydrolysis products of DDMMP was measured. First, when the compound 1 sikyeoteul hydrolyzed in acidic conditions (HCl 6N, 103 ℃, 12 hour reaction), N - methyl-phenylalanine and 2-hydroxy-3-methyl-butyric acid (butyric acid) are generated ¹ H NMR and ESIMS data analysis was used. The resulting N - optical activity of the methyl-phenylalanine _{^{([α] 23 D 20 o}} , c 0.05, 1M HCl) is a reference material R - figures methyl phenylalanine _{^{([α] 25 D 24.7 o}} , c 1.6, 1M HCl) - N Correspondingly, it can be seen that C-3 of Compound 1 has an R orientation. In a similar manner, the optical activity ([α] ²³ _D 50 ^o , c 0.02, CHCl ₃ ) of the resulting 2-hydroxy-3-methyl butyric acid was reported in the literature as S -2-hydroxy-3-methyl butyl. When compared with the acid value ([α] ²⁵ _D + 19 ^o , c 1, CHCl ₃ ), it can be seen that C-6 of MMPPD has an R orientation. On the other hand, the optical activity ([α] ²³ _D 48.8 ^o , c 0.43, CHCl ₃ ) of MMPPD is the value of the synthesized (3 S , 6 S ) -baciatin isomer ([α] ²³ _D 24.3 ^o , c 1.0, CHCl ₃ )

In addition, in order to clearly distinguish the planar and relative structures of the MMPPD isolated in the present invention, the NMR measurement data of the MMPPD of the present invention and the NMR data of the vaciatin isomer reported in the literature were examined closely. NMR measurement data of MMPPD of the present invention, in particular, the chemical shift value of H-3 and H-6 is different from the reported NMR data of (3 R , 6 R ) or (3 S , 6 S ) isomer have. On the other hand, the NMR measurement data of the compound 2 can be seen that is consistent with the NMR data of the reported (3 R , 6 R ) or (3 S , 6 S ) isomer. Thus, substantially reported synthetic isomers of vaciatin are not isocyanates, but open compounds with diketomorphine rings, ie N-methyl-[(1-hydroxy-2-methyl) propyl] phenyl It can be seen that it is an alanate. Further reported optical activity of 4-methyl-6- (1-methylethyl) -3-phenylmethyl-1,4-peroxoxazine-2,5-dione as (3 R , 6 R ) -baciatin isomer The value corresponds to the value of compound 2 ([α] ²³ _D + 25 ^o , c 0. 12, CHCl ₃ ). Thus, the overall tertiary structure, including the absolute orientation of compound 1, is (3 R , 6 R ) -4-methyl-6- (1-methylethyl) -3-phenylmethyl-1,4-perhydroxyxazine-2 Clearly identified as, 5-dione.

Example 3 Nuclear Staining with DAPI / PI

⁶ -well HL-60 cells at 1 × 10 ⁶ cells / ml, with or without 10 nmol / L MMPPD in RPMI 1640 medium containing 10% fetal bovine serum Incubated for 4 hours in a 6-well dishes. After incubation, 1 ml of cell suspension was centrifuged at 1,000 rpm for 3 minutes to precipitate cells. Thereafter, 100% of formalin buffered with 4% neutral buffer was fixed to the cell precipitate. Immobilized 50 μl of cell suspension was transferred to slides and dried at room temperature. The immobilized cells were washed with PBS, then dried and stained for 1 minute with DNA-specific fluorochrom DAPI (4,6-Diamidino-2-phenylindole) / PI. Attached cells were washed with PBS, then dried and mounted with 90% glycerol. Slides were observed using a fluorescence microscope. The results are shown in FIG.

As shown in FIG. 1, MMPPD caused nuclear condensation, nucleation and apoptosis body as a typical morphological feature of cell death through apoptosis.

Example 4 DNA Fragmentation Analysis

1 × 10 ⁶ cells / ml of HL-60 cells were treated with various concentrations of MMPPD for 6 hours. Then, MMPPD-treated cells were Winsim separated for 3 minutes at 1,000rpm, washed with PBS. To the cell precipitate, lysis buffer (50 mM Tris-HCl, pH8.0, 10 mM EDTA, 0.5% SDS, 0.5 mg / ml proteinase K (Sigma Chemical Co., St. louis. MO. USA)) was added and room temperature It was made to react by 30 minutes and dissolved. Then, 25 mg / ml of RNaes A was added, followed by reaction at 37 ° C. for 30 minutes, followed by addition of protein precipitation solution (40% ammonium sulfate), followed by centrifugation at 1,300 rpm at 4 ° C. for 10 minutes. All proteins precipitated out. The supernatant was then extracted with phenol / chloroform (1: 1, v / v) and the aqueous phase (aqueous phaes) was extracted once more with chloroform / isoamyl alcohol (24: 1, v / v). Then, double volume of isopropanol was added to precipitate the DNA. DNA pellets recovered by centrifugation were washed with 70% ethanol, dried in air and then dissolved in TE buffer consisting of 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. After quantification of DNA, 10 μg of DNA was electrophoresed with 1.5% agarose gel at 5V / cm. Then, after staining the DNA with EtBr (ethidium bromide), observed under ultraviolet light, the results are shown in FIG.

At about 180-200 bp positions in cells treated with MMPPD, a number of oligonucleosomal DNA fragments were identified, indicating that apoptosis occurred in MMPPD treated cells.

Example 4 Flow Cytometric Analysis

Cells with exponential growth are suspended in RPMI 1640 medium to a density of 1 × 10 ⁶ cells / ml, treated with 0, 5, 20, 50 or 100 μg / ml of MMPPD and incubated at 37 ° C. for 6 hours. It was.

1 × 10 ⁶ cells / well of HL-60 cells treated with MMPPD were fixed with 80% chilled ethanol for 30 minutes, washed with PBS, and 0.5% tryptone X- containing 1 mg / ml RNase A. 0.25 ml of 100 solution was treated at 37 ° C for 30 minutes. Then, 0.25 ml of a PI solution of 50 mg / ml concentration was added, and left in the dark for 30 minutes. Thereafter, the cells were passed through a FACS Vantage (Becton Dickinson, San jose, Calif., USA) flow cytometer to determine the number of cells from the sample, and the DNA amount of the sub-G1 phase was measured from the sample cells. The results are shown in FIG.

As shown in FIG. 3, as the concentration of MMPPD increased, the apoptosis peak of the sub-G1 phase also increased. These results indicate that MMPPD induces apoptosis of HL-60 cells in a concentration dependent manner.

As described above, (3R, 6R) -4-methyl-6- (1-methylethyl) -3-phenylmethyl-1,4-perhydrooxazine- 2,5-dione is very excellent in apoptosis inducing activity in cancer cells, so that a composition containing it can be used as an apoptosis inducing agent composition, in particular an anticancer agent.

References

1.Tanono F, Kikuchi Y, Fushiya S, Hojo H, Nozoe S, Yahagi N, Kondo Y. The culture fluid of Isaria japonica Yasuda augments anti-sheep red blood cell antibody response in mice. Biological & Pharmaceutical Bulletin 1996; 19: 641-3

Bok JW, Lermer L, Chilton J, Klingeman HG, Neil Towers GH. Antitumor sterols from the mycelia of Cordyceps sinensis . Phytochemistry 1999; 51: 891-8

Cory JG, Suhadolink RJ, Resnick B, Rich MA. Incorporation of cordycepin (3'-deoxyadenosine) into ribonucleic acid and deoxyribonucleic acid of human tumor cells. Biochimica et Biophysica Acta 1965; 103: 646-53

4.Furuya T, Hirotani M, Matsuzawa M. N ^6- (2-hydroxyethyl) adenosine, A biologically active compound from cultured mycelia of Cordyceps and Isaria species. Phytochemistry 1983; 22: 2509-12

Fusita T, Inoue K, Yamamoto S, Ikumoto T, Sasaki S, Toyama R, Chiba K, Hoshino Y, Okumoto T. Fungal metabolites. Part II. A potent immunosuppressive activity found in Isaria sinclairii metabolites. Journal of Antibiotics 1994; 47: 208-15

6. Story M, Kodym R. Signal transduction during apoptosis: Implications for cancer therapy. Frontiers in Bioscience 1998; 3: 365-75

7. McConkey DJ. Biochemical determinants of apoptosis and necrosis. Toxicology Letters 1998; 99: 157-68

8.Fadeel B, Orrenius, S, Zhivotovsky, B. Apoptosis in human disease: A new skin for the old ceremony? Biochemical and Biophysical Research Communications 1999; 266: 699-717

9.Oh GS, Hong, KW, Oh H, Pae HO, Seo WG, Kim IK, Kim NY, Kwon TO, Shin MK, Chung HT. 4-Acetyl-12,13-epoxyl-9-trichothecene-3,15-diol isolated from the fruiting bodies of Isaria japonica Yasuda induces apoptosis of human leukemia cells (HL-60). Biological & Pharmaceutical Bulletin 2001; 24: 785-9

10. Hasumi K, Shinohara C, Iwanaga T, Endo A. Lateritin, a new inhibitor of acyl-CoA: Cholesterol acyltransferase produced by Gibberella lateritium IFO 7188. Journal of Antibiotics 1993; 46: 1782-7

11.Kamgamizono T, Nishino E, Matsumoto K, Kawashima A, Kishimoto M, Sakai N, He BM, Chen ZX, Adachi T, Morimoto S, Hanada K. Bassiatin, a new platelet aggregation inhibitor produced by Beauveria basiana K-717. Journal of Antibiotics 1995; 48: 1407-12

Claims (5)

(3R, 6R) -4-methyl-6- (1-methylethyl) -3-phenylmethyl-1,4-perhydrooxazine-2,5-dione represented by the following formula (1). [Formula 1] 2. (3R, 6R) -4-methyl-6- (1-methylethyl) -3-phenylmethyl-1,4-perhydrooxazine-2, characterized in that it is separated from the fruiting bodies of the Snow Cordyceps. , 5-dione. An apoptosis inducer composition comprising the compound according to claim 1 or 2 as an active ingredient. 4. The apoptosis inducer composition of claim 3, wherein the apoptosis inducer composition induces apoptosis in cancer cells. The apoptosis according to claim 4, wherein the cancer cells are cancer cells selected from the group consisting of leukemia cells, breast cancer cells, liver cancer cells, cervical cancer cells, gastric cancer cells, ovarian cancer cells, bladder cancer cells, lung cancer cells and epithelial cancer cells. Inducer composition.