JPH07233071A - Cancer gene function inhibitor - Google Patents

Abstract

PURPOSE:To obtain an anti-cancer agent capable of preventing growth of cancer cell, comprising a compound isolated from Ervatamia microphylla kerr of a tropical plant as an active ingredient. CONSTITUTION:This anti-cancer agent contains a compound of the formula extracted from Ervatamia microphylla kerr of an apocynaceous plant as an active ingredient. When the compound of the formula is given to a cancer cell having an oncogene such as ras cancer gene, the shape of the cancer cell is normalized and growth of the cancer cell is suppressed. The compound can be pharmaceutically manufactured into a dosage form such as powder, granule, tablet, capsule, injection, etc. A dose is 0.1-100mg/kg/day in the case of oral administration. The compound of the formula has 794 molecular weight and slightly yellowish powdery substance.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anticancer agent containing a compound having an oncogene function suppressing activity as an active ingredient.

[0002]

2. Description of the Related Art In the process of carcinogenesis and promotion of cells,
It is known that oncogenes play an important role. Oncogenes are generated when abnormalities such as point mutations, translocations, and amplifications occur in proto-oncogenes on the chromosomal DNA of normal cells, and more than 70 types have been found so far. Among them, the ras oncogene is found in colorectal cancer, breast cancer, leukemia, etc., and accounts for 20% of all human cancer tissues.
It is a typical thing that is said to exist in the rank. Therefore,
A substance that inhibits the action of this ras oncogene product is expected as a novel therapeutic drug for cancer. To date, such inhibitors include oxanosine isolated from actinomycetes (N. Shimada et al., The Journal of Ant.
ibiotics, 34, 1216-1218, 1981), compactin isolated from mold (O. Ito et al., Cancer Research, 49,996-100).
0,1989) is known.

[0003]

However, all of the ras oncogene action inhibitor substances discovered so far have problems in stability and toxicity, and none of them are clinically applicable yet. Therefore, there is a demand for the development of a clinically applicable drug having a mechanism of action not found in known inhibitors.

[0004]

[Means for Solving the Problems] In order to obtain an inhibitor that can be clinically applied, the present inventors have screened by using the action of normalizing the morphology of cancer cells as an index,
As a result of searching for substances that are plant-derived and inhibit the action of the ras oncogene product, r was found in the leaf extract of the tropical plant Ervatamia microphylla Kerr.
The present invention has been completed by discovering that it has an activity of inhibiting the action of the as oncogene product and identifying the active ingredient to obtain the compound represented by the chemical formula (1).

[Chemical 2] That is, the present invention provides an anticancer agent containing a compound represented by the chemical formula (1) as an active ingredient and optionally a pharmaceutically acceptable carrier and the like.

The substance of the present invention isolated from Ervatamia microfila has a molecular weight of 794. Hereinafter, in the present specification, this substance is referred to as “III-121C” for convenience. III-121C is a slightly yellow powdery substance, and in the silica gel thin layer chromatography, the developing system chloroform: methanol = 20: 1, hexane: ethyl acetate =
At 1: 2 and toluene: acetone = 2: 1, the Rf values are 0.56, 0.25, and 0.52, respectively. FAB
Mass spectrum positive and negative results are shown in FIGS. 1 and 2. From the result of mass spectrum, it was found that the molecular weight was 794.

The ultraviolet absorption spectrum is as shown in FIG. 3 and the infrared absorption spectrum is as shown in FIG. 4, and a conjugated double bond and a carbonyl group are present. ¹ H-NMR spectrum and ¹³
The results of the C-NMR spectrum are shown in FIG. 5 and FIG. 6, respectively.
Is the street. In addition, III-121C was subjected to silica gel thin layer chromatography, and chloroform: methanol = 20:
After development in 1, various color reactions were conducted to examine the properties. In the 2,4-dinitrophenylhydrazine reaction, R
The position of f = 0.56 is colored red to indicate the presence of a carbonyl group, there is no peptide bond in the Rydon-Smith reaction, the presence of an amino group is not recognized in the ninhydrin reaction, and in the α-naphthol / sulfuric acid reaction. No coloration of sugar was observed, and the anisaldehyde reaction suggested the absence of sugar and steroid. Further, as a result of repeated studies, it was revealed that the structure of the compound of the present invention isolated from Ervatamia microfila was a structure represented by the above chemical formula (1).

When III-121C was given to a cancer cell having an oncogene such as the ras oncogene, the morphology of the cancer cell was normalized and its growth was inhibited. Therefore, this substance acts as an oncogene product. It has been found to have an inhibiting effect.
III-121C is a concentration (I
C ₅₀ ) varies depending on the type of cancer cell, but is about 0.2 to
It was 0.5 μg / ml (Table 1).

[Table 1]

Examples of pharmaceutically acceptable carriers used in the anticancer agent of the present invention include natural or synthetic aluminum silicate, microcrystalline cellulose, talc, dextrin,
Examples include excipients such as starch and lactose, and diluents such as vegetable oil and propylene glycol. In addition to this, pharmaceutically acceptable stabilizers, coating agents, suspending agents, emulsifying agents, solubilizing agents, preservatives, buffering agents, sweetening agents and the like may be present as other optional ingredients. As these additives, known additives may be used in appropriate combination. The dosage form may be any form such as powder, granules, tablets, capsules and injections. The anticancer agent of the present invention is mainly orally administered, but the present invention is not limited by the administration method. The daily dose for an adult varies depending on the administration method and medical condition. In the case of oral administration, which is the main method of administration, the usual dose is 0.1 to 100 mg / kg body weight / day. However, the present invention is not limited by dose.

Ervatami Microfila
a microphylla Kerr) is a small tree of the Oleander family, which grows naturally in Southeast Asia or is partly cultivated. The raw material plant may be dried for reasons such as storage and transportation. In that case, it is preferable to dry it in the sun or in an oven at 60 ° C. or lower. In addition, crushed ones are preferable when extracting the components. The solvent used for extraction is preferably a non-polar solvent, particularly preferably a hydrocarbon solvent or a halogenated hydrocarbon solvent. Examples of the hydrocarbon solvent include pentane, hexane, heptane, octane, benzene, toluene and xylene, and hexane is particularly preferable. Examples of the halogenated hydrocarbon solvent include chloroform, methylene dichloride, carbon tetrachloride and the like, and chloroform is particularly preferable. The most preferred solvent is chloroform. Of course, the solvent is not limited to these solvents, and any solvent that can stably extract III-121C can be used. Although the amount of the solvent is not particularly limited, it may be any amount as long as III-121C can be extracted, and is generally 500 to 1500 ml per 100 g of dry and pulverized Ervatamia microfila. The extraction temperature is also not particularly limited, but may be any temperature at which the compound can be stably extracted without decomposition, and is preferably room temperature to 60 ° C. The extraction time varies depending on the temperature, but is 2 to 20 hours for extraction at room temperature, for example. The extraction may be repeated twice or more depending on the extraction rate.

III-121C is purified from the extract obtained. Purification can be performed by, for example, chromatography after once concentrating the extract. Chromatography may preferably include column chromatography, gel filtration chromatography, high performance liquid chromatography, liquid-liquid countercurrent partition chromatography, thin layer chromatography or combinations thereof. For column chromatography, for example, Merck silica gel 60 (manufactured by Merck) as a carrier and chloroform: methanol = 100: 1 as a solvent can be used. For gel filtration chromatography, Toyopearl HW-40 (manufactured by Tosoh Corporation) as a carrier and methanol as a solvent can be used. However, the types of chromatography, carrier and solvent are not limited to those mentioned above. Then, as a result of analyzing the structure of III-121C, it was found to be represented by the chemical formula (1). Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the examples.

[0011]

[Example] 1. Extraction of active substances from Ervatamia microfila
After crushing the leaves 85g of Eruvatamia and micro filler was dried at 60 ° C. oven out, placed in a 2l Erlenmeyer flask and stirred for 5 hours at room temperature chloroform was added to 850 ml. The solvent was refreshed, the same operation was repeated, and extraction was carried out three times in total. The three extracts were combined, the chloroform solvent was distilled off under reduced pressure, and the obtained extract was further freeze-dried to obtain 10.5 g of a brown powder. This powder was used as a raw material for the next purification.

2. Purification of the active substance from the extract 1.34 g of the extract was dissolved in a small amount of chloroform and 10 g of silica gel packed in a column (trade name: Merck sili
cagel 60, made by Merck), first charge 100
Impurities were eluted with% chloroform. Next, it was eluted with chloroform: methanol = 100: 1, and fractionated by about 10 ml each. Thin-layer chromatography of each fraction (silica gel, solvent chloroform: methanol = 2
(0: 1, UV330 nm), Rf = 0.56, and blue fractions were collected and dried under reduced pressure, and the weight was 83.6 mg. When this fraction was subjected to a cell morphological change induction test, morphological normalizing activity was observed.

Subsequently, the active substance was dissolved in a small amount of methanol to remove the insoluble precipitate, and then charged on Toyopearl HW-40 previously equilibrated with methanol for gel filtration chromatography. Fractions eluted with 100% methanol and collected in 3 ml aliquots were analyzed by thin layer chromatography, chloroform: methanol = 20: 1.
The fractions of blue spots developed at Rf = 0.56 were collected and dried under reduced pressure, and the weight was 18.
It was 8 mg (hereinafter this substance is referred to as III-121C). When the purity of III-121C was confirmed by thin layer chromatography, it was a single spot. When this fraction was subjected to a cell morphological change induction test, morphological normalizing activity was observed.

3. Physicochemical Properties of Purified Product Various physicochemical properties of the purified product III-121C were measured, and various spectral analyzes were performed. The results are shown below. 1) Physicochemical properties III-121C is a pale yellow powdery substance. Development system on silica gel TLC (trade name: Merck 5715, manufactured by Merck); chloroform: methanol = 20: 1, hexane: ethyl acetate = 1: 2, toluene: acetone = 2: 1.
In, the Rf value was 0.56 0.25 0.52. It was also soluble in chloroform, ethyl acetate and methanol, and sparingly soluble in hexane. When the melting point was measured, it was burnt black at 206 to 210 ° C. and decomposed.

2) Analysis of Mass Spectrum The measurement results of FAB mass spectrum are shown in FIGS. 1 and 2. In the positive spectrum of Fig. 1, a characteristic peak at 795.4 (m / z) was obtained. Further, since a peak was found at 793.3 (m / z) in the negative spectrum of FIG. 2, it was found that the molecular weight was 794. 3) Analysis of UV spectrum The measurement result of UV absorption spectrum λ _max (E 1% / 1 cm) is
As shown in FIG. 330 nm (224) under neutral conditions (MeOH),
330nm (203) 245n under acidic condition (HCl / MeOH)
m (195) 339 nm under basic conditions (NaOH / MeOH)
(182) Absorption was observed at 254 nm (180). There was no significant change with pH. 4) Analysis of IR spectrum The result of IR spectrum measurement is shown in FIG. 2930 cm ^-1
Absorption seems to be derived from the methylene groups is seen, also a carbonyl group absorption of 1676cm ^-1, the presence of conjugated double bonds by the absorption of 1610 cm ^-1 was suggested to.

[0016] 5) The results of the measurements of the analysis ¹ H-NMR spectrum of ¹ H-NMR spectrum in FIG. 0.
A peak at 75 ppm was derived from a methyl group, multiple peaks at 2.0 to 3.7 ppm were considered to be derived from a methylene group, and a peak at 3.8 ppm was derived from a methoxy group. When deuterium substitution was performed, 5.1 ppm singlet disappeared, suggesting the existence of exchangeable protons. 6) ¹³ C-NMR spectrum analysis ¹³ results of measurement of C-NMR spectra are shown in Figure 6.
The peak at 6.2 ppm is probably due to the methyl group, the multiple peaks at 40 to 60 ppm are probably due to the methylene group, and the peak at 100 to 140 ppm is probably due to the carbon of the double bond. .

7) Various color reaction a. Iodine color reaction Purified sample was applied to silica gel TLC plate (trade name: Me
rck 5715, manufactured by Merck & Co., Inc.) and developed with chloroform: methanol = 20: 1. After confirming that the solvent was completely removed, this TLC plate was left to react in a closed container filled with vaporized iodine for about 5 minutes. After 5 minutes, the Rf = 0.56 position where the spot of the purified product was present was colored yellow. This suggested that the purified product had an unsaturated double bond. b. 2,4-dinitrophenylhydrazine (2,4-Dinitrophe
nyl-hydrazine) Color reaction a. 2,4-Dinitrophenylhydrazine (0.1 ml / 100 ml 2N HCl) was sprayed onto the TLC plate developed in the same manner as above, and this was heated on a hot plate. After a few minutes, Rf
The position of 0.56 was colored red. This suggested the presence of a carbonyl group in the purified product.

C. Rydon-Smith Color Reaction, a. The TLC plate developed in the same manner as the above was sprayed with a 1% sodium hypochlorite solution and then 100% ethanol to completely dry it. Further, a mixed solution of 1% soluble starch and 1% potassium iodide was sprayed, and the state of color development was observed. As a result, no color development was confirmed, suggesting that the purified product had no peptide bond. d. Ninhydrin Color Reaction The above a. A ninhydrin solution (mixture of ninhydrin 3 mg / 100 ml n-BuOH and CH ₃ COOH 3 ml) was sprayed on the TLC plate developed in the same manner as above, and this was heated on a hot plate to observe the state of color development. As a result, no color was observed, which suggested that the purified product did not have an amino group.

E. α-Naphthol color reaction: a. Α-naphthol solution (15% α-naphthol / EtOH 10.5ml, H ₂ SO ₄ 6.5m
1, a mixed solution of EtOH 40.5 ml and H ₂ O 4.0 ml) was sprayed, and this was heated on a hot plate to observe the state of color development. As a result, no color was observed, which suggested that the purified product did not contain sugar. f. Anisaldehyde Color Reaction The above a. Anisaldehyde solution (p-anisaldehyde 0.5ml / 50ml CH ₃ COOH and H ₂ SO ₄ was applied to the TLC plate developed in the same manner as above.
A mixed solution with 1 ml) was sprayed, and this was heated on a hot plate to observe the state of color development. As a result, no coloration was observed, which suggested that the purified product was free of sugar and steroid.

[0020] 4. Morphology of III-121C for various cells
Effect of change inducer K-ras ^ts -N, rat renal cells transformed with temperature-sensitive Kirsten sarcoma virus and Rous sarcoma virus
RK cells (NIH in the United States) and RSV ^ts -NRK cells (Institute of Medical Science, University of Tokyo) are 5% fetal calf serum (Gibco / BRL) at 33 ° C. showing the morphology of cancer cells and 39 ° C. showing the morphology of normal cells. Dulbecco's modified Eagle medium (Nissui Pharmaceutical Co., Ltd.) containing glutamine 0.6 g / l, kanamycin 0.2 g / l, and NaHCO ₃ 2.25 g / l was used as a culture medium to obtain 5% CO ₂ Cultured in an incubator. In addition, normal rat kidney cells, NRK-4
9 cells (Dainippon Pharmaceutical Co., Ltd.), mouse NIH3T3 cells (JCRB cell bank), Kirsten sarcoma virus and H
arvey sarcoma virus-transformed K-ras-NIH
3T3 cells, H-ras-NIH3T3 cells and Nr
As-NIH3T3 cells (Institute of Medical Science, University of Tokyo)
Similarly cultured at 37 ° C. III- in various cells
The effect of 121C is that each cell is seeded on a 24-well plastic plate (trade name: Coaster 3524, manufactured by Coaster Co., Ltd.) at 3.0 × 10 ⁴ cells / ml of medium / well, and 5% CO _{2 is} added.
After culturing in the incubator for 1 day, the III-121C sample was added, and the cell morphology was observed until the 3rd day thereafter.

(1) K-ras ^ts -NRK cells K-ras ^ts -NRK cells show several thin projections from the cells at 33 ° C., and the cells overlap each other to form a morphology of cancer cells, which is 39 ° C. Then, the cells are flat, show no protrusions, do not overlap, and show the morphology of normal cells. Then, when 0.3 μg / ml of III-121C was added at 33 ° C. and the cells were cultured for 2 days, normal cells having no protrusions and flat and not overlapping were formed. Also, at 39 ° C,
The morphology was similar to that without III-121C.

(2) K-ras-NRK cells K-ras-NRK cells, which are cancer cells, have a rounded and rounded shape, and III-121C at 0.3 μg / m.
When 1 was added and the cells were cultured for 2 days, the cell shape became flat and the adhered area increased, resulting in normal cell morphology.

(3) K, H, N-ras-NIH3T3
K-ras-NIH3T3 cells and Hr which are cancer cells
Each of the as-NIH3T3 cells has an elongated protrusion shape, and the cells are overlapped with each other. In addition, N-ras-NIH3T3 cells are cells with elongated elongated protrusions. III-121 in these cells
When C was added at 0.3 μg / ml and cultured for 2 days,
It was observed that the shape became flat and the adhesion area increased, and the cells were in the form of normal cells, NIH3T3.

(4) RSV ^ts -NRK cells RSV ^ts -NRK cells, like K-ras ^ts -NRK cells, show elongated rhomboid cancer cell morphology at 33 ° C and are flat and adherent at 39 ° C. It is a temperature-sensitive strain transformed with the src oncogene showing a wide normal cell morphology. When III-121C (0.3 μg / ml) was added thereto and cultured for 2 days, the morphology of the cells was rounded at both 33 ° C. and 39 ° C., showing a shape similar to that of normal NRK-49 cells.

5. III-121C access to various cells
Chin filament composition effect After washing with 100% ethanol, dry-sterilized 18φ cover glass (manufactured by Matsunami Glass Industry Co., Ltd.) in a 12-well plastic plate, 3.0 x 10 ⁴ cells / DME of each cell
M 1 ml / well was seeded and cultured for 1 day in a 5% CO ₂ incubator. The next day, III-121C 1 each
After adding 1 μg / ml and culturing for 1 day, the cells were fixed with paraformaldehyde. Rhodamine phalloidin reagent (Molecular Probes) was reacted with this, and the cell morphology was observed using a G filter (excitation 546 nm) with a fluorescence microscope. K-ras ^ts -NRK cells, K-ras-
NRK cells, K-ras-NIH3T3 cells, and H-
As a result of observation of ras-NIH3T3 cells, it was observed that linear actin filaments were running in the long axis direction of the cells, and the cells were in the form of normal cells.

6. Cells for various cells of III-121C
Proliferation inhibitory effect K-ras ^ts -NRK cells were spread on a 24-well plastic plate (trade name: Coaster 3524, manufactured by Coaster Co., Ltd.) at 1.0 × 10 ⁴ cells / DMEM 1 ml / well,
After culturing for 1 day in a 5% CO ₂ incubator, III-12
1C was added and cultured. After removing the medium, the cells were detached using trypsin, and DMEM was added to homogenize the cells. Coulter counter (ZM type)
When measured using, the results shown in Fig. 7 are obtained,
A cell growth inhibitory effect was observed in a concentration-dependent manner. Next, various cells were put into a 12-well plastic plate (Coaster 35
21), 2.0 × 10 ⁴ cells / DMEM 1 ml / well, and the cells were cultured in a 5% CO ₂ incubator for 1 day. The next day, III-121C was added, and after culturing for another 3 days,
The number of cells was measured in the same manner. The concentration at which cell growth was inhibited by 50% (IC ₅₀ ) was determined. The results are as shown in Table 1.

7. The LD ₅₀ value of III-121C for acute toxicity to mice was as follows:
It was confirmed that the toxicity was weaker than that of existing plant alkaloid anticancer agents. III-121C 24.3mg / kg Vincristine sulfate 2.5mg / kg Vindesine sulfate 9.1mg / kg Vinblastine sulfate 15.2mg / kg

8. Growth inhibition of III-121C in mice
Control effect 1 x 1 subcutaneously on the back of 6 nude mice per group
0 were implanted with ^six of K-ras-NRK cells. A small tumor was confirmed 4 days later, so this was set as day 0, and thereafter 0,
III-121C 0.01mg / mouse, 0.1mg / mouse on the 2nd and 4th day
e, 0.5 mg / mouse was intraperitoneally administered to nude mice in each group. The amount of tumor was observed as shown in FIG. 8, showing that the growth was suppressed in a dose-dependent manner.

9. III-121C Alkaloid anticancer drug vinblastine, etc.
It will be described below with reference to FIGS. 9 to 14 that the mechanism of action is different from that of C.

(1) When 1 μg / ml of III-121C was added to K-ras ^ts -NRK cells at 33 ° C., the morphology in which the cells with protrusions were overlapped (FIG. 9) was flat and overlapped after 1 day. The morphology was absent (FIG. 10) and became more prominent after 2 days (FIG. 11). In contrast, vinblastine, which is known as a microtubule formation inhibitor, vin
When 0.1 μg each of cristine and colchicine was added, the number of round cells or elongated cells apparently in the M phase of the cell cycle increased after 1 day (Fig. 1 respectively).
2, FIG. 13, FIG. 14). This indicates that III-121C has an anti-ras action but no cell division inhibitory action.

(2) Further, K-ras-NRK during proliferation
Add III-121C or vinblastine to cells 3
After culturing at 3 ° C., and after 24 hours, cells were fractionated by flow cytometry to examine the relationship between the amount of DNA and the number of cells.
This relationship is shown in FIG.
8 shows. DN when vinblastine is added (Fig. 18)
The number of cells with a large amount of A is increasing, and the block of G ₂ / M phase of the cell cycle is clear, but III-121C addition (0.3 μg
Fig. 16 for addition of 1 ml / ml and Fig. 17 for addition of 1 μg / ml D
The NA distribution was the same as that of the cells before addition (FIG. 15), and mitotic inhibition did not occur. From the above, it was found that the known alkaloid anticancer agent vinblastine and the like and III-121C have completely different mechanisms of action.

[0032]

The compound of the present invention extracted from the tropical plant Ervatamia microfila represented by the chemical formula (1) has the activity of inhibiting the growth of cancer cells and normalizing its morphology, and is effective as an anticancer agent. Can be used for

[Brief description of drawings]

FIG. 1 Positive spectrum of FAB mass spectrum

[FIG. 2] Measurement result of negative spectrum of FAB mass spectrum

[Fig. 3] Measurement result of UV absorption spectrum λ _max (E 1% / 1 cm)

FIG. 4 IR spectrum measurement results

FIG. 5: Results of ¹ H-NMR spectrum measurement

FIG. 6 Measurement results of ¹³ C-NMR spectrum

FIG. 7: Cell growth inhibitory effect on K-ras ^ts -NRK cells in medium

FIG. 8: Cell growth inhibitory effect on K-ras-NRK cells in nude mice

FIG. 9: Before addition of K-ras ^ts -NRK cells III-121C

FIG. 10: Day 1 after addition of K-ras ^ts -NRK cells III-121C

FIG. 11: 2 days after addition of K-ras ^ts -NRK cells III-121C

FIG. 12: Day 1 after addition of K-ras ^ts -NRK cells vinblastine

FIG. 13: Day 1 after addition of K-ras ^ts -NRK cell vincristine

FIG. 14: Day 1 after addition of K-ras ^ts -NRK cells colchicine

FIG. 15: Relationship between DNA amount and cell number (before addition of drug)

FIG. 16: Relationship between DNA amount and cell number (III-121C
0.3 μg / ml added)

FIG. 17 Relationship between DNA amount and cell number (III-121C added at 1 μg / ml)

FIG. 18: Relationship between DNA amount and cell number (vinblastine
0.1 μg / ml added)

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hiroaki Takahashi 1500 Inoguchi, Nakai-cho, Ashigaragami-gun, Kanagawa Terumo Corporation (72) Inventor Takashi Yamamoto 1500 Inoguchi, Nakai-cho, Ashigagami-gun, Kanagawa Terumo Corporation

Claims (1)

[Claims] 1. An oncogene function inhibitor comprising a compound represented by the chemical formula (1) as an active ingredient. [Chemical 1]