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WO1997008161A1 - Oncogene function depressant - Google Patents

Oncogene function depressant Download PDF

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Publication number
WO1997008161A1
WO1997008161A1 PCT/JP1996/002411 JP9602411W WO9708161A1 WO 1997008161 A1 WO1997008161 A1 WO 1997008161A1 JP 9602411 W JP9602411 W JP 9602411W WO 9708161 A1 WO9708161 A1 WO 9708161A1
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WO
WIPO (PCT)
Prior art keywords
cells
ras
aglaiastatin
cell
solvent
Prior art date
Application number
PCT/JP1996/002411
Other languages
French (fr)
Japanese (ja)
Inventor
Kazuo Umezawa
Takashi Koyano
Hiroaki Takahashi
Takashi Yamamoto
Original Assignee
Terumo Kabushiki Kaisha
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP7219288A external-priority patent/JPH0967375A/en
Priority claimed from JP7219289A external-priority patent/JPH0967360A/en
Priority claimed from JP7228355A external-priority patent/JPH0967376A/en
Application filed by Terumo Kabushiki Kaisha filed Critical Terumo Kabushiki Kaisha
Publication of WO1997008161A1 publication Critical patent/WO1997008161A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
    • C07D491/14—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/93—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems condensed with a ring other than six-membered

Definitions

  • the present invention relates to a novel compound having oncogene function inhibitory activity, and an oncogene function inhibitor and an anticancer agent containing the compound as an active ingredient.
  • oncogenes play an important role in the process of carcinogenesis and promotion of cells. Oncogenes are caused by abnormalities such as point mutations, translocations, and amplifications in proto-oncogenes on the chromosomal DNA of normal cells, and over 70 species have been found so far. . Among them, the ras oncogene is colorectal cancer,? It is found in L cancers, leukemias, etc., and is said to be present in about 20% of all human cancer tissues. Therefore, a substance that inhibits the function of the ras oncogene is expected to be a new therapeutic agent for cancer of concebut.
  • inhibitors include oxanosine isolated from actinomycetes (0.1 I to et al., Cancer Research, vol. 4 9.996-1000, 1989) and compactin isolated from mold. (N. Matsuda et al., Cell Pharma Pharmacology, vol. 1. 219-223. 1994) and the like.
  • the present inventors have conducted a search for a substance that inhibits ras oncogene function from plants by conducting screening using an effect of normalizing cancer cell morphology as an index to obtain a clinically applicable inhibitor.
  • the activity of inhibiting the function of the ras oncogene was found in the extract of the leaves of the tropical plant Aglaia odorata LOUR, and the active ingredients were identified to give the formulas (1) to (3).
  • the present invention has been completed.
  • the present invention provides a compound represented by any one of the chemical formulas (1) to (3) as an active ingredient, and an anticancer agent containing the compound as an active ingredient and an oncogene function inhibitor or, optionally, a pharmaceutically acceptable carrier.
  • an anticancer agent containing the compound as an active ingredient and an oncogene function inhibitor or, optionally, a pharmaceutically acceptable carrier.
  • Figure 1 shows the measurement results of the ultraviolet absorption spectrum of aglaiastatin A under neutral conditions (MeOH).
  • Fig. 2 shows the measurement results of aglaiastatinA under ultraviolet absorption spectrum acidic conditions (0. IN HCl-eOH).
  • FIG. 3 shows the measurement results of aglaiastatin A under the ultraviolet absorption spectrum basic condition (0.1N NaOH-MeOH).
  • FIG. 4 shows the infrared absorption spectrum measurement results of aglaiastatinA.
  • the numbers in the graph represent the peak numbers.
  • FIG. 5 shows the 1 H-NMR spectrum measurement results of aglaias tinA.
  • Figure 6 shows a 1 3 CN R scan Bae spectrum measurement result of AglaiastatinA.
  • Fig. 7 shows the measurement results of aglaiastatin B under the ultraviolet absorption spectrum neutral condition (MeOH).
  • Fig. 8 shows the measurement results of aglaias tinB under ultraviolet absorption spectrum acidic conditions (0.1N HCl-MeOH).
  • FIG. 9 shows the measurement results of aglaias tinB under the ultraviolet absorption spectrum basic condition (0. IN NaOH-MeOH).
  • FIG. 10 shows the results of infrared absorption spectrum measurement of aglaiastatinB.
  • the numbers in the graph represent peak numbers.
  • FIG. 11 shows the measurement results of 1 H-NMR spectrum of aglaiastatinB.
  • FIG. 12 shows the results of 13 C-NMR spectrum measurement of aglaiastatinB.
  • Figure 13 shows the results of measurement of aglaiastatin C under neutral conditions (MeOH) in the ultraviolet absorption spectrum.
  • Figure 14 shows the measurement results of aglaiastatin C under the ultraviolet absorption spectrum acidic condition (0. IN HCl-MeOH).
  • Figure 15 shows the measurement results of aglaiastatin C under basic conditions of ultraviolet absorption spectrum (0. IN NaOH-MeOH).
  • Figure 16 shows the measurement results of infrared absorption spectrum of aglaiastatinC.
  • the numbers in the graph represent peak numbers.
  • Figure 17 shows the results of 1 H-MR spectrum measurement of aglaiastatinC.
  • FIG. 18 shows the results of ' 3 C-NMR spectrum measurement of aglaiastatinC.
  • Agura Odra Isago is a small tree of the family Graceae, which grows naturally in Southeast Asia and is partially cultivated for ornamental use and fragrance.
  • the compounds according to the present invention, aglaias tinA, aglaiastatinB, and aglaiastatin C, were each isolated from Agraya and Odra.
  • & 81 & 1 & 3 118 is a colorless powder having a molecular weight of 4 3 4 (measured by EI-S spectrum).
  • the ultraviolet absorption spectrum of aglaias tinA is shown in Fig. 1 to Fig. 3, the infrared absorption spectrum. Is shown in Figure 4.
  • the measurement results of the 1 H-NMR spectrum and the ' 3 C-NMR spectrum are as shown in FIGS. 5 and 6.
  • the plant used as the raw material of aglaiastatin A may be dried for reasons such as preservation and transportation, and in that case, it is preferable to dry it in the sun or in an oven at 60 ° C or lower.
  • pulverized one is preferable for extraction.
  • the solvent used for the extraction any solvent can be used as long as it can stably extract aglaiasta tin A, but a non-polar solvent is preferable, and a hydrocarbon solvent or a halogenated hydrocarbon solvent is particularly preferable.
  • the hydrocarbon solvent include pentane, hexane, heptane, octane, benzene, toluene, xylene and the like, with toluene being particularly preferred.
  • the halogenated hydrocarbon solvent include, for example, chloroform, methylene dichloride, carbon tetrachloride, etc., and particularly preferred is glo-form. These solvents may be used alone or in combination of two or more.
  • the operation such as the amount of solvent, temperature, and time during extraction is not particularly limited as long as aglaiastatinA can be obtained stably without decomposition and in a high yield.
  • the method for isolating and purifying aglaias tinA is not particularly limited as long as the compound can be obtained stably from the extract.
  • An example of a suitable isolation and purification method is chromatography. Chromatography may preferably include column chromatography, gel filtration chromatography, high performance liquid chromatography, thin layer chromatography or a combination thereof. Column chromatography Silica gel, alumina, or the like can be used as a carrier for the laffy, and a hydrocarbon-based solvent, halogenated hydrocarbon, alcohol, or the like can be used as a developing solvent.
  • TOPEARL HW-40 manufactured by Tosoh Corporation
  • alcohols can be used as a solvent.
  • the types of the chromatographic carrier and the solvent are not limited to those described above.
  • aglaias tinA When given to cancer cells having the ras oncogene and the like, aglaias tinA has the effect of normalizing the cell morphology, suppressing its growth, and inhibiting the function of the oncogene.
  • normalizing the morphology of cancer cells means, for example, returning cancer cells that have protruded or proliferate due to overlapping cells to normal cell morphology without any prominence.
  • Inhibiting the growth of cancer cells refers to inhibiting growth by 50% at very low concentrations (eg, on the order of ngZml).
  • the effect of inhibiting oncogene function is morphologically It refers to the effect of turning cancer cells into normal cells.
  • Aglaias TINA inhibits 50% growth of the cancer cell concentration (IC 5 e) is a different but 2. 4 ⁇ 7.5ng / ml depending on the type of cancer cells.
  • Pharmaceutically acceptable carriers used in the anticancer agent of the present invention include, for example, excipients such as natural or synthetic aluminum gateate, microcrystalline cellulose, talc, dextrin, starch, lactose, and vegetable oils, propylene glycol and the like. Diluent I can do it.
  • pharmaceutically acceptable stabilizers, coatings, suspending agents, emulsifiers, solubilizers, preservatives, buffers, sweeteners and the like may be present as other optional ingredients.
  • Known additives may be used in appropriate combination as these additives.
  • the composition may be used in any form such as powders, granules, tablets, capsules, and injections.
  • the anticancer agent containing aglaias tin A of the present invention as an active ingredient is mainly orally administered, and the present invention is not limited by the administration method.
  • the daily dose for adults differs depending on the administration method and medical condition.
  • oral administration which is the main method of administration, the usual dosage is 100 to 500 mg per adult per day.
  • the present invention is not limited by dosage.
  • the second compound of the present invention is a colorless powder having a molecular weight of 524 (by FAB-MS spectrum measurement).
  • the ultraviolet absorption spectrum is shown in FIGS. 7 to 9, and the infrared absorption spectrum is shown in FIG.
  • the measurement results of the 1 H-NMR spectrum and the 13 C-NMR spectrum are as shown in FIGS. 11 and 12.
  • the plant used as the raw material of aglaiastatin B may be dried for reasons such as preservation and transportation, and in that case, it is preferable to dry it in the sun or in an oven at 60 ° C or lower.
  • pulverized one is preferable for extraction.
  • the solvent used for extraction is aglaiasta Any solvent can be used as long as it can stably extract tin B, but a non-polar solvent is preferable, and a hydrocarbon solvent or a halogenated hydrocarbon solvent is particularly preferable.
  • the hydrocarbon solvent include pentane, hexane, heptane, octane, benzene, toluene, xylene and the like, with toluene being particularly preferred.
  • As the halogenated hydrocarbon solvent for example, chloroform, methylene dichloride, carbon tetrachloride and the like can be mentioned, and chloroform is particularly preferable. These solvents may be used alone or in combination of two or more
  • the operation such as the amount of the solvent, the temperature, and the time during the extraction is not particularly limited as long as aglaiastatinB can be obtained stably without decomposition and in a high yield.
  • the method for isolating and purifying aglaias tinB is not particularly limited as long as the compound can be stably obtained from the extract.
  • An example of a suitable isolation and purification method is chromatography. Chromatography may preferably include column chromatography, gel filtration chromatography, high performance liquid chromatography, thin layer chromatography or a combination thereof. Silica gel, alumina or the like can be used as a carrier for the column chromatography, and a hydrocarbon solvent, halogenated hydrocarbon, alcohol, or the like can be used as a developing solvent.
  • As gel filtration chromatography Toyopearl HW-40 (trade name, manufactured by Tosoh Corporation) can be used as a carrier, and alcohols can be used as a solvent.
  • aglaiastatin B is given to a cancer cell having a ras oncogene or the like, it has the effect of normalizing the cell morphology, suppressing its growth, and inhibiting the function of the oncogene.
  • Aglaiastatin B inhibits 50% growth of the cancer cell concentration (IC 5.) is a different but 3. 6-8. 9 ng / ml depending on the type of cancer cells.
  • an oncogene function inhibitor or an anticancer agent can be provided using aglaiastatin B, a novel compound according to the present invention, as an active ingredient.
  • Pharmaceutically acceptable carriers used in the anticancer agent of the present invention include, for example, excipients such as natural or synthetic aluminum gateate, microcrystalline cellulose, talc, dextrin, starch, lactose, and vegetable oils, propylene glycol And diluents.
  • pharmaceutically acceptable stabilizers, coating agents, suspending agents, emulsifiers, solubilizers, preservatives, buffers, sweeteners and the like may be present as other optional ingredients.
  • Known additives may be used in appropriate combination as these additives.
  • the composition may be used in any form such as powders, granules, tablets, capsules, and injections.
  • the anticancer agent containing aglaiastatin B as an active ingredient of the present invention is mainly orally administered, but the present invention is not limited by the administration method.
  • the daily dose for adults differs depending on the administration method and medical condition. In the case of oral administration, which is the main method of administration, the daily dose for adults is 100 to 500 mg, the usual dosage.
  • the present invention is not limited by dosage.
  • the third compound of the present invention is a colorless powder having a molecular weight of 526 (by FAB-MS spectrum measurement).
  • molecular formula is C 3 1 H 30 N 2 ⁇ 6.
  • the ultraviolet absorption spectrum is as shown in FIGS. 13 to 15, and the infrared absorption spectrum is as shown in FIG.
  • the measurement results of the 1 H-NMR spectrum and the 13 C-NMR spectrum are as shown in FIGS. 17 and 18.
  • the plant used as the raw material for aglaiastatin C may be dried for reasons such as preservation and transportation, and in that case, it is preferable to dry it in the sun or in an oven at 60 ° C or lower.
  • pulverized one is preferable for extraction.
  • any solvent can be used as long as it can stably extract aglaias tin C, but a non-polar solvent is preferable, and a hydrocarbon solvent or a halogenated hydrocarbon solvent is particularly preferable.
  • hydrocarbon solvent examples include pentane, hexane, heptane, octane, benzene, toluene, xylene and the like, with toluene being particularly preferred.
  • halogenated hydrocarbon solvent for example, chloroform, methylene dichloride, carbon tetrachloride and the like can be mentioned, and chloroform is particularly preferable. One or more of these may be used in combination.
  • the operation such as the amount of solvent, temperature, and time during extraction is not particularly limited as long as aglaiastatin C can be obtained stably without decomposition and in a high yield.
  • the isolation and purification method of aglaiastatin C is a method whereby this compound can be obtained stably from the extract. If there is, it is not particularly limited.
  • An example of a suitable isolation and purification method is chromatography. Chromatography may preferably include column chromatography, gel filtration chromatography, high performance liquid chromatography, thin layer chromatography or a combination thereof. Silica gel, alumina or the like can be used as a carrier for the column chromatography, and a hydrocarbon solvent, halogenated hydrocarbon, alcohol, or the like can be used as a developing solvent. In gel filtration chromatography, Toyopearl is used as a carrier.
  • aglaiastatin C When aglaiastatin C is given to a cancer cell having a ras oncogene or the like, it has the effect of normalizing the cell morphology, suppressing its growth and inhibiting the function of the oncogene.
  • concentration of a glaiastatin C that inhibits the growth of cancer cells by 50% varies depending on the type of cancer cell, and is 1.0 to 5.6 ng Zml.
  • an oncogene function inhibitor or an anticancer agent can be provided using aglaiastatin C, a novel compound according to the present invention, as an active ingredient.
  • Pharmaceutically acceptable carriers used in the antimicrobial agents of the present invention include, for example, excipients such as natural or synthetic aluminum gateate, microcrystalline cellulose, talc, dextrin, starch, lactose, and vegetable oils, propylene Diluents such as glycols.
  • excipients such as natural or synthetic aluminum gateate, microcrystalline cellulose, talc, dextrin, starch, lactose, and vegetable oils
  • propylene Diluents such as glycols.
  • other optional ingredients include pharmaceutically acceptable stabilizers, Coatings, suspending agents, emulsifiers, solubilizers, preservatives, buffers, sweeteners and the like can also be present.
  • Known additives may be used in appropriate combination as these additives.
  • the composition may be used in any form such as powders, granules, tablets, capsules, and injections.
  • the anticancer agent of the present invention containing aglaiastatin C as an active ingredient is mainly administered orally.
  • the present invention is not limited by the administration method.
  • the daily dose for adults differs depending on the administration method and medical condition.
  • oral administration which is the main method of administration, the usual dosage is 100 to 50 Omg per adult per day.
  • the present invention is not limited by dosage.
  • aglaiastatins A, B and C all have very similar functions on cancer cells. This is thought to be due to the structure common to aglaiastatin A, B, and C.
  • aglaiastatin A, B, and C can be used as an oncogene function inhibitor or an anticancer agent effective for suppressing the function of the ras oncogene.
  • the active fraction was added to Toyopearl HW-40 previously equilibrated with methanol and subjected to gel filtration chromatography to fractionate about 2 ml each. Further, the active fraction was collected and subjected to HPLC fractionation (column: PEGASIL 0DS, manufactured by Senshu Ichigaku Co., Ltd., elution solvent: 70% methanol, flow rate: 4 ml / min, detection: UV210 nm), and about 10 ml And fractionated. When an activity test was performed, the activity was confirmed in the fraction eluted at a retention time of 58 minutes. The fractions were collected to obtain a colorless powder (aglaias tinA 4.6 mg). This fraction was a single spot when subjected to silica gel thin layer chromatography.
  • K - In ras ts -NRK cells (US NIH) is 3 9 ° C showing the form status of 33 ° C and normal cells showing the morphology of the cancer cells, base Dar containing 5% calf serum (Gibco Laboratories, Inc.) Tsu co modified Eagle's medium glutamine (Nissui Pharmaceutical Co., Ltd.) 0. 6 g / l, kanamycin 0.2g / l, NaHC0 3 2.25g / l, culture medium plus (hereinafter, referred to as DMEM as also), it was cultured in 5% C0 2 incubator scratch.
  • DMEM culture medium plus
  • cells are calcium-magnesium-free phosphate buffer (PBS (-):.. NaCl 8. Og / 1 Na 2 P0 4 ⁇ 12 ⁇ 2 0 2. 31g / l H 2 P0 4 0.2g / l After washing with pH 7.4)
  • PBS (-) calcium-magnesium-free phosphate buffer
  • NaCl 8. Og / 1 Na 2 P0 4 ⁇ 12 ⁇ 2 0 2. 31g / l H 2 P0 4 0.2g / l
  • O trypsin - EDTA solution trypsin 0.5g / l, NaCl 8.0g / L glucose 1.0g / l 'Phenol red 0.02g / l, KH 2 P0 4 0.35g / l, EDTA 0.2g / l
  • Ri by culture flasks Cells were detached, passaged by transferring them to fresh and culture flasks.
  • Each cell was seeded on a 48-well plastic plate (manufactured by Coaster) at 1.5 ⁇ 10 4 Z DME at 0.5 ml / well, and cultured in a 5% CO 2 incubator for 1 day. The test substance was added the next day, and cell morphological changes were observed up to the third day.
  • K-ras ts -NRK cells show a number of small protrusions from the cells, and the cells overlap and show the morphology of cancer cells.At 39 ° C, the cells are flat with protrusions. It shows normal cell morphology without overlapping.
  • K-ras-NRK cells which are cancer cells, have a rounded shape, but after adding 10 ng / ml of aglaiastatinA and culturing at 37 ° C for 3 days, the cell morphology becomes flat and the adhesive surface It also showed normal cell morphology.
  • K-ras-NIH3T3 cells which are cancer cells, have an elongated protruding morphology, and the cells overlap. Add 10 ng / ml of aglaiastatin A to this at 37 ° C After culturing for 3 days, it was observed that the cells became flat and the adherent surface increased, and that they were in the form of normal NIH3T3 cells.
  • Each cell was seeded on a 48-well plastic plate (manufactured by Course Yuichi) at 1.0 ⁇ 10 4 cells / ⁇ EM 0.5 ml / we 11 and cultured in a 5% CO 2 incubator at an appropriate temperature for 1 day. The next day further cultured for 3 days plus aglaiastatin A, PBS (-) was washed twice with added trypsin by 100 ⁇ l / well, the cells are detached in minutes 0% C0 2 under the conditions of the thermostat, DMEM Was added at 900 1 / well to make it uniform. The cell concentration of this solution was measured using a Coulter counter (ZM type) to determine the concentration that inhibits cell growth by 50% (IC 5 ). The results were as shown in Table 1.
  • aglaiastatinA the IC 5. Since ng / m 1 is on the order of ng / m 1 and ordinary cytotoxic substances are on the order of / ig / m 1, cell proliferation was suppressed at an extremely low concentration. Moreover, its concentration is higher for tumor cells than for normal cells. Among the cell names shown in Table 1, those containing ras are tumor cells.
  • Acute toxicity of Aglaias TINA compared ICR male mice (5 weeks old), LD 50 is at 300 mg / kg or more, acute toxicity low les, it was confirmed.
  • HP LC fractionation of this active fraction (column: PEGASIL 0DS, manufactured by Senshu Ichigaku Co., Ltd., elution solvent: 65% methanol, flow rate: 5 ml / min. Detection: UV210 nm) was performed, and fractions of about lml were performed. did.
  • an activity test was performed, the activity was confirmed in the fraction eluted at a retention time of 56 minutes. This fraction was collected to obtain a colorless powder (aglaiastatin B 5.2 mg). This fraction was a single spot when subjected to silica gel thin layer chromatography.
  • K-ras ts -NRK cells is the 39 hand showing the form of 33 ° C and normal cells showing the morphology of the cancer cells, 5% calf serum Dulbecco's modified Eagle medium containing (Gibco BRL) ( Nissui Pharmaceutical Co., Ltd.) to glutamine 0. 6 g / l, kanamycin 0.2g / K NaHC0 3 2.25g / l , as the culture liquid plus, were cultured in 5% C0 2 incubator evening one.
  • the cells were washed with a calcium-magnesium-free phosphate buffer (PBS (-): 8. Og / 1 NaCl, 2.31 g / l Na2P04-12H20, 0.2 g / l KH2P04; pH 7.4) After that, culture with trypsin-EDTA solution (0.5 g / l trypsin, 8. Og / 1 NaCl, 1. Og / 1 glucose, 0.02 g / l Phenol red, 0.35 g / l KH2P04, 0.2 g / l EDTA) The cells were detached from the flask, transferred to a flask containing a fresh culture medium, and transferred.
  • PBS (-) 8. Og / 1 NaCl, 2.31 g / l Na2P04-12H20, 0.2 g / l KH2P04; pH 7.4
  • trypsin-EDTA solution 0.5 g / l trypsin
  • Each cell, 4 to 8-well plastic plates (Costar) were seeded at 1. 5 X 10 4 cells Z DMEM 0.5 ml / well, and cultured for one day in 5% C0 2 incubator scratch. The test substance was added the next day, and cell morphological changes were observed up to the third day.
  • K-ras' S- NRK cells show several small protrusions from the cells at 33, and the cells overlap to show the morphology of cancer cells. Shows a normal cell morphology without any overlap and no projection.
  • K-ras-NRK cells which are cancer cells, have a rounded shape, but after adding aglai astatin B 20 ng / ml and culturing at 37 ° C for 3 days, the cell morphology becomes flat. However, the adhesion surface was increased to show normal cell morphology.
  • K-ras-NIH3T3 cells which are cancer cells, have an elongated protruding morphology, and the cells overlap each other.
  • 20 ng / ml of aglaiastatin B was added thereto and cultured at 37 ° C for 3 days, it was observed that the cells became flat, the adhesion surface was increased, and the cells were in the form of normal NIH3T3 cells.
  • Each cell was seeded on a 48-well plastic plate (manufactured by Coaster) at 1.0 ⁇ 10 4 cells: DMEM 0.5 ml / we 11 and cultured in a 5% CO 2 incubator at an appropriate temperature for 1 day. Next day was added and incubated an additional 3 days aglaiastatinB, PBS (-) was washed twice with added trypsin by 100 1 / well, the cells are detached in minutes 0% C0 2 under the conditions of the thermostat, the DMEM The cells were added at 900 ⁇ l / well so that the cells were uniformly dispersed.
  • LD 50 is at 300mg / kg or more, and acute toxicity is low record, it has been confirmed this and force.
  • the active fraction was subjected to HPLC fractionation (column: PEGASIL 0DS, manufactured by Senshi Kagaku Co., Ltd., elution solvent: 65% methanol, flow rate: 5 ml / min, detection: UV210 hidden), and about 1 ml each. Fractionated. When an activity test was performed, the activity was confirmed in the fraction eluted at a retention time of 42 minutes. This fraction was collected to obtain a colorless powder (aglaiastatin C 2. lmg). This fraction was a single spot when subjected to silica gel thin layer chromatography.
  • K-ras ts -NRK cells (US NIH) is 39 ° C showing a 33 ° configuration of the C and normal cells showing the morphology of the cancer cells, varying Dal Beck co containing 5% calf serum (Gibco Laboratories, Inc.) law Eagle medium (Nissui Pharmaceutical Co., Ltd.) to glutamine 0.6 g / l, as the culture liquid plus kanamycin 0.2g / l, NaHC0 3 2.25g / K, in 5% C0 2 in Kyubeta one Cultured.
  • rat kidney cells Normal rat kidney cells, NRK-49 cells (Dainippon Pharmaceutical Co., Ltd.), mouse NI H3T3 cells (JCRS cell bank), and cancer cells, K-ras-NRK cells (Showa Pharmaceutical University), K_ras -NIH3T3 cells, H-ras-NIH3T3 cells, and N-ras-N ⁇ H3T3 cells (Institute of Medical Science, The University of Tokyo) were similarly cultured at 37 ° C. Also, cells are calcium-magnesium-free phosphate buffer (PBS (-): NaCl 8.
  • PBS (-) calcium-magnesium-free phosphate buffer
  • trypsin-EDTA solution trypsin 0.5g / l, NaCl 8.Og / 1, glucose 1.0g / l, phenol red 0.02g / L KH 2 P0 4 0.35g / L EDTA 0.2g /
  • the cells were detached from the culture flask in l) and subcultured by transferring to a flask containing a new culture solution.
  • Each cell was spread on a 48-well plastic plate (manufactured by Coaster) at 1.0 ⁇ 10 4 cells / DMEM 0.5 ml / we 11 and cultured in a 5% CO 2 incubator for 1 day. The test substance was added the next day, and cell morphological changes were observed up to the third day.
  • K-ras ts- NRK cells show several small protrusions The vesicles overlap and show the morphology of cancer cells. At 39 ° C, the cells are flat with no protrusions and no overlap, showing the morphology of normal cells.
  • K-ras-NRK cells which are cancer cells, have a rounded shape, but after adding aglai astatin C 20 ng / ml and culturing at 37 ° C for 3 days, the cell morphology becomes flat. However, the adhesion surface was increased to show normal cell morphology.
  • K-ras-NIH3T3 cells which are cancer cells, have an elongated protruding morphology, and the cells are overlapped.
  • 20 ng / ml of aglaias tin C was added thereto and cultured at 37 ° C for 3 days, it was observed that the cells became flat, the adhesion surface was increased, and the cells were in the form of normal NIH3T3 cells.
  • Each cell was spread on a 48-well plastic plate (manufactured by Coaster) with 1.0 ⁇ 10 4 DMEM at 0.5 ml / well, and cultured in a 5% CO 2 incubator at an appropriate temperature for 1 day. And further cultured for 3 days plus next day aglaiastatinC, PBS (-) was washed twice with added trypsin by 100 1 / wd l, peel the cells in a few minutes 0% C0 2 under the conditions of the thermostat, DMEM Was added at 900 1 / well so that the cells were uniformly dispersed. The cell concentration of this solution was measured using a Kohl-Izu counter (ZM type), and the concentration (IC 50 ) that inhibited cell growth by 50% was determined. The results are shown in Table 3. iastatinC IC s . Is from 1.0 to 5.6 n gZm 1. Among the cell names shown in Table 3, those containing ras are tumor cells.
  • the compound of the present invention isolated from the tropical plant agrobacterium represented by the chemical formulas (1), (2) and (3) has an activity of inhibiting the growth of cancer cells and normalizing the form thereof. It can be effectively used as an oncogene function inhibitor or an anticancer agent.

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Abstract

Novel compounds having the activity of depressing oncogene functions, i.e., aglaiastatins A (represented by chemical formula (1)), B and C, isolated from Aglaia odorata LOUR; and oncogene function depressants and anticancer drugs containing the same as the active ingredient.

Description

明細書 発明の名称  Description Title of Invention
癌遺伝子機能抑制剤 技術分野  Oncogene function inhibitor Technical field
本発明は、 癌遺伝子機能抑制活性を有する新規化合物、 および該化合物を有効 成分として含有する癌遺伝子機能抑制剤および抗癌剤に関する。 背景技術  The present invention relates to a novel compound having oncogene function inhibitory activity, and an oncogene function inhibitor and an anticancer agent containing the compound as an active ingredient. Background art
細胞の発癌やプロモーションの過程で、 癌遺伝子が重要な役割を果たしている ことが知られている。 癌遺伝子は、 正常細胞の染色体 D NA上にあるプロト癌遺 伝子に点突然変異、 転座、 増幅等の異常が起こることによって発生し、 これまで に 7 0種あまりが見出されている。 その中でも r a s癌遺伝子は大腸癌、 ？ L癌、 白血病等から見出され、 ヒト癌組織全体の 2 0 %位に存在するといわれる代表的 なものである。 そこで、 この r a s癌遺伝子の機能を阻害する物質が、 新しいコ ンセブトの癌治療薬として期待されている。 現在までに、 そのような阻害物質と しては放線菌から単離されたォキザノシン （0. I to ら、 Cancer Research, vol. 4 9. 996-1000, 1989 ) 、 かびから単離されたコンパクチン (N. Matsuda ら、 Cel lul ar Pharmacology, vol. 1. 219-223. 1994) 等が知られている。  It is known that oncogenes play an important role in the process of carcinogenesis and promotion of cells. Oncogenes are caused by abnormalities such as point mutations, translocations, and amplifications in proto-oncogenes on the chromosomal DNA of normal cells, and over 70 species have been found so far. . Among them, the ras oncogene is colorectal cancer,? It is found in L cancers, leukemias, etc., and is said to be present in about 20% of all human cancer tissues. Therefore, a substance that inhibits the function of the ras oncogene is expected to be a new therapeutic agent for cancer of concebut. To date, such inhibitors include oxanosine isolated from actinomycetes (0.1 I to et al., Cancer Research, vol. 4 9.996-1000, 1989) and compactin isolated from mold. (N. Matsuda et al., Cell Pharma Pharmacology, vol. 1. 219-223. 1994) and the like.
しかし、 これまでに発見された r a s癌遺伝子機能阻害物質は、 いずれも安定 性や毒性に問題があり、 臨床応用可能なものはまだない。 そのため、 現在までに 知られている阻害物質には見られない作用機構を有し且つ、 臨床応用が可能な薬 剤の開発が望まれている。 発明の開示 However, all of the ras oncogene function inhibitors discovered so far have problems with stability and toxicity, and there are no clinically applicable ones yet. Therefore, to date There is a demand for the development of a drug that has an action mechanism not found in known inhibitors and that can be applied clinically. Disclosure of the invention
本発明者らは、 臨床応用可能な阻害物質を得るために、 癌細胞の形態を正常化 させる作用を指標とするスクリーニングにより、 植物由来で r a s癌遺伝子機能 を阻害する物質の探索を行った結果、 熱帯植物アグラィァ ·ォドラー夕 （Aglaia odorata LOUR ) の葉の抽出物中に r a s癌遺伝子の機能を阻害する活性を見出 し、 活性成分を特定することにより化学式 ( 1 ) 〜 （3 ) で表される化合物を得 、 本発明を完成させた。  The present inventors have conducted a search for a substance that inhibits ras oncogene function from plants by conducting screening using an effect of normalizing cancer cell morphology as an index to obtain a clinically applicable inhibitor. The activity of inhibiting the function of the ras oncogene was found in the extract of the leaves of the tropical plant Aglaia odorata LOUR, and the active ingredients were identified to give the formulas (1) to (3). Thus, the present invention has been completed.
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すなわち、 本発明は有効成分として化学式 （1) 〜 （3) で表される化合物、 及び該化合物を有効成分とし、 癌遺伝子機能抑制剤、 または任意に医薬上許容可 能な担体を含有する抗癌剤が提供される £ 図面の簡単な説明 That is, the present invention provides a compound represented by any one of the chemical formulas (1) to (3) as an active ingredient, and an anticancer agent containing the compound as an active ingredient and an oncogene function inhibitor or, optionally, a pharmaceutically acceptable carrier. Will be offered £ BRIEF DESCRIPTION OF THE FIGURES
図 1はアグラィアスタチン （aglaiastatin) Aの紫外線吸収スペクトル中性条 件（MeOH) での測定結果を示す。  Figure 1 shows the measurement results of the ultraviolet absorption spectrum of aglaiastatin A under neutral conditions (MeOH).
図 2は aglaiastatinAの紫外線吸収スぺクトル酸性条件 (0. IN HCl- eOH ) で の測定結果を示す。  Fig. 2 shows the measurement results of aglaiastatinA under ultraviolet absorption spectrum acidic conditions (0. IN HCl-eOH).
図 3は aglaiastatinAの紫外線吸収スぺクトル塩基性条件（0. 1N NaOH-MeOH) での測定結果を示す。  FIG. 3 shows the measurement results of aglaiastatin A under the ultraviolet absorption spectrum basic condition (0.1N NaOH-MeOH).
図 4は aglaiastatinAの赤外線吸収スぺクトル測定結果を示す。 グラフ中の数 字はピ一ク番号を表すものである。  FIG. 4 shows the infrared absorption spectrum measurement results of aglaiastatinA. The numbers in the graph represent the peak numbers.
図 5は aglaias tinAの1 H-NMRスぺクトル測定結果を示す。 FIG. 5 shows the 1 H-NMR spectrum measurement results of aglaias tinA.
図 6は aglaiastatinAの1 3 C-N Rスぺクトル測定結果を示す。 Figure 6 shows a 1 3 CN R scan Bae spectrum measurement result of AglaiastatinA.
図 7は aglaiastatinBの紫外線吸収スぺクトル中性条件 (MeOH) での測定結果 を示す。  Fig. 7 shows the measurement results of aglaiastatin B under the ultraviolet absorption spectrum neutral condition (MeOH).
図 8は aglaias tinBの紫外線吸収スぺクトル酸性条件 (0. 1N HCl-MeOH) での 測定結果を示す。  Fig. 8 shows the measurement results of aglaias tinB under ultraviolet absorption spectrum acidic conditions (0.1N HCl-MeOH).
図 9は aglaias tinBの紫外線吸収スぺクトル塩基性条件 (0. IN NaOH-MeOH)で の測定結果を示す。  FIG. 9 shows the measurement results of aglaias tinB under the ultraviolet absorption spectrum basic condition (0. IN NaOH-MeOH).
図 1 0は aglaiastatinBの赤外線吸収スペクトル測定結果を示す。 グラフ中の 数字はピーク番号を表すものである。  FIG. 10 shows the results of infrared absorption spectrum measurement of aglaiastatinB. The numbers in the graph represent peak numbers.
図 1 1は aglaiastatinBの1 H-NMRスペクトル測定結果を示す。 FIG. 11 shows the measurement results of 1 H-NMR spectrum of aglaiastatinB.
図 1 2は aglaiastatinBの1 3 C-NMRスぺクトル測定結果を示す。 図 1 3は aglaiastatinCの紫外線吸収スぺク トル中性条件 （MeOH) での測定結 果を示す。 FIG. 12 shows the results of 13 C-NMR spectrum measurement of aglaiastatinB. Figure 13 shows the results of measurement of aglaiastatin C under neutral conditions (MeOH) in the ultraviolet absorption spectrum.
図 1 4は aglaiastatinCの紫外線吸収スぺク トル酸性条件 (0. IN HCl-MeOH) で の測定結果を示す。  Figure 14 shows the measurement results of aglaiastatin C under the ultraviolet absorption spectrum acidic condition (0. IN HCl-MeOH).
図 1 5は aglaiastatinCの紫外線吸収スぺク トル塩基性条件 (0. IN NaOH-MeOH) での測定結果を示す。  Figure 15 shows the measurement results of aglaiastatin C under basic conditions of ultraviolet absorption spectrum (0. IN NaOH-MeOH).
図 1 6は aglaiastatinCの赤外線吸収スペク トル測定結果を示す。 グラフ中の 数字はピーク番号を表すものである。  Figure 16 shows the measurement results of infrared absorption spectrum of aglaiastatinC. The numbers in the graph represent peak numbers.
図 1 7は aglaiastatinCの1 H- MRスペク トル測定結果を示す。 Figure 17 shows the results of 1 H-MR spectrum measurement of aglaiastatinC.
図 1 8は aglaiastatinCの' 3 C - NMRスぺク トル測定結果を示す。 発明を実施するための最良の形態 FIG. 18 shows the results of ' 3 C-NMR spectrum measurement of aglaiastatinC. BEST MODE FOR CARRYING OUT THE INVENTION
アグラィァ .ォドラ一夕はセンダン科の小木であり、 東南アジアに自生し、一 部観賞用、 香料等として栽培されている。 このアグラィァ，ォドラ一夕から、 本 発明に係る化合物である aglaias tinA、 aglaiastatinB、 および aglaiastatin Cがそれぞれ単離された。  Agura Odra Isago is a small tree of the family Graceae, which grows naturally in Southeast Asia and is partially cultivated for ornamental use and fragrance. The compounds according to the present invention, aglaias tinA, aglaiastatinB, and aglaiastatin C, were each isolated from Agraya and Odra.
以下本発明について詳細に説明する。  Hereinafter, the present invention will be described in detail.
&81&1&3 11八は分子量4 3 4 (EI- S スぺクトル測定による） の無色の粉末 である。 シリカゲル薄層クロマトグラフィーにおける R f値は 0. 58 (展開溶媒 クロ口ホルム： メタノール =40： 1 )、 0.58 (展開溶媒 トルエン：ァセトン= 2 : 1 ) である。  & 81 & 1 & 3 118 is a colorless powder having a molecular weight of 4 3 4 (measured by EI-S spectrum). The Rf values in silica gel thin-layer chromatography are 0.58 (developing solvent: methyl form: methanol = 40: 1) and 0.58 (developing solvent: toluene: acetone = 2: 1).
aglaias tinAの紫外線吸収スぺクトルは図 1から図 3、 赤外線吸収スぺクト ルは図 4の通りである。 また1 H— NMRスペクトル及び' 3 C— NMRスぺクト ルの測定結果は、 図 5及び図 6の通りである。 The ultraviolet absorption spectrum of aglaias tinA is shown in Fig. 1 to Fig. 3, the infrared absorption spectrum. Is shown in Figure 4. The measurement results of the 1 H-NMR spectrum and the ' 3 C-NMR spectrum are as shown in FIGS. 5 and 6.
検討の結果、 アグラィ了 ·ォドラー夕から単離された aglaiastatinAは前記化 学式 （ 1 ) で表される構造であることが支持された。  As a result of the study, it was supported that aglaiastatinA isolated from Agrai-Oddler-Yu had the structure represented by the chemical formula (1).
aglaiastatin Aの原料となる植物は、 保存、 運搬等の理由から乾燥してもよ く、 その場合は天日で、 あるいは 60°C以下のオーブンで乾燥するのが好ましい。 また、 抽出に際しては粉砕したものが好ましい。 抽出に用いる溶媒は aglaiasta tin Aを安定に抽出し得る溶媒であればいかなるものでも使用することができる が、 非極性溶媒が好ましく、 特に炭化水素系溶媒またはハロゲン化炭化水素溶媒 が好ましい。 炭化水素系溶媒としてはペンタン、 へキサン、 ヘプタン、 ォク タン、 ベンゼン、 トルエン、 キシレン等が挙げられ、 特にトルエンが好ましい。 またハロゲン化炭化水素溶媒としては、 例えばクロ口ホルム、 二塩化メチレン、 四塩化炭素等が挙げられ、 特にグロ口ホルムが好ましい。 これらの溶媒は 1種ま たは 2種以上を組み合わせて用いてもよい。  The plant used as the raw material of aglaiastatin A may be dried for reasons such as preservation and transportation, and in that case, it is preferable to dry it in the sun or in an oven at 60 ° C or lower. In addition, pulverized one is preferable for extraction. As the solvent used for the extraction, any solvent can be used as long as it can stably extract aglaiasta tin A, but a non-polar solvent is preferable, and a hydrocarbon solvent or a halogenated hydrocarbon solvent is particularly preferable. Examples of the hydrocarbon solvent include pentane, hexane, heptane, octane, benzene, toluene, xylene and the like, with toluene being particularly preferred. Examples of the halogenated hydrocarbon solvent include, for example, chloroform, methylene dichloride, carbon tetrachloride, etc., and particularly preferred is glo-form. These solvents may be used alone or in combination of two or more.
抽出時の溶媒量、 温度、 時間などの操作は、 aglaiastatinAが分解を起こすこ となく安定に、 且つ高収量で得られる範囲であれば特に限定されるものでは ない。  The operation such as the amount of solvent, temperature, and time during extraction is not particularly limited as long as aglaiastatinA can be obtained stably without decomposition and in a high yield.
aglaias tinAの単離精製法は、 本化合物が抽出物から安定に得られる方法で あれば特に限定されるものではない。 好適な単離精製法の例としてクロマトグラ フィ一を挙げることができる。 クロマトグラフィーは好ましくはカラムクロマト グラフィ一、 ゲル濾過クロマトグラフィ一、高速液体クロマトグラフィ一、薄層 クロマトグラフィーまたはこれらの組み合わせを包含し得る。 カラムクロマトグ ラフィ一に用いる担体としてはシリカゲル、 アルミナなどが使用でき、 展開溶媒 としては炭化水素系溶媒、 ハロゲン化炭化水素、 アルコール類などを用いること ができる。 またゲル濾過クロマトグラフィ一としては担体としてトョパール HW - 40 (東ソ一社製） 、 溶媒としてアルコール類を用いることができる。 しかしクロ マトグラフィー担体及び溶媒の種類は上記のものに限定されない。 単離精製され た aglaias tinAの構造を解析した結果、 化学式 （1 ) で表されることが判明し た。 The method for isolating and purifying aglaias tinA is not particularly limited as long as the compound can be obtained stably from the extract. An example of a suitable isolation and purification method is chromatography. Chromatography may preferably include column chromatography, gel filtration chromatography, high performance liquid chromatography, thin layer chromatography or a combination thereof. Column chromatography Silica gel, alumina, or the like can be used as a carrier for the laffy, and a hydrocarbon-based solvent, halogenated hydrocarbon, alcohol, or the like can be used as a developing solvent. For gel filtration chromatography, TOPEARL HW-40 (manufactured by Tosoh Corporation) can be used as a carrier and alcohols can be used as a solvent. However, the types of the chromatographic carrier and the solvent are not limited to those described above. As a result of analyzing the structure of the isolated and purified aglaias tinA, it was found that it was represented by the chemical formula (1).
aglaias tinAは、 r a s癌遺伝子等を有する癌細胞に与えると細胞の形態を 正常化し、 且つその増殖を抑制し、 癌遺伝子の機能を阻害する効果を有する。 本発明において、 癌細胞の形態を正常化するとは、 例えば、 突起が出たり細胞 同士が重なり合って増殖している癌細胞を、 平らで突起も見られなレ、正常細胞の 形態に戻すことを指し、 癌細胞の増殖を抑制するとは、 非常に低濃度 （例えば n gZm lのオーダ一） で増殖を 5 0 %抑制することを指し、 癌遺伝子の機能を阻 害する効果とは、 形態的に癌細胞を正常細胞にする効果を指す。  When given to cancer cells having the ras oncogene and the like, aglaias tinA has the effect of normalizing the cell morphology, suppressing its growth, and inhibiting the function of the oncogene. In the present invention, normalizing the morphology of cancer cells means, for example, returning cancer cells that have protruded or proliferate due to overlapping cells to normal cell morphology without any prominence. Inhibiting the growth of cancer cells refers to inhibiting growth by 50% at very low concentrations (eg, on the order of ngZml). The effect of inhibiting oncogene function is morphologically It refers to the effect of turning cancer cells into normal cells.
aglaias tinAが癌細胞の増殖を 50%抑制する濃度 （ I C 5 e) は、 癌細胞の種 類によって異なるが 2. 4 〜7.5ng/mlである。 Aglaias TINA inhibits 50% growth of the cancer cell concentration (IC 5 e) is a different but 2. 4 ~7.5ng / ml depending on the type of cancer cells.
このような機能に基づき、 本発明に係る新規化合物であるアグラィアスタチン (aglaiastatin) Aを有効成分とし、 癌遺伝子機能抑制剤または抗癌剤を提供す ることが出来る。  Based on such a function, it is possible to provide an oncogene function inhibitor or an anticancer agent using aglaiastatin A, a novel compound according to the present invention, as an active ingredient.
本発明の抗癌剤に使用される医薬上許容可能な担体としては、 例えば天然もし くは合成ゲイ酸アルミニウム、 微結晶セルロース、 タルク、 デキストリン、 澱粉、 乳糖などの賦形剤、 及び植物油、 プロピレングリコールなどの希釈剤が挙 げられる。 これに加えて、 その他の任意成分として、 医薬上許容可能な安定剤、 コーティング剤、 懸濁化剤、 乳化剤、 溶解補助剤、 保存剤、 緩衝剤、 甘味剤など も存在させ得る。 これらの添加剤としては、 公知のものを適宜組み合わせて使用 し得る。 また剤形としては粉剤、 顆粒剤、 錠剤、 カプセル剤、 注射剤等の任意の 形態で用いられる。 Pharmaceutically acceptable carriers used in the anticancer agent of the present invention include, for example, excipients such as natural or synthetic aluminum gateate, microcrystalline cellulose, talc, dextrin, starch, lactose, and vegetable oils, propylene glycol and the like. Diluent I can do it. In addition, pharmaceutically acceptable stabilizers, coatings, suspending agents, emulsifiers, solubilizers, preservatives, buffers, sweeteners and the like may be present as other optional ingredients. Known additives may be used in appropriate combination as these additives. The composition may be used in any form such as powders, granules, tablets, capsules, and injections.
本発明の aglaias tin Aを有効成分とする抗癌剤は主として経口投与される 、 本発明は投与方法によって限定されない。 成人一日あたりの投与量は投与方 法及び病状等によって異なる。 主たる投与方法である経口投与の場合は、 成人 1 日当たり 100〜500mg が通常の投与量である。 し力、し、 本発明は投与量によって 限定されない。  The anticancer agent containing aglaias tin A of the present invention as an active ingredient is mainly orally administered, and the present invention is not limited by the administration method. The daily dose for adults differs depending on the administration method and medical condition. In the case of oral administration, which is the main method of administration, the usual dosage is 100 to 500 mg per adult per day. The present invention is not limited by dosage.
本発明の第 2の化合物である aglaias tinBは分子量 524 (FAB-MSスぺクトル 測定による） の無色の粉末である。 シリ力ゲル薄層クロマトグラフィーにおける R f値は 0.43 (展開溶媒 クロ口ホルム： メタノール =20： 1 ) 、 0.46 (展開溶 媒 トルエン ：アセトン = 1 ： 1 ) であった。  The second compound of the present invention, aglaias tinB, is a colorless powder having a molecular weight of 524 (by FAB-MS spectrum measurement). The Rf values in the silica gel thin-layer chromatography were 0.43 (developing solvent: chlorophyll form: methanol: 20: 1) and 0.46 (developing solvent: toluene: acetone = 1: 1).
紫外線吸収スぺクトルは図 7から図 9、 赤外線吸収スぺクトルは図 1 0の通り である。 また1 H— NMRスペク トル及び1 3 C— NMRスペク トルの測定結 果は、 図 1 1及び図 1 2の通りである。 The ultraviolet absorption spectrum is shown in FIGS. 7 to 9, and the infrared absorption spectrum is shown in FIG. The measurement results of the 1 H-NMR spectrum and the 13 C-NMR spectrum are as shown in FIGS. 11 and 12.
検討の結果、 了グライア 'ォドラー夕から単離された本発明化合物は前記化学 式 （2 ) で表される構造であることが支持された。  As a result of the study, it was supported that the compound of the present invention, which was isolated from Ria gliadora, had the structure represented by the chemical formula (2).
aglaiastatin Bの原料となる植物は、 保存、 運搬等の理由から乾燥してもよ く、 その場合は天日で、 あるいは 60°C以下のオーブンで乾燥するのが好ましい。 また、 抽出に際しては粉砕したものが好ましい。 抽出に用いる溶媒は aglaiasta tin Bを安定に抽出し得る溶媒であればいかなるものでも使用することができる が、 非極性溶媒が好ましく、 特に炭化水素系溶媒またはハロゲン化炭化水素溶媒 が好ましい。 炭化水素系溶媒としてはペンタン、 へキサン、 ヘプタン、 ォク タン、 ベンゼン、 トルエン、 キシレン等が挙げられ、 特にトルエンが好ましい。 またハロゲン化炭化水素溶媒としては、 例えばクロ口ホルム、 二塩化メチレン、 四塩化炭素等が挙げられ、 特にクロ口ホルムが好ましい。 これらの溶媒は 1種ま たは 2種以上を併用してもよい。 The plant used as the raw material of aglaiastatin B may be dried for reasons such as preservation and transportation, and in that case, it is preferable to dry it in the sun or in an oven at 60 ° C or lower. In addition, pulverized one is preferable for extraction. The solvent used for extraction is aglaiasta Any solvent can be used as long as it can stably extract tin B, but a non-polar solvent is preferable, and a hydrocarbon solvent or a halogenated hydrocarbon solvent is particularly preferable. Examples of the hydrocarbon solvent include pentane, hexane, heptane, octane, benzene, toluene, xylene and the like, with toluene being particularly preferred. As the halogenated hydrocarbon solvent, for example, chloroform, methylene dichloride, carbon tetrachloride and the like can be mentioned, and chloroform is particularly preferable. These solvents may be used alone or in combination of two or more.
抽出時の溶媒量、 温度、 時間などの操作は、 aglaiastatinBが分解を起こすこ となく安定に、 且つ高収量で得られる範囲であれば特に限定されるものでは ない。  The operation such as the amount of the solvent, the temperature, and the time during the extraction is not particularly limited as long as aglaiastatinB can be obtained stably without decomposition and in a high yield.
aglaias tinBの単離精製法は、 本化合物が抽出物から安定に得られる方法で あれば特に限定されるものではない。 好適な単離精製法の例としてクロマトグラ フィ一を挙げることができる。 クロマトグラフィーは好ましくはカラムクロマト グラフィ一、 ゲル濾過クロマトグラフィー、 高速液体クロマトグラフィー、 薄層 クロマトグラフィーまたはこれらの組み合わせを包含し得る。 カラムクロマトグ ラフィ一に用いる担体としてはシリカゲル、 アルミナなどが使用でき、 展開溶媒 としては炭化水素系溶媒、 ハロゲン化炭化水素、 アルコール類などを用いること ができる。 またゲル濾過クロマトグラフィーとしては担体としてトヨパール HW- 40 (商品名、 東ソ一社製） 、 溶媒としてアルコール類を用いることができる。 し かしクロマトグラフィー担体及び溶媒の種類は上記のものに限定されない。 単離 精製された aglaiastatinBの構造を解析した結果、 化学式 （2 ) で表されること が判明した。 aglaiastatin Bは、 r a s癌遺伝子等を有する癌細胞に与えると細胞の形態 を正常化し、 且つその増殖を抑制し、 癌遺伝子の機能を阻害する効果を有する。 aglaiastatin Bが癌細胞の増殖を 50%抑制する濃度 （ I C 5。） は、 癌細胞の種 類によって異なるが 3. 6〜8. 9ng/mlである。 The method for isolating and purifying aglaias tinB is not particularly limited as long as the compound can be stably obtained from the extract. An example of a suitable isolation and purification method is chromatography. Chromatography may preferably include column chromatography, gel filtration chromatography, high performance liquid chromatography, thin layer chromatography or a combination thereof. Silica gel, alumina or the like can be used as a carrier for the column chromatography, and a hydrocarbon solvent, halogenated hydrocarbon, alcohol, or the like can be used as a developing solvent. As gel filtration chromatography, Toyopearl HW-40 (trade name, manufactured by Tosoh Corporation) can be used as a carrier, and alcohols can be used as a solvent. However, the types of the chromatography carrier and the solvent are not limited to those described above. Analysis of the structure of the isolated and purified aglaiastatinB revealed that it was represented by the chemical formula (2). When aglaiastatin B is given to a cancer cell having a ras oncogene or the like, it has the effect of normalizing the cell morphology, suppressing its growth, and inhibiting the function of the oncogene. Aglaiastatin B inhibits 50% growth of the cancer cell concentration (IC 5.) is a different but 3. 6-8. 9 ng / ml depending on the type of cancer cells.
このような機能に基づき、 本発明に係る新規化合物であるアグラィアスタチン (aglaiastatin) Bを有効成分とし、 癌遺伝子機能抑制剤または抗癌剤を提供す ることが出来る。  Based on such a function, an oncogene function inhibitor or an anticancer agent can be provided using aglaiastatin B, a novel compound according to the present invention, as an active ingredient.
本発明の抗癌剤に使用される医薬上許容可能な担体としては、 例えば天然もし くは合成ゲイ酸アルミニウム、 微結晶セルロース、 タルク、 デキストリン、 澱粉、 乳糖などの賦形剤、 及び植物油、 プロピレングリコ一ルなどの希釈剤が挙 げられる。 これに加えて、 その他の任意成分として、 医薬上許容可能な安定剤、 コ一ティング剤、 懸濁化剤、 乳化剤、 溶解補助剤、 保存剤、 緩衝剤、 甘味剤など も存在させ得る。 これらの添加剤としては、 公知のものを適宜組み合わせて使用 し得る。 また剤形としては粉剤、 顆粒剤、 錠剤、 カプセル剤、 注射剤等の任意の 形態で用いられる。  Pharmaceutically acceptable carriers used in the anticancer agent of the present invention include, for example, excipients such as natural or synthetic aluminum gateate, microcrystalline cellulose, talc, dextrin, starch, lactose, and vegetable oils, propylene glycol And diluents. In addition, pharmaceutically acceptable stabilizers, coating agents, suspending agents, emulsifiers, solubilizers, preservatives, buffers, sweeteners and the like may be present as other optional ingredients. Known additives may be used in appropriate combination as these additives. The composition may be used in any form such as powders, granules, tablets, capsules, and injections.
本発明の aglaiastatin Bを有効成分とする抗癌剤は主として経口投与される が、 本発明は投与方法によって限定されない。 成人一日あたりの投与量は投与方 法及び病状等によって異なる。 主たる投与方法である経口投与の場合は、 成人 1 日当たり 100〜500mg力、'通常の投与量である。 し力、し、 本発明は投与量によって 限定されない。  The anticancer agent containing aglaiastatin B as an active ingredient of the present invention is mainly orally administered, but the present invention is not limited by the administration method. The daily dose for adults differs depending on the administration method and medical condition. In the case of oral administration, which is the main method of administration, the daily dose for adults is 100 to 500 mg, the usual dosage. The present invention is not limited by dosage.
本発明の第 3の化合物である aglaiastatin Cは分子量 5 2 6 (FAB-MSスぺク卜 ル測定による） の無色の粉末である。 シリカゲル薄層クロマトグラフィーにおけ る R f値は 0. 42 (展開溶媒 クロ口ホルム ： メタノール： =20： 1 ) 、 0. 49 (展開 溶媒 トルエン：アセトン = 1 ： 1 ) であった。 また、 元素分析の結果、 分子式 は C 3 1 H30N2 〇6 である。 The third compound of the present invention, aglaiastatin C, is a colorless powder having a molecular weight of 526 (by FAB-MS spectrum measurement). In silica gel thin layer chromatography The R f values were 0.42 (developing solvent: chloroform: methanol: = 20: 1) and 0.49 (developing solvent: toluene: acetone = 1: 1). As a result of elemental analysis, molecular formula is C 3 1 H 30 N 2 〇 6.
紫外線吸収スぺク トルは図 1 3から図 1 5、 赤外線吸収スぺク トルは図 1 6の通 りである。 また' H— NMRスペクトル及び1 3 C— NMRスペク トルの測定結果 は、 図 1 7及び図 1 8の通りである。 The ultraviolet absorption spectrum is as shown in FIGS. 13 to 15, and the infrared absorption spectrum is as shown in FIG. The measurement results of the 1 H-NMR spectrum and the 13 C-NMR spectrum are as shown in FIGS. 17 and 18.
検討の結果、 アグラィァ ·ォドラ一夕から単離された本発明化合物は前記化学 式 （3 ) で表される構造であることが支持された。  As a result of the study, it was supported that the compound of the present invention isolated from Agraya dora overnight had the structure represented by the chemical formula (3).
aglaiastatin Cの原料となる植物は、 保存、 運搬等の理由から乾燥してもよ く、 その場合は天日で、 あるいは 60°C以下のオーブンで乾燥するのが好ましい。 また、 抽出に際しては粉砕したものが好ましい。 抽出に用いる溶媒は aglaias tin Cを安定に抽出し得る溶媒であればいかなるものでも使用することができる が、 非極性溶媒が好ましく、 特に炭化水素系溶媒またはハロゲン化炭化水素溶媒 が好ましい。 炭化水素系溶媒としてはペンタン、 へキサン、 ヘプタン、 ォク タン、 ベンゼン、 トルエン、 キシレン等が挙げられ、 特にトルエンが好ましい。 またハロゲン化炭化水素溶媒としては、 例えばクロ口ホルム、 二塩化メチレン、 四塩化炭素等が挙げられ、 特にクロ口ホルムが好ましい。 これらの 1種または 2 種以上を併用してもよい。  The plant used as the raw material for aglaiastatin C may be dried for reasons such as preservation and transportation, and in that case, it is preferable to dry it in the sun or in an oven at 60 ° C or lower. In addition, pulverized one is preferable for extraction. As the solvent used for the extraction, any solvent can be used as long as it can stably extract aglaias tin C, but a non-polar solvent is preferable, and a hydrocarbon solvent or a halogenated hydrocarbon solvent is particularly preferable. Examples of the hydrocarbon solvent include pentane, hexane, heptane, octane, benzene, toluene, xylene and the like, with toluene being particularly preferred. As the halogenated hydrocarbon solvent, for example, chloroform, methylene dichloride, carbon tetrachloride and the like can be mentioned, and chloroform is particularly preferable. One or more of these may be used in combination.
抽出時の溶媒量、 温度、 時間などの操作は、 aglaiastatinCが分解を起こすこ となく安定に、 且つ高収量で得られる範囲であれば特に限定されるものでは ない。  The operation such as the amount of solvent, temperature, and time during extraction is not particularly limited as long as aglaiastatin C can be obtained stably without decomposition and in a high yield.
aglaiastatinCの単離精製法は、 本化合物が抽出物から安定に得られる方法で あれば特に限定されるものではない。 好適な単離精製法の例としてクロマトグラ フィ一を挙げることができる。 クロマトグラフィ一は好ましくはカラムクロマト グラフィ一、 ゲル濾過クロマトグラフィー、 高速液体クロマトグラフィー、 薄層 クロマトグラフィーまたはこれらの組み合わせを包含し得る。 カラムクロマトグ ラフィ一に用いる担体としてはシリカゲル、 アルミナなどが使用でき、 展開溶媒 としては炭化水素系溶媒、 ハロゲン化炭化水素、 アルコール類などを用いること ができる。 またゲル濾過クロマトグラフィ一としては担体としてトヨパールThe isolation and purification method of aglaiastatin C is a method whereby this compound can be obtained stably from the extract. If there is, it is not particularly limited. An example of a suitable isolation and purification method is chromatography. Chromatography may preferably include column chromatography, gel filtration chromatography, high performance liquid chromatography, thin layer chromatography or a combination thereof. Silica gel, alumina or the like can be used as a carrier for the column chromatography, and a hydrocarbon solvent, halogenated hydrocarbon, alcohol, or the like can be used as a developing solvent. In gel filtration chromatography, Toyopearl is used as a carrier.
HW-40東ソ一社製） 、 溶媒としてアルコール類を用いることができる。 しかしク ロマトグラフィ一担体及び溶媒の種類は上記のものに限定されない。 そして、 aglaiastatin Cの構造を解析した結果、 化学式 （3 ) で表されることが判明し た。 HW-40 manufactured by Tosoh Corporation) and alcohols can be used as a solvent. However, the types of the chromatographic carrier and the solvent are not limited to those described above. As a result of analyzing the structure of aglaiastatin C, it was found that it was represented by the chemical formula (3).
aglaiastatin Cは、 r a s癌遺伝子等を有する癌細胞に与えると細胞の形態 を正常化し、 且つその増殖を抑制し癌遺伝子の機能を阻害する効果を有する。 a glaiastatin Cが癌細胞の増殖を 5 0 %抑制する濃度 （ I C 5。） は、 癌細胞の種 類によって異なる力く、 1 . 0〜5. 6 n gZm lである。 When aglaiastatin C is given to a cancer cell having a ras oncogene or the like, it has the effect of normalizing the cell morphology, suppressing its growth and inhibiting the function of the oncogene. The concentration of a glaiastatin C that inhibits the growth of cancer cells by 50% (IC 5 ) varies depending on the type of cancer cell, and is 1.0 to 5.6 ng Zml.
このような機能に基づき、 本発明に係る新規化合物であるアグラィアスタチン (aglaiastatin) Cを有効成分とし、 癌遺伝子機能抑制剤または抗癌剤を提供す ることが出来る。  Based on such a function, an oncogene function inhibitor or an anticancer agent can be provided using aglaiastatin C, a novel compound according to the present invention, as an active ingredient.
本発明の抗瘙剤に使用される医薬上許容可能な担体としては、 例えば天然もし くは合成ゲイ酸アルミニウム、 微結晶セルロース、 タルク、 デキストリン、 澱粉、 乳糖などの賦形剤、 及び植物油、 プロピレングリコールなどの希釈剤が挙 げられる。 これに加えて、 その他の任意成分として、 医薬上許容可能な安定剤、 コーティ ング剤、 懸濁化剤、 乳化剤、 溶解補助剤、 保存剤、 緩衝剤、 甘味剤など も存在させ得る。 これらの添加剤としては、 公知のものを適宜組み合わせて使用 し得る。 また剤形としては粉剤、 顆粒剤、 錠剤、 カプセル剤、 注射剤等の任意の 形態で用いられる。 Pharmaceutically acceptable carriers used in the antimicrobial agents of the present invention include, for example, excipients such as natural or synthetic aluminum gateate, microcrystalline cellulose, talc, dextrin, starch, lactose, and vegetable oils, propylene Diluents such as glycols. In addition to this, other optional ingredients include pharmaceutically acceptable stabilizers, Coatings, suspending agents, emulsifiers, solubilizers, preservatives, buffers, sweeteners and the like can also be present. Known additives may be used in appropriate combination as these additives. The composition may be used in any form such as powders, granules, tablets, capsules, and injections.
本発明の aglaiastatin Cを有効成分とする抗癌剤は主として経口投与される 力 \ 本発明は投与方法によって限定されない。 成人一日あたりの投与量は投与方 法及び病状等によって異なる。 主たる投与方法である経口投与の場合は、 成人 1 日当たり 1 0 0〜5 0 O m gが通常の投与量である。 し力、し、 本発明は投与量に よって限定されない。  The anticancer agent of the present invention containing aglaiastatin C as an active ingredient is mainly administered orally. The present invention is not limited by the administration method. The daily dose for adults differs depending on the administration method and medical condition. In the case of oral administration, which is the main method of administration, the usual dosage is 100 to 50 Omg per adult per day. The present invention is not limited by dosage.
本発明に係る新規化合物であるアグラィアスタチン （aglaiastatin) A、 B、 および Cは、 いずれも癌細胞に対して、 極めて類似した機能を有する。 これは、 アグラィアスタチン （aglaiastatin) A、 B、 および Cに共通する構造に起因す るものと考えられる。  The novel compounds according to the present invention, aglaiastatins A, B and C, all have very similar functions on cancer cells. This is thought to be due to the structure common to aglaiastatin A, B, and C.
アグラィアスタチン （aglaiastatin) A、 B、 および Cは、 これらの機能によ り、 r a s癌遺伝子の機能の抑制に対して有効な癌遺伝子機能抑制剤または抗癌 剤として用いることが出来る。 実施例  With these functions, aglaiastatin A, B, and C can be used as an oncogene function inhibitor or an anticancer agent effective for suppressing the function of the ras oncogene. Example
以下、 実施例により本発明をさらに詳細に説明するが、 本発明はこれらの実施 例に限定されるものではない。  Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.
I . aglaiastatinA  I. aglaiastatinA
1 . アグラィァ ·ォドラ一夕からの活性物質の抽出 60°Cのオーブンで乾燥したアグラィァ ·ォドラ一夕の葉 167gを粉砕した後、 2 Lの三角フラスコに入れ、 クロ口ホルム 1000ml を加えて室温で 5時間撹拌し た。 溶媒を新しくして同様の操作を繰り返し、 合計 3回の抽出を行った。 3回分 の抽出液を合わせて減圧下でクロ口ホル厶を留去し、 暗緑色シラップ 5.7 gを得 た。 このシラップを次の精製の原料とした。 1. Extraction of active substance from Agraya dora After 167 g of Agrilla odora leaves dried in a 60 ° C. oven were crushed, the mixture was placed in a 2 L Erlenmeyer flask, added with 1000 ml of black-mouthed form, and stirred at room temperature for 5 hours. The same operation was repeated with a new solvent, and a total of three extractions were performed. The extracts from the three times were combined, and the chloroform was distilled off under reduced pressure to obtain 5.7 g of dark green syrup. This syrup was used as a raw material for the next purification.
2. 抽出物からの活性物質の精製 2. Purification of the active substance from the extract
クロ口ホルム抽出物 3.9gを少量のトルェンに溶かし、 力ラムに充塡した 120gの シリカゲル （Merck silicagel 60, メルク社製） の上に添加し、 トルエン ： ァセ トン =20： 1 , 16： 1 , 6 : 1, 3 : 1, 2 : 1， 1 : 1の溶媒で順次溶出し、 約 20mlずつ分画した。 各画分を後述の 「4. aglaiastatinAの各種細胞に対する 形態変化誘導効果」 で用いた細胞形態変化誘導試験に付したところ、 トルエン ： アセトン =16 ： 1溶出画分に形態正常化作用が認められた。 活性画分を集めた ところ重量は 387mgであつた。  Dissolve 3.9 g of black-mouthed form extract in a small amount of toluene and add it to 120 g of silica gel (Merck silicagel 60, manufactured by Merck) filled in a column of toluene. Toluene: acetone = 20: 1,16: The mixture was eluted sequentially with a solvent of 1, 6: 1, 3: 1, 2: 1, 1: 1 and fractionated by about 20 ml. Each fraction was subjected to the cell morphological change induction test used in “4. Morphological change-inducing effect of aglaiastatinA on various cells” described below. As a result, a morphological normalizing effect was observed in the toluene: acetone = 16: 1 eluted fraction. Was. The active fraction was collected and weighed 387 mg.
続いてこの活性画分に 80%メタノールを加え溶解し、 不溶物を取り除いた。 その後カラムに充塡した Merck silicagel 60 (silanised ) に添加し 80%メ 夕ノ一ルで溶出し約 4 mlずつ分画した。 各画分を先の細胞形態変化誘導試験に再 び付し、 活性が認められた画分を集めた。 活性画分の重量は 20 Omgで あった。  Subsequently, 80% methanol was added to the active fraction to dissolve it, and insolubles were removed. Thereafter, the mixture was added to Merck silicagel 60 (silanised) packed in a column, and eluted with 80% methanol to fractionate about 4 ml each. Each fraction was re-subjected to the above-mentioned cell morphological change induction test, and fractions showing activity were collected. The weight of the active fraction was 20 Omg.
次に、 活性画分を予めメタノールで平衡化したトヨパール HW— 40に添加し てゲル濾過クロマトグラフィーを行い約 2m 1ずつ分画した。 さらにこの活性画 分を集め、 HPLC分取 （カラム： PEGASIL 0DS、 (株） センシュ一科学製、 溶 出溶媒： 70%メタノール、 流速：4 ml/min、 検出： U. V.210nm)を行い約 10mlず つ分画した。 活性試験を行ったところリテンションタイム 5 8分に溶出される画 分に活性が確認された。 この画分を集め、 無色の粉末 （aglaias tinA 4. 6mg) を得た。 この画分はシリカゲル薄層クロマトグラフィーを行ったところ単一 スポットであった。 Next, the active fraction was added to Toyopearl HW-40 previously equilibrated with methanol and subjected to gel filtration chromatography to fractionate about 2 ml each. Further, the active fraction was collected and subjected to HPLC fractionation (column: PEGASIL 0DS, manufactured by Senshu Ichigaku Co., Ltd., elution solvent: 70% methanol, flow rate: 4 ml / min, detection: UV210 nm), and about 10 ml And fractionated. When an activity test was performed, the activity was confirmed in the fraction eluted at a retention time of 58 minutes. The fractions were collected to obtain a colorless powder (aglaias tinA 4.6 mg). This fraction was a single spot when subjected to silica gel thin layer chromatography.
3. aglaiastatinAの物理化学的性質  3. Physicochemical properties of aglaiastatinA
無色粉末 （aglaiastatinA) の各種物理化学的性質を測定した。 本化合物のシ リカゲル薄層クロマトグラフィーにおける R f値は 0.58 (展開溶媒： クロ口ホル ム： メタノール =40： 1 ) 、 0.58 (展開溶媒： トルエン：アセトン = 2 ： 1 ) で あった。 融点は 6 8〜7 1 °Cであった。  Various physicochemical properties of the colorless powder (aglaiastatinA) were measured. The Rf values of this compound in silica gel thin-layer chromatography were 0.58 (developing solvent: pore form: methanol = 40: 1) and 0.58 (developing solvent: toluene: acetone = 2: 1). Melting point was 68-71 ° C.
また各種スぺクトルの測定を行った。 結果を図 1から図 6に示す。  Various spectra were also measured. The results are shown in FIGS.
4 . aglaias tinAの各種細胞に対する形態変化誘導効果  4. Induction effect of aglaias tinA on morphological changes of various cells
a . 各種細胞の培養 a. Culture of various cells
K - rast s-NRK細胞 （米国 N I H) が癌細胞の形態を示す 33°C及び正常細胞の形 態を示す 3 9 °Cにおいて、 5 %仔牛血清 （Gibco Laboratories社製） を含むダル べッコ変法イーグル培地 （日水製薬 （株） 製） にグルタミン 0. 6g/l、 カナマ イシン 0.2g/l、 NaHC03 2.25g/l、 を加えたものを培養液 （以下、 DMEMと称 することもある） として、 5 % C02インキュベータ一中で培養した。 正常なラッ ト腎細胞である NRK-49細胞 （大日本製薬 （株） ） 、 および瘙細胞である K-ras - NRK細胞、 K-ras-NIH3T3細胞、 H-ras- NIH3T3細胞、 N- ras_NIH3T3細胞 （東京大 学医科学研究所） は、 37°Cで同様に培養した。 K - In ras ts -NRK cells (US NIH) is 3 9 ° C showing the form status of 33 ° C and normal cells showing the morphology of the cancer cells, base Dar containing 5% calf serum (Gibco Laboratories, Inc.) Tsu co modified Eagle's medium glutamine (Nissui Pharmaceutical Co., Ltd.) 0. 6 g / l, kanamycin 0.2g / l, NaHC0 3 2.25g / l, culture medium plus (hereinafter, referred to as DMEM as also), it was cultured in 5% C0 2 incubator scratch. Normal rat kidney cells, NRK-49 cells (Dainippon Pharmaceutical Co., Ltd.), and 瘙 cells, K-ras-NRK cells, K-ras-NIH3T3 cells, H-ras-NIH3T3 cells, N-ras_NIH3T3 Cells (Tokyo University Medical Research Institute) were similarly cultured at 37 ° C.
また、 細胞は、 カルシウム ·マグネシウム不含リン酸塩緩衝液 （PBS (-)： NaCl 8. Og/1 . Na2P04 ·12Η20 2. 31g/l . H2P04 0.2g/l ； pH 7.4) で洗浄した後、 O トリプシン- EDTA溶液 （trypsin 0.5g/l , NaCl 8.0g/L glucose 1.0g/l' Phenol red 0.02g/l , KH2P04 0.35g/l , EDTA 0.2g/l) で培養フラスコよ り細胞をはがし、 新しし、培養液を入れたフラスコ中に移すことにより継代した。 b . 形態変化誘導効果の観察 Also, cells are calcium-magnesium-free phosphate buffer (PBS (-):.. NaCl 8. Og / 1 Na 2 P0 4 · 12Η 2 0 2. 31g / l H 2 P0 4 0.2g / l After washing with pH 7.4) O trypsin - EDTA solution (trypsin 0.5g / l, NaCl 8.0g / L glucose 1.0g / l 'Phenol red 0.02g / l, KH 2 P0 4 0.35g / l, EDTA 0.2g / l) in Ri by culture flasks Cells were detached, passaged by transferring them to fresh and culture flasks. b. Observation of morphological change inducing effect
各細胞を、 4 8穴プラスチックプレ一ト （コースター社製）に 1. 5 X 104 個 Z DME 0.5ml/well でまき、 5 % C02インキュベータ一中で 1日培養した。 翌日被 検物質を加え、 その後 3日目までの細胞の形態変化を観察した。 Each cell was seeded on a 48-well plastic plate (manufactured by Coaster) at 1.5 × 10 4 Z DME at 0.5 ml / well, and cultured in a 5% CO 2 incubator for 1 day. The test substance was added the next day, and cell morphological changes were observed up to the third day.
i ) K-ras t s-NRK細胞に対する aglaiastatinAの効果 i) Effect of aglaiastatinA on K-ras ts- NRK cells
K-ras t s- NRK細胞は、 33°Cでは細胞からいくつもの細い突起が見られ、 また細 胞同士が重なり合つて癌細胞の形態を示し、 39°Cでは細胞は平で突起も見られず 重なり合うこともなく正常細胞の形態を示す。 At 33 ° C, K-ras ts -NRK cells show a number of small protrusions from the cells, and the cells overlap and show the morphology of cancer cells.At 39 ° C, the cells are flat with protrusions. It shows normal cell morphology without overlapping.
そこで、 33°Cで aglaiastatinA 10ng/mlを添加して 3日間培養した結果、 突起 もなく平で重なり合うこともない正常細胞の形態を示した。 また、 39°Cでは、 細 胞の形態に大きな変化はなかった。  Therefore, as a result of adding 3 ng / ml of aglaiastatinA at 33 ° C and culturing for 3 days, it showed a normal cell morphology without projections and without overlapping. At 39 ° C, there was no significant change in cell morphology.
ii) K-ras- NRK細胞に対する aglaias tinAの効果 ii) Effect of aglaias tinA on K-ras- NRK cells
癌細胞である K-ras-NRK細胞は、 形が丸く盛り上がっているが、 これに aglaiastatinA 10ng/mlを添加して 37°Cで 3日間培養した結果、 細胞の形態が平 になり、 接着面も増して正常細胞の形態を示した。  K-ras-NRK cells, which are cancer cells, have a rounded shape, but after adding 10 ng / ml of aglaiastatinA and culturing at 37 ° C for 3 days, the cell morphology becomes flat and the adhesive surface It also showed normal cell morphology.
ii i ) K-ras-NIH3T3細胞に対する aglaias tin Aの効果 ii i) Effect of aglaias tin A on K-ras-NIH3T3 cells
癌細胞である K-ras- NIH3T3細胞は、 細長い突起状の形態をしており、 さらに 細胞同士が重なり合っている.。 これに aglaiastatin Aを 10ng/ml添加して 37°C で 3日間培養したところ、 平になり接着面も増大し、 正常細胞である NIH3T3細胞 の形態になつているのが観察された。 K-ras-NIH3T3 cells, which are cancer cells, have an elongated protruding morphology, and the cells overlap. Add 10 ng / ml of aglaiastatin A to this at 37 ° C After culturing for 3 days, it was observed that the cells became flat and the adherent surface increased, and that they were in the form of normal NIH3T3 cells.
5. aglaiastatinAの各種細胞に対する細胞増殖抑制効果  5. Cell growth inhibitory effect of aglaiastatinA on various cells
各細胞を、 4 8穴プラスチックプレート （コース夕一社製） に 1.0 X104 個/ Ί EM 0.5ml /we 11 でまき、 1日適温 5%C02 インキュベータ一中で培養した。 翌日 aglaiastatin Aを加え更に 3日間培養し、 PBS (-)で 2回洗った後、 トリプシン を 100 ^l/wellずつ加え、 数分 0% C02条件下の恒温機中で細胞をはがし、 DMEM を 900 1/wellずつ加えて均一にした。 この溶液の細胞濃度をコール夕一カウン ター （ZM型） を用いて測定し、 細胞増殖を 50%阻害する濃度 （ I C5。） を求め た。 結果は表 1に示した通りであった。 すなわち、 aglaiastatinAは I C5。が n g/m 1のオーダーであり、 通常の細胞毒性物質が/ i g/m 1のオーダーである ので、 極めて低濃度で細胞増殖を抑制した。 しかもその濃度は正常細胞よりも腫 瘍細胞に対して大きい。 なお、 表 1に示した細胞名のうち ras を含むものは腫瘍 細胞である。 Each cell was seeded on a 48-well plastic plate (manufactured by Course Yuichi) at 1.0 × 10 4 cells / ΔEM 0.5 ml / we 11 and cultured in a 5% CO 2 incubator at an appropriate temperature for 1 day. The next day further cultured for 3 days plus aglaiastatin A, PBS (-) was washed twice with added trypsin by 100 ^ l / well, the cells are detached in minutes 0% C0 2 under the conditions of the thermostat, DMEM Was added at 900 1 / well to make it uniform. The cell concentration of this solution was measured using a Coulter counter (ZM type) to determine the concentration that inhibits cell growth by 50% (IC 5 ). The results were as shown in Table 1. In other words, aglaiastatinA the IC 5. Since ng / m 1 is on the order of ng / m 1 and ordinary cytotoxic substances are on the order of / ig / m 1, cell proliferation was suppressed at an extremely low concentration. Moreover, its concentration is higher for tumor cells than for normal cells. Among the cell names shown in Table 1, those containing ras are tumor cells.
〔表 1〕 aglaiastatinAの各種細胞に対する増殖抑制効果 細胞名 I C so (ng/ml ) [Table 1] Growth inhibitory effect of aglaiastatinA on various cells Cell name I C so (ng / ml)
K-ras-NRK 5.0  K-ras-NRK 5.0
K-ras-瞧 T3 7.0  K-ras- 瞧 T3 7.0
H-ras-画 3T3 5.3  H-ras-art 3T3 5.3
N-ras-NIH3T3 7.5  N-ras-NIH3T3 7.5
NRK - 4 9 10  NRK-4 9 10
K-ras 1 5 -NRK (33°C) 4. 6 K-ras 15 -NRK (33 ° C) 4.6
K-ras , s -NRK (39°C) 2.4 K-ras , s -NRK (39 ° C) 2.4
6 . 急性毒性  6. Acute toxicity
aglaias tinAの急性毒性は、 ICR系雄性マウス （5週令） に対し、 L D 50は 300mg/kg以上であり、 急性毒性が低レ、ことが確認された。 Acute toxicity of Aglaias TINA, compared ICR male mice (5 weeks old), LD 50 is at 300 mg / kg or more, acute toxicity low les, it was confirmed.
I I . aglaiastatinB I I. AglaiastatinB
1 . アグラィァ 'ォドラー夕からの活性物質の抽出  1. Extraction of Active Substances from Agraya Odler Evening
60°Cのオーブンで乾燥したアグラィァ 'ォドラー夕の葉 167gを粉砕した後、 2 Lの三角フラスコに入れ、 クロ口ホルム 1000ml を加えて室温で 5時間撹拌し た。 溶媒を新しくして同様の操作を繰り返し、 合計 3回の抽出を行った。 3回分 の抽出液を合わせて減圧下でクロ口ホルムを留去し、 暗緑色シラップ 5. 7 gを得 た。 このシラップを次の精製の原料とした。  After 167 g of Agra'dola evening leaves dried in an oven at 60 ° C. were crushed, 167 g of the leaves were put into a 2 L Erlenmeyer flask, and 1000 ml of black-mouthed form was added, followed by stirring at room temperature for 5 hours. The same operation was repeated with a new solvent, and a total of three extractions were performed. The extracts from the three extractions were combined, and the chloroform was distilled off under reduced pressure to obtain 5.7 g of dark green syrup. This syrup was used as a raw material for the next purification.
2. 抽出物からの活性物質の精製 クロ口ホルム抽出物 3.9 gを少量のトルエンに溶かし、 カラムに充塡した 120 gのシリカゲル （Merck silicagel 60, メルク社製） の上に添加し、 トルエン： アセトン =20 : 1, 16 : 1, 6 : 1, 3 : 1, 2 : 1, 1 ： 1の溶媒で順次溶出 し、 約 20mlずつ分画した。 各画分を後述の 「4. aglaiastatinBの各種細胞に対 する形態変化誘導効果」 で用いた細胞形態変化誘導試験に付したところ、 トルエン ： アセトン = 3 ： 1から 1 ： 1溶出画分に形態正常化作用が認めら れた。 活性画分を集めたところ重量は 639mgであった。 2. Purification of the active substance from the extract Dissolve 3.9 g of black-mouthed form extract in a small amount of toluene, add to 120 g of silica gel (Merck silicagel 60, manufactured by Merck) packed in a column, and add toluene: acetone = 20: 1, 16: 1, The column was eluted sequentially with a solvent of 6: 1, 3: 1, 2: 1, 1: 1 and fractionated by about 20 ml. Each fraction was subjected to the cell morphological change induction test used in “4. Effect of aglaiastatin B on morphological change in various cells” described below. It was found that toluene: acetone = 3: 1 to 1: 1 eluted fraction A normalizing effect was observed. The active fraction was collected and weighed 639 mg.
この活性画分を、 メタノールで平衡化したトョパール冊-40で約21111ずっ分画 し、 さらにカラムに充塡した erck silicagel 60 (silanised ) に添加し 70 %メタノ一ルで溶出し約 4 mlずつ分画した。 各画分を先の細胞形態変化誘導試験 に再び付し、 活性が認められた画分を集めた。  Approximately 21111 fractions of this active fraction were fractionated with Toyopearl volume-40 equilibrated with methanol, added to erck silicagel 60 (silanised) packed in a column, and eluted with 70% methanol. Fractionated. Each fraction was again subjected to the above-described cell morphological change induction test, and fractions showing activity were collected.
さらにこの活性画分の HP LC分取 （カラム: PEGASIL 0DS、 （株） センシュ一 科学製、 溶出溶媒： 65%メタノール、 流速： 5ml/min. 検出： U.V.210nm ) を行 い約 lmlずつ分画した。 活性試験を行ったところリテンションタイム 56分に溶 出される画分に活性が確認された。 この画分を集め、 無色の粉末 （aglaiastatin B 5.2mg) を得た。 この画分はシリカゲル薄層クロマトグラフィーを行ったとこ ろ単一スポットであった。  Further, HP LC fractionation of this active fraction (column: PEGASIL 0DS, manufactured by Senshu Ichigaku Co., Ltd., elution solvent: 65% methanol, flow rate: 5 ml / min. Detection: UV210 nm) was performed, and fractions of about lml were performed. did. When an activity test was performed, the activity was confirmed in the fraction eluted at a retention time of 56 minutes. This fraction was collected to obtain a colorless powder (aglaiastatin B 5.2 mg). This fraction was a single spot when subjected to silica gel thin layer chromatography.
3. aglaiastatinBの物理化学的性質  3. Physicochemical properties of aglaiastatinB
無色粉末 （aglaiastatinB) の各種物理化学的性質を測定した。 本化合物のシ リカゲル薄層クロマトグラフィーにおける R f値は 0.43 (展開溶媒： クロ口ホル ム ： メタノール =20： 1 ) 、 0.46 (展開溶媒： トルエン ：アセトン = 1 ： 1 ) で あった。 融点は 142〜145てであった。 また各種スぺクトルの測定を行った。 結果を図 7から図 1 2に示す。 Various physicochemical properties of the colorless powder (aglaiastatinB) were measured. The R f values of this compound in silica gel thin-layer chromatography were 0.43 (developing solvent: pore-form: methanol = 20: 1) and 0.46 (developing solvent: toluene: acetone = 1: 1). The melting point was 142-145. Various spectra were also measured. The results are shown in FIGS. 7 to 12.
4 . aglaiastatinBの各種細胞に対する形態変化誘導効果  4. Induction effect of aglaiastatinB on morphological changes of various cells
a . 各種細胞の培養  a. Culture of various cells
K-ras t s -NRK細胞 （米国 N I H) が癌細胞の形態を示す 33°C及び正常細胞の 形態を示す 39てにおいて、 5 %仔牛血清 （Gibco BRL社製） を含むダルベッコ変 法イーグル培地 （日水製薬 （株） 製） にグルタミン 0. 6g/l 、 カナマイシン 0.2g/K NaHC03 2.25g/l、 を加えたものを培養液として、 5 % C02インキュベー 夕一中で培養した。 正常なラット腎細胞である NRK- 49細胞 （大日本製薬 （株))、 および癌細胞である K-ras-NIH3T3細胞、 H-ras_NIH3T3細胞、 N-ras- NIH3T3細胞 (東京大学医科学研究所） は、 37°Cで同様に培養した。 K-ras ts -NRK cells (US NIH) is the 39 hand showing the form of 33 ° C and normal cells showing the morphology of the cancer cells, 5% calf serum Dulbecco's modified Eagle medium containing (Gibco BRL) ( Nissui Pharmaceutical Co., Ltd.) to glutamine 0. 6 g / l, kanamycin 0.2g / K NaHC0 3 2.25g / l , as the culture liquid plus, were cultured in 5% C0 2 incubator evening one. Normal rat kidney cells, NRK-49 cells (Dainippon Pharmaceutical Co., Ltd.), and cancer cells, K-ras-NIH3T3 cells, H-ras_NIH3T3 cells, N-ras-NIH3T3 cells (The Institute of Medical Science, The University of Tokyo) ) Was similarly cultured at 37 ° C.
また、 細胞は、 カルシウム 'マグネシウム不含リン酸塩緩衝液 (PBS(-) :8. Og/1 NaCl， 2.31g/l Na2P04-12H20, 0.2g/l KH2P04 ； pH 7. 4) で洗浄した後、 トリ プシン- EDTA溶液 (0.5g/l trypsin, 8. Og/1 NaCl， 1. Og/1 glucose, 0. 02g/l Phenol red, 0.35g/l KH2P04, 0.2g/l EDTA)で培養フラスコより細胞をはがし、 新しレ、培養液を入れたフラスコ中に移すことにより継代した。  The cells were washed with a calcium-magnesium-free phosphate buffer (PBS (-): 8. Og / 1 NaCl, 2.31 g / l Na2P04-12H20, 0.2 g / l KH2P04; pH 7.4) After that, culture with trypsin-EDTA solution (0.5 g / l trypsin, 8. Og / 1 NaCl, 1. Og / 1 glucose, 0.02 g / l Phenol red, 0.35 g / l KH2P04, 0.2 g / l EDTA) The cells were detached from the flask, transferred to a flask containing a fresh culture medium, and transferred.
b . 形態変化誘導効果の観察 b. Observation of morphological change inducing effect
各細胞を、 4 8穴プラスチックプレート （コースター社製）に 1. 5 X 104 個 Z DMEM 0.5ml/well でまき、 5 % C02インキュベータ一中で 1日培養した。 翌日被 検物質を加え、 その後 3日目までの細胞の形態変化を観察した。 Each cell, 4 to 8-well plastic plates (Costar) were seeded at 1. 5 X 10 4 cells Z DMEM 0.5 ml / well, and cultured for one day in 5% C0 2 incubator scratch. The test substance was added the next day, and cell morphological changes were observed up to the third day.
i ) K-ras t s -NRK細胞に対する aglaiastatinBの効果 i) Effect of aglaiastatinB on K-ras ts- NRK cells
K-ras ' S-NRK細胞は、 33ででは細胞からいくつもの細い突起が見られ、 また細 胞同士が重なり合って癌細胞の形態を示し、 39°Cで同様に培養した場合は、 細胞 は平で突起も見られず重なり合うこともなく正常細胞の形態を示す。 K-ras' S- NRK cells show several small protrusions from the cells at 33, and the cells overlap to show the morphology of cancer cells. Shows a normal cell morphology without any overlap and no projection.
そこで、 33°Cで aglaiastatinB 10ng/mlを添加して 3日間培養した結果、 突起 もなく平で重なり合うこともない正常細胞の形態を示した。 また、 39°Cでは、 細 胞の形態に大きな変化はなかった。  Then, as a result of adding aglaiastatinB 10 ng / ml at 33 ° C and culturing for 3 days, it showed a normal cell morphology without protrusions and without overlapping. At 39 ° C, there was no significant change in cell morphology.
i i) K- ras-NRK細胞に対する aglaiastatinBの効果 i i) Effect of aglaiastatinB on K-ras-NRK cells
癌細胞である K-ras- NRK細胞は、 形が丸く盛り上がっているが、 これに aglai astatin B 20ng/mlを添加して 37°Cで 3日間培養した結果、 細胞の形態が平にな り、 接着面も増して正常細胞の形態を示した。  K-ras-NRK cells, which are cancer cells, have a rounded shape, but after adding aglai astatin B 20 ng / ml and culturing at 37 ° C for 3 days, the cell morphology becomes flat. However, the adhesion surface was increased to show normal cell morphology.
i i i ) K-ras-NIH3T3細胞に対する aglaiastatin Bの効果 iii) Effect of aglaiastatin B on K-ras-NIH3T3 cells
癌細胞である K-ras- NIH3T3細胞は、 細長い突起状の形態をしており、 さらに 細胞同士が重なり合つている。 これに aglaiastatin Bを 20ng/ml添加して 37°C で 3日間培養したところ、 平になり接着面も増大し、 正常細胞である NIH3T3細胞 の形態になつているのが観察された。  K-ras-NIH3T3 cells, which are cancer cells, have an elongated protruding morphology, and the cells overlap each other. When 20 ng / ml of aglaiastatin B was added thereto and cultured at 37 ° C for 3 days, it was observed that the cells became flat, the adhesion surface was increased, and the cells were in the form of normal NIH3T3 cells.
5 . aglaiastatin Bの各種钿胞に対する細胞増殖抑制効果 5. Cellular growth inhibitory effect of aglaiastatin B on various cells
各細胞を、 4 8穴プラスチックプレート （コースター社製） に 1.0 X 104 個： DMEM 0.5ml /we 11 でまき、 1日適温 5 %C02 インキュベータ一中で培養した。 翌 日 aglaiastatinBを加え更に 3日間培養し、 PBS (-)で 2回洗った後、 トリプシン を 100 1/wellずつ加え、 数分 0 % C02条件下の恒温機中で細胞をはがし、 DMEM を 900 〃l/wellずつ加えて細胞が均一に分散するようにした。 この溶液の細胞濃 度をコール夕一カウンター （ZM型） を用いて測定し、 細胞増殖を 50%阻害する 濃度（ I C 50) を求めた。 結果は表 2に示した通りであった。 なお、 表 2に示し た細胞名のうち ras を含むものは腫瘍細胞である。 〔表 2〕 aglaiastatinBの各種細胞に対する増殖抑制効果 細胞名 I C50 (ng/ml ) Each cell was seeded on a 48-well plastic plate (manufactured by Coaster) at 1.0 × 10 4 cells: DMEM 0.5 ml / we 11 and cultured in a 5% CO 2 incubator at an appropriate temperature for 1 day. Next day was added and incubated an additional 3 days aglaiastatinB, PBS (-) was washed twice with added trypsin by 100 1 / well, the cells are detached in minutes 0% C0 2 under the conditions of the thermostat, the DMEM The cells were added at 900 μl / well so that the cells were uniformly dispersed. The cell concentration of this solution was measured using a Kohl-Yuichi counter (ZM type) to determine the concentration (IC 50 ) that inhibited cell growth by 50%. The results were as shown in Table 2. Among the cell names shown in Table 2, those containing ras are tumor cells. [Table 2] Growth inhibitory effect of aglaiastatinB on various cells Cell name IC 50 (ng / ml)
K-ras-NRK 8.0  K-ras-NRK 8.0
K-ras-画 3T3 8.9  K-ras-drawing 3T3 8.9
H-ras-NIH3T3 7.8  H-ras-NIH3T3 7.8
N-ras-NIH3T3 8.0  N-ras-NIH3T3 8.0
NRK- 49 9.8  NRK- 49 9.8
K-ras ts -匪 （33°C) 5.1 K-ras ts -Marauder (33 ° C) 5.1
K-ras ts -NRK (39°C) 3.6 K-ras ts -NRK (39 ° C) 3.6
6. 急性毒性  6. Acute toxicity
aglaias tinBの急性毒性は、 ICR系雄性マウス （5週令） に対し、 LD50は 300mg/kg以上であり、 急性毒性が低レ、こと力確認された。 The acute toxicity of aglaias tinB, compared ICR male mice (5 weeks old), LD 50 is at 300mg / kg or more, and acute toxicity is low record, it has been confirmed this and force.
I I I. aglaiastatinC I I I. aglaiastatinC
1. アグラィァ 'ォドラー夕からの活性物質の抽出  1. Extraction of Active Substances from Agraya Oddler Evening
60°Cのオーブンで乾燥したアグラィァ ·ォドラ一夕の葉 167gを粉砕した後、 2 Lの三角フラスコに入れ、 クロ口ホルム 1000ml を加えて室温で 5時間撹拌し た。 溶媒を新しくして同様の操作を繰り返し、 合計 3回の抽出を行った。 3回分 の抽出液を合わせて減圧下でクロ口ホルムを留去し、 暗緑色シラップ 5.7 gを得 た。 このシラップを次の精製の原料とした。  After 167 g of Agrilla odora leaves dried in a 60 ° C. oven were crushed, the mixture was placed in a 2 L Erlenmeyer flask, added with 1000 ml of black-mouthed form, and stirred at room temperature for 5 hours. The same operation was repeated with a new solvent, and a total of three extractions were performed. The extracts from the three extractions were combined, and the chloroform was removed under reduced pressure to obtain 5.7 g of dark green syrup. This syrup was used as a raw material for the next purification.
2. 抽出物からの活性物質の精製 クロ口ホルム抽出物 3. 9gを少量のトルエンに溶かし、 カラムに充填した 120gの シリカゲル （Merck si l icagel 60, メルク社製） の上に添加し、 トルエン：ァセ トン =20： 1 , 16： 1 , 6 ： 1 , 3 ： 1 , 2 ： 1， 1 : 1の溶媒で順次溶出し、 約 20mlずつ分画した。 各画分を後述の 「4 . aglaiastatinCの各種細胞に対する 形態変化誘導効果」 で用いた細胞形態変化誘導試験に付したところ、 トルエン ： アセトン = 3 ： 1から 1 : 1溶出画分に形態正常化作用が認められた。 活性画分 を集めたところ重量は 639mgであった。 2. Purification of the active substance from the extract Dissolve 3.9 g of black-mouthed form extract in a small amount of toluene, and add it to 120 g of silica gel (Merck silica gel 60, manufactured by Merck) packed in a column. Toluene: acetone = 20: 1, 16 : 1,6: 1,3: 1,2: 1,1: 1 eluted sequentially and fractionated about 20 ml each. Each fraction was subjected to the cell morphological change induction test used in “4. Effect of aglaiastatin C on morphological change induction on various cells” described below. Toluene: acetone = 3: 1 to 1: 1 morphological normalization An effect was observed. The active fraction was collected and weighed 639 mg.
この活性画分を、 メタノールで平衡化したトヨパール HW-40で約 2 mlずつ分画 し、 さらにカラムに充塡した Merck si licagel 60 (silanised) に添加し 70 % メタノ一ルで溶出し約 4 mlずつ分画した。 各画分を先の細胞形態変化誘導試験に 再び付し、 活性が認められた画分を集めた。  Approximately 2 ml of this active fraction was fractionated with Toyopearl HW-40 equilibrated with methanol, further added to Merck silicagel 60 (silanised) packed in a column, and eluted with 70% methanol to elute about 4 ml. It was fractionated by ml. Each fraction was again subjected to the above-described cell morphological change induction test, and fractions showing activity were collected.
さらにこの活性画分を H P L C分取 （カラム: PEGASIL 0DS、 （株） センシユー 科学製、 溶出溶媒： 65%メタノール、 流速： 5 ml/min、 検出： U. V.210隱 ) を行 レ、約 1 mlずつ分画した。 活性試験を行つたところリテンションタイム 42分に溶 出される画分に活性が確認された。 この画分を集め、 無色の粉末 （aglaiastatin C 2. lmg) を得た。 この画分はシリカゲル薄層クロマトグラフィーを行ったとこ ろ単一スポットであった。  Further, the active fraction was subjected to HPLC fractionation (column: PEGASIL 0DS, manufactured by Senshi Kagaku Co., Ltd., elution solvent: 65% methanol, flow rate: 5 ml / min, detection: UV210 hidden), and about 1 ml each. Fractionated. When an activity test was performed, the activity was confirmed in the fraction eluted at a retention time of 42 minutes. This fraction was collected to obtain a colorless powder (aglaiastatin C 2. lmg). This fraction was a single spot when subjected to silica gel thin layer chromatography.
3. aglaiastatinCの物理化学的性質 3. Physicochemical properties of aglaiastatinC
無色粉末 （aglaiastatinC) の各種物理化学的性質を測定した。 本化合物のシ リカゲル薄層クロマトグラフィーにおける R f 値は 0.42 (展開溶媒： クロ口ホル ム ： メタノール =20： 1 ) 、 0.49 (展開溶媒： トルエン ：アセトン = 1 ： 1 ) で あった。 融点は 157〜160 てであった。 また各種スぺクトルの測定を行った。 結果を図 1 3から図 1 8に示す。 Various physicochemical properties of the colorless powder (aglaiastatinC) were measured. The R f values of this compound in silica gel thin-layer chromatography were 0.42 (developing solvent: pore form: methanol = 20: 1) and 0.49 (developing solvent: toluene: acetone = 1: 1). The melting point was 157-160. Various spectra were also measured. The results are shown in FIGS. 13 to 18.
4. aglaiastatinCの各種細胞に対する形態変化誘導効果  4. Effect of aglaiastatin C on morphological changes of various cells
a. 各種細胞の培養  a. Culture of various cells
K-ras ts -NRK細胞 （米国 N I H) が癌細胞の形態を示す 33°C及び正常細胞の 形態を示す 39°Cにおいて、 5%仔牛血清 （Gibco Laboratories社製） を含むダル べッコ変法イーグル培地 （日水製薬 （株） 製） にグルタミン 0.6g/l 、 カナマイ シン 0.2g/l、 NaHC03 2.25g/K を加えたものを培養液として、 5 % C02イン キュベータ一中で培養した。 正常なラット腎細胞である NRK- 49細胞 （大日本製 薬 （株)）、 およびマウス N I H3T3細胞 （JCRS細胞バンク） ならびに癌細 胞である K-ras-NRK細胞 （昭和薬科大学） 、 K_ras-NIH3T3細胞、 H-ras- NIH3T3細 胞、 N-ras-N〖H3T3細胞 （東京大学医科学研究所） は、 37°Cで同様に培養した。 また、 細胞は、 カルシウム ·マグネシウム不含リン酸塩緩衝液 （PBS (-)： NaCl 8. Og/1 ， Na2P04-12H20 2.31g/l ， KH2P04 0.2g/l； pH 7.4) で洗浄した後、 トリプシン- EDTA溶液（trypsin 0.5g/l, NaCl 8. Og/1, glucose 1.0g/l, phenol red 0.02g/L KH2P04 0.35g/L EDTA 0.2g/l) で培養フラスコより細胞を はがし、 新しい培養液を入れたフラスコ中に移すことにより継代した。 In K-ras ts -NRK cells (US NIH) is 39 ° C showing a 33 ° configuration of the C and normal cells showing the morphology of the cancer cells, varying Dal Beck co containing 5% calf serum (Gibco Laboratories, Inc.) law Eagle medium (Nissui Pharmaceutical Co., Ltd.) to glutamine 0.6 g / l, as the culture liquid plus kanamycin 0.2g / l, NaHC0 3 2.25g / K, in 5% C0 2 in Kyubeta one Cultured. Normal rat kidney cells, NRK-49 cells (Dainippon Pharmaceutical Co., Ltd.), mouse NI H3T3 cells (JCRS cell bank), and cancer cells, K-ras-NRK cells (Showa Pharmaceutical University), K_ras -NIH3T3 cells, H-ras-NIH3T3 cells, and N-ras-N 〖H3T3 cells (Institute of Medical Science, The University of Tokyo) were similarly cultured at 37 ° C. Also, cells are calcium-magnesium-free phosphate buffer (PBS (-): NaCl 8. Og / 1, Na 2 P0 4 -12H 2 0 2.31g / l, KH 2 P0 4 0.2g / l; After washing with pH 7.4), trypsin-EDTA solution (trypsin 0.5g / l, NaCl 8.Og / 1, glucose 1.0g / l, phenol red 0.02g / L KH 2 P0 4 0.35g / L EDTA 0.2g / The cells were detached from the culture flask in l) and subcultured by transferring to a flask containing a new culture solution.
b. 形態変化誘導効果の観察 b. Observation of morphological change-inducing effect
各細胞を、 48穴プラスチックプレート （コースター社製）に 1.0X104 個/ DMEM 0.5ml /we 11 でまき、 5% C02インキュベータ一中で 1日培養した。 翌日被 検物質を加え、 その後 3日目までの細胞の形態変化を観察した。 Each cell was spread on a 48-well plastic plate (manufactured by Coaster) at 1.0 × 10 4 cells / DMEM 0.5 ml / we 11 and cultured in a 5% CO 2 incubator for 1 day. The test substance was added the next day, and cell morphological changes were observed up to the third day.
i ) K-ras ,s -NRK細胞に対する aglaias tinCの効果 i) Effect of aglaias tinC on K-ras , s- NRK cells
K-ras ts-NRK細胞は、 33°Cでは細胞からいくつもの細い突起が見られ、 また細 胞同士が重なり合つて癌細胞の形態を示し、 39°Cでは細胞は平で突起も見られず 重なり合うこともなく正常細胞の形態を示す。 At 33 ° C, K-ras ts- NRK cells show several small protrusions The vesicles overlap and show the morphology of cancer cells. At 39 ° C, the cells are flat with no protrusions and no overlap, showing the morphology of normal cells.
そこで、 33°Cで aglaiastatinC 10ng/mlを添加して 3日間培養した結果、 突起 もなく平で重なり合うこともない正常細胞の形態を示した。 また、 39°Cでは、 細 胞の形態に大きな変化はなかった。  Therefore, as a result of adding aglaiastatin C 10 ng / ml at 33 ° C and culturing for 3 days, it showed a normal cell morphology without protrusions and without overlapping. At 39 ° C, there was no significant change in cell morphology.
i i) K- ras-NRK細胞に対する aglaias tinCの効果 i i) Effect of aglaias tinC on K-ras-NRK cells
癌細胞である K-ras-NRK細胞は、 形が丸く盛り上がっているが、 これに aglai astatin C 20ng/mlを添加して 37°Cで 3日間培養した結果、 細胞の形態が平にな り、 接着面も増して正常細胞の形態を示した。  K-ras-NRK cells, which are cancer cells, have a rounded shape, but after adding aglai astatin C 20 ng / ml and culturing at 37 ° C for 3 days, the cell morphology becomes flat. However, the adhesion surface was increased to show normal cell morphology.
i i i ) K-ras-隱 3T3細胞に対する aglaiastatin Cの効果 iii) Effect of aglaiastatin C on K-ras-Oki 3T3 cells
癌細胞である K-ras- NIH3T3細胞は、 細長い突起状の形態をしており、 さらに 細脗同士が重なり合つている。 これに aglaias tin Cを 20ng/ml添加して 37°C で 3日間培養したところ、 平になり接着面も増大し、 正常細胞である NIH3T3細胞 の形態になつているのが観察された。  K-ras-NIH3T3 cells, which are cancer cells, have an elongated protruding morphology, and the cells are overlapped. When 20 ng / ml of aglaias tin C was added thereto and cultured at 37 ° C for 3 days, it was observed that the cells became flat, the adhesion surface was increased, and the cells were in the form of normal NIH3T3 cells.
5 . aglaiastatinCの各種細胞に対する細胞増殖抑制効果  5. Cell growth inhibitory effect of aglaiastatinC on various cells
各細胞を、 4 8穴プラスチックプレート （コースター社製） に 1. 0 X 104 個ノ DMEM 0.5ml/well でまき、 1日適温 5 %C02 インキュベータ一中で培養した。 翌 日 aglaiastatinCを加え更に 3日間培養し、 PBS (-)で 2回洗った後、 トリプシン を 100 1/wd lずつ加え、 数分 0 % C02条件下の恒温機中で細胞をはがし、 DMEM を 900 1/wellずつ加えて細胞が均一に分散するようにした。 この溶液の細胞濃 度をコール夕一カウンター （ZM型） を用いて測定し、 細胞増殖を 50%阻害する 濃度（I C 50) を求めた。 結果は表 3に示した通りであり、 癌細胞に対する agla iastatinCの I Cs。は 1. 0〜5· 6 n gZm 1である。 なお、 表 3に示した細 胞名のうち r a sを含むものは腫瘍細胞である。 Each cell was spread on a 48-well plastic plate (manufactured by Coaster) with 1.0 × 10 4 DMEM at 0.5 ml / well, and cultured in a 5% CO 2 incubator at an appropriate temperature for 1 day. And further cultured for 3 days plus next day aglaiastatinC, PBS (-) was washed twice with added trypsin by 100 1 / wd l, peel the cells in a few minutes 0% C0 2 under the conditions of the thermostat, DMEM Was added at 900 1 / well so that the cells were uniformly dispersed. The cell concentration of this solution was measured using a Kohl-Izu counter (ZM type), and the concentration (IC 50 ) that inhibited cell growth by 50% was determined. The results are shown in Table 3. iastatinC IC s . Is from 1.0 to 5.6 n gZm 1. Among the cell names shown in Table 3, those containing ras are tumor cells.
〔表 3〕 aglaiastatinCの各種細胞に対する増殖抑制効果 細胞名 I Cso (ng/ml )  [Table 3] Growth inhibitory effect of aglaiastatinC on various cells Cell name I Cso (ng / ml)
K-ras-NRK 2.8  K-ras-NRK 2.8
K-ras-麵 T3 5.6  K-ras- 麵 T3 5.6
H-ras-NIH3T3 1.0  H-ras-NIH3T3 1.0
N-ras-NIH3T3 1.8  N-ras-NIH3T3 1.8
NRK - 49 2.1  NRK-49 2.1
NIH3T3 4.1  NIH3T3 4.1
6. 急性毒性  6. Acute toxicity
aglaiastatinCの急性毒性は、 ICR系雄性マウス （5週令） に対し、 LD50は 300mg'/kg以上であり、 急性毒性が低いことが確認された。 産業上の利用の可能性 Acute toxicity of aglaiastatinC, compared ICR male mice (5 weeks old), LD 50 is at 300 mg '/ kg or more, acute toxicity is low was confirmed. Industrial applicability
化学式 （ 1 ) 、 （2) 、 および （3) で示される熱帯植物アグラィァ . ォドラ一タから単離された本発明に係る化合物は、 癌細胞の増殖を阻止しその形 態を正常化させる活性を有し、 癌遺伝子機能抑制剤または抗癌剤として有効に使 用できる。  The compound of the present invention isolated from the tropical plant agrobacterium represented by the chemical formulas (1), (2) and (3) has an activity of inhibiting the growth of cancer cells and normalizing the form thereof. It can be effectively used as an oncogene function inhibitor or an anticancer agent.

Claims

請求の範囲 The scope of the claims
( 1 ) 化学式 ( 1 ) で表される化合物アグラィアスタチン (aglaiastatin) Ac (1) The compound represented by the chemical formula (1), aglaiastatin A c
Figure imgf000029_0001
Figure imgf000029_0001
(2) 化学式 (1) で表される化合物アグラィアスタチン （aglaiastatin) A を有効成分として含有する癌遺伝子機能抑制剤。 (2) An oncogene function inhibitor comprising, as an active ingredient, the compound aglaiastatin A represented by the chemical formula (1).
(3) 化学式 （2)で表される化合物アグラィアスタチン（aglaiastatin)B。 (3) Compound aglaiastatin B represented by chemical formula (2).
Figure imgf000030_0001
Figure imgf000030_0001
(4) 化学式 （2)で表される化合物アグラィアスタチン (aglaiastatin)Bを 有効成分として含有する癌遺伝子機能抑制剤。 (4) An oncogene function inhibitor comprising, as an active ingredient, the compound aglaiastatin B represented by the chemical formula (2).
(5) 化学式 (3)で表される化合物アグラィアスタチン (aglaiastatin)C。  (5) Compound aglaiastatin C represented by chemical formula (3).
Figure imgf000030_0002
Figure imgf000030_0002
(6) 化学式 (3)で表される化合物アグラィアスタチン (aglaiastatin)Cを 有効成分として含有する癌遺伝子機能抑制剤。 (6) An oncogene function inhibitor comprising, as an active ingredient, the compound aglaiastatin C represented by the chemical formula (3).
(7) 化学式 (1)で表される化合物アグラィアスタチン (aglaiastatin)A、 化学式 （2)で表される化合物アグラィアスタチン （aglaiastatin) B、 お よび化学式 (3)で表される化合物アグラィアスタチン (aglaiastatin)Cか らなる群から選ばれる少なくとも 1つの化合物を有効成分として含有する抗 癌剤 o  (7) The compound aglaiastatin A represented by the chemical formula (1), the compound aglaiastatin B represented by the chemical formula (2), and the compound aglaiastatin B represented by the chemical formula (3) An anti-cancer agent containing at least one compound selected from the group consisting of astatin (aglaiastatin) C as an active ingredient o
PCT/JP1996/002411 1995-08-28 1996-08-28 Oncogene function depressant WO1997008161A1 (en)

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JP7/219288 1995-08-28
JP7/219289 1995-08-28
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