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WO1997008161A1 - Agent deprimant une fonction oncogenique - Google Patents

Agent deprimant une fonction oncogenique 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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WIPO (PCT)
Prior art keywords
cells
ras
aglaiastatin
cell
solvent
Prior art date
Application number
PCT/JP1996/002411
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English (en)
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/ja
Priority claimed from JP7219289A external-priority patent/JPH0967360A/ja
Priority claimed from JP7228355A external-priority patent/JPH0967376A/ja
Application filed by Terumo Kabushiki Kaisha filed Critical Terumo Kabushiki Kaisha
Publication of WO1997008161A1 publication Critical patent/WO1997008161A1/fr

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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

L'invention, qui porte sur de nouveaux composés à activité déprimant des fonctions oncogéniques, des aglaiastatines A (représentées par la formule chimique (1), B et C, isolées à partir d'Aglaia odorata (Lour), concerne également des agents déprimants de fonctions oncogéniques et des médicaments anticancéreux contenant ces composés comme ingrédient actif.
PCT/JP1996/002411 1995-08-28 1996-08-28 Agent deprimant une fonction oncogenique WO1997008161A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
JP7/219289 1995-08-28
JP7219288A JPH0967375A (ja) 1995-08-28 1995-08-28 癌遺伝子機能抑制剤
JP7219289A JPH0967360A (ja) 1995-08-28 1995-08-28 癌遺伝子機能抑制剤
JP7/219288 1995-08-28
JP7/228355 1995-09-05
JP7228355A JPH0967376A (ja) 1995-09-05 1995-09-05 癌遺伝子機能抑制剤

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WO2000007579A2 (fr) * 1998-08-05 2000-02-17 Bayer Aktiengesellschaft UTILISATION DE DERIVES DE CYCLOPENTABENZOFURANNE POUR LUTTER CONTRE DES MALADIES DEPENDANT DU NF-λB
WO2000008007A2 (fr) * 1998-08-05 2000-02-17 Bayer Aktiengesellschaft Derives de cyclopentabenzofuranne et leur utilisation
US6710075B2 (en) 2000-07-05 2004-03-23 The Government Of The State Of Sarawak, Malaysia Therapeutic compounds and methods
US9387192B2 (en) 2008-11-20 2016-07-12 Dkfz Deutchers Krebsforschungszentrum Combination of rocaglamide and apoptosis inducing substances for the treatment of cancer

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000007579A2 (fr) * 1998-08-05 2000-02-17 Bayer Aktiengesellschaft UTILISATION DE DERIVES DE CYCLOPENTABENZOFURANNE POUR LUTTER CONTRE DES MALADIES DEPENDANT DU NF-λB
WO2000008007A2 (fr) * 1998-08-05 2000-02-17 Bayer Aktiengesellschaft Derives de cyclopentabenzofuranne et leur utilisation
WO2000008007A3 (fr) * 1998-08-05 2000-05-11 Bayer Ag Derives de cyclopentabenzofuranne et leur utilisation
WO2000007579A3 (fr) * 1998-08-05 2000-09-08 Bayer Ag UTILISATION DE DERIVES DE CYCLOPENTABENZOFURANNE POUR LUTTER CONTRE DES MALADIES DEPENDANT DU NF-λB
US6420393B1 (en) * 1998-08-05 2002-07-16 Bayer Aktiengesellschaft Cyclopentabenzofuran derivatives and their use
US6518274B1 (en) 1998-08-05 2003-02-11 Bayer Aktiengesellschaft Use of cyclopentabenzofuran-derivatives for combating (NF-κB)-dependent diseases
US6943182B2 (en) 1998-08-05 2005-09-13 Bayer Aktiengesellschaft Cyclopentabenzofuran derivatives and their use
US6710075B2 (en) 2000-07-05 2004-03-23 The Government Of The State Of Sarawak, Malaysia Therapeutic compounds and methods
US9387192B2 (en) 2008-11-20 2016-07-12 Dkfz Deutchers Krebsforschungszentrum Combination of rocaglamide and apoptosis inducing substances for the treatment of cancer
EP2364148B1 (fr) * 2008-11-20 2018-07-18 DKFZ Deutsches Krebsforschungszentrum Combinaison de rocaglamide et de substances induisant l'apoptose pour le traitement d'un cancer

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