JPH04275284A - Antitumor substance be-23372m and its production - Google Patents
Antitumor substance be-23372m and its productionInfo
- Publication number
- JPH04275284A JPH04275284A JP6260791A JP6260791A JPH04275284A JP H04275284 A JPH04275284 A JP H04275284A JP 6260791 A JP6260791 A JP 6260791A JP 6260791 A JP6260791 A JP 6260791A JP H04275284 A JPH04275284 A JP H04275284A
- Authority
- JP
- Japan
- Prior art keywords
- culture
- culturing
- formula
- strain
- source
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
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- 230000000259 anti-tumor effect Effects 0.000 title claims description 11
- 238000004519 manufacturing process Methods 0.000 title claims description 10
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- 239000000463 material Substances 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- HAWPXGHAZFHHAD-UHFFFAOYSA-N mechlorethamine Chemical class ClCCN(C)CCCl HAWPXGHAZFHHAD-UHFFFAOYSA-N 0.000 description 1
- 229960004961 mechlorethamine Drugs 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 235000010981 methylcellulose Nutrition 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- GOQYKNQRPGWPLP-UHFFFAOYSA-N n-heptadecyl alcohol Natural products CCCCCCCCCCCCCCCCCO GOQYKNQRPGWPLP-UHFFFAOYSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 238000004810 partition chromatography Methods 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229920001515 polyalkylene glycol Polymers 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 239000012286 potassium permanganate Substances 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 150000003377 silicon compounds Chemical class 0.000 description 1
- 201000010153 skin papilloma Diseases 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 235000010339 sodium tetraborate Nutrition 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
Abstract
Description
【０００１】0001
【産業上の利用分野】本発明は医薬の分野で有用であり
、さらに詳細には腫瘍細胞の増殖を阻害し、制癌効果を
発揮する新規化合物、その製法及びその用途並びに該新
規物質を産生する新規な微生物に関するものである。[Industrial Application Field] The present invention is useful in the field of medicine, and more specifically, a novel compound that inhibits the proliferation of tumor cells and exhibits an anticancer effect, its production method, its use, and the production of the new substance. It is related to a new microorganism.
【０００２】0002
【従来の技術】癌化学療法の分野においては、ブレオマ
イシン（Ｂｌｅｏｍｙｃｉｎ）及びアドリアマイシン（
Ａｄｒｉａｍｙｃｉｎ）等の多くの微生物代謝産物を臨
床的に応用することが試みられ、またこれらは実際に臨
床において使用されている。しかしながら、様々な種類
の腫瘍に対してその効果は必ずしも充分ではなく、より
優れた新規な抗腫瘍性物質に関しては不断の希求がある
のが現状である。BACKGROUND OF THE INVENTION In the field of cancer chemotherapy, bleomycin and adriamycin (
Attempts have been made to clinically apply many microbial metabolites such as Adriamycin, and these are actually used clinically. However, their effects on various types of tumors are not necessarily sufficient, and there is a constant desire for new and better antitumor substances.
【０００３】0003
【発明が解決しようとする課題】このような現状に鑑み
、本発明者らは広く微生物代謝産物をスクリーニングす
ることにより、優れた新規抗腫瘍性物質を見出すことを
課題とする。[Problems to be Solved by the Invention] In view of the current situation, the present inventors have set themselves the task of discovering excellent new antitumor substances by screening a wide range of microbial metabolites.
【０００４】0004
【課題を解決するための手段】本発明者らは、ヒト癌細
胞に対する増殖阻止作用を指標に、広く微生物代謝産物
をスクリーニングすることにより、埼玉県上尾市の銀杏
の樹皮より分離した真菌Ｆ−２３３７２株が、強い阻害
活性を有する物質を産生していることを発見し、この物
質を抽出精製、単離し、構造決定を行った結果、後記式
（１）で表される新規な化合物が優れた抗腫瘍活性を有
することを明らかにし、本発明を完成した。[Means for Solving the Problems] The present inventors screened a wide range of microbial metabolites using their growth-inhibiting effect on human cancer cells as an indicator, and discovered that the fungus F- It was discovered that the 23372 strain produced a substance with strong inhibitory activity, and as a result of extracting and purifying this substance, isolating it, and determining its structure, the novel compound represented by the following formula (1) was found to be superior. The present invention has been completed based on the results of the present invention.
【０００５】即ち、本発明は、式That is, the present invention provides the formula
【０００６】[0006]
【化４】
で表される抗腫瘍性物質ＢＥ−２３３７２Ｍ、その製造
法及びその抗腫瘍剤としての用途並びにＢＥ−２３３７
２Ｍを産生する新規微生物に関するものである。Antitumor substance BE-23372M represented by [Chemical formula 4], its production method, its use as an antitumor agent, and BE-2337
This invention relates to a new microorganism that produces 2M.
【０００７】本発明に係わる化合物について、その理化
学的性状を以下に示す。
ＢＥ−２３３７２Ｍの理化学的性状
性状：赤橙色固体
分子式：Ｃ１７Ｈ１２Ｏ６
マススペクトル：ＨＲＦＡＢ−ＭＳ（ｍ／ｚ）；３１２
．０６２５［Ｍ］＋
ＵＶスペクトル：λｍａｘ（メタノール）ｎｍ（ε）；
２６６（８，８００），４２６（２０，４００）ＩＲス
ペクトル：ν（ＫＢｒ，ｃｍ−１）；３２９８，１７５
２，１６０５，１５１８，１４５８，１３４４，１３０
２，１２６３，１１７３，１１２２，１０６５，９４２
１Ｈ−ＮＭＲスペクトル：（３００ＭＨｚ，（ＣＤ３）
２ＣＯ，δｐｐｍ）；６．９３（１Ｈ，ｄ，Ｊ＝８．３
Ｈｚ），６．９４（１Ｈ，ｄ，Ｊ＝８．３Ｈｚ），７．
１０（１Ｈ，ｓ），７．１５（１Ｈ，ｓ），７．２４（
１Ｈ，ｄｄ，Ｊ＝８．３，２．１Ｈｚ）７．２６（１Ｈ
，ｄｄ，Ｊ＝８．３，２．１Ｈｚ），７．３２（１Ｈ，
ｄ，Ｊ＝２．１Ｈｚ），７．３３（１Ｈ，ｄ，Ｊ＝２．
１Ｈｚ），８．５（２Ｈ，ｂｒ ｓ）１３Ｃ−ＮＭＲ
スペクトル：（１００ＭＨｚ，（ＣＤ３）２ＣＯ，δｐ
ｐｍ）；９９．０，１１２．９，１１６．５，１１６．
７，１１７．５，１１８．８，１２１．５，１２３．３
，１２４．９，１２８．６，１３４．５，１４６．３×
２，１４８．５，１４８．７，１５６．７，１７０．２
溶解性：ジメチルスルホキシドに易溶、アセトン、メタ
ノールに可溶で、クロロホルム、酢酸エチルに難溶、ｎ
−ヘキサンに不溶
酸性、中性、塩基性物質の区別：酸性物質Ｒｆ値：０．
２７［メルク社製、キーゼルゲル６０Ｆ２５４使用、展
開溶媒；クロロホルム／メタノール／ギ酸（２０：２：
１）］
呈色反応：塩化第二鉄反応
陽性過マンガン酸カリウム反応 陽性
培養癌細胞に対する作用
ＢＥ−２３３７２Ｍのヒト癌細胞に対する増殖阻止作用
を決定するため、ｉｎｖｉｔｒｏで試験を行った。細胞
は、ヒト類表皮癌細胞Ａ４３１及びヒト胃癌細胞ＭＫＮ
７を使用した。細胞培養用培地は、Ａ４３１には牛胎児
血清１０％含有ＤＭＥＭ培地を、ＭＫＮ７には牛胎児血
清１０％含有ＲＰＭＩ１６４０培地を用いた。癌細胞増
殖阻害の検定は、２×１０３個の癌細胞を含む細胞培養
用培地２００μｌを９６穴のマイクロプレートに分注し
、３７℃で２４時間、５％ＣＯ２下で培養したのち、Ｂ
Ｅ−２３３７２Ｍを加え、３７℃で更に７２時間、５％
ＣＯ２下で培養したのち、細胞を５０％ＴＣＡで固定し
、０．４％スルホローダミンＢで染色した。染色された
細胞から１０ｍＭトリス液を用いて色素を抽出し、５４
０ｎｍにおける吸光度を測定して対照群と比較した。
その結果、ＢＥ−２３３７２ＭはＡ４３１に対して２．
６μｇ／ｍｌのＩＣ５０を示し、ＭＫＮ７に対して７．
６μｇ／ｍｌのＩＣ５０を示した。The physicochemical properties of the compound according to the present invention are shown below. Physical and chemical properties of BE-23372M Properties: Red-orange solid Molecular formula: C17H12O6 Mass spectrum: HRFAB-MS (m/z); 312
．． 0625 [M] + UV spectrum: λmax (methanol) nm (ε);
266 (8,800), 426 (20,400) IR spectrum: ν (KBr, cm-1); 3298,175
2,1605,1518,1458,1344,130
2,1263,1173,1122,1065,942 1H-NMR spectrum: (300MHz, (CD3)
2CO, δppm); 6.93 (1H, d, J = 8.3
Hz), 6.94 (1H, d, J=8.3Hz), 7.
10 (1H, s), 7.15 (1H, s), 7.24 (
1H, dd, J = 8.3, 2.1Hz) 7.26 (1H
, dd, J=8.3, 2.1Hz), 7.32 (1H,
d, J=2.1Hz), 7.33(1H, d, J=2.
1Hz), 8.5 (2H, br s) 13C-NMR
Spectrum: (100MHz, (CD3)2CO, δp
pm); 99.0, 112.9, 116.5, 116.
7,117.5,118.8,121.5,123.3
,124.9,128.6,134.5,146.3×
2,148.5,148.7,156.7,170.2 Solubility: Easily soluble in dimethyl sulfoxide, soluble in acetone and methanol, slightly soluble in chloroform and ethyl acetate, n
- Distinction between acidic, neutral and basic substances insoluble in hexane: Acidic substance Rf value: 0.
27 [manufactured by Merck, using Kieselgel 60F254, developing solvent: chloroform/methanol/formic acid (20:2:
1)] Color reaction: ferric chloride reaction
Positive Potassium Permanganate Reaction Positive Effect on Cultured Cancer Cells In order to determine the growth inhibiting effect of BE-23372M on human cancer cells, an in vitro test was conducted. The cells are human epidermoid carcinoma cell A431 and human gastric cancer cell MKN.
7 was used. As the cell culture medium, DMEM medium containing 10% fetal bovine serum was used for A431, and RPMI1640 medium containing 10% fetal bovine serum was used for MKN7. For assay of cancer cell growth inhibition, 200 μl of cell culture medium containing 2×103 cancer cells was dispensed into a 96-well microplate, cultured at 37°C for 24 hours under 5% CO2, and then B.
Add E-23372M and 5% at 37°C for an additional 72 hours.
After culturing under CO2, cells were fixed with 50% TCA and stained with 0.4% sulforhodamine B. The dye was extracted from the stained cells using 10mM Tris solution, and 54
The absorbance at 0 nm was measured and compared with the control group. As a result, BE-23372M was 2.2% higher than A431.
It showed an IC50 of 6 μg/ml and 7.7 μg/ml against MKN7.
It showed an IC50 of 6 μg/ml.
【０００８】次に、本発明化合物であるＢＥ−２３３７
２Ｍの製造法について説明する。Next, BE-2337, which is a compound of the present invention,
The manufacturing method of 2M will be explained.
【０００９】本発明の抗腫瘍性物質ＢＥ−２３３７２Ｍ
の製造に使用する微生物又はその変異株は、抗腫瘍性物
質ＢＥ−２３３７２Ｍを産生するものならばいずれでも
良いが、例えば以下の菌学的性状を有する微生物Ｆ−２
３３７２株を挙げることができる。
１．形態
Ｆ−２３３７２株のコロニーは菌糸のみで、分生子・菌
核他の耐久細胞や耐久組織は形成されない。菌糸はこげ
茶ないし淡茶色で、その直径は７．２〜８．５μｍで、
隔壁が多い。菌糸表面は滑面ないしやや粗面であり、不
規則ないぼ状突起を持つ菌糸も混在する。
２．培養性状
各種寒天培地を用い、２８℃で７日間培養した結果を以
下の表に示す。表中（）内はメツエン・ハンドブック・
オブ・カラー［（Ｍｅｔｈｕｅｎ Ｈａｎｄｂｏｏｋ
ｏｆ Ｃｏｌｏｕｒ）第３版、１９８４年］によ
る色名の記載である。Antitumor substance BE-23372M of the present invention
The microorganism or its mutant strain used in the production of the microorganism may be any one that produces the antitumor substance BE-23372M, but for example, microorganism F-2 having the following mycological properties may be used.
There are 3372 stocks. 1. Colonies of form F-23372 strain are only hyphae, and durable cells such as conidia and sclerotia and durable tissues are not formed. The hyphae are dark brown or light brown, with a diameter of 7.2 to 8.5 μm.
There are many bulkheads. The hyphae surface is smooth or slightly rough, and hyphae with irregular wart-like protrusions are also present. 2. Culture properties The results of culturing at 28° C. for 7 days using various agar media are shown in the table below. The information in parentheses in the table is the Metzen Handbook.
Of Color [(Methuen Handbook
of Color) 3rd edition, 1984].
【００１０】0010
【表１】
上記の表でコーンミール寒天培地を除く全ての培地上で
、３日目にはコロニーがシャーレ一杯に広がる。また気
生菌糸はやや淡色であるが、コロニーの表面、裏面とも
、多量の分泌液（可溶性色素）のため同一色に見える。[Table 1] In the table above, on all the media except cornmeal agar, colonies spread to fill the Petri dish on the third day. Although aerial hyphae are slightly pale in color, both the front and back sides of the colony appear to be the same color due to the large amount of secreted liquid (soluble pigment).
【００１１】Ｆ−２３３７２株の生育ｐＨ範囲はｐＨ３
．５〜１２．０、至適ｐＨ範囲はｐＨ４．５〜６．０で
ある。また生育温度範囲は１４〜４３℃、至適温度範囲
は２８〜３５℃である。[0011] The growth pH range of strain F-23372 is pH 3.
．． 5 to 12.0, and the optimum pH range is pH 4.5 to 6.0. The growth temperature range is 14-43°C, and the optimum temperature range is 28-35°C.
【００１２】なお、本菌株は通商産業省工業技術院微生
物工業技術研究所に寄託されており、その微工研受託番
号は微工研菌寄第１２０７７号（ＦＥＲＭ Ｐ−１２
０７７）である。[0012] This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and its accession number is FERM P-12.
077).
【００１３】本発明で使用する抗腫瘍性物質ＢＥ−２３
３７２Ｍを産生する微生物の変異株は、例えばＸ線若し
くは紫外線等の照射処理、例えばナイトロジェン・マス
タード、アザセリン、亜硝酸、２−アミノプリン若しく
はＮ−メチル−Ｎ’−ニトロ−Ｎ−ニトロソグアニジン
（ＮＴＧ）等の変異誘起剤による処理、ファージ接触、
形質転換、形質導入又は接合等の通常用いられる菌種変
換処理方法により抗腫瘍性物質ＢＥ−２３３７２Ｍ産生
菌を変異させた微生物である。Antitumor substance BE-23 used in the present invention
Mutant strains of microorganisms that produce 372M can be treated with irradiation such as X-rays or ultraviolet rays, such as nitrogen mustard, azaserine, nitrous acid, 2-aminopurine or N-methyl-N'-nitro-N-nitrosoguanidine ( Treatment with mutagenic agents such as NTG), phage contact,
This is a microorganism obtained by mutating the antitumor substance BE-23372M-producing bacterium using commonly used bacterial species conversion treatment methods such as transformation, transduction, or conjugation.
【００１４】本発明のＢＥ−２３３７２Ｍを製造するに
あたり、ＢＥ−２３３７２Ｍの生産菌株を栄養源含有培
地に接種して好気的に発育させることにより、ＢＥ−２
３３７２Ｍを含む培養物が得られる。栄養源としては、
真菌の栄養源として公知のものが使用できる。例えば、
炭素源としては、市販されているブドウ糖、グリセリン
、麦芽糖、デンプン、庶糖、糖蜜又はデキストリンなど
が単独又は混合物として用いられる。窒素源としては、
市販されている大豆粉、コーンスティープリカー、肉エ
キス、酵母エキス、綿実粉、ペプトン、小麦胚芽、魚粉
、無機アンモニウム塩又は硝酸ナトリウムなどが単独又
は混合物として用いられる。無機塩としては、市販され
ている炭酸カルシウム、塩化ナトリウム、塩化カリウム
、硫酸マグネシウム又は各種リン酸塩などを使用するこ
とができる。その他必要に応じて、鉄、マンガン又は亜
鉛などの重金属塩を微量添加することもできる。また、
発泡の著しい時には、消泡剤として、例えば大豆油又は
亜麻仁油等の植物油、オクタデカノール等の高級アルコ
ール類、各種シリコン化合物等を適宜添加しても良い。
これらのもの以外でも、該生産菌が利用し、ＢＥ−２３
３７２Ｍの生産に役立つものであれば、いずれも使用す
ることができる。In producing BE-23372M of the present invention, BE-23372M production strain is inoculated into a nutrient-containing medium and grown aerobically.
A culture containing 3372M is obtained. As a source of nutrients,
Known nutritional sources for fungi can be used. for example,
As the carbon source, commercially available glucose, glycerin, maltose, starch, sucrose, molasses, dextrin, and the like can be used alone or as a mixture. As a nitrogen source,
Commercially available soybean flour, corn steep liquor, meat extract, yeast extract, cottonseed flour, peptone, wheat germ, fish meal, inorganic ammonium salt, sodium nitrate, and the like can be used alone or as a mixture. As the inorganic salt, commercially available calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, or various phosphates can be used. In addition, a small amount of heavy metal salts such as iron, manganese, or zinc may be added as necessary. Also,
When foaming is significant, antifoaming agents such as vegetable oils such as soybean oil or linseed oil, higher alcohols such as octadecanol, various silicon compounds, etc. may be appropriately added. In addition to these, the producing bacteria also utilize BE-23.
Anything useful in the production of 372M can be used.
【００１５】培養方法としては、一般の微生物代謝産物
の生産方法と同様に行えばよく、固体培養でも液体培養
でもよい。液体培養の場合は、静置培養、攪拌培養、振
盪培養又は通気培養などのいずれを実施してもよいが、
特に振盪培養又は深部通気攪拌培養が好ましい。培養温
度は２０℃〜３７℃が適当であるが、好ましくは２５℃
〜３０℃である。好ましい培地のｐＨは４〜８の範囲で
、培養時間は２４時間〜１９２時間、好ましくは４８時
間〜１４４時間である。[0015] The culturing method may be the same as a general method for producing microbial metabolites, and solid culture or liquid culture may be used. In the case of liquid culture, static culture, agitation culture, shaking culture, or aerated culture may be used, but
In particular, shaking culture or deep aeration agitation culture is preferred. The appropriate culture temperature is 20°C to 37°C, preferably 25°C.
~30°C. The preferred pH of the medium is in the range of 4 to 8, and the culture time is 24 to 192 hours, preferably 48 to 144 hours.
【００１６】培養物から目的とするＢＥ−２３３７２Ｍ
を採取するには、微生物の生産する代謝物を培養物から
採取するのに通常使用される分離手段が適宜利用される
。Target BE-23372M from culture
To collect the metabolites produced by microorganisms, separation means commonly used for collecting metabolites produced by microorganisms from cultures may be used as appropriate.
【００１７】ＢＥ−２３３７２Ｍは主に菌体中に存在す
るので、菌体より通常の分離手段、例えば溶媒抽出法、
イオン交換樹脂法又は吸着若しくは分配クロマトグラフ
ィー法及びゲル濾過法等を単独又は組合せて行うことに
より精製できる。また高速液体クロマトグラフィーや薄
層クロマトグラフィーなども抽出精製に利用可能である
。BE-23372M mainly exists in bacterial cells, so it can be separated from bacterial cells by conventional means such as solvent extraction,
It can be purified by ion exchange resin method, adsorption or partition chromatography method, gel filtration method, etc. alone or in combination. High performance liquid chromatography and thin layer chromatography can also be used for extraction and purification.
【００１８】好ましい分離−精製の例としては次の方法
が挙げられる。まず培養液を濾過し、菌体を得る。得ら
れた菌体からメタノール又はアセトン等の有機溶媒を用
いて抽出する。抽出液を留去して得られた残渣を酢酸エ
チルに溶解し、酸性水で洗った後、濃縮する。ここで得
られた粗物質をシリカゲルクロマトグラフィー（クロロ
ホルム／メタノール／ギ酸）、次いでゲル濾過カラムク
ロマトグラフィー（クロロホルム／メタノール／エタノ
ール／水）に付せば、ＢＥ−２３３７２Ｍの赤橙色固体
を得ることができる。[0018] Examples of preferred separation and purification include the following method. First, the culture solution is filtered to obtain bacterial cells. The obtained bacterial cells are extracted using an organic solvent such as methanol or acetone. The residue obtained by distilling off the extract is dissolved in ethyl acetate, washed with acidic water, and then concentrated. If the crude material obtained here is subjected to silica gel chromatography (chloroform/methanol/formic acid) and then gel filtration column chromatography (chloroform/methanol/ethanol/water), a reddish-orange solid of BE-23372M can be obtained. can.
【００１９】本発明の化合物ＢＥ−２３３７２Ｍは腫瘍
細胞の増殖を阻害し、制癌効果を有するが、本発明化合
物を抗腫瘍剤として使用する際の投与形態としては各種
の形態を選択でき、例えば錠剤、カプセル剤、散剤、顆
粒剤若しくは液剤等の経口剤、又は例えば溶液若しくは
懸濁液等の殺菌した液状の非経口剤が挙げられる。The compound BE-23372M of the present invention inhibits the proliferation of tumor cells and has an anticancer effect, but when the compound of the present invention is used as an antitumor agent, various forms can be selected, such as Examples include oral preparations such as tablets, capsules, powders, granules, or liquid preparations, and sterile liquid parenteral preparations such as solutions or suspensions.
【００２０】固体の製剤は、そのまま錠剤、カプセル剤
、顆粒剤又は粉末の形態として製造することもできるが
、適当な添加物を使用して製造することもできる。その
ような添加物としては、例えば乳糖若しくはブドウ糖等
の糖類、例えばトウモロコシ、小麦若しくは米等の澱粉
類、例えばステアリン酸等の脂肪酸、例えばメタケイ酸
アルミン酸マグネシウム若しくは無水リン酸カルシウム
等の無機塩、例えばポリビニルピロリドン若しくはポリ
アルキレングリコール等の合成高分子、例えばステアリ
ン酸カルシウム若しくはステアリン酸マグネシウム等の
脂肪酸塩、例えばステアリルアルコール若しくはベンジ
ルアルコール等のアルコール類、例えばメチルセルロー
ス、カルボキシメチルセルロース、エチルセルロース若
しくはヒドロキシプロピルメチルセルロース等の合成セ
ルロース誘導体、その他、水、ゼラチン、タルク、植物
油、アラビアゴム等通常用いられる添加物が挙げられる
。[0020] Solid preparations can be manufactured as they are in the form of tablets, capsules, granules, or powders, but they can also be manufactured using appropriate additives. Such additives include, for example, sugars such as lactose or glucose, starches such as corn, wheat or rice, fatty acids such as stearic acid, inorganic salts such as magnesium aluminate metasilicate or anhydrous calcium phosphate, e.g. polyvinyl Synthetic polymers such as pyrrolidone or polyalkylene glycols, fatty acid salts such as calcium stearate or magnesium stearate, alcohols such as stearyl alcohol or benzyl alcohol, synthetic cellulose derivatives such as methylcellulose, carboxymethylcellulose, ethylcellulose or hydroxypropylmethylcellulose , and other commonly used additives such as water, gelatin, talc, vegetable oil, and gum arabic.
【００２１】これらの錠剤、カプセル剤、顆粒剤及び粉
末等の固形製剤は一般的には０．１〜１００重量％、好
ましくは５〜１００重量％の有効成分を含む。[0021] These solid preparations such as tablets, capsules, granules and powders generally contain 0.1 to 100% by weight, preferably 5 to 100% by weight of the active ingredient.
【００２２】液状製剤は、水、アルコール類又は例えば
大豆油、ピーナツ油若しくはゴマ油等の植物由来の油等
液状製剤において通常用いられる適当な添加物を使用し
、懸濁液、シロップ剤若しくは注射剤等の形態として製
造される。[0022] Liquid preparations can be prepared using appropriate additives commonly used in liquid preparations, such as water, alcohols, or vegetable-derived oils such as soybean oil, peanut oil, or sesame oil, to form suspensions, syrups, or injections. It is manufactured in the form of
【００２３】特に、非経口的に筋肉内注射、静脈内注射
又は皮下注射で投与する場合の適当な溶剤としては、例
えば注射用蒸留水、塩酸リドカイン水溶液（筋肉内注射
用）、生理食塩水、ブドウ糖水溶液、エタノール、静脈
内注射用液体（例えばクエン酸及びクエン酸ナトリウム
等の水溶液）若しくは電解質溶液（点滴静注及び静脈内
注射用）等、又はこれらの混合溶液が挙げられる。In particular, suitable solvents for parenteral administration by intramuscular injection, intravenous injection or subcutaneous injection include, for example, distilled water for injection, aqueous lidocaine hydrochloride solution (for intramuscular injection), physiological saline, Examples include an aqueous glucose solution, ethanol, a liquid for intravenous injection (for example, an aqueous solution of citric acid and sodium citrate), an electrolyte solution (for intravenous drip infusion and intravenous injection), or a mixed solution thereof.
【００２４】これらの注射剤は予め溶解したものの他、
粉末のまま或いは適当な添加物を加えたものを用時溶解
する形態もとり得る。これらの注射液は、通常０．１〜
１０重量％、好ましくは１〜５重量％の有効成分を含む
。[0024] In addition to pre-dissolved injections,
It may be in the form of a powder or may be dissolved with appropriate additives before use. These injection solutions usually have a concentration of 0.1 to
It contains 10% by weight of active ingredient, preferably 1-5% by weight.
【００２５】また、経口投与の懸濁剤又はシロップ剤等
の液剤は、０．５〜１０重量％の有効成分を含む。Liquid preparations such as suspensions or syrups for oral administration contain 0.5 to 10% by weight of the active ingredient.
【００２６】本発明の化合物の実際に好ましい投与量は
、配合された組成物の種類、適用頻度及び治療すべき特
定部位、腫瘍によって変化することに注意すべきである
。例えば、１日当りの成人１人当りの投与量は、経口投
与の場合、１０〜５００ｍｇであり、非経口投与、好ま
しくは静脈内注射の場合、１日当り１０〜１００ｍｇで
ある。なお、投与回数は投与方法及び症状により異なる
が、１回ないし５回である。It should be noted that the actual preferred dosage of the compounds of this invention will vary depending on the type of composition formulated, the frequency of application and the particular site and tumor to be treated. For example, the dosage per adult per day is 10 to 500 mg for oral administration, and 10 to 100 mg per day for parenteral administration, preferably intravenous injection. The number of administrations varies depending on the administration method and symptoms, but is 1 to 5 times.
【００２７】次に実施例を挙げ、本発明を具体的に説明
する。しかしながら、本発明は実施例に限定されるもの
ではなく、実施例の修飾手段はもちろん、本発明によっ
て明らかにされたＢＥ−２３３７２Ｍの性状に基づいて
、公知の手段を用いてＢＥ−２３３７２Ｍを生産、濃縮
、抽出、精製する方法すべてを包含する。Next, the present invention will be specifically explained with reference to Examples. However, the present invention is not limited to the examples, and BE-23372M can be produced using known means based on the properties of BE-23372M revealed by the present invention as well as modified means of the examples. , concentration, extraction, and purification methods.
【００２８】[0028]
【実施例】実施例１
斜面寒天培地に培養した真菌Ｆ−２３３７２株をポリペ
プトン０．３％、グルコース１％、小麦胚芽１．０％、
グルテンミール０．５％、麦芽エキス０．３％、マルト
ース３．０％、塩化ナトリウム０．２％、硝酸ナトリウ
ム０．１％、リン酸一カリウム０．１％、硫酸マグネシ
ウム０．０５％、硫酸第一鉄０．０００２％、塩化第二
銅０．００００４％、塩化マンガン０．００００４％、
塩化コバルト０．００００４％、硫酸亜鉛０．００００
８％、ホウ酸ナトリウム０．００００８％及びモリブデ
ン酸アンモニウム０．０００２４％からなる培地（ｐＨ
６．０）１００ｍｌを含む５００ｍｌ容の三角フラスコ
４本に接種し、２８℃で７２時間、回転振盪機（毎分１
８０回転）上で培養した。この培養液を２ｍｌずつ、上
記の培地を１００ｍｌ含む５００ｍｌ容の三角フラスコ
１４０本に接種し、２８℃で７２時間、回転振盪機（毎
分１８０回転）上で培養した。[Example] Example 1 Fungal strain F-23372 cultured on a slanted agar medium was treated with 0.3% polypeptone, 1% glucose, 1.0% wheat germ,
Gluten meal 0.5%, malt extract 0.3%, maltose 3.0%, sodium chloride 0.2%, sodium nitrate 0.1%, monopotassium phosphate 0.1%, magnesium sulfate 0.05%, Ferrous sulfate 0.0002%, cupric chloride 0.00004%, manganese chloride 0.00004%,
Cobalt chloride 0.00004%, zinc sulfate 0.0000
8%, sodium borate 0.00008% and ammonium molybdate 0.00024% (pH
6.0) Inoculate four 500 ml Erlenmeyer flasks containing 100 ml, and shake at 28°C for 72 hours using a rotary shaker (1 minute per minute).
80 revolutions). 2 ml of this culture solution was inoculated into 140 500 ml Erlenmeyer flasks containing 100 ml of the above medium, and cultured on a rotary shaker (180 revolutions per minute) at 28° C. for 72 hours.
【００２９】得られた培養液（約１４Ｌ）を９０℃で１
０分間加熱処理した後セライトを加え、濾過法により濾
過し、得られた菌体にアセトン（１２Ｌ）を加え室温で
１時間撹拌した。濾過法によってアセトン抽出液を得た
。アセトン抽出液を減圧下に濃縮し、アセトンを除去し
、得られた濃縮液（約１．８Ｌ）を１Ｎ ＨＣｌでｐ
Ｈを３に調整した後、酢酸エチル１．８Ｌで２回抽出し
た。酢酸エチル層を合わせ、無水硫酸ナトリウムを用い
て脱水した後、濃縮乾固した。このものをクロロホルム
１０ｍｌに溶解し、１５ｇのシリカゲル（メルク社製キ
ーゼルゲル６０）のカラムに吸着させ、クロロホルムで
カラムを洗浄した後、クロロホルム／メタノール／ギ酸
（１００：１０：１）の混液で溶出した。ＢＥ−２３３
７２Ｍを含む分画を集め、濃縮乾固し、得られた粗物質
をクロロホルム／メタノール／エタノール／水（５：２
：２：１）の混液５ｍｌに溶解し、同混液で予め平衡化
した、セファデックスＬＨ−２０（ファルマシア社製）
のカラム（２．５×５０ｃｍ）に付し、同混液で展開し
た。ＢＥ−２３３７２Ｍを含む分画を集め濃縮乾固し、
粗物質１６ｍｇを得た。この粗物質を再び上記と同様に
セファデックスＬＨ−２０カラムに付すことにより、Ｂ
Ｅ−２３３７２Ｍ ５．７ｍｇを赤橙色固体として得
た。[0029] The obtained culture solution (approximately 14 L) was incubated at 90°C for 1
After heat treatment for 0 minutes, Celite was added and filtered by a filtration method. Acetone (12 L) was added to the obtained bacterial cells and stirred at room temperature for 1 hour. An acetone extract was obtained by a filtration method. The acetone extract was concentrated under reduced pressure to remove acetone, and the resulting concentrate (approximately 1.8 L) was diluted with 1N HCl.
After adjusting H to 3, the mixture was extracted twice with 1.8 L of ethyl acetate. The ethyl acetate layers were combined, dehydrated using anhydrous sodium sulfate, and then concentrated to dryness. This material was dissolved in 10 ml of chloroform, adsorbed onto a 15 g column of silica gel (Kieselgel 60 manufactured by Merck & Co.), and after washing the column with chloroform, it was eluted with a mixture of chloroform/methanol/formic acid (100:10:1). . BE-233
Fractions containing 72M were collected, concentrated to dryness, and the resulting crude material was dissolved in chloroform/methanol/ethanol/water (5:2
Sephadex LH-20 (manufactured by Pharmacia), dissolved in 5 ml of a mixture of 2:1) and equilibrated in advance with the same mixture.
column (2.5 x 50 cm) and developed with the same mixture. Fractions containing BE-23372M were collected and concentrated to dryness,
16 mg of crude material was obtained. By applying this crude material again to the Sephadex LH-20 column in the same manner as above, B
5.7 mg of E-23372M was obtained as a red-orange solid.
【００３０】[0030]
【発明の効果】本発明に記載するＢＥ−２３３７２Ｍは
、ヒトの癌細胞の増殖を強く抑制することから、医薬の
分野で癌の治療剤として有用である。EFFECTS OF THE INVENTION BE-23372M described in the present invention strongly suppresses the proliferation of human cancer cells and is therefore useful as a therapeutic agent for cancer in the pharmaceutical field.
Claims (4)
薬上許容されうる塩。Claims 1: An antitumor substance BE-23372M represented by the formula: [Image Omitted] or a pharmaceutically acceptable salt thereof.
薬上許容されうる塩を有効成分とする抗腫瘍剤。[Claim 2] An antitumor agent comprising an antitumor substance BE-23372M represented by the formula [Image Omitted] or a pharmaceutically acceptable salt thereof as an active ingredient.
し、ＢＥ−２３３７２Ｍを産生する微生物又はその変異
株を培養して、ＢＥ−２３３７２Ｍを蓄積させ、採取す
ることを特徴とする製法。[Claim 3] When producing the antitumor substance BE-23372M represented by the formula [Chemical formula 3], a microorganism that produces BE-23372M or a mutant strain thereof is cultured to accumulate and collect BE-23372M. A manufacturing method characterized by
養することを特徴とする第３請求項記載の製造法。4. The production method according to claim 3, which comprises culturing the fungal strain F-23372 or a mutant strain thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6260791A JPH04275284A (en) | 1991-03-04 | 1991-03-04 | Antitumor substance be-23372m and its production |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6260791A JPH04275284A (en) | 1991-03-04 | 1991-03-04 | Antitumor substance be-23372m and its production |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04275284A true JPH04275284A (en) | 1992-09-30 |
Family
ID=13205173
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6260791A Pending JPH04275284A (en) | 1991-03-04 | 1991-03-04 | Antitumor substance be-23372m and its production |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04275284A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995006032A1 (en) * | 1993-08-20 | 1995-03-02 | Banyu Pharmaceutical Co., Ltd. | Tyrosine kinase inhibitor |
-
1991
- 1991-03-04 JP JP6260791A patent/JPH04275284A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1995006032A1 (en) * | 1993-08-20 | 1995-03-02 | Banyu Pharmaceutical Co., Ltd. | Tyrosine kinase inhibitor |
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