JP5641716B2 - Novel compounds produced by microorganisms belonging to the genus Streptomyces, microorganisms producing the compounds, and pharmaceutical preparations containing the compounds as active ingredients - Google Patents
Novel compounds produced by microorganisms belonging to the genus Streptomyces, microorganisms producing the compounds, and pharmaceutical preparations containing the compounds as active ingredients Download PDFInfo
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- JP5641716B2 JP5641716B2 JP2009168886A JP2009168886A JP5641716B2 JP 5641716 B2 JP5641716 B2 JP 5641716B2 JP 2009168886 A JP2009168886 A JP 2009168886A JP 2009168886 A JP2009168886 A JP 2009168886A JP 5641716 B2 JP5641716 B2 JP 5641716B2
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Description
本発明は、微生物が産生する新規化合物、その化合物を産生する微生物、及びその化合物を有効成分とする医薬に関する。より詳細には、ストレプトミセス（Streptomyces）属に属する微生物によって産生される複数の新規化合物、それらの化合物を産生する上記微生物、及びそれらの化合物を有効成分とする医薬製剤に関する。 The present invention relates to a novel compound produced by a microorganism, a microorganism producing the compound, and a pharmaceutical comprising the compound as an active ingredient. More specifically, the present invention relates to a plurality of novel compounds produced by microorganisms belonging to the genus Streptomyces, the microorganisms producing these compounds, and pharmaceutical preparations containing these compounds as active ingredients.
従来、Verrucosisporaに属する公知の放線菌株が、下記式（１）で表されるアビソマイシン（Abyssomicin）Ｃを産生すること、及びVerrucosispora 株AB-18-032は、abyssomicin Ｃの類縁体であり、下記式（２）〜（６）でそれぞれ表されるabyssomicin Ｂ，Ｄ，atrop-Ｃ，Ｇ及びＨを産生することも報告されている（非特許文献１参照）。また、Streptomycesに属する公知の放線菌株が、下記式（７）で表されるabyssomicin Eを生産することも報告されている（非特許文献２参照）。 Conventionally, a known actinomycete belonging to Verrucosispora produces Abyssomicin C represented by the following formula (1), and Verrucosispora strain AB-18-032 is an analog of abyssomicin C. It has also been reported that abyssomicin B, D, atrop-C, G and H represented by formulas (2) to (6) are produced (see Non-Patent Document 1). It has also been reported that a known actinomycete belonging to Streptomyces produces abyssomicin E represented by the following formula (7) (see Non-Patent Document 2).
上述した放線菌がポリケチド型抗生物質であるabyssomicin を産生することは報告されているが、それら以外の微生物がabyssomicin Ｃ又はその類縁体を産生することは知られていない（非特許文献１、２参照）。
さらに、この化合物は抗菌性を示すとともに、細菌から発見された最初のｐ−アミノ安息香酸の生合成阻害剤であることは報告されている（非特許文献１参照）が、他の生物活性については知られていない。
また、abyssomicin Ｃ以外の上記類縁体にabyssomicin Ｃと同等の抗菌性があるか否か、及びabyssomicin Ｃを含むこれらの類縁体に他の生物活性があるか否かについては報告されていない。
It has been reported that the actinomycetes described above produce abyssomicin, which is a polyketide type antibiotic, but it is not known that other microorganisms produce abyssomicin C or its analogs (Non-Patent Documents 1 and 2). reference).
Furthermore, it is reported that this compound is antibacterial and is the first biosynthesis inhibitor of p-aminobenzoic acid discovered from bacteria (see Non-Patent Document 1). Is not known.
Moreover, it has not been reported whether the above analogs other than abyssomicin C have antibacterial activity equivalent to abyssomicin C, and whether these analogs containing abyssomicin C have other biological activities.
また、従来、癌治療においては、癌細胞の増殖を阻害する薬剤が使用されてきており、こうした薬剤としては、ムスチン、シクロホスファミド、クロラムブシル等のアルキル化剤、アクチノマイシンＤ、ブレオマイシン、ドキソルビシン等の細胞毒抗生物質、ビンクリスチン、ビンブラスチン等のビンカアルカロイド、葉酸拮抗薬であるメトトレキセート、抗ピリミジン薬であるフルオロウラシルやシタラビン、抗プリン薬であるメルカプトプリンその他の代謝拮抗剤等を挙げることができる。
これらの薬剤は、細胞の増殖を抑制する作用を有するものであるが、増殖した癌細胞の基底膜への浸潤又は細胞遊走を抑制する作用を有するものではない。
一方で、癌細胞は、原発巣で増殖し、基底膜に浸潤した癌細胞は、血流に乗って他の部位へ移動することによって転移を起こすことが知られている。
Conventionally, drugs that inhibit the growth of cancer cells have been used in the treatment of cancer. Examples of such drugs include alkylating agents such as mustine, cyclophosphamide, chlorambucil, actinomycin D, bleomycin, doxorubicin. Vinca alkaloids such as vincristine and vinblastine, methotrexate as a folic acid antagonist, fluorouracil and cytarabine as an antipyrimidine drug, mercaptopurine as an antipurine drug and other antimetabolite agents.
These drugs have an action of suppressing cell growth, but do not have an action of suppressing the invasion or cell migration of the proliferated cancer cells into the basement membrane.
On the other hand, it is known that cancer cells proliferate in the primary lesion, and cancer cells that have infiltrated the basement membrane undergo metastasis by moving to other sites in the bloodstream.
しかし、癌の化学療法において使用される上記の薬剤は、それらの細胞傷害活性を利用するものであるから、使用される薬剤が癌細胞に対してある程度の選択作用を示すとはいえ、正常細胞も傷害される。特に、骨髄、胃腸管上皮、毛嚢等の増殖の速い正常細胞に対してはダメージが大きく、副作用が問題とされている。 However, since the drugs used in cancer chemotherapy utilize their cytotoxic activity, normal cells can be used even though the drugs used exhibit some degree of selective action on cancer cells. Will also be injured. Particularly, fast-growing normal cells such as bone marrow, gastrointestinal tract epithelium, hair follicles are seriously damaged, and side effects are a problem.
一方で、化学療法剤でたたききれなかった癌細胞が原発巣で急激に増殖し、周辺組織へ浸潤し、転移を起こして全身状態が悪化するという問題も生じている。周辺組織への浸潤又は転移を阻害することができれば、病状の急激な悪化を抑制できる可能性が高くなり、患者のＱＯＬ（Quality of health）も維持しやすい。
以上のような状況の下では、細胞傷害作用以外に、周辺組織への浸潤や細胞遊走による転移を抑制できる癌の治療剤の開発に対しては、強い社会的な要請がある。
On the other hand, cancer cells that could not be beaten by a chemotherapeutic agent rapidly proliferate at the primary lesion, infiltrate surrounding tissues, cause metastasis, and deteriorate the general condition. If the infiltration or metastasis to surrounding tissues can be inhibited, the possibility of suppressing a rapid deterioration of the disease state is high, and the patient's QOL (Quality of health) is easily maintained.
Under the circumstances as described above, there is a strong social demand for the development of a therapeutic agent for cancer that can suppress metastasis due to invasion to surrounding tissues and cell migration in addition to the cytotoxic action.
本発明者は、以上のような状況の下で、上記のような活性を示す新規化合物を産生するStreptomyces属に属する微生物を見出し、本願発明を完成したものである。 Under the circumstances as described above, the present inventor has found a microorganism belonging to the genus Streptomyces that produces a novel compound exhibiting the above activity, and has completed the present invention.
すなわち、本発明は、特許生物寄託センターに受託番号NITE P-779として寄託されたストレプトミセス（Streptomyces）属に属する微生物である。
また、本発明はまた、前記の微生物が産生し、下記式（I）で表される構造を有する新規化合物である。
That is, the present invention is a microorganism belonging to the genus Streptomyces deposited at the Patent Organism Depositary under the accession number NITE P-779.
The present invention is also a novel compound produced by the above microorganism and having a structure represented by the following formula (I).
式中、Ｒ1、Ｒ４及びＲ５は、それぞれ独立にメチル基を表し；Ｒ2は、酸素原子が結合してカルボニル基となるか、又は水酸基を表し；Ｒ3は、水素原子又はアシル基を表す。また、波線は一重結合又は二重結合を表わす。
また、上記式（I）で表される化合物は、下記式（II）で表される化合物であることが好ましい。ここで、Ｒ１〜Ｒ5 及び波線は、上記の通りである。
In the formula , R 1 , R 4 and R 5 each independently represent a methyl group ; R 2 represents a carbonyl group by binding an oxygen atom or a hydroxyl group; R 3 represents a hydrogen atom or an acyl Represents a group. The wavy line represents a single bond or a double bond.
The compound represented by the above formula (I) is preferably a compound represented by the following formula (II). Wherein, R 1 to R 5 and wavy line are as described above.
また、上記式（I）又は（II）で表される化合物は、下記式（III）で表される、基底膜浸潤阻害活性、細胞遊走阻害活性、細胞傷害活性を有する新規化合物であることが好ましい。 In addition, the compound represented by the above formula (I) or (II) is a novel compound having a basement membrane invasion inhibitory activity, cell migration inhibitory activity, or cytotoxic activity represented by the following formula (III). preferable.
本発明はまた、上記式（I）〜（III）のいずれかで表される化合物、生理学的に許容されるそれらの塩、及び生理学的に許容されるそれらの水和物からなる群から選ばれるいずれかを有効成分として含有する抗癌剤である。ここで、上記式（I）〜（III）のいずれかで表される化合物の生理学的に許容されるそれらの塩、及び生理学的に許容されるそれらの水和物を、「上記式（I）〜（III）のいずれかで表される化合物の塩等」という。 The present invention is also selected from the group consisting of the compounds represented by any of the above formulas (I) to (III), physiologically acceptable salts thereof, and physiologically acceptable hydrates thereof. It is an anticancer agent containing any of the above as an active ingredient. Here, physiologically acceptable salts of the compounds represented by any one of the above formulas (I) to (III) and physiologically acceptable hydrates thereof are referred to as “the above formula (I ) To (III), and the like.
本発明は、さらにまた、上記式（I）〜（III）のいずれかで表される化合物及びそれらの塩等からなる群から選ばれるいずれかを有効成分として含有する、癌細胞の基底膜浸潤阻害剤である。
さらに、本発明は、上記式（I）〜（III）のいずれかで表される化合物及びそれらの塩等からなる群から選ばれるいずれかを有効成分として含有する、癌細胞の遊走阻害剤である。
本発明はさらにまた、上記式（I）又は（II）で表される化合物が、下記式（IV）又は（Ｖ）で表される、新規化合物であることが好ましい。
Furthermore, the present invention also provides a basement membrane infiltration of cancer cells, containing as an active ingredient any one selected from the group consisting of compounds represented by any one of the above formulas (I) to (III) and salts thereof. An inhibitor.
Furthermore, the present invention is a cancer cell migration inhibitor comprising as an active ingredient any one selected from the group consisting of compounds represented by any one of the above formulas (I) to (III) and salts thereof. is there.
In the present invention, the compound represented by the formula (I) or (II) is preferably a novel compound represented by the following formula (IV) or (V).
式中、Ｒ3は水素原子又はアシル基を表す。 In the formula, R 3 represents a hydrogen atom or an acyl group.
上記式（Ｉ）、（IV）又は（Ｖ）で表される化合物は、基底膜浸潤阻害活性、細胞遊走阻害活性、及び細胞傷害活性をさらに有するものであることが好ましい。
本発明はまた、上記式（Ｉ）、（IV）又は（Ｖ）で表される化合物、生理学的に許容されるそれらの塩、及び生理学的に許容されるそれらの水和物からなる群から選ばれるいずれかを有効成分として含有する抗癌剤である。ここで、上記式（Ｉ）、（IV）又は（Ｖ）のいずれかで表される化合物の生理学的に許容されるそれらの塩、及び生理学的に許容されるそれらの水和物を、「上記式（I）、（IV）又は（Ｖ）のいずれかで表される化合物の塩等」という。
The compound represented by the above formula (I), (IV) or (V) preferably further has a basement membrane invasion inhibitory activity, a cell migration inhibitory activity, and a cytotoxic activity.
The present invention also includes a compound represented by the above formula (I), (IV) or (V), physiologically acceptable salts thereof, and physiologically acceptable hydrates thereof. It is an anticancer agent containing any one selected as an active ingredient. Here, physiologically acceptable salts of the compounds represented by any of the above formulas (I), (IV) or (V), and physiologically acceptable hydrates thereof are referred to as “ It refers to a salt of a compound represented by any one of the above formulas (I), (IV) or (V).
また、本発明は、上記式（Ｉ）、（IV）又は（Ｖ）で表される化合物及びそれらの塩等からなる群から選ばれるいずれかを有効成分として含有する、癌細胞の基底膜浸潤阻害剤である。
本発明はさらにまた、上記式（Ｉ）、（IV）又は（Ｖ）のいずれかで表される化合物及びそれらの塩等からなる群から選ばれるいずれかを有効成分として含有する、癌細胞の遊走阻害剤であることが好ましい。
また、本発明は、上記式（Ｉ）、（IV）又は（Ｖ）のいずれかで表される化合物及びそれらの塩等からなる群から選ばれるいずれかを有効成分として含有する、抗菌剤であることが好ましい。
Further, the present invention provides a basement membrane infiltration of cancer cells containing as an active ingredient any one selected from the group consisting of compounds represented by the above formula (I), (IV) or (V) and salts thereof. An inhibitor.
The present invention further provides a cancer cell comprising as an active ingredient any one selected from the group consisting of compounds represented by any one of the above formulas (I), (IV) or (V) and salts thereof. A migration inhibitor is preferred.
The present invention also provides an antibacterial agent containing as an active ingredient any one selected from the group consisting of compounds represented by any one of the above formulas (I), (IV) or (V) and salts thereof. Preferably there is.
本発明において、上述したストレプトミセス（Streptomyces）属に属する微生物は、上記式（I）〜（IV）で表される新規な化合物を産生する。そして、これらの化合物のうち、式（II）及び（III）で表される化合物は、基底膜浸潤阻害活性、細胞遊走阻害活性、及び細胞傷害活性及び細胞遊走阻害活性を有し、上記式（IV）又は（Ｖ）で表される化合物は、さらに抗菌活性をも有する。 In the present invention, the above-mentioned microorganism belonging to the genus Streptomyces produces novel compounds represented by the above formulas (I) to (IV). Among these compounds, the compounds represented by the formulas (II) and (III) have a basement membrane invasion inhibitory activity, a cell migration inhibitory activity, and a cytotoxic activity and a cell migration inhibitory activity. The compound represented by IV) or (V) further has antibacterial activity.
本発明に係る微生物（TP-A0877）は、富山県射水市で採取された岩石より分離した放線菌Streptomyces fragilisである。この微生物は、以下のようにして単離することができる。
富山県射水市で採取された岩石を、乳鉢で細かく粉砕し、例えば、ＹＳ培地等の所定の培地で懸濁し、試料を調製する。
次いで、平板培地上にこれらの試料を塗布し、恒温器中で培養し、平板上に出現したコロニーを採取することにより、菌を分離することができる。ここで使用する平板培地としては、例えば、Ｂｎ２培地、ＨＭＧ培地等に塗布した試料を、恒温器中にて、約３０℃〜６４℃の間の所望の温度で培養することにより、設定した温度で生育可能な菌を分離することができる。
The microorganism (TP-A0877) according to the present invention is Streptomyces fragilis isolated from rocks collected in Imizu City, Toyama Prefecture. This microorganism can be isolated as follows.
A rock collected in Imizu City, Toyama Prefecture is finely pulverized in a mortar and suspended in a predetermined medium such as a YS medium to prepare a sample.
Subsequently, these samples are applied on a plate medium, cultured in a thermostatic chamber, and colonies appearing on the plate are collected, whereby bacteria can be separated. The plate medium used here is, for example, a temperature set by culturing a sample coated on a Bn2 medium, HMG medium, or the like at a desired temperature between about 30 ° C. and 64 ° C. in a thermostat. Can be isolated.
上記のようにして得られた微生物を、顕微鏡で形態を観察するとともに、寒天培地における生育状態を観察することによって、いずれの属に属するものであるかを決定することができる。
例えば、形態学的に、ループを形成する気菌糸と連鎖する楕円球の胞子とが認められる場合には、前記微生物は放線菌であると判断することができ、その場合には、生育状態の確認に、ＩＳＰ培地 No. ３（オートミール寒天培地、３２℃培養）、ＩＳＰ培地 No. ４（スターチ・無機塩寒天培地、３２℃培養）、ＩＳＰ培地 No. ７（チロシン寒天培地、３２℃培養）、ＩＳＰ培地 No. ２（イースト・麦芽寒天培地、３２℃培養）、及びＩＳＰ培地 No. ５（グリセリン・アスパラギン寒天培地、３２℃培養）を使用して、これらの上における生育状態を確認することにより、この微生物を同定することができる。
The microorganisms obtained as described above can be determined to belong to which genus by observing the morphology with a microscope and observing the growth state in an agar medium.
For example, when morphologically observing an elliptical spore linked to an aerial hypha that forms a loop, it can be determined that the microorganism is an actinomycete. For confirmation, ISP medium No. 3 (oatmeal agar medium, 32 ° C. culture), ISP medium No. 4 (starch / inorganic salt agar medium, 32 ° C. culture), ISP medium No. 7 (tyrosine agar medium, 32 ° C. culture) Use ISP medium No. 2 (yeast / malt agar medium, 32 ° C. culture) and ISP medium No. 5 (glycerin / asparagine agar medium, 32 ° C. culture) to confirm the growth state on these. This microorganism can be identified.
次に、同定した微生物を増殖させ、どのような二次代謝産物を産生するかの検討を行う。
一般的な手順としては、まず、上記の菌を、種母培地に接種して、例えば、約28〜32℃の温度で３〜５日間振とう培養により、前培養する。こうした種母培地としては、例えば、V-22液体培地を使用することが好ましい。次いで、前培養後の培地から所定量を取って他の培地に接種し、さらに数日間、例えば、上記と同じ温度で振とう培養し、種母培養液を得る。ここで、振とう培養は、例えば、150〜250rpm/分の範囲で行うことができ、約200rpmで行うことが菌の生育の面から好ましい。
Next, the identified microorganisms are grown to examine what secondary metabolites are produced.
As a general procedure, first, the above-mentioned bacteria are inoculated into a seed medium and pre-cultured by, for example, shaking culture at a temperature of about 28 to 32 ° C. for 3 to 5 days. As such a seed medium, for example, a V-22 liquid medium is preferably used. Next, a predetermined amount is taken from the pre-cultured medium and inoculated into another medium, and further cultured with shaking at the same temperature as described above for several days, for example, to obtain a seed culture solution. Here, the shaking culture can be performed, for example, in the range of 150 to 250 rpm / minute, and is preferably performed at about 200 rpm from the viewpoint of the growth of bacteria.
次に、上記のようにして得た種母培養液から所定量を取り、所定量の生産培地を入れたＫ型フラスコに移植し、所定の培養条件（例えば、25〜35℃にて、６〜８日間）、振とう培養する。これによって、上記の微生物に二次代謝産物を産生させることができる。
培養終了後、各フラスコに１−ブタノールを加えて、約１時間、振とう培養器上で、撹拌抽出を行う。これを遠心し、有機相と水相（菌体を含む）とに分ける。得られた有機相を減圧濃縮し、抽出物を得る。これをカラムクロマトグラフィーに供する。このため、抽出に使用する有機溶媒としては、１−ブタノール以外には、酢酸エチル、塩化メチレン等が好ましい。
Next, a predetermined amount is taken from the seed culture solution obtained as described above, transplanted to a K-type flask containing a predetermined amount of production medium, and subjected to predetermined culture conditions (for example, 25-35 ° C., 6 Incubate with shaking for ~ 8 days). As a result, the above-mentioned microorganism can produce a secondary metabolite.
1-butanol is added to each flask after completion | finish of culture | cultivation, and stirring extraction is performed on a shaking incubator for about 1 hour. This is centrifuged and divided into an organic phase and an aqueous phase (including bacterial cells). The obtained organic phase is concentrated under reduced pressure to obtain an extract. This is subjected to column chromatography. For this reason, as an organic solvent used for extraction, in addition to 1-butanol, ethyl acetate, methylene chloride and the like are preferable.
ここで使用するカラムクロマトグラフィーとしては、各種の吸着性担体を使用することができるが、分離効率の面から、順相シリカゲルを使用することが好ましい。
次に、カラムから吸着物質を溶出させる溶離液を調製する。この溶出は、溶離液の極性を変えながら行うため、所定の割合で上記有機溶媒を含有する溶離液を数種類用意して、ステップグラジエント法によって行ってもよく、連続的な濃度勾配となるようグラジエント法で行ってもよい。
ステップグラジエント法による場合には、例えば、有機溶媒としてクロロホルムとメタノールを使用し、1:0、20:1、10:1、4:1、2:1、1:1、0:1というようにメタノールの量を増加させた溶離液を調製し、これらの溶離液を用いて順次溶出させることによって分画する。
その後、各溶出画分を高速液体クロマトグラフィー（ＨＰＬＣ）に供して分画し、各画分にどのような二次代謝産物が含まれるかを確認する。
As the column chromatography used here, various adsorbent carriers can be used, but normal phase silica gel is preferably used from the viewpoint of separation efficiency.
Next, an eluent for eluting the adsorbed substance from the column is prepared. Since this elution is carried out while changing the polarity of the eluent, several types of eluents containing the above-mentioned organic solvent at a predetermined ratio may be prepared and performed by the step gradient method. You may go by law.
In the case of the step gradient method, for example, chloroform and methanol are used as organic solvents, and 1: 0, 20: 1, 10: 1, 4: 1, 2: 1, 1: 1, 0: 1, etc. Fractionation is carried out by preparing eluents with increasing amounts of methanol and sequentially eluting with these eluents.
Thereafter, each eluted fraction is subjected to high performance liquid chromatography (HPLC) and fractionated, and what secondary metabolites are contained in each fraction is confirmed.
ここで使用するＨＰＬＣ用カラムとしては、例えば、COSMOSIL 5C18-AR-II4.6×250mm（nacalai tesque 社製）、cadenza CD-C18 75 ×4.6mm（Imtakt社製）、MICROSORB-MV 100×4.6mm（RAININ INSTRUMENT COMPANY. INC製）等を使用することができ、MICROSORB-MV 100×4.6mmを使用することが好ましい。
化合物の存在が認められた画分をそれぞれ減圧濃縮することにより、本発明の化合物の粗精製物を得ることができる。
次いで、上記各粗精製物を逆相シリカゲルカラムに供することにより、精製物を得ることができる。精製用のシリカゲル担体としては、例えば、COSMOSIL 75C18-Prep （nacalai tesque 社製）を使用することができる。
以上のようにして、本発明の新規化合物を得ることができる。これらの化合物を公知の方法に従って処理し、所望の塩や水和物を得ることができる。
As the HPLC column used here, for example, COSMOSIL 5C18-AR-II 4.6 × 250 mm (manufactured by nacalai tesque), cadenza CD-C18 75 × 4.6 mm (manufactured by Imtakt), MICROSORB-MV 100 × 4.6 mm (Manufactured by RAININ INSTRUMENT COMPANY. INC) can be used, and it is preferable to use MICROSORB-MV 100 × 4.6 mm.
A crude product of the compound of the present invention can be obtained by concentrating the fractions in which the presence of the compound is recognized under reduced pressure.
Next, the purified product can be obtained by subjecting each of the above crude purified products to a reverse phase silica gel column. As a silica gel carrier for purification, for example, COSMOSIL 75C18-Prep (manufactured by nacalai tesque) can be used.
As described above, the novel compound of the present invention can be obtained. These compounds can be processed according to known methods to obtain the desired salts and hydrates.
また、本発明は、上述した方法で得られた化合物で得られるものであり、その一般式は、下記式（Ｉ）又は（II）に示す新規化合物である。ここで、各式中、Ｒ1、Ｒ４及びＲ５は、それぞれ独立にメチル基を表し；Ｒ2は、酸素原子が結合してカルボニル基となるか、又は水酸基を表し；Ｒ3は、水素原子又はアシル基を表すものであることが好ましい。また、波線は一重結合又は二重結合を表わす。 Moreover, this invention is obtained with the compound obtained by the method mentioned above, The general formula is a novel compound shown to following formula (I) or (II). Here, in each formula, R 1 , R 4 and R 5 each independently represents a methyl group ; R 2 represents a carbonyl group by combining an oxygen atom; or R 3 represents a hydroxyl group; It preferably represents a hydrogen atom or an acyl group. The wavy line represents a single bond or a double bond.
また、上記式（Ｉ）又は（II）で表される化合物は、本発明の微生物によって産生される化合物であり、下記式（III）で表されるものであることが好ましい。 The compound represented by the above formula (I) or (II) is a compound produced by the microorganism of the present invention, and is preferably represented by the following formula (III).
また、上記式（III）で表される化合物は、基底膜浸潤阻害活性、細胞遊走阻害活性、及び細胞傷害活性をさらに有するものであることが好ましい。
さらに、上記式（I）又は（II）で表される化合物は、下記式（IV）で表されるものであることが好ましい。
The compound represented by the above formula (III) preferably further has a basement membrane invasion inhibitory activity, a cell migration inhibitory activity, and a cytotoxic activity.
Furthermore, the compound represented by the formula (I) or (II) is preferably represented by the following formula (IV).
式中、Ｒ3は、炭素数１〜８の直鎖状または分岐鎖状の置換または非置換のアルキル基、アルケニル基、アルキニル基、及びアシル基からなる群から選ばれる置換基を表すものであることが好ましい。より好ましくは、上記式（IV）で表される化合物は、下記式（Ｖ）で表される化合物である。 In the formula, R 3 represents a substituent selected from the group consisting of a linear or branched substituted or unsubstituted alkyl group, alkenyl group, alkynyl group, and acyl group having 1 to 8 carbon atoms. Preferably there is. More preferably, the compound represented by the above formula (IV) is a compound represented by the following formula (V).
また、上記式（IV）又は（Ｖ）で表される化合物は、基底膜浸潤阻害活性、細胞遊走阻害活性、細胞傷害活性及び抗菌活性をさらに有するものであることが好ましい。 The compound represented by the above formula (IV) or (V) preferably further has a basement membrane invasion inhibitory activity, a cell migration inhibitory activity, a cytotoxic activity and an antibacterial activity.
ここで、上記式（IV）で表される化合物は、本発明の微生物によって産生される化合物である式（III）を下記のように修飾することによって得られるものである。
すなわち、上記のようにして得られた上記式（III）で表される化合物を、適当な有機溶媒に溶解し、酸化剤を加えて攪拌処理することにより、上記式（Ｖ）で表される化合物を得ることができる。有機溶媒としては、クロロホルム、ジクロルメタン等を挙げることができ、酸化剤としては、二酸化マンガン等を上げることができる。攪拌処理は、室温で、約20〜30時間行うことが、収量の点から好ましい。
反応が終了した反応液を濾過して酸化剤を除き、濾液を減圧濃縮する。その後、溶離液として、例えば、所定の割合のヘキサン−酢酸エチルを用いてシリカゲルカラムクロマトグラフィーを行うことにより、上記式（Ｖ）で表される化合物を得ることができる。
Here, the compound represented by the formula (IV) is obtained by modifying the formula (III), which is a compound produced by the microorganism of the present invention, as follows.
That is, the compound represented by the above formula (III) obtained as described above is dissolved in an appropriate organic solvent, added with an oxidizing agent, and stirred, and then represented by the above formula (V). A compound can be obtained. Examples of the organic solvent include chloroform and dichloromethane, and examples of the oxidizing agent include manganese dioxide. The stirring treatment is preferably performed at room temperature for about 20 to 30 hours from the viewpoint of yield.
The reaction solution after completion of the reaction is filtered to remove the oxidizing agent, and the filtrate is concentrated under reduced pressure. Thereafter, the compound represented by the above formula (V) can be obtained by performing silica gel column chromatography using, for example, a predetermined ratio of hexane-ethyl acetate as an eluent.
本発明はさらにまた、上記式（Ｉ）〜（III）のいずれかで表される化合物、生理学的に許容されるそれらの塩、及びそれらの水和物からなる群から選ばれる、いずれかを有効成分とする、癌治療用の医薬製剤である。ここで、生理学的に許容される塩としては、ナトリウム塩、カリウム塩、カルシウム塩、塩酸塩、硝酸塩、硫酸塩、炭酸塩等を挙げることができ、それらの水和物としては、一水和物、二水和物等を挙げることができる。
上記の化合物の塩又はそれらの水和物を使用する場合には、ナトリウム塩、カリウム塩、塩酸塩を使用することが、溶解性及び薬物の代謝の面から好ましい。また、一水和物又は二水和物を、同様の理由から好適に使用することができる。
本発明の医薬製剤は、癌細胞の基底膜浸潤阻害剤、及び癌細胞の遊走阻害剤としても作用するものである。
The present invention further includes any one selected from the group consisting of compounds represented by any one of the above formulas (I) to (III), physiologically acceptable salts thereof, and hydrates thereof. It is a pharmaceutical preparation for cancer treatment as an active ingredient. Here, examples of physiologically acceptable salts include sodium salts, potassium salts, calcium salts, hydrochlorides, nitrates, sulfates, carbonates, and the like. Product, dihydrate and the like.
When using a salt of the above compound or a hydrate thereof, it is preferable to use a sodium salt, a potassium salt, or a hydrochloride from the viewpoints of solubility and drug metabolism. Moreover, a monohydrate or a dihydrate can be used conveniently for the same reason.
The pharmaceutical preparation of the present invention also acts as an inhibitor of cancer cell basement membrane invasion and an inhibitor of cancer cell migration.
上記の各官能基は、Ｒ1及びＲ3が水素原子、Ｒ2がヒドロキシル基、Ｒ4及びＲ5がメチル基であることが好ましい。これらの官能基を有する化合物は上記式（III）で表されるものとなるが、癌細胞の基底膜浸潤阻害作用及び癌細胞の遊走阻害作用が発揮されることによる。
本明細書中において、「基底膜」とは、上皮細胞の底面や筋細胞・脂肪細胞・Schwann細胞を取り巻く無構造な細胞外構造で、選択的フィルタであるとともに、構造的形態形成的機能を有する。基底膜は、透明板・緻密板・繊維細網板の３層構造とコラーゲン基質と数種の糖タンパクとからなる。
In each of the above functional groups, R 1 and R 3 are preferably a hydrogen atom, R 2 is a hydroxyl group, and R 4 and R 5 are preferably a methyl group. The compound having these functional groups is represented by the above formula (III), and is due to the effect of inhibiting cancer cell basement membrane invasion and cancer cell migration.
In this specification, the “basement membrane” is an unstructured extracellular structure surrounding the bottom surface of epithelial cells and muscle cells / adipocytes / Schwann cells, and is a selective filter and has a structural morphogenic function. Have. The basement membrane is composed of a three-layer structure of a transparent plate, a dense plate, and a fiber reticular plate, a collagen matrix, and several types of glycoproteins.
「浸潤」とは、隣接組織を破壊等することによって、癌細胞（悪性新生物）が局部的に広がることをいい、悪性新生物が上皮性腫瘍の場合には、その直下にある上皮基底膜に入り込むことをいうが、本明細書においては、癌細胞が、血管の外膜、中膜、及び内膜を通過することをも含むものとする。
また、「細胞遊走」は、器官・形態形成、及び癌の浸潤・転移等の他、動脈硬化等の各種の生理的・病的なプロセスに関与することが知られている作用である。本明細書においては、原発巣を形成している癌細胞が基底膜を通り抜け、転移巣を形成するまでのプロセスに関与する、能動的な移動の全体を「癌細胞の遊走」という。
“Invasion” means that cancer cells (malignant neoplasms) spread locally by destroying adjacent tissues, etc. When the malignant neoplasm is an epithelial tumor, the epithelial basement membrane directly below it In the present specification, it is intended to include that cancer cells pass through the outer membrane, the media, and the inner membrane of blood vessels.
“Cell migration” is an action known to be involved in various physiological and pathological processes such as arteriosclerosis, in addition to organ / morphogenesis, invasion / metastasis of cancer, and the like. In the present specification, the entire active migration involved in the process of cancer cells forming the primary lesion passing through the basement membrane and forming a metastatic lesion is referred to as “cancer cell migration”.
本発明はまた、上記式（Ｉ）、（IV）、（Ｖ）のいずれかで表される化合物、生理学的に許容されるそれらの塩、及びそれらの水和物からなる群から選ばれる、いずれかを有効成分とする、癌治療用の医薬製剤である。ここで、生理学的に許容される塩及び水和物は上述した通りである。
上記の化合物の塩又はそれらの水和物を使用する場合には、ナトリウム塩、カリウム塩、塩酸塩を使用することが、溶解性及び薬物の代謝の面から好ましい。また、一水和物又は二水和物を、同様の理由から好適に使用することができる。
本発明の上記式（Ｉ）、（IV）、（Ｖ）のいずれかで表される医薬製剤は、癌細胞の基底膜浸潤阻害剤、癌細胞の遊走阻害剤、又は抗菌剤としても作用するものである。
The present invention is also selected from the group consisting of compounds represented by any one of the above formulas (I), (IV), (V), physiologically acceptable salts thereof, and hydrates thereof. It is a pharmaceutical preparation for cancer treatment containing any one of the active ingredients. Here, the physiologically acceptable salts and hydrates are as described above.
When using a salt of the above compound or a hydrate thereof, it is preferable to use a sodium salt, a potassium salt, or a hydrochloride from the viewpoints of solubility and drug metabolism. Moreover, a monohydrate or a dihydrate can be used conveniently for the same reason.
The pharmaceutical preparation represented by any one of the above formulas (I), (IV), and (V) of the present invention also acts as a cancer cell basement membrane infiltration inhibitor, cancer cell migration inhibitor, or antibacterial agent. Is.
上記の各官能基は、Ｒ1及びＲ3が水素原子、Ｒ2が酸素原子、Ｒ4及びＲ5がメチル基であることが好ましい。これらの官能基を有する化合物は上記式（Ｖ）で表されるものとなるが、上述した細胞傷害作用、癌細胞の基底膜浸潤阻害作用、癌細胞の遊走阻害作用に加えて、抗菌作用が発揮されることによる。
また、上述した本発明の癌治療用医薬製剤、癌細胞の浸潤阻害剤、癌細胞の遊走阻害剤、及び抗菌剤が、上記化合物の塩を含む場合には、ナトリウム塩又はカリウム塩を含むものであることが好ましく、それらの水和物を含む場合には、一水和物又は二水和物を含むものであることが、上述した薬理効果が高いことから好ましい。
ここで、上記の医薬製剤中における前記有効成分の含量は、製剤の１用量当たり０．１〜１，０００ｍｇであることが好ましく、０．５〜５００ｍｇであることがより好ましい。
In each of the above functional groups, R 1 and R 3 are preferably hydrogen atoms, R 2 is an oxygen atom, and R 4 and R 5 are preferably methyl groups. The compound having these functional groups is represented by the above formula (V), but has an antibacterial action in addition to the above-described cytotoxic action, cancer cell basement membrane invasion inhibitory action, and cancer cell migration inhibitory action. By being demonstrated.
In addition, when the above-described pharmaceutical preparation for cancer treatment, cancer cell invasion inhibitor, cancer cell migration inhibitor, and antibacterial agent of the present invention contain a salt of the above compound, it contains a sodium salt or a potassium salt. In the case where these hydrates are included, it is preferable that they include monohydrate or dihydrate because the pharmacological effect described above is high.
Here, the content of the active ingredient in the pharmaceutical preparation is preferably 0.1 to 1,000 mg, more preferably 0.5 to 500 mg per dose of the preparation.
また、上記癌治療用医薬製剤は、経口投与、静脈内投与、又は腹腔内投与が可能な剤形であることが好ましく、錠剤、散剤、カプセル剤、顆粒剤、丸剤、トローチ剤、及び液剤からなる群から選ばれるものであることが好ましい。
上記の化合物を有効成分する抗癌剤は、上記以外の粉剤その他の固形剤としてもよく、注射剤用の凍結乾燥製剤、リポソーム剤等、各種の剤形とすることもできる。
上述したように製造したこれらの化合物およびそれらの塩類を用いて製剤を製造する場合には、常法に従って、粉末とした後に散剤としてもよく、公知の賦形剤、崩壊剤等とともに打錠し、錠剤、トローチ剤等にしてもよい。錠剤の場合には、必要に応じて白糖その他の糖等を用いて、単層又は複数の層でコーティングを行い、糖衣錠としてもよい。また、矯味・矯臭剤を添加してもよい。
The above-mentioned pharmaceutical preparation for cancer treatment is preferably in a dosage form that can be administered orally, intravenously, or intraperitoneally. Tablets, powders, capsules, granules, pills, troches, and solutions It is preferably selected from the group consisting of
The anticancer agent containing the above-mentioned compound as an active ingredient may be a powder or other solid agent other than those described above, and may be various dosage forms such as a freeze-dried preparation for injection and a liposome.
When a preparation is produced using these compounds produced as described above and their salts, it may be powdered after being powdered according to a conventional method, and tableted together with known excipients, disintegrants, etc. , Tablets, troches and the like. In the case of a tablet, a sugar-coated tablet may be obtained by coating with a single layer or a plurality of layers using sucrose or other sugars as necessary. Moreover, you may add a taste-masking agent.
また、上記の化合物及びそれらの塩類を適当な溶媒に溶解し、常法に従って乾燥させ、顆粒剤としてもよい。さらに、上記のような粉剤、顆粒剤を所定の大きさの軟カプセル又は硬カプセルに充填し、カプセル剤とすることもできる。
また、液剤とする場合には、必要に応じて、ｐＨ調整剤、分散剤等を添加することもできる。リポソーム剤とする場合には、適当なリン脂質を選択し、溶液中でこれらとともに懸濁することによって、製造することができる。
上記医薬製剤中における前記有効成分の含量は、製剤の１用量当たり０．０５〜８００ｍｇであることが好ましく、０．１〜４００ｍｇであることがより好ましい。
Further, the above-mentioned compounds and salts thereof may be dissolved in a suitable solvent and dried according to a conventional method to form granules. Furthermore, the above powders and granules can be filled into soft capsules or hard capsules of a predetermined size to form capsules.
Moreover, when setting it as a liquid agent, a pH adjuster, a dispersing agent, etc. can also be added as needed. In the case of a liposome preparation, it can be produced by selecting appropriate phospholipids and suspending them together in a solution.
The content of the active ingredient in the pharmaceutical preparation is preferably 0.05 to 800 mg, more preferably 0.1 to 400 mg per dosage of the preparation.
上記医薬製剤は、経口投与、静脈内投与、又は腹腔内投与が可能な剤形であることが好ましく、錠剤、散剤、カプセル剤、顆粒剤、丸剤、トローチ剤、及び液剤からなる群から選ばれるものであることが好ましい。また、上記医薬製剤は、粉剤その他の上記以外の固形剤としてもよく、注射剤用の凍結乾燥製剤、リポソーム剤その他の各種の剤形とすることができる。こうした剤形の製造は、上記と同様にして行うことができる。
ここで、上記の医薬製剤に含有される前記有効成分の含量は、各製剤の１用量当たり０．０１〜１００ｍｇであることが好ましく、０．０２〜３００ｍｇであることがより好ましい。具体的な投与量は、投与される患者の身長、体重、性別、年齢、疾患の進行の程度等を勘案して定めることができる。
なお、上述した癌細胞の浸潤阻害剤、癌細胞の遊走阻害剤、及び抗菌剤を製造する場合にも、上記の医薬製剤の場合と同様にして行うことができる。
The pharmaceutical preparation is preferably a dosage form that can be administered orally, intravenously, or intraperitoneally, and is selected from the group consisting of tablets, powders, capsules, granules, pills, troches, and solutions. It is preferable that The pharmaceutical preparation may be a powder or other solid preparations other than those described above, and may be a freeze-dried preparation for injection, a liposome preparation or other various dosage forms. Such dosage forms can be produced in the same manner as described above.
Here, the content of the active ingredient contained in the pharmaceutical preparation is preferably 0.01 to 100 mg, more preferably 0.02 to 300 mg, per dose of each preparation. The specific dose can be determined in consideration of the height, weight, sex, age, degree of disease progression, etc. of the patient to be administered.
It should be noted that the above-described pharmaceutical preparation can also be carried out when producing the above-described cancer cell invasion inhibitor, cancer cell migration inhibitor, and antibacterial agent.
（実施例１）菌の探索と分離・同定
（１−１）試薬等
以下の試薬を使用した。
塩化カルシウム、炭酸カルシウム、ブドウ糖、グリセロール、硫酸マグネシウム７水和物（MgSO4・7H2O）、硫酸鉄７水和物（FeSO4・7H2O）、塩化マンガン（MnCl2・4H2O）４水和物、硫酸ニッケル４水和物（NiSO4・4H2O）、硫酸亜鉛４水和物（ZnSO4・4H2O）、水酸化ナトリウム、塩化ナトリウム、ＮＺケース（NZ Case）、NZアミン（NZ Amine）、溶性でんぷん（Soluble Starch）、メタノール、アセトン、アセトニトリル、リン酸水素２カリウム（K2HPO4）、リン酸水素２ナトリウム12水和物（Na2HPO4・12H2O）、リン酸水素２ナトリウム、ギ酸、塩酸、二酸化マンガン、ジシクロヘキシルアミド、ジメチルアミノピリジン、塩化メチレン、ドデシル硫酸ナトリウム（SDS）、ゲランガム（Gellan gum）、Cell Counting Kit及び寒天は、和光純薬工業（株）より購入した。
(Example 1) Search and separation / identification of bacteria (1-1) Reagents etc. The following reagents were used.
Calcium chloride, calcium carbonate, glucose, glycerol, magnesium sulfate heptahydrate (MgSO 4 · 7H 2 O), iron sulfate heptahydrate (FeSO 4 · 7H 2 O), manganese chloride (MnCl 2 · 4H 2 O) Tetrahydrate, nickel sulfate tetrahydrate (NiSO 4 · 4H 2 O), zinc sulfate tetrahydrate (ZnSO 4 · 4H 2 O), sodium hydroxide, sodium chloride, NZ case (NZ Case), NZ Amine (NZ Amine), soluble starch (Soluble Starch), methanol, acetone, acetonitrile, dipotassium hydrogen phosphate (K 2 HPO 4 ), disodium hydrogen phosphate 12 hydrate (Na 2 HPO 4 · 12H 2 O) , Disodium hydrogen phosphate, formic acid, hydrochloric acid, manganese dioxide, dicyclohexylamide, dimethylaminopyridine, methylene chloride, sodium dodecyl sulfate (SDS), Gellan gum, Cell Counting Kit and agar are manufactured by Wako Pure Chemical Industries, Ltd. Purchase from It was.
肉エキスはDIFCOより、トリプトン及び酵母エキスはBacto Laboratories Pty Ltd.より購入した。(R)-MTPAと(S)-MTPAは、シグマ−アルドリッチジャパン（株）（SIGMA-ALDRICH Japan K.K.）より購入した。
ファーマメディア（Pharmamedia）はTraders Protein社より購入した。
NMR用重メタノールと重クロロホルムは関東化学（株）より購入した。
Meat extract was purchased from DIFCO, and tryptone and yeast extract were purchased from Bacto Laboratories Pty Ltd. (R) -MTPA and (S) -MTPA were purchased from Sigma-ALDRICH Japan KK.
Pharmamedia was purchased from Traders Protein.
NMR heavy methanol and chloroform were purchased from Kanto Chemical Co., Inc.
10％ウシ胎児血清（FBS）はJRH Biosciences社より、RPMI1640培地はInvitrogen-Gibco社より、それぞれ購入した。Colon 26 L-5は、富山大学和漢薬研究所病態生理学研究室にて済木育夫教授らにより樹立されたマウス大腸癌由来細胞であり、済木育夫教授より供与を受けた。MOPSは、同仁化学（株）より購入し、ヘマトキシリン及びエオシンは、武藤化学（株）より購入した。 10% fetal bovine serum (FBS) was purchased from JRH Biosciences, and RPMI1640 medium was purchased from Invitrogen-Gibco. Colon 26 L-5 is a mouse colorectal cancer-derived cell established by Prof. Ikuo Seki et al. At the Department of Pathophysiology, University of Toyama. MOPS was purchased from Dojin Chemical Co., Ltd., and hematoxylin and eosin were purchased from Muto Chemical Co., Ltd.
（１−２）菌の探索
探索源として、富山県射水市で採取された岩石を、乳鉢で細かく粉砕したものを使用した。
粉砕した上記の岩石１ｇを、10mLのＹＳ培地に懸濁し、ボルテックスミキサーで１分間攪拌した後に、室温にて30分間静置した。ＹＳ培地の組成を下記表１に示す。
(1-2) Search for Bacteria As a search source, rocks collected in Imizu, Toyama Prefecture were finely crushed with a mortar.
1 g of the crushed rock was suspended in 10 mL of YS medium, stirred for 1 minute with a vortex mixer, and allowed to stand at room temperature for 30 minutes. The composition of the YS medium is shown in Table 1 below.
静置30分後、この懸濁液をＹＳ培地で、順次10倍に段階希釈した。適当な濃度に調整した懸濁液0.1mLをＢｎ２平板培地又はＨＭＧ平板培地２枚に、コンラージ棒にて塗布し、各平板培地を表１に示す温度に設定した恒温器（恒温器：ADVANTEC INCUBATOR CI-612）に入れ、２〜４週間培養し、出現したコロニーから釣菌し、菌を単離した。上記の試料から、約90株が得られた。
下記の表２及び３に、Ｂｎ２培地及びＨＭＧ培地の組成を示す。
After 30 minutes of standing, this suspension was serially diluted 10 times with YS medium. Apply 0.1 mL of suspension adjusted to appropriate concentration to 2 Bn2 plate medium or HMG plate medium with a congeal rod, and set each plate medium to the temperature shown in Table 1 (Incubator: ADVANTEC INCUBATOR CI-612), and cultured for 2 to 4 weeks. From the colonies that appeared, the fungus was isolated and the fungus was isolated. About 90 strains were obtained from the above sample.
Tables 2 and 3 below show the compositions of the Bn2 medium and the HMG medium.
ここで、上記ＨＭＧ培地で使用した金属溶液の組成及びフミン酸溶液の組成を下記表４及び５に示す。 Here, the composition of the metal solution used in the HMG medium and the composition of the humic acid solution are shown in Tables 4 and 5 below.
（１−３）菌の同定
本菌株の分類的性質は下記の通りである。実験方法は『放線菌の分類と同定』（日本放線菌学会 2001年）に従って行った。
菌の同定に使用したＩＳＰ（International Streptomyces Project）培地Ｎｏ.２及び同Ｎｏ.４は、DIFCO社より購入した。また、ＩＳＰ培地Ｎｏ.３、同Ｎｏ.５、及び同Ｎｏ.７培地としては、日本放線菌学会規格放線菌培地ダイゴＮｏ.３、Ｎｏ.５、及び同Ｎｏ.７を日本製薬（株）より購入した。
色調は標準として、『新色事典』（財団法人日本色彩研究所 1987年）を用いて決定し、色標名とともに括弧内にそのコードを併せて記した。32℃、４週間培養後の各種培地における観察結果を示した。
(1-3) Identification of bacteria The taxonomic properties of this strain are as follows. The experimental method was performed according to “Classification and identification of actinomycetes” (Japan Society for Actinomycetes 2001).
ISP (International Streptomyces Project) Medium No. 2 and No. 4 used for the identification of the fungus were purchased from DIFCO. In addition, as the ISP medium No. 3, No. 5, and No. 7 medium, the standard actinomycetes medium Daigo No. 3, No. 5, and No. 7 of the Japanese Actinomycetes Society are designated as Nippon Pharmaceutical Co., Ltd. We purchased more.
As a standard, the color tone was determined using “New Color Encyclopedia” (Japan Color Research Institute, 1987), and the code was also written in parentheses along with the color standard name. The observation results in various media after culturing at 32 ° C. for 4 weeks are shown.
本菌株の分類学的性質は下記の通りであった。
（１）形態学的性質
図１に、TP-A0877菌株の走査型電子顕微鏡写真を示す。図１に示すように、気菌糸はループを形成し、胞子は表面がシワ状の楕円球の胞子が連鎖するものであった。
（２）寒天培地における生育状態
TP-A0877菌株の各種寒天培地上の生育状態を表６に示した。色調は標準として、『新色事典』（財団法人日本色彩研究所 1987年）を用いて決定し、色標名とともに括弧内にそのコードを併せて記した。観察は32℃、4週間目の各種培地における結果である。
本菌株はIPS2、ISP3、ISP4上では良く生育したが、ISP5上では余り良く生育しなかった。ISP2及びISP4上では、気菌糸は見られなかった。また拡散色素は、ISP2上以外では見られなかった。
The taxonomic properties of this strain were as follows.
(1) Morphological properties FIG. 1 shows a scanning electron micrograph of the TP-A0877 strain. As shown in FIG. 1, the aerial hyphae formed a loop, and the spores were linked with wrinkled elliptical spherical spores.
(2) Growth state on agar medium
Table 6 shows the growth state of the TP-A0877 strain on various agar media. As a standard, the color tone was determined using “New Color Encyclopedia” (Japan Color Research Institute, 1987), and the code was also written in parentheses along with the color standard name. The observation is the result in various media at 32 ° C. for 4 weeks.
This strain grew well on IPS2, ISP3, and ISP4, but did not grow well on ISP5. No aerial hyphae were seen on ISP2 and ISP4. Also, no diffusing dye was seen except on ISP2.
（３）生理的性質
（ａ）生育温度範囲：ベネット寒天培地において10℃〜39℃の温度範囲で生育し、31℃〜34℃付近で良好に生育した。
（ｂ）メラニン様色素の生成：陽性であった。
（４）炭素原の利用
利用可能な炭素源は、D-グルコース、D-キシロース、L-アラビノースであった。一方、利用可能でない糖は、D-フラクトース、シュクロース、L-ラムノース、ラフィノース、イノシトール、及びD-マンニトールであった。
（５）菌体分析
全菌体加水分解物中のジアミノピメリン酸はLL型を含んでいた。また、全菌体糖としては、グルコースとマンノースを含んでいた。
（６）16S rDNA配列の解析
この菌の16S rDNAの全塩基配列（1460塩基対)を解析し、DNAデータベース登録番号FM875937(EMBL Nucleotide Sequence Database)と比較したところ、Streptomyces fragilisに99.8%の相同性を示した
以上の分類学的性質から本菌株をStreptomyces fragilisと同定した。
(3) Physiological properties (a) Growth temperature range: Grow in a temperature range of 10 ° C to 39 ° C on a Bennett agar medium, and grow well in the vicinity of 31 ° C to 34 ° C.
(B) Formation of melanin-like pigment: positive.
(4) Use of carbon source Available carbon sources were D-glucose, D-xylose, and L-arabinose. On the other hand, the unavailable sugars were D-fructose, sucrose, L-rhamnose, raffinose, inositol, and D-mannitol.
(5) Cell analysis Diaminopimelic acid in the whole cell hydrolyzate contained LL type. Moreover, glucose and mannose were contained as whole microbial sugar.
(6) Analysis of 16S rDNA sequence The entire base sequence (1460 base pairs) of 16S rDNA of this bacterium was analyzed and compared with DNA database accession number FM875937 (EMBL Nucleotide Sequence Database). This strain was identified as Streptomyces fragilis from the above taxonomic characteristics.
（実施例２）上記微生物が産生する二次代謝産物の探索
100ｍLの種母培地Ｖ-22液体培地（溶性でんぷん 1.0％、グルコース 0.5％、NZ-case 0.3％、酵母エキス 0.2％、Tryptone 0.5％、KH2PO4 0.1％、MgSO4・7H2O 0.05％、CaCO3 0.3％を含む）を入れたＫ型フラスコに、TP-A0877を接種し、200rpm、30℃で、ロータリーシェーカー（いわしやサンキ社製）を用いて、４日間培養し、種母培養液を得た。
得られた種母培養液を３mLずつ、100ｍLの生産培地A-3M（グルコース 0.5％、グリセロール 2.0％、溶性でんぷん 2.0％、Pharmamedia 1.5％、酵母エキス 0.3％、Diaion HP-20 1.0％を含む）を入れたＫ型フラスコ（20個）に移植し、200rpm、30℃で、ロータリーシェーカー（いわしやサンキ社製）を用いて、６日間振とう培養した。
(Example 2) Search for secondary metabolites produced by the microorganism
100mL seed medium V-22 liquid medium (soluble starch 1.0%, glucose 0.5%, NZ-case 0.3%, yeast extract 0.2%, Tryptone 0.5%, KH 2 PO 4 0.1%, MgSO 4 · 7H 2 O 0.05% Inoculate TP-A0877 into a K-type flask containing CaCO 3 0.3%) and incubate at 200rpm, 30 ° C for 4 days using a rotary shaker (Iwashiya Sanki). Seed culture A liquid was obtained.
3 mL each of the obtained seed culture solution, 100 mL of production medium A-3M (containing 0.5% glucose, 2.0% glycerol, 2.0% soluble starch, 1.5% Pharmamedia, 0.3% yeast extract, 1.0% Diaion HP-20) The mixture was transplanted into K-shaped flasks (20 pieces) containing sucrose, and cultured with shaking at 200 rpm and 30 ° C. using a rotary shaker (manufactured by Iwashi and Sanki) for 6 days.
培養終了後、それぞれのＫ型フラスコから培地を集め、２Ｌの培養液に等量のブタノールを加えて１時間、30℃で、ロータリーシェーカーを用いて攪拌した。この溶液を3,000回転（遠心機：HITACHI himac CR20、ロータ：HITACHI R12A、いずれも日立製作所（株）製）で、25℃にて10分間遠心し、固相（菌体部分）と液相（ブタノール相）とに分離した。
得られたブタノール相をロータリーエバポレーター（イワキ社製）を用いて減圧濃縮し、抽出物（1.4ｇ）を得た。
次いで、抽出物を順相シリカゲルカラムクロマトグラフィーに供し、下記表７に示す溶出溶媒を順次用いてステップグラジエントで、溶出し分画した（カラムサイズ：２ｘ１５ｃｍ、フラクションボリューム：２０ｍＬ）。
After completion of the culture, the medium was collected from each K-shaped flask, and an equal amount of butanol was added to 2 L of the culture solution and stirred for 1 hour at 30 ° C. using a rotary shaker. This solution was centrifuged at 3,000 rpm (centrifuge: HITACHI himac CR20, rotor: HITACHI R12A, both manufactured by Hitachi, Ltd.) for 10 minutes at 25 ° C., and the solid phase (cell part) and liquid phase (butanol) Phase).
The obtained butanol phase was concentrated under reduced pressure using a rotary evaporator (manufactured by Iwaki Co., Ltd.) to obtain an extract (1.4 g).
Subsequently, the extract was subjected to normal phase silica gel column chromatography, and eluted and fractionated with a step gradient using the elution solvents shown in Table 7 below (column size: 2 × 15 cm, fraction volume: 20 mL).
上記の順相シリカゲルカラムクロマトグラフィーの溶出画分を、ＨＰＬＣにて分析した。ＨＰＬＣ条件は下記の通りとした。
＜HPLC条件＞
装置：HEWLETTPACKARD 1090
溶離液：アセトニトリル：0.15% KH2PO4水溶液(pH 3.5)=15：85〜85：15
カラム：MICROSORB-MV 75ｘ4.6mm
流 速：1.2mL/min
カラム温度：室温
The elution fraction of the above normal phase silica gel column chromatography was analyzed by HPLC. The HPLC conditions were as follows.
<HPLC conditions>
Device: HEWLETTPACKARD 1090
Eluent: Acetonitrile: 0.15% KH 2 PO 4 aqueous solution (pH 3.5) = 15: 85 to 85:15
Column: MICROSORB-MV 75x4.6mm
Flow rate: 1.2mL / min
Column temperature: room temperature
得られた画分を分析した結果、クロロホルム：メタノール＝２：１で溶出した画分に、abyssomicin Iが存在することが確認された。このため、この画分を減圧濃縮し、粗精製物（126.5mg）を得た。
次に、この粗精製物を以下の条件で、逆相ODSカラムクロマトグラフィーを用いて分画した。
＜クロマトグラフィー条件＞
カラムサイズ：4.5ｘ20ｃｍ
担体：COSMOSIL 75C18-Prep （nacalai tesque 社製）
ポンプ：PUMP540（山善）
フラクションサイズ：300ｍＬ
溶出溶媒：アセトニトリル：0.1％ギ酸水溶液＝2:8,3:7,4:6,5:5,6:4,7:3,8:2
As a result of analyzing the obtained fraction, it was confirmed that abyssomicin I was present in the fraction eluted with chloroform: methanol = 2: 1. Therefore, this fraction was concentrated under reduced pressure to obtain a crude product (126.5 mg).
Next, this crude product was fractionated using reverse phase ODS column chromatography under the following conditions.
<Chromatography conditions>
Column size: 4.5x20cm
Carrier: COSMOSIL 75C18-Prep (manufactured by nacalai tesque)
Pump: PUMP540 (Yamazen)
Fraction size: 300mL
Elution solvent: Acetonitrile: 0.1% formic acid aqueous solution = 2: 8, 3: 7, 4: 6, 5: 5, 6: 4, 7: 3, 8: 2
次に、得られた各溶出画分を以下の条件でHPLCにより分析した。
＜HPLC条件＞
装置：HEWLETTPACKARD 1090
溶離液：アセトニトリル：0.15％KH2PO4水溶液(pH3.5)＝15：85〜85：15
流 速：1.2mL/min
カラム：MICROSORB-MV 75ｘ4.6mm
カラム温度：室温
Next, each obtained elution fraction was analyzed by HPLC under the following conditions.
<HPLC conditions>
Device: HEWLETTPACKARD 1090
Eluent: Acetonitrile: 0.15% KH 2 PO 4 aqueous solution (pH 3.5) = 15: 85 to 85:15
Flow rate: 1.2mL / min
Column: MICROSORB-MV 75x4.6mm
Column temperature: room temperature
その結果、アセトニトリル：0.1％ギ酸水溶液＝4：6画分にabyssomicin Iが存在することが確認された。このため、この画分をさらに減圧濃縮し、酢酸エチルで抽出し、有機相を減圧濃縮することにより、abyssomicin I（31.3mg）を得た。
Abyssomicin Iは、無色油状物質として得られた。紫外吸収スペクトルでは215nm及び255nmに吸収極大を示した。赤外吸収スペクトルでは、3389cm-1にOH伸縮振動に由来する吸収帯が、1743-1、1670-1、1604cm-1にカルボニル伸縮振動に由来する吸収帯が観測された。また、高分解能ESI-TOFMSにより、[M-H]-がm/z 347.1508に検出された。以上より、abyssomicin Iの分子式をC19H24O6と決定した。
Abyssomicin Iの物理化学的性質を以下の表８に示す。
As a result, it was confirmed that abyssomicin I was present in the acetonitrile: 0.1% formic acid aqueous solution = 4: 6 fraction. Therefore, this fraction was further concentrated under reduced pressure, extracted with ethyl acetate, and the organic phase was concentrated under reduced pressure to obtain abyssomicin I (31.3 mg).
Abyssomicin I was obtained as a colorless oil. The ultraviolet absorption spectrum showed absorption maximums at 215 nm and 255 nm. The infrared absorption spectrum, absorption bands derived from the OH stretching vibration 3389Cm -1 is 1743 -1, 1670 -1, the absorption band derived from the carbonyl stretching vibration 1604Cm -1 was observed. Further, by high resolution ESI-TOFMS, [MH] - it was detected in m / z 347.1508. Based on the above, the molecular formula of abyssomicin I was determined as C 19 H 24 O 6 .
The physicochemical properties of Abyssomicin I are shown in Table 8 below.
また、abyssomicin IをBruker Avance 500 NMRスペクトロメーター（500MHz、ブルカー社製)を用いて1H NMR及び二次元NMRスペクトルを、並びにBruker Avance 400 NMRスペクトロメーター（100 MHz、ブルカー社製)を用いて13C NMRスペクトルを分析した。
1H-NMRでは、メチルが３個、メチレンが３個、メチンが７個に相当するプロトンシグナルが観測された。13C-NMRでは、0〜55ppmの高磁場に９本のシグナル、70〜90ppmには酸素の結合した炭素シグナルが４本、さらに低磁場にはオレフィン炭素のシグナルが３本、酸素の結合したオレフィン炭素もしくはカルボニル炭素が３本、合計１９本のシグナルが観測された（CD3OD）。各スペクトルデータを、下記表９に示す（CD3OD）。
Further, Abyssomicin I a Bruker Avance 500 NMR spectrometer (500 MHz, manufactured by Bruker) 1 H NMR and two-dimensional NMR spectra with and Bruker Avance 400 NMR spectrometer (100 MHz, manufactured by Bruker) using 13 C NMR spectra were analyzed.
In 1 H-NMR, proton signals corresponding to 3 methyl, 3 methylene and 7 methine were observed. 13 C-NMR shows 9 signals in a high magnetic field of 0 to 55 ppm, 4 carbon signals with oxygen bound at 70 to 90 ppm, and 3 signals of olefin carbon with oxygen bound in a low magnetic field. A total of 19 signals were observed for 3 olefinic or carbonyl carbons (CD 3 OD). Each spectrum data is shown in Table 9 below (CD 3 OD).
次いで、炭素と水素の直接結合をHMQCスペクトルにより決定した（図２参照）。COSYスペクトルにより、図３に太線で示すプロトン間のつながりを決定した。
さらにHMBCスペクトルの解析により、図３に示すプロトン−炭素間の相関が観測された。観測されたHMBC相関のすべてを表８に示した。
さらにNOESY相関が図４に示すように観測されたことより、abyssomicin Iの相対立体配置を図２に示すように決定した。この構造は、高分解能EST-TOFMS分析の結果とも一致した。図２に、abyssomicin Iの構造（絶対配置）およびナンバリングを示した。
以上より、上記のようにして得られた化合物は、abyssomicinの新規類縁体であることから、これをabyssomicin Iと命名した。
The direct bond between carbon and hydrogen was then determined by HMQC spectrum (see FIG. 2). Based on the COZY spectrum, the connection between protons indicated by the bold line in FIG. 3 was determined.
Further, the proton-carbon correlation shown in FIG. 3 was observed by analysis of the HMBC spectrum. All the observed HMBC correlations are shown in Table 8.
Furthermore, since the NOESY correlation was observed as shown in FIG. 4, the relative configuration of abyssomicin I was determined as shown in FIG. This structure was consistent with the results of high-resolution EST-TOFMS analysis. FIG. 2 shows the structure (absolute configuration) and numbering of abyssomicin I.
From the above, since the compound obtained as described above is a novel analog of abyssomicin, it was named abyssomicin I.
（実施例３）abyssomicin Iの誘導体の合成
上記のようにabyssomicin Iの絶対配置の決定を行った後に、二酸化マンガンを用いて、abyssomicin Iの7位の水酸基を選択的にケトンへ酸化し、誘導体であるabyssomicin I-2を得た。
(Example 3) Synthesis of derivative of abyssomicin I After determining the absolute configuration of abyssomicin I as described above, manganese dioxide was used to selectively oxidize the hydroxyl group at the 7-position of abyssomicin I to a ketone. I got abyssomicin I-2.
Abyssomicin I（15mg, 0.04mmol）をクロロホルム（８mL）に溶解し、二酸化マンガン（0.10ｇ）を加えて、室温で24時間、卓上スターラーを用いて激しく撹拌した。次に、この反応液をセライト上で濾過して二酸化マンガンを除去し、濾液を、ロータリーエバポレーターを用いて減圧濃縮した。
減圧濃縮した残渣を、シリカゲルカラムクロマトグラフィー（溶離液：ヘキサン−酢酸エチル＝10：１〜１：２）により精製し、abyssomicin I-2（7.6mg、収率55％）を得た。
Abyssomicin I (15 mg, 0.04 mmol) was dissolved in chloroform (8 mL), manganese dioxide (0.10 g) was added, and the mixture was vigorously stirred using a desktop stirrer at room temperature for 24 hours. Next, this reaction solution was filtered on celite to remove manganese dioxide, and the filtrate was concentrated under reduced pressure using a rotary evaporator.
The residue concentrated under reduced pressure was purified by silica gel column chromatography (eluent: hexane-ethyl acetate = 10: 1 to 1: 2) to obtain abyssomicin I-2 (7.6 mg, yield 55%).
実施例２で使用した装置を用いて、1H NMR及び13C NMRのスペクトルデータ、及び質量分析（HR-ESI-TOFMS）データを得た。以下に、それぞれのデータを示す。
1H NMR (500 MHz, CDCl3)δ 1.09(3H, d, J = 6.8 Hz, H-17), 1.11(3H, d, J = 7.6 Hz, H-19), 1.53(1H, dd, J = 12.6, 2.6 Hz, H-14), 1.57(1H, dddd, J = 16.1, 4.5, 4.5, 4.5 Hz, H-5), 1.63(3H, s, H-18), 1.90(1H, dddd, J = 16.3, 12.9, 3.7, 3.7 Hz, H-5), 2.38(1H, m, H-6), 2.43(1H, ddd, J = 13.5, 13.1, 4.3 Hz, H-4), 2.66(1H, ddq, J = 11.0, 2.6, 7.6 Hz, H-13), 2.75(1H, dd, J = 12.6, 11.0 Hz, H-14), 3.15(1H, dd, J = 6.9, 2.2 Hz, H-10), 3.21(1H, ddd, J = 13.5, 4.5, 4.0 Hz, H-4), 3.44(1H, d, J = 4.2 Hz, OH-11), 3.81(1H, br.s, H-11), 5.80(1H, d, J = 16.7 Hz, H-8), 6.35(1H, dd, J = 16.7, 6.9 Hz, H-9)
Using the apparatus used in Example 2, 1 H NMR and 13 C NMR spectral data and mass spectrometry (HR-ESI-TOFMS) data were obtained. Each data is shown below.
1 H NMR (500 MHz, CDCl 3 ) δ 1.09 (3H, d, J = 6.8 Hz, H-17), 1.11 (3H, d, J = 7.6 Hz, H-19), 1.53 (1H, dd, J = 12.6, 2.6 Hz, H-14), 1.57 (1H, dddd, J = 16.1, 4.5, 4.5, 4.5 Hz, H-5), 1.63 (3H, s, H-18), 1.90 (1H, dddd, J = 16.3, 12.9, 3.7, 3.7 Hz, H-5), 2.38 (1H, m, H-6), 2.43 (1H, ddd, J = 13.5, 13.1, 4.3 Hz, H-4), 2.66 (1H , ddq, J = 11.0, 2.6, 7.6 Hz, H-13), 2.75 (1H, dd, J = 12.6, 11.0 Hz, H-14), 3.15 (1H, dd, J = 6.9, 2.2 Hz, H- 10), 3.21 (1H, ddd, J = 13.5, 4.5, 4.0 Hz, H-4), 3.44 (1H, d, J = 4.2 Hz, OH-11), 3.81 (1H, br.s, H-11 ), 5.80 (1H, d, J = 16.7 Hz, H-8), 6.35 (1H, dd, J = 16.7, 6.9 Hz, H-9)
13C NMR (100 MHz, CDCl3)δ 16.0(C-17), 16.3(C-19), 19.7(C-18), 28.3(C-13), 30.2(C-5), 34.4(C-14), 42.2(C-6), 42.4(C-4), 51.1(C-10), 75.4(C-11), 79.9(C-15), 88.1(C-12), 104.2(C-2), 135.2(C-8), 136.2(C-9), 169.4(C-1), 183.6(C-16), 194.5(C-3), 204.6(C-7)
HR-ESI-TOFMS m/z 345.1349 [M-H]- (calc for C19H23O6 345.1344)
13 C NMR (100 MHz, CDCl 3 ) δ 16.0 (C-17), 16.3 (C-19), 19.7 (C-18), 28.3 (C-13), 30.2 (C-5), 34.4 (C- 14), 42.2 (C-6), 42.4 (C-4), 51.1 (C-10), 75.4 (C-11), 79.9 (C-15), 88.1 (C-12), 104.2 (C-2 ), 135.2 (C-8), 136.2 (C-9), 169.4 (C-1), 183.6 (C-16), 194.5 (C-3), 204.6 (C-7)
HR-ESI-TOFMS m / z 345.1349 [MH] - (calc for C 19 H 23 O 6 345.1344)
（実施例４）Abyssomicin I-2のMTPAエステル誘導体の合成
（１）Abyssomicin I-2の(R)-MTPAエステル誘導体の合成
Abyssomicin I-2（１mg, 2.8mmol）の無水塩化メチレン（100μL）溶液に、(R)-メトキシトリフルオロフェニル酢酸（(R)-MTPA, 1.5mg, 6.4μmol）、ジシクロヘキシルアミド（２mg, 9.7μmol）、ジメチルアミノピリジン（１mg, 8.2μmol）を加えて、室温で24時間反応させた。
反応液をシリカゲルカラムクロマトグラフィー（溶離液：ヘキサン：酢酸エチル＝10：１〜１：２）により精製し、(R)-MTPAエステル誘導体（１mg）を得た。
(Example 4) Synthesis of MTPA ester derivative of Abyssomicin I-2 (1) Synthesis of (R) -MTPA ester derivative of Abyssomicin I-2
To a solution of Abyssomicin I-2 (1 mg, 2.8 mmol) in anhydrous methylene chloride (100 μL), (R) -methoxytrifluorophenylacetic acid ((R) -MTPA, 1.5 mg, 6.4 μmol), dicyclohexylamide (2 mg, 9.7 μmol) ) And dimethylaminopyridine (1 mg, 8.2 μmol) were added and allowed to react at room temperature for 24 hours.
The reaction solution was purified by silica gel column chromatography (eluent: hexane: ethyl acetate = 10: 1 to 1: 2) to obtain (R) -MTPA ester derivative (1 mg).
実施例２で使用した装置を用いて、1H NMRのスペクトルデータ、及び質量分析（HR-ESI-TOFMS）データを得た。以下に、それぞれのデータを示す。 Using the apparatus used in Example 2, 1 H NMR spectrum data and mass spectrometry (HR-ESI-TOFMS) data were obtained. Each data is shown below.
1H NMR (500 MHz, CDCl3)δ 1.078(3H, d, J = 7.2 Hz, H-19), 1.126(3H, d, J = 6.8 Hz, H-17), 1.481(3H, s, H-18), 1.570(1H, m, H-14), 1.625(1H, m, H-5), 1.893(1H, m, H-5), 2.320(1H, m, H-13), 2.427(1H, m, H-6), 2.482(1H, td, J = 13.2, 4.4 Hz, H-4), 2.534(1H, dd, J = 13.8, 10.0 Hz, H-14), 3.034(1H, dd, J = 6.7, 2.2 Hz, H-10), 3.179(1H, ddd, J = 13.5, 4.9, 3.8 Hz, H-4), 5.036(1H, d, J = 2.2 Hz, H-11), 5.995(1H, d, J = 16.5 Hz, H-8), 6.335(1H, dd, J = 16.5, 7.0 Hz, H-9)
HR-ESI-TOFMS m/z 561.1742 [M-H]- (calc for C29H28F3O8 561.1742)
1 H NMR (500 MHz, CDCl 3 ) δ 1.078 (3H, d, J = 7.2 Hz, H-19), 1.126 (3H, d, J = 6.8 Hz, H-17), 1.481 (3H, s, H -18), 1.570 (1H, m, H-14), 1.625 (1H, m, H-5), 1.893 (1H, m, H-5), 2.320 (1H, m, H-13), 2.427 ( 1H, m, H-6), 2.482 (1H, td, J = 13.2, 4.4 Hz, H-4), 2.534 (1H, dd, J = 13.8, 10.0 Hz, H-14), 3.034 (1H, dd , J = 6.7, 2.2 Hz, H-10), 3.179 (1H, ddd, J = 13.5, 4.9, 3.8 Hz, H-4), 5.036 (1H, d, J = 2.2 Hz, H-11), 5.995 (1H, d, J = 16.5 Hz, H-8), 6.335 (1H, dd, J = 16.5, 7.0 Hz, H-9)
HR-ESI-TOFMS m / z 561.1742 [MH] - (calc for C 29 H 28 F 3 O 8 561.1742)
（２）Abyssomicin I-2の(S)-MTPAエステル誘導体の合成
(R)-MTPAエステル誘導体と同様にして、abyssomicin I-2の(S)-MTPAエステル誘導体を合成した。
実施例２で使用した装置を用いて、1H NMRのスペクトルデータ、及び質量分析（HR-ESI-TOFMS）データを得た。以下に、それぞれのデータを示す。
(2) Synthesis of (S) -MTPA ester derivatives of Abyssomicin I-2
The (S) -MTPA ester derivative of abyssomicin I-2 was synthesized in the same manner as the (R) -MTPA ester derivative.
Using the apparatus used in Example 2, 1 H NMR spectrum data and mass spectrometry (HR-ESI-TOFMS) data were obtained. Each data is shown below.
1H NMR (500 MHz, CDCl3)δ 0.928(3H, d, J = 7.2 Hz, H-19), 1.139(3H, d, J = 6.9 Hz, H-17), 1.427(3H, s, H-18), 1.520(1H, m, H-14), 1.898(1H, m, H-5), 2.282(1H, m, H-13), 2.38(2H, m, H-4 and H-5), 2.440(1H, dd, J = 12.9, 11.0 Hz, H-14), 2.555(1H, m, H-6), 2.959(1H, dd, J = 7.4, 2.4 Hz, H-10), 4.868(1H, d, J = 2.4 Hz, H-11), 5.926(1H, dd, J = 11.7, 7.3 Hz, H-4), 6.034(1H, d, J = 16.7 Hz), 6.570(1H, dd, J = 16.7, 7.3 Hz, H-9)
HR-ESI-TOFMS m/z 585.1704 [M+Na]+ (calc for C29H29F3NaO8 585.1707)
1 H NMR (500 MHz, CDCl 3 ) δ 0.928 (3H, d, J = 7.2 Hz, H-19), 1.139 (3H, d, J = 6.9 Hz, H-17), 1.427 (3H, s, H -18), 1.520 (1H, m, H-14), 1.898 (1H, m, H-5), 2.282 (1H, m, H-13), 2.38 (2H, m, H-4 and H-5 ), 2.440 (1H, dd, J = 12.9, 11.0 Hz, H-14), 2.555 (1H, m, H-6), 2.959 (1H, dd, J = 7.4, 2.4 Hz, H-10), 4.868 (1H, d, J = 2.4 Hz, H-11), 5.926 (1H, dd, J = 11.7, 7.3 Hz, H-4), 6.034 (1H, d, J = 16.7 Hz), 6.570 (1H, dd , J = 16.7, 7.3 Hz, H-9)
HR-ESI-TOFMS m / z 585.1704 [M + Na] + (calc for C 29 H 29 F 3 NaO 8 585.1707)
1H-NMRデータを解析した結果、(S)-MTPAエステルの化学シフト値δSと(R)-MTPAエステルの化学シフト値δRの差Δδは、MTPAの結合した11位の左側と右側で逆符合となった（図５参照）。このため、11位の絶対配置はRと決定され、abyssomicin I-2およびIの絶対配置が、下記式（Ｖ）に示すように決定された。
以上のようにして得たabyssomicin Iの誘導体を、abyssomicin I-2と命名した。
As a result of analysis of 1 H-NMR data, the difference Δδ between the chemical shift value δ S of the ( S ) -MTPA ester and the chemical shift value δ R of the (R) -MTPA ester is the left side and the right side of the 11th position where MTPA is bound. The sign was reversed (see FIG. 5). For this reason, the absolute configuration at the 11th position was determined to be R, and the absolute configurations of abyssomicin I-2 and I were determined as shown in the following formula (V).
The derivative of abyssomicin I obtained as described above was named abyssomicin I-2.
（実施例５）Abyssomicin I及びabyssomicin I-2の生物活性
以上のようにして得られた新規化合物abyssomicin I及びabyssomicin I-2の生物活性を、以下のようにして検討した。
（１）抗菌活性
検定菌として、下記の表１０に示す細菌（２種）及び酵母（３種）、並びに培地を使用して、abyssomicin I及びabyssomicin I-2の抗菌活性を検討した。
(Example 5) Biological activity of Abyssomicin I and abyssomicin I-2 The biological activities of the novel compounds abyssomicin I and abyssomicin I-2 obtained as described above were examined as follows.
(1) Antibacterial activity The antibacterial activity of abyssomicin I and abyssomicin I-2 was examined using the bacteria (2 types) and yeasts (3 types) shown in Table 10 below and a medium as the test bacteria.
これらの菌株を、寒天平板上で培養してコロニーを形成させ、各コロニーを白金耳にかき取り、８mLの上記各液体培地に懸濁した後、30℃にて20時間振とう培養（120rpm）した。その後、遠心分離により集菌し（3000rpm, ５分）、生理食塩水に懸濁し、OD660を測定した。検量線をもとに希釈倍率を決め、培地にて最終濃度が、１×105 cells/mLの菌液を調製した。
Abyssomicin I及びabyssomicin I-2それぞれの標準溶液（10mＭ、100倍溶液）を、DMSOを用いて調製した。
96ウェル滅菌平底プレートに上記の各培地を100μＬずつ分注し、各ウェルに100倍濃度とした上記の各標準溶液を、各濃度当たり3ウェルずつに、１〜0.5％添加した。このプレート上にはコントロールとして、化合物非添加のウェルを16個設けた。
These strains are cultured on an agar plate to form colonies. Each colony is scraped into a platinum loop and suspended in 8 mL of each liquid medium, and then shaken at 30 ° C. for 20 hours (120 rpm). did. Thereafter, cells were harvested by centrifugation (3000 rpm, 5 minutes), and suspended in physiological saline was measured OD 660. The dilution factor was determined based on the calibration curve, and a bacterial solution having a final concentration of 1 × 10 5 cells / mL was prepared in the medium.
Abyssomicin I and abyssomicin I-2 standard solutions (10 mM, 100-fold solution) were prepared using DMSO.
100 μL of each medium described above was dispensed into a 96-well sterilized flat bottom plate, and each standard solution having a 100-fold concentration was added to each well at 1 to 0.5% in 3 wells per concentration. As a control, 16 wells without compound addition were provided on this plate.
各ウェルに、上記のように濃度調整をした菌液を10％添加（最終濃度：１×104 cells/mL）し、撹拌した。その後、大腸菌は37℃で、また、大腸菌以外の菌及び酵母は30℃で、それぞれ20時間、恒温槽中にて静置培養した。
培養後の菌液が均一になるよう、Biomek2000（Beckman社製）を用いて撹拌した後に、各ウェルにおける菌の増殖を、Ｅmax（Molecular Devices社製）を用いて、OD650で測定した。
コントロール（化合物非添加区）の増殖を100％として、化合物添加区の増殖程度を決定した。結果を表１１に示す。
10% of the bacterial solution whose concentration was adjusted as described above was added to each well (final concentration: 1 × 10 4 cells / mL) and stirred. Thereafter, E. coli was cultured at 37 ° C., and bacteria and yeasts other than E. coli were cultured at 30 ° C. for 20 hours in a thermostatic bath.
After stirring using Biomek2000 (Beckman) so that the bacterial solution after culture was uniform, the growth of the bacteria in each well was measured at OD 650 using Emax (Molecular Devices).
The growth degree of the compound addition group was determined with the growth of the control (non-compound addition group) as 100%. The results are shown in Table 11.
表１１に示したように、abyssomicin Iはいずれの微生物に対しても抗菌活性を示さなかった。
一方、abyssomicin I-2はグラム陽性細菌であるMicrococcus luteusに対してのみ活性を示した。
As shown in Table 11, abyssomicin I did not show antibacterial activity against any microorganism.
On the other hand, abyssomicin I-2 showed activity only against Micrococcus luteus, a Gram-positive bacterium.
（２）基底膜浸潤阻害活性
Abyssomicin I及びabyssomicin I-2の基底膜浸潤阻害活性の測定を、membrane invasion culture system (MICS, Hendrix et al (1985) Clin. Exp. Metastasis 3:221-223)(癌と化学療法 31(4)：512−517 2004)を用いて行った。
トランスウェル・カルチャー・チャンバー（Transwell cell culture chamber, Corning Coster社製）に、メンブランフィルタ（孔径8.0μm：Nucleopore社製）を接着し、メンブランフィルタの外側に１μｇのフィブロネクチン（Iwaki社製）をコーティングした。クリーンベンチ内で２〜３時間、フィブロネクチンコーティングを乾燥させた。
(2) Basement membrane infiltration inhibitory activity
Membrane invasion culture system (MICS, Hendrix et al (1985) Clin. Exp. Metastasis 3: 221-223) (cancer and chemotherapy 31 (4) : 512-517 2004).
A membrane filter (pore size 8.0 μm: manufactured by Nucleopore) was adhered to a Transwell cell culture chamber (manufactured by Corning Coster), and 1 μg of fibronectin (manufactured by Iwaki) was coated on the outside of the membrane filter. . The fibronectin coating was dried in a clean bench for 2-3 hours.
その後、メンブレンフィルタの内側に、マトリゲル（Matrigel、BD Science社製）１μｇをコーティングし、クリーンベンチ内で終夜乾燥させた。24ウェルプレートの各ウェルに、DMSOに溶解した上記の化合物をそれぞれ試料として加え、600μLの0.1％牛血清アルブミン（BSA）含有RPMI1640培地を各ウェルに入れた。
マウス大腸癌由来Colon 26-L5細胞を４×104 cells/100μLとなるよう各ウェルに加え、DMSOで溶解したサンプルを、24ウェルプレートの各ウェルにおける最終濃度になるよう加えた。この細胞懸濁液を、フィブロネクチンとマトリゲルとを上記のようにしてコーティングしたトランスウェル・チャンバー内に、100μLずつ分注した。
Thereafter, 1 μg of Matrigel (Matrigel, manufactured by BD Science) was coated on the inner side of the membrane filter and dried overnight in a clean bench. To each well of a 24-well plate, the above compound dissolved in DMSO was added as a sample, and 600 μL of 0.1% bovine serum albumin (BSA) -containing RPMI1640 medium was added to each well.
Mouse colon cancer-derived Colon 26-L5 cells were added to each well so as to be 4 × 10 4 cells / 100 μL, and a sample dissolved in DMSO was added to a final concentration in each well of a 24-well plate. 100 μL of this cell suspension was dispensed into a transwell chamber coated with fibronectin and Matrigel as described above.
このトランスウェル・チャンバーを24ウェルプレートに設置し、５％CO2インキュベータ中で37℃にて８時間培養した。培養終了後、設置したチャンバーを24ウェルプレートから外し、エタノールに１分間つけて、細胞を固定した。次いで、ヘマトキシリンに３分間、エオシンに30秒間、それぞれ浸漬して、細胞を染色した。水洗後、綿棒でチャンバー内をふき取り、染色液が付着しなかった細胞を除去した。
このようにして得られたメンブラン表面を乾燥させ、光学顕微鏡でメンブランの中心とその周辺４視野の計５視野について、基底膜バリアーを破って浸潤した癌細胞の数をカウントした。その平均値を、基底膜の浸潤阻害活性の指標とした。
コントロールとなるウェルにはDMSOのみを添加し、コントロールを100％としたときの各試料の各濃度における基底膜浸潤阻害活性を測定した。
The transwell chamber was placed in a 24-well plate and cultured at 37 ° C. for 8 hours in a 5% CO 2 incubator. After completion of the culture, the installed chamber was removed from the 24-well plate and placed in ethanol for 1 minute to fix the cells. Next, the cells were stained by immersion in hematoxylin for 3 minutes and in eosin for 30 seconds. After washing with water, the inside of the chamber was wiped off with a cotton swab to remove cells to which the staining solution did not adhere.
The membrane surface thus obtained was dried, and the number of cancer cells infiltrated by breaking through the basement membrane barrier was counted for a total of 5 fields including the center of the membrane and 4 fields around it by an optical microscope. The average value was used as an index of the basement membrane infiltration-inhibiting activity.
Only DMSO was added to control wells, and the basement membrane invasion inhibitory activity at each concentration of each sample when the control was taken as 100% was measured.
Abyssomicin Iは、マウス大腸癌由来colon 26-L5細胞のマトリジェルへの浸潤をIC50値11μMで阻害した。一方、abyssomicin I-2のIC50値は0.21μMであり、浸潤阻害活性はabyssomicin Iに比べて約50倍上昇していた。
この結果は、先の抗菌活性と同様に共役二重結合が重要であることを示唆する。また、誘導体合成により、天然物よりも強力な浸潤阻害剤を得られることが示された。
以上より、abyssomicin Iの構造は、癌細胞の浸潤阻害剤の新しいリード母核と考えられる。
Abyssomicin I inhibited the invasion of mouse colon cancer-derived colon 26-L5 cells into matrigel with an IC 50 value of 11 μM. On the other hand, the IC 50 value of abyssomicin I-2 was 0.21 μM, and the invasion inhibitory activity was about 50 times higher than that of abyssomicin I.
This result suggests that conjugated double bonds are as important as the previous antimicrobial activity. In addition, it was shown that a stronger invasion inhibitor than natural products can be obtained by derivative synthesis.
Based on the above, the structure of abyssomicin I is considered to be a new lead nucleus of cancer cell invasion inhibitor.
（３）細胞遊走阻害活性
細胞遊走阻害試験は、メンブランフィルターの内側にマトリゲルをコーティングしていない点を除いて、基底膜浸潤阻害活性測定と同様の方法により行った。
その結果、abyssomicin Iはマウス大腸癌由来colon 26-L5細胞の遊走を、10μg/mLで30％阻害したが、IC50値は0.87μMであった。
この結果、abyssomicin I-2では、細胞遊走阻害活性も増強されていることが示された。
(3) Cell migration inhibitory activity The cell migration inhibitory test was performed by the same method as the measurement of the basement membrane invasion inhibitory activity, except that the inside of the membrane filter was not coated with matrigel.
As a result, abyssomicin I inhibited the migration of mouse colon cancer-derived colon 26-L5 cells by 30% at 10 μg / mL, but the IC 50 value was 0.87 μM.
As a result, abyssomicin I-2 was shown to have enhanced cell migration inhibitory activity.
（４）細胞傷害活性
WST-1細胞を使用するCell counting Kit（和光純薬工業（株））を用いて、細胞傷害活性の測定を行った。10％牛胎児血清を含有するRPMI1640培地に、マウス大腸癌由来Colon 26 Ｌ−５細胞を10×104 cells/mLになるように懸濁した。
DMSOで溶解した上記の化合物を10〜1000μg/mLでこの細胞懸濁液に添加し、96ウェルマイクロプレートに、100μLずつ加えた（化合物の終濃度は、0.1〜10μg/mL）。
５％CO2インキュベータ中にて、37℃で24時間培養した後に、各ウェルにWST-1（和光純薬Cell counting Kit）を10μLずつ加え、37℃でさらに２時間培養した。
その後、ウェルプレートリーダー（サンライズクラシック、和光純薬工業（株））を用いて、450nmの吸光強度を測定した。コントロールにはDMSOのみを添加し、コントロールを100％としたときの各サンプルの細胞傷害活性を測定した。
(4) Cytotoxic activity
Cytotoxic activity was measured using a Cell counting Kit (Wako Pure Chemical Industries, Ltd.) using WST-1 cells. Mouse colon cancer-derived Colon 26 L-5 cells were suspended in RPMI1640 medium containing 10% fetal bovine serum so as to be 10 × 10 4 cells / mL.
The above compound dissolved in DMSO was added to this cell suspension at 10 to 1000 μg / mL, and 100 μL each was added to a 96-well microplate (the final concentration of the compound was 0.1 to 10 μg / mL).
After culturing at 37 ° C. for 24 hours in a 5% CO 2 incubator, 10 μL of WST-1 (Wako Pure Chemical Cell counting Kit) was added to each well, followed by further culturing at 37 ° C. for 2 hours.
Thereafter, the absorbance intensity at 450 nm was measured using a well plate reader (Sunrise Classic, Wako Pure Chemical Industries, Ltd.). Only DMSO was added to the control, and the cytotoxic activity of each sample was measured when the control was taken as 100%.
Abyssomicin Iは、マウス大腸癌由来colon 26-L5細胞の増殖を、10μg/mlで全く阻害しなかった。一方、abyssomicin I-2は、10μg/mlで細胞増殖を45％阻害しており、細胞傷害活性も、abyssomicin I-2で高くなっていた。
以上より、abyssomicin Iとその類縁体であるabyssomicin I-2とでは、構造の相違が生物活性に反映しているものと推察された。
Abyssomicin I did not inhibit the proliferation of mouse colon cancer-derived colon 26-L5 cells at 10 μg / ml. On the other hand, abyssomicin I-2 inhibited cell proliferation by 45% at 10 μg / ml, and its cytotoxic activity was also higher with abyssomicin I-2.
From the above, it was speculated that the structural difference between abyssomicin I and its analog abyssomicin I-2 is reflected in biological activity.
上述したabyssomicin Ｂ, Ｃ, atrop-Ｃ，Ｄ, Ｅ, Ｇ, 及びＨは、上記式（１）〜（７）で示すように、すべて４位にメチル基を有するが、12位にはメチル基を有していない。
一方、本願発明のabyssomicin Iは、逆に、４位にメチル基を有しておらず、12位にメチル基を有している。この相違は、生合成前駆体の取り込み様式が異なることによるためであり、abyssomicin Iの生産菌が他の生産菌とは異なる生合成上の特徴を有することに起因するものであることが上記の試験結果から結論付けられた。
本発明によれば、細胞傷害活性、基底膜浸潤阻害活性、細胞遊走阻害活性を有する新規な化合物を産生する微生物が提供される。
また、上記の新規化合物に加えて、細胞傷害活性、基底膜浸潤阻害活性、細胞遊走阻害活性を有する新規な化合物及び抗菌活性を有するその類縁体が提供される。
The abyssomicin B, C, atrop-C, D, E, G, and H described above all have a methyl group at the 4-position, as shown by the above formulas (1) to (7). Does not have a group.
On the other hand, abyssomicin I of the present invention, on the other hand, does not have a methyl group at the 4-position and has a methyl group at the 12-position. This difference is due to the difference in the biosynthetic precursor uptake mode, and it is due to the fact that the abyssomicin I producing bacteria have different biosynthetic characteristics from other producing bacteria. It was concluded from the test results.
ADVANTAGE OF THE INVENTION According to this invention, the microorganisms which produce the novel compound which has cytotoxic activity, basement membrane invasion inhibitory activity, and cell migration inhibitory activity are provided.
In addition to the above novel compounds, there are provided novel compounds having cytotoxic activity, basement membrane invasion inhibiting activity, cell migration inhibiting activity and analogs thereof having antibacterial activity.
本発明の微生物は、有用な二次代謝産物を産生し、医薬の分野において有用である。 The microorganism of the present invention produces a useful secondary metabolite and is useful in the field of medicine.
NITE P-779 NITE P-779
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