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

Description

  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.

  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).

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.

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.

S. Keller et al, Abyssomicin G, H, atrop-C from the Marine Verrucosispora Strain AB-18-032, J. Antibiot. (2007) 60: 391-394 X-M. Niu et al, Abyssomicin E, a highly functionalized polycyclic metabolite from Streptomyces species, Org. Lett. (2007) 9: 2437-2440

  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.

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.

  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.

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).

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.

  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.

  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.

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).

In the formula, R ³ represents a hydrogen atom or an acyl group.

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).

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.

  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.

FIG. 1 is a scanning electron micrograph of a microorganism belonging to the genus Streptomyces, TP-A0877. FIG. 2 is a diagram showing the structure (absolute configuration) and numbering of abyssomicin I, which is a novel compound of the present invention. FIG. 3 is a diagram showing the COZY correlation and HMBC correlation of the structure of abyssomicin I of the present invention. FIG. 4 is a diagram showing the NEOSY correlation of the structure of abyssomicin I of the present invention. FIG. 5 is a diagram when the absolute structure of abyssomicin I-2 of the present invention is determined by the new Mosher method.

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.

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.

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.

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.

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.

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.

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.

  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).

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).

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).

  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.

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.

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.

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.

“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”.

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.

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.

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.

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.

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.

(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.

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% 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) 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.

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.

* 1, * 2: Details are shown in Table 4 and Table 5 below.

  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) 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.

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) 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.

(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.

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).

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

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

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

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.

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).

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.

(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 (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%).

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)

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)

(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).

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.

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) 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.

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)

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.

(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.

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.

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.

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) 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.

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.

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 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) 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) 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 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.

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.

  The microorganism of the present invention produces a useful secondary metabolite and is useful in the field of medicine.

  NITE P-779

Claims (13)

A novel compound represented by the following formula (I).
(Wherein 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 a hydroxyl group; R ³ represents a hydrogen atom or Represents an acyl group, and a wavy line represents a single bond or a double bond. ) The novel compound according to claim 1, wherein the compound represented by the formula (I) is represented by the following formula (II).
(Wherein 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 a hydroxyl group; R ³ represents a hydrogen atom or Represents an acyl group, and a wavy line represents a single bond or a double bond. ) The compound represented by the formula (I) or (II) is represented by the following formula (III) and has at least a basement membrane invasion inhibitory activity or a cell migration inhibitory activity. New compound.
  Containing as an active ingredient any one selected from the group consisting of the compound according to any one of claims 1 to 3, physiologically acceptable salts thereof, and physiologically acceptable hydrates thereof, Pharmaceutical preparation for cancer treatment.   Containing as an active ingredient any one selected from the group consisting of the compound according to any one of claims 1 to 3, physiologically acceptable salts thereof, and physiologically acceptable hydrates thereof, An inhibitor of cancer cell basement membrane invasion.   Containing as an active ingredient any one selected from the group consisting of the compound according to any one of claims 1 to 3, physiologically acceptable salts thereof, and physiologically acceptable hydrates thereof, Cancer cell migration inhibitor. The novel compound according to claim 1 or 2, wherein the compound represented by the formula (I) or (II) is represented by the following formula (IV).
(In the formula, R ³ represents a hydrogen atom or an acyl group.) The compound represented by the above formula (IV) is represented by the following formula (V), and is at least one selected from the group consisting of antibacterial activity, basement membrane invasion inhibitory activity, cell migration inhibitory activity, and cytotoxic activity. The novel compound according to claim 7, further having activity.
  A pharmaceutical preparation for treating cancer, comprising as an active ingredient any one selected from the group consisting of the compound according to claim 7 or 8, a physiologically acceptable salt thereof, and a physiologically acceptable hydrate thereof. .   Basement membrane infiltration of cancer cells containing as an active ingredient any one selected from the group consisting of the compound according to claim 7 or 8, a physiologically acceptable salt thereof, and a physiologically acceptable hydrate thereof Inhibitor.   9. A cancer cell migration inhibitor comprising as an active ingredient any one selected from the group consisting of the compound according to claim 7 or 8, a physiologically acceptable salt thereof, and a physiologically acceptable hydrate thereof. .   An antibacterial agent comprising as an active ingredient any one selected from the group consisting of the compound according to claim 7 or 8, a physiologically acceptable salt thereof, and a physiologically acceptable hydrate thereof.   A microorganism that produces a novel compound represented by the above formula (I) (Accession No. NITE P-779).