JPH09110689A - Agent for inhibiting production of venous cell-adhering molecule-1 and napyradiomycin sc - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new inhibiting agent containing a napyradiomycin compound represented by a specific formula as an active ingredient, capable of inhibiting the cell-adhesive molecule production of human venous endothelial cells within a range not expressing cytotoxicity, and useful as a cancer metastasis-inhibiting agent. SOLUTION: This agent for inhibiting the venous cell-adhesive molecule-1 production contains a napyradiomycin compound of formula I (R<1> is Cl, H; R<2> is H; R<3> , R<4> are each methyl, OH, or R<3> , R<4> form a ethylidene group together; R<5> is Cl, Br), preferably napyradiomycin SC of formula II, as an active ingredient. The napyradiomycin compound is obtained e.g. by culturing Streptomyces cyaneus (FERM P-15233) in a liquid culture medium in culture conditions comprising 30-37 deg.C and a pH of 7-8 under an aerobic condition for 12hr to 10 days, extracting the microorganism culture product or the culture supernatant with an organic solvent and further separating the napyradiomycin compound by a chromatography.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a drug and a novel compound which have an action of inhibiting the production of venous cell adhesion molecule-1 (VCAM-1) by venous endothelial cells, including a cancer metastasis inhibitor and an anti-inflammatory agent. It is expected to be used as a therapeutic drug for arteriosclerosis, transplant rejection, rheumatoid arthritis, sarcoidosis, etc.

[0002]

2. Description of the Related Art VCAM-1 is a venous cell adhesion molecule (VC
AM), which is a glycoprotein with a molecular weight of 90,000 newly discovered in 1989 (Osborn, L., et al, Cell 59, 12).
03, 1989). Expression of VCAM-1 is induced by stimulating vascular endothelial cells with inflammatory cytokines such as TNF (tumor necrosis factor) and IL-1 (interleukin-1), and is a ligand for vascular endothelial cells and VCAM-1. V
Cells expressing LA-4 (lymphocytes, macrophages,
Eosinophils, basophils, melanoma, etc.). Recently, expression in follicular dendritic cells and the like has also been confirmed.

In addition to VCAM-1 in the inflammatory response, P
-Adhesion molecules such as selectin, ELAM-1 and ICAM-1 are involved. Among them, the adhesion via VCAM-1 and ICAM-1 is particularly strong, and these functions play an important role in actual diseases. It is said that Upregulation of VCAM-1 expression in vascular endothelial cells in a wide range of lesions such as arteriosclerosis, allograft rejection, cancer metastasis such as melanoma, bronchial asthma, and various acute and chronic inflammatory diseases has been reported. . Therefore, VCAM-of this venous endothelial cell
A substance having an action of suppressing the production of 1 is considered to be effective as a therapeutic drug for these diseases. The present inventors have already reported some substances that exhibit such an action (Workshop “Molecular Target of Cancer Chemotherapy” 039, 1994, Ministry of Education, Research Center for Cancer Priority Research), but found better substances. Is desired.

On the other hand, in the medical field, although inflammatory diseases are not directly life-threatening, there are many patients suffering from symptoms due to various causes, and more effective therapeutic agents with fewer side effects are always required. In addition, despite the fact that cancer metastasis is a major cause of cancer death, at present, there are few established methods for preventing or treating cancer.

By the way, microorganisms belonging to the genus of Chainia (for example, Chainia rubra)
) MG802-AF1 (Ministry of Industrial Science, Microbiology No. 8022))
From the culture solution of

[0006]

Embedded image

A series of substances represented by the following formula have been isolated and named as napyradiomycins (K. Shiomi, et al.,
J. Antibiotics, 494, 39, 1986; ibid., 1213, 40, 19
87). However, these substances have only been reported to have antibacterial action, cytotoxicity, and estrogen receptor antagonist action, and are not known to have VCAM-1 production inhibitory activity.

[0008]

Therefore, an object of the present invention is to provide a novel VCAM-1 production inhibitor.

[0009]

Means for Solving the Problems As a result of searching for components contained in a culture solution of a marine-derived microorganism, the present inventors have found that napyradiomycins isolated from a culture solution of a microorganism belonging to the genus Streptomyces are venous endothelial cells. The present invention has been completed by finding that it exhibits an action of inhibiting the production of VCAM-1 by V.

That is, the present invention provides the following structural formula (1)

[0011]

Embedded image

(In the formula, R ¹ represents a chlorine atom or a hydroxyl group, R ² represents a hydrogen atom, or R ¹ and R ² together form a single bond. R ³ and R ⁴ represent , Each independently represent a methyl group or a hydroxyl group, or together represent a methylidene group. R ⁵ represents a chlorine atom or a bromine atom.) A molecule-1 (VCAM-1) production inhibitor is provided.

Further, the following structural formula

[0014]

Embedded image

The present invention provides napyradiomycin SC represented by:

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The napyradiomycins according to the present invention can be separated from a culture solution of a microorganism belonging to, for example, Streptomyces or Kainia.

The microorganism used in the present invention may be any of those belonging to the genus Streptomyces or the genus Cainia and capable of producing the napyradiomycins represented by the general formula (1). Can be newly separated from.

As an example of a microorganism belonging to the genus Streptomyces, the present inventors newly isolated it as a microorganism capable of producing napyradiomycins, and identified it as Streptomyces cyaneus (SCRC-).
A64 can be mentioned. This Streptomyces
Sianeus SCRC-A64 is a FERM P-
Deposited as 15233 (Deposit date 1995 1995)
(October 11th).

This strain was isolated using the medium having the composition shown in Table 1 as follows.

[0020]

[Table 1] ─────────────────────────────── a. Medium Yeast extract 0.4% Malt extract 1.0% Glucose 0.4% Agar 2.0% 75% Artificial seawater, pH 7.3 SZ medium Glucose 1.0% Bactopeptone 0.5% Yeast extract 0.1% Iron (III) phosphate 0.01% 75% Artificial seawater, pH 7.4 ───────────────────────────────

A. Of the above composition The agar plate of the medium was inoculated with a sample obtained from various seas, which was appropriately diluted with sterilized physiological saline, and cultured at 35 ° C. for 7 to 14 days. The colonies appearing on this agar medium were picked up on a slant medium having the same medium composition to isolate a large number of strains.

Next, 5 mL of SZ medium was dispensed into each test tube and sterilized in the same manner, and each strain was 35 ° C. in this liquid medium.
The cells were shake-cultured for 7 days.

Streptomyces cyaneus SCRC which produces the napyradiomycins of the present invention in this manner
-A64 was obtained.

This strain has the following mycological properties.

1. a Colony morphology on agar medium Color: Brown Gloss: None Shape: Irregular, trapezoidal, pleated shape Size: Diameter 7-8 mm (7 days) Spore formation: None (in starch mineral salt medium It may form a spiral spore chain when cultured for 12 days.)

2. Biochemical properties Growth temperature: 25-37 ° C (+), 45 ° C (-) Catalase: Positive oxidase: Negative Gram stain: Positive OF test: Negative

3. Chemical taxonomic properties Cell wall diamino acid: LL diaminopimelic acid Major fatty acid type: 12-methyltetradecanoic acid,
14-Methylpentadecanoic acid, 14-Methylhexadecanoic acid

From the above results, SCRC-A64 belongs to the genus Streptomyces, and as a result of a more detailed species identification test, this strain was identified as Streptomyces cyaneus.

It is also possible to mutate the bacterium to obtain a strain with higher productivity. Further, a gene involved in the production of napyradiomycins present in the cells of this strain is excised, and this is inserted into an appropriate vector, for example, a plasmid, and using this vector, an appropriate host, for example, Escherichia coli (Escherichia coli) is used. ) Or a heterologous host such as yeast, or a homologous host such as Streptomyces, it is possible to artificially create the napyradiomycin-producing bacterium of the present invention.

The strain of the present invention can be preserved according to a conventional method, for example, on an agar slant or by a freeze-drying method. As the agar slant medium, a medium commonly used for the storage of Streptomyces spp.
For example, the medium described above for the isolation of bacteria can be used. Freeze-drying can also be performed according to a conventional method.

When the above-mentioned microorganisms are cultured to produce the napyradiomycins of the present invention, any basal nutrient medium may be used as long as the microorganisms of the present invention can grow therein. This medium contains one or more kinds of nitrogen sources, such as yeast extract, peptone, and meat extract. If necessary, various sugars can be added to this medium as a carbon source. It is necessary to add sodium chloride or natural seawater or artificial seawater to this medium.

The culture may use either a solid medium or a liquid medium, but in order to obtain a large amount of a target substance, a liquid medium is used, and static culture or shaking culture, aeration or aeration
It is preferable to carry out the reaction under aerobic conditions by stirring culture or the like.
The culture temperature may be any temperature within the temperature range in which the bacteria grow and the napyradiomycins of the present invention are produced,
It is preferably 25 to 45 ° C, more preferably 30 to 45 ° C.
37 ° C. The pH ranges from 6 to 9, preferably from 7 to 8. The culture time may be selected so that a harvestable amount of napyradiomycins is produced, and preferably 12 hours to 10 days. The napyradiomycins represented by the above general formula (1) of the present invention include, for example, microbial culture cells of Streptomyces or culture supernatant extracted with an organic solvent,
Further separation by chromatography can be obtained by a usual method.

Examples of the solvent used for extraction include methanol, ethanol, ethyl acetate, chloroform and the like. Column and thin layer chromatography are used for chromatography. As column chromatography, in addition to silica gel, Sephadex LH20 and reversed phase system are used. RP-1
8 is used, and as the thin layer chromatography, RP-18 is used in addition to silica gel.

Specific examples of the compound contained in the general formula (1) in the present invention include:

[0035]

Embedded image The napyradiomycins represented by

The VCAM-1 production inhibitor containing the above compound of the present invention as an active ingredient can be provided by oral administration, parenteral administration (subcutaneous, intravenous, intramuscular, sternum injection, etc.) or rectal administration. The dose of the above compound varies depending on the patient's symptoms, age, administration method, etc., but is usually 0.0
It is about 1 to 500 mg / kg / day. For administration, the above compound can be used as it is, but usually it is used in the form of a preparation. For formulation, pharmacologically and pharmaceutically acceptable components of the formulation such as conventional diluents, carriers, fillers, additives, etc. may be used, and the methods usually used in this field may be applied. it can. Further, it is also possible to prepare a sustained-release preparation by a known technique. As the form of the preparation, tablets, granules, fine granules, powders, capsules, syrups, elixirs, injections, eye drops, eye ointments or suppositories are used. The amount of active ingredient contained in the drug product is 0.0
It is about 1 to 99.99%.

Examples of the above-mentioned components for preparation include internal preparations (oral preparations), injectable preparations (injection preparations), mucosal administration preparations (buccal, troches, suppositories, etc.), external preparations (ointments, patches, etc.), etc. Appropriate components are used depending on the route of administration. For example, in the case of oral preparations and mucous membrane preparations, excipients (eg, starch, lactose, crystalline cellulose, calcium lactose, magnesium aluminometasilicate, anhydrous silicic acid), disintegrants (eg, carboxymethylcellulose, carboxy) Ingredients such as methyl cellulose calcium), lubricants (eg magnesium stearate, talc), coating agents (eg hydroxyethyl cellulose), flavoring agents, etc., and in the case of injectable agents, they can constitute aqueous injectable agents. Dissolving agents or solubilizing agents (eg distilled water for injection, physiological saline, propylene glycol), suspending agents (eg polysorbate 8)
0, etc.), pH adjusters (eg organic acids or metal salts thereof), stabilizers and other formulation ingredients, and in the case of external preparations, aqueous or oily solubilizers or solubilizers (eg: Alcohol, fatty acid esters), adhesives (carboxyvinyl polymers, polysaccharides), emulsifiers (eg:
Pharmaceutical ingredients such as surfactants) are used.

The above-mentioned preparation can be prepared by a known production method, for example, the method described in the Japanese Pharmacopoeia, 10th Edition General Rules for Preparation or a method with appropriate modification.

[0039]

The present invention will be described below in detail with reference to examples and test examples.

Example 1. Production of Napyradiomycins by Culturing Streptomyces cyaneus SCRC-A64 Streptomyces cyaneus SCRC-A64 was aerobically cultured at 25 ° C. for 7 days in the SZ medium shown in Table 1. The culture solution (55 L) was mixed with half volume of methanol-chloroform (1:
Extracted once with 2), then twice with chloroform, combined and concentrated to give 2 g of a dark brown crude extract.

Example 2. Separation and purification of napyradiomycins in culture broth Example 1. 80% methanol-water and n
-Partitioned with hexane. Hexane-soluble fraction (360 m
g) was obtained. A glass column with an inner diameter of 1.5 cm was filled with YMC ODS-AQ suspended in methanol to a height of 16 cm, the solvent was replaced with 70% methanol-water, and this hexane fraction was adsorbed on a small amount of the same ODS resin. Put it on the column. 70% methanol-water 70m in this column
L, then 80% methanol-water (50 mL), 90% methanol-water (60 mL) and methanol (100 mL) in this order, and the first half of the methanol elution portion was collected and concentrated to 68 mg.
To obtain a concentrate.

A glass column having an inner diameter of 12 mm was filled with Sephadex LH-20 swollen with n-hexane-chloroform-methanol (4: 5: 1) at a height of 28 cm. Dissolve 68 mg of the concentrate in a small amount of the same mixed solvent, load it on this column, and elute with the same solvent, 20 to 30 mL
The eluates of the eyes were combined and concentrated to obtain 32 mg of a brown oily substance.

The above oily substance was made into 0.8 mL of 80% methanol solution, and 100 μL each was applied to high-performance liquid chromatography (column: Shiseido Capcel).
Pak C-8 7 mm x 150 mm; Solvent: 80%
Methanol-water, flow rate 0.6 mL / min. ; Detection: U
V210 nm) and the retention time was 20 to 27 minutes. This portion was collected and concentrated to obtain 7.
2 mg was again injected into high performance liquid chromatography (same as above, but the solvent was 70% methanol-water), and the retention time from 60 minutes to 67 minutes was fractionated and concentrated to give 4.1 mg of napyradiomycin B1 (1a) as a brown glass. Obtained as a substance. It was identified by NMR spectrum analysis.

A retention time of 10 to 17 minutes of the high performance liquid chromatography eluted with the above 80% methanol-water was fractionated and similarly separated by high performance liquid chromatography using 70% methanol-water to retain the retention time of 25 to 29 minutes. Up to the minute was collected. This was concentrated to give 0.6 mg of napyradiomycin B2 (1b). It was identified by NMR spectrum analysis.

A glass column having an inner diameter of 2.8 cm was packed with YMC ODS-AQ suspended in methanol to a height of 23 cm, the solvent was replaced with 70% methanol-water, and the crude extract was dissolved in 80% methanol. A fraction (1.3 g) was adsorbed on a small amount of the same ODS resin and loaded on the column. 70% methanol-water, 80% methanol-water,
90% methanol-water and methanol (300 mL each) were sequentially flowed, and the first half of the methanol elution portion was collected and concentrated.
72 mg of concentrate was obtained.

A glass column having an inner diameter of 12 mm was filled with Sephadex LH-20 swollen with n-hexane-chloroform-methanol (4: 5: 1) at a height of 28 cm. 172 mg of the concentrate was dissolved in a small amount of the same mixed solvent and loaded on this column, and eluted with the same solvent, 20 to 30 m
The L-th eluate was combined and concentrated to give a yellow oily substance 25 mg.
I got This was recrystallized from methanol to obtain 5 mg of napyradiomycin B4 (1c) as pale yellow needle crystals. It was identified by NMR spectrum analysis and X-ray crystallographic analysis.

In the ODS column chromatography of the 80% methanol-soluble fraction, the first half of the 90% methanol eluate was collected and concentrated to obtain 82 mg of a concentrate. This was dissolved in about 5 mL of methanol and allowed to stand at room temperature to give 21 mg of novel napyradiomycin SC (1
d) was obtained as yellow plate crystals.

Molecular formula: C ₂₅ H ₃₂ O ₇ Cl ₂ ¹ H-NMR (CD ₃ OD): δ 0.40 (3H, s, 17-Me), 0.68 (3H, s, 17-M)
e), 1.19 (3H, s, 13-Me), 1.27 (3H, s, 2-Me), 1.40 (1H, dd,
J = 16.4, 1.7Hz, H-11), 1.47 (3H, s, 2-Me), 1.56 (1H, m, H-
14), 1.60 (1H, dd, J = 6.8, 1.7Hz, H-12), 1.76 (1H, m, H-1
4), 1.8 (1H, m, H-15), 1.94 (1H, m, H-15), 2.19 (1H, dd, J = 1
3.5, 11.2Hz, H-4), 2.22 (1H, dd, J = 13.5, 5.1Hz, H-4),
2.56 (1H, dd, J = 16.4, 6.9Hz, H-11), 3.69 (1H, dd, J = 12.0,
4.1Hz, H-16), 4.46 (1H, dd, J = 11.2, 5.1Hz, H-3), 6.64 (1
H, d, J = 2.4Hz, H-7), 7.08 (1H ,, J = 2.4Hz, H-9).

¹³ C-NMR (CD ₃ OD): δ14.5 (17-Me), 20.3 (2-M
e), 22.8 (13-Me), 27.3 (17-Me), 27.4 (2-Me), 29.8 (C-1
5), 33.1 (C-11), 39.9 (C-17), 40.5 (C-14), 40.5 (C-4),
48.6 (C-12), 57.5 (C-3), 70.2 (C-13 / 16), 70.3 (C-16 / 1
3), 78.6 (C-2 / 4a), 79.7 (C-4a / 2), 83.6 (C-10a), 107.0
(C-5a), 107.78 (C-7 / 9), 107.83 (C-9 / 7), 134.9 (C-9a),
164.4 (C-6 / 8), 166.2 (C-8 / 6), 193.3 (C-5 / 10), 199.0 (C
-10/5).

EI-MS (m / Z): 514 (M ⁺ ). [Α] _D ²⁵ : -62 ° (c 0.2, MeOH) MP: 168.5-170.5 ° C

Test Example 1. Human VCAM-1 Production Inhibition Test Normal human umbilical vein endothelial cells (CS-VE) were supplemented with 10 ng / mL of human recombinant basic FGF.
% CHF-MCBD131 medium containing fetal bovine serum,
Prepared to 1 × 10 ⁴ cells / mL and treated with collagen I 9
0.15 mL of each was dispensed into a 6-well plate and cultured in a CO ₂ incubator (CO ₂ 5%, humidity 100%, 37 ° C.) until confluence was reached. Human recombinant TNF-
CHL containing 10% fetal bovine serum supplemented with 25 U / mL α
100 μL of medium that had been replaced with 100 μL of MCBD131 medium and subsequently added with the test compound to a predetermined concentration
L was added to the CO ₂ incubator (CO ₂ 5%, humidity 100%,
The cells were cultured at 37 ° C for 18 hours. 0.3% after removing the medium
100 μL of cooled methanol containing hydrogen peroxide was added, and the cells were fixed by allowing them to stand at room temperature for 30 minutes to prepare anti-human VCAM-1.
VCAM-1 was quantified by the two antibody method using an antibody, horseradish peroxidase-labeled mouse IgG1 antibody and 3,3 ′, 5,5′-tetramethylbenzidine as a substrate. The 50% production inhibitory concentration (IC ₅₀ ) was determined from the percentage of the measured VCAM-1 amount relative to that of the control group. Table 2 shows the results.

[0052]

[Table 2]

A cytotoxicity test was conducted on the above compound by the following method, and it was confirmed that no toxicity was observed at the above concentration. That is, 1.1 mg / g was added to a 96-well plate in which the sample was added and cultured under the same conditions as in Test Example 1.
0.05 mL of MTT aqueous solution was dispensed, and viable cells were measured by the MTT method. The 50% growth inhibitory concentration (IC ₅₀ ) was determined from the percentage of the measured value with respect to the control group. Table 3 shows the results.

[0054]

[Table 3]

[0055]

INDUSTRIAL APPLICABILITY Napyradiomycins, which are active ingredients of the venous cell adhesion molecule-1 production inhibitor of the present invention, produce cell adhesion molecule (VCAM-1) by human venous endothelial cells within a range that does not show cytotoxicity. Inhibit. Therefore, the inhibitor of the present invention includes a cancer metastasis inhibitor, an anti-inflammatory agent, bronchial asthma,
It has applications as a therapeutic drug for arteriosclerosis, transplant rejection, rheumatoid arthritis, sarcoidosis, etc.

Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI Technical display location A61K 31/35 ADU A61K 31/35 ADU C07D 311/92 101 C07D 311/92 101 // C12P 17/06 C12P 17 / 06 (C12P 17/06 C12R 1:01)

Claims (2)

[Claims] [Claim 1] The following general formula (1) (In the formula, R ¹ represents a chlorine atom or a hydroxyl group, R ² represents a hydrogen atom, or R ¹ and R ² together form a single bond. R ³ and R ⁴ are different from each other. Is a methyl group or a hydroxyl group, or together represents a methylidene group. R ⁵ represents a chlorine atom or a bromine atom.) A venous cell adhesion molecule containing napyradiomycin as an active ingredient-1. Production inhibitor. 2. The following structural formula: Napyradiomycin SC represented by