JPH07228583A - Abratuvolactams, producing microorganisms and production methods thereof, and anti-inflammatory agents - Google Patents

Abstract

(57) [Summary] [Object] To provide an antitumor substance and / or an anti-inflammatory substance having a novel structure from a culture solution of a marine microorganism. [Structure] The following general formula, obtained by extracting and purifying a microbial culture, Abratuvolactams represented by the formula (wherein R ¹ and R ² independently represent a hydrogen atom or a hydroxyl-protecting group, and R ³ and R ⁴
Independently represent a hydrogen atom or a lower alkyl group), a method for producing Streptomyces microorganisms and abratuvolactams capable of producing them, and an anti-inflammatory agent. EFFECT Aburatsubo lactams, growth inhibitory activity against murine lymphocytic leukemia cells (P388), O ₂ of human neutrophil ^- production inhibitory activity, PAF production inhibitor of intracellular phospholipase A ₂ inhibitory activity and human neutrophils It has activity and is expected to be used as a drug such as an antitumor agent and an anti-inflammatory agent.

Description

Detailed Description of the Invention

[0001]

Aburatsubo with production inhibitory activity, PAF production inhibitory activity of intracellular phospholipase A ₂ inhibitory activity and / or human neutrophil ^- This invention relates to anti-tumor activity, O ₂ of human neutrophils The present invention relates to lactams, microorganisms that produce abratuvolactams, a method for producing abratuvoractams, and anti-inflammatory agents containing the same.

[0002]

BACKGROUND OF THE INVENTION Many antitumor agents derived from natural substances including microorganisms are known, such as adriamycin and mitomycin, but there are still problems such as unsatisfactory therapeutic results and serious side effects. In the medical field, there is always a demand for drugs that are more effective and have fewer side effects.

In addition, the inflammatory reaction is a kind of biological defense effect which is activated when a harmful stimulus enters the body, but as a result, swelling, pain, organ dysfunction and the like are involved,
It is often fatal. Specifically, from acute inflammation associated with type 1 allergy to external effects to chronic inflammation caused by nephritis and rheumatic diseases, the causes, onset processes, and symptoms are extremely wide-ranging and complicated.
What is called an anti-inflammatory agent is used as a symptomatic therapeutic agent, and it is roughly classified into a steroid anti-inflammatory agent and a non-steroid anti-inflammatory agent, and various glucocorticoids, indomethacin, etc. are representative. However, steroidal anti-inflammatory drugs have a protein synthesis inhibitory action including various protein mediators, and although they have a wide range of pharmacological effects and a large curative effect, they are known to cause serious side effects. It was recently revealed that the separation of the two is almost impossible. On the other hand, the effects of non-steroidal anti-inflammatory drugs are limited because the cyclooxygenase inhibition (prostaglandin production inhibition) action is the main pharmacological action. Therefore and development of anti-inflammatory agent is required based on a new pharmacological action, O ₂ ^- production inhibitory activity, intracellular phospholipase A ₂ inhibitory activity, the compounds having a PAF production inhibitory activity is considered a substance that meets the purpose. That is, various reactive oxygen species are the most central mediators involved in tissue damage during inflammation, but their production is leukocyte (neutrophil, macrophage)
Due to production ^- O ₂ by. In addition, intracellular phospholipase A2 is an enzyme whose existence was recently revealed,
It is known that its activation commonly initiates the enzymatic production of prostaglandins, leukotrienes, and PAF, which are the major inflammation-inducing mediators. Also PAF
Is a potent inflammatory mediator along with prostaglandins and leukotrienes, but its synthetic inhibitors are unknown.

[0004]

The object of the present invention is therefore to discover an antitumor substance and / or an anti-inflammatory substance having a novel structure from a culture solution of marine microorganisms.

[0005]

As a result of searching the components contained in the culture solution of marine actinomycetes, the inventors of the present invention have the following general formula (1), which is separated from a novel microbial culture solution belonging to the genus Streptomyces, (2) and Aburatsubo lactams represented by (3), in vitro growth inhibitory activity against P388, the human neutrophil O ₂ ^- production inhibitory activity, intracellular phospholipase a ₂ inhibitory activity and human neutrophils It was found that they have a PAF production inhibitory activity and completed this study.

That is, the present invention has the general formula

[0007]

[Chemical 10]

[Chemical 11]

Or

[0009]

[Chemical 12]

(Wherein R ¹ and R ² independently represent a hydrogen atom or a lower alkyl group, and R ³ and R ⁴ independently represent a hydrogen atom or a hydroxyl-protecting group). I will provide a.

The present invention also provides, as a common partial structure,

[0012]

[Chemical 13]

[Chemical 14]

And

[0014]

[Chemical 15]

(Wherein R ¹ and R ² independently represent a hydrogen atom or a lower alkyl group, and R ³ represents a hydrogen atom or a hydroxyl-protecting group), and

(A) The following physical properties: Molecular formula: C ₃₀ H ₄₀ N ₂ O ₅ ¹ H-NMR (C ₅ D ₅ N): δ 0.7 (1H, m), 0.80 (3H, t), 0.90 (3H ,
d), 1.0 (1H, m), 1.05 (1H, m), 1.25 (1H, m), 1.35 (1H, m),
1.55 (1H, m), 1.6 (1H, m), 1.63 (1H, m), 1.71 (1H, m), 1.
88 (1H, m), 1.9 (1H, m), 1.9 (1H, m), 2.23 (1H, m), 2.28 (1
H, m), 2.4 (1H, m), 3.13 (1H, m), 3.19 (1H, m), 3.3 (3H, s),
4.05 (1H, m), 4.20 (1H, bs), 4.79 (1H, bd), 5.6 (1H, dd),
5.95 (1H, m), 6.08 (1H, dd), 6.12 (1H, dd), 6.12 (1H, d),
6.32 (1H, d), 7.55 (1H, dd), 7.68 (1H, dd);

(B) The following physical properties and molecular formula: C ₃₀ H ₄₀ N ₂ O ₆ ¹ H-NMR (C ₅ D ₅ N): δ 0.8 (3H, t), 0.8 (1H, m), 0.85 (3H ,
d), 0.95 (1H, m), 1.05 (1H, m), 1.2 (1H, m), 1.25 (1H, m),
1.5 (1H, m), 1.85 (1H, m), 1.9 (1H, m), 2.0 (1H, m), 2.0 (1
H, m), 2.2 (1H, m), 2.4 (1H, m), 2.5 (1H, m), 2.5 (1H, m),
2.7 (1H, m), 2.85 (1H, dd), 3.15 (1H, m), 3.25 (3H, s), 3.
5 (1H, m), 3.5 (1H, m), 4.0 (1H, m), 4.1 (1H, d), 4.8 (1H,
m), 5.3 (1H, m), 5.5 (1H, m), 6.6 (1H, bd), 6.7 (1H, bd),
7.4 (1H, bdd), 7.7 (1H, bdd);

Or (c) the following physical properties: molecular formula: C ₃₀ H ₄₀ N ₂ O ₅ ¹ H-NMR (C ₅ D ₅ N): δ 0.82 (3H, t), 0.84 (1H, m), 0.87 ( 3H,
d), 1.0 (1H, m), 1.05 (1H, m), 1.25 (1H, m), 1.28 (1H, m),
1.48 (1H, m), 1.50 (1H, m), 1.64 (1H, m), 1.88 (1H, m),
1.92 (1H, m), 2.02 (1H, m), 2.02 (1H, m), 2.12 (1H, m), 2.
5 (1H, m), 2.58 (1H, m), 2.6 (1H, m), 2.75 (1H, bdd), 2.89
(3H, s), 3.13 (1H, m), 3.20 (1H, m), 3.28 (1H, m), 3.58 (1
H, m), 3.60 (1H, bd), 3.70 (1H, m), 5.50 (1H, m), 5.60 (1
H, m), 6.75 (2H, d), 7.48 (1H, dd), 7.60 (1H, dd);

Furthermore, the present invention provides a microorganism producing the above-mentioned abratuvolactams, a method for producing the above-mentioned abratuvolactams, and an anti-inflammatory agent containing the same as an active ingredient.

Examples of the hydroxyl-protecting group in the above general formulas (1), (2) and (3) include acyl groups such as acetyl group, propanoyl group and benzoyl group, trimethylsilyl group,
Examples thereof include a silyl group such as t-butyldimethylsilyl group, an acetal-type protecting group such as methoxymethyl group, and tetrahydropyranyl group. Examples of the lower alkyl group include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, pentyl group and hexyl group.

The microorganism of the present invention belongs to the genus Streptomyces and produces the above-mentioned abratuvolactams, and such microorganism can be newly isolated from nature.

As an example of a microorganism belonging to the genus Streptomyces, Streptomyces s newly identified by the present inventors as a microorganism having the ability to produce abratuvolactams and identified as a novel microorganism belonging to the genus Streptomyces
p. SCRC-A20 can be mentioned. This new strain, Streptomyces sp. SCRC-A20
Has been deposited as FERM P-13915 at the Patent Microorganism Depositary Center, Institute of Biotechnology, Institute of Industrial Science and Technology.

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

[0024]

[Table 1]

A. Of the above composition The agar plate of the medium was inoculated with a sample collected from various seas, which was appropriately diluted with sterilized physiological saline, and cultured at 25 ° 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 25 ° C. in this liquid medium.
The cells were shake-cultured for 7 days.

Thus, Streptomyces sp. Which produces the abratuvolactams of the present invention. SCRC-
A20 was obtained from a snail snail collected at an oil pot in Miura City, Kanagawa Prefecture.

This new strain has the following mycological properties.

1. Morphology of colonies on yeast-malt agar medium Color: Light yellow Gloss: None Shape: Circular and regular, trapezoidal ridges and folds are formed Size: Diameter 1-1.5 mm Spore formation: None (starch (It may form a spiral spore chain when cultured in an inorganic salt medium for 12 days.)

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

3. Growth status on various agar media (37 ℃,
Cultivation for 15 days) Salt polymyxin + -BTB Teibol-MacConkey-Stahiro No. 110 ++ Potato dextrose-Sabouraud + Galactose agar +-Marine agar + Malt agar- Tryptosoy agar + SZ agar ^* +++ (blue dye production) (* 2% agar added to SZ liquid medium)

4. 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-A20 is similar to the genus Nocardia in that the morphological characteristics of the bacterium, the absence of sporulation, and the fact that hyphae are easily cleaved, but the chemical properties of SCRC-A20 are similar to those of the genus Nocardia. This is not the case, and the properties are close to those of the genus Streptomyces. Therefore, this strain was identified as a novel microorganism belonging to the genus Streptomyces.

It is also possible to mutate the bacterium to obtain a strain with higher productivity. In addition, a gene involved in the production of abratuvolactams present in the cells of this strain is excised and inserted into an appropriate vector such as a plasmid, and using this vector, an appropriate host, such as Escherichia coli (Escherichia coli ) Or a heterologous host such as a yeast, or a homologous host such as a Streptomyces genus, it is possible to artificially create the abratuvolactam-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, it is possible to use the medium commonly used for the preservation of Streptomyces sp., For example, the medium described above for the isolation of the fungus. Freeze-drying can also be performed according to a conventional method.

In the case of culturing the above-mentioned microorganism to produce the abratuvolactams of the present invention, any basal nutrient medium may be used as long as the microorganism of the present invention can grow therein. This medium contains, as a nitrogen source, one or more kinds of yeast extract, peptone, meat extract and the like. 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.

For the culture, either a solid medium or a liquid medium may be used, but in order to obtain a large amount of the target substance, a liquid medium is used, and static culture or shaking culture, aeration
It is preferably carried out under aerobic conditions by stirring culture or the like.
The culturing temperature may be any temperature as long as the bacterium grows and is a temperature range in which the abratuvolactams of the present invention are produced,
It is preferably 25 to 45 ° C, more preferably 30 to 45 ° C.
37 ° C. The pH is in the range of 6-9, preferably 7-8. The culturing time may be selected such that a harvestable amount of abratuvolactam is produced, preferably 12 hours to
10 days.

The above-mentioned abratuvolactams of the present invention can be obtained by an ordinary method, for example, by extracting a microbial cell culture of Streptomyces or a culture supernatant with an organic solvent and further separating by chromatography.

Examples of the solvent used for extraction include methanol, ethanol, ethyl acetate, chloroform and the like. Column chromatography and thin layer chromatography are used for chromatography. As column chromatography, besides 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 abratuvolactams of the present invention include:

[0041]

[Chemical 16]

Abratu lactam A represented by

[0043]

[Chemical 17]

Abratu lactam B represented by

[0045]

[Chemical 18]

Abratuvolactam C represented by

The anti-inflammatory agent of the present invention can be administered orally or parenterally for treatment. As the orally-administered agent, solid preparations such as powder, granules, capsules and tablets, or liquid preparations such as syrups and elixirs can be used. Moreover, an injection can be prepared as a parenteral preparation.

These preparations are manufactured according to a conventional method by adding pharmacologically and pharmaceutically acceptable manufacturing aids to the active ingredient. Further, it is also possible to prepare a sustained-release preparation by a known technique. When the manufacturing aid is used, the content of Abratuvolactam Cs in the anti-inflammatory agent of the present invention is usually 0.1 to 10% by weight, preferably 0.2 to 5% by weight.

The above-mentioned additives can be used as oral preparations (oral preparations), injectable preparations (injection preparations), and mucosal administration preparations (buccal, trocar).
Appropriate components for formulation depending on the administration route such as H., suppositories) and external preparations (ointments, patches, etc.) are used. For example, in the case of oral preparations and mucous membrane preparations, excipients (eg starch,
Lactose, crystalline cellulose, calcium lactose, magnesium aluminometasilicate, silicic acid anhydride), disintegrants (eg: carboxymethyl cellulose, carboxymethyl cellulose calcium), lubricants (eg: magnesium stearate, talc), co- In the case of injectable preparations, such as tong agents (eg, hydroxyethyl cellulose), flavoring agents, and the like, solubilizing agents or solubilizing agents (eg, distilled water for injection, which can constitute an aqueous injectable agent). Formulation components such as physiological saline, propylene glycol), suspending agents (eg, surfactants such as polysorbate 80), pH adjusters (eg, organic acid or its metal salt), stabilizers, etc. For external preparations, aqueous or oily solubilizers or solubilizers (eg:
Pharmaceutical ingredients such as alcohols, fatty acid esters), adhesives (eg carboxyvinyl polymers, polysaccharides), emulsifiers (eg surfactants) are used.

The anti-inflammatory agent of the present invention having the above-mentioned constitution can be produced by a known production method, for example, the method described in the Japanese Pharmacopoeia Method 10th Edition General Rules for Preparation or a method with appropriate modification.

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples.

[0052]

【Example】

Example 1. Production of abratuvolactams by culturing SCRC-A20 Streptomyces sp. SCRC-A20 was added to a 3 / 4-concentration artificial seawater medium (pH 7.4) containing glucose 1.0%, bactopeptone 0.5%, yeast extract 0.1%, and iron (III) phosphate 0.01%. The culture was performed aerobically at 7 ° C for 7 days. The culture broth (10 liters (L)) is suction-filtered with filter paper and Celite to separate the cells and the supernatant,
Extracted 3 times with mL of methanol. The extracts were combined, concentrated to about 100 mL with an evaporator, and extracted with ethyl acetate and chloroform three times in sequence to combine. When an insoluble matter appeared, it was separated by filtration and combined with the above extract as a methanol solution. The supernatant was extracted 3 times with half volume of ethyl acetate,
Extract with chloroform, concentrate with the extract,
1.9 g of crude extract was obtained. This is repeated until the culture solution 10
From 0 L, 19 g of a crude extract was obtained.

Example 2. Separation of abratuvolactams in culture medium Silica gel (100% suspended in a glass column having an inner diameter of 3.5 cm x 40 cm with 4% methanol-chloroform).
g) was charged and the crude extract (3.7 g) dissolved in a small amount of 4% methanol-chloroform was adsorbed. 4% methanol-chloroform solution 500 in this column
mL, 8% methanol-chloroform solution 600 mL,
A 20% methanol-chloroform solution (700 mL) was poured, and the fractions eluted with 8% methanol-chloroform and the fractions eluted with 20% methanol-chloroform were collected and concentrated. The remaining crude extract was treated in the same manner to obtain a total of 830 mg and 6.4 g of concentrates from each fraction.

Example 3. Abratu bolactam A and B
Isolation and Purification of Sephadex LH-20 swollen with 20% chloroform-methanol was filled in a glass column having an inner diameter of 1.8 cm at a height of 55 cm, and the concentrate of the fraction eluted with the above 20% methanol-chloroform was collected. 1.5 g of this was dissolved in a small amount of 20% chloroform-methanol and adsorbed. 150 mL of 20% chloroform-methanol was passed through this column, and about 20 mL was collected from the 10th mL,
Concentrated. The rest was treated in the same manner to obtain a total of 4.1 g of concentrate.

A glass column having an inner diameter of 3.5 cm was filled with RP-18 suspended in methanol to a height of 30 cm, and the solvent was replaced with 70% methanol-water. The above concentrate 3.
1 g was adsorbed on a small amount of RP-18 and placed on the column. Sequentially to this column 70% methanol-water 300m
L, 85% methanol-water 300 mL, methanol 30
0 mL and 300 mL of chloroform were poured, and the latter half of the methanol elution part and the first half of the chloroform elution part were combined and concentrated to obtain 440 mg of a concentrate.

A glass column having an inner diameter of 1.8 cm was filled with RP-18 suspended in methanol to a height of 25 cm, and the solvent was replaced with 80% methanol-water. Previous RP-18
260 mg of the concentrate obtained by column treatment was added to a small amount of RP-1.
8 and adsorbed on a column. Sequentially 8 to this column
0% methanol-water 100 mL, 90% methanol-water 100 mL, methanol 100 mL, chloroform 1
00 mL was poured, and the fraction eluted with methanol was allowed to stand, and the precipitated yellow needle crystals were filtered off to obtain 1 mg of abratuvolactam A.

Molecular formula: C ₃₀ H ₄₀ N ₂ O ₅ Melting point: 255 to 258 ° C. ¹ H-NMR (C ₅ D ₅ N): δ 0.7 (1H, m, H-9), 0.80 (3H, t, H -29),
0.90 (3H, d, H-27), 1.0 (1H, m, H-28), 1.05 (1H, m, H-11),
1.25 (1H, m, H-10), 1.35 (1H, m, H-7), 1.55 (1H, m, H-7),
1.6 (1H, m, H-28), 1.63 (1H, m, H-12), 1.71 (1H, m, H-24),
1.88 (1H, m, H-14), 1.9 (1H, m, H-9), 1.9 (1H, m, H-13), 2.2
3 (1H, m, H-24), 2.28 (1H, m, H-14), 2.4 (1H, m, H-8), 3.13
(1H, m, H-6), 3.19 (1H, m, H-25), 3.3 (3H, s, H-Nb-Me), 4.
05 (1H, m, H-25), 4.20 (1H, bs, H-22), 4.79 (1H, bd, H-23),
5.6 (1H, dd, H-5), 5.95 (1H, m, H-15), 6.08 (1H, dd, H-4),
6.12 (1H, dd, H-16), 6.12 (1H, d, H-18), 6.32 (1H, d, H-
2), 7.55 (1H, dd, H-17), 7.68 (1H, dd, H-3), 9.2,9.6 (1H, -OH / -NH respectively).

¹³ C-NMR (C ₅ H ₅ N): δ 13.0, 19.6, 26.5, 2
9.0, 32.4, 36.0, 38.0, 40.8, 41.8, 45.7, 47.9, 49.
8, 50.5, 54.5, 56.7, 71.6, 74.0, 101.9, 121.5, 12
6.2, 127.0, 132.4, 134.8, 139.4, 143.7, 144.2, 16
6.5, 173.4, 174.3, 192.7. EI-MS (m / z): 508 (M ⁺ ) [α] _D ²⁰ : + 868 ° (c 0.05, C ₅ H ₅ N) Furthermore, this substance was analyzed by X-ray crystallography. Was determined to have the above structure.

The white solid precipitated in the 90% methanol eluate obtained by the second RP-18 column treatment was filtered off to obtain 8 mg of abratuvolactam B.

50 mg of the concentrate obtained by the first RP-18 column was adsorbed on a preparative silica gel thin layer plate (thickness: 0.5 mm, 20 × 20 cm), and chloroform: diethylamine: isopropyl alcohol (7:
1: 3) for 2 hours, scrape off the part with Rf value of 0.4 to 0.5, add chloroform: methanol: water (2: 1: 1), shake vigorously and let stand, then lower layer Even after separating and concentrating, 8 mg of abratuvolactam B was obtained.

Molecular formula: C ₃₀ H ₄₀ N ₂ O ₆ Melting point: 239 to 242 ° C. (brown) ¹ H-NMR (C ₅ D ₅ N): δ 0.8 (3H, t, H-29), 0.8 (1H, m, H-9),
0.85 (3H, d, H-27), 0.95 (1H, m, H-28), 1.05 (1H, m, H-11),
1.2 (1H, m, H-13), 1.25 (1H, m, H-10), 1.5 (1H, m, H-28),
1.85 (1H, m, H-24), 1.9 (1H, m, H-12), 2.0 (1H, m, H-9), 2.
0 (1H, m, H-13), 2.2 (1H, m, H-24), 2.4 (1H, m, H-17), 2.5
(1H, m, H-17), 2.5 (1H, m, H-14), 2.7 (1H, m, H-7), 2.85 (1
H, dd, H-18), 3.15 (1H, m, H-8), 3.25 (3H, s, H-Nb-Me), 3.
5 (1H, m, H-18), 3.5 (1H, m, H-25), 4.0 (1H, m, H-25), 4.1
(1H, d, H-22), 4.8 (1H, m, H-23), 5.3 (1H, m, H-16), 5.5 (1
H, m, H-15), 6.6 (1H, bd, H-5), 6.7 (1H, bd, H-2), 7.4 (1H,
bdd, H-3), 7.7 (1H, bdd, H-4), 9.6 (1H, -OH / -NH).

¹³ C-NMR (C ₅ D ₅ N): δ 12.8 (C-29), 17.8 (C-
27), 26.4 (C-28), 27.8 (C-17), 28.3 (C-Nb-Me), 32.2 (C
-24), 37.7 (C-18), 37.7 (C-25), 38.6 (C-9), 41.1 (C-1
3), 43.9 (C-8), 48.9 (C-11), 52.2 (C-12), 54.4 (C-10),
55.3 (C-14), 65.6 (C-7), 70.8 (C-23), 72.5 (C-22), 10
2.2 (C-20), 131.4 (C-16), 133.6 (C-15), 134.4 (C-2), 1
36.7 (C-5), 137.8 (C-4), 138.9 (C-3), 166.7 (C-1), 17
5.8 (C-26), 189.8 (C-19), 192.5 (C-21), 201.0 (C-6).

SI-MS (m / z): 525 (M + H ⁺ ), 547 (M + Na ⁺ ). [Α] _D ²⁰ : + 197 ° (c 0.43, C ₅ H ₅ N)

Example 4. Purification of Abratuvolactam C 80 mg of the concentrate of the fraction eluted with the above 8% methanol-chloroform was adsorbed on two preparative silica gel thin layer plates (thickness 0.5 mm, 20 × 20 cm), and chloroform: diethylamine (4 hours) at (7: 3),
The portion having an Rf value of 0.25 to 0.45 was scraped off, chloroform: methanol: water (2: 1: 1) was added, and the mixture was vigorously shaken and then left standing, and the lower layer was separated and concentrated. The rest is processed in the same way and the total fractions containing the target substance as the main component is 260m.
g was obtained. 50 mg of this was adsorbed on a preparative silica gel thin layer plate (thickness: 0.5 mm, 20 × 20 cm), developed with chloroform: diethylamine (7: 3) for 3 hours, and had an Rf value of 0.3 to 0.4. The portion was scraped off, chloroform: methanol: water (2: 1: 1) was added, and the mixture was vigorously shaken and allowed to stand, and the lower layer was separated and concentrated. The rest is processed in the same manner, and the total amount of Abratuvolactam C is 140 mg,
Obtained as a colorless glass.

Molecular formula: C ₃₀ H ₄₀ N ₂ O ₅ ¹ H-NMR (C ₅ D ₅ N): δ 0.82 (3H, t, H-29), 0.84 (1H, m, H-9),
0.87 (3H, d, H-27), 1.0 (1H, m, H-28), 1.05 (1H, m, H-11),
1.25 (1H, m, H-10), 1.28 (1H, m, H-13), 1.48 (1H, m, H-2
4), 1.50 (1H, m, H-28), 1.64 (1H, m, H-24), 1.88 (1H, m, H-
23), 1.92 (1H, m, H-12), 2.02 (1H, m, H-9), 2.02 (1H, m, H-
13), 2.12 (1H, m, H-23), 2.5 (1H, m, H-17), 2.58 (1H, m, H-
14), 2.6 (1H, m, H-17), 2.75 (1H, bdd, H-7), 2.89 (3H, s, N
b-Me), 3.13 (1H, m, H-18 / 25), 3.20 (1H, m, H-8), 3.28 (1
H, m, H-25 / 18), 3.58 (1H, m, H-18 / 25), 3.60 (1H, bd, H-2
2), 3.70 (1H, m, H-25 / 18), 5.50 (1H, m, H-16), 5.60 (1H,
m, H-15), 6.75 (2H, d, H-2,5), 7.48 (1H, dd, H-3 / 4), 7.60
(1H, dd, H-4 / 3), 9.1 (1H, b, NH / OH).

¹³ C-NMR (C ₅ D ₅ N): δ 12.8 (C-29), 17.8 (C-
27), 23.2 (C-24), 26.53 (C-23), 26.58 (C-28), 26.7 (Nb
-Me), 28.3 (C-17), 38.9 (C-9), 39.8 (C-25 / 18), 41.3 (C
-18/25), 41.5 (C-13), 44.1 (C-8), 48.7 (C-11), 52.
0 (C-12), 53.3 (C-10), 54.3 (C-14), 64.3 (C-22), 6
5.2 (C-7), 102.1 (C-20), 132.4 (C-16), 133.2 (C-15), 1
35.4 (C-2 / 5), 135.4 (C-5 / 2), 136.4 (C-3 / 4), 139.4 (C-4
/ 3), 166.5 (C-1), 175.3 (C-26), 193.4 (C-19), 195.3 (C
-21), 201.1 (C-6).

HR-FAB-MS (m / z): 509.2995 (M + H ⁺ ). C ₃₀ H
₄₀ N ₂ O ₅ (calcd. 509.3015). [Α] _D ²⁰ : 136 ° (c 0.27, pyridine).

Test Example 1. Mouse Lymphocytic Leukemia Cell (P388) Cell Growth Inhibitory Activity Measurement Mouse lymphocytic leukemia cell (P388) was treated with 2-hydroxyethyl disulfide 5 μM and kanamycin sulfate 100.
RPMI containing 10% fetal bovine serum supplemented with μg / mL
Add 1 × 10 ⁴ cells / mL to -1640 medium
Prepared above, and added the above-mentioned Abratuvolactam A, B, or C to a predetermined concentration, and added to a CO ₂ incubator (CO ₂
The cells were cultured at 5%, humidity 100%, 37 ° C.) for 4 days. MT
The number of surviving cells was measured by the T colorimetric method, and the 50% cell growth inhibitory concentration (IC ₅₀ ) was determined from the growth inhibition rate relative to the control group. The results are shown in Table 2.

[0069]

[Table 2]

Test Example 2. O ₂ in human neutrophils ^- production inhibitory activity measuring human acute myelogenous leukemia cells HL-60, containing 10% fetal calf serum and 1.4% dimethylsulfoxide RPM
The cells were suspended in I-1640 medium at 3 × 10 ⁵ cells / mL and cultured in a CO ₂ incubator (37 ° C., CO ₂ 5.0%, humidity 100%) for 4 days to give neutrophils. Differentiated into. Differentiated human neutrophils were added to MEM medium without addition of phenol red, the cell concentration was adjusted to 2 × 10 ⁶ cells / mL, and 25 μL thereof was dispensed into a 96-well plate. Abratuvolactam A, B, or C was used as a predetermined 200-fold concentration methanol solution, and 200 μL of the same medium added with 2 μL was added to the above cell solution in an amount of 25 μL and incubated in a CO ₂ incubator for 15 minutes. At this time, 25 μL of the same medium was added to the sample-free group and the 100% inhibition control group. 100% after culturing for 15 minutes
For the inhibition control group, 1 mg of TPA 200 μg / mL ethanol solution was added to 4 mg / mL physiological saline solution of cytochrome C.
μL / mL and SOD15000U / mL (= 4.2mg
/ ML) 50 μL of a solution to which 40 μL / mL of a physiological saline solution was added, and 50 μL of another to which cytochrome C solution was added with TPA alone at the same concentration, and the mixture was incubated in a CO ₂ incubator for 1 hour to develop color. 550nm with plate reader
(Control, 570 nm) absorbance measured by the O ₂ in ^- to quantify the reduced cytochrome C produced by, O ₂ relative to the control group ^- 50% production inhibition The inhibitory concentration from production inhibition rate (I
_C50 ) was determined. The results are shown in Table 3.

[0071]

[Table 3]

Test Example 3. Phospholipase A ₂ Inhibitory Activity Measurement As phospholipase A ₂ (PLA ₂ ), 85 kDa cytosolic PLA 2 (cPLA 2) purified from rabbit platelets based on a previous report (FEBS Lett., 282, 326-330, 1991) was used, and its inhibitory activity against this enzyme was used. It measured as follows.
Abratu voractam C was dissolved in methanol and used as a test solution. 25 μl of 1M Tris-hydrochloric acid (pH 9.0)
L, 50 mM calcium chloride solution 20 μL of mixed buffer solution was mixed with the test solution and cPLA2 solution to make 200 μL, and 3
The reaction was carried out at 7 ° C for 20 minutes. Then, the substrate 1-palmitoyl-2- [ ¹⁴ C] arachidonoyl-glycerophosphoethanolamine (0.5 nmol / 50000 d
(pm / 50 μL) was added, and the mixture was further reacted at 37 ° C. for 20 minutes. Dole reagent (isopropanol: heptane: 1N
Sulfuric acid = 10: 40: 1) (1.25 mL) was added to stop the reaction, the [ ¹⁴ C] arachidonic acid fraction was collected by the method of Dole, and its radioactivity was measured by a liquid scintillation counter to measure the enzyme activity. It was measured. The results are shown in Table 4.

[0073]

[Table 4]

Test Example 4 Measurement of PAF production inhibitory activity Human acute myeloid leukemia cells HL-60 were differentiated into neutrophils in the same manner as in Test Example 1, and 7.7 × 10 ⁶ cells /
The suspension was suspended in Eagle MEM medium so as to have a volume of mL. 260 μL of this cell suspension and 100 μL of a test solution diluted with MEM medium were dispensed into glass spitzs and kept at 37 ° C., and 40 μL of 20 μM A23187 solution was added and kept for 15 minutes. The reaction was stopped by adding 1.5 mL of chloroform: methanol (1: 2), further adding 0.5 mL each of chloroform and water to separate into two layers, and the lower layer was collected to extract total lipids. PAF was separated and extracted from total lipids by TLC (developing solvent, chloroform: methanol: acetic acid: water = 50: 20: 8: 4), dissolved in 20 μL of methanol and 80 μL of Tyrode's solution to prepare a sample, and the following rabbit platelets were used. PAF was quantified by a bioassay based on agglutination.

Rabbit platelets obtained by centrifugation of peripheral blood collected from ACD blood from the ear vein of a rabbit were subjected to 50 mM HEPES.
Buffer solution (pH 7.2), 0.25% gelatin and 2.4%
2.5 × 10 ⁸ / mL in Tyrode's solution containing citric acid
It was suspended so that Add 240 μL of this to a cuvette and dilute 1/20 to 1/200 of the above sample.
μL was added and the permeability was measured with an aglycometer to examine the platelet aggregation ability, and PAF in the sample was quantified by comparing with the PAF standard solution. The results are shown in Table 5.

[0076]

[Table 5]

[0077]

INDUSTRIAL APPLICABILITY Since the abratuvolactams of the present invention have an in vitro growth inhibitory activity on mouse lymphocytic leukemia cells (P388), they can be used as an antitumor agent, and can also be used as an O of human neutrophils. _{It has a 2} ^- production inhibitory activity, an intracellular phospholipase A ₂ inhibitory activity and a human neutrophil PAF production inhibitory activity, and is expected to be used as a drug such as an anti-inflammatory agent. Furthermore, according to the present invention, these abratuvolactams can be easily and constantly produced by using a microorganism.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12P 17/18 B 7432-4B // (C12N 1/20 C12R 1: 465) (C12P 17/18 (72) Inventor Kazuyoshi Yazawa 1-28-10 Unomori, Sagamihara City, Kanagawa Prefecture (72) Inventor Tomoko Tsuji 6-12-2 Nomidai, Kanazawa-ku, Yokohama City, Kanagawa Prefecture (72) Inventor ▲ Pei ▼ Myeong Ai 2-34 Daemyung-dong, South District, Daejeong-si, South Korea (4/5)

Claims (7)

[Claims] 1. A general formula: [Chemical 2] Or [Chemical 3] Abratuvolactam represented by the formula (wherein R ¹ and R ² independently represent a hydrogen atom or a lower alkyl group, and R ³ and R ⁴
Represents independently a hydrogen atom or a hydroxyl-protecting group). 2. A general formula: [Chemical 5] Or Abratuvolactam represented by the formula (wherein R ¹ and R ² independently represent a hydrogen atom or a lower alkyl group, and R ³ and R ⁴
Represents independently a hydrogen atom or a hydroxyl-protecting group). 3. A common partial structure is as follows: [Chemical 8] And (In the formula, R ¹ and R ² independently represent a hydrogen atom or a lower alkyl group, and R ³ represents a hydrogen atom or a protective group for a hydroxyl group.)
And (a) the following physical properties: Molecular formula: C ₃₀ H ₄₀ N ₂ O ₅ ¹ H-NMR (C ₅ D ₅ N): δ 0.7 (1H, m), 0.80 (3H, t), 0.90 (3H,
d), 1.0 (1H, m), 1.05 (1H, m), 1.25 (1H, m), 1.35 (1H, m),
1.55 (1H, m), 1.6 (1H, m), 1.63 (1H, m), 1.71 (1H, m), 1.
88 (1H, m), 1.9 (1H, m), 1.9 (1H, m), 2.23 (1H, m), 2.28 (1
H, m), 2.4 (1H, m), 3.13 (1H, m), 3.19 (1H, m), 3.3 (3H, s),
4.05 (1H, m), 4.20 (1H, bs), 4.79 (1H, bd), 5.6 (1H, dd),
5.95 (1H, m), 6.08 (1H, dd), 6.12 (1H, dd), 6.12 (1H, d),
6.32 (1H, d), 7.55 (1H, dd), 7.68 (1H, dd); (b) The following physical properties and molecular formula: C ₃₀ H ₄₀ N ₂ O ₆ ¹ H-NMR (C ₅ D ₅ N): δ 0.8 (3H, t), 0.8 (1H, m), 0.85 (3H,
d), 0.95 (1H, m), 1.05 (1H, m), 1.2 (1H, m), 1.25 (1H, m),
1.5 (1H, m), 1.85 (1H, m), 1.9 (1H, m), 2.0 (1H, m), 2.0 (1
H, m), 2.2 (1H, m), 2.4 (1H, m), 2.5 (1H, m), 2.5 (1H, m),
2.7 (1H, m), 2.85 (1H, dd), 3.15 (1H, m), 3.25 (3H, s), 3.
5 (1H, m), 3.5 (1H, m), 4.0 (1H, m), 4.1 (1H, d), 4.8 (1H,
m), 5.3 (1H, m), 5.5 (1H, m), 6.6 (1H, bd), 6.7 (1H, bd),
7.4 (1H, bdd), 7.7 (1H, bdd); or (c) the following physical properties: molecular formula: C ₃₀ H ₄₀ N ₂ O ₅ ¹ H-NMR (C ₅ D ₅ N): δ 0.82 (3H, t), 0.84 (1H, m), 0.87 (3H,
d), 1.0 (1H, m), 1.05 (1H, m), 1.25 (1H, m), 1.28 (1H, m),
1.48 (1H, m), 1.50 (1H, m), 1.64 (1H, m), 1.88 (1H, m),
1.92 (1H, m), 2.02 (1H, m), 2.02 (1H, m), 2.12 (1H, m), 2.
5 (1H, m), 2.58 (1H, m), 2.6 (1H, m), 2.75 (1H, bdd), 2.89
(3H, s), 3.13 (1H, m), 3.20 (1H, m), 3.28 (1H, m), 3.58 (1
H, m), 3.60 (1H, bd), 3.70 (1H, m), 5.50 (1H, m), 5.60 (1
H, m), 6.75 (2H, d), 7.48 (1H, dd), 7.60 (1H, dd); 4. A microorganism of the genus Streptomyces that produces the abratuvolactam according to claim 1, 2. 5. The microorganism is Streptomyces s
p. The microorganism according to claim 4, which is SCRC-A20. 6. A method for producing abratuvolactams, which comprises culturing the microorganism according to claim 4 and collecting the abratuvolactams according to claim 1, 2 or 3 from the culture solution. 7. An anti-inflammatory agent comprising the abratuvolactam according to claim 1, 2 or 3 as an active ingredient.