JPH1045731A - Benzoxazole compounds - Google Patents

Abstract

(57) [Summary] [Object] To provide a novel physiologically active substance having an ELAM-1 adhesion inhibitory action. [Constitution] Formula [Formula 6] (Wherein R is a hydrogen atom or a methoxy group), and a compound represented by the formula: A compound represented by the formula:

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel physiologically active substance having a cell adhesion inhibitory action.

[0002]

2. Description of the Related Art In recent years, research on cell adhesion molecules has progressed,
Inflammation, allergy, arteriosclerosis, cancer metastasis, autoimmune disease,
It has been clarified to play a very important role in the formation of pathological conditions such as the presentation of self antigens. Although many types of cell adhesion molecules have been found so far, it has become clear that each has a shared function and its expression is strictly controlled. Among them, ELAM-1 is
Autoimmunity is known to induce neutrophils, monocytes, and T cells that play important roles in inflammation to the site of inflammation, and is characterized by chronic inflammation by autoimmune mechanisms such as rheumatoid arthritis. Its importance has also been revealed in diseases. Already in various inflammation animal models,
It has been shown that administration of an anti-ELAM-1 monoclonal antibody or a partial peptide of ELAM-1 has a remarkable anti-inflammatory effect. It has also been shown to be useful as a drug for asthma, psoriasis and rheumatoid arthritis.

[0003]

SUMMARY OF THE INVENTION Cell adhesion molecule ELAM
-1 is a protein, and it is difficult to convert these high molecular compounds such as monoclonal antibodies into drugs. Therefore, EL
A low molecular weight compound having a new mechanism of action called AM-1 adhesion inhibitory action is desired. An object of the present invention is to provide an ELAM
-1 to provide a novel physiologically active substance having an adhesion inhibitory action.

[0004]

Means for Solving the Problems The present inventors have isolated a large number of strains from soil and plants to achieve the above object,
As a result of various studies on metabolites of the strain, it was found that a certain strain produced a novel bioactive substance having an ELAM-1 adhesion inhibitory effect, and the present invention was completed.

That is, the present invention provides

[0006]

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A compound represented by the formula (hereinafter abbreviated as AI-070):

[0008]

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(Hereinafter abbreviated as AI-071) and a compound represented by the formula

[0010]

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(Hereinafter, abbreviated as AI-072).

The novel physiologically active substance AI-070 of the present invention
072-producing strain is a strain newly isolated from nature by the present inventors, and has a microorganism name of “ Microtetraspor
a spiralis TA-0294 '' and the microorganism deposit number `` FERM P
-15757 "at the Institute of Biotechnology and Industrial Technology.

The mycological properties of this strain are shown below. A. Morphological characteristics This strain grows well on yeast malt extract agar medium and oatmeal agar medium and has good sporulation, but growth on other mediums tends to be poor overall, and sporulation is also observed. I can't. Observation of the colonies grown on the oatmeal agar medium under a microscope reveals that the underlying mycelium is well developed and diverges, but no division is observed. Through a short spore stalk (about 0.3-3.0 μm)
A spore chain consisting of 30 spores is formed. Rarely, a second spore stalk is formed on the spore stalk, and a spore chain similar to the above is formed at the tip. The spore chain is spiral, usually 2 to
5 rotations. The spores are 1.1 to 1.6 μm in length, 0.8 to 1.2 μm in width, oval to barrel, and wrinkled on the surface. In addition, sporangia, sclerotium and spore motility are not observed.

B. Properties on Various Media The following Table 1 shows the results of macroscopic observations when the cells were cultured on various media at 28 ° C. for 21 days. In addition, the color display quoted the system color name of the Japan Standards Association and JIS color name book (1985).

[0015]

[Table 1]

C. Physiological properties Growth temperature range: 20-38 ° C., optimum growth temperature: 28-34 ° C.

Liquefaction of gelatin: negative Coagulation of skim milk: negative Peptoneization of skim milk: false positive Production of melanin-like pigment: negative Hydrolysis of starch: negative Use of carbon source (28 ° C. on Pridham Götrib agar medium) L-arabinose: w Inositol: w D-xylose: w D-mannitol: w D-glucose: + maltose: + D-fructose: w D-mannose: + sucrose: w glycerol: + L-lamulose : W dulcitol:-raffinose: w lactose:-(+: grows w: grows weakly-: hardly grows).

D. Chemical taxonomic properties Presence / absence of diaminopimelic acid and optical isomer: diaminopimelic acid of meso type was detected. Amino acid composition of cell wall: Alanine and glutamic acid were detected. Glycine was not detected. Types of reducing sugars in all cells: ribose, madulose, mannose, galactose and glucose were detected. Menaquinone: MK-9 (H ₄ ) was detected as the main component. MK-9 (H ₂ ) and MK-9 (H ₆ ) were also detected in small amounts. Phospholipid: phosphatidylinositol mannoside,
Diphosphatidylglycerol, hydroxyphosphatidylethanolamine and ninhydrin positive glycolipids were detected. Also, trace amounts of phosphatidylglycerol were detected.

The TA-0294 strain was prepared as described above 1).
The diaminopimelic acid is detected in meso form, and the cell wall amino acid does not contain glycine (cell wall type I
II). 2) Contains madulose as a whole cell sugar component (sugar pattern B). 3) menaquinones MK-9 _(H 4)
As a main component. 4) The phospholipid is of the PIV type. Based on these results, this strain is a member of the genus Microtetraspora.
It became clear that it was a bacterial species. This strain is called "BERGEY'S
MANUAL OF Systematic Bacteriology Vol.4 ", when searching for species, Microtetraspora formed spiral spore chains and formed white colonies.
It turned out to be similar to spiralis or M. fastidiosa . This strain has a wrinkled surface structure of spores, and the color of the colony is white to pale yellow.
It is similar to Microtetraspora spiralis and has many similarities in other properties. Furthermore, the growth characteristics of M. spiralis JCM 3286 on 9 kinds of agar medium were very similar. Based on the above results, this strain was transformed with Microtetraspora spiralis TA-
It was named 0294.

The production of AI-070,071 and 072, according to the case of producing general fermentation products generally, in a medium containing various nutrients Microtetraspora spiralis
This is performed by culturing TA-0294 under aerobic conditions. The medium is mainly a liquid medium, carbon source, nitrogen source,
Consisting of inorganic salts, vitamins, precursors,
An antifoam can be added and the pH is adjusted around 7.
As the carbon source, for example, glucose, maltose, dextrin, glycerin, starch or the like is used alone or as a mixture. Nitrogen sources include, for example, yeast extract, peptone, meat extract, soy flour, corn steep liquor,
Urea, ammonium salt or the like is used alone or as a mixture. As the inorganic salt, for example, monopotassium phosphate, magnesium sulfate, sodium chloride, calcium carbonate and the like are used alone or as a mixture. Adecanol, silicon compounds, and the like can be used as the defoaming agent.

Aerobic culture such as shaking culture and aeration and stirring culture is suitable as the culture method.
4 to 7 days at 5 ° C, desirably pH 5 to 7, 27 to 32
Incubate at 6 ° C for 6-7 days.

AI-070 produced by this culture,
071 and 072 may be isolated according to a general method for collecting fermentation products. That is, after completion of the culture, a culture filtrate is obtained by centrifugation or filtration, adsorbed on a polystyrene resin such as Diaion HP-20 (trade name, manufactured by Mitsubishi Kasei), and then eluted with an organic solvent such as lower alcohol or acetone. Let it. The eluate is concentrated under reduced pressure to remove the organic solvent, and then transsolved in a water-insoluble organic solvent such as ethyl acetate, benzene, methylene chloride, etc., and concentrated under reduced pressure to form a syrup. This syrup is again dissolved in an organic solvent such as ethyl acetate, benzene, methylene chloride, acetone, methanol, etc., and column chromatography using silica gel, column chromatography packed with silica gel ODS for reverse phase distribution, Sephadex LH-
AI-070, 071 and 072 can be purified and isolated by subjecting them to gel filtration chromatography such as 20.

The target substances of the present invention, AI-070, 071, and 072, obtained by the above purification, are analyzed according to their molecular weight, ultraviolet absorption spectrum, ¹ H-NMR spectrum, ¹³ C-NMR spectrum and the like. The chemical structures were determined as shown in Chemical formulas 3, 4 and 5, respectively.

The physical and chemical properties of AI-070 are as follows. (A) Appearance: yellow powder (b) Melting point: 126-130 ° C. (c) Molecular weight: 416 (d) Molecular formula: C ₂₀ H ₂₀ N ₂ O ₈ (e) HRFAB mass spectrum Actual value 455.0856 Theoretical value 455.0856 (Calculated as C ₂₀ H ₂₀ N ₂ O ₈ K) (f) FAB mass spectrum: m / z 417 (M + H) ⁺ (g) Specific rotation ⁺ 82.7 ° (c = 0.09, measured in methanol solution) (H) UV absorption spectrum: λmax 215 nm (ε = 35700) 272 nm (ε = 8800) 280 nm (ε = 8800) 289 nm (ε = 7400) (measured in methanol solution) (i) Infrared absorption spectrum: KBr method FIG. 1 shows the results of the measurement.

(J) ¹ H-NMR spectrum: CDCl ₃
The result measured at 400 MHz is shown in FIG.

(K) ¹³ C-NMR spectrum: CDCl
₃ shows the results measured at 100 MHz in FIG.

(L) Solubility in solvent: Soluble in chloroform, methanol, ethanol, acetonitrile Insoluble in n-hexane and benzene (m) Color reaction; Positive: H ₂ SO ₄ , ammonium molybdate sulfate, I ₂ negative : Ninhydrin, FeCl ₃ , vanillin sulfate, phosphomolybdenum (n) Basic, acidic, neutral distinction: neutral.

The physicochemical properties of AI-071 are as follows. (A) Appearance: yellow powder (b) Melting point 90-91 ° C. (c) Molecular weight: 446 (d) Molecular formula: C ₂₁ H ₂₂ N ₂ O ₉ (e) HRFAB mass spectrum Observed value 447.1431 Theoretical value 447.1404 (Calculated as C ₂₁ H ₂₃ N ₂ O ₉ ) (f) FAB mass spectrum: m / z 447 (M + H) ⁺ (g) Specific rotation +92.3 ゜ (c = 0.05, measured in methanol solution) (H) UV absorption spectrum: λmax 215 nm (ε = 36800) 239 nm (ε = 15600) 273 nm (ε = 9900) 280 nm (ε = 10000) 288 nm (ε = 9100) 312 nm (ε = 4100) (measured in methanol solution) (I) Infrared absorption spectrum: FIG. 4 shows the results measured by the Neat method.

(J) ¹ H-NMR spectrum: CDCl ₃
FIG. 5 shows the results measured at 500 MHz.

(K) ¹³ C-NMR spectrum: CDCl
₃ shows the results measured at 125 MHz in FIG.

(L) Solubility in solvent: Soluble in chloroform, methanol, ethanol, acetonitrile Insoluble in n-hexane and benzene (m) Color reaction; Positive: H ₂ SO ₄ , ammonium molybdate sulfate, I ₂ negative : Ninhydrin, FeCl ₃ , vanillin sulfate, phosphomolybdenum (n) Basic, acidic, neutral distinction: neutral.

The physical and chemical properties of AI-072 are as follows. (A) Appearance: pale yellow powder (b) Melting point: 114-115 ° C. (c) Molecular weight: 402 (d) Molecular formula: C ₁₉ H ₁₈ N ₂ O ₈ (e) HRFAB mass spectrum Observed value 403.1125 Theoretical value 403 .1141 (calculated as C ₁₉ H ₁₉ N ₂ O ₈ ) (f) FAB mass spectrum: m / z 403 (M + H) ⁺ (g) Specific rotation ⁺ 19.6 ° (c = 0.12, in methanol solution) (H) UV absorption spectrum: λmax 216 nm (ε = 32600) 238 nm (ε = 9400) 247 nm (ε = 5100) 280 nm (ε = 3200) 288 nm (ε = 2900) (measured in methanol solution) (i) Infrared absorption spectrum: FIG. 7 shows the result measured by the Neat method.

(J) ¹ H-NMR spectrum: DMSO
Among -d _6, shows the results of measurement by 400MHz in FIG.

(K) ¹³ C-NMR spectrum: DMSO
FIG. 9 shows the results of measurement at −100 MHz during −d ₆ .

(L) Solubility in solvents: Soluble in chloroform, methanol, ethanol, acetonitrile Insoluble in n-hexane and benzene (m) Color reaction; Positive: ammonium molybdate sulfate, I ₂ , phosphomolybdenum Negative: H ₂ SO ₄ , ninhydrin, FeCl ₃ , vanillin sulfate (n) Basic, acidic, neutral distinction: neutral.

[0036]

Industrial Applicability The compounds of the present invention exhibit ELAM-1 cell adhesion inhibitory activity and are useful as anti-inflammatory, anti-rheumatic and anti-asthmatic agents.

[0037]

The present invention will be described in more detail with reference to the following Examples and Test Examples. Example Soluble starch 2.0%, glucose 0.5%, NZ case (Wako Pure Chemical) 0.3%, yeast extract 0.2%, meat extract (Mikuni Chemical Industry) 0.5%, calcium carbonate 0.5% 2 mL of a pH 7 liquid medium containing 3% was placed in a 24 mL test tube, and sterilized at 120 ° C. and 2 atm for 20 minutes. Then, the sterile liquid medium was inoculated with Microtetraspora spiralis TA-0294 strain, and cultured at 28 ° C. for 6 days on a rotary shaker (200 RPM) to obtain a seed culture solution. Next, 70 Erlenmeyer flasks having a capacity of 500 mL were filled with 2.0% oatmeal, 2.0% glucose, 0.3% meat extract, and 0.3% calcium carbonate.
%, Sodium chloride 0.3%, ferric sulfate 0.04%, and 100 mL of a production medium containing 0.04% of manganese chloride. The mixture is adjusted to pH 7.0 and sterilized. After inoculation, aeration and agitation culture at 28 ° C for 6 days (20
0 RPM). After completion of the culture, 7 L of the obtained culture solution
Was separated into a filtrate and cells by centrifugation. The filtrate was adsorbed on 350 mL of Diaion HP-20 (trade name, manufactured by Mitsubishi Kasei Corporation) and then eluted twice with 1 L of 80% aqueous acetone. These were combined, and the solvent was distilled off under reduced pressure. The obtained aqueous layer was extracted three times with 300 ml of ethyl acetate. The ethyl acetate layers were combined, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 322 mg of a brown syrup-like substance. .

320 mg of this brown syrup was dissolved in methylene chloride, and 40 mL of silica gel [Silica gel 60 (Merck)] prepared with methylene chloride was used.
Column. Methylene chloride-methanol (1
00: 0) Elution was performed with 100 mL to remove these fractions. Then methylene chloride-methanol (99: 1),
The mixture was eluted sequentially with 100 mL of a mixed solvent of (98: 2) and (95: 5), and the obtained fraction was concentrated to dryness to give a crude product of 189 m
g was obtained.

185 mg of this crude product was dissolved in a small amount of methanol, and 20% methanol (methanol: water = 1: 1) was dissolved.
The mixture was adsorbed on 60 ml of the ODS column [Chromatorex (manufactured by Senshu-Kagaku)] prepared in 4) and eluted sequentially with 20% methanol and 30% methanol. A 20% methanol fraction was concentrated to dryness, and 67 mg of the crude product obtained was packed with Sephadex LH-20 (trade name; manufactured by Pharmacia) prepared with chloroform: methanol: n-hexane = 5: 1: 5. Gel filtration was performed using the same solvent using a 280 ml column, and the active fraction was concentrated under reduced pressure to dryness to obtain 24.4 mg of AI-072. A 30% methanol fraction was concentrated to dryness, and 26 mg of the crude product obtained was filled with Sephadex LH-20 (trade name; manufactured by Pharmacia) prepared with chloroform: methanol: n-hexane = 5: 1: 5. Gel filtration was carried out in the same manner using a 110 mL column, and AI-070 was added as an active substance to 12.3.
mg of AI-071 was obtained. To check the purity,
Column: YMC-PackAM-302 ODS-AM
(4.6mm x 150mm), UV absorption 215nm
While monitoring with 30% CH ₃ CN, flow rate 1.0 ml /
min. AI-070: Rt 9.1 min, A
I-071 is Rt10.1 min, AI-072 is Rt
It was 4.2 min.

Test Example 1 [Test of Huvec-Colo201 cell adhesion inhibitory effect] The ELAM-1 cell adhesion inhibitory effect was determined by the Huvec-Col.
It was evaluated using the o201 cell adhesion inhibitory test. That is, Huvec cells, which are human umbilical vein vascular endothelial cells, are cultured in a solid phase and stimulated with TNF-α to induce adhesion molecules (ELAM-1) on the cells. Fluorescently labeled human colorectal cancer cells (Colo201 cells, SLex expression) were simultaneously added to the samples, and evaluation was performed by measuring the amount of adhesion between the cells. (Specimen) AI-070, 071 and 0 obtained in Examples
72 was dissolved in DMSO and used as a sample. (Test cells) Huvec cells (human umbilical vein vascular endothelial cells) Fluorescently labeled Colo201 cells (human colon cancer cells) (Test method) Huvec cells were collected at 2 × 10 ⁴ cells /
The cells are seeded on a collagen-coated 96-well plate so as to form wells, and cultured overnight at 37 ° C. in a 199 medium. After removing the medium, a medium (10% FCS-RPMI) containing rhTNF-α (100 u / ml) is added at 50 ul / well, and then a sample is added, followed by culturing at 37 ° C. for 6 hours. In addition, 1 × 10 ⁵ cells of fluorescently labeled Colo201 cells
ls / 50 ul / well and add the plate to 37
Shaking culture was performed at 50 ° C. and 50 rpm for 20 minutes to complete the adhesion. After removing the medium and washing with 0.25 mM Ca ²⁺ PBS buffer, the adhered Colo201 cells were removed with 1% NP-
After dissolving at 40 (50 ul / well), the fluorescence intensity was measured. The results were expressed as adhesion inhibition rates.

(Results) The results are shown in Table 2.

[0042]

[Table 2]

[Brief description of the drawings]

FIG. 1 shows an infrared absorption spectrum of AI-070 measured by a KBr method.

FIG. 2 shows AI- measured at 400 MHz in CDCl ₃ .
The ¹ H-NMR spectrum of 070.

FIG. 3 shows AI- measured at 100 MHz in CDCl ₃ .
070 shows a ¹³ C-NMR spectrum.

FIG. 4 shows an infrared absorption spectrum of AI-071 measured by the Neat method.

FIG. 5 shows AI- measured at 500 MHz in CDCl ₃ .
2 shows the ¹ H-NMR spectrum of ^No. 071.

FIG. 6 shows AI- measured at 125 MHz in CDCl ₃ .
1 shows the ¹³ C-NMR spectrum of 071.

FIG. 7 shows an infrared absorption spectrum of AI-072 measured by the Neat method.

[8] Among DMSO-d _6, was measured at 400 MHz A
¹ shows the ¹ H-NMR spectrum of I-072.

[9] Among DMSO-d _6, was measured at 100 MHz A
1 shows a ¹³ C-NMR spectrum of I-072.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification number Office reference number FI Technical indication (C12P 17/14 C12R 1:01) (C12P 17/16 C12R 1:01) (72) Inventor Tsutomu Ando 3-24-1, Takada, Toshima-ku, Tokyo Inside Taisho Pharmaceutical Co., Ltd. (72) Inventor Akie Yamaguchi 3- 24-1, Takada, Toshima-ku, Tokyo Inside Taisho Seiyaku Co., Ltd.

Claims (2)

[Claims] (1) Formula (1) (In the formula, R is a hydrogen atom or a methoxy group.) 2. The formula: A compound represented by the formula: