JPH09143118A - Polyketides, their production and icam-1 expression-inhibiting agent containing the same as active ingredient - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain new polyketides useful as active ingredients for expression- inhibiting medicines against ICAM-1 especially playing an important role on the adhesion of leukocytes to vascular endothelial cells as a cell-adhesive factor, and finally used as a new antlaliergic medicine. SOLUTION: These polyketides are expressed by formulas I, II, III and IV. The polyketides are obtained by culturlng Phialomyces macrosporus, an imperfect fungus belonging to the genus Phialomyces and having an ability to produce the polyketides, and subsequently separating and purifying the culture product with various kinds of column chromatography.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel polyketide, a method for producing the same, and an IC containing the same as an active ingredient.
It relates to an AM-1 expression inhibitor.

[0002]

2. Description of the Related Art Adrenal cortex hormones, antimetabolites, alkylating agents, alkaloids, antibiotics, salicylic acid derivatives, pyrazolidine derivatives, indomethacin, etc. have been known as immunosuppressants, anti-inflammatory agents, and antiallergic agents. Used as a therapeutic agent for autoimmune diseases (collagen disease, chronic rheumatism, psoriasis, etc.), allergic diseases, rejection after organ transplantation, sepsis, inflammatory bowel disease, asthma, nephritis, various arthritis, multiple organ failure, etc. Has been. However,
These therapeutic agents are not always satisfactory in terms of efficacy, sustainability, side effects, etc., and further development of excellent immunosuppressive agents, anti-inflammatory agents, and antiallergic agents is required.

By the way, ICAM- which is a cell adhesion molecule
1 (Intercellular adhesion m
It has become clear that olecule-1) plays an important role in the development of inflammation. (Carlo
S.T. M. et al (1994) Leukocyt
e-endotheliaal adhesion mo
rules Blood 84 , 2068-210
1; Springer, T .; A. et al. , (19
94) Traffic signals for ly
mphocyte recirculation an
d leukocyte emulation: th
e mulistep paradigm Cell
301-314). That is, ICAM-1 plays an important role in adhesion between leukocytes and vascular endothelial cells, and this step is considered to be very important for the development of inflammation. Therefore, substances that suppress the expression of ICAM-1 are expected to be immunosuppressive agents, anti-inflammatory agents, and antiallergic agents with a new mechanism.

However, as a substance that suppresses ICAM-1 expression, inhibitors of Proteasome such as MG132 represented by the following formula (A) are currently reported (R
ead, M .; A. et al. , Immunity,
2 , 493 (1995), the development of a new substance having an ICAM-1 expression inhibitory action is desired.

[0005]

Embedded image

[0006]

SUMMARY OF THE INVENTION The present invention has been made from the above point of view, and its ultimate purpose is to develop new types of immunosuppressive agents, anti-inflammatory agents, and antiallergic agents. It is an object to provide an inhibitor.

[0007]

[Means for Solving the Problems] The present inventors focused on the fact that microorganisms produce physiologically active substances such as antibiotics, obtained a large number of microorganisms from nature and cultured them As a result of repeated studies on the physiological activity of various kinds of products, IC was found in the culture of microorganisms belonging to imperfect bacteria.
It was found that a substance having an AM-1 expression suppressing action was contained, the structure of the substance was clarified, and the present invention was completed. That is, according to the present invention, the following formulas (I) to (IV)

[0008]

Embedded image The polyketides represented by are provided. According to another aspect of the present invention, which belongs to an imperfect bacterium and has the general formula (I) to (I
There is provided a method for producing a polyketide, which comprises culturing a microorganism having the ability to produce the polyketide represented by V) and collecting the polyketide from the culture. Further, according to a preferred embodiment of the present invention, there is provided the above method, wherein the microorganism belonging to the incomplete bacterium is a microorganism belonging to the genus Phialomyces; and the microorganism belonging to the genus Phialomyces is Phialomyces macrosporus. It According to still another aspect of the present invention, an ICA containing the polyketides of the general formulas (I) to (IV) as an active ingredient.
An M-1 expression inhibitor is provided.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The polyketides of the present invention are compounds represented by the above formulas (I) to (IV). Since the compound of the present invention represented by the above formulas (I) to (IV) has an asymmetric carbon, various isomers exist, and all of these isomers are contained in the compound of the present invention. When the compound of the present invention is used as a medicine, it can be made into a preparation according to the administration route together with a usual preparation carrier. For example, tablets for oral administration,
It is prepared in the form of capsules, granules, powders, liquids and the like. In preparing a solid preparation for oral administration, conventional excipients, binders, lubricants, other coloring agents, disintegrating agents and the like can be used. Examples of the excipient include lactose, starch, talc, magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate, gum arabic, etc., and polyvinyl alcohol as a binder,
Examples include polyvinyl ether, ethyl cellulose, gum arabic, shellac, sucrose, and the like, and examples of lubricants include magnesium stearate, talc, and the like. In addition, commonly known colorants and disintegrants can be used. The tablets may be coated by a known method. The liquid preparation may be an aqueous or oily suspension, solution, syrup, elixir, etc.,
It is prepared by a commonly used method. When preparing an injection, a pH adjusting agent, a buffer, a stabilizer, an isotonicity agent, a local anesthetic, etc. are added to the compound of the present invention, and subcutaneously added by a conventional method.
Intramuscular and intravenous injections can be manufactured. As a base for producing a suppository, for example, an oleaginous base such as cacao butter, polyethylene glycol, lanolin, fatty acid triglyceride, Witepsol (registered trademark Dynamite Nobel) can be used.

The dose of the preparation thus prepared varies depending on the patient's symptoms, body weight, age and the like and cannot be taken uniformly, but usually the compound of the present invention is in the range of about 1 to 2000 mg per day for an adult. It is preferable that the amount is set, and it is usually preferable to administer it in divided doses 1 to 4 times a day.

Next, a method for producing the compound of the present invention will be described. For example, the polyketides represented by the above formulas (I) to (IV) of the present invention can be chemically synthesized, but usually, a microorganism having an ability to produce the polyketide is cultured to obtain a culture, that is, It is obtained by isolating polyketides from bacterial cells and / or culture supernatant. The microorganism used in the method for producing the polyketide of the present invention,
It is not particularly limited as long as it is a microorganism having the ability to produce the polyketides, Fiaromaisesu (Phi
microorganisms belonging to the genus Alomyces , for example, Phialomyces macrosporus ( Phialomyces)
macrosporus ) and the like, and more preferably Phialomyces macrosporus MCI32 described below.
There are 26 strains. The culture of the above microorganisms, isolation and purification of polyketides will be described in detail below.

(1) Culture In the present invention, an imperfect bacterium, for example, a microorganism belonging to the genus Phialomyces and having the ability to produce polyketides is cultured in a medium containing nutrients that can be utilized by ordinary microorganisms. To do. As a nutrient source, a known one conventionally used for culturing fungi can be used. For example, rice, glucose, starch syrup, dextrin, starch, molasses, animal / vegetable oil, etc. may be used as the carbon source. As the nitrogen source, soybean flour, wheat germ, corn steep liquor, cottonseed meal, meat extract, peptone, yeast extract, ammonium sulfate, sodium nitrate, urea and the like can be used. In addition, if necessary, it is also effective to add inorganic salts capable of producing sodium, potassium, calcium, magnesium, cobalt, chlorine, phosphoric acid, sulfuric acid and other ions. In addition, organic substances and inorganic substances that help the growth of bacteria and promote the production of polyketides can be added as appropriate.

As the culture method, a culture method under aerobic conditions,
Particularly, the solid culture method and the deep culture method are suitable. The temperature suitable for culturing is 15 to 35 ° C., more preferably 20.
Incubate at around -30 ° C. The production of polyketides varies depending on the culture medium and culture conditions, but the maximum accumulation is usually reached within 5 to 30 days in any of solid culture, shaking culture, and tank culture. When the accumulated amount of polyketides in the culture reaches the maximum, the culture is stopped, and the target substance is isolated and purified from the culture solution.

(2) Isolation and Purification of Polyketides Since the polyketides represented by the above formulas (I) to (IV) of the present invention are fat-soluble substances, they should be isolated and purified from the culture. It can be done by utilizing the characteristics. That is, for example, a solvent extraction method using ethyl acetate, chloroform, etc .; a synthetic adsorbent such as silica gel, alumina, octadecyl silica gel, Diaion HP-20 (manufactured by Mitsubishi Chemical Co., Ltd.), Sephadex LH-20 (manufactured by Pharmacia), etc. Column chromatography using a gel filter, etc., or high performance liquid chromatography; preparative thin layer chromatography using silica gel as a carrier, and recrystallization with water or various organic solvents are effective in some cases.

(3) Novel strain used for the production of polyketides As a microorganism belonging to the genus Phialomyces that produces the above polyketides, the incomplete bacterium Fiaromai newly isolated from the litter of plant by the present inventors The C. macrosporus MCI3226 strain (hereinafter abbreviated as "this strain" or "MCI3226 strain") will be described. The microbiological properties of this strain are as follows.

1. Morphological properties Colonies grow on potato dextrose agar (P
DA), it diffuses rapidly after culturing at 27 ° C. for 7 days.
It is velvety, initially grayish blue, then gradually dark green, then dark black. The back surface of the colony is yellowish brown to dark brown. The basal hyphae are branched, have septa, are colorless and have a width of 5.6 to 7.8 μm. Aerial hyphae are branched, have septa and are colorless. Conidia spores are born from basal hyphae or aerial hyphae. Many branches do not branch even if they are unbranched or branched, and have a partition wall. It has a height of 1 mm and a width of 7.8 to 9.4 μm, and is colorless and forms a phialide at the tip. One to three phialides are formed at the tip of the conidia peduncle and have a subcylindrical shape or a bottle shape. Size is 21
˜31 μm × 8.4-13.1 μm, and is colorless. Conidia are Waialo-type conidia, which are spindle-shaped or lemon-shaped, projecting sharply at the upper end, cutting at the lower end, dark yellowish brown, and having wart-like projections on the surface. . The size is 20 to 34 μm × 16 to 22 μm,
A conidium chain is formed by wedge-shaped connection with the conidia formed above at the upper end protruding portion.

2. Physiological properties Growth temperature (1 week culture on PDA): 20-30 ° C, 3
No growth at 7 ° C Optimal temperature: 27 ° C Growth pH (27 ° C, 1 week on LCA liquid medium): 3 to
8 Optimum pH: 7-8

3. Taxonomic considerations: This strain (MCI3226) has 1) conidia stalk usually unbranched or slightly branched, 2) conidia are Waialo-type conidia, spindle-shaped or lemon-shaped, 3 )
The upper end of the conidia protrudes sharply, and the projection at the lower end is cut-out, and the conidia formed at the upper end is wedge-shaped to form a conidial chain. 4) The surface has wart-like projections. It is dark yellowish brown and has a size of 20 to 34 μm × 16 to 22 μm. Based on the above properties, this strain (MCI3226) belongs to the genus Phialomyces of the subdivision of incomplete fungi-incomplete filamentous fungi-Fiaro type conidia forming group. M. B. Ellis (1971) Dematia
According to ceous Hyphomycetes, Ph
The genus ialomyces is P. It is a genus of a single type founded on macrosporus type. This strain (M
Various properties of conidia of CI 3226) are described in P. macros
It matched well with that of porus . Therefore, this strain
P. It was identified as macrosporus, Fiaromaisesu-Makurosuporusu (Phialomyces mac
Rosporus ) MCI3226.

The MCI3226 strain has been deposited as FERM P-15263 at the Institute of Biotechnology, Institute of Biotechnology, AIST. In general, the genus Phialomyces is likely to change its properties as seen in the case of other fungi, but mutant strains derived from the MCI3226 strain (naturally occurring or artificially induced) are also used in the present invention. be able to.
In addition, even a zygote or a gene recombinant derived from this strain can be used in all the methods of the present invention as long as it has the ability to produce polyketides.

[0020]

EXAMPLES Examples of the present invention will be shown below, but various production methods can be devised based on the properties of polyketides. Therefore, the present invention is not limited to the present example, and not only the modification means of the example but also known methods based on the properties of polyketides to produce, concentrate, extract, and purify all methods. Comprehensive.

Example 1 <1> Culture of MCI3226 strain 60 g of rice and 20 ml of tap water were added to a 500 ml Erlenmeyer flask 1
Each of them was dispensed into 00 and autoclaved at 121 ° C. for 20 minutes. This strain was dispersed in several places from a slant in this medium and inoculated, followed by static culture at 27 ° C. for 14 days.

<2> Purification of Polyketides The solid culture containing the obtained bacterial cells was added to 5 per flask.
Add 160 ml of 0% acetone water, extract with stirring, and add 13 L
To obtain an aqueous acetone solution. It was concentrated under reduced pressure to 7.2 L. This was extracted with 7.2 L of ethyl acetate, and the ethyl acetate layer was distilled off under reduced pressure to obtain 2.37 g of a residue. This residue was dissolved in methanol, insoluble materials were removed by filtration, and the filtrate was evaporated under reduced pressure to give 2.08 g of residue.

This was subjected to reverse layer partition chromatography using octadecyl silica gel. This residue is MCI
Sprinkle on GEL ODS IMY (manufactured by Mitsubishi Chemical),
After drying under reduced pressure, MCI GEL ODS IMY 47g
Was loaded onto a column equilibrated with water. The column was 250 ml of water and 250 m of methanol-water mixture (1: 4).
1, washed with 50 ml of a methanol-water mixture (2: 3), and then eluted with 100 ml of a methanol-water mixture (2: 3), and the solvent was distilled off from the eluate under reduced pressure to obtain 0.12 g of fraction A.
-1 was obtained. Furthermore, a methanol-water mixture (2: 3) 10
Elution was performed with 0 ml and 50 ml of a methanol-water mixture (1: 1), the eluates were collected, and the solvent was distilled off under reduced pressure to obtain 0.16 g of fraction A-2. Furthermore, a methanol-water mixture (1:
1) 200 ml, methanol-water mixture (3: 2) 50 m
elution was performed, the eluates were collected, and the solvent was distilled off under reduced pressure.
35 g of fraction A-3 was obtained.

The fraction A-3 obtained above was further added to In
ertsil PREP-ODS column (50 mm x 2
It was purified by preparative high performance liquid chromatography equipped with 50 mm) (manufactured by GL Science). Fraction A-3 was dissolved in 6 ml of methanol, 3 ml of which was subjected to high performance liquid chromatography. After washing with 1500 ml of a methanol-water mixture (1: 1), the mixture was eluted with 175 ml of a methanol-water mixture (1: 1). The remaining 3 ml of methanol solution was similarly fractionated, the eluate was collected, and the solvent was distilled off under reduced pressure to obtain a residue of 0.17 g. Further, this residue was treated with a CAPCELL PAK C ₁₈ column (30
mm × 250 mm) (manufactured by Shiseido Co., Ltd.) and purified by preparative high performance liquid chromatography. This residue was dissolved in 1 ml of methanol, 0.5 ml of which was
The column was subjected to high performance liquid chromatography equilibrated with a methanol-water mixture (1: 1). After washing with 630 ml of a mixture of methanol and water (1: 1), the mixture was eluted with 81 ml of a mixture of methanol and water (1: 1). The remaining 0.5 ml of methanol solution was similarly fractionated, the eluate was collected, the solvent was distilled off under reduced pressure, and the fraction containing Compound 1 0.11
g was obtained. This is recrystallized from a mixed solution of ether-n-hexane to give a compound represented by the formula (I ′) (“compound 1”).
To obtain 96.6 mg of powder.

Next, the fraction A-2 was transferred to Inertsil PR.
It was purified by preparative high performance liquid chromatography equipped with an EP-ODS column (50 mm × 250 mm). Fraction A-2 was dissolved in 6 ml of methanol, 3 ml of which was dissolved
Was subjected to high performance liquid chromatography in which the column was equilibrated with a mixed solution of methanol and water (2: 3). After washing with 1350 ml of a methanol-water mixture (2: 3), elution was performed with 225 ml of a methanol-water mixture (2: 3). The remaining 3 ml of methanol solution was similarly fractionated, the eluate was collected, and the solvent was distilled off under reduced pressure to obtain a residue of 22.6 mg. Furthermore, this residue is used for J'sphere ODS-H8
It was purified by preparative high performance liquid chromatography equipped with 0 column (30 mm × 250 mm) (manufactured by YMC Co., Ltd.). Dissolve this residue in 1 ml of methanol,
The column was subjected to high performance liquid chromatography equilibrated with a mixed solution of methanol and water (2: 3). After washing with 684 ml of a mixture of methanol and water (2: 3), elution was performed with 54 ml of a mixture of methanol and water (2: 3). The solvent was distilled off from this eluate under reduced pressure to obtain 3.8 mg of fraction B-1. Further, elution was performed with 63 ml of a methanol-water mixture (2: 3)
The solvent was distilled off from the eluate under reduced pressure to obtain 2.5 mg of fraction B-2.
I got

Fraction B-1 was further purified by preparative high performance liquid chromatography. Fraction B-1 was dissolved in 1 ml of acetonitrile, and YMC-Pack P
polymer C ₁₈ column (30 mm x 300 mm)
(Manufactured by YMC Co., Ltd.) is attached, and acetonitrile-
It was subjected to high performance liquid chromatography equilibrated with a water mixture (1: 1). Acetonitrile-water mixture (1: 1) 54
After washing with 9 ml, an acetonitrile-water mixture (1:
1) Elute with 45 ml and evaporate the solvent under reduced pressure,
2.2 m of the compound represented by the formula (II ′) (“compound 2”)
g was obtained.

Fraction B-2 was also purified by preparative high performance liquid chromatography. Fraction B-2 was dissolved in 1 ml of acetonitrile, and YMC-Pack Pol was used.
It was attached to a ymer C ₁₈ column (30 mm × 300 mm) and subjected to high performance liquid chromatography equilibrated with an acetonitrile-water mixture (1: 1). Acetonitrile-
After washing with 603 ml of a water mixture (1: 1), elution was performed with 36 ml of an acetonitrile-water mixture (1: 1), the solvent was distilled off from the eluate under reduced pressure, and the compound represented by the formula (II ″) (“ Compound 3 ") was obtained.

Next, the fraction A-1 was dissolved in 2 ml of methanol, 1 ml of which was dissolved in J'sphere ODS-H8.
It was attached to a 0 column (30 mm × 250 mm) and subjected to high performance liquid chromatography equilibrated with a methanol-water mixture (3: 7). Methanol-water mixture (3: 7) 79
After washing with 2 ml, methanol-water mixture (3: 7) 5
Elution with 4 ml gave fraction C-1. After further washing with 63 ml of a methanol-water mixture (3: 7), it was eluted with 63 ml of a methanol-water mixture (3: 7) to give a fraction C-2. After further washing with 36 ml of a methanol-water mixture (3: 7), it was eluted with 63 ml of a methanol-water mixture (3: 7) to give a fraction C-3. Similarly, the remaining 1 ml of methanol solution is also separated, the obtained eluates are combined with the previously separated eluates, and the solvent is distilled off under reduced pressure.
Fraction C-1 8.3 mg, Fraction C-2 18.2 mg,
Fraction C-3 5.3 mg was obtained.

Fraction C-2 was further separated by high performance liquid chromatography. Fraction C-2 was dissolved in 0.8 ml of methanol, and YMC-Pack Pol was used.
A ymer C ₁₈ column (30 mm × 300 mm) was attached and subjected to high performance liquid chromatography equilibrated with a methanol-water mixture (2: 3). After washing with 423 ml of a methanol-water mixture (2: 3), elution was performed with 45 ml of a methanol-water mixture (2: 3), and the solvent was distilled off from the eluate under reduced pressure to obtain 5.1 mg of a residue. Further, this residue was dissolved in 1 ml of methanol, and CAPCELL PA was used.
A K C ₁₈ column (30 mm × 250 mm) was attached and subjected to high performance liquid chromatography equilibrated with an acetonitrile-water mixture (1: 4). After washing with 432 ml of acetonitrile-water mixture (1: 4), acetonitrile-
Elution was performed with 36 ml of a water mixed solution (1: 4), and the solvent of the eluate was evaporated under reduced pressure to obtain 3.1 mg of the compound represented by the formula (III ′) (“compound 4”).

Next, the fraction C-3 was separated.
Fraction C-3 was dissolved in 1 ml of methanol, and YMC-Pa
ck Polymer C ₁₈ column (30mm x 300
mm), and subjected to high performance liquid chromatography equilibrated with a methanol-water mixture (2: 3). After washing with 441 ml of a methanol-water mixture (2: 3), elution was performed with 36 ml of a methanol-water mixture (2: 3), and the solvent was distilled off from the eluate under reduced pressure to give the compound represented by the formula (III ″). 3.0 mg of a compound (“compound 5”) was obtained.

Fraction C-1 was also separated by high performance liquid chromatography. Fraction C-1 was dissolved in 1 ml of methanol, and YMC-Pack Polymer was added.
A C ₁₈ column (30 mm × 300 mm) was attached and subjected to high performance liquid chromatography equilibrated with a methanol-water mixture (2: 3). Methanol-water mixture (2: 3) 6
After washing with 48 ml, mixed solution of methanol and water (2: 3)
Elution was carried out with 54 ml, and the solvent was distilled off from the eluate under reduced pressure to give 1.9 mg of the compound represented by the formula (IV ′) (“compound 6”).
I got

<3> Structure Determination of Polyketides The physicochemical properties of the compounds 1 to 6 obtained above were measured by various methods and their structures were analyzed. The results were as follows.

(1) Structure of Compound 1 Compound 1 was determined to be a compound represented by the following formula (I ′) based on the following physicochemical properties.

[0034]

Embedded image

Physicochemical Properties of Compound 1 1) EIMS, m / z: 238 (M ⁺ ), 220 ([M
-H ₂ O] ⁺ ), 195, 164, 136 HREIMS, C ₁₃ H ₁₈ O ₄ ([M-H ₂ O] ⁺ , ob
sd. m / z 238.1150, calcd. m / z2
38.1205) 2) UV (in methanol) λ _max (nm): 232, 3
21

3) Optical rotation (C = 0.98, in methanol)

(Equation 1)

4) ¹ H-NMR (5 in deuterated chloroform)
00 MHz) δ (ppm): 0.94 (3H, t, J =
7.5 Hz), 1.31 (3 H, s), 1.59 (2
H, qt, J = 7.5, 7.5 Hz), 2.20 (2
H, m), 2.78 (1H, m), 3.68 (1H,
d, J = 10.3 Hz), 3.73 (1H, dd, J =
13.4, 10.8Hz), 4.75 (1H, dd, J
= 10.6, 5.4 Hz), 5.48 (1 H, s),
5.66 (1 H, d, J = 2.1 Hz) 5) ¹³ C-NMR (in deuterated chloroform, 125 MHz)
δ (ppm): 13.62 (q), 18.93 (q),
20.01 (t), 36.56 (t), 37.79
(D), 68.73 (t), 75.22 (d), 77.
29 (s), 100.69 (d), 113.49
(D), 152.52 (s), 168.80 (s), 2
0.07 (s) 6) IR (KBr method) ν _max (cm ⁻¹ ): 3364,2
961,1663,1593,1456,1289,1
221, 1148, 1100, 871

(2) Structure of Compound 2 Based on the following physicochemical properties, Compound 2 was determined to be a compound represented by the following formula (II ').

[0039]

Embedded image

Physicochemical properties of compound 2 1) EIMS, m / z: 236 (M ⁺ ), 218 ([M
-H ₂ O] _{^{+), 201,175 HREIMS, C 13}} H 14 O 3 ( [M-H ₂ O] ^+, ob
sd. m / z 218.0955, calcd. m / z2
18.0943) 2) UV (in methanol) λ _max (nm): 232, 2
80,300 (sh)

3) Optical rotation (C = 0.12, in methanol)

(Equation 2)

4) ¹ H-NMR (in deuterated acetonitrile,
500 MHz) δ (ppm): 1.18 (3H, t, J
= 7.6 Hz), 1.28 (3H, s), 2.60 (2
H, q, J = 7.5 Hz), 2.99 (1H, dd, J
= 2.9, 17.9 Hz), 3.24 (1H, dd, J
= 3.4, 17.8 Hz), 4.12 (1H, dd, J
= 3.2, 3.2 Hz), 6.67 (1H, s), 7.
71 (1H, s) 5) ¹³ C-NMR (in deuterated acetonitrile, 125 MH
z) δ (ppm): 14.24 (q), 23.24
(Q), 23.31 (t), 34.11 (t), 75.
33 (d), 77.47 (s), 115.74 (d),
123.42 (s), 129.09 (d), 130.9
1 (s), 141.39 (s), 161.69 (s),
200.48 (s) 6) IR (KBr method) ν _max (cm ⁻¹ ): 3410,1
672, 1607, 1582, 1283, 1105, 1
055,1011

(3) Structure of Compound 3 Compound 3 was determined to be a compound represented by the following formula (II ″) based on the following physicochemical properties.

[0044]

[Chemical 6]

Physicochemical Properties of Compound 3 1) EIMS, m / z: 236 (M ⁺ ), 218 ([M
-H ₂ O] _{^{+), 201,175 HREIMS, C 13}} H 14 O 3 ( [M-H ₂ O] ^+, ob
sd. m / z 218.0876, calcd. m / z2
18.0943) 2) UV (in methanol) λ _max (nm): 231,2
80,300 (sh)

3) Optical rotation (C = 0.12, in methanol)

(Equation 3)

4) ¹ H-NMR (in deuterated acetonitrile,
500 MHz) δ (ppm): 1.18 (3H, t, J
= 7.6 Hz), 1.20 (3H, s), 2.60 (2
H, q, J = 7.5 Hz), 2.83 (1H, dd, J
= 10.8, 16.7 Hz), 3.09 (1H, dd,
J = 5.6, 16.8 Hz), 3.97 (1H, dd,
J = 5.7, 10.8 Hz), 6.70 (1H, s),
7.72 (1H, s) 5) ¹³ C-NMR (in deuterated acetonitrile, 125 MH
z) δ (ppm): 14.35 (q), 18.47
(Q), 23.55 (t), 35.50 (t), 73.
70 (d), 78.59 (s), 115.55 (d),
123.32 (s), 129.83 (d), 131.6
6 (s), 142.07 (s), 162.03 (s),
200.48 (s) 6) IR (KBr method) ν _max (cm ⁻¹ ): 3420,1
667, 1607, 1580, 1273, 1107, 1
074, 1044

(4) Structure of Compound 4 From the following physicochemical properties, Compound 4 has the following formula (III ′)
It was determined to be a compound represented by.

[0049]

Embedded image

Physicochemical Properties of Compound 4 1) EIMS, m / z: 240 (M ⁺ ), 222 ([M
-H ₂ O] _{^{+) HREIMS, C 13 H 18}} O 3 ( [M-H ₂ O] ^+, ob
sd. m / z 222.1209, calcd. m / z2
22.1256) 2) UV (in methanol) λ _max (nm): 246

3) Optical rotation (C = 0.28, in methanol)

(Equation 4)

4) ¹ H-NMR (5 in deuterated chloroform,
00 MHz) δ (ppm): 0.94 (3H, t, J =
7.5 Hz), 1.26 (3 H, s), 1.64 (2
H, qt, J = 7.5, 7.5 Hz), 1.78 (3
H, s), 2.46 (4H, m), 2.56 (1H, d
d, J = 5.8, 18.3 Hz), 3.38 (1H,
d, J = 16.5 Hz), 3.45 (1H, d, J = 1)
6.5 Hz), 3.78 (1H, brs), 3.97
(1 H, dd, J = 6.0, 10.0 Hz) 5) ¹³ C-NMR (in deuterated chloroform, 125 MHz)
δ (ppm): 11.72 (q), 13.61 (q),
17.23 (t), 17.72 (q), 37.50
(T), 45.20 (t), 48.53 (t), 72.
28 (d), 77.19 (s), 130.57 (s),
148.58 (s), 201.82 (s), 205.1
9 (s) 6) IR (KBr method) ν _max (cm ⁻¹ ): 3449,1
713, 1671, 1634, 1456, 1426, 1
366, 1302, 1196, 1096, 1047, 1
009,980

(5) Structure of Compound 5 From the following physicochemical properties, Compound 5 has the following formula (III ″)
It was determined to be a compound represented by.

[0054]

Embedded image

Physicochemical Properties of Compound 5 1) EIMS, m / z: 240 (M ⁺ ), 222 ([M
-H ₂ O] ⁺ ) HREIMS, C ₁₃ H ₂₀ O ₄ (M ⁺ , obsd.m / z
240.1328, calcd. m / z 240.136
0) 2) UV (in methanol) λ _max (nm): 248

3) Optical rotation (C = 0.30, in methanol)

(Equation 5)

4) ¹ H-NMR (in deuterated chloroform, 5
00 MHz) δ (ppm): 0.93 (3H, t, J =
7.4 Hz), 1.35 (3 H, s), 1.26 (2
H, qt, J = 7.3, 7.3 Hz), 1.80 (3
H, s), 2.47 (2H, t, J = 7.2 Hz),
2.50 (1H, brd, J = 20.0Hz), 2.7
3 (1H, brs), 2.83 (1H, brd, J = 1
9.0 Hz), 3.25 (1H, d, J = 16.6H
z), 3.55 (1H, d, J = 16.5Hz), 4.
02 (1H, brs), 4.10 (1H, dd, J =
2.0, 3.7 Hz) 5) ¹³ C-NMR (in deuterated chloroform, 125 MHz)
δ (ppm): 11.48 (q), 13.61 (q),
17.18 (t), 23.03 (q), 36.29
(T), 45.07 (t), 48.71 (t), 72.
87 (d), 75.94 (s), 129.49 (s),
146.37 (s), 201.48 (s), 205.7
9 (s) 6) IR (KBr method) ν _max (cm ⁻¹ ): 3453,1
713, 1672, 1638, 1420, 1379, 1
308, 1065, 937, 901

(6) Structure of Compound 6 Based on the following physicochemical properties, Compound 6 was determined to be a compound represented by the following formula (IV ′).

[0059]

Embedded image

Physicochemical Properties of Compound 6 1) EIMS, m / z: 252 (M ⁺ ), 209, 18
1 HREIMS, C ₁₃ H ₁₆ O ₅ (M ⁺ , obsd.m / z
252.0994, calcd. m / z 252.099
8) 2) UV (in methanol) λ _max (nm): 218,2
66

3) Optical rotation (C = 0.19, in methanol)

(Equation 6)

4) ¹ H-NMR (in deuterated acetonitrile,
500 MHz) δ (ppm): 0.96 (3H, t, J
= 7.4 Hz), 1.26 (3 H, s), 1.68 (2
H, qt, J = 7.3, 7.3 Hz), 2.77 (1
H, dd, J = 9.0, 17.8 Hz), 2.78 (2
H, t, J = 7.3 Hz), 3.40 (1H, dd, J
= 5.3, 17.8 Hz), 3.74 (1H, br
s), 4.03 (1H, dd, J = 5.1, 8.8H)
z), 8.42 (1H, s) 5) ¹³ C-NMR (in deuterated acetonitrile, 125 MH
z) δ (ppm): 13.92 (q), 18.15
(T), 18.47 (q), 29.03 (t), 43.
35 (t), 74.71 (d), 79.07 (s), 1
27.32 (s), 135.82 (s), 147.62
(S), 154.71 (d), 189.06 (s), 1
96.41 (s) 6) IR (KBr method) ν _max (cm ⁻¹ ): 3449,1
680, 1589, 1524, 1422, 1078, 9
32

Example 2 ICAM-1 Expression Inhibitory Effect of Polyketides The polyketides obtained above were analyzed by T. Willi
ams et al. (Anal. Biochem., 17
6 , 28-32 (1989)), according to ICAM-1
The expression inhibitory activity was measured. That is, human ICAM-1
A vector expressing the luciferase gene was constructed under the control of the gene expression regulatory region, and this was introduced into the human lung adenocarcinoma cell line A-549 (ATCC CCL185). This cell line is a tumor necrosis factor-alpha (Tumor Necr
luciferase is expressed in response to the stimulus of the oxidase factor-α; TNF-α), and by measuring the enzyme activity, ICAM-1 expression can be quantitatively measured.

In the actual measurement, the above-mentioned A-549 was inoculated and cultured on a plate, and the above polyketides (final concentration 10 ⁻⁵
M), and added TNF-α (final concentration 2000 U / m
I) was stimulated for 6 hours. Further, after the cell lysate treatment, the luciferase enzyme activity was measured using a luminometer using luciferin as a substrate. As a result, the compound 1 had an I of 62% as compared with the case where the test drug was not added.
It showed a CAM-1 expression inhibitory action.

[0065]

INDUSTRIAL APPLICABILITY The polyketides of the present invention can be used at low concentration
Since it has an AM-1 expression inhibitory action, an IC containing the same
The AM-1 expression inhibitor is expected as an immunosuppressant, anti-inflammatory agent, antiallergic agent and the like.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location C07C 49/747 9049-4H C07C 49/747 C C07D 307/87 C07D 307/87 311/76 311 / 76 C12P 7/26 C12P 7/26 17/04 17/04 17/06 17/06 // (C12P 7/26 C12R 1: 645) (C12P 17/04 C12R 1: 645) (C12P 17/06 C12R 1 : 645) (72) Inventor Noriko Chiba 1000 Kamoshida-cho, Aoba-ku, Yokohama-shi, Kanagawa Mitsubishi Chemical Corporation Yokohama Research Institute (72) Inventor Takashi Mikawa 1000 Kamoshida-cho, Aoba-ku, Yokohama-shi, Kanagawa Mitsubishi Chemical Corporation Yokohama Inside the research institute

Claims (5)

[Claims] 1. Polyketides represented by the following formulas (I) to (IV). Embedded image 2. A method for producing a polyketide, which comprises culturing a microorganism belonging to an incomplete bacterium and having the ability to produce the polyketide according to claim 1, and collecting the polyketide from the culture. 3. The method according to claim 2, wherein the incomplete bacterium is a microorganism belonging to the genus Phialomyces. 4. The method according to claim 3, wherein the microorganism belonging to the genus Phialomyces is Phialomyces macrosporus. 5. An ICAM-1 expression inhibitor comprising the polyketide according to claim 1 as an active ingredient.