WO1995026969A1

WO1995026969A1 - Moranoline derivative

Info

Publication number: WO1995026969A1
Application number: PCT/JP1995/000609
Authority: WO
Inventors: Akira Hasegawa; Makoto Kiso; Yoshiaki Yoshikuni
Original assignee: Nippon Shinyaku Co., Ltd.
Priority date: 1994-03-31
Filing date: 1995-03-30
Publication date: 1995-10-12

Abstract

A compound represented by general formula (I), wherein R1 represents C¿8?-C30 alkyl, C8-C30 alkenyl or C8-C30 alkynyl; and R?2 and R3¿ represent each independently galactosyl, sialylgalactosyl or fucosyl. It is useful for treating inflammation and accompanying thrombus formation, asthma and rheumatism, and preventing and treating immunopathy and cancer, because it has a cell adhesion inhibitor activity and antagonistically inhibit secretin.

Description

CT / JP9S / 00609

1

Description Moranoline derivatives

The present invention relates to novel molanolin derivatives. The compounds according to the present invention are useful in the field of medicine, for example, as anti-inflammatory agents and anti-viral agents. Background art

According to recent research, the sugar chains of sialic acid-containing glycolipids and glycoproteins have receptor functions for hormones, bacterial toxins, viruses, and others, and also recognize, differentiate, proliferate, and adhere to cells. It is becoming clear that it is deeply involved in basic and dynamic life phenomena such as cancer, immunity, and aging.

Accordingly, sialic acid derivatives have been attracting attention as useful substances having physiological activity, and researches on therapeutic, diagnostic and prophylactic agents for various diseases have been made.

For example, as antiviral agents, there are JP-A-63-139193, JP-A-3-251593, and the like. — There are Japanese Patent Application Laid-Open Nos. 243074 and JP-A-62-20994. Furthermore, as an application to cancer diagnosis and treatment, there is JP-A-61-370000 and the like.

The present inventors have intensively studied various 耱 -chain antigen derivatives containing sialic acid, and have found novel sugar chain antigen derivatives containing moranolin, which have excellent pharmacological actions as pharmaceuticals, and have already filed an international patent application. (WO93 / 15098).

However, although these sialic acid derivatives exist in the natural world as minor components, it has been extremely difficult to obtain these compounds as single pure compounds from living organisms. Therefore, research on sialic acid derivatives as pharmaceuticals has only just begun in recent years, and its clinical application is greatly expected. Disclosure of the invention

An object of the present invention is to provide a moranolin sugar chain antigen derivative which is useful as a medicament and has a novel structure.

The present inventors have conducted further studies and conducted intensive studies. As a result, the present inventors have found that the compound represented by the general formula [I] is suitable for the above object, and have completed the present invention.

[I]

In the formula, R ¹ represents a C8-C30 alkyl, a C8-C30 alkyl, or a C8-C30 alkyl, and R ² and R ³ are different and represent a galactosyl, sialyl galactosyl, or fucosyl group.

The compound according to the present invention is a novel compound that has not been described in the literature, and exhibits excellent pharmacological actions as described below.

The alkyl represented by R ¹ in the general formula (I) is preferably a straight-chain or branched alkyl having 8 to 30 carbon atoms, for example, octyl, isooctyl, nonyl, isononyl, decyl, isodecyl, pendecyl, isopendecyl, Dodecyl, isododecyl, tridecyl, isotridecyl, tetradecyl, isotetradecyl, pentadecyl, isopentadecyl, hexadecinole, isohexadecyl, heptadecyl, isoheptadecyl, occtadecyl, isooctadecyl, nonadecyl, isononadecyl, etc. it can.

The alkenyl is preferably a straight-chain or branched one having 8 to 30 carbon atoms, for example, octyl, nonenyl, decenyl, pendenyl, dodecenyl, tridecyl, tetradecenyl, pentadecenyl, hexadeceyl. Heptadecenyl, octadecyl, nonadecel, icosenyl, and the like. Alkyl is preferably a straight-chain or branched one having 8 to 30 carbon atoms, for example, octyl, noryl, decinyl, pendecyl, dodecyl, tridecyl, tetradecyl. Pentadecyl, hexadecyl, hebutadecyl, octadecyl, nonadecyl, and icosyl. Examples of the compound according to the present invention include the following compounds in addition to the compounds described in the examples relating to the production method described below, but these are examples of one compound of the present invention. Inventive compounds are not limited to these.

0— (5-acetamido-3,5-dideoxy-D-glycero α-D-galacto-2-nonuloviranosylic acid) one (2 → 3) -0- (; 9-D-galactobilanosyl) one (1 → 4 ) 1 0— C (α—L-fucopyranosyl) 1 (1 → 3)] 1-octyl-1,5-doxy-1,5-imimino D—glucitol

Ο— (5-acetamido 3,5-dideoxy D-glycero α-D-galacto-2-nuroviranosylic acid) 1 (2 → 3) 1 0— (i? -1 D—galactobilanosyl) 1 (1 → 4 ) 1 O— [(α-L-fucoviranosyl) 1 (1 → 3)] 1-dodecyl-1,5-dideoxy 1,5-imimino D-glucitol

Ο— (5-acetamido 3,5-dideoxy D-glycerol α-D-galacto-2-nonuloviranosylic acid) one (2 → 3) one O— (-D-galactobilanosyl) one (1 → 4) -0— [(α-L-fucoviranosyl) 1 (1 → 3)] 1-tetradecyl-1,5 dideoxy 1,1,5-1 f mino-D-glucitol

0— (5-acetamido-3,5-dideoxy D-glycemic α-D-galacto-2-nonuloviranosylic acid)-(2 → 3) -0- (^ -D-galactoviranosyl) one (1 → 4) 1Ο— [(α-L-fucopyranosyl) 1 (1 → 3)] 1Νhexadecyl-1,5 1, didoxy 1,5—imimino D—glucitol

-0— (5-acetamido-3,5-dideoxy D-glycero α-D-galacto-2-nonurobilanosylate) 1 (2 → 3) — Ο— (/ 3-D—galactobilanosyl) 1 ( 1 → 4) 1Ο— C (α— L—fucopyranosyl) 1 (1 → 3)) 1Ν—octadecyl 1, 5 I-doxy-1,5-Iminnow D-Glucitol

O— (5-acetamido-3,5-dideoxy D—glycerol α—D—galacto-2-nonuloviranosylic acid) 1 (2 → 3) 1 0— (/ 9—1 D-galactobilanosyl) 1 (1 → 4) 1Ο— [(α-L-fucoviranosyl) 1 (1 → 3)] 1-icosyl-1,5-dioxy-1,5-imimino D-glucitol

Ο— (5-acetamido-3,5-dideoxy D—glycerol α—D—galacto-2-nonuloviranosylate) 1 (2 → 3) -0- (−D—galactoviranosyl) 1 (1 → 4 ) -0- [(α-L-fucoviranosyl) 1 (1 → 3)] 1 N-octyl 1,5 dideoxy 1,1,5-imimino D-glucitol

O— (5-acetamido-3,5-dideoxy D—glycerol α—D—galacto-2-nonuloviranosylic acid) 1 (2 → 3) — Ο— (3-D—galactobilanosyl) 1 (1 → 4) 1 0— [(α-L-fucoviranosyl) 1 (1 → 3)] 1Ν-Dodeshrew 1,5 dideoxy 1,5—Iminor D—glucitol

Ο— (5-acetamido 3,5-dideoxy D-glycero α-D-galacto-2-nonurobiranosylate) 1 (2 → 3) -0- (jS—D-galactoviranosyl) 1 (1 → 4) 1 O— [(α-L-fucoviranosyl) 1 (1 → 3)] 1-tetradecenyl-1,5-dideoxy 1,5-iminon D-glucitol

0— (5-acetamido-3,5-dideoxy D—glycerol α—D—galacto-2-nonuloviranosylic acid) 1 (2 → 3) -0— (3—D—galactobilanosyl) 1 (1 → 4) 1 0 — [(α-L-fucopyranosyl) 1 (1 → 3)] 1-hexadeshru 1,5-dideoxy 1,5 Minnow D—glucitol

Ο— (5-acetamido-3,5-dideoxy D-glycero α-D-galacto-2-nonurobilanosylate) 1 (2 → 3) — Ο— (/ 3—D-galactobilanosyl) 1 (1 → 4) One 0— [(α-L-fucopyranosyl) one (1 → 3)] One N-octadecyl-1,5-dideoxy-1,5-imino D-glucitol

0— (5—acetami.do 3,5—dideoxy D—glycerol α—D—galacto 2 1- (2 → 3) — O — (^-D—galacto-vilanosyl) 1 (1 → 4) 1 0—C (α-L-fucoviranosyl) 1 (1 → 3)] 1 Ν—Ikosh-Lu 1,5 Idoxy 1,5—Iminnow D—Glucitol

0— (5-acetamido-3,5-dideoxy-D-glycerol α-D-galacto-2-nonuloviranosylic acid) one (2 → 3) one O— (iS—D-galactobilanosyl) one (1 → 3) 1-0— ((α-L-fucopyranosyl) 1 (1 → 4)) 1-octyl-1,5-doxy-1,5-imimin D-glucitol

Ο— (5-acetamido 3,5-dideoxy D—glycemic α-D—galacto-2-nonurobilanosylic acid) 1 (2 → 3) 1 Ο— (3-D-galactobilanosyl) 1 (1 → 3) 1 0— ((α-L-fucopyranosyl) 1 (1 → 4)) 1-dodecyl-1,5-dideoxy 1,5-iminin D-glucitol

Ο— (5-acetamido-3,5-dideoxy D—glycerol α—D—galacto-2-nonuloviranosylic acid) one (2 → 3) -0- (/ 3—D-galactobilanosyl) one (1 → 3) 1 0— [(α-L-fucoviranosyl) 1 (1 → 4)] 1 N-tetradecyl 1,5 1-dideoxy 1,5-imino D-glucitol

O— (5-acetamido 3,5-dideoxy D-glycero α-D-galacto-2-nonuloviranosylic acid) 1 (2 → 3) — Ο— (-D-galactobilanosyl) 1 (1 → 3) One 0—C (α—L-fucoviranosyl) One (1 → 4)] One Ν-hexadecyl-1,5 One-dideoxy 1,5-Imino D—Glucitol

Ο— (5-acetamido-3,5-dideoxy-D-glycerol α-D-galacto-2-nonurobiranosylate) 1 (2 → 3) 1 0 — (^ 1 D-galactoviranosyl) — (1 → 3) 1-0 — [(α-L-fucopyranosyl) 1 (1 → 4)] 1-octadecyl-1,1,5 dideoxy-1,5, iminor D-glucitol

0— (5-acetamido-3,5-dideoxy D—glycerol α—D—galactose 2-nonuloviranosylate) 1 (2 → 3) 1 一 — (^ 1 D—galactobilanosyl) 1 (1 → 3) 1 0— [(a-L-fucopyranosyl) 1 (1 → 4)] 1Ν-icosyl- 1, 5-di Deoxy 1,5—Iminnow D—Gnoresitol

0— (5-acetamido 3,5-dideoxy D—glycerol α—D—galacto-2-nonurobilanosylate) 1 (2 → 3) — 0— (/ 9—1 D—galactobilanosyl) 1 ( 1 → 3) 1 0-[(α-L-fucoviranosyl) 1 (1 → 4)] 1-octyl 1,5 1-dideoxy 1,5-Iminault D-glucitol

Ο— (5-acetamido-3,5-dideoxy-D-glycerol α-D-galacto-2-nonurobiranosylic acid) one (2 → 3) -0- (^ one D-galactobilanosyl) one (1 → 3) — 0— [(α-L-fucopyranosyl) 1 (1 → 4)] 1Ν-dodecyl-1,5-didoxy 1,5—Imino D—glucitol

Ο— (5-acetamido 3,5-dideoxy-D-glycerol α-D-galacto-2-nonuloviranosylic acid) 1 (2 → 3) 1 0-(/ 3—D-galactobilanosyl) ― (1 → 3) 1 0— [(α-L-fucopyranosyl) 1 (1 → 4)] 10-tetradecyl-1,5-dideoxy 1,5 1 ^ Τmino-D-glucitol

O— (5-acetamido-3,5-dideoxy-D-glycerol α-D-galacto-2-nonuloviranosylic acid) one (2 → 3) -0- (-D-galactoviranosyl) one ( 1 3) — 0— C (α-L-fucoviranosyl) 1 (1 → 4)] 1 一 -hexadesinyl 1, 5-dideoxy 1, 5-imino-D-glucitol

0— (5-acetamido-3,5-dideoxy D—glycero α-D—galacto-2-nonuloviranosylic acid)-(2 → 3) 1 Ο— (/ 3-D—galactobilanosyl) 1 (1 → 3) 1-0— ((α-L-fucoviranosyl) 1 (1 → 4)) 1-octadecenyl-1,5-dideoxy 1,5-imiminol D-glucitol

0— (5-acetamido 3,5-dideoxy D—glycerol α—D—galacto-2-nonuloviranosylate) — (2—3) —O— () 9-D-galactoviranosyl) ( 1 → 3) 1-0— ((α-L-fucopyranosyl) 1 (1 → 4)) 100-Icosyl-Lu 1,5 Didoxy 1,5-imino D-Glucitol

The molanolin derivative represented by the general formula (I) can be produced, for example, by the following method. Can be

Compound of the following formula (II)

AcNH

["]

(In the formula, Ac represents an acetyl group, B z represents a benzoyl group, and Me represents a methyl group, and the same applies hereinafter.) In a solvent such as ethyl acetate, after adding a small amount of drunk acid and water, ~ Condensed with aldehydes, followed by catalytic hydrogenation in the presence of a catalyst to give a compound of formula (III)

95/0060

AcNH

[HI]

(Wherein, R ¹ is the same as described above, and is represented similarly below.)

The added amounts of acetic acid and water are preferably 1/100 to 1/10 of the solvent. The reaction temperature is preferably from 0 to 50 ° C. The reaction time varies depending on the reaction temperature, but is usually 0.5 to 24 hours. As the catalyst, palladium hydroxide on carbon is preferable, but is not limited thereto.

Next, after all o-acyl (acetyl and benzoyl) groups of the compound [III] were eliminated with an alkoxide in an alcohol solvent, an alkali aqueous solution was added thereto to hydrolyze and neutralize the methyl ester of the carboxylic acid. Thereafter, a tetravalent 锆 chain comprising the compound of the present invention, molanolin, of the present invention can be produced. AcNH

The reaction temperature is preferably from 0 to 50 ° C. The reaction time varies depending on the reaction temperature, but is usually 0 to 48 hours. The alkoxide is preferably sodium methoxide, and the alkaline aqueous solution is preferably an aqueous hydration aqueous solution, but is not limited thereto.

Further, similarly to the above, from the compound [IV] of the following formula,

[IV] The compound of the present invention of the following formula can be produced.

Further, in the same manner as described above, from a compound [V] of the following formula,

The compound of the present invention of the following formula can be produced,

The compound [II], the compound [IV], and the compound [V] used as a starting material for the production of the compound of the present invention can be produced by the method described in WO93 / 15098.

Compounds that have cell adhesion inhibitory activity inhibit the adhesion of leukocytes or cancer cells to endothelial cells by antagonistically inhibiting selectins present in endothelial cells.Thus, inflammation and thrombosis associated with inflammation, rheumatism, It is useful for the prevention and treatment of infectious diseases, immune diseases, AIDS and cancer.

Therefore, the compound according to the present invention, which exhibits a better cell adhesion inhibitory activity than the compound described in WO93 / 15098, is a better antiviral agent, anti-inflammatory agent, therapeutic agent for thrombosis, therapeutic agent for rheumatism It is useful as a therapeutic agent for infectious diseases, anti-AIDS, immune disease and cancer.

When the compound of the present invention is administered as a medicament, the compound of the present invention is used as such or in a pharmaceutically acceptable nontoxic and inert carrier, for example, 0.1% to 99.5%, preferably 0.5% to 90%. As a composition, it is administered to animals, including humans.

As the carrier, one or more solid, semi-solid, or liquid diluents, fillers, and other formulation aids are used. The pharmaceutical compositions are desirably administered in unit dosage form. The pharmaceutical composition of the present invention can be administered orally, intraosseously, topically (e.g., transdermally), or rectally. Can be administered. Needless to say, it is administered in a dosage form suitable for these administration methods. For example, intragroup administration is particularly preferred.

It is desirable to adjust the dose as an anti-inflammatory therapeutic agent in consideration of the patient's condition such as age, body weight, etc., administration route, nature and extent of the disease, etc. As the amount of the active ingredient of the invention, the range of 100 ms to 3 gZ days per day for humans is preferably

500 mg to lgZ days The human range is common. In some cases, lower doses may be sufficient, and conversely, higher doses may be required. It is desirable to administer the drug in two or three divided doses a day.

〔Example〕

The present invention will be described in more detail with reference to examples of the present invention. The measured optical rotation temperatures are all 25 ^eC .

Example 1

O— (5-acetamido-3,5-dideoxy D—glycerol α—D—galacto-2-nonurobilanosylic acid) one (2 → 3) -0- (^ one D-galactobilanosyl) one (1 → 4) Manufacture of 1Ο — [(α-L-fucopyranosyl) 1 (1 → 3)] 1-decyl-1,5-dideoxy-1,5-imino D-glucitol

Step 1 Ο— (Methyl 5-acetamido-4,7,8,9-tetra-0-acetyl-1,3,5-dideoxy D-glycero α-D-galacto-2-nonuroviranosilone) 1 (2 → 3) — Ο— (2, 4, 6-tree Ο-benzoyl-; 3-D-galactovilanosyl) 1 (1 → 4) -1 0— [(α-L-fucoviranosyl) 1 (1 → 3)] -2- 0-Acetyl-Ν-decyl-1,5-dideoxy-1,5-imino D-glucitol Obtained according to the method described in WO93 / 15098 载 (methyl-5-acetamido 4,7,8,9-tetra-0-acetyl- 3,5-dideoxy D-glycerol α-D-galacto-2-nonuloviranosylonate 1 (2 → 3) -0— (2,4,6-tree 0 1-benzoylol 3-D-galactovirano Syl) 1 (1 → 4) 1 0— C (α—L-fucopyranosyl) 1 (1 → 3)) 1 2— Ο—acetyl-1, 5 Jidokishi 1, 5-Imino P95 0 9

13

(1) Dissolve D-glucitol (30 mg) in ethyl acetate (5 ml), add acetic acid (0.1 ml), water (0.05 ml), and 1-decanal (0.5 ml). Washed palladium hydroxide solvent (30 mg) was added thereto, and the mixture was stirred at room temperature for 10 hours to perform hydrogenation reaction. After confirming the anti-iW completion by TLC, 触媒 ft ^ the catalyst and wash with methanol? The combined residue was concentrated under reduced pressure, and the resulting residue was subjected to column chromatography-I (Kogel C-200, effluent; dichloromethane: methanol = 20: 1) to give the target compound (50 mg, 60% ).

Sex

Optical rotation [α] _D +4.30 (C = 0.372, dichloromethane)

Elemental analysis value (as C ₇₁ H ₉₄ N ₂ 0 ₂₉ )

Calculated value C = 59.24% H = 6.58% N- l. 95%

Measured value C = 59. 1 1% H = 6.45% N = 1.85%

Step 2 O— (5-acetamido-3,5-dideoxy D—glycerol α—D—galactol 2-nonuloviranosylic acid) 1 (2 → 3) -0- (/ 3-D—galactobilanosyl) 1 (1 → 4) 1 O— [(α-L-fucoviranosyl) 1 (1 → 3)] 1-decyl-1,5-dideoxy 1,5-iminin D-glucitol

The compound (65 mg) obtained in Step 1 was dissolved in methanol (10 ml), sodium methoxide was added until pH = 12, and the mixture was stirred at room temperature for one hour. Then, a 0.2 N aqueous KOH solution (lml) was added, and the mixture was stirred at room temperature overnight. After confirming the completion of the reaction by TLC, the mixture was neutralized with ion-exchange resin Amberlite IR-120 (H +), the resin was separated by a furnace, and washed with methanol and water. The combined solution and washing solution were subjected to decompression lysis, and the resulting residue was subjected to gel filtration (Sephadex LH-20, effluent; methanol: water = 2: 1) to give the target compound (41 ms, quantitative ) Got.

Physical property values

Optical rotation [α] _D -43.00. (C-0.800, methanol: water = 2: 1)

Elemental analysis [C ₃₉ H ₇ . N ₂ 〇 ₂₁ )

Calculated value C = 5 1.88% H = 7.81% N = 3.10% Measured value C = 5 1.74% H = 7.67% N = 3.00%

Example 2

O— (5-acetamido-3,5-dideoxy D-glycero α-D-galacto-2-nonuloviranosylic acid) 1 (2-3) — Ο2 ”(^ 1 D-galactobilanosyl) 1 ( 1 → 3) 1 0—C (α-L-fucopyranosyl) 1 (1 → 4)] 1-Decyl-1,5-dideoxy-1,5-imino D-glucitol

Step 10 0— (Methyl 5-acetamido-4,7,8,9-tetra-0-acetyl-1-3,5-dideoxy-1 D-glycero α-D-galacto-2-nonurobiranosylone) 1 (2 → 3 ) — Ο— (2, 4, 6—tree Ο—Benzoru) 9-1 D—Galactovilla nosyl) 1 (1 → 3) — Ο— C (α—L-fucoviranosyl) 1 (1 → 4)) 1 2— Ο One acetyl-Ν-decyl 1, 5-dideoxy 1, 5-imino D-glucitol

09- (methyl 5-acetamido-4,7,8,9-tetra-)-acetyl-3,5-dideoxy D-glycer α-D-galactor 2-nonulovilla obtained according to the method described in W093 eight 5098 Nosilonate) 1 (2 → 3) — 0— (2, 4, 6—tree 0 1 Benzoylou)? One D—galactobilanosyl) One (1 → 3) — Ο— ((α-L-fucovilanosyl)-(1 → 4)) One 2-O-acetyl-1,5-dideoxy-1,5-imino Dissolve the D-glucitol compound (20 mg) in ethyl acetate (5 ml), add acetic acid (0.5 ml), water

(0.1 ml), 1-decanal (0.5 ml), catalytic hydrogenation in advance, palladium hydroxide on carbon (20 mg) washed with ethyl acetate, catalytic hydrogenation at room temperature, and stirring for 1 hour did. After confirming the completion of the reaction by TLC, the catalyst was separated and washed with methanol. The base solution and the washing solution were combined to perform decompression and the resulting residue was subjected to column chromatography (ヮ -gel C-200, effluent; dichloromethane: methanol = 20: 1) to obtain the target compound (llmg , 56%).

Physical property values

Optical rotation [α] _D + 2.52 ° (C = 0.238, dichloromethane)

Elemental analysis value (as C ₇₁ H ₉₄ N ₂ 0 ₂₉ ) Calculated value C = 59.24% H = 6.58% N = 1.95%

Measured value C-59. 18% H = 6.44% N = l. 93%

Step 2 O— (5-acetamido-3,5-dideoxy-D-glycerol α-D-galacto-2-nonuloviranosylic acid) one (2 → 3) -0- (^ one D-galactoviranosyl) one (1 → 3) 1 O— [(α-L-fucoviranosyl) 1 (1 → 4)] 1Ν-decyl-1,5-dideoxy-1,1,5-imino-D-glucitol

Compound obtained in Step 1 (52 mg) was dissolved in methanol (10 ml), was added sodium methoxide until _P H = 12, and one晚挽stirred at room temperature. Thereafter, a 0.2 N aqueous KOH solution (1 ml) was added, and the mixture was stirred at room temperature for 1 hour. After confirming the completion of the reaction by TLC, the mixture was neutralized with ion-exchange resin Amberlite IR-120 (H +), the resin was separated, and washed with methanol and then with water. The filtrate and washing solution were combined and subjected to lysis under reduced pressure. The resulting residue was subjected to gel filtration (Sephadex LH-20, efflux: methanol: water = 2: 1) to obtain the desired compound (33 mg, quantitative). Was.

Physical property values

Optical rotation [α] _D — 34.95. (C = 0.618, methanol: water = 2: 1)

Elemental analysis [C ₃₉ H ₇ . (As N ₂ 0 ₂₁ )

Calculated value C = 5 1.88% H = 7.8 1% N = 3.10%

Measured value C = 5 1.69% H = 7.68% N = 2.93%

„[Test Example]

E— s e 1 e c t i n dependency H L 60 ZHU V E C

Harvested and cultured 5th generation human vascular endothelial cells according to standard methods

HUVEC) were seeded at 2 × 10 ⁴ cells / well in a 96-well microphone-mouth plate coated with 1% gelatin. After one晚cultured in C0 ₂ Inkyubeta of 37 ° C, the cell layer 100 RPMI / FCS / HEPES medium (RPMMI-1640, 10% FCS , 25mM HEPES, pH7.4) and washed twice with, 10 U / RPMμΙΟΟ of RPMI / FCS / HEPES containing IL-1β was added, and the mixture was incubated at 4 o'clock (activation). Measure cell adhesion in non-activated cells In order to do this, we created a Basal with only RPMI / FCS / HEFES at the same time. Cultured human leukemia cells HL60 were washed twice with RPMI-1640 (RPMI / HEPES) without FCS, suspended in 10 ml of RPMI / FCS / HEPES medium with 0.5% glutaraldehyde, and placed on ice. Fixed for 20 minutes. After fixation, the cells were washed 3 times with RPMI / HEPES, cell numbers were diluted with RPMI / FCS / HEPES at 2's 10 ⁶ cells / ml, and stored in permanent until use.

After activation, HUVECs were washed three times with 100 μl of RPMI / FCS / HEPES and 50 μl of RPMI / FCS / HEPES (Control) or a compound of 501 dissolved in RPMI / FCS / HEPES ( (300 g / ml) and incubated for 30 minutes at room temperature. Next, the immobilized HL60 cells (50 μl) were added to each well and incubated at room temperature for 45 minutes. Each well was filled with RFMI / FCS / HEPES, sealed using a microplate seal to prevent air bubbles from entering, the plate was inverted, and left undisturbed for 1 hour to remove unbound HL60 cells.

Method for measuring the number of adherent cells

The number of adherent cells was determined by the activity of myelin peroxidase (ΜΡΟ), an enzyme present in HL60 cells. Add 50 μl of a phosphate buffer (50 mM, ρΗ6.0) containing 0.5% hexadecyltrimethylammonium bromide (ΗΤΑΒ) to each well, and incubate for 30 minutes at room temperature to remove ΜΡΟ from the cells. Was solubilized. At the same time, a known number of HL60 cells treated in the same manner were prepared in a row of 96 ゥ elburate as a standard. Then 0.6mM Jianishijin dihydrochloride, 0.4 mM H ₂ 0 ₂ a Complex free phosphate buffer solution (100 mM, pH 5.4) to 200 mu 1 was added and allowed to react at room temperature for 20 minutes, BIO-RAD Inc. Model 3550 The absorbance at 450 nm was measured using MICRO PLATE READER. A standard curve was prepared from the absorbance obtained from the standard cells, and the number of adherent cells was determined. The experiment was performed using 6 μl for each treatment. From the number of adherent cells in each well

The cell adhesion rate in each treatment when the value of Control was set to 100% was determined, and the average value and the standard error between 6 μl were determined. The results are shown in Table 1. table 1

Cell adhesion rate

Control 100.0 +3.4

Basal 12.0 + 0.6 *

Compound of the present invention Example 1 58.8 + 2.6 *

Compound of Invention Example 2 36.6 + 1.7 *

*: P <0.01, vs Control

[Results] The compound of Example 1 of the present invention inhibited cell adhesion by 41% (P <0.01). The compound of Example 2 of the present invention inhibited cell adhesion by 63% (F <0.01). As described above, the compound according to the present invention exhibited a strong cell adhesion inhibitory action. From these results, it was shown that the compound according to the present invention is useful as a medicament for preventing and treating diseases caused by cell adhesion, such as inflammation, immune disease, and viral infection.

Claims

The scope of the claims

1. Moranolin derivatives represented by the following general formula [I].

In the formula, R ¹ represents a C8-C30 alkyl, C8-C30 alkyl, or C8-C30 alkyl; R ² and R ³ are each different and represent a sialylgalactosyl, galactosyl, or fucosyl group <<

2. Moranolin derivative represented by the following formula.

ACNH

0, C00H

10 ^π 21

Moranolin derivative represented by

C10H21

AcNH