WO1999047533A1

WO1999047533A1 - Compounds having immunomodulatory effects

Info

Publication number: WO1999047533A1
Application number: PCT/JP1999/001369
Authority: WO
Inventors: Shigeki Nunomura; Ryoichi Osawa; Masami Iida; Isao Suda
Original assignee: Nissin Food Products Co., Ltd.
Priority date: 1998-03-19
Filing date: 1999-03-18
Publication date: 1999-09-23
Also published as: JPH11269190A

Abstract

Compounds represented by the structural formula: nonulonic acid derivative →monosaccharide→ceramide or salts thereof which have immunomodulatory effects, in particular, a high immunopotentiating activity and a high antitumor activity.

Description

Description Compound having immunomodulatory action

The present invention relates to a glycosphingolipid compound having an immunomodulatory activity, particularly an immunostimulatory activity and an antitumor activity. Background art

With cancer always occupying the top of adult diseases, the search for compounds with immunostimulatory and antitumor activity has always been a challenge. Recently, it has been discovered that sponge glycosphingolipids derived from sponges have immunostimulatory and antitumor activities, and more active glycosphingolipids have been synthesized with reference to their structures.

(JP-A-5-59081 and JP-A-5-913).

JP-A-5-59081 and JP-A-5-91193 disclose a glycosphingolipid in which ceramide is glycoside-linked to galactose as the best compound. In order to improve the water solubility of the compound, a monosaccharide is added to the galactose portion (WO94 / 24,142). In such a case, the activity is increased. Not shown in the experimental results. Disclosure of the invention

A series of inventions in this field were made with the primary objective of increasing the activity of the compound, in which case it was necessary to design the compound to further enhance its immunostimulatory and antitumor activities. However, the present inventors have found that the activity may be increased by regioselectively adding a monosaccharide, which was not recognized to have increased activity in the above literature. Significantly increased activity when bound And completed the present invention.

More specifically, according to the present invention, the following compounds and medicaments are provided.

(1) Nonuronic acid derivative A compound represented by the structural formula of monosaccharide → ceramide or a salt thereof.

(2) The compound or a salt thereof according to (1), wherein the nonuronic acid derivative is sialic acid or KDN.

(3) The compound according to (1) or (2) or a salt thereof, wherein the monosaccharide is galactose.

(4) The ceramide is a compound having 24 carbon atoms.

(3) The compound according to any of the above or a salt thereof.

(5) The ceramide is a saturated compound (Cer (C24, Sat.))

(4) The compound of the above or a salt thereof.

(6) The compound or a salt thereof according to any one of (1) to (5), wherein the nonuronic acid derivative is bonded to the 6-position of galactose.

(7) A compound represented by the structural formula of NeuAcA2 → 6 (Βη- »2) Ga1α → 1'Cer (C24) or a salt thereof.

(8) A compound represented by the structural formula of NeuAc 2 → 6 Gal → l′ Cer (C24, Sat.) or a salt thereof. (C24, Sat.) indicates a saturated compound having 24 carbon atoms.

(9) A medicament comprising an effective amount of the compound according to any one of (1) to (8) or a salt thereof.

(10) The medicament according to (9), which is an immunomodulator.

(11) The medicament according to (9), which is an immunopotentiator.

(12) The medicament according to (9), which is an antitumor agent.

As used herein, the term "nonuronic acid" is synonymous with the commonly used nonuronic acid, and refers to an acidic sugar having 9 carboxyl groups at the 1-position. Follow The term “nonuronic acid derivative” used in the present specification includes, of course, neuraminic acid (5-amino-3,5-dideoxy-D-glycerol-D-galacto-nonuronic acid) and neuraminic acid derivative. included. Further, “sialic acid” described later is also included in the “nonuronic acid derivative” because it is an acyl derivative of neuraminic acid.

As used herein, “KDN” refers to 2-keto-3-deoxy-D-glycero-2-nononic acid. I do. The term “sialic acid” used in the present specification means a generic name for a series of derivatives of a substance having a basic structure of neuraminic acid (Yasuo Inoue Baifukan, “Chemistry of Biomolecules 1 Chemistry of Carbohydrates” P80-81 ), Which includes acyl derivatives, specifically those such as N-acetylneuraminic acid and N-glycolylneuraminic acid.

A “salt” is required to neutralize the carboxylic acid / sulfonic acid in the molecule, such as a sodium salt or a potassium salt, and is bound to a cation that does not decrease its activity. The cation to be bound may be any cation that does not impair the activity of the compound of the present invention. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flowchart showing the steps for producing the example compounds according to the present invention.

FIG. 2 is a flowchart showing a production process of an example compound according to the present invention.

FIG. 3 is a graph showing the mouse lymphocyte proliferation promoting effect of the example compounds of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

As shown in the experimental examples described below, the compound of the present invention exhibits a lymphocyte proliferation-promoting activity, that is, an immunostimulatory activity in a mouse lymphocyte blastogenesis reaction. Therefore, the administration of an antitumor agent or an anti-cancer agent based on the immunostimulatory activity or radiation It can be used as a therapeutic agent for myelosuppression caused by treatment, and for various infections and immunodeficiencies. In addition, for immunocompetent cells Since it acts, it can be used as an immunomodulator, for example, as a therapeutic agent for autoimmune diseases such as rheumatoid arthritis.

The compound of the present invention as a drug can be administered by any suitable route of administration, specifically, in the case of animals, such as intraperitoneal administration, subcutaneous administration, intravenous administration to a vein or artery, and local administration by injection. For humans, intravenous administration, intraarterial administration, local administration by injection, intraperitoneal / thoracic administration, oral administration, subcutaneous administration, intramuscular administration, sublingual administration, transdermal absorption or rectal administration Can be administered. When the compound of the present invention is administered as a drug, it may be administered in the form of injections, suspensions, tablets, granules, powders, capsules, ointments, creams, etc., depending on the administration method and administration purpose. Monkey To produce these preparations, solvents, solubilizers, tonicity agents, preservatives, antioxidants, excipients, binders, lubricants, stabilizers and the like can be added. Solvents include, for example, water, physiological saline, etc., solubilizers include, for example, ethanol, polysorbates, cremophor, etc., and excipients, for example, lactose, starch, crystalline cellulose, mannitol, maltose, phosphoric acid Calcium hydrogen, light anhydrous silicic acid, calcium carbonate, etc., as binders, for example, starch, polyvinylpyrrolidone, hydroxypropyl cellulose, ethyl cellulose, carboxymethyl cellulose, gum arabic, etc., as disintegrants, For example, starch, calcium carboxymethylcellulose, etc., as lubricants, for example, magnesium stearate, talc, hydrogenated oil, etc., as stabilizers, for example, lactose, mannitol, maltose, polysorbates, macrogol , Polyoxy Ethylene hydrogenated castor oil and the like. Also, if necessary, glycerin, dimethylacetamide, 70% sodium lactate, surfactant, basic substance (for example, sodium hydroxide, ethylenediamine, ethanolamine, sodium carbonate, arginine, nugglemin, tris) (Aminomethane). These components can be used to produce injections, tablets, granules, capsules and the like. The dose of the compound of the present invention is determined in consideration of the results of animal experiments and various situations so that the total dose does not exceed a certain amount when administered once and repeatedly. The specific dosage is Needless to say, it varies according to the method of administration, the condition of the patient or treated animal, such as age, body weight, sex, sensitivity, diet (diet), administration time, concomitant drug, patient or the degree of the disease. Under the above conditions, the appropriate dose and number of doses must be determined by a specialist's appropriate dose determination test based on the above guidelines. Example

Compound (51 mg, 43 mol) was dissolved in toluene (5 mL), and 1: 2 = molecular sieve 3A: 4A (600 mg) _s 1: 1 = mercuric bromide: mercuric cyanide (179 mg) was added thereto, and the mixture was stirred at 0 ° C for 1 hour under a stream of argon gas. Then, sialic acid donor 丄 (74 mg, 0.14 麵 ol) was added, and the mixture was stirred at 0 ° C at room temperature for 3 hours. The reaction solution was neutralized by adding triethylamine, diluted with ethyl acetate, filtered through celite, and washed successively with saturated aqueous sodium hydrogen carbonate and saturated brine. After the ethyl acetate layer was dried over magnesium sulfate, the solvent was distilled off under reduced pressure. The residue was purified by silica gel columnography (1: 1 toluene: ethyl acetate) to obtain Compound A (43 mg, 60%).

Compound 丄 (R.kuhn, Chem. Ber., 99 (1966, 611-617))

Compound (Same application number NN 1-00)

Rf = 0.56 (toluene: ethyl acetate = 1: 1)

Ή-ΝΜΕ (ϋϋϋ13) ό ·: 5.608 (1H, d, J = 8.8Hz, NH), 4.880 (1H, m, H-4b), 4.771 (1H, d, J = 3.3Hz, H-la), 4.651 (1H, d, J = 11.5Hz, CH ^ Ph), 4.575 (1H, d, J = 12.1Hz, CH ^ Ph), 3.763 (3H, s, OMe), 2.586 (1H, dd, J = 4.8 , 12.8Hz, H-3be, 2.129 , 2.123, 2.033, 2.017, 1.887 (15H, 5s, 5Ac), 1.365, 1.310 (6H, 2s, CH 3), 1.049 (9H, s, ί - Bu), 0.881 ( 6H, t, J = 7.0Hz, 2CH ₃ ). <Example 2>

Compound A (43 mg, 26 mol) was dissolved in 1,2-dichloroethane (3 mL), 75% trifluoroacetic acid (1.0 mL) was added, and the mixture was stirred at 0 ° C for 3 hours. Direct silica gel column reaction Purification by luffy (2: 1 toluene: acetone) was performed to obtain stereoglycoside _1 (21 mg, 59%) in a stereoselective manner.

Rf: 0.19 (toluene: acetone = 2: 1)

^{1 H-NMR (CDC13) c5} ': 7.38-7.31 (5H, m, Arom.H), 6.336 (1H, d, J = 7.7Hz, NH), 5.674 (1H, dt, J = 7.7, 15.4Hz, 5Cer), 5.378 (1H, m, H-8b), 5.311 (1H, dd, J = 2.2, 7.7Hz, H-7b), 5.164 (1H, d, J = 9.5Hz, NH), 4.882 (1H, m, H-4b), 4.812 (1H, d, J = 3.7Hz, H-la), 4.787 (1H, d, J = 11.7Hz, CH ^ Ph), 4.732 (1H, d, J = 11.7Hz, Ph), 3.800 (3H, s, OMe), 2.555 (1H, dd, J = 4.4, 13.2Hz, H-3beg), 2.150, 2.127, 2.038, 2.030, 1.880 (15H, 5s, 5Ac), 0.880 (6H , T, J = 6.6 Hz, 2CH ₃ ).

Compound! _ (7 mg, 5 / mol) was dissolved in 1: 1 methanol: tetrahydrofuran (0.6 mL), 1N aqueous sodium hydroxide solution (0.3 mL) was added, and the mixture was stirred at room temperature for 1 day, and the reaction solution was directly added to Sephadex LH Column purification was performed using -20 (5: 5: 1 chloroform: methanol: water) to obtain a compound (6 mg, qu.).

Rf = 0.55 (cloth form: methanol: water = 12: 8: 1)

'H-NMR (CD30D-CDC13) 0-: 7.44-7.25 (5H, m, Arom.H), 5.628 (1H ₅ dt, J = 7.7, 15.4Hz, 5Cer) ₃ 5.453 (1H, dd, J = 7.3) , 15.0Hz, 4Cer), 4.903 (1H, d, J = 3.3Hz, H-la), 4.777 (1H, d, J = 11.4Hz, CH ^ Ph), 4.705 (1H, d, J = 11.4Hz, CH ^ Ph), 2.799 (1H , dd, J = 2.6, 12.5Hz, H- 3be, 2.010 (3H, s, NHAc) 3 0.894 (6H, t, J = 7.0Hz, 2CH 3).

Example 4>

Dissolve the compound (14 mg, 10 zmol) in 4: 1 = methanol: water (5 mL), add 20 palladium hydroxide-carbon (15 mg), replace with hydrogen gas, and perform catalytic reduction at room temperature for 19 hours. Was. The reaction solution was filtered through celite, and the filtrate was distilled off. The residue was dissolved in 1: 1 methanol: tetrahydrofuran (0.6 mL), and a 1N aqueous sodium hydroxide solution (0.3 mL) was added. After stirring at room temperature for 1 day, the reaction solution was directly purified with a column of Sephadex LH-20 (5: 5: 1 column: methanol: water) to obtain a compound (10 g, 87%).

Rf = 0.40 (cloth form: methanol: water = 12: 8: 1)

! H-NMR (00300-00013) 0 ·: 4.711 (1H, br-s, H-la), 2.708 (1H, br-dd, H-la)

3be, 1.914 (3H, s, NHAc), 0.795 (6H, t, J = 7.0Hz, 2CH 3).

Lymphocytes collected from the spleen of a BALB / c mouse (female, 8 to 10 weeks old, Nippon S.L.I.) according to a standard method were used.

0 was prepared to ⁰ / ml. Using a flat-bottomed 96-well plate, to the cell suspension 100〃 1, samples were added 10 1 prepared in various concentrations with physiological saline, 3 days, 3 7 ° C - the culture in a 5% C0 ₂ Conditions After performing the above, the cell proliferation was measured by MTT assay.

The results are shown in Fig. 3. Existing compound: Compound and Compound showed remarkable lymphocyte proliferation promoting action as compared to L. Thus, the compounds of the present invention represented by Compound Nos. I and ^ _ are useful in that they have immunostimulatory activity and antitumor activity.

1) Immunostimulatory activity

As shown in the above experimental examples, the compound of the present invention exhibited a lymphocyte proliferation-promoting activity, ie, an immunostimulatory activity, in mouse lymphocyte blastogenesis.

2) Immunomodulators, especially immunostimulants and antitumor agents

Thus, it was revealed that the compounds of the present invention show immunostimulatory activity. Therefore, the compound of the present invention can be used as an immunostimulating agent or an antitumor agent based on an immunostimulating action. Since the compound of the present invention has immunostimulatory activity, it can be used as a therapeutic drug for bone marrow suppression caused by administration of an anticancer drug or radiation therapy, and a therapeutic drug for various infectious diseases and immunodeficiency diseases. Further, since it acts on immunocompetent cells, it can be used as an immunomodulator, for example, as a therapeutic agent for autoimmune diseases such as rheumatoid arthritis.

Claims

The scope of the claims

1. Nonuronic acid derivative Compound represented by the structural formula monosaccharide ceramide or its compound

¾πι

2. The compound or a salt thereof according to claim 1, wherein the nonuronic acid derivative is sialic acid or KDN.

3. The compound or a salt thereof according to claim 1 or 2, wherein the monosaccharide is galactose.

4. The compound or a salt thereof according to any one of claims 1 to 3, wherein the ceramide is a compound having 24 carbon atoms.

5. The compound or a salt thereof according to claim 4, wherein said ceramide is a saturated compound (C er (C24, Sat.)).

6. The compound or a salt thereof according to any one of claims 1 to 5, wherein the nonuronic acid derivative is bonded to position 6 of galactose.

7. A compound represented by the structural formula of NeuAca2 → 6 (Bn → 2) Gal 1′Cer (C24) or a salt thereof.

8. A compound represented by the structural formula of NeuAc2 → 6Gal → l'Cer (C24, Sat.) or a salt thereof.

9. Contains an effective amount of the compound according to any one of claims 1 to 8 or a salt thereof.

10. The medicament according to claim 9, which is an immunomodulator.

1 1. The medicament according to claim 9, which is an immunostimulant.

1 2. The medicament according to claim 9, which is an antitumor agent.