JPH059193A - New glycosphingolipid, its production and use thereof - Google Patents

Abstract

PURPOSE:To provide a new glycosphingolipid having antitumor activity and immuno potentiation activity and useful as an antitumor agent and immuno potentiating agent. CONSTITUTION:The glycosphingolipid of formula I [R3 is group of formula II or III when R1 is alpha-D-galactopyranosyl and R2 is (OH2)2-1-OH3, and R2 is (CH2)21-CH3 of (CH2)22CH3 when R1 is beta-D-glucopyranosyl and R3 is group of formula IV], e.g. the compound of formula V. The compound can be produced by extracting Agelas mauritianus (a kind of porifera) with an organic solvent (preferably acetone, ethyl acetate, etc.), usually fractionating the extract by counter-current partition, etc., and purifying the roughly purified product by high-performance liquid chromatography, etc. The extraction with organic solvent is preferably carried out at about room temperature for 12-36hr under agitation.

Description

Detailed Description of the Invention

BACKGROUND OF THE INVENTION

TECHNICAL FIELD The present invention relates to a novel glycosphingolipid having an effective antitumor activity and immunostimulatory activity, a method for producing the same, and uses thereof.

[0002]

[Outline of Invention]

[0003]

The physiological activity of a chemical substance largely depends on its chemical structure, and it can be said that there is a constant need for novel compounds having antitumor activity or immunostimulatory activity. The present invention aims to meet the above-mentioned needs.

[0004]

[Means for Solving the Problems] The present inventors have found that a specific glycosphingolipid is extracted from the sponge Agelus Mauritianus and found that it has antitumor activity and immunostimulatory activity. It was created, and the present invention was completed based on this knowledge. That is,
The novel glycosphingolipid according to the present invention is represented by the following formula (I).

[Chemical 2] Here, when R ₁ is α-D-galactopyranosyl and R ₂ is the following group (a), R ₃ is the following group (b) or (c), and R ₁ is When β-D-glucopyranosyl and R ₃ is the group (d) below,
R ₂ is the following group (a) or (e). _{(A) - (CH 2)} 21 -CH 3 (b) -CH (OH) - (CH 2) 11 -CH (CH 3)
_{2 (c) -CH (OH)} - (CH 2) 11 -CH (CH 3)
_{-CH 2 -CH 3 (d) -CH} = CH- (CH 2) 2 -CH = C (C
_{H 3) -CH = CH- (CH} 2) 6 -CH 3 (e) - (CH 2) 22 -CH 3 present invention also relates to the preparation of compounds of formula (I). That is, the method for producing the glycosphingolipid represented by the above formula (I) according to the present invention comprises collecting the sponge Agelus mauritianus and subjecting it to an extraction operation with an organic solvent. I)
The sphingoglycolipid represented by is collected. Further, the compound of the present invention represented by the formula (I) can be chemically synthesized according to the step formula or reaction route formula described later. The invention also relates to the use of the compounds of formula (I). That is, the antitumor agent and immunostimulant according to the present invention are one or two glycosphingolipids represented by the above formula (I).
It contains one or more species as active ingredients.

[Detailed Description of the Invention] <Glycosphingolipid> The glycosphingolipid according to the present invention is represented by the formula (I) as described above, and the structural formulas of these specific compounds are: Equation (1)-
It can be shown as (4).

[Chemical 3]

[Chemical 4]

[Chemical 5]

[Chemical 6]

<Method for Producing Compound of the Present Invention> The compound according to the present invention, that is, the glycosphingolipid represented by the above formula (I) can be obtained by extraction from a sponge, but is induced by chemical modification of a related compound. Alternatively, it can be wholly synthesized by a chemical synthesis means in which various general chemical reactions necessary for synthesizing glycosphingolipid are combined. Compounds of formula (I) may be prepared using conventional chemical synthetic procedures for glycosphingolipids, for example, Agricultural and Biological Chemistry.
ical Chemistry), 54, No. 3, pp. 663-667, 1
The method described in 990 can be applied mutatis mutandis. As an example of such a synthesis, for example, the compound represented by the formula (I) can be synthesized through the following steps.

[Chemical 7]

[Chemical 8] In the above process formula, the following abbreviations are used. Bn: benzyl Bz: benzoyl Tr: trityl Ms: methanesulfonyl TsOH: p-toluenesulfonic acid DMAP: 4-dimethylaminopyridine MS-4A: Molecular sieves -4A (dehydrating agent) R _4: an alkyl or alkenyl -CH (OH) - _{(CH 2) 2 -R 5:} R in formula (I)
R ₆ corresponding to ₃ : A more specific synthetic method of the compound of the present invention corresponding to R ₂ in the formula (I) can be shown by the following reaction route formula. This reaction pathway formula is specifically shown for the above-mentioned compound (1), but the compound (3) according to the present invention is also synthesized according to this method (for details of this in the synthetic examples of the experimental examples described later). can do.

[Chemical 9]

[Chemical 10] The abbreviations used in this formula are the same as in the case of the above process formula.

The method of obtaining from sponges is basically A)
Sponge collection step, B) extraction step of contacting sponge with at least one suitable organic solvent to obtain a crude extract containing the compound of the present invention, C) organic solvent obtained from step B) The step of collecting the compound of the present invention from the crude extract. (Step A)) This step is a step of collecting sponges. A preferred example of the sponge is Agelas mauritianus , which can be collected from the sea of Kume Island, Okinawa Prefecture, for example. (Step B)) This step includes at least 1 step from a sponge.
This is a step of extracting the compound of the present invention as a crude extract by using an appropriate organic solvent or water. An organic solvent is preferable as the extraction solvent. Suitable organic solvents for extraction may be those capable of extracting the compound of the present invention from sponges, and examples thereof include esters such as ethyl acetate and butyl acetate, methanol, ethanol and isopropyl alcohol. Alcohols such as, heptane, hexane, aliphatic hydrocarbons such as isooctane, ketones such as acetone and methyl ethyl ketone, aromatic compounds such as benzene and toluene, ethers such as diethyl ether and t-butyl methyl ether, methylene chloride, chloroform, 1 Preferred examples include substituted compounds of lower aliphatic hydrocarbons such as 1,2-dichloroethane. In the present invention, these may be used alone or in a mixture of a plurality of them. Among the above-mentioned organic solvents, more preferable ones are ethanol, acetone, ethyl acetate and the like, and as preferable examples of a combination of a plurality of kinds,
Examples include methanol and chloroform, methanol and methylene chloride, acetone and toluene. As the extraction method, animal and plant materials, known methods for extracting physiologically active substances from biological materials such as microbial materials, particularly glycosphingolipids, for example, Liebigs Annalen der Chemie, 1 volume, 51 pages,
The method described in 1990, tetrahedron (Tetrahedro
n), Vol. 45, No. 12, pp. 3897, 1989, or Zeitschrift fuer Natur.
forschung Teil B) Volume 42, No. 11, page 1476, 19
The method described in 1987 and the like can be applied correspondingly, and these extraction methods can be used alone or in combination of plural kinds. Specifically, for example, a sponge animal may be used as it is, or may be subjected to a pretreatment such as homogenization and freeze-drying, and the extraction temperature is 0 to 80.
C, preferably near room temperature, extraction time 1-72 hours,
The extraction operation is preferably performed under extraction conditions for 12 to 36 hours, preferably with stirring. If necessary, the above extraction operation can be repeated a desired number of times. (Step C)) This step is a step of collecting the compound of the present invention from the crude extract containing the compound of the present invention obtained in step B). As the collection method, known methods for fractionation and isolation of physiologically active substances, in particular glycosphingolipids, from various biological materials as described above can be used. For a general description of such methods, see, for example, Liebigs Annalen der Chemie, Vol. 1, p. 51, 199.
You can refer to year 0, etc. Specifically, as the fractionation method, for example, a fractionation method utilizing a difference in solubility (for example, by combining water-containing methanol and methanol), a distribution method utilizing a difference in distribution ratio (including a countercurrent distribution method) ( (For example, a combination of ethyl acetate and water), etc., and the crude extract described above is treated by these fractionation methods,
The compound of the present invention represented by the formula (I) can be obtained as a crude product by collecting the target fraction. In order to further purify the crude purified product of the compound of the present invention thus obtained, the above-mentioned fractionation method may be combined with the isolation method as described below and carried out a required number of times. In addition, if necessary, the crude extract obtained from step B) can be subjected to an operation of an appropriate isolation method a required number of times to obtain a purified product of the compound of the present invention. Examples of such an isolation method include a method of eluting by chromatography such as adsorption chromatography, partition chromatography, thin layer chromatography, high performance liquid chromatography, and gel filtration. Specific examples of the chromatography include silica gel, ODS, Toyopearl HW as the stationary phase.
-40 (Tosoh), Sephadex LH-20 (Pharmacia) and the like, and a mixture of the organic solvent, water and the like as described above in the step B) as a mobile phase alone or in combination of two or more thereof. Column chromatography used in. Preferred specific examples of the eluate include methanol and chloroform for a single substance, methanol and chloroform for a mixture, methanol and water,
And so on.

<Use of the Compound of the Present Invention> The compound of the present invention represented by the formula (I) is useful in that it has the following physiological activities, that is, antitumor activity and immunostimulatory activity. 1) Antitumor activity As shown in Experimental Example 2 described later, the compound of the present invention showed antitumor activity against B16 mouse melanoma cells intraperitoneally and subcutaneously in mice. 2) Immunostimulatory activity The compound of the present invention, as shown in Experimental Example 2 below, is a mouse lymphocyte mixed culture reaction, mouse natural killer (NK).
It showed an activating effect in the test of cell activation enhancing reaction and macrophage activation reaction. 3) Antitumor Agent and Immunostimulatory Agent As described above, it was revealed that the compound of the present invention exhibits antitumor activity and immunostimulatory activity. Therefore, the compound of the present invention can be used as an antitumor agent or an immunostimulant. The compound of the present invention as an antitumor agent or immunostimulant can be administered by any purposeful administration route and in a dosage form determined by the administration route adopted. The drug is usually in a form diluted with a pharmaceutically acceptable carrier or diluent. When the compound of the present invention is used as an antitumor agent or an immunostimulant, it can be orally or parenterally administered to humans or mammals. For example, the compound of the present invention can be dissolved or suspended in an appropriate injectable solvent (for example, distilled water for injection) and administered by intravenous injection, intramuscular injection, subcutaneous injection, or the like. In addition, a suitable diluent (eg, starch, sucrose, lactose, calcium carbonate, or any other compound usually used for this purpose) or a lubricant (eg, stearic acid, sodium benzoate, boric acid, silica, polyethylene glycol). (Pharmaceutical ingredients such as any compound commonly used for this type of purpose) are added to powders, tablets, granules, capsules,
It can be orally administered in the form of a troche or a dry syrup. The dose of the compound of the present invention is determined in consideration of the results of animal tests and individual circumstances so that the total dose does not exceed a certain amount when administered continuously or intermittently. The specific dose may vary depending on the administration method, the condition of the patient or the animal to be treated, such as age, weight, sex, sensitivity, diet, administration time, drugs used in combination, the patient or the degree of the disease. Needless to say, the appropriate dose and the number of administrations under certain conditions should be determined by a dose determination test by a specialist based on the above guidelines. The minimum dose required for expression of the activity of the compound of the present invention is generally 0.1 per 1 kg of host body weight.
It is about 01 mg.

<Experimental example>

EXAMPLES The present invention will be explained in more detail below by way of experimental examples, but the present invention is not limited by these experimental examples. Experimental Example 1 : Preparation of Compound A sponge animal ( Agelas mauritianus ) collected in the sea of Kume Island, Okinawa Prefecture was homogenized and freeze-dried (346.5 g). The first solvent was extracted with methanol-chloroform (1: 1) to obtain an extract, which was concentrated under reduced pressure to obtain a residue (43.67 g). The residue was partitioned between ethyl acetate and water as the first partition solvent to obtain the crude active fraction, the upper ethyl acetate layer was dehydrated with anhydrous sodium sulfate, and the lower aqueous layer was extracted with 1-butanol. . Compound (1),
The ethyl acetate-dissolved fraction containing (2), (3) and (4) and the 1-butanol-dissolved fraction were combined and concentrated under reduced pressure to obtain a residue (36.77 g).
The residue was washed with 30% aqueous methanol, extracted with methanol, and the extract was concentrated under reduced pressure to give a brown solid 4.6.
6 g was obtained. This solid was treated with silica gel (Wako Gel C-2
00) Column chromatograph, using chloroform first and then eluting with slow addition of methanol to separate. The active fraction was obtained from the fraction eluted with chloroform containing 5% to 8% methanol (224.3 mg), extracted again with methanol and concentrated under reduced pressure to give a brown solid. This solid was applied to a Toyopearl HW-40 column, and an active fraction was obtained from the fraction eluted with chloroform-methanol (1: 1) (161.8 mg), which was subjected to reverse phase high performance liquid chromatography (Rp-HPLC; YMC-D). -ODS-7 (manufactured by YMC Co., Ltd.), 100% methanol, 11 ml
/ Min (detection is by a differential refractometer)) as a colorless powder of the compound (1) (8.4 mg) of the present invention, compound (2) (31.7 mg), compound (3) (6.8 mg) , Compound (4) (9.9 mg) for 50 minutes, 53 minutes, 5 minutes, respectively.
Obtained in order with an elution time of 7 minutes and 61 minutes. Compound (1), compound (2), compound (3) and compound (4) showed the following spectrum data. Compound (1) [α] ²⁸ _D = + 65.4 ° (1-PrOH, c = 1.
0) MS: FDMS 846 (M + H) + negative FABMS 844 (M-H) - UV: (λmax (nm), MeOH) 202 (ε2200) IR: (cm -1, KBr) 3400,2950,287
0, 1645, 1535, 1475, 1080. mp: 198-200 ° C NMR: ¹ H (500 MHz, C ₅ D ₅ N; 27 ° C) δ
(ppm) 8.50 (1H, d, J = 9.2Hz), 7.5
5 (1H, bs), 7.01 (1H, bs), 6.69
(1H, d, J = 6.7Hz), 6.65 (1H, b
s), 6.52 (1H, bs), 6.30 (1H, b
s), 6.08 (1H, d, J = 6.1Hz), 5.59
(1H, d, J = 3.7Hz), 5.28 (1H, m),
4.64 (2H, m), 4.58 (1H, m), 4.5
3 (1H, m), 4.48 (2H, m), 4.39 (1
H, m), 4.34 (2H, m), 4.32 (1H,
m), 4.27 (1H, m), 2.29 (1H, m),
2.19 (1H, m), 1.99 (1H, m), 1.8
8 (2H, m), 1.73 (1H, m), 1.66 (2
H, m) 1.46 (2H, m), 1.42-1.10
(55H, m), 0.88 (9H, m). ¹ H (500 MHz, C ₅ D ₅ N + D ₂ O; 55 ° C) δ
(ppm) 5.46 (1H, d, J = 3.7Hz), 5.
14 (1H, m), 4.62 (1H, m), 4.58
(2H, m), 4.48 (2H, m), 4.43 (1
H, dd, J = 3.7, 10.4 Hz), 4, 32 (2
H, m), 4.30 (2H, m), 4.26 (1H,
m), 2.21 (2H, m), 2.01 (1H, m),
1.90 (2H, m), 1.75 (1H, m), 1.7
0 (2H, m), 1.55 (2H, m), 1.50-
1.15 (55H, m), 0.92 (9H, m). ¹³ C (125 MHz, C ₅ D ₅ N; 55 ° C) δ (ppm)
175.9 (s), 101.2 (d), 76.1
(D), 72.9 (d), 72.8 (d), 72.8
(D), 71.5 (d), 70.9 (d), 70.2
(D), 68.6 (t), 62.7 (t), 51.2
(D), 39.6 (t), 35.7 (t), 34.1
(T), 32.3 (t), 30.6 (t), 30.5
(T), 30.2 (t), 30.2 (t), 30.1
(T), 29.8 (t), 28.5 (d), 28.0
(T), 26.8 (t), 26.1 (t), 23.1
(T), 23.0 (q), 14.5 (q). Compound _{^{(2) [α] 28 D}} = -1.6 ° (1-PrOH, c = 1.0 ) MS: FDMS 838 (M + H) + negative FABMS 836 (M-H) - UV: (λmax (nm) , MeOH) 236 (ε5800) IR: (cm ^-1 , KBr) 3350, 2920, 285
0, 1640, 1530, 1470, 1080. mp: 141.0-142.0 ° C. NMR: ¹ H (500 MHz, C ₅ D ₅ N; 27 ° C.) δ
(ppm) 8.33 (1H, d, J = 9.2Hz), 7.6
1 (1H, d, J = 5.5Hz), 7.22 (1H, b
s), 7.12 (1H, bs), 6.86 (1H, d,
J = 4.9 Hz), 6.35 (1H, bs), 6.19
(1H, d, J = 15.3Hz), 5.99 (1H, d
d, J = 6.7, 15.9 Hz), 5.90 (1H, d
t, J = 6.1, 15.9 Hz), 5.64 (1H, d
t, J = 7.3, 15.3 Hz), 5.49 (1H, b
t, J = 6.7 Hz), 4.89 (1H, d, J = 7.9)
Hz), 4.79 (1H, m), 4.74 (1H, m),
4.69 (1H, dd, J = 6.1, 10.4Hz),
4.56 (1H, m), 4.49 (1H, bd, J = 1
1.0Hz), 4.34 (1H, m), 4.21 (1H,
m), 4.19 (2H, m), 4.01 (1H, m),
3.88 (1H, m), 2.21 (2H, m), 2.1
5 (2H, m), 2.11 (2H, m), 2.00 (1
H, m), 1.79 (1H, m), 1.75 (3H,
s), 1.70 (1H, m), 1.45 to 1.05 (4
9H, m), 0.84 (3H, t, J = 6.7Hz),
0.84 (3H, t, J = 6.7Hz). ¹³ C (125 MHz, C ₅ D ₅ N; 27 ° C) δ (ppm)
175.5 (s), 135.3 (d), 134.2
(S), 132.1 (d), 131.9 (d), 12
9.9 (d), 127.8 (d), 105.5 (d),
78.4 (d), 78.4 (d), 75.0 (d), 7
2.5 (d), 72.3 (d), 71.6 (d), 7
0.0 (t), 62.7 (t), 54.6 (d), 3
5.6 (t), 33.1 (t), 32.7 (t), 3
2.0 (t), 32.0 (t), 29.9 (t), 2
9.8 (t), 29.5 (t), 29.4 (t), 2
9.4 (t), 28.3 (t), 25.8 (t), 2
2.8 (t), 14.2 (q), 12.7 (q). Compound (3) [α] ²⁸ _D = + 62.8 ° (1-PrOH, c = 1.
0) MS: FDMS 860 (M + H) + negative FABMS 858 (M-H) - UV: (λmax (nm), MeOH) 201 (ε2300) IR: (cm -1, KBr) 3350,2940,285
0, 1660, 1545, 1495, 1100. mp: 189.5-190.5 ° C NMR: ¹ H (500 MHz, C ₅ D ₅ N; 27 ° C) δ
(ppm) 8.47 (1H, d, J = 9.2Hz), 7.5
1 (1H, d, J = 4.9Hz), 6.96 (1H, d,
J = 4.9Hz), 6.64 (1H, d, J = 6.7H)
z), 6.60 (1H, d, J = 5.0Hz), 6.48
(1H, bt, J = 5.1Hz), 6.24 (1H, d,
J = 3.7 Hz), 6.04 (1H, d, J = 6.1H)
z), 5.56 (1H, d, J = 3.7Hz), 5.24
(1H, m), 4.60 (2H, m), 4.56 (1
H, m), 4.50 (1H, bs), 4.45 (2H,
m), 4.35 (1H, m), 4.31 (3H, m),
4.24 (1H, m), 2.26 (1H, m), 2.1
8 (1H, m), 1.98 (1H, m), 1.87 (2
H, m), 1.73 (1H, m), 1.65 (2H,
m), 1.42-1.10 (59H, m), 0.88
(9H, m). ¹ H (500 MHz, C ₅ D ₅ N + D ₂ O; 55 ° C) δ
(ppm) 5.43 (1H, d, J = 3.7Hz), 5.0
4 (1H, m), 4.67 (1H, dd, J = 4.0,
7.4 Hz), 4.56 (1 H, m), 4.54 (1 H,
m), 4.46 (1H, m), 4.45 (1H, m),
4.44 (1H, m), 4.38 (1H, dd, J =
4.9, 5.0 Hz), 4.33 (1 H, m), 4.3
1 (1H, m), 4.28 (1H, m), 4.25 (1
H, m), 2.19 (2H, m), 2.07 (1H,
m), 1.95 (1H, m), 1.75 (2H, m),
1.50-1.20 (61H, m), 1.00 (9H,
m). ¹³ C (125 MHz, C ₅ D ₅ N; 27 ° C) δ (ppm)
175.0 (s), 101.2 (d), 76.5
(D), 72.9 (d), 72.4 (d), 72.4
(D), 71.6 (d), 70.9 (d), 70.2
(D), 68.3 (t), 62.6 (t), 50.6
(D), 36.8 (t), 35.5 (t), 34.5
(D), 34.4 (t), 32.1 (t), 30.3
(T), 30.3 (t), 30.1 (t), 30.0
(T), 29.8 (t), 29.7 (t), 29.5.
(T), 27.3 (t), 26.4 (t), 25.8.
(T), 22.9 (t), 19.3 (q), 14.2
(Q), 11.5 (q). ¹³ C (125 MHz, C ₅ D ₅ N; 55 ° C) δ (ppm)
176.4 (s), 101.2 (d), 75.9
(D), 73.0 (d), 73.0 (d), 73.0
(D), 71.6 (d), 71.0 (d), 70.3
(D), 68.9 (t), 62.8 (t), 51.5
(D), 37.4 (t), 35.8 (t), 35.1
(D), 33.6 (t), 32.6 (t), 30.8
(T), 30.6 (t), 30.4 (t), 30.4
(T), 30.4 (t), 30.3 (t), 30.2
(T), 30.0 (t), 27.8 (t), 26.9.
(T), 26.1 (t), 23.3 (t), 19.9.
(Q), 14.7 (q), 12.0 (q). Compound _{^{(4) [α] 28 D}} = -2.0 ° (1-PrOH, c = 1.0 ) MS: FDMS 852 (M + H) + negative FABMS 850 (M-H) - UV: (λmax (nm) , MeOH) 236 (ε5500) IR: (cm ^-1 , KBr) 3350, 2920, 285
0, 1640, 1530, 1470, 1080. mp: 158.5-159.5 ° C. NMR: ¹ H (500 MHz, C ₅ D ₅ N; 27 ° C.) δ
(ppm) 8.34 (1H, d, J = 9.2Hz), 7.6
2 (1H, d, J = 5.5Hz), 7.22 (1H, d,
J = 3.7 Hz), 7.13 (1H, bs), 6.87
(1H, d, J = 4.9 Hz), 6.36 (1H, bt,
J = 6.1 Hz), 6.19 (1H, d, J = 15.3H)
z), 5.99 (1H, dd, J = 6.7, 15.9H)
z), 5.89 (1H, dt, J = 6.1, 15.9H)
z), 5.63 (1H, dt, J = 7.3, 15.3H)
z), 5.49 (1H, bt, J = 6.7Hz), 4.8
9 (1H, d, J = 7.9Hz), 4.78 (1H,
m), 4.74 (1H, m), 4.69 (1H, dd,
J = 6.1, 10.4Hz), 4.56 (1H, m),
4.49 (1H, m), 4.34 (1H, m), 4.2
1 (1H, m), 4.19 (2H, m), 4.01 (1
H, m), 3.88 (1H, m), 2.21 (2H,
m), 2.15 (2H, m), 2.11 (2H, m),
2.00 (1H, m), 1.79 (1H, m), 1.7
5 (3H, s), 1.70 (1H, m), 1.45
1.05 (51H, m), 0.84 (3H, t, J =
6.7 Hz), 0.84 (3H, t, J = 6.7 Hz). ¹³ C (125 MHz, C ₅ D ₅ N; 27 ° C) δ (ppm)
175.5 (s), 135.5 (d), 134.2
(S), 132.0 (d), 131.9 (d), 12
9.9 (d), 127.8 (d), 105.4 (d),
78.4 (d), 78.3 (d), 75.0 (d), 7
2.5 (d), 72.3 (d), 71.6 (d), 6
9.9 (t), 62.8 (t), 54.6 (d), 3
5.6 (t), 33.1 (t), 32.6 (t), 3
2.0 (t), 31.9 (t), 29.9 (t), 2
9.8 (t), 29.5 (t), 29.4 (t), 2
9.3 (t), 28.3 (t), 25.8 (t), 2
2.8 (t), 14.2 (q), 12.6 (q).

[0010]Experimental example 2:Antitumor activity of compounds of the present invention 1. Paired with B16 mouse melanoma cells in the abdominal cavity
Antitumor activity 6 week old female purchased from Japan SLC, Inc.
BDF₁The experiment was conducted with 6 mice per group. B16
Mouse melanoma cells 8 × 10^FivePer mouse intraperitoneally
Transplanted (transplantation day 0), 1, 5 and 9 after transplantation
On the day, weigh 20g mouse sample prepared for each dose
0.2 ml / peritoneal injection and measure the number of days until death
Decided The results are shown in Table 1. Each compound shows remarkable antitumor activity
did. 2. Subcutaneously to B16 mouse melanoma cells
Antitumor activity 6 week old female purchased from Japan SLC, Inc.
BDF₁The experiment was conducted with 6 mice per group. B16
1 x 10 mouse melanoma cells⁶Individual / Mouse subcutaneously
15 days after the planting (day 15), the tumor body under the dorsal skin
Product (major axis x minor axis x height) / 2 was measured and the average values were almost the same.
Grouping was done so that they would be the same. 15 samples
7, 19, 21, 23 days later (day 15, 17, 19,
Twenty-three, 23) was administered intravenously via the tail vein,
Tumor 2,26 days later (day 15, 19, 22, 26)
The volume was measured.   Table 2     Sample doseTumor volume (mm3)   (Compound) (mg / kg) day15 day19 day22 day26     Control group-340 1073 1749 4283       (1) 0.1 318 577 921 2222                          (6.5)^*  (46.2) (47.3) (48.1)       (3) 0.1 310 617 667 1335                          (9.0) (42.6) (61.9) (68.8)   As the results shown in Table 2, the compound (1) and the compound
(3) markedly suppressed tumor growth.Immunostimulatory activity of the compound of the present invention 1. Mixed lymphocyte reaction Extracted from the spleen of C57BL / 6 mouse according to a conventional method
Treated spleen cells with mitomycin C 50 μg / ml
Target cells, while BALB / c mouse phosphorus
Lymphocytes taken out from the node according to the usual method are called reactive cells.
did. 10% FCS of the above-mentioned spleen cells and lymphocytes
2 x 10 each using RPMI 1640 as medium⁶Pieces / ml
Was prepared. Using a round bottom 96-well plate, float both cells above
50 μl / well each of the eluents and samples (10 μl / wel
l) was added and the temperature was 37 ° C-5% CO for 4 days.₂Culture under the conditions
After doing³H-thymidine (³H-TdR) 0.
5 μCi / well was added. After 8 hours,
Do the best with a liquid scintillation counter³
Uptake of H-TdR was measured.     Table 3                                     ³H-TdR incorporation                               (% Of control)       Sample / concentration (μg / ml)_Ten-1_Ten-2_Ten-3_Ten-Four _Ten-Five     (Compound)           (1) 236 220 184 189 179           (2) 169 134 142 127 147           (3) 255 209 179 164 168     (4) 202 182 141 117 127 From the results shown in Table 3, all samples were 10^-Fiveμg /
Strong lymphocyte mixed culture reaction enhancing activity even at a low concentration of ml
showed that. 2. Natural killer (NK) cell activity enhancing action (in
vivo) 6 week old female purchased from Japan SLC, Inc.
C57BL / 6 mice were used for the experiment. Sample each 0.
Intraperitoneal administration of 1 mg / kg for 2 consecutive days followed by 3 days
The spleen was removed from the mouse. From the removed spleen
To remove the splenocytes and make ammonium chloride-Tris isotonic
1x10 after removing red blood cells with buffer⁷It will be pieces / ml
Fermented in 10% FCS RPMI 1640.
100 cells of this cell suspension was added to a 96-well round bottom microplate.
Dispense μl each (effector cells 1 x 10⁶Pieces / we
ll). On the other hand⁵¹Cr-labeled YAC-1 cells
1 x 10⁶10% FCS RPM so that the number becomes 1 / ml
Suspended in I 1640 and used as target cells 10 μl
Each was dispensed into wells. In this case, the E / T ratio (effector
-Cell number / target cell number) is 100: 1
Ratio by changing the number of target cells to 50: 1, 25:
Changed to 1. Reaction system of the above target cells and effector cells
For 4 hours at 37 ° C-5% CO₂Culture under the conditions of. 4:00
After a while, add 70 μl of supernatant from each well to the counter
And collect the target cells with a gamma counter.
Was measured.     Table 4                             Specific cytotoxicity (%) *       _{sample    E / T ratio  25: 1 50: 1 100: 1}       (Compound)         Reference 0.5 1.3 2.3         (1) 14.0 23.9 39.8         (2) 3.6 8.0 18.7         (3) 18.5 33.5 55.4         (4) 8.3 17.5 37.2 From the results shown in Table 4, all samples were tested for N in vivo.
It exhibited a K cell activity enhancing action. 3. Macrophage activation (in vivo) Eight week old BALB / c mice were used in the experiments. Compound
0.1 mg / kg of (3) was intraperitoneally administered for 2 consecutive days.
Remove peritoneal exudate cells (PEC) from mice on day
It was Furthermore, this PEC is attached to a plastic dish.
Cells divided into non-adherent cells and MethA cells
The growth inhibitory effect was examined. PEC or plastic
5 x 10 dish-adherent cells and non-adherent cells⁶Pieces / ml
Suspended in 10% FCS RPMI 1640
Let Add this cell suspension to a 96-well round bottom microplate
Dispense 100 μl of each into effector cells (effector cells 5 × 10
^FivePieces / well). On the other hand, 5 x 1 of MethA as a target cell
0^Five10% FCS RPMI 164 so that the number becomes 1 / ml
Float to 0 and add 10 μl of this cell suspension to each well.
Dispensed. In this case, the E / T ratio is 100: 1, but
Change the number of vector cells and change the ratio as shown in Tables 5 and 6.
It was The target cell-effector cell reaction system described above is used.
Time 37 ° C-5% CO₂After culturing under the conditions of³
H-TdR 0.5 μCi / well was added. And 6 o'clock
After a period of time, the cells are harvested and liquid scintillation filters are used.
By unter³Uptake of H-TdR was measured.
Note that only effector cells³Uptake of H-TdR
Since it was less than 1% of the target cell uptake,
Calculation was performed by the formula.     Table 5                               Tumor cell growth inhibitory effect (%)     E / T ratio 100: 1 50: 1 25: 1       PEC vs. 68.2 30.5-3.8     Compound (3) 95.7 82.7 41.2     Table 6                               Tumor cell growth inhibitory effect (%)     E / T ratio 20: 1 10: 1 5: 1       Non-adherent cell reference 9.7 1.4 -11.4     Compound (3) 12.6 0.2-6.3       Adherent cell reference 41.4 15.4 1.6     Compound (3) 77.0 54.9 23.5 Mice treated with compound (3) as shown in Table 5
Of PEC showed a remarkable cytostatic effect. And the table
As is clear from 6, this cytostatic effect is
Sticky dish-adherent cells (mainly macro fur
It was shown to be due to (i). The results of Table 5 and Table 6
Compound (3) is a prominent macrophage in vivo
It became clear that it has a diactivating effect.

Experimental Example 3 : Synthetic Example The synthetic method of compound (1) and its physicochemical properties are as follows (for the compound number in the synthetic method, refer to the above reaction route formula). In addition, NMR data describes the main peak. (i) Synthesis of Compound 1 Compound 1 is an Agricultural and Biological Chemistry.
mistry), Vol. 54, No. 3, pages 663-667, 1990. (ii) Synthesis of Compound 3 To 15.0 g of Wittig salt 2 (Compound 2) was added 60 ml of tetrahydrofuran, and the atmosphere was replaced with argon. At −10 ° C., add 2N-butyllithium-hexane solution to 14.
4 ml was added and stirred for 30 minutes. Aldehyde 1 (Compound 1)
5.74 g was dissolved in 10 ml of tetrahydrofuran and added. Further, 8 ml of tetrahydrofuran was added. It returned to room temperature and stirred for 15 hours. The mixture was concentrated, brine was added, and the mixture was extracted twice with ethyl acetate. It was washed with brine and concentrated. Purify with a silica gel column (Wakogel C-200, 200 g, hexane: ethyl acetate = 5: 1), and use alcohol 3
(Compound 3) was obtained. Yield 5.06g. Yield 67.6
%. Data of compound 3 ¹ H-NMR (500 MHz, CDCl ₃ ) δ (ppm) :
7.20-7.35 (15H, m), 5.71 (1H,
m), 5.44 (1H, dd, J = 10.7, 9.5H)
z) 5.37 (1H, m), 5.11 (1H, m),
4.30-4.70 (6H, m), 4.42 (1H, d
d, J = 9.5, 5.8 Hz), 4.06 (1H, m),
3.55 (1H, dd, J = 5.8, 3.0Hz), 3.
50 (2H, d, J = 6.1Hz), 2.99 (1H,
m), 1.85-2.00 (2H, m), 1.51 (1
H, m), 1.10-1.40 (12H, m), 0.8
5 & 0.84 (each 3H, d, J = 6.1 Hz). (iii) Synthesis of compound 4 To 0.19 g of alcohol 3 was added 1.9 ml of pyridine,
50.3 μl of methanesulfonyl chloride was added at −5 ° C., and the mixture was stirred at room temperature for 15 hours. It was concentrated and azeotroped with toluene. Diethyl ether was added and the mixture was washed with brine. Concentrate to a silica gel column (Wako Gel C-200, 10
g, hexane: acetone = 6: 1) to obtain a mesyl derivative 4 (compound 4). Yield 0.21g. Yield 97.4
%. Data of compound 4 ¹ H-NMR (500 MHz, CDCl ₃ ) δ (ppm) :
7.25-7.40 (15H, m), 5.79 (1H,
m), 5.48 (1H, t like, J = 10.4H)
z), 5.36 (1H, m), 5.00-5.12 (2
H, m), 4.75 (1H, d, J = 11.6Hz),
4.35-4.55 (6H, m), 3.76 (1H,
m), 3.66 (1H, dd, J = 10.7, 6.7H
z), 3.50 (1H, dd, J = 10.7, 3.0H
z), 2.93 (3H, s), 1.51 (1H, m),
1.10-1.40 (12H, m), 0.85 & 0.8
4 (each 3H, d, J = 6.7 Hz). FDMS 663 (M + H) ⁺ . (iv) Synthesis of Compound 5 5.8 of mesyl derivative 4 was added to 0.58 g of palladium black.
4 g was dissolved in 50 ml of ethyl acetate and added. The atmosphere was replaced with hydrogen, and the mixture was stirred at room temperature for 15 hours. It was filtered through Celite and concentrated (triol 5 (compound 5)). Yield 3.37g. Yield 96.6%. Data of compound 5 ¹ H-NMR (500 MHz, CDCl ₃ ) δ (ppm) :
5.04 (1H, m), 4.04 (2H, m), 3.6
1 (2H, bs), 3.20 (3H, s), 1.1-
1.4 (23H, m), 0.87 (6H, d, J = 6.
7Hz). (v) Synthesis of compound 6 To 3.37 g of triol 5 was added dimethylformamide 6
7.4 ml and sodium azide 4.91 g were added. 10
The mixture was stirred at 0 ° C for 15 hours. The mixture was concentrated, brine was added, and the mixture was extracted with ethyl acetate. The extract was washed with brine, concentrated, and purified with a silica gel column (Wakogel C-200, 110 g, hexane: acetone = 3: 1) to obtain an azide body 6 (compound 6). Yield 2.09g. Yield 71.6%. Data of compound 6 ¹ H-NMR (500 MHz, CDCl ₃ ) δ (ppm) :
3.97 (1H, dd, J = 11.6, 4.9Hz),
3.90 (1H, dt, J = 11.6, 4.3Hz),
3.77 (2H, m), 3.62 (1H, m), 1.5
2 (1H, m), 1.1-1.4 (22H, m), 0.
86 (6H, d, J = 6.7Hz). (vi) Synthesis of compound 7 To 211 mg of azide body 6, 1.1 ml of pyridine and 257 mg of triphenylmethyl chloride were added, and the mixture was stirred at 50 ° C for 15 hours. It was concentrated and azeotroped with toluene. Purification with a silica gel column (Wako Gel C-200, 15 g, hexane: ethyl acetate = 5: 1) gave 0.30 g of trityl compound 7 (compound 7). Yield 84.0%. (vii) Synthesis of Compound 8 To 0.30 g of trityl compound 7, 3.0 ml of pyridine was added,
0.18 ml of benzoyl chloride and 5.8 mg of 4-dimethylaminopyridine were added and stirred. After 15 hours, a small amount of ethanol was added, and the mixture was stirred and concentrated. It was azeotropically distilled with toluene and purified with a silica gel column (Wakogel C-200, 15 g, hexane: ethyl acetate = 12: 1) to give a benzoyl compound 8.
0.40 g of (Compound 8) was obtained. Yield quantitative. (viii) Synthesis of compound 9 To 0.40 g of benzoyl compound 8 was added 8 ml of methylene chloride and 4 ml of methanol. 48.5 mg of p-toluenesulfonic acid monohydrate was added, and the mixture was stirred at room temperature for 3 days. The mixture was concentrated, ethyl acetate and aqueous sodium hydrogen carbonate were added, and the layers were separated. Washed with saline solution,
Concentrated. Silica gel column (Wakogel C-200,
Purification with 10 g, hexane: ethyl acetate = 3: 1) gave 0.22 g of alcohol 9 (compound 9). Yield 79.
7%. Data of compound 9 ¹ H-NMR (500 MHz, CDCl ₃ ) δ (ppm) :
8.02 (2H, d, J = 7.3), 7.98 (2H,
d, J = 6.7 Hz), 7.60 (1H, t, J = 7.3)
Hz), 7.55 (1H, t, J = 7.6Hz), 7.46
(2H, t, J = 7.6Hz), 7.41 (2H, t, J
= 7.9 Hz), 5.51-5.56 (2H, m), 3.
98 (1H, m), 3.79 (2H, m), 0.84
(6H, d, J = 6.7Hz). (ix) Synthesis of compound 10 To 5 mg of palladium black, 38 mg of alcohol 9 was dissolved in 1 ml of tetrahydrofuran and added. Replace with hydrogen,
Stir for 15 hours at room temperature. It was filtered through Celite and concentrated (amine 10 (compound 10)). It was directly used for the next reaction. (x) Synthesis of Compound 11 To amine 10 obtained in the above reaction, 41 mg of p-nitrophenyl ester of α-R-acetoxytetracosanoic acid dissolved in 2.0 ml of tetrahydrofuran was added. After stirring at room temperature for 1 day, concentrate and concentrate on a silica gel column (Wako Gel C-2
The product was purified with 00,10 g and hexane: acetone = 3: 1) to obtain 25.5 mg of ceramide 11 (compound 11). Yield from alcohol 9 39.6%. Data of compound 11 ¹ H-NMR (500 MHz, CDCl ₃ ) δ (ppm) :
8.06 & 7.96 (each 2H, d, J = 7.3)
Hz), 7.64 (1H, t, J = 7.3 Hz), 7.54
(1H, t, J = 7.6 Hz), 7.50 & 7.39 (e
ach 2H, t, J = 7.9 Hz), 7.05 (1H,
d, J = 9.2 Hz), 5.45 (1H, dd, J = 9.
1,2.4 Hz), 5.38 (1H, dt, J = 9.8,
3.1Hz), 5.20 (1H, t, J = 6.1Hz),
4.36 (1H, m), 3.55-3.70 (2H,
m), 1.51 (1H, m), 0.89 (3H, t, J
= 6.7 Hz), 0.86 (6H, d, J = 6.7 Hz). FDMS 935 (M + H) ⁺ . (xi) Synthesis of compound 12 3.0 mg of ceramide 11 was added with 3.0 ml of tetrahydrofuran,
Stannous chloride 42.9 mg, silver perchlorate 46.9 mg, and molecular sieves 4A powder 300 mg were mixed and stirred for 30 minutes. Cool to −10 ° C. and benzyl fluorosugar 78.
4 mg was dissolved in tetrahydrofuran (1.2 ml) and added.
It was gradually returned to room temperature and stirred for 15 hours. Stannous chloride 4
2.9 mg, silver perchlorate 46.9 mg, and molecular sieves 4A powder 300 mg were added, and 4 hours later, benzyl fluoride galactose 70 mg was added to tetrahydrofuran 0.9.
It was dissolved in ml and added. After a further 20 hours, the same amounts of stannous chloride and silver perchlorate were added, and the mixture was stirred for 24 hours. The mixture was filtered through Celite, and the residue was extracted with a small amount of acetone. Water was added and the mixture was extracted 3 times with ethyl acetate. The mixture was concentrated and purified with a silica gel column (Wako Gel C-200, 15 g, hexane: ethyl acetate = 6: 1) to obtain 50.3 mg of α-glycoside 12 (compound 12). Yield 35.8%. Raw material 11
Was recovered at 36.8 mg (recovery rate 40.9%). Data of compound 12 ¹ H-NMR (500 MHz, CDCl ₃ ) δ (ppm) :
8.02 & 7.90 (each 2H, d, J = 7.9)
Hz), 7.59 & 7.50 (each 1H, t, J =
6.4 Hz), 7.45 (1H, t, J = 7.6 Hz),
7.16-7.35 (22H, m), 5.78 (1H,
dd, J = 9.8, 2.6 Hz), 5.39 (1H,
m), 5.09 (1H, dd, J = 7.6, 5.2H
z), 4.87, 4.73, 4.66, 4.63, 4.
55, 4.48 & 4.40 (each 1H, d, J =
11.3Hz), 4.732 (1H, d, J = 4.3H)
z), 4.07 (1H, t, J = 7.2Hz), 3.98
(1H, dd, J = 10.4, 3.3Hz), 3.92
(1H, m), 3.87 (1H, dd, J = 11.9,
2.4 Hz), 3.81 (1H, dd, J = 9.8, 2.
4Hz), 3.59 (1H, dd, J = 12.1, 2.3)
Hz), 3.52 (1H, dd, J = 8.9, 6.4H
z) 3.45 (1H, dd, J = 9.2, 6.7H
z), 2.02 (3H, s), 0.88 (3H, t, J
= 7.0 Hz), 0.85 (6H, d, J = 6.7 Hz). FDMS 1456 (M ⁺ ). (xii) Synthesis of compound 14 (compound (1)) 50.1 mg of α-glycoside 12 was added with 3.0 ml of ethyl acetate.
And 8.0 mg of palladium black were added thereto, the atmosphere was replaced with hydrogen, and the mixture was stirred at room temperature for 15 hours. The mixture was filtered through Celite and concentrated to give 33.2 mg of tetraol 13 (compound 13). Yield 8
8.1%. FDMS 1096 (M ⁺ ). To 33.0 mg of tetraol 13 was added 3 ml of methanol and 0.3 ml of 1N sodium methoxide methanol solution, and the mixture was left for 3 hours. Resin (DOWEX 50W X
8) was added to adjust the pH to 7, and the mixture was filtered. The residue was well extracted with chloroform-methanol (1: 1) and concentrated. Purification with a silica gel column (Wako Gel C-200, 5 g, chloroform: methanol = 5: 1) yielded 13.2 mg of compound 14 (compound (1)). Yield 51.8%. Data of compound 14 ¹ H-NMR, ¹³ C-NMR and FDMS data were the same as the data of compound (1) obtained from a natural product.

[0012]

The compound of the present invention is a glycosphingolipid having excellent antitumor activity and immunostimulatory activity, and can be expected as an antitumor agent and an immunostimulatory agent. The fact that the compound of the present invention has such physiological activity is understood to be unexpected to those skilled in the art.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical indication location C07H 15/04 E 7822-4C (72) Inventor Isao Akimoto 3 Miyahara-cho, Takasaki-shi, Gunma Prefecture Kirin Brewery Co., Ltd. Pharmaceutical Research Institute

Claims (4)

[Claims] 1. A glycosphingolipid represented by the following formula (I). [Chemical 1] Here, when R ₁ is α-D-galactopyranosyl and R ₂ is the following group (a), R ₃ is the following group (b) or (c), and R ₁ is When β-D-glucopyranosyl and R ₃ is the group (d) below,
R ₂ is the following group (a) or (e). _{(A) - (CH 2)} 21 -CH 3 (b) -CH (OH) - (CH 2) 11 -CH (CH 3)
_{2 (c) -CH (OH)} - (CH 2) 11 -CH (CH 3)
_{-CH 2 -CH 3 (d) -CH} = CH- (CH 2) 2 -CH = C (C
_{H 3) -CH = CH- (CH} 2) 6 -CH 3 (e) - (CH 2) 22 -CH 3 2. The sponge characterized by collecting sponge Agelus mauritianus and subjecting it to an extraction operation with an organic solvent, and collecting the glycosphingolipid according to claim 1 from the extract. Production method of glycolipid. 3. An antitumor agent containing one or more compounds of claim 1 as an active ingredient. 4. An immunostimulant containing, as an active ingredient, one or more of the compounds according to claim 1.