Sialosylceramides and Production Method Thereof

The present invention relates to novel sialosylceramides and a production method thereof.

The glycolipid of mammalian cells comprises a lipid structure expressed as a ceramide in which a fatty acid is amido-bonded to sphinogosine of a long chain amino alcohol and to which sugars such as glucose, galactose, N-acetylglucosamine, N-acethylgalactosamine, fucose, and sialic acid are bonded through glicosidic linkage in various combinations, and belongs to the category of so-called sphingoglycolipids. In particular, the glycolipid having sialic acid is called ganglioside.

Most of these compounds are generally located in the outer molecular layer of the two molecular layers of cell membrane and it has been thought from recent investigations that the compounds play an important role in the function as a reception and response receptor of recognition and information, differentiation, proliferation, malignant change, or behavior in cells.

However, the function of ganglioside-based glycolipid as a component of cell membrane has not been sufficiently elucidated and it is difficult to isolate ganglioside from an organism and purify it.

Although precision synthesis of GM.sub.3 (GM: ganglioside monosialo) and GM.sub.4 has been successfully achieved, the synthesis of an unnatural compound (GM.sub.5) of the present invention which has a similar structure to that of the above compounds is necessary and indispensable to the elucidation of the function of the ganglioside-based glycolipid as a component of cell membrane.

An object of the present invention is to provide novel sialosylceramides and a production method thereof.

The present invention relates to a compound expressed by the following formula (1): ##STR5## [wherein R.sup.1 denotes a hydrogen atom or a CH.sub.3 CO group, R.sup.2 denotes --COOR.sup.4 (R.sup.4 denotes Na or a methyl group) or ##STR6## (R.sup.5 denotes a hydrogen atom, --COC.sub.6 H.sub.5 or --Si(C.sub.6 H.sub.5).sub.2 C(CH.sub.3).sub.3), and R.sup.3 denotes ##STR7## (R.sup.5 denotes a hydrogen atom, --COC.sub.6 H.sub.5 or --Si(C.sub.6 H.sub.5).sub.2 C(CH.sub.3).sub.3) when R.sup.2 is --COOR.sup.4 or denotes --COOR.sup.4 when R.sup.2 is ##STR8## (R.sup.4 denotes Na or a methyl group)] and a production method thereof.

Typical examples of the compound expressed by the formula (1) of the present invention are indicated below. ##STR9##

A production method of the present invention will be described in detail below with reference to schemes I to III.

The compound (V) is prepared from N-acethylneuraminic acid methyl ester peracetate by the method of Kuhn and others (refer to Chem. Ber., 99, 611-617 (1966)).

The compound (VI) which is a ceramide portion is produced by the method disclosed in Japanese Patent Application No. 44913/1984 [Japanese Patent Disclosure No. 190745/1985].

The compound (IX) which is also a ceramide portion is produced by the method disclosed in Japanese Patent Disclosure No 190745/1985 or Japanese Patent Application No. 248981/1986 (See scheme III).

The compound (VI) or (IX), a glycosidation catalyst such as silver trifurate, a solvent such as dichloroethane, a mixed solution of tetrahydrofuran and chloroform, nitromethane, tetrahydrofuran or chloroform are added to M.S. (molecular sieves) 4A or AW300, and the mixture is agitated at room temperature for about 1 to 6 hours. Then, a solution containing the compound (V), and a solvent such as dichloroethane, a mixed solution of tetrahydrofuran and chloroform, nitromethane, tetrahydrofuran or chloroform is added to the resulting mixture under cooling, for example, with ice-methanol, and agitated at room temperature for about 10 to 20 hours after being allowed to stand for about 0.5 to 2 hours. The reaction solution is filtered and chloroform is added to the filtrate which is then washed with a saturated aqueous solution of sodium hydrogen carbonate and saturated salt water, dried with anhydrous magnesium sulfate, and subjected to vacuum distillation. The residue is purified by a known means such as a silica gel column to obtain the compounds (I) and (II) or compounds (VII) and (VIII).

The compound (I) is dissolved in methanol and sodium methoxide is added to the methanol solution and agitated at room temperature for about 3 to 12 hours. After the reaction has been completed, the reaction solution is subjected to vacuum distillation and methanol, tetrahydrofuran, and distilled water are added to the residue and agitated at room temperature for about 10 to 20 hours. The reaction solution is neutralized with IRC-50, filtered, and then subjected to vacuum distillation. The residue is purified by a known means such as a Sephadex LH-20 column to obtain the compound (III).

The compound (III) can be obtained by treating the compound (VII) in the same manner as the above.

The compound (IV) can be obtained by treating the compound (II) or (VIII) in the same manner as the above. ##STR10##

All the compounds (I), (II), (III), (IV), (VII) and (VIII) which are synthesized by the above-mentioned scheme are novel compounds.

Usefulness

The above novel compounds of the present invention are useful as a tumor maker, a molecular marker for cells having the ability of differential induction, or an intermediate of the synthesis thereof.

The present invention is described in detail below with reference to examples.

100 mg (0.13 milimol) of the compound (VI), 400 mg (1.5 milimol) of silver triflate, 3 ml of dichloroethane were added to 1 g of activated molecular sieves 4A and stirred at room temperature for 3 hours under argon atmosphere. Then, 1 ml of dichloroethane solution containing 50 mg (0.1 milimol) of the compound (V) was added to the resulting solution under cooling with ice-methanol bath and stirred at room temperature for 15 hours after being allowed to stand for 1 hour. The resulting reaction solution was filtered and chloroform was added to the filtrate which was then washed with a saturated aqueous solution of sodium hydrogencarbonate and saturated sodium chloride water, dried over anhydrous magnesium sulfate, and concentrated under vacuum. The residue was purified by a silica gel column (Wako gel C-300, 25 g, toluene:ethyl acetate=1:2, chloroform:methanol=10:1) to obtain 17 mg (14%) of the compound (I) and 7 mg (6%) of the compound (II). (Physical property of the compound (I))

Rf=0.30 (toluene:ethyl acetate=1:2)

[.alpha.].sub.D.sup.20 -1.33 (C=0.75, chloroform)

Elementary analysis: Calculated: C, 69.16; H, 9.32; N, 2.12; (+C.sub.6 H.sub.5 CH.sub.3). Measured: C, 68.69; H, 9.73; N, 2.25.

.sup.1 H-NMR(CDCl.sub.3): .delta.H 3.772 (s, 3H, OMe), 2.107, 2.024, 1.961, 1.881, 1.885 (Ac.times.5), 0.879 (t, 6H, CH.sub.2 CH.sub.3 .times.2)

(Physical property of the compound (II))

Rf=0.29 (toluene:ethyl acetate=1:2)

[.alpha.].sub.D.sup.20 -2.40 (C=0.25, chloroform)

Elementary analysis: Calculated: C, 69.16; H, 9.32; N, 2.12; (+C.sub.6 H.sub.5 CH.sub.3). Found: C, 69.22; H, 9.87; N, 2.18.

.sup.1 H-NMR(CDCl.sub.3): .delta.H 3.559 (s, 3H, OMe), 2.068, 2.033, 2.027, 2.023, 1.880 (Ac.times.5), 0.879 (t, 6H, CH.sub.2 CH.sub.3 .times.2)

100 mg (0.13 milimol) of the compound (VI), 400 mg (1.5 milimol) of silver triflate, 3 ml of a mixed solution of tetrahydrofuran and chloroform (1:1) were added to 1 g of molecular sieves 4A and stirred at room temperature for 3 hours under argon atmosphere. Then, 1 ml of a mixed solution of tetrahydrofuran and chloroform (1:1) containing 100 mg (0.2 milimol) of the compound (V) was added to the resulting solution under cooling with ice-methanol bath and agitated at room temperature for 15 hours after being allowed to stand for 1 hour. The obtained reaction solution was filtered and chloroform was added to the filtrate which was then washed with a saturated aqueous solution of sodium hydrogencarbonate and saturated sodium chloride water, dried over anhydrous magnesium sulfate, and concentrated under vacuum. The residue was purified by a silica gel column (Wako gel C-300, 25 g, toluene:ethyl acetate=1:2, chloroform:methanol=10:1) to obtain 70 mg (29%) of the compound (I) and 10 mg (42%) of the compound (II).

(Physical property of the compound (I))

Same as those of Example 1.

(Physical property of the compound (II))

Same as those of Example 1.

100 mg (0.13 milimol) of the compound (VI), 400 mg (1.5 milimol) of silver triflate, 3 ml of nitromethane were added to 1 g of molecular sieves AW300 and stirred at room temperature for 3 hours under argon atmosphere. Then, 1 ml of a nitromethane solution containing 100 mg (0.2 milimol) of the compound (V) was added to the resulting solution under cooling with ice-methanol bath and stirred at room temperature for 15 hours after being allowed to stand for 1 hour. The obtained reaction solution was filtered, the filrate was diluted with chloroform and then washed with a saturated aqueous solution of sodium hydrogencarbonate and saturated sodium chloride water, dried over anhydrous magnesium sulfate, and concentrated under vacuum. The residue was purified by a silica gel column (Wako gel C-300, 25 g, toluene:ethyl acetate=1:2, chloroform:methanol=10:1) to obtain 55 mg (23%) of the compound (I) and 60 mg (25%) of the compound (II).

(Physical property of the compound (I))

Same as those of Example 1.

(Physical property of the compound (II))

Same as those of Example 1.

100 mg (0.13 milimol) of the compound (VI), 400 mg (1.5 milimol) of silver triflate, 3 ml of tetrahydrofuran were added to 1 g of molecular sieves AW300 and stirred at room temperature for 3 hours under argon atmosphere. Then, 1 ml of a tetrahydrofuran solution containing 100 mg (0.2 milimol) of the compound (V) was added to the resulting solution under cooling with ice-methanol bath and stirred at room temperature for 15 hours after being allowed to stand for 1 hour. The obtained reaction solution was filtered and chloroform was added to the filtrate which was then washed with a saturated aqueous solution of sodium hydrogencarbonate and saturated sodium chloride water, dried over anhydrous magnesium sulfate, and subjected to vacuum distillation. The residue was purified by a silica gel column (C-300, 25 g, toluene:ethyl acetate=1:2, chloroform:methanol=10:1) to obtain 70 mg (29%) of the compound (I) and 72 mg (30% of the compound (II).

(Physical property of the compound (I))

Same as those of Example 1.

(Physical property of the compound (II))

Same as those of Example 1.

14 mg (0.012 milimol) of the compound (I) was dissolved in 8 ml of methanol and 50 .mu.l sodium methoxide was added to the obtained solution and stirred at room temperature for 6 hours. The reaction solution was subjected to vacuum distillation and 1 ml of methanol, 1 ml of tetrahydrofuran, and 0.5 ml of distilled water were added to the residue and stirred at room temperature for 15 hours. The reaction solution was neutralized with IRC-50, filtered, and then subjected to vacuum distillation. The residue was purified by a Sephadex LH-20 column (chloroform:methanol:water=60:30:4.6) to obtain 4.7 mg (43%) of the compound (III).

(Physical property of the compound (III))

Rf=0.63 (butanol:ethanol:water=2:1:1)

.sup.1 H-NMR .delta.H (d.sub.6 DMSO:D.sub.2 O=49:1, 30.degree.); 1.858 (S, 3H, NHCOCH.sub.3); 0.853 (t, 6H, --CH.sub.2 CH.sub.3).

Decomposition point: 217.degree.-222.degree. C.

2.0 mg (50%) of the compound (IV) was obtained in the similar manner to that of Example 5 except that 30 .mu.l of sodium methoxide was added to 5 mg (0.004 milimol) of the compound (II).

(Physical property of the compound (IV))

Rf=0.63 (butanol:ethanol:water=2:1:1)

.sup.1 H-NMR .delta.H (d.sub.6 DMSO:D.sub.2 O=49:1, 30.degree.); 1.884 (S, 3H, NHCOCH.sub.3); 0.855 (t, 6H, --CH.sub.2 CH.sub.3).

Decomposition point: 74.degree.-78.degree. C.

According to the method described in Japanese Patent Application No. 248981/1986, the compound (IX) was prepared from the ceramide compound (1a).

The ceramide compound (1a) 1.360 g (2.09 m mol) was dissolved in pyridine 30 ml, and TrCl 722.8 mg (2.77 m mol) was added to the pyridine solution followed by stirring at 50.degree. C. for 12 hours. Pyridine was removed from the reaction mixture and the residue was dissolved in chloroform. The chloroform solution was washed with water and saturated aqueous solution of sodium chloride, dried with anhydrous magnesium sulfate, and subjected to vacuum distillation. The residue was purified by a silica gel column (Wako gel C-300, 85 g, toluene:ethyl acetate=5:1 containing 2% Et.sub.3 N) to obtain 909 mg (45%) of the compound (1b).

DMF 15 ml, ClSi(C.sub.6 H.sub.5).sub.2 C(CH.sub.3).sub.3 370 mg (1.36 m mol) and imidazole 183 mg (2.69 m mol) were added to the compound (1b) 800 mg (0.90 m mol) followed by stirring at room temperature for 12 hours. Ether was added to the obtained reaction solution. The obtained ether solution was washed with water and a saturated aqueous solution of sodium chloride, dried over anhydrous magnesium sulfate, and concentrated under vacuum. The residue was purified by a silica gel column (Wako gel, C-300, 80 g, hexane:ethyl acetate=10:1, containing 2% Et.sub.3 N) to obtain 1.006 g (99%) of the compound (1c).

The compound (1c) 1.0 g (0.88 m mol) was dissolved in a mixed solution of dichloroethane 20 ml and methanol 1 ml, and TsOH (p-toluensulfonic acid monohydrate) 67 mg (0.35 m mol) was added to the reaction mixture followed by stirring at room temperature for 1 hour. A saturated aqueous solution of NaHCO.sub.3 was added to the obtained reaction solution to neutralize the solution. After adding chloroform to the solution, the obtained chloroform solution was washed with water and a saturated aqueous solution of sodium chloride, dried over anhydrous magnesium sulfate, and subjected to vacuum distillation. The residue was purified by a silica gel column (Wako gel, C-300, 25 g, hexane:ethyl acetate=5:1) to obtain the compound (IX) 650 mg (83%).

Physical properties of the compound (IX)

Rf=0.113 (hexane:ethyl acetate=5:1)

[.alpha.].sub.D.sup.25 13.79 (C=0.425, EtOAc)

400 MHz NMR CDCl.sub.3, TMS, ppm 0.879, 6H, t, J=6.5, --CH.sub.2 CH.sub.3 .times.2, 1.066, 9H, s, t-butyl, 1.252, 62H, s, --CH.sub.2 --, 1.574, 2H, m, H-3', 1.869, 2H, m, H-6, 1.962, 2H, m, H-2', 3.621, 1H, d, d, d, J=2.9, 7.2, 11.0, H-1, 3.831, 1H, m, H-2, 3.887, 1H, d, d, d, J=2.6, 4.4, 11.1, H-1, 4.335, 1H, t, J=3.6, H-3, 5.370, 1H, dd, J=15.2, 5.5, H-4, 5.406, 1H, dt, J=15.2, 5.6, H-5, 7.34-7.66, 10H, m, aromatic protons

The compound (IX) 100 mg (0.113 m mol) was dissolved in THF 2 ml and AgOTf 505 mg (1.96 m mol) dissolved in THF 1 ml were added to activated molecular sieves 4A 1 g followed by stirring for 10 minutes. The compound (V) 202 mg (0.34 m mol) dissolved in THF 1 ml was added slowly to the obtained reaction mixture at -20.degree. C. followed by agitation at room temperature for 18 hours. The reaction mixture was filtered by using celite, the filtrate was diluted with EtOAc, washed with a saturate aqueous solution of sodium hydrogencarbonate, water and a saturate aqueous solution of sodium chloride, dried with anhydrous MgSO.sub.4, and subjected to vacuum distillation. vacuum The residue was purified by a silica gel column (Wako gel, C-300, 25 g, toluene:EtOAc=1:1) to obtain the compounds (VII) (28.5 mg, 18.6%) and (VIII) (36.4 mg, 23.8%).

Physical properties of the compound (VII) Rf=0.46 (toluene:EtOAc=1:1) Elementary analysis: Calculated: C, 68.79; H, 9.47; N, 2.06; Found: C, 68.20; H, 9.34; N, 2.05.

NMR 400 MHz ppm CDCl.sub.3 TMS, 0.879 (6H, s, CH.sub.3 33 2, J=7.0), 1.045 (9H, s, t-Bu), 1.919, 2.003, 2.031, 2.094, 2.138 (s, Ac.times.5), 2.404 (1H, 3-Heq, dd, J=12.70, 7.82), 3.75 (3H, s, OCH.sub.3), 5.05 (m, 1H, H-4), 7.3-7.7 (m, 10H, Ph)

Physical properties of the compound (VIII)

Rf=0.42 (toluene:EtOAc=1:1)

Elementary analysis: Calculated: C, 67.01; H, 9.52; N, 2.00 (+2H.sub.2 O). Found: C, 66.95; H, 9.14; N, 2.18.

NMR 400 MHz ppm CDCl.sub.3 TMS, 0.874 (6H, t, CH.sub.3 .times.2, J=7.0), 1.031 (9H, s, t-Bu), 1.884, 2.028, 2.041, 2.093, 2.128 (s, OAc.times.5), 2.532 (1H, dd, J=12.82, 4.76, H-3eq), 3.728 (3H, s, OCH.sub.3) 4.858 (1H, m, H-4), 7.3-7.7 (10H, m, Ph).