Sialosylceramides and production method thereof

This invention provides a novel compound expressed by the following formula (1): ##STR1## [wherein R.sup.1 denotes a hydrogen atom or CH.sub.3 CO--, R.sup.2 denotes --COOR.sup.4 (R.sup.4 denotes Na or a methyl group) or ##STR2## (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 ##STR3## (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 (R.sup.4 denotes Na or a methyl group) or denotes --COOR.sup.4 when R.sup.2 is ##STR4## and a method of preparation thereof. The above novel compounds of 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. 'Y

BACKGROUND OF THE INVENTION 
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. 
SUMMARY OF THE INVENTION 
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.

EXAMPLES 
The present invention is described in detail below with reference to 
examples. 
EXAMPLE 1 
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) 
EXAMPLE 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. 
EXAMPLE 3 
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. 
EXAMPLE 4 
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. 
EXAMPLE 5 
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. 
EXAMPLE 6 
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. 
EXAMPLE 7 
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 
Example 8 
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).