Glycolipid containing N-glycolylneuraminic acid and method of producing the same

The present invention provides ganglioside-related compounds expressed by the following formula: ##STR1## (wherein R.sub.1 denotes a hydrogen atom or SiR.sub.3 R.sub.4 R.sub.5 (wherein R.sub.3 and R.sub.4 each denotes a methyl or phenyl group, and R.sub.5 denotes a tertiary butyl or dimethylphenylmethyl group), and R.sub.2 denotes a hydrogen atom, a trityl group, or ##STR2## (wherein M denotes an alkali metal atom)) and methods of producing the same. These ganglioside-related compounds are useful as markers for the early detection of cancer and in the immunotherapy for cancer.

BACKGROUND OF THE INVENTION 
(1) Field of the Invention 
The present invention relates to ganglioside-related compounds which 
exhibit Hanganatziu-Deicher antigen activity (referred to as "H--D 
antigen" hereinafter) and methods of producing the same. 
The present invention also relates to ceramide-related compounds which are 
intermediates for synthesizing the above-described ganglioside-related 
compounds. 
(2) Prior Art 
The glycolipids of mammalian cells are the glycosidic linkages between 
lipid structures called ceramides in which fatty acids are amido-bonded to 
long-chain amino alcohols called sphingosines and sugars, such as glucose, 
galactose, N-acetylglucosamine, N-acetylgalactosamine, fucose, and sialic 
acid, in various combinations, and belong to the category of so-called 
sphingoglycolipids. Of these glycolipids, substances containing sialic 
acid are specifically called gangliosides. 
H--D antigens are antigens which were discovered separately by Hanganatziu 
and Deicher in patients which had been injected with horse antiserums, for 
the purpose of curing their diseases, during the 1920s, and are known as 
antigens which react with the infected serums injected, as well as the red 
blood corpuscles of various animals such as sheep, horses, pigs, rabbits, 
and guinea pigs. H--D antigens have also recently been extracted as 
gangliosides from the red blood corpuscles of horses and have been 
purified to form simple molecules. In addition, it has been proven that 
gangliosides exhibiting H--D antigen activity are principal glycolipid 
components of the red blood corpuscles of horses which have been called 
"hematosides". 
It is assumed that the chemical structure of H--D antigens is Gd Neu(2-3) 
Gal(1-4) Glc-Cer. 
Furthermore, at present, H--D antigens have often been detected in the 
serum of diseased patients, regardless of whether heterologous antiserums 
have been administered. It has also been recognized that these antigens 
appear on the surfaces of cancerous lymphocyte cells of humans or domestic 
fowl. 
Since it seems likely that H--D antigens could be used as markers, not only 
in the early detection of cancer, but also in immunotherapy for cancer, it 
is expected that they will be applied to the field of the prevention and 
treatment of cancer. 
Most of such ganglioside-related compounds generally reside in the outer 
molecular layer of the two molecular layers of a cell membrane, and it is 
considered from the results of recent research that they play important 
roles in the discrimination and the acceptance and response of information 
in cells, in a receptor function, in differentiation, and in the 
proliferation, malignant change, and behavior of cells. 
However, it is very difficult to isolate and purify oligosaccharide chains 
containing sialic acid from living bodies. It has therefore become 
necessary and indispensable, in order to elucidate the correlation between 
the accurate biological information on these oligosaccharide chains 
containing sialic acid and the molecular structures thereof, that these 
chains should be accurately synthesized. 
The inventors have previously developed a method of synthesizing such a 
ceramide portion of a glycolipid in a stereoselective manner with a good 
yield (Japanese Patent Laid-Open No. 190745/1985). 
When subjected to glycosylation with a sugar chain portion, the 
above-described ceramide (I) has conventionally been changed into a 
benzoylated compound (IV) by the method described below: 
The compound (I) is treated with trityl chloride in pyridine to produce a 
tritylated compound (II) which is then treated with benzoyl chloride and 
dimethylaminopyridine to obtain a trityl-benzoylated compound (III). This 
compound is treated with para-toluenesulfonic acid to separate a trityl 
group and produce the benzoylated compound (IV) of the ceramide. In this 
series of reactions, the benzoylation can be performed without isolating 
the compounds (II) and (III) ((I)-(IV)). 
However, the glycosylation using the above-described benzoylated compound 
(IV) has the disadvantages described below: 
The benzoylated compound (IV) exhibits poor solubility in solvents, such as 
methylene chloride, benzene, or nitromethane, which are generally used for 
glycosylation, and thus the glycosylation can be performed only at a low 
concentration, and the yield of the product obtained from the 
glycosylation is insufficient (about 30 to 35%). 
SUMMARY OF THE INVENTION 
The inventors have conducted intensive investigations with a view to 
solving the above-described problems. As a result, they unexpectedly 
discovered that the replacement of the benzoyl group in the 
above-described compound (IV) by various types of silyl moieties produced 
remarkable effects such as an increase in the solubility of the compound 
(IV) in solvents, even under the conditions of the above-described 
glycosylation, and thus they have determined that the reaction could, for 
example, be performed at a high concentration (of more than about 50%), as 
well as provide a large increase in the yield of the product. These 
findings led to the achievement of the present invention. 
##STR3## 
It is an object of the present invention to provide novel intermediate 
ganglioside-related compounds which concern the above-described various 
medical fields, and a method of producing the same. 
It is another object of the present invention to provide novel 
ceramide-related compounds which are intermediates for the synthesis of 
the above-described ganglioside-related compounds. 
The present invention relates to ganglioside-related compounds of the 
following formula: 
##STR4## 
(wherein R.sub.1 denotes a hydrogen atom or SiR.sub.3 R.sub.4 R.sub.5 
(wherein R.sub.3 and R.sub.4 each denote a methyl or phenyl group, and 
R.sub.5 denotes a tertiary butyl or dimethylphenylmethyl group), and 
R.sub.2 denotes a hydrogen atom, trityl group (Tr), or 
##STR5## 
(wherein M denotes an alkali metal atom). 
The present invention also relates to a method of producing 
ganglioside-related compounds characterized by hydrolyzing compounds of 
the following formula: 
##STR6## 
(wherein =OCOCH.sub.3 and Ac=COCH.sub.3) to produce compounds of the 
following formula: 
##STR7## 
(wherein M denotes an alkali metal atom). 
The present invention further relates to a method of producing 
galglioside-related compounds characterized by acetylating compounds of 
the following formula: 
##STR8## 
(wherein Si.sup.t BuPh.sub.2 denotes a diphenyl-t-butylsilyl group) to 
obtain compounds of the following formula: 
##STR9## 
which are then hydrolyzed to obtain compounds of the following formula: 
##STR10## 
DESCRIPTION OF THE PREFERRED EMBODIMENTS 
The present invention is described in detail below with reference to 
Production Schemes I to V, wherein, in the chemical formulae, denotes 
OCOCH.sub.3 and denotes OCH.sub.2 C.sub.6 H.sub.5. 
(1) Production of Compound (1) 
Compound (1) is produced in accordance with the following method: 
Formula 
##STR11## 
The known compound (A) is prepared by the method described in Monatsh Chem. 
97, 654 (1966) (refer to Reference Examples 1 and 2). 
(2) Production of Compound (2) 
The above-described compound (1) is first added to a solvent such as acetyl 
chloride, and hydrogen chloride gas is added to the solvent while it is 
cooled with ice. After being agitated for 24 hours, the thus-obtained 
solution is concentrated under reduced pressure, and a solvent such as 
toluene is added to the residue. The obtained solution is subjected to 
azeotropic distillation to produce compound (2). 
(3) Production of Compounds (3) and (4) 
Compounds (3) and (4) are produced by reacting the above-described compound 
(2) with a compound (B) under the reaction conditions described below. 
Examples of catalysts, include HgBr.sub.2, Hg(CN).sub.2, AgClO.sub.4, 
AgCO.sub.3, AgOTf (wherein Tf denotes triflic acid: abbreviated the same 
way hereinafter), and silver silicate. AgOTf or a mixture of HgBr.sub.2 
and Hg(CN).sub.2 in a ratio of between 1:3 and 1:1 is preferably used as 
the catalyst. In addition, CH.sub.2 Cl.sub.2, benzene, toluene, 
chloroform, CH.sub.3 CN, CH.sub.3 NO.sub.2, or tetrahydrofuran can be used 
as a solvent. It is preferable to use CH.sub.2 Cl.sub.2 or tetrahydrofuran 
as the solvent. 
The reaction can be performed at a temperature of within the range of about 
-25.degree. C. to about 90.degree. C. under ice cooling, but preferably at 
room temperature. 
The reaction is performed under agitation for about 30 minutes to about 24 
hours. It is preferable to have an agitated reaction of about 24 hours. 
The thus-obtained reaction product is purified by a conventional method 
such as column chromatography. 
(4) Production of Compounds (5) and (6) 
Compounds (5) and (6) are obtained by acetylating the above-described 
compounds (3) and (4), respectively, under the reaction conditions given 
below. 
CH.sub.3 COCl or Ac.sub.2 O can be used as a reagent of this reaction, but 
Ac.sub.2 O is preferable. 
Pyridine, TEA, dichloromethane, dichloroethane, or THF can be used as a 
solvent, and a catalytic amount of dimethylaminopyridine dissolved in 
pyridine is preferably added to the reaction solution. 
The reaction temperature is within the range of about 0.degree. C. to about 
100.degree. C., but is preferably about 60.degree. C. 
The reaction can be performed under agitation for about 30 minutes to 24 
hours, preferably under agitation for 24 hours. 
(5) Production of Compounds (7) and (8) 
Compounds (7) and (8) are obtained by reacting the above-described 
compounds (5) and (6), respectively, under the reaction conditions given 
below. 
Pd--C, Pd(OH).sub.2, or PtO.sub.2 can be used as a catalyst for the 
reaction in an atmosphere of hydrogen. A reductant such as HCOOH-MeOH can 
be also used as a catalyst, but 10% Pd--C is preferable. 
Methanol, a mixed solvent of methanol and water, a mixed solvent of 
methanol and AcOH, or AcOH can be used as a solvent, but methanol is 
preferable. 
The reaction temperature is within the range of ice-cooling temperature to 
about 60.degree. C., but is preferably room temperature. 
The reaction time is within the range of about 1 hour to about 24 hours, 
but is preferably about 24 hours. The reaction is preferably performed 
under agitation. 
The thus-obtained reaction product is purified by filtration. 
(6) Production of Compounds (9) and (10) 
Compounds (9) and (10) are obtained by acetylating the above-described 
compounds (7) and (8), respectively, under the reaction conditions given 
below. 
Ac.sub.2 O or CH.sub.3 COCl can be used as a reagent for the reaction, but 
Ac.sub.2 O is preferable. 
Pyridine, TEA, dichloromethane, dichloroethane, DMF, or THF can be used as 
a solvent, but a catalytic amount of dimethylaminopyridine dissolved in 
pyridine is preferably added to the reaction solution. 
The reaction temperature is within the range of about 0.degree. C. to about 
80.degree. C., but is preferably about 60.degree. C. 
The reaction time is within the range of about 30 minutes to about 24 
hours, but is preferably about 24 hours. It is preferable to agitate the 
reaction solution. 
The thus-obtained reaction product is purified by a conventional method 
such as column chromatography. 
(7) Production of Compounds (11) and (12) 
Compounds (11) and (12) are obtained by deacetylating the above-described 
compounds (9) and (10), respectively, under the reaction conditions given 
below. 
NH.sub.2 NH.sub.2 AcOH can be used as a reagent for the reaction. 
DNF can be used as a solvent. 
The reaction temperature is within the range of room temperature to about 
80.degree. C., but is preferably about 60.degree. C. 
The reaction time is within the range of about 5 minutes to about 1 hour, 
preferably about 20 minutes. It is preferable to agitate the reaction 
solution. 
The thus-obtained reaction product is purified by a conventional method 
such as column chromatography. 
(8) Production of Compounds (13) and (14) 
Compounds (13) and (14) are obtained by reacting the above-described 
compounds (11) and (12), respectively, with Cl.sub.3 CCN under the 
reaction conditions given below. 
CCl.sub.3 CN--DBU, CCl.sub.3 CN--NaH, CCl.sub.3 CN--K.sub.2 CO.sub.3, or 
CCl.sub.3 CN--BuLi can be used as a catalyst for the reaction, but 
CCl.sub.3 CN--DBU is preferable. 
Dichloroethane, benzene, toluene, dichloromethane, or chloroform can be 
used as a solvent, but dichloromethane is preferable. 
The reaction temperature is within the range of about -25.degree. C. to 
about 50.degree. C., but is preferably about 0.degree. C. 
The reaction time is within the range of about 30 minutes to about 12 
hours, but is preferably about 4 hours. It is preferable to agitate the 
reaction solution. 
The thus-obtained reaction product is purified by a conventional method 
such as column chromatography. 
(9) Production of Compounds (15) and (16) 
Compounds (15) and (16) are obtained by reacting the above-described 
compounds (13) and (14), respectively, with a compound (C) under the 
reaction conditions given below. 
BF.sub.3 Et.sub.2 O, TMS triflate, TiCl.sub.4, AlCl.sub.3, or SnCl.sub.4 
can be used as a catalyst for the reaction, but BF.sub.3 Et.sub.2 O is 
preferable. 
CH.sub.2 Cl.sub.2, C.sub.2 H.sub.4 Cl.sub.2, THF, benzene, toluene, 
CH.sub.3 CN, CH.sub.2 NO.sub.2, or ether can be used as a solvent, but 
CH.sub.2 Cl.sub.2 is preferable. 
The reaction temperature is within the range of about -25.degree. C. to 
about 60.degree. C., but is preferably a temperature under cooling with 
ice and methanol. 
The reaction time is within the range of about 1 hour to about 24 hours, 
but is preferably about 24 hours. It is preferable to agitate the reaction 
solution. 
(10) Production of Compounds (17) and (18) 
Compounds (17) and (18) are obtained by reacting the above-described 
compounds (15) and (16), respectively, under the reaction conditions given 
below. 
Bu.sub.4 NF or HF can be used as a catalyst for the reaction, but Bu.sub.4 
NF is preferably used. 
THF, CH.sub.3 CN, CH.sub.3 NO.sub.2, EtOAc, CH.sub.2 Cl.sub.2, CHCl.sub.3, 
DMF, ether, benzene, or toluene can be used as a solvent, but THF is 
preferable. 
The reaction temperature is within the range of about 0.degree. C. to about 
50.degree. C., but is preferably room temperature. 
The reaction time is within the range of about 30 minutes to about 48 
hours, and reaction is preferably performed under agitation for about 48 
hours. 
Then, the following reaction is carried out under the reaction conditions 
given below. 
Ac.sub.2 O or CH.sub.3 COCl can be used as a reagent for the reaction, but 
Ac.sub.2 O is preferable. 
Pyridine, TEA, or dimethylaminopyridine can be used as a solvent, but a 
catalytic amount of dimethylaminopyridine dissolved in pyridine is 
preferably added to the reaction solution. 
The reaction temperature is within the range of about 0.degree. C. to about 
80.degree. C., but is preferably about 60.degree. C. 
The reaction time is within the range of about 30 minutes to about 24 
hours, but the reaction is preferably performed under agitation for about 
6 hours. 
The thus-obtained reaction product is purified by a conventional method 
such as column chromatography. 
(11) Production of Compounds (19) and (20) 
Compounds (19) and (20) are obtained by deacetylating the above-described 
compounds (17) and (18), respectively, under the reaction conditions given 
below. 
NaH-MeOH, K.sub.2 CO.sub.3 -MeOH, TEA-MeOH, KOH-MeOH, or NaOH-MeOH can be 
used as a catalyst for the reaction, but NaOCH.sub.3 is preferable and 
0.1N NaOCH.sub.3 is more preferable. 
Methanol, ethanol, propanol, THF, or dioxane can be used as a solvent, but 
methanol is preferable. 
The reaction temperature is within the range of about -10.degree. C. to 
about 50.degree. C., but is preferably room temperature. 
The reaction time is within the range of about 30 minutes to about 24 
hours, but the reaction is preferably performed under agitation for about 
6 hours. 
Then, the following reaction is carried out under the reaction conditions 
given below. 
NaOH, KOH, or LiOH can be used as a catalyst for the reaction, but NaOH is 
preferable. 
MeOH--THF, MeOH--dioxane, ethanol--THF, ethanol-dioxane, propanol-dioxane, 
or propanol-THF can be used as a solvent, and MeOH-THF is preferable. 
The reaction temperature is within the range of about 0.degree. C. to about 
50.degree. C., but is preferably room temperature. 
The reaction time is within the range of about 30 minutes to about 24 
hours, but is preferably about 24 hours. It is preferable to agitate the 
reaction solution. 
The starting raw material for the ceramide-related compounds which are 
intermediates of the ganglioside-related compounds of the present 
invention is the above-described compound (22). The ceramide-related 
compounds can be obtained in accordance with the steps described below. 
(1) Production of Compound (1) of the Following Formula: 
##STR12## 
(wherein R.sub.3, R.sub.4 and R.sub.5 each denotes the same as that 
described above) 
Compounds (23), (25), and (27) are obtained from compound (22) under the 
reaction conditions given below. 
Ph.sub.2.sup.t BuSiCl, Ph.sub.2 (CMe.sub.2 Ph)SiCl, or Me.sub.2 (CMePh)SiCl 
can be used as a reagent used for each reaction. It is preferable that 
each reaction is performed in the presence of a chlorine such as 
imidazole. 
Dimethylformamide (DMF), tetrahydrofuran (THF), chloroform, pyridine, or 
collidine can be used as a solvent, but DMF is preferable. 
The reaction temperature is within the range of about 0.degree. C. to about 
100.degree. C., but is preferably within the range of room temperature to 
40.degree. C. 
The reaction time is within the range of about 1 hour to 2 nights, and but 
is preferably 1 to 2 nights. It is preferable to agitate the reaction 
solution. 
The thus-obtained reaction product is purified by a conventional method 
such as column chromatography. 
(2) Production of Compounds (24), (26), and (28) of the Following Formula: 
##STR13## 
(wherein R.sub.1, R.sub.2, and R.sub.3 each denotes the same as that 
described above) 
Toluenesulfonic acid (abbreviated to TsOH hereinafter), CF.sub.3 COOH, 
CH.sub.3 COOH (abbreviated to AcOH hereinafter), HCOOH, HCl can be used as 
a catalyst used in each reaction, but TsOH is preferable. 
MeOH-CH.sub.2 Cl.sub.2, MeOH--C.sub.2 H.sub.4 Cl.sub.2, CHCl.sub.3 --MeOH, 
AcOH-MeOH, THF-MeOH, dioxane-MeOH, an aqueous AcOH solution, an aqueous 
THF solution, or an aqueous dioxane solution can be used as a solvent, but 
MeOH--CH.sub.2 Cl.sub.2 is preferable. 
The reaction temperature is within the range of about 0.degree. C. to about 
80.degree. C., but is preferably room temperature. 
The reaction time is within the range of about 30 minutes to one night, but 
is preferably 1 to 6 hours. It is preferable to agitate the reaction 
solution. 
The thus-obtained reaction product is purified by a conventional method 
such as column chromatography. 
(Availability of the Invention) 
The ganglioside-related compounds are useful as markers for the early 
detection of cancer and in the immunotherapy for cancer. 
In addition, the ceramide-related compounds are advantageously used for 
synthesizing ceramide portions when glycolipids and the above-described 
ganglioside-related compounds, which are useful as tumor markers, are 
synthesized. 
The present invention is described in detail below with reference to 
Reference Examples and Examples.