CA1269366A - Galactose derivatives and preparation thereof - Google Patents
Galactose derivatives and preparation thereofInfo
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- CA1269366A CA1269366A CA000555846A CA555846A CA1269366A CA 1269366 A CA1269366 A CA 1269366A CA 000555846 A CA000555846 A CA 000555846A CA 555846 A CA555846 A CA 555846A CA 1269366 A CA1269366 A CA 1269366A
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Abstract
ABSTRACT OF THE DISCLOSURE
The invention is concerned with new galac-tose derivatives having the formula:
wherein R1 is benzyl or
The invention is concerned with new galac-tose derivatives having the formula:
wherein R1 is benzyl or
Description
~6~
This application is a divisional of Canadian application No. 486,085 filed ~une 28, 1985.
The invention relates to sialic acid deriva-tives and galactose derivatives, more particularly gan-gliosides and intermediate compounds for use in producing gangliosides, and to methods for producing the same.
Gycolipids found in mammal cells are glycosides between ceramides, which are sphingosines (long chain amino alcohols) to which aliphatic acids have been attached through an amide linkage, and one or more sugars such as glucose, galactose, N-acetyl, glucosamine, N-acetyl galactosamine, fucose, sialic acid, etc. Among these glycosides, those containing sialic acid are called gangliosides.
Gangliosides exist mainly in the outer m~ole-cular layer of double molecular layers of the mammal cell membrane. Recent studies show that gangliosides play important roles in reception and recognition of, and response to, information in cells, receptor mechan-ism, differentiation, cell propagation, malignant cell transformation, cell behavior, etc.
However, it is very difficult to isolate sialic acid residue-containing oligosaccharides from an organ-ism. Therefore precise synthesis of such sialic acid residue-containing oligosaccharides is necessary for - 1 - ~
3~i6 the elucidation of the precise correla-tion between bio-logical information and the molecular s-tructure of the oligosaccharides.
It is an object of the invention to provide novel gangliosides, novel sialic acid derivatives for use in producing the gangliosides and methods for pro-ducing the same.
It is another object of the invention to pro-vide novel galactose and lactose derivatives which can be used as intermediate compounds in producing the gan-gliosides and methods for producing the same.
The novel sialic acid derivatives provided in accordance with the invention of the parent applica-tion are represented by the general formula:
R10 ORl . R2 R3 (1) Ac~N
ORl wherein Rl is hydrogen or acetyl group, one of R2 and R3 is -COOR4 wherein R4 is hydrogen, alkali metal such as sodium and potassium, alkaline earth metal such as calcium or methyl group, the other of R2 and R3 is 3~i6 OR~
L ORs \~ o -- ~`~`\ OR7 ORs or --O
~ o R~ O ~ ~ OR7 ORs .
wherein Rs is hydrogen, acetyl or benzyl group, R6 is hydrogen or acetyl group, R7 is hydrogen, acetyl or benzyl group, or OR
R~ O
This application is a divisional of Canadian application No. 486,085 filed ~une 28, 1985.
The invention relates to sialic acid deriva-tives and galactose derivatives, more particularly gan-gliosides and intermediate compounds for use in producing gangliosides, and to methods for producing the same.
Gycolipids found in mammal cells are glycosides between ceramides, which are sphingosines (long chain amino alcohols) to which aliphatic acids have been attached through an amide linkage, and one or more sugars such as glucose, galactose, N-acetyl, glucosamine, N-acetyl galactosamine, fucose, sialic acid, etc. Among these glycosides, those containing sialic acid are called gangliosides.
Gangliosides exist mainly in the outer m~ole-cular layer of double molecular layers of the mammal cell membrane. Recent studies show that gangliosides play important roles in reception and recognition of, and response to, information in cells, receptor mechan-ism, differentiation, cell propagation, malignant cell transformation, cell behavior, etc.
However, it is very difficult to isolate sialic acid residue-containing oligosaccharides from an organ-ism. Therefore precise synthesis of such sialic acid residue-containing oligosaccharides is necessary for - 1 - ~
3~i6 the elucidation of the precise correla-tion between bio-logical information and the molecular s-tructure of the oligosaccharides.
It is an object of the invention to provide novel gangliosides, novel sialic acid derivatives for use in producing the gangliosides and methods for pro-ducing the same.
It is another object of the invention to pro-vide novel galactose and lactose derivatives which can be used as intermediate compounds in producing the gan-gliosides and methods for producing the same.
The novel sialic acid derivatives provided in accordance with the invention of the parent applica-tion are represented by the general formula:
R10 ORl . R2 R3 (1) Ac~N
ORl wherein Rl is hydrogen or acetyl group, one of R2 and R3 is -COOR4 wherein R4 is hydrogen, alkali metal such as sodium and potassium, alkaline earth metal such as calcium or methyl group, the other of R2 and R3 is 3~i6 OR~
L ORs \~ o -- ~`~`\ OR7 ORs or --O
~ o R~ O ~ ~ OR7 ORs .
wherein Rs is hydrogen, acetyl or benzyl group, R6 is hydrogen or acetyl group, R7 is hydrogen, acetyl or benzyl group, or OR
R~ O
2 0 ~~ OR9 wherein R8 is hydrogen, acetyl or benzyl group, Rg is hydrogen, benzyl, allyl, monochloroacetyl, -C(CC13)=N~ or :
- ' :.
_ 10 - C~12 ~ \ C, ~127 NllCOCz~H~7 wherein Rlo is hydrogen or benzoyl group, or R2 and R3 show together I r O B n ~ O
-CO-O ~ ~ ~ OBn OBn wherein Bn is benzyl group.
The invention of the present divisional appli-cation provides galactose derivatives represented ~y the formula:
l 4 ~ OR
OR~
....
i6 where~n Rl is benzyl or 1~0 f~ O~s I, O
R and R2 are hydrogen or benzyl when Rl is benzyl, otherwise R and R2 are hydrogen, acetyl or benzyl, R3 and .R4 are hydrogen or R3 together w.i-th R o.r R~
~orm isopropylidene, Rs is benzyl or allyl, and R6 is hydrogen, acetyl or benzyl.
As seen ~rom the above, some of the galactose derivatives provided by the inven~ion are also lactose derivatives.
The invention will now be explained in detail.
(a) Synthesis of ceramide moiety Ceramide moieties of gangliosides can be prepared by the method as shown in Scheme lb. Compound (I) can be prepared by the method as shown in Scheme la (see the specification of Japanese Patent Laid-Open No. 60-190745.).
. - 5 -~ 6 Compound (ii) is obtained by refluxing overnight an alkyl halide such as l-bromotetradecane and triphenylphosphine in a solvent such as xylene.
1,2-O-isopropylidene-~-D-xylo-pentodialdo-1,4-furanose (iJ is reacted with the compound (ii) in a solvent, e.g.
tetrahydrofuran (THF) and hexane in the presence of BuLi to obtain 4-alkylvinyl derivative (iii). The reaction temperature and time are preferably in the range of -15C to 25C and 0.5 to 24 hours, respectively.
The compound (iii) is treated with methanesulfonylchloride in dry pyridine to obtain 3-methanesulfonyl derivative (iv). The reaction temperature and tim2 are preferably in the range of 0C to 25C and 2 to 24 hours, respectively.
The compound (iv) is treated in acetic acid - water to remove isopropylidene group. Diol derivative (v) is obtained.
The reaction temperature and time are preferably in the range of 70C to 9QC and 0.5 to 5 hours, respectively.
The compound (v) is treated with an oxldizing agent (e.g. sodium metaperiodate) in a solvent (e.g~ ethanol) to cleave the diol and then treated with a reducing agent (e.g.
sodium borohydride) to obtain diol (vi). The oxidation reaction is preferably conducted at 0C to 25C for 0.5 to 24 hoursO The reduction reaction is preferably conducted at oC
to 10C for 0.5 to 2 hours.
The compound (vi) is reacted with an alkyl vinyl ether such as ethyl vinyl ether in a solvent such as dichIoromethane ....
~ 3 ~
in the presence of a catalyst such as p-toluenesulfonate to obtain di-alkyl vinyl ether (vii). This reaction is preferably conducted at oC to 30C for 0~5 to 24 hours.
The compound (vii) is treated with an azide such as S sodium a~ide in a solvent such as dimethylrormamide (DMF) to obtain azide (viii). This reaction is preferably carried out at 70C to 120C for 8 hours to six days.
The azide (viii) is reduced by a reducing agent such as sodium borohydride and Lindler catalyst/H2 in a solvent such as ethanol and isopropanol to give amine (ix). The reaction is carried out at a re~lux temperature ~or one to six days when sodium borohydride is used and at 0C to 30C for 2 to 24 hours at a hydrogen pressure of l to 4 atms. when Lindler catalyst/H2 is used.
The amine (ix) is reacted with an acyl halide in the presence of an amine such as pyridine and dimethylaminopyridine to obtain amide (x) or (xi). This reaction is preferably carried out at 0C to 30C for 0.5 to 24 hours. Alternatively, the amine (ix) dissolved in a solvent such as dichlorométhane is reacted with an aliphatic acid in the presence of ~-chloro-l-methylpyridinium iodide and tri-n-butylamine to obtain the amide (x) or (xi). This reaction is preferably carried out at a reflux temperature for 0.5 to 13 hours under an inert atmosphere.
~5 The amide (x) or (xi) is treated with pyridinium p~
toluenesulfonate, AMsERLIST A-15 (traclemark), etc. in a solvent such as methanol or dichloromethane to obtain ceramide (xii) or (I).
- ' :.
_ 10 - C~12 ~ \ C, ~127 NllCOCz~H~7 wherein Rlo is hydrogen or benzoyl group, or R2 and R3 show together I r O B n ~ O
-CO-O ~ ~ ~ OBn OBn wherein Bn is benzyl group.
The invention of the present divisional appli-cation provides galactose derivatives represented ~y the formula:
l 4 ~ OR
OR~
....
i6 where~n Rl is benzyl or 1~0 f~ O~s I, O
R and R2 are hydrogen or benzyl when Rl is benzyl, otherwise R and R2 are hydrogen, acetyl or benzyl, R3 and .R4 are hydrogen or R3 together w.i-th R o.r R~
~orm isopropylidene, Rs is benzyl or allyl, and R6 is hydrogen, acetyl or benzyl.
As seen ~rom the above, some of the galactose derivatives provided by the inven~ion are also lactose derivatives.
The invention will now be explained in detail.
(a) Synthesis of ceramide moiety Ceramide moieties of gangliosides can be prepared by the method as shown in Scheme lb. Compound (I) can be prepared by the method as shown in Scheme la (see the specification of Japanese Patent Laid-Open No. 60-190745.).
. - 5 -~ 6 Compound (ii) is obtained by refluxing overnight an alkyl halide such as l-bromotetradecane and triphenylphosphine in a solvent such as xylene.
1,2-O-isopropylidene-~-D-xylo-pentodialdo-1,4-furanose (iJ is reacted with the compound (ii) in a solvent, e.g.
tetrahydrofuran (THF) and hexane in the presence of BuLi to obtain 4-alkylvinyl derivative (iii). The reaction temperature and time are preferably in the range of -15C to 25C and 0.5 to 24 hours, respectively.
The compound (iii) is treated with methanesulfonylchloride in dry pyridine to obtain 3-methanesulfonyl derivative (iv). The reaction temperature and tim2 are preferably in the range of 0C to 25C and 2 to 24 hours, respectively.
The compound (iv) is treated in acetic acid - water to remove isopropylidene group. Diol derivative (v) is obtained.
The reaction temperature and time are preferably in the range of 70C to 9QC and 0.5 to 5 hours, respectively.
The compound (v) is treated with an oxldizing agent (e.g. sodium metaperiodate) in a solvent (e.g~ ethanol) to cleave the diol and then treated with a reducing agent (e.g.
sodium borohydride) to obtain diol (vi). The oxidation reaction is preferably conducted at 0C to 25C for 0.5 to 24 hoursO The reduction reaction is preferably conducted at oC
to 10C for 0.5 to 2 hours.
The compound (vi) is reacted with an alkyl vinyl ether such as ethyl vinyl ether in a solvent such as dichIoromethane ....
~ 3 ~
in the presence of a catalyst such as p-toluenesulfonate to obtain di-alkyl vinyl ether (vii). This reaction is preferably conducted at oC to 30C for 0~5 to 24 hours.
The compound (vii) is treated with an azide such as S sodium a~ide in a solvent such as dimethylrormamide (DMF) to obtain azide (viii). This reaction is preferably carried out at 70C to 120C for 8 hours to six days.
The azide (viii) is reduced by a reducing agent such as sodium borohydride and Lindler catalyst/H2 in a solvent such as ethanol and isopropanol to give amine (ix). The reaction is carried out at a re~lux temperature ~or one to six days when sodium borohydride is used and at 0C to 30C for 2 to 24 hours at a hydrogen pressure of l to 4 atms. when Lindler catalyst/H2 is used.
The amine (ix) is reacted with an acyl halide in the presence of an amine such as pyridine and dimethylaminopyridine to obtain amide (x) or (xi). This reaction is preferably carried out at 0C to 30C for 0.5 to 24 hours. Alternatively, the amine (ix) dissolved in a solvent such as dichlorométhane is reacted with an aliphatic acid in the presence of ~-chloro-l-methylpyridinium iodide and tri-n-butylamine to obtain the amide (x) or (xi). This reaction is preferably carried out at a reflux temperature for 0.5 to 13 hours under an inert atmosphere.
~5 The amide (x) or (xi) is treated with pyridinium p~
toluenesulfonate, AMsERLIST A-15 (traclemark), etc. in a solvent such as methanol or dichloromethane to obtain ceramide (xii) or (I).
3~;6 Scheme l a R R
~ la1 Pvrldine ~6,~. OH
~ O ~ Ib)Aco~/~2o (i)(iii~ R = C13H27 ~v) R = Cl3H27 3-(CH2)13-P~3~(ph)3-Br~3(iv) R - Cl3H27 (ii)R'= SO2CH3 ~a) Ethanol Uvls Na metaperiodateR
Ib) PyridiniUm/ ~. CH OR' p-toluenesulfonate, 2 Ethyl vinyl ether OR' ,(vi) R = C lsH27 R'= H
(vii) R = C13H27 ~aI NaN3 ~b)I~aBH~
IC)Pyridine/C1sH31COCQ~ R' (a) NaN3 ~ CH2~EE
Ib)Lindler cat /~2 (c~ C23H47COOH OEE
(viii) R = Cl3H27 R'= N3 (ix) R=Cl3H27 R'= N H2 (x ) R = Cl 3 H2 7 R'= NHCOClsH
(xi) R = Cl3Hz 7 R'= ~HCOC23H~7 ~yridinium/
p-toluenesulfonate or Jl ~MBE~LTST A-lS~ HI~ R
H 27Cl 3 ~ (~H2()H
OH
(xii) R = C 15 H3l (I) R = C 2 3 ~I~7 The compound (I) thus obtained is treated with trityl chloride in pyridine to obtain trityl derivative (II) which is then treated with benzoyl chloride and dimethylaminopyridine to obtain trityl - benzoyl derivative (III) which is then treated with p-toluenesulfonic acid to remove the trityl group. Benzoyl ceramide (IV) is obtained. The compound (IV) can be obtained without the isolation of the compounds (II) and (III)~
c~
~-- N _, O ~
N O
N ~
S _ ~
N
CY ~
m R
~4 o~
,",o^~ < ~
(b) Synthesis of lactose derivatives and galactose derivatives Lactose and galactose derivatives which can be used in producing gangliosides of this invention can be produced by the processes as shown in Schemes 2a and 2b.
D-Lactose octaacetate (1) is treated with tri (n-butyl) tin allyloxide in a solvent such as ethylene chloride in the presence of a catalyst such as tin tetrachloride to obtain allyl derivative (2) which is then deacetylated by a conventional manner, e.g. by NaOMe/MeOH to obtain the deacetylated compound (3) which is further reacted with 2,2-dimethoxypropane and p-toluenesulfonic acid in acetone~DMF.
3', 4'-0-isopropylidene derivative (4) and 4', 6'-0 isopropylidene derivative (5) are obtained. With or without the isolation of the compounds (4) and (5), they are treated with benzyl bromide in DMF in the presence of NaH to obtain penta-O-benzyl derivatives (6) and (7) which are then treated with 90% acetic acid in water to remove isopropylidene group.
~o The compounds (8) and (9) are obtained.
The compound (4) is acetylated by acetic anhydride/pyridine, followed by the treatment with 90% C~3COOH
to obtain the compound (11).
Benzyl 3', 4'-0-isopropylidene lactose ~F) is treated in DMF with benzyl bromide in the presence of ~aH to obtain benzyl derivative (G) which is then treated with aqueous acetic acid solution to remove isopropylidene group. Hexa-O-benzyl derivative (H) is obtained.
. . .. . . . .. .
3~
Galactose derivatives which can be used in producing the sialic acid derivatives of this invention can be prepared as follows. Ben2yl galactoside (A) suspended in acetone is reacted with 2,2-dimethoxypropane in the presence of p-S toluenesulfonic acid to obtain 3,4-0-isopropylidene derivative (B~ which is then reacted with benzyl bromide in a solvent, e.g. DMF in the presence of NaH to convert it into tribenzyl derivative (C~ which is subsequently treated with aqueous acetic acid solution to remove isopropylidene group. The compound (D) is obtained~
o O ~
r O
C~ t~ Q
3~i~
. __ ~, ~
o~
Q ~t ~ ><O , Z ~ ¦ m I L~ m .c ~ Z C) m ~ ~ ~ ~ .
I ~ ~
93~
(c) Synthesis of sialic acid derivatives The compound (8), (9), (ll), (D) or (H) is reacted with N-acetyl neuraminic acid acetate methyl ester (E) which can be produced by the Kuhn's method, if necessary, followed by the removal o~ protective groups, to obtain sialic acid derivatives of this invention.
The reaction between the compound (8), (9), (ll~, (D) or (H) and the compound (F) is carried out in a solvent such as dichloromethane or 1,2-dichloroethane in the presence of a glycosidation catalyst such as Hg(CN)2~ HgBr2, molecular sieve (hereinafter referred to as MS), Ag2CO3, AgClO4, AgOSO2CF3, (CH3)3COSO2CF3, etc. at -20C to 150C for l to 120 hours.
Trisaccharide (12), (13), (14), (45), (46) or (47) or disaccharide (31), (32) or (33) is obtained. ~he removal o~
protective groups of these compounds gives the desired compounds, respectively. The compound (13) is acetylated ~y acetic anhydride-pyridine to give the compound (15) which is then treated with PdCl2 and AcONa/AcOH to remove allyl group.
The resulting compound (16) is treated with monochloroacetic anhydride-pyridine to obtain the compound (17). Catalytic reduction of the compound (17) and subsequent acetylation give the compound (18) which is then treated sodium acetate and thiourea in a sol~ent such as ethanol to give the compound (l9)- Treatment of the compound (l9) with trichloro-acetonitrile in the presence of Na~ in a solvent such asCH2Cl2 gives the compound (20). This compound is reacted with the ceramide (IV) in the presence of MS4A and ~F~-Et~O to give -the compound (21). Deacetylation and debenzoylation or the compound (21) give the compound (22) or ganglioside GL~3.
The compound (23) is treated in a similar manner to give a epimer of ganglioside G~3 (28) (see scheme 5).
Alternatively, ganglioside GM3 (the compound (22)) may be produced as follows:
The compound (47) is acetylated by acetic anhydride-pyridine to obtain the compound (52). Catalytic reduction of this compound is carried out in a solvent such as MeOH usi~g 10% Pd-C to remove benzyl groups. The resulting debenzylated compound (53) is acetylated by acetic anhydride-pyridine to obtain peracetate (54) which is then treated with hydrazinium acetate in a solvent such as DMF at 30C to 80C for 5 to 30 minutes to obtain the compound (19) which is subsequently reacted with trichloroacetonitrile in a solvent such as methylene chloride in the presence of NaH under ice-cooled condition. The resulting compound (20) is reacted with the ceramide ~IV) under an inert gas atmosphere such as argon in the presence of a glycosidation catalyst such as BF3-Et2O/molecular sieve 4A. The compound (21) thus obtained is treated in a conventional manner to remove acetyl and benzoyl groups and neutralized by AMBERLIST A-15 to obtain ganglioside GM3 (22).
Specific examples of the processes oE this invention are shown in Schemes 3 to 10 as set below.
6~
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~t - ~ ,k I I --¢
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U I o _ ~ ~ R~
NS o I (~ (~ IN
:" ~D 00 0 +
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~ S n < ~ 0 ~ ~ I O
O ~ Z _ ~ ) ~0 ~:) O
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O ~ W
~_~ r N ' 3 t~ ~ ~ I 8 0 1 (1) N n~ 1 ~ . I ) U~ _ N ~ ~ ~
--~ O ~ I / I
I O ~ I O
O ~' O ~ 3 c~
o ~
l rl ~ oz ~ CS 1 t~ C~ t`~ C`l 3~
O O
z ,~ t ~
~ ~3 ~ o a~3 I~ ~
O , ~ I r 6 ~:
:~ I I Q a) , + ~ â
3~i~
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o~ ~ o /
Q~
tn ~ O I t) ,~ u X
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~3 ~3 o~l ~J~ ~ ) o ~ ~ ~
~ -$-~ U ~ ~ ~ ~
~a ~ ~ .~, o o _ ~ I I I
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¢ ;~ ~N o D~ CO
O
IO
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6~
O
O
O O
g 8 o~
O ~ / 3 ~ 0 5 ~
O O
I
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~ ul~ ~
o~ 5 Z ~
~ ~ . .
`
' ~ 3 ~ )--' ~1 ~ t~ N
j~ + I .
r n _, ~ _ O O~ \ o >
v~ ~ o o~
o n n ~ o ~ o O$~o a a ~ C ~ . b~
S ~ Y ~o ~U
~ c ~ n 11 o ~
~ C C N ~ Z / 1 N
~ 1l o n ~ l c,~ O ~
O _ _ ~: _ c c r c~
~ 24 ~
All the compounds thus obtained (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), ~21), (23), (~4), (25), (26~, (27), (28), (B), (C), (D), (G), (~), (31), (32), (33), (34), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (50), (51), (52), (53) and (54) are novel.
The new compounds of the invention may be employed as tumor markers, di~ferentiation markers of cells having differentiation potency, or useful intermediates for the synthesis of various gangliosides.
The inventions of both the parent and divisional applications will now be illus-trated with reference to the following non-limiting examples.
An outline of Examples is as follows:
Reference Example 1 (I) ~ (~I) Tritylation 2 (II) -~ (III) Benzoylation 3 (III) , (IV) Detritylation
~ la1 Pvrldine ~6,~. OH
~ O ~ Ib)Aco~/~2o (i)(iii~ R = C13H27 ~v) R = Cl3H27 3-(CH2)13-P~3~(ph)3-Br~3(iv) R - Cl3H27 (ii)R'= SO2CH3 ~a) Ethanol Uvls Na metaperiodateR
Ib) PyridiniUm/ ~. CH OR' p-toluenesulfonate, 2 Ethyl vinyl ether OR' ,(vi) R = C lsH27 R'= H
(vii) R = C13H27 ~aI NaN3 ~b)I~aBH~
IC)Pyridine/C1sH31COCQ~ R' (a) NaN3 ~ CH2~EE
Ib)Lindler cat /~2 (c~ C23H47COOH OEE
(viii) R = Cl3H27 R'= N3 (ix) R=Cl3H27 R'= N H2 (x ) R = Cl 3 H2 7 R'= NHCOClsH
(xi) R = Cl3Hz 7 R'= ~HCOC23H~7 ~yridinium/
p-toluenesulfonate or Jl ~MBE~LTST A-lS~ HI~ R
H 27Cl 3 ~ (~H2()H
OH
(xii) R = C 15 H3l (I) R = C 2 3 ~I~7 The compound (I) thus obtained is treated with trityl chloride in pyridine to obtain trityl derivative (II) which is then treated with benzoyl chloride and dimethylaminopyridine to obtain trityl - benzoyl derivative (III) which is then treated with p-toluenesulfonic acid to remove the trityl group. Benzoyl ceramide (IV) is obtained. The compound (IV) can be obtained without the isolation of the compounds (II) and (III)~
c~
~-- N _, O ~
N O
N ~
S _ ~
N
CY ~
m R
~4 o~
,",o^~ < ~
(b) Synthesis of lactose derivatives and galactose derivatives Lactose and galactose derivatives which can be used in producing gangliosides of this invention can be produced by the processes as shown in Schemes 2a and 2b.
D-Lactose octaacetate (1) is treated with tri (n-butyl) tin allyloxide in a solvent such as ethylene chloride in the presence of a catalyst such as tin tetrachloride to obtain allyl derivative (2) which is then deacetylated by a conventional manner, e.g. by NaOMe/MeOH to obtain the deacetylated compound (3) which is further reacted with 2,2-dimethoxypropane and p-toluenesulfonic acid in acetone~DMF.
3', 4'-0-isopropylidene derivative (4) and 4', 6'-0 isopropylidene derivative (5) are obtained. With or without the isolation of the compounds (4) and (5), they are treated with benzyl bromide in DMF in the presence of NaH to obtain penta-O-benzyl derivatives (6) and (7) which are then treated with 90% acetic acid in water to remove isopropylidene group.
~o The compounds (8) and (9) are obtained.
The compound (4) is acetylated by acetic anhydride/pyridine, followed by the treatment with 90% C~3COOH
to obtain the compound (11).
Benzyl 3', 4'-0-isopropylidene lactose ~F) is treated in DMF with benzyl bromide in the presence of ~aH to obtain benzyl derivative (G) which is then treated with aqueous acetic acid solution to remove isopropylidene group. Hexa-O-benzyl derivative (H) is obtained.
. . .. . . . .. .
3~
Galactose derivatives which can be used in producing the sialic acid derivatives of this invention can be prepared as follows. Ben2yl galactoside (A) suspended in acetone is reacted with 2,2-dimethoxypropane in the presence of p-S toluenesulfonic acid to obtain 3,4-0-isopropylidene derivative (B~ which is then reacted with benzyl bromide in a solvent, e.g. DMF in the presence of NaH to convert it into tribenzyl derivative (C~ which is subsequently treated with aqueous acetic acid solution to remove isopropylidene group. The compound (D) is obtained~
o O ~
r O
C~ t~ Q
3~i~
. __ ~, ~
o~
Q ~t ~ ><O , Z ~ ¦ m I L~ m .c ~ Z C) m ~ ~ ~ ~ .
I ~ ~
93~
(c) Synthesis of sialic acid derivatives The compound (8), (9), (ll), (D) or (H) is reacted with N-acetyl neuraminic acid acetate methyl ester (E) which can be produced by the Kuhn's method, if necessary, followed by the removal o~ protective groups, to obtain sialic acid derivatives of this invention.
The reaction between the compound (8), (9), (ll~, (D) or (H) and the compound (F) is carried out in a solvent such as dichloromethane or 1,2-dichloroethane in the presence of a glycosidation catalyst such as Hg(CN)2~ HgBr2, molecular sieve (hereinafter referred to as MS), Ag2CO3, AgClO4, AgOSO2CF3, (CH3)3COSO2CF3, etc. at -20C to 150C for l to 120 hours.
Trisaccharide (12), (13), (14), (45), (46) or (47) or disaccharide (31), (32) or (33) is obtained. ~he removal o~
protective groups of these compounds gives the desired compounds, respectively. The compound (13) is acetylated ~y acetic anhydride-pyridine to give the compound (15) which is then treated with PdCl2 and AcONa/AcOH to remove allyl group.
The resulting compound (16) is treated with monochloroacetic anhydride-pyridine to obtain the compound (17). Catalytic reduction of the compound (17) and subsequent acetylation give the compound (18) which is then treated sodium acetate and thiourea in a sol~ent such as ethanol to give the compound (l9)- Treatment of the compound (l9) with trichloro-acetonitrile in the presence of Na~ in a solvent such asCH2Cl2 gives the compound (20). This compound is reacted with the ceramide (IV) in the presence of MS4A and ~F~-Et~O to give -the compound (21). Deacetylation and debenzoylation or the compound (21) give the compound (22) or ganglioside GL~3.
The compound (23) is treated in a similar manner to give a epimer of ganglioside G~3 (28) (see scheme 5).
Alternatively, ganglioside GM3 (the compound (22)) may be produced as follows:
The compound (47) is acetylated by acetic anhydride-pyridine to obtain the compound (52). Catalytic reduction of this compound is carried out in a solvent such as MeOH usi~g 10% Pd-C to remove benzyl groups. The resulting debenzylated compound (53) is acetylated by acetic anhydride-pyridine to obtain peracetate (54) which is then treated with hydrazinium acetate in a solvent such as DMF at 30C to 80C for 5 to 30 minutes to obtain the compound (19) which is subsequently reacted with trichloroacetonitrile in a solvent such as methylene chloride in the presence of NaH under ice-cooled condition. The resulting compound (20) is reacted with the ceramide ~IV) under an inert gas atmosphere such as argon in the presence of a glycosidation catalyst such as BF3-Et2O/molecular sieve 4A. The compound (21) thus obtained is treated in a conventional manner to remove acetyl and benzoyl groups and neutralized by AMBERLIST A-15 to obtain ganglioside GM3 (22).
Specific examples of the processes oE this invention are shown in Schemes 3 to 10 as set below.
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All the compounds thus obtained (4), (5), (6), (7), (8), (9), (10), (11), (12), (13), (14), (15), (16), (17), (18), (19), (20), ~21), (23), (~4), (25), (26~, (27), (28), (B), (C), (D), (G), (~), (31), (32), (33), (34), (35), (36), (37), (38), (39), (40), (41), (42), (43), (44), (45), (46), (47), (48), (49), (50), (51), (52), (53) and (54) are novel.
The new compounds of the invention may be employed as tumor markers, di~ferentiation markers of cells having differentiation potency, or useful intermediates for the synthesis of various gangliosides.
The inventions of both the parent and divisional applications will now be illus-trated with reference to the following non-limiting examples.
An outline of Examples is as follows:
Reference Example 1 (I) ~ (~I) Tritylation 2 (II) -~ (III) Benzoylation 3 (III) , (IV) Detritylation
4 (I) -~ (IV) Ben20ylation Example ~ (2) Allylation ~ (2) -~ (3) Deacetylation 3 (3) _~ (4)~(5) Isopropylidene deriv.
4 (4~+(5) ~ (6)~(7) Benzylation
4 (4~+(5) ~ (6)~(7) Benzylation
5 (6) ~ (8) Deisopropylidene
6 (7) -~ (9) Deisopropylidene
7 (4) ~ (10) Acetylation a (lo) ~ (11) Deisopropylidene 9 ~E)+(8) -~ (12)+(13)+(14) Glycosidation 10 (13) _~ (15) Acetylation 11 (15) -~ (16) Deacetylation 12 (16) -~ (17) Monochloroacetylation 13 (17) -~ (18) Debenzylation Acetylation 14 (18) -~ (19) Demonochloroacetylation 15 (19) -~ (20) -C(NH~-CC13 deriv~
16 (IV)+(20)-~ (21) Ceramide 17 (21) -~ (22) Deacetylation and - Debenzoylation 18 (E)+(ll) -~ (23) Glycos.idation 19 (23) -~ (24) Acetylation 20 (24) -~ (25) Deallylation 21 (25) -~ (26) -C(N~I)-CC13 deriv.
22 (26) -~ (27) Ceramide 23 (27) -~ (28) Deacetylation and Debenzoylation 24 (A) -~ (B) Isopropylidene derlv.
25 (B) ~~ (C) Benzylation 26 (C) ~~ (D) Deisopropylidene 27 (P~ ~~ (G) Benzylation 28 (G) -~ (H) Deisopropylidene 29 (E)+(D) -~ (31)~(32)+(33) Glycosidation 30 (31) -~ (34) Deacetylation 31 (31~ 35) Opening of lactone ring 32 (35) -~ (36) Debenzylation 33 (32) -~ (37) Deacetylation ~iS3;~
34 (37) -~ (38) Debenzylation 35 (33) -~ ~39) Deacetylation 36 (39) -~ (40) Debenzylation 37 (32) -~ (41) Acetylation 38 (41) -~ ~42) Debenzylation Acetylation 39 (33) -~ (43) Acetylation 40 (43) -~ (44) Debenzylation Acetylation 41 (E)+(~ (45)+(46)~(47) Glycosidation 42 (46) -~ (4S) Deacetylation 43 (48) -~ (49) Debenzylation 44 (47) -~ tS0) ~eacetylation 45 (50) -~ (51) Debenzylation 46 (47) -~ (52) Acetylation 47 (52) -~ (53) -~ (54) Debenzylation Acetylation 48 (54) -~ (19) Deacetylation 49 (19) -~ (20) -C(NH)-CC13 deriv.
50 (20) -~ (21) Ceramide 51 (21) -~ (22) Deacetylation and Deben20ylation Reference Example 1 The compound (I) (325 mg, 0.5 mmol) was dissolved in dry pyridine (5 ml). Trityl chloride (TrCl) (278 mg, 1.0 mmol) was added. The mixture was stirred at room temperature for 24 hours and further at 55C for 4 hours. The solvent was evaporated in vacuo. The residue was dissolved in chloroform, washed with water, dried on MgSO4 and concentrated in vacuo.
The resulting residue was subjected to column chromatography (Wakogel C-300, 50g, hexane-ethylacetate- 4:i) to glve the compound (II) (297 mg, 66.6~).
(The compound (II)) (~)22 _o 94 (C = 0.96, CHCi3) Analysis:
Calcd. C,82.09 H,10.96 N,1.57 for (C~lHg7NO3) Found C,82.00 H,11.17 N,1.49 Rf 0.34 (hexane-ethylacetate 4:1) Reference Example 2 The compound (II) (224 mg, 0.25 mmol) was dissolved in pyridine (2 ml). Benzoyl chloride (70 mg, 0.5 mmol) and dimethylaminopyridine (30 mg) were added and the mixture was stirred at room temperature for 24 hours and at 55C for one hour, and concentrated in vacuo. The residue was dissolved in chloroform, washed with water, dried on MgSO4 and concentrated in vacuo. The resulting residue was subjected to column chromatography (Robar column Li Chroprep Si60 Gro~e A, hexane-ethylacetate = 4:1) to give the compound (III) (183 mg, 88.2%) and the compound (II) (38 mg~
(The compound (III)) (~)19 5.93 (C~C13, C = 0.86) Analysis:
Calcd. C,81.96 H,10.22 N,1.41 (for C68HlOlN04) Found C,82.01 H,10.17 N,1.36 Rf 0.34 (hexane: ethylacetate 4:1) Reference Example 3 The compound (III) (163 mg, 0.164 mmol) was dissolved in CH2C12 ~5 ml) and MeOH (2.5 ml). Paratoluenesulfonic acid monohydrate (16 mg) was added. The mixture was stirred at room temperature for 24 hours and concentrated in vacuo~ The residue was subjected to column chromatography (WAKOGEI, C-300~*
g) and eluted with 3% methanol-containing chloroform to give the compound (IV) (104 mg, 84.3%).
(The compound (IV)) (~)23 +16 5o (CHC13, C = 1.1 ) Analysis:
Calcd. C,78.03 H,11.63 N,1.86 2~ (~or C49H87N4) Found C,77.85 H,11.54 N,1.84 Reference Example 4 The compound (I) ~975 mg, 1.5 mmol) was dissolved in pyridine (15 ml). TrCl (625 mg, 2.25 mmol) was added~ The mixture was stirred at 55C for 4 hours and at room temperature for 24 hours. Benzoylchloride (315 mg, 2.25 mmol) * Trademark and dimethylaminopyridine ~1~3 mg, 1.5 mmol) were added and stirred at room temperature for 5 hours. The reaction mixture was concentrated in vacuo and the residue was dissolved in ethyl acetate. The solution was washed with water, dried on MgS04 and concentrated in vacuo. The residue was dissolved in CH2C12 (10 ml). Para-toluene sulfonic acid (100 mg) was added and stirred at room temperature for 24 hoursO The reaction mixture was concentrated in vacuo. The residue was dissolved in ethyl acetate, washed with a saturated solution of sodiu~
bicarbonate in diluted hydrochloric acid and then a saturated solution of sodium chloride in water, dried on MgSO4 and concentrated in vacuo. The residue was subjected to column chromatography (WAKOGEL C-300~ 10 g) and eluted with 2 methanol-containing chloroform to give the compound (IV) (801.6 mg, 70.9%).
Example 1 n-Bu3Sn-O-C~2C~C~2 (80.7g, 0.23 mol) was dissolved i~
ethylene chloride (500 ml). Tin tetrachloride (31.0 ml) was added under ice-cooled condition. To this solution, there was added 250 ml of a solution of D-lactose octaacetate (1) (1429v 0.21 mol) in ethylene chloride~ The reaction mixture W2S
stirred at room temperature for 2.5 hours and then poured into a saturated KF solution. Insoluble products precipitated were filtered. The filtrate was washed with a saturated NaCl solution, dried on MgSO4 and concentrated in vacuo. The residue was subjected to column chromatography (silica gel kg) and eluted with toluene-ethylacetate (1:1) to give the compound (2) (85.8 g, 57.8~).
(The compound (2)) NMR 90MHz CHC13 Sppm (TMS) 1.96, 2.04, 2.12, 2.06 (OCOCH3 x 7) 5.64 ~ 6.00 lHm -CH2-CH=~H2 Example 2 The compound (2) (R5.8 9, 0.127 mol) was dissolved in methanol (600 ml~. N-NaOCH3 solution (10 ml) was added and stirred at room temperature for 2 hours. Precipitated crystals were collected by filtration. The compound (3) (41.8 g, 86.2%) was obtained.
(The compound (3)) Analysis:
Calcd. C,47.12 H,6.85 (for C15H26ll) Found C,46.92 H,7.01 Example 3 The compound (3) (44.4 g, 0.116 mol) was suspended in acetone (550 ml) and DMF (550 ml). Para-toluene sulfonic acid (2.32 g) and 2,2-dimethoxypropane (25.4 g) were added and stirred at room temperature for 2 days. Triethylamine (10 ml) was added to the reaction mixture and then, concentrated in vacuo. Ethyl acetate was added to the residue to precipita~e ~93~i~
and collect a mixture of the compounds (4) and (5) (41.4 g, 91.1%)~ Rf 0.56 (CHC13: MeOH 5 : 1) (The compound (4)) CMR D20 (dioxane) 25.952, 27.740, -CH3, 101.695 102.727 anomeric carbon 118.981 =CH2 134.096 -CH=
(The compound (5)) C~ D~O (dioxane) 18.421, 28.770, -C~13, 101.695 103~321 anomeric carbon 119.090 =CH2 134.043 -CH=
Example 4 The mixture of the compounds (4) and (5) (2.2 g, 5.2 mmol) was dissolved in DMF (50 ml). NaH (50% in oil) (1.87 g) was added and stirred at room temperature for 30 minutes.
Under ice-cooled condition, there was added benzylbromide (6.67 g, 39.0 mmol) and stirred at room temperature for 24 hours. A small amount of methanol was added under ice-cooled condition. The reaction mixture was then stirred for 30 minutes and concentrated in vacuo. The residue was dissolved in ethyl acetate~ washed with water, dried on MgSO4 and concentrated in vacuo. The resultlng residue was subjected to silica gel column chromatography (WAKOGEL C-300, 120 9) to give the compound (6) (2.47 g, 56.9%) and the compound (7) (1.66 9, 38.2%).
(The compound (6)) S Analysis:
Calcd. C,72.91 H;6.92 (for C53H60ll) Found C,72.79 H,6.87 (The compound (7)) Analysis:
Calcd. C,72.91 - H,6.92 (for Cs3H60oll) Found C,72.95 H,6.93 Example 5 15The compound (6) (2.47 g, 2.8 mmol) was dissolved in -~ 60 ml of 90% acetic acid solution in water and stirred at 60C
for 3 hours. The reaction mixture was concentrated in vacuo.
The residue was recrystallized from ether - hexane to give the compound (8) (1.24 g, 52.6~) as needle crystals.
~The compound (8)) m.p. 112 - 3C
(~)20 +l9.o (CHC13/ C - 1.50) Analysis:
Calcd. C,72.09 H,6.78 ~fo~ C50H56ll) Found C,72.10 H,6.80 Example 6 The compound (7) (1.66g, 1.9 mmol) was dissolved in 30 ml of 90% acetic acid solution in water and stirred at 60C
for 3 hours. The reaction mixture was concentrated in vacuo.
The residue was recrystallized from chloroform-hexane to give the compound (9) ~1.01g, 57.6%).
(The compound (9)) m.p. 159 - 160C
(~)20 +26.3 (CHC13, C - 1.07) Analysis: `
Calcd. C,72.09 H,6.78 (Eor C50H56ll) Found C,72.06 H,6.79 Example 7 -' The compound (4) (2.11g, 5.0 mmol) was dissolved in acetic anhydride (15 ml) and pyridine (li ml). The reaction mixture was stirred at room temperature for 24 hours and concentrated in vacuo. The residue was subjected to silicagel column chromatography ~wAKOGEL C-300, 250g) and eluted with 3.5% MeOH-containing chloroform to give the compound (10) (2.31g, 73.1%).
(The compound (10)) (~)22 ~6.70 (CHC13, C = 1.15) Analysis: (for C28H4016 1/2 H2O) Calcd. C, 52.41 H, 6.44 Found. C, 52.31 H, 6.24 9;~
Example 8 The compound (10) tl5.5g, 23~9 mmol~ was dissolved in 90~ CF3COOH solution and stirred at room temperature for 20 minutes. The reaction mixture was conce~trated in vacuo. The residue was dissolved in ethyl acetate. The solution was washed with NaHCO3 solution and -then saturated NaCl solution, dried on MgSO4 and concentrated in vacuo. The residue was subjected to silicagel column chromatography ~KOGEL C-300, 300g) and eluted with ~% MeOH-containing chloroform to give the compound (11) (11.0g).
.
(The compound (11)) (~)D2 -3.380 CHC13, C = 1.42 NMR CDC13 ~ppm (TMS) 2.04, 2.12, OCOCH3 x 5 5.64 ~ 6.00 1 Hm CH=CH2 -~ CMR 25 MH~ CDC13 ppm 20.664, CH3CO-, 99.373 9 100.933, anomeric carbon, 117.552,=CH2, 113O343, -CH=CH2 169.603, 170.626, 170.821, 171.016, 171~211, -COCH3 Example 9 To activated molecular sieves 4A (2.2 g~, there were added Hg(CN)2 (504 mg), HgBr2 (720 mg), the compound (8) ~833 mg, 1.0 mmol) and ethylene chloride (3 ml) and the mixture was stirred for one hour under argon atmosphere. To this solution, there was added a solution of the compound (E) (506 mg, 1.0 mmol) prepared by the Kuhn method in ~ ~ ~9;~3~
dichloroethane (3 ml). The mixture was stirred at room temperature for 24 hours and then filtered. The insolubles were washed with ethyl acetate. The filtrate and th~ washings were combined, washed with water, dried on MgS04 and concentrated in vacuo. The residue W2S subjected to silioagel column chromatography (WAKOGEI. C-300, 80 g) and eluted with toluene-ethylacetate (1:2) to give the compound (12) (99 mg, 7.6%~, the compound (13) (91 mg, 7.0%) and the compound (14) (37 mg, 2.8~).
(The compound (14)) N~ 400MHz CDC13 (TMS)ppm 1.750 lHtJ=12.94, (H-3 ax) 1.991, 2.063, 2.075, 2.101, s, OCOCH3, 2.409, lH, dd, J=12.94, 4.63(H-3 eq), 3.685, 3H, s, OCH3, 7.183 ~ 7.447 25H
(aromatic proton) (The compound (13)) NMR 400MHz CDC13 (TMS)ppm 1.871, 1.882, 1.984, 2.014, 2.090, s, OCOC~3, 2.500, lH dd J=12.94, 4.64(H-3 eq), 3 753, 3HS, OCH3, 7.207 ~ 7.413 25H (aromatic proton) (The compound (12)) NMR 400MHz CDC13 TMS ppm 1.720, 1.979, 1.988, 2.08~, 2.122, s, OCOC~3, 2.528, lH dd J=13.67, 4.39(H-3 eq), 3.663, 3H, s, OCH3, 7.223 ~- 7.43,0 25H (aromatic proton) Example 10 The compound (13) (605 mg, 0.57 mmol) was dissolved in pyridine (10 ml) and acetic anhydride (10 ml). To this soLution, there was added dimethylamino-pyridine (70 mg). The mixture was stirred at room temperature for 24 hours and then concentrated in vacuo. The residue was subjected to silicagel column chromatography (WAKOGEL C-300, 80 g) and eluted with lS toluene-ethylacetate (1:2) to give the compound (15) (452 my, 7~.6%).
(The compound (15)) NMR 90MHz CDC13 ~ppm 1076, 1.85, 1.96, 2.00, 2.01, 2.08 -OCOCH3, 3.83, s, OCH3, 7.06 ~ 7~40, aromatic proton Example 11 The compound (15) (410 mg, 0.34 mmol) was dissolved in 90~ AcOH. To this solution, there were added AcONa ~500 mg) and palladium chloride (540 mg). The mixture was stirred for 2 hours in an ultrasonic stirrer and then concentrated in 33~
vacuo. The residue was dissolved in ethylacetate. The solution was washed with water, dried on MgSO4 and concentrated in vacuo. The residue was subjected to silicagel column chromatography (WAKOGEL C-300, 509) and eluted wlth 10~
5 l~eOH-containing isopropyl ether to gi-ve the compound (lo) (353 mg, 89%).
(The compound (16)) Analysis:
Calcd. C, 63.53 H, 5.95, N, 1.07 (for C69H77N024) Found C, 63,12 H, 5.99, N, 0.98 Example 12 The compound (16) (312 mg, 0.24 mmol) was dissolved in pyridine (7 ml). Dry monochloroacetic acid (312 mg) was added and stirred at room temperature for one hour, to which ethylacetate was added to dilute it. The solution was washed with saturated NaHCO3 solution, diluted HCl and saturated NaCl solution, dried on MgSO~ and concentrated in vacuo. The residue was subjected to silicagel column chromatography (WAKOGEL C-300, 409) and eluted with 10% MeOff-containing isopropyl ether to give the compound (17) (257 mg, 77.6%).
(The compound (17)) NMR CDC13 ~ppm TMS 1.80, 1~86, 1.97, 2.03, 2.04, 2.06, OCOCH3~ 2.60 ~x~
lH m H-3 eq 3.84, OCH3 7.10 ~ 7.40 (aromatic proton) Example 13 The compound (17) (91 mg, 0.065 mmol) was dissolved in methanol (3 ml). 10% Pd/C (50 mg) was added and catalytic reduction was carried out at room temperature for 24 hours.
The reaction mixture was filtered to remove Pd/C and the filtrate was concentrated in vacuo. To the residue, there were added acetic anhydride (1.0 ml) pyridine ~1.0 ml)~ The mixture was stirred at room ~temperature for 2.5 hours and concentrated in vacuo. The residue was subjected to silicagel column chromatography ~WAKOGEL C-300, 10 g) and eluted with 4~
MeOH-containing chloroform to give the compound ~18) (61 mg,
16 (IV)+(20)-~ (21) Ceramide 17 (21) -~ (22) Deacetylation and - Debenzoylation 18 (E)+(ll) -~ (23) Glycos.idation 19 (23) -~ (24) Acetylation 20 (24) -~ (25) Deallylation 21 (25) -~ (26) -C(N~I)-CC13 deriv.
22 (26) -~ (27) Ceramide 23 (27) -~ (28) Deacetylation and Debenzoylation 24 (A) -~ (B) Isopropylidene derlv.
25 (B) ~~ (C) Benzylation 26 (C) ~~ (D) Deisopropylidene 27 (P~ ~~ (G) Benzylation 28 (G) -~ (H) Deisopropylidene 29 (E)+(D) -~ (31)~(32)+(33) Glycosidation 30 (31) -~ (34) Deacetylation 31 (31~ 35) Opening of lactone ring 32 (35) -~ (36) Debenzylation 33 (32) -~ (37) Deacetylation ~iS3;~
34 (37) -~ (38) Debenzylation 35 (33) -~ ~39) Deacetylation 36 (39) -~ (40) Debenzylation 37 (32) -~ (41) Acetylation 38 (41) -~ ~42) Debenzylation Acetylation 39 (33) -~ (43) Acetylation 40 (43) -~ (44) Debenzylation Acetylation 41 (E)+(~ (45)+(46)~(47) Glycosidation 42 (46) -~ (4S) Deacetylation 43 (48) -~ (49) Debenzylation 44 (47) -~ tS0) ~eacetylation 45 (50) -~ (51) Debenzylation 46 (47) -~ (52) Acetylation 47 (52) -~ (53) -~ (54) Debenzylation Acetylation 48 (54) -~ (19) Deacetylation 49 (19) -~ (20) -C(NH)-CC13 deriv.
50 (20) -~ (21) Ceramide 51 (21) -~ (22) Deacetylation and Deben20ylation Reference Example 1 The compound (I) (325 mg, 0.5 mmol) was dissolved in dry pyridine (5 ml). Trityl chloride (TrCl) (278 mg, 1.0 mmol) was added. The mixture was stirred at room temperature for 24 hours and further at 55C for 4 hours. The solvent was evaporated in vacuo. The residue was dissolved in chloroform, washed with water, dried on MgSO4 and concentrated in vacuo.
The resulting residue was subjected to column chromatography (Wakogel C-300, 50g, hexane-ethylacetate- 4:i) to glve the compound (II) (297 mg, 66.6~).
(The compound (II)) (~)22 _o 94 (C = 0.96, CHCi3) Analysis:
Calcd. C,82.09 H,10.96 N,1.57 for (C~lHg7NO3) Found C,82.00 H,11.17 N,1.49 Rf 0.34 (hexane-ethylacetate 4:1) Reference Example 2 The compound (II) (224 mg, 0.25 mmol) was dissolved in pyridine (2 ml). Benzoyl chloride (70 mg, 0.5 mmol) and dimethylaminopyridine (30 mg) were added and the mixture was stirred at room temperature for 24 hours and at 55C for one hour, and concentrated in vacuo. The residue was dissolved in chloroform, washed with water, dried on MgSO4 and concentrated in vacuo. The resulting residue was subjected to column chromatography (Robar column Li Chroprep Si60 Gro~e A, hexane-ethylacetate = 4:1) to give the compound (III) (183 mg, 88.2%) and the compound (II) (38 mg~
(The compound (III)) (~)19 5.93 (C~C13, C = 0.86) Analysis:
Calcd. C,81.96 H,10.22 N,1.41 (for C68HlOlN04) Found C,82.01 H,10.17 N,1.36 Rf 0.34 (hexane: ethylacetate 4:1) Reference Example 3 The compound (III) (163 mg, 0.164 mmol) was dissolved in CH2C12 ~5 ml) and MeOH (2.5 ml). Paratoluenesulfonic acid monohydrate (16 mg) was added. The mixture was stirred at room temperature for 24 hours and concentrated in vacuo~ The residue was subjected to column chromatography (WAKOGEI, C-300~*
g) and eluted with 3% methanol-containing chloroform to give the compound (IV) (104 mg, 84.3%).
(The compound (IV)) (~)23 +16 5o (CHC13, C = 1.1 ) Analysis:
Calcd. C,78.03 H,11.63 N,1.86 2~ (~or C49H87N4) Found C,77.85 H,11.54 N,1.84 Reference Example 4 The compound (I) ~975 mg, 1.5 mmol) was dissolved in pyridine (15 ml). TrCl (625 mg, 2.25 mmol) was added~ The mixture was stirred at 55C for 4 hours and at room temperature for 24 hours. Benzoylchloride (315 mg, 2.25 mmol) * Trademark and dimethylaminopyridine ~1~3 mg, 1.5 mmol) were added and stirred at room temperature for 5 hours. The reaction mixture was concentrated in vacuo and the residue was dissolved in ethyl acetate. The solution was washed with water, dried on MgS04 and concentrated in vacuo. The residue was dissolved in CH2C12 (10 ml). Para-toluene sulfonic acid (100 mg) was added and stirred at room temperature for 24 hoursO The reaction mixture was concentrated in vacuo. The residue was dissolved in ethyl acetate, washed with a saturated solution of sodiu~
bicarbonate in diluted hydrochloric acid and then a saturated solution of sodium chloride in water, dried on MgSO4 and concentrated in vacuo. The residue was subjected to column chromatography (WAKOGEL C-300~ 10 g) and eluted with 2 methanol-containing chloroform to give the compound (IV) (801.6 mg, 70.9%).
Example 1 n-Bu3Sn-O-C~2C~C~2 (80.7g, 0.23 mol) was dissolved i~
ethylene chloride (500 ml). Tin tetrachloride (31.0 ml) was added under ice-cooled condition. To this solution, there was added 250 ml of a solution of D-lactose octaacetate (1) (1429v 0.21 mol) in ethylene chloride~ The reaction mixture W2S
stirred at room temperature for 2.5 hours and then poured into a saturated KF solution. Insoluble products precipitated were filtered. The filtrate was washed with a saturated NaCl solution, dried on MgSO4 and concentrated in vacuo. The residue was subjected to column chromatography (silica gel kg) and eluted with toluene-ethylacetate (1:1) to give the compound (2) (85.8 g, 57.8~).
(The compound (2)) NMR 90MHz CHC13 Sppm (TMS) 1.96, 2.04, 2.12, 2.06 (OCOCH3 x 7) 5.64 ~ 6.00 lHm -CH2-CH=~H2 Example 2 The compound (2) (R5.8 9, 0.127 mol) was dissolved in methanol (600 ml~. N-NaOCH3 solution (10 ml) was added and stirred at room temperature for 2 hours. Precipitated crystals were collected by filtration. The compound (3) (41.8 g, 86.2%) was obtained.
(The compound (3)) Analysis:
Calcd. C,47.12 H,6.85 (for C15H26ll) Found C,46.92 H,7.01 Example 3 The compound (3) (44.4 g, 0.116 mol) was suspended in acetone (550 ml) and DMF (550 ml). Para-toluene sulfonic acid (2.32 g) and 2,2-dimethoxypropane (25.4 g) were added and stirred at room temperature for 2 days. Triethylamine (10 ml) was added to the reaction mixture and then, concentrated in vacuo. Ethyl acetate was added to the residue to precipita~e ~93~i~
and collect a mixture of the compounds (4) and (5) (41.4 g, 91.1%)~ Rf 0.56 (CHC13: MeOH 5 : 1) (The compound (4)) CMR D20 (dioxane) 25.952, 27.740, -CH3, 101.695 102.727 anomeric carbon 118.981 =CH2 134.096 -CH=
(The compound (5)) C~ D~O (dioxane) 18.421, 28.770, -C~13, 101.695 103~321 anomeric carbon 119.090 =CH2 134.043 -CH=
Example 4 The mixture of the compounds (4) and (5) (2.2 g, 5.2 mmol) was dissolved in DMF (50 ml). NaH (50% in oil) (1.87 g) was added and stirred at room temperature for 30 minutes.
Under ice-cooled condition, there was added benzylbromide (6.67 g, 39.0 mmol) and stirred at room temperature for 24 hours. A small amount of methanol was added under ice-cooled condition. The reaction mixture was then stirred for 30 minutes and concentrated in vacuo. The residue was dissolved in ethyl acetate~ washed with water, dried on MgSO4 and concentrated in vacuo. The resultlng residue was subjected to silica gel column chromatography (WAKOGEL C-300, 120 9) to give the compound (6) (2.47 g, 56.9%) and the compound (7) (1.66 9, 38.2%).
(The compound (6)) S Analysis:
Calcd. C,72.91 H;6.92 (for C53H60ll) Found C,72.79 H,6.87 (The compound (7)) Analysis:
Calcd. C,72.91 - H,6.92 (for Cs3H60oll) Found C,72.95 H,6.93 Example 5 15The compound (6) (2.47 g, 2.8 mmol) was dissolved in -~ 60 ml of 90% acetic acid solution in water and stirred at 60C
for 3 hours. The reaction mixture was concentrated in vacuo.
The residue was recrystallized from ether - hexane to give the compound (8) (1.24 g, 52.6~) as needle crystals.
~The compound (8)) m.p. 112 - 3C
(~)20 +l9.o (CHC13/ C - 1.50) Analysis:
Calcd. C,72.09 H,6.78 ~fo~ C50H56ll) Found C,72.10 H,6.80 Example 6 The compound (7) (1.66g, 1.9 mmol) was dissolved in 30 ml of 90% acetic acid solution in water and stirred at 60C
for 3 hours. The reaction mixture was concentrated in vacuo.
The residue was recrystallized from chloroform-hexane to give the compound (9) ~1.01g, 57.6%).
(The compound (9)) m.p. 159 - 160C
(~)20 +26.3 (CHC13, C - 1.07) Analysis: `
Calcd. C,72.09 H,6.78 (Eor C50H56ll) Found C,72.06 H,6.79 Example 7 -' The compound (4) (2.11g, 5.0 mmol) was dissolved in acetic anhydride (15 ml) and pyridine (li ml). The reaction mixture was stirred at room temperature for 24 hours and concentrated in vacuo. The residue was subjected to silicagel column chromatography ~wAKOGEL C-300, 250g) and eluted with 3.5% MeOH-containing chloroform to give the compound (10) (2.31g, 73.1%).
(The compound (10)) (~)22 ~6.70 (CHC13, C = 1.15) Analysis: (for C28H4016 1/2 H2O) Calcd. C, 52.41 H, 6.44 Found. C, 52.31 H, 6.24 9;~
Example 8 The compound (10) tl5.5g, 23~9 mmol~ was dissolved in 90~ CF3COOH solution and stirred at room temperature for 20 minutes. The reaction mixture was conce~trated in vacuo. The residue was dissolved in ethyl acetate. The solution was washed with NaHCO3 solution and -then saturated NaCl solution, dried on MgSO4 and concentrated in vacuo. The residue was subjected to silicagel column chromatography ~KOGEL C-300, 300g) and eluted with ~% MeOH-containing chloroform to give the compound (11) (11.0g).
.
(The compound (11)) (~)D2 -3.380 CHC13, C = 1.42 NMR CDC13 ~ppm (TMS) 2.04, 2.12, OCOCH3 x 5 5.64 ~ 6.00 1 Hm CH=CH2 -~ CMR 25 MH~ CDC13 ppm 20.664, CH3CO-, 99.373 9 100.933, anomeric carbon, 117.552,=CH2, 113O343, -CH=CH2 169.603, 170.626, 170.821, 171.016, 171~211, -COCH3 Example 9 To activated molecular sieves 4A (2.2 g~, there were added Hg(CN)2 (504 mg), HgBr2 (720 mg), the compound (8) ~833 mg, 1.0 mmol) and ethylene chloride (3 ml) and the mixture was stirred for one hour under argon atmosphere. To this solution, there was added a solution of the compound (E) (506 mg, 1.0 mmol) prepared by the Kuhn method in ~ ~ ~9;~3~
dichloroethane (3 ml). The mixture was stirred at room temperature for 24 hours and then filtered. The insolubles were washed with ethyl acetate. The filtrate and th~ washings were combined, washed with water, dried on MgS04 and concentrated in vacuo. The residue W2S subjected to silioagel column chromatography (WAKOGEI. C-300, 80 g) and eluted with toluene-ethylacetate (1:2) to give the compound (12) (99 mg, 7.6%~, the compound (13) (91 mg, 7.0%) and the compound (14) (37 mg, 2.8~).
(The compound (14)) N~ 400MHz CDC13 (TMS)ppm 1.750 lHtJ=12.94, (H-3 ax) 1.991, 2.063, 2.075, 2.101, s, OCOCH3, 2.409, lH, dd, J=12.94, 4.63(H-3 eq), 3.685, 3H, s, OCH3, 7.183 ~ 7.447 25H
(aromatic proton) (The compound (13)) NMR 400MHz CDC13 (TMS)ppm 1.871, 1.882, 1.984, 2.014, 2.090, s, OCOC~3, 2.500, lH dd J=12.94, 4.64(H-3 eq), 3 753, 3HS, OCH3, 7.207 ~ 7.413 25H (aromatic proton) (The compound (12)) NMR 400MHz CDC13 TMS ppm 1.720, 1.979, 1.988, 2.08~, 2.122, s, OCOC~3, 2.528, lH dd J=13.67, 4.39(H-3 eq), 3.663, 3H, s, OCH3, 7.223 ~- 7.43,0 25H (aromatic proton) Example 10 The compound (13) (605 mg, 0.57 mmol) was dissolved in pyridine (10 ml) and acetic anhydride (10 ml). To this soLution, there was added dimethylamino-pyridine (70 mg). The mixture was stirred at room temperature for 24 hours and then concentrated in vacuo. The residue was subjected to silicagel column chromatography (WAKOGEL C-300, 80 g) and eluted with lS toluene-ethylacetate (1:2) to give the compound (15) (452 my, 7~.6%).
(The compound (15)) NMR 90MHz CDC13 ~ppm 1076, 1.85, 1.96, 2.00, 2.01, 2.08 -OCOCH3, 3.83, s, OCH3, 7.06 ~ 7~40, aromatic proton Example 11 The compound (15) (410 mg, 0.34 mmol) was dissolved in 90~ AcOH. To this solution, there were added AcONa ~500 mg) and palladium chloride (540 mg). The mixture was stirred for 2 hours in an ultrasonic stirrer and then concentrated in 33~
vacuo. The residue was dissolved in ethylacetate. The solution was washed with water, dried on MgSO4 and concentrated in vacuo. The residue was subjected to silicagel column chromatography (WAKOGEL C-300, 509) and eluted wlth 10~
5 l~eOH-containing isopropyl ether to gi-ve the compound (lo) (353 mg, 89%).
(The compound (16)) Analysis:
Calcd. C, 63.53 H, 5.95, N, 1.07 (for C69H77N024) Found C, 63,12 H, 5.99, N, 0.98 Example 12 The compound (16) (312 mg, 0.24 mmol) was dissolved in pyridine (7 ml). Dry monochloroacetic acid (312 mg) was added and stirred at room temperature for one hour, to which ethylacetate was added to dilute it. The solution was washed with saturated NaHCO3 solution, diluted HCl and saturated NaCl solution, dried on MgSO~ and concentrated in vacuo. The residue was subjected to silicagel column chromatography (WAKOGEL C-300, 409) and eluted with 10% MeOff-containing isopropyl ether to give the compound (17) (257 mg, 77.6%).
(The compound (17)) NMR CDC13 ~ppm TMS 1.80, 1~86, 1.97, 2.03, 2.04, 2.06, OCOCH3~ 2.60 ~x~
lH m H-3 eq 3.84, OCH3 7.10 ~ 7.40 (aromatic proton) Example 13 The compound (17) (91 mg, 0.065 mmol) was dissolved in methanol (3 ml). 10% Pd/C (50 mg) was added and catalytic reduction was carried out at room temperature for 24 hours.
The reaction mixture was filtered to remove Pd/C and the filtrate was concentrated in vacuo. To the residue, there were added acetic anhydride (1.0 ml) pyridine ~1.0 ml)~ The mixture was stirred at room ~temperature for 2.5 hours and concentrated in vacuo. The residue was subjected to silicagel column chromatography ~WAKOGEL C-300, 10 g) and eluted with 4~
MeOH-containing chloroform to give the compound ~18) (61 mg,
8`1%).
.~
(The compound (18)) NMR (90MHz, CDC13 Sppm) 1.88 ~ 2.04 OCOCH3 x 11 3.86, s, 3H, ~OCH3 Example 14 The compound (18) (61 mg, 0.053 mmol) was dissolved in ethanol. To this solution, there were added thiourea (20 mg) and sodium acetate (4 mg). The mixture was heated and refluxed for 5 hours. The reaction mixture was concentrated in vacuo. The residue was dissolved in 4% MeOH-containing 6~
chloroform and subjected to silicagel column chromatography (WAKOGEL C-300, 10 g) to give the compound (19) (21 mg). The compound (18) (40 mg) was recovered.
(The compound (19)) Rf 0.42 (5% MeOH-containing chloroform) N~R 90MHz 1.88, 2.02, 2.10, 2.16, 2.24, 5 OCOCH3, 3.85, s, OCH3 Example 15 The compound (19) (20-mg, 0.0187 mmol) was dissolved in methylene chloride t0.5 ml) and trichloroacetonitrile (13.5 mg) was added. To this, there was added NaH (60% in oil) (lo O
mg) under ice-cooled condition and stirred for 2 hours. The mixture was concentrated in vacuo. The residue was subjected to silic~gel column chromatography (wAKOGEL C-300, 5 g) and eluted with ethyl acetate to give the compound (20) (10.0 mg)~
(The compound (20)) Rf 0.33 (ethyl acetate) NMR 90MHz ~ ~ppm) 8.60, NH, 5.50, d J=4.0 anomeric proton, 3.88, s, OCH3, 2.60, lH, dd, J=4.0, J=13.0 (in CDC13) Example 16 To activated molecular sieves 4A (2009), there were added the compound (20) (10 mg, 0.009 mmol), the compound (IV) (12 mg, 0.018 mmol) and CHC13 (0.2 ml). To this mixture, there was added BF3.Et2O (2.0 ~ 1) with stirring~ The mixture was stirred at room temperature for 24 hours. Insolubles were filtered and washed with CHC13 The filtrate and the washings were combined and concentrated in vacuo~ The residue was subjected to silicagel column chromatography (C-300, 10 g) and eluted with 4% MeOH-containing~`chloroform to give the compound (21) (4 mg).
(The compound (21)) Rf 0.29 3% MeOH-containing chloroform Ana'lysis:
Calcd. C,61.91 H,8.16 N,1.56 (for C93~146N2032) Found~ C,62.41 H,8.Ql ~,1.48 Example 17 The compound (21) (4.0 mg) was dissolved in a mixed solvent of methanol and THF (1:1) (0.5 ml). N-NaOMe (0~1 ml) was added and stirred at room temperature for 2 hours. The mixture was concentrated. To the residue, there were added water (0.1 ml) and MeOH-THF (1:1) (0.5 ml) and stirred at room temperature for 2 hours. AMBERLIST A-15 was added ' : ' , to neutralize the mixture and then filtered. AMBERLIST A-15 was washed with methanol. The solution combined w2S
concentrated in vacuo. The residue was washed with ether and dried to give the compound t22) (ganglioside GM3) (1.7 mg).
(The compound (22)) Analysis:
Calcd. C,60.64 H, 9.32 N, 2.18 (for C6sHllgN2o2lNa) Found C, 60.21 H, 8.97 N, 2.10 Example 18 To activated molecular sieves 4A (20 g), there were added Hg(CN)2 (5.04 g), HgBr2 (7.~0 g), the compound (11) (5.~3g, 10 mmol) and dichloroethane (20 ml). The mixture was stirred for 30 minutes under argon atmosphere. The compound (E) (5.06 g, 10 mmol) prepared by the Kuhn method was dissolved in 10 ml of dichloroethane. Each of one fifth of this solution was added at 30 minute intervals. After the addition was completed, the mixture was stixred for 24 hours.
The reaction mixture was filtered and insolubles were washed with ethyl acetate. The filtrate and the washings were combined, washed with water, dried on MgSO~ and concentrated in vacuo. The residue was subjected to silicagel column chromatography (W~KOGEL C-300, 300 g) and eluted with 4~ MeO~I-containing chloroform to give a fraction containing the compound (23). The fraction was further subjected to silicagel coLumn chromatography (WAKOGEL C-300, 100 9) to give the compound (23) ~634 mg, 6~).
(The compound (23)) S (~)D -8.94(CHC13, C = 0.94) NMR 400MHz(CDC13, ~ ppm) 1~867, 1.982, 2.021, 2.027, 2.034, 2.056, 2.070, 2.~77.
2.136, 2.140, OCOCH3, 2.637, lH, dd J=4.8g, 13.43 Hz H-~3eq, 3.832, 3H, s, -OCH3, 5.777 ~ 5.862 lH, m, -OCH2 -CH=CH2 -Example 19 To the compound (23) (266 mg, 0.25 mmol), there were added acetic anhydride (1.0 ml) and pyridine (1.0 ml). The mixture was s~irred at room temperature for 24 hours and concentrated in vacuo. The residue was subjected to column chromatography ~Robar column, size ~ ) and eluted with 4~ MeOH-containing chloroform to give the compound (24) (257 mg, 20g2.7%).
(The compound (24)) (~)D2 -6.94 (CHC13, C = 0.72) NMR 400MHz (CDC13, S ppm) 1.795, lH, t, J=12.21 Hz 3Hax, 2.429, lH, dd, J-4.63, 13.42 Hz, 3H, eq, 3.84, 3H, s, OCH3, 5.790 ~ 5.872, m, lH, -CH2 -CH=CH2 ' Example 20 The compound (24) (216 mg, 0.195 mmol) was dissolved in 9o% AcOH (3.0 ml). To this solution, there were added palladium chloride (41 my) ancl sodium acetate (38 mg)~ The mixture was stirred for 5 hours in an ultrasonic stirrer, filtered and concentrated in vacuo. The residue was dissolved in ethyl acetate. The solution was washed with saturated NaHCO3 solution and NaCl solution, dried on MgSO4 and concentrated in vacuo. The residue was subjected to silicagel column chromatography (WAI~OGEL C-300, 10 9) and eluted with 4 MeOH-containing chloro~orm to give the compound (25) (117 mg, 56~).
(The compound (25)) NMR 90MHz (CDC13, ~ ppm) 1.92 ~ 2.32, OCOCH3 x ~1, 3.86 -OCH3 Example 21 Trichloroacetonitrile (68 mg) and NaH (60~ in oil) (5 mg) were added under lce-cooled condition with stirring to a solution of the compound (25) (125 mg, 0~117 mmol) in dichloromethane (1.0 ml). The mixture was stirred for 2 hours, filtered through C3LITE (trademark) and concentrated.
The residue was subjected to silicagel column chromatography (WAKOGEL C-300, 5 g) and eluted with ethyl acetate to give the compound (26) (99 mg, 69.7%).
(The compound ~26)) NMR 90MHz (CDC13, ~ ppm) 3.88, s, -OCH3, 6.46, 1~ d, anomeric proton, 8.63, s, =N~
Example 22 To activated molecular sieves 4A (0.5 g), there were added the compound (26) (43 mg, 0.035 mmol), the compound (IV) (27 mg, 0. 035 mmol) and CHC13 (0 .5 ml). BF3 OEt2 (S,~l) was added to the mixture with stirring under ice-cooled condition.
The mixture was stirred at room temperature for 2~ hours.
After the filtration, insolubl~ès were washed with chloroform~
The filtrate and the washings were combined and concentrated in vacuo. The residue was subjected to silicagel column chromatography (WAKOGEL C-300, 12 9) and eluted with 3% MeO~-15 containing chloroform to give the compound (27) (23 mg).
(The compound (27) ) (~ ~ 25 +4 15 (C = 0.65, CHC13) NP~R 400MHz (CDC13, ~ppm) 0.87 - CH2C~3~
1.25 - (CH2)n -, 1.90, 1.96, 1.98, 2.02, 2.03, 2.04, 2.05, 2.05, 2.07, 2.08, 2.29, OCOCH3, 3.82, --OCH3, 7~30 ^~ 8.00 (aromatic proton) :25 Example 23 The compound (27) (27 mg, 0.015 mmol) was dissolved in methanol (0.5 ml). ~-NaOMe (0.036 ml) was added to th1s solution and stirred at room temperature for 4 hours. T~F
(0.5 ml) was added to dissolve the crystals precipitated. The mixture was stirred for additional two hours and concentrated in vacuo. To the residue, there were added 80% MeOH solution (3.0 ml) in water and THF (3.0 ml). The mixture was stirred at room temperature for 5 hours and concentrated in vacuo.
Water was added to the residue. Insolubles were collected to give the compound (28) (12.7 mg, 65.8%).
(The compound t28)) (a)25 -7.65 (C~C13 -MeOH 1:1 C = 0.55) NMR 400MHz (d.6 DMSO D20 98/?) ~ppm, O.85, -CH2CH3, 1.23 -~CH2 ~, 1.85 NHCOCH3 _ _ _ 4.17 anomeric proton Example 24 Benzyl galactoside (the compound A) (8~] g, 30 mmol) was suspended in acetone (150 ml). To this suspension, there were added paratoluenesulfonic acid (600 mg) and 2,2-dimethoxypropane (4.32 g). The mixture was stirred at room temperature for 2 hours. After triethylamine (2 ml) was added, the reaction mixture was concentrated in vacuo. The residue was subjected to silicagel column chromatography ~SiO2 C-300, 300 g) and eluted with toluene-ethyl acetate (1:2) to give the compound B (6 2 g, 66.7%).
(The compound B) Rf=0.58 (EtOAc) (~)D4 -2.38 (CHC13, C = 1.08) Analysis:
Calcd. C, 61.9 H, 7.15 (for C16~22Q6) Found C, 61.63 H, 7.13 Example 25 50~ NaH (46 mg) was washed with hexane and added to a solution of the compound B (100 mg, 0.32 mmol) in DMF (3 ml).
The solution was stirred at room temperature for 30 minutes and then ice-cooled, to which benzylbromide (165 mg) was added. The mixture was stirred at room temperature for one hour and MeOH (1 ml) was added. Ethyl acetate and saturated NaCl solution were added and shaked. The ethyl acetate layer was dried on MgSO4 an'd concentrated in vacuo. The residue was subjectd to silicagel column chromatography ~SiO~ C-300, 10 g) and eluted with toluene-ethyl acetate (10:1) to give the compound C (111 mg, 70.2%).
(The compound C) Rf 0.55 (toluene-ethyl acetate=l:l) (~)25 +7.25 (CHC13, C = 1.02) Analysis:
Calcd. C, 73.45 H, 6.99 (for C30H346) Found C, 73.38 H, 6.98 , ~ :
-Example 26 The compound C (12.3 g, 25 mmol) was dissolved in 80~AcOH (50 ml) and stirred at 60C for 3 hours. The reaction mixture was concentrated in vacuo. The residue was washed with ether to give the compound D as needle crystals (5.2 g, 46~).
(The compound D) m.p. 107 -108C
Rf 0.17 (toluene-ethyl acetate 10:1) (a)D -15.1 (CHC1`3-C = 1.00) Analysis:
Calcd. C, 71.98 H, 6.71 (for C27H306) Found C, 72.41 ~, 6.75 Example 27 .
50~ NaH (1.07 g) was washed with hexane and suspended in DMF (10 ml), to which the isopropylidene derivative F (1.41 g, 3.00 mmol) dissolved in DMF (20 ml) was added and stirred at 0C for 30 minutes, to which benzylbromide (2.7 ml) was added. The mixture was stirred at room temperature o-Jernight and then cooled to 0C. Methanol was added thereto. Dl~F was distilled off. The residue was dissolved in ethyl acetate.
The solution was washed with water, dried on MgSO~, concentrated in vacuo, subjected to silicagel column chromatography (sio2 C-300, 70 g) and eluted with toluene-ethylacetate (9:1) to give the compound G (1.725 g, 62.1 (The compound G) Rf 0.64 (toluene-ethyl acetate = 4:1) (~)22 ~9 4O (CHC13, C = 1.49) Example 28 The compound G (5.11 g, 5.53 mmol) was dissolv~d in AcOH (so ml~, to which water (10 ml) was added. The mixture was stirred at 60C for 2 hours and concentrated in vacuo.
The resulting solid was suspended in and washed with he~ane to obtain the compound ~ (4.82 g, 98.5%).
(The compound H) m.p. 86 ~ 88C
(a) D2 ~19 . 5 (CHC13, C = 1.20) Rf 0.35 (toluene-ethyl acetate = 4:1) Analysis:
Calcd. C, 73.45 H, 6.62 (for C54H58ll) Found C, 73.52 H, 6.50 Example 29 To activated molecular sieves 4A (15 g), there were added Hg(CN)2 (3.03 9), HgBr2 (1.44 g), the compound D (1.80 g, 4 mmol3 and dichloroethane (6 ml). The mixture was stirred for one hour under argon atmosphere. To this solution, there was added one half of a solution of the compound E prepared from N-acetyl neuraminic acid acetate methyl e5ter (2.13 g, 4 mmol) in dichloroethane (6 ml), after one hour, the other half ~4g -thereof and the mixture was stirred at room temperature overnight. The reaction mixture was filtered. Insolubles were washed with ethyl acetate. The ~iltrate and -the washings were combined, washed with saturated NaCl solution three times, dried on MgSO4 and concen~rated in vacuo. The residue was subjected to silicagel column chromatography ~SiO2 C-300, 80 9) and eluted with toluene-ethyl acetate (1:2) to give the compound (31) (82 mg) and a mixture of the compounds (32) and (33) (15~ from N-acetyl neuraminic acid acetate methyl ester).
The mixture was subjected to Robar column (~-siæe) and eluted with 10% MeOH-containing toluene to separate the compound (33) ( -anomer, 190 mg) and the compound (32) (~-anomer, 300 mg).
(The compound (31)) m.p. 147 ~ 152C
(~)23 +11.3(C - 1.80, CHC13) Analysis:
Calcd.C, 61~32 H, 6.04 N, 1.55 (for C46H53N171/2 H2O) FoundC, 61.30 H, 5.99 N, 1.53 (The compound ~32) ~-anomer) (~)D -25.4 ~C = 1.40, CHC13) Analysis:
Calcd.C, 61.09 H, 6.21 N, 1.52 (for C47H57N018) FoundC, 60.90 H, 6.27 N, 1.48 Rf 0.24 ~toluene-methanol 10:1) (The compoun~ (33) ~ -anomer) (~)29 -21.7 (C = 1~15, CHC13) Analysis:
Calcd. C, 61.09 H, 6.21 N, 1.52 Found C, 60.92 H, 6.25 N, 1~54 Rf 0.20 (toluene-methanol 10:1) Example 30 The compound (31) (60 mg) was dissolved in MeOH (~ 0 ml). N-NaOCH3 (0.3 ml) was added. The mixture was stirred at room temperature for 24 hours, then neutrali2ed hy AMBERL~ST
A-15 and filtered. The filtrate was concentrated in vacuo to givè the compound (34) as crystalline powder (33 mg, 67.8~).
(The compound (34)) Analysis:
Calcd. C, 61.53 H, 6.39 N, 1~89 (for C38H45N013 ~ H20) Found C, 61.52 H, 6.29 N, 1.82 Rf 0.88 (BuOH : EtOH : H2O = 4:2:2j Example 31 The compound (31) (200 mg) was dissolved in MeOH (12 ml)~ to which N-NaOMe (1.1 ml) was added. The mixture was stirred at room temperature ~or 24 hours, neutralized by AMBERLITE CG-50 and then filtered through CELITE
~6~3~
(Trade mark). The residue was subjected to column chromatography (silanized silicagel, 10 g) and eluted with MeOH - H2O (1:2) to give the compound (35) (109 mg, 63.6~).
(The compound (35)) Rf 0~69 (BuOH : EtOH : H2O - 4:2:2) NMR 400MH~ D20 ~ ppm, (acetone) 1.656, lH t (H-3 ax), 2.664, lHd (H-3 eq) 2.019 3H S NHCOCH3, 6.979 ~ 7.314 15H ~aromatic proton) Example 32 The compound (35) (108 mg) was dissolved in MeOH - ~2 (9:1) (5 ml). Cata~tic reduction was carried out at room temperature for 24 hours and then at 60C ~or 5 hours. The reaction mixture was concentrated in vacuo. The residue was subjected to Robar column (PR-8, size A) and eluted with MeOH
- H2O (80:1) to give the compound (36) (23.1 mg, 33.1%).
(The compound (36)) Rf 0.34 (BuOH : EtOH : H2O - 4:2:2)~
NMR 400MHz D2O Sppm, (acetone) 2.054, NHCOCH3, 1.618 ~ 1.704, lHm H-3 ex 2.494 ~ 2.607, lH m H-3 eq Example 33 The compound (32) (106 mg, 0.11 mmol) was dissolved in MeOH (3 ml), to which N-NaOMe (0.3 ml) was addedO The mixture was stirred at room temperature for 24 hours, neutralized by S AMBERLIST A-15, and then concentrated in vacuo.
The residue was subjected to Robar column (RP-18, size ~ ) and eluted with MeOH -H2O (3:1) to give the compound (37) (60 mg, 70.5~)-(The compound (37)) Rf 0.34 (~uOH : EtOH : H2O = 4:2:2) Analysis: (for C3gH47NO4) Calcd. C, 61.53 H, 6.39 N, 1.89 Found C, 61.20 II~ 6.31 N, 1.52 Example 34 The compound (37) (63 mg) was dissolved in MeOH (2.0 ml), to which 10% Pd-C (63 mg) was added. Catalytic reduction was carried out for 24 hours. The reaction mixture was filtered to remove Pd-C and concentrated in vacuo to give almost quantitatively the compound (38).
(The compound (38)) Rf 0.26 (BuOH : EtOH : H2O = 4:2:2) (~)19 +11.0 (C = 0.30, water) Example 35 The compound (33) (260 mg, 0.28 mmol) was dissolved in MeOH (5 ml), to which N-NaOMe (1012 ml) was added. The mixture was stirred at room temperature for 24 hours, neutralized by A~IBERLIST A-15, filtered and concentrated in vacuo. The residue was subjected to Robar column ~RP-18, size ~ ) and eluted with MeOH - H2O (3:1) to give the compound (39) (120 mg, 57.5%).
(The compound (39)) Rf 0.64 (BuOH : EtOH : H2O - 4:2:2) (~)25 -25.9 (C = 1.62, CH30H) Analysis:
Calcd. C, 61.53 H, 6.39 N, 1.89 (for c38H47No4) Found C, 61.15 H, 6.05 N, 1.94 Example 36 The compound (39) (108 mg, 0.146 mmol) was dissolved in MeOH (5 ml), to which 10~ Pd-C (200 mg) was added.
Catalytic reduction was carried out for 24 hours. The mixture was filtered to remove Pd-C and concentrated in vacuo to give the compound (40) (68 mg, about 100~).
(The compound (40)) Rf 0.37 (BuOH : EtOH : H2O = 4:2:2) (~)1 ~23.1 (C = 1.18, H2O) Example 37 --The compound ~32) (577 mg, 0.625 mmol) was dissolved in acetic anhydride (10 ml) and pyridine (10 ml). The mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated in vacuo. The residue was subjec~ed to silicagel column chromatography (SiO2 C-300, 80 g) and eluted wlth toluene-ethyl acetate (1:2) to give the compound ~41) (477 mg, 79.1%).
(The compound (41)) Rf 0.56 (15% MeOH-containing isopropylether) (~)23 -23.9 (CHC13, C = 1.03) Analysis: -Calcd. C, 60.92 H, 6.16 N, 1.45 (for c49H59NOl9) Found C, 60.69 ~, 6.18 N, 1.41 Example 38 The csmpound (41) (379 mg, 0.39 mmol) was dissolved in MeQ~ (15 ml), to which 10% Pd-C (200 mg) was added. Catalytic reduction was carried out at room temperature for 24 hours.
After filtration, the mixture was concentrated in vacuo. The residue was dissolved in a mixed solvent o~ acetic anhydride (5 ml) and pyridine (5 ml) and stirred at room temperature for 3 hours. The reaction mixture was concentrated in vacuo. The residue was dissolved in ethyl acetate, washed with water, dried on MgSO4 and concentrated in vacuo~ The residue was 3~
subjected to silicagel column chromatography tC-300, 20 g) and eluted with 15% MeOH-containing isopropylether to give the compound (42) (181 mg, 61.7%).
(The compound (42)) Rf 0.20 (3~ MeOH-containing chloroform) NMR 400MHz CDC13 ~ppm, (TMS), 1.799, lH t J=11.96, H-3 ex 2.585 lH dd J-13.19, 4.64, H-3 eq, 3.748 Example 39 The compound (33) (195 mg, 0.211 mmol) was dissolved in acetic anhydride (10 ml) and pyridine (10 ml). The mixture was stirred at room temperature for 24 hours and concentrated in vacuo. The residue was subjected to silicagel column and Robar column ~-size) and eluted with toluene-ethylacetate (1:2) to give the compound (43) (148 mg, 72.6%).
(The compound (43)) (~)23 _30.7o (CHC13, C=1.25) Analysis:
Calcd. C, 60.92 H, 6.16 N, 1.45 (~or c4gHsgNOl9) Found C, 61.65 H, 6.28 N, 1.33 3~
Example 40 The compound ~43) (g9 mg, 0.10 mmol) was dissolved in MeOH (5 ml), to which 10~ Pd-C (100 mg) was added. Catalytic reduction was carried out at room temperature for 24 hours.
The reaction mixture was filtered to remove Pd-C and concentrated in vacuo. The residue was dissolved in acetic anhydride (1 ml) and pyridine (1 ml), stirred at room temperature for 24 hours and concentrated in vacuo. The residue was subjected to Robar column (size A) and eluted with ethyl acetate - toluene (2:1) to give quantitatively the compound ~44) (77 mg).
(The compound (44)) Rf 0.25 (3% MeOH-containing chloroform) NMR 400MHz CDC13 Sppm, (TMS), 1.711, lH t J=12.45, H-3 ax 1.856 ~ 2.229, 27H, OCOCH3, 2.583 ~ 2.642 m, 1~, H-3ax, 3.863, s, OCH3 Example 41 The compound H (3.85 g, 4.4 mmol), Hg(CN)2 (1.36 g) and HgBr2 (648 mg) were added to molecular sieves 4A (7.2 g), to which dichloroethane (5 ml) was added under argon atmosphere and stirred at room temperature for one hour. To this solution, there were added one half of a solution of N-acetyl-D-acetyl neuraminyl chloride (the compound E) (916 mg, 1.8 mmol) in dichloroethane (5 ml) and after one hour, the remaining half of the solution. The reaction mixture was stirred at room temperature for 2 days and filtered.
Insolubles were washed with ethyl acetate. The filtrate and the washings were combined, washed with water, dried on MgSO4 and concentrated in vacuo. The residue was subjected to silicagel column chromatography (C-300, 180 g) and eluted with toluene - ethyl acetate (1:2) to give the compound (45) (93 mg, 3.8%) and a mixture of the compounds (~6) and (47) (425 mg, 17.4~). The mixture (425 mg) was subjected to Robar column (size ~) and eluted with 10~ MeOH-containing toluene to give the compound (46) (~-anomer, 255 mg~ and the compound (47) (~-anomer, 100 mg).
(The compound (45)) (Rf 0.49, 15% MeOH-containing isopropylether) Analysis:
Calcd. C, 65.52 H, 6.32 N, 1.03 (for C74H85N023) Found C, 65.27 H, 6.07 N, 1.00 (The compound (46)) (~)D +3.10 (C = 1.065, CHcl3) (Rf 0.31, 10~ MeOH-containing toluene) Analysis:
Calcd. C, 65.52 H, 6.32 N, 1.03 (for c74HgsNo23) Found C, 65.64 H, 6.33 N, 1.05 $
(The compound (47)) (~)D +5.84 (C = 0.925, CHC13) (Rf 0.26, 10% MeOH-containing toluene) Analysis:
Calcd. C, 65.52 H, 6.32 N, 1.03 ( f or C7 4H8 5N2 3 ) Found C, 65.40 ~, 6.25 N, 1.10 Example 42 The compound (46) (136 mg, 0.1 mmol) was dissolved in MeOH (5 ml), to which N-NaOMe (0.8 ml) was added. The mixture was stirred at room temperature Eor 24 hours, neutralized by AMBERLIST A-15, flltered to remove the resin and concentrated in vacuo. 0.1 N-NaOH solution tl-0 ml) was adced to the residue. The mixture was stirred at room temperature for 24 hours, neutralized by Amberlist A-15 (tradename), filtered and concentrated in vacuo to give the compound (48) 196 mg, 80.6%).
(The compound (48)) Rf 0.70 BuOH : EtOH : H2O (4 : 2 : 2) (~)19 ~6.62 (C = 0.71, MeOH) Example 43 ~5 The compound (48) (84 mg, 0.071 mmol) was dissolved in ~eO~
(5 ml), to which 10% Pd-C (100 mg) was added. Catalytic reduction was carried out at room temperature for 2 days. The .
mixture was filtered to remove Pd-C and concentrated to give the compound (49) (42 mg, 93.7%).
(The compound (48)) Rf 0.19 BuOH : EtOH : H2O (4 : 2 : 2) (~)19 +11.6 (C = 1.08, H2O) Example 44 The compound (47) (136 mg, 0.1 mmol) was dissolved in MeOH (5 ml), to whicb N-NaO~e (0.6 ml) was added. The mixture was stirred at room temperature for 24 hours, neutralized by AMBERLIST A-15 , filtered and c.oncentrated in -.
vacuo. The residue was mixed ..with 0.1 N-NaOH solution (1.0 ml) and MeOH (2.0 ml) and stirred at room temperature for 7 hours. The reaction mixture was neutralized by AMBERLI~T A-15 and concentrated in vacuo to give the compound (50) (97 mg, 81.4~)-(The compound (50)) Rf 0.65 BuOH : EtOH : H2O (4 : 2 : 2) (a)20 +6.44 ~C = 0.87, MeOH) Example 45 The compound (50) t80 mg, 0.067 mmol) was dissolved in MeOH (5 ml), to which 10% Pd-C (100 mg) was added. Catalytic reduction was carried out at room temperature for 24 hours, filtered to remove Pd-C and concentrated in vacuo to give quantitatively the compound (51) (42 mg).
(The compound (51)) Rf 0.34 BuOH : EtOH : H2O (4 : 2 : 2) ( )19 +19 2 (C = 1.53, H2O) Example 46 The compound (47) ~507 mg, 0.373 mmol) was dissol~ed in acetic anhydride (5.0 ml) and pyridine (5.0 ml) and stirred at room temperature for 24 hours~ The reaction mixture was subjected to silicagel column chromatography (wAKoGEL C-300, 50 9) and eluted with 10% MeOH-containing toluene to give the compound (52) (484 mg, 93~), (The compound (52)) (a) D ~3-33 (C = 1.17, CHC13) Analysis:
Calcd. C, 65.27 H, 6.27 N, 1.00 (for C76H37N024) Found C, 65.25 H, 6.47 N~ 1.03 PMR 400MHz CDC13 ppm (TMS) 1.762, 1.855, 1.973, 2.000, 2.013, 2~073, s, C~3CO, 2.598, q, J=12.69, 4.64, H-3c, eq, 3.834, OCH3, s Example 47 The compound (52) was dissolved in MeOH (15 ml), to which 10% Pd-C (200 mg) was added. Catalytic reduction was carried out. Ater the reduction was completed, the reaction mixture which contained the compound (53) (TLC, BuOH-EtOH-H2O
4:2:2, Rf 0.55) was filtered to remove Pd-C and concentrated in vacuo. The residue was dissolved in acetic anhydride (5 ml) and pyridine (5 ml) and stirred at room temperature for 2 hours. The mixture was concentrated in vacuo. The residue was subjected to silicagel column chromatography (C-300, 10 g) and eluted with ethyl acetate to give the compound (54) (235 mg, 69.3~.
(The compound (54)) Rf 0.28 (EtOAc) Analysi 5:
Calcd. C, 49.77 H, 5.72 N, 1.26 (for c46H63No3o) Found C, 49.85 H, 5.77 N, 1.45 P~R 400 MHz CDC13 ppm (TMS) 1.68, t, J=12.45, H-3c, ax, 1.86 ~ 2.25, CH3CO, 36H, 2.58, m, H-3c, eq, 3.84, 3.85, s, OCH3 Example 48 The compound (54) (190 mg, 0.171 mmol) was dissolved in DMF (1.0 ml) and heated to 50C, to which hydrazinium acetate (19 mg) was added and stirred for 5 minutes. After cooled, there was added ethyl acetate (10 ml) to the reaction mixture and stirred for 30 minutes. The mixture was diluted with ethyl acetate and washed with saturated NaCl solution.
The organic layer was dried on MgSO4 and concentrated in . - 62 -~3~$~
Yacuo. The residue was subjected to silicagel column chromatography ~Wakogel C-300, 10 9) and eluted with 1~ MeOH-containing ethyl acetate to give the compound (19) (14R ~g, 81.0%).
(The compound (1~)) Rf 0.29 1~ MeOH-containing ethyl acetate Analysis:
Calcd. C, 4~.48 H, 5.76 N, 1.31 (for C44H61N29) Found C, 49.25 H, 5.81 N, 14 56 Example 49 The compound (19) (145 mg, 0.136 mmol) was dissolved in methylene chloride (1.0 ml). To this, there was added trichloroacetonitrile (54 ~ 1) and NaH (60% in oil) (7.0 mg) under ice-ccoled condition and stirred for 2 hours. The mixture was concentrated in vacuo. The residue was subjected to silicagel column chromatography (wA~OGEL C-300, 10 g) and eluted with ethyl acetate to give the compound (20) (110 mg, 66.8%).
(The compound (20)) PMR 400MHz CDC13 ppm (TMS) 1.68, t, J=12.45, H-3c, ax, 1.86~ 2.10, CH3COO, 33H, 2.58, q, J=12.69, 4.63, H-3c, eq, 3.848, s, OCH3, 6.49, d, J=3.67 H-la, 8.66, s =NH
CMR 25MHz CDC13 ppm ~3.13, C-la, 96.88, c-2c, 101.15, c-lb, 161.13, -O-C=NH
Example 50 Activated molecular sieves 4A (0.5 g), the compound (20) (60 mg, 0.049 mmol), and the compound (IV) (37 mg, 0.049 mmol) were dissolved in CHC13 (0.5 ml) under argon atmosphere and ice-cooled condition. To this mixture, there was added BF3 . Et2O (7 ~1) with stirring. The mixture was stirred at ice-cooled condition for two hours and at room temperature Eor 24 hours. The reaction mixture was diluted with CHC13, filtered through Celite. Insolubles were filtered. The filtrate was concentrated in vacuo. The residue was subjected to silicagel column chromatography (WAKOGEL C-300, 12 9) and eluted with 3% MeOH-containing chloroform to give the compound (21) (32 mg, 36%).
(The compound (21)) (~)D5 +4.51 (CHC13, C = 1.13) Rf 0.39 EtOAc Analysis:
Calcd. C, 61.91 Ht 8.16 N, 1.55 (for C93H146N2032) Found C, 61.93 H, 8.22 N, 1.58 PMR 400 MHz CDCl3 ppm TMS 0.88, t, J=6.35, -CH3 x 2, 1.25, -CH2- x 32, , , 1.67, ~, J=12.46, H-3c, ax, 1.86 ~ 2.22, CH3COO x 11, 2.57, q, J=12.69, 4.67, H-3c, eq, 3.84, s, CH3-O , 7.44, m, 2H, 7.60, m ,lH, 7.99, m, 2H, aromatic proton CMR 22.5MHz CDC13, ppm, 96.33, C-2c, 100.56 C-la, 101.05, C-lb.
Example 51 The compound (21) (25 mg, 0.014 mmol) was dissolved in methanol (0.5 ml). 1 N-NaOMe (56 ~ 1) was added and stirred at room temperature for 24 hours. The mixture was concentrated.
To the residue, there were added water (0.1 ml), ~HF (0.5 ml) and MeOH (0.5 ml) and stirred for 5 hours. AMBERLIST A-15 was added to neutralize the mixture and then filtered. The filtrate was concentrated in vacuo. The residue was recrystallized from MeOH to give the compound (22) (11 mg, 61.7%).
(The compound (22)) PMR 400MHz Me2SO-d6-D20 (98:2 V/V~ TMS, ppm 1.23, s, 64H, -CH2-, 1.89, s, 3H, NHCOCH3, 1.92, bs, 2H, H-6', 1.37, H-3c, ax, 2.75, H-3c, eq, 4.16, d, J=7.57, H-la, 4.19, d, J=7.56, H-lb - 65 ~
.~
(The compound (18)) NMR (90MHz, CDC13 Sppm) 1.88 ~ 2.04 OCOCH3 x 11 3.86, s, 3H, ~OCH3 Example 14 The compound (18) (61 mg, 0.053 mmol) was dissolved in ethanol. To this solution, there were added thiourea (20 mg) and sodium acetate (4 mg). The mixture was heated and refluxed for 5 hours. The reaction mixture was concentrated in vacuo. The residue was dissolved in 4% MeOH-containing 6~
chloroform and subjected to silicagel column chromatography (WAKOGEL C-300, 10 g) to give the compound (19) (21 mg). The compound (18) (40 mg) was recovered.
(The compound (19)) Rf 0.42 (5% MeOH-containing chloroform) N~R 90MHz 1.88, 2.02, 2.10, 2.16, 2.24, 5 OCOCH3, 3.85, s, OCH3 Example 15 The compound (19) (20-mg, 0.0187 mmol) was dissolved in methylene chloride t0.5 ml) and trichloroacetonitrile (13.5 mg) was added. To this, there was added NaH (60% in oil) (lo O
mg) under ice-cooled condition and stirred for 2 hours. The mixture was concentrated in vacuo. The residue was subjected to silic~gel column chromatography (wAKOGEL C-300, 5 g) and eluted with ethyl acetate to give the compound (20) (10.0 mg)~
(The compound (20)) Rf 0.33 (ethyl acetate) NMR 90MHz ~ ~ppm) 8.60, NH, 5.50, d J=4.0 anomeric proton, 3.88, s, OCH3, 2.60, lH, dd, J=4.0, J=13.0 (in CDC13) Example 16 To activated molecular sieves 4A (2009), there were added the compound (20) (10 mg, 0.009 mmol), the compound (IV) (12 mg, 0.018 mmol) and CHC13 (0.2 ml). To this mixture, there was added BF3.Et2O (2.0 ~ 1) with stirring~ The mixture was stirred at room temperature for 24 hours. Insolubles were filtered and washed with CHC13 The filtrate and the washings were combined and concentrated in vacuo~ The residue was subjected to silicagel column chromatography (C-300, 10 g) and eluted with 4% MeOH-containing~`chloroform to give the compound (21) (4 mg).
(The compound (21)) Rf 0.29 3% MeOH-containing chloroform Ana'lysis:
Calcd. C,61.91 H,8.16 N,1.56 (for C93~146N2032) Found~ C,62.41 H,8.Ql ~,1.48 Example 17 The compound (21) (4.0 mg) was dissolved in a mixed solvent of methanol and THF (1:1) (0.5 ml). N-NaOMe (0~1 ml) was added and stirred at room temperature for 2 hours. The mixture was concentrated. To the residue, there were added water (0.1 ml) and MeOH-THF (1:1) (0.5 ml) and stirred at room temperature for 2 hours. AMBERLIST A-15 was added ' : ' , to neutralize the mixture and then filtered. AMBERLIST A-15 was washed with methanol. The solution combined w2S
concentrated in vacuo. The residue was washed with ether and dried to give the compound t22) (ganglioside GM3) (1.7 mg).
(The compound (22)) Analysis:
Calcd. C,60.64 H, 9.32 N, 2.18 (for C6sHllgN2o2lNa) Found C, 60.21 H, 8.97 N, 2.10 Example 18 To activated molecular sieves 4A (20 g), there were added Hg(CN)2 (5.04 g), HgBr2 (7.~0 g), the compound (11) (5.~3g, 10 mmol) and dichloroethane (20 ml). The mixture was stirred for 30 minutes under argon atmosphere. The compound (E) (5.06 g, 10 mmol) prepared by the Kuhn method was dissolved in 10 ml of dichloroethane. Each of one fifth of this solution was added at 30 minute intervals. After the addition was completed, the mixture was stixred for 24 hours.
The reaction mixture was filtered and insolubles were washed with ethyl acetate. The filtrate and the washings were combined, washed with water, dried on MgSO~ and concentrated in vacuo. The residue was subjected to silicagel column chromatography (W~KOGEL C-300, 300 g) and eluted with 4~ MeO~I-containing chloroform to give a fraction containing the compound (23). The fraction was further subjected to silicagel coLumn chromatography (WAKOGEL C-300, 100 9) to give the compound (23) ~634 mg, 6~).
(The compound (23)) S (~)D -8.94(CHC13, C = 0.94) NMR 400MHz(CDC13, ~ ppm) 1~867, 1.982, 2.021, 2.027, 2.034, 2.056, 2.070, 2.~77.
2.136, 2.140, OCOCH3, 2.637, lH, dd J=4.8g, 13.43 Hz H-~3eq, 3.832, 3H, s, -OCH3, 5.777 ~ 5.862 lH, m, -OCH2 -CH=CH2 -Example 19 To the compound (23) (266 mg, 0.25 mmol), there were added acetic anhydride (1.0 ml) and pyridine (1.0 ml). The mixture was s~irred at room temperature for 24 hours and concentrated in vacuo. The residue was subjected to column chromatography ~Robar column, size ~ ) and eluted with 4~ MeOH-containing chloroform to give the compound (24) (257 mg, 20g2.7%).
(The compound (24)) (~)D2 -6.94 (CHC13, C = 0.72) NMR 400MHz (CDC13, S ppm) 1.795, lH, t, J=12.21 Hz 3Hax, 2.429, lH, dd, J-4.63, 13.42 Hz, 3H, eq, 3.84, 3H, s, OCH3, 5.790 ~ 5.872, m, lH, -CH2 -CH=CH2 ' Example 20 The compound (24) (216 mg, 0.195 mmol) was dissolved in 9o% AcOH (3.0 ml). To this solution, there were added palladium chloride (41 my) ancl sodium acetate (38 mg)~ The mixture was stirred for 5 hours in an ultrasonic stirrer, filtered and concentrated in vacuo. The residue was dissolved in ethyl acetate. The solution was washed with saturated NaHCO3 solution and NaCl solution, dried on MgSO4 and concentrated in vacuo. The residue was subjected to silicagel column chromatography (WAI~OGEL C-300, 10 9) and eluted with 4 MeOH-containing chloro~orm to give the compound (25) (117 mg, 56~).
(The compound (25)) NMR 90MHz (CDC13, ~ ppm) 1.92 ~ 2.32, OCOCH3 x ~1, 3.86 -OCH3 Example 21 Trichloroacetonitrile (68 mg) and NaH (60~ in oil) (5 mg) were added under lce-cooled condition with stirring to a solution of the compound (25) (125 mg, 0~117 mmol) in dichloromethane (1.0 ml). The mixture was stirred for 2 hours, filtered through C3LITE (trademark) and concentrated.
The residue was subjected to silicagel column chromatography (WAKOGEL C-300, 5 g) and eluted with ethyl acetate to give the compound (26) (99 mg, 69.7%).
(The compound ~26)) NMR 90MHz (CDC13, ~ ppm) 3.88, s, -OCH3, 6.46, 1~ d, anomeric proton, 8.63, s, =N~
Example 22 To activated molecular sieves 4A (0.5 g), there were added the compound (26) (43 mg, 0.035 mmol), the compound (IV) (27 mg, 0. 035 mmol) and CHC13 (0 .5 ml). BF3 OEt2 (S,~l) was added to the mixture with stirring under ice-cooled condition.
The mixture was stirred at room temperature for 2~ hours.
After the filtration, insolubl~ès were washed with chloroform~
The filtrate and the washings were combined and concentrated in vacuo. The residue was subjected to silicagel column chromatography (WAKOGEL C-300, 12 9) and eluted with 3% MeO~-15 containing chloroform to give the compound (27) (23 mg).
(The compound (27) ) (~ ~ 25 +4 15 (C = 0.65, CHC13) NP~R 400MHz (CDC13, ~ppm) 0.87 - CH2C~3~
1.25 - (CH2)n -, 1.90, 1.96, 1.98, 2.02, 2.03, 2.04, 2.05, 2.05, 2.07, 2.08, 2.29, OCOCH3, 3.82, --OCH3, 7~30 ^~ 8.00 (aromatic proton) :25 Example 23 The compound (27) (27 mg, 0.015 mmol) was dissolved in methanol (0.5 ml). ~-NaOMe (0.036 ml) was added to th1s solution and stirred at room temperature for 4 hours. T~F
(0.5 ml) was added to dissolve the crystals precipitated. The mixture was stirred for additional two hours and concentrated in vacuo. To the residue, there were added 80% MeOH solution (3.0 ml) in water and THF (3.0 ml). The mixture was stirred at room temperature for 5 hours and concentrated in vacuo.
Water was added to the residue. Insolubles were collected to give the compound (28) (12.7 mg, 65.8%).
(The compound t28)) (a)25 -7.65 (C~C13 -MeOH 1:1 C = 0.55) NMR 400MHz (d.6 DMSO D20 98/?) ~ppm, O.85, -CH2CH3, 1.23 -~CH2 ~, 1.85 NHCOCH3 _ _ _ 4.17 anomeric proton Example 24 Benzyl galactoside (the compound A) (8~] g, 30 mmol) was suspended in acetone (150 ml). To this suspension, there were added paratoluenesulfonic acid (600 mg) and 2,2-dimethoxypropane (4.32 g). The mixture was stirred at room temperature for 2 hours. After triethylamine (2 ml) was added, the reaction mixture was concentrated in vacuo. The residue was subjected to silicagel column chromatography ~SiO2 C-300, 300 g) and eluted with toluene-ethyl acetate (1:2) to give the compound B (6 2 g, 66.7%).
(The compound B) Rf=0.58 (EtOAc) (~)D4 -2.38 (CHC13, C = 1.08) Analysis:
Calcd. C, 61.9 H, 7.15 (for C16~22Q6) Found C, 61.63 H, 7.13 Example 25 50~ NaH (46 mg) was washed with hexane and added to a solution of the compound B (100 mg, 0.32 mmol) in DMF (3 ml).
The solution was stirred at room temperature for 30 minutes and then ice-cooled, to which benzylbromide (165 mg) was added. The mixture was stirred at room temperature for one hour and MeOH (1 ml) was added. Ethyl acetate and saturated NaCl solution were added and shaked. The ethyl acetate layer was dried on MgSO4 an'd concentrated in vacuo. The residue was subjectd to silicagel column chromatography ~SiO~ C-300, 10 g) and eluted with toluene-ethyl acetate (10:1) to give the compound C (111 mg, 70.2%).
(The compound C) Rf 0.55 (toluene-ethyl acetate=l:l) (~)25 +7.25 (CHC13, C = 1.02) Analysis:
Calcd. C, 73.45 H, 6.99 (for C30H346) Found C, 73.38 H, 6.98 , ~ :
-Example 26 The compound C (12.3 g, 25 mmol) was dissolved in 80~AcOH (50 ml) and stirred at 60C for 3 hours. The reaction mixture was concentrated in vacuo. The residue was washed with ether to give the compound D as needle crystals (5.2 g, 46~).
(The compound D) m.p. 107 -108C
Rf 0.17 (toluene-ethyl acetate 10:1) (a)D -15.1 (CHC1`3-C = 1.00) Analysis:
Calcd. C, 71.98 H, 6.71 (for C27H306) Found C, 72.41 ~, 6.75 Example 27 .
50~ NaH (1.07 g) was washed with hexane and suspended in DMF (10 ml), to which the isopropylidene derivative F (1.41 g, 3.00 mmol) dissolved in DMF (20 ml) was added and stirred at 0C for 30 minutes, to which benzylbromide (2.7 ml) was added. The mixture was stirred at room temperature o-Jernight and then cooled to 0C. Methanol was added thereto. Dl~F was distilled off. The residue was dissolved in ethyl acetate.
The solution was washed with water, dried on MgSO~, concentrated in vacuo, subjected to silicagel column chromatography (sio2 C-300, 70 g) and eluted with toluene-ethylacetate (9:1) to give the compound G (1.725 g, 62.1 (The compound G) Rf 0.64 (toluene-ethyl acetate = 4:1) (~)22 ~9 4O (CHC13, C = 1.49) Example 28 The compound G (5.11 g, 5.53 mmol) was dissolv~d in AcOH (so ml~, to which water (10 ml) was added. The mixture was stirred at 60C for 2 hours and concentrated in vacuo.
The resulting solid was suspended in and washed with he~ane to obtain the compound ~ (4.82 g, 98.5%).
(The compound H) m.p. 86 ~ 88C
(a) D2 ~19 . 5 (CHC13, C = 1.20) Rf 0.35 (toluene-ethyl acetate = 4:1) Analysis:
Calcd. C, 73.45 H, 6.62 (for C54H58ll) Found C, 73.52 H, 6.50 Example 29 To activated molecular sieves 4A (15 g), there were added Hg(CN)2 (3.03 9), HgBr2 (1.44 g), the compound D (1.80 g, 4 mmol3 and dichloroethane (6 ml). The mixture was stirred for one hour under argon atmosphere. To this solution, there was added one half of a solution of the compound E prepared from N-acetyl neuraminic acid acetate methyl e5ter (2.13 g, 4 mmol) in dichloroethane (6 ml), after one hour, the other half ~4g -thereof and the mixture was stirred at room temperature overnight. The reaction mixture was filtered. Insolubles were washed with ethyl acetate. The ~iltrate and -the washings were combined, washed with saturated NaCl solution three times, dried on MgSO4 and concen~rated in vacuo. The residue was subjected to silicagel column chromatography ~SiO2 C-300, 80 9) and eluted with toluene-ethyl acetate (1:2) to give the compound (31) (82 mg) and a mixture of the compounds (32) and (33) (15~ from N-acetyl neuraminic acid acetate methyl ester).
The mixture was subjected to Robar column (~-siæe) and eluted with 10% MeOH-containing toluene to separate the compound (33) ( -anomer, 190 mg) and the compound (32) (~-anomer, 300 mg).
(The compound (31)) m.p. 147 ~ 152C
(~)23 +11.3(C - 1.80, CHC13) Analysis:
Calcd.C, 61~32 H, 6.04 N, 1.55 (for C46H53N171/2 H2O) FoundC, 61.30 H, 5.99 N, 1.53 (The compound ~32) ~-anomer) (~)D -25.4 ~C = 1.40, CHC13) Analysis:
Calcd.C, 61.09 H, 6.21 N, 1.52 (for C47H57N018) FoundC, 60.90 H, 6.27 N, 1.48 Rf 0.24 ~toluene-methanol 10:1) (The compoun~ (33) ~ -anomer) (~)29 -21.7 (C = 1~15, CHC13) Analysis:
Calcd. C, 61.09 H, 6.21 N, 1.52 Found C, 60.92 H, 6.25 N, 1~54 Rf 0.20 (toluene-methanol 10:1) Example 30 The compound (31) (60 mg) was dissolved in MeOH (~ 0 ml). N-NaOCH3 (0.3 ml) was added. The mixture was stirred at room temperature for 24 hours, then neutrali2ed hy AMBERL~ST
A-15 and filtered. The filtrate was concentrated in vacuo to givè the compound (34) as crystalline powder (33 mg, 67.8~).
(The compound (34)) Analysis:
Calcd. C, 61.53 H, 6.39 N, 1~89 (for C38H45N013 ~ H20) Found C, 61.52 H, 6.29 N, 1.82 Rf 0.88 (BuOH : EtOH : H2O = 4:2:2j Example 31 The compound (31) (200 mg) was dissolved in MeOH (12 ml)~ to which N-NaOMe (1.1 ml) was added. The mixture was stirred at room temperature ~or 24 hours, neutralized by AMBERLITE CG-50 and then filtered through CELITE
~6~3~
(Trade mark). The residue was subjected to column chromatography (silanized silicagel, 10 g) and eluted with MeOH - H2O (1:2) to give the compound (35) (109 mg, 63.6~).
(The compound (35)) Rf 0~69 (BuOH : EtOH : H2O - 4:2:2) NMR 400MH~ D20 ~ ppm, (acetone) 1.656, lH t (H-3 ax), 2.664, lHd (H-3 eq) 2.019 3H S NHCOCH3, 6.979 ~ 7.314 15H ~aromatic proton) Example 32 The compound (35) (108 mg) was dissolved in MeOH - ~2 (9:1) (5 ml). Cata~tic reduction was carried out at room temperature for 24 hours and then at 60C ~or 5 hours. The reaction mixture was concentrated in vacuo. The residue was subjected to Robar column (PR-8, size A) and eluted with MeOH
- H2O (80:1) to give the compound (36) (23.1 mg, 33.1%).
(The compound (36)) Rf 0.34 (BuOH : EtOH : H2O - 4:2:2)~
NMR 400MHz D2O Sppm, (acetone) 2.054, NHCOCH3, 1.618 ~ 1.704, lHm H-3 ex 2.494 ~ 2.607, lH m H-3 eq Example 33 The compound (32) (106 mg, 0.11 mmol) was dissolved in MeOH (3 ml), to which N-NaOMe (0.3 ml) was addedO The mixture was stirred at room temperature for 24 hours, neutralized by S AMBERLIST A-15, and then concentrated in vacuo.
The residue was subjected to Robar column (RP-18, size ~ ) and eluted with MeOH -H2O (3:1) to give the compound (37) (60 mg, 70.5~)-(The compound (37)) Rf 0.34 (~uOH : EtOH : H2O = 4:2:2) Analysis: (for C3gH47NO4) Calcd. C, 61.53 H, 6.39 N, 1.89 Found C, 61.20 II~ 6.31 N, 1.52 Example 34 The compound (37) (63 mg) was dissolved in MeOH (2.0 ml), to which 10% Pd-C (63 mg) was added. Catalytic reduction was carried out for 24 hours. The reaction mixture was filtered to remove Pd-C and concentrated in vacuo to give almost quantitatively the compound (38).
(The compound (38)) Rf 0.26 (BuOH : EtOH : H2O = 4:2:2) (~)19 +11.0 (C = 0.30, water) Example 35 The compound (33) (260 mg, 0.28 mmol) was dissolved in MeOH (5 ml), to which N-NaOMe (1012 ml) was added. The mixture was stirred at room temperature for 24 hours, neutralized by A~IBERLIST A-15, filtered and concentrated in vacuo. The residue was subjected to Robar column ~RP-18, size ~ ) and eluted with MeOH - H2O (3:1) to give the compound (39) (120 mg, 57.5%).
(The compound (39)) Rf 0.64 (BuOH : EtOH : H2O - 4:2:2) (~)25 -25.9 (C = 1.62, CH30H) Analysis:
Calcd. C, 61.53 H, 6.39 N, 1.89 (for c38H47No4) Found C, 61.15 H, 6.05 N, 1.94 Example 36 The compound (39) (108 mg, 0.146 mmol) was dissolved in MeOH (5 ml), to which 10~ Pd-C (200 mg) was added.
Catalytic reduction was carried out for 24 hours. The mixture was filtered to remove Pd-C and concentrated in vacuo to give the compound (40) (68 mg, about 100~).
(The compound (40)) Rf 0.37 (BuOH : EtOH : H2O = 4:2:2) (~)1 ~23.1 (C = 1.18, H2O) Example 37 --The compound ~32) (577 mg, 0.625 mmol) was dissolved in acetic anhydride (10 ml) and pyridine (10 ml). The mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated in vacuo. The residue was subjec~ed to silicagel column chromatography (SiO2 C-300, 80 g) and eluted wlth toluene-ethyl acetate (1:2) to give the compound ~41) (477 mg, 79.1%).
(The compound (41)) Rf 0.56 (15% MeOH-containing isopropylether) (~)23 -23.9 (CHC13, C = 1.03) Analysis: -Calcd. C, 60.92 H, 6.16 N, 1.45 (for c49H59NOl9) Found C, 60.69 ~, 6.18 N, 1.41 Example 38 The csmpound (41) (379 mg, 0.39 mmol) was dissolved in MeQ~ (15 ml), to which 10% Pd-C (200 mg) was added. Catalytic reduction was carried out at room temperature for 24 hours.
After filtration, the mixture was concentrated in vacuo. The residue was dissolved in a mixed solvent o~ acetic anhydride (5 ml) and pyridine (5 ml) and stirred at room temperature for 3 hours. The reaction mixture was concentrated in vacuo. The residue was dissolved in ethyl acetate, washed with water, dried on MgSO4 and concentrated in vacuo~ The residue was 3~
subjected to silicagel column chromatography tC-300, 20 g) and eluted with 15% MeOH-containing isopropylether to give the compound (42) (181 mg, 61.7%).
(The compound (42)) Rf 0.20 (3~ MeOH-containing chloroform) NMR 400MHz CDC13 ~ppm, (TMS), 1.799, lH t J=11.96, H-3 ex 2.585 lH dd J-13.19, 4.64, H-3 eq, 3.748 Example 39 The compound (33) (195 mg, 0.211 mmol) was dissolved in acetic anhydride (10 ml) and pyridine (10 ml). The mixture was stirred at room temperature for 24 hours and concentrated in vacuo. The residue was subjected to silicagel column and Robar column ~-size) and eluted with toluene-ethylacetate (1:2) to give the compound (43) (148 mg, 72.6%).
(The compound (43)) (~)23 _30.7o (CHC13, C=1.25) Analysis:
Calcd. C, 60.92 H, 6.16 N, 1.45 (~or c4gHsgNOl9) Found C, 61.65 H, 6.28 N, 1.33 3~
Example 40 The compound ~43) (g9 mg, 0.10 mmol) was dissolved in MeOH (5 ml), to which 10~ Pd-C (100 mg) was added. Catalytic reduction was carried out at room temperature for 24 hours.
The reaction mixture was filtered to remove Pd-C and concentrated in vacuo. The residue was dissolved in acetic anhydride (1 ml) and pyridine (1 ml), stirred at room temperature for 24 hours and concentrated in vacuo. The residue was subjected to Robar column (size A) and eluted with ethyl acetate - toluene (2:1) to give quantitatively the compound ~44) (77 mg).
(The compound (44)) Rf 0.25 (3% MeOH-containing chloroform) NMR 400MHz CDC13 Sppm, (TMS), 1.711, lH t J=12.45, H-3 ax 1.856 ~ 2.229, 27H, OCOCH3, 2.583 ~ 2.642 m, 1~, H-3ax, 3.863, s, OCH3 Example 41 The compound H (3.85 g, 4.4 mmol), Hg(CN)2 (1.36 g) and HgBr2 (648 mg) were added to molecular sieves 4A (7.2 g), to which dichloroethane (5 ml) was added under argon atmosphere and stirred at room temperature for one hour. To this solution, there were added one half of a solution of N-acetyl-D-acetyl neuraminyl chloride (the compound E) (916 mg, 1.8 mmol) in dichloroethane (5 ml) and after one hour, the remaining half of the solution. The reaction mixture was stirred at room temperature for 2 days and filtered.
Insolubles were washed with ethyl acetate. The filtrate and the washings were combined, washed with water, dried on MgSO4 and concentrated in vacuo. The residue was subjected to silicagel column chromatography (C-300, 180 g) and eluted with toluene - ethyl acetate (1:2) to give the compound (45) (93 mg, 3.8%) and a mixture of the compounds (~6) and (47) (425 mg, 17.4~). The mixture (425 mg) was subjected to Robar column (size ~) and eluted with 10~ MeOH-containing toluene to give the compound (46) (~-anomer, 255 mg~ and the compound (47) (~-anomer, 100 mg).
(The compound (45)) (Rf 0.49, 15% MeOH-containing isopropylether) Analysis:
Calcd. C, 65.52 H, 6.32 N, 1.03 (for C74H85N023) Found C, 65.27 H, 6.07 N, 1.00 (The compound (46)) (~)D +3.10 (C = 1.065, CHcl3) (Rf 0.31, 10~ MeOH-containing toluene) Analysis:
Calcd. C, 65.52 H, 6.32 N, 1.03 (for c74HgsNo23) Found C, 65.64 H, 6.33 N, 1.05 $
(The compound (47)) (~)D +5.84 (C = 0.925, CHC13) (Rf 0.26, 10% MeOH-containing toluene) Analysis:
Calcd. C, 65.52 H, 6.32 N, 1.03 ( f or C7 4H8 5N2 3 ) Found C, 65.40 ~, 6.25 N, 1.10 Example 42 The compound (46) (136 mg, 0.1 mmol) was dissolved in MeOH (5 ml), to which N-NaOMe (0.8 ml) was added. The mixture was stirred at room temperature Eor 24 hours, neutralized by AMBERLIST A-15, flltered to remove the resin and concentrated in vacuo. 0.1 N-NaOH solution tl-0 ml) was adced to the residue. The mixture was stirred at room temperature for 24 hours, neutralized by Amberlist A-15 (tradename), filtered and concentrated in vacuo to give the compound (48) 196 mg, 80.6%).
(The compound (48)) Rf 0.70 BuOH : EtOH : H2O (4 : 2 : 2) (~)19 ~6.62 (C = 0.71, MeOH) Example 43 ~5 The compound (48) (84 mg, 0.071 mmol) was dissolved in ~eO~
(5 ml), to which 10% Pd-C (100 mg) was added. Catalytic reduction was carried out at room temperature for 2 days. The .
mixture was filtered to remove Pd-C and concentrated to give the compound (49) (42 mg, 93.7%).
(The compound (48)) Rf 0.19 BuOH : EtOH : H2O (4 : 2 : 2) (~)19 +11.6 (C = 1.08, H2O) Example 44 The compound (47) (136 mg, 0.1 mmol) was dissolved in MeOH (5 ml), to whicb N-NaO~e (0.6 ml) was added. The mixture was stirred at room temperature for 24 hours, neutralized by AMBERLIST A-15 , filtered and c.oncentrated in -.
vacuo. The residue was mixed ..with 0.1 N-NaOH solution (1.0 ml) and MeOH (2.0 ml) and stirred at room temperature for 7 hours. The reaction mixture was neutralized by AMBERLI~T A-15 and concentrated in vacuo to give the compound (50) (97 mg, 81.4~)-(The compound (50)) Rf 0.65 BuOH : EtOH : H2O (4 : 2 : 2) (a)20 +6.44 ~C = 0.87, MeOH) Example 45 The compound (50) t80 mg, 0.067 mmol) was dissolved in MeOH (5 ml), to which 10% Pd-C (100 mg) was added. Catalytic reduction was carried out at room temperature for 24 hours, filtered to remove Pd-C and concentrated in vacuo to give quantitatively the compound (51) (42 mg).
(The compound (51)) Rf 0.34 BuOH : EtOH : H2O (4 : 2 : 2) ( )19 +19 2 (C = 1.53, H2O) Example 46 The compound (47) ~507 mg, 0.373 mmol) was dissol~ed in acetic anhydride (5.0 ml) and pyridine (5.0 ml) and stirred at room temperature for 24 hours~ The reaction mixture was subjected to silicagel column chromatography (wAKoGEL C-300, 50 9) and eluted with 10% MeOH-containing toluene to give the compound (52) (484 mg, 93~), (The compound (52)) (a) D ~3-33 (C = 1.17, CHC13) Analysis:
Calcd. C, 65.27 H, 6.27 N, 1.00 (for C76H37N024) Found C, 65.25 H, 6.47 N~ 1.03 PMR 400MHz CDC13 ppm (TMS) 1.762, 1.855, 1.973, 2.000, 2.013, 2~073, s, C~3CO, 2.598, q, J=12.69, 4.64, H-3c, eq, 3.834, OCH3, s Example 47 The compound (52) was dissolved in MeOH (15 ml), to which 10% Pd-C (200 mg) was added. Catalytic reduction was carried out. Ater the reduction was completed, the reaction mixture which contained the compound (53) (TLC, BuOH-EtOH-H2O
4:2:2, Rf 0.55) was filtered to remove Pd-C and concentrated in vacuo. The residue was dissolved in acetic anhydride (5 ml) and pyridine (5 ml) and stirred at room temperature for 2 hours. The mixture was concentrated in vacuo. The residue was subjected to silicagel column chromatography (C-300, 10 g) and eluted with ethyl acetate to give the compound (54) (235 mg, 69.3~.
(The compound (54)) Rf 0.28 (EtOAc) Analysi 5:
Calcd. C, 49.77 H, 5.72 N, 1.26 (for c46H63No3o) Found C, 49.85 H, 5.77 N, 1.45 P~R 400 MHz CDC13 ppm (TMS) 1.68, t, J=12.45, H-3c, ax, 1.86 ~ 2.25, CH3CO, 36H, 2.58, m, H-3c, eq, 3.84, 3.85, s, OCH3 Example 48 The compound (54) (190 mg, 0.171 mmol) was dissolved in DMF (1.0 ml) and heated to 50C, to which hydrazinium acetate (19 mg) was added and stirred for 5 minutes. After cooled, there was added ethyl acetate (10 ml) to the reaction mixture and stirred for 30 minutes. The mixture was diluted with ethyl acetate and washed with saturated NaCl solution.
The organic layer was dried on MgSO4 and concentrated in . - 62 -~3~$~
Yacuo. The residue was subjected to silicagel column chromatography ~Wakogel C-300, 10 9) and eluted with 1~ MeOH-containing ethyl acetate to give the compound (19) (14R ~g, 81.0%).
(The compound (1~)) Rf 0.29 1~ MeOH-containing ethyl acetate Analysis:
Calcd. C, 4~.48 H, 5.76 N, 1.31 (for C44H61N29) Found C, 49.25 H, 5.81 N, 14 56 Example 49 The compound (19) (145 mg, 0.136 mmol) was dissolved in methylene chloride (1.0 ml). To this, there was added trichloroacetonitrile (54 ~ 1) and NaH (60% in oil) (7.0 mg) under ice-ccoled condition and stirred for 2 hours. The mixture was concentrated in vacuo. The residue was subjected to silicagel column chromatography (wA~OGEL C-300, 10 g) and eluted with ethyl acetate to give the compound (20) (110 mg, 66.8%).
(The compound (20)) PMR 400MHz CDC13 ppm (TMS) 1.68, t, J=12.45, H-3c, ax, 1.86~ 2.10, CH3COO, 33H, 2.58, q, J=12.69, 4.63, H-3c, eq, 3.848, s, OCH3, 6.49, d, J=3.67 H-la, 8.66, s =NH
CMR 25MHz CDC13 ppm ~3.13, C-la, 96.88, c-2c, 101.15, c-lb, 161.13, -O-C=NH
Example 50 Activated molecular sieves 4A (0.5 g), the compound (20) (60 mg, 0.049 mmol), and the compound (IV) (37 mg, 0.049 mmol) were dissolved in CHC13 (0.5 ml) under argon atmosphere and ice-cooled condition. To this mixture, there was added BF3 . Et2O (7 ~1) with stirring. The mixture was stirred at ice-cooled condition for two hours and at room temperature Eor 24 hours. The reaction mixture was diluted with CHC13, filtered through Celite. Insolubles were filtered. The filtrate was concentrated in vacuo. The residue was subjected to silicagel column chromatography (WAKOGEL C-300, 12 9) and eluted with 3% MeOH-containing chloroform to give the compound (21) (32 mg, 36%).
(The compound (21)) (~)D5 +4.51 (CHC13, C = 1.13) Rf 0.39 EtOAc Analysis:
Calcd. C, 61.91 Ht 8.16 N, 1.55 (for C93H146N2032) Found C, 61.93 H, 8.22 N, 1.58 PMR 400 MHz CDCl3 ppm TMS 0.88, t, J=6.35, -CH3 x 2, 1.25, -CH2- x 32, , , 1.67, ~, J=12.46, H-3c, ax, 1.86 ~ 2.22, CH3COO x 11, 2.57, q, J=12.69, 4.67, H-3c, eq, 3.84, s, CH3-O , 7.44, m, 2H, 7.60, m ,lH, 7.99, m, 2H, aromatic proton CMR 22.5MHz CDC13, ppm, 96.33, C-2c, 100.56 C-la, 101.05, C-lb.
Example 51 The compound (21) (25 mg, 0.014 mmol) was dissolved in methanol (0.5 ml). 1 N-NaOMe (56 ~ 1) was added and stirred at room temperature for 24 hours. The mixture was concentrated.
To the residue, there were added water (0.1 ml), ~HF (0.5 ml) and MeOH (0.5 ml) and stirred for 5 hours. AMBERLIST A-15 was added to neutralize the mixture and then filtered. The filtrate was concentrated in vacuo. The residue was recrystallized from MeOH to give the compound (22) (11 mg, 61.7%).
(The compound (22)) PMR 400MHz Me2SO-d6-D20 (98:2 V/V~ TMS, ppm 1.23, s, 64H, -CH2-, 1.89, s, 3H, NHCOCH3, 1.92, bs, 2H, H-6', 1.37, H-3c, ax, 2.75, H-3c, eq, 4.16, d, J=7.57, H-la, 4.19, d, J=7.56, H-lb - 65 ~
Claims (14)
1. The galactose derivatives having the general formula:
wherein R1 is benzyl or R and R2 are hydrogen or benzyl when R1 is benzyl, otherwise R and R2 are hydrogen, acetyl or benzyl, R3 and R4 are hydrogen or R3 together with R or R4 form isopropylidene, R5 is benzyl or allyl and R6 is hydrogen, acetyl or benzyl.
wherein R1 is benzyl or R and R2 are hydrogen or benzyl when R1 is benzyl, otherwise R and R2 are hydrogen, acetyl or benzyl, R3 and R4 are hydrogen or R3 together with R or R4 form isopropylidene, R5 is benzyl or allyl and R6 is hydrogen, acetyl or benzyl.
2. A compound according to claim 1, having the formula:
wherein R1 is allyl or benzyl, R2 is hydrogen, benzyl or acetyl, R3 is hydrogen or benzyl R4 is hydrogen, R5 is hydrogen, benzyl or acetyl, or R4 together with R3 or R5 form isopropylidene.
wherein R1 is allyl or benzyl, R2 is hydrogen, benzyl or acetyl, R3 is hydrogen or benzyl R4 is hydrogen, R5 is hydrogen, benzyl or acetyl, or R4 together with R3 or R5 form isopropylidene.
3. A compound according to claim 1, having the formula:
wherein R1 is allyl or benzyl and R2 is benzyl or acetyl.
wherein R1 is allyl or benzyl and R2 is benzyl or acetyl.
4. A compound according to claim 1, having the formula:
wherein R1 is allyl and R2 is-benzyl.
wherein R1 is allyl and R2 is-benzyl.
5. A process for the preparation of a compound of the formula:
wherein R1 is allyl, R2 is benzyl, R3 is hydrogen or benzyl, R4 is benzyl when R3 is hydrogen or R4 is hydrogen when R3 is benzyl, which comprises reacting allyl D- lactoside with 2,2-dimethoxypropane to form 3',4'- or 4',6'-0-isopropylidene derivative which is then reacted with benzyl bromide to form 2,3,6,2',6'-or 2,3,6,2',3'-penta-0-benzyl derivative whieh is then treated with an aqueous solution of an organic acid to remove the isopropylidene group.
wherein R1 is allyl, R2 is benzyl, R3 is hydrogen or benzyl, R4 is benzyl when R3 is hydrogen or R4 is hydrogen when R3 is benzyl, which comprises reacting allyl D- lactoside with 2,2-dimethoxypropane to form 3',4'- or 4',6'-0-isopropylidene derivative which is then reacted with benzyl bromide to form 2,3,6,2',6'-or 2,3,6,2',3'-penta-0-benzyl derivative whieh is then treated with an aqueous solution of an organic acid to remove the isopropylidene group.
6. A process for the preparation of a compound of the formula:
wherein R1 is allyl and R2 is acetyl, which comprises treating allyl D-3',4'-0-isopropylidene-laetoside with acetic anhydride-pyridine to form 2,3,6,2',6'-penta-0-acetyl derivative which is then treated with an aqueous solution of an organic acid to remove the isopropylidene group.
wherein R1 is allyl and R2 is acetyl, which comprises treating allyl D-3',4'-0-isopropylidene-laetoside with acetic anhydride-pyridine to form 2,3,6,2',6'-penta-0-acetyl derivative which is then treated with an aqueous solution of an organic acid to remove the isopropylidene group.
7. A process according to claims 5 or 6, wherein the solution of organic acid used to remove the iso-propylidene group is an aqueous solution of acetic acid or trifluoroacetic acid.
8. A process for the preparation of a compound of the formula:
wherein R1 is benzyl, which comprises reacting benzyl D-3',4'-isopropylidene lactoside with benzyl bromide to form 2,3,6,2',6'-penta-0-benzyl derivative which is then treated with an aqueous solution of an organic acid to remove the isopropylidene group.
wherein R1 is benzyl, which comprises reacting benzyl D-3',4'-isopropylidene lactoside with benzyl bromide to form 2,3,6,2',6'-penta-0-benzyl derivative which is then treated with an aqueous solution of an organic acid to remove the isopropylidene group.
9. A process for the preparation of benzyl D-2,6-di-O-benzyl galactoside, which comprises reacting benzyl D-galactoside with 2,2-dimethoxypropane to form 3,4-0-isopropylidene derivative which is then reacted with benzyl bromide to form 2,6-di-0-benzyl derivative which is then treated with an aqueous solution of an organic acid to remove the isopropylidene group.
10. A process according to claims 8 or 9, wherein the solution of organic acid used to remove the iso-propylidene group is an aqueous solution of acetic acid or trifluoroacetic acid.
11. A compound of the formula:
wherein R1 is allyl, R2 is benzyl, R3 is hydrogen or benzyl, R4 is benzyl when R3 is hydrogen or R4 is hydrogen when R3 is benzyl.
wherein R1 is allyl, R2 is benzyl, R3 is hydrogen or benzyl, R4 is benzyl when R3 is hydrogen or R4 is hydrogen when R3 is benzyl.
12. A compound of the formula:
wherein R1 is allyl and R2 is acetyl.
wherein R1 is allyl and R2 is acetyl.
13. A compound of the formula:
wherein R1 is benzyl.
wherein R1 is benzyl.
14. Benzyl D-2,6-di-O-benzyl galactoside.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000555846A CA1269366A (en) | 1984-06-28 | 1988-01-05 | Galactose derivatives and preparation thereof |
Applications Claiming Priority (6)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP133881/1984 | 1984-06-28 | ||
| JP59133882A JPS6112697A (en) | 1984-06-28 | 1984-06-28 | Galactose derivative |
| JP133882/1984 | 1984-06-28 | ||
| JP59133881A JPS6112695A (en) | 1984-06-28 | 1984-06-28 | Sialic acid derivative |
| CA000486085A CA1262129A1 (en) | 1984-06-28 | 1985-06-28 | Sialic acid derivatives, galactose derivatives and method for producing the same |
| CA000555846A CA1269366A (en) | 1984-06-28 | 1988-01-05 | Galactose derivatives and preparation thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1269366A true CA1269366A (en) | 1990-05-22 |
Family
ID=27167534
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000555846A Expired - Fee Related CA1269366A (en) | 1984-06-28 | 1988-01-05 | Galactose derivatives and preparation thereof |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1269366A (en) |
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1988
- 1988-01-05 CA CA000555846A patent/CA1269366A/en not_active Expired - Fee Related
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