Note: Descriptions are shown in the official language in which they were submitted.
WO 9S/00527 216 4 9 61 PCT/SE94100604
FUCO8YI~TED GLYC08I~5R A8 IN}IIBITO}?.8 OF BACTERIl~I, AnRF~ R
FlELD OF THE INVENTION
The present invention relates to the use of 1-fucose-containing
glycoside derivatives for the preparation of pharmaceutical
compositions for the treatment or prophylaxis of conditions
involving gastrointestinal infections by Helicobacter pylori, a
method of treating such conditions using the derivatives, as
well as novel glycoside derivatives.
BACKGRoUND OF THE INVENTION
Helicobacter pylori is a microaerophilic spiral shaped organism
(originally assigned to the genus Campylobacter) which is found
in the stomach and generally appears to have an exclusive
habitat in the human gastric mucosa. It has been estimated that
this bac~erium infects the gastric mucosa of more than 60% of
adult humans by the time they are 60 years old. Moreover, H.
20 pyl ori has been implicated as a contributing factor in a number
of pathological conditions, including acute (type B) gastritis,
gastric and duodenal ulcers, atrophic gastritis, and gastric
adenocarc:inoma.
Tissue tropism of bacteria is partly governed by the ability of
a bacterial strain to adjust to the local chem
ical environment in its specific habitat. In addition,adherence
is a nec~ss~y prerequisite for colonization in order to
prevent removal from the new habitat, e.g. through peristalsis
in the gastrointestinal tract. In mammals, bacteria adhere to
proteins or glycoconjugates (glycosphingolipids, glycoproteins)
on or in the vicinity of epithelial cell surfaces (mucus), and
a number of specific bacterial adhesin-protein and
adhesin-carbohydrate interactions have been described in the
,35 literature.
With respect to H. pylori, studies in model systems such as
mouse adrenal Y-l cells (see D. G. Evans, D. J., Jr. Evans, and
SUB~ JTE SHEEr
wo gs/~527 ~ 9 6 ~ PCT/SE94/00604
--2--
D. Y. Graham, (1989) Infect. Immun. 57, 2272-2278) have
suggested that surface-associated flexible fibrillar structures
that surround this bacterium function as adhesins or
colonization factor antigens to mediate the binding of H.
5 pyl ori to cellular sialic acid-containing glycoprotein
receptors.
SUMMARY OF THE INVENTION
The invention concerns the use of mono-, di-, tri- or
oligosaccharide glycoside derivatives having at least one
terminal group Y, as defined below, derived from L-fucose, said
derivatives being compounds of the general formula Ia, Ib, Ic,
Id, Ie or If
Y-Z1-R A-Z2-R A-Z3-B-Z4-R
Ia Ib Ic
A--Z5--B--Z6-c--z7--R A-Z8-B-Zg-C-zlo~D-zll--R
Id Ie
A--Zl2--B--Zl3-C-Zl4-D-Zls~E~Zl6~R
wherein
Zl, Z2~ Z3~ Z4~ Z5~ Z6~ Z7~ Zg~ Zg~ Zlo, Zll~ Zl2~ Zl3~ Zl4~
Z15 and Z16 independently are O, S, CH2, or NR25, where R2s
is hydrogen, Cl_24-alkyl, C2_24-alkenyl, C1_24-alkylcarbonyl,
or benzoyl optionally substituted with hydroxy, amino,
Cl_4-alkyl, C1_4-alkoxy, nitro, halogen, phenoxy, or mono-
or di-halogen-Cl_4-alkyl;
SUBs ~ JTE SHEET
wo 95/00527 2 1 6 ~ 9 6 1 PCT/æ94/00604
y ~ C~,~ A i~
R2A RIA
R" ~ R-C
B i9 R3~ ; C 19 ~ ;
R2~ R~- RX R1C
10 R'~ ~ E i8 '~ ~_
~ R~
R2D R1D R2E R-E
15wherein
the wavy line in Y, A, B, C, D and E signifies a bond
which is either in the ~- or in the ~-configuration;
~Rl, R2, and R3 each in~ep~n~ntly are H, halogen,
~zido, guanidinyl, br~h~ or unbrAn~he~ Cl_24-alkyl,
c2_2g-alkenyl, c2_24-alkynyl, c3-8-CYClalkYl ~
3-8 cycloalkyl-Cl_24-alkyl, or Cl_l2-alkoxy-Cl_l2-alkyl
group which is optionally substituted with hydroxy,
amino, halogen, or oxo; aryl or aryl-Cl_4-alkyl
optionally substituted in the aryl moiety with hydroxy,
amino, Cl_4-alkyl, Cl_4-alkoxy, nitro, halogen, phenoxy,
or mono- or di-halogen-Cl_4-alkyl;
tri(Cl_4-alkyl)silylethyl; oxo;
a ~LoU~ =CR4Rs wherein R4 and R5 independently are H,
or cl_4-alkYl;
or a ~l~U~ XR10 wherein X is O, S, NR20, or =N-, and Rlo
~s H, branched or unbranched Cl_24-alkyl, C2_24-alkenyl,
C2-24-alkYnYl, C3-8-cycloalkyl,
3-8 cycloalkyl-Cl_24-alkyl, or Cl_l2-alkoxy-C1 12-alkyl
group which is optionally substituted with hydroxy,
amino, halogen, or oxo; aryl, aryl-Cl_4-alkyl, or
heterocyclyl-Cl_4-alkyl optionally substituted in the
aryl or heterocyclyl moiety with hydroxy, amino,
SUB~ JTE SHEE~T
WOg5/~7 ~6 49~ ~ PCT/SE94100604
C1_4-alkyl, Cl_4-alkoxy, nitro, halogen, phenoxy, or
mono- or di-halogen-C1_4-alkyl; tri (Cl_4
alkyl)silylethyl; tri(C1_4-alkyl)silyl;
tri(C1_4-alkyl)silylethoxymethyl; the acyl residue of a
naturally occurring amino acid; C1_24-alkylcarbonyl;
C2_24-alkenylcarbonyl;
C3_8-cycloalkyl-C1_24-alkylcarbonyl; arylcarbonyl; or
terpenyl; and
R20 is H, C1_24-alkyl~ C2_24-alkenyl,
C1_24-alkylcarbonyl, or benzoyl or phthaloyl optionally
substituted in the benzene ring with hydroxy, amino,
Cl_4-alkyl, Cl_4-alkoxy, nitro, halogen, phenoxy, or
mono- or di-halogen-C1_4-alkyl;
1A ~ R2A~ R3A~ R4A~ Rls~ R2s~ R3B~ R4B~ Rlc~ R2c~ R3c~ R4c~
R1D~ R2Dr R3D~ R4D~ R1E~ R2E~ R3E~ and R4E each
in~eren~ently is as defined for Rl, R2, and R3 above,
or is a group of the formula VII
YZl VII
wherein Y and Zl are as defined above;
with the provisos
that one of R1B~ R2B~ R3B~ or R4B is Z3, Z5, Z8 or Z12~
~hat one of R1c~ R2C~ R3C, or R4c is Z6~ Z9 or Z13, that
one of R1D~ R2Dr R3D~ or R4D is ZlO~ or Z14, that one of
R1E~ R2E' R3E' or R4E is Z15, that at least one and at
the most five of R1A~ R2A~ R3A~ R4A~ R1B' R2B~ R3B~ R4B~
R1C~ R2C~ R3C~ R4c~ R1D~ R2D~ R3D~ R4D~ R1E~ R2E~ R3E~ and
3 R4E is a group of the formula VII, and
that the configurations of the substituents R1A~ R2A'
R3A~ and R4ACH2 in A, the configurations of the
substituents R18, R2B, R38, and R48CH2 in B, the
configurations of the substituents R1C, R2C' R3c~ and
R4CCH2 in C, the configurations of the substituents
R1D ~ R2D' R3Dr and R4DCH2 in D, and the configurations
of the substituents R1E, R2E' R3E' and R4ECH2 in E
independently are D-gluco, L-gluco, D-galacto,
SUB~ ~ .JTE SHEET
-
W095l~s27 21 6 ~ 9 61 PCT/SE94/00604
--5--
L-galacto, D-manno, L-manno, D-talo, L-talo, D-allo,
L-allo, D-altro, L-altro, D-gulo, L-gulo, D-ido, or
L-ido;
~ 5 is a branched or unbranched C1_24-alkyl, C2_24-alkenyl,
c2-24-alkynyl, C3-8-cycloalkyl,
C3_8-cycloalkyl-Cl_24-alkYl, Cl-l2-alkXY-Cl-l2-alkYl,
Cl_24-alkylcarbonyl, C2_24-alkenylcar~onyl, or
C3_8-cycloalkyl-Cl_24-alkylcarbonyl group which is
optionally substituted with hydroxy, amino, halogen, or
oxo; an aryl, aryl-Cl_4-alkyl, arylcarbonyl or
aryl-Cl_4-alkylcarbonyl group optionally substituted in
the aryl moiety with hydroxy, amino, C1_4-alkyl,
Cl_4-alkoxy, nitro, halogen, phenoxy, or mono- or
di-halogen-Cl_4-alkyl; terpenyl;
tri(Cl_4-alkyl)silylethyl; heterocyclyl;
e t heterocyclyl-C1_4-alkyl; or
-~ heterocyclyl-C1_4-alkylcarbonyl;
a group o~ the formula II or IIa
R3o~(CH2)q~StO)m-CH2CH2- II
tR3O~(CH2)q~S(O)m~CH2~2CH~CH2~IIa
wherein R30 is H, carboxy, C1_4-alkoxycarbonyl,
hy~-o~, amino, or a matrix MA, q is an integer
from 1 to 24, and m is O or 2; or
a group of the formula III or IIIa
Phe-s(o)m-cH2cH2- III
tPhe-S(O)m-CH2]2CH~CH2~ IIIa
wherein m is as defined above, and each Phe is
35 phenyl optionally substituted with hydroxy, amino,
Cl_4-alkyl, Cl_4-alkoxy, nitro, halogen, phenoxy or
mono- or di-halogen Cl_4-alkyl; or phenyl-C1_4-alkyl
optionally monosubstituted in the phenyl moiety
SUB~ JTE SHEET
W095/00S27 2 ~ 6 4 9 6 ~ PCT/SE94100604
with hydroxy, amino, C1_4-alkyl, Cl_4-alkoxy, nitro,
halogen, phenoxy, or mono- or
di-halogen-C1_4-alkyl;
a group of the formula IV
R40cH2cH(cH2Rso)cH2- IV
wherein R40 and R50 independently are halogen; or0
a group Q-(Spacer) r~, where r is an integer O or l, and
Q is a matrix MA or a group -COO-MA;
in therapy, especially for the treatment or prophylaxis in
humans of conditions involving infection by Helicobacter pylori
of human gastric mucosa. Another aspect of the invention
relates to the use of said compounds for the preparation of
pharmaceutical compositions for use against the above mentioned
conditions.
DETAILED DESCRIPTION OF THE INVENTION
In the present context, the terms "Cl_4-alkyl", "C1_8-alkyl" and
"Cl_24-alkyl" as a separate group or as part of a group
designates alkyl ~LOu~ with 1-4, 1-8 or 1-24 carbon atoms
which may be straight or branched such as methyl, ethyl,
propyl, isopropyl, butyl, isobutyl, tert.butyl, dimethylbutyl,
pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl,
hexadecyl, octadecyl, etc.
In the carbon chain the definition "Cl_24-alkyl" is used
herein, but also shorter number of carbon atoms in the carbon
chain is possible as "Cl_8-alkyl" or "Cl_4-alkyl".
The term "Cl_4-alkyl" is used herein when substituents are
defined.
The term "C3_8-cycloalkyl" as a group or as part of a group
designates a cyclic alkyl group with 3-8 carbon atoms such as
SUB~ JTE SHEET
WOg5/~7 21 6 ~ ~ ~1 PCT/SE94/00604
_7_
cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,
or cyclooctyl.
The term "C2_24-alkenyl" designates unsaturated alkyl groups
with 2-24 carbon atoms which may be straight or branched,
preferably straight, in which the double bond may be present
anywhere in the chain, for example vinyl, l-propenyl,
2-propenyl, hexenyl, decenyl, hexadecenyl, octadecenyl. The
term "C2_24-alkynyl" designates an alkyl group with 2-24 carbon
atoms and incorporating a triple bond, e.g. ethynyl,
1-p~o~yl.yl, 2-propynyl, 2-butynyl etc. The term "halogen"
designates Cl, Br, I and F, preferably F and Cl.
The terms "C1_4-alkoxy" and "Cl_24-alkoxy" designate yLou~
comprising an oxa function substituted with an alkyl group as
defined above.
The terms "aryl" and "aryloxy", either as a separate group or
as part of a group, designates phenyl or naphthyl, preferably
phenyl.
The term "aryl-amide" defines either aryl-NH-C(O)- e.g.
anilids, or aryl-C(O)-NH- e.g.benzamide.
The term ~terpenyl moiety" designates ~G~ derived from some
of the various unsaturated hydrocarbon compounds generically
known as the terpenes, namely the monoterpenes and the
sesquiterpenes, as well as hydroxy or oxo derivatives thereof.
Examples of such ~.ou~ are myrcenyl, (-)-limonenyl,
terpineloyl, (+)-~-pinenyl, geraniolyl, (-)-mentholyl,
(-)-camphoryl, farnesolyl, ~-eudesmolyl, and manoolyl.
In the present context, the term "oligosaccharide" designates
an oligosaccharide containing 4-10 monosaccharide units,
preferably 4-7 monosaccharide units, the monosaccharide units
being selected from aldohexo-ec (i.e. D-glucose, L-glucose,
D-galactose, L-galactose, D-mannose, L-mannose, D-talose,
L-talose, D-allose, L-allose, D-altrose, L-altrose, D-gulose,
L-gulose, D-idose, or L-idose) or their derivatives, where the
oligosaccharide may be linear or branched with the proviso that
SlJB~ I 11 ~E SHEEr
woss/~7 PCT/SE94/006W
2I'S~'9~ 8-
there are no more than sëven monosaccharide units in the
longest chain in the oligosaccharide.
As indicated above, the wavy lines on the carbon atoms
S neighbouring the ring oxygen atoms in groups Y, A, B, C, D, and
E signify that the bonds in question which are glycosidic bonds
have either the ~- or the ~-configuration. It is clear that
each of the bonds in question on a particular group Y, A, B, C,
D, and E may assume the ~- or the ~-configuration independent
of the corresponding bonds on the other groups.
A mono- or di-halogen-Cl_4-alkyl group may be substituted in
any position and if substituted with 2 halogen atoms, the
halogen atoms may be the same or different.
The term ~'heterocyclyl~' designates a monocyclic 5- or
6-membered, or a fused bicyclic (each ring being 5- or
6-membered), aromatic or partly or fully saturated heterocyclic
group containing from one to four hetero atoms per ring, the
heteroatoms being selected independently from O, S and N and
bound either via a carbon atom or v~a a nitrogen atom. Typical
but non-limiting examples of such groups may comprise pyrrolyl,
pyrazolyl, pyridinyl, thienyl, thiazolyl, oxazolyl, imidazolyl,
isoxazolyl, isothiazolyl, furyl, pyrazinyl, pyrimidinyl,
pyridazinyl, 2H-1,3-oxazinyl, 4H-1,3-oxazinyl, 6H-1,3-oxazinyl,
2H-1,3-thiazinyl, 4H-1,3-thiazinyl, 6H-1,3-thiazinyl,
lH-1,2,3-triazolyl, 2H-1,2,3-triazolyl, lH-1,2,4-triazolyl,
4H-1,2,4-triazolyl, indolyl, purinyl, piperidyl or piperidino,
morpholinyl or morpholino, piperazinyl, tetrahydrofuryl,
thiazolidinyl, oxazolidinyl, imidazolidinyl, isoxazolidinyl,
isothiazolidinyl, pyrrolidinyl, lH-tetrazolyl, or
2H-tetrazolyl.
The term "acyl residue of a naturally occurring amino acid"
designates the acyl residue of the L-amino acids occurring in
proteins in nature, e.g. alanyl, valyl, leucyl, isoleucyl,
prolinyl, phenylalanyl, tryptophanyl, methionyl, glycyl, seryl,
threonyl, cysteinyl, ~yLG~yl, asparagyl, glutamyl, lysyl,
SUB~ 111 IJTE SHEET
W095/~ ~ 6~g~6~ PCT/SE94/00604
_g_
arginyl, histidyl and the acyl residues of aspartic acid and
glutamic acid, the acyl residue referring both to the carboxy
group next to the amino function as well as the carboxy group
at the end of the respective side chains, preferably, however,
the carboxy ~lo~ next to the amino functions.
The term "sphingoid" refers to D-erythro-2-amino-1,3-
octadecanediol, its homologs and stereoisomers and to hydroxy
and unsaturated derivatives thereof, including ceramide (see
further definitions in Journ. of Lipid Research, vol. 19,
(1978), 617-631).
The term ~steroid~ refers to well-known steroids as
cholesterol, cortisone, hydrocortisone, corticosterone,
betamet~asone, prednosolone, prednisone etc.
The term "matrix" as used herein and designated as "MA"
signifies any organic or inorganic, polymeric or macromolecular
structure to which the aglycon part of the O-, S-, C-, or
N-glycosidic compound of the formula Ia, Ib, Ic, Id, Ie or If
is attached either covalently or by e.g. hydrophobic
interaction. Examples of such matrices are residues of
proteins, glyco~G~eins, polypeptides, polysaccharides,
liposomes, emulsions, plastic polymers and inorganic materials.
Residues of proteins are preferably bonded through nucleophilic
y~OU~S in the proteins, e.g. groups such as amino, hydroxyl and
mercapto groups. Proteins or polypeptides themselves may be any
of a wide variety of substances, in particular biologically
compatible proteins such as globulins, albumins such as human
serum a~bumin (HSA), bovine serum albumin (BSA) or sheep serum
albumin (SSA), ovalbumin, fibrins, or "key-hole" limpet
haemocyanin (KLH), glycoproteins such as bovine or human whole
casein or lectins, and the like. Other examples of such
matricee~ are synthetic polymers where one or several amino
acids are coupled to a polymer of defined size(s), e.g.
polylysi.ne or oligolysine. In the various proteins or
polypeptides, the linkage to the remainder of the group R may
be through amino groups or through carboxyl groups.
SUB~ JTE SHEEr
woss/~s27 2 1 6 ~9 `~ 1 PCT/SE94100604
--10--
The polysaccharides, to which the o-, S-, C-, or N-glycosidic
compounds ~re attached, may be any of a wide variety of
polysaccharides. The aglycon part of the compound of formula
Ia, Ib, Ic, Id, Ie or If may be bonded through hydroxyl groups
on ordinary polysaccharides such as cellulose, sepharose,
starch or glycogen, through amino groups on amino saccharides
such as chitosane or aminated sepharose, and through mercapto
gLou~s of thio-modified polysaccharides.
Liposomes may be any biocompatible, biodegradable microesicular
system compose of one or several bilayers surrounding aqueous
compartments, within which a variety of agents can be
encapsulated: hydrophobic agents in the lipid bilayers and
hydrophilic agents in the inner aqueous space. The
lS physicochemical properties of the liposomes are mainly
dependent on the lipid composition.
L~posomes are composed of phospholipids, such as egg yolk
phospholipids, soya phospholipids, synthetic
phosphatidylcholine e.g dimyritoylphosphatidylcholine (DMPC)
and/or dipalmtoylphosphartidylchlorine (DPPC) or purified
phosphatidylcholines of vegetable origin or other lipids, such
as galactolipids, sphingolipids or glycosphingolipids.
Emulsions are heterogenous mix~u~e~ of two or more imiscible
liquids. To stabilize these systems an emulsifier is added. The
emulsifier is oriented at the interface of the imisible liquids
and usually only one phase persist in dropted form.
Emulsions fall into two general categories. The heterogenous
system described by droplets of an organic liquid dispersed in
a continuous water phase is called oil-in-water emulsion to/w).
Alternatively, the heterogenous system described by droplets of
water dispersed in a continuous oil phase is called water-in-
oil emulsion (w/o).
Any vegetable oil such as soybean oil, safflower oil, sesameoil, peanut oil, cottonseed oil, borago oil, sunflower oil,
SUB~ ITE SHEET
wo 95/00527 2 1 6 ~ 9 61 PCT/SE94/00604
--11--
corn oil, olive oil, medium chain triglycerides (such as
Miglyol R ), or acetylated monoglycerides may be used as
internal or continuous phase.
Examples of plastics to which the aglycon part of the compounds
of the formula Ia, Ib, Ic, Id, Ie or If may be attached are
aminated latex, thiolated, aminated, or hydroxylated
polys~y~ene, polyacrylamide and polyvinyl alcohol. Other
possible carriers are beads and gels of carbohydrate origin or
polymers where carbohydrates are used in combination with other
polymeric materials such as sephacryl. These gels are further
substituted with ~,ou~ such as amino, thiols, cyano, active
esters and disulfides. The plastics in question may be in the
form of e.g. beads or film.
lS Examples of inorganic material, to which the aglycon part of
the compounds of the formula Ia, Ib, Ic, Id, Ie or If may be
attached are silicon oxide materials such as silica gel,
zeolite, diatomaceous earth, or the surface of various glass or
silica gel types such as thiolated or aminated glass, where the
silica gel or the glass may be in the form of e.g. beads.
Another example of an inorganic material is aluminium oxide.
Particularly preferred matrix MA is human serum albumin (HSA),
bovine serum albumin (BSA) and polyacrylamide (PAA).
An interesting embodiment of the invention i8 when the compound
of formula Ia, Ib, Ic, Id, Ie or If comprises a matrix MA, said
matrix incorporating a multiplicity (i.e. 2 or more, such as
2-lOO when the matrix is a protein such as BSA or HSA, or
lO-lO,OOO when the matrix is a polymer such as polyacrylamide)
of moieties of the formula Ia, Ib, Ic, Id, Ie and If. It is
contemplated that the presence of several such moieties will
substantially enhance the inhibiting effect of the entire
compound due to a multivalency-effect thereof on the bacteria.
It is also possible that the presence of several moieties of
the formula Ia, Ib, Ic, Id, Ie and If may even lead to
agglutination of the bacteria.
SUB~ ~ JTE SHEEr
wo gs/00s27 2`1~6 ~ 6 ~ PCT/SE94/00604~
--12--
When, in connection with the definition of formulas Ia, Ib, Ic,
Id, Ie and If, it is stated that the configurations of the
UbStitUentS R1A~ R2A~ R3A~ and R4ACH2 in A, the configurations
of the æubstituents R1B~ R2B~ R3B~ and R4sCH2 ~ ..
configurations of the substituents Rlc, R2C~ R3C~ and R4CCH2 in
C, the configurations of the substituents R1D' R2D' R3D~ and
R4DCH2 in D, and the configurations of the substituents R1E'
R2~ ~3~, and R4ECH2 in E independently are D-gluco, L-gluco,
D-galacto, L-galacto, D-manno, L-manno, D-talo, L-talo, D-allo,
L-allo, D-altro, L-altro, D-gulo, L-gulo, D-ido, or L-ido, this
is intended to mean that the stereochemical substitution
patterns that can be assumed by the various R-groups or
R-group-containing groups on the cyclic groups A, B, C, D and E
correspond to the stereochemical patterns formed by the 2-, 3-,
and 4-hydroxy groups and the 5-hydroxymethyl group in
D-glucose, L-glucose, D-galactose, L-galactose, D-mannose,
L-mannose, D-talose, L-talose, D-allose, L-allose, D-altrose,
L-alLIvsc, D-gulose, L-gulose, D-idose, or L-idose,
respectively.
It will be clear that the ylOUp~ Rl, R2, R3 and CH3 in the
group Y are arranged in such a configuration to give a
L-galacto-pyranosyl unit and that the group Y therefore is a
L-fucose unit or a derivative thereof.
In the compounds of the formula Ia, Ib, Ic, Id, Ie or If, it is preferred that Zl~ Z2~ Z3~ Z4~ Z5~ Z6~ Z7~ Z8~ Zg, Z1O~ Zll~ Z12
Z13, Z14, Z15 and Z16 are 0.
It is also preferred that at the most four, more preferably at
the most three, in particular one or two of R1A~ R2A~ R3A~ R4A~
R1B' R2B' R3B~ R4B~ R1C~ R2C, R3C, R4C, R1D~ R2D, R3D, R4D, R1E~
R2Er R3E, or R4E is a group of formula VII.
35 It is also preferred that R1A is a group VII in the
~-configuration.
SUB~ UTE SHEET
W095l00527 1~q961 PCT/SE94/00604
-13-
It is also preferred that the configuration f R1A' R2A~ R3A and
R4ACH2 in A are D-galacto, A being in the ~-configuration.
Particularly preferred compounds are those wherein R1A is a
--5 group VII in the a-configuration and the configuration of R1A'
R2A' R3A and R4ACH2 in A are D-galacto, A being in the
~-configuration, especially A is Fuc~1-2Gal~.
It is also preferred that R2~ is Z3, Z5~ Z8~ or Z12~ and the
gUration of R1B~ R2B, R3B~ and R4gCH2 in B are D-gluco B
being in the ~-configuration.
It is al~;o preferred that R1B is an acetamido group.
Particularly preferred compounds are those wherein R1A is a
group VII in the ~-configuration; the configuration of RlA,
R2A ~ ~3A and R4ACH2 in A are D-galacto, A being in the
~-configuration; R2B is Z3, Z5, Z8~ or Z12; and h
configuration of R1B~ R2B~ R3B~ and R4BCH2 in B are D-gluco, B
being in the ,B-configuration and R1B is an acetamido group.
Especially interesting are those compounds in which the A-Z3-B
is Fuc~1-2Gal~1-3GlcNAc~ or Fuc~1-2Gal~1-3(Fuc~1-4)GlcNAc~,
those com~oull~s in which A-Z5-B-Z6-C is
Fuc~1-2Gal~1-3GlcNAc~1-3Gal~ or
Fuc~1-2Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~, those compounds in which
A-Za-B-Zg C-Zlo~D is
GalNAc~1-3(Fuc~1-2)Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~ or
Fucel-2Ga~l-3(Fuc~l-4)GlcNAc~l-3Gal~l-4Glc~and those cu~ounds
in which A-Z12-B-Zl3-c-Zl4-D-zls-E is
GalNAc~1-3(Fuc~1-2)Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~l-4Glc~.
It is also preferred that R3B is a group of the formula VII in
the ~-configuration.
Particularly preferred compounds are those wherein the
Configurations f R1A~ R2A~ R3A~ and R4ACH2 in A and f R1B~ R2B~
R3B~ and R~sBCH2 in B are D-galacto, and the configurations of
SU~ UTE SHFET
PCT/SE~/a~O~
W095/00527 2 1 6 4 ~ 6 1.
-14-
Rlc~ R2CI R3C, and R4CCH2 in C are D-gluco, A being in the
~-configuration, and B and C being in the ~-configuration, and
in which R1B and R3C are groups of the formula VII in the
~-configuration, and in which R1A and R~c are acetamido groups,
and R2B iS Z5, Z8 or Z12 ~ and R2C is Z6 ~ Z9 or Z13.
An interesting class of compounds is that in which the
carbohydrate moiety contains the structure Y-Zl-A- where Zl is
O and the L-fucose unit Y is linked to the 2-position of A.
Examples of interesting basic carbohydrate structures in this
class are those having the following formulae where the
substituents Rl, R2, R3, R1A~ R2A' R3A' and R4A each
indicated as OH, although this is not to be construed as
limiting the definitions of the R-substituents in this manner;
rather~ Rl~ R2, R3, R1A~ R2A~ R3A~ and R4A should be considered
as being able to assume all the meanings defined above in
connection with the formulae Ia, Ib, Ic, Id, Ie and If. Thus,
the structure Y-Zl-A- may be
Fuc~1-2All~1-
Fuc~1-2Alt~1-
Fuc~1-2Glc~1-
Fuc~1-2Man~l-
Fuc~1-2Gul~l-
Fuc~1-2Ido~1-
Fuc~1-2Gal~1-
Fuc~1-2Tal~l-
When the groups Rl, R2, R3, R~, RZA' R3A' R4A' R1B' R2B' R3B'
R4B~ Rlc~ R2c~ R3c~ R4cr R1D' R2D~ R3D~ R4D~ R1E' R2E~ R3E~ or R4E
in Y, A, B, C, D, and E are not hydroxyl, they may preferably
be selected among the following:
H, Cl, F, azido, guanidyl, methyl, ethyl, propyl, vinyl, allyl,
prop-l-enyl, ethynyl, prop-2-ynyl, prop-1-ynyl, acetyl,
cyclopropyl, cyclopropylmethyl, methoxymethyl, hydroxymethyl,
phenyl, oxo, methylene, thiol, amino, methoxy, ethoxy, propoxy,
butoxy, hexyloxy, decyloxy, tetradecyloxy, octadecyloxy,
vinyloxy, allyloxy, 1-propen-1-yloxy, crotyloxy,
SVB~ 111 ~JTE SHE~
O 951~527 1 ~ 1 9 61 ~ PCT/SE94/00604
--15--
3-buten-1-yloxy, 2-hexen-1-yloxy, 5-hexen-1-yloxy,
S-decen-l-yloxy, 9-decen-1-yl~y, ll-tetradecen-1-yloXy~
oleoyl, ethynyloxy, 2-propyn-1-yloxy, 1-propyn-1-yloxy,
methylthio, methylamino, dimethylamino, cyclopropoxy,
cyclopropylmethoxy, methoxymethoxy, phenoxy, benzyloxy,
2-furylmethoxy, 2-thienylmethoxy, 2-pyridylmethoxy,
trimethylsilyloxy, trimethylsilylethoxy, acetoxy, propionyloxy,
butyryloxy, h~xAnoyloxy, decanoyloxy, tetradecanoyloxy,
octadecanoyloxy~ acetamido, N-methylacetamido, acetylthio,
glycyloxy, or alanyloxy.
Interesting examples of aglycon groups R are the following:
Methyl, ethyl, propyl, isopropyl, butyl, sec.butyl, isobutyl,
tert.butyl, pentyl, isopentyl, 2-methylbutyl, 1-methylbutyl,
1-ethylpropyl, hexyl, isohexyl, 3-methylpentyl, 2-methylpentyl,
l-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, heptyl, isoheptyl,
4-methylhexyl, 3-methylhexyl, 2-methylhexyl, 1-methylhexyl,
3-ethylpentyl, 2-ethylpentyl, l-ethylpentyl, 1-propylbutyl,
octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl,
tetracosyl, cyclG~ro~yl, cyclopropylethyl, cyclobutyl,
cyclobutylmethyl, cyclopentylmethyl, cyclopentylprop-3-yl,
cyclohexyl, cyclohexylmethyl, cyclohexylprop-3-yl, cycloheptyl,
phenyl, 4-nitrophenyl, benzyl, 4-phenylprop-1-yl,
3-hexylthio-2-(hexylthio)methylprop-1-yl,
3-hexylsulfonyl-2-(hexylsulfonyl)methylprop-1-yl,
3-decylthio-2-(decylthio)methylprop-1-yl,
3-decylsulfonyl-2-(decylsulfonyl)methylprop-1-yl,
8-amino-3l6-dioxaoct-1-yl, 1,3-dihydroxyprop-2-yl,
1,3-diaminoprop-2-yl, 3-hydroxy-2-(hydroxymethyl)prop-1-yl,
2-phenylthioethyl or trimethylsilylethyl.
In a group R comprising a matrix MA, the linkage between the
matrix MA and the remainder of R may typically be through any
of the spacers well known in the field of protein conjugates,
cf. for example J.H. Pazur, Adv. Carbohydr. Chem. Biochem., Vol
39, (1980), 40S-447; Y.C. Lee & R.T. Lee, "Glycoconjugates~,
Vol. 4 Part B, 57-83, Ed. Horowitz, Academic Press, N.Y.
(1982); and G. Magnusson, FEMS Symposium, 215-228 (1986). In
SUB~ 11- ~JTE SHEEt'
W095/00527 21 ~ ~ 9 G 1 --7 6-- PCT/SE94/006~
the present context, the term "Spacer" is intended to mean a
molecule moiety which links the active substance to a carrier.
A spacer molecule is designed to have two different
~unctionalities, each reacting specifically with another
functionality, a linear moiety being placed between these two
functionalities. By li~ki~g the active substance to a carrier
v~a a Spacer, one makes the active substance more accessible,
e.g. to H. pylori adhesins or colonization factor antigens.
The Spacer can be defined as (W)v-S'-P', wherein S' is an Cl_
alkyl~ an C2-24 alkenyl, an Cl_24alkylaryl, an arylCl_24alkyl an
arylC1_24alkylaryl, an Cl_24alkylarylcl-24alkyl group which
groups may be interrupted by carbonyl, thiocarbonyl,
oxycarbonyl, carbonyloxy, carbonylamino, aminocarbonyl, aza,
oxa or ~hia groups; an aryl group, an aryloxy, an C1_24alkoxy,
a polyethyleneglycol group, a steroid group, a sphingoid group;
all groups may be substituted with carboxyl, Cl_4alkylcarbonyl,
amide, hydroxy, alkoxy, aryloxy, phenoxy;
P' is NH--C(S), NH--C(O), C(O), NH, C(S), C(O)O, (O)CO, SO, SO2,
SO3, SO4, PO3, PO4;
W iS N~-C(S), NR-C(O), C(O), C(S), C(O)O, (O)CO, SO, S02, SO3,
SO4, PO2, PO3 ~ PO4 '
with the proviso that when Zl~ Z2 ~ Z4 ~ Z7 ~ Zll or Z16 are CH2
then W cannot be P02,
with the proviso that when Zl~ Z2~ Z4~ Z7~ Zll or Z16 are O or S
then W cannot be ~O)CO, SO4 or PO4, and with the proviso that
when Zl, Z2~ Z4~ Z7~ Z11 or Z16 are NH then W cannot be NH-C(S),
. NH-C(O) ~ (O)CO, SO4, PO4; and v is an integer O or 1.
The atom of the sugar moiety which linkages to the spacer is
selected among from the following: -O-, -S-, -NH-, -CH2-
preferably -O-.
In the compounds of the formulas Ia, Ib, Ic, Id, Ie and If, the
various groups R carrying the matrix MA may themselves comprise
the spacer and the linkage. Specific and typical examples of
linkages are those formed through amino group- or keto group-
SUB~ JTE SHEEI-
W095/00~7 21 6 ~ 9 ~1 PCT/SE94100604
-17-
containing matrices.: Such linkages between the spacer and the
matrix may have the following general structures:
-NH-C(S)N~- or -C(O) -NH-, or -NH-C(OJ-
-` 5
wherein ~he atoms marked bold and italic orginate from the
given matrix.
The number of structures of the formulas Ia, Ib, Ic, Id, Ie or
If on each matrix unit may be mono- or multivalent and may vary
between ~ to lO,OOO, depending on the nature of the matrix.
Below follow a non-limiting list of examples of spacers
suitable between Q and the remainder of R
spacer 1 ~~~~~
spacer 2 ~~ ~~ O~'~
OH CH~
spacer 3 ; ~H~\ ~ Cl 1~1 ~ Nl;
spacer 4 ~ spacer 5 ~ ~ ~
In the li~t of spacers given above and below, the atoms marked
in bold italics originate from the matrix in question.
The vertical wavy lines on the left and right ends in the
spacer above signify that there are bonds at the ends.
As examples of compounds of the general formulas Ia, Ib, Ic,
Id, Ie or If comprising matrix moieties, the following may be
mentioned-
SUB:~ 111 UTE SHEET
WO 95/00527 216 4 9 6 I PCT/SE94/00604~
--18--
~0~ --~NHCH~-BSA
' m'
O
--O ~ ~ NH C-polyacrylam~d~
. ..~.
-o
\ ~NH C-poJ~c~JJm;d~
\~o~ <~ OH
H O ~ cHzNHc~ ~cr~l~mld~
~.~ OH
H ~
- O~ - OH
HO~ ~-- --C~NH-N~-~S~
H OH
-\~o~ f' I ~ ~Jl( ~ GSA
SUE3~ JTE SHEET
WO 95/00527 21 6 ~ PCT/SEg4/00604
--19--
~0 (-CH2 )p CNH-8SA
~_ O (-CH2-)p ~--C--NH-HSA
.0 0
\ ~_O (-CH2-)p--~--C--PAA
~~ \ o_ ~_8 ~o~ ~_C_NH-HSA
--N _ 8~o --N - C - PAA
~ ~0~
~ ~C~
O
SUB~ 111 ~JTE SHEET
wogs/~7 PCT/SE94/00604
2`1G`~9 6~ -20-
When ~ is used in the examples above, this has the
meaning of mono-, di-, tri- or oligosaccharide as specified in
the text, and m' is an integer 0-5 and p is an integer 0-13.
When the matrix above is exemplified by BSA, HSA and
polyacrylamide(PAA) this can be any other protein or peptide or
other matrix specified in the text.
Specific examples of interesting compounds of the formula Ia,
Ib, Ic, Id, Ie or If are the following:.
Fuc~1-2Gal~l-O-propyl
Fuc~1-2Gal~1-O-iso~r~pyl
Fuc~1-2Gal~l-O-butyl
Fuc1-2Gal~l-O-tert-butyl
Fuc~1-2Gal~l-O-hexyl
Fuc~l-2Gal~l-O-octyl
Fuc~1-2Gal~l-O-decyl
Fuc~1-2Gal~l-O-tetradecyl
Fuc~1-2Gal~l-O-octadecyl
Fuc~l-2Gal~1-O-(C6bissulfide)
Fuc~1-2Gal~l-o-(C1Obissulfide)
Fuc~1-2Gal~l-O-(C6bissulfone)
Fuc~1-2Gal~l-O-(ClObissulfone)
Fuc~1-2Gal~l-O-(8-amino-3,6-dioxaoct-1-yl)
Fuc~1-2Gal~1-3GlcNAc~l-O-propyl
Fuc~1-2Gal~1-3GlcNAc~l-o-isopropyl
Fuc~1-2Gal~1-3GlcNAc~l-O-butyl
Fuc~1-2Gal~1-3GlcNAc~l-O-tert.butyl
Fuc~1-2Gal~1-3GlcNAc~l-O-hexyl
Fuc~1-2Gal~1-3GlcNAc~1-O-octyl
Fuc~1-2Gal~1-3GlcNAc~l-O-decyl
35 Fuc~1-2Gal~1-3GlcNAc~l-O-tetradecyl
Fuc~1-2Gal~1-3GlcNAc~1-O-octadecyl
Fuc~1-2Gal~1-3GlcNAc~-1-O-(C6bissulfide)
SlJ~ .ITE SHEET
wos5l0os27 Q PCT/SE94/00604
_2~ 6~`~Gl
Fuc~1-2Gal~1-3GlcNAc~-1-O-(C6bissulfone)
Fuc~1-2.al~1-3GlcNAc~-1-0-(8-amino-3,6-dioxaoct-1-yl)
Fuc~1-2Gal~1-3Glc~l-O-propyl
Fuc~1-2Gal~1-3Glc~1-O-isopropyl
Fuc~1-2Gal~1-3Glc~1-O-butyl
Fuc~1-2Gal~1-3Glc~1-O-tert.butyl
Fuc~1-2Gal~1-3Glc~1-O-hexyl
Fuc~1-2Gal~1-3Glc~l-O-octyl
Fuc~1-2Gal~1-3Glc~1-O-tetradecyl
Fuc~1-2Gal~1-3Glc~1-O-octadecyl
Fuc~l-2Gal~l-3Glc~1-O-(C6bissulfide)
Fuc~1-2Gal~1-3Glc~1-O-(C6bissulfone)
Fuc~1-2C~al~1-3Glc~1-O-(8-amino-3,6-dioxaoctyl)
wherein
C6bissulfide - 3-hexylthio-2-(hexylthio)methylprop-1-yl-
C~Obissulfide ~ 3-decylthio-2-(decylthio)methylprop-1-yl-
C6bissulfone ~ 3-hexylsulfonyl-2-(hexylsulfonyl)methylprop
--1--yl--
ClObissulfone ~ 3-decylthio-2-(decylthio)methylprop-1-yl-
. 25 Further interesting compounds are:
Fuc~1-2Gal~1-O-Me
Fuc~1-3Glc~1-O-Me
Fuc~1-3GlcNAc~1-O-Me
Fuc~1-3GlcNAc~l-Spacer l-BSA
Fuc~1-3GlcNAc~l-O tetradecyl
Fuc~1-4GlcNAc~l-O-Me
Fuc~1-4GlcNAc~1-Spacer 2-polyacrylamide
Fuc~1-4GlcNAc~1-O-tetradecyl
Fuc~1-4Gal~l-o-Me
Fuc1-6Gal~l-O-Me
Fuc~1-6Gal~1-Spacer 2-polyacrylamide
Fuc~1-2Gal~l-Spacer 2-polyacrylamide
SUB~ ~ JTE SHEET
wo g5,00527 2 1 ~ 4 ~ 6 ~ -22- PCT/SEg4/00604
Fuc~1-2Gal~l-Spacer-1-BSA
Fuc1-2Gal~l-Spacer 1-HSA
Fuc~1-2Gal~1-Spacer 4-BSA
Fuc~1-2Gal~1-Spacer 4-HSA
Fuc~1-2Gal~l-spacer s-polyacrylamide .
Fuc~1-2Gal~1-0-tetradecyl
Fuc~1-2Gal~1-3GlcNAc~1-Spacer 5-polyacrylamide
Fuc~l-2Gal~l-3Glc~Ac~l-spacer 4-BSA
Fuc~1-2Gal~1-3GlcNAc~l-Spacer 4-HSA
Fuc~l-2Gal~l-3GlcNAc~l-spacer 2-polyacrylamide
Fuc~l-2Gal~l-3GlcNAc~l-spacer 1-HSA
Fuc~l-2Gal~l-3GlcNAc~l-spacer 1-~SA
Fuc~l-2Gal~l-3GlcNAc~l-o-tetrade
Fuc~1-2Gal~1-3Glc~1-Spacer 1-HSA
Fuc~1-2Gal~1-3Glc~1-Spacer 1-BSA
Fuc~l-2Gal~l-3Glc~l-spacer 4-HSA
Fuc~1-2Gal~1-3Glc~1-spacer 4-BSA
Fuc~l-2Gal~l-3Glc~l-spacer 2-polyacrylamide
Fuc~l-2Gal~l-3Glc~l-spacer s-polyacrylamide
Fuc~1-2Gal~1-3(Fuc~1-4)Glc~1-Spacer 1-HSA
Fuc~1-2Gal~1-3(Fuc~1-4)Glc~1-Spacer 1-BSA
Fuc~1-2Gal~1-3(Fuc~1-4)Glc~1-Spacer 4-HSA
Fuc~1-2Gal~1-3(Fucal-4)Glc~l-Spacer 4-BSA
Fuc~1-2Gal~1-3(Fuc~1-4)Glc~1-Spacer 2-polyacrylamide
Fuc~1-2Gal~1-3(Fuc~1-4)Glc~l-Spacer 5-polyacrylamide
Fucel-2Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~1-Spacer 3-BSA
Fuc~1-2Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~1-Spacer 2-polyacrylamide
Fuc~1-2Gal~1-3(Fuc~1-4)GlcN~c~1-3Gal~1-Spacer 5-polyacrylamide
Fuc~1-2Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~1-0-tetradecyl
Fuc~1-2Gal~1-4Glc~1-Spacer l-BSA
Fuc~1-2Gal~1-4Glc~1-Spacer 2-polyacrylamide
Fuc~1-2Gal~1-4Glc~l-0-tetradecyl
Gal~1-4(Fuc~1-3)GlcNAc~1-Spacer 1-BSA
Gal~1-4(Fuc~1-3)GlcNAc~1-Spacer 2-polyacrylamide
Gal~1-4(Fuc~1-3)GlcNAc~1-O-tetradecyl
Fuc~1-2Gal~1-3GlcNAc~1-3Gal~1-Spacer 3-BSA
Fuc~1-2Gal~1-3GlcNAc~1-3Gal~1-Spacer 3-HSA
Fuc~1-2Gal~1-3GlcNAc~1-3Gal~1-Spacer 5-polyacrylamide
SUB~ I ~ ~ LITE SHEET
Wog5/00~7 -23~ PCT/SE94/00604
~Uc~l-2Gal~l-3GlcNAc~l-3Gal~l-spacer l-HSA
Fuc~1-2~al~1-3GlcNAc~1-3Gal~l-Spacer 5-BSA
Fuc~1-2Gal~1-3GlcNAc~1-3Gal~l-Spacer 4-HSA
Fuc~1-2Gal~1-3GlcNAc~1-3Gal~1-Spacer 4-BSA
Fucal-2~al~1-3GlcNAc~1-3Gal~l-Spacer 2-polyacrylamide
Fuc~1-2Gal~1-3GlcNAc~1-3Gal~l-o-tetradecyl
GalNAc~1-3~Fuc~l-2)3Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~l-Spacer-
3-8SA
GalNAc~1-3(Fucal-2)3Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~l-Spacer-
2-polyacrylamide
GalNAcal-3tFuc~1-2)3Gal~l-3(Fucc1-4)GlcNAc~1-3Gal~1-0-tetra-
decyl
Fucal-2~al~1-4(Fuc~1-3jGlc~l-Spacer l-BSA
Fucal-2Gal~1-4(Fuc~1-3)Glc~l-Spacer 2-polyacrylamide
15. Fucal-2Gal~1-4(Fuc~1-3)Glc~l-0-tetradeCyl
Fucal-2(3-o-methyl)Gal~1-0-tetradecyl
~UC~1-2(3-O-methyl)Gal~l-Spacer 1-BSA
Fucal-2(3-o-methyl)Gal~1-spacer 2-polyacrylamide
Fucal-2(3-o-allyl)Gal~1-spacer 1-BSA
Fucal-2(3-o-allyl)Gal~l-spacer 2-polyacrylamide
Fucal-2(3-O-allyl)Gal~1-0-tetradecyl
Fucal-2(3-o-propyl)Gal~l-Spacer l-HSA
Fucal-2(3-O-propyl)Gal~l-Spacer l-BSA
Fuc~l-2(3-o-propyl)Gal~l-spacer 2-polyacrylamide
~ucal-2(3-O-propyl)Gal~l-Spacer 4-HSA
Fucal-2(3-o-propyl)Gal~l-Spacer 4-BSA
Fucal-2t3-O-propyl)Gal~l-Spacer 5-polyacrylamide
Fucal-2(3-O-butyl)Gal~l-Spacer 1-BSA
Fucal-2(3-o-butyl)Gal~l-Spacer 2-polyacrylamide
Fucal-2(3-O-butyl)Gal~l-0-tetradecyl
Fucal-2(3-o-methyl)Gal~1-3GlcNAc~l-Spacer l-BSA
Fuc1-2(3-o-methyl)Gal~1-3GlcNAc~l-Spacer 2-polyacrylamide
. Fucal-2(3-0-methyl)Gal~1-3GlcNAc~l-0-tetradecyl
Fucal-2(3-o-allyl)Gal~1-3GlcNAc~l-Spacer l-BSA
Fuc~1-2(3-O-allyl)Gal~1-3GlcNAc~l-Spacer 2-polyacrylamide
Fucal-2(3-o-allyl)Gal~1-3GlcNAc~l-0-tetradecyl
Fucal-2(3-o-propyl)Gal~1-3GlcNac~l-Spacer l-HSA
Fucal-2t3-o-propyl)Gal~l-3GlcNac~l-Spacer l-BSA
SUB~ JTE SHEEr
WO 95/~527 216 ~ 9 6 I PCT/SE94100604
-24-
Fucal-2(3-O-propyl~Gal~1-3GlcNac~1-Spacer 2-polyacrylamide
Fuc~1-2(3-O-propyl)Gal~1-3GlcNac~1-Spacer 4-HSA
Fuc~1-2(3-O-propyl)Gal~1-3GlcNac~1-SpaCer 4-BSA
Fucal-2(3-o-propyl)Gal~1-3GlcNac~1-Spacer 5-polyacrylamide
Fucal-2(3-O-butyl)Gal~1-3GlcNAc~1-Spacer 1-BSA ..
Fucal-2(3-o-butyl)Gal~l-3GlcNAc~l-spacer 2-polyacrylamide
Fucal-2(3-O-butyl)Gal~1-3GlcNAc~l-O-tetradecyl
Fucal-2(3-O-propyl)Gal~1-3(Fucal-4)GlcNac~l-Spacer l-HSA
Fucal-2(3-o-propyl)Gal~1-3(Fucal-4)GlcNac~l-SpaCer l-BSA
Fucal-2(3-O-propyl)Gal~1-3(Fuc~1-4)GlcNac~l-Spacer 2-
polyacrylamide
Fucal-2(3-o-propyl)Gal~1-3(Fucal-4)GlcNac~l-Spacer 4-HSA
Fucal-2(3-o-propyl)Gal~1-3(Fuc~1-4)GlcNac~l-Spacer 4-BSA
Fucal-2(3-o-propyl)Gal~1-3(Fucal-4)GlcNac~1-Spacer 5-
polyacrylamide
Fucal-2(3-O-methyl)Gal~1-4GlcNAc~l-Spacer 1-BSA
Fucal-2(3-O-methyl)Gal~1-4GlcNAc~1-Spacer 2-polyacrylamide
Fuc1-2(3-O-methyl)Gal~1-4GlcNAc~l-O-tetradecyl
Fucal-2(3-O-allyl)Gal~1-4GlcNAc~1-Spacer 1-BSA
Fucal-2(3-O-allyl)Gal~1-4GlcNAc~1-Spacer 2-polyacrylamide
Fucal-2(3-O-allyl)Gal~1-4GlcNAc~1-O-tetradecyl
Fuc~1-2(3-O-butyl)Gal~1-4GlcNAc~1-Spacer l-BSA
Fucal-2(3-O-butyl)Gal~1-4GlcNAc~l-Spacer 2-polyacrylamide
Fucal-2(3-O-butyl)Gal~1-4GlcNAc~l-O-tetradecyl
Fucal-2(3-O-methyl)Gal~1-3Glc~1-Spacer 1-BSA
Fucal-2(3-o-methyl)Gal~1-3Glc~l-Spacer 2-polyacrylamide
Fucal-2(3-O-methyl)Gal~1-3Glc~1-O-tetradecyl
Fucal-2(3-O-allyl)Gal~1-3Glc~1-Spacer 1-BSA
Fucal-2(3-O-allyl)Gal~1-3Glc~l-Spacer 2-polyacrylamide
Fucal-2(3-O-allyl)Gal~1-3Glc~l-O-tetradecyl
Fuc~1-2(3-O-butyl)Gal~1-3Glc~l-Spacer l-BSA
Fucal-2(3-O-butyl)Gal~1-3Glc~l-Spacer 2-polyacrylamide
Fucal-2(3-O-butyl)Gal~1-3Glc~l-O-tetradecyl
Fucal-2Gal~1-4GlcNAc~1-Spacer 1-BSA
Fuc~1-2Gal~1-4GlcNAc~l-spacer 2-polyacrylamide
Fuc~1-2Gal~1-4GlcNAc~l-O-tetradecyl
Gal~1-3(Fucal-2)Gal~l-Spacer 1-BSA
Galal-3(Fucal-2)Gal~l-Spacer 2-polyacrylamide
SUB~ JTE SHEEr
wo 95/00s27 ~?1 6~9 S PCT/SEg4/00604
Gal~1-3tFuc~1-2)Gal~1-0-tetradecyl
GalNAc~l-3(Fuc~l-2)Gal~l-4Glc~l-spacer 1-BSA
GalNAcel-3(Fuc~l-2)Gal~l-4Glc~l-spacer 2-polyacrylamide
GalNAcal-3(Fuc~1-2)Gal~1-4Glc~1-0-tetradecyl
Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~1-4Glc~1-Spacer 1-BSA
Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~1-4Glc~l-Spacer 2-polyacrylamide
Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~1-4Glc~1-0-tetradecyl
Gal~1-3(Fuc~1-4)GlcNAc~l-Spacer l-BSA
Gal~1-3(Fuc~1-4)GlcNAc~l-Spacer 2-polyacrylamide
Gal~1-3(Fuc~1-4)GlcNAc~1-0-tetradecyl
In the present application, such as the list above, specific
compounds or parts of compounds may be named or represented in
a condensed form corresponding to the recommendations
concerning nomenclature of glycoproteins, glycopeptides, and
peptidoglycans made by the Joint Commission on Biochemical
Nomenclature under the International Union of Pure and Applied
Chemistry and the International Union of Biochemistry (cf. Pure
& Applied Chem., Vol. 60, No. 9, pp 1389-1394, 1988).
In another aspect, the invention concerns a pharmaceutical
composition comprising a compound of the formula Ia, Ib, Ic,
Id, Ie or If as defined above or a mixture thereof in
combination with at least one anti-ulcer medicament, or with at
least one antibacterially active compound, or mixtures thereof,
as well as a pharmaceutically acceptable carrier.
The term "anti-ulcer medicament" is intended to denote any
substance or composition which is able to reduce or participate
in reducing gastrointestinal ulcerations, in particular
ulcerations in the stomach or duodenum. Pharmaceutical
compositions according to the invention containing such
substances or compositions have the potential advantage of
being able to provide a dual effect by on the one hand reducing
the ulceration and on the other hand simultaneously lowering
the degree of infection in the stomach by H. pylori by
preventing or inhibiting the adhesion of the bacterium onto the
gastric or duodenal mucosa, thereby further promoting the
SUB~ 111 ~JTE SHEET~
.
Wogsl~7 2 1 ~ ~ 9 6 I PCT/SE94/00604
-26-
healing of an ulcer; Suitable types of anti-ulcer medicaments
are gastric secretion inhibiting compounds (primarily acid
secretion inhibiting compounds) and antacids.
In a preferred aspect of the use according to the invention,
the pharmaceutical composition prepared is adapted to be
administered in combination with a preparation for s~ Ard
therapy of gastritis or ulcus, such as preparations containing
anti-ulcer or anti-gastritis medicaments, e.g. selected among
gastric secretion inhibiting compounds such as omeprazole,
- cimetidine, ranitidine, lansoprazole, pantoprazole, sucralfate,
famotidine, or nizatidine, or antacids such as magnesium
hydroxide, aluminium hydroxide, calcium carbonate, sodium
carbonate, sodium hydrogen carbonate, simethicone or aluminium
magnesium hydroxide or a hydrate thereof (such as the
monohydrate known as magaldrate).
In another preferred aspect of the use according to the
invention, the pharmaceutical composition prepared is adapted
to be administered in combination with a preparation for a
course of therapy with an antibacterial agent, such as an
antibacterial agent selected from those listed above, in
particular preparations containing ~-lactam antibiotics such as
amoxicillin, ampicillin, cephalothin, cefaclor or cefixime; or
macrolides such as erythromycin, or clarithromycin; or
tetracyclines such as tetracycline or doxycycline; or
aminoglycosides such as gentamycin, k~A~ycin or amikacin; or
quinolones such as norfloxacin, ciprofloxacin or enoxacin; or
- others such as metronidazole, nitrofurantoin or
chloramphenicol; or preparations containing bismuth salts such
as bismuth subcitrate, bismuth subsalicylate, bismuth
subcarbonate, bismuth subnitrate or bismuth subgallate.
In a further aspect, the invention concerns all novel compounds
2mong those having the formula Ia, Ib, Ic, Id, Ie or If defined
above.
SU~ .JTE SHEET
WOs5/00~7 -27- 2 1 ~ ~ 9 6 1 PCT/SE94100604
The compounds of formula Ia, Ib, Ic, Id, Ie or If can be
prepare~ according to several general methods using
monosaccharides or oligosaccharides as starting materials.
Functional group transformations can be performed before or
S after the formation of glycoside bonds. To ensure
transformations of the functional group in a certain position,
the use of reactions which are regiospecific or the protection
with protective yLoup~ may optionally be neceRs~ry. The
protective y~OU~S can be removed or can form part of the
compoun~ in question.
The compounds of the invention can e.g. be prepared as shown in
the scheme below. In the scheme, al~ho~gh specific
substit~ents or configuration may be shown, it is to be
lS understood that to the extent that it is appropriate, the
various groups shown may assume the full varia~ility range as
defined for the general formulae Ia, Ib, Ic, Id, Ie, and If.
S~IB~ ITE SHEET
WO 95/00527 PCTISE94100604
L96~ ~
--28--
MONOSACCHARIDES
Step 1
H~\~ Ra ~\ ~Ra
~ c OH OH
Step 2
H~\~ Ra ~ ~ Rs
Rb Rb
Step 3
CH3 ~o ~< ~~ ~~
HO \ ~~ \ ~z Ra
Rb /n \ Rb / m
Step 4
CH3 ~ o ~< ~~
HO \ ~~ \ ~z Rc
Rb /n \ /m
SUB~ I 11 ~JTE SHEET
2ls~96~
wos~loQ~27 PCT/SE94/00604
-29-
In the first step (step 1) a monosaccharide, e.g. L-fucose,
D-galactose, D-glucose, 2-deoxy-2-phthalimido-D-glucose,
2-deoxy-2-phthalimido-D-galactose~ D-mannose, is converted to a
glycoside, with aglycons (Ra), e.g. SEt, SPh, OTMSEt, O-allyl
or OBn (~nown aglycons in the art), to form the Ra-glycoside
derivati~e in such a way that the Ra-glycoside is possible to
~ transfor~ to a glycosyl donator by activation of the anomeric
centre. 'rhe Ra-glycosides can be prepared as follows: A
monosacc~aride as above is per-O-acylated with acetic anhydride
in pyridine or with acetic anhydride-sodium acetate or with
benzoyl chloride in pyridine. The monosaccharide per-O-acylate
is reacted with, e.g. hydrogen bromide or hydrogen chloride in
a suitable solvent such as, e.g. acetic acid or
dichloro~ethane~ to form per-O-acylated glycosyl bromide or
chloride (e.g. on O-acylation and glycosyl halide synthesis,
see M. L. Wolfrom and A. Thompson, Methods in Carbohydrate
Chemistr~, Vol. 2, 211-215, edited by R. L. Whistler and M. L.
Wolfrom, Academic Press, New York, 1963, G. Hewit and G.
Fletcher Jr., ibid, 226-228, and R. U. Lemieux, ibid, 223-224).
The aglycon (Ra) is trans~erred to the monosaccharide by
reacting a suitable thio~ or alcohol, e.g. HSEt, HSPh, HOTMSEt,
HO-allyl, or HOBn with the monosaccharide per-O-acylate using a
Lewis acid such as boron trifluoride etherate (see e.g. R. J.
Ferrier and R. H. Furneaux, Car~ohydr. Res. 52 (1976), 63-68,
J. Dahmén, T. Frejd, G. Gronberg, T. Lave, G. Magnusson, and G.
Noori, Carbohydr. Res. 116 (1983), 303-307), or trimethylsilyl
trifluoromethanesulfonate (see T. Ogawa, K. Beppu, S.
N~k~h~yashi, Carbohydr. Res. 93 (1981), C6-C9) as promoters.
The reac~ion is carried out in a suitable solvent such as
chloroform, dichloromethane and/or toluene. When the
monosaccl~aride derivative in question is a per-O-acylated
glycosyl bromide or chloride, promoters such as silver
trifluoromethanesulfonate or mercury(II) salts (see e.g. H.
Paulsen, Angew. Chem. Int. Ed. Engl. 21 (1982), 155-173) can be
. 35 used, and the reactions are carried out in suitable solvents
such as dichloromethane and/or toluene. The monosaccharide
Ra-glycosides is obtained after de-O-acylation using sodium
methoxide (see e.g. A. Thompson, M. L. Wolfrom, and E. Pascu,
SUB~ JTE SHEET
W095l00527 ~16 ~ 9 ~1 pcTlæs4loo6o4~
Methods in Carbohydrate Chemistry, Vol. 2, 215-220, edited by
R. L. Whistler and M. L. Wolfrom, Academic Press, New York,
1963) in methanol or in methanol containing a co-solvent such
as dichloromethane or tetrahydrofuran.
In the second step (step 2) the monosaccharide Ra-glycoside is
~urther derivatized. New functional groups (Rb) which will form
part of the ~inal product or act as protective groups during
the subsequent glycosylation steps are introduced. Examples of
functional group transformations are: OH-groups to ethers or
esters (see e.g. Protective Groups in Organic Synthesis edited
by T. W. Greene and P. G. M. Wuts, John Wiley & Sons, Inc., New
York, 1991), OH-groups to carbonates (see e.g. J. March,
Advanced Organic Chemistry - Reaction MechAni-cms~ and
Structure, 347, 3rd Ed., John Wiley & Sons, New York, 1985, and
references cited herein), reductive removal or OH-groups via
halides, sulfonates or other routes (see e.g. J. March,
Advanced Organic Chemistry - Reaction ~eC~A~ i -sms, and
Structure, 389-392, 394, 3rd Ed., John Wiley & Sons, New York,
1985, and references cited herein, and H. H. Baer, Pure Appl.
Chem. 61~7) (1989), 1217-1234, and references cited herein),
OH-groups to halogen (see e.g. J. March, Advanced Organic
Ch~ çtry - Reaction NechAnicms~ and Structure, 381-286, 3rd
Ed., John Wiley & Sons, New York, 1985, and references cited
herein), OH-groups to azido groups (see e.g. J. March, Advanced
organic Chemistry - ~eaction Mech~ni~cms~ and Structure, 380,
3rd Ed., John Wiley & Sons, New York, 1985, and references
cited herein, and H. H. Baer, Pure Appl. Chem. 61~7) (1989),
1217-1234, and references cited herein), OH-groups to amino
groups via azides or other routes (see e.g. J. March, Advanced
Organic ~ri-ctry - Reaction MechAnicms~ and Structure,
798-800. 1106, 3rd Ed., John Wiley & Sons, New York, 1985, and
references cited herein, and H. H. Baer, Pure Appl . Chem . 61 ( 7 )
(1989), 1217-1234, and references cited herein), OH groups to
keto groups (oxo) (see e.g. J. March, Advanced Organic
Chemistry - ~eaction Mec~An;~ms, and structure, 1048-1120, 3rd
Ed., John W~ley & Sons, New York, 1985, and references cited
herein). OH groups to exomethylene derivatives via keto groups
SU~ JTE SHEEr
W095/~52Y ~9 6 ~31- PCT/SE94/00604
or other routes (see e.g. J. March, Advanced Organic Chemistry
- ~eact~on ~e~hAn;sms, and Structure, 400-404, 407, 845-854,
3rd Ed. r John Wiley & Sons, New York, 1985, and references
cited herein), OH groups to alkyl groups via exomethylene
derivatives and subsequent hydrogenation or via other routes
(æee e.g. H. O. H. House, Modern synthetic Reactions, 1-130,
2nd Ed.~ W. A. Benjamin, Inc., Menlo Park, C.A., 1972, and
references cites herein, or J. Yoshimura, Adv. Car~ohydr. Chem.
BiochemO ~2 il984), 69-134), and PYchA~ge of OH groups for
heterocyclie ~GU~D via different routes (see e.g. A. R.
Xatrizky, Handbook of Heterocyclic Chemistry, Pergamon Press,
Oxford, 1985).
.
In the third step (step 3), condensation of the Ra-glycosides
substituted with functional ~L OU~ (Rb) (protective groups
known inn the ar~) from a~ove are performed. For O-glycosidic
linkages: One Ra-glycoside derivative is transformed to a
glycosyl donor by activation at the anomeric centre, and
reacted with another Ra-glycoside which has been transformed to
a glycosyl acceptor by removing one or several protective
~L OU~ ( see e.g. H. Paulsen, Angew. Chem. Int. Ed. Engl. 21
(1982), 155-173, R. R. Schmidt, Angew. Chem. Int. Ed. Engl. 25
(1986), 212-235, P. F~gedi, P. J. Garegg, H. Lonn, and T.
Norberg, Glycoconj. J. ~ (1987), 97-108, Protective Groups in
Organic Synthesis edited by T. W. Greene and P G. M. Wuts, John
Wiley & Sons, Inc., New York, 1991). For C-glycosidic linkages
see e.g. R. R. Schmidt, and G. Effenberger, Liebigs Ann. Chem.
(1987), 82S-831, S. Czernecki, and G. Ville, J. Org. Chem. 5
(1989), 610-612, R. Preuss, and R. R. Schmidt, J. Carbohydr.
Chem. 10l5) (1992), 887-900, o. Martin, and W. Lai, J. Org.
Chem. 5~ (1993), 176-185, or C. R. Bertozzi, P. D. Hoeprich,
Jr., and M. D. Bednarski, J. Org. Chem. 57 (1992), 6092-6094.
For S-glycosidic linkages see e.g. L-X Wang, N. Sakairi, and H.
Kuzuhars, J. Chem. Soc. Perkin ~rans. 1 (1990), 1677-1982, or
M. Blanc-Meusser, L. Vigne, H. Driguez, J. Lehman, J. Streck,
and K. Urbahns, Carbohydr. Res. 22~ (1982), 59-71.
SUB~ JTE SHEEi-
W095/~27 2 ~ 6 ~ 32- PCT/SE94/00604
Further glycosidic linkages may be introduced by repeating the
third step.
In the fourth step (step 4) the substituent (Rc) at the
reducing end is introduced. Rc is defined as (Zl-z16)-~ ~
wherein R and Zl-z16 have the definition given for compounds
Ia, Ib, Ic, Id, Ie and If. The term "(Zl-Z16)-R" shall be read
as Zl-R, 22-~, Z3-~...... Z16-~- Activation of an
oligosaccharide Ra-glycoside derivative from step 3 at the
anomeric centre of the reducing end and reaction with a
suitable nucleophile leads to O-, C-, S-, or N-glycosidic
derivatives, respectively. A final product i5 obtained after
removal of protective groups, if necessary. When the compound
o~ the invention is in the form of a conjugate with a
particular matrix, the Rc-glycoside derivative is further
transformed via different routes to the final product (see e.g.
Y. G. Lee, and R. T. Lee, Glycoconjugates, 121-164, edited by
H. J. Allen, and E. C. Kisailus, Dekker, New York, 1992, R.
Roy, F. D. Tropper, and A. Romanowska, J. Soc., Chem. Commun.
(1992), 1611-1613, or C. P. Sotwell and Y. C. Lee, Adv.
Carbohydr. Chem. Biochem. , Vol. 37 tl980), 225-281).
Copolymerisation reactions for preparation o~ copolymers of
acrylamide and the mono-, di-, tri- or oligosaccharide
glycosides with or without a spacer are performed by known
methods, for example as described in E. Xallin, H. Lonn, T.
Norberg and M. Elofsson, J. Carbohydr. Chemistry 8(4), 597-611
(1989) or ~. Andersson and S. Oscarsson, Bioconjugate
Chemistry, vol. 4(3), 246-247 (1g93). The general strategy for
preparation of these conjugates has been to attach an olefinic
yL~ to a carbohydrate, and then copolymerize this derivative
with acrylamide. The olefinic group has been introduced into
the car~ohydrate molecule either as an allyl glycoside at an
early stage by acryloylation of an amino function of a mono-,
di-, tri- or oligosaccharide derivative or by other known
methods.
SIJB~ JTE SHEET
W095/~52~ 21 6 ~ g 6 1 PCT/SE94/00604
As indicated above, pharmaceutical preparations containing the
compou~ds of the general formula Ia, Ib, Ic, Id, Ie or If
constitute a further aspect of the invention.
The compounds of the invention can be administered systemically
or locally and are preferably administered orally or by
injection, by the rectal route, by the transdermal route, by
infusion or by inhalation in the form of a pharmaceutical
preparation comprising the active ingredient in the form of the
original compound or in the form of a pharmaceutically
acceptable salt thereof in association with a pharmaceutically
acceptable carrier which may be a solid, semi-solid or liquid
diluent or an ingestible capsule, and such preparations
compri e a further aspect of the invention. Pharmaceutically
acceptable carriers must, of course, be of sufficiently high
purity and sufficiently low toxicity to render them suitable
for administration to human or mammals being treated. The
compounds may also be used without carrier material. As
examples of pharmaceutical preparations may be mentioned
tablets, capsules, dragees, solutions, drops, such as nasal
~s, aerosols for inhalation, nasal spray, liposomes, etc.
Usually the active substance will comprise between O.Ol and 99
% by weight of the preparation, e.g. between 0.5 and 20% by
weight for preparations intended for injection and between O.l
and So~ by weight for preparations intended for oral
administration.
~he preparations are preferably in unit dosage form, whether as
single dosage units or as multiple dosage units.
To produce pharmaceutical preparations in the form of dosage
units for oral application containing a compound of the
invention, the active ingredient may be mixed with
conventionally used solids, pulverulent carriers, e.g. lactose, 35 saccharose, sorbitol, mannitol, a starch such as potato starch,
corn starch, amylopectin, laminaria powder or citrus pulp
powder, a cellulose derivative or gelatine and also may include
lubricants such as magnesium or calcium stearate or a Carbowax~
SUB~ ITE SHEFJ-
-
WO95/~527
216 4 9 61 pcTJsæ94loo6o4
-34-
or other polyethylene glycol waxes and compressed to form
tablets or cores for drag~es. If drag~es are required, the
cores may be coated with e.g. concentrated sugar solutions
which may contain gum arabic, talc and/or titanium dioxide, or
, alternatively~ with a film forming agent dissolved in easily
volatile organic solvents or mixtures of organic solvents.
Dyestuffs can be added to these coatings, e.g. to distinguish
between different contents of active substance. For the
preparation of soft gelatine capsules consisting of gelatine
and, e.g. glycerol and a plasticizer, or similar closed
capsules, the active substance may be admixed with a Carbowax~
or a suitable oil such as e.g. sesame oil, olive oil, or
arachis oil. Hard gelatine capsules may contain granulates of
the active substance with solid, pulverulent carriers such a~
lS lactose, saccharose, sorbitol, mannitol, starches, e.g. potato
starch or corn starch, or amylopectin, cellulose derivatives or
gelatine, and may also include magnesium stearate or stearic
acid as lubricants.
The compositions of the invention may be formulated æo as to
provide quic~, sustained or delayed release of the active
ingredient after administration to the patient by employing
procedures well known in the art.
By using several layers of the active drug, separated by slowly
dissolving coatings, sustained release tablets are obtained.
Another way of preparing sustained release tablets is to divide
the dose of the active drug into granules with coatings of
different thicknesæ and compress the granules into tablets
together with the carrier substance.
The active substance can also be incorporated in slowly
dissolving tablets made of e.g. fat and wax substances or
evenly distributed in a tablet of an insoluble substance such
as a physiologically inert plastic substance.
Liquid preparations for oral application may be in the form of
elixirs, syrups or suspensions, e.g. solutions cont~ from
SUE~s ~ JTE SHEET
W095/~27 16~9 ~1 PCT/SE94/00604
-35-
about 0.1% to 20~ by weight of the active substance, sugar and
a mixture of ethanol, water, glycerol, propylene glycol and
optionally aroma, saccharin and/or carboxymethylCellUlose as
dispersing agents. The formulations can additionally include
~ 5 wetting agent, emulsifying and suspending agents, preserving
agents and sweeteni~q agents.
For parenteral application by injection, preparations may
comprise an aqueous solution of the active drug or a
physiologically acceptable salt thereof, desirably in a
concentration of 0.5-20% and optionally also a stabilizing
agent and/or buffer substances in aqueous solution. Dosage
units of the solution may advantageously be enclosed in
ampoules.
There is limited knowledge of compounds that inhibit the
adherence of Helicobacter pylori to mucosal surfaces such that
the compounds are useful in the prevention or treatment of
gastrointestinal disorders and ~i ~^A~95 caused or mediated by
20 Helicobacter pylori. Because of this limited knowledge, the
dosage at which the active ingredients may be administered may
vary within a wide range and will depend on various factors
such as e.g. the severity 4f the infection, the age of the
patient etc. and may have to be individually adjusted.
The phar~aceutical compositions of the subject invention
preferably contain from about 1 mg to about SO g, more
preferably from about lO mg to about 5 g per day of the active
ingredient and may be divided into multiple doses.
The invention is further illustrated by the following,
non-limi~ing examples.
SUB~ JTE SHEET
PCTISE94/00604
WO 95t00527
2i~ 36-
Ph~o~ R Ph/~o,~ ~HO~C R
CH~ ~ 't OBn ~ CH3 ~ O; OBn
~ / g~ OBn
B o OBn~ BnO
3 R=OMe
1 R=SEt
6R= ~
2 R=OMe
9R= O o~ O~N~
5R= ~~
8 R = O o - - o - N3
OH
\
J HNAc
CH~ ~ OH
OH
HO
4 R = OMe
7R= --1~
10 R ~ ~~o~ ~\NH2 x CH3COOH
OH
HO~O~O o~O--NlN~ BSA
H H
_I HN~C
CH3~ 0 ~ OH
rOH
HO 1S-18
SUB~ )TE SHEEr
WO 95/00527 215~961 PCT/SE94/00604
OH OBz
- HO~--o R Ho~~5~ R
HO~ BzO~ OBn
H3C~5_SEl
12 R = O Si(CH3)3 0 14
OBz =OJ~Ph 08n= OCH2Ph
08n HO
I OBn ¦ OH
H3C ~08n OB H3C ~OH OH
O~R H~R
~ c O NI~Ac
.~
R ~ --Si(CH3)3 16 R ~ --Si(CH3)3
O O
OE~z _ O~Ph NHAc = HN~CH3
OBn ~ OCH2Ph
SUB~ 1 1 1 ulTe SHEET
WO 95/OO!j27 PCT/SE94/00604
21 6~61 -38-
OAC ~OH
PCO~VR HO~R
~o o~O
17 R = SEt 18 R = SEt
OAC = O~CH3
OBn H3C~OBn
ACO- ~Ro HO~
19 R5 SEt 21 R5 OMe
20 R OMe OBn= OCH2Ph
23 R- O~_ O
/~ NHAC = HN CH3
OBn = OCH2Ph
OH
OAC ~ O~CH3 H3C !~OH
HO;;~,R
NHAC
22 R = OMe
24 R ~ ~
SUB~ I z I ~JTE SHEET'
WO 95/00527 PCT/SE94/00604
~ -39- 21 6~96 ~
'' BnO~ \,.~SEt BnO~$\\vOR2
ORl OR1
R~_Ac 27 R~- Bz, R2s -CH2CH2N3
26 R~ = Bz 28 R~ s H, R2 ~ -CH2CH2N3
32 R~ - Bz, R2= -(CH2CH20)2CH2CH2N3
33 R~ = H, R2- -(CH2CH20)2CH2CH2N3
R~O OR
~Bn R10-~\ \v
BnO
14 ~O~OR
R10
29 R, =Bn, R2 - -CH2CH2N3
30 R~, H, R2- -CH2CH2NH2
57 Rl . H, R2 ~ -CH2CH2NHCOCH2
34 R1 - Bn, R2 - -(CH2CH20)2CH2CH2N3
35 R, =H, R2 - -(CH2CH20)2CH2CH2NH2
37 R~ ~ H, R2 s -(Cl~2CH20~2CH2CH2NHCOCHCH2
~ o~H NHCN~HSA /HO~ O o~O--~NHCN~HSA
\r / ~ OH
31 36
. = .
:
- SUt~ 1 ITE SHEET~
WO 95/00527 PCT/SE94/00604
%i~49~ -40-
Ph~_ BnO~
HO NPhlh N3 BnO ~\ ~ R2 N3
43
o 44 Rl ~ AcR2 ~ NPh~h
NPhth ~ N~ 45R~ =OH R2 5 NHAc
Or
R~ ~O RRo~_O
R10 ~\ - NHAc R2
CHa~ ~OR~
R~
46 Rl s Bn R2 s N3 R3 = CHPh
47 R~ = Bn R2 = NHCOCF~ R3 = CHPh
48 Rl = H R2 = NHCOCF3 R3 = H
49 Rl 5 H R2 = NHCOCHCH2 R3 = H
SUB~ JTE SHEEr
wo gsl00s27
PCT/SE94/00604
-41- 216q9
BnO OBn OR3
~_0 R40~_0
BnO ~ OR~ R2 N3
45 R~ = H R2 = NHAc R3, R4 = CHPh
51 R1=H R2=NHAc R4=H R3 =OBn
R~O
¦ OR~
CH35~oR,
1 ~ 0--~
R10 ~0\ NHAc R2
CH3~ OR,
R~O
52 R, = Bn R2 = N3 R4 ~ Bn
53 R~. Bn R2= NHCOCF3 R4 =Bn
54 R~ z H R2 = NHCOCF3 R4 = H
55 R~ _H R2- NHCOCH2 R4 ~ H
SUB~ 111 L)TE SHEEr
wo gsl00s27
PCT/SE94/00604
2~G 4~6~ -
.H
. ~ H~
CH3~H
OH
OH
Fuca1 -2Gal~1 -3(Fuc1 -4)GlcNAc~1 -3Gal~1 -4Glc~ -R
39 R1 - OH
40 Rl = NH2
41 Rl = NHCOCHCH2
42 R2 = Fuca1-2Gal~1~3(Fuc~1~4)GlcNAc~1~3Gal~1~4Glc~l -NH H2N--C~
38 R2 = Fuca1-2Gal~1-O(CH2CH20)2CH2CH2NH R2~C---
58 R2 = Fuc1-2Gal,B1-CH2CH2NH H2N--C--
50 R2 = Fuc~1-2Gal~1-3GlcNAc~1-CH2CH2NH
56 R2 s Fuc1-ZGal~1-3(Fuc~1-4)GlcNAc~1-CH2CH2NH
SUB~ 111 ~JTE SHEET
woss/~s27 ~ pcTlsEs4loo6o4
-43-
General metho~o
lH and 13C NMR spectra in examples 1 to 6 were recorded on a
Varian Gemini 300 spectrometer and on a Varian Unity 400 MHz
- 5 spectrometer. In examples 1 to 6 the following reference
signals were used: CHC13, ~ 7.25 (lH in CDCl3); CHCl3, ~ 77.9
(13C in CDCl3); (CH3)2CO, ~ 2.24 or CHD2OH ~ 3.31 (1H in D2O);
tCH3)2CO, ~ 33.19 or CHD2OH, ~ 51.89 (13C in D2O); CHD2OH,
3.31 (lH in CD30D). lH andl3C NMR spectra in all other
examples were recorded at 25C in CDC13 (using
tetramethylsilane as internal st~nAArd for lH, CDCl3 ~ 77.0 for
3C) and in D2O (HDO ~ 4.765 for lH, us~ng aceton ~ 30.0 as
internal st~n~rd for 13C). NMR spectra recorded for all
compounds were in agreement with the structures postulated and
only selected data are reported. Masspectra to determine the
degree of substitution of carbohydrate component vs. protein
were performed on a VG TOFSPEC linear time of flight
masspectrometer. Fab-NS was run on a Nermag 1010L, with an
Iontech FAB gun and a matrix of thioglycerol. Optical rotations
were measured using a Perkin Elmer 241 polarimeter. Thin layer
chromatography (TLC) was performed on Nerck DC-Fertigplatten
(Kiselgel 60 F254 0.25 mm) and spots were visualized by W or
by spraying with 10% sulphuric acid followed by charring at
elevated temperature, or by spraying with phospohomolybdic acid
or ninhydrin in n-butanol (0.5%). Silica gel 60 (40-63 Am) and
Amicon Matrex2 Silica Si 0.35-0.70 m was used for column
chromatography.
Separations were also performed on a Chromatotron rotary TLC
using 1--2 mm layers of Silica Gel 60 PF254 with gipsum. All
Biogel P-2 column were eluated with 1% n-buthanol in deionized
water if not otherwise stated.
BXAMPLB 1
~ethyl ~-acet~mi~o-2-deoxy-3-O-a-L-fucG~ ~osyl-~-D-
glu~o~y~oside (~ )
SUB~ TE SHEET
W095/00~7 2 1 6 ~`9 6 1 PCT/SE94/00604
-44-
(i) Methyl 4,6-0-benzylidene-3-0-(tri-0-benzyl-
~-L-fucopyranosyl)-2-deoxy-2-phthalimido-~-D-glucopyranoside
(2)
Trifluoromethanesulfonic acid (2 ~1, 0.023 mmol) was added to a
stirred mixture of ethyl 3-o-(tri-0-benzyl-~-L-
fucopyranosyl)-4~6-o-benzylidene-2-deoxy-2-
phthalimido-l-thio-~-D-glucopyranoside (1) (100 mg, 0.117
mmol), (prepared according to H. Lonn, Carbohydr. Res. 139
(1985), 105-113) methanol (7 ~1, 0.175 mmol), N-iodosuccinimide
(40 mg, 0.175 mmol) and ground molecular sieves (100 mg, 3A) in
dichloromethane-diethyl ether (3 ml, 2:1) at -30C. After 45
min the reaction mixture was filtered through a layer of Celite
into an aqueous solution of sodium hydrogen carbonate and
sodium bisulphite. The organic layer was separated, washed with
aqueous sodium chloride, and concentrated. Column
chromatography (toluene-ethyl acetate, 20:1) of the residue
gave amorphous (2) (93 mg, 97 %), ~a~D -16.2 (c 1.0, CHCl3).
1H NMR data (CDC13, ~): 7.80 to 7.00 (24H, benzyl and
phthaloyl), 5.29 (d, lH, J 8.6 Hz, H-l), 4.84 to 4.25 (SH,
CH2Ph), 4.84 (bs, lH, H-l'), 4.66 (dd, lH, J 8.5 and 10.3 Hz,
H-3), 4.48 to 4.43 ~m, lH, H-3'), 4.35 (dd, lH, J 8.6 and 10.3
Hz, H-2), 4.08 (bdd, lH, J 6.4 and 13.0 Hz, H-5'), 3.91 to 3.81
(2H), 3.78 to 3.66 (4H), 3.51 to 3.47 (lH), 3.48 (s, 3H, OCH3),
0.90 (d, 3H, CH3).
3C NMR data (CDCl3, ~): 168.0 (CO), 138.8 to 123.0 (benzyl),
101.1 (CHPh), 99.7 (C-l'), 99.4 (C-l), 82.1, 79.5, 78.0, 75.7,
75.5, 74.6, 73.0, 72.5, 68.6, 67.2 (C-5'), 66.1, 56.9 (OCH3),
55.5 (C-2), 16.3 (CH3).
(ii) Methyl 2-acetamido-3-0-(2,3,4-tri-0-benzyl-~-L-
fucopyranosyl)-4~6-o-benzylidene-2-deoxy-~-D-glucopyranoside
35 (3)
A solu~ion of (2) (1.13 g, 1.36 mmol) and hydrazine hydrate
(3.3 ml, 68 mmol) in agueous 95% ethanol was boiled under
SUB;~ JTE SHEEI-
W095/~27 1 6~.9 61 PCTISE94/00604
-45-
reflux for 20 h, ccoled, and concentrated. The residue was
acetylated with acetic anhydride-pyridine (50 ml, 1:1)
overnight. The solution was concentrated, and the residue was
subjected to column chromatography (heptane-ethyl acetate, 1:1)
S to give crude (3) ~.hich was used directly in the next step.
H NMR data (CDC13, ~): 7.50 to 7.25 (20H, benzyl), 5.71 (d,
lH, J 7.4, NH), 5.52 (s, lH, CH2Ph), 5.09 (d, lH, H-1'), 4.85
to 4.58 (6H, CH2Ph), 4.82 (d, lH, H-l), 4.37 )dd, lH, J 4.6 and
10.4 Hz, H-6), 4-.28 (bt, lH, H-3), 4.12 to 4.05 (2H, H-2' and
H-5'), 3.95 (dd, lH, J 2.6 and 10.2 Hz, H-3'), 3.78 (bt, lH,
H-6), 3.63 (bs, lH, H-4'=, 3.60 (bt, lH, H-4), 3.53 (m, lH,
H-5), 3.48 (s, 3H, OCH3), 3.42 (ddd, lH, J 7.2, 8.2 and 9.5 Hz,
H-2), 1.67 (s, 3H, NHAc), 0.84 (d, 3H, CH3).
13C NMR data (CDC13, ~): 170.6 (C0), 138.6 to 126.2 (benzyl),
101.8 (~-1), 101.6 (CHPh), 98.4 (C-l'), 80.8 (C-4), 79.8
(C-3'), 77.6 (C-4'), 77.0 (C-2' or C-5'), 75.1 (C-3), 74.9
(CH2Ph), 72.5 (CH2Ph), 68.8 (C-6), 66.9 (C-2' or C-5'), 66.2
(C-5), 58.1 (C-2), 57.0 (OCH3), 23.2 (NHAc), 16.3 (CH3).
(~ii) Methyl 2-acetamido-2-deoxy-3-O-~-L-fucopyranosyl-~-D-
glucopyranoside (4)
2S A solution of crude (3) (1.05 g) in acetic acid-ethyl acetate-
water (9:5:1, 120 ml) was hydrogenolysed at 200 kPa over 10%
Pd/C (1 g) over night. The mixture was filtered through a layer
of Celite and conc~ntrated. Column chromatography (chloroform-
methanol-water, 65:35:6) of the residue gave amorphous ~ (469
mg, 90% calculated from (2), t~D -116.0 (C 1.0, water).
lH NMR data (D20, acetone ref., ~): 4.99 (d, lH, J 4.0 Hz,
H-l'), ~.46 (d, lH, J 8.7 Hz, H-l), 4.33 (bdd, lH, H-5'), 3.98
to 3.45 (9H), 3.51 (s, 3H, OCH3), 2.03 (s, 3H, NHAc), 1.17 )d,
3H, CH3).
SUB~ I 11 ~JT~ SHEET
WOg5/~7 2 1 6 ~ 9 6 1 PCT/SE94/00604 ~
-46-
13C NMR data (D20, acetone ref., ~3: 177.6 (C0), 104.7 (C-l),
102.9 (C-l'), 83.5, 78.8, 74.7, 72.5, 71.6, 70.9, 69.9, 63.7,
60.0, 59.1 (C-2), 25.2 (NHAc), 18.1 (CH3).
~SAMP~E 2
3,3-D~Qthylbutyl 2-acet~ido-2-~eoxy-3-0-~-L-fUcopyr~no~yl-~-
D-glucG~.anos~- (7)
(i) 3,3-Dimethylbutyl 3-0-(2,3,4-tri-0-benzyl-~-L-
fucopyranosyl)-4~6-o-benzylidene-2-deoxy-2-phthalimido-~-D-
gluco~Lanoside (5)
Trifluoromethanesulfonic acid (30 ~1, 0.35 mmol) was added to a
stirred mixture of (1), 3,3-dimethyl-butan-1-ol (317 ~1, 2.62
mmol~, N-iodosuccinimide (602 mg, 2.62 mmol), and ground
molecular sieves (1.5 g, 3A) in dichloromethane-diethyl ether
(2:1, 45 ml) at -30C. After 45 min the reaction mixture was
filtered through a layer of Celite into an aqueous solution of
sodium hydrogen carbonate and sodium bisulphite. The organic
layer was separated, washed with aqueous sodium chloride, and
concentrated. Column chromatography (heptane-ethyl acetate,
6:1) of the residue gave amorphous (5) (1.42 g, 90~), t~]D
-22.2 (c 1.0, CHC13).
lH NMR data (CDC13, ~): 7.80 to 7.0 (24 H, benzyl and
phthaloyl), 5.57 (s, lH, CHPh), 5.35 (d, lH, J 8.5 Hz, H-1),
4.84 (bs, lH, H-l'), 4.83 to 4.24 (5H, CH2Ph), 4.65 (dd, lH, J
8.3 and 10 3 Hz, H-3), 4.34 (dd, lH, J 8.5 and 10.4 Hz, H-2),
4.07 (dd, lH, J 5.5 and 10.4 Hz, H-5'), 3.96 to 3.66 (7 H,
inter alia OCH2), 3.53 to 3.45 (2H, inter alia OCH2), 1.46 to
1.29 (m, 2H, CH2C(CH3)3), 0.88 (d, 3H, J 6.4 Hz, CH3), 0.73 (s,
9H, CH2C(CH3)3)-
13C NMR data (CDC13, ~): 168.0 (C0), 138.8 to 123.0 (benzyl and
phthaloyl), 101.1 (CHPh), 99.4 (C-l'), 98.9 (C-l), 82.1, 79.5,
76.0, 75.7 (C-3), 75.6, 74.6, 73.0, 72.6, 68.7, 67.3 (OCH2),
SUB:~ 111 LITE SHEET
woss/005~7 216~961 PCT/SE94/00604
67.2 (C-5), 66.2, 55.8 (C-2), 42.4 (CH2C(CH3)3), 30.8
(CH2C(C~3)3), 29.4 (CH2C(CH3)3), 16-3 (CH3).
(ii) 3,.~-Dimethylbutyl 3-0-(2,3,4-tri-O-benzyl-~-L-
fucopyranosyl)-2-acetamido-4~6-o-benzylidene-2-deoxy-
~-D-glucopyranoside (6)
A solution of (5) (1.42 g, 1.58 mmol) and hydrazine hydrate
(3.9 ml, 79 mmol) in aqueous 90% ethanol (100 ml) was boiled
under reflux for 20 h, cooled, and concentrated. The residue
was acetylated with acetic anhydride-pyridine (50 ml, 1:1)
overnight. The solution was concentrated. Column chromatography
(heptane-ethyl acetate, 3:1, containing 1% methanol) of the
residue qave amorphous (6) (1.16 g, 90%), t~]D ~74-7 (c 1.0,
CHC13).
H NMR data (CDC13, ~): 7.50 to 7.20 (20 H, benzyl), 5.63 (d,
lH, J 7O3 Hz, NH), 5.51 (s, lH, CH2Ph), 5.07 (d, lH, J 3.1 Hz,
H-l'). 4.93 to 4.57 (6H, CH2Ph), 4.92 (d, lH, H-1), 4.39 to
4.29 (2I~, H-6 and H-3), 4.11 to 4.04 (2H, H-2'and H-5'), 3.98
to 3.85 (2H, H-3'and OCH2), 3.77 (bt, lH, H-6), 3.61 (bs, lH,
H-4'), 3.55 (bt, lH, H-4), 3.59 to 3.44 (2H, H-5 and OCH2),
3.33 (bdd, lH, H-2), 1.63 (s, 3H, OAc), 1.56 to 1.40 (m, 2H,
CH2C(CH3)3), 0.89 (s, 9H, CH2C(CH3)3), 0.82 (d, 3H, CH3).
3C NMR data (CDC13, ~): 170.4 (Co), 138.6 to 126.0 (benzyl),
101.6 (CHPh), 100.7 (C-l), 98.1 (C-l'), 80.9 (C-4), 79.8
(C-3'), 77.6 (C-4'), 77.0 (C-2'or C-5'), 74.9 (C-3), 74.8
(CH2Ph) r 74.0 (CH2Ph), 72.5 (CH2Ph), 68.9 (OCH2 or H-6), 67.5
(OCH2 or H-6), 66.8 (C-5' or C-2'), 66.2 (C-5), 58.6 (C-2),
42-7 (CII2C(CH3)3)~ 29-7 (CH2C(CH3)3), 29.6 (C(CH3)3), 23.2
(NHAc), 16.2 (CH3).
(iii) 3l3-Dimethylbutyl 2-acetamido-2-deoxy-3-O-~-L-
fUcopyranosyl-~-D-glu~G~y ~noside (7)
A solution of the compound (6) (1.08 g, 1.33 mmol) in acetic
acid:ethyl acetate:water, 9:5:1 (120mL) was hydrogenolysed at
SU~ JTE SHEEr
wo gs/00s27 2 1 6 ~ 9 6 ~ - 48- PCT/SE94/00604
200 kPa over 10% Palladium on charcoal (Pd/C) (1 g) over night.
The mixture was filtered through a layer of Celite and
concentrated. Column chromatography (chloroform-methanol-water,
100:30:3) of the residue gave amorphous (7) (566 mg, 94%), t~]D
S -109-7 (c 1.0, water).
H NMR data (D20, acetone ref., ~): 4.99 (d, lH, H-l'), ~.84
(g, lH, CHPh), 4.34 (bdd, lH, H-5'), 4.02 to 3.43 (llH), 2.01
(s, 3H, NHAc), 1.57 ti 1.41 (m, 2H, CH2C(CH3)3), 1.17 (d 3H,
CH3), 0.90 (8, 9H, C(CH3)3)-
3C NMR data (D20, acetone ref., ~): 177.3 (C0), 103.6 (C-l),
102.8 (C-l'), 83.6, 78.8, 74.8, 72.5, 71.6, 70.9, 69.8, 63.7,
58.1 (C-2), 44.9 (CHC(CH3)3), 31.9 (C(CH3)3), 31-9 )C(CH3)3),
25.2 (NHAc), 18.1 (CH3).
~XAM~g 3
~Ucal-3GlcNAc~1-0-~p-~r~ 1-B8A-con~ugate (11)
(i) 8-Azido-3,6-dioxaoctyl 3-0-(2,3,4-tri-0-benzyl-~-L-
~u~y.anosyl)-4,6-0-benzylidene-2-deoxy-2-phthalimido-~-D-
glucopyranoside (8)
Trifluoromethanesulfonic acid ~24 ~1, 0.27 mmol) was added to a
stirred mixture of (1) (1.15 g, 1.34 mmol), 8-azido-3,6-
dioxaoctan-1-ol (352 ~1, 2.01 mmol) (prepared according to P.
H. Amvam-Zollo and P. Sina~, Carbohydr. Res. 150 (1986), 199-
212), N-iodosuccinimide (461 mg, 2.01 mmol) and ground
molecular sieves (1.15 g, 3A) in dichloromethane-diethyl ether
(30 ml, 2:1) at -30C. After 1 h the reaction mixture was
filtered through a layer of Celite into a aqueous solution of
sodium hydrogen car~onate and sodium bisulphite. The organic
layer was separated, washed with aqueous sodium chloride, and
concentrated. Column chromatography (heptane-ethyl acetate,
2:1) of the residue gave amorphous (8) (1.03 g, 79%), t~]D
-21-7 (c 1.0, CHC13).
SUBS I ~ I ~JTE SHEET
W095/~527 21 B~ 9 61 pcTlsæ94loo6o4
-49-
lH NMR data (CDC13~ 7.80 to 7.05 (24H, Bzl, Phth), 5.59 (s,
lH, CHPh), 5.44 (d, lH, J 8.6 Hz, H-l), 4.83 (bs, lH, H-l'),
4.83 to 4.24 (SH, CH2Ph), 4.65 (dd, lH, J 8.5 and 10.3 Hz,
H-3), 4.45 to 4.41 (lH, H-3'), 4.39 (dd, lH, J 8.6 and 10.3 Hz,
~ 5 H-2), 4.09 (dd, lH, J 6.4 and 12.6 Hz, H-5'), 3.99 to 3.83 (3H,
inter alia OCH2), 3.81 to 3.68 (5H, inter alia OCH2), 3.61 to
3.30 (llH, inter alia OCH2 and CH2N3), 0.90 (d, 3H, J 6.4 Hz,
CH3)-
13C NMR data (CDC13, ~): 169.0 (C0), 138.9 to 123.1 (Bzl,
Phth), 101.1 (CH2Ph), 99.4 (C-l'), 98.9 (C-l), 82.1, 79.6,
78.0, 75.6, 75.5, 74.7, 73.1, 72.6, 70.5, 70.4, 70.1, 69.9,
69.~, 68.7, 67.2, 66.2, 55.7 (C-2), 50.6 (CH2N3), 16.4 (CH3).
(ii) 8 Azido-3,6-dioxaoctyl 2-acetamido-3-0-(2,3,4-tri-0-
benzyl ~-L-fucopyranosyl)-4,6-O-benzylidene-2-deoxy-~-D-
glucopyranoside (9)
A solution of (8) (633 mg, 0.65 mmol) and hydrazine hydrate
(1.6 ml, 33 mmol) in aqueous 90% ethanol (45 ml) was boiled
under reflux for 24 h, cooled, and concentrated. The residue
was acetylated with acetic anhydride-pyridine (50 ml, 1:1)
overnight. The solution was concentrated. Column chromatography
(chloroform-acetone, 9:1) and re-chromatography (ethyl acetate-
heptane, 3:1) of the residue gave amorphous (9) (440 mg, 77~),
t~D -72.6 (c 1.0, CHC13).
H NMR data (CDC13, ~): 7.50 to 7.25 (20H, benzyl), 7.92 (d,
lH, J S.9 Hz, NH), 5.51 (s, lH, CHPh), 5.16 (d, lH, J 3.5 Hz,
H-l ~, 4.93 (d, lH, J 7.7, H-l), 4.95 to 4,56 (6H, CH2Ph), 4.34
(dd, lH, J 4.9 and 10,4 Hz, H-6), 4.26 (bt, lH, H-3), 4.12
(bdd, lH, H-5'), 4.06 tdd, lH, J 3.5 and 10.1 Hz, H-2'), 3.95
(dd, lH, J 2.7 and 10.1 Hz, H-3'), 3.90 (bt, lH, H-6), 3.81 to
3.45 (14H, inter alia OCH2), 3.40 (m, 2H, CH2N3), 1.74 (s, 3H,
NHAc), 0.84 (d, 3H, J 6.4 Hz, CH3).
3C NMR data (CDC13, ~): 170.4 (C0), 138.7 to 126.1 (benzyl),
101.5 (CHPh), 101.2 (C-l), 97.8 (C-l'), 80.6 (C-4), 79.6
SIJB~ JT~ SHEET
wos5/~27 pcTlsæ94loo6o4-
21~49~1 -so~
(C-3'), 77.7 (C-4 ~ 76.7 (C-2~ or C-5~), 74.9 (C-3), 74.6
(CH2Ph), 73.7 (CH2Ph), 72.2 (CH2Ph), 70.6 (CH20), 70.6 (CH2O),
70.4 (CH20), 69.9 (CH2O), 68.8 (CH2O), 68.8 (H-6), 66.7 (C-2'
or C-5'), 66.2 (C-5), 57.8 (C-2), 50.6 (CH2N3), 23.2 (NHAc),
16.2 (CH3)-
(ii~) 8-Amino-3,6-dioxaoctyl 2-acetamido-2-deoxy-3-O-~-L-
fucopyranosyl-~-D-glucopyranoside acetic acid salt (10)
A solution of (9) (57 mg, 0.065 mmol) in acetic acid-water
(9:1, 30 ml) was hydrogenolysed at 200 kPa over 10% Pd/C (100
~ mg) over night. The mixture was filtered through a layer of
Celite and concentratéd. The residue was first subjected to
column chromatography on silica gel (chloroform-methanol-water,
4:4:1) and then on A12O3 ~Merck, basic, 0.063-0.200 mm,
chloroform-methanol-water, 4:4:1) to give amorphous (10) (18
mg, 51~), t~D -70.6 (c 0.2, water).
1 H NMR data (D2O, acetone ref., ~): 4.98 (d, lH, J 4.0 Hz,
H-l'). 4.53 (d, lH, J 8.6 Hz, H-l). 4.32 (bdd, lH, H-5'), 4.05
to 3.42 (19H), 3.19 (m, 2H, CH2NH2), 2.01 (s, 3H, NHAc), 1.88
(CH3COOH), 1.14 (d, 3H, J 6.6 HZ, CR3).
13C NMR data (DzO, acetone ref., ~): 184.2 (CH3COOH), 103.8
(C-l), 102.8 (C-l'), 83.3, 78.8, 74.8, 72.6, 72.5 72.4, 72.0,
71.5, 70.9, 69.8, 69.4, 63.7, 58.1 (C-2), 42.0 (CH2NH2), 26.3
(CH3COOH), 25.2 (NHAc), 18.1 (CH3).
tiV) Fuc~1-3GlcNAc~l-o-Spacer l-BSA-conjugate (11)
Thiophosgene (67 ~1, 0.856 mmol) in acetone (6 ml) was added
dropwise to an ice-cold solution of (10) (120 mg, 0.214 mmol)
in water-ethanol-0.1 N phosphate buffer pH 7 (1:1:1, 30 ml).
The pH was kept at 6-7 with aqueous sodium hydroxide (1 M)
during the reaction. After 20 min the mixture was extracted
with diethyl ether (30 ml), concentrated to a volume of 10 ml,
and added to a solution of bovine serum albumin (695 mg, 10.7
mmol) in a~ueous sodium hydrogen carbonate (15 ml, 0.1 M, pH
SUB~ ~ .JTE SHEET
W095/~27 21 6 ~ 9 61 PCT/SE94/00604
--51--
9.3). During the addition, pH was adjusted to g with aqueous
sodium hydroxide (1 M). After 24 h the reaction mixture was
desalted by ultrafiltration (Filtron, omegacell 150, 10 K) and
freeze-dried to give (11) (672 mg). The degree of substitution
S was determined by sugar analysis (see M. A. Jermyn, Anal. Chem.
68 (1975), 332-335) to 15-18 mol disaccharide/mol protein.
EgAMPL~ 4
2-Trimethylgilylethyl 2-acot~mi~o-2-deoxy-~-O-~-L-fuco-
pyr~nosyl-~-D-glucGy~.~no~ide ~6)
(i) Trimethylsilylethyl 3,6-di-O-benzoyl-2-deoxy-2-phthalimido-
~-D-glucopyranoside (13)
2-Trimethylsilylethyl-2-deoxy-2-phthalimido-~-D-glucopyranoside
(12) (1.64 g, 4.0 mmol) (prepared as described by K. Jansson,
S. Ahlfors, T. Frejd, J. Rilhberg, G. Magnusson, J. Dahmén, G.
Noori, and K. Stenvall, J. Org. Chem. 53 (1988), 5629-5647),
was ~ clved in pyridine-dichloromethane (3:1, 24 ml) and
cooled to -45C. A mixture of benzoyl chloride (1060 ml, 9.1
mmol) and pyridine (900 ml) was added during 30 minutes. The
reaction was completed after 3 hours and methanol (40 ml) was
added. The solvents were evaporated and the residue was
co-evaporated with toluene 3 times. The residue was
chromatographed (SiO2, heptane/ethyl acetate 2~ to give
pure (13) (2.38 g, 96%). ~D20 l69.4 (c 1.2, CHCl3).
lH NMR data (CDC13, ~): d 7.3-8.2 (m, 14H, 2 O-benzyl,
N-phthalamoyl), 5.88 (dd, lH, J 7.1, 8.3 Hz, H-3~, s.46 td, lH,
J 8.5 Hz, H-l), 4.79 (dABq, lH, J 4.2, 12.4 Hz, H-6), 4.67
(dABq, lH, J 1.9, 11.7 Hz, H-6), 4.45 (dd, lH, J 8.4, 10.8 Hz,
H-2), 3.8-4.1 (m, 3H, H-4, H-5, OCH2CH2), 3.57 (dt, lH, J 7.2,
9.7 Hz, OCH2CH2), 0.7-1.0 (m, 2H, CH2Si), -0.15 (s, 6H, SiMe3).
(ii) 2-Trimethylsilylethyl 3,6-di-O-benzoyl-4-O-(2,3,4-tri-o-
benzyl-~-L-fucopyranosyl)-2-deoxy-2-phthalimido-~-D-
glucopyranoside (15)
.~
SUB~ TE SHEEr
Wog~/00527 216 ~ 9 G 1 PCT/SE94/00604
-52-
Compound (13) was dissolved in dichloromethane-N,N-
dimethylformamide (8 ml, 5:3) and tetrabutylammonium bromide
(664 mg, 2.06 mmol) and molecular sieves (4 A, 4 g, activated)
was added. To a solution of thioethyl 2,3,4-tri-O-benzyl-1-
thio-~-L-fucopyranoside (14) (986 mg, 2.06 mmol) (prepared
according to H. L~nn, Carbohydr. Res. 139 (1985), 105-113) in
dichloromethane (8 ml) was added bromine (122 ml, 2.37 mmol) in
dtchloromethane (2 ml). After 15 min stirring, cyclohexene
(distilled) was added dropwise until the bromine colour
disappeared. This solution was then added to the mixture above
containing compound (13) and stirred for 48 h. The mixture was
then filtered through Celite, the solvents were evaporated and
the residue was co-concentrated with toluene three times.
Column chromatography of the residue (heptane/ethyl acetate,
S:l~ 1:1) gave (15) (896 mg, 85%), t~]D20 + 14.6 (c 1.2,
CHC13).
lH NMR data (CDC13, ~): 5.44 (d, lH, J 8.5 Hz, H-l), 4.80 (d,
lH, J 3.6 Hz, H-1').
(iii) 2-Trimethylsilylethyl 2-acetamido-2-deoxy-4-O-(~-L-fuco-
pyranosyl)-~-D-glucopyranoside (16)
Compound (15) (760 mg, 0.74 mmol) was dissolved in methanol (7
ml) and sodium methoxide (220 ml, 2 M in methanol) was added.
The solution was stirred over night at room temperature and
then neutralized with Amberlite IR-120(H). Filtration and
evaporation of the solvents gave a syrup. The syrup was
dissolved in acetic acid (15 ml) and 10% Pd/C (860 mg) was
~0 added. After l.S h hydrogenolysis (lO0 kPa), the mixture was
filtered and the solvents evaporated. The resulting syrup was
dissolved in ethanol (18 ml) and hydrazine hydrate was added.
The solution was refluxed for 3 h. Evaporation of the solvents
and co-evaporation with ethanol S times gave a syrup that was
dissolved in methanol-water mixture (5:1, 60 ml). Acetic
anhydride (5 ml) was added and the solution was stirred for 1.5
h. The solvents were evaporated. Column chromatography (sio2~
SUB~ 111 ~TE SHEET
woss/oos~l 1 ~ 9 61 PCT/SE94/00604
-53-
dichloromethane/methanol, 5:1) gave (16) (lOo mg, 29%), t~]D20
-113.5 (c 0.7, H20).
H NMR data (D20, ~): d 4.93 ~d, lH, J 3.66 Hz, H-1'), 4.52 (d,
~ 5 lH, J 8.06 Hz, H-1).
EXAMPLE S
Mathyl 2-acet~mi~o-2-deoYy-6-O-~-~-
fu~G~y~nosy~ D-glucG~y~nosi~ ~22)
(i) Ethyl 2-deoxy-2-phthalimido-1-thio-~-D-glUCopyranoside (18)
Ethyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-1-thio-~-D-
glucopyranoside (17) (5.79 g, 12 mmol) (prepared according to
H. Lonn, Car~ohy~r. Res. (1985) 139, 105-113) was dissolved in
methanol (250 ml) and methanolic sodium methoxide (0.2 M, 2.5
ml) was added. The mixture was stirred for 15 h. Neutralization
with acidic cation exchange resin (Bio-Rad AG~ 50W-X8),
filtration, evaporation and crystallization from water ~ave
(18) (3083 g, 89%), m.p. approx. 96C; m.p. after
recrystallization 159-161C; ta3D22 +9.8 (c 0.9, methanol).
lH NMR data (CD30D, CHD2OD ref., ~) d: 7.91-7.79 t5H), 5.32 (d,
lH, J 10.5 Hz, H-l), 4.28 (dd, lH, J lo and 8 Hz, H-3), 4.05
(t, lH, J 10.5 Hz, H-2), 3.93 (dd, lH, J 12 and 2 Hz, H-6),
3.73 (dd, lH, J 12 and 5.5 Hz, H-6), 3.46 (ddd, lH, J 10, 5.5
and 2 Hz, H-5), 3.40 (dd, lH, J 10 and 8 Hz, H-4), 2.74 (dg,
lH, J 12.5 and 7.5 Hz, SCH), 2.63 (dq, lH, J 12.5 and 7.5 Hz,
SCH) ancl 1.17 (t, 3H, J 7.5 Hz, CH3CH2).
(ii) Ethyl 3,4-di-O-acetyl-6-O-
(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-
2-deoxy 2-phthalimido-1-thio-~-D-glucopYranOSide (19)
3s
Bromine (0.485 ml, 9.4 mmol) was added to a solution of ethyl
2,3,4-tri-O-benzyl-l-thio-~-L-fucopyranoside (14) (4.5 g, 9.4
mmol) in dichloromethane (70 ml) at 0C. The mixture was
SUB~ JT~ SHEET
W095l~7 ~ 21~9GI PCT/SE94/0060 ~
-54-
stirred for 35 min and was then evaporated twice with benzene.
Cyclohexene (0.5 ml) was added and the mixture was again
evaporated with benzene. The residue was dissolved in
dichloromethane (2S ml) and then added during 1 h, to a stirred
mixture of compound (18) (3.32 g, 9.4 mmol), powdered molecular --
sieves (20 g, 4 A) and tetraethylammonium bromide (3.5 g) in
dimethylformamide (75 ml). The reaction mixture was stirred for
2 h at 0C, then for 2 h at room temperature followed by
filtering through Celite. The filtrate was partitioned between
dichloromethane and saturated aqueous sodium hydrogen
carbonate. The aqueous phase was extracted with dichloromethane
and the combined organic phases were washed with water, and
concentrated. The residue was chromatographed (ethyl acetate-
heptane; 1:1-3:1) to give a crude product, which was
O-acetylated by stirring in acetic anhydride (50 ml) and
pyridine (75 ml) for 17 h at room temperature. Evaporation with
toluene and chromatography (ethyl acetate-heptane; 1:2-1:3)
gave tl9) (3.3 g, 40S), t~]D22 -4.6 (c 1.4, chloroform).
lH-NMR data (CHC13, ~): 7.90-7.83 (2H), 7.79-7.71 (2H),
7.45-7.24 (lSH), 5.83 (dd, lH, J 10 and 9.5 Hz, H-3), 5.43 (d,
lH, J 10.5 Hz, H-1), 5.09 (dd, lH, J 10 and 9.5 Hz, H-4), 4.99
and 4.67 (2H, AB-system, J 11.5 Hz, benzylic H), 4.97 (d, lH, J
3.5 Hz, 8-1'), 4.89 and 4.77 (2H, AB-system, J 12 Hz, benzylic
H), 4.79 and 4.73 (2H, AB-system, J 12 Hz, benzylic H), 4.39
(t, lH, J 10.5 Hz), 4.06 (dd, lH, J 10 and 3.5 Hz), 3.97-3.87
(3H), 3.76 (dd, lH, J 12 and 6 Hz), 3.70- 3.62 (2H), 2.67 (dq,
lH, J 12 and 7.5 Hz, SCH), 2.56 (dq, lH, J 12 and 7.5 Hz, SCH),
1.98 (s, 3H, CH3C0), 1.87 (s, 3H, CH3C0), 1.15 (t, 3H, J 7.5
Hz, C~3CH2), 1.13 (d, 3H, J 7.5 Hz, FUC-CH3).
Calc. for C47HslN012S: C 66.1; H 6.02; N 1.64; S 3.75. Found: C
66.4; H 6.1; N 1.55; S 3.25.
(iii) Methyl 3,4-di-O-acetyl-6-O-(2,3,4-tri-
-O-benzyl-~-L-fucopyranosyl)-2-deoxy-2-phthalimido-~-D-glucopyr
anoside (20j
..
SUt~ JTE SHEET
wos5t~527 21 6~9~1 pcT/sæ94loo6o4
To a mixture of (19.) (853 mg, 1 mmol), methanol (0.102 ml, 2.5
mmol), N-iodosuccinimide (344 mg, 1.52 mg) and ground molecular
sieves (0.9 g, 4 A) in dichloromethane-diethyl ether (2:1, 25
ml) at -30C, was added trifluoromethanesulphonic acid (0.030
- 5 ml, 0.3 mmol). After 2.5 h. the reaction mixture was filtered
through Celite into an aqueous solution of sodium hydrogen
carbona~e and aqueous sodium bisulphite. The organic phase was
separated and washed with saturated aqueous sodium chloride,
and concentrated. Chromatography (ethyl acetate- heptane; 2:3)
of the residue gave (20) (778 mg, 94S), ta]D22 -2.8 (c 1.1,
chloroform).
lH NMR data (CHC13, ~): 7.90-7.82 (2H), 7.78-7.70 (2H),
7.45-7.24 (15H), 5.79 (dd, lH, J 11 and 9 Hz, H-3), 5.26 (d,
lH, J 805 Hz, H-1), 5.08 (dd, lH, J 10 and 9 Hz, H-4), 4.99 and
4.67 (A~-system, 2H, J 11.5 Hz, benzylic H), 4.96 (d, 1~, J 3.5
Hz, H-l~), 4.88 and 4.77 (AB-system, 2H, J 12 Hz, benzylic H),
4.81 and 4.69 (AB system, 2H, J~12 Hz, benzylic H), 4.28 (dd,
lH, J 1~ and 8.5 Hz, H-2), 4.07 (dd, lH, J 10 and 3.5 Hz),
3.99-3.85 (3H), 3.77 (dd, lH, J 12 and 6 Hz), 3.71-3.64 (2H),
3.34 (s~ 3H, CH30), 2.00 (s, 3H, CH3CO), 1.86 (s, 3H, CH3CO),
1.14 (dr 3H, J 6.5 Hz, FUC-CH3).
Calc. for C46H49N0l3: C 67.06; H 5.99; N 1.70. Found: C 67.0; H
6.1; N 1.65.
(iv) Methyl 2-acetamido-6-0-(2,3,4-tri-0-benzyl-~-L-
-fucopyranosyl)-2-deoxy-~-D-glucopyranoside (21)
Compound( 20) was deacetylated in methanolic sodium methoxide
(9.5 mM, 31.5 ml) for 1.5 h. Neutralization with acidic cation
eYchAnge resin (Bio-Rad AG~ 50W-X8), filtration and
concentration gave a residue which was dissolved in methanol
(20 ml). Hydrazine monohydrate (0.8 ml) was added and the
mixture was heated under reflux for 4 h and then cooled to
10C. Water (15 ml) and acetic anhydride (5 ml) were added and
the reaction mixture was stirred at room temperature. After 20
min a white precipitate was obtained. Addition of methanol (10
SUB~ 1-1 ~JTE SHEET
WO 95/00527 PCT/SE~,'C0 60~
2~9~_s6_ ~
ml) facilitated stirring. After additional 2.5 h, pyridine (2
ml) was added which resulted in a clear solution. The mixture
was then stirred for 30 min. The methanol was evaporated and
the aqueous residue was extracted with dichloromethane. The
organic phase was washed with 1 M HCl and saturated aqueous
sodium hydrogen carbonate, and concentrated. Chromatography
(ethyl acetate-methanol, 10:1) of the residue gave (21) (435
mg, 77~). An analytical sample was crystallized from ethanol,
m.p. 224-226OC (d), t~]D22 -75.4 (c 0.9, CHC13).
H NMR data (CDC13-CD30D, 3:1, CHD20D ref., ~) d: 7.41-7.20
tl5H), 4.91 and 4.61 (AB-system, 2H, J 11.5 Hz, benzylic H),
4.80 and 4.70 (AB-system, 2H, J 11.5 Hz, benzylic H), 4.78 (d,
lH, J 3 Hz, ~-1'), 4.75 (s, 2H, benzylic H), 4.24 (d, lH, J 8.5
Hz, H-1), 4.09-3.97 (2H), 3.92 (dd, lH, J 10 and 2.5 Hz), 3.86
(dd, lH, J 11 and 2 Hz), 3.77-3.56 (3H), 3.42 (dd, lH, J 9.5
and 8.5 Hz) 3.36 (s, 3H, CH30), 1.97 (s, 3H, CH3CO), 1.07 (d,
3H, J 6.5 Hz, Fuc-CH3).
(v) Methyl 2-acetamido-2-deoxy-6-0-~-L-fu~oy~LanoSyl-~-D-
glucopyranoside (221
A solution of (21) (362 mg, 0.56 mmol) in acetic acid (50 ml)
was hydrogenolysed at 230 kPa over 10% Pd/C (160 mg) over
night. The mixture was filtered through a layer of Celite and
concentrated. Column chromatography (chloroform-methanol-water,
65:40:10) of the residue gave amorphous (22) (192 mg, 90%,),
t~]D22 -106 (c 1.1, H20).
lH NMR data (D20, CH30H ref., ~) : 4.95 ( d, lH, J 4 Hz, H-l'),
4.46 (d, lH, J 8.5 Hz, H-l), 4.15 (q, lH, J 6.5 Hz), 4.02 (dd,
lH, J 12 and 1.5 Hz), 3.92 (dd, lH, J 10.5 and 3.5 HZ),
3.84-3.67 (4H), 3.62-3.49 (6H), 3.51 (s, CH30), 1.24 (d, 3H, J
6.5 Hz, Fuc-CH3).
13C NMR data (D20, CH30H ref., ~) : 177.7, lOS.0, 102.4, 78.0,
76.9, 74.8, 72.9, 72.5, 71.2, 70.3, 69.7, 60.0, 58.5, 25.2,
18.3.
SUB~ 11~ I.JTE SHEET
Wos5l~527 21 6 ~ 9 61 PCT/SE94/00604
-57-
BXAMPLE 6
3,3-Dl~thylbutyl 2-~cet~mi~o-2-deoYy-6-0-~-L-fucopyr~nosyl-~-
D-glucolJ~.~noside ~24)
r 5
(i) 3,3--Dimethylbutyl 3,4-di-0-acetyl-6-0-(2,3,4-tri-0-benzyl-
~-L-fucopyranosyl)-2-deoxy-2-phthalimido-~-D-glucopyranoside
(23)
To a stirred mixture of (19) (8S3 mg, 1 mmol), 3,3
dimethylbutan-1-ol (0.182 ml, 1.5 mmol), N-iodosuccinimide (344
mg, 1.52 mmol) and powdered molecular sieves (0.9 g, 4 A) in
dichloromethane-diethyl ether (2:1; 25 ml) at -30C, was added
trifluoromethanesulphonic acid (0.017 ml, 0.19 mmol). After 1 h
additional 3,3-dimethylbutan-1-ol (O.loO ml, 0.82 mmol) and
trifluoromethanesulphonic acid (0.015 ml, 0.17 mmol) were added
and stirring was continued for 2.S h. The reaction mixture was
then filtered through Celite into an aqueous solution of sodium
hydrogen carbonate and sodium bisulphite. The organic phase was
w~cheA with agueous sodium chloride and concentrated. The
residue was submitted to chromatography (ethyl acetate-heptane;
2:5) to give (23) (740 mg, 83%), t~]D22 -8.5 (c 1.3, CHCl3).
lH NMR data (CHC13, ~): 7.89-7.82 (2H), 7.78-7.70 (2H),
7.44-7.24 (15 H), 5.79 (dd, lH, J 11 and 9 Hz, H-3), 5.32 (d,
lH, J 8.5 Hz, H-l), 5.08 (dd, lH, J 10 and 9 Hz, H-4), 4.99 and
4.66 (AB-system, 2H, J ll.S HZ, benzylic H), 4.91 (d, lH, J 3.5
Hz, H-l'), 4.88 and 4.77 (AB-system, 2H, J 12 Hz, benzylic H),
4.80 and 4.69 (AB system, 2H, J=12 Hz, benzylic H), 4.29 (dd,
lH, J 11 and 8.5 Hz, H-2~, 4.05 (dd, lH, J 10 and 3.5 Hz),
3.99-3.73 (6H), 3.70-3.63 (2H), 3.38 ~m, lH), 1.98, (s, 3H,
CH3CO), 1.87 (s, 3H, CH3C0), 1.30 (m; 2H, OCH2CH2), 1. 13 (d,
3H, J 6.5 Hz, Fuc-CH3), 0.69 (s, 9H).
Calc. for C51H49NO13: C 69.3; H 5.59; N 1.59. Found: C 68.4; H
6.65; N 1.75.
SU13~ ~ JTE SHEET
wos5loo527 PCT/SE94/00604
21 6~9 ~1 -58-
(ii) 3,3-Dimethylbu~yl
2-acetamido-2-deoxy-6-o-~-L-fucopyranosyl-~-D-glucopyranoside
(24)
S Compound (23) (680 mg, ?6 mmol) was dissolved in methanol (25
ml). Methanolic sodium methoxide (0.2 M, 1 ml) was added and
the mixture was stirred for 3.5 h. Neutralization with acidic
cation exchange resin (Bio-Rad AG~ 50W-X8), filtration and
evaporatiOn gave a residue which was dissolved in methanol (20
ml). Hydrazine monohydrate (O.S ml, 10.3 mmol) was added and
the mixture was heated under reflux for 3.5 h and then cooled
to 10C. Water (10 ml), methanol (2 ml) and acetic acid
anhydride (2.5 ml) were added and the mixture was stirred at
room temperature for 2.5 h during which time additional
portions of acetic acid anhydride (2.0 and 0.5 ml) were added.
The methanol was then evaporated, and the aqueous residue was
partitioned between dichloromethane and water. The aqueous
phase was extracted with dichloromethane and the organic phase
was concentrated. Chromatography of the residue (ethyl
acetate-methanoli 20:1) gave a product which was dissolved in
acetic acid (S0 ml). 10% Pd/C (160 mg) was added and the
mixture was hydrogenolyzed at 230 kPa for 4 h at room
temperature. The mixture was filtered through a layer of Celite
and concentrated. Column chromatography of the residue
(chloroform-methanol-water, 150:40:3~65:40:10) of the residue
gave amorphous (24) (275 mg, 80%,), t~]D22 -87.2 (c 0.95,
H20) .
lH NMR data (D20, CH30H ref., ~) : 4.94 (d, lH, J 4 Hz, H-l'),
4.S4 (d, lH, J 8.5 Hz, H-l), 4.15 tq, lH, J 6.5 Hz), 4.03-3.88
(8H), 2.03 (s, 3H, CH3CON), 1.58-1.40 (2H, OCH2CH2), 1.24
(d, 3Hr J 6.5 Hz, FUC-CH3), 0.90 (s, 9H).
13C NMR data (D20, CH30B ref., ~) : 177.S, 104.0, 102.4, 77.9,
76.9, 74.9, 73.0, 72.6, 71.2, 71.0, 69.7, 58.6, 45.0, 31.9,
25.2, 18.4.
SUB~3 111 ~JTE SHEET
W095/~7 21 6~9 61 PCT/SE94/00604
Calc. for C20H37NO1o~ C 53.2; H 8.26; N 3.10. Found: C 51.2; H
8.25; N 3.2.
~XAMP~E 7
,, 5
Fuc~1-2~al~1-O-spacer ~-~8A ~31)
..
i) Ethyl 2-0-acetyl-3,4,6-tri-0-benzyl-1-thio-~-D-
galactopyranoside (25)
Compound (25) was prepared from acetobromogalactose (70.73 g,
0.172 mmol), according to proceedure described by S Nilsson, H
L~nn and T Norberg, Glycoconjugate J., 1989, 6, 21-34. Yield of
(25) wa$ 26.13 g t28%).
TLC: Rf 0.33 (heptane:ethyl acetate, 9:2)
13C-NMR (CDC13) ~: 170.2 (CO), 139.2, 138.6, 138.4 (aromatic
C), 84.2, 82.1 78.1, 75.0, 74.1, 73.6, 72.6, 70.3, 69.2, (C-
1,2,3,4,5,6, 3x CH2Ph), 24.1 (SCH2CH3), 21.6 (OCOCH3), 15.4
(SCH2CH3~.
lH-NMR (CDCl3) ~: 5.43 (bt, lH, J2,3 9-7 Hz, H-2), (d lH~ Jl,2
11.9 Hz, H-l), 4.01 (bd, lH, J3,4 2.9 Hz, H-4), 3.55 (dd, lH,
H-3).
2S
(ii) Ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-l-thio-~-D-
galactopyranoside (26)
Ethyl 2-O-acetyl-3,4,6-tri-O-~enzyl-l-thio-~-D-
galactopyranoside (25) was deacetylated with sodium methoxide
~n methanol (50 ml, pH 12) and subsequently benzoylated with
benzoylchloride (1.96 gr., 14 mmol) in pyridine (20 ml)
according to st~Ard procedures. Crystalline 26 was obtained
in almos~ quantitative yield (3.44 gr., 97%).
NMR (CDCl3) lH: ~ 5.70 (lH, t, 9.8Hz, H-2) 2.60-2.80 (2H, m, -
CH2cH3) ~
SIJB~ ~ JTE SHEET
WO 95t~S27 2 1 6 4 9 6 I PCT/SE94/00604~
-60-
13C: ~ 14.8, 23.6 (~Et), 68.6, 70.2, 71.7, 72.8, 73.6, 74.4,
76.6, 127.5-138.6 (aromatic C), 165.4 (C=O)
(iii) 2-~zidoethyl 3,4,6-tri-O-benzyl-~-D-galactOPYranoside
(28)
To a stirred suspension of the thioglycoside (26) (700 mg, 1.17
mmol), 2-azidoethanol (204 mg, 2.34 mmol; prepared according to
A. Ya. Chernyak et al. and A.V. Rama Rao, Carbohydr. Res.,
1992, 223, 303-309), N-iodosuccinimide (395 mg, 1.75 mmol,) and
ground molecular sieves (3A, 400 mg) in dichloromethane (25 ml)
was added at 0C trifluoromethanesulfonic acid (TfOH; 35 mg,
0.23 mmol; according to method published by G.H. Veeneman, S.H.
Van Leeuwen, J.H. Van Boom, Tetrahedron Lett., 1990, 31, 1331).
When TLC (toluene:ethyl acetate, 6:1) showed complete
conversion ( ~ 15 minutes), reaction was quenched by addition
of triethylamine at 0C. The solution was filtered through a
layer of celite, diluted with dichloromethane and washed twice
with aqueous Na2S2O3 (10~) and finally with water.
The organic phase was dried over magnesium sulfate, filtered
and concentrated and the residue was immediately subjected to
TLC (toluene:ethyl acetate, 15:1). Solvent removal left 672 mg
of 2-Azidoethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-~-D-
galactopyranoside (27) as a colourless oil (92%), which was
treated with sodium methoxide in methanol (pH 11) at room
temperature for 6 hours. The solution was neutralized with
Dowex 50 H+ resin, filtered and concentrated. The crystalline
product (28) (540 mg, 89% from 2) was used without further
purification for the preparation of disaccaride (29).
Compound (27): NMR (CDC13) lH: ~5.66 (lH, dd, 10.0, 7.9 HZ, H-
2) 4.57 (lH, d, 7.8 Hz, H-1)
13C: ~ 50.7 (CH2N3, 67.3, 68.7, 71.9, 72.5, 73.6, 73.9, 74.5,
101.4 (C-l), 127.6-137.8 (aromatic C), 165.3 (C=O)
Compound (28): 13C: ~ 50.7 (CH2N3), 68.4, 68.7, 71.4, 72.6,
73.0, 73.6, 73.9, 74.5, 81.7, 103.4 (C-1), 125.3-138.4
(aromatic C)
SUB~ ITE SHEEi-
=
wo9s/~27 216 4 9 61 pcTlsæ94loo6o4
-61-
(iv) 2-Azidoethyl 3,4,6-tri-0-benzyl-2-0-(2,3,4-tri-0-benzyl-~-
L-fucopyranosyl)-~-D-galactopyranoside (29)
To a solution of thioethylglycoside (14) (400 mg, 0.836 mmol)
- 5 in dichloromethane (10 ml), bromine (134 mg, 0.836 mmol) was
added at 0C. After about S minutes at 0C, the solution was
allowed to attain room temperature and the solvent was
evaporated. After co-evaporation with toluene the residue was
dissolved in dichloromethane (2 ml) and added at room
temperature to a suspension of tetraethylammonium bromide (176
mg, 0.835 mmol; prepared according to R.U. Lemieux, K.B.
Hendriks, R.V. Stick, K. James, J. Am. Chem. Soc. 1975, 97:14,
4056), compound (28) (290 mg, 0.558 mmol) and ground molecular
sieves (3A, 300 mg) in CH2Cl2:DMF (4:1, 7 ml). TLC
(toluene:ethyl acetate, 6:1) showed complete conversion after
stirring for 20 hours. The mixture was filtered, diluted with
dichloromethane and washed with water. The organic phase was
dried over magnesium sulfate and filtered and concentrated in
vacuo. Preparative TLC yielded the title compound (29) as a
viscous oil (407 mg, 78~).
NMR (CDC13) 13C: ~ 16.5, 33.6, 50.9, 66.4, 66.9, 68.8, 71.4,
72.0, 7208, 72.9, 73.5, 73.6, 74.4, 74.8, 75.7, 78.1, 79.6,
84.3, 97.3, 102.0, 125.3-129.0 (aromatic C), 138.0, 138.3,
139Ø
(v) 2-Aminoethyl 2-o-~-L-fucopyranosyl-~-D-galactopyranoside
(30)
The protected disaccaride derivative (29) (80 mg, 85 ~mol) was
dissolve~ in ethanol (abs., 10 ml) and water (1 ml) and Pd/C
(10%, lO0 mg) was added. The mixture was hydrogenated and
stirred rapidly at room temperature at 50 PSI. When reaction
was not completed within 60 hours, the mixture was filtered and
the product formed was isolated (TLC, ethyl
acetate:~ethanol:acetic acid:water, 5:3:3:1, Rf=O.lS). After
concentration in vacuo, the residue was resolved in a buffer of
aqueous pyridine/acetic acid (2.5%/1%, pH 5.4) and eluated
r
SUBS 111 ~JTE SHEET
WO 95/00527 2 1 6 4 9 61 PCT/SE94/00604
--62--
through a Bio Gel P-2 column. Evaporation and freeze drying
gave 14 mg (44%) of the title compound (30) as a white powder.
NMR (CDC13) 13C: ~ 16.8, 39.8, 61.1, 66.4, 67.1, 68.7, 69.6,
71.9, 73.0, 7s.0, 78.4, 100.0, 101.7
(vi) Fuc~1-2Gal~l-O-spacer 4-HSA (31)
To a stirred ice-cooled solution of thiophosgene (10 eq.) in
tetrahydrofuran t2 ml), the amino derivative (30) (30 ~mol) in
sodium borate buffer (0.85 M, 2 ml, pH 8.5) was added. The
solution was stirred at room temperature for 10 minutes and
then extracted with diethylether (3 x 2 ml). The aqueous phase
containing the isothiocyanate derivative was added to a
solution of Human Serum Albumine (HSA) (1/30 eq.) in the same
buffer system (0.5 ml). pH was adjusted to 8.5 with aqueous
sodium hydroxide (0.25 M) and the mixture was stirred at room
temperature for 48 hours. Freeze drying of the reaction mixture
was followed by ultracentrifugation purification with
Centriprep tubes (lOKO). Freeze drying of the purified
solutions gave the ~SA-conjugates (31) in excellent yield (18
mg).The degree of substitution was determined by Time of Flight
masspectroscopy to 8 mol disaccharide/mol protein.
EXAMPL~ 8
Fuc~1-2Gal~l-o-spacer -~8A ~36)
(i) 8-Azido-3,6-dioxaoctyl 3,4,6-tri-O-benzyl-~-D-
galactopyranoside (33)
The azidoderivative (33) was synthesized from the thioglycoside
t26) (1004 mg, 1.68 mmol) and 1-azido-8-hydroxy-3,6-dioxaoctane
(686 mg, 3.35 mmol; prepared according to C.R. Bertozzi, M.D.
R~nArski, J. Org. Chem., 1991, 56, 4326-4329) according to a
procedure similar to the one used for synthesis o~ derivative
~28) (TLC; toluene:EtOAc 6:1) showed complete conversion within
40 minutes. Similar workup and deacylation of 8-azido-3,6-
SlJBs I ~ I JTE SHEEI
W095/00527 2 1 6 ~ 9 6 ~ PCT/SE94/00604
-63-
dioxaoctyl 2-o-benzoyl-3,4,6-tri-0-benzyl-~-D-galactopyranoside
(32) yielded 933 mg (78~ from 26) of the title compound (33) as
a viscous ~
-~- 5 Compound (32); NMR (CDC13): 1H: ~ 5.64 (lH, dd, 10.0, 7.9 Hz,
H-2).
13C: ~ 50.6 (CH2N3), 68.7, 68.9, 69.8, ~0.3, 70.5, 70.7, 71.8,
71.9, 72.6, 73.6, 73.8, 74.6, 80.0, 101.6 (C-l)
Compound (33); 13C:~ 50.6 (CH2N3), 68.7, 68.8, 70.0, 70.2,
70.5, 7~.6, 71.4, 72.6, 73.3, 73.5, 73.8, 74.5, 81.9, 103.8 (C-
1)
(ii) 8-Azido-3,6-dioxaoctyl 3,4,6-tri-0-benzyl-2-0-(2,3,4-tri-
O-benzyl-~-L-fucopyranosyl)-~-D-galactopyranoside (34)
Disaccaride (34) was synthesized from compound (33) (500 mg,
0.82 mmol) and thioethylglycoside (14) (512 mg, 1.07 mmol)
according to the proce~llre described for the corresponding
derivative (29). Preparative ~LC gave 683 mg (81%) of the title
compound (34) as an oil.
MMR (CDCl3) 13C: ~ 18.3, 50.2, 66.2, 68.2, 68.8, 70.0, 70.2,
70.3, 70.6, 71.2, 72.0, 72.3, 72.5, 73.0, 73.3, 73.6, 74.4,
74,6, 75.8, 78.0, 79.7, 84.2, 98.6, 102.0
(iii) 8-~mino-3,6-dioxaoctyl 2-0-~-L-fucopyranosyl-~-D-
galactopyranoside (35)
8-Azido-3,6-dioxaoctyl 3,4,6-tri-0-benzyl-2-0-(2,3,4 tri-O-
benzyl-~L-fucopyranosyl)-~-D-galactopyranOSide (34) (35 mg, 34
~mol) was dissolved in a mixture of ethyl acetate:ethanol:water
in 1:2:2 (vol., 12 ml) and acidified with 20 ~l HOAc
(according to method published by S. Nilsson, Doctoral
dissertation, Lund University, April 1992). The solution was
hydrogenated at 50 PSI on 10% Pd/C (140 mg) at room temperature
overnight and when TLC (ethyl acetate:methanol:acetic
acid:water, 5:3:3:1) showed complete deprotection, the mixture
SUB~ JTE SHEET
wos~/~s27 PCT/SE94/00604
~16~G~-64- --
was filtered and evaporated. Purification on a Bio-Gel~ P-2
column taq. pyridine:acetic acid, 2.5:1 by vol., pH 5.4)
concentration and freeze drying gave the title compound (35) as
a white powder (14 mg, 90%).
NMR (CDC13) 13C: ~ 15.2 (CH3), 38.9, 60.7, 66.1, 66.5, 68.1,
68.s, 68.7, 69.2, 69.3, 69.4, 69.6, 71.7, 73.4, 74.8, 76.6,
99.2 (C-l'), 101.4 (C-1).
tiV) Fuc~1-2Gal~1-o-spacer 1-HSA (36)
To a stirred ice-cooled solution of thiophosgene (10 eq.) in
tetrahydrofuran (2 ml), the amino derivative (35) (30 ~mol) in
sodium borate buffer (0.85 M, 2 ml, pH 8.5) was added. The
solution was stirred at room temperature for lO minutes and
then extracted with diethylether (3 x 2 ml). The aqueous phase
containing the isothiocyanate derivative was added to a
solution of Human Serum Albumine (HSA) (1/30 eq.) in the same
buffer system (0.5 ml). pH was adjusted to 8.5 with aqueous
sodium hydroxide (0.25 M) and the mixture was stirred at room
temperature for 48 hours. Freeze drying of the reaction mixture
was followed by ultracentrifugation purification with
Centriprep tubes (lOKO). Freeze drying of the purified
solutions gave the HSA-conjugates 36 in excellent yields (33
mg).
The degree of substitution was determined by Time of Flight
masspectroScopy to 5 mol disaccharide/mol protein.
EXA~P~E 9
Fuc~-2Gal~1-o-~pacer 2-~AA (38)
(i) 8-N-acrylamido-3.6-dioxaoctyl 2-0-~-L-fucopyranosyl-~-D-
galactopyranoside (37)
0.8 ml deaerated 0.5 M sodiumborate aq. buffer (pH 8.5) and 2.4
ml deaerated methanol was added to 15 mg of the compound (35).
The reaction mixture was flushed with nitrogen and cooled to
SUB~ JTE SHEET
W095l~5~ 2 1 6 ~ 9 6 ~ PCT/SE94/00604
-65-
0C. 3.3 ~1 of acryioylchloride was added and stirring was
continued for 10 minutes. The reaction mixture was concentrated
at room temperature to about a third of its original volume.
Purification on a Bio-Gel- P2 column and lyophilization gave
S the title compound (37) ~14 mg, 83%)
,
NMR-data: 13C (D2O): ~ 15.0 (CH3), 38.54 (CH2N), 60.51, 66.31,
67.87, 68.19, 68.30, 68.50, 68.98, 69.10, 69.12, 69.43, 71.50,
73.25, 74.55, 76.08 (C-2,3,4,5,6; C-2,3,4,5; 5xCH2O) 98.88 (C-
1'), 101.16 (C-l), 126.92 and 129.43 (CH=CH2).
(ii) Fuc~1-2Gal~l-O-spacer 2-PAA (38)
To a solution of the compound (373 (14 mg, 0.027 mmol) and
acrylamide (9.7 mg, 0,14 mmol) in deaerated water (1 ml) was
added first N,N,N~N~-tetramethylendiamine (6 ~1) and then
ammonium persulphate (3.5 mg). The mixture was stirred at
roomtemperature over night. The polymer (38) obtained was
purified by gel chromatography on a Bio-Gel- P2 column.
Freeze-drying of the purified solutions gave the PAA conjugate
in excellent yield (17.9 mg). lH-NMR showed an average
i..~oL~oration of 1 oligosaccharide per 7 acrylamide units.
.
E~MPr~E 1 0
FUC~1--2G~ 3 ~FUCt~ )GlCNAC,B--3G~l,~ Glc,Bl--N~l--PAA (~2)
(i) Fuc~l-2Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~1-4Glc~l-NH2 (40).
Solid ammonium bicarbonate was added until æaturation to a
solution of Fuc~1-2Gal~1-3(Fuc~1-4)GlcNAc~1-3Gal~1-4Glcl-OH
(Lewis B hexasackaride (39), purchased from Iso Sep AB, 25 mg
in water (1.25 mL). The mixture was stirred in an open vessel
at room kemperature for 6 days. Ammonium bicarbonate was added
at intervals, saturation was assured by always keeping a
portion of solid salt present in the mixture. When TLC
- 35 indicated no more conversion, the mixture was diluted with
water (5 mL) and concentrated to half the original volume. The
residue was diluted to 20 mL with water and concentrated to 5
mL. This process was repeated once, then the residue was
SUBs ~ JTE SHEEr
wos5/oos27 PCT/SE94/00604
21649~1 ~
-66-
diluted to 10 mL and lyophilized. The crude product was put on
a Bio-gel P2-column, and the fraction containing Lewis B
glycosylamine (40) was collected, (20 mg 80~).
NMR data: 13C (D2O): ~ 84.66 (C-NR2), 97.54, 99.33, 100.39,
100.72, 102.98 (C-1 carbons of the nonreducing sugarunits),
15.08, 15.13 (2xCH3-fucose), 21.95 (CH3-CON-Glc~Ac).
(ii) Fuc~1-2Gal~1-3(Fuc~l-4)GlcNAc~1-3Gal~1-4GlC~1-NH-CO-CH-CH2
(41)
Sodium carbonate (50 mg) and deaerated methanol (O.5 mL) was
~e~ to a solution of the glycosylamine (40) (20 mg, 0.02
mmol) in water (0.5 mL). The mixture was stirred at 0C while
acryloyl chloride (60 ~L., 0.74 mmol) in tetrahydrofuran (0.5
mL) was added during 5 min. After 10 min. the solution was
diluted with water (3 mL) and concentrated to 2 mL. The
solution was again diluted with water (2 mL), 200 ~L
tetrahydrofuran (inhibitor solution) was added, and the
solution was concentrated to 1-2 mL. This solution was purified
by gel filtration on a Bio-Gel~ P2 column. Appropriate
fractions were pooled and lyophilized to obtain the title
compound (41) (14 mg, 67%).
NMR data: 13C (D2O): ~ 81.28 (C-NHCOCHCH2), 97.42, 99.18,
100.25, 102.59, 102.85 (C-1 carbons of the nonreducing
sugaruni~s)~ 14.99, 15.06 (2xCH3-fucose), 21.92 (CH3CON-
GlcNAc), 125.93, 130.32,(CH=CH2).
Fab ms: pseudomolecular ion m/z; 1053 (M+H) and 1075 (M+Na)+.
(iii) Fuc~1-2Gal~l-3(Fuc~1-4)GlcNAc~-3Gal~1-4GlC~l-NH-PAA (42)
Copolymerization of N-Acryloylglycosylamine with acrylamide.
A solution of the N-acryloylglycosylamine (41) (13 ~mol) and
acrylamide (53 ~mol, 3.7 mg) in distilled water (200 ~L) was
deaerated by flushing with nitrogen for 20 min. The solution
was then stirred at 0C and N,N,N,',N'-
tetramethylethylenediamine (2 ~L) and ammonium persulfate (1
SUt~ ITE SHEET
W095/00527 21 ~ ~ 9 61 . ~ PCT/SE94100604
-67-
mg) wer~ added. The mixture was slowly stirred at 0C for 2
hours and then at room temperature overnight. The viscous
solution was diluted with water (1 mL) and purified by gel
filtration on Bio-Gel P2 column eluated with aqueous n-
- 5 buthanol (1~). Fractions containing polymer were pooled and
lyophilized. Yield: 3mg.
H-NMR ~ ws presence of approximately
1 Lewis ~ unit per 5 CHCH2 units.
ESANPL~ 11
Fuc1-2~ 3çT.~t~fll-o-~p~c~r 5-PAA (S0)
(i) 2-azidoethyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-~-D-
glucopyranoside (43)
Ethyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-1-thio-~-D-
glucopyranoside (prepared according to H L~nn, Carbohydr. Res.,
139 (198S), 105-113) (O.S g, 1.1 mmol) was dissolved in 20 ml
of dichloromethane and 2-azidoethanol (prepared according to
Chernyak A.Y. et al. Carbohydr. Res., 1992, 223, (303-309)
(0.148 g, 1.7 mmol) crushed 4A molecular sieves were added and
the mixture stirred for 30 min. Dimethyl(methylthio)sulfonium
2S triflate (DMTST) (0.439 g, 1.7 mmol; prepared according to P.
F~gedi and P.J. Garegg, Carbohydr. Res., 149 (1989), 9-12) was
added at room temperature and stirring was continued for 4
hours. Analysis by TLC (toluene-ethylacetate) show no starting
material, and to the reaction mixture was added 1 ml of
triethylamine and stirring was continued for another 30 min.
The reaction mixture was transfered to a silica gel column and
eluted with toluene:ethylacetate 6:1 to give (372 mg, 72%) of
the title compound (43).
NMR-data: 13C (CDC13): ~ 50.38 (CH2-N); 56.42 (CH-N); 66.2,
(CH-O); 68.44 (CH2O); 68.50 (CH-O; 68.53 (CH2-O); 82.05 (CH-O);
98,89 (C-1); 101.83 (PhCH).
SUB~ JTE SHEET
wogs/00~7 PCT/SE94/00604
2l6~961_68- --
(ii) 2-Azidoethyl 3-0-(2-0-acetyl-3,4,6-tri-0-benzyl-~-D-
galcatopyranosyl)-4,6-o-benzylidene-2-deoxy-2-phthalimido-~-D-
glucopyranoside (44)
S Ethyl 2-0-acetyl-3,4,6-tri-0-benzyl-1-thio-~-D-
galactopyranoside (25) (818 mg, 1.5 mmol) and the compound (43)
(395 mg, 0.85 mmol) were dissolved in 30 ml of dichloromethane,
crushed 4A molecular sieves were added and the mixture stirred
for 20 min. The reaction was flushed with nitrogen and DMTST
(787 mg, 3.05 mmol, dissolved in 5 ml of dichloromethane) was
added dropwise to the reaction mixture and the dropfunnel
rinsed with 6 ml of dichloromethane. After 2 hours 1 ml of
triethylamine was added and stirred for 30 min., filtration,
concentration and column chromatography (toluene:ethylacetate
10:1 gave three fractions. Fraction 1 the ~-product (97.32,
98.89; (C-l and C-l'). Fraction 2 almost pure 44 (306 mg, 39~).
NMR-data: 13C (CDC13 ref. tetramethylsilane 0 ppm): ~ 20.32
(CH3C0), 50.47 (CH2N), S5.13 (CH-N), 66.57, 68.15, 68.20,
68.65, 71.58, 71.71, 72.17, 72.94, 73.46, 74.38, 75.15, 80.47,
81.08 (C-3,4,5,6 C-2,3,4,5,6; 3xCH2Ph; CH2-0), 98.86 (C-l),
100.75, 101.23 (C-l and CH Ph), 168.84 (C=0).
~iii) 2-azidoethyl 2-acetamido-3-0-(3,4,6-tri-0-benzyl-~-D-
galactopyranosyl)-4~6-o-benzylidene-2-deoxy-~-D-glucopyranoside
(45)
To compound (44) (525 mg, 0.56 mmol) was added 50 ml of ethanol
and 1.1 ml of hydrazinhydrate reflux over night and TLC
(toluene:ethylacetate 1:2) showed a new product. Concentration
and coevaporation with toluene, followed and then dissolving in
45 ml of dichloromethane and washing with an equal amount of
water, coevaporation with toluene, gave the crude monohydroxy
amine. This crude product was dissolved in
dichloromethane:methanol (1:1, 15 ml) and 1.5 ml of acetic
anhydride was added. After 3 hours no starting material was
left (TLC). Concentration and chromatography (toluene-
SUB~ 111 ~JTE SHEET
W095/~527 ~ 6 1 PCT/SE94/00604
-69-
ethylacetate 1:2) ga~e (204 mg, 45~) of the title compound
(45).
NMR-data: 13C (CDC13): ~ 23.59 (NHCOCH3), 50.59 (C-N), 56.89
(C-N), 6G.40, 68.28, 68.56, 70.55, 72.44, 73.21, 73.40, 73.53,
74.60, 76.08, 79.75, 81.71 (C-3,4,5,6; C-2',3',4',5',6';
3xCH2Ph; CH2O) 100.97, 101.25, 103.48 (C-1, C-11 and CHPh),
171.67 (C=O).
(iv) 2-azidoethyl 2-acetamido-3-O-(3,4,6-tri-O-benzyl-2-O-
(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-~-D-galctopyranosyl]-
4,6-O-benzylidene-2-deoxy-~-D-glucopyranoside (46)
The compound (45) (137 mg, 0.17 mmol) and the compound (14)
(162 mg, 0.34 mmol) were dissolved in dichloromethane (75 ml),
and molecular sieves (4A) were added, and the mixture was
stirred for 20 min. DMTST (96 mg, 0.37 mmol) was added and
stirring was continued for 1.5 hours. 1 ml of triethylamine was
added and stirring was continued for another 20 min. Filtration
through celite, concentration and column chromatography
(toluene:ethylacetate 1:1) gave (46) (101 mg, 49%).
NMR-data: 13C (CDC13): ~ 16.83 (C83 fucose), 23.30 (NHCOCH3),
50.66 (CH2-N), 57.40 (C-2), 66.55, 67.17, 67.96, 68.60, 72.31,
72.91, 72.99, 73.04, 73,10, 73.51, 74.48, 74.6s, 76.05, 76.29,
76.62, 77.60, 79.43, 79.53, 83.21 (C-3,4,5,6; C-2',3',4',5~,6~;
C-2", 3",4",5"; 6xCH2Ph), 97.67 (C-l"), 100.94, 101.07, 102.13
tC-1, C-l', CHPh), 170.92 (C=O).
(v) 2-trifluoracetamidoethyl 2-acetamido-3-O-t3,4,6-tri-O-
benzyl-2-o-(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-~-D-
galactopyranosyl]-4~6-o-benzylidene-2-deoxy-~-D-glucopyranoside
(47).
The compound (46) (135 mg, 0.11 mmol) was dissolved in 11 mL of
ethanol and 10% Pd/C (140 mg) was added. The reaction mixture
was hydrogenated at atmospheric pressure for 15 minutes.
Analysis by Tlc (ethyl acetate:methanol:acetic acid:water
SUB~ JTE SHEET
W095/00~7 PCT/SE94/00604
216~9~1 ~70- -
12:3:3:1) showed no starting material, but one ninhydrin
positive product. The mixture was filtered through celite,
concentrated and dissolved in dichloromethane (7 mL), and
pyridine (3.5 mL), flushed with nitrogen and cooled to 0C.
Trifluoroacetic anhydride (31 ~1, 0.22 mmol) was added. After
one hour the mixture was concentrated and coevaporated with 2
ml of toluene twice. Column chromatography (toluene:ethyl
acetate, 1:3) gave 47 (73 mg, 52%)
NMR-data: 13C (CDC13): ~ 17.06 (CH3 fucose) 22.66 (NHCOCH3),
39.59 (CH2-N), 54.83 (C-2), 65.56, 66.77, 67.78, 68.44, 68.69,
72.80, 73.05, 73.24, 2x73.46, 74.46, 74.66, 76.38, 77.08,
77.80, 78.91, 79.96, 80.01, 82.07, (C-3,4,5,6; C-
2',3',4~,S~,6~; C-2",3",4",5"; 6xCH2Ph) 98.61 (C-1"), 101.22,
lol.9s~ 102.35 (C-l, C-l~, CHPh), 171.64 (NHCOCH3).
(vi) 2-trifluoroacetamidoethyl 2-acetamido-2-deoxy-3-O-t2-O-
(~-L-fucopyranosyl)-~-D-galactopyranosyl]-~-D-glucopyranoside
(48)
Trisaccharide (47) (73mg, S6.2 ~mol) was dissolved in absolute
ethanol (7 ml) with water (0.25 ml) and glacial acetic acid (2
~1). The solution was hydrogenated over 10% Pd/C (152 mg) at 50
PSI at room temperature for 1 hour. When TLC (ethyl
acetate:acetic acid:methanol:water 12:3:3:1; R~ = 0.14 for the
compound (48) showed complete conversion, the reaction mixture
was filtered through a layer of celite and concentrated. The
crude, solid residue (46 mg) was used in the next reaction
without further purification.
NMR-data: 13C (D2O): ~ 16.59 (CH3 fucose), 21,85 (NHCOCH3),
39.44 (CH2-N), 54.56 (C-2), 60.45-76.9S (C3,4,5,6,
C2',3',4',s',6', 2",3",4",5") 99.29, 99.92, 101.28 (C-1, C-1',
C-l''), 173.48 (NHCOCH3).
SUB~ 111 ~TE SHEET
woss/~527 ~ 61 PCT/SE94/00604
-71- ~
(vii) 2-acrylamidoethyl 2-acetamido-2-deoxy-3-0-2-0-~-L-
fucopyranosyl-~-D-galactopyranosyl-~-D-glucopyranoside (49)
The crude compound (48) (46 mg) was dissolved in aqueous
~-~ 5 ammonia (25%, 4 ml) and stirred at room temperatur. The
reaction was complete within 1 hour and yielded the free amino
~ derivat~ve exclusively. (TLC ethy~ acetate:acetic acid:methanol
:water 5:3:3:1). Cor.centration and co-concentration with
toluene was followed by purification on a Bond-Elut (SCX, H~-
form) cation exchange resin 0.5 g cartridge. The sample was
dissolved in 3 ml of water and pH was adjusted to pH 6 with
aqueous acetic acid. The sample was put on the column and then
eluted with 2M ammonia in methanol:water, 1:1 (5 ml). The
fractions containing free amine (ninhydrin positive) were
pooled, concentrated and lyophilized to give (30 mg, 0.05 mmol)
crude amine.
1 ml deaerated 0.5 M sodiumborate (aq buffer (pH 8.5) and
deaerated methanol (3 ml) was added to the crude amine. The
reactio~ mixture was flushed with nitrogen and cooled to 0C,
6.4 ~1 (0.078 mmol) acryloylchloride was added and stirring was
continued for 10 minutes. The reaction mixture was concentrated
at room temperature to about a third of its original volume.
Purification on a Bio-Gel P2 column and lyophilization gave 49
of the title compound (49) (30 mg, 86~ from (47)).
NMR-data: 13C (D20): ~ 14.99 (-CH3, fucose), 21.99 (NHCOCH3),
39.10 (CH2N), 54.58 (C-2) 99.25, 99.93, 101.39 (C-1, C'-1, C"-
1), 127.27, 129.65 (CH=CH2).
(viii) Fuc~1-2Gal~1-3GlcNAc~1-0-spacer 5-PAA (50)
Copolymerization of 2-acrylamidoethyl 2-acetamido-2-deoxy-3-o-
2-o-~-L-fucopyranosyl-~-D-galactopyranosyl-~-D-glucopyranoside
(49) with acrylamide.
To acrylamide (10 mg, 144 ~mol) was added at room temperature a
solution of the trisaccharide (49) (18 mg, 29 ~mol) in
SUB~ JTE SHEEr
Wo 9~/00s27 PCT/SE94/00604
2l~4~ 72- --
deaerated water (1 ml). To this slowly stirred solution (kept
in the dark and under nitrogen) was added at oC~ first
N,N,N~,N~-tetramethylethylenediamine (6 ~1), and then ammonium
persulphate (3.5 mg). The mixture was stirred at room
5 temperature over night. TLC (ethyl acetate:acetic acid:methanol
:water 5:3:3:2) showed that almost all of the compound (49) was
consumed and that a charring baseline product had been formed.
The polymer was purified by gel chromatography on a Bio-Gel P-
2 column eluted with aqueous n-butanol (1%). Freeze-drying of
10 the polymeric fraction eluted in the void volume gave 13.1 mg
of the polymer (So) were the lH N~ analysis of the product
showed an average incorporation of 1 trisaccharide per 7.6
acrylamide units, and 11.9 mg of polymer (50) were the lH NMR
analysis of the product showed an average incorporation of 1
15 trisaccharide per 10.3 acrylamide units.
B~ PI~IS 12
Fuc~l-2Gzll~Bl-3lFuc~ )alGNAc~Bl-o-sp~c~r 5--PAA(55)
(i) 2-azidoethyl 2-acetamido-6-0-benzyl-3-0-t3,4,6-tri-0-
benzyl-~B-D-galactopyranosyl)-2-deoxy-~B-D-glucopyranoside (51).
Diethyl ether saturated with hydrogen chloride was added, at
25 roomtemperature, to a stirred mixture of 2-azidoethyl 2-
acetamido-3-o-3~4~6-tri-o-benzyl-~B-D-galactopyranosyl-4~6
benzylidene-2-deoxy-,~-D-glucopyranoside (45) (420 mg, 0.52
mmol), sodium cyanoborohydrids (200 mg, 3,2 mmol) and molecular
sieves 3A in tetrahydrofuran (20 ml) until the mixture was
30 acidic (as determined with indicator paper; method according to
M. Nilsson and T. Norberg Carbohydr. Res., 183 (1988 71-82).
The mixture was stirred for 20 min. at roomtemperature and then
triethylamine (0.30 mL) was added. The mixture was filtered
through Celite, washed with water, dried and evaporated. The
35 crude product was purified by column chromatography (toluene:
ethyl acetate, 6:1) to give pure compound (51) (266 mg, 0.32
mmol, 65%).
SIJB-~ 1 l l ~JTE SHEET
W095/~527 2 1 6 ~ 9 6 f PCT/SE94/00604
-73-
NMR-data: 13C (cDCl,): ~ 23.41 (NHCOCH3), 50, 30 (CH2N), 56, 81
(C-N), 66,3-81,9 (C-3,4,5,6; C-2',3',4',5',6'; 4xCH2Ph; CH2O)
100.90, 103.21 (C-1, C-l'), 173,4 (CO)
(ii) 2 azidoethyl 2-acetamido-2-deoxy-4-0-(2,3,4-tri-O-benzyl-
~-L-fucopyranosyl)-3-O-t3,4,6-tri-O-benzyl-2-O-(2,3,4-tri-O-
benzyl-~-L-fucopyranosyl)-~-D-galactopyranosyl]-~-D
glucopyranoside (52).
The compound (51) (157 mg, 0.19 mmol) and the compound (14)
(362 mg, 0.76 mmol) were dissolved in dichloromethane (100 ml),
and 3g ~A molecular sieve (MS) were added and stirred for 20
min. Dimethyl (methylthio)sulfonium triflate (DMTST) (207 mg,
0.80 mmol) was added and stirring waæ continued for 1.5 hour. 2
ml of triethylamine was added and stirring was continued for
another 20 min. Filtration through celite, concentration and
column chromatography (toluen:ethylacetate, 1:1) gave the title
compound (52) (142 mg, 0.086 mmol, 45%).
NMR-data: 13C (CDC13): ~ 17.01, 16.81 (2xCH3 fucose), 23.20
(NHCOCH3), 50.35 (CH2-N), 57.21 (C-2), 98.31, 99.70, 101.14,
102.30 (C1, C1', 2xC1-fucose), 170.30 (C=O).
(iii) 2-trifluoroacetamidoethyl 2-acetamido-2-deoxy-4-O-
(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-3-0-~3,4,6 tri-O-benzyl-
2-O-(2,3,4-tri-O-benzyl-~-L-fucopyranosyl)-~-D-
galactopyranosyl]-~-D-glycopyranoside (53).
The compound (52) (140 mg, 0.084 mmol) was dissolved in 11 ml
ethanol and 10% Pd/C (150 mg) was added. The reaction mixture
was hydrogenated at atmospheric pressure for 15 minutes.
Analysis by TLC (ethyl acetate:methanol:acetic acid:water
12:3:3:1) showed no starting material, but one ninhydrin
positive product. The mixtures was filtered through celite,
concentrated and dissolved in dichloromethane (10 ml) and
pyridine (3.5 ml), flushed with nitrogen and cooled to 0C.
SUB~ JT~ SHEET
wos5/00527 PCT/SE94/00604
2 1 6~ 6~ _74_
Trifluoroacetic anhydride (31 ~1, 0.22 mmol) was added. After
one hour the mixture was concentrated and coevaporated with 2
ml of toluene twice. Column chromatography (toluene:ethyl
acetate 1:2~ gave the compound (53) (82.8 mg, 0.053 mmol, 63%).
.
NMR-data: 13C (CDC13): ~ 17.33, 16.93 ( 2xCH3 fucose), 22.30
(NHCOCH3), 39.25 (CH2-N), 54.48 (C-2), 99.03, 99.98, 101.63,
102.75, (Cl, Cl', 2xCl-fucose), 171.73 (NHCOC~3).
(iv) 2-trifluoroacetamidoethyl 2-acetamido-2-deoxy-3-0-(2-O-~-
L-fucopyranosyl-~-D-galactopyranosyl)-4-o-~-L-fucopyran
D-glucopyranoside (54).
The tetrasaccharide (53) (78 mg, 0.05 mmol) was dissolved in
absolute ethanol (8 ml) with water (0.25 ml) and glacial acetic
acid (2 ~L). The solution was rapidly stirred with 10~ Pd/C
(150 mg) under hydrogen (50 PSI) at room temperature for 1
hour. When TLC (ethyl acetate:acetic acid:methanol:water 12:3
3:1; showed complete conversion, the reaction mixture was
filtered thorugh a layer of celite and concentrated. The crude
compound (54) (35 mg) was used in the next reaction without
further purification.
NMR-data: 11C (CDC13): ~ 16.91, 16.53 (2xCH3 fucose), 22.15
(NHCOCH3), 39.14 (CH2N), 54.20 (C-2), 99.33, 100.03, 101.73,
102.95 (C-l, C-l', 2xC-1 fucose), 173.30 (NHCOCH3) there were
no 13C signals in the "aromatic region".
(v) 2-acrylamidoethyl 2-acetamido-2-deoxy-3-O-(2-O-(~-L-
fucopyranosyl-~-D-galactopyranosyl)-4-O-~-L-fucopyranosyl-~-D-
glucopyranoside (5S).
35 mg of the crude compound (54) was dissolved in aqueous
ammonia (25%, 4 ml) and stirred at room tPmrPrature. The
reaction was complete within 1 hour and yielded the free amino
derivative exclusively. TLC (ethylacetate:acetic acid:
methanol:water, 5:3:3:2), concentration and co-concentration
SUB~ 111 JTE SHEET
wos5l0o~27 PCTISE94/00604
~ 2l6~96I_75 ''~
with toluene was foilowed by purification on a Bond-Elut
cartridge (SCX, H+-form) cation exchange resin. The sample was
dissolved in 3 ml of water and pH was adjusted to 6 with
aqueous acetic acid. The sample was put on the column and then
eluted with 2M ammonia in methanol:water, 1:1 (5 ml). The
fraction~ containing free amine (ninhydrin positive) were
pooled, concentrated and lyophilized to give crude amine (20
mg). 1 ml Deaerated 0.5 M sodiumborate (aq) buffer (pH 8.5) and
deaerated methanol (3 ml) was added to the crude amine. The
reaction mixture was flushed with nitrogen and cooled to 0C. 6
~L acryloylchloride was added and stirring was continued for 10
min. The reaction mixture was co~c~ntrated at room temperature
to about a third of its original volume. Purification on a Bio-
Gel- P2 column and lyophilization gave pure title compound (55)
(15 mg).
NMR-data: 13C (D2O): ~ 16.90, 16.45 (2xCH3 fucose), 21.95
(NHCOCH3) 39.51 (CH2N), 54.31 (C-2) g9.21, 99.95, 101.56,
102.87 (C-l, C-l', 2xC-1 fucose), 127.21, 129.57 (CH=CH2)
173.27 (NHCOCH3).
(vi) Fuc~1-2Gal~1-3(Fuc~1-4)GlcNAc~l-O-spacer 5-PAA (56)
Copolymerization of 2-acrylamidoethyl 2-acetamido-2-deoxy-3-O-
2-O-~-L-fucopyranosyl-~-D-galactopyranosyl-4-~-L-fUcopyranosyl-
~-D-glucopyranoside (55) and acrylamide.
To acrylamide (8.3 mg, 120 ~mol) was added at room temperature
a solution of tetrasaccharide (54) tl5 mg, 20 ~mol) in
deaerated water (1 ml). To this slowly stirred solution (kept
in the dark and under nitrogen atmosphere was added at 0OC,
first N,N,N',N'-tetramethylethylenediamine (6 ~1), and then
ammonium persulphate (3.5 mg). The mixture was stirred at room
temperature over night. TLC (ethyl acetate:acetic acid:methanol
:water 5:3:2) showed that all of compound (49) was consumed and
that a charring baseline product had been formed. The polymer
was purified by gel chromatography on a Bio-Gel P-2 column
SUB~ JTE SHEEl-
Woss/oo~27 2 1 6 ~ 9 G 1 PCTISE94/00604
-76-
eluted with aqueous n-butanol (1%). Freeze-drying of the
polymeric fraction eluted in the void volume gave 20 mg of
polymer (s6).
S A lH-NMR analysis of the product showed an average
incorporation of 1 trisaccharide per 6 acrylamide units.
.,
~XAMP~ 3
Fuc~1-2 Gal~1-O-~F~ S-PAA (58)
(i) 2-acrylamidoethyl 2-O-~-L-fucopyranosyl-~-D-
galactopyranoside (57)
lS 0.3 ml deaerated 0.5 M sodiumborate (aq) buffer (pH 8.5) and
methanol (0.9 ml) was added to 6.4 mg of the compound (30). The
reaction mixture was flushed with nitrogen and cooled to 0C.
2 ~1 acryloyl chloride was added and stirring was continued for
10 minutes. The reaction mixture was concentrated at room
temperature to about a third of its original volume.
Purification on a Bio-Gel~ P2 column and lyophilization gave
the compound (57) (4 mg, 57%).
NMR-data: 1H (D2O): ~ 1.2 (d, CH3 fucose) 4.52 (dd, H-l), s.22
(m, H-l'), S.80 (dd, CH=CH2), 6.25 (m, CH=CH2).
(ii) Fuc~1-2 Gal~1-O-spacer 5-PAA (58)
To a solution of the compound (57) (4 mg, 9 ~mol) and
acrylamide (3.3 mg, 47 ~mol) in deaerated water (0.7S ml) was
added first N,N,N',N'-tetramethylenediamine (2 ~1) and then
ammonium persulphate (1.5 mg). The mixture was stirred at room
temperature over night. The polymer (58) was purified on a Bio-
Gel P2 column (9.1 mg).
NMR-data: lH (D20) showed an average incorporation of 1
oligosaccharide per 12.3 acrylamide units.
SUI~s 111 ~JTE SHEE~
WO 95/00527 21 6 ~ 9 61 PCT/SE94/00604
--77--
BIOLOGICAL EXPERIMENTS
Materials and Methods
- S In situ adherence assay for Helicobacter pylori
Non-infected samples from normal adult human gastric tissue
(o~tained from Huddinge Sjukhus, Sweden) were used to study
Helicobacter pylori adherence. All samples were fixed in 4%
formalin and subsequently e~h~ed in paraffin.
Sect~ons, 4 ~m thick, were placed on glass slides and used for
Steiner~s silver staining (to identify the cell types present
in gastric units, and to verify that the tissue samples have no
pathologic changes) and/or subsequent adherence assay.
Four clinical isolates, A4, A5, A7, and A8 (obtained from
H~ inge Sjukhus) of Helicobacter pylo~i were used.
Nelicobacter pylori was cultured at 37C on Brucella Agar
supplemented with 10% bovine blood and l~ IsoVitalex (Becton
Dickinson Microbiology System, Cockeyville, MD) under
microaerophilic conditions (5~ 2~ 10% C02, 85% N2) and 98%
humidity. Five days after inoculation, bacteria from one
full-grown plate were resuspended by gentle pipetting in 25 ml
of 0.lM NaCl/ 0.lM sodium carbonate, pH 9Ø 250 ~l of a
freshly prepared l0 mg/ml solution of fluorescein
~sothiocyanate (FITC, Sigma Chemical Co.) in dimethylsulfoxide
was added to the suspension of bacteria which was then
~ h~ted for l hour at room temperature in the dark. The
bacteria were recovered by centrifugation at 3000 x g for l0
minutes, and then resuspended in phosphate buffered saline
(PBS) + 0.05% polyoxyethylene sorbitan monolaurate (Tween 20)
by gentle pipetting and subsequently pelleted by centrifugation
as aboveO The wash procedure was repeated 3 times and the
; 35 suspension was finally resuspended to an Optical Density of
0.2. The intensity of FITC-labelling of all bacterial strains
was similar as ~udged by inspection of comparable numbers of
organisms by fluorescence microscopy. Aliquots of l ml were
SUB~ JT~ SHEET
W095/005Z7 pcTlsE9~l~aD~o1
2 ~ 78- ~
taken from the final suspensions and utilized immediately or
stored at -20OC until use. No difference in binding pattern was
observed between strains labelled and used immediately and
strains that were frozen and thawed once before use.
Slide-mounted tissue sections were deparaffinized in Bio-Clear
(Bio-Optica SpA) and absolute alcohol, 95% alcohol followed by
70S alcohol, rinsed in water followed by P8S and then incubated
for 45 minutes in blocking buffer (1% gelatin/0.05% Tween 20 in
PBS). FITC labelled bacterial suspension (OD about 0.200-0.2s0)
was mixed with equal amount of a concentrated solution of the
compound. The mixture was preincubated for 2 hours at room
temperature in the dark, 200 ~l of the mixture was placed on a
slide-mounted tissue section and incubated for 1 hour at room
temperature in a humidified chamber. The slides were
subsequently washed 6 times with PBS prior to inspection under
fluorescence microscope.
y~li8
The in situ adherence assay was used to ascertain binding of
Helicobacter pylori to human gastric tissue and to demonstrate
inhibition of Helicobacter pylori with terminal
L-fucose-cont~ining compounds, e.g. LNFl-HSA.
To analyze the ability of terminal L-fucose-containing
compounds to inhibit binding. FITC labelled bacterial
suspension tO.D. about 0.200-0.250) was mixed with equal amount
of a concentrated solution of the compound. The mixture was
preinc~lhAted for 2 hours at room temperature in the dark. 200~1
of the mixture was placed on a slide-mounted tissue section and
was incubated for l hour at room temperature. After inc~hAtion,
the treated tissue sections were washed 6 times with PBS before
analysis of the tissue sections.
Comparison tissue sections treated with test compound with
untreated tissue sections using fluorescence microscopy and
image analysis (Neotech Image Grabber 24/1.1 to transfer the
SUB~ l l l ~)TE SHEEr
W095/~527 PCT/SE94/00604
6~9 61 -79-
visual microscope ~mage to a computer screen and optilab
24/2.1.1 Grafted, to count the adhered bacteria).
The given values in the table are the average number of adhered
` 5 bacteria on three different areas per section comparing treated
(with compound) with untreated tissue sections.
SU~ JTE SHEET
WO 95/005~7 PCT/SE!)~ G~50 1
~6b~9~ -80- ~
T~ble
5COMPOUND CONC INHIB
(average value)
tFuc~1-2Gal~1-spacer 1]s-HSA 2 mM 34%
"
tFucal-2Gal~l-spacer 4]8-HSA 2 mM 35%
tFuc~1-2Gal~1-spacer 2]n~PAA 2 mM 45%
n=1 per 12.3 acrylamide moieties
tFUc~l-2Gal~l-spacer 5~-PAA 1 mM 53%
n=1 per 5 acrylamide moleties
tFucal-2Gal~l-3GlcNAc~l-spacer 2]n-PAA 2 mM 40%
nsl per 7.6 acrylamide moieties
[Fucal-2Gal~1-3GlcNAc~1-Gal~1-spacer 3]35-
-HSA (LNF1-HSA) 0.2 mM72%
tPurch~ from Iso Sep AB, Sweden)
tFucal-2Gal~l-3(Fuc~l-4)GlcNAc~l-Ga~
-spacer 3]32-HSA (LND1-HSA) 0.2 mM71%
(Purchased from Iso Sep AB, Sweden)
tFUcal-2Gal~1-3Fuc~1-4)GlcNac~l-Gal~1-
-spacer 3]n-PAA
n=1 per 18 acrylamide moieties 0.2 mM67~
n-1 per 5 acrylamide moieties 0.2 mM84%
n=1 per 6 acrylamide moieties 0.2 mM93%
tGalNAcal-3(Fucal-2)Gal~l-3(Fucal-4)- 0.2 mM80%
GlcNac~l-Gal~l-spacer 3]22-HSA (A-hepta-HSA)
(Purchased from Iso Sep AB, Sweden)
SUB~ JTE SHEEr
