Note: Descriptions are shown in the official language in which they were submitted.
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Compounds and Their Uses
Field of the Invention
The present invention relates to compounds and their uses, and in particular
to
compounds which have a mimetic or antagonistic property of an inositol
phosphoglycan or a free GPI precursor of an IPG, and the uses of these
compounds,
e.g. to treat a condition ameliorated by administration of an IPG second
messenger or
an IPG antagonist thereof.
Background of the Invention
Many of the actions of growth factors or hormones on cells are thought to be
mediated
by a family of inositol phosphoglycan (IPG) second messengers ~'3~. It is
thought that
the source of IPGs is a "free" form of glycosyl phosphatidylinositol (GPI)
situated in
cell membranes. IPGs are thought to be released by the action of
. phosphatidylinositol-specific phospholipases following binding of growth
factors to
receptors on the cell surface. There is evidence that IPGs mediate the action
of a large
~,,.; .
number of growth factors including insulin, nerve growth factor, hepatocyte
growth
factor, insulin-like growth factor I (IGF-I), fibroblast growth factor,
transforming
growth factor j3, the action of IL-2 on B-cells and T-cells, ACTH signalling
of
adrenocortical cells, IgE, FSH and hCG stimulation of granulosa cells,
thyrotropin
stimulation of thyroid cells, cell proliferation in the early developing ear
and rat
mammary gland.
Partially characterised inositolphosphoglycans (IPGs) have been postulated to
mediate
the action of a number of growth factors and hormones including insulin and
insulin-
like growth factor I (IGF-I) ~1~. Despite their isolation from several tissues
type, the
precise chemical structures of these IPGs are, however, still unknown and two
main
structural groups have been proposed on the basis of the chemical composition
X2.3]
which display different biological activity and tissue distribution ~4~; the
family of
glucosamine-myo-inositol containing IPGs (IPG-A) and the family of clZiro-
inositol-
galactosamine containing IPGs (IPG-P).
SUBSTITUTE SHEET (RULE 26)
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2
In an attempt to establish the minimal structural requirements for biological
activity, a
number of compounds containing some of the basic structural motifs that have
been
postulated for IPG mediators have been synthesised in the art ~5~. These
synthetic
compounds include O-(2-amino-2-deoxy-D-glucopyranosyl)-a(1~6)-chino-inositol 1-
phosphate and O-(2-amino-2-deoxy-D-glucopyranosyl)-a(1~6)-myo-inositol 1-
phosphate ~6~.
US Patent No: 6,004,938 (Hoechst) discloses a group of synthetic inositol
glycans
having insulin-like action. The compounds are based on 2-6 monsaccharide units
linked to an inositol moiety. The examples in the patent all employ myo-
inositol and
are composed of 5 or 6 units apart from two pseudo-trisaccharide compounds G
and
H. Compounds G and H are HO-PO(H)O-6Man-a(1~4)-GIuN-a(1~6)-(L)inositol-
1,2(cyclic) phosphate and HO-PO(H)O-6Man-a(1~4)-GIuN-a(1~6)-(L)inositol,
otherwise known as O-(6-hydrogenphosphonate-a-D-mannopyranosyl)-(1~4)-(2-
ammonio-2-deoxy-a-D-glucopyranosyl)-(1~6)-L-myo-inositol-1,2-cyclic phosphate
and O-(6-hydrogenphosphonate-a-D-mannopyranosyl)-(1~4)-(2-amino-2-deoxy-a-D-
glucopyranosyl)-L-myo-inositol. The properties of exemplified compounds are
investigated in lipogenesis and glucose transport assays employing rat fat
cells.
W096/14075 (University of Virginia) discloses a generic family of compounds D-
hexosamines linked to an inositol via a (31,4-linkage. The inositols can be
myo or
chiYO-inositol or pinitol, while the hexosamines are glucosamine or
galactosamine.
However, this application describes the synthesis of just two compounds 4-O-(2-
deoxy-2-amino-(3-D-galactopyranosyl)-D-pinitol and 4-O-(2-deoxy-2-amino-(3-D-
galactopyranosyl)-D-chino-inositol, or in IUPAC notation O-(2-amino-2-deoxy-(3-
D-
galactopyranosyl)-(1~4)-D-pinitol and O-(2-amino-2-deoxy-/3-D-
galactopyranosyl)-
(1 ~4)-D-chino-inositol.
W099/06421 (University of Virginia) describes synthetic insulin mimetic
substances
and includes a general formula I showing (31,4-linked disaccharides. However,
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despite this the compounds synthesised in this application are exactly the
same as
those disclosed in the applicant's earlier application, W096/14075.
A mufti-step synthesis of a IPG-P mimetic from glucose has been previously
reported
in Jaramillo et al ~6~, which discloses a compound called C4, 1-D-6-O-(2-amino-
2-
deoxy-a-D-glucopyranosyl)-chino-inositol 1-phosphate. A further synthesis of
C4 is
described in our co-pending International Patent Application PCT/GB99/03715
(Rademacher Group Limited). Zapata et al ~'6~ discloses three other compounds
C1-
C3 which are:
C 1 1-D-4-O-(2-amino-2-deoxy-a-D-glucopyranosyl)-myo-inositol 1-phosphate.
C2 1-D-6-O-(2-amino-2-deoxy-a-D-glucopyranosyl)-myo-inositol 1-phosphate.
C3 1-D-6-O-(2-amino-2-deoxy-a-D-glucopyranosyl)-myo-inositol 1,2 cyclic-
phosphate.
C4 1-D-6-O-(2-amino-2-deoxy-a-D-glucopyranosyl)-chino-inositoll-phosphate.
It remains a significant problem in the art to produce synthetic compounds
which can
mimic one or more of the activities of inositol phosphoglycans or which act as
antagonists of IPGs.
Summary of the Invention
Broadly, the present invention relates to IPG mimetic and antagonist compounds
and
to methods of producing the compounds and to theix medical uses. The compounds
disclosed herein are useful as synthetic mimetics of IPG-P or IPG-A second
messengers and/or growth factors whose action is mediated by IPGs, as
synthetic
mimetics of GPI precursors of IPGs, or as competitive antagonists of IPGs. In
particular, in preferred embodiments, the compounds are based on the 1,6
linkage of a
sugar residue and a cyclitol.
Accordingly, in a first aspect, the present invention provides a compound
represented
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by the general formula:
X-1,6-cyclitol
wherein:
X represents a sugar residue;
the sugar residue is unsubstituted or substituted with between one and four
groups, and the cyclitol is unsubstiiuted or is further substituted with
between one and
four groups, the group or groups being as defined herein;
with the proviso that the compound is not 1-D-4-O-(2-amino-2-deoxy-a-D-
glucopyranosyl)-myo-inositol 1-phosphate, 1-D-6-O-(2-amino-2-deoxy-a-D-
glucopyranosyl)-myo-inositol 1-phosphate, 1-D-6-O-(2-amino-2-deoxy-a-D-
glucopyranosyl)-myo-inositol 1,2 cyclic-phosphate, 1-D-6-O-(2-amino-2-deoxy-a-
D-
glucopyranosyl)-chino-inositol 1-phosphate, O-(6-hydrogenphosphonate-a-D-
mannopyranosyl)-(1~4)-(2-ammonio-2-deoxy-a-D-glucopyranosyl)-(1--~b)-L-myo-
inositol-1,2-cyclic phosphate or O-(6-hydrogenphosphonate-a-D-mannopyranosyl)-
(1 ~4)-(2-amino-2-deoxy-a-D-glucopyranosyl)-L-myo-inositol.
In a further aspect, the present invention provides the present invention
provides a
compound represented by the general formula:
X-a 1, 6-cyclitol
wherein:
X represents a sugar residue;
the sugar residue is unsubstituted or substituted with between one and four
groups, and the cyclitol is unsubstituted or is further substituted with
between one and
four groups, the group or groups being as defined herein;
with the proviso that the compound is not 1-D-4-O-(2-amino-2-deoxy-a-D-
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glucopyranosyl)-myo-inositol 1-phosphate, 1-D-6-O-(2-amino-2-deoxy-a-D-
glucopyranosyl)-myo-inositol 1-phosphate, 1-D-6-O-(2-amino-2-deoxy-a-D-
glucopyranosyl)-myo-inositol 1,2 cyclic-phosphate, 1-D-6-O-(2-amino-2-deoxy-a-
D-
glucopyranosyl)-chino-inositol 1-phosphate, O-(6-hydrogenphosphonate-a-D-
5 mannopyranosyl)-( 1 ~4)-(2-ammonio-2-deoxy-a-D-glucopyranosyl)-( 1 ~ 6)-L-
myo-
inositol-1,2-cyclic phosphate or O-(6-hydrogenphosphonate-a-D-mannopyranosyl)-
( 1 ~ 4)-(2-amino-2-deoxy-a-D-glucopyranosyl)-L-myo-inositol.
In a further aspect, the present invention provides the present invention
provides a
compound represented by the general formula:
X-~i1,6-cyclitol
wherein:
X represents a sugar residue;
the sugar residue is unsubstituted or substituted with between one and four
groups, and the cyclitol is unsubstituted or is further substituted with
between one and
four groups, the group or groups being as defined herein.
Preferably, the sugar residue is a hexose or a pentose, and may be an aldose
or a
ketose. The sugar residue can be a member of the D or L series and can include
amino
sugars, deoxy sugars and their uronic acid derivatives. Preferably, where the
sugar
residue is a hexose, it is selected from the group consisting of glucose,
galactose or
mannose, or substituted hexose sugar residues such as an amino sugar residue
such as
hexosamine, galactosamine or glucosamine, and more preferably D-glucosamine (2-
amino-2-deoxy-D-glucose) or D-galactosamine (2-amino-2-deoxy-D-galactose).
Preferred pentose sugar residues include arabinose, fucose and ribose. The
sugar
residue is optionally substituted at one, two, three or four positions, other
than the
position of linkage to the cyclitol moiety.
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The cyclitol moiety is preferably selected from myo-inositol, chino-inositol
or pinitol
(3-O-methyl-chino-inositol), in either their D or L forms, and is optionally
substituted
at one or more of the positions other than the position of linkage to the
sugar radical,
or in the case of pinitol additionally the 3-position. The sugar radical is
optionally
substituted at one, two, three or four positions other than at the position of
linkage to
the inositol moiety or the anomeric position. Where the cyclitol moiety is
substituted
at the 3-position (e.g. is a pinitol or a related compound), preferably the
substituent is
Ci-to alkyl, and may be a substituted or unsubstituted primary, secondary or
tertiary
alkyl group. Examples of substituted groups include CF3, X(CHZ)n O- (where X
is
hydrogen, or substituted or unsubstituted alkyl), CHF20-. A preferred alkyl
group is
methyl when the cyclitol is D or L-pinitol (3-O-methyl-chino-inositol), and is
optionally substituted at one or more of the positions other than the 3-
position or the
position of linkage to the sugar residue. In further embodiments, the cyclitol
may
have one or more of the hydroxyl groups through which the substituents
described
above are removed so that any substituent(s) are linked to the ring carbon
atom. The
sugar residue is optionally substituted at one, two, three, four or five
positions other
than at the position of linkage to the inositol moiety. The compounds of the
invention
may be either a or (3 linked.
To avoid confusion, the numbering system used herein is clarified with
reference to
the following structures. Importantly, as the chino-inositol molecule contains
a C2
plane of symmetry and positions 1 and 6 are equivalents. Therefore, for
instance,
compounds containing a (3(1,6) linkage can be regarded as (i(1,1) if there are
no other
substituents to define priority in the numbering system. Monosaccharide
residues and
oligosaccharides are named and numbered according to the recommendation
proposed
by the Joint Commission on Biochemical Nomenclature, International Union of
Pure
and Applied Chemistry and International Union of Biochemistry and Molecular
Biology.
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HO
3 HO 2 1
HO "' 6 chiro-Inositol
HO OH
OH
HO
3 HO 2 1
Me0 ~ 6 Pinitol:3-O-Methyl-chiro-inositol
HO OH
4 OH
g HO HO 4
HO ~ 10H myo-Inositol
OH
HO
., 2 3
In these and other aspects of the invention, preferably the substituent group
or groups
of the cyclitol moiety and the sugar residue are independently selected from:
(a) phosphoryl groups such as phosphate -O-P(O)(OH)z; thiophosphate -O-
P(S)(OH)Z; phosphate esters -O-P(O)(OR)2; thiophosphate esters -O-
P(S)(OR)z; phosphonate -O-P(O)OHR; thiophosphonate -O-P(S)OHR;
substituted phosphonate -O-P(O)OR1R2; substituted thiophosphonate -O-
P(S)OR1R2; -O-P(S)(OH)(SH); cyclic phosphate;
(b) other phosphorus containing compounds such as phosphoramidite -O-P(OR)-
NR1R2 and phosphoramidate -O-P(O)(OR)-NR,Rz;
(c) sulphur groups such as -O-S(O)(OH), -SH, -SR, -S(~O)-R, -S(O)ZR, RO-
S(O)i , -O-S02NH2, -O-SOZRIR2 or sulphamide -NHSOZNH2;
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(d) amino groups such as -NHR, -NR,RZ, -NHAc, -NHCOR, -NH-O-COR, -
NHS03-, -NHS02R, -N(SO2R)2, andlor amidino groups such as -NH-
C(=NH)NHZ and/or ureido groups such as -NH-CO-NR,R2 or thiouriedo
groups such as -NH-C(S)-NHZ;
(e) hydroxy groups and substituted hydroxy groups such as -OR3, where R3 is
C,_,o
unsubstituted or substituted alkyl, e.g. CHFZ or CF3, alkoxyalkyl,
aryloxyalkyl,
cycloalkyl, alkenyl (unsubstituted alkyl), alkylene (C3_~ cycloalkyl), -OCOR,
aryl, heteroaryl, acetal, or where two hydroxyl groups are joined as a ketal;
(f) halogen substituents such as fluorine or chlorine;
(g) hydrogen, e.g. to provide a deoxy sugar;
wherein R, R, and Rz are independently hydrogen or C,_,o unsubstituted or
substituted alkyl or aryl.
The compounds may be provided as racemic or diasteromeric mixtures, resolved
or
partially resolved optical isomers, and as pharmaceutically acceptable salts,
esters and
derivatives as discussed in more detail below.
In a further aspect, the present invention provides a compound represented by
the
general formula:
X-a1,6-myo-inositol
wherein:
X represents a sugar residue;
the sugar residue is unsubstituted or substituted with between one and four
groups, and the inositol is unsubstituted or is further substituted with
between one and
four groups, the group or groups being as defined herein;
with the proviso that the compound is not 1-D-4-O-(2-amino-2-deoxy-a-D-
glucopyranosyl)-myo-inositol 1-phosphate, 1-D-6-O-(2-amino-2-deoxy-a-D-
glucopyranosyl)-myo-inositol 1-phosphate, 1-D-6-O-(2-amino-2-deoxy-a-D-
glucopyranosyl)-myo-inositol 1,2 cyclic-phosphate.
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Examples of compounds within this aspect of the invention are RGL1023,
RGL1027,
RGL1029, RGL1105, RGL1115, RGL1116, RGL1125, RGL1126, RGL1134,
RGLl 133, RGL1135 and RGL1130.
In a further aspect, the present invention provides a compound represented by
the
general formula:
X-a1,6-chino-inositol
wherein:
X represents a sugar residue;
the sugar residue is unsubstituted or substituted with between one and four
groups, and the inositol is unsubstituted or is further substituted with
between one and
four groups, the group or groups being as defined herein;
with the proviso that the compound is not 1 D-6-O-(2-amino-2-deoxy-a-D-
glucopyranosyl)-D-chino-inositol 1-phosphate.
Examples of compounds within this aspect of the invention are RGL1017, RGLl
121
and derivatives thereof.
In a further aspect, the present invention provides a compound represented by
the
general formula:
X-a1,6-pinitol
wherein:
X represents a sugar residue;
the sugar residue is unsubstituted or substituted with between one and four
groups, and the pinitol is unsubstituted or is further substituted with
between one and
four groups, the group or groups being as defined herein.
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Examples of compounds within this aspect of the invention are RGL1024 and
RGL1025.
In a further aspect, the present invention provides a compound represented by
the
5 general formula:
X-~i 1,6-myo-inositol
wherein:
X represents a sugar residue;
10 the sugar residue is unsubstituted or substituted with between one and four
groups, and the inositol is unsubstituted or is further substituted with
between one and
four groups, the group or groups being as defined herein.
Examples of compounds within this aspect of the invention are RGL1002 and
RGL1124.
In a further aspect, the present invention provides a compound represented by
the
general formula:
2o X-X31,6-chino-inositol
wherein:
X represents a sugar residue;
the sugar residue is unsubstituted or substituted with between one and four
groups, and the inositol is unsubstituted or is further substituted with
between one and
four groups, the group or groups being as defined herein.
Examples of compounds within this aspect of the invention are RGL1018, RGL1019
and RGL1120 and derivatives thereof.
In a further aspect, the present invention provides a compound represented by
the
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general formula:
X-~i 1,6-pinitol
wherein:
X represents a sugar residue;
the sugar residue is unsubstituted or substituted with between one and four
groups, and the inositol is unsubstituted or is further substituted with
between one and
four groups, the group or groups being as defined herein.
Examples of compounds within this aspect of the invention are RGL1015, RGLl
119
and derivatives thereof.
In preferred embodiments, the present invention provides a compound, or a
substituted form thereof as defined above, selected from the group consisting
of:
RGL1023 O-(2'-amino-2'-deoxy-6'-phosphate-D-glucopyranosyl)-a(1,6)-D-myo-
inositol.
RGL1027 O-(2'-amino-2'-deoxy-4'-phosphate-D-glucopyranosyl)-a(1,6)-D-myo-
inositol.
RGL1029 O-(2'-amino-2'-deoxy-3'-phosphate-D-glucopyranosyl)-a(1,6)-D-myo-
inositol.
RGL1017 O-(2'-amino-2'-deoxy-D-glucopyranosyl)-a(1,6)-D-chino-inositol.
RGL1024 O-(2-amino-2-deoxy-D-glucopyranosyl)-a(1,6)-D-3-O-methyl-chiro-
inositol.
RGL1025 O-(2-amino-2deoxy-D-galactopyranosyl)-a(1,6)-D-3-O-methyl-chiro-
inositol.
RGL1018 O-(2'-amino-2'-deoxy-D-glucopyranosyl)-(3(1,6)-D-chino-inositol.
RGL1019 O-(2'-amino-2'-deoxy-D-glucopyranosyl)-a(1,6)-D-chino-inositol-1-
phosphate.
RGL1015 O-(2-amino-2-deoxy-D-glucopyranosyl)-(3(1,6)-3-O-methyl-chiro-
inositol.
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RGL1105 1 "-D-4'-O-(6"-phosphate-a-D-mannopyranosyl)-[1'-D-6-O-(2'-
amino-2'-deoxy-a-D-glucanopyranosyl)-myo-inositol] .
RGL1115 1'-D-6-D-(2'-amino-2'-deoxy-a-D-glucopyranosyl)-5-O-phosphate-
myo-inositol.
RGL1121 1'-D-1-O-(2'-amino-2'-deoxy-a-D-galactopyranosyl)-D-chiro-
inosito1.
RGL 1120 1'-D-6-O-(2'-amino-2'-deoxy-(3-D-glucopyranosyl)-D-chiro-
inositol.
RGL 1129 1'-D-2-O-(2'-amino-2'-deoxy-a-D-galactopyranosyl)-D-chino-
inosito1.
RGL 1122 1'-D-5-O-(2'-amino-2'-deoxy-a-D-glucopyranosyl)-D-chino-
inositol.
RGL1115 1'-D-6-O-(2'-amino-2'-deoxy-a-D-glucanopyranosyl)-5-O-phosphate-
myo-inositol.
RGL 1116 1'-D-6-O-(2'-amino-2'-deoxy-a-D-glucopyranosyl)-D-5-O-acetyl-myo-
inositol.
RGLl 117 1'-D-5-O-(2'-amino-2'-deoxy-a-D-glucopyranosyl)-D-6-O-acetyl-myo-
inositol.
RGL1119 1'-D-6-O-(2'-amino-2'-deoxy-(3-D-galactopyranosyl)-3-O-methyl-D-
chino-inositol.
RGL1124 1'-D-6-O-(2'-amino-2'-deoxy-(3-D-glucopyranosyl)-5-O-acetyl-myo-
inositol.
RGL1125 1'-D-6-O-(2'-amino-2'-deoxy-a-D-glucopyranosyl)-5-O-butyryl-myo-
inositol.
RGLl 126 1'-D-6-O-(2'-amino-2'-deoxy-a-D-glucopyranosyl)-5-O-palmityl-
myo-inositol.
RGL1134 1'-D-6-O-(2'-amino-3'-O-benzyl-4'-O-phosphate-2'-deoxy-a-D-
glucopyranosyl)-3,4,5-tri-O-benzyl-myo-inositol-1,2-cyclic phosphate.
RGL1133 1'-D-6-O-(2'-amino-3'-O-benzyl-4',6'-di-O-sulphate-2'-deoxy-a-D-
glucopyranosyl)-3,4,5-tri-O-benzyl-myo-inositol-1,2-di-O-sulphate.
RGL1135 1'-D-6-O-(2'-amino-3'-O-benzyl-4',6'-di-O-cyclic phosphate-2'-
deoxy-a-D-glucopyranosyl)-3,4,5-tri-O-benzyl-myo-inositol-1,2-cyclic
phosphate.
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RGL 113 0 1' -D-6-O-(2'-amino-4'-O-phosphate-2'-deoxy-a-D-glucopyranosyl)-
myo-inositol-1,2-cyclic phosphate.
1'-D-6-O-(2'-amino-6'-O-phosphate-2'-deoxy-a-D-glucopyranosyl)-
myo-inositol-1,2-cyclic phosphate.
In a further aspect, the present invention provides methods for making the
compounds
of the invention or their intermediates as set out in the following
experimental
description and the schemes. In a further related aspect, the present
invention further
relates to compounds which are the novel intermediates described herein.
In a further aspect, the present invention provides one or more of the above
compounds for use in a method of medical treatment. The compounds may be
useful
as IPG mimetics or IPG antagonists, e.g. as competitive antagonists.
In a further aspect, the present invention provides the use of one or more of
the above
compounds for the preparation of a medicament for the treatment of a condition
ameliorated by the administration of an inositol phosphoglycan (IPG) second
messenger or an IPG antagonist. Examples of such conditions are set out in the
pharmaceutical uses section below.
In a further aspect, the present invention provides a method of treating a
condition in a
mammal ameliorated by an inositol phosphoglycan (IPG) second messenger or an
IPG
antagonist, the method comprising administering to the mammal a
therapeutically
effective amount of one or more of the above compounds.
Embodiments of the invention will now be described by way of example and not
limitation with reference to the accompanying drawings.
Brief Description of the Figures
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Scheme 1 shows the coupling of diol 2 with trichloroacetimidate 1 resulting in
6-O-
glycosylation. Subsequent manipulation of protective groups afforded compound
4.
Scheme 2 shows the production of compounds RGL1023 and RGL1027 from
intermediate 4.
Scheme 3 shows the production of compound RGL1029 by coupling acceptor 2 with
trichloroacetimidate 11.
Scheme 4 describes the preparation of RGL1019.
Scheme 5 describes the preparation of the precursors for RGL1017 and RGL1018,
which are shown in Scheme 6.
' 'Scheme 7 shows the synthesis of building block 7 by bis-protection of the
trans-
diequatorially oriented hydroxyl groups of D-pinitol (5) as cyclohexane 1,2-
diacetal
and the cis-oriented hydroxyl groups as isopropylidene acetal.
Scheme 8 shows the glycosylation of D-pinitol building block 7 with the 2-
azido-2-
deoxy-D-glucopyranosyl trichloroacetimidate 8 to give the IPG-like compounds 1
and
2.
Scheme 9 shows the glycosylation of D-pinitol building block 7 with the 2-
azido-2-
deoxy-D-galactopyranosyl trichloroacetimidate 13 to give the IPG-like
compounds 3
and 4.
Scheme 10 shows the synthesis of RGLl 105.
Scheme 11 shows the synthesis of RGL1115 and RGL1116
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Scheme 12 shows the synthesis of RGL1117.
Scheme 13 shows the synthesis of RGLl 124.
5 Scheme 14 shows the synthesis of RGL1125.
Scheme 15 shows the synthesis of RGL1126.
Scheme 16 shows the synthesis of RGL1119.
Scheme 17 shows the synthesis of RGL1134.
Scheme 18 shows the synthesis of RGLl 135.
Scheme 19 shows the synthesis of RGL1133.
Scheme 20 show the synthesis of RGLl 130.
Figure 1 shows a graph of basal lipogenesis stimulation of exemplary compounds
of
the invention.
Figure 2 shows a graph of glucose stimulated lipogenesis stimulation of
exemplary
compounds of the invention.
Figure 3 shows a graph of the PKA inhibition of exemplary compounds of the
invention.
Figure 4 shows the results of a G6Pase inhibition assay testing the effect of
compound
RGLl 133 against a known inhibitor, sodium O-vanadate.
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Detailed Description
Inositol~hospho~lycans (IPGs)
IPG-A mediators modulate the activity of a number of insulin-dependent enzymes
such as CAMP dependent protein kinase (inhibits), adenylate cyclase (inhibits)
and
cAMP phospho-diesterases (stimulates). In contrast, IPG-P mediators modulate
the
activity of insulin-dependent enzymes such as pyruvate dehydrogenase
phosphatase
(stimulates) and glycogen synthase phosphatase (stimulates). The A-type
mediators
mimic the lipogenic activity of insulin on adipocytes, whereas the P-type
mediators
mimic the glycogenic activity of insulin on muscle. Both A-and P-type
mediators are
mitogenic when added to fibroblasts in serum free media. The ability of the
mediators
to stimulate fibroblast proliferation is enhanced if the cells are transfected
with the
EGF-receptor. A-type mediators can stimulate cell proliferation in the chick
cochleovestibular ganglia.
Soluble IPG fractions having A-type and P-type activity have been obtained
from a
variety of animal tissues including rat tissues (liver, kidney, muscle, brain,
adipose,
heart) and bovine liver. IPG-A and IPG-P biological activity has also been
detected in
human liver and placenta, malaria parasitized RBC and mycobacteria. The
ability of
an anti-inositolglycan antibody to inhibit insulin action on human placental
cytotrophoblasts and BC3H1 myocytes or bovine-derived IPG action on rat
diaphragm
and chick cochleovestibular ganglia suggests cross-species conservation of
many
structural features. However, it is important to note that although the prior
art
includes these reports of IPG-A and IPG-P activity in some biological
fractions, the
purification or characterisation of the agents responsible for the activity is
not
disclosed.
IPG-A substances are cyclitol-containing carbohydrates, also containing Zn2+
ions and
phosphate and having the properties of regulating lipogenic activity and
inhibiting
CAMP dependent protein kinase. They may also inhibit adenylate cyclase, be
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17
mitogenic when added to EGF-transfected fibroblasts in serum free medium, and
stimulate lipogenesis in adipocytes.
IPG-P substances are cyclitol-containing carbohydrates, also containing Mn2+
and/or
Zn2+ ions and phosphate and having the properties of regulating glycogen
metabolism
and activating pyruvate dehydrogenase phosphatase. They may also stimulate the
activity of glycogen synthase phosphatase, be mitogenic when added to
fibroblasts in
serum free medium, and stimulate pyruvate dehydrogenase phosphatase.
Methods for obtaining A-type and P-type mediators are set out in Caro et al,
1997, and
in W098/11116 and W098/11117. Protocols for determining characteristic IPG
biological activities such as PDH activation, PKA inhibition, acetylCoA
activation,
fructose-1,6-bis-phosphatase activity and lipogenesis are well known in the
art or
provided in the experimental section below.
Drug Formulation
The compounds of the invention may be derivatised in various ways. As used
herein
"derivatives" of the compounds includes salts, coordination complexes with
metal
ions such as Mnz+ and Zn2+, esters such as in vivo hydrolysable esters, free
acids or
bases, hydrates, prodrugs or lipids, coupling partners.
Salts of the compounds of the invention are preferably physiologically well
tolerated
and non toxic. Many examples of salts are known to those skilled in the art.
Compounds having acidic groups, such as phosphates or sulfates, can form salts
with
alkaline or alkaline earth metals such as Na, K, Mg and Ca, and with organic
amines
such as triethylamine and Tris (2-hydroxyethyl)amine. Salts can be formed
between
compounds with basic groups, e.g. amines, with inorganic acids such as
hydrochloric
acid, phosphoric acid or sulfuric acid, or organic acids such as acetic acid,
citric acid,
benzoic acid, fumaric acid, or tartaric acid. Compounds having both acidic and
basic
groups can form internal salts.
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18
Esters can be formed between hydroxyl or carboxylic acid groups present in the
compound and an appropriate carboxylic acid or alcohol reaction partner, using
techniques well known in the art.
Derivatives which as prodrugs of the compounds are convertible in vivo or in
vitro
into one of the parent compounds. Typically, at least one of the biological
activities
of compound will be reduced in the prodrug form of the compound, and can be
activated by conversion of the prodrug to release the compound or a metabolite
of it.
An example of prodrugs are glycolipid derivatives in which one or more lipid
moieties are provided as substituents on the sugar residue or the cyclitol
moieties,
leading to the release of the free form of the compound by cleavage with a
phospholipase enzyme. Examples of prodrugs include the use of protecting
groups
which may be removed in situ releasing active compound or serve to inhibit
clearance
of the drug in vivo. Protecting groups are well known in the art and are
discussed
1 S further below. An example of a suitable protecting group that might be
used as a
prodrug is the azido group used in the synthesis below, e.g. on the 2-position
of the
sugar moiety.
Other derivatives include coupling partners of the compounds in which the
compounds is linked to a coupling partner, e.g. by being chemically coupled to
the
compound or physically associated with it. Examples of coupling partners
include a
label or reporter molecule, a supporting substrate, a carrier or transport
molecule, an
effector, a drug, an antibody or an inhibitor. Coupling partners can be
covalently
linked to compounds of the invention via an appropriate functional group on
the
compound such as a hydroxyl group, a carboxyl group or an amino group. Other
derivatives include formulating the compounds with liposomes.
Pharmaceutical Compositions
The compounds described herein or their derivatives can be formulated in
pharmaceutical compositions, and administered to patients in a variety of
forms, in
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19
particular to treat conditions which are ameliorated by the administration of
inositol
phosphoglycan second messengers or IPG antagonists such as competitive
antagonist.
Pharmaceutical compositions for oral administration may be in tablet, capsule,
powder
or liquid form. A tablet may include a solid carrier such as gelatin or an
adjuvant or
an inert diluent. Liquid pharmaceutical compositions generally include a
liquid
carrier such as water, petroleum, animal or vegetable oils, mineral oil or
synthetic oil.
Physiological saline solution, or glycols such as ethylene glycol, propylene
glycol or
polyethylene glycol may be included. Such compositions and preparations
generally
contain at least 0.1 wt% of the compound.
Parental administration includes administration by the following routes:
intravenous,
cutaneous or subcutaneous, nasal, intramuscular, intraocular, transepithelial,
intraperitoneal and topical (including dermal, ocular, rectal, nasal,
inhalation and
aerosol), and rectal systemic routes. For intravenous, cutaneous or
subcutaneous
injection, or injection at the site of affliction, the active ingredient will
be in the form
of a parenterally acceptable aqueous solution which is pyrogen-free and has
suitable
pH, isotonicity and stability. Those of relevant skill in the art are well
able to prepare
suitable solutions using, for example, solutions of the compounds or a
derivative
thereof, e.g. in physiological saline, a dispersion prepared with glycerol,
liquid
polyethylene glycol or oils.
In addition to one or more of the compounds, optionally in combination with
other
active ingredient, the compositions can comprise one or more of a
pharmaceutically
acceptable excipient, carrier, buffer, stabiliser, isotonicizing agent,
preservative or
anti-oxidant or other materials well known to those skilled in the art. Such
materials
should be non-toxic and should not interfere with the efficacy of the active
ingredient.
The precise nature of the carrier or other material may depend on the route of
administration, e.g. orally or parentally.
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Liquid pharmaceutical compositions are typically formulated to have a pH
between
about 3.0 and 9.0, more preferably between about 4.5 and 8.5 and still more
preferably
between about 5.0 and 8Ø The pH of a composition can be maintained by the
use of
a buffer such as acetate, citrate, phosphate, succinate, Tris or histidine,
typically
5 employed in the range from about 1 mM to 50 mM. The pH of compositions can
otherwise be adjusted by using physiologically acceptable acids or bases.
Preservatives are generally included in pharmaceutical compositions to retard
microbial growth, extending the shelf life of the compositions and allowing
multiple
10 use packaging. Examples of preservatives include phenol, meta-cresol,
benzyl
alcohol, para-hydroxybenzoic acid and its esters, methyl paraben, propyl
paraben,
benzalconium chloride and benzethonium chloride. Preservatives are typically
employed in the range of about 0.1 to 1.0 % (wlv).
15 Preferably, the pharmaceutically compositions are given to an individual in
a
"prophylactically effective amount" or a "therapeutically effective amount"
(as the
case may be, although prophylaxis may be considered therapy), this being
sufficient to
show benefit to the individual. Typically, this will be to cause a
therapeutically useful
activity providing benefit to the individual. The actual amount of the
compounds
20 administered, and rate and time-course of administration, will depend on
the nature
and severity of the condition being treated. Prescription of treatment, e.g.
decisions
on dosage etc, is within the responsibility of general practitioners and other
medical
doctors, and typically takes account of the disorder to be treated, the
condition of the
individual patient, the site of delivery, the method of administration and
other factors
known to practitioners. Examples of the techniques and protocols mentioned
above
can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A.
(ed),
1980. By way of example, and the compositions are preferably administered to
patients in dosages of between about 0.01 and 100mg of active compound per kg
of
body weight, and more preferably between about 0.5 and l Omg/kg of body weight
.
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21
The composition may further comprise one or more other pharmaceutically active
agents, either further compounds of the invention, inositol phosphoglycans,
growth
factors such as insulin, NGF or other growth factors listed below, or other
drugs, e.g.
those in use for the treatment of diabetes or other conditions set out below.
Medical Uses
As set out above, IPGs are second messengers for a range of different growth
factors,
including insulin, nerve growth factor, hepatocyte growth factor, endothelial
growth
factor, insulin-like growth factor I (IGF-I), fibroblast growth factor,
transforming
growth factor J3, the action of IL-2 on B-cells and T-cells, ACTH signalling
of
adrenocortical cells, IgE, FSH and hCG stimulation of granulosa cells,
thyrotropin
stimulation of thyroid cells, cell proliferation in the early developing ear
and rat
mammary gland. Consequently, IPGs or their antagonists can be used in the
treatment
or amelioration of disorders mediated by the growth factors or to mimic
specific
growth factor biological activities.
Examples of conditions which can be treated using IPGs or IPG antagonists
include,
diabetes, obesity, dyslipidaemia, pre-eclampsia, neurotrophic disorders,
hepatic
damage and adrenal atrophy.
W098/10791 discloses that pre-eclampsia is characterised by elevated levels of
IPG-P
and that it can be treated using an IPG-P antagonist. Compounds of the
invention
which are IPG-P antagonists, e.g. antagonists which compete with wild-type IPG-
P
but lack one or more of its activities, could be used in the treatment of pre-
eclampsia.
The use of both IPG-P and IPG-A and IPG-A antagonists in the diagnosis and
treatment of diabetes is disclosed in W098/11435. This application discloses
that in
some forms of diabetes the ratio of P:A-type IPGs is imbalanced and can be
corrected
by administering a medicament containing an appropriate ratio of IPG-P, IPG-A
or
antagonists) thereof. In particular, it describes the treatment of obese type
II diabetes
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22
(NIDDM) patients with a P-type IPG and/or an A-type IPG antagonist and the
treatment of IDDM or lean type II diabetes (body mass index < 27) with a
mixture of
P- and A-type IPGs, typically in a P:A ratio of about 6:1 for males and 4:1
for
females. The compounds and compositions of the present invention can be
employed
in such types of treatment. More particularly, the compounds are likely to be
of use in
the treatment of various form of diabetes and diabetic complications including
diabetes due to insulin resistance, insulin resistance in type I diabetes and
brittle
diabetes, obese or lean type II diabetes, and of conditions associated with
insulin
resistance or insulin underproduction, such as neurotrophic disorders or
polycystic
ovary syndrome, lipodystrophy, age-related memory loss, and post-ischaemic
damage
secondary to stroke or post-transplant complications.
The compounds of this invention are also likely to be of use in controlling
neuron
proliferation or neurite outgrowth, either in vitro or in vivo, e.g. acting as
a nerve or
neurite growth factor mimetic second messenger. They may thus have
applications in
the treatment and/or diagnosis of any condition related to neuron
proliferation or
neurite differentiation. W099138516 discloses that IPG-A and synthetic
mimetics
thereof cause neuron proliferation, mimicking the action of the growth factor
IGF-I.
In contrast, IPG-P and synthetic mimetics thereof such as compound C4 cause
neurite
outgrowth. The neurons may be central (brain and spinal cord) neurons,
peripheral
(sympathetic, parasympathetic, sensory and enteric) neurons, e.g. the
compounds used
in the regeneration of peripheral nerves, or motor neurons. Treatments may
involve
the treatment of damage to nerve, spinal cord or central nervous system damage
secondary to trauma, or autoimmune or metabolic damage, or post-ischaemic
damage
secondary to stroke or post-transplant complications, motor neuron disease,
neurodegenerative disorders or neuropathy. Damage to the nervous system
includes
the results of trauma, stroke, surgery, infection (e.g. by viral agents),
ischernia,
metabolic disease, toxic agents, or a combination of these or similar causes.
Motor
neuron disease includes conditions involving spinal muscular atrophy,
paralysis or
amyotrophic lateral sclerosis. Neurodegenerative disorders include Parkinson's
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23
disease, Alzheimer's disease, epilepsy, multiple sclerosis, Huntingdon's
chorea and
Meniere's disease.
The compounds of the invention may also be useful as hepatocyte growth factor
mimetic second messengers, e.g. in the preparation of medicaments for the
treatment
of hepatic damage caused by infection, alcohol abuse, drug sensitivity, or
autoimmunity. The compounds may also be useful as fibroblast growth factor
mimetic second messengers or epidermal growth factor mimetic second
messengers,
e.g. in the preparation of medicaments for the promotion of wound healing
following
surgery or trauma or tissue damage induced by ischaemia or autoimmunity.
In other embodiments, the compounds of the invention may be useful as adrenal
cell
growth factor mimetic second messengers or ACTH mimetic second messengers in
the preparation of a medicament for the treatment of disease states involving
adrenal
atrophy.
The compounds of the invention can readily be tested using the assays
identified
herein to determine their suitability for some or all of the medical uses
described
above. Thus, even compounds with a relatively low activity in one of the
enzymes
assays disclosed herein, may be useful by virtue of possessing a different
activity, and
moreover the pattern of activities can be used to rapidly screen the compounds
for
suitability in the various medical applications disclosed herein.
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Activity Diabetes Diabetes ObesityAlzheimer'sNeurotrophics
I II
PDH Kinase Inhibit Inhibit No Inhibit No effect
Effect
PDH Activate Activate No No effect No effect
Phosphatase effect
Acetyl CoA Activate No effectNo No effect No effect
caxboxylase effect
I
* found in liver and adipose cytosol.
Methods of Making the Compounds
Based on the disclosure herein, the knowledge in the art and in references ~5-
"~, the
skilled person could couple sugar residues and cyclitols together, optionally
with one
or more substituents. Examples of further compounds of the invention made by
analogous syntheses include RGL1115, RGL1121, RGL1120, RGLl 129, RGL1122,
RGL1116 and RGL1117.
Useful guidance on the synthesis of the exemplified compounds and for
introducing
the substituents set out herein is provided by the papers by Gigg & Gigg,
Khiar &
Martin-Lomas ~5~ and Baeschlin et al ~'g~ and the references cited therein.
Phosphoryl groups such as phosphate, cyclic phosphate or substituted phosphate
or
cyclic phosphate can be substituted into the compounds of the invention by the
phosphate or phosphoramidite method, Bannwath et al, Flelvetica C'heinica
Acta,
70:175-186, 1987 and Yu & Fraser-Reid, Tetrahedron Lett., 29:979-982, 1988.
Phosphate protecting groups can also be synthesized according to the methods
disclosed in Hoeben-Weyl, Methods of Organic Chemistry, volume 12/1 or 12/2,
Teilheimer, Synthetic Methods of Organic Chemistry, Vol 45. Protecting groups
for
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the OH of sugars include menthoxycarbonyl (MntCO), acetal (in particular, two
R
groups may together represent a bridging acetal such as O-cyclohexylidene, O-
isopropylidene or O-benzylidene), tent-butyldimethylsilyl (TBDMS), benzyl
(Bn),
tent-butyldiphenylsilyl (TBDPS). Many protecting groups suitable for use in
the
5 syntheses and reactions of saccharides are known and are well documented in
standard
reference works. The choice depends in part on the route by which the compound
is
synthesised and/or on the uses to which it is to be put, including the
reactions which it
is subsequently intended to undergo.
10 Bioactivi Assay
The compounds of the invention can be tested for one or more the
characteristic IPG-
P andlor IPG-A activities mentioned above to determine whether they will be
suitable
for use a IPG mimetics or antagonists. Preferred assays measure the effect of
the
compounds on PDH phosphatase, PKA or lipogenesis. Protocols for these assays
are
15 provided in Caro et al ~'4~. The compounds can also be tested to determine
whether
they activate or inhibit other enzymes involved in insulin signalling
mechanism, such
as glucose-6-phosphatase.
Examples
20 General Methods.
All reactions were run under an atmosphere of dry argon using oven-dried
glassware
and freshly distilled and dried solvents. THF and diethyl ether were distilled
from
sodium benzophenone ketyl. Dichloromethane and acetonitrile were distilled
from
calcium hydride. TLC was performed on Silica gel GFZSa (Merck) with detection
by
25 charring with phosphomolibdic acidBtOH. For flash chromatography, Silica
Gel
(Merck 230-400 mesh) was used. Columns were eluted with positive air pressure.
Chromatographic eluents are given as volume to volume ratios (v/v). Routine
NMR
spectra were recorded with Bruker Avance DPX300 ('H, 300 MHz), Bruker Avance
DRX400 ('H, 400 MHz), and Bruker Avance DRX500 ('H, 500 MHz) spectrometers.
Chemical shifts are reported in ppm, and coupling constants are reported in
Hz.
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26
Spectra were referenced to the residual proton or carbon signals of the
solvent. High-
resolution mass spectra were recorded on a Kratos MS-80RFA 241-MC apparatus.
Optical rotations were determined with a Perkin-Elmer 341 polarimeter.
Elemental
analyses were performed using a Leco CHNS-932 apparatus. The organic extracts
were dried over anhydrous sodium sulfate and concentrated in vacuo.
Myo-inositol containing compounds
The synthesis of compounds RGL 1023, 1027 and 1029 involved the preparation of
a
glycosyl acceptor with position 6 free for reaction with the corresponding
glycosyl
donor. Diol 2 was chosen as the myo-inositol acceptor ~"~ as it can be
regioselectively
glycosylated at position 6. Regio- and stereoselective glycosylation of diol 2
is most
conveniently performed using the trichloroacetimidate procedure with 2-azido-2-
deoxy glycosyl donors bearing protective group patterns compatible with the
further
transforamtions required and designed as to provide an acceptable reactivity-
selectivity balance in the forthcoming glycosylation reaction. Thus, use of
trichloroacetimidate 1 was the glycosyl donor of choice. Coupling of 1 with 2
in
diethyl ether at -20°C using TMS triflate as promoter resulted in 6-O-
glycosylation
with good yield. Manipulation of protective groups afforded compound 4, a key
intermediate for compounds RGL 1023 and RGL 1027 (Scheme 1). Selective opening
of the benzylidene protecting group on positions 4' and 6' of the hexosamine
residue
afforded 5 and 6 in good yields. These compounds, precursors of phosphorylated
species RGL 1023 and RGL 1027, were subjected to treatment with
phosphorylating
reagent dibenzyloxi(diisopropylamino)phosphine, which afforded the
corresponding
phosphite derivatives. Oxidation to the phosphate form was achieved ih situ by
reaction with MPBCA in dichloromethane at room temperature for two hours with
good yield. Trifluoracetic acid treatment of compounds 7 and 8 in
di~hloromethane at
room temperature for 4 hours afforded diols 9 and 10 with moderate to good
yields.
Compounds RGL1023 and RGL1027 were obtained after hydrogenolytic
debenzylation and concomitant azide reduction of 9 and 10 in buffered medium
(Scheme 2).
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27
Synthesis of compound RGL1029 required that the glycosyl donor to be used
contained a protecting group in position 3 different from a benzyl group, to
be
introduced later on in the glycosyl acceptor residue. Thus,
trichloroacetimidate 11,
bearing ap-methoxy-benzyl group in position 3 was used. Coupling with acceptor
2 in
S the conditions described above afforded compound 12 in good yield.
Trifluoracetic
acid hydrolysis and subsequent treatment with benzyl chloride in the presence
of NaH
in DMF afforded the benzylated derivative 13, which afforded precursor 14 in
good
yield after oxidative cleavage of thep-methoxy-benzyl group with 2,3-dichloro-
5,6-
dicyano-1,4-benzoquinone in dichloromethane.
Treatment of compound 14 with dibenzyl(diisopropyl)phosphoramidite followed by
oxidation of the phosphite group afforded compound 15 (7S%). Deprotection of
the
ketal group on the inositol moiety was achieved by acid hydrolysis to yield
compound
16 (6S%), which was then subjected to catalytic hydrogenolysis to produce
RGL1029
1 S quantitatively.
1'-D-6-O-(2'-azido-3'-O-benzyl-4',6'-O-benzylidene-2'-deoxy-a-D-
glucopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4-O-
(1,1,3,3-tetraisopropyldisiloxanyl)-myo-inositol (3)
To a solution of trichloroacetimidate 2-azido-3-O-benzyl-4,6-O-benzylidene-2-
deoxy-
D-glucopyranoside 1 (1.698 g, 3.217 mmol) and 1-D-1,2-O-(L-1,7,7-
trimethyl[2.2.1~-
bicyclohept-2-ylidene)-3,4-O-(1,1,3,3-tetraisopropyldisiloxanyl)-myo-inositol2
(2.51
g, 4.507 mmol) in EtaO (SO mL) at -20°C, trimethylsilyl
trifluromethanesulfonate (29
ml, 0.160 mmol) was added. The mixture was warmed to room temperature over S
h,
2S quenched by addition of Et3N (0.S mL), filtered through celite and
concentrated. The
residue was purified by flash chromatography (Hexane/EtOAc 9S/S to. 7S/2S) to
give
three disaccharides : a(1~6) 3 as a white solid (SS% yield).
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28
1'-D-6-O-(2'-azido-3'-O-benzyl-4',6'-O-benzylidene-2'-deoxy-a-D-
glucopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4,5-
tri-
O-benzyl-myo-inositol (4)
To a solution of disaccharide a(1~6) 3 (1.60 g, 1.735 mmol) in THF (20 mL) at
0°C,
tetrabutyl ammonium fluoride was added (1 M solution in THF, 3.81 mL). After
15
min the solution was concentrated and the residue re-dissolved in DMF (15 mL).
The
solution was cooled to 0°C and 60 % sodium hydride in mineral oil (312
mg, 7.801
mmol) and benzyl bromide (0.93 mL, 7.801 mmol) was added. After 2 h methanol
was added. The mixture was diluted with CHzCl2 (100mL), washed with sat. NaCI
(2x100 mL), dried over MgS04 and concentrated. Flash chromatography of the
crude
(hexane/AcOEt 95/5) gave product 4 (1.410 g, 1.484 mmol, 85% over two steps).
1'-D-6-O-(2'-azido-3',6'-di-O-benzyl-2'-deoxy-a-D-glucopyranosyl)-1,2-O-(L-
1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4,5-tri-O-benzyl-myo-inositol
(5)
To a solution of disaccharide 4 (1.363 g, 1.431 mmol) in THF (40 mL) sodium
cyanoborohydride ( 1 M solution in THF, 21.5 mL) was added. After 15 min at
r.t.
hydrochloric acid (1M solution in ether) was added until evolution of hydrogen
ceased. The mixture was stirred at r.t. for 2 h, diluted with CHZClz (60 mL)
and
washed with sat. NaHC03 (2x100 mL). The aqueous layer was extracted with
CHZC12
(2x50 mL) and the combined organic layer was dried over MgS04 and
concentrated.
Flash chromatography of the crude (hexane/AcOEt 9/1) gave the compound 5 (1.0
g,
1.050 mmol, 73 %).
1'-D-6-O-(2'-azido-3',4'-di-O-benzyl-2'-deoxy-a-D-glucopyranosyl)-1,2-0-(L-
1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4,5-tri-O-benzyl-myo-inositol
(6)
To a solution of disaccharide 4 (460 mg, 0.484 mmol) and borane-dimethylamine
complex (115 mg, 1.952 mmol) in CHZCIz (40 mL) at 0°C, boron
trifluoride diethyl
etherate (254 mL, 1.963 mmol) was added dropwise. After 30 min, the stirring
was
continued at r.t. for 1 hour, and then the reaction quenched with sat. NaHC03
(15
mL). The crude material was diluted with CHZCIz (60 mL), washed with sat. NaCI
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29
(3x100 mL), dried over MgS04 and concentrated. Flash chromatography of the
crude
(hexanelAcOEt 6/1) gave compound 6 (276 mg, 0.290 mmol, 60 %).
1'-D-6-O-(2'-azido-3',6'-di-O-benzyl-2'-deoxy-4'-dibenzyl-phosphate-a-D-
glucopyranosyl)-1,2-D-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4,5-
tri-
O-benzyl-myo-inositol (7)
To a solution of disaccharide 5 (100 mg, 0.105 mmol) and 1-H-tetrazole (30 mg,
0.428 mmol) in anh. CHzCl2(10 mL) at 0°C, dibenzyl
diisipropylphosphoramidite
(141 mL, 0.420 mmol) was added dropwise. After the addition was completed, the
icebath was removed and the solution stirred for 2 h 30 min. The mixture was
cooled
to -40°C and a solution of 70% 3-chloroperbenzoic acid (65 mg, 0.264
mmol) in
CHzClz (4 mL) was added. The mixture was stirred for 2 h 30 min, diluted with
CHZCl2 (30 mL), washed with sat. NazS03 (2x50 mL), sat. NaHC03 (2x50 mL) and
sat. NaCI (2x50 mL), dried over MgSO~ and concentrated. Flash chromatography
of
the crude mixture (hexane/AcOEt 4/1) gave compound 7 (100 mg, 0.082 mmol,
78%).
1'-D-6-O-(2'-azido-3',4'-di-O-benzyl-2'-deoxy-6'-dibenzyl-phosphate-a-D-
glucopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4,5-
tri-
O-benzyl-myo-inositol (8)
To a solution of disaccharide 6 (150 mg, 0.157 mmol) and 1-H-tetrazole (44 mg,
0.628 mmol) in CHaCl2 (15 mL) at 0°C, dibenzyl
diisipropylphosphoramidite (212
mL, 0.630 mmol) was added dropwise. After the addition was completed, the
solution was stirred at r.t. for 3 h. The mixture was cooled to -40°C
and a solution of
70% 3-chloroperbenzoic acid (97 mg, 0.393 mmol) in anh. CHZC12 (5 mL) was
added.
The mixture was stirred for 45 min, diluted with CHaCl2 (30.mL), washed with
sat.
NaHS03 (2x50 mL), sat. NaHCO3 (2x50 mL) and sat. NaCI (2x50 mL), dried over
MgS04 and concentrated. Flash chromatography of the crude mixture
(hexane/AcOEt
4/1) gave compound 8 (152 mg, 0.125 mmol, 80%).
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1'-D-6-O-(2'-azido-3',6'-di-O-benzyl-2'-deoxy-4'-dibenzyl-phosphate-a-D-
glucopyranosyl)-3,4,5-tri-O-benzyl-myo-inositol (9)
To a solution of disaccharide 7 (100 mg, 0.082 mmol) in CHZCIa (10 mL) H20
(0.09
mL, 5 mmol), and trifluoroacetic acid (0.38 mL, 4.949 mmol) were added and the
reaction stirred for 4 h at r.t. The mixture was then diluted with CHZCIz (40
rnL),
washed with sat. NaHC03 (2x50 mL), sat. NaCI (3x50 mL), dried over MgS04 and
concentrated. Flash chromatography of the crude mixture(hexane/AcOEt 1/1 to
1/2
and finally AcOEt 100%) gave compound 9 (60 mg, 0.056 mmol, 68%).
10 1'-D-6-O-(2'-azido-3',4'-di-O-benzyl-2'-deoxy-6'-dibenzyl-phosphate-a-D-
glucopyranosyl)-3,4,5-tri-O-benzyl-myo-inositol (10)
To a solution of disaccharide 8 (110 mg, 0.091 mmol) in CHZCIz (10 mL) H20
(0.1
mL, 5.551 mmol), and trifluoroacetic acid (0.42 mL, 5.470 mmol) were added and
the
reaction stirred for 4 h at r.t. The mixture was then diluted with CHZC12 (40
mL),
15 washed with sat. NaHC03 (2x50 mL), sat. NaCI (3x50 mL), dried over MgS04
and
concentrated. Flash chromatography of the crude mixture (hexane/AcOEt 111 to
1/2
and finally AcOEt 100%) gave compound 10 (84 mg, 0.078 mmol, 86%) as a white
solid.
20 1'-D-6-O-(2'-amino-2'-deoxy-4'-phosphate-a-D-glucopyranosyl)-myo-inositol
(RGL1027)
To a suspension of disaccharide 9 (16 mg, 14.840 mmol) in EtOH (0.6 mL) 10%
Pd/C
(3.2 mg, 0.003 mmol) was added and the reaction stirred under hydrogen
atmosphere
at r.t, for 36 h. The solvent was evaporated, the crude suspended in H20
dest., filtered
25 through celite and the filtrate lyophilized to give RGL1027 (4.6 mg, 10.919
mmo~,
74%). 'H-NMR (D20, 500 MHz): 8 5.25 (broad s, 1H, HI,), 3.96 (m, 1H, H4,),
3.91
(m, 2H, H3,+H2), 3.84 (m, 1H, H5,), 3.82 (m, 1H, Hb,b), 3.61 (m, 3H,
Hb,a+H~+H6), 3.52
(t, J-- 8.85 Hz, 1H, H4), 3.41 (broad d, J 8.85 Hz, 1H, H3), 3.27 (d, J= 8.85
Hz, 1H,
HS), 3.18 (m, 1H, H2,). '3C_NMR (DZO, 500 MHz): d 97.40 (C1,), 80.94 (C6),
73.03
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(Cs), 72.91 (C4), 72.68 (C2), 72.25 (Cs,), 72.08 (C~), 71.99 (C4,), 71.32
(C3), 70.79
(C3,), 60.36 (C6,a+C6,b), 55.07 (C2,).
1'-D-6-O-(2'-amino-2'-deoxy-6'-phosphate-a-D-glucopyranosyl)-myo-inositol
(RGL1023)
To a suspension of disaccharide 10 (16 mg, 14.840 mmol) in a mixture
MeOH/HZO/AcOH 9/1/0.1 (0.5 mL) 10% PdIC (3.2 mg, 0.003 mmol) was added, and
the reaction stirred under hydrogen atmosphere at r.t. for 18 h. The crude was
filtered
through celite with an aqueous wash and the filtrate lyophilized to give
RGL1023 (5.5
mg, 13.055 mmol, 88%). 'H-NMR (D20, 500 MHz): 8 5.23 (broad s, 1H, H,,), 4.00
(m, 1H, Hs,), 3.99 (m, 1H, H6,a), 3.90 (broad s, 1H, Hz), 3.78 (m, 1H, H6,6),
3.73
(broad t, J-- 9.1 Hz, 1H, H3,), 3.61 (m, 2H, H,+H6), 3.60 (m, 1H, H4,), 3.53
(t, J= 9.1
Hz, 1 H, H4), 3.40 (dd, JI= 2.3 Hz, Jz= 9.1 Hz, 1 H, H3), 3.26 (broad t, J--
8.9 Hz, 1 H,
Hs), 3.14 (m, 1H, H2,). '3C-NMR (D20, 500 MHz): d 97.49 (C~,), 81.14 (C6),
73.69
(Cs), 72.90 (C4), 72.72 (C2), 72.69 (Cs,), 72.0 (C,), 71.27 (C3), 69.19 (C4,),
62.5
(C6,a+C6,b), 56.65 (C3,), 55.0 (C2,).
1'-D-6-O-(2'-azido-3'-O-(para)methoxybenzyl-4',6'-O-benzylidene-2'-deoxy-a-D-
glucopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4-O-
(1,1,3,3-tetraisopropyldisiloxanyl)-myo-inositol (12)
To a solution of trichloroacetimidate 2-azido-3-O-(para)methoxybenzyl-4,6-O-
benzylidene-2-deoxy-D-glucopyranoside 11 (166 mg, 0.298 mmol) and 1-D-1,2-O-(L-
1,7, 7-trimethyl [2.2.1 ]-bicyclohept-2-ylidene)-3,4-O-( 1,1,3,3-
tetraisopropyldisiloxanyl)-myo-inositol 2 (166 mg, 0.298 mmol) in Et~O.(3 mL)
at r.t.,
trimethylsilyl trifluoromethanesulfonate (1 mL, 5.534 mmol) was added. After
20
minutes, the reaction was quenched by addition of Et3N (0.1 mL), filtered
through
celite, concentrated and purified by flash chromatography (hexane/AcOEt 95/5)
to
give disaccharide 12 (171 mg, 0.180 mmol, 60%).
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1'-D-6-O-(2'-azido-3'-O paramethoxybenzyl-4',6'-O-benzylidene-2'-deoxy-a-D-
glucopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4,5-
tri-
O-benzyl-myo-inositol (13)
To a solution of disaccharide IZ (120 mg, O.I26 mmol) in THF (1.5 mL) at
0°C,
tetrabutylammonium fluoride (1 M solution in THF, 284 mL) was added. The
solution
was warmed up to r.t. over 1 hour and then concentrated. The residue was re-
dissolved in DMF (2 mL), cooled to 0°C and 60% sodium hydride in
mineral oil (23
mg, 0.575 mmol) and benzyl bromide (67 mL, 0.563 mmol) were added. After
stirring at r.t. overnight under argon atmosphere, the excess of base was
destroyed by
addition of methanol, the mixture concentrated to dryness, diluted with CHZCIz
(25
mL), washed with sat. NaCI (3x25 mL), dried over MgS04 and the solvent
evaporated
to dryness. Flash chromatography of the crude mixture (hexane/AcOEt 9:1)
compound I3 (98 mg, O.I00 mmol, 79%).
1'-D-6-O-(2'-azido-4',6'-O-benzylidene-2'-deoxy-a-D-glucopyranosyl)-1,2-O-(L-
1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4,5-tri-O-benzyl-myo-inositol
(14)
To a solution of disaccharide 13 (120 mg, 0.122 mmol) in a CH2C12/H20 9/1
mixture
(1.5 mL), 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (35 mg, 0.154 mol) was
added,
and stirred for 30 min at r.t. The reaction was diluted with CHZCla (25 mL),
filtered
over celite, washed with sat. NaHC03 (2x25 mL), sat. NaCI (25 mL), dried over
Na2SO4 and concentrated. Flash chromatography of the crude mixture
(hexane/AcOEt
Ell) gave dissaccharide 14 (87 mg, 0.101 mmol , 83%).
1'-D-6-O-(2'-azido-4',6'-O-benzylidene-3'-dibenzyl-phosphate-2'-deoxy-a-D-
glucopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4,5-
tri-
O-benzyl-myo-inositol (15)
To a solution of disaccharide 14 (70 mg, 0.081 mmol) and 1-H-tetrazole (23 mg,
0.328 mmol) in CHZCIa (7 mL) at 0 ° C, dibenzyl
diisipropylphosphoramidite (110 mL,
0.327 mmol) was added dropwise. After the addition, the solution stirred for 3
h at
r.t.. The mixture was then cooled to -40°C and a solution of 70% 3-
chloroperbenzoic
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33
acid (50 mg, 0.203 mmol) in CHZC12 (3 mL) was added. The mixture was stirred
for 2
h 30 min, diluted with CHZCIz (40 mL), washed with sat. NaHS03 (2x50 mL), sat.
NaHC03 (2x50 mL) and sat. NaCI (2x50 mL), dried over MgSO~ and concentrated.
Flash chromatography (hexane/AcOEt 4:1) gave compound 15 (68 mg, 0.061 mmol,
75%).
1'-D-6-O-(2'-azido-3'-dibenzyl-phosphate-2'-deoxy-a-D-glucopyranosyl)-3,4,5-
tri-O-benzyl-myo-inositol (16)
To a solution of disaccharide 15 (30 mg, 0.027 mmol) in CHZCIz (3 mL), H20
(0.06
mL, 3.330 mmol) and trifluoroacetic acid (249 mL, 3.243 mmol) were added, and
the
reaction stirred for 18 h at r.t. The mixture was then diluted with CHZC12 (25
mL),
washed with sat. NaHC03 (2x25 mL), sat. NaCI (3x25 mL), dried over MgS04 and
concentrated. Flash chromatography (hexane/AcOEt 1/3 to AcOEt 100% and finally
AcOEt/MeOH 9/1) gave compound 16 (16 mg, 0.018 mmol, 67%).
1'-D-6-O-(2'-amino-2'-deoxy-3'-phosphate-a-D-glucopyranosyl)-myo-inositol
(RGL1029)
To a suspension of disaccharide 16 (8 mg, 8.910 mmol) in a mixture MeOH/Hz0
4/1
(0.3 mL), 10% Pd/C (2.1 mg, 0.002 mmol) was added and stirred under hydrogen
atmosphere at r.t. for 18 h. The MeOH was evaporated, the crude suspended in
dest.
H20, filtered through celite and the filtrate lyophilized to give RGL1029 (3.6
mg,
8.545 mmol, 96%) as a white solid. 'H-NMR (D20, 500 MHz): 8 5.30 (broad s, 1H,
HI,), 4.22 (broad s, 1H, H3,), 3.97 (m, 1H, H5,), 3.90 (broad s, 1H, HZ), 3.70
(m, 2H,
Hb,a+H6,b), 3.66 (broad d, 1H, HI), 3.60 (broad t, J 9.1 Hz, 1H, H6), 3.54 (m,
1H, H4,),
3.51 (t, J 9.1 Hz, H, H4), 3.41 (dd, Jl= 9.1 Hz, JZ 4.1 Hz, 1H, H3), 3.31 (t,
J-- 9.1
Hz, 1H, HS), 3.25 (m, 1H, Hz,). '3C-NMR (D20, 500 MHz): d 97.20 (C,>), 80.88
(C6),
73.42 (C3,),72.88 (CS), 72.82 (C4), 72.76 (CZ), 72.0 (C5,), 71.91 (C,), 71.25
(C3), 69.52
(C4,), 60.25 (C6,a+C6,6), 54.59 (C2,).
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34
ClZiro-inositol containing com ounds
Compound 3 was obtained when the glycosylation of 1 was carried out using a
glycosyl donor such as 2 (Scheme 1). Deallylation of 3 using hydrogen
activated
[Ir(COD) (Ph2MeP)z] PF6 catalytic isomerisation and subsequent NBS-H20
promoted
cleavage yielded 5 that was phosphorylated using the phosphoramidite procedure
to
give compound 6 in 87% yield. Hydrogenation of 6 in the presence of 10%
palladium
on charcoal gave 7 (RGL 1019) in quantitative yield.
The synthesis of D-chino-inositol containing IPG like compounds bearing more
complex oligosaccharide structures was envisaged using the
trichloroacetimidate
derivative 8 (Scheme 2) as glycosyl donor. Using this glycosyl donor, building
blocks
were also prepared to be used for the synthesis of pseudodisaccharides RGL
1017 and
RGL1018 (Scheme 3).
Glycosylation of 1 with 8 afforded an a/(3 mixture of pseudodisaccharides 9
and 10 in
70% overall yield (Scheme 2). These compounds were separated and treated as
indicated in Scheme 3. Selective reductive opening of the benzylidene acetals
in 9 and
10 with NaBH3CN-HCl afforded the partially protected derivatives 11 and 12
respectively in good yield. Deallylation of II gave I3 that after catalytic
hydrogenation gave 14 in quantitative yield. A similar route from 12 yielded
16
(RGL1018) through 15.
6-O-Allyl-1-O-(3,4,6-tri-O-benzyl-2-azido-2-deoxy-(3-D-glucopyranosyl)-2,3,4,5-
tetra-O-benzyl-D-clairo--inositol (3) and 6-O-allyl-1-O-(3,4,6-tri-O-benzyl-2-
azido-
2-deoxy-a-D-glucopyranosyl), 2,3,4,5-tetra-O-benzyl-D-chino--inositol (4)
A mixture of compound 1 (73 mg, 0.125 mmol) and compound 2 (49 mg, 0.078
mmol) was coevaporated two times with toluene and then re-disolved in CHZC12
and
treated with TMSOTf (0.1 M solution in CHZC12, 50 p.L) at -25°C. After
30 min the
reaction mixture was allowed to warm during 10 min., then quenched by addition
of
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Et3N and evaporated to dryness. The residue was purified by column
chromatography
[Hexane AcOEt (7:3-X3.1)] to give 4 (35 mg, 35%) and 3 (25 mg, 25%) as syrups.
Data for 3: 'H NMR (CDCl3, 500 MHz): 8 3.29-3.38 (m, 3H, H-2', H-3', H-5'),
3.55
(t, 1H, J9.2 Hz, H-4'), 3.63 (d, 2H, H-6'a, H-6'b), 3.79-3.86 (m, 3H), 3.88-
3.95 (m,
5 3 H, A 11 ), 4.00 (t, 1 H, J 3 .9, 3 .1 Hz), 4.09-4.14 (m, 1 H, A 11 ), 4.45-
4.5 5 (m, 4H, H-1',
CH2Ph), 4.60-4.68 (m, 3H, CHzPh), 4.76-4.92 (m, 8H, CHzPh), 5.05-5.16 (m, 2H,
A11), 5.73-5.81 (m, 1H, A11), 7.14-7.38 (m, 35H, arH). HRFABMS: Calcd. for C6a
H6., N3 O,o Na (M+Na+) 1060.472416; Found 1060.476982. Anal. Calcd. for C64
H6~
N3 Olo: C, 74.04; H, 6.50; N, 4.05. Found: C, 73.54; H, 6.88; N, 4.10.
1-O-(2-Azido-2-deoxy-3,4,6-tri-O-benzyl-[3-D-glucopyranosyl)-2,3,4,5, tetra-O-
benzyl-D-chiro--inositol (5)
A solution of the indium catalyst in anhydrous THF (5.9 x 10-3 M solution, 173
pL)
previously treated under a hydrogen atmosphere for 30 minutes was added over a
solution of 3 (35 mg, 0.034 mmol) in anhydrous THF (0.4 mL). The mixture was
stirred at roon temperature for 1h under Argon and then NBS (9 mg, 0.051 mmol)
and
distilled water (131 p,L) were added and the mixture stirred again for 2h,
treated with
a saturated solution of sodium hydrogen carbonate (0.3 mL). The reaction
mixture
was then extracted with AcOEt (2 x 10 mL), washed with saturated sodium
chloride
solution (2 x 20 mL), dried over Mg S04 and evaporated to dryness. The residue
was
purified by column chromatography (Hex6- AcOEt 1-~ Hex 4- AcOEt 1) to give
pure
5 (30 mg, 88%) as a colourless oil. 'H NMR (CDCl3, 500 MHz): 8 7.4-7.1 (m,
35H,
ArH), 4.91-4.76 (m, 8H, AB System), 4.71-4.47 (m, 6H, AB System), 4.53 (d, J=
7.0
Hz, 1H, Hl.), 4.29 (broad t, J= 3.5 Hz, 1H, H,), 4.22 (broad t, J= 3.4 Hz, 1H,
H6), 4.0
(t, J-- 9.5 Hz, 1 H, H4), 3 .92 (dd, J, = 2.8 Hz, Jz 9.9 Hz, 1 H, HS), 3 . 8 5
(dd, JI = 3 .1 Hz,
J2 9.4 Hz, 1H, HZ), 3.76 (t, J= 9.4 Hz, 1H, H3), 3.66 (m, 2H, H6.a + H6.b),
3.60 (m,
1 H, H4.), 3 .3 5 (m, H2. + H3.), 3 .31 (dt, J~ = 3 .1 Hz, Jz 9. 8 Hz, 1 H,
H5.), 2.44 (s, 1 H,
OH). '3C NMR (CDCl3, 125 MHz): 8 139.04, 138.99, 138.92, 138.09, 137.95,
128.48,
128.47, 128.45, 128.44, 128.35, 128.30, 128.28, 128.17, 128.04, 127.95,
127.92,
127.88, 127.84, 127.76, 127.74, 127.44, 127.30, 102.95 (C,.), 82.95, 81.70,
81.30,
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36
80.26, 79.56, 77.52, 76.29, 75.83, 75.76, 75.54, 75.04, 74.90, 73.53, 73.35,
73.07,
68.96, 68.56, 66.81.
6-O-(2-Azido-2-deoxy-3,4,6-tri-O-benzyl-j3-D-glucopyranosyl)-2,3,4,5-tetra-O-
benzyl-1-O-(dibenzyloxyphosphoryl)-D-clziro--inositol (6)
A solution of 5 (16 mg, 0.016 mmol) in a 1:1 mixture of dichloromethane-
acetonitrile
(0.4 mL) was treated with N, N diisopropyl phosphoramidite (12 p.L, 0.036
mmol) and
tetrazole (5. I mg, 0.072 mmol). The mixture was stirred at room temperature
for 1.5
hr. and then treated with tent-butyl hydroperoxide (30 p.L) and the stirring
continued
for 1h. The solution was then evaporated to dryness and the residue was
purified on
column chromatography (Hex 7- AcOEt 2) to give 6 as colourless syrup (17.5 mg,
87%). 'H NMR (CDC13, 500 MHz): ~ 7.37-7.10 (m, 45H, ArH), 5.19 (ddd, J,= 2.9
Hz, JZ 4.1 Hz, J3 7.6 Hz, 1H, HI), 4.73-4.93 (m, 12H, AB System), 4.66 (d, J--
12.0
Hz, 1 H, AB System), 4.54 (m, 4H, AB System), 4.42 (d, 1 H, AB System), 4.39
(d, J--
7.9 Hz, 1H, Hl,), 4.17 (broad t, J= 3.7 Hz, 1H, H6), 3.92 (m, 2H, HZ + H4),
3.79 (dd,
Jl = 3 .0 Hz, JZ 9.8 Hz, 1 H, HS), 3 .73 (t, J= 9.6 Hz, 1 H, H3), 3 .71 (m, 1
H, H6,b), 3 .67
(m, IH, H3,), 3.64 (dd, JI= 1.8 Hz, JZ I 1.0 Hz, IH, Hb,a), 3.36-3.29 (m, 3H,
Hz, + H4,
+ H5,). '3C NMR (CDCl3, 125 MHz): b 138.90, 138.64, 138.09, 138.04, 137.98,
137.95, 135.93, 128.59, 128.50, 128.47, 128.43, 128.40, 128.39, 128.37,
128.34,
128.33, 128.29, 128.27, 128.25, 128.20, 128.14, 128.01, 127.99, 127.92,
127.90,
127.84, 127.75, 127.69, 127.62, 127.57, 127.49, 127.46, 127.42, 127.36, 103.32
(C,,),
82.81 (C3,), 81.39, 80.92, 78.73, 78.21 (C2, J~P 42 Hz), 76.03 (C6, J~p 3.4
Hz), 75.76,
75.53, 75.07, 75.05, 74.10, 74.06 (C,, J~P 5.9 Hz), 73.70, 72.99, 72.67, 69.50
(POCHZ Ar, J~P 5.9 Hz), 69.1 I (POCHZ Ar, J~p 5.9 Hz), 68.61 (C6,), 66.76
(Ca,).
3'P NMR (CDCl3, 202 MHz): 8 -2.31.
6-O-(2-Ammonio-2-deoxy-(3-D-glucopyranosyl)-D-cltiro--inositol-1-phosphate (7)
To a mixture of 6 (17.5 mg, 13.9 pm) in methanol (1.6 mL) and AcOH/AcONa
buffer
(I.6 mL) was added 10% Pd/C (21.5 mg). The mixture was stirred under hydrogen
for
24h and then filtered over Celite, washed with a 1:1 mixture of EtOH/H20 and
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liophylized. The residue was purified on Sephadex G-10 (10% aqueous EtOH) to
afford pure 7 (RGL 1019) (5 mg, 100%). 'H NMR (D20, 500 MHz): 8 4.66 (d, J=
8.2
Hz, 1 H, H, ~), 4.5 8 (ddd, J, = 3.0 Hz, JZ 4.2 Hz, J3 8.5 Hz, 1 H, H, ), 4.19
(t, J= 3 . 8
Hz, 1 H, H6), 3 .93 (dd, J, = 9.3 Hz, JZ 3.3 Hz, 1 H, HS), 3.9 (dd, J, = 12.7
Hz, JZ 2.2
Hz, 1 H, H6-b), 3.71 (dd, J, = 12.7 Hz, Jz 5.7 Hz, 1 H, H6.a), 3.64 (m, 2H, HZ
+ H3), 3 .54
(t, J= 9.4 Hz, 1 H, H4), 3.47 (ddd, J, = 2.2 Hz, Jz 5.6 Hz, J3 9.6 Hz, 1 H,
H5,), 3 .45 (t,
J= 9.4 Hz, 1 H, H3,), 3 .3 6 (t, J= 9.6 Hz, 1 H, Hø,), 2.77 (dd, J, = 9.4 Hz,
JZ 8.2 Hz, 1 H,
H2,). 3'P NMR (D20, 202 MHz): ~ 3.02.
1-O-(2-Azido-2-deoxy-3-O-benzyl-4,6-O-benzylidene-a- and-(3-D-
glucopyranosyl)-6-O-allyl-2,3,4,5-tetra-O-benzyl-D-clairo--inositol (9 and 10)
A mixture of 8 (520 mg, 0.985 mmol) and 1 (382 mg, 0.657 mmol) was dissolved
in
anhydrous CHZC12 (6.6 mL) and treated with a solution (2.50 ~L) of
trimethylsilyl
triflate (80 ~L) in CHZC12 (2 mL). The mixture was stirred at room temperature
for 1.5
h and then 100 ~L of the above solution of TMSOTf was added. After an
additional
hour with stirring (174 mg, 0.328 mmol) in CHZC12 (1.5 mL) was added and
stirring
was continued for 2h. The mixture was the treated with Et3N, evaporated to
dryness
and the residue fractionated on column chromatography (Hexane 8: AcoEt 1) to
yield
9 (130.5 mg, 49%) and 10 (302 mg, 21%). Data for 9: 'H NMR (CDC13, 500 MHz): 8
7.47-7.21 (m, 30H, ArH), 5.79 (ddt, Jl = 5.6 Hz, Jz 10.5 Hz, J3 17.1 Hz, 1 H,
OCHZCH--CHZ), 5.51 (s, 1H, CH benzyliden), 5.17 (dd, Jl= 1.5 Hz, Jz 17.2 Hz,
1H,
OCHZCH=CHH), 5.13 (dd, J,= 1.5 Hz, JZ=10.4 Hz, 1H, OCHZCH=CHIC, 4.97-4.76
(m, I OH, AB System), 4.70 (d, J-- 3.8 Hz, I H, H~,), 4.25-4.17 (m, 2H, H5, +
OCHHCH=CHZ), 3.99 (t, JI= 9.4 Hz, 1H, H3,), 3.97 (m, 1H, O-CH-H CH= CHZ), 3.95
(m, 1H, H6,eq), 3.97-3.74 (m, 6H, ChiroIns), 3.64 (t, J~= 9.3 Hz, 1H, H4,),
3.56 (t, J=
10.3 Hz, 1 H, H6,~, 3.49 (dd, J, = 3.7 Hz, Jz 9.8 Hz, 1 H, HZ,). Data for 10:
'H NMR
(CDCl3, 500 MHz): b 7.49-7.20 (m, 30H, ArH), 5.74 (ddt, J,= 5.6 Hz, J2 10.4
Hz,
J3 17.4 Hz, 1H, OCHZCH-- CHZ), 5.54 (s, 1H, CH benzyliden), 5.15 (broad dd,
Jl=
17.4 Hz, JZ 1.5 Hz, 1H, OCHZCH=CHH), 5.11 (broad dd, J~= 10.4 Hz, JZ 1.5 Hz,
1H, OCHzCH= CHI, 4.49-4.78 (m, 7H, AB System), 4.72 (d, J= 11.7 Hz, 1H, AB
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38
System), 4.62 (d, J= 11.7 Hz, 1 H, AB System), 4.60 (d, ,J--11.7 Hz, 1 H, AB
System),
4.54 (d, J= 8.1 Hz, 1H, HI,), 4.21 (dd, J,= 5.1 Hz, JZ 10.4 Hz, 1H, H6,eq),
4.13 (broad
dd, J, = 5.6 Hz, Jz 13.0 Hz, 1 H, OCHHCH=CHZ), 3.92-3.74 (m, 6H, ChiroIns), 3
.7
(t, J= 10.3 Hz, 1 H, Hb,aX), 3.61 (t, J= 9.3 Hz, 1 H, H4,), 3.46 (t, J= 9.4
Hz, 1 H, H3.),
3.28 (t, J= 8.3 Hz, 1 H, H~.), 3.23 (dt, J, = 5.1 Hz, Ja 9.8 Hz, 1 H, H5,).
1-O-(2-Azido-2-deoxy-3,6-di-O-benzyl-a-D-glucopyranosyl)-6-O-allyl-2,3,4,5-
tetra-O-benzyl-D-chiro--inositol (11)
To a solution of 9 (716 mg, 0.757 mmol) in THF (19 mL) 4~ molecular sieves
were
added and the mixture stirred for 30 min. Then a 1M solution of sodium
cyanoborohydride in THF (15 mL, 15.14 mmol) and a 1M solution of HCl in ether
was added until the evolution of gas ceased. The mixture was then treated with
saturated aqueous solution of NaHC03 and the organic layer washed with
saturated
NaCI, dried over NazS04 and evaporated. The residue was purified by column
chromatography (Hexane 4: AcoEt 1) to give 11 (575 mg, 80%). 'H NMR (CDCl3,
500 MHz): 8 7.44-7.23 (m, 30H, Ar-H), 5.82 (ddt, J,= 5.6 Hz, JZ 10.4 Hz, J3
17.2
Hz, 1H, OCHzCH--CHZ), 5.21 (broad dd, J,= 1.6 Hz, JZ 17.2 Hz, 1H,
OCHZCH=CHf~, 5.16 (broad dd, Jl = 1.6 Hz, JZ 10.4 Hz, 1 H, OCHZCH=CHH),
4.96-4.65 (m, 10H, AB System), 4.74 (d, J= 3.6 Hz, 1H, H~,), 4.44 (d, J= 12.0
Hz,
1 H, AB System), 4.32 (d, J= 12.1 Hz, 1 H, AB System), 4.22 (broad, dd, J~ =
5.4 Hz,
Ja 13.0 Hz, 1 H, OCHHCH=CH2), 4.12 (m, 1 H, H5,), 4.0 (m, 1 H, OCHHCH=CHZ),
4.04-3.78 (m, 6H, ChiroIns), 3.76 (m, 2H, H3. + +H4,), 3.45 (dd, J, = 3.6 Hz,
JZ 10.0
Hz, 1 H, HZ,), 3.3 8 (dd, J, = 3.5 Hz, JZ 10.4 Hz, 1 H, H6,a), 3.27(dd, J, =
4.2 Hz, Jz
10.4 Hz, 1 H, H6,~, 2.39 (d, J-- 1.6Hz, 1 H, OH4.).
1-O-(2-Azido-2-deoxy-3,6-di-O-benzyl-[3-D-glucopyranosyl)-6-O-allyl-2,3,4,5-
tetra-O-benzyl-D-clziro--inositol (12)
A solution of 10 (290 mg, 0.307 mmol) in anhydrous THF (7.7 mL) was stirred
with
4~ molecular sieves for 30 min. Then a 1M solution of sodium cyanoborohydride
in
THF (5.6 mL) and 1M solution of HCl in ether were added and the mixture
stirred
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39
until evolution of gas ceased. The mixture was then treated with saturated
aqueous
NaHC03 and the organic layer washed with saturated aqueous NaCI, dried over
Na2S0~ and evaporated. The residue was purified by column chromatography
(Hexane
5: AcoEt 1) to give pure 12 (39.2 rng, 95%). 'H NMR (CDC13, 500 MHz): 8 7.41-
7.21
(m, 30H, Ar-H), 5.76 (ddt, J,=5.6 Hz, Ji 10.4 Hz, J3=17.2 Hz, 1H, OCHZCH--
CHZ),
5.12 (dd, J, = 1.6 Hz, JZ 17.2 Hz, 1 H, OCHzCH=CHIC, 5.04 (broad dd, J, = 1.6
Hz,
JZ=10.3 Hz, 1 H, OCHZCH=CHH), 4.96-4.49 (m, 12H, AB System), 4.48 (d, J= 8.0
Hz, 1 H, H,,), 4.11 (broad, dd, J, = 5.4 Hz, JZ 13.0 Hz, 1 H, OCHHCH=CHZ),
3.99-
3.78 (m, 6H, ChiroIns), 3.89 (m, 1H, OCHHCH=CHZ), 3.67 (dd, J,= 4.0 Hz, Jz
10.3
Hz, 1 H, H6,6), 3 .62 (dd, J, = 5.2 Hz, JZ 10.3 Hz, 1 H, H6,~, 3 . 51 (dt, J,
= 2.3 Hz, JZ
9.6 Hz, 1 H, H4,), 3.29 (m, 1 H, H5.), 3 .25 (t, J=8.0 Hz, H2,), 3.16 (t, J, =
8.8 Hz, 1 H,
H3,), 2.50 (d, J, = 2.3 Hz, 1 H, OH4.).
1-O-(2-Azido-2-deoxy-3,6-di-O-benzyl-a-D-glucopyranosyl)-2,3,4,5-tetra-O-
benzyl-D-clairo--inositol (13)
A solution of the iridium catalyst in anhydrous THF (5.8 x 10-'3 M solution,
158 p,1)
previously treated under a hydrogen atmosphere for 30 minutes was added over a
solution of 11 (29 mg, 0.030 mmol) in anhydrous THF (0.3 mL). The mixture was
then stirred at room temperature for 45 minutes, and cooled to 0°C. NBS
(7.7 mg,
0.043 mmol) and water (106 p,L), and THF (1.75 mL) were added and the mixture
was stirred for 15 min. and treated with a saturated solution of NaHC03. The
mixture
was extracted with CHzCl2 and the organic layer dried over NazS04 and
evaporated.
The residue was purified by column chromatography (Hex 4: AcOEt 1 ~ Hex 3:
AcOEt 1) to give pure 13 (22 mg, 79%). 'H NMR (CDC13, 500 MHz): 8 7.41-7.19
(m,
30 H, ArH), 4.94-4.66 (m, 10H, AB System), 4.78 (d, J= 3.6 Hz, 1H, H,,), 4.37
(d, J=
12.0 Hz, 1H, AB System), 4.25 (d, J= 12.1 Hz, 1H, AB System), 4.14. (broad t,
J= 3.4
Hz, 1H, H6), 4.08 (m, 1H, H5,), 4.04 (broad t, J= 3.6 Hz, 1H, H,), 3.95 (dd,
J,= 3.2
Hz, Jz 9.2 Hz, 1 H, H~), 3 .86 (dd, J, = 2.7 Hz, JZ 9.5 Hz, 1 H, HS), 3.82 (t,
J= 9.1 Hz,
1 H, H4), 3 .77 (t, J= 9.1 Hz, 1 H, H3), 3 .72 (m, 2H, H3, + H4,), 3.42 (ad,
J, = 3.6 Hz,
Jz=10.0 Hz, 1H, H2,), 3.28 (dd, J,= 3.4 Hz, Jz 10.3 Hz, 1H, H6,6), 3.18 (dd,
J,= 4.3
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Hz, Jz 10.3 Hz, 1 H, H6. J, 2.5 (s, 1 H, OH,), 2.41 (s, 1 H, OH4,). '3C NMR
(CDC13, 125
MHz): 8 138.83, 138.79, 138.53, 138.15, 138.10, 137.72, 128.57, 128.52,
128.42,
128.36, 128.34, 128.29, 128.19, 128.13, 128.10, 127.98, 127.96, 127.79,
127.71,
127.67, 127.52, 127.43, 127.36, 97.39 (C,,), 81.74, 81.20, 80.00, 79.99,
78.38, 76.13,
5 75.76, 75.23, 73.52, 73.26, 72.99, 72.46, 70.03, 69.13, 67.25, 63.28.
1-O-(2-Azido-2-deoxy-3,6-di-O-benzyl-[3-D-glucopyranosyl)-2,3,4,5-tetra-O-
benzyl-D-chiro--inositol (15)
A solution of the iridium catalyst in anhydrous THF (5.8 x 10-3, 178 p,1)
previously
10 treated under a hydrogen atmosphere for 30 minutes was added over a
solution 12
(32.7 mg, 0.034 mmol) in anhydrous THF (0.3 mL). The mixture was then stirred
at
room temperature for 45 minutes and cooled to 0 °C. NBS (8.8 mg, 0.049
mmol)
water (120 p,1) and THF (2 ml) were added and the mixture stirred for 15 min.
The
reaction mixture was treated with saturated solution of NaHC03, extracted with
1 S CHzCl2 and the organic layer dried and evaporated. The residue was
purified by
column chromatography (Hex 4: AcOEt 1-~ Hexane 3 AcOEt 1) to give pure 15
(22.5
mg, 72%). 'H NMR (CDC13, 500 MHz): 8 7.39-7.22 (m, 30 H, ArH), 4.93-4.50 (m,
12H, AB System), 4.53 (d, J-- 8.1 Hz, 1H, Hl,), 4.19 (m, 2H, H6 + Hl), 3.98
(t, J= 9.4
Hz, 1H, H3), 3.90 (dd, JI=2.6 Hz, Jz 9.9 Hz, 1H, HZ), 3.80 (dd, J,= 2.8 Hz, Jz
9.4
20 Hz, 1 H, HS), 3.75 (t, J-- 9.2 Hz, 1 H, H4), 3.65 (m, 2H, Hb,b + H6,~, 3
.54 (dt, J, = 2.2
Hz, JZ 9.2 Hz, 1H, H4), 3.28 (m, 2H, H2, + H5,), 3.16 (t, J= 9.2 Hz, 1H, H3,),
2.54 (d,
J= 2.2 Hz, 1H, OH4,), 2.43 (s, 1H, OH). '3C NMR (CDCl3, 125 MHz): 8 139.02,
138.98, 138.93, 138.19, 138.09, 137.69, 128.64, 128.52, 128.44, 128.43,
128.35,
128.29, 128.27, 128.15, 128.12, 128.06, 127.90, 127.87, 127.86, 127.82,
127.67,
25 127.44, 127.42, 127.31, 102.98 (C,'), 82.29, 81.69, 81.29, 80.21, 79.56,
76.36, 75.82,
75.76, 75.11, 73.89, 73.71, 73.38, 73.11, 71.68, 70.01, 69.02, 66.20.
6-O-(2-Amino-2-deoxy-a-D-glucopyranosyl)-D-chiro--inositol (14, RGL 1017)
To a solution of 13 (8 mg, 0.009 mmol) in methanol (2 mL) was added 10% PdIC
(28
30 mg) and a drop of acetic acid. The mixture was stirred under hydrogen
atmosphere for
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41
8 h and then filtered over Celite and liophylized. The residue was purified
using a
Dowex 50 H+ resin using methanol, water and 1% ammonium hydroxide to obtain
pure 14 (RGL1017) (2.5 mg, 83%). 'H NMR (D20, 500 MHz): 8 5.0 (d, J= 3.6 Hz,
1 H, H,' ), 4.12 (t, J= 3 . 6 Hz, 1 H, H6), 4. 01 (t, J= 3 .6 Hz, 1 H, H, ), 3
. 97 (ddd, J, = 2. 5
Hz, JZ 4.5 Hz, J3= 9.6 Hz, 1 H, H5,), 3 .82 (dd, J, = 3.6 Hz, Jz 9.6 Hz, 1 H,
HZ), 3.80
(m, 1 H, Hb.b), 3.76 (m, 1 H, Hb,a), 3.70 (dd, J, = 3.3 Hz, JZ 9.5 Hz, 1 H,
HS), 3.61 (t, J=
9.7 Hz, 1 H, H3.), 3.59 (t, J= 8.9 Hz, 1 H, H3), 3.55 (t, J= 8.4 Hz, 1 H, H~),
3.40 (t, J=
9.6 Hz, 1 H, H4,), 2.79 (dd, J, = 9.7 Hz, JZ 3.6 Hz, H2,).
6-O-(2-Amino-2-deoxy-[3-D-glucopyranosyl)-D-chiro--inositol (16, RGL 1018)
To a solution of 15 (4.5 mg, 0.004 mmol) in methanol ( 1.1 mL) 10% PdIC ( 16
mg)
and a drop of AcOH were added. The mixture was stirred for 8h under a hydrogen
atmosphere and the filtered over Celite, washed with methanol and liophylized.
The
residue was purified over a Dowex 50 H+ column using methanol, water and 1%
ammonium hydroxide to give pure 16 (RGL 1018) (1.6 mg, 100%). 'H NMR (DaO,
500 MHz): 8 4.57 (d, J= 8.2Hz, 1H, H,.), 4.26 (t, J= 3.6 Hz, 1H, H,), 4.06 (t,
J= 3.6
Hz, 1 H, H6), 3 .92 (dd, J, = 2.3 Hz, J2 12.4 Hz, 1 H, H6,b), 3 .84 (dd, J, =
3 .2 Hz, J2 9.7
Hz, 1 H, HS), 3 .77 (dd, J, = 3 .2 Hz, JZ 9.5 Hz, 1 H, HZ), 3 .74 (dd, J,
=12.4 Hz, Ja 5.6
Hz, 1 H, H6-a), 3.62 (t, J= 9.5 Hz, 1 H, H4), 3.57 (t, J= 9.5 Hz, 1 H, H3),
3.47 (m, 1 H,
H5,), 3.41 (t, J=9.5 Hz, 1H, H3,), 3.37 (t, J= 9.3 Hz, 1H, H4~), 2.71 (t, J--
8.8 Hz, 1H,
Hz,).
Pinitol containing compounds
The synthesis of compounds 1-4 involved the preparation of a glycosyl acceptor
with
position 6 differentiated to be reactive with the corresponding glycosyl
donor.
Protection of the pinitol unit as cyclohexane-1,2-diacetal as proposed_by Ley
~8~ was
attempted. The selectivity of the reaction of 5 with 1,1,2,2-
tetramethoxycyclohexane
arises from the stabilising influence of the four anomeric effects in the
resulting acetal
6 and the equatorial arrangement of all four sterically demanding alkyl
substituents of
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42
the central 1,4-dioxane unit (Scheme 1). Treatment of 6 with 2,2-
dimethoxypropane
in the presence of TsOH gave 7 in 80% yield.
Glycosylation of 7 with 2-azido-2-deoxy-3,4,6-tri-O-benzyl-D-glucopyransol-
trichloroacetimidate 8 ~9~ prepared following a well established
procedure'°~66~, in
dichloromethane and using TMSOTf as promoter ~"~ gave a 2:1 mixture of the a
(9)
and ~3 (10) linked pseudodisaccharides in 54% yield (Scheme 2). The acetal
groups
were removed ~'z~ to give 11 and 12 respectively which were subjected to
hydrogenolysis to afford finally 1 and 2 in quantitative yield.
Glycosylation of 7 with 2-azido-2-deoxy-3,4,6-tri-O-benzyl-D-galactopyranose
(13)
under different conditions gave a mixture of a- and j3-linked
pseudodisaccharides 14
and 15 in moderate yield (Scheme 3). Removal of the acetal groups and
subsequent
hydrogenolysis afforded 3 and 4.
4,5-O-(1',2'-dimethoxycyclohexane-1',2'-diyl)-3-O-methyl-D-chiro-inositol (6)
3-O-methyl-D-chino-inositol (D-pinitol) (283 mg, 1.457 mmol, 1 equiv) was
dissolved
in methanol (15 mL), then 1,2-cyclohexane diacetal (507 mg, 2.458 mmol, 1.7
equiv),
trimethyl orthoformiate (200 mL, 1.752 mmol, 1.2 equiv) and 1-(S~-(+)-10-
camphorsulfonic acid (24 mg, 0.102 mmol, 0.07 equiv) were added. The reaction
mixture was heated at 70°C for 24 h whereupon it was diluted with MeOH
and
quenched with solid NaHC03. The residue was concentrated and purified by flash
chromatography (Hex/EtOAc 1:15) to give 6 (222 mg, 0.664 mmol, 45%; 89% based
on 134 mg of recovered D-pinitol). Rf (Hex/EtOAc 1:20): 0.22; [a]z°~ -
22.9 (c =
1.05, CHCl3); 'H NMR (CDC13, 300 MHz): d = 4.12 (Ys, 1H, H,), 4.10 (s, 2H, H4,
HS), 4.07 (Ys, 1 H, H6), 3. 81 (dt, 1 H, Jz_3 = 9.0 Hz, Jz_, = 3.6 Hz, Jz_o~.
=1.8 Hz, Hz),
3.47 (t, 1H, J= 9.0 Hz, H3), 3.23 (s, 3H, OCH3), 3.21 (s, 3H, OCH3), 2.76 (d,
IH,JcH_z
=1.8 Hz, OHz), 2.60 (s, 1H, OH,), 2.56 (s, 1H, OH_6), 1.86-1.78 (m, 1H, CDA),
1.77-
1.66 (m, 3H, CDA), 1.57-1.50 (m, 2H, CDA), 1.42-1.34 (m, 2H, CDA);'3C-NMR
(CDCl3, 75 MHz); d = 98.9 (C), 98.0 (C), 80.2 (C3), 71.5(Cz), 70.8, 70.4, 70.3
(C~, C4,
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C6), 68.7 (CS), 61.1 (OCH3), 47.0(OCH3, CDA), 46.8 (OCH3, CDA), 27.1 (CH2),
27.0
(CHZ), 21.5 (CHz), 21.4 (CHZ); HRFABMS Calcd. for C,SH26Og: 357.3571 found:
357.1528; MALDI-TOF Calcd. for C,5Hz60$+Na+: 357.4, found: 357.5, calcd. for
C,SH260g+K+: 373.5, found: 374.1
4,5-O-(1',2'-dimethoxycyclohexane-1',2'-diyl)-3-O-methyl-1,2-O-isopropyliden-D-
chino-inositol (7)
4,5-O-(1',2'-dimethoxycyclohexane-1',2'-diyl)-3-O-methyl-D-chino-inositol (6)
(326
mg, 0.975 mmol, 1 equiv) and 2,2-dimethoxypropane (1,321 mL, 1.072 mmol, 1.1
equiv) in acetone (4 mL) were treated with p-toluenesulfonic acid monohydrate
(9.3
mg, 0.049 mmol, 0.05 equiv). The reaction was stirred for 1 h, whereupon it
was
quenched with solid NaHC03, the solvent evaporated and the residue purified by
flash
chromatography (Hex/AcOEt 1:l) to give 7 (292 mg, 0.873 mmol, 81%) as a white
solid. Rf(Hex/EtOAc 3:1): 0.71; [a]2°~ -18.0 (c = 0.30, CHCl3); 'H NMR
(CDC13,
500 MHz): d = 4.27 (dd, 1H, J,_2 = 7.3 Hz, J,_6 = 3.2 Hz, H,), 4.24 (t, 1H, J=
3.2 Hz,
H6), 4.16 (t, 1 H, J = 7.3 Hz, Hz), 4.06 (t, 1 H, J = 10.2 Hz, H4), 3 .94 (dd,
1 H, J5~ _
10.2 Hz, J$_6 = 3.2 Hz, HS), 3.61 (s, 3H, OCH3), 3.39 (dd, 1H, J3~ =10.2 Hz,
J3_2 = 7.3
Hz, H3), 3.23 (s, 3H, OCH3, CDA), 3.22 (s, 3H, OCH3, CDA), 1.86-1.78 (m, 1H,
CDA), 1.77-1.66 (m, 3H, CDA),1.52 (s, 3H, CH3), 1.57-1.51 (m, 2H, CDA), 1.36
(s,
3H, CH3),1.41-1.33 (m, 2H, CDA);'3C-NMR (CDC13, 125 MHz): d=109.0 (C), 98.4
(C, CDA), 97.8 (C, CDA), 82.7 (C3), 79.6 (CZ), 76.4 (C,), 68.5 (CS), 68.3
(C6),
67,4(C4), 60.2 (OCH3), 47.1 (OCH3, CDA), 46.9 (OCH3, CDA), 27.0 (CH3), 27.1
(CHZ), 26.9 (CHZ), 26.0 (CH3); 21.4 (2CH2); Anal. Calcd. for C,8H3°Og:
C, 57.74%; H,
8.08%; found: 57.46%; H, 7.86%; HRFABMS Calcd. for C,8H3°Og+Na+:
397.1838,
found: 397.1854; MALDI-TOF Calcd. for C,8H3°O8+Na+ : 397.4, found:
397.3 calcd.
for C,8H3°Og+K+ : 413.5, found: 413.8
Glycosylation reaction of 7 and 8
To a solution of 8 (809 mg, 1.305 mmol, 1.5 equiv) in CHZC12 (4 mL), compound
7
0
(295 mg, 0.788 mmol, 1 equiv) and freshly activated 4 A molecular sieves were
added
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44
and the mixture stirred for 1 h under Argon. Then, TMSOTf (I2.6 mL, O.I04
mmol,
0.08 equiv) was added and the reaction mixture stirred for 24 h. The
suspension was
filtered through celite and the solvent evaporated under vacuum to provide a
mixture
of two disaccharides (a/(3 = 2:1) which can be separated by flash
chromatography
(Hex/EtOAc 4:1) to obtain 9 (235 mg, 0.282 mmol, 36%) and 10 (118 mg, 0.142
mmol, 18%).
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-D-glucopyranosyI-a(1-~6)-4,5-O-(I',2'-
dimethoxycyclohexane-1',2'-diyl)-1,2-O-isopropyliden-3-O-methyl-D-cl:iro-
inositol (9)
Rf (Hex/EtOAc 3:1): 0.17; [a]2°D +48.8° (c = 0.25, CHC13);'H
NMR (CDC13, 500
MHz): d = 7.43-7.22 (m, 15H, Ph), 4.94 (d, 1H, J,,_z, = 3.2 Hz, H~,), 4.83 (d,
1H, J=
13.0 Hz, CHPh), 4.82 (AB syst., 2H, CHzPh), 4.58 (d, 1H, J=12.0 Hz, CHPh),
4.58
(m,1H, H5,), 4.57 (d, 1H, J=13.0 Hz, CHPh), 4.42 (d, 1H, J-- 12.0 Hz, CHPh),
4.25
(t, 1 H, J = 6.4 Hz, HZ), 4.18 (t, 1 H, J = 2.5 Hz, H, ), 4.16 (t, 1 H, J =
2.5 Hz, H6), 4.11
(t, 1 H, J =10.5 Hz, H4), 3.96 (t, 1 H, J=10.1 Hz, H3' ), 3 .90 (dd, 1 H, J5~
=10.5 Hz, JS
6 = 2.5 Hz, HS), 3.79 (t, 1 H, J = 10.1 Hz, H4,), 3.71 (dd, 1 H, J6a'-6b' -
10.8 Hz, J6~,_s, _
2.4 Hz, H68,), 3.60 (s, 3H, OCH3), 3.59 (dd, 1H, J6a'-6b' = 10.8 Hz, Jbb'_5'
=1.6 Hz, Hbb'),
3.41 (dd, 1H, J3~ = 10.5 Hz, J3_2 = 6.4 Hz, H3), 3.36 (dd, 1H, Ja,_3, = 10.2
Hz, J2,_,, _
3.2 Hz, H2,), 3.19 (s, 6H, 2 OCH3, CDA), 1.83-1.78 (m, 1H, CDA), 1.72-1.61 (m,
3H,
CDA), 1.52 (s, 3H, CH3), 1.55-1.43 (m, 2H, CDA), 1.40-1.30 (m, 2H, CDA), 1.36
(s,
3H, CH3); MALDI-TOF Calcd. for C45HS,N3O12+Na+ : 855.0, found: 854.3, calcd.
for
C~sHs~NsOiz+I~+ : 871.1, found: 870.5
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-D-glucopyranosyI-j3(1-~6)-4,5-O-(1',2'
dimethoxycyclohexane-1',2'-diyl)-1,2-O-isopropyliden-3-O-methyl-D-clairo-
inositol (10)
Rf(Hex/EtOAc 3:1 ): 0.20; [a]2°D -8.0° (c = 1.18, CHC13);'H
NMR (CDC13, 500
MHz): d 7.40-7.24 (m, 13H, Ph), 7.21-7.13 (m, 2H, Ph), 4.97 (d, 1H, J,,_a, =
8.1 Hz,
H,,), 4.92 (d, 1H, J= 10.8 Hz, CHPh), 4.82 ( d, 1H, J= 10.8 Hz, CHPh), 4.78
(d, 1H,
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J=10.8 Hz, CHPh), 4.62 (d, 1H, J=10.8 Hz, CHPh), 4.58 (AB syst., 2H, CH2Ph),
4.49 (t, 1H, J= 2.5 Hz, H6,), 4.30 ( dd, 1H, J,_2 = 5.1 Hz, J,_6 = 2.5 Hz,
H,), 4.20 (t,
1 H, J = 10.8 Hz, H4), 4.17 ( t, 1 H, J = 5.1 Hz, HZ), 3.98 (dd, 1 H, J5~ =
10.8 Hz, JS_s =
2.5 Hz, HS), 3.71-3.57 (m, 3H, HS,, H6,, H4,), 3.61 (s, 3H,OCH3), 3.47-3.39
(m, 3H,
5 H3,, H3, H6,), 3.30 (dd, 1H, J= 8.1 Hz, H2,), 3.23 (s, 3H, OCH3, CDA), 3.18
(s, 3H,
OCH3, CDA), 1.83-1.77 (m, 1H, CDA), 1.80-1.43 (m, SH, CDA), 1.52 (s, 3H, CH3),
1.41-1.28 (m, 2H, CDA), 1.33 (s, 3H, CH3); '3C-NMR (CDC13, 125 MHz): d = 138.5
(C), 138.4 (C), 138.3 (C), 128.9 (2CH), 128.8 (2CH), 128.8 (CH), 128.8 (2CH),
128.4
(2CH), 128.3 (2CH), 128.3 (CH), 128.2 (2CH), 128.1 (CH), 109.9 (C), 101.3
(C,,),
10 98.4 (C, CDA), 98.0 (C, CDA), 83.9 (C3,),83.6 (C3),78.1 (C4,),76.7
(C,),76.0
(CHZ),75.9 (C6,),75.5 (CHZ),75.4 (CHZ),73.9 (C6),72.6 (C5,),68.8 (C5),68.7
(C2),68.1
(C4), 67.5 (C2,), 60.4 (OCH3),47.4 (OCH3, CDA),47.3 (OCH3, CDA),28.4
(CH3),27.5
(CHZ),27.4 (CH2),26.3 (CH3),21.8 (2CH2); HRFABMS Calcd. for C45HS~N3O12
854.3840, found = 854.3872; MALDI-TOF Calcd. for C45HS.,N3012+Na+ : 855.0,
15 found: 854.3, calcd. for C45HS~N3O12+K'~ : 871.1, found: 870.4
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-D-galactopyranosyl-a(1-~ 6)-4,5-O-(1',2'-
dimethoxycyclohexane-1',2'-diyl)-1,2-O-isopropyliden-3-O-methyl-D-chiro-
inositol (14)
20 To a solution of 13 (84 mg, 0.14 mmol, 1.3 equiv), 7 (39 mg, 0.10 mmol, 1.0
equiv)
0
in CH2CIz:Hex, 1:3 (2 mL) and freshly activated 4 A molecular sieves were
added
and the mixture stirred for 1 h under Argon. Then, TMSOTf (2.43 mL, 0.02 mmol,
0.15 equiv) was added at -40°C and the reaction mixture stirred for 24
h at 0°C. The
suspension was filtered through celite and the solvent removed under vacuum to
25 provide the crude material. Flash chromatography (Hex/ EtOAc 4:1) afforded
14 (38
mg, 0.046 mmol, 46%). Rf (Hex/EtOAc 1:1): 0.67; [a]2°D +59.7° (c
=1.44, CHC13);
'H NMR (CDC13, 300 MHz) : d = 7.46-7.28, (m, 15H, Ph), 4.98 (d, 1H, J= 3.3 Hz,
HI,), 4.89 (d, 1H, J= 11.2 Hz, CHPh), 4.78 (d, 1H, J=11.1 Hz, CHPh), 4.69 (m,
1H,
H5,), 4.67 (d, 1H, J= 11.1 Hz, CHPh), 4.59 (d, 1H, J=11.2 Hz, CHPh), 4.46, (AB
30 syst., 2H, CH2Ph),4.28 (t, 1H, J= 6.3 Hz, Hz), 4.20 (m, 2H, H,, H6), 4.15
(bs, 1H,
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Ha,), 4.11 (m, 1 H, Ha), 4.04 (dd, 1 H, J3,_2, = 10.6 Hz, J3,~, = 2.4 Hz,
H3,), 3.92 (dd, 1 H,
Ja_s = 10.8 Hz, Js_6 = 2.1 Hz, Hs), 3.86 (dd, 1H, J2,_3, = 10.6 Hz, J2,_,, =
3.3 Hz, H2,),
3.66-3.60 (m, 1 H, H66,), 3.63 (s, 3H, OCH3), 3.50 (dd, 1 H, J6a'-66' = 8.1
Hz, Jsa,_s, = 5.4
Hz, Hba,), 3.42 (dd, 1H, J3_4 = 10.2 Hz, J3_2 = 6.3 Hz, H3), 3.20 (s, 3H,
OCH3, CDA),
3.18 (s, 3H, OCH3, CDA), 1.90-1.63 (m, 4H, CDA), 1.54 (s, 3H, CH3), 1.60-1.33
(m,
4H, CDA),1.40 (s, 3H, CH3);'3C-NMR (CDCl3, 75 MHz): 8 139.0 (C). 138.5 (C),
138.0 (C), 110.2 (C), 98.3 (C, CDA), 98.2 (C, CDA), 97.8 (C,,), 83.6 (C3),
80.6 (CZ),
77.2 (C3,), 75.2 (C6), 75.0 (CHZ), 73.9 (Ca,), 73.8 (CHZ), 72.8 (C,), 72.7
(CHz), 69.9
(Cs,), 68.4 (C6,), 68.1 (Ca), 67.1 (Cs), 60.5 (OCH3), 60.0 (CZ,), 47.2 (20CH3,
CDA),
28.5 (CH3), 27.4 (CHZ), 27.0 (CHZ), 26.7 (CH3), 21.9 (2 CHa); MALDI-TOF Calcd.
for CasHs~N30~2+Na+ : 855.0 Found: 854.0 Calcd. for CasHs~N3O12+R+ : 871.1
Found: 870.1
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-D-glucopyranosyl-all ~ 6)-3-O-methyl-D-
chino-inositol (11)
Disaccharide (9) was dissolved in a mixture of trifluoroacetic acid/water
(20:1, 3.7
mL) and stirred at room temperature for 40 min. Then, the reaction mixture was
diluted with CHZCl2 (10 mL) and immediately poured into an ice-cold,
vigorously
stirred solution of saturated aqueous sodium bicarbonate (90 mL). The layers
were
separated, and the aqueous phase extensively extrated (CHZCIz, 4x 30 ml),
dried over
Na2SOa and concentrated under vacuum. Purification by flash chromatography
(C12CH2/MeOH, 11:1 and then CHzCIz/MeOH, 15:1) afforded 11 (4 mg, 6.138 mmol,
56%). Rf (C12CH2/MeOH, 9:1): 0.3; [a]2°D +38.5° (c = 0.19,
CHCl3);'H NMR
(CDCl3, 500 MHz): d = 7.34-7.16 (m,lSH, Ph), 4.94 (d, 1H, J= 3.5 Hz, H,), 4.84
AB syst., 2H, CHzPh), 4.79 (d, 1H, J= 11.0 Hz, CHPh), 4.56 (d, 1H, J= 11.0 Hz,
CHPh), 4.50 (d, 1H, J=11.0 Hz, CHPh), 4.47 (d, 1H, J= 11.0 Hz, CHPh), 4.16
(bs,
1H, H1), 4.12 (m, 1H, Hs,), 4.04 (bs, 1H, H6), 3.92 (m, 1H, HZ), 3.87-3.84 (m,
2H, H3,,
Hs), 3.71 (Yt, 1H, J= 8.0 Hz, Ha), 3.56 (m, 3H, Ha., 2H6.), 3.63 (s, 3H,
OCH3), 3.44
(dd, 1H, JZ,_3, = 10.0 Hz, J,,_z, = 3.5 Hz, H2,), 3.37 (Yt, 1H, J= 8.0 Hz,
H3).
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2-Azido-2-deoxy-3,4,6-tri-O-benzyl-D-glucopyranosyl-[3(1-~6)-3-O-methyl-D-
chiro-inositol (12)
Compound 12 (28 mg, 43.0 mmol, 71 %) was obtained via the general procedure
described above for compound 11. Purification by flash chromatography
(Hex/EtOAc
1:20). Rf(EtOAc): 0.47; 'H NMR (CDCl3, 500 MHz): d = 7.34-7.26 (m, 13H, Ph),
7.19-7.10 (m, 2H, Ph), 4.83 (AB syst., 2H, CHZPh), 4.78 (d, 1H, J=11.0 Hz,
CHPh),
4.54 (AB syst., 2H, CHZPh), 4.52 (d, 1H, J= 11.0 Hz, CHPh), 4.46 (d, 1H, J=
8.0 Hz,
H~,), 4.21 (bs, 1H, H,), 4.10 (bs, 1H, H6), 3.94-3.90 (m, 2H, H2, HS), 3.80
(t, 1H, J=
8.5 Hz, H4), 3.69-3.59 (m, 2H, 2H6,), 3.62 (s, 3H, OCH3), 3.58 (t, 1H, J= 9.5
Hz, H4,),
3.47 (t, 1H, J= 9.5 Hz, H3,), 3.46-3.36 (m, 3H, HZ', HS,, H3), 3.35-3.27 (bs,
1H, OH),
3.27-3.15 (bs, 1H, OH), 3.15-2.94 (2bs, 2H, 20H).
2-Azido-2-deoxy-3,4,6-tri-O-benzyl-D-galactopyranosyl-all ~ 6)-3-O-methyl-D-
chiro-inositol (16)
Compound 16 (7 mg, 10.7 mmol, 41 %) was obtained via the general procedure
described above for compound 11, using purification by flash chromatography
(CHzCl2/MeOH, 10:1). Rf(CHZCIz/MeOH 9:1): 0.48; 'HNMR (CDCl3, 500 MHz): d
= 7.36-7.21 (m, 15H, Ph), 4.96 (d, 1H, JI_2 = 3.5 Hz, H,'), 4.85 (d, 1H, J=
11.5 Hz,
CHPh), 4.71 (d, 1H, J= 11.0 Hz, CHPh), 4.67 (d, 1H, J=11.0 Hz, CHPh), 4.49 (d,
1H, J= 11.0 Hz), 4.49 (d, 1H, J= 11.5 Hz, CHPh), 4.40 (d, 1H, J= 11.0 Hz,
CHPh),
4.20 (dd, 1H, J4,_5, = 7.5 Hz, JS'_6, = 4.5 Hz, H5,), 4.15 (t, 1H, J= 4.0 Hz,
H,), 4.01 (t,
1H, J= 4.0 Hz, H6), 3.95-3.90 (m, 2H, HZ,, H4,), 3.87 (m, 1H, HZ), 3.84 (dd,
JZ'_3'=
10.5 Hz, J3,~, = 2.5 Hz, H3,), 3.82-3.77 (m, 1H, HS), 3.65 (t, 1H, J= 8.2 Hz,
H4), 3.62
(s, 3H, OCH3), 3.58 (dd, 1H, J6a'-6b' = 9.5 Hz, JS'_sa' = 7.5 Hz, H6a,), 3,38
(dd, 1H, J6a'-sb'
= 9.5 Hz, J5,_66' = 4.5 Hz, H6b,), 3.35 (t, 1H, J= 8.2 Hz, H3).
2-Amino-2-deoxy-D-glucopyranosyl-a(1~6)-3-O-methyl-D-chino-inositol (1)
Compound 11 (3.8 mg, 5.831 mmol, 1.0 equiv) and 10% Pd/C (10 mg, 9.398 mmol)
were stirred in methanol under a hydrogen atmosphere for 24 h. The slurry was
filtered, washed with water and the filtrate was concentrated and lyophilized
to give
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the fully deprotected disaccharide 1 (2.6 mg, 7.3 mmol, quantitative). Rf
(EtOAc/MeOH/H20/AcOH 2:2:1:1): 0.44;[a]2°D +55.2° (c = 0.125,
H20). 'H NMR
(D20, 500 MHz): 8 = 5.22 (d, 1H, H,,), 4.16 (t, 1H, H,), 4.19 (t, 1H, H6),
4.07 (dt, 1H,
H5,), 3.90 (dd, 1H, HS), 3.85-3.75 (m, 3H, H4,, H6a,, H66,), 3.80 (dd, 1H,
Hz), 3.70 (t,
1 H, H3,), 3.64 (s, 3H, OCH3), 3.52 (t, 1 H, H4), 3.36 (t, 1 H, H3), 3.24 (dd,
1 H, HZ').
2-Amino-2-deoxy-D-glucopyranosyl-[3(1-~6)-3-O-methyl-D-elziro-inositol (2)
Compound 12 (24 mg, 67.54 rnmol, 98%) was treated as described above to afford
compound 2. Rf (EtOAc/MeOH/Hz0/AcOH 2:2:1:1 ): 0.42, 'H NMR (D20, 500
MHz): d = 4.70 (d, 1H, J= 8.5 Hz, H,,), 4.32 (Yt, 1H, J= 3.5 Hz, H,), 4.12
(Yt, 1H, J
= 3.5 Hz, H6), 3 .96 (dd, 1 H, Jba'-sb' =9.5 Hz, J6a'-s' =1.7 Hz, H6a'), 3.92
(dd, 1 H, J5~ _
8.5 Hz, JS_6 = 3.5 Hz, HS), 3.88 (dd, 1H, Jz_3 = 9.8 Hz, JI_2 6.6 Hz, HZ),
3.82 (dd, 1H,
Jsa'-sn' = 9.5 Hz, JS'_6b' = 4.0 Hz, H66'), 3.78 (t, 1H, J= 9.5 Hz, H4'), 3.66
(s, 3H, OCH3),
3.48 (t, 1H, J= 9.8 Hz, H4,), 3.57-3.51 (m, 2H, H3,, H5,), 3.40 (t, 1H, J= 9.8
Hz, H3),
2.86 (t, 1H, J= 8.5 Hz, H2,).
2-Amino-2-deoxy-D-glucopyranosyl-a(1~6)-3-O-methyl-D-clziro-inositol (14)
Compound 14 (5.3 mg, 14.9 mmol, quantitative) was obtained via the general
procedure described above for compound 12. Rf (C12CH2/MeOH 9/1): 0.071;
[a]2°n
(c = 0.305, H20): +70.1;'H NMR (DzO, 500 MHz): 8 = 5.31 (d, 1H, J= 3.5 Hz,
Hl,),
4.29 (t, 1 H), 4.19 (tl't, 1 H, H 1 ), 4.14 (~I't, 1 H, H6), 4.05 (~~'s, 1 H,
H4'), 3.92 (dd, 1 H),
3.77 (d, 3H), 3.72 (t, 1H), 3.65 (s, 3H, OCH3), 3.62 (m, 1H), 3.52 (dd, 1H,
JZ,_3' = 11.0
Hz, Jl,_Z'= 3.5 Hz, HZ'), 3.37 (t, 1H, J= 10.0 Hz, H3).
Synthesis of RGL1105
1 "-D-4'-O-(2",3",4"-tri-O-benzyl-6"-tertbutyldimethylsilyl-a-D-.
mannopyranosyl)-[1'-D-6-O-(2'-azido-3',6'-di-O-benzyl-2'-deoxy-a-D-
glucopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4,5-
tri-
O-benzyl-myo-inositol] (3)
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A mixture of compounds 1 (284 mg, 0.298 mmol) and 2 (340 mg, 0.408 mmol), was
dissolved in Toluene and the solvent removed (3x10 mL). To the water-free
solid
mixture, Et20 anh. ( 10 mL) and 41~ powdered molecular sieves was added and
allowed to dissolve at room temperature under Argon atmosphere. After ~ min,
TMSOTf (12 ~L, 0.066 mmol) was added and the reaction allowed to proceed for
90
min. Then, the reaction was quenched with Et3N (2 mL) and after 5 min
stirring, the
solvents are removed and the residue purified by column chromatography (Si02,
hexane/AcOEt 19:1 ), to obtain 3 (363 mg, 75%). 'H NMR (CDCl3, 500 MHz): d
7.72-
7.58 (m, SH, ArH), 7.42-7.13 (m, 39H, ArH), 7.07 (m, SH, ArH), 6.94 (t, J=7.5
Hz,
1H, ArH), 5.58 (d, J=3.0 Hz, 1H, H anom.), 5.261 (d, J=2.9 Hz, 1H, H anom.),
4.95
(d, J=11 Hz, 1H, H benc.), 4.87 (d, J=11 Hz, 1H, H benc.), 4.75-4.63 (m, 7H),
4.62-
4.49 (m, 4H), 4.42 (m, 2H), 4.46 (m, 3H), 4,7 (d, J=12.0 Hz, 1H, H benc.),
4.12 (m,
3H), 4.1 (dd, J~=3Hz, Jz 11.5 Hz, 1H), 3.98-3.91 (m, 3H), 3.78-3.89 (m, 4H),
3.69
(m, 1H), 3.49-3.61 (m, 2H), 3.44 (m, 1 H), 1.99 (m, 2H), 1.74-1.85 (m, 2H),
1.55 (d,
J=12.5 Hz, 1 H),1.47 (m, 1 H), 1.27-1.3 8 (m, 1 H), 1.10 (s, 3 H), 0.95 (s, 3
H), 0.92 (s,
3H).
1"-D-4'-O-(2",3",4"-tri-O-benzyl-a-D-mannopyranosyl)-[1'-D-6-O-(2'-azido
3',6'-di-O-benzyl-2'-deoxy-a-D-glucopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]
bicyclohept-2-ylidene)-3,4,5-tri-O-benzyl-myo-inositol] (4)
A solution of 3 (290 mg, 0.179 mmol) in THF (15 mL) under Argon atmosphere,
was
treated with TBAF (1.0 M in THF, 1.8 mL, 1.800 mmol) at 0°C. The
reaction mixture
was allowed to reach room temperature and kept stirring for 66 h. Then, the
solvents
were removed, the remaining material redissolved in AcOEt (50 mL), washed with
NaCI s.s. (3 x 50 mL), dried over MgS04 and the solvents evaporated. The
residue
was purified by column chromatography (Si02, hexane/AcOEt 9:1 to 8:1 to 6:1
and
finally 4:1), to obtain 4 (206 mg, 83%).'H NMR (CDCl3, S00 MHz): 7.25-7.44 (m,
37H, ArH), 7.11-7.20 (m, 3H, ArH), 5.68 (d, J 3.5 Hz, 1H, H anom.), 5.32 (d, J-
-2
Hz, 1H, H anom.), 4.95 (m, 1H), 4.55-4.86 (m, 12H), 4.50 (d, J 12 Hz, 1H, H
benc.),
4.40 (d, J 12 Hz, 1H, H benc.),4.34 (m, 1H), 4.20 (d, J--12 Hz, 1H, H benc.),
4.10 (m,
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3H), 3.82-4.03 (m, 6H), 3.64-3.76 (m, SH), 3.47-3.58 (m, 2H), 3.41 (dd, J,=3.5
Hz,
Jz 10 Hz, 1H), 2.4 (s,lH, OH), 1.97 (m, 2H), 1.74-1.84 (m, 2H), 1.53 (d, J--13
Hz,
1H), 1.47 (m, 1H), 1.24-1.35 (m, 1H), 1.13 (s, 3H), 0.94 (s, 3H), 0.93 (s,
3H).
5 1 "-D-4'-O-(2",3",4"-tri-D-benzyl-6"-dibenzylphosphate-a-D-mannopyranosyl)-
[1'-D-6-O-(2'-azido-3',6'-di-O-benzyl-2'-deoxy-a-D-glucopyranosyl)-1,2-O-(L-
1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4,5-tri-O-benzyl-myo-inositol]
(5)
To a solution of trisaccharide 4 (190 mg, 0.137 mmol) and 1H Tetrazole in
anhydrous
CHZCl2 (10 mL) under Argon atmosphere and at 0°C, dibenzyl
10 diisipropylphosphoramidite (DBPA, 0.1 mL, 0.298 mmol) was added. The
reaction
mixture was stirred for 3h, while allowing to reach room temperature. Then,
the
reaction mixture was cooled to 0°C and a solution of 70% 3-
chloroperbenzoic acid (85
mg, 0.345 mmol) in anh. CHzCl2 (5 mL) was added. The mixture was stirred for
1h,
diluted with CHZC12 (25 mL), washed with sat. Na~S03 (2x50 mL), sat. NaHC03
15 (2x50 mL) and sat. NaCI (2x50 mL), dried over MgS04 and concentrated. Flash
chromatography of the crude mixture (hexanelAcOEt 9/l, 8/1, 711, 6/1, 511 and
4/1)
gave compound 5 (210 mg, 93%). 'H-NMR (CDCl3, 500 MHz): d 7.13-7.42 (m, 49H,
ArH), 7.08 (m, 1 H, ArH), 5.66 (d, J--3.5 Hz, 1 H, H anom.), 5.28 (d, J 2 Hz,
1 H, H
anom.),. 4.90-5.03 (m, 6H), 4.75-4.81 (m, 4H), 4.72 (d, J 12 Hz, 1 H, H
benc.), 4.65
20 (m, 2H), 4.52-4.61 (m, 3H), 4.45 (m, 2H), 4.34 (m, 2H), 4.15-4.29 (m, 3H),
4.03-4.12
(m, 4H), 3.80-3.94 (m, SH), 3.72 (m, 2H), 3.56 (m, 2H), 3.49 (m, 1H), 3.57
(dd,
J~=3.5 Hz, J2 10 Hz, 1H), 1.97 (m, 2H), 1.78 (m, 2H), 1.52 (d, J--12.5 Hz,
1H), 1.46
(m, 1H), 1.28 (m, 1H), 1.12 (s, 3H), 0.93 (s, 3H), 0.89 (s, 3H). 3'P-NMR
(CDCl3, 202
MHz): d -1.72.
1"-D-4'-O-(2",3",4"-tri-O-benzyl-6"-dibenzylphosphate-a-D-mannopyranosyl)-
(1'-D-6-O-(2'-azido-3',6'-di-O-benzyl-2'-deoxy-a-D-glucopyranosyl)-3,4,5-tri-O-
benzyl-myo-inositol] (6)
To a solution of trisaccharide 5 (200 mg, 0.122 mmol) in CHzCIa (15 mL) HZO
(0.2
mL, 11.1 mmol), and trifluoroacetic acid (0.6 mL, 7.81 mmol) were added and
the
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reaction stirred for 18 h at r.t. The mixture was then diluted with AcOEt (50
mL),
washed with sat. NaHC03 (2x50 mL), sat. NaCI (3x50 mL), dried over MgS04 and
concentrated. Flash chromatography of the crude mixture (hexane/AcOEt 4/1,
2/1,
1/1 y 1l2) gave compound 6 (142 mg, 77%). 'H-NMR (CDC13, 500 MHz): d 7.10-
7.39 (m, SOH), 5.53 (d, J--3.5 Hz, 1H, H anom.), 5.24 (d, J--2.5 Hz, 1H, H
anom.),
4.87-5.03 (m, 8H), 4.72-4.79 (m, 4H), 4.63 (m, 2H), 4.57 (d, J--l2Hz, 1H, H
benc.),
4.49 (d, J--12 Hz, 1H, H benc.), 4.25-4.38 (m, 3H), 4.09-4.26 (m, 4H), 4.01-
4.09 (m,
3H), 3.95 (m, 1H), 3.84 (m, 3H), 3.69 (m, 3H), 3.6 (m, 1H), 3.5 (m, 1H), 3.43
(m,
2H), 3.3 7 (m, 1 H), 3.29 (m, 1 H), 2.94 (wide s., 1 H, OH). 3'P-RMN (CDCl3,
202
MHz): d -1.79.
1"-D-4'-O-(6"-phosphate-a-D-mannopyranosyl)-[1'-D-6-O-(2'-amino-2'-deoxy-
a-D-glucopyranosyl)-myo-inositol] (RGL 1105)
To a suspension of trisaccharide 6 (115 mg, 0.076 mmol) in a mixture of
MeOH/H20
(5 mL, 9:1) 10% Pd/C (162 mg, 0.152 mmol) was added and the reaction stirred
under
hydrogen atmosphere at r.t. for 24 h. The solvent was evaporated, the crude
suspended in H20 (10 mL), filtered through celite and the filtrate lyophilized
to give
RGL1105 (45 mg, quant.). 'H-RMN (DZO, 500 MHz): d 5.46 (d, 3.5 Hz, 1H, H
anom.), 5.31 (s, 1H, H anom.), 4.0-4.27 (m, 6H), 3.72-3.92 (m, 8H), 3.68 (t, J-
-9.S Hz,
1H), 3.54 (m, 1H), 3.41 (m, 1H). 3'P-RMN (DzO, 202 MHz): d 0.66.
Synthesis of RGL1115
Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-4',6'-O-benzylidene-2'-deoxy-a-D
glucosopyranosyl)-1,2-O-(L-1,7,7-trimethyl(2.2.1]-bicyclohept-2-ylidene)-3,4-O
(1,1,3,3; tetraisopropyldisiloxanyl)-5-O-di-benzylphosphate-myo-inosito112
To a solution of a-1,6 anomer 11 (0.16 mmol) and 1-H tetrazole (0.63 mmol) in
anhydrous C12CH2 (15 ml) at 0 °C (ice bath) was added dropwise dibenzyl
diisopropylphosphoramidite (0.63 mmol). After the addition was completed the
ice-
bath was removed and the mixture left to stir under an inert atmosphere whilst
being
monitored by TLC analysis (hexane:EtOAc [4:1]). After 72 hours the a-1,6
anomer 11
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starting material had been consumed. A solution of mCPBA (0.39 mmol) in
anhydrous DCM (5 ml) was added to the reaction vessel and the mixture left to
stir for
3 hours at R.T. under an inert atmosphere. The mixture was diluted with DCM
(30
ml), washed with sat. Na2S03 (2 x 25 ml), sat. NaHC03 (2 x 25 ml) and brine (2
x 25
ml). The organic layer was then dried over MgS04 and concentrated to dryness
in
vacuo. The crude product was purified by column chromatography (hexane:EtOAc
[4:1]) to yield phosphate 12 as a pale yellow oil (60 %). 8H (CDC13: 360 MHz)
4.6
(2H, dd, PhCH20), 5.3 (1H, d, Hl'), 5.4 (1H, s, PhCHO); 8P (CDC13: 146 MHz)
0.2
(1 P°).
Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-4',6'-O-benzylidene-2'-deoxy-a-D-
glucosopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-5-O-
di-
benzylphosphate-myo-inositol 13
To a solution of phosphate 12 (0.09 mmol) in anhydrous THF (1.3 ml) at
0°C (ice
bath) was added TBAF (1.0 M solution in THF) (0.2 ml) and the reaction left to
stir at
R.T. under an inert atmosphere. After 45 minutes TLC analysis (hexane:EtOAc
[3:2])
indicated that all phosphate 12 starting material had been consumed. The
reaction
mixture was concentrated to dryness irc vacuo before being purified by column
chromatography (hexane:EtOAc [3:2]) to yield de-silylated saccharide 13 as a
pale
yellow oil (44 %). 8H (d4-MeOH: 360 MHz) 5.4 (1H, d, H1'), 5.5 (1H, s, PhCHO);
8P
(d4-MeOH: 146 MHz) 1.1 (1P°).
Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-2'-deoxy-a-D-glucosopyranosyl)-5-
O-di-benzylphosphate-myo-inositol 14
To a solution of de-silylated saccharide 13 (0.04 mmol) in DCM (5.0 ml) was
added
distilled water (44 ml) then TFA (187 ml) and the mixture stirred at R.T. for
4 hours
whilst being monitored by TLC analysis (EtOAc). The reaction mixture was
concentrated to dryness in vacuo before the crude product was purified by
column
chromatography (EtOAc [100 %] then EtOAc:MeOH [10:1]) to yield the de-
protected
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53
saccharide 14 (89 %) as a pale yellow oil. 8H (d4-MeOH: 360 MHz) 5.3 (1H, d,
Hl');
8P (d4-MeOH: 146 MHz) 0.9 (1P'').
Synthesis of 1'-D-6-O-(2'-amino-2'-deoxy-a-D-glucosopyranosyl)-5-O-
phosphate-myo-inosito115
To a suspension of de-protected saccharide 14 (0.04 mmol) in a mixture of HPLC
grade methanol:distilled water [4:1] (1 ml) was added 10 % wt. PdlC (10 mg)
and the
mixture stirred under an atmosphere of hydrogen at R.T. for 72 hours. The
methanol
was evaporated in vacuo before the crude product was suspended in distilled
water
then filtered through celite. The filtrate was then evaporated to dryness in
vacuo to
yield a yellow oil. 'H-NMR analysis of this oil indicated benzyl groups
remained on
the pseudo-disaccharide. Consequently, the oil was re-suspended in a mixture
of
HPLC grade methanol:distilled water [4:1] (1 ml). 10 % wt. Pd/C (10 mg) was
added
and the mixture stirred under an atmosphere of hydrogen at R.T. for a further
20
hours. The methanol was evaporated in vacuo, the crude product suspended in
distilled water before being filtered through celite. The filtrate was then
evaporated
to dryness ivc vacuo to yield de-protected phosphate 15 as a dark yellow oil.
'H-NMR
analysis indicated that the product 15 was slightly contaminated with
impurities. The
product 15 was therefore re-dissolved in distilled water and washed with
EtOAc. The
aqueous layer was then concentrated to dryness in vacuo to give pure de-
protected
phosphate 15 (11 mg) as a light brown oil. 8H (D20: 360 MHz) 5.6 (1H, d, H1');
~P
(D20: 146 MHz) 2.4 (1P'~.
Synthesis of RGL 1116
Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-4',6'-O-benzylidene-2'-deoxy-a-D-
glucosopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4-O-
(1,1,3,3; tetraisopropyldisiloxanyl)-5-O-acetyl-myo-inositol 16
a-1,6 Anomer 11 (0.16 mmol) was dissolved in acetic anhydride (2 ml) before
pyridine (4 ml) was added. The reaction mixture was then stirred at R.T.
overnight.
TLC analysis (hexane:EtOAc [9:1]) of the reaction mixture suggested that only
c.a. 50
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54
of the a-1,6 anomer 11 starting material had been consumed. The reaction was
therefore heated to 45 °C for 8 hours at which point TLC analysis
(hexane:EtOAc
[9:1 ]) indicated complete consumption of the a-1,6 anomer 11 starting
material. The
reaction mixture was concentrated to dryness in vacuo, using toluene to
azeotropically
remove the pyridine. The crude product was then purified by column
chromatography (hexane:EtOAc [8:1]) to yield acetate I6 (89 %) as a pale
yellow oil.
8H (CDCl3: 360 MHz) 5.1 (2H, dd, PhCHzO), 5.3 (1H, dd, HS), 5.7 (1H, d, H1'),
5.8
(1H, s, PhCHO).
I0 Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-4',6'-O-benzyIidene-2'-deoxy-a-
D-
glucosopyranosyl)-1,2-O-(L-I,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-5-O-
acetyl-myo-inositol 18
To a solution of acetate 16 (0.14 mmol) in anhydrous THF (2.0 ml) at
0°C was added
TBAF (1.0 M solution in THF) (310 ml) and the reaction left to stir at R.T.
under an
inert atmosphere. After 1.5 hours TLC analysis (hexane:EtOAc [3:2]) indicated
that
the acetate 16 starting material had been consumed. The reaction mixture was
concentrated to dryness in vacuo before being purified by column
chromatography
(hexane:EtOAc [3:2]) to yield de-silylated acetate 18 as a pale yellow oil (42
%). 8H
(CDCl3: 360 MHz) 4.7 (1H, dd, HS), 4.8 (2H, dd, PhCH~O), 5.3 (1H, d, H1'), 5.5
(1H,
s, PhCHO).
Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-2'-deoxy-a-D-glucosopyranosyl)-5-
O-acetyl-myo-inositol 20
To a solution of de-silylated acetate 18 (0.06 mmol) in DCM (3.0 ml) was added
distilled water (64 ml) then TFA (273 ml) and the mixture stirred at R.T. for
4.5 hours
whilst being monitored by TLC analysis (EtOAc:MeOH [10:1]). The. reaction
mixture was concentrated to dryness ih vacuo before the crude product was
purified
by column chromatography (EtOAc (100 %) then EtOAc:MeOH [10:1]) to yield the
de-protected acetate 20 (55 %) as an orange oil. 8H (d4-MeOH: 360 MHz) 4.8
(3H, m,
HS and PhCH20), 5.4 (1H, d, H1').
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Synthesis of 1'-D-6-O-(2'-amino-2'-deoxy-a-D-glucosopyranosyl)-5-O-acetyl-
myo-inositol 22
To a suspension of de-protected acetate 20 (0.03 mmol) in a mixture of HPLC
grade
methanol:distilled water [10:1] (1 ml) was added 10 % wt. Pd/C (5 mg) and the
mixture stirred under an atmosphere of hydrogen at R.T. for 24 hours. The
methanol
was evaporated in vacuo before the crude product was suspended in distilled
water
then filtered through celite. The filtrate was then evaporated to dryness in
vacuo to
yield de-protected acetate 22 as a colourless oil (7 mg).
10 Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-4',6'-O-benzylidene-2'-deoxy-(3-
D-
glucosopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4-O-
(1,1,3,3,-tetraisopropyldisiloxanyl)-5-O-acetyl-myo-inositol 17
(3-1,6 Anomer 9 (0.12 mmol) was dissolved in acetic anhydride (2 ml) before
pyridine
(4 ml) was added. The reaction mixture was then stirred at R.T. overnight. TLC
15 analysis (hexane:EtOAc [9:1]) of the reaction mixture suggested that only
c.a. 50 % of
the [3-1,6 anomer 9 starting material had been consumed. The reaction was
therefore
heated to 45°C for 9 hours at which point TLC analysis (hexane:EtOAc
[9:1])
indicated that only a small quantity of the (3-1,6 anomer 9 starting material
remained
unreacted. The reaction mixture was concentrated to dryness i~ vacuo, using
toluene
20 azeotropically remove the pyridine. The crude product was then purified by
column
chromatography (hexane:EtOAc [8:1]) to yield acetate 17 (63 %) as a pale
yellow oil.
8H (CDCl3: 360 MHz) 4.8 (2H, dd, PhCH20), 5.0 (1H, dd, HS), 5.3 (1H, d, H1'),
5.5
(1H, s, PhCHO).
25 Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-4',6'-O-benzylidene-2'-deoxy-[3-
D-
glucosopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-5-O-
acetyl-myo-inositol 19
To a solution of acetate 17 (0.08 mmol) in anhydrous THF (2.0 ml) at
0°C was added
TBAF (1.0 M solution in THF) (165 ml) and the reaction left to stir at R.T.
under an
30 inert atmosphere. After 1.5 hours TLC analysis (hexane:EtOAc [3:2])
indicated that
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56
the acetate 17 starting material had been consumed. The reaction mixture was
concentrated to dryness in vacuo before being purified by column
chromatography
(hexane:EtOAc [3:2]) to yield de-silylated acetate 19 as a pale yellow oil (53
%). 8H
(CDCl3: 360 MHz) 4.8 (3H, m, PhCH20 and HS), 5.5 (1H, d, H1').
Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-2'-deoxy-(3-D-glucosopyranosyl)-5-
O-acetyl-myo-inositol 21
To a solution of de-silylated acetate 19 (0.04 mmol) in DCM (3.0 ml) was added
distilled water (46 ml) then TFA (200 ml) and the mixture stirred at R.T. for
4.5 hours
whilst being monitored by TLC analysis (EtOAc). The reaction mixture was
concentrated to dryness i~z vacuo before the crude product was purified by
column
chromatography (EtOAc (100 %) then EtOAc:MeOH [10:1]) to yield the de-
protected
acetate 21 (67 %) as an orange oil. 8,.~ (d4-MeOH: 360 MHz) 4.8 (3H, m, PhCH20
and HS), 5.4 (1H, d, H1').
Synthesis of 1'-D-6-O-(2'-amino-2'-deoxy-[3-D-glucosopyranosyl)-5-O-acetyl-
myo-inositol 23
To a suspension of de-protected acetate 21 (0.03 mmol) in a mixture of HPLC
grade
methanol:distilled water [10:1] (1 ml) was added 10 % wt. PdIC (6 mg) and the
mixture stirred under an atmosphere of hydrogen at R.T. for 48 hours. The
methanol
was evaporated i~z vacuo before the crude product was suspended in distilled
water
then filtered through celite. The filtrate was then evaporated to dryness in
vacuo to
yield de-protected acetate 23 as a colourless oil (7 mg).
Synthesis of RGL1117
Synthesis of 1'-D-5-O-(2'-azido-3'-O-benzyl-4',6'-O-benzylidene-2'-deoxy-a-D-
glucopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4-O-
(1,1,3,3, tetraisopropyldisiloxanyl)-6-O-acetyl-myo-inosito124
a-1,5 Anomer 10 (0.31 mmol) was dissolved in acetic anhydride (2 ml) before
pyridine (4 ml) was added. The reaction mixture was then heated at 45
°C overnight
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57
until TLC analysis (hexane:EtOAc [9:1]) indicated complete consumption of the
a-
1,5 anomer 10 starting material. The reaction mixture was concentrated to
dryness in
vacuo, using toluene to azeotropically remove the pyridine. The crude product
was
then purified by column chromatography (hexane:EtOAc [9:1]) to yield acetate
24 (97
%) as a pale yellow oil. 8H (d4-MeOH: 360 MHz) 4.9 (2H, dd, PhCHzO), 5.3 ( 1
H, dd,
HS), 5.8 (2H, m, H1' and PhCHO).
Synthesis of 1'-D-5-O-(2'-azido-3'-O-benzyl-4',6'-O-benzylidene-2'-deoxy-a-D-
glucopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-6-O-
acetyl-myo-inosito125
To a solution of acetate 24 (0.30 mmol) in anhydrous THF (4.2 ml) at 0
°C was added
TBAF (1.0 M solution in THF) (663 ~,l) and the reaction left to stir at R.T.
under an
inert atmosphere. After 1 hour TLC analysis (hexane:EtOAc [3:2]) indicated
that the
acetate 24 starting material had been consumed. The reaction mixture was
concentrated to dryness in vacuo before being purified by column
chromatography
(hexane:EtOAc [3:2]) to yield de-silylated acetate 25 as a pale yellow oil (78
%). 8H
(d4-MeOH: 360 MHz) 4.8 (2H, dd, PhCHzO), 5.1 (1H, dd, HS), 5.5 (1H, d, H1'),
5.6
(1H, s, PhCHO).
Synthesis of 1'-D-5-O-(2'-azido-3'-O-benzyl-2'-deoxy-a-D-glucopyranosyl)-6-O-
acetyl-myo-inositol 26
To a solution of de-silylated acetate 25 (0.24 mmol) in DCM (30.0 ml) was
added
distilled water (260 ~.1) then TFA (1.l ml) and the mixture stirred at R.T.
for 4 hours
whilst being monitored by TLC analysis (EtOAc). The reaction mixture was
concentrated to dryness in vacuo before the crude product was purified by
column
chromatography (EtOAc (100 %) then EtOAc:MeOH [10:1]) to yield~~the de-
protected
acetate 26 (35 %) as a pale yellow oil. 8H (d4-MeOH: 360 MHz) 4.8 (2H, dd,
PhCHzO), 5.2 (1H, dd, HS), 5.4 (1H, d, H1').
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Synthesis of 1'-D-5-O-(2'-amino-2'-deoxy-oc-D-glucopyranosyl)-6-O-acetyl-myo-
inositol 27 (RGL1117)
To a suspension of de-protected acetate 26 (0.03 mmol) in a mixture of HPLC
grade
methanol:distilled water:AcOH [10:1:0.1] (1 ml) was added 10 % wt. Pd/C (5 mg)
and the mixture stirred under an atmosphere of hydrogen at R.T. for 16 hours.
The
methanol was evaporated in vacuo before the crude product was suspended in
distilled
water then filtered through celite. The filtrate was then evaporated to
dryness in
vacuo to yield de-protected acetate 27 as a colourless oil (91 %). 8H (D20:
360 MHz)
4.9(lH,dd,HS),5.4(lH,d,Hl').
~nthesis of RGL1124
Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-4',6'-O-benzylidene-2'-deoxy-(3-D-
glucopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4-O-
(1,1,3,3; tetraisopropyldisiloxanyl)-5-O-acetyl-myo-inositol 17
(3-1,6 Anomer 9 (0.12 mmol) was dissolved in acetic anhydride (2 ml) before
pyridine
(4 ml) was added. The reaction mixture was then stirred at R.T. overnight. TLC
analysis (hexane:EtOAc [9:1]) of the reaction mixture suggested that only c.a.
50 % of
the (3-1,6 anomer 9 starting material had been consumed. The reaction was
therefore
heated to 45 °C for 9 hours at which point TLC analysis (hexane:EtOAc
[9:1])
indicated that only a small quantity of the (3-1,6 anomer 9 starting material
remained
unreacted. The reaction mixture was concentrated to dryness in vacuo, using
toluene
to azeotropically remove the pyridine. The crude product was then purified by
column chromatography (hexane:EtOAc [8:1]) to yield acetate 17 (63 %) as a
pale
yellow oil. 8H (CDC13: 360 MHz) 4.8 (2H, dd, PhCH20), 5.0 (1H, dd, H5), 5.3
(1H, d, H1'), 5.5 (1H, s, PhCHO).
Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-4',6'-O-benzylidene-2'-deoxy-(3-D-
glucopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-5-O-
acetyl-myo-inositol 19
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59
To a solution of acetate 17 (0.08 mmol) in anhydrous THF (2.0 ml) at 0
°C was added
TBAF (1.0 M solution in THF) (165 p,I) and the reaction left to stir at R.T.
under an
inert atmosphere. After 1.5 hours TLC analysis (hexane:EtOAc [3:2]) indicated
that
the acetate 17 starting material had been consumed. The reaction mixture was
concentrated to dryness in vacuo before being purified by column
chromatography
(hexane:EtOAc [3:2]) to yield de-silylated acetate 19 as a pale yellow oil (53
%). 8H
(CDC13: 360 MHz) 4.8 (3H, m, PhCHzO and HS), 5.5 (1H, d, H1').
Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-2'-deoxy-[3-D-glucopyranosyl)-5-O-
acetyl-myo-inosito121
To a solution of de-silylated acetate 19 (0.04 mmol) in DCM (3.0 ml) was added
distilled water (46 ~1) then TFA (200 p,1) and the mixture stirred at R.T. for
4.5 hours
whilst being monitored by TLC analysis (EtOAc). The reaction mixture was
concentrated to dryness in vacuo before the crude product was purified by
column
chromatography (EtOAc (100 %) then EtOAc:MeOH [10:1]) to yield the de-
protected
acetate 21 (67 %) as an orange oil. 8H (d4-MeOH: 360 MHz) 4.8 (3H, m, PhCH20
and HS), 5.4 (1H, d, H1').
Synthesis of 1'-D-6-O-(2'-ami-no-2'-deoxy-(3-D-glucopyranosyl)-5-O-acetyl-myo-
inositol 23 (RGL 1124)
To a suspension of de-protected acetate 21 (0.03 mmol) in a mixture of HPLC
grade
methanol:distilled water [10:1] (1 ml) was added 10 % wt. Pd/C (6 mg) and the
mixture stirred under an atmosphere of hydrogen at R.T. for 48 hours.
The methanol was evaporated in vacuo before the crude product was suspended in
distilled water then filtered through celite. The filtrate was then evaporated
to
dryness iu vacuo to yield de-protected acetate 23 as a colourless oil (7~mg).
Synthesis of RGL1125
Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-4',6'-O-benzylidene-2'-deoxy-a-D-
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WO 01/85740 PCT/GBO1/02088
glucopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4-O-
(1,1,3,3; tetraisopropyldisiloxanyl)-5-O-butyryl-myo-inositol 28
a-1,6 Anomer 1I (0.22 mmol) was dissolved in pyridine (1.5 ml) before butyric
anhydride (71 p,1) was added. The reaction mixture was then heated at 45
°C
5 overnight at which point TLC analysis (hexane:EtOAc [10:1]) indicated only
slight
consumption of the a-1,6 anomer 11 starting material. Butyric anhydride (284
p,1)
was subsequently added and the reaction mixture stirred at 55 °C for 18
hours. TLC
analysis (hexane:EtOAc [10:1]) indicated that approximately 30 % of the a-1,6
anomer 11 starting material had been consumed at this stage. A further amount
of
10 butyric anhydride was then added (to take the concentration to 15 molar
equivalents)
and the mixture heated at 80 °C overnight. TLC analysis (hexane:EtOAc
[10:1])
indicated that approximately 50 % of the a-1,6 anomer 11 starting material
remained.
Therefore, the reaction mixture was heated to 110 °C for 6 hours until
it was
determined by TLC analysis (hexane:EtOAc [10:1]) that approximately 30 % of
the a-
15 1,6 anomer 11 starting material remained. Butyric anhydride (5 equivalents)
and
pyridine (1.5 ml) were added and the reaction mixture heated at 110 °C
overnight.
TLC analysis (hexane:EtOAc [10:1]) then indicated that the a-1,6 anomer 11
starting
material had been consumed. The reaction mixture was concentrated to dryness
in
vacuo, using toluene to azeotropically remove the pyridine. The crude product
was
20 then purified by column chromatography (hexane:EtOAc [9:1]) to yield
butyrate 28 as
a yellow oil. The product 28 was observed by'H-NMR to be contaminated with
butyric anhydride. Further purification via column chromatography
(hexane:EtOAc
[9:1]) was attempted but the 213 mg of isolated butyrate 28 still retained a
slight
contamination of butyric anhydride. 8H (CDCl3: 360 MHz) 4.9 (PhCH20), 5.1
(HS),
25 5.7 (H1'), 5.8 (PhCHO).
Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-4',6'-O-benzylidene-2'-deoxy-a-D-
glucopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-5-O-
butyryl-myo-inositol 29
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61
To a solution of butyrate 28 (0.22 mmol) in anhydrous THF (3.0 ml) at 0
°C was
added TBAF (1.0 M solution in THF) (480 p,1) and the reaction left to stir at
R.T.
under an inert atmosphere. After 1 hour TLC analysis (hexane:EtOAc [3:2])
indicated that the butyrate 28 starting material had been consumed. The
reaction
mixture was concentrated to dryness in vacuo before being purified by column
chromatography (hexane:EtOAc [3:2]) to yield de-silylated butyrate 29 as a
pale
yellow oil (160 mg) [slight butyric anhydride impurity present in product 29].
SH
(CDCl3: 360 MHz) 4.7 (2H, m, PhCH20), 5.0 (1H, m, HS), 5.4 (1H, d, Hl'), 5.5
(1H,
s, PhCHO).
Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-2'-deoxy-a-D-glucopyranosyl)-5-O-
butyryl-myo-inositol 30
To a solution of de-silylated butyrate 29 (0.22 mmol) in DCM (15.0 ml) was
added
distilled water (240 p1) then TFA (1.0 ml) and the mixture stirred at R.T.
After 20
hours TLC analysis (EtOAc) indicated consumption of butyrate 29 starting
material.
The reaction mixture was then concentrated to dryness in vacuo before the
crude
product was purified by column chromatography (EtOAc (100 %) then EtOAc:MeOH
[10:1]) to yield the de-protected butyrate 30 (20 mg).
Synthesis of 1'-D-6-O-(2'-amino-2'-deoxy-oc-D-glucopyranosyl)-5-O-butyryl-
myo-inositol 31 (RGL 1125)
To a suspension of de-protected butyrate 30 (0.04 mmol) in a mixture of HPLC
grade
methanol:distilled water:AcOH [10:1:0.1] (1.5 ml) was added 10 % wt. Pd/C (8
mg)
and the mixture stirred under an atmosphere of hydrogen at R.T. for 60 hours.
The
methanol was evaporated in vacuo before the crude product was suspended in
distilled
water then filtered through celite. The filtrate was then evaporated to
dryness i~
vacuo to yield de-protected butyrate 31 as a colourless oil (14 mg).
Synthesis of RGL 1126
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62
Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-4',6'-O-benzylidene-2'-deoxy-a-D-
glucopyranosyl)-1,2-O-(L-1,7,7-trimethyl(2.2.1]-bicyclohept-2-ylidene)-3,4-O-
(1,1,3,3; tetraisopropyldisiloxanyl)-5-O-palmityl-myo-inositol 32
a-1,6 Anomer 11 (0.14 mmol) was dissolved in pyridine (2.5 ml) before palmitic
anhydride (2.1 mmol) was added. The reaction mixture was then heated to reflux
for
80 hours until TLC analysis (hexane:EtOAc [10:1]) indicated that the a-1,6
anomer
11 starting material had been consumed. The reaction mixture was concentrated
to
dryness in vacuo, using toluene to azeotropically remove the pyridine. The
crude
product was then purified by column chromatography (hexane:EtOAc [9:1]) [using
a
wide column to ensure separation of the large excess of palmitic anhydride] to
yield
palmitate 32 as a pale yellow oil (87 %). 8H (CDCl3: 360 MHz) 4.7 (2H, m,
PhCH20),
4.9 (1H, m, HS), 5.4 (1H, d, H1'), 5.5 (1H, s, PhCHO).
Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-4',6'-O-benzylidene-2'-deoxy-a-D-
glucopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-5-O-
palmityl-myo-inositol 33
To a solution of palmitate 32 (0.12 mmol) in anhydrous THF (1.7 ml) at 0
°C was
added TBAF (1.0 M solution in THF) (280 p,1) and the reaction left to stir at
R.T.
under an inert atmosphere. After 1 hour TLC analysis (hexane:EtOAc [3:2])
indicated that the palmitate 32 starting material had been consumed. The
reaction
mixture was concentrated to dryness in vacuo before being purified by column
chromatography (hexane:EtOAc [3:2]) to yield de-silylated palmitate 33 as a
pale
yellow oil (92 %). 8H (CDC13: 360 MHz) 4.7 (2H, m, PhCH2O), 5.2 (1H, d, H1'),
5.4
(1H, s, PhCHO).
Synthesis of 1'-D-6-O-(2'-azido-3'-O-benzyl-2'-deoxy-a-D-glucopyranosyl)-5-O-
palmityl-myo-inositol 34
To a solution of de-silylated palmitate 33 (0.11 mmol) in DCM (9.0 ml) was
added
distilled water (1 SO p,1) then TFA (630 p1) and the mixture stirred at R.T.
After 18
hours TLC analysis (EtOAc) indicated consumption of palmitate 33 starting
material.
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The reaction mixture was then concentrated to dryness i~ vacuo before the
crude
product was purified by column chromatography (EtOAc (I00 %) then EtOAc:MeOH
[10:1]) to yield the de-protected palmitate 34 (25 %).
Synthesis of 1'-D-6-O-(2'-amino-2'-deoxy-cc-D-glucopyranosyl)-5-O-palmityl-
myo-inositol 35 (RGL 1126)
To a suspension of de-protected palmitate 34 (0.25 mmol) in a mixture of HPLC
grade methanol:distilled water:AcOH [10:1:0.1] (1.5 mI) was added 10 % wt.
Pd/C (6
mg) and the mixture stirred under an atmosphere of hydrogen at R.T. for 60
hours.
The methanol was evaporated in vacuo before the crude product was suspended in
distilled water and filtered through celite. The filtrate was then evaporated
to dryness
in vacuo to yield a colourless oil (7 mg).
Synthesis of RGL1119
2-Deoxy-3,4,6-tri-O-acetyl-2-trichloroacetamide-D-galactopyranosyl-(3-(1~6)-
1,2-
diisopropylidene-3,4-(1',2'-dimethoxycyclohexane-1',2'-diyl)-3-O-methyl-D-
chiro-inositol (3)
A 10 mL round-bottomed flash was charged with donor 1 (81 mg, 0.14 mmol, 1.6
eq.), acceptor 2 (32 mg, 0.08 mmol, 1.0 eq), freshly 4 ~ molecular sieves and
CHZCIz
(2 mL) and the mixture was stirred for 1 h under Argon. Then TfOTMS was added
(3.1 mL, 0.02 mmol, 0.2 eq) at 0°C and the reaction mixture was stirred
for 5 h. The
suspension was filtered through a short pad of celite and the solvent removed
to
provide the crude material. Flash chromatography (Hex/EtOAc 2:1) afforded 9b
(42
mg, 0.054 mmol, 63%). Rf (Hex/EtOAc 1:3): 0.52; [a]°2°-27.5 (c
=1.19, CHC13);'H
NMR (CDCI3, 500 MHz) : b = 6.59 (d, 1H, J~_~,= 9.0 Hz, NH), 5.35 (d, 1H, J =
2.8
Hz, H4,), 5.28 (d, 1H, J = 8.0 Hz, Hl,), 5.16 ( dd, 1H, JH3'-H2' I 1.0
Hz,"JH3,-H4,=2.8 Hz,
H3,), 4.46 (s, 1H, H6), 4.26 (m, 1H, H~),4.20-4.10 (m, 3H, H2,, Hba,, H66,),
4.07 ( t, 1H,
J = 6.0 Hz, Ha), 4.02 (t, 1H, J =10.0 Hz, H4), 3.95 (m, 1H, HS), 3.85 (m, 1H,
H5,), 3.53
(s, 3H, OCH3), 3.33 ( t, 1H, J = I0.0 Hz, H3), 3.22 (s, 3H, OCH3, CDA), 3.20
(s, 3H,
OCH3, CDA), 2.16 (s, 3H, CH3C0), 2.02 (s, 3H, CH3C0), 1.98 (s, 3H, CH3C0),
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1.72-1.60 (m, 4H, 2CH2), 1.52-1.48 (m, 2H, CHZ), 1.51 (s, 1H, CH3), 1.36-1.31
(m,
2H, CH2), 1.34 (s, 3H, CH3); '3C NMR (CDC13, 125 MHz) : 8 = 170.7 (CH3C0),
170.3 (CH3C0), 170.2 (CH3C0), 162.2 (CC13C0),109.6 (CC13C0), 99.4 (C,,), 97.9
(C, CDA), 97.8 (C, CDA), 92.2 (C), 83.5 (C3), 79.6 (CZ), 76.0 (C,), 72.2 (C6),
71.2
(CS>), 70.1 (C3,), 68.2 (CS), 67.7(C4), 66.8 (C~,), 61.4 (C6,), 59.8 (OCH3),
53.1 (C2,),
47.3 (OCH3, CDA), 46.9 (OCH~, CDA), 28.5 (2CH2), 27.2 (CH3), 27.0 (CH3), 26.2
(2CH2), 21.4 (CH3C0), 20.7 ( CH3C0), 20.6 (CH3C0).
2-Deoxy-2-trichloroacetamido-3,4,6-tri-O-acetyl-n-galactopyranosyl-~3-(1~6)-3-
O-
methyl-D-chino-inositol(4)
Pseudodisaccharide 3 (36 mg, 0.05 mmol, 1.0 eq) was dissolved in a mixture of
trifluoroacetic acid/water (15:1, 3.2 mL) and stirred for 3 h at room
temperature. The
solvent was removed under vacuum and the crude was purified by flash
chromatography to provide 4 (22 mg, 0.03 mmol, 75%) as a white solid. Rf
(C12CHZlMeOH 7:1): 0.15; [a]p2°-1.7 (c = 0.72, MeOH); 'H NMR (MeOD, 500
MHz)
8 = 5.36 (d, 1 H, JH4'-HS'- 3.5 Hz, H4,), 5.24 ( dd, 1 H, JH3,_~,=11.1 Hz,
JH3,_Ha,= 3.5 Hz
H4,), 5.01 (d, 1H, J = 8.5 Hz, Hl>), 4.16 (m, 2H, H6a,>H66'), 4.08 (dd, 1H,
J~>_H3~ 11.2
Hz, Jm,_Hl>= 8.5 Hz, H2,), 4.05-4.02 (m, 2H, HS,, H6), 3.98 (t, 1H, J= 3.0 Hz,
H~), 3.79
(dd, 1H, JHS_H4 9~5 Hz, JHS-H6 3.0 Hz, HS), 3.70 (dd, 1H, J~_H3= 9.7 Hz,
J~_Hl= 3.0
Hz, H~)> 3.58 (m, 1H, H4), 3.56 (s, 3H. OCH3), 3.22 (t, 1H, J = 9.7 Hz, H3),
2.16 (s,
3H, CH3CO), 2.05 (s, 3H, CH3C0), 1.93 (s, 3H, CH3C0); '3C NMR (MeOD, 125
MHz) : 8 = 170.8 (CH3C0), 170.6 (CH3C0), 170.1 (CH3C0), 163.2 (C13CC0), 101.3
(C,>), 92.5 (C13CCO), 83.3 (C3), 79.1 (C6), 72.4 (C4), 71.4 (C,), 71.0 (CS),
70.6 (CS>),
70.4 (C2), 70.3 (C3,), 66.9 (C4>), 61.3 (C6>), 58.9 (OCH3), 52.5 (CZ>), 19.2,
19.1, 19.0
(3CH3C0); anal. calcd. for CZ~H3oNO,4Cl3: C, 40.24%; H, 4.82%; N, 2.23%;
found:
38.89%; H, 5.17%; N, 1.94%.
2-amino-2-deoxy-D-galactopyranosyl-[3-(1-~6)-3-O-methyl-D-claim-inositol (5,
RGL 1119)
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Compound 4 (6 mg, 10 mmol) and Ba(OH)2 (8 mg, 0.094 mmol, 4.8 eq.) was stirred
in water/EtOH 1:1 (4 mL) for 1 h at 90°C, whereupon the solvent was
removed under
vacuum. Cold water was added to the residue and filtered through a paper
filter to
eliminate Ba(OH)2.. The filtrate was removed under vacuum and the residue was
5 redissolved in the minimum amount of MeOH and loaded onto a Varian CBA
(carboxylic acid) cartridge (3 cm3/500 mg) preequilibrated with MeOH. After
elution
with some lengths of MeOH to remove barium salts, pseudodisaccharide was
neutralized loading it directly onto a Varian PSA (ethylenediamine-N-propyl)
cartridge preequilibrated with MeOH to give RGL 1119 (3 mg, 8 mmol, 79%). Rf
10 (EtOAc/MeOH/H20/AcOH 2:2:1:1): 0.29; [a]DZ~ +8.1 (c = 0.28, H20);'H NMR
(D20, 500 MHz): 8 = 4.32 (d, 1H, J = 9.0 Hz, H,,), 4.15 (bt, 1H, J = 3.5 Hz,
H6), 3.90
(bt, 1H, J = 3.5 Hz, H~), 3.75-3.71 (m, 3H, H5, Ha, H4,), 3.68-3.53 (m, 4H,
H6a,, H6b'
HS,, H4), 3.47 (s,3H, OCH3), 3.42 (dd, 1H, J~,_HZ,= 10.0 Hz, JH3'-H4'- 3.5 Hz,
H3,), 3.21
(t, 1 H, J = 9.5 Hz, H3), 2.74 (t, 1 H, J = 9.0 Hz Hz,); i3C NMR (CDC13, 125
MHz) : 8 =
15 100.8 (C,,), 83.5 (C3), 82.2 (C,), 75.9 (C4), 73.9 (C3,), 73.1 (C5,), 71.3
(C6), 70.8 (C2),
70.0 (CS), 68.2 (C4,), 62.4 (C6,), 53.7 (CZ,). CI HRMS calcd. for
C,3HZSN0~°(M~+H):
356.1556, found: 356.1548.
Synthesis of RGL 1134
20 1'-D-6-O-(2'-azido-3'-O-benzyl-2'-deoxy-oc-D-glucopyranosyl)-3,4,5-tri-O-
benzyl-
myo-inositol (2)
To a mixture of the disaccharide 1 (0.72 g, 0.75 mmol) in DCM (45 ml) was
added
water (0.82 ml, 0.82 g, 45.6 mmol) and trifluoroacetic acid (5.18 g, 3.5 mmol,
45.4
mmol) at room temperature for 1.25 h. The mixture was diluted with DCM (100
ml)
25 and washed with NaHC03 (3 x 50 ml) and brine (1 x 50 ml). The solvent was
dried
(MgS04), filtered and concentrated in vacuo to yield a crude oil (0.84 g). The
tetrol 2
(0.37 g, 67%) was isolated after flash chromatography (silica, 60~80~100%
ethyl
acetate/hexanes).
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1'-D-6-O-(2'-azido-3'-O-benzyl-6'-O-DMT-2'-deoxy-a-D-glucopyranosyl)-3,4,5-
tri-O-benzyl-myo-inositol (3)
The tetrol 2 (110 mg, 0.15 mmol) was co-evaporated with pyridine (2 x 1 ml)
and
dissolved in pyridine (2 mI). To the solution was added DMTCI (78 mg, I.5 eq)
and
the reaction stirred for 16 h. The solvent was removed in vacuo and the
residue
dissolved in DCM (30 ml). The solution was washed with NaHC03 (sat, 2 x 20
ml),
dried (MgS04), filtered and concentrated in vacuo. The residual pyridine was
removed by co-evaporation with toluene. The product (100 mg, 97 pmol) was
isolated by flash chromatography (silica, 10%~50% ethyl acetatelhexanes).
1'-D-6-O-(2'-azido-3'-O-benzyl-4'-O-phosphate-6'-O-DMT-2'-deoxy-a-D-
glucopyranosyl)-3,4,5-tri-O-benzyl-myo-inositol-1,2-cyclic phosphate, sodium
salt
(4)
Methyldichlorophosphate (30 p,1, 0.29 mmol, 3 eq) was added to pyridine (630
p,1)
with stirring under an atmosphere of nitrogen at room temperature. After 30
min the
triol 3 (I00 mg, 0.10 mmol) in pyridine (100 p.1) was added and the reaction
stirred for
a further 4 h. The reaction was diluted with DCM (I0 ml) and quenched by
addition
of NaHC03 solution. The resulting emulsion was separated by the addition of
brine
and the aqueous phase extracted with DCM (1 x 10 ml). The combined organic
solvents were dried (Na2S0ø), filtered and concentrated in vacuo to yield the
xequired
product 4 (17 mg, 13.8 p,mol, I4%, RfØ04 20% ethyl acetate/hexanes).
1'-D-6-O-(2'-azido-3'-O-benzyl-4'-O-phosphate-2'-deoxy-a-D-glucopyranosyl)-
3,4,5-tri-O-benzyl-myo-inositol-1,2-cyclic phosphate, sodium salt (5)
To a stirred solution of compound 4 (17 mg, 13.4 ~mol) in DCM (2 ml) was added
a
solution of TFA in DCM (2 %, 2 ml) at 0°C. TLC indicated the reaction
proceeded to
completion in ca. 1 min. The xeaction was quenched by addition of NaHC03 (sat.
5
ml), diluted with DCM (10 ml). The phases were separated and the organic phase
dried (NaZS04), filtered and concentrated i~ vacuo. The residue was purified
by
column chromatography (silica, 1:l ethyl acetate/hexanes~ethyl acetate) to
yield the
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product as a colourless solid (10 mg, 10.8 p,mol, 81%). 'H-NMR (250 MHz), 8
(ppm): 7.25 (m, 20 H); 5.3 (d, 1 H, H-1'); 5.0 (d, 1 H); 4.7 (m); 7.6 (bs, 1
H); 3.9 (m,
2 H); 3.64 (m); 3.3 (m); 2.62 (s, 1 H); 2.22 (d, 1 H).
1'-D-6-O-(2'-amino-3'-O-benzyl-4'-O-phosphate-2'-deoxy-a-D-glucopyranosyl)-
3,4,5-tri-O-benzyl-myo-inositol-1,2-cyclic phosphate, sodium salt (6, RGL
1134)
Pd/C (10%, 5 mg) was added to a solution of compound 5 (10 mg, 10.8 qmol) in
methanol/THF/H20 (1:1:1, 3 ml) and stirred at room temperature overnight under
an
atmosphere of hydrogen. The mixture was filtered through celite and the celite
washed with methanol (2 x 5 ml) and water (2 x 5 ml). The solution was
concentrated
in vacuo to yield a colourless solid. Residual celite was removed by
filtration through
cotton wool and the crude product RGL 1134 (12 mg) was isolated after
evaporation
in vacuo. 'H-NMR (250 MHz), 8(ppm): 7.4 (m, 20 H); 5.38 (d, 1 H, H-1'); 5.0
(d, 1
H); 4.8 (m); 4.18 (bs, 1 H); 3.9 (m, 2 H); 3.8 (bd, 1 H); 3.6 (m, 2 H); 3.22
(m, 4 H);
2.65 (m, 1 H).
Synthesis of RGL1135
1'-D-6-O-(2'-azido-3'-O-benzyl-4',6'-di-O-cyclic phosphate-2'-deoxy-a-D-
glucopyranosyl)-3,4,5-tri-O-benzyl-myo-inositol-1,2-cyclic phosphate,
triethylammonium salt (2)
Methyldichlorophosphate (30 ~,1, 0.29 mmol, 3 eq) was added to pyridine (630
p1)
with stirring under an atmosphere of nitrogen at room temperature. After 30
min
compound 1 (100 mg, 0.14 mmol) in pyridine (100 p,1) was added and the
reaction
stirred for a further 4 h. The reaction was diluted with DCM (10 ml) and
quenched by
addition of NaHC03 solution. The resulting emulsion was separated by the
addition of
brine and the aqueous phase extracted with DCM (1 x 10 ml). The combined
organic
solvents were dried (Na2S04), filtered and concentrated in vacuo. The crude
material
was purified by preparative TLC (methanol/DCM/Et3N 10:90:1 ) to yield compound
2
(11 mg, 7.5%).
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1'-D-6-O-(2'-amino-3'-O-benzyl-4',6'-di-O-cyclic phosphate-2'-deoxy-oc-D-
glucopyranosyl)-3,4,5-tri-O-benzyl-myo-inositol-1,2-cyclic phosphate,
triethylammonium salt (3, RGL 1135)
Compound 2 (11 mg, 10.4 ~,mol) in THF/ethanol/water (1:1:l, 3 ml) was stirred
at
room temperature for 22 h under an atmosphere of hydrogen. The mixture was
filtered
through celite and washed with methanol. The solvent was removed in vacuo to
yield
RGL 1135 (8 mg, 71 %). 'H-NMR (250 MHz), ~ (ppm): 7.25 (m, 20 H); 5.6 (d, 1 H,
H-1'); 5.0 (d, 1 H); 4.8 (m); 4.4 (m); 4.2 (m); 4.0 (m); 3.7 (m); 3.45 (m, 1
H); 3.25
(dd, 1 H); 3.1 (c); 2.1 (s, 1 H); 1.3 (t).
Synthesis of RGL 1133
1'-D-6-O-(2'-azido-3'-O-benzyl-4',6'-di-O-sulphate-2'-deoxy-oc-D-
glucopyranosyl)-3,4,5-tri-O-benzyl-myo-inositol-1,2-di-O-sulphate, sodium salt
(2).
A mixture of the tetrol 1 (88 mg, 0.12 mmol), sulfur trioxide-trimethylamine
complex
(0.32 g, 2.30 mmol, 20 eq) in DMF was heated for 16 h at 50°C. The DMF
was
removed in vacuo and the residue dissolved in MeOHlwater (9:1, 5 ml). Portions
(1
ml) were taken for prep. TLC (R f 0.2, 4:1 EtOH/0.88 ammonia solution). Each 1
ml
portion yielded ca. 15 mg (11 % per pTLC). The ammonium salts were dissolved
in
MeOH/water (9:1) and passed through Dowex (Na+) to give a quantitative return
of
the sodium salt after concentration i~ vacuo, re-dissolution in water and
lyophilisation.'H-NMR (250 MHz), 8(ppm): 7.35 (m 20 H); 5.85 (d, 1 H), 5.55
(s, 1
H); 5.15 (d, 1 H), 4.8 (m), 4.25 (m); 3.95 (c, 2 H); 3.48 (m, 3 H); 3.25 (m);
2.95. IR
(crri'): 3460.44; 2111.27; 1633.18; 1496.32; 1453.97; 1213.66; 1123.57;
1047.01;
1016.31; 922.85; 802.42; 747.37; 696.33; 582.86
1'-D-6-O-(2'-amino-3'-O-benzyl-4',6'-di-O-sulphate-2'-deoxy-a-D-
glucopyranosyl)-3,4,5-tri-O-benzyl-myo-inositol-1,2-di-O-sulphate, sodium salt
(3)
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A mixture of the tetrasulfate 2 (17 mg, 15 pmol), Pd/C (10%, 13 mg), ammonium
acetate (5 mg) in THF/ethanol/water (1:1:1, 3 ml) was stirred at room
temperature for
22 h. The mixture was filtered through celite and the celite washed with
methanol.
The solvent was removed iu vacuo and the residue dissolved in water. Compound
3,
RGL 1133 (14 mg, 84 %) was isolated after freeze drying.'H-NMR (250 MHz), ~
(ppm): 7.4, (m, 20 H); 5.5, (s, 1 H); 5.18 (s, 1 H); 4.8 (m, 8 H), 4.1 (m, 6
H); 3.6 (m, 7
H). IR (crri'): 2924.62; 1573.89; 1496.74; 1453.79; 1214.25; 1130.12; 1048.06;
996.84; 925.29; 802.77; 748.57; 696.19; 579.54.
Synthesis of RGL 1130
1'-D-6-O-(2'-azido-3',6'-di-O-benzyl-2'-deoxy-a-D-glucopyranosyl)-1,2-O-(L-
1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4,5-tri-O-benzyl-myo-inositol
and
1'-D-6-O-(2'-azido-3',4'-di-O-benzyl-2'-deoxy-a-D-glucopyranosyl)-1,2-O-(L-
1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4,5-tri-O-benzyl-myo-inositol
(2)
Compound 1 (150 mg, 0.16 mmol) was dissolved in dry THF (3.75 ml) and sodium
cyanoborohydride (2.07 ml, 1 M in THF) was added. The mixture was stirred at
room
temperature for 15 min and hydrogen chloride (ca. 1.8 ml, 1 M in diethyl
ether) added
until gas evolution ceased. The turbid mixture was stirred for a further 1.5
h. The
mixture was diluted with DCM (10 ml) and washed with NaHC03 (sat, 2 x 25 ml).
The aqueous phase was extracted with DCM (2 x 20 ml). The combined organic
extracts were dried (MgS04), filtered and concentrated i~ vacuo. The resulting
yellow
material was purified by column chromatography (silica, 15%~20% ethyl
acetatelhexanes) yielding the desired product (110 mg, 73%, 1:1 mixture of 4'
and 6'
ethers).
1'-D-6-O-(2'-azido-3',6'-di-O-benzyl-4'-O-(dibenzyl)phosphate-2'.~deoxy-a-D-
glucopyranosyl)-1,2-O-(L-1,7,7-trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4,5-
tri-
O-benzyl-myo-inositol and 1'-D-6-O-(2'-azido-3',4'-di-O-benzyl-6'-O-
(dibenzyl)phosphate 2'-deoxy-a-D-glucopyranosyl)-1,2-O-(L-1,7,7-
trimethyl[2.2.1]-bicyclohept-2-ylidene)-3,4,5-tri-O-benzyl-myo-inositol (3)
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To a solution of the disaccharides 2 (100 mg, 0.1 mmol) and tetrazole (38 mg,
0.55 mol) in dry DCM (10 ml) at 0°C was added dibenzyl
diisopropylphosphramidite
(140p,1). After 2.5 h, the mixture was cooled to -40 °C and mCPBA (60
mg) in DCM
(4 ml) was added. The reaction mixture was warmed to room temperature and
stirred
5 for 2.5 h. The mixture was diluted with DCM (20 ml), washed with sodium
metabisulfite (2 x 10 mI), NaHC03 (sat. 2 x 10 mI) and brine (2 x 10 ml). The
solution was dried (MgS04) and concentrated in vacuo. The mixture of products
(100
mg, 78%) was isolated after column chromatography (silica, 20% ethyl
acetate/hexanes).
1'-D-6-O-(2'-azido-3',6'-di-O-benzyl-4'-O-(dibenzyl)phosphate-2'-deoacy-a-D-
glucopyranosyl)-3,4,5-tri-O-benzyl-myo-inositol and 1'-D-6-O-(2'-azido-3',4'-
di-
O-benzyl-6'-O-(dibenzyl)phosphate 2'-deoxy-a-D-glucopyranosyl)-3,4,5-tri-O-
benzyl-myo-inositol (4)
To a mixture of Compounds 3 (110 mg, 89.6 ~mol) in DCM (45 ml) was added water
(approx. 1 ml) and TFA (3.5 ml) at room temperature and kept stirring for 2 h.
The
mixture was diluted with DCM (100 ml) and washed with NaHC03 (3 x 50 ml) and
brine (50 ml). The solvent was dried (MgSOø), filtered and concentrated to
yield
compounds 4 (80 mg, 82%), which were isolated after column chromatography
(silica, 50%~60% ethyl acetate/hexanes).
I'-D-6-O-(2'-amino-4'-O-phosphate-2'-deoxy-a-D-glucopyranosyl)-myo-inositol-
1,2-cyclic phosphate and 1'-D-6-O-(2'-amino-6'-O-phosphate-2'-deoxy-a-D-
glucopyranosyl)-myo-inositol-1,2-cyclic phosphate (5, RGL 1130)
Dichloromethylphosphate (40 ~,1) was added to pyridine (700 ~l) and stirred
for 30
min. Compounds 4 (80 mg, 73.1 pmol) were dissolved in a minimum of pyridine
and
added to the dichloromethylphosphate solution and stirred for 2.5 h. NaHC03
(sat. 2
ml) was added and the solvent removed in vacuo. The residue was taken up in
water
(10 ml) and acidified (pH 1) with dilute HCI. The aqueous solution was
extracted
with ethyl acetate (2 x 50 ml), dried (MgS04) and concentrated under reduced
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pressure. The oil was dissolved in THF/EtOH/water (1:1:1, 15 ml) and the
resulting
solution was degassed and flushed with nitrogen. Pd/C (10 %, 150 mg) and
ammonium acetate (40 mg) were added and the reaction placed under an
atmosphere
of hydrogen. The mixture was stirred for 24 h and the reaction mixture
filtered
through celite and the celite was washed with water and ethanol. The solvent
was
removed in vacuo and the residue co-evaporated with ethanol (2 x) and toluene
(3 x)
to give compound 5, RGL 1130 as a crude solid (30 mg). 'H-NMR (500 MHz), 8
(ppm): 5.38 (bs); 4.7 (m); 4.5 (dt); 4.17-3.17 (m); 2.7 (s); 2.4-2.28 (m);
2.23-2.03 (m);
1.13-1.19 (m).
Assay Data
PDH activation: 100wM
RGL 1023 14%
RGL1027 132%
RGL1029 361%
RGL1015 38%
RGL1024 65%
RGL1025 17%
PKA inhibition:
0.1~.M 1.O~.M lOwM
RGL1027 18% 10%
RGL1029 59% 33%
RGL1018 19% -
RGL1019 32% 31%
RGL1015 14% 15% 12%
RGL 1024 17% 34% 2%
RGL1025 48% -13%
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Figure 4 shows the result of an assay measuring the effect of compound RGL1133
in
inhibiting glucose 6-phosphatase, as compared to an known inhibitor, sodium O-
vanadate. The results show that RGL1133 is a good inhibitor of G6Pase activity
and
would be useful for decreasing hepatic glucose output in diabetic subjects.
This
suggests that this compound would be useful in the treatment of both type I
and type II
diabetes.
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73
References:
The references mentioned herein are all expressly incorporated by reference.
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1997; d)
Jones & Varela-Nieto, Int. J. Biochem. Cell Biol., 30:313-326, 1998.
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[12] Once et al, Chem. Eur. J., 3:431-440, 1997.
[13] Rademacher et al, Brazilian J. Med. Biol. Res., 27:327-341, 1994.
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W098/11116 and W098/11117 (Rademacher Group Limited).
W098/11435 and WO98/10791 (Rademacher Group Limited).
W099/38516 (Rademacher Group Limited).
