Note: Descriptions are shown in the official language in which they were submitted.
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GLYCOSAMINOGLYCAN (GAG) MIMETICS
TECHNICAL FIELD
The invention that is the subject of this application lies in the area of
compounds that
mimic the structure of certain carbohydrates. More particularly, the invention
lies in the area
of glycosaminoglycan (GAG) mimetics.
Specifically, the invention relates to compounds comprising at least one
charged group
that are designed to mimic the structure of GAGS. The invention also relates
to methods for
the preparation of the compounds, compositions comprising the compounds, and
use of the
compounds and compositions thereof for the antiangiogenic, antimetastatic,
anti-inflammatory,
anticoagulant, antithrombotic, and/or antimicrobial treatment of a mammalian
subject. The
invention further relates to the use of the compounds and compositions thereof
in the treatment
of a mammalian subject having a condition amenable to treatment with such
agents.
BACKGROUND ART
Glycosaminoglycans (GAGs) are linear, polyanionic polysaccharides that are
produced
by most animal cells and are usually found attached to a protein core [1,2].
GAGs occur
abundantly (as proteoglycans) and are extruded by cells to the cell surface
and into the
extracellular matrix (ECM) [3]. It has been recognised that GAGS, especially
those belonging
to the heparan sulfate (HS) family (HS-GAGs), mediate numerous physiological
processes.
For example, HS-GAGS play key roles in cell growth and development,
angiogenesis,
coagulation, tumour metastasis, cell adhesion, activation of growth factors,
binding of
cytolcines and chemolcines, and infection by bacteria and viruses [4-6]. In
recent years there
has been a dramatic increase in the list of proteins that interact with GAGS
and the list
continues to grow. The emerging view is that unique sequences of extracellular
GAGS bind
specifically to important proteins and by doing so influence fundamental
biological processes.
It has been shown that molecules that mimic the structure of certain GAGs-
which
molecules are referred to as "GAG mimetics"-can bind to GAG-binding proteins
and
modulate their biological activity: e.g., the activation of AT-III by various
pentasaccharides
[7,8], or the activation of fibroblast growth factors (FGFs) by sucrose
octasulfate [9].
Similarly, it has been shown that GAG mimetics can antagonise the binding of a
GAG to its
target protein and in so doing inhibit that protein's biological or disease
function. For example,
anticancer agents that have been developed to target HS-binding angiogenic
growth factors
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include polysulfonated compounds [10], suramin and the related suradistas
[11], and sulfated
oligosaccharides [12,13].
The present invention relates to novel, small molecule GAG mimetics that bind
to
GAG-binding proteins and modulate their functions. The compounds incorporate
at least one
negatively charged group (preferably a sulfo group) to interact with the
positively charged
residues in the GAG-binding site of the target proteins, and also contain one
or more
substituents to form interactions with other protein residues in and around
the above-mentioned
binding site. Important and distinguishing features of the compounds described
herein are that
they have fewer sulfo groups and are of lower molecular weight than previously
described
polysulfated GAG mimetics such as the sulfated oligosaccharides [12,13].
Another important
feature is that their structures are based on cyclic scaffolds (e.g., a
monosaccharide) with sulfo
groups and other substituents placed in specific, pre-defined orientations
about the ring, thus
differing significantly from the simple, randomly charged GAG mimetics
described by
Kisilevsky [14]. The binding of the compounds described herein to a selection
of HS-binding,
angiogenic growth factors is demonstrated via a surface plasmon resonance
(SPR) solution
affinity assay. Additionally, a selection of compounds are shown to inhibit
the HS-mediated
infection of cells and cell-to-cell spread by herpes simplex virus.
One aspect of the present invention is the utilisation of the Ugi reaction
[15,16] to
provide a diverse array of GAG mimetics. The capacity for variation in the
manner in which
the individual charged structures are connected to one another or to other
functional groups as
well as the scope of application to mimic the diverse structural variation of
GAGS is
demonstrated. As will be apparent to those skilled in the art, the
functionalisation of the cyclic
scaffolds is not limited to the Ugi reaction. For example, the use of many
other reactions such
as alkylation, acylation and cycloaddition is demonstrated.
SUMMARY OF THE INVENTION
It is an object of the invention to provide novel charged compounds that have
utility as
GAG mimetics. .
It is a further object of the invention to provide effective synthetic routes
for the
preparation of the subject compounds.
According to a first embodiment of the invention, there is provided a compound
of the
formula
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XR5
R4X . R6
R3X n XR~
XR2
wherein:
n is an integer of from 0 to 2;
Z is N, N(O), O, S, S(O), S(O)Z, P, P(O), P(O)2, Si, Si(O), or Si(O)2;
each X is independently C, C(O), N, N(O), O, S, S(O), S(O)2, P, P(O), P(O)2,
Si, Si(O),
or Si(O)2 or is a bond; and
each of Rl to R6 is independently a bond or is selected from the group
consisting of:
hydrogen;
halogen;
straight chain, cyclic, branched, substituted, heterocyclic, heteroatom
substituted
or unsubstituted alkyl, alkenyl, alkynyl, aryl, or heteroaryl;
phosphoryl groups such as phosphate, thiophosphate -O-P(S)(OH)2; phosphate
esters -O-P(O)(OR)2; thiophosphate esters -O-P(S)(OR)2; 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);
and cyclic phosphate;
other phosphorus containing compounds such.as phosphoramidite
-O-P(OR)-NR1R2; and phosphoramidate -O-P(O)(OR)-NR1R2;
sulfur groups such as -O-S(O)(OH), -SH, -SR, -S(--~O)-R, S(O)2R, RO-S(O)2 ,
-O-S02NH2, -O-S02R1R2 or sulfamide NHS02NH2;
amino groups such as NHR, -NR1R2, -NHAc, -NHCOR, -NH-O-COR, -
NHS03, -NHS02R, -N(S02R)2, and/or amidino groups such as -NH-
C(--NH)NH2 and/or ureido groups such as -NH-CO-NR1R2 or thiouriedo groups
such as -H-C(S)-NH2;
another unit of the structure I, attached through any position, where Z, X and
Rl
to R6 are as defined above; or
a substructure based upon a group of the following formula:
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O YR~o
R7Y~N~N YR~~
H Y Y ~ II
i
Ra Rs
wherein:
Y is a bond or is selected from the group consisting of: straight chain,
cyclic, branched, substituted, heterocyclic, heteroatom substituted or
unsubstituted alkyl; straight chain, cyclic, branched, substituted,
heterocyclic, heteroatom substituted or unsubstituted acyl; and aryl,
substituted aryl, heteroaryl;
and
each of R~ to Rl l is independently at least one structure according to
formula I, or a structure according to formula II;
with the provisos that:
when Z is O, and X is O or a bond, then all of Rl to RS are not H or CH20H; or
when Z is N and X is O or a bond, then all of Rl to R6 are not H.
According to a second embodiment of the invention, there is provided a
pharmaceutical
or veterinary composition for the prevention or treatment in a mammalian
subject of a disorder
resulting from angiogenesis, metastasis, inflammation, coagulation,
thrombosis, and/or
microbial infection, which composition comprises at least one compound
according to the first
embodiment together with a pharmaceutically or veterinarially acceptable
carrier or diluent for
said at least one compound.
According to a third embodiment of the invention, there is provided the use of
a
compound according to the first embodiment in the manufacture of a medicament
for the
prevention or treatment in a mammalian subject of a disorder resulting from
angiogenesis,
metastasis, inflammation, coagulation, thrombosis, and/or microbial infection.
According to a fourth embodiment of the invention there is provided a method
for the
prevention or treatment in a mammalian subject of a disorder resulting from
angiogenesis,
metastasis, inflammation, coagulation, thrombosis, and/or microbial infection,
which method
comprises administering to the subject an effective amount of at least one
compound according
to the first embodiment, or a composition comprising said at least one
compound.
In other embodiments of the invention, there are provided processes for
synthesising
the compounds according to the first embodimrent as defined above.
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With further regard to the compounds of the first embodiment, if not otherwise
specified, alkyl, aryl and other substituent groups are used in accordance
with their usual
meaning in the art. For example, alkyl and aryl groups would normally have
from 1 to 10
carbon atoms. Additionally, two of the groups Rl to RS may be connected to
each other to form
a bicyclic strucure; or the cyclic structure of formula I may contain a double
bond, i.e., two
contiguous XRl to XRS groups may be bonds.
Preferred compounds of the invention have the general structures of formulae
III-VI, as
defined in Tables 1-4 below.
In order that the invention may be more readily understood and put into
practice, one or
more preferred embodiments thereof will now be described, by way of example
only.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The following abbreviations are used herein:
GAG glycosaminoglycan
HS hepaxan sulfate
FGF fibroblast growth factor
aFGF acidic fibroblast growth factor (or FGF-1)
bFGF basic fibroblast growth factor (or FGF-2)
VEGF vascular endothelial growth factor
SPR surface plasmon resonance
HSV herpes simplex virus
The present inventors have found that a broad range of compounds with GAG
mimetic
properties can be synthesised using a number of different strategies as
illustrated below in the
examples. These compounds have utility in the prevention or treatment in
mammalian subjects
of a disorder resulting from angiogenesis, metastasis, inflammation, microbial
infections,
coagulation or thrombosis. This utility results from the ability of the
compounds to modulate
the activity of GAG-binding proteins responsible for disease processes.
The GAG mimetics of the invention, as indicated above, can be synthesised
using a
number of different routes, including the Ugi reaction, and generally
incorporating sulfonation
in the process.
Preferred compounds according to the first embodiment of the invention as
defined
above include those embraced by generic structures I and II and those included
in Tables 1-4
below.
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As indicated above, the compounds according to the invention have utility in
the
prevention or treatment in mammalian subjects of a disorder resulting from
angiogenesis,
metastasis, inflammation, microbial infection, coagulation or thrombosis. The
compounds
have particular utility in the treatment of the foregoing disorders in humans.
The compounds
are typically administered as a component of a pharmaceutical composition as
described in the
following paragraphs.
Pharmaceutical compositions for oral administration can be in tablet, capsule,
powder
or liquid form. A tablet can include a solid carrier such as gelatine or an
adjuvant or an inert
diluent. Liquid pharmaceutical compositions generally include a liquid carrier
such as water,
petroleum, animal or vegetable oils, a mineral oil or a synthetic oil.
Physiological saline
solution, or glycols such as ethylene glycol, propylene glycol or polyethylene
glycol may be
included. Such compositions and preparations will generally contain at least
0.1 wt% of the
compound.
Parenteral administration includes administration by the following routes:
intravenously, cutaneously or subcutaneously, nasally, intramuscularly,
intraocularly,
transepithelially, intraperitoneally and topically. Topical administration
includes dermal,
ocular, rectal, nasal, as well as administration by inhalation or by aerosol
means. For
intravenous, cutaneous or subcutaneous injection, or injection at a site where
treatment is
desired, 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
skill in the art
will be well able to prepare suitable solutions using, for example, solutions
of the subject
compounds or derivatives thereof.
In addition to the at least one compound and a carrier or diluent,
compositions
according to the invention can further include a pharmaceutically or
veterinarially acceptable
excipient, buffer, stabiliser, isotonicising agent, preservative or
antioxidant or any other
material known to those of skill in the art. It will be appreciated by the
person of skill that such
materials should be non-toxic and should not interfere with the efficacy of
the compound(s).
The precise nature of any additive may depend on the route of administration
of the
composition: that is, whether the composition is to be administered orally or
parenterally.
With regard to buffers, aqueous compositions typically include such substances
so as to
maintain the composition at a close to physiological pH or at least within a
range of about pH
5.0 to about pH 8Ø
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Compositions according to the invention can also include active ingredients in
addition
to the at least one compound. Such ingredients will be principally chosen for
their efficacy as
antiangiogenic, antimetastatic, anti-inflammatory, anticoagulant,
antithrombotic, antimicrobial
agents but can be chosen for their efficacy against any associated condition.
A pharmaceutical or veterinary composition according to the invention will be
administered to a subject in either a prophylactically effective or a
therapeutically effective
amount as necessary for the particular situation under consideration. The
actual amount of at
least one compound administered by way of a composition, and rate and time-
course of
administration, will depend on the nature and severity of the condition being
treated or the
prophylaxis required. Prescription of treatment such as decisions on dosage
and the like will
be within the shill of the medical practitioner or veterinarian responsible
for the care of the
subject. Typically however, compositions for administration to a human subject
will include
between about 0.01 and 100 mg of the compound per kg of body weight and more
preferably
between about 0.1 and 10 mg/kg of body weight.
The compounds can be included in compositions as pharmaceutically or
veterinarially
acceptable derivatives thereof. As used herein "derivatives" of the compounds
includes salts,
coordination complexes with metal irons such as Mn2+ and Zn2+, esters such as
ih vivo
hydrolysable esters, free acids or bases, hydrates, or prodrugs. 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 also be formed between compounds with basic groups, such as
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 acid basic groups can form internal salts.
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 that
will be well known to those of skill in the art.
Prodrug derivatives of the compounds of the invention can be transformed ivc
vivo or in
vita°o into the parent compounds. Typically, at least one of the
biological activities of a parent
compound may be suppressed in the prodrug form of the compound, and can be
activated by
conversion of the prodrug to the parent compound or a metabolite thereof.
Examples of
prodrugs are glycolipid derivatives in which one or more lipid moieties are
provided as
substituents on the moieties, leading to the release of the free form of the
compound by
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cleavage with an enzyme having phospholipase activity. Prodrugs of compounds
of the
invention include the use of protecting groups which may be removed in vivo to
release the
active compound or serve to inhibit clearance of the drug. Suitable protecting
groups will be
lcnown to those of skill in the art and include an acetate group.
As also indicated above, compounds according to the invention have utility in
the
manufacture of a medicament for the prevention or treatment in a mammalian
subject of a
disorder resulting from angiogenesis, metastasis, inflammation, coagulation,
thrombosis and/or
microbial infection. Processes for the manufacture of such medicaments will be
known to
those of skill in the art and include the processes used to manufacture the
pharmaceutical
compositions described above.
The compounds falling within the scope of the invention have been found to
have bind
growth factors. In particular, it has been established that the compounds have
affinity for
aFGF, bFGF and VEGF. The compounds thus have utility as antiangiogenic,
antimetastatic
and/or anti-inflammatory agents in the treatment of mammalian subjects
including humans.
The uses of the compounds include the treatment of angiogenesis-dependent
diseases such as
angiogenesis associated with the growth of solid tumours, and proliferative
retinopathies, as
well as the treatment of inflammatory diseases and conditions such as
rheumatoid arthritis. The
compounds may also activate the growth factors and could thus be used in
cardiovascular
treatments.
As further indicated above, the compounds of the invention additionally have
utility as
anticoagulant or antithrombotic agents. The compounds can therefore be used
for both the
prophylaxis and treatment of many thrombotic and cardiovascular diseases, the
most notable of
these being deep venous thrombosis, pulmonary embolism, thrombotic stroke,
peripheral
arterial thrombosis, unstable angina and myocardial infarction. Since
compositions of the
charged aminoacid compounds can be delivered orally, the compounds are an
attractive
alternative to warfarin, a widely used oral anticoagulant with severe side
effects.
The compounds of the invention additonally have been found to inhibit viral
infection
and thus have utility as antiviral agents in the treatment or prevention of
many viral infections.
The compounds of the invention are particularly suited for the treatment or
prevention of
infection resulting from pathogens which utilise HS as an attachment/entry
receptor [6], for
example, HSV, HIV, Dengue virus, Yellow fever virus, Cytomegalovirus and
Hepatitis C
virus. Similarly, the compounds of the invention are also suited for the
treatment or prevention
of infection resulting from non-viral microbial pathogens which utilise HS as
an
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attachment/entry, for example, Plasmodium (malaria). Most notable is the
inhibition by the
compounds of the invention of the cell-to-cell spread of HSV-1 and HSV-2.
Having broadly described the invention, non-limiting examples of the
compounds, their
synthesis, and their biological activities, will now be given with reference
to the accompanying
Tables which will be briefly described in the following section of this
specification.
General Procedures
Genes°al pf~ocedure fo~~ alkylation ahd dep~otectioh of diols
The diol (1 eq.) in DMF was added dropwise to a cooled (0°), stirred
suspension of pre-
washed (hexane) NaH (5 eq.) in DMF. Once the addition was complete, stirring
was
maintained (0°~r.t., 20 min). The mixture was cooled (0°, 5 min)
and the alkyl halide (2 eq.)
was introduced dropwise with continued stirring (0°~r.t., o/n). The
mixture was cooled once
again (0°) and MeOH (5 mL) was introduced with continued stirring (5
min). The solvent was
evaporated and the residue subjected to workup (EtOAc) and flash
chromatography to
homogeneity (TLC). This residue was co-evaporated (2 x 10 mL MeCN). The crude
mixture
and p-TsOH~H20 (50 mg) in MeOH/MeCN (1:1) was heated under reflux (1 h). The
mixture
was cooled (r.t.) and Et3N (100 ~,L) was added prior to evaporation of the
solvent. The residue
was subjected to flash chromatography (EtOAc/hexane) to yield the diol.
Geue~al p~~ocedu~e fog sulfouation of alcohols
A mixture of the alcohol and S03~trimethylamine (2 eq per hydroxyl group) in
DMF
was heated (60°, o/n). The cooled (r.t.) reaction mixture was treated
with MeOH and then
made basic (to pH>10) by the addition of Na2C03 (10% w/w). The mixture was
filtered and
the filtrate evaporated and co-evaporated (H20). Where deacylation of the
sulfated product
was required, the crude product was taken up in water and 1M NaOH was added (2
eq per acyl
group). When deprotection was complete the product was caxried through to the
next stage.
The crude sulfated material in H20 was subjected to size exclusion
chromatography. The pure
fractions were evaporated and co-evaporated (H20) and then lyophilised (H20)
to yield the
sulfated product. When required, after lyophilisation the product was passed
through an ion
exchange resin column (AG°-SOW-X8, Na+ form, 1 x4 cm, deionized HaO, 15
mL) in order to
transfer the product uniformly into the sodium salt form. The solution
collected was
evaporated and lyophilised to give the final product as a colourless glass or
white power.
Size exclusion chromatography
Size exclusion chromatography (SEC) was performed over Bio-Gel P-2 in a 5 x
100 cm
column with a flow rate of 2.8 mLlmin of 0.1 M NH4HC03, collecting 2.8 min
(7.8 mL)
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fractions. Fractions were analysed for carbohydrate content by TLC (charring)
and/or for poly-
charged species by the dimethyl methylene blue test, and then for purity by
capillary
electrophoresis (CE) and those deemed to be free of salt were pooled and
lyophilised.
In the cases of the presence of undersulfated by-products or other salt
contaminants
(normally only small amounts, but often detected), an LH20 SEC step (2 ~ 95
cm, deionized
water, 1.2 mL/min, 3.5 min per vial) was applied to remove them completely.
Dimethyl nzethylene blue Test
Dimethyl methylene blue (DMB) reagent was prepared by dissolving 16 mg of DMB
in
1 L of deionized water containing 3.04 g of glycine, 2.37 g of NaCI. 0.1 M HCl
(95 ml) was
added to adjust the pH to ,3Ø The stock solution was stored in a brown
coloured bottle at r.t.
(the solution was stable for at least 3 months under such conditions).
A 96-well microtitre plate was loaded with 10 ~,L of fraction solution per
well. 55 ~,L
of DMB stock solution was added into each used well. An instant colour change
from blue to
pink indicated the presence of polycharged species, i.e., sulfated product
fractions.
General p~oceduf°e fog NIS glycosylatiohs
Glycosyl acceptor (1 eq), thioglycoside donor (1.l eq), 500 mg of freshly
activated
powdered 3A molecular sieves and 10 mL of dry DCM were stirred at -20°
for 20 min before
1.3 eq of NIS and 1 drop of TfOH were added. Stirring was continued at -
20° until the reaction
was complete by TLC (~l h) before 400 ~.L of Et3N was added. Evaporation (in
vacuo) onto
silica gel and flash chromatography yielded the glycosylated product.
General procedure for Ugi four-component reaction
Solutions of the acid (1 eq), amine (1 eq), carbonyl compound (1 eq) and
isocyanide (1
eq) in MeOH, MeOH-THF (varied ratios) or CHC13 were transferred into a
reaction vial (final
concentration: 0.1-0.5 M). When D-glucuronic acid was the acid component, it
was added as a
_ solid. In the case of bis-acid, bis-amine, bis-aldehyde or bis-isocyanide,
the amount was 0.5 eq.
The mixture was stirred or shaken at r.t. or 60 °C for 1 h to 5 days.
The progress of the reaction
was monitored by TLC. The mixture was evaporated and the residue was purified
by flash
chromatography or dried completely under high vacuum followed by direct
peracetylation and
purification by flash chromatography.
General pr~oceduf~e for acetylation of hydroxyl groups:
The corresponding alcohol was dissolved in DCM-pyridine (15:1 v/v, 0.15 M)
containing DMAP (0.42 mol%). Acetic anhydride (2 eq per hydroxyl) was added
and the
mixture was stirred at r.t. o/n. The mixture was poured into ice-chilled 0.5 M
HCl and
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extracted with CHC13. The organic phase was separated and washed with cold 0.5
M HCl (x2),
brine, and dried (MgS04). The solution was filtered and evaporated. The
residue was purified
by flash chromatography (gradient elution with hexane-EtOAc) to give pure
product.
General procedure for Zernplerc deacetylatiorrldebenzoylation:
A solution of the acetate/benzoate in anhydrous MeOH (0.1 M) was treated with
a
solution of sodium methoxide in MeOH (1.35 M, 0.2-0.6 eq). The mixture was
stirred at r.t.
for 1-3 h (monitored by TLC). Acidic resin AGE-SOW-X8 (H+ form) was added to
adjust the
pH to 6-7, the mixture was filtered and the resin was rinsed with MeOH. The
combined filtrate
and washings were evaporated in vacuo and thoroughly dried to give the poly-of
product.
General pr°ocedure for deprotectiorc of benzyl ether°s via
hydrogenolysis
To a solution of the benzyl ether-protected compound (0.03 mmol) in MeOH or
EtOH
(2 mL) was added 5% Pd/C or 20% Pd(OH)2 on charcoal (30 mg or excess). The
mixture was
loaded in a miniclave (Biichi AG, Uster/Switzerland) and stirred under
hydrogen atmosphere
(50 psi) for 2-10 h. Alternatively, the mixture was bubbled with hydrogen gas
for 1 h then
stirred at r.t. under 1 atmosphere of hydrogen for 1-5 days. The reaction was
monitored by
TLC (EtOAc or MeCN-water 10:1). The mixture was filtered and rinsed with MeOH,
or
EtOH. The filtrate was evaporated and dried under high vacuum, checked by 1H
NMR, freeze-
dried and used directly for sulfonation.
Methylatiorc of hydr°oxyl groups
The dried poly-of was dissolved in anhydrous DMF (0.04 M) under argon and
stirred
with NaH (60% suspension in mineral oil, 1.2 eq per hydroxyl) at r.t. for 1 h.
Iodomethane
(1.2 eq per hydroxyl) was added and stirring continued oln. MeOH was added and
the mixture
was evaporated onto silica and purified by flash chromatography.
General procedure for Huisgen cycloadditioh reactions.
The sulfated sugar azide was dissolved in water (0.75 M) and a solution of
acetylene in
t-butanol (0.9 M, 1 eq) was added. To this mixture was added a solution of
copper (II) sulfate
(0.3 M in water, 5 mol%) and a solution of sodium ascorbate (1 M, in water, 20
mol%). The
mixture was shaken on a minishalcer at r.t. o/n, and purified by column
chromatography (silica
1x18 cm, gradient elution with EtOAc-MeOH-H20 50:2:1, 20:2:1 to 10:2:1) to
give the
corresponding triazole product.
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Example 1: PG2038
Step a: Methyl 3, 4, 6-ty~i-O-acetyl-2-O-be~zyl-a-D-galactopyf~anosyl-(1 ~4)-
~, 3, 6-
tai-O-benzyl-(i-D-glucopyy~a~coside.
Methyl 2, 3, 6-tai-O-benzyl-(3-D-glucopyra~coside [17] (150 mg, 322 ~,mol),
methyl 3, 4, 6-
tr~i-O-acetyl-~-O-benzyl-1-thio-[3-D-galactopy~anoside [18] (151 mg, 354
~.mol) and 200 mg of
3~ molecular sieves were subjected to the general NIS glycosylation procedure
using 95 mg
(422 ~.mol) of NIS. Flash chromatography (gradient elution 20:80 to 25:75
EtOAc:hexanes)
yielded 274 mg of partially deacetylated material. To this mixture was added
10 mL of DCM,
200 ~,L of acetic anhydride, 200 of ~,L Et3N and 2 mg of DMAP, and the
solution was stirred
for 1 h before evaporation and flash chromatography (gradient elution 25:75 to
30:70
EtOAc:hexanes) to give 180 mg (66%) Of the title compound as a colourless
glass. 1H n.m.r.
(400 MHz, CDC13) 8: 7.05-7.35 (m, 20H, 4~Ph), 5.74 (d, 1H, J1,2= 4.0, H1II),
5.29 (dd, 1H, J3,4
= 3 .2, Jq,S = 1.2, H4II), 5.23 (dd, 1 H, Jz,3 = 10.8, H3II), 4.92 (d, 1 H,
Jgen, = 12.0, PhCH2), 4.85 (d,
1 H, Jge,n = 10. 8, PhCH2), 4. 5 5-4.69 (m, 4H, PhCH2), 4.42 (AB, 1 H, Jgen, =
12.0, PhCH2), 4.40
(AB, 1H, PhCH2), 4.32 (d, 1H, J1,2= 7.6, Hli), 4.10 (dt, 1H, J5,6= 6.8, HSII),
3.88-3.96 (m, H,
HSI+H6I), 3.82 (dd, 1H, Jgen,= 11.1, H6II), 3.70-3.76 (m, 4H,
H2~H6I+H2II+H3I), 3.56 (s, 3H,
OMe), 3.5-3.6 (m, 1H, H4I), 3.45 (dd, 1H, J2,3 = 9.0, H2I), 2.02 (s, 3H, Ac),
1.93 (s, 3H, Ac),
1.88 (s, 3H, Ac). 13C n.m.r. (100 MHz, CDCl3) 8: 170.17, 170.06, 169.87,
138.82, 138.20,
138.13, 137.68, 128.33, 128.25, 128.19, 128.04, 127.61, 127.57, 127.49,
127.46, 127.01,
126.39, 104.42, 97.12, 84.49, 82.26, 74.46, 74.27, 73.69, 73.31, 73.28, 73.02,
69.47, 69.17,
68.35, 66.60, 61.62, 56.96, 20.69, 20.63, 20.58.
Step b: Methyl 3, 4, 6-tri-O-acetyl-a-D-galactopy~a~cosyl-(I ~4)-(3-D-
glucopyr~a~coside.
Pearlman's catalyst (20 mg) and 20 ~,L of acetic acid were added to a solution
of 90 mg
(106 ~.mol) of methyl 3, 4, 6-t~°i-O-acetyl-2-O-benzyl-a-D-
galactopyranosyl-(1--~4)-2, 3, 6-
tri-O-benzyl-(3-D-glucopyranoside in 10 mL of MeOH. An atmosphere of hydrogen
was
applied with 3 vacuum purges and the suspension was stirred for 3 days. After
filtration,
evaporation and co-evaporation with PhMe the residue was subjected to flash
chromatography
(gradient elution 100:0 to 100:3 EtOAc:MeH) to yield 47 mg (91%) of the title
compound. 1H
n.m.r. (400 MHz, CD30D) 8: 5.39 (br d, 1H, J3,4= 3.0, H4II), 5.32 (d, 1H,
J1,2= 3.8, Hlii), 5.10
3 0 (dd, 1 H, J2,3 = 10.6, H3II), 4.3 8 (br t, 1 H, J5,6 = 6.8, HSII), 4.20
(d, 1 H, Jl,a = 7.8, H 1 I), 4.09 (app
d (ABX), 2H, J5,6 = 6.5,' H6II), 4.00 (dd, 1 H, H2II), 3 .92 (dd, 1 H, J5,6A =
1.7, Jgen, = 12.2, H6AI),
3.80 (dd, 1H, J5,6B= 4.8, H6BI), 3.62 (dis t, 1H, J2,3~3,4= 9.1, H3I), 3.56
(part obs t, 1H, J3,4,.,4,s=
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9.3, H4I), 3.53 (s, 3H, OMe), 3.42 (ddd, 1H, J4,s = 9.4, HSI), 3.22 (dd, 1H,
J2,3 = 9.1, H2I), 2.10
(s, 3H, Ac0), 2.05 (s, 3H, Ac0), 2.00 (s, 3H, Ac0).
Step c: Methyl 2-O-sulfo-a-D-galactopyranosyl-(1 ~4)-2, 3, 6-tai-O-sulfo-(3-D-
glucopy~anoside,
tetrasodium salt (PG203~)
The above disaccharide (32.2 mg, 0.667 mmol), was subjected to the standard
sulfonation and deacetylation procedures to give the title compound as a white
foam (4.0 mg,
7.8%, 96% purity, CE: 7.18 min). 1H NMR (D20, 400 MHz): 5.473 (d, 1H, Jln-an=
3.6, H1II),
4.833 (d, 1H, Jl-zr = 2.8, H1I), 4.60 (overlapped with water, 1H, H3I), 4.551
(m, 1H, H2I),
4.306 (dd, 1H, JZn-sn = 10.2, H2II), 4.17-4.06 (m, 4H, H4I, HSI and H6I),
3.902 (d, 1H, J3n-4II =
3.6, H4II), 3.867 (dd, 1H, H3II), 3.616 (dd, 1H, J6~I-6e9n = 12.0, Jsn-6~II =
7.2, H6axII), 3.564
(dd, 1H, JSII-6eqII= 5.2, H6eqII), 3.363 (dd, 1H, HSII), 3.343 (s, 3H, CH30).
Example 2: PG2046 and PG2047
Step a: ~ Azido-3, 4, 6-tai-O-benzoyl-2-deoxy-a-D-glucopyf°a~cosyl-(1-
>4)-1, 6-afahyd~~o-2-azido-
2-deoxy-3-O-benzyl-(3-D-glucopyf°anose
A solution of 3, 4, 6-tr°i-O-acetyl-2-azido-2-deoxy-D-
glucopyf°anosyl tf°ichloro-
acetiynidate [19] (201 mg, 0.453 mmol) and l, 6-ahhyd~o-2-azido-3-O-benzyl-2-
deoxy-(3-D-
glucopy~~anose [20] (84 mg, 0.302 mmol) in 1,2-DCE (5 mL) was stirred in the
presence of
activated mol. sieves (300 mg of 3A powder) under an atmosphere of argon
(r.t., 30 min). The
mixture was cooled (-20°) with continued stirring (10 min) and TBDMSOTf
(21 ~,L,
0.091 mmol) was introduced drop-wise and stirring maintained (-20°, 10
min). Et3N (100 ~,L)
was introduced and the mixture filtered and evaporated. The residue was
subjected to aqueous
worlcup (EtOAc) and flash chromatography (10-40% EtOAc/hexanes) to yield a
pale yellow
coloured oil (130 mg). This residue was co-evaporated (2 x 10 mL MeCN) then
subjected to
the Zemplen deacetylation general procedure. The product was subjected to
aqueous workup
(EtOAc) to yield a colourless oil (98 mg). This residue was co-evaporated (2 x
10 mL MeCN).
BzCI (210 ~,L, 1.81 mmol) was added to a solution of the crude product (0.302
mmol, max.)
and pyridine (2 mL) in 1,2-DCE (3 mL) and the combined mixture stirred (r.t.,
o/n). The
mixture was cooled (0°) and MeOH (2 mL) was introduced with continued
stirring (0°--~r.t.,
2 min) before evaporation and co-evaporation (toluene) of the solvent. The
residue was
subjected to aqueous worlcup (EtOAc) and flash chromatography (10-30%
EtOAc/hexanes) to
yield two compounds.
Firstly, the title compound as a colourless foam (101 mg, 46%,~ 3 steps). 1H
NMR (400
MHz, CDC13) 8 3.11 (s, 1 H; H-2I), 3.41 (dd, 1 H, J1,2 3.7, J2,3 10.7 Hz; H-
6I), 3.61 (s, 1 H; H-
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3I), 3.39 (s, 1 H; H-4I), 4.05 (d, 1 H, J6,6 7.3 Hz; H-6I), 4.41-4.49 (m, 2 H;
H-6II), 4.55, 4.68
(AB quartet, JA,B 11.9 Hz; CH2Ph), 4.79 (ddd, 1 H, J4,5 10.3, Js,s 2.9, 5.9
Hz; H-SII), 4.91 (br d,
1 H, J5,6 5.5 Hz; H-SI), 5.08 (d, 1 H, J1,2 3.6 Hz; H-lII), 5.51 (dd, 1 H,
J3,4 9.5, J4,5 10.2 Hz, H-
4a), 5.60 (s, 1 H,; H-lI), 6.10 (dd, 1 H, J2,3 10.7, J3,4 9.3 Hz; H-3II), 7.29-
7.55, 7.89-8.03 (2 m,
20 H; ArH). 13C NMR (100 MHz, CDCl3) 8 58.74, 61.47, 63.30, 64.84, 69.19,
69.49, 70.52,
73.17, 74.62, 78.12, 79.57 (11 C; C-2I-6I, C2II-6II, CH2ph), 100.71, 101.16 (2
C; C-lI, C-lII),
128.10, 128.42, 128.57, 128.61, 128.64, 128.81, 128.88, 129.15, 129.86,
129.90, 130.06,
130.17, 133.43, 133.53, 133.74, 137.48 (Ar), 165.61, 165.62, 166.26 (3 C;
C=O).
Next, 2-azido-3, 4, 6-tai-O-benzoyl-2-deoxy-(3-D-glucopy~~ahosyl-(1-~4)-1, 6-
anhyd~o-2-
azido-2-deoxy-3-O-be~czyl-(3-D-glucopy~anose as a colourless oil (27 mg, 12%,
3 steps).
1H NMR (400 MHz, CDCl3) ~ 3.19 (s, 1 H; H-2I), 3.74 (dd, 1 H, J5,6 6.2, J6,6
7.1 Hz; H-6I),
3.79-3.88 (m, 2 H; H-2II, H-3I), 3.88 (ddd, J4,5 9.2, J5,6 3.1, 4.7 Hz; H-
SII), 3.95 (br s, 1 H; H-
4I), 4.10 (d, 1 H, J6,6 7.3 Hz; H-6I), 4.34 (dd, 1 H; J5,6 4.9, J6,6 12.2 Hz;
H-6II), 4.50 (dd, 1 H,
Js,6 3.1, Jg 6 12.3 Hz, H-6II), 4.52, 4.59 (AB quartet, JA,B 12.0 Hz; CH2Ph),
4.65 (d, 1 H, Jl,a
7.9 Hz; H-lII), 4.69 (br d, 1 H, J5,6 5.5 Hz; H-SI), 5.44 (t, 1 H, JZ,s=3,4
9.7 Hz; H-3II), 5.49 (br s,
1 H; H-l I), 5.51 (t, 1 H, J3 4=q,5 9.6 Hz; H-4II), 7.23-7.50, 7.84-7.96 (2 m,
20 H; ArH).
Step b: 2-Deoxy-2-sulfarrzido-a-D-glucopyr~anosyl-(1-j4)-1, 6-anhydf°o-
2-deoxy-~-sulfamido-3-
O-benzyl-(3-D-glucopy~anose, disodium salt (PG2046)
A mixture of 2-azido-3, 4, 6-tri-O-benzoyl-2-deoxy-a-D-glucopy~anosyl-(1-~4)-
l, 6-
a~ehydro-2-azido-2-deoxy-3-O-benzyl-[3-D-glucopyr~a~coside (127 ~,mol),
Pearhnan's catalyst
(11 mg), and ammonium formate (300 mg) in 2:1 MeOH:EtOAc (7 mL) was heated to
65°
under argon until complete by TLC. The mixture was cooled to r.t., filtered
(0.2 Vim) and
evaporated. The crude amine was purified by SPE (300 mg C18 Waters cartridge,
equilibrated
with 5:95 MeOH:H20, gradient eluted 5:95 to 100:0 MeOH:H20) to yield 53 mg of
the
diamine (58%). Without further purification, to the diamine was added DMF (5
mL),
S03~Me3N (41 mg, 295 ~mol) and NaHC03 (40 mg, 475 ~mol). The mixture was
heated to
60° for 1 h then cooled to rt and quenched with ice and Na2C03 (sat.
aqueous). This suspension
was stored at -18° o/n and the sample was filtered. The filtrate was
evaporated. Water (1 mL)
and NaOH (250 ~,L, 1M) were added and the solution was stirred overnight then
loaded
directly onto the SEC column (general procedures) to yield 22 mg (28 % over
three steps) of
the title compound. 1H NMR (400 MHz, DZO, solvent suppressed) 8: 7.35-7.21 (m,
SH, ArH),
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5.43 (br s, 1H, H11), 5.18 (d, 1H, Jl_2 = 3.6, H111), 4.72-4.691 (m, 1H, H51),
4.54-4.521 (m, 2H,
ArCH2), 4.05 (d, 1H, Jgem = 7.9, H6A1), 3.85 (br s, 1H, H31), 3.76-3.58 (m,
SH), 3.51 (dd, 1H,
J2-3 = 10.4, J3_4 = 9.1, H311), 3.34 (t, 1H, J3_4~4-s = 9.2, H411), 3.23 (br
s, 1H, H21), 3.12 (dd, 1H,
H211). 1sC NMR (100 MHz, CDC13) 8: 133.3, 124.8, 124.6, 124.4, 96.9, 95.1,
72.7, 71.5, 70.8,
68.3, 68.2, 67.2, 66.0, 61.0, 56.6, 54.0, 49.8.
Step c: 2-Deoxy-2-sulfamido-a-D-glucopyranosyl-(1-->4)-1, 6-anhyd~o-2-deoxy-~-
sulfamido-(3-
D-glucopy~anoside, dasodium salt (PG2047)
A mixture of 2-deoxy-2-sulfamido-a-D-glucopy~anosyl-(1 ~4)-1, 6-
anhydf°o-2-deoxy-2-
sulfamido-3-O-be~czyl-(3-D-glucopyrahoside, disodium salt (12.9 mg, 20.8 ~mol)
and
Pearlman's catalyst (5 mg) in purified water (2 mL) was subjected to 50 psi HZ
overnight. The
mixture was filtered and lyophilised to yield 10.7 mg (98 %) of the title
compound. 1H NMR
(400 MHz, D2O) 8: 5.47 (br s, 1H, H11), 5.20 (d, 1H, Jl_Z = 3.5, H111), 4.68
(br d, 1H, JS_4 = 5.5,
HS), 4.07 (d, 1H, Jgem = 7.6, H6A1), 3.98 (br s, 1H, H31), 3.75-3.64 (m, 4H),
3.52 (t, 1H,
JZ_3~3~ = 9.3, H311), 3.34 (t, 1H, J3_4,..4-5 = 9.3, H411), 3.13 (obs. dd2,
1H, H211), 3.11 (br s, 1H,
H21).
Example 3: PG2039 and PG2037
Step a: Methyl 3, 4-di-O-acetyl-2, 6-di-O-be~czyl-a-D-galactopy~anosyl-(1-~~)-
2, 3, 6-
tri-O-benzyl-(3-D-glucopyranoside
Methyl 2, 3, 6-tai-O-benzyl-[i-D-glucopyranoside (287 mg; 618 ~.mol), 302 mg
(618
~,mol) of ethyl 3, 4-di-O-acetyl-2, 6-O-dibenzyl-1-thio-(3-D-galactopyranoside
[21 ] and 700 mg
of 3~ molecular sieves were subjected to the general NIS glycosylation
procedure using 181
mg (803 ~,mol, l.3eq) of NIS. Flash chromatography (2.5 x 20 cm, gradient
elution 1:5 to 1:3
EtOAc:Hexanes) yielded the title compound as a colourless gum (176 mg, 32%).
1H NMR
(400 MHz, CDC13, 400 MHz): 7.40-7.12 (m, 25H, Ph), 5.818 (d, 1H, Jlll-21I =
3.6, H111), 5.481
(d, 1H, J4II-31I = 3.2, H411), 5.309 (dd, 1H, J311-21I = 10.8, J311-41I = 3.2,
H311), 4.980 (d, 1H, Jgem =
11.6, a-PhCH2), 4.904 (d, 1H, Jgem = 11.2, b-PhCHa), 4.748 (d, 1H, Jgem =
11.6, a-PhCH2),
4.67-4.57 (m, 3H, b-PhCH2 and e-PhCHa), 4.479 (d, 1H, Jgem = 12.8, d-PhCH2),
4.443 (d, 1H,
Jgen, = 12. 8, d-PhCH2), 4.415 (d, 1 H, Jge,n = 11.6, e-PhCH2), 4.3 60 (d, 1
H, Jl1-2i = 8.0, H 11),
4.201 (d, 1H, Jge,n = 12.6, e-PhCH2), 4.153 (t, 1H, J511-saxll = 7.2, J511-
6eq11= 6.0, HSII), 4.072 (t,
1H, J~1_31= 9.0, JqI-SI = 9.0, H41), 3.858 (dd, 1H, J211-31I = 10.8, J211-lII
= 3.6, H211), 3.82-3.76 (m,
3H, H31, H6ax1 and H6 eql), 3.597 (s, 3H, OMe), 3.62-3.57 (m, 1H, H51), 3.507
(t, 1H, Jal_sl =
' Affected by the solvent suppression signal.
z Partially obscured by H2I.
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8.4, J2I-II = 8.0, H2I), 3.347 (dd, 1H, J(eqII-6axII = 9.2, J6eqII-SII = 6.0,
H6eqII), 3.291 (dd, 1H,
J6axII-6eqII= 9.2, JgaxII-5II= 7.2, H6axII), 1.958 (s, 3H, OAc), 1.930 (s, 3H,
OAc). I3C NMR (100
MHz, CDC13, 100 MHz): 169.94 (CO), 169.78 (CO), 138.88, 138.35, 138.24, 137.73
and
137.64 (5x ipso-Ph), 128.25, 128.22, 128.21, 128.15, 128.12, 128.00, 127.80,
127.57, 127.52,
127.46, 127.41, 127.37, 126.93, 126.37, 104.40, 97.16, 84.63, 82.31, 74.42,
74.30, 73.70,
73.25, 73.16, 73.14, 72.98, 69.73, 69.07, 68.89, 67.64, 67.50, 56.86, 20.71,
20.55.
Step b: Methyl 3, 4-di-O-acetyl-a-D-galactopy~ahosyl-(1--~4)-/3-D-
glucopyranoside.
Following the standard debenzylation procedure, methyl 3, 4-di-O-acetyl-2, 6-
di-
O-be~zyl-a-D-galactopy~ahosyl-(1-~4)-2, 3, 6-tr°i-O-benzyl-[3-D-
glucopy~a~coside (88 mg, 98.8
~.mol) was deprotected to give the title compound as a colourless powder (42
mg, 97%). IH
NMR (D20, 400 MHz): 5.394 (d, 1H, JIII-2II= 3.6, H1II), 5.294 (d, 1H, J4II-
sII= 3.2, H4II), 4.953
(dd, 1H, J3II-2II= 10.4, J3II-4II= 3.2, H3II), 4.229 (d, 1H, JII-aI= 8.4,
H1I), 4.080 (t, 1H, JSII-6~II=
6.4, JSII-6eqII = 6.0, HSII), 3.965 (dd, 1 H, J2II-3II = 10.4, J2II-lII = 3.6,
H2II), 3.803 (dd, 1 H, J6eqI-seql
= 12.0, J(eqI-SI = 1.6, H6eqI), 3.67-3.59 (m, 2H, H6axI and H3I), 3.54-3.40
(m, 4H, H4I, HSI and
H6II), 3.407 (s, 3H, OMe), 3.134 (dd, 1H, J2I-3I = 9.2, J2I-II = 8.4, H2I),
2.012 (s, 3H, OAc),
1.909 (s, 3H, OAc). I3C NMR (D20, 100 MHz): 173.57, 173.47, 103.20, 99.71,
77.12, 76.30,
74.57, 73.09, 70.89, 69.94, 69.04, 66.45, 60.82, 60.18, 57.31, 20.34, 20.09.
Step c: Methyl 2, 6-di-O-sulfo-a-D-galactopyranosyl-(1-~4)-2, 3, 6-tai-O-sulfo-
(3-D-
glucopy~anoside, pentasodium salt (PG2039)
Following the standard sulfonation/deacetylation procedures, 42 mg (95.4
~.mol) of
methyl 3, 4-di-O-acetyl-a-D-galactopyr~av~osyl-(1-->4)-(i-D-glucopyranoside
was converted to
the title compound as a white powder (14.8 mg, 18%, CE: 6.12 min). IH NMR
(D20, 400
MHz): 5.404 (d, 1H, JIII-zII= 3.6, H1II), 4.756 (d, 1H, JII-aI= 3.6, H1I),
4.60 (overlappped with
water, 1H, H3I), 4.448 (dd, 1H, J2I-3I = 3.2, H2I), 4.296 (dd, 1H, J2II-3II =
10.0, H2II), 4.23-4.00
(m, 7H, H6I, HSI, H6II, H4I and HSII), 3.958 (dd, 1H, J3II-4II = 3.6, J4II-sII
= 0.8, H4II), 3.930 (dd,
1H, H3II), 3.367 (s, 3H, CH30).
Step d: Methyl 2, 6-di-O-benzyl-3, 4-di-O-methyl-a-D-galactopyr~anosyl-(1->4)-
2, 3, 6-t~°i-O-
benzyl-(3-D-glucopyr~avcoside
Following the standard deacetylation and methylation procedures, methyl 3, 4-
di-O-
acetyl-2, 6-di-O-beuzyl-a-D-galactopyr~anosyl-(1-~4)-~, 3, 6-t~~i-O-benzyl-(3-
D-glucopy~anoside
(72 mg, 80.8 ~,mol) was converted into the title compound as colourless gum
(62.7 mg, 93%).
IH NMR (CDCl3, 400 MHz): 7.35-7.08 (m, 25H, Ph), 5.717 (d, 1H, JIn-an = 3.6,
H1II), 4.856
(d, 1H, Jgen, = 11.2, a-PhCH2), 4.843 (d, 1H, Jgen, = 10.8, b-PhCH2), 4.695
(d, 2H, Jge,n = 12.0,
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a-PhCH2 and c-PhCH2), 4.631 (d, 1H, Jgen, = 12.4, d-PhCH2), 4.571 (d, 1H,
Jgen, = 10.8,
b-PhCH2), 4.500 (d, 1H, Jgen,= 12.4, d-PhCH2), 4.450 (d, 1H, Jge,n= 11.6, c-
PhCH2), 4.433 (d,
1 H, Jgem = 11.2, e-PhCH2), 4.3 59 (d, 1 H, Jge", = 11.2, e-PhCH2), 4.3 03 (d,
1 H, JII-2I = 7.6, H 1 I),
3.949 (t, 1H, J4I-3I = 9.0, J4I-SI = 9.0, H4I), 3.871 (dd, 1H, JSII-6axII =
7.2, JSII-6eqII = 6.4, HSII),
3.791 (dd, 1H, J2II-3II = 10.4, J2II-III = 3.6, H2II), 3.77-3.68 (m, 4H, H4II,
H3I, H6axI and H6eqI)),
3.59-3.53 (m, 2H, HSI and H6II), 3.551 (s, 3H, OMe), 3.51-3.40 (m, 3H, H3II,
H6II and H2I),
3.492 (s, 3H, OMe), 3.433 (s, 3H, OMe).
Step e: Methyl 3, 4-di-O-methyl-a-D-galactopyf°anosyl-(1-~4)-(3-D-
glucopyranoside
Following the standaxd debenzylation procedure, methyl 2, 6-di-O-benzyl-3, 4-
di-O-
methyl-a-D-galactopyranosyl-(1-~4)-2, 3, 6-tai-O-benzyl-(3-D-glucopy~anoside
(62.7 mg, 75.1
~mol) was deprotected to give the title compound as colourless gum (28 mg,
97%). IH NMR
(D20, 400 MHz): 5.232 (d, 1H, JIII-aII= 4.4, H1II), 4.217 (d, 1H, JII-aI= 8.0,
H1I), 3.83-3.75 (m,
3H, H4II, HSII and H6I), 3.682 (dd, 1H, J2II-3II = 10.4, J2II-III = 4.4,
H2II), 3.64-3.52 (m, 4H, H6I,
H3II and H6II), 3.47-3.38 (m, 3H, H4I, HSI and H3II), 3.400 (s, 3H, OMe),
3.340 (s, 6H,
2xOMe), 3.117 (dd, 1H, J2I-3I = 9.6, J2I-II = 8.0, H2I). I3C NMR (D20, 100
MHz): 103.20,
99.64, 79.35, 76.92, 76.34, 75.35, 74.65, 73.06, 72.03, 68.00, 61.02, 60.89,
60.86, 57.30, 56.94.
Step f Methyl 3, 4-di-O-methyl-2, 6-di-O-sulfo-a-D-galactopy~anosyl-(1-~4)-2,
3, 6-tri-O-
sulfo-(3-D-glucopy~anoside, pentasodium salt (PG2037)
Following the standard sulfonation procedure, methyl 3, 4-di-O-methyl-a-D-
galactopyranosyl-(1-~4)-(3-D-glucopyranoside (28 mg, 72.8 ynol) gave the title
compound
(3.2 mg, 4.9%). IH NMR (400 MHz, Da0): 5.357 (d, 1H, JIII-aII= 3.2, H1II),
4.766 (d, 1H, JII-2I
= 3.6, HlI), 4.60 (overlappped with water, 1H, H3I), 4.455 (dd, 1H, J2I-3I=
2.8, H2I), 4.304 (dd,
1H, J2II-3II = 10.0, H2II), 4.22-3.99 (m, SH, HSI, H6I, H4I and HSII), 4.002
(d, 2H, JSII-6II = 6.8,
H6II), 3.886 (d, 1H, J3II-4II= 3.2, H4II), 3.667 (dd, 1H, H3II), 3.398 (s, 3H,
CH30), 3.367 (s, 3H,
CH30), 3.356 (s, 3H, CH30).
Example 4: PG2053 and PG2042
Methyl 4-O-Allyl-~, 3-di-O-sulfo-a-L-rhamnoside, disodium salt (PG2053).
The title compound was obtained from methyl 2, 3-O-isopropylidene-a-L-
y~harnnopy~anoside [22] via the general alkylation (with allyl bromide) and
deprotection
procedure followed by the general sulfonation procedure, as a colourless
powder. CE tm _
10.48 min. IH NMR (400 MHz, DZO) ~ 1.19 (d, 3 H, J5,6 6.4 Hz; H-6), 3.26 (s, 3
H; OMe);
3.29-3.40 (m, 1 H; H-4), 3.59-3.67 (m, 1 H; H-5), 4.00-4.05, 4.18-4.22 (2 m, 2
H; OCHa),
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4.41-4.42 (m, 1 H; H-3), 4.63-4.64 (m, 2 H; H-2), 4.83 (s, 1 H; H-1), 5.07-
5.21 (m, 2 H;
=CH2), 5.76-5.88 (m, 1 H; =CH).
When a reduced quantity (1 eq.) of S03~trimethylamine was employed, ~rzethyl 4-
O-
allyl-2-O-sulfo-a-La°hamvcoside, sodium salt (PG2042) was exclusively
obtained. CE tm >
25.00 min. 1H NMR (400 MHz, D20) 81.19 (d, 3 H, J5,6 6.4 Hz; H-6), 3.16 (t, 1
H, J3,4 3.1,
J4,5 9.7 Hz; H-4), 3.25 (s, 3 H; OMe), 3.54-3.58 (m, 1 H; H-5), 3.76 (dd, 1 H,
Ja,3 9.7 Hz; H-3),
4.03-4.18 (m, 2 H; OCHZ), 4.34-4.35 (m, 1 H; H-2), 4.80 (s, 1 H; H-1), 5.08-
5.22 (m, 2 H;
=CHZ), 5.78-5.88 (m, 1 H; =CH).
Example 5: PG2024
Methyl 4-O-Benzyl-2, 3-di-O-sulfo-a L-rhamnoside, disodium salt (PG2024)
The title compound was obtained from methyl 2, 3-O-isopr~opylidene-a-L-
r~hamnopyrafzoside via the general alkylation (with benzyl bromide) and
deprotection
procedure followed by the general sulfonation procedure, as a colourless
powder. CE
tm = 10.82 min. 1H NMR (400 MHz, D20) d 1.00 (d, 3 H, J5,6 6.8 Hz; H-6), 3.23
(s, 3 H;
OMe); 3.78-3.80 (m, 1 H; H-4), 3.88-3.94 (m, 1 H; H-5), 4.41-4.43 (m, 1 H; H-
2), 4.52-4.56
(m, 2 H; H-3), 4.54, 4.78 (AB quartet, JA,B 12.0 Hz; CHaPh), 4.90 (dd, 1 H,
J1,2 1.2 Hz; H-1),
7.20-7.36 (m, 5 H; ArH).
Example 6: PG2054
Step a: Methyl 4-O-behzoyl-a L-rhamhoside
A solution of methyl 2, 3-O-isopr~opylidehe-a-L-f~hamhopyf°auoside (200
mg, 920
~mol), benzoyl chloride (193 mg, 1.38 mmol) and Et3N (364 ~L, 2.76 mmol) in
DCM (10 mL)
was stirred overnight. The resulting suspension (Et3N~HCl precipitates) was
diluted with DCM
(50 mL) and washed with NaHC03 (sat. aqueous), water then brine, dried (MgSO4)
and
evaporated. The residue was taken up in 50 mL of 1:1 MeCN:H2O and p-TsOH (10
mg) was
added. The resulting solution was stirred until the reaction was complete
(TLC, ~4 h),
evaporated and subjected to flash chromatography (1:1 EtOAc:hexanes) to give
165 mg (64
over two steps) of the title compound as a colourless solid. 1H NMR (400 MHz,
CDC13) 8:
8.03-8.00 (m, 2H, Hortho), 7.55 (tt, 1H, Jgp_Hm = 7.5, JHp_Ho = 1.3, Hpara),
7.43-7.39 (m, 2H,
Hmeta), 5.09 (dis t, 1H, J4_3--4-5 = 9.3, H4), 4.73 (br s, 1H, H1), 4.01-3.97
(m, 2H, H2+H3),
3.91 (dq, 1H, J~_5 = 9.7, JS_6 = 6.4, HS), 3.53-3.46 (br s, 2H, OH), 3.38 (s,
3H, OMe), 1.25 (d,
3H, H6). 13C NMR (100 MHz, CDCl3) 8: 167.1, 133.3, 129.8, 129.5, 128.3, 100.6,
75.7, 70.8,
70.1, 65.7, 55.0, 17.4.
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Step b: Methyl 4-O-Benzoyl-2, 3-di-O-sulfo-a L-rhamnoside, Disodium salt
(PG2D54)
The title compound was obtained from methyl 4-O-behzoyl-a L-rhamnoside via the
general sulfonation procedure as a colourless powder. CE tm = 11.14 min. 1H
NMR (400
MHz, DZO) 81.14 (d, 3 H, Js,6 6.3 Hz; H-6), 3.33 (s, 3 H; OMe), 3.99-4.07 (m,
1 H; H-5),
4.66-4.73 (m, 2 H; H-2, -3), 4.95 (d, 1 H, J1,2 1.4 Hz; H-1), 5.04 (t, 1 H, J3
~=q g 9.6 Hz; H-4),
7.35-7.41, 7.53-7.55, 7.92-7.93 (3 m, 5 H; Ph).
Example 7: PG2041
Step a: 4,6-O-Be~czylide~ce-1,2-dihyd~o-D-glucal.
A mixture of tri-O-acetyl-D-glucal (1.7 g, 6.25 mmol), AcOH (50 ~.L) and
Pd(OH)2/C
(100 mg) in MeOH (15 mL) was vigorously stirred under H2 (1 atm.) overnight.
The mixture
was filtered, the solvent evaporated and the residue subjected to flash
chromatography (10-
50% EtOAc/hexanes) to yield tai-O-acetyl-1,2-dihyd~o-D-glucal as a colourless
oil. This
residue was co-evaporated (2 .x 10 mL MeCN) then subjected to the Zemplen
deacetylation
general procedure to yield l, 2-dihyd~o-D-glucal as a colourless oil (825 mg,
89%). This
residue was co-evaporated (2 x 10 mL MeCN).
p-TsOH.H20 (50 mg) was added to a solution of the 1, 2-dihya'~°o-D-
glucal (495 mg,
3.34 mmol) and a,a-dimethoxytoluene (753 p,L, 5.01 mmol) in DMF (5 mL) and the
combined
mixture stirred (60°, 1 h). Et3N (100 p,L) was introduced and the
solvent was evaporated. The
residue was subjected to flash chromatography (0-5% MeOH/CHC13) to yield the
title
compound as colourless needles (503 mg, 64%). 1H NMR (400 MHz, CDC13) 81.72-
2.01 (m,
2 H; H-2), 3.27-3.33 (m, 1 H; H-5), 3.41 (dd, 1 H, J3,4 8.8, Js,6 9.1 Hz; H-
4), 3.49-3.56 (m, 1 H;
H-3), 3.67 (t, 1 H, Jg 6=6,6 10.3 Hz; H-6), 3.81-3.87, 3.93-3.98 (2 m, 2 H; H-
1), 4.25 (dd, 1 H;
Js,6 4.9 Hz; H-6), 5.53 (s, 1 H; CHPh), 7.23-7.52 (m, 5 H, CHPh). 13C NMR (100
MHz,
CDC13) 8 33.47 (C-2); 66.46, 69.07, 69.64, 71.32 (4 C; C-1,-4,-5,-6), 84.14,
(C-3), 102.16
(CHPh), 126.43, 128.55, 129.34, 137.50 (4 C; Ph).
Step b: 3-O-Benzyl-4,6-di-O-sulfo-1,2-dihyd~o-D-glucal, Disodium salt
(PG2041).
4,6-O-Benzylidene-1,2-dihyd~o-D-glucal was subjected to the alkylation (benzyl
bromide), de-protection and sulfonation general procedures to yield the title
compound as a
colourless powder. CE tm = 15.40 min. 1H NMR (400 MHz, CDC13) ~ 1.48-1.53,
1.97-2.03 (2
m, 2 H; H-2), 3.30-3.71 (m, 1 H; H-1), 3.52-3.57 (m, 1 H; H-5), 3.60-3.66 (m,
1 H; H-3), 3.78-
3.83 (m, 1 H; H-1), 3.97 (dd, 1 H, Js,6 8.0, J6,6 11.4 Hz; H-6), 3.98 (t, 1 H,
J3,~-4,s 8.9 Hz; H-4),
4.34 (dd, 1 H, Js,6 2.3 Hz; H-6), 4.52-4.67 (m, 2 H; CHZPh), 7.21-7.36 (m, 5
H; Ph).
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Example 8: PG2030
Step a: 1, 6 Auhyd~o-3-O-methyl-(3-D-glucopy~ahose.
p-Toluenesulfonyl chloride (790 mg, 4.14 mmol) was added to a cooled
(0°) suspension
of 3-O-methyl-D-glucopyranose (804 mg, 4.14 mmol) in pyridine (10 mL) and the
reaction
mixture stirred (0°-~r.t, 1.5 h). Ac20 (1.5 mL, 15 mmol) and N,N
dimethylaminopyrdine
(50 mg) were then introduced and stirring continued (r.t., 4 h). The mixture
was then cooled
(0°) and MeOH (3 mL) was added and stirring maintained (10 min) prior
to evaporation of the
solvent. The residual oil was dissolved (EtOAc) and subjected to workup
yielding the tosylate
as a pale yellow coloured oil (1.93 g). A mixture of the crude tosylate (1.93
g) and NaOH
(20 mL of 1.0 M, 20 mmol) in EtOH (20 mL) was heated (80°, 1 h). The
mixture was
neutralised with acetic acid and the solvent evaporated and co-evaporated
(toluene). The crude
residue was treated with pyridine (10 mL), Ac20 (5 mL) and N,N
dimethylaminopyridine
(50 mg) and the combined mixture stirred (r.t., o/n). The mixture was treated
with ice-water
(10 mL) and stirring continued (r.t., 3 h) before being subjected to workup
(EtOAc). The
residual oil was subjected to flash chromatography (20-50% EtOAc/hexanes) to
yield an
inseparable mixture of 2, 4-di-O-acetyl-l, 6-ayzhyd~o-3-O-methyl-(3-D-
glucopyy~anose (a) and
l, 2, 4-ti~i-O-acetyl-3-O-methyl-6-O-tosyl-a-D-glucopyranose (b) (in a ratio
of 3:1) as a pale
yellow oil (466 mg). The ratio was determined by integration of the H-1 and 3-
OMe signals
observed in the 1H NMR spectrum. Partial 1H NMR (400 MHz, CDCl3) 8 3.32 (s, 3
H;
OMe b), 3.45 (s, 3 H; OMe a); 5.22 (br s, 1 H; H-1 b), 5.42 (br s, 1 H; H-1
a). The mixture of
the two compounds (456 mg) was subjected to the Zemplen deacetylation general
method and
the residue subjected to flash chromatography (0-5% MeOH/EtOAc) to yield the
title
compound as a colourless oil (162 mg, 33%, 3 steps). 1H NMR (400 MHz, CDCl3):
8 3.27-
3.30 (m, 1 H; H-3), 3.38 (s, 3 H; OMe), 3.57-3.59 (m, 1 H; H-2), 3.63-3.65 (m,
1 H; H-4), 3.70
(dd, 1 H, J5,6 = 5.6, J6,6 = 7.2 Hz; H-6), 4.06 (d, 1 H, J6,6 = 7.2 Hz; H-6),
4.48-4.51 (m, 1 H; H-
5), 4.39 (br s, 1 H; H-1).
Step b: 1, 6 Anhydr~o-4-O-benzyl-3-O-methyl-(3-D-glucopy~a~cose.
A mixture of 1, 6-anhyd~o-3-O-methyl-(3-D-glucopyr~anose (155 mg, 0.88 mmol)
and
Bu~,SnO (241 mg, 0.97 mmol) in toluene (18 mL) was heated under reflux (with
azeotropic
removal of water) until the solution was one-half the original volume. The
mixture was cooled
(80°), BnBr (104 ~,L, 0.88 mmol) and Bu4NBr (567 mg, 1.76 mmol) were
introduced and
stirring continued (o/n). The mixture was treated with MeOH (2 mL) and Ha0 (1
mL) with
continued stirring (10 min) prior to evaporation of the solvent. The residue
was subjected to
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workup (EtOAc) and flash chromatography (20-60% EtOAc/ hexanes) to yield two
compounds.
Firstly, the title compound was produced as a colourless oil (94 mg, 40%). 1H
NMR
(400 MHz, CDCl3): 8 2.58 (d, 1 H, J2 OH 6.4 Hz; OH), 3.32-3.43 (m, 5 H; H-3, H-
4, OMe),
3:52-3.57 (m, 1 H; H-2), 3.68-3.72, 4.01-4.04 (2 m, 2 H; H-6), 4.55-4.58 (m, 1
H; H-5), 4.64
(s, 2 H; CH2Ph), 5.39-5.40 (m, 1 H; H-1), 7.28-7.36 (m, 5 H; ArH).
Secondly, l, 6-anhydro-2-O-benzyl-3-O-methyl-(3-D-glucopyr~auose was afforded
as a
colourless oil (91 mg, 39%). 1H NMR (400 MHz, CDCl3): ~ 2.90 (br s, 1 H; OH),
3.31-3.34
(m, 4 H; H-2, OMe), 3.36-3.38 (m, 1 H; H-3), 3.58 (br s, 1 H; H-4), 3.68 (dd,
1 H, J5,6 6.0 Hz,
J6,6 7.2 Hz; H-6), 4.08 (dd, 1 H, J5,6 0.8 Hz, J6,6 7.2 Hz; H-6), 4.47-4.49
(m, 1 H; H-5), 4.56,
4.62 (AB quartet, JA,B 12.0 Hz; CH2Ph), 5.40-5.41 (m, 1 H; H-1), 7.26-7.36 (m,
5 H; ArH).
Step c: l, 6 Anhyd~o-4-O-benzyl-3-O-methyl-2-O-sulfo-(3-D-glucopy~anose,
sodium salt
(PG2030)
1, 6 Anhyd~o-4-O-benzyl-3-O-methyl-(3-D-glucopy~ahose (84 mg, 0.32 mmol) was
sulfonated according to the general procedure and subjected to flash
chromatography
(50/2/1-X10/2/1 EtOAc/MeOH/H20) prior to SEC to yield the title compound as a
pale yellow
coloured powder (70 mg, 60%); CE tm = 5.62 min; 1H NMR (400 MHz, DZO) ~ 3.19
(s, 3 H;
OCH3); 3.43-3.45 (m, 1 H; H-4), 3.52-3.53 (m, 1 H; H-3), 3.57 (dd, 1 H, J5,6 =
5.9 Hz,
J6,6 = 7.8 Hz; H-6), 3.82 (dd, 1 H, J5,6 =1.1 Hz, J6,6 = 7.8 Hz; H-6), 3.97-
3.99 (m, 1 H; H-5),
4.59-4.61 (m, 3 H; H-2, CH2Ph), 5.41 (br s, 1 H; H-1), 7.22-7.34 (m, 5 H;
ArH).
Example 9: PG2012 and PG2013
Step a: N-benzyl-N-(cyclohexylacetamido)-1, 2, 3, 4-tet~~a-O-acetyl-D-
glucuronamide
Following the general procedure for the Ugi reaction, D-glucuronic acid (0.950
g, 4.89
mmol), and solutions of each of the following three reagents: benzylamine (2 M
in MeOH,
2.45 mL, 4.89 mmol), formaldehyde (2 M in MeOH, 2.45 mL, 4.89 mmol) and
cyclohexylisocyanide (1 M in MeOH, 4.89 mL, 4.89 mmol) were loaded into a
reaction vessel
and the mixture stirred at r.t. for 19 h. The volatiles were removed under
reduced pressure and
dried under high vacuum to afford N-benzyl-N-(cyclohexylacetamido)-D-
glucut°ouamide as a
yellow foam.
Following the general procedure for acetylation, the above crude Ugi product
was
peracetylated to give the title compound as pale-yellow foam 1.929 g, 66% (two
steps, Rf =
0.37, hexane-EtOAc 1:1) after flash chromatography (gradient elution with
hexanes-EtOAc 2:1
to 1:1). 1H NMR (CDC13, 400 MHz) was very complicated due to the presence of
anomers and
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rotamers. The spectrum was not simplified after the temperature was raised to
55 °C.
However, in pyridine-d6 at 100 °C, each set of rotamers was coalesced
in some degree into
much more simplified structure, thus two anomers were clearly observed (a: (3
ratio = 69:31 ).
1H NMR (CDC13, 400 MHz, 25 °C): 7.41-7.14 (m, SH, Ph), 6.337 (d, 0.39H,
J= 3.6), 6.300 (d,
0.29H, J= 3.6), 5.969 (br d, 0.52H, J= 8), 5.823 (br d, 0.09H, J= 8.4), 5.66-
5.41 (m, 2.25H),
5.28-5.09 (m, 1.3H), 4.92-4.58 (m, 2.25H), 4.411 (d, J = 10) and 4.395 (d, J =
14, O.SSH),
4.271 (d, O.11H, J= 9.6), 4.219 (d, 0.09H, J= 17.2), 4.125 (d, 0.17H, J= 14),
4.098 (d, 0.17H,
J = 14.4), 3.994 (d, J = 15.2) and 3.963 (d, J = 14.8, 0.89H), 3.82-3.59 (m,
2.04H), 2.190,
2.111, 2.038, 2.033, 2.025, 2.023, 2.016, 2.014, 2.008, 1.998, 1.983, 1.944
and 1.927 (all
singlet, 12H, Ac), 1.89-1.55 (m, SH, cyclohexyl-CH2), 1.41-0.83 (m, SH,
cyclohexyl-CH2). 1H
NMR (CDCl3, 400 MHz, 55 °C): 7.38-7.16 (m, SH, Ph), 6.336 (d, J= 3.2, )
and 6.153 (d, J=
3.2, 0.7H ), 5.939 (br d, 0.6H, J = 6.8), 5.721 (br d, 0.2H, J = 7.2), 5.65-
5.55 (m, 1.3H),
5.52-5.41 (m, 1H,), 5.28-5.10 (m, 1.4H), 4.85-4.58 (m, 2.4H), 4.48-4.40 (m,
0.6H) , 4.318 (d,
O.1H, J= 9.2), 4.205 (d, O.1H, J= 17.6), 4.02-3.93 (m, 0.9H), 3.84-3.62 (m,
2.2H), 2.179,
2.090, 2.021, 2.012, 2.001, 1.989, 1.983, 1.975, 1.968, 1.959 and 1.935 (all
singlet, 12H, Ac),
1.88-1.56 (m, SH, cyclohexyl-CHZ), 1.42-0.88 (m, SH, cyclohexyl-CHZ). 1H NMR
(pyridine-
d6, 400 MHz, X7.22, 100 °C): only typical sugar protons are given; the
remaining signals
(except acetate singlets) were complicated and appeared as broad lumps. a,-
anomer, 6.672 (d, J
= 3.6, glu-H1 ), 5.456 (dd, J = 9.6, 3.6, glu-H2); [3-anomer, 6.206 (d, J =
8.0, glu-H1 ), 5.741 (t,
J= 9.2, glu-H4 or HS), 5.515 (dd, J= 8.8, 8.0, glu-H2).
Step b: N-benzyl-N-(cyclohexylacetamido)-l, 2, 3, 4-tett~a-O-sulfo-a-D-
glucuronamide,
tetrasodium salt (PG2012) and N-beuzyl-N-(cyclohexylacetamido)-1,2,3-t~~i-O-
sulfo-a-D-
glucm~ohamide, tf°isodium salt (PG2013)
Following the general procedure for deacetylation, the above tetraacete (0.441
g, 0.747
mmol) was deacetylated to give N-behzyl-N-(cyclohexylacetamido)-D-
glucu~~onamide as pale-
yellow glass (0.316 g, 100%).
Following the general procedure for sulfonation, the above tetrol (0.257 g,
0.608 mmol)
was sulfonated (using sulfur trioxide pyridine complex, 60 °C, 19 h).
The residue was co-
evaporated with toluene and purified by flash chromatography [2.5 x 20cm,
eluted with
EtOAc, MeCN, MeCN-Et3N (10:1), MeCN-Et3N-H20 (110:2:11)]. The fractions were
divided
into two parts according to TLC and CE. The less polar part was purified again
by flash
chromatography, LH20 (x2) and ion exchange chromatography to give trisulfate
PG2013 as
white fluffy powder after lyophilisation (19.3 mg, 4.4%). 1H NMR (DaO, 400
MHz): two
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rotamers in a ratio of 56:44. major rotamer, 8 7.36-7.11 (m, SH, Ph), 5.946
(d, 1H, J= 3.2, Hl),
4.894 (d, 1H, J= 9.6, HS), 4.748 (d, 1H, J= 16, a-CH2), 4.685 (d, 1H, J= 16, a-
CH2), 4.502 (t,
1H, J= 10.4, 9.6, H3), 4.306 (dd, 1H, J= 9.6, 3.6, H2), 4.005 (t, 1H, J= 9.6,
8.8, H4), 3.869 (s,
2H, b-CHZ), 3.42-3.32 (m, 1H, cyclohexyl-CHN), 1.64-1.36 (m, SH, cyclohexyl-
CH2),
1.20-0.92 (m, SH, cyclohexyl-CH2); minor rotamer, 7.36-7.11 (m, SH, Ph), 5.905
(d, 1H, J=
3.2, H1), 4.578 (d, 1H, J= 10, HS), 4.523 (s, 2H, c-CH2), 4.478 (t, 1H, J=
10.4, 9.6, H3), 4.321
(d, 1H, J= 17.6, d-CH2), 4.281 (dd, 1H, J= 9.6, 3.2, H2), 4.039 (t, 1H, J=
9.6, 9.2, H4), 3.900
(d, 1H, J = 17.6, d-CH2), 3.42-3.32 (m, 1H, cyclohexyl-CHN), 1.64-1.36 (m, SH,
cyclohexyl-CH2), 1.20-0.92 (m, SH, cyclohexyl-CH2). 13C NMR (D20, 100 MHz, no
reference): double-up of each signals due to two rotamers, 169.96 (amide-CON),
169.75
(amide-CON), 168.91 (amide-CON), 168.85 (amide-CON), 135.45 (ipso-Ph), 135.20
(ipso-
Ph), 129.16, 129.07, 128.47, 128.32, 128.20 and 128.13 (meta-, o~tho- and
pay°a-Ph), 95.67
and 95.65 (glu-C1), 77.84 and 77.75 (glu-C3), 73.76 and 73.71 (glu-C2), 70.66
and 70.18 (glu-
C4), 69.23 and 68.70 (glu-CS), 52.87 (a-CH2), 51.12 (c-CH2), 50.32 (d-CH2),
50.02 (b-CH2),
49.25 and 49.00 (cyclohexyl-CHN), 31.89 and 31.86, 25.08 and 25.05, 24.47 and
24.38
(cyclohexyl-CHZ). ES-LRMS (+ve, m/z): C21H2~N2Na3016S3 required 728.02, found
751
(M+Na+), 729 (M+H~); ES-HRMS (+ve, m/z): M+Na+, CZ1H2~N2Naq.016S3 required
751.0113,
found 750.1087; M+H+, C21H28NZNa3016S3 required 729.0294, found 729.0242.
The polar part was purified by LH20 column (x2) and ion exchange column to
give
tetrasulfate PG2012 as an off white powder after lyophilisation (7.6 mg,
1.5%). 1H NMR
(D20, 400 MHz): two rotamers in a molax ratio of 70:30. Major rotamer, 8 7.34-
7.16 (m, SH,
Ph), 5.954 (d, 1 H, J = 3 .6, H 1 ), 5.23 5 (d, 1 H, J = 9.6, HS), 4.904 (d, 1
H, J = 15.6, a-CHa),
4.67-4.57 (overlapped with water, 1H, H3), 4.536 (t, 1H, J= 9.6, 8.8, H4),
4.466 (d, 1H, J=
15.6, a-CH2), 4.394 (dd, 1H, J= 9.8, 3.4, H2), 3.927 (d, 1H, J= 16.8, b-CH2),
3.749 (d, 1H, J=
16.8, b-CH2), 3.30-3.20 (m, 1H, cyclohexyl-CHN), 1.65-1.35 (m, SH, cyclohexyl-
CH2),
1.18-0.92 (m, SH, cyclohexyl-CH2); minor rotamer, 7.34-7.16 (m, SH, Ph), 5.912
(d, 1H, J=
3.4, Hl), 4.77-4.72 (m, 2H, HS and H3 or H4), 4.689 (d, 1H, J= 15.2, c-CH2),
4.67-4.56
(overlapped with water, 1H, H4 or H3), 4.373 (dd, 1H, J= 9.8, 3.4, H2), 4.256
(d, 1H, J= 18.4,
d-CHZ), 4.215 (d, 1 H, J = 15.2, c-CH2), 3 .93 6 (d, 1 H, J = 18.4, d-CH2), 3
.3 6-3 .26 (m, 1 H,
cyclohexyl-CHN), 1.65-1.35 (m, SH, cyclohexyl-CH2), 1.18-0.92 (m, SH,
cyclohexyl-CH2).
i3C NMR (DaO, 100 MHz, no reference): major rotamer, 172.35 (amide-CON),
171.69 (amide-
CON), 137.22 (ipso-Ph), 131.71 and 131.58 (meta- and ortho-Ph), 131.11 (par~a-
Ph), 97.68
(glu-C1), 78.08 (glu-C4), 77.60 (glu-C3), 76.43 (glu-C2), 70.10 (glu-CS),
55.81 (a-CHZ), 53.17
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(b-CH2), 51.77 (cyclohexyl-CHN), 34.17 (cylcohexyl-CH2), 27.56 (cylcohexyl-
CH2), 27.13
(cylcohexyl-CH2); minor rotamer (only typical peaks shown), 97.65 (glu-C1),
78.22 (glu-C4),
77.77 (glu-C3), 76.33 (glu-C2), 71.01 (glu-CS), 53.26 (d-CHa), 53.79 (c-CH2),
52.10
(cyclohexyl-CHN), 34.28 (cylcohexyl-CH2), 27.52 (cylcohexyl-CH2), 27.08
(cylcohexyl-CH2).
ES-MS (+ve, m/z): C21Hz6N2Na~0i9S4 required 829.96, found 853 (M+Na~), 831
(M+H'). ES-
HRMS (+ve, m/z): M+Na+, C21H26N2NasOi9S4 required 852.9501, found 852.9334;
M+H+,
C21H2~N2Na~019S4 required 830.9682, found 830.9635.
Example 10: PG2064
Step a: 2-(N-acetyl-N-cyclohexyl)amiv~o-N-(methyl 2, 3, 4-tai-O-benzyl-6-deoxy-
a-D-
mannopy~anos-6 yl)acetamide
Following the general procedure for the Ugi reaction, a solution of each of
the
following four reagents: acetic acid (2 M in MeOH, 60 ~,L, 119 qmol),
cyclohexylamine (2 M
in MeOH, 60 ~,L, 119 ~.mol), formaldehyde (2 M in MeOH, 60 ~.L, 119 ~,mol) and
methyl
2, 3, 4-tai-O-berzzyl-6-deoxy-6-isocyaho-oc-D-manv~opyf°anoside (0.721
M in CHC13, 150 ~,L, 108
qmol) was loaded into a 4 mL sample vial and the mixture stirred at 60
°C for 19 h. The
volatiles were removed under reduced pressure and purified by flash
chromatography (gradient
elution with hexane-EtOAc 4:1 to 1:4) to afford the title compound as a
colourless gum, 42 mg,
60% (Rf = 0.49, EtOAc). 1H NMR (CDCl3, 400 MHz): two rotamers in a ratio of
72:28. ~
7.38-7.26 (m, 15H, 3 x C6H5), 6.933 (t, 72% x 1H, J= 4.4, NH in major
rotamer), 6.357 (t,.
28% x 1H, J= 5.8, NH in minor rotamer), 4.91-4.41 (m, 7H, sugar-H1 and 3 x
PhCH2), 4.04-
3.42 (m, 9H, sugar-H2-6, NCH2C0 and cyclohexyl-CH), 3.305 (s, 72% x 3H, CH30
in major
rotamer), 3.266 (s, 28% x 3H, CH30 in minor rotamer), 2.067 (s,'72% x 3H,
CH3C0 in major
rotamer), 2.012 (s, 28% x 3H, CH3C0 in major rotamer), 1.85-1.00 (m, 10H,
cyclohexyl-CH2).
Step b: 2-(N-acetyl-N-cyclohexyl)ami~zo-N-( methyl 6-deoxy -2, 3, 4-tai-O-
sulfo-a,-D-
nZauuopyranos-6 yl)acetamide, trisodium salt (PG2064)
Following the general procedure for deprotection of benzyl ethers, a mixture
of the
above tribenzyl ether (42 mg, 0.065 mmol), 20% palladium on activated charcoal
(22 mg) in
MeOH (2 mL) was stirred under hydrogen atmosphere at 50 psi for 10 h. General
work-up
gave the triol intermediate as a colourless gum. Following the general
procedure for
sulfonation, the triol was sulfonated (sulfur trioxide trimethylamine complex,
60 °C, 19 h) and
the crude was evaporated. The residue was purified via sequential SEC (Bio Gel
P-2 followed
by LH20). The pure product was converted to the sodium salt by passing through
an ion
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exchange column to give the title compound as a white fluffy powder after
lyophilisation (3.1
mg, 7.0%, two steps). 1H NMR (D20, int. ref. acetone at 2.05, 400 MHz): two
rotamers in a
ratio of 63:37. b 4.844 (s, 1H, sugar-H1), 4.678 (s, 1H, sugar-H2), 4.51-4.45
(m, 1H,
sugar-H3), 4.240 (t, 37% x 1H, J= 9.6, sugar-H4 in minor rotamer), 4.214 (t,
63% x 1H, J=
9.6, sugar-H4 in major rotamer), 3.980 (s, 37% x 2H, COCH2N in minor rotamer),
3.825 (s,
63% x 2H, COCH2N in major rotamer), 3.78-3.59 (m, 3H, sugar-H5, one sugar-H6
and
cyclohexyl-CH), 3.31-3.17 (m, 4H, one sugar-H6 and CH30 [3.253, s, 3H]), 2.060
(s, 67% x
3H, CH3C0 in major rotamer), 1.900 (s, 33% x 3H, CH3C0 in minor rotamer), 1.70-
0.88 (m,
10H, cyclohexyl-CH2).
Example 11: PG2068
Step a
Following the general procedure for the Ugi reaction, monomethyl succinate
(15.7 mg,
0.119 mmol) and a solution of each of the following three reagents: ethylamine
(2 M in MeOH,
60 ~L, 119 ~mol), formaldehyde (2 M in MeOH, 60 ~L, 119 ~,mol) and ~Zetlzyl 2,
3, 4-tai-O-
be~zyl-6-deoxy-6-isocya~co-a-D-mahhopyranoside (0.721 M in CHCl3, 150 ~.L, 108
~,mol) was
loaded into a 2 mL sample vial and the mixture stirred at r.t. for 19 h. The
volatiles were
removed under reduced pressure and purified by flash chromatography (gradient
elution with
hexane-EtOAc 1:1 to 1:4 then EtOAc) to afFord pure product as a colourless gum
(46.8 mg,
65%). 1H NMR (CDC13, 400 MHz): two rotamers in a ratio of 73:27. 8 7.38-7.25
(m, 15H,
Ph), 6.598 (t, 73% x 1H, J= 6, NH), 6.529 (t, 27% x 1H, J= 6, NH), 4.93-4.61
(m, 7H,
sugar-H1 and 3 x PhCH2), 4.14-3.24 (m, 16H, sugar 6 x H, NCHZCO, 2 x CH3O
[singlets at
3.660 and 3.301, 73%; 3.648 and 3.276, 27%] and ethyl-CH2), 2.70-2.42 (m, 4H,
COCH2CH2C0), 1.138 (t, 73% x 3H, J= 7, ethyl-CH3), 1.014 (t, 27% x 3H, J= 7,
ethyl-CH3).
i3C (100 MHz, CDC13, 8 77.0): major rotamer, 173.32, 171.73, 168.94, 138.20,
138.17, 138.09,
128.22, 128.18, 128.10, 127.76, 127.63, 127.49, 127.46, 98.94, 79.97, 75.38,
75.01, 74.84,
73.01, 72.01, 70.10, 54.63, 51.63, 49.84, 43.59, 39.63, 28.99, 27.24, 13.45.
minor rotamer
(only non-overlapped peaks), 173.26, 171.54, 168.01, 128.27, 127.69, 127.55,
98.98, 79.85,
75.14, 74.40, 72.86, 71.92, 69.97, 54.74, 50.91, 49.72, 42.10, 28.90, 27.83,
12.29.
Step b (PG2068)
Following the general procedure for deprotection of benzyl ethers, a mixture
of the
above tribenzyl ether (46.8 mg, 0.0706 mmol), 20% palladium on activated
charcoal (30 mg) in
MeOH (3 mL) was stirred under hydrogen atmosphere at 50 psi for 2 h. General
work-up gave
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the triol intermediate as a colourless gum. Following the general procedure
for sulfonation, the
triol was sulfonated (sulfur trioxide trimethylaxnine complex, 60 °C,
19 h). The residue was
dissolved in 1M NaOH (3 mL, 0.16 M). The mixture was stirred at room
temperature
overnight and concentrated under reduced pressure. The residue was purified
via sequential
SEC (Bio-Gel P-2 followed by LH20). The pure product was converted into the
sodium salt by
passing through an ion exchange column to give the PG2068 as a white powder
(4.9 mg, 9.8%,
two steps). 1H NMR (D20, int. ref. acetone at 2.05, 400 MHz): two rotamers in
a ratio of
70:30. 8 4.86-4.84 (m, 1H, sugar-H1), 4.69-4.67 (m, 1H, sugar-H2), 4.50-4.46
(m, 1H,
sugar-H3), 4.28-4.19 (m, 1H, sugax-H4), 4.072 (s, 30% x 2H, 2H of COCH2N in
minor
rotamer), 3.987 (d, 35% x 2H, J= 16.8, 1H of COCH2N in major rotamer), 3.815
(d, 35% x
2H, J= 16.8, 1H of COCH2N in major rotamer), 3.79-3.69 (m, 2H, sugar-HS and
one sugar-
H6), 3.44-3.18 (m, 6H, one sugar-H6, ethyl-CHZ and CH30 [3.245, s, 3H]), 2.633
(t, J= 6.8)
and 2.45-2.37 (m, total 4H, COCH2CHZC02), 1.054 (t, 70% x 3H, J= 7.2, CH30),
0.906 (t,
30% x 3H, J= 7.2, CH30).
Example 12: PG2075
Step a
3-Chlorophenylacetic acid (223 mg, 1.307 mmol) was dissolved in MeCN (3 mL).
Ammonia solution (28%, 0.26 mL, 3.8 mmol) was added. The mixture was swirled
for a while
and evaporated in vacuo. The residue was suspended in MeCN (3 mL), filtered
and the white
solid was washed with MeCN and freeze-dried to afford ammonium 3-
chlorophenylacetate
(0.195 g, 80%).
Following the general procedure for the Ugi reaction, the above ammonium salt
(22.5
mg, 0.120 mmol) and a solution of each following two reagents: formaldehyde (2
M in MeOH,
60 ~.L, 119 ~.mol) and 2-isocyanoethyl 2, 3, 4, 6-tet~~a-O-bev~zyl-a,-D-
mannopyranoside (0.762 M
in CHC13, 157 ~.L, 120 ~,mol) was loaded into a 2 mL sample vial and the
mixture stirred at r.t.
for 19 h. The volatiles were removed under reduced pressure and the residue
purified by flash
chromatography to give the product as a colourless gum (34.8 mg, 37%). 1H NMR
(CDCl3,
400 MHz): 8 7.39-7.06 (m, 24H, 4 x C6H5 and 1 x C6H4), 6.731 (t, 1H, J= 6.0,
NH), 4.878 (d,
1H, J= 10.8, a-CH2), 4.844 (d, 1H, J= 2.0, sugar-H1), 4.770 (d, 1H, J= 12.4, b-
CH2), 4.723 (d,
1H, J= 12.4, b-CH2), 4.640 (s, 2H, c-CH2), 4.589 (d, 1H, J= 11.6, d-CH2),
4.535 (d, 1H, J=
11.6, d-CHZ), 4.505 (d, 1H, J= 10.8, a-CHZ), 4.446 (d, 1H, J= 14.8, e-CHa),
4.362 (d, 1H, J=
14.8, e-CH2), 3.90-3.51 (m, 12H), 3.38-3.29 (m, 1H). 13C (100 MHz, CDC13, 8
77.0): 169.67,
166.81, 138.27, 138.11, 137.70, 135.16, 134.31, 129.82, 129.40, 128.34,
128.31, 128.01,
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_2'J-
127.85, 127.78, 127.70, 127.68, 127.60, 127.53, 127.46, 98.90, 79.96, 75.08,
75.04, 74.71,
73.58, 72.73, 72.19, 72.13, 69.78, 68.49, 63.17, 40.25, 39.27.
Step b (PG2075)
Following the general procedure for deprotection of benzyl ethers, a mixture
of the
above tetrabenzyl ether (34.8 mg, 0.0439 mmol), 20% palladium on activated
charcoal (26 mg)
in MeOH (2 mL) was stirred under hydrogen atmosphere at 50 psi for 2 h.
General work-up
gave the tetrol intermediate as a colourless gum. Following the general
procedure for
sulfonation, the above tetrol was sulfonated . The residue was purified via
SEC (Bio-Gel P-2).
The pure product was converted into the sodium salt by passing through an ion
exchange
column to give PG2075 as a white fluffy powder after lyophilisation (10.6 mg,
28%, two
steps). 1H NMR (D20, int. ref. acetone at 2.05, 400 MHz): 8 7.30-7.11 (m, 4H,
Ar), 5.00-4.97
(m, 1H, sugar-H1), 4.72-4.28 (m, 3H, sugar-H2, H3 and H4), 4.21-3.30 (m, 11H,
sugar-H5, H6
and 4 x CH2).
Example 13: PG2014
Step a
Following the general procedure for the Ugi reaction, 2-(benzyl3, 4, 6-tai-O-
benzyl-a-D-
manv~opyy~anoside-2 yl)acetic acid (50 mg, 0.0835 mmol) and a solution of each
following
three reagents: benzylamine (2 M in Me.OH, 41.8 ~.L, 0.0835 mmol),
formaldehyde (2 M in
MeOH, 41.8 ~,L, 0.0835 mmol) and 2-isocyanoethyl 2, 3, 4, 6-tetra-O-be~zyl-a,-
D-
manhopy~anoside (0.415 M in MeOH, 201.4 ~L, 0.0835 mmol) was loaded into a 2
mL sample
vial and the mixture stirred at r.t. for 19 h. General work-up gave the
product as a colourless
gum (38.9 mg, 36%). 1H NMR (400 MHz): two rotamers around amide CO-NH single
bond in
a ratio of 66:34. 8 7.40-7.05 (m, 45H, 9 x C6H5), 6.66 (t, 0.66H, J= 5.6, CONH-
major rotamer)
and 6.40 (t, 0.34H, J = 5.4, CONH-minor rotamer), 5.104 (d, 0.66H, J = 1.2,
H1I-major
rotamer) and 5.046 (s, 0.34H, Hli-minor rotamer), 4.86-4.20 (m, 21H), 3.97-
3.48 (m, 16H),
3.36 (q, 0.66H, J= 5.6, major rotamer) and 3.26 (q, 0.34H, J= 5.6, minor
rotamer).
Step b (PG2014).
Following the general procedure for the deprotection of benzyl ethers, a
mixture of the
above octabenzyl ether (35 mg, 0.0267 mmol) and 20% palladium on activated
charcoal (10
mg) in EtOH (4 mL) was stirred under hydrogen atmosphere at 50 psi for 2 h.
General work-
up gave the octol intermediate as a colourless gum. Following the general
procedure for
sulfonation, the above octol was sulfonated (sulfur trioxide trimethylamine
complex, 60 °C, 19
h). The residue was purified via SEC (Bio-Gel P-2). The pure product was
converted into the
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sodium salt by passing through an ion exchange column to give PG2014 as a
white powder
(16.6 mg, 44%, two steps). 1H NMR (D20, 400 MHz, complicated due to two
rotamers): 8
7.32-7.14 (m, SH, Ph), 5.80-5.66 (m, O.SH), 5.44-5.39 (m, O.SH), 5.04-4.96 (m,
1.5H), 4.80-
4.20 (m, 9H, overlapped with water), 4.18-3.78 (m, 7.5H), 3.76-3.46 (m, 1.5H),
3.42-2.98 (m,
3.5H).
Example 14: PG2016
Step a
Following the general procedure for the Ugi reaction, tans-1,4-
diaminocyclohexane
(6.3 mg, 0.055 mmol) and a solution of each following three reagents: 2-
(methyl 2, 3, 4-ti-O-
be~czyla-D-mahnopyranoside-6 yl)acetic acid (0.91 M in MeOH, 121 ~,L, 0.11
mmol),
formaldehyde (2 M in MeOH, 55 ~.L, 0.11 mmol) and cyclohexylisocyanide (1 M in
MeOH,
110 ~.L, 0.11 mmol) was loaded into a 2 mL sample vial and the mixture stirred
at r.t. for 5
days. The volatiles were removed under reduced pressure and purified by flash
chromatography (gradient elution with hexanes-EtOAc 2:1 to 1:4) to give the
product as a
colourless gum, 33.0 mg, 43% (Rf = 0.24, DCM-MeOH 95:5 or Rf = 0.48, MeCN-
EtOAc 1:1).
1H NMR (CDCl3, 400 MHz, very complicated due to rotamers): 8 7.50-7.20 (m,
30H, Ph),
6.62 (br s, 1.1H), 6.48 (br s, 0.38H), 5.96 (br d, 0.26H, J= 8), 5.79 (br d,
0.26H, J= 10),
4.92-4.85 (m, 2H), 4.78-4.56 (m, 12H), 4.28-4.22 (m, 2.8H), 4.35-4.06 (m,
1.6H), 3.94-3.56
(m, 19.6H), 3.28 (s, 6H, OMe), 1.88-1.42 (m, 16H), 1.36-1.00 (m, 12H).
Step b. (PG2016).
Following the general procedure for the deprotection of benzyl ethers, a
mixture of the
above hexabenzyl ether (33 mg, 0.0235 mmol) and 20% palladium on activated
charcoal (65
mg) in MeOH (2.8 mL) was stirred under hydrogen atmosphere at 1 atm for 5
days. General
work-up gave the hexol intermediate as a colourless gum. Following the general
procedure for
sulfonation, the above hexol was sulfonated. The residue was purified via
sequential column
chromatography (SEC on Bio-Gel P-2 followed by ion exchange column) to give
PG2016 as a
white powder (12.2 mg, 35%). 1H NMR (D20, 400 MHz): 8 4.97-4.92 (m, 2H, man-
Hl), 4.77-
4.75 (m, 2H, man-H2), 4.58-4.52 (m, 2H, man-H3), 4.46-4.08 (m, 6H, containing
man-H4 at
4.46-4.36, and OCHaCO), 3.98-3.80 (m, 8H, man-H5, man-H6 and NCH2C0), 3.80-
3.32 (m,
6H, containing man-H6 at 3.80-3.64, and cyclohexyl-CH), 3.318 (s, 6H, OMe),
1.82-1.34 (m,
18H, cyclohexyl-CHZ), 1.26-1.00 (m, 10H, cyclohexyl-CHa). ES-MS (+ve, m/z):
CaoH6aN4Na60saSs required 1472.10, found 1495 (M+Na+), 1473 (M+H+). ES-HRMS
(+ve,
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nZ/z): M+Na+, C4oH62N4Na~034S6 required 1495.0853, found 1495.0957; M+H+,
C4oH63NaNa6034S6 required 1473.1034, found 1473.1082.
Example 15: PG2015
Step a
Following the general procedure for the Ugi reaction, 3,3-dimethylglutaric
acid (7.1
mg, 0.0443 mmol) and a solution of each following three reagents: 3-
aminops°opyl 2, 3, 4, 6-
tetra-O-benzyl-a-D-mahnopyf°anoside (0.642 M in MeOH, 138 ~.L, 0.0886
mmol),
formaldehyde (2 M in MeOH, 44.3 ~.L, 0.0886 mmol) and cyclohexylisocyanide (1
M in
MeOH, 88.6 ~.L, 0..0886 mmol) were loaded into a 2 mL sample vial and the
mixture stirred at
r.t. for 5 days. The volatiles were removed under reduced pressure and
purified by flash
chromatography (gradient elution with hexanes-EtOAc 2:1 to 1:4) to give the
product as a
colourless gum, 32.3 mg, 46% (Rf = 0.45, hexane-EtOAc 1:3). 1H NMR (CDC13, 400
MHz,
very complicated due to rotamers): S 7.38-7.21 (m, 40H, Ph), 7.003 (d, 0.41H,
J= 7.7), 6.930
(br s, 0.21H), 6.726 (d, 0.4H, J= 8.4), 6.597 (d, 0.73H, J= 8.8), 6.487 (br s,
0.25H), 4.85-4.78
(m, 4H), 4.76-4.59 (m, 10H), 4.54-4.45 (m, 4H), 4.00-3.62 (m, 20H), 3.46-3.14
(m, 6H),
2.52-2.22 (m, 4H), 1.90-1.52 (m, 15H), 1.34-1.00 (m, 15H).
Step b (PG2015).
Following the general procedure for the deprotection of benzyl ethers, a
mixture of the
above hexabenzyl ether (32.3 mg, 0.0202 mmol), 20% palladium on activated
charcoal (41 mg)
in MeOH (2.8 mL) was stirred under hydrogen atmosphere at 1 atm for 5 days.
General work-
up gave the octol intermediate as a colourless gum. Following the general
procedure for
sulfonation, the above octol was sulfonated. The residue was purified via SEC
(Bio-Gel P-2)
to give PG2015 as a white powder (12.6 mg, 37%). 1H NMR (D2O, 400 MHz): ~
5.020 (d, 2H,
J = 1.6, man-H1), 4.759 (br s, 2H, man-H2), 4.66-4.56 (m, 2H, man-H3,
overlapped with
water), 4.46-4.41, (m, 2H, man-H6), 4.265 (t, 2H, J = 9.6, 9.2, man-H4), 4.10-
3.96 (m, 4H,
man-HS and man-H6), 4.05-3.14 (m, 14H, NCHaCO, cyclohexyl-CH and NCH2CH2CH20),
2.50-2.11 (m, 4H, CCHZCO), 1.84-1.42 (m, 14H, cyclohexyl-CH2 and NCH2CH2CHa0),
1.24-0.93 (m, 16H, cyclohexyl-CH2 and Me). ES-MS (+ve, m/z): C41H93N1103757 (7
x
S03NH4) required 1556, found 1578 (M+Na~, 1556 (M+H+). ES-HRMS (+ve, m/z):
M+H+
C41H93N1103~5~ required 1556.3857, found 1556.3783.
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Example 16: PG2155.
Ethyl 2, 6-Di-O-be~rzyl-3, 4-di-O-sulfo-~ D-galactopyra~coside, disodium salt
(PG2155)
The title compound was obtained from ethyl 2, 6-di-O-benzyl-3, 4-di-O-sulfo-
,~i D
galactopyf°anoside [23] nia the general sulfonation procedure as a
colourless powder. 1H NMR
(400 MHz, D20) ~ 1.10 (dd, 3 H; CH2CH3); 2.49-2.66 (m, 2H, CHZCH3); 3.59 (dd,
1 H, Jl,aa,s
9.7 Hz; H-2); 3.65 (dd, 1 H, J5,6a 3.3, J6~,66 11.0 Hz; H-6a); 3.68 (dd, 1H,
.15,66 4.0 Hz; H-6b);
3.83 (m, 1 H; H-5); 4.36 (dd, 1 H, J3,4 3.0 Hz; H-3); 4.46 (s, 2 H; CHZPh);
4.48 (d, 1 H; H-1);
4.60, 4.75 (AB quartet, J 10.3 Hz; CH2Ph); 4.85 (dd, 1 H; J4,5 0.0 Hz; H-4);
7.22-7.29,
7.37-7.39 (2 m, 10 H; ArH).
Example 17: PG2163.
Step a: Methyl 4-O-allyl-6-azido-6-deoxy-2, 3-di-O-isop~opylidene-a D-
mavcnopyranoside
A solution of methyl 6-azido-6-deoxy-a D-mahnopy~a~coside (311 mg, 1.419 mmol)
in
2,2-dimethoxypropane (4.7 mL, 0.3 M) was treated with (~)-camphor-10-sulfonic
acid (16 mg,
0.0709 mmol, 5 mol%). The mixture was stirred at r.t. for 1 h. TLC indicated
the complete
conversion to the product (Rf = 0.40, EtOAc-hexane = 17:83). The mixture was
basified by
addition of sat. NaZC03 (aq. sol.) and evaporated under vacuum. The residue
was extracted
with EtOAc (30 mL) and the EtOAc solution washed with brine, dried (MgS04).
Filtration and
evaporation gave a gum, which was co-evaporated with toluene once. The final
colourless
gum was dissolved in anhydrous DMF (3.5 mL, 0.4 M) and stirred with NaH (60%
dispersion
in mineral oil, 163 mg, 4.257 mmol, 3 eq) for 1h. Allyl bromide (360 ~,L,
4.257 mmol, 3 eq)
was added and the mixture stirred at r.t. for another 6 h, treated with
methanol (1 mL) and
evaporated to dryness. The residue was purified by silica column
chromatography (2.5x18 cm,
eluted with EtOAc-hexane 1:10 to 1:6) to give the title compound as a
colourless gum (0.281
mg, 66% over 2 steps). 1H NMR (CDC13, 400 MHz): 5.85 (m, 1H, allyl-2'), 5.23
(ddd, 1H,
J2_3'trans= 17.2, .I3'-gern= 3.6, Jl~_3~= 1.6, H3'trans)~ 5.17-5.13 (m, 1H,
H3'~;s), 4.89 (s, 1H, H1), 4.35
(dddd, 1H, Jl~gem = 12.4, Jl~_2~ = 5.2, J= 1.6, H1'), 4.16 (dd, 1H, J2_3 =
5.6, J3_4 = 7.2, H3), 4.09
(d, 1H, H2), 4.05 (dddd, 1H, H1'), 3.67 (ddd, 1H, J4_5= 10.4, J5_6~= 6.8,
J5_6en= 2.4, HS), 3.48
(dd, 1H, J6~_6ea = 13.2, H6eq), 3.40 (dd, 1H, J6~_seq = 13.2, JS_6~ = 6.8,
H6ax), 3.33 (dd, 1H,
HS), 1.50 (s, 3H, Me), 1.30 (s, 3H, Me). 13C NMR (CDC13, 100 MHz): 134.4,
117.1, 109.2,
98.0, 78.2, 76.2, 75.6, 71.5, 68.0, 54.9, 51.6, 27.8, 26.1.
Step b: Methyl 4-O-allyl-6-azido-6-deoxy-a D-mauuopyy~av~oside
Methyl 4-O-allyl-6-azido-6-deoxy-2, 3-di-O-isopr~opylide~ce-a D-
mannopyr~auoside (56
mg, 0.187 mmol) was dissolved in MeCN-MeOH-HZO (3 mL, 3 mL and 0.2 mL
respectively)
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and treated with p-toluenesulfonic acid monohydrate (7 mg, 0.0374 mmol, 20
mol%). The
mixture was stirred at r.t. for 5 h and triethylamine (0.4 mL) added. The
mixture was
evaporated and the residue purified by column chromatography (silica 1x18 cm,
eluted with
EtOAc-hexane 1:6 to 2:1) to give the product as a colourless waxy solid (34.8
mg, 72%). 1H
NMR (CDC13, 400 MHz): 5.91 (m, 1H, allyl-H2'), 5.28 (ddd, 1H, J2~_3'trans =
16.8, J3~trans-3'c~s =
3.0, Jl~_3~ = 1.6, allyl-H3'trans), 5.20 (ddd, 1H, J2~_3'cIs = 10.0, J3~trans-
3'cIs = 3.0, Jl~_3~ = 1.6,
allyl-H3'cis), 4.72 (ddd, 1H, Jl_2 = 2.0, H1), 4.28 (dddd, 1H, Jgem = 12.4,
Jl~_2~ = 5.6, J= 1.6,
allyl-1'), 4.13 (dddd, 1H, allyl-1'), 3.92 (dd, 1H, J2_3 = 3.6, H2), 3.88 (dd,
1H, J3_4 = 9.6, H3),
3.70 (ddd, 1 H, J4_5 = 9.6, JS_6~ = 5.6, J5_6eq = 2.8, HS), 3.54-3.44 (m, 3H,
H4, H6ax and H6eq),
3.39 (s, 3H, Me0), 2.62 (br s, 2H, OH).
Step c: Methyl 4-O-allyl-6-azido-2-O-benzyl-6-deoxy-2, 3-di-O-sulfohato-a D-
mannopy~ahoside disodium salt (PG2163)
Methyl 4-O-allyl-6-azido-6-deoxy-a D-man~copyranoside was sulfonated according
to
the standard procedure to yield the title compound as a white powder, 55 mg
(89%). Rf = 0.20
(EtOAc-MeOH-H20=10:2:1). 1H NMR (D20, 400 MHz): 5.88-5.76 (m, 1H, allyl-2'),
5.20 (d,
1H, J2~_3'trans = 17.2, allyl-3'trans), 5.12 (d, 1H, J2~_3'cis = 10.0, allyl-
3'cis), 4.91 (s, 1H, H1), 4.65
(br s, 1 H, J2_3 = 3.2, H2), 4.48 (dd, 1 H, J2_ = 3.2, J3_4 = 9.2, H3 ), 4.21
(dd, 1 H, Jgem = 11.6, Jl~_2~ _
5.6, allyl-1'), 4.00 (dd, 1H, Jl~_2~= 6.4, allyl-1'), 3.71-3.67 (m, 1H, HS),
3.58 (dd, 1H, Jgea_6a~c=
13.6, Jg_6eq = 2.0, H6eq), 3.58 (dd, 1H, J4_5 = 9.6, H4), 3.46 (dd, 1H, J6eq-
s~ = 13.6, JS_6~ = 5.6,
H6ax), 3.30 (s, 3H, Me0). 13C NMR (D20, 100 MHz, internal MeOH at 49.05 ppm):
133.9,
119.2, 98.4, 76.1, 75.3, 74.2, 73.1, 70.7, 55.4, 50.8.
Example 18: PG2160, PG2161 and PG2173.
Step a: Methyl 6-azido-6-deoxy-2, 3-di-O-behzylide~ce-a D-maunopyranoside
Methyl 6-azido-6-deoxy-a-D-mannopyranoside (1.011 g, 4.61 mmol) was dissolved
in
anhydrous DMF (9 mL) and acetonitrile (9 mL). Benzaldehyde dimethyl acetal
(1.38 mL, 9.22
mmol, 2 eq) and (~)-camphor-10-sulfonic acid (214 mg, 0.922 mmol, 20 mol%)
were added in
that order. The mixture was stirred under house vacuum at 60 °C
(external) overnight, and the
volatile materials were removed on rotavap. The residue was loaded on silica
gel and purified
by column (silica 2.5x20 cm, gradient elution with hexane-ethyl aceate 10:1,
9:1, 7:1, 5:1, 4:1,
3:1 to 2:1). The fractions were pooled into two parts. The less polar part (Rf
= 0.55 and 0.51,
hexane-EtOAc 3:1) was a mixture of 4-acetals and the polar part was mainly the
4-OH
product. The mixtures were combined and evaporated. The residue was re-
dissolved in
dichloromethane (50 mL) and stirred with 1M ammonium chloride solution (50
mL). The less
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polar spots were slowly disappeared and converted into the polar product.
However, the
conversion was not further improved after 40 min: Thus the dichloromethane
phase was
separated and stirred with 0.5 M HCl solution (50 mL) for another 20 min. TLC
indicated no
further change. The DCM phase was separated and washed with brine-1M NaOH,
dried
(MgS04). The dried DCM solution was filtered, evaporated and the residue was
purified by
silica column as above to give the title compound as a colourless gurniny
solid (0.543 g, 38%,
Rf = 0.29, EtOAc-hexane = 1:3). 1H NMR (CDC13, 400 MHz): two benzylidene
epimers in a
ratio of 1:1. 7.49-7.36 (m, SH, C6Hs), 6.126 and 5.902 (2xs, 1H, benzylidene-
CH), 5.066 and
4.987 (2xs, 1H, sugar-Hl), 4.396 (dd, O.SH, J= 6.4, 5.6, sugar-H3), 4.248 (dd,
O.SH, J= 6.4,
6.0, sugar-H3), 4.214 (dd, O.SH, J = 6.0, sugar-H2), 4.102 (dd, O.SH, J = 4.8,
sugar-H2),
3.86-3.38 (m, 4H), 3.466 and 3.424 (2xs, 3H, CH30), 2.984 (br s, 1H, OH). 13C
NMR (CDC13,
100 MHz): 138.01, 136.41, 129.38, 128.99, 128.24, 128.12, 126.41, 125.86,
103.85, 102.60,
97.74, 97.53, 79.36, 77.75, 77.47, 74.81, 70.00, 68.91, 68.42, 66.97, 54.78,
51.18 and 51.10.
Step b: Methyl 6-azido-3-O-benzyl-6-deoxy-a D-mannopyf°anoside and
methyl 6-azido-2-O-
benzyl-6-deoxy-a D-mannopy~anoside
A solution of methyl 6-azido-6-deoxy-2,3-di-O-benzylidene-a-D-mannopyranoside
(240 mg, 0.781 mmol) in DMF (7.8 mL, 0.1 M) was treated with sodium
cyanoborohydride
(589 mg, 9.37 mmol, 12 eq) and molecular sieve 3A (1 g). The mixture was
stirred at r.t. for
min, then at 70 °C while TFA (0.361 mL, 4.686 mmol, 6 eq) was added
slowly. After
20 addition, the mixture was stirred at 70 °C for 6 h, cooled to 0
°C and basified by addition of
solid Na2C03. The cold mixture was filtered and the cake washed with EtOAc.
The filtrate
and washings was extracted once with sat. Na2C03. Evaporation gave a gum,
which was
purified by column chromatography (silica 2.5x18 cm, eluted with EtOAc-hexane
1:4 to 1:1) to
give methyl 6-azido-3-O-benzyl-6-deoxy-a D-mannopy~anoside (colourless gum, 64
mg, 26%,
Rf = 0.45, EtOAc-hexane=1:1); 1H NMR (CDC13, 400 MHz): 7.41-7.32 (m, SH, Ph),
4.78 (d,
1 H, Jl _Z = 1.6, H 1 ), 4.70 (d, 1 H, Jgen, = 12.0, CH2), 4.56 (d, 1 H, CH2),
4.02 (dd, 1 H, J2_3 = 3.2,
H2), 3.78 (dd, 1H, J3_4 = 8.4, J4_s = 10.0, H4), 3.73 (ddd, 1 H, Js_6~ = 6.0,
Js_6e9 = 3.2, HS), 3.64
(dd, 1H, H3), 3.52 (dd, 1H, J6~_6eq = 13.2, H6eq), 3.47 (dd, 1H, J6~_6e9 =
13.2, Js_6~ = 6.0,
H6ax), 3.40 (s, 3H, Me0), 2.21 (br s, 2H, 2xOH) and methyl 6-azido-2-O-benzyl-
6-deoxy-a D-
mannopy~anoside (colourless waxy solid, 62 mg, 26%, Rf = 0.27, EtOAc-
hexane=l:l). 1H
NMR (CDCl3, 400 MHz): 7.38-7.28 (m, SH, Ph), 4.78 (s, 1H, H1), 4.71 (d, 1H, J=
11.6, CHa),
4.53 (d, 1H, J= 11.6, CH2), 3.75-3.61 (m, 4H, H2, H3, H4 and HS), 3.53 (dd,
1H, J= 13.2, 1.5,
H6eq), 3.46-3.40 (dm, 1H, J= 13.2, H6ax), 3.38 (s, 3H, Me0), 2.83 (br s, 2H,
2xOH).
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Step c: Methyl 6-azido-~-O-benzyl-6-deoxy-2, 3-di-O-sulfonato-a D-
ynannopyranoside
disodium salt (PG2160)
Methyl 6-azido-2-O-benzyl-6-deoxy-a D-mannopyranoside was sulfonated according
to
the standard procedure to yield the title compound as a white powder, 64 mg,
59%. 1H NMR
(D20, 400 MHz): 7.38-7.26 (m, SH, Ph), 4.72 (d, 1H, Jgen, = 12.0, CH2Ph), 4.59
(d, 1H, Jl_2 =
2.0, H1), 4.58 (d, 1H, CH2Ph), 4.49 (d, 1H, JZ_3 = 2.8, J3_4 = 9.6, H3), 4.44
(dd, 1H, J4_5 = 9.6,
H4), 4.07 (dd, 1H, H2), 3.78 (ddd, 1H, JS_6~ = 5.6, Jg_6eq = 2.4, HS), 3.62
(dd, 1H, J6~_6eq =
13.2, H6eq), 3.51 (dd, 1H, J6~_6eq= 13.2, Js-sue= 5.6, H6ax), 3.22 (s, 3H,
Me0).
Step d: Methyl 6-azido-3-O-benzyl-6-deoxy-2, 4-di-O-sulfonato-a D-
nZannopyranoside
disodiunz salt (PG2161)
Methyl 6-azido-3-O-benzyl-6-deoxy-a D-mannopyranoside (64 mg) was sulfonated
according to the standard procedure to yield the title compound as a white
powder, 58 mg,
66%. 1H NMR (D20, 400 MHz): 7.42-7.21 (m, SH, Ph), 4.92 (d, 1H, Jl_Z = 2.4,
Hl), 4.67 (d,
1H, Jge°,= 12.4, CH2Ph), 4.60 (d, 1H, CH2Ph), 4.56 (dd, 1H, J2_3= 3.2,
H2), 4.35 (dd, 1H, J3_4=
9.6, J4_5 = 9.6, H4), 3.80 (dd, 1H, H3), 3.75 (ddd, 1H, JS_6~ = 6.0, JS_6ea =
2.8, HS), 3.66 (dd,
1 H, J6~_6eq = 13.4, H6eq), 3.54 (dd, 1 H, J6~_6eq = 13.4, J5_6~ = 6.0, H6ax),
3.29 (s, 3H, Me0).
Step e: Methyl 6-~l '-(4 phenyl)triazolylJ-2-O-benzyl-6-deoxy-2, 3-di-O-
sulfonato-a D-
mannopyranoside disodium salt (PG2173)
Methyl 6-azido-2-O-benzyl-6-deoxy-2, 3-di-O-sulfonato-a D-mannopyranoside
disod
ium salt was subjected to the Huisgen reaction general procedure using phenyl
acetylene to
yield the title compound as a white powder 6.8 mg, 67%, Rf = 0.34, EtOAc-MeOH-
H20 =
10:2:1. 1H NMR (D20, 400 MHz): 8.26 (s, 1H, triazole), 7.64-7.60 (m, 2H), 7.37-
7.15 (m,
8H), 4.89 (dd, 1H, J= 14.4, 2.8, H3), 4.67 (d, 1H, J= 12.0, PhCH2), 4.57-4.39
(m, SH, PhCH2,
H 1, H4 and H6eq and H6ax), 4.04 (dd, 1 H, J = 2.8, 2.0, H2), 3 .99 (ddd, 1 H,
J = 9.2, 8. 8, 2.4,
HS), 2.83 (s, 3H, Me0).
Example 19: PG2170
Allyl 6-azido-2, 3-O-disulfonato-6-deoxy-4-O-(1-naphthylmethyl)-a D-
mannopyranoside
disodium salt (containing 10% of 2-naphthylmethyl isomer) (PG2170)
The title compound, prepared analogously to PG2163 beginning with allyl 6-
azido-6-
deoxy-a D-mannopyranoside, was obtained as a white powder 87.5 mg, 87%, Rf =
0.28
(major), 0.22 (minor), EtOAc-MeOH-Ha0 =10:2:1. 1H NMR (DaO, 400 MHz): 7.87 (d,
1H, J
= 8.4, naphthyl), 7.65-7.54 (m, 2H, naphthyl), 7.33-7.19 (m, 4H, naphthyl),
5.68 (ddt, 1H,
Jallyl2'-3'trans = 22.0, Jallyl2'-3'cis = 10.8, Jauym-z' = 6.0, allyl-2'),
5.23 (AB quartet, 2H, Jgem = 12.0,
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naphthyl-CH2), 5.17-5.02 (m, 3H, allyl-3' and H1), 4.74 (dd, 1H, Jl_Z= 2.0,
J2_3 = 3.2, H2), 4.64
(d, 1H, J3_4= 9.6, H3), 3.90 (dd, 1H, Jallyll'gem= 13.2, J= 6.0, allyl-1'),
3.81 (dd, 1H, allyl-1'),
3 .64 (t, 1 H, J~_s = 9.6, H4), 3.3 9 (ddd, 1 H, Jg_6eq = 2.0, Js-s~ = 5.2,
HS), 2.78 (dd, 1 H, Jge9_6ax =
13.6, H6eq), 2.68 (dd, 1H, Js_6~= 5.2, J(eq_6~= 13.6, H6ax). Typical signals
for minor isomer
2-naphthylmethyl derivative: 4.84 (AB quartet, 2H, Jgem= 11.2, naphthyl-CH2),
3.52 (ddd, 1H,
J4_s= 10.0, Js_6eq= 2.4, Js-sue= 6.0, HS), 3.07 (dd, 1H, J6eq_6~= 13.6, H6eq),
2.96 (dd, 1H,
J6eq-6ax= 13.6, Js_6~= 6.0, H6ax). The source of the minor isomer is the
commercial 9:1
mixture of 1- and 2-bromomethylnapthalene used in step a.
Additional compounds were synthesized using appropriate modifications of the
syntheses detailed above in Examples 1 to 19. These additional compounds are
included in the
tables giving the results of biological testing of compounds according to the
invention.
Example 20
Biological Testing of Compounds
Methods
1. Growth Factor Binding
Binding affinities of ligands for the growth factors were measured using a
surface
plasmon resonance (SPR) based solution affinity assay. The principle of the
assay is that
heparin immobilised on a sensorchip surface distinguishes between free and
bound growth
factor in an equilibrated solution of the growth factor and a ligand. Upon
injection of the
solution, the free growth factor binds to the immobilised heparin, is detected
as an increase in
the SPR response and its concentration thus determined. A decrease in the free
growth factor
concentration as a function of the ligand concentration allows for the
calculation of the
dissociation constant, K~. It is important to note that ligand binding to the
growth factors can
only be detected when the interaction involves the hepaxin binding site, thus
eliminating the
chance of evaluating non-specific binding to other sites on the protein. A 1:1
stoichiometry has
been assumed for all protein:ligand interactions.
The preparation of heparin-coated sensorchips, via immobilisation of
biotinylated BSA-
heparin on a streptavidin-coated sensorchip, has been described [24]. Heparin
has also been
immobilised via aldehyde coupling using either adipic acid dihydrazide or 1,4-
diaminobutane.
For each Kd measurement, solutions were prepared containing a fixed
concentration of protein
and varying concentrations of the ligand in buffer. Ligands binding to FGF-1
and VEGF were
measured in HBS-EP buffer (10 mM HEPES, pH 7.4, 150 mM NaCI, 3.0 mM EDTA and
0.005% (v/v) polysorbate 20), while binding to FGF-2 was measured in HBS-EP
buffer
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containing 0.3 M NaCI [24]. Prior to injection, samples were maintained at 4
°C to maximise
protein stability. For each assay mixture, 50-200 ~,L of solution was injected
at 5-40 ~.L/min
and the relative binding response measured. All surface binding experiments
were performed
at 25 °C. The surface was regenerated by injection of 40 ~.L of 4M NaCI
at 40 ~L/min,
followed by injection of 40 ~,L of buffer at 40 ~.L/min.
Sensorgram data were analysed using the BIAevaluation software (BIAcore).
Background sensorgrams were subtracted from experimental sensorgrams to
produce curves of
specific binding, and baselines were subsequently adjusted to zero for all
curves. The relative
binding response for each injection was converted to free protein
concentration using the
equation
LPJ = LPJtotal
y~m
where t° is the relative binding response and ~"t is the maximal
binding response.
Binding equilibria established in solution prior to injection were assumed to
be of 1:l
stoichiometry. Therefore, for the equilibrium,
P+L ~ P~L
where P corresponds to the growth factor protein, L is the ligand, and P~L is
the protein:ligand
complex, the equilibrium equation is
K = ~P~~L~
a ~P.L~
and the binding equation [24] can be expressed as
~P~ _ LP.lorar - (Ka + ~L~ro~r ~' ~P~roral ) + ~Ka + LL~ro~a4l + ~P~~orar ~2 _
[L~mrar LP~roral
The K~ values given are the values fit, using the binding equation, to a plot
of [P]
versus [L]totat. Where Kd values were measured°in duplicate, the values
represent the average
of the duplicate measurements. It has been shown that GAG mimetics that bind
tightly to these
growth factors elicit a biological response in vivo [24].
2. Antiviral Assays.
Selected compounds were tested against two types of herpes simplex virus
(HSV), i.e.,
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HSV-1 and HSV-2, in two assays for inhibition of viral infectivity and cell-to-
cell spread, as
described by Nyberg et al. [25]. Monolayer cultures of African green monkey
kidney cells
(GMK AH1) [26] cultivated in 6-well cluster plates, were used. The viral
strains used were
herpes simplex virus type 1 (HSV-1) KOS321 strain [27] and HSV-2 strain 333
[28].
In both assays the compounds were tested at 200 ~.M.
(i) In the assay of HSV infectivity, the compounds were mixed with the virus,
incubated for 10 min at room temperature and then the mixture was added to
cells,
and kept on cells for 1h only to allow (or not) the virus attachment to/entry
into the
cells. Thus this assay reflects whether or not the compound in question has
the
ability to bind to the virus particles and block its attachment to/entry into
the cells.
An inhibition is manifested as a decreased number of viral plaques.
(ii) The next assay, termed HSV spread, relies on the addition of compound to
the cells
after the virus attachment/entry steps have already occuiTed. This assay
reflects
whether the examined compound has the ability to inhibit virus transmission
from
an infected to an uninfected cell (cell-to-cell spread) and in addition
whether the
compound has the ability to enter the cells and inhibit viral replication.
Lack of
compound activity in the assay of virus infectivity but some activity in the
virus
spread assay suggests that the compound acts by entering the cells and
inhibition of
viral replication step(s). An inhibition is manifested as a reduction in the
size of
viral plaques.
The results (see Table 5) are expressed as % of control, ie., as the number
(infectivity
assay) or the size (spread assay) of viral plaques developed in the presence
of compound
relative to the mock-treated controls (no compound).
Results
The results of the tests as described in the preceding section are presented
in Tables 1 to
5.
Table 1
Re
I
III
RA
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PG Kd aFGFKd bFGFKd VEGF Kd
# FGF-4
RA,Rr,RH,RI=OMe; RB,Re,RE=OS03Na;2019 218 657 912 ~,M
~M ~M
RD=CHZOS03Na; RG=H
RA,Rr,RH=OMe; RB,R~,RE,RI=OS03Na;2037 47.7 507 645 ~M
~,M ~.M
RD=CHZOS03Na; RG=H
Rr,RH,Ri=OH; RA=OMe; RB,Re,RE=OS03Na;2038 77.9 2.10 368 ~M
~,M mM
RD=CHZOS03Na; RG=H
RA=OMe; RF,RH=OH; RB,Re,RE,RI=OS03Na;2039 21.8 3.50 1.27 mM
~M mM
RD=CHZOS03Na; RG=H
Rr,RG,Ri=OH; RD-RA=-CH20-; RB,RE=NHS03Na;2046 6.35 3.70 1.50 mM
mM mM
R~=OBn; RH=H
Rr,RG,RI,Re=OH; RD-RA=-CHZO-; 2047 388 1.95 2.55 mM
RB,RE=NHS03Na; ~,M mM
RH=H
RF,RH,RI=OH; RA=OMe; RB,R~,RE=OS03Na;2063 1.39 2.35 2.59 mM
mM mM
RD,RG=H
Table 2
RPRR, Rs
RQ",.. ~p IV
RN .,~~R~
'. R
~e
RoR~ RM
PG # Kd aFGF Kd bFGF Kd VEGF
RK=OMe; RM,Ra=OS03Na; RQ=OBn; RR=CH3;2023 1.76 4.90 mM 2.27
mM mM
Ra~RL~Rrr~Rr~Rs=H
RK=OMe; RM,Ro=OS03Na; Rr=OBn; RR=CH3;2024 4.73 3.65 mM 6.40
mM mM
RJ,RL,RN,RQ,Rs=H
RK=OMe; RM,RN=OBz; RQ=OS03Na; Rs=CHZOS03Na;
2028 1.10 9.25 mM 1.65
mM mM
RJ,RL,Ro,Rr,RR=H
RK=OMe; RM,Ro=OS03Na; RQ=Oallyl; 2029 1.34 > 10.00 236
RR=CH3; mM mM ~tM
RJ,RL,RN,Rr,Rs=H
Rs-RK=-CH20-; RM=OS03Na; RN=OMe;
RQ=OBn; 2030 317 ~M 4.61 mM
RJ,RL,Ro,Rr,RR=H
RN=O(CHZ)30Ph;RQ=OS03Na; Rs=CHZOS03Na;2040 12.9 7.50 mM 2.44
mM mM
Ra~RK~RL~RM~Ro~Rr~RR=H
RN=OBn;RQ=OS03Na; Rs=CHZOS03Na; 2041 9.38 5.10 mM 1.04
mM mM
Ra~Rx~RL~RM~Ro~Rr~RR=H
RK=OMe; RM=OS03Na; Ro=OH; Rr=Oallyl;
RR=CH3; 2042 3.05 10.7 mM 2.59
mM mM
RJ,RL,RN,RQ,Rs=H
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#
RN=OMe;RQ=OS03Na; RS CHZOS03Na; 2043 6.43 mM 17.4 1.73
mM mM
Ra~Rx~Rr.~RM~Ro~Rr~RR=H
Rx =OMe; RM,Ro=OS03Na; RP=OOCCHZCHZPh;2044 366 ~M 1.55 1.65
RR CH3; mM mM
RJ,RL,RN,RQ,Rs=H
RJ/Rx=H/OMe (anomeric mixture); Rs-RN=-CHZO-;2045 392 ~.M 3.40 1.07
mM mM
RM=OS03Na; RQ=OBn; RL,Ro,Rr,RR=H
Rx=OMe; RN,RM=OH; RQ=OS03Na; Rs=CHzOS03Na;
2048 233 ~M 5.30 796 ~,M
mM
RJ,RL,Ro,RP,RR=H
Rx=OMe; RN,RM=OBn; RQ=OS03Na; Rs=CHZOSO3Na;
2049 1.51 mM 60.0 2.72
~M mM
RJ,RL,Ro,RP,RR=H
Rx=OMe; RM =OSO3Na; RN,RQ=OBn; 2050 3.31 mM 8.25 ~ 10.00
mM mM
Ra~RL~Ro~Rr~RR~Rs=H
Rx=OMe; RM,RN=OBn; RQ=OS03Na; 2051 2.46 mM > 20.4 4.63
mM mM
RJ,RL,Ro,RP,RR,Rs=H
Rx=OMe; RM,Ro=OS03Na; RP=OOCCHZOPh;
RR=CH3; 2052 5.92 mM 4.50 686 ~M
mM
RJ,RL,RN,RQ,Rs=H
Rx=OMe; RM,Ro=OS03Na; RP=Oallyl; 2053 1.30 mM 5.17 343 ~M
RR=CH3; mM
Ra~RL~Rrr~~~Rs=H
Rx=OMe; RM,Ro=OSO3Na; RP=OBz; RR=CH3;2054 454 ~tM 2.73 403 ~.M
mM
Rr~Rr.~Rrr~~~Rs=H
Rx=OMe; RM,Ro=OS03Na; RP=OOCPh(p-OMe);2056 797 ~M 2.45 485 ~,M
RR=CH3; mM
Rr~RL~Rrr~~~Rs=H
Rs-RN= -CH20-; RL=OS03Na; Rp=OBn; 2079 1.92 mM ~ 3.45 1.73
mM mM
RJ~Rx~RM~Ro~Rr~RR=H
Rs-RN= -CHzO-; RM=OS03Na; RQ=OBn; 2080 1.62 mM ~ 11.7 1.36
mM mM
RJ~Rx~RL~Ro~Rr~RR=H
Rx=OMe; RM,Ro=OS03Na; RP=OCHZCyclohexyl;
RR=CH3; 2085 9.60 mM ~ 17.4 8.90
mM mM
RJ,RL,RN,RQ,Rs=H
Rx=OMe; RM,Ro=OS03Na; RP=O(CHZ)30Ph;
RR=CH3; 2086 3.05 1.50
mM mM
RJ,RL,RN,RQ,Rs=H
Rx=OMe; RM,Ro=OS03Na; RP=OH; RR=CH3;2087 566 ~M 2.35 899 ~M
mM
RJ,RL,RN,RQ,Rs=H
Rx=OMe; RM,Ro=OS03Na; RP=O(CHZ)3Ph;
RR=CH3; 2088 676 ~M 3.00 761 ~M
mM
RJ,RL,RN,RQ,Rs=H
Rx=OMe; RM,Ro=OS03Na; RP=OCHz(2-Napthyl);
2089 1.20 mM 2.15 2.33
mM mM
Rx=CHs RJ~RL~Rrr~~~Rs=H
Rx=OMe; RM,Ro=OS03Na; RP=OCHZ(E)CH=CHPh;2090 3.85 mM 2.50 3.02
mM mM
RR=CHs Ra~RL~RN~~~Rs=H
Rx=OMe; RM,Ro=OS03Na; Rr=OCHZ(1-Napthyl);
2091 1.37 mM 1.50 1.98
mM mM
RR=CHs RJ~RL~RN~~~Rs=H
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#
Rx=OMe; RM,Ro=OS03Na; RP=OCHZPh(p-Me);2092 2.70 mM 2.85 mM 2.86
RR=CH3; mM
RJ,RL,RN,RQ,Rs=H
Rx=OMe; RM,Ro=OS03Na; RP=OOCPh(p-NOZ);2093 110 ~,M 1.66 mM 856 ~.M
RR=CH3;
RJ,RL,RN,RQ,Rs=H
Rx=NHCOGHzOPh(2,4-di-Cl); RM,RN,RQ=OS03Na;
2096 35.7 pM 141 pM 20.4 pM
Rs=CHzOS03Na; RJ,RL,Ro,RP,RR=H
RJ=OMe; RL,RN=OBz; RQ=OSO3Na; Rs=CHzOS03Na;2097 127 ~.M 2.05 mM 267 pM
Rx~RM~Ro~Rr~RR=H
Rx=OMe; RM,Ro=OSO3Na; RP=OCHZPh(p-CF3);2098 ~ 7.85 2.65 mM 2.60
RR=CH3; mM mM
RJ,RL,RN,RQ,Rs=H
Rx=OMe; RM,Ro=OS03Na; RP OCHZPh(m-CF3);2099 1.30 mM 2.85 mM 5.70
RR=CH3; mM
RJ,RL,RN,RQ,Rs=H
RJ OMe; RL,RQ=OS03Na; RN=Oallyl; 2100 1.25 mM N 18.1 mM 146
Rs=CHzOallyl; ~M
Rx~RM~Ro~Rr~RR=H
RJ=OMe; RL,RN=OH; Rp=OS03Na; Rs=CHZOSO3Na;2101 77.3 ~M 188 ~.M
Rx~RNt~Ro~Rr~RR=H
Rx=OMe; RM,Ro=OS03Na; RP=OMe; RR=CH3;2102 116 ~,M 1.30 mM 206 ~M
Ra~RL~RN~~~Rs=H
Rx=OMe; RM,Ro=OS03Na; RP=OCHZCyclopropyl;
2103 5.50 mM 4.20 mM 3.00
mM
RR CH3; RJ,RL,RN,RQ,Rs=H
Rx=N3; RM,Ro=OS03Na; RP=OBn; RR=CH3;2104 1.80 mM 2.45 mM 3.30
mM
RJ,RL,RN,RQ,Rs=H
RJ=N3; RM=OS03Na;Ro=OH; RP=OBn; 2105 1.85 mM 2.10 mM 8.30
RR=CH3; mM
Rx~RL~Rrr~~~Rs=H
Rx=OBn; RM,Ro=OS03Na; RP=OMe; RR=CH3;2106 1.31 mM 3.43 mM 1.45
mM
Rr,RL,RN,RQ,Rs=H
Rx=OMe; RM,RN=OOCPh(p-OMe); RQ=OS03Na;
2107 645 ~M
Rs=CHZOS03Na; RJ,RL,Ro,RP,RR=H
Rx=OMe; RM,RN=OCH2(E~CH=CHPh; RQ=OS03Na;2108 563 ~M
Rs=CHZOS03Na; RJ,RL,Ro,RP,RR=H
Rx=OMe; RM,RN=OOCPh(p-NOz); R~,=OS03Na;
2109 441 ~M 1.00 mM
Rs=CHZOS03Na; RJ,RL,Ro,RP,RR=H
Rx-Rs=-OCHZ-; RM=3-phenyl-[1,2,3]triazol-1-yl;
2110 2.80 mM
RN,RQ=OS03Na; RJ,RL,Ro,RP,RR=H
Rx=OMe; RM,Ro=OS03Na; RP=OOCPh(3,4-di-Cl);2111 1.30 mM 1.60 mM
Rx=CHs~ Rr~RL~RN~~~Rs=H
Rx=OMe; RM,Ro=OS03Na; RP=OOCCHZPh(rn-Cl);
2112 2.30 mM 2.55 mM 4.10
mM
RR=CH3; RJ,RL,RN,RQ,Rs=H
Rx=OMe; RM,Ro=OS03Na; RP=OOCCHzPh(3,4-di-Cl);
2113 1.45 mM 1.60 mM 1.60
mM
RR CH3; RJ,RL,RN,RQ,Rs=H
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PG Kd aFGF Kd bFGF Kd VEGF
#
Rx=OMe; RM,Ro=OS03Na; Rr=OOCCHzPh(p-CF3);2114 3.85 mM 2.30 mM 888 pM
Rx=CHs RJ~RL~RN~~~Rs=H
RJ=OMe; RL=OMe; RN=Oallyl; RQ=OS03Na;2129 5.40 mM 9.10 mM 3.00
mM
Rs=CHzOS03Na; Rx,RM,RO,Rr,RR=H
RJ=OMe; RL,RN=OS03Na; RQ=OMe; Rs=CHZOallyl;
2 130 1.05 mM 7.85 mM 361 pM
Rx~RM~Ro~Rr~RR=H
Rx=OMe; RM,Ro=OS03Na; Rr=OEt; RR=CH3;2131 8.30 mM 7.20 mM ~ 21.8
mM
RJ,RL,RN,RQ,Rs=H
RJ=OMe; RL,RN,Rp=OAc; Rs=CHZOS03Na;2137 > 20.0 ~ 8.80 mM
mM
Rx~RM~Ro~Rr~RR=H
RJ OMe; RL,RQ=OS03Na; RN=Oallyl;1~=CHZOC(Ph3);2139 884 ~M ~ 2.70 mM 383
. pM
Rx~RNt~Ro~Rr~RR=H
RJ=OMe; RL=OS03Na; RN=Oallyl; Rp=OCHZ(CH3)C=CH2;
2140 1.90 mM 862 pM
Rs=CHZOCHZ(CH3)C=CH2; Rx,RM,Rc,Rr,RR=H
RJ=OMe; RL,RQ=OS03Na; RN=Oallyl;
Rs=CHZOMe; 2141 4.90 mM 613 pM
Rx~RM~Ro~Rr~RR=H
RJ=OMe; RL,RQ=OS03Na; RN=Oallyl; 2142 3.00 mM 481 ~,M
Rs=CHZOCHZ(CH3)C=CHZ; Rx,RM,RO,Rr,RR=H
Rx=Oallyl; RM=OMe;RN=4-phenyl-[1,2,3]triazol-1-
2143 1.10 mM 398 pM
yI,RQ=OS03Na; Rs=CHZOS03Na; RJ,RL,Ro,Rr,RR=H
Rx=NHCOCHzOPh(2,4-di-Cl); RM,RN,RQ=OH;2144 6.00 mM 3.00 mM
Rs=CHZOS03Na; RJ,RL,RO,Rr,RR=H
RJ=OMe; RL,RQ=OS03Na; RN=Oallyl;
Rs=CHZOH; 2145 ~ 5.95 ~ 23.0 mM 1.60
mM mM
Rx,RM,Ra,Rr,RR=H
RJ=OMe; RL,RQ=OS03Na; RN=Oallyl;
Rs=CHZOBn; 2147 1.50 mM 9.10 mM 2.10
mM
Rx~RM~Ro~Rr~Rx=H
RJ=OMe; RL,RQ=OS03Na; RN=Oallyl;
Rs=CHzOCH2(3- 2148 2.40 mM ~ 13.0 mM 981
pM
rid 1 ' Rx,RM,Ro,Rr,RR=H
pY Y )~
RJ=OMe; RL,R~,=OS03Na; RN=OCHZ(2-napthyl);2149 2.50 mM ~ 6.70 mM 709
~.M
Rs=CHZOH; Rx,RM,RO,Rr,RR=H
RJ=OMe; RL,Rp=OS03Na; RN=OBn; Rs=CHZOH;2150 6.00 mM ~ 24.2 mM 7.80
mM
Rx~RM~Ro~Rr~RR=H
RJ=OMe; RL,RQ=OS03Na; RN=OBn; Rs=CHzOallyl; ,
2151 3.50 mM ~ 9.90 mM 935
pM
Rx,RM,Ro,Rr,RR=H
Rx=OMe; RM,Ro=OS03Na; Rr=OPr; RR=CH3;2152 ~ 6.30 3.40 mM
mM
Ra~RL~RN~~~Rs=H
RJ=OMe; RL,Rp=OH; RN=OS03Na; Rs=CHzN3;2153 ~ 11.5 42.1 mM ~ 11.1
mM mM
Rx~RM~Ro~Rr~RR=H
RJ=OMe; RL,RN=-OCHPhO-; RQ=OS03Na;
Rs=CHZN3; 2154 ~ 3.70 ~ 26.7 mM 2.15
mM mM
Rx,RM,Ra,Rr,RR=H
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PG Kd aFGF Kd bFGF Kd VEGF
#
RK=SEt; Rr,,r=OBn; RN,RP=OS03Na; 2155 188 ~M 1.06 448 ~,M
Rs=CHZOBn; mM
RJ~Rr.~Ro~~~Rx=H
RJ=OMe; RL,RQ=OSO3Na; RN=Oallyl; 2156 360 ~M 1.65 1.04
Rs=CHzN3; mM mM
Rx~RM~Ro~Rr~RR=H
RJ=OMe; RL,RN=OS03Na; RQ=OBn; Rs=CHZN3;2157 444 ~,M 3.30 144 ~.M
mM
RK,RM,Ro,Rr,RR=H
RJ=OMe; RL=OH;RN=OS03Na; RQ=OCHz(2-napthyl);
2158 1.55 mM ~ 10.3 985 ~M
mM
Rs=CHZN3; RK,RM,Ro,RP,RR=H
RJ=OMe; RL,RQ=OH; RN=OS03Na; Rs=CHzNHz;2159 1.90 mM -~- 62.36.40
mM mM
RK,RM,Ro,Rr,RR=H
R,r=OMe; RL=OBn; RN,RQ=OS03Na; Rs=CHzN3;2160 4.40 mM 2.50 ~ 12.9
mM mM
RK~RM~Ro~RP~Rx=H
RJ=OMe; RL,RQ=OS03Na; RN=OBn; Rs=CHzN3;2161 ~ 18.2 ~ 10.6
mM mM
Rx~RM~Ro~Rr~RR=H
RJ=OMe; RL,RN=OS03Na; RQ=Oallyl; 2163 396 ~M 4.80 61.2
Rs=CHZN3; mM ~,M
RK~RM~Ro~Rr~RR H
RJ OMe; RL,RN=OS03Na; RQ=OCHz(2-napthyl);
2164 1.35 mM 1.70 1.30
mM mM
Rs=GHzN3; RK,RM,Ro,RP,RR=H
RJ/RK=H/OMe (anomeric mixture); Rs-RN=2165 1.13 mM ~ 25.5 1.70
-CH20-; mM mM
RM=OBn; RQ=OS03Na; RJ,RK,RL,Ro,RP,RR=H
RK=SMe; RM,Ro=OS03Na; RP=OBn; RR=CH3;2166 3.60 mM 1.90 2.70
mM mM
RJ,RL,RN,R~,Rs=H
RK=OBn; RM,Ro=OS03Na; RP=OBn; RR=CH3;2168 1.90 mM 1.20
mM
Rr~Rr.~Rrr~~~Rs=H
RJ=Oallyl; RL,RN=OS03Na; RQ=OCHz(1-napthyl);2170 277 ~M 1.35 106 ~,M
mM
Rs=CHzNs~ Rx~RM~Ro~Rr~RR=H
RJ=OMe; RL,RQ=OS03Na; RN=OBn; Rs=CHzNHz;2171 845 pM ~ 18.9 2.00
mM mM
Rx~RNt~Ro~Rr~RR H
RJ=OMe; RL=OBn; RN,RQ=OS03Na; Rs=CHzNHz;
2172 3.80 mM ~ 5.90 2.30
mM mM
RK,RM,Ro,Rr,RR=H
RJ=OMe; RL=OBn; RN,RQ=OS03Na; Rs=CHz(4-phenyl-
2173 18.9 ~M 918 ~M 89.3
~M
[1,2,3]triazol-1-yl); RK,RM,Ro,RP,RR=H
RJ=Oallyl; RL=OH; RN=OS03Na; RQ=OBz;
Rs=CHZN3; 2174 278 pM ~ 19.3 846 ~M
mM
RK,RM,Ro,RP,RR=H
RJ=OMe; RL,RN=OS03Na; RQ=Oallyl;
Rs=CHZNHz; 2175 465 ~M 136 pM
RK,RM,Ro,RP,RR=H
RJ=OMe; RL,RN=OS03Na; RQ=Oallyl; 2176 693 ~M 256 ~.M
Rs=CHz(4-
(CHZNHz)-[1,2,3]triazol-1-yl); RK,RM,Ro,Rr,RR=H
RK=OMe; RM,Ro=OS03Na; RP=OCO-Cyclohexyl;2177 ~ 9,40
RR=CH3; mM
Rr~RL~Rrr~~~Rs=H
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#
R~=OMe; RL,RN=OSO3Na; RQ=Oallyl;
Rs=CHz(4-
(CHZNHCO(2-napthyl))-[1,2,3]triazol-1-yl);2178 130 ~M 1.20 mM 60.9 ~M
Rx~RM~Ro~Rr~RR=H
R~=OMe; RL,RN=OSO3Na; RQ=Oallyl;
Rs=CHz(4-
(CHZNHCO-Cyclohexyl)-[1,2,3]triazol-1-yl);2179 52.9 ~M 143 ~M 10.3 ~,M
RK,RM,Ro,Rr,RR=H
RJ=OMe; RL,RN=OSO3Na; RQ=Oallyl;
Rs=CHz(4-
(CHzNHCO-Ph(p-OMe))-[1,2,3]triazol-1-yl);2180 91.9 ~,M 1.90 mM
RK,RM,Ro,Rr,RR=H
RJ=OMe; RL,RN=OS03Na; RQ=Oallyl;
Rs=CHz(4-
(CHzNHCOCHZOPh)-[1,2,3]triazol-1-yl);2181 365 ~M 203 ~M
RK,RM,Ro,Rr,RR=H
RJ=OMe; ~RL,RN=OSO3Na; RQ=Oallyl; 2182 107 ~M 847 ~M
Rs=CHz(4-
(CHzNHCOPh)-[1,2,3]triazol-1-yl);
RK,RM,Ro,Rr,RR=H
RJ=OMe; RL,RN=OS03Na; RQ=Oallyl;
Rs=CHz(4-(CHz-N 2183 324 ~M 82.7 pM
phthalimido)-[1,2,3]triazol-1-yl);
RK,RM,Ro,Rr,RR=H
RJ=OMe; RL,RN=OS03Na; RQ=Oallyl;
Rs=CHz(4-
(CHzNHSO2Ph(p-Me))-[1,2,3]triazol-1-yl);2184 388 ~M 1.6 mM
Rx~RM~Ro~Rr~RR H
RJ=OMe; RL,RN=OSO3Na; RQ=Oallyl; 2185 421 ~M 440 pM
Rs=CHz(4-Ph-
[1,2,3]triazol-1-yl); RK,RM,Ro,Rr,RR=H
RJ=OMe; RL,RN=OS03Na; RQ=Oallyl; 2186 88.6 ~M >4.6 mM
Rs=CHz(4-t-butyl-
[1,2,3]triazol-1-yl); RK,RM,Ro,Rr,RR=H
RJ=OMe; RL=OS03Na; RN=OH; RQ=OCHz(2-napthyl);
2187 320 pM
Rs=CHzNs~ RK~RM~Ro~Rr~RR=H
RJ=OMe; RL,RN=OS03Na; Rp=OCHz(2-napthyl);
2188 1.22 mM 821 ~M
Rs=CHZN3; Rh,RM,Ro,Rr,RR=H
RJ=OMe; RL,RN=OS03Na; RQ=Oallyl;
Rs=CHZNHSOZMe; 2189 191 1tM 35.0 ~M
RK,RM,Ro,Rr,RR=H
RJ=OMe; RL,RN=OSO3Na; RQ=Oallyl;
Rs=CHzNHCOCH3; 2190 408 ~,M 98.0 ~M
RK,RM,Ro,Rr,RR=H
RJ=OMe; RL,RN=OS03Na; RQ=Oallyl;
Rs=CHzNHBz; 2191 1.39 mM 317 ~M
RK,RM,Ro,Rr,RR=H
R~=OMe; RL,RN=OS03Na; RQ=Oallyl;
Rs=CHzNHCOPh(p- 2192 1.85 mM 222 ~M
OMe); RK,RM,Ro,Rr,RR=H
RJ=OMe; RL,RN=OS03Na; RQ=Oallyl;
Rs=CHz(4-
(CHzNHCO(o-COzNa)phenyl)-[1,2,3]triazol-1-yl);2193 1.20 mM 270 ~M
Rx~RM~Ro~Rr~Rx=H
RJ=OMe; RL,RN=OS03Na; RQ=Oallyl;
Rs=CHz(4-
(CHzNHCOPh(3,4,5-tri-OMe))-[1,2,3]triazol-1-yl);2194 1.25 mM >2.8 mM
Rx~RM~Ro~Rr~RR=H
RJ=OPh(p-OMe); RL,RN=OS03Na; RQ=OBn;
Rs=CHzN3; 2195 1.05 mM 751 ~M
Rx~RM~Ro~Rr~Rx=H
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PG # Kd aFGF Kd bFGF Kd VEGF
RK=OBn; RM,Ro=OS03Na; RP=OBn; RR=CH3; 2196 170 ~M 2.50 mM
RJ,RL,RN,RQ,Rs H
RJ=OMe; RL,RN=OS03Na; RQ=Oallyl; Rs=CHZ(4-
(CHZOCHzPh(p-OMe))-[1,2,3]triazol-1-yl); 2197 131 ~M 381 ~M
Rx~RM~Ro~Rr~RR H
Table 3
R~ H
RT''N N'R V
w
Ru O
PG # Kd aFGF Kd bFGF Kd VEGF
RT=1,2,3,4-tetra-O-sodimn sulfonato-D-glucuronoyl;2007 2.34 mM
RU=CHZCHZOS03Na; RW=cyclohexyl;
R~=H
RT=1-O-Me-2,3,4-tri-O-sodium sulfonato-a-D-
mannopyranos-6-yl-acetyl; RU=CHzCH20S03Na;2008 296 ~M 551 ~M 335 ~M
R~,=H ;
RW=cyclohexyl
RT=Ac; RU=2-(2,3,4,6-tetra-O-sodium
sulfonato-a-D-
mannopyranos-1-O-yl-)-ethyl; R~=H; 2009 428 ~M
RW=2-(2,3,4,6-tetra-
O-sodium sulfonato-a-D-mamiopyranos-1-O-yl-)-ethyl
RT=3-(2,3,4,6-tetra-O-sodium sulfonato-a-D-
mannopyranos-1-O-yl-)-propyl; RU=COCHZCHZPh;2010 556 ~M
RW=cyclohexyl; R~=H
RT=1,2,3,4-tetra-O-sodium sulfonato-D-glucuronoyl;
2011 62.4 ~M
RU=Bn; Rw=cyclohexyl; R~=Ph
RT=1,2,3,4-tetra-O-sodium sulfonato-a-D-glucuronoyl;2012 122 ~M 505 ~M
RU=Bn; RW=cyclohexyl; R~=H
RT=1,2,3-tri-O-sodium sulfonato-a-D-glucuronoyl;
RU=Bn; 2013 587 ~M 1.16 mM
RW=cyclohexyl; R~=H
RT=1,3,4,6-tetra-O-sodium sulfonato-a-D-mannopyranos-2--
yl-acetyl; RU=Bn; R~,=H; RW=2-(2,3,4,6-tetra-O-sodium2014 5.09 ~M 85.1 ~M 8.82
~,M
sulfonato-a-D-mannopyranos-1-O-yl-)-ethyl
RT=3-(2,3,4,6-tetra-O-sodium sulfonato-a-D-
mannopyranos-1-O-yl-)-propyl; RU= 2018
CO(CHZ)3Ph; R.r-H;
RW=cyclohexyl
RT=1,2,3,4-tetra-O-sodium sulfonato-a-D-glucuronoyl;
2020 104 ~M 206 ~M 437 ~,M
RU,Rw=Bn; R~=H
RT=1-O-Me-2,3,4-tri-O-sodium sulfonato-a-D-2032 260 ~M 201 ~M 705 ~M
mannopyranos-6-yl-acetyl; RU=Bn;
RW=cyclohexyl; R~=H
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PG # Kd aFGF Kd bFGF Kd VEGF
RT=1-O-Me-2,3,4-tri-O-sodium sulfonato-a-D-
mannopyranos-6-yl-acetyl; RU=Bn; 2034 37.6 ~M 16.5 ~M 115 ~M
R~=H; Rw=2-(2,3,4,6-
tetra-O-sodium sulfonato-a-D-mannopyranos-1-O-yl-)-ethyl
RT=Ac; RU=Bn; R~=H; RW=2-(2,3,4,6-tetra-O-sodium2035 24.8 ~M 287 ~M 76.6 ~M
sulfonato-a-D-mannopyranos-1-O-yl-)-ethyl
RT=Ac; RU=Bn; R~=H; RW=2-(2,3,4,6-tetra-O-sodium
2036 118 pM 2.50 mM 1.10 mM
sulfonato-(3-D-mannopyranos-1-O-yl-)-ethyl
RT=Ac; RU=CHZCHzPh; R~=H; RW=6-deoxy-1-O-Me-2,3,4-
2058 224 ~M 682 ~M 109 ~M
tri-O-sodium sulfonato-a-D-mannopyranos-6-yl
RT=Ac; RU=CHZCHzPh; R~=H; Rw=6-deoxy-1-O-Me-2,3,4-
2058 224 ~M 682 ~M 109 ~M
tri-O-sodium sulfonato-a-D-mannopyranos-6-yl
RT=Ac; RU=Bn; R~=H; RW=6-deoxy-1-O-Me-2,3,4-tri-O-
2059 140 1tM 192 pM 77.0 ~.M
sodium sulfonato-a-D-mannopyranos-6-yl
RT=Ac; RU=Bn; R~=H; Rw=6-deoxy-1-O-Me-2,3,4-tri-O-
2059 140 ~M 192 ~M 77.0 ~M
sodium sulfonato-a-D-mannopyranos-6-yl
RT=Ac; RU=Ph; R~=H; RW=6-deoxy-1-O-Me-2,3,4-tri-O-2060 196 ~.M 481 ~M 76.3 ~M
sodium sulfonato-a-D-mannopyranos-6-yl
RT Ac; RU=cyclohexyl; R~=H; Rw=6-deoxy-1-O-Me-2,3,4-2064 314 pM 413 pM 1.70 mM
tri-O-sodium sulfonato-a-D-mannopyranos-6-yl
RT=Ac; RU=CHZCHZOS03Na; R~=H; RW=6-deoxy-1-O-2065 94.8 ~M 241 ~.M 283 ~M
Me-2,3,4-tri-O-sodium sulfonato-a-D-mannopyranos-6-yl
RT=m-CIPhCHZCO; RU=H; R~=H; RW=6-deoxy-1-O-Me-
2066 37.4 ~M 433 ~M 45.3 ~M
2,3,4-tri-O-sodium sulfonato-a-D-mannopyranos-6-yl
RT=Et; RU=CO(CHZ)zCOONa; R~=H; RW=6-deoxy-1-O-
2068 338 pM 291 ~M 207 ~M
Me-2,3,4-tri-O-sodium sulfonato-a-D-mamzopyranos-6-yl
RT=Et; RU=CO(CHZ)WCOONa; R~=H; Rw=6-deoxy-1-O-
2069 160 ~M 477 ~M 104 ~,M
Me-2,3,4-tri-O-sodium sulfonato-a-D-maniiopyranos-6-yl
RT=Et; RU=CO(CHZ)90S03Na; R~=H; Rw=6-deoxy-1-O-
2070 111 ~M 243 ~,M 545 pM
Me-2,3,4-tri-O-sodium sulfonato-a-D-mannopyranos-6-yl
RT=Et; RU=CO(CHZ)WCOONa; R~=H; Rw=6-deoxy-1-O-
Me-2,3,4-tri-O-sodium sulfonato-a-D-mannopyranos-6-yl,2071 119 ~M 2.24 mM 161
~M
mixture of mono- and di- sulfates
RT=Ac; RU=cyclohexyl; R~=H; RW=6-deoxy-1-O-Me-2,3,4-
tri-O-sodium sulfonato-a-D-mannopyranos-6-yl,2072 264 ~M 2.98 mM 277 ~M
mixture of
mono- and di-sulfates
RT=Ac; RU=Ph; R~,=H; RW=2-(2,3,4,6-tetra-O-sodium2073 97.4 ~M 236 pM 402 ~M
sulfonato-a-D-mannopyranos-1-O-yl-)-ethyl
RT=Ac; RU=(CHZ)zPh; R~=H; Rw=2-(2,3,4,6-tetra-O-
2074 11.8 ~M 113 ~,M 28.8 ~M
sodium sulfonato-a-D-mannopyranos-1-O-yl-)-ethyl
RT= m-CIPhCH2C0; RU,R~=H; Rw=2-(2,3,4,6-tetra-O-
2075 171 ~.M 837 ~M 90.8 ~,M
sodium sulfonato-a-D-mannopyranos-1-O-yl-)-ethyl
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PG # Kd aFGF Kd bFGF Kd VEGF
RT=Et; RU=CO(CHz)ZCOZNa; R~=H; RW=2-(2,3,4,6-tetra-
2076 43.4 pM 118 p,M 40.3 ~,M
O-sodium sulfonato-a-D-mannopyranos-1-O-yl-)-ethyl
RT=Et; RU=CO(CHZ)4COZNa; R~=H; R~2-(2,3,4,6-tetra-
2077 43.6 pM 188 pM 81.1 pM
O-sodium sulfonato-a-D-mannopyranos-1-O-yl-)-ethyl
RT=Et; RU=CO(CHZ)90S03Na; R~=H;
RW=2-(2,3,4,6-tetra- 2078 20.0 pM 157 p,M 49.61tM
O-sodium sulfonato-a-D-mannopyranos-1-O-yl-)-ethyl
RT=Bz; RU=Et; R~=H; Rw=6-deoxy-1-O-Me-2,3,4-tri-O-
2081 366 pM 480 ~M 1.10 mM
sodimn sulfonato-a-D-mannopyranos-6-yl
RT=CO(CHZ)ZPh; RU=Et; R~,=H; RW=6-deoxy-1-O-Me-
2082 596 1tM
2,3,4-tri-O-sodium sulfonato-a-D-mannopyranos-6-yl
RT=CO(CHZ)3Ph; RU=Et; R~,=H; RW=6-deoxy-1-O-Me-
2083 453 pM 403 ~M 80.7 ~.M
2,3,4-tri-O-sodium sulfonato-a-D-mannopyranos-6-yl
RT=COCHZOS03Na; RU=Et; R~,=H; Rw=6-deoxy-1-O-Me-
2084 161 ~M 192 pM 277 pM
2,3,4-tri-O-sodium sulfonato-a-D-mannopyranos-6-yl
Rw cyclohexyl; RU=Ac; R~=H; RT=6-deoxy-1-O-Me-2,3,4-
2094 246 pM 565 pM 1.10 mM
tri-O-sodium sulfonato-a-D-mannopyranos-6-yl
RW=cyclohexyl; RU=Ac; R~=H; RT=6-deoxy-1-O-Me-2,3-
O-benzylidene,4-O-sodium sulfonato-a-D-mannopyranos-6-2115 5.10 mM 3.90 mM
y1
RT Ac; RU=cycloheptyl; R~=H; RW=6-deoxy-1-O-Me-
2116 369 ~M 411 ~M 3.00 mM
2,3,4-tri-O-sodium sulfonato-a-D-mannopyranos-6-yl
RT=Ac; RU=cycloheptyl; R~=H; R~,,~=6-deoxy-1-O-Me-2,4-2117 1.50 mM 2.90 mM
di-O-sodium sulfonato-a-D-mannopyranos-6-yl
RT=Ac; RU=cyclooctyl; R~=H; Rw=6-deoxy-1-O-Me-2,4-di-2120 1.60 mM ~ 11.0 mM
O-sodium sulfonato-a-D-mamiopyranos-6-yl
RT=COCHZPh(p-CF3); RU=Et; R~=H; 2122 3.70 mM 1.20 mM
RW=6-deoxy-1-O-
Me-2,4-di-O-sodium sulfonato-a-D-mannopyranos-6-yl
RT=COCHZPh(p-CF3); RU=Et; R~=H;
Rw=6-deoxy-1-O- 2124 242 pM 570 ~M
Me-2,3-di-O-sodium sulfonato-a-D-mannopyranos-6-yl
RW=cyclohexyl; RU=Ac; R~=H; RT=6-deoxy-1-O-Me-4-O-2125 26.6 mM ~ 166 mM
sodium sulfonato-a-D-mannopyranos-6-yl
RT=Ac; RU=cyclododecyl; R~=H; Rw=6-deoxy-1-O-Me-
2126 265 pM 483 ~M 1.20 mM
2,3,4-tri-O-sodium sulfonato-a-D-mannopyranos-6-yl
RT Ac; RU=4-t-butylcyclohexyl; R~=H;
RW=6-deoxy-1-O- 2132 243 ~M 544 pM 1.00 mM
Me-2,3,4-tri-O-sodium sulfonato-a-D-mannopyranos-6-yl
RT=Ac; RU=1-(1-adamantyl)-ethyl;
R~=H; Rw=6-deoxy-1- 2133 398 ~M
O-Me-2,4-di-O-sodium sulfonato-a-D-mannopyranos-6-yl
RT=CO(CHZ)3Ph; RU=Et; R~,=H; R,~6-deoxy-1-O-Me-2,4-2135
di-O-sodium sulfonato-a-D-mannopyranos-6-yl
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PG # Kd aFGF Kd bFGF Kd VEGF
RT=6-deoxy-1-O-Me-2,3,4-tri-O-sodium sulfonato-a-D-
mannopyranos-6-yl; RU=CHzCONHcyclohexyl; 2138 418 ~.M 449 ~,M 1.34 mM
RW=cyclohexyl; Rv=H
RW=cyclohexyl; RU=CHO; Rv=H; RT=6-deoxy-1-O-Me- 2162 1.20 mM
2,3,4-tri-O-sodium sulfonato-a-D-maimopyranos-6-yl
Table 4
H H
N N, R~ N VI
O RY RY O
PG Kd aFGF Kd bFGF Kd VEGF
#
Rx=COCHZC(CH3)ZCHzCO; R~3-(2,3,4,6-tetra-O-sodium2015 2.94 ~M 7.56 267 nM
~,M
sulfo-a-D-mannopyranos-1-O-yl)-propyl
Rx=1,4-traps-cyclohexyl; RY=1-O-Me-2,3,4-tri-O-sodium2016 32.6 ~.M 81.8
931 nM
~M
sulfo-a-D-mannopyranos-6-yl-acetyl
Rx=COCHZC(CH3)ZCHZCO; Ry=2-(2,3,4,6-tetra-O-sodium2057 18.8 ~M 61.6 55.6
~M ~M
sulfo-a-D-mannopyranos-1-O-yl)-ethyl,
undersulfated
Rx=COCH2C(CH3)ZCHZCO; Ry=2-(2,3,4,6-tetra-O-sodium2062 10.3 ~M 29.7 17.3
~M ~M
sulfo-a-D-mannopyranos-1-O-yl)-ethyl
Table 5: Antiviral Testing
Results
PG# HSV-1 InfectivityHSV-2 Infectivity HSV-1 SpreadHSV-2 Spread
2000 100.8 NT (= not tested) 82.1 NT
2001 85.6 NT 88.3 NT
2002 89.2 NT 113.8 NT
2003 97 NT 95.2 NT
2005 102.8 NT 131.7 NT
2040 106.6 NT 86.2 NT
2041 108.3 NT 60 NT
2042 92 NT 107.6 NT
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PG# HSV-1 InfectivityHSV-2 InfectivityHSV-1 Spread HSV-2 Spread
2000 100.8 NT (= not tested)82.1 NT
2001 85.6 NT 88.3 NT
2002 89.2 NT 113.8 NT
2003 97 NT 95.2 NT
2005 102.8 NT 131.7 NT
2040 106.6 NT 86.2 NT
2041 108.3 NT 60 NT
2044 73.5 NT 77.9 NT
2085 99.2 NT 64.1 52.8
2091 81.8 135.6 74.9 58.9
2092 90.3 101.4 75.4 50
2093 90.3 121.6 74.9 55.5
2097 101.8 111.5 45 42.2
2098 81.8 116.3 68.4 54.4
2099 89.5 115.9 76 43.3
2103 80.8 112 94.2 70
2111 100.3 105.3 57.9 55.5
2112 95.6 90.9 74.9 76.7
2113 95.1 91.3 46.2 31.1
2114 86.4 99 71.3 68.9
2139 86.8 81.8 47.8 20
2146 108.5 92 56.2 52.8
2145 92.1 76 73.7 77.8
2151 100 75.5 84.5 86.1
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The results presented in Tables 1 to 4 demonstrate that the broad range of
compounds
embraced by the invention have strong affinity for GAG-binding growth factors
and may thus
serve as modulators of their activity. The results in presented in Table 5
demonstrate that the
compounds do indeed possess in vivo activity.
The foregoing embodiments are illustrative only of the principles of the
invention, and
various modifications and changes will readily occur to those skilled in the
art. The invention is
capable of being practiced and carried out in various ways and in other
embodiments. It is also
to be understood that the terminology employed herein is for the purpose of
description and
should not be regarded as limiting.
The term "comprise" and variants of the term such as "comprises" or
"comprising" are
used herein to denote the inclusion of a stated integer or stated integers but
not to exclude any
other integer or amy other integers, unless in the context or usage an
exclusive interpretation of
the term is required.
Any reference to publications cited in this specification is not an admission
that the
disclosures constitute common general knowledge in Australia.
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