Note: Descriptions are shown in the official language in which they were submitted.
WO92/18~KPCT/US92/03~2
., .
2 ~ ~ 8 3 u ~
.~ SULFATED POLYSACCHARIDES AS INHIBITORS OF
` 10SMOOTH MUSC~E CELL PROLIFERATION
':
. .
~: .
:~, 15
. Te$hnical Fiel~
The invention relates to the use of
i; carbohydrate preparations as therapeutic and diagnostic
compositions. In particular, the invention relates to
; polysaccharides having six or more saccharide units and
~ compositions containing such polysaccharides which are
; useful in treating diseases and conditions characterized
by excessive smooth muscle cell proliferation.
. 25
Abbreviat~ns
In the representations of oligomers produced
synthetically and those deri~ed from heparin, the follow-
ing abbreviations are used: D-glucuronic acid ~ GlcA;
~-iduronic acid ~ IdoA; D-glucosamine ~ GlcNH2;
N-acetyl-D-glucosamine - GlcNAc; D-glucogamine N-sulfate
. GlcNS; 2,5-anhydromannose ~ Man~2,5);
2,5-anhydromannitol ~ ManH~2,5); D-xylose . Xyl;
glycosaminoglycan ~ GAG.
3S
p
-
.' ~ . , .
W092/~8~ PCT/US92/03~2
,f~
21~8~6~
-2-
The location of the 0-linked sulfate residue~
is indicated by l~S~ and the number of the position of
; sulfation where the sulfate residue is linked to oxygen
on the sugar residue. In these designations, also, the
alpha and beta anomeric linkages are as those conven-
tionally found in heparin and the indicated D or L
configurations as conventionally found pertains. The
locations of the ~ulfates are shown below the
abbreviation for the sugar to which they apply, thus,
for example,
IdoA-GlcNS
2S 6S
refer to L-iduronic acid and D-glucosamine N-sulfate with
sulfates connected respectively at the ~ and 6 positions
of the sugar residues.
~açkground Ar~
Proliferation of smooth muscle cells in blood
vessel walls occurs in regponse to vascular injury, and
in association with certain disease gtates (Austin, G.E.,
- et al., J A~ Col~ Caxd~Ql (19a5) 6:369-375). The prolif-
eration of these cells can have negative effects due to
the production of excess proteins or other matrix
molecules, which, along with the cells them9elve~, form
pathologic le8ions of, for example, athero8ClerOsiS,
renal hypertension, pulmonary hypextengion, vasculiti~,
and po8t~surgical va~cular retinosig. These results are
distinguished ~rom the acute responge to trauma charac-
terized by blood clotting.
Glycosaminoglycan~ ~GAG) are copolymers of
alternating hexo8amine and aldouronic acid residues which
are ~ound in sul~ated f orms and are synthesized as
proteoglycans. They have collectively been called muco-
. r.~ .
~"
WO92/18~6 PCT/US92/0309~
~.
.
2~ a~5~!
.
-3-
polysaccharides, and tho9e in heparin are more precisely
called glycosaminoglycuronans.
To place the compositions discussed below in
context, it may be noted that heparin and heparan sulfate
are members of the GAG family which are classified by the
nature of the hexosamine/aldouronic acid repeating units.
For example, in chondroitin sulfates, the aldouronic acid
i8 primarily D-glucuronic acid, and the hexosamine is
N-acetylated 2-amino-2-deoxy-D-galactose, more commonly
0 known as N-acetyl galactosamine and abbreviated as
GalNAc.
In dermatan sulfate (chondroitin sulfate ~) the
aldouronic acid i9 mostly L-iduronic acid and the hexo-
~; samine is GalNAc. In keratan sulfate, the aldouronic
acid is replaced by D-galactose, and the hexosamine is
mostly N-acetylated 2-amino-2-deoxy-D-glucose, more
; commonly known as N-acetyl glucosamine and abbreviated as
GlcNAc.
In the compositions of interest herein,
heparan sulfate and heparin, the hexosamine is mostly
N~acetylated or N-sulfated glucosamine (GlcN), and the
aldouronic acid is mostly L-iduronic in heparin and
mostly D-glucuronic acid in heparan sulfate. Heparan
sulfate i9 commonly considered to have a higher
proportion of glucuronic acid than heparin.
Problems of heterogeneity in preparations of
heparan sulfate or heparin isolated from tissues make
sharp distinctlons di~icult, gince these oligosaccha-
rides are related by the biosynthesis pathway, as
explained below. Conveneional heparin (used as an
antlcoagulant) has a molecular weight of 5-25 kDa and is
extracted as a mixture o~ various chain lengths by
~onventional procedures. These procedures involve
autolysis and extraction of suitable tissues, such as
` 35
. .
WO92/18~ PCT/US92/03092
, . . ( ,
21~56~
-4-
beef or porcine lung, intestine, or liver, and removal of
other GAGs as well as nonpolysaccharide components.
The molecular weight of the chains in the
extract is significantly lower than the 60-100 kd known
to exist in the polysaccharide chains of the heparin
proteoglycan synthesized in the tissue. The GAG moiety
i~ synthesized bound to a peptide matrix at a serine
- re~idue through a tetrasaccharide lin~age region of the
: sequence D-GlcA-D-Gal-D-Gal-D-Xyl ~ protein, which is
then elongated at the D-GlcA re~idue with alternate
additions of GlcNAc and GlcA.
The polysaccharide sidechains are modified by a
series of enzymes which sequentially deacetylate the
N-acetyl glucosamine and replace the acetyl group with
sulfate, epimerize the hydroxyl at C5 of the D-glucuronic
acid re~idue (to convert it to L-iduronic acid), sulfate
the 0-2 of the resulting ~-iduronic acid and the 0-6 of
the glucosamine residue. Some of the chains are further
sulfated at the 0-3 of the glucosamine residue, either at
the heparan or heparin stage. This latter sulfation
generates the active sequence required for anti-
thrombin III binding and thus anticoagulation activity.
Other chemically possible sulfation sites are on the 0-2
of D-glucuronic acid.
Due to their ob~ious chemical similarity,
isolated "heparinll may contain considerable amounts of
what might otherwise be classified as heparan sulfate.
There is an exten9ive body of art concerning
depolymerization of heparin/heparan sulfate chains and
separation of productg by size. Particularly relevant is
the report of Guo, ~. et al., nal ~iochem ~198~)
L~Q:54-62 which discloses the results of structure
determination after the 2,5-anhydromannose at the
3S
.~
W092/18~ PCT/US92/O~g2
`~ 2~0~9
,
-5-
reducing terminus is reduced to the corre~ponding
2,5-anhydromannitol.
The involvement of heparin or heparan sulfate
or degradatlon products thexeof in smooth muscle prolif-
eration has been recognized for some time. Heparin andheparan sulfate can slow or arrest the vascular smooth
muscle cell proliferation associated with injury
described hereinabove (Clowe~, A.W., et al., Nature
(1977) 265:625-626). The effect of heparan sulfate and
heparin on smooth muscle cell proliferation is also
described by Marcum, J.A., et al. in ~ioloqv of
Proteoqlycan, Academic Press (1987) pp. 301-343. The
inhibition of vascular smooth muscle cell growth by
heparin was further described by Castellot, J.J., ~r., et
al., J ~iol Chem (1982) 257:11256-11260 and the effect of
heparin on vascular smooth muscle cell growth in fetal
tissue was described by Benitz, W.E., et al., J C~ll
Physiol (19B6) 127:1 7. The effect of heparin as an
inhibitor of both pericyte and smooth muscle cell pro-
liferation was shown by Orlidge, A., et al.,
Microvascular Research (1986) 31:41-53, and these authors
further showed that chondroitin sulfate and dermatan
sulfate do not have this e~fect. A review of the effects
o~ heparin and heparan 8ulfate on the proliferation of
smooth muscle cells ig by ~enitz, W.E. in ~The Pulmonary
Clrculation: Normal and Abnormal", Flshman, ~.P., ed.,
University o~ Pennsylvania Pre~s ~1908).
It is not clear by what mechanism these glycos-
aminoglycans operate, or to what extent they interact
! 30 with other growth factors such as epithelial and fibro-
blast growth factors. It has been proposed that a 3-0
sulfate on glucosamine in an oligogaccharide of at least
5 sugar~ is important in this procegs and that both 0-
and N-sulfation is important ~castellot, J.J., et al., J
,
, .
, .
WO92/18~6 PCT/US92/0~2
f ', ' f
. .
2 1 ~ 6-
Cell Physlol (1984) -20:315-320; Castellot, J.J., et al.,
J Cell Biol (1986) 102:1979-1984). Hexasaccharides-
decasaccharides obtained from partial nitrous acid diges-
tion of heparin bind to acidic fibroblast growth faccor
and aid its mitogenic activity in fibroblasts, but
inhibit the proliferation of endothelial cells under some
conditions (~arzu, T., et al., J Cell Physiol (1989)
14o:s3~-s4a). The effecti~e hexasaccharide was stated to
have the structure:
IdoA-GlcNS-IdoA-GlcNS-IdoA-Man(2,5)
2S 6S 2S 6S 2S 6S
Others have indicated that the presence of 2-
0-sulfate glucuronic acid is not necessary for antipro-
liferative activit~ (Wright, Jr., T.C., et al., J ~iol
Chem ~1989) ~~:1534-1542). In this article, size
separated fragments of defined length prepared by nitrous
acid cleavage and gel filtration were further separated
according to charge for some assays. Partially digested
heparin separated only according to size was tested with
respect to stimulation of the growth of smooth muscle
cells and epithelial cells. Similar results were found
in both cases, although the results were not identical.
~5 Tetrasaccharides of the type tested were shown to have
~ery low antiproliferati~e acti~ity; hexasaccharides,
octasaccharides, and decagaccharides were shown to be
active to approximately the same level on a weight/volume
concentration basis. Also tested was a synthetic
pentapeptlde which repregentg a unigue sequence of the
heparin required for the binding of heparin to
antithrombin III; this pentapeptide was active in
inhibiting proliferation for smooth muscle but not
epithelial cells. The size-separated fraceions were
~, .
.~:
. ~ ,
WO92/18~6 PCT/US92/03~92
, 6 ~
-7-
then treated chemically to produce "o-oversulfation" and
this treatment enhanced the inhibitory acti~ity; indeed,
oversul~ation of the tetrasaccharide frasment prepara-
tion yielded a tetrasaccharide fraction which was active
in inhibiting proliferation. The converse proce~s,
comprising desulfation and reacetylation of the amino
groups of glucosamine results in a redùction in
antiproliferati~e activity. These fragments could,
however, be made more active by subsequent oversulfation.
Also capable of reducing the acti~ity of the
heparin fragments was reduction of the carboxyl groups so
as to reduce the total negati~e charge. O-oversulfation
partially restores this acti~ity. These results with
N-desulfated, N-acetylated fragments which are lacking in
antiproliferative activity is distinguishable from
pre~ious results wherein similarly treated heparin
retains the capacity to prevent cell di~ision because of
the size dependency of the antiproliferative acti~ity--
larger fragments being more powerful in general than
smaller ones.
When the size separated fraction was further
fractionated according to charge, it was f ound that the
most highly charged fraction~ showed the greatest
activlty. Furthermore, it was shown that although the
synthetic pentasaccharide identified as the antithrombin
III binding site is capable of inhibiting proliferation
in 8mooth muscle cells, any treatment of heparin which
would destroy the sequence corresponding to this
pentapeptide ~i.e., periodate treatment) does not destroy
a~tiproliferacive activity.
Methods o~ synthesizing oligosaccharides are
di~closed in U.S. Patent 4,943,630 issued July 14, l990
which is incorporated herein by reference to disclose
such methods.
_5
.,
:'~
J.
~,
~ W092/18~ ~CT/US92/03092
. .. ..
21 08~69
-8-
The present inventors have now found that an
enhanced antiproliferative activity with respect to
smooth muscle cells is associated with an oligosaccharide
$ portion of the heparin or heparan sulfate G~Gs which is
highly sulfated and contains 6 or 8 saccharide units and
have provided synthesls mechanisms for making
- polysaccharide~ containing 6 or more sugar residues,
which oligosaccharides have enhanced antiproliferative
activity with respect to smooth muscle cells.
Disclosure of the Invention
The invention provides a low molecular weight
glycosaminoglycan (GAG) composition which has superior
specific antiproliferative activity with regard to smooth
muscle cells. The existence of this acti~ity in a low
molecular weight GAG provides the opportunity for effec-
tive pharmaceutical compositions which can be prepared by
synthesis or by isolation of the composition from natural
sources.
Accordingly, in one aspect, the invention is
directed to a process to prepare a sulfated
polysaccharide having antiproliferative activity. The
polysaccharides o~ the invention may be produced
synthetically using a se~uence of chemical reactions as
di8closed herein or obtained by digesting hepaxin and
carrying out separation procedures based on size and
charge as disclosed herein.
In order to produce the polysaccharide
compounds of the invention synthetically it is first
neCessary~ to synthegize an iduronic acid synthon. Next,
a glucosamine synthon is produced. The iduronic acid
synehon and glucosamine gynthon are reacted to produce a
di9accharide synthon. The disaccharide unit~ can be
reacted to form oligogaccharides containing 4, 6, 8 or
W~92/1~6 PCT/US92/0~92
2 1 ~
g
any multiple thereof of monosaccharide units and/or can
be reacted with either an lduronic or glucosamine re-
action synthon to provide oligosaccharides containing any
odd number of saccharide units.
In order to obtain the oligosaccharide
compounds by digestion, the heparin is obtained from a
natural source and subjected to dige~tion with nitrous
acid under condition~ which favor the formation of an
oligosaccharide mixture containing large amounts of hexa-
and octasaccharides. Following the digestion, the
mixture is separated according to size and those factions
corresponding to hexa- and octasaccharides are combined
and recovered. The reco~ered portion~ are then separated
according to charge in order to obtain the more highly
lS charged fractions. These fractions will contain
oligosaccharides which are highly sulfated. Polysacchar-
ides sulfated at the 0-3 position of the GlcN (associated
with anticlotting activity) are not encompassed by the
present in~ention.
~0 The in~ention is also directed to
pharmaceutical compositions comprised of the
oligosaccharides of the invention either alone or in
combination with excipients, i.e., pharmaceutically
acceptable materials with no pharmacological effect.
Such compositions may be administered to a patient in
order to regulate smooth muscle cell proliferation.
A prlmary object of the present invention i9 to
provide synthetically produced oligosaccharides
containing 6 or more monosaccharide units, which are
highly sulfated at particular positions other than 0-3
po~itions of the GlcNg and which effect smooth muscle
cell proliferation.
Another important object of ~he present inven-
- tion is to provide a method o obtaining hexa- and
~5
....
.,
.
.
WO92/18~ PCT/US92/0~92
~,
: . J,
2 1 ~ 9
-10-
octasaccharide units from natural heparin and heparan
sul~ate which hexa- and octasaccharide units are
effective in regulating smooth muscle cell prolifera~ion
and which do not process any significant degree of
anticlotting activity.
An advantage of the present in~ention is that
the oligosaccharide units can be formulated into
pharmaceutical compositions which can be administered to
aid in the regulation of smooth muscle cell
proliferation.
A feature of the present invention is that the
oligosaccharide units include monosaccharide residues
which are sulfated at particular positions (other than
the 0-3 position) which effected the ability of the
oligosaccharide to regulate smooth muscle cell
proliferation.
These and other objects, advantages and
features of the present invention will become apparent to
those persons skilled in the art upon reading the details
of the structure, synthesig and usage ag more fully set
forth below, reference being made to the accompanying
figures and general structural formulag forming a part
herein wherein like symbols refer to like molecular
moietie9 throughout.
f Description Qf the Drawings
Flgures 1~ and l~ show the elution profiles
rom gel ~iltration chromatography of reaction mixture9
produced using varying amounts of nitrou9 acid.
Figure 2 shows the growth inhibition acti~itY
of the various sized fractions.
Figures 3A and 3~ ghow the elution profiles of
hexasaccharide and octasaccharide subunits, respecti~ely,
from DEAE-Toyopearl chroma~ography.
~5
~,,
~ .
. .
. .
W092/18~ PCT/US92/0~92
. .....
2~5~
-11-
Figures 4A and ~ show the growth inhibition
activity of various fractions collected in the elution
profiles of Figures 3A and 3B.
Figure SA shows the elution profile from
s re~erse-phase ion-pairing HPLC for the S-6 fraction shown
in Fi~ure 3A.
Figure SB shows a comparable profile for the
total hexasaccharide fraction.
Figures 6A, 68 and 6C are charts showing pos-
sible sulfated positions for fifty-seven octasaccharides
of the invention.
Detailed Description of Preferred Embodiments
~efore the present oligosaccharides and
processes for making and formulating such are described,
it i9 to be understood that this invention is not limited
to the particular oligosaccharides, formulations or
processes described as such compounds, compositions and
methods may, of course, vary. It is also to be
understood that the terminology used herein is for the
purpose of describing particular embodiments only, and is
not intended to be limiting since the scope of the
present ln~ention will be limited only by the appended
claims.
It muct be noted that as used in this
~pecification a~d the appended claims, the singular forms
"a", "an" and ~the~ include plural referents unless the
content clearly dictates otherwise. Thus, ~or example,
reference to "an oligosaccharide~ includes mixtures of
oligosaccharideg and, reference to "an octasaccharide"
include~ reference to mixtures of octasaccharides of the
type described herein and reference to "the process step"
or "the process" includes reference to various steps and
processes o~ the type described herein which will be
, ,,
~ .
WO92/18~6 PCT/US92/0~2
,,
2~0~
-12-
known to those skilled in the art or which will become
apparent to those skilled in the art upon reading this
disclo~ure and so forth.
Deflnitions
~ y ~heparin/heparan sulfate~ or ~heparin~ i5
meant a preparation obtained from tissue~ in a manner
conventional for the preparation of heparin as an anti-
coagulant or otherwise synthesized and corresponding to
that obtained from ti~sue. See Conrad, ~.E., Heparin and
Related Polysaccharides, Vol. 56, p. 18 of Annals of
N.Y., Academy of Sc., June 7, 19~9, incorporated herein
by reference. This preparation may include residues of
D-glucuronic acid (GlcA), as characteristic of heparan
sulfate as well as iduronic acid (IdoA) as characteristic
of heparin. However, both GlcA and IdoA are present in
both, they are present in different proportional amounts.
The (IdoA)/GlcA ratio increases as heparan sulfate
becomes more heparin-like. As described in the
~ackground section above, the conversion of D-glucuronic
acid to L-iduronic acid i9 a result of epimerization at
the 5 carbon of GlcA residues in a heparan-type inter-
mediate. This sequence of steps involved in such
epimerization and conversion is understood in the art.
To the extent that full conversion has not been made,
heparan sulfate characteristicg remain in the prepa-
ration. ~ecause the precise nature of the polymeric
chains in the preparations of heparin is not generally
determined, and varies from preparation to preparation,
the term "heparin/heparan sulfatel' or ~heparin" is
intended to cover the range of mixtures encountered.
Perhaps the main feature which distinguishes heparan
sulfate ~rom heparin i9 that the latter has anti-
coagulant activity.
W092/18~K PCT/US92/03092
,. ,, ~ , .
.
-13-
The ~heparin/heparan sulfate~ preparation can
be obtained from a variety of mamm~lian tissues,
including, i~ desired, human tissue. Generally, porcine
or bovine sources are u~ed, and vascularized tissues are
-`; 5 preferred. A preferred source of heparin/heparan sulfate
starting material i9 porcine intestinal mucosa, and
preparations labeled ~heparin~ prepared from this tissue
source are commercially available. In general, the
heparin/heparan sulfate starting material is prepared
from the selected tissue gource by allowing the tissue to
undergo autolysis and extracting the tissue with alkali,
followed by coagulation of the protein, and then
precipitation of the heparin-protein complex from the
supernatant by acidification. The complex i9 recovered
by reprecipitation with a polar nonaqueoug solvent, such
as ethanol or acetone or their mixtures, and the fats are
removed by extraction with an organic solvent such as
ethanol and proteins by treatment with a proteolytic
enzyme, such as trypsin. Suitable procedures for the
preparation of the heparin gtarting material are found,
for example, in Charles, A.F., et al., ~iç~h~m J (1936)
30:1927-1933, and modificationg of thig bagic procedure
are also known, 9uch as those disclo~ed by Coyne, E., in
C~Qmi5t~Y ~D~_aiolQg~o~ a~i~, Elsevier Publisher9,
~orth Holland, New York, Lunblad, R.L., et al., eds.
(19E1).
The ~ynthetic oligosaccharides of the present
lnvention include at leagt 6 gaccharide regidue units and
have the following general structural formula:
. .
WO g2/18546 Pcr/us92/o3og2
2:~856~ -14- r
0 ~
~} 0~,~
~3*,
~ / ~
~
O
. 25
~ ~0 ~
~,o1~
~
O~
W092/l8~ 2 1 ~ ~ ~ a ~ PCT/US92/0~92
-15-
(The hydroxyl groups on the 3-position;of the sugars have
been omitted for greater clarity) and the ~ adjacent the
carbon substituted with COOH indicates (here and
throughout the application) undetermined stereochemistry
which may be any possible stereochemistry for the
molecule; wherein each of the variables A, B, C and D are
independently hydrogen or SO3R with the proviso that at
least 2 of the variables are SO3R and each R is
independently H~, Na+, or other suitable cation; and
wherein Rl and R2 are each independently hydrogen, or one
or more repeating units having the following structure:
Fo~nula I(a)
CH2C~ E COOH
~n ~o~c~F
HNSO3-
wherein when the unit of structural Formula I(a) is con-
nected at one end the hydrogen is not present and the
hydrogen at the end not connected is present and further
wherein each of the variables E and F is independently
hydxogen or SO3R.
Some preferred embodiments of the present
invention include compounds Or structural Formula I
wherein each o~ A, ~, C and D are -SO 3 and either Rl or
R2 is the unit o~ structural Formula I(a). Another
pre~-rxed embodiment includes compounds of structural
Formula I wherein each of A, B, C and D is -SO 3 and both
W092/18~ PCT/US92/0~2
~;~.
2las~
-16-
Rl and R2 are the unit of structural Formula I(a) and
wherein each E and F of the unit I(a) i9 -SO 3 .
Polysaccharldes Derived from Heparin and/or Heparan
Sulfate
Preferably, the heparin/heparan sulfate prepa-
ration u~ed as a starting material i9 first purified by
extraction with a solvent in which the heparin is in-
soluble, such as ethanol or acetone. The purified start-
ing material i9 then depolymerized.
Depolymerization in general can use various re-
agents, such as nitrous acid, heparinase or periodate.
The antiproliferative compositions of the invention are
obtainable when partial nitrous acid digestion i5
conducted under conditions which m2ximize formation of
hexasaccharide and octasaccharide fragments.
; In typical procedures, the nitrous acid is
- prepared in situ by acidification of a solution of sodium
nitrite at a concentration of 50 mM, and the reagent is
used to treat the heparin at a concentration of about 60-
180 mg/ml, at a pH of about 1.0 to about 2.0, preferably
about 1.5. The reaction is conducted at room temper-
ature and can be neutralized by addition of a suitable
reagent at the desired gtage of digestion. Other
depolymerization methods can be used as long as they
produce active componeneg, i.e., componentg which ~1) are
predominantly hexa- and octasaccharides; (2) are heavily
sulfated; ~3) have subgtantial antiproliferation activity
with respect to smooth mugcle cellg; and (4) have in-
slgn1ficant or no anticlotting ac~ivity.
Isolated fragmentg can then be ~egted for theirabilley to inhibit smooth mugcle cell proliferation.
Fragmentq with high activity with regpect to inhibiting
the proliferacion of smooth mugcle cellg and low activity
,
WO92~18~6 ~ ~CT/US92/03092
~ 2 1 0 ~
.~ , .
-17-
with respec~ to their ability to inhibit blood
coagulation (relative to commercial heparin) are
preferred.
The depolymerization re~ults in a mixture of
.~ 5 fragments that is then separated on the basis of size. A
variety of size separation techniques are available,
including gel permeation, density gradient
centrifugation; especially preferred is gel filtration
chromatography using a Sephadex or polyacrylamide gel
system with a fractionation range of about 100-3500
daltons. A particularly preferred gel permeation resin
is Biogel Pl0, and upon separation usi~g this method,
fragments which are disaccharides, tetrasaccharides,
hexasaccharides, octa~accharides, and oligosaccharides of
higher molecular weights are effectively separated.
The fractions containing predominantly hexa-
and octasaccharide units show enhanced acti~ity in
inhibiting the proliferation of smooth muscle cells.
Verification of this property can be obtained using
standard assays, such as those described in Castellot,
J.J. Jr., et al., J ~lL ~iol ~19a6) 1Q~:1979-1984.
Other a3say methods, such as those of ~enitz, W.E., et
al., ~_~ (1986) ~2~:1-7 can also be used.
; The hexasaccharide ~ragments thus obtained are
o~ the formula:
:'
. A
.~ .
,~
.,
WO 92/18546 : PCI/US92/03(~92
--1 8-- '' -
21 08ato3
O
o O--
o Q
0~
V / o
- 20 ~ ~o3 o,~O
C~
2 5 . ~ O ~
; ~ o~
3 0 O o~
s~
~ .
W092/18~K 2 ~ 9 ~ 3 ~ ~ PCT/US92/0~92
~: --19--
wherein each of the variables A, B, C and D is independ-
ently H or S03R and each R is independently H or a
cation, with the proviso that at least two of the
variables A, B, C or D is -S03R. It is pointed out that
hydroxyl groups on 3-positions of the sugars have been
omitted for greater clarity and the * adjacent the COOH
indicates undetermined stereochemistry.
In the compounds of formula (II) the sugar at
the reducing terminus is deaminated to form the
2,5-anhydromannose shown. When this compound is further
reduced, the CHO shown becomes -CH20H; however, this
reduction does not occur in the depolymerization reaction
er se. This reduced form is a compound of the present
invention as shown below in formula (IIa).
; .
, . .
.
WO 92/18546 PCI`/IJS92/03092
--20-- ,r .
, 2108~gg,^ , ~ ~
O
~1~
~ y
O~ ~
O
o~ /
V I O
d~Z
O ~
~ o o~O
O ' ,~
;~
2S ~ ~/ O~
~ l/ Z
3 O o.. )
_~
~Q
:C
' .
. .
: '~
~ .~
'.~ ' ' .
WO92/18~6 PCT/US92/03092
.`, .. ~., .
2 ~L O ~3 r 6 ;~
-21-
In the -S03R, the cations represented by R can
either be inorganic cations such as sodium, potassium,
calcium, or ammonium ion or can be organic cations such
as those obtained from quaternary aminesi these salts are
formed by simple neutralization.
Based on the above formula (II) it can be seen
that there are eleven different possible configurations
with respect to the position~ of -S03R moieties when 2 or
more are present. These configurations are schematically
shown in the following table wherein an "X" indicates a
-SO3R is present at the indicated A, B, C or D position.
,~
.
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.
,. ~
. ~ , .
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W092/18~ PCT/VS92/03~2
f
- 2~ 9 ~ 5 ~ 9 -22-
Table 1
" ,
A ~ C D = S03
, .
1. X X X X
2. X X X
3. X X X
4. X X ~X
5. X X X
6. X X
7. x X
8. X X
9. X X
10. X X
11. X X
I~ that each ~R~ can be any cation, the above eleven pos-
sible structures represent a significantly larger number
of compounds, i.e., the acid and salt forms.
The basic structure of the eleven possible
configurations shown by formula (1) and the above table
are put forth below. Regarding the structures II(1) -
II~11) it is pointed OUt that hydroxyl groups on the
3~positio~ of the sugars have been omitted for greater
clarity.
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WO92/18~6 2 1 ~ P~T/US92/O~
-27-
Representative compounds of the invention,
wherein R is as above defined are set forth as follows.
In these representations, the following abbreviations are
used: L-iduronic acid = IdoA; D-glucosamine = GlcNH2; N-
acetyl-D-glucosamine = GlcNAc; D-glucosamine N-sulfate =
GlcNS; 2,5-anhydromannose = Man(2,5); 2,5-anhydromannitol
= ManH(2,5). The location of the O-linked sulfate
residues is indicated by "S" and the number of the posi-
tion of sulfation where the S03R residue is linked to
oxygen. In the designations below, the alpha and beta
anomeric linkages are as those shown in formula l above
and the indicated D or L configurations as set forth
above pertains. The locations of the sulfates are shown
below the abbreviation for the sugar to which they apply.
lS
The hexasaccharide and octasaccharide fragments
obtained by digesting heparin and following the
above-described procedures are of the formula:
` 35
:
`~:
. .
.
W092/18~6 .: . ~ PCT/US92/~3092
-28- ~.
21 ~ 8 ~ ~ ~ Formula m
COOH ~CH2O- A COOH \ r
~0 , ~ ,~_~0 ,~
OSO3- ~~O \~O- B
\ HNSO3- /
n
CH20SO3
~CH2O- C COOH
2 o HNso ~CHO
wherein n is 1 or 2, each of the variables A, B, C and D
. i8 independently H or SO~R, wherein each R is
independently H or a cation, with the proviso that at
least two o~ said A, B, 0 and D are S03R. As in the
Formulas I and II above, the hydroxyl groups in the 3
positlon of the sugars have been omitted for greater
¢larity and the asterisk next to the position of the
carboxyl group indicates that the stereochemistry thereof
3 i8 undetermlned.
In the compounds o~ formula (III) the sugar at
the reducing tarminus is deaminated to form the
2,5-anhydromanno~e shown. When this sugar is further
reduced, the CH0 ~hown becomes -CH20H; however, the
W092/18~6 2 ~ PCT/US92/0~92
-29-
reduction does not occur in the depolymerization reaction
per se. The reduced compound is part of the present
invention and is shown below as formula (IIIa) wherein
each of the variables is defined as in formula III above.
Fonnula III(a)
COOH / ~H20- A COOH \
(H) OSO3.~0/~O- B~
HNSO3-
n
CH20SO3-
~CH2O- C COOH
~\/~o D~\cHo
03-
The cations represented by R can either be
inor~anic cations such as sodium, potassium, calcium, or
ammoniu~ ion or can be organic cations such as those
obtained from quaternary amines and these salts are
formed by simple neutrallzation. Aæ above, the hydroxyls
at the 3 positions are not shown in the structure, but
are understood to be present, and the asterisk adjacent
the positlon o~ the carboxyl groups indicates that the
stereochemistry at these positions is undetermined.
3S
,
WO92/18~ ` PCT/US92/03092
2 1 ~ ~ r~ ~ ~ ~ 30- ~ ~
Based on the above formula (III) it can be seen
that there are fifty-seven different possible configura-
tions with respect to the position of the -SO3R moieties
when 2 or more are p~esent. These configurations are
schematically shown in Figures 6A, 6B and 6C wherein an
"X" indicates a -SO3R is present at the indicated A, B,
A', B', C or D position, wherein A' and B' represent the
embodiments of A and B in the parenthesized disaccharide
unit proximal to the dehydromannose or dehydromannitol
residue.
In that each "R" can be H or a cation, the
fifty-seven possible structures represent a significantly
larger number of compounds, i.e., the acid and salt
forms.
The basic structural formulae of the
fifty-seven configurations are not put forth herein.
However, these formulae can be deduced from formula III
and Figures 6A, 6B and 6C by referring to the formulae
II(l) - II(ll~ above.
Preferred compounds of the invention are the
hexasaccharides. However, the preferred octasaccharides
include octasaccharides having antiproliferative activity
with smooth muscle cells which have the formula
IdoA-GlcNS-IdoA-GlcNS-IdoA-GlcNS-IdoA-Man(2,5)
2S 6S
wherein at least two of the six sugars in the middle
~GlcNS-IdoA-GlcNS-IdoA-GlcNS-IdoA- group include a
sulfate; or a phy~iologically acceptable salt thereof.
Especially preferred among these are
octasaccharides wherein at least two IdoA-GlcNY units are
IdoA-GlcNS-IdoA-GlcNS
2S 6S 2S 6S
Thus, preferred octasaccharides of the
invention include any of the following octasaccharides,
WO92/1B~6 2 ~ O ~ ~ 6 3 PCT/US92/0~2
31-.
their pharmaceutically acceptable salts and mixtures of
two or more of such octasaccharides and their salts
IdoA-GlcNS-IdoA-GlcNS-IdoA-GlcNS-IdoA-Man(2,5);
2S 6S 2S 6S 2S 6S 2S 6S
IdoA-GlcNS-IdoA-GlcNS-IdoA-GlcNS-IdoA-Man(2,5);
2S 6S 2S 6S 2S 6S 6S
IdoA-GlcNS-IdoA-GlcNS-IdoA-GlcNS-IdoA-Man(2,5);
2S 6S 2S 6S 2S 2S 6S
IdoA-GlcNS-IdoA-GlcNS-IdoA-GlcNS-IdoA-Man(2,5);
2S 6S 2S 6S 6S 2S 6S
IdoA-GlcNS-IdoA-GlcNS-IdoA-GlcNS-IdoA-Man(2,5);
2S 6S 2S 2S 6S 2S 6S
IdoA-GlcNS-IdoA-GlcNS-IdoA-GlcNS-IdoA-Man(2,5); and
2S 6S 6S 2S 6S 2S 6S
IdoA-GlcNS-IdoA-GlcNS-IdoA-GlcNS-IdoA-Man(2,5).
2S 2S 6S 2S 6S 2S 6S
State~ent of ~ti 1i~Y
The oligosaccharide compositions of the invention
are use~ul in therapeutic applications for treat~ent of
conditions or diseases which are characterized by exces-
sive and destructive smooth muscle cell proliferation.
: These conditions frequently occur where the subject has
been exposed to trauma, such as in the case of surgical
patients. T~e trauma caused by wounds or surgery results
in vascular damage and secondary smooth muscle cell pro-
liferation, which secondary proliferation re~ults in
va~cul~r resenosis. This undesirable result can occur
a~ter va~cular grart surgery, heart transplantation, bal-
loon or laser angiopla~ty, arterial traumatic injury,po~t~urgical repair o~ muscular arteries, long-term
in-dwelling of arterial catheters, invasive arterial
diagno~tic procedures, kidney, lung or liver transplants,
coronary artery bypass surgery, carotid artery bypass
W092/l8~6 PCT/US92/03092
2~ ~8~ 32- ~-
surgery, femoral popliteal bypass surgery, and intra-
cranial arterial bypass surgery.
In addition to secondary smooth muscle cell
proliferation events occurring as a result o~ trauma,
certain diseases are associated with unwanted vascular
proliferation, although in these cases, too, it is
assumed that some internal unknown injury has caused the
secondary result. These disease states include
Goodpasture syndrome, acute glomerulonephritis, neonatal
pulmonary hypertension, asthma, congestive heart failure,
adult pulmonary hypertension, and renal vascular
h~pertension.
For all these diseases and conditions, administra-
tion of suitable amounts of the compositions of the
invention is useful in treatment. Administration is by
typical routes appropriate for polysaccharide composi-
tions, and generally includes systemic administration,
such as by injection. Particularly preferred is intra-
venous injection, as continuous injection over long time
periods can be easily continued. Typical dosage ranges
are in the range of 0.1-10 mg/kg/hr on a constant basis
over a period of 5-30, preferably 7-14, days. Particu-
larly pre~erred dosage is about 0.3 mg/kg/hr, or, for a
70 kg adult, 21 mg/hr or 540 mg/day.
Other modes of administration are less preferred
but may be more convenient. Injection subcutaneously at
a lower do~e or administered orally at a ælightly higher
do~e than intravenous in~ection, or by transmembrane or
tran8dermal or other topical administration for localized
in~ury may also be erfective. Localiæed administration
through a continuous release device, such as a supporting
matrix, perhaps included in a vascular gra~t material, is
particularly u~e~ul where the location of the trauma is
accossible.
W092/18~ 2 ~ PCT/USg~/0~2
, --
-33-
Formulations suitable for the foregoing modes of
administration are known in the art, and a ~uitable
compendium of formulations is found in Reminaton's
Pharmaceutical Sciences, Mack Publishing Company, Easton,
PA, latest edition.
; - The compositions of the invention may also be
labeled using typical methods such as radiolabeling,
fluorescent labeling, chromophores or enzymes, and used
in a competitive assay for the amount of
lo antiproliferative component in a biological sample.
Suitable protocols for competitive assays of analytes in
biological samples are well known in the art, and
generally involve treatment of the sample, in admixture
with the labeled competitor, with a specific binding
partner which is reactive with the analyte such as,
typically, an immunoglobulin or fragment thereof. The
antibodies prepared according to the invention are useful
for this purpo~e. The bindinq of analyte and co~petitor
to the antibody can be measured by removing the bound
complex and assaying either the complex or the
supernatant for the label. The separation can be made
more facile by preliminary conjugation o~ the specific
binding partner to a solid support. Such techniques are
well known in the art, and the protocols available for
such competitive assaya are too numerous and too well
known to be Get forth in detail here.
The antibodies of the invention are use~ul in
immunoassays, not only o~ the type described above
involving competition between labeled composition and the
analyte antiproli~eration ~actor in the sample, but also
for direct immunoassay ~or the ractOr. ~lternate
protocol~ involving direct assays are also of wide
variaty and well known. Typically, the analyte bound to
antibody is detected by means of an additional reactive
:, .
WO92/18~K 2 ~ ~ 8 ~ ~ ~ PCT/US92/0~2
-34-
partner which bears a label or other means of detection.
Thus, in typical sandwich assays, for example, the
binding of the antibodies of the invention to analyte can
be detected by further reaction with a labeled
preparation of these same antibodies or by labeled
antibody immunoreactive with this preparation by virtue
of species differences.
The antibodies of the invention can also be formu-
lated into pharmaceutical compositions and used to
stimulate the growth of smooth muscle cells in subjects
for which this result is desirable.
EXAMPLES
The following examples are put forth so as to
provide those of ordinary skill in the art with a
complete disclosure and description of how to make the
compounds and compositions of the invention and are not
intended to limit the scope of what the inventors regard
as their invention. Efforts have been made to insure
accuracy with respect to numbers used (e.g., amounts,
temperature, etc.), but some experimental errors and
deviations should be accounted for. Unless indicated
otherwise, parts are parts by weight, temperature is in
degrees centigrade, and pressure is at or near
atmospheric.
Exam~le 1
Pre~ar~ion o~ ~ex~- and Octasaccharide
Heparin Fraoments
To 20 g o~ heparin dissolved in 160 ml water was
added 690 mg solid NaNO2 to give a ~inal HONO
concentration o~ 50 mM in the deamination mixture.
Approximately 70 ml 6 M HCl was added dropwise while the
mixture was stirred with a maqnetic stirrer. The pH
, .
.,
W092/18~6 2 :} ~ PCT/US92/0~92
^~ -35-
dropped slowly to 1.5 and was maintained at 1.5 by
dropwise addition of either 6 M HCl or 2 M Na2C03.
Initially, the addition of acid caused the reaction
mixture to tur~ yellow, but, as the reaction reached
completion (a~out 6 min, when N2 evolution ceased), the
solution became clear. When the reaction was complete,
2 M Na2C03 was added to bring the final pH up to 8.5. A
fine white precipitate which sometimes appeared was
removed by centrifugation and the supernatant was
decanted, degassed under vacuum, and then loaded directly
onto a BioGel P10 column.
For the BioGel chromatography, two columns were
connected in tandem, each approximately 5 cm in diameter,
128 cm in length, were packed with a total of 5 1. of
BioGel P10. The columns were prepared and run in 0.5 M
NH4~C03 at a flow rate of 0.7 ml per min. The
deamination mixture was loaded onto the column in the
smallest possible volume (less than 160 ml). Fractions
of 18 ml were collected and analyzed by the carbazole
procedure. Fractions in individual peaks were combined
and dried by extensive lyophilization to remove the
NH4HC03. Peaks containing mixtures of di-, tetra-,
hexa-, octa-, deca-, and higher oligosaccharides were
obtained, with the higher oligosaccharides eluting early
and the disaccharides eluting last.
Exam~le 2
~eQt on Smooth Muscle Proliferation
Solutions to be tested were made up in "complete
medium", whic~ i8 DMEM medium containing 10% retal calf
sorum ~nd penicillin/streptomycin.
Bovine smooth muscle cells (SMC) were isolated ~rom
~' bovine pulmonary artery by the method of Ross, R.J., Cell
~iQl (1971) 172-186. SMC from passage 3-10 were plated
. . .
f,
i
'
WO92/18~ - PCT/US92/0~2
2 1 G 8 ~ 36- ~_
at 350-700 c~lls per well in 96-well microtiter plates in
the medium above and allowed to attach for 2-4 hr. The
complete medium was replaced with DMEM supplemented with
0.1% fetal calf serum, and the cells were incubated for
an additional period of about 24 to 72 hr to arrest cell
growth. The low-serum medium was then replaced with
complete medium containing the test samples.
The cells were allowed to grow for up to 7 days
with replicate plates sampled at regular intervals. Cell
number was determined by removing the medium and washing
the cells with phosphate-buffered saline, adding
75-150 ul lysis buffer, and assaying for lactate
dehydrogenase (LDH) activity, as described by Brandley,
B., et al., J ~iol Chem (1987) 262:6431. The activity of
LDH is proportional to cell number.
The results of one such assay on oligosaccharides
ranging in size from tetra- to tetradecasaccharide
fractions are shown in Figure 2. These results show that
hexasaccharide and octasaccharide fractions are active in
the antiproliferative assay; the tetrasaccharide fraction
appears substantially less active. While high molecular
weight fragments are also active in this assay, fragments
of hexasaccharide length and longer have comparable
activity to heparin on a concentration weight/volume
basis and shorter oligosaccharides are more conveniently
amenable to de novo synthesis. Hence, the minimal unit
capable o~ antiproli~erative activity is of interest.
As ~hown in Figure 2, 80~ inhibition of prolif-
eration is ~ound at concentrations of the octasaccharide
rractiOn as low as 15 ug/ml. Comparable inhibition is
8hown by the hexasaccharide ~raction at about 60 ug/ml.
WO92/18~K 2 ~ G 8 ~ ~ ~ PCT/US92/03092
... . .
-37-
Exam~le 3
Anion Exchanae SeParation of
Hexasaccharides and Octasaccharides
The hexasaccharide and octasaccharide fractions
obtained according to Example 1 were subjected to anion
exchange chromatography on a 1 x 7 cm column of DEAE-
toyopearl packed in 0.1 M NH4HCO3 and developed with a
linear gradient from .01 M to 1.0 M NH4HCO3 (total volume
= 600 ml). Approximately 20 mg of each oligosaccharide
mixture was loaded onto the column.
The results are shown in Figure 3A and 3B for the
hexasaccharides and octasaccharides respectively. The
eluate was divided into six fractions of e~uivalent mass
as shown in Figures 3A and 3B and assayed according to
the method of Example 2 for ability to inhibit smooth
muscle proliferation. The results of this assay are
shown in Figures 4A and 4B.
As shown in Figure 4A, the ability to inhibit
smooth muscle proliferation appears correlated with
charge, as the highest charged ~ractions are considerably
more e~ective. Fractions eluting early in either column
do not appear to have substantial antiproliferative
activity. ~owever, fractions with high affinity for the
anion exchanger are quite e~ective. For example, in the
ca~e of the hexasaccharide mixture, concentrations of
roughly 75 ug/ml of any of the four highest charged
~ractions gave inhibition o~ smooth muscle proliferation
in the a~say oquivalent to 60% Or the maximum inhibition
obtainable with commercial heparin;the highe~t charged
four rractions o~ the octasaccharide anion exchange
oeparation were capabla o~ approximately 60-80%
inhibition at 15-30 ug/ml.
The DEAE-toyopearl chromatography was run on a
larger scale using a 5 cm x 27 cm column packed in 0.3 M
~;
; ~ .
WO 92/18546 2 1 0 8 ~ ~ ~ PCr/lJS92/03092
-38- ''
NH4HC03. Up to 3 g of oligosaccharide mixture was loaded
onto this column and the column was washed successively
with 2 1 volumes of o . 3 M, 0 . 5 M, . 06 M, 0 . 9 M, and 1.2 M
NH4HC03. The fraction emerging in 0.9 M NH4HC03,
equivalent to the most highly sulfated pool from the
- smaller column, is recovexed in yields of approximately
150 mg/g of starting oligosaccharide mixture.
Example 4
lo Reverse Phase Ion Pairinq HPLC
The unresolved hexasaccharides and the hexasac-
charide fraction which is of the highest charged
character obtained from the DEAE-Toyopearl column in
Example 3 were subjected to reversed phase ion paring
HPLC as described by Guo, Y., et al., Anal Biochem (1988)
168:54-62. The elution patterns for these procedures are
shown in Figures 5A and 5~.
Figure 5A shows the elution profile from the
highest charged fragment of DEAE-Toyopearl; Figure 5B
shows the results using the total hexasaccharide
fraction. As a comparison of these profiles will
indicate, the charge separated fraction is a greatly
simplified mixture. The individual components of this
simplified mixture are expected to have antiproliferative
actiVitY-
The more highly charged ~raqments generally show
(as compared with less highly charged ~ragments and/or
¢ommercial h~parin) ~1) a greater ability to inhibit the
prolif~ration of ~mooth muscla cells and (2) a lesser
ability to act as an anticoagulant. Further
~r~tionation/separation processing can be carried out
which improve ractOrs (1) and (2) and also simultaneously
aid in eliminating fragments which include
oligosaccharides sulfated at the 3-position. It is
W092/18~ 2 ~ ~ 8 ~ ~ ~ PCT/US92/0~W2
-39-
pointed out that i~ order for a glucosamine to have
anticoagulant activity it must be sulfated at the
3-position. Preferred oligosaccharide fragments of the
in~ention possess characteristic (1) and (2) and (3) are
highly charged and (4) include a very low (or no) amounts
of saccharides sulfated at the 3-position as compared
with fragments of commercial heparin. In order to obtain
such preferred oligosaccharides, it is preferable to
produce them synthetically rather than obtain them from
lo digestion of heparin.
Example 5
Svnthesis of Preferred Oli~osaccharides
As indicated above, the oligosaccharides of the
invention which are particularly preferred have a number
of distinct characteristics such as greater ability to
inhibit proliferation of smooth muscle cells, lesser
ability to act an as anticoagulant, high degrees of
sulfation, and lack of sulfation at the 3-position.
Although it i9 possible to obtain such particularly
preferred oligosaccharides by the digestion of heparin
and thereafter separation of the fragments obtained in
accordance with methods described above, it is preferable
to obtain such particularly preferred fragments using
chemical synthesis methodologies. Chemical synthesis
methodologies makes it possible to obtain highly pure
reaction products all of which have the same structure
and therefore characteristics. The following i5 a flow
diagram which shows the synthesis of particularly
prererred oligosaccharides. At the end of the structural
synthesi~ ~chemes put rorth below, a written description
is provided which describes methods of carrying out such
synthe~is.
WO92/18~6. PCT/US92/03~2
~ .
2~085~
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Schematic Structural Retrosynthe~is for Making
Partlcularly Preferred Oligosaccharides
OR ¦
S~,~o~lOl
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n = 2, 3. 4
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~vo 92~18~6 2 ~ 0 8 ~ 5 ~ PCT/US92/0~92
,
~51-
Retrosynthesis of Partlcularly Preferred Oli~osaccharides
Oligosaccharides, regardles~ of their size or
whether their glucosamine unit is sulfated at 0-6, can be
retrosynthesized to a common protected disaccharide unit.
This disaccharide unit is open for chain extension in
both directions; its thioglyco~ide function permits
chain-extension towards the reducing end, whereas
protection of its 0-4' position by the temporary
chloracetyl group allows further chain-extensions towards
the non-reducing end. The ~mino groups of the target
compounds are masked as azido groups, which a~sure
advantageous stereocontrol in glycosylation reactions.
Benzyl (Bn) groups are used as permanent blocking groups
for the hydroxyls which are nonsulfated in the target
compounds, and the semi-permanent benzoyl (~z) group is
used for the OH~s to be sulfated. The ~-methoxybenzyl
(M~n) group stand~ for hydroxyl groups the sulfation of
which i9 optional in the target compounds.
The disaccharide synthon should be available by
glycosylating the protected 2-azido-2-deoxy-
glucopyranosyl derivative with the iduronosyl ~romide.
Though all the target oligosaccharides can be
synthesized from this single disaccharide synthon, for
the particular case o~ the reducing end this synthon can
be substituted by another d~saccharide unit (shown in
brackets), which bears a methyl glycoside at the reducing
end, and an amino group which i9 protected by a
benzyloxycarbonyl (Z) group. This digaccharide can be
synthesized by coupling the same iduronic acid donor with
a glucosamine derivative shown in brackets.
This retrosynthegis in~olves a novel and
; particularly advancageoug blocking group strategy, which,
- in contrast to previoug gyntheses of sulfated oligosac-
charides, permits the preparat'on of oligosaccharideS
~,
,
~; .
WO92tl8~6 PCT/US92/03092
~.'' ~,,
2 1 0 8 ~ 6 ~ s2-
with different sulfation patterns from the same protected
derivative.
The combination of an acyl-type (benzoyl)
protecting group with the ~-methoxybenzyl group allows
specific deprotection and subsequent sulfation in any
order, leadiny to structures which have sulfate groups in
positions: a) masked by benzoyl groups in the protected
derivative; b) masked by ~-methoxy~enzyl groups in the
- protected derivative; and c) masked by both benzoyl and
~-methoxybenzyl groups in the protected derivative.
A further advantage of the use of the ~-
methoxybenzyl group is that if selective removal of this
group i9 not required it can be removed by catalytic
hydrogenation in the same step as the permanent benzyl
groups, thereby reducing the number of required synthetic
steps .
For the syntheqis of the iduronic acid donor 3-0-
benzyl-L-idose ~van ~oeckel, C.A.A. et al., Carbohydr
~h~m ~1985) 4:293, incorporated herein by reference) was
tritylated with trityl chloride in pyridine and the
product, without i~olation, was directly benzoylated by
the addition of benzoyl chloride to the reaction mixture.
The trityl group was removed by acid hydrolygis, and the
primary hydroxyl group was oxidized by chromic acid,
~ollowed by esteri~ication of the resulting carboxyl
group with diazomethane. Conversion to the glycosyl
bromide wa~ achieved with titanium~IV)bromide.
For the synthesig of the glucosamine synthon 2-
azldo~2~deoxy-D-glucose peracetace (Paulsen, H. et al.,
Chem ~er ~197a) L}1:2~34, incorporated herein by
re~erence) was converted into the thioglycoside in three
steps by selective deacetylation with hydrazine ace~ate
~Excoffier, G. ee al., Carbohydr Res (1978) ~:368,
incorporated herein by reference), followed by conversion
`'
:
W~92/18~6 PCT/US92/03~2
.. . .
2 ~ ~ 8 ~ ~ ~
-53-
of the resulting hemiacetal lnto the chloride, and
subseouent thioglycosidation. The acetyl groups were
removed by Zemplen-deacetylation, and the 4,6-0-~-
methoxybenzylidene acetal was prepared by an acetal-
exchange reaction. The 3-OH group was benzylated, and
the 4,6-0-acetal ring wa~ reducti~ely opened with
NaCN9H3-trifluoroacetic acid (Johansson, R. et al.,
J Chem Soc (19~4) 1:2371, incorporated hexein by
reference).
The glucosamine synthon for the reducing end was
synthesized by an analogous sequence from methyl 2-
benzyloxycarbonylamino-2-deoxy-~-D-glucopyranoside
(Heyns, K. et al., Chem ~er (1955) 88:188, incorporated
herein by reference) by ~-methoxybenzylidenation,
benzylation, and reductive ring opening.
Coupling of the 2-azido- and 2-benzyloxycarbonyl-
amino-2-deoxy-D-glucose derivatives with the same
iduronosyl bromide was performed using silver triflate,
in combination with collidine as a buffer of the reaction
medium, and resulted in the disaccharides.
E30th disaccharides were further functionalized to
the disaccharide synthons. The compounds were
debenzoylated and a single benzoyl group was introduced
at the 0-2' position. In the ca~e of the disaccharide
thioglycoside derivative, the benzoylation wag followed
by chloroacetylation o~ the 0-4' position.
The two disaccharide synthons were coupled by using
dlmethyl(methylthio)sulfonium tri~late (F~gedi, P. et
al., Car~ohydr Res (19~6) ~ C9, incorporated herein by
re~erence) (DMTST) to give the tetragaccharide with the
required ~-interglycosidic linkage.
Selective removal of the chloroacetyl group
~ followed by glycosylation with the game disaccharide
; donor would give the protected hexagaccharide, which can
,
;
. WO 92/18546 PCI`/US92/030g2
f-ir .
~:~Q8~69
-54-
be sulfated and deprotected in dif~erent ways as
discussed previously.
Additlonal methods of synthesis may become apparent
to those skilled in the art upon reviewing the above
disclosure by itself and/or in combination with U.S.
Patent 4,943,630 issued ~uly 24, 1990, which patent is
incorporated herein by reference to disclose methods of
synthesizing oligosaccharides.
While the present invention has been deccribed with
reference to specific embodiments thereof, it snould be
understood by those skilled in the art that various
changes may be made and equivalence may be submitted
without departing from the true spirit and scope of the
invention. In addition, many modifications may be made
to adape a particular situation, material, composition of
matter, process, process step or steps, to the objective,
spirit and scope of the invention. All such
modifications are intended to be within the scope of the
claims appended hereto.
,~ ~5
