CA2108569A1 - Sulfated polysaccharides as inhibitors of smooth muscle cell proliferation - Google Patents

Abstract

Highly sulfated oligosaccharides in the form of hexasaccharide and octasaccharide compounds which have antiproliferative activity with respect to smooth muscle cells are useful in treatment of conditions characterized by unwanted smooth muscle cell proliferation such as a result of trauma or disease states such as asthma, congestive heart failure and hypertension. The oligosaccharides have increased ability to inhibit the proliferation of smooth muscle cells and decreased ability to act as an anticoagulant as compared with commercial heparin and/or unseparated fragments of heparin.

Description

WO92/18~KPCT/US92/03~2 ., .

2 ~ ~ 8 3 u ~

.~ SULFATED POLYSACCHARIDES AS INHIBITORS OF
` 10SMOOTH MUSC~E CELL PROLIFERATION

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:~, 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.

p -.' ~ . , .

W092/~8~ PCT/US92/03~2 ,f~
21~8~6~

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

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.~ .

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WO92/18~6 PCT/US92/0309~
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2~ a~5~!
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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~

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 .~

W092/18~ PCT/US92/O~g2 `~ 2~0~9 , 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

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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) 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 ~, .
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. ~ , WO92/18~6 PCT/US92/03~92 , 6 ~

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.
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~, ~ W092/18~ ~CT/US92/03092 . .. ..
21 08~69 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 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~

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~

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 ,. ,, ~ , .

.

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:

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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 (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

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~

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 ~
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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
.~ .
,~
., 6 : 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~
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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).

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WO 92/18546 PCI`/IJS92/03092 --20-- ,r .
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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
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WO92/18~6 PCT/US92/03092 .`, .. ~., .
2 ~L O ~3 r 6 ;~

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.

~ 30 '``'`' ` .
~ 35 ;

., $~
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''~

WO g2/1~546 PCI /US92/03092 ~ 2108~

3~ o ~0 15 ~0~

~0, ~0, ~

~- ~0, ~,_ \y --` ~
:~;
'''' ,-. . ., ~ !. .:
` 2108~9 ~o~ ~"o, ~,o 25 ~ ~o ~O

30 ~Z ~Z ~2 ct~ O~ O~

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. s WO 92/18~46 PCI/US92/03092 . --25--~ 2~ 0~
, ,.. ~, .

P:~ sY

~; ~0 m~~ ~, 25 ~ ~O 8_~o ~o ~
o=~
~-' O~ O~
P~ .
X
3 5 t` 00 , , .
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wo 92/1854~ ` ` . PCr/US92/03~g2 2 1 0 8 ~ 6 ~

10 ~9 ~3 15 0~ Z ~ $, ~

o\ o.l o\

h O, C~ _ WO92/18~6 2 1 ~ P~T/US92/O~

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 :
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W092/18~6 .: . ~ PCT/US92/~3092 -28- ~.
21 ~ 8 ~ ~ ~ Formula m COOH ~CH2O- A COOH \ r ~0 , ~ ,~_~0 ,~

OSO3- ~~O \~O- B
\ HNSO3- /
n ~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 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~

n ~CH2O- C COOH

~\/~o D~\cHo 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.

, 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) 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

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);

IdoA-GlcNS-IdoA-GlcNS-IdoA-GlcNS-IdoA-Man(2,5);

IdoA-GlcNS-IdoA-GlcNS-IdoA-GlcNS-IdoA-Man(2,5);

IdoA-GlcNS-IdoA-GlcNS-IdoA-GlcNS-IdoA-Man(2,5);

IdoA-GlcNS-IdoA-GlcNS-IdoA-GlcNS-IdoA-Man(2,5);

IdoA-GlcNS-IdoA-GlcNS-IdoA-GlcNS-IdoA-Man(2,5); and IdoA-GlcNS-IdoA-GlcNS-IdoA-GlcNS-IdoA-Man(2,5).

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 , --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 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 ... . .

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 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 ~ .
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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 ~ ~ ~

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 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

Claims (17)

What is claimed is:

1. A compound capable of inhibiting the proliferation of smooth muscle cells, having the following structural formula:

Formula I

wherein each of A, B, C, and D is independently H or SO3R
and each R is independently H or a cation, with the proviso that at least two of A, B, C or D is -SO3R;
noting that hydroxyl groups on the sugars have been omitted in Formulas I and I(a) for greater clarity and the * adjacent the carbon substituted with COOH indicates any possible stereochemistry in either I or I(a); R1 and R2 are each independently hydrogen or one or more repeating units having the following structure:

Formula I(a) wherein when a unit of Formula I(a) is connected at one end, the hydrogen at that end is not present; and each of E and F is independently hydrogen or SO3R and noting that the hydroxyl group on the 3-position on each sugar is present but has been omitted for greater clarity.

2. The compound of claim 1, wherein at least three of A, B, C or D is -SO3R.

3. The compound of claim 2, wherein all of A, B, C and D are -SO3R.

4. The compound of claim 1, wherein R1 and R2 are H, and each R is H, Na, K, Ca or NH4.

5. A mixture of hexasaccharide and octasaccharide compounds capable of inhibiting the proliferation of smooth muscle cells, the compound in the mixture having the following structural formula:

Formula III

wherein n is 1 or 2 and each of A, B, C and D is independently H or SO3R, wherein each R is independently H or a cation, with the proviso that at least two of said A, B, C and D are SO3R; and wherein a hydroxyl group present on the 3 position of each sugar is present but has been omitted from the depiction; and wherein the asterisk on the carbons substituted with carboxyl indicates that the stereochemistry is not determined.

6. The mixture of claim 5, wherein at least three of A, B, C or D is -SO3R.

7. The mixture of claim 5, wherein at least four of A, B, C or D is -SO3R.

8. The mixture of claim 5, wherein at least five of A, B, C or D is -SO3R.

9. The mixture of claim 5, wherein all of A, B, C
and D are -SO3R.

10. The mixture of claim 5, wherein each R is H, Na, K, Ca or NH4.

11. A mixture of hexasaccharide and octasaccharide compounds capable of inhibiting the proliferation of smooth muscle cells, the compound in the mixture having the following structural formula:

Formula III(a) wherein n is 1 or 2 and each of A, B, C and D is independently H or SO3R, wherein each R is independently H or a cation, with the proviso that at least two of said A, B, C and D are SO3R; and wherein a hydroxyl group present on the 3 position of each sugar is present but has been omitted from the depiction; and wherein the asterisk on the carbons substituted with carboxyl indicates that the stereochemistry is not determined.

12. The mixture of claim 11, wherein at least three of A, B, C or D is -SO3R.

13. The mixture of claim 11, wherein at least four of A, B, C or D is -SO3R.

14. The mixture of claim 11, wherein at least five of A, B, C or D is -SO3R.

15. The mixture of claim 11, wherein all of A, B, C and D are -SO3R.

16. The mixture of claim 11, wherein each R is H, Na, K, Ca or NH4.

17. A pharmaceutical composition useful in the treatment of conditions characterized by unwanted smooth muscle cell proliferation, comprising:
a pharmaceutically effective amount of a heparin digest derivative in the form of oligosaccharide fragments selected from the group consisting of hexasaccharide and octasaccharide or pharmaceutically acceptable salt thereof which fragments have an increased ability to inhibit smooth muscle cell proliferation and a decreased ability to act as an anticoagulant as compared with commercial heparin; and a pharmaceutically acceptable carrier.