AU2008202261A1 - Derivatives of partially desulphated glycosaminoglycans as heparanase inhibitors, endowed with antiangiogenic activity and devoid of anticoagulating effect - Google Patents

Description

Australian Patents Act 1990 Regulation 3.2A 00 0 00 (N ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title "Derivatives of partially desulphated glycosaminoglycans as heparanase inhibitors, endowed with antiangiogenic activity and devoid of anticoagulating effect" The following statement is a full description of this invention, including the best method of performing it known to us:- Q \OPER\MAL\2OO8Way\35462DO Div 2005doc -1- O 00 SDerivatives of partially desulphated glycogaminoglycans as heparanase inhibitors, endowed with antiangiogenic activity and devoid of anticoagulating effect This is a divisional of Australian Patent Application No.

2001292231, the entire contents of which are incorporated herein by reference.

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SThe invention described herein relates to partly desulphated o glycosaminoglycan derivatives, particularly heparins, to processes

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c for their preparation, to their use as active ingredients for the preparation of medicaments useful in pathological conditions, like tumors, included the metastatic forms, and for any therapeutic indication gaining benefit from the inhibition of the heparanase, and to pharmaceutical compositions containing them.

State of the art Studies performed in the Tumor Biological Research Unit of the Hadassah-Hebrew University Hospital-Israel (Isr. Med. Assoc. J.

2000, 2, 37-45; J. Med. Chem. 2000, 43, 2591-600; Invasion Metastasis 1994-95, 14, 290-302; Exp. Cell Res. 1992, 201, 208-15;) focus on the involvement of heparin-binding growth factors, heparan sulphate and heparan sulphate-degrading enzymes (heparanase) in tumor angiogenesis and metastasis. These studies have been applied to screening and to the identification of heparin derivatives and heparin/heparan sulphate mimetics with potent heparanase inhibiting activity (Nature Med. 1999, 5, 735-6; Science, 1999, 285, 33-4].

Tumor cells release the enzyme heparanase, an endo-p-Dglucuronidase which degrades the polysaccharide chain of heparan 00 2 0 C' sulphate proteoglycans on cell surfaces and in the extracellular matrix.

Involvement in tumor angiogenesis of heparanase has been correlated with the ability to release bFGF (FGF-2) and other growth S5 factors from its storage within the ECM (extracellular matrix). These

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growth factors provide a mechanism for induction of 00 neovascularization in normal and pathological situations.

Heparanase may thus facilitate not only tumor cell invasion and metastasis but also tumor angiogenesis, both critical steps in tumor progression.

Specific inhibitors of the heparanase enzyme prevent release and activation of growth factors stored by heparan sulphate as well as disruption of the ECM, and are regarded as a very promising approach to develop anticancer drugs.

So, one of possible therapeutic approaches for an antiangiogenic drug is the development of a potent and selective heparanase inhibitor.

For a discussion of angiogenesis, reference may be made to WO 01/55221, in the name of the present applicant.

Another important involvement of heparanase is both inflammation and autoimmunity. In fact, heparanase activity correlates also with the ability of activated cells of the immune system to leave the circulation and elicit both inflammatory and autoimmune responses. Interaction of platelets, granulocytes, T and 00 3 o c' B lymphocytes, macrophages and mast cells with the subendothelial ECM is associated with degradation of heparan sulphate by heparanase activity. The enzyme is released from intracellular compartments lysosomes, specific granules) in response to various activation signals, suggesting its regulated involvement and

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c presence in inflammatory sites and autoimmune lesions. Treatment

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00 of experimental animals with heparanase inhibitors nonq anticoagulant species of low molecular weight heparin LMWH) markedly reduced the incidence of experimental autoimmune encephalomyelitis (EAE), adjuvant arthritis and graft rejection in experimental animals, indicating that heparanase inhibitors may beapplied to inhibit autoimmune and inflammatory disease.

Heparin Heparin is a heterogeneous mixture of naturally occurring polysaccharides of various lengths and various degrees of sulphation which possesses anticoagulant activity and is secreted by the connective tissue mast cells present in the liver (from which it was first isolated), in the muscles, lungs, thymus and spleen.

In addition to the regular sequence, a sequence corresponding to the active site for antithrombin activity has been identified in heparin.

The antitumor and antimetastatic activity of heparin and its derivatives is due to its ability to inhibit heparanase, to block growth factors and to regulate angiogenesis.

00 4 0 c-i Heparan sulphates (HS) Heparan sulphates (HS) are ubiquitous protein ligands. The c-i proteins bind to the HS chains for a variety of actions from simple immobilisation or protection against the proteolytic degradation action to specific modulations of biological activities, such as angiogenesis.

oo The carbohydrate skeleton, in both heparin and the heparan 0c sulphates consists in an alternation of D-glucosamine (GlcN) and hexuronic acids (GlcA or IdoA).

In heparin, the GlcN residues are mainly N-sulphated, whereas in HS they are both N-sulphated and N-acetylated, with a small amount of unsubstituted NH2 groups.

HS is also on average less O-sulphated than heparin.

The use of heparin in the treatment of angiogenesis disorders, such as tumours, particularly metastases, is substantially limited by the anticoagulant activity of heparin.

Chemical modifications have been made to heparin so as to reduce its anticoagulant capacity, at the same time preserving its antitumor properties.

The opening of a unit of glucuronic acid in the antithrombin site reduces the affinity of heparin for antithrombin: in this way, heparins can be used with reduced anticoagulant effects, but still retaining antiangiogenic properties.

00 0 c Heparanases Heparanases are enzymes belonging to a family of c endoglycosidases (an endo-P-D-glucuronidase) that hydrolyse the internal glycoside bonds of the chains of heparan sulphates (HS) and heparin.

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g These endoglycosidases are involved in the proliferation of 00 tumour cells, in metastases and in the neovascularisation of tumours. These enzymes are biological targets for antiangiogenic activity. In the scientific literature there are a large number of structure/activity correlation studies (see, for example, Lapierre F. et al., Glycobiology, vol. 6, 355-366, 1996). Though many aspects have still to be clarified, studies have been reported regarding the inhibition of heparanases by heparin and its derivatives, using specific tests which have led to the emergence of considerations of a structural type which may serve as guides for obtaining new, more selective derivatives.

In the above-mentioned work of Lapierre et al., heparin derivatives are described as obtained by 2-0 desulphation or by "glycol split" (oxidation with periodate and subsequent reduction with sodium borohydride). These derivatives, defined here as desulphated heparin" and "RO-heparin", respectively, have partly maintained the antiangiogenic activity of heparin as assessed by means of the CAM test in the presence of corticosteroids (ibid. page 360).

00 6

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CN N-acyl heparin derivatives, which are closer mimics of heparan sulphate than heparin, have been reported to inhibit heparanase c-i only somewhat less than N-sulphate derivatives. (Irimira T., c- Biochemistry 1986, 25, 5322-5328; Ishai-Michaeli et al, IN 5 Biochemistry 1992, 31, 2080-2088).

g? Heparins and FGF c- 00 FGFs regulate multiple physiological processes such as cell c growth and differentiation, but also functions involved in pathological processes such as tumour angiogenesis.

FGFs are growth factors (a family of more than polypeptides, of which the acid (FGF-1) and basic FGFs (FGF-2) are the ones which have been most studied, which require a polysaccharide cofactor, heparin or HS, to bind to the FGF receptor (FGFR) and activate it.

Though the precise mechanism whereby heparin and HS activate FGFs is unknown, it is known, however, that heparin/FGF/FGFR form a "trimolecular" or "ternary" complex.

One mechanism postulated is that heparin and HS induce socalled sandwich dimerisation of FGF, and the latter, thus dimerised, forms a stable complex with FGFR.

Antimetastatic activity of heparin derivatives The ability of a primary tumour to generate metastatic cells is perhaps the main problem facing anticancer therapy.

Heparin derivatives with a substantial ability to block 00 7

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heparanase seem to be equally capable of inhibiting angiogenesis a both in primary tumours and in metastases.

c In addition, the inhibition of heparanase reduces the migration ability of tumour cells from the primary tumour to other organs.

The antimetastatic activity in animal models has been found to CN correlate with the heparanase-inhibiting ability of heparin and 00 heparin derivatives (Bitan M. et al, Isr. J. Med. Sci. 1995, 31, 106- S108) as well as other sulphated polysaccharides (Miao, H. Q. et al, Int. J. Cancer 1999, 83, 424-431, and references therein). Studies on the molecular-weight dependence of the antimetastatic activity indicated that also very low-MW heparins (Sciumbata, et al, Invasion Metastasis 1996, 16, 132-143) and oligosaccharide polysulphates (Parish, et al, Cancer Res. 1999, 59, 3433-3441) retain significant antimetastatic activity. Although in general removal of N-sulphate groups (N-desulphation) decreases the antimetastatic potential of heparins, this activity is partially restored upon N-acylation (N-acetylation, N-hexanoylation (Bitan 1995), and N-succinylation (Sciumbata, 1996) of resulting free NH 2 groups. The antimetastatic activity of heparins was found to be inversely correlated to their degrees of O-sulphation. (Bitan M., 1995). However, selective 2-O-desulphation of iduronic acid residues did not involve a strong reduction of the antimetastatic activity of heparin (Lapierre, Glycobiology 1996, 6, 355-366).

In general, both the heparanase-inhibiting and the 00 8

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CN antimetastatic activity of heparins and other sulphated polysaccharides decrease with decreasing molecular weight and (C degree of sulphation (Bitan 1995; Parish, 1999). However, these activities also depend on the carbohydrate backbone of the polysaccharide (type of residues and position of glycosydic linkages) (Parish, C. 1999). Since the tridimensional structure of the active 0 0 site of heparanase is not yet known, it is difficult to predict which c- polysaccharide backbones and sulphation patterns most effectively inhibit the enzyme.

On the basis of the present knowledge, the structural requirements of heparin-like molecules that favour the angiogenesisinhibiting action can be grouped in two categories on the basis of the target one intends to block: a) inhibition of heparanase: although this enzyme recognizes and cleaves heparin and HS sequences of at least eight monosaccharide units containing N-acyl-glucosamine-glucuronic acid (or N-sulphated glucosamine residues see, for example, D.

Sandback-Pikas et al. J. Biol. Chem., 273, 18777-18780 (1998) and references cited), its inhibition can be efficiently accomplished by heparin fragments longer than tetradecasaccharide (Bitan 1995) or by extensively sulphated, shorter oligosaccharides, such as maltohexaose sulphate (MHS) and phosphomannopentaose sulphate (PI-88) (Parish, C. 1999). However, both long heparin fragments and heavily sulphated oligosaccharides are anticoagulant, a property 00 9 ci that should be avoided for potential antimetastatic drugs; b) inhibition of angiogenic growth factors (fibroblast type: FGF- C 1 and FGF-2; vascular endothelium type: VEGF; vascular permeability type: VPF): to this end the heparin-like compounds preferably have sequences at least five monosaccharide units long, c containing 2-sulphated iduronic acid and glucosamine N,6- 00 sulphated (see, for example, M. Maccarana et al. J. Biol. Chem., 268, S23989-23905 (1993)).

The literature discloses small peptides (5-13 amino acids) with antiangiogenic activity (US Patent 5,399,667 of the University of Washington) which act by binding to a thrombospondin receptor, or longer peptides (50 amino acids approx.).

Modified platelet factors are known (EP 0 589 719, Lilly), capable of inhibiting endothelial proliferation, with ICso=7 nM.

Oligosaccharide fragments with antiangiogenic activity have also been amply described: it has been found, in fact, that by varying the carbohydrate sequence the interaction selectivity can be increased.

In addition, heparin can be used as a vehicle for substances which are themselves antiangiogenic, such as some steroids, exploiting the affinity of heparin for vascular endothelial cells; see, for example, WO 93/18793 of the University of Texas and Imperial Cancer Research Technology, where heparins are claimed with acidlabile linkers, such as adipic acid hydrazine, bound to cortisol. The 00 0 c antiangiogenic effect of the conjugated molecules is greater than that of the unconjugated molecules, even when administered c-i simultaneously.

In Biochim. Biophys. Acta (1996), 1310, 86-96, heparins bound to steroids cortisol) are described with a hydrazone group in C- C 20 that present greater antiangiogenic activity than the two 00 unconconjugated molecules.

c EP 0 246 654 by Daiichi Sc. describes sulphated polysaccharides with antiangiogenic activity with Phase II studies.

0o EP 0 394 971 by Pharmacia Upjohn Harvard Coll. describes hexa-saccharides heparin fragments with low sulphation, capable of inhibiting the growth of endothelial cells and angiogenesis stimulated by FGF-1. EP 0 618 234 by Alfa Wasserman describes a method for preparing semisynthetic glycosaminoglycans with a heparin or heparan structure bearing a nucleophilic group. WO 95/05182 by Glycomed describes various sulphated oligosaccharides with anticoagulant, antiangiogenic and antiinflammatory activity. US 5,808,021 by Glycomed describes a method for preparing substantially non-depolymerised 2-0, desulphated heparin with a desulphation percentage in positions 2of the iduronic acid 2-0) and in position 3 of the glucosamine unit 3-0) ranging from approximately 99 to approximately 75% of the original percentage. This method envisages desulphation conducted in the presence of a cation of a bivalent metal, exemplified by 00 11

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Cl calcium or copper, followed by lyophilisation of the product obtained. The desulphated heparins have antiangiogenic activity. EP Ci 0 251 134, Yeda Res Dev Co Ltd et al, discloses the use of subcoagulant dosages of heparin or its derivatives for preventing IN 5 allograft rejection and treating autoimmune diseases. The activity of heparin is given by inhibition of heparanase. WO 88/05301, Univ.

oo Australian Nat., discloses antimetastatic and/or antiinflammatory c' compositions containing a sulphated polysaccharide, which is heparanase inhibitor. Heparin, fucoidan, pentosan sulphate, dextran 0o sulphate are provided. WO 92/01003, Univ. Texas System, discloses the use of a heparin derivative, which is devoid of anticoagulation activity, as heparanase inhibitor. These derivatives have sulphamino or O-sulphate groups, M.W. 1000-15000 and each terminal monomeric unit is a monomeric repeating unit with a terminal O atom bound to a blocking group. WO 94/14851 and WO 96/06867, Glycomed, provide 2-0, 3-O-de-sulphated mucosal heparin, or fragments thereof, being at least 96.7% de-sulphated at the position and ate least 75% desulphated at the 3-0 position useful as non-anticoagulant heparanase inhibitors. WO 95/09637 and WO 96/09828, Glycomed, discloses highly sulphated maltooligosaccharide compounds with heparin like properties. WO 95/30424, Glycomed, provides 6-O-desulphated heparin or fragments thereof with heparanase inhibiting activity. WO 96/33726, Univ. Australian Nat., discloses sulphated 00 12

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NC oligosaccharides as heparan mimetics having heparanase inhibiting activity. WO 01/35967, Knoll AG, provides a method for treating ci cardiac insufficiency and related conditions by administering an heparanase inhibitor, among which, heparin which has partly S 5 reduced COOH groups, or is at least partly N-desulphated and Nacetylated or is at least partly N,O-desulphated and N-resulphated 00 or is O-acetylated is mentioned.

Nc The aim of the invention described herein is to find optimal glycosaminoglycan structures for generating antiangiogenic activity based on heparanase inhibition and/or FGF growth factor inhibition mechanisms. An additional aim of the invention described herein is to provide a medicament with antiangiogenic activity which is essentially devoid of the typical side effects of heparin derivatives, such as, for example, anticoagulant activity.

WO 01/55221, in the name of the applicant, discloses glycosaminoglycans, particularly a desulphated heparin, with a desulphation degree not greater than 60% of the total uronic units.

These derivatives are provided with antiangiogenic activity and are devoid of anticoagulant activity. Said compounds exert their antiangiogenic activity based on the inhibition of FGF. No activity was foreseen for inhibition of heparanase.

In quite general terms, WO 01/55221 also provides a modified heparin, containing glycosamine residues with different degrees of N-desulphation and optional subsequent total or partial acetylation.

00 13 0 CN The general teaching of said reference does not explicitly describe the N-desulphation and optional subsequent total or partial c- acetylation steps.

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Abstract of the invention I 5 It has now been found that on subjecting a glycosaminoglycan, Ssuch as a heparin-like glycosaminoglycan, heparin or modified 0 o heparin, containing glucosamine residues with different degrees of N-desulphation and optional subsequent total or partial N-acylation (preferably N-acetylation), to controlled 2-O-desulphation treatment to of the iduronic units up to a degree of desulphation not greater than of the total uronic units, the growth-factor-mediated angiogenic properties are maintained.

Surprisingly, heparin 2-O-desulphated disclosed in the above mentioned WO 01/55221 are also inhibitors of heparanes. This property was found to be further enhanced upon glycol splitting of non-sulphated uronic acid residues. Glycol-splitting, a chemical modification leading to a dramatic loss of anticoagulant activity (Casu et al, Arzneim. Forsch. (Drug Res.) 1986, 36, 637-642) was also found to dramatically enhance the heparanase-inhibiting properties of partially N-acetylated heparins obtained through N-desulphation followed by N-acetylation of the resulting free amino groups and of the 2-O-desulphated compounds.

The desulphation carried out in the conditions described in the present invention also produces the formation of iduronic units with 00 14 San oxyranic ring in position 2,3. The opening of the oxyranic ring in the conditions described in the present invention gives rise to Lcl iduronic or L-galacturonic units.

It is an object of the invention described herein the use of said 0 5 glycosaminoglycan derivatives for the preparation of a medicament 0 having heparanase and/or FGF growth factor inhibiting activity.

0 According to the present invention, said glycosaminoglycan lN derivative is preferably a heparin-like glycosaminoglycan. Still according to the present invention, said glycosaminoglycan to derivative is a modified heparin, containing glycosamine residues with different degrees of N-desulphation and optional subsequent total or partial N-acetylation.

In one particular embodiment, the invention described herein refers to a formula compound

CO

2

NHR

U

00 Ni where the U ring may have the following meanings: C0 2 0 0 00 0 H -0 C A 0 0 0 B

C

X and which can be the same or different, are an aldehyde group or the -CH 2 -D group, where D is hydroxyl or an amino acid, a peptide or a residue of a carbohydrate or oligosaccharide; R and R1, which can be the same or different, are an SO 3 a Cj- C8 acyl residue, optionally bearing at least a further carboxy group; acetyl, hexanoyl, succinyl, pivaloyl are the preferred acyl residues; n and m, which can be the same or different, may vary from 1 to 40; the sum of n+m ranges from 6 to 40; the m:n ratio ranges from 10: 2 to 1: 1; The symbol (/indicates that the units marked m and n are statistically distributed along the polysaccharide chain and are not is necessarily in sequence.

Examples Of Cl-C8 acyl residue, optionally bearing at least a further carboxy group are acetyl, propionyl, butyryl, pentanoyl, 00 16

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c-i hexanoyl, heptanoyl, and all the possible isomers, oxalyl, malonyl, succinyl, pivaloyl, glutaroyl; acetyl, hexanoyl, pivaloyl are the c-i preferred acyl residues.

When R or Ri are N-acyl groups, they preferentially range from IND 5 40 to 60% of the sum R Ri. Preferably, m is greater than or equal to n. Preferably n ranges from 40 to 60% of the sum m+n.

00 The compounds of formula above, wherein R and R 1 are C 1 c- or C 3

-C

8 acyl residue are new.

The compounds which are the subject matter of the invention to described herein, are characterized by a high power of inhibiting heparanase with interesting antiangiogenic properties, and are therefore useful as active ingredients for the preparation of medicaments for the treatment of pathologies gaining benefit from the inhibition of the heparanase, pathologies based on abnormal angiogenesis, and particularly for the treatment of metastases.

The compounds according to the present invention also inhibit FGFs.

Advantageously, the compounds according to the present invention show reduced, if not non-existent anticoagulant properties, thus avoiding or reducing the side effects typical of the heparins. A further advantage stems from the fact that. the compounds according to the invention can be characterised with instrumental analytical techniques, such as NMR spectroscopy, thus allowing process control which is absolutely desirable from the 00 17 CN industrial point of view.

t Also in the case of modified heparins, molecular weight (MW) c- has a very important function when making angiogenesis inhibitors.

It is well known, in fact, that a reduction in molecular weight (MW) 5 up to values corresponding to penta-saccharide units does not lead to a loss of antiangiogenic activity. On the other hand, it has been 00 established that, whereas beyond a certain length the heparin C chains favour rather than inhibit activation of FGF, they are even better inhibitors of heparanase than shorter chains. However, the 0o optimal chain length for inhibition of heparanase depends on the structure of the inhibitor (carbohydrate backbone, positional linkages, sulphation pattern) and should be established for any new type of potential inhibitors.

Detailed description of the invention is The compounds according to the present invention containing glycosamine residues with different degrees of N-desulphation and optional subsequent total or partial acetylation are herein specifically disclosed and claimed as new compounds.

What is meant by desulphation degree is the percentage of non-sulphated iduronic acids in relation to total uronic acids originally present in the starting heparin. One initial preferred range for the desulphation percentage is from approximately 40 to approximately Among the formula compounds, a first preferred compound OO 18 C-I is a heparin partially 2-O-desulphated with a molecular weight (MW) of 11200, a polydispersion index D of 1.3, a desulphation degree of c 1.99 (expressed as the S0 3 -:COO- molar ratio), a percentage of modified uronic acids compared to total uronic acids of \0 5 approximately 50%. Said compound (hereinafter also called ST1514) is comprised in formula where, among the other corresponding 0 0 definitions, m:n=l:l and the units marked m and n are distributed Salong the polysaccharide chain in a regular, alternating manner.

A second preferred compound is an LMW heparin partially 2- O-desulphated with a molecular weight (MW) of 3050, a polydispersion index of 2.2, a desulphation degree of 1.99 (expressed as the SO3-:COO- molar ratio), a percentage of modified uronic acids compared to total uronic acids of approximately 50%. Said compound (hereinafter also called ST2010) is comprised in formula where, among the other corresponding definitions, m:n=1:1 and the units marked m and n are distributed along the polysaccharide chain in a regular, alternating manner. This compound is obtained by nitrous acid depolymerization of ST1514, followed by reduction of aldehyde groups, therefore most of its reducing end residues are anhydromannose residues:

CH

2

OR

O CH 2

OH

H HO H H ,O H 00 19

O

CN A third preferred compound is an LMW heparin partially desulphated with a molecular weight of Mn=5800, Mw=7520, a c polydispersion index of 1.294, a percentage of modified uronic acids compared to total uronic acids of approximately 50%. Said ND 5 compound (hereinafter also called ST2184) is comprised in formula where, among the other corresponding definitions, m:n=l:l and 0 0 the units marked m and n are distributed along the polysaccharide CI chain in a regular, alternating manner. This compound is obtained by nitrous acid depolymerization of ST1514, followed by reduction of aldehyde groups, therefore most of its reducing end residues are anhydromainose residues.

A fourth preferred compound is a partially N-desulphated and N-reacetylated heparin with a molecular weight (MW) of 11200, a polydispersion index of 1.3, a desulphation degree of 1.6 (expressed as the SO3-:COO- molar ratio), a percentage of modified uronic acids compared to total uronic acids of approximately 30%. Said compound (hereinafter also called ST1518) is comprised in formula where, among the other corresponding definitions, the 50% of the sum of R and Ri is N-acetyl.

A fifth preferred compound is an LMW partially N-desulphated and N-reacetylated heparin with a molecular weight of Mn=4780, Mw=10000, a polydispersion index of 2.092, a percentage of modified uronic acids compared to total uronic acids of approximately 30%. Said compound (hereinafter also called ST2168) 00 C-i is comprised in formula where, among the other corresponding definitions, the 50% of the sum of R and Ri is N-acetyl.

(c A sixth preferred compound is a partially N-desulphated and N-reacetylated heparin with a molecular weight of Mn=10890, N 5 Mw=22370, a polydispersion index of 2.054. Said compound (hereinafter also called ST2037) is comprised in formula where, 00 among the other corresponding definitions, the 27% of the sum of R c-i and RI is N-acetyl.

A seventh preferred compound is a partially N-desulphated and N-reacetylated heparin with a molecular weight of Mn=10210, Mw=21270, a polydispersion index of 2.083. Said compound (hereinafter also called ST2038) is comprised in formula where, among the other corresponding definitions, the 39% of the sum of R and RI is N-acetyl.

An eighth preferred compound is a partially N-desulphated and N-reacetylated heparin with a molecular weight of Mn=11070, Mw=22000, a polydispersion index of 1.987. Said compound (hereinafter also called ST2041) is comprised in formula where, among the other corresponding definitions, the 64% of the sum of R and R 1 is N-acetyl.

A ninth preferred compound is. a partially N-desulphated and N-reacetylated heparin, a percentage of modified uronic acids compared to total uronic acids of approximately 30%. Said compound is comprised in formula where, among the other 00 21 CN corresponding definitions, the 27% of the sum of R and RI is Nacetyl (ST2185).

cl A tenth preferred compound is a partially N-desulphated and N-reacetylated heparin, a percentage of modified uronic acids 5 compared to total uronic acids of approximately 30%. Said compound (hereinafter also called ST2186) is comprised in formula 0 0 where, among the other corresponding definitions, the 39% of the Cl sum of R and Ri is N-acetyl.

A eleventh preferred compound is a partially N-desulphated 0o and N-reacetylated heparin, a percentage of modified uronic acids compared to total uronic acids of approximately 30%. Said compound (hereinafter also called ST2187) is comprised in formula where, among the other corresponding definitions, the 64% of the sum of R and Ri is N-acetyl.

A twelfth preferred compound is a partially 2-O-desulphated heparin with a molecular weight (MW) of 12900 D, a polydispersion index D of 1.5, a desulphation degree of 1.9 (expressed as S03-:COOmolar ratio), percentage of modified uronic acids compared to total uronic acids: 5% epoxide groups, 29% oxidated and reduced uronic residues. Said compound (hereinafter also called ST1513) is comprised in formula where, among the other corresponding definitions, m:n= 1: and the units marked m and n are distributed along the polysaccharide chain in a regular, alternating manner.

A thirteenth preferred compound is a partially 00 22

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cN- desulphated heparin with a molecular weight (MW) of 9200 D, a polydispersion index D of 1.5, percentage of modified uronic acids c-i compared to total uronic acids: 11% epoxide groups, 27.5% oxidated and reduced uronic residues. Said compound (hereinafter also called ST1515) is comprised in formula where, among the other

(N

corresponding definitions, m:n=l:1l and the units marked m and n 0 0 are distributed along the polysaccharide chain in a regular, C-i alternating manner.

A fourteenth preferred compound is a partially desulphated heparin with a molecular weight (MW) of 11000 D, a polydispersion index D of 1.5, a desulphation degree of 1.93 (expressed as S0 3 -:COO- molar ratio), a percentage of modified uronic acids compared to total uronic acids: 5% epoxide groups, 29% oxidated and reduced uronic residues.

The preparation of compound ST1514, ST1513, ST1516 and ST1515 are specifically disclosed in WO 01/55221.

The partially 2-0-desulphated derivatives according to the invention described herein are prepared as disclosed in the above mentioned WO 01/55221.

As far as the N-desulphated and optionally N-acetylated glycosaminoglycans according to the present invention, they can be prepared by means of a process, enabling also the preparation of the desulphated heparins, comprising: a) N-desulphation by solvolytic hydrolysis of sulphamino 23 Cl residues in DMSO:H20 95:5 v:v at ambient temperature for Sa time ranging from 0.5 to 8 h, and even more preferably for capproximately 2 h, to give the total or partial elimination of sulphate groups at position 2 of the glucosamine residues; I 5 b) N-acylation of said totally or partially desulphated groups at position 2 of the glucosamine residues by treatment in 0 alkaline aqueous solution (pH 8-9) with an acylating agent, NC such as acyl anhydrides, to give totally or partially acylated groups at position 2 of the glucosamine residues; then submitting the obtained compounds to steps d) or e) and f-g) below, or alternatively directly to step f) below; c) basic treatment at a temperature ranging from ambient temperature to approximately 100*C, preferably from 50 to 0 C, and even more preferably at approximately which leads to the elimination of a controlled percentage of sulphate groups in position 2 of the iduronic acid and to the formation of epoxide groups; and, if desired d) opening of said epoxide ring at approximately pH 7, at a temperature ranging from approximately 50 0 C to approximately 100*C, preferably at approximately 70°C, to yield residues of galacturonic acid; or, alternatively e) opening of said epoxide ring at a temperature ranging from approximately 0 0 C to 30°C, preferably at approximately to yield residues of iduronic acid; and, if desired 00 24 C f) oxidation of the diols with sodium periodate, to yield the opening of the glycoside ring and the formation of two caldehyde groups per modified residue; and, if desired; g) reduction of said aldehyde groups to primary alcohol and, if IN 5 desired, transformation of the D group to a group other than hydroxyl, as envisaged in the different meanings assigned in 0 formula CI h) optional acid hydrolysis of compounds obtained in step g) to obtain oligosaccharides corresponding to the regular sequences, preferably by deamination with nitrous acid.

This reaction, which is usually applied to obtain LMW heparin by cleaving the linkage between N-sulphate glucosamine residues and the next uronic acid, leads to a LMW compound having at the non reducing end a residue consisting of an uronic acid and at the reducing end a residue of anhydro mannose, this latter can be further modified to anhydromannitol by reduction with borohydride.

The obtained LMW compounds contain at least one residue of glycol-split iduronic acid; or alternatively i) submitting the products obtained in step g) to partial enzymatic hydrolysis with an enzyme selected from the group consisting of lyase, heparinase, heparitinase, or equivalent of to yield oligosaccharides, preferably tetra- or octa-saccharides, with the non-reducing terminal residue 00 0 CI consisting of unsaturated iduronic acid, the reducing residue consisting of an N-sulphoglucosamine and c-I containing at least one residue of open iduronic acid.

i) optionally the compound obtained in step c) or the product N 5 obtained in step d) is treated by partial enzyme hydrolysis; and, if desired o 0 j) subjection of the products obtained in one of steps c), C- and f) to partial 6-O-desulphation; or, alternatively, k) subjection of the starting heparin partially or totally 6desulphated to steps c) and f).

The 2-O-desulphated derivatives according to the present invention are obtained with the process above disclosed by omitting steps a) and b).

The process according to the present invention is also illustrated by the schemes below: N- 1 F lo HT 0 l r t H P ff'sSSds PrsK^S^^

"S^SS^'

n a (81HH) ST 1| RedOx fSphIltIng on nwn-sudphated Idu ionic acids oil ely present In tha Stalling itepedni Starting Heparin RO-Heparin 1) Py S2) DMSO/Mei N.OH Epox Hyrlyi 2-O-desulphate partially 2-O-des-RO-H, fincra of 2-O-desuiphatIan degree) Hyroyis N-Desulfated-Heparln LW2" sR-ea~ 11) AciO N20H Hydrolysis ex 2) NeCO,IHO Epox 2-0-desuiphate Rex I ehresse ofi2-Q-desulphatlon degree)...

N-acyl-Heparln NAy-OHpd RedOx I Hydrolys ISPl~ttng on non-suiphated uranic acids owth nzyme origInally present In the Starting Hepadfni O ihHO LMW-N-AcyI.RO-Hoparln According to the invention described herein, the preferred compound are: heparin partially 2-0-desuiphated, obtainable by the process described above, where steps a) and b) are omitted, step c) is conducted for 45 min at 60*C, and step d) at at pH 7, and having a molecular weight (MW) of 11200, a polydispersion index D of 1.3, a desuiphation degree of 1.99 (expressed as the S03-:COO- molar ratio), percentage of modified uronic acid compared to total uronic acid of approximately 50% (hereinafter also called ST 1514); LMW heparin partially 2-0-desuiphated, obtainable by the process described above, where steps a) and b) are omitted, step c) is conducted for 45 min at 60'C and step d) at 70 0

C

at pH 7, followed by step f) g) and h) conducted by 0 CN deamination and having a molecular weight (MW) of 3050, a polydispersion index D of 2.2, a desulphation degree of 1.99 c-i (expressed as the S03-:COO- molar ratio), a percentage of modified uronic acid compared to total uronic acid of IN 5 approximately 50% (hereinafter also called ST2010); c¢- LMW heparin partially 2-O-desulphated, obtainable by the 0 0 process described above, where steps a) and b) are omitted, Ci step c) is conducted for 45 min at 60°C and step d) at at pH 7, followed by step f) g) and h) conducted by deamination and having a molecular weight Mn=5800, Mw -7520, a polydispersion index D of 1.294, a percentage of modified uronic acid compared to total uronic acid of approximately 50% (hereinafter also called ST2184); heparin N-acetyl obtainable by the process described above, where step a) is conducted for 2h at room temperature and step b) for 2h at 4°C, steps e) are omitted, step f) is conducted at 4*C for one night, step g) for 3 h at room temperature and having a molecular weight (MW) of 11200, a polydispersion index D of 1.3, a desulphation degree of 1.6 (expressed as the SOa-:COOmolar ratio), percentage of modified uronic acids compared to total uronic acids of approximately 30% (hereinafter also called ST1518).

LMW heparin N-acetyl obtainable by the process 00 28 0 NC described above, where step a) is conducted for 2h at room i temperature and step b) for 2h at 4°C, steps e) are c- omitted, step f) is conducted at 4"C for one night, step g) for 3 h at room temperature, step h) is conducted by nitrous acid deamination at 4°C for 17 min, followed by reduction of aldehyde groups with borohydride at room temperature for 0 3 h, and having a molecular weight of Mw=4780, c- Mn= 10000, a polydispersion index D of 2.092, a percentage of modified uronic acids compared to total uronic acids of approximately 30% (hereinafter also called ST2168).

heparin N-acetyl obtainable by the process described above, where step a) is conducted for 2h at room temperature and step b) for 2h at 4*C, steps g), h) are omitted, and having a molecular weight of Mn= 10890, Mw=22370, a polydispersion index D of 2.054 (hereinafter also called ST2037).

heparin N-acetyl obtainable by the process described above, where step a) is conducted for 2h at room temperature and step b) for 2h at 4°C, steps g), h) are omitted, and having a molecular weight of Mn= 10210, Mw=21270, a polydispersion index D of 2.083 (hereinafter also called ST2038).

heparin N-acetyl obtainable by the process described above, where step a) is conducted for 2h at room 00 29 NC temperature and step b) for 2h at 4"C, steps g), Sh) are omitted, and having a molecular weight of Mn=11070, c-i Mw=22000, a polydispersion index D of 1.987 (hereinafter also called ST2041).

S- heparin N-acetyl obtainable by the process described above, where step a) is conducted for 2h at room 0 temperature and step b) for 2h at 4°C, steps g), Ci h) are omitted, and having a percentage of modified uronic acids compared to total uronic acids of approximately (hereinafter also called ST2185).

heparin N-acetyl obtainable by the process described above, where step a) is conducted for 2h at room temperature and step b) for 2h at 4°C, steps g), h) are omitted, and having a percentage of modified uronic acids compared to total uronic acids of approximately (hereinafter also called ST2186).

-heparin N-acetyl obtainable by the process described above, where step a) is conducted for 2h at room temperature and step b) for 2h at 4°C, steps g), h) are omitted, and having a percentage of modified uronic acids compared to total uronic acids of approximately (hereinafter also called ST2187).

The preparation of compounds ST1514, ST1513, ST1516 and ST1515 are specifically disclosed in WO 01/55221.

00 c-I The molecular weights are determined by HPLC-GPC (high performance liquid chromatography gel permeation c-i chromatography). The desuphation degree is determined by conductimetry and the percentage of modified uronic acids by 13C_ IND 5 NMR.

C-i C-i MW is the molecular weight, and D is the polydispersion index 00 expressed as MW/Mn.

C-i According to the invention described herein, the starting products are glycosaminoglycans of various origins, preferably naturally occurring heparins. It is also possible to use chemically modified heparins with a percentage content of N,6 disulphate ranging from 0 to 100%. Starting from products with a different sulphated glucosamine content, it is possible to modulate the length of the regular sequences between one open iduronic acid and another. The glycosaminoglycans according to the invention that present opening of the glycoside ring are conventionally called RO derivatives by those skilled in the field, meaning by this that the glycoside ring has been opened by means of an oxidation action, followed by a reduction (Reduction-Oxidation RO). This opening of the glycoside ring is also conventionally called "glycol split", so-called because of the formation of the two primary hydroxy present on the open ring. The compounds referred to herein will also be called "RO" or "Glycol Split" derivatives.

In a further embodiment of the invention described herein, the

O

00 31 c- aldehydes and primary hydroxy derived from the opening reaction described above ("glycol split") also lend themselves to the c-i subsequent functionalisation. Therefore, formula compounds may also bear equal or different groups, as defined above for X and X', 5 on the primary hydroxy deriving from glycol split, for example, oligosaccharide or peptide groups, ranging from a single saccharide 00 or amino acid to more than one unit of length, preferably 2 or 3 0c units.

Formula compounds where X and X' are -CH20H can also be used as vehicles for other types of drugs, by means of suitable binding with the heparin portion which is capable of providing a stable bond in normal conditions of manufacture and storage of a formulated drug, which, however, releases the transported drug in the body, preferably in the vicinity of the target organ. Examples of drugs that can be transported are steroidal and non-steroidal antiinflammatory drugs, corticosteroids, and other drugs with an antimetastatic action, in which case there will be an advantageous enhancement of the antimetastatic effect as a result of the sum of the separate intrinsic activities of the compounds according to the invention and the antimetastatic agent bound thereto, with the related advantages of a greater target selectivity and lower systemic toxicity. Examples of these drugs are the metalloproteinase inhibitors. Other drugs which can be usefully transported are those that act at the endothelial level. Formula compounds where X and 00 32 X' are other than hydroxy or aldehyde can also be used as vehicles Sfor drugs, in which case the X and X' groups will act as "spacers" between the transported molecule, that is to say the glycosaminoglycan of the present invention and the molecule acting D 5 as the vehicle, in those cases where this may be desirable for

IN

reasons of pharmacokinetics or pharmacodynamics.

00 In the case of compounds according to the invention deriving from heparin, these are prepared starting from heparin as such by means of N-desulphation followed by N-acylation using techniques known to the technical experts in the field. For example, the Ndesulphation is conducted by solvolysis in DMSO:H 2 0 solution 95:5 v:v at room temperature for time ranging from 0.5 to 8 h followed by N-acylation in alkaline condition with, for example, acylanhydrides acetyl, hexanoyl, succinyl, pivaloyl).

The following 2-O-desulphation is conducted in the presence of alkaline agents, such as sodium hydroxide, at temperatures ranging from ambient temperature to 100*C, preferably from 50 to 70°C, for example at 600C, for a sufficiently long period to obtain the desired 2-O-desulphation. The 2-O-desulphation is controlled by acting on the process parameters, such as the concentrations of reactants, the temperature and the reaction times. One preferred example consists in maintaining constant concentrations of substrate (glycosaminoglycan) at 80 mg/ml and of NaOH at 1 M, a constant temperature of 60°C and controlling the desulphation with a 00 33 Cl reaction time from 15 to 60 min. The expert in the field may vary the conditions, for example by raising the reaction temperature and c shortening the reaction time, on the basis of normal trial and error in experimental practice and on the basis of his or her general 5 knowledge of the subject.

The treatment with alkaline agents gives rise to an 00 intermediate product characterised by the presence of an epoxide C-i ring on the desulphated unit. In a thoroughly surprising manner, these intermediates have proved to be endowed with heparanase 0o inhibiting properties similar to those of the formula compounds.

Therefore, a further object of the invention described herein is a derivative of partially 2-O-desulphated heparin, and therefore heparin with a reduced charge, particularly heparin not desulphated more than 60%, characterised by an epoxide ring on the desulphation site. Said compounds characterised by an epoxide ring also belong to the whole scope covered by the present invention.

Subsequent to the formation of the epoxide ring, the latter is opened, again resorting to known techniques. The percentage of epoxide formed is calculated from the ratio between the areas of the 13C-NMR signals at approximately 55 ppm, characteristic of carbons 2 and 3 of the uronic acid ring containing the epoxide and the total number of anomeric signals (Cl of the glucosamine and uronic acid residues). If the opening is conducted hot, a galacturonic acid residue is obtained, whereas, if the opening of the epoxide ring is 00 34 cI conducted cold, an iduronic acid residue is obtained. Preferred i examples of compounds containing an epoxide ring are those c-I obtainable by the process described above and having epoxidated

C-I

uronic acid contents of 14% (hereinafter ST1509), 24% (hereinafter IND 5 ST1525) and 30% (hereinafter ST1526), respectively.

c-I SThe partially desulphated heparin is then subjected to "glycol- 0 split" (RO for short), according to the process defined above and CI Smith degradation (SD for short).

Alternatively, formula compounds can. also be obtained without passing via the epoxide intermediate, that is to say by direct glycol split and subsequent Smith degradation.

The process described so far leads to formula compounds in which the X and X' groups are both For X and X' other than -CH20H, methods are available to the expert in the field for transforming the hydroxyl group with other groups envisaged in the definitions given above (see for example Scheme on page 26, compounds ST1828, ST1829, ST1917 and ST1919). For example, the conjugation with amino acids or peptides can be done by treating the intermediate aldehyde derived from the glycol-split reaction with a reductive amination reaction (Hoffmann J. et al. Carbohydrate Research, 117, 328-331 (1983)), which can be conducted in aqueous solvent and is compatible with maintenance of the heparin structure.

If desired, and this constitutes a further object of the invention 00 3 described herein, the formula compounds can be further degraded with acid agents in suitable pH conditions, e.g. at pH 4, to yield a c-i mixture of oligosaccharides that maintain the antiangiogenic properties.

In the same way, objects of the present invention are the c-i compounds obtained by one of the steps i) and j) of the 00 C) process described above.

Objects of the invention described herein are pharmaceutical compositions containing as their active ingredient at least one formula compound, alone or in combination with one or more formula compounds, or, said formula compound or compounds in combination with the N-acyl-desulphated heparins described above, e.g. the epoxidated intermediates; the latter can also be used alone as active ingredients in the pharmaceutical compositions. The active ingredient according to the present invention will be in a mixture with suitable vehicles and/or excipients commonly used in pharmaceutical technology, such as, for instance, those described in "Remington's Pharmaceutical Sciences Handbook", latest edition.

The compositions according to the present invention will contain a therapeutically effective quantity of the active ingredient. The doses will be determined by the expert in the field, e.g. the clinician or primary care physician according to the type of disease to be treated and the patient's condition, or concomitantly with the administration of other active ingredients. By way of an example, 036 Cl doses ranging from 0.1 to 100 mg/kg may be indicated.

Examples of pharmaceutical compositions are those that can cN be administered orally or parenterally, intravenously, c-i intramuscularly, subcutaneously, transdermally or in the form of N0 5 nasal or oral sprays. Pharmaceutical compositions suitable for the c¢- Spurpose are tablets, hard or soft capsules, powders, solutions, 0 suspensions, syrups, and solid forms for extemporary liquid Ci preparations. Compositions for parenteral administration are, for example, all the intramuscular, intravenous and subcutaneous injectable forms as well as solutions, suspensions and emulsions.

Liposome formulations should also be mentioned. The tablets also include forms for the controlled release of the active ingredient whether as oral administration forms, tablets coated with suitable layers, microencapsulated powders, complexes with cyclodextrins, depot forms, for example, subcutaneous forms, such as depot injections or implants.

The compounds according to the invention described herein possess anti-heparanase and antiangiogenic activity. This makes them suitable for the preparation of medicaments useful for the treatment of subjects, generally mammals, and particularly human subjects, suffering from altered angiogenesis or subjects who need a treatment inhibiting the heparanasic activity.

Examples of diseases treated with the medicament which is the object of the present invention are primary tumours, metastases, 00 37 0 Cl diabetic retinopathies, psoriasis, retrolenticular fibroplasia, restenosis after angioplasty, coronary by-pass, inflammation, ci arthritis, autoimmune diseases, allograft rejection, cardiovascular diseases, fibro-proliferative disease, diseases elicited by abnormal N 5 platelet aggregation, diseases elicited by smooth muscle proliferation, Goodpasture syndrome, acute glomerulonephritis, 0 0 neonatal pulmonary hypertension, asthma, congestive heart failure, Nc adult pulmonary hypertension, renal vascular hypertension, proliferative retinopathies, experimental autoimmune encephalomyelitis, multiple sclerosis, insulin dependent diabetes, inflammatory bowel disease, ulcerative colitis, Crohn's disease.

Advantageously, the compounds according to the present invention are substantially devoid of the side effects typical of heparin. In particular, the compounds according to the invention are substantially devoid of anticoagulant activity. By substantially devoid of such 'activity the expert in the field means no or only negligible activity from the point of view of clinical use.

The heparanase inhibiting activity was determined according to a method established by Vlodavsky's group (Bitan M. et al, 1995).

The method is based on evaluation of the extent of fragmentation of the heparan sulphate chains of heparan sulphate proteoglycans (HSPG) caused by heparanase. Sulphate labelled extracellular matrix (ECM) is most commonly used as a source of HSPG. Sulphate labelled ECM is incubated with recombinant heparanase at pH 6.2 00

O

0

(N

t?

(N

38 in the absence and in the presence of increasing concentrations of the test compound. To evaluate the occurrence of proteoglycan degradation, the incubation medium is collected and applied for gel filtration on Sepharose 6B columns (0.9 x 30 cm). Fractions (0,2 ml) 5 are eluted with PBS at a flow rate of 5 ml/h and counted for radioactivity. The excluded volume (Vo) is marked by blue dextran and the total included volume (Vt) by phenol red. Degradation fragments of HS side chains are eluted from Sepharose 6B at 0.5 Kav 0.8 (peak II). Under the reported experimental conditions, good heparanase inhibitors inhibit fragmentation of HS at concentrations of 10 pg/ml or less.

The results are shown in Table 1. below.

Table 1 at dose ranging from 25 qg/ml to g/ml HeDaranase inhibition Inhibition Dose 25 pg/ml 10 pg/ml 5 pg/ml Heparin 100% n.d. >100% ST1516 Heparin 40 RO 100% n.d. ST1514 Heparin -50 RO 100% 100% ST1515 Heparin 27.5 RO 100% 100% 100% heparin ST1518 100% 100% 00 39

O

C Worthy to be noted, ST1518 has a high inhibition activity even at the concentration of 1 lig/ml.

c-i The compounds according to the present invention, and in particular new one, were tested for their activity with respect to IN 5 FGF's growth factors, with the same experimental model as described in WO 01/55221 and showed an activity comparable with 00 the ones disclosed in the cited reference.

c-i The following examples further illustrate the invention.

EXAMPLE I ST1518 An excess of pyridine was added to an aqueous solution of 1 g of heparin, previously eluted from a column of Amberlite IR 120. The .solution was evaporated under reduced pressure; the resulting pyridine salt of the heparin was dissolved in 50 ml of a mixture of

DMSO/H

2 0 95:5 and stirred at 20°C for 2 hours, in order to obtain a desulphation degree of about Then, the solution was diluted with an equal volume of a saturated solution of NaHCO 3 The solution was dialysed against distilled water in membranes (cut-off 1000-2000D). The final product was isolated by evaporation under reduced pressure.

N-acetylated heparin was prepared by N-acetylation of 50% Ndesulphated heparin. 1 g of heparin was dissolved in 10 ml of distilled water; the solution was cooled to 4 0 C and saturated with 00 C sodium hydrogen carbonate; 625 gl of acetic anhydride were added to this solution and the mixture was stirred for 2 hours at 4°C.

(q During the reaction, pH was controlled and maintained at about 8 by adding sodium hydrogen carbonate. Then, the solution obtained 5 was dialysed against distilled water in membranes (cut-off 2000g 1000 D).

00 1 g of heparin 50% N-acetylated heparin is dissolved in 25 ml c of distilled water and cooled to 4°C. after the addition of 25 ml of a solution of NaI04 0.2 M, the solution is left to stir in the dark for to hours, and the reaction is stopped by adding ethylene glycol and the salts are eliminated by tangential ultrafiltration. 400 mg of NaBH 4 subdivided in several portions, are added to the desalted solution.

The solution is left to stir for 3 hours at ambient temperature, then neutralized with diluted HC1 and desalted by tangential ultrafiltration.

The 13C NMR spectrum of the compound is shown in figure 1.

EXAMPLE 2 ST2010 and ST2184 g of heparin is dissolved in 63 ml of a solution NaOH 1N. The solution is left to stir for 45 min at 60*C, cooled and neutralized with diluted HC1. Then, the solution was stirred for 48 h at 70°C, cooled and dialysed against water in membranes (cut-off 2000-1000 2 g of 2-O-desulphated heparin is dissolved in 50 ml of distilled water and cooled to 4 0 C. after the addition of 50 ml of a solution of 00 41

O

C NaI04 0.2 M, the solution is left to stir in the dark for 20 hours, and C the reaction is stopped by adding ethylene glycol and the salts are c eliminated by tangential ultrafiltration. 800 mg of NaBH 4 subdivided in several portions, are added to the desalted solution. The solution is left to stir for 3 hours at ambient temperature, then neutralized c with diluted HC1 and desalted by tangential ultrafiltration.

00 400 mg of oxidated-reducted heparin are dissolved in 25 ml of distilled water. After the addition of 7 mg NaN02, the pH is adjusted to 2 with diluted HC1, and the solution is left to stir for 17 min at 4°C. The reaction is stopped by neutralization. 60 mg of NaBH 4 subdivided in several portions, are added to the desalted solution.

The solution is left to stir for 3 hours at ambient temperature, then neutralized with diluted HC1 and fractionated by gel filtration. Two fractions with different molecular weights were isolated: ST2010 having a Mw=3050 and ST2184 having a Mn=5800, Mw=7520.

The 13C NMR spectrum of the compound ST2010 is shown in figure 2.

EXAMPLE 3 ST2041 An excess of pyridine was added to an aqueous solution of 2 g of heparin, previously eluted from a column of Amberlite IR 120. The solution was evaporated under reduced pressure; the resulting pyridine salt of the heparin was dissolved in 100 ml of a mixture of P:\OPER\MALU208 305462) Dw doc.21/)l lSX 00 -42- 95:5 and stirred at 20'C for 4 hours, in order to obtain a C desulphation degree of about 64%.

Then, the solution was diluted with an equal volume of a saturated solution of NaHCO3. The solution was dialysed against distilled water in N 5 membranes (cut-off 1000-2000D). The final product was isolated by evaporation under reduced pressure.

00 The 13C NMR spectrum of the compound ST2041 is shown in figure 3.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Claims (1)

1. 2.092, a percentage of modified uronic acids compared to total uronic acids of approximately 30%, the 50% of the sum of R and R 1 is N-acetyl; partially N-desulphated and N-reacetylated heparin with a molecular weight of Mn=10890, Mw=22370, a polydispersion index of 2.054, the 27% of the sum of R and R 1 is N-acetyl; partially N-desulphated and N-reacetylated heparin with a molecular weight of Mn=10210, Mw=21270, a polydispersion index of 2.083, the 39% of the sum of R and R 1 is N-acetyl; partially N-desulphated and N-reacetylated heparin, a percentage of modified uronic acids compared to total uronic acids of approximately the 27% of the sum of R and R 1 is N-acetyl; partially N-desulphated and N-reacetylated heparin, a percentage of modified uronic acids compared to total uronic acids of approximately the 39% of the sum of R and Ri is N-acetyl. 3) Process for the preparation of the compound of claim 1 comprising the following steps: a) N-desulphation by solvolytic hydrolysis of sulphamino residues in DMSO:H20 95:5 v:v at ambient temperature for a time ranging from to 8 h, to give the total or partial elimination of sulphate groups at position 2 of the glucosamine residues; b) N-acylation of said totally or partially desulphated groups at position 2 of the glucosamine residues by treatment in alkaline aqueous P:OPER\MAL\200830546200 DL doc-21/05/2008 00 0 solution (pH 8-9) with an acylating agent, to give totally or partially 1 acylated groups at position 2 of the glucosamine residues; then submitting the obtained compounds to steps d) or e) and f-g) below, or alternatively directly to step f) below; N 5 c) basic treatment at a temperature ranging from ambient temperature to approximately 100 0 C, which leads to the elimination of a 00 controlled percentage of sulphate groups in position 2 of the iduronic acid 0and to the formation of epoxide groups; and, if desired d) opening of said epoxide ring at approximately pH 7, at a temperature ranging from approximately 50°C to approximately 100'C, to yield residues of galacturonic acid; or, alternatively e) opening of said epoxide ring at a temperature ranging from approximately 0°C to 30°C, to yield residues of iduronic acid; and, if desired f) oxidation of the diols with sodium periodate, to yield the opening of the glycoside ring and the formation of two aldehyde groups per modified residue; g) reduction of said aldehyde groups to primary alcohol and, if desired, transformation of the D group to a group other than hydroxyl, as envisaged in the different meanings assigned in formula h) optional acid hydrolysis of compounds obtained in step g) to obtain oligosaccharides corresponding to the regular sequences; or alternatively i) submitting the products obtained in step g) to partial enzymatic hydrolysis with an enzyme selected from the group consisting of lyase, heparinase, heparitinase, or equivalent of to yield oligosaccharides, preferably tetra or octa-saccharides, with the non-reducing terminal residue consisting of unsaturated iduronic acid, the reducing residue consisting of an N-sulphoglucosamine and containing at least one residue of open iduronic acid; j) optionally the compound obtained in step c) or the product P \OPER\MALU\2I080546200 Dv doc.21A)5/2005 00 0 -46- obtained in step d) is treated by partial enzyme hydrolysis; and, if desired k) subjection of the products obtained in one of steps and f) to partial 6-O-desulphation; or, alternatively, 1) subjection of the starting heparin partially or totally N 5 desulphated to steps c) and f). 00 4) Compounds obtained by the process of claim 3. Compounds obtainable by the process of claim 3, selected from the group consisting of: partially N-desulphated and N-reacetylated heparin with a molecular weight (MW) of 11250 a polydispersion index of 1.66, a desulphation degree of 1.7 (expressed as S0 3 -:COO- molar ratio), a percentage of modified uronic acids compared to total uronic acids of approximately 30%, the 50% of the sum of R and R 1 is N-acetyl; partially N-desulphated and N-reacetylated LMW heparin with a molecular weight of Mn=4780, Mw=10000, a polydispersion index D of 2.092, a percentage of modified uronic acids compared to total uronic acids of approximately 30%, the 50% of the sum of R and Ri is N-acetyl; partially N-desulphated and N-reacetylated heparin with a molecular weight of Mn=10890, Mw=22370, a polydispersion index of 2.054, the 27% of the sum of R and Ri is N-acetyl; partially N-desulphated and N-reacetylated heparin with a molecular weight of Mn=10210, Mw=21270, a polydispersion index of 2.083, the 39% of the sum of R and R 1 is N-acetyl; partially N-desulphated and N-reacetylated heparin, a percentage of modified uronic acids compared to total uronic acids of approximately the 27% of the sum of R and R 1 is N-acetyl; partially N-desulphated and N-reacetylated heparin, a percentage of modified uronic acids compared to total uronic acids of approximately the 39% of the sum of R and R 1 is N-acetyl. P.OPERIMAL2UIX8 3DS46200 Dv doc.2 00 0 -47- C 6) Pharmaceutical compositions containing at least one compound of claims 1, 2 or 4 as an active ingredient in admixture with pharmaceutically acceptable vehicles and excipients. IND 7) Use of a compound of any one of claims 1, 2, 4, or 5 in the 00 preparation of a medicament. 8) Use of a compound of any one of claims 1, 2, 4, or 5 as a vehicle for a drug. 9) Use according to claim 8, wherein said drug is selected from the group consisting of: steroidal and non-steroidal anti-inflammatory drugs, corticosteroids, drugs with antimetastatic action, drugs that act at the endothelial level. Use according to claim 9, wherein said antimetastatic drug is a metalloproteinase inhibitor. 11) A method of treating primary tumours, metastases, diabetic retinopathies, psoriasis, retrolenticular fibroplasia, restenosis after angioplasty, coronary by-pass, inflammation, arthritis, autoimmune diseases, allograft rejection, cardiovascular diseases, fibro-proliferative disease, diseases elicited by abnormal platelet aggregation, diseases elicited by smooth muscle proliferation, Goodpasture syndrome, acute glomerulonephritis, neonatal pulmonary hypertension, asthma, congestive heart failure, adult pulmonary hypertension, renal vascular hypertension, proliferative retinopathies, multiple sclerosis, experimental autoimmune encephalomyelitis, insulin dependent diabetes, inflammatory bowel disease, ulcerative colitis, or Crohn's disease including the step of administering to a patient a medicament comprising a modified heparin of 2008202261 22 May 2008