SYNTHETIC HEXASACCHARIDES MIMICS OF HEPARIN SHOWING HEPARANASE INHIBITION ACTIVITY

Abstract

The present invention is directed to hexasaccharides mimetics of heparins presenting remarkable heparanase inhibition activity. The compounds have formula (I), wherein R.sup.1 is selected from the group consisting of NH.sub.2, NHSO.sub.3Na, NHAc; R.sup.2 is selected from the group consisting of NH.sub.2, NHSO.sub.3Na, NHAc; R.sup.3 is selected from the group consisting of Bn, H; R.sup.4 is selected from the group consisting of Ac, H, SO.sub.3Na; R.sup.5 is CO.sub.2Na; R.sup.6 is selected from the group consisting of Me, Ethyl, Alkyl, alkyl azide, alkynyl, cholestanol aglycon; R.sup.7 is selected from the group consisting of CH.sub.2OH, COOH.

##STR00001##

Claims

1. An hexasaccharide having formula ##STR00016## wherein R.sup.1 is selected from the group consisting of NH.sub.2, NHSO.sub.3Na, NHAc; R.sup.2 is selected from the group consisting of NH.sub.2, NHSO.sub.3Na, NHAc; R.sup.3 is selected from the group consisting of Bn, H; R.sup.4 is selected from the group consisting of Ac, H, SO.sub.3Na; R.sup.5 is CO.sub.2Na; R.sup.6 is selected from the group consisting of Me, Ethyl, Alkyl, alkyl azide, alkynyl, cholestanol aglycon; R.sup.7 is selected from the group consisting of CH.sub.2OH, COOH.

2. The hexasaccharide according to claim 1 wherein the hexasaccharide has formula: ##STR00017##

3. The hexasaccharide according to claim 1 wherein the hexasaccharide has formula: ##STR00018##

4. The hexasaccharide according to claim 1 wherein the hexasaccharide has formula ##STR00019## wherein R.sup.6 is selected from the group consisting of Me, Ethyl, Alkyl, alkyl azide, alkynyl, cholestanol aglycon; R.sup.7 is selected from the group consisting of CH.sub.2OH, COOH; R.sup.8 is selected from the group consisting of SO.sub.3Na, Ac.

5. The hexasaccharide according to claim 4 wherein the hexasaccharide has formula: ##STR00020##

6. The hexasaccharide according to claim 4 wherein the hexasaccharide has formula: ##STR00021##

Description

DETAILED DESCRIPTION OF THE INVENTION

[0008] The hexasaccharides according to the present invention are obtainable by glycosplit reaction of a uronic acid ring of the following hexasaccharide:

##STR00003##

Wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 and R.sup.7 have the above defined meanings. The hexasaccharide is composed of alternating rings of glucosamine sulfate and uronic acid. The term uronic acid comprises both iduronic acid and glucuronic acid.

[0009] In a preferred embodiment, the hexasaccharides have formula:

##STR00004##

[0010] In another preferred embodiment, the hexasaccharides have formula:

##STR00005##

[0011] In another preferred embodiment, the hexasaccharides have formula:

##STR00006##

Wherein R.sup.6 and R.sup.7 have the above defined meanings, R.sup.8 is selected from the group consisting of SO.sub.3Na, Ac.

[0012] When the gs ring is a glucuronic acid ring, the hexasaccharides have formula:

##STR00007##

[0013] When the gs ring is an iduronic acid ring, the hexasaccharides have formula:

##STR00008##

[0014] The compounds according to the invention can be prepared starting from the pivotal hexasaccharide 12,13 and 16 (Scheme 1) that after functionalization presents a single residue (glucuronic acid) susceptible to periodate oxidation, and from which different target structures (13-18) can be derived.

[0015] To prepare 12 we adopted (Scheme 1) a classical disaccharide building-block approach (C. Tabeur et al., Oligosaccharides corresponding to the regular sequence of heparin: chemical synthesis and interaction with FGF-2. Bioorg. Med. Chem. 1999, 7, 2003-2012) based on the trichloroacetimidate glycosylation method (R. R. Schmidt, W. Kinzy, Adv. Carbohydr. Chem. Biochem. 1992, 50, 21-123). The fully orthogonally protected hexasaccharide 9 was thus obtained from the known (M. Petitou et al., Synthesis of Heparin Fragments: A -methyl Pentaoside with High Affinity for Antithrombin II, Carbohy. Res. 1987, 167, 67-75) 3 that was first coupled with the known 2 (C. A. A. van Boeckel et al., Synthesis of a Pentasaccharide Corresponding to the Antithrombin III Binding Fragment of Heparin, Journal of Carbohydrate Chemistry, 1985, 4 (3), 293-321, https://doi.org/10.1080/07328308508070182), in the presence of tert-butyldimethylsilyl triflate, in dichloromethane at 30 C. to give the tetrasaccharide 4 in 65% yield. The hexasaccharide sequence was completed by reaction under similar conditions with the trichloroacetimidate disaccharide 8 itself classically obtained from 6. A mixture of the two anomers was formed (56%, /=4/1). After saponification to remove the acetyl groups and cleave the methyl esters, preparative HPLC allowed to separate 10 from its -anomer. Following O-sulfation (11) and hydrogenolysis, 12 was in part N-sulfated by sulfur trioxide pyridine complex in aqueous sodium hydrogen carbonate to obtain 13 (78%), it was also in part N-acetylated by acetic anhydride in aqueous sodium hydrogen carbonate to give 16 (75%). The glycol-split derivatives were then obtained (Scheme 2). After periodate oxidative cleavage of 13 the resulting dialdehyde, that was not isolated, was reduced into 14 using sodium borohydride, or further oxidized by NaClO.sub.2 in the presence of NaH.sub.2PO.sub.4 buffer at pH 5 (Pinnick oxidation) to give 15 (yield: 39%, 2 steps) after purification by an anionic exchanging column Mono Q, desalting and lyophilization. The same oxidation method yielded 17 and 18 from 16. All the structures of final products were confirmed by 1H- and 13C-NMR analysis (HSQC-dept, COSY, TOCSY, HMBC) and LC-MS. The inhibition of heparanase was evaluated using an assay based on the cleavage of the synthetic heparin pentasaccharide fondaparinux (Arixtra; Aspen). Cleavage of fondaparinux by heparanase, yields a disaccharide that is assayed by colorimetry. The assay was essentially performed as described by Hammond (E. Hammond, C. P. Li, V. Ferro, Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening, Anal. Biochem. 396 (2010), 112-116, http://doi.10.1016/j.ab.2009.09.007.

TABLE-US-00001 Scheme 1 [00009] [00010]

##STR00011##

[0016] Serial dilutions of the compounds and of the reference roneparstat were tested. IC.sub.50 values were ultimately determined using GraphPad software. The results are reported Table 1. Inhibition by hexasaccharides 12, 13 and 16 (Scheme 1), comprising an intact glucuronic acid unit was hardly detectable over the concentration range tested, with a tendency however for a better inhibition when the glucosamine units are N-sulfated rather than N-acetylated. This tendency was again observed after periodate cleavage (14 vs. 17) and particularly after introduction of the two carboxylic acid functions (15 and 18). Thus, the N-sulfated hexasaccharide 15 displayed an IC.sub.50 of 70 nM (Scheme 2). Comparing the activity of 12 and 16 with other derivatives, our results clearly demonstrate the critical role played by gs and gs-ox uronic acid units. Comparison of the activities of 15 and 18, it appears that N-sulfated glucosamine are preferred over N-acetylated ones, despite the fact that in roneparstat N-acetyl groups only are present.

TABLE-US-00002 TABLE 1 Compound IC.sub.50 No. g/ml M Roneparstat 0.006 12 50 30 13 >50 >26 14 10 5 15 0.15 0.07 16 inactive 17 inactive 18 38 21

[0017] In order to estimate the inhibition properties of a iduronic gs ring when compared to a glucuronic gs ring, trisaccharides corresponding to the three central rings of the structure (gs ring plus one ring on each side) have been prepared both with an iduronic ring and a glucuronic ring. The synthetic route is shown in Scheme 3.

TABLE-US-00003 Scheme 3 [00012] [00013] [00014] [00015]

[0018] Trisaccharide 21a and 21b were obtained by coupling monosaccharide 19 (Minghong Ni et al., Investigating Glycol-Split-Heparin-Derived Inhibitors of Heparanase: A Study of Synthetic Trisaccharides, Molecules 2016, 21, 1602;) to 20a (M. Petitou et al., Synthesis of heparin fragments: A methyl -pentaoside with high affinity for antithrombin III, Carbohydr. Res. 167 (1987), 67-75,) or 20b which was epimerized by DBU in DMF from 20a. The activator used in glycosylation reaction was TMSOTf/CH2Cl2/20 C. Given the oligosaccharide's structure, it is needed to protect and deprotect orthogonally the hydroxyl group from time to time (V. Dimakos, et al, Site-Selective Functionalization of Hydroxyl Groups in Carbohydrate Derivatives, Chem. Rev. 118 (2018) 11457-11517). This may result in cumbersome multi-steps preparation. Among selective methods allowing to protect or deprotect one position among several bearing the same function, Magnesium methoxide in methanol (G. Tiruchinapally et al., Divergent Heparin Oligosaccharide Synthesis with Preinstalled Sulfate Esters, Chemistry, 17 (2011), 10106-10112) was chosen and optimized (4 equiv. Mg(OH)2 at 10 C. for 3-4 h) to selective deacetylate 21a and 21b in a satisfied yield 45-55%. After O-sulfation, saponification, hydrogenolysis and N-sulfation, 24a and 24b were obtained. 24a and 24b were subsequently oxidized by sodium periodate, reduced by sodium borohydride (called gs) to give 25a and 25b. 24a and 24b were oxidized by sodium periodate and further oxidized by NaClO2 (Pinnick oxidation) (called gs,ox) to give 26a and 26b.

[0019] The activity of the different compounds is shown in Table 2. It is possible to note that the compounds containing a glucuronic ring are in general more active than compounds comprising a iduronic ring. It is expected that this trend is also valid for hexasaccharide structures according to the invention.

TABLE-US-00004 TABLE 2 Trisaccharide IC.sub.50 Compound (g/ml) M (nmol/ml) Roneparstat 0.006 24a (gluco) 39 40.0 25a (gluco gs) 37 37.9 26a (gluco di-carbossi) Inactive 24b (ido) Inactive 25b (ido gs) 75 76.8 26b (ido di-carbossi) 75 71.6