Synthetic hexasaccharide mimics of heparin comprising a cholestanol moiety showing heparanase inhibitory activity

Abstract

The present invention is directed to synthetic hexasaccharide mimics of heparin having formula (I) wherein: R1 is selected from the group consisting of NH2, NHSO3 -, NHAc; R2 is selected from the group consisting of H, SO3 -, Bn, 2-Naphthylmethyl, preferably H; R3 is selected from the group consisting of H, Ac, SO3 -; R4 is CO2 -; R5 is Me, Et, C3-C30 linear or branched alkyl, optionally comprising one or more isolated or fused rings, PEG-OMe; R6 is selected from the group consisting of H, Ac, SO3 -; R is a C10-C30 linear or branched alkyl optionally comprising one or more isolated or fused rings. In another embodiment, the invention is directed to the use as a drug of hexasaccharides having the above formula, wherein R, R1, R2, R3, R4 and R6 have the above defined meaning, while R5 is selected from the group consisting of Me, Et, C3-C30 linear or branched alkyl, optionally comprising isolated or fused rings, PEG-OMe.

Description

Synthetic hexasaccharide mimics of heparin comprising a cholestanol moiety showing heparanase inhibitory activityField of the invention

[0001] The present invention is directed to synthetic hexasaccharides comprising a cholestanol or alkyl moiety exhibiting remarkable heparanase inhibitory activity.Background of the invention

[0002] Heparan sulfate (HS) refers to a family of glycosaminoglycan chains present in about 20 glycoproteins, the proteoglycans (PG), distributed mainly in the extracellular matrix and at the cell surface, but also inside the cell (Thibault Annaval et al., Heparan Sulfate Proteoglycans Biosynthesis and Post Synthesis Mechanisms Combine Few Enzymes and Few Core Proteins to Generate Extensive Structural and Functional Diversity, Molecules, 2020, 25, 4215; doi: 10.3390 / molecules25184215). PGs exert their biological functions by interacting with a vast array of protein ligands including the most recently discovered SARS-CoV-2 (Thomas Mandel Clausen et al, SARS-CoV-2 Infection Depends on Cellular Heparan Sulfate and ACE2. Cell 183, 1-15, November 12, 2020).

[0003] Heparanase (Hpal) is the only P-D-endoglucuronidase in mammals capable of cleaving the HS chains of proteoglycans present on the cell membranes and in the ECM, facilitating HS turnover and recycling [Xu D.; Esko J.D.; Demystifying heparan sulfate-protein interactions, Ann. Rev. Biochem. 2014, 83,129-157. https: / / doi.10.1146 / annurev-biochem- 060713-035314], Cleavage of the HS chains results in the release and activation of growth factors, cytokines and other bioactive molecules anchored to the HS chains, promoting angiogenesis and tumor growth [Vlodavsky I.; Ilan N.; Naggi A.; Casu B.. Heparanase: Structure, Biological Function, and Inhibition by Heparin-Derived Mimetics of Heparan Sulfate, Current Pharmaceutical Design, 2007,13, 2057-2073. https: / / doi.10.2174 / 138161207781039742]. In fact, over-expression of Hpal correlates with the aggressiveness of carcinomas, sarcomas and hematological malignancies. Decades of studies encourage the search for effective heparanase inhibitors, either natural or synthetic compounds such as modified heparins, sulfated oligosaccharides, small molecules and anti-Hpal antibodies [i. McKenzie E. A.; Heparanase: A target for drug discovery in cancer and inflammation, Br. J. Pharmacol. 2007, 151, 1-14, ii. Coombe D. R.; Gandhi N. S.; Heparanase: A challenging Cancer Drug Target, Front. Oncol. 2019, 9, 1316],

[0004] WO 2015 / 062951 discloses a process for the preparation of a glucosaminoglycan derivative inhibiting heparanase, comprising: N-desulfation of from 25% to 100% of the N-sulfated residues of a glucosaminoglycan; oxidation, preferably by periodate at a pH of from 5.5 to 10.0, of from 25% to 100% of the 2-N-, 3-O-non-sulfated glucosamine residues and of the 2-0- non-sulfated uronic acid residues of said glucosaminoglycan, under conditions effective to convert adjacent diols and adjacent OH / NH2 to aldehydes; reduction, preferably by sodium borohydride, of said oxidized glucosaminoglycan, under conditions effective to convert said aldehydes to alcohols. The document discloses that, by reducing Mw, IC50 increases up to 750 ng / ml for a Mw of about 5 kDa.

[0005] A previous patent by the same Applicant, WO 2024 / 012972, disclosed that synthetic O-methylated hexasaccharide heparin mimetics exhibit heparanase inhibitory activity. In particular, when 2-0 non-sulfated uronic acid was glycosplit by sodium periodate and oxidized (abbreviated as gs,ox) to form tricarboxyl glucuronate, the resulting product demonstrated higher anti -heparanase activity compared to its non-glycosplit counterparts. The gs,ox compounds were tested in the Extracellular matrix (ECM) degradation assay showing an heparanese inhibitory activity at 10 pg, with 50 ng of Heparanase after 2 hours of incubation at 37 °C of about 30-40%. The ECM is a reliable assay since it employs a naturally produced substrate, which better predicts the inhibitory effect in vivo.

[0006] It would be important to develop new amphiphilic hexasaccharide heparin mimetics exhibiting improved heparanase inhibition activity in the ECM assay.Summary of the invention

[0007] The present invention is directed to a new class of hexasaccharides heparin mimetics comprising a cholestanol group or a C10-C30 linear or branched alkyl group, and presenting a significant heparanase inhibitory activity in the ECM assay. The compounds have structure:IWherein R1=NH2, NHSCE', NHAc; R2=H, SCE', Bn, 2-Naphthylmethyl, preferably H; R3=H, Ac, SCE'; R4=CO2-, R5= Me, Et, C3-C30 linear or branched alkyl, optionally containing one or more isolated or fused cycloalkyl rings, PEG-OMe; R6=H, Ac, SCE'; R= C10-C30 linear or branched alkyl, optionally containing one or more isolated or fused cycloalkyl rings.

[0008] . The present invention is also directed to hexasaccharide mimics of heparin for use as a medicament having formula:IWherein R1=NH2, NHSCE', NHAc; R2=H, SCE', Bn, 2-Naphthylmethyl, preferably H; R3=, H, Ac, SCE'; R4=CO2;R5= Me, Et, C3-C30 linear or branched alkyl, optionally containing isolated or fused rings, PEG-OMe; R6=H, Ac, SCE'; R= C10-C30 linear or branched alkyl group optionally containing isolated or fused ringsDetailed description of the invention

[0009] . The hexasaccharides according to the present invention are heparin mimetics comprising a Cio-C3O linear or branched alkyl group optionally containing isolated or fused rings and presenting a high heparanase inhibitory activity in the ECM assay. The general formula of these compounds is the following:Wherein R, R1, R2, R3, R4, R5and R6have the above defined meanings. In a preferred embodiment R is a sterol group having formula CnEhsO (gonane structure: cyclopentanoperhydrophenanthrene) preferably modified with other functional groups (such as for example side aliphatic chain in carbon Cl 7) and / or modified rings (such as presence of double bonds). Preferably, R is a cholestanol group of formula:

[0010] . The hexasaccharide is composed of alternating rings of glucosamine sulfate and uronic acid. The term uronic acid comprises both iduronic acid and glucuronic acid. The number of sulfated groups is equal to or less than 8, that means that on average at least one uronic acid residue is not sulfated.

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

[0012] WO 2024 / 012972 discloses hexasaccharides of general formula:Wherein R1is selected from the group consisting of NH2, NHSChNa, NHAc; R2is selected from the group consisting of NH2, NHSChNa, NHAc; R3is selected from the group consisting of Bn, H; R4is selected from the group consisting of Ac, H, SChNa; R5is CChNa; R6is selected from the group consisting of Me, Ethyl, Alkyl, alkyl azide, alkynyl, cholestanol; However, these compounds are disclosed only as an intermediate in the preparation of the compounds of formula:Wherein R1to R6have the above defined meanings and-R7is selected from the group consisting of CH2OH, CO2Na. Table 1 of WO2024 / 012972 shows that, while compounds of formula IV are active in heparanase inhibition, compounds of formula I (13 and 16) are not.

[0013] It has been surprisingly found that the compounds of formula I are even more active than the compounds of formula IV in the extracellular matrix (ECM) degradation assay as shown in figure 1-4.

[0014] The strong inhibitory activity of heparanase makes these compounds particularly useful in the treatment of all those pathologies that manifest themselves through an overproduction of heparanase. These pathologies include cancer.

[0015] The following is a scheme of the reactions used in the preparation of compounds of formula I:

[0016] Scheme 1 : Reagents and conditions: a) CI3CCN, CS2CO3, CH2Q2, RT, 92%. b) NH2NH2.HOAC, Py-AcOH, RT, 30 min, 68%. c) TMSOTf, CH2CI2, 20 °C, 90%. d) NH3(gas), THF-MeOH, O °C, 2 h. e) CI3CCN, Cs2CO3, CH2CI2, RT, 71% (2 steps), f) NH3(gas), THF- MeOH, O °C, 2 h. g) CI3CCN, Cs2CO3, CH2CI2, RT, 64% (2 steps), h) 5a-Cholestan-3P-ol, TMSOTf, -10 °C, dry Toluene, P-epimer: 51%, a-epimer: 25%. i) NH2NH2.HOAC, Py-AcOH,RT, 2.5h, 68%. j) TMSOTf, CH2CI2, 30 °C, 74%. k) LiOH, H2O2, THF-MeOH-H2O, 24h, NaOH, RT, 48 h, 92%. 1) SO3.Et3N, DMF, 55 °C, 22 h, 57%. m) H2, 10% Pd / C, t-BuOH-H2O, RT, 54 h, 94%. n) SO3.Py, aq. NaHCO3, 0 °C - RT, 41%.

[0017] To obtain fully protected hexasaccharide 13, a classical disaccharide building-block approach was adopted [C. Tabeur, J.M. Mallet, F. Bono, J.M. Herbert, M. Petitou, P. Sinay, 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 chemical synthesis is illustrated as scheme 1. The critical step consists in introducing lipophilic cholestanyl group into reducing-end and selectively forming P- anomer. 10 activated as trichloroacetimidate with commercially available 5-a-Cholestan-3-P-ol (abbreviated as Choi). A mixture of two anomers was formed. After optimisation of the reaction and separation conditions, reaction was caried out in the presence of TMSOTf promotor at -10°C in dry toluene, pure P-epimer 11 was obtained with 51% yield. The ratio p / a anomers was 2 / 1. The sequence of building hexasaccharide backbone was different from our patent WO 2024 / 012972 where a tetrasaccharide was first prepared from non-reducing end. Due to the low yield and the difficulty preparing disaccharide 12, it was preferred to insert this disaccharide 12 into the backbone of the hexasaccharide in the last step. Trichloroacetimidate disaccharide 2 was first coupled at the non-reducing end with acceptor 4 in the presence of TMSOTf in di chloromethane at -20 °C to give tetrasaccharide 5 in 89.5% yield without forming P-epimer. The tetrasaccharide 7 was coupled with 12 to give protected hexasaccharide 13 (yield: 74.4%). Classical methods were adopted to deprotect and functionalize oligosaccharide 13 although slight modifications were applied due to the amphiphilic character of product. The lead compound Hexa-Ido-OChol (IC-1) was given.

[0018] Similar reactions as published in patent WO 2024 / 012972 were used to prepare the second series Hexa-Ido-OMe with methyl group at reducing end (IM-1) with intact iduronic acid. The third series containing glucuronic acid instead of iduronic acid and methyl (GM-1) was prepared as described in WO 2024 / 012972. The chemical structures were shown in formula V.IM-1, R= a-OMe, (Sodium Iduronate)IC-1, R= P-OChol. (Sodium Iduronate)GM-1, R= a-OMe (Sodium Glucuronate)

[0019] For comparison, their glycosplit (gs) version were prepared, compound GM-2, IM-2, IC-2 and glycosplit oxidized (gs,ox) version: compound GM-3, IM-3 and IC-3.IM-2, Rl= a-OMe, R2= CH2OH, (Sodium Iduronate)IC-2, R1 = P-OChol, R2= CH2OH, (Sodium Iduronate)GM-2, Rl= a-OMe, R2= CH2OH, (Sodium Glucuronate)IM-3, Rl= a-OMe, R2= CO2Na, (Sodium Iduronate)IC-3, R1 = P-OChol. R2= CO2Na, (Sodium Iduronate)GM-3, Rl= a-OMe, R2= CO2Na, (Sodium Glucuronate)Heparanase inhibitory assays in vitro

[0020] The inhibition of heparanase was evaluated in vitro by Arixtra and ECM degradation assay methods.Arixtra assay

[0021] The Arixtra assay is based on the cleavage of a synthetic pentasaccharide fondaparinux (Arixtra®, Aspen) in the presence of an inhibitor. The cleavage of fondaparinux by heparanase produces a disaccharide that is quantified colorimetrically [Hammond E, Ferro V.; An Enzymatic Activity Assay for Heparanase That Is Useful for Evaluating Clinically Relevant Inhibitors and Studying Kinetics. Methods Mol Biol. 2023, 2619:227-238. doi: 10.1007 / 978-1- 0716-2946-8 16. PMID: 36662473], Roneparstat was used as reference. The inhibition results of nine compounds are reported in table 1.Table 1. Heparanase inhibition IC50 values [pM (pg / ml)] of Arixtra assay

[0022] The series containing the cholestanol moiety (IC-1, IC-2 and IC-3) showed higher activity, regardless of the structure of the non-sulfated iduronic acid residue (intact, gs or gs-ox). Additionally, their IC50 values resulted comparable to that of the Hexa-G(gs-ox)-OMe (GM-3) falling within the range of 0.2-0.3 pg / ml. The other two hexasaccharides of this latter series (gs and intact G residue) (GM-1 and GM-2) along with the iduronic acid containing OMe- hexasaccharides (IM-1, IM-2 and IM-3) demonstrated very lower or no activity.35Sulfate-labeled ECM degradation assay

[0023] Extracellular matrix (ECM) degradation assay based on: [35S]-labelled ECM was incubated (in buffer solution, 2h, 37°C) with the heparanase in the absence and presence of the inhibitory molecule. To evaluate the occurrence of proteoglycan degradation, the incubation medium was collected, applied to gel filtration and detected by counting radioactivity [Li L, Barash U., et al, A new synthesized dicarboxylated Oxy-heparin efficiently attenuates tumor growth and metastasis, Cells, 2024,13,211], The inhibition results are shown in figure 1 to 4.

[0024] Figure 1 shows Heparanase inhibitory activity (ECM assay) of Iduronic acid containing O-Cholestanol-hexasaccharides (IC) series at 1 pg, with 50 ng of Heparanase after 2 hours of incubation at 37° C, while figure 2 is performed at 10 pg. Line with A indicates heparanase only; line with > indicates IC-1; line with x indicates IC-2; line with * indicates IC-3. In all figures the x axis reports fractions while the y axis reports sulfate labelled material in CPM.

[0025] Figure 3 shows heparanase inhibitory activity (ECM assay) of glucuronic acid containing O-methyl-hexasaccharides at Ipg, with 50 ng of heparanase after 2 hours of incubation at 370C, while figure 4 is performed at 10 pg. Line with A indicates heparanase only; line with > indicates GM-2; line with • indicates GM-3.

[0026] All IdoA containing OChol-hexasaccharide (Ido-Choi) series showed activity in the ECM assay, achieving 60 % and 100 % of inhibition at concentration of i pg (Fig.1) and 10 pg (Fig.2), respectively, with no significant difference among the three versions.

[0027] Surprisingly, both hexa-G(gs)-OMe and hexa-G(gs,ox)-OMe, which were found to be active in the Arixtra assay, were inactive in ECM assay at 1 pg (Fig. 3), with hexa-G(gs,ox)- OMe showing only partial activity at 10 pg (Fig. 4). It is believed that the ECM assay is better than the Arixtra assay. Arixtra assay uses a synthetic pentasaccharide substrate, while the ECM assay employs a naturally produced substrate, which better predicts the inhibitory effect in vivo.In vitro Cytotoxicity assay for cell line MDA-MB-231

[0028] . To investigate the effect of Hexa-Ido oligosaccharides on breast cancer cell proliferation, the breast cancer cell line MDA-MB231, a highly aggressive, invasive, and poorly differentiated triple-negative breast cancer (TNBC) were seeded in well plates and incubated in the presence or absence of compounds for 24, 48, or 72 hours using four treatment conditions: control (no treatment), 5 pg / mL, 10 pg / mL, 50 pg / mL of hexasaccharides. Absorbance values (570 nm) were used as an indicator of cell viability (MTT Assay). Experiment repeated three times, in triplicate (Fig 5-7).

[0029] . IC-1 (Hexa-Ido-OChol) exhibits strong, dose-dependent cytotoxicity, with concentrations significantly reducing cell viability over time. The strongest effects occur at 72 hours, where both 10 pg / ml and 50 pg / ml exhibit strong inhibitory effects, furthermore the dose of 10 and 50 pg / ml is effective already at 12 and 48 hours. The results are shown in Fig. 5. IC-2 (Hexa-Ido(gs)-OChol) shows cytotoxic effect in a dose- and time-dependent manner, with the most significant cell viability reduction at 50 pg / ml, after 48 and 72 hours, while the 10 pg / ml dose is effective only at 78 hours. The results are shown in Fig. 6. IM-l(Hexa-Ido-OMe) was inactive for each dose or time tested. The results are shown in Fig. 7In vivo mesothelioma model test

[0030] . IC-1 (termed compound S5776) was tested its effectiveness in tumor model mesothelioma. Luciferase-labelled mesothelioma 487 cells were inoculated i.p (3xl06) in NOD / SCID mice (n=18). Nine days after cell inoculation, mice were randomly assigned into two groups (9 mice for control and 9 mice for treatment)) receiving vehicle (PBS) and compound S5776 (0.5 mg / mouse, twice a week). Tumor growth was weekly inspected by IVIS (In Vivo Imaging System) imaging following administration of luciferin until 48th day after cell inoculation. The results are shown in figure 8 and figure 9. The results demonstrate that IC-1 attenuates mesothelioma growth under these experimental conditions. They strongly imply that IC-1 exhibits anti-tumorigenic potential.

Claims

Claims1. A hexasaccharide for use as a medicament, the hexasaccharide having the formula:wherein:Ri is selected from NIL, NHSCri , NHAc;R2 is selected from H, SO3 ", Bn, 2-naphthylmethyl, preferably H;R3 is selected from H, Ac, SO3 ;R4 is CO2 ;Rs is selected from Me, Et, linear or branched C3-C30 alkyl optionally comprising one or more isolated or fused rings, PEG-OMe;Re is selected from H, Ac, SO3 ;R is selected from either a linear or branched C10-C30 alkyl group optionally containing one or more isolated or fused rings.

2. The hexasaccharide according to claim 1, wherein the R group is a gonane group preferably modified with other functional groups comprising an aliphatic side chain on carbon C17 and / or having modified rings comprising the presence of double bonds.

3. The hexasaccharide according to claim 2, wherein R has the formula:

4. The hexasaccharide according to any one of claims 1-3 for use in the treatment of cancer.

5. A hexasaccharide having formula:wherein:Ri is selected from NIL, NHSOs , NHAc;R2 is selected from H, SO3 ", Bn, 2-naphthylmethyl;R3 is selected from H, Ac, SO3 ;R4 is CO2 ;Rs is Me;Re is selected from H, Ac, SO3 ;R is selected from either a linear or branched C10-C30 alkyl group optionally comprising one or more isolated or fused rings.

6. The hexasaccharide according to claim 5, wherein the R group is a gonane group preferably modified with other functional groups comprising an aliphatic side chain on carbon C17 and / or having modified rings comprising the presence of double bonds.

7. The hexasaccharide according to claim 6, wherein the R group has the formula:

8. The hexasaccharide according to any one of claims 5-7, wherein the R2 group is H.

9. The hexasaccharide according to claim 5, wherein the hexasaccharide has the formula:

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