Synthetic hexasaccharide mimetics of heparin showing heparanase inhibitory activity

Abstract

The present invention relates to hexasaccharide mimetics of heparin, which exhibit significant heparanase inhibitory activity. The compounds have the formula (I) wherein R1 is selected from the group consisting of NH2, NHSO3Na, NHAc; r2 is selected from the group consisting of NH2, NHSO3Na and NHAc; r3 is selected from a group consisting of Bn and H; r4 is selected from the group consisting of Ac, H and SO3Na; r < 5 > is CO2Na; r6 is selected from the group consisting of Me, ethyl, alkyl, alkyl azide, alkynyl, cholanol aglycone; and R7 is selected from a group consisting of CH2OH and COOH. # imgabs0 #

Description

Field of the Invention

[0001] The present invention relates to synthetic hexasaccharides showing significant heparanase inhibitory activity. Background Art

[0002] Heparan sulfate (HS) refers to a family of glycosaminoglycan chains, i.e., proteoglycans (PGs), present in about 20 glycoproteins, which are mainly distributed in the extracellular matrix and on the cell surface, but also distributed 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). PG exerts its biological function by interacting with a large number of protein ligands, including the 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 (HPSE) is a β-D-endoglycosidase that can cleave the HS chain of proteoglycans and thereby regulate the functions of many proteins that interact with HS. Heparanase cleavage of HS can affect a variety of biological processes by releasing and activating growth factors, cytokines and other bioactive molecules, such as inflammation, angiogenesis and cell migration (R. Goldberg et al., Versatile role of heparanase inflammation, Matrix. Biology, 2013, 32 (5), 234-240), ultimately leading to tumor growth and metastasis (I. Vlodavsky et al., Heparanase: Structure, Biological Function, and Inhibition by Heparin-Derived Mimetics of Heparan Sulfate, Current Pharmaceutical Design, 13 (2007), 2057-2073). HPSE is also associated with diabetes and arteriosclerosis. Elevated levels of HPSE have recently been found in Covid-19 patients (B. Buijsers et al., C, Front. Immunol. 11:545047. doi:10.3389 / fimmu.2020.545047). HPSE is the only enzyme in mammals with this activity. A second heparanase has been discovered, heparanase-2, but it lacks enzymatic activity.

[0004] WO 2015 / 062951 discloses a method for preparing a glucosaminoglycan derivative that inhibits heparanase, comprising: N-desulfurizing 25% to 100% of the N-sulfated residues of the glucosaminoglycan; oxidizing 25% to 100% of the 2-N-, 3-O-non-sulfated glucosamine residues and 2-O-non-sulfated uronic acid residues of the glucosaminoglycan by periodate, preferably at a pH of 5.5 to 10.0, under conditions that effectively convert adjacent diols and adjacent OH / NH2 into aldehydes; reducing the oxidized glucosaminoglycan, preferably by sodium borohydride, under conditions that effectively convert the aldehydes into alcohols. The document discloses that by reducing the Mw, for an Mw of about 5 kDa, IC 50 The increase was up to 750ng / ml.

[0005] Minghong Ni et al. (Investigating Glycol-Split-Heparin-Derived Inhibitors of Heparanase: A Study of Synthetic Trisaccharides Molecules 2016, 21, 1602; doi: 10.3390 / molecules21111602) disclosed trisaccharides with heparanase inhibitory properties; however, the compound GlcN(Ns, 6s)-GlcA(gs)-GlcN(Ns, 1, 6anh) showed an IC 50 It is 2000ng / ml.

[0006] Therefore, it is very important to develop synthetic compounds that show good HPSE inhibitor activity. Summary of the invention

[0007] The present invention relates to a new class of heparin hexasaccharide mimetics, which exhibit significant heparanase inhibitory activity. The compound has the following structure:

[0008]

[0009] Among them, R 1 Selected from the group consisting of NH2, NHSO3Na, NHAc; R 2 Selected from the group consisting of NH2, NHSO3Na, NHAc; R 3 Selected from the group consisting of Bn, H; R 4 Selected from the group consisting of Ac, H, SO3Na; R 5 CO2Na; R 6 Selected from the group consisting of Me, ethyl, alkyl, alkyl azide, alkynyl, cholesteryl aglycone; R 7 Selected from the group consisting of CH2OH, COOH. DETAILED DESCRIPTION

[0010] The hexasaccharide according to the present invention can be obtained by the following glycosplit reaction of the uronic acid ring of the hexasaccharide:

[0011]

[0012] Among them, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and R 7has the meaning defined above. The hexasaccharide consists of alternating rings of glucosamine sulfate and uronic acid. The term uronic acid includes iduronic acid and glucuronic acid.

[0013] In a preferred embodiment, the hexasaccharide has the formula:

[0014]

[0015] In another preferred embodiment, the hexasaccharide has the formula:

[0016]

[0017] In another preferred embodiment, the hexasaccharide has the formula:

[0018]

[0019] Among them, R 6 and R 7 has the meaning defined above, R 8 Selected from the group consisting of SO3Na, Ac.

[0020] When the gs ring is a glucuronic acid ring, the hexasaccharide has the formula:

[0021]

[0022] When the gs ring is an iduronic acid ring, the hexasaccharide has the formula:

[0023]

[0024] The compounds according to the invention can be prepared starting from the key hexasaccharides 12, 13 and 16 (Scheme 1), which after functionalization present a single residue (glucuronic acid) that is susceptible to periodate oxidation and from which different target structures (13-18) can be derived.

[0025] To prepare 12, we adopted (Scheme 1) - a classical disaccharide building block method (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 trichloroacetimidyl ester glycosylation method (RR 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: Aα-methyl Pentaoside with High Affinity for Antithrombin II, Carbohy. Res. 1987, 167, 67-75) 3, which was first coupled with the known 2 (CAA 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 dichloromethane at -30°C in the presence of tert-butyldimethylsilyl trifluoromethanesulfonate to give the tetrasaccharide 4 in 65% yield. The hexasaccharide sequence was completed by reaction with the trichloroacetimidate disaccharide 8 itself, classically obtained from 6, under similar conditions. A mixture of two anomers was formed (56%, α / β=4 / 1). Preparative HPLC allowed the separation of 10 from its β-anomer after saponification to remove the acetyl group and cleavage of the methyl ester. After O-sulfation (11) and hydrogenolysis, 12 was partially N-sulfated with sulfur trioxide pyridine complex in aqueous sodium bicarbonate to give 13 (78%), which was also partially N-acetylated with acetic anhydride in aqueous sodium bicarbonate to give 16 (75%). The diol cleavage derivative was then obtained (Scheme 2).After periodate oxidative cleavage of 13, the resulting dialdehyde (not isolated) was reduced to 14 using sodium borohydride or further oxidized by NaClO2 in the presence of NaH2PO4 buffer at pH 5 (Pinnick oxidation) to afford 15 (yield: 39%, 2 steps) after purification by anion exchange column Mono Q, desalting and lyophilization. The same oxidation procedure produced 17 and 18 from 16. All structures of the final products were recognized. 1 H- and 13 C-NMR analysis (HSQC-dept, COSY, TOCSY, HMBC) and LC-MS were used to confirm the results. Inhibition of heparanase was evaluated by assaying the cleavage of fondaparinux (Aspen). Heparanase cleaves fondaparinux to produce a disaccharide that is measured colorimetrically. The assay was performed essentially as described by Hammond (E. Hammond, CP 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).

[0026]

[0027]

[0028] Serial dilutions of the compounds and the reference roneparstat were tested. IC 50 The values ​​were finally determined using GraphPad software. The results are reported in Table 1. Hexasaccharides 12, 13 and 16 (Scheme 1) comprising intact glucuronic acid units showed almost no detectable inhibition in the concentration range tested, however, when the glucosamine unit was N-sulfated rather than N-acetylated, there was a trend towards better inhibition. This trend was observed again after periodate cleavage (14 vs. 17), and especially after the introduction of two carboxylic acid functional groups (15 and 18). Therefore, the N-sulfated hexasaccharide 15 showed an IC 5070 nM (Scheme 2). Comparing the activities of 12 and 16 with other derivatives, our results clearly demonstrate the key role played by the gs and gs-ox uronic acid units. Comparing the activities of 15 and 18, it seems that N-sulfated glucosamine is superior to N-acetylated glucosamine, despite the fact that only N-acetyl groups are present in roneparstat.

[0029] Table 1

[0030]

[0031] In order to evaluate the inhibitory properties of the iduronic acid gs ring when compared to the glucuronic acid gs ring, a trisaccharide corresponding to the three central rings of the structure (gs ring plus one ring on each side) has been prepared with both iduronic acid and glucuronic acid rings. The synthetic route is shown in Scheme 3.

[0032]

[0033] Trisaccharides 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) with 20a (M. Petitou et al., Synthesis of heparin fragments: Amethylα-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 activating agent used in the glycosylation reaction was TMSOTf / CH2Cl2 / -20°C. Given the structure of oligosaccharides, orthogonal protection and deprotection of hydroxyl groups are required 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-step preparation. In a selective method that allows protection or deprotection of one of several positions with the same function, magnesium methoxide in methanol (G. Tiruchinapally et al., Divergent Heparin Oligosaccharide Synthesis with Preinstalled Sulfate Esters, Chemistry, 17 (2011), 10106–10112) (4 equivalents of Mg(OH)2 at -10°C for 3-4h) was selected and optimized to selectively deacetylate 21a and 21b in satisfactory yields of 45-55%. After O-sulfation, saponification, hydrogenolysis and N-sulfation, 24a and 24b were obtained. 24a and 24b were subsequently oxidized by sodium periodate and reduced by sodium borohydride (referred to as gs) to give 25a and 25b. 24a and 24b were oxidized by sodium periodate and further oxidized by NaClO2 (Pinnick oxidation) (referred to as gs,ox) to give 26a and 26b.

[0034] The activities of the different compounds are shown in Table 2. It can be noted that the compounds comprising a glucuronic acid ring are generally more active than the compounds comprising an iduronic acid ring. This trend is expected to be valid also for the hexasaccharide structures according to the present invention.

[0035] Table 2

[0036]

Claims

1. A hexasaccharide having the formula in, R 1 Selected from the group consisting of NH2, NHSO3Na, NHAc; R 2 Selected from the group consisting of NH2, NHSO3Na, NHAc; R 3 Selected from the group consisting of Bn, H; R 4 Selected from the group consisting of Ac, H, SO3Na; R 5 is CO2Na; R 6 Selected from the group consisting of Me, ethyl, alkyl, alkyl azide, alkynyl, cholestanol aglycone; R 7 Selected from the group consisting of CH2OH, COOH.

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

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

4. The hexasaccharide according to claim 1, wherein The hexasaccharide has the formula Among them, R 6 Selected from the group consisting of Me, ethyl, alkyl, alkyl azide, alkynyl, cholestanol aglycone; R 7 Selected from the group consisting of CH2OH, COOH; R 8 Selected from the group consisting of SO3Na, Ac.

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

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

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