Synthetic hexasaccharide mimics of heparin exhibiting heparanase inhibitory activity

Abstract

The present invention relates to a hexasaccharide mimetic of heparin that exhibits significant heparanase inhibitory activity. The compound has the formula: 【Chemical 1】 JPEG2025522036000020.jpg29159 and in the formula, R 1 is selected from the group consisting of NH2, NHSO3Na, and NHAc; R 2 is selected from the group consisting of NH2, NHSO3Na, and NHAc; R 3 is selected from the group consisting of Bn and H; R 4 is selected from the group consisting of Ac, H, and SO3Na, R 5 is CO2Na, and R 6 is selected from the group consisting of Me, ethyl, alkyl, alkyl azide, alkynyl, and cholestanol aglycon, and R 7 is selected from the group consisting of CH2OH and COOH.

Description

Technical Field

[0001] Field of the Invention The present invention relates to a synthetic hexasaccharide exhibiting remarkable heparanase inhibitory activity.

Background Art

[0002] Heparan sulfate (HS) refers to a family of glycosaminoglycan chains present in approximately 20 glycoproteins, proteoglycans (PGs), which are mainly distributed in the extracellular matrix and cell surface, but also in the cell interior (Thibault Annaval et al., Heparan Sulfate Proteoglyc 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 wide range 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 chains of proteoglycans, thereby regulating the functions of many proteins that interact with HS. Degradation of HS by heparanase can affect diverse biological processes such as inflammation, angiogenesis, and cell migration through the release and activation of growth factors, cytokines, and other bioactive molecules (R. Goldberg et al., Versatile role of heparanase inflammation, Matrix. Biology, 2013, 32(5), 234 - 240), and the ultimate outcome is 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 involved in diabetes and atherosclerotic diseases. Elevated levels of HPSE have recently been discovered 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 capable of this activity. A second heparanase, heparanase-2, has been discovered, but it has no enzymatic activity.

[0004] International Publication No. WO 2015 / 062951 discloses a method for preparing a glucosaminoglycan derivative that inhibits heparanase, the method comprising: N-desulfation of 25% to 100% of the N-sulfated residues of a glucosaminoglycan; preferably, oxidation with periodate at a pH of 5.5 to 10.0 of 25% to 100% of the 2-N-, 3-O-unsulfated glucosamine residues and the 2-O-unsulfated uronic acid residues of said glucosaminoglycan under conditions effective to convert adjacent diols and adjacent OH / NH2 to aldehydes; preferably, reduction with sodium borohydride of said oxidized glucosaminoglycan under conditions effective to convert said aldehydes to alcohols. This document discloses that by decreasing the Mw, the IC 50 increases up to 750 ng / ml for an Mw of about 5 kDa.

[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) disclose trisaccharides having heparanase inhibitory properties, but the compound GlcN(Ns,6s)-GlcA(gs)-GlcN(Ns,1,6anh) exhibits an IC 50 of 2000 ng / ml.

[0006] Therefore, it would be important to develop synthetic compounds that exhibit excellent activity as HPSE inhibitors.

Summary of the Invention

[0007] The present invention relates to a new class of hexasaccharides that are heparin mimetics and exhibit significant heparanase inhibitory activity. The compounds have the following structure:

Chemical Formula

Mode for Carrying Out the Invention

[0008] The hexasaccharide according to the present invention is the following hexasaccharide:

Chemical formula

[0009] In a preferred embodiment, the hexasaccharide has the following formula:

Chemical formula

[0010] In another preferred embodiment, the hexasaccharide has the following formula:

Chemical formula

[0011] In another preferred embodiment, the hexasaccharide has the following formula: [Chemistry] In the formula, R 6 and R 7 have the meanings defined above, and R 8 is selected from the group consisting of SO3Na and Ac.

[0012] When the gs ring is a glucuronic acid ring, the hexasaccharide has the following formula: [Chemistry]

[0013] When the gs ring is an iduronic acid ring, the hexasaccharide has the following formula: [Chemistry]

[0014] The compounds according to the present invention can be prepared starting from very important hexasaccharides 12, 13, 16 (Scheme 1) that exhibit a single residue (glucuronic acid) that is susceptible to oxidation by periodic acid after functionalization, and different target structures (13 - 18) can be derived therefrom.

[0015] To prepare 12, we adopted a classical disaccharide substructure synthesis 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 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 compound 3 (M. Petitou et al., Synthesis of Heparin Fragments: A α - methyl Pentaoside with High Affinity for Antithrombin II, Carbohy. Res. 1987, 167, 67 - 75), which 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 dichloromethane at - 30 °C in the presence of tert - butyldimethylsilyl triflate to yield the tetrasaccharide 4 in 65% yield. The hexasaccharide sequence was completed by reaction of 6 with the trichloroacetimidate disaccharide 8 obtained by classical methods under similar conditions. A mixture of the two anomers was formed (56%, α / β = 4 / 1). After saponification to remove the acetyl groups and cleave the methyl esters, preparative HPLC separated 10 from its β - anomer. After O - sulfation (11) and hydrogenolysis, 12 was partially N - sulfated with sulfur trioxide pyridine complex in aqueous sodium bicarbonate solution to give 13 (78%).It was also acetylated in part with acetic anhydride in aqueous sodium bicarbonate solution to give 16 (75%). A glycol-split derivative was then obtained (Scheme 2). After periodate oxidation cleavage of 13, the resulting dialdehyde was not isolated but reduced to 14 using sodium borohydride or further oxidized with NaClO2 in the presence of NaH2PO4 buffer at pH 5 (Pinick oxidation), and after purification, desalting, and lyophilization on an anion exchange column Mono Q, 15 was obtained (yield: 39%, 2 steps). The same oxidation method gave 17 and 18 from 16. The structures of all the final products were confirmed by H- and C-NMR analysis (HSQC-dept, COSY, TOCSY, HMBC) and LC-MS. Heparanase inhibition was evaluated using an assay based on the cleavage of the synthetic heparin pentasaccharide fondaparinux (Arixtra®; Aspen). Cleavage of fondaparinux by heparanase produces disaccharides, which are measured colorimetrically. The assay was basically carried out 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.). 1 H- and 13 C-NMR analysis (HSQC-dept, COSY, TOCSY, HMBC) and LC-MS. Heparanase inhibition was evaluated using an assay based on the cleavage of the synthetic heparin pentasaccharide fondaparinux (Arixtra®; Aspen). Cleavage of fondaparinux by heparanase produces disaccharides, which are measured colorimetrically. The assay was basically carried out 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.). Scheme 1

Chem.

Chem.

[0016] Compounds and serial dilutions of the reference compound roneparstat were tested. IC 50The values were finally determined using GraphPad software. The results are reported in Table 1. Inhibition by hexasaccharides 12, 13, 16 (Scheme 1) containing unmodified glucuronic acid units was hardly detectable over the concentration range tested, although there was a tendency for better inhibitory effects when the glucosamine unit was N-sulfated rather than N-acetylated. This tendency was also observed after periodate cleavage (14 vs 17), especially after the introduction of two carboxylic acid functional groups (15 and 18). Thus, N-sulfated hexasaccharide 15 showed an IC 50 of 70 nM (Scheme 2). Comparing the activities of 12 and 16 with those of other derivatives, the results of this study clearly show the important role played by the uronic acid units of gs and gs-ox. Comparing the activities of 15 and 18, N-sulfated glucosamine appears to be preferred over N-acetylated glucosamine, despite the fact that roneparstat contains only N-acetyl groups.

Table 1

Chem.

[0017] Trisaccharides 21a and 21b were obtained by attaching 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 α-pentaoside with high affinity for antithrombin III, Carbohydr. Res. 167 (1987), 67-75,) or 20b epimerized from 20a with DBU in DMF. The activator used in the sugar chain synthesis reaction was TMSOTf / CH2Cl2 / −20 °C. Considering the structure of the oligosaccharide, it was necessary to protect and deprotect the hydroxyl groups orthogonally as appropriate (V. Dimakos, et al, Site-Selective Functionalization of Hydroxyl Groups in Carbohydrate Derivatives, Chem. Rev. 118 (2018) 11457-11517). This can lead to a complicated multi-step preparation. Among the selective methods that enable protecting or deprotecting one of multiple positions with the same functional group, the magnesium methoxide method in methanol (G. Tiruchinapally et al., Divergent Heparin Oligosaccharide Synthesis with Preinstalled Sulfate Esters, Chemistry, 17 (2011), 10106-10112) was selected and optimized (4 equivalents of Mg(OH)2 at −10 °C for 3-4 h), selectively deacetylating 21a and 21b in a satisfactory 45-55% yield. After O-sulfation, saponification, hydrogenolysis, and N-sulfation, 24a and 24b were obtained. 24a and 24b were then oxidized with sodium periodate and reduced with sodium borohydride to yield 25a and 25b (referred to as gs).24a and 24b were oxidized by sodium periodate and further oxidized by NaClO2 (pinic oxidation) to yield 26a and 26b (referred to as gs, ox).

[0018] The activities of different compounds are shown in Table 2. It can be generally said that compounds containing a glucuronic acid ring are more active than compounds containing an iduronic acid ring. This tendency is also expected to apply to the hexasaccharide structure according to the present invention. [Table 2]

Claims

1. formula: 【Chemistry 1】 A hexasaccharide having the following characteristics, R １ NH ２ NHSO ３ Selected from the group consisting of Na and NHAc, R ２ NH ２ NHSO ３ Selected from the group consisting of Na and NHAc, R ３ It is selected from the group consisting of Bn and H, R ４ is selected from the group consisting of Ac, H, SO ３ Na, and R ５ CO ２ It is Na, R ６ It is selected from the group consisting of Me, ethyl, alkyl, alkyl azide, alkynyl, and cholestanol aglycone. R ７ CH ２ Selected from the group consisting of OH and COOH, Hexasaccharide.

2. formula: 【Chemistry 2】 The hexasaccharide according to claim 1, having the following characteristics.

3. formula: 【Transformation 3】 The hexasaccharide according to claim 1, having the following characteristics.

4. formula: 【Chemistry 4】 It has, in the formula, R ６ It is selected from the group consisting of Me, ethyl, alkyl, alkyl azide, alkynyl, and cholestanol aglycone. R ７ CH ２ Selected from the group consisting of OH and COOH, R ８ SO ３ Selected from the group consisting of Na and Ac, The hexasaccharide according to claim 1.

5. formula: 【Transformation 5】 The hexasaccharide according to claim 4, having the properties of the hexasaccharide.

6. formula: 【Transformation 6】 The hexasaccharide according to claim 4, having the properties of the hexasaccharide.

7. Formula: 【Transformation 7】 A hexasaccharide having the following characteristics, R1 is selected from the group consisting of NH2, NHSO3Na, and NHAc. R2 is selected from the group consisting of NH2, NHSO3Na, and NHAc. R3 is selected from the group consisting of Bn and H. R4 is selected from the group consisting of Ac, H, SO3Na, R5 is CO2Na, R 6 is selected from the group consisting of Me, ethyl, alkyl, alkyl azide, alkynyl, and cholestanol aglycone. R7 is selected from the group consisting of CH₂OH and COOH. Hexasaccharide.

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