Reinvestigation of Synthesis of Enoxaparin Under PTC Conditions

Abstract

Heparin is a mixture of Glycosaminoglycan (GAG) chains originating from porcine intestinal mucosa. It is used therapeutically as an anticoagulant for the treatment and prevention of thrombosis. Glycosaminoglycans such as Heparin (H) and Heparan Sulfate (HS) are considered attractive therapeutic agents because they modulate many biological processes and have been implicated in numerous pathologies, including cardiovascular, cancer, inflammation, metabolic, and neurodegenerative diseases and viral infections. These biological functions are believed to be dependent on the interaction of these linear polysaccharides with key proteins such as growth factors, cytokines, proteases, adhesion proteins, lipid binding proteins, etc., which have a heparin binding domain in common and are termed Heparin Binding Proteins (HBPs). The variability in unfractionated heparin’s pharmacokinetic properties and pharmacological effects led to the development of Low MW Heparin (LMWH), which is a degraded product of heparin using chemical or enzymatic cleavage techniques. The most common form of LMWH in the U.S. is enoxaparin, which is produced by β-eliminative cleavage of the benzyl esters of porcine mucosal heparin under alkaline conditions. This cleavage process leads to the generation of unnatural structures in enoxaparin. We present herein the purification strategies used to generate hexasaccharides that were further evaluated in vitro for their affinity for these protein targets, as well as heparanase inhibition. The hexasaccharides contain the same (L-iduronic acid-D glucosamine) carbohydrate backbone but varying substitution patterns. We present here a new purification process of Enoxaparin with good yield.

Keywords: Heparin; Enoxaparin; Low molecular weight; Anticoagulant

Introduction

The anticoagulant drug Heparin has been used to treat thrombosis for a propos 80 years [1]. The drug was originally isolated from dog liver and demonstrated to possess anticoagulant activity in 1916 [2]. During the 1930s, heparin was successfully prepared from bovine lung, and this drug source was later developed as a pharmaceutical product in the United States [3,4]. Heparin is a linear polysaccharide composed of a repeating disaccharide building block of alternating β-1,4-linked Hexuronic Acid (HexA) and Glucosamine residue (GlcN). The HexA can be either β-D-Glucuronic Acid (GlcA) or a-LIduronic Acid (IdoA) at which the C-2 position can be substituted by an O-sulfo group. The GlcN may be modified by an N-acetyl group (GlcNAc), an N-sulfo group (GlcNS), or can be un-substituted, whereas O-sulfo group substitution can occur at its C-3 and/or C-6 positions [5,6]. A pentasaccharide sequence of GlcNAc/NS(6S)-GlcAGlcNS( 3S,6S)-IdoA(2S)-GlcNS(6S) is the structural motif for heparin that specifically binds to antithrombin III (ATIII) and inactivates the blood clotting process [7].

Heparin molecule comes under the family of Glycosaminoglycans (GAGs) and consists of the heterogeneous mixture of polymer due to this the molecule has been referred to as “Unfractionated Heparin (UFH) amongst clinicians and researchers [8]. Heparin possesses biological functionality towards angiogenesis and hostpathogen interactions [9-11]. Furthermore, Heparin is popular in the pharmaceutical industry for its anticoagulant properties, and its de-polymerized version, termed Low Molecular Weight Heparins (LMWH), has gained much attention in the recent past. Different types of LMWH are derived based on differing de-polymerization methods. These molecules are similar to UFH in monosaccharide composition and oligosaccharide sequence. LMWH possess several advantages over UFH due to their lower molecular mass, including prolonged antithrombotic effect and better bioavailability. Given that LMWH do not bind to plasma proteins and endothelial cells, they have a longer half-life in circulation [12]. Due to the often-reported side effects causing Heparin Induced Thrombocytopenia (HIT), LMWHs have been explored as anticoagulants13. These molecules are considered more potent compared to Unfractionated Heparin (UFH) [14].

In this paper, we describe the significant procedure for the synthesis of the Enoxaparin, leading to a simplification and shortening of the process. The synthesis steps have been shown in Scheme-1. We have used a method for the de-polymerization of Heparin sodium to get Enoxaparin, and have characterized the synthetic intermediates in their production. It is our hope that the data presented here and the convenience of our method will facilitate further investigation of this important class of compounds. The spectral and analytical data strongly supported the structure of heparin.

Results and Discussion

Routes used to de-polymerization of the compound described in this work, are depicted in Scheme 1. Key intermediates 2, 3 were prepared from corresponding Heparin sodium 1 by the complexation, separation, reduction.

    

    

    Figure 1: Key intermediates 2,3 were prepared from corresponding Heparin sodium 1 by the complexation, separation, reduction.