A review on synthetic methods for 2-Deoxy-D-glucose

2-Deoxy-D-glucose (2-DG) is a non-metabolizable glucose analog that has shown promising pharmacological activities and has been used to study the role of glucose in cancer cells. 2-DG is an inhibitor of glycolysis, potential Energy Restriction Mimetic agent and inhibits pathogen-associated molecular patterns. Its radioisotope derivatives have application as tracers. Recently, 2-DG has been used as an anti-COVID-19 drug lowering the need for supplemental oxygen. In this review, different synthetic strategies for preparation of 2-DG including enzymatic synthesis have been discussed. The understanding of these methods would help in developing therapeutics or diagnostic agents aimed at exploring therapeutic targets related with energy metabolism.


Introduction
Energy is an essential requirement for sustaining life in all living organisms. From primitive life to multicellular organized living organisms, glucose is the primary source of the energy. Diseases such as cancer, viral infections and COVID-19 are found to do rewiring in cell metabolism such that the cell start using primarily glycolysis (Warburg effect) 1 to fulfill high energy demand required for high rate of proliferation of diseased cell or virus. In principle, these infected or diseased cells can be targeted by interfering with glycolysis, 2 as the normal cells can rely on fatty acid and keto bodies such as β-hydroxybutyrate metabolism to get essential energy. Lipid metabolism produces acetyl CoA that enters in to citric acid cycle and oxidative phosphorylation. 3 2-Deoxy-D-glucose (2-DG, 2-deoxy-D-arabino-hexopyranose, 1) was realized as non-metabolizable glucose analog and a competitive inhibitor of glycolysis 4 in which the 2-hydroxyl group of glucose is replaced by hydrogen. 5 Researchers has exploited its application in diagnostics, [6][7][8] potential in different biological activities including herbicidal activity, 9 as an adjunct to chemotherapy and radiotherapy in the treatment of solid tumors, 10 as an antiviral treatment in herpes simplex patients, and as an antiepileptic in temporal lobe epilepsy patients ( Figure 1). Recently, 2-DG has attracted attention due to its application in COVID-19 treatment to lower the need for supplemental oxygen. 2,11,12  This review discusses structure, druggability, analysis, estimation, toxicology, handling and preparation of 2-DG. We have focusses on various synthetic strategies for synthesis of 2-DG and isotopic 2-DG. Derivatives of 2-DG have not been considered in this review article.

Structure, Physical Properties and Characterization of 2-DG
Structure of 2-DG has been depicted in Figure 2, and it has been assigned CAS number: 154-17-6. Its synonyms are 2-deoxy-D-Glucose, 2-Deoxy-D-arabino-hexose; D-Arabino-2-deoxyhexose. 2-DG is a crystalline white to offwhite solid with a melting point at 146 o C. 13 15 The α-pyranose form of the reducing aldose 2-deoxy-D-arabino-hexose (2-Deoxy-D-arabinohexopyranose) adopts a 4 C1 conformation, with the anomeric hydroxy group in axial and the other substituents in equatorial positions. A three-dimensional hydrogen-bonded network is created in the crystal as a result of the four hydroxy groups each serving as an intermolecular hydrogen-bond donor function. 16 2-DG is found in nature. 14,17 2-deoxy-α-D-arabino-hexopyranose, and 2-deoxy-β-D-arabinohexopyranose have been isolated from the carbohydrate portion of the solid-state fermentation extract of Actinosynnema pretiosum ssp. auranticum ATCC 31565. 17  Table 1, and 13

Analysis of 2-Deoxy-D-glucose
2-DG reduces Fehling's solution and gives a positive Keller-Kiliani reaction. 14 2-Deoxy-D-glucose (2-DG) concentration and purity can be measured in a sample of crystalline or liquid by HPLC with accuracy and precision suitable for analysis of active pharmaceutical ingredients and drug products. 21 The method is suitable for the standardization and quality control of the APIs and drugs. 21 UV-HPLC (195 nm) has been used to detect and quantify 2-DG using μBondapak 10 μm NH2 column and a Varian Micropak 10 μm NH2 column. The retention time is usually four minutes with eluent 85% MeCN/H2O. 22 Polymer-based amino column (HILICpak VG-50 4E column) and Shodex SUGAR SC1011 columns have also been used in the separation of 2-DG and glucose. Pharmacokinetic studies of 2-DG involves estimation of 2-Deoxyglucose in the plasma. 23 For this purpose, precolumn fluorescent derivatization was achieved by reductive amination of 2-DG using sodium cyanoborohydride and 2-aminobenzoic acid. 23

Druggability of 2-DG
2-DG blocks the activity of different enzymes involved in glycolysis, leading to cell death. 2-DG has a molecular weight of 164.158 Da, logP of-1.525, five Hydrogen Bond Acceptors (HBA), and four Hydrogen Bond donors (HBD). Thus, there are four matching Lipinski Rules. The polar Surface Area (PSA) of 2-DG was 90.15, and it consisted of one Rotatable Bond (RotB). Thus, two matching Veber Rules exist. Approval 11 of 2-DG for emergency uses in hospitals to treat COVID-19 patients requiring supplemental oxygen in India highlighted 2-DG, and also a new hope is evolved for development of safe drug to end current pandemic. Mutation in the virus is of much concern, which results in deactivation of available drugs and monoclonal vaccines. Thus, a target which is not directly affected via mutation has its own value. Targeting glycolysis in energy hungry infected cells will stop the multiplication of virus. [24][25][26][27] Different studies has used variable amount of 2-DG, however maximum tolerable dose reported is 250 mg/kg body weight (BW). 28

Toxicology and Handling of 2-DG
2-Deoxyglucose (2-DG) is a toxic glucose analog. 2-DG has a pleiotropic mechanism of action. 29-31 2-DG contains a structural resemblance with glucose and mannose. Due to this resemblance with mannose, 2-DG strongly interferes in the N-linked glycosylation process, 29 resulted to halt protein synthesis and cause endoplasmic reticulum (ER) stress. 29,32 2-DG stimulates autophagy, enhances oxidative stress, and suppresses N-linked glycosylation. 33 Ketogenic Diet increases tolerance against glycolysis inhibitors. 3 2-DG should be handled with hand protection and mask. Exposure to moisture should be avoided.

Methods for Synthesis of 2-DG
A good number of methods are reported in the literature for the synthesis of 2-DG. These methods suffer from different operational problems, such as low yield, tedious workup or purification, low purity of the product due to formation of diastereomeric mixture or racemic mixture.

From Glucal and its derivatives
Glucal is the glycal formed from glucose and is one of the common starting materials for the synthesis of 2-DG. A general conversion involves bromination (or halogenation) of Glycal at C-2 followed by the replacement of bromine with hydrogen. Bromination takes place in nucleophilic solvent using molecular bromine. A number of reagents have been used to replace bromine attached to C-2 with hydrogen are shown in Table 2. Binkley et. al. reported photolysis of α and β anomers of 7 to yield α and β anomers of 8. Treatment of 8 with Baker ion exchange resin ANGA-542 in methanol produced 2-DG in 78% yield. 35 The compound 7 was synthesized by nucleophilic bromination of 4 followed by hydrolysis and acetylation (Scheme 1). 35
Mereyala and coworkers reported an economical and high-yielding process for the synthesis of 2-DG with high purity. (R)-D-Glycal is used as a starting material (Scheme 2). 37

Conclusions
The molecule 2-DG follows the Lipinski rule of five and has several activities such as antagonist of glucose metabolism, inhibition of sugar uptake, antiviral uses, antiinflammation activity, and anticancer activity. It is used as a metabolite inhibitor and tracer. 2-DG is a dual D-glucose and D-mannose mimetic. Recent interest due to its use in COVID-19 has again risen. As the present synthetic methods are tedious, and expensive, there is scope for developing a new economical process using more mild conditions to synthesize 2-deoxy-D-glucose with good yield and purity. It will open up development of new antiviral drugs and treatment for hyperglycemic patients.