Title of Invention

"AN IMPROVED PROCESS FOR PREPARATION OF 2-DEOXY-D-GLUCOSE"

Abstract

An improved process for the preparation of 2-deoxy-D-glucose (2-DG) comprising converting D-glucose to glucose pentaacetate, brominating said glucose pentaacetate to obtain acetobromoglucose, reducing said acetobromoglucose to glucal triacetate, subjecting said glucal triacetate to the step of deesterification to obtain D-glucal, hydrating said D-glucal to 2 deoxy-D-glucose characterized in that said step of conversion being carried out by reacting D-glucose with acetic anhydride and acetic acid using sulphuric acid as catalyst and said step of bromination, reduction, deestrification and hydration being carried out in the manner as herein described, and then subjecting said 2-deoxy-D-glucose to the step of purification.

Full Text

FIELD OF INVENTION The present invention relates to an improved process for the preparation of the 2-deoxy-D glucose(2-DG). BACKGROUND INFORMATION 2-DG is a glucose analogue and has been extensively used as a tool to study the glucose transport and regulation of glucose metabolism in a variety of cellular systems and intact organisms including primates and humans. 2-DG has been extensively used in patients of different tumours, in patients of gastric ulcer diseases to test for completeness of vagotomy and also for differential diagnosis of hypoglycemia in children without any untoward side effects. The main goal of a successful cancer therapy is to completely remove the neoplastic cells without causing any appreciable damage to the normal tissues of the host. Many of the compounds such as S-2-3-propylamine ethyl phosphorothioic acid (WR 2721), 2-mercapto propionyl glycine(MPG) have been widely used as radio-protector for normal cells. However, clinical trials have shown that repeated dose of this can result in severe unpleasant side effects. 5-Bromouracil (BU) and 5-bromo deoxyuridine (BudR) as radio sensitisers have been used in the clinical trials with a view that their configurational similarity is matched with thymine. Hypoxic cells are glucose dependent for their survival. Two glucose analogues namely, 2-deoxy-D-glucose and 5-thio-D-glucose have thus been tested for their cytotoxic and radio sensitising activity. It has been reported that 2-DG can preferentially inhibit repair processes in cells with high glycolytic activity and is able to inhibit post irradiation DNA repair and cell recruitment processes differentially in systems which depend largely on glycolysis for their energy supply. The prognosis of patients suffering from malignant cerebral gliomas (heterogeneous tumours) has remained dismal despite application of multimodel therapy and many advances in medical radiation technology. The failure of radiotherapy in cerebral gliomas is primarily due to the presence of hypoxic, intrinsically radioresistant, and repair proficient subpopulations of cells in the tumour. In living organisms, damage to DNA is the major cause of cell death and cell loss induced by ionizing radiations, it has been demonstrated that the cellular processes leading to the repair and fixation of radiation damage require continuous flow of metabolic energy supplied by the respiratory and/or glycolytic pathways. Because glucose usage in transformed cells and tumours is increased and tumour cells derive a large part of their matabolic energy (ATP) from the glycolytic pathway, it was predicted that inhibitors of glucose transport and glycolysis could differentially inhibit repair processes in these cells, leading to an enhancement of the radiation damage. Several studies have demonstrated that presence of 2-DG, an inhibitor of glucose transport and glycolysis, during the first few hours following irradiation could indeed inhibit the repair of DNA lesions and potentially lethal damage, thereby enhancing the radiation damage in various cellular systems with high rates of glycolysis like the cancer cells under euoxic as well as hypoxic conditions. Thus combination of ionizing radiations with 2-DG provides a unique opportunity to selectively destroy tumours by differentially enhancing the radiation damage in cancer cells and at the same time preventing radiation injuries to normal tissue. PRIOR ART Bolliger et al have prepared 2-DG by the reaction of 2-ethylthiotetra benzoyl-D-glucose diethyl mercaptal with cadmium carbonate and mercuric chloride. The disadvantage of the above process is that this process is a nine-step process and time consuming. Another disadvantage of the above process is that the yield is very low of the order of 2 to 5%. Further disadvantage of the above process is that the product obtained is impure. Stanek et al have also prepared 2-DG by the reaction of triacetyl D-glucal and silver salt of benzoyl iodide ((Bz02)IAg) followed by reduction of 1-benzoyl 3,4,6-triacetyl 2-deoxy-2-iodo-a-D-glucopyranose. The above mentioned process has also the disadvantage that it is a seven-step process with very low-yield. Cramer F.B has prepared 2-DG using D-glucose as starting material. The D-glucose is converted to glucose pentaacetate using acetic anhydride in the presence of sulphuric acid as catalyst. The reaction mixture is cooled and Hydrogen bromide (HBr) gas from a HBr gas cylinder, is passed into the reaction mixture. The acetobromoglucose obtained thereby is reduced by a reducing mixture. After processing, the glucal triacetate so formed is de-esterified to D-glucal using sodium metal followed by addition of dry ice (solid carbon dioxide). Finally D-glucal is hydrated to 2-deoxy-D-glucose (2-DG) using sulphuric acid in aqueous medium. One of the disadvantage of the above process is that the product obtained is impure. Another disadvantage of the above process is that the yield is low. Further disadvantage of the above process is that the reaction of D-glucose with acetic anhydride being exothermic, temperature goes up to 300°C which results in boiling of the mixture leading to splashing out of the unreacted acetic anhydride and acetic acid which is formed as a byproduct, which may cause serious hazards to users and environment as well. Still further disadvantage of the above process is that due to intense heat generated as a result of uncontrolled exothermic reaction of D-glucose with acetic anhydride, the D-glucose gets partially burnt which leads to impurities and low-yield. Yet futher disadvantage of the above process is that the hydrogen bromide gas cylinder used in this process is expensive and not available in certain countries like India. Even further disadvantage of the above process is that the reaction of hydrogen bromide with glucose pentaacetate is very slow at ambient temperature and major volume of Hydrogen bromide passes out unreacted, from the reaction vessel, which causes serious hazards to the users as it is toxic and it penetrates into the human skin and damages the muscle tissues. Further disadvantage of the above process is that during reduction of acetobromoglucose to glucal triacetate, the temperature has to be strictly and constantly maintained at zero degree centigrade which is difficult to maintain during the entire period of the reaction. Still further disadvantage of the above process of the known art is that sodium metal requires about two days of time for deesterification of glucal triacetate, which leads to an impure product. Still further disadvantage of the above process is that the process uses dry ice i.e. solid carbon dioxide which is expensive. NEED FOR THE INVENTION There is a need for an improved process for the preparation of 2-deoxy-D-glucose (2-DG) which provides higher yield of 2-DG with higher purity and at the same time is cost effective. The process should not be hazardous to the users and need to be eco-friendly and should overcome the drawbacks/disadvantages of the processes known in the art. OBJECTS OF THE INVENTION The primary object of the present invention is to propose an improved process for preparation of 2-deoxy-D-glucose (2-DG). Another object of the present invention is to propose an improved process for preparation of 2-DG which provides higher yield of 2-DG. Still another object of the present invention is to propose a process for the preparation of 2-DG which provides 2-DG of purity of the order of higher than 99.5%. Yet another object of the present invention is to propose an improved process for the preparation of 2-DG which is eco-friendly. Further object of the present invention is to propose a cost-effective process for preparation of 2-DG which prepares hydrogen bromide, using cheaper chemicals as compard to known process which uses highly expensive hydrogen bromide gas cylinder. Still further object of the present invention is to propose a cost-effective process for preparation of 2-DG in which carbon-dioxide is prepared by the use of cheaper chemicals as compared to known process which uses expensive dry ice i.e. solid carbon dioxide. Yet further object of the present invention is to propose an improved process for the preparation of 2-DG in which the high reaction temperature due to exothermic reaction of D-glucose and acetic anhydride is controlled by the use of acetic acid thereby preventing splashing out of toxic vapours of acetic anhydride and acetic acid. Even further object of the present invention is to propose an improved process for the preparation of 2-DG which takes significantly less time of the order of 1-2 hours for deesterif ication as compared to the known processes which take over 2 days, only for the step of deesterif ication. Still even further object of the present invention is to propose an improved process for the preparation of 2-DG in which optimum proportion of sulphuric acid is used during hydration of glucal into 2-DG. Yet further object of the present invention is to propose an improved process for the preparation of 2-DG which uses a reducing mixture comprising of constituents mixed in optimised proportions which leads to complete reduction of acetobromoglucose to glucal triacetate which in turn eliminates impurities and leads to higher yield. Still further object of the present invention is to propose an improved process for the preparation of 2-DG which uses multi-step purification process which leads to 2-DG of very high purity. BRIEF DESCRIPTION OF INVENTION. According to this invention there is provided an improved process for the preparation of 2-deoxy-D-glucose (2-DG) comprising converting D-glucose to glucose pentaacetate, brominating said glucose pentaacetate to obtain acetobromoglucose, reducing said acetobromoglucose to glucal triacetate, subjecting said glucal triacetate to the step of deesterification to obtain D-glucal, hydrating said D-glucal to 2 deoxy-D-glucose characterised in that said step of conversion being carried out by reacting D-glucose with acetic anhydride and acetic acid using sulphuric acid as catalyst and said step of bromination, reduction, deestrification and hydration being carried out in the manner as herein described, and then subjecting said 2-deoxy-D-glucose to the step of purification. The process provides the compound 2-DG of very high purity of order higher than 99.5% and at the same time provides higher yield of product as compared to known processes. During the first step of conversion of D-glucose to glucose pentaacetate, which is an exothermic reaction,in addition to sulphuric acid used as catalyst in the known process, an additional chemical namely acetic acid is used which controls the reaction temperature and prevents, the boiling of constituents and splashing out of toxic chemical vapours. The process prepares hydrogen bromide gas instead of using highly-expensive hydrogen bromide gas cylinders which are not available in countries like India. The proportionate quantities of the constituents in the reducing mixture have also been optimised to lead to completion of the reduction of acetobromoglucose to glucal triacetate. The carbon-dioxide required during deesterification is prepared by reaction of relatively cheaper chemicals namely sodium carbonate and hydrochloric acid as compared to the known process which uses expensive dry ice i.e. solid car ban dioxide. Besides, instead of sodium metal used in the known process which requires 2~days time for completion of reaction, lN-sodium methoxide is used which enables completion of reaction in about half an hour. During hydration of D-glucal, an optimum quantity of the sulphuric acid is used which leads to completion of reaction and eliminates impurities. The process uses multi-step purification involving filteration and evaporation after treatment with barium carbonate, crystallization in isopropyl alcohol and filtration. After dissolving, the crude 2-DG in water and then passing through membrane filter followed by passing through cation and anion exchage resins successively. DESCRIPTION OF PROCESS According to the present invention, the proposed process for the preparation of 2-deoxy-D-glucose comprises of the following steps:- 1. Conversion of D-glucose to glucose penta-acetate 20 to 25 wt% of D-glucose mixed with 60 to 65 wt% of acetic anhydride and 15 to 20 wt% of acetic acid are taken in a reaction vessel. To this mixture, about 0.1 to 0.2 ml of H2S04 which serves as catalyst, is added. The reaction is exothermic and the acetic acid restrains the temperature of reaction mixture to about 100°C and thus prevents the burning of glucose and splashing out of reactants. The mixture is stirred under atmospheric pressure at room temperature for about 1 hour. A transparent solution of glucose penta-acetate in acetic acid solution is obtained. 2. Bromination of glucose pentaacetate to acetobromoqlucose In a separate vessel, tetralin in 50-55% of the wt of glucose pentaacetate and bromine in the wt% of 85-90% of glucose pentaacetate are taken. Hydrogen bromide gas, thus generated is passed into the reaction mixture of step-I. The tetralin and bromine are in the ratio of 3:5. When the weight of reaction mixture increases by about 35-40% of its weight, the passing of hydrogen bromide gas is discountinued. The vessel is kept in a cool place for carrying out the next reaction. 3. Reduction of acetobromoglucose to glucal triacetate The acetobromoglucose obtained by the step-2 is reduced to glucal triacetate by reducing mixture comprising of four different constituents. The constituents of reducing mixture are activated zinc, sodium acetate, acetic acid and copper sulphate in aqueous medium at -i°C to l°C. The quantities of these constituents as wt% of glucose pentaacetate are 25-30%, 50 to 55%, 50 to 55% and 2 to 3% respectively. The different constituents of the reducing mixture are in the simple ratio of 1:2:2:0.1. After reduction is over, the mixture is filtered, diluted with water to about two times of its volume and is extracted with benzene or hexane, Benzene/hexane is removed under vacuum and viscous material left is diluted with 95% ethyl alcohol. The quantity of ethyl alcohol taken is about half of the volume of the product obtained after extraction and vacuum evaporation. The yield of crystallised glucal tri-acetate thus obtained is in the order of higher than 70% of acetobromoglucose. 4. De-esterification of glucal triacetate to D-qlucal The glucal triacetate obtained is dissolved in dry methanol and to this is added IN-sodium methoxide. The quantity of dry methanol taken is four times the wt of glucal triacetate and quantity of IN-sodium methoxide is about 2 0% of the weight of glucal triacetate. The mixture is refluxed for about 15 minutes. In a separate vessel, sodium carbonate is taken and concentrated hydrochloric acid is poured over it through a pressure equalising funnel. The carbon-dioxide thus generated is passed into the reaction mixture till the reaction mixture becomes neutral to slightly acidic. The methanol is removed under vacuum and the product obtained namely D-glucal is used as such for next step. The yield at this stage is about 30-35% of the glucal triacetate. 5• Hydration of D-glucal to 2-deoxy-D-qlucose The product obtained is diluted with distilled water and to this 1N-H2S04 is added. The quantity of water taken is about 2 0 times the weight of the product and the quantity of IN-sulphuric acid (H2S04) taken is about 8 0% of the weight of the product. The mixture is kept for 16-18 hours for completion of hydration. Excess acid is neutralised by barium carbonate. After filtration and evaporating the water under vacuum, the viscous liquid obtained is crystallised in dry isopropyl alcohol. After filtration, 2-DG is obtained. Its yield is about 60% of the D-glucal. 6. Purification The crude 2-DG obtained after step-5, is normally contaminated with barium sulphate. This impurity of barium sulphate is removed by dissolving the crude 2-DG in water and filtered through membrane filter 0.45 microns. This is then passed through cation exchange resin like Dowex 50w. The quantity of resin taken is about 15 to 2 0% of the weight of crude 2-DG. It is then passed through anion exchange resin like Amberlite IRA-400OH. The quantity of this resin taken is about 30 to 35% of the weight of crude 2-DG. After this, a shiny fluffy compound 2-DG is obtained which contains less than 2ppm of barium. The invention is now illustrated with a working example which is intended to illustrate the working of the process with a typical example and is not to be taken restrictively to imply any limitation on the scope of the invention in any way. Example: In a l litre round bottom three necked flask equipped with mechanical stirrer were placed anhydrous D-glucose (0.66M) and acetic anhydride (3.30M). Glacial acetic acid (100ml) was added in the above mixture. To it the catalyst prepared by l-l/2ml H2S04 in 5ml glacial acetic acid (lml) in the mixture was added and stirred at room temperature. After 15 minutes exothermic reaction started and mixture became transparent. Water was used to cool the reaction. Solution was stirred for one hour to ensure the complete reaction. The HBr gas was generated using tetralin and bromine and was passed through tube in the same vessel under stirring. When absorption was completed, vessel was kept in a cool place for not more than one hour. The product acetobromoglucose (468gm) was further reduced using activated zinc (150 gm) , sodium acetate(300gm), acetic acid (300ml) and copper sulphate(15gm) in aqueous medium at -1° to l°C. Chemicals were used in this reaction in a simple ratio of 1:2:2:0.1 respectively. When reduction was over, the whole mixture was filtered through Buckner funnel. It was diluted with distilled water and extracted with benzene/hexane. Benzene was removed under vacuum and viscous material left was diluted with 95% ethyl alcohol to get the crystallised product in the order of yield of>70%. Thus glucal triacetate was formed. The product (130gm, 0.66M) formed was dissolved in dry methanol (500ml) and 2 0ml of IN-sodium methoxide was added and the mixture was refluxed for 15 minutes. After cooling the contents, 100ml of H2O was added and carbon dioxide, generated by the reaction of sodium carbonate and hydrochloric acid was passed. The contents become slightly acidic and D-glucal was formed. It could be used as such for further step. The yield was in the range of 50-52 gm (-35%). The product so formed was diluted with one litre of distilled water. To this 40ml of 1N-H2S04 was added and kept for 16-18 hours for hydration. Excess acid was neutralized by barium carbonate. Upon filtering and evaporating of water under vacuum, the viscous liquid was crystallised in dry isopropyl alcohol, which upon removal gave 30-32 gms of crude-2deoxy-D-glucose. This subsequently was charcoalised to produce 28 gm of off-white 2-deoxy-D-glucose containing barium sulphate in the range of 2 to 3% as impurity which is toxic to mammals. The crude 2-DG was dissolved in distilled water and passed through membrane filter 0.45 microns. Barium sulphate left after filtration was finally removed using 5gm cation exchange resin (Dowex 50w, 1.7 meg/ml) followed by lOgm of anion exchange resin (Amberlite IRA-400OH), 1.1.meg/ml). A shiny fluffy compound was obtained containing barium On toxicity evaluation it had been confirmed that the LD5 0 value obtained in mice from the prepared 2-DG by this method was higher i.e. 6gm/kg body weight as compared to the reported value of 4.5gm/kg body weight (Lazlo et al: J. Clin. Invest 40:171-176, 1960), thus ensuring better purity. Purity of 2 DG was determined to be higher than 99.5% by High Performance Liquid chromatography using carbohydrate column and refractive index detector. It is to be understood that the above description of the present invention is suceptible to considerable modifications, change and adaptation by those skilled in the art, such modifications are intended to be considered to be within the scope of the present invention, which is set forth by following claims:- WE CLAIM: 1. An improved process for the preparation of 2- deoxy-D-glucose (2-DG) comprising converting D-glucose to glucose pentaacetate, brominating said glucose pentaacetate to obtain acetobromoglucose, reducing said acetobromoglucose to glucal triacetate, subjecting said glucal triacetate to the step of deesterification to obtain D-glucal, hydrating said D-glucal to 2 deoxy-D- glucose characterised in that said step of conversion being carried out by reacting D-glucose with acetic anhydride and acetic acid using sulphuric acid as catalyst and said step of bromination, reduction, deestrification and hydration being carried out in the manner as herein described, and then subjecting said 2- deoxy-D-glucose to the step of purification. 2. An improved process as claimed in claim 1 wherein the said step of bromination is carried out by passing hydrogen bromide gas. 3. An improved process as claimed in claim 1 wherein the said step of reduction is carried out by using a reducing mixture comprising of activated zinc, sodium acetate, acetic acid and copper sulphate at a temperature of -l'C to l'C. 4. An improved process as claimed in claims l and 4 wherein said reducing mixture comprises activated zinc, sodium acetate, acetic acid and copper sulphate in the ratio of 1:2:2:0.1 respectively. 5. An improved process as claimed in claim l wherein said step of de-esterification is carried out by de-esterification with IN-sodium methoxide in methanol followed by passing carbon dioxide which is freshly prepared in a separate vessel by reaction of sodium carbonate with concentrated hydrochloric acid. 6. An improved process as claimed in claim 1 wherein said step of hydration is carried out by addition of 80% distilled water and 1N-H SO by weight of said D-glucal 2 4 and keeping the same for 16-18 hours. 7. An improved process as claimed in claim l wherein said step of purification comprises filtering said 2-DG, through a membrane filter of 0.45 microns and passing the same through a cation exchange resin like Dowex 50w, followed by passing through anion exchange resin like Ambertite IRA-4000H. 8. An improved process as claimed in claim 5 wherein said IN-sodium methoxide used during de-esterification is about 20% by weight of glucal triacetate. 9. An improved process as claimed in claims 1 and 6 wherein a neutralising agent such as barium carbonate is used during said step of hydration. 10. An improved process as claimed in claims 1 and 6 wherein a crystallizing medium such as dry isopropyl alcohol is used during said step of hydration. 11. An improved process for the preparation of 2-DG substantially as herein described and illustrated in the examples.

Documents:

358-del-1998-abstract.pdf

358-del-1998-claims.pdf

358-del-1998-complete specification (granted).pdf

358-del-1998-correspondence-others.pdf

358-del-1998-correspondence-po.pdf

358-del-1998-description (complete).pdf

358-del-1998-form-1.pdf

358-del-1998-form-2.pdf

358-del-1998-form-3.pdf

358-del-1998-form-4.pdf

358-del-1998-pa.pdf