FORM 2

THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION:
An improved process for the preparation of,b, bl-1, 2-Dihydroxy-l, 2-cyclobutenediacrylic acid -di - g- lactone
2. APPLICANT (S):
(a) NAME: FDC Ltd.
(b)NATIONALITY: Indian company incorporated under the Companies Act 1956
(c) ADDRESS: 142-48, S.V. Road, Jogeshwari (W), Mumbai - 400 102, Maharashtra, India.
3. PREAMBLE TO THE DESCRIPTION
The following specification particularly describes the invention and the manner in which it is to be performed.

Field of the Invention
The present invention relates to an improved process for preparation of substantially pure b, bl -1, 2-Dihydroxy-l, 2-cyclobutenediacrylic acid -di - g- lactone in good yields. b, bl -1, 2-Dihydroxy-l, 2-cyclobutenediacrylic acid -di - g- lactone is known in the art as Anemonin, which is a potential candidate of choice for treating thalessemic disorder.
Background & Prior Art
Thalassemia is a dreaded disease among children. The disease is caused due to hereditary disorders connected with defective hemoglobin synthesis characterized by hypochromia, microcytosis haemolysis and a variable degree of anemia. Thalassemia involves a heterogeneous group of molecular defects with a wide spectrum of clinical expressions.
Patients suffering from thalassemia suffer from anemias with decrease or absence of synthesis of a globins chain of normal hemoglobin. The patients of thalassemia are broadly classified into two major groups according to the affected globins chain. Alpha (a) thalassemia is associated with decreased or absence of alpha chain synthesis. Beta (p) thalassemia is associated with decrease or absence of beta chain synthesis.
Thalassemia major also known as Cooley's anemia, Mediterranean anemia and Von-Jackass's anemia is characterized by marked anemia (ranging from 1 to 6 gm/dl of hemoglobin), severe hemolysis and ineffective erthropoiresis. In the case of thalassemia major, iron in the hemoglobin also breaks down and gets deposited in the vital organs of the patients e.g. liver, kidney, spleen, heart etc. This is also known as iron overloading in the body and the life span of the child suffering from thalassemia major becomes unpredictable. Every year out of 1,00,000 children born with thalassemia major in the world, 10,000 are born in India.
The anemonin (compound of the formula I) posses the properties of chelating the iron present in the body. Thus the excess of iron present in the body of the patients is removed thereby enhancing the life span of the patients.
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US 5,665,392 disclose a pharmaceutical formulation useful for treating patients suffering from thalassemia, which comprises powder of Anemonin.
J.A.C.S (1955) 77,2332 discloses process for synthesis of Anemonin (I) from Levulinic Acid as starting material. The process has disadvantages such as use of costly starting material and also has inherent problem of safety as liquid Bromine is used in this process. Liquid bromine is corrosive with potential symptoms of overexposure leading to dizziness, headache, lacrimation, sever burns of eyes and skin etc and is a potential hazard. Use of bromine therefore is unsafe and hazardous and needs extra care when required to handle in industrial manufacture. The resultant poor Yield is another issue, which makes this process as a secondary choice. This process is narrated in GB896949 with modifications.
Synthetic Communications 20(17), 2607 (1990) discloses the synthesis of Protoanemonin a precursor of b, bl -1, 2-Dihydroxy-l, 2-cyclobutenediacrylic acid -di - y- lactone from 5-hydroxy methyl furfural. The use of 5-hydroxy methyl furfural has inherent drawback, which indirectly enhances the manufacturing cost.
J.Chem.Soc.Commun, 1990, 1079 describes a process that involves catalytic vapor phase oxidation of 2-methyl furan in the presence of Vanadium-Molybdenum catalyst on corundizium support. This process has inherent drawbacks, as it involves a troublesome process step that requires vapor phase reaction condition which enhances the cost as well as safety concerns. The process also requires special infrastructure of highly specialized catalyst. The use of vapor phase conditions is difficult to achieve on larger scale and also pose safety problems.
Solution of these problems associated with prior art becomes the objects of the present invention. Hence, the present invention provides industrially viable and economically feasible process thereby eliminating the all the above-mentioned shortcomings.
The present invention is a modification of a process appeared in Synthetic Communications 33(21), 3727,(2003 ), wherein the process now is much more simplified
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with better yields, to suit industrial manufacture by using cheaper starting material and avoiding the isolation, crystallization and purifications of intermediates thus making the process in-situ resulting in better yields than reported. This makes the process more suitable for industrial scale-up and is thus cost efficient and robust.
Objective of the invention:
It is an object of the invention to provide an improved, industrially viable and cost effective process for preparation of b, bl -1, 2-Dihydroxy-l, 2-cyclobutenediacrylic acid -di - g- lactone by use of 2-Deoxy -D-ribose and other cheaper and easily available raw materials that obviates the drawbacks of prior art processes.
Summary of the Invention
The present invention provides an in-situ synthesis of b,bl-l, 2-Dihydroxy-l, 2-cyclobutenediacrylic acid -di - g- lactone (formula I). 2-Deoxy-D-Ribose is subjected to glycosilation with Methanolic Hydrochloride to afford methyl furanoside. The resulting mixture of methyl furanoside is concentrated and the isolated oily compound is tosylated with p-Toluoyl Chloride to give 3, 5-di-O-p-Toluoyl methyl furanoside (IV). Oxidation of 3,5-di-O-p-Toluoyl methyl furanoside (IV) gives the 3,5-di-O-p-Toluoyl -2-deoxy-D-Ribono-1, 4-lactone (V). 3,5-di-O-p-Toluoyl -2-deoxy-D-Ribono-l, 4-lactone (V) undergo elimination reaction to give 5-Methylene-2- (5H)-furanone (Protanemonin) (II) which on dimerisation in aerobic condition yields b,bl-1,2-Dihydroxy-l, 2-cyclobutenediacrylic acid -di - g- lactone (I).
The invention further provides pharmaceutical formulations containing Anemonin prepared by the process described herein above.
Detailed Description of the Invention
The present invention describes an in-situ synthesis for preparation of b,bl-1,2-Dihydroxy-1, 2-cyclobutenediacrylic acid -di - g- lactone, Anemonin (I) with better yields. The synthetic preparation of Anemonin is given in scheme I.
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Reaction Scheme Of b,bl -1, 2-Dihydroxy-l, 2-cyclobutenediacrylic acid -di - y- lactone (Scheme I)

In accordance with the above scheme, the synthesis for preparation of p,p' -1,2-Dihydroxy-1,2-cyclobutenediacrylic acid -di - y- lactone (I) comprising the steps of:
a) glycosilating 2-deoxy-D-Ribose using alcoholic hydrochloride;
b) toluoylating of the crude glycosilated product to yield 3,5-di-O-p-Toluoyl methyl furanoside (IV);
c) oxidizing 3,5-di-O-p-Toluoyl methyl furanoside (IV) with m-chloroperbenzoic acid and BF3 Etherate to give 3,5-di-O-p-Toluoyl -2-deoxy-D-Ribono-1,4-lactone (V);
d) purifying the oxidized product, 3,5-di-O-p-Toluoyl -2-deoxy-D-Ribono-l, 4-lactone (V) using aliphatic solvents;
e) detoluoylating 3,5-di-0-p-Toluoyl-2-deoxy-D-Ribono-l, 4-lactone (V) to give 5-Methylene-2- (5H)-furanone (Protoanemonin) (II);
f) purifying 5-Methylene-2- (5H)-furanone (Protoanemonin) (II) by column chromatography;
g) dimerizing 5-Methylene-2- (5H)-furanone (II) to give the final product, p,p'-1,2-Dihydroxy-l, 2-cyclobutenediacrylic acid -di - y- lactone (I); and
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h) purifying b,bl -1, 2-Dihydroxy-l, 2-cyclobutenediacrylic acids -di - g-lactone (I) from suitable solvent.
Various solvents with different constitution are used which are process specific, product specific. Hydroxylic solvents are to be construed to mean solvents whose molecular formula has hydroxyl group as electronegative part of molecule. The process of glycosilation may be facilitated by the use of diverse group of solvents with heteroatom present in the molecular formula. The solvents used for the above process are selected from Dichloromethane, Methanol, Diisopropyl Ether, Pet Ether, Chloroform and Water either alone or a mixture thereof.
The present invention provides use of Methanolic HC1 as acid catalyst in glycosilation step instead of Acetyl Chloride which is flammable liquid, extremely irritating to eyes, corrosive, causes severe burns. The use of acid catalyst (hydrogen chloride) employed in the glycosilation process step gives quantitative yields and is adequately elaborated in following examples.
The tosylation of crude methyl furanoside provides the 3,5-di-O-p-Toluoyl methyl furanoside (the compound of formula IV) in high yield and crude product has purity in the range of 88-90%.
The oxidation of 3,5-di-O-p-Toluoyl methyl furanoside (IV)using m-chloroperbenzoic acid in the presence of BF3 Etherate provides 3,5-di-O-p-Toluoyl -2-deoxy-D-Ribono-l, 4-lactone (V) in high yield. In this oxidation reaction, the additional amount of (25%) BF3 Etherate is necessary for completion of reaction.
The use of di-isopropyl ether instead of Dichloromethane for extraction of compound of formula (V) simplifies the workup process avoiding the formation of inevitable emulsion problem, which is a potential problem associated with chlorinated solvents. This modification also improves the yield of intermediate (compound of formula V) by avoiding the probable loss of the product in emulsion.
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Further the invention provides novel method of purification for purifying the intermediate compound, 3,5-di-0-p-Toluoyl-2-deoxy-D-Ribono-l,4-lactone of formula (V), which is accomplished by re-pulping the isolated product in aliphatic solvents like Pet Ether, Hexane, and Cyclohexane, preferably in Pet. Ether, to obtain the product with a purity in the range of 90-95% without use of crystallization.
Further, the elimination reaction(detoluylation) is carried out on 3,5-di-O-p-Toluoyl -2-deoxy-D-Ribono-1, 4-lactone (V) using Triethyl Amine as a base at a temperature of 25°-30°c for 3 to 4 hrs to obtain 5-Methylene-2- (5H)-furanone (II) as yellow colored oil, which is purified by column chromatography to yield pure 5-Methylene-2- (5H)-furanone (II) as pale yellow colored oil. The detoluylation of 3,5-di-0-p-Toluoyl-2-deoxy-D-Ribono-1,4-lactone (V) to obtain Protoanemonin (II) is accomplished by using lesser amount of triethylamine, when compared to prior art methods, thus making process more cost effective.
The invention provides a simple method of dimerization of 5-Methylene-2- (5H)-furanone (Protoanemonin) (II) under anerobic conditions at ambient temperature to obtain the desired product b,bl -1, 2-Dihydroxy-l, 2-cyclobutenediacrylic acid -di - y- lactone the compound of formula (I) in high yield and purity. The compound, 5-Methylene-2-(5H)-furanone (II) dimerises on standing at ambient temperature to yield b,bl-1,2-Dihydroxy-1,2-cyclobutenediacrylic acid -di - y- lactone( I).
Further b,bl-1,2-Dihydroxy-l, 2-cyclobutenediacrylic acid -di - y- lactone (Anemonin)(I) is purified by crystallization using suitable solvent selected from Dichloromethane, Methanol, Diisopropyl Ether, Pet Ether, Chloroform and Water either alone or a mixture thereof. The preferable solvent mixture used for crystallization is Methanol and Chloroform in a ratio of 1:2.
Analysis of b,bl -1,2-Dihydroxy-l, 2-cyclobutenediacrylic acid -di - y- lactone (Anemonin)(I) is carried our using simple isocratic HPLC mode with C-18 column(250mm x 4.6mm,3um) with mobile phase ACN:H20 (20:80) Flow rate 0.7ml/ min, wavelength :225 nm,R.T. of Anemonin= 15-16 mins(Approx).
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Thus the process for preparation of b,bl -1, 2-Dihydroxy-l, 2-cyclobutenediacrylic acid -di - g- lactone (Anemonin) of the present invention is robust, easy to scale up and industrially reproducible in high yield and purity.
The present invention further discloses pharmaceutical formulations comprising Anemonin prepared by the process described hereinabove.
The following examples further illustrate the present invention but are not construed limiting in any manner to the scope of the invention as substantially described.
Example 1.
Preparation of compound 3,5-di-O-p-Toluoyl methyl furanoside (the compound of
formula IV).
To a solution of 80.0 gms (0.596 mol) of 2-Deoxy -D-Ribose(III) in MeOH (960 ml) added Methanolic hydrogen chloride (160 ml, 5%). The reaction mixture was stirred at 25-40°c for 15 mins and neutralized by adding solid sodium bicarbonate (32 gms). After filtration, methanol was removed by repeated co-evaporation with pyridine (100 ml x 2 times) under reduced pressure. The residual syrup was dissolved in pyridine (500 ml), cooled to 0°c and p-Toluoyl Chloride (180 ml, 1.16 mol) was added dropwise. The solution was stirred under nitrogen at 25-40°c for 18-24 hrs. The reaction mixture diluted with saturated aqueous sodium bicarbonate solution and extracted three times with saturated aqueous solution, then with 5% aqueous solution of Potassium Di-hydrogen Orthophosphate. The organic layers were dried on Sodium Sulphate then evaporated and co-evaporated with Toluene under reduced pressure for complete removal of Pyridine to yield a syrupy residue. Yield of product is 220gms.
Example 2
Preparation of compound 3,5-di-O-p-Toluoyl -2-deoxy-D-Ribono-l,4-lactone (formula
V)
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To the above resulting syrup added 500 ml Dichloromethane and cooled the reaction mixture to 10-15°c .To the reaction mixture added 320 ml (50%) BF3 Etherate and m-Chloroperbenzoic Acid 320gms (50-75%). The reaction mixture was stirred under nitrogen for 6 hrs at 25-40°c. After 6 hrs, 80ml (50%) of BF3 etherate was added and the reaction mixture was stirred at room temperature for 18 hrs.The solution was diluted with sodium bicarbonate 0.5M and stirred for 15-30 mins.The mixture was extracted with di-isopropyl ether (500 ml x 3 times ). The combined layers were washed with 0.5M sodium bicarbonate (500 ml) and dried on sodium sulphate. After evaporation of the solvent under reduced pressure the solid obtained was repulped with Pet. Ether (250 ml x 3 times) at room temperature. The product was dried under reduced pressure to yield a white colored solid.
Yield of product: 194 Gms (88%). Melting point =108°-110°c
Example 3.
Preparation of 5-Methylene-2- (5H)-furanone (Formula II)
The white colored 190 gm solid of 3,5-di-O-p-Toluoyl -2-deoxy-D-Ribono-l, 4-lactone (V) was dissolved in dichloromethane 400 ml, to this added triethyl amine (190 ml). The reaction mixture was stirred 25-40°c temperature for 3-4 hrs. After completion of the reaction, the reaction mixture was washed with 1M Sodium Bicarbonate (500 ml x 4 times) and dried on Sodium Sulphate. The solvent, dichloromethane was distilled out under reduced pressure to obtain yellow color oil. The oil was then purified on chromatographic column on silica gel (pentane/Dichloromethane: 1/2 eluent) afforded pure 5-Methylene-2- (5H)-furanone (Protoanemonin) in 65% yield.
Example 4
Preparation of b,bl -1,2-Dihydroxy-l, 2-cyclobutenediacrylic acid -di - g- lactone
(Anemonin)(Formula I)
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The compound 5-Methylene-2- (5H)-furanone (Protoanemonin) (formula II) 30.0 gm was subjected to dimerisation in aerobic condition 25-55°c temperature to yield 30.0gm of b,bl -1,2-Dihydroxy-l, 2-cyclobutenediacrylic acid -di -g- lactone (Anemonin). Anemonin was then extracted using a mixture of hot methanol: chloroform in the ratio of 1:2 and further concentrated till to dryness to obtain the product. The purity was checked by using isocratic method of HPLC. Yield of the product: 50%. Purity: greater than 80%.
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We Claim:
1. A Process for preparation of b,bl –1,2-Dihydroxy-l, 2-cyclobutenediacrylic
acid -di - g- lactone comprising the steps of:
a) glycosilating 2-deoxy-D-Ribose using alcoholic hydrochloride;
b) toluoylating of the crude glycosilated product to yield 3,5-di-O-p-toluoyl methyl furanoside (IV);
c) oxidizing 3,5-di-O-p-Toluoyl methyl furanoside (IV) with m-chloroperbenzoic acid and BF3 Etherate to give 3,5-di-O-p-Toluoyl -2-deoxy-D-Ribono-1,4-lactone (V);
d) purifying the oxidized product, 3,5-di-O-p-Toluoyl -2-deoxy-D-Ribono-1, 4-lactone (V) using aliphatic solvents;
e) detoluoylating 3,5-di-0-p-Toluoyl-2-deoxy-D-Ribono-l, 4-lactone (V) to give 5-Methylene-2- (5H)-furanone (Protoanemonin) (II);
f) dimerizing 5-Methylene-2- (5H)-furanone (II) to give the final product,
b,bl -1,2-Dihydroxy-l, 2-cyclobutenediacrylic acid -di - y- lactone (I); and
g) purifying b,bl-l, 2-Dihydroxy-l, 2-cyclobutenediacrylic acids -di - g-
lactone (I).
2. The process as claimed in claim 1, wherein the glycosilation of 2-Deoxy-D-Ribose is carried out using Methanolic Hydrogen Chloride (l%-5%).
3. The process as claimed in claim 1, wherein the oxidation of 3,5 di-O-p-Toluoyl methyl furanoside is carried out by using m-Chloroperbenzoic acid in presence of chlorinated solvent preferably in Dichloromethane and Boron Trifluoride Etherate as a catalyst.
4. The process as claimed in claim 1,wherein the oxidation of 3,5 di-O-p-Toluoyl methyl furanoside is carried out in presence of excess Boron Trifluoride Etherate.
5. The process as claimed in claim 1,wherein purification of 3,5-di-O-p-Toluoyl -2-deoxy-D-Ribono-l, 4-lactone (V) is carried out by using aliphatic solvents selected from Pet Ether, Hexane, and Cyclohexane preferably Pet Ether.
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6. The process as claimed in claim 1,wherein the detoluylation of 3,5-di-O-p-Toluoyl -2-deoxy-D-Ribono-l, 4-lactone (V) is carried out using Tertiary Amine as a base.
7. The process as claimed in claim 1,wherein the dimesrisation of 5-Methylene-2-(5H)-furanone to 3, b,bl -1,2-Dihydroxy-l, 2-cyclobutenediacrylic acid -di - g-lactone is subjected in aerobic condition at ambient temperature.
8. The process as claimed in claim 1 .wherein the purification of b,bl -1,2-Dihydroxy-1, 2-cyclobutenediacrylic acid -di - g- lactone is carried out by extracting crude solid in hot methanol and chloroform mixture.
9. The process as claimed in claim 8, wherein the methanol and chloroform mixture is used in a ratio of 1:2.
10. The process as claimed in claim 1,wherein the said reactions are carried out in suitable organic solvent selected from the group of Dichloromethane, Methanol, Diisopropyl Ether, Pet Ether, Chloroform and Water either alone or a mixture thereof.
11. A Process for preparation of b,bl -1,2-Dihydroxy-1,2-cyclobutenediacrylic acid -di - g- lactone as substantially described herein with reference to the foregoing example 1 to 4.
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Dated this 29th day of January 2007


ABSTRACT:
The present invention discloses an in-situ synthesis of b,bl -1,2-Dihydroxy-l, 2-cyclobutnediacrylic acid -di - g- lactone (I). Comprising the steps of dimerisation of 5-Methylene-2 (5H)-furanone (II) in aerobic condition & purifying the same in a suitable solvent(s). 2-Deoxy-D-Ribose (III) on glycosilation and further protection with p-Toluoyl Chloride gives 3,5-di-O-p-Toluoyl methyl furanoside (IV). Conversion of (IV) into 3,5-di-O-p-Toluoyl -2-deoxy-D-Ribono-l, 4-lactone (V) using m-Chloroperbenzoic acid and BF3 Etherate and followed by elimination of (V) yields 5-Methylene-2 (5H)-furanone (II).
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