FORM 2
THE PATENT ACT, 1970
(39 OF 1970)
AND
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION:
IMPROVED PROCES S FOR PREPARATION OF 2,2' -CYCLOCYTIDINE
HYDROCHLORIDE
2. APPLICANT:
a. NAME: SURAJLOK CHEMICALS PRIVATE LIMITED
b. NATIONALITY: INDIAN
c. ADDRESS: 128 CD, Charkop Cooperative Industrial Estate Ltd,
Near Hindustan Naka, Charkop, Kandivali (West), Mumbai - 400067 Maharashtra, India.
3. PREAMBLE TO THE DESCRIPTION:
The following specification particularly describes the invention and the manner in which it is to be performed.

Title:
Improved Process For Preparation of 2,2'-Cyclocytidine Hydrochloride
Technical Field of the Invention:
The present invention relates to an improved process for preparation of 2,2'-cyclocytidine hydrochloride of formula (I), a key intermediate of Cytarabine, from Cytidinc.

Formula (I)
Background and Prior art:
Cytarabine which is also chemically known as 4-amino-l-[(2R,3S,4R,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrim- idin-2-one, is a chemotherapy agent used mainly in the treatment of cancers of white blood cells such as acute myeloid leukemia (AML) and non-Hodgkin lymphoma. It kills cancer cells by interfering with DNA synthesis. Cytarabine has the following chemical structure:

HO OH
The most common synthetic method for Cytarabine is converting cytidine into 2,2'-cyclocytidine hydrochloride under the action of a dehydrating agent, and further ring-opening to obtain cytarabine hydrochloride.
2

Hence 2,2'cyclocytidine hydrochloride of formula (I) is the key intermediate of Cytarabine.
Journal of Organic Chemistry, 32, 1462 (1967) disclosed synthesis of 2,2'-cyclocytidine hydrochloride from Uridine using a process comprising 6 steps. In the final step 2,2'-cyclocytidine hydrochloride was obtained in 57% yield. Therefore the overall yield of 2,2'-cyclocytidine hydrochloride from Uridine can be expected to be 10-20%.
The process disclosed in Journal of Medicinal Chemistry, 10, 331 (1967) used 2,4-dithio-2',3',5'-triacetylarabinofuranosyluracil as a starting material which was produced in 7 stages of operation to obtain 2,2'cyclocytidine hydrochloride.
Journal of Organic Chemistry, 32, 816 - 817 (1967) disclosed a method for synthesizing cytarabine hydrochloride, where Cytidinewas converted into 2,2'-
cyclocytidinehydrochloride under the action of polyphosphoric acid which is corrosive and can produce highly toxic phosphorus oxide fumes when decomposed by heat.
Tetrahedron Lett., 1970, 869 disclosed synthesis of hydrochloride and fumarate salt of 2,2'-cyclocytidine. However, the process involves column chromatography for separation of these salts and crude 2,2'-cyclocytidine was obtained only in 30.4%.
Bulletin of the chemical society Japan, vol. 48(2), 505 - 507 (1975) disclosed synthesis of cyclocytidine by converting cytidine into 2',3,-0-sulfinyl cytidine using thionyl chloride in acetonitrile and water. 2',3'-0-sulfinyl cytidine on heating in an aqueous acid solution was converted into 2,2'-cyclocytidine hydrochloride. However, the process involves column chromatography for isolation of the product.
The patent number US3755296 disclosed preparation of 2,2'-cyclocytidine by
reacting Cytidine with a reagent formed from an acid chloride such as thionyl
chloride, phosphorus oxychloride or phosgene in the presence of dimethylformamide
(DMF). The process requires use of ion exchange chromatography for purification
'aT which makes the process unsuitable for commercial scale. Also purity of the product
^ is not mentioned in the patent.
_2
jz The patent number US5610292 disclosed the synthesis of Cytarabine in which
N Methanol, Cytidine and dibutyl tin oxide suspension was refluxed at room
| temperature for 12 hours; followed by reaction with triethylamine and p-
Ifc toluenesulfonyl chloride for 12 hours at room temperature; concentration and
g> refluxing in chloroform. The resulting white precipitate was filtered and dried to get
crude 2,2'-cyclocytidine hydrochloride which was purified using ethanol to provide purified product in 29% yield.The process involves longer reaction period and use of
CM
^-
o
CM *t CM
O
CO J
©
CO JO
CM
^ 1 PO MltMRA T ft- 3 - ft h - j f) 7 4. 1 7 * 7 p;
c - - -
"p CO
o

hazardous chemicals. Use of p-toluenesulfonyl chloride and methanol is potential source for genotoxic impurities.
Dibutyltin oxide is highly toxic and irritating to human eyes and skin. It has a strong irritating effect at high concentration. It causes severe headache, nausea, vomiting, drowsiness, and even coma. In addition, these substances have high boiling points and are difficult tocontrol within the limit in 2,2'-cyclocytidine hydrochloride. Therefore the resulting products have low purity and are likely to cause side effects when used on the human body.
The patent number US3856777 disclosed a procedure in which a mixture of Cytidine hydrochloride and ethylene carbonate was heated at 150°C for 50 minutes. The reaction mixture was dissolved in water and passed through a column packed with active charcoal. The product was obtained in 51% yield. However, purity of the product was not mentioned. Elution of product by water, followed by concentration of water for isolation (very high E-Factor) of the crude product and purification using ethanol-water system make the process unsuitable for commercial scale.
The patent number SU1569334 disclosed synthesis of 2,2'-cyclocytidine hydrochloride by treating Cytidine in acetonitrile with thionyl chloride and heating the resulting 2',3'-0-sulfinylcytidine hydrochloride with 1.3-1.5 excess phthalimide for 2 h at 100°C followed by crystallization from lower alcohol. However, due to difficulty in phthalimide removal from the reaction and high waste generation the process unsuitable for commercial scale.
The patent number SU610380 disclosed synthesis of 2,2'-cyclocytidine hydrochloride by treating cytidine with 5-6 parts of phosphorus oxychloride and DMF mixture in DMF-MeCN (1:1) for 5-6 h at 20-30°C and treatment of the reaction mixture with an equivalent amount of water at 20-40° for 1 h and then with water or alcohol at reflux for 0.5 h. However, phosphorus oxychloride is a dangerous reagent that decomposes violently in contact with water, generating a large amount of heat and smoke, causing phosphorus toxicity and even explosion.

CD G)
Q.
CD
CN
E o
LL
CN O
CO CN ^-
O
CN ^-CN
O CN
CO ^-
o
CO CN
o
CN C
"p CO
o

The patent number CN109422788 disclosed synthesis of 2,2'cyclocytidine hydrochloride by acetylation of Cytidine using acetylsalicylic acid chloride to provide acetyl cyclocytidine; followed by deprotection of acetyl cyclocytidine. Acetylsalicylic acid chloride is a costly reaction and generates aspirin as a by-product. Due to high water solubility and acidity of aspirin, it is very difficult to remove from the reaction mixture. Also, protection - deprotection reactions may lead to yield loss and lower atomic efficiency.
0 2 4
M U- M B

The patent application number US20230399354 disclosed synthesis of 2,2'-cyclocytidine hydrochloride from cytidine using diphenyl carbonate (247 mmol), DMF and sodium hydroxide. However, the process is not reproducible and did not go for completion; very less product formation is observed on TLC. Also the process is unsafe as diphenyl carbonate (247 mmol) leads to formation of phenol (in quantity higher than permissible level) as a by-product which is genotoxic.
The patent application number IN2014MU0218 disclosed synthesis of 2,2'-cyclocytidine hydrochloride by converting cytidine into 2',3'-0-sulfinyl cytidine using thionyl chloride; followed by conversion of 2',3'-0-sulfinyl cytidine into 2,2'cyclocytidine hydrochloride. However, the yield of 2,2'-cyclocytidine hydrochloride is low when compared with the theoretical yield. Also the conversion of 2',3'-0-sulfinyl cytidine into 2,2'-cyclocytidine hydrochlorideis cumbersome, involves distillation of water for isolation of cyclocytidine hydrochloride. This may lead to degradation of 2,2'-cyclocytidine hydrochloride to Cytidine during commercial scale.
The prior art search reveals ample data for preparation of 2,2'-cyclocytidine hydrochloride. However, the process mentioned in the prior art are deficient with respect to number of stages (which eventually lead to loss of yield and increase time cycle) and require silica, resin, charcoal columns for isolation and purification. Furthermore* the prior art processes involve use of aqueous solutions for ion exchange chromatography which has its own drawback on commercial scale,
Solution of these problems associated with prior art becomes the objects of the present invention. Hence, the present invention provides an industrially viable and economically feasible process thereby eliminating all the above-mentioned shortcomings with better yields, to suit industrial manufacture by using cheaper starting material and avoiding the column chromatography, distillation or purifications of the product thus making the process 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 therefore an object of the invention is to overcome or ameliorate atleastone disadvantage of the prior art or to provide a useful alternative.
Another object of the invention is to provide an improved, industrially viable and cost effective process for preparation of 2,2'-cyclocytidine hydrochloride avoiding use of column chromatographic techniques.
5

Yet anotherobject of the invention is to provide the process for manufacture of 2,2'-cyclocytidine hydrochloride in better yield and purity with high atomic efficiency and low e-factor.
Summary of the invention:
In accordance with the above objectives, the present invention provides concise, economic, industrially feasible and environmentally friendly process for the preparation of 2,2'-cyclocytidine hydrochloride using safe, mild, inexpensiveand easy to handle reagents.
Other features, objects and advantages of the inventions will be apparent from the appended examples and claims.
Detailed description of the invention:
Unless specified otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, to which this invention belongs. Although any methods and material or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described. To describe the invention, certain terms are defined herein specified as follows:
Unless stated to the contrary, any of the words 'having', 'including', 'includes', 'comprising' and 'comprises' mean 'including without limitations' and shall not be construed to limit any general statement that it follows to the specific or similar items or matters immediately following it. Embodiments of the invention are not mutually exclusive, but may be implemented in various combinations. The described embodiments of the invention and the disclosed examples are given for the purpose illustration rather than limitation of the invention as set forth the appended claims.
Accordingly, the present invention provides an improved process to prepare highly pure 2,2'-cyclocytidine hydrochlorideof formula (I)
OH
X^-o HCI
Formula (I)
comprisingtreating2'33'-0-sulfinyl cytidine hydrochloride of formula (II) with at least one solvent.
6

Formula (II)
In a preferred embodiment the reaction is carried out in presence of a solvent selected from dimethylformamide, dimethylacetamide, N-methylpyrrolidone, organic carbonate, dioxane, ether, alcohol, aliphatic hydrocarbons, aromatic hydrocarbon, ester, haloalkanes or mixture thereof.
The carbonate is a compound of formula (III)
C
< A ^
Formula (III)
wherein R and R are alkyl containing 1 to 6 cai'bon atoms, cycloalkyl containing 1 to 6 carbon atoms, aryl or substituted aryl.
Preferably R and R are independently methyl or phenyl.
Advantageously the reaction is carried out in non-aqueous medium.
The reaction is optionally carried out in presence of a base and catalyst.
The catalyst is a carbonate of formula (III)
The carbonate is a compound of formula (III)
O
R!
Formula (III)

1 0
wherein R and R are alkyl containing 1 to 6 carbon atoms, cycloalkyl containing 1 to 6 carbon atoms,aryl or substituted aryl.
In a preferred embodiment Rl and R2 are independently methyl or phenyl.
The carbonate catalyst is conveniently used in an amount, relative to 25,3'-0-sulfmyl cytidine, preferably in a range between 0.05 to 1.5 equivalents, more preferably 0.20 to 0.50 equivalents.
The base is organic or inorganic. Examples of organic base include primary amines, tertiary amines and secondary amines. Examples of amines are triethylamine, N-methylmorpholine, diisopropyl ethyl amine, N-methylpiperidine. Examples of inorganic base include alkali metal carbonate, alkaline earth metal carbonate, alkali metal bicarbonate, alkaline earth metal bicarbonate, alkali metal hydroxide, alkaline earth metal hydroxide and mixture thereof. Examples of alkali metal carbonate include sodium carbonate and potassium carbonate. Examples of alkali metal bicarbonate include sodium bicarbonate and potassium bicarbonate. Examples of alkaline earth metal carbonate include calcium carbonate and magnesium carbonate. Examples of alkaline earth metal bicarbonate include calcium bicarbonate and magnesium bicarbonate. Examples of alkali metal hydroxide include potassium hydroxide and sodium hydroxide.
The base is conveniently used in an amount, relative to 2,,3'-0-sulfinyl cytidine, preferably in a range between 0.02 to 1.0 equivalent, more preferably 0.05 to 0.60 equivalent.
The process of the present invention may be carried out at suitable temperature. To minimize the decomposition of products and impurity formation the reaction is carried out at 60°C to 130°C, more preferably at 70°C to 120°C. The most preferred reaction temperature for is 75°C to 100°C.
The process of the present invention further comprises reacting cytidine with thionyl chloride or sulfuryl chloride in presence of a catalyst and solvent to provide 2\3'-0-sulfinyl cytidine of formula (II)*
The catalyst used in the synthesis of 2',3'-0-sulfinyl cytidineis amide e.g. dimethy lformamide.
The solvent used in the synthesis of 2'53'-0-sulfinyl cytidine is selected from acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, toluene, dichloromethane, carbon tetrachloride or mixture thereof

The process for preparation of 2\35-0-sulfmyl cytidine may be carried out at suitable temperature. To minimize the decomposition of products and impurity formation, the process for preparation of 2',3'-0-sulfmyl cytidineis carried out at -5°C to 25°C, more preferably at -5°C to 10°C. The most preferred reaction temperature for synthesis of ?',V-0-sn1finyl cytidine is -5°C to 5°C.
On completion of the reaction 2.2'-cyclocytidine hydrochloride can be recovered from the reaction mass in a convenient manner. Optionally the workup of the reaction involves distillation of the solvent followed-bypH adjustment using-concentrated - -hydrochloric acid to isolate 2.2'-cyclocytidinehydrochloride. Therefore the process of the present invention avoids the tedious process of column chromatography and purification of the final product; and has better atomic efficiency.
2.2'-cyclocytidine hydrochloride is subjected to hydrolysis to obtain Cytarabine by methods known in the prior art.
Further details of the process of the present invention will be apparent from the examples presented below. Examples presented are purely illustrative and are not limited to the particular embodiments illustrated herein but include the permutations, which are obvious as set forth in the description.
Example-1
Preparation of 2,2' -cyclocytidine hydrochloride
A clean and dry 100 ml round bottom flask was charged with dimethylformamide (30.0 ml), diphenyl carbonate (2.04 gm,0.0095 mol) and Sodium hydroxide (0.152 gm, 0.0038 mol) at 85 to 90°C. To this hot solution 2',3'-0-sulfinyl cytidine hydrochloride (10.0 g 0.030 mol) was added and the mixture was maintained for 2 to 3 hours at 85 to 90°C. The reaction progress was monitored by HPLC. After completion of the reaction N,N-Dimethylformamide was distilled completely under vacuum at 85 to 90°C. The resulting residue was acidified with concentrated hydrochloric acid in presence of methanol to obtain solid. The solid was separated by filtration and dried under vacuum to obtain 2,2'-cyclocytidine hydrochloride (yield 7.0gm, HPLC purity 99.06%).
Example - 2
A clean and dry 100 ml round bottom flask was charged with dimethylformamide (30.0 ml), dimethyl carbonate (0.69 gm, 0.0077 mol) and Sodium hydroxide (0.12gm, 0.003 mol) and the reaction mixture was heated to 80 to 90°C.To this hot solution 2',3'-0-sulfinyl cytidine hydrochloride (10.0 g 0.030 mol) was added and the mixture

was maintained for 0.5 hour at 80 to 90°C. The reaction progress was monitored by HPLC. The reaction mass was stirred overnight at room temperature. Dimethylformamide was distilled completely under vacuum at 70 to 80°C.The resulting residue was acidified with concentrated hydrochloric acid to obtain solid. The solid was separated by filtration and dried under vacuum to obtain 2,2'= cyclocytidine hydrochloride (yield 6.9gm, HPLC purity 99.62%).
Example - 3
Preparation of 2,2'-cyclocytidine hydrochloride
A clean and dry 100 ml round bottom flask was charged with 2',3'-0-sulfinyl cytidine hydrochloride (10 gm, 0.030 mol) and dimethylformamide (30.0 ml). The reaction mass heated to 90 to 95°C for 2.0 to 3.0 hours. Progress of the reaction was monitored by HPLC. After completion of the reaction dimethylformamide was distilled completely under vacuum at 85 to 90°C. The resulting residue was acidified with concentrated hydrochloric acid in presence of methanol to obtain solid. The solid was separated by filtration and dried under vacuum to obtain 2,2'-cyclocytidine hydrochloride (yield 6.3 gm, HPLC purity 99.28%).
Example - 4
Preparation of 2',3'-0-sulfinyl cytidine hydrochloride
A clean and dry 250 ml round bottom flask was charged withacetonitrile (90 ml) and Cytidine (30.0 g, 0.12 moles) at room temperature. Dimethylformamide (2.0 ml) was added to this solution and the reaction mixture was cooled to 0 to 5°C. Thionyl chloride (51.0 g, 0.43 moles) was added to the cooled reaction mixture and maintained for 2 to 3 hours at 0 to 5 °C. The reaction progress was monitored by HPLC. The reaction mass was quenched with ice-cold mixture of methanol and water. The resulting solid was filtered off, washed by chilled methanol (30.0 ml) and dried under vacuum to obtain 2\3'-0-sulfinyl cytidine hydrochloride (yield 31.4gm, HPLC purity 98.46%).

We claim,

1. Process for preparing 2,2'-cyclocytidine hydrochloride of formula (I)

0 Nrr^NH
Formula (I)
comprisingtreating2,3,-o-sulfmyl cytidine hydrochloride of formula (II) with atleast one solvent.

Formula (II)
2. The process for preparing 2,2'-cyclocytidine hydrochloride as claimed in claims 1
wherein the solvent is selected from dimethylformamide, dimethylacetamide, N-
methylpyrrolidone, organic carbonate, dioxane, ether, alcohol, aliphatic
hydrocarbons, aromatic hydrocarbon, ester, haloalkanes or mixture thereof.
3. The process for preparing 2,2'-cyclocytidine hydrochloride as claimed in claim
*v 1 wherein the carbonate is of formula (III)

o
1
R.

R2

Formula (III)
g wherein R and R are alkyl containing 1 to 6 carbon atoms, cycloalkyl containing 1
to 6 carbon atoms,aryl or substituted aryl.

4. The process for preparing 2,2'-cyclocytidine hydrochloride as claimed in claim 3
1 0
wherein R and R are independently methyl or phenyl.
5. The process for preparing 2,2'-cyclocytidine hydrochloride as claimed in claim 1 wherein the process is carried out in presence of a base and catalyst.
6. The process for preparing 2,2'-cyclocytidine hydrochloride as claimed in claim 5 -wherein-the baseis organic or inorganic.- - ~ - -_ _
7. The process for preparing 2,2'-cyclocytidine hydrochloride as claimed in claims 6 wherein the base is selected from primary amines, secondary amines, tertiary amines, alkali metal carbonate, alkaline earth metal carbonate, alkali metal bicarbonate, alkaline earth metal bicarbonate, alkali metal hydroxide,alkaline earth metalhydroxide and mixture thereof.
8. The process for preparing 2,2'-cyclocytidine hydrochloride as claimed in claim 7 wherein the base is selected from triethylamine, N-methylmorpholine, diisopropylethyl amine, N-methylpiperidine,sodiumcarbonate, potassiumcarbonate, sodium bicarbonate, potassium bicarbonate, calcium carbonate,magnesiumcarbonate, calcium bicarbonate, magnesium bicarbonate, potassium hydroxide andsodiumhydroxide.
9. The process for preparing 2,2'-cyclocytidine hydrochloride as claimed in claim 5 wherein the catalyst is carbonate of formula (III)
O
Formula (III) wherein R and R" are alkyl containing 1 to 6 carbon atoms, cycloalkyl containing 1 to 6 carbon atoms,aryl or substituted aryl.
10. The process for preparing 2,2'-cyclocytidine hydrochloride as claimed in claim 9 wherein R and R are independently methyl or phenyl.
11. The process for preparing 2,2'-cyclocytidine hydrochloride as claimed in claim 1 further comprising reacting cytidine with thionyl chloride or sulfuryl chloride in presence of a catalyst and solvent to provide 2',3'-0-sulfinyl cytidine hydrochloride of formula (II);

12. The process for preparing 2,2'-cyclocytidinehydrochloride as claimed in claim 11 wherein thecatalyst is selected from amides.
13. The process for preparing 2,2'-cyclocytidine hydrochloride as claimed in claim 12 wherein the catalyst is dimethylformamide.
14. The process for preparing 2,2'-cyclocytidine hydrochloride as claimed in claim 11 wherein the solvent is selected from acetonitrile, tetrahydrofuran, 2-methyltetrahydrofuran, toluene, dichloromethane, carbon tetrachloride or mixture thereof.