Description:Related Application

This is an application for patent of addition to the co-pending application no. 202121043551 filed on 25th September, 2021.

Field of Invention
The present invention relates to an improved, cost-effective process for conversion of bisnoralcohol into 25-OH 7-DHC (provitamin of 25-OH vitamin D3(Calcifediol).

Background of the Invention
The metabolite of vitamin D3, 25-hydroxycholecalciferol (Calcifediol) is more potent anti- rachitic agent than vitamin D itself therefore development of a facile method for the synthesis of vitamin D3 and its analogue Calcifediol is highly important.

Various synthetic routes for the production of cholesterol and 7-dehydrocholesterol, 25-OH cholesterol and 7-dehydrocholesterol from phytosterol are reported in WO2020225830 and WO2021005618. However, the processes known have the disadvantage that high conversion of phytosterol to vitamin D3 and its analogues is dependent on content of stigma sterol present in the phytosterol.

The Indian patent application no. 202121043551 have addressed this problem by providing the preparation of vitamin D3 and its analogues by fully utilizing the phytosterol that consists various sterols such as stigmasterol, sitosterol and campesterol etc. In said Indian patent application, to utilize fully the phytosterol consisting of stigmasterol, sitosterol and campesterol etc. the bisnoralcohol was prepared from phytosterol (soya sterol) by fermentation method which was then converted to 7-dehydrocholesterol / 25-OH 7-dehydrocholesterol in good yield and purity. Furthermore, in the process disclosed in IN’551 for construction of 25-hydroxy side chain, the intermediate 20(S)-20-tosyloxymethyl-3ß-tertbutyldimethylsilyloxy-pregna-5,7-diene is coupled with Grignard reagent to yield3ß-tert-Butyldimethylsilyloxy-25-triethylsilyloxycholesta-5,7-diene.

However, for scaling up the reaction to industrial level i.e. in bulk quantity the reagent 4-bromo-2-methyl-2-[(trimethylsilyl)oxy] butane is not easily available commercially and cost wise is not economical. The in-house preparation of said reagent includes three steps starting from Grignard reaction of ethyl-3-bromopropionate with methyl magnesium iodide/ bromide followed by protection of hydroxyl group with trimethyl silyl chloride and purification of reagent by distillation. The process is tedious, cumbersome and not economical on an industrial scale.

The present inventors observed that there remains a scope to provide an improved and cost-effective, scalable process for preparation of / 25-OH 7-dehydrocholesterol and further preparation to 25-OH vitamin D3 (Calcifediol) from bisnoralcohol. This remains the objective of the invention.

Summary of the invention
In accordance with the above, the present invention provides an improved and cost-effective scalable process for synthesis of / 25-OH 7-dehydrocholesterol from bisnoralcohol comprising;
i. Converting Bisnoralcohol to (20S)-20-tosyloxymethyl-3ß-tertbutyldimethylsilyloxy-pregna-5,7-diene (1a) by the known process;
ii. Reacting (20S)-20-tosyloxymethyl-3ß-tertbutyldimethylsilyloxy-pregna-5,7-diene (1a) with sodium iodide in dry acetone to yield 20S)-20-Iodomethyl-3ß-tert-butyldimethylsilyloxypregna-5,7-diene(2a);
iii. Reacting (20S)-20-Iodomethyl-3ß-tert-butyldimethylsilyloxypregna-5,7-diene(2a) with ethyl acrylate in presence of zinc and nickel salt at a temperature in the range of 50-60ºC followed by cooling to room temperature and reacting within 0.5hours with a solution of (20S)-20-Iodomethyl-3ß-tert-butyldimethylsilyloxypregna-5,7-diene (2a) at room temperature to yield Ethyl-3ß-tert-Butyldimethylsilyloxy-chola-5,7-diene-24-carboxylate (3a);
iv. Subjecting ice cooled ethyl-3ß-tert-Butyldimethylsilyloxy-chola-5,7-diene-24-carboxylate (3a) to Grignard reaction in THF to yield crude 3ß-tert-Butyldimethylsilyloxy-25-hydroxychola-5,7-diene(4a) followed by recrystallization to obtain pure compound (4a);
v. Deprotecting 3ß-tert-Butyldimethylsilyloxy-25-hydroxychola-5,7-diene(4a) with Tetrabutylammonium fluoride trihydrate to obtain 3ß-25-Dihydroxycholesta-5,7-diene (pro vitamin of 25-Hydroxy vitamin D3).

The (20S)-20-tosyloxymethyl-3ß-tertbutyldimethylsilyloxy-pregna-5,7-diene (1a) is obtained from bisnoralcohol by the process described in the Indian Patent application no. 202121043551.

In another aspect, the 3ß-25-Dihydroxycholesta-5,7-diene (pro vitamin of 25-Hydroxy vitamin D3) is converted to 25-OH vitamin D3(Calcifediol).

In an aspect, the present invention thus provides a process for synthesis of 25-OH vitamin D3 (Calcifediol) from bisnoralcohol via synthesis of 3ß-25-Dihydroxycholesta-5,7-diene (pro vitamin of 25-Hydroxy vitamin D3) as shown above.

Detailed Description of the invention
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

The initial material as used in the present invention soya sterol for the production of bisnoralcohol was procured from Hubei Goto biotechnology Co Ltd, China.

In accordance with the above, the present invention relates to an improved and cost-effective process for synthesis of / 25-OH 7-dehydrocholesterol from bisnoralcohol comprising;
i. Converting Bisnoralcohol to (20S)-20-tosyloxymethyl-3ß-tertbutyldimethylsilyloxy-pregna-5,7-diene (1a) by the known process;
ii. Reacting (20S)-20-tosyloxymethyl-3ß-tertbutyldimethylsilyloxy-pregna-5,7-diene (1a) with sodium iodide in dry acetone to yield 20(S)-20-Iodomethyl-3ß-tert-butyldimethylsilyloxypregna-5,7-diene(2a);

iii. Reacting 20(S)-20-Iodomethyl-3ß-tert-butyldimethylsilyloxypregna-5,7-diene(2a) with ethyl acrylate in presence of zinc and nickel salt at a temperature in the range of 50-60ºC followed by cooling to room temperature and reacting within 0.5hours with a solution of (20S)-20-Iodomethyl-3ß-tert-butyldimethylsilyloxypregna-5,7-diene (2a) at room temperature to yield Ethyl-3ß-tert-Butyldimethylsilyloxy-chola-5,7-diene-24-carboxylate (3a);

iv. Subjecting ice cooled ethyl-3ß-tert-Butyldimethylsilyloxy-chola-5,7-diene-24-carboxylate (3a) to Grignard reaction in THF to yield crude 3ß-tert-Butyldimethylsilyloxy-25-hydroxychola-5,7-diene(4a) followed by recrystallization to obtain pure compound (4a);

v. Deprotecting 3ß-tert-Butyldimethylsilyloxy-25-hydroxychola-5,7-diene(4a) with Tetrabutylammonium fluoride trihydrate to obtain 3ß-25-Dihydroxycholesta-5,7-diene (pro vitamin of 25-Hydroxy vitamin D3).

According to step (i), to (20S)-20-tosyloxymethyl-3ß-tertbutyl dimethyl silyloxy pregna-5,7-diene (1a) obtained from bisnoralcohol was added sodium iodide and dry acetone. The mixture was refluxed for 3-5 hours and cooled. The mixture was poured in to water and extracted in a solvent, washed and the solvent extract was dried and concentrated to yield (20S)-20-Iodomethyl-3ß-tert-butyldimethylsilyloxypregna-5,7-diene (2a) as an off-white solid.

The step (ii) comprises adding nickel salt selected from NiC12.6H20 to a vigorously stirred mixture of zinc dust and ethyl acrylate in a base. The mixture was heated to 50-55oC, whereupon an exotherm ensued, and stirring was continued at 60-70oC for about 30 min. The resulting reddish-brown mixture was cooled to 25oC and treated during 0.5 h with a solution of (20S)-20-Iodomethyl-3ß-tert-butyldimethylsilyloxypregna-5,7-diene (2a), in a solvent at a rate so as to maintain the temperature below 25oC. The mixture was stirred at 25oC for 3 to 4 hours, poured into the solvent and filtered, washed with suitable solvent, filtered. The filtrate were washed several times. Dried the mixture and evaporated the solvent to give off white solid of Ethyl-3ß-tert-Butyldimethylsilyloxy-chola-5,7-diene-24-carboxylate (3a) which was sufficiently pure and used directly in the next step.

The third step (iii) of the present process comprised of subjecting the compound (3a) to Grignard reaction. Accordingly, to a stirred, cooled ice bath (0-5oC solution of Ethyl-3ß-tert-Butyldimethylsilyloxy-chola-5,7-diene-24-carboxylate(3a) in dry THF under nitrogen atmosphere was added methyl magnesium bromide during a period of about 30 min. The mixture was stirred at ice bath temperature for about 2hr and at room temperature for about 18 hour. The reaction was monitored by TLC, cooled to 0oC, and carefully quenched with saturated NH4Cl solution. The mass was extracted in a suitable solvent, washed, dried evaporated in vacuo to give crude 3ß-tert-Butyldimethylsilyloxy-25-hydroxychola-5,7-diene(4a).

The crude mass was further stirred with lower alcohol, filtered, dried to get desired product as a colorless solid. Recrystallization was carried out in a suitable solvent to give 3ß-tert-Butyldimethylsilyloxy-25-hydroxychola-5,7-diene (4a) as white crystalline solid.

According to step (4), to 3ß-tert-Butyldimethylsilyloxy-25-hydroxychola-5,7-diene (4a) was added Bu4NF•3H2O in THF and the mixture was stirred under argon at 25-30oC for about 12 h. This was followed by the addition of suitable solvent and the mixture was washed. The organic phase was separated, dried and the solvent removed under reduced pressure. The residue was crystallized from suitable solvent to obtain 3ß-25-Dihydroxycholesta-5,7-diene (pro vitamin of 25-Hydroxy vitamin D3).

The solvent for the process was selected from lower alcohols such as methanol, ethanol, isopropyl alcohol; ethers such as DMF, THF, MTBE and the like; ketones such as acetone; esters such as ethyl acetate; alone or combination thereof.

The base was selected from ethylamine, triethylamine, pyridine, alkali or alkaline earth metal carbonates and bicarbonates and the like alone or combination thereof.

In an embodiment, the compound (20S)-20-tosyloxymethyl-3ß-tertbutyl dimethyl silyloxy pregna-5,7-diene (1a) was obtained from bisnoralcohol by the process disclosed in Indian patent application no. 202121043551.

Accordingly, the process for preparation of (20S)-20-tosyloxymethyl-3ß-tertbutyl dimethyl silyloxy pregna-5,7-diene (1a) from bisnoralcohol comprises the steps of;
i. Reacting bisnoralcohol with p-Chloranil in presence of an alcohol selected from C1 to C4 alcohols to obtain (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one;
ii. Tosylating the (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one to obtain
(20S)-20-tosyloxymethyl-pregna-4,6-dien-3-one;
iii. Acylating the (20S)-20-tosyloxymethyl-pregna-4,6-dien-3-one in presence of acylating agent selected from acetic anhydride, acetyl chloride or mixture thereof to obtain (20S)-20-tosyloxymethyl-3ß-acetoxy-pregna-1,3,5- triene;
iv. Reacting the (20S)-20-tosyloxymethyl-3ß-acetoxy-pregna-1,3,5-triene using a reductant selected from Sodium borohydride, LiAlH4 or Calcium borohydride (Ca (BH4)2 at a temperature of 0-10°C to obtain (20S)-20-tosyloxymethyl-3ß- hydroxy-pregna-5,7-diene;
v. Protecting the (20S)-20-tosyloxymethyl-3ß-hydroxy-pregna-5,7-diene by
reacting with tert-butyldimethyl silylchloride/imidazole to obtain (20S)-20- tosyloxymethyl-3ß- tertbutyldimethylsilyloxy-pregna-5,7-diene (1a).

The starting material Bisnoralcohol was prepared from soya sterol by fermentation method. In this method, the entire soya sterol got converted into Bisnoralcohol in greater than 95%.

In yet another embodiment, the present invention provides a process for preparation of 25-OH vitamin D3(Calcifediol) comprising.
i. Irradiating 3ß-25-Dihydroxycholesta-5,7-diene (pro vitamin of 25-Hydroxy vitamin D3) obtained as above in presence of an antioxidant and photosensitizer 5-(3-pyridyl)-2, 2’-bithiophene under medium pressure mercury lamp of wavelength 250-310nm;
ii. Concentrating the irradiated solution of step (i), cooling and crystallizing out the unreacted mass;
iii. Filtering and washing the unreacted mass of step (i) and reusing for irradiation;
iv. Evaporating the filtrate under vacuum to obtain crude product, dissolving the crude product in solvent, refluxing, concentrating under vacuum to obtain reddish orange mass followed by purifying.
v. Repeating the step (iv) of concentration of the filtrate severally to obtain quantitative yield of the pure product.

The present process offers an improvement over the process disclosed in IN’551 wherein construction of 25-hydroxy side chain is carried out in a convenient manner and which can be scaled up to industrial level in a cost effective manner. The side chain construction is effected through coupling Ni(0) complex derived from ethyl acrylate with 20S)-20-Iodomethyl-3ß-tert-butyldimethylsilyloxypregna-5,7-diene (2a) in presence of Zinc and pyridine to get corresponding ester (3a).Synthesis of 3ß-25-Dihydroxycholesta-5,7-diene (pro vitamin of 25-Hydroxy vitamin D3) was accomplished by treating ester(3a) with commercially available methyl magnesium bromide to give 3ß-tert-Butyldimethylsilyloxy-25-hydroxychola-5,7-diene(4a) followed by desilylation with n-Bu4N+F.

In an embodiment, the present invention not only provides an improved and cost-effective process for preparation of 3ß-25-Dihydroxycholesta-5,7-diene (pro vitamin of 25-Hydroxy vitamin D3) from bisnoralcohol but also the preparation of 25-OH vitamin D3(Calcifediol) with comparative yield and purity.

The process of the present invention provides a promising route that offers economical and efficient strategies for potential large-scale production of 25-OH 7- dehydrocholesterol. Moreover, the present process can be carried out on an industrial scale for preparation of pro vitamin of 25-Hydroxy vitamin D3 and 25-OH vitamin D3(Calcifediol).

Examples:
Example 1: Preparation of (20S)-20-Iodomethyl-3ß-tert-butyldimethylsilyloxypregna-5,7-diene ((3ß-tert-Butyldimethylsilyloxy-22-iodo-23,24-bisnorchola-5,7-diene)) (2a)

A mixture of 50g (0.0836mole) of (20S)-20-tosyloxymethyl-3ß-tertbutyldimethylsilyloxypregna-5,7-diene (1a), prepared from bisnoralcohol, sodium iodide (24.9g, 0.167 mole) and 1000mL of dry acetone were heated at reflux for 3-4 hrs and cooled. The mixture was poured into water and extracted with ethyl acetate. The ethyl acetate extract was washed with 2% sodium thiosulfate solution (2x500mL) followed by washing with water, brine solution and finally the ethyl acetate extract was dried over anhydrous sodium sulfate. The extract was concentrated to yield (20S)-20-Iodomethyl-3ß-tert-butyldimethylsilyloxypregna-5,7-diene (2a) as an off-white solid.
Analytical data:
Yield: 42.0g (90.7%)
Appearance: Off-White solid
M.pt: 140-143oC
GC analysis: ~ 95% (RT: 19.5)
HPLC analysis: 96.8% (RT: 26.4)

Example 2: Preparation of Ethyl-3ß-tert-Butyldimethylsilyloxy-chola-5,7-diene-24-carboxylate(3a):

To a vigorously stirred mixture of 18.7g (0.288mole) of zinc dust and 23mL (0.216 mol) of ethyl acrylate in 80 mL of pyridine was added 17.1g (0.072 mol) of NiC12 .6H20. The mixture was heated to 50-55oC, whereupon an exotherm ensued, and stirring was continued at 65oC for 30 min. The resulting reddish-brown mixture was cooled to 25oC and treated during 0.5 h with a solution of 40g (0.072 mol) of (20S)-20-Iodomethyl-3ß-tert-butyldimethylsilyloxypregna-5,7-diene (2a), in 120 mL of THF at a rate so as to maintain the temperature below 25oC. The mixture was stirred at 25oC for 3 to 4 h, poured into 600 mL of EtOAc, and filtered through a pad of Celite. The pad was washed with EtOAc (2 x 200 mL), and the filtrate and washings were washed with 1.0 N HC1 (4 x200 mL), 500 mL of a solution of EDTA (80.0 g EDTA + 80 g NaHCO3 in 1.0 L of H2O), and brine (2 x 200 mL). Dried the mixture over Na2SO4 and evaporated to give off white solid of Ethyl-3ß-tert-Butyldimethylsilyloxy-chola-5,7-diene-24-carboxylate (3a) which was sufficiently pure and used directly in the next step.
Yield: 35.0g (92%)
An analytical sample was prepared by an additional purification by silicagel column chromatography using EtOAc: n-heptane (2:98) as an eluent. Concentration of collected pure fractions yielded desired product as white solid.
Appearance: White solid
M.Pt: 97-100oC
GC purity: ~94% (RT: 28.87)
HNMR: d 0.02-0.01 (6H, s) 0.77 (9H, s),0.97(3H, d),1.16(3H, t), 2.23 (2H, t), 2.43 (1H, dd), 2.71(1H, dd),4.3(2H, q),4.23(1H, dddd), 5.97-6.10(2H, dd).
Mass spectrum (m/e): 529(M+1)

Example 3: Preparation of 3ß-tert-Butyldimethylsilyloxy-25-hydroxychola-5,7-diene(4a)

To a stirred, cooled ice bath (0-5oC solution of 30.0g (0.056mol) of Ethyl-3ß-tert-Butyldimethylsilyloxy-chola-5,7-diene-24-carboxylate(3a) in 200mL of dry THF under nitrogen atmosphere was added 65mL of methyl magnesium bromide (3.0 M in ether) during 30 min. The mixture was stirred at ice bath temperature for 2hr and at room temperature for 18 h, reaction was monitored by TLC, cooled to 0oC, and carefully quenched with saturated NH4Cl solution. The mass was extracted with 500mL of EtOAc, washed with brine (2x500 mL), dried over Na2SO4, and evaporated in vacuo to give 28 g of crude 3ß-tert-Butyldimethylsilyloxy-25-hydroxychola-5,7-diene.

The crude mass was further stirred with methanol, filtered, dried to get 25.5g (~87%) of desired product as a colorless solid. Recrystallization was carried out in methanol to give 3ß-tert-Butyldimethylsilyloxy-25-hydroxychola-5,7-diene (4a) as white crystalline solid.

Analytical Data:
Appearance: Colorless solid
GC purity: >95%
NMR: d 0.02-0.01 (6H, s) 0.77 (9H, s), 1.10(3H, d), 2.26 (1H, dd),2.35(1H, dd) 2.58 (1H, dd), 2.72(1H, dd),4.3(1H, dddd), 5.97-6.01 (2H, dd).
Mass spectrum (m/e): 515(M+1)

Example 4: Preparation of 3ß-25-Dihydroxycholesta-5,7-diene (pro vitamin of 25-Hydroxy vitamin D3)

To a solution of 3ß-tert-Butyldimethylsilyloxy-25-hydroxychola-5,7-diene (4a) in THF (25.0g, 0.057, mole) Bu4NF•3H2O (0.114mole, 36g) in THF (300 mL) was added and the mixture was stirred under argon at 25-30oC for 12 h. This was followed by the addition of EtOAc (500 mL) and the mixture washed with brine (100x3mL). The organic phase was separated, dried over Na2SO4 and the solvent removed under reduced pressure. The residue was crystallized from MeOH.
Analytical Data:
Yield: 18.0g (80%)
GC: 96% (RT: 10.05)
M.Pt: 155–158°C.
1H-NMR (400 MHz, CDCl3): d= 0.61 (s, 3 H), 0.94 (s, 3 H), 0.97 (d, 3 H), 1.20 (s, 6 H), 3.7 (m, 1 H), 5.40 (d, 1 H), 5.55 (d, 1 H).
The 1H NMR data correspond to those known from the literature.
13C-NMR: (100 MHz, CDCl3): d141.4, 139.8, 119.5, 115.3,71.1, 70.5, 55.7, 54.4, 46.1, 44.4, 43.0, 40.8, 39.2, 38.4, 37.0,36.3, 36.1, 32.1, 29.3, 29.2, 28.0, 23.1, 21.0, 20.6, 18.8, 16.3,11.6.

Example 5: Preparation of 25-OH vitamin D3(Calcifediol):

In a 0.75 liter of tetrahydrofuran was dissolved 25g of 25-OH 7-dehydrocholesterol of example 4, and the mixture was stirred in a magnetic stirrer under nitrogen atmosphere. To the mixture was added 0.5g of butylated hydroxy toluene (BHT) and 0.5g of sensitizer 5-(3-pyridyl)-2,2'-bithiophene and stirred for a while to obtain clear solution. Falling film apparatus was used to irradiate the solution continuously for 180 minutes at room temperature and the irradiation was carried out using ultraviolet rays from the medium pressure mercury lamp of wavelength 250-310nm. The content of the pre vitamin D derivative of the intended product was determined and monitored by high performance liquid chromatography (HPLC). After, 180-minute irradiation reaction mixture was cooled to 0-5oC for 30 minutes. The solids separated out was filtered as first crop containing 25-OH 7-Dehydrocholesterol (50-60%).
The filtrate was concentrated to 20% of the original volume, cooled to 0-5°C for 24hrs and the separated solids were filtered as second crop that contained 25-OH 7-Dehydrocholesterol (15-20%).
The first and second crops were combined and reused in subsequent batches.
The filtrate was then evaporated under vacuum at 40-45°C to get the crude compound which was then dissolved in 200mL toluene and refluxed for 1-2hr. The solvent was concentrated completely under vacuum to get crude reddish orange sticky mass (resin).
Yield :9.8g
The residue was purified by column chromatography over silica gel with Toluene: methyl ketone 1:99, 2:98, 4:96 & 5: 95 to isolate pure 25-OH Vitamin D3 /Calcifediol crystals which were recrystallized in acetone/water twice, filtered and dried to get highly pure crystals of Calcifediol.
Yield: 3.6g
The Filtrate was further concentrated and kept for 2nd crystallization and the process of crystallization using acetone/water was carried out severally to isolate pure Calcifediol.

Analytical Data:
Appearance: White crystalline solid
HPLC: 99.6% (RT: 13.49)
KF: 4.16 % (limit: 3.8% to 5.0%)
1H NMR (400 MHz, CDCl3): d 6.23 (d, 1H), 6.03(d, 1H), 5.05 (br, 1H), 4.82 (br, 1H), 3.95 (m, 1H),1.21 (s, 6H), 0.93 (d, 3H), 0.54 (s, 3H).
13C-NMR (100 MHz, CDCl3): d 145.1,142.2, 135.0, 122.4, 117.4, 112.4, 71.1, 69.1, 56.5, 56.3, 45.9, 45.8, 44.3, 40.5, 36.3. 36.1,35.1, 31.9, 29.3, 29.1 28.9, 27.6, 23.5, 22.2, 20.8, 18.7, 11.9.

, Claims:1. An improved and cost effective process for synthesis of 7-dehydrocholesterol/25-OH7-dehydrocholesterol from bisnoralcohol comprising;

i. Converting Bisnoralcohol to (20S)-20-tosyloxymethyl-3ß-tertbutyldimethylsilyloxy-pregna-5,7-diene (1a) by the known process;

ii. Reacting (20S)-20-tosyloxymethyl-3ß-tertbutyldimethylsilyloxy-pregna-5,7-diene (1a) with sodium iodide in dry acetone to yield 20(S)-20-Iodomethyl-3ß-tert-butyldimethylsilyloxypregna-5,7-diene(2a);

iii. Reacting 20(S)-20-Iodomethyl-3ß-tert-butyl dimethyl silyl oxypregna-5,7-diene(2a) with ethyl acrylate in presence of zinc and nickel salt at a temperature in the range of 50-60ºC followed by cooling to room temperature and reacting within 0.5hours with a solution of (20S)-20-Iodomethyl-3ß-tert-butyldimethylsilyloxypregna-5,7-diene (2a) at room temperature to yield Ethyl-3ß-tert-Butyldimethylsilyloxy-chola-5,7-diene-24-carboxylate (3a);

iv. Subjecting ice cooled ethyl-3ß-tert-Butyldimethylsilyloxy-chola-5,7-diene-24-carboxylate (3a) to Grignard reaction in THF to yield crude 3ß-tert-Butyldimethylsilyloxy-25-hydroxychola-5,7-diene(4a) followed by recrystallization to obtain pure compound (4a);

v. Deprotecting 3ß-tert-Butyldimethylsilyloxy-25-hydroxychola-5,7-diene(4a) with Tetrabutylammonium fluoride trihydrate to obtain 3ß-25-Dihydroxycholesta-5,7-diene (pro vitamin of 25-Hydroxy vitamin D3).

2. The process as claimed in claim 1, wherein 3ß-25-Dihydroxycholesta-5,7-diene obtained in step (v) is converted to vitamin D3 (calcifediol) by the process comprising;

i. Irradiating 3ß-25-Dihydroxycholesta-5,7-diene (pro vitamin of 25-Hydroxy vitamin D3) in presence of an antioxidant and photosensitizer 5-(3-pyridyl)-2, 2’-bithiophene under medium pressure mercury lamp of wavelength 250-310nm;

ii. Concentrating the irradiated solution of step (i), cooling and crystallizing out the unreacted mass;

iii. Filtering and washing the unreacted mass of step (i) and reusing for irradiation;

iv. Evaporating the filtrate under vacuum to obtain crude product, dissolving the crude product in solvent, refluxing , concentrating under vacuum to obtain reddish orange mass followed by purifying;

v. Repeating the step (iv) of concentration of the filtrate severally to obtain quantitative yield of the pure product.

3. The process as claimed in any one of the claim 1 and 2, wherein the process is carried out in solvent selected from lower alcohols such as methanol, ethanol, isopropyl alcohol; ethers such as DMF, THF, MTBE and the like; ketones such as acetone; esters such as ethyl acetate; alone or combination thereof.

4. The process as claimed in claim 1, wherein the process is carried out in presence of base selected from ethylamine, triethylamine, pyridine, alkali or alkaline earth metal carbonates and bicarbonates and the like alone or combination thereof.