Description:FIELD OF THE INVENTION:
The present invention relates to plant based vitamin D3 (Cholecalciferol) i.e. Green DOT Vitamin D3. The present invention further relates to a commercial process for preparation of plant sourced vitamin D3 with improved yield and purity, wherein the starting material is soyabean oil deodorizer distillate (SODD).

BACKGROUND OF THE INVENTION:
Vitamin D3, known as Cholecalciferol, is a type of Vitamin D i.e. Calciferol. It is a fat‑soluble vitamin and is known for its antirachitic activity. It is needed for the maintenance of normal blood levels of calcium and phosphate that are required for mineralization of bone, muscle contraction, nerve conduction, and general cellular function in all cells of the body. It is also known to play a beneficial role in muscle strength, immunity, cardiovascular diseases, diabetes, pregnancy & lactation, psoriasis, certain cancers, multiple sclerosis.

Vitamin D deficiency is prevalent in as significant as approximately 1 billion people around the world, consisting of all ethnicities, age groups, sex and geographies. [Adapted from source: Am. J. Clin. Nutr. 2008;87(4):1080S–1086S.] In India, Vitamin D deficiency is prevalent in all age groups with most of the studies reporting a prevalence of deficiency among 80%–90% population. [Adapted from source: J Family Med Prim Care. 2018 Mar-Apr;7(2):324–330].

The major source of Vitamin D3 is synthesis in the skin upon exposure to sunlight. Main natural dietary sources of Vitamin D3 are fish and egg yolk, which is considered as non-vegetarian food. Vegetables and grains are poor sources of Vitamin D3.

A New Research Center 2021 Survey indicated that approximately 81% of Indians impose certain limits on their diet, such as not eating non-vegetarian food on particular days, and approximately 39% described themselves as ‘vegetarian’. A Rakuten Insight Survey 2024 indicated that more than 46% of Indians follow a vegetarian or vegan diet. This shows that a significant percentage of Indian population is vegetarian or vegan for various reasons including cultural and religious reasons, and among those who consume non-vegetarian diet, a large number of people do not consume it to the extent that is sufficient to meet the body’s Vitamin D3 requirements.

Currently, Vitamin D3 is derived from sheep’s wool, like merino sheep, and therefore it is of animal origin. In India, as per the norms of Food Safety and Standards Authority of India (FSSAI), such animal-sourced Vitamin D3 is classified as non-vegetarian and products containing it therefore bear a red dot as per FSSAI norms. Such animal-sourced Vitamin D3 may not be therefore suitable for the aforesaid vegetarian and vegan population.

Further, the total number of sheep across the globe has also been declining significantly because of which the starting material for production of Vitamin D3 from animal source is also threatened.

In view of the above, it was a need and necessity to find a sustainable, scalable, alternate, plant-sourced starting material for production of Vitamin D3. The purpose of this current invention i.e. plant-sourced Vitamin D3 is to provide the missing nutritional source of Vitamin D3 to a large number of vegetarian and vegan population as aforesaid.

The present invention uses raw material which is derived from plant source that does not compete with food and is derived from waste by-product strain of soyabean oil refining process. When soyabean oil is refined, the refined oil is used for edible food purpose, and remaining residue which is called as Soya Oil Deodorized Distillate (‘SODD’) is further purified to obtain Vitamin E and Phytosterols. Through bio-transformation process, Phytosterols are converted to Bisnoralchohol. Such Bisnoralchohol as a starting material is the objective of the present invention to produce plant-sourced Vitamin D3.
Having starting material of the present invention sourced from largely cultivated soyabean plant such as SODD and which does not compete with food, makes it sustainable, scalable, and economical. Such a plant-sourced Vitamin D3 can therefore meet needs of a significant number of people who are deficient in Vitamin D3 at almost epidemic level including vegetarian/vegan population in India and globally as well.

SUMMARY OF THE INVENTION
In accordance with the above, the present invention provides a plant based/sourced vitamin D3 and the process for preparation thereof.

Provided herein is the process for preparation of Vitamin D3 (cholesterol) from plant sourced bisnoralcohol. The bisnoraclcohol is obtained from soyabean oil deodorizer distillate (SODD) which is purified to obtain Vitamin E and Phytosterols. The phytosterols isolated are then converted to bisnoralcohol through bio-transformation process known in the art.

The Bisnoralcohol (1) obtained from SODD is further processed to vitamin D3 as follows.

Accordingly, the process for preparation of vitamin D3 from plant sourced bisnoralcohol comprises the steps of
i. Oxidizing bisnorlacohol (1) to Bisnordiene alcohol (2);

ii. Protecting the 20-hydroxyl group of compound (2) to obtain protected compound (3)

iii. Protecting the ketonic group of the compound (3) to obtain the ketal of formula (4)

iv. Reacting compound (4) with Grignard reagent to compound (5)

v. Deprotecting compound (5) to compound (6);

vi. Acylating compound (6) to compound (7) ;

vii. Reducing compound (7) to 7-DHC; and

viii. Irradiating 7-DHC to said vitamin D3

DESCRIPTION OF THE FIGURES
Fig 1: Depicts the FTIR of Vitamin D3 prepared from the plant source of the present invention and standard cholecalciferol.
Fig 2: Depicts (a) the HPLC chromatogram of Blank solution (b) System suitability HPLC Chromatogram of Pre-cholecalciferol, Trans-cholecalciferol and Cholecalciferol (c) HPLC Chromatogram of standard cholecalciferol
Fig 3: Depicts (a) the HPLC chromatogram of Blank solution (b) and (c) Vitamin D3 prepared from the plant source by the present invention.
Fig 4: Depicts the HPLC chromatogram of the residual solvents used in the present process (a) of Blank solution (b) and (c) of the standard solution 01 and 02 respectively.

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 the invention belongs. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances here said event or circumstance occurs and instances where it does not.

Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps.

The present invention meets the long felt need in the health food and nutritional supplementary industry to provide a vegetarian (vegan) vitamin D3 derived exclusively from the plant source. The development of vitamin D3 from the plant source avoids or ameliorates the problems and concerns with vitamin D3 obtained from animal, fish or livestock derived cholesterol.

The vegan vitamin D3 obtained by the process of the present invention is economically viable, meets the pharmacopeia standard, and is competitive to the vitamin D3 sourced from animal or other livestock.

In an embodiment, the present invention relates to plant based vitamin D3 having pharmacopeia compliant HPLC chromatogram (Fig 2 and 3) with green dot status prepared by a process starting from bisnoralcohol obtained from SODD comprising;
i. Oxidizing bisnoralcohol (1) suspended in the solvent with 2,3-Dichloro-5,6-dicyano-p-benzoquinone (DDQ) or p-chloranil to obtain (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one (2);
ii. Reacting (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one (2) of step (i) with acetylating agent selected from p-toluene sulphonyl chloride, Methane sulphonyl chloride, Trifluoromethanesulfonic anhydride to obtain Bisnordiene tosylate or Bisnordiene mesylate or Bisnordiene Triflate (3);
iii. Reacting Bisnordiene tosylate or Bisnordiene mesylate or Bisnordiene Triflate (3) of step (ii) with Ethane1,2-dithiol, 2-mercaptoethanol, ethylene glycol or Propane1,3 diol in presence of trimethyl orthoformate followed by reaction with p-toluene sulphonic acid, methane sulfonic acid or Trifluoromethanesulfonic anhydride to obtain 3-Ketal Bisnordiene Tosylate or 3-Ketal Bisnordiene mesylate or 3-Ketal Bisnordiene triflate (4);
iv. Reacting 3-Ketal Bisnordiene Tosylate or 3-Ketal Bisnordiene mesylate or 3-Ketal Bisnordiene triflate (4) of step (iii) with a Grignard reagent to yield 3- ketal 4,6- Cholestadiene (5);
v. Deketalizing 3- ketal 4,6- Cholestadiene (5), of step (iv) with deketalizing agent to obtain Cholest-4,6-diene-3-one (6);
vi. Acylating Cholest-4,6-diene-3-one (6) with the acylating agent to obtain 3-Acetoxy- 3,5,7 cholesta triene (7);
vii. Reducing 3-Acetoxy- 3,5,7 cholesta triene (7) of step (vi) with a reducing agent to obtain 7 –Dehydrocholesterol (7-DHC); and
viii. Irradiating the mixture 7-DHC of step (vii), optionally with plant sourced cholesterol, butylated hydroxyl toluene and 2% aq. Sodium hydroxide or 2% potassium hydroxide or 2% sodium carbonate preferably with 2% sodium hydroxide dissolved in the solvent using UV lamp to obtain vitamin D3.

DETAILS OF THE PROCESS:
Step (i): Dehydrogenation of Bisnoralcohol (1) obtained from SODD to (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one i.e. Bisnordiene alcohol (2).
To the bisnoralcohol (1) suspended in the solvent selected from C1-C5 alcohol, aromatic hydrocarbon such as Toluene was added p-chloranil at a temperature in the range of 25-35°C. The reaction mass was heated to 60-65°C for a period of about 4 hours till the TLC analysis indicated absence of starting material. The solvent was distilled out and to the residue was charged dichloromethane and washed with 5% KOH solution followed by further washing with water. Evaporating the dichloromethane layer to obtain the crude residue (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one, Bisnordiene alcohol (2).

To the above residue was added heptane and the mixture was stirred for 30-60 minutes at 25-30°C, filtered and washed with heptane and dried the solid.

In an alternate embodiment, to the bisnoralcohol (1) suspended in 1,4 dioxane was added the oxidizing agent 2,3-Dichloro-5,6-dicyano-p-benzoquinone (DDQ) at a temperature of 25oC to 35oC. The reaction mass was heated at the same temperature of 25oC to 35oC for 24-48 hours till TLC indicated absence of the starting material. The solvent 1,4 dioxane was distilled out and to the residue was added dichloromethane and washed with 5% KOH solution. This was followed by washing with water and the dichloromethane layer was evaporated to yield the crude product (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one i.e. Bisnordiene alcohol (2).

To the crude product was added heptane and the mixture was stirred for 30-60 minutes at 25-30°C, filtered and washed with heptane and dried the solid.

Step (ii): Conversion of (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one (2) i.e. Bisnordiene alcohol (2) to obtain Bisnordiene tosylate or bisnordiene mesylate or bisnordiene Triflate (3).
To the Bisnordiene alcohol (2) obtained in step (i) above was added p-toluene sulphonyl chloride (1.7-2.0 mole equivalent) in the presence of base and the solvent at 25-35°C. The reaction mixture was stirred for 12-18 hours till the TLC analysis indicated the absence of starting material. the residue was washed with 1:1 aq. HCl followed by washing with water and finally with 8% sodium bicarbonate solution. The solvent was evaporated to obtain the crude residue Bisnordiene Tosylate (3).

The crude residue was optionally crystallized from methanol or acetone to obtain the pure compound (3).

In an embodiment, Bisnordiene mesylate (3) is obtained by the process described above.

The solvent for the process is selected from C1-C5 alcohols, toluene, halogenated hydrocarbons such as chloroform or dichloromethane, imidazole, ethyl acetate or mixtures thereof.

The base may be selected from organic base such as pyridine, triethylamine, ethylamine and the like.

In an alternate process, to the Bisnordiene alcohol (2) obtained in step (i) above was added the base pyridine and the solvent selected from dioxane or dichloromethane or mixture thereof followed by addition of trifluoromethanesulfonic anhydride. The reaction mass was stirred at a temperature in the range of 25-35°C for 12-18 hours till the TLC analysis indicated the absence of starting material. The residue was washed with 1:1 aq. HCl followed by washing with water and finally with 8% sodium bicarbonate solution. The solvent layer was then evaporated to yield Bisnordiene Triflate (3).

To the crude product was added heptane and the mixture was stirred for 30-60 minutes at 25-30°C, filtered and washed with heptane and dried the solid to obtain pure Bisnordiene Triflate (3”).

Step (iii): Conversion of Bisnordiene tosylate or Bisnordiene mesylate or Bisnordiene Triflate (3) to 3-Ketal Bisnordiene tosylate or 3-Ketal Bisnordiene mesylate or 3-Ketal Bisnordiene triflate (4).
Bisnordiene tosylate (3) obtained in step (ii) above was suspended in suitable solvent along with the carbonyl protecting agent selected from ethane1,2-dithiol to 2-mercapto thiol at room temperature followed by charging of trimethyl orthoformate to obtain the cyclic ether. This was followed by addition of p- toluene sulphonic acid and the reaction mixture was stirred at same temperature for 12-18 hours till the TLC analysis indicated the absence of starting material. The suitable base was added to the mixture to adjust the pH in the range of 8-9 followed by addition of water and finally 10% sodium chloride solution. The dichloromethane layer was evaporated to obtain 3-Ketal Bisnordiene Tosylate (4).
The 3-Ketal Bisnordiene mesylate or 3-Ketal Bisnordiene triflate (4) may be prepared from Bisnordiene mesylate or Bisnordiene Triflate (3) by the process described above.

Step (iv): Conversion of 3-Ketal Bisnordiene Tosylate (4) to 3-Ketal 4,6- Cholestadiene (5).
To the magnesium turnings in presence of THF or methyl-tert butyl ether or 2-Methyl THF or diethyl Ether at 40-50°Cunder nitrogen atmosphere was added drop wise Isopentyl bromide, optionally in presence of 1,2 dibromoethane to initiate the reaction, at same temperature and stirred for about 20minutes. The reaction mass was cooled to 0-10°C. The catalyst cuprous iodide or cuprous bromide and the compound 3-Ketal Bisnordiene Tosylate (4) obtained in step (iii) above dissolved in suitable solvent were added to the mixture and stirred till the TLC indicated the completion of reaction. The reaction mass was cooled to 10°C, quenched the reaction mass with 10%ammonium chloride solution and then extracted in the solvent. The entire solvent layer was evaporated under vacuum to obtain the residue 3-Ketal 4,6- Cholestadiene (5).

The residue was further purified in the solvent selected from methanol, ethyl alcohol, isopropyl alcohol, acetone or toluene.

The residue was further crystallized from said solvent at 25-30°C, chilled to 5-10°C filtered, washed with chilled solvent, filtered and dried to obtain pure product.

The 3-Ketal Bisnordiene mesylate or 3-Ketal Bisnordiene triflate (4) may be converted to 3-Ketal 4,6- Cholestadiene by the process described above.

Step (v): Conversion of 3-Ketal 4,6- Cholestadiene (5) to Cholest-4,6-diene-3-one (6)
To 3-Ketal 4,6- Cholestadiene (5) obtained in step (iv) above was added p-toluene sulfonic acid, methane sulfonic acid, potassium bisulfate or acetic acid and water and the reaction mass was stirred for 3-8 hrs till TLC indicated completion of the reaction. The mixture was washed with water and then extracted with ethyl acetate. The entire ethyl acetate layer was then evaporated under vacuum to obtain the residue Cholest-4,6-diene-3-one (6).
The reaction was carried out in presence of the solvent selected from methanol, ethyl acetate, acetone or toluene.

The crude product may be crystallized from methanol or acetone at 25-30°C, chilled to 5-10°C, filtered, further washed with chilled methanol or acetone filtered and dried to obtain the pure compound.

Step (vi): Conversion of Cholest-4,6-diene-3-one (6) to 3-Acetoxy- 3,5,7 cholesta triene (7).
To Cholest-4,6-diene-3-one (6) obtained in step (v) above was suspended in acylating agents selected from acetic anhydride and acetyl chloride mixture, Trifluoromethanesulfonic anhydride and Trifluoromethanesulfonyl chloride mixture, sulphuric acid and isopropenyl acetate mixture, benzoic anhydride and benzoyl chloride mixture, propionic anhydride and propionyl chloride mixture, Isobutyric anhydride and Isobutyl chloride mixture, Trifluroacetic anhydride and trifluroacetyl chloride at 25-35°C. The reaction mass was stirred for 6-8 hours at 105-110°C till the TLC analysis indicated the absence of the starting material. Cooled the reaction mass to ambient temperature. Quenched the reaction mass in ammonium hydroxide solution followed by water and finally extracted in a solvent selected from dichloromethane or ethyl acetate and finally washed with 10% sodium bicarbonate solution. The solvent layer was evaporated to get the residue.

The residue was optionally crystallized from C1-C5 alcohols, stirred at 25-30°C, chilled to 5-10°C, filtered, washed with said chilled solvent, filtered and dried the product to obtain pure 3-Acetoxy- 3,5,7 cholestatriene(7).

Step (vii): Preparation of 7-DHC from 3-Acetoxy- 3,5,7 cholestatriene(7)
To the solvent selected from lower alcohols such as methanol or ethanol cooled to 5-10°C was added reducing agent selected from lithium borohydride, sodium borohydride, sodium triacetoxyborohydride, optionally in presence of calcium chloride and the reaction mass was stirred for about 30mins at 0-10°C. This was followed by addition of 3-Acetoxy- 3,5,7cholestatriene(7) obtained in step (vi). Toluene was added and the mixture was stirred at 0-10°C till TLC indicated completion of the reaction. After completion of the reaction, the reaction mass was quenched in ice cold water and extracted in suitable solvent, washed with 2N HCl solution to yield crude product 7-DHC.

In an alternate process, to 3-Acetoxy- 3,5,7 cholestatriene(7) obtained in step (vi) dissolved in a mixture of methanol and toluene was added the reducing agent selected from lithium borohydride or sodium borohydride at 25-30°C under pressure. The reaction completion was monitored on TLC. After completion of the reaction, the reaction mass was filtered, washed with water followed by washing with 2NHCl solution to yield crude 7-DHC.

Step (viii): Irradiation of 7-DHC
The mixture of 7-DHC obtained in step (vii), antioxidant, Butylated hydroxyl toluene and 2% aq. NaOH or 2% aq KOH or 2% aq sodium carbonate preferably 2% aq NaOH was dissolved in ethanol at 75-85°C and the mixture was irradiated by UV lamp at 80-85°C for 180 minutes preferably. The reaction mass was cooled to 25-30°C. The solids separated out were filtered as first crop which contains 7-Dehydrocholesterol.

The filtrate was concentrated to 20% of the original volume, cooled to 20-25°C and the separated solids were filtered as second crop that contains 7-Dehydrocholesterol.

The filtrate was again concentrated under vacuum, methyl tert butyl ether was added, washed with 1:1 ethanol water and distilled under vacuum. Acetone was added to the residue, cooled to 5- 10°C and the separated solids were filtered as the third crop that contains 7-Dehydrocholesterol. All these first, second and third crops were combined and reused in subsequent batches.
The filtrate was then evaporated under vacuum at 40-45°C and the crude Vitamin D3 (resin) thus obtained was purified by either of the following methods:
a) Converting the resin to its ester like Acetate or Propionate or Butyrate or Valerate or 2-Nitrobenzoate or 4-Nitrobenzoate more preferably Butyrate, crystallized and finally saponified with a base such as Sodium Hydroxide or Potassium hydroxide or Sodium carbonate or Potassium carbonate Sodium methoxide or Sodium Ethoxide or Potassium butoxide or Lithium Aluminium Hydride and finally crystallized from acetone or methyl formate.
OR
b) Purifying the Crude resin by column chromatography using Silica gel or Alumina or Alumina with 2-10% water and using Toluene: 2-Butanone as an eluent or Toluene: Acetone as an eluent.

In an optional embodiment, the irradiation process of conversion of 7-DHC to plant based vitamin D3 may be carried out in presence of a photosensitizer such as 5-(3-Pyridyl)-2,2’-bithiophene.

In another embodiment, the irradiation of 7-DHC may be carried out optionally with plant sourced cholesterol.

In an embodiment, the vitamin D3 prepared by the process of the present invention from plant sourced bisnoralcohol meets the Indian, European and USP pharmacopeia as well as standard cholecalciferol as shown in the FTIR spectrum and HPLC chromatogram assay in Figs 1 to Fig 3.

In an embodiment, the plant based vitamin D3 of 40MIU of the present invention that meets the pharmacopeia standard provides the nutritional source to a large number of vegetarian and vegan population.

Other features and embodiments of the invention will become apparent by the following examples which are given for illustration of the invention rather than limiting its intended scope. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art.

Examples:
Example 1: Preparation of (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one i.e. Bisnordiene alcohol (2) from Bisnoralcohol.
General Procedure:
Bisnoralcohol (303 millimole) charged in methanol was reacted with p-chloranil (1.1-1.59 mole equivalent) preferably 1.27 mole equivalent at 40-65oC, preferably at 60-65oC for 4-18 hours preferably 4 hrs to synthesise Bisnordiene alcohol (2).

Example 1(a): 100gms (303 millimole) of Bisnoralcohol was suspended in 300 ml of methanol along with 95gms (386 millimole) of para- chloranil at 25-35°C. The reaction mass was heated to 60-65°C for 4 hours till the TLC analysis indicated absence of starting material, Distilled out methanol, charged Dichloromethane (1000 ml) and washed with 1000 ml 5% KOH solution followed by washing with 500ml water and Dichloromethane layer was evaporated to obtain the residue Bisnordiene alcohol (2).
Yield: 90gm; Percentage Yield: 90%
HPLC purity: 85%.

Purification:
To the above residue Bisnordiene alcohol (2) was added 1.0lit heptane, stirred for 30-60 min at 25-30°C. The mixture was filtered, washed with heptane and dried the solid.
Yield: 87 gm
Purity: 96%

Example 1(b): 100 gms (303 millimole) of Bisnoralcohol was suspended in 300 ml toluene along with 100 gms(406 millimole) of para- chloranil at 25-35°C. The reaction mass was heated to 60-65°C for 4 hours till the TLC analysis indicated absence of starting material. Distilled out toluene, charged Dichloromethane (1000 ml), washed with 1000 ml 5% KOH solution followed by washing with 500 ml water and Dichloromethane layer evaporated to obtain the residue Bisnordiene alcohol (2).
Yield: 92gm; Percentage Yield: 92.9%
HPLC purity: 84%

Example 1(c): 100 gms (303 millimole) of Bisnoralcohol was suspended in 300 ml of Isopropyl alcohol along with 100 gms(406 millimole) of para- chloranil at 25-35°C. The reaction mass was heated to 40-45°C for 12hours till the TLC analysis indicated absence of starting material. Distilled out methanol and charged dichloromethane (1000 ml). Washed with 1000 ml 5% NaOH solution followed by washing with 500ml water and Dichloromethane layer was evaporated to obtain the residue Bisnordiene alcohol (2).
Yield: 95gm; Percentage Yield: 95%
HPLC purity: 84%

Example 1(d): 100 gms (303 millimole) of Bisnoralcohol was suspended in 300 ml of tertiary butanol along with 100 gms(406 millimole) of para- chloranil at 25-35°C. The reaction mass was heated to 60-65°C for 4 hours till the TLC analysis indicated absence of starting material. Distilled out t- butanol and charged methylene dichloride (1000 ml). Washed with 1000 ml 5% KOH solution followed by washing with 500ml water and Dichloromethane layer was evaporated to obtain the residue Bisnordiene alcohol (2) (90gm).

Yield: 90gm; Percentage Yield: 90%
HPLC purity: 85%

The residue was further crystallized in methanol 100 ml stir at 25-30°C chill to 5-10°C filter it wash by chill methanol 25 ml, dry the filtered product to obtain the solid Bisnordiene alcohol (2).

Yield: 50gm; Percentage Yield 50%
HPLC purity: 96%

Example 1(e): 100gms (303 millimole) of Bisnoralcohol was suspended in 300 ml of 1,4Dioxane along with 95gms (418 millimole) 2,3-Dichloro-5,6-dicyano-p-benzoquinone (DDQ) at 25-35°C. The reaction mass was heated to 25-35°C for 24-48 hours till the TLC analysis indicated absence of starting material, Distilled out 1,4 Dioxane, charged Dichloromethane (1000 ml) and washed with 1000 ml 5% KOH solution followed by washing with 500ml water and Dichloromethane layer was evaporated to obtain the residue Bisnordiene alcohol (2).
Yield: 90gm; Percentage Yield: 90%
HPLC purity: 95%.

Example 2: Preparation of Bisnordiene Tosylate or Binordiene mesylate Binordiene Triflate (3)
General Procedure:
Bisnordienealcohol (2) dissolved in Dichloromethane was treated with p-toluene sulphonyl chloride (1.7-2.0 mole equivalent) preferably 1.7 mole equivalent in presence of pyridine (4-20 mole equivalent) preferably 4.1 mole equivalent at 10-40oC, preferably at 30-40oC for 8-16 hrs preferably 12 hrs to synthesise Bisnordiene Tosylate (3).

Example 2(a): 100 gms (304.8 millimole) of Bisnordiene alcohol (2) was suspended in 500 gm (6944 millimole) of pyridine and 500 ml Dichloromethane along with 120 gms (630 millimole) of para toluene sulphonyl chloride at 25-35°C. The reaction mass was stirred for 12-18 hours till the TLC analysis indicated the absence of starting material. Washed the reaction mass with 500ml 1:1 HCl solution followed by 500ml water and finally with 500 ml of 8% sodium bicarbonate solution. The dichloromethane layer was evaporated to get the residue Bisnordiene Tosylate (3).
Yield: 134gm; Percentage Yield: 93%
HPLC purity: 92%

Example 2(b): 100gms (304.8milimole) of Bisnordienealcohol (2) was suspended in 100gm (1388 millimole) of pyridine and 500 ml Dichloromethane along with 120gms (630 millimole) of para toluene sulphonyl chloride at 25-35°C. The reaction mass stirred for 12-18 hours till the TLC analysis indicated the absence of starting material. Washed the mixture with 500ml 1:1 HCl solution followed by 500ml water and finally with 500 ml of 8% sodium bicarbonate solution. The dichloromethane layer evaporated to get residue Bisnordiene Tosylate (3).
Yield: 138gm; Percentage Yield: 94.5 %
HPLC purity: 91%
Purification:
To the residue Bisnordiene alcohol (2) obtained above was added 1.0 lit heptane, stirred for 30-60 min 25-30°C, filtered, washed with heptane and dried to obtain the solid.
Yield: 135 gm
Purity: 96%

Example 2(c): 1gm(3.04milimole) of Bisnordienealcohol (2) was suspended in 5gm (69.44 millimole) of pyridine and 50ml chloroform along with 1.8gm (9.46 millimole) of para toluene sulphonyl chloride at 25-35°C. The reaction mass was stirred for 12-18 hours till the TLC analysis indicated the absence of starting material. Washed the mixture with 50ml 1:1 HCl solution followed by 50ml water and finally with 50 ml of 8% sodium bicarbonate solution. The dichloromethane layer was evaporated to get residue Bisnordiene Tosylate (3).
Yield: 1.4gm; Percentage Yield 97%.
HPLC purity: 86%
The residue was then crystalized from methanol.
The residue was further crystallized in methanol 5 ml stirred at 25-30°C, chilled to 5-10°C filtered and washed with chilled methanol (2 ml), dried the filtered product to obtain the solid
Yield: 1.0gm; Percentage Yield: 69%
HPLC purity: 96%

Example 2(d): 1gm (3.04milimole) of Bisnordienealcohol (2) was suspended in 5gm (69.44 millimole) of pyridine along with 1.5 gms (7.88 millimole) of para toluene sulphonyl chloride at 25-35°C. The reaction mass stirred for 12-18 hours till the TLC analysis indicated the absence of starting material. Washed the mixture with 50ml 1:1 HCl solution followed by 50ml water and finally with 50 ml of 8% sodium bicarbonate solution. The dichloromethane layer evaporated to get residue Bisnordiene Tosylate (3).
Yield: 1.4gm; Percentage Yield: 97%.
HPLC purity: 85%
The residue was crystallized from methanol.
The residue was further crystallized in methanol 5 ml stirred at 25-30°C, chilled to 5-10°C filtered and washed with chilled methanol (2 ml), dried the filtered product to obtain the solid.
Yield: 1.1 gm; Percentage Yield: 76%
HPLC purity: 92%

Example 2 (e): 1gm (3.04milimole) of Bisnordienealcohol (2) was suspended in 5gm (49.41 millimole) of Triethyl amine and 50ml Dichloromethane along with 1.1 gm (5.77 millimole) of para toluene sulphonyl chloride at 25-35°C. The reaction mass stirred for 12-18 hours till the TLC analysis indicated the absence of starting material. Washed the mixture with 50ml 1:1 HCl solution followed by 50 ml water and finally with 50ml of 8% sodium bicarbonate solution. The dichloromethane layer evaporated to get residue Bisnordiene Tosylate (3).
Yield: 1.4gm; Percentage Yield 97%
HPLC purity: 85%
The residue was crystallized from acetone
The residue was further crystallized in acetone 5 ml stirred at 25-30°C, chilled to 5-10°C filtered and washed with chilled acetone (2 ml), dried the filtered product to obtain the solid.
Yield: 1.0 gm; Percentage Yield 69%
HPLC purity: 93%

Example 2(f): 1gm (3.04milimole) of Bisnordienealcohol (2) was suspended in 5gm (73.44 millimole) of Imidazole and 50ml Ethyl Acetate along with 1.1 gms(5.77 millimole) of para toluene sulphonyl chloride at 25-35°C. The reaction mass stirred for 12-18 hours till the TLC analysis indicated the absence of starting material wash with 50ml 1:1 HCl solution followed by 50 ml water and finally with 50 ml of 8% sodium bicarbonate solution. The ethyl acetate layer evaporated to get residue Bisnordiene Tosylate (3).
Yield: 1.4gm; Percentage Yield 97%
HPLC purity: 83%
The residue was crystallized from methanol.
The residue was further crystallized in methanol 5 ml stirred at 25-30°C, chilled to 5-10°C filtered and washed with chilled methanol (2 ml), dried the filtered product to obtain the solid.
Yield: 0.8 gm; Percentage Yield 55%
HPLC purity: 91%

Example 2(g): 1gm (3.04milimole) of Bisnordienealcohol (2) was suspended in 5gm (38.68 millimole) of Diisopropylethylamine and 50 ml Dichloromethane along with 1.1 gms(5.77 millimole) of para toluene sulphonyl chloride at 25-35°C. The reaction mass stirred for 12-18 hours till the TLC analysis indicated the absence of starting material. Washed the mixture with 50ml 1:1 HCL solution followed by 50ml water and finally with 50ml of 8% sodium bicarbonate solution. The dichloromethane layer was evaporated to get residue Bisnordiene Tosylate (3).
Yield: 1.2 gm; Percentage Yield: 83%
HPLC purity: 84%

Example 2(h): 1gm (3.04milimole) of Bisnordienealcohol (2) was suspended in 5gm (69.44 millimole) of pyridine along with 1.5 gms(7.88 millimole) of para toluene sulphonyl chloride at 25-35°C. The reaction mass stirred for 12-18 hours till the TLC analysis indicated the absence of starting material. Add dichloromethane 50 ml. Washed with 50ml 1:1 HCl solution followed by 50 ml water and finally with. 50 ml of 8% sodium bicarbonate solution. The dichloromethane layer evaporated to get residue Bisnordiene Tosylate (3).
Yield: 1.4gm; Percentage Yield: 97%
HPLC purity: 85%

Example 2(i): 1gm (3.04milimole) of Bisnordienealcohol (2) was suspended in 5gm (69.44 millimole) of pyridine along with 1.5 gms(13.09 millimole) of Methane sulphonyl chloride at 25-35°C. The reaction mass stirred for 12-18 hours till the TLC analysis indicated the absence of starting material. Washed the mixture with 50ml 1:1 HCl solution followed by 50 ml water and finally with 50 ml of 8% sodium bicarbonate solution. The dichloromethane layer evaporated to get residue Bisnordiene mesylate (3).
Yield: 1gm; Percentage Yield: 69 %
HPLC purity: 83%

Example 2(j): 100 gms (304.8 millimole) of Bisnordiene alcohol (2) was suspended in 500 gm (6944 millimole) of pyridine and 500 ml Dichloromethane along with 120 gms (425 millimole) of Trifluoromethanesulfonic anhydride at 25-35°C. The reaction mass was stirred for 12-18 hours till the TLC analysis indicated the absence of starting material. Washed the residue with 500ml 1:1 HCl solution followed by 500ml water and finally with 500 ml of 8% sodium bicarbonate solution. The dichloromethane layer was evaporated to get the residue Bisnordiene Triflate (3).
Yield: 140gm; Percentage Yield: 96%
HPLC purity: 95%

Example 2(k): 100 gms (304.8 millimole) of Bisnordiene alcohol (2) was suspended in 500 gm (6944 millimole) of pyridine and 500 ml Dioxane along with 120 gms (425 millimole) of Trifluoromethanesulfonic anhydride at 25-35°C. The reaction mass was stirred for 12-18 hours till the TLC analysis indicated the absence of starting material. Washed the residue with 500ml 1:1 HCl solution followed by 500ml water and finally with 500 ml of 8% sodium bicarbonate solution. The dichloromethane layer was evaporated to get the residue Bisnordiene Triflate (3).

Yield: 138gm; Percentage Yield: 93%
HPLC purity: 96%

Example 3: Preparation of (2S)-2-(9a,11a-dimethyl-1,2,3,3a,3b,8,9,9a,9b,10,11,11a-dodecahydrospiro[cyclopenta[a]phenanthrene-7,2'-[1,3]dioxolan]-1-yl)propyl 4-methyl benzene-1-sulfonate i.e. 3-Ketal Bisnordiene Tosylate or 3-Ketal Bisnordiene Mesylate or 3-Ketal Bisnordiene Triflate (4)
General Procedure:
Bisnordiene Tosylate (3) of example 2 in MDC was treated with ethylene glycol (7.82 to 15.63 mole equivalence), preferably 15.53 mole equivalence in presence of trimethyl orthoformate (2.28 to 9.14 mole equivalence) preferably 6.88 mole equivalence, in presence of p- toluene sulphonic acid(0.05 to 0.1 mole equivalence) preferably 0.05 mole equivalence at 25 to 120oC preferably at 25-32oC to synthesise 3-Ketal Bisnordiene Tosylate (4).

Example 3(a):100 gms (273.85 millimole) of Bisnordiene Tosylate (3) was suspended in 500 ml dichloromethane along with 200gm (3222 millimole) of ethylene glycol 25-35°C and charged 200 gm (1884 millimole) of trimethyl orthoformate. Added 2.0gm (10.5 millimole) p- toluene sulphonic acid the reaction mass stirred for 12-18 hours till the TLC analysis indicated the absence of starting material. Added Triethylamine to adjust pH 8-9 followed by addition of 100 ml water and finally 100 ml of 10% sodium chloride solution. The dichloromethane layer evaporated to get residue 3-Ketal Bisnordiene Tosylate (4).
Yield: 106gm; Percentage Yield: 97%
HPLC purity: 87%

Example 3(b): 100 gms (273.85millimole) of Bisnordiene Tosylate (3) was suspended 500 ml toluene along with 200gm (3222 millimole) of ethylene glycol at 25-35°C. Then charged 2.0 gm of para toluene sulphonic acid (10.5 millimole). The reaction mass stirred for 18-24 hours at reflux with Dean stark apparatus till the TLC analysis indicated the absence of starting material. Added triethylamine to adjust pH to 8-9 followed by addition of 10 ml water and finally 100 ml of 10% sodium chloride solution. The Toluene layer evaporated to get residue 3-Ketal Bisnordiene Tosylate (4).
Yield: 95gm; Percentage Yield: 87%
HPLC purity: 85%

Example 3(c): 1gm (2.73 millimole) of Bisnordiene Tosylate (3) was suspended in 20ml Dichloromethane along with 2gm(32.22 millimole) of Ethylene Glycol at reflux temperature. Charged 2 gm(18.84 millimole) of trimethyl orthoformate. Added 0.02gm of para toluene sulphonic acid the reaction mass stirred for 6-12 hours till the TLC analysis indicated the absence of starting material. Added triethylamine to adjust pH to 8-9 followed by addition of 10 ml water and finally with 10 ml of 10% sodium chloride solution. The dichloromethane layer evaporated to get residue 3-Ketal Bisnordiene Tosylate (4).
Yield: 1.0gm; Percentage Yield: 91%
HPLC purity: 90%

Example 3(d): 1 gm (2.73 millimole) of Bisnordiene Tosylate (3) was suspended in 20 ml Dichloromethane along with 2gm (26.28 millimole) of Propane1,3 diol 25-35°C. Charged 2 gm (18.84 millimole) of trimethyl orthoformate. The reaction mass stirred for 12-18 hours till the TLC analysis indicated the absence of starting material. Added triethylamine to adjust pH to alkaline followed by 10 ml water and finally with 10 ml of 10% sodium chloride solution. The dichloromethane layer evaporated to get residue 3-Ketal Bisnordiene Tosylate (4).
Yield: 0.9 gm; Percentage Yield: 82%
HPLC purity: 92%

Example 3(f): 1 gm (2.07 millimole) of Bisnordiene Tosylate (3) was suspended in 20 ml Toluene along with 1gm (16.1 millimole) of Ethylene Glycol at 25-35°C. Charged 0.5 gm(4.71 millimole) of trimethyl orthoformate. The reaction mass stirred for 6-18 hours till the TLC analysis indicated the absence of starting material. Added triethylamine to adjust pH 8-9 followed by addition of 10 ml water and finally 10 ml of 10% sodium chloride solution. The Toluene layer evaporated to get residue 3-Ketal Bisnordiene Tosylate (4).
Yield: 1.03gm; Percentage Yield: 94%.
HPLC purity:.80%
The residue was purified from toluene.
The residue was further crystallized in toluene 5 ml stir at 25-30°C chill to 5-10°C filter it wash by chill toluene 2 ml, dry the filtered product
Yield: 0.8gm; Percentage Yield 73%
HPLC purity: 95%

Example 3(g): 1 gm (2.73 millimole) of Bisnordiene Tosylate (3) was suspended in 20 ml Toluene along with 2 gm(32.22 millimole) of Ethylene Glycol at reflux temperature. Charged 2 gm(18.84 millimole) of trimethyl orthoformate. Added 0.02 gm of sulphuric acid. The reaction mass was stirred for 6-12 hours till the TLC analysis indicated the absence of starting material. Added triethylamine to adjust pH 8-9 followed by 10 ml water and finally 10 ml of 10% sodium chloride solution. The toluene layer was evaporated to get residue 3-Ketal Bisnordiene Tosylate (4).
Yield: 1.0gm; Percentage Yield: 91 %
HPLC purity: 90%

Example 3(h): 1 gm (2.73 millimole) of Bisnordiene Tosylate (3) was suspended in 20 ml heptane along with 2 gm(32.22 millimole) of Ethylene Glycol at reflux temperature. Charged 2 gm(13.49 millimole) of Triethyl ortho formate. Added 0.02 gm of sulphuric acid the reaction mass stirred for 6-12 hours till the TLC analysis indicated the absence of starting material. Added pyridine to adjust pH 8-9 followed by addition of 10ml water and finally with 10 ml of 10% sodium chloride solution. The Hepatane layer evaporated to get residue 3-Ketal Bisnordiene Tosylate (4).
Yield: 0.8gm; Percentage Yield: 73%
HPLC purity: 92%
Example 3(i): 1 gm (2.07 millimole) of Bisnordiene Tosylate (3) was suspended 20 ml Toluene along with 1 gm (16.1 millimole) of Ethylene Glycol 25-35°C. Charged 0.5 gm(4.71 millimole) of trimethyl orthoformate,add 0.1 gm Para toluene sulphonic acid , The reaction mass stirred for 6-18 hours till the TLC analysis indicated the absence of starting material. Added triethylamine to adjust pH to alkaline followed by addition of 10 ml water and finally with 10 ml of 10% sodium chloride solution. The Toluene layer evaporated to get residue 3-Ketal Bisnordiene Tosylate (4).
Yield: 1.06gm; Percentage Yield: 95%
HPLC purity: 84%
The residue was further purified using methanol
The residue was further crystallized in methanol 5 ml stir at 25-30°C chill to 5-10°C filter it wash by chill methanol 2 ml, dry the filtered product
Yield: 1gm; Percentage Yield: 91 %
HPLC purity: 93%

Example 3(j):
100 gms (273.85 millimole) of Bisnordiene Tosylate (3) was suspended in 500 ml dichloromethane along with 200gm (2123 millimole) of Ethane1,2-dithiol 25-35°C and charged 200 gm (1884 millimole) of trimethyl orthoformate. Added 2.0gm (10.5 millimole) p- toluene sulphonic acid the reaction mass stirred for 12-18 hours till the TLC analysis indicated the absence of starting material. Added Triethylamine to adjust pH 8-9 followed by addition of 100 ml water and finally 100 ml of 10% sodium chloride solution. The dichloromethane layer evaporated to get residue 3- Ketal Bisnordiene Tosylate (4).

Yield: 115gm; Percentage Yield: 97%
HPLC purity: 98%

Example 3(k):
100 gms (273.85 millimole) of Bisnordiene Tosylate (3) was suspended in 500 ml dichloromethane along with 200gm (2560 millimole) of 2-mecapto ethanol 25-35°C and charged 200 gm (1884 millimole) of trimethyl orthoformate. Added 2.0gm (10.5 millimole) p- toluene sulphonic acid the reaction mass stirred for 12-18 hours till the TLC analysis indicated the absence of starting material. Added Triethylamine to adjust pH 8-9 followed by addition of 100 ml water and finally 100 ml of 10% sodium chloride solution. The dichloromethane layer evaporated to get residue 3-Ketal Bisnordiene Tosylate (4).

Yield: 115gm; Percentage Yield: 97%
HPLC purity: 98%
The compound Bisnordiene Tosylate (3’) can be converted to 3-Ketal Bisnordiene mesylate (4’) by the process illustrated above.

Example 4: Preparation of 3-Ketal 4,6- Cholestadiene (5)
General procedure:
3-Ketal Bisnoralcohol Tosylate (4) was treated with Isopentyl Magnesium Bromide (2 to 8 mole equivalence) preferably in 4 mole equivalence in THF at 0-25oC preferably at 15oC with cuprous (1) catalyst about 0.0097 mole equivalence to yield 3-Ketal 4,6- Cholestadiene (5)

Example 4(a): 100gms (4166 millimole) of Magnesium turnings was suspended in 500ml of Dry Tetrahydrofuran at 40-50°Cunder nitrogen atmosphere. 1.0gm (6.62 millimole) of isopentyl bromide was added at 40-50°C drop wise to initiate the reaction with help of 1,2 dibromoethane. Once the vigorous effervescence started was added slowly 200 gm(1324 millimole) of Isopentyl bromide and reaction mass was stirred for 60 minutes. The reaction mass was cooled to 0-10°C. Catalyst cuprous iodide 5.0 gm(26.25 millimole) and 100 gm(190.11 millimole) of the compound 3-Ketal Bisnordiene Tosylate (4) obtained in example 3 dissolved in 300 ml tetrahydrofuran were added simultaneously and stirred till the TLC indicated the completion of reaction. The reaction mass was cooled to 10°C, quenched the reaction mass with 500ml of 10% ammonium chloride solution and then extracted in 500 ml ethyl acetate. The entire ethyl acetate layer was then evaporated under vacuum to obtain the residue 3-Ketal 4,6- Cholestadiene (5).
Yield: 65.0gm; Percentage Yield: 81%
HPLC purity: 85%
Example 4(b): 50 gms (2083 millimole) of magnesium turnings was suspended in 500ml of dry tetrahydrofuran at 50-60°C under nitrogen atmosphere. 1gm ( 6.62 millimole) of isopentyl bromide was added at 40-50°C drop wise to initiate the reaction. Once the vigorous effervescence started slowly added 100 gm(662 millimole) of Isopentyl bromide and reaction mass was stirred for 60 minutes. The reaction mass was then cooled to 0-10°C. Catalyst cuprous bromide 1.0 gm(34.85milimole) and 100 gm(190.11 millimole) of the compound 3-Ketal Bisnordiene Tosylate (4) dissolved in 150ml Tetrahydrofuran were added simultaneously and the mixture was stirred till the TLC indicated the completion of reaction. The reaction mass was cooled to 10°C, quenched the reaction mass with 500 ml of 10%ammonium chloride solution and then extracted in 500ml ethyl acetate. The entire ethyl acetate layer was then evaporated under vacuum to obtain the residue 3-Ketal 4,6- Cholestadiene (5).
Yield: 75 gm; Percentage Yield: 93%
HPLC purity: 80%
The residue was further yield purified in acetone.
The residue was further crystallized in methanol 100 ml stir at 25-30°C chill to 5-10°C filter it wash by chill Acetone 25 ml, dry the filtered product
Yield: 25gm; Percentage Yield: 31%
HPLC purity: 92%

Example 4(c): 5.0 gms (208.4 millimole) of magnesium turnings was suspended in 50ml of methyl-tert butyl ether at 40-50°Cunder nitrogen atmosphere. 1gm(6.62 millimole) of isopentyl bromide was added at 40-50°C drop wise to initiate the reaction with help of 1,2 dibromoethane. Once the vigorous effervescence started slowly added 15gm (97.37 millimole) of Isopentyl bromide and reaction mass was stirred for 60 minutes. The reaction mass was cooled to 0-10°C. The catalyst cuprous bromide dimethyl sulfide 0.1 gm(0.486milimole) and 10 gm(19.0milimole) of the compound 3-Ketal Bisnordiene Tosylate (4) dissolved in 50 ml methyl-tert Butyl ether were added simultaneously and the mixture was stirred till the TLC indicated the completion of reaction. The reaction mass was cooled to 10°C. Quenched the reaction mass with 10% ammonium chloride solution in 50 ml water and then extracted in Methyl-tert butyl Ether 50 ml. The entire organic layer was then evaporated under vacuum to obtain the residue 3-Ketal 4,6- Cholestadiene (5).
Yield: 3.2gm; Percentage Yield: 80%
HPLC purity: 85%
The residue was crystalized using Isopropyl alcohol.
The residue was further crystallized in isopropyl alcohol 10 ml stir at 25-30°C chill to 5-10°C filter it wash by chill Isopropyl alcohol 5 ml, dry the filtered product
Yield: 1.5 gm ; Percentage Yield: 37 %
HPLC purity: 91%

Example 4(d): 5.0 gms (208.4 millimole) of magnesium turnings was suspended in 50ml of dry Diethyl Ether at 40-50°Cunder nitrogen atmosphere. 1gm(6.62 millimole) of isopentyl bromide was added at 28-32°C drop wise to initiate the reaction with help of 1,2 dibromoethane. Once the vigorous effervescence started slowly added 15 gm(97.37 millimole) of Isopentyl bromide and reaction mass was stirred for 60 minutes. The reaction mass was then cooled to 0-10°C. The catalyst cuprous bromide dimethyl sulfide.1 gm(0.486milimole) and10 gm(190 millimole) of the compound 3-Ketal Bisnordiene Tosylate (4) dissolved in 50ml diethyl ether were added simultaneously and stirred till the TLC indicated the completion of reaction. The reaction mass was cooled to 10°C. Quenched the reaction mass with with 10% ammonium chloride solution in 50 ml water and then extracted in Diethyl Ether 50 ml, The organic layer was then evaporated under vacuum to obtain the residue 3-Ketal 4,6- Cholestadiene (5).
Yield: 3.0 gm; Percentage Yield: 75%
HPLC purity: 87%
The residue was crystalized ethyl alcohol.
The residue was further crystallized in ethanol 15 ml stir at 25-30°C chill to 5-10°C filter it wash by chill ethanol 5 ml, dry the filtered product
Yield: 2.0 gm; Percentage Yield: 50%
HPLC purity: 92%

Example 4(e): 5.0 gms (208 millimole) of magnesium turnings was suspended in 50ml of 2- Methyl Tetra hydrofuran at 55-60°Cunder nitrogen atmosphere. 1 gm(6.62 millimole) of isopentyl bromide was added at 55-60°C drop wise to initiate the reaction with help of 1,2 dibromoethane. Once the vigorous effervescence started slowly added 15gm(97.37 millimole) of Isopentyl bromide and reaction mass was stirred for 60 minute. The reaction mass was cooled to 0-10°C. The catalyst cuprous bromide dimethyl sulfide 0.1 gm(0.486milimole) and 10 gm(190 millimole) of the compound 3-Ketal Bisnordiene Tosylate (4) was dissolved in 50 ml diethyl ether were added simultaneously and stirred till the TLC indicated the completion of reaction. The reaction mass was cooled to 10°C. Quenched the reaction mass with 10% ammonium chloride solution in 50 ml water and then extracted in Diethyl Ether 50 ml, The organic layer was then evaporated under vacuum to obtain the residue 3-Ketal 4,6- Cholestadiene (5).
Yield: 3.0 gm; Percentage Yield 75%
The residue was crystalized using acetone
The residue was further crystallized in acetone 10 ml stir at 25-30°C chill to 5-10°C filter it wash by chill acetone 5 ml, dry the filtered product.
Yield: 1.0 gm; Percentage Yield: 25%
HPLC purity: 87%

Example 4(f): 5.0 gms (208.4 millimole) of magnesium turnings was suspended in 50ml of Tetrahydrofuran at 40-50°Cunder nitrogen atmosphere. 1 gm(6.62 millimole) of isopentyl bromide was added at 40-50°C drop wise to initiate the reaction with help of 1,2 dibromoethane. Once the vigorous effervescence started slowly added 15gm(97.37 millimole) of Isopentyl bromide and reaction mass was stirred for 60 minutes. The reaction mass was cooled to 0-10°C. The catalyst cuprous chloride 0.1 gm(1.01milimole) and 10 gm (19 milimole) of the compound-Ketal Bisnordiene Tosylate (4) dissolved in 50 ml tetrahydrofuran were added simultaneously and stirred the mixture till the TLC indicated the completion of reaction. The reaction mass was cooled to 10°C. Quenched the reaction mass with 10% ammonium chloride solution in 50 ml water and then extracted in Methyl-tert butyl Ether.50 ml. The entire organic layer was then evaporated under vacuum to obtain the residue 3-Ketal 4,6- Cholestadiene (5).
Yield: 3.0gm; Percentage Yield: 75%
HPLC purity: 85%
The residue was crystalized using methanol
The residue was further crystallized in methanol 10 ml stir at 25-30°C chill to 5-10°C filter it wash by chill methanol 5 ml, dry the filtered product
Yield: 1.3gm; Percentage Yield: 32%
HPLC purity: 91%

Example 4(g): 5.0 gms (208.4 millimole) of magnesium turnings was suspended in 50ml of Tetrahydrofuran at 40-50°Cunder nitrogen atmosphere. 1 gm(6.62 millimole) of isopentyl bromide was added at 40-50°C drop wise to initiate the reaction with help of 1,2 dibromoethane. Once the vigorous effervescence started slowly added 15gm(97.37 millimole) of Isopentyl bromide and reaction mass was stirred for 60 minutes. The reaction mass was cooled to 0-10°C. The catalyst cuprous iodide 0.1gm and 10 gm(19 milimole) of the compound 3-Ketal Bisnordiene Tosylate (4) dissolved in 50 ml tetrahydrofuran was added simultaneously and the mixture was stirred till the TLC indicated the completion of reaction. The reaction mass was cooled to 10°C. Quenched the reaction mass with 10% ammonium chloride solution in 50 ml water and then extracted in Toluene 50 ml . The entire organic layer was then evaporated under vacuum to obtain the residue 3-Ketal 4,6- Cholestadiene (5).
Yield: 3.0gm; Percentage Yield: 83%
HPLC purity: 86%
The residue was crystalized using alcohol.
Yield 1.1 gm; Percentage Yield: 30%
HPLC purity: 90%
Example 4(h): To 10 gm(19.0milimole) of the compound 3-Ketal Bisnordiene Tosylate (4) obtained in example 3 dissolved in 50 ml tetrahydrofuran was added catalyst cuprous iodide 0.1gm followed by simultaneous addition of 2.0 mole equivalent of isopentyl magnesium bromide solution in 0.5M THF, stirred till the TLC indicated the completion of reaction. The reaction mass was cooled to 10°C. Quenched the reaction mass with 10% ammonium chloride solution in 50 ml water and then extracted in Toluene 50 ml. The entire organic layer was then evaporated under vacuum to obtain the residue 3-Ketal 4,6- Cholestadiene (5).
Yield: 3.0gm; Percentage Yield: 75%
HPLC purity: 85%
The residue was crystalized using Toluene
The residue was further crystallized in toluene 10 ml stir at 25-30°C chill to 5-10°C filter it wash by chill methanol 5 ml, dry the filtered product
Yield: 1.1 gm; Percentage Yield: 13%
HPLC purity: 90%
The compound 3-Ketal Bisnordiene mesylate (4) or 3-Ketal Bisnordiene triflate (4) can be converted to the compound (5) by the process illustrated above.

Example 5: Preparation of Cholest-4,6-diene-3-one (6)
General Procedure:
3-Ketal 4,6- Cholestadiene (5) was treated with p-Toluene sulphonic acid at about 0.62 mole equivalence at room temperature to obtain Cholest-4,6-diene-3-one (6).

Example 5(a): To the 100 gm compound 3-Ketal 4,6- Cholestadiene (5) obtained from example 4 ,added 500 ml ethyl acetate was added 25 gm (131.4milimole) para toluene sulphonic acid monohydrate in 100 ml water and stirred the mass for 3-8 hrs till TLC indicated completion of the reaction. The mixture was washed with 200ml of water and then extracted in 1000 ml of ethyl acetate. The entire ethyl acetate layer was then evaporated under vacuum to obtain the residue Cholest-4,6-diene-3-one (6).
Yield: 80 gm; Percentage Yield: 89%
HPLC purity: 87%

Example 5(b): To 5.0gm (10.62 millimole) of the compound 3-Ketal 4,6- Cholestadiene (5) was added 50 ml of ethyl acetate, 2.5 ml HCl and stirred the mass for 3-8 hrs till TLC indicated completion of the reaction. The reaction mass was then washed with 20ml of water and extracted in 20 ml of ethyl acetate. The entire ethyl acetate layer was then evaporated under vacuum to obtain the residue Cholest-4,6-diene-3-one (6).
Yield: 3.9 gm; Percentage Yield: 87%
HPLC purity: 82%
The residue was crystallized with Acetone
The residue was further crystallized in Acetone 10 ml stir at 25-30°C chill to 5-10°C filter it wash by chill Acetone 5 ml, dry the filtered product
Yield: 1.5 gm; Percentage Yield: 33%
HPLC purity: 95%

Example 5(c): To 50 gm (106.21 millimole) of the compound 3-Ketal 4,6- Cholestadiene (5) was added 100 ml of ethyl acetate, 25 ml HC1 and the mixture was stirred for 3 hrs till TLC indicated completion of the reaction. The mass was washed with 20ml of water and extracted in 20ml ethyl acetate. The entire ethyl acetate layer was then evaporated under vacuum and residue to obtain the residue Cholest-4,6-diene-3-one (6).
Yield: 41 gm, Percentage Yield: 93%
HPLC purity: 80%
The residue was crystallized from methanol.
The residue was further crystallized in methanol 100 ml stir at 25-30°C chill to 5-10°C filter it wash by chill methanol 25 ml, dry the filtered product
Yield: 30gm; Percentage Yield: 67%
HPLC purity: 90%

Example 5(d): To 50 gm (106.21 millimole) of the compound 3-Ketal 4,6- Cholestadiene (5) was added 100 ml Toluene, 25 ml HCl and the mixture was stirred for 3 hrs till TLC indicated completion of the reaction. The mixture was then washed with 20ml of water and extracted in Toluene. The entire Toluene layer was then evaporated under vacuum to obtain the residue Cholest-4,6-diene-3-one (6).
Yield: 40 gm; Percentage Yield: 89%
HPLC purity: 85%

Example 5(e): To 50 gm (106.21 millimole) of the compound 3-Ketal 4,6- Cholestadiene (5) was added 100 ml acetone, 25 ml HCl and the reaction mixture was stirred for 3 hrs till TLC indicated completion of the reaction. The mixture was then washed with 20ml of water and then extracted in 20 ml of ethyl acetate. The entire ethyl acetate layer was then evaporated under vacuum to obtain the residue Cholest-4,6-diene-3-one (6).
Yield: 38 gm; Percentage Yield: 85%
HPLC purity: 86%

Example 5(f): To 50 gm (106.21 millimole) of the compound 3-Ketal 4,6- Cholestadiene (5) was added 100 ml methanol, 25 ml HCl. The reaction mixture was stirred for 3 hrs till TLC indicated completion of the reaction. The mixture was then washed with washed with 50ml of waste and then extracted in 20ml ethyl acetate. The entire ethyl acetate layer was then evaporated under vacuum to obtain the residue Cholest-4,6-diene-3-one (6).
Yield: 36 gm, Percentage Yield: 80%
HPLC purity: 85%
Example 5(g): To the 100-gm compound 3-Ketal 4,6- Cholestadiene (5) obtained from example 4 was added 25 gm (260.1 smilimole) Methanesulfonic acid in 100 ml water and stirred the mass for 3-8 hrs till TLC indicated completion of the reaction. The mixture was washed with 200ml of water and then extracted in 1000 ml of ethyl acetate. The entire ethyl acetate layer was then evaporated under vacuum to obtain the residue Cholest-4,6-diene-3-one (6).
Yield: 80 gm; Percentage Yield: 89%
HPLC purity: 95%

Example 5(h): To the 100-gm compound 3-Ketal 4,6- Cholestadiene (5) obtained from example 4 was added 25 gm (183.6 smilimole) Potassium bisulfate in 100 ml water and stirred the mass for 3-8 hrs till TLC indicated completion of the reaction. The mixture was washed with 200ml of water and then extracted in 1000 ml of ethyl acetate. The entire ethyl acetate layer was then evaporated under vacuum to obtain the residue Cholest-4,6-diene-3-one (6).
Yield: 80 gm; Percentage Yield: 89%
HPLC purity: 95%

Example 5(i): To the 100-gm compound 3-Ketal 4,6- Cholestadiene (5) obtained from example 4 was added 25 gm (416.3 smilimole) Acetic acid in 100 ml water and stirred the mass for 3-8 hrs till TLC indicated completion of the reaction. The mixture was washed with 200ml of water and then extracted in 1000 ml of ethyl acetate. The entire ethyl acetate layer was then evaporated under vacuum to obtain the residue Cholest-4,6-diene-3-one (6).
Yield: 80 gm; Percentage Yield: 89%
HPLC purity: 95%

Example 6: Preparation of 3-Acetoxy- Cholest-3,5-diene (7).
General procedure:
Cholest-4,6-diene-3-one (6) was treated with acylating agents to form enol esters, acetylating agents in an amount of 11.34 to 22.68 mole equivalence, preferably 11.34 mole equivalence in presence of acid catalyst at 100-110oC to yield 3-Acetoxy- Cholest-3,5-diene(7).

Example 6(a): 100 gms (261 millimole) of Cholest-4,6-diene-3-one (6) obtained from example 5 was suspended in 300gm (2938 millimole) of acetic anhydride and 100 gm(1274 millimole) of acetyl chloride at 25-35°C. The reaction mass was stirred for 6-8 hours at 105-110°C till the TLC analysis indicated the absence of the starting material. Cooled the reaction mass to ambient temperature. Quenched the reaction mass in ice cold water solution followed by 500ml water and finally extracted with 500 ml of Dichloromethane. Washed the organic layer with 250 ml of 8% sodium bicarbonate solution. The dichloromethane layer was evaporated to get the residue 3-Acetoxy- Cholest-3,5-diene(7).
Yield: 90 gm; Percentage Yield: 81%
HPLC purity: 82%.
The residue was purified with acetone.
The residue was further crystallized in Acetone 200 ml stir at 50-60°C chill to 10°C filter it wash by chill Acetone 50 ml, dry the filtered product
Yield: 50gm; Percentage Yield: 45%
HPLC purity: 91%

Example 6(b): 50 gms (130 millimole) of Cholest-4,6-diene-3-one (6) was suspended in 100 gm (998.8 millimole) isopropenyl acetate and 1 ml of sulphuric acid at 25-35°C. The reaction mass was stirred for 12-18 hours at 100°C till the TLC analysis indicated the absence of starting material. Cooled the reaction mass to ambient temperature. Quenched the reaction mass in ice cold water solution followed by extraction with 500 ml Dichloromethane and finally washing with 50 ml of 10% sodium bicarbonate solution. The dichloromethane layer was evaporated to get residue 3-Acetoxy- Cholest-3,5-diene(7).
Yield:.52.0 gm; Percentage Yield: 94%;
HPLC purity: 80%
The residue obtained was further isolated from silica column chromatography with 5% acetone in Toluene as the eluent.
Yield: 20 gm; Percentage Yield: 36%
HPLC purity: 88%

Example 6(c): 1.0 gm (2.6 millimole) of Cholest-4,6-diene-3-one (6) was suspended in 10 gm (9.9 millimole) acetic anhydride and 5 gm of acetyl chloride at 25-35°C. The reaction mass was stirred for 6-8 hours at 100°C till the TLC analysis indicated the absence of starting material. Cooled the reaction mass to ambient temperature. Quenched the reaction mass in ice cold water solution followed by extraction with Dichloromethane and finally washed with 50 ml of 10% sodium bicarbonate solution. The dichloromethane layer was evaporated to get residue 3-Acetoxy- 3,5,7 cholesta triene (7).
Yield: 1.0 gm; Percentage Yield: 90%
The residue was crystalized with isopropyl alcohol.
Yield: 0.5gm; Percentage Yield: 45%
HPLC purity: 90%

Example 6(d): 1.0 gm(2.6 millimole) of Cholest-4,6-diene-3-one (6) was suspended in 10gm (44.20 millimole) Benzoic anhydride and 5 gm (35.56 millimole ) of benzoyl chloride at 25-35°C. The reaction mass was stirred for 6-8 hours at 100°C till the TLC analysis indicated the absence of starting material. Cooled the reaction mass to ambient temperature. Quenched the reaction mass in ice cold water solution followed by extraction with ethyl acetate and finally washed with 50 ml of 10% sodium bicarbonate solution. The Ethyl acetate layer was evaporated to get residue 3-Acetoxy- 3,5,7 cholesta triene (7).
Yield: 1.1 gm; Percentage Yield: 99%
HPLC purity: 79%
The residue was crystallised from ethanol.
The residue was further crystallized in ethanol 10 ml stir at 25-30°C chill to 5-10°C filter it wash by chill ethanol 5 ml, dry the filtered product
Yield: 0.4gm; Percentage Yield: 36%
HPLC purity: 90%

Example 6(e): 1.0 gm (2.6 millimole) of Cholest-4,6-diene-3-one (6) was suspended in 10 gm (76.86 millimole) propionic anhydride, 5gm(54.04) of propionyl chloride at 25-35°C. The reaction mass was stirred for 6-12 hours at 100°C till the TLC analysis indicated the absence of starting material. Cooled the reaction mass to ambient temperature, quenched the reaction mass in ice cold water solution followed by extraction with Dichloromethane and finally washed with 50 ml of 10% sodium bicarbonate solution. The dichloromethane layer evaporated to get residue 3-Acetoxy- 3,5,7 cholesta triene (7).
Yield: 0.8gm; Percentage Yield: 72 %
HPLC purity: 75%
The residue was crystalized from isopropyl alcohol.
The residue was further crystallized in isopropyl alcohol 10 ml stir at 25-30°C chill to 5-10°C filter it wash by chill isopropyl alcohol 5 ml, dry the filtered product
Yield: 0.5gm; Percentage Yield: 45%
HPLC purity: 90%

Example 6(f): 1.0 gm (2.6 millimole) of Cholest-4,6-diene-3-one (6) was suspended in 10 gm (9.9 millimole) Isobutyric anhydride, 5 gm(63.21 millimole) of Isobutyl chloride at 25-35°C. The reaction mass was stirred for 6-8 hours at 100°C till the TLC analysis indicated the absence of starting material. Cooled the reaction mass to ambient temperature, quenched the reaction mass in ice cold water solution followed by extraction with Dichloromethane and finally washed with 50 ml of 10% sodium bicarbonate solution. The dichloromethane layer was evaporated to get residue 3-Acetoxy- 3,5,7 cholesta triene (7).
Yield: 1.0 gm; Percentage yield: 85%
HPLC purity: 80%

Example 6(g): 1.0 gm (2.6 millimole) of Cholest-4,6-diene-3-one (6) was suspended in 10 gm (47.61 millimole) Trifluroacetic anhydride, 5 gm(37.74 millimole) of trifluroacetyl chloride at 25-35°C. The reaction mass stirred for 12-24 hours at 100°C till the TLC analysis indicated the absence of starting material. Cooled the reaction mass to ambient temperature, quenched the reaction mass in ice cold water solution followed by extraction with Dichloromethane and finally washed with 50 ml of 10% sodium bicarbonate solution. The dichloromethane layer evaporated to get residue 3-Acetoxy- 3,5,7 cholesta triene (7).
Yield: 1.1 gm; Percentage yield: 90%
HPLC purity: 88%

Example 6(i): 100 gms (261 millimole) of Cholest-4,6-diene-3-one (6) obtained from example 5 was suspended in 300gm (1063 millimole) of Trifluoromethanesulfonic anhydride and 100 gm(593 millimole) of Trifluoromethanesulfonyl chloride at 25-35°C. The reaction mass was stirred for 6-8 hours at 105-110°C till the TLC analysis indicated the absence of the starting material. Cooled the reaction mass to ambient temperature. Quenched the reaction mass in ice cold water solution followed by 500ml water and finally extracted with 500 ml of Dichloromethane. Washed the organic layer with 250 ml of 8% sodium bicarbonate solution. The dichloromethane layer was evaporated to get the residue 3-Acetoxy- 3,5,7 cholesta triene (7).
Yield: 90 gm; Percentage Yield: 81%
HPLC purity: 90%.

Example 6(j): 100 gms (261 millimole) of Cholest-4,6-diene-3-one (6) obtained from example 5 was suspended in 300gm (2938 millimole) of acetic anhydride and 100 gm(1274 millimole) of acetyl chloride at 25-35°C. The reaction mass was stirred for 6-8 hours at 105-110°C till the TLC analysis indicated the absence of the starting material. Cooled the reaction mass to ambient temperature. Quenched the reaction mass in ice cold water solution followed by 500ml water and finally extracted with 500 ml of Ethyl acetate. Washed the organic layer with 250 ml of 8% sodium bicarbonate solution. The Ethyl Acetate layer was evaporated to get the residue 3-Acetoxy- 3,5,7 cholesta triene (7).
Yield: 90 gm; Percentage Yield: 81%
HPLC purity: 95%.

Example 6(k): 100 gms (261 millimole) of Cholest-4,6-diene-3-one (6) obtained from example 5 was suspended in 300gm (2938 millimole) of acetic anhydride and 100 gm(1274 millimole) of acetyl chloride at 25-35°C. The reaction mass was stirred for 6-8 hours at 105-110°C till the TLC analysis indicated the absence of the starting material. Cooled the reaction mass to ambient temperature. Quenched the reaction mass in Amonium hydroxide solution followed by 500ml water and finally extracted with 500 ml of Ethyl acetate. Washed the organic layer with 250 ml of 8% sodium bicarbonate solution. The Ethyl Acetate layer was evaporated to get the residue 3-Acetoxy- 3,5,7 cholesta triene (7).
Yield: 90 gm; Percentage Yield: 81%
HPLC purity: 95%.

Example 6(l): 100 gms (261 millimole) of Cholest-4,6-diene-3-one (6) obtained from example 5 was suspended in 300gm (2938 millimole) of acetic anhydride and 100 gm(1274 millimole) of acetyl chloride at 25-35°C. The reaction mass was stirred for 6-8 hours at 105-110°C till the TLC analysis indicated the absence of the starting material. Cooled the reaction mass to ambient temperature. Quenched the reaction mass in 8% sodium bicarbonate solution followed by 500ml water and finally extracted with 500 ml of Ethyl acetate. Washed the organic layer with 250 ml of 8% sodium bicarbonate solution. The Ethyl Acetate layer was evaporated to get the residue 3-Acetoxy- 3,5,7 cholesta triene (7).
Yield: 90 gm; Percentage Yield: 81%
HPLC purity: 95%.

Example 6(m): 100 gms (261 millimole) of Cholest-4,6-diene-3-one (6) obtained from example 5 was suspended in 300gm (2938 millimole) of acetic anhydride and 100 gm(1274 millimole) of acetyl chloride at 25-35°C. The reaction mass was stirred for 6-8 hours at 105-110°C till the TLC analysis indicated the absence of the starting material. Cooled the reaction mass to 18-20°C temperature. Filter the mass and wash with water followed by methanol.
Yield: 60 gm; Percentage Yield: 66%
HPLC purity: 96%.

Example 6(n): 100 gms (261 millimole) of Cholest-4,6-diene-3-one (6) obtained from example 5 was suspended in 300gm (2938 millimole) of acetic anhydride and 100 gm (1274 millimole) of acetyl chloride at 25-35°C. The reaction mass was stirred for 6-8 hours at 105-110°C till the TLC analysis indicated the absence of the starting material. Distilled out reaction mass up to 75%. Cooled the reaction mass to ambient temperature. Quenched the reaction mass in ice cold water solution followed by 500ml water and finally extracted with 500 ml of Ethyl acetate. Washed the organic layer with 250 ml of 8% sodium bicarbonate solution. The Ethyl Acetate layer was evaporated to get the residue 3-Acetoxy- 3,5,7 cholesta triene (7).
Yield: 90 gm; Percentage Yield: 81%
HPLC purity: 96%.

Example 7: Preparation of 7-dehydrocholesterol (7-DHC)
General Procedure:
3-Acetoxy- 3,5,7 cholesta triene (7) obtained from example 6 was treated with in- situ generation of calcium borohydride (reacting sodium borohydride 2.44 mole equivalence with calcium chloride anhydrous 1 mole equivalents ) at 0-5oC for 4-12 hours to yield 7 Dehydrocholesterol as major product and Epi -7 -Dehydrocholesterol as side product.

Alternately, 3-Acetoxy- 3,5,7 cholesta triene (7) obtained from example 6 can be reduced directly using suitable reducing agent and carrying out the process as described above.

Example 7(a): To 60 gm (540.6 millimole) of calcium chloride was charged absolute ethanol (600 ml) and stirred. Cooled the reaction mass to 5-10oC followed by slow addition of 50 gm(1321 millimole) of sodium borohydride and allowed to stir for 30 min at 5-10oC. This was followed by slow addition of 100 gm (235 millimole ) of 3-Acetoxy- Cholest-3,5,7-triene(7) dissolve in 100ml MDC at 0-5°C, then stirred at 0-5oC for 4-12 hrs till TLC indicated completion of the reaction. Quenched the reaction mass in 1.0 lit of ice cold water and extracted with1.0 lit Dichloromethane. Then washed with 250 ml of 50% acetic acid to yield crude product 7-DHC.
Yield: 84 gm, Percentage Yield: 96%
HPLC purity: 65% ; epi-7Dehydrocholetserol 10 %
Purification:
Dissolved the above crude in 250 ml of 20% toluene in methanol solution. Heated to 65°C and maintained the temperature for 30 minutes. Allowed to cool to 25-30°C gradually, chilled to 10-15°C, filtered and washed with 20ml methanol. Dried the solid product under vacuum at 50°C.
Yield: dry weight 40 gm; Percentage Yield: 44 %
HPLC purity: 96% ; Epi- 7- Dehydrocholesterol: 2%
Example 7(b): Charged 60gm calcium chloride to ethanol (300 ml), cooled to 0-5°C, then slowly added sodium borohydride 50 gm(1321 millimole) and allowed to stir for 30 min at 0-5oC. 60gm (141 millimole ) of the compound 3-Acetoxy-Cholest-3,5-diene(7) dissolved in 100 ml methylene dichloride (MDC) was slowly added at 0-10 °C for 12 hrs and the until reaction compliance was checked on TLC. After completion of the reaction the reaction mass was quenched in 1.0 lit ice cold water and extracted with 1000 ml DCM and finally washed with 250 ml of 2N HCl solution to yield crude product 7-DHC.
Yield: 80 gm; Percentage Yield: 88%
HPLC purity: 70% , epi-7dehydrocholestrol 10 %.

Purification by column chromatography:
Dissolved the crude in toluene and loaded on packed 60-200 mesh size silica column in Toluene. Eluted with 5% Acetone in Toluene and elute separation analysed by TLC to separate the Epi-7-dehydrocholesterol (8 gm)
Final main fraction crystalized in 200 ml methanol;

Yield: Dry weight 50 gm; Percentage Yield: 55%
HPLC purity 96.5%,

Example 7(c): To 6 gm (54.06 millimole) of calcium chloride was charged methanol (100 ml), stirred, cooled the reaction mass to 5-10oC, then slowly added 5.0 gm(132.1 millimole) sodium borohydride and allowed to stir for 30 min at 40-45oC. This was followed by slow addition of 10 gm (23.58 millimole ) of compound 3-Acetoxy-Cholest-3,5-diene(7) dissolved in 10 m Toluene at 40-45°C then stirred for 12-18 hrs and the reaction completion checked on TLC. After the reaction, the reaction mass was quenched in ice cold water, extracted with 50 ml Toluene and washed with 20 ml of 2 N HCl solution to yield crude product 7-DHC.
Yield: 3.0 gm; Percentage Yield: 55%
HPLC purity: 65%; Epi -7-Dehydrocholesterol: 25%
The final pure compound was obtained by column chromatography as described in example 7(b).
Yield: 1.2 gm; Percentage Yield: 12%

Example 7(d): To 100 ml of methanol, stirred, cooled to 5-10oC was slowly added 5.0 gm(132.1 millimole) sodium borohydride and allowed to stir for 30 min at 0-10oC. This was followed by slow addition of 10 gm (23.58 millimole) of compound 3-Acetoxy-Cholest-3,5-diene(7) dissolved in 10 ml Toluene at 0-10°C, then stirred for 12-18 hrs and the reaction completion checked on TLC. After completion of the reaction, the reaction mass was quenched in ice cold water, extracted with 50 ml Toluene, washed with 20 ml of 2N HCl solution to yield crude product 7-DHC.
Yield: 3.0 gm; Percentage Yield: 92.%
HPLC purity 65% ; Epi- 7 -Dehydrocholesterol 20%

The final purified 7-DHC was obtained by column chromatography as described in example7(b).
Yield: 1gm 10%

Example 7(e): To 100 ml of ethanol, stirred, cooled to 5-10oC was slowly added sodium triacetoxyborohydride 5.0 gm(23.59 millimole) and allowed to stir for 30 min at 0-10oC. This was followed by slow addition of 10 gm (23.58 millimole ) of compound 3-Acetoxy-Cholest-3,5-diene(7) dissolved in 10 ml toluene at 0-10 °C, then stirred for 12-18 hrs and the reaction completion checked on TLC. After completion of the reaction, the reaction mass was quenched in ice cold water and extracted with 50 ml Toluene. Washed with 20 ml of 2N HCl solution to yield crude product 7-DHC.
Yield: 6.0 gm; Percentage Yield: 66.%
HPLC purity: 65%; Epi- 7- Dehydrocholesterol 25%

Example 7(f): To 100 ml of ethanol, stirred, cooled the mass to 5-10oC followed by slowly adding lithium borohydride 5.0 gm(210.26 millimole) and allowed to stir for 30 min at 0-10oC. 10 gm (23.58 millimole) of 3-Acetoxy-Cholest-3,5-diene(7) dissolved in 10 ml Toluene was slowly added to the mixture at 0-10°C and then stirred for 12-18 hrs.The reaction completion was checked on TLC. After completion of the reaction the reaction mass was quenched in ice cold water and extracted with 50 ml Toluene. Washed with 20 ml 2N HCl solution to yield crude product 7-DHC.
Yield: 6.0 gm; Percentage Yield: 66 %
HPLC purity: 68% ;Epi- 7 -Dehydrocholesterol 12%.

Example 7(g): To 25 ml Methanol and 100 ml Toluene followed by 10 gm (23.58 millimole) of 3-Acetoxy-Cholest-3,5-diene(7) dissolved with addition of sodium borohydride at 25-30°C in pressure reactor maintain hydrogen pressure. The reaction completion was checked on TLC. After completion of the reaction the reaction mass was filter through filter aid. Wash with water followed by 20 ml 2N HCl solution to yield crude product 7-DHC.
Yield: 7.0 gm; Percentage Yield:70 %
HPLC purity: 75% ;Epi- 7 -Dehydrocholesterol 2%.

Example 8: Irradiation of 7-DHC to vitamin D3
Example 8a
37.5 gms of 7-Dehydrocholesterol, 1 gm of Butylated hydroxyl toluene and 25 ml of 2% Aq. Sodium hydroxide solution were dissolved in 1250 ml of Ethanol at 75-85°C and the mixture was irradiated by UV lamp at 80-85°C for 180 minutes preferably. The reaction mass was cooled to 25-30°C. The solids separated out were filtered as first crop which contains 7-Dehydrocholesterol
The filtrate was concentrated to 20% of the original volume, cooled to 20-25°C and the separated solids were filtered as second crop that contains 7-Dehydrocholesterol
The filtrate was again concentrated under vacuum. 2000 ml Methyl tert butyl ether was added, washed with 2 *50 ml 1:1 ethanol water and distilled under vacuum. 100 ml Acetone was added to the residue, cooled to 5- 10°C and the separated solids were filtered as the third crop that contains 7-Dehydrocholesterol. All these first, second and third crops are combined and reused in subsequent batches.
The filtrate was then evaporated under vacuum at 40-45°C and the crude Vitamin D3 (resin) thus obtained can be purified by either of the following methods:
1. Converting the resin to its ester like Acetate or Propionate or Butyrate or Valerate or 2-Nitrobenzoate or 4-Nitrobenzoate more preferably Butyrate, crystallized and finally saponified by a base like Sodium Hydroxide or Potassium hydroxide or Sodium carbonate or Potassium carbonate Sodium methoxide or Sodium ethoxide or Potassium butoxide or Lithium Aluminium Hydride and finally crystallized from acetone or methyl formate.
2. Purification of the Crude resin by column chromatography using Silica gel or Alumina or Alumina with 2-10% water and using Toluene: 2-Butanone as an eluent or Toluene: Acetone as an eluent.

Example: 8b
163 gms of 7-Dehydrocholesterol (0.423) was suspended in 4L Ethanol at 28-30°C. 0.6 gms of Butylated hydroxy toluene was added at room temperature. Subsequently 2.5 gms of sodium hydroxide was added at room temperature followed by addition of 25.0gms of 5-(3-Pyridyl)-2,2’-bithiophene. The above reaction mass was heated to 80-85°C and irradiated at 80-85°C using UV lamp for 200 minutes. The reaction mass was evaporated under vacuum and the residue obtained.
The residue was purified over column chromatography to isolate the pure crystalline Vitamin D3 using Toluene: Methyl ethyl ketone 1:99, 2: 98, 3: 97, 4: 96 & 5:95 as a eluent over silica gel or Alumina respectively.
Yield: 24.45 gms
% Yield: 15%

Example: 8c
96 gms of 7-Dehydrocholesterol, 1 gm of Butylated hydroxyl toluene and 25 ml of 2% Aq. Sodium hydroxide solution were dissolved in 1250 ml of THF at 25-35°C and the mixture was irradiated using UVlamp at 25-35°C for 180-360 minutes preferably. The reaction mass was cooled to 10-15°C. The solids separated out were filtered as first crop which contains 7-Dehydrocholesterol
The filtrate was concentrated to 20% of the original volume, cooled to 20-25°C and the separated solids were filtered as second crop that contains 7-Dehydrocholesterol
The filtrate was again concentrated under vacuum. 2000 ml Heptane was added, washed with 2 *50 ml 1:1 ethanol water and distilled under vacuum. 200 ml 2-Butanone (methyl ethyl ketone) was added to the residue, cooled to 10°C and the separated solids were filtered as the third crop that contains Vitamin D3 + 7-Dehydrocholesterol. All these first, second and third crops are combined and reused in subsequent batches.
The filtrate was then evaporated under vacuum at 40-45°C and the crude Vitamin D3 (resin) which can be isolated by known method as explain in example 8a.
Plant based Vitamin D3 obtained by photosynthesis of plant based 7-Dehydrocholesterol is characterised as per pharmacopeia analysis.

Example 8d
37.5 gms of 7-Dehydrocholesterol and 37.5 gms Cholesterol (especially obtained from plant source like phytosterol, Bisnoralcohol), 1gm of Butylated hydroxyl toluene and 25 ml of 2% Aq. Sodium hydroxide solution were dissolved in 1250 ml of Ethanol at 75-85°C and the mixture was irradiated by UV lamp at 80-85°C for 180 minutes preferably. The reaction mass was cooled to 25-30°C. The solids separated out were filtered as first crop which contains 7-Dehydrocholesterol with Cholesterol.
The filtrate was concentrated to 20% of the original volume, cooled to 20-25°C and the separated solids were filtered as second crop that contains 7-Dehydrocholesterol with Cholesterol.
The filtrate was again concentrated under vacuum. 2000 ml petroleum ether was added, washed with 2 *50 ml 1:1 ethanol water and distilled under vacuum. 100 ml Acetone was added to the residue, cooled to 5- 10°C and the separated solids were filtered as the third crop that contains 7-Dehydrocholesterol. All these first, second and third crops are combined and reused in subsequent batches.
The filtrate was then evaporated under vacuum at 40-45°C and the crude Vitamin D3 (resin) thus obtained can be purified by either of the following methods:
1. Converting the resin to its ester like Acetate or Propionate or Butyrate or Valerate or 2-Nitrobenzoate or 4-Nitrobenzoate more preferably Butyrate, crystallized and finally saponified by a base like Sodium Hydroxide or Potassium hydroxide or Sodium carbonate or Potassium carbonate Sodium methoxide or Sodium Ethoxide or Potassium butoxide or Lithium Aluminium Hydride and finally crystallized from acetone or methyl formate.
2. Purification of the Crude resin by column chromatography using Silica gel or Alumina or Alumina with 2-10% water and using Toluene: 2-Butanone as an eluent or Toluene: Acetone as an eluent.

Example 9: Characterization
A. FTIR analysis:
The plant based Vitamin D3 of the present invention is characterized by FTIR at wavelength as depicted in Fig 1.The Fig 1 also depicts the FTIR of standard cholecalciferol. As observed from the Fig 1, vitamin D3 obtained from the plant source in the present invention meets the wavelength of standard cholecalciferol.
B. HPLC Chromatogram
Fig 2 and Fig 3 depicts the HPLC chromatogram of (i) Blank solution (ii) Cholecalciferol and (iii) vitamin D3 obtained from the plant source of the present invention. The HPLC chromatogram complies the system suitability and the USP assay method for vitamin D3 of the present invention is observed to meet the USP pharmacopeia of Indian, European and American standard.
C. Product Analysis




Although the invention has been described in detail in the foregoing for the purpose of illustration, it is to be understood that such detail is solely for that purpose and that variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention except as it may be limited by the claims.
, Claims:
1. Plant based Vitamin D3 having pharmacopeia compliant HPLC chromatogram (Fig 1 to Fig 3) prepared by a process starting from bis-noralcohol obtained from SODD comprising;
i. Oxidizing bisnoralcohol (1) suspended in the solvent with 2,3-Dichloro-5,6-dicyano-p-benzoquinone (DDQ) or p-chloranil to obtain (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one (2);
ii. Reacting (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one (2) of step (i) with acetylating agent selected from p-toluene sulphonyl chloride, Methane sulphonyl chloride, Trifluoromethanesulfonic anhydride to obtain Bisnordiene tosylate or Bisnordiene mesylate or Bisnordiene Triflate (3);
iii. Reacting Bisnordiene tosylate or Bisnordiene mesylate or Bisnordiene Triflate (3) of step (ii) with Ethane1,2-dithiol or 2-mercaptoethanol or ethylene glycol or Propane1,3 diol in presence of trimethyl orthoformate followed by reaction with p- toluene sulphonic acid, methane sulfonic acid or Trifluoromethanesulfonic anhydride to obtain 3-Ketal Bisnordiene Tosylate or 3-Ketal Bisnordiene mesylate or 3-Ketal Bisnordiene triflate (4);
iv. Reacting 3-Ketal Bisnordiene Tosylate or 3-Ketal Bisnordiene mesylate or 3-Ketal Bisnordiene triflate (4) of step (iii) with a Grignard reagent to yield 3- ketal 4,6- Cholestadiene (5);
v. Deketalizing 3-ketal 4,6-Cholestadiene (5) of step (iv) with deketalizing agent to obtain Cholest-4,6-diene-3-one (6);
vi. Acylating Cholest-4,6-diene-3-one (6) with the acylating agent to obtain 3-Acetoxy- 3,5,7 cholesta triene (7);
vii. Reducing 3-Acetoxy- 3,5,7 cholesta triene (7) of step (vi) with a reducing agent to obtain 7 –Dehydrocholesterol (7-DHC); and
viii. Irradiating the mixture 7-DHC of step (vii), optionally with plant sourced cholesterol, butylated hydroxyl toluene and 2% aq. Sodium hydroxide or 2% potassium hydroxide or 2% sodium carbonate preferably with 2% sodium hydroxide dissolved in the solvent using UV lamp to obtain vitamin D3.

2. The process as claimed in claim 1, wherein oxidation of bisnoralcohol with DDQ or with p-chloranil of step (i) is carried out in the solvent selected from 1,4 dioxane, C1-C5 alcohol or toluene at a temperature in the range of 25-65°C.

3. The process as claimed in claim 1, wherein the solvent for step (ii) selected from chloroform or dichloromethane, ethyl acetate, dioxane or mixtures thereof; and the base selected from pyridine, triethylamine, imidazole or Diisopropylethylamine.

4. The process as claimed in claim 1, wherein the solvent for step (iii) selected from chloroform or dichloromethane, aromatic or aliphatic hydrocarbon such as toluene or heptane and at room temperature or at reflux temperature.

5. The process as claimed in claim 1, wherein the Grignard reaction of step (iv) is carried out in THF, methyl–tert butyl ether,, dry diethyl ether, 2- Methyl Tetra hydrofuran or mixture thereof.

6. The process as claimed in claim 1, wherein the deketalization of step (v) is carried out in the solvent selected from water, methanol, toluene, ethylacetate, acetone, acetic acid or mixture thereof.

7. The process as claimed in claim 1, wherein the acylating agent of step (vi) selected from acetic anhydride and acetyl chloride mixture, Trifluoromethanesulfonic anhydride and Trifluoromethanesulfonyl chloride mixture, sulphuric acid and isopropenyl acetate mixture, benzoic anhydride and benzoyl chloride mixture, propionic anhydride and propionyl chloride mixture, Isobutyric anhydride and Isobutyl chloride mixture or Trifluroacetic anhydride and trifluroacetyl chloride mixture.

8. The process as claimed in claim 1, wherein the reducing agent of step (vii) selected from lithium borohydride, sodium borohydride, sodium triacetoxyborohydride, optionally in presence of calcium chloride; and the solvent selected from lower alcohols, methylene dichloride, toluene or mixture thereof.

9. The process as claimed in claim 1, wherein the solvent for irradiation in step (viii) selected from ethanol or THF or mixture thereof and optionally in presence of the photosensitizer.

10. The process as claimed in claim 1, wherein the intermediates and the product obtained may be purified by crystallization from the solvent or by column chromatography.