DESC:FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
AND
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

1. TITLE OF THE INVENTION:

“PROCESS FOR PREPARATION OF 7-DEHYDROCHOLESTEROL”

2. APPLICANT:

(a) NAME: FERMENTA BIOTECH LIMITED

(b) NATIONALITY: Indian Company incorporated under the Companies
Act, 1956

(c) ADDRESS: ‘DIL’ Complex, Ghodbunder Road, Majiwada,
Thane (West) - 400610, Maharashtra, India.

3.PREAMBLE TO THE DESCRIPTION:
The following specification particularly describes the invention and the manner in which it is to be formed:

Technical filed:
This invention relates to an improved cost-effective process for preparation of 7-Dehydrocholesterol of formula I with good yield and purity.

Background and prior art:
7-Dehydrocholesterol is an important precursor of vitamin D3 and its other derivatives. 7-dehydrocholesterol under photoreaction produces vitamin D3. 7-Dehydrocholesterol has been synthesized in the prior art using variety of synthetic procedures. Steroidal synthetic route is always a topic of interest for the synthesis of 7-dehydrocholesterol.

Formula I
Allylic substitution of cholesterol and its derivatives at 7-position and subsequent elimination reaction produces 7-dehydro derivatives as shown in scheme 1.

For example, Tachibana, Y. et al (Bull. Chem. Soc. Jpn. 1978, 51, 3085) discloses allylic bromination of cholesterol at 7-position followed by dehydrobromination to produce 7-dehydrocholestrol. The process reported by Tachibana, Y. et al has the drawback of formation of side product 4, 6-dehydrosterol. This problem was solved to certain extent and the yield of the reaction improved by adding some intermediate steps by Confalone, P.N et al. as reported in J. Org. Chem. 1981, 46, 1030. However, the method reported by Confalone, P.N et al. involves expensive reagents and hence is not viable for large scale production.

Also different procedures have been adopted for the allylic oxidation. For example, Cholesterol is oxidized at allylic position using chromium trioxide and further reduced with sodium borohydride to obtain 7-hydroxycholesterol, which on subsequent elimination reaction to obtain 7-dehydrocholesterol.

In yet another process, (Li, Z.; Tan, T. China Patent Association 101220075, 2008-1-25) cholesterol-3-acetate is converted to 7-oxocholesterol-3-acetate, which is reacted with tosylhydrazine to produce 7-tosylhydrozone cholesterol-3-acetate, which is further converted to 7-dehydrocholesterol-3-acetate by Bamford- Steven’s reaction. This route also involves some transition metal catalyzed steps and expansive experimental design and therefore not suitable for large scale production.

Hexacarbonyl molybdenum and other molybdenum containing catalysts are also reported for carbon-carbon bond formation at allylic position.

Dugas, D et al (J. Mol. Catal. A: Chem. 2006, 253, 119) reported Palladium catalyzed elimination of 7-acetoxycholesterol derivatives to 7-dehydrocholesterol derivatives, however, due to the high prices of palladium catalysts as well as the requirement of complex experimental conditions makes this process difficult to practice on commercial scale.

In the light of the foregoing there remains a need in the art to provide cost-efficient process for preparation of 7-Dehydrocholesterol of formula I. Accordingly, it is an objective of the invention to provide a robust process for preparation of 7-Dehydrocholesterol with improved yield and purity that can be easily scaled to commercial production.

Summary of the invention:
The present invention discloses an improved process for preparation of 7-Dehydrocholesterol with good yield and purity over the process reported by Confalone, et al. in J. Org. Chem. 1981, 46, 1030-1032.

Accordingly, the process for preparation of 7-Dehydrocholesterol with good yield and purity which comprises:
a) protecting hydroxyl group of cholesterol with a suitable protecting group in presence of an organic base;
b) brominating the 3-protected cholesterol in presence of catalytic amounts of pyridine or its derivatives to obtain 7-(a+ß) bromo protected cholesterol;
c) Epimerizing the 7(a+ß) bromo protected cholesterol using tetrabutyl ammonium bromide in toluene or a ketonic solvent or combinations thereof to obtain predominantly 7 a - bromo 3-protected cholesterol;
d) Reacting 7 a - bromo 3-protected cholesterol with substituted or unsubstituted thiophenol or its salt in presence of liquor ammonia or any organicbase to obtain predominantly 7 ß -thiophenyl 3-protected cholesterol;
e) Oxidizing 7 ß -thiophenyl 3-protected cholesterol in presence of Cumene hydrogen peroxide and Titanium tetraisopropoxide to obtain 7-phenyl sulfoxide 3-protected cholesterol;
f) Converting 7-phenyl sulfoxide 3-protected cholesterol into 7-Dehydro 3-protected cholesterol in presence of a base;
g) crystallizing 7-Dehydro 3-protected cholesterol from an organic solvent and
h) Deprotecting the 7-Dehydro 3-protected cholesterol by treating with alkali in presence of methanol followed by crystallization from an organic solvent.

The protection groups are ester protecting groups and may be selected from the group consisting of acetate, propionate, butyrate, benzoate, valerate etc. This reaction results in an yield of 94-98% and HPLC purity of >90%. The organic base used in protection reaction is 4-Dimethylaminopyridine and the reaction is carried at a temperature range of 25-35ºC.
The bromination reaction is conducted using brominating agent selected from N-Bromosuccinimide (NBS) or 1,3-Dibromo-5,5-Dimethylhydantoin(DDH) and the reaction is conducted at reflux temperature of the solvent used. The solvent for the bromination reaction is selected from hydrocarbon solvents such as petroleum ether(40-60°C).

The epimerization reaction is performed in presence of tetrabutyl ammonium bromide and in an organic solvent selected from toluene or a suitable ketone selected from Acetone, 2-butanone, Methyl isobutyl ketone, Di-isobutyl ketone or a mixture of solvents namely toluene and acetone, Toluene and 2-Butanone, Toluene and methyl isobutyl ketone, Toluene and Di-isobutyl ketone. The epimerization reaction is performed at a temperature of -30ºC to -10ºC.

The thiol reaction of epimerized bromo derivative is conducted at a temperature of 0-5ºC with Thiophenol or substituted Thiophenols or the salt of thiophenol or the salt of substituted thiophenols is selected from the group consisting of sodium, Potassium and Lithium salts. The substituted or unsubstituted thiophenols according to the invention are selected from the group consisting of 3-Aminothiophenol; 3-Methyl thiophenol; 4-Chloro thiophenol; 4-Bromo thiophenol; 2-Bromo thiophenol; 4-Bromo-2-Trifluoromethoxy thiophenol; 4-Methoxy thiophenol; 3-Methoxy thiophenol; 2,4-Difluoro thiophenol; 3,4-Difluoro thiophenol; 3,4-DiMethoxy thiophenol; 4-(Trifluoromethyl) thiophenol; 2-Amino-4-chloro thiophenol; 2,3-Dichloro thiophenol; 2-Nitro thiophenol.

The oxidation reaction is conducted at a temperature range of -10 to 0ºC in a solvent selected from hydrocarbon solvent or halogenated hydrocarbon solvent such as toluene; dichloromethane, or dichloroethane.

The conversion of 7-phenyl sulfoxide 3-protected cholesterol into 7-Dehydro 3-protected cholesterol in presence of a base such as Sodium acetate Triethyl amine, sodium acetate, N,N-diisopropylethyl amine or any other organic amines is carried out in toluene at a temperature range of 70-75ºC. The crude 7-Dehydro 3-protected cholesterol can be purified by crystallizing from an alcoholic solvent selected from methanol, Ethanol or Isopropanol or a ketonic solvent selected from the group consisting of Acetone, 2-Butanone, Diisobutyl ketone, Methyl isobutyl ketone.

The deprotection reaction is conducted at a temperature of 40-50ºC followed by crystallization of 7-dehydro cholesterol from an organic solvent. the 7-Dehydrocholesterol is purified from a solvent selected from the group consisting of methanol; ethanol or 2-propanol, Acetone, 2-Butanone, Methyl isobutylketone or Di-isobutylketone or from a mixture of solvents like Dichloromethane and Methanol or Toluene and methanol or Methanol and Di-isopropyl ether.

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

A cost-effective and robust process for preparation of 7-Dehydrocholesterol with good yield and purity is disclosed in the present invention.

According to the present invention, the cost effective process includes use of cheaper and efficient reagents in appropriate molar amounts at each stage of the synthesis of 7-Dehydrocholesterol, which make the process robust and efficiently manageable on industrial scale without compromising on yields and purity.

In a preferred embodiment, the invention provides process for synthesis of 7-Dehydrocholesterol which comprises;
a) protecting hydroxyl group of cholesterol with a suitable protecting group in presence of an organic base;
b) brominating the 3-protected cholesterol in presence of catalytic amounts of pyridine or its derivatives to obtain 7-(a+ß) bromo protected cholesterol;
c) Epimerizing the 7(a+ß) bromo protected cholesterol using tetrabutyl ammonium bromide in toluene or a ketonic solvent or combinations thereof to obtain predominantly 7 a - bromo 3-protected cholesterol;
d) Reacting 7 a - bromo 3-protected cholesterol with substituted or unsubstituted thiophenol or its salt in presence of liquor ammonia or any other organic base to obtain predominantly 7 ß -thiophenyl 3-protected cholesterol;
e) Oxidizing 7 ß -thiophenyl 3-protected cholesterol in presence of Cumene hydrogen peroxide and Titanium tetraisopropoxide to obtain 7-phenyl sulfoxide 3-protected cholesterol;
f) Converting 7-phenyl sulfoxide 3-protected cholesterol into 7-Dehydro 3-protected cholesterol in presence of a base;
g) purifying the 7-Dehydro 3-protected cholesterol from an organic solvent and
h) Deprotecting the 7-Dehydro 3-protected cholesterol by treating with alkali in presence of methanol followed by crystallizing from an organic solvent.

The protection groups are ester protecting groups selected from the group consisting of acetate, propionate, butyrate, benzoate, valerate etc. This reaction results in an yield of 94-98% and HPLC purity of >90%.

The bromination reaction is conducted using brominating agent selected from NBS or DDH and the reaction is conducted at reflux temperature of the solvent used. The solvent for the bromination reaction is selected from hydrocarbon solvents such as n-Hexane, n-Heptane or petroleum ether.

The epimerization reaction is performed in presence of tetrabutyl ammonium bromide and in an organic solvent selected from toluene or a suitable ketone selected from acetone, 2-Butanone, Di-isobutylketone or methyl isobutyl ketone or a mixture of solvents namely toluene and acetone, Toluene and 2-Butanone, Toluene and methyl isobutyl ketone, Toluene and Di-isobutyl ketone. The epimerization reaction is performed at a temperature of -30ºC to -10ºC. The epimerization reaction of the instant invention avoids the use of anhydrous lithium bromide; instead it uses tetrabutyl ammonium bromide as a cost-effective alternative to anhydrous lithium bromide.

The thiol reaction of epimerized bromo derivative is conducted at a temperature of 0-5º-30ºC in presence of Liq. ammonia. The use of Liq. Ammonia instead of triethyl amine further reduces the production cost. The substituted or unsubstituted thiophenols as used in the invention are selected from the group consisting of 3-Aminothiophenol; 3-Methyl thiophenol; 4-Chloro thiophenol; 4-Bromo thiophenol; 2-Bromo thiophenol; 4-Bromo-2-Trifluoromethoxy thiophenol; 4-Methoxy thiophenol; 3-Methoxy thiophenol; 2,4-Difluoro thiophenol; 3,4-Difluoro thiophenol; 3,4-DiMethoxy thiophenol; 4-(Trifluoromethyl) thiophenol; 2-Amino-4-chloro thiophenol; 2,3-Dichloro thiophenol; 2-Nitro thiophenol. Alternately, instead of thiophenol the salts of substituted or unsubstituted thiophenol can be used. Such salts are selected from the group consisting of sodium, Potassium and Lithium salts of thiophenol.

The oxidation reaction is conducted at a temperature range of -10 to 25ºC using Cumene hydrogen peroxide and Titanium tetraisopropoxide, which further reduces the cost over the use of m-chloroperbenzoic acid as taught by Confalone, et al.

The conversion of 7-phenyl sulfoxide 3-protected cholesterol into 7-Dehydro 3-protected cholesterol is carried in presence of a base such as Triethyl amine, sodium acetate, N,N-diisopropylethyl amine or any other organic amines; in toluene at a temperature range of 70-75ºC,, also substantially reduces the cost of the final product, 7-Dehydrocholesterol of formula I.

The crude 7-Dehydro 3-protected cholesterol can be purified by crystallizing from an alcoholic solvent selected from methanol, Ethanol or Isopropanol or a ketonic solvent such as Acetone, 2-Butanone, Diisobutyl ketone, Methyl isobutyl ketone.

Finally the deprotection reaction is conducted at a temperature of 40-50ºC using a suitable alkali in methanol instead of DNS (denatatured spirit) also reduces the cost of the final product, 7-Dehydrocholesterol of formula I, which is further crystallized from an organic solvent. The 7-Dehydrocholesterol is purified from a solvent selected from the group consisting of methanol; ethanol or 2-propanol, Acetone, 2-Butanone, Methyl isobutylketone or Di-isobutylketone or from a mixture of solvents like Dichloromethane and Methanol or Toluene and methanol or Methanol and Di-isopropyl ether.

Thus the instant invention successfully reduced the cost at each and every stage of the synthesis of 7-Dehydrocholesterol of formula I, which make the process robust and efficiently manageable on industrial scale without compromising on yields and purity.

The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

Examples:
Example 1
Synthesis of Cholesterol Acetate(1a)

11gms (0.0285M) of Cholesterol was suspended in 40 ml of petroleum ether along with 0.067gms of 4-Dimethylaminopyridine at 25-35ºC. 6.5-7.5 gms(0.07352M) of Acetic anhydride was then added and the reaction mass refluxed for 2-4 hours till the TLC analysis indicates the absence of starting material. The reaction mass was washed with 50ml water/ saturated salt solution followed by 50 ml of 10% sodium bicarbonate solution and finally with 50ml water.

The organic layer was dried over anhydrous sodium sulphate and distilled under vacuum to yield 11.5-12gms of white solid.

1H NMR and Mass spectra indicates the formation of Cholesterol Acetate(1a).
HPLC analysis: >90%.
Yield: 94-99%.

Similarly Cholesterol was treated with Butyric anhydride, propionic anhydride, valeric anhydride and benzoic anhydride and converted to its cholesterol butyrate(1b), cholesterol propionate(1c), cholesterol valerate(1d) and cholesterol benzoate(1e) derivative respectively with an yield of 94-98% and HPLC purity of >90%.

Example 2
Synthesis of 7-bromo-cholesterol-3-acetate(2a)

12gms (0.02871M) of Cholesterol-3-acetate(1a) was dissolved in 50ml of petroleum ether at 28-35ºC. 0.5ml of pyridine was added at 28-35ºC. Finally 5.6gms (0.0195M) of DDH was added and the reaction mass was refluxed for 180 minutes or till the TLC indicates the completion of reaction. The reaction mass was cooled to 15ºC, filtered, washed with 200ml of petroleum ether. The entire solvent was then evaporated under vacuum and the residue was used as such in the next step. i.e. Epimerization.
Similarly, by using the above procedure, 7-bromo-cholesterol-3- butyrate(2b); 7-bromo-cholesterol-3- propionate(2c); 7-bromo-cholesterol-3- valerate(2d) and 7-bromo-cholesterol-3- benzoate(2e).

Example 3
Epimerization to 7-alpha-bromo-cholesteryl-3-acetate(3a)

The crude bromo derivative was dissolved in 120ml Toluene and/or 108ml acetone/ Methyl ethyl ketone/methyl isobutyl ketone or 100ml tetrahydrofuran, cooled to -10ºC to 30ºC. 2gms of tetrabutylammonium bromide was added and the reaction mass was stirred at -10ºC to 30ºC for 2-4 hours and epimerized to 7a-Bromocholesteryl-3-acetate(3a). After 2-4 hours the reaction mass was used as such in the next step.

Similarly 7-Bromocholesteryl-3-butyrate (2b), 7-Bromocholesteryl-3-propionate (2c), 7-Bromocholesteryl-3-valerate (2d) and 7-Bromocholesteryl-3-benzoate(2e) is prepared from its respective derivatives and subsequently epimerized predominantly to 7a-Bromocholesteryl-3-butyrate(3b), 7a-Bromocholesteryl-3-propionate(3c), 7a-Bromocholesteryl-3-valerate(3d) and 7a-Bromocholesteryl-3- benzoate(3e) respectively.

Example 4
Synthesis of 7ß-Thiophenyl-Cholesteryl-3-acetate(4a)

The reaction mass containing the epimerized bromo derivative predominantly 7a-Bromo-Cholesteryl-3-acetate (3a), from the previous step was cooled to 0-5ºC. 2.5gms (0.036M) of 25% Liquor Ammonia was added at 0-5ºC followed by 4 gms (0.036M) of Thiophenol and the reaction mass was stirred at 0-5º-30ºC for 2-5 hours. After 5 hours the reaction mass was washed with a) 10ml water b) 20ml of 1N Hydrochloric acid and c) 10ml of saturated salt solution. The organic phase is then evaporated under vacuum and the concentrated reaction mass consisting predominantly of 7ß-Thiophenyl-Cholesteryl-3-acetate(4a),is used as such in the next step i.e Oxidation.

Similarly, the 7ß-Thiophenyl-Cholesteryl-3-butyrate(4b), 7ß-Thiophenyl-Cholesteryl-3-propionate(4c),7ß-Thiophenyl-Cholesteryl-3-valerate(4d) and 7ß-Thiophenyl-Cholesteryl-3-benzoate(4e) were prepared. The HPLC analysis of 3-Butyrate, propionate, valerate and benzoate Derivatives indicate similar conversion as the 3-Acetate thiol derivative.

HPLC analysis: 75-91% of 7ß-Isomer and 2-11% of 7a-isomer
Example 5
Synthesis of 7ß-Thiophenyl-Cholesteryl-3-acetate (Alternative route)

The reaction mass containing the epimerized bromo derivative, predominantly 7a-Bromo-Cholesteryl-3-acetate, from the previous step was cooled to 0-5ºC. 0.5gms (0.0072M) of 25% Liquor Ammonia was added at 0-5ºC followed by 4.8gms (0.36M) of sodium salt of Thiophenol. The reaction mass was stirred at 0-5º-30ºC for 2-5 hours. After 5 hours, the reaction mass was washed with 1)10ml water 2) 20ml of 1N Hydrochloric acid 3) and finally with 10ml of saturated salt solution.

The solvent was then evaporated under vacuum and the reaction mass used as such in the next step i.e Oxidation.

HPLC analysis: 75-91% of 7ß-Isomer and 2-11% of 7a-isomer
The above reaction can also be performed by using Potassium and Lithium salts of Thiophenol.

Example 6: Oxidation
Synthesis of 7-(phenylsulfoxide)-Cholesteryl-3-acetate(5a)

The crude concentrated Thiol derivative (4a) was dissolved in 60ml toluene, cooled to -30 to 50ºC preferably cooled to -10 to 20ºC and more preferably cooled to -10 to 0ºC. 8gms (0.0281M) of Titanium tetraisopropoxide was added and the entire reaction mass was stirred at -10 to 0ºC for 1-2 hours. After 2 hours, 8gms(0.0424M) of 60-90% preferably 80% Cumene hydrogen peroxide was added gradually in 1-4 hours preferably between 1-2 hours maintaining the temperature at -10 to 0ºC and the reaction mass stirred at -5 to 5ºC for 1-4hours or till the TLC/HPLC analysis indicates the completion of reaction. After 3 hours 50ml of water was added at 0ºC. The entire reaction mass was then filtered over Hy-flo; the organic and the aqueous layers are separated; the organic layer was washed with 50ml of 1N sodium metabisulphite solution and finally with 30ml water; dried over anhydrous sodium sulphate and the solvent was removed under vacuum. After complete evaporation of the solvent, the residue- 7-(phenylsulfoxide)-Cholesteryl-3-acetate(5a)was used as such in the next reaction.

Similarly 7-(phenylsulfoxide)-Cholesteryl-3-butyrate(5b), 7-(phenylsulfoxide)-Cholesteryl-3-propionate(5c),7-(phenylsulfoxide)-Cholesteryl-3-valerate(5d)
7-(phenylsulfoxide)-Cholesteryl-3-benzoate(5e) was prepared from their respective thiol derivatives with similar purity levels as the 3-Acetate derivative.
HPLC analysis: 7-(phenylsulfoxide)-Cholesteryl-3-acetate >90%

Example 7:Elimination
Synthesis of 7-Dehydrocholesterol acetate(6a)

The crude concentrated sulfoxide(5a) derivative was dissolved in 60-90ml of Toluene. 1gms of anhydrous sodium acetate was added and the reaction mass heated at 70ºC for 8-32 hours preferably 10-25 hours or more preferably 14-18 hours or till the HPLC analysis indicates the absence of starting material i.e Sulfoxide. The reaction mass was cooled to room temperature and washed with 3 x 25ml water. The organic layer was then dried over anhydrous sodium sulphate; the solvent evaporated under vacuum at 50-60ºC and the crude concentrated residue-7-Dehydrocholesterol acetate(6a) was used as such in the next step.

Similarly using the above procedure, the compounds 5b-e are converted to 6b-e and also all the substituted thiophenol sulphoxide derivatives (23-37a-e), are converted to 6a-e. HPLC analysis: Crude 7-Dehydrocholesterol acetate 55-85%

Example 8
Synthesis of 7-Dehydrocholesterol(7a)

The crude 7-Dehydrocholesterol acetate(6a) was dissolved in 50ml toluene at 28-30ºC. 2gms(0.05M) of sodium hydroxide dissolved in 20ml water and 100ml methanol was added to the reaction mass and heated to 55-70ºC for 2-4 hours or till the TLC and HPLC analysis indicates the absence of starting material. After the completion of reaction, the reaction mass was cooled to 28-30ºC and washed with a)10ml water b) 3x15ml saturated salt solution or till the pH of the aqueous layer is 7(neutral pH). The solvent was evaporated under vacuum at 55-70ºC and the residue was purified by crystallization either from a mixture of solvents like Dichloromethane and Methanol; Toluene and methanol, Methanol and Di-isopropyl ether or from lone solvent such as methanol; ethanol or 2-propanol. The product was filtered and dried at 55-70ºC till constant weight.

Appearance: White solid
Yield: 7.5-8.5gms
%Yield (wrt cholesterol):70-78%
HPLC purity: >94%
Similarly the compounds 6b-e are saponified to 7-Dehydrocholesterol (7a) with similar yields and purity.
The saponification can also be performed by Potassium hydroxide, Sodium borohydride and n-Butyl Lithium.

Example 8 & Example 9 can be done also in one step as insitu reaction.
Example 9: Elimination and saponification:
Reaction:

The crude concentrated sulfoxide(5a) derivative was dissolved in 60-90ml of Toluene. 1gms of anhydrous sodium acetate was added and the reaction mass was heated at 70ºC for 8-32 hours preferably 10-25 hours or more preferably,14-18 hours or till the HPLC analysis indicates the absence of starting material i.e Sulfoxide. The reaction mass was cooled to 50-70°C. 2gms(0.05M) of sodium hydroxide dissolved in 2ml water and 10ml methanol was added to the reaction mass and heated to 55-70ºC for 2-4 hours or till the TLC and HPLC analysis indicates the absence of starting material. After the completion of reaction, the reaction mass was cooled to 28-30ºC and washed with a)100ml water b) 3x150ml saturated salt solution or till the pH of the aqueous layer is 7(neutral pH). The solvent was evaporated under vacuum at 55-70ºC and the residue was purified by crystallization either from a mixture of solvents like Dichloromethane and Methanol or Toluene and methanol or Methanol and Di-isopropyl ether or from lone solvent such as methanol; ethanol or 2-propanol, Acetone, 2-Butanone, Methyl isobutylketone or Di-isobutylketone. The product (7a) was filtered and dried at 55-70ºC till constant weight.

Appearance: White solid
Yield: 7.5-8.5gms
%Yield (wrt cholesterol):70-78%
HPLC purity: >94%

Similarly the compounds 6b-e and 23-37a-e, are converted to 7-dehydrocholesterol (7a) in a one pot reaction with similar yields and purity.

Example 10
Purification of crude 7-Dehydrocholesterol Acetate(6a)
As per J. Org. Chem. 1981, 46, 1030 the crude 7-Dehydrocholesterol acetate is purified by using a short column chromatography using silica gel and a mixture of solvents Methylene dichloride and n-Hexane as the mobile phase. We have purified the crude 7-Dehydrocholesterol acetate by suspending the crude mass in either Acetone, methyl ethyl ketone, Methyl isobutyl ketone or methanol, ethanol, isopropanol or in a mixture of solvents such methylene dichloride and methanol/Ethanol/isopropanol or Di-isopropyl ether and methanol/Ethanol/isopropanol.

The crude 7-Dehydrocholesterol acetate(6a) is suspended in 2-3 volumes of any of the above mentioned solvents and cooled to -10°C. After 3-4 hours the solids are filtered, washed with 1-4 volumes of any of the above mentioned solvents, dried under vacuum at 30-80°C.
Appearance: white crystalline solid
Yield: 9-10gms
HPLC purity: >90%.

Similarly the compounds 6b-e can be purified and isolated in pure form.
The purified compounds 6a-e can be further saponified as mentioned in example 8 to obtain 7-Dehydrocholesterol(7a).
Appearance: white crystalline solid
Yield: 7.5-8.5gms
HPLC purity: >94%.

Example 11
Synthesis of 7ß-(2’-nitrothiophenyl)-Cholesteryl-3-acetate(8a)
Reaction:

The reaction mass containing the epimerized bromo derivative predominantly 7a-Bromo-Cholesteryl-3-acetate (3a), from the previous step was cooled to 0-5ºC. 2.5gms (0.036M) of 25% Liquor Ammonia was added at 0-5ºC followed by 5.6gms (0.036M) of 2-Nitro thiophenol and the reaction mass was stirred at 0-5º-30ºC for 2-5 hours. After 5 hours the reaction mass was washed with a) 10ml water b) 20ml of 1N Hydrochloric acid and c) 10ml of saturated salt solution. The organic phase is then evaporated under vacuum and the concentrated reaction mass consisting predominantly of 7ß-(2’-nitrothiophenyl)-Cholesteryl-3-acetate(8a) is used as such in the next step i.e Oxidation.
Similarly, the 7ß-(2’-nitrothiophenyl)-Cholesteryl-3-butyrate(8b), 7ß-(2’-nitrothiophenyl)-Cholesteryl-3-propionate(8c),7ß-(2’-Nitrothiophenyl)-Cholesteryl-3-valerate(8d) and 7ß-(2’-Nitrothiophenyl)-Cholesteryl-3-benzoate(8e) were prepared. The HPLC analysis of 3-Butyrate and benzoate Derivatives indicate similar conversion as the 3-Acetate thiol derivative.

HPLC analysis: 75-91% of 7ß-Isomer and 2-11% of 7a-isomer

Similarly the following compounds are prepared:
1. 7ß-(3’-Aminothiophenyl)-Cholesteryl-3-acetate(9a) by treating 7a-Bromo-Cholesteryl-3-acetate(3a) with 3-Aminothiophenol.
2. 7ß-(4’-Methylthiophenyl)-Cholesteryl-3-acetate(10a) by treating 7a-Bromo-Cholesteryl-3-acetate(3a) with 3-Methyl thiophenol.
3. 7ß-(4’-Chlorothiophenyl)-Cholesteryl-3-acetate(11a) by treating 7a-Bromo-Cholesteryl-3-acetate(3a) with 4-Chloro thiophenol.
4. 7ß-(4’-Bromothiophenyl)-Cholesteryl-3-acetate(12a) by treating 7a-Bromo-Cholesteryl-3-acetate(3a) with 4-Bromo thiophenol.
5. 7ß-(2’-Bromothiophenyl)-Cholesteryl-3-acetate(13a) by treating 7a-Bromo-Cholesteryl-3-acetate(3a) with 2-Bromo thiophenol.
6. 7ß-(4’-Bromo-2’-trifluoromethoxy thiophenyl)-Cholesteryl-3-acetate(14a) by treating 7a-Bromo-Cholesteryl-3-acetate(3a) with 4-Bromo-2-Trifluoromethoxy thiophenol.
7. 7ß-(4’-Methoxy thiophenyl)-Cholesteryl-3-acetate(15a) by treating 7a-Bromo-Cholesteryl-3-acetate(3a) with 4-Methoxy thiophenol.
8. 7ß-(3’-Methoxy thiophenyl)-Cholesteryl-3-acetate(16a) by treating 7a-Bromo-Cholesteryl-3-acetate(3a) with 3-Methoxy thiophenol.
9. 7ß-(2’,4’-Difluoro thiophenyl)-Cholesteryl-3-acetate(17a) by treating 7a-Bromo-Cholesteryl-3-acetate(3a) with 2,4-Difluoro thiophenol
10. 7ß-(3’,4’-Difluoro thiophenyl)-Cholesteryl-3-acetate(18a) by treating
7a-Bromo-Cholesteryl-3-acetate(3a) with 3,4-Difluoro thiophenol.
11. 7ß-(3’,4’-Dimethoxy thiophenyl)-Cholesteryl-3-acetate(19a) by
treating 7a-Bromo-Cholesteryl-3-acetate(3a) with 3,4-DiMethoxy
thiophenol.
12. 7ß-(4’-Trifluoromethyl thiophenyl)-Cholesteryl-3-acetate(20a) by
treating 7a-Bromo-Cholesteryl-3-acetate(3a) with 4-(Trifluoromethyl)
thiophenol.
13. 7ß-(2’-Amino-4’-chloro thiophenyl)-Cholesteryl-3-acetate(21a) by
treating 7a-Bromo-Cholesteryl-3-acetate(3a) with 2-Amino-4-chloro
thiophenol.
14. 7ß-(2’,3’-Dichlorothiophenyl)-Cholesteryl-3-acetate(22a) by treating
7a-Bromo-Cholesteryl-3-acetate(3a) with 2,3-Dichloro thiophenol

Similarly the following butyrate, propionate, valerate and benzoate derivatives are prepared:
1. 7ß-(2’-nitrothiophenyl)-Cholesteryl-3-butyrate(8b) by treating 7a-Bromo-Cholesteryl-3-butyrate(3b) with 2-Nitro thiophenol.
2. 7ß-(2’-nitrothiophenyl)-Cholesteryl-3-propionate(8c) by treating 7a-Bromo-Cholesteryl-3-propionate(3c) with 2-Nitro thiophenol.
3. 7ß-(2’-nitrothiophenyl)-Cholesteryl-3-valerate(8d) by treating 7a-Bromo-Cholesteryl-3-valerate(3d) with 2-Nitro thiophenol.
4. 7ß-(2’-nitrothiophenyl)-Cholesteryl-3-benzoate(8e) by treating 7a-Bromo-Cholesteryl-3-valerate(3e) with 2-Nitro thiophenol.
5. 7ß-(3’-Aminothiophenyl)-Cholesteryl-3-butyrate(9b) by treating 7a-Bromo-Cholesteryl-3-butyrate(3b) with 3-Aminothiophenol.
6. 7ß-(3’-Aminothiophenyl)-Cholesteryl-3-propionate(9c) by treating 7a-Bromo-Cholesteryl-3-propionate(3c) with 3-Aminothiophenol.
7. 7ß-(3’-Aminothiophenyl)-Cholesteryl-3-valerate(9d) by treating 7a-Bromo-Cholesteryl-3-valerate(3d) with 3-Aminothiophenol.
8. 7ß-(3’-Aminothiophenyl)-Cholesteryl-3-benzoate(9e) by treating 7a-Bromo-Cholesteryl-3-benzoate(3e) with 3-Aminothiophenol.
9. 7ß-(4’-Methylthiophenyl)-Cholesteryl-3-butyrate(10b) by treating 7a-Bromo-Cholesteryl-3-butyrate(3b) with 3-Methyl thiophenol.
10. 7ß-(4’-Methylthiophenyl)-Cholesteryl-3-propionate(10c) by treating 7a-Bromo-Cholesteryl-3-propionate(3c) with 3-Methyl thiophenol.
11. 7ß-(4’-Methylthiophenyl)-Cholesteryl-3-valerate(10d) by treating 7a-Bromo-Cholesteryl-3-valerate(3d) with 3-Methyl thiophenol.
12. 7ß-(4’-Methylthiophenyl)-Cholesteryl-3-benzoate(10e) by treating
7a-Bromo-Cholesteryl-3-benzoate(3e) with 3-Methyl thiophenol.
13. 7ß-(4’-Chlorothiophenyl)-Cholesteryl-3-butyrate(11b) by treating
7a-Bromo-Cholesteryl-3-butyrate(3b) with 4-Chloro thiophenol
14. 7ß-(4’-Chlorothiophenyl)-Cholesteryl-3-propionate(11c) by treating 7a-Bromo-Cholesteryl-3-propionate(3c) with 4-Chloro thiophenol.
15. 7ß-(4’-Chlorothiophenyl)-Cholesteryl-3-valerate(11d) by treating
7a-Bromo-Cholesteryl-3-valerate(3d) with 4-Chloro thiophenol.
16. 7ß-(4’-Chlorothiophenyl)-Cholesteryl-3-benzoate(11e) by treating
7a- Bromo-Cholesteryl-3-benzoate(3e) with 4-Chloro thiophenol.
17.7ß-(4’-Bromothiophenyl)-Cholesteryl-3-butyrate(12b) by treating
7a-Bromo-Cholesteryl-3-butyrate(3b) with 4-Bromo thiophenol.
18. 7ß-(4’-Bromothiophenyl)-Cholesteryl-3-propionate(12c) by treating
7a-Bromo-Cholesteryl-3-propionate(3c) with 4-Bromo thiophenol.
19.7ß-(4’-Bromothiophenyl)-Cholesteryl-3-valerate(12d) by treating
7a-Bromo-Cholesteryl-3-valerate(3d) with 4-Bromo thiophenol.
20. 7ß-(4’-Bromothiophenyl)-Cholesteryl-3-benzoate(12e) by treating
7a-Bromo-Cholesteryl-3-benzoate(3b) with 4-Bromo thiophenol.
21.7ß-(2’-Bromothiophenyl)-Cholesteryl-3-butyrate(13b) by treating
7a-Bromo-Cholesteryl-3-butyrate(3b) with 2-Bromo thiophenol.
22. 7ß-(2’-Bromothiophenyl)-Cholesteryl-3-propionate(13c) by treating
7a-Bromo-Cholesteryl-3-propionate(3c) with 2-Bromo thiophenol.
23.7ß-(2’-Bromothiophenyl)-Cholesteryl-3-valerate(13d) by treating
7a-Bromo-Cholesteryl-3-valerate(3d) with 2-Bromo thiophenol.
24. 7ß-(2’-Bromothiophenyl)-Cholesteryl-3-benzoate(13e) by treating
7a-Bromo-Cholesteryl-3-benzoate(3e) with 2-Bromo thiophenol.
25. 7ß-(4’-Bromo-2’-trifluoromethoxy thiophenyl)-Cholesteryl-3- butyrate(14b) by treating 7a-Bromo-Cholesteryl-3-butyrate(3b) with 4-Bromo-2-Trifluoromethoxy thiophenol.
26. 7ß-(4’-Bromo-2’-trifluoromethoxy thiophenyl)-Cholesteryl-3-propionate(14c) by treating 7a-Bromo-Cholesteryl-3-propionate(3c) with 4-Bromo-2-Trifluoromethoxy thiophenol.
27. 7ß-(4’-Bromo-2’-trifluoromethoxy thiophenyl)-Cholesteryl-3-valerate(14d) by treating 7a-Bromo-Cholesteryl-3-valerate(3d) with 4-Bromo-2-Trifluoromethoxy thiophenol.
28. 7ß-(4’-Bromo-2’-trifluoromethoxy thiophenyl)-Cholesteryl-3-benzoate(14e) by treating 7a-Bromo-Cholesteryl-3-benzoate(3c) with 4-Bromo-2-Trifluoromethoxy thiophenol.
29. 7ß-(4’-methoxy thiophenyl)-Cholesteryl-3-butyrate(15b) by treating 7a-Bromo-Cholesteryl-3-butyrate(3b) with 4-methoxy thiophenol.
30. 7ß-(4’-methoxy thiophenyl)-Cholesteryl-3-propionate(15c) by treating 7a-Bromo-Cholesteryl-3-propionate(3c) with 4-methoxy thiophenol.
31. 7ß-(4’-methoxy thiophenyl)-Cholesteryl-3-valerate(15d) by treating 7a-Bromo-Cholesteryl-3-valerate(3d) with 4-methoxy thiophenol.
32. 7ß-(4’-methoxy thiophenyl)-Cholesteryl-3-benzoate(15e) by treating 7a-Bromo-Cholesteryl-3-benzoate(3e) with 4-methoxy thiophenol.
33. 7ß-(3’-methoxy thiophenyl)-Cholesteryl-3-butyrate(16b) by treating 7a-Bromo-Cholesteryl-3-butyrate(3b) with 3-methoxy thiophenol.
34. 7ß-(3’-methoxy thiophenyl)-Cholesteryl-3-propionate(16c) by treating 7a-Bromo-Cholesteryl-3-propionate(3c) with 3-methoxy thiophenol.
35. 7ß-(3’-methoxy thiophenyl)-Cholesteryl-3-valerate(16d) by treating 7a-Bromo-Cholesteryl-3-valerate(3d) with 3-methoxy thiophenol.
36. 7ß-(3’-methoxy thiophenyl)-Cholesteryl-3-benzoate(16e) by treating 7a-Bromo-Cholesteryl-3-benzoate(3e) with 3-methoxy thiophenol.
37. 7ß-(2’,4’-Difluoro thiophenyl)-Cholesteryl-3-butyrate(17b) by treating 7a-Bromo-Cholesteryl-3-butyrate(3b) with 2,4-Difluoro thiophenol.
38. 7ß-(2’,4’-Difluoro thiophenyl)-Cholesteryl-3-propionate(17c) by treating 7a-Bromo-Cholesteryl-3-propionate(3c) with 2,4-Difluoro thiophenol.
39. 7ß-(2’,4’-Difluoro thiophenyl)-Cholesteryl-3-valerate(17d) by treating 7a-Bromo-Cholesteryl-3-valerate(3d) with 2,4-Difluoro thiophenol.
40. 7ß-(2’,4’-Difluoro thiophenyl)-Cholesteryl-3-benzoate(17e) by treating 7a-Bromo-Cholesteryl-3-benzoate(3e) with 2,4-Difluoro thiophenol.
41. 7ß-(3’,4’-Difluoro thiophenyl)-Cholesteryl-3-butyrate(18b) by treating 7a-Bromo-Cholesteryl-3-butyrate(3b) with 3,4-Difluoro thiophenol.
42. 7ß-(3’,4’-Difluoro thiophenyl)-Cholesteryl-3-propionate(18c) by treating 7a-Bromo-Cholesteryl-3-propionate(3c) with 3,4-Difluoro thiophenol.
43. 7ß-(3’,4’-Difluoro thiophenyl)-Cholesteryl-3-valerate(18d) by treating 7a-Bromo-Cholesteryl-3-valerate(3d) with 3,4-Difluoro thiophenol.
44. 7ß-(3’,4’-Difluoro thiophenyl)-Cholesteryl-3-benzoate(18e) by treating 7a-Bromo-Cholesteryl-3-benzoate(3e) with 3,4-Difluoro thiophenol.
45. 7ß-(3’,4’-Dimethoxy thiophenyl)-Cholesteryl-3-butyrate(19b) by treating 7a-Bromo-Cholesteryl-3-butyrate(3b) with 3,4-Dimethoxy thiophenol.
46. 7ß-(3’,4’-Dimethoxy thiophenyl)-Cholesteryl-3-propionate(19c) by treating 7a-Bromo-Cholesteryl-3-propionate(3c) with 3,4-Dimethoxy thiophenol.
47. 7ß-(3’,4’-Dimethoxy thiophenyl)-Cholesteryl-3-valerate(19d) by treating 7a-Bromo-Cholesteryl-3-valerate(3d) with 3,4-Dimethoxy thiophenol.
48. 7ß-(3’,4’-Dimethoxy thiophenyl)-Cholesteryl-3-benzoate(19e) by treating 7a-Bromo-Cholesteryl-3-benzoate(3d) with 3,4-Dimethoxy thiophenol.
49. 7ß-(4’-trifluoromethyl thiophenyl)-Cholesteryl-3-butyrate(20b) by treating 7a-Bromo-Cholesteryl-3-butyrate(3b) with 4-Trifluoromethyl thiophenol.
50. 7ß-(4’-trifluoromethyl thiophenyl)-Cholesteryl-3-propionate(20c) by treating 7a-Bromo-Cholesteryl-3-propionate(3c) with 4-Trifluoromethyl thiophenol.
51. 7ß-(4’-trifluoromethyl thiophenyl)-Cholesteryl-3-valerate(20d) by treating 7a-Bromo-Cholesteryl-3-valerate(3d) with 4-Trifluoromethyl thiophenol.
52. 7ß-(4’-trifluoromethyl thiophenyl)-Cholesteryl-3-benzoate(20e) by treating 7a-Bromo-Cholesteryl-3-benzoate(3e) with 4-Trifluoromethyl thiophenol.
53. 7ß-(2’-amino-4’-chloro thiophenyl)-Cholesteryl-3-butyrate(21b) by treating 7a-Bromo-Cholesteryl-3-butyrate(3b) with 2-Amino-4-chloro thiophenol.
54. 7ß-(2’-amino-4’-chloro thiophenyl)-Cholesteryl-3-propionate(21c) by treating 7a-Bromo-Cholesteryl-3-propionate(3c) with 2-Amino-4-chloro thiophenol.
55. 7ß-(2’-amino-4’-chloro thiophenyl)-Cholesteryl-3-valerate(21d) by treating 7a-Bromo-Cholesteryl-3-valerate(3d) with 2-Amino-4-chloro thiophenol.
56. 7ß-(2’-amino-4’-chloro thiophenyl)-Cholesteryl-3-benzoate(21e) by treating 7a-Bromo-Cholesteryl-3-benzoate(3e) with 2-Amino-4-chloro thiophenol.
57. 7ß-(2’, 3’-Dichloro thiophenyl)-Cholesteryl-3-butyrate(22b) by treating 7a-Bromo-Cholesteryl-3-butyrate(3b) with 2-Amino-4-chloro thiophenol.
58. 7ß-(2’, 3’-Dichloro thiophenyl)-Cholesteryl-3-propionate(22c) by treating 7a-Bromo-Cholesteryl-3-propionate(3c) with 2-Amino-4-chloro thiophenol.
59. 7ß-(2’, 3’-Dichloro thiophenyl)-Cholesteryl-3-valerate(22d) by treating 7a-Bromo-Cholesteryl-3-valerate(3d) with 2-Amino-4-chloro thiophenol.
60. 7ß-(2’, 3’-Dichloro thiophenyl)-Cholesteryl-3-benzoate(22e) by treating 7a-Bromo-Cholesteryl-3-benzoate(3e) with 2-Amino-4-chloro thiophenol.

All the above thiophenol derivatives 4a-e, 10a-e, 11a-e, 12a-e, 13a-e, 14a-e, 15a- e, 16a-e, 17a-e can be synthesized by using the sodium, potassium and lithium salts of the respective thiophenols. The Yields and the purity are similar to that obtained by using free thiophenols.

In the above experiments 7-bromo-3-cholesterol esters are treated with Thiophenols and substituted thiophenols in presence of a base. In this case we have used 10-30% liquor ammonia. As per Confalone, et al. in J. Org. Chem. 1981, 46, 1030-1032, the above reactions have been performed by using triethyl amine as a base. We have even used N, N-Diisopropyl ethyl amine and the results are similar. It is cost effective to use 10-30% liquor ammonia than any other organic amines.

Example 12
Synthesis of 7-(2’-nitro thiophenylsulfoxide)-Cholesteryl-3-acetate(23a)
Reaction:

The crude concentrated Thiol derivative(8a) was dissolved in 60ml toluene, cooled to -30 to 50ºC preferably cooled to -10 to 20ºC and more preferably cooled to -10 to 0ºC. 8gms (0.0287M) of Titanium tetraisopropoxide was added and the entire reaction mass was stirred at -10 to 0ºC for 1-2 hours. After 2 hours, 8gms(0.424M) of 60-90% preferably 80% Cumene hydrogen peroxide was added gradually in 1-4 hours preferably between 1-2 hours maintaining the temperature at -10 to 0ºC and the reaction mass stirred at -5 to 5ºC for 1-4hours or till the TLC/HPLC analysis indicates the completion of reaction. After 3 hours 50ml of water was added at 0ºC. The entire reaction mass was then filtered over Hy-flo; the organic and the aqueous layers are separated; the organic layer was washed with 50ml of 1N sodium metabisulphite solution and finally with 30ml water; dried over anhydrous sodium sulphate and the solvent was removed under vacuum. After complete evaporation of the solvent, the residue- 7-(2-Nitrothiophenylsulfoxide)-Cholesteryl-3-acetate (23a) was used as such in the next reaction.

Similarly 7-(2’-Nitro-thiophenylsulfoxide)-Cholesteryl-3-butyrate(23b),
7-(2’-Nitro-thiophenylsulfoxide)-Cholesteryl-3-propionate (23c), 7-(2’-Nitro thiophenylsulfoxide)-Cholesteryl-3-valerate(23d) and
7-(2-Nitro-thiophenylsulfoxide)-Cholesteryl-3-benzoate(23e) was prepared from their respective thiol derivatives with similar purity levels as the 3-Acetate derivative.
HPLC analysis: 7-(phenylsulfoxide)-Cholesteryl-3-acetate >92%

Similarly the following compounds are prepared:
1. 7-(3’-amino thiophenylsulfoxide)-Cholesteryl-3-acetate(24a) is prepared by oxidizing 7ß-(3’-Aminothiophenyl)-Cholesteryl-3-acetate(9a).
2. 7-(4’-methyl thiophenylsulfoxide)-Cholesteryl-3-acetate(25a) is prepared by oxidizing 7ß-(4’-Methylthiophenyl)-Cholesteryl-3-acetate(10a).
3. 7-(4’-Chloro thiophenylsulfoxide)-Cholesteryl-3-acetate(26a) is
prepared by oxidising 7ß-(4’-Chlorothiophenyl)-Cholesteryl-3
-acetate(11a).
4. 7-(4’-Bromo thiophenylsulfoxide)-Cholesteryl-3-acetate(27a) is
prepared by oxidising 7ß-(4’-Bromothiophenyl)-Cholesteryl-3-
acetate(12a).
5. 7-(2’-Bromo thiophenylsulfoxide)-Cholesteryl-3-acetate(28a) is
prepared by oxidising 7ß-(2’-Bromothiophenyl)-Cholesteryl-3-
acetate(13a).
6. 7-(4’-Bromo-2’-trifluoromethoxy thiophenylsulfoxide)-Cholesteryl-3-acetate(29a) is prepared by oxidizing 7ß-(4’-Bromo-2’-trifluoromethoxy thiophenyl)-Cholesteryl-3-acetate(14a).
7. 7-(4’-Methoxy thiophenylsulfoxide)-Cholesteryl-3-acetate(30a) is prepared by oxidizing 7ß-(4’-Methoxy thiophenyl)-Cholesteryl-3-acetate(15a).
8. 7-(3’-Methoxy thiophenylsulfoxide)-Cholesteryl-3-acetate(31a) is prepared by oxidizing 7ß-(3’-Methoxy thiophenyl)-Cholesteryl-3-acetate(16a).
9. 7-(2’,4’-Difluoro thiophenylsulfoxide)-Cholesteryl-3-acetate(32a) is prepared by oxidising 7ß-(2’,4’-Difluoro thiophenyl)-Cholesteryl-3-acetate(17a).
10. 7-(3’,4’-Difluoro thiophenylsulfoxide)-Cholesteryl-3-acetate(33a) is
prepared by oxidizing 7ß-(3’,4’-Difluoro thiophenyl)-Cholesteryl-3-
acetate(18a).
11. 7-(3’,4’-Dimethoxy thiophenylsulfoxide)-Cholesteryl-3-acetate(34a) is
prepared by oxidizing 7ß-(3’,4’-Dimethoxy thiophenyl)-Cholesteryl
-3-acetate(19a).
12. 7-(4’-Trifluoromethyl thiophenylsulfoxide)-Cholesteryl-3-
acetate(35a) is prepared by oxidising 7ß-(4’-Trifluoromethyl
thiophenyl)- Cholesteryl-3-acetate(20a) .
13. 7-(2’-Amino-4’-chloro thiophenylsulfoxide)-Cholesteryl-3-
acetate(36a) is prepared by oxidizing 7ß-(2’-Amino-4’-chloro
thiophenyl)-Cholesteryl-3-acetate(21a).
14. 7-(2’,3’-Dichlorothiophenylsulfoxide)-Cholesteryl-3-acetate(37a) is prepared by oxidizing 7ß-(2’,3’-Dichlorothiophenyl)-Cholesteryl-3-
acetate(22a).
Similarly the following butyrate, propionate, valerate and benzoate derivatives are prepared:
1. 7-(3’-amino thiophenylsulfoxide)-Cholesteryl-3-butyrate(24b) is prepared by oxidizing 7ß-(3’-Aminothiophenyl)-Cholesteryl-3-butyrate(9b).
2. 7-(3’-amino thiophenylsulfoxide)-Cholesteryl-3-propionate(24c) is prepared by oxidizing 7ß-(3’-Aminothiophenyl)-Cholesteryl-3-propionate(9c).
3. 7-(3’-amino thiophenylsulfoxide)-Cholesteryl-3-valerate(24d) is prepared by oxidizing 7ß-(3’-Aminothiophenyl)-Cholesteryl-3-valerate(9d).
4. 7-(3’-amino thiophenylsulfoxide)-Cholesteryl-3-benzoate(24e) is prepared by oxidizing 7ß-(3’-Aminothiophenyl)-Cholesteryl-3-benzoate(9e).
5. 7-(4’-methyl thiophenylsulfoxide)-Cholesteryl-3-butyrate(25b) is prepared by oxidizing 7ß-(4’-Methylthiophenyl)-Cholesteryl-3-acetate(10b).
6. 7-(4’-methyl thiophenylsulfoxide)-Cholesteryl-3-propionate(25c) is prepared by oxidizing 7ß-(4’-Methylthiophenyl)-Cholesteryl-3-proponate(10c).
7. 7-(4’-methyl thiophenylsulfoxide)-Cholesteryl-3-valerate(25d) is prepared by oxidizing 7ß-(4’-Methylthiophenyl)-Cholesteryl-3-valerate(10d).
8. 7-(4’-methyl thiophenylsulfoxide)-Cholesteryl-3-benzoate(25e) is prepared by oxidizing 7ß-(4’-Methylthiophenyl)-Cholesteryl-3-benzoate(10e).
9. 7-(4’-Chloro thiophenylsulfoxide)-Cholesteryl-3-butyrate(26b) is
prepared by oxidising 7ß-(4’-Chlorothiophenyl)-Cholesteryl-3
-butyrate(11b).
10. 7-(4’-Chloro thiophenylsulfoxide)-Cholesteryl-3-propionate(26c) is
prepared by oxidising 7ß-(4’-Chlorothiophenyl)-Cholesteryl-3
-propionate(11c).
11. 7-(4’-Chloro thiophenylsulfoxide)-Cholesteryl-3-valerate(26d) is
prepared by oxidising 7ß-(4’-Chlorothiophenyl)-Cholesteryl-3
-valerate(11d).
12. 7-(4’-Chloro thiophenylsulfoxide)-Cholesteryl-3-benzoate(26e) is
prepared by oxidising 7ß-(4’-Chlorothiophenyl)-Cholesteryl-3
-acetate(11a).
13.7-(4’-Bromo thiophenylsulfoxide)-Cholesteryl-3-butyrate(27b) is
prepared by oxidising 7ß-(4’-Bromothiophenyl)-Cholesteryl-3-
butyrate(12b).
14.7-(4’-Bromo thiophenylsulfoxide)-Cholesteryl-3-propionate(27c) is
prepared by oxidising 7ß-(4’-Bromothiophenyl)-Cholesteryl-3-
propionate(12c).
15.7-(4’-Bromo thiophenylsulfoxide)-Cholesteryl-3-valerate(27d) is
prepared by oxidising 7ß-(4’-Bromothiophenyl)-Cholesteryl-3-
acetate(12d).
16.7-(4’-Bromo thiophenylsulfoxide)-Cholesteryl-3-benzoate(27e) is
prepared by oxidising 7ß-(4’-Bromothiophenyl)-Cholesteryl-3-
valerate(12e).
17.7-(2’-Bromo thiophenylsulfoxide)-Cholesteryl-3-butyrate(28b) is
prepared by oxidising 7ß-(2’-Bromothiophenyl)-Cholesteryl-3-
butyrate(13b).
18.7-(2’-Bromo thiophenylsulfoxide)-Cholesteryl-3-propionate(28c) is
prepared by oxidising 7ß-(2’-Bromothiophenyl)-Cholesteryl-3-
acetate(13c).
19. 7-(2’-Bromo thiophenylsulfoxide)-Cholesteryl-3-valerate(28d) is
prepared by oxidising 7ß-(2’-Bromothiophenyl)-Cholesteryl-3-
valerate(13d).
20.7-(2’-Bromo thiophenylsulfoxide)-Cholesteryl-3-benzoate(28e) is
prepared by oxidising 7ß-(2’-Bromothiophenyl)-Cholesteryl-3-
benzoate(13e).
21.7-(4’-Bromo-2’-trifluoromethoxy thiophenylsulfoxide)-Cholesteryl-
3-butyrate(29b) is prepared by oxidizing 7ß-(4’-Bromo-2’-
trifluoromethoxy thiophenyl)-Cholesteryl-3-butyrate(14b).
22.7-(4’-Bromo-2’-trifluoromethoxy thiophenylsulfoxide)-Cholesteryl-
3- propionate(29c) is prepared by oxidizing 7ß-(4’-Bromo-2’
-trifluoromethoxy thiophenyl)-Cholesteryl-3-butyrate(14c).
23.7-(4’-Bromo-2’-trifluoromethoxy thiophenylsulfoxide)-Cholesteryl-3-
valerate(29d) is prepared by oxidizing 7ß-(4’-Bromo-2’-trifluoromethoxy
thiophenyl)-Cholesteryl-3-valerate(14d).
24.7-(4’-Bromo-2’-trifluoromethoxy thiophenylsulfoxide)-Cholesteryl-3-
benzoate(29e) is prepared by oxidizing 7ß-(4’-Bromo-2’-
trifluoromethoxy thiophenyl)-Cholesteryl-3-benzoate(14e).
25.7-(4’-Methoxy thiophenylsulfoxide)-Cholesteryl-3-butyrate(30b) is prepared by oxidizing 7ß-(4’-Methoxy thiophenyl)-Cholesteryl-3-butyrate(15b).
26.7-(4’-Methoxy thiophenylsulfoxide)-Cholesteryl-3-propionate(30c) is
prepared by oxidizing 7ß-(4’-Methoxy thiophenyl)-Cholesteryl-3-
propionate(15c).
27. 7-(4’-Methoxy thiophenylsulfoxide)-Cholesteryl-3-valerate(30d) is
prepared by oxidizing 7ß-(4’-Methoxy thiophenyl)-Cholesteryl-3-
valerate(15d).
28.7-(4’-Methoxy thiophenylsulfoxide)-Cholesteryl-3-benzoate(30e) is
prepared by oxidizing 7ß-(4’-Methoxy thiophenyl)-Cholesteryl-3-
benzoate(15e).
29. 7-(3’-Methoxy thiophenylsulfoxide)-Cholesteryl-3-butyrate(31b) is
prepared by oxidizing 7ß-(3’-Methoxy thiophenyl)-Cholesteryl-3
-butyrate(16b).
30. 7-(3’-Methoxy thiophenylsulfoxide)-Cholesteryl-3-propionate(31c) is prepared by oxidizing 7ß-(3’-Methoxy thiophenyl)-Cholesteryl-3-propionate(16c).
31. 7-(3’-Methoxy thiophenylsulfoxide)-Cholesteryl-3-valerate(31d) is prepared by oxidizing 7ß-(3’-Methoxy thiophenyl)-Cholesteryl-3-valerate(16d).
32. 7-(3’-Methoxy thiophenylsulfoxide)-Cholesteryl-3-benzoate(31e) is prepared by oxidizing 7ß-(3’-Methoxy thiophenyl)-Cholesteryl-3-benzoate(16e).
33. 7-(2’,4’-Difluoro thiophenylsulfoxide)-Cholesteryl-3-butyrate(32b) is prepared by oxidising 7ß-(2’,4’-Difluoro thiophenyl)-Cholesteryl-3-butyrate(17b).
34. 7-(2’,4’-Difluoro thiophenylsulfoxide)-Cholesteryl-3-propionate(32c) is prepared by oxidising 7ß-(2’,4’-Difluoro thiophenyl)-Cholesteryl-3-propionate(17c).
35. 7-(2’,4’-Difluoro thiophenylsulfoxide)-Cholesteryl-3-valerate(32d) is prepared by oxidising 7ß-(2’,4’-Difluoro thiophenyl)-Cholesteryl-3-valerate(17d).
36. 7-(2’,4’-Difluoro thiophenylsulfoxide)-Cholesteryl-3-benzoate(32e) is prepared by oxidising 7ß-(2’,4’-Difluoro thiophenyl)-Cholesteryl-3-benzoate(17e).
37. 7-(3’,4’-Difluoro thiophenylsulfoxide)-Cholesteryl-3-butyrate(33b) is
prepared by oxidizing 7ß-(3’,4’-Difluoro thiophenyl)-Cholesteryl-3-
butyrate(18b).
38.7-(3’,4’-Difluoro thiophenylsulfoxide)-Cholesteryl-3-propionate(33c)
is prepared by oxidizing 7ß-(3’,4’-Difluoro thiophenyl)-Cholesteryl-3-
propionate(18c).
39.7-(3’,4’-Difluoro thiophenylsulfoxide)-Cholesteryl-3-valerate(33d) is
prepared by oxidizing 7ß-(3’,4’-Difluoro thiophenyl)-Cholesteryl-3-
valerate(18d).
40.7-(3’,4’-Difluoro thiophenylsulfoxide)-Cholesteryl-3-benzoate(33e) is
prepared by oxidizing 7ß-(3’,4’-Difluoro thiophenyl)-Cholesteryl-3-
benzoate(18e).
41.7-(3’,4’-Dimethoxy thiophenylsulfoxide)-Cholesteryl-3-butyrate(34b) is
prepared by oxidizing 7ß-(3’,4’-Dimethoxy thiophenyl)-Cholesteryl
-3-butyrate(19b).
42. 7-(3’,4’-Dimethoxy thiophenylsulfoxide)-Cholesteryl-3-propionate(34c) is prepared by oxidizing 7ß-(3’,4’-Dimethoxy thiophenyl)-Cholesteryl
-3-propionate(19c).
43. 7-(3’,4’-Dimethoxy thiophenylsulfoxide)-Cholesteryl-3-valerate(34d) is
prepared by oxidizing 7ß-(3’,4’-Dimethoxy thiophenyl)-Cholesteryl
-3-valerate(19d).
44. 7-(3’,4’-Dimethoxy thiophenylsulfoxide)-Cholesteryl-3-benzoate(34e) is
prepared by oxidizing 7ß-(3’,4’-Dimethoxy thiophenyl)-Cholesteryl
-3-benzoate(19e).
45. 7-(4’-Trifluoromethyl thiophenylsulfoxide)-Cholesteryl-3-
butyrate(35b) is prepared by oxidising 7ß-(4’-Trifluoromethyl
thiophenyl)- Cholesteryl-3-butyrate(20b).
46.7-(4’-Trifluoromethyl thiophenylsulfoxide)-Cholesteryl-3-
propionate(35c) is prepared by oxidising 7ß-(4’-Trifluoromethyl
thiophenyl)- Cholesteryl-3-propionate(20c).
47.7-(4’-Trifluoromethyl thiophenylsulfoxide)-Cholesteryl-3-
valerate(35d) is prepared by oxidising 7ß-(4’-Trifluoromethyl
thiophenyl)- Cholesteryl-3-valerate(20d).
48.7-(4’-Trifluoromethyl thiophenylsulfoxide)-Cholesteryl-3-
benzoate(35e) is prepared by oxidising 7ß-(4’-Trifluoromethyl
thiophenyl)- Cholesteryl-3-benzoate(20e) .
49.7-(2’-Amino-4’-chloro thiophenylsulfoxide)-Cholesteryl-3-
butyrate(36b) is prepared by oxidizing 7ß-(2’-Amino-4’-chloro
thiophenyl)-Cholesteryl-3-butyrate(21b).
50.7-(2’-Amino-4’-chloro thiophenylsulfoxide)-Cholesteryl-3-
propionate(36c) is prepared by oxidizing 7ß-(2’-Amino-4’-chloro
thiophenyl)-Cholesteryl-3-acetate(21c).
51.7-(2’-Amino-4’-chloro thiophenylsulfoxide)-Cholesteryl-3-
valerate(36d) is prepared by oxidizing 7ß-(2’-Amino-4’-chloro
thiophenyl)-Cholesteryl-3-valerate(21d).
52.7-(2’-Amino-4’-chloro thiophenylsulfoxide)-Cholesteryl-3-
benzoate(36e) is prepared by oxidizing 7ß-(2’-Amino-4’-chloro
thiophenyl)-Cholesteryl-3-acetate(21e).
53.7-(2’,3’-Dichlorothiophenylsulfoxide)-Cholesteryl-3-butyrate(37b) is prepared by oxidizing 7ß-(2’,3’-Dichlorothiophenyl)-Cholesteryl-3-
butyrate(22b).
54.7-(2’,3’-Dichlorothiophenylsulfoxide)-Cholesteryl-3-propionate(37c) is prepared by oxidizing 7ß-(2’,3’-Dichlorothiophenyl)-Cholesteryl-3-
propionate(22c).
55. 7-(2’,3’-Dichlorothiophenylsulfoxide)-Cholesteryl-3-valerate(37d) is prepared by oxidizing 7ß-(2’,3’-Dichlorothiophenyl)-Cholesteryl-3-
valerate(22d).
56. 7-(2’,3’-Dichlorothiophenylsulfoxide)-Cholesteryl-3-benzoate(37e) is prepared by oxidizing 7ß-(2’,3’-Dichlorothiophenyl)-Cholesteryl-3-
benzoate(22d). ,CLAIMS:We claim,

1. An improved cost-effective process for synthesis of 7-Dehydrocholesterol with good yield and purity comprising:
a) epimerizing 7(a+ß) bromo protected cholesterol in presence of tetrabutyl ammonium bromide in in toluene or a ketonic solvent or combinations thereof to obtain predominantly 7 a - bromo 3-protected cholesterol;
b) Reacting 7 a - bromo 3-protected cholesterol with substituted or unsubstituted thiophenol or its salts in presence of a liquor ammonia to obtain predominantly 7 ß -thiophenyl 3-protected cholesterol;
c) Oxidizing 7 ß -thiophenyl 3-protected cholesterol in presence of Cumene hydrogen peroxide and Titanium tetraisopropoxide to obtain 7-phenyl sulfoxide 3-protected cholesterol;
d) Converting 7-phenyl sulfoxide 3-protected cholesterol into 7-Dehydro 3-protected cholesterol in presence of base;
e) purifying 7-Dehydro 3-protected cholesterol by suspending in a suitable organic solvent; and
f) Deprotecting the 7-Dehydro 3-protected cholesterol by treating with alkali in presence of methanol to obtain 7-Dehydrocholesterol followed by purification of 7-Dehydrocholesterol from an organic solvent.

2. The process according to claim 1, wherein, the epimerization is performed at a temperature of -30ºC to -10ºC in a solvent selected from toluene, acetone, 2-butanone, Methyl isobutyl ketone, Di-isobutyl ketone or a mixture of solvents such as toluene and acetone, Toluene and 2-Butanone, Toluene and methyl isobutyl ketone, Toluene and Di-isobutyl ketone.

3. The process according to claim 1, wherein, the substituted or unsubstituted thiophenols are selected from the group consisting of 3-Aminothiophenol; 3-Methyl thiophenol; 4-Chloro thiophenol; 4-Bromo thiophenol; 2-Bromo thiophenol; 4-Bromo-2-Trifluoromethoxy thiophenol; 4-Methoxy thiophenol; 3-Methoxy thiophenol; 2,4-Difluoro thiophenol; 3,4-Difluoro thiophenol; 3,4-DiMethoxy thiophenol; 4-(Trifluoromethyl) thiophenol; 2-Amino-4-chloro thiophenol; 2,3-Dichloro thiophenol; 2-Nitro thiophenol.

4. The process according to claim 1, wherein, the salt of Thiophenol is selected from the group consisting of sodium, Potassium and Lithium salts.

5. The process according to claim 1, wherein, the oxidation using cumene hydroperoxide and Titanium isopropoxide is carried out at a temperature range of -10 to 0ºC in a solvent selected from hydrocarbon solvent or halogenated hydrocarbon solvent such as toluene; dichloromethane, or dichloroethane.

6. The process according to claim 1, wherein, the conversion of 7-phenyl sulfoxide 3-protected cholesterol into 7-Dehydro 3-protected cholesterol is carried out in toluene at a temperature range of 70-75ºC in presence of a base selected from the group consisting of Triethyl amine, sodium acetate, N,N-diisopropylethyl amine or any other organic amines.

7. The process according to claim 1, wherein, the 7-Dehydro 3-protected cholesterol is purified by crystallizing from a solvent selected from Acetone, methyl ethyl ketone, Methyl isobutyl ketone or methanol, ethanol, isopropanol or in a mixture of solvents such methylene dichloride and methanol/Ethanol/isopropanol or Di-isopropyl ether and methanol/Ethanol/isopropanol.

8. The process according to claim 1, wherein, the deprotection is conducted at a temperature of 40-50ºC.

9. The process according to claim 1, wherein, the 7-Dehydrocholesterol is purified from a solvent selected from the group consisting of methanol; ethanol or 2-propanol, Acetone, 2-Butanone, Methyl isobutylketone or Di-isobutylketone or from a mixture of solvents like Dichloromethane and Methanol or Toluene and methanol or Methanol and Di-isopropyl ether.