Claims:
1. A process for preparation of 7-Dehydrocholesterol comprising;
a) Reacting bisnoralcohol with p-Chloranil in presence of an alcohol to obtain (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one;
b) Tosylating the (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one to obtain (20S)-20-tosyloxymethyl-pregna-4,6-dien-3-one;
c) Acylating the (20S)-20-tosyloxymethyl-pregna-4,6-dien-3-one in presence of acylating agent to obtain (20S)-20-tosyloxymethyl-3ß-acetoxy-pregna-1,3,5-triene;
d) reacting the (20S)-20-tosyloxymethyl-3ß-acetoxy-pregna-1,3,5-triene using a reductant at a temperature of 0-10°C to obtain (20S)-20-tosyloxymethyl-3ß-hydroxy-pregna-5,7-diene;
e) Protecting the (20S)-20-tosyloxymethyl-3ß-hydroxy-pregna-5,7-diene by reacting with tert-butyldimethyl silylchloride/imidazole to obtain (20S)-20-tosyloxymethyl-3ß- tertbutyldimethylsilyloxy-pregna-5,7-diene;
f) Subjecting the (20S)-20-tosyloxymethyl-3ß-tertbutyldimethylsilyloxy-pregna-5,7-diene to Grignard reaction in presence of isopentyl bromide to obtain 3ß- tertbutyldimethylsilyloxy -cholesta-5,7-diene; and
g) Deprotecting the 3ß- tertbutyldimethylsilyloxy-cholesta-5,7-diene using a deprotectant at a temperature range of 35 to 50°Cto obtain Cholest-5,7-diene (7-Dehydrocholesterol).
2. The process as claimed in claim 1, wherein, the alcohol used in step a) may be selected from C1 to C4 alcohols, viz., methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, etc.
3. The process as claimed in claim 1, wherein, the reaction of step a) is conducted at reflux temperature of the solvent used.
4. The process as claimed in claim 1, wherein, the acylating in step c) may be selected from the group consisting of acetic anhydride, acetyl chloride or mixture thereof.
5. The process as claimed in claim 1, wherein, the reductant used in step d) is selected from Sodium borohydride, Li AlH4, Calcium borohydride (Ca (BH4)2 etc.
6. The process as claimed in claim 5, wherein, the reductant is Sodium borohydride in solvent mix of THF & t-butanol.
7. The process as claimed in claim 1, wherein, the deprotect is selected from the group consisting of tetrabutylammonium floride solution (TABF in THF), Tetrabutyl ammonium tribromide in MeOH etc.
8. A process for preparation of 3ß-25-Dihydroxycholesta-5,7-diene (provitamin of 25-Hydroxy vitamin D3) which process comprises;
a) Reacting (20S)-20-tosyloxymethyl-3ß-tertbutyldimethylsilyloxy-pregna-5,7-diene with 4-bromo-2-methyl-2-[(trimethylsilyl)oxy]butane under Grignard conditions to obtain ß-tert-Butyldimethylsilyloxy-25-triethylsilyloxycholesta-5,7-diene; and
b) Deprotecting the ß-tert-Butyldimethylsilyloxy-25-triethylsilyloxycholesta-5,7-diene in presence of a deprotectant to obtain 3ß-25-Dihydroxycholesta-5,7-diene.
9. The process as claimed in claim 8, wherein the deprotectant is selected from the group consisting of tetrabutylammonium fluoride solution (TABF in THF), Tetrabutyl ammonium tribromide in MeOH etc.
, Description:Technical filed:
The present invention relates to a process for conversion of bisnoralcohol into 7-DHC (provitamin of vitamin D3) & 25-OH 7-DHC (provitamin of 25-OH vitamin D3(Calcifediol).

Background and prior art:
The metabolite of vitamin D3, 25-hydroxycholecalciferol (Calcifediol) is more potent anti- rachitic agent than vitamin D itself therefore development of a facile method for the synthesis of vitamin D3 and its analogue Calcifediol is highly important. Earlier the present inventors have reported various synthetic routes for the production of cholesterol and 7-dehydrocholesterol (WO2021005618) from phytosterol and 25-OH cholesterol and 7dehydrocholesterol from phytosterol (WO2020225830), which is a cheaper raw material.

However, these processes which start from phytosterol has a limitation i.e., the conversion of vitamin D3 and its analogues are solely dependent on the content of stigmasterol present in the phytosterol.

Therefore, it is an objective of the present invention to overcome the above limitation by fully utilizing the phytosterol that consists various sterols such as stigmasterol, sitosterol and campesterol etc., thereby providing a cost-effective process for the preparation of vitamin D3 and its analogues.

Summary of the invention:
In line with the above objective, the present invention provides a process for preparation of vitamin D3 and its analogues starting from bisnoralcohol.

To utilise full conversion of phytosterol (that consisting of stigmasterol, sitosterol and campesterol etc.) the present inventors have prepared the starting material, bisnoralcohol from phytosterol (soya sterol) by fermentation method.
In this method, the entire soya sterol got converted into Bisnoralcohol in greater than 95%. The process is shown in below scheme.

In another aspect, the bisnoralcohol thus obtained was converted into 7-dehydrocholesterol / 25-OH 7-dehydrocholesterol in good yield and purity directly without involving production stages of cholesterol/25-OH cholesterol, which makes the process more economical and industrially viable. The 7-dehydrocholesterol/25-OH 7-dehydrocholesterol thus obtained can be converted into vitamin D3/ Calcifediol by irradiating under high-pressure mercury lamp in a solvent in presence of a sensitizer as per the process reported in our earlier patent applications. WO2021005618, WO2020225830 and WO2021005619A1.

Thus, the present invention provides an efficient synthetic method for producing 7-dehydrocholesterol(7-DHC) through multiple reactions starting from cheap and commercially available bisnoralcohol (BA). The process reported in the present invention provides a promising route that offers economical and efficient strategies for potential large-scale production of 7-DHC & 25-OH 7- dehydrocholesterol.

Detailed description of the invention:
The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated. The initial material as used in the present invention soya sterol for the production of bisnoralcohol was procured from Hubei Goto biotechnology co ltd, China.

In accordance with the above, the present invention provides a process for preparation of 7-Dehydrocholesterol, which process comprises;
a) Reacting bisnoralcohol with p-Chloranil in presence of an alcohol to obtain (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one;
b) Tosylating the (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one to obtain (20S)-20-tosyloxymethyl-pregna-4,6-dien-3-one;
c) Acylating the (20S)-20-tosyloxymethyl-pregna-4,6-dien-3-one in presence of acylating agent to obtain (20S)-20-tosyloxymethyl-3ß-acetoxy-pregna-1,3,5-triene;
d) reacting the (20S)-20-tosyloxymethyl-3ß-acetoxy-pregna-1,3,5-triene using a reductant at a temperature of 0-10°C to obtain (20S)-20-tosyloxymethyl-3ß-hydroxy-pregna-5,7-diene;
e) Protecting the (20S)-20-tosyloxymethyl-3ß-hydroxy-pregna-5,7-diene by reacting with tert-butyldimethyl silylchloride/imidazole to obtain (20S)-20-tosyloxymethyl-3ß- tertbutyldimethylsilyloxy-pregna-5,7-diene;
f) Subjecting the (20S)-20-tosyloxymethyl-3ß-tertbutyldimethylsilyloxy-pregna-5,7-diene to Grignard reaction in presence of isopentyl bromide to obtain 3ß- tertbutyldimethylsilyloxy -cholesta-5,7-diene; and
g) Deprotecting the 3ß- tertbutyldimethylsilyloxy-cholesta-5,7-diene using a deprotectant at a temperature range of 35 to 50°Cto obtain Cholest,5,7-diene (7-Dehydrocholesterol).

The alcohol that can be used for the reduction of bisnoralcohol may be selected from C1 to C4 alcohols, viz., methanol, ethanol, n-propanol, isopropanol, n-butanol, t-butanol, etc.

The acylating may be selected from the group consisting of acetic anhydride, acetyl chloride or mixture thereof.

The reductant used for the synthesis of (20S)-20-tosyloxymethyl-3ß-hydroxy-pregna-5,7-diene is selected from Sodium borohydride, LiAlH4 and Calcium borohydride Ca(BH4)2 etc. More preferably, the reductant is Sodium borohydride in solvent mix of THF & t-butanol.
The deprotect is selected from the group consisting of tetrabutylammonium fluoride solution (TABF in THF), Tetrabutylammonium tribromide in MeOH, etc.
The synthesis of Cholest,5,7-diene (7-Dehydrocholesterol) is shown in below scheme.
Scheme:

Reagents & conditions:
Reagents & conditions: 1) chloranil, t-BuOH, reflux 2) Tosyl-Cl, pyridine, MDC 3) Ac2O, AcCl, 100oC 4) NaBH4, THF /MeOH, H2O 5) TBDMS-Cl, Imidazole, I2, MDC RT 6) Mg, CuBr.Me2S, THF, 0oC-RT 7) TBAF, THF, RT 8) Photo irradiation/ Heat

In another embodiment, the invention provides a process for preparation of 3ß-25-Dihydroxycholesta-5, 7-diene (25-OH 7- dehydrocholesterol), which process comprises;
a) Reacting (20S)-20-tosyloxymethyl-3ß-tertbutyldimethylsilyloxy-pregna-5,7-diene with 4-bromo-2-methyl-2-[(trimethylsilyl)oxy]butane under Grignard conditions to obtain ß-tert-Butyldimethylsilyloxy-25-triethylsilyloxycholesta-5,7-diene; and
b) Deprotecting the ß-tert-Butyldimethylsilyloxy-25-triethylsilyloxycholesta-5,7-diene to obtain 3ß-25-Dihydroxycholesta-5,7-diene.

The deprotectant used in the synthesis of 3ß-25-Dihydroxycholesta-5,7-diene is tetrabutylammonium fluoride solution (TABF in THF solvent)

Accordingly, in an embodiment, Bisnoralcohol was dissolved in t-butanol under stirring. p-Chloranil was then charged, and the reaction mixture heated to 1000 C and maintained at this temperature for 3-4h. The mixture was then cooled to 50 C and the resulting solid was filtered to obtain (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one as light brown solid with good yields and purity.

In the second stage, (20S)-hydroxymethyl-pregna-4,6-dien-3-one thus obtained with reacted with p-toluenesulfonylchloride in MDC in presence of pyridine as a base (at and the reaction mixture was stirred at RT for 12h. After the completion of the reaction, the mass was quenched with water, stirred and layers were separated. The (20S)-20-tosyloxymethyl-pregna-4,6-dien-3-one was recovered in good yields from MDC by washing with water followed by brine solution and finally dried over anhydrous Na2SO4.

In the third stage, (20S)-20-tosyloxymethyl-pregna-4,6-dien-3-one was acylated by reacting with the mixture of Acetic anhydride and acetyl chloride under stirring at 95-100oC for 16hr until completion of starting material. The reaction mixture was quenched by pouring in ice water mixture and the solid product obtained filtered washed water, dissolved in MDC and washed MDC layer thoroughly with water and brine solution and finally dried over anhydrous Na2SO4 to obtain (20S)-20-tosyloxymethyl-3ß-acetoxy-pregna-1,3,5-triene in good yields.
In a fourth stage, the (20S)-20-tosyloxymethyl-3ß-acetoxy-pregna-1,3,5-triene thus obtained was dissolved in a mix solvent (THF & t-butanol) and reacted with sodium borohydride at a temperature of 0-5oC. After completion of the reaction, the reaction mixture was slowly poured into ice water mixture under stirring, pH was adjusted to 3-4 by slow addition of dil HCl. The product was extracted into EtOAc and EtOAc layer was washed with brine solution (250mL x2) and dried over anhydrous Na2SO4 , filtered and concentrated to get crude product which upon stirring in n-heptane to get the (20S)-20-tosyloxymethyl-3ß-hydroxy-pregna-5,7-diene in good yields and purity.

In the fifth stage, (20S)-20-tosyloxymethyl-3ß-hydroxy-pregna-5,7-diene was taken in THF and imidazole and tert-butyldimethyl silylchloride followed by catalytic Iodine. The Reaction mixture was stirred at RT for 4h until completion of starting material. THF removed under vacuo and to the residue water added and extracted the reaction mass with MDC, washed MDC layer with saturated salt solution and dried over anhydrous Na2SO4 to obtain crude product which was stirred in n-heptane, to obtain (20S)-20-tosyloxymethyl-3ß-tertbutyldimethylsilyloxy-pregna-5,7-diene in good yields and purity, which was filtered and dried.

In the sixth stage, (20S)-20-tosyloxymethyl-3ß-tertbutyldimethylsilyloxy-pregna-5,7-diene was reacted with isopentyl bromide under Grignard conditions under nitrogen atmosphere in presence of CuBr.Me2S as catalyst under stirring at room temperature for 2h. The reaction mixture was quenched by pouring into a saturated cold solution of ammonium chloride and then extracted with ethyl acetate and the ethyl acetate layer was washed with saturated brine solution and solvent concentrated using vacuo to get crude product which upon stirring in methanol solid precipitated, filtered, and dried to obtain 3ß-tertbutyldimethylsilyloxy-cholesta-5,7-diene as colourless solid in very good yield, which was used as such for next reaction without further purification.
In the last stage, 3ß-tertbutyldimethylsilyloxy-cholesta-5,7-diene was deprotected by reacting with TBAF in THF under stirring at RT for 3h at 40-450 C for 2 h till completion of starting material. The Reaction was quenched by adding water and extracted with EtOAc, washed with water followed by saturated salt solution and the organic layer was dried over anhydrous Na2SO4, concentrated under vacuum. The product thus obtained was dissolved in MDC, charcoal was added, stirred at 50oC for 15min, filtered over hyflow bed, solvent removed using vacuo to get crude product, which was stirred in methanol for 15 mins, filtered and dried under vacuo to get Cholest,5,7-diene (7-Dehydrocholesterol) as colourless product in good yields and purity.

The beauty of the process of the present invention is that the process yields 7-Dehydrocholesterol in an overall yield of 75 to 80%, without involving the production stage of cholesterol, in highly economical manner. The process of the present invention thus can be carried out on industrial scale.
In another embodiment, the invention provides a process for preparation of 3ß-25-Dihydroxycholesta-5,7-diene (provitamin of 25-Hydroxy vitamin D3). This process can be conducted in two stages.

In the first stage, ß-tert-Butyldimethylsilyloxy-25-triethylsilyloxycholesta-5,7-diene was synthesised by reacting 3ß-tert-Butyldimethylsilyloxy-22-tosyloxy-23,24-bisnorchola-5,7-diene with 4-bromo-2-methyl-2-[(trimethylsilyl)oxy]butane under Grignard conditions. The Grignard conditions include magnesium turnings in THF and few drops of 1,2-dibromoethane in presence of CuBr•Me2S as catalyst under nitrogen atmosphere and stirring at 500 C for 2-3 h, the reaction mixture was poured into saturated aqueous NH4Cl at 0 °C and the aqueous layer was extracted with ethyl acetate (EtOAc), and the layer was washed with saturated aqueous NH4Cl, saturated aqueous NaHCO3 and brine, and dried over MgSO4. The oily residue thus obtained was stirred in acetone to afford ß-tert-Butyldimethylsilyloxy-25-triethylsilyloxycholesta-5,7-diene as white solid, filtered, and dried and used as such for next reaction without further purification.
In the second stage, the mixture of 3ß-tert-Butyldimethylsilyloxy-25-trimethylsilyloxycholesta-5,7-diene and Bu4NF•3H2O in THF were stirred under argon atmosphere at 40 °C for 4-5 h. After completion of the reaction, EtOAc was added, the mixture washed with brine and the organic phase was separated, dried over Na2SO4 and the solvent removed under reduced pressure. The residue was crystallized from MeOH to obtain 3ß-25-Dihydroxycholesta-5,7-diene.

The beauty of the process of the present invention is that the process results in 3ß-25-Dihydroxycholesta-5,7-diene with a overall yields of 75 to 85%, without involving the production stage of 25-OH cholesterol, in highly economical manner. The process of the present invention thus can be carried out on industrial scale.
The starting material Bisnoralcohol was prepared from soya sterol by fermentation method. In this method, the entire soyasterol got converted into Bisnoralcohol in greater than 95%.

The following examples are presented to further explain the invention with experimental conditions, which are purely illustrative and are not intended to limit the scope of the invention.

Example 1:
Step 1: Synthesis of (20S)-20-hydroxymethyl-pregna-4,6-dien-3-one

Bisnoralcohol (100g, 0.302mol) was charged to a reaction vessel followed by t-butanol (1L) with stirring. p-Chloranil (80g, 0.325mol) was then charged and the reaction mixture heated to 100oC and maintained at this temperature for 3-4h. The mixture was then cooled to 5oC and held that temperature for 2h. The resulting solid was filtered, and filter-cake washed with t-butanol. Filtrate was concentrated in vacuo. The residue was dissolved in MDC (2L) and washed with water(500mLX2), 3% KOH (250mlX2) again washed with water(500mL) followed by saturated brine solution(500mL). MDC layer was dried over anhy. Na2SO4 and concentrated in vacuo to crude product which upon stirring in n-heptane gave the light brown solid (75g, 75% Y with 97% GC purity)
M.Pt: 135-140oC
HPLC purity:
GC purity: 97%
Mass spectra: M+1: 329, 353(Na+ adduct)
M-1: 327

dH(400MHz, DMSO); 6.18-6.13(2H,C7-CH,C6-CH), 5.60(1H,s,C4-CH), 4.28-4.26(1H,t,), 3.42(1H,dd), 3.10-3.04(1H,p), 2.59-2.54(1H,ddd), 2.27-2.21(1H,m), 2.19(1H,brt), 1.95-1.16(16H), 1.11(3H,s), 1.06(3H,d), 0.78 (3H,s)
C13NMR (400MHz): 198, 163.2, 141.2, 127.5, 122.9, 65.5, 52.7, 52.0, 50.1, 42.9, 40.1, 38.5, 37.1, 35.4, 33.5, 33.3, 27.2, 23.3, 20.2, 16.9, 15.9, 11.7

Step 2: Synthesis of (20S)-20-tosyloxymethyl-pregna-4,6-dien-3-one

To a solution of (20S)-hydroxymethyl-pregna-4,6-dien-3-one (50g, 0.152mol) in MDC (700mL), pyridine(50mL) was added followed by p-toluenesulfonylchloride(50g,0.262mol) at 0oC.The reaction was stirred at RT for 12h. The reaction was quenched with water(2L) stirred and layer separated. aqueous layer was washed with MDC (250mLx2). The combined organic layer was washed with water(500mL) followed by brine solution(500mL) and finally dried over anhydrous Na2SO4. Solvent concentrated in vacuo to get desired product as light brown solid (56g, Y 76%),
M.Pt: 176-179oC
HPLC purity: 99.24(RT: 17.57)
Mass spectra: M+1: 483, (Na+ adduct): 505,
M-1: 482

dH(400MHz,DMSO);7.79-7.77(2H,Ar-H),7.49-7.47(2H,Ar-H),6.186.11(2H,m,C6H&C7H,) ,5.60(1H,s,C4H),3.93-3.90(1H,dd,C22H),3.81-3.77(1H,dd,C22H),2.57-2.52(1H,ddd,C2H) ,2.42(4H,m,C2HandTs-CH3),2.17(1H,t),2.01-1.96(2H,m)-1.70-1.34(6H),1.22-1.08(6H),1.04 (3H,s,C19H),0.911-0.895 (3H,d,C21H),0.669(3H,s,C18H)
C13NMR(400MHz): 198,163.1,144.8,141,132.3,130.1,127.6,127.5,122.9,75.6,52.5.50.9,49.9,42.9,40.1,38.8,38.5,37.0,35.4,35.3,33.5,33.3,26.6,23.1,21.0,20.1,16.5,15.9,11.5

Step 3: Synthesis of (20S)-20-tosyloxymethyl-3ß-acetoxy-pregna-1,3,5-triene

(20S)-tosyloxymethyl-pregna-4,6-dien-3-one (50g, 0.102 mol) was charged to a reaction vessel followed by Acetic anhydride(1L) and acetyl chloride (350 mL). The reaction mixture was stirred at 95-100oC for 16hr until completion of starting material. The reaction mixture was quenched by pouring slowly in ice water mixture(3L) with constant stirring for 30mins. Solid product obtained filtered washed wet cake with water (250mLX2). Solid was dissolved in MDC and washed MDC layer thoroughly with water and brine solution and finally dried over anhydrous Na2SO4. MDC layer concentrated in vacuo to get crude sticky solid which upon stirring in n-heptane for 30mins light brown solid precipitated, filtered, and dried in vacuo. (47g, Y 86%,)
M.Pt: 143-147oC
HPLC purity: 95.28(RT: 22.55)
dH(400MHz,DMSO);7.79-7.77(2H,Ar-H),7.50-7.48(2H,Ar-H),5.75(1H),5.60-5.59(1H,d), 5.49-5.47(m,1H),3.92-3.90(1H,dd),3.83-3.79(1H,dd),2.42(4H,S), 2.17(1H,t), 2.04(s,3H)2.01-1.96(2H,m)-1.70-1.34(6H),1.22-1.08(6H),1.04(3H,s,C19H),0.911-0.895 (3H,d,C21H), 0.669 (3H,s,C18H)
C13NMR(400MHz): 168.7,148.4,144.8,142.2,137.4,132.3,130.1,127.6,119.5,117.3,115.7,50.7,43.9,42.4,40.1,39.9,39.0,38.8,37.8,35.6,35.3,34.0,26.5,24.6,22.5,21.0,20.7,20.1,16.6,15.8,11.5.

Step 4: Synthesis of (20S)-20-tosyloxymethyl-3ß-hydroxy-pregna-5,7-diene

Step 3 material 50g (0.095mol) dissolved in a mix solvent (THF & t-butanol (600mL: 500mL), 1.2: 1 ratio) was taken in a reaction vessel, cooled to 0-5oC. To the reaction mixture sodium borohydride (40g, 1.05mol) was added portion wise slowly over a period of 30mins maintaining the temperature followed by slow water(50mL) addition. The reaction mixture was stirred at same temperature for 2h and then at room temperature for 16h until completion of starting material. Reaction mixture was slowly poured into ice water mixture (3L) under stirring, pH was adjusted to 3-4 by slow addition of dil HCl. It was then extracted with EtOAc (500mL X2). EtOAc layer was washed with brine solution (250mL x2) and dried over anhydrous Na2SO4 , filtered and concentrated using vacuo to get crude product which upon stirring in n-heptane ( 250 mL) solid obtained, filtered and dried in vacuo to get off white solid.( 40g, Y : 87%, purity by HPLC : > 90%)
M.Pt: 156-159oC
HPLC purity: 98.9% (RT: 16.93)
dH (400MHz, DMSO);7.79-7.77(2H, Ar-H),7.50-7.48(2H, Ar-H),5.46-5.47(1H, d),5.39(1H, d),4.66-4.65(d,1H),3.92-3.91(1H, d),3.83-3.82(1H, dd),3.33(1H),2.42(3H, S),2.2-1.17(20H), 0.93-0.91(d,3H),0.84(3H, S).
C13NMR (400MHz): 144.8,140.6,139.7,132.3,130.1,127.6,118.6,116.2,75.5,68.5,53.4,50.8,45.5,42.4,40.6,40.1,38.8,38.1,37.8,36.4,35.6,31.7,26.5,22.5,21.0,20.5,16.6,15.90,11.4.
Step 5 Synthesis of (20S)-20-tosyloxymethyl-3ß-tertbutyldimethylsilyloxy-pregna-5,7-diene

To a solution of step 4 material (50g,0.095mol) in THF (500 mL), imidazole (25g,0.36 mol) was added. To the reaction mixture tert-butyldimethyl silylchloride(30g,0.2mol) was added slowly portion wise followed by catalytic Iodine (1g). Reaction mixture was stirred at RT for 4h until completion of starting material. THF removed under vacuo and to the residue water(1L) added and extracted with MDC (500mLX2), washed MDC layer with saturated salt solution and dried over anhydrous Na2SO4. Solvent removed using vacuo and obtained crude product stirred in 500mL n-heptane, solid filtered and dried (46g, Y: 74.6%, Purity by HPLC 94.7%)
M.Pt: 163-166oC, a= -62o (c= 0.5, EtOAc)
GC purity: 92%,(RT: 6.2)

Step 6: Synthesis of 3ß-tertbutyldimethylsilyloxy-cholesta-5,7-diene

To a stirred magnesium turnings (40g, 1.67mol) in THF (300mL) few drops of 1,2-dibromoethane was added under nitrogen atmosphere followed by few drops of isopentyl bromide and heated to 50oC for few minutes to initiate reaction then a remaining solution of isopentyl bromide (80mL, mol) was added dropwise under nitrogen. After being stirred at 50oC for 60mins the reaction mixture was cooled to 0oC and a suspension of CuBr.Me2S (200mg, mol) was added and a solution of step 5 material (50g, 0.084mol) in THF (300 mL) was added dropwise with slow stirring over a period of 30mins. The reaction mixture was stirred at room temperature for 2h. The reaction mixture was quenched by pouring into a saturated cold solution of ammonium chloride and then extracted with ethyl acetate (500mL X2). The combined organic layer was washed with saturated brine solution (500 mL) and solvent concentrated using vacuo to get crude product which upon stirring in methanol(200mL) solid precipitated, filtered, and dried to get colourless solid (40g, Y: 95%, purity by GC: 96.75%, RT: 14.43) which was used as such for next reaction without further purification.

Step 7: synthesis of Cholest,5,7-diene (7-Dehydrocholesterol)

To a solution of step 6(32g, 0.064 mol) in THF (400 mL), TBAF (50g, .158 mol) dissolved in THF (200mL) was added in one lot at room temperature. Reaction mixture was stirred at RT for 3h at 40-45oC for 2h till completion of starting material. Reaction was quenched by adding water (1.5L) and extracted with EtOAc(500mLX2). Combined organic layer was washed with water(1L) followed by saturated salt solution(500ml). Organic layer dried over anhydrousNa2SO4. The solvent was concentrated using vacuo to get crude product which was dissolved in MDC (500mL) 5g charcoal added stirred at 50oC for 15min, filtered over hyflow bed solvent removed using vacuo to get crude product which was stirred in methanol(200mL) for 15 mins. Filtered and dried under vacuo to get colourless product (18g, Y: 75%, 97.41% purity, RT 11.13, 94% assay purity, GC purity: 97.7%).
M.Pt: 148-152oc
HNMR and 13C NMR data correspond to those known from the literature.

Analytical Data:
Yield: 18g(75%)
M.Pt: 148-152oC
HPLC: 97.4% (RT: 11.13)
GC: 97.71% (RT: 7.41)
1H-NMR: (400 MHz, CDCl3): d= 0.60 (s, 3 H), 0.94 (s, 3 H), 0.96 (d, 3 H), 1.19 (s, 6 H), 3.66 (m, 1 H), 5.39 (d, 1 H), 5.55 (d, 1 H).
13C-NMR (100 MHz, CDCl3): d 141.4, 139.7, 119.6, 116.2, 70.4, 55.9, 54.5, 46.2, 42.9, 40.7, 39.5, 39.2, 38.3, 37.0. 36.1, 36.1, 31.9, 28.0, 28.0, 23.8, 23.0, 22.8, 22.5, 21.1, 18.5, 16.2; 11.8.

Example 2:
Synthesis of 3ß-25-Dihydroxycholesta-5,7-diene (provitamin of 25-Hydroxy vitamin D3)
Stage 1: Synthesis of ß-tert-Butyldimethylsilyloxy-25-triethylsilyloxycholesta-5,7-diene:

To stirred magnesium turnings (8.0 g, 0.326 mol, 10 eq.) in THF (100ml) few drops of 1,2-dibromoethane were added under nitrogen atmosphere followed by few drops of 4-bromo-2-methyl-2-[(trimethylsilyl)oxy]butane and heated to 50oC for few minutes to initiate the reaction. This was followed by addition of remaining solution of 4-bromo-2-methyl-2-[(trimethylsilyl)oxy]butane(77.0 g, 0.326 mol) in THF (100 mL) drop wise under N2 atmosphere. After being stirred at the same temperature i.e. 50oC for 30 min, the reaction mixture was cooled at 0°C and a suspension of CuBr•Me2S (6.68g, 0.0326 mol, 1.0 eq.) in THF (10ml) was added followed by dropwise addition of solution of 3ß-tert-Butyldimethylsilyloxy-22-tosyloxy-23,24-bisnorchola-5,7-diene (20g, 0.0326 mol, 1.0 eq.), obtained in THF (200 mL) at 0 °C under N2. After being stirred at room temperature for 2-3 h, the reaction mixture was poured into saturated aqueous NH4Cl at 0 °C and the aqueous layer was extracted twice with ethyl acetate (EtOAc). The combined organic layer was washed with saturated aqueous NH4Cl, saturated aqueous NaHCO3 and brine, and dried over MgSO4. The obtained mixture was filtered and concentrated in vacuo. The oily residue was stirred in acetone to afford ß-tert-Butyldimethylsilyloxy-25-triethylsilyloxycholesta-5,7-diene as white solid, filtered and dried and used as such for next reaction without further purification.
Analytical Data:
Yield: 16.4g(78%)
Appearance: White solid
M.Pt: 115-118oC
GC: ~ 94% (RT: 25.78)

Stage 2: Synthesis of 3ß-25-Dihydroxycholesta-5,7-diene:

Compound(A) 3ß-tert-Butyldimethylsilyloxy-25-trimethylsilyloxycholesta-5,7-diene (16.4, 0.0254 mol) and Bu4NF•3H2O (25 g, 0.079 mol) in THF (250 mL) were stirred under argon atmosphere at 40 °C for 4-5 h. EtOAc (500 mL) was then added, and the mixture washed with brine (100x3mL). The organic phase was separated, dried over Na2SO4 and the solvent removed under reduced pressure. The residue was crystallized from MeOH.
Analytical Data:
Yield: 9.0g(85%)
Appearance: White solid
M.Pt: 154–157°C.
GC: ~98% (RT: 10.0)

1H-NMR (400 MHz, CDCl3): d= 0.60 (s, 3 H), 0.94 (s, 3 H), 0.96 (d, 3 H), 1.19 (s, 6 H), 3.66 (m, 1 H), 5.39 (d, 1 H), 5.55 (d, 1 H).
The 1H NMR data correspond to those known from the literature.
13C-NMR: (100 MHz, CDCl3): d141.3, 139.8, 119.5, 116.3,71.1, 70.4, 55.8, 54.4, 46.2, 44.4, 42.9, 40.8, 39.1, 38.3, 37.0,36.3, 36.0, 32.0, 29.3, 29.2, 28.1, 23.0, 21.1, 20.8, 18.8, 16.3,11.8.