Heptadecane-Steroid-Dienyne Derivative from Estrone and OTBS-estrone

Several bicycle-derivatives have been prepared using different protocols; nevertheless, expensive reagents and special conditions are required. The aim of this study was synthesize a dioxa-diazaspiro[bicyclo[9.4.2]heptadecane-steroid-dienyne derivative by a series of reactions which involving; a) alkinilization of estrone or OTBS-estrone with 5-hexyn-1-ol to form two propargyl alcohol derivatives (3 or 4); b) esterification 3 or 4 with succinic acid to form two dioxaspiro-steroidcyclotridecan derivatives (5 or 6); c) preparation of diazaspiro[bicycle[9.4.2]heptadecane-steroid-4 amino complex (7 or 8) by reaction of 5 or 6 with ethylenediamine; d) removal of silyl fragment of 8 via hydrofluoric acid to form the compound diazaspiro[bicycle[9.4.2] heptadecane-steroid-3 ́-ol (9); e) preparation of diazaspiro[bicycle[9.4.2]heptadecane-steroid-4-oxobutanoic acid (10) via esterification of 9 with succinic acid; f) amidation of 10 with ethylenediamine to form diazaspiro[bicyclo[9.4.2] heptadecane-steroid-4-aminobutanoate (11); g) synthesis of dioxadiazaspiro [bicyclo[9.4.2] heptadecane-steroid-dienyne (12) via pyrrolization of 11 using boric acid. The chemical structure of compounds was confirmed by NMR spectroscopic data.


Removal of silyl fragment of 8 via hydrofluoric acid to form 9
A solution of 8 (200 mg 0.34 mmol), in 5 ml hydrofluoric acid was stirring for 12 h to room temperature.The reaction mixture was evaporated to dryness under reduced pressure.After, the residue was purified by crystallization from methanol:water  A solution of 9 (200 mg 0.42 mmol), succinic acid (100 mg, 0.85 mmol), 1,3dicyclohexylcarbodiimide (120 mg, 0.58 mmol), p-toluensulfonic acid monohydrate 260 mg (1.36 mmol) in acetonitile:methanol 6 ml (1:2) was stirring for 72 h to room temperature.The reaction mixture was evaporated to dryness under reduced pressure.After, the residue was purified by crystallization from methanol:water:hexane (3:1)

Reduction of amide group of 7 to form 11
A solution of 7 (200 mg 0.34 mmol), NaBH 3 CN (30 mg, 0.48 mmol) in 10 ml of dioxane:water (3:2) was stirring for 72 h to room temperature.The reaction mixture was evaporated to dryness under reduced pressure.After, the residue was purified by crystallization from methanol:water (4:2).

RESULTS AND DISSCUSION
T h e r e a r e r e p o r t s w h i c h i n d i c a t e the preparation of diverse bicycle derivatives; nevertheless, expensive reagents and special conditions are required.Therefore, in this study a dioxa-diazaspiro[bicyclo[9.4.2]heptadecane-steroiddienyne derivative from estrone and OTBS-estrone was synthetized using several strategies.

Propargylic-alcohols derivatives via reaction of terminal Alkynes with ketone group (3 or 4)
There are several reports which showed the preparation of some propargylic-alcohols using different methods and reagents such as disulfide-oxazolidine 10 , Ti(O-i-Pr) 4 -BINOL complex 11 , chiral diamine-coordinated tin(II) triflate 12 , P(PhCH 2 NCH 2 CH 2 ) 3 N 13 and others; however some of these reagents are difficult to handle require and special conditions.Therefore, in this work the estrone was reacted with 5-Hexyn-1-ol in basic medium (Scheme 2).The mechanism of reaction involves a mechanism via SN 2 .The 1 H NMR spectrum of 3 showed several signals at 0.92 ppm for methyl group; at 1.22-1.50,1.70-2.10,2.24-2.76 and 6.48-7.10ppm for steroid moiety; at 1.58-1.60,2.20, and 3.66 ppm for methylene groups involved in the arm bound to both D-ring of steroid and alkyne group; at 5.54 ppm for hydroxyl groups.The 13

Esterification 3 or 4 with succinic acid to form two dioxaspiro-steroid-cyclotridecan derivatives (5 or 6)
There are diverse reagents used as catalyst to preparing of ester derivatives 14,15 ; however, most of the conventional methods are of limited use for some compounds.Therefore, in this study the method reported by Erlanger and coworkers 16 for esterification of other compounds was used.Thus, compounds 5 or 6 were prepared by the reaction of 3 or 4 with succinic acid using 1,3dicyclohexylcarbodiimide (DCC) as coupling reagent.It is important to mention that when DCC is used alone as a condensing agent in ester synthesis, the yield of esters is often unsatisfactory due to formation of an N-acylurea by-product.Some reports showed that addition of a catalytic amount of a strong acid to the esterification reaction in the presence of DCC considerably increases the yield of esters and decreases the formation of N-acylurea 3 .Therefore, p-toluenesulfonic acid was used to increase the yield of 5 or 6 in the esterification of 3 or 4 with succinic acid in the presence of DCC (Scheme 3).The 1 H NMR spectrum of 5 showed several signals at 0.98 ppm for methyl group; at 1,08, 1.50, 2.24 and 4.10 ppm for methylene groups bound to both alkyne and ester groups; at 2.54-2.58ppm for methylene groups bound to both ester groups; at 1. 22  30-173.70ppm for carbons of ester groups; at 174.12 ppm for carboxyl group.Finally, the presence of compound 5 was confirmed with mass spectrum which showed a molecular ion at 550.25.
On the other hand, the 1 H NMR spectrum of 6 showed several signals at 0.28 and 1.10 ppm for tert-buthyldimethylsylane fragment; at 0.98 ppm for methyl group; at 1,08, 1.50, 2.24 and 4.10 ppm for methylene groups bound to both alkyne and ester groups; at 2.54-2.58ppm for methylene groups bound to both ester groups; at 1.22-1.44,1.58-2.22,2.38-2.46,2.80-2.82and 6.84-7.28ppm for steroid moiety; at 2.54-2.58ppm for methylene groups bound to ester an carboxyl groups.The 13 C NMR spectra displays chemical shifts at -4.44, 18.16 and 25.72 ppm for tert-buthyldimethylsylane fragment; at 10.90 for methyl group; at 19.20, 28.60, 32.10 and 64.82 ppm for methylene bound to both ester and alkyne groups; at 30.00-31.72 ppm for methylene groups bound to both ester groups; at 23.66, 26.74-27.70,29.60, 34.32-52.80,80.99 and 117.20-153.42ppm for steroid moiety; at 79.10 and 89.02 ppm for alkyne group; at 172.04-173.70 ppm for carbons of ester groups.In addition, the presence of compound 6 was confirmed with mass spectrum which showed a molecular ion at 564.32.

Preparation of diazaspiro[bicycle[9.4.2] heptadecane-steroid-4 amino complex (7 or 8).
There are several methods for preparation of ether derivatives which involve the use of different reagents such hexyl bromide/sodium cyanide17, m-chloroperoxybenzoic acid 18 , hydrazonyl chloride 19 , N,N-dimethylbarbituric acid 20 and others.In this study, the compounds 7 or 8 were prepared via formation of imino group from 5 or 6 with ethylenediamine in presence of boric acid (Scheme 4).It is also noteworthy, that dditionally compound 7 involves the formation of an amide group participating in the arm attached to A-ring of steroid.The 1 H NMR spectrum of 7 showed several signals at 0.98 ppm for methyl group; at 1,08, 1.70, 1.98 and 3.12 ppm for methylene groups bound to both alkyne and ester groups; at 1.22-1.64,1.72-1.90,2.00-2.24,2.34, 2.68-2.76 and 6.70-7.28ppm for steroid moiety; at 2.30-2.40ppm for methylene groups bound to ester an carboxyl groups; at 2.96-3.02and 4.34 ppm for methylene groups bound to imino groups; at 5.96 ppm for carboxyl group.The 13  Finally, the presence of compound 7 was confirmed with mass spectrum which showed a molecular ion at 573.32.
On the other hand, the 1 H NMR spectrum of 8 showed several signals at 0.28 and 1.04 ppm for tert-buthyldimethylsylane fragment; at 0.98 ppm for methyl group; at 1.08, 1.70, 1.98 and 3.12 ppm for methylene groups bound to both alkyne and ester groups; at 1. 22 ppm for imino groups.In addition, the presence of compound 8 was confirmed with mass spectrum which showed a molecular ion at 588.37.

Removal of silyl fragment of 8 via hydrofluoric acid to form 9
There are several reagent for removal of silyl protecting groups from hydroxyl such as ammonium fluoride 21,20 , tris(dimethylamino) sulfonium/difluorotrimethylsilicate 22 , hydrofluoric acid 23 and others.In this study, hydrofluoric acid was used to removal of silyl-protecting group from hydroxyl of the compound 8 to form 9 (Scheme 5).The 1 H NMR spectrum of 9 showed several signals at 0.98 ppm for methyl group; at 1.10, 1.70, 1.98 and 3.12 ppm for methylene groups bound to both alkyne and ester groups; at 1. 22 T h e c o m p o u n d 1 0 w a s p r e p a r e d by the reaction of 9 with succinic acid using 1,3-dicyclohexylcarbodiimide in presence of p-toluenesulfonic acid (Scheme 5).The 1 H NMR spectrum of 10 showed several signals at 0.98 ppm for methyl group; at 1.10, 1.70, 1.98 and 3.12 ppm for methylene groups bound to both alkyne and ester groups; at 1.22-1.62,1.72-1.92,2.00-2.34 and 6.74-7.28ppm for steroid moiety; at 2.60, 2.90 ppm for methylene groups bound to both ester and   Many procedures for the formation of amide groups are known in the literature, the most widely practiced method employs carboxylic acid chlorides as the electrophiles which react with the amine in the presence of an acid scavenger 24 .Despite its wide scope, this protocol suffers from several drawbacks; most notable are the limited stability of many acid chlorides and the need for hazardous reagents for their preparation (thionyl chloride) 25 .In this work two different methods for amide formation were employed, in the first one the technique reported by Pingwah 26 for boric acid catalyzed amidation of carboxylic acids and amine was used.Therefore, boric acid was used as catalyst in the reaction of 10 with ethylenediamine to form the compounds 7 (Scheme 5).Here, is important to mention that with this method, the yield of 7 was higher compared to the reaction of 5 with ethylenediamine.

Reduction of amide group of 7 to form 11
Several reagents have been used for reduction of amides to form amino groups such as Fe 3 (CO) 12 27 , LiAlH 28 , borohydride derivative 29 and others.In this study, 7 was reacted with NaBH 3 CN to form 11. The 1 H NMR spectrum of 11 showed several signals at 0.98 ppm for methyl group; at 1,08, 1.70, 1.98 and 3.12 ppm for methylene groups bound to both alkyne and ester groups; at 1. 22

Synthesis of dioxa-diazaspiro[bicyclo[9.4.2] hepta-decane-steroid-dienyne (12) via pyrrolization of 11
There are different methods for the preparation of pyrrole derivatives [30][31][32][33] ; nevertheless, some protocols that require hazardous reagents as well as different experimental conditions for the preparation of these compounds.In this study 11 was reacted with ethylenediamine in presence of boric acid to form an imino group and consequently bring formation of the pyrrole ring involved in the compound 12 (Scheme 6).The 1 H NMR spectrum of 12 showed several signals at 0.98 ppm for methyl group; at 1.08, 1.70, 1.98 and 3.12 ppm for methylene groups bound to both alkyne and ester groups; at 1.22-1.62,1.72-1.90,2.00, 2.24-2.76,2.80 and 6.90-7.28ppm for steroid moiety; at 2.10-2.18,2.77, 2.84 and 4.14-4.20 ppm for pyrrole ring; at 2.96-3.02ppm for methylene groups bound to imino groups.The 13
form the compound diazaspiro[bicyclo[9.4.2] h e p t a d e c a n e -s t e ro i d 4 -a m i n o bu t a n o a t e derivative (11
C NMR spectra displays chemical shifts at 11.80 for methyl group; at 19.34, 25.44-25.50and 67.82 ppm for methylene bound to both ester and alkyne groups; at 23.34, 28.00, 47.45-50.85ppm for methylene groups bound to imino groups; at 24.00, 27.70-27.74,29.70, 33.70-45.20,53.57, 80.00 and 118.90-149.40ppm for steroid moiety; at 30.00-31.78 ppm for methylene groups bound to both ester and carboxyl groups; at 81.00 and 86.62 ppm for alkyne group; at 164.40 and 173.70 ppm for imino groups; at 169.16 ppm for ester group; at 177.52 ppm for carboxyl group.

). Following, 11 was reacted with ethylenediamine/boric acid to form 12 carboxyl
groups; at 2.96-3.00 and 4.34 ppm for methylene groups bound to imino groups at 8.96 ppm for hydroxyl group; at 8.66 ppm for carboxyl group.The 13 C NMR spectra displays chemical shifts at 11.80 ppm for methyl group; at 19.36, 25.42-25.50and 67.82 ppm for methylene bound to both ester and alkyne groups; at 23.30, 28.00, 47.46-50.88ppm for methylene groups bound to imino groups; 24.00, 27.70-27.74,29.70, 33.70-45.18,53.57, 79.00 and 119.32-149.72 ppm for steroid moiety; at 81.00-81.62ppm for alkyne group; at 164.42-173.70 ppm for imino groups; at 171.28 ppm for ester group at 174.12 ppm for carboxyl group.In addition, the presence of compound 10 was confirmed with mass spectrum which showed a molecular ion at 574.30.
C NMR spectra displays chemical shifts at 15.82 for methyl group; at 19.34, 25.44-25.50and 67.82 ppm for methylene bound to both ester and alkyne groups; at 23.34, 28.00, 47.45-50.85ppm for methylene groups bound to imino groups; at 23.76, 25.58-27.90,33.70-44.11,52.22, 79.00 and 115.76-155.50ppm for steroid moiety; at 26.32, 30.80 and 40.64 ppm for methylene groups bound to both ester and amino groups; at 81.00 and 86.62 ppm for alkyne group; at 164.40 and 173.70 ppm for imino groups.Finally, the presence of compound 12 was confirmed with mass spectrum which showed a molecular ion at 541.33.