"Sulfatation of Estrogen Mixtures"

Patentamt Europaisches ||| || 1 1| || || || || || || || || || 1 1| (19) J European Patent Office

Office europeen des brevets (1 1 ) EP0 875 515 A2

(12) EUROPEAN PATENT APPLICATION

(43) Date of publication:ation: (51) |nt. ci.6: C07J 31/00, A61 K 31/565 04.11.1998 Bulletin 1998/45

(21) Application number: 98201385.6

(22) Date of filing: 28.04.1998

(84) Designated Contracting States: (72) Inventors: AT BE CH CY DE DK ES Fl FR GB GR IE IT LI LU • Raijmakers, P. H. MC NL PT SE 5406 VD Uden (NL) Designated Extension States: • Hofstraat, R. G. AL LT LV MK RO SI 5346 JA OSS (NL) • Boom van den, H. P. A. J. M. (30) Priority: 02.05.1997 EP 97201333 5408 AA Volkel (NL)

(71 ) Applicant: Akzo Nobel N.V. (74) Representative: Kraak, Hajo et al 6824 BM Arnhem (NL) N.V. Organon, Postbus 20 5340 BH OSS (NL)

(54) "Sulfatation of estrogen mixtures"

(57) The invention relates to a method for the preparation of a mixture of sulfated estrogens containing delta(8,9)-dehydro estrone [delta(8,9)DHE] or derivatives thereof.

CM < LO LO LO co o Q_ LU Printed by Xerox (UK) Business Services 2.16.3/3.4 1 EP 0 875 51515 A2 2

Description according to the present invention has the advantage that in a single reaction mixture sulfated estrogens can The invention relates to a method for the prepara- be obtained in a specific ratio. It has surprisingly been tion of a mixture of sulfated estrogens, more particularly found that although the estrogens present in the natural to the sulfatation of a mixture containing 3-hydroxy- s conjugated estrogen preparations have different physi- estra-1 ,3,5(10),8(9)-tetra-en-1 7-one [delta(8,9)-dehy- cal properties, such as crystallization behavior and sol- dro estrone; delta(8,9)DHE; delta 8 estrone; 8,9 dehy- ubility, the ratio of sulfated products in the sulfatation dro estrone; CAS no. 61612-83-7]. reaction mixture reflects the amounts of the input com- The sodium sulfate of the delta(8,9) derivative of ponents. Therefore, a one pot reaction suffices to pre- estrone [delta(8,9)DHES] is present in minor amounts 10 pare the sulfated estrogen mixture. In addition, this of about 3-4% in natural conjugated estrogen composi- reaction can, when appropriate, be coupled directly to tions, for instance in the commercially available product the isomerization reaction by which delta(8,9)DHE is Premarin® which is being used in hormone replace- prepared, i.e. isomerization of equilin or a derivative ment therapy. thereof to said derivative. In this reaction equilin or a In addition to estrone sodium sulfate, several com- is derivative thereof is treated with a lithium salt of ethylen- ponents have been identified in natural conjugated ediamine or with lithium amide in dimethylsulfoxide. estrogen compositions a/o. the sodium sulfates of equi- Thus, the present invention offers the first easy and lin (in amounts of 22.5-30.5%), 17 alfa dihydro equilin inexpensive method of production of sulfated steroid (13.3-19.5%), 17 beta dihydro equilin (0.5-4.0%), 17 mixtures containing delta(8,9)DHE through sulfatation alfa estradiol (2.5-9.5%) 1 7 beta estradiol (< 4.5%) and 20 of an estrogen mixture containing delta(8,9)DHE or delta(8,9)-dehydro estrone (< 12.5%) (US Pharmaco- derivatives thereof which can be obtained by isomeriza- poeia, 1995, p. 627). tion of equilin or a derivative thereof. It has been suggested in SCRIP no. 2049 (1995), p. Accordingly mixtures of estrogens comprising a 15 that minor amounts of delta(8,9)DHES could have a compound according to the general formula I significant contribution to the effect of conjugated estro- 25 gens. It has further been suggested that delta(8,9)DHES, which has a relatively low affinity to the estrogen receptor, has a high functional activity, which may play a role in the reported LDL-cholesterol-reduc- ing properties and cardiovascular effects of conjugated 30 estrogens, in particular of Premarin®. Data reveal that delta(8,9)DHES contributes to about 18% of Premarin's circulating estrogens. It is therefore of importance to obtain an easy method of production of sulfated mixtures of delta(8,9)DHE. 35 Apart from cumbersome total synthesis, J.C. Jac- quesy et al., Chem. Abstr. 76 (1972), 154000f disclosed isomerization of equilin in hyperacidic media. Conver- wherein R1 is H, sion to delta(8,9)DHE was achieved by using hydrogen R2 is H and R3 is O-acyl; or fluoride or hydrogen fluoride/antimony fluoride at -30 40 R3 is H and R2 is O-acyl; or °C. It is evident that such dangerous reaction conditions R2 and R3 together represent O can be sulfated in are completely unsuitable and unacceptable for large admixture with one or more compounds taken from scale production of delta(8,9)DHE. Moreover, in US pat- the group of compounds of general formula II ent 5,395,831, wherein the method of Jacquesy is applied, it has been disclosed that said hydrogen fluo- 45 ride method does not provide pure delta(8,9)DHE, but in addition thereto 10 % of the unwanted delta(9,11)-iso- mer. Methods of production which are commercially acceptable, whether or not through isomerization of equilin, thus have not been disclosed. so Synthesis of sulfated esters of steroids has been described in the literature. As summarized by Jenkins and Sandberg (Methods in Enzymology, 15, 351-358, 1969) one of the methods involves the sulfatation of mono- and dihydroxy C-18 and C-19 steroid com- ss wherein R1 , R2 and R3 have the previously defined pounds utilizing pyridine sulfuric acid complexes. This meanings and the dotted line at position 7-8 repre- process, however, has been applied to individual, i.e. sents an optional double bond. free from other, steroid compounds. The process

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In a preferred embodiment of the invention R2 and when (co)solvents are added. Lithium amide in dimeth- R3 in formula I and/or II represent O. More preferably, ylsulfoxide (DMSO) also provides mixtures of delta 8,9 estrone is sulfated in admixture with equilin. delta(8,9)DHE and equilin or derivatives thereof, which According to another embodiment of the invention can be converted according to the present invention into compounds of general formula II are one or more of the s their sodium sulfates, to be used in the manufacture of precursors of the minor components as present in natu- pharmaceutical compositions containing conjugated ral conjugated estrogen mixtures such as sulfate esters estrogens. of 1 7 alfa dihydro equilin, 1 7 beta dihydro equilin, 1 7 alfa Preferably, the C3 position is occupied by a tetrahy- estradiol and 17 beta estradiol. dropyranyl ether because such an ether can easily be The ratio of the compounds in the reaction mixture to prepared on an aromatic group, is stable under the is not critical but for economical reasons the preferred isomerization conditions, and, after isomerization, can ratio is the ratio as present in natural mixtures. easily be removed to prepare a hydroxyl group for sulfa- The compounds of general formula I can be pre- tation. pared by isomerization of equilin and said derivatives If R1 in formula III is silyl(alkyl)3, the isomerization is which are indicated in Formula III: ts preferably performed at a temperature of between about 0 and 90 °C, and with more preference at about 30 °C if equilin or a derivative thereof is treated with a lithium salt of ethylenediamine or about 65 °C if equilin or a derivative thereof is treated with lithium amide in 20 dimethylsulfoxide. If on the other hand, R-| is tetrahydropyranyl a mixture of the substrate and an aforemen- tioned solvent is treated with a lithium salt of ethylenediamine at a temperature of between about -78 °C and 50 °C, preferably between approximately 0 °C 25 and -20 °C using THF as the cosolvent. If the isomerization reaction is performed only partially a mixture of compounds of general formula III and delta 8,9 derivatives is formed which can be further wherein R-| is silyl(alkyl)3 or O-tetrahydropyranyl, processed to obtain sulfate conjugated estrogens mix- R2 and R3 together represent O; or 30 tures. Optionally also derivatives of general formula II R2 and R3 together represent acetal or cyclic can be added and in a single reaction all compounds acetal. can be sulfated simultaneously. Preferred compounds to be isomerized and sulfated according to the present The term alkyl, as used in the definition of the for- invention are those wherein R2 and R3 in the formulas mulas, means a branched or unbranched alkyl group 35 represent O. having preferably 1-7 carbon atoms, like hexyl, isobutyl, If in the compound of formula 1 1 1 , Ri is silyl(alkyl)3 or tertiary butyl, propyl, isopropyl, ethyl, and methyl. Pref- tetrahydropyranyl, the derivatives can be isomerized erably, silyl(alkyl)3 is Si(Me)2tert.butyl. The term acyl and subsequently hydrolyzed resulting in compounds means an acyl group derived from an alkylcarboxylic according to formula I or a mixture of compounds acid, the alkyl moiety having the meaning given previ- 40 according to formulas I and III wherein R-| is H by treat- ously, or derived from formic acid. Acetals are derived ment with mild acid such as dilute (< 0.2 N) hydrochloric from alcohols having preferably 1-6 carbon atoms. acid, 50 % acetic acid with cosolvents like THF, ace- With the term tetrahydropyranyl also equivalent tone, methylene chloride, ethanol or by treatment of the mixed acetals or mixed hemithioacetals are meant such derivatives under neutral conditions like trimethylsilyl as e.g. ethoxyethyl, methoxyethyl(MOM), methylmeth- 45 iodide/trimethylsilyl bromide in methylene chloride; oxyethyl, methoxyethoxymethyl (MEM), tetrahydrofura- methyl iodide in acetone, H20, NaHC03; pyridinium p- nyl, methylthiomethyl, tetrahydrothiopyranyl, toluenesulfonate, tert-butanol; tetrabutylammonium flu- tetrahydrothiofuranyl or ethers as methyl and tert-butyl oride in methylene chloride; AgN03 in acetone as as described in Protective Groups in Organic Synthesis, described in Protective Groups in Organic Synthesis, by by Greene, Th. and Wuts, P. (1991), chapter 2, p. 14-87. so Greene, Th. and Wuts, P. (1991), chapter 2, p.14-87. The isomerization can be performed using lithium The thus obtained estrogen derivative mixture can salts of ethylenediamine. This method results into the be sulfated according to the invention in admixture with production of very pure delta(8,9)DHE. Such lithium one or more mono acyl derivatives of the group of 1 7 salts can be prepared by treatment of ethylenediamine alfa dihydro equilin, 17 beta dihydro equilin, 17 alfa with lithium or with alkyllithium, preferably with methyl- 55 estradiol and 17 beta estradiol. lithium. (Co)solvents like tetrahydrofuran, dimethylsul- The following examples are illustrative for the inven- foxide, and the like may be added. Usually mixtures of tion and should in no way be interpreted as limiting the derivatives of delta(8,9)DHE and equilin are obtained scope of the invention.

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Example 1 100 °C and the mixture was stirred for 30 min. at 100 °C. The reaction mixture was cooled to 23 °C , after which 4 Lithium (13 g) was added portionwise to 920 ml of g of 17p-dihydroequilin were added at a temperature of ethylenediamine under an atmosphere of nitrogen at 95 < 30 °C. The mixture was stirred for another 4 h at 30 °C and the mixture was stirred for 30 min. at 1 00 °C. The 5 °C. The suspension was poured into 250 ml of ice water reaction mixture was cooled to 23 °C, after which 100 g and at a temperature of < 25 °C acetic acid was added of equilin were added at a temperature of < 30 °C. The until pH 7. The suspension was cooled to 5 °C and the mixture was stirred for another 2 h at 30 °C. The sus- crystals were filtered off. The crystals were suspended pension was poured into 2.5 1 of ice water and at a tem- in 150 ml of water and 100 ml of ethyl acetate were perature < 25 °C acetic acid was added until pH 7. The 10 added. The layers were separated and the ethyl acetate aqueous layer was extracted three times with 2.5 I of solution was evaporated under vacuum until a volume of ethyl acetate. The organic layer was washed with water, 20 ml. The suspension was stirred at -1 5 °C for 1 h, after 5 g of active carbon (Norit®) were added and the sus- which the crystals were filtered off, washed with ethyl pension was stirred at 21 °C for 30 min. The suspension acetate and dried under vacuum at 40 °C, to obtain 2.5 was filtered over dicalite and the filtrate was evaporated is g of 8,9-dehydro-17p-estradiol, having a purity of > 95 under vacuum until a volume of about 500 ml. The sus- %. pension was stirred for 1 h at 0 °C, after which the crystalline material was filtered off, washed with ethyl Example 5 acetate and dried under vacuum at 40 °C, to obtain 81 g of delta(8,9)-dehydro estrone, having a purity of about 20 A solution of methyllithium-lithiumbromide (20 ml, 95%. 2.1 M) in diethylether was added in 10 min. to 40 ml of The contents of delta(8,9)DHE and equilin were ethylenediamine under an atmosphere of nitrogen at 36 determined using 1H-NMR spectroscopy, characteristic °C. The temperature of the mixture was raised to 55 °C peaks of which are 0.90 ppm (C18) for delta(8,9)DHE and diethylether was distilled off. The mixture was and 5.53 ppm (C7) and 0.79 ppm (C18) for equilin. 25 stirred for 1 h at 55 °C. The reaction mixture was cooled to 3 °C , after which 2 g of equilin-3-methylether were Example 2 added at a temperature of < 10 °C. The mixture was stirred for another 2 h at 1 2 °C, after which 200 ml of ice Lithium amide (5 g) was added to a mixture of 5 g of water were added. To the mixture acetic acid was added equilin in 150 ml of DMSO. The mixture was heated to 30 until pH 8. The suspension was stirred for 1 h at 15 °C, 65 °C and stirred for 70 min. The reaction mixture was after which the crystals were filtered off, washed with poured into 500 ml of water and acidified to pH 6.5 using water and dried under vacuum at 45 °C, to obtain 2.0 g 4N hydrochloric acid. The crystals were filtered off, of 8,9-dehydro-estrone-3-methylether, having a purity of washed with water and dried under vacuum at 40 °C to approx. 80 %. obtain 5 g of a 4:5 mixture of equilin and delta(8,9)- 35 dehydro estrone. Example 6

Example 3 According to the procedure described in example 5, 17p-dihydroequilin 3,17-diacetate, was treated with A 6% solution of methyllithium-lithiumbromide com- methyllithium/ethylenediamine at 30 °C to give quantita- plex in diethylether (23.5 ml) was added during approx- tively 8,9-dehydro-17p-estradiol having a purity of imately 15 minutes to 46 ml ethylenediamine under an approx. 90 %. atmosphere of nitrogen at a temperature of approximately 25°C. The temperature of the mixture was raised Example 7 to 55°C and diethylether was distilled off. Subsequently 45 the reaction mixture was stirred for 1 h at 55°C. The mix- According to the procedure described in example 4, ture was cooled to 20°C and 2.5 g of equilin was added. equilin-17-neopentylacetal was treated with lithium/eth- The mixture was stirred for another 90 minutes at 30°C. ylenediamine at 20 °C to give in a yield of 90 % 8,9- The suspension was poured into ice water and the dehydro-estron-17-neopentylacetal having a purity of mixture was extracted with ethyl acetate. After evapora- 50 approx. 90 %. tion of the ethyl acetate extract until a volume of 20 ml was reached and cooling to 0°C, 2 g of crystalline delta Example 8 8-estrone was isolated. According to the procedure described in example 4, Example 4 1 7p-dihydroequilin-3,1 7-di(trimethylsilylether) was treated with lithium/ethylenediamine to give quantita- Lithium (1,1 g) was added portionwise to 80 ml of tively 8,9-dehydro-17p-estradiol having a purity of ethylenediamine under an atmosphere of nitrogen at approximately 90 %.

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Example 9 to 20°C and a mixture of n-butanol (0.65 I) and water (0.15 I) was added. Methanol was evaporated in vac- To a suspension of equilin (5 g) in 80 ml of methyl- uum and the organic layer was washed with aq 5% ene chloride were added 13.3 ml of 3,4-dihydro-2H- sodium chloride and water. After evaporation of the pyran and 36 mg of p-toluenesulfonic acid at 0 °C. The s organic layer to dryness, the residue was dissolved in mixture was stirred for 90 min at 0 °C, after which 1 .3 ml 100% ethanol (0.29 I) at 60 °C and the mixture was of triethylamine was added. The reaction mixture was stirred in the presence of active carbon (0.6 g) for 30 washed with water, dried with sodium sulfate and evap- min at 60 °C. The suspension was filtered and the fil- orated to dryness. The crude equilin-3-tetrahydropyran- trate was partially concentrated in vacuum. The solution ylether (6.5 g) was used for isomerization. w was cooled to 10 °C and triturated with diethylether (0.27 I). The precipitate was filtered off, washed with Example 10 diethylether and dried in vacuum to yield 15.5 g of a 1 :5 mixture of delta(8,9)-dehydro estrone sodiumsulfate A solution of methyllithium-lithiumhromide (8.1 ml, and 17a-dihydro equilin sodiumsulfate. GLC analysis 2.1 M) in diethylether was added in 10 min to 16.2 ml of is indicated full recovery of all steroids charged. ethylenediamine under an atmosphere of nitrogen at 36 °C. The temperature of the mixture was raised to 55 °C Example 13 and diethylether was distilled off. The mixture was further stirred for 1 h at 55 °C, then cooled to 3 °C. After According to the procedure described in example addition of 34.4 ml of tetrahydrofuran the mixture was 20 12, a mixture of delta(8,9)-dehydro estrone (12.5 g) and cooled to -10 °C and equilin-3-tetrahydropyranylether equilin (12.5 gram) were sulfated with sulfuric acid / ace- (1 . 1 5 g) was added at a temperature of < -5 °C. The mix- tic anhydride / pyridine. The mixture of crude pyridi- ture was stirred for another 3 h at -10 °C, after which 45 nesulfates was saponified and converted to the ml of ice water was added. Then, tetrahydrofuran was corresponding mixture of sodiumsulfates with sodium distilled off using vacuum, and the residual suspension 25 hydroxide in methanol as described in example 12, was stirred for 1 h at 15 °C. The crystals were filtered yielding 27.5 g of a 1:1 mixture of delta(8,9)-dehydro off, washed with water and dried under vacuum at 45 estrone sodiumsulfate and equilin sodiumsulfate. GLC °C, to yield delta(8,9)-dehydro-estrone-3-tetrahydro- analysis indicated full recovery of the steroids charged. pyranylether (1 .0 g). 30 Example 14 Example 1 1 According to the procedure described in example A suspension of 0.73 g of delta(8,9)-dehydro 12, a mixture of delta (8,9)-dehydro estrone (6.1 g), estrone-3-tetrahydropyranylether in acetic acid / ace- 17a-dihydro equilin monoacetate (35.8 g), 17p-dihydro tone / water (4:2:1, 35.7 ml) was heated to reflux and 35 equilin monoacetate (3.5 g), 1 7

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of compounds according to formula III wherein R1 = silyl(alkyl)3 or tetrahydropyranyl.

7. Method for the preparation of a sulfated estrogen 5 mixture comprising the steps of

a. partial isomerization of compounds with the general formula III

wherein R1 is H, 15 R2 is H and R3 is O-acyl; or R3 is H and R2 is O-acyl; or R2 and R3 together represent O in admixture with one or more compounds taken from the group of compounds of general formula II

wherein R-| is silyl(alkyl)3 or O-tetrahydropyranyl, 25 R2 and R3 together represent O; or R2 and R3 together represent acetal or cyclic acetal with a lithium salt of ethylenediamine, optionally in admixture with tetrahydro- 30 furan, or with lithium amide in dimethylsulfoxide to compounds with general formula I

wherein R-j, R2 and R3 have the previously defined meanings and the dotted line at position 7-8 represents an optional double bond.

2. Method according to claim 1 wherein R2 and R3 in formula I and/or II represent O.

3. Method according to claim 2 wherein delta 8,9 estrone is sulfated in admixture with equilin.

4. Method according to claim 1 wherein delta 8,9 wherein R-|, R2 and R3 have the previously estrone is sulfated in admixture with at least one of defined meanings the compounds selected from the group of mono 45 acyl derivatives of 17 alfa dihydro equilin, 17 beta b. conversion of the compounds obtained from dihydro equilin, 17 alfa estradiol and 17 beta estra- step a whereby the Ri substituent becomes H diol. and R2 and R3 together O. c. sulfatation of the compounds obtained from 5. Method according to claim 4 characterized in that so step b, optionally in admixture with one or more the mixture is sulfated in a ratio as present in natu- mono acyl derivatives of the group of 1 7 alfa ral occurring conjugated estrogen compositions. dihydro equilin, 17 beta dihydro equilin, 17 alfa estradiol and 17 beta estradiol. 6. Method for the preparation of a sulfated estrogen mixture characterized in that prior to conversion of 55 8. Method for the preparation of a sulfated estrogen the mixture into the corresponding sulfates accord- mixture comprising the steps of ing to claim 1-5, at least the compounds according to formula I wherein R-| = H have been formed out a. isomerization of compounds with the general

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formula III

15 wherein R-| is silyl(alkyl)3 or O-tetrahydropyranyl, R2 and R3 together represent O; or R2 and R3 together represent acetal or cyclic acetal 20 with a lithium salt of ethylene-diamine or with lithium amide in dimethylsulfoxide to compounds with general formula I

wherein R1 , R2 and R3 have the previously 35 defined meanings

b. conversion of the compounds obtained from step a whereby the R1 substituent becomes H and R2 and R3 together O. 40 c. sulfatation of the compound obtained from step b in admixture with one or more mono acyl derivatives of the group of 1 7 alfa dihydro equilin, 17 beta dihydro equilin, 17 alfa estradiol and 1 7 beta estradiol. 45

Method according to claim 7 or 8 wherein R2 and R3 together represent O.