Synthesis of 13C-labeled steroid hormones Gijsbert
Zomer and Jobannes
F.C. Stavenuiter
National Institute of Public Health and Environmental The Netherlands
Protection,
Bilthoven,
The synthesis of ‘3C-labeled steroid hormones is reviewed. Two general approaches are highlighted: partial synthesis in which part of the steroid nucleus is replaced with ‘3C-labeled synthons, and total synthesis. Examples from both approaches, leading to (3--‘C)-, (4-“C)-, (3,4-‘“C,)-, und (I ,2,3,4-‘.‘C,)and cortisol), are presented. labeled steroid hormones (e.g.. testosterone, estradiol, progesterone, (Steroids 55440-442. 1990)
Keywords:
steroid(s);
‘jC-labeled
steroids:
synthesis;
stable
Introduction 13C-Labeled steroid hormones are needed in the development of definitive gas chromatography/mass spectrometry quantitation methods. They also find application as tracers for in vivo metabolic and kinetic studies. The advantages of “C over 2H include stability of incorporation of the label and better nuclear magnetic resonance visibility. Furthermore, 13Catoms alter the properties of the molecule much less than deuterium. The isotope effects caused by “C both in vitro (e.g., gas chromatography retention times, fragmentation in the mass spectrometer) and in vivo are much smaller than deuterium isotope effects. This paper reviews the synthesis of “C-labeled steroids. Preparation of 13C-labeled steroid hormones by degradation of existing steroids followed by incorporation of the 13C label(s) The procedure described below, pioneered by Turner,’ is depicted in Scheme I. The required enol lactone can be prepared by ozonolysis of the @-unsaturated ketone function, followed by lactonization of the resulting keto acid. The anion of this enol lactone, formed by reaction with sodium hydride, is reacted with phenyl [l-‘3C]acetate. Ring opening of the resulting product, followed by decarboxylation, gives a 1,5-diketone that on treatment with acid or base, gives the (3-‘3C)-labeled steroid. Obviously,
Address reprint requests to Dr. Gijsbert Zomer, of Public Health and Environmental Protection, BA Bilthoven, The Netherlands.
440
Steroids,
1990, vol. 55, October
National Institute P.O. Box I. 3720
isotope
labeling
(4-‘3C)-labeled steroids can be prepared in the same way by substituting phenyl [l- 13C]acetate for phenyl [2-“Clacetate. An improved method was reported by Fujimoto.’ This method involves the Grignard addition of labeled methyl iodide to Turner’s enol lactone, followed by alkali treatment (Scheme 2); it is simpler and milder and gives higher yields (45% to 50%) than Turner’s method. The method was also successfully applied to the synthesis of [4-‘3C]progesterone.3 In this case, the C-20 ketone function was protected as the enol acetate. The synthesis has been slightly modified to allow for the introduction of two 13C atoms (Scheme 3).4 The enolate anion formed by the reaction of Turner’s lactone with a base was allowed to react with [1,2-“C,] acetyl chloride. For the synthesis to be successful, the choice of the base used in generating the enolate anion proved critical: the best results were obtained with either lithium bis(trimethylsilyl)amide or lithium N-isopropylcyclohexylamide. Hydrolysis, decarboxylation, and ring closure could be effected in one step using either concentrated hydrochloric acid in acetic acid or methanolic potassium hydroxide. Overall yields of c@-unsaturated ketones were approximately 40%. [3,4‘3Cz]Cholest-4-en-3-one could be converted to [3,4‘3CZ]cholesterol in 50% yield by silylating the enone to yield the trimethylsilyl enol ether, which was subsequently reduced with sodium borohydride. A similar procedure was used to prepare estrone and estradiol (Scheme 4): IPnortestosterone benzoate was converted to an enol lactone by ozonolysis, followed by treatment of the resulting keto acid with a mixture of acetyl chloride and acetic anhydride. Treatment of the enolate anion with [I ,2-“C,]acetyl chloride, followed 0
1990 Butterworth-Heinemann
13C-Labeled steroid synthesis: Zomer and Stavenuiter
Scheme 1
Synthesis of [3-13C]-labeled steroids.
by reaction with acid, gave [3,4-‘3C2]estr-4-en-3-one 17-benzoate, a key compound in the synthesis of [3,4r3C,]estradiol and [3,4-‘3C,]estrone. [1,2-13C,]Estradiol was obtained by aromatization (CuBr! , LrBr, acetonitrile), followed by hydrolysis. [ 1,2- 3C,]Estrone was prepared by hydrolysis of the benzoate ester, followed by oxidation of the 17-hydroxyl group and aromatization using the same conditions as with [I ,2‘3C2]estradiol. [3,4-‘3C,]Progesterone was also synthesized by this general approach. The C-20 ketone function was protected as the ethylenedioxy ketal. Acylation using [ 1,2‘3C,]acetyl chloride and treatment of the acetylated product with strong acid gave [ 1,2-r3C,]progesterone.
Preparation of WC-labeled steroid hormones by total synthesis
R=IvJ\ Scheme 2
R= ),/&
Scheme 3
oro$n,
*=“C
Synthesis of [4-13C]-labeled steroids.
orO&Ph
Total synthesis of r3C-labeled steroids offers a number of advantages. First, a clever design of the synthetic route toward the r3C-labeled compounds gives the opportunity to prepare more than one labeled steroid from the same precursor molecule. Second, there is the possibility of introducing more than one or two r3C atoms into the steroid molecule. An example of a total synthetic approach is shown in Scheme 5j? reaction of 1-iodo-3,3_ethylenedioxy-
‘=“C
Synthesis of [3,4-‘3C,]-labeled
steroids.
. ii “C Scheme 6
P
Synthesis of [1,2,3,4-13C,]-labeled
(EtObPOH -
‘,*.oH
9 .,*.oA
0
KH -
steroids. 0
.A.,~.,/,
1,“” II B’,.,*,oH
-
B++a
Br. . ,*.o”
NaOH
(EtOLPQ I&’ I
(OWzOP..
Scheme4
Synthesis of [3,4-‘3C,]estradiol
and [3,4-13C,]estrone.
/*.
r7 4 ,o . .‘\.
Scheme 6 Synthesis of 13C-labeled pentanone ethylene ketal.
Steroids,
5-(diethylphosphono)-2-
1990, vol. 55, October
441
Papers OH
1) H+C(CH&,
H’
2) OH 0 3)NaOAC i AC20 0
PO(OEt)r Z)on’/MOOH
.=‘C
Scheme 7
Synthesis
of multiple
‘3C-labeled
steroids.
[ 1,2,3,4-13C,]butane with the anion of des-A- 17p-t-butoxy-9-estren-5-one gave des-A-17P-t-butoxy-9-(3,3ethylenedioxy[ 1,2,3,4-‘3C,]butyl)-9-estren-5-one. This material could be converted to [ 1,2,3,4-‘3C,]testosterone by reductive methylation (lithium metal in liquid ammonia using methyl iodide), followed by treatment with acid. This material could also be converted to [I ,2,3,4-‘3C,]estradiol by deketalization using pyridinium p-toluenesulfonate in refluxing wet acetone, followed by ring closure using potassium t-amylate and aromatization using acetyl bromide and acetic anhydride. Finally, this material was transformed into [ 1,2,3,4-‘3C,]cortisol acetate in 11 steps. This scheme offers the possibility of introducing more 13Catoms into the steroid. In this example, four carbon atoms have been replaced with their stable isotopes; however, if necessary, this can be easily extended to five carbon atoms by using 13C-labeled methyl iodide in the case of testosterone and cortisol or more carbon atoms by using a ‘3C-labeled Cz-unit , e.g., acetylene, to construct the dihydroxyacetone side chain of cortisol. The starting material for this synthesis, I -iodo3,3_ethylenedioxy-[ 1,2,3,4-‘3C,]butane is easily prepared in 41% overall yield from [ 1,2-‘3C,]ethylene and Des-A-17/W-butoxy-9-es[l 2-13C2a] cetyl chloride. trin-5-one can be prepared in 30% overall yield from commercially available (7aS)-( +)-2,3,7,7a_tetrahydro7a-methyl- 1H-indene- IS-(6H)-dione (Aldrich Chemie, Brussels, Belgium).’ Subsequently, another approach to total synthesis was published. 8 Following the work of Henrick et al. ,9
442
Steroids,
1990, vol. 55, October
a synthesis for ‘“C-labeled 5-(diethylphosphono)-2-pentanone ethylene ketal was devised according to Scheme 6: [2,3-“C,]acetic acid was esterified and condensed using potassium hydride to give ethyl 2-0x0-[1,2,3,4‘3C,]butanoate. Bromination of [ 1,2-‘3CJacetic acid, followed by reduction and ring closure, gave [1,2‘3C2]ethylene oxide. Reaction of ethyl 2-0x0-I I ,2,3,4‘3C,]butanoate with [ 1,2-‘3CJethylene oxide gave 2acetylbutyrolactone labeled with “C at all the carbon atoms. Reaction with hydrogen bromide afforded 5bromo-2-l I ,2,3,4,5-“C,]pentanone. Ketalization and reaction of the result’ng ketal with the sodium salt of diethyl phosphite gave 5-(diethylphosphono)-2[ 1,2,3,4,5-‘3C,]pentanone ethylene ketal. The use of this C, synthon in the synthesis of multiple ‘3C-labeled steroids can be envisioned as shown in Scheme 7.’ The optically pure precursor compound is readily available. “’ This synthetic route affords the same intermediate as the one obtained in Scheme 5, albeit in this case with the possibility of introducing five 13Catoms into the steroid.
References I. 2.
3. 4. 5. 6. 7.
8.
Turner RB (1950). Steroids labeled with isotopic carbon: cholestenone and testosterone. J Am Chem SW 72:579-585. Fujimoto GI (1951). Labeling of steroids in the 4-position. J Am Churn Sot 73: 1856. Fujimoto G, Prager J (1953). A synthesis of progesterone labeled in position 4. J Am Chem SOL. 75:3259-3261. Yuan S (1982). Svnthesis of 3.4-“C, steroids. Steroids 39:279-289. Zomer G, Wynberg H, Drayer NM (1984). [ 1,2,3,4-“C,ITestosterone and I I ,2.3.4-“C,lestradiol. Steroids 44:283-292. Zomer G. Wynberg H, Diayer NM (1984). The synthesis of I I ,2.3,4-“C,]cortisol. Steroids 44:293-300. Hajos ZG, Parrish DR (1984). ( + )-(laS)-2,3,7,7a_Tetrahydro(I H-indene-I ,5(6H)-dila-methyl-l H-indene-I ,5-(6H)-dione one. 2,3.7.7a-tetrahydro-7a-methyl-, (S)-). Org SW 63:26-36. DeGraw JI. Christie PH. Cairns T (1982). Svnthesis of carbon13-labeled S-(diethylphosphono)-2:pentzmdne ethylene ketal. a reagent for synthesis of multi carbon-13 labeled steroids. ./
Lahelled 9.
IO.
Compd
Rudiopharm
19~945-952.
Henrick CA, BZihme E, Edwards JA, Fried JH (1968). The reaction of phosphoranes and phosphonate anions with enol lactones. A new method for the preparation of cyclic a,/~?unsaturated ketones. J Am Chem Sot 90:5926-5927. Velluz L. Nomine G. Amiard G, Torelli V, CC&de J (1963). Progress in total steroid synthesis. C R Acud Sci [III]
257:3086-3088.
Zomer and Jobannes
F.C. Stavenuiter
National Institute of Public Health and Environmental The Netherlands
Protection,
Bilthoven,
The synthesis of ‘3C-labeled steroid hormones is reviewed. Two general approaches are highlighted: partial synthesis in which part of the steroid nucleus is replaced with ‘3C-labeled synthons, and total synthesis. Examples from both approaches, leading to (3--‘C)-, (4-“C)-, (3,4-‘“C,)-, und (I ,2,3,4-‘.‘C,)and cortisol), are presented. labeled steroid hormones (e.g.. testosterone, estradiol, progesterone, (Steroids 55440-442. 1990)
Keywords:
steroid(s);
‘jC-labeled
steroids:
synthesis;
stable
Introduction 13C-Labeled steroid hormones are needed in the development of definitive gas chromatography/mass spectrometry quantitation methods. They also find application as tracers for in vivo metabolic and kinetic studies. The advantages of “C over 2H include stability of incorporation of the label and better nuclear magnetic resonance visibility. Furthermore, 13Catoms alter the properties of the molecule much less than deuterium. The isotope effects caused by “C both in vitro (e.g., gas chromatography retention times, fragmentation in the mass spectrometer) and in vivo are much smaller than deuterium isotope effects. This paper reviews the synthesis of “C-labeled steroids. Preparation of 13C-labeled steroid hormones by degradation of existing steroids followed by incorporation of the 13C label(s) The procedure described below, pioneered by Turner,’ is depicted in Scheme I. The required enol lactone can be prepared by ozonolysis of the @-unsaturated ketone function, followed by lactonization of the resulting keto acid. The anion of this enol lactone, formed by reaction with sodium hydride, is reacted with phenyl [l-‘3C]acetate. Ring opening of the resulting product, followed by decarboxylation, gives a 1,5-diketone that on treatment with acid or base, gives the (3-‘3C)-labeled steroid. Obviously,
Address reprint requests to Dr. Gijsbert Zomer, of Public Health and Environmental Protection, BA Bilthoven, The Netherlands.
440
Steroids,
1990, vol. 55, October
National Institute P.O. Box I. 3720
isotope
labeling
(4-‘3C)-labeled steroids can be prepared in the same way by substituting phenyl [l- 13C]acetate for phenyl [2-“Clacetate. An improved method was reported by Fujimoto.’ This method involves the Grignard addition of labeled methyl iodide to Turner’s enol lactone, followed by alkali treatment (Scheme 2); it is simpler and milder and gives higher yields (45% to 50%) than Turner’s method. The method was also successfully applied to the synthesis of [4-‘3C]progesterone.3 In this case, the C-20 ketone function was protected as the enol acetate. The synthesis has been slightly modified to allow for the introduction of two 13C atoms (Scheme 3).4 The enolate anion formed by the reaction of Turner’s lactone with a base was allowed to react with [1,2-“C,] acetyl chloride. For the synthesis to be successful, the choice of the base used in generating the enolate anion proved critical: the best results were obtained with either lithium bis(trimethylsilyl)amide or lithium N-isopropylcyclohexylamide. Hydrolysis, decarboxylation, and ring closure could be effected in one step using either concentrated hydrochloric acid in acetic acid or methanolic potassium hydroxide. Overall yields of c@-unsaturated ketones were approximately 40%. [3,4‘3Cz]Cholest-4-en-3-one could be converted to [3,4‘3CZ]cholesterol in 50% yield by silylating the enone to yield the trimethylsilyl enol ether, which was subsequently reduced with sodium borohydride. A similar procedure was used to prepare estrone and estradiol (Scheme 4): IPnortestosterone benzoate was converted to an enol lactone by ozonolysis, followed by treatment of the resulting keto acid with a mixture of acetyl chloride and acetic anhydride. Treatment of the enolate anion with [I ,2-“C,]acetyl chloride, followed 0
1990 Butterworth-Heinemann
13C-Labeled steroid synthesis: Zomer and Stavenuiter
Scheme 1
Synthesis of [3-13C]-labeled steroids.
by reaction with acid, gave [3,4-‘3C2]estr-4-en-3-one 17-benzoate, a key compound in the synthesis of [3,4r3C,]estradiol and [3,4-‘3C,]estrone. [1,2-13C,]Estradiol was obtained by aromatization (CuBr! , LrBr, acetonitrile), followed by hydrolysis. [ 1,2- 3C,]Estrone was prepared by hydrolysis of the benzoate ester, followed by oxidation of the 17-hydroxyl group and aromatization using the same conditions as with [I ,2‘3C2]estradiol. [3,4-‘3C,]Progesterone was also synthesized by this general approach. The C-20 ketone function was protected as the ethylenedioxy ketal. Acylation using [ 1,2‘3C,]acetyl chloride and treatment of the acetylated product with strong acid gave [ 1,2-r3C,]progesterone.
Preparation of WC-labeled steroid hormones by total synthesis
R=IvJ\ Scheme 2
R= ),/&
Scheme 3
oro$n,
*=“C
Synthesis of [4-13C]-labeled steroids.
orO&Ph
Total synthesis of r3C-labeled steroids offers a number of advantages. First, a clever design of the synthetic route toward the r3C-labeled compounds gives the opportunity to prepare more than one labeled steroid from the same precursor molecule. Second, there is the possibility of introducing more than one or two r3C atoms into the steroid molecule. An example of a total synthetic approach is shown in Scheme 5j? reaction of 1-iodo-3,3_ethylenedioxy-
‘=“C
Synthesis of [3,4-‘3C,]-labeled
steroids.
. ii “C Scheme 6
P
Synthesis of [1,2,3,4-13C,]-labeled
(EtObPOH -
‘,*.oH
9 .,*.oA
0
KH -
steroids. 0
.A.,~.,/,
1,“” II B’,.,*,oH
-
B++a
Br. . ,*.o”
NaOH
(EtOLPQ I&’ I
(OWzOP..
Scheme4
Synthesis of [3,4-‘3C,]estradiol
and [3,4-13C,]estrone.
/*.
r7 4 ,o . .‘\.
Scheme 6 Synthesis of 13C-labeled pentanone ethylene ketal.
Steroids,
5-(diethylphosphono)-2-
1990, vol. 55, October
441
Papers OH
1) H+C(CH&,
H’
2) OH 0 3)NaOAC i AC20 0
PO(OEt)r Z)on’/MOOH
.=‘C
Scheme 7
Synthesis
of multiple
‘3C-labeled
steroids.
[ 1,2,3,4-13C,]butane with the anion of des-A- 17p-t-butoxy-9-estren-5-one gave des-A-17P-t-butoxy-9-(3,3ethylenedioxy[ 1,2,3,4-‘3C,]butyl)-9-estren-5-one. This material could be converted to [ 1,2,3,4-‘3C,]testosterone by reductive methylation (lithium metal in liquid ammonia using methyl iodide), followed by treatment with acid. This material could also be converted to [I ,2,3,4-‘3C,]estradiol by deketalization using pyridinium p-toluenesulfonate in refluxing wet acetone, followed by ring closure using potassium t-amylate and aromatization using acetyl bromide and acetic anhydride. Finally, this material was transformed into [ 1,2,3,4-‘3C,]cortisol acetate in 11 steps. This scheme offers the possibility of introducing more 13Catoms into the steroid. In this example, four carbon atoms have been replaced with their stable isotopes; however, if necessary, this can be easily extended to five carbon atoms by using 13C-labeled methyl iodide in the case of testosterone and cortisol or more carbon atoms by using a ‘3C-labeled Cz-unit , e.g., acetylene, to construct the dihydroxyacetone side chain of cortisol. The starting material for this synthesis, I -iodo3,3_ethylenedioxy-[ 1,2,3,4-‘3C,]butane is easily prepared in 41% overall yield from [ 1,2-‘3C,]ethylene and Des-A-17/W-butoxy-9-es[l 2-13C2a] cetyl chloride. trin-5-one can be prepared in 30% overall yield from commercially available (7aS)-( +)-2,3,7,7a_tetrahydro7a-methyl- 1H-indene- IS-(6H)-dione (Aldrich Chemie, Brussels, Belgium).’ Subsequently, another approach to total synthesis was published. 8 Following the work of Henrick et al. ,9
442
Steroids,
1990, vol. 55, October
a synthesis for ‘“C-labeled 5-(diethylphosphono)-2-pentanone ethylene ketal was devised according to Scheme 6: [2,3-“C,]acetic acid was esterified and condensed using potassium hydride to give ethyl 2-0x0-[1,2,3,4‘3C,]butanoate. Bromination of [ 1,2-‘3CJacetic acid, followed by reduction and ring closure, gave [1,2‘3C2]ethylene oxide. Reaction of ethyl 2-0x0-I I ,2,3,4‘3C,]butanoate with [ 1,2-‘3CJethylene oxide gave 2acetylbutyrolactone labeled with “C at all the carbon atoms. Reaction with hydrogen bromide afforded 5bromo-2-l I ,2,3,4,5-“C,]pentanone. Ketalization and reaction of the result’ng ketal with the sodium salt of diethyl phosphite gave 5-(diethylphosphono)-2[ 1,2,3,4,5-‘3C,]pentanone ethylene ketal. The use of this C, synthon in the synthesis of multiple ‘3C-labeled steroids can be envisioned as shown in Scheme 7.’ The optically pure precursor compound is readily available. “’ This synthetic route affords the same intermediate as the one obtained in Scheme 5, albeit in this case with the possibility of introducing five 13Catoms into the steroid.
References I. 2.
3. 4. 5. 6. 7.
8.
Turner RB (1950). Steroids labeled with isotopic carbon: cholestenone and testosterone. J Am Chem SW 72:579-585. Fujimoto GI (1951). Labeling of steroids in the 4-position. J Am Churn Sot 73: 1856. Fujimoto G, Prager J (1953). A synthesis of progesterone labeled in position 4. J Am Chem SOL. 75:3259-3261. Yuan S (1982). Svnthesis of 3.4-“C, steroids. Steroids 39:279-289. Zomer G, Wynberg H, Drayer NM (1984). [ 1,2,3,4-“C,ITestosterone and I I ,2.3.4-“C,lestradiol. Steroids 44:283-292. Zomer G. Wynberg H, Diayer NM (1984). The synthesis of I I ,2.3,4-“C,]cortisol. Steroids 44:293-300. Hajos ZG, Parrish DR (1984). ( + )-(laS)-2,3,7,7a_Tetrahydro(I H-indene-I ,5(6H)-dila-methyl-l H-indene-I ,5-(6H)-dione one. 2,3.7.7a-tetrahydro-7a-methyl-, (S)-). Org SW 63:26-36. DeGraw JI. Christie PH. Cairns T (1982). Svnthesis of carbon13-labeled S-(diethylphosphono)-2:pentzmdne ethylene ketal. a reagent for synthesis of multi carbon-13 labeled steroids. ./
Lahelled 9.
IO.
Compd
Rudiopharm
19~945-952.
Henrick CA, BZihme E, Edwards JA, Fried JH (1968). The reaction of phosphoranes and phosphonate anions with enol lactones. A new method for the preparation of cyclic a,/~?unsaturated ketones. J Am Chem Sot 90:5926-5927. Velluz L. Nomine G. Amiard G, Torelli V, CC&de J (1963). Progress in total steroid synthesis. C R Acud Sci [III]
257:3086-3088.
