Research Article Received 17 November 2014,
Revised 03 December 2014,
Accepted 04 December 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3257
Synthesis of a [14C]-steroid intermediate: an application of a nonstabilized Horner– Wadsworth–Emmons olefination approach Nelo R. Rivera,* Sumei Ren, and David Hesk Radiolabeled steroid derivative 1 was successfully prepared using a Horner–Wadsworth–Emmons approach: a [14C]-label was efficiently incorporated into the C-18 position of the molecule. Previously published procedures employing other olefination methods are either not applicable due to unavailability of [14C]-precursors or suffer from poor reactivity. Keywords: steroid; carbon-14; Horner–Wadsworth–Emmons (HWE); olefination
Introduction We recently needed to prepare [14C]-radiolabeled steroid derivative 1 to be used as a synthon for [14C]-labeled steroid compounds. The synthesis of unlabeled 1 has been previously disclosed by other groups (Scheme 1).1 Based on their general synthetic strategy, we wanted to pursue a late-stage introduction of the radiolabel at the C-18 position via olefination with an appropriate [14C] reagent.
Results and discussion
20
The direct conversion of ketone 3 to the corresponding unlabeled olefin 4 was accomplished using either Peterson or Wittig conditions (Scheme 1, conditions iv or v, respectively).1 The requisite [14C]-trimethylsilylmethyl lithium or equivalent reagent needed for the Peterson olefination, however, is not easily accessible. On the other hand, the [14C]-Wittig reagent is readily prepared from [14C]-methyl iodide and triphenylphosphine.2 Unfortunately, we were unable to efficiently carry out the direct transformation of ketone 3 to olefin 4 with [14C]-methyltriphenylphosphonium iodide. Less than 5% conversion of starting material to product was observed despite using an excess of the [14C]-Wittig reagent. This led us to consider an alternative Horner–Wadsworth– Emmons (HWE) approach. The addition of HWE reagents such as dimethyl methylphosphonate, without a carbanion stabilizing group at the β-position, to ketones results in β-hydroxyphosphonates which do not readily eliminate to give the desired olefin product. As a consequence, olefination using nonstabilized HWE reagents has been viewed as a difficult transformation. However, a report by Reichwein and Pagenkopf showed that the resulting hydroxyphosphonate undergoes elimination via a saponification–dehydration sequence.3 To our delight, this was indeed the case when we subjected our substrate to the reported conditions using unlabeled dimethyl methylphosphonate. Encouraged by this initial result, we
J. Label Compd. Radiopharm 2015, 58 20–22
prepared the [14C]-HWE reagent via the Michaelis–Arbuzov reaction.4 Addition of the [14C]-HWE reagent to ketone 3 gave the corresponding hydroxyphosphonate 5 in 46% radiochemical yield based on the phosphonate reagent. We also found that quenching the reaction at low temperature was key to the success of this reaction. Formation of the desired product was not observed when the reaction was allowed to warm to room temperature; only starting material was observed by HPLC analysis. Saponification using methanolic aqueous NaOH gave the corresponding monophosphonic acid 6; and dehydration using diisopropylcarbodiimide followed by acetal deprotection under acidic conditions gave the desired [14C]-steroid intermediate 1 in 78% overall yield from 5 (Scheme 2).
Conclusion We have successfully demonstrated the use of a nonstabilized HWE reagent for the introduction of a [14C]-label into an advance steroid intermediate. Other olefination approaches such as Peterson and Wittig reactions that have been used for the preparation of the unlabeled steroid 1 were not applicable in our case due to the unavailability of the [14C]precursor (Peterson approach) or lack of reactivity of the [14C]-reagent (Wittig approach). The slow elimination of the HWE adduct was successfully overcome by applying the twostep sequence involving a partial hydrolysis followed by dehydration.
Department of Process Chemistry, Merck Research Laboratories, 126 E. Lincoln Ave., PO Box 2000, Rahway, NJ 07065, USA *Correspondence to: Nelo R. Rivera, Department of Process Chemistry, Merck Research Laboratories, 126 E. Lincoln Ave., PO Box 2000, Rahway, NJ, 07065, USA. E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
N. R. Rivera et al.
Scheme 1. Synthesis of unlabeled steroid derivative 1. Conditions: (i) p-TsOH, 1,2-ethanedithiol, MeOH, reflux, 72%; (ii) ethylene glycol, triethylorthoformate, p-TsOH, CH2Cl2, 40 °C then 20 °C, 96%; (iii) pyridinium dichromate, DMF, 30 °C, 89%; (iv) Mg, I2, chloromethyl trimethylsilane, dibromoethane, THF, toluene, reflux, 87% (Peterson condition); (v) methyl triphenylphosphonium bromide, KOtBu, toluene, 90 °C, 68% (Wittig condition); (vi) H2SO4, water, acetone, 20 °C, 83%.
Experimental Chemicals and solvents were obtained from standard commercial sources and used without further purification. [14C]-methyl iodide (SA = 56.0 mCi/mmol) was obtained from Amersham, Piscataway, NJ. Ketone 3 as well as an authentic sample of unlabeled 1 were provided to us by Merck Process Chemistry Group. Mass spectrometry determinations were performed on an Agilent 1100 CDMSD instrument in ES positive ionization mode. Agilent Poroshell 120 EC-C18 column (2.7 um, 50 × 3.0 mm), 1.0 mL/min, 40 °C. Gradient from 10% Acetonitrile [0.1% Formic Acid] / 90% Water [0.1% Formic Acid] to 95% Acetonitrile [0.1% Formic Acid] / 5% Water [0.1% Formic Acid] from 0 to 5.2 min. Six minutes run time. High-performance liquid chromatography condition used to monitor reaction progress and radiochemical purity (RCP) is shown below. Retention times of products obtained are consistent with that of authentic unlabeled material. Supelco Ascentis Express C18 column (2.7 μm, 50 × 4.6 mm), 210 nm, 1.8 mL/min, 40 °C. Gradient from 10% Acetonitrile [0.1% Formic Acid] / 90% Water [0.1% Formic Acid] to 95% Acetonitrile [0.1% Formic Acid] / 5% Water [0.1% Formic Acid] from 0 to 6.5 min. Eight minutes run time. Compound 5 To a round bottom flask containing [14C]-dimethyl methylphosphonate (SA = 28.9 mCi/mmol; 92.7 mCi) was added a THF solution of ketone 3 (670 mg; 1.593 mmol) in THF (4 mL), then cooled to < 70 °C. Lithium diisopropylamide (LDA) (1.68 M; 1.52 mL) was added slowly via syringe over a period of 10–15 min. The reaction was aged for 1 hr, and after completion as indicated by HPLC analysis, it was quenched at < 70 °C with
Aq NH4Cl (6.0 mL). Upon warming to 10 °C, the reaction mixture was extracted with EtOAc (10 mL), and the resulting organic layer was washed with Aq NH4Cl (6 mL) followed by Aq NaCl (6 mL). The aqueous layers were re-extracted with EtOAc (10 mL), and the resulting combined organic layers were dried over MgSO4, filtered and purified by column chromatography (80% EtOAc/Hexanes + 0.01% Et3N) to give 5 (42.9 mCi, 46.3% yield; 99.0% RCP). Liquid chromatography–mass spectrometry (LC–MS) (EI+) m/z 547 (M + H)+. Compound 6 To a round bottom flask containing hydroxyphosphonate 5 (42.9 mCi; 1.49 mmol) in MeOH (13 mL) was added 4 M NaOH (1.195 mL; 4.78 mmol) and stirred at room temperature overnight. The MeOH solution was partially concentrated, and the solution was adjusted to pH 2 with 1 M HCl (ca 4 mL). The reaction was then extracted with dichloromethane (10 mL, then 3 × 6.5 mL). The organic layers were dried over MgSO4, filtered and concentrated and used directly in the next step. Crude product 6 had an RCP of 98.5%. LC–MS (EI+) m/z 533 (M + H)+. Compound 1 To a round bottom flask containing β-hydroxy phosphonic acid 6 (42.9 mCi; 1.49 mmol) in chloroform (7.5 mL) was added diisopropylcarbodiimide (0.620 mL, 3.98 mmol) and aged at room temperature for approximately 1 hr after which analysis showed complete conversion to acetal intermediate 4. MeOH (1 mL) was added, and the crude reaction was concentrated down and rediluted with MeOH (10 mL). To the resulting slurry was added 6 M HCl (0.531 mL, 3.19 mmol) and stirred at room temperature overnight. After reaction completion, the slurry was concentrated down and redissolved in 20 mL EtOAc. The thin slurry was passed through a filter, and the filtered solid was washed down with
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Scheme 2. Horner–Wadsworth–Emmons olefination approach to [ C]-labeled steroid derivative 1.
J. Label Compd. Radiopharm 2015, 58 20–22
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org
N. R. Rivera et al. dichloromethane. The combined filtrate was concentrated and purified by column chromatography, eluting with dichloromethane, to give 1. (33.6 mCi; 99.8 RCP; SA = 29.7 mCi/ mmol; 78% overall yield from 5). LC–MS (EI+) m/z 377 (M + H)+.
Conflict of Interest The authors did not report any conflict of interest.
References
Acknowledgements The authors thank Mr. Steven Staskiewicz, Dr. David Schenk and Dr. David Waterhouse for providing analytical support. We are also grateful to Dennis Rots for providing us with necessary intermediates and to Dr. Cheol Chung for his assistance in preparing this manuscript.
[1] M. Ostendorf. WO Patent No: WO 2013135744, 2013. [2] For example: S. Rhee, J. Malerich, J. Lee, M. Tanga, J. Label. Compd. Radiopharm. 2012, 55, 186–187. [3] J. Reichwein, B. Pagenkopf, J. Am. Chem. Soc. 2003, 125, 1821–1824. [4] (a)B. Arbuzov, Pure Appl. Chem. 1964, 9, 307; (b)W. Bechtold, A. Dahl, J. Label. Compd. Radiopharm. 1985, 22, 1181–1186.
22 www.jlcr.org
Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 20–22
Revised 03 December 2014,
Accepted 04 December 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3257
Synthesis of a [14C]-steroid intermediate: an application of a nonstabilized Horner– Wadsworth–Emmons olefination approach Nelo R. Rivera,* Sumei Ren, and David Hesk Radiolabeled steroid derivative 1 was successfully prepared using a Horner–Wadsworth–Emmons approach: a [14C]-label was efficiently incorporated into the C-18 position of the molecule. Previously published procedures employing other olefination methods are either not applicable due to unavailability of [14C]-precursors or suffer from poor reactivity. Keywords: steroid; carbon-14; Horner–Wadsworth–Emmons (HWE); olefination
Introduction We recently needed to prepare [14C]-radiolabeled steroid derivative 1 to be used as a synthon for [14C]-labeled steroid compounds. The synthesis of unlabeled 1 has been previously disclosed by other groups (Scheme 1).1 Based on their general synthetic strategy, we wanted to pursue a late-stage introduction of the radiolabel at the C-18 position via olefination with an appropriate [14C] reagent.
Results and discussion
20
The direct conversion of ketone 3 to the corresponding unlabeled olefin 4 was accomplished using either Peterson or Wittig conditions (Scheme 1, conditions iv or v, respectively).1 The requisite [14C]-trimethylsilylmethyl lithium or equivalent reagent needed for the Peterson olefination, however, is not easily accessible. On the other hand, the [14C]-Wittig reagent is readily prepared from [14C]-methyl iodide and triphenylphosphine.2 Unfortunately, we were unable to efficiently carry out the direct transformation of ketone 3 to olefin 4 with [14C]-methyltriphenylphosphonium iodide. Less than 5% conversion of starting material to product was observed despite using an excess of the [14C]-Wittig reagent. This led us to consider an alternative Horner–Wadsworth– Emmons (HWE) approach. The addition of HWE reagents such as dimethyl methylphosphonate, without a carbanion stabilizing group at the β-position, to ketones results in β-hydroxyphosphonates which do not readily eliminate to give the desired olefin product. As a consequence, olefination using nonstabilized HWE reagents has been viewed as a difficult transformation. However, a report by Reichwein and Pagenkopf showed that the resulting hydroxyphosphonate undergoes elimination via a saponification–dehydration sequence.3 To our delight, this was indeed the case when we subjected our substrate to the reported conditions using unlabeled dimethyl methylphosphonate. Encouraged by this initial result, we
J. Label Compd. Radiopharm 2015, 58 20–22
prepared the [14C]-HWE reagent via the Michaelis–Arbuzov reaction.4 Addition of the [14C]-HWE reagent to ketone 3 gave the corresponding hydroxyphosphonate 5 in 46% radiochemical yield based on the phosphonate reagent. We also found that quenching the reaction at low temperature was key to the success of this reaction. Formation of the desired product was not observed when the reaction was allowed to warm to room temperature; only starting material was observed by HPLC analysis. Saponification using methanolic aqueous NaOH gave the corresponding monophosphonic acid 6; and dehydration using diisopropylcarbodiimide followed by acetal deprotection under acidic conditions gave the desired [14C]-steroid intermediate 1 in 78% overall yield from 5 (Scheme 2).
Conclusion We have successfully demonstrated the use of a nonstabilized HWE reagent for the introduction of a [14C]-label into an advance steroid intermediate. Other olefination approaches such as Peterson and Wittig reactions that have been used for the preparation of the unlabeled steroid 1 were not applicable in our case due to the unavailability of the [14C]precursor (Peterson approach) or lack of reactivity of the [14C]-reagent (Wittig approach). The slow elimination of the HWE adduct was successfully overcome by applying the twostep sequence involving a partial hydrolysis followed by dehydration.
Department of Process Chemistry, Merck Research Laboratories, 126 E. Lincoln Ave., PO Box 2000, Rahway, NJ 07065, USA *Correspondence to: Nelo R. Rivera, Department of Process Chemistry, Merck Research Laboratories, 126 E. Lincoln Ave., PO Box 2000, Rahway, NJ, 07065, USA. E-mail: [email protected]
Copyright © 2015 John Wiley & Sons, Ltd.
N. R. Rivera et al.
Scheme 1. Synthesis of unlabeled steroid derivative 1. Conditions: (i) p-TsOH, 1,2-ethanedithiol, MeOH, reflux, 72%; (ii) ethylene glycol, triethylorthoformate, p-TsOH, CH2Cl2, 40 °C then 20 °C, 96%; (iii) pyridinium dichromate, DMF, 30 °C, 89%; (iv) Mg, I2, chloromethyl trimethylsilane, dibromoethane, THF, toluene, reflux, 87% (Peterson condition); (v) methyl triphenylphosphonium bromide, KOtBu, toluene, 90 °C, 68% (Wittig condition); (vi) H2SO4, water, acetone, 20 °C, 83%.
Experimental Chemicals and solvents were obtained from standard commercial sources and used without further purification. [14C]-methyl iodide (SA = 56.0 mCi/mmol) was obtained from Amersham, Piscataway, NJ. Ketone 3 as well as an authentic sample of unlabeled 1 were provided to us by Merck Process Chemistry Group. Mass spectrometry determinations were performed on an Agilent 1100 CDMSD instrument in ES positive ionization mode. Agilent Poroshell 120 EC-C18 column (2.7 um, 50 × 3.0 mm), 1.0 mL/min, 40 °C. Gradient from 10% Acetonitrile [0.1% Formic Acid] / 90% Water [0.1% Formic Acid] to 95% Acetonitrile [0.1% Formic Acid] / 5% Water [0.1% Formic Acid] from 0 to 5.2 min. Six minutes run time. High-performance liquid chromatography condition used to monitor reaction progress and radiochemical purity (RCP) is shown below. Retention times of products obtained are consistent with that of authentic unlabeled material. Supelco Ascentis Express C18 column (2.7 μm, 50 × 4.6 mm), 210 nm, 1.8 mL/min, 40 °C. Gradient from 10% Acetonitrile [0.1% Formic Acid] / 90% Water [0.1% Formic Acid] to 95% Acetonitrile [0.1% Formic Acid] / 5% Water [0.1% Formic Acid] from 0 to 6.5 min. Eight minutes run time. Compound 5 To a round bottom flask containing [14C]-dimethyl methylphosphonate (SA = 28.9 mCi/mmol; 92.7 mCi) was added a THF solution of ketone 3 (670 mg; 1.593 mmol) in THF (4 mL), then cooled to < 70 °C. Lithium diisopropylamide (LDA) (1.68 M; 1.52 mL) was added slowly via syringe over a period of 10–15 min. The reaction was aged for 1 hr, and after completion as indicated by HPLC analysis, it was quenched at < 70 °C with
Aq NH4Cl (6.0 mL). Upon warming to 10 °C, the reaction mixture was extracted with EtOAc (10 mL), and the resulting organic layer was washed with Aq NH4Cl (6 mL) followed by Aq NaCl (6 mL). The aqueous layers were re-extracted with EtOAc (10 mL), and the resulting combined organic layers were dried over MgSO4, filtered and purified by column chromatography (80% EtOAc/Hexanes + 0.01% Et3N) to give 5 (42.9 mCi, 46.3% yield; 99.0% RCP). Liquid chromatography–mass spectrometry (LC–MS) (EI+) m/z 547 (M + H)+. Compound 6 To a round bottom flask containing hydroxyphosphonate 5 (42.9 mCi; 1.49 mmol) in MeOH (13 mL) was added 4 M NaOH (1.195 mL; 4.78 mmol) and stirred at room temperature overnight. The MeOH solution was partially concentrated, and the solution was adjusted to pH 2 with 1 M HCl (ca 4 mL). The reaction was then extracted with dichloromethane (10 mL, then 3 × 6.5 mL). The organic layers were dried over MgSO4, filtered and concentrated and used directly in the next step. Crude product 6 had an RCP of 98.5%. LC–MS (EI+) m/z 533 (M + H)+. Compound 1 To a round bottom flask containing β-hydroxy phosphonic acid 6 (42.9 mCi; 1.49 mmol) in chloroform (7.5 mL) was added diisopropylcarbodiimide (0.620 mL, 3.98 mmol) and aged at room temperature for approximately 1 hr after which analysis showed complete conversion to acetal intermediate 4. MeOH (1 mL) was added, and the crude reaction was concentrated down and rediluted with MeOH (10 mL). To the resulting slurry was added 6 M HCl (0.531 mL, 3.19 mmol) and stirred at room temperature overnight. After reaction completion, the slurry was concentrated down and redissolved in 20 mL EtOAc. The thin slurry was passed through a filter, and the filtered solid was washed down with
21
14
Scheme 2. Horner–Wadsworth–Emmons olefination approach to [ C]-labeled steroid derivative 1.
J. Label Compd. Radiopharm 2015, 58 20–22
Copyright © 2015 John Wiley & Sons, Ltd.
www.jlcr.org
N. R. Rivera et al. dichloromethane. The combined filtrate was concentrated and purified by column chromatography, eluting with dichloromethane, to give 1. (33.6 mCi; 99.8 RCP; SA = 29.7 mCi/ mmol; 78% overall yield from 5). LC–MS (EI+) m/z 377 (M + H)+.
Conflict of Interest The authors did not report any conflict of interest.
References
Acknowledgements The authors thank Mr. Steven Staskiewicz, Dr. David Schenk and Dr. David Waterhouse for providing analytical support. We are also grateful to Dennis Rots for providing us with necessary intermediates and to Dr. Cheol Chung for his assistance in preparing this manuscript.
[1] M. Ostendorf. WO Patent No: WO 2013135744, 2013. [2] For example: S. Rhee, J. Malerich, J. Lee, M. Tanga, J. Label. Compd. Radiopharm. 2012, 55, 186–187. [3] J. Reichwein, B. Pagenkopf, J. Am. Chem. Soc. 2003, 125, 1821–1824. [4] (a)B. Arbuzov, Pure Appl. Chem. 1964, 9, 307; (b)W. Bechtold, A. Dahl, J. Label. Compd. Radiopharm. 1985, 22, 1181–1186.
22 www.jlcr.org
Copyright © 2015 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2015, 58 20–22
