The synthesis of an internal standard for bicalutamide
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Date
2008
Authors
Jordaan, Maryam Amra
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Publisher
University of the Free State
Abstract
(R,S)-Bicalutamide [N-(4-cyano-3-trifluoromethylphenyl)-α-methyl-α-hydroxy-β-(4- fluorophenylsulfonyl)propanamide], sold as Casodex®, is the leading antiandrogen currently used to treat prostate cancer. It binds to androgen receptors and blocks cancer growth. This work aims to develop internal standards for the bio-analytical component of clinical trials that are required to detect bicalutamide and derivatives. An internal standard is added to the body fluid sample (mostly blood) at the beginning of the sample work up at about the same concentration of the analyte to be quantified. An ideal internal standard has a similar extraction recovery and a similar retention time in HPLC. For quantification with mass spectrometry it should have a difference of at least 3 mass units from the analyte and a similar ionization response. The internal standard is used to calibrate the total ion current of the metabolite. We did not have access to deuterium labeled starting materials and investigated structural analogues as an alternative strategy to obtain internal standards. De novo synthesis of structural analogues failed because we could not deprotonate the methyl sulfone in the presence of aromatic amides. We ascribed this to incomplete disclosure in the patented methods. Treatment of bicalutamide with palladium on activated charcoal under the right conditions did not give the usually produced amide but gave smooth reduction of the C≡N group to a CH3 group. This unusual reduction gave ready access to a good internal standard in good yield. Elimination of the tertiary aliphatic hydroxy group of bicalutamide would give an alkene with similar polarity that could serve as an internal standard. Acid catalysis using 1M HCl or p-toluene sulfonic acid failed, but treatment of bicalutamide with H2SO4 in benzene gave hydrolysis of the nitrile to an amide. This provides a second internal standard in good yield. Bicalutamide did not react with weak base. Strong base such as LDA led to fission of the aliphatic moiety and isolation of aromatic sulfone and amide fragments. Derivitization of the tertiary aliphatic hydroxy group of bicalutamide with 3- nitrobenzoyl chloride gave a benzoyl ester that allows facile thermal elimination of nitrobenzoic acid at 40 ºC to form an alkene. This represents a third potential internal standard. The NOESY experiment proves that the alkene has a Z-configuration. This indicates that the pro-R aliphatic hydrogen of bicalutamide was eliminated stereoselectively via a syn-periplanar cyclic transition state. Efforts to eliminate the hydroxy group of bicalutamide photolytically at 300 nm in ethanol yielded an unexpected replacement of the aromatic CF3 group with an ethoxy group. We could not find a similar transformation in the literature and believe it to be a novel reaction. We used 19F NMR to prove the absence or presence of fluorine and CF3 moieties in the products. We also used the magnitude of 13C-19F coupling constants in proton decoupled 13C spectra for accurate resonance assignment and structure elucidation. We will test these novel analogues of bicalutamide in cancer bioassays and use them as internal standards for the quantification of bicalutamide and analogues in body fluids.
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Dissertation (M.Sc. (Chemistry))--University of the Free State, 2008, Antiandrogens, Drugs -- Analysis, Medicine -- Research