The synthesis of an internal standard for bicalutamide

[摘要] English: (R,S)-Bicalutamide [N-(4-cyano-3-trifluoromethylphenyl)-α-methyl-α-hydroxy-β-(4-fluorophenylsulfonyl)propanamide], sold as Casodex®, is the leading antiandrogencurrently used to treat prostate cancer. It binds to androgen receptors and blockscancer growth.This work aims to develop internal standards for the bio-analytical component ofclinical trials that are required to detect bicalutamide and derivatives. An internalstandard is added to the body fluid sample (mostly blood) at the beginning of thesample work up at about the same concentration of the analyte to be quantified. Anideal internal standard has a similar extraction recovery and a similar retention time inHPLC. For quantification with mass spectrometry it should have a difference of atleast 3 mass units from the analyte and a similar ionization response. The internalstandard is used to calibrate the total ion current of the metabolite. We did not have access to deuterium labeled starting materials and investigatedstructural analogues as an alternative strategy to obtain internal standards. De novosynthesis of structural analogues failed because we could not deprotonate the methylsulfone in the presence of aromatic amides. We ascribed this to incomplete disclosurein the patented methods.Treatment of bicalutamide with palladium on activated charcoal under the rightconditions did not give the usually produced amide but gave smooth reduction of theC≡N group to a CH3 group. This unusual reduction gave ready access to a goodinternal standard in good yield.Elimination of the tertiary aliphatic hydroxy group of bicalutamide would give analkene with similar polarity that could serve as an internal standard. Acid catalysisusing 1M HCl or p-toluene sulfonic acid failed, but treatment of bicalutamide withH2SO4 in benzene gave hydrolysis of the nitrile to an amide. This provides a secondinternal standard in good yield. Bicalutamide did not react with weak base. Strong base such as LDA led to fission ofthe 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 ofnitrobenzoic acid at 40 ºC to form an alkene. This represents a third potential internalstandard. The NOESY experiment proves that the alkene has a Z-configuration. Thisindicates that the pro-R aliphatic hydrogen of bicalutamide was eliminatedstereoselectively via a syn-periplanar cyclic transition state. Efforts to eliminate the hydroxy group of bicalutamide photolytically at 300 nm inethanol yielded an unexpected replacement of the aromatic CF3 group with an ethoxygroup. We could not find a similar transformation in the literature and believe it to bea novel reaction. We used 19F NMR to prove the absence or presence of fluorine and CF3 moieties inthe products. We also used the magnitude of 13C-19F coupling constants in protondecoupled 13C spectra for accurate resonance assignment and structure elucidation.We will test these novel analogues of bicalutamide in cancer bioassays and use themas internal standards for the quantification of bicalutamide and analogues in bodyfluids.

[发布机构] University of the Free State