Steroid 2,4-Dinitrophenylhydrazones: their preparation, separation, and characterization
Koehler, Walter Robert
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2,4-Dinitrophenylhydrazone (DNPH) derivatives of twelve ketonic steroids were prepared and characterized. The steroids were chosen to include adrenal, placental and testicular hormanes and metabolites of these hormones. It was found with the exception of adrenosterone-bis-DNPH that the DNPH's could be classified into four well defined groups on the basis of their ultraviolet spectra and that this classification could be correlated with the structures of the DNPH's. Within each group the compounds were similar to each other with respect to their melting points, color, solubilities and infrared spectra as well as their ultraviolet absorption spectra. Group 1a. - DNPH's of steroids having a single carbonyl group at the 17 or 20 position, having no double bond conjugated with the carbonyl group and having no adjacent hydroxyl groups. The steroids studied were pregnenolone, androsterone and dehydroisoandrosterone. These compounds were yellow, λmax. = 368-370 mµ, εmax = 24,250 ± 550) and had melting points of 249°- 255° ± 9°C. Group 1b. - DNPH's of monoketonic steroids having the Δ4-3-keto grouping as in testosterone and methyl testosterone. These compounds were red (λmax. = 392-394, εmax. = 31,000) and had melting points in the range 216-238°C. Group 2a. - DNPH's of diketonic steroids, one of whose carbonyl was of the type 1a. and the other of type 1b. The steroids studied were Δ4-androstenedione, progesterone and 11α-hydroxyprogesterone. These compounds were red-orange to red (λmax = 380-382, εmax. = 49,300 ± 500) and decomposed before melting. Group 2b. - DNPH's of diketonic steroids, as in group 2a., but with a hydroxyl group adjacent to the C-20 carbonyl. These compounds were red-orange (λmax. = 386, εmax. = 47,500 ± 2,600) and decomposed before melting. Evidence from the ultraviolet and infrared absorption spectra is presented suggesting that in this group the product isolated is one of a pair of geometrical isomers, namely the one in which the DNPH group at 0-20 is syn with respect to the adjacent hydroxyl group and that the bathochromic shift in the ultraviolet spectra is due to stabilization of an excited resonance state by hydrogen bonding. Adrenosterone-bis-DNPH did not fall into any of the above groups and was considered separately. It was red-orange (λmax. = 376, εmax. = 43,600) and decomposed without melting. Infrared absorption spectra of all compounds are presented. These spectra indicate whether the compound is a mono- or bis- dinitrophenylhydrazone, whether the steroid had any hydroxyl groups and whether the original steroid had any carbonyl groups in unreactive positions, such as the C-11 position. In the case of the mono-dinitrophenylhydrazones the infrared spectra were detailed enough to identify the specific steroid empirically. Results are presented for the separation of mixtures of steroid DNPH's by various chromatographic systems. The system finally adopted consisted of alumina, previously treated with a solution of 1 per cent ethanol in benzene, as the stationary phase and benzene, methylene chloride as eluents. This system was used to separate a metabolite of testosterone as the dinitrophenylhydrazone, after incubation with human serum, from the un-metabolized substrate. By means of melting point determination, ultraviolet absorption spectra and infrared absorption spectra this metabolite was identified as Δ4-androstenedione, which is in agreement with similar results previously published.
Thesis (M.A.)--Boston University