Metabolism of 17α-ethynyl-19-nortestosterone and 17α-ethynyl estrenol in Vivo

Abstract

Studies were made on the metabolism of two popular progestins, 17α-ethynyl-19-nortestosterone (ENT) and 17α-ethynyl-estrenol (EEL).
ENT in a dose of 100 mg per day, ³H-ENT or ¹⁴C-ENT was administered to post menopausal women who were operated on for uterine cancer. After the administration, urine samples were pooled every 24 hours and stored. The total radioactivity of each 24 hours' urine smple was estimated by counting in a Packard Tricarb liquid scintillation spectrometer (Fig. 1).
In every three cases, about 30% of radioactivity was found to be excreted in the urine during 5 days. The urines were hydrolysed and extracted as indicated in Table 2. Using Girard-T reagent, the crude extract was further separated into ketonic and non-ketonic fractions. The extract of each fraction was absorbed on alumina and eluted successively with 0.1, 0.3, 0.5, 5.0 and 30% methanol in benzene. From this column, four radioactive peaks were eluted (Fig. 2 and 3).
The first radioactive peak was eluted in a similar fraction as 17α-ethynyl-5α-19-norandrostane-17β-ol-3-one. However, these two compounds were not identical in Rf values on paper chromatography, silicagel thin layer chromatography (T.L.C.), and from retention time (Rt) in gasliquid chromatography (GL.C.). It was highly probable from this chromatographic behavior that the radioactive metabolite was 17α-ethynyl-5β-19-norandrostane-17β-ol-3-one.
The second radioactive peak corresponded in the column chromatographic behavior to ENT which was eluted with 0.1 % methanol in benzene. This was applied to a paper and run in benzene-ligroin-methanol-water system. It became a single radioactive peak corresponding in it mobility to authentic ENT. After several repeated paper chromatographies, UV absorption and sulfuric acid chromogen spectra of this radioactive material were measured. Identical spectra of this material with those of authentic ENT were obserbed (Fig. 10). To an aliquot was added carrier ENT and crystallized several times from aqueous ethanol. The specific activity remained constant throughout the above mentioned crystallization (Table 3). These results suggested that this radioactive material was ENT.
The third radioactive peak was eluted in 0.3% methanol in benzene and was compared to 17α-ethynyl-5α-19-norandrostane-3β, 17β-diol which was obtained from an authentic sample of 17α-ethynyl-5α-19-norandrostane-17β-ol-3-one by its reduction with sodimu borohydride. Paper chromatography, silicagel T.L.C. and G.L.C. were carried out with these two compounds. UV absorption and sulfulic acid chromogen spectra were also measured. All these results indicated the identity of these two compounds (Fig. 12, 13 and 14). Since, in addition to the above results, the oxidation products of the radioactive metabolite were identical with authentic 17α-ethynyl-5α-19-norandrostane-17β-ol-3-one in their chromatographic and spectrometric properties; this radioactive material was identified as 17α-ethynyl-5α-19-norandrostane-3β, 17β-diol.
The fouth radioactive material was eluted in 5% methanol in benzene from the column and was separated into 4 to 5 radioactive compounds on the paper chromatography in benzene-methanol-water system (Fig. 20).
A similar experiment with the urine of patients who were administered EEL, revealed that this compound converted in vivo into metabolites identical with those of ENT. This indicated that EEL first metabolized to ENT and further to those metabolites.
Overall metabolism of EEL and ENT in vivo was indicated as in Fig. 19.
Relationship between previously reported androgenic activity and the metabolism of EEL and ENT were also discussed.