Use of the hyphenated LC-MS/MS technique and NMR/IR spectroscopy for the identification of exemestane stress degradation products during the drug development.

Exemestane (6-Methyleneandrosta-1,4-diene-3,17-dione) active pharmaceutical ingredient (EE-3) was subjected to thermal, photolytic, oxidative, acidic and base stress conditions prescribed by the ICH (International Conference on Harmonization) guideline Q1A(R2). EE-3 was found to degrade in base, acidic and oxidative conditions. Eleven new degradation products of EE-3 were characterized by the LC-MS/MS technique. One of these impurities was isolated and identified by the LC-MS/MS, NMR and IR techniques. The LC-MS/MS studies were carried out to establish fragmentation pathways of EE-3 and its new impurity. Based on the results obtained from different spectroscopic studies, this impurity was characterized as 3-hydroxy-1,6-dimethyl-oestratetraen-(1, 3, 5(10), 6)-17-one (EE-3Z). The degradation pathway of EE-3 leading to the generation of eleven products was proposed and this has not been reported so far. The separation of EE-3 from its impurities (process-related and degradants) was achieved using a Gemini C18 column (150mm×4.6mm×3µm) with gradient elution. The degradation products were well resolved from the main peak and its impurities, thus proving the method's stability and indicating power of the method. The method was validated according to the ICH guidelines for parameters such as specificity, limit of detection, limit of quantitation, precision, linearity, accuracy, robustness and system suitability.

Synthesis and photo-conversion of androsta- and pregna-5,7-dienes to vitamin D3-like derivatives.

Calcitriol (3beta,5Z,7E)-9,10-secocholesta-5,7,10(19)-trien-1alpha,3beta,25-triol) is a powerful oncostatic form of vitamin D3 that is of limited clinical utility due to hypercalcemic (toxic) effects. Since the removal of the side chain reduces or eliminates the calcemic activity of vitamin D3, secosteroidal compounds lacking or with a shortened side chain are good candidates for anti-cancer drugs. In addition, 5,7-steroidal dienes without a side chain can be generated in vivo under pathological conditions. A series of androsta- and pregna-5,7-dienes was efficiently synthesized from their respective 3-acetylated 5-en precursors by bromination-dehydrobromination and deacetylation reactions. Ultraviolet B (UVB) irradiation was used to generate corresponding 9,10-secosteroids with vitamin D-like structures. Additional products with tachysterol-like (T-like) structures or 5,7-dienes with inverted configuration at C-9 and C-10 (lumisterol, L-like) were also detected. Different doses of UVB resulted in formation of various products. At low doses, previtamin D-, T- or L-like compounds were formed as the main products, while higher doses induced further isomerization, with formation of potentially oxidized derivatives. In summary, we describe dynamic UVB induced conversion of androsta- and pregna-5,7-dienes into vitamin D-like compounds and their rearranged analogues; additionally novel T-like and L-like structures were also produced and characterized. Further biological evaluation of newly synthesized compounds should help to select the best candidate(s) for potential treatment of hyperproliferative diseases including cancer.

Isolation and structure elucidation of the major photodegradation products of loteprednol etabonate.

Photodegradation of loteprednol etabonate (5), a steroid anti-inflammatory drug, in the solid state, in aqueous suspension, and in aqueous acetonitrile solution has been investigated. Analysis by HPLC showed that the profile of photodegradation products in the solid state was qualitatively similar to that in the aqueous suspension, although the profile in the aqueous acetonitrile solution was considerably different. The major photodegradation products were isolated from the aqueous suspension and the aqueous acetonitrile solution by using preparative reversed-phase HPLC and their structures were elucidated on the basis of spectroscopic data. Photolysis in the solid state and in aqueous suspension yielded three rearrangement products, chloromethyl 17alpha-ethoxycarbonyloxy-11beta-hydroxy-5alpha-methyl-2-oxo-19-norandrosta-1(10),3-diene-17beta-carboxylate (8), chloromethyl 17alpha-ethoxycarbonyloxy-11beta-hydroxy-1-methyl-3-oxo-6(5-->10alpha)-abeo-19-norandrosta-1,4-diene-17beta-carboxylate (9), and chloromethyl 1beta,11beta-epoxy-17alpha-ethoxycarbonyloxy-2-oxo-10alpha-androsta-4-ene-17beta-carboxylate (10). In aqueous acetonitrile solution, 10 was the major product, however, 8 and 9 were not obtained. Pathways for the formation of these compounds from loteprednol etabonate (5) are proposed.

Noncommunicating photoreaction paths in some pregna-1,4-diene-3,20-diones.

The photochemistry of three pregna-1,4-diene-3,20-diones bearing a hydroxy or alkoxy group at C(17) (4-6) has been examined. Irradiation at 254 or 366 nm, where absorption by the cross-conjugated ketone moiety in ring A is predominant or exclusive, causes the 'lumiketone' rearrangement of this chromophore in low to medium quantum yield (Phi(r) 0.05 to 0.31). On the contrary, irradiation at 310 nm, where the isolated ketone at C(20) absorbs a large portion of light causes Norrish-I fragmentation of that chromophore with a higher Phi(r) (0.11-0.83). This leads to end-products arising from the conversion of the C(17) alkyl radical, in a way depending on the structure and the medium (reduction by hydrogen donating solvent, addition of oxygen when present). No intramolecular T-T energy transfer between the separated chromophores occurs. The 'lumiketone' rearrangement occurs independently from the irradiation wavelength (Phi(r) 0.06-0.18) with the strictly related androsta-1,4-dien-3-one 8 lacking the C(20) ketone function.

Light-evoked recovery from wortmannin-induced inhibition of catecholamine secretion and synaptic transmission.

Wortmannin (WT) is known to inhibit catecholamine (CA) secretion in chromaffin cells. This effect was found to be sensitive to UV light in experiments designed to perform simultaneous monitoring of changes in [Ca2+]i and CA secretion in perfused rat adrenal medullas. When the change in [Ca2+]i was measured using calcium green-1 (490 nm excitation), a 35-min treatment with 10 microM WT caused a 69% inhibition of CA secretion evoked by excess (30 mM) extracellular K+ and a moderate inhibition of the [Ca2+]i response. In contrast, the same treatment of fura-2-loaded cells with WT caused only an 11% inhibition of the high-K+-evoked secretion and no significant attenuation of the [Ca2+]i response. However, during interruption of fluorometry with fura-2, the inhibitory effect of WT developed at a rate similar to that exhibited in calcium green-1-loaded cells. The WT-induced inhibition of high-K+- or bradykinin-evoked secretory responses, which was otherwise irreversible, was reversed by exposing WT-treated chromaffin cells to 380-nm light. When WT was reapplied to the cells of which the secretory ability had been restored by light irradiation, the secretory response was inhibited with a time course similar to that shown during the initial treatment with WT. The photosensitive effect of WT was also demonstrated using bullfrog sympathetic ganglia in which WT-induced inhibition of synaptic transmission was reversed by irradiation with 380-nm light. These results suggest that UV light removes the inhibitory effects of WT by disrupting the covalent bond formed between WT and a target molecule which remains to be determined, although myosin light chain kinase has been reported as the target molecule in both cases examined in this study.