Screening approach, optimisation and scale-up for chiral liquid chromatography of cathinones.

Screening approaches adopted in pharmaceutical companies for chiral LC method development may be quite complicated and sophisticated in order to guarantee a high success rate. However in other environments it may be of more value to assess how simple a screen might be used to still have a good chance of achieving success. The genuine need to develop chiral separations for the former 'legal-high' drug mephedrone and related cathinones of topical interest presented a good opportunity to develop this theme. In initial work on mephedrone itself, no chiral separation was observed on Chirobiotic V, Cyclobond I 2000 DNP, Whelk-O1 and AmyCoat using reversed phase mobile phases. However, using normal phase solvents, chiral separation was observed on all the chiral stationary phases (CSP) used except Chiralcel OJ-H. Of the chiral separations observed on RegisPack, RegisCell and Whelk-O1, some optimisation work was carried out on the latter two which had showed the greatest enantioselectivity. Following optimisation, the best enantioselectivity (1.59) and enantioresolution (5.90) was found with a 250 mm × 4.6 mm I.D. Whelk-O1 column using a propan-2-ol (IPA)-hexane-trifluoroacetic acid (TFA)-triethylamine (TEA) (10:90:0.05:0.05, v/v/v/v) mobile phase. Subsequent screening on other cathinones was restricted to RegisPack, RegisCell and Whelk-O1 or equivalent phases with two mobile phases and this gave a very good success rate. Indeed it was possible to separate all six cathinones on one column, RegisCell, with one mobile phase, propan-2-ol-hexane-TFA (15:85:0.1, v/v/v) but obviously it had been necessary to go through the 3-column screen to arrive at this finding. While Whelk-O1 was not so successful, ease of optimisation on this phase was again a feature. To illustrate the applicability of these separations, it was shown that, as a basis for semi-preparative work, the optimsed mephedrone separation on Whelk-O1 could be scaled-up to a 2000µl injection of a 1.0 mg ml(-1) solution in mobile phase (2.0mg on-column) while still using the 250 mm × 4.6 mm I.D. analytical column.

Ethynyl and Propynylpyrene Inhibitors of Cytochrome P450.

The single-crystal X-ray structures and in vivo activities of three aryl acetylenic inhibitors of cytochromes P450 1A1, 1A2, 2A6, and 2B1 have been determined and are reported herein. These are 1-ethynylpyrene, 1-propy-nylpyrene, and 4-propynylpyrene. To investigate electronic influences on the mechanism of enzyme inhibition, the experimental electron density distribution of 1-ethynylpy-rene has been determined using low-temperature X-ray diffraction measurements, and the resulting net atomic charges compared with various theoretical calculations. A total of 82,390 reflections were measured with Mo Kα radiation to a (sinθ/λ)(max) = 0.985 Å(-1). Averaging symmetry equivalent reflections yielded 8,889 unique reflections. A least squares refinement procedure was used in which multipole parameters were added to describe the distortions of the atomic electron distributions from spherical symmetry. A map of the model electron density distribution of 1-ethynylpyrene was obtained. Net atomic charges calculated from refined monopole population parameters yielded charges that showed that the terminal acetylenic carbon atom (C18) is more negative than the internal carbon (C17). Net atomic charges calculated by ab initio, density functional theory, and semi-empirical methods are consistent with this trend suggesting that the terminal acetylenic carbon atom is more likely to be the site of oxidation. This is consistent with the inhibition mechanism pathway that results in the formation of a reactive ketene intermediate. This is also consistent with assay results that determined that 1-ethynylpyrene acts as a mechanism-based inhibitor of P450s 1A1 and 1A2 and as a reversible inhibitor of P450 2B1. Crystallographic data: 1-ethynylpyrene, C(18)H(10), P2(1)/c, a = 14.571(2) Å, b = 3.9094(5) Å, c = 20.242(3) Å, ß = 105.042(2)°, V = 1,113.5(2) Å(3); 1-propynylpyrene, C(19)H(12), P2(1)/n, a = 8.970(2) Å, b = 10.136(1) Å, c = 14.080(3) Å, ß = 99.77(2)°, V = 1,261.5(4) Å(3); 4-propynylpyrene, C(19)H(12), Pbca, a = 9.904(1) Å, b = 13.174(2) Å, c = 19.401(1) Å, V = 2,531.4(5) Å(3).