No abstract

T-type Ca2ϩ channels are believed to play an important role in pain perception, and anesthetic steroids such as alphaxalone and allopregnanolone, which have a 5␣-configuration at the steroid A, B ring fusion, are known to inhibit T-type Ca 2ϩ channels and cause analgesia in a thermal nociceptive model (Soc Neurosci Abstr 29:657.9, 2003). To define further the structure-activity relationships for steroid analgesia, we synthesized and examined a series of 5␤-reduced steroids for their ability to induce thermal antinociception in rats when injected locally into the peripheral receptive fields of the nociceptors and studied their effects on T-type Ca 2ϩ channel function in vitro. We found that most of the steroids completely blocked T-type Ca 2ϩ currents in vitro with IC 50 values at a holding potential of Ϫ90 mV ranging from 2.8 to 40 M. T current blockade exhibited mild voltage-dependence, suggesting that 5␤-reduced neuroactive steroids stabilize inactive states of the channel. For the most potent steroids, we found that other voltage-gated currents were not significantly affected at concentrations that produce nearly maximal blockade of T currents. All tested compounds induced dose-dependent analgesia in thermal nociceptive testing; the most potent effect (ED 50 , 30 ng/100 l) obtained with a compound [(3␤,5␤,17␤)-3-hydroxyandrostane-17-carbonitrile] that was also the most effective blocker of T currents. Compared with previously studied 5␣-reduced steroids, these 5␤-reduced steroids are more efficacious blockers of neuronal T-type Ca 2ϩ channels and are potentially useful as new experimental reagents for understanding the role of neuronal T-type Ca 2ϩ channels in peripheral pain pathways.

The unnatural enantiomers of dehydroepiandrosterone sulfate (1), pregnenolone sulfate (2), and (3alpha,5beta)-3-hydroxypregnan-20-one sulfate (3), compounds 4-6, respectively, were prepared by total steroid synthesis. The enantioselectivity of the compounds as negative modulators of the GABAA receptors present in cultured rat hippocampal neurons was examined using electrophysiological methods. Enantioselectivity was found for the inhibitory actions of the dehydroepiandrosterone enantiomers. The IC50s for compounds 1 and 4 were 11 +/- 1 and 80 +/- 14 microM, respectively. Little, if any, enantioselectivity was found for the other two pairs of steroid sulfate inhibitors. The IC50s for compounds 2 and 5 were 82 +/- 12 and 76 +/- 27 microM, respectively. The IC50s for compounds 3 and 6 were 39 +/- 7 and 46 +/- 2 microM, respectively. The results suggest that the sites of action for the androstane and pregnane series of steroid sulfate blockers of GABA-mediated current are different. The observed enantioselectivity for the actions of dehydroepiandrosterone sulfate indicates that its inhibitory actions are mediated via a chiral recognition site and provides new evidence in support of the earlier hypothesis that there is a binding site for this compound on GABAA receptors. Conversely, the failure to observe enantioselectivity for the actions of pregnenolone sulfate and steroid sulfate 3 indicates that a chiral recognition site for these steroids does not exist on GABAA receptors and suggests that the effects of these compounds on this receptor's function may arise indirectly as a consequence of steroid-induced membrane perturbation.

A series of 7-azabicyclo[2.2.l]hept-5-ene complexes are prepared from [0s(NH3)5(q2-L)l2+ (L = pyrrole, 1-methylpyrrole, 2,5-dimethylpyrrole, 1,2,5-trimethylpyrrole, or 1-(trimethylsily1)pyrrole) and various dipolarophiles (e.g., acrylonitrile, methyl acrylate, a-methylene-y-butyrolactone, dimethyl maleate, dimethyl fumarate, N-phenyl maleimide, cyclopentene-1,2-dicarboxylic acid anhydride, and (E>-and (a-methyl 3-(3'-pyridyl)acrylate). The cycloaddition is promoted by coordination of the pyrrole with [Os(NH&]*+ across C3 and C4, transforming the uncoordinated portion of the pyrrole nucleus into an azomethine ylide capable of undergoing 1,3-dipolar cycloadditions.The metal serves not only to activate the pyrrole ring but also to stabilize the resulting 7-azabicyclo[2.2.l]heptene ligands. A number of organic 7-azabicyclo[2.2. llheptanes, including analogs of the alkaloid epibatidine, have been synthesized by this methodology. For the cases examined, the cycloaddition favors ex0 stereochemistry of the electronwithdrawing substituent when the pyrrole nitrogen is unsubstituted. Crystal structures have been determined for the complexes obtained from the reactions of pyrrole with N-phenylmaleimide @a), 2,5dimethylpyrrole with dimethyl maleate (13a), and 2,5-dimethylpyrrole with a-methylene-y-butyrolactone (22a).

A number of steroids seem to have anesthetic effects resulting primarily from their ability to potentiate currents gated by gamma-aminobutyric acidA (GABAA) receptor activation. One such compound is (3alpha,5alpha, 17beta)-3-hydroxyandrostane-17-carbonitrile [(+)-ACN]. We were interested in whether carbonitrile substitution at other ring positions might result in other pharmacological consequences. Here we examine effects of (3beta,5alpha, 17beta)-17-hydroxyestrane-3-carbonitrile [(+)-ECN] on GABAA receptors and Ca2+ channels. In contrast to (+)-ACN, (+)-ECN does not potentiate GABAA-receptor activated currents, nor does it directly gate GABAA-receptor mediated currents. However, both steroids produce an enantioselective reduction of T-type current. (+)-ECN blocked T current with an IC50 value of 0.3 microM with a maximal block of 41%. (+)-ACN produced a partial block of T current (44% maximal block) with an IC50 value of 0.4 microM. Block of T current showed mild use- and voltage-dependence. The (-)-ECN enantiomer was about 33 times less potent than (+)-ECN, with an IC50 value of 10 microM and an amount of maximal block comparable to (+)-ECN. (+)-ECN was less effective at blocking high-voltage-activated Ca2+ current in DRG neurons (IC50 value of 9. 3 microM with maximal block of about 27%) and hippocampal neurons. (+)-ECN (10 microM) had minimal effects on voltage-gated sodium and potassium currents in rat chromaffin cells. The results identify a steroid with no effects on GABAA receptors that produces a partial inhibition of T-type Ca2+ current with reasonably high affinity and selectivity. Further study of steroid actions on T currents may lead to even more selective and potent agents.