Effects of channel modulators on cloned large-conductance calcium-activated potassium channels.

Through expression of the cloned mouse (mSlo) or human (hSlo) large-conductance (BK) Ca(2+)-activated K+ channel in Xenopus laevis oocytes and HEK 293 cells, we characterized the effects of reported blockers and openers of BK channels to initiate the study of the molecular determinants of BK channel modulation. In oocytes, iberiotoxin and charybdotoxin, peptidyl scorpion toxins, were both equally effective blockers of BK current, although iberiotoxin was significantly more potent than charybdotoxin. The structurally related peptide kaliotoxin was not a potent blocker of BK current. Paxilline, a fungal tremorgenic alkaloid, was an effective but complex blocker of BK current. Tetrandrine, a putative blocker of type II BK channels, and ketamine were relatively ineffective. The putative BK openers NS004 and NS1619, phloretin, niflumic acid, flufenamic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) increased BK current in oocytes at microM concentrations; many of these produced biphasic concentration-response relationships. Coapplication of representative blockers and openers revealed several patterns of interaction, including competitive and noncompetitive antagonism. NS1619, niflumic acid, and phloretin were tested by using excised inside-out membrane patches from HEK 293 cells and were found to increase the activity of hSlo BK channels and produce a leftward shift in the G/Gmax-versus-voltage relationship of these channels. These results represent the first comprehensive examination of the molecular pharmacology of BK channels.

Nonprostanoid prostacyclin mimetics. 5. Structure-activity relationships associated with [3-[4-(4,5-diphenyl-2-oxazolyl)-5- oxazolyl]phenoxy]acetic acid.

cis-[3-[2-(4,5-Diphenyl-2-oxazolyl)ethenyl]phenoxy]acetic acid (3) was previously identified as a nonprostanoid prostacyclin (PGI2) mimetic that potently inhibits ADP-induced aggregation of human platelets with an IC50 of 0.18 microM. As part of an effort to further explore structure-activity relationships for this class of platelet inhibitor and to provide additional insight into the nonprostanoid PGI2 mimetic pharmacophore, the effect of constraining the cis-olefin moiety of 3 into various ring systems was examined. Incorporation of the cis-olefin of 3 into either an oxazole (26) or an unsubstituted pyrazole (35) heterocycle provided compounds that are equipotent with progenitor 3. However, the oxazole 11f, which is isomeric with 26, inhibits ADP-induced human platelet aggregation in vitro with an IC50 of 0.027 microM, 6-fold more potent than 3, 26, or 35. These results suggest that the central oxazole ring of 11f is functioning as more than a simple scaffold that provides optimal stereodefinition for interaction with the PGI2 receptor. The nitrogen atom of the central heterocycle of 11f is postulated to engage in hydrogen-bond formation with a donor moiety in the PGI2 receptor protein, an interaction not available to 26 due to the markedly different topology. In support of this contention, the crystal structures of 11f and 26 contain strong intermolecular hydrogen bonds between the carboxylic acid hydrogen atom and the nitrogen atom of the central oxazole ring. Although 11f and 26 are exact isosteres and could, in principle, adopt the same molecular packing arrangement in the solid state, this is not the case, and the intermolecular hydrogen-bonding interactions in 11f and 26 are accommodated by entirely different molecular packing arrangements. Incorporation of the olefin moiety of 3 into a benzene ring provided a compound, 40, over 60-fold weaker with an IC50 of 11.1 microM. The affinities of 11f, 26, 31, 32, and 40 for the human platelet PGI2 receptor, determined by displacement of [3H]iloprost, correlated with inhibition of platelet function. The solid-state structures of 11f, 26, 31, 32, and 40 were determined and revealed that the more potent compounds 11f and 26 adopt a relatively planar overall topography. In contrast, the central phenyl ring and the phenoxy ring of the weakly active compound 40 are rotated by 53 degrees from planarity. The chemical shifts of the protons of the phenoxy rings of 3, 11f, 18, 26, 31, 32, and 40 suggest that in solution 3, 11f, 18, and 26 adopt a planar conformation while 40 does not.(ABSTRACT TRUNCATED AT 400 WORDS)

Nonprostanoid prostacyclin mimetics. 3. Structural variations of the diphenyl heterocycle moiety.

4,5-Diphenyl-2-oxazolenonanoic acid (2) and 2-[3-[2-(4,5-diphenyl-2-oxazolyl)ethyl]phenoxy]acetic acid (3) were previously identified as nonprostanoid prostacyclin (PGI2) mimetics that inhibit ADP-induced aggregation of human platelets in vitro. The effects on biological activity of substitution and structural modification of the 4- and 5-phenyl rings of 3 was examined. Potency showed a marked sensitivity to the introduction of substituents to these aromatic rings and only the bis-4-methyl derivative 9j, IC50 = 0.34 microM, demonstrated enhanced potency compared to the parent structure 3, IC50 = 1.2 microM. Substitution at the ortho or meta positions of the phenyl rings, replacement by thiopheneyl or cyclohexyl moieties, or constraining in a planar phenanthrene system resulted in compounds that were less effective inhibitors of ADP-induced platelet aggregation. In contrast, variation of the heterocycle moiety revealed a much less stringent SAR and many 5- and 6-membered heterocycles were found to effectively substitute for the oxazole ring of 2 and 3. The diphenylmethyl moiety functioned as an effective isostere for 4,5-diphenylated heterocycles since 13aad showed similar platelet inhibitory activity to 3. With the exception of the 3,4,5-triphenylpyrazole derivative 13g, compounds presenting the (m-ethylphenoxy)acetic acid side chain discovered with 3 demonstrated enhanced potency compared to the analogously substituted alkanoic acid derivative. The structure-activity findings led to a refinement of a model of the nonprostanoid PGI2 mimetic pharmacophore.