Dualistic actions of cromakalim and new potent 2H‐1,4‐benzoxazine derivatives on the native skeletal muscle K_ATP channel

Abstract

1 New 2H-1,4-benzoxazine derivatives were synthesized and tested for their agonist properties on the ATP-sensitive K + channels (K ATP ) of native rat skeletal muscle fibres by using the patch-clamp technique. The novel modifications involved the introduction at position 2 of the benzoxazine ring of alkyl substituents such as methyl ( -CH 3 ), ethyl ( -C 2 H 5 ) or propyl ( -C 3 H 7 ) groups, while maintaining pharmacophore groups critical for conferring agonist properties. 2 The effects of these molecules were compared with those of cromakalim in the presence or absence of internal ATP (10 À4 M). In the presence of internal ATP, all the compounds increased the macropatch K ATP currents. The order of potency of the molecules as agonists wasBell-shaped dose -response curves were observed for these compounds and cromakalim indicating a downturn in response when a certain dose was exceeded. Abbreviations: -CH 3 , methyl; -C 2 H 5 , ethyl; -C 3 H 7 , propyl; DMSO, dimethylsulphoxide; FDB, flexor digitorum brevis; K ATP , ATP-sensitive K + channel; KCOs, potassium channel openers; Kir6.2, inward rectifier K + channel 6.2 subunit; log D, logarithm of the distribution coefficient at a particular pH; log P, logarithm of the partition coefficient; pK a , negative logarithm of the dissociation constant; SUR2A, sulphonylureas receptor 2A subunit

Molecular Determinants for the Activating/Blocking Actions of the 2H-1,4-Benzoxazine Derivatives, a Class of Potassium Channel Modulators Targeting the Skeletal Muscle KATP Channels

“…DMSO did not affect K ATP current even in the absence of internal nucleotide. The drugs were tested in the presence or in the absence of internal ATP (10 Ϫ4 M) with no added Mg 2ϩ ions to reduce possible ATPase activity in the patches (Russ et al, 2003;Tricarico et al, 2003). Synthesis of the New 2H-1,4-Benzoxazine Derivatives.…”

“…The key intermediate in the synthetic pathway of all benzoxazine derivatives 2-9 ( Fig. 1) was the appropriate 2-substituted-6-chloro-2H-1,4-benzoxazine-3-one, which was condensed with 3-aminopyridine either in refluxing dry toluene in the presence of TiCl 4 and anisole as previously reported for derivative 1 (compounds 3 and 9) (Tricarico et al, 2003), or in dry acetonitrile in the presence of triethyl amine and POCl 3 (compounds 2 and 4-8). In addition, the preparation of benzoxazinones followed two different synthetic pathways.…”

“…The concentration -response relationships of the K ATP currents constructed in the presence of internal ATP fits the product of two equations describing the interaction of a ligand with two sites mediating opposite effects, the stimulatory effect or the inhibitory effect (Rovati and Nicosia, 1994;Tricarico et al, 2003). However, the concentration-response relationships of the K ATP currents versus drug concentrations constructed in the absence of ATP are well fitted by one inhibitory term.…”

“…A downturn in response is observed with this compound when a certain dose is exceeded, suggesting that its effectiveness is reduced at higher concentrations. Furthermore, the same compound in the absence of internal ATP, caused a 41% inhibition of the K ATP channels at 10 Ϫ4 M concentration, which seems to be mediated by interaction with the Kir6.2 subunit (Tricarico et al, 2003;Rolland et al, 2006). Dual hypotheses have been proposed to explain this peculiar behavior: first, the drug action is dependent on the ability to bind two distinct sites of skeletal muscle K ATP channel complex modulating opposite actions; second, the drug binds to a single site whose affinity and/or effect are modulated by ATP or generally by tissue metabolism (Tricarico et al, 2003).…”

“…1), a novel modulator of the muscular K ATP channels, in the presence of internal ATP leads to 54% activation of the fast-twitching muscle K ATP channels at 10 Ϫ7 M concentration (Tricarico et al, 2003). A downturn in response is observed with this compound when a certain dose is exceeded, suggesting that its effectiveness is reduced at higher concentrations.…”