Dualistic actions of cromakalim and new potent 2H-1,4-benzoxazine derivatives on the native skeletal muscle K(ATP) channel

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 (-CH3), ethyl (-C2H5) or propyl (-C3H7) 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(-4) 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 was -C3H7 (DE50 = 1.63 x 10(-8) M) > -C2H5 (DE50 = 1.11 x 10(-7) M) > -CH3 (DE50 = 2.81 x 10(-7) M) > cromak-slim (DE50 = 1.42 x 10(-5) M). Bell-shaped dose-response curves were observed for these compounds and cromakalim indicating a downturn in response when a certain dose was exceeded. 3 In contrast, in the absence of internal ATP, all molecules including cromakalim inhibited the K-ATP currents. The order of increasing potency as antagonists was cromakalim (IC50 = 1.15 x 10(-8) M) greater than or equal to -CH3 (IC50 = 2.6 x 10(-8) M) > -C2H5 (IC50 = 4.4 x 10(-8) M) > -C3H7 (IC50 = 1.68 x 10(-7) M) derivatives. 4 These results suggest that the newly synthesized molecules and cromakalim act on muscle K-ATP channel by binding on two receptor sites that have opposite actions. Alternatively, a more simple explanation is to consider the existence of a single site for potassium channel openers regulated by ATP which favours the transduction of the channel opening. The alkyl chains at position 2 of the 2H-1,4-benzoxazine nucleus is pivotal in determining the potency of benzoxazine derivatives as agonists or antagonists.