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(-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 -C(3)H(7) (DE(50)=1.63 x 10(-8) M) >-C(2)H(5) (DE(50)=1.11 x 10(-7) M)>-CH(3) (DE(50)=2.81 x 10(-7) M)>cromak-slim (DE(50)= 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 (IC(50)=1.15 x 10(-8) M)> or =-CH(3) (IC(50)=2.6 x 10(-8) M)>-C(2)H(5) (IC(50)=4.4 x 10(-8) M)>-C(3)H(7) (IC(50)=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.

Figure 1

Chemical structures of cromakalim and of the newly synthesised benzoxazine derivatives. The 2H-1,4-benzoxazine derivatives are structural analogues of benzopyrans, the difference being the replacement of the carbon atom at position 4 with a nitrogen atom. The new molecules contain different alkyl substituents of variable length such as the methyl, ethyl and propyl at position 2 of the benzoxazine nucleus. All the synthesized molecules have a pyridylamine group with hydrogen bond forming capacity at position 3 and an electron-withdrawing group such as the chlorine atom at position 6 of the benzoxazine nucleus. The introduction of different alkyl substituents at position 2 of the benzoxazine ring gives the opportunity to evaluate the influence of the increase of lipophilicity and/or of alkyl chain length in this part of the molecule.

Figure 2

Effects of K⁺ channel openers on K_ATP channels of rat skeletal muscle fibres in the presence of internal ATP. (a) Digital average of K_ATP current recorded in the excised inside-out macropatches, at −60 mV (V m), with high cytosolic KCl solutions on both sides of the membrane, in the absence (controls) (n=31 macropatches), in the presence of 10⁻⁴ M concentration of ATP (n=31 macropatches), in the presence of 10⁻⁴ M ATP+the 2H-1,4-benzoxazine derivatives having the methyl (n=6 macropatches), ethyl (n=5 macropatches) or propyl (n=7 macropatches) groups at position 2 of the benzoxazine ring or in the presence of 10⁻⁴ M ATP+cromakalim (n=4 macropatches). C and O in the traces indicate closed and open channel levels, respectively, (b) Concentration–response relation of the 2H-1,4-benzoxazine derivatives having the–CH₃, –C₂H₅ or –C₃H₇ groups at position 2 of the benzoxazine ring, and of cromakalim on the K_ATP currents inhibited by a 10⁻⁴ M concentration of internal ATP. All the compounds under investigation including cromakalim produced a concentration-dependent increase of the K_ATP current at the lower doses, in contrast inhibiting it at the highest doses, (c) Log P plot versus the DE₅₀ of the 2H-1,4-benzoxazine derivatives and cromakalim. An inverse relation was observed between the log P, as index of lipophilicity, and DE₅₀ of the compounds under investigation showing a coefficient of correlation of −0.97.

Figure 3

Effects of K⁺ channel openers on K_ATP channels of rat skeletal muscle fibres in the absence of internal ATP. (a) Digital average of K_ATP current recorded in the excised inside-out macropatches, at −60 mV (V m), with high cytosolic KCl solutions on both sides of the membrane, in the absence (controls) (n=26 macropatches), or in the presence of the 2H-1,4-benzoxazine derivatives having the methyl (n=5 macropatches), ethyl (n=4 macropatches) or propyl (n=6 macropatches) groups at position 2 on the benzoxazine ring or in the presence of cromakalim (n=7 macropatches). C and O in the traces indicate closed and open channel levels, respectively. (b) Concentration–response relations of the 2H-1,4-benzoxazine derivatives having the –CH₃, –C₂H₅ or –C₃H₇ groups at position 2 of the benzoxazine ring, and of cromakalim on the K_ATP currents in the absence of ATP. All the compounds under investigation produced a concentration-dependent inhibition of the K_ATP current, (c) Log P plot versus the IC₅₀ of the 2H-1,4-benzoxazine derivatives and cromakalim. The calculated coefficient of correlation between the log P and IC₅₀ of the compounds under investigation was 0.8.