Structural requisites of 2‐(p‐chlorophenoxy)propionic acid analogues for activity on native rat skeletal muscle chloride conductance and on heterologously expressed CLC‐1

Abstract

1 The 2-(p-chlorophenoxy)propionic acid (CPP) modulates in a stereoselective manner the macroscopic chloride conductance (gCl), the electrical parameter sustained by the CLC-1 channel, of skeletal muscle. In order to determine the structural requirements for modulating native gCl and to identify high-affinity ligands, the effects of newly synthesised CPP analogues have been evaluated on gCl of rat EDL muscle fibres by means of the two-microelectrode current-clamp technique. 2 Each type of the following independent modification of CPP structure led to a three-to 10-fold decrease or to a complete lack of gCl-blocking activity: replacement of the electron-attractive chlorine atom of the aromatic ring, substitution of the oxygen atom of the phenoxy group, modification at the chiral centre and substitution of the carboxylic function with a phosphonate one. 3 The analogues bearing a second chlorophenoxy group on the asymmetric carbon atom showed a significant gCl-blocking activity. Similar to racemate CPP, the analogue with this group, spaced by an alkyl chain formed by three methylenic groups, blocked gCl by 45% at 100 mm.4 These latter derivatives were tested on heterelogously expressed CLC-1 performing inside-out patch-clamp recordings to further define how interaction between drug and channel protein could take place. Depending on the exact chemical nature of modification, these derivatives strongly blocked CLC-1 with K D values at À140 mV ranging from about 4 to 180 mm. 5 In conclusion, we identified four molecular determinants pivotal for the interaction with the binding site on muscle CLC-1 channels: (a) the carboxylic group that confers the optimal acidity and the negative charge; (b) the chlorophenoxy moiety that might interact with a hydrophobic pocket; (c) the chiral centre that allows the proper spatial disposition of the molecule; (d) an additional phenoxy group that remarkably stabilises the binding by interacting with a second hydrophobic pocket.

Molecular switch for CLC-K Cl − channel block/activation: Optimal pharmacophoric requirements towards high-affinity ligands

“…In the last few years, the study of 2-(p-chlorophenoxy)propionic acid (CPP) derivatives and fenamates, allowed us to open the way toward a pharmacological characterization of heterologously expressed CLC-K channels (12,13,(17)(18)(19)(20). Particularly, we discovered the presence of an activating and a blocking extracellular drug-binding site.…”

“…In the last few years, working with two structurally unrelated classes of compounds (CPP derivatives and fenamates), we identified CLC-K activators and inhibitors with moderate affinity (12,13,(17)(18)(19)(20). The only CLC-K opener identified was niflumic acid (NFA).…”

Elucidation of the enantioselective recognition mechanism of a penicillin G acylase‐based chiral stationary phase towards a series of 2‐aryloxy‐2‐arylacetic acids

“…Finally, to investigate the elution order of the enantiomers of this series, some compounds (1-3, 12, 15, 16) were synthesized in optically active form. For these derivatives, the absolute configuration was already known (1-3, 15) 25 or assigned by chemical correlation (12,16).…”

Investigations of Pharmacologic Properties of the Renal CLC-K1 Chloride Channel Co-expressed with Barttin by the Use of 2-(p-Chlorophenoxy)Propionic Acid Derivatives and Other Structurally Unrelated Chloride Channels Blockers

“…In this regard, it is interesting to notice that the activity of derivative N11 is specific for the renal CLC-K1 channel. Indeed, this derivative binds CLC-1 with a much smaller potency with respect to CPP, producing only a slight inhibition of native gCl of rat skeletal muscle fibers as well as of expressed CLC-1 channel (19). Recently, the inhibitor binding site for 9-AC and the more simple clofibrate derivative p-chlorophenoxy-acetic acid (CPA) have been mapped on CLC-1 (27).…”

“…All tested compounds were synthesized in our laboratory as racemate mixtures according to procedures previously reported: CPP (N1) (15); CPV (N2) and CPPA (N12) (16); N11 (17); N3, N6, N9, and N10 (18); N4, N5, and N7 (19). Derivative N8 was Figure 1.…”