Synthesis of Trifluoromethylaryl Diazirine and Benzophenone Derivatives of Etomidate that Are Potent General Anesthetics and Effective Photolabels for Probing Sites on Ligand-Gated Ion Channels

Abstract

To locate the binding sites of general anesthetics on ligand-gated ion channels, two derivatives of the intravenous general anesthetic etomidate (2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate), in which the 2-ethyl group has been replaced by photoactivable groups based on either aryl diazirine or benzophenone chemistry, have been synthesized and characterized pharmacologically. TDBzl-etomidate (4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl 1-(1-phenylethyl)-1H-imidazole-5-carboxylate) and BzBzl-etomidate (4-benzoylbenzyl-1-(1-phenylethyl)-1H-imidazole-5-carboxylate are both potent general anesthetics with half-effective anesthetic concentrations of 700 and 220 nM, respectively. Both agents resembled etomidate in enhancing currents elicited by low concentrations of GABA on heterologously expressed GABAA receptors and in shifting the GABA concentration-response curve to lower concentrations. They also allosterically enhanced the binding of flunitrazepam to mammalian brain GABAA receptors. Both agents were also effective and selective photolabels, photoincorporating into some, but not all, subunits of the Torpedo nicotinic acetylcholine receptor to a degree that was allosterically regulated by an agonist or a noncompetitive inhibitor. Thus, they have the necessary pharmacological and photochemical properties to be useful in identifying the site of etomidate-induced anesthesia.

Identification of Binding Sites in the Nicotinic Acetylcholine Receptor for TDBzl-etomidate, a Photoreactive Positive Allosteric Effector

“…Nonradioactive TDBzl-etomidate and [ 3 H]TDBzl-etomidate (16 Ci/mmol) were synthesized as described previously (23 )] ϩ f ns where f(x) is the current or total radioligand bound in the presence of inhibitor concentration x, f 0 is the current or specific radioligand bound in the absence of inhibitor, f ns is the leak current in the absence of ACh or nonspecific radioligand binding, and IC 50 is the concentration of inhibitor associated with the inhibition of 50% of ACh-induced currents or radioligand binding.…”

“…We report here that TDBzl-etomidate, a photoreactive general anesthetic developed to provide an improved definition of etomidate binding sites in GABA A Rs (23) (Fig. 1), acts as a novel positive allosteric modulator of muscle-type Torpedo nAChR.…”

Mechanisms of neurosteroid interactions with GABAA receptors

“…Photoaffinity labeling has successfully been used to locate the benzodiazepine (Davies et al, 1996;Duncalfe et al, 1996;Sawyer et al, 2002;Wieland et al, 1992) and muscimol (Smith & Olsen, 1994) binding sites on the GABA A receptor, and sites for etomidate (Husain et al, 2006;Ziebell et al, 2004) and octanol (Pratt et al, 2000) on the Torpedo nicotinic acetylcholine receptor. In principle photoaffinity labeling techniques should provide a comprehensive search of all possible protein targets and should not be confounded by either multiple regions of protein contributing to a binding site or by the existence of multiple binding sites.…”

Convenient Synthesis of a [1‐14C]Diazirinylbenzoic Acid as a Photoaffinity Label for Binding Studies of V‐ATPase Inhibitors

“…The 14 C-labeled benzoic acid 11 is generally suitable for attachment to alcohols and amines, yet it can also lead to a photoaffinity label for carboxylic acids, since the carboxy moiety in 11 can easily be reduced to a hydroxymethyl group. [26] We expect that the convenient synthesis of 11 together with the long half-life of 14 C will make it the photoaffinity label of choice in several future binding studies.…”