Allyl m-Trifluoromethyldiazirine Mephobarbital: An Unusually Potent Enantioselective and Photoreactive Barbiturate General Anesthetic

Abstract

We synthesized 5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (14), a trifluoromethyldiazirine-containing derivative of general anesthetic mephobarbital, separated the racemic mixture into enantiomers by chiral chromatography, and determined the configuration of the (+)-enantiomer as S by x-ray crystallography. Additionally, we obtained the 3H-labeled ligand with high specific radioactivity. R-(−)-14 is an order of magnitude more potent than the most potent clinically used barbiturate, thiopental, and its general anesthetic EC50 approaches those for propofol and etomidate, whereas S-(+)-14 is tenfold less potent. Furthermore, at concentrations close to its anesthetic potency, R-(−)-14 both potentiated GABA-induced currents and increased the affinity for the agonist muscimol in human α1β2/3γ2L GABAA receptors. Finally, R-(−)-14 was found to be an exceptionally efficient photolabeling reagent, incorporating into both α1 and β3 subunits of human α1β3 GABAA receptors. These results indicate R-(−)-14 is a functional general anesthetic that is well-suited for identifying barbiturate binding sites on Cys-loop receptors.

Specificity of Intersubunit General Anesthetic-binding Sites in the Transmembrane Domain of the Human α1β3γ2 γ-Aminobutyric Acid Type A (GABAA) Receptor*

“…The NH of barbituric acid was pointing down, 4 Å from ␥2Phe-304. 2) at anesthetic concentrations and compared the patterns of subunit photolabeling to those seen for the ␣1␤3 GABA A R (15,16). When GABA A R subunits were resolved by SDS-PAGE after photolabeling, the two preparations appeared essentially the same based upon Coomassie Blue stain, with three bands migrating at ϳ56, ϳ59, and ϳ61 kDa ( Fig.…”

“…GABA A R-R-mTFD-MPAB acts as a potent tadpole general anesthetic, characterized by an EC 50 of 3.7 M and as a GABA A R potentiator at anesthetic concentrations, whereas S-mTFD-MPAB was 10-fold less potent as an anesthetic and potentiated weakly GABA A R responses (16). In the absence of GABA, R-and S-mTFD-MPAB each produced a concentration-dependent inhibition of R- [ 3 H]mTFD-MPAB photolabeling, characterized by IC 50 values of 1.4 Ϯ 0.2 and 34 Ϯ 6 M, respectively, with high concentrations of either enantiomer inhibiting subunit incorporation by Ͼ95% (Fig.…”

“…3 H] R-mTFD-MPAB (38 Ci/mmol) were synthesized as described (16). Racemic MPAB and MPPB (5-propyl-1-methyl-5-phenylbarbituric acid) were synthesized by phenylation of diethyl allylmalonate and diethyl propylmalonate, respectively, with diphenyliodonium trifluoroacetate.…”

“…Additionally, R-and S-MPPB were prepared from the corresponding isomers of MPAB by catalytic reduction with hydrogen gas on Pd/C. Racemic mBr-MPAB (5-allyl-1-methyl-5-(m-bromophenyl-barbituric acid) was synthesized analogously to the recently described methods (16) by reaction of 5-allyl-1-methyl barbiturate with (3-bromophenyl)(4-methoxyphenyl)iodonium. Other barbiturates were from commercial sources.…”

“…The etomidate-binding sites, identified by photoaffinity labeling of amino acids in ␤M3 and ␣M1, are in the two ␤ ϩ -␣ Ϫ subunit interfaces about 50 Å below the agonist sites (14,15). We reported recently that the R-enantiomer of 5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (mTFD-MPAB) is an extremely potent, photoreactive barbiturate that rivals etomidate in potency and stereoselectivity (16). Here, we report that R- [ 3 H]mTFD-MPAB photolabels new anesthetic-binding sites in human ␣1␤3␥2 GABA A Rs at the ␣ ϩ -␤ Ϫ and ␥ ϩ -␤ Ϫ subunit interfaces.…”