p-Trifluoromethyldiazirinyl-etomidate: a potent photoreactive general anesthetic derivative of etomidate that is selective for ligand-gated cationic ion channels

Abstract

We synthesized the R- and S-enantiomers of ethyl 1-(1-(4-(3-((trifluoromethyl)-3H-diazirin-3-yl)phenyl)ethyl)-1H-imidazole-5-carboxylate (trifluoromethyldiazirinyl-etomidate), or TFD-etomidate, a novel photoactivable derivative of the stereoselective general anesthetic etomidate (R-(2-ethyl 1-(phenylethyl)-1H-imidazole-5-carboxylate)). Anesthetic potency was similar to etomidate's, but stereoselectivity was reversed and attenuated. Relative to etomidate, TFD-etomidate was a more potent inhibitor of the excitatory receptors, nAChR (nicotinic acetylcholine receptor) ((alpha1)(2)beta1delta1gamma1) and 5-HT(3A)R (serotonin type 3A receptor), causing significant inhibition at anesthetic concentrations. S- but not R-TFD-etomidate enhanced currents elicited from inhibitory alpha1beta2gamma2L GABA(A)Rs by low concentrations of GABA, but with a lower efficacy than R-etomidate, and site-directed mutagenesis suggests they act at different sites. [(3)H]TFD-etomidate photolabeled the alpha-subunit of the nAChR in a manner allosterically regulated by agonists and noncompetitive inhibitors. TFD-etomidate's novel pharmacology is unlike that of etomidate derivatives with photoactivable groups in the ester position, which behave like etomidate, suggesting that it will further enhance our understanding of anesthetic mechanisms.

Figure 1

Etomidate and TFD-etomidate modulation of GABA-induced currents through human wild type α1β2 2L GABA_A receptors expressed in Xenopus oocytes. GABA concentration response curves were measured before and after the addition of drug, with 2 – 3 recordings done at each concentration. Current recordings were normalized to the maximal GABA response. A. Data shown for the experiment with S-TFD-etomidate represents experiments on 3 different oocytes. Nonlinear least squares fitting gave (EC50 in μM and Hill coefficient, n_H): for the GABA control (■), EC₅₀ = 17 ± 2, n_H = 1.5 ± 0.1, I_max = 0.9 ± 0.02, and in the presence of 10 μM S-TFD-etomidate (▲), EC₅₀ = 6.4 ± 1.6, n_H = 0.9 ± 0.2, I_max = 0.90 ± 0.05. B. The experiment with R-TFD-etomidate was done on one oocyte due to the high concentration of reagent needed. For the GABA control (■), EC₅₀ = 25 ± 1, n_H =1.4 ± 0.1, I_max = 1.01 ± 0.01. In the presence of 34 μM R-TFD-etomidate (▼), EC₅₀ = 30 ± 2.5, n_H = 1.3 ± 0.04, I_max = 0.73 ± 0.02. C. S-TFD-etomidate modulates an etomidate-insensitive mutant. The top line of current traces show the effects of the applications of S-TFD-etomidate and R-etomidate on 3 μM GABA currents elicited from wild type (α1β2γ2L) GABA_A receptors using 5 and 10 μM S-TFD-etomidate and then 5 μM R-etomidate. The middle line of current traces shows the same series of applications of drugs on 1 μM GABA currents elicited from the mutant (α1β2M286Wγ2L) GABA receptor.

Figure 2

TFD-etomidate does not interact allosterically with GABA_A receptor ligands. Effect of TFD-etomidate and etomidate on: A. 1 nM [³H]flunitrazepam binding; B. 5 nM [³H]muscimol binding both to cerebral cortex membranes.

Figure 3

Actions on the 5-HT_3A receptor of TFD-etomidate and etomidate. A. Anesthetic concentration-dependence of the percent peak current amplitude evoked by 100 μM 5-HT. For both agents, the percent peak current amplitude decreased with anesthetic concentration. The IC₅₀s were 60 ± 5 μM and 9 ± 1 μM and n_H = 1.5 ± 0.2 and 0.9 ± 0.1 for etomidate and TFD-etomidate, respectively. The inset shows representative current traces (100 μM 5-HT) obtained in the absence of anesthetic (grey) and in the presence of 10 μM TFD-etomidate. B. Anesthetic concentration-dependence for currents activated in the absence of agonist. The ordinate is normalized to the current elicited by 100 μM 5-HT. For both etomidate and TFD-etomidate, this activation current increased linearly with anesthetic concentration with slopes of 3.9 ± 0.4 μM⁻¹ and 38 ± 0.8 μM⁻¹ for etomidate and TFD-etomidate, respectively. The inset shows a representative current trace obtained upon application of 10 μM TFD-etomidate alone.

Figure 4

TFD-etomidate is a more potent inhibitor of ACh-induced currents than etomidate. Oocytes expressing wild type Torpedo nAChRs were tested with 10 μM ACh (~EC₁₀) and then with 10 μM ACh plus increasing amounts of etomidate or derivatives. The current traces in the top right inset show one oocyte’s current response to 10 μM ACh plus increasing amounts of R-etomidate (0, 3, 10, 30, 100, and 300 μM and 1 and 3 mM). The effect of TFD-etomidate on 10 μM ACh currents is shown in the bottom left inset (0, 3, 10, 30, 100 and 300 μM TFD-etomidate). Nonlinear least squares analysis of the curves yielded: for R-etomidate (○), IC₅₀ = 21 ± 3 μM, n_H = 0.9 ± 0.1 (3 oocytes); for TFD-etomidate, IC₅₀ = 4.1 ± 0.5 μM, n_H =1.1 ± 0.07 (data not shown, 1 oocyte, each response was tested at least three times at each TFD-etomidate concentration); for R-TFD-etomidate (▼), IC₅₀ = 11.3 ± 1.6 μM, n_H = 0.7 ± 0.1 (2 oocytes with 2 – 3 recordings at each concentration), and for S-TFD-etomidate (▲): IC₅₀ = 4.7 ± 0.5 μM, n_H = 0.8 ± 0.1). Currents were normalized to the 10 μM ACh response. Graphs for R- and S-TFD-etomidate represent data from 2 oocytes with at least 2 – 3 recordings at each drug concentration.

Figure 5

Photoincorporation of [³H]TFD-etomidate into Torpedo nAChR. nAChR-rich membranes were photolabeled with 0.3 μM [³H]TFD-etomidate as described in Experimental Procedures. A, Polypeptides were resolved by SDS-PAGE, visualized by Coomassie Blue Stain (lane 1), and processed for fluorography (14 day exposure, lanes 2–8). Membrane suspensions were photolabeled with [³H]TFD-etomidate in the absence (lane 2) or the presence of 200 μM carbamylcholine (lane 3), 200 μM carbamylcholine and 100 μM proadifen (lane 4), 100 μM proadifen (lane 5), 100 μM tetracaine (lane 6), 200 μM carbamylcholine and 100 μM PCP (lane 7), or 100 μM PCP (lane 8). The stained polypeptide bands corresponding to the nAChR α, β, γ, and δ subunits, rapsyn (Rsn), the α subunit of the Na⁺/K⁺-ATPase (αN/K, 90 kDa), calelectrin (37K) and the mitochondrial voltage-dependent anion channel (VDAC) are indicated on the left. B, the ³H incorporation in the excised nAChR α, β, γ, and δ subunit gel bands was determined by liquid scintillation counting (average and range for two photolabelings performed in parallel).

Figure 6

Does the Meyer-Overton rule correctly predict TFD-etomidate’s anesthetic potency? The EC₅₀ concentration for loss of righting reflexes (LoRR) in tadpoles is plotted against the octanol/water partition coefficient for 25 general anesthetics. The conventional general anesthetics are shown as black circles, and the photoactivable general anesthetics as blue circles except for R- and S-TFD-etomidate, which are shown as red squares. The solid line is the least squares fit to all 25 general anesthetics and the dotted line to agents 1–13. The slope was constrained to be −1 as demanded by the Meyer-Overton rule and the intercepts (± standard deviation) are 1.42 ± 0.12 and −1.13 ± 0.07 respectively. Key #, Agent: 1, Methanol; 2, Ethanol; 3, Propanol; 4, Diethylether; 5, Butanol; 6, Pentanol; 7, Hexanol; 8, Amobarbital; 9, Heptanol; 10, Halothane; 11, Methoxyflurane; 12, Pentobarbital; 13, Octanol; 14, 3-Azioctanol; 15, S(−)-Azietomidate; 16, Propofol; 17, R(+)-Azietomidate; 18, BzBzl-etomidate; 19, TDBzl-etomidate; 20, S-TFD-etomidate; 21, R-TFD-etomidate; 22, R(+)-etomidate; 23, R(+)-Etomidate azide; 24, S(−)-Etomidate azide; 25, S(−)-Etomidate. Data from ^{, , , , ,}

Scheme 1

Photoactivable derivatives of the general anesthetic etomidate

Scheme 2

Synthesis of TFD-etomidate. (a) tert-butyldimethylsilyl chloride, DBU, dichloromethane; (b) (i) n-butyllithium, THF, (ii) diethyl trifluoroacetamide, THF; (iii) water/ammonium chloride; (c) hydroxylamine hydrochloride, pyridine; (d) p-toluene sulfonyl chloride, triethylamine, dimethylamino pyridine, dichloromethane; (e) liquid ammonia, ether; (f) iodine, triethylamine, dichloromethane; (g) triphenylphosphine dibromide, dichloromethane; (h) ammonia, methanol; (i) ethyl chloroacetate, triethylamine, DMF; (j) formic acid, diisopropyl carbodiimide, pyridine, dichloromethane; (k) (i) paraffinic sodium suspension, THF, ethanol, ethyl formate, (ii) conc. HCl, KSCN, water; (l) sodium nitrite, nitric acid, water, chloroform; (m) (i) tetrabutylammonium fluoride, THF, (ii) ammonium chloride. Mitsunobu reaction: (n) ethyl 1H-imidazole-5-carboxylate, THF; (o) triphenylphosphine, THF; (p) tert-butylazodicarboxylate, THF.